CN111454361A - Antibodies that bind 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 bind 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 expressed on antigen presenting cells such as B cells, macrophages and dendritic cells binding of this protein to CD 40L (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 functions (Ara A et al (2018) Immunotargets The 7: 55-61).

On the other hand, CD40 is also present on non-immune cells and tumors (Costello et al, (1999) ImmunoToday 20 (11): 488-493; Tong et al, (2003) Cancer Gene Ther 10 (1): 1-13; L ee et al, (2014) Curr Cancer Drug Targets 14 (7): 610-620; Ara A et al, (2018) supra), and is reported to be associated with various immune diseases (including autoimmune diseases), atherosclerotic thrombi, Cancer and respiratory diseases. for example, CD40/CD 40L expression is upregulated in atheroma-associated cells CD40 is expressed in almost all B cell malignancies as well as in solid tumors up to 70%, and CD40 ligand binding in certain B cell malignancies increases expression of various apoptosis factors that protect tumor cells from apoptosis (Acstello et al, L: Nat 9136, Nat 9136).

Although the role of CD40 in tumorigenesis is very complex, several CD40 antibodies have been developed for potential tumor therapy. CP-870893, developed fully human IgG 2-activated CD40 antibodies, can activate dendritic cells and have shown clinical utility in a variety of background advanced Cancer patients (Vonderheide et al, (2007) J ClinOncol 25 (7): 876-. Dacetuzumab, also known as SGN-40, is a humanized IgG 1-activated CD40 antibody developed by Seattles genetics that exhibits anti-tumor activity upon weekly intravenous administration, particularly in patients with diffuse large B-cell lymphoma. Preclinical data also show Da_cetuz_uThe synergistic effect of mab with other drugs such as rituximab CD20 (L apolombella et al., (2009) Br J Haematol 144 (6): 848-855; Hussein et al., (2010) Haematologica 95 (5): 845-848; de Vos et al., (2014) J He-matol Oncol 7: 44). Chi L ob 7/4, chimeric IgG1 agonist anti-human CD40 developed by the United kingdom Cancer Research UK (Cancer Research UK) is undergoing initial clinical testing 11 of 21 patients for stable disease without partial or complete remission (Chokury et al., (2014) Cancer quinonese 2 (229): 229) 229, CD40 antagonist also shows effects in human leukemia and chronic lymphoma cells (Bengale et al.) (Bengale et al) in leukemia cells (BEemal 75-8678; Bensql et al., (3658: 8678).

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 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 of the invention or antigen-binding portions thereof specifically bind to human or monkey CD40, blocking or promoting CD40-CD 40L interaction the antibodies of the invention are agonistic CD40 antibodies, activate the CD40 signaling pathway, promote maturation of immune cells such as dendritic cells.

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 of the invention, or an antigen-binding portion thereof, 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, kidney cancer, ovarian cancer, cervical cancer, breast cancer, lung cancer, and nasopharyngeal cancer in some embodiments, the method comprises administering a composition, bispecific molecule, immunoconjugate, PD-T cell, or oncolytic virus encoding or carrying an antibody of the invention in some embodiments, at least one other anti-cancer antibody may be administered with an antibody of the invention or an antigen-binding portion thereof, such as a VISTA antibody, a PD-1 antibody, a PD-L antibody, a L AG-3 antibody, and/or a CT 32A-4 antibody in another embodiment, the antibody of the invention or an antigen-binding portion thereof may be administered with a cytokine (e.g., an I-36 antibody, a CD-L-antibody, a chemotherapeutic agent, such as a chemotherapeutic agent, or a chemotherapeutic agent, such as a humanized antibody of human origin.

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-CD 40L interaction, wherein antibodies 16a6, 7B4 and 13a2 promote CD40-CD 40L interaction (a) and antibody 92F6 inhibits CD40-CD 40L 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 I L-12 (C).

Figure 13 shows the binding affinity of chimeric or humanized CD40 antibodies 7B4(a), 7B4-VH0V L0 (B), 7B4-VH2V L2 (C), 7B4-VH2V L03 (D), 7B4-VH3V L2 (E), 7B4-VH3V L3 (F), 13a2(G), 13a2-VH0V L0 (H), 13a2-VH2V L2 (I), 13a2-VH2V L3 (J), 13a2-VH3V L2 (K), and 13a2-VH3V L3 (L) as measured by SPR, as well as the reference antibodies RO7009789(M) and ADC1013(N) to human CD 40.

