BR122024005601A2 - ISOLATED ANTIBODY FRAGMENT, AUTONOMOUS KNOB DOMAIN PEPTIDES, PHARMACEUTICAL COMPOSITION, POLYNUCLEOTIDE, VECTOR, HOST CELL AND METHOD FOR PRODUCING ISOLATED ANTIBODY FRAGMENT - Google Patents

Abstract

The present description relates to isolated antibody fragments, in particular to isolated knob domains of ultra-long bovine CDR-H3 or portions thereof that bind to an antigen of interest, and formulations comprising the same. The description further relates to the use of the isolated antibody fragments and formulations in therapy. The present description also extends to methods of preparing said isolated antibody fragments.

Description

Field of invention

[0001] The present description relates to isolated antibody fragments, in particular to isolated knob domains of ultra-long bovine CDR-H3 or portions thereof that bind to an antigen of interest, and formulations comprising the same. The description further relates to the use of the isolated antibody fragments and formulations in therapy. The present description also extends to methods of preparing said isolated antibody fragments. Background of the invention

[0002] The high specificity and affinity of antibodies make them ideal diagnostic and therapeutic agents. Standard full-length monoclonal antibodies have a size of 150 kDa. Advances in the field of recombinant antibody technology have resulted in the production of antibody fragments, such as Fv, Fab, Fab’ and F(ab’)2 fragments. These smaller molecules retain the antigen-binding activity of whole antibodies and may also exhibit altered biodistribution, tissue penetration and pharmacokinetic properties compared to whole immunoglobulin molecules. Indeed, antibody fragments are proving to be versatile therapeutic agents. To date, the smallest naturally occurring, functional, autonomous antibody fragment reported has been the VHH fragment derived from camelids (Hamers-Casterman, C. et al. Naturally occurring antibodies devoid of light chains. Nature 363, 446-448 (1993)) and the VNAR (Variable New Antigen Receptor) fragment from sharks (Greenberg, A. S. et al. A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks. Nature 374, 168-173 (1995)), resulting in heavy chain variable region fragments of some 12 to 15 kDa.

[0003] Although such fragments appear to exhibit several advantages over whole immunoglobulins, they also suffer from an increased rate of clearance from serum since they lack the Fc domain that imparts a long in vivo lifetime. Furthermore, the availability of recombinant production methods and systems is limiting in antibody production and may present technical challenges, for example in terms of DNA engineering, cell productivity, etc. Furthermore, miniaturizing antibody fragments may allow them to be produced without antibody expression. The smallest fragments may be amenable to chemical synthesis and remove the need for DNA or cells altogether.

[0004] Therefore, there is a need to provide novel therapeutic products, including novel antibody-derived therapeutic products, having improved properties useful in therapy, notably having improved pharmacokinetic properties (e.g. biodistribution, bioavailability, cell and tissue penetration, clearance) and/or improved biological function (e.g. specificity, binding affinity, neutralization, cell cytotoxicity) and/or the ability to be manufactured by chemical synthesis, independent of cells or cellular machinery.

[0005] There is also a need to identify new methods of producing highly diverse and specific antibody-derived therapeutic products.

[0006] Some bovine antibodies have been distinguished by unusually long CDR-H3 (so-called “ultra-long bovine CDR-H3”) with lengths of up to 69 residues, which contribute to the high diversity of the antibody repertoire. The ultra-long bovine CDR-H3 has been distinguished by a very unusual three-dimensional structure comprising a “stalk domain” and a “knob domain”, for example by Stanfield et al. (Stanfield, R. L., Wilson, I. A. & Smider, V. V. Conservation and diversity in the ultralong third heavy-chain complementarity-determining region of bovine antibodies. Sci Immunol 1, (2016) (hereinafter “Stanfield et al. (supra)”). For example, WO2013/106485 describes humanized antibodies comprising an ultralong CDR-H3, in particular wherein a heterologous polypeptide is inserted into or replaces at least a portion of the knob domain of the ultralong CDR-H3. So far, the ultralong bovine CDR-H3 has only been distinguished when associated with additional domains of the full antibody structure, notably when integrated as part of a Fab fragment as described in Stanfield et al. mentioned above.

Summary of the invention

[0007] The present inventors show for the first time that bovine antibody knob domains are capable of autonomously binding antigen with high affinity in the absence of the ultra-long CDR-H3 stalk region, neighboring CDRs or Fab infrastructure.

[0008] In particular, the invention provides isolated knob domains of ultra-long bovine CDR-H3 and portions thereof, which surprisingly retain functionality and are capable of binding their antigen of interest outside the full-length bovine antibody framework, i.e., when expressed on their own.

[0009] The present invention provides improved antibody fragments, notably bovine antibody fragments having a high specificity for their antigen of interest and having improved properties, notably pharmacokinetic properties (e.g. biodistribution, bioavailability, cell and tissue penetration, clearance) and/or biological properties (e.g. specificity, binding affinity, neutralization, cell cytotoxicity) useful in therapy.

[0010] Advantageously, the antibody fragments as disclosed herein effectively bridge a molecular weight gap between camelid-derived VHH antibody domains and chemical macrocycles, with potential for therapeutic utility. Furthermore, by virtue of their low molecular weight, the invention provides an antibody fragment that can be manufactured by chemical synthesis, without the requirement for a cellular mechanism. Accordingly, the invention also provides peptides encoding antibody fragments according to the invention, which are not isolated from bovine, but are produced synthetically.

[0011] Furthermore, the invention provides a novel antibody format that may be useful in multiple applications, based on the diversity of antigens, diseases and disorders that can be targeted. Advantageously, the novel antibody format may lead to the discovery of novel epitopes on an antigen of interest, as well as novel biological pathways and mechanisms of action associated therewith.

[0012] Thus, in one aspect, an isolated antibody fragment is provided, wherein the fragment is a knob domain of a bovine ultra-long CDR-H3 or portion thereof that binds to an antigen of interest.

[0013] In one embodiment, the isolated antibody fragment is the knob domain of the ultra-long bovine CDR-H3. In one embodiment, the isolated antibody fragment comprises at least two, or at least four, or at least six, or at least eight, or at least ten cysteine residues. In one embodiment, the isolated antibody fragment comprises at least one, or at least two, or at least three, or at least four, or at least five disulfide bonds. In one embodiment, the isolated antibody fragment comprises a (Z1) X1 C X2 motif at its N-terminal end, wherein: a. Z1 is present or absent and when Z1 is present, Z1 represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids; and, b. X1 is any amino acid residue, preferably selected from the list consisting of Serine, Threonine, Asparagine, Alanine, Glycine, Proline, Histidine, Lysine, Valine, Arginine, Isoleucine, Leucine, Phenylalanine and Aspartic acid; and, c. C is cysteine; and, d. X2 is an amino acid selected from the list consisting of Proline, Arginine, Histidine, Lysine, Glycine and Serine.

[0014] In one embodiment, the isolated antibody fragment comprises an (AB)n and/or (BA)n motif, wherein A is any amino acid residue, B is an aromatic amino acid selected from the group consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and wherein n is 1, 2, 3, or 4.

[0015] In one embodiment, the isolated antibody fragment is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length or more, and is up to 55 amino acids in length. In one embodiment, the isolated antibody fragment is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

[0016] In one embodiment, the isolated antibody fragment comprises a sequence that is a variant of a naturally occurring sequence.

[0017] In one embodiment, the isolated antibody fragment according to the invention further comprises a bridged moiety between two amino acids. In one embodiment, the bridged moiety comprises a trait selected from the group consisting of a disulfide bond, an amide (lactam) bond, a thioether bond, an aromatic ring, an unsaturated aliphatic hydrocarbon chain, a saturated aliphatic hydrocarbon chain, and a triazole ring.

[0018] In one embodiment, the isolated antibody fragment is wholly bovine. In one embodiment, the isolated antibody fragment is chimeric. In one embodiment, the isolated antibody fragment is synthetic.

[0019] In one embodiment, the isolated antibody fragment binds to the complement component C5, i.e. the antigen of interest is C5. In one embodiment, the isolated bovine antibody fragment has a sequence selected from the list consisting of SEQ ID NO: 157 to SEQ ID NO: 310, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326 to SEQ ID NO: 331, SEQ ID NO: 334, SEQ ID NO: 336, SEQ ID NO: 339, SEQ ID NO: 341 to SEQ ID NO: 350, SEQ ID NO: 352, and SEQ ID NO: 572 to SEQ ID NO: 609, or any one thereof with at least 95%, 96%, 97%, 98%, or 99% accuracy. similarity or identity.

[0020] In one embodiment, the isolated antibody fragment binds human serum albumin, i.e. the antigen of interest is human serum albumin. In one embodiment, the isolated antibody fragment has the sequence SEQ ID NO: 510.

[0021] In one embodiment, there is provided a polypeptide comprising at least one isolated antibody fragment according to the invention. In one embodiment, there is provided a polypeptide comprising at least two isolated antibody fragments according to the invention, wherein the antibody fragments are linked together, optionally via a linker, e.g. a cleavable linker. In one embodiment, the at least two isolated antibody fragments bind to the same antigen. In another embodiment, the at least two isolated antibody fragments bind to different antigens. In one embodiment, the polypeptide comprises at least one bridged moiety between two amino acids.

[0022] In one embodiment, the isolated antibody fragment or polypeptide according to the invention is fused to one or more effector molecules, optionally via a linker, for example a cleavable linker. In one embodiment, the effector molecule is an antibody. In one embodiment, the effector molecule is a full-length IgG. In another embodiment, the effector molecule is selected from the list consisting of a Fab, a VHH, a VH, a VL, a scFv and a dsscFv. In one embodiment, the effector molecule comprises an albumin binding domain. In one embodiment, the effector molecule is albumin or a protein comprising an albumin binding domain. In one embodiment, the albumin binding domain comprises SEQ ID NO: 435 for CDR-H1, SEQ ID NO: 436 for CDR-H2, SEQ ID NO: 437 for CDR-H3, SEQ ID NO: 430 for CDR-L1, SEQ ID NO: 431 for CDR-L2, and SEQ ID NO: 432 for CDR-L3; or a heavy chain variable domain selected from SEQ ID NO: 434 and SEQ ID NO: 444 and a light chain variable domain selected from SEQ ID NO: 429 and SEQ ID NO: 443.

[0023] In another embodiment, the invention also provides pharmaceutical compositions comprising an isolated antibody fragment or a polypeptide according to the present invention, in combination with one or more pharmaceutically acceptable excipients.

[0024] In another embodiment, the invention also provides an isolated antibody fragment or a polypeptide according to the present invention, for use in therapy.

[0025] In another embodiment, the invention also provides a polynucleotide encoding an isolated antibody fragment or a polypeptide according to the invention. In another embodiment, the invention provides a vector comprising a polynucleotide according to the invention. In another embodiment, the invention provides a host cell comprising a polynucleotide or vector of the invention. In another embodiment, the invention provides a process for producing an isolated antibody fragment or polypeptide according to the invention, said process comprising expressing an isolated antibody fragment or a polypeptide of the invention from a host cell of the invention.

[0026] In another aspect, the invention provides methods of producing an isolated antibody fragment or a polypeptide as defined in the present description, said method comprising a chemical synthesis step. In one embodiment, the chemical synthesis comprises a step of incorporating a coupling reagent with a radioisotope. In one embodiment, the radioisotope is an alpha-emitting radioisotope, preferably Astatin 211.

[0027] The present disclosure also provides new methods of screening antibody fragments and therapeutic polypeptides derived therefrom, comprising immunizing a bovine with an antigen of interest.

[0028] Thus, in one aspect, there is provided a method for producing an isolated antibody fragment or polypeptide of the invention, said method comprising: a) immunizing a bovine with an immunogenic composition, and; b) isolating antigen-specific memory B cells, and; c) sequencing the CDR-H3 cDNA or portions thereof, and; d) expressing or synthesizing the ultra-long CDR-H3 knob domain or portion thereof,

[0029] wherein the immunogenic composition comprises an antigen of interest or immunogenic portions thereof or DNA encoding the same.

[0030] In one embodiment, the method further comprises a step of screening, e.g. for binding to said antigen of interest, wherein, optionally, the screening step is preceded by a step of reforming the ultra-long CDR-H3 or the knob domain of the ultra-long CDR-H3 or portion thereof into a screening format. In one embodiment, the step of reforming the ultra-long CDR-H3 or the knob domain of the ultra-long CDR-H3 or portion thereof into a screening format comprises fusing the ultra-long CDR-H3 or the knob domain of the ultra-long CDR-H3 or portion thereof, to a carrier, optionally via a linker, e.g. a cleavable linker. In one embodiment, the carrier is an Fc polypeptide. In one embodiment, the Fc polypeptide is a scFc.

[0031] In another aspect, a library comprising at least one isolated antibody fragment of the invention is provided. In one embodiment, the library is a synthetic library. In one embodiment, the library is a phage library. In one embodiment, the phage library is a naive library. In one embodiment, the phage library is an immune library. In one embodiment, the library is prepared from bovine.

[0032] In another aspect, the invention provides a phage display library comprising a plurality of recombinant phages; each of the plurality of recombinant phages comprising an expression vector comprising M13, wherein the expression vector comprising M13 comprises a polynucleotide sequence encoding an isolated antibody fragment of the invention, optionally displayed within the complete ultra-long CDR-H3 sequence. In one embodiment, the isolated antibody fragment, optionally displayed within the complete ultra-long CDR-H3 sequence, is fused to the sequence encoding the pIII coat protein of the M13 phage, directly or via a spacer.

[0033] The invention also provides a method for generating a phage display library of ultra-long CDR-H3 sequences, said method comprising: a) immunizing a bovine with an immunogenic composition, and; b) isolating total RNA from PBMC or secondary lymphoid organ, and; c) amplifying the ultra-long CDR-H3 cDNA sequences, and; d) fusing the sequences obtained in c) with the sequence coding for the pIII protein of an M13 phage within a phagemid vector, and; e) transforming host bacteria with the phagemid vector obtained in step d) in combination with a helper phage co-infection, and; f) culturing the bacteria obtained in step e), and; g) recovering the phages from the culture medium of the bacteria,

[0034] wherein the immunogenic composition comprises an antigen of interest or immunogenic portions thereof or DNA encoding the same.

[0035] In one embodiment, step c) comprises: a) a primary PCR with primers flanking CDR-H3, annealing with the conserved VH frame 3 and frame 4, to amplify all CDR-H3 sequences, and b) a second round of PCR using stalked primers to specifically amplify ultra-long sequences from the primary PCR.

[0036] In one embodiment, the primers used in step a) comprise or consist of SEQ ID NO: 446 and SEQ ID NO: 447 and/or the primers used in step b) are selected from the group consisting of SEQ ID NO: 482 to SEQ ID NO: 494.

[0037] The invention also provides a method for producing an isolated antibody fragment of the invention that binds to an antigen of interest, said method comprising: a) generating a phage display library of ultra-long CDR-H3 sequences, and, b) enriching the phage display library against the antigen of interest to produce an enriched population of phage that binds the antigen of interest; and, c) sequencing an ultra-long CDR-H3 from the enriched phage population obtained in step b); and, d) expressing or synthesizing an isolated antibody fragment derived from the ultra-long CDR-H3 obtained in step c).

[0038] The invention also provides a method for producing an isolated antibody fragment of the invention that binds to an antigen of interest, said method comprising: a) generating a phage display library of isolated antibody fragments of the invention; and, b) enriching the phage display library against the antigen of interest to produce an enriched population of phage that binds the antigen of interest; and, c) sequencing an isolated antibody fragment from the enriched phage population obtained in step b); and, d) expressing or synthesizing an isolated antibody fragment obtained in step c).

[0039] It will be understood that the ability to exploit the diversity of the bovine immunological repertoire, for the ability to chemically synthesize the product, links chemistry and biology in a new way and offers significant advantages over current procedures in immunotherapy. Detailed Description of the Invention Isolated antibody fragments

[0040] In one aspect, the present disclosure provides an isolated antibody fragment, wherein the fragment is the knob domain of a bovine ultra-long CDR-H3 or a portion thereof that binds to an antigen of interest.

[0041] An “isolated” antibody fragment is one that has been separated (e.g. by purification means) from a component of its natural environment. In the context of the present description, an “isolated” antibody fragment may be obtained from bovine and optionally engineered to produce any variant according to the invention or may be produced recombinantly or synthetically, for example by chemical synthesis. The term “knob domain peptide” may be used to refer to an isolated antibody fragment as described in the present description.

[0042] Antibody fragments for use in the context of the present disclosure encompass entire knob domains of bovine ultra-long CDR-H3 and any portion thereof, notably any functionally active portion thereof (i.e., any portion of a knob domain of a bovine ultra-long CDR-H3 that contains an antigen-binding domain that specifically binds an antigen of interest).

[0043] Whole antibodies also known as “immunoglobulins (Ig)” generally refer to intact or full-length antibodies that is, comprising the elements of two heavy chains and two light chains, interconnected by disulfide bonds, which assemble to define a characteristic Y-shaped three-dimensional structure. Classical natural whole antibodies are monospecific in that they bind one type of antigen and bivalent in that they have two independent antigen-binding domains. The terms “intact antibody”, “full-length antibody” and “full antibody” are used interchangeably to refer to a monospecific bivalent antibody having a structure similar to a native antibody structure, including an Fc region as defined herein.

[0044] Each light chain is comprised of a light chain variable region (herein abbreviated as VL) and a light chain constant region (CL). Each heavy chain is comprised of a heavy variable region (herein abbreviated as VH) and a heavy chain constant region (CH) consisting of three constant domains CH1, CH2 and CH3 or four constant domains CH1, CH2, CH3 and CH4, depending on the Ig class. The “class” of an Ig or antibody refers to the type of constant region and includes IgA, IgD, IgE, IgG and IgM and several of these may be further divided into subclasses, for example IgG1, IgG2, IgG3, IgG4. The constant regions of antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system.

[0045] The term “constant domain(s)”, “constant region”, as used herein are used interchangeably to refer to the domain(s) of an antibody that are outside of the variable regions. Constant domains are identical in all antibodies of the same isotype, but are different from one isotype to another. Typically, the constant region of a heavy chain is formed, from the N- to C-terminus, by the CH1-hinge-CH2-CH3-optionally CH4, which comprises three or four constant domains.

[0046] “Fc”, “Fc fragment”, “Fc region” are used interchangeably to refer to the C-terminal region of an antibody comprising the constant region of an antibody excluding the first domain of the constant region. Thus, Fc refers to the last two constant domains, CH2 and CH3 of IgA, IgD and IgG or the last three constant domains of IgE and IgM and the flexible hinge N-terminus for these domains.

[0047] The VH and VL regions of a full-length antibody can be further subdivided into regions of hypervariability (or “hypervariable regions”) determining antigen recognition, called complementarity determining regions (CDRs), interspersed with regions that are more structurally conserved, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs together form a variable region. By convention, the CDRs in the heavy chain variable region of an antibody or antigen-binding fragment thereof are referred to as CDR-H1, CDR-H2, and CDR-H3 and in the light chain variable region as CDR-L1, CDR-L2, and CDR-L3. They are numbered sequentially in the direction from the N-terminus to the C-terminus of each chain.

[0048] CDRs are conventionally numbered according to a system devised by Kabat et al. This system is set forth in Kabat et al., 1991, in Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (hereinafter “Kabat et al. (supra)”). This numbering system is used in this specification except where otherwise indicated. Kabat residue designations do not always correspond directly with linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or additional amino acids than the strict Kabat numbering corresponding to a shortening of, or insertion into, a structural component, either frame or complementarity determining region (CDR), of the basic variable domain structure. The correct Kabat numbering of residues can be determined for a given antibody by aligning homologous residues in the antibody sequence with a “standard” Kabat numbered sequence.

[0049] The CDRs of the heavy chain variable domain are located at residues 31 to 35 (CDR-H1), residues 50 to 65 (CDR-H2) and residues 93 to 102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (Chothia, C. and Lesk, A. M. J. Mol. Biol., 196, 901-917 (1987)), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus, unless otherwise indicated 'CDR-H1' as used herein is intended to refer to residues 26 to 35, as described by a combination of the Kabat numbering system and Chothia's topological loop definition. The CDRs of the light chain variable domain are located at residues 24 to 34 (CDR-L1), residues 50 to 56 (CDR-L2) and residues 89 to 97 (CDR-L3) according to the Kabat numbering system. Based on sequence alignments of different members of the immunoglobulin family, numbering schemes have been proposed and are for example described in Kabat et al., 1991 and Dondelinger et al., Frontiers in Immunology, Vol 9, article 2278 (2018).

[0050] Different species exhibit a diversity of CDR-H3 lengths. Some bovine antibodies have been distinguished by unusually long CDR-H3s (so-called “ultra-long bovine CDR-H3s”) with lengths up to 69 residues, representing 1 to 15% of the bovine repertoire, whereas more conventional bovine antibodies have CDR-H3s of around 23 residues. Camelid single-chain antibodies have up to 24 residues and shark IgNAR antibodies have up to 27 residues. CDR-H3s are too long to be accommodated by any of these numbering schemes, but alternative systems have been used, such as that discussed in Stanfield et al. (supra).

[0051] “Bovine CDR-H3” as used herein encompasses all CDR-H3s found in cattle, including regular bovine CDR-H3 and ultra-long bovine CDR-H3.

[0052] The term “ultra-long bovine CDR-H3” refers to the subset of CDR-H3s that have the features of distinguished ultra-long CDR-H3s as defined herein below, notably comprising a duplication of the IGHVI-7 gene segment. Ultra-long CDR-H3 has been found in bovine IgG of all classes.

[0053] The ultra-long bovine CDR-H3 was distinguished by a very unusual three-dimensional structure comprising a “stalk domain” and a “knob domain”. The stalk domain is composed of two antiparallel β-strands (each strand generally corresponding to about 12 residues). The knob domain is a disulfide-rich domain comprising a loop motif and lies at the top of the stalk, which serves as a bridge to connect the knob domain with the main bovine antibody scaffold.

[0054] CDR-H3 is derived from DNA rearrangement of variable (V), diversity (D) and joining (J) gene segments. The ultra-long CDR-H3s are encoded by the VHBUL (Bovine Ultra Long), DH2 and JH1 gene segments and their length is due to an unusually long DH2 segment. The ultra-long CDR-H3 was distinguished by a duplication of the IGHVI-7 gene segment.

[0055] The isolated antibody fragment of the present description does not comprise the stalk domain of the ultra-long bovine CDR-H3.

[0056] The “stalk domain” of ultra-long bovine CDR-H3 has been distinguished notably by its structure. The skilled person will appreciate that the definition of a “stalk domain” may rely on analysis of the crystal structure and/or sequencing information, notably since they will appreciate that the position and structure of the stalk domain may vary slightly from one ultra-long CDR-H3 to another, for example in terms of size. The term “stalk domain” will generally be appreciated by the skilled person to correspond to the antiparallel β-strands that bridge the knob domain with the scaffold of the primary bovine antibody. The length of the stalk β-strands may differ, notably from long β-strands (12 or more residues) to shorter β-strands.

[0057] The skilled person will appreciate that the definition of a knob domain can rely on crystal structure analysis and/or sequencing information, notably since he/she will understand that the position and structure of the knob domain may vary slightly from one ultra-long CDR-H3 to another, for example in terms of size, cysteine content, disulfide bond content. In particular, the sequence of ultra-long CDR-H3 can be determined by well-known sequencing methods and the skilled person will be able to identify the minimal sequence that defines a knob domain, based for example on a comparative analysis, with well-distinguished ultra-long CDR-H3 as well as the stem and knob domains thereof, for example by alignment with well-known and/or standard nucleic acid and/or amino acid sequences and/or based on crystal structure analysis.

[0058] As mentioned above, ultra-long CDR-H3s are too long to be accommodated by any of the existing numbering schemes, but alternative systems have been used, such as the one discussed in Stanfield et al. (supra). Structural analysis has also been provided for example by Wang et al. (Wang, F. et al. Reshaping antibody diversity. Cell 153, 1379-1393 (2013)).

[0059] The conserved Cysteine at position 92 (Kabat) and the conserved Tryptophan at position 103 (Kabat) respectively define the beginning and end of CDR-H3, as illustrated in Figure 14. The germline encoded VHBUL DH2 JH1 has the following sequence:

[0060] CTTVHQSCPDGYSYGYGCGYGYGCSGYDCYGYGGYGGYGGYGYSSY SYSYTYEYYVDAWGQGLLVTVSS

[0061] (VHBUL; followed by the DH2 gene region in bold; followed by the JH1 gene region underlined; The sequence encoding CDR-H3 is in italics, between positions 92 and 103 according to Kabat)

[0062] The Kabat numbering system can be used for heavy chain residues 1 to 100 and 101 to 228, but residues between 100 and 101 (corresponding to the residues encoded by the DH2 and JH1 genes) do not accommodate the Kabat numbering system and can be numbered differently, for example sequentially with a D identifier, as described in Stanfield et al. (supra), with the conserved Cysteine residue at the beginning of DH2 being "D2", followed by D3, D4, etc.). For purposes of illustration, Figure 14 indicates identifiers D2, D10, D20, D30, and D40 within the DH2 segment.

[0063] Following Cys H92, the common motif TTVHQ (positions 93 to 97 in germline VHBUL, according to Kabat) begins with the ascending strand of the β-stalk region of CDR-H3. The length between the end of VHBUL and the conserved “CPD” motif in DH2 is variable due to differences in junctional diversity formed through V-D recombination. In Stanfield, these junctional residues are referred to as “a,b,c” following residue H100, depending on their length (e.g., as illustrated in Figure 14, the bovine CDR-H3 BLV1H12 comprises 3 residues following H100, referred to as a, b, and c).

[0064] The DH2 region was distinguished to encode the knob domain and part of the descending strand of the stalk region. DH2 begins with a conserved Cysteine that is part of a conserved “CPD” motif in the germline sequence, which distinguishes the beginning of the knob domain. The knob domain ends at the beginning of the descending strand of the β-stalk region. The descending strand of the β-stalk region was distinguished by alternating aromatic-aliphatic residues in some ultra-long CDR-H3. The descending strand of the β-stalk region ends with the residues encoded by the genetic J region, followed by residue H101, H102 according to Kabat.

[0065] In the context of the present description, the minimal sequence that can define a knob domain corresponds to the portion of the ultra-long CDR-H3 encapsulated by the disulfide bonds, more particularly the minimal sequence of the knob domain starts from the first cysteine residue of an ultra-long CDR-H3 and ends with the last cysteine residue of the ultra-long CDR-H3. Therefore, a minimal knob domain typically comprises at least two cysteines. In one embodiment, the sequence of the knob domain starts from a residue preceding the first cysteine residue of an ultra-long CDR-H3 and ends after the residue subsequent to the last cysteine residue of the ultra-long CDR-H3. Additional amino acids can be present at the N-terminal end and/or at the C-terminal end of the knob domain sequence, preferably up to 5 additional amino acids can be present at the N-terminal end and/or at the C-terminal end.

[0066] As an example, the ultra-long bovine CDR-H3 sequence BLV1H12 is italicized in the following sequence comprising VHBUL, DH2 (underlined), JH1 (Cys92 and Trp103 of Kabat are in bold):

[0067] CTSVHQ ETKKYQ SCPDGYRERSDCSNRPACGTSDCCRVSVFGNCLTTLPVSYSYTYNYEW HVDVWGQGLLVTVSS

[0068] The knob domain of this sequence can therefore be defined as the following sequence: SCPDGYRERSDCSNRPACGTSDCCRVSVFGNCL

[0069] (i.e., from a residue preceding the first cysteine to the residue subsequent to the last cysteine residue of the ultra-long CDR-H3; cysteine residues in bold):

[0070] Another example is provided below with the ultra-long CDR-H3 K149 (SEQ ID NO: 1 of this patent application):

[0071] TSVLQSTKPQKSCPDGFSYRSWDDFCCPMVGRCLAPRNTYTTEFTIEA

[0072] The knob domain that can be defined according to the present application for this sequence is in bold, starting from a residue preceding the first cysteine residue of the ultra-long CDR-H3 and ending after the residue subsequent to the last cysteine residue of the ultra-long CDR-H3.

[0073] In one embodiment, the isolated antibody fragment consists of the knob domain of a bovine ultra-long CDR-H3, i.e. a full-length knob domain, notably comprised between the upstem and downstem of the ultra-long CDR-H3.

[0074] In one embodiment, the isolated antibody fragment comprises or consists of a portion of the knob domain of a bovine ultra-long CDR-H3 that binds to an antigen of interest.

[0075] In one embodiment, the isolated antibody fragment comprises at least two, or at least four, or at least six, or at least eight, or at least ten cysteine residues. In one embodiment, the isolated antibody fragment comprises at least two cysteine residues. In one embodiment, the isolated antibody fragment comprises at least four cysteine residues. In one embodiment, the isolated antibody fragment comprises at least six cysteine residues. In one embodiment, the isolated antibody fragment comprises at least eight cysteine residues. In one embodiment, the isolated antibody fragment comprises at least ten cysteine residues.

[0076] In one embodiment, the isolated antibody fragment comprises two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or fourteen cysteine residues. In one embodiment, the isolated antibody fragment comprises between two cysteine residues and ten cysteine residues. In one embodiment, the isolated antibody fragment comprises between four cysteine residues and eight cysteine residues.

[0077] Two cysteine residues can bridge together to form a disulfide bond within the knob domain.

[0078] In one embodiment, the isolated antibody fragment comprises at least one, or at least two, or at least three, or at least four, or at least five disulfide bonds. In one embodiment, the isolated antibody fragment comprises one, two, three, four, five, six, or seven disulfide bonds. In one embodiment, the isolated antibody fragment comprises between one disulfide bond and five disulfide bonds. In one embodiment, the isolated antibody fragment comprises between two disulfide bonds and four disulfide bonds.

[0079] It will be appreciated that an increased content of cysteine residues will increase the possibility for forming disulfide bonds within the isolated antibody fragment. Such disulfide bonds contribute to forming a loop motif within the isolated antibody fragment, which may be advantageous for increasing the stability and/or rigidity and/or binding specificity and/or binding affinity of the isolated antibody fragment.

[0080] In one embodiment, the isolated antibody fragment comprises a (Z1) X1 C X2 motif at its N-terminal end, wherein: a. Z1 is present or absent and when Z1 is present, Z1 represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids; and, b. X1 is any amino acid residue; and, c. C is cysteine; and, d. X2 is an amino acid selected from the list consisting of Proline, Arginine, Histidine, Lysine, Glycine, and Serine. Z1 as defined herein represents any amino acid or any sequence of 2, 3, 4, or 5 independently selected amino acids which may be the same or different. In one embodiment, Z1 is 1 amino acid. In another embodiment, Z1 has 2 amino acids, which may be the same or different. In another embodiment, Z1 has 3 amino acids, which may be the same or different. In another embodiment, Z1 has 4 amino acids, which may be the same or different. In another embodiment, Z1 has 5 amino acids, which may be the same or different.

[0081] In one embodiment, X1 is selected from the list consisting of Serine, Threonine, Asparagine, Alanine, Glycine, Proline, Histidine, Lysine, Valine, Arginine, Isoleucine, Leucine, Phenylalanine, and Aspartic acid. Thus, in one aspect, the invention provides an isolated antibody fragment, wherein the knob domain or portion thereof comprises a (Z1) X1 C X2 motif at its N-terminal end, wherein: a. Z1 is present or absent and when Z1 is present, Z1 represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids; and, b. X1 is any amino acid residue, preferably selected from the list consisting of Serine, Threonine, Asparagine, Alanine, Glycine, Proline, Histidine, Lysine, Valine, Arginine, Isoleucine, Leucine, Phenylalanine, and Aspartic acid; and, c. C is cysteine; and, d. X2 is an amino acid selected from the list consisting of Proline, Arginine, Histidine, Lysine, Glycine and Serine.

[0082] In one embodiment, the isolated antibody fragment comprises a (Z1)X1 C X2 motif at its N-terminal end, wherein C is cysteine; and X1 is selected from the list consisting of Serine (S), Threonine (T), Asparagine (N), Alanine (A), Glycine (G), Proline (P), Histidine (H), Lysine (K), Valine (V), Arginine (R), Isoleucine (I), Leucine (L), Phenylalanine (F), and Aspartic acid (D) and X2 is selected from the list consisting of Proline (P), Arginine (R), Histidine (H), Lysine (K), Glycine (G), and Serine (S) and wherein Z1 is present or absent and when Z1 is present, Z1 represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids.

[0083] In one embodiment, the N-terminal end of the isolated antibody fragment comprises a motif comprising 3 amino acid residues, corresponding to an X1C X2 motif, selected from the list consisting of SCP, TCP, NCP, ACP, GCP, CRP, HCP, SCR, KCP, VCP, TCH, RCP, ICP, ICR, HCR, LCR, SCK, SCG, NCP, TCS, DCP, and FCR.

[0084] Preferably, the N-terminal end of the isolated antibody fragment is initiated by a motif selected from the list consisting of (Z1)SCP, (Z1)TCP, (Z1)NCP, (Z1)ACP, (Z1)GCP, (Z1)HCP, (Z1)KCP, (Z1)VCP, (Z1)RCP, (Z1)ICP, (Z1)DCP, wherein Z1 is present or absent and when Z1 is present, Z1 represents 1 amino acid or 2, 3, 4 or 5 independently selected amino acids.

[0085] In one embodiment, the isolated antibody fragment comprises an (AB)n and/or (BA)n motif, wherein A is any amino acid residue, B is an aromatic amino acid selected from the group consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H), and wherein n is 1, 2, 3, or 4.

[0086] In one embodiment, A is an aliphatic amino acid residue. An aliphatic amino acid is an amino acid containing an aliphatic side chain functional group. Aliphatic amino acid residues include Alanine, isoleucine, leucine, proline, and valine.

[0087] In one embodiment, the isolated antibody fragment comprises a 2 to 8 amino acid motif that is rich in aromatic and/or aliphatic amino acids. In one embodiment, the knob domain comprises a 2 to 8 amino acid motif comprising at least 2, or at least 3, or at least 4, or at least 5 amino acids selected from the group consisting of: tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H).

