CN111454362B - Antibodies that specifically bind to CD40 and uses thereof - Google Patents

Abstract

The present invention provides an isolated monoclonal antibody that specifically binds to human CD 40. Nucleic acid molecules encoding the antibodies are also provided, as are expression vectors, host cells, and methods for expressing the antibodies. The invention also provides immunoconjugates, bispecific molecules, chimeric antigen receptors, oncolytic viruses and pharmaceutical compositions comprising the antibodies, and methods of treatment using the antibodies of the invention.

Description

Antibodies that specifically bind to CD40 and uses thereof

This application is a division of chinese patent application CN201910160181.7 entitled "antibodies that bind CD40 and uses thereof" filed 3, 4, 2019.

Technical Field

The present invention relates to an antibody specifically binding to human CD40, and its preparation and use, in particular its use in the treatment of human diseases associated with CD40, such as cancer, inflammatory diseases, infectious diseases, atherosclerotic thrombi, and respiratory diseases.

Background

CD40, also known as tumor necrosis factor receptor superfamily member 5 or TNFR5, is a transmembrane costimulatory protein that is expressed on antigen presenting cells such as B cells, macrophages, and dendritic cells. Binding of this protein to CD40L (CD154), a major ligand expressed primarily by activated T lymphocytes and platelets, activates antigen-presenting cells, stimulates multiple downstream signaling pathways, including immune cell activation and proliferation, and cytokine and chemokine production, enhancing cellular and immune function (Ara a et al, (2018) Immunotargets Ther 7: 55-61).

On the other hand, CD40 is also present on non-immune cells and tumors (Costello et al, (1999) immunological Today 20 (11): 488-493; Tong et al, (2003) Cancer Gene Ther 10 (1): 1-13; Lee et al, (2014) Curr Cancer Drug Targets 14 (7): 610-620; Ara A et al, (2018) supra), and has been reported to be associated with various immune diseases (including autoimmune diseases), atherosclerotic thrombi, cancers and respiratory diseases. For example, CD40/CD40L expression is up-regulated in atheroma-associated cells. CD40 is expressed in almost all B cell malignancies and up to 70% of solid tumors, and CD40 ligand binding in certain B cell malignancies contributes to increased expression of various factors that protect tumor cells from apoptosis (Lee et al, (1999) Proc Natl Acad Sci USA 96: 9136-9141).

Although the role of CD40 in tumorigenesis is very complex, several CD40 antibodies have been developed for potential tumor therapy. CP-870,893, a fully human IgG 2-activated CD40 antibody developed, can activate dendritic cells and has shown clinical utility in advanced Cancer patients in a variety of settings (Vonderheide et al, (2007) J Clin Oncol 25 (7): 876-. Dacetuzumab, also known as SGN-40, is a humanized IgG 1-activated CD40 antibody developed by Seattle Genetics that exhibits anti-tumor activity upon weekly intravenous administration, particularly in patients with diffuse large B-cell lymphoma. Preclinical data also show the synergistic effect of Dacetuzumab with other drugs such as CD20 mab rituximab (Lapalombella et al, (2009) BT J Haematol 144 (6): 848-. A chimeric IgG1 agonist anti-human CD40 antibody developed by Chi Lob 7/4, the british Cancer Research UK, is undergoing initial clinical testing. 11 of 21 patients were stable without partial or complete remission (Chowdhury et al, (2014) Cancer Immunol Res 2 (3): 229-. In addition, antagonistic CD40 antibodies also showed anti-tumor effects in human multiple myeloma and chronic lymphocytic leukemia (Bensinger W et al, (2012) Br J Haematol.159 (1): 58-66; Mohammad Luqman et al, (2008) Blood 112: 711-.

More CD40 antibodies with better pharmacological properties are needed.

Disclosure of Invention

The present invention provides an isolated monoclonal antibody, e.g., a murine, human, chimeric or humanized monoclonal antibody that binds to CD40 (e.g., human CD40 and monkey CD 40). It may be an agonistic CD40 antibody that activates the CD40 signaling pathway.

The antibodies of the invention can be used in a variety of applications, including detection of CD40 protein, treatment or prevention of CD 40-associated diseases, such as cancer, inflammatory diseases, infectious diseases, atherosclerotic thrombi, and respiratory diseases.

Accordingly, in one aspect, the invention relates to an isolated monoclonal antibody (e.g., a humanized antibody), or antigen binding portion thereof, comprising a heavy chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, wherein the CDR1, CDR2 and CDR3 regions comprise (1) the amino acid sequence of SEQ ID NOs: 1. 8 and 15; (2) SEQ ID NOs: 1. 9 and 15; (3) SEQ ID NOs: 2. 9 and 15; (4) SEQ ID NOs: 3. 10 and 16; (5) SEQ ID NOs: 4. 11 and 17; (6) SEQ ID NOs: 5. 12 and 18; (7) SEQ ID NOs: 6. 13 and 19; or (8) SEQ ID NOs: 7. 14 and 20; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to these sequences, respectively, wherein the antibody, or antigen-binding portion thereof, binds to CD 40.

In one aspect, an isolated monoclonal antibody, or antigen binding portion thereof, of the invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NOs: 37. 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, or an amino acid sequence comprising at least 80%, 85%, 90%, 95%, 98%, or 99% identity to these sequences, wherein the antibody or antigen-binding portion thereof binds to CD 40.

In one aspect, an isolated monoclonal antibody, or antigen-binding portion thereof, of the invention comprises a light chain variable region comprising the CDR1 region, CDR2 region and CDR3 region, wherein the CDR1 region, CDR2 region and CDR3 region comprise (1) the amino acid sequence of SEQ ID NOs: 21. 27 and 31; or (2) SEQ ID NOs: 22. 28 and 32; (3) SEQ ID NOs: 23. 29 and 33; (4) SEQ ID NOs: 24. 27 and 34; (5) SEQ ID NOs: 25. 27 and 35; or (6) SEQ ID NOs: 26. 30 and 36; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to these sequences, respectively, wherein the antibody, or antigen-binding portion thereof, binds to CD 40.

In one aspect, an isolated monoclonal antibody, or antigen binding portion thereof, of the invention comprises a light chain variable region comprising the amino acid sequence of SEQ ID NOs: 51. 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61, or a sequence comprising at least 80%, 85%, 90%, 95%, 98%, or 99% identity to these sequences, wherein the antibody or antigen-binding portion thereof binds to CD 40.

In one aspect, an isolated monoclonal antibody or antigen-binding portion thereof of the invention comprises a heavy chain variable region and a light chain variable region, each comprising a CDR1 region, a CDR2 region, and a CDR3 region, wherein the heavy chain variable region CDR1 region, CDR2 region, and CDR3 region, and the light chain variable region CDR1 region, CDR2 region, and CDR3 region comprise (1) the amino acid sequence set forth in SEQ ID NOs: 1. 8, 15, 21, 27 and 31; (2) SEQ ID NOs: 1. 9, 15, 21, 27 and 31; (3) SEQ ID NOs: 2. 9, 15, 21, 27 and 31; (4) SEQ ID NOs: 3. 10, 16, 22, 28 and 32; (5) SEQ ID NOs: 4. 11, 17, 23, 29, and 33; (6) SEQ ID NOs: 5. 12, 18, 24, 27 and 34; (7) SEQ ID NOs: 6. 13, 19, 25, 27 and 35; or (8) SEQ ID NOs: 7. 14, 20, 26, 30 and 36; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to these sequences, respectively, wherein the antibody, or antigen-binding portion thereof, binds to CD 40.

In one embodiment, the isolated monoclonal antibody, or antigen binding portion thereof, of the invention comprises a heavy chain variable region and a light chain variable region comprising, respectively, (1) SEQ ID NOs: 37 and 51; (2) SEQ ID NOs: 38 and 52; (3) SEQ ID NOs: 39 and 53; (4) SEQ ID NOs: 40 and 54; (5) SEQ ID NOs: 41 and 55; (6) SEQ ID NOs: 44 and 58; (7) SEQ ID NOs: 45 and 59; (8) SEQ ID NOs: 46 and 60; (9) SEQ ID NOs: 46 and 61; (10) SEQ ID NOs: 47 and 60; (11) SEQ ID NOs: 47 and 61; (12) SEQ ID NOs: 48 and 59; (13) SEQ ID NOs: 49 and 60; (14) SEQ ID NOs: 49 and 61; (15) SEQ ID NOs: 50 and 60; or (16) SEQ ID NOs: 50 and 61; or an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% identity to these sequences, respectively, wherein the antibody, or antigen-binding portion thereof, binds to CD 40.

In one embodiment, the isolated monoclonal antibody or antigen-binding portion thereof of the invention comprises a heavy chain comprising a heavy chain variable region and a heavy chain constant region, and a light chain comprising a light chain variable region and a light chain constant region, wherein the heavy chain variable region and the light chain variable region comprise the amino acid sequences described above, and the heavy chain constant region comprises the amino acid sequence set forth in SEQ ID NOs: 62. 63 or 64, and a light chain constant region comprising the amino acid sequence of SEQ ID NOs: 65 or 66, or an amino acid sequence comprising at least 80%, 85%, 90%, 95%, 98%, or 99% identity to such sequences, wherein the antibody, or antigen-binding portion thereof, binds to CD 40.

The antibodies of the invention in some embodiments comprise or consist of two heavy chains and two light chains, wherein each heavy chain comprises a heavy chain constant region sequence, a heavy chain variable region sequence, or a CDR sequence as described above, and each light chain comprises a light chain constant region sequence, a light chain variable region sequence, or a CDR sequence as described above. The antibody of the invention may be a full length antibody, for example of the IgG1, IgG2 or IgG4 subtype. In some embodiments, the antibodies of the invention may be single chain antibodies, or may be comprised of antibody fragments, such as Fab or Fab' 2 fragments.

The antibodies or antigen binding portions thereof of the invention specifically bind to human or monkey CD40, blocking or promoting CD40-CD40L interactions. The antibodies of the invention are agonistic CD40 antibodies, activate the CD40 signaling pathway, and promote maturation of immune cells, such as dendritic cells. The antibodies of the invention, or antigen-binding portions thereof, have in vivo anti-tumor effects that are comparable to or better than prior art CD40 antibodies, and comparable or lower toxicity. The tumor no longer grows, or even disappears completely, after the administration of the antibody has ceased.

The invention also provides immunoconjugates comprising an antibody or antigen-binding portion thereof of the invention linked to a therapeutic agent, such as a cytotoxin. The invention also provides bispecific molecules comprising an antibody or antigen-binding portion thereof of the invention, to which is attached a second functional group, e.g., a second antibody, having a binding specificity that is different from that of the antibody or binding portion thereof of the invention. In another aspect, an antibody of the invention, or an antigen-binding portion thereof, can be part of a Chimeric Antigen Receptor (CAR). The antibody or antigen-binding portion thereof of the present invention may be encoded by or carried by an oncolytic virus for use.

The invention also provides a composition comprising an antibody or antigen-binding portion thereof, or an immunoconjugate or bispecific molecule, or CAR of the invention, and a pharmaceutically acceptable carrier.

The invention also includes nucleic acid molecules encoding the antibodies or antigen binding portions thereof of the invention, as well as expression vectors comprising the nucleic acids and host cells comprising the expression vectors. The present invention also provides a method for producing a CD40 antibody using a host cell containing the above expression vector, comprising the steps of: (i) expressing the antibody in a host cell, and (ii) isolating the antibody from the host cell or culture thereof.

In another aspect, the invention provides a method of enhancing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, of the invention. In some embodiments, the methods comprise administering a composition, bispecific molecule, immunoconjugate, CAR-T cell, or oncolytic virus encoding or carrying an antibody of the invention.

In another aspect, the invention provides a method of treating an inflammatory disease, an infectious disease, atherosclerotic thrombus, or a respiratory disease in a subject comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion of the invention. In some embodiments, the methods comprise administering a composition, bispecific molecule, immunoconjugate, CAR-T cell, or oncolytic virus encoding or carrying an antibody of the invention. In some embodiments, other drugs, such as anti-inflammatory and antimicrobial agents, may be administered with the antibodies of the invention or antigen binding portions thereof. In some embodiments, the inflammatory disease comprises an autoimmune disease.

In another aspect, the invention provides a method of preventing, treating, or ameliorating a cancer disease in a subject, comprising administering to the subject a therapeutically effective amount of an antibody, or antigen-binding portion thereof, of the invention. The cancer may be a solid or non-solid tumor, including, but not limited to, B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, melanoma, intestinal adenocarcinoma, pancreatic cancer, intestinal cancer, gastrointestinal cancer, prostate cancer, bladder cancer, renal cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, and nasopharyngeal cancer. In some embodiments, the method comprises administering a composition, bispecific molecule, immunoconjugate, CAR-T cell, or oncolytic virus encoding or carrying an antibody of the invention. In some embodiments, at least one other anti-cancer antibody can be administered with an antibody of the invention or an antigen-binding portion thereof, e.g., a VISTA antibody, a PD-1 antibody, a PD-L1 antibody, a LAG-3 antibody, and/or a CTLA-4 antibody. In another embodiment, the antibodies of the invention, or antigen binding portions thereof, are administered with a cytokine (e.g., IL-2 and/or IL-21) or a costimulatory antibody (e.g., CD137 antibody and/or GITR antibody). In another embodiment, an antibody or antigen-binding portion thereof of the invention can be administered with a chemotherapeutic agent, which can be a cytotoxic agent, such as epirubicin, oxaliplatin, and/or 5-fluorouracil (5-FU). The antibodies of the invention may be, for example, murine, human, chimeric or humanized antibodies.

Other features and advantages of the present disclosure will be more apparent based on the following detailed description and examples, which should not be construed as limiting. The contents of all documents, Genbank records, patents and published patent applications cited in this application are expressly incorporated herein by reference.

Description of the drawings

Figure 1 shows a ranking of CD40 signaling pathway agonistic activity for 108 hybridoma clones.

Figure 2 shows the promoting or inhibiting effect of CD40 antibody on CD40-CD40L interaction, wherein antibodies 16a6, 7B4 and 13a2 promote CD40-CD40L interaction (a) and antibody 92F6 inhibits CD40-CD40L interaction (B).

Figure 3 shows CD40 signaling pathway agonistic activity of CD40 antibody.

Fig. 4 shows the promoting effect of CD40 antibody on dendritic cell maturation as measured by staining with CD86(a), CD80(B), and CD83 (C).

FIG. 5 shows the binding of chimeric CD40 antibodies to HEK 293A/human CD40 cells (A) or HEK 293A/monkey CD40 cells (B).

Figure 6 shows CD40 signaling pathway agonistic activity of a chimeric CD40 antibody.

Figure 7 shows the promotion of dendritic cell maturation by CD40 antibody as measured by CD86 staining.

Figure 8 shows the binding force of chimeric and humanized CD40 antibody to human or monkey CD40, with chimeric and humanized 13a2 antibody (a) and humanized 7B4 antibody (B) binding to human CD40, and chimeric and humanized 13a2 antibody (C) and humanized 7B4 antibody (D) binding to monkey CD 40.

Fig. 9 shows CD40 signaling pathway agonistic activity of chimeric and humanized 7B4 antibody (a) and chimeric and humanized 13a2 antibody (B).

Fig. 10 shows the promoting effect of CD40 antibody on dendritic cell maturation in donor 1 as measured by staining with CD86(a), CD80(B), and CD83 (C).

Figure 11 shows the promotion of dendritic cell maturation by CD40 antibody in donor 2 as measured by staining with CD86(a) and CD80 (B).

FIG. 12 shows the promoting effect of CD40 antibody on dendritic cell maturation in donor 3 as measured by staining with CD86(A), CD80(B), and IL-12 (C).

Figure 13 shows the binding affinity of chimeric or humanized CD40 antibodies 7B4(a), 7B4-VH0VL0(B), 7B4-VH2VL2(C), 7B4-VH2VL3(D), 7B4-VH3VL2(E), 7B4-VH3VL3(F), 13a2(G), 13a2-VH0VL0(H), 13a2-VH2VL2(I), 13a2-VH2VL3(J), 13a2-VH3VL2(K), and 13a2-VH3VL3(L) as measured by SPR, SPR 70089 (M) and ADC (N) to human CD 40.

Figure 14 shows the binding affinity of the chimeric or humanized CD40 antibody to full length CD40 ECD or a truncation thereof (a) and to full length CD40 ECD or a mutant thereof (B).

Figure 15 shows the binding specificity of humanized CD40 antibodies 7B4-VH2VL2(a) and 13a2-VH3VL3(B) to human CD 40.

Figure 16 shows the effect of genetically engineered CD40 antibody on dendritic cell maturation as measured by staining with CD86(a), CD80(B), and CD83 (C).

Fig. 17 shows the average tumor volume (a) of each group to which the humanized CD40 antibody or control drug was administered, and the individual tumor volumes of the solvent group (B), 7B4VH2VL2 group (C), 13A2VH3VL3 group (D), RO7009789 group (E), and APX005(F) group.

Figure 18 shows the average animal body weight for each group administered with the humanized CD40 antibody or control drug.

Figure 19 shows the effect of humanized CD40 antibody on tumor infiltrating CD4+ T cells (a) and CD8+ T cells (B) proliferation.

Figure 20 shows the promotion of tumor-infiltrating dendritic cell (CD45+ CD11c + cells) maturation by humanized CD40 antibody measured by staining with CD80, CD83, and CD 86.