Figure 14 shows the binding affinity of the chimeric or humanized CD40 antibody to full length CD40ECD or a truncation thereof (a) and to full length CD40ECD or a mutant thereof (B).

Figure 15 shows the binding specificity of humanized CD40 antibodies 7B4-VH2V L2 (a) and 13a2-VH3V L3 (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 the control drug was administered, and the individual tumor volumes of the solvent group (B), 7B4VH2V L2 group (C), 13A2VH3V L3 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, which 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 being linked by a heavy chain variable region (abbreviated V), and any antigen-binding fragment (i.e., antigen-binding portion) or single chain thereof_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 demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. 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 prepared by techniques known in the artCommonly used techniques known to the person are available and fragments can be functionally screened in the same way 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".

As used herein, "specifically binds to humanAn antibody to CD40 "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 or less, more preferably 5.0x10^-9M is less than or equal to M.

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

The term "high affinity" for IgG antibodies means a KD for the antigen of 1.0x10^-6M or less, preferably 5.0x10^-8M or less, more preferably 1.0x10^-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^-6M 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, while the term "antagonistic CD40 antibody" refers to blocking or inhibiting the CD40 signaling pathway that can be primed by CD 40L 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 1x10^-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 V_HOr 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, independently of CDR1 and/or CDR2, can determine the binding specificity of an antibody to the same antigen individually and that it is predicted that a plurality of antibodies with the same binding specificity can be generated based on this CDR3 sequence, see, e.g., Klimka et al, British J.of Cancer 83(2) 252. Sci 260 (2000); Beibo et al, J.mol.biol.296: 833. 849 (2000); Rader et al, Proc.Natl.Acad.Sci.US.A.95: 8910. 8915 (1998); Barbas et al, J.am.Chem.Soc.116: 2161. Bufonic 2162 (1994); Barbas et al, Proc.Natl.Acad.Sci.S.A.92: 1995: 2533; Proc.Natl.Acad.Sci.7. J.31: 2161. J.J.J.11, J.31. J.31: 14, J.11. J.31: 14. J.11, J.11. J.11, J.J.11, J.11. J.11, J.11. J.11, J.J.11, J.11.

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; deWildt 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; kelleyand 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), B-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 antibody of the present invention may be provided with one or more V of the CD40 antibody of the present invention_H/V_LAntibodies of sequence were used as starting material to prepare genetically modified antibodies. Antibodies can be made by modifying one or both variable regions (i.e., V)_HAnd/or V_L) One or more residues within (e.g., in one or more CDR regions and/or one or more framework regions) are genetically modified to improve binding affinity and/or increase similarity to antibodies naturally occurring in 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. Because the 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 invention is directed to an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2 and CDR3 having the sequences described above and/or a light chain variable region comprising CDR1, CDR2 and CDR3 having the sequences 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 Genbank accession numbers for the heavy chain germline sequences in the following HCo7HuMAb 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 3-23fAJ 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) B L AST (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, it is beneficial to mutate residues in the framework regions to maintain or enhance the antigen binding properties of the 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 present invention, or one, two, three, four or five amino acid substitutions, deletions or additionsThe amino acid sequence of (a); (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_LFor example, those that make genetic modifications to alter antibody properties. 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, such as low fucosyl antibodies with reduced fucose residue amounts, or antibodies with increased bisecting GlcNac structure altered glycosylation patterns have been demonstrated to increase the ADCC activity of the antibodies.such modifications of glycosylation can be made by expressing the antibodies in host cells altered in the glycosylation system cells with altered glycosylation systems are known in the art and can be used as host cells expressing the recombinant antibodies of the invention to make antibodies with altered glycosylation.for example, cell lines Ms704, Ms705 and Ms lack the fucosyltransferase gene FUT8(α (1, 6) -fucosyltransferase) such that the antibodies expressed in cell lines of Ms704, Ms and Ms709 lack the fucosyltransferase gene FUT8 in their sugars and thus make antibodies with a deletion of fucose 8 residues in their sugars.704, Ms705 and Ms 8-/-cell lines by targeted disruption of the fucose 8 gene in CHO/DG44 cells using two alternative vectors for the deletion of fucose 8 residues in their glycoproteins, such as a fucose-transferase gene linked to a transgenic cell line of fucose-expressing antibodies with reduced fucose residue levels in rat glycoprotein I-fucose transferase gene expression in transgenic cells such as described by the transgenic antibody production of transgenic antibody lines of fucose-transgenic antibody laid-transgenic mouse.