[0088] In one embodiment, the isolated antibody fragment is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length or more. In one embodiment, the isolated antibody fragment is up to 50 amino acids in length or up to 55 amino acids in length. In one embodiment, the isolated antibody fragment is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length or more, and is up to 55 amino acids in length. In one embodiment, the isolated antibody fragment is a portion of a knob domain of a bovine ultra-long CDR-H3 that is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids in length. In one embodiment, the isolated antibody fragment is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length. In one embodiment, the isolated antibody fragment is a knob domain of a bovine ultra-long CDR-H3 that is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

[0089] In one embodiment, the knob domain of the ultra-long CDR-H3, when expressed by itself, binds to an antigen of interest with a binding affinity that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of that of the ultra-long CDR-H3 comprising said knob domain or portion thereof, for example when the knob domain of the ultra-long CDR-H3 is expressed or synthesized as part of the entire ultra-long CDR-H3.

[0090] As mentioned above, the isolated antibody fragment can be synthetically produced, for example by chemical synthesis.

[0091] In one aspect, the invention provides a peptide that binds an antigen of interest comprising or consisting of the sequence of formula (I):

[0092] where:

[0093] C represents a cysteine residue; and,

[0094] Z1 is present or absent and when Z1 is present, Z1 represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids; and,

[0095] X1 is present or absent and when X1 is present, X1 is any amino acid residue, preferably selected from the list consisting of Serine, Threonine, Asparagine, Alanine, Glycine, Proline, Histidine, Lysine, Valine, Arginine, Isoleucine, Leucine, Phenylalanine and Aspartic acid; and,

[0096] X2 is selected from the list consisting of Proline, Arginine, Histidine, Lysine, Glycine and Serine; and,

[0097] Z2 is present or absent and when Z2 is present, Z2 represents 1 amino acid or 2, 3, 4, or 5 independently selected amino acids; and,

[0098] n2, n4, n6, n8, n10, n12, n14, and n16 are independently 0 or 1; and,

[0099] Y represents any amino acid or any sequence of amino acids which may be the same or different; and,

[0100] n1, n3, n5, n7, n9, n11, n13, n15 and n17 represent the number of amino acids in Y and are independently selected from 0 to 22, preferably from 1 to 15; and,

[0101] at least one of n1, n3, n5, n7, n9, n11, n13, n15 and n17 is not equal to 0; and,

[0102] X3 is present or absent and when X3 is present, X3 represents any amino acid, preferably selected from the list consisting of Leucine, Serine, Glycine, Threonine, Tryptophan, Asparagine, Tyrosine, Arginine, Isoleucine, Aspartic acid, Histidine, Glutamic acid, Valine, Lysine, Proline; and,

[0103] wherein the peptide is up to 55 amino acids in length.

[0104] Z1 represents any amino acid or any sequence of 2, 3, 4, or 5 independently selected amino acids which may be the same or different. In one embodiment, Z1 is 1 amino acid. In another embodiment, Z1 has 2 amino acids, which may be the same or different. In another embodiment, Z1 has 3 amino acids, which may be the same or different. In another embodiment, Z1 has 4 amino acids, which may be the same or different. In another embodiment, Z1 has 5 amino acids, which may be the same or different.

[0105] Z2 represents any amino acid or any sequence of 2, 3, 4, or 5 independently selected amino acids that may be the same or different. In one embodiment, Z2 has 1 amino acid. In another embodiment, Z2 has 2 amino acids, which may be the same or different. In another embodiment, Z2 has 3 amino acids, which may be the same or different. In another embodiment, Z2 has 4 amino acids, which may be the same or different. In another embodiment, Z2 has 5 amino acids, which may be the same or different.

[0106] Z1 and Z2 may comprise any amino acid as long as the properties of the otherwise defined peptide are retained, for example the binding capabilities to an antigen of interest.

[0107] In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (I) is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length. In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (I) is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

[0108] Parentheses are generally used for optional residues or sequences. For example, (C) generally indicates an optional Cysteine residue, in the context of the present disclosure.

[0109] In one embodiment, the peptide comprises 2 Cysteine residues. Therefore, in a particular aspect, the invention provides a peptide that binds an antigen of interest comprising or consisting of the sequence of formula (II):

[0110] wherein Z1, X1, C, X2, Y, n1, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0111] In such an embodiment, n1 may be comprised between 1 and 20 amino acids. In one embodiment, n1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0112] In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (II) is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length. In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (II) is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

[0113] In another embodiment, the peptide comprises 4 Cysteine residues. Therefore, in a particular aspect, the invention provides a peptide that binds an antigen of interest comprising or consisting of the sequence of formula (III):

[0114] wherein Z1, X1, C, X2, Y, n1, n3, n5, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0115] In an embodiment, n1 is comprised between 3 and 15 and/or n3 is comprised between 4 and 12 and/or n5 is comprised between 1 and 14. In an embodiment, n1 is 3, 5, 7, 8, 10, 11, 14, or 15. In an embodiment, n3 is 4, 5, 6, 8, 10, 11, or 12. In an embodiment, n5 is 3, 4, 5, 6, 7, 9, 10, 11, or 14.

[0116] In another embodiment, n1 and/or n3 and/or n5 is equal to 0 and two or three Cysteine residues are contiguous.

[0117] In one embodiment, the peptide has the sequence of formula (IIIa):

[0118] wherein Z1, X1, C, X2, Y, n5, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0119] In one embodiment, the peptide has the sequence of formula (IIIb):

[0120] wherein Z1, X1, C, X2, Y, n1, n5, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0121] In one embodiment, the peptide has the sequence of formula (IIIc): (

[0122] wherein Z1, X1, C, X2, Y, n1, n3, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0123] In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (III), (IIIa), (IIIb), or (IIIc) is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length. In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (III), (IIIa), (IIIb), or (IIIc) is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

[0124] In another embodiment, the peptide comprises 6 Cysteine residues. Therefore, in a particular aspect, the invention provides a peptide that binds an antigen of interest comprising or consisting of the sequence of formula (IV):

[0125] wherein Z1, X1, C, X2, Y, n1, n3, n5, n7, n9, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0126] In an embodiment, n1 = 2 to 9 and/or n3 = 1 to 10 and/or n5 = 2 to 9 and/or n7 = 1 to 15 and/or n9 = 1 to 14. In an embodiment, n1 = 2, 3, 4, 5, 6, 7, 8, or 9. In an embodiment, n3 = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In an embodiment, n5 = 2, 3, 4, 5, 6, 7, 8, or 9. In an embodiment, n7 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In an embodiment, n9 = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14.

[0127] In one embodiment, n1 = 0 and/or n3 = 0 and/or n5 = 0 and/or n7 = 0 and/or n9 = 0 and two or three Cysteine residues are contiguous. In one embodiment, the peptide has the sequence of formula (IVa):

[0128] wherein Z1, X1, C, X2, Y, n5, n7, n9, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0129] In one embodiment, the peptide has the sequence of formula (IVb):

[0130] wherein Z1, X1, C, X2, Y, n1, n5, n7, n9, X3, and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0131] In one embodiment, the peptide has the sequence of formula (IVc):

[0132] wherein Z1, X1, C, X2, Y, n1, n3, n7, n9, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0133] In one embodiment, the peptide has the sequence of formula (IVd):

[0134] wherein Z1, X1, C, X2, Y, n1, n3, n5, n9, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0135] In one embodiment, the peptide has the sequence of formula (IVe):

[0136] wherein Z1, X1, C, X2, Y, n1, n3, n5, n7, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0137] In one embodiment, the peptide has the sequence of the formula (IVf):

[0138] wherein Z1, X1, C, X2, Y, n1, n7, n9, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0139] In one embodiment, the peptide has the sequence of formula (IVg):

[0140] wherein Z1, X1, C, X2, Y, n1, n3, n9, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0141] In one embodiment, the peptide has the sequence of the formula (IVh):

[0142] wherein Z1, X1, C, X2, Y, n1, n3, n5, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0143] In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (IV), (IVa), (IVb), (IVc), (IVd), (IVe), (IVf), (IVg), or (IVh), is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length. In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (IV), (IVa), (IVb), (IVc), (IVd), (IVe), (IVf), (IVg), or (IVh), is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

[0144] In another embodiment, the peptide comprises 8 Cysteine residues. Therefore, in a particular aspect, the invention provides a peptide that binds an antigen of interest comprising or consisting of the sequence of formula (V):

[0145] wherein Z1, X1, C, X2, Y, n1, n3, n5, n7, n9, n11, n13, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0146] In one embodiment, n1 = 0 and/or n3 = 0 and/or n5 = 0 and/or n7 = 0 and/or n9 = 0 and/or n11 = 0, and/or n13 = 0 and two or three Cysteine residues are contiguous.

[0147] In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (V) is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length. In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (V) is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

[0148] In another embodiment, the peptide comprises 10 Cysteine residues. Therefore, in a particular aspect, the invention provides a peptide that binds an antigen of interest comprising or consisting of the sequence of formula (VI): (

[0149] wherein Z1, X1, C, X2, Y, n1, n3, n5, n7, n9, n11, n13, n15, n17, X3 and Z2 are defined as above and wherein the peptide is up to 55 amino acids in length.

[0150] In one embodiment, n1 = 0 and/or n3 = 0 and/or n5 = 0 and/or n7 = 0 and/or n9 = 0 and/or n11 = 0, and/or n13 = 0 and/or n15 = 0 and/or n17 = 0 and two or three Cysteine residues are contiguous.

[0151] In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (VI) is 5 amino acids in length or more, 10 amino acids in length or more, 15 amino acids in length or more, 20 amino acids in length or more, 25 amino acids in length or more, 30 amino acids in length or more, 35 amino acids in length or more, 40 amino acids in length or more, 45 amino acids in length. In one embodiment, the peptide that binds an antigen of interest comprising or consisting of the sequence of formula (VI) is between 5 and 55, or between 15 and 50, or between 20 and 45, or between 25 and 40 amino acids in length.

[0152] Preferably, the isolated antibody fragment of the present invention specifically binds to an antigen of interest, i.e., comprises a binding domain specific to an antigen of interest. "Specifically" as used herein is intended to refer to a binding domain that only recognizes the antigen for which it is specific or a binding domain that has significantly higher binding affinity to the antigen for which it is specific compared to the affinity for antigens for which it is not specific, for example 5, 6, 7, 8, 9, 10 times higher binding affinity.

[0153] Preferably, the isolated antibody fragment of the present invention has a specific binding affinity (as measured by its dissociation constant KD) for its cognate antigen of 10-5 M or less, 10-6 M or less, 10-7 M or less, 10-8 M or less, 10-9 M or less, 10-10 M or less, or 10-11 M or less. In one embodiment, the isolated antibody fragment of the present invention has a specific binding affinity (as measured by its dissociation constant KD) for its cognate antigen between 1. 10-7 M and 1. 10-8 M, or between 1. 10-8 M and 1. 10-9 M, or between 1. 10-9 M and 1. 10-10 M.

[0154] Affinity can be measured by known techniques such as surface plasmon resonance techniques including Biacore®. Affinity can be measured at room temperature, 25°C or 37°C. Affinity can be measured at physiological pH, i.e. around pH 7.4. In one embodiment, affinity values as described above are measured using Biacore, notably Biacore 8K, at pH 7.4.

[0155] It will be appreciated that the affinity of the antibody fragments provided by the present invention can be altered using any suitable method known in the art.

Fragment of isolated antibody variants

[0156] In some aspects, the isolated antibody fragment comprises a sequence that is a variant of a naturally occurring sequence of a knob domain of a bovine ultra-long CDR-H3.

[0157] In other words, the present disclosure provides variants of isolated antibody fragments as described above, comprising non-naturally occurring sequences, i.e. which have been further engineered, for example to improve at least one pharmacokinetic and/or biological function. In such aspects, the isolated antibody fragment comprising a naturally occurring sequence may be referred to as a “precursor”.

[0158] The present invention also includes antibody fragments, i.e., ultra-long bovine CDR-H3 knob domains or portions thereof, comprising sequences that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% similar or identical to a sequence given herein. “Identity”, as used herein, indicates that at any particular position in the aligned sequences, the amino acid residue is identical between the sequences. “Similarity”, as used herein, indicates that at any particular position in the aligned sequences, the amino acid residue is of a similar type between the sequences. For example, leucine may be substituted in place of isoleucine or valine. Other amino acids that can often be substituted in place of one another include, but are not limited to: - phenylalanine, tyrosine, and tryptophan (amino acids having aromatic side chains); - lysine, arginine, and histidine (amino acids having basic side chains); - aspartate and glutamate (amino acids having acidic side chains); - asparagine and glutamine (amino acids having amide side chains); and - cysteine and methionine (amino acids having sulfur-containing side chains).

[0159] The degrees of identity and similarity can be easily calculated by well-known methods, for example the BLAST® software available from NCBI.

[0160] In one embodiment, antibody fragments of the present disclosure are processed to provide improved affinity for a target antigen or antigens. Such variants can be obtained by various affinity maturation protocols including mutating the CDRs, chain shuffling, use of mutator strains of E. coli, DNA shuffling, phage display and sex PCR. Vaughan et al (Nature Biotechnology, 16, 535-539, 1998) discusses these affinity maturation methods. Another method useful in the context of the present disclosure for improving the binding of the isolated antibody fragment at a binding site on the antigen of interest is a method as described in WO2014/198951. Improved affinity as used herein in this context refers to an improvement over the starting isolated antibody fragment. Affinity can be measured as described above.

[0161] In one embodiment, the isolated antibody fragment is a variant of a parent bovine antibody fragment that has an affinity that is at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% higher than the affinity of the parent bovine antibody fragment, as measured for example by Biacore.

[0162] “Truncated variants” when referring to antibody fragments are those with one or more amino acids in the native or starting amino acid sequence removed from each terminus of the polypeptide.

[0163] In some embodiments, the isolated antibody fragment is a variant that has been engineered to comprise a disulfide bond that is in a position that does not occur naturally. This can be engineered into the molecule by introducing cysteine(s) within the amino acid chain at the required position or positions. This unnatural disulfide bond is in addition to or as an alternative to the natural disulfide bond(s) that may be present in the isolated parent antibody fragment. The cysteine(s) at the natural positions can be replaced with an amino acid such as serine that is incapable of forming a disulfide bridge. Introduction of engineered cysteines can be accomplished using any method known in the art. These methods include, but are not limited to, PCR extension overlap mutagenesis, locus-directed mutagenesis, or cassette mutagenesis (see, generally, Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing & Wiley-Interscience, NY, 1993). Locus-directed mutagenesis kits are commercially available, for example the QuikChange® Locus-Directed Mutagenesis kit (e.g., Stratagene, La Jolla, CA). Cassette mutagenesis can be performed based on Wells et al., 1985, Gene, 34: 315-323. Alternatively, mutants can be made by whole-gene synthesis by annealing, ligation, and PCR amplification and cloning of overlapping oligonucleotides.

[0164] In one aspect, it may be useful to decrease or remove cysteine residues and/or disulfide bonds in an isolated antibody fragment of the disclosure, for example to decrease the risk of immunogenicity, i.e. of side reactions occurring during or after administration to a patient. In such an aspect, one or all of the cysteine(s) in the natural positions may be replaced with an amino acid such as serine that is incapable of forming a disulfide bond. It will be appreciated that alternative bridging moieties may be used to stabilize and/or form a cyclized isolated antibody fragment in the absence of cysteine residues. In one embodiment, the isolated antibody fragment is a variant that has been engineered to remove cysteine residues and comprising at least one bridging moiety as defined herein. In one embodiment, the isolated antibody fragment is a variant that has been engineered to contain only one, or only two, or only three, or only four, cysteine residues and/or contain only one, or only two disulfide bonds and that optionally further comprises at least one bridged moiety as defined herein.

[0165] Additional modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of tyrosinyl, seryl or threonyl residues, methylation of alpha-amino groups of lysine, arginine and histidine side chains (Croitoon, T. E., Proteins: Structure and Molecular Properties, W. H. Freeman and Co., San Francisco, 1983, pp. 79-86).

[0166] The isolated antibody fragment of the invention may be cyclized. Cyclization may be advantageous to confer more resistance to proteolysis, notably resulting in improved stability.

[0167] Therefore, in one embodiment, the isolated antibody fragment of the present disclosure additionally comprises a bridged moiety between two amino acids.

[0168] Cyclized antibody fragments include any antibody fragments that have as part of their structure one or more cyclic features such as a loop, bridging portion, and/or an internal bond. As used herein, the term “bridging portion” refers to one or more components of a bridge formed between two adjacent or non-adjacent amino acids, unnatural amino acids, or non-amino acids in an isolated antibody fragment. Bridging portions can be of any size or composition.

[0169] In one embodiment, a bridged moiety may be between the amino acid residue at the N-terminal position and the amino acid residue at the C-terminal position such as to create a head-to-tail cyclization. In one embodiment, a bridged moiety may be between amino acids that are not at the terminal position.

[0170] In one embodiment, the isolated antibody fragment comprises only one bridged moiety between two amino acids. In another embodiment, the isolated antibody fragment comprises more than one bridged moiety between two amino acids, for example two or three or five bridged moieties, each being between two amino acids.

[0171] In one embodiment, the bridging moiety comprises a trait selected from the group consisting of a disulfide bond, an amide (lactam) bond, a thioether bond, an aromatic ring, an unsaturated aliphatic hydrocarbon chain, a saturated aliphatic hydrocarbon chain, and a triazole ring.

[0172] In one embodiment, the disulfide bond is formed between two naturally occurring cysteine residues. In another embodiment, the disulfide bond is formed between cysteine residues, with at least one cysteine residue being engineered, as described above.

[0173] In some embodiments, the bridging moieties can comprise one or more chemical bonds between two adjacent or non-adjacent amino acids, unnatural amino acids, non-amino acid residues, or combinations thereof. In some embodiments, such chemical bonds can be between one or more functional groups on the adjacent or non-adjacent amino acids, unnatural amino acids, non-amino acid residues, or combinations thereof. The bridging moieties can comprise one or more traits including, but not limited to, an amide (lactam) bond, disulfide bond, thioether bond, aromatic ring, triazole ring, and hydrocarbon chain. In some embodiments, bridging moieties comprise an amide bond between an amine functionality and a carboxylate functionality, each present on an amino acid side chain, unnatural amino acid, or non-amino acid residue. In some embodiments, the amine or carboxylate functionalities are part of a non-amino acid residue or unnatural amino acid residue. In some cases, bridging moieties may comprise bonds formed between residues that may include, but are not limited to, (S)-2-amino-5-azidopentanoic acid, (S)-2-amino-hept-6-enoic acid, (S)-2-aminopent-4-ynoic acid, and (S)-2-aminopent-4-enoic acid. Bridging moieties may be formed through cyclization reactions using olefin metathesis. In some cases, such bridging moieties may be formed between (S)-2-aminopent-4-enoic acid and (S)-2-amino-hept-6-enoic acid residues. In some embodiments, the bridging moiety comprises a disulfide bond formed between two thiol-containing residues. In some embodiments, the bridging moiety comprises one or more thioether bonds. Such thioether bonds may include those found in cyclothioalkyl compounds. These bonds are formed during a chemical cyclization reaction between chloroacetic acid, N-terminal modified groups, and cysteine residues. In some cases, bridging moieties comprise one or more triazole rings. Such triazole rings may include, but are not limited to, those formed by the cyclization reaction between (S)-2-amino-5-azidopentanoic acid and (S)-2-aminopent-4-ynoic acid. In some embodiments, bridging moieties comprise non-protein or non-polypeptide based moieties, including but not limited to cyclic rings (including but not limited to aromatic ring structures (e.g., xylyls)). Such bridging moieties may be introduced by reaction with reagents containing multiple reactive halides, including, but not limited to, poly(bromomethyl)benzenes, poly(bromomethyl)pyridines, poly(bromomethyl)alkylbenzenes, and/or (E)-1,4-dibromobut-2-ene.

[0174] In one embodiment, the antibody fragment of the invention is wholly bovine. In such an embodiment, each and every residue is derived from a bovine germline sequence. In some embodiments, each and every residue is derived from a bovine germline sequence that may have undergone affinity maturation to an antigen.

[0175] In one embodiment, the isolated antibody fragment of the invention is chimeric.

[0176] The term “chimeric” refers to an antibody fragment comprising at least two portions, one being derived from a particular source or species, such as bovine, while the other portion is derived from a different source or species, such as human. In one embodiment, the antibody fragment is human/bovine chimeric. In one embodiment, the antibody fragment comprises at least one residue derived from a human sequence.

[0177] In one embodiment, the isolated antibody fragment of the invention is synthetic. The term “synthetic” refers to an isolated antibody fragment that has been produced de novo by synthesis, notably by chemical synthesis as described herein. Antigens of Interest

[0178] Antigens of interest may be any medically relevant protein such as those proteins upregulated during disease or infection, for example receptors and/or their corresponding ligands. Particular examples of antigens include cell surface receptors such as T-cell or B-cell signaling receptors, costimulatory molecules, checkpoint inhibitors, natural killer cell receptors, immunoglobulin receptors, TNFR family receptors, B7 family receptors, adhesion molecules, integrins, cytokine/chemokine receptors, GPCRs, growth factor receptors, kinase receptors, tissue-specific antigens, cancer antigens, pathogen recognition receptors, complement receptors, hormone receptors or soluble molecules such as cytokines, chemokines, leukotrienes, growth factors, hormones or enzymes or ion channels, epitopes, fragments and post-translationally modified forms thereof.

[0179] In one embodiment, an antigen of interest bound by the isolated antibody fragment provides the ability to recruit effector functions, such as complement pathway activation and/or effector cell recruitment.

[0180] Recruitment of the effector function may be direct wherein the effector function is associated with a cell, said cell bearing a recruitment molecule on its surface. Indirect recruitment may occur where binding of an antigen to an antigen-binding domain on the antibody fragment isolated according to the present disclosure to a recruitment polypeptide causes the release of, for example, a factor which in turn may directly or indirectly recruit the effector function or may be via activation of a signaling pathway. Examples include IL2, IL6, IL8, IFNY, histamine, C1q, opsonin and other members of the classical and alternative complement activation cascades such as C2, C4, C3 convertase and C5 to C9.

[0181] As used herein, “a recruitment polypeptide” includes an FCYR such as FCYRI, FCYRII, and FCYRIII, a complement pathway protein such as, but not limited to, C1q and C3, a CD (Cluster of Differentiation marker) protein, or a fragment thereof that retains the ability to recruit cell-mediated effector function directly or indirectly. A recruitment polypeptide also includes immunoglobulin molecules such as IgG1, IgG2, IgG3, IgG4, IgE, and IgA that possess effector function.

[0182] In one embodiment an antigen-binding domain in the antibody fragment isolated according to the present disclosure has specificity for a complement pathway, with C5 being particularly preferred.

[0183] Additionally, isolated antibody fragments of the present disclosure can be used to chelate radionuclides by virtue of a single domain antibody that binds to a nuclide chelating protein. Such fusion proteins are of use in imaging or radionuclide targeting methods for therapy.

[0184] In one embodiment, an antigen-binding domain within an isolated antibody fragment according to the disclosure has specificity for a serum carrier protein, a circulating immunoglobulin molecule, or CD35/CR1, for example to provide an extended half-life for the isolated antibody fragment with specificity for said antigen of interest by binding to said serum carrier protein, circulating immunoglobulin molecule, or CD35/CR1.

[0185] As used herein, “serum carrier proteins” include thyroxine binding protein, transthyretin, α1-acid glycoprotein, transferrin, fibrinogen, and albumin or a fragment of any of them.

[0186] As used herein, a “circulating immunoglobulin molecule” includes IgG1, IgG2, IgG3, IgG4, sIgA, IgM, and IgD or a fragment of any of them.

[0187] CD35/CR1 is a protein present on red blood cells that has a half-life of 36 days (normal range 28 to 47 days; Lanaro et al., 1971, Cancer, 28(3): 658-661).

[0188] In one embodiment, the antigen of interest for which the isolated antibody fragment has specificity is a serum carrier protein, such as a human serum carrier, such as human serum albumin. Isolated antibody fragments that bind to C5

[0189] In one aspect, the present invention provides an isolated antibody fragment of the invention that binds to the C5 component of Complement.

[0190] In one embodiment, the isolated antibody fragment of the invention specifically binds to the C5 component of Complement.

[0191] The complement system is a component of the innate immune response and includes about 20 circulating complement component proteins, including C5. Activation occurs via a proteolytic cleavage pathway initiated by pathogen recognition and leading to pathogen destruction. Three such pathways are known in the complement system and are referred to as the classical pathway, the lectin pathway, and the alternative pathway. Complement component C5 is cleaved by the C5 convertase complex into C5a and C5b. C5a, much like C3a, diffuses into the circulation and promotes inflammation by acting as a chemoattractant for inflammatory cells. C5b remains attached to the cell surface where it triggers the formation of MAC through interactions with C6, C7, C8, and C9. The MAC is a hydrophilic pore that spans the membrane and promotes the free flow of fluid in and out of the cell, thereby destroying the cell.

[0192] In one embodiment, the isolated antibody fragment is the knob domain of a bovine ultra-long CDR-H3, wherein the ultra-long bovine CDR-H3 has a sequence selected from the list consisting of SEQ ID NO: 1 through SEQ ID NO: 154. In one embodiment, the isolated antibody fragment is the knob domain of a bovine ultra-long CDR-H3, wherein the ultra-long bovine CDR-H3 has a sequence that is a variant of any of SEQ ID NO: 1 through SEQ ID NO: 154 with at least 95, 96, 97, 98, or 99% similarity or identity.

[0193] In one embodiment, the isolated antibody fragment comprises or consists of a truncated variant of any one of the sequences SEQ ID NO: 1 to SEQ ID NO: 154.

[0194] In one embodiment, the isolated antibody fragment corresponds to the knob domain of a bovine ultralong CDR-H3 or portion thereof that binds to C5 and has a sequence selected from the list consisting of SEQ ID NO: 157 to SEQ ID NO: 310, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326 to SEQ ID NO: 331, SEQ ID NO: 334, SEQ ID NO: 336, SEQ ID NO: 339, SEQ ID NO: 341 to SEQ ID NO: 350, and SEQ ID NO: 352. In one embodiment, the isolated antibody fragment has the sequence SEQ ID NO: 450. In one embodiment, the isolated antibody fragment corresponds to the knob domain of a bovine ultralong CDR-H3 or portion thereof that binds to C5 and comprises a sequence of any one of SEQ ID NO: 157 through SEQ ID NO: 310, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 318, SEQ ID NO: 320, SEQ ID NO: 322, SEQ ID NO: 324, SEQ ID NO: 326 through SEQ ID NO: 331, SEQ ID NO: 334, SEQ ID NO: 336, SEQ ID NO: 339, SEQ ID NO: 341 through SEQ ID NO: 350, SEQ ID NO: 352, and SEQ ID NO: 450, or any one thereof with at least 95%, 96%, 97%, 98% or 99% similarity or identity.

[0195] In one embodiment, the isolated antibody fragment is the knob domain of a bovine ultra-long CDR-H3, wherein the ultra-long bovine CDR-H3 has a sequence of any one of SEQ ID NO: 521 through SEQ ID NO: 571. In one embodiment, the isolated antibody fragment is the knob domain of a bovine ultra-long CDR-H3, wherein the ultra-long bovine CDR-H3 has a sequence that is a variant of any one of SEQ ID NO: 521 through SEQ ID NO: 571 with at least 95, 96, 97, 98, or 99% similarity or identity.

[0196] In one embodiment, the isolated antibody fragment comprises or consists of a truncated variant of any one of the sequences SEQ ID NO: 521 to SEQ ID NO: 571.

[0197] In one embodiment, the isolated antibody fragment corresponds to the knob domain of a bovine ultra-long CDR-H3 or portion thereof that binds to C5, in particular to human C5, and comprises a sequence of any one of SEQ ID NO: 572 to SEQ ID NO: 609 or any one thereof with at least 95%, 96%, 97%, 98%, or 99% similarity or identity. In one embodiment, the isolated antibody fragment comprises or consists of a truncated variant of any one of the sequences SEQ ID NO: 572 to SEQ ID NO: 609 that binds to C5.

[0198] In one embodiment, the isolated antibody fragment corresponds to the knob domain of a bovine ultra-long CDR-H3 or portion thereof that binds to human C5 and rat C5 and comprises a sequence of any one of SEQ ID NO: 572 through SEQ ID NO: 578 or a sequence of any one of SEQ ID NO: 594 through SEQ ID NO: 599 or any one thereof with at least 95%, 96%, 97%, 98%, or 99% similarity or identity.

[0199] In one embodiment, the isolated antibody fragment of the present invention is cross-reactive between species. This represents a technical advantage in the context of the development of a therapeutic product since it allows testing in in vivo models that generate reliable and reproducible results, predictive of the situation in humans for the same molecule. In one embodiment, the antibody fragment of the present invention binds human C5 and at least one of rabbit C5, murine C5, rat C5 or cynomolgus C5. In one embodiment, the antibody fragment of the present invention binds human C5, rabbit C5 and murine C5 and optionally rat C5 and/or cynomolgus C5. In one embodiment, the antibody fragment of the present invention binds human C5, rabbit C5, murine C5, rat C5 and cynomolgus C5. Isolated antibody fragments that bind to Albumin

[0200] In one embodiment, the present disclosure provides an isolated antibody fragment of the invention that binds to human serum albumin, i.e. the antibody fragment as defined herein comprises an albumin binding domain. In one embodiment, the isolated antibody fragment specifically binds to human serum albumin. In such embodiments, the isolated antibody fragment may have an extended serum half-life. Furthermore, where the isolated antibody fragment of the invention is fused to an effector molecule, it may be useful for prolonging the serum half-life of said effector molecule.

[0201] In one embodiment, the isolated antibody fragment of the present invention binds cynomolgus serum albumin, murine serum albumin, and/or rat serum albumin.

[0202] In one embodiment, the isolated antibody fragment is the knob domain of a bovine ultra-long CDR-H3, wherein the ultra-long bovine CDR-H3 has a sequence of any one of SEQ ID NO: 497 through SEQ ID NO: 508. In one embodiment, the isolated antibody fragment is the knob domain of a bovine ultra-long CDR-H3, wherein the ultra-long bovine CDR-H3 has a sequence that is a variant of any one of SEQ ID NO: 497 through SEQ ID NO: 508 with at least 95, 96, 97, 98, or 99% similarity or identity.

[0203] In one embodiment, the isolated antibody fragment comprises or consists of a truncated variant of any one of the sequences SEQ ID NO: 497 to SEQ ID NO: 508.

[0204] In one embodiment, the isolated bovine antibody fragment of the present invention has a sequence of any one of SEQ ID NO: 509 to SEQ ID NO: 520 or any one thereof with at least 95%, 96%, 97%, 98%, or 99% similarity or identity. In one embodiment, the isolated antibody fragment comprises or consists of a truncated variant of any one of the sequences SEQ ID NO: 509 to SEQ ID NO: 520 that binds serum albumin. In one embodiment, the isolated bovine antibody fragment of the present invention specifically binds murine serum albumin and comprises or has the sequence SEQ ID NO: 509. In one embodiment, the isolated bovine antibody fragment of the present invention specifically binds human serum albumin and comprises or has the sequence SEQ ID NO: 510. In one embodiment, the isolated bovine antibody fragment of the present invention binds murine and rat albumin. In one embodiment, the isolated bovine antibody fragment of the present invention binds to murine and rat albumin and comprises or has the sequence of any one of SEQ ID NO: 511 to SEQ ID NO: 520. Isolated Antibody Fragment Fusion Proteins

[0205] In some embodiments, the isolated antibody fragment of the invention is fused to one or more effector molecules, optionally via a linker, e.g., a cleavable linker.

[0206] In the context of the present description, the terms “Fused to”, “inserted into” and “conjugated to” may be used interchangeably. Thus, antibody fragment fusion proteins encompass molecules comprising an isolated antibody fragment of the invention inserted into an exogenous protein, for example a second antibody.

[0207] Antibody fragment fusion proteins also encompass isolated antibody fragments conjugated to an effector molecule, for example by chemical conjugation.

[0208] In one embodiment, the isolated antibody fragment of the invention is genetically fused to one or more effector molecules, optionally via a linker. In one embodiment, the isolated antibody fragment of the invention is genetically fused to one or more effector molecules directly, i.e., without a linker. In another embodiment, the isolated antibody fragment of the invention is genetically fused to one or more effector molecules via a linker. In one embodiment, the isolated antibody fragment of the invention is genetically fused to one or more effector molecules directly and additionally genetically fused to one or more effector molecules via a linker.

[0209] In one embodiment, the linker is a peptide linker. The term “peptide linker” as used herein refers to a peptide comprised of amino acids. A range of suitable peptide linkers will be known to the person skilled in the art. In one embodiment, the linker is a flexible linker. In one embodiment, the linker is selected from a sequence comprised in the list consisting of SEQ ID NO: 361 to SEQ ID NO: 427. Table 1. Flexible Linker Sequences (S) is optional in sequences 361 and 365 to 369. Table 2. Articulation linking sequences

[0210] Examples of rigid linkers include the peptide sequences GAPAPAAPAPA (SEQ ID NO: 412), PPPP (SEQ ID NO: 413), and PPP.

[0211] In one embodiment the binding peptide is an albumin binding peptide.

[0212] Examples of albumin-binding peptides are provided in WO2007/106120 and include: Table 3. Albumin-binding peptides

Effector molecules

[0213] The term “effector molecule” as used herein includes, for example, biologically active proteins, e.g. enzymes, polypeptides, peptides, other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof e.g. DNA, RNA and fragments thereof, radionuclides, particularly radioiodides, radioisotopes, chelated metals, nanoparticles, and reporter groups such as fluorescent compounds or compounds that can be detected by NMR or ESR spectroscopy.

[0214] Particular radioisotopes of interest are alpha-emitting radioisotopes, in particular short-lived alpha-emitting isotopes such as Astatin isotopes. In one embodiment, the effector molecule is Astatin 211. Astatin 211 may be advantageously used for targeted alpha particle therapy (TAT) in particular in the treatment of cancer, with a potential to deliver radiation in a highly localized and toxic manner, while advantageously having a low half-life of 7.2 hours. Thus, in one aspect, the present disclosure provides an isolated antibody fragment conjugated to Astatin 211. Radiochemical methodologies using coupling agents have been described.