Detailed Description

For a better understanding of the present invention, certain terms are first defined. Other definitions are listed throughout the detailed description section.

The term "CD 40" refers to member five of the tumor necrosis factor superfamily. The term includes variants, homologs, orthologs, and paralogs. For example, an antibody specific for human CD40 may in some cases cross-react with a CD40 protein of another species, such as monkey. In other embodiments, an antibody specific for human CD40 protein may be completely specific for human CD40 protein without cross-reacting with other species or other types of proteins, or may cross-react with CD40 protein of some other species but not all other species.

The term "human CD 40" refers to a CD40 protein having a human amino acid sequence, for example, the amino acid sequence of Genbank accession No. NP _001241.1 (SEQ ID No.: 68). The terms "monkey CD 40" and "murine CD 40" refer to CD40 proteins having monkey and mouse sequences, respectively, e.g., sequences having Genbank accession No. NP _001252791.1(SEQ ID No.: 70) and Genbank accession No. NP _035741.2(SEQ ID No.: 72), respectively.

The term "antibody" herein is intended to include full-length antibodies and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof. Full-length antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains, the heavy and light chains being linked by disulfide bonds. Each heavy chain is composed of heavy chain variable region (V)_H) And a heavy chain constant region. The heavy chain constant region is composed of three domains, i.e., C_H1、C_H2And C_H3. Each light chain is composed of light chain variable region (V)_L) And a light chain constant region. The light chain constant region consists of a domain C_LAnd (4) forming. V_HAnd V_LRegions may also be divided into hypervariable regions, known as Complementarity Determining Regions (CDRs), which are separated by more conserved Framework Regions (FRs). Each V_HAnd V_LIs composed of three CDRs and four FRs, and is arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from amino terminal to carboxyl terminal. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various immune system cells (e.g., effector cells) and the first component of the classical complement system (C1 q).

The term "antigen-binding portion" of an antibody (or simply antibody portion), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (e.g., CD40 protein). It has been confirmed that the antigen binding activity of an antibodyCan be performed by fragments of full-length antibodies. Examples of binding fragments comprised in the "antigen-binding portion" of an antibody include (i) a Fab fragment consisting of V_L、V_H、C_LAnd C_H1A monovalent fragment of (a); (ii) f (ab')₂A fragment, a bivalent fragment comprising two Fab fragments linked by a hinge region disulfide bridge; (iii) from V_HAnd C_H1A constituent Fd fragment; (iv) from antibody single-armed V_LAnd V_H(iii) a Fv fragment of (i); (v) from V_HThe constituted dAb fragment (Ward et al, (1989) Nature 341: 544-546); (vi) an isolated Complementarity Determining Region (CDR); and (vii) a nanobody, a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, despite the two domains V of the Fv fragment_LAnd V_HEncoded by different genes, which can be joined by recombinant means via a synthetic linker which makes both single protein chains, where V_LAnd V_HThe regions pair to form monovalent molecules (known as single chain Fc (scFv); see, e.g., Bird et al, (1988) Science 242: 423-. These single chain antibodies are also intended to be included within the term meaning. These antibody fragments can be obtained by conventional techniques known to those skilled in the art, and the fragments can be functionally screened in the same manner as intact antibodies.

The term "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies having different antigenic specificities. For example, an isolated antibody that specifically binds to CD40 protein is substantially free of antibodies that specifically bind to antigens other than CD40 protein. However, an isolated antibody that specifically binds to human CD40 protein may have cross-binding properties to other antigens, such as CD40 protein from other species. Furthermore, the isolated antibody is substantially free of other cellular material and/or chemicals.

The term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

The term "mouse-derived antibody" refers to an antibody in which the variable region framework and CDR regions are derived from mouse germline immunoglobulin sequences. In addition, if the antibody contains constant regions, it is also derived from mouse germline immunoglobulin sequences. The murine antibodies of the invention may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences, such as mutations introduced by random or point mutations in vitro or by somatic mutations in vivo. However, the term "murine antibody" does not include antibodies having CDR sequences from other mammalian species inserted into the mouse framework sequences.

The term "chimeric antibody" refers to an antibody obtained by combining genetic material of non-human origin with genetic material of human origin. Or more generally, a chimeric antibody refers to an antibody that combines genetic material of one species with genetic material of another species.

The term "humanized antibody" refers to an antibody that is derived from a non-human species but whose protein sequence has been modified to increase its similarity to a naturally occurring human antibody.

The terms "antibody recognizing an antigen" and "antibody specific for an antigen" are used herein interchangeably with the term "antibody specifically binding to an antigen".

Herein, an antibody that "specifically binds to human CD 40" refers to an antibody that binds to human CD40 (and possibly CD40 of other non-human species) but does not substantially bind to non-CD 40 protein. Preferably, the antibody binds human CD40 protein, i.e., K, with "high affinity_DThe value was 5.0x10^-8M or less, preferably 1.0x10^-8M is less than or equal to M, more preferably 5.0x10^-9M is less than or equal to M.

The term "does not substantially bind" to a protein or cell means that it does not bind to a protein or cell, or does not bind to it with high affinity, i.e., binds to a protein or cell with a KD of 1.0x10^-6M is more than or equal to, more preferably 1.0x10^-5M is more than or equal to, more preferably 1.0x10^-41.0x10 of M or more^-3M is more than or equal to, more preferably 1.0x10^-2M is more than M.

The term "high affinity" for an IgG antibody refers to K for the antigen_DIs 1.0x10^-6M is less, preferably 5.0x10^-8M or less, more preferably 1.0 x 10^-8M below, 5.0x10^-9M or less, more preferably 1.0x10^-9M is less than or equal to M. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding of the IgM subtype means K_DIs 10^{- 6}M is less, preferably 10^-7M is less, more preferably 10^-8M is less than or equal to M.

The term "K_assoc"or" K_a"refers to the binding rate of a particular antibody-antigen interaction, and the term" K_dis"or" K_d"refers to the rate of dissociation of a particular antibody-antigen interaction. The term "K_D"refers to the dissociation constant, from K_dAnd K_aRatio (K)_d/K_a) Obtained and expressed in molar concentration (M). K of antibody_DThe values may be determined by methods known in the art. Preferred antibodies K_DMeasured using a Surface Plasmon Resonance (SPR), preferably using a biosensing system such as Biacore^TMAnd (5) measuring by the system.

The term "EC₅₀"half maximal effect concentration, also called, refers to the concentration of antibody that causes 50% of the maximal effect.

The term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals, such as non-human primates, sheep, dogs, cats, cows, and horses, are preferred.

The term "agonistic CD40 antibody" as used herein refers to a CD40 antibody that is capable of binding to CD40 and activating or priming the CD40 signaling pathway to promote immune cell activation and proliferation and cytokine and chemokine production. The term "antagonistic CD40 antibody" refers to blocking or inhibiting the CD40 signaling pathway that can be triggered by CD40L binding.

The term "therapeutically effective amount" refers to an amount of an antibody of the invention sufficient to prevent or alleviate symptoms associated with a disease or disorder (e.g., cancer) and/or to reduce the severity of the disease. The therapeutically effective amount is related to the disease to be treated, wherein the actual effective amount can be readily determined by one skilled in the art.

Aspects of the invention are described in more detail below.

The CD40 antibody has binding specificity to human CD40 and other beneficial functional characteristics

The antibodies of the invention specifically bind human CD40, e.g., K, with high affinity_DValue of 1x 10^-8M is less than or equal to M. The antibody also has cross-reactivity with monkey CD40 and does not bind mouse CD 40.

The antibodies of the invention are agonistic CD40 antibodies that activate or elicit the CD40 signaling pathway and are involved in immune cell activation and proliferation and cytokine and chemokine production.

The antibodies of the invention, compared to the prior art CD40 antibody, have comparable or better in vivo anti-tumor effects, comparable or lower toxicity, than the prior CD40 antibody. After stopping antibody administration, the tumor stops growing, or even completely eliminates.

Preferred antibodies of the invention are monoclonal antibodies. Furthermore, the antibody may be, for example, a murine, chimeric or humanized monoclonal antibody.

CD40 monoclonal antibody

Preferred antibodies of the invention are monoclonal antibodies whose structural and chemical properties are described below. V of CD40 antibody_HThe amino acid sequence is SEQ ID NOs: 37. 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. V of CD40 antibody_LThe amino acid sequence is SEQ ID NOs: 51. 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61. The variable heavy/light chain sequences of the antibodies are listed in Table 1 below, and some antibodies have the same VH or V_L. Preferred antibody heavy chain constant region amino acid sequences are SEQ ID NOs: 62. 63 or 64, and the amino acid sequence of the light chain constant region is SEQ ID NOs: 65 or 66.

V of other CD40 antibodies that bind to human CD40_HAnd/or V_LSequences (or CDR sequences) that can be compared to the V of an antibody of the invention_HAnd/or V_LSequences (or CDR sequences) "mix and pair". Preferably, when V_HAnd V_L(or CDRs therein) are mixed and paired, a specific V_H/V_LV in the pairing_HV whose sequence can be approximated by a structure_HAnd (4) sequence substitution. Similarly, particular V is preferred_H/V_LV in the pairing_LV with sequence approximated by structure_LAnd (4) sequence substitution.

Thus, in one embodiment, an antibody or antigen-binding portion thereof of the invention comprises:

(a) a heavy chain variable region comprising an amino acid sequence set forth in table 1; and

(b) a light chain variable region comprising an amino acid sequence set forth in Table 1, or a V of another CD40 antibody_LWherein the antibody specifically binds to human CD 40.

In another embodiment, an antibody or antigen-binding portion thereof of the invention comprises:

(a) CDR1, CDR2, and CDR3 of the heavy chain variable regions listed in table 1; and

(b) the CDRs 1, CDR2 and CDR3 of the light chain variable regions listed in table 1, or the CDRs of another CD40 antibody, wherein the antibody specifically binds human CD 40.

In another embodiment, the antibody or antigen-binding portion thereof of the invention comprises the heavy chain variable region CDR2 of the CD40 antibody as well as other CDRs of an antibody that binds human CD40, e.g., the heavy chain variable region CDR1 and/or CDR3, and/or the light chain variable region CDR1, CDR2, and/or CDR3 of another CD40 antibody.

Furthermore, it is well known in the art that the CDR3 domain, independent of the CDR1 and/or CDR2, can determine the binding specificity of an antibody to the same antigen individually, and that multiple antibodies with the same binding specificity can be predicted to be generated based on this CDR3 sequence. See, e.g., Klimka et al, British j.of Cancer 83 (2): 252-260 (2000); beiboer et al, j.mol.biol.296: 833-849 (2000); rader et al, proc.natl.acad.sci.u.s.a.95: 8910-8915 (1998); barbas et al, j.am.chem.soc.116: 2161-2162 (1994); barbas et al, proc.natl.acad.sci.u.s.a.92: 2529 2533 (1995); ditzel et al, j.immunol.157: 739-; berezov et al, BIAjournal 8: scientific Review 8 (2001); igarashi et al, j. biochem (Tokyo) 117: 452-7 (1995); bourgeous et al, j.virol 72: 807-10 (1998); levi et al, Proc.Natl.Acad.Sci.U.S.A.90: 4374-8 (1993); polymenis and Stoller, j.immunol.152: 5218 5329(1994) and Xu and Davis, Immunity 13: 37-45 (2000); U.S. Pat. nos.6,951, 646; 6,914,128, respectively; 6,090,382; 6,818,216, respectively; 6,156,313, respectively; 6,827,925, respectively; 5,833,943, respectively; 5,762,905, and 5,760,185. These references are all incorporated herein by reference in their entirety.

In another embodiment, the antibody of the invention comprises the CDR2 of the heavy chain variable region of the CD40 antibody and at least the CDR3 of the heavy and/or light chain variable region of the CD40 antibody, or the CDR3 of the heavy and/or light chain variable region of another CD40 antibody, wherein the antibody is capable of specifically binding to human CD 40. Preferably, these antibodies (a) compete for binding to CD 40; (b) functional characteristics are reserved; (c) binds to the same epitope; and/or (d) has a similar binding affinity as the CD40 antibody of the invention. In another embodiment, the antibody may further comprise the light chain variable region CDR2 of a CD40 antibody, or the light chain variable region CDR2 of another CD40 antibody, wherein the antibody specifically binds human CD 40. In another embodiment, an antibody of the invention can include the heavy chain/light chain variable region CDR1 of a CD40 antibody, or the heavy chain and/or light chain variable region CDR1 of another CD40 antibody, wherein the antibody specifically binds to human CD 40.

Conservative modifications

In another embodiment, the antibody of the invention comprises a heavy and/or light chain variable region sequence or CDR1, CDR2 and CDR3 sequences that are conservatively modified with one or more of the CD40 antibodies of the invention. It is known in the art that some conservative sequence modifications do not abolish antigen binding. See, e.g., Brummell et al, (1993) Biochem 32: 1180-8; de Wildt et al, (1997) prot. eng.10: 835-41; komissarov et al, (1997) J.biol.chem.272: 26864-26870; hall et al, (1992) j.immunol.149: 1605-12; kelley and O' Connell (1993) biochem.32: 6862-35; Adib-Conquy et al, (1998) int.immunol.10: 341-6and Beers et al, (2000) Clin.Can.Res.6: 2835-43.

Thus, in one embodiment, the antibody comprises a heavy chain variable region and/or a light chain variable region comprising CDR1, CDR2, and CDR3, respectively, wherein:

(a) heavy chain variable region CDR1 comprises the sequences listed in table 1, and/or conservative modifications thereof; and/or

(b) Heavy chain variable region CDR1 comprises the sequences listed in table 1, and/or conservative modifications thereof; and/or

(c) Heavy chain variable region CDR3 comprises the sequences listed in table 1, and/or conservative modifications thereof; and/or

(d) The light chain variable region CDR1, and/or CDR2, and/or CDR3 comprises the sequences listed in table 1, and/or conservative modifications thereof; and is

(e) The antibody specifically binds to human CD 40.

The antibodies of the invention have one or more of the following functional characteristics, such as high affinity for human CD40, and the ability to elicit ADCC or CDC of a CD40 expressing cell.

In various embodiments, the antibody can be, for example, a murine, human, chimeric, or humanized antibody.

The term "conservative sequence modification" as used herein refers to amino acid modifications that do not significantly affect or alter the binding properties of the antibody. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as point mutations and PCR-mediated mutations. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains are known in the art. These groups of amino acid residues include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a CDR region of an antibody of the invention can be replaced with other amino acid residues of the same side chain set, and the resulting antibody can be tested for retained function (i.e., the function described above) using the functional assays described herein.

Genetically modified antibodies

The antibodies of the invention may be prepared as genetically modified antibodies using antibodies having one or more of the VH/VL sequences of the CD40 antibodies of the invention as starting material. Antibodies can be genetically modified by modifying one or more residues within one or both variable regions (i.e., VH and/or VL) (e.g., in one or more CDR regions and/or one or more framework regions) to improve binding affinity and/or increase similarity to antibodies naturally produced by certain species. For example, the framework regions are modified to provide humanized antibodies. Alternatively, the antibody may be genetically modified by modifying residues in the constant region, for example to alter the effector function of the antibody.

In certain embodiments, CDR region implantation can be used to genetically modify the variable region of an antibody. Antibodies interact with the target antigen primarily through amino acid residues located in the six heavy and light chain Complementarity Determining Regions (CDRs). For this reason, amino acid residues within a CDR are more diverse between individual antibodies than sequences outside the CDR. Since CDR sequences are responsible for the major antibody-antigen interactions, recombinant antibodies that mimic the properties of a particular native antibody can be expressed by constructing expression vectors containing CDR sequences of the particular native antibody implanted into the framework sequences of different antibodies of different properties (Riechmann et al, (1998) Nature 332: 323-.

Accordingly, another embodiment of the present inventionThe formula (ii) relates to an isolated monoclonal antibody or antigen binding portion thereof comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 having the sequences of the invention described above and/or a light chain variable region comprising CDR1, CDR2 and CDR3 having the sequences of the invention described above. Although these antibodies comprise V of the monoclonal antibody of the invention_HAnd V_LCDR sequences, they can contain different framework sequences.

Such framework sequences can be obtained from public DNA databases or public references including germline antibody gene sequences. For example, germline DNA sequences for human heavy and light chain variable region genes can be found in the Vbase human germline sequence database (www.mrc-cpe.cam.ac.uk/Vbase) and Kabat et al., (1991), supra; tomlinson et al, (1992) j.mol.biol.227: 776-798; and Cox et al, (1994) eur.j.immunol.24: 827 and 836. As another embodiment, germline DNA sequences for human heavy and light chain variable region genes are available in the Genbank database. For example, the following Genbank accession numbers for the heavy chain germline sequences in HCo7 HuMAb mice are 1-69 (NG-0010109, NT-024637 & BC070333), 3-33 (NG-0010109 & NT-024637), and 3-7 (NG-0010109 & NT-024637). As another example, the following germline sequences of the heavy chains from Hco12 HuMAb mice have Genbank accession numbers 1-69 (NG-0010109, NT-024637 & BC070333), 5-51 (NG-0010109 & NT-024637), 4-34 (NG-0010109 & NT-024637), 3-30.3(CAJ556644), and 3-23(AJ 406678).