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, in a manner such that one or more PEG groups are attached to the antibody or antibody fragmentAnd (3) reacting under the condition. 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 c-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)_m、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 chain constant 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 scFv genes, encoding V_HAnd V_LThe DNA fragment of (A) may be operably linked to a DNA fragment encoding a flexible linker, e.g., encoding an amino acid sequence (Gly4-Ser)₃Such that the VH and V L sequences 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 sequences" includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of antibody genes, such regulatory sequences are described, for example, in Goeddel (Gene expression technology. methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)).

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 operableGround connection, 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 cross-linked with therapeutic agents to form immunoconjugates, e.g., antibody-drug cross-linkers (ADCs). 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 ADCs, the antibodies and therapeutic agents are preferably cross-linked by a linker, e.g., a peptide linker, a disulfide linker, or a hydrazone linker more preferably the linker is a peptide linker, e.g., Val-Cit, Ala-Val, Val-Ala-Val, L ys-L ys, Pro-Gly-Val-Va 1, Ala-Val-Asn, Val-L eu-L ys, Ala-Ala-Asn, Git-Git, L ys, L ys, Cit, Val, or Val-L eu-L ys, WO-Asp-Gl-35, WO-366326, WO 07/051,081, WO 7,129,261, WO 07/051,081, WO 6326, WO 07/051,081, WO 6326, and WO 07/051,081.

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 Chemist7y, 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.

Excipients that may be used include carriers, surfactants, thickening or emulsifying agents, solid binders, dispersing or suspending agents, solubilizers, coloring agents, flavoring agents, coatings, disintegrants, lubricants, sweeteners, preservatives, isotonicity agents, and combinations thereof The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (L ippincott 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, the monoclonal antibodies of the invention may be formulated to ensure proper in vivo distribution, for example, to ensure that the therapeutic antibodies of the invention cross the blood-brain barrier, the antibodies 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; 5,416,016; and 5,399,331; V.V.Ranade (1989) J.Clin.Pharmacol.29: 685; Umezawai et al, (1988) biochem.biophysis.Res.Commun.153: 1038; Bloeman et al., (1995) FEBS L et.357: 140; M.Owai et al, (1995) antimicrob.Agents Chemother.39: 180; Briscot et al 1995, Amio.J.Phys.1233: 1233: 1994: Biotech. Cheyne et al (1994: 90) and Biotech. 2. 1994: 90. Biotech. J.3. Chellen et al.

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

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 L AG-3 antibody, a PD-1 antibody, a PD-L antibody, and/or a CT L a-4 antibody.

For example, CD40 signaling pathway activation can be combined with CT L A-4 and/or L AG-3 and/or PD-1 blockade, and chemotherapy.

Optionally, the combination of CD40 with one or more other antibodies (e.g., CT L A-4 antibody and/or L AG-3 antibody and/or PD-1 antibody) can 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(I L-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 the amino acid sequences SEQ ID NOs: 68, 70 and 72, respectively) were synthesized and enzymatically cloned into p L V-EGFP (2A) -Puro vectors (Beijing Ongheng Biotech Co., Ltd., China). the resulting p L V-EGFP (2A) -Puro-CD40 was transfected into HEK293T cells (Nanjing family Bai Co., China) by means of lipofection with psPAX and pMD2.G plasmids to produce lentiviruses, specifically in accordance with the procedure described for L off amine 3000(Thermo Fisher Scientific, USA). after three days of transfection, cell culture medium of HEK293T cells (DMEM # (SH 5, Gibco) supplemented with 10% of HET GFD #, 500 # gene #) was harvested from HEK293 cells cultured in a DMEM 3648 medium (DMEM # SH 5, Gibco) containing antibodies derived from HEK293 cells expressing human, mouse CD 293, mouse.

