BR122024005246A2 - METHOD OF PURIFICATION OF A BINDING PROTEIN PRODUCED BY A HOST CELL - Google Patents

Abstract

"METHOD OF PURIFYING A BINDING PROTEIN PRODUCED BY A HOST CELL". The present invention relates to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein has dual variable domains that have a cross-over orientation and wherein a second pair of polypeptides forming the binding protein has a single variable domain. The invention also provides methods for preparing trispecific and/or trivalent binding proteins and uses of such binding proteins.

Description

REFERENCES TO RELATED APPLICATIONS

[001] This Application claims priority benefit of U.S. Provisional Application Serial No. 62/322,036, filed April 13, 2016; U.S. Provisional Application Serial No. 62/331,191, filed May 3, 2016; Provisional Patent Application No. 62/412,187, filed October 24, 2016; and EP Application EP17305298.6, filed March 17, 2017; all of which are incorporated herein by reference in their entirety.

PRESENTATION OF SEQUENCE LISTING IN ASCII TEXT FILE

[002] The contents of the following ASCII text file presentation are incorporated herein by reference in their entirety: a computer-readable form (CRF) of the Sequence Listing (file name: 183952027140SEQLISTING.txt, date registered: April 11, 2017, size: 200 KB).

FIELD OF INVENTION

[003] The invention relates to trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein has dual variable domains that have a cross-over orientation and wherein a second pair of polypeptides forming the binding protein has a single variable domain. The invention also relates to methods for preparing trispecific and/or trivalent binding proteins and uses of such binding proteins.

BACKGROUND

[004] Monoclonal antibody-based biotherapeutics have become an important avenue for novel drug development. Monoclonal antibody technology offers specific targeting, precise delivery of signaling and/or payload to a specific cell population, and confers a long-lasting biological effect through its Fc functions. Efforts in antibody engineering have enabled the development of bispecific antibodies that combine the specificities of two monoclonal antibodies for various biological applications, expanding the scope of antibody drug development. Newly discovered neutralizing antibodies with enhanced breadth and potency may provide more options for the development of biotherapeutic agents to treat complex diseases such as cancer, arthritis, and/or inflammatory disorders.

BRIEF SUMMARY

[005] Provided herein are multispecific binding proteins (e.g., antibodies) that form three antigen-binding sites. These binding proteins can specifically bind to one, two, or three antigenic targets or target proteins.

[006] In one embodiment, the invention provides a binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more antigenic targets or target proteins, wherein a first polypeptide chain has a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[007] and a second polypeptide chain has a structure represented by the formula: VH1-L3-VH2-L4-CH1[II]

[008] and a third polypeptide chain has a structure represented by the formula: VH3-CH1 [III]

[009] and a fourth polypeptide chain has a structure represented by the formula: VL3-CL [IV]

[0010] where:

[0011] VL1 is a first immunoglobulin light chain variable domain;

[0012] VL2 is a second immunoglobulin light chain variable domain;

[0013] VL3 is a third immunoglobulin light chain variable domain;

[0014] VH1 is a first immunoglobulin heavy chain variable domain;

[0015] VH2 is a second immunoglobulin heavy chain variable domain;

[0016] VH3 is a third immunoglobulin heavy chain variable domain;

[0017] CL is an immunoglobulin light chain constant domain;

[0018] CH1 is an immunoglobulin CH1 heavy chain constant domain; and

[0019] L1, L2, L3 and L4 are amino acid linkers;

[0020] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[0021] In some embodiments, the second and/or third polypeptide chain further comprises a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains.

[0022] In another embodiment, the invention provides a binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[0023] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[0024] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3 [III]

[0025] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[0026] where:

[0027] VL1 is a first immunoglobulin light chain variable domain;

[0028] VL2 is a second immunoglobulin light chain variable domain;

[0029] VL3 is a third immunoglobulin light chain variable domain;

[0030] VH1 is a first immunoglobulin heavy chain variable domain;

[0031] VH2 is a second immunoglobulin heavy chain variable domain;

[0032] VH3 is a third immunoglobulin heavy chain variable domain;

[0033] CL is an immunoglobulin light chain constant domain;

[0034] CH1 is an immunoglobulin heavy chain CH1 constant domain;

[0035] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[0036] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[0037] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[0038] L1, L2, L3 and L4 are amino acid linkers;

[0039] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[0040] In some embodiments, the binding protein is trispecific and capable of specifically binding to three different antigenic targets. In some embodiments, the binding protein is trivalent but bispecific and capable of specifically binding to three antigenic targets, two of which are identical. In some embodiments, the binding protein of the present invention is trivalent but monospecific and capable of specifically binding to three antigenic targets, all of which are identical. In some embodiments, the binding protein is capable of inhibiting the function of one or more target proteins. In some embodiments, the binding protein is trispecific and capable of specifically binding to three different antigenic targets.

[0041] In some embodiments, a binding protein of the present invention comprises one, two, or three antigen-binding sites that specifically bind to a target protein selected from A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, the IgE receptor), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GI- TRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, , IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as integrin b4), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STE-AP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more of the above antigenic targets are human antigenic targets. In some embodiments, the binding protein of the present invention is trispecific and capable of specifically binding to three different antigenic targets selected from the list above. In some embodiments, the binding protein of the present invention is trivalent but bispecific and capable of specifically binding to three antigenic targets selected from the list above, two of which are identical. In some embodiments, the binding protein of the present invention is trivalent but monospecific and capable of specifically binding to three antigenic targets selected from the list above, all of which are identical. In some embodiments, the binding protein specifically binds to three target proteins corresponding to two target proteins on T cells and one target tumor protein. In some embodiments, one of said target proteins on T cells is CD3. In some embodiments, one of said target proteins on T cells is CD28. In some embodiments, said target tumor protein is CD38. In some embodiments, the binding protein specifically binds to three target proteins corresponding to two target proteins on T cells and a target protein selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, quiti- nase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, trifosfato difosfo-hidrolase 1 de ectonucleosídeo, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, 7, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1.

[0042] In another embodiment, the invention provides a binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain has a structure represented by the formula: V L2-L1-VL1-L2-CL[I]

[0043] and a second polypeptide chain has a structure represented by the formula: VH1-L3-VH2-L4-CH1[II]

[0044] and a third polypeptide chain has a structure represented by the formula: V H3-CH1 [III]

[0045] and a fourth polypeptide chain has a structure represented by the formula: V L3-CL [IV]

[0046] where:

[0047] VL1 is a first immunoglobulin light chain variable domain;

[0048] VL2 is a second immunoglobulin light chain variable domain;

[0049] VL3 is a third immunoglobulin light chain variable domain;

[0050] VH1 is a first immunoglobulin heavy chain variable domain;

[0051] VH2 is a second immunoglobulin heavy chain variable domain;

[0052] VH3 is a third immunoglobulin heavy chain variable domain;

[0053] CL is an immunoglobulin light chain constant domain;

[0054] CH1 is an immunoglobulin CH1 heavy chain constant domain; and

[0055] L1, L2, L3 and L4 are amino acid linkers;

[0056] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[0057] where:

[0058] (a) VL1, VL2, and VL3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 10, 14, 18, 22, 115;

[0059] (b) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions of a variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 43-59, 123-125;

[0060] (c) VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 151, 153, 155, 157, 159, 161, 163, 165, and 167;

[0061] (d) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 43-59, 123-125, 138-140, and 149; or

[0062] (e) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions and/or a variable domain sequence shown in Tables 2-5; and

[0063] where:

[0064] (a) VH1, VH2, and VH3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 9, 13, 17, 21, 114;

[0065] (b) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions of a variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-42, 120-122;

[0066] (c) VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 150, 152, 154, 156, 158, 160, 162, 164, and 166;

[0067] (d) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-42, 120-122, and 126-128; or

[0068] (e) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions and/or a variable domain sequence shown in Tables 2-5.

[0069] In some embodiments, the second and/or third polypeptide chain further comprises a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains.

[0070] In another embodiment, the invention provides a binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[0071] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[0072] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3 [III]

[0073] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[0074] where:

[0075] VL1 is a first immunoglobulin light chain variable domain;

[0076] VL2 is a second immunoglobulin light chain variable domain;

[0077] VL3 is a third immunoglobulin light chain variable domain;

[0078] VH1 is a first immunoglobulin heavy chain variable domain;

[0079] VH2 is a second immunoglobulin heavy chain variable domain;

[0080] VH3 is a third immunoglobulin heavy chain variable domain;

[0081] CL is an immunoglobulin light chain constant domain;

[0082] CH1 is an immunoglobulin CH1 heavy chain constant domain;

[0083] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[0084] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[0085] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[0086] L1, L2, L3 and L4 are amino acid linkers;

[0087] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[0088] where:

[0089] (a) VL1, VL2, and VL3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 10, 14, 18, 22, 115;

[0090] (b) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions of a variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 43-59, 123-125;

[0091] (c) VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 151, 153, 155, 157, 159, 161, 163, 165, and 167;

[0092] (d) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 43-59, 123-125, 138-140, and 149; or

[0093] (e) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions and/or a variable domain sequence shown in Tables 2-5;

[0094] where:

[0095] (a) VH1, VH2, and VH3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 9, 13, 17, 21, 114;

[0096] (b) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions of a variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-42, 120-122;

[0097] (c) VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 150, 152, 154, 156, 158, 160, 162, 164, and 166;

[0098] (d) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-42, 120-122, and 126-128; or

[0099] (e) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions and/or a variable domain sequence shown in Tables 2-5.

[00100] In some embodiments, VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 151, 153, 155, 157, 159, 161, 163, 165, and 167; and VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 150, 152, 154, 156, 158, 160, 162, 164, and 166. In some embodiments, VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 43-59, 123-125, 138-140, and 149; and (d) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-42, 120-122, and 126-128.

[00101] In some embodiments of any of the binding proteins described herein, (a) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45; (b) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45; (c) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57; (d) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57; (e) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 42; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 58, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59; (f) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 42; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 58, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59; (g) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 126, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 127 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 128; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 138, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 139 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 140; (h) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 126, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 127, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 128; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 138, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 139, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 140; (i) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 120, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 121, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 122; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125; or (j) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 120, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 121 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 122; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125. In some embodiments, (a) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[00102] In some embodiments, the binding protein comprises an antigen-binding site that specifically binds to a protein on the surface of a T cell and another antigen-binding site that specifically binds to a target antigen, e.g., a target tumor protein. In some embodiments, the binding protein comprises an antigen-binding site that specifically binds to CD3, an antigen-binding site that specifically binds to CD28, and an antigen-binding site that specifically binds to a target tumor protein selected from the group consisting of CD19, CD20, CD38, Her2, and LAMP1. In some embodiments, VH1 and VL1 form a first antigen-binding site that specifically binds to human CD3, VH2 and VL2 form a second antigen-binding site that specifically binds to human CD28, and VH3 and VL3 form a third antigen-binding site that specifically binds to a human tumor target protein. In some embodiments, VH1 and VL1 form a first antigen-binding site that specifically binds to human CD28, VH2 and VL2 form a second antigen-binding site that specifically binds to human CD3, and VH3 and VL3 form a third binding site that specifically binds to a human tumor target protein. In some embodiments, the antigen-binding site specifically binds to a human tumor target protein selected from the group consisting of CD19, CD20, CD38, Her2, and LAMP1. In some embodiments, the antigen-binding site that specifically binds to CD3 comprises: (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 152 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 153; or (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155. In some embodiments, the antigen-binding site that specifically binds to CD3 comprises six CDRs or a light chain and a heavy chain variable domain shown in Tables 2-5. In some embodiments, the antigen-binding site that specifically binds to CD28 comprises: (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 161; or (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 163. In some embodiments, the antigen-binding site that specifically binds to CD28 comprises six CDRs or a light chain and a heavy chain variable domain shown in Tables 2-5. In some embodiments, the antigen-binding site that specifically binds to a target tumor protein comprises: (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 156 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 157; (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 159; (c) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 165; (d) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 150 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 151; or (e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 166 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, the antigen-binding site that specifically binds to a target tumor protein comprises six CDRs or a light chain and a heavy chain variable domain shown in Tables 2-5. In some embodiments, the antigen-binding site that specifically binds to a target tumor protein comprises six CDRs or one light chain and one heavy chain variable domain of an anti-Her2, anti-CD19, anti-CD20, anti-CD38, or anti-LAMP1 binding domain shown in Tables 2-5.

[00103] In another embodiment, the invention provides a binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain has a structure represented by the formula: V L2-L1-VL1-L2-CL[I]

[00104] and a second polypeptide chain has a structure re-presented by the formula: VH1-L3-VH2-L4-CH1[II]

[00105] and a third polypeptide chain has a structure re-presented by the formula: V H3-CH1 [III]

[00106] and a fourth polypeptide chain has a structure represented by the formula: V L3-CL [IV]

[00107] where:

[00108] VL1 is a first immunoglobulin light chain variable domain;

[00109] VL2 is a second immunoglobulin light chain variable domain;

[00110] VL3 is a third immunoglobulin light chain variable domain;

[00111] VH1 is a first immunoglobulin heavy chain variable domain;

[00112] VH2 is a second immunoglobulin heavy chain variable domain;

[00113] VH3 is a third immunoglobulin heavy chain variable domain;

[00114] CL is an immunoglobulin light chain constant domain;

[00115] CH1 is an immunoglobulin CH1 heavy chain constant domain; and

[00116] L1, L2, L3 and L4 are amino acid linkers;

[00117] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[00118] where:

[00119] (a) VL1, VL2, and VL3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 61, 63, 69, 71, 74, 76, 82, 86, 88, 94; or

[00120] (b) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions of a variable domain of at least one amino acid sequence set forth in any one of SEQ ID NOs: 61, 63, 69, 71, 74, 76, 82, 86, 88, 94;

[00121] (c) VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 169, 171, and 173;

[00122] (d) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-147, 178, and 179; or

[00123] (e) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions and/or a variable domain sequence shown in Tables 2-5; and

[00124] where:

[00125] (a) VH1, VH2, and VH3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 60, 62, 68, 73, 75, 81, 85, 87, 93; or

[00126] (b) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions of a variable domain of at least one amino acid sequence set forth in any one of SEQ ID NOs: 60, 62, 68, 73, 75, 81, 85, 87, 93;

[00127] (c) VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 168, 170, and 172;

[00128] (d) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 129-137; or

[00129] (e) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions and/or a variable domain sequence shown in Tables 2-5.

[00130] In some embodiments, the second and/or third polypeptide chain further comprises a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains.

[00131] In another embodiment, the invention provides a binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00132] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3 [II]

[00133] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3 [III]

[00134] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[00135] where:

[00136] VL1 is a first immunoglobulin light chain variable domain;

[00137] VL2 is a second immunoglobulin light chain variable domain;

[00138] VL3 is a third immunoglobulin light chain variable domain;

[00139] VH1 is a first immunoglobulin heavy chain variable domain;

[00140] VH2 is a second immunoglobulin heavy chain variable domain;

[00141] VH3 is a third immunoglobulin heavy chain variable domain;

[00142] CL is an immunoglobulin light chain constant domain;

[00143] CH1 is an immunoglobulin CH1 heavy chain constant domain;

[00144] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[00145] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00146] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00147] L1, L2, L3 and L4 are amino acid linkers;

[00148] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[00149] where:

[00150] (a) VL1, VL2, and VL3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 61, 63, 69, 71, 74, 76, 82, 86, 88, 94;

[00151] (b) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions of a variable domain of at least one amino acid sequence set forth in any one of SEQ ID NOs: 61, 63, 69, 71, 74, 76, 82, 86, 88, 94;

[00152] (c) VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 169, 171, and 173;

[00153] (d) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-147, 178, and 179; or

[00154] (e) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions and/or a variable domain sequence shown in Tables 2-5;

[00155] where:

[00156] (a) VH1, VH2, and VH3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 60, 62, 68, 73, 75, 81, 85, 87, 93;

[00157] (b) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions of a variable domain of at least one amino acid sequence set forth in any one of SEQ ID NOs: 60, 62, 68, 73, 75, 81, 85, 87, 93;

[00158] (c) VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 168, 170, and 172;

[00159] (d) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 129-137;

[00160] (e) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions and/or a variable domain sequence shown in Tables 2-5.

[00161] In some embodiments, VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any of SEQ ID NOs: 169, 171, and 173; and VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any of SEQ ID NOs: 168, 170, and 172. In some embodiments, VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-147, 178, and 179; and VH1, VH2 and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 129-137.

[00162] In some embodiments of any of the binding proteins described herein, (a) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; (b) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; (c) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; (d) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; (e) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; (f) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; (g) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; (h) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; (i) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; or (j) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144. In some embodiments, one or more of VH1, VL1, VH2, VL2, VH3, and VL3 comprises one, two, or three CDR sequences of an antibody set forth in Tables 2-5.

[00163] In some embodiments, the binding protein comprises three antigen-binding sites, where one, two, or three of the antigen-binding site(s) specifically binds to a target cytokine protein selected from the group consisting of IL-4, IL-13, and TNFα. In some embodiments, (a) VH1 and VL1 form a first antigen-binding site that specifically binds to human TNFα, VH2 and VL2 form an antigen-binding site that specifically binds to human IL13, and VH3 and VL3 form an antigen-binding site that specifically binds to human IL14; (b) VH1 and VL1 form a first antigen-binding site that specifically binds to human TNFα, VH2 and VL2 form a second antigen-binding site that specifically binds to human IL14, and VH3 and VL3 form a third antigen-binding site that specifically binds to human IL13; (c) VH1 and VL1 form a first antigen-binding site that specifically binds to human IL14, VH2 and VL2 form a second antigen-binding site that specifically binds to human TNFα, and VH3 and VL3 form a third antigen-binding site that specifically binds to human IL13; (d) VH1 and VL1 form a first antigen-binding site that specifically binds to human IL14, VH2 and VL2 form a second antigen-binding site that specifically binds to human IL13, and VH3 and VL3 form a third antigen-binding site that specifically binds to human TNFα; (e) VH1 and VL1 form a first antigen-binding site that specifically binds to human IL13, VH2 and VL2 form a second antigen-binding site that specifically binds to human IL14, and VH3 and VL3 form a third antigen-binding site that specifically binds to human TNFα; or (f) VH1 and VL1 form a first antigen-binding site that specifically binds to human IL13, VH2 and VL2 form a second antigen-binding site that specifically binds to human TNFα, and VH3 and VL3 form a third antigen-binding site that specifically binds to human IL14. In some embodiments, the antigen-binding site that specifically binds to human TNFα comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 169. In some embodiments, the antigen-binding site that specifically binds to human IL14 comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 170 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 171. In some embodiments, the antigen-binding site that specifically binds to human IL13 comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 172 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 173.

[00164] In some embodiments of any of the binding proteins described herein, the second and/or third polypeptide chain further comprises a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains. In some embodiments, at least one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments, L1, L2, L3, or L4 is independently at least one amino acid in length. In some embodiments, the binding protein is trispecific and capable of specifically binding to three different antigenic targets. In some embodiments, the binding protein is trispecific and capable of specifically binding to three different antigenic targets. In some embodiments, the binding protein is capable of inhibiting the function of one or more target proteins.

[00165] In some embodiments of any of the binding proteins described herein, at least one of L1, L2, L3, or L4 is independently 0 amino acids in length. In some embodiments, L1, L2, L3, or L4 is independently at least one amino acid in length. In some embodiments, one, two, three, or all four of L1, L2, L3, and L4 are between 0 and 15 amino acids in length. In some embodiments, at least two of L1, L2, L3, and L4 are between 1 and 15 amino acids in length. In some embodiments, (a) L1, L2, L3, and L4 are each independently zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148); or (b) L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 175), L2 comprises the sequence TKGPS (SEQ ID NO: 106), L3 comprises the sequence S, and L4 comprises the sequence RT; L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; L1 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; or L1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 105), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 105), and L4 is 0 amino acids in length.

[00166] In some embodiments of any of the binding proteins described herein, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the second polypeptide chain further comprises a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and wherein the third polypeptide chain further comprises a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the first and/or second Fc regions comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; and wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments, the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the CH3 domains of the second and third polypeptide chains both comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the second polypeptide chain further comprises a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the third polypeptide chain further comprises a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; and wherein only one of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the CH3 domains of the second and third polypeptide chains are human IgG1 CH3 domains, and wherein only one of the CH3 domains comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the first and/or second Fc regions are human IgG4 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the CH3 domains of the second and third polypeptide chains are human IgG4 CH3 domains, and wherein the CH3 domains each comprise amino acid substitutions at positions corresponding to human IgG4 positions 228 and 409 according to the EU index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the second polypeptide chain further comprises a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains; wherein the third polypeptide chain further comprises a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains; wherein the first and/or second Fc regions are human IgG4 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the CH3 domains of the second and third polypeptide chains are human IgG4 CH3 domains, and wherein the CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the first and/or second Fc regions are human IgG1 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the CH3 domains of the second and third polypeptide chains are human IgG1 CH3 domains, and wherein the CH3 domains each comprise amino acid substitutions at positions corresponding to human IgG1 positions 234 and 235 according to the EU index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[00167] In some embodiments of any of the binding proteins described herein, the CL domain of the first polypeptide chain is a human kappa CL domain and the CL domain of the fourth polypeptide chain is a human lambda CL domain; or the CL domain of the first polypeptide chain is a human lambda CL domain and the CL domain of the fourth polypeptide chain is a human kappa CL domain. In some embodiments, the first polypeptide chain comprises a human lambda CL domain; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F; and wherein the fourth polypeptide chain comprises a CL kappa domain. In some embodiments, the first polypeptide chain comprises a CL lambda domain; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F; and wherein the fourth polypeptide chain comprises a kappa CL domain. In some embodiments, the first polypeptide chain comprises a lambda CL domain; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354, 366, 435, and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C, T366W, H435R, and Y436F; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the fourth polypeptide chain comprises a CL kappa domain. In some embodiments, the first polypeptide chain comprises a CL kappa domain; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R and Y436F; and wherein the fourth polypeptide chain comprises a lambda CL domain. In some embodiments, the second and/or third polypeptide chains comprise a human IgG1 or IgG4 Fc region.

[00168] In another embodiment, the invention provides a binding protein comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain wherein:

[00169] (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2;

[00170] (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2;

[00171] (c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14;

[00172] (d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14;

[00173] (e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 18

[00174] (f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 18;

[00175] (g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22;

[00176] (h) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22;

[00177] (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61;

[00178] (j) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61;

[00179] (k) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 71;

[00180] (l) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 76 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 76; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 75 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 75; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74;

[00181] (m) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 82; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 81 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 81; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74;

[00182] (n) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 88 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 88; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 87; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86;

[00183] (o) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 94 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 93 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 93; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86;

[00184] (p) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74;

[00185] (q) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86;

[00186] (r) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74;

[00187] (s) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86;

[00188] (t) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115; or

[00189] (u) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115.

[00190] In another embodiment, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the binding protein or polypeptide thereof according to any of the above embodiments. In another embodiment, the invention provides an expression vector comprising the nucleic acid molecule according to one of the above embodiments. In another embodiment, the invention provides an isolated host cell comprising the nucleic acid molecule according to any of the above embodiments. In another embodiment, the invention provides an isolated host cell comprising the expression vector according to any of the above embodiments. In some embodiments, the isolated host cell is a mammalian cell or an insect cell. In one embodiment, the invention provides a vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of a binding protein according to any of the above embodiments. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the one or more vectors are expression vectors. In one embodiment, the invention provides an isolated host cell comprising the vector system according to any of the above embodiments. In one embodiment, the invention provides a method of producing a binding protein, the method comprising: a) culturing a host cell according to any of the above embodiments under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. In one embodiment, the invention provides a pharmaceutical composition comprising the binding protein according to any of the above embodiments and a pharmaceutically acceptable carrier.

[00191] In another embodiment, the invention provides a method of preventing and/or treating cancer in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein or pharmaceutical composition according to any of the above embodiments. In another embodiment, the invention provides a binding protein or pharmaceutical composition according to any of the above embodiments for use in preventing and/or treating cancer in a patient. In another embodiment, the invention provides a binding protein according to any of the above embodiments for the manufacture of a medicament for preventing and/or treating cancer in a patient. In some embodiments, the binding protein comprises an antigen-binding site that specifically binds to a protein on the surface of a T cell and another antigen-binding site that specifically binds to a target tumor protein. In some embodiments, the binding protein comprises an antigen-binding site that specifically binds to CD3, an antigen-binding site that specifically binds to CD28, and an antigen-binding site that specifically binds to a target tumor protein selected from the group consisting of CD19, CD20, CD38, Her2, and LAMP1. In some embodiments, the at least one binding protein is co-administered with a chemotherapeutic agent. In some embodiments, the patient is a human. In some embodiments, the binding protein is capable of inhibiting the function of one or more target proteins selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, quiti- nase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, trifosfato difosfo-hidrolase 1 de ectonucleosídeo, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, , ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD- 1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TLR, , TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1.

[00192] In another embodiment, the invention provides a method of preventing and/or treating an inflammatory disease or disorder in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein or pharmaceutical composition according to any of the above embodiments. In another embodiment, the invention provides a binding protein or pharmaceutical composition according to any of the above embodiments for use in preventing and/or treating an inflammatory disease or disorder in a patient. In another embodiment, the invention provides a binding protein according to any of the above embodiments for the manufacture of a medicament for preventing and/or treating an inflammatory disease or disorder in a patient. In some embodiments, the binding protein comprises three antigen-binding sites that each specifically bind to a target cytokine protein selected from the group consisting of IL-4, IL-13, and TNFα. In some embodiments, two of the three binding sites specifically bind to a target cytokine protein selected from the group consisting of IL-4, IL-13, and TNFα. In some embodiments, the at least one binding protein is co-administered with an anti-inflammatory agent. In some embodiments, the patient is a human. In some embodiments, the binding protein is capable of inhibiting the function of one or more target proteins selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, 3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptina, LPFS2, MHC classe II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, receptor plaquetário, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, quinase Syk, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1.

[00193] In another embodiment, the invention provides a method of purifying a binding protein produced by a host cell comprising:

[00194] (a) producing, in a host cell, a binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more antigenic targets or target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00195] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[00196] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1[III]

[00197] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[00198] where:

[00199] VL1 is a first immunoglobulin light chain variable domain;

[00200] VL2 is a second immunoglobulin light chain variable domain;

[00201] VL3 is a third immunoglobulin light chain variable domain;

[00202] VH1 is a first immunoglobulin heavy chain variable domain;

[00203] VH2 is a second immunoglobulin heavy chain variable domain;

[00204] VH3 is a third immunoglobulin heavy chain variable domain;

[00205] CL is an immunoglobulin light chain constant domain;

[00206] CH1 is an immunoglobulin CH1 heavy chain constant domain;

[00207] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[00208] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00209] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00210] L1, L2, L3 and L4 are amino acid linkers;

[00211] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair; wherein only one of the CH3 domains of the second polypeptide chain and the CH3 domain of the third polypeptide chain comprise amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F;

[00212] (b) contacting the binding protein produced in (a) with Protein A; and

[00213] (c) eluting the Protein A binding protein under conditions suitable to isolate the binding protein from binding proteins comprising 0 or 2 CH3 domains comprising the amino acid substitutions H435R and Y436F.