Antibody protein sequences were compared to protein sequence databases using one of the sequence similarity search methods known in the art as space (gap) BLAST (Altsch μ l et al, (1997), supra).

Preferred framework sequences for use in antibodies of the invention are those that are structurally similar to the framework sequences used in antibodies of the invention. V_HThe CDR1, CDR2, and CDR3 sequences can be embedded in framework regions having the same sequence as the germline immunoglobulin gene from which the framework sequences were derived, or the CDR sequences can be embedded in framework regions comprising one or more mutations compared to the germline sequence. For example, in some cases, in the framework regionsIt may be beneficial to mutate residues to maintain or enhance the antigen binding properties of an antibody (see, e.g., U.S. Pat. No. 5,530,101; 5,585,089; 5,693,762, and 6,180,370).

Another class of variable region modifications is to modify V_HAnd/or V_LAmino acid residues within the CDR1, CDR2, and/or CDR3 regions are mutated to improve one or more binding properties (e.g., affinity) of the antibody of interest. Point mutations or PCR-mediated mutations can be made to introduce mutations, and their effect on antibody binding or other functional properties can be evaluated in vitro or in vivo assays known in the art. Preferably, conservative modifications known in the art are introduced. The mutation may be an amino acid substitution, addition or deletion, but is preferably a substitution. In addition, typically no more than one, two, three, four, or five residues within a CDR region are altered.

In another embodiment, the present invention provides an isolated CD40 monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, comprising: (a) v_HA CDR1 region comprising a sequence of the invention, or an amino acid sequence with one, two, three, four, or five amino acid substitutions, deletions, or additions; (b) v_HA CDR2 region comprising a sequence of the invention, or an amino acid sequence with one, two, three, four, or five amino acid substitutions, deletions, or additions; (c) v_HA CDR3 region comprising a sequence of the invention, or an amino acid sequence with one, two, three, four, or five amino acid substitutions, deletions, or additions; (d) v_LA CDR1 region comprising a sequence of the invention, or an amino acid sequence with one, two, three, four, or five amino acid substitutions, deletions, or additions; (e) v_LA CDR2 region comprising a sequence of the invention, or an amino acid sequence with one, two, three, four, or five amino acid substitutions, deletions, or additions; and (f) V_LA CDR3 region comprising a sequence of the invention, or an amino acid sequence with one, two, three, four, or five amino acid substitutions, deletions, or additions.

Genetically engineered antibodies of the invention are included at V_HAnd/or V_LMaking a genetic modification in the framework residues ofFor example, to alter the properties of the antibody. Typically, these framework modifications are used to reduce the immunogenicity of the antibody. For example, one approach is to "back mutate" one or more framework residues into the corresponding germline sequence. More specifically, an antibody undergoing somatic mutation may contain framework residues that differ from the germline sequence of the resulting antibody. These residues can be identified by comparing the antibody framework sequences to the germline sequences of the resulting antibody.

Another class of framework modifications comprises mutating one or more residues of the framework regions, or even one or more CDR regions, to remove T cell epitopes and thereby reduce the potential immunogenicity of the antibody. This method, also known as "deimmunization," is described in more detail in U.S. patent publication 20030153043.

Furthermore, as an alternative to modifications within the framework or CDR regions, the antibodies of the invention may be genetically engineered to include genetic modifications in the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antibody-dependent cellular cytotoxicity. In addition, the antibodies of the invention may be chemically modified (e.g., one or more chemical functional groups may be attached to the antibody), or modified to alter glycosylation, to alter one or more functional properties of the antibody.

In one embodiment, C_H1The hinge region of (a) is modified, for example, the number of cysteine residues in the hinge region is increased or decreased. This process is further described in U.S. Pat. No. 5,677,425. Change C_H1Cysteine residues in the hinge region, for example, to facilitate assembly of the heavy chain light chain or to increase/decrease stability of the antibody.

In another embodiment, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C of the Fc hinge fragment_H2-C_H3A linking region such that the antibody has reduced SpA binding relative to native Fc-hinge domain SpA binding. This method is described in more detail in U.S. Pat. No. 6,165,745.

In another embodiment, the glycosylation of the antibody is modified. For example, deglycosylated antibodies (i.e., antibodies lacking glycosylation) can be made. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such modifications of glycation can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that location. Such deglycosylation can increase the affinity of the antibody for the antigen. See, for example, U.S. Pat. Nos.5,714,350 and 6,350,861.

Furthermore, antibodies with altered glycosylation patterns can be prepared, such as low fucosyl antibodies with reduced amounts of fucose residues, or antibodies with increased bisecting GlcNac structures. Altered glycosylation patterns have been shown to increase the ADCC activity of an antibody. Such modifications of glycation can be performed, for example, by expressing the antibody in a host cell with an altered glycosylation system. Cells with altered glycosylation systems are known in the art and can be used as host cells for expression of recombinant antibodies of the invention to produce antibodies with altered glycosylation. For example, the cell lines Ms704, Ms705 and Ms709 lack the fucosyltransferase gene FUT8(α (1, 6) -fucosyltransferase), such that antibodies expressed in the Ms704, Ms705 and Ms709 cell lines lack fucose in their sugars. Ms704, Ms705 and Ms709FUT 8-/-cell lines were prepared by targeted disruption of the FUT8 gene in CHO/DG44 cells using two alternative vectors (see U.S. patent publication 20040110704 and Yamane-Ohnuki et al, (2004) Biotechnol Bioeng 87: 614-22). As another example, EP 1,176,195 describes a cell line with a disrupted FUT8 gene function, which encodes a fucosyltransferase, whereby antibodies expressed in the cell line exhibit low fucosylation by reducing or eliminating the alpha-1, 6 linkage related enzymes. EP 1,176,195 also describes a cell line which has a low or no activity for adding fucose to N-acetylglucosamine which binds to the Fc region of antibodies, for example the rat myeloma cell line YB2/0(ATCC CRL 1662). WO 03/035835 describes a CHO variant cell line, Lec13 cell, which has a reduced ability to add fucose to an Asn (297) -related sugar, resulting in low fucosylation of the antibody expressed in the host cell (see Shield et al, (2002) J.biol.chem.277: 26733-. Antibodies with altered glycosylation patterns can also be prepared in chicken eggs, as described in WO 06/089231. Alternatively, antibodies with altered glycosylation patterns can be made in plant cells such as duckweed. WO 99/54342 discloses a cell line genetically engineered to express a glycosyltransferase that modifies a glycoprotein (e.g.,. beta. (1, 4) -N-acetylglucosaminyltransferase III (GnTIII)), such that antibodies expressed in the genetically engineered cell line exhibit increased bisecting GlcNac structure that results in enhanced ADCC activity by the antibody (Umana et al., (1999) nat. Biotech.17: 176-180). Alternatively, fucose residues of an antibody can be cleaved using a fucosidase, e.g., an α -L-fucosidase that removes fucose residues from an antibody (Tarentino et al, (1975) biochem.14: 5516-23).

Another modification of the antibodies herein is pegylation (pegylation). The antibody can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), e.g., a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation is performed by an acylation reaction or alkylation reaction with a reactive PEG molecule (or similar reactive water-soluble polymer). The term "polyethylene glycol" as used herein includes any form of PEG used to derivatize other proteins, such as mono (C)₁-C₁₀) Alkoxy-or aryloxy-polyethylene glycols or polyethylene glycol maleimides. In certain embodiments, the antibody requiring pegylation is a deglycosylated antibody. Methods of PEGylating proteins are known in the art and can be applied to the antibodies of the invention. See, e.g., EPO 154316 and EP 0401384.

Physical Properties of antibodies

The antibodies of the invention may be characterized by their various physical properties to detect and/or distinguish their classification.

For example, an antibody may comprise one or more glycosylation sites in the light or heavy chain variable region. These glycosylation sites may result in increased immunogenicity of the antibody, or altered pK values of the antibody due to altered antigen binding (Marshall et al (1972) Annu Rev Biochem 41: 673-. Glycosylation is known to occur in motifs containing N-X-S/T sequences. In some cases, it is preferred that the CD40 antibody does not contain variable region glycosylation. This can be achieved by selecting antibodies that do not contain glycosylation motifs in the variable region or by mutating residues of the glycosylation region.

In a preferred embodiment, the antibody does not comprise an asparagine isomerization site. Deamidation of asparagine may occur in the N-G or D-G sequence, creating isoaspartic acid residues that introduce kinks into the polypeptide chain and reduce its stability (isoaspartic acid effect).

Each antibody will have a unique isoelectric point (pI) that falls substantially within the pH range of 6-9.5. The pI of the IgG1 antibody generally falls within a pH range of 7-9.5, while the pI of the IgG4 antibody substantially falls within a pH range of 6-8. It is speculated that antibodies with pI outside the normal range may have some unfolded structure and be unstable under in vivo conditions. Therefore, it is preferred that the pI value of the CD40 antibody falls within the normal range. This can be achieved by selecting antibodies with pI in the normal range or by mutating uncharged surface residues.

Nucleic acid molecules encoding the antibodies of the invention

In another aspect, the invention provides nucleic acid molecules encoding the heavy and/or light chain variable regions or CDRs of the antibodies of the invention. The nucleic acid may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially pure" when purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques. The nucleic acids of the invention may be, for example, DNA or RNA, and may or may not comprise intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

The nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by hybridomas (e.g., hybridomas prepared from transgenic mice carrying human immunoglobulin genes), cdnas encoding the light and heavy chains of the antibodies prepared by the hybridomas can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), nucleic acids encoding such antibodies can be collected from the gene libraries.

Preferred nucleic acid molecules of the invention include V encoding the CD40 monoclonal antibody_HAnd V_LThose of sequences or CDRs. Once the code V is obtained_HAnd V_LThe DNA fragments of (1), which can be further manipulated by standard recombinant DNA techniques, for example, to convert the variable region gene into a full-length antibody chain gene, a Fab fragment gene or a scFv gene. In these operations, V is encoded_HOr V_LIs operably linked to another DNA segment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" means that two DNA segments are linked together such that the amino acid sequences encoded by both DNA segments are in reading frame.

Code V_HIsolated DNA of the region may be operably linked to V_HCoding DNA and coding heavy chain constant region (C)_H1、C_H2And C_H3) Into the full-length heavy chain gene. The sequence of the human heavy chain constant region gene is known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but is preferably an IgG1 or IgG4 constant region. For the Fab fragment heavy chain gene, V is encoded_HThe DNA of the region may be operably linked to a DNA encoding only heavy chain C_H1Another DNA molecule of the constant region is linked.

Code V_LIsolated DNA of the region may be operably linked to V_LCoding DNA and coding light chainConstant region C_LInto a full-length light chain gene. The sequence of the human light chain constant region gene is known in the art, and DNA fragments comprising these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a kappa and lambda constant region.

To create the scFv genes, DNA fragments encoding VH and VL may be operably linked to a linker encoding a flexible linker such as the amino acid sequence (Gly4-Ser)₃Is connected to another fragment of, thereby V_HAnd V_LThe sequence may be expressed as a continuous single chain protein, wherein V_HAnd V_LThe regions are connected by this flexible linker (see, e.g., Bird et al, (1988) Science 242: 423-.

Preparation of monoclonal antibodies of the invention

The monoclonal antibody of the present invention can be produced using a monoclonal antibody produced by Kohler and Milstein (1975) Nature 256: 495 was prepared by somatic cell hybridization (hybridoma) technique. Other embodiments for making monoclonal antibodies include viral or oncogenic transformation of B lymphocytes and phage display techniques. Chimeric or humanized antibodies are also well known in the art. See, for example, U.S. Pat. nos. 4,816,567; 5,225,539; 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

Generation of transfectomas for preparing the monoclonal antibodies of the invention

Antibodies of the invention can also be produced in host cell transfectomas using, for example, recombinant DNA technology in conjunction with gene transfection methods (e.g., Morrison, S. (1985) Science 229: 1202). In one embodiment, DNA encoding partial or full length light and heavy chains obtained by standard molecular biology techniques is inserted into one or more expression vectors such that the genes are operably linked to transcriptional and translational regulatory sequences. In this context, the term "operably linked" refers to the linkage of the antibody genes into a vector such that transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody genes.

The term "regulatory sequence" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of an antibody gene. Such regulatory sequences are described, for example, in Goeddel (Gene Expression technology. methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)). Preferred regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers from Cytomegalovirus (CMV), simian virus 40(SV40), adenoviruses, such as the adenovirus major late promoter (AdMLP), and polyoma virus. Alternatively, non-viral regulatory sequences may be used, such as the ubiquitin promoter or the beta-globin promoter. In addition, the regulatory elements are composed of sequences of different origins, such as the SRa promoter system, which comprises the sequence from the SV40 early promoter and the long terminal repeat of the human T-cell leukemia type I virus (Takebe et al, (1988) mol.cell.biol.8: 466-. The expression vector and expression control sequences are selected to be compatible with the expression host cell used.

The antibody light chain gene and the antibody heavy chain gene may be inserted into the same or different expression vectors. In a preferred embodiment, the variable regions are constructed as full length antibody genes by insertion into expression vectors that already encode the heavy and light chain constant regions of the desired subtype, such that V_HWith C in the carrier_HIs operably connected to, V_LWith C in the carrier_LAre operatively connected. Alternatively, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry other sequences, such as sequences that regulate replication of the vector in a host cell (e.g., an origin of replication) and a selectable marker gene. Selectable marker genes can be used to select host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216; 4,634,665 and 5,179,017). For example, typically a selectable marker gene confers drug resistance, e.g., G418, hygromycin, or methotrexate resistance, on a host cell into which the vector has been introduced. Preferred selectable marker genes include the dihydrofolate reductase (DHFR) gene (for methotrexate selection/amplification of DHFR host cells) and the neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vectors encoding the heavy and light chains are transfected into the host cell by standard techniques. The term "transfection" in its various forms encompasses a variety of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, e.g., electroporation, calcium phosphate precipitation, DEAE-dextrose transfection, and the like. Although it is theoretically possible to express the antibodies of the invention in prokaryotic or eukaryotic host cells, it is preferred that the antibodies are expressed in eukaryotic cells, most preferably mammalian host cells, since eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

Preferred mammalian host cells for expression of recombinant antibodies of the invention include Chinese hamster ovary (CHO cells) (including DHFR-CHO cells administered with a DHFR selectable marker as described in, for example, R.J.Kaufman and P.A.Sharp (1982) J.mol.biol.159: 601-621), NSO myeloma cells, COS cells and SP2 cells, described in Urlaub and Chasin, (1980) Proc.Natl.Acad.Sci.USA 77: 4216-4220. Another preferred expression system, particularly when using NSO myeloma cells, is the GS gene expression system described in WO 87/04462, WO 89/01036 and EP 338,841. When a recombinant expression vector encoding an antibody gene is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or preferably sufficient to allow secretion of the antibody into the medium in which the host cell is grown. Antibodies can be recovered from the culture medium using protein purification methods.

Immunoconjugates

The antibodies of the invention may be crosslinked with a therapeutic agent to form an immunoconjugate, e.g., an antibody-drug conjugate (ADC). Suitable therapeutic agents include cytotoxins, alkylating agents, DNA minor groove binding molecules, DNA intercalators, DNA cross-linkers, histone deacetylase inhibitors, nuclear export inhibitors, proteasome inhibitors, inhibitors of topoisomerase I or II, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics, and antimitotic agents. In an ADC, the antibody and therapeutic agent are preferably cross-linked by a linker that is cleavable, e.g., a peptidic, disulfide, or hydrazone linker. More preferably, the linker is a peptide linker, such as Val-Cit, Ala-Val, Val-Ala-Val, Lys-Lys, Pro-Val-Gly-Val-Val, Ala-Asn-Val, Val-Leu-Lys, Ala-Ala-Asn, Cit-Git, Val-Lys, Cit, Ser or Glu. ADCs may be as in us patent 7,087,600; 6,989,452, respectively; and 7,129,261; PCT publications WO 02/096910; WO 07/038,658; WO 07/051,081; WO 07/059,404; WO 08/083, 312; and WO 08/103,693; U.S. patent publication 20060024317; 20060004081, respectively; and 20060247295.

Bispecific molecules

In another aspect, the invention relates to bispecific molecules comprising one or more antibodies of the invention linked to at least one other functional molecule, such as another peptide or protein (e.g., another antibody or receptor ligand), to generate bispecific molecules that bind to at least two different binding sites or targeting molecules. The term "bispecific molecule" includes molecules with three or more specificities.

In embodiments, the bispecific molecule has a third specificity in addition to the Fc binding specificity and the CD40 binding specificity. The third specificity may be for an Enhancer Factor (EF), such as a molecule that binds to a surface protein involved in cytotoxic activity and thereby increases the immune response against the target cell. For example, the enhancer antibody can bind to cytotoxic T cells (e.g., via CD2, CD3, CD8, CD28, CD4, CD40, or ICAM-1) or other immune cells, causing an enhanced immune response against the target cells.

Bispecific molecules can occur in a variety of forms and sizes. At one end of the size spectrum, the bispecific molecule remains in the traditional antibody format, except that it has two binding arms, each with a different specificity, instead of having two binding arms of the same specificity. At the other extreme, bispecific molecules are composed of two single-chain antibody fragments (scFv) connected via a peptide chain, called Bs (scFv)₂Constructs. Bispecific molecules of intermediate size comprise two different f (ab) fragments linked by a peptide linker. These and other forms of bispecific molecules can be prepared by genetic engineering, somatic hybridization, or chemical methods. See, e.g., Kufer et al, supra; cao and Suresh, Bioconjugate Chemistry, 9(6), 635-644 (1998); and van Spriel et al, Immunology Today, 21(8), 391-.