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 BA L B/C mice (Wintolite, China) were immunized by cross-injection of recombinant human CD40(ECD) -his protein (Yiqiao Shenzhou, China, Cat: 10774-H08H) and monkey CD40(ECD) -hFc protein (Yiqiao Shenzhou, China, Cat: 90097-C02H) as shown in Table 2, human CD40(ECD) -his protein and monkey CD40(ECD) -hFc protein were emulsified with equal volumes of complete Freund adjuvant (Cat #: F5881-10 x 10M L, Sigma, USA), incomplete Freund adjuvant (Cat #: F5506-6 x 10M L, Sigma, USA) or PBS.

TABLE 2 immunization procedure

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

Based on the results of the E L ISA test and FACS test 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, mice were sacrificed and spleens were removed and prepared into single cell suspensions in PBS splenocytes were washed 3 times with DMEM medium (Cat #: SH30243.01B, Hyclone, USA.) the mouse myeloma cells SP2/0(CR L-1581, ATCC, USA) in logarithmic growth phase were mixed with the above isolated mouse splenocytes in a ratio of 1: 4 and washed 2 times with DMEM after cell fusion was performed by means of fusion with PEG (Cat #: P7181, Sigma, USA.) the fused cells were washed 3 times with DMEM and resuspended in cell growth medium (RPMI1640+ 10% FBS +1 XHAT). The cell suspensions were plated on 96-well plates with 200. mu.l/well, 5 × 10X HAT⁴For each well, cells were plated at 37 ℃ with 5% CO₂The culture medium was changed to a fresh medium (DMEM + 10% FBS +1X HAT) on day 7, 2-3 days later, cell culture supernatant was aspirated, and hybridomas were screened by E L ISA and FACS.

Screening of hybridoma cell lines by E L ISA

Hybridoma clones that bind to human CD40 were first screened by a high throughput E L ISA binding assay the hybridoma clones that bind to human CD40 were further tested for their ability to bind to monkey or mouse CD 40.

For E L ISA detection, 96-well E L ISA plates were plated with human CD40(ECD) -his (0.5. mu.g/ml, Cat #: 10774-H08H, Hookea, China), monkey CD40(ECD) -hFc (0.5. mu.g/ml, Cat #: 90097-C02H, Hookea, China) or mouse CD40-his (0.5. mu.g/ml, Cat #: 50324-M03H, Hookea, China) 100. mu.l per well, overnight at room temperature E L ISA plates were washed with PBST (PBS + 0.05% Tween 20) solution for 3 times, 200. mu.l of blocking solution (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20) for 2 hours at room temperature, washed with PBST solution for 3 times, supernatant with hybridoma cell culture medium (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20) for 2 hours, washed with PBS + PBS for 3 times, diluted with PBS + 1% goat serum + 1% PBS + 100. mu.10. mu.1. mu.10. mu.1. mu.10 hours, and diluted PBS for 100. mu.10. mu.1. mu.10 hours, and added with PBS for 100. mu.10 hours, and after incubation of goat serum, the test

234 hybridoma cell lines with specific binding force to human and monkey CD40 were selected by the E L ISA.

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/mouse CD40 cells from example 1 were added to each assay well of a 96-well plate.hybridoma culture supernatant was diluted 10-fold with a diluent (PBS containing 1% BSA, 1% goat serum, 0.01% Tween 20) and added to the sample assay wells, 100. mu.l per well was incubated at 4 ℃ for 1 hour, then washed 3 times with FACS wash (PBS + 1% BSA + 0.01% Tween 20). thereafter, the cells were washed with a resuspension wash, and 500-fold diluted APC # -goat anti-mouse IgG secondary antibody (Cat: 405308, Bio L egen, USA) was added for incubation at 4 ℃ for 1 hour, and the plate was washed 3 times with FACS and then examined for cell fluorescence using a 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.

Producing antibodies to CD40Subcloning of hybridoma cells

The above 162 hybridoma clones were subjected to 2 rounds of subcloning, during which multiple subclones (n > 3) of each clone were selected and characterized by the above-described E L ISA and FACS assays.