[00214] In some embodiments, the CL domain of the first polypeptide chain is a human kappa CL domain and the CL domain of the fourth polypeptide chain is a human lambda CL domain; or the CL domain of the first polypeptide chain is a human lambda CL domain and the CL domain of the fourth polypeptide chain is a human kappa CL domain, and the method further comprises: (d) contacting the binding protein eluted in (c) with a light kappa chain affinity medium; and (e) eluting the binding protein from the light kappa chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only lambda CL domains. In some embodiments, the method further comprises, after (e), (f) contacting the binding protein eluted in (e) with a lambda light chain affinity medium; and (g) eluting the binding protein from the lambda light chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only CL kappa domains. In some embodiments, the CL domain of the first polypeptide chain is a human CL kappa domain and the CL domain of the fourth polypeptide chain is a human CL lambda domain; or the CL domain of the first polypeptide chain is a human CL lambda domain and the CL domain of the fourth polypeptide chain is a human CL kappa domain, and the method further comprises: (d) contacting the binding protein eluted in (c) with a lambda light chain affinity medium; and (e) eluting the binding protein from the lambda light chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only CL kappa domains. In some embodiments, the method further comprises, after (e), (f) contacting the binding protein eluted in (e) with a light kappa chain affinity medium; and (g) eluting the binding protein from the light kappa chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only lambda CL domains. In some embodiments, the first polypeptide chain comprises a lambda CL domain; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to the EU index, wherein the amino acid substitutions are S354C and T366W; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F; and wherein the fourth polypeptide chain comprises a CL kappa domain. In some embodiments, the binding protein is detected in one or more of (c) and (e) using hydrophobic interaction chromatography (HIC). In some embodiments, the CH3 domains and/or Fc regions of the second and third polypeptide chains are CH3 domains and/or Fc regions of human IgG1 or IgG4.

[00215] It should be understood that one, some or all of the properties of the different embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to those skilled in the art. These and other embodiments of the invention are further described by the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[00216] Figures 1A-1C show schematic representations of trispecific binding proteins comprising four polypeptide chains that form three antigen-binding sites that specifically bind three target proteins, wherein a first pair of polypeptides have dual variable domains that have a cross-orientation (VH1-VH2 and VL2-VL1) forming two antigen-binding sites, and wherein a second pair of polypeptides have a single variable domain (VH3 and VL3) forming a single antigen-binding site. Figure 1A shows a trispecific binding protein comprising a "knobs-into-holes" mutation, wherein the "knob" is in the second pair of polypeptides with a single variable domain. Figure 1B shows a trispecific binding protein comprising a "knobs-into-holes" mutation, wherein the "knob" is in the first pair of polypeptides with the cross-orientation. Figure 1C shows the orientation of the variable domains in the polypeptide chains and the "knob"/"hole" orientation for the binding proteins shown in Tables 1-3. "Heavy chain A" (e.g., a third polypeptide chain of the present invention) indicates the variable domain of heavy chain A. "Light chain A" (e.g., a fourth polypeptide chain of the present invention) indicates the variable domain of light chain A. "Heavy chain B" (e.g., a second polypeptide chain of the present invention) indicates variable domain 1 and variable domain 2 of heavy chain B. "Light chain B" (e.g., a first polypeptide chain of the present invention) indicates variable domain 1 and variable domain 2 of light chain B.

[00217] Figure 2 shows the results of an ELISA assay determining the binding of an anti-Her2 x CD28 x CD3 IgG4 trispecific antibody (Binding Protein 1) or isotype control antibody to human CD3, CD28, and Her2. Bound antibodies were detected using a horseradish peroxidase (HRP)-conjugated anti-Fab secondary antibody.

[00218] Figures 3A-3C show the results of antibody-mediated specific killing of Her2+ breast cancer cells using an anti-Her2 x CD28 x CD3 IgG4 trispecific antibody (referred to herein as "Binding Protein 1"), an anti-CD28 x CD3 IgG4 bispecific antibody (huCD28 x CD3), an anti-Her2 IgG1 antibody, or a control antibody (human IgG1), using human PBMC at E:T = 10. Figure 3A shows the results of trispecific antibody-mediated specific killing of ZR-75-1 cells. Figure 3B shows the results of trispecific antibody-mediated specific killing of AU565 cells. Figure 3C shows the results of FACS analysis determining cell surface expression of the indicated markers on ZR-75-1 and AU565 cells.

[00219] Figures 4 and 5 show the results of antibody-mediated specific killing of Her2+ breast cancer cells using an anti-her2 x CD28 x CD3 (Binding Protein 1) trispecific IgG4 antibody, an anti-CD28 x CD3 (huCD28 x CD3) bispecific antibody, an anti-Her2 IgG1 antibody, or a control antibody (human IgG1). Figure 4 shows the results of specific antibody-mediated killing of ZR-75-1 cells using human peripheral blood mononuclear cells (PBMCs) from donor KP45926 at E:T = 10. Figure 5 shows the results of specific antibody-mediated killing of AU565 cells using human PBMCs from donor KP45944 at E:T = 10. Figures 4 and 5 confirm similar cell killing results as shown in Figures 3A-3C using PBMCs from a different donor.

[00220] Figures 6A and 6B show the activation (CD69+) and proliferation of human T cells treated with anti-Her2 x CD28 x CD3 trispecific IgG4 binding protein (HER2/CD28sup xCD3mid; referred to herein as "Binding Protein 1"), anti-Her2 x CD28 x CD3 trispecific IgG4 binding protein lacking the anti-CD28 binding domain (HER2/ΔCD28sup xCD3mid), anti-Her2 x CD28 x CD3 trispecific IgG4 binding protein lacking the anti-CD3 binding domain (HER2/CD28sup xΔCD3mid), anti-Her2 x CD28 x CD3 trispecific IgG4 binding protein lacking both the anti-CD28 and anti-CD3 binding domains (HER2/Δ(CD28sup xΔCD3mid), antibody anti-CD3 control monoclonal antibody or IgG4 control antibody. Figure 6A shows the activation (CD69+) of human CD4+ T cells from three donors. Figure 6B shows the activation (CD69+) of human CD8+ T cells from three donors. CD28sup : anti-CD28 superagonist antibody. CD3mid : anti-CD3 antibody.

[00221] Figures 7A-C show activation of the IL-2, NFkB, and nuclear factor of activated T cells (NFAT) pathway via anti-CD3 and anti-CD28 signaling, as measured by luciferase assay using human Jurkat T cells with an IL-2 promoter-luciferase construct (Figure 7A), an NFKB promoter-luciferase construct (Figure 7B), or an NFAT promoter-luciferase construct (Figure 7C). Antibodies tested were those described above in reference to Figures 6A-6D.

[00222] Figure 8 shows the results of an ELISA assay that determines the binding of an anti-CD19 x CD28 x CD3 IgG4 trispecific antibody (referred to herein as "Binding Protein 3") or isotype control antibody to CD3, CD28, and CD19. Bound antibodies were detected using a horseradish peroxidase (HRP)-conjugated anti-Fab secondary antibody.

[00223] Figures 9a-9N show the results of specific antibody-mediated killing of CD19+ human GCB lymphoma cells using an anti-CD19 x CD28 x CD3 IgG4 trispecific antibody (referred to herein as "Binding Protein 3") or the indicated controls using human PBMCs as effector cells at E:T = 10. Figure 9A shows the results of specific antibody-mediated killing of OCI-LY19 cells. Figure 9B shows the results of FACS analysis determining cell surface expression of the indicated markers on OCI-LY19 cells. Figure 9C shows the results of specific antibody-mediated killing of OCI-LY19 cells using PBMCs from donor KP48572 at E:T = 10. Figure 9D shows the results of specific antibody-mediated killing of OCI-LY19 cells using PBMCs from donor KP48573 at E:T = 10. Figure 9E shows the results of specific antibody-mediated killing of KARPASS-422 human lymphoma cells using PBMCs from donor KP48572 at E:T = 10. Figure 9F shows the results of specific antibody-mediated killing of KARPASS-422 cells using PBMCs from donor KP48573 at E:T = 10. Figure 9G shows the results of specific antibody-mediated killing of MeC1 human chronic B-cell leukemia cells using PBMCs from donor KP48572 at E:T = 10. Figure 9H shows the results of specific antibody-mediated killing of human multiple myeloma cells RPMI8226 using PBMCs from donor KP48775 at E:T = 10. Figure 9I shows the results of specific antibody-mediated killing of human Burkitt's lymphoma cells Raji using PBMCs from donor KP48572 at E:T = 10. Figure 9J shows the results of specific antibody-mediated killing of human diffuse large B-cell lymphoma cells HBL1 using PBMCs from donor KP48775 at E:T = 10. Figure 9K shows the results of specific antibody-mediated killing of human large cell lymphoma cells SUDHL8 using PBMCs from donor KP48572 at E:T = 10. Figure 9L shows the results of specific antibody-mediated killing of SUDHL8 cells using PBMCs from donor KP48573 at E:T = 10. Figure 9M shows the results of specific antibody-mediated killing of ARH77 human B-cell lymphoma cells using PBMCs from donor KP48775 at E:T = 10. Figure 9N shows the results of specific antibody-mediated killing of OCI-Ly3 cells using PBMCs from donor KP48775 at E:T = 10.

[00224] Figure 10 shows the results of an ELISA assay determining the binding of an anti-CD38 x CD28 x CD3 trispecific IgG4 antibody (Binding Protein 5) or isotype control antibody to CD3, CD28, and CD38. Antibody binding was detected using a horseradish peroxidase (HRP)-conjugated anti-Fab secondary antibody.

[00225] Figures 11A-11D show the results of antibody-mediated specific killing of CD38+ human multiple myeloma cancer cells using an anti-CD38 x CD28 x CD3 trispecific IgG4 antibody (Binding Protein 5), an anti-CD28 x CD3 bispecific IgG4 antibody (huCD28 x CD3), an anti-CD38 IgG1 antibody, or a control antibody (human IgG1). Figure 11A shows the results of specific antibody-mediated killing of MOLP-8 cells using human PBMCs as effector cells at E:T = 10. Figure 11B shows the results of specific antibody-mediated killing of RPMI-8226 cells using human PBMCs as effector cells at E:T = 10. Figure 11C shows the results of specific antibody-mediated killing of KMS-12-B cells using human PBMCs as effector cells at E:T = 10. Figure 11D shows the results of FACS analysis determining the cell surface expression of the indicated markers on MOLP-8, RPMI-8226, and KMS-12-BM cells.

[00226] Figures 12A-12D show the results of specific antibody-mediated killing of CD38+ human multiple myeloma cancer cells using an anti-CD38 x CD28 x CD3 trispecific IgG4 antibody (Binding Protein 5), an anti-CD28 x CD3 bispecific IgG4 antibody (huCD28 x CD3), an anti-CD38 IgG1 antibody, or a control antibody (human IgG1) using human PBMCs as effector cells at E:T = 10. Figure 12A shows the results of specific antibody-mediated killing of NCI-H929 cells. Figure 12B shows the results of specific antibody-mediated killing of MM.1S cells. Figure 12C shows the results of specific antibody-mediated killing of MM.1R cells. Figure 12D shows the results of FACS analysis determining cell surface expression of the indicated markers in NCI-H929, MM.1S, and MM.1R cells.

[00227] Figures 13A-13D show the results of antibody-mediated specific killing of CD38+ human multiple myeloma cancer cells using an anti-CD38 x CD28 x CD3 trispecific IgG4 antibody (Binding Protein 5), an anti-CD28 x CD3 bispecific IgG4 antibody (huCD28 x CD3), an anti-CD38 IgG1 antibody, or a control antibody (human IgG1) using human PBMCs as effector cells at E:T = 10. Figure 13A shows the results of antibody-mediated specific killing of OPM-2 cells. Figure 13B shows the results of antibody-mediated specific killing of KMS-26 cells. Figure 13C shows the results of antibody-mediated specific killing of U266 cells. Figure 13D shows the results of FACS analysis determining cell surface expression of the indicated markers in OPM-2, KMS-26, and U226 cells.

[00228] Figures 14A-14C show the results of specific antibody-mediated killing of CD38+ human lymphoma cancer cells using an anti-CD38 x CD28 x CD3 (Binding Protein 5) IgG4 trispecific antibody, an anti-CD28 x CD3 (huCD28 x CD3) IgG4 bispecific antibody, an anti-CD38 IgG1 antibody, or a control antibody (human IgG1) using human PBMCs as effector cells at E:T = 10. Figure 14A shows the results of specific antibody-mediated killing of SUDHL-8 cells. Figure 14B shows the results of specific antibody-mediated killing of OCI-LY19 cells. Figure 14C shows the results of FACS analysis determining the cell surface expression of the indicated markers on SUDHL-8 and OCI-LY19 cells.

[00229] Figures 15A-15D show the results of specific antibody-mediated killing of ALL CD38+ cancer cells using a trispecific IgG4 anti-CD38 x CD28 x CD3 antibody (Binding Protein 5), a bispecific IgG4 anti-CD28 x CD3 antibody (huCD28 x CD3), an IgG1anti-CD38 antibody, or a control antibody (human IgG1) using human PBMCs as effector cells at E:T = 10. Figure 15A shows the results of specific antibody-mediated killing of KOPN-8 cells. Figure 15B shows the results of specific antibody-mediated killing of HAL-1 cells. Figure 15C shows the results of specific antibody-mediated killing of CCRF-SB cells. Figure 15D shows the results of FACS analysis determining cell surface expression of the indicated markers in KOPN-8, HAL-1, and CCRF-SB cells.

[00230] Figure 16 shows the results of specific antibody-mediated killing of CD38+ myeloma cancer cells using anti-CD38 x CD28 x CD3 IgG4 trispecific antibodies (referred to herein as "Binding Protein 5" and "Binding Protein 6" depending on the specific anti-CD28 binding domain used), an anti-CD28 x CD3 bispecific IgG4 antibody (huCD28 x CD3), an anti-CD38 IgG1 antibody, a control anti-CD38 IgG1 antibody, or a control antibody (human IgG1) using PBMCs from donor PK45926 at E:T = 10.

[00231] Figures 17A and 17B show the activation pathway of IL-2, NFkB, and nuclear factor of activated T cells (NFAT) via anti-CD3 and anti-CD28 signaling, as measured by luciferase assay using human Jurkat T cells with an IL-2 promoter-luciferase construct (Figure 17A) or an NFAT promoter-luciferase construct (Figure 17B). The antibodies tested were anti-CD38 x CD28 x CD3 trispecific IgG4 antibody (Binding Protein 5, denoted as “Tri-Ab”), anti-CD38 x CD28 x CD3 trispecific IgG4 antibody lacking the CD28-binding domain (denoted as “Tri-Ab (ΔCD28)), anti-CD38 x CD28 x CD3 trispecific IgG4 antibody lacking the CD3-binding domain (denoted as “Tri-Ab (ΔCD3)), and anti-CD38 x CD28 x CD3 trispecific IgG4 antibody lacking the CD3 and CD28-binding domains (denoted as “Tri-Ab (ΔCD28x ΔCD3)). Luciferase assay was performed in duplicate for each of the indicated Tri-Abs.

[00232] Figures 18A-18E show the results of a dose-escalation toxicity study using the anti-Her2 x CD28 x CD3 trispecific IgG4 antibody (referred to herein as "Binding Protein 1") in non-human primates (dose escalation from 0.1, 0.5, 2.5, 5, 10 to 100 μg/kg, animals referred to as "409" and "410"). Figure 18A shows the results of the percentage of circulating CD4+ T cells in each animal 6 hours after administration of the anti-Her2 x CD28 x CD3 trispecific antibody. Figure 18B shows the results of the percentage of circulating CD8+ T cells in each animal 6 hours after administration of the anti-Her2 x CD28 x CD3 trispecific antibody. Figure 18C shows the results of activation (CD69+) of circulating CD4+ T cells 6 hours after dosing. Figure 18D shows the results of activation (CD69+) of circulating CD8+ T cells 6 hours after dosing. Figure 18E shows the release of inflammatory cytokines observed 6 hours after administration of the anti-Her2 x CD28 x CD3 trispecific antibody at each dose.

[00233] Figures 19A-B show the in vivo anti-tumor activity of the anti-Her2 x CD28 x CD3 trispecific IgG4 antibody (referred to herein as "Binding Protein 1") in the humanized NSG mouse model with CD34+ umbilical cord blood cells implanted with BT474 cells. Figure 19A shows the change in body weight of mice treated with the indicated concentrations of the anti-Her2 x CD28 x CD3 trispecific binding protein or PBS control. Figure 19B shows the change in tumor volume in mice treated with the indicated concentrations of the anti-Her2 x CD28 x CD3 trispecific binding protein or PBS control.

[00234] Figures 20A-20H show the in vivo anti-tumor activity of the anti-Her2 x CD28 x CD3 trispecific IgG4 antibody (referred to herein as "Binding Protein 1") in the humanized NSG mouse model with human PBMCs implanted with BT474 cells. Figure 20A shows the effect of administration of the indicated concentrations of the anti-Her2 x CD28 x CD3 trispecific binding protein, the indicated concentrations of Herceptin, or vehicle control on the body weight of mice. Figure 20B shows the dose-dependent anti-tumor activity of the anti-Her2 x CD28 x CD3 trispecific binding protein, Herceptin, or the indicated controls in individual mice. Figure 20C shows the mean tumor volume in mice following administration of the indicated concentrations of the anti-Her2 x CD28 x CD3 trispecific binding protein or PBS control. Figure 20D shows the mean tumor volume in mice after administration of the indicated concentrations of Herceptin or PBS control. Figure 20E shows bar graphs of the mean tumor volume on day 34 in mice after administration of the indicated concentrations of the anti-Her2 x CD28 x CD3 trispecific binding protein, the indicated concentrations of Herceptin, or PBS control. Figure 20F shows the mean tumor weight on day 34 in mice after administration of the indicated concentrations of the anti-Her2 x CD28 x CD3 trispecific binding protein, the indicated concentrations of Herceptin, or PBS control. Figure 20G shows human CD45+, CD3+, CD4+, and CD8+ cells in the blood of mice at the end of the study. Figure 20H shows human CD45+, CD3+, CD4+, CD8+ cells in the spleens of mice at the end of the study.

[00235] Figures 21A-F show the results of a dose-escalation toxicity study using the anti-CD38 x CD28 x CD3 (Binding Protein 5) trispecific IgG4 antibody in non-human primates (dose escalation from 0.1, 0.5, 2.5, 5, 10, to 100 μg/kg). Figure 21A shows the activation of (CD69+) T cells (line graph) and the proliferation (bar graph) of circulating CD4+ T cells following administration of the anti-CD38 x CD28 x CD3 trispecific antibody. Figure 21B shows the activation of (CD69+) T cells (line graph) and the proliferation (bar graph) of circulating CD8+ T cells following administration of the anti-CD38 x CD28 x CD3 trispecific antibody. Figure 21C shows the release of IL6 in animals given the anti-CD38 × CD28 × CD3 trispecific antibody 6 hours after each dose per individual animal. Figure 21D shows the release of IL-10 in animals given the anti-CD38 × CD28 × CD3 trispecific antibody 6 hours after each dose per individual animal. Figure 21E shows the release of TNFα in animals given the anti-CD38 × CD28 × CD3 trispecific antibody. Figure 21F shows the release of IFNγ in animals given the anti-CD38 × CD28 × CD3 trispecific antibody 6 hours after each dose per individual animal.

[00236] Figures 22A-22C show the in vivo anti-tumor activity of the anti-CD38 x CD28 x CD3 trispecific IgG4 antibody (Binding Protein 5) in the humanized NSG mouse model with CD34+ umbilical cord blood cells implanted with RPMI-8226 multiple myeloma cells transduced with CD38 and PD-L1. As a pilot study, this experiment determined the working dose range for Binding Protein 5. Figure 22A shows the in vivo tumor growth curve in groups with the indicated concentrations for the controls or anti-CD38 x CD28 x CD3 trispecific binding proteins. Figure 22B shows tumor-infiltrating human CD8+ T cells in mice receiving the anti-CD38 x CD28 x CD3 trispecific binding protein or the indicated controls. Figure 22C shows tumor-infiltrating human CD4+ T cells in mice receiving anti-CD38 × CD28 × CD3 trispecific binding protein or the indicated controls.

[00237] Figures 23A-23D show the in vivo activity of the anti-CD38 x CD28 x CD3 trispecific IgG4 antibody (Binding Protein 5) in the humanized NSG mouse model with CD34+ umbilical cord blood cells implanted with RPMI-8226 cells transduced with CD38 and PD-L1. Figure 23A shows the change in body weight of mice treated with the indicated concentrations of the anti-CD38 x CD28 x CD3 trispecific binding protein or PBS control. Figure 23B shows the change in tumor volume in mice treated with the indicated concentrations of the anti-CD38 x CD28 x CD3 trispecific binding protein or PBS control. Figure 23C shows the tumor volumes in each group on Day 19. The tumor volumes in all treated groups showed a marked reduction that is statistically different from the PBS control group. Figure 23D shows the serum concentration of inflammatory cytokines IFN-γ, TNF, and IL-2 in mice four hours after the first dose of the indicated concentrations of anti-CD38 x CD28 x CD3 trispecific binding protein or PBS control.

[00238] Figures 24 and 25 show the in vivo activation of T cells in the humanized NSG mouse model with CD34+ umbilical cord blood cells by administration of an anti-CD38 x CD28 x CD3 trispecific IgG4 antibody (Binding Protein 5; triangle), anti-CD28 x CD3 bispecific IgG4 antibody (square), or an anti-CD28 IgG4 antibody (circle) by determining the increase in the percentage of CD69+ T cells. Figure 24 shows the in vivo activation of CD4+ T cells. Figure 25 shows the in vivo activation of CD8+ T cells.

[00239] Figures 26A-26C show in vivo activation of T cells in the humanized NSG mouse model with CD34+ umbilical cord blood cells by administration of a trispecific IgG4 anti-CD38 x CD28 x CD3 antibody (Binding Protein 5; triangle), a bispecific IgG4 anti-CD28 x CD3 antibody (square), or an IgG4 anti-CD28 antibody (circle) when determining serum levels of inflammatory cytokines. Figure 26A shows serum IL-2 levels. Figure 26B shows serum TNF levels. Figure 26C shows serum IFN-γ levels.

[00240] Figures 27A and 27B show the purification of the indicated proteins by size exclusion chromatography. Figure 27A shows the purification of Binding Proteins 9-15 by size exclusion chromatography. Figure 27B shows the purification of Binding Proteins 16-19 by size exclusion chromatography.

[00241] Figure 28A depicts the trispecific binding protein used in experiments to optimize a purification scheme and configuration of optional binding protein features (e.g., kappa/lambda light chains, knob/hole mutations, and H435R/Y436F mutations).

[00242] Figure 28B shows each of the configurations tested.

[00243] Figure 29 shows representative chromatograms from analytical hydrophobic interaction chromatography (HIC), demonstrating that trispecific binding proteins were distinguishable from unpaired species.

[00244] Figures 30A and 30B show the successful purification of a binding protein with a lambda light chain for the CODV arm, kappa light chain for the Fab arm, knob mutations in the CODV arm, hole mutations in the Fab arm, and RF mutations in the Fab arm via purification steps on Protein A followed by Kap-paSelect (GE Healthcare). Successful purification of the mismatched species binding protein was demonstrated by hydrophobic interaction chromatography (HIC; Figure 30A) and SDS-PAGE (Figure 30B).

DETAILED DESCRIPTION

[00245] The invention provides trispecific and/or trivalent binding proteins comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, wherein a first pair of polypeptides forming the binding protein has dual variable domains that have a cross-over orientation and wherein a second pair of polypeptides forming the binding protein has a single variable domain.

General Definitions

[00246] As used in accordance with the present invention, the following terms, unless otherwise indicated, shall be understood to have the following meanings. Unless otherwise required by the context, terms in the singular shall include the plural and terms in the plural shall include the singular.

[00247] The term "polynucleotide" as used herein refers to single-stranded or double-stranded nucleic acid polymers of at least 10 nucleotides in length. In certain embodiments, the nucleotides comprising the polynucleotide can be ribonucleotides or deoxyribonucleotides, or a modified form of either type of nucleotide. Such modifications include base modifications such as bromuridine, ribose modifications such as arabinoside and 2',3'-dideoxyribose, and internucleotide bond modifications such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranyladate, and phosphoramidate. The term "polynucleotide" specifically includes single-stranded and double-stranded forms of DNA.

[00248] An "isolated polynucleotide" is a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, that: (1) is not associated with all or part of a polynucleotide in which the isolated polynucleotide is found in nature, (2) is linked to a polynucleotide to which it is not linked in nature, or (3) does not occur in nature as part of a larger sequence.

[00249] An "isolated polypeptide" is one that: (1) is free of at least some other polypeptides with which it would normally be found, (2) is essentially free of other polypeptides from the same source, e.g., the same species, (3) is expressed by a cell of a different species, (4) has been separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is associated in nature, (5) is not associated (by covalent or noncovalent interaction) with portions of a polypeptide with which the "isolated polypeptide" is associated in nature, (6) is operatively associated (by covalent or noncovalent interaction) with a polypeptide with which it is not associated in nature, or (7) does not occur in nature. Such an isolated polypeptide may be encoded by genomic DNA, cDNA, mRNA or other RNA of synthetic origin, or any combination thereof. Preferably, the isolated polypeptide is substantially free of polypeptides or other contaminants that are found in its natural environment that will interfere with its use (therapeutic, diagnostic, prophylactic, research or otherwise).

[00250] Naturally occurring antibodies typically comprise a tetramer. Each such tetramer is typically composed of two identical pairs of polypeptide chains, each pair having a full-length "light" chain (which typically has a molecular weight of about 25 kDa) and a full-length "heavy" chain (which typically has a molecular weight of about 5070 kDa). The terms "heavy chain" and "light chain" as used herein refer to any immunoglobulin polypeptide that has a variable domain sequence sufficient to confer specificity for a target antigen. The amino-terminal portion of each light and heavy chain typically includes a variable domain of about 100 to 110 or more amino acids which typically is responsible for antigen recognition. The carboxy-terminal portion of each chain typically defines a constant domain responsible for effector function. Thus, in a naturally occurring antibody, a full-length heavy-chain immunoglobulin polypeptide includes a variable domain (VH) and three constant domains (CH1, CH2, and CH3), where the VH domain is at the amino terminus of the polypeptide and the CH3 domain is at the carboxy terminus, and a full-length light-chain immunoglobulin polypeptide includes a variable domain (VL) and a constant domain (CL), where the VL domain is at the amino terminus of the polypeptide and the CL domain is at the carboxy terminus.

[00251] Human light chains are typically classified as kappa and lambda light chains, and human heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody isotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses including, but not limited to, IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is further divided into subclasses including, but not limited to, IgA1 and IgA2. Within full-length light and heavy chains, the variable and constant domains are typically joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See, for example, FUNDAMENTAL IMMUNOLOGY (Paul, W., ed., Raven Press, 2nd ed., 1989), which is incorporated by reference in its entirety for all purposes. The variable regions of each light/heavy chain pair typically form an antigen-binding site. The variable domains of naturally occurring antibodies typically exhibit the same general structure of relatively conserved framework (FR) regions joined by three hypervariable regions, also called complementarity-determining regions, or CDRs. The CDRs of both chains of each pair are typically aligned by the framework regions, which may allow binding to a specific epitope. From amino to carboxy terminus, both light and heavy chain variable domains typically comprise the FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 domains.

[00252] The term "CDR set" refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., PROTEIN SEQUENCES OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs can be referred to as Kabat CDRs. Chothia et al. (Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-17; Chothia et al., 1989, Nature 342: 877-83) found that certain subportions within the Kabat CDRs adopt nearly identical peptide backbone conformations despite having great diversity at the amino acid sequence level. These subportions were designated L1, L2, and L3. or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions can be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs have been described by Padlan, 1995, FASEB J. 9: 133-39; MacCallum, 1996, J. Mol. Biol. 262 (5): 732-45; and Lefranc, 2003, Dev. Comp. Immunol. 27: 55-77. Still other definitions of CDR boundaries may not strictly follow one of the systems here, but will still overlap with the Kabat CDRs, although they may be shortened or expanded in light of predictions or experimental findings that particular residues or groups of residues or even entire CDRs do not have a significant impact on antigen binding. The methods used here may use CDRs defined according to either of these systems, although certain embodiments use CDRs defined by Kabat or Chothia. The identification of predicted CDRs using amino acid sequence is well known in the field, such as in Martin, A.C. "Protein Sequence And Structure Analysis of Antibody Variables Domains" in Antibody Engineering, Vol. 2. Kontermann R., Dübel S., eds. Springer-Verlag, Berlin, pages 33–51 (2010). The amino acid sequence of the heavy and/or light chain variable domain can also be inspected to identify CDR sequences by other conventional methods, for example, by comparison with known amino acid sequences of other heavy and light chain variable regions to determine regions of sequence hypervariability. The numbered sequences can be aligned by eye or by using an alignment program, such as one of the CLUSTAL suites of programs as described in Thompson, 1994, Nucleic Acids Res. 22: 4673–80. Molecular models are conventionally used to correctly delineate framework regions and CDRs and thus correct sequence-based assignments.