Oncolytic viruses encoding or carrying antibodies

Oncolytic viruses preferentially infect and kill cancer cells. The antibodies of the invention are used with oncolytic viruses. Furthermore, an oncolytic virus encoding an antibody of the invention can be introduced into a human.

Pharmaceutical composition

In another aspect, the invention provides a pharmaceutical composition comprising one or more antibodies of the invention formulated together with a pharmaceutically acceptable carrier. The composition may optionally comprise one or more other pharmaceutically active ingredients, such as another antibody or drug, e.g. a VISTA antibody. The pharmaceutical compositions of the invention may be administered in combination therapy with, for example, another anti-cancer agent, another anti-inflammatory agent, or an antimicrobial agent.

The pharmaceutical composition may comprise any number of excipients. Excipients that may be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersing or suspending agents, solubilizers, coloring agents, flavoring agents, coatings, disintegrating agents, lubricants, sweetening agents, preservatives, isotonic agents and combinations thereof. Selection and use of suitable excipients is described in Gennaro, ed., Remington: the Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003).

Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g. by injection or bolus injection). Depending on the route of administration, the active ingredient may be encapsulated in a material to protect it from acids and other natural conditions that might inactivate it. "parenteral administration" means a mode other than enteral and topical administration, and is typically performed by injection, including but not limited to intravenous, intramuscular, intraarterial, intramembranous, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, supradural, and intrasternal injection and bolus injection. Alternatively, the antibodies of the invention may be administered by a parenteral route, e.g., topical, epidermal or mucosal administration, e.g., intranasal, oral, vaginal, rectal, sublingual or topical.

The pharmaceutical compositions may be in the form of a sterile aqueous solution or dispersion. They may also be formulated in microemulsions, liposomes or other ordered structures suitable for high concentrations of drug.

The amount of active ingredient that is formulated with a carrier material into a single dosage form will vary with the host treated and the particular mode of administration, and is essentially the amount of composition that produces a therapeutic effect. The amount is from about 0.01 to about 99%, by percentage, of the active ingredient in combination with a pharmaceutically acceptable carrier, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of the active ingredient.

The dosage regimen is adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a rapid perfusion agent may be administered, multiple divided doses may be administered over time, or the dose may be decreased or increased in proportion to the criticality of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage units for convenient administration and uniform dosage. Dosage unit form refers to physically discrete units suitable for single administration to a subject; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the pharmaceutical carrier. Alternatively, the antibody may be administered in a slow release formulation, in which case the frequency of administration required is reduced.

For administration of the antibody, the dosage may be about 0.001-100mg/kg of host body weight, more usually 0.01-5 mg/kg. For example, the dose may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, or 10mg/kg body weight, or in the range of 1-10mg/kg body weight. Exemplary treatment regimens involve administration once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months. Preferred dosing regimens for CD40 of the invention include intravenous administration, 1mg/kg body weight or 3mg/kg body weight, with the antibody being administered on one of the following dosing schedules: (i) administering six times every four weeks, and then once every three months; (ii) once every three weeks; (iii) once at 3mg/kg body weight, then once every three weeks at 1mg/kg body weight. In some methods, the dose is adjusted to achieve a blood concentration of about 1-1000 μ g/ml, and in some methods about 25-300 μ g/ml.

A "therapeutically effective amount" of a CD40 antibody of the invention results in a reduction in the severity of disease symptoms, an increase in the frequency and duration of the asymptomatic phase, or the ability to prevent injury or disability resulting from an infectious disease. For example, for the treatment of a subject with a tumor, a "therapeutically effective amount" preferably inhibits tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and more preferably by at least about 80% as compared to an untreated subject. A therapeutically effective amount of a therapeutic antibody can reduce tumor size, or alleviate a symptom in a subject, which can be a human or another mammal.

The pharmaceutical composition may be a slow release agent including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, for example, Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Pharmaceutical compositions can be administered via medical devices, such as (1) needleless hypodermic injection devices (e.g., U.S. Pat. Nos.5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) micro infusion pumps (us patent 4,487,603); (3) transdermal drug delivery devices (us patent 4,486,194); (4) bolus devices (U.S. Pat. nos. 4,447,233 and 4,447,224); and (5) infiltration equipment (U.S. Pat. Nos. 4,439,196 and 4,475,196).

In certain embodiments, a monoclonal antibody of the invention can be formulated to ensure proper in vivo distribution. For example, to ensure that a therapeutic antibody of the invention crosses the blood brain barrier, the antibody may be formulated in liposomes, which may additionally contain targeting functional groups to enhance selective delivery to specific cells or organs. See, for example, U.S. Pat. nos. 4,522,811; 5,374,548, respectively; 5,416,016; and 5,399,331; v. ranade (1989) j.clin.pharmacol.29: 685 of raw materials; umezawa et al, (1988) biochem. biophysis. res. commun.153: 1038; bloeman et al, (1995) FEBS lett.357: 140 of a solvent; m.owas et al, (1995) antimicrob. ingredients chemither.39: 180 of the total weight of the composition; briscoe et al, (1995) am.j.physiol.1233: 134; schreier et al, (1994) j.biol.chem.269: 9090; keinanen and Laukkanen (1994) FEBS Lett.346: 123; and Killion and Fidler (1994) immunology 4: 273.

uses and methods of the invention

The antibodies (compositions, bispecific molecules and immunoconjugates) of the invention have a variety of in vitro and in vitro applications, relating to the treatment and/or prevention of, for example, cancer, inflammatory diseases or infectious diseases. The antibodies can be administered to a human subject to inhibit tumor growth, for example, in vivo.

In view of the ability of the CD40 antibodies of the invention to inhibit tumor cell proliferation and survival, the invention provides methods of inhibiting tumor cell growth in a subject comprising administering to the subject an antibody of the invention, whereby tumor growth is inhibited in the subject. Non-limiting examples of tumors that can be treated by the antibodies of the invention include, but are not limited to, B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, melanoma, intestinal adenocarcinoma, pancreatic cancer, intestinal cancer, gastrointestinal cancer, prostate cancer, bladder cancer, renal cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, and nasopharyngeal cancer, whether primary or metastatic. In addition, refractory or recurrent malignancies may be inhibited with the antibodies of the present invention.

In another aspect, the invention provides a method of treating an inflammatory disease, an infectious disease, atherosclerotic thrombus, and a respiratory disease in a subject comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding portion of the invention. In some embodiments, an anti-inflammatory agent, antimicrobial agent, or other therapeutic agent may be administered with an antibody of the invention or antigen-binding portion thereof.

In general, the antibodies of the invention can be used to enhance an immune response in a subject.

These and other methods of the present invention are discussed further below.

Combination therapy

The present invention provides combination therapies in which a CD40 antibody or antigen-binding portion thereof of the invention is administered with one or more other antibodies effective to inhibit tumor growth in a subject. In one embodiment, the invention provides a method of inhibiting tumor growth in a subject comprising administering to the subject a CD40 antibody and one or more other antibodies, e.g., a VISTA antibody, a LAG-3 antibody, a PD-1 antibody, a PD-L1 antibody, and/or a CTLA-4 antibody. In certain embodiments, the subject is a human.

CD40 signaling pathway activation may additionally be combined with standard cancer therapy. For example, activation of the CD40 signaling pathway can be combined with CTLA-4 and/or LAG-3 and/or PD-1 blockade, and chemotherapy. For example, a chemotherapeutic agent may be administered with the CD40 antibody, and the chemotherapeutic agent may be a cytotoxic agent. For example, epirubicin, oxaliplatin, and/or 5-FU can be administered to a patient receiving CD40 antibody therapy.

Optionally, the combination of CD40 and one or more other antibodies (e.g., CTLA-4 antibodies and/or LAG-3 antibodies and/or PD-1 antibodies) may also be combined with immunogenic agents, such as cancer cells, purified tumor antigens (including recombinant proteins, peptides, and sugar molecules), and cells transfected with genes expressing immunostimulatory cytokines (He et al, (2004) j.immunol.173: 4919-28). Non-limiting examples of tumor vaccines that can be used include melanoma antigen peptides, such as gp100 peptide, MAGE antigen, Trp-2, MART1, and/or tyrosinase, or tumor cells transfected to express the cytokine GM-CSF.

Other therapies that may be combined with the CD40 antibody include, but are not limited to, interleukin 2(IL-2) administration, radiation therapy, surgery, or hormone ablation.

The combination of therapeutic agents discussed herein can be administered simultaneously as a single composition in a pharmaceutically acceptable carrier, or simultaneously as separate compositions, wherein each agent is in a pharmaceutically acceptable carrier. In another embodiment, the combination of therapeutic agents may be administered sequentially.

Furthermore, if multiple combination therapy administrations are performed and the agents are administered sequentially, the order of sequential administration at each time point may be reversed or remain the same, sequential administration may be combined with simultaneous administration or any combination thereof.

The invention is further described by the following examples, which should not be construed as limiting. All figures and all references, Genebank sequences, patents and published patent applications cited throughout this application are incorporated herein by reference in their entirety.

Examples

Example 1 construction of HEK293A cell line stably expressing human, monkey or mouse CD40

cDNA sequences encoding human, monkey or mouse CD40(SEQ ID NOs: 67, 69 and 71, encoding amino acid sequences SEQ ID NOs: 68, 70 and 72, respectively) were synthesized and cloned into pLV-EGFP (2A) -Puro vector (Beijing England flourishing Biotech Co., Ltd., China) by restriction enzyme digestion. The resulting pLV-EGFP (2A) -Puro-CD40 was transfected into HEK293T cells (Nanjing Kebai, China) with psPAX and pMD2.G plasmids by lipofection to generate lentiviruses, in a manner exactly identical to the protocol of Lipofectamine 3000(Thermo Fisher Scientific, USA). Three days after transfection, lentiviruses were harvested from cell culture medium (DMEM medium (Cat #: SH30022.01, Gibco) supplemented with 10% FBS (Cat #: FND500, Excell)) of HEK293T cells. HEK293A cells (south kyoto bai, china) were then transfected with lentiviruses to obtain HEK293A cells (HEK 293A/hCD40, HEK293A/rhCD40, HEK293A/muCD40, respectively) stably expressing human, monkey, or mouse CD 40. Transfected HEK293A cells were cultured in DMEM + 10% FBS medium containing 0.2. mu.g/ml puromycin (Cat #: A11138-03, Gibco) for 7 days. Expression of human and monkey CD40 was analyzed by FACS analysis by flow analyzer using a commercially available human CD40 antibody (PE-anti-human CD40 antibody, Cat #313006, Biolegend, usa). Similarly, expression of mouse CD40 was confirmed by FACS using a commercially available mouse CD40 antibody (PE-mouse CD40 antibody, Cat #124609, Biolegend, usa).

EXAMPLE 2 preparation of hybridoma cell lines secreting human CD40 antibody

The mouse anti-human CD40 monoclonal antibody is obtained by conventional hybridoma fusion technology, and the scheme is slightly changed.

Inoculation of

13 BALB/C mice (Wintonlihua, China) were immunized by cross-injection of recombinant human CD40(ECD) -his protein (Yiwangshizhou, China, Cat: 10774-H08H) and monkey CD40(ECD) -hFc protein (Yiwangshizhou, China, Cat: 90097-C02H), the specific immunization protocol being shown in Table 2. Human CD40(ECD) -his protein and monkey CD40(ECD) -hFc protein were sonicated with equal volumes of complete Freund adjuvant (Cat #: F5881-10 × 10ML, Sigma, USA), incomplete Freund adjuvant (Cat #: F5506-6 × 10ML, Sigma, USA) or PBS.

TABLE 2 immunization procedure

At 1 week after each booster immunization, 50. mu.l of serum was taken from each mouse and titer was measured by ELISA, specifically using recombinant human CD40-hFc (Cat #: 10774-H02H, Yi Qiao Shen, China) and monkey CD40(ECD) -hFc (Cat #: 90097-C02H, Yi Qiao Shen, China) for binding test. Titre testing was also performed by FACS using HEK293A cells expressing human, monkey, mouse CD40 prepared in example 1.

Based on the results of ELISA and FACS assays after the last boost, 7 mice with higher serum titers were selected for the next hybridoma cell line preparation.

Preparation of hybridoma cell lines

Hybridoma cell lines were prepared by conventional hybridoma fusion techniques, with minor modifications to the protocol.

Four days after the last boost, the spleens were removed from the mice after sacrifice and single cell suspensions were prepared in PBS. Splenocytes were washed 3 times with DMEM medium (Cat #: SH30243.01B, Hyclone, USA). Mouse myeloma cell SP2/0(CRL-1581, ATCC, USA) in logarithmic growth phase was mixed with the above-isolated mouse spleen cell at a ratio of 1: 4 and washed 2 times with DMEM. Cell fusion was performed by means of PEG (Cat #: P7181, Sigma, USA) fusion. The fused cells were washed 3 times with DMEM and resuspended in cell growth medium (RPMI 1640+ 10% FBS +1X HAT). The cell suspension was plated on 96-well culture plates at 200. mu.l/well, 5X 10⁴For each well, cells were plated at 37 ℃ with 5% CO₂The humidified cell culture chamber of (2) was cultured for 7 days. On day 7, the medium was changed to fresh medium (DMEM + 10% FBS +1X HAT). After 2-3 days, cell culture supernatants were aspirated and hybridomas were screened by ELISA and FACS.

Screening of hybridoma cell lines by ELISA

Hybridoma clones that bind to human CD40 were first screened by high throughput ELISA binding assays. Hybridoma clones that bind to human CD40 were further tested for their ability to bind to monkey or mouse CD 40.

For ELISA assays, 96-well ELISA plates were plated with human CD40(ECD) -his (0.5. mu.g/ml, Cat #: 10774-H08H, Yiqiaoshengzhou, China), monkey CD40(ECD) -hFc (0.5. mu.g/ml, Cat #: 90097-C02H, Yiqiaoshengzhou, China) or mouse CD40-his (0.5. mu.g/ml, Cat #: 50324-M03H, Yiqiaoshengzhou, China), 100. mu.l per well overnight at room temperature. ELISA plates were washed 3 times with PBST (PBS + 0.05% Tween 20) solution, blocked with 200. mu.l blocking solution (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20) for 2 hours at room temperature, and washed 3 times with PBST solution. The hybridoma cell culture medium supernatant was diluted 10-fold with a diluent (PBS + 1% BSA, + 1% goat serum + 0.01% Tween 20), and then 100. mu.l of the diluted solution was pipetted into the sample test wells. After 1 hour incubation at room temperature, plates were washed 3 times with PBST. Mu.l goat anti-mouse Fc-HRP (1: 5000, Cat #: A9309-1ml, Sigma, USA) was added to each well, incubated at room temperature for 1 hour, washed 3 times with PBST, and developed with 80. mu.l TMB. After 5-10 minutes, the coloration was stopped by adding 80. mu.l of 0.16M sulfuric acid and the OD450 reading was determined using a microplate reader (SpectraMaxR i3X, molecular. mu. lar. Devies, USA)

234 hybridoma cell lines were selected for specific binding to human and monkey CD40 by ELISA as described above.

Screening of hybridoma cell lines by FACS detection

The 234 hybridoma cell lines selected were further tested for their binding ability to human, monkey or mouse CD40 expressed by HEK293A cells. Firstly 10⁵The preparation of HEK 293A/human CD40 cells, HEK 293A/monkey CD40 cells, or HEK 293A/murine CD40 cells from example 1 were added to each assay well of a 96-well plate. Hybridoma culture supernatant was diluted 10-fold with diluent (PBS containing 1% BSA, 1% goat serum, 0.01% tween 20) and added to sample test wells at 100 μ l per well. After incubation at 4 ℃ for 1 hour, the cells were washed 3 times with FACS wash (PBS + 1% BSA + 0.01% Tween 20). Thereafter, the cells were resuspended in FACS wash, and a 500-fold dilution of APC goat anti-mouse IgG secondary antibody (Cat #: 405308, BioLegen, USA) was added for 4-degree incubation for 1 hour, and the plate was washed 3 times with PBS and then cell fluorescence was detected on FACS detector (BD).

Based on the FACS screening described above, 162 hybridoma clones were obtained with high binding capacity to HEK 293A/human CD40 cells and HEK 293A/monkey CD40 cells, but not to HEK-293A/mouse CD40 cells.

Subcloning of hybridoma cells producing CD40 antibody

The 162 hybridoma clones were subjected to 2 rounds of subcloning. During subcloning, multiple subclones (n > 3) of each clone were selected and characterized by ELISA and FACS detection as described above. The subclones obtained by this procedure were identified as monoclonal hybridoma cell lines. 108 subclones exhibiting high binding to human and monkey CD40 were finally obtained, each subclone being derived from a different original parent clone.

Screening of hybridoma cell lines by HEK-Blue Activity detection

108 subclones selected above were plated in 96-well plates and cultured for 5 days. Supernatants were collected for HEK-Blue activity assay to identify CD40 antibodies with agonistic activity on the human CD40 signaling pathway.