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, 4 × 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 DMEM medium was added to each well to replace the original medium, after 7 hours of incubation, DMEM medium was replaced with HEK-Blue assay buffer (Invivogen, US, Cat #: hb-det3), 100. mu. L per well and 100u L hybridoma supernatant per well were added to the resulting mixture at 37 ℃ with 5% CO.₂The readings at OD630 were determined using a SpectraMax microplate reader (molecular μ lar Devices, USA), where the negative control was HE L antibody (L ifeTein, LL C, US, Cat. #: L T12031), RO7009789 (an agonistic antibody made according to the amino acid sequence disclosed in US7338660B2 and having a human IgG 2/kappa constant region)) And CD 40L (Cat #: 10239-H08E, Protoyoho, China, natural ligand and activator of CD40) 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 (pH7.0), 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/1)

Antibodies

Subtype of cell

Expression Titers (mg/l)

13A2

Mouse IgG1/K

24.744

77D9

Mouse IgG

1/kappa

12.22

16A6

Mouse IgG1/K

31.111

79D7

Mouse IgG

1/kappa

20.39

29A10

Mouse IgG

1/kappa

33.889

142F7

Mouse IgG1/K

17.22

7B4

Mouse IgG1/K

18.667

89D11

Mouse IgG1/K

95.73

9A7

Mouse IgG

1/kappa

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 IgG

1/kappa

39.97

35C9

Mouse IgG

1/kappa

11.667

82D3

Mouse IgG2a/K

16.27

16F4

Mouse IgG2b/K

14.000

23B8

Mouse IgG2a/K

32.33

50F6

Mouse IgG

1/kappa

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 the E L ISA to determine its binding affinity to recombinant human, monkey or mouse CD40 protein.

E L ISA detection plates were coated with 500ng/ml human CD40(ECD) -his (Cas: 10774-H08H, Chi) at 4 ℃ overnight, wells were blocked with 200 μ l of blocking solution (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20) at room temperature for 2 hours, then 100 μ l of CD40 antibody (40 μ g/ml maximum) was added, incubated at room temperature for 1 hour, plates were washed 3 times with PBST (PBS + 0.05% Tween 20) and then 5000-fold diluted goat anti-mouse IgG-HRP (Simga, U.S., Cat #: A9309-1ml) was added, incubated at room temperature for 1 hour, plates were developed with freshly prepared Ultra-TMB (BD, U.S., Cat # No.: 555214) at room temperature for 5 minutes, and then spectragax was used^Ri3X (Molecular devices, USA) reads at 450 nm.

The species cross-reactivity of 20 CD40 monoclonal antibodies to monkey or mouse CD40 was further tested by direct E L ISA specifically, 500ng/ml monkey CD40(ECD) -hFc protein (see, warburg, china, Cat: 90097-C02H) or mouse CD40-hFc (see, warburg, china, Cat: 50324-M03H) was coated in 96-well E L ISA plates and incubated with 100 μ l of CD40 antibody (highest concentration 40 μ g/ml) diluted in a gradient, after which HRP-goat anti-mouse IgG # (Sigma, US, Cat: a 09-1ml), CD4 antibody R07009789 and ADC1013 (prepared according to the amino acid sequence disclosed in US2016/0311916a1 and having a human IgG1/κ constant region) were used as reference.

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 with mouse CD40And (4) reacting.

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, 10⁵HEK293A cells were plated in 96-well plates and a gradient dilution of CD40 antibody (maximum concentration 40. mu.g/ml) was added after incubation for 1 hour at 4 ℃ the plates were washed 3 times with PBST after which a 500-fold dilution of APC-goat anti-mouse IgG (Bio L egen, USA, Cat #: 405308) was added after incubation for 1 hour at 4 ℃ the cells were washed 3 times with PBS and the 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-CD 40L

Purified CD40 antibodies were further tested for their ability to block or promote binding of human CD 40L to human CD 40. briefly, 96-well E L ISA plates were coated overnight at 4 ℃ with 500ng/ml human CD 40L (Cat #: 10239-H08E, china, see.) after which the plates were blocked with 200 μ l blocking solution (PBS + 2% BSA) at room temperature for 2 hours.gradient diluted CD40 antibody (maximum concentration of 40 μ g/ml) was mixed with 2 μ g/ml human CD40-hFc (Cat #: 10774-H02H, see. after incubation for 1 hour at 37 ℃ and the resulting mixture was then added to the detection wells, incubated for 1 hour at room temperature the plates were washed 3 times with PBST (mgpbs + 0.05% tween 20) and anti-human IgG FC-5000 fold diluted FC-5000 (siaa, usa, Cat 0170-1M L hours), washed with dev # 5 μ g/ml PBST (M5-80M) after incubation for 1 hour, the addition of M5 μ g/ml HRP, read with 5 μ g-HRP (M) after incubation for 1 minute, M5 μ g-80 min.