[0215] In one embodiment, the isolated antibody fragment comprises a chemical cage for halogen capture. The ability to chemically synthesize the isolated antibody fragment of the invention allows for the incorporation of coupling reagents within the synthesis itself, eliminating the need for drug or radioisotope conjugation to biologically produced antibodies, where controlling the substitution ratio can be difficult and can easily result in high, risky solubility and activity of the antibody and low, risky inefficient product.

[0216] An example would be the incorporation of a boron cage, such as decaborate, directly into the synthesis of an isolated antibody fragment, so that the product would be easily labeled with astatin-211 in the clinic, immediately prior to administration. This would simplify the current labeling conditions, which involve two steps, the first of which is the coupling of a bifunctional linker to a biologically produced antibody, usually using succinimide chemistry to target amines or maleimide groups to target sulfhydryl groups on the antibody, followed by labeling with the radioisotope. Astatin-211 emits alpha particles and is being tested in immunotherapy, where its high energy and short path length are attractive in targeted cell killing. Its half-life is only 7.2 hours, so simplification and shortening of the labeling procedure would be beneficial in allowing the optimal dose to be administered to patients.

[0217] Thus, in one embodiment, the isolated antibody fragment or polypeptide as defined herein is produced by chemical synthesis comprising a step of incorporating a coupling reagent with a radioisotope. In one embodiment, the radioisotope is an alpha-emitting radioisotope. In one embodiment, the radioisotope is Astatine 211.

[0218] Enzymes of interest include, but are not limited to, proteolytic enzymes, hydrolases, lyases, isomerases, transferases. Proteins, polypeptides and peptides of interest include, but are not limited to, immunoglobulins, toxins such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin, a protein such as insulin, a-interferon, b-interferon, nerve growth factor, platelet-derived growth factor or tissue plasminogen activator, a thrombotic agent or an antiangiogenic agent, for example angiostatin or endostatin, or a biological response modifier such as a lymphokine, interleukin-1 (IL-1), interleukin-2 (IL-2), granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), nerve growth factor (NGF) or other growth factor, and immunoglobulins.

[0219] Other effector molecules may include detectable substances useful for example in diagnosis. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive nuclides, positron-emitting metals (for use in positron emission tomography) and non-radioactive paramagnetic metal ions.

[0220] In another embodiment the effector molecule may increase the half-life of the isolated antibody fragment in vivo, and/or reduce the immunogenicity of the isolated antibody fragment and/or enhance the delivery of an isolated antibody fragment across an epithelial barrier to the immune system. Examples of suitable effector molecules of this type include Fc fragments, polymers, albumin, albumin-binding proteins or albumin-binding compounds such as those described in WO05/117984. In one embodiment, the effector molecule is palmitic acid. Palmitic acid has the advantageous property to bind albumin and enhance interaction with cells. In one embodiment, the effector molecule is an activated form of palmitic acid such as palmitoyl.

[0221] Where the effector molecule is a polymer it may, in general, be a synthetic or naturally occurring polymer, for example an optionally substituted straight or branched chain polyalkylene, polyalkenylene or polyoxyalkylene polymer or a branched or unbranched polysaccharide, for example a homo- or hetero-polysaccharide.

[0222] Specific optional substituents that may be present in the above synthetic polymers include one or more hydroxy, methyl, or methoxy groups.

[0223] Specific examples of synthetic polymers include optionally substituted straight or branched chain poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol) or derivatives thereof, especially optionally substituted poly(ethylene glycol) such as methoxypoly(ethylene glycol) or derivatives thereof.

[0224] Specific naturally occurring polymers include lactose, amylose, dextran, glycogen, or derivatives thereof.

[0225] “Derivatives” as used herein is intended to include reactive derivatives, for example thiol-selective reactive groups such as maleimides and the like. The reactive group may be attached directly or through a linker segment to the polymer. It will be appreciated that the residue of such a group will in some cases form part of the product as the linker group between the antibody fragment and the polymer.

[0226] The size of the polymer may be varied as desired, but will generally be in a range of average molecular weight from 500 Da to 50,000 Da, for example from 5,000 to 40,000 Da such as from 20,000 to 40,000 Da. Suitable polymers include a polyalkylene polymer such as a poly(ethylene glycol) or especially a methoxypoly(ethylene glycol) or a derivative thereof and especially having a molecular weight in the range from about 15,000 Da to about 40,000 Da.

[0227] In one embodiment, antibodies for use in the present disclosure are attached to poly(ethylene glycol) (PEG) moieties. In a particular example, the PEG molecules may be attached via any available amino acid side chain or terminal amino acid functional group located on the isolated antibody fragment, for example any free amino, imino, thiol, hydroxyl or carboxyl group. Such amino acids may occur naturally in the isolated antibody fragment or may be engineered into the fragment using recombinant DNA methods. Suitably, the PEG molecules are covalently attached via a thiol group of at least one cysteine residue located on the isolated antibody fragment.

[0228] According to the present description, the isolated antibody fragment can be modified by the addition of one or more conjugative groups.

[0229] As used herein, a “conjugate” refers to any molecule or moiety attached to another molecule. In the present invention, conjugates may be polypeptide (amino acid) based or not. Conjugates may comprise lipids, small molecules, RNA, DNA, polypeptides, polymers, or combinations thereof. Functionally, conjugates may serve as targeting molecules or may serve as payloads to be delivered to a cell, organ, or tissue. Conjugates are typically covalent modifications introduced by reacting targeted amino acid residues or the termini of the polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Such modifications are within the ordinary versatility of the art and are accomplished without undue experimentation.

[0230] The conjugation process may involve PEGylation, lipidation, albumination, biotinylation, desthiobiotinylation, the addition of other polypeptide tails, or grafting onto antibody Fc domains, intact antibody CDR regions, or antibody domains produced by any number of means. The conjugate may include anchors including a cholesterol oleate moiety, a cholesteryl laurate moiety, an α-tocopherol moiety, a phytol moiety, an oleate moiety or an unsaturated cholesterol ester moiety or a lipophilic compound selected from acetanilides, anilides, aminoquinolines, benzhydryl compounds, benzodiazepines, benzofurans, cannabinoids, cyclic polypeptides, dibenzazepines, digitalis glycosides, ergot alkaloids, flavonoids, imidazoles, quinolines, macrolides, naphthalenes, opiates (such as, but not limited to, morphines or other psychoactive drugs), oxazines, oxazoles, phenylalkylamines, piperidines, polycyclic aromatic hydrocarbons, pyrrolidines, pyrrolidinones, stilbenes, sulfonylureas, sulfones, triazoles, tropanes, and vinca alkaloids. In one embodiment, the conjugation process involves palmitoylation. Palmitoylation can be used to improve the pharmacokinetics of an isolated antibody fragment or polypeptide as described herein.

[0231] In one embodiment, the effector molecule is albumin. In one embodiment, the effector molecule is human serum albumin. In one embodiment, the effector molecule is rat serum albumin. In one embodiment, the isolated antibody fragment is fused to the N- and/or C-terminal end of albumin. In one embodiment, the isolated antibody fragment is inserted into albumin. In such an embodiment, the isolated antibody fragment is preferably inserted at a position distal to the site of interaction of albumin with FcRn. In one embodiment, the isolated antibody fragment is inserted into human serum albumin. Residues in albumin distal to the interaction with FcRn can be selected as sites for inserting the isolated antibody fragment of the invention, for example Alanine 59, Alanine 171, Alanine 364, Aspartic acid 562 into human serum albumin. In one embodiment, the isolated antibody fragment is inserted into albumin, optionally via one or more, e.g. two, linker(s). For example, the isolated antibody fragment can be inserted into albumin via two linkers, one linker at the N-terminal end of the isolated antibody fragment and the other linker at the C-terminal end of the isolated antibody fragment. A suitable linker can be a flexible linker as described herein. In one embodiment, the linker or at least one of the linkers is SGGGS.

[0232] In one embodiment, the invention provides a human serum albumin knob domain fusion protein (i.e. a fusion protein comprising an isolated antibody fragment of the invention and human serum albumin) comprising or having a sequence selected from the list consisting of SEQ ID NO: 452, SEQ ID NO: 454, SEQ ID NO: 456, SEQ ID NO: 458, SEQ ID NO: 460, SEQ ID NO: 462, SEQ ID NO: 464, and SEQ ID NO: 466.

[0233] In one embodiment, the effector molecule is an Fc fragment or any derivative thereof that can increase the half-life of the isolated antibody fragment in vivo. Examples of derivatives of Fc fragments include Fc variants, multimers of Fc fragments, Fc polypeptide such as scFc.

[0234] In one embodiment, the effector molecule is an Fc fragment. In one embodiment, the effector molecule is an Fc fragment of a human IgG1. The Fc region of the human IgG1 heavy chain is defined herein to comprise residues C226 to its carboxyl terminus, wherein the numbering is according to the EU index as in Kabat. In the context of human IgG1, the lower hinge refers to positions 226 to 236, the CH2 domain refers to positions 237 to 340, and the CH3 domain refers to positions 341 to 447 according to the EU index as in Kabat. The corresponding Fc region of other immunoglobulins can be identified by the sequence alignments.

[0235] In one embodiment, the isolated antibody fragment according to the invention is fused to an Fc fragment. In one embodiment, the isolated antibody fragment is fused to the N- and/or C-terminal end of an Fc fragment. In one embodiment, the isolated antibody fragment according to the invention is inserted into an Fc fragment. In such an embodiment, the isolated antibody fragment is preferably inserted at a position distal to the Fc interaction site with FcRn. In one embodiment, the isolated antibody fragment according to the invention is inserted into an Fc fragment of a human IgG1. Residues in the Fc, distal to the interaction with FcRn, can be selected as sites for inserting the isolated antibody fragment of the invention, for example Alanine 327, Glycine 341, Asparagine 384, Glycine 402 in the Fc fragment of the IgG1. In one embodiment, the isolated antibody fragment is inserted into an Fc fragment of a human IgG1, optionally via one or more, e.g. two, linker(s). For example, the isolated antibody fragment can be inserted into the Fc fragment of the IgG1 via two linkers, one linker at the N-terminal end of the isolated antibody fragment and the other linker at the C-terminal end of the isolated antibody fragment. A suitable linker can be a flexible linker as described herein. In one embodiment, the linker or at least one of the linkers has the sequence SEQ ID NO: 365.

[0236] In one embodiment, the invention provides a human IgG1 Fc knob domain fusion protein (i.e. a fusion protein comprising an isolated antibody fragment of the invention and human IgG1 Fc fragment) comprising or having a sequence selected from the list consisting of SEQ ID NO: 471 to SEQ ID NO: 474.

[0237] In one embodiment, the effector molecule is an antibody.

[0238] The antibody for use as an effector molecule in the context of the present description includes full antibodies as defined above and functionally active fragments thereof (i.e., molecules that contain an antigen-binding domain that specifically binds an antigen, also called antigen-binding fragments). The antibody may be (or derived from) monoclonal, multivalent, multispecific, bispecific, fully human, humanized, bovine, or chimeric.

[0239] The antibody constant region domains, if present, may be selected taking into account the proposed function of the antibody and in particular the effector functions that may be required. For example, the constant region domains may be human IgG1, IgG2 or IgG4 domains. In particular, human IgG constant region domains may be used, especially from the IgG1 isotype when the antibody molecule is intended for therapeutic uses and antibody effector functions are required. Alternatively, the IgG2 and IgG4 isotypes may be used when the antibody is intended for therapeutic purposes and antibody effector functions are not required. It will be appreciated that sequence variants of these constant region domains may also be used. For example, IgG4 molecules in which the serine at position 241 (numbered according to the Kabat numbering system) has been changed to proline as described in Angal et al. (Angal et al., 1993. A single amino acid substitution that abolishes the heterogeneity of the chimeric mouse/human antibody (IgG4) as observed during SDS-PAGE analysis Mol Immunol 30, 105-108) and herein termed IgG4P, can be used.

[0240] The human IgG1 heavy chain Fc region is defined herein to comprise residues C226 to its carboxyl terminus, wherein the numbering is according to the EU index as in Kabat. In the context of human IgG1, the lower hinge refers to positions 226 to 236, the CH2 domain refers to positions 237 to 340 and the CH3 domain refers to positions 341 to 447 according to the EU index as in Kabat. The corresponding Fc region of other immunoglobulins can be identified by sequence alignments.

[0241] In one embodiment, the effector molecule is a full-length IgG. In one embodiment, the effector molecule is a full-length IgG1. In one embodiment, the effector molecule is a full-length IgG4.

[0242] In another embodiment, the effector molecule is an antigen-binding fragment of an antibody.

[0243] Antigen-binding fragments of antibodies generally comprise at least one variable light (VL) or variable heavy (VH) domain and include: single chain antibodies (e.g. a full-length heavy chain or light chain), Fab, modified Fab, Fab’, modified Fab’, F(ab’)2, Fv, Fab-Fv, Fab-dsFv, single domain antibodies (sdAb, e.g. VH or VL or VHH), scFv, dsscFv, Bis-scFv, diabodies, trigobodies, triabodies, tetrabodies and epitope-binding fragments of any of the above (see for example Holliger and Hudson, 2005, Nature Biotech. 23(9): 1126-1136; Adair and Lawson, 2005, Drug Design Reviews - Online 2(3), 209-217). Methods for creating and manufacturing such antibody binding fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181). For example, antibody binding fragments can be obtained from any whole antibody, especially a whole monoclonal antibody, using any suitable cleavage and/or enzymatic digestion techniques, for example pepsin treatment. Alternatively, antibody starting material can be prepared by use of recombinant DNA techniques involving the manipulation and re-expression of variable and/or constant regions of antibody-encoding DNA. Standard molecular biology techniques can be used to modify, add or delete amino acids or domains as desired. Any changes to the variable or constant regions are further encompassed by the terms ‘variable’ and ‘constant’ regions as used herein. The starting material for the antibody fragment may be obtained from any species including, for example, mouse, rat, rabbit, hamster, camel, llama, goat or human. Portions of the antibody fragment may be obtained from more than one species; for example, the antibody fragments may be chimeric. In one example, the constant regions are from one species and the variable regions from another. The starting material for the antibody fragment may also be modified. In another example, the variable region of the antibody fragment has been created using recombinant DNA engineering techniques. Such engineered versions include those created for example from natural antibody variable regions by insertions, deletions or changes in or to the amino acid sequences of the natural antibodies. Particular examples of this type include those engineered region variable domains containing at least one CDR and, optionally, one or more framework amino acids from one antibody and the remainder of the variable region domain from a second antibody.

[0244] Antigen-binding fragments of antibodies include single chain antibodies (e.g. scFv and dsscfv), Fab, Fab', F(ab')2, Fv, single domain antibodies or nanobodies (e.g. VH or VL or VHH or VNAR). Other antibody fragments for use in the present invention include the Fab and Fab' fragments described in international patent applications WO2011/117648, WO2005/003169, WO2005/003170 and WO2005/003171. Methods for creating and manufacturing such antibody fragments are well known in the art (see for example Verma et al., 1998, Journal of Immunological Methods, 216, 165-181).

[0245] The term “Fab fragment” as used herein refers to an antibody fragment comprising a light chain fragment comprising a VL (variable light) domain and a constant domain of a light chain (CL) and a VH (variable heavy) domain and a first constant domain (CH1) of a heavy chain.

[0246] A typical “Fab’ fragment” comprises a heavy and a light chain pair wherein the heavy chain comprises a VH variable region, a CH1 constant domain and a natural or modified hinge region and the light chain comprises a VL variable region and a CL constant domain. Dimers of a Fab’ according to the present disclosure create an F(ab’)2 where, for example, dimerization may be via the hinge.

[0247] The term “single domain antibody” as used herein refers to an antibody fragment consisting of a single monomeric variable antibody domain. Examples of single domain antibodies include VH or VL or VHH or V-NAR.

[0248] The “Fv” refers to two variable domains, for example cooperative variable domains, such as a cognate pair or affinity-matured variable domains, i.e. a VH and VL pair.

[0249] “Single chain variable fragment” or “scFv” as used herein refers to a single chain variable fragment comprising or consisting of a heavy chain variable domain (VH) and a light chain variable domain (VL) that is stabilized by a peptide linker between the VH and VL variable domains. The VH and VL variable domains may be in any suitable orientation, for example the C-terminus of VH may be linked to the N-terminus of VL or the C-terminus of VL may be linked to the N-terminus of VH.

[0250] “Disulfide-stabilized single-chain variable fragment” or “dsscFv” as used herein refers to a single-chain variable fragment that is stabilized by a linker peptide between the VH and VL variable domain and also includes an interdomain disulfide bond between VH and VL.

[0251] In one embodiment, the effector molecule is a multispecific antibody. Multispecific antibody as used herein refers to an antibody having at least two binding domains, i.e., two or more binding domains, e.g., two or three binding domains, wherein the at least two binding domains independently bind two different antigens or two different epitopes on the same antigen. Multispecific antibodies encompass monovalent and multivalent multispecific antibodies, e.g., bivalent, trivalent, tetravalent multispecific antibodies. In one embodiment, the effector molecule is a bispecific antibody. Bispecific antibody as used herein refers to an antibody with two antigen specificities. In one embodiment, the effector molecule is a trispecific antibody. Trispecific antibody as used herein refers to an antibody with three antigen specificities.

[0252] A variety of multispecific antibody formats have been generated and can be used in the present invention, for example bispecific IgG, appended IgG, multispecific (e.g., bispecific) antibody fragments, multispecific (e.g., bispecific) fusion proteins, and multispecific (e.g., bispecific) antibody conjugates, as described for example in Spiess et al. (Spiess et al., Alternative molecular formats and therapeutic applications for bispecific antibodies. Mol Immunol. 67(2015): 95-106).

[0253] Preferred multispecific antibodies for use as an effector molecule in the present invention include appended IgG and appended Fab, wherein a full-length IgG or a Fab fragment, respectively, is engineered by the attachment of at least one additional antigen-binding domain (e.g. two, three or four additional antigen-binding domains), for example a single domain antibody (such as VH or VL or VHH), a scFv, a dsscFv, a dsFv to the N- and/or C-terminus of the heavy and/or light chain of said IgG or Fab, for example as described in WO2009/040562, WO2010035012, WO2011/030107, WO2011/061492, WO2011/061246 and WO2011/086091 all incorporated herein by reference. In particular, the Fab-Fv format was first described in WO2009/040562 and the disulfide stabilized version thereof, the Fab-dsFv, was first described in WO2010/035012. A unique Fab-dsFv linker, wherein the dsFv is connected to the Fab via a single linker between the VL or VH domain of the Fv and the C-terminus of the LC or HC of the Fab, was first described in WO2014/096390, incorporated herein by reference. An appended IgG comprising a full-length IgG1 engineered by the attachment of a dsFv to the C-terminus of the IgG heavy or light chain, was first described in WO2015/197789, incorporated herein by reference.

[0254] Another preferred antibody for use as an effector molecule in the present invention comprises a Fab linked to two scFvs or dsscFvs, each scFv or dsscFv binding the same or a different target (e.g., a scFv or dsscFv binding a therapeutic target and a scFv or dsscFv that increases half-life by binding, for example, albumin). Such antibody fragments are described in International Patent Application Publication No. WO2015/197772, which is hereby incorporated by reference in its entirety. Another preferred antibody for use as an effector molecule in the fragment of the present invention comprises a Fab linked only to a scFv or dsscFv, as described for example in WO2013/068571 incorporated herein by reference and Dave et al., Mabs, 8(7) 1319-1335 (2016).

[0255] In one embodiment, the effector molecule is selected from the list consisting of a Fab, a single domain antibody (a VHH or a VH or a VL), a scFv, and a dsscFv.

[0256] In one embodiment, the effector molecule is a VHH, i.e. the invention provides a fusion protein between a VHH and an isolated antibody fragment of the invention. An isolated antibody fragment of the invention can be inserted into the loops of the frame of a VHH antibody, at the end opposite the CDRs, to make a bispecific single chain antibody. In one embodiment, the VHH is the hC3nb1 VHH, which binds C3 and C3b. In one embodiment, the VHH comprises or has the sequence SEQ ID NO: 351. In one embodiment, a hC3nb1 VHH-knob fusion protein comprises or has a sequence selected from the list consisting of SEQ ID NO: 353 to SEQ ID NO: 357. In another embodiment, the invention provides an ultra-long hC3nb1 VHH-CDR-H3 fusion protein comprising or having a sequence SEQ ID NO: 359 or SEQ ID NO: 360.

[0257] In another embodiment, the effector molecule is a Fab, i.e. the invention provides a fusion protein between a Fab and an isolated antibody fragment of the invention. In one embodiment, the isolated antibody fragment is inserted into the CDR-H3 of a Fab. In one embodiment, the Fab comprises a heavy chain having the sequence SEQ ID NO: 311, which is paired with the light chain of SEQ ID NO: 325. In one embodiment, the Fab-knob domain fusion protein has a sequence selected from the list consisting of SEQ ID NO: 312, SEQ ID NO: 314, SEQ ID NO: 316, SEQ ID NO: 319, SEQ ID NO: 321, and SEQ ID NO: 323.

[0258] In one embodiment, the antibody, i.e. the effector molecule comprises an albumin binding domain.

[0259] In one embodiment, the effector molecule is albumin or a protein comprising an albumin-binding domain.

[0260] “Albumin binding domain” as used herein refers to a portion of a protein that specifically interacts with serum albumin. In particular in the context of antibodies used as an effector molecule, it refers to a portion of the antibody, comprising a part or all of one or more variable domains, for example a pair of VH and VL variable domains, that specifically interact with albumin. An albumin binding domain may comprise a single domain antibody. As such, an albumin binding domain according to the present disclosure may refer to a VH, VL or VH/VL pair that binds to albumin.

[0261] In one embodiment, the antibody comprising an albumin binding domain comprises a light and/or heavy chain sequence; and/or a light and/or heavy chain variable domain sequence; and/or at least one of CDR-L1, CDR-L2, and CDR-L3 sequences; and/or at least one of CDR-H1, CDR-H2, and CDR-H3 selected from below (CDRs in bold):

[0262] Fab light chain CA645 (gL5):

[0263] DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLI YEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTK VEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 428)

[0264] VL domain (gL5):

[0265] DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLI YEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTK VEIK (SEQ ID NO: 429)

[0266] CDR-L1: QSSPSVWSNFLS (SEQ ID NO: 430)

[0267] CDR-L2: EASKLTS (SEQ ID NO: 431)

[0268] CDR-L3: GGGYSSISDTT (SEQ ID NO: 432)

[0269] Fab CA645 heavy chain (gH5):

0270 QTYICNVNHKPSNTKVDKKV EPKSC (SEQ ID NO: 433)

[0271] VH domain (gH5):

[0272] EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIG IIWASGTTFYATWAKGRFTISRDNSKNTVILQMNSLRAEDTAVYYCARTVPGYSTAP YFDLWGQGTLVTVSS (SEQ ID NO: 434)

[0273] CDR-H1: GIDLSNYAIN (SEQ ID NO: 435)

[0274] CDR-H2: IIWASGTTFYATWAKG (SEQ ID NO: 436)

[0275] CDR-H3: TVPGYSTAPYFDL (SEQ ID NO: 437)

[0276] Additional VH and VL sequences useful in the context of the present disclosure are listed below:

[0277] CA645 VH domain (gH1):

[0278] EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIG IIWASGTTFYATWAKGRFTISRDSTTVILQMNSLRAEDTAVYYCARTVPGYSTAPYF DLWGQGTLVTVSS (SEQ ID NO: 438)

[0279] CA645 VH domain (gH37):

[0280] EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIG IIWASGTTA

[0281] YATWAKGRFTI SRDNSKNTV ILQMNSLRAEDTAVYYCARTVPGYSTAPYFDLWGQ

[0282] GTLVTVSS (SEQ ID NO: 439)

[0283] CA645 VH domain (gH47):

[0284] EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKGLEWIG IIWASGTTFYATWAKGRFTISRDNSKNTVILQMNSLRAEDAVYYCARTVPGYSAAPY FDLWGQGTLVTVSS (SEQ ID NO: 440)

[0285] CA645 VL Domain (gL1):

[0286] DIVMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLI YEASKLTS GVPSRFKGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGGGTKVEIK (SEQ ID

[0287] NO: 441)

[0288] VL Domain CA645 (gL4):

[0289] DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLI YEASKLTS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC GGGYSSISD TTFGGGTKVEIK

[0290] (SEQ ID NO: 442)

[0291] In some embodiments, the albumin binding domain comprises variants of the VL and VH domains that bind human serum albumin as described above (SEQ ID NO: 429, SEQ ID NO: 434, and SEQ ID NO: 442 respectively) that comprise an additional cysteine residue such that a disulfide bond can be formed between the VL and VH domains. The additional cysteine-containing variants can have the following sequences (wherein the additional cysteine residues are underlined):

[0292] CA645-Cys (gL5) VL domain:

[0293] DIQMTQSPSSVSASVGDRVTITCQSSPSVWSNFLSWYQQKPGKAPKLLI YEASKLTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCGGGYSSISDTTFGCGTK VEIK (SEQ ID NO: 443)

[0294] CA645-Cys VH domain (gH5):

[0295] EVQLLESGGGLVQPGGSLRLSCAVSGIDLSNYAINWVRQAPGKCLEWIG IIWASGTTFYATWAKGRFTISRDNSKNTVILQMNSLRAEDTAVYYCARTVPGYSTAP YFDLWGQGTLVTVSS (SEQ ID NO: 444)

[0296] VL CA645-Cys (gL4):

[0297] diqmtqspssvsasvgdrvtitcqsspsvwsnflswyqqkpgkapklliyeaskltsgvpsrfsgsgsg tdftltisslqpedfatyycgggyssisdttfgCgtkveikRT (SEQ ID NO: 445)

[0298] In some embodiments, the VH framework of the albumin binding domain is human (e.g. VH3, such as VH3 1-3 3-23) and comprises for example 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as amino acids that are donor residues. In such embodiments, the VH can have a sequence set forth in SEQ ID NO: 434, SEQ ID NO: 438, SEQ ID NO: 439, SEQ ID NO: 440, SEQ ID NO: 444, or a variant of any of them with at least 95, 96, 97, 98, or 99% identity similarity.

[0299] In some embodiments, the VL framework of the albumin binding domain is human (e.g. VK1, such as 2-1-(1)L5) and comprises for example 1, 2, 3, 4, 5, or 6 amino acid substitutions, such as amino acids that are donor residues. In such embodiments, the VL can have a sequence set forth in SEQ ID NO: 429, SEQ ID NO: 441, SEQ ID NO: 442, SEQ ID NO: 443, SEQ ID NO: 445, or a variant of any of them with at least 95, 96, 97, 98, or 99% identity similarity.

[0300] In some embodiments, the albumin binding domain comprises VH and VL sequences selected from the combinations SEQ ID NO: 434 and SEQ ID NO: 429 or SEQ ID NO: 444 and SEQ ID NO: 443 or a variant or variants of any of them with at least 95, 96, 97, 98, or 99% similarity or identity.

[0301] In some embodiments, the VH and VL sequences of the albumin binding domain are SEQ ID NO: 434 and SEQ ID NO: 429, respectively. In some embodiments, the VH and VL sequences of the albumin binding domain are SEQ ID NO: 444 and SEQ ID NO: 443, respectively.

[0302] In one embodiment, the albumin binding domain comprises SEQ ID NO: 435 for CDR-H1, SEQ ID NO: 436 for CDR-H2, SEQ ID NO: 437 for CDR-H3, SEQ ID NO: 430 for CDR-L1, SEQ ID NO: 431 for CDR-L2, and SEQ ID NO: 432 for CDR-L3; or a heavy chain variable domain selected from SEQ ID NO: 434 and SEQ ID NO: 444 and a light chain variable domain selected from SEQ ID NO: 429 and SEQ ID NO: 443.

[0303] In one embodiment, the effector molecule is a Fab that binds human serum albumin, i.e. the Fab comprises an albumin-binding domain. Thus, in one aspect, the invention provides a Fab that binds serum albumin, wherein an isolated antibody fragment according to the invention is inserted into its frame, for example the frame 3 (FW3) region of the V domain, notably the VH domain as described in WO2020/011868 (published January 16, 2020). As explained, in addition to three CDR loops, antibody light and heavy chains, both conventional and single-chain camelid VHH, have a fourth loop that is formed by frame 3. The Kabat numbering system defines frame 3 as positions 66 to 94 in a heavy chain and positions 57 to 88 in a light chain.

[0304] Thus, in one aspect, the invention also provides a bispecific antibody format, in particular stable and capable of simultaneously binding two antigens. Advantageously, an isolated CA645 Fab-antibody fragment fusion protein (which may also be called a CA645 Fab-knob fusion protein) as described herein can simultaneously bind C5 and albumin, which may confer an increased serum half-life to the isolated antibody fragment.

[0305] In one embodiment, the invention provides an isolated antibody fragment of the invention, inserted into FW3 of the VH of a 645 Fab. In one embodiment, the 645Fab comprises a heavy chain having the sequence SEQ ID NO: 334, which is paired with a light chain of SEQ ID NO: 329. In one embodiment, the invention provides a CA645 Fab-knob fusion protein comprising a sequence selected from the list consisting of SEQ ID NO: 332, SEQ ID NO: 333, SEQ ID NO: 335, SEQ ID NO: 337, SEQ ID NO: 338, and SEQ ID NO: 340.

[0306] Polypeptides comprising isolated antibody fragments

[0307] In one aspect, the present disclosure provides a polypeptide comprising at least one antibody fragment isolated according to the invention.

[0308] In one aspect, the present disclosure provides a polypeptide comprising at least two isolated antibody fragments according to the invention, wherein the isolated antibody fragments are linked together, optionally via a linker, e.g., a cleavable linker.

[0309] In one embodiment, the at least two isolated antibody fragments bind to the same antigen including binding to the same epitope on the antigen or binding to different epitopes on the antigen.

[0310] In another embodiment, the at least two isolated antibody fragments bind to different antigens.

[0311] The polypeptide can be monospecific, multispecific, multivalent, bispecific.

[0312] “Monospecific polypeptide” as used herein refers to a polypeptide comprising at least two isolated antibody fragments of the present disclosure, wherein the polypeptide binds to only one antigen of interest.

[0313] “Multispecific polypeptide” as used herein refers to a polypeptide comprising at least two isolated antibody fragments of the present disclosure, wherein the polypeptide comprises at least two antigen-binding domains, i.e., two or more antigen-binding domains, e.g., two or three antigen-binding domains, wherein the at least two antigen-binding domains independently bind two different antigens or two different epitopes on the same antigen. Multispecific polypeptides may be monovalent for each specificity (antigen). The multispecific polypeptides described herein encompass both monovalent and multivalent multispecific polypeptides, e.g., bivalent, trivalent, tetravalent, as well as multispecific polypeptides having different valencies for different epitopes (e.g., a multispecific polypeptide that is monovalent for a first antigen specificity and bivalent for a second antigen specificity that is different from the first).

[0314] In one embodiment, the polypeptide is monospecific and bivalent. In another embodiment, the polypeptide is bispecific.

[0315] “Bispecific polypeptide” as used herein refers to a polypeptide with two antigen specificities. In one embodiment, the polypeptide comprises two antigen-binding domains wherein one binding domain binds ANTIGEN 1 and the other binding domain binds ANTIGEN 2, i.e., each binding domain is monovalent for each antigen. In one embodiment, the antibody is a tetravalent bispecific polypeptide, i.e., the polypeptide comprises four antigen-binding domains, wherein, for example, two binding domains bind ANTIGEN 1 and the other two binding domains bind ANTIGEN 2. In one embodiment, the polypeptide is a trivalent bispecific polypeptide.

[0316] It will be appreciated that a polypeptide of the invention comprising at least two isolated antibody fragments may be produced, for example, synthetically or recombinantly and may comprise bovine or chimeric or synthetic isolated antibody fragments or a combination thereof. For example, a polypeptide according to the invention may comprise two isolated antibody fragments, both being synthetic or one being synthetic and the other being bovine. In one embodiment, the polypeptide according to the invention comprises only synthetic isolated antibody fragments.

[0317] In one aspect, the polypeptide comprising at least two isolated antibody fragments is cyclized. In some embodiments, the polypeptide comprising at least two isolated antibody fragments comprises at least one bridged moiety between two amino acids.

[0318] When the polypeptide is cyclic and has no terminal amino acids, it may be referred to as a macrocycle.

[0319] The definitions of the bridging moiety described above and connection to the cyclized antibody fragments also apply to cyclized polypeptides of the present disclosure.

[0320] In particular, in one embodiment, the bridging moiety comprises a trait selected from the group consisting of a disulfide bond, an amide (lactam) bond, a thioether bond, an aromatic ring, an unsaturated aliphatic hydrocarbon chain, a saturated aliphatic hydrocarbon chain, and a triazole ring. Production Methods

[0321] The isolated antibody fragment or polypeptide of the invention may be produced by any suitable method, such as recombinant expression and/or chemical synthesis.

[0322] In one aspect, the present disclosure also provides methods of producing an isolated antibody fragment of the invention or a polypeptide of the invention, said method comprising a chemical synthesis step.

[0323] Chemical synthesis approaches have been described, such as solid phase polypeptide synthesis (see, for example, Coin, I et al. (2007); Nature Protocols 2(12):3247-56).

[0324] In one embodiment, the isolated antibody fragment of the invention is produced by solid phase polypeptide synthesis.

[0325] In one embodiment, the isolated antibody fragment of the invention is produced using standard solid phase Fmoc/tBu methods. Such methods are, for example, described in Atherton and Sheppard 1989, Fluorenylmethoxycarbonylpolyamide solid phase peptide synthesis: general principles and development. In Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Eynsham, Oxford, pp,25-37.); and Merrifield R.B. “Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide”. J. Am. Chem. Soc. 85 (14): 2149 2154 (1963).

