CD40 agonist antibodies and methods of use

Abstract

Provided in the present disclosure are anti-CD40 agonist antibodies, nucleic acid molecules encoding the anti-CD40 antibodies, expression vectors and host cells for expressing the anti-CD40 antibodies. The present disclosure also provides methods for verifying antibody function in vitro and antibody efficacy in vivo. The antibodies of the present disclosure provide a very effective agent for treating cancer by modulating immune function.

Description

[0001] Cross-references

[0002] This application claims priority to international patent application PCT / CN2020 / 110536, filed on August 21, 2020, the entire contents of which are incorporated herein by reference.

[0003] sequence list

[0004] This application contains a sequence list, the entire contents of which are incorporated herein by reference. Technical Field

[0005] This application generally relates to antibodies. More specifically, this application relates to fully human monoclonal antibodies against CD40, methods for their preparation, and their uses. Background Technology

[0006] Immune checkpoint blockade (anti-CTLA-4, PD-1, and PD-L1 mAbs) holds promise for the treatment and remission of a variety of cancers, including common cancers such as lung and breast cancer. However, despite its broad applicability, the majority (over 80%) of cancer patients are treatment-resistant or refractory to treatment. [1] Tumor necrosis factor receptor superfamily member 5 (TNFRSF5, also known as CD40) is an immune checkpoint protein that plays a crucial role in regulating T cell immunity by activating antigen-presenting cells (APCs). CD40 is widely expressed in hematopoietic cells (dendritic cells, B cells, monocytes). [4-6] On non-hematopoietic cells [7-8] The interaction between CD40 on APCs and its ligand CD40L expressed on activated effector T cells activates APCs, thereby upregulating cytokine production and the expression of antigen-presenting molecules, co-stimulatory molecules, and adhesion molecules. [10-11] Furthermore, CD40 is a proximal regulator of other TNF family signaling receptors on T cells. [12-13] CD40 signaling leads to IL-12 production and upregulation of CD80, CD86, OX40 ligands, 4-1BB ligands, and GITR ligands on APCs. CD8 + Stimulation of the corresponding receptors on T cells, combined with IL-12 and type I IFN, induces robust CD8+. + T cell activation, proliferation, and effector function, as well as tumor-specific CD8. + The formation and maintenance of T cell memory

[14] .

[0007] In multiple preclinical mouse tumor models and clinical trials, CD40 agonists have shown to be a very promising strategy for treating cancer. Pharmaceutical companies such as Pfizer, Roche, AbbVie, and Apexigen have developed a variety of CD40-targeting agonists. Pfizer's CD40 agonist antibody (CP-870, 893, currently known as Selicrelumab or RO7009789) is a fully human IgG2 antibody that strongly activates dendritic cells and induces T cells to secrete IFN-γ.

[15] CP-870 and 893, alone or in combination with other drugs such as gemcitabine and the anti-CTLA-4 antibody tremlimimab, have shown clinical efficacy in patients with advanced cancer. [16-18] Apexigen is developing the CD40 agonist APX005M, a humanized IgG1 monoclonal antibody against FC mutations. In an interim analysis of a small phase Ib study, 20 out of 24 evaluable patients (83%) with metastatic pancreatic ductal adenocarcinoma showed tumor shrinkage after receiving APX005M in combination with gemcitabine and albumin-bound paclitaxel (with or without the anti-PD-1 antibody nivolumab).

[19] CellDex's CD40 antibody, CDX-1140, has lower agonist activity levels than CP-870 and 893, but exhibits potent antitumor activity in xenograft models. It is currently in Phase 1 trials to determine the maximum tolerated dose and further evaluate its tolerability and efficacy.

[20] In addition, Alligator is collaborating with Johnson & Johnson to develop ADC-1013 as a potential treatment for advanced solid tumors.

[0008] There is still significant room for improvement in using antibodies targeting CD40 as therapeutic agents. As agonist antibodies targeting co-stimulatory receptors, toxicity is likely the most concerning issue (e.g., cytokine release syndrome), as it limits clinical application. For the currently most potent agonist, CP-870,893, the most common side effect is cytokine release syndrome, which manifests shortly after infusion with chills, fever, and other symptoms. In a combination study of APX005M and nivolumab, 54% of patients (13 out of 24) experienced adverse events leading to treatment discontinuation, and 10 patients (42%) experienced treatment-related serious adverse events.

[19] Two dose-limiting toxicities were observed during treatment: grade 3 and grade 4 febrile neutropenia.

[0009] In this disclosure, fully human antibodies against CD40 are generated that not only possess appropriate agonistic activity against the CD40 pathway but also exhibit minimal safety risk. The antibodies of this disclosure can bind to human CD40 protein with high affinity; exhibit weak or no ADCC effect and stimulate cytokine release much less than CP-870,893; and effectively modulate immune responses in vitro and in vivo. Invention Overview

[0011] In a broader sense, this disclosure relates to compounds, methods, compositions, and articles of an antibody that improves efficacy. The benefits provided by this disclosure are broadly applicable to the fields of antibody therapy and diagnostics, and can be used in combination with antibodies capable of reacting with a wide range of targets.

[0012] This disclosure provides an antibody targeting CD40, a nucleic acid molecule encoding an anti-CD40 antibody, an expression vector and host cell for expressing the anti-CD40 antibody, and methods for validating antibody function in vitro and in vivo. The antibody disclosed herein provides a highly effective agent for treating various cancers by modulating human immune function.

[0013] In some aspects, this disclosure includes isolated antibodies or antigen-binding moieties thereof that target CD40, such as human CD40 or cynomolgus monkey CD40. Preferably, the isolated antibody or antigen-binding moieties thereof have agonist activity on CD40.

[0014] In some embodiments, the isolated antibody or its antigen-binding portion comprises:

[0015] A) One or more heavy chain CDRs (HCDRs) selected from the following group:

[0016] (i)HCDR1, which contains SEQ ID NO: 1; (ii)HCDR2, which contains SEQ ID NO: 2; and (iii)HCDR3, which contains SEQ ID NO: 3;

[0017] B) One or more light chain CDRs (LCDRs) selected from the group consisting of: (i) LCDR1, which contains SEQ ID NO: 7 or a variant thereof; (ii) LCDR2, which contains SEQ ID NO: 5; and (iii) LCDR3, which contains SEQ ID NO: 6; or

[0018] C) One or more HCDRs of A) and B) One or more LCDRs.

[0019] In some embodiments, variants of SEQ ID NO: 7 include substitutions at no more than two amino acid positions in SEQ ID NO: 7, preferably substitutions at one amino acid position in SEQ ID NO: 7. In some further embodiments, the substitution occurs at one of the “NNG” positions in SEQ ID NO: 7. In at least one embodiment, a variant of SEQ ID NO: 7 is shown as SEQ ID NO: 4, which differs from SEQ ID NO: 7 only in the substitution of G to A in “NNG”.

[0020] In some embodiments, the isolated antibody or its antigen-binding portion comprises:

[0021] A) One or more heavy chain CDRs (HCDRs) selected from the following groups: (i) HCDR1 as shown in SEQ ID NO: 1; (ii) HCDR2 as shown in SEQ ID NO: 2; and (iii) HCDR3 as shown in SEQ ID NO: 3;

[0022] B) One or more light chain CDRs (LCDRs) selected from the group consisting of: (i) LCDR1 as shown in SEQ ID NO: 4 or 7; (ii) LCDR2 as shown in SEQ ID NO: 5; and (iii) LCDR3 as shown in SEQ ID NO: 6; or

[0023] C) One or more HCDRs of A) and B) One or more LCDRs.

[0024] In some embodiments, the isolated antibody or its antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0025] The VH comprises: (i) HCDR1 as shown in SEQ ID NO: 1; (ii) HCDR2 as shown in SEQ ID NO: 2; and (iii) HCDR3 as shown in SEQ ID NO: 3; and

[0026] The VL includes: (i) LCDR1 as shown in SEQ ID NO: 4 or 7; (ii) LCDR2 as shown in SEQ ID NO: 5; and (iii) LCDR3 as shown in SEQ ID NO: 6.

[0027] In some embodiments, the isolated antibody or its antigen-binding portion comprises:

[0028] (A) Heavy chain variable region (VH):

[0029] (i) It contains the amino acid sequence shown in SEQ ID NO: 8;

[0030] (ii) It contains at least 85%, 90%, or 95% of the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 8; or

[0031] (iii) It comprises an amino acid sequence having one or more added, deleted, and / or substituted amino acids compared to the amino acid sequence shown in SEQ ID NO: 8; and / or

[0032] (B) Light chain variable region (VL):

[0033] (i) It contains an amino acid sequence as shown in SEQ ID NO:9 or 10;

[0034] (ii) It contains at least 85%, at least 90%, or at least 95% of the same amino acid sequence as shown in SEQ ID NO: 9 or 10; or

[0035] (iii) It comprises an amino acid sequence having one or more added, deleted and / or substituted amino acids compared to the amino acid sequence shown in SEQ ID NO:9 or 10.

[0036] In some embodiments, the isolated antibody or its antigen-binding portion comprises a heavy chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 8 and a light chain variable region comprising an amino acid sequence as shown in SEQ ID NO: 9 or 10.

[0037] In some embodiments, the isolated antibody or its antigen-binding portion comprises a heavy chain variable region as shown in SEQ ID NO: 8 and a light chain variable region as shown in SEQ ID NO: 9 or 10.

[0038] In some embodiments, the isolated antibody or its antigen-binding portion disclosed herein further comprises an IgG constant domain, such as a human IgG constant domain. The human IgG constant domain may be a human IgG1 or IgG2 constant domain, preferably a human IgG2 constant domain.

[0039] In some embodiments, such as the isolated antibody or its antigen-binding moiety disclosed herein, one or more of the following properties are present:

[0040] (a) Strongly binds to human CD40 protein or cynomolgus monkey CD40 protein on the cell surface, with EC50 values ​​as measured by FACS that are comparable to or better than those of the reference antibody;

[0041] (b) It specifically binds to human CD40 and has no cross-reactivity with human OX40, 4-1BB, GITR and BCMA;

[0042] (c) It effectively competes with human CD40L for binding to CD40 with an IC50 in the nM range and an inhibition rate of approximately 100%;

[0043] (d) It induces concentration-dependent activation of NFκB and effectively enhances B cell proliferation in a dose-dependent manner, and both effects are more moderate than those of BMK4 (i.e., CP-870,893);

[0044] (e) Induces moderate levels of IL-12p40 secretion and upregulation of CD80 and CD86;

[0045] (f) It does not mediate or weakly mediates ADCC activity in human B cells, such as CD40-positive B cells.

[0046] (g) Does not stimulate human PBMCs to release large amounts of cytokines IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A; and

[0047] (h) exhibits significant antitumor activity and is tolerated in treated mice at all dose levels.

[0048] In some embodiments, the isolated antibody or its antigen-binding portion is a chimeric antibody, a humanized antibody, or a fully human antibody. Preferably, the antibody is a fully human monoclonal antibody.

[0049] In some embodiments, the isolated antibody or its antigen-binding portion comprises a heavy chain having the amino acid sequence of SEQ ID No:14 and a light chain having the amino acid sequence of SEQ ID No:15.

[0050] In some respects, this disclosure relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and / or light chain variable regions of isolated antibodies as disclosed herein.

[0051] In some respects, this disclosure relates to vectors comprising nucleic acid molecules encoding antibodies or antigen-binding portions thereof as disclosed herein.

[0052] In some respects, this disclosure relates to host cells containing expression vectors as disclosed herein.

[0053] In some respects, this disclosure relates to pharmaceutical compositions comprising at least one antibody or antigen-binding portion thereof as disclosed herein, and a pharmaceutically acceptable carrier.

[0054] In some aspects, this disclosure relates to a method for preparing an anti-CD40 antibody or an antigen-binding portion thereof, the method comprising expressing the antibody or the antigen-binding portion thereof in a host cell and isolating the antibody or the antigen-binding portion from the host cell.

[0055] In some aspects, this disclosure relates to a method for modulating a CD40-related immune response in a subject, comprising administering to the subject an antibody or an antigen-binding portion thereof as disclosed herein, thereby modulating a CD40-related immune response in the subject.

[0056] In some aspects, this disclosure relates to methods for treating tumor cell growth in a subject, comprising administering to the subject an effective amount of an antibody or antigen-binding portion thereof as disclosed herein, or a pharmaceutical composition as disclosed herein.

[0057] In some aspects, this disclosure relates to methods for treating or preventing cancer in a subject, including administering to the subject an effective amount of an antibody or antigen-binding portion thereof, as disclosed herein, or a pharmaceutical composition. In some embodiments, the cancer may be selected from breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, NSCLC, non-Hodgkin's lymphoma, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma. In one embodiment, the cancer is colon cancer. In another embodiment, the cancer is melanoma.

[0058] In some respects, this disclosure relates to the use of antibodies or antigen-binding portions thereof as disclosed herein in the preparation of medicaments for the treatment or prevention of cancer.

[0059] In some respects, this disclosure relates to antibodies or antigen-binding portions thereof as disclosed herein for the treatment or prevention of cancer.

[0060] In some respects, this disclosure relates to kits or devices and related methods that employ antibodies or antigen-binding portions thereof as disclosed herein, or pharmaceutical compositions as disclosed herein.

[0061] The above is an overview and therefore includes simplifications, generalizations, and omissions of details where necessary; thus, those skilled in the art will recognize that this overview is merely illustrative and not intended to be limiting in any way. Other aspects, features, and advantages of the methods, compositions, and / or devices and / or other subjects described herein will become apparent from the teachings presented herein. This overview is provided to simply introduce some alternative concepts, which will be further described in the detailed description below. This overview is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. Brief description of the attached diagram

[0063] Figure 1-3 This illustrates the effect of W3525-1.9.16-P5-uIgG2K on human CD40-engineered cells as determined by FACS. Figure 1), RAJI cells ( Figure 2 ) and A431 cells ( Figure 3 The result of the combination of ).