[00253] The term "Fc" as used herein refers to a molecule comprising the sequence of a non-antigen-binding fragment resulting from the digestion of an antibody or produced by other means, whether in monomeric or multimeric form, and may contain the hinge region. The original immunoglobulin source of the native Fc is preferably of human origin and may be any of the immunoglobulins, although IgG1 and IgG2 are preferred. Fc molecules are comprised of monomeric polypeptides that may be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules varies from 1 to 4 depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of an Fc is a disulfide-linked dimer resulting from papain digestion of an IgG. The term "native Fc" as used herein is generic to the monomeric, dimeric, and multimeric forms.

[00254] An F(ab) fragment typically includes a light chain and the VH and CH1 domains of a heavy chain, wherein the -CH1 portion of the heavy chain VH of the F(ab) fragment cannot form a disulfide bridge with another heavy chain polypeptide. As used herein, an F(ab) fragment may also include a light chain containing two variable domains separated by an amino acid linker and a heavy chain containing two variable domains separated by an amino acid linker and a CH1 domain.

[00255] F(ab') fragment typically includes a light chain and a portion of a heavy chain that contains more of the constant region (between the CH1 and CH2 domains), so that an interchain disulfide bond can be formed between two heavy chains to form an F(ab')2 molecule.

[00256] The term "binding protein" as used herein refers to a non-naturally occurring (or recombinant or engineered) molecule that specifically binds to at least one target antigen and that comprises four polypeptide chains that form at least three antigen-binding sites, wherein the first polypeptide chain has a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00257] and a second polypeptide chain has a structure re-presented by the formula: VH1-L3-VH2-L4-CH1[II]

[00258] and a third polypeptide chain has a structure re-presented by the formula: V H3- CH1 [III]

[00259] and a fourth polypeptide chain has a structure represented by the formula: V L3- CL [IV]

[00260] where:

[00261] VL1 is a first immunoglobulin light chain variable domain;

[00262] VL2 is a second immunoglobulin light chain variable domain;

[00263] VL3 is a third immunoglobulin light chain variable domain;

[00264] VH1 is a first immunoglobulin heavy chain variable domain;

[00265] VH2 is a second immunoglobulin heavy chain variable domain;

[00266] VH3 is a third immunoglobulin heavy chain variable domain;

[00267] CL is an immunoglobulin light chain constant domain;

[00268] CH1 is an immunoglobulin CH1 heavy chain constant domain; and

[00269] L1, L2, L3 and L4 are amino acid linkers;

[00270] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[00271] A "recombinant" molecule is one that has been prepared, expressed, created, or isolated by recombinant means.

[00272] One embodiment of the invention provides binding proteins with biological and immunological specificity for between one and three target antigens. Another embodiment of the invention provides nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Another embodiment of the invention provides expression vectors comprising nucleic acid molecules comprising nucleotide sequences encoding polypeptide chains that form such binding proteins. Yet another embodiment of the invention provides host cells expressing such binding proteins (i.e., comprising nucleic acid molecules or vectors encoding polypeptide chains that form such binding proteins).

[00273] The term "interchangeability" as used herein refers to the interchangeability of variable domains within the binding protein format and the retention of folding and final binding affinity. "Full interchangeability" refers to the ability to interchange the order of both the VH1 and VH2 domains, and thus the order of the VL1 and VL2 domains, in the polypeptide chain of formula I or the polypeptide chain of formula II (i.e., to reverse the order) while maintaining full functionality of the binding protein, as evidenced by retention of binding affinity. Furthermore, it should be noted that the VH and VL designations refer only to the location of the domain in a particular protein chain in the final format. For example, VH1 and VH2 may be derived from the VL1 and VL2 domains in parental antibodies and placed in the VH1 and VH2 positions of the binding protein. Similarly, VL1 and VL2 can be derived from VH1 and VH2 domains in parental antibodies and placed in the VH1 and VH2 positions in the binding protein. Thus, the VH and VL designations refer to the current location and not to the original location in a parental antibody. The VH and VL domains are therefore "interchangeable."

[00274] The term "antigen" or "target antigen" or "antigenic target" as used herein refers to a molecule or portion of a molecule that is capable of being bound by a binding protein and is further capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen. A target antigen may have one or more epitopes. For each target antigen recognized by a binding protein, the binding protein is capable of competing with an intact antibody that recognizes the target antigen.

[00275] The term "Her2" refers to human epidermal growth factor receptor 2 which is a member of the epidermal growth factor receptor family.

[00276] "CD3" is a differentiation factor 3 polypeptide and is a protein on the surface of T cells which is typically part of the T cell receptor (TCR) complex.

[00277] "CD28" is a cluster of differentiation of 28 polypeptides and is a protein on the surface of T cells that provides costimulatory signals for T cell activation and survival.

[00278] "CD19" is a cluster of differentiation of 19 polypeptides and is located on B cells.

[00279] "CD20" is a cluster of differentiation of 20 polypeptides and is an activated glycosylated phosphoprotein expressed on the surface of B cells.

[00280] "CD38" is a cluster of differentiation 38 polypeptides and is a glycoprotein found on the surface of many immune cells.

[00281] "LAMP1" is a lysosome-associated membrane protein 1.

[00282] "IL-4" is interleukin 4 and is a cytokine that induces the differentiation of naïve helper T cells.

[00283] "IL-13" is interleukin 13 and is a cytokine secreted by many types of cells, such as T cells.

[00284] "TNFa" is tumor necrosis factor alpha and is a cytokine involved in systemic inflammation.

[00285] The term "T cell engaging agent" refers to binding proteins directed to a host's immune system, more specifically to the cytotoxic activity of T cells, as well as directed to a target tumor protein.

[00286] The term "monospecific binding protein" refers to a binding protein that specifically binds to an antigenic target.

[00287] The term "monovalent binding protein" refers to a binding protein that has one antigen-binding site.

[00288] The term "bispecific binding protein" refers to a binding protein that specifically binds to two different antigenic targets.

[00289] The term "bivalent binding protein" refers to a binding protein that has two binding sites.

[00290] The term "trispecific binding protein" refers to a binding protein that specifically binds to three different antigenic targets.

[00291] The term "trivalent binding protein" refers to a binding protein that has three binding sites. In particular embodiments, the trivalent binding protein can bind to one antigenic target. In other embodiments, the trivalent binding protein can bind to two antigenic targets. In other embodiments, the trivalent binding protein can bind to three antigenic targets.

[00292] An "isolated" binding protein is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the binding protein is purified: (1) from greater than 95 wt % antibody, as determined by the Lowry method, and more preferably greater than 99 wt %, (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by use of a rotating cup sequencer, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain. Isolated binding proteins include the binding protein in situ within recombinant cells, since at least one component of the binding protein's natural environment will not be present.

[00293] The terms “substantially pure” or “substantially purified,” as used herein, refer to a compound or species that is the predominant species present (i.e., on a molar basis, is more abundant than any other individual species in the composition). In some embodiments, a substantially purified fraction is a composition in which the species comprises at least about 50% (on a molar basis) of all macromolecular species present. In other embodiments, a substantially pure composition will comprise greater than about 80%, 85%, 90%, 95%, or 99% of all macromolar species present in the composition. In still other embodiments, the species is purified to essential homogeneity (contaminant species cannot be detected in the composition via conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

[00294] A "neutralizing" binding protein, as used herein, refers to a molecule that is capable of blocking or substantially reducing an effector function of a target antigen to which it binds. As used herein, "substantially reducing" means a reduction of at least about 60%, preferably at least about 70%, more preferably at least about 75%, even more preferably at least about 80%, most preferably at least about 85%, or at least about 90% of an effector function of the target antigen.

[00295] The term "epitope" includes any determinant, preferably a polypeptide determinant, capable of specifically binding to an immunoglobulin or T-cell receptor. In certain embodiments, epitopic determinants include chemically active surface clusters of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural features and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody or binding protein. In certain embodiments, a binding protein is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules. In some embodiments, a binding protein is said to specifically bind to an antigen when the equilibrium dissociation constant is < 10-8 M, more preferably when the equilibrium dissociation constant is < 10-9 M, and most preferably when the dissociation constant is < 10-10 M.

[00296] The dissociation constant (KD) of a binding protein can be determined, for example, by surface plasmon resonance. In general, surface plasmon resonance analysis measures real-time binding interactions between the ligand (a target antigen on a biosensor matrix) and the analyte (a binding protein in solution) by surface plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor; Piscataway, NJ). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). The term "KD" as used herein refers to the dissociation constant of the interaction between a specific binding protein and a target antigen.

[00297] The term "binds specifically" as used herein refers to the ability of a binding protein or antigen-binding fragment thereof to bind an antigen that contains an epitope with a Kd of at least about 1 x 10-6 M, 1 x 10-7 M, 1 x 10-8 M, 1 x 10-9 M, 1 x 10-10 M, 1 x 10-11 M, 1 x 10-12 M or more, and/or to bind an epitope with an affinity that is at least twice its affinity for a non-specific antigen.

[00298] The term "linker" as used herein refers to one or more amino acid residues inserted between immunoglobulin domains to confer sufficient mobility for the light and heavy chain domains to cross-fold on double variable region immunoglobulins. A linker is inserted at the transition between variable domains or between variable and constant domains, respectively, at the sequence level. The transition between domains can be identified because the approximate size of immunoglobulin domains is well understood. The precise location of a domain transition can be determined by locating peptide stretches that do not form secondary structural elements, such as beta sheets or alpha helices, as demonstrated by experimental data or as may be assumed by secondary structure modeling or prediction techniques. The linkers described herein are referred to as L1, which is located on the light chain between the C-terminus of the VL2 domain and the N-terminus of the VL1 domain; and L2, which is located on the light chain between the C-terminus of the VL1 domain and the N-terminus of the CL domain. The heavy chain linkers are known as L3, which is located between the C-terminus of the VH1 domain and the N-terminus of the VH2 domain; and L4, which is located between the C-terminus of the VH2 domain and the N-terminus of the CH1 domain.

[00299] The term "vector", as used herein, refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information into a host cell. The term "vector" includes a nucleic acid molecule that is capable of carrying another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule into which additional DNA segments can be inserted. Another type of vector is a viral vector, in which additional DNA segments can be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomic mammalian vectors) can be integrated into the genome of a host cell after introduction into the host cell and thus replicate along with the host genome. In addition, certain vectors are capable of controlling the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" may be used interchangeably here, since a plasmid is the most commonly used form of vector. However, the invention is intended to include other forms of expression vectors, such as viral vectors (e.g., retroviruses, adenoviruses, and replication-defective adeno-associated viruses), which serve equivalent functions.

[00300] The term "recombinant host cell" (or "host cell"), as used herein, refers to a cell into which a recombinant expression vector has been introduced. A host cell or recombinant host cell is intended to refer not only to the cell in question, but also to the progeny of such cell. Since certain modifications may occur in successive generations due to mutation or environmental influences, such progeny may not in fact be identical to the parent cell, but such cells are still included within the scope of the term "host cell" as used herein. A wide variety of host cell expression systems can be used to express the binding proteins, including bacterial, yeast, baculovirus, and mammalian expression systems (as well as phage display expression systems). An example of a suitable bacterial expression vector is pUC19. To express a binding protein recombinantly, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the polypeptide chains of the binding protein, such that the polypeptide chains are expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the binding protein can be recovered.

[00301] The term "transformation" as used herein refers to a change in the genetic characteristics of a cell, and a cell that has been transformed is when engineered to contain new DNA. For example, a cell is transformed where it is genetically modified from its native state. After transformation, the transforming DNA may recombine with that of the cell, physically integrating into a chromosome of the cell, or it may be transiently maintained as an episomal element without being replicated, or it may replicate independently as a plasmid. A cell is considered to be stably transformed when the DNA is replicated upon cell division. The term "transfection" as used herein refers to the uptake of exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced into the cell membrane. Various methods of transfection are well known in the art. Such methods can be used to introduce one or more exogenous DNA molecules into suitable host cells.

[00302] The term "naturally occurring," as used herein and applied to an object, refers to the fact that the object can be found in nature and has not been manipulated by man. For example, a polynucleotide or polypeptide that is present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally modified by man is naturally occurring. Similarly, "non-naturally occurring," as used herein, refers to an object that is not found in nature or that has been structurally modified or synthesized by man.

[00303] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids; unnatural amino acids and analogues, such as α,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids, may also be suitable components for the polypeptide chains of binding proteins. Examples of unconventional amino acids include: 4-hydroxyproline, carboxyglutamate, ε-N,N,N-trimethyl-lysine, ε-N-acetyl-lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methyl-histidine, 5-hydroxylysine, α-N-methylarginine, and other similar amino acids and amino acids (e.g., 4-hydroxyproline). In the polypeptide notation used here, the left-hand direction is the amino-terminal direction and the right-hand direction is the carboxyl-terminal direction, in accordance with standard usage and convention.

[00304] Naturally occurring residues can be divided into classes based on common side chain properties: (1) hydrophobic: Met, Ala, Val, Leu, Ile, Phe, Trp, Tyr, Pro; (2) polar hydrophilic: Arg, Asn, Asp, Gln, Glu, His, Lys, Ser, Thr; (3) aliphatic: Ala, Gly, Ile, Leu, Val, Pro; (4) aliphatic hydrophobic: Ala, Ile, Leu, Val, Pro; (5) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (6) acidic: Asp, Glu; (7) basic: His, Lys, Arg; (8) residues that influence chain orientation: Gly, Pro; (9) aromatic: His, Trp, Tyr, Phe; and (10) aromatic hydrophobic: Phe, Trp, Tyr.

[00305] Conservative amino acid substitutions may involve the exchange of a member of one of these classes for another member of the same class. Non-conservative substitutions may involve the exchange of a member of one of these classes for a member of another class.

[00306] Those skilled in the art will be able to determine suitable variants of the polypeptide chains of binding proteins using well-known techniques. For example, those skilled in the art can identify suitable areas of a polypeptide chain that can be altered without destroying activity by targeting regions that are thought to be unimportant for activity. Alternatively, those skilled in the art can identify residues and portions of the molecules that are conserved among similar polypeptides. Furthermore, even areas that may be important for biological activity or structure can be subjected to conservative amino acid substitutions without destroying biological activity or adversely affecting the polypeptide structure.

[00307] The term "patient" as used herein includes both humans and animals.

[00308] The terms "treatment" or "treating" as used herein refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those who have a disorder, as well as those prone to having the disorder or those in whom the disorder is to be prevented. In particular embodiments, the binding proteins can be used to treat humans with cancer or humans susceptible to cancer, or ameliorate cancer in a human. The binding proteins can also be used to prevent cancer in a human patient. In specific embodiments, the cancer is multiple myeloma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, lymphoma, breast cancer such as Her2+ breast cancer, germline B-cell lymphoma, or B-cell acute lymphoblastic leukemia. In other embodiments, the binding proteins can be used to treat humans with inflammatory disorders or humans susceptible to inflammatory disorders, or ameliorate inflammatory disorders in a human.

[00309] The terms "pharmaceutical composition" or "therapeutic composition" as used herein refer to a compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

[00310] The term "pharmaceutically acceptable carrier" or "physiologically acceptable carrier" as used herein refers to one or more formulation materials suitable for effecting or enhancing the delivery of a binding protein.

[00311] The terms "effective amount" and "therapeutically effective amount", when used in reference to a pharmaceutical composition comprising one or more binding proteins, refer to an amount or dose sufficient to produce a desired therapeutic result. More specifically, a therapeutically effective amount is an amount of a binding protein sufficient to inhibit, for some period of time, one or more of the clinically defined pathological processes associated with the condition being treated. The effective amount may vary depending on the specific binding protein being used and also depends on a variety of factors and conditions related to the patient being treated and the severity of the disorder. For example, if the binding protein is administered in vivo, factors such as the age, weight, and health of the patient, as well as dose-response curves and toxicity data obtained in preclinical animal work, would be some of the factors considered. The determination of an effective amount or therapeutically effective amount of a given pharmaceutical composition is well within the capability of those skilled in the art.

[00312] One embodiment of the invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of a binding protein. Trispecific and/or Trivalent Binding Proteins

[00313] In one embodiment, the binding protein of the invention is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more (e.g., three) different antigenic targets or target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00314] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1[II]

[00315] and a third polypeptide chain comprises a structure represented by the formula: V H3-CH1 [III]

[00316] and a fourth polypeptide chain comprises a structure represented by the formula: V L3-CL [IV]

[00317] where:

[00318] VL1 is a first immunoglobulin light chain variable domain;

[00319] VL2 is a second immunoglobulin light chain variable domain;

[00320] VL3 is a third immunoglobulin light chain variable domain;

[00321] VH1 is a first immunoglobulin heavy chain variable domain;

[00322] VH2 is a second immunoglobulin heavy chain variable domain;

[00323] VH3 is a third immunoglobulin heavy chain variable domain;

[00324] CL is an immunoglobulin light chain constant domain;

[00325] CH1 is an immunoglobulin CH1 heavy chain constant domain; and

[00326] L1, L2, L3 and L4 are amino acid linkers;

[00327] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[00328] In one embodiment, the binding protein of the invention is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more (e.g., three) antigenic targets or target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00329] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1[II]

[00330] and a third polypeptide chain comprises a structure represented by the formula: V H3-CH1[III]

[00331] and a fourth polypeptide chain comprises a structure represented by the formula: V L3-CL [IV]

[00332] where:

[00333] VL1 is a first immunoglobulin light chain variable domain;

[00334] VL2 is a second immunoglobulin light chain variable domain;

[00335] VL3 is a third immunoglobulin light chain variable domain;

[00336] VH1 is a first immunoglobulin heavy chain variable domain;

[00337] VH2 is a second immunoglobulin heavy chain variable domain;

[00338] VH3 is a third immunoglobulin heavy chain variable domain;

[00339] CL is an immunoglobulin light chain constant domain;

[00340] CH1 is an immunoglobulin CH1 heavy chain constant domain; and

[00341] L1, L2, L3 and L4 are amino acid linkers;

[00342] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[00343] In one embodiment, the binding protein of the invention is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more (e.g., three) different antigenic targets or target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: V L2-L1-VL1-L2-CL[I]

[00344] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[00345] and a third polypeptide chain comprises a structure represented by the formula: V H3-CH1-hinge-CH2-CH3 [III]

[00346] and a fourth polypeptide chain comprises a structure represented by the formula: V L3- CL [IV]

[00347] where:

[00348] VL1 is a first immunoglobulin light chain variable domain;

[00349] VL2 is a second immunoglobulin light chain variable domain;

[00350] VL3 is a third immunoglobulin light chain variable domain;

[00351] VH1 is a first immunoglobulin heavy chain variable domain;

[00352] VH2 is a second immunoglobulin heavy chain variable domain;

[00353] VH3 is a third immunoglobulin heavy chain variable domain;

[00354] CL is an immunoglobulin light chain constant domain;

[00355] CH1 is an immunoglobulin heavy chain CH1 constant domain;

[00356] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[00357] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00358] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00359] L1, L2, L3 and L4 are amino acid linkers;

[00360] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[00361] In one embodiment, the binding protein of the invention is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more (e.g., three) antigenic targets or target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: V L2-L1-VL1-L2-CL[I]

[00362] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[00363] and a third polypeptide chain comprises a structure represented by the formula: V H3-CH1-hinge-CH2-CH3 [III]

[00364] and a fourth polypeptide chain comprises a structure represented by the formula: V L3- CL [IV]

[00365] where:

[00366] VL1 is a first immunoglobulin light chain variable domain;

[00367] VL2 is a second immunoglobulin light chain variable domain;

[00368] VL3 is a third immunoglobulin light chain variable domain;

[00369] VH1 is a first immunoglobulin heavy chain variable domain;

[00370] VH2 is a second immunoglobulin heavy chain variable domain;

[00371] VH3 is a third immunoglobulin heavy chain variable domain;

[00372] CL is an immunoglobulin light chain constant domain;

[00373] CH1 is an immunoglobulin heavy chain CH1 constant domain;

[00374] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[00375] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00376] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00377] L1, L2, L3 and L4 are amino acid linkers;

[00378] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[00379] In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross-linked orientation that forms two distinct antigen-binding sites. In some embodiments, the VH1 and VL1 domains form a binding pair and form the first antigen-binding site. In some embodiments, the VH2 and VL2 domains form a binding pair and form the second antigen-binding site. In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen-binding site. In some embodiments, the VH3 and VL3 domains form a binding pair and form the third antigen-binding site.

[00380] In one embodiment, the binding protein of the invention is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more (e.g., three) antigenic targets or target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: VD1-L1-VD2-L2-CL[I]

[00381] and a second polypeptide chain comprises a structure represented by the formula: VD3-L3-VD4-L4-CH1-hinge-CH2-CH3[II]

[00382] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3 [III]

[00383] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL

[00384] where:

[00385] VD1 is a heavy or light chain variable domain of a first immunoglobulin;

[00386] VD2 is a heavy or light chain variable domain of a second immunoglobulin;

[00387] VD3 is a heavy or light chain variable domain of a third immunoglobulin;

[00388] VD4 is a heavy or light chain variable domain of a fourth immunoglobulin;

[00389] VH3 is an immunoglobulin heavy chain variable domain;

[00390] VL3 is an immunoglobulin light chain variable domain;

[00391] CL is an immunoglobulin light chain constant domain;

[00392] CH1 is an immunoglobulin heavy chain CH1 constant domain;

[00393] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[00394] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00395] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00396] L1, L2, L3 and L4 are amino acid linkers;

[00397] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[00398] In some embodiments, the binding protein of the invention comprises three antigen-binding sites that specifically bind to one, two, or three antigenic targets or target proteins. In some embodiments, the binding protein binds to three antigenic targets. In some embodiments, the binding protein binds to three different antigenic targets. In some embodiments, two of the antigen-binding sites bind to the same antigenic target. In these embodiments, the binding protein comprises the same binding domains twice or different binding domains and/or specifically binds to different antigens or epitopes on the same antigenic target. In some embodiments, three of the antigen-binding sites bind to the same antigenic target. In these embodiments, the binding protein comprises the same binding domains three times or different binding domains and/or specifically binds to different antigens or epitopes on the same antigenic target.

[00399] In some embodiments, VL1, VL2, and VL3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 10, 14, 18, 22, or 115; and VH1, VH2, and VH3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 3, 9, 13, 17, 21, or 114. In other embodiments, VL1, VL2, and VL3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 61, 63, 69, 71, 74, 76, 82, 86, 88, or 94; and VH1, VH2, and VH3 are each independently a variable domain derived from an amino acid sequence as set forth in any one of SEQ ID NOs: 60, 62, 68, 73, 75, 81, 85, 87, or 93. In other embodiments, VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions of a variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 43-59, 123-125; and VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions of a variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-42, 120-122. In other embodiments, VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions of a variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 61, 63, 69, 71, 74, 76, 82, 86, 88, or 94; and VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions of a variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 60, 62, 68, 73, 75, 81, 85, 87, or 93. In some embodiments, VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions and/or a variable domain sequence set forth in Tables 2-5.

[00400] In some embodiments, VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any of SEQ ID NOs: 169, 171, and 173; and/or VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any of SEQ ID NOs: 168, 170, and 172. In some embodiments, VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-147, 178, and 179; and/or VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 129-137. In some embodiments, VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 151, 153, 155, 157, 159, 161, 163, 165, and 167; and/or VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 150, 152, 154, 156, 158, 160, 162, 164, and 166. In some embodiments, VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions that comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 43-59, 123-125, 138-140, and 149; and/or VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions of a variable domain comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-42, 120-122, and 126-128. In some embodiments, VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions and/or a variable domain sequence set forth in Tables 2-5.

[00401] In particular embodiments, the order of the VH1 and VH2 domains, and therefore the order of the VL1 and VL2 domains, in the polypeptide chain of formula I or in the polypeptide chain of formula II (i.e., to reverse the order) are swapped.

[00402] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 1; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 2.

[00403] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 1; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 2.

[00404] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 13; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 14.

[00405] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 13; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 14.

[00406] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 17; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 18 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 18.

[00407] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 17; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 18 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 18.

[00408] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 21; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22 optionally comprising CDRs that are 100% identical to CDRs of the polypeptide chain of SEQ ID NO: 22.

[00409] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 21; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 22.

[00410] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 63; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 60; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 61.

[00411] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 60; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 61.

[00412] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 60; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 71 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 71.

[00413] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 76 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 76 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 76; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 75 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 75 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 75; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 74.

[00414] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 82 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 82; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 81 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 81 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 81; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 74.

[00415] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 88 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 88 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 88; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 87 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 87; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 86.

[00416] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 94 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 94; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 93 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 93 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 93; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 86.

[00417] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 74.

[00418] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 86.

[00419] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 63; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 74.

[00420] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 63; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 86.

[00421] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 115.

[00422] In some embodiments, the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 115.

[00423] In other embodiments, the binding protein of the invention is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more (e.g., three) different target proteins, wherein a first polypeptide chain has a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00424] and a second polypeptide chain has a structure re-presented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3(hole)[II]

[00425] and a third polypeptide chain has a structure re-presented by the formula: VH3-CH1-hinge-CH2-CH3 (knob) [III]

[00426] and a fourth polypeptide chain has a structure represented by the formula: VL3-CL[IV]

[00427] where:

[00428] VL1 is a first immunoglobulin light chain variable domain;

[00429] VL2 is a second immunoglobulin light chain variable domain;

[00430] VL3 is a third immunoglobulin light chain variable domain;

[00431] VH1 is a first immunoglobulin heavy chain variable domain;

[00432] VH2 is a second immunoglobulin heavy chain variable domain;

[00433] VH3 is a third immunoglobulin heavy chain variable domain;

[00434] CL is an immunoglobulin light chain constant domain;

[00435] CH1 is an immunoglobulin CH1 heavy chain constant domain; and

[00436] L1, L2, L3 and L4 are amino acid linkers;

[00437] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[00438] In other embodiments, the binding protein of the invention is a trispecific and/or trivalent binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more (e.g., three) target proteins, wherein a first polypeptide chain has a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00439] and a second polypeptide chain has a structure re-presented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3(hole)[II]

[00440] and a third polypeptide chain has a structure re-presented by the formula: VH3-CH1-hinge-CH2-CH3 (knob) [III]

[00441] and a fourth polypeptide chain has a structure represented by the formula: VL3-CL[IV]

[00442] where:

[00443] VL1 is a first immunoglobulin light chain variable domain;

[00444] VL2 is a second immunoglobulin light chain variable domain;

[00445] VL3 is a third immunoglobulin light chain variable domain;

[00446] VH1 is a first immunoglobulin heavy chain variable domain;

[00447] VH2 is a second immunoglobulin heavy chain variable domain;

[00448] VH3 is a third immunoglobulin heavy chain variable domain;

[00449] CL is an immunoglobulin light chain constant domain;

[00450] CH1 is an immunoglobulin heavy chain CH1 constant domain; and

[00451] L1, L2, L3 and L4 are amino acid linkers;

[00452] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[00453] In some embodiments, the first polypeptide chain and the second polypeptide chain have a cross-linked orientation that forms two distinct antigen-binding sites. In some embodiments, the VH1 and VL1 domains form a binding pair and form the first antigen-binding site. In some embodiments, the VH2 and VL2 domains form a binding pair and form the second antigen-binding site. In some embodiments, the third polypeptide and the fourth polypeptide form a third antigen-binding site. In some embodiments, the VH3 and VL3 domains form a binding pair and form the third antigen-binding site. In some embodiments, the second polypeptide chain and the third polypeptide chain comprise one or more modifications. In some embodiments, the second polypeptide chain and the third polypeptide chain of a binding protein are different, e.g., have different CH1, CH2, and/or CH3 domains (such as those including a modification described herein). In some embodiments, the first polypeptide chain and the fourth polypeptide chain comprise one or more modifications. In some embodiments, the first polypeptide chain and the fourth polypeptide chain of a binding protein are different, e.g., have different CL domains (such as those including a modification described herein and/or lambda vs. kappa CL domains).