[0326] The synthesis is typically carried out in a sequential manner in the C to N direction on robotic synthesizers. The synthesis can be initiated on appropriate polystyrene supports with the first amino acid attached via a bond to the support. An example synthesis protocol is described in the Example section of the present description. It will be appreciated by the skilled person that other protocols can be used, e.g. using different reagents, protecting groups, other experimental conditions and the skilled person will be able to adapt the protocol depending on the nature of the desired peptide and synthetic strategy.

[0327] The chemical synthesis of the isolated antibody fragments of the invention advantageously comprises the formation of disulfide bonds between two cysteine residues, which lead to the cyclization of the isolated antibody fragments. Cyclic peptides can be produced by the formation of a disulfide bond between two cysteine residues or by cyclization from the head to the tail or side chain, forming an amide bond. Using special groups, it is possible to cyclize between two specific cysteines in a peptide, in this way it is possible to have more than one disulfide in a peptide. Different methods are available and comprise a site-directed method as described in the Example section of the present description. The choice of protecting groups to be used in an orthogonal protection strategy may vary. Cyclization between two cysteine residues can alternatively be achieved by using thermodynamically controlled air oxidation to obtain the lowest energy form of the disulfides in the sequence, using a mixture of reduced and oxidized glutathione, for example, as described in the Examples.

[0328] In one aspect, a boron cage, such as decaborate, is directly incorporated into the isolated antibody fragment during chemical synthesis. The isolated antibody fragment can thus be readily labeled, just prior to administration, with a radioisotope, for example, with astatine-211. Therefore, in one aspect the invention provides a method for producing an isolated antibody fragment or a polypeptide as defined in the present disclosure, said method comprising a chemical synthesis step and wherein the chemical synthesis comprises a step of incorporating a coupling reagent with a radioisotope. In one embodiment, the radioisotope is an alpha-emitting radioisotope. In one embodiment, the radioisotope is Astatine-211.

[0329] The present disclosure also provides a polynucleotide encoding the isolated antibody fragment or polypeptide of the present invention. The polynucleotide (i.e., DNA sequence) of the present invention may comprise synthetic DNA, e.g., produced by chemical processing, cDNA, genomic DNA, or any combination thereof.

[0330] It will be appreciated that in the context of a polypeptide comprising at least two isolated antibody fragments of the invention, the DNA may be synthetic and include in a single DNA sequence the sequence encoding as to the at least two isolated antibody fragments. Alternatively, the polypeptide comprising at least two isolated antibody fragments of the invention may use two separate non-synthetic or synthetic DNA sequences, each encoding one of the at least two isolated antibody fragments, which will then be conjugated or ligated together after expression.

[0331] DNA sequences encoding an isolated antibody fragment of the present invention can be obtained by methods well known to those skilled in the art.

[0332] The present invention also relates to a cloning or expression vector comprising one or more polynucleotides or DNA sequences of the present invention. Accordingly, there is provided a cloning or expression vector comprising one or more polynucleotides encoding an isolated antibody fragment or polypeptide of the present invention. In the context of a polypeptide comprising at least two isolated antibody fragments of the invention, the cloning or expression vector comprises at least two polynucleotides, encoding the at least two isolated antibody fragments of the present invention, respectively, and suitable signal sequences.

[0333] General methods by which vectors can be constructed, transfection methods, and culture methods are well known to those skilled in the art.

[0334] Also provided is a host cell comprising one or more cloning or expression vectors comprising one or more polynucleotides encoding an isolated antibody fragment of the present invention or one or more vectors comprising the same. Any suitable host cell/vector system may be used for the expression of CDR-H3 polynucleotide sequences encoding the isolated antibody fragment or polypeptide according to the present disclosure. Bacterial, e.g., E. coli and other microbes may be used or eukaryotic, e.g., mammalian, host cell expression systems may also be used. Suitable mammalian host cells include CHO, myeloma, or hybridoma cells. Suitable types of Chinese Hamster Ovary (CHO cells) for use in the present invention may include CHO and CHO-K1 cells including dhfr-CHO cells, such as CHO-DG44 cells and CHO-DXB11 cells, which may be used with a DHFR selectable marker, or CHOK1-SV cells which may be used with a glutamine synthetase selectable marker. Other cell types for use in antibody expression include lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells, COS cells.

[0335] In one aspect, there is provided a process for producing an isolated antibody fragment or polypeptide of the invention, said process comprising expressing an isolated antibody fragment or polypeptide of the invention from a host cell as defined in the present disclosure.

[0336] In one aspect, a method is provided for producing an isolated antibody fragment or polypeptide as described herein, said method comprising: a) immunizing a bovine with an immunogenic composition, and; b) isolating antigen-specific memory B cells, and; c) sequencing the CDR-H3 cDNA or portions thereof, and; d) expressing or synthesizing the ultra-long CDR-H3 knob domain or portion thereof,

[0337] wherein the immunogenic composition comprises an antigen of interest or immunogenic portions thereof or DNA encoding the same. Step a)

[0338] An “immunogenic composition” refers to a composition that is capable of generating an immune response in a bovine administered with said composition. An immunogenic composition typically allows for the expression of an immunogenic antigen of interest in the administered bovine, against those bovine antibodies that may be raised as part of the immune response.

[0339] “Protein immunization” refers to the technique of administering an immunogenic protein comprising an antigen of interest or immunogenic portion of said protein, comprising said antigen of interest or immunogenic portion thereof.

[0340] In one embodiment, the immunogenic composition comprises a full-length protein. In another embodiment, the immunogenic composition comprises an immunogenic portion of a protein.

[0341] “DNA immunization” refers to the technique of direct administration into bovine cells of a genetically engineered nucleic acid molecule encoding a full-length protein or an immunogenic portion thereof comprising an antigen of interest (also referred to as nucleic acid vaccine or DNA vaccine herein) to produce an immune response in said cells against said antigen of interest. DNA immunization uses the host cell machinery to express peptide(s) corresponding to the administered nucleic acid molecule and/or achieve the expected effect, in particular, antigen expression at the cellular level and, in addition, immunotherapeutic effect(s) at the cellular level or within the host organism.

[0342] “Cellular immunization” refers to the technique of administering cells naturally expressing or transfected with an immunogenic protein comprising an antigen of interest or immunogenic portion of said protein, comprising said antigen of interest or immunogenic portion thereof. In one embodiment, the immunization in step a) is performed using cell immunization with fibroblasts transfected with an immunogenic protein comprising an antigen of interest or immunogenic portion of said protein, comprising said antigen of interest or immunogenic portion thereof.

[0343] By “Immunogenic portion” is meant a portion of the protein or antigen of interest that retains the ability to induce a response in the bovine animal administered with said portion of the protein or antigen of interest or DNA encoding the same, in order to allow the production of antibody fragments of the invention as disclosed herein.

[0344] In one embodiment, immunization step a) may be performed using protein immunization, DNA immunization, or cell immunization, or any combination thereof.

[0345] The immunization step a) may be performed using a prime-boost immunization protocol which entails a first administration (the prime immunization or initial administration) of the immunogenic composition and then at least one additional administration (the booster immunization or booster administration) which is separated in time from the first administration within the course of the immunization protocol. Booster immunizations comprise one, two, three or more administrations.

[0346] In one embodiment, immunization step a) is performed using a primer-boost immunization protocol comprising an initial immunization with an antigen of interest in the presence of a first adjuvant, followed by at least one booster immunization with said antigen of interest in the presence of a second adjuvant.

[0347] In one embodiment, the immunogenic composition is administered by subcutaneous injection, e.g., into the shoulder. In one embodiment, the antigen of interest is the Complement component C5.

[0348] “Adjuvant” refers to an immune stimulant. Adjuvants are substances well known in the art. Traditional adjuvants, which act as immune stimulants or antigen delivery systems or both, include, for example, Alum, polysaccharides, liposomes, nanoparticles based on biodegradable polymers, lipopolysaccharides. For example, the adjuvant may be a Freund's adjuvant, a Montanide adjuvant or a Fama adjuvant. Step b)

[0349] Methods for isolating antigen-specific memory B cells are well known and generally comprise isolating B cells from PBMC (Peripheral Blood Mononuclear Cells) or from secondary lymphoid organs, i.e., from the lymphoid node or the spleen. In one embodiment, the isolation of antigen-specific memory B cells is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after the immunization step a). In one embodiment, isolation of antigen-specific memory B cells is performed 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours after immunization step a). In one embodiment, step b) comprises sorting the antigen-specific B cells by flow cytometry. Step c)

[0350] Step c) generally comprises a first step of obtaining cDNA from the memory B cells obtained in step b), using methods well known in the art, for example, methods comprising an RT-PCR performed on memory B cell lysate. Methods for sequencing cDNA are well known in the art. Step c) comprises sequencing the CDR-H3 cDNA or portions thereof to identify ultra-long CDR-H3. Analysis of the sequences in step c) allows identification of ultra-long CDR-H3 in comparison to standard CDR-H3 as disclosed herein based on the size of the CDR-H3 sequence and/or using alternative methods such as sequence alignments with well-known nucleic or amino acid sequences and/or ultra-long CDR-H3 standard. The knob domain can then be defined as described in the present description and its sequences isolated.

[0351] For example, a method for amplifying CDR-H3 cDNA as described in the Examples may be used. The method may comprise a first round of RT-PCR performed on the lysate of memory B cell isolates in step b). The method may comprise a primary polymerase chain reaction (PCR) with primers flanking CDR-H3, annealing to the conserved VH 3 frame and 4 frame, to amplify all CDR-H3 sequences, regardless of their length or amino acid sequence. The method may further comprise a second round of PCR to barcode the CDR-H3 sequences for ion stream sequencing, as described in the Examples.

[0352] In one embodiment, the method for sequencing CDR-H3 cDNA or portions thereof comprises: 1) a primary PCR with primers flanking CDR-H3, annealing with the conserved VH 3 frame and 4 frame, to amplify all CDR-H3 sequences, and 2) a second round of PCR to barcode the CDR-H3 sequences for ion stream sequencing.

[0353] In one embodiment, the primers used in step 1) comprise or consist of SEQ ID NO: 446 and SEQ ID NO: 447. In one embodiment, the primers used in step 2) comprise or consist of SEQ ID NO: 448 and SEQ ID NO: 449). Step d)

[0354] Step d) may be carried out according to well-known methods for expressing polypeptides, notably using cloning, expression vectors and host cells as described above.

[0355] Step d) may alternatively comprise the chemical synthesis of the ultra-long CDR-H3 knob domain or portion thereof, which may be carried out according to well-known methods including as described above.

[0356] In one embodiment, the method for producing an isolated antibody fragment of the invention further comprises a screening step, e.g., for binding to an antigen of interest.

[0357] Optionally, the screening step is preceded by a step of reforming an ultra-long CDR-H3 or the knob domain of the ultra-long CDR-H3 or portion thereof into a screening format.

[0358] In one embodiment, the step of reforming the ultra-long CDR-H3 or the ultra-long CDR-H3 knob domain or portion thereof in a screening format comprises fusing the ultra-long CDR-H3 or the ultra-long CDR-H3 knob domain or portion thereof, to a carrier, optionally via a linker, e.g., a cleavable linker.

[0359] It will be appreciated that the screening step may be performed before or after step d). For example, the isolated antibody fragments of the invention (i.e. the ultra-long CDR-H3 knob domain or portion thereof) may be expressed in a host cell according to step d), then recovered and evaluated in vitro for binding to the antigen of interest, optionally following a step of reforming the ultra-long CDR-H3 knob domain or portion thereof in a screening format as described in the present description.

[0360] Alternatively, the knob domain of the ultra-long CDR-H3 may be expressed or synthesized as part of the entire ultra-long CDR-H3 after step c) and assessed for binding to the antigen of interest prior to step d) optionally after a step of reforming the ultra-long CDR-H3 in a screening format as described herein. In such an alternative, the knob domains or portions thereof comprised in an ultra-long CDR-H3 that have been found to specifically bind to the antigen of interest may be expressed or synthesized in step d).

[0361] In one embodiment, the carrier is an Fc polypeptide. An “Fc polypeptide” as used herein is a polypeptide comprising an Fc fragment. In one embodiment, the Fc polypeptide is a scFc. “Single-chain Fc polypeptide” or “scFc” as used herein refers to a single-chain polypeptide comprising two CH2 domains and two CH3 domains distinguished wherein said CH2 and CH3 domains form a functional Fc domain within the chain. The functional Fc domain in the single-chain polypeptides of the present invention is not formed by dimerization of two chains, i.e., the two CH2 domains and two CH3 domains are present in a single chain and form a functional Fc domain within the single chain. The term 'functional' as used herein refers to the ability of the Fc domain formed within the single-chain polypeptide to provide one or more actuator functions usually associated with Fc domains although it is envisaged that other functions may be engineered into such domains.

[0362] In one embodiment, the carrier is a scFc and comprises the sequence SEQ ID NO: 155. In one embodiment, the carrier is a scFc and the fusion protein comprises a linker, wherein the linker comprises a TEV protease cleavage site and a Gly-Ser linker. In one embodiment, the carrier is a scFc and the fusion protein comprises the sequence SEQ ID NO: 156.

[0363] Additional scFc sequences and variants useful in the context of the present disclosure have been described in WO2008/012543.

[0364] In one aspect, the invention provides a method for producing an isolated antibody fragment as described in the present disclosure, said method comprising: a) immunizing a bovine with an immunogenic composition, and; b) isolating total RNA from PBMC or secondary lymphoid organ, and; c) amplifying the cDNA of the ultra-long CDR-H3, and; d) sequencing an ultra-long CDR-H3 or portion thereof, and, e) expressing or synthesizing the knob domain of the ultra-long CDR-H3 or portion thereof,

[0365] wherein the immunogenic composition comprises an antigen of interest or immunogenic portions thereof or DNA encoding the same.

[0366] Step a) is as described above.

[0367] Step b) Methods for isolating total RNA from PBMC or secondary lymphoid organ are well known in the art.

[0368] It will be appreciated that step c) generally comprises a first step of obtaining cDNA from the total RNA obtained in step b) using RT-PCR. Advantageously, in step c) a method for directly amplifying the cDNA of the ultra-long CDR-H3 and discriminated from standard CDR-H3 can be used. The method may comprise a primary polymerase chain reaction (PCR) with primers flanking CDR-H3, annealing with the conserved VH frame 3 and frame 4, to amplify all CDR-H3 sequences, regardless of their length or amino acid sequence. The method may additionally comprise a second round of PCR with stalked primers to specifically amplify the ultra-long sequences of the primary PCR. This method is advantageous when it allows to directly clone an ultra-long CDR-H3 sequence of interest into expression vectors.

[0369] In one embodiment, the method for amplifying CDR-H3 cDNA comprises: a) a primary PCR using primers flanking CDR-H3, annealing with the conserved VH frame 3 and frame 4, to amplify all CDR-H3 sequences and b) a second round of PCR using stalked primers to specifically amplify ultra-long sequences from the primary PCR.

[0370] In one embodiment, the primers used in step 1) comprise or consist of SEQ ID NO: 446 and SEQ ID NO: 447. In one embodiment, the primers used in step 2) are selected from the group consisting of SEQ ID NO: 482 through SEQ ID NO: 494. It will be appreciated that the primers used in step 2) comprise an upstream primer and a downstream primer, i.e., the primers may comprise an upstream primer of any one of SEQ ID NO: 482 through SEQ ID NO: 488 and a downstream primer of any one of SEQ ID NO: 489 through SEQ ID NO: 494.

[0371] Step d) comprises sequencing the CDR-H3 cDNA or portion thereof in order to identify the ultra-long CDR-H3 knob domain peptide or portions thereof. Step d) may be performed according to methods well known in the art, such as direct nucleotide sequencing.

[0372] Step e) is as described above.

[0373] In one embodiment, the method for producing an isolated antibody fragment of the invention further comprises a step of screening, e.g., for binding to an antigen of interest. Optionally, the screening step is preceded by a step of reforming the ultra-long CDR-H3 or the knob domain of the ultra-long CDR-H3 or portion thereof in a screening format as described herein. The screening step may be performed before or after step e). For example, isolated antibody fragments of the invention (i.e., the knob domain of the ultra-long CDR-H3 or portion thereof) may be expressed in a host cell according to step e), then recovered and evaluated in vitro for binding to the antigen of interest, optionally following a step of reforming the knob domain of the ultra-long CDR-H3 or portion thereof in a screening format as described herein.

[0374] Alternatively, the knob domain of the ultra-long CDR-H3 may be expressed or synthesized as part of the entire ultra-long CDR-H3 after step d) and assessed for binding to the antigen of interest prior to step e) optionally after a step of reforming the ultra-long CDR-H3 in a screening format as described herein. In such an alternative, the knob domains or portions thereof comprised in the ultra-long CDR-H3 that have been found to specifically bind to the antigen of interest may be expressed or synthesized in step e).

[0375] In another aspect, the invention provides a method for producing an isolated antibody fragment as described in the present disclosure, said method comprising: a) immunizing a bovine with an immunogenic composition, and; b) isolating antigen-specific memory B cells, and; c) amplifying the ultra-long CDR-H3 cDNA, and; d) sequencing the ultra-long CDR-H3; and, e) expressing or synthesizing the knob domain of the ultra-long CDR-H3 or portion thereof,

[0376] wherein the immunogenic composition comprises an antigen of interest immunogenic portions thereof or DNA encoding the same.

[0377] Steps a) to e) are as described above.

[0378] In one embodiment, the method for producing an isolated antibody fragment as described in the present disclosure further comprises a screening step, e.g. for binding to an antigen of interest, which is as described above and notably may be performed before or after step e). Antibody fragment libraries

[0379] In one aspect, the disclosure provides immune libraries and methods for generating immune libraries, comprising a plurality of isolated antibody fragments of the invention, notably ultra-long bovine CDR-H3 knob domains or portions thereof, or a plurality of DNA or RNA sequences encoding the same.

[0380] Advantageously, the invention provides novel methods of discovering antibody fragments and therapeutic polypeptides derived therefrom, comprising immunizing cattle with an antigen of interest as described above. It may be possible to generate extensive immune libraries of isolated bovine antibody fragments, notably ultra-long bovine CDR-H3 knob domains and portions thereof, and to evaluate and select for those knob domains having a desired effect, e.g., for their binding and/or binding affinity to an antigen of interest, in particular by display technologies.

[0381] Antibody fragment immune repertoires are generated by using genetic information encoding bovine antibody fragments of the present disclosure, which may be derived from B cells isolated from cattle administered an antigen of interest. Immune libraries may be screened using bovine antibody fragment display technologies, e.g., using in vitro display technologies (such as phage display, bacterial display, yeast display, ribosome display, mRNA display). Mammalian cell display may be advantageous for displaying disulfide-rich proteins, such as ultralong bovine CDR-H3 or fragment thereof, e.g., as described in Crook, Z. R. et al. Publisher Correction: Mammalian display screening of diverse cystine-dense peptides for difficult to drug targets. Nat Commun 9, 1072, (2018).

[0382] In one embodiment, the invention provides libraries of isolated antibody fragments of the invention expressed on the surface of mammalian cell fusion proteins, such as Fc polypeptide fusion proteins. In one embodiment, the invention provides libraries of ultra-long bovine CDR-H3 knob domains.

[0383] In one embodiment, the invention provides immune libraries or naive libraries of isolated antibody fragments of the invention, prepared from animals that have not been administered immunogen. In one embodiment, the invention provides phage display libraries of isolated antibody fragments of the invention. In such an embodiment, the isolated antibody fragments of the invention may be expressed directly on the surface of phage using any suitable method.

[0384] In one aspect, the invention provides libraries of ultra-long CDR-H3 sequences, i.e., libraries of isolated antibody fragments of the invention, when expressed as part of the complete CDR-H3 sequence (i.e., comprising the knob and stalk domains). In one embodiment, the libraries are naive libraries. In one embodiment, the naive libraries are prepared from bovine. In another embodiment, the libraries are immune libraries. In one embodiment, the libraries are prepared from immunized cattle. In a specific aspect, the disclosure provides a phage display library of isolated antibody fragments of the invention, optionally displayed within the complete CDR-H3 sequences. In one embodiment, the phage display library is an M13 phage display library. In one embodiment, isolated antibody fragments of the invention, optionally displayed within the complete CDR-H3 sequences, are fused directly to the pIII coat protein of phage M13. In one embodiment, isolated antibody fragments of the invention, optionally displayed within the complete CDR-H3 sequences, are fused to the pIII coat protein of phage M13 via a linker (or “spacer”). A suitable linker may be a linker that allows to separate the cysteine-rich domain from the cysteines of pIII, notably to ensure that pIII and the knob domain peptide fold independently and correctly. Methods for producing a phage display library are well known. Phagemid vectors, for example, have been described in Hoogenboom HR et al. (Hoogenboom HR, Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 1991;19(15):4133-4137). In one embodiment, the phage display library of isolated antibody fragments of the invention comprises CDR-H3 of sequence SEQ ID NO: 477 and/or SEQ ID NO: 104 and/or SEQ ID NO: 13 and/or SEQ ID NO: 1.

[0385] In one aspect, the disclosure provides a phage display library comprising a plurality of recombinant phages; each of the plurality of recombinant phages comprising an expression vector comprising M13, wherein the expression vector comprising M13 comprises a polynucleotide sequence encoding an isolated antibody fragment as disclosed herein, optionally displayed within the complete ultra-long CDR-H3 sequence. In one embodiment, the isolated antibody fragment, optionally displayed within the complete ultra-long CDR-H3 sequence, is fused to the sequence encoding the pIII coat protein of the M13 phage, directly or via a spacer.

[0386] In one aspect, the disclosure provides methods for generating phage display libraries of ultra-long CDR-H3 sequences, i.e., libraries of isolated antibody fragments of the invention, displayed within the complete CDR-H3 sequence. For example, a method as described in Example 12 may be used, wherein the complete ultra-long CDR-H3 sequence is fused directly to the pIII coat protein of phage M13.

[0387] In one aspect, a method is provided for generating a phage display immune library of ultra-long CDR-H3 sequences, said method comprising: a) immunizing a bovine with an immunogenic composition, and; b) isolating total RNA from PBMC or secondary lymphoid organ, and; c) amplifying the ultra-long CDR-H3 sequences, and; d) fusing the sequences obtained in c) to the sequence coding for the pIII protein of an M13 phage within a phagemid vector, and; e) transforming host bacteria with the phagemid vector obtained in step d) in combination with a helper phage co-infection, and; f) culturing the bacteria obtained in step e), and; g) recovering the phages from the culture medium of the bacteria,

[0388] wherein the immunogenic composition comprises an antigen of interest or immunogenic portions thereof or DNA encoding the same.

[0389] Steps a) to g) are methods well known in the art. For example, steps a) to g) may be performed as described in Example 12. In particular, for step c), a method for amplifying CDR-H3 cDNA as described in Example 12 may be used. The method may comprise a primary PCR with primers flanking CDR-H3, annealing with the conserved VH frame 3 and frame 4, to amplify all CDR-H3 sequences, regardless of their length or amino acid sequence. The method may further comprise a second round of PCR with stalked primers to specifically amplify ultra-long sequences from the primary PCR.

[0390] In one embodiment, the method for amplifying the CDR-H3 cDNA comprises: 1) a primary PCR using primers flanking CDR-H3, annealing with the conserved VH frame 3 and frame 4, to amplify all CDR-H3 sequences, and 2) a second round of PCR using stalked primers to specifically amplify ultra-long sequences from the primary PCR.

[0391] In one embodiment, the primers used in step 1) comprise or consist of SEQ ID NO: 446 and SEQ ID NO: 447). In one embodiment, the primers used in step 2) are selected from the group consisting of SEQ ID NO: 482 through SEQ ID NO: 494. It will be appreciated that the primers used in step 2) comprise an upstream primer and a downstream primer, i.e., the primers may comprise an upstream primer of any one of SEQ ID NO: 482 through SEQ ID NO: 488 and a downstream primer of any one of SEQ ID NO: 489 through SEQ ID NO: 494.

[0392] In one aspect, the invention provides a method for producing an isolated antibody fragment of the invention that binds to an antigen of interest, said method comprising: a) generating an ultra-long CDR-H3 phage display library; and, b) enriching the phage display library against the antigen of interest to produce an enriched population of phage that binds the antigen of interest; and, c) sequencing an ultra-long CDR-H3 from the enriched phage population obtained in step b); and, d) expressing or synthesizing an isolated antibody fragment (i.e. the knob domain of the ultra-long CDR-H3 or portion thereof) derived from the ultra-long CDR-H3 obtained in step c).

[0393] Steps a) to d) are methods well known in the art. For example, steps a) to d) may be performed as described in Example 12. For example, enrichment of the phage display library against the antigen of interest in step b) may be performed by panning the library obtained in step a) against the antigen of interest. The enriched sublibraries may be further evaluated by monoclonal phage screening ELISA as described in the Examples.

[0394] In step c), the sequence of the ultra-long CDR-H3 sequences can be amplified using PCR using appropriate primers, e.g., sequencing primers by annealing to the phagemid vector. In one embodiment, the primers used comprise or consist of SEQ ID NO: 495 and/or SEQ ID NO: 496.

[0395] In another aspect, the invention provides a method for producing an isolated antibody fragment of the invention that binds to an antigen of interest, said method comprising: a) generating a phage display library of isolated antibody fragments of the invention; and, b) enriching the phage display library against the antigen of interest to produce an enriched population of phage that binds the antigen of interest; and, c) sequencing an isolated antibody fragment from the enriched phage population obtained in step b); and, d) expressing or synthesizing an isolated antibody fragment (i.e. the knob domain of the ultra-long CDR-H3 or portion thereof) obtained in step c).

[0396] Steps a) may be performed according to methods as disclosed in this description.

[0397] Steps b) to d) are as disclosed above.

[0398] Advantageously, the description provides a way to

[0399] bypass cell sorting and deep sequencing for bovine antibody fragment discovery, as libraries of CDR-H3 sequences can be cloned and evaluated for binding to an antigen or panel of antigens using in vitro display technologies.

[0400] A library will generally contain at least 102 members, more preferably at least 106 members, and most preferably at least 10' members (e.g., any of the mRNA-polypeptide complexes). In some embodiments, the library will include at least 1012 members or at least 1014 members. In general, the members will differ from each other; however, it is expected that some degree of redundancy will exist in any library.

[0401] In another aspect, the disclosure provides synthetic libraries and methods for generating synthetic libraries, comprising a plurality of isolated antibody fragments of the invention or a plurality of DNA or RNA sequences encoding the same.

[0402] Synthetic libraries may comprise isolated antibody fragments expressed on the surface of cells. Synthetic libraries may be screened using display technologies, for example, using in vitro display technologies (such as phage display, bacterial display, yeast display, ribosome display, mRNA display). Mammalian cell display may also be used.

[0403] In one aspect, the disclosure provides synthetic libraries and methods for generating synthetic libraries, comprising isolated antibody fragments of the invention fused to (or inserted into) a suitable framework, preferably a protein framework. A suitable protein framework may be another antibody fragment, for example, an antigen-binding fragment of an antibody, such as a VHH, a VH, a VL, a Fab, a scFv, and a dsscFv, or any other suitable framework. For example, isolated antibody fragments of the invention may be inserted into a VH or a VL domain, more particularly into a 3' frame region of a VH or VL, for example, as described in WO2020/011868 incorporated herein by reference.

[0404] Synthetic libraries can be screened using display technologies, for example, using in vitro display technologies (such as phage display, bacterial display, yeast display, ribosome display, mRNA display) in which each isolated antibody fragment of the invention is expressed as part of a fusion protein with a suitable protein structure, such as a binding to the antigen fragment.

[0405] In one embodiment, the fusion protein consists of an isolated antibody fragment of the invention fused to a suitable protein structure, such as an antigen-binding fragment, optionally via one or more, e.g., two, linker(s). In another embodiment, the fusion protein comprises an isolated antibody fragment of the invention, optionally displayed within the full CDR-H3 sequences or portion thereof, fused to a suitable protein structure, such as an antigen-binding fragment, optionally via one or more, e.g., two, linker(s).

[0406] In one aspect, the disclosure provides a synthetic phage display library of isolated antibody fragments of the invention, wherein each of the isolated antibody fragments is displayed as part of a fusion protein with a binding antigen fragment of an antibody. In one embodiment, the fusion protein comprises an isolated antibody fragment of the invention optionally displayed within the full CDR-H3 sequence or portion thereof. In one embodiment, the binding antigen fragment of an antibody is a VHH. Therefore, the invention provides a phage display library comprising isolated antibody fragments of the invention, optionally displayed within the full CDR-H3 sequences or portion thereof, expressed on the surface of phage, as VHH fusion proteins. In one embodiment, each isolated antibody fragment of the invention, optionally displayed within the full CDR-H3 sequence or portion thereof, is inserted into a VHH, e.g., into the non-binding VH frame 3, optionally via one or more linkers. A suitable linker can enhance the independent and correct folding of the isolated antibody fragment or full CDR-H3 sequence and the VHH. In one embodiment, the VHH phage display library is a VHH M13 phage display library. In one embodiment, the VHH fusion proteins are fused directly to the pIII coat protein of phage M13. In one embodiment, the VHH fusion proteins are fused to the pIII coat protein of phage M13 via a linker. In one embodiment, the VHH comprises or has the sequence SEQ ID NO: 351. In one embodiment, the phage display library of isolated antibody fragments of the invention comprises VHH fusion proteins of the sequences SEQ ID NO: 476 and/or SEQ ID NO: 478 and/or SEQ ID NO: 479 and/or SEQ ID NO: 480.

[0407] Methods for producing a phage display library are well known. The method described in the present disclosure for producing VHH-Phage libraries in which CDR-H3 are inserted into VHH may be used as an example. Pharmaceutical compositions and medical uses

[0408] In one aspect, the invention provides a pharmaceutical composition comprising an isolated antibody fragment or polypeptide as defined in the present description, in combination with one or more pharmaceutically acceptable excipients.

[0409] The term “pharmaceutically acceptable excipient” as used herein refers to a pharmaceutically acceptable formulation carrier, solution, or additive to enhance the desired characteristics of the compositions of the present disclosure. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Solutions or suspensions may be encapsulated in biodegradable liposomes or microspheres. The formulation will generally be provided in a substantially sterile form utilizing sterile manufacturing processes. This may include production and sterilization by filtration of the buffered solvent solution used for the formulation, aseptic suspension of the isolated antibody fragment in the sterile buffered solvent solution, and dispensing of the formulation into sterile receptacles by methods familiar to those of ordinary skill in the art.

[0410] The pharmaceutically acceptable carrier must not, by itself, induce the production of antibodies harmful to the individual receiving the composition and must not be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles.

[0411] Pharmaceutically acceptable salts may be used, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates and sulfates or organic acid salts such as acetates, propionates, malonates and benzoates.

[0412] Pharmaceutically acceptable carriers in therapeutic compositions may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances such as wetting or emulsifying agents or pH buffering substances may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, pastes and suspensions, for ingestion by the patient.

[0413] The isolated antibody fragments or polypeptides of the disclosure may be released dispersed in a solvent, for example in the form of a solution or a suspension. These may be suspended in an appropriate physiological solution, for example physiological saline, a pharmacologically acceptable solvent or buffered solution.

[0414] A thorough discussion of pharmaceutically acceptable carriers is available in Remington's Pharmaceutical Sciences (Mack Publishing Company, N.J. 1991).

[0415] The pharmaceutical compositions suitably comprise a therapeutically effective amount of the isolated antibody fragments or polypeptides of the invention. The term “therapeutically effective amount” as used herein refers to an amount of a therapeutic agent required to ameliorate or prevent a targeted disease or condition or to exhibit a detectable therapeutic or preventative effect. For any antibody fragment, the therapeutically effective amount can be estimated initially in cell culture assays or in animal models, usually in rodents, rabbits, dogs, pigs or primates. The animal model can also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. The precise therapeutically effective amount for a human subject will depend on the severity of the disease state, the general health of the subject, the age, weight and gender of the subject, diet, timing, and frequency of administration, drug combination(s), reaction sensitivities and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgment of the clinician.

[0416] The compositions may be administered individually to a patient or may be administered in combination (e.g., simultaneously, sequentially, or separately) with other agents, drugs, or hormones. Agents as used herein refer to an entity that when administered has a physiological effect. Drug as used herein refers to a chemical entity that at a therapeutic dose has an appropriate physiological effect.

[0417] The dose at which the isolated antibody fragment or polypeptide of the present disclosure is administered depends on the nature of the condition being treated, the extent of inflammation present, and whether the isolated antibody fragment is being used prophylactically or to treat an existing condition.

[0418] The frequency of dosing will depend on the half-life of the isolated antibody fragment or polypeptide and the duration of its effect. If the isolated antibody fragment or polypeptide has a short half-life (e.g., 2 to 10 hours) it may be necessary to give one or more doses per day. Alternatively, if the isolated antibody fragment or polypeptide has a long half-life (e.g., 2 to 15 days) it may only be necessary to give a single dose per day, once a week, or even every 1 to 2 months.

[0419] The pharmaceutical compositions of this invention may be administered by any number of routes, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, transdermal, transcutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, intravaginal, or rectal routes. Hyposprays may also be used to administer the pharmaceutical compositions of the invention.

[0420] Suitable forms for administration include forms suitable for parenteral administration, for example by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and this may contain formulatory agents, such as suspending agents, preservatives, stabilizers and/or dispersing agents. Alternatively, the isolated antibody fragment may be in dry form, for reconstitution before use with a suitable sterile liquid.

[0421] Direct delivery of the compositions will generally be accomplished by injection, subcutaneously, intraperitoneally, intravenously, or intramuscularly, or delivered to the interstitial space of a tissue. The compositions may also be administered to a specific tissue of interest. The dosing regimen may be a single dose schedule or a multiple dose schedule.

[0422] In one embodiment, the formulation is provided as a formulation for topical administrations including inhalation.

[0423] Suitable inhalable preparations include inhalable powders, metered aerosols containing propellant gases or inhalable solutions free of propellant gases (such as nebulizable solutions or suspensions). The inhalable powders according to the description containing the active substance may consist of the above-mentioned active substances or of a mixture of the above-mentioned active substances with a physiologically acceptable excipient. The propellant gases which can be used to prepare the inhalable aerosols are known in the art. Suitable propellant gases are selected from hydrocarbons, such as n-propane, n-butane or isobutane and halohydrocarbons, such as chlorinated and/or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The above-mentioned propellant gases may be used by themselves or in mixtures thereof.