Interestingly, the data show that 6 mouse antibodies (13a2, 16a6, 7B4, 50F6, 142F7, and 101C12) promote human CD40-CD 40L interactions, whereas 3 antibodies (23B8, 92F6, 82D3) block CD40-CD 40L interactions, the remaining few antibodies have no significant effect on CD40-CD 40L binding the results of 4 representative clones are shown in fig. 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) 4 × 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 SpectraMaxRi3X microplate reader (molecular. mu. larDevices, USA).

EC₅₀Values are summarized in table 6and curves for representative mabs are shown in figure 3. As can be seen, the 20 antibodies show different degrees of agonistic activity in the functional HEK-Blue assay, indicating that the 20 antibodies can stimulate CD40 downstream signalsAnd (4) a passage.

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

Briefly, 96-well E L ISA assay plates were coated with 5. mu.g/ml of R07009789 or ADC1013, overnight at 4 ℃, these wells were blocked with 200. mu.l of blocking solution (PBS + 1% BSA + 1% goat serum + 0.05% Tween 20) at room temperature for 2 hours 0.5. mu.g/ml human CD40(ECD) -his protein (Cat #: 10774-H08H, state of Hokkera, China), incubation continued at room temperature for 1 hour for 3 times, plates were washed with PBST, 1. mu.g/ml purified antibody was added, incubated at room temperature for 1 hour, E L ISA plates were washed with PBST for 3 times, then added with 20000-fold diluted anti-mouse Fc-HRP (Cat #: A9309-1MC, Sica, USA), read at room temperature for 1 hour, washed with PBST wash solution for 3 times, incubated with fresh Fc-HRP (Cat # A9309-1MC, Sica, USA), and developed with Mozhuchi-S # 25 min and developed using a color developing instrument (TMR X nm).

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 E L ISA test

+: there is competition; -: absence of competition

Example 9 agonistic CD40 antibodies promote dendritic cell maturation

Briefly, PBMC from a blood sample from a healthy human donor were collected by gradient density centrifugation and resuspended in RPMI1640 medium for 2 hours at 37 deg.C, adherent cells, i.e., mononuclear cells, were collected and cultured in RPMI1640 medium containing 100ng/ml recombinant human GM-CSF (R & D; U.S.; Cat: 7954-GM), 100ng/ml recombinant human I L-4 (R & D; U.S.; Cat #: 6507-I L), and 10% FBS 1640 medium after 3 days, the medium was half-refreshed, and after 5 days of cell culture, different concentrations of CD40 antibody (10ug/ml or 1ug/ml) and control antibody were added to the cell culture and continued for 48 hours of culture.

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.

The variable domains of 7B4 and 13A2 were subjected to three-dimensional structural modeling to determine key framework amino acid residues that might play an important role in maintaining the CDR loops, thereby designing the back-mutations of humanized antibodies briefly, the selected structural templates had the same type of L-CDR 1, L-CDR 2, L-CDR 3, H-CDR1, H-CDR2, and H-CDR3 loops as 7B4 and 13A2, respectively, the structural models of humanized 7B4 and 13A2 were constructed using the selected structural templates by replacing the mouse framework with human germline heavy and light chain framework sequences.

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), 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 antibody was further tested for binding to HEK 193A/human CD40 and HEK 293A/monkey CD40 according to the method steps in example 5. chimeric and humanized CD40 antibody was further tested for its ability to activate CD40 signaling pathway by HEK-Blue according to example 7. humanized antibody was further tested for its ability to promote dendritic cell maturation according to the method steps in example 9, dendritic cells were derived from blood samples from 3 different healthy human donors, secretion of I L-12 (p40) in dendritic cells was tested using I L-12 (p40) test kit (BD, US, Cat #: 551116), with the specific experimental steps being in full agreement with the specification.

As shown in figures 8, 9, 10 (donor 1), 11 (donor 2) and 12 (donor 3), all humanized antibodies bound to human and monkey OX40 proteins, did not bind to mouse OX40, activated CD40 signaling pathway, promoted dendritic cell maturation, and 13a2-VH3V L2, 13a2-VH3V L3 and 7B4VH2V L2 showed the highest binding, agonistic and functional activity.