[00454] In some embodiments, a binding protein of the present invention comprises an antigen-binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 150, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 150, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 151, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 151. In some embodiments, .... embodiments, a binding protein of the present invention comprises an antigen-binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 152, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 152, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 153, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 153. In some embodiments, a binding protein of the present invention comprises an antigen-binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 153. The binding site of the present invention comprises an antigen-binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 154, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 154, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 155, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 155. In some embodiments, a protein A binding protein of the present invention comprises an antigen-binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 156, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 156, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 157, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 157. In some embodiments, a binding protein of the present invention comprises an antigen-binding site comprising a heavy ... comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 158, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 158, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 159, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 159. In some embodiments, a binding protein of the present invention comprises an antigen-binding site comprising a heavy chain variable domain heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 160, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 160, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 161, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 161. In some embodiments, a binding protein of the present invention comprises an antigen-binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 162, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 162, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 163, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 163. In some embodiments, a binding protein of the present invention comprises an antigen binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 164, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 164, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 165, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 165. In some embodiments, a binding protein of the present invention comprises an antigen binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 166, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 166, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 167, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 167. In some embodiments, a binding protein of the present invention comprises an antigen binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 168, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 168, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 169, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 169. In some embodiments, a binding protein of the present invention comprises an antigen binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 170, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 170, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 171, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 171. In some embodiments, a binding protein of the present invention comprises an antigen binding site comprising a heavy chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 172, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 172, and/or a light chain variable domain comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO: 173, optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 173.

[00455] In some embodiments, a binding protein of the present invention binds to one, two, or three antigenic targets with an equilibrium dissociation constant (KD) that is less than or equal to 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, or 1 nM. Exemplary assays for determining KD are known in the art. For example, in some embodiments, KD is determined by measuring binding kinetics between 0 °C and 37 °C (e.g., at 0 °C, 4 °C, 25 °C, or 37 °C) using the techniques described in Example 1 (e.g., SPR or ELISA).

[00456] In some embodiments, a binding protein of the present invention activates CD4 and/or CD8 T cells in vitro and/or induces in vitro antibody-mediated cell killing of a cell expressing one or more antigenic targets of one or more binding domains of the binding protein. Exemplary in vitro cell killing and T cell activation assays are known in the art. For example, in some embodiments, in vitro cell killing and/or T cell activation is assayed using the techniques described in Example 1.

[00457] In some embodiments, a binding protein of the present invention specifically binds to and/or blocks signaling mediated by one or more cytokines. Exemplary cytokine release assays are known in the art. For example, in some embodiments, cytokine release is assayed using the techniques described in Example 1.

[00458] In some embodiments, a binding protein of the present invention comprises a first antigen-binding site that specifically binds to a target protein on T cells, a second antigen-binding site that specifically binds to a target protein on T cells, and a third antigen-binding site that specifically binds to a target antigen or target protein. In some embodiments, a binding protein of the present invention comprises a first antigen-binding site that specifically binds to a target protein on T cells, a second antigen-binding site that specifically binds to a target protein on T cells, and a third antigen-binding site that specifically binds to a target tumor protein. In some embodiments, a binding protein of the present invention comprises a first antigen-binding site that specifically binds to a target protein on T cells, a second antigen-binding site that specifically binds to a target protein on T cells, and a third antigen-binding site that specifically binds to a target human tumor protein. In some embodiments, the first and second antigen-binding sites specifically bind to the target tumor protein, e.g., selected from CD3 and CD28, respectively. In some embodiments, the first and second antigen-binding sites specifically bind to the target tumor protein, e.g., selected from CD28 and CD3, respectively. In some embodiments, the third antigen-binding site specifically binds to CD19, CD20, CD38, Her2, or LAMP1. Other examples of such targets and target proteins are provided infra.

[00459] In some embodiments, a binding protein of the present invention comprises a first antigen-binding site that specifically binds to CD3, a second antigen-binding site that specifically binds to CD28, and a third antigen-binding site that specifically binds to a target antigen or target protein. In some embodiments, a binding protein of the present invention comprises a first antigen-binding site that specifically binds to CD28, a second antigen-binding site that specifically binds to CD3, and a third antigen-binding site that specifically binds to a target antigen or target protein. Further examples of such antigenic targets or target proteins are provided infra. In some embodiments, a binding protein of the present invention comprises a first antigen-binding site that specifically binds to CD3, a second antigen-binding site that specifically binds to CD28, and a third antigen-binding site that specifically binds to a target tumor protein. In some embodiments, a binding protein of the present invention comprises a first antigen-binding site that specifically binds to human CD3, a second antigen-binding site that specifically binds to human CD28, and a third antigen-binding site that specifically binds to a human target tumor protein. In some embodiments, a binding protein of the present invention comprises a first antigen-binding site that specifically binds to CD28, a second antigen-binding site that specifically binds to CD3, and a third antigen-binding site that specifically binds to a human target tumor protein. In some embodiments, a binding protein of the present invention comprises a first antigen-binding site that specifically binds to human CD28, a second antigen-binding site that specifically binds to human CD3, and a third antigen-binding site that specifically binds to a human target tumor protein. In some embodiments, the third antigen-binding site specifically binds to CD19, CD20, CD38, Her2, or LAMP1. Other examples of such tumor antigenic targets or tumor target proteins are provided infra.

[00460] In some embodiments, the antigen-binding site that specifically binds to CD3 comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 152 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 153; or a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155. Suitable VH, VL, and/or CDR sequences of antibodies that specifically bind to CD3 for use in any of the binding proteins described herein can be found in International Publication No. WO2016/116626, which is incorporated by reference herein in its entirety. In some embodiments, the antigen-binding site that specifically binds to CD3 comprises six CDRs or one light chain and one heavy chain variable domain shown in Tables 2-5. In some embodiments, the antigen-binding site that specifically binds to CD3 comprises (i) three heavy chain CDRs of SEQ ID Nos. 34, 35, and 36, respectively, and three light chain CDRs of SEQ ID Nos. 52, 53, and 54, respectively; or (ii) three heavy chain CDRs of SEQ ID Nos. 34, 35, and 36, respectively, and three light chain CDRs of SEQ ID Nos. 149, 53, and 54, respectively. In some embodiments, the antigen-binding site that specifically binds to CD3 is part of the polypeptide chain comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 3. In some embodiments, the antigen-binding site that specifically binds to CD3 is part of the polypeptide chain comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 4.

[00461] In some embodiments, the antigen-binding site that specifically binds to CD28 comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 161; or a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 163. In some embodiments, the antigen-binding site that specifically binds to CD28 comprises six CDRs or one light chain and one heavy chain variable domain shown in Tables 2-5. In some embodiments, the antigen-binding site that specifically binds to CD28 comprises (i) three heavy chain CDRs of SEQ ID Nos. 28, 29, and 30, respectively, and three light chain CDRs of SEQ ID Nos. 46, 47, and 48, respectively; or (ii) three heavy chain CDRs of SEQ ID Nos. 31, 32, and 33, respectively, and three light chain CDRs of SEQ ID Nos. 49, 50, and 51, respectively. In some embodiments, the antigen-binding site that specifically binds to CD28 is part of the polypeptide chain comprising the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 3. In some embodiments, the antigen-binding site that specifically binds to CD28 is part of the polypeptide chain comprising the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4 optionally comprising CDRs that are 100% identical to the CDRs of the polypeptide chain of SEQ ID NO: 4.

[00462] In some embodiments, a binding protein of the present invention comprises a first antigen-binding site that specifically binds to CD3, a second antigen-binding site that specifically binds to CD28, and a third antigen-binding site that specifically binds to CD38, or a first antigen-binding site that specifically binds to CD28, a second antigen-binding site that specifically binds to CD3, and a third antigen-binding site that specifically binds to CD38, wherein: • the antigen-binding site that specifically binds to CD3 comprises (i) three heavy chain CDRs of SEQ ID Nos. 34, 35, and 36, respectively, and three light chain CDRs of SEQ ID Nos. 52, 53, and 54, respectively; or (ii) three heavy chain CDRs of SEQ ID Nos. 34, 35, and 36, respectively, and three light chain CDRs of SEQ ID Nos. 149, 53, and 54, respectively; and • the antigen-binding site that specifically binds to CD28 comprises (i) three heavy chain CDRs of SEQ ID Nos. 28, 29, and 30, respectively, and three light chain CDRs of SEQ ID Nos. 46, 47, and 48, respectively; or (ii) three heavy chain CDRs of SEQ ID Nos. 31, 32, and 33, respectively, and three light chain CDRs of SEQ ID Nos. 49, 50, and 51, respectively; and • the antigen-binding site that specifically binds to CD38 comprises (i) three heavy chain CDRs of SEQ ID Nos. 40, 41, and 42, respectively, and three light chain CDRs of SEQ ID Nos. 58, 44, and 59, respectively.

[00463] In some embodiments, the antigen-binding site that specifically binds to a target tumor protein comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 156 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 157; a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 159; a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 165; a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 150 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 151; or a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 166 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 167. In some embodiments, the antigen-binding site that specifically binds to a target tumor protein comprises six CDRs or a light chain and a heavy chain variable domain set forth in Tables 2-5. In some embodiments, the antigen-binding site that specifically binds to a target tumor protein comprises six CDRs of an anti-Her2, anti-CD19, anti-CD20, anti-CD38, or anti-LAMP1 antibody set forth in Tables 2-5.

[00464] In some embodiments, a binding protein of the present invention comprises four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00465] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[00466] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3[III]

[00467] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[00468] where:

[00469] VL1 is a first immunoglobulin light chain variable domain;

[00470] VL2 is a second immunoglobulin light chain variable domain;

[00471] VL3 is a third immunoglobulin light chain variable domain;

[00472] VH1 is a first immunoglobulin heavy chain variable domain;

[00473] VH2 is a second immunoglobulin heavy chain variable domain;

[00474] VH3 is a third immunoglobulin heavy chain variable domain;

[00475] CL is an immunoglobulin light chain constant domain;

[00476] CH1 is an immunoglobulin heavy chain CH1 constant domain;

[00477] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[00478] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00479] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00480] L1, L2, L3 and L4 are amino acid linkers;

[00481] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[00482] where:

[00483] VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 169, 171, and 173; and

[00484] where:

[00485] VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 168, 170, and 172.

[00486] In some embodiments, a binding protein of the present invention comprises four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00487] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[00488] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3[III]

[00489] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[00490] where:

[00491] VL1 is a first immunoglobulin light chain variable domain;

[00492] VL2 is a second immunoglobulin light chain variable domain;

[00493] VL3 is a third immunoglobulin light chain variable domain;

[00494] VH1 is a first immunoglobulin heavy chain variable domain;

[00495] VH2 is a second immunoglobulin heavy chain variable domain;

[00496] VH3 is a third immunoglobulin heavy chain variable domain;

[00497] CL is an immunoglobulin light chain constant domain;

[00498] CH1 is a CH1 heavy chain constant domain in the globulin; of immuno-

[00499] CH2 is a CH2 heavy chain constant domain in the globulin; of immuno-

[00500] CH3 is a constant domain of CH3 heavy chain inglobulin; of immuno-

[00501] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00502] L1, L2, L3 and L4 are amino acid linkers;

[00503] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[00504] where:

[00505] VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-147, 178, and 179; and

[00506] where:

[00507] VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 129-137.

[00508] In some embodiments, a binding protein of the present invention comprises four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00509] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[00510] and a third polypeptide chain comprises a structure represented by the formula:

[00511] VH3-CH1-hinge-CH2-CH3 [III]

[00512] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[00513] where:

[00514] VL1 is a first immunoglobulin light chain variable domain;

[00515] VL2 is a second immunoglobulin light chain variable domain;

[00516] VL3 is a third immunoglobulin light chain variable domain;

[00517] VH1 is a first immunoglobulin heavy chain variable domain;

[00518] VH2 is a second immunoglobulin heavy chain variable domain;

[00519] VH3 is a third immunoglobulin heavy chain variable domain;

[00520] CL is an immunoglobulin light chain constant domain;

[00521] CH1 is an immunoglobulin heavy chain CH1 constant domain;

[00522] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[00523] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00524] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00525] L1, L2, L3 and L4 are amino acid linkers;

[00526] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[00527] where:

[00528] VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 151, 153, 155, 157, 159, 161, 163, 165, and 167; and

[00529] where:

[00530] VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 150, 152, 154, 156, 158, 160, 162, 164, and 166.

[00531] In some embodiments, a binding protein of the present invention comprises four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00532] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[00533] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3[III]

[00534] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[00535] where:

[00536] VL1 is a first immunoglobulin light chain variable domain;

[00537] VL2 is a second immunoglobulin light chain variable domain;

[00538] VL3 is a third immunoglobulin light chain variable domain;

[00539] VH1 is a first immunoglobulin heavy chain variable domain;

[00540] VH2 is a second immunoglobulin heavy chain variable domain;

[00541] VH3 is a third immunoglobulin heavy chain variable domain;

[00542] CL is an immunoglobulin light chain constant domain;

[00543] CH1 is an immunoglobulin CH1 heavy chain constant domain;

[00544] CH2 is an immunoglobulin heavy chain CH2 constant domain;

[00545] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00546] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00547] L1, L2, L3 and L4 are amino acid linkers;

[00548] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[00549] where:

[00550] VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 43-59, 123-125, 138-140, and 149; and

[00551] where:

[00552] VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-42, 120-122, and 126-128.

[00553] In some embodiments, a binding protein of the present invention comprises an antigen-binding site that specifically binds to CD3, an antigen-binding site that specifically binds to CD28, and an antigen-binding site that specifically binds to a target antigen other than CD3 or CD28. In some embodiments, a binding protein of the present invention comprises an antigen-binding site that specifically binds to human CD3, an antigen-binding site that specifically binds to human CD28, and an antigen-binding site that specifically binds to a human antigenic target other than CD3 or CD28. In some embodiments, a binding protein of the present invention comprises (a) an antigen-binding site that specifically binds to CD3, wherein the antigen-binding site that specifically binds to CD3 comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 152 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 153, (ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155, (iii) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36, and a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54 or (iv) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36 and a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 149, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; (b) an antigen-binding site that specifically binds to CD28, wherein the antigen-binding site that specifically binds to CD28 comprises (i) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 161, (ii) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 163, (iii) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30 and a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48 or (iv) a heavy chain variable domain comprising a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable domain comprising a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; and (c) an antigen-binding site that specifically binds to an antigenic target other than CD3 or CD28. In some embodiments, a binding protein of the present invention comprises a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 4 or 10, a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 3 or 9, and a third and a fourth polypeptide chains, wherein the third and fourth polypeptide chains form an antigen-binding domain that specifically binds to an antigenic target other than CD3 or CD28. In some embodiments, the antigen-binding site that specifically binds to an antigenic target other than CD3 or CD28 binds to a target antigen selected from A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD19, CD20, CD23 (also known as FCER2, the IgE receptor), CD24, CD27, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as integrin b4), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a coreceptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5/CCXCR1).

[00554] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[00555] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[00556] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[00557] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57.

[00558] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57.

[00559] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 42; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 58, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59.

[00560] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 42; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 58, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59.

[00561] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 126, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 127, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 128; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 138, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 139, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 140.

[00562] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 126, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 127, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 128; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 138, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 139, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 140.

[00563] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142.

[00564] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142.

[00565] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142.

[00566] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147.

[00567] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147.

[00568] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144.

[00569] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144.

[00570] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147.

[00571] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144.

[00572] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147.

[00573] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144.

[00574] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 120, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 121, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 122; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

[00575] In some embodiments, VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 120, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 121, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 122; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

Antigenic Targets

[00576] In some embodiments, a binding protein of the present invention binds to one or more (e.g., one, two, or three) of the following antigenic targets or target proteins: A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, the IgE receptor), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, 17, IL17Rb (also known as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as integrin b4), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STEAP2, Syk kinase, TACI, TDO, T14, TI-GIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more of the above antigenic targets are human antigenic targets.

[00577] In one embodiment, the binding proteins specifically bind to one or more tumor antigenic targets (e.g., target proteins). In other embodiments, the binding proteins specifically bind to one or more tumor target proteins and one or more T cell target proteins, including a T cell receptor complex. These T cell-engaging binding proteins are capable of transiently recruiting T cells to the target cells and, at the same time, activating the cytolytic activity of the T cells. Examples of T cell target proteins include, but are not limited to, CD3 and CD28, among others. Other examples of such antigenic targets or target proteins are provided supra. In some embodiments, trispecific binding proteins can be generated by combining the antigen-binding domains of two or more monospecific antibodies (parent antibodies) into one antibody. In some embodiments, a binding protein of the present invention binds to one or more (e.g., one, two, or three) of the following antigenic targets: CD3, CD19, CD20, CD28, CD38, Her2, LAMP1, IL-4, IL-13, and TNFα.

[00578] In some embodiments of the invention, the trivalent binding protein is capable of binding to three antigenic targets. In some embodiments of the invention, the trivalent binding protein is capable of binding to three different antigenic targets. In one embodiment, the binding protein is trispecific and a light chain-heavy chain pair is capable of binding to two different antigenic targets or epitopes and a light chain-heavy chain pair is capable of binding to one target antigen or epitope. In another embodiment, the binding protein is capable of binding to three tumor antigenic targets. In another embodiment, the binding protein is capable of binding to three different tumor antigenic targets. In other embodiments, the binding protein is capable of inhibiting the function of one or more of the antigenic targets.

[00579] In some embodiments, a binding protein of the present invention binds one or more target tumor proteins. In some embodiments, the binding protein is capable of specifically binding to three epitopes on a single target tumor protein. In some embodiments, the binding protein is capable of specifically binding to three different epitopes on a single target tumor protein. In some embodiments, the binding protein is capable of binding to two different epitopes on a first target tumor protein and one epitope on a second target tumor protein. In some embodiments, the first and second target tumor proteins are different. In some embodiments, the binding protein is capable of specifically binding to three different target tumor proteins.

[00580] In some embodiments, a binding protein of the present invention binds to one or more cytokine target proteins. In some embodiments, the binding protein is capable of specifically binding to three epitopes on a single cytokine target protein. In some embodiments, the binding protein is capable of specifically binding to three different epitopes on a single cytokine target protein. In some embodiments, the binding protein is capable of binding to two different epitopes on a first cytokine target protein and one epitope on a second cytokine target protein. In some embodiments, the first and second cytokine target proteins are different. In some embodiments, the binding protein is capable of specifically binding to three different cytokine target proteins. In some embodiments, the one or more cytokine target proteins are one or more of IL-4, IL-13, and/or TNFα. Other examples of cytokine target proteins are provided infra.

[00581] In some embodiments, a binding protein of the present invention binds one or more target tumor proteins and one or more T cell target proteins. In some embodiments, the binding protein is capable of specifically binding to a target tumor protein and two different epitopes on a single target T cell protein. In some embodiments, the binding protein is capable of specifically binding to a target tumor protein and two different T cell target proteins (e.g., CD28 and CD3). In some embodiments, the binding protein is capable of specifically binding to a target T cell protein and two different epitopes on a single target tumor protein. In some embodiments, the binding protein is capable of specifically binding to a target T cell protein and two different target tumor proteins. In some embodiments, the first and second polypeptide chains of the binding protein form two antigen-binding sites that specifically bind to two T cell target proteins and the third and fourth polypeptide chains of the binding protein form one antigen-binding site that specifically binds to one target tumor protein. In some embodiments, the first and second polypeptide chains of the binding protein form two antigen-binding sites that specifically target two target tumor proteins and the third and fourth polypeptide chains of the binding protein form one antigen-binding site that specifically binds to one target T cell protein. In some embodiments, the one or more target tumor proteins are one or more of CD3, CD19, CD20, CD28, CD38, Her2, LAMP1, IL-4, IL-13, and/or TNFα. In some embodiments, the one or more T cell target proteins are one or more of CD3 and CD28. Other examples of tumor target proteins and T cell target proteins are provided supra.

[00582] In some embodiments, a binding protein of the present invention binds, independently of one another, the same or different, one, two, or three antigenic targets or target proteins selected from cytokine target proteins, tumor antigens or target tumor proteins, T cell target proteins, immune checkpoint inhibitors, immune checkpoint modulators, immune checkpoint costimulatory molecules, and/or target molecules on the surface of an immune cell. In some embodiments, a binding protein of the present invention is trivalent but bispecific and capable of specifically binding twice to the same antigenic targets or target proteins. In some embodiments, a binding protein of the present invention is capable of specifically binding to two different epitopes on a single cytokine target protein, tumor antigen or tumor protein target, T-cell target protein, immune checkpoint inhibitor, immune checkpoint modulator, immune checkpoint costimulatory molecule, and/or target molecule on the surface of an immune cell. Other examples of such antigenic targets or target proteins are provided supra.

[00583] The binding proteins of the invention can be prepared using domains or sequences obtained or derived from any human or non-human antibody including, for example, human, murine or humanized antibodies.

Binders

[00584] In some embodiments, the linkers L1, L2, L3, and L4 range from no amino acids (length = 0) to about 100 amino acids in length or less to 100, 50, 40, 30, 20, or 15 amino acids or less. The linkers can also be 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in length. L1, L2, L3, and L4, in a binding protein, can all have the same amino acid sequence or can all have different amino acid sequences.

[00585] Examples of suitable linkers include a single glycine residue (Gly); a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycine residues (Gly-Gly-Gly-Gly; SEQ ID NO: 98); a peptide with five glycine residues (Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 99); a peptide with six glycine residues (Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 100); a peptide with seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly; SEQ ID NO: 101); a peptide with eight glycine residues (Gly-Gly-Gly-Gly- Gly-Gly-Gly-Gly; SEQ ID NO: 102). Other combinations of amino acid residues may be used, such as the Gly-Gly-Gly-Gly-Gly-Ser peptide (SEQ ID NO: 103), the Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser peptide (SEQ ID NO: 104), the Gly-Gly-Gly-Gly-Ser peptide (SEQ ID NO: 103), the Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser peptide (SEQ ID NO: 104), the Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser peptide (SEQ ID NO: 105), and the Gly-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Gly peptide (SEQ ID NO: 148). Other suitable linkers include a single Ser and Val residue; the dipeptide Arg-Thr, Gln-Pro, Ser-Ser, Thr-Lys, and Ser-Leu; Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 106), Thr-Val-Ala-Ala-Pro (SEQ ID NO: 107), Gln-Pro-Lys-Ala-Ala (SEQ ID NO: 108), Gln-Arg-Ile-Glu-Gly (SEQ ID NO: 109); Ala-Ser-Thr-Lys-Gly-Pro-Ser (SEQ ID NO: 110), Arg-Thr-Val-Ala-Ala-Pro-Ser (SEQ ID NO: 111), Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 112) and His-Ile-Asp-Ser-Pro-Asn-Lys (SEQ ID NO: 113). The examples listed above are not intended to limit the scope of the invention in any manner and linkers comprising amino acids randomly selected from the group consisting of valine, leucine, isoleucine, serine, threonine, lysine, arginine, histidine, aspartate, glutamate, asparagine, glutamine, glycine and proline have been shown to be suitable in the binding proteins. For further descriptions of linker sequences see, for example, WO2012135345.

[00586] The identity and sequence of amino acid residues in the linker can vary depending on the type of secondary structural element needed to be achieved in the linker. For example, glycine, serine, and alanine are best for linkers with maximum flexibility. Some combination of glycine, proline, threonine, and serine are useful if a more rigid and extended linker is needed. Any amino acid residue can be considered as a linker in combination with other amino acid residues to build larger peptide linkers as needed, depending on the desired properties.

[00587] In some embodiments, the length of L1 is at least twice the length of L3. In some embodiments, the length of L2 is at least twice the length of L4. In some embodiments, the length of L1 is at least twice the length of L3 and the length of L2 is at least twice the length of L4. In some embodiments, L1 is 3 to 12 amino acid residues in length, L2 is 3 to 14 amino acid residues in length, L3 is 1 to 8 amino acid residues in length, and L4 is 1 to 3 amino acid residues in length. In some embodiments, L1 is 5 to 10 amino acid residues in length, L2 is 5 to 8 amino acid residues in length, L3 is 1 to 5 amino acid residues in length, and L4 is 1 to 2 amino acid residues in length. In some embodiments, L1 is 7 amino acid residues in length, L2 is 5 amino acid residues in length, L3 is 1 amino acid residue in length, and L4 is 2 amino acid residues in length. In some embodiments, L1 is 10 amino acid residues in length, L2 is 10 amino acid residues in length, L3 is 0 amino acid residues in length, and L4 is 0 amino acid residues in length. In some embodiments, L1, L2, L3, and L4 each independently have a length selected from 0 to 15 amino acids (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids), wherein at least two of the linkers have a length of 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids). In some embodiments, L1, L2, L3, and L4 are each 0 amino acids in length.

[00588] In some embodiments, L1, L2, L3, and/or L4 comprise the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 525). In some embodiments, L1 comprises the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 525). In some embodiments, L3 comprises the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 525).

[00589] In some embodiments, L1, L2, L3, and/or L4 comprise a sequence derived from a naturally occurring sequence at the junction between an antibody variable domain and an antibody constant domain (e.g., as described in WO2012/135345). For example, in some embodiments, the linker comprises a sequence found at the transition between an endogenous VH and CH1 domain or between an endogenous VL and CL domain (e.g., kappa or lambda). In some embodiments, the linker comprises a sequence found at the transition between an endogenous human VH and CH1 domain or between an endogenous human VL and CL domain (e.g., human kappa or lambda).

[00590] In some embodiments, L1, L2, L3, and/or L4 comprise the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 175). In some embodiments, L1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 175). In some embodiments, L1 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 175), L2 comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: 176), L3 comprises the sequence Ser, and L4 comprises the sequence Arg-Thr. In some embodiments, L3 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 175). In some embodiments, L1 comprises the sequence Ser, L2 comprises the sequence Arg-Thr, L3 comprises the sequence Gly-Gln-Pro-Lys-Ala-Ala-Pro (SEQ ID NO: 175), and L4 comprises the sequence Thr-Lys-Gly-Pro-Ser-Arg (SEQ ID NO: 176).

[00591] In some embodiments, L1, L2, L3, and L4 each independently comprise a sequence selected from (GGGGS)n (where n is an integer between 0 and 5; SEQ ID NO: 174), GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148). In some embodiments, L1 comprises the sequence GQPKAAP (SEQ ID NO: 175), L2 comprises the sequence TKGPS (SEQ ID NO: 106), L3 comprises the sequence S, and L4 comprises the sequence RT. In some embodiments, L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. In some embodiments, L1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 105), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 105), and L4 is 0 amino acids in length. In some embodiments, L1 and L2 are zero amino acids in length and L3 and L4 each independently comprise a sequence selected from (GGGGS)n (where n is an integer between 0 and 5; SEQ ID NO: 174), GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148). In some embodiments, L3 and L4 are zero amino acids in length and L1 and L2 each independently comprise a sequence selected from (GGGGS)n (where n is an integer between 0 and 5; SEQ ID NO: 174), GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148).

Fc Regions and Constant Domains

[00592] In some embodiments, a binding protein of the present invention comprises a second polypeptide chain further comprising a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains. In some embodiments, a binding protein of the present invention comprises a third polypeptide chain further comprising a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains. In some embodiments, a binding protein of the present invention comprises a second polypeptide chain further comprising a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains, and a third polypeptide chain further comprising a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains.