[0064] Figure 4 The binding of W3525-1.9.16-P5-uIgG2K to cynomolgus monkey CD40 on the cell surface, as determined by FACS, is illustrated.

[0065] Figure 5 The results of antibody competitive binding to CD40 by CD40L, as determined by FACS, are illustrated.

[0066] Figure 6 The binding results of W3525-1.9.16-P5-uIgG2K to a member of the TNFR superfamily (which is homologous to CD40) are illustrated.

[0067] Figure 7 Comparative binding results of W3525-1.9.16-P5-uIgG2K to human, cynomolgus monkey, mouse, rat and canine CD40 proteins, as measured by ELISA, are presented.

[0068] Figure 8-9 Examples are given based on Ramos cells ( Figure 8 ) and U937 cells ( Figure 9 The results of the NFκB reporter gene assay.

[0069] Figure 10 The results of B cell proliferation stimulated by W3525-1.9.16-P5-uIgG2K in an in vitro B cell proliferation assay are illustrated.

[0070] Figure 11-15 An example is shown in the in vitro DC activation assay, which induced IL-12P40 secretion by W3525-1.9.16-P5-uIgG2K. Figure 11 CD80 Figure 12 CD86 Figure 13 CD54 Figure 14 ) and CD83 ( Figure 15 The result expressed.

[0071] Figure 16 The ADCC effect of the antibody on human primary B cells is illustrated.

[0072] Figure 17-18 The results of antibody-mediated tumor growth inhibition are illustrated in the MC38 mouse colon cancer model derived from CD40 humanized mice. Figure 17 ) and weight change results ( Figure 18 The arrows indicate the time of administration.

[0073] Figures 19-20 The tumor growth inhibition results of the antibody are illustrated in a CD40 humanized mouse B16F10 mouse melanoma model. Figure 19 ) and weight change results ( Figure 20 The arrows indicate the time of administration.

[0074] Figure 21 The survival curves of mice bearing B16F10 melanoma after antibody treatment are shown. Invention Details

[0076] While the invention can be embodied in many different forms, what is disclosed herein are specific illustrative embodiments that demonstrate the principles of the invention. It should be emphasized that the invention is not limited to the specific embodiments illustrated herein. Furthermore, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter.

[0077] Unless otherwise defined herein, scientific and technical terms used in conjunction with this invention will have the meaning commonly understood by one of ordinary skill in the art. Furthermore, unless the context otherwise requires, singular terms shall include plural forms, and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context explicitly indicates otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a protein” includes multiple proteins; reference to “a cell” includes a mixture of cells, etc. In this application, unless otherwise stated, the use of “or” means “and / or.” Furthermore, the use of the term “comprising” and other forms such as “including” and “containing” is not limiting. Moreover, the scope provided in the specification and the appended claims includes all values ​​between endpoints and breakpoints.

[0078] Generally, the terms and techniques used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well-known and commonly used in the art. Unless otherwise stated, the methods and techniques disclosed herein are generally performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Abbas et al., Cellular and Molecular Immunology, 6th ed., WBSaunders Company (2010); Sambrook J. & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). The terminology, laboratory procedures, and techniques used in analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein are well-known and commonly used in the field.

[0079] definition

[0080] To better understand this invention, the definitions and explanations of relevant terms are provided below.

[0081] As used herein, the term "antibody" or "Ab" generally refers to a Y-shaped tetrameric protein comprising two heavy chains (H) and two light chains (L) held together by covalent disulfide bonds and non-covalent interactions. The light chains of an antibody can be divided into κ and λ light chains. The heavy chains can be divided into μ, δ, γ, α, and ε, which define the antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. In both the light and heavy chains, the variable region is linked to the constant region via a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The VH and VL regions can be further divided into hypervariable regions (called complementarity-determining regions (CDRs)) separated by relatively conserved regions (called framework regions (FRs)). Each VH and VL consists of 3 CDRs and 4 FRs in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the N-terminus to the C-terminus. The variable regions (VH and VL) of each heavy / light chain pair form antigen-binding sites. The distribution of amino acids in the various regions or domains follows the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al., (1989) Nature 342:878-883. Antibodies can have different antibody isotypes, such as IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.

[0082] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody, which may be used interchangeably in the context of this application, refers to a polypeptide containing a fragment of a full-length antibody that retains the ability to specifically bind to an antigen that specifically binds to the full-length antibody, and / or competes with the full-length antibody for binding to the same antigen. Generally, see Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, NY (1989), which is incorporated herein by reference for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Under certain conditions, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, biantibodies, and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide. Antigen-binding fragments of antibodies can be obtained from a given antibody (e.g., the monoclonal anti-human CD40 antibody provided in this application) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods) and can be screened for specificity in the same manner as intact antibodies.

[0083] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody molecule composed of a single molecule. Monoclonal antibodies exhibit binding specificity and affinity for specific antigens.

[0084] The term "humanized antibody" refers to an antibody in which a CDR sequence derived from another mammalian species, such as a mouse, has been transplanted onto a human frame sequence. Further frame region modifications can be performed within the human frame sequence.

[0085] As used herein, the term "recombinant antibody" refers to an antibody prepared, expressed, produced, or isolated by recombinant means, such as an antibody isolated from an animal that is transgenic with respect to the immunoglobulin gene of another species, an antibody expressed using a recombinant expression vector transfected into a host cell, an antibody isolated from a library of recombinant combined antibodies, or an antibody prepared, expressed, produced, or isolated by any other means involving splicing an immunoglobulin gene sequence into another DNA sequence.

[0086] As used herein, the term "fully human" or "completely human" in relation to an antibody or antigen-binding domain means an antibody or antigen-binding domain having or consisting of the following amino acid sequences, which correspond to the amino acid sequences of antibodies produced by humans or human immune cells, or derived from non-human sources such as transgenic non-human animals using human antibody reservoirs or other antibody-encoding sequences. In some embodiments, fully human antibodies do not contain amino acid residues (particularly antigen-binding residues) derived from non-human antibodies.

[0087] The terms “CD40,” “CD40 antigen,” or “CD40 protein” are used interchangeably throughout this document. CD40 is a single-chain glycoprotein known as a member of the tumor necrosis factor / nerve growth factor superfamily. CD40 is expressed by antigen-presenting cells (e.g., B cells, macrophages, and dendritic cells) as well as by non-immune cells and tumors. CD40L is the natural ligand of CD40, a type II 39 kDa membrane glycoprotein. CD40 and CD40L are a pair of co-stimulatory molecules, and their interaction has been found to be essential in mediating many immune and inflammatory responses, including T cell-dependent immunoglobulin type switching, memory B cell formation, and germinal center formation.

[0088] As used herein, the terms "anti-CD40 antibody," "CD40 antibody," or "antibody against CD40" refer to an antibody as defined herein that is capable of binding to CD40, such as the human CD40 protein. In some embodiments, the anti-CD40 antibody disclosed herein is a CD40 agonist.

[0089] The term "agonist" or "stimulatory" includes any molecule that can enhance or stimulate the biological activity of a naturally occurring peptide sequence. Suitable agonist molecules specifically include agonist peptides, agonist antibodies or antibody fragments, fragments of natural peptides, or amino acid sequence variants. The term "CD40 agonist" refers to a molecule capable of stimulating, activating, or otherwise enhancing CD40 activity by, for example, binding to CD40 and stimulating CD40 activity, or by binding to one or more CD40 inhibitors and preventing the interaction between the inhibitor and CD40. Agonists include, but are not limited to, antibodies and their antigen-binding fragments, proteins, peptides, glycoproteins, glycopeptides, glycolipids, polysaccharides, oligosaccharides, nucleic acids, biological organic molecules, peptide mimics, pharmacological agents and their metabolites, small molecules, fusion proteins, receptor molecules and derivatives, as well as antisense molecules, RNA aptamers, and ribozymes targeting CD40 inhibitors.

[0090] As used herein, the term "Ka" is intended to denote the association rate of a specific antibody-antigen interaction, while the term "Kd" is intended to denote the dissociation rate of a specific antibody-antigen interaction. The Kd value of an antibody can be determined using methods well-established in the art. As used herein, the term "K" is... D "This aims to represent the dissociation constant of a specific antibody-antigen interaction, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and expressed as a molar concentration (M). A preferred method for determining antibody Kd is through surface plasmon resonance, preferably using a biosensor system such as..." system.

[0091] As used in this article, the term "high affinity" for IgG antibodies refers to a affinity of 1 × 10⁻⁶ against a target antigen (e.g., CD40). -7 M or lower, preferably 5×10 -8 M or lower, or even better, 1×10 -8 M or lower K D Antibodies.

[0092] As used in this article, the term "EC" 50 "Effective concentration," also known as "half-maximum concentration," refers to the concentration of a drug, antibody, or agent that induces a 50% response between baseline and maximum after a specific exposure time. In the context of this application, EC... 50 It can be expressed in units of "nM".

[0093] As used herein, “blocking binding” refers to the ability of an antibody or its antigen-binding fragment to block or inhibit the binding of two molecules to any detectable level. In some embodiments, the binding of CD40 and CD40L can be inhibited by at least 50% by an antibody or its antigen-binding fragment as disclosed herein. In some embodiments, this inhibition can be greater than 60%, greater than 70%, greater than 80%, or greater than 90%.

[0094] As used herein, the term "separated" refers to a state obtained artificially from the natural state. If a "separated" substance or component exists naturally, it may be due to natural changes, separation from its natural state, or both. For example, if an unseparated polynucleotide or polypeptide exists naturally in a living organism, the same high-purity polynucleotide or polypeptide separated from that natural state is called a separated polynucleotide or polypeptide. The term "separated" does not exclude either artificially created or synthetic substances or other impurities that do not affect the activity of the separated substance.

[0095] As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to the CD40 protein is substantially free of antibodies that specifically bind to antigens other than the CD40 protein). However, isolated antibodies that specifically bind to the human CD40 protein may exhibit cross-reactivity with other antigens, such as CD40 proteins from other species. Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0096] As used herein, the term "vector" refers to a nucleic acid medium in which polynucleotides can be inserted. When a vector allows the expression of a protein encoded by the polynucleotides inserted therein, the vector is called an expression vector. This vector can be used to express the carried genetic material elements in host cells through transformation, transduction, or transfection. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, bacteriophages, granules, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and multivacuolar papillomaviruses (such as SV40). A vector may contain multiple elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain an origin of replication.

[0097] As used herein, the term "host cell" refers to a cellular system that can be engineered to produce proteins, protein fragments, or peptides of interest. Host cells include, but are not limited to, cultured cells, such as mammalian cultured cells derived from rodents (rats, mice, guinea pigs, or hamsters), such as CHO, BHK, NSO, SP2 / 0, YB2 / 0; or human tissue or hybridoma cells, yeast cells, and insect cells, as well as cells contained within transgenic animals or cultured tissues. The term covers not only the specific test cell but also the progeny of such cells. Such progeny may differ from the parent cell due to mutations or environmental influences that may result in certain modifications, but are still included within the scope of the term "host cell."

[0098] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by alignment and comparison of sequences. "Percentage identity" refers to the percentage of identical residues among amino acids or nucleotides in the compared molecules, calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably addressed using a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or peptides include those described in Computational Molecular Biology (edited by Lesk, AM), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects (edited by Smith, DW), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I (edited by Griffin, AM and Griffin, HG), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer (edited by Gribskov, M. and Devereux, J.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0099] As used herein, the term "immunogenicity" refers to the ability of an organism to stimulate the formation of specific antibodies or sensitized lymphocytes. It refers not only to the property of an antigen to stimulate the activation, proliferation, and differentiation of specific immune cells to ultimately produce immune effector substances such as antibodies and sensitized lymphocytes, but also to the specific immune response of antibodies or sensitized T lymphocytes that can be formed in the organism's immune system after stimulation with an antigen. Immunogenicity is the most important characteristic of an antigen. Whether an antigen can successfully induce an immune response in the host depends on three factors: the nature of the antigen, the host's reactivity, and the immunization method.

[0100] As used herein, the term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques used for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and are disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, ibid.; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197. In one specific embodiment of the invention, the human CD40 gene is transfected into 293F cells.

[0101] As used herein, the terms “hybridoma” and “hybridoma cell line” are used interchangeably. When referring to the terms “hybridoma” and “hybridoma cell line,” they also include subclones and progeny cells of the hybridoma.

[0102] As used herein, the term “SPR” or “surface plasmon resonance” refers to and includes optical phenomena that allow for the analysis of real-time, biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden, and Piscataway, NJ). For a detailed description, see the examples and... U. et al. (1993) Ann. Biol. Clin. 51: 19-26; U. et al. (1991) Biotechniques 11:620-627; Johnson, B. et al. (1995) J. Mol. Recognit. 8:125-131; and Johnson, B. et al. (1991) Anal. Biochem. 198:268-277.

[0103] As used herein, the term "fluorescence-activated cell sorting" or "FACS" refers to a specific type of flow cytometry. It provides a method for sorting a heterogeneous mixture of biological cells, one cell at a time, into two or more containers based on the specific light scattering and fluorescence characteristics of each cell (FlowMetric. "Sorting Out Fluorescence Activated Cell Sorting". 2017-11-09). Instruments used to perform FACS are known to those skilled in the art and are commercially available to the public. Examples of such instruments include the FACSStar Plus, FACScan, and FACSort instruments from Becton Dickinson (Foster City, CA), the EpicsC from Coulter Epics Division (Hialeah, FL), and the MoFlo from Cytomation (Colorado Springs, Colorado).