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

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

Specifically, 100-200RU (reaction unit) human CD40(ECD) -his protein (Cat: 10774-H08H, Chi-Qiao-Ho, China) was coupled to a CM5 biochip (Cat #: BR-1005-30, GE L ife Sciences, USA), and then the unreacted groups of the chip were blocked with 1M aminoethanol, and the antibody diluted in gradient (concentration from 0.3. mu.M to 10. mu.M) was injected into the SPR reaction solution (HBS-EP buffer, pH7.4, Cat #: BR-1006-69, GE L ife Sciences, USA), the binding rate was controlled at 30. mu. L/min. when the binding force of the antibody was calculated, the binding rate of RU. in the blank control well was subtracted (k.84 binding rate (k. L/min)_a) And dissociation rate (kd) was calculated 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	1.07E+5	1.83E-4	1.71E-9
ADC1013	1.2E+6	3.48E-2	2.9E-08
				13A2	4.84E+05	1.73E-03	3.58E-09
13A2-VH0VL0	3.96E+05	1.14E-02	2.88E-08
				13A2-VH2VL2	8.23E+05	1.71E-03	2.08E-09
13A2-VH2VL3	7.35E+05	2.61E-03	3.55E-09
				13A2-VH3VL2	8.25E+5	2.42E-3	2.93E-09
13A2-VH3VL3	5.98E+5	4.59E-3	7.68E-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 the E L ISA.

The extracellular domain (ECD) of CD40 contained 4 cysteine-rich domains (CRDs) designated CRD1, CRD2, CRD3 and CRD 4. based on the structure of CD40ECD, a full-length CD40ECD, five CD40ECD truncations and four CD40ECD mutants were constructed, and the information of these recombinant proteins is shown in Table 10 below. these several proteins were linked at the N-terminus to a signal peptide (SEQ ID NO: 83) for protein expression and secretion, and at the C-terminus to an mFc tag (SEQ ID NO: 84) for E L ISA detection, synthesis of DNA sequences encoding these recombinant proteins and cloning into pcDNA3.1 vector recombinant protein expression and purification was performed according to the method steps described in example 10. E L ISA detection was performed according to the method steps described in example 2 to assess the binding of monoclonal antibody to recombinant CD40 proteins.

As shown in a of figure 14, all antibodies bound to full-length CD40ECD 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 CD40ECD truncations and mutants

Note: CRD Δ 1 represents a truncated CRD1 domain

Example 17 specific binding of humanized CD40 antibody to human CD40

To examine the binding specificity of the antibody to CD40, the binding capacity of the humanized antibody to human CD40 and other members of the TNFRSF family with higher sequence homology was examined by the E L ISA method 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-H H, Qian Shen, China), and human TNFRSF11, Cat L-H5240, ACR #: ACR 52O, ACR 96, ACR H522, ACR, Chinese was examined.

As shown in fig. 15, neither 13A2VH3V L3 nor 7B4VH2V L2 bound to human OX40(TNFRSF4), HVEM (TNFRSF14), 4-1BB (TNFRSF9), NGFR (TNFRSF16), DR6(TNFRSF21), or RANK (TNFRSF11), indicating the binding specificity of the 13A2VH3V L3 and 7B4VH2V L2 antibodies to human CD 40.

Example 18 the genetically modified CD40 antibody has better agonistic activity

Studies have shown that optimal biological and anti-tumor effects of agonistic CD40 antibodies require the co-action of Fc receptors (fcrs) (Richman and von dehyde, (2014) cancer immunolres 2 (1): 19-26). thus, CD40 antibodies were prepared with the heavy/light chain variable region of 13A2VH3V L3 or 7B4VH2V L2 and the human IgG 1/kappa constant region, and the human IgG1 constant region carrying the S267E and L328F mutations (mutated IgG1 constant region amino acid sequences such as SEQ ID No.: 64). the resulting antibodies were designated 13A2-VH3V L3-IgG 1 (SE/L F) and 7B4-VH2V L2-IgG 1 (SE/L F), respectively, and further tested for their activities that promote dendritic cell maturation according to the method steps in example 9. RO 70089, ADC 7001013 and APX005 are used as controls.