An HEK-Blue reporter cell line (HEK-Blue/CD40) expressing this fusion protein was constructed by infecting HEK-Bluenull 1_ v cells (InvivoGen, USA) with a lentivirus (prepared as in example 1) expressing human CD40(SEQ ID NO.: 68) and pressure-screening with 10. mu.g/ml puromycin.

For HEK-Blue/CD40 reporter system detection, 4X 10⁴HEK-Blue/CD40 cells were resuspended in 200. mu.l of cell culture medium (DMEM medium (Hyclone, USA, Cat #: SH30243.01) + 10% FBS (Excell, China, Cat #: FND500) + 10. mu.g/ml puromycin (GIBCO, USA, Cat #: A11138-03) + 100. mu.g/ml Normocin^TM(Invivogen, USA, Cat #: ant-nr-2) + 100. mu.g/ml bleomycin (Invivogen, USA, Cat #: ant-Zn-5), plated in 96-well plates and incubated overnight at 37 ℃. The next day, 200. mu.l of DMEM medium was added to each well, replacing the original medium. After 7 hours of incubation, DMEM medium was replaced with HEK-Blue detection buffer (Invivogen, US, Cat #: hb-det3), 100. mu.L per well, and 100. mu.L of hybridoma supernatant was added per well. The resulting mixture was 5% CO at 37 deg.C₂Incubate until blue. The reading at OD630 was determined using a SpectraMax microplate reader (molecular μ lar Devices, USA). Among them, the negative control was HEL antibody (LifeTein, LLC, US, Cat. #: LT 12031). RO7009789 (an agonistic antibody made according to the amino acid sequence disclosed in US7338660B2 and having a human IgG 2/kappa constant region) and CD40L (Cat #: 10239-H08E, Natural ligand and activator of CD40, Chinesian, China) were used as positive controls.

As shown in fig. 1, 38 clones showed varying degrees of CD40 agonistic activity.

Example 3 purification of mouse CD40 monoclonal antibody

Based on the above HEK-Blue assay, a total of 20 clones with high HEK-Blue activity (see Table 3 below for details) were picked for further study. Monoclonal mouse antibodies of 20 selected clones were first purified. Briefly, each subcloned hybridoma cell was grown in T175 cell culture flasks, each containing 100ml of fresh serum-free hybridoma medium (Gibco, Cat #: 12045-. Cells were incubated at 37 ℃ with 5% CO₂Cultured in an incubator for 10 days. The culture was collected, centrifuged at 3500rpm for 5 minutes, and cell debris was removed by filtration through a 0.22 μm filter. Monoclonal antibodies were enriched and purified by pre-equilibrated protein-A affinity column (Cat #: 17040501, GE, USA). Then, elution was carried out with an elution buffer (20mM citric acid, pH3.0 to pH 3.5). Thereafter, the antibody was stored in PBS (pH 7.0), and the antibody concentration was detected by NanoDrop.

The subtype of the purified antibody was determined by using kappa and lambda-mouse Rapid typing kits (Thermal, USA, Cat #: 26179) and mouse monoclonal antibody typing reagents (Sigma, USA, Cat #: IS02-1KT), and the detection procedure was in accordance with the kit instructions. The subtypes and expression titers of the 20 selected clonal antibodies are summarized in table 3.

TABLE 3 subtype and expression Titers of CD40 antibody

Antibodies	Subtype of cell	Expression Titers (mg/l)	Antibodies	Subtype of cell	Expression Titers (mg/l)
						13A2	Mouse IgG1/k	24.744	77D9	Mouse IgG1/k	12.22
16A6	Mouse IgG1/k	31.111	79D7	Mouse IgG1/k	20.39
						29A10	Mouse IgG1/k	33.889	142F7	Mouse IgG1/k	17.22
7B4	Mouse IgG1/k	18.667	89D11	Mouse IgG1/k	95.73
						9A7	Mouse IgG1/k	7.778	91E4	Mouse IgG2a/k	4.47
19H4	Mouse IgG1/k	10.000	101C12	Mouse IgG1/k	18.94
						37G10	Mouse IgG1/k	65.333	92F6	Mouse IgG1/k	39.97
35C9	Mouse IgG1/k	11.667	82D3	Mouse IgG2a/k	16.27
						16F4	Mouse IgG2b/k	14.000	23B8	Mouse IgG2a/k	32.33
50F6	Mouse IgG1/k	12.778	51F7	Mouse IgG1/k	1.44

EXAMPLE 4 purified mouse CD40 monoclonal antibody binds to human and monkey CD40

The purified mouse CD40 monoclonal antibody was first tested by ELISA to determine its binding affinity to recombinant human, monkey or mouse CD40 protein.

ELISA assay plates were coated overnight at 4 ℃ with 500ng/ml human CD40(ECD) -his (Cas, Chi, China, Cat: 10774-H08H). Each well was blocked with 200. mu.l of blocking solution (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20) at room temperature for 2 hours, then 100. mu.l of a gradient diluted CD40 antibody (maximum concentration 40. mu.g/ml) was added and incubated at room temperature for 1 hour. The plates were washed 3 times with PBST (PBS + 0.05% Tween 20), then 5000-fold diluted goat anti-mouse IgG-HRP (Simga, USA, Cat #: A9309-1ml) was added and incubated for 1 hour at room temperature. The plates were developed with freshly prepared Ultra-TMB (BD, USA, Cat # No.: 555214) for 5 minutes at room temperature. Then using SpectraMax^Ri3X (Molecular devices, USA) reads at 450 nm.

Species cross-reactivity of 20 CD40 monoclonal antibodies to monkey or mouse CD40 was further tested by direct ELISA. Specifically, 500ng/ml monkey CD40(ECD) -hFc protein (Yiqian Shenzhou, China, Cat: 90097-C02H) or mouse CD40-hFc (Yiqian Shenzhou, China, Cat: 50324-M03H) was coated in a 96-well ELISA plate and incubated with 100. mu.l of CD40 antibody (maximum concentration 40. mu.g/ml) in a gradient dilution. HRP-goat anti-mouse IgG (Sigma, Cat #: A9309-1ml) was then used. The CD4 antibodies RO7009789 and ADC1013 (prepared according to the amino acid sequence disclosed in US2016/0311916A1 and having a human IgG 1/kappa constant region) were used as references.

EC of the above binding assay₅₀The values are summarized in table 4. It can be seen that of the 20 antibodies, except 51F7, all cross-reacted with monkey CD40 but not mouse CD 40.

TABLE 4.20 binding of mouse CD40 mAbs to human, monkey, or mouse CD40

Example 5 binding of the mouse CD40 monoclonal antibody to human and monkey CD40 expressed by HEK293A cells

To further determine whether the CD40 antibody binds to human, monkey or mouse CD40 expressed by HEK293A cells, FACS cell binding assays were performed using HEK293A cells stably expressing human, monkey or mouse CD40, respectively (see example 1). Briefly, 10⁵Individual HEK293A cells were plated in 96-well plates and a gradient of CD40 antibody (maximum concentration 40 μ g/ml) was added. After incubation for 1 hour at 4 ℃, plates were washed 3 times with PBST. Thereafter, 500-fold dilutions of APC-goat anti-mouse IgG (BioLegen, USA, Cat #: 405308) were added. After 1 hour incubation at 4 ℃, cells were washed 3 times with PBS and then cell fluorescence was monitored using a FACS detector (BD).

As shown in table 5, all 20 mouse CD40 monoclonal antibodies showed high binding to human and monkey CD40, but did not bind to mouse CD40 (data not shown).

TABLE 5 binding affinities of CD40 antibodies to human and monkey CD40

Example 6 mouse CD40 antibody inhibits or promotes the interaction between human CD40-CD40L

The purified CD40 antibody was further tested for its ability to block or promote binding of human CD40L to human CD 40. Briefly, 96-well ELISA plates were coated with 500ng/ml human CD40L (Cat #: 10239-H08E, Chinesian, China) overnight at 4 ℃. Thereafter, the plates were blocked with 200. mu.l of blocking solution (PBS + 2% BSA) for 2 hours at room temperature. The CD40 antibody (highest concentration 40. mu.g/ml) was mixed with 2. mu.g/ml human CD40-hFc (Cat #: 10774-H02H, Cassia, Chinesia), and incubated at 37 ℃ for 1 hour, and the resulting mixture was subsequently added to the assay wells and incubated at room temperature for 1 hour. The plates were washed 3 times with PBST (PBS + 0.05% Tween 20), and 5000-fold diluted anti-human IgG FC-HRP (Simga, USA, Cat #: A0170-1ML) was added and incubated at room temperature for 1 hour. The plate was washed 3 times with PBST wash solution and 80. mu.l of TMB was added to each well for color development. After 5-10 minutes, the reaction was stopped by adding 80. mu.l of 0.16M sulfuric acid, followed by OD450 readings using SpectraMaxR i3X (molecular. mu. lar. Devices, USA).

Interestingly, the data indicate that 6 mouse antibodies (13a2, 16a6, 7B4, 50F6, 142F7 and 101C12) promote human CD40-CD40L interactions, whereas 3 antibodies (23B8, 92F6, 82D3) block CD40-CD40L interactions, the remaining few antibodies have no apparent effect on CD40-CD40L binding. The results of 4 representative clones are shown in figure 2.

Example 7 mouse CD40 antibody activates CD40 signaling pathway activity

To determine whether the selected mouse CD40 antibody has CD40 signaling pathway agonistic activity, a HEK-Blue activity assay was performed. Briefly, the HEK-Blue/CD40 cells prepared in example 2 were incubated in DMEM medium (Hyclone, U.S.A., Cat #: SH30243.01) further containing 10% FBS (Excell, China, Cat #: FND500), 10. mu.g/ml puromycin (GIBCO, U.S., Cat #: A11138-03), 100. mu.g/ml Normocin^TM(Invivogen, Cat #: ant-nr-2, USA), 100 μ g/ml bleomycin (Invivogen, Cat #: ant-Zn-5, USA). Will be 4X 10⁴Each HEK-Blue/CD40 cell was aliquoted into 200. mu.l of cell culture medium in a 96-well plate and cultured overnight (about 12 hours) at 37 ℃. The medium was replaced with 200. mu.l of fresh DMEM medium and incubation was continued for 7 hours. Thereafter, the DMEM medium in each well was replaced with 100. mu.l HEK Blue assay buffer (Invivogen, Cat #: hb-det 3; U.S.) containing various concentrations of CD40 antibody (ranging from 100. mu.g/ml to 0.01 ng/ml). The cells were continued to grow at 37 ℃ until blue color appeared. OD630 readings were measured using a SpectraMaxR i3X microplate reader (molecular μ lar Devices, USA).

EC₅₀Values are summarized in table 6and curves for representative mabs are shown in figure 3. It can be seen that these 20 antibodies showed varying degrees of agonistic activity in the functional HEK-Blue assay, indicating that they are able to stimulate the signaling pathway downstream of CD 40.

TABLE 6 agonistic activity of CD40 antibody

Antibodies	HEK Blue EC50(ng/mL)	Antibodies	HEK Blue EC50(ng/mL)
				RO7009789	23.26	142F7	64.77
ADC1013	1034	89D11	91.06
				13A2	12.61	91E4	152.7
16A6	11.37	82D3	285.4
				7B4	26.14	23B8	3373
50F6	42.16	51F7	6173
				101C12	175.2	92F6	263.7
77D9	22.26	19H4	92.79
				35C9	86.08	16F4	252.1
37G3	146.1	29A10	139.8
				79D7	55.29	9A7	61.96

Example 8 epitope Competition

Antigen binding epitope competition between antibodies was detected by means of competition ELISA. Briefly, 96-well ELISA assay plates were coated with 5. mu.g/ml of R07009789 or ADC1013 overnight at 4 ℃. The wells were blocked with 200 μ l of blocking solution (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20) for 2 hours at room temperature. 0.5. mu.g/ml human CD40(ECD) -his protein (Cat #: 10774-H08H, Chinesian, Chinese) was added to the assay plate and incubation continued for 1 hour at room temperature. The plate was washed 3 times with PBST, 1. mu.g/ml purified antibody was added and incubated for 1 hour at room temperature. ELISA plates were washed 3 times with PBST and then incubated for 1 hour at room temperature with 20000-fold dilutions of anti-mouse Fc-HRP (Cat #: A9309-1MC, Simga, USA). After further washing with PBST wash 3 times, the plates were developed with freshly prepared Ultra-TMB (Huzhou Yingchuang, China, Cat #: TMB-S-003) for 5 minutes at room temperature and read at 450nm with a SpectraMax microplate reader (Molecular Devices; US; SpectraMaxR i 3X).

The results are summarized in table 7. The 7 mouse antibodies (101C12, 142F7, 89D11, 13a2, 16a6, 7B4, and 50F6) were epitope-competitive with the two reference antibodies, while antibodies 9a7, 92F6, 19H4, 16F4, and 51F7 were not epitope-competitive with either of the reference antibodies. The remaining antibodies compete for epitopes present in one of the two reference antibodies.

TABLE 7 epitope Competition for Competition ELISA assays

+: there is competition; -: absence of competition

Example 9 agonistic CD40 antibodies promote dendritic cell maturation

Dendritic cell maturation experiments were performed to further confirm the agonistic activity of the mouse CD40 antibody. Briefly, PBMCs from blood samples from a healthy human donor were collected by gradient density centrifugation and resuspended in RPMI1640 medium and cultured at 37 ℃ for 2 hours to collect adherent cells, i.e., mononuclear cells. The above cells were cultured in RPMI1640 medium containing 100ng/ml recombinant human GM-CSF (R & D; U.S.; Cat: 7954-GM), 100ng/ml recombinant human IL-4(R & D; U.S.; Cat #: 6507-IL) and 10% FBS. After 3 days the medium was half-changed and the cells were cultured for 5 days, different concentrations of CD40 antibody (10ug/ml or 1ug/ml) and control antibody were added to the cell culture and the culture was continued for 48 hours. Dendritic cell activation markers were stained with mouse anti-human CD83 (BD; USA; Cat #: 556910), PE mouse anti-human CD86 (BD; USA; Cat #: 555658), and BV650 mouse anti-human CD80 (BD; USA; Cat #: 564158) and detected by flow cytometry.

The results for representative antibodies are shown in figure 4. Compared to Hel controls, mouse CD40 antibodies 16a6, 29a10, 7B4, and 13a2 increased the expression of CD86(a biomarker of mature dendritic cells), while antibodies 16a6, 29a10, 7B4, and 13a2 significantly upregulated the expression of CD80 and CD83 (both costimulatory molecules).

Example 10 expression and purification of chimeric CD40 antibodies

8 antibodies ((13A2, 16A6, 7B4, 29A10, 92F6, 77D9, 50F 6and 142F7) were selected for further study.A hybridoma cell of these 8 antibodies was first cloned for the sequence of the variable region by the RT-PCR method using the primers mentioned in the literature (Juste, Muzard, & Billiald, (2006), Anal biochem., 1; 349 (1): 159-61). expression vectors were constructed by inserting sequences encoding the variable region and the respective human IgG 2/kappa constant regions (the amino acid sequences of the heavy and light constant regions are listed in SEQ ID NOs: 63 and 65, respectively) into the restriction sites XhoI/BamHI of pCDNA3.1(Invitrogen, USA). the amino acid sequences encoding the variable regions are summarized in Table 1.

The expression vector obtained above was transfected into HEK-293F cells (Cobioer, China). In particular, HEK-293F cells were in Free Style^TM293 expression medium (Gibco, Cat #: 12338-018) and cells transfected with each expression vector by means of Polyethyleneimine (PEI) in a ratio of 1: 3, 1.5. mu.g of DNA per ml of cell culture. Transfected HEK-293F cells at 37 ℃ in 5% CO₂The cultivation was carried out in an incubator at 120 RPM. After 10-12 days, cell culture supernatants were collected and monoclonal antibodies were purified according to the procedure of example 3.

Example 11 binding of chimeric monoclonal antibody to CD40 to human or monkey CD40 expressed by HEK293A cells

The chimeric antibodies were tested for their binding to HEK 293A/human CD40 cells, HEK 293A/monkey CD40 cells, and HEK 293A/mouse CD40 cells prepared in example 1, according to the method steps of example 5. RO7009789 and ADC1013 antibodies as positive controls

As shown in fig. 5, the chimeric antibody had high affinity to both human and monkey CD40, but did not bind to mouse CD40 (data not shown).

Example 12 chimeric CD40 antibodies have CD40 signaling pathway agonism and promote dendritic cell maturation

The chimeric antibodies were further tested for their activation effect on the CD40 signaling pathway by HEK-Blue assay and dendritic maturation assay according to the method steps of example 7 and example 9. RO7009789, APX005 and/or ADC1013 were used as positive control antibodies, where APX005 has a human IgG 1/kappa constant region, prepared according to the amino acid sequence in WO2014/070934a 1.

As shown in fig. 6,8 chimeric antibodies showed similar functional activity to their parent mab. Figure 7 shows that all chimeric antibodies tested were able to upregulate the expression of CD86 protein (a biomarker for mature dendritic cells) on dendritic cells, indicating its promotion of dendritic cell maturation.

Example 13 humanized engineering of CD40 antibody

Based on the above-described related functional assays, two antibodies, 7B4 and 13a2, were selected for humanization engineering and further study. Humanization of mouse-derived antibodies was performed by Complementarity Determining Region (CDR) grafting (U.S. Pat. No. 5,225,539), as described in detail below.