[0104] As used herein, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cytotoxic form in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely necessary for this killing. The primary cells mediating ADCC, NK cells, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of molecules of interest, in vitro ADCC assays can be performed, such as those described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells that can be used for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Optionally or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example in animal models such as those published by Clynes et al. in PNAS (USA) 95:652-656 (1998).

[0105] The term "subject" includes any human or non-human animal, preferably a human.

[0106] As used in this article, the term “cancer” refers to any tumor or malignant cell growth, proliferation, or metastasis that causes medical conditions, including solid tumors and non-solid tumors such as leukemia.

[0107] The terms "treatment" and "curing" as used in this article generally refer to treatments and therapies for humans or animals that achieve some desired therapeutic effect, such as inhibiting disease progression, including a slowdown in the rate of progression, a halt in the rate of progression, disease regression, disease improvement, and disease cure. Treatment as a preventative measure (i.e., prevention) is also included. For cancer, "treatment" may refer to inhibiting or slowing the growth, proliferation, or metastasis of tumors or malignant cells, or some combination thereof. For tumors, "treatment" includes removing all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor development, or some combination thereof.

[0108] As used herein, the term "effective amount" refers to the amount of an active compound or a material, composition, or dosage form containing the active compound that, when administered according to the desired treatment regimen, is effective in producing certain desired therapeutic effects commensurate with a reasonable benefit / risk ratio. For example, when used in conjunction with the treatment of CD40-related diseases or conditions, "effective amount" refers to the amount or concentration of an antibody or its antigen-binding portion that is effective in treating said disease or condition.

[0109] As used in this article, the terms “prevention,” “avoidance,” or “stopping” in relation to a disease condition in mammals refer to preventing or delaying the onset of a disease or the manifestation of its clinical or subclinical symptoms.

[0110] As used herein, the term “pharmaceuticalally acceptable” means that the carrier, diluent, excipient and / or salt thereof is chemically and / or physically compatible with the other components in the formulation and is physiologically compatible with the recipient.

[0111] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active agent, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th edition. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and ionic strength enhancers include, but are not limited to, sodium chloride.

[0112] As used herein, the term "adjuvant" refers to a nonspecific immune enhancer that, when delivered to an organism along with or before an antigen, can enhance the organism's immune response to the antigen or alter the type of immune response. Various adjuvants exist, including but not limited to aluminum adjuvants (e.g., aluminum hydroxide), Freund's adjuvants (e.g., complete and incomplete Freund's adjuvants), Corynebacterium breve, lipopolysaccharides, cytokines, etc. Freund's adjuvants are currently the most commonly used adjuvants in animal experiments. Aluminum hydroxide adjuvants are more commonly used in clinical trials.

[0113] Anti-CD40 antibody

[0114] In some aspects, the present invention includes isolated antibodies against CD40 or antigen-binding portions thereof.

[0115] In the context of this application, "antibody" can include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized and primate-derived antibodies, CDR transplantation antibodies, human antibodies, recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotype antibodies, synthetic antibodies, including their mutant proteins and variants; and their derivatives (including Fc fusion proteins and other modifications), as well as any other immunoreactive molecule that exhibits preferential binding or association with the CD40 protein. Furthermore, unless the context otherwise specifies, the term also includes all classes of antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and all subclasses (i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In a preferred embodiment, the antibody is a monoclonal antibody. In a more preferred embodiment, the antibody is a humanized or fully human monoclonal antibody.

[0116] Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, and transgenic animals (e.g., transgenic animals). Monoclonal antibodies can be produced using hybridomas and well-established biochemical and genetic engineering techniques, as described in detail in An, Zhigiang (ed.), Therapeutic Monoclonal Antibodies: From Bench to Clinic, John Wiley and Sons, 1st ed., 2009; Shire et al. (eds.), Current Trends in Monoclonal Antibody Development and Manufacturing, Springer Science+Business Media LLC, 1st ed., 2010; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed., 1988; Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), each of which is incorporated herein by reference in its entirety. In some embodiments, the antibodies disclosed herein are obtained using hybridoma technology and genetically engineered OmniRat (developed by Open Monoclonal Technology (OMT)). It should be understood that the selected binding sequence can be further modified, for example, to increase affinity for the target, humanize the target binding sequence, improve its production in cell cultures, reduce its immunogenicity in vivo, generate multispecific antibodies, etc., and antibodies containing modified target binding sequences are also antibodies of the present invention. In a preferred embodiment, anti-human CD40 monoclonal antibodies are prepared by using hybridomas. The generation of hybridomas is well known in the art. See, for example, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York.

[0117] In some embodiments of this disclosure, a series of high-throughput screenings were performed to identify positive hybridoma cell lines. The aim of the screening process was to discover candidate high-affinity binders of human and cynomolgus monkey CD40 with suitable functional activity along the CD40 pathway. The antibody sequence can be further optimized (e.g., by removing post-translational modification sites (PTMs)) to obtain a lead antibody with high binding affinity and suitable agonist activity.

[0118] Anti-CD40 agonist antibody

[0119] Antagonistic anti-CD40 antibodies have been developed, but CD40 agonists have been found to be a promising strategy for treating cancer. Several CD40-targeting agonists are currently in clinical trials, such as CP-870,893 (Pfizer), APX005M (Apexigen), CDX-1140 (CellDex), and ADC-1013 (Alligator), all of which are agonistic CD40 antibodies.

[0120] In cancer treatment, the main mechanism of CD40 agonist antibodies is to activate APCs to induce an anti-tumor T cell response, without requiring CD40 expression on tumor cells. CD40 agonist antibodies can replace CD40L on activated T cells to enhance immunity. CD40-activated B cells enter a proliferative state, thereby enhancing the T cell response.

[0121] As demonstrated in the examples, the anti-CD40 antibody disclosed herein effectively enhances B cell proliferation (in vitro proliferation assay), induces concentration-dependent activation of NFκB (RGA assay), and induces significantly lower levels of cytokine production, such as IL-2, IL-4, IL-6, and TNF, compared to a reference antibody. Therefore, the risk of cytokine release syndrome (CRS) induced by the anti-CD40 antibody disclosed herein is significantly lower, and CRS is the most frequently reported adverse event in clinical trials involving agonistic anti-CD40 antibodies.

[0122] Anti-CD40 antibodies with certain properties

[0123] The antibodies disclosed herein are characterized by specific functional features or properties. Based on their mechanism of action against the target, the in vitro functional properties and pharmacological activities of the antibodies have been adequately evaluated at both the molecular and cellular levels. In some embodiments, the isolated antibody or its antigen-binding moiety possesses one or more of the following properties:

[0124] (a) Strongly binds to human CD40 protein or cynomolgus monkey CD40 protein on the cell surface, with EC50 values ​​as measured by FACS that are comparable to or better than those of the reference antibody;

[0125] (b) It specifically binds to human CD40 and has no cross-reactivity with human OX40, 4-1BB, GITR and BCMA;

[0126] (c) It effectively competes with human CD40L for binding to CD40 with an IC50 of about 1.4 nM and an inhibition rate of about 100%;

[0127] (d) Shows cross-reactivity with human and cynomolgus monkey CD40, but no cross-reactivity with mouse, rat and dog CD40;

[0128] (e) It induces concentration-dependent activation of NFκB, with a more moderate effect than BMK4;

[0129] (f) It effectively enhances B cell proliferation in a dose-dependent manner, and the effect is more moderate than that of BMK4;

[0130] (g) Induces moderate levels of IL-12p40 secretion and upregulation of CD80 and CD86;

[0131] (h) When in human IgG2 form, it does not mediate or weakly mediates ADCC activity on human B cells;

[0132] (i) It does not stimulate human PBMCs to release large amounts of cytokines IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A; and

[0133] (j) It exhibits significant antitumor activity and is tolerated in treated mice at all dose levels.

[0134] The antibodies disclosed herein bind to human and cynomolgus monkey CD40 with high affinity. The binding of the antibodies of this invention to CD40 can be assessed using one or more techniques established in the art, such as ELISA. The binding specificity of the antibodies of this invention can also be determined, for example, by monitoring the binding of the antibody to cells expressing the CD40 protein using flow cytometry. For example, the antibody can be tested by flow cytometry in which the antibody reacts with a cell line expressing human CD40, such as CHO K1 cells transfected to express CD40 on their cell surface. Alternatively or optionally, the binding of the antibody can be tested in a BIAcore binding assay, including binding kinetics (e.g., Kd value). Other suitable binding assays include ELISA assays, for example using recombinant CD40 protein. For example, the antibodies of this disclosure bind to CD40 with 10 nM or less Kd. D Combined with human CD40, at 9.5 nM or lower K D Combined with human CD40, at 9 nM or lower K D Combined with human CD40, at 8.5 nM or lower K D Binds to human CD40 protein with 8 nM or less K D Binds to human CD40 protein at 7.5 nM or lower K D Binds to human CD40 protein, or with 7 nM or less K D It binds to human CD40 protein, as determined by the BIAcore binding assay.

[0135] Furthermore, the antibody disclosed herein can block the binding of CD40L to CD40. The prominent role of the CD40 ligand / CD40 pathway in immune regulation and homeostasis is well known. The binding of CD40 and CD40 ligands (transiently expressed on T cells and other non-immune cells under inflammatory conditions) is involved in regulating numerous molecular and cellular processes, including the initiation and progression of cellular and humoral adaptive immunity. The antibody disclosed herein has been shown to effectively compete with human CD40L for binding to CD40 with an IC50 in the nM range and an inhibition rate of approximately 100%.

[0136] Anti-CD40 antibodies containing CDR

[0137] In some implementations, the isolated antibody or its antigen-binding portion comprises:

[0138] A) One or more heavy chain CDRs (HCDRs) selected from the following group:

[0139] (i)HCDR1, which contains SEQ ID NO: 1;

[0140] (ii)HCDR2, which contains SEQ ID NO: 2; and

[0141] (iii)HCDR3, which contains SEQ ID NO: 3;

[0142] B) One or more light chain CDRs (LCDRs) selected from the following group:

[0143] (i)LCDR1, which contains SEQ ID NO: 7 or a variant thereof;

[0144] (ii) LCDR2, which includes SEQ ID NO: 5; and

[0145] (iii) LCDR3, which contains SEQ ID NO: 6; or

[0146] C) One or more HCDRs of A) and B) One or more LCDRs.

[0147] In some embodiments, variants of SEQ ID NO: 7 include substitutions at no more than two amino acid positions in SEQ ID NO: 7, preferably at no more than one position. In some further embodiments, the substitution occurs at one of the amino acids “NNG” in SEQ ID NO: 7, for example by mutating the amino acid “G” to any other amino acid. In one embodiment, the variant of SEQ ID NO: 7 is as shown in SEQ ID NO: 4, which differs from SEQ ID NO: 7 in that the amino acid “G” in “NNG” is replaced with “A”.

[0148] Variable regions and CDRs in antibody sequences can be identified according to general rules already developed in the art (as described above, such as the Kabat numbering system) or by comparing the sequence with a database of known variable regions. Methods for identifying these regions are described in Kontermann and Dubel (eds.), *Antibody Engineering*, Springer, New York, NY, 2001, and Dinarello et al., *Current Protocols in Immunology*, John Wiley and Sons Inc., Hoboken, NJ, 2000. Exemplary databases of antibody sequences are described in and available from the “Abysis” website (maintained by A.C. Martin of the Department of Biochemistry & Molecular Biology, University College London, London, England) at www.bioinf.org.uk / abs and the VBASE2 website www.vbase2.org, as described in Retter et al., *Nucl. Acids Res.*, 33 (Database issue): D671-D674 (2005). The Abysis database is preferred for sequence analysis. It integrates sequence data from Kabat, IMGT, and the Protein Database (PDB) with structural data from the PDB. See Dr. Andrew C. Martin's *Protein Sequence and Structure Analysis of Antibody Variable Domains*, in *Antibody Engineering Lab Manual* (Ed.: Duebel, S. and Kontermann, R., Springer-VerCD40, Heidelberg, ISBN-13:978-3540413547, also available at bioinforg.uk / abs). The Abysis database website also includes general rules developed for identifying CDRs that can be used in accordance with the teachings herein. Unless otherwise stated, all CDRs described herein are obtained according to the Kabat numbering system.

[0149] In some implementations, the isolated antibody or its antigen-binding portion comprises:

[0150] A) One or more heavy chain CDRs (HCDRs) selected from the following group:

[0151] (i) HCDR1 as shown in SEQ ID NO: 1; (ii) HCDR2 as shown in SEQ ID NO: 2; and (iii) HCDR3 as shown in SEQ ID NO: 3;

[0152] B) One or more light chain CDRs (LCDRs) selected from the following group:

[0153] (i) LCDR1 as shown in SEQ ID NO: 4 or 7; (ii) LCDR2 as shown in SEQ ID NO: 5; and (iii) LCDR3 as shown in SEQ ID NO: 6; or

[0154] C) One or more HCDRs of A) and B) One or more LCDRs.

[0155] In one particular implementation, the isolated antibody or its antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0156] (a) VH includes: (i) HCDR1 as shown in SEQ ID NO: 1; (ii) HCDR2 as shown in SEQ ID NO: 2; and (iii) HCDR3 as shown in SEQ ID NO: 3; and

[0157] (b) VL includes: (i) LCDR1 as shown in SEQ ID NO: 4 or 7; (ii) LCDR2 as shown in SEQ ID NO: 5; and (iii) LCDR3 as shown in SEQ ID NO: 6.