[00593] In some embodiments, a binding protein of the present invention includes one or two Fc variants. The term "Fc variant" as used herein refers to a molecule or sequence that is modified from a native Fc, but that still comprises a binding site for the salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fc variants, and their interaction with the salvage receptor, are known in the art. Thus, the term "Fc variant" can comprise a molecule or sequence that is humanized from a native, non-human Fc. Furthermore, a native Fc comprises regions that can be removed because they provide structural features or biological activity that are not required for the antibody-like binding proteins of the invention. Thus, the term "Fc variant" encompasses a molecule or sequence that lacks one or more native Fc sites or residues, or in which one or more Fc sites or residues have been modified, which affect or are involved in: (1) disulfide bonding, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

[00594] To improve the yields of binding proteins, the CH3 domains can be altered by the "knob-into-holes" technology which is described in detail with several examples, for example, in International Publication No. WO 96/027011, Ridgway et al. al., 1996, Pro-tein Eng. 9: 617-21; and Merchant et al., 1998, Nat. Biotechnol. 16: 67781. Specifically, the interaction surfaces of the two CH3 domains are altered to enhance the heterodimerization of both heavy chains containing these two CH3 domains. Each of the two CH3 domains (of the two heavy chains) can be the "knob", while the other is the "hole". Introduction of a disulfide bridge further stabilizes the heterodimers (Merchant et al., 1998; Atwell et al., 1997, J. Mol. Biol. 270: 26-35) and increases yield. In particular embodiments, the knob is on the second pair of polypeptides with a single variable domain. In other embodiments, the knob is on the first pair of polypeptides that have the cross-over orientation. In still other embodiments, the CH3 domains do not include a knob-into-hole.

[00595] In some embodiments, a binding protein of the present invention comprises a "knob" mutation on the second polypeptide chain and a "hole" mutation on the third polypeptide chain. In some embodiments, a binding protein of the present invention comprises a "knob" mutation on the third polypeptide chain and a "hole" mutation on the second polypeptide chain. In some embodiments, the "knob" mutation comprises substitution(s) at positions corresponding to positions 354 and/or 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C, T366W, T366Y, S354C and T366W, or S354C and T366Y. In some embodiments, the knob mutation comprises substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are S354C and T366W. In some embodiments, the hole mutation comprises substitution(s) at positions corresponding to positions 407 and, optionally, 349, 366, and/or 368 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are Y407V or Y407T and, optionally, Y349C, T366S, and/or L368A. In some embodiments, the hole mutation comprises substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index. In some embodiments, the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

[00596] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and, optionally, 354 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are T366W or T366Y and, optionally, S354C; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and, optionally, 349, 366, and/or 368 of and of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y407V or Y407T and, optionally, Y349C, T366S, and/or L368A.

[00597] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 407 and, optionally, 349, 366, and/or 368 of and human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y407V or Y407T and, optionally, Y349C, T366S, and/or L368A; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366 and, optionally, 354 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are T366W or T366Y and, optionally, S354C.

[00598] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises an amino acid substitution at the position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V.

[00599] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises amino acid substitution(s) at positions corresponding to positions 366, 368, and/or 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are T366S, L368A, and/or Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises an amino acid substitution at a position corresponding to position 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitution is T366W.

[00600] In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V. In some embodiments, the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[00601] In some embodiments, a binding protein of the present invention comprises one or more mutations to improve serum half-life (see, e.g., Hinton, P.R. et al. (2006) J. Immunol. 176(1): 346-56). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and a third polypeptide chain further comprising a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first and/or second Fc regions comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are M428L and N434S. In some embodiments, a binding protein of the present invention comprises knob and hole mutations and one or more mutations to improve serum half-life. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[00602] In some embodiments, a binding protein of the present invention comprises one or more mutations to improve the stability of, e.g., the hinge region and/or dimeric interface of IgG4 (see, e.g., Spiess, C. et al. (2013) J. Biol. Chem. 288:26583-26593). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and a third polypeptide chain further comprising a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K. In some embodiments, a binding protein of the present invention comprises knob and hole mutations and one or more stability-improving mutations. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[00603] In some embodiments, a binding protein of the present invention comprises one or more mutations to improve purification, e.g., by modulating affinity for a purification reagent. For example, it is known that heterodimeric binding proteins can be selectively purified from their homodimeric forms if one of the two Fc regions of the heterodimeric form contains mutation(s) that reduce(s) or eliminate binding to Protein A, since the heterodimeric form will have intermediate affinity in Protein A-based purification than the homodimeric form and can be selectively eluted from Protein A, e.g., using different pHs (see, e.g., Smith, E.J. et al. (2015) Sci. Rep. 5: 17943). In some embodiments, the mutation comprises substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and a third polypeptide chain further comprising a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; and wherein only one of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, a binding protein of the present invention comprises "knob" and "hole" mutations and one or more mutations to improve purification. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions.

[00604] In some embodiments, a binding protein of the present invention comprises one or more mutations to reduce effector function, e.g., Fc receptor-mediated antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or antibody-dependent cellular cytotoxicity (ADCC). In some embodiments, the second polypeptide chain further comprises a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains; wherein the third polypeptide chain further comprises a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG1 according to the EU index, wherein the amino acid substitutions are L234A and L235A. In some embodiments, the second polypeptide chain further comprises a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to human IgG1 positions 234, 235, 329 according to the EU index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the Fc regions of the second and third polypeptide chains are human IgG1 Fc regions, and wherein the Fc regions each comprise amino acid substitutions at positions corresponding to human IgG1 positions 234, 235, and 329 according to the EU index, wherein the amino acid substitutions are L234A, L235A, and P329A. In some embodiments, the mutation comprises substitutions at positions corresponding to human IgG4 positions 234 and 235 according to the EU index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, the binding protein comprises a second polypeptide chain further comprising a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and a third polypeptide chain further comprising a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A. In some embodiments, a binding protein of the present invention comprises "knob" and "hole" mutations and one or more mutations to reduce effector function. In some embodiments, the first and/or second Fc regions are human IgG1 Fc regions. In some embodiments, the first and/or second Fc regions are human IgG4 Fc regions. For further description of mutations in Fc at position 329, see, e.g., Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591-6604 and WO1999051642.

[00605] In some embodiments, the types of mutations described above may be combined in any order or combination. For example, a binding protein of the present invention may comprise two or more of the "knob" and "hole" mutations, one or more mutations to improve serum half-life, one or more mutations to improve IgG4 stability, one or more mutations to improve purification, and/or one or more mutations to reduce effector function described above.

[00606] In certain embodiments, a binding protein of the present invention comprises: a first polypeptide chain comprising a lambda CL domain; a CH3 domain of a second polypeptide chain comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to the EU index, wherein the amino acid substitutions are S354C and T366W; a CH3 domain of a third polypeptide chain comprising amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F; and a fourth polypeptide chain comprising a kappa CL domain. In some embodiments, the first polypeptide chain comprises a lambda CL domain; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to the EU index, wherein the amino acid substitutions are S354C and T366W; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F; and wherein the fourth polypeptide chain comprises a kappa CL domain. In some embodiments, the first polypeptide chain comprises a lambda CL domain; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354, 366, 435, and 436 of human IgG1 according to the EU index, wherein the amino acid substitutions are S354C, T366W, H435R, and Y436F; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V; and wherein the fourth polypeptide chain comprises a CL kappa domain. In some embodiments, the first polypeptide chain comprises a CL kappa domain; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to the EU index, wherein the amino acid substitutions are S354C and T366W; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435 and 436 of human IgG1 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R and Y436F; and wherein the fourth polypeptide chain comprises a lambda CL domain.

[00607] In some embodiments, a binding protein of the present invention is purified via Protein A affinity chromatography, kappa light chain affinity chromatography (e.g., using a KappaSelect resin according to the manufacturer's instructions; GE Healthcare), and optionally, lambda light chain affinity chromatography (e.g., using a LambdaFabSelect resin according to the manufacturer's instructions; GE Healthcare). In some embodiments, a binding protein of the present invention is purified via Protein A affinity chromatography, lambda light chain affinity chromatography (e.g., using a LambdaFabSelect resin according to the manufacturer's instructions; GE Healthcare), and optionally, kappa light chain affinity chromatography (e.g., using a KappaSelect resin according to the manufacturer's instructions; GE Healthcare). In some embodiments, the binding protein comprises two Fc regions, each comprising a CH3 domain and only one of the CH3 domains comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F. In some embodiments, a binding protein of the present invention is purified via Protein A affinity chromatography, then kappa light chain affinity chromatography (e.g., using a KappaSelect resin according to the manufacturer's instructions; GE Healthcare), then optionally lambda light chain affinity chromatography (e.g., using a LambdaFabSelect resin according to the manufacturer's instructions; GE Healthcare) in sequence. In some embodiments, a binding protein of the present invention is purified via Protein A affinity chromatography, then lambda light chain affinity chromatography (e.g., using a LambdaFabSelect resin according to the manufacturer's instructions; GE Healthcare), then optionally kappa light chain affinity chromatography (e.g., using a Kap-paSelect resin according to the manufacturer's instructions; GE Healthcare) in sequence. For example, in some embodiments, the binding protein is contacted with Protein A, eluted from Protein A under conditions suitable to isolate the binding protein from binding proteins comprising 0 or 2 CH3 domains comprising the amino acid substitutions are H435R and Y436F, contacted with a light kappa chain affinity medium (e.g., as used in KappaSelect resin; GE Healthcare), and eluted from the light kappa chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only lambda CL domains (e.g., according to the manufacturer's instructions). Suitable conditions for eluting Protein A are known in the art including, without limitation, a stepwise elution gradient from a pH of 4.5-2.8. In some embodiments, Protein A or a Protein A variant useful for protein purification is employed. In some embodiments, Protein A is attached to a substrate or resin, e.g., as part of a chromatography medium. In some embodiments, after elution from the light kappa chain affinity medium, the binding protein is contacted with a lambda light chain affinity medium (e.g., as used in LambdaFabSelect resin; GE Healthcare) and eluted from the lambda light chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only CL kappa domains (e.g., according to the manufacturer's instructions). In some embodiments, a binding protein of the present invention is detected using HIC chromatography. In some embodiments, the binding protein comprises: a first polypeptide chain comprising a lambda CL domain; a CH3 domain of a second polypeptide chain comprising amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; a CH3 domain of a third polypeptide chain comprising amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F; and a fourth polypeptide chain comprising a CL kappa domain. In some embodiments, the binding protein is produced by a host cell. In some embodiments, the binding protein is purified from cell culture medium or host cell extract. In some embodiments, the binding proteins are secreted by a host cell or produced and extracted from a host cell (e.g., prior to being contacted with Protein A). In some embodiments, the binding protein is in a cell culture medium or host cell extract when contacted with Protein A. In some embodiments, the binding protein is purified from other binding proteins, polypeptides, and/or other cellular components.

[00608] In some embodiments, CH1, CH2, CH3, and CL of the trispecific binding proteins described herein can comprise any of the sequences of CH1, CH2, CH3, and CL of binding proteins 1-53.

Nucleic Acids

[00609] Standard recombinant DNA methodologies were employed to construct the polynucleotides encoding the polypeptides that form the binding proteins, incorporate these polynucleotides into recombinant expression vectors, and introduce these vectors into host cells. See, for example, Sambrook et al., 2001, MOLECULAR CLONING: A LABORATORY MANUAL (Cold Spring Harbor Laboratory Press, 3rd ed.). Enzymatic reactions and purification techniques may be performed according to the manufacturer's specifications, as commonly achieved in the art, or as described herein. Unless specific definitions are provided, the nomenclature used in connection with and the laboratory procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Likewise, conventional techniques may be used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, distribution, and patient treatment.

[00610] Other aspects of the present invention relate to isolated nucleic acid molecules comprising a nucleotide sequence encoding any of the binding proteins described herein. In some embodiments, the isolated nucleic acid is operably linked to a heterologous promoter to control transcription of the nucleic acid sequence encoding the binding protein. A promoter can refer to nucleic acid control sequences that control the transcription of a nucleic acid. A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a sequence encoding a binding protein if the promoter affects the transcription or expression of the coding sequence. Examples of promoters may include, but are not limited to, promoters obtained from the genomes of viruses (such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, simian virus 40 (SV40), and so on), heterologous eukaryotic promoters (such as the actin promoter, an immunoglobulin promoter, heat shock promoters, and so on), the CAG promoter (Niwa et al., Gene 108 (2): 193-9, 1991), the phosphoglycerate kinase (PGK) promoter, a tetracycline-inducible promoter (Masui et al., Nucleic Acids Res. 33: e43, 2005), the lac system, the trp system, the tac system, the trc system, the major operator and PCR regions, and so on. phage lambda promoter, the 3-phosphoglycerate kinase promoter, the yeast acid phosphatase promoters, and the yeast alpha-mating factor promoter. The polynucleotides encoding binding proteins of the present invention may be under the control of a constitutive promoter, an inducible promoter, or any other suitable promoter described herein or another suitable promoter that is readily recognized by those skilled in the art.

[00611] In some embodiments, the isolated nucleic acid is incorporated into a vector. In some embodiments, the vector is an expression vector. Expression vectors can include one or more regulatory sequences operably linked to the polynucleotide to be expressed. The term "regulatory sequence" includes promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Examples of suitable enhancers can include, but are not limited to, enhancer sequences from mammalian genes (such as globin, elastase, albumin, α-fetoprotein, insulin, and so on) and enhancer sequences from a eukaryotic cell virus (such as the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, adenovirus enhancers, and so on). Examples of suitable vectors may include, for example, plasmids, cosmids, episomes, transposons, and viral vectors (e.g., adenoviral, vaccinia virus, Sindbis-viral, measles-pox, herpes viral, lentiviral, retroviral, adeno-associated viral vectors, etc.). Expression vectors may be used to transfect host cells such as, for example, bacterial cells, yeast cells, insect cells, and mammalian cells. Biologically functional viral DNA vectors and plasmids capable of expression and replication in a host are known in the art and may be used to transfect any cell of interest.

[00612] Other aspects of the present invention relate to a vector system comprising one or more vectors encoding a first, second, third, and fourth polypeptide chain of any of the binding proteins described herein. In some embodiments, the vector system comprises a first vector encoding the first polypeptide chain of the binding protein, a second vector encoding the second polypeptide chain of the binding protein, a third vector encoding the third polypeptide chain of the binding protein, and a fourth vector encoding the fourth polypeptide chain of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and second polypeptide chains of the binding protein and a second vector encoding the third and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and third polypeptide chains of the binding protein and a second vector encoding the second and fourth polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first and fourth polypeptide chains of the binding protein and a second vector encoding the second and third polypeptide chains of the binding protein. In some embodiments, the vector system comprises a first vector encoding the first, second, third, and fourth polypeptide chains of the binding protein. The one or more vectors of the vector system can be any of the vectors described herein. In some embodiments, the one or more vectors are expression vectors.

Isolated Host Cells

[00613] Other aspects of the present invention relate to an isolated host cell comprising one or more isolated polynucleotides, vectors, and/or vector systems described herein. In some embodiments, the host cell is a bacterial cell (e.g., an E. coli cell). In some embodiments, the host cell is a yeast cell (e.g., an S. cerevisiae cell). In some embodiments, the host cell is an insect cell. Examples of insect host cells may include, for example, Drosophila cells (e.g., S2 cells), Trichoplusia ni cells (e.g., High Five™ cells), and Spodoptera frugiperda cells (e.g., Sf21 or Sf9 cells). In some embodiments, the host cell is a mammalian cell. Examples of mammalian host cells may include, for example, human embryonic kidney cells (e.g., 293 or 293 cells subcloned for growth in suspension culture), Expi293TM cells, CHO cells, baby hamster kidney cells (e.g., BHK, ATCC CCL 10), mouse Sertoli cells (e.g., TM4 cells), monkey kidney cells (e.g., CV1 ATCC CCL 70), African green monkey kidney cells (e.g., VERO-76, ATCC CRL-1587), human cervical carcinoma cells (e.g., HELA, ATCC CCL 2), canine kidney cells (e.g., MDCK, ATCC CCL 34), rat liver cells (e.g., BRL 3A, ATCC CRL 1442), human lung cells (e.g., W138, ATCC CCL 75), human liver cells (e.g. e.g., Hep G2, HB 8065), mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51), TRI cells, MRC 5 cells, FS4 cells, a human hepatoma cell line (e.g., Hep G2), and myeloma cells (e.g., NS0 and Sp2/0 cells).

[00614] Other aspects of the present invention relate to a method of producing any of the binding proteins described herein. In some embodiments, the method includes a) culturing a host cell (e.g., any of the host cells described herein) comprising an isolated nucleic acid, vector, and/or vector system (e.g., any of the isolated nucleic acids, vectors, and/or vector systems described herein) under conditions such that the host cell expresses the binding protein; and b) isolating the binding protein from the host cell. Methods for culturing host cells under conditions to express a protein are well known to those of skill in the art. Methods of isolating proteins from cultured host cells are well known to those of skill in the art including, for example, via affinity chromatography (e.g., two-step affinity chromatography comprising protein A affinity chromatography followed by size exclusion chromatography).

Uses for Binding Proteins

[00615] Binding proteins may be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays for the detection and quantification of one or more target antigens. Binding proteins bind to one or more target antigens with an affinity that is appropriate for the assay method to be employed.

[00616] For diagnostic applications, in certain embodiments, binding proteins may be labeled with a detectable moiety. The detectable moiety may be any that is capable of producing, directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 3H, 14C, 32P, 35S, 125I, 99Tc, 111In, or 67Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase.

[00617] Binding proteins are also useful for in vivo imaging. A binding protein labeled with a detectable moiety can be administered to an animal, preferably into the bloodstream, and the presence and localization of the labeled antibody in the host assayed. The binding protein can be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other means of detection known in the art.

[00618] Binding proteins can also be used for cell activation, tumor targeting, neutralization of cytokine activity, neutralization of viral infection, combination of multiple signaling events, treatment of cancer, arthritis and/or inflammatory disorders. For example, in some embodiments, a binding protein specifically binds to one, two, or three antigenic targets selected from A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4 (also known as VTCN1), B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2 (also known as MCP-1), CCL3 (also known as MIP-1a), CCL4 (also known as MIP-1b), CCL5 (also known as RANTES), CCL7 (also known as MCP-3), CCL8 (also known as mcp-2), CCL11 (also known as eotaxin), CCL15 (also known as MIP-1d), CCL17 (also known as TARC), CCL19 (also known as MIP-3b), CCL20 (also known as MIP-3a), CCL21 (also known as MIP-2), CCL24 (also known as MPIF-2/eotaxin-2), CCL25 (also known as TECK), CCL26 (also known as eotaxin-3), CCR3, CCR4, CD3, CD19, CD20, CD23 (also known as FCER2, the IgE receptor), CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80 (also known as B7-1), CD86 (also known as B7-2), CD122, CD137 (also known as 41BB), CD137L, CD152 (also known as CTLA4), CD154 (also known as CD40L), CD160, CD272, CD273 (also known as PDL2), CD274 (also known as PDL1), CD275 (also known as B7H2), CD276 (also known as B7H3), CD278 (also known as ICOS), CD279 (also known as PD-1), CDH1 (also known as E-cadherin), chitinase, CLEC9, CLEC91, CRTH2, CSF-1 (also known as M-CSF), CSF-2 (also known as GM-CSF), CSF-3 (also known as GCSF), CX3CL1 (also known as SCYD1), CXCL12 (also known as SDF1), CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13Ra1, , IL13Ra2, IL15, IL17, IL17Rb (also known as a receptor for IL25), IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4 (also known as integrin b4), ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2 (also known as a receptor for IL33), STE-AP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP (also known as a co-receptor for IL7Ra), TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM, and XCR1 (also known as GPR5/CCXCR1). In some embodiments, one or more of the above antigenic targets are human antigenic targets.

[00619] In some embodiments, a binding protein of the present invention is administered to a patient in need thereof for the treatment or prevention of cancer. For example, in some embodiments, the binding protein comprises an antigen-binding site that specifically binds to a protein on the surface of a T cell and another antigen-binding site that specifically binds to a target tumor protein (e.g., two antigen-binding sites that specifically bind to proteins on the surface of T cells and one antigen-binding site that specifically binds to a target tumor protein, or two antigen-binding sites that specifically bind to target tumor proteins and one antigen-binding site that specifically binds to a protein on the surface of a T cell). In certain embodiments, the binding protein comprises an antigen-binding site that specifically binds to CD3, an antigen-binding site that specifically binds to CD28, and an antigen-binding site that specifically binds to a target tumor protein selected from CD19, CD20, CD38, Her2, and LAMP1. In some embodiments, the binding protein is co-administered with a chemotherapeutic agent. In some embodiments, the patient is a human.

[00620] In some embodiments, a binding protein of the present invention is administered to a patient in need thereof for the treatment or prevention of an inflammatory disease or disorder. In some embodiments, the binding protein comprises three antigen-binding sites that each specifically bind to a target cytokine protein selected from IL-4, IL-13, and TNFα. In some embodiments, the binding protein is co-administered with an anti-inflammatory agent. In some embodiments, the patient is a human.

[00621] The present invention also relates to a kit comprising a binding protein and other reagents useful for detecting levels of target antigen in biological samples. Such reagents may include a detectable marker, blocking serum, positive and negative control samples, and detection reagents. In some embodiments, the kit comprises a composition comprising any binding protein, polynucleotide, vector, vector system, and/or host cell described herein. In some embodiments, the kit comprises a container and a label or informational insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials, such as glass or plastic. The container contains a composition which is, by itself or in combination with another composition, effective for treating, preventing, and/or diagnosing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial pierceable by a hypodermic injection needle). In some embodiments, the label or package insert indicates that the composition is used to prevent, diagnose, and/or treat the condition of choice. Alternatively, or additionally, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. Therapeutic Compositions of Binding Proteins and Administration Thereof

[00622] Therapeutic or pharmaceutical compositions comprising binding proteins are within the scope of the invention. Such therapeutic or pharmaceutical compositions may comprise a therapeutically effective amount of a binding protein or binding protein conjugate, in admixture with a pharmaceutically or physiologically acceptable formulating agent selected to suit the method of administration.

[00623] Acceptable formulation materials are preferably non-toxic to recipients at the dosages and concentrations employed.

[00624] The pharmaceutical composition may contain formulation materials to modify, maintain or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring, and diluent agents, emulsifying agents, polymers, and so on. hydrophilic compounds (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as Pluronics; PEG; sorbitan ethers; polysorbates such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or Tyloxapal), stability-enhancing agents (such as sucrose or sorbitol), tonicity-enhancing agents (such as alkali metal halides—preferably sodium or potassium chloride—or mannitol and sorbitol), delivery vehicles, diluents, excipients, and/or pharmaceutical adjuvants; see, for example, REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A. R. Gennaro, Org., Mack Publishing Company 1990), and subsequent editions thereof, incorporated herein by reference for any purpose).

[00625] The ideal pharmaceutical composition will be determined by those skilled in the art depending on, for example, the intended route of administration, delivery format, and desired dosage. Such compositions may influence the physical state, stability, in vivo release rate, and in vivo clearance rate of the binding protein.

[00626] The primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a vehicle or carrier suitable for injection may be water, physiological saline, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration. Neutral buffered physiological saline or saline mixed with serum albumin are other examples of vehicles. Other exemplary pharmaceutical compositions comprise Tris buffer having a pH of about 7.0-8.5 or acetate buffer having a pH of about 4.0-5.5, which may further include sorbitol or a suitable substitute. In one embodiment of the invention, binding protein compositions may be prepared for storage by mixing the selected composition of the desired degree of purity with formulating agents in the form of a lyophilized cake or an aqueous solution. Additionally, the binding protein can be formulated as a lyophilizate using appropriate excipients, such as sucrose.

[00627] The pharmaceutical compositions of the invention may be selected for parenteral or subcutaneous delivery. Alternatively, the compositions may be selected for inhalation or for delivery via the digestive tract, such as orally. The preparation of such pharmaceutically acceptable compositions is within the skill in the art.

[00628] The components of the formulation are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at a physiological pH or slightly lower pH, typically within a pH range of about 5 to about 8.

[00629] When parenteral administration is contemplated, therapeutic compositions for use may be in the form of a parenterally acceptable, pyrogen-free aqueous solution comprising the desired binding protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water in which a binding protein is formulated as a suitably preserved, sterile isotonic solution. Yet another preparation may involve formulating the desired molecule with an agent, such as injectable microspheres, bioerodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), spheres or liposomes, which allow for controlled or sustained release of the product which may then be delivered via a depot injection. Hyaluronic acid may also be used and this may have the effect of promoting a sustained duration in circulation. Other suitable means for introducing the desired molecule include implantable drug delivery devices.

[00630] In one embodiment, a pharmaceutical composition may be formulated for inhalation. For example, a binding protein may be formulated as a dry powder for inhalation. Binding protein inhalation solutions may also be formulated with a propellant for aerosol administration. In yet another embodiment, the solutions may be nebulized.

[00631] It is also contemplated that certain formulations may be administered orally. In one embodiment of the invention, binding proteins that are administered in this manner may be formulated with or without carriers customarily used in compounding solid dosage forms such as tablets and capsules. For example, a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and systemic degradation is minimized. Additional agents may be included to facilitate absorption of the binding protein. Diluents, flavorings, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be used.

[00632] Another pharmaceutical composition may involve an effective amount of binding proteins in admixture with non-toxic excipients that are suitable for the manufacture of tablets. By dissolving the tablets in sterile water, or other suitable vehicle, solutions may be prepared in unit dose form. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binding agents such as starch, gelatin or acacia; or lubricating agents such as magnesium stearate, stearic acid or talc.

[00633] Additional pharmaceutical compositions of the invention will be apparent to those skilled in the art, including formulations involving binding proteins in sustained or controlled delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery media, such as liposomal carriers, bioerodible microparticles or porous spheres, and depot injections, are also known to those skilled in the art. Additional examples of controlled release preparations include semipermeable polymeric matrices in the form of shaped articles, e.g., films or microcapsules. Sustained release matrices can include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), ethylene vinyl acetate, or poly-D(-)-3-hydroxybutyric acid. Sustained release compositions may also include liposomes, which may be prepared by any of several methods known in the art.

[00634] Pharmaceutical compositions to be used for in vivo administration should typically be sterile. This may be achieved by filtration through sterile filtration membranes. When the composition is lyophilized, sterilization using this method may be conducted before or after lyophilization and reconstitution. The composition for parenteral administration may be stored in lyophilized form or in a solution. In addition, parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[00635] Once the pharmaceutical composition has been formulated, it may be stored in sterile vials as a solution, suspension, gel, emulsion, solid or as a powder, dehydrated or lyophilized. Such formulations may be stored in a ready-to-use form or in a form (e.g. lyophilized) that requires reconstitution prior to administration.

[00636] The present invention also encompasses kits for the production of a single dose administration unit. Each of the kits may contain both a first container with a dry protein and a second container with an aqueous formulation. Also included within the scope of the present invention are kits containing pre-filled single or multi-chamber syringes (e.g., liquid syringes and mixers).

[00637] The effective amount of a pharmaceutical composition of the binding protein to be used therapeutically will depend, for example, on the therapeutic context and objectives. Those skilled in the art will understand that appropriate dosage levels for treatment will vary depending, in part, on the molecule delivered, the indication for which the binding protein is being used, the route of administration, and the size (body weight, body surface area, or organ size) and condition (age and general health) of the patient. Accordingly, the physician may titrate the dosage and modify the route of administration to obtain the optimal therapeutic effect.

[00638] The frequency of dosing will depend on the pharmacokinetic parameters of the binding protein in the formulation to be used. Typically, a physician will administer the composition until a dosage is reached that achieves the desired effect. The composition may therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely done by those skilled in the art and is within the scope of tasks routinely performed by them. Appropriate dosages can be determined using suitable dose-response data.

[00639] The route of administration of the pharmaceutical composition is in accordance with known methods, for example, orally; by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal or intralesional injection; by sustained release systems; or by implant devices. When desired, the compositions may be administered by bolus injection or continuously by infusion or through an implant device.

[00640] The composition may also be administered locally by implantation of a membrane, sponge or other suitable material in which the desired molecule has been absorbed or encapsulated. When an implant device is used, the device may be implanted in any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed release bolus or continuous administration.