[0424] The inhalable aerosols containing propellant gas may also contain other ingredients, such as co-solvents, stabilizers, surface active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All these ingredients are known in the art. The inhalable aerosols containing propellant gas according to the invention may contain up to 5% by weight of active substance. The aerosols according to the invention contain, for example, 0.002 to 5% by weight, 0.01 to 3% by weight, 0.015 to 2% by weight, 0.1 to 2% by weight, 0.5 to 2% by weight or 0.5 to 1% by weight of active.

[0425] Alternatively, topical administrations to the lung may also be by administration of a liquid solution or suspension formulation, for example, using a device such as a nebulizer, for example, a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus(R) nebulizer connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc., Richmond, Va.).

[0426] In one embodiment, the formulation is provided as separate ampoules containing a single dose for delivery by nebulization.

[0427] In one embodiment the isolated antibody fragment or polypeptide is provided in lyophilized form, for reconstitutions or, alternatively, as a suspension formulation.

[0428] The isolated antibody fragment or polypeptide of the present disclosure may be released dispersed in a solvent, for example, in the form of a solution or a suspension. This may be suspended in an appropriate physiological solution, for example, physiological saline, a pharmacologically acceptable solvent or a buffered solution. Buffered solutions known in the art may contain from 0.05 mg to 0.15 mg of disodium edetate, 8.0 mg to 9.0 mg of NaCl, 0.15 mg to 0.25 mg of polysorbate, 0.25 mg to 0.30 mg of anhydrous citric acid and 0.45 mg to 0.55 mg of sodium citrate per 1 ml of water in order to achieve a pH of about 4.0 to 5.0. As mentioned above, a suspension may be made, for example, from lyophilized isolated antibody fragment or polypeptide.

[0429] The nebulizable formulation according to the present description may be provided, for example, as single dose units (e.g., sealed plastic containers and vials) packaged in foil envelopes. Each vial contains a unit dose in a volume, for example, 2 ml, of solvent/buffer solution.

[0430] The isolated antibody fragments or polypeptides of the present disclosure are thought to be suitable for delivery via nebulization.

[0431] The present invention also provides a process for preparing a pharmaceutical or diagnostic composition comprising adding and mixing the isolated antibody fragment or polypeptide of the present invention together with one or more pharmaceutically acceptable excipients, diluents or carriers.

[0432] The present invention also provides methods and compositions for the delivery of the isolated antibody fragments as described herein by gene therapy, particularly by adeno-associated virus (AAV) vector.

[0433] Accordingly, the present invention provides a pharmaceutical composition comprising a viral vector having a viral capsid and an artificial genome comprising an expression cassette flanked by inverted terminal repeats (ITRs) wherein the expression cassette comprises a transgene comprising a polynucleotide sequence encoding the isolated antibodies as described herein. The ITR sequences can be used to package the artificial genome comprising the polynucleotide sequences encoding the isolated antibody fragment or polypeptide as described herein into the virion of the viral vector.

[0434] The transgene in the expression cassette is operably linked to expression control elements, such as promoters, that will control expression of the transgene in human cells.

[0435] The viral vector is preferably AAV-based viral vectors. A variety of AAV capsids have been described in the art. Methods of generating AAV vectors have also been described extensively in the literature (e.g., WO 2003/042397; WO 2005/033321, WO 2006/110689; U.S. Patent No. 7,588,772 B2). The source of AAV capsids can be selected from an AAV that targets a desired tissue. For example, suitable AVV may include, for example, AAV9 (U.S. Patent No. 7,906,111; US 2011-0236353-A1), rh10 (WO 2003/042397), and/or hu37 (US 7,906,111B2; US20110236353). However, other AAV, including, for example, AAV1, AAV2, AAV-TT, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV.PHP.B (or variants thereof), and others may also be selected.

[0436] Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art (US7,790,449B2; US7,282,199B2; WO 2003/042397; WO 2005/033321, WO 2006/110689; and US7,588,772 B2).

[0437] In one aspect, the invention provides an isolated antibody fragment or polypeptide as defined in the present description, for use in therapy.

[0438] The isolated antibody fragments and polypeptides of the invention are useful in the treatment of diseases or disorders including inflammatory diseases or disorders, immune diseases and disorders, complement related diseases and disorders, autoimmune diseases, vascular indications, neurological diseases and disorders, kidney related indication, eye diseases.

[0439] In some embodiments, the isolated antibody fragments, polypeptides, and pharmaceutical compositions thereof according to the present invention may be useful in the treatment of diseases, disorders, and/or conditions in which cleavage of C5 leads to progression of the disease, disorder, and/or condition. Such diseases, disorders, and/or conditions may include, but are not limited to, immune and autoimmune, neurological, cardiovascular, pulmonary, and ocular diseases, disorders, and/or conditions.

[0440] Immune and autoimmune diseases and/or disorders may include, but are not limited to, Acute Disseminated Encephalomyelitis (ADEM), Acute Necrotizing Hemorrhagic Leukoencephalitis, Addison's Disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing Spondylitis, Acute Antibody-Mediated Rejection Following Organ Transplantation, Anti-GBM/Anti-TBM Nephritis, Antiphospholipid Syndrome (APS), Autoimmune Angioedema, Autoimmune Aplastic Anemia, Autoimmune Dysautonomia, Autoimmune Hepatitis, Autoimmune Hyperlipidemia, Autoimmune Immunodeficiency, Autoimmune Inner Ear Disease (AIED), Autoimmune Myocarditis, Autoimmune Pancreatitis, Autoimmune Retinopathy, Autoimmune Thrombocytopenic Purpura (ATP), Autoimmune Thyroid Disease, Autoimmune Urticaria, Axonal Neuropathies and neuronal, Bacterial sepsis and septic shock, Balo's disease, Behcet's disease, Bullous pemphigus, Cardiomyopathy, Castleman's disease, Celiac disease, Chagas disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigus/benign mucosic pemphigus, Crohn's disease, Cogans syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Type I diabetes, Discoid lupus, Dressler syndrome, Endometriosis, Esophagitis eosinophilic, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Glomerulonephritis, Goodpasture syndrome, Granulomatosis with polyangiitis (GPA) see Wegener's, Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia (including atypical hemolytic uremic syndrome and atypical hemolytic uremic syndrome resistant to plasma therapy), Henoch-Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Diabetes insulin-dependent (type 1), Interstitial cystitis, Juvenile arthritis, Juvenile diabetes, Kawasaki syndrome, Lambert-Eaton syndrome, Large vessel vasculopathy, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Conjunctivitis lignea, Linear IgA disease (LAD), Lupus (SLE), Lyme disease, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple endocrine neoplasia syndromes, Multiple sclerosis, Multifocal motor neuropathy, Myositis, Myasthenia gravis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial pemphigus, Optic neuritis, Osteoarthritis, Palindromic rheumatism, PANDAS (Pain-dependent motor disorders) Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perennial encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Autoimmune polyglandular syndromes types I, II, and III, Polyendocrinopathies, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Red cell aplasia pure, Raynauds phenomenon, Reactive arthritis, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Shiga toxin producing Hemolytic-Uremic Syndrome due to Escherichia Coli Hemolytic-Uremic Syndrome (STEC-HUS), Sjogren's syndrome, Small vessel vasculopathy, Sperm and testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac syndrome, Sympathetic ophthalmia, Takayasu arteritis, Temporal arteritis/Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Tubular autoimmune disorder, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Uveitis, Vesiculobullous dermatosis, Vasculitis, Vitiligo and Wegener's granulomatosis (also known as Granulomatosis with Polyangiitis (GPA)).

[0441] Neurological diseases, disorders and/or conditions may include, but are not limited to, Alzheimer's disease, Parkinson's disease, Lewy body dementia and Multiple Sclerosis.

[0442] Cardiovascular diseases, disorders and/or conditions may include, but are not limited to, atherosclerosis, myocardial infarction, stroke, vasculitis, trauma (surgery), and conditions arising from cardiovascular intervention (including, but not limited to, cardiac bypass surgery, arterial grafting, and angioplasty).

[0443] Lung diseases, disorders and/or conditions may include, but are not limited to, asthma, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD) and adult respiratory distress syndrome.

[0444] Ocular related applications include, but are not limited to: corneal transplantation, corneal ulcers, cytomegalovirus retinitis, dry eye syndrome, endophthalmitis, Fuch's disease, Glaucoma, immune complex vasculitis, inflammatory conjunctivitis, ischemic retinal disease, keratitis, macular edema, ocular parasitic infestation/migration, retinitis pigmentosa, scleritis, Stargardt's disease, subretinal fibrosis, uveitis, vitreoretinal inflammation, and Vogt-Koyanagi-Harada disease.

[0445] In one embodiment, the invention provides an isolated antibody fragment or polypeptide or pharmaceutical composition as defined herein for use in the prevention and/or treatment of a pathological disease, disorder or condition selected from the group consisting of infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infection, arthritis, such as rheumatoid arthritis, asthma, such as severe asthma, chronic obstructive pulmonary disease (COPD), pelvic inflammatory disease, Alzheimer's disease, inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, Celiac disease, Gallbladder disease, Pilonidal disease, Peritonitis, Psoriasis, Vasculitis, Surgical adhesions, Stroke, Type I diabetes, Lyme disease, meningoencephalitis, autoimmune uveitis, immune-mediated inflammatory disorders of the central and peripheral nervous system, such as multiple sclerosis, lupus (such as systemic lupus erythematosus) and Guillain-Barr syndrome, Atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's disease, IgA nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune disorders, pancreatitis, trauma (surgery), graft-versus-host disease, transplant rejection, heart disease including ischemic diseases such as myocardial infarction as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis, and hypochlordiasis.

[0446] Isolated antibody fragments to enable small molecule drug discovery

[0447] In another aspect, the present invention relates to an improved method for identifying compounds of therapeutic interest using target antibody-protein interactions to help present and/or maintain the protein in a conformation that exposes or presents a binding site that has the potential to modify the function of the protein and that may be occluded in the "natural" conformation. Such a method has been described in WO2014/001557 published January 3, 2014 and Lawson, A. Nat Rev Drug Discovery 11,519-525, (2012).

[0448] In this way, the isolated antibody fragments of the present description can be used as a tool to facilitate chemical drug discovery.

[0449] Thus, in one aspect there is provided a method of identifying compounds capable of binding to a functional conformational state of a protein of interest or protein fragment thereof, said method comprising the steps of: (a) Binding an isolated function-modifying antibody fragment of the invention to the target protein of interest or a fragment thereof to provide an antibody-bound protein or protein fragment, wherein the isolated antibody fragment has binding kinetics with the protein or fragment that are such that it has a low dissociation rate constant, (b) Providing a test compound having a low molecular weight, (c) Evaluating whether the test compound from step b) binds the antibody-bound protein or fragment, and (d) Selecting a compound from step c) based on the ability to bind to the protein or fragment thereof.

[0450] In one embodiment, the method comprises an additional step of assessing the binding of an analogue of a compound selected in step d) to bind to an antibody-restricted protein or fragment prepared in step a).

[0451] In one embodiment, the method further comprises the step of performing synthetic chemical methods to modify or elaborate a first test compound selected in step d).

[0452] In one embodiment, the method comprises an additional step of generating three-dimensional structural information, e.g., using X-ray crystallography between step c) and step d) or following step d) to gain structural information on the binding of a test compound.

[0453] In one embodiment, the antibody-bound protein or fragment is used to generate three-dimensional structural information, e.g., using X-ray crystallography in the presence of a bound compound identified in step c), and optionally comprises the additional step of performing computational modeling based on the three-dimensional structural information obtained therefrom. 3D structural representation

[0454] In one embodiment a three-dimensional structural representation of at least one such isolated antibody fragment in complex with the target protein is subsequently generated in order to obtain information about where the isolated antibody fragment is binding to the target protein, the functional conformational state of the target protein and which amino acid residues and hence atoms in the target protein and the isolated antibody fragment are in contact with each other or interact with each other. This may be performed prior to step a) or step b) if desired.

[0455] The functional conformational state revealed by structural analysis may be previously known or unknown. In one example, the functional conformational state revealed by structural analysis of an antibody target protein is novel. In one example, structural analysis of the antibody target protein reveals previously occluded structural features that are not available in the unconstrained protein. In one example, these previously occluded features may be suitable targets for small molecule binding.

[0456] In some embodiments, the invention provides the use of an isolated antibody fragment of the invention to identify functional binding sites for small molecules on proteins and/or to define biologically relevant conformations; conformations in which the proteins are incompetent in binding or signaling, that stabilize the complex or that induce signaling.

[0457] Any suitable method known in the art can be used to generate the three-dimensional structural representation of an antibody:target protein complex. Examples of such methods include X-ray crystallography, NMR (Nuclear Magnetic Resonance) spectroscopy, and hydrogen deuterium mass spectroscopy in solution. Preferably, X-ray crystallography is used. As set forth hereinabove, the target protein can be the mature protein or a suitable fragment or derivative thereof. Chemical Compound Screening

[0458] In the method of the present invention, candidate compounds, compound fragments or isolated antibody fragments may each be tested for their effect on the biological activity of the target protein. For example, isolated antibody fragments and identified compounds that bind to the target protein may be introduced via standard screening formats into biological assays to determine the inhibitory or stimulatory activity of the isolated compounds or antibody fragment, or alternatively or in addition, binding assays to determine binding or blocking, such as ELISA, BIAcore, protein X-ray crystallography and NMR-based screening may be appropriate, alternatively or in addition, the ability of the isolated antibody fragment or compound to induce structural changes may be identified using, for example, FRET-based assays as described in WO2014/001557.

[0459] Thus, in some embodiments, the invention provides for the use of an isolated antibody fragment of the invention to screen in vitro for novel chemical material at specific functional sites on a protein by techniques such as Forster resonance energy transfer/fluorescence resonance energy transfer (FRET).

[0460] In the method of the present invention, the ability of test compound fragments to bind the isolated antibody fragment-bound protein is determined. In one example, the compound selected in step (d) of the method does not bind the unbound target protein. In one example, the compound selected in step (d) of the method does not bind the isolated antibody fragment in the absence of target protein. In one example, the compound selected in step (d) of the method does not bind the unbound target protein or the isolated antibody fragment only. In one example of the method of the present invention, step c) further comprises assessing whether the test compound of step b) binds the protein or fragment in the absence of isolated antibody fragment and step (d) further comprises selecting a compound of step c) based on the ability of the test compound to bind only the isolated antibody fragment-bound protein or fragment and not the unbound protein or fragment.

[0461] Typically, at later screening stages following further elaboration of the compound fragments identified by the method and once the potency of the chemical compound has reached a level appropriate for binding the target protein is sufficient to allow the compound to bind the target protein in the absence of the isolated antibody fragment.

[0462] “Comprising” in the context of the present description is intended to mean including. Where technically appropriate, embodiments of the invention may be combined. Embodiments are described herein as comprising certain features/elements. The description also extends to separate embodiments consisting of or consisting essentially of said features/elements.

[0463] Technical references, such as patents and applications, are incorporated herein by reference.

[0464] Any of the modalities specifically and explicitly set forth herein may form the basis of a waiver alone or in combination with one or more additional modalities.

[0465] The present description is further described by way of illustration only in the following examples, referring to the attached Figures in which: Brief description of the figures

[0466] Figure 1: Single-cycle SPR kinetics of PGT121 Fab-knob domain fusion proteins binding to C5. Vertical axis: RU (Refractive Index Unit); horizontal axis: time in seconds.

[0467] Figure 2: Chromatogram showing the purification of the K57 knob domain peptide from the 645 Fab and TEV protease proteins by hydrophobic interaction chromatography. Vertical axis: AU

[0468] (Arbitrary Unit); Horizontal axis; has in minutes.

[0469] Figure 3: SPR single-cycle kinetics of knob domain binding to proteolytically cleaved C5 from 645 Fab. Vertical axis: RU (Refractory Index Unit); horizontal axis: time in seconds.

[0470] Figure 4: Single-cycle kinetic SPR sensorgrams showing the knob domain binding peptides K8 and K92 to mouse and rabbit C5. Vertical axis: RU (Refractory Index Unit); horizontal axis: time in seconds.

[0471] Figure 5: Complement Activation ELISAs. Vertical axis: Complement Activation inhibition in %; horizontal axis: knob domain peptide concentration in nM. C5b n/e: C5b neoepitope. Fig. 5A: Classical Pathway K8 inhibition; Fig. 5B: Alternative Pathway K8 inhibition; Fig. 5C: Classical Pathway K57 inhibition; Fig. 5D: Alternative Pathway K57 inhibition; Fig. 5E: Classical Pathway K92 inhibition; Fig. 5F: Alternative Pathway K92 inhibition; Fig. 5G: Classical Pathway K149 inhibition; Fig. 5H: Alternative Pathway K149 inhibition.

[0472] Figure 6: Example curves from the Alternative and Classical Pathway bacterial killing assays. Vertical axis: Escherichia Coli survival in %; horizontal axis: micromolar concentration of knob domain peptide. Fig 6A: K8, K57 and K92 evaluated in the Classical Pathway bacterial killing assay; Fig. 6B: K57, K92 and K8 evaluated in the Alternative Pathway bacterial killing assay.

[0473] Figure 7: Binding of synthetic knob domain peptides to C5 by single-cycle kinetics. Vertical axis: RU (Refractory Index Unit); horizontal axis: time in seconds.

[0474] Figure 8: Vertical axis: Inhibition of complement activation in %; horizontal axis:

[0475] knob domain peptide concentration in nM. Fig. 8A: Example curves for chemically derived knob domain peptides in the Classical Pathway C5b neoepitope ELISA. Fig. 8B: Example curves for chemically derived knob domain peptides in the Alternative Pathway C5b neoepitope ELISA.

[0476] Figure 9: Crystal structure of the K8 peptide in complex with C5. The K8 peptide is shown as a mesh surface.

[0477] Figure 10: The K8 peptide interacts with the MG8 domain of C5. The MG8 domain of C5 is shown in isolation with the K8 peptide. Certain important K8 residues that participate in H-bonding or salt bridge interactions are shown.

[0478] Figure 11: The K8 peptide interacts with the MG8 domain of C5. The MG8 domain of C5 is shown in isolation with the K8 peptide. Certain important C5 residues that participate in H-bonding or salt bridge interactions are shown.

[0479] Figure 12: Binding of disulfide arrangements for the K8 peptide.

[0480] Figure 13: Vertical axis: inhibition of complement activation in %; horizontal axis:

[0481] knob domain peptide concentration in nM. Fig. 13A: Example curves for hC3nb1 - K57 constructs in the Alternative Pathway C5b neoepitope ELISA. Fig. 13B:

[0482] Example curves for hC3nb1-K57 in the Classical Pathway C5b ELISA neoepitope.

[0483] Figure 14: Traits and sequence numbering system of bovine CDR-H3 ultra-long sequence: sequence alignment of BLV1H12 with germline-encoded VHBUL, DH2, JH1 segments. Cysteine residues are in bold, the conserved cysteine at position 92 Kabat in the VHBUL segment (H92), the conserved Tryptophan at position 103 Kabat in the JH1 segment (H103) and the conserved cysteine at the beginning of DH2, are in bold and in a rectangle.

[0484] Figure 15: Two different views of the crystal structure of the Human serum albumin, neonatal Fc receptor (FcRn), human IgG1 Fc ternary complex (single chain only) (PDB accession code: 4N0F). The residues of human serum albumin, distal to the interface with FcRn, that were selected as insertion sites for the knob domain peptides K57 and K92 are highlighted (Alanine 59, Alanine 171, Alanine 364, Aspartic acid 562).

[0485] Figure 16: Crystal structure of human serum albumin, neonatal Fc receptor (FcRn), human IgG1 Fc (single chain, CH2 and CH3 domains only) ternary complex (PDB accession code: 4N0F). Residues in the Fc, distal to the interaction with FcRn, were selected as sites for engineering the knob domain peptide K149 (Alanine 327, Glycine 341, Asparagine 384, Glycine 402).

[0486] Figure 17: Phage display of ultralong CDR-H3 as estimated by ELISA. Horizontal axis: Optical density OD 630 nm; Vertical axis; Biotinylated C3, biotinylated C5, C5, C3, anti-myc used to estimate binding. Constructs used were hC3nb1-K149 CDR-H3, hC3nb1-K92 CDR-H3, hC3nb1-K57 CDR-H3, hC3nb1-K8 CDR-H3, hC3nb1 (without any insert).

[0487] Figure 18: Complement Inhibition ELISA data for CP- and AP-driven assays for K8chemFE, K57chem FE, and K92chemFE. Vertical axis: Inhibition of complement activation in %; horizontal axis: Log concentration of knob domain peptide in nM. Fig. 18A: CP ELISA. Fig. 18B: AP ELISA.

[0488] Figure 19: Hemolysis assays specific for alternative (AP) or classical (CP) pathway activation for K8chemFE, K8chemFEcyclic, K57chem FE and K92chemFE, RA101295-14 and His-SOBI002. Vertical axis: Hemolysis inhibition (in %); horizontal axis: Log concentration of knob domain peptide in nM. Fig. 19A: CP hemolysis assay. Fig. 19B: AP hemolysis assay.

[0489] Figure 20: Plasma stability. Vertical axis: knob domain concentration in plasma (in ng/mL); horizontal axis: time (hours). Fig. 20A: K57chemFE. Fig. 20B: K57chemFE-Palmitoyl. Fig. 20C: K8chemFE.

[0490] Figure 21: In vivo pharmacokinetics following intravenous dosing to Sprague Dawley rats for K8chemFE, K57chemFE, and K57chemFE-Palmitoyl. Vertical axis: knob domain concentration (in ng/mL); horizontal axis: time (hours).

[0491] Figure 22: Crystal structure of peptide K92 in complex with C5. Fig. 22A: Peptide K92 in complex with C5. Fig. 22B: Cysteine arrangements for peptide K92. Fig. 22C: Position of K92 mutations. Examples Example 1: Generation of bovine antibody fragments from immunization of cows with Complement component C5 and biological activity

[0492] As described below, cows were immunized with C5, immune material was then isolated and cell sorting of antigen-specific memory B cells was performed from a draining lymph node taken close to the site of immunization. Flow cytometry was used to identify memory B cells that were double positive for two fluorescently labeled populations of C5 and a polyclonal mixture of antigen-enriched B cells was collected. 1. Immunization of Holstein Friesians with Complement C5 and isolation of antigen-specific memory B cells

[0493] Two adult Friesian cows were immunized with Complement C5 (full-length recombinant C5 protein, obtained from CompTech). Three subcutaneous injections were made into the shoulder at one-month intervals with 1.25 mg C5 mixed 1:1 (v/v) with Fama Adjuvant (GERBU Biotechnik). A fourth injection into the shoulder was made three weeks later with 1.25 mg C5 emulsified 1:1 with Freund's complete adjuvant (Sigma). A final injection into the shoulder was made one month later with 1.25 mg C5 emulsified 1:1 with Montanide (Seppic). Serum bleeds were taken ten days after each injection to check serum antibody titers. Collection of immune material

[0494] 500 mL samples of whole blood were collected post-C5 immunization. PBMCs were isolated using Leucosep tubes (Griener Bio-a) as per the manufacturer's instructions. In addition, a draining lymph node from the neck adjacent to the immunization site and a portion of spleen were collected. Tissues were homogenized using a gentle MACS tissue dissociator (Miltenyi), passed through a 40 μm cell strainer, and collected in RPMI 10% fetal calf serum. Cells were frozen in fetal calf serum 10% DMSO. Sorting of antigen-specific memory B cells by flow cytometry

[0495] A draining lymph node sample was thawed at 37°C and resuspended in warm RPMI, 10% FCS (v/v), 1 mM EDTA. Cells were centrifuged for 5 minutes at 400 g and the supernatant removed. The cell pellet was disrupted and resuspended in Assay Buffer (AB) comprising PBS, 1 mM EDTA, 1% BSA (w/v), 25 mM Hepes, at room temperature. Cells were centrifuged as before and resuspended in 2 mL of ice-cold AB containing 2 μg/mL each of C5-AF488 (fluorochrome Alexa Fluor 488) and C5-AF647 (fluorochrome Alexa Fluor 647) and incubated for 30 minutes on ice. Cells were then centrifuged, the supernatant removed, and the pellet washed in cold AB. An aliquot was taken for counting. Cells were centrifuged again, the supernatant removed, and cells resuspended to 5 x 106/mL in ice-cold AB before filtering through a 40 μm mesh. DAPI was added to a final concentration of 1 μg/mL just prior to acquisition on a BD Biosciences FACSAria III cell sorter (San Jose). Cells were identified by forward and side scatterers, and DAPI-positive dead cells were removed from analysis. Single cells were identified by pulse processing of scatterer height and area parameters. Cells positive for both C5-AF488 and C5-AF647 were then identified and sorted into 1.5 mL eppendorfs containing 1 mL PBS, 20% FCS, 25 mM Hepes maintained at 4°C.

[0496] AF488 was excited by a 488 nm laser and collected through a 530/30 BP filter and AF647 was excited by a 640 nm laser and collected through a 660/20 BP filter. DAPI was excited by a 407 nm laser and collected through a 450/40 BP filter. 2. Deep sequencing of C5-enriched CDR-H3 library revealed ultra-long CDR-H3 clonotypes.

[0497] Similar to camelid VHH verified from heavy chain only antibodies, there is no requirement for the heavy (HC) and light (LC) chain pair, since the extended CDR-H3 of the knob domain contains the antigen binding components; consequently, sequencing of antigen-enriched CDR-H3 polyclonal libraries has been used as a rapid method for finding knob domains.

[0498] The antigen-rich pool of memory B cells was lysed and RT-PCR was performed directly on the lysate. A primary polymerase chain reaction (PCR) with primers flanking CDR-H3, annealing to the conserved VH frame 3 and frame 4, was used to amplify all CDR-H3 sequences, regardless of their length or amino acid sequence. A second round of PCR was used to barcode the CDR-H3 sequences for ion stream sequencing. Methods: RT PCR on B cell lysate

[0499] C5-specific memory B cells from FACS were pelleted by centrifugation at 10,000 g in a centrifuge at 4°C. Cells were resuspended and lysed with 120 μL of an ice-cold solution of NP-40 detergent (0.5% v/v) and RNasin (Promega) at 1 U/μL. An RT PCR mixture was prepared using Super Script IV villous Master Mix (Invitrogen), comprising 32 μL of cell lysate and 8 μL of Master Mix. The reaction mixture was incubated at 25°C for 10 minutes, 50°C for an additional 10 minutes, and finally heated to 85°C for 5 minutes. Primary PCR

[0500] A primary PCR was used to specifically amplify IgG CDR3 cDNA sequences. The forward primer anneals to the conserved 3 frame sequence of the heavy chain variable domain, VH, and the reverse primer sequence anneals to the conserved 4 frame sequence of VH. The PCR product when read 5' to 3' therefore encodes the CDR3 sequence regardless of the length, amino acid sequence or composition of V, D, J gene segments. The PCR mixture was prepared using a Hot-start KOD master mix kit (Merck Millipore) as per the manufacturer's instructions. The primers used were 5'-GGACTCGGCCACMTAYTACTG-3' (SEQ ID NO: 446) and 5'- GCTCGAGACGGTGAYCAG-3' (SEQ ID NO: 447) and 2 μL of cDNA template was used per 50 μL of PCR. The reaction mixture was heated for 2 minutes at 96°C and then subjected to thirty cycles of: 96°C for 30 seconds, 55°C for 30 seconds and 68°C for 60 seconds. Finally, the mixture was heated at 68°C for 5 minutes. Gel purification

[0501] The PCR product contained a polyclonal mixture of CDR-H3 sequences, comprising regular and ultralong CDR-H3, which was visualized on an analytical gel. An excision was performed with transposition of approximately 250 to 500 base pairs, based on the marker. A QiaQUICK gel extraction kit (Qiagen) was used to extract DNA from the excised portion of the gel, as per the manufacturer's instructions. PCR Barcoding and Gel Purification

[0502] A secondary PCR was performed for barcoded sequences for ion torrent sequencing. Primers were as used before, but with the addition of adapter (italics) and barcoded sequence (bold):

[0503] 5'CCATCTCATCCCTGCGTGTCTCCGACTCAGTAAGGAGAACGGACTC GGCCACMTAYTACTG-3'(SEQ ID NO: 448) and

[0504] 5’-CCTCTCTATGGGCAGTCGGTGATGCTCGAGACGGTGAYCAG- 3’(SEQ ID NO: 449). Secondary PCR was performed using a KOD Master Mix kit as described for primary PCR. The secondary PCR product was gel purified and concentrated. Finally, samples were purified using Beckman Coulter AMP magnetic beads as per the manufacturer’s instructions. Ion torrent deep sequencing of CDR3 libraries

[0505] The purified DNA sample was diluted to 20 ng/μL (1 μg total) and stored at -20oC. Deep sequencing of all samples was performed using a commercial Ion Torrent PGM technology service by MACROGEN on a 318 chip.

[0506] In summary, approximately 1.1 Gb of data were obtained per chip, which translates to 6613415 raw reads of an average length of 170 base pairs. The first processing step consisted of the conversion of FASTQ to FASTA followed by the demultiplexing of the latter to classify the sequences according to their barcodes and to generate individual FASTA files. Each FASTA file was then translated into all three reading frames and concatenated into a file containing all frames. Using Perl scripts, only sequences of interest, flanked between the conserved FR3 and FR4 protein sequences (the DSATYY and LL[V,I]TVSS motifs were used), were kept in a single file. Finally, all sequences were exported to excel files with frequencies: the number of copies of each sequence checked. The number of significant reads, after processing, for each sample was found to be ~680000 and 530000 for barcodes 1 and 2 respectively. Results:

[0507] Deep sequencing of the CDR-H3 library revealed ultra-long CDR-H3 sequences in 4.3 percent of the total sequences. The ultra-long CDR-H3s were easily identified by their length (>90 base pairs) and by a characteristic duplication of the IGHV1-7 gene segment, which has been reported as a universal trait of ultra-long CDR-H3. After filtering, 3559 unique CDR-H3 sequences were obtained from the single draining lymph node sample. Of these, 154 were ultra-long CDR-H3, the full list is shown in Table 4. Table 4. Ultra-long CDR-H3 sequences derived from C5-immunized cows 3. The ultra-long CDR-H3 can function autonomously to bind Human Complement Component C5 independently of supporting the antibody infrastructure (scaffold).

[0508] To screen for C5 knob binding domains, from 154 identified ultra-long CDR-H3 sequences, ultra-long CDR3 sequences were selected based on clonotype, copy number, and cysteine pattern to make a representative set of 52 sequences are described in Table 5. Table 5. 52 ultra-long CDR3 sequences selected for screening

Expression of Knob-TEV-HKH-ScFc fusion proteins:

[0509] We took full-length ultra-long CDR-H3 spanning H93 to H102 (Kabat) and expressed it recombinantly with a single-chain Fc (ScFc) tag at the C-terminus and expressed as CDR-H3-ScFc fusions as transient transfections of 2 mL of Expi293F cells. Standard methods were used to clone the sequences into a useful vector for mammalian expression.

[0510] To the CDR-H3 sequences described in Table 5, a C-terminal tag comprising Gly-Ser linkers (italics), a TEV protease cleavage site (underlined), a 10x polyhistidine sequence, and a single-chain Fc was appended.

[0511] The seven amino acid recognition site for TEV protease is Glu-Asn-Leu-Tyr-Phe-Gln-Gly with cleavage occurring between Gln and Gly.

[0512] The sequence of the scFc sequence is as follows (CH2-CH3-linker in italics-CH2-CH3):

0513 kttppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslsl spgkggsstasgsgsggsgtagssggagssggsttaggsasgsgstgsgtggassggasgasgepkssdkthts ppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyrvvs vltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdiavewesn gqpennykttppvldsdgsfflyskltvd ksrwqqgnvfscsvmhealhnhytqkslslspgk (SEQ ID NO: 155)

[0514] The C-terminal Label sequence is shown below:

(0515) qvsltclvkgfypsdiavewesngqpennykttppvldsdgsffl yskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkggsstasgsgsggsgtagssggagssggsttaggs asgsgstgsgtggassggasgasgepkssdkthtsppcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvsh edpevkfnwyvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqpre pqvytlppsrdeltknqvsltclvkgfyps diavewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfsc svmhealhnhytqkslslspgk (SEQ ID NO: 156)

[0516] Plasmid DNA for each construct was amplified using miniprep kits (Qiagen). Individual 2.0 mL Expi293F cell cultures at 3 x 106 cells/mL for construct were plated into 48-well cell blocks using Expifectamine 293 Transfection kits (Invitrogen) as per the manufacturer's instructions. Cells were cultured for four days and centrifuged at 2500 rpm for 30 minutes. C5 Binding ELISA:

[0517] Cell supernatants were screened in a C5 binding ELISA according to the following method: 96-well ELISA plates (Nunc Maxisorp) were coated with a 2 μg/mL solution of C5, in carbonate-bicarbonate buffer (Sigma). All washing steps comprised four wash cycles with PBS, 0.05% Tween 20. Blocking buffer was PBS, 1% BSA (w/v). Cell supernatants were plated as 1:10 and 1:100 dilutions in Assay Buffer (PBS, 0.05% Tween 20, 0.1% BSA [w/v]). For development, a 1/5,000 dilution of a goat anti-human Fc, HRP secondary antibody (Thermo Scientific) was used with 3,3',5,5' Tetramethylbenzidine ‘One Step’ (Thermo Scientific). The reaction was stopped by the addition of a 2% (w/v) NaF solution and OD measured at 630 and 390 nm wavelength using a BMG labtech plate reader. Results:

[0518] 14 C5 binders were identified. The hit sequences are shown in Table 6. Table 6

[0519] For the hits, purified proteins were prepared according to the method described as follows. Using an Akta pure (GE Healthcare) Hi-Trap Nickel excel (GE Healthcare) column they were equilibrated with 10 column volumes (CV) of PBS. Cell supernatants were loaded and the column was washed with 7x CV of PBS, 0.5 M NaCl. The column was then washed with 7x CV of Buffer A (0.5 M NaCl, 0.02 M Imidazole, PBS pH 7.3). Protein samples were eluted by isocratic elution with 10x CV of Buffer B (0.5 M NaCl, 0.25 M Imidazole, PBS pH 7.3) as fractions. The column was washed with 0.1 M NaOH and re-equilibrated in PBS before subsequent loading. After elution, protein-containing fractions were pooled and buffer exchanged into PBS using PD-10 columns (GE Healthcare).