[0158] Anti-CD40 antibodies containing heavy chain variable regions and light chain variable regions

[0159] In some implementations, the isolated antibody or its antigen-binding portion comprises:

[0160] (A) Heavy chain variable region (VH):

[0161] (i) Contains the amino acid sequence shown in SEQ ID NO: 8;

[0162] (ii) Contains at least 85%, 90%, or 95% of the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 8; or

[0163] (iii) Contains an amino acid sequence having one or more added, deleted, and / or substituted amino acids compared to the amino acid sequence shown in SEQ ID NO: 8; and / or

[0164] (B) Light chain variable region (VL):

[0165] (i) Contains the amino acid sequence shown in SEQ ID NO: 9 or 10;

[0166] (ii) Contains at least 85%, at least 90%, or at least 95% identical amino acid sequences to those shown in SEQ ID NO: 9 or 10; or

[0167] (iii) An amino acid sequence having one or more added, deleted and / or substituted amino acids compared to the amino acid sequence shown in SEQ ID NO: 9 or 10.

[0168] The percentage identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table with a vacancy length penalty of 12 and a vacancy penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix with vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0169] Alternatively or additionally, the protein sequence of the present invention can be further used as a “query sequence” to perform a search against a public database to, for example, identify relevant sequences. Such a search can be performed using the XBLAST program (version 2.0) of Altschul et al. (1990) J.MoI.Biol. 215:403-10. A BLAST protein search with a score of 50 and a word length of 3 can be performed using the XBLAST program to obtain amino acid sequences homologous to the antibody molecule of the present invention. For vacancy alignment for comparative purposes, vacancy BLAST, as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402, can be used. When using the BLAST and vacancy BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

[0170] In one particular embodiment, the isolated antibody or its antigen-binding portion comprises: a heavy chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 8; and a light chain variable region comprising or consisting of the amino acid sequence of SEQ ID NO: 9 or 10.

[0171] In other embodiments, the amino acid sequences of the heavy chain variable region and / or the light chain variable region may be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, having 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the respective sequences described above.

[0172] In some further embodiments, the isolated antibody or its antigen-binding moiety may contain one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4, or 5) conserved substitutions or modifications of amino acids in the variable regions of the heavy and / or light chains. It is understood in the art that certain conserved sequence modifications that do not eliminate antigen binding can be made. See, for example, Brummell et al. (1993) Biochem 32:1180-8; de Wildt et al. (1997) Prot. Eng. 10:835-41; Komissarov et al. (1997) J. Biol. Chem. 272:26864-26870; Hall et al. (1992) J. Immunol. 149:1605-12; Kelley and O'Connell (1993) Biochem. 32:6862-35; Adib-Conquy et al. (1998) Int. Immunol. 10:341-6 and Beers et al. (2000) Clin. Can. Res. 6:2835-43.

[0173] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the fundamental properties of a protein / peptide comprising an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, such as substitutions of physically or functionally similar residues (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.) to the corresponding amino acid residue. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of amino acids are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997), which are incorporated herein by reference).

[0174] Antibody constant domain containing Fc region

[0175] The anti-CD40 antibody and antigen-binding fragment provided herein further include a human IgG constant domain. The human IgG constant domain may be a human IgG1 or IgG2 constant domain, preferably a human IgG2 constant domain. In some embodiments, the Fc region is a human IgG2 Fc region. For example, the Fc region may be a wild-type Fc region, or the Fc region may contain one or more amino acid substitutions that alter antibody-dependent cytotoxicity (ADCC) or other effector function.

[0176] Human IgG2 is the weakest binding isoform to FcγRIIB and FcγRIIIA, and it can induce agonistic activity independent of Fc-mediated cross-linking, thereby reducing the potential toxicity of hyperclustering caused by Fc and FcγR interactions. Furthermore, one CD40L trimer naturally interacts with two CD40 receptors.

[24] Similarly, the antibodies with a human IgG2 backbone disclosed herein can aggregate CD40 to form dimers, mimicking the interaction between CD40L trimers and CD40.

[0177] As illustrated in the examples, W3525-1.9.16-P5-uIgG2K and BMK4 in the human IgG2 form do not mediate or weakly mediate ADCC activity on human B cells, and are therefore unlikely to trigger ADCC on CD40-positive B cells. In contrast, BMK5 (with a human IgG1 Fc backbone) can effectively induce ADCC effects on human B cells in a dose-dependent manner.

[0178] Nucleic acid molecules encoding the antibodies of this invention

[0179] In some aspects, the present invention relates to isolated nucleic acid molecules comprising nucleic acid sequences encoding heavy chain variable regions and / or light chain variable regions of isolated antibodies as disclosed herein.

[0180] The nucleic acids of this 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, as further described below), the light and heavy chains of the antibody prepared via hybridoma can be obtained by standard PCR amplification or cDNA cloning techniques. For antibodies obtained from immunoglobulin gene libraries (e.g., using phage display technology), the nucleic acid encoding such antibody can be recovered from the gene library.

[0181] By operatively linking the nucleic acid encoding VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3), the isolated nucleic acid encoding the VH region can be converted into a full-length heavy chain gene. The sequences of human heavy chain constant region genes are known in the art (see, for example, Kabat et al. (1991), ibid.), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region, but more preferably an IgG1 or IgG4 constant region.

[0182] By operatively linking DNA encoding the VL region to another DNA molecule encoding the light chain constant region CL, isolated nucleic acids encoding the VL region can be converted into a full-length light chain gene (and a Fab light chain gene). The sequences of human light chain constant region genes are known in the art (see, for example, Kabat et al., ibid.), and DNA fragments containing these regions can be obtained by standard PCR amplification. In a preferred embodiment, the light chain constant region may be a κ or λ constant region.

[0183] Once the DNA fragments encoding the VH and VL regions are obtained, these fragments can be further manipulated using standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, the DNA fragment encoding VL or VH is operatively linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. The term "operatively linked" as used herein is intended to mean that two DNA fragments are linked such that the amino acid sequences encoded by both fragments remain within the frame.

[0184] Nucleic acid sequences encoding the heavy and light chains of the anti-CD40 antibody as described herein can be cloned into an expression vector, with each nucleotide sequence operatively linked to a suitable promoter. In one embodiment, each nucleotide sequence encoding the heavy and light chains is operatively linked to a different promoter. Alternatively, the nucleotide sequences encoding the heavy and light chains can be operatively linked to a single promoter, such that both the heavy and light chains are expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0185] In some implementations, the nucleotide sequences encoding the two chains of the antibody are cloned into two vectors, which can be introduced into the same or different cells. When the two chains are expressed in different cells, each chain can be isolated from the host cell expressing them, and the separated heavy and light chains can be mixed and incubated under suitable conditions that allow for antibody formation.

[0186] In some embodiments, the present invention relates to isolated nucleic acid molecules comprising a heavy chain variable region or a nucleic acid sequence of the heavy chain encoding an isolated antibody as disclosed herein.

[0187] In some specific implementations, the isolated nucleic acid molecule encoding the heavy chain variable region of the isolated antibody comprises a nucleic acid sequence selected from the following:

[0188] (A) Nucleic acid sequence encoding the heavy chain variable region shown in SEQ ID NO: 8;

[0189] (B) The nucleic acid sequence shown in SEQ ID NO: 11; or

[0190] (C) A nucleic acid sequence that hybridizes with the complementary strand of a nucleic acid sequence of (A) or (B) under highly stringent conditions.

[0191] In some embodiments, the present invention relates to isolated nucleic acid molecules comprising a light chain variable region or a nucleic acid sequence of a light chain encoding an isolated antibody as disclosed herein.

[0192] In some specific implementations, the isolated nucleic acid molecule encoding the light chain variable region of the isolated antibody comprises a nucleic acid sequence selected from the following:

[0193] (A) A nucleic acid sequence encoding the light chain variable region shown in SEQ ID NO: 9 or 10;

[0194] (B) The nucleic acid sequence shown in SEQ ID NO: 12 or 13; or

[0195] (C) A nucleic acid sequence that hybridizes with the complementary strand of a nucleic acid sequence of (A) or (B) under highly stringent conditions.

[0196] For example, the nucleic acid molecule consists of SEQ ID NO: 11, 12, or 13. Alternatively, the nucleic acid molecule has at least 80% (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with SEQ ID NO: 11, 12, or 13. In some specific embodiments, the percentage of identity is derived from the degeneracy of the genetic code, and the encoded protein sequence remains unchanged.

[0197] Exemplary high-strict conditions involve hybridization at 45°C in 5X SSPE and 45% formamide, followed by a final wash at 65°C in 0.1X SSC. It should be understood in the art that equivalent strict conditions can be achieved by variations in temperature and buffer or salt concentration, as described in Ausubel et al. (Eds.), Protocols in Molecular Biology, John Wiley & Sons (1994), pp. 6.0.3–6.4.10. Modifications to the hybridization conditions can be determined empirically or precisely calculated based on probe length and the percentage of guanosine / cytosine (GC) base pairings. Hybridization conditions can be calculated as described in Sambrook et al. (Eds.), Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York (1989), pp. 9.47–9.51.

[0198] host cells

[0199] The host cells disclosed in this disclosure can be any cells suitable for expressing the antibodies of this disclosure, such as E. coli cells, yeast cells, insect cells, and mammalian cells. Mammalian host cells used for expressing the antibodies of this disclosure include Chinese hamster ovary (CHO) cells (including dhfr-CHO cells, as described in Urlaub and Chasin, (1980) Proc. Natl. Acad. ScL USA 77:4216-4220, used with DHFR selection markers, for example, as described in RJ Kaufman and PA Sharp (1982) J. MoI. Biol. 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. In particular, for use with NSO myeloma cells, another expression system is the GS gene expression system disclosed in WO 87 / 04462, WO89 / 01036, and EP 338,841. When a recombinant expression vector encoding an antibody is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period of time to allow the antibody to be expressed in the host cells or by secreting the antibody into the culture medium in which the host cells are cultured. The antibody can be recovered from the culture medium using standard protein purification methods.

[0200] Pharmaceutical Composition

[0201] In some aspects, the present invention relates to pharmaceutical compositions comprising at least one antibody or antigen-binding moiety thereof as disclosed herein and a pharmaceutically acceptable carrier.

[0202] Components of the composition

[0203] The pharmaceutical composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or drug. The pharmaceutical compositions of the present invention may also be administered in combination with, for example, another immunostimulant, anticancer agent, antiviral agent, or vaccine, such that the anti-CD40 antibody enhances the immune response to the vaccine. Pharmaceutically acceptable carriers may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending / dispersing agents, chelating agents, diluents, adjuvants, excipients, or non-toxic excipients, combinations or more of various components known in the art.

[0204] Suitable components may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavorings, thickeners, colorants, emulsifiers, or stabilizers such as sugars and cyclodextrins. Suitable antioxidants may include, for example, methionine, ascorbic acid, EDTA, sodium thiosulfate, platinum, catalase, citric acid, cysteine, mercaptoglycerol, mercaptoacetic acid, mercaptosorbitol, butylated methyl anisole, butylated hydroxytoluene, and / or propyl gallate. As disclosed in this invention, antibodies or antigen-binding fragments containing compositions disclosed herein can be oxidized in solvents containing one or more antioxidants such as methionine that reduce the antibody or its antigen-binding fragment. Redox reactions can prevent or reduce the decrease in binding affinity, thereby enhancing antibody stability and extending shelf life. Therefore, in some embodiments, this invention provides compositions comprising one or more antibodies or their antigen-binding fragments and one or more antioxidants such as methionine. The present invention further provides various methods in which an antibody or its antigen-binding fragment is mixed with one or more antioxidants such as methionine, thereby preventing oxidation of the antibody or its antigen-binding fragment, thereby extending its shelf life and / or increasing its activity.

[0205] To further illustrate, pharmaceutically acceptable carriers may include, for example, aqueous carriers such as sodium chloride injection, Ringer's injection, isotonic dextran injection, sterile water injection, or dextran and lactated Ringer's injection; non-aqueous carriers such as plant-derived fixed oils, cottonseed oil, corn oil, sesame oil, or peanut oil; antibacterial agents or antifungal concentrations of antimicrobial agents; isotonic agents such as sodium chloride or glucose; buffers such as phosphate or citrate buffers; antioxidants such as sodium bisulfate; local anesthetics such as procaine hydrochloride; suspending and dispersing agents such as sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone; emulsifiers such as polysorbate 80 (TWEEN-80); barrier agents or chelating agents such as EDTA (ethylenediaminetetraacetic acid) or EGTA (ethylene glycol tetraacetic acid); ethylene glycol; polyethylene glycol; propylene glycol; sodium hydroxide; hydrochloric acid; citric acid; or lactic acid. Antimicrobial agents used as carriers can be added to pharmaceutical compositions containing phenol or cresol, mercury preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzyl chloride in multi-dose containers. Suitable excipients may include, for example, water, saline, dextran, glycerol or ethanol. Suitable non-toxic adjuvants may include, for example, wetting agents or emulsifiers, pH buffers, stabilizers, solubility enhancers or agents such as sodium acetate, sorbitol monolaurate, triethanolamine oleate or cyclodextrin.

[0206] Application, formulation and dosage

[0207] The pharmaceutical compositions of the present invention can be administered to subjects in need via various routes, including but not limited to oral, intravenous, intra-arterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous and intrathecal, or implantation or inhalation. The compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalers, and aerosols. Appropriate formulations and routes of administration can be selected based on the intended application and treatment regimen.

[0208] Suitable formulations for enteral administration include hard or soft gelatin capsules, pills, tablets, including coated tablets, elixirs, suspensions, syrups, or inhalers and their controlled-release formulations.