To select the humanized acceptor framework of murine antibodies 7B4 and 13A2, the light and heavy chain variable region sequences of 7B4 and 13A2 were aligned with the human immunoglobulin gene database of the NCBI website (http:// www.ncbi.nlm.nih.gov/igblast /). The human germline IGVH and IGVK with the highest degree of homology to 7B4 and 13a2 were selected as frameworks for humanization engineering. The light chain germline acceptor sequence selected was human IGKV2-30 x 02 and the heavy chain germline acceptor sequence selected was human IGHV4-28 x 06, see table 8 in particular.

Three-dimensional structural modeling of the variable domains of 7B4 and 13a2 was performed to identify key framework amino acid residues that may play an important role in maintaining the CDR loop structures, in order to design back mutations of humanized antibodies. Briefly, the selected structural templates have the same types of L-CDR1, L-CDR2, L-CDR3, H-CDR1, H-CDR2, and H-CDR3 cyclic structures as 7B4 and 13A2, respectively. Using the selected structural templates, a structural model of humanized 7B4 and 13a2 was constructed by replacing the mouse framework with human germline heavy and light chain framework sequences. Three-dimensional structural modeling was then performed to identify key framework amino acid residues that might play an important role in maintaining the CDR loop structure or heavy and light chain linkages. When the murine antibody framework and the human germline acceptor framework possess the same amino acid residues at a certain position, the human germline amino acid residues are retained. On the other hand, when the murine framework and the human germline acceptor framework have different amino acid residues at a certain position, the importance of the residue is evaluated by structural simulation. If an amino acid residue within the framework of the human germline receptor is found to interact with and affect a CDR residue, that residue will revert back to a murine residue.

TABLE 8 structural templates for antibody structural simulation

Antibody chains	PDB encoding of template structures	Sequence identity	Sequence similarity
				13A2 heavy chain	5E2T	71％	83％
13A2 light chain	1DLF	84％	92％
				7B4 heavy chain	5E2T	87％	90％
7B4 light chain	1DLF	87％	95％

Based on the above structural modeling, the 13a2 heavy chain identified 5 potential back mutations (I49M, V68I, M70I, K44N, G45K), and the light chain identified 5 potential back mutations (M4L, R51L, F76L, Y92F, Q105S). The 7B4 heavy chain identified 5 potential back mutations (I49M, V68I, M70I, K44N, G45K) and the light chain identified 4 potential back mutations (M4L, R51L, Y92F, Q105S).

As shown in table 1, a total of three humanized heavy chain variable regions and three humanized light chain variable regions were designed for 13a2, resulting in a total of 5 humanized antibodies. Similarly, for 7B4, a total of three humanized heavy chain variable regions and three humanized light chain variable regions were designed, resulting in a total of 5 humanized antibodies.

Synthesizing the sequence encoding the humanized heavy chain variable region plus the constant region of human IgG2 and the sequence encoding the humanized light chain variable region plus the constant region of human kappa, the amino acid sequences of the heavy chain constant region and the light chain constant region are set forth in SEQ ID NOs: 63, and 65, and cloned into the pcdna3.1(+) expression vector (Invitrogen, usa) using BamH I and Xho I restriction sites. All expression constructs were confirmed by sequencing. The HEK293F expression system (Invitrogen, usa) was transfected with heavy and light chain expression vectors and transiently expressed 10 humanized CD40 antibodies (5 7B4 antibodies, 5 13a2 antibodies) with the method steps as described in example 10. The humanized antibody was purified as described in example 3.

Example 14 characterization of chimeric and humanized CD40 antibodies

Chimeric and humanized CD40 antibodies were further tested for binding to HEK 193A/human CD40 and HEK 293A/monkey CD40 according to the method steps in example 5. Referring to example 7, chimeric and humanized CD40 antibodies were further tested for their activation of the CD40 signaling pathway by HEK-Blue. Humanized antibodies were further tested for their effect in promoting maturation of dendritic cells derived from blood samples from 3 different healthy human donors according to the method steps in example 9. The secretion of IL-12(p40) in dendritic cells was detected by IL-12(p40) detection kit (BD, US, Cat #: 551116), and the specific experimental procedures were completely in accordance with the instructions.

As shown in figures 8, 9, 10 (donor 1), 11 (donor 2) and 12 (donor 3), all humanized antibodies bound to human and monkey CD40 proteins, did not bind to mouse CD40, activated the CD40 signaling pathway, promoted dendritic cell maturation, and showed the highest binding, agonistic and functional activity of 13a2-VH3VL2, 13a2-VH3VL3 and 7B4VH2VL 2.

Example 15 affinity of chimeric and humanized CD40 antibodies to human CD40

By BIAcore^TM8K (GE Life Sciences, USA) to quantify the binding affinity of chimeric or humanized CD40 antibody to human CD 40.

Specifically, 100-200RU (reaction unit) of human CD40(ECD) -his protein (Cat: 10774-H08H, Chi Qiao, China) was coupled to a CM5 biochip (Cat #: BR-1005-30, GE Life Sciences, USA), followed by blocking of the unreacted groups of the chip with 1M aminoethanol. The antibody was injected into the SPR reaction solution (HBS-EP buffer, pH7.4, Cat #: BR-1006-69, GE Life Sciences, USA) at 30. mu.L/min in a gradient dilution (concentration from 0.3. mu.M to 10. mu.M). Binding of the antibody was calculated by subtracting RU from the blank control well. Binding Rate (k)_a) And dissociation rate (k)_d) Calculations were performed using the formula for the 1: 1 pairing model in the BIA evaluation software. Equilibrium dissociation constant K_DThrough k_d/k_aAnd (4) calculating. The antibody binding dissociation curves determined by SPR are shown in fig. 13, and table 9 shows the binding affinity data of the chimeric antibody and the humanized antibody. RO7009789 and ADC1013 antibodies as positive controls.

TABLE 9 binding affinity of CD40 antibody to human CD40

Antibodies	ka	kd	KD
				RO700789	1.07 E+5	1.83 E-4	1.71 E-9
ADC1013	1.2 E+6	3.48 E-2	2.9 E-08
				13A2	4.84E+05	1.73 E-03	3.58 E-09
13A2-VH0VL0	3.96 E+05	1.14 E-02	2.88 E-08
				13A2-VH2VL2	8.23E+05	1.71 E-03	2.08 E-09
13A2-VH2VL3	7.35 E+05	2.61 E-03	3.55 E-09
				13A2-VH3VL2	8.25 E+5	2.42 E-3	2.93 E-09
13A2-VH3VL3	5.98 E+5	4.59 E-3	7.68 E-09
				7B4	1.00E+06	3.6E-03	3.6E-09
7B4-VH0VL0	2.75E+06	6.8E-03	2.47E-09
				7B4-VH2VL2	2E+06	5.81E-03	2.91E-09
7B4-VH2VL3	1.78E+06	6.39E-03	3.6E-09
				7B4-VH3VL2	1.46E+06	6.15E-03	4.2E-09
7B4-VH3VL3	2.13E+06	4.88E-03	2.99E-09

Example 16 antigen binding epitope identification of chimeric and humanized CD40 antibodies

Chimeric and humanized CD40 antibodies were tested for antigen binding epitopes by ELISA.

The extracellular region (ECD) of CD40 contains 4 cysteine-rich domains (CRDs), designated CRD1, CRD2, CRD3, and CRD4, respectively. Based on the structure of CD40 ECD, one full-length CD40 ECD, five CD40 ECD truncations, and four CD40 ECD mutants were constructed, and the information for these recombinant proteins is shown in table 10 below. The N-terminal of each of these proteins was linked to a signal peptide (SEQ ID NO: 83) for protein expression and secretion, and the C-terminal thereof was linked to an mFc tag (SEQ ID NO: 84) for ELISA detection. DNA sequences encoding these recombinant proteins were synthesized and cloned into pcDNA3.1 vector. Recombinant protein expression and purification was performed according to the method steps in example 10. The binding of the monoclonal antibodies to recombinant CD40 protein was assessed by performing an ELISA assay according to the method steps described in example 2.

As shown in a of figure 14, all antibodies bound to full-length CD40 ECD and none bound to the truncation, indicating that the CRD1 domain is involved in and important for antibody binding. B of fig. 14 shows that the 13a2 chimeric antibody, three humanized antibodies, and ADC1013 did not bind to CD40 mutants 2-4, indicating that these five antibodies have the same or similar binding epitopes. RO7009789 did not bind to mutants 1, 2 or 4 and APX005 did not bind to mutants 2 or 4.

TABLE 10 CD40 ECD truncations and mutants

Note: CRD Δ 1 represents a truncated CRD1 domain

Example 17 specific binding of humanized CD40 antibody to human CD40

To determine the binding specificity of the antibodies to CD40, the binding of the humanized antibodies to human CD40 and other members of the TNFRSF family with higher sequence homology were determined by ELISA, according to the method steps described in example 2.

Specifically, the binding affinity of the humanized antibody to human CD40(ECD) -his (TNFRSF4, Cat #: 10774-H08H, Yi Qian Shen, China), human OX40-his (TNFRSF5, Cat #: 10481-H08H, Yi Qian Shen, China), human HVEM-mFc (TNFRSF14, Cat #: HVM-H5255, ACRO, China), human 4-1BB (TNFRSF9, Cat #: 41B-H522a, TNFRSF14, China), human NGFR (TNFRSF16, Cat #: 13184-H08H, Yi Qian Shen, China DR6(TNFRSF21, Cat #: 10175-H08H, Qian Shen, China), and human RANK (TNFRSF11, Cat #: RAL 5240, ACR #: ACR 48, ACR, Chinese was examined.

As shown in figure 15, neither 13A2VH3VL3 nor 7B4VH2VL2 bound to human OX40(TNFRSF4), HVEM (TNFRSF14), 4-1BB (TNFRSF9), NGFR (TNFRSF16), DR6(TNFRSF21), or RANK (TNFRSF11), indicating the binding specificity of the 13A2VH3VL3 and 7B4VH2VL2 antibodies to human CD 40.

Example 18 the genetically modified CD40 antibody has better agonistic activity

Studies have shown that the best biological and antitumor effects of the agonistic CD40 antibody require a co-action of Fc receptors (FcR) (Richman and Vonderheide, (2014) Cancer Immunol Res 2 (1): 19-26). Thus, the CD40 antibody was prepared to have a heavy/light chain variable region of 13A2VH3VL3 or 7B4VH2VL2 and a human IgG 1/kappa constant region, with the human IgG1 constant region carrying the S267E and L328F mutations (mutated IgG1 constant region amino acid sequence as set forth in SEQ ID No.: 64). The resulting antibodies were designated 13A2-VH3VL3-IgG1(SE/LF) and 7B4-VH2VL2-IgG1(SE/LF), respectively, and further tested for dendritic cell maturation-promoting activity according to the procedure of example 9. RO7009789, ADC1013 and APX005 were used as positive controls and Hel was used as negative control.

As shown in fig. 16, both 13a2-VH3VL3-IgG1(SE/LF) and 7B4-VH2VL2-IgG1(SE/LF) showed higher agonistic activity in promoting dendritic cell maturation compared to the parent antibody, and 13a2-VH3VL3-IgG1(SE/LF) had the highest activity among all tested antibodies.

Example 19 humanized antibodies have anti-tumor effects in vivo

In vivo antitumor activity of CD40 antibody having the heavy/light chain variable region of 13A2VH3VL3 or 7B4VH2VL2 and the mouse IgG 1/kappa constant region (the amino acid sequence of the mouse IgG 1/kappa constant region is shown in SEQ ID NOs: 62 and 66) was studied, and the animal model used was established by implanting MC38 mouse intestinal adenocarcinoma into a transgenic mouse (GemPharmatech Co. Ltd, China) humanized for the CD40 target. Antibodies the mouse IgG 1/kappa constant region was used to boost Fc function of the antibodies in a mouse model.

Each mouse was injected subcutaneously in one flank on day 0 at 1X 10⁶MC38 cells. When the tumor grows to 80mm³At this time, five groups of 8 were randomly divided. Mice were intraperitoneally injected with 13a2-VH3VL3, 7B4-VH2VL2, control antibody (RO7009789 or APX005), or PBS on days 4,7, 11, 14, 18, and 21, 10 mg/kg/day, where RO7009789 and APX005 were both engineered to have the amino acid sequence as set forth in SEQ ID NOs: 62 and 66, mouse IgG 1/kappa constant region.

Mice body weight change and tumor size were followed over time. The long (D) and short (D) edges of the tumor were measured with a vernier caliper every other day and by the formula TV-0.5 x D²Tumor volume was calculated. The tumor reached 3.5cm in the antibody group³The experiment was stopped. Tumor volume differences were determined by one-way anova.

On day 25, 4 larger tumor mice were taken from each group for T cell analysis, and the other mice continued to be tumor size measured. Mice for T cell analysis were sacrificed and tumors were immediately taken and placed in Hanks buffer with collagenase. The tumor tissue was cut into small pieces with scissors and the cut tumor tissue was further incubated in Hanks buffer containing collagenase and gently shaken for 30 minutes at 37 ℃. Thereafter, 10ml of RPMI1640 + 10% FBS was added to each sample to terminate collagenase activity and maintain immune cell viability. The sample was filtered through a 70 μm cell filter (Corning, Cat #: 352350) and placed in a fresh centrifuge tube. Samples were centrifuged and resuspended in PBSF buffer (PBS + 2% FBS) at a cell density of 1X 10⁷Cells/ml. The samples were washed 2 times with PBSF and then divided into two portions, to one of which was added a fluorescently labeled CD45 antibody (Brilliant Violet 785)^TMMouse CD45 antibody, Biolegend, US, Cat #: 103149), CD8 antibody (APC mouse CD8a antibody, Biolegend, US, Cat #: 100712), CD3 antibody (FITC mouse CD3 antibody, Biolegend, US, Cat #: 100203) and CD4 antibody (PerCP mouse CD4 antibody, Biolegend, US, Cat #: 100432), to another portion of the mixture was added a fluorescently labeled CD11c antibody (APC mouse CD8a antibody, Biolegend, US, Cat #: 100712), CD80 antibody (APC mouse CD11c antibody, Biolegend, US, Cat #: 117310), CD83 antibody (E/Cy7 mouse CD83 antibody, Biolegend, US, Cat #: 121518) and CD86 antibody (FITC mouse CD86 antibody, Biolegend, US, Cat #: 105005) mixtures. The resulting mixture was incubated at 4 ℃ for half an hour. Cells were washed 2 times with PBSF and then analyzed by FACS machine (BD).

As shown in figure 17, treatment with the CD40 antibody significantly slowed or inhibited tumor growth compared to the negative control group, despite the different individual responses. Tumor growth inhibition was observed in all mice in the 7B4VH2VL2 and APX005 groups, and most mice exhibited tumor growth inhibition in the 13A2VH3VL3 and RO7009789 groups. On day 28, tumors in 4 remaining mice completely disappeared in the 7B4VH2VL 2-administered group, and in the APX005 group, tumors in 2 of 4 mice completely disappeared.

CD40 antibody treatment may cause weight loss in mice due to antibody toxicity problems. As shown in FIG. 18 and Table 11, tumor weight was subtracted (approximately 1000mm for 25 days tumor)³1.2g), the body weight of the 13A2VH3VL 3-treated group mice on day 25 was slightly increased from the initial body weight, without the problem of substantial weight loss associated with treatment with, for example, RO7009789, and had less effect on body weight than 7B4VH2VL2 and APX005, indicating that the 13A2VH3VL3 antibody was less toxic.

TABLE 11 body weights (mean. + -. SE (g) for groups of 8 mice each)

Group/day of the day

4

6

8

11

14

18

21

25

13A2-VH3VL3

22.9±0.5

20.6±0.5

22.5±0.5

24.1±0.5

24.7±0.5

25.2±0.5

24.7±0.5

25.4±0.8

7B4-VH2VL2

23.0±0.3

21.2±0.2

23.4±0.2

23.8±0.2

22.7±0.5

24.4±0.2

22.2±0.5

22.9±0.5

RO7009789

23.9±0.3

21.5±0.4

22.9±0.4

22.6±0.3

22.2±0.4

22.1±0.5

21.8±0.4

19.6±0.5

APX005

22.6±0.3

20.8±0.3

22.9±0.3

23.1±0.4

23.4±0.6

24.1±0.4

21.5±0.6

22.0±0.7

PBS

23.3±0.4

23.2±0.3

24.2±0.4

23.7±0.3

25.1±0.4

26.5±0.4

27.8±0.5

30.7±0.6

Figure 19 shows that antibody 7B4VH2VL2 significantly elevated CD45⁺CD45 in cells⁺CD3⁺CD8⁺Cells and CD45⁺CD3⁺CD4⁺Percentage of cells. CD45⁺CD3⁺CD8⁺The percentage of cells also increased in the 13A2VH3VL3 treated group. In addition, figure 20 shows that 7B4VH2VL2 treatment significantly increased tumor infiltrating dendritic cells (CD 45)⁺CD11c⁺Cells) indicating its strong agonistic activity in promoting dendritic cell maturation.

The sequences of CD40 antibody 77D9 of the present application are summarized below.

While the invention has been described in connection with one or more embodiments, it is to be understood that the invention is not limited to those embodiments, and the above description is intended to cover all other alternatives, modifications, and equivalents included within the spirit and scope of the appended claims. All documents cited herein are incorporated by reference in their entirety.