[0520] The purified proteins were titrated in an ELISA experiment for binding to C5 and a negative control of bovine serum albumin. These data surprisingly show that certain ultra-long CDR-H3s are viable even in the absence of the supporting framework of the parent antibody. Example 2: Antibody fragments isolated according to the invention confer binding to Human Complement Component C5 when inserted into the CDR-H3 of a Fab. 1. Production of a Fab comprising a knob domain inserted into its CDR-H3

[0521] To assess the importance of the β-stem, the knob domains were separated from the stem and inserted into the CDR-H3 of a Fab, flanked by TEV protease sites to allow the knob domain peptide to be excised. We considered a knob domain peptide fragment, from the residue preceding the first cysteine residue of CDR-H3 to the residue following the last cysteine residue of CDR-H3, where the ultra-long CDR-H3 has a minimum of two cysteines. The knob domain peptides for the 154 CDR-H3s of SEQ ID NO: 1 through 154 are shown in bold in Table 4 and listed in Table 7 below: Table 7

[0522] The regions flanking the knob domain fragment are shown in italics in Table 4.

[0523] For expression of knob domain peptides, the human Fab, PGT121, was selected as the vehicle. The CDR-H3 of PGT121, which binds complex-type N-glycans within the HIV gp120 envelope, contains an extended antiparallel β-stem that was an ideal platform on which to present the knob domain peptide, while the natural antigen was unlikely to inappropriately contribute to C5 binding. The amino residues of Kabat H100c to H100h (Gly-Ile-Val-Ala-Phe-Asn) were deleted and replaced with a knob domain peptide sequence between two TEV protease sites. From the set of 14 binders, six knob domains of diverse peptide sequences, with different numbers and arrangements of cysteines, were reformatted as PGT121-cleavable knob fusion proteins.

[0524] The PGT-121 heavy chain sequence, with a C-terminal His tag, one of the TEV protease cleavage sites in bold and GS motif underlined, is as follows, the deleted CDR-H3 residues are shown in italics:

0525 pvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdk rvepkscdktleenlyfqgsggshhhhhhhhhh (SEQ ID no: 311)

[0526] The heavy chain sequences of the PGT-121-knob fusions were as follows, inserted sequences are shown in italics, with TEV protease cleavage sites in bold and GS motif underlined:

[0527] PGT-121-K149

(0528) qvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfpa vlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdktleenlyfqgsggshhhhhhhhhh (SEQ ID no: 312)

[0529] K149 (portion)

[0530] scpdgfsyrswddfccpmvgrclaprn (SEQ ID no: 313)

[0531] PGT-121-K136

0532 ngtqvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfp avlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdktleenlyfqgsggshhhhhhhhhh (SEQ ID no: 314)

[0533] K136

[0534] tcpdnyrevdgcdpydcclttwctnsyctryi (SEQ ID no: 315)

[0535] PGT-121-K92

(0536) fymdvwgngtqvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvsw nsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdktleenlyfqgsggshh hhhhhhhh (SEQ ID no: 316)

[0537] K92 vtcpegwsecgvaiygyecgrwgcghflnsgpnispyvtt (SEQ ID no: 317)

[0538] For K92, a Serine was mutated to Threonine T at the underlined position in the initial sequence of the ultra-long CDR-H3, with no impact on binding properties.

[0539] The same assays can be reproduced with the starting sequence: vtcpegwsecgvaiygyecgrwgcghflnsgpnispyvSt (SEQ ID NO: 318)

[0540] PGT-121-K57

0541 mdvwgngtqvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvsw nsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdktleenlyfqgsggshh hhhhhhhh (SEQ ID no: 319)

[0542] K57 sgcppgyksgvdcspgseckwgcyavdgrryggygadsgvGS (SEQ ID no: 320)

[0543] For K57, the GS was removed from the C-terminus and the TEV protease site attached directly to the C-terminus, without a GS linker.

[0544] PGT-121-K8

0545 qgsewftyfymdvwgngtqvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvs wnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdktleenlyfqgsggsh hhhhhhhhh (SEQ ID NO: 321)

[0546] K8 vcpdgfnwgygcaagssrfctrhdwccyderadshtygfctgnrv (SEQ ID no: 322)

[0547] PGT-121-K60

0548 dvwgngtqvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvswn sgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkrvepkscdktleenlyfqgsggshhh hhhhhhh (SEQ ID no: 323)

[0549] K60 kscregyidgggcclpgscrgcacsyydwlkcprdcrgtsee (SEQ ID no: 324)

[0550] The heavy chains were paired to the PGT-121 light chain sequence as follows:

[0551] qsvltqppdisvapgetariscgekslgsravqwyqhragqapsliiynnqdrpsgiperfsgspdspf gttatltitsveagdeadyychiwdsrvptkwvfgggttltvlgqpkaapsvtlfppsseelqankatlvclisdfypgavtv awkadsspvkagvetttpskqsnnkyaassilsltpeqwkshrsyscqvthegstvektvaptecs (SEQ ID NO: 325) 2. Biacore analysis of binding to C5

[0552] Binding to C5 was measured using surface plasmon resonance (SPR) single-cycle kinetics as described below. C5 has been shown to be activated by pH extremes and thus single-cycle kinetics, in which sequential injections of increasing concentrations of analyte are performed in the absence of stringent regeneration steps, was used to maintain the integrity of the C5 protein on the surface of the sensor chip.

[0553] Biacore Single-Cycle Kinetics. Using a Biacore 8K (GE Healthcare), C5 was immobilized onto a CM5 chip by amine coupling. Flow cells were activated using EDC/NHS (flow rate, 10 μL/min; contact time, 30 s). C5, at 1 μg/mL in pH 4.5 sodium acetate buffer, was immobilized on only two flow cells (flow rate, 10 μL/min; contact time, 420 s). Finally, ethanolamine was applied to both flow cells (flow rate, 10 μL/min; contact time, 420 s). A final immobilization level of approximately 2,000 response units was obtained.

[0554] Single-cycle kinetics were measured using titrations in HBS-EP buffer. A high flow rate of 40 μL/min was used, with a contact time of 230 s and a dissociation time of 900 s. Binding to the reference surface was subtracted and the data were fitted to a single-site binding model using Biacore evaluation software. Results

[0555] As fusion proteins, the knob domains confer high affinity C5 binding to the PGT121 Fab.

[0556] By SPR, five PGT121-knobs bound C5 with affinities in the picomolar to nanomolar range (Figure 1 and Table 8), with only the K60 knob domain found to be nonfunctional after reformation. Table 8. Biacore single-cycle kinetic data on the PGT121-knob domain fusion protein

[0557] Summary of kinetics for n = 3

[0558] PGT121-knob domain fusion proteins were counter-screened to bind to the human complement component C3. C3 is the closest human homologue to C5, sharing a conserved fold and a sequence homology of 26.5%. In these experiments, no cross-reactivity was detected. 3. Purification of knob domain peptides from Fab fusion proteins

[0559] To obtain knob domain peptides, TEV protease was used to proteolytically excise the knob domains from the CDR-H3 of PGT121 Fab. Fab-knob peptide fusion proteins (10 mg/mL) were incubated with TEV protease, at a ratio of 1:100 (w/w), for a minimum of 2 hours at room temperature. Peptides can be purified and isolated using a Waters UV-driven FractionLynx system with a Waters XBridge Protein BEH C4 OBD Prep Column (300 Å, 5 μm, 19 mm x 100 mm). An aqueous solvent of water, 0.1% trifluoroacetic acid (TFA), and an organic solvent of 100% MeCN was used. The column was run at 20 mL/min at 40°C with a gradient from 5 to 50% organic solvent in 10.8 min. The column was cleaned with three steep ramps from 5 to 95% organic solvent. Fractions containing knob peptide were pooled and lyophilized using a Labconco Freezone freeze dryer. For storage at -80°C and subsequent analysis, the peptides were resuspended in 20 mM Tris pH 7.4. Table 9. The sequences of the six isolated knob domain peptides obtainable after cleavage are as follows: Example 3: Antibody fragments isolated according to the invention confer binding to Human Complement Component C5 when inserted within the 3-frame region of the albumin-binding VH domain of Fab (645Fab)

[0560] As described in WO2020/011868 (published January 16, 2020), Fab antibody fragments comprising an insert polypeptide within the 3-frame region of the V domain, notably the VH domain, may provide a novel bispecific antibody format, in particular stable and capable of simultaneously binding two antigens. Advantageously, the CA645 Fab-knob fusion proteins as described herein may simultaneously bind C5 and albumin, which may confer an increased serum half-life for the knob domain peptide.

[0561] In addition to three CDR loops, the antibody heavy and light chains, both conventional and single-chain camelid VHH, have a fourth loop that is formed by the 3-frame. The Kabat numbering system defines the 3-frame as positions 66 to 94 in a heavy chain and positions 57 to 88 in a light chain.

[0562] The same methods as described in Example 2 were used to reform the six knob domains of the ultra-long CDR-H3 that bind to C5 as cleavable CA645 Fab-knob fusion proteins.

[0563] The light chain V region or light chain of the CA645 Fab fusion proteins described in the following Examples comprised or had SEQ ID NO: 429 (CA645 VL domain (gL5)) or SEQ ID NO: 428 (CA645 gL5 light chain) respectively. Alternative light chains or light V regions may be used, for example the light V regions comprising the VL domain of SEQ ID NO: 441 or SEQ ID NO: 442.

[0564] Sequences of heavy chain 645 Fab-knob domain fusions:

[0565] The 645 Fab heavy chain (gH5), with a K8 knob peptide (italics) inserted within its 3' frame region, with a C-terminal His tag, TEV protease cleavage sites and GS motif (Bold)

[0566] evqllesggglvqpggslrlscavsgidlsnyainwvrqapgkglewigiiwasgttfyatwakgrftisrd nsggggsenlyfqgsvcpdgfnwgygcaagssrfctrhdwccyderadshtygfctgnrvenlyfqgsgggskntvil qmnslraedtavyycartvpgystapyfdl wgqgtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtv swnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkschhhhhhhhhh (SEQ ID NO: 332)

[0567] seq. K8 vcpdgfnwgygcaagssrfctrhdwccyderadshtygfctgnrv (SEQ ID NO: 322)

[0568] The 645 Fab heavy chain, with a K57 knob peptide (italics) inserted within its 3' frame region, with a C-terminal His tag, TEV protease cleavage sites and GS motif (bold)

[569] lvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvs wnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkschhhhhhhhhh (SEQ ID NO: 333)

[0570] SEQ. K57 gcppgyksgvdcspgseckwgcyavdgrryggygadsgv (SEQ ID NO: 334)

[0571] The 645 Fab heavy chain, with a K60 knob peptide (italics) inserted within its 3' frame region, with a C-terminal His tag, TEV protease cleavage sites and GS motif (bold)

[572] lvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtvsw nsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkschhhhhhhhhh (SEQ ID NO: 335)

[0573] K60 kscregyidgggcclpgscrgcacsyydwlkcprdcrgtsee (SEQ ID no: 336)

[0574] The 645 Fab heavy chain, with a K92 knob peptide (italics) inserted within its 3' frame region, with a C-terminal His tag, TEV protease cleavage sites and GS motif (bold)

[0575] evqllesggglvqpggslrlscavsgidlsnyainwvrqapgkglewigiiwasgttfyatwakgrftisrd nsggggsenlyfqGSgsvtcpegwsecgvaiygyecgrwgcghflnsgpnispyvttgsenlyfqgsgggskntvil qmnslraedtavyycartvpgystapyfdlwgq gtlvtvssastkgpsvfplapsskstsggtaalgclvkdyfpepvtv swnsgaltsgvhtfpavlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkschhhhhhhhhh (SEQ ID NO: 337)

[0576] SEQ. K92 vtcpegwsecgvaiygyecgrwgcghflnsgpnispyvtt (SEQ ID no: 317)

[0577] The 645 Fab heavy chain, with a K136 knob peptide (italics) inserted within its 3' frame region, with a C-terminal His tag, TEV protease cleavage sites and GS motif (bold)

[0578] evqllesggglvqpggslrlscavsgidlsnyainwvrqapgkglewigiiwasgttfyatwakgrftisrd nsggggsenlyfqgstcpdnyrevdgcdpydcclttwctnsyctryienlyfqgsgggskntvilqmnslraedtavyy cartvpgystapyfdlwgqgtlvtvss astkgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfp avlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkschhhhhhhhhh (SEQ ID NO: 338)

[0579] seq. k136 stcpdnyrevdgcdpydcclttwctnsyctryi (SEQ ID NO: 339)

[0580] The 645 Fab heavy chain, with a K149 knob peptide (italics) inserted within its 3' frame region, with a C-terminal His tag, TEV protease cleavage sites and GS motif (bold)

[0581] evqllesggglvqpggslrlscavsgidlsnyainwvrqapgkglewigiiwasgttfyatwakgrftisrd nsggggsenlyfqgsgscpdgfsyrswddfccpmvgrclaprngsenlyfqgsgggskntvilqmnslraedtavyy cartvpgystapyfdlwgqgtlvtvssast kgpsvfplapsskstsggtaalgclvkdyfpepvtvswnsgaltsgvhtfp avlqssglyslssvvtvpssslgtqtyicnvnhkpsntkvdkkvepkschhhhhhhhhh (SEQ ID NO: 340)

[0582] seq. k149 scpdgfsyrswddfccpmvgrclaprn SEQ ID no: 313

[0583] The knob domain peptides were purified and isolated as described above, the sequences of the isolated peptides are provided below in Table 10: Table 10

[0584] An example chromatography trace of the preparative scale purification of the K57 peptide is shown in Figure 2. Binding to human C5

[0585] We measured the individual rate constants (kon and koff) and equilibrium dissociation constants (KD) for the knob domain peptides binding to C5 by the Biacore single-cycle chemical kinetics method described above.

[0586] The peptides exhibit high affinity binding, K < 40 nM, when measured by single-cycle SPR kinetics (Figure 3 and Table 11). The only knob domain peptide found not to bind was K60. Table 11. Biacore single-cycle kinetics data on isolated knob domain peptides

[0587] Summary of kinetics for n = 3

[0588] The knob domain peptides were counterscreened for cross-reactivity to human complement component C3 and ovalbumin, with no evidence of binding to either protein observed. Binding to mouse and rabbit C5 protein

[0589] Isolated knob domain peptides K8, K57, K92, and K149 were tested for cross-reactivity against mouse and rabbit C5 protein. C5 was purified from human serum or animal serum (TCS biosciences), using methods described for example in Macpherson et al, J Biol Chem. 2018 Sep 7;293(36):14112-14121. Cross-reactivity to mouse C5 was observed for knob domain peptides K8 and K92 (Figure 4). Peptides K57 and K149 were specific for human C5 and did not cross-react with mouse or rabbit proteins. We report individual rate constants (kon and koff) and equilibrium dissociation constants (KD) for the binding of K8 and K92 to mouse and rabbit C5 in Table 12. Table 12. Single-cycle Biacore kinetics data of knob domain peptides.

[0590] Data from n = 1 experiment.

Complement activation assays

[0591] To elucidate the functional consequences of the binding of knob domain peptides to human C5, we performed complement activation assays using the SVAR complement activation ELISA that measured CP (classical pathway) and AP (alternative pathway) activation in human serum through the formation of the C5b neo-epitope. We also developed orthogonal ELISA assays that measured total and AP-specific complement-mediated C3b and MAC (membrane attack complex) deposition. For all activation conditions, we also tracked C5a release by ELISA.

Methods

[0592] For C3 and C9 ELISAs, microtiter plates (e.g. MaxiSorp; Nunc) were incubated with 50 ml of a solution containing 2.5 μg/ml aggregated human IgG (Sigma-Aldrich) for the CP or 20 mg/ml zymosan (Sigma-Aldrich) in 75 mM sodium carbonate (pH 9.6) for the AP, overnight at 4°C. As a negative control, wells were coated with 1% (w/v) BSA/PBS. Microtiter wells were washed four times with 250 ml of wash buffer (50 mM Tris-HCl, 150 mM NaCl, and 0.1% Tween 20 (pH 8) between each step of the procedure. Wells were blocked using 250 μl of 1% (w/v) BSA/PBS for 2 h at room temperature. Normal human serum (NHS) was diluted in Gelatin veronal buffer with calcium and magnesium (0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN3, 0.15 mM calcium chloride, 1 mM magnesium chloride, pH 7.3; for CP) or Mg-EGTA (2.5 mM Veronal buffer [pH 7.3] containing 70 mM NaCl, 140 mM glucose, 0.1% gelatin, 7 mM MgCl2 and 10 mM EGTA; for AP). NHS was used at a concentration of 1% in CP or 5% in AP. NHS was mixed with serially diluted concentrations of peptides (16 μM to 15.6 nM) in appropriate buffer and preincubated on ice for 30 min. The peptide-NHS solutions were then incubated in the wells of microtiter plates for 35 min for CP/LP (both C3b and C9 detection) or 35 min for AP (C3b) or 60 min for AP (C9). Complement activation was assessed by detection of deposited complement activating factors using specific Abs against C3b (rat anti-human C3d; e.g. from Hycult) and MAC (goat anti-human C9; e.g. CompTech). Bound primary Abs were detected with HRP-conjugated goat anti-mouse (Abcam) or rabbit anti-goat (Dako) secondary Abs. Bound HRP-conjugated antibodies were detected using TMB A solution (Eco-TEK) with absorbance measured at OD450.

[0593] For the C5b ELISA, assays were conducted using the CP and AP Complement functional ELISA kits (SVAR) as per the manufacturer's protocol. For sample preparation: serum was diluted as per the respective protocol for the CP and AP assays. Peptide titrations were prepared and allowed to incubate for 15 minutes at room temperature prior to plating.

[0594] For C5a ELISA assays, assays were conducted using the Human Complement C5a ELISA kit (Invitrogen) as per the manufacturer's protocol. For sample preparation: at the end of the 37°C incubation of the serum/peptide samples in the C5b ELISA assay plate, 50 μL of the diluted, activated serum was transferred to a C5a ELISA assay plate containing 50 μL/well of Assay Buffer. All subsequent experimental steps were performed as described in the protocol. Results

[0595] These assays revealed that the knob domain peptide K57 is a potent and fully effective inhibitor of C5 activation, preventing C5a release, C5b neo-epitope formation, and MAC. There was no effect on C3b deposition, suggesting that the peptide is inhibiting complement activation downstream of C3. In contrast, the K149 peptide is a silent, high-affinity binder to C5 with no detectable effect on C5a release, C5b neo-epitope formation, or MAC deposition, even at peptide concentrations in excess of 100 x KD.

[0596] We also identified knob domain peptides that exert more nuanced allosteric effects on C5. Peptide K92 was a noncompetitive C5 inhibitor preventing C5 activation by AP. We observed decreased C5a release, decreased C5b neo-epitope formation, and decreased MAC deposition in assays where the AP component was isolated. Intriguingly, no effect was observed in assays for which the AP component was not isolated or in assays for which the CP component was isolated, suggesting that peptide K92 does not inhibit C5 activation by CP C5 convertase, but it does partially inhibit activation by AP C5 convertase.

[0597] Similarly, the K8 peptide was a non-competitive inhibitor of AP, but also demonstrated non-competitive inhibition of CP; decreasing C5a release, C5b neo-epitope formation, and MAC deposition in both CP- and AP-driven assays. For both the K8 and K92 peptides, no effect on C3b deposition was detected. Our complement ELISA data are shown in Figure 5 and Tables 13 to 18. Table 13. C5b deposition ELISA by the classical pathway.

[0598] Data from n = 3 experiments unless specified. *ND = Not detected. a Data are an average of n = 6 experiments Table 14. C5b deposition ELISA by the alternative pathway

[0599] Data from n = 3 independent titrations unless specified. *ND = not detected. a Data are mean of n = 5 independent titrations b Data are mean of n = 4 independent titrations Table 15. Inhibition of C5a release mediated by the classical pathway.

[0600] Data from n = 3 independent titrations, unless otherwise stated *ND = Not detected. a Data are an average of n = 5 independent titrations Table 16. Inhibition of C5a release mediated by the alternative pathway

[0601] Data from n = 3 independent titrations unless otherwise stated *ND = Not detected. a Data are an average of n = 4 independent titrations Table 17. Inhibition of C9 deposition mediated by the classical pathway.

[0602] Data from n = 3 independent titrations unless otherwise stated. *ND = not detected. a Data from n = 1 titration. Table 18. Inhibition of C9 deposition mediated by the alternative pathway

[0603] Data from n = 3 independent titrations *ND = not detected. a Data from n = 1 titration. Inhibition of 'killing assay' knob domain peptides by complement-mediated bacterial lysis

[0604] We explored the effects of our peptides in bacterial killing assays, using the Escherichia coli strain DC10B which is susceptible to complement-mediated lysis when incubated even in diluted human serum (as described for example in (as described for example in Monk IR, et al.; Transforming the untransformable: application of direct transformation to genetically manipulate Staphilococcus aureus and Staphilococcus epidermidis. mBio. Vol. 3 (2); 2012 and commercially available).

Methods

[0605] Normal human serum (NHS) was prepared using fresh blood drawn from 8 healthy donors using vacutainer serum collection tubes (BD). Blood was kept at room temperature and allowed to clot for 30 min followed by a 1 h incubation on ice. After two rounds of centrifugation (700 X g; 4°C, 8 min), serum fractions were collected, pooled, and stored in aliquots at -80°C. Heat-inactivated sera (DNHS) were prepared by incubation at 56°C for 30 min.

[0606] Escherichia coli DC10B strains were grown to exponential phase (OD600 0.4 to 0.5) in LB broth with shaking (180 rpm) at 37°C. Bacteria were harvested by centrifugation, washed once in PBS, and resuspended at an OD600 = 0.1 (correlating with approximately 107 CFU/ml) in Gelatin Veronal Buffer with Calcium and Magnesium (“GVB++”; 5 mM veronal buffer [pH 7.3], 0.1% [w/v] gelatin, 140 mM NaCl, 1 mM MgCl2, and 0.15 mM CaCl2). Peptides at specific concentrations or PBS (control) were incubated with varying percentages of pooled NHS in GVB++ buffer for 30 min on ice. Then, 50 μL of bacteria were added to 50 μL of NHS peptide/PBS solutions and incubated for 20 min at 37°C. Following incubation, aliquots were removed, serially diluted, and spread onto LB agar plates. The plates were incubated for 18 h at 37°C, after which the remaining CFU were enumerated and survival calculated compared with the CFU at time 0. Controls consisted of DNHS and serum treated with the complement inhibitor C5 OmCI (10 mg/ml; 0.625 mM).

Results

[0607] The knob domain peptide K57 was fully effective in preventing complement-mediated lysis. We report IC50 value of K57 of 115 nM and 1.22 μM for CP and AP-mediated killing assay, respectively, the data are shown in Tables 19 to 20 and Figure 6.

[0608] The K8 knob domain was able to inhibit both CP- and AP-directed killing assays; we reported K8 IC50 values of 16.9 μM and 25.8 μM for the CP- and AP-mediated killing assay, respectively. Consistent with our pathway-specific ELISA data, the K92 peptide was only able to inhibit the AP-directed killing assay, achieving an IC50 of 2.465 μM and an Emax of 91.0%. Table 19. Inhibition of classical pathway-mediated bacterial lysis.

[0609] Data from n = 3 independent titrations *ND = not detected. Table 20. Inhibition of bacterial lysis mediated by the alternative pathway

[0610] Data from n = 3 independent titrations

[0611] data from n = 1 experiment. Example 4: Production of isolated antibody fragments of the invention by chemical synthesis, notably solid phase, of peptide

[0612] As shown below, isolated functional knob domains of ultra-long CDR-H3 can be chemically synthesized by solid-phase peptide synthesis. In order to form a disulfide bond between two cysteine residues within the knob domain peptides, the knob domain peptides were synthesized by two methods: 1) a site-directed method, whereby the cysteines were specifically protected and deprotected to form disulfide bonds in a pre-ordered manner; and 2) by a free energy method, whereby the formation of disulfide bonds was left under free energy in the presence of glutathione redox buffer. Peptide Synthesis

[0613] All peptides were synthesized using solid phase synthesis, utilizing the Fmoc technique (e.g. described in Atherton, E. and R. C. Sheppard. 1989. Fluorenylmethoxycarbonylpolyamide solid phase peptide synthesis: general principles and development. In Solid Phase Peptide Synthesis: A Practical Approach. IRL Press, Eynsham, Oxford, pp. 25-37). Synthesis was carried out in a sequential manner in the C to N direction on a robotic synthesizer (Symphony, Protein Technologies). Syntheses were initiated on appropriate polystyrene supports (nova biochem) with the first amino acid attached via a Wang linkage to the carboxyl, 0.3 mM/g substitution. Chain elongation was facilitated using a twenty-minute double coupling strategy with a 3-fold molar excess of resin loading reagents containing N-alpha-protected amino acids dissolved in DMF (side chains orthogonally protected with protecting groups suitable for Fmoc chemistry) and the coupling reagent TBTU, in the presence of DIPEA. Temporary amino protection was removed by two 5-minute treatments with 20% piperidine in DMF. After the peptide sequences were complete, the peptidyl resins were treated with a mixture of TFA, ethane dithiol, and triisopropyl silane, 95:3:2 for 3 hours to cleave the peptides and all protecting groups. The peptides were isolated by filtration and trituration with diethyl ether. The peptides were dissolved in acetonitrile and water and freeze-dried prior to purification. 1) Formation of disulfide bonds by a site-directed method

[0614] Using special protecting groups, it is possible to cyclize between two specific cysteines in a peptide, thus it is possible to have more than one disulfide in a peptide. Only the K149 method was synthesized by the site-directed method to create two different disulfide-linked forms, K149A and K149B. The site-directed disulfide bond formation was as follows:

[0615] K149A

[0616] SC1PDGFSYRSWDDFC1C2PMVGRC2LAPRN (SEQ ID NO: 347)

[0617] K149B

[0618] SC1PDGFSYRSWDDFC2C1PMVGRC2LAPRN (SEQ ID NO: 348)

[0619] Peptide K149 was synthesized using an orthogonal protection strategy for cysteine residues. For K149A, residues C2 and C15 were protected using ACM (acetamidomethyl) in the side chain while C16 and C22 were protected with a trityl group in the side chain. For K149B, residues C2 and C16 were protected using ACM (acetamidomethyl) in the side chain while C15 and C22 were protected with a trityl group in the side chain.

[0620] The peptide was synthesized using standard Fmoc solid phase chemistry as described above. After cleavage with TFA (trifluoroacetic acid), which removed all side chain protecting groups with the exception of ACM, the linear peptide was purified using rHPLC on a C18 column.

[0621] The first cyclization reaction was carried out under mild oxidizing conditions using Potassium Ferracyanide and the progress of the reaction between C16 and C22 (K149A) or C15 and C22 (K149B) was monitored by LC-MS. When this was observed to have gone to completion the peptide was purified to remove any residual linear peptide and then treated with an excess of Iodine. Iodine promoted the concomitant removal of the ACM protecting groups and the final oxidation of cysteine residues C2 and C15 (K149A) or C2 and C16 (K149B). The reaction was monitored by LC-MS and the dicyclic peptide repurified by HPLC. 2) Synthesis and formation of disulfide bonds by the free energy method. The peptides are shown below in Table 21. Table 21

[0622] It was found that the most expedient and high throughput mode to obtain these cyclic peptides with more than two disulfide bonds was to purify by RP-HPLC the linear sequence and immediately start the cyclization without a freeze-drying step, otherwise a batch of insoluble polymeric material resulted.

[0623] Cyclization was achieved using controlled thermodynamic air oxidation to obtain the lowest energy form of the disulfides in the sequence using a mixture of reduced and oxidized glutathione.

[0624] Crude peptides were dissolved in DMSO/water and treated with TCEP to ensure that cysteine residues were fully reduced. Peptides were purified by RP-HPLC, on a Varian Prostar system equipped with two 210 pumps and a 355 UV spectrophotometer. Driving buffers were for pump A, solvent A, 0.1% (v/v ammonium acetate in water, pH 7.5 to 7.8) and for pump B, solvent B, 100% acetonitrile. The peptide was loaded onto a prep RP-HPLC column (C18 Axia, 22 mm x 250 mm, 5 micron partical, 110 angstrom pore size, Phenomenex) and the linear sequence eluted from the column by conducting a gradient between solvents A and B, 5% B to 65% B in 60 min. The linear peptide was identified by ESMS.

[0625] The linear peptide solution (approximately 50 mL) was added to 500 mL of a cyclization buffer, (0.2 M phosphate buffer, pH 7.5, containing 1 mM EDTA, 5 mM reduced glutathione and 0.5 mM oxidized glutathione). The solution was stirred at room temperature for 48 hours. After that, a small sample was analyzed by analytical HPLC to assess the level of cyclization.

[0626] When cyclization was judged sufficiently complete, the entire buffer containing the peptide was pumped onto a preparative RP HPLC column (C 18 Axia as above). The cyclic peptide was eluted using a gradient between solvent A (0.1% TFA in water) and solvent B (0.1% TFA in acetonitrile) from 5% B to 65% B in 60 minutes. Fractions identified as the correct compound were freeze-dried prior to analysis. Binding to C5

[0627] We used single-cycle SPR kinetics to measure binding to human C5 protein. The results are presented in Figure 7 and Table 22 below.

[0628] For the peptides produced by glutathione cyclization, all examples bound to C5 with broadly comparable affinities to when recombinantly expressed. These data suggest that functional knob domain peptides can be derived by chemical synthesis, whereby the lowest energy disulfide bond form is obtained. For the site-directed methods, both the K149A and K149B peptides bound to C5, but the K149A peptide was 41-fold more potent than K149B and equipotent to K149 produced by glutathione cyclization. The disulfide arrangement of K149A is both the lowest energy form and the form for which the binding free energy to C5 is lowest. Table 22. Binding of chemically derived knob domain peptides to C5 by single-cycle SPR kinetics

[0629] Data from n = 1 experiment.

Functional activity tests

[0630] The functional activity of chemically derived knob domain peptides was confirmed in a complement activation ELISA that measured inhibition of C5b neoepitope deposition. These data are displayed in Figure 8 and Tables 23 and 24. Table 23. ELISA of the classical pathway of C5b deposition.

[0631] Data from n = 3 independent titrations unless specified. Table 24. C5b neoepitope alternative pathway ELISA

[0632] Data from n = 3 independent titrations unless specified. a Data are from n = 2 independent titrations

Example 5: Crystal structure

[0633] To elucidate the structural mechanisms for the allosteric modulation of C5 by a knob domain peptide, we solved the crystal structure of the C5-K8 complex. As an example, a resolution of 2.3 Å can be used.

[0634] C5 at 6.1 mg/ml (20 mM Tris, 75 mM NaCl, pH 7.35) was mixed in a 1:1 molar ratio with knob domain peptide K8. The drops were adjusted by vapor diffusion method at 18°C with a 1:1 mixture of mother liquor (v/v). Crystals were grown in a mother liquor of: 0.1 M ADA, 14% Ethanol (v/v), pH 6.0. Prior to snap freezing in liquid nitrogen, C5-K8 crystals were cryoprotected in mother liquor with 30% MPD (v/v).

[0635] Data were collected at the Diamond Light Source (Harwell, UK). The structure of C5-K8 was solved using the Balbes automated molecular replacement pipeline (F. Long, et al. “BALBES: a Molecular Replacement Pipeline“ Acta Cryst. D64 125-132 (2008)) using the apo C5 structure (PDB 3CU7), minus the C345c domain. A backbone model of the K8 peptide was produced using ARP-wARP (a software suite commonly used for automated model building in X-ray crystallography) that informed manual model building in Coot, within the CCP4 (Collaborative Computational Project, Number 4) suite (M. D. Winn et al. Acta. Cryst. D67, 235-242 (2011)). For example, Langer G, Cohen SX, Lamzin VS, Perrakis A. (2008) Automated macromolecular model building for x-ray crystallography using ARP/wARP version 7. Nat. Protoc. 3, 1171-1179. The model was subjected to multiple rounds of refinement in Phenix.

[0636] The structural data are shown in Figures 9, 10, and 11. The structure shows that the K8 peptide binds to a previously unrecognized site for regulating C5 in the MG8 domain of the α chain of C5. The K8 peptide mediated a crystal contact that ensured low B-site factors, clearly enunciating the disulfide bond arrangement and the intra- and inter-chain interactions of the peptide backbone and side chains. The disulfide bond arrangement of the K8 peptide has been elucidated and is shown in Figure 12. PDBePISA (Interactive service at the European Bioinformatics Institute) was used to examine the molecular interface between the K8 peptide and C5. The hydrogen bonding and salt bridge interactions are listed in Tables 25 to 28. Table 25. K8 residues participating in the surface C5-K8 interactions Table 26. C5 residues participating in the C5-K8 interaction surface Table 27. Hydrogen bonding interactions of side chains and main chain with C5 Table 28. K8 residues participating in salt bridge interactions with C5 Example 6: Antibody fragments isolated according to the invention confer binding to Human Complement Component C5 when inserted into the N- or C-terminus or the frame loops of a VHH to create a bispecific single chain antibody

[0637] We produced VHH domain knob fusion proteins by inserting the K57 knob domain peptide into the scaffold loops (loop 1, loop 2, and loop 3) of a VHH antibody, at the end opposite the CDRs, to make a bispecific single-chain antibody. The K57 knob domain was recombinantly fused to the hC3nb1 VHH, which binds C3 and C3b and for which a crystal structure has been published (Protein Data Bank (PDB) code: 6EHG).