[0209] Formulations suitable for parenteral administration (e.g., by injection) include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions) in which the active ingredient is dissolved, suspended, or otherwise provided (e.g., in liposomes or other microparticles). These liquids may additionally contain other pharmaceutically acceptable components, such as antioxidants, buffers, preservatives, stabilizers, antibacterial agents, suspending agents, thickeners, and solutes that make the formulation isotonic with the intended recipient's blood (or other relevant bodily fluids). Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, etc. Examples of isotonic carriers suitable for such formulations include sodium chloride injection, Ringer's solution, or lactated Ringer's solution. Similarly, specific dosing regimens (including dose, time, and repetition) will depend on the specific individual and their medical history, as well as empirical considerations such as pharmacokinetic parameters (e.g., half-life, clearance, etc.).

[0210] The frequency of administration can be determined and adjusted during treatment, and is based on reducing the number of proliferating or tumorigenic cells, maintaining this reduction in tumor cells, reducing tumor cell proliferation, or delaying the development of metastasis. In some embodiments, the administered dose can be adjusted or reduced to control potential side effects and / or toxicity. Alternatively, a continuously releasing formulation of the therapeutic composition of the present invention may be suitable.

[0211] Those skilled in the art will understand that appropriate dosages can vary from patient to patient. Determining the optimal dosage typically involves balancing the level of therapeutic benefit with any risks or adverse side effects. The chosen dosage level will depend on a variety of factors, including but not limited to the activity of the specific compound, administration, timing of administration, compound clearance rate, duration of treatment, other drugs, compounds and / or materials used in combination, severity of the condition, and species, the patient's sex, age, weight, condition, general health status, and previous medical history. The amount of compound and route of administration are ultimately determined by a physician, veterinarian, or clinician, but a dosage is typically chosen to achieve a local concentration at the site of action to achieve the desired effect without causing substantial harmful or adverse side effects.

[0212] Typically, the antibodies or antigen-binding portions of the present invention can be administered in a variety of ranges. These include about 5 μg / kg body weight to about 100 mg / kg body weight per dose; about 50 μg / kg body weight to about 5 mg / kg body weight per dose; and about 100 μg / kg body weight to about 10 mg / kg body weight per dose. Other ranges include about 100 μg / kg body weight to about 20 mg / kg body weight per dose and about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, the dose is at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, and at least about 10 mg / kg body weight.

[0213] In any case, the antibody or its antigen-binding portion of the present invention is preferably administered to subjects in need as required. Those skilled in the art can determine the frequency of administration, for example, based on considerations by the attending physician regarding the condition being treated, the age of the subject being treated, the severity of the condition being treated, and the general health condition of the subject being treated.

[0214] In some preferred embodiments, the treatment process involving the antibody or antigen-binding portion thereof of the present invention will comprise multiple doses of the selected pharmaceutical product administered over several weeks or months. More specifically, the antibody or antigen-binding portion thereof of the present invention may be administered daily, every two days, every four days, weekly, every ten days, every two weeks, every three weeks, monthly, every six weeks, every two months, every ten weeks, or every three months. In this regard, it is understood that the dosage or interval may be varied or adjusted based on patient response and clinical practice.

[0215] The dosage and regimen of the disclosed therapeutic composition may also be determined empirically in individuals receiving one or more administrations. For example, an incremental dose of the therapeutic composition as described herein may be administered to an individual. In selected embodiments, the dosage may be gradually increased, decreased, or mitigated based on empirical determination or observed side effects or toxicities. To assess the efficacy of the selected composition, biomarkers of a specific disease, condition, or disease can be tracked as previously described. For cancer, these include direct measurement of tumor size by palpation or visual observation, indirect measurement of tumor size by X-ray or other imaging techniques; improvement assessed by direct tumor biopsy and microscopic examination of tumor samples; reduction of pain or paralysis by measurement of indirect tumor biomarkers (e.g., PSA for prostate cancer) or tumorigenic antigens; improvement of tumor-related speech, vision, breathing, or other disabilities; increased appetite; or improvement in quality of life or prolonged survival as measured by administered tests. Those skilled in the art will understand that the dosage will vary depending on the individual, the type of tumor, the stage of the tumor, whether the tumor has begun to metastasize to other sites within the individual, and past and concurrent treatments.

[0216] Compatible formulations for parenteral administration (e.g., intravenous injection) may comprise an antibody or antigen-binding moiety thereof disclosed herein at a concentration of about 10 μg / ml to about 100 mg / ml. In some selected embodiments, the concentration of the antibody or antigen-binding moiety thereof will include 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, 500 μg / ml, 600 μg / ml, 700 μg / ml, 800 μg / ml, 900 μg / ml, or 1 mg / ml. In other preferred embodiments, the concentration of the antibody or its antigen-binding portion will include 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 8 mg / ml, 10 mg / ml, 12 mg / ml, 14 mg / ml, 16 mg / ml, 18 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 60 mg / ml, 70 mg / ml, 80 mg / ml, 90 mg / ml, or 100 mg / ml.

[0217] Application of the present invention

[0218] The antibodies, antibody compositions, and methods of the present invention have numerous in vitro and in vivo uses, including, for example, the detection of CD40 or the enhancement of immune responses. For example, these molecules can be administered in vitro or ex vivo to cultured cells, or, for example, in vivo to human subjects, to enhance immunity in various situations. Immune responses can be modulated, for example, enhanced, stimulated, or upregulated.

[0219] For example, subjects may include patients who require enhanced immune responses. The method is particularly suitable for treating patients with conditions that can be treated by enhancing immune responses, such as T-cell-mediated immune responses. In one specific embodiment, the method is particularly suitable for in vivo treatment of cancer. To achieve antigen-specific enhancement of the immune response, anti-CD40 antibodies may be administered together with the antigen of interest, or the antigen may already be present in the subject to be treated (e.g., a subject carrying a tumor or virus). When CD40 antibodies are administered together with another agent, the two can be administered in any order or simultaneously.

[0220] The present invention further provides a method for detecting the presence of human CD40 antigen in a sample or measuring the amount of human CD40 antigen, comprising contacting a sample and a control sample with a human monoclonal antibody or its antigen-binding portion that specifically binds to human CD40, under conditions allowing the formation of a complex between the antibody or a portion thereof and human CD40. The formation of the complex is then detected, wherein differential complex formation between the samples compared to the control sample indicates the presence of human CD40 antigen in the sample. Furthermore, the anti-CD40 antibody of the present invention can be used to purify human CD40 by immunoaffinity purification.

[0221] Treatment of diseases including cancer

[0222] In some aspects, the present invention provides a method for treating a symptom or disease in mammals, comprising administering a therapeutically effective amount of an antibody or its antigen-binding portion, as disclosed herein, to a patient (e.g., a human) requiring treatment. The symptom or disease may be cancer.

[0223] The methods provided in this disclosure can be used to treat or prevent a variety of cancers involving CD40, whether malignant or benign, and whether primary or secondary. Cancers can be solid tumors or hematologic malignancies. Examples of these cancers include lung cancers such as bronchial carcinomas (e.g., non-small cell lung cancer, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, and adenocarcinoma), alveolar cell carcinoma, bronchial adenoma, chondromalacia (non-cancerous), and sarcoma (cancerous); cardiac cancers such as myxoma, fibroma, and rhabdomyosarcoma; bone cancers such as osteochondroma, chondroma, chondroblastoma, chondromyoid fibroma, osteoid osteoma, giant cell tumor, chondrosarcoma, multiple myeloma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, Ewing's tumor (Ewing's sarcoma), and reticulum cell sarcoma; and brain cancers such as gliomas (e.g., glioblastoma multiforme), anaplastic astrocytoma, and astrocytoma. Tumors, oligodendrogliomas, medulloblastomas, chordomas, schwannomas, ependymomas, meningiomas, pituitary adenomas, pineal tumors, osteomas, hemangioblastomas, craniopharyngiomas, chordomas, germ cell tumors, teratomas, dermoid cysts, and hemangiomas; cancers of the digestive system such as colon cancer, leiomyomas, epidermoid carcinomas, adenocarcinomas, leiomyosarcomas, gastric adenocarcinomas, intestinal lipomas, enterofibromas, intestinal fibromas, colorectal polyps, and colorectal cancer; liver cancers such as hepatocellular adenomas, hemangiomas, hepatocellular carcinomas, fibrolamellar carcinomas, bile duct carcinomas, hepatoblastomas, and angiosarcomas; kidney cancers such as renal adenocarcinomas, renal cell carcinomas, adrenoid adenomas, and transitional cell carcinomas of the renal pelvis; bladder cancer; blood cancers. Systemic cancers such as acute lymphoblastic (lymphoblastic) leukemia, acute myeloid (myeloid, myeloblastic, myelomonocytic) leukemia, chronic lymphocytic leukemia (e.g., Sezary syndrome and hairy cell leukemia), chronic myeloid (myeloid, myeloblastic, granulocytic) leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, mycosis fungoides, and myeloproliferative disorders (including myeloproliferative disorders such as polycythemia vera, myelofibrosis, thrombocytosis, and chronic myeloid leukemia); skin cancers such as basal cell carcinoma, squamous cell carcinoma, melanoma, Kaposi's sarcoma, and Paget's disease. Cancers of the head and neck; eye-related cancers such as retinoblastoma and intraocular melanoma; male reproductive system cancers such as benign prostatic hyperplasia, prostate cancer, and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, and choriocarcinoma); breast cancer; female reproductive system cancers such as uterine cancer (endometrial cancer), cervical cancer (cervical tumor), ovarian cancer (ovarian tumor), vulvar cancer, vaginal cancer, fallopian tube cancer, and hydatidiform mole; thyroid cancers (including papillary, follicular, anaplastic, or medullary carcinoma); pheochromocytoma (adrenal gland); non-cancerous growths of the parathyroid gland; pancreatic cancer; and hematologic cancers such as leukemia, myeloma, non-Hodgkin's lymphoma, and Hodgkin's lymphoma. In one specific implementation, the cancer is colon cancer. In another specific implementation, the cancer is melanoma.

[0224] In some implementations, examples of cancer include, but are not limited to, B-cell lymphomas (including low-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade disseminated NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-splitting cell NHL; massive disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia; and other cancers. Allergic leukemia (ALL); hairy cell leukemia; chronic myeloid leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal angiogenesis associated with scarring, edema (e.g., associated with brain tumors), B-cell proliferative disorders, and Meig syndrome. More specific examples include, but are not limited to, relapsed or refractory NHL, first-line low-grade NHL, stage III / IV NHL, chemotherapy-resistant NHL, precursor B-cell lymphoblastic leukemia and / or lymphoma, small lymphocytic lymphoma, B-cell chronic lymphocytic leukemia and / or... Or prolymphocytic leukemia and / or small lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma and / or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma, marginal zone B-cell lymphoma, splenic marginal zone lymphoma, extranodal marginal zone-MALT lymphoma, lymph node marginal zone lymphoma, hairy cell leukemia, plasmacytoma and / or plasmacytic myeloma, low-grade / follicular lymphoma, intermediate-grade / follicular NHL, mantle cell lymphoma, follicular central lymphoma (follicular), intermediate-grade diffuse NHL, diffuse large B-cell lymphoma, invasive Invasive NHL (including aggressive frontline NHL and aggressive relapsed NHL), relapsed or refractory NHL after autologous stem cell transplantation, primary mediastinal large B-cell lymphoma, primary exudative lymphoma, high-grade immunoblastic NHL, high-grade lymphoblastic NHL, high-grade small non-splitting cell NHL, blistering NHL, Burkitt lymphoma, precursor (peripheral) large granular lymphocytic leukemia, mycosis fungoides and / or Sezary syndrome, cutaneous (skin) lymphoma, anaplastic large cell lymphoma, angiogenic lymphoma.

[0225] In some implementations, examples of cancer further include, but are not limited to, B-cell proliferative disorders, which further include, but are not limited to, lymphomas (e.g., B-cell non-Hodgkin lymphoma (NHL)) and lymphocytic leukemias. Such lymphomas and lymphocytic leukemias include, for example, a) follicular lymphomas, b) small unmitotic cell lymphomas / Burkitt lymphomas (including endemic Burkitt lymphoma, sporadic Burkitt lymphoma, and non-Burkitt lymphoma), c) marginal zone lymphomas (including extranodal marginal zone B-cell lymphomas (mucosa-associated lymphoid tissue lymphoma, MALT), nodular marginal zone B-cell lymphomas, and splenic marginal zone lymphomas), d) mantle cell lymphomas (MCL), and e) large cell lymphomas (including diffuse large cell B-cell lymphoma (DLCL)). Diffuse mixed cell lymphoma, immunoblastic lymphoma, primary mediastinal B-cell lymphoma, angiocentric lymphoma (pulmonary B-cell lymphoma), f) hairy cell leukemia, g) lymphocytic lymphoma, Waldenstrom macroglobulinemia, h) acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL) / small lymphocytic lymphoma (SLL), B-cell prolymphocytic leukemia, i) plasma cell vegetations, plasma cell myeloma, multiple myeloma, plasmacytoma and / or j) Hodgkin's disease.

[0226] Stimulation of the immune response

[0227] In some aspects, the present invention also provides a method for enhancing (e.g., stimulating) an immune response in a subject, comprising administering to the subject an antibody of the present invention or an antigen-binding portion thereof to enhance the subject's immune response. For example, the subject is a mammal. In a particular embodiment, the subject is a human.