Sequence listing

<110> Beijing Tiankuang-Shi Biotech Co., Ltd

Beijing Huafang Tianshi biopharmaceutical Co.,Ltd.

<120> antibodies that bind to CD40 and uses thereof

<130> 55566 00006

<160> 84

<170> PatentIn version 3.5

<210> 1

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2/7B4-HV-CDR1

<400> 1

Thr Asn Tyr Tyr Trp Asn

1 5

<210> 2

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> 16A6-HV-CDR1

<400> 2

Thr Asn Tyr His Trp Asn

1 5

<210> 3

<211> 6

<212> PRT

<213> Artificial sequence

<220>

<223> 29A10-HV-CDR1

<400> 3

Ser His Tyr Tyr Met Tyr

1 5

<210> 4

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> 92F6-HV-CDR1

<400> 4

Asp Thr Tyr Met His

1 5

<210> 5

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> 77D9-HV-CDR1

<400> 5

Asn Tyr Ala Met Ser

1 5

<210> 6

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> 50F6-HV-CDR1

<400> 6

Thr Tyr Trp Ile Asn

1 5

<210> 7

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> 142F7-HV-CDR1

<400> 7

Asn Tyr Leu Ile Glu

1 5

<210> 8

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2-HV-CDR2

<400> 8

Tyr Ile Asn Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Asn

1 5 10 15

<210> 9

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> 7B4/16A6-HV-CDR2

<400> 9

Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu Lys Asn

1 5 10 15

<210> 10

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> 29A10-HV-CDR2

<400> 10

Thr Ile Ser Asp Ala Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val Lys

1 5 10 15

Gly

<210> 11

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> 92F6-HV-CDR2

<400> 11

Arg Ile Asp Pro Ala Asn Gly Asn Thr Asn Tyr Asp Pro Lys Phe Gln

1 5 10 15

Gly

<210> 12

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> 77D9-HV-CDR2

<400> 12

Glu Val Ser Gly Ser Gly Tyr Tyr Thr Tyr Tyr Pro Asp Thr Val Thr

1 5 10 15

Gly Arg Phe

<210> 13

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> 50F6-HV-CDR2

<400> 13

Arg Ile Ser Pro Gly Ser Gly Ser Thr His Tyr Asn Glu Met Phe Lys

1 5 10 15

Gly

<210> 14

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> 142F7-HV-CDR2

<400> 14

Asn Pro Gly Thr Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys Asp

1 5 10 15

<210> 15

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2/7B4/16A6-HV-CDR3

<400> 15

Leu Asp Tyr

1

<210> 16

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> 29A10-HV-CDR3

<400> 16

Gly Gly Tyr Trp Phe Phe Asp Val

1 5

<210> 17

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> 92F6-HV-CDR3

<400> 17

Trp Gly Tyr Asp Trp Tyr Phe Asp Val

1 5

<210> 18

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> 77D9-HV-CDR3

<400> 18

Arg Ala Tyr

1

<210> 19

<211> 3

<212> PRT

<213> Artificial sequence

<220>

<223> 50F6-HV-CDR3

<400> 19

Asn Asp Tyr

1

<210> 20

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> 142F7-HV-CDR3

<400> 20

Gly Gly Ser Gly Phe Ala Tyr

1 5

<210> 21

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2/7B4/16A6-LV-CDR1

<400> 21

Arg Ser Ser Gln Ser Leu Glu Asn Ser Asn Gly Asn Thr Phe Leu Asn

1 5 10 15

<210> 22

<211> 17

<212> PRT

<213> Artificial sequence

<220>

<223> 29A10-LV-CDR1

<400> 22

Glu Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 23

<211> 10

<212> PRT

<213> Artificial sequence

<220>

<223> 92F6-LV-CDR1

<400> 23

Ser Ala Ser Ser Ser Val Ser Tyr Ile His

1 5 10

<210> 24

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> 77D9-LV-CDR1

<400> 24

Arg Ser Ser Gln Ser Ile Val Leu Thr Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 25

<211> 16

<212> PRT

<213> Artificial sequence

<220>

<223> 50F6-LV-CDR1

<400> 25

Arg Ser Ser Gln Ser Ile Val Asn Ser Asn Gly Asn Thr Tyr Leu Glu

1 5 10 15

<210> 26

<211> 11

<212> PRT

<213> Artificial sequence

<220>

<223> 142F7-LV-CDR1

<400> 26

Arg Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn

1 5 10

<210> 27

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2/7B4/16A6/77D9/50F6-LV-CDR2

<400> 27

Lys Val Ser Asn Arg Phe Ser

1 5

<210> 28

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> 29A10-LV-CDR2

<400> 28

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 29

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> 92F6-LV-CDR2

<400> 29

Thr Thr Ala Asn Leu Ala Ser

1 5

<210> 30

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> 142F7-LV-CDR2

<400> 30

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 31

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2/7B4/16A6-LV-CDR3

<400> 31

Leu Gln Val Thr His Val Pro Phe Thr

1 5

<210> 32

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> 29A10-LV-CDR3

<400> 32

Gln Gln Tyr Tyr Arg Ser Pro Leu Thr

1 5

<210> 33

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> 92F6-LV-CDR3

<400> 33

Gln Gln Arg Ser Asn Tyr Pro Phe Thr

1 5

<210> 34

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> 77D9-LV-CDR3

<400> 34

Phe Gln Gly Ser His Val Pro Tyr Thr

1 5

<210> 35

<211> 9

<212> PRT

<213> Artificial sequence

<220>

<223> 50F6-LV-CDR3

<400> 35

Phe Gln Gly Ser His Val Pro Leu Thr

1 5

<210> 36

<211> 7

<212> PRT

<213> Artificial sequence

<220>

<223> 142F7-LV-CDR3

<400> 36

Gln Gln Gly Asn Thr Leu Pro

1 5

<210> 37

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2-HV

<400> 37

Glu Val Lys Leu Glu Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

100 105 110

<210> 38

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 7B4-HV

<400> 38

Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

100 105 110

<210> 39

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 16A6-HV

<400> 39

Glu Val Gln Leu Glu Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr His Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

100 105 110

<210> 40

<211> 123

<212> PRT

<213> Artificial sequence

<220>

<223> 29A10-HV

<400> 40

Gln Val Lys Leu Glu Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Asp Ala Gly Ser Tyr Thr Tyr Tyr Ser Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Asp Asp Thr Ala Met Tyr Phe Cys

85 90 95

Ala Arg Thr Tyr Tyr Arg Gly Asp Gly Gly Tyr Trp Phe Phe Asp Val

100 105 110

Trp Gly Ala Gly Thr Ala Val Thr Val Ser Ser

115 120

<210> 41

<211> 118

<212> PRT

<213> Artificial sequence

<220>

<223> 92F6-HV

<400> 41

Gln Val Gln Leu Glu Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Asn Tyr Asp Pro Lys Phe

50 55 60

Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Gly Tyr

65 70 75 80

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Tyr Asp Trp Tyr Phe Asp Val Trp Gly Ala Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 42

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 77D9-HV

<400> 42

Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asn Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ser Pro Gly Glu Arg Leu Glu Trp Val

35 40 45

Ala Glu Val Ser Gly Ser Gly Tyr Tyr Thr Tyr Tyr Pro Asp Thr Val

50 55 60

Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Leu Tyr

65 70 75 80

Leu Glu Val Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Thr Ser Arg Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala

100 105 110

<210> 43

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 50F6-HV

<400> 43

Gln Val Gln Leu Glu Gln Ser Gly Asp Asp Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Ile Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Arg Ile Ser Pro Gly Ser Gly Ser Thr His Tyr Asn Glu Met Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Ile Gln Leu Ser Ser Leu Ser Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Thr Arg Asn Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

100 105 110

<210> 44

<211> 116

<212> PRT

<213> Artificial sequence

<220>

<223> 142F7-HV

<400> 44

Glu Val Gln Leu Glu Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr

20 25 30

Leu Ile Glu Trp Gly Ile Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Asn Pro Gly Thr Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Gly Ser Gly Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val

100 105 110

Thr Val Ser Ala

115

<210> 45

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2-VH0VL0-HV

<400> 45

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Asn Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 46

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2-VH2VL2/VH2VL3-HV

<400> 46

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 47

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2-VH3VL2/VH3VL3-HV

<400> 47

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 48

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 7B4-VH0VL0-HV

<400> 48

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 49

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 7B4-VH2VL2/VH2VL3-HV

<400> 49

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

35 40 45

Met Gly Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 50

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 7B4-VH3VL2/7B4-VH3VL3-HV

<400> 50

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Asp

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Tyr Ser Ile Thr Thr Asn

20 25 30

Tyr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Asn Lys Leu Glu Trp

35 40 45

Met Gly Tyr Ile Lys Tyr Asp Gly Ser Asn Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Asn Arg Ile Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

100 105 110

<210> 51

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2-LV

<400> 51

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Leu

50 55 60

Asp Arg Phe Ser Gly Thr Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 52

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 7B4-LV

<400> 52

Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Leu

50 55 60

Asp Arg Phe Ser Gly Thr Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 53

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 16A6-LV

<400> 53

Asp Ile Val Leu Thr Gln Ser Thr Leu Ser Leu Ser Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Leu

50 55 60

Asp Arg Phe Ser Gly Thr Gly Ser Gly Thr Asp Leu Thr Leu Thr Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 54

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> 29A10-LV

<400> 54

Asp Ile Val Ile Thr Gln Ser Thr Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Glu Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Ala Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Arg Ser Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 55

<211> 106

<212> PRT

<213> Artificial sequence

<220>

<223> 92F6-LV

<400> 55

Asp Ile Val Ile Thr Gln Ser Thr Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Thr Thr Ala Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Phe Thr

85 90 95

Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 56

<211> 113

<212> PRT

<213> Artificial sequence

<220>

<223> 77D9-LV

<400> 56

Asp Ile Val Met Thr Gln Ser Pro Thr Leu Ser Leu Pro Val Ser Leu

1 5 10 15

Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Leu

20 25 30

Thr Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Arg Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

65 70 75 80

Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln

85 90 95

Gly Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 57

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 50F6-LV

<400> 57

Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val Asn Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 58

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> 142F7-LV

<400> 58

Asp Ile Val Leu Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 59

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2-VH0VL0/7B4-VH0VL0-LV

<400> 59

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 60

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2-VH2VL2/VH3VL2/7B4-VH2VL2/VH3VL2-LV

<400> 60

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 61

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> 13A2-VH2VL3/VH3VL3/7B4-VH2VL3/VH3VL3-LV

<400> 61

Asp Val Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Glu Asn Ser

20 25 30

Asn Gly Asn Thr Phe Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val

85 90 95

Thr His Val Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 62

<211> 324

<212> PRT

<213> Artificial sequence

<220>

<223> mouse IgG1 heavy chain constant region

<400> 62

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

<210> 63

<211> 326

<212> PRT

<213> Artificial sequence

<220>

<223> human IgG2 heavy chain constant region

<400> 63

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 64

<211> 330

<212> PRT

<213> Artificial sequence

<220>

<223> human IgG1 heavy chain constant region, containing the S267E and L328F mutations

<400> 64

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Glu His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

325 330

<210> 65

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> human constant region of Kl chain

<400> 65

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 66

<211> 107

<212> PRT

<213> Artificial sequence

<220>

<223> mouse constant region of Kl chain

<400> 66

Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu

1 5 10 15

Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe

20 25 30

Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg

35 40 45

Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu

65 70 75 80

Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser

85 90 95

Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

100 105

<210> 67

<211> 834

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 67

atggttcgtc tgcctctgca gtgcgtcctc tggggctgct tgctgaccgc tgtccatcca 60

gaaccaccca ctgcatgcag agaaaaacag tacctaataa acagtcagtg ctgttctttg 120

tgccagccag gacagaaact ggtgagtgac tgcacagagt tcactgaaac ggaatgcctt 180

ccttgcggtg aaagcgaatt cctagacacc tggaacagag agacacactg ccaccagcac 240

aaatactgcg accccaacct agggcttcgg gtccagcaga agggcacctc agaaacagac 300

accatctgca cctgtgaaga aggctggcac tgtacgagtg aggcctgtga gagctgtgtc 360

ctgcaccgct catgctcgcc cggctttggg gtcaagcaga ttgctacagg ggtttctgat 420

accatctgcg agccctgccc agtcggcttc ttctccaatg tgtcatctgc tttcgaaaaa 480

tgtcaccctt ggacaagctg tgagaccaaa gacctggttg tgcaacaggc aggcacaaac 540

aagactgatg ttgtctgtgg tccccaggat cggctgagag ccctggtggt gatccccatc 600

atcttcggga tcctgtttgc catcctcttg gtgctggtct ttatcaaaaa ggtggccaag 660

aagccaacca ataaggcccc ccaccccaag caggaacccc aggagatcaa ttttcccgac 720

gatcttcctg gctccaacac tgctgctcca gtgcaggaga ctttacatgg atgccaaccg 780

gtcacccagg aggatggcaa agagagtcgc atctcagtgc aggagagaca gtga 834

<210> 68

<211> 277

<212> PRT

<213> Intelligent people

<400> 68

Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu

20 25 30

Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val

35 40 45

Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu

50 55 60

Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His

65 70 75 80

Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr

85 90 95

Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr

100 105 110

Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly

115 120 125

Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys

145 150 155 160

Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln

165 170 175

Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu

180 185 190

Arg Ala Leu Val Val Ile Pro Ile Ile Phe Gly Ile Leu Phe Ala Ile

195 200 205

Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Thr Asn

210 215 220

Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Pro Asp

225 230 235 240

Asp Leu Pro Gly Ser Asn Thr Ala Ala Pro Val Gln Glu Thr Leu His

245 250 255

Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile Ser

260 265 270

Val Gln Glu Arg Gln

275

<210> 69

<211> 837

<212> PRT

<213> Kiwi berry (Macaca mulatta)