[0638] The sequences are shown below, the constructs were expressed with a C-terminal single-chain Fc tag (not shown):

[0639] hC3nb1

[0640] QVQLVETGGGLVQAGGSLRLSCAASGIFSLNAMGWFRQAPGKEREFV ATINRSGGRTYYADSVKGRFTISRDNGKNMVILQMHSLKPEDTAIYYCAAGTGWSP QTDNEYNYWGQGTQVTVSS (SEQ ID NO: 351)

[0641] Peptide K57 (linker in italics)

[0642] SGGGSGCPPGYKSGVDCSPGSECKWGCYAVDGRRYGGYGADSGVSG GGS (SEQ ID NO: 352)

[0643] hC3nb1 - K57 loop 1 (peptide and linker in italics)

[0644] QVQLVETGGGLVQASGGGSGCPPGYKSGVDCSPGSECKWGCYAVDG RRYGGYGADSGVSGGGSGSLRLSCAASGSIFSLNAMGWFRQAPGKEREFVATINR SGGRTYYADSVKGRFTISRDNGKNMVILQMHSLKPEDTAIYYCAAGTGWSPQTDNE YNYWGQGTQVTVSS (SEQ ID NO: 353 )

[0645] hC3nb1 - K57 loop 2 (peptide and linker in italics)

[0646] QVQLVETGGGLVQAGGSLRLSCAASGSIFSLNAMGWFRQAPSGGGSG CPPGYKSGVDCSPGSECKWGCYAVDGRRYGGYGADSGVSGGGSKEREFVATINR SGGRTYYADSVKGRFTISRDNGKNMVILQMHSLKPEDTAIYYCAAGTGWSPQTDNE YNYWGQGTQVTVSS (SEQ ID NO: 354)

[0647] hC3nb1 - K57 loop 2 delta proline (peptide and linker in italics)

[0648] QVQLVETGGGLVQAGGSLRLSCAASGSIFSLNAMGWFRQASGGGSGC PPGYKSGVDCSPGSECKWGCYAVDGRRYGGYGADSGVSGGGSKEREFVATINRS GGRTYYADSVKGRFTISRDNGKNMVILQMHSLKPEDTAIYYCAAGTGWSPQTDNEY NYWGQGTQVTVSS (SEQ ID NO: 355)

[0649] hC3nb1 - K57 loop 3 (peptide and linker in italics)

[00650] 6)

[0651] hC3nb1- K57 C-terminus (peptide and linker in italics)

(0652) ID NO: 357)

[0653] Additionally, constructs were fabricated where the entire ultra-long K57 CDR-H3 was fused as the N- and C-terminus as follows:

[0654] K57 CDR-H3 ultra-long

[0655] TTVHQRTIKSGCPPGYKSGVDCSPGSECKWGCYAVDGRRYGGYGADS GVGSTYTHEFYVDAWGQG (SEQ ID NO: 358)

[0656] K57 ultralong CDR-H3-hC3nb1 (CDR-H3 and linker in italics)

[0657] TTVHQRTIKSGCPPGYKSGVDCSPGSECKWGCYAVDGRRYGGYGADS GVGSTYTHEFYVDAWGQGSSSSGSSSSGQVQLVETGGGLVQAGGSLRLSCAASG SIFSLNAMGWFRQAPGKEREFVATINRSGGRTYYADSVKGRFTISRDNGKNMVILQ MHSLKPEDTAIYYCAAGTGWSPQTDNEYNYW GQGTQVTVSS (SEQ ID NO: 359)

[0658] hC3nb1- K57 ultra-long CDR-H3 (CDR-H3 and linker in italics)

[ 0659 ] DAWGQG (SEQ ID NO: 360)

[0660] We measured binding to C3 and C5 by multicycle SPR kinetics and we report complete binding kinetics and equilibrium dissociation constants (KD) for binding to both proteins. Table 29. Summary of C3 binding kinetics from Biacore single-cycle kinetics.

[0661] Data from n = 3 independent experiments Table 30. Summary of C5 binding kinetics from Biacore single-cycle kinetics.

[0662] Data from n = 3 independent experiments.

[0663] Finally, we tested our constructs in AP and CP complement activation ELISAs, which measured C5b neoepitope deposition. These data are shown in Figure 13. Example 7: Antibody fragments isolated according to the invention fused to effector molecules to improve in vivo half-life

[0664] As described, the antibody fragments isolated according to the invention may be fused to an effector molecule that may increase the half-life of the isolated antibody fragment in vivo. Examples of suitable effector molecules of this type include Fc fragments and albumin.

[0665] In the present example, knob domain peptides have been inserted into albumin and Fc fragments. Advantageously, the resulting fusion proteins may confer an improved half-life to the knob domain peptides, useful in therapy. 7.1. Human and Rat Albumin Knob Domain Fusion Proteins

[0666] Because of the proximity of the N- and C-termini, knob domain peptides can be engineered within loop or turn motifs in the middle of polypeptide chains without disturbing the overall protein fold.

[0667] Fusion proteins were expressed that incorporated the knob domain peptides K57 and K92, flanked by a linker sequence, at various sites throughout the polypeptide chain of the serum albumin proteins from Homo sapiens and Rattus norvegicus, as shown in Figure 14. The sites were selected on the basis that they were unlikely to prevent neonatal Fc receptor binding. Based on this, these fusion proteins may exhibit binding to the human C5 protein and a prolonged serum half-life in vivo.

[0668] K57 knob domain (shown in italics):

[0669] GCPPGYKSGVDCSPGSECKWGCYAVDGRRYGGYGADSGV (SEQ ID NO: 334)

[0670] K92 knob domain (shown in italics):

[0671] TCPEGWSECGVAIYGYECGRWGCGHFLNSGPNISPYVST (SEQ ID NO: 450)

[0672] Linker (shown in bold): SGGGS

[0673] Rat serum albumin - K57 site 1

[0674] RGVFRREAHKSEIAHRFKDLGEQHFKGLVLIAFSQILQKCPYEEHIKLVQE VTDFAKTCVADENASGGGSGCPPGYKSGVDCSPGSECKWGCYAVDGRRYGGYG ADSGVSGGGSENCDKSIHTLFGDKLCAIPKLRDNYGELADCCAKQEPERNECFLQH KDDNPNLPPFQRPEAEAMCTSFQENPTS FLGHILHEVARRHPYFYAPELLYYAEKY NEVLTQCCTESDKAACLTPKLDAVKEKALVAAVRQRMKCSSMQRFGERAFKAWAV ARMSQRFPNAEFAEITKLATDVTKINKECCHGDLLECADDRAELAKYMCENQATISS KLQACCDKPVLQKSQCLAEIEHDNIPADLPSIAADFVEDKEVCKNYAEAKDVFLGTFL YEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEGDPPACYGTVLAEFQPLVEEPKNL VKTNCELYEKLGEYGFQNAVLVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEA QRLPCVEDILSAILNRLCVLHEKTPVSEKVTKCCSGSLVERRPCFSALTVDETYVPKE FKAETFTFHSDICTLPDKEKQIKKQTALAELVKHK PKATEDQLKTVMGDFAQFVDKC CKAADKDNCFATEGPNLVARSKEALA (SEQ ID NO: 451)

[0675] Human serum albumin - K57 site 1

[0676] RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQILQQCPFEDHVKLVN EVTEFAKTCVADESASGGGSGCPPGYKSGVDCSPGSECKWGCYAVDGRRYGGY GADSGVSGGGSENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKILY EIARRHPYFYAPELLFFAKR YKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWA VARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSIS SKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG MFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQ NLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHP EAKRMPCAEDILSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYV PKEFNAETFTFHADICTLSEKERQIKKQ TALVELVKHKPKATKEQLKAVMDDFAAFV EKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 452)

[0677] Rat serum albumin - K92 site 1

[0678] RGVFRREAHKSEIAHRFKDLGEQHFKGLVLIAFSQILQKCPYEEHIKLVQE VTDFAKTCVADENASGGGSTCPEGWSECGVAIYGYECGRWGCGHFLNSGPNISP YVSTSGGGSENCDKSIHTLFGDKLCAIPKLRDNYGELADCCAKQEPERNECFLQHK DDNPNLPPFQRPEAEAMCTSFQENPTSFLGH ILHEVARRHPYFYAPELLYYAEKYN EVLTQCCTESDKAACLTPKLDAVKEKALVAAVRQRMKCSSMQRFGERAFKAWAVA RMSQRFPNAEFAEITKLATDVTKINKECCHGDLLECADDRAELAKYMCENQATISSK LQACCDKPVLQKSQCLAEIEHDNIPADLPSIAADFVEDKEVCKNYAEAKDVFLGTFLY EYSRRHPDYSVSLLLRLAKKYEATLEKCCAEGDPPACYGTVLAEFQPLVEEPKNLV KTNCELYEKLGEYGFQNAVLVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEAQ RLPCVEDILSAILNRLCVLHEKTPVSEKVTKCCSGSLVERRPFCSALTVDETYVPKEF KAETFTFHSDICTLPDKEKQIKKQTALAELVKH KPKATEDQLKTVMGDFAQFVDKCC KAADKDNCFATEGPNLVARSKEALA (SEQ ID NO: 453)

[0679] Human serum albumin - K92 site 1

[0680] RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQILQQCPFEDHVKLVN EVTEFAKTCVADESASGGGSTCPEGWSECGVAIYGYECGRWGCGHFLNSGPNISP YVSTSGGGSENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQH KDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKILYE IARRHPYFYAPELLFFAKRYK AAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVA RLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSK LKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFL YEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLI KQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAK RMPCAEDILSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICTLSEKERQIKKQTALVE LVKHKPKATKEQLKAVMDDFAAFVEKC CKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 454)

[0681] Rat serum albumin - K57 site 2

[0682] RGVFRREAHKSEIAHRFKDLGEQHFKGLVLIAFSQILQKCPYEEHIKLVQE VTDFAKTCVADENAENCDKSIHTLFGDKLCAIPKLRDNYGELADCCAKQEPERNEC FLQHKDDNPNLPPFQRPEAEAMCTSFQENPTSFLGHILHEVARRHPYFYAPELLYY AEKYNEVLTQCCTESGGGSGCPPGY KSGVDCSPGSECKWGCYAVDGRRYGGYG ADSGVSGGGSSDKAACLTPKLDAVKEKALVAAVRQRMKCSSMQRFGERAFKAWA VARMSQRFPNAEFAEITKLATDVTKINKECCHGDLLECADDRAELAKYMCENQATIS SKLQACCDKPVLQKSQCLAEIEHDNIPADLPSIAADFVEDKEVCKNYAEAKDVFLGT FLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEGDPPACYGTVLAEFQPLVEEPKN LVKTNCELYEKLGEYGFQNAVLVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPE AQRLPCVEDILSAILNRLCVLHEKTPVSEKVTKCCSGSLVERRPCFSALTVDETYVPK EFKAETFTFHSDICTLPDKEKQIKKQTALAEL VKHKPKATEDQLKTVMGDFAQFVDK CCKAADKDNCFATEGPNLVARSKEALA (SEQ ID NO: 455)

[0683] Human serum albumin - K57 site 2

[0684] RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQILQQCPFEDHVKLVN EVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKILYEIARRHPYFYAPELLFF AKRYKAAFTECCQASGGGSGCPPGYKSGVDC SPGSECKWGCYAVDGRRYGGYG ADSGVSGGGSADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAV ARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISS KLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGM FLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQN LIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPE AKRMPCAEDILSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVP KEFNAETFTFHADICTLSEKERQIKKQTAL VELVKHKPKATKEQLKAVMDDFAAFVE KCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 456)

[0685] Rat serum albumin - K92 site 2

[0686] RGVFRREAHKSEIAHRFKDLGEQHFKGLVLIAFSQILQKCPYEEHIKLVQE VTDFAKTCVADENAENCDKSIHTLFGDKLCAIPKLRDNYGELADCCAKQEPERNEC FLQHKDDNPNLPPFQRPEAEAMCTSFQENPTSFLGHILHEVARRHPYFYAPELLYY AEKYNEVLTQCCTESGGGSTCPEGWSE CGVAIYGYECGRWGCGHFLNSGPNISP YVSTSGGGSSDKAACLTPKLDAVKEKALVAAVRQRMKCSSMQRFGERAFKAWAV ARMSQRFPNAEFAEITKLATDVTKINKECCHGDLLECADDRAELAKYMCENQATISS KLQACCDKPVLQKSQCLAEIEHDNIPADLPSIAADFVEDKEVCKNYAEAKDVFLGTFL YEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEGDPPACYGTVLAEFQPLVEEPKNL VKTNCELYEKLGEYGFQNAVLVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEA QRLPCVEDILSAILNRLCVLHEKTPVSEKVTKCCSGSLVERRPCFSALTVDETYVPKE FKAETFTFHSDICTLPDKEKQIKKQTALAELVKHK PKATEDQLKTVMGDFAQFVDKC CKAADKDNCFATEGPNLVARSKEALA (SEQ ID NO: 457)

[0687] Human serum albumin - K92 site 2

[0688] RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQILQQCPFEDHVKLVN EVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKILYEIARRHPYFYAPELLFF AKRYKAAFTECCQASGGGSTCPEGWSECGVAIY GYECGRWGCGHFLNSGPNISP YVSTSGGGSADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVA RLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSK LKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFL YEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLI KQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAK RMPCAEDILSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICTLSEKERQIKKQTALVE LVKHKPKATKEQLKAVMDDFAAFVEKC CKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 458)

[0689] Rat serum albumin - K57 site 3

[0690] RGVFRREAHKSEIAHRFKDLGEQHFKGLVLIAFSQILQKCPYEEHIKLVQE VTDFAKTCVADENAENCDKSIHTLFGDKLCAIPKLRDNYGELADCCAKQEPERNEC FLQHKDDNPNLPPFQRPEAEAMCTSFQENPTSFLGHILHEVARRHPYFYAPELLYY AEKYNEVLTQCCTESDKAACLTPK LDAVKEKALVAAVRQRMKCSSMQRFGERAFK AWAVARMSQRFPNAEFAEITKLATDVTKINKECCHGDLLECADDRAELAKYMCENQ ATISSKLQACCDKPVLQKSQCLAEIEHDNIPADLPSIAADFVEDKEVCKNYAEAKDVF LGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAESGGGSGCPPGYKSGVDCSP GSECKWGCYAVDGRRYGGYGADSGVSGGGSGDPPACYGTVLAEFQPLVEEPKNL VKTNCELYEKLGEYGFQNAVLVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEA QRLPCVEDILSAILNRLCVLHEKTPVSEKVTKCCSGSLVERRPFCSALTVDETYVPKE FKAETFTFHSDICTLPDKEKQIKKQTALAELVKHKP KATEDQLKTVMGDFAQFVDKC CKAADKDNCFATEGPNLVARSKEALA (SEQ ID NO: 459)

[0691] Human serum albumin - K57 site 3

[0692] RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQILQQCPFEDHVKLVN EVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKILYEIARRHPYFYAPELLFF AKRYKAAFTECCQAADKAACLLPKLDELRDE GKASSAKQRLKCASLQKFGERAFKA WAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQD SISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAASGGGSGCPPGYKSGVDCS PGSECKWGCYAVDGRRYGGYGADSGVSGGGSADPHECYAKVFDEFKPLVEEPQ NLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHP EAKRMPCAEDILSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYV PKEFNAETFTFHADICTLSEKERQIKKQTALVE LVKHKPKATKEQLKAVMDDFAAFV EKCCKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 460)

[0693] Rat serum albumin - K92 site 3

[0694] RGVFRREAHKSEIAHRFKDLGEQHFKGLVLIAFSQILQKCPYEEHIKLVQE VTDFAKTCVADENAENCDKSIHTLFGDKLCAIPKLRDNYGELADCCAKQEPERNEC FLQHKDDNPNLPPFQRPEAEAMCTSFQENPTSFLGHILHEVARRHPYFYAPELLYY AEKYNEVLTQCCTESDKAACLTPK LDAVKEKALVAAVRQRMKCSSMQRFGERAFK AWAVARMSQRFPNAEFAEITKLATDVTKINKECCHGDLLECADDRAELAKYMCENQ ATISSKLQACCDKPVLQKSQCLAEIEHDNIPADLPSIAADFVEDKEVCKNYAEAKDVF LGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAESGGGSTCPEGWSECGVAIY GYECGRWGCGHFLNSGPNISPYVSTSGGGSGDPPACYGTVLAEFQPLVEEPKNLV KTNCELYEKLGEYGFQNAVLVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEAQ RLPCVEDILSAILNRLCVLHEKTPVSEKVTKCCSGSLVERRPCFSALTVDETYVPKEF KAETFTFHSDICTLPDKEKQIKKQTALAELVKHKP KATEDQLKTVMGDFAQFVDKCC KAADKDNCFATEGPNLVARSKEALA (SEQ ID NO: 461)

[0695] Human serum albumin - K92 site 3

[0696] RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQILQQCPFEDHVKLVN EVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKILYEIARRHPYFYAPELLFF AKRYKAAFTECCQAADKAACLLPKLDELRDE GKASSAKQRLKCASLQKFGERAFKA WAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQD SISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAASGGGSTCPEGWSECGVAIY GYECGRWGCGHFLNSGPNISPYVSTSGGGSADPHECYAKVFDEFKPLVEEPQNLI KQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAK RMPCAEDILSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKE FNAETFTFHADICTLSEKERQIKKQTALVELVK HKPKATKEQLKAVMDDFAAFVEKC CKADDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 462)

[0697] Rat serum albumin - K57 site 4

[0698] RGVFRREAHKSEIAHRFKDLGEQHFKGLVLIAFSQILQKCPYEEHIKLVQE VTDFAKTCVADENAENCDKSIHTLFGDKLCAIPKLRDNYGELADCCAKQEPERNEC FLQHKDDNPNLPPFQRPEAEAMCTSFQENPTSFLGHILHEVARRHPYFYAPELLYY AEKYNEVLTQCCTESDKAACLTPK LDAVKEKALVAAVRQRMKCSSMQRFGERAFK AWAVARMSQRFPNAEFAEITKLATDVTKINKECCHGDLLECADDRAELAKYMCENQ ATISSKLQACCDKPVLQKSQCLAEIEHDNIPADLPSIAADFVEDKEVCKNYAEAKDVF LGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEGDPPACYGTVLAEFQPLVEE PKNLVKTNCELYEKLGEYGFQNAVLVRYTQKAPQVSTPTLVEAARNLGRVGTKCCT LPEAQRLPCVEDILSAILNRLCVLHEKTPVSEKVTKCCSGSLVERRPCFSALTVDETY VPKEFKAETFTFHSDICTLPDKEKQIKKQTALAELVKHKPKATEDQLKTVMGDFAQF VDKCCKAASGGGSGCPPGYKSGVDCSPGS ECKWGCYAVDGRRYGGYGADSGVS GGGSDKDNCFATEGPNLVARSKEALA (SEQ ID NO: 463)

[0699] Human serum albumin - K57 site 4

[0700] RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQILQQCPFEDHVKLVN EVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKILYEIARRHPYFYAPELLFF AKRYKAAFTECCQAADKAACLLPKLDELRDE GKASSAKQRLKCASLQKFGERAFKA WAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQD SISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEE PQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCK HPEAKRMPCAEDILSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA AFVEKCCKADSGGGSGCPPGYKSGVDCSP GSECKWGCYAVDGRRYGGYGADSG VSGGGSDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 464)

[0701] Rat serum albumin - K92 site 4

[0702] RGVFRREAHKSEIAHRFKDLGEQHFKGLVLIAFSQILQKCPYEEHIKLVQE VTDFAKTCVADENAENCDKSIHTLFGDKLCAIPKLRDNYGELADCCAKQEPERNEC FLQHKDDNPNLPPFQRPEAEAMCTSFQENPTSFLGHILHEVARRHPYFYAPELLYY AEKYNEVLTQCCTESDKAACLTPK LDAVKEKALVAAVRQRMKCSSMQRFGERAFK AWAVARMSQRFPNAEFAEITKLATDVTKINKECCHGDLLECADDRAELAKYMCENQ ATISSKLQACCDKPVLQKSQCLAEIEHDNIPADLPSIAADFVEDKEVCKNYAEAKDVF LGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEGDPPACYGTVLAEFQPLVEE PKNLVKTNCELYEKLGEYGFQNAVLVRYTQKAPQVSTPTLVEAARNLGRVGTKCCT LPEAQRLPCVEDILSAILNRLCVLHEKTPVSEKVTKCCSGSLVERRPCFSALTVDETY VPKEFKAETFTFHSDICTLPDKEKQIKKQTALAELVKHKPKATEDQLKTVMGDFAQF VDKCCKAASGGGSTCPEGWSECGVAIYGY ECGRWGCGHFLNSGPNISPYVSTSG GGSDKDNCFATEGPNLVARSKEALA (SEQ ID NO: 465)

[0703] Human serum albumin - K92 site 4

[0704] RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQILQQCPFEDHVKLVN EVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNE CFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKILYEIARRHPYFYAPELLFF AKRYKAAFTECCQAADKAACLLPKLDELRDE GKASSAKQRLKCASLQKFGERAFKA WAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQD SISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVF LGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEE PQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCK HPEAKRMPCAEDILSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDE TYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA AFVEKCCKADSGGGSTCPEGWSECGVAIYGYECGRWGCGHFLNSGPNISPYVST SGGGSDKETCFAEEGKKLVAASQAALGL (SEQ ID NO: 466) 7.2. Human and mouse IgG1 Fc domain knob fusion proteins

[0705] The fusion proteins were designed such that they incorporated the K149 knob domain peptide, flanked by a linker sequence, at various sites in the middle of the Homo sapiens and Rattus norvegicus immunoglobulin gamma-1 heavy chain constant region polypeptide chain, as shown in Figure 15. The sites were selected on the basis that they were unlikely to impede binding to the neonatal Fc receptor. Based on this, these K149-IgG1 fusion proteins may exhibit binding to the human C5 protein and a prolonged serum half-life in vivo.

[0706] K149 sequence (shown in italics): SCPDGFSYRSWDDFCCPMVGRCLAPRN (SEQ ID NO: 313)

[0707] Linker sequences (shown in bold): SGGGGS

[0708] Rat IgG1 heavy chain constant region Site 1 - K149

[0709] AETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSG VHTFPAVLQSGLYTLTSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRNCGGD CKPCICTGSEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISQDDPEVHFSWFVDDVEV HTAQTRPPEEQFNSTFRSVSELPILHQ DWLNGRTFRCKVTSASGGGGSSCPDGFS YRSWDDFCCPMVGRCLAPRNSGGGGSAFPSPIEKTISKPEGRTQVPHVYTMSPTK EEMTQNEVSITCMVKGFYPPDIYVEWQMNGQPQENYKNTPPTMDTDGSYFLYSKL NVKKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 467)

[0710] Rat IgG1 heavy chain constant region Site 2 - K149

[0711] AETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSG VHTFPAVLQSGLYTLTSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRNCGGD CKPCICTGSEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISQDDPEVHFSWFVDDVEV HTAQTRPPEEQFNSTFRSVSELPILHQ DWLNGRTFRCKVTSAAFPSPIEKTISKPES GGGGSSCPDGFSYRSWDDFCCPMVGRCLAPRNSGGGGSGRTQVPHVYTMSPTK EEMTQNEVSITCMVKGFYPPDIYVEWQMNGQPQENYKNTPPTMDTDGSYFLYSKL NVKKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 468)

[0712] Rat IgG1 heavy chain constant region Site 3 - K149

[0713] AETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSG VHTFPAVLQSGLYTLTSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRNCGGD CKPCICTGSEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISQDDPEVHFSWFVDDVEV HTAQTRPPEEQFNSTFRSVSELPILHQ DWLNGRTFRCKVTSAAFPSPIEKTISKPEG RTQVPHVYTMSPTKEEMTQNEVSITCMVKGFYPPDIYVEWQMNSGGGGSSCPDG FSYRSWDDFCCPMVGRCLAPRNSGGGGSGQPQENYKNTPPTMDTDGSYFLYSKL NVKKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 469)

[0714] Rat IgG1 heavy chain constant region Site 4 - K149

[0715] AETTAPSVYPLAPGTALKSNSMVTLGCLVKGYFPEPVTVTWNSGALSSG VHTFPAVLQSGLYTLTSSVTVPSSTWPSQTVTCNVAHPASSTKVDKKIVPRNCGGD CKPCICTGSEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISQDDPEVHFSWFVDDVEV HTAQTRPPEEQFNSTFRSVSELPILHQ DWLNGRTFRCKVTSAAFPSPIEKTISKPEG RTQVPHVYTMSPTKEEMTQNEVSITCMVKGFYPPDIYVEWQMNGQPQENYKNTPP TMDTDGSGGGGSSCPDGFSYRSWDDFCCPMVGRCLAPRNSGGGGSSYFLYSKL NVKKEKWQQGNTFTCSVLHEGLHNHHTEKSLSHSPGK (SEQ ID NO: 470)

[0716] Human IgG1 heavy chain constant region Site 1 - K149

[0717] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKASGGGGS SCPDGFSYRSWDDFCCPMVGRCLAPRNSGGGGSLPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRW

[0718] QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 471)

[0719] Human heavy chain constant region Site 2 - K149

0720 SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGSGGGGSSCPDGFSYRSWDDFCCPMVGRCLAPRNSGGGGSQPREPQVY TLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRW

[0721] QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 472)

[0722] Human heavy chain constant region Site 3 - K149

0723 SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNSGGGGSS CPDGFSYRSWDDFCCPMVGRCLAPRNSGGGGSGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRW

[0724] QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 473)

[0725] Human heavy chain constant region Site 4 - K149

0726 SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSGGGGSSCPDGFSYRSWDDFCCPMVGRCLAPRNSGGGGSSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 47 4) 7.3. Fusion protein synthesis, expression in Expi293F cells and binding to C5 synthesis and expression in Expi293F cells:

[0727] Custom synthesis and cloning into a pMH expression vector, containing a human cytomegalovirus (CMV) promoter and an in-frame C-terminal 10x histidine tag, can be performed by ATUM. For all constructs, a Mus musculus immunoglobulin heavy chain leader sequence can be used: MEWSWVFLFFLSVTTGVHS (SEQ ID NO: 475).

[0728] Plasmid DNA is amplified using QIAGEN Plasmid Plus Giga Kits and quantified by A260. Individual Expi293F cell cultures, at 3 x 106 cells/mL per construct, are established using Expifectamine 293 Transfection Kits (Invitrogen) as per the manufacturer's instructions. Cells are grown for four days, centrifuged at 4000 rpm for one hour and filtered through a 0.22 μm filter. Using Akta pure (GE Healthcare), Hi-Trap Nickel excel columns (e.g. GE Healthcare) are equilibrated with 10 column volumes (CV) of PBS. Cell supernatants are loaded at 1.0 mL/min and the column is washed with 7x CV of PBS, 0.5 M NaCl. The column is then washed with 7x CV of Buffer A (0.5 M NaCl, 0.02 M Imidazole, PBS pH 7.3). Protein samples are eluted by isocratic elution with 10x CV of Buffer B (0.5 M NaCl, 0.25 M Imidazole, PBS pH 7.3). After elution, protein-containing fractions are pooled and buffer exchanged into PBS using PD-10 columns (GE Healthcare) or Slide-a-lyzer dialysis cassettes (Thermo Fisher). Proteins are visualized by SDS-PAGE, quantified by A280, and aliquoted for storage at -80°C. Biacore single-cycle kinetics:

[0729] The ability of fusion proteins to specifically bind C5 can be assessed by Biacore. Using a Biacore 8K (GE Healthcare), C5 protein is immobilized on a CM5 S-Series sensor chip by amine coupling. Flow cells are activated using a minimal immobilization protocol: EDC/NHS was mixed at a 1:2 ratio (flow rate, 10 μL/min; contact time, 30 s). C5, at 1 μg/mL in pH 4.5 sodium acetate buffer, is immobilized on only the two flow cells (flow rate, 10 μL/min; contact time, 420 s). Finally, ethanolamine is applied to both flow cells (flow rate, 10 μL/min; contact time, 420 s). A final immobilization level of approximately 100 to 200 response units is achieved.

[0730] Single-cycle kinetics are measured using a 3-fold, 7-point titration of a highest concentration of 1 μM (spanning a range of 1 μM to 1.4 nM) in HBS-EP buffer (GE healthcare). A flow rate of 40 μL/min is used, with a contact time of 230 s and a dissociation time of 1800 s. Binding to the reference surface is subtracted and the data are fitted to a single-site binding model using Biacore evaluation software. Example 8: Phage display libraries of antibody fragments isolated according to the invention

[0731] Phage display libraries of knob domain peptides as disclosed herein may be generated by any suitable method, such as ligation of the knob domains directly or via a spacer to pIII of M13, for example within the entire bovine CDR-H3 or alternatively within the framework of another protein, such as an antibody fragment for example scFv, Fab or VHH.

[0732] To enable phage display of ultra-long CDR-H3 sequences on pIII of filamentous bacteriophage M13, hC3nb1, a camelid VHH that bound complement component C3, was engineered to accommodate bovine ultra-long CDR-H3 sequences. The CDR-H3 sequences were inserted between residues H74 and H75 (Kabat numbering system), within a non-binding VH 3-frame loop. When displayed on phage, the modified hC3nb1 retained binding to C3 via its canonical CDRs, in a manner that is well explained by the cocrystal structure (pdb accession code: 6EHG). In most cases binding to C5 was also observed. The sequences, methods and results are presented below. Sequences:

[0733] Sequence of hC3nb1, with insertion site shown in bold:

[0734] QVQLVETGGGLVQAGGSLRLSCAASGSIFSLNAMGWFRQAPGKEREFV ATINRSGGRTYYADSVKGRFTISRDNGKNMVILQMHSLKPEDTAIYYCAAGTGWSP QTDNEYNYWGQGTQVTVSS (SEQ ID NO: 351)

[0735] hC3nb1-K8

[ 0736 ] NO: 476)

[0737] K8 CDR-H3:

[0738] TVQQKTHQVCPDGFNWGYGCAAGSSRFCTRHDWCCYDERADSHTYG FCTGNRVTNTYEFHADA (SEQ ID NO: 477)

[0739] hC3nb1-K57

0740 SEQ ID NO: 478)

[0741] K57 CDR-H3:

[0742] TTVHQRTIKSGCPPGYKSGVDCSPGSECKWGCYAVDGRRYGGYGADS GVGSTYTHEFYVDA (SEQ ID NO: 104)

[0743] hC3nb1-K92

[ 0744 ] Q ID NO: 479)

[0745] K92 CDR-H3:

[0746] SIVHQKAHTSVTCPEGWSECGVAIYGYECGRWGCGHFLNSGPNISPYV STHKYEWYVDA (SEQ ID NO: 13)

[0747] hC3nb1-K149

[00748] )

[0749] K149 CDR-H3:

[0750] TSVLQSTKPQKSCPDGFSYRSWDDFCCPMVGRCLAPRNTYTTEFTIEA (SEQ ID NO: 1) Methods

[0751] Bovine CDRH3, hC3nb1 and hC3nb1-CDR-H3 ultra-long have been cloned into a phagemid vector by TWIST Biosciences. The phagemid vector contains a pelB leader sequence, ahead of the CDRH3, hC3nb1 or hC3nb1-CDR-H3 ultra-long sequence for display. This is followed by a polyhistidine and c-myc-Tag, which is fused directly to pIII. The entire pIII fusion protein is under the control of the glucose-repressible lac promoter. Upon superinfection with helper phage, an M13 origin of replication will result in the synthesis of single-stranded phagemid DNA, encoding the pIII fusion display construct. Each construct was prepared with and without coexpression of FKBP-type peptidyl-prolyl cis-trans isomerase (FkpA), accession number: P45523 (FKBA_ECOLI).

[0752] The constructs were transformed into TG-1 cells using a heat shock method. Briefly, 1 μL of plasmid DNA was added to 50 μL of competent cells, mixed lightly, and incubated on ice for 30 minutes. The cells were heat shocked at 42°C for 30 seconds and incubated on ice for 2 minutes. 200 μL of medium was added, and 50 μL was plated on 2TY medium + 100 μg/ml Carbenicillin + 2% Glucose and incubated overnight at 37°C.

[0753] Individual colonies were picked from the plates and plated into individual cultures of 5.0 mL of 2TY medium + 100 μg/ml Carbenicillin + 2% Glucose. The culture was incubated at 37°C with shaking until an OD of ~0.5 was reached, at which time they were superinfected with M13K07 helper phage at an MOI of 10 (New England Biolabs). The cultures were centrifuged and resuspended in the absence of glucose (2TY medium + 100 μg/ml Carbenicillin + 50 μg/ml Kanamycin) allowing leakage of the lac promoter and thus expression of the recombinant VHH-pIII fusion protein from the phagemid. Finally, the cells were grown overnight at 30°C.

[0754] Complements C5 and C3 were not site-specifically labeled with biotin to allow immobilization on an ELISA plate. Stocks of amine-reactive EZ-binding biotin (e.g. Thermo Scientific) were prepared in DMSO and frozen at -20°C. Biotin was added in a ten-fold molar excess to C5 and C3 in PBS. The solution was incubated for 60 minutes at room temperature. Unreacted biotin was removed using two sequential 0.5 mL Zeba desalting columns (Thermo Fisher) as per the manufacturer's instructions.

[0755] 96-well ELISA plates were coated with 100 μL/well of: anti c-Myc antibody (e.g. Novus clone #9E10), C3 protein or C5 protein in PBS (10 μg/mL). Additional plates were coated with streptavidin in PBS (5 μg/mL) and incubated overnight at 2 to 8°C. After coating, overnight phage recovery cultures were centrifuged at 4,500 rpm for 10 minutes. To block phage, 500 μL of supernatant was removed and placed into a fresh block containing 500 μL per well of 6% (w/v) Marvel milk powder in PBS. The coating solution was removed from the ELISA plate by washing with four cycles of Wash Buffer (PBS, 0.1% Tween 20) at 300 μL per well using a plate washer (BMG labtech). Blocking Buffer (PBS, 3% Marvel milk powder (w/v)) was added (100 μL/well) and the plate was incubated for 1 hour at room temperature. The blocked streptavidin plates were washed again and 100 μL/well of C5-biotin (5 μg/mL), C3-biotin (5 μg/mL) or Assay Buffer (1x PBS) was added and incubated for 30 minutes. All plates were then washed and the blocked phage supernatant was added (100 μL/well) and incubated for 1 hour with shaking (400 rpm). The plates were washed again and an anti-M13 HRP Ab, at a dilution of 1:11000 in Blocking Buffer (PBS, 3% Marvel milk powder (w/v), was added (100 μL/well); and incubated for 1 hour at room temperature with shaking (400 rpm). Finally, the plates were washed and 100 μL per well of ‘One Step’ TMB (Thermo Scientific) was added and incubated for approximately 5 minutes. The plate was read on a BMG Labtech plate reader at 630 nm. Results

[0756] The results are presented in Figure 16. The hC3nb1 VHH was successfully displayed on phage, as indicated by the anti-myc label and binding to human C3, both when directly immobilized to the plate and when biotinylated C3 was captured on a streptavidin plate. No cross-reactivity with the C5 protein was observed.