[0228] The term "enhanced immune response" or its grammatical variations mean any response that stimulates, induces, increases, improves, or strengthens the mammalian immune system. An immune response can be a cellular response (i.e., cell-mediated, such as cytotoxic T lymphocyte-mediated) or a humoral response (i.e., an antibody-mediated response), and can be a primary or secondary immune response. Examples of enhanced immune responses include increased CD4+. +This method enhances the production of helper T cell activity and cytolytic T cells. The enhancement of the immune response can be assessed using a number of in vitro or in vivo measurements known to those skilled in the art, including but not limited to cytotoxic T lymphocyte assays, cytokine release (e.g., IL-2 production or IFN-γ production), tumor regression, survival of tumor-carrying animals, antibody production, immune cell proliferation, expression of cell surface markers, and cytotoxicity. Typically, the methods of this disclosure enhance the immune response in mammals compared to the immune response in untreated mammals or untreated mammals not treated with the methods disclosed herein. In one embodiment, the immune response is cytokine production, particularly IFN-γ production or IL-12 production. In another embodiment, the immune response is enhanced B cell proliferation.

[0229] For agonistic antibodies targeting co-stimulatory receptors, toxicity such as cytokine release syndrome limits their clinical application. Therefore, to avoid high cytotoxicity or excessive release of cytokines, moderate or moderate levels of agonistic activity may be preferred. The antibody disclosed in this paper enhances IL-12 secretion and activation marker expression in dendritic cells (DCs), but to a more moderate degree than BMK4.

[0230] Antibodies or their antigen-binding portions can be used alone as a monotherapy or in combination with chemotherapy or radiotherapy.

[0231] Used in combination with chemotherapy

[0232] Antibodies or their antigen-binding portions can be used in combination with anticancer agents, cytotoxic agents, or chemotherapy agents.

[0233] The terms "anticancer agent" or "antiproliferative agent" refer to any agent that can be used to treat cell-proliferating conditions such as cancer, and include, but are not limited to, cytotoxic agents, cell inhibitors, anti-angiogenic agents, radiotherapy and radiotherapy agents, targeted anticancer agents, BRMs, therapeutic antibodies, cancer vaccines, cytokines, hormone therapy, radiotherapy and anti-metastatic agents, and immunotherapy agents. It should be understood that, in selected embodiments as described above, such anticancer agents may comprise conjugates and may be bound to a disclosed site-specific antibody prior to administration. More specifically, in some embodiments, a selected anticancer agent is linked to an unpaired cysteine ​​residue of an engineered antibody to provide an engineered conjugate as described herein. Therefore, such engineered conjugates are explicitly contemplated within the scope of this invention. In other embodiments, the disclosed anticancer agent is administered in combination with a site-specific conjugate comprising the various therapeutic agents described above.

[0234] As used herein, the term "cytotoxic agent" refers to a substance that is toxic to cells and reduces or inhibits cell function and / or causes cell damage. In some embodiments, the substance is a naturally occurring molecule derived from a living organism. Examples of cytotoxic agents include, but are not limited to, small molecule toxins or enzymatically active toxins from bacteria (e.g., diphtheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcus enterotoxin A), fungi (e.g., α-acin, localized aspergillin), plants (abrusatil, ricin, saccharitoxin, quercetin, American pokeweed antiviral protein, saponins, white tree toxin, momoridin, trichosanthes pollen protein, barley toxin, tung oil (Aleurites fordii) protein, caryophyllin protein, Phytolacca mericana protein (PAPI, PAPII and PAP-S), bitter melon inhibitor, jatropha toxin, croton toxin, salsa inhibitor, white tree toxin, mitegellin, localized aspergillin, phenolmycin, neomycin and trichothecene compounds), or animals (e.g., small molecule toxins or enzymatically active toxins from cytotoxic RNases, such as extracellular pancreatic RNase; DNase I, including its fragments and / or variants).

[0235] For the purposes of this invention, "chemotherapy agents" include chemical compounds (e.g., cytotoxic agents or cell inhibitors) that nonspecifically reduce or inhibit the growth, proliferation, and / or survival of cancer cells. These chemical agents typically target intracellular processes required for cell growth or division, and are therefore particularly effective for cancer cells that typically grow and divide rapidly. For example, vincristine depolymerizes microtubules, thereby inhibiting cells from entering mitosis. Generally, chemotherapy agents can include any chemical agent that inhibits or is designed to inhibit cancer cells or cells that may become cancerous or produce tumorigenic progeny (e.g., TIC). These agents are often used in combination, and combinations are often the most effective, for example, in regimens such as CHOP or FOLFIRI.

[0236] Examples of anticancer agents (as components of site-specific conjugates or in an unconjugated state) that can be used in combination with the site-specific constructs of the present invention include, but are not limited to, alkylating agents, alkyl sulfonates, aziridine, ethyleneimine and methylmelamine, acetogenins, camptothecin, lichenin, callystatin, CC-1065, cryptophycins, dolastatin, pyruvicin, eleutherobin, and pancratistatin. Sarcodictyin, spongistatin, nitrogen mustard, antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, esporamycin, chromophores of enediyne antibiotics, aclacinomysins, actinomycin, atrazoserine, bleomycin, actinomycin C, carabicin, erythromycin, carcinomalacin, chromomycin, daunorubicin, detoxin, 6-diazo-5-oxo-L-leucine. Doxorubicin, Epirubicin, Isorubicin, Idarubicin, Emaciclomycin, Mitomycin, Mycophenolic acid, Nogamycin, Oligomycin, Pepromycin, Potfiromycin, Puromycin, Quelamycin, Rodobicin, Streptomycin, Streptozotocin, Tuberculin, Ubenimex, Netostatin, Zolarubicin; Antimetabolites, Erlotinib, Vemurafenib, Crizotinib, Sorafenib, Ibrutinib, Enzalutamide, Folic acid analogs, Purine analogs, Androgens, Anti-adrenergics, Folic acid supplements such as frolinic acid, acetodextrin, Aldoxycycline glycosides Aminolevulinic acid, emuramicin, acridine, bestrabucil, bismuth subcitrate, edaraxazole, defofamine, colchicine, diacinone, elfornithine, eletyl acetate, epoxigerulone, etoposide, gallium nitrate, hydroxyurea, lentinan, chlordamine, maytansinoids, mitoxantrone, mitoxantrone, mopidanmol, nitraerine, pentostatin, methamidophos, pirarubicin, loxoantrone, podophyllic acid, 2-ethylhydrazine, procarbazine. Polysaccharide complexes (JHS Natural Products, Eugene, OR), razorbenzan; crizolam; germanespiramine; tinuzonic acid; triaminoquinone; 2,2',2”-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, baculosporin A, and anguidine); urethane; vinblastine; dacarbazine; mannomustine; dibromomannitol; dibromoeusine; piperobromethane; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxanes; chloranbucil; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum analogs; Vincristine; Platinum; Etoposide (VP-16); Ifosfamide; Mitoxantrone; Vincristine Vinorelbine; Norfloxacin; Teniposide; Idatraxa; Daunorubicin; Aminopterin; Xeloda; Ibandronate; Irinotecan (Camptosar, CPT-11); topoisomerase inhibitor RFS 2000; Difluoromethylornithine; Retinoids; Capecitabine; Cobbutatin; Leucovorin; Oxaliplatin; Inhibitors of PKC-α, Raf, H-Ras, EGFR, and VEGF-A (which reduce cell proliferation), and pharmaceutically acceptable salts, acids, or derivatives of any of the above. This definition also includes anti-hormonal agents used to regulate or inhibit the hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators, aromatase inhibitors that inhibit aromatase, which regulates estrogen production in the adrenal glands, and anti-androgens; and Traxatabine (a 1,3-dioxane cytosine analog); antisense oligonucleotides, ribozymes such as VEGF expression inhibitors and HER2 expression inhibitors; vaccines, rIL-2; Topoisomerase 1 inhibitors; rmRH; vinorelbine and esporomycin, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0237] Used in combination with radiotherapy

[0238] This invention also provides combinations of antibodies or their antigen-binding portions with radiotherapy (i.e., any mechanism for locally inducing DNA damage within tumor cells, such as gamma irradiation, X-rays, UV irradiation, microwaves, electron emission, etc.). Combined therapies using the targeted delivery of radioisotopes to tumor cells are also contemplated, and the disclosed antibodies can be used in combination with targeted anticancer agents or other targeted agents. Typically, radiotherapy is administered in pulses over a period of about one to two weeks. Radiotherapy can be administered to subjects with head and neck cancer for about six to seven weeks. Optionally, radiotherapy can be administered as a single dose or as multiple sequential doses.

[0239] diagnosis

[0240] This invention provides in vitro and in vivo methods for detecting, diagnosing, or monitoring proliferative disorders, as well as methods for screening cells from patients to identify tumor cells, including tumorigenic cells. Such methods include identifying an individual with cancer for treatment or monitoring cancer progression, including contacting the patient or a sample obtained from the patient (in vivo or in vitro) with an antibody as described herein and detecting the presence or absence of the antibody in the sample, or the binding level, of a bound or free target molecule. In some embodiments, the antibody will comprise a detectable marker or reporter molecule as described herein.

[0241] In some implementations, the binding of an antibody to specific cells in a sample may indicate that the sample may contain tumorigenic cells, thereby suggesting that an individual with cancer can be effectively treated with the antibodies described herein.

[0242] Samples can be analyzed using a variety of assays, such as radioimmunoassay, enzyme immunoassay (e.g., ELISA), competitive binding assay, fluorescence immunoassay, immunoblotting, Western blot analysis, and flow cytometry. Compatible in vivo diagnostic or diagnostic assays may include imaging or monitoring techniques known in the art, such as magnetic resonance imaging, computed tomography (e.g., CAT scan), positron emission tomography (e.g., PET scan), radiography, ultrasound, etc., as known to those skilled in the art.

[0243] Drug packaging and reagent kits

[0244] Pharmaceutical packages and kits containing one or more containers of an antibody or its antigen-binding moiety containing one or more doses are also provided. In some embodiments, a unit dose is provided, wherein the unit dose contains a predetermined amount of a composition comprising, for example, an antibody or its antigen-binding moiety, with or without one or more other reagents. For other embodiments, such a unit dose is supplied in a single-use, pre-filled syringe. In other embodiments, the composition contained in the unit dose may comprise saline, sucrose, or the like; buffers, such as phosphates; and / or formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder, which can be reconstituted upon addition of a suitable liquid (e.g., sterile water or saline solution). In some preferred embodiments, the composition comprises one or more substances that inhibit protein aggregation, including but not limited to sucrose and arginine. Any label on or associated with the container indicates that the packaged composition is intended for the treatment of selected oncological conditions.

[0245] The present invention also provides a kit for generating single- or multiple-dose administration units of antibodies and optionally one or more anticancer agents. The kit includes a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of a variety of materials, such as glass or plastic, and contains a pharmaceutically effective amount of the disclosed conjugated or unconjugated form of antibody. In other preferred embodiments, the container includes a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a hypodermic needle). Such kits typically contain a pharmaceutically acceptable formulation of the antibody in a suitable container and optionally one or more anticancer agents in the same or different containers. The kit may also contain other pharmaceutically acceptable formulations for diagnostic or combination therapy. For example, in addition to the antibody or its antigen-binding portion of the present invention, such a kit may contain any one or more anticancer agents, such as chemotherapeutic or radiotherapy agents; anti-angiogenic agents; anti-metastatic agents; targeted anticancer agents; cytotoxic agents; and / or other anticancer agents.

[0246] More specifically, the kits may have a single container containing the disclosed antibody or its antigen-binding moiety, with or without additional components, or they may have different containers for each desired reagent. In the case of provided combination therapeutic agents, a single solution may be premixed in molar equivalents or in a manner where one component is more than another. Alternatively, the antibody and any optional anticancer agent in the kit may be stored separately in different containers prior to administration to the patient. The kit may also include a second / third container for containing sterile, pharmaceutically acceptable buffers or other diluents such as sterile water for injection (BWFI), phosphate-buffered saline (PBS), Ringer's solution, and glucose solution.

[0247] When the reagent kit components are provided as one or more liquid solutions, the liquid solutions are preferably aqueous solutions, particularly sterile aqueous solutions or saline solutions. However, the reagent kit components may also be provided as dry powders. When reagents or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is conceivable that the solvent may also be provided in a separate container.

[0248] As briefly described above, the kit may also contain tools for administering antibodies or their antigen-binding portions and any optional components to a patient, such as one or more needles, IV bags or syringes, or even eye drops, pipettes or other similar devices through which the preparation can be injected or introduced into an animal or applied to a diseased area of ​​the body. The kits of the present invention typically also include devices for containing vials or the like, and other tightly sealed components for commercial sale, such as injection or blow-molded plastic containers, in which the desired vials and other devices are placed and held.

[0249] Sequence List Overview

[0250] This application is accompanied by a sequence listing containing several nucleic acid and amino acid sequences. Tables A, B, C, and D below provide an overview of the included sequences.

[0251] The final lead anti-CD40 antibody used in this example is called “W3525-1.9.16-P5-uIgG2K” or simply “W3525” antibody. Its parent antibody, “W3525-1.9.16-uIgG2K”, differs from W3525 in that one amino acid substitution is made in the light chain CDR1. The table below lists the amino acid sequences of CDR, VH, and VL, as well as the nucleotide sequences encoding the variable region.

[0252] Table A: CDR amino acid sequence

[0253]

[0254] Table B: Amino acid sequences of the variable region

[0255]

[0256]

[0257] Table C: Nucleotide Sequence of Variable Region

[0258]

[0259]

[0260]

[0261] Table D Full-Length Sequence

[0262] Example

[0263] The invention generally described herein will be more readily understood by referring to the following embodiments, which are provided by way of example and are not intended to limit the invention. These embodiments are not intended to represent all or only the experiments conducted below.

[0264] Example 1: Preparation of materials, reference antibody and cell line

[0265] 1.1 Preparation of materials

[0266] Table 1 provides information on the commercial materials used in the examples.

[0267] Table 1

[0268]

[0269]

[0270]

[0271] Table 2 below lists the information on the material codes used in the experiment.