<400> 69

Ala Thr Gly Gly Thr Thr Cys Gly Thr Cys Thr Gly Cys Cys Thr Cys

1 5 10 15

Thr Gly Cys Ala Gly Thr Gly Cys Gly Thr Cys Cys Thr Cys Thr Gly

20 25 30

Gly Gly Gly Cys Thr Gly Cys Thr Thr Gly Cys Thr Gly Ala Cys Cys

35 40 45

Gly Cys Thr Gly Thr Cys Thr Ala Thr Cys Cys Ala Gly Ala Ala Cys

50 55 60

Cys Ala Cys Cys Cys Ala Cys Thr Gly Cys Ala Thr Gly Cys Ala Gly

65 70 75 80

Ala Gly Ala Ala Ala Ala Ala Cys Ala Gly Thr Ala Cys Cys Thr Ala

85 90 95

Ala Thr Ala Ala Ala Cys Ala Gly Thr Cys Ala Gly Thr Gly Cys Thr

100 105 110

Gly Thr Thr Cys Thr Thr Thr Gly Thr Gly Cys Cys Ala Gly Cys Cys

115 120 125

Ala Gly Gly Ala Cys Ala Gly Ala Ala Ala Cys Thr Gly Gly Thr Gly

130 135 140

Ala Gly Thr Gly Ala Cys Thr Gly Cys Ala Cys Ala Gly Ala Gly Thr

145 150 155 160

Thr Cys Ala Cys Cys Gly Ala Ala Ala Cys Ala Gly Ala Ala Thr Gly

165 170 175

Cys Cys Thr Thr Cys Cys Thr Thr Gly Cys Ala Gly Thr Gly Ala Ala

180 185 190

Ala Gly Cys Gly Ala Ala Thr Thr Cys Cys Thr Ala Gly Ala Cys Ala

195 200 205

Cys Cys Thr Gly Gly Ala Ala Thr Ala Gly Ala Gly Ala Gly Ala Cys

210 215 220

Ala Cys Gly Cys Thr Gly Cys Cys Ala Cys Cys Ala Gly Cys Ala Cys

225 230 235 240

Ala Ala Ala Thr Ala Cys Thr Gly Cys Gly Ala Cys Cys Cys Cys Ala

245 250 255

Ala Cys Cys Thr Ala Gly Gly Gly Cys Thr Thr Cys Gly Gly Gly Thr

260 265 270

Cys Cys Ala Gly Cys Ala Gly Ala Ala Gly Gly Gly Cys Ala Cys Cys

275 280 285

Thr Cys Ala Gly Ala Ala Ala Cys Ala Gly Ala Cys Ala Cys Cys Ala

290 295 300

Thr Cys Thr Gly Cys Ala Cys Cys Thr Gly Thr Gly Ala Ala Gly Ala

305 310 315 320

Ala Gly Gly Cys Cys Thr Gly Cys Ala Cys Thr Gly Thr Ala Thr Gly

325 330 335

Ala Gly Thr Gly Ala Gly Thr Cys Cys Thr Gly Thr Gly Ala Gly Ala

340 345 350

Gly Cys Thr Gly Thr Gly Thr Cys Cys Cys Gly Cys Ala Cys Cys Gly

355 360 365

Cys Thr Cys Ala Thr Gly Cys Thr Thr Gly Cys Cys Thr Gly Gly Cys

370 375 380

Thr Thr Thr Gly Gly Gly Gly Thr Cys Ala Ala Gly Cys Ala Gly Ala

385 390 395 400

Thr Thr Gly Cys Thr Ala Cys Ala Gly Gly Gly Gly Thr Thr Thr Cys

405 410 415

Thr Gly Ala Thr Ala Cys Cys Ala Thr Cys Thr Gly Thr Gly Ala Gly

420 425 430

Cys Cys Cys Thr Gly Cys Cys Cys Gly Gly Thr Cys Gly Gly Cys Thr

435 440 445

Thr Cys Thr Thr Cys Thr Cys Cys Ala Ala Thr Gly Thr Gly Thr Cys

450 455 460

Ala Thr Cys Thr Gly Cys Thr Thr Thr Thr Gly Ala Ala Ala Ala Gly

465 470 475 480

Thr Gly Thr Cys Gly Cys Cys Cys Thr Thr Gly Gly Ala Cys Ala Ala

485 490 495

Gly Cys Thr Gly Thr Gly Ala Gly Ala Cys Cys Ala Ala Ala Gly Ala

500 505 510

Cys Cys Thr Gly Gly Thr Thr Gly Thr Gly Cys Ala Ala Cys Ala Gly

515 520 525

Gly Cys Ala Gly Gly Cys Ala Cys Ala Ala Ala Cys Ala Ala Gly Ala

530 535 540

Cys Thr Gly Ala Thr Gly Thr Thr Gly Thr Cys Thr Gly Thr Gly Gly

545 550 555 560

Thr Cys Cys Cys Cys Ala Gly Gly Ala Thr Cys Gly Gly Cys Ala Gly

565 570 575

Ala Gly Ala Gly Cys Cys Cys Thr Gly Gly Thr Gly Gly Thr Gly Ala

580 585 590

Thr Cys Cys Cys Cys Ala Thr Cys Thr Gly Cys Thr Thr Gly Gly Gly

595 600 605

Gly Ala Thr Cys Cys Thr Gly Thr Thr Thr Gly Thr Cys Ala Thr Cys

610 615 620

Cys Thr Cys Cys Thr Cys Thr Thr Gly Gly Thr Gly Cys Thr Gly Gly

625 630 635 640

Thr Cys Thr Thr Thr Ala Thr Cys Ala Ala Ala Ala Ala Gly Gly Thr

645 650 655

Gly Gly Cys Cys Ala Ala Gly Ala Ala Gly Cys Cys Ala Ala Ala Cys

660 665 670

Gly Ala Thr Ala Ala Gly Gly Cys Cys Cys Cys Cys Cys Ala Cys Cys

675 680 685

Cys Cys Ala Ala Gly Cys Ala Gly Gly Ala Ala Cys Cys Cys Cys Ala

690 695 700

Gly Gly Ala Gly Ala Thr Cys Ala Ala Thr Thr Thr Thr Cys Thr Gly

705 710 715 720

Gly Ala Cys Gly Ala Thr Cys Thr Thr Cys Cys Thr Gly Gly Cys Thr

725 730 735

Cys Cys Ala Ala Cys Cys Cys Thr Gly Cys Cys Gly Cys Thr Cys Cys

740 745 750

Ala Gly Thr Gly Cys Ala Gly Gly Ala Gly Ala Cys Thr Thr Thr Ala

755 760 765

Cys Ala Thr Gly Gly Ala Thr Gly Cys Cys Ala Ala Cys Cys Ala Gly

770 775 780

Thr Cys Ala Cys Cys Cys Ala Gly Gly Ala Gly Gly Ala Thr Gly Gly

785 790 795 800

Cys Ala Ala Ala Gly Ala Gly Ala Gly Thr Cys Gly Cys Ala Thr Cys

805 810 815

Thr Cys Ala Gly Thr Gly Cys Ala Gly Gly Ala Gly Ala Gly Ala Cys

820 825 830

Ala Gly Thr Gly Ala

835

<210> 70

<211> 278

<212> PRT

<213> Kiwi berry

<400> 70

Met Val Arg Leu Pro Leu Gln Cys Val Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val Tyr Pro Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu

20 25 30

Ile Asn Ser Gln Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val

35 40 45

Ser Asp Cys Thr Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Ser Glu

50 55 60

Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr Arg Cys His Gln His

65 70 75 80

Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr

85 90 95

Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly Leu His Cys Met

100 105 110

Ser Glu Ser Cys Glu Ser Cys Val Pro His Arg Ser Cys Leu Pro Gly

115 120 125

Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys

145 150 155 160

Cys Arg Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln

165 170 175

Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Gln

180 185 190

Arg Ala Leu Val Val Ile Pro Ile Cys Leu Gly Ile Leu Phe Val Ile

195 200 205

Leu Leu Leu Val Leu Val Phe Ile Lys Lys Val Ala Lys Lys Pro Asn

210 215 220

Asp Lys Ala Pro His Pro Lys Gln Glu Pro Gln Glu Ile Asn Phe Leu

225 230 235 240

Asp Asp Leu Pro Gly Ser Asn Pro Ala Ala Pro Val Gln Glu Thr Leu

245 250 255

His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile

260 265 270

Ser Val Gln Glu Arg Gln

275

<210> 71

<211> 869

<212> DNA

<213> mouse (Mus musculus)

<400> 71

atggtgtctt tgcctcggct gtgcgcgcta tggggctgct tgttgacagc ggtccatcta 60

gggcagtgtg ttacgtgcag tgacaaacag tacctccacg atggccagtg ctgtgatttg 120

tgccagccag gaagccgact gacaagccac tgcacagctc ttgagaagac ccaatgccac 180

ccatgtgact caggcgaatt ctcagcccag tggaacaggg agattcgctg tcaccagcac 240

agacactgtg aacccaatca agggcttcgg gttaagaagg agggcaccgc agaatcagac 300

actgtctgta cctgtaagga aggacaacac tgcaccagca aggattggag gcatgtgctc 360

agcacacgcc ctgtatccct ggctttggag ttatggagat ggccactgag accactgata 420

ccgtctgtca tccctgccca gtcggcttct tctccaatca gtcatcactt ttcgaaaagt 480

gttatccctg gacaagctgt gaggataaga acttggaggt cctacagaaa ggaacgagtc 540

agactaatgt catctgtggt ttaaagtccc ggatgcgagc cctgctggtc attcctgtcg 600

tgatgggcat cctcatcacc attttcgggg tgtttctcta tatcaaaaag gtggtcaaga 660

aaccaaagga taatgagatc ttaccccctg cggctcgacg gcaagatccc caggagatgg 720

aagattatcc cggtcataac accgctgctc cagtgcagga gacgctgcac gggtgtcagc 780

ctgtcacaca ggaggatggt aaagagagtc gcatctcagt gcaggagcgg caggtgacag 840

acagcatagc cttgaggccc ctggtctga 869

<210> 72

<211> 289

<212> PRT

<213> mice

<400> 72

Met Val Ser Leu Pro Arg Leu Cys Ala Leu Trp Gly Cys Leu Leu Thr

1 5 10 15

Ala Val His Leu Gly Gln Cys Val Thr Cys Ser Asp Lys Gln Tyr Leu

20 25 30

His Asp Gly Gln Cys Cys Asp Leu Cys Gln Pro Gly Ser Arg Leu Thr

35 40 45

Ser His Cys Thr Ala Leu Glu Lys Thr Gln Cys His Pro Cys Asp Ser

50 55 60

Gly Glu Phe Ser Ala Gln Trp Asn Arg Glu Ile Arg Cys His Gln His

65 70 75 80

Arg His Cys Glu Pro Asn Gln Gly Leu Arg Val Lys Lys Glu Gly Thr

85 90 95

Ala Glu Ser Asp Thr Val Cys Thr Cys Lys Glu Gly Gln His Cys Thr

100 105 110

Ser Lys Asp Cys Glu Ala Cys Ala Gln His Thr Pro Cys Ile Pro Gly

115 120 125

Phe Gly Val Met Glu Met Ala Thr Glu Thr Thr Asp Thr Val Cys His

130 135 140

Pro Cys Pro Val Gly Phe Phe Ser Asn Gln Ser Ser Leu Phe Glu Lys

145 150 155 160

Cys Tyr Pro Trp Thr Ser Cys Glu Asp Lys Asn Leu Glu Val Leu Gln

165 170 175

Lys Gly Thr Ser Gln Thr Asn Val Ile Cys Gly Leu Lys Ser Arg Met

180 185 190

Arg Ala Leu Leu Val Ile Pro Val Val Met Gly Ile Leu Ile Thr Ile

195 200 205

Phe Gly Val Phe Leu Tyr Ile Lys Lys Val Val Lys Lys Pro Lys Asp

210 215 220

Asn Glu Ile Leu Pro Pro Ala Ala Arg Arg Gln Asp Pro Gln Glu Met

225 230 235 240

Glu Asp Tyr Pro Gly His Asn Thr Ala Ala Pro Val Gln Glu Thr Leu

245 250 255

His Gly Cys Gln Pro Val Thr Gln Glu Asp Gly Lys Glu Ser Arg Ile

260 265 270

Ser Val Gln Glu Arg Gln Val Thr Asp Ser Ile Ala Leu Arg Pro Leu

275 280 285

Val

<210> 73

<211> 173

<212> PRT

<213> Artificial sequence

<220>

<223> full Length OD40 ECD

<400> 73

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr

20 25 30

Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 74

<211> 133

<212> PRT

<213> Artificial sequence

<220>

<223> truncation 1

<400> 74

Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr His

1 5 10 15

Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val Gln

20 25 30

Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu Gly

35 40 45

Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg Ser

50 55 60

Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser Asp

65 70 75 80

Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser

85 90 95

Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu

100 105 110

Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro

115 120 125

Gln Asp Arg Leu Arg

130

<210> 75

<211> 90

<212> PRT

<213> Artificial sequence

<220>

<223> truncation 2

<400> 75

Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys

1 5 10 15

Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala

20 25 30

Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe

35 40 45

Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys

50 55 60

Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp

65 70 75 80

Val Val Cys Gly Pro Gln Asp Arg Leu Arg

85 90

<210> 76

<211> 50

<212> PRT

<213> Artificial sequence

<220>

<223> truncation 3

<400> 76

Glu Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu

1 5 10 15

Lys Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln

20 25 30

Gln Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg

35 40 45

Leu Arg

50

<210> 77

<211> 156

<212> PRT

<213> Artificial sequence

<220>

<223> truncation 4

<400> 77

Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr Glu

1 5 10 15

Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu Asp

20 25 30

Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp Pro

35 40 45

Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr

50 55 60

Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys Glu

65 70 75 80

Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys Gln

85 90 95

Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val Gly

100 105 110

Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp Thr

115 120 125

Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn Lys

130 135 140

Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

145 150 155

<210> 78

<211> 150

<212> PRT

<213> Artificial sequence

<220>

<223> truncation 5

<400> 78

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Pro Cys Gly Glu Ser Glu Phe Leu Asp Thr Trp Asn Arg Glu Thr

20 25 30

His Cys His Gln His Lys Tyr Cys Asp Pro Asn Leu Gly Leu Arg Val

35 40 45

Gln Gln Lys Gly Thr Ser Glu Thr Asp Thr Ile Cys Thr Cys Glu Glu

50 55 60

Gly Trp His Cys Thr Ser Glu Ala Cys Glu Ser Cys Val Leu His Arg

65 70 75 80

Ser Cys Ser Pro Gly Phe Gly Val Lys Gln Ile Ala Thr Gly Val Ser

85 90 95

Asp Thr Ile Cys Glu Pro Cys Pro Val Gly Phe Phe Ser Asn Val Ser

100 105 110

Ser Ala Phe Glu Lys Cys His Pro Trp Thr Ser Cys Glu Thr Lys Asp

115 120 125

Leu Val Val Gln Gln Ala Gly Thr Asn Lys Thr Asp Val Val Cys Gly

130 135 140

Pro Gln Asp Arg Leu Arg

145 150

<210> 79

<211> 173

<212> PRT

<213> Artificial sequence

<220>

<223> mutant 1

<400> 79

Glu Pro Pro Thr Ala Cys Ala Ala Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr

20 25 30

Glu Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 80

<211> 173

<212> PRT

<213> Artificial sequence

<220>

<223> mutant 2

<400> 80

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Ala

20 25 30

Ala Phe Thr Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 81

<211> 173

<212> PRT

<213> Artificial sequence

<220>

<223> mutant 3

<400> 81

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr

20 25 30

Glu Ala Ala Glu Thr Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 82

<211> 173

<212> PRT

<213> Artificial sequence

<220>

<223> mutant 4

<400> 82

Glu Pro Pro Thr Ala Cys Arg Glu Lys Gln Tyr Leu Ile Asn Ser Gln

1 5 10 15

Cys Cys Ser Leu Cys Gln Pro Gly Gln Lys Leu Val Ser Asp Cys Thr

20 25 30

Glu Phe Thr Ala Ala Glu Cys Leu Pro Cys Gly Glu Ser Glu Phe Leu

35 40 45

Asp Thr Trp Asn Arg Glu Thr His Cys His Gln His Lys Tyr Cys Asp

50 55 60

Pro Asn Leu Gly Leu Arg Val Gln Gln Lys Gly Thr Ser Glu Thr Asp

65 70 75 80

Thr Ile Cys Thr Cys Glu Glu Gly Trp His Cys Thr Ser Glu Ala Cys

85 90 95

Glu Ser Cys Val Leu His Arg Ser Cys Ser Pro Gly Phe Gly Val Lys

100 105 110

Gln Ile Ala Thr Gly Val Ser Asp Thr Ile Cys Glu Pro Cys Pro Val

115 120 125

Gly Phe Phe Ser Asn Val Ser Ser Ala Phe Glu Lys Cys His Pro Trp

130 135 140

Thr Ser Cys Glu Thr Lys Asp Leu Val Val Gln Gln Ala Gly Thr Asn

145 150 155 160

Lys Thr Asp Val Val Cys Gly Pro Gln Asp Arg Leu Arg

165 170

<210> 83

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Signal peptide

<400> 83

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 84

<211> 324

<212> PRT

<213> Artificial sequence

<220>

<223> mFc-tag

<400> 84

Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala

1 5 10 15

Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu

50 55 60

Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val

65 70 75 80

Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys

85 90 95

Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro

100 105 110

Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu

115 120 125

Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser

130 135 140

Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu

145 150 155 160

Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr

165 170 175

Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn

180 185 190

Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro

195 200 205

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln

210 215 220

Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val

225 230 235 240

Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val

245 250 255

Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln

260 265 270

Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn

275 280 285

Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val

290 295 300

Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His

305 310 315 320

Ser Pro Gly Lys

Claims (20)

1. An isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, and a light chain variable region comprising a CDR1 region, a CDR2 region and a CDR3 region, wherein the amino acid sequences of the heavy chain CDR1 region, the CDR2 region and the CDR3 region, and the light chain CDR1 region, the CDR2 region and the CDR3 region are set forth in SEQ ID NOs: 5. 12, 18, 24, 27, and 34, wherein the antibody, or antigen-binding portion thereof, binds to CD40,wherein said antigen binding portion is selected from the group consisting of Fab fragments, F (ab')₂Fragments, Fv fragments, and scFv fragments.

2. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 42.

3. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 56.

4. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 2, wherein the heavy chain variable region and the light chain variable region comprise, respectively, SEQ ID NOs: 42 and 56.

5. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, which is an IgG1, IgG2, or IgG4 subtype.

6. The isolated monoclonal antibody, or antigen-binding portion thereof, of claim 1, comprising a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region and light chain constant region comprise, respectively, the amino acid sequences set forth in (1) SEQ ID NOs: 62 and 66; (2) SEQ ID NOs: 63 and 65; or (3) SEQ ID NOs: 64 and 65, respectively.

7. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1 that (a) binds to human CD 40; (b) binds to monkey CD 40; (c) does not bind to mouse CD 40; (d) activating the CD40 signal path; and (e) promoting dendritic cell maturation.

8. The isolated monoclonal antibody, or antigen binding portion thereof, of claim 1, which is a murine, chimeric, or humanized antibody.

9. A bispecific molecule, immunoconjugate, chimeric antigen receptor, genetically modified T cell receptor, or oncolytic virus comprising the isolated monoclonal antibody, or antigen-binding portion thereof, of any one of claims 1-8.

10. A composition comprising the isolated monoclonal antibody, or antigen-binding portion thereof, of any one of claims 1-8, and a pharmaceutically acceptable carrier.

11. The composition of claim 10, further comprising a secondary antibody, and/or a cytokine.

12. The composition of claim 11, wherein the second antibody is selected from the group consisting of a VISTA antibody, a PD-1 antibody, a PD-L1 antibody, a LAG-3 antibody, a TIM-3 antibody, a STAT3 antibody, and a ROR1 antibody.

13. The composition of claim 11, wherein the cytokine is GM-CSF and/or IL-4.

14. A nucleic acid encoding the isolated monoclonal antibody or antigen binding portion thereof of any one of claims 1-8.

15. An expression vector comprising the nucleic acid of claim 14.

16. A host cell comprising the expression vector of claim 15.

17. Use of the isolated antibody or antigen-binding portion thereof of any one of claims 1-8, or the composition of any one of claims 10-13, in the manufacture of a medicament for treating cancer.

18. The use of claim 17, wherein the cancer is selected from the group consisting of B-cell lymphoma, chronic lymphocytic leukemia, multiple myeloma, melanoma, pancreatic cancer, gastrointestinal cancer, prostate cancer, bladder cancer, renal cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, and nasopharyngeal cancer.

19. Use according to claim 18, wherein the gastrointestinal cancer is intestinal cancer.

20. The use according to claim 19, wherein the intestinal cancer is intestinal adenocarcinoma.

Images