[00641] In some embodiments, the present invention relates to a method of preventing and/or treating a proliferative disease or disorder (e.g., cancer). In some embodiments, the method comprises administering, to a patient, a therapeutically effective amount of at least one of the binding proteins described herein. In some embodiments, the patient is a human. In some embodiments, the at least one binding protein is administered in combination with one or more anti-cancer therapies (e.g., any anti-cancer therapy known in the art). In some embodiments, the at least one binding protein is administered prior to one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered concurrently with one or more anti-cancer therapies. In some embodiments, the at least one binding protein is administered subsequent to one or more anti-cancer therapies.

[00642] In some embodiments, the present invention relates to a method of preventing and/or treating an inflammatory disease or disorder (e.g., cancer). In some embodiments, the method comprises administering to a patient a therapeutically effective amount of at least one of the binding proteins described herein. In some embodiments, the patient is a human. In some embodiments, the at least one binding protein is administered in combination with one or more anti-inflammatory therapies (e.g., any anti-inflammatory therapy known in the art). In some embodiments, the at least one binding protein is administered prior to one or more anti-inflammatory therapies. In some embodiments, the at least one binding protein is administered simultaneously with one or more anti-inflammatory therapies. In some embodiments, the at least one binding protein is administered following one or more anti-inflammatory therapies.

[00643] Without limiting the present invention, a number of embodiments of the present invention are described below for purposes of illustration.

[00644] Item 1. A binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: V L2-L1-VL1-L2-CL[I]

[00645] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1[II]

[00646] and a third polypeptide chain comprises a structure represented by the formula: V H3-CH1[III]

[00647] and a fourth polypeptide chain comprises a structure represented by the formula: V L3-CL [IV]

[00648] where:

[00649] VL1 is a first immunoglobulin light chain variable domain;

[00650] VL2 is a second immunoglobulin light chain variable domain;

[00651] VL3 is a third immunoglobulin light chain variable domain;

[00652] VH1 is a first immunoglobulin heavy chain variable domain;

[00653] VH2 is a second immunoglobulin heavy chain variable domain;

[00654] VH3 is a third immunoglobulin heavy chain variable domain;

[00655] CL is an immunoglobulin light chain constant domain;

[00656] CH1 is an immunoglobulin CH1 heavy chain constant domain; and

[00657] L1, L2, L3 and L4 are amino acid linkers;

[00658] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[00659] Item 2. A binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00660] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[00661] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3[III]

[00662] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[00663] where:

[00664] VL1 is a first immunoglobulin light chain variable domain;

[00665] VL2 is a second immunoglobulin light chain variable domain;

[00666] VL3 is a third immunoglobulin light chain variable domain;

[00667] VH1 is a first immunoglobulin heavy chain variable domain;

[00668] VH2 is a second immunoglobulin heavy chain variable domain;

[00669] VH3 is a third immunoglobulin heavy chain variable domain;

[00670] CL is an immunoglobulin light chain constant domain;

[00671] CH1 is an immunoglobulin heavy chain CH1 constant domain;

[00672] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[00673] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00674] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00675] L1, L2, L3 and L4 are amino acid linkers;

[00676] and wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair.

[00677] Item 3. A binding protein according to item 1, wherein the second and/or third polypeptide chains further comprise a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains.

[00678] Item 4. A binding protein according to item 1 or item 2, wherein at least one of L1, L2, L3, or L4 is independently 0 amino acid in length.

[00679] Item 5. A binding protein according to item 1 or item 2, wherein L1, L2, L3, or L4 is independently at least one amino acid in length.

[00680] Item 6. A binding protein according to item 1 or item 2, wherein (a) L1, L2, L3, and L4 are each independently zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148); or (b) L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148).

[00681] Item 7. A binding protein according to item 1 or item 2, wherein:

[00682] (a) L1 comprises the sequence GQPKAAP (SEQ ID NO: 175), L2 comprises the sequence TKGPS (SEQ ID NO: 106), L3 comprises the sequence S, and L4 comprises the sequence RT;

[00683] (b) L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L3 is 0 amino acids in length, and L4 is 0 amino acids in length;

[00684] (c) L1 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; or

[00685] (d) L1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 105), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 105), and L4 is 0 amino acids in length.

[00686] Item 8. A binding protein according to item 1 or item 2, wherein the binding protein is trispecific and capable of specifically binding to three different antigenic targets.

[00687] Item 9. A binding protein according to any one of items 1-8, wherein the binding protein specifically binds to three target proteins corresponding to two target proteins on T cells and a target tumor protein.

[00688] Item 10. A binding protein according to item 9, wherein one of said target proteins on T cells is CD3.

[00689] Item 11. A binding protein according to item 9 or item 10, wherein one of said target proteins on T cells is CD28.

[00690] Item 12. A binding protein according to any one of items 9-11, wherein said target tumor protein is CD38.

[00691] Item 13. A binding protein according to any one of items 1-8, wherein the binding protein specifically binds to three target proteins corresponding to two target proteins on T cells and one target protein selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, 2, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17Rb, , IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1.

[00692] Item 14. A binding protein according to item 1 or item 2, wherein the binding protein is capable of inhibiting the function of one or more target proteins.

[00693] Item 15. A binding protein according to item 14, wherein the one or more target proteins are selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, CSF-3, CX3CL1, 12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, lep- tina, LPFS2, MHC classe II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, receptor plaquetário, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, quinase Syk, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1.

[00694] Item 16. A binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: V L2-L1-VL1-L2-CL[I]

[00695] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1[II]

[00696] and a third polypeptide chain comprises a structure represented by the formula: V H3-CH1[III]

[00697] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[00698] where:

[00699] VL1 is a first immunoglobulin light chain variable domain;

[00700] VL2 is a second immunoglobulin light chain variable domain;

[00701] VL3 is a third immunoglobulin light chain variable domain;

[00702] VH1 is a first immunoglobulin heavy chain variable domain;

[00703] VH2 is a second immunoglobulin heavy chain variable domain;

[00704] VH3 is a third immunoglobulin heavy chain variable domain;

[00705] CL is an immunoglobulin light chain constant domain;

[00706] CH1 is an immunoglobulin CH1 heavy chain constant domain; and

[00707] L1, L2, L3 and L4 are amino acid linkers;

[00708] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[00709] where:

[00710] (a) VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 151, 153, 155, 157, 159, 161, 163, 165, and 167; or

[00711] (b) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 43-59, 123-125, 138-140, and 149; and

[00712] where:

[00713] (a) VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 150, 152, 154, 156, 158, 160, 162, 164, and 166; or

[00714] (b) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-42, 120-122, and 126-128.

[00715] Item 17. A binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00716] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[00717] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3[III]

[00718] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[00719] where:

[00720] VL1 is a first immunoglobulin light chain variable domain;

[00721] VL2 is a second immunoglobulin light chain variable domain;

[00722] VL3 is a third immunoglobulin light chain variable domain;

[00723] VH1 is a first immunoglobulin heavy chain variable domain;

[00724] VH2 is a second immunoglobulin heavy chain variable domain;

[00725] VH3 is a third immunoglobulin heavy chain variable domain;

[00726] CL is an immunoglobulin light chain constant domain;

[00727] CH1 is an immunoglobulin heavy chain CH1 constant domain;

[00728] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[00729] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00730] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00731] L1, L2, L3 and L4 are amino acid linkers;

[00732] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[00733] where:

[00734] (a) VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 151, 153, 155, 157, 159, 161, 163, 165, and 167; or

[00735] (b) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 43-59, 123-125, 138-140, and 149; and

[00736] where:

[00737] (a) VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 150, 152, 154, 156, 158, 160, 162, 164, and 166; or

[00738] (b) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 25-42, 120-122, and 126-128.

[00739] Item 18. A binding protein according to item 16, wherein the second and/or third polypeptide chains further comprise a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin CH2 and CH3 heavy chain constant domains.

[00740] Item 19. A binding protein according to item 16 or item 17, wherein at least one of L1, L2, L3, or L4 is independently 0 amino acid in length.

[00741] Item 20. A binding protein according to item 16 or item 17, wherein L1, L2, L3, or L4 is independently at least one amino acid in length.

[00742] Item 21. A binding protein according to item 16 or item 17, wherein (a) L1, L2, L3, and L4 are each independently zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148); or (b) L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148).

[00743] Item 22. A binding protein according to item 16 or item 17, wherein:

[00744] (a) L1 comprises the sequence GQPKAAP (SEQ ID NO: 175), L2 comprises the sequence TKGPS (SEQ ID NO: 106), L3 comprises the sequence S, and L4 comprises the sequence RT;

[00745] (b) L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L3 is 0 amino acids in length, and L4 is 0 amino acids in length;

[00746] (c) L1 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; or

[00747] (d) L1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 105), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 105), and L4 is 0 amino acids in length.

[00748] Item 23. A binding protein according to item 16 or item 17, wherein:

[00749] (a) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45;

[00750] (b) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 25, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 26, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 27; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45;

[00751] (c) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57;

[00752] (d) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 37, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 38, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 39; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 55, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 56, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 57;

[00753] (e) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 42; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 58, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59;

[00754] (f) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 40, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 41, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 42; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 58, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 59;

[00755] (g) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 126, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 127, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 128; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 138, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 139, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 140;

[00756] (h) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 126, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 127, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 128; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 138, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 139, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 140;

[00757] (i) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 120, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 121, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 122; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125; or

[00758] (j) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 31, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 32, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 33; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 49, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 50, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 51; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 120, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 121, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 122; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

[00759] Item 24. A binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: V L2-L1-VL1-L2-CL[I]

[00760] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1[II]

[00761] and a third polypeptide chain comprises a structure represented by the formula: V H3-CH1[III]

[00762] and a fourth polypeptide chain comprises a structure represented by the formula: V L3-CL [IV]

[00763] where:

[00764] VL1 is a first immunoglobulin light chain variable domain;

[00765] VL2 is a second immunoglobulin light chain variable domain;

[00766] VL3 is a third immunoglobulin light chain variable domain;

[00767] VH1 is a first immunoglobulin heavy chain variable domain;

[00768] VH2 is a second immunoglobulin heavy chain variable domain;

[00769] VH3 is a third immunoglobulin heavy chain variable domain;

[00770] CL is an immunoglobulin light chain constant domain;

[00771] CH1 is an immunoglobulin heavy chain CH1 constant domain; and

[00772] L1, L2, L3 and L4 are amino acid linkers;

[00773] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[00774] where:

[00775] (a) VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 169, 171, and 173; or

[00776] (b) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-147, 178, and 179;

[00777] where:

[00778] (a) VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 168, 170, and 172; or

[00779] (b) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 129-137.

[00780] Item 25. A binding protein comprising four polypeptide chains that form three antigen-binding sites, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL[I]

[00781] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3[II]

[00782] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1-hinge-CH2-CH3[III]

[00783] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL[IV]

[00784] where:

[00785] VL1 is a first immunoglobulin light chain variable domain;

[00786] VL2 is a second immunoglobulin light chain variable domain;

[00787] VL3 is a third immunoglobulin light chain variable domain;

[00788] VH1 is a first immunoglobulin heavy chain variable domain;

[00789] VH2 is a second immunoglobulin heavy chain variable domain;

[00790] VH3 is a third immunoglobulin heavy chain variable domain;

[00791] CL is an immunoglobulin light chain constant domain;

[00792] CH1 is an immunoglobulin heavy chain CH1 constant domain;

[00793] CH2 is an immunoglobulin CH2 heavy chain constant domain;

[00794] CH3 is an immunoglobulin CH3 heavy chain constant domain;

[00795] hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and

[00796] L1, L2, L3 and L4 are amino acid linkers;

[00797] wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair;

[00798] where:

[00799] (a) VL1, VL2, and VL3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 169, 171, and 173; or

[00800] (b) VL1, VL2, and VL3 each independently comprise light chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 141-147, 178, and 179;

[00801] where:

[00802] (a) VH1, VH2, and VH3 each independently comprise a variable domain sequence as set forth in any one of SEQ ID NOs: 168, 170, and 172; or

[00803] (b) VH1, VH2, and VH3 each independently comprise heavy chain complementarity determining regions comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 129-137.

[00804] Item 26. A binding protein according to item 24, wherein the second and/or third polypeptide chains further comprise a CH1-linked Fc region, the Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3.

[00805] Item 27. A binding protein according to item 24 or item 25, wherein at least one of L1, L2, L3, and L4 is independently 0 amino acid in length.

[00806] Item 28. A binding protein according to item 24 or item 25, wherein L1, L2, L3, and L4 are independently at least one amino acid in length.

[00807] Item 29. A binding protein according to item 24 or item 25, wherein (a) L1, L2, L3, and L4 are each independently zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148); or (b) L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148).

[00808] Item 30. A binding protein according to item 24 or item 25, wherein:

[00809] (a) L1 comprises the sequence GQPKAAP (SEQ ID NO: 175), L2 comprises the sequence TKGPS (SEQ ID NO: 106), L3 comprises the sequence S, and L4 comprises the sequence RT;

[00810] (b) L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L3 is 0 amino acids in length, and L4 is 0 amino acids in length;

[00811] (c) L1 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; or

[00812] (d) L1 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 105), L2 is 0 amino acids in length, L3 comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 105), and L4 is 0 amino acids in length.

[00813] Item 31. A binding protein according to item 24 or item 25, wherein:

[00814] (a) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142;

[00815] (b) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142;

[00816] (c) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147;

[00817] (d) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147;

[00818] (e) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144;

[00819] (f) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 129, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 130, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 131; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 141, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 178, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 142; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144;

[00820] (g) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147;

[00821] (h) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144;

[00822] (i) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; or

[00823] (j) VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 135, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 136, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 137; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 145, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 146, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 147; VH3 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 132, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 133, and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 134; and VL3 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 143, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 179 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 144.

[00824] Item 32. A binding protein according to any one of items 1, 16 and 24, wherein the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, and Y407V.

[00825] Item 33. A binding protein according to any one of items 1, 16 and 24, wherein the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the first Fc region comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V; and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, wherein the second Fc region comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W.

[00826] Item 34. A binding protein according to any one of items 1, 16, 24, 32 and 33, wherein the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3, and wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the first and second Fc regions comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are M428L and N434S.

[00827] Item 35. A binding protein according to any one of items 2, 17 and 25, wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W; and wherein the CH3 domains of the third polypeptide chain comprise amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V.

[00828] Item 36. A binding protein according to any one of items 2, 17 and 25, wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368 and 407 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A and Y407V; and wherein the CH3 domains of the third polypeptide chain comprise amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are S354C and T366W.

[00829] Item 37. A binding protein according to any one of items 2, 17, 25, 35 and 36, wherein the CH3 domains of the second and third polypeptide chains both comprise amino acid substitutions at positions corresponding to positions 428 and 434 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are M428L and N434S.

[00830] Item 38. A binding protein according to any one of items 1, 16, 24, and 32-34, wherein the second polypeptide chain further comprises a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the third polypeptide chain further comprises a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the first and second Fc regions are human IgG1 or IgG4 Fc regions; and wherein only one of the first and second Fc regions comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F.

[00831] Item 39. A binding protein according to any one of items 2, 17, 25 and 35-37, wherein the CH3 domains of the second and third polypeptide chains are CH3 domains of human IgG1 or IgG4 and wherein only one of the CH3 domains comprises amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F.

[00832] Item 40. A binding protein according to any one of items 1, 16, 24, 32-34 and 38, wherein the second polypeptide chain further comprises a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the third polypeptide chain further comprises a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K.

[00833] Item 41. A binding protein according to any one of items 2, 17, 25, 35-37 and 39, wherein the CH3 domains of the second and third polypeptide chains are CH3 domains of human IgG4 and wherein the CH3 domains each comprise amino acid substitutions at positions corresponding to positions 228 and 409 of human IgG4 according to the EU index, wherein the amino acid substitutions are S228P and R409K.

[00834] Item 42. A binding protein according to any one of items 1, 16, 24, 32-34, 38 and 40, wherein the second polypeptide chain further comprises a first Fc region linked to CH1, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the third polypeptide chain further comprises a second Fc region linked to CH1, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the first and second Fc regions are human IgG4 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A.

[00835] Item 43. A binding protein according to any one of items 2, 17, 25, 35-37, 39 and 41, wherein the CH3 domains of the second and third polypeptide chains are human IgG4 CH3 domains and wherein the CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are F234A and L235A.

[00836] Item 44. A binding protein according to any one of items 1, 16, 24, 32-34, 38 and 40, wherein the second polypeptide chain further comprises a first CH1-linked Fc region, the first Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the third polypeptide chain further comprises a second CH1-linked Fc region, the second Fc region comprising an immunoglobulin hinge region and immunoglobulin heavy chain constant domains CH2 and CH3; wherein the first and second Fc regions are human IgG1 Fc regions; and wherein the first and second Fc regions each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are L234A and L235A.

[00837] Item 45. A binding protein according to any one of items 2, 17, 25, 35-37, 39 and 41, wherein the CH3 domains of the second and third polypeptide chains are human IgG1 CH3 domains and wherein the CH3 domains each comprise amino acid substitutions at positions corresponding to positions 234 and 235 of human IgG4 according to the EU index, wherein the amino acid substitutions are L234A and L235A.

[00838] Item 46. A binding protein according to any one of items 2, 17, 35-37, 39, 41, 43 and 45, wherein VH1 and VL1 form a first antigen-binding site that specifically binds to human CD3, wherein VH2 and VL2 form a second antigen-binding site that specifically binds to human CD28, and wherein VH3 and VL3 form a third antigen-binding site that specifically binds to a human target tumor protein.

[00839] Item 47. A binding protein according to any one of items 2, 17, 35-37, 39, 41, 43 and 45, wherein VH1 and VL1 form a first antigen-binding site that specifically binds to human CD28, wherein VH2 and VL2 form a second antigen-binding site that specifically binds to human CD3, and wherein VH3 and VL3 form a third antigen-binding site that specifically binds to a human target tumor protein.

[00840] Item 48. A binding protein according to item 46 or item 47, wherein the third antigen-binding site specifically binds to a human target tumor protein selected from the group consisting of CD19, CD20, CD38, Her2, and LAMP1.

[00841] Item 49. A binding protein according to any one of items 46-48, wherein the antigen-binding site that specifically binds to CD3 comprises:

[00842] (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 152 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 153; or

[00843] (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155.

[00844] Item 50. A binding protein according to any one of items 46-49, wherein the antigen-binding site that specifically binds to CD28 comprises:

[00845] (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 161; or

[00846] (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 163.

[00847] Item 51. A binding protein according to any one of items 46-50, wherein the antigen-binding site that specifically binds to a target tumor protein comprises:

[00848] (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 156 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 157;

[00849] (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 159;

[00850] (c) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 165;

[00851] (d) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 150 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 151; or

[00852] (e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 166 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 167.

[00853] Item 52. A binding protein according to any one of items 2, 17, 35-37, 39, 41, 43 and 45, wherein:

[00854] (a) VH1 and VL1 form a first antigen-binding site that specifically binds human TNFα, VH2 and VL2 form a second antigen-binding site that specifically binds human IL13, and VH3 and VL3 form a third antigen-binding site that specifically binds human IL14;

[00855] (b) VH1 and VL1 form a first antigen-binding site that specifically binds human TNFα, VH2 and VL2 form a second antigen-binding site that specifically binds human IL14, and VH3 and VL3 form a third antigen-binding site that specifically binds human IL13;

[00856] (c) VH1 and VL1 form a first antigen-binding site that specifically binds to human IL14, VH2 and VL2 form a second antigen-binding site that specifically binds to human TNFα, and VH3 and VL3 form a third antigen-binding site that specifically binds to human IL13;

[00857] (d) VH1 and VL1 form a first antigen-binding site that specifically binds to human IL14, VH2 and VL2 form a second antigen-binding site that specifically binds to human IL13, and VH3 and VL3 form a third antigen-binding site that specifically binds to human TNFα;

[00858] (e) VH1 and VL1 form a first antigen-binding site that specifically binds to human IL13, VH2 and VL2 form a second antigen-binding site that specifically binds to human IL14, and VH3 and VL3 form a third antigen-binding site that specifically binds to human TNFα; or

[00859] (f) VH1 and VL1 form a first antigen-binding site that specifically binds to human IL13, VH2 and VL2 form a second antigen-binding site that specifically binds to human TNFα, and VH3 and VL3 form a third antigen-binding site that specifically binds to human IL14.

[00860] Item 53. A binding protein according to item 52, wherein the antigen-binding site that specifically binds to human TNFα comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 168 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 169.

[00861] Item 54. A binding protein according to item 52 or item 53, wherein the antigen-binding site that specifically binds to human IL14 comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 170 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 171.

[00862] Item 55. A binding protein according to any one of items 52-54, wherein the antigen-binding site that specifically binds to human IL13 comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 172 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 173.

[00863] Item 56. A binding protein according to any one of items 1-55, wherein:

[00864] (a) the CL domain of the first polypeptide chain is a human kappa CL domain and the CL domain of the fourth polypeptide chain is a human lambda CL domain; or

[00865] (b) the CL domain of the first polypeptide chain is a human lambda CL domain and the CL domain of the fourth polypeptide chain is a human kappa CL domain.

[00866] Item 57. A binding protein according to any one of items 2, 17, 25, 35-37, 39, 41 and 43-55, wherein the first polypeptide chain comprises a lambda CL domain; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to the EU index, wherein the amino acid substitutions are S354C and T366W; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435 and 436 of human IgG1 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R and Y436F; and wherein the fourth polypeptide chain comprises a CL kappa domain.

[00867] Item 58. A binding protein comprising a first polypeptide chain, a second polypeptide chain, a third polypeptide chain, and a fourth polypeptide chain wherein:

[00868] (a) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2;

[00869] (b) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 1; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 2;

[00870] (c) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14;

[00871] (d) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 13; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 14;

[00872] (e) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 18;

[00873] (f) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 17; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 18;

[00874] (g) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22;

[00875] (h) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 21; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 22;

[00876] (i) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61;

[00877] (j) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 61 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 61;

[00878] (k) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 60 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 60; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 71 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 71;

[00879] (l) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 76 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 76; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 75 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 75; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74;

[00880] (m) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 82 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 82; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 81 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 81; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74;

[00881] (n) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 88 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 88; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 87 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 87; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86;

[00882] (o) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 94 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 94; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 93 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 93; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86;

[00883] (p) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74;

[00884] (q) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 69 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 69; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 68 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 68; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86;

[00885] (r) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 73 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 73; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 74 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 74;

[00886] (s) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 63 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 63; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 62 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 62; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 85 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 85; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 86 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 86;

[00887] (t) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 4 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 4; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 3; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115; or

[00888] (u) the first polypeptide chain comprises the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10; the second polypeptide chain comprises the amino acid sequence of SEQ ID NO: 9 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 9; the third polypeptide chain comprises the amino acid sequence of SEQ ID NO: 114 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 114; and the fourth polypeptide chain comprises the amino acid sequence of SEQ ID NO: 115 or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 115.

[00889] Item 59. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a binding protein according to any one of items 1-58.

[00890] Item 60. An expression vector comprising the nucleic acid molecule according to item 59.

[00891] Item 61. An isolated host cell comprising the nucleic acid molecule according to item 59.

[00892] Item 62. An isolated host cell comprising the expression vector according to item 60.

[00893] Item 63. An isolated host cell according to item 61 or item 62, wherein the host cell is a mammalian cell or an insect cell.

[00894] Item 64. A pharmaceutical composition comprising binding protein according to any one of items 1-58 and a pharmaceutically acceptable carrier.

[00895] Item 65. A method of preventing and/or treating cancer in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein according to any one of items 1-23 and 32-58 or the pharmaceutical composition according to item 64.

[00896] Item 66. The method of item 65, wherein the binding protein comprises an antigen-binding site that specifically binds to a protein on the surface of a T cell and another antigen-binding site that specifically binds to a target tumor protein.

[00897] Item 67. The method of item 66, wherein the binding protein comprises a first antigen-binding site that specifically binds to CD3, a second antigen-binding site that specifically binds to CD28, and a third antigen-binding site that specifically binds to a target tumor protein selected from the group consisting of CD19, CD20, CD38, Her2, and LAMP1.

[00898] Item 68. The method of any one of items 65-67, wherein the at least one binding protein is co-administered with a chemotherapeutic agent.

[00899] Item 69. A method of preventing and/or treating an inflammatory disease or disorder in a patient comprising administering to the patient a therapeutically effective amount of at least one binding protein according to any one of items 1-15, 24-45 and 52-58 or the pharmaceutical composition according to item 64.

[00900] Item 70. The method of item 69, wherein the binding protein comprises three antigen-binding sites that each specifically bind to a target cytokine protein selected from the group consisting of IL-4, IL-13, and TNFα.

[00901] Item 71. The method of item 69 or item 70, wherein the at least one binding protein is co-administered with an anti-inflammatory agent.

[00902] Item 72. The method according to any one of items 65-71, wherein the patient is a human being.

[00903] Item 73. The method of item 65 or item 69, wherein the binding protein is capable of inhibiting the function of one or more target proteins selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, CSF-2, -3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, IL18, 2, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, AP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1.

[00904] Item 74. A binding protein according to any one of items 1-23 and 32-58 or the composition according to item 64 wherein it is for use in the prevention and/or treatment of cancer in a patient.

[00905] Item 75. A binding protein for use or the composition for use according to item 74, wherein the binding protein comprises an antigen-binding site that specifically binds to a protein on the surface of a T cell and another antigen-binding site that specifically binds to a target tumor protein.

[00906] Item 76. A binding protein for use or the composition for use according to item 75, wherein the binding protein comprises a first antigen-binding site that specifically binds to CD3, a second antigen-binding site that specifically binds to CD28, and a third antigen-binding site that specifically binds to a target tumor protein selected from the group consisting of CD19, CD20, CD38, Her2, and LAMP1.

[00907] Item 77. A binding protein for use or composition for use according to any one of items 74-76, wherein the binding protein is co-administered with a chemotherapeutic agent.

[00908] Item 78. A binding protein according to any one of items 1-15, 24-45 and 52-58 or the pharmaceutical composition according to item 64 wherein it is for use in the prevention and/or treatment of an inflammatory disease or disorder in a patient.

[00909] Item 79. A binding protein for use or the composition for use according to item 78, wherein the binding protein comprises three antigen-binding sites that each specifically bind to a target cytokine protein selected from the group consisting of IL-4, IL-13, and TNFα.

[00910] Item 80. A binding protein for use or the composition for use according to item 78 or item 79, wherein the binding protein is co-administered with an anti-inflammatory agent.

[00911] Item 81. A binding protein for use or composition for use according to any one of items 74-80, wherein the patient is a human.

[00912] Item 82. A binding protein for use or the composition for use according to item 74 or item 78, wherein the binding protein is capable of inhibiting the function of one or more target proteins selected from the group consisting of A2AR, APRIL, ATP-Dase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CSF-1, CSF-2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, EN- TPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17Rb, , IL18, IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, ST2, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1.

[00913] Item 83. A method of purifying a binding protein produced by a host cell comprising:

[00914] (a) producing a binding protein according to any one of items 2, 17, 25, 35-37, 41, 43, 45 and 46-55 in a host cell, wherein only one of the CH3 domains of the second polypeptide chain and the CH3 domain of the third polypeptide chain comprise amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F;

[00915] (b) contacting the binding protein produced in (a) with Protein A; and

[00916] (c) eluting the Protein A binding protein under conditions suitable to isolate the binding protein from binding proteins comprising 0 or 2 CH3 domains comprising the amino acid substitutions H435R and Y436F.

[00917] Item 84. The method of item 83, wherein the CL domain of the first polypeptide chain is a human CL kappa domain and the CL domain of the fourth polypeptide chain is a human CL lambda domain; or the CL domain of the first polypeptide chain is a human CL lambda domain and the CL domain of the fourth polypeptide chain is a human CL kappa domain, and the method further comprises:

[00918] (d) contacting the binding protein eluted in (c) with a light kappa chain affinity medium; and

[00919] (e) eluting the binding protein from the light kappa chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only lambda CL domains.

[00920] Item 85. The method according to item 84, which further comprises, after (e):

[00921] (f) contacting the binding protein eluted in (e) with a lambda light chain affinity medium; and

[00922] (g) eluting the binding protein from the lambda light chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only CL kappa domains.