[0272] Table 2 Material Codes

[0273]

[0274]

[0275] 1.2 Antigen production

[0276] The antigens W352-hPro1.ECD.hFc (NP_001241.1, 21-193), W352-mPro1.ECD.hFc (NP_035741.2, 20-193), W352-hPro1.ECD.His (NP_001241.1, 21-193), W352-mPro1.ECD.His (NP_035741.2, 20-193), W352-cynoPro1.ECD.His (XP_005569274.1, 21-193), and W352-hpro1L1.ECD.mFc (NP_000065.1, 47-261) were expressed and produced in human Expi-293F cells. The antigen proteins were purified and stored at -80°C.

[0277] 1.3 Production of reference antibodies

[0278] In the following studies, anti-human CD40 antibodies BMK3, BMK4, BMK5, and BMK7 were used as reference antibodies. Their variable domains were synthesized according to the sequences disclosed in their respective patents, the information of which is summarized in Table 3 below. BMK4 and BMK7 were constructed by fusing the variable domain with a constant domain of human IgG2 (κ light chain), while BMK3 and BMK5 were constructed by fusing the variable domain with a constant domain of human IgG1 (κ light chain). Human IgG1 / IgG2-independent antibodies were used as isotype control antibodies. All of the above antibodies were stored at -80°C.

[0279] Table 3. Reference Antibody Information

[0280] Antibody code company Patent number Molecular name WBP352-BMK3.uIgG1K Novartis US20140205602 CHIR-12.12 WBP352-BMK4.hIgG2K Pfizer US20160152713 21.4.1 / CP-870,893 WBP352-BMK5.hIgG1K Apexigen US20160208007 APX005 WBP352-BMK7-uIgG2K CellDex WO2017184619 3C3 / CDX-1140

[0281] 1.4 Generation of Cell Pools / Cell Lines

[0282] The human CD40-expressing cell line W352-CHOK1.hPro1.A7 was generated using CHO-K1 cells transfected with full-length human CD40 (NM_001250.5, NP_001241.1).

[0283] The cell line W352-CHOK1.mPro1.B3, which expresses mouse CD40, was generated using CHO-K1 cells transfected with full-length mouse CD40 (NP_035741.2).

[0284] The reporter cell pool W352-Ramos.NFkBRE.luc was generated by transfection with a vector containing 5 copies of the NF-κB response element, which drives the transcription of the unstable form of the Nanoluc luciferase fusion protein (JQ513377.1).

[0285] The reporter gene cell pool W352-U937.hPro1.NFkBRE.luc was generated by co-transfection with two vectors. One vector contained the full-length human CD40 gene (NM_001250.5, NP_001241.1), and the other contained 5 copies of the NF-κB response element to drive the transcription of the unstable form of the Nanoluc luciferase fusion protein (JQ513377.1).

[0286] The reporter gene cell line Jurkat-NFAT-CD16.A5 was generated by co-transfection with two vectors: one containing the V158 variant of full-length human FcγRIIIa (NM_000569.7, NP_000560.6), and the other containing an NFAT response element to drive the expression of firefly luciferase (DQ904462).

[0287] Example 2: Generation, screening, and optimization of antibody hybridomas

[0288] 2.1 Immunity

[0289] OmniRat (developed by Open Monoclonal Technology) is a gene carrier that delivers a chimeric human / rat IgH locus (containing 22 human V genes). H All people D and J H The segment is connected to rat C in its native conformation. H Transgenic rats containing the IgL locus (12 Vκ linked to Jκ-Cκ and 16 Vλ linked to Jλ-Cλ) and fully human IgL locus (12 Vκ linked to Jκ-Cκ and 16 Vλ linked to Jλ-Cλ). [2-3] The endogenous Ig locus was silenced using a designed zinc finger nuclease. OmniRat rats can produce antibodies with human idiotypes as efficiently as wild-type rats.

[0290] Will Two 1-week-old OmniRat rats (one male and one female) were immunized with 40 μg of W352-hPro1.ECD.hFc together or alternately with W352-mPro1.ECD.hFc. Immunization was repeated approximately weekly or every two weeks for a total of 182 days.

[0291] 2.2 Serum titer detection

[0292] The titer of anti-human / mouse CD40 antibodies in serum samples was determined by ELISA. Microplates were coated with 100 μl of coating buffer (0.02 M Na₂CO₃ and 0.18 M NaHCO₃, pH 9.2) containing 0.5 μg / mL W352-hPro1.ECD.His or W352-mPro1.ECD.His per well and incubated overnight at 4°C. On the day of assay, diluted rat serum samples (first 1:100, then 3-fold diluted with 1×PBS / 2% BSA) and negative controls were added to plates blocked with 1×PBS / 2% BSA for 1 hour, and the plates were incubated at ambient temperature for 2 hours. After washing three times with 1×PBST (PBS containing 0.05% Tween-20), HRP-labeled goat anti-rat IgG Fc was added and the plates were incubated at ambient temperature for 1 hour. After removing unbound material, TMB (3,3',5,5'-tetramethylbenzidine) substrate was added, and the reaction was terminated with 2M HCl. The absorbance at 450 nm was measured using a microplate spectrophotometer.

[0293] Serum titers of immunized OMT rats are shown in Table 4. Rats #1 were selected for final antigen enhancement after the second blood draw and were euthanized; their lymph nodes were collected and used for fusion.

[0294] Table 4 Serum titers of anti-CD40 antibodies

[0295]

[0296] 2.3 Hybridoma formation

[0297] Lymph nodes were collected from OMT rats under sterile conditions and dissociated into single-cell suspensions. The isolated cells were then mixed with myeloma cells SP2 / 0 at a ratio of 1:1.2. Electrocytogenic fusion was performed using a BTX 2001 electrocytometer according to the manufacturer's instructions. After fusion, the cell suspension from the fusion chamber was immediately transferred to a sterile tube containing more culture medium and incubated at 37°C, 5% CO2 for at least 24 hours. The cell suspension was then mixed and transferred to 96-well plates (1×10⁻⁶). 4 Cells / well. The 96-well plates were incubated at 37°C and 5% CO2, with periodic monitoring. When the clones reached approximately 80% confluence in the wells, 100 μl of supernatant was transferred from the tissue culture plate to the 96-well assay plate for antibody screening.

[0298] 2.4 Antibody screening and subcloning

[0299] The high-throughput screening process included preliminary screening via ELISA (screening for human CD40 binders), confirmatory screening via FACS / ELISA (screening for human and mouse CD40 binders via FACS, screening for cynomolgus monkey CD40 binders via ELISA, and screening for antibodies that block human CD40 / CD40L interaction), and functional screening via NFκB reporter gene assay (screening for antibodies that can activate the CD40 pathway via RGA). Based on binding, competition, and functional activity, 30 hybridoma lines were identified for subcloning.

[0300] Dilute each hybridoma cell line in the logarithmic growth phase to 150-200 cells per 1.5 mL of semi-solid HAT medium. Gently mix the cell suspension on a vortex mixer and then inoculate into the wells of a 6-well plate. When cell clusters grow, pick each visible single colony and transfer it to a 96-well plate containing DMEM medium supplemented with 10% fetal bovine serum. After culturing for 2-3 days, collect the supernatant from the single colonies for purification.

[0301] Following subcloning, a total of 500 single clones were obtained, all of which proceeded to the next round of high-throughput screening. Twenty-six positive clones were selected for purification, sequencing, and further characterization.

[0302] 2.5 Antibody Optimization

[0303] 2.5.1 IgG Conversion

[0304] After sequence analysis and functional screening, candidates were selected for the construction of fully human antibodies.

[0305] The DNA sequences of the variable domains of these candidates were synthesized and cloned into a pCI vector containing human IgG2 Fc. After sequence confirmation, an expression vector containing complete IgG of fully human antibody was used for transient transfection to produce antibodies.

[0306] 2.5.2 Eliminating PTM

[0307] The amino acid “NG” in the light chain CDR1 of candidate W3525-1.9.16-uIgG2K was identified as a high-risk deamidation site. Therefore, antisense mutagenic nucleotides were designed to introduce some mutations into the light chain of “W3525-1.9.16-uIgG2K”.

[0308] Based on a comparison of binding affinity and agonistic activity, W3525-1.9.16-P5-uIgG2K (referred to as the W3525 antibody) was selected from the variants as the final lead antibody.

[0309] Example 3: In vitro characterization of the W3525 antibody

[0310] 3.1 Human CD40 binding assay (FACS)

[0311] Place W352-CHOK1.hPro1.A7(1×10 5 (cells / well), Raji (1×10) 5 (cells / well) or A431 (5×10) 4 Cells (cells / well) were incubated at 4°C for 1 hour with various concentrations of W3525 antibody (serial dilutions of 3-fold from 100 nM to 0.0051 nM, or serial dilutions of 5-fold from 100 nM to 0.00128 nM). After washing with 1×PBS / 1% BSA, Alex647-labeled goat anti-human IgG (1:250) or R-PE-labeled goat anti-human IgG (1:150) as secondary antibodies were added, and the cells were incubated at 4°C in the dark for 1 hour. Anti-human CD40 antibodies BMK4 and BMK5 were used as positive controls. Human IgG1 and IgG2 isotype antibodies were used as isotype controls. Cells were then washed and resuspended in 1×PBS / 1% BSA. MFI of cells was measured by flow cytometry and analyzed by FlowJo. GraphPad Prism was used to plot the logarithm of antibody concentration (X-axis) against MFI (Y-axis). EC50 values ​​were determined using a four-parameter dose-response curve model.

[0312] The bonding results of W3525 on W352-CHOK1.hPro1.A7, Raji, and A431 are respectively... Figure 1 , 2As shown in Figures 3 and 4, W3525 strongly binds to human CD40 on the cell surface with an EC50 of 1.1 nM; strongly binds to Raji cells with an EC50 of 0.36 nM; and strongly binds to A431 cells with an EC50 of 0.16 nM, which is comparable to or better than the reference antibody.

[0313] 3.2 CD40 binding assay in cynomolgus monkeys (FACS)

[0314] 293F cells (2 × 10⁻⁶) transiently transfected with cynomolgus monkey CD40 were used. 5 Cells / well were incubated with W3525 antibody at various concentrations (serial 3-fold dilutions from 200 nM to 0.0102 nM) at 4°C for 1 hour. After washing with 1×PBS / 1% BSA, R-PE-labeled goat anti-human IgG (1:150) was added, and the cells were incubated with the cells in the dark at 4°C for 1 hour. The cells were washed and resuspended, and MFI and EC50 values ​​were determined as described above.

[0315] The binding results of W3525 to cynomolgus CD40 on the cell surface were as follows: Figure 4 As shown in the figure, W3525 strongly binds to cynomolgus monkey CD40 on the cell surface, with an EC50 of 2.4 nM, comparable to the reference antibody.

[0316] 3.3 Ligand Binding Competition Assay (FACS)

[0317] W352-CHOK1.hPro1.A7 cells were injected at a rate of 1×10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 96-well plates. A constant concentration (5 μg / ml) of W352-hPro1L1.ECD.mFc diluted in 1×PBS / 1% BSA was added to the cells, followed by serially diluted antibody (3-fold serial dilutions from 60 nM to 0.0274 nM in 1×PBS / 1% BSA) and mixed thoroughly. The plates were incubated at 4°C for 1 hour.

[0318] The results of the competition between human CD40 and CD40L are as follows: Figure 5 As shown. W3525 can effectively compete with human CD40L to bind CD40, with an IC50 of 1.4 nM and achieving 100% inhibition, while BMK4 can only partially compete with CD40L to bind CD40. Note: %inhibition = (MFI) top -MFI bottom ) / MFI top ×100%.

[0319] 3.4 CD40 homologous protein binding assay (ELISA)

[0320] Pre-coat each well of the microplate with 100 μl of coating buffer (0.02 M Na2CO3 and 0.18 M NaHCO3, pH 9.2) containing 1 μg / ml recombinant human CD40, OX40, 4-1BB, GITR, or BCMA extracellular domain proteins, and incubate overnight at 4°C. The next day, wash the plate once with 1×PBST. Block with 200 μL of 1×PBS / 2% BSA for 1 hour, then wash the plate three times with 1×PBST. Add W3525 to the plate at a concentration of 66.7 nM and incubate at ambient temperature for 1 hour. Anti-CD40 antibodies BMK4 and BMK5, anti-OX40 antibody MEDI0562, anti-4-1BB antibody BMS-663513, anti-GITR antibody INCAGN01876, and anti-BCMA antibody EM801 were used as positive controls. Human IgG2 isotype antibody was used as an isotype control. After incubation, the plate was washed three times with 1×PBST. HRP-labeled goat anti-human IgG antibody diluted in 1×PBS / 2% BSA was added at a ratio of 1:5000, and the plate was incubated for 1 hour. After washing six times with 1×PBST, color development was initiated by adding 100 μl of TMB substrate, and the reaction was stopped by adding 100 μL of 2M HCl. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer. Antibody concentration (X-axis) was plotted against absorbance (Y-axis) using a GraphPad Prism. All samples were tested in duplicate.

[0321] The binding results of W3525 to CD40 homologs of TNFR superfamily members are as follows: Figure 6 As shown, the W3525 antibody can specifically bind to human CD40, but has no cross-reactivity with human OX40, 4-1BB, GITR, and BCMA.

[0322] 3.5 Protein binding of cross-species (ELISA)

[0323] Each well of the microplate was pre-coated with 100 μl of coating buffer containing 1 μg / ml of recombinant human CD40, cynomolgus monkey CD40, mouse CD40, canine CD40, or rat CD40 extracellular domains, and incubated overnight at 4°C. The ELISA procedure was then performed as described above. Anti-human CD40 antibodies BMK4 and BMK5 were used as positive controls. Human IgG1 and IgG2 isotype antibodies were used as isotype controls.