[0757] The hC3nb1-K8, hC3nb1-K57 and hC3nb1-K92 proteins all exhibited binding to human C5, both when directly immobilized to the plate and when biotinylated C5 was captured on a streptavidin plate. This suggests that the ultralong CDR-H3 adopts a native fold over the VHH, achieving successful surface display. Only the hC3nb1-K149 protein did not bind either C3 or C5.

[0758] Coexpression of FkpA is not a prerequisite for ultralong CDR-H3 display with hC3nb1 fusion proteins but may provide a modest increase in display levels, potentially accounting for a small increase in signal in the phage ELISA (data not shown). Example 9: Forster resonance energy transfer (FRET) assays

[0759] To provide additional evidence for high affinity binding of knob domain peptides, steady-state Forster resonance energy transfer (FRET) assays were developed. This was achieved by labeling C5 with a terbium chelate donor fluorophore and each of the active PGT121 knob fusion proteins as described in Example 2 with an AlexaFluor 647 acceptor fluorophore. Titrations of the PGT121 knob fusion proteins in the presence and absence of a saturating concentration of unlabeled C5 protein were used to derive apparent KD (KD app) for the interaction with C5 after a 24-hour incubation. Methods:

[0760] Forster resonance energy transfer (FRET) of C5 purified from serum was labeled with an amine-reactive terbium chelate (Molecular Probes, Life Technologies) as per the manufacturer's instructions. Briefly, C5 at 1.15 mg/mL was buffer exchanged into a buffer of 50 mM Bicine, 100 mM NaCl, pH 8.2 using a zeba column (Thermo Scientific). Terbium was reconstituted in DMSO to 5 mM and added at 1% final (v/v) to create an approximate 10-fold molar excess of C5. After a one-hour incubation at room temperature, unbound dye was removed by two sequential buffer exchanges into 20 mM Tris, 100 mM NaCl, pH 7.4, again using zeba columns (Thermo Scientific). The labeling ratio was quantified by UV spectroscopy, the final molar ratio of dye to protein was 4:1. Next, PGT121 knob domain fusion proteins as described in Example 2 were labeled with an amine-reactive AlexaFluor 647 (AF647) dye (molecular probe, life technologies), using the same protocol as above, but with a 30 min incubation with dye. After removal of unbound dye, UV spectroscopy quantified the final molar ratio of dye to protein as 2:1.

[0761] For determination of PGT121 KD knob domain fusion protein, C5 Tb was plated in a black, low-volume 384-well assay plate (Corning) to give a final assay concentration (FAC) of 1 nM, HBS-EP buffer (GE healthcare) or unlabeled C5 (1 μM FAC) was added. Eight-point, three-fold titrations of PGT121 knob domain fusion protein were prepared in HBS-EP buffer to give a range of 100 nM to 0.046 nM or 500 nM to 0.22 nM (FAC). Plates were wrapped in aluminum foil and incubated for 48 hours with shaking. Plates were read on an Envision plate reader (Perkin Elmer) at 2, 24 and 48 hour intervals (HTRF laser, Excitation 330 nm and Emission 665/615 nm). For adjustment, background was subtracted and curves fitted using prism software to a 4-parameter logistic model. Results:

[0762] Despite the modification of both proteins by labeling, the PGT121 fusion proteins bound C5 with high affinity (Tables 40 and 41), in a manner consistent with our SPR experiments. Table 40. KD app FRET assay for the PGT121 fusion proteins binding to C5-Tb after a 2-hour incubation Table 41. KD app FRET assay for PGT121 fusion proteins binding to C5-Tb after a 24-hour incubation

[0763] Although the fusion proteins PGT121-K92 and PGT121-K136, which exhibited particularly tight binding on the biacore mediated by a slow koff, had lower affinity by the steady-state method. Competition Assay

[0764] Knob domain titrations were then tested in a competition assay format, using displacement of the precursor PGT121 knob domain fusion protein as a readout for binding.

[0765] C5-Tb was plated at 1 nM (FAC) and knob domain peptide titrations prepared in HBS-EP buffer, to give a range from 1000 nM to 0.46 nM (FAC). PGT121 knob domain AF647 fusion proteins were prepared to give the following concentrations, which are equal to the KD app measured in the previous experiment: PGT121-K8 AF647 5 nM (FAC), PGT121-K57 AF647 3 nM (FAC), PGT121-K92 AF647 12 nM (FAC), PGT121-136 AF647 40 nM (FAC) and PGT121-149 AF647 66 nM (FAC). The plates were wrapped in aluminum foil, incubated for 24 hours with shaking and read on an Envision plate reader (HTRF laser, Excitation 330 nm and Emission 665/615 nm). The curves were fitted using prism software to a 4-parameter logistic model. The IC50 values were converted to inhibitory constants (Ki)

[0766] IC50 values were measured and inhibition constants (Ki) were derived for each of the knob domain peptides using the Cheng-Prusoff equation:

[0767] The results are presented in Table 42 below. Table 42. Competition FRET Assay 24-Hour Incubation

[0768] These provide additional evidence for high affinity, sub-50 nM interactions and consolidate the SPR observations in Example 2. In all FRET experiments the Hill slopes were within the expected range (0.5 to 2), i.e. indicative of reversible binding as defined by the law of mass action, while the nM shift of the PGT121 knob domain fusion proteins suggests that the interactions are indeed site specific. Example 10:

[0769] Additional experiments were conducted to characterize knob domains produced by chemical peptide synthesis. 1 - Knob domain peptides

[0770] K149A and K149B and their production method are described in Example 4 (sometimes suffixed chemSD for Site Directed). K8, K57, K92, K149 were produced using the free energy method and are shown below in Table 43 (sometimes suffixed chemFE for Free Energy). For the peptides produced by both methods, liquid chromatography/mass spectrometry (LC/MS) confirmed that masses compatible with the predicted amino acid sequences with complete bond formation were unanimously present. Table 43: Sequences of the knob domain tested in the present example

Chemical variants

[0771] Cyclic K8 chemFE: To create a small, cyclic antibody fragment, head-to-tail cyclization of the K8chemFE knob domain was performed, resulting in a cyclic peptide referred to as “cyclic K8 chemFE”). The N- and C-termini of the knob domains are in close proximity and this may mean that among the antibody fragments, they are uniquely amenable to cyclization.

[0772] K92 chemFE W21A/ W6A/ Y14A/ Y16A/ F26A: A series of π-π stacking interactions span one side of K92, spanning residues: Y14, Y16, F26, H25, W21, W6, and P3. To assess the importance of stacking interactions for maintaining tertiary structure, a series of alanine mutants, Y14A, Y16A, F26A, H25A, W21A, and W6A, were synthesized.

[0773] K92 chemFE W21H: Based on the structure, it was considered that a W21H mutation would introduce an additional electrostatic interaction between the polar nitrogen of the histidine imidazole ring and N77C5 (numbering based on the mature C5 sequence), possibly leading to improved binding affinity.

[0774] K57 chemFE -Palmitoyl: To explore the effect of fatty acid conjugation, a palmitoylated form of K57chem was synthesized. 2 - Binding to C5

[0775] Binding of knob domain peptides to human C5 was measured by SPR, using a multiple cycle kinetic method as follows:

[0776] Kinetics were measured using a Biacore 8K (GE Healthcare) with a CM5 chip, which was prepared as follows: EDC/NHS was mixed at a 1:1 ratio (flow rate, 10 μL/min; contact time, 30 seconds), and human C5 protein at 1 μg/mL in sodium acetate buffer pH 4.5 was injected into a single flow cell (flow rate, 10 μL/min; contact time, 60 seconds). Final immobilization levels in the range of 2000 to 3000 response units (RU) were obtained, to yield theoretical Rmax values of ~50 to 60 RU. Serial dilutions of peptide were prepared in HBS-EP buffer and injected (flow rate, 30 μL/min; contact time, 240 s; dissociation time, 6000 s). After each injection, the surface was regenerated with two sequential injections of 2 M MgCl2 (flow rate, 30 μL/min; contact time, 30 s). Binding to the reference surface was subtracted, and the data were fitted to a single-site binding model using Biacore evaluation software.

[0777] The results are presented in Table 44 below. Table 44: Summary of Biacore multiple cycle kinetics data from n = 3 experiments *The stoichiometric ratio of the mean is calculated by dividing the measured Rmax value with the theoretical Rmax

[0778] The chemical knob domains bound C5 with high affinity (Table 44), equivalent to values previously reported for biological peptides. For K149, which has two disulfide bonds, the adjacent cysteines C15 and C16 are unable to pair, giving rise to only two potential disulfide bond arrangements: K149chemSD-A (C2-C15, C16-C22) and K149chemSD-B (C2-C16, C15-C22). Although both forms bind C5, K149B exhibited approximately 35-fold lower affinity. When produced by the free energy method (hereafter suffixed chemFE), K149 (K149chemFE) bound C5 with affinity equal to the higher affinity K149chemSD-A form.

[0779] Since the disulfide bond mismatch of K149 was tolerated to some degree, the effect of removing the disulfide bonds entirely was next tested by reducing and capping cysteines with iodoacetamide (IAM). Following LC/MS analysis, to confirm that uniform capping had occurred, binding to C5 was again measured by SPR. For K149chem, loss of disulfide bonds completely abolished binding, while for K92 there was a substantial drop in affinity from 411 pM to 2 μM, primarily mediated by prolongation of the association rate. Loss of disulfide bonds at K57 also affected affinity, but a KD of 76 nM was retained. In both cases, decreases in affinity were predominantly mediated by a decrease in the association rate, potentially due to a loss of tertiary structure. Chemical mutants:

[0780] Cyclic K8 chemFE: This fragment bound human C5.

[0781] K92 chemFE W21A/ W6A/ Y14A/ Y16A/ F26A: When tested for binding to C5 by SPR, removal of any aromatic residue was highly detrimental to binding, while the H25A mutation completely prevented peptide folding. All mutations decreased affinity relative to wild-type K92chemFE, including residues distal to the paratrope such as W6 and residues that do not support molecular interactions with C5 such as Y16; highlighting the importance of non-covalent tertiary structure in maintaining binding integrity.

[0782] K92 chemFE W21H: Although this mutation did not actually improve the affinity of K92chemFE, replacing W21 with a histidine was tolerated such that the mutant would fold and produce a much lower loss of affinity relative to removing the aromatics at the other sites; suggesting that the histidine was partially able to maintain the stacking interactions required for folding and maintenance of tertiary structure. Notably, W21H was able to sustain affinity due to the greatly improved kon value relative to the other mutants.

[0783] K57 chemFE -Palmitoyl: binding to C5 was not affected by conjugation at the N-terminus. 3 - Functional Activity

[0784] Having demonstrated binding to C5, biological function was assessed in a range of complement ELISAs and erythrocyte hemolysis assays that were specific for activation of either the alternative (AP) or classical (CP) pathway.

[0785] Complement ELISA: Assays were conducted using CP and AP Complement ELISA functional kits (SVAR, COMPL 300 RUO) as per the manufacturer's protocol. For sample preparation, serum was diluted as per the respective protocol for CP and AP assays; serial dilutions of peptides were prepared and allowed to incubate with serum for 15 minutes at room temperature prior to plating.

[0786] Hemolysis assay: For AP, 50 μL of 24% normal human sera (Complement Technology), 50 μL of 20 mM MgEGTA (Complement Technology), and 48 μL of GVB0 buffer (0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN3, pH 7.3, Complement Technology) were aliquoted into a single well of a 96-well tissue culture plate (USA Scientific) and then mixed with 2 μL of inhibitors serially diluted in DMSO. Following equilibration for 15 minutes at room temperature, 50 μL of rabbit erythrocytes (Complement Technology), at 2.5 x107 per well, were added to the plates, which were then incubated at 37°C for 30 minutes. The plates were centrifuged at 1,000 x g for 3 min and 100 μL of supernatant was collected, transferred to another 96-well tissue culture treated plate and absorbance was measured at 412 nm. For CP, 50 μL of 4% normal human sera, 48 μL of GVB++ buffer (0.1% gelatin, 5 mM Veronal, 145 mM NaCl, 0.025% NaN3, pH 7.3 with 0.15 mM CaCl2 and 0.5 mM MgCl2, Complement Technology) and 2 μL of inhibitors serially diluted in DMSO were aliquoted into a single well and equilibrated as described. Next, 100 μL of antibody-sensitized sheep red blood cells (Complement Technology) at 5 x 107 per well were added to the plate, which was then incubated at 37°C for 1 hour. Samples were subsequently processed as described.

Results

[0787] The results of the CP ELISA assays are presented in Figure 18A. The results of the AP ELISA assays are presented in Figure 18B. The results of the CP and AP hemolysis assays are presented in Figure 19A and Figure 19B respectively.

[0788] In complement activation ELISAs that screened for C5b deposition, K57chemFE was a potent and fully effective inhibitor of both CP and AP; chemical K8 (K8chemFE) was a partial inhibitor of both CP and AP; while chemical K92 (K92chemFE) partially inhibited AP and showed modest, dose-dependent enhancement of CP. Consistent with results obtained with biologically derived K149, K149chemFE was a silent, nonfunctional binder of C5.

[0789] The behavior in the erythrocyte hemolysis assays was consistent with the ELISAs. K57chemFE was a potent and fully effective inhibitor of complement-mediated cell lysis, K92chemFE was active only in the AP-directed hemolysis assay, and K8 was a partial inhibitor for CP and weakly active in the AP assay. Importantly, these ELISA and hemolysis assay observations closely mirrored those previously reported with the biological forms of the peptides. In the hemolysis assays, K57chemFE was broadly equivalent to the two clinical C5 inhibitors: RA101295-14, a close analogue of the macrocyclic peptide Zilucoplan from UCB-Ra Pharma, which is currently in Phase III trials, and SOBI002, an affibody from Swedish Orphan Biovitrum, which was discontinued after showing transient adverse effects in a Phase 1 trial.

[0790] Cyclic K8chemFE: was a functionally active complement inhibitor, with only a modest loss of potency relative to K8chemFE (Figure 19). 4 - Cross-reactivity

[0791] As target binding may influence pharmacokinetics, we tested the knob domains for cross-reactivity with Rattus norvegicus (rat) C5 protein.

Method

[0792] Kinetics were measured using a Biacore 8K (GE Healthcare) with a CM5 chip, which was prepared as follows: EDC/NHS was mixed at a 1:1 ratio (flow rate, 10 μL/min; contact time, 30 seconds), and rat C5 protein at 1 μg/mL in pH 4.5 sodium acetate buffer was injected into a single flow cell (flow rate, 10 μL/min; contact time, 60 seconds). Final immobilization levels in the range of 2000 to 3000 response units (RU) were obtained, to yield theoretical Rmax values of ~50 to 60 RU. Serial dilutions of peptide were prepared in HBS-EP buffer and injected (flow rate, 30 μL/min; contact time, 240 s; dissociation time, 6000 s). After each injection, the surface was regenerated with two sequential injections of 2 M MgCl2 (flow rate, 30 μL/min; contact time, 30 s). Binding to the reference surface was subtracted, and the data were fitted to a single-site binding model using Biacore evaluation software.

Results

[0793] By SPR, K8chem was cross-reactive with rat C5 protein as well as C5 from another species (data not shown), while K57chem was specific for human C5. 5 - Stability

[0794] To test whether knob domains are resistant to proteolysis by virtue of their abundant disulfide bonds, we used mass spectrometry to track the stability of K8chemFE, K57chemFE, and K57chemFE-palmitoyl in human, rat, and Mus musculus (mouse) plasma over a 24-hour period.

Method

[0795] Stability in rat/mouse/human Lithium Heparinized plasma was evaluated at a concentration level of 1.25 μg/mL for K57chemFE-Palmitoyl, 6.25 μg/mL for K57 and 8 ng/mL for K8, over a 24 hour period at room temperature. A calibration line and suitable quality control samples were prepared and frozen at -80°C, along with additional separate boosts for evaluation. These separate boosts were placed in the freezer after the original calibration line at time intervals of 0.116, 0.25, 0.5, 1, 2, 4, 6 and 24 hours. At a minimum of 1 hour freeze for the final 24 hour boost. These were extracted via protein precipitation along with the original calibration line.

Results

[0796] The unmodified knob domains were exceptionally stable in human plasma, with >75% of peptide remaining intact after 10 hours (Figure 20). 6 - Pharmacokinetics

[0797] The pharmacokinetics of K8chemFE, K57chemFE, and K57chemFE-palmitoyl were measured following intravenous bolus dosing to Sprague Dawley rats (Figure 21). Method

[0798] Plasma pharmacokinetics were studied for each peptide in male Sprague-Dawley rats of body weight between 324 and 425 g. The drug was administered intravenously via the tail vein. The doses administered were 10 mg/kg for the peptides K8chemFE, K57chemFE and 1 mg/kg for K57chemFE-Palmitoyl. Blood samples were taken at 7 minutes, 15 minutes, 30 minutes, followed by 1, 2, 4, 8 and 24 hours. Blood was collected into Li Heparin tubes and spun down to prepare plasma samples for bioanalysis. Bioanalytical data were analyzed on an individual animal basis using Pharsight Phoenix 64 Build 8.1. Non-compartmental analysis was conducted and mean pharmacokinetic parameters for each drug calculated.

Results

[0799] Following administration at 10 mg/kg, K57chemFE underwent renal clearance and was eliminated in a rapid manner typical of low molecular weight peptides and proteins (t1/2 = 17 mins/plasma clearance [Clp] = 10.6 ml/min/kg). In contrast, K8chemFE bound tightly to rat C5 protein and adopted target-like kinetics, resulting in markedly enhanced exposure (t1/2 = ~9 hours/Clp = 3.3 ml/min/kg). Due to reduced solubility, K57chemFE-palmitoyl was tested at a dose of 1 mg/kg; conjugation of a palmitic fatty acid prolonged exposure relative to unmodified K57chem (t1/2 = 1.6 hours/Clp = 0.8 ml/min/kg). This suggested that conjugation, potentially in combination with cyclization or other chemical modifications, is a viable route to prolong the biological exposure of chemical knob domains.

Example 11: Crystal structure of the C5-K92 complex

[0800] A crystal structure of the K92 knob domain isolated from bovine (K92bio) or produced by chemical synthesis (K92chemFE) in complex with C5 was also solved at a resolution of 2.75 Å and 2.57 Å respectively as described in Example 5.

[0801] A simulated mFo-DFc annealing exclusion map of the C5-K92chemFE complex showed clear electron density for the peptide, while the final structure shows that the fold and disulfide bond arrangement of C5-K92chemFE are contiguous with K92bio. Analysis with the PDBPiSA macromolecular structure analysis tool confirmed that the molecular interactions supporting binding to C5 are compatible between K92chemFE and K92bio.

[0802] Figure 22A shows K92chemFE in complex with C5. Figure 22B shows the disulfide arrangement at K92. Figure 22C shows the position of the K92 mutations mentioned in the previous example. Example 12: Construction of ultralong CDR-H3 phage libraries for the discovery of knob domain peptides

[0803] The following method allows for the generation of antigen-enriched ultra-long CDR-H3 libraries that can be performed for any antigen of interest. Ultra-long CDR-H3 phage display libraries would be generated by any suitable method, such as ligation to ultra-long CDR-H3 directly or via a spacer to pIII of M13, e.g. fused to hC3nb1 VHH.

[0804] As an example, to enable the creation of antigen-enriched CDR-H3 libraries, immunizations of cattle with human C5 and with human and mouse serum albumin proteins were performed. The ultra-long CDR-H3 sequences were specifically amplified and fused directly to the bacteriophage minor coat protein gene g3p (or pIII) to enable enrichment against antigen using phage display. The phagemid vector contains a pelB leader sequence, ahead of the ultra-long CDR-H3 sequence for display. This is followed by a polyhistidine and c-myc tag, which is fused directly to g3p. The entire open reading frame encoding the fusion protein is under the control of the glucose-repressible lac promoter. Upon superinfection with helper phage, an M13 origin of replication will result in the synthesis and packaging of single-stranded phagemid DNA encoding the phage display construct into the phage virion. By this method, ultra-long CDRH3 sequences can be displayed on the surface of phage virions to retain genotype-to-phenotype linkage, and through phage biopanning, these antibody fragments that bind the immunized antigens were isolated.

Methods Immunization:

[0805] Holstein Friesian cattle were immunized with purified human C5 or serum albumin protein antigens, one cow per immunogen, with one prime and two boosters at four-week intervals. For C5, two adult Friesian cows were immunized with C5 Complement (CompTech). For the initial immunizations, 1.25 mg C5 was mixed 1:1 (v/v) with Fama Adjuvant (GERBU Biotechnik). Three subcutaneous injections into the shoulder were performed at 1-month intervals. Ten days after immunization, 10 mL of blood was drawn to allow testing of serum antibody titer. For subsequent boosters, 1.25 mg C5 was emulsified 1:1 with complete Freund's adjuvant (Sigma)—and, for the final dose, Montanide (Seppic)—immediately prior to subcutaneous injection into the shoulder. For human and mouse serum albumin (HSA/MSA respectively), three immunizations were performed at four-week intervals, where 1 mg total protein, 0.5 mg each HSA and MSA, was homogeneously mixed 1:1 (v/v) with Montanide adjuvant (Seppic) prior to administration into the left shoulder.

Collection of immunological material and ELISA assay:

[0806] Seven days following the second and third immunizations, serum samples were taken to assess target-specific responses using ELISA on protein adsorbed directly to Nunc Maxisorb plates at 2 μg/ml in PBS. Serum was diluted in PBS supplemented with 1% BSA (w/v) and the response determined using an anti-Bovine H+L - HRP-conjugated secondary antibody (Stratech). After determination of a selective serum titer response, ten days later the final additional booster sample was taken from these cows: 500 mL of blood, a spleen sample ~2 cm3 and a single draining lymph node taken close to the immunization site. Identification of ultra-long CDR-H3 sequences binding to the antigen of interest:

[0807] An RNeasy plus midi kit (Qiagen) was used to purify total RNA from 5 x 107 cells purified from lymph nodes, as per the manufacturer's protocol. The RNA was immediately used in an RT-PCR reaction using Super script IV villus Master Mix (Invitrogen).

Primary PCR

[0808] The cDNA encoding CDR-H3 regions was selectively amplified by PCR. The primers annealed to the 3-frame and 4-frame of the heavy chain, thereby amplifying the CDR-H3 region regardless of changes in length (standard or ultra-long CDR-H3). The primers used (read from 5’ to 3’) were: forward primer: GGACTCGGCCACMTAYTACTG (SEQ ID NO: 446) and reverse primer: GCTCGAGACGGTGAYCAG (SEQ ID NO: 447). PCR was performed using Phusion Green Hotstart II Master mix (Thermo scientific). DNA from the primary PCR was column purified before being used in a secondary PCR.

Secondary PCR

[0809] Primer sets derived from the upstream and downstream stem sequences of the ultra-long CDR-H3 were used to specifically amplify ultra-long sequences from the primary PCR DNA. 7 upstream stem primers were used individually in separate PCR reactions while 6 downstream stem primers were pooled. The upstream primers were used at a concentration of 10 μM and each downstream primer was at a concentration of 10 μM in the pooled solution. The primer sets also contained SfiI and NotI restriction enzyme sites that allowed ligation to the vector.

[0810] The primers used (read from 5’ to 3’) were as follows: Upstream primer set:

[0811] CTCGCGGCCCAGCCGGCCATGGCCACTACTGTGCACCAAAAAACA (SEQ ID NO: 482)

[0812] CTCGCGGCCCAGCCGGCCATGGCCACTACTGTGCACCAAAGAACC (SEQ ID NO: 483)

[0813] CTCGCGGCCCAGCCGGCCATGGCCACTACTGTGCACCAAAAAACG (SEQ ID NO: 484)

[0814] CTCGCGGCCCAGCCGGCCATGGCCACTACTGTGCACCAACAAACT (SEQ ID NO: 485)

[0815] CTCGCGGCCCAGCCGGCCATGGCCACTACTGTGCACCAACAGACC (SEQ ID NO: 486)

[0816] CTCGCGGCCCAGCCGGCCATGGCCACTACTGTGGTCCAGAAAACA (SEQ ID NO: 487)

[0817] CTCGCGGCCCAGCCGGCCATGGCCACTACTGTAGTCCAACGAACA (SEQ ID NO: 488) Descendant primer set:

[0818] TGATGGCGGCCGCGGCATCGACGTACCATTCGTA (SEQ ID NO: 489)

[0819] TGATGGCGGCCGCGGTATCGACGTACCATTCGTA (SEQ ID NO: 490)

[0820] TGATGGCGGCCGCGGCTTCGACGTACAATTCGTA (SEQ ID NO: 491)

[0821] TGATGGGCGGCCGCGGCATTGACGTAGAATTCGTA (SEQ ID NO: 492)

[0822] TGATGGGCGGCCGCGGCCTCGATGTCAAATTCGTA (SEQ ID NO: 493)

[0823] TGATGGCGGCCGCGGTTTCGACGTGGTATTCGTA (SEQ ID NO: 494)

[0824] PCR was performed using Phusion Green Hotstart II Master mix (Thermo scientific). The secondary PCR product was column purified before being used for cloning into the phagemid vector. Library Construction

[0825] A phagemid vector (derived from pUC119) was used throughout the research. Both the phagemid vector and the secondary PCR product were digested using NotI and SfiI. Once digested, the vector and CDR-H3 inserts were ligated using a 1:3 molar ratio of vector to insert. The precipitated ligation was used to transform E. coli TG1 cells (Lucigen) using electroporation. Recovered samples were plated on the selective medium, 2TY Agar supplemented with 1% Glucose, 100 μg/mL Carbenicillin and incubated at 30°C overnight. Phage Recovery

[0826] The culture was harvested by adding selective liquid medium, 2TY supplemented with 1% Glucose and 100 μg/mL Carbenicillin and scraping the biomass into the liquid culture. The harvested culture was used to plate fresh selective medium at an OD600 of 0.1 AU. The culture was incubated at 37°C until it reached an OD600 of approximately 0.5 AU. Helper phage M13K07 was added at a multiplicity of infection (MOI) of 20 and the culture was allowed to stand at 37°C for 1 hour. The culture was then centrifuged and the pellet resuspended in 2TY medium supplemented with 50 μg/mL Kanamycin and 100 μg/mL Carbenicillin and incubated at 30°C overnight.

[0827] Following overnight incubation, the culture supernatant was recovered by centrifugation, the phage pellet resuspended in 20 mL of PBS. An additional round of precipitation was performed and the phage pellet resuspended in PBS supplemented with 20% glycerol to a final concentration of approximately 1012 PFU/mL. The purified phage aliquots were stored at -80°C until required.

Phage Bio-Panning

[0828] For the C5 libraries, a single round of enrichment was performed with human or mouse C5 protein. For the albumin library two rounds of enrichment were performed with human and mouse serum albumin. Phages were blocked for 30 minutes. Biotinylated antigen was added to the blocked phage at a concentration of 100 nM and incubated at room temperature with mixing.

[0829] Streptavidin Dynabeads (Thermofisher) were resuspended in the blocked phage solution, incubated for 10 min, and washed four times with 1 mL of 0.1% Tween 20 in PBS. After washing, the beads were pelleted and the supernatant removed. 500 μL of 0.1 M hydrochloric acid was added to elute the phage from the beads prior to incubation with mid-log E. coli TG1 cells to allow bacterial infection.

[0830] Cells were cultured on the solid selective medium, 2TY Agar supplemented with 1% glucose and 100 μg/mL Carbenicillin for the recovery of enriched sublibraries and for single colony screening. Monoclonal Phage Screening ELISA

[0831] Colonies were picked onto a 96-well culture plate containing 2TY selective medium supplemented with 1% Glucose and 100 μg/ml Carbenicillin and were incubated at 37°C until the wells reached mid-log phase of growth prior to the addition of M13K07 helper phage. The block was incubated without shaking for 1 hour to obtain infection and phage production using continued culture on selective medium supplemented with 50 μg/ml Kanamycin and 100 μg/ml Carbenicillin.

[0832] Antigen binding was assessed by monoclonal phage ELISA, Nunc MaxiSorp 96-well flat bottom plates (Thermofisher) were coated with a 2.5 mg/ml solution of human, mouse or rat serum albumin, human C5 or mouse C5 diluted with PBS and left overnight at 4°C. Negative control plate and Anti-myc label plate were also prepared.

[0833] The recovered monoclonal phage supernatants were blocked with an equal volume of a 2% (w/v) milk in PBS solution for serum albumin proteins or 2% BSA and 2% milk (w/v) in PBS solution. The coating solution was removed from the coated Nunc plates and each plate was blocked with 1% BSA in PBS (for C5 libraries) or 1% milk powder (for albumin library). Both phage and Nunc plates were blocked for 1 hour at room temperature. Nunc plates were washed using a 96-well microplate washer (BioTek) with a PBS solution containing 0.1% Tween20 (Sigma Aldrich). 100 μL of the blocked phage solution was added to each well of the Nunc MaxiSorp plate and allowed to shake at room temperature for 1 hour. The plates were washed and dried as previously described. 100 μL of an anti-M13-horseradish peroxidase-conjugated antibody (GE Healthcare) diluted (1:5,000) in a 1% BSA or milk solution was added to each well and allowed to shake at room temperature for 1 h. The plates were washed once more and 50 μL of TMB solution (Merck Millipore) was added to each well. The plates were allowed to shake for 10 min. Absorbance was measured at 630 nm and 490 nm. Binding signals were obtained where the absorbance at 630 nm was >3 times the signal from the irrelevant antigen.

[0834] For sequencing, 0.5 μL of cell containing enriched monoclonal recovery block media was added to the corresponding wells on the PCR plate containing: 20.75 μL of DPEC treated water; 2.5 μL of 10x Standard Taq buffer (New England Biolabs); 0.5 μL of 10 μM forward and reverse primer stock and 0.25 μL of Taq DNA polymerase (5000 u/mL - New England Biolabs).

[0835] The primers used to amplify the insert within the phagemid vector and annealing to the phagemid vector (read from 5’ to 3’) were as follows:

[0836] Advanced: GTTGGCCGATTCATTAATGCAG (SEQ ID NO: 495)

[0837] Reverse: ACAGACAGCCCTCATAGTTAGC (SEQ ID NO: 496)

[0838] The plate was heated to 95°C for five minutes in a thermal cycler and then heated for thirty-five cycles of: (95°C for 40 seconds; 55°C for 40 seconds; 68°C for 100 seconds and 72°C for 2 minutes). Finally, 1 μL of Illustra ExoProStar was added to each well to remove unused dNTPs and primers prior to sequencing. The plate was placed in a thermal cycler at 37°C for 40 minutes and 80°C for 15 minutes, prior to Sanger sequencing, performed at Macrogen.

Results

[0839] By monoclonal phage ELISA the following ultra-long CDR-H3 sequences were identified as binders (OD > 0.2 AU): Table 45: Ultra-long CDR-H3 from albumin antisera Table 46: Albumin-specific ultralong CDR-H3 knob domain sequences Table 47: Anti-C5 ultra-long CDR-H3 Table 48: Anti-C5 ultralong CDR-H3 knob domains

[0840] The minimal sequence of knob domains as defined in this application are highlighted in bold in the Table below.

Claims (10)

1. Isolated antibody fragment, characterized in that the fragment is a variant of the knob domain of a bovine ultra-long CDR-H3 or a portion thereof that binds to an antigen of interest: a) comprising a disulfide bond in a position that does not occur naturally, b) having a cysteine replaced in a natural position by another amino acid, for example serine, or c) being cyclized. 2. Polypeptide, characterized by the fact that it comprises at least two isolated antibody fragments, in which the fragments are the knob domains of an ultra-long bovine CDR-H3 or a portion thereof that binds to an antigen of interest, and in which the antibody fragments are linked to each other, optionally through a linker, for example, a cleavable linker. 3. Polypeptide, characterized by the fact that it comprises at least two isolated antibody fragments as defined in claim 1, in which the antibody fragments are linked to each other, optionally through a linker, for example, a cleavable linker. 4. Polypeptide according to claim 2 or 3, characterized in that at least two isolated antibody fragments bind to (a) the same antigen or (b) different antigens. 5. Pharmaceutical composition, characterized by the fact that it comprises an isolated antibody fragment as defined in claim 1, or a polypeptide as defined in any one of claims 2 to 4, in combination with one or more pharmaceutically acceptable excipients, diluents or carriers. 6. Isolated antibody fragment according to claim 1, or polypeptide according to any one of claims 2 to 4, characterized in that it is for use in therapy. 7. Polynucleotide, characterized by the fact that it encodes an isolated antibody fragment as defined in claim 1 or a polypeptide as defined in any one of claims 2 to 4. 8. Vector, characterized by the fact that it comprises a polynucleotide as defined in claim 7. 9. A host cell comprising the polynucleotide as defined in claim 7 or the vector as defined in claim 8. 10. A method for producing an isolated antibody fragment that binds to an antigen of interest as defined in claim 1, comprising: a) generating a phage display library of ultra-long CDR-H3 sequences; and, b) enriching the phage display library against the antigen of interest to produce an enriched population of phages that bind to the antigen of interest; and, c) sequencing an ultra-long CDR-H3 from the enriched population of phages obtained in step b); and, d) expressing or synthesizing an isolated antibody fragment derived from the ultra-long CDR-H3 obtained in step c).