[00923] Item 86. The method of item 83, wherein the CL domain of the first polypeptide chain is a human CL kappa domain and the CL domain of the fourth polypeptide chain is a human CL lambda domain; or the CL domain of the first polypeptide chain is a human CL lambda domain and the CL domain of the fourth polypeptide chain is a human CL kappa domain, and the method further comprises:

[00924] (d) contacting the binding protein eluted in (c) with a lambda light chain affinity medium; and

[00925] (e) eluting the binding protein from the lambda light chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only CL kappa domains.

[00926] Item 87. The method according to item 86, which further comprises, after (e):

[00927] (f) contacting the binding protein eluted in (e) with a light kappa chain affinity medium; and

[00928] (g) eluting the binding protein from the light kappa chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only lambda CL domains.

[00929] Item 88. The method of any one of items 83-87, wherein the first polypeptide chain comprises a lambda CL domain; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to the EU index, wherein the amino acid substitutions are S354C and T366W; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435, and 436 of human IgG1 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R, and Y436F; and wherein the fourth polypeptide chain comprises a CL kappa domain.

[00930] Item 89. The method of any one of items 83-88, wherein the binding protein is detected in one or more of (c) and (e) using hydrophobic interaction chromatography (HIC).

[00931] Item 90. The method of any one of items 83-89, wherein the CH3 domains of the second and third polypeptide chains are CH3 domains of human IgG1 or IgG4.

EXAMPLES

[00932] The following Examples are illustrative of specific embodiments of the invention and various uses thereof. They are provided for explanatory purposes only and should not be construed as limiting the scope of the invention in any way.

Example 1: Materials and Methods

[00933] The following materials and methods were used for the experiments described in Examples 2-5. Design of Trispecific Antibodies

[00934] A schematic illustration of the design of the general trispecific antibody illustrated in Figures 1A-1C. Individual trispecific antibodies were designed based on 5 parameters: 1) Selection of antibody binding sites; 2) Consideration of the position of each binding site; 3) Choice of ligands for the bispecific binding arm (i.e., heavy chain/light chain B in Figure 1C); 4) Integration of the “Knob” and “Hole” mutation into the respective antibody halves; 5) Choice of Fc isotype (IgG1 or IgG4). After assembly of the amino acid sequences for each trispecific molecule, four genes for each trispecific Ab were synthesized using human-preferred codons (CambrY Applied Biosciences, Cambridge, MA, USA) and cloned into a eukaryotic expression vector. Production and Purification of Trispecific Antibodies

[00935] Trispecific antibodies were produced by transient transfection of 4 expression plasmids into Expi293 cells using the ExpiFectamine™ 293 Transfection Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Briefly, 25% (w/w) of each plasmid was diluted in Opti-MEM, mixed with prediluted ExpiFectamine reagent for 20-30 minutes at room temperature (RT), and added to Expi293 cells (2.5 x 106 cells/ml). Transfection optimization to determine the best plasmid ratio was frequently used to produce the trispecific antibody in good yield and purity.

[00936] 4-5 days after transfection, the supernatant of the transfected cells was collected and filtered through a 0.45 μm filtration unit (Nalgene). The trispecific antibody in the supernatant was purified using a 3-step procedure. First, Protein A affinity purification was used and the bound Ab was eluted using IgG Elution Buffer (Thermo Fisher Scientific). Second, the product was dialyzed against PBS (pH 7.4) overnight with 2 buffer changes of PBS. Any precipitate was clarified by filtration through a 0.45 μm filter unit (Nalgene) prior to the next step. Third, purification by size exclusion chromatography (SEC) (Hiload 16/600 Superdex 200pg, or Hiload 26/600 Superdex 200pg, GE Healthcare) was used to remove aggregates and different species in the preparation. Fractions were analyzed on reduced and nonreduced SDS-PAGE to identify fractions containing the monomeric trispecific antibody before pooling. Purified antibody can be aliquoted and stored at −80 °C long-term.

ELISA assays

[00937] The binding properties of the purified antibodies were analyzed using ELISA or SPR methods. For ELISA, antigens corresponding to each binding site on the trispecific antibody were coated onto a 96-well Immuno Plate (Nunc 439454, Thermo Fisher Scientific) overnight at 4 °C using 2 μg/ml of each antigen in PBS (pH 7.4). The coated plate was blocked using 5% skim milk + 2% BSA in PBS for one hour at room temperature, followed by washing with PBS + 0.25% Tween 20 three times (Aqua Max 400, Molecular Devices). Serial dilution of antibodies (trispecific and control Abs) was prepared and added to ELISA plates (100 μl/well in duplicate), incubated at room temperature for one hour, followed by washing 5 times with PBS + 0.25% Tween 20.

[00938] After washing, HRP-conjugated secondary anti-human Fab (1:5000, Cat. No. 109-035-097, Jackson ImmunoResearch Inc.) was added to each well and incubated at room temperature for 30 minutes. After washing 5 times with PBS + 0.25% Tween 20, 100 μL of TMB Microwell Peroxidase Substrate (KPL, Gaithersburg, MD, USA) was added to each well. The reaction was terminated by the addition of 50 μL of 1 M H2SO4 and the OD450 was measured using SpectraMax M5 (Molecular Devices) and analyzed using SoftMax Pro6.3 software (Molecular Devices). The final data were transferred to GraphPad Prism software (GraphPad Software, CA, USA) and graphed as shown. EC50 was calculated using the same software.

SPR tests

[00939] Two pairs of heavy and light chains were selected for full kinetic analysis. Kinetic characterization of purified antibodies was performed using surface plasmon resonance (SPR) technology on a BIACORE 3000 (GE Healthcare). A capture assay employing tag-specific antibody capture and orientation of the antibodies investigated was used. For capture of protein constructs containing Fc, the Human Antibody Capture Kit (GE Healthcare) was used, for capture of protein constructs containing the His tag, the His Capture Kit (GE Healthcare) was used. The capture antibody was immobilized via primary amine groups (11000 RU) on a research-grade CM5 chip (GE Life Sciences) using standard procedures. The assayed antibody was captured at a flow rate of 10/min with an adjusted RU value that would typically result in a minimum analyte binding signal of 30 RU.

[00940] For an exemplary assay, recombinant human IL13 (Catalog # IL012) and human IL4 (Catalog # IL004) were purchased from Millipore, recombinant human TNFα (Catalog # H8916) was purchased from Sigma Aldrich. Binding kinetics were measured for recombinant human IL4 and IL13 over a range of concentrations between 0.1 and 3 nM for IL4 and 0.8 to 25 nM for IL13. For human TNFα, a concentration range of 3 to 100 nM was used. As the assay buffer, HBS EP (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% Surfactant P20) was used at a flow rate of 30 µL/min. The chip surfaces were regenerated with the regeneration solution of the respective capture kit. Kinetic parameters were analyzed and calculated in the BIAevaluation v4.1 software package using a flow cell without captured antibody as a reference and the 1:1 Langmuir binding model with mass transfer. To study simultaneous antigen binding, trispecific antibodies were captured by an anti-human antibody capture surface. The antigens were used at single concentrations with 3 nM IL4, 25 nM IL13, and 100 nM TNFα. To show simultaneous binding of all three antigens, a mixture of IL4, IL13, and TNFα was injected. In separate analysis cycles, IL13 was injected alone, followed by IL4 or TNFα, and followed by either an IL4/TNFα coinjection or a TNFα/IL4 coinjection. The final response measured in each cycle was compared to show the similarity of consecutive binding of two or three antigens and the simultaneous binding of a mixture of all three antigens.

In Vitro T Cell Activation and Proliferation Assays

[00941] Human PBMCs were purified from buffy coat purchased from Blood Research Component (Brookline, MA, USA) using the Ficoll-Paque Plus method. Briefly, fresh buffy coat was first diluted 1:3 in PBS (pH 7.4), and mixed with Ficoll-Paque Plus solution (Ficoll) thoroughly before use by inverting the vial several times. 15 ml of density gradient medium was added to each Leucosep® tube and centrifuged for 30 s at 1000 x g, RT. The medium is now located below the porous barrier. 30-40 mL of diluted buffy coat was then carefully poured into each Leucosep tube and centrifuged at 800 x g for 15 min at room temperature with brake off and Max Accel at 5. The plasma was removed, and the remaining supernatant, which contained the enriched PBMCs, was transferred to a new tube (Leucosep tube was not kept inverted for longer than 2 s). The enriched PBMCs were washed with 45 mL of PBS and centrifuged at 250 x g for 10 min at room temperature. The wash was repeated and multiple tubes were combined into one tube. The cells were resuspended in 20 mL of PBS and counted using a Bio-Rad TC20.

[00942] To set up the in vitro T cell activation assay, purified human PBMCs were resuspended in culture medium (RPMI1640 with 10% FBS and supplemented with glutamine/Streptomycin) (Thermo Fisher Scientific) (106 cells/ml). Indicated concentrations of different trispecific and control antibodies were added to each well or used to coat the plate prior to use as described in Stebbings, R. et al. (2007) J. Immunol. 179: 3325-3331 and incubated for 16-24 hours in a tissue culture incubator. Cells were centrifuged and the supernatant was collected to measure cytokine release or discarded. Cells were stained with fluorescently labeled antibodies for T cell markers (CD3, CD4, CD8, etc.) and activation markers (CD69, CD62L, etc.) and analyzed by running the samples on a Fortessa flow cytometer (Beckton Dickinson, San Jose, CA), followed by analysis using Flowjo software (FlowJo v10) and plotted as shown.

[00943] To set up the in vitro T cell proliferation assay, purified human PBMCs were resuspended in culture medium (RPMI1640 with 10% FBS and supplemented with glutamine/Streptomycin) (Thermo Fisher Scientific) (106 cells/ml). The indicated concentrations of different trispecific and control antibodies were added to each well and incubated for 1-7 days in a tissue culture incubator. The cells were centrifuged and the supernatant was collected to measure cytokine release or discarded. Cells were stained with fluorescently labeled antibodies for T cell markers (CD3, CD4, CD8, etc.) and activation markers (CD69, CD62L, etc.) and analyzed by running the samples on a Fortessa flow cytometer (Beckton Dickinson, San Jose, CA), followed by analysis using Flowjo software (FlowJo v10) and plotted as shown.

In Vitro Cell Death Assay

[00944] Purified human PBMCs were used for in vitro killing assays against various cancer cells using different trispecific antibodies. Briefly, the killing assay was set up in a 96-well V-bottom plate. To each plate, 40 ml of PBMCs from each donor were plated at 2 x 106 cells/ml and 30 ml of PKH26-labeled target cells (Sigma # MINI26) at 2.5 x 105 cells/ml (4 μL of dye to stain up to 1 x 107 cells) were prepared. First, 20 μL/well of test proteins at various concentrations or PMA were added to each well, followed by the addition of 80 μL/well of labeled target cells to each well (2 x 104 cells/well). 100 μL of PBMCs were then added to each well, achieving E:T = 10:1 per well (2 x 105 cells/well) and incubated for 24 h at 37 °C in a 5% CO2 incubator. Cells were centrifuged and the supernatant was collected to measure cytokine release or discarded. Cells were stained with Vivid LIVE/DEAD™ Fixable Violet Dead Cell Staining Buffer (Life Technology # L34955) (staining buffer was prepared by adding 60 μL Vivid reagent to 60 ml PBS). Cells were resuspended in 100 μL staining buffer by incubating for 15 min at room temperature in the dark. After washing the cells with 1x PBS, the cells were resuspended in 200 μL of PBS with 0.5% paraformaldehyde, and PKH26+Vivid+ cancer cells were collected by a Fortessa flow cytometer (Beckton Dickinson, San Jose, CA), followed by analysis using Flowjo software. The percentage of killing is calculated as “specific killing – spontaneous/total cell killing” and represented as shown.

Cytokine Release Assay

[00945] For measurement of inflammatory cytokine concentrations in the in vitro activation assays, in vitro killing assays, activation assays in humanized NSG mice with in vivo cord blood CD34+ cells and the toxicity study, cell culture supernatant was collected and serum samples were diluted according to the manufacturer's protocol using the Milliplex Human High Sensitivity T Cell 13-Plex (EMD Millipore). These were subsequently analyzed by the EMD Millipore MAGPIX System and MILLIPLEX Analyst 5.1 software.

In Vivo Mouse Models and Efficacy Studies

[00946] NSG mice engrafted with human CD34+ hematopoietic stem cells (hu-CD34) have been used as an independently vivo model of GC. These mice develop multilineage human immune cells and are a validated platform for efficacy studies in immuno-oncology (see, e.g., Shultz, L.D. et al. (2014) Cold Spring Harb. Protoc. 2014: 694-708). Hu-CD34+ NSG mice are produced by injection of CD34+ hematopoietic stem cells and show effective engraftment of multiple lineages of human immune cell populations, including T cells, B cells, and a few other populations (McDermott, S.P. et al. (2010) Blood 116: 193-200). Multilineage hematopoiesis occurs within 12 weeks. Development has been stable for over a year without graft-versus-host disease.

[00947] For the efficacy study using hu-CD34 NSG mice, mice were purchased from The Jackson Laboratory (Maine, USA) and human cell populations were validated prior to use. In general, 5 x 106 tumor cells mixed in Matrigel (BD Biosciences) (50% v/v) were used to inoculate a tumor into each mouse. Once the tumor size reached the range of 100-150 mm3, mice were selected and randomly divided into each study group. Antibodies were administered intravenously in doses administered 3 times per week. Body weight was monitored 1-3 times per week. Tumor size was measured by tumor calipers 1-3 times/week. All mice were sacrificed when the tumor size reached 1,500 mm3 or 24 hours after the last dose. Final blood samples (0.3 mL) were collected in serum separation tubes, mixed by gentle inversion five times, and placed in a tube rack. Terminal tumors were also collected and weighed before being placed in fixative for immunohistochemical analysis.

[00948] Humanized NSG mice with hu-human PBMCs (PBMC) were used as another in vivo model. These mice are produced by injection of purified human PBMCs from healthy donors, which have a faster engraftment rate using adult peripheral blood mononuclear cells and allow for short-term studies requiring strong effector and memory cell function and are suitable for short-term (3-4 weeks) efficacy study due to graft-versus-host disease.

[00949] For the efficacy study using hu-PBMC NSG mice, 8-10 week old NSG mice (Cat. No: 005557, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) were purchased from Jackson Laboratory (Maine, USA). Each mouse was inoculated with 5 x 10 6 tumor cells mixed in Matrigel (BD Biosciences) (50% v/v). Once the tumor size reached the range of 50 -100 mm3, 10 x 10 6 human PBMCs from a healthy donor were reconstituted for each mouse. The reconstitution of human cells was validated the next day. Once the tumor size reached the range of 100-150 mm3, mice were selected and randomized into each group for study. Antibodies were administered intravenously in doses given 3 times per week. Body weight was monitored 1–3 times per week. Tumor size was measured by tumor calipers 1–3 times/week. All mice were sacrificed when tumor size reached 1,500 mm3 or 24 h after the last dose. Final blood samples (0.3 mL) were collected in serum separator tubes, mixed by gentle inversion five times, and placed in a tube rack. Terminal tumors were also collected and weighed before being placed in fixative for immunohistochemical analysis. NHP Tolerability and Pharmacokinetic Study

[00950] All NHP studies were performed by Covance (Princeton, New Jersey, USA) according to the Covance ICUCA protocol. Drug-naïve and protein-naïve male cynomolgus monkeys were used in all studies. Based on the study design, monkeys were selected and grouped for each trispecific antibody. An antibody was administered via intravenous infusion over 1 hour via the saphenous vein. Escalating doses were given on consecutive days for low doses (<10 μg/kg) but with a 1-2 day interval for higher doses (>10 μg/kg) for observation purposes. Blood samples were collected at 0 hour (Day 1 only), 0.5 hour (halfway through the infusion), 1 and 6 hours from the start of the infusion for all animals after each dose, as specified. Additional unscheduled blood samples were collected at the discretion of the study director, pathologist, and/or clinical veterinarian. All animals were returned to the colony on day 60. PBMCs and serum from the blood samples were prepared using standard methods and preserved for future analysis.

Luciferase Reporter Assay

[00951] GloResponse™ IL2-luc2P Jurkat Cells, Thaw and Use (Pro- mega part # CS187002) and Jurkat Cell GloResponse™ NFAT-Luc2 (Promega Cat. # CS176401) were purchased from Promega (WI, USA) and prepared for use according to the manufacturer's protocol.

[00952] Briefly, cells were thawed for 2 min in a 37 °C water bath and gently transferred to a 15 mL conical centrifuge tube containing 10 mL of pre-warmed R10 medium. The tube was centrifuged at 300 g for 5 min at RT. The supernatant was removed and the cells were resuspended in 20 mL of pre-warmed R10 medium and transferred to a 75 cm2 culture flask, followed by incubation at 37 °C in a tissue culture incubator until the cells began to grow and were stable (~3-4 days). Cells were split twice a week to 0.1 and 6 cells/mL. Cells were maintained in R10 medium + Hygromycin B for selection. Cells were used for assays ~7 days after thawing.

[00953] For antibody stimulation, trispecific or control antibodies were prepared at various concentrations and serially diluted in PBS. 25 μL of antibodies were distributed per well. For plate-bound Abs, a Maxisorp plate was used and incubated at 4 °C overnight. For soluble Abs, a U-bottom plate was used. Reporter cells were resuspended at 0.3-0.5 and 6 μg/mL and 175 μL of cells were added to each well and incubated in a 37 °C, 5% CO2 incubator for 6 hours. The plate was then removed from the incubator and allowed to equilibrate to room temperature (10-15 min). Next, 50 μL of Bio-Glo™ Reagent (Promega Cat. # G7941) (room temperature) was added to each well of the assay plate. After incubation for 5 min, luminescence activity was measured using the MicroBeta2 LumiJET microplate counter (Perkin Elmer; 1 s reading time). Data were plotted using GraphPad Prism software. Conformational Stability

[00954] Thermostability measurements (e.g., melting points, Tm) were determined using differential scanning fluorimetry (DSF). Samples were diluted in D-PBS buffer (Invitrogen) to a final concentration of 0.2 μg/μL including a 4x concentrated solution of SYPRO-Orange dye (Invitrogen, 5000x stock in DMSO) in D-PBS in 96-well plates with a white half-skirt (BIORAD). All measurements were performed in duplicate using a MyiQ2 real-time PCR instrument (BIORAD). First negative derivative curves (-d(RFU)/dT) of the melting curves were generated in iQ5 Software v2.1 (BIORAD). Data were then exported to Microsoft Excel for Tm determination and graphical display of the data.

IC50 measurements Detection of antibody activity against IL-4 and IL-13 with a reporter cell line

[00955] The activities of bispecific antibodies or derivatives against the cytokines IL4 and IL13 were determined in commercially available HEK-Blue IL-4/IL-13 reporter cells (InvivoGen). HEK-Blue IL-4/IL-13 cells are designed to monitor activation of the STAT6 pathway by IL-4 or IL13. Stimulation of the cells with either cytokine results in the production of secreted embryonic alkaline phosphatase (SEAP) by the reporter gene that can be measured in the culture supernatant with the QUANTI-Blue assay. To test the activities of antibodies against IL4 or IL13, the cytokines were preincubated for 1 hour with different concentrations of the antibodies and added to 50,000 HEK-Blue IL-4/IL-13 cells. Cytokine-mediated induction of SEAP after 24 h of incubation was measured in cell culture supernatant with the QUANTI-Blue assay (InvivoGen). Each experiment was performed with n = 3 data points for each antibody concentration. The half-maximal inhibitory concentration (IC50) for each antibody was calculated using the in-house Biostat-Speed V2.0 application (Sanofi). Detection of antibody activity against TNFα with a reporter cell line

[00956] The activities of bispecific antibodies or derivatives against TNFα were determined using commercially available HEK-Blue TNF-α reporter cells (InvivoGen). HEK-Blue TNF-α cells are designed to detect bioactive TNFα by monitoring activation of the NFκB pathway via expression of the secreted embryonic alkaline phosphatase (SEAP) reporter gene that can be measured in culture supernatant with a QUANTI Blue assay (InvivoGen). To determine antibody activities against TNFα, cytokines were preincubated for 1 hour with different concentrations of antibodies and added to 50,000 HEK Blue TNF-α cells. Cytokine-mediated induction of SEAP was measured after 24 hours in culture supernatant with the QUANTI-Blue assay (InvivoGen). Each experiment was performed with n = 3 data points for each antibody concentration. The half-maximal inhibitory concentration for each antibody was calculated. Example 2: Overview of Trispecific Binding Proteins

[00957] A novel strategy was developed for the generation of trispecific binding proteins. The trispecific proteins comprised four polypeptides that formed three target-binding sites (Figures 1A-C). Each target-binding site comprised the VH and VL domains of an antibody that targeted a distinct human antigenic target (see, for example, Table 1). The trispecific binding proteins contained a first pair of polypeptides that possessed dual variable domains that have a cross-orientation, forming two distinct antigen-binding sites (designated by the CODV Ig format) and a second pair of polypeptides, each with a single variable domain that formed a third antigen-binding site (Figures 1A and 1B). Table 1: Heavy and light chain SEQ ID NOs for binding proteins 1-21 and the target antigens to which the binding proteins are directed

[00958] The first pair of polypeptides (which had dual variable domains) comprised a first polypeptide having the structure VL2-Linker-VL1-Linker-Immunoglobulin light chain constant domain and a second polypeptide having the structure VH1-Linker-VH2-Linker-Immunoglobulin heavy chain constant domain CH1, resulting in a pair of polypeptides that had a cross-linked orientation that formed two distinct antigen-binding sites: VH1-VL1 and VH2-VL2 (Figure 1C, see B heavy and light chains). Table A provides a summary of the design of the bispecific arm (i.e., the arm comprising the B heavy and light chains) of IgG1 and IgG4 variants of representative trispecific binding proteins, including an indication of the various combinations of linkers used in the bispecific arm of the trispecific binding proteins. The second pair of polypeptides (each possessing a single variable domain) comprised a first polypeptide having the structure VH3-immunoglobulin heavy chain constant domain CH1 and a second polypeptide having the structure VL3-immunoglobulin light chain constant domain, resulting in a pair of polypeptides that formed a third antigen-binding site: VH3-VL3 (Figure 1C, see Heavy and Light Chains A). In addition, the trispecific binding proteins were constructed so that either CH3 domain could include a "knob" or "hole" modification to facilitate antibody heterodimerization (Figure 1). Table A: Summary of the design of the bispecific arm of trispecific binding proteins such as IgG1 ("Hole") or IgG4 ("Hole") * [ ,] /[GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGS (SEQ ID NO: 104)]; or [GGGGSGGGGSGGGS (SEQ ID NO: 105), ]/[GGGGSGGGSGGGGS (SEQ ID NO: 105)]

Example 3: In Vitro Binding Activity and Antibody-Mediated Specific Maturation of T Cell Coupling Agents

[00959] This example describes in vitro assays to characterize the activities of T cell engaging agents.

[00960] Using the approach described in Example 2 above for trispecific binding protein design, four trispecific binding proteins (Binding Proteins 1, 3, 5, and 6) were generated. These trispecific binding proteins were created by grafting onto a trispecific binding protein backbone the VH and VL domains isolated from antibodies that target distinct human proteins: CD3, CD19, CD28, CD38, or Her2. Binding Protein 1 was constructed such that the first pair of polypeptides (which formed two antigen-binding sites) targeted CD28 and CD3 and the second pair of polypeptides (which formed the single antigen-binding site) targeted Her2 (Binding Protein 1 = Her2 x (CD28 x CD3)). Binding Protein 3 was constructed so that the first pair of polypeptides (which formed two antigen-binding sites) targeted CD28 and CD3 and the second pair of polypeptides (which formed the only antigen-binding site) targeted CD19 (Binding Protein 3 = CD19 x (CD28 x CD3)). Binding Protein 5 was constructed so that the first pair of polypeptides (which formed two antigen-binding sites) targeted CD28 and CD3 and the second pair of polypeptides (which formed the only antigen-binding site) targeted CD38 (Binding Protein 5 = CD38 x CD28 x CD3)). Binding Protein 6 was constructed so that the first pair of polypeptides (which formed two antigen-binding sites) targeted CD28 and CD3 and the second pair of polypeptides (which formed the single antigen-binding site) targeted CD38 (Binding Protein 6 = CD38 x (CD28 x CD3)). In Vitro Assays Using T-Specific Binding Proteins Comprising Anti-Her2

[00961] To test the ability of the trispecific binding proteins to target and bind to three different human antigens, the specificity of Binding Protein 1 for its targets was first examined via an ELISA assay. Binding Protein 1 was able to bind to three of its target proteins - CD3, CD28, and Her2 (Figure 2) - indicating that each binding domain in the trispecific format retained its function.

[00962] ZR-75-1, AU565 (Her2+), ARH-77 (CD19+), MOLP-8, RPMI-8226, KMS-12_BM, NCI-H929, MM.1.S, MM.1., ROPM-2, KMS-26, and U266 (CD38+) cells were labeled with the membrane dye PKH-26 (Sigma) and used as target cells in a cytotoxicity assay. These labeled cell lines were co-cultured at an E:T ratio of 10:1 with enriched human Pan T cells in the presence of increasing concentrations of a trispecific antibody, bispecific antibody, or control proteins for 24 hours. The extent of cell lysis in target cells was determined by staining with a live/dead cell marker (Life Technologies) and measuring the number of dead cells in the labeled target cell population by passing the samples through a Fortessa flow cytometer (Beckton Dickinson, San Jose, CA), followed by analysis using Flowjo software (FlowJo v10).

[00963] Her2+, CD19+, CD38+ tumor cell lines were stained with fluorescently conjugated antibodies against human CD3, CD28, CD19, CD38, LAMP1, and/or Her2 (Biolegend). Staining with respective isotype-matched control antibodies was also included. Cells were then acquired on the Fortessa instrument (Beckton Dickinson, San Jose, CA). Flow analysis was performed on FlowJo v10. The results of mediated killing of various binding proteins are shown in Figures 3A-5, 9A, 9B, and 11A-16.

[00964] The ability of Binding Protein 1 to induce antibody-mediated cell death of tumor cells expressing HER2 proteins on their surface was tested. Not only was Binding Protein 1 able to bind to all three of its target proteins, but it was also able to induce antibody-mediated cell death of Her2+ cell lines (Figures 3A-4). Binding Protein 1 exhibited potent antibody-mediated cell killing activities, whereas anti-Her2 antibodies and anti-CD3/CD28 bispecific Abs showed minimal killing activities (Figures 3A, 3B, 4, and 5), demonstrating the efficacy of using the trispecific Ab to engage tumor cells with T cells via a tumor antigen (HER2) and T cell markers (CD3 and CD28). Anti-CD3/CD28 is not only important for T cell recruitment, but also allows more effective T cell activation and survival signaling, potentially improving efficacy.

[00965] In addition, in vitro studies on T cell activation and proliferation, as well as cytokine production, were performed using the anti-Her2 x CD28 x CD3 trispecific antibody (Binding Protein 1). Binding Protein and control variants having one or two binding domains inactivated by site-directed mutagenesis (ΔCD28: inactivated anti-CD28; ΔCD3: inactivated anti-CD3; Δ(CD3x CD28): both inactivated anti-CD3 and anti-CD28 antibodies) were used in the in vitro activation assay of human PBMCs as described in Example 1. The results showed that Binding Protein 1 activated primary human CD4 T cells and CD8 T cells effectively in vitro. Inactivation of the anti-CD28 antibody reduced the potency of activation, indicating the importance of the anti-CD28 co-signaling pathway. Inactivation of the anti-CD3 binding site conferred Binding Protein 1 minimal activity, suggesting that anti-CD3 antibody enabled primary T cell activation signaling (Figures 6A and 6B). Similar results were obtained using IL2 and NFAT reporter human T cell lines (Jurkat-IL2 and Jurkat-NFAT) (Figures 7A–7C). In Vitro Assays Using Trispecific Binding Proteins Comprising Anti-CD19

[00966] The anti-CD28 x CD3 x CD19 trispecific binding protein was able to bind to its target antigens (Figure 8), indicating that each binding domain in the trispecific format retained its function.