As shown in fig. 16, both 13a2-VH3V L3-IgG 1 (SE/L F) and 7B4-VH2V L2-IgG 1 (SE/L F) showed higher agonistic activity in promoting dendritic cell maturation compared to the parent antibody, and 13a2-VH3V L3-IgG 1 (SE/L F) had the highest activity among all tested antibodies.

Example 19 humanized antibodies have anti-tumor effects in vivo

The in vivo anti-tumor activity of CD40 antibodies having the heavy/light chain variable region of 13A2VH3V L3 or 7B4VH2V L2 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 using animal models established by implanting MC38 mouse intestinal adenocarcinoma into a transgenic mouse humanized with a CD40 target (GemPharmatech Co. L td, China). the antibodies use mouse IgG 1/kappa constant regions to enhance the Fc function of the antibodies in the mouse model.

Each mouse was injected subcutaneously in one flank on day 0 with 1 × 10⁶MC38 cells. When the tumor grows to 80mm³At day 4,7, 11, 14, 18 and 21, mice were intraperitoneally injected with 13A2-VH3V L3, 7B4-VH2V L2, control antibody (RO7009789 or APX005), or PBS, 10 mg/kg/day, with RO7009789 and APX005 both engineered to have a mouse IgG 1/kappa constant region as shown in SEQ ID NOs: 62 and 66.

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 the formula TV ═ 0.5 xdxdxdxdxdxdxd²Tumor 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 were followedTumor size measurement is continued after mice for T cell analysis are sacrificed, immediately after the tumors are placed in collagenase-containing Hanks buffer, the tumor tissue is cut into small pieces with scissors, and the cut tumor tissue is further incubated in collagenase-containing Hanks buffer, gently shaken at 37 ℃ for 30 minutes, after which 10ml of RPMI1640+ 10% FBS is added to each sample, collagenase activity is terminated, and immune cell viability is maintained, the samples are filtered through a 70 μm cell filter (Corning, Cat #: 352350), and placed in new centrifuge tubes, the samples are resuspended in PBSF buffer (PBS + 2% FBS) after centrifugation, with a cell density of 1 × 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 fig. 17, treatment with the CD40 antibody significantly slowed or inhibited tumor growth compared to the negative control group, although individual responses were different in the 7B4VH2V L2 and APX005 groups, tumor growth inhibition was observed in all mice, most of the mice showed tumor growth inhibition in the 13A2VH3V L3 and RO7009789 groups, at day 28, tumors in the 7B4VH2V L2 administration group in 4 remaining mice completely disappeared, 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 1000m for 25 days tumor)m³1.2g), the body weight of the 13A2VH3V L3-treated group mice on day 25 was slightly increased from the initial body weight, with no problem of substantial weight loss, such as with RO7009789 treatment, and had less effect on body weight than 7B4VH2V L2 and APX005, indicating less toxicity of the 13A2VH3V L3 antibody.

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 7B4VH2V L2 significantly elevated CD45⁺CD45 in cells⁺CD3⁺CD8⁺Cells and CD45⁺CD3⁺CD4⁺Percentage of cells. CD45⁺CD3⁺CD8⁺The percentage of cells also rose in the 13A2VH3V L3 treated group, in addition, fig. 20 shows that 7B4VH2V L2 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 142F7 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 Hua Yintian Shi biological pharmacy Limited liability company

<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

2025 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

3540 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

5055 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

6570 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 ValSer 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 PhePro 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 GluLys 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 GluThr 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 CysAla 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 GlyAla 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 GlyThr 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 CysAla 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 4045

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 4045

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 GlyVal 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 ThrCys 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 ThrPhe 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 (18)

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: 7. 14, 20, 26, 30 and 36, wherein the antibody, or antigen-binding portion thereof, binds to CD 40.

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: 44, or a pharmaceutically acceptable salt thereof.

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: 58.

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: 44 and 58.

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 CD40, (b) binds to monkey CD40, (c) does not bind to mouse CD40, (d) promotes binding of CD40-CD 40L, (e) activates the CD40 signaling pathway, (f) promotes dendritic cell maturation, and (g) promotes proliferation of CD8+ and/or CD4+ T cells.

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 L AG-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 I L-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, 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.

Images