[00967] The anti-CD28 x CD3 x CD19 trispecific binding protein was also capable of inducing antibody-mediated cell killing of CD19+ cells (Figures 9A-9N). Similarly, the anti-CD19 x CD28 x CD3 trispecific binding protein exhibited potent killing activity against human lymphoma cells, whereas both anti-CD3/CD28, anti-CD19, and the isotype control antibodies exhibited minimal killing activities, demonstrating the efficacy of using the trispecific Ab to engage tumor cells with T cells via a tumor antigen (CD19) and T cell markers (CD3 and CD28). In Vitro Assays Using Trispecific Binding Proteins Comprising Anti-CD38

[00968] As observed with Binding Proteins 1 and 3, Binding Protein 5 was able to bind to all three of its target proteins (CD3, CD28, and CD38) as assessed by ELISA assay (Figure 10), indicating that each binding domain in the tri-specific format maintained its function.

[00969] Binding Protein 5 was also found to induce antibody-mediated cell killing of cells (Figures 11A-15D) against 9 human multiple myeloma cell lines with various levels of CD38 and CD28 expression (see Figures 11D, 12D & 13D). Similarly, the trispecific Binding Protein 5 exhibited potent killing activity against human multiple myeloma cells, whereas both anti-CD38 antibodies and isotype controls showed minimal killing activities, demonstrating the effectiveness of using the trispecific antibody to engage tumor cells with T cells via tumor antigens (CD38 and CD28) and T cell markers (CD3 and CD28). The bispecific anti-CD3/CD28 control antibody also showed marginal killing activity against CD28+ MM cells; see Figures 11B, 12A-C, and 13A-C).

[00970] These results demonstrate that the trispecific antibody platform described here provides the possibility of integrating binding sites for two tumor markers, or two binding sites for T cell markers, allowing flexibility for scientific modeling and various applications. Binding Protein 5 was also effective against 5 CD38+ human lymphoma cell lines (Figures 14C and 15D), showing potent killing activities (Figures 14A-B and 15A-C).

[00971] Antibody-mediated cell killing against multiple RPMI8226 myeloma cell lines using Binding Proteins 5 and 6 was tested and their EC50 values were calculated and compared with those of a bispecific CODV antibody format that targets CD28 and CD3 (Figure 16 and Table B). Binding Proteins 5 and 6 differ only in the anti-CD28 antibody binding domain; Binding Protein 5 contains a superagonist anti-CD28 antibody, whereas Binding Protein 6 contains a conventional anti-CD28 antibody. Binding Protein 5 showed more potent killing activity. Table B: Calculated EC50 values for bispecific and trispecific binding proteins

[00972] The activity of the trispecific anti-CD38 x CD28 x CD3 Binding Protein 5 and control variants that have one or two binding domains inactivated by site-directed mutagenesis (ΔCD28: inactivated anti-CD28; ΔCD3: inactivated anti-CD3; Δ(CD3x CD28): both inactivated anti-CD3 and anti-CD28 antibodies) were tested using human IL2 and NFAT reporter T cell lines (Jurkat-IL2 and Jurkat-NFAT) in the in vitro activation assay as described in Example 1. The results showed that Binding Protein 5 activated the human IL2 and NFAT promoters effectively in vitro (Figures 17A and 17B). Knockdown of anti-CD28 reduced the potency of activation, which was most prominent for the IL2 reporter, indicating the importance of the anti-CD28 co-signaling pathway. Knockdown of the anti-CD3 binding site conferred minimal Binding Protein 5 activity, suggesting that anti-CD3 antibody enabled activation signaling of primary T cells.

Example 4: In Vivo Activity of T Cell Coupling Agents

[00973] This example describes experiments that characterize the properties and activities of anti-Her2 or anti-CD38 antibody containing T cell engaging agents in vivo. In Vivo Assays Using Trispecific Binding Proteins Comprising Anti-Her2

[00974] A dose-escalation study using the Her2 x CD28 x CD3 trispecific antibody was performed in non-human primates (Figures 18A-18E) as described in Example 1. All three binding domains in Binding Protein 1 are cross-reactive with monkey CD3/CD28/HER2. A dose-escalation toxicity study was designed to assess the potential toxicity profile of the molecule. Blood samples were collected for isolation of serum and PBMCs. Circulating T cell populations were investigated after each dosing (Figures 18A and 18B), along with activation of the T cell subpopulation (CD69+) (Figures 18C and 18D). The percentage of circulating CD4 and CD8 T cells was increased with low-dose escalation, but eventually decreased with high-dose escalation. Significant activation of CD4 and CD8 T cells was prominent only at a dose of 100/kg, suggesting a relatively high tolerable dose. Serum levels of several cytokines were also measured. Significant cytokine release was observed only at the highest dose (100/kg; Figure 18E).

[00975] Next, the effect of the anti-Her2 x CD28 x CD3 trispecific Binding Protein 1 antibody on tumor growth in humanized mouse models was analyzed as described in Example 1 (Figures 19A-20H). Figures 19A & 19B summarize the results obtained using the human CD34+ NSG (hu-CD34) hematopoietic stem cell-engrafted mouse model inoculated with the HER2+ human breast cancer cell line BT474. Significant antitumor activities were evident in all dose groups. The antitumor activity was dose-dependent, which was statistically different compared to the control group at 25 μg/kg. There was no significant body weight loss in any of the treated groups.

[00976] A 2nd in vivo study using an NSG mouse model reconstituted with human PBMCs inoculated with the HER2+ human breast cancer cell line BT474 was also performed (Figures 20A-20H). Significant antitumor activities were observed in high dose groups (100 and 500 μg/kg). Tumor shrinkage was observed in 40% of mice in the 500 μg/kg group. Antitumor activity was dose dependent. Antitumor activity in groups treated with doses of 100 and 500 μg/kg was significantly better than in groups treated with anti-HER2 (0.1 to 10 mg/kg), indicating superior antitumor activity of Binding Protein 1. No significant body weight loss was observed in any treated group. In Vivo Assays Using Trispecific Binding Proteins Comprising Anti-CD38

[00977] A dose-escalation study was conducted in non-human primates using the anti-CD38 x CD28 x CD3 trispecific antibody (Binding Protein 5) as described in Example 1 (Figures 21A-21F). Two of the three binding domains in Binding Protein 5 are cross-reactive with those of monkey CD3 and CD28. A dose-escalation toxicity study was designed to assess the potential toxicity profile of the molecule. Blood samples were collected for isolation of serum and PBMCs. Circulating T cell populations were investigated after each dosing (Figures 21A and 21B, bar graphs), along with activation of the T cell subpopulation (CD69+) (Figures 21A and 21B, line graphs). The percentage of circulating CD4 and CD8 T cells increased at low-dose escalation, but eventually decreased at high-dose escalation. Significant activation of CD4 and CD8 T cells was prominent only at a dose of 100/kg, suggesting a relatively high tolerable dose. Serum levels of several cytokines were also measured. Significant cytokine release was observed only at the highest dose (100/kg; Figures 21C-21F).

[00978] The in vivo activity of the anti-CD38 x CD28 x CD3 trispecific antibody was then tested in humanized mice (Figures 22A-23D) as described in Example 1. Figures 22A-22C summarize the results of a dose-finding pilot study using the hematopoietic NSG (Hu-CD34) mouse model engrafted with human CD34+ hematopoietic stem cells implanted with the human MM cell line RPMI-8226 transduced with CD38 and PD-L1, treated with Binding Protein 5 at doses of 5, 50, and 100 µg/kg. Significant antitumor activity was evident only in the 5 µg/kg group (Figure 22A). CD8 T cell infiltration was observed in mice treated with Binding Protein 5 (5 μg/kg) (Figures 22B and 22C).

[00979] A follow-up study in the same model was performed using Binding Protein 5 at doses of 0.04-5 μg/kg (Figures 23A-23D). Significant antitumor activity was demonstrated in all Binding Protein 5-treated groups (Figure 23B), which were statistically different from control at the end of the study (Figure 23C). No significant body weight loss was observed in any of the treated groups (Figure 23A). Dose-dependent induction of serum inflammatory cytokines IFN-Y, TNF-α, and IL-2 four hours after the first dose was observed in mice treated with the indicated concentrations of Binding Protein 5 or PBS control (Figure 23D), indicating effective T-cell activation by the trispecific Binding Protein 5 in vivo.

[00980] Humanized CD34+ NSG mice (n = 3) were injected iv with 100 mcg/kg of trispecific Ab (triangle), bispecific Ab (square), or unispecific Ab (circle). CD4+ or CD8+ T cell activation was measured at 0 (pre-injection), 1, 24, and 72 hours post-Ab injection by determining the mean increase in %CD69, %decrease in CD62L, and/or plasma inflammatory cytokine concentration at each time point by Luminex xMAP multiplexing technology. The T cell activation results of various trispecific antibodies are shown in Figures 24-26C.

[00981] Systemic T cell activation in vivo was studied in the NSG mouse model engrafted with human CD34+ hematopoietic stem cells (hu-CD34) after administration of Binding Protein 5, anti-CD3/CD28_IgG4 bispecific antibody and anti-CD28 IgG4 antibody controls (Figure 24). 100/kg of Binding Protein 5 and control antibodies were administered to 3 mice/group. Blood samples were collected at pre, 1 hour, 24 hours and 72 hours after administration. Sera of mice and human T cells were isolated from the blood and preserved for analysis of T cell activation and for measurement of serum cytokine levels. Figures 24 and 25 show that both human CD4 and CD8 T cells were activated 1 hour after antibody infusion, which returned to baseline by 72 hours. Figures 26A-26C show the elevation of serum release of IFN-Y, TNF-α, and IL-2 in the same mice, which was observed 1 hour after infusion, and returned to baseline by 24 hours. These results demonstrate that Binding Protein 5 and the anti-CD3/CD28 IgG4 bispecific antibody are effective in activating T cells in the given animal model, making it suitable for in vivo efficacy study.

Example 5: Characterization of trivalent trispecific and bispecific cytokine-targeted binding proteins

[00982] The following example describes experiments that characterize the stability, binding properties, and activities of novel trivalent bispecific and trispecific proteins that target human cytokines for binding.

[00983] Trispecific binding proteins (e.g., which bind three different target proteins; Binding Proteins 9-15) as well as trivalent bispecific binding proteins (e.g., which bind one antigen bivalently over one antigen monovalently; Binding Proteins 16-19) were designed (Table C). With the exception of Binding Protein 11, where a kappa constant domain was used in both the CODV-LC and Fab-LC arms, all other Binding Proteins (9-10 and 12-19) were produced with a kappa constant domain in the CODV-LC arm and a lambda constant domain in the Fab-LC arm. As the main Fc part, the IgG1 sequence was used. Whereas CODV-HC harbors the RF-knob mutations (S354C, T366W; H435R and Y436F), the Fab-HC arm contains the hole mutations (Y349C, T366S, L368A, Y407V). Table C: Summary of trispecific/trivalent binding proteins targeting anti-IL-4/IL-13/TNFα

[00984] Trivalent trispecific and bispecific binding proteins were produced and purified as described above (Figure 27). Table D: SEC purification of binding proteins 16-19

[00985] In order to assess the stability of the trispecific binding proteins, their melting point was assessed by DSF and compared to the thermal stability of the parental antibodies (Table E). Table E: Summary of thermal stability by DSF and percentage of monomers from preparative size exclusion chromatography for various trispecific binding proteins nd = not determined

[00986] To assess the binding affinity of each binding domain within the trispecific antibody format, SPR analysis for each individual antigen was performed as previously described. The results were compared to the affinities of the parental antibodies (Tables F, G, and H). Table F: Summary of surface plasmon resonance results for IL-4 to various trispecific binding proteins Table G: Summary of surface plasmon resonance results for IL-13 for various trispecific binding proteins Table H: Summary of surface plasmon resonance results for TNFα for various trispecific binding proteins

[00987] In order to evaluate the neutralization activity of the trispecific binding proteins, a cellular assay was performed using different HEK Blue kits (InvivoGen). Cytokines were pre-incubated with different concentrations of anti-cytokine antibodies for 30 minutes at room temperature in a 96-well plate. Controls included the use of cytokine only or antibody only. 50,000 HEK Blue cells (HEK Blue TNFa/IL1β cells (InvivoGen, Cat. # Hkb-tnfil1; HEK Blue STAT-6 cells (InvivoGen, Cat. # Hkb.stat6) were added to a cytokine/antibody mixture and incubated for 23 hours at 37 °C, 5% CO2 in an incubator, QuantiBlue Reagent was added to each culture well and incubated for 2 hours at 37 °C. OD was measured at 620 nm and IC50 was calculated using BioStat Speed 2.0. The results of the HEK Blue Reporter Cell Assay of various trispecific antibodies are shown in Tables I and M.

[00988] Next, IC50 values were calculated for Binding Proteins 9-15 and measured against the individual parental antibodies (Table I). Table I: Summary of HEK Blue Reporter Assays (IC50 Data) for Various Trispecific Binding Proteins

[00989] The thermal stability of the trivalent bispecific binding proteins was measured by differential scanning fluorimetry (DSF; Table J). Table J: Summary of DSF thermal stability for various trivalent binding proteins

[00990] The binding affinity and number of target proteins bound by each of the trivalent bispecific binding proteins were measured for IL-4 (Table K) and IL-13 (Tables K and L) activity. Table K: Summary of surface plasmon resonance results for IL-4 for various trivalent binding proteins Table L: Summary of surface plasmon resonance results for IL-13 for various trivalent binding proteins

[00991] Finally, IC50 values were calculated for Binding Proteins 16-19 (Table M). Table M: Summary of HEK Blue Reporter assays (IC50 data) for various trivalent binding proteins Example 6: Trispecific Binding Protein Shape Optimization

[00992] A problem with many existing heterodimeric binding protein formats (e.g., bispecific antibodies and their variants) is that it can be difficult to purify only the desired heterodimeric species without also including any of the homodimeric species. Thus, a process for the efficient purification of the desired heterodimeric binding protein is of great interest, e.g., for industrial-scale production.

[00993] As described herein, the binding proteins of the present invention may include various optional features including, without limitation, "knob" and "hole" mutations (e.g., to promote proper heterodimer formation) and mutations to improve purification. In addition, these binding proteins include two light chains, leading to four potential configurations: two kappa light chains, two lambda light chains, a kappa light chain in the dual variable domain arm (the "CODV arm") and a lambda light chain in the traditional antibody arm (the "Fab arm"), and a lambda light chain in the CODV arm and a kappa light chain in the Fab arm.

[00994] Therefore, experiments were performed to identify a process that allows for efficient purification of the desired binding protein of interest. Binding protein variants were also tested for their purification efficiency.

[00995] Figure 28A shows a diagram of an exemplary binding protein of the present invention, indicating variations that lead to unique configurations. These experiments tested the effect of the placement of: kappa and lambda light chains (e.g., two kappa, two lambda, one kappa in the CODV arm and lambda in the Fab arm, and lambda in the CODV arm and kappa in the Fab arm), knob and hole mutations (e.g., mutations in the CODV arm and mutations in the Fab arm or mutations in the CODV arm and knob mutations in the Fab arm), and H435R/Y436F mutations (“RF mutations”, e.g., RF mutations in the CODV or Fab arm or no RF mutation). A total of 18 different variants were tested, as shown in Figure 28B. For these experiments, the CODV arm had antigen-binding sites specific for TNFα (i.e., VH and VL sequences of SEQ ID NOs: 168 and 169, respectively) and IL4 (i.e., VH and VL sequences of SEQ ID NOs: 170 and 171, respectively), whereas the Fab arm had an antigen-binding site specific for IL13 (i.e., VH and VL sequences of SEQ ID NOs: 172 and 173, respectively). S354C and T366W were used for the knob mutations and Y349C, T366S, L368A, and Y407V were used for the hole mutations.

[00996] Several processing steps were tested for the ability to monitor correct CODV and Fab arm pairing (e.g., as opposed to CODV or Fab homodimers), as well as correct heavy-light chain pairing (e.g., as opposed to pairing between the Fab arm light chain and the CODV arm heavy chain or between the Fab arm heavy chain and the CODV arm light chain). Analytical size exclusion chromatography (SEC) was found to be ineffective in distinguishing correct heavy chain and light chain pairing; Fab arm light chain binding proteins unpaired to the CODV heavy chain and homodimeric binding proteins with two Fab arms were found to co-elute with the desired trispecific binding proteins. However, analytical hydrophobic interaction chromatography (HIC) was found to decompose the trispecific binding proteins of binding proteins with the unpaired Fab arm light chain to the homodimeric heavy chain binding proteins and CODV with two Fab arms (Figure 29).

[00997] The 18 binding protein configurations shown in Figure 28B were purified by Protein A affinity chromatography and then purified with KappaSelect (GE Healthcare). Species were monitored by HIC chromatography. One binding protein configuration was efficiently purified without inclusion of mismatched species: lambda light chain for the CODV arm, kappa light chain for the Fab arm, knob mutations in the CODV arm, hole mutations in the Fab arm, and RF mutations in the Fab arm. HIC chromatography (Figure 30A), SDS-PAGE (Figure 30B), and intact mass analysis demonstrated that a single species corresponding to the desired trispecific binding protein was purified.

[00998] These results identify a binding protein configuration that allows for more efficient purification of proteins of interest from unpaired binding species. Furthermore, it was shown that the Protein A purification process followed by the KappaSelect purification steps allows for efficient separation of binding proteins of interest from unpaired species.

[00999] Although the description includes several embodiments, it should be understood that variations and modifications will occur to those skilled in the art. Therefore, it is intended that the appended claims cover all such equivalent variations that fall within the scope of the description. Furthermore, the section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[001000] Each embodiment described herein may be combined with any other embodiment or embodiments unless clearly indicated otherwise. In particular, any feature or embodiment indicated as being preferred or advantageous may be combined with any other feature or features or embodiment or embodiments indicated as being preferred or advantageous unless clearly indicated otherwise.

[001001] All references cited in this Patent Application are expressly incorporated herein by reference. Table 1: Heavy and Light Chain SEQ ID NOs for Binding Proteins 1-21 and the Target Antigens to Which the Binding Proteins Are Directed Table 2: Sequences of heavy and light chains of binding proteins specifically directed to Her2, CD3, CD28, CD19 and/or CD20. CDR sequences are in bold and italics Note: CDR sequences are in bold and italics in the amino acid sequences above.

Claims (17)

1. A method of purifying a binding protein produced by a host cell, comprising: (a) producing in a host cell a binding protein comprising four polypeptide chains that form three antigen-binding sites that specifically bind to one or more antigenic targets or target proteins, wherein a first polypeptide chain comprises a structure represented by the formula: VL2-L1-VL1-L2-CL [I] and a second polypeptide chain comprises a structure represented by the formula: VH1-L3-VH2-L4-CH1-hinge-CH2-CH3 [II] and a third polypeptide chain comprises a structure represented by the formula: VH3-CH1 [III] and a fourth polypeptide chain comprises a structure represented by the formula: VL3-CL [IV] wherein: VL1 is a first light chain variable domain of immunoglobulin; V L2 is a second immunoglobulin light chain variable domain; V L3 is a third immunoglobulin light chain variable domain; V H1 is a first immunoglobulin heavy chain variable domain; V H2 is a second immunoglobulin heavy chain variable domain; VH3 is a third immunoglobulin heavy chain variable domain; CL is an immunoglobulin light chain constant domain; CH1 is an immunoglobulin CH1 heavy chain constant domain; CH2 is an immunoglobulin CH2 heavy chain constant domain; CH3 is an immunoglobulin CH3 heavy chain constant domain; hinge is an immunoglobulin hinge region that connects the CH1 and CH2 domains; and L1, L2, L3, and L4 are amino acid linkers; wherein the polypeptide of formula I and the polypeptide of formula II form a cross-linked light chain-heavy chain pair, wherein only one of the CH3 domains of the second polypeptide chain and the CH3 domain of the third polypeptide chain comprise amino acid substitutions at positions corresponding to positions 435 and 436 of human IgG1 or IgG4 according to the EU index, wherein the amino acid substitutions are H435R and Y436F; (b) contacting the binding protein produced in (a) with Protein A; and (c) eluting the binding protein from Protein A under conditions suitable to isolate the binding protein from binding proteins comprising 0 or 2 CH3 domains comprising the amino acid substitutions are H435R and Y436F, thereby purifying the binding protein. 2. The method of claim 1, wherein the CL domain of the first polypeptide chain is a human kappa CL domain, and the CL domain of the fourth polypeptide chain is a human lambda CL domain; or the CL domain of the first polypeptide chain is a human lambda CL domain, and the CL domain of the fourth polypeptide chain is a human kappa CL domain, and the method further comprises: (d) contacting the binding protein eluted from (c) with a light kappa chain affinity medium; (e) eluting the binding protein from the light kappa chain affinity medium under conditions suitable for isolating the binding protein from binding proteins comprising only lambda CL domains; wherein, optionally, the method further comprises, after (e): (f) contacting the binding protein eluted from (e) with a lambda light chain affinity medium; and (g) eluting the binding protein from the lambda light chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only CL kappa domains. 3. The method of claim 1, wherein the CL domain of the first polypeptide chain is a human CL kappa domain, and the CL domain of the fourth polypeptide chain is a human CL lambda domain; or the CL domain of the first polypeptide chain is a human CL lambda domain, and the CL domain of the fourth polypeptide chain is a human CL kappa domain, and the method further comprises: (d) contacting the binding protein eluted from (c) with a lambda light chain affinity medium; (e) eluting the binding protein from the lambda light chain affinity medium under conditions suitable for isolating the binding protein from binding proteins comprising only CL kappa domains; wherein, optionally, the method further comprises, after (e): (f) contacting the binding protein eluted from (e) with a light kappa chain affinity medium; and (g) eluting the binding protein from the light kappa chain affinity medium under conditions suitable to isolate the binding protein from binding proteins comprising only lambda CL domains. 4. The method of any one of claims 1 to 3, wherein the first polypeptide chain comprises a lambda CL domain; wherein the CH3 domain of the second polypeptide chain comprises amino acid substitutions at positions corresponding to positions 354 and 366 of human IgG1 according to the EU index, wherein the amino acid substitutions are S354C and T366W; wherein the CH3 domain of the third polypeptide chain comprises amino acid substitutions at positions corresponding to positions 349, 366, 368, 407, 435 and 436 of human IgG1 according to the EU index, wherein the amino acid substitutions are Y349C, T366S, L368A, Y407V, H435R and Y436F; and wherein the fourth polypeptide chain comprises a kappa CL domain. 5. Method according to any one of claims 1 to 4, characterized in that the binding protein is detected in one or more of (c) and (e) using hydrophobic interaction chromatography (HIC). 6. Method according to any one of claims 1 to 5, characterized in that the CH3 domains of the second and third polypeptide chains are CH3 domains of human IgG1 or IgG4. 7. The method of any one of claims 1 to 6, wherein (a) L1, L2, L3, and L4 are each independently zero amino acids in length or comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148); or (b) L1, L2, L3, and L4 each independently comprise a sequence selected from the group consisting of GGGGSGGGGS (SEQ ID NO: 104), GGGGSGGGGSGGGGS (SEQ ID NO: 105), S, RT, TKGPS (SEQ ID NO: 106), GQPKAAP (SEQ ID NO: 175), and GGSGSSGSGG (SEQ ID NO: 148). 8. The method of any one of claims 1 to 6, wherein: (a) L1 comprises the sequence GQPKAAP (SEQ ID NO: 175), L2 comprises the sequence TKGPS (SEQ ID NO: 106), L3 comprises the sequence S, and L4 comprises the sequence RT; (b) L1 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L2 comprises the sequence GGGGSGGGGS (SEQ ID NO: 104), L3 is 0 amino acids in length, and L4 is 0 amino acids in length; or (c) L1 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L2 comprises the sequence GGSGSSGSGG (SEQ ID NO: 148), L3 is 0 amino acids in length, and L4 is 0 amino acids in length. 9. Method according to any one of claims 1 to 8, characterized in that the binding protein is trispecific and capable of specifically binding to three different antigenic targets. 10. Method according to any one of claims 1 to 9, characterized in that the binding protein specifically binds to three target proteins corresponding to two target proteins on T cells and to a tumor target protein, wherein optionally one of said target proteins on T cells is CD3; wherein optionally one of said target proteins on T cells is CD28; wherein optionally said tumor target protein is CD38. 11. The method of any one of claims 1 to 10, wherein the binding protein specifically binds to three target proteins corresponding to two target proteins on T cells and to a target protein selected from the group consisting of A2AR, APRIL, ATPDase, BAFF, BAFFR, BCMA, BlyS, BTK, BTLA, B7DC, B7H1, B7H4, B7H5, B7H6, B7H7, B7RP1, B7-4, C3, C5, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL15, CCL17, CCL19, CCL20, CCL21, CCL24, CCL25, CCL26, CCR3, CCR4, CD3, CD19, CD20, CD23, CD24, CD27, CD28, CD38, CD39, CD40, CD70, CD80, CD86, CD122, CD137, CD137L, CD152, CD154, CD160, CD272, CD273, CD274, CD275, CD276, CD278, CD279, CDH1, chitinase, CLEC9, CLEC91, CRTH2, CSF-1, 2, CSF-3, CX3CL1, CXCL12, CXCL13, CXCR3, DNGR-1, ectonucleoside triphosphate diphosphohydrolase 1, EGFR, ENTPD1, FCER1A, FCER1, FLAP, FOLH1, Gi24, GITR, GITRL, GM-CSF, Her2, HHLA2, HMGB1, HVEM, ICOSLG, IDO, IFNα, IgE, IGF1R, IL2Rbeta, IL1, IL1A, IL1B, IL1F10, IL2, IL4, IL4Ra, IL5, IL5R, IL6, IL7, IL7Ra, IL8, IL9, IL9R, IL10, rhIL10, IL12, IL13, IL13Ra1, IL13Ra2, IL15, IL17, IL17Rb, , IL22, IL23, IL25, IL27, IL33, IL35, ITGB4, ITK, KIR, LAG3, LAMP1, leptin, LPFS2, MHC class II, NCR3LG1, NKG2D, NTPDase-1, OX40, OX40L, PD-1H, platelet receptor, PROM1, S152, SISP1, SLC, SPG64, STEAP2, Syk kinase, TACI, TDO, T14, TIGIT, TIM3, TLR, TLR2, TLR4, TLR5, TLR9, TMEF1, TNFa, TNFRSF7, Tp55, TREM1, TSLP, TSLPR, TWEAK, VEGF, VISTA, Vstm3, WUCAM and XCR1. 12. Method according to claim 10 or 11, characterized in that L1, L2, L3 and/or L4 comprise the sequence Asp-Lys-Thr-His-Thr (SEQ ID NO: 525). 13. The method of any one of claims 1 to 6 to 11, wherein VH1 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30; VL1 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 46, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 47 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 48; VH2 comprises a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 34, a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 35 and a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 36; VL2 comprises a CDR-L1 comprising the amino acid sequence of SEQ ID NO: 52, a CDR-L2 comprising the amino acid sequence of SEQ ID NO: 53 and a CDR-L3 comprising the amino acid sequence of SEQ ID NO: 54. 14. The method of any one of claims 1 to 6, wherein VH1 and VL1 form a first antigen-binding site that specifically binds to human CD28, wherein VH2 and VL2 form a second antigen-binding site that specifically binds to human CD3, and wherein VH3 and VL3 form a third antigen-binding site that specifically binds to a human tumor target protein; wherein optionally the third antigen-binding site specifically binds to a human tumor target protein selected from the group consisting of CD19, CD20, CD38, Her2, and LAMP1. 15. The method of claim 14, wherein the antigen-binding site that specifically binds to CD3 comprises: (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 152 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 153; or (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 154 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 155. 16. The method of claim 14 or 15, wherein the antigen-binding site that specifically binds to CD28 comprises: (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 160 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 161; or (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 162 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 163. 17. The method of any one of claims 14 to 16, wherein the antigen-binding site that specifically binds to a target protein comprises: (a) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 156 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 157; (b) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 158 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 159; (c) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 164 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 165; (d) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 150 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 151; or (e) a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 166 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO: 167.