[0324] Comparative binding results of W3525 to human CD40, cynomolgus monkey CD40, mouse CD40, canine CD40 and rat CD40 proteins are as follows: Figure 7 As shown, W3525 showed cross-reactivity with human and cynomolgus monkey CD40, but no cross-reactivity with mouse, rat, and canine CD40, while BMK4 showed cross-reactivity with canine CD40.

[0325] 3.6 Affinity to human CD40 (SPR)

[0326] Surface plasmon resonance (SPR) was used to perform full binding kinetic analysis to quantitatively determine the binding affinity of W3525 for the extracellular domain of recombinant CD40. SPR allows for real-time, label-free detection of biomolecular interactions. SPR occurs when polarized light strikes a conductive surface at the interface between two media. This generates an electron charge density wave called a plasma, which reduces the intensity of the reflected light from a specific angle called the resonance angle, which is proportional to the mass on the sensor surface.

[0327] The affinity of W3525 for human CD40 was determined using a Biacore 8K. An activator was first prepared by mixing 400 mM EDC and 100 mM NHS, and was freshly prepared before use. The CM5 sensor chip was activated with the activator at a flow rate of 10 μL / min for 420 seconds. Then, goat anti-human Fc IgG (30 μg / ml diluted in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 10 μL / min for 420 seconds. The chip was then inactivated by injecting 1 M ethanolamine-HCl at a flow rate of 10 μL / min for 420 seconds. After fixation, antibody W3525 was diluted to 66.7 nM in running buffer (1×HBS-EP+) and captured onto channel Fc4 at a flow rate of 10 μL / min for 30 seconds. Seven concentrations (0, 1.5625, 3.125, 6.25, 12.5, 25, and 50 nM) of the analyte W352-hPro1.ECD.His were sequentially injected into the channel at a flow rate of 30 μL / min, with a binding time of 90 seconds followed by a dissociation time of 300 seconds. After dissociation, a glycine solution (10 mM, pH 1.5) was injected as a regeneration buffer to regenerate the chip.

[0328] The affinity of BMK4 and BMK5 for human CD40 was determined using a Proteon XPR36. First, an activator was prepared by mixing 40 mM EDC and 100 mM sulfonated NHS, freshly prepared for each use. The GLM sensor chip was activated with the activator at a flow rate of 30 μL / min for 300 seconds. Then, goat anti-human Fc IgG (30 μg / ml diluted in 10 mM NaAc, pH 4.5) was injected into the channel at a flow rate of 30 μL / min for 300 seconds. The chip was then inactivated with 1 M ethanolamine-HCl at a flow rate of 30 μL / min for 300 seconds. Antibody BMK5 was diluted to 13.34 nM in running buffer (1×HBS-EP+) and injected into the L4 channel at a flow rate of 30 μL / min for 100 seconds. The chip was rotated 90° and washed with running buffer until baseline stability was achieved. Six concentrations (10, 5, 2.5, 1.25, 0.625, and 0 nM) of analyte W352-hPro1.ECD.His were sequentially injected into channels A1-A6 at a flow rate of 100 μL / min, with a binding time of 120 seconds followed by a dissociation time of 240 seconds. After dissociation, glycine solution (10 mm, pH 1.5) was injected as a regeneration buffer to regenerate the chip. After regeneration, antibody BMK4 was diluted to 13.34 nM in running buffer (1×HBS-EP+) and injected into channel L3 at a flow rate of 30 μL / min for 80 seconds. The chip was rotated 90° and washed with running buffer until the baseline stabilized. Six concentrations (40, 20, 10, 5, 2.5, and 0 nM) of analyte W352-hPro1.ECD.His were sequentially injected into channels A1-A6 at a flow rate of 100 μL / min, with a binding time of 240 seconds followed by a dissociation time of 600 seconds. After each cycle, a glycine solution (10 mm, pH 1.5) was injected as a regeneration buffer to regenerate the chip.

[0329] After subtracting the signals from the reference channel and buffer channel from the signal of the test sample, the experimental data were fitted based on a 1:1 binding model.

[0330] The molar concentration of W352-hPro1.ECD.His was calculated based on its molecular weight of 29 kDa. The kinetic affinity of W3525 for W352-hPro1.ECD.His is shown in Table 5, with an affinity constant of 7.12 nM.

[0331] Table 5 Affinity to human CD40 free protein

[0332]

[0333] 3.7 Affinity (SPR) to cynomolgus monkey CD40

[0334] The affinity of BMK4 and BMK5 for cynomolgus monkey CD40 was determined using a Biacore 8K. The immobilization procedure was the same as described above. Antibodies BMK4 and BMK5 were diluted to 33.35 nM in running buffer (1×HBS-EP+) and captured onto Fc2 of channels 6 and 7, respectively, at a flow rate of 10 μL / min for 30 seconds. Seven concentrations (0, 1.563, 3.125, 6.25, 12.5, 25, and 50 nM) of analyte W352-cynoPro1.ECD.His were sequentially injected into Fc1 and Fc2 of channels at a flow rate of 30 μL / min for 240 seconds of binding followed by a 600-second dissociation period. After dissociation, glycine solution (10 mm, pH 1.5) was injected as a regeneration buffer to regenerate the chip.

[0335] The affinity of W3525 for cynomolgus monkey CD40 was determined using a Biacore T200. The immobilization procedure was identical to that of the Biacore 8K. Antibody W3525 was diluted to 33.35 nM in run buffer (1×HBS-EP+) and captured onto Fc2 at a flow rate of 10 μl / min for 60 seconds. Seven concentrations (0, 1.56, 3.13, 6.25, 12.5, 25, and 50 nM) of analyte W352-cynoPro1.ECD.His were sequentially injected into Fc1 and Fc2 channels at a flow rate of 30 μl / min for 240 seconds of binding followed by a 300-second dissociation time. After each cycle, glycine solution (10 mm, pH 1.5) was injected as a regeneration buffer to regenerate the chip.

[0336] The molar concentration of W352-cPro1.ECD.His was calculated based on its molecular weight of 29 kDa. The kinetic affinity of W3525 for W352-cPro1.ECD.His is shown in Table 6, with an affinity constant of 8.47 nM.

[0337] Table 6. Affinity to CD40 free protein in cynomolgus monkeys

[0338] Antibody ka(1 / Ms) kd(1 / s) KD(nM) BMK4 1.55E+05 1.07E-03 6.94 BMK5 5.68E+05 8.82E-03 15.5 W3525 4.44E+05 3.77E-03 8.47

[0339] 3.8NFκB Reporting Determination

[0340] Two NFκB luciferase reporter gene cell systems, W352-Ramos.NFκBRE.luc and W352-U937.hPro1.NFκBRE.luc, were developed to evaluate the agonist activity of W3525 against the CD40 pathway. Cells were cultured at 4 × 10⁶ cells / year. 4Cells were seeded at a density of 50 μL per well in a 96-well plate. Various concentrations of W3525 (serially diluted 10-fold from 100 nM to 0.00001 nM) were then added to the wells in 50 μL increments. Anti-human CD40 antibodies BMK4 and BMK5 were used as positive controls. Human IgG1 and IgG2 isotype antibodies were used as isotype controls. The plate was incubated at 37°C and 5% CO2 for 5–6 hours. Recombinant luciferase substrate was added to each well (50 μL / well) and thoroughly mixed. Luciferase intensity was read using a microplate spectrophotometer (Envision). A graph of the logarithm of antibody concentration (X-axis) versus fold change in luciferase intensity (Y-axis) was plotted using a GraphPad Prism. EC50 values ​​were determined using a four-parameter dose-response curve model. The experiment was repeated three times, with each sample tested in duplicate.

[0341] To directly confirm CD40 activation, two luciferase reporter cell lines (one based on the B-lymphoma cell line Raji, and the other on the monocytic leukemia cell line U937) were used to quantify the in vitro potency of W3525. Data are as follows: Figure 8 and Figure 9 As shown, the efficacy data are summarized in Table 7. W3525 was able to induce concentration-dependent activation of NFκB and was shown to be more effective than BMK4.

[0342] Table 7 Antibody potency in NFκB reporting assays

[0343]

[0344] 3.9 In vitro B cell proliferation assay

[0345] Use EasySep according to the manufacturer's plan. TM Human CD19 Positive Selection Kit II isolates human B cells from human peripheral blood mononuclear cells (PBMCs) using magnetic selection. Freshly isolated human B cells are then processed at 6 × 10⁻⁶. 4Cells per well were added at a density of 100 μl. Subsequently, antibodies of various concentrations (serially diluted 10-fold from 100 nM to 0.001 nM) were added to the wells at 100 μl volumes. Anti-human CD40 antibodies BMK4 and BMK5 were used as positive controls. Human IgG1 and IgG2 isotype antibodies were used as isotype controls. The plates were incubated at 37°C and 5% CO2 for 5 days, and B cell proliferation levels were measured using CellTiter-Glo according to the manufacturer's instructions. Luciferase intensity was read using a microplate spectrophotometer (M5e). A graph of the logarithm of antibody concentration (X-axis) versus relative optical units (Y-axis) was plotted using a GraphPad Prism. EC50 values ​​were determined using a four-parameter dose-response curve model. The experiment was repeated twice, and all samples were tested in triplicate.

[0346] CD40 agonist antibodies can replace CD40L in acting on activated T cells to promote immunity. CD40-activated B cells enter a proliferative state, which in turn enhances the T cell response. The results of B cell proliferation stimulated by W3525 are as follows: Figure 10 As shown in Table 8, the efficacy data are summarized. W3525 effectively enhances B cell proliferation in a dose-dependent manner, and the effect is more moderate than that of BMK4.

[0347] Table 8. Antibody potency in B cell proliferation assay

[0348]

[0349] 3.10 In vitro DC activation assay

[0350] Human monocytes were isolated from human PBMCs using human CD14 magnetic beads according to the manufacturer's protocol via magnetic selection. The freshly isolated monocytes were adjusted to 2 × 10⁶ cells / mL in complete RPMI-1640 medium. 6 The culture medium was supplemented with 800 U / ml recombinant human GM-CSF and 50 ng / ml IL-4. Cells were cultured for 4 or 5 days to induce dendritic cells. Dendritic cells were then cultured in 96-well plates at a rate of 1 × 10⁶ cells / mL. 5Cells were plated at a density of 100 μL per well. Antibodies of various concentrations (from 30 nM to 0.1235 nM in 3-fold serial dilutions, or from 10 nM to 0.0412 nM in 3-fold serial dilutions) were then added to the wells in 100 μL volumes. Anti-human CD40 antibodies BMK4 and BMK5 were used as positive controls. Human IgG1 and IgG2 isotype antibodies were used as isotype controls. The plates were incubated at 37°C with 5% CO2 for 3 days. The supernatant was collected to measure IL-12p40 by ELISA, and cells were harvested to detect the expression of CD80, CD86, CD83, and / or CD54 by FACS. The experiment was performed three times, with all samples tested in triplicate.

[0351] The following steps were taken to measure human IL-12p40 secretion using the Human IL-12 (p40) ELISA Set kit: Recombinant human IL-12p40 was used as a standard. A series of concentrations of 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.03125 ng / ml were used for the standard curve. Plates were pre-coated with 50 μl of a specific capture antibody for human IL-12p40 (diluted 1:250 in coating buffer), then sealed and incubated overnight at 4°C. The next day, the plates were washed once with 1×PBST. After blocking with 1×PBS / 2% BSA for 1 hour, 50 μL of standard or sample was pipetted into each well and incubated at ambient temperature for 2 hours. After incubation, the plates were washed three times with 1×PBST. After removing unbound material, the working assay (biotinylated detection antibody and streptavidin-HRP, diluted 1:250 in 1×PBS / 2% BSA) was added to the wells and incubated at ambient temperature for 1 hour. After incubation, the plate was washed 6 times with 1×PBST. Color development was initiated by adding 50 μl of TMB substrate solution, and the reaction was stopped by adding 50 μl of 2M HCl. Absorbance was read at 450 nm and 540 nm using a microplate spectrophotometer (M5e). The concentration of IL-12p40 in the supernatant was calculated by back-calculating from the standard curve using Softmax Pro software built into the M5e. GraphPadPrism was used to plot the logarithm of antibody concentration (X-axis) against IL-12p40 concentration (Y-axis). An sigmoid curve was fitted using a four-parameter dose-response model.

[0352] The expression of CD80, CD86, CD83, and CD54 was measured by FACS using the corresponding commercial fluorescent antibodies. Cells were transferred from culture plates to FACS plates and washed once with 1×PBS / 1% BSA. The fluorescent antibodies were diluted 20-fold (for CD83) or 100-fold (for CD80, CD86, and CD54) in 1×PBS / 1% BSA and added to cells at 100 μL / well. The plates were incubated in the dark at 4°C for 1 hour. Cells were then washed and resuspended in 1×PBS / 1% BSA. The MFI of cells was measured by flow cytometry and analyzed by FlowJo. The logarithm of antibody concentration (X-axis) was plotted against MFI (Y-axis) using GraphPad Prism. An sigmoid curve was fitted using a four-parameter dose-response curve model.

[0353] CD40 is expressed on APCs such as monocytes and dendritic cells. Conjugation of CD40 in monocytes and dendritic cells via CD40L or CD40 agonist antibodies leads to the secretion of various cytokines (e.g., IL-12) and the upregulation of activation markers (e.g., CD80, CD86, CD54, and CD83). The results of W3525-induced IL-12p40 secretion are shown in the figure. Figure 11 As shown. The results of CD80, CD86, CD54, and CD83 expression are shown in... Figure 12 , 13 In 14 and 15, the potency data for IL-12p40 release and CD80, CD86, CD54, and CD83 expression are summarized in Table 9. Compared with BMK4, W3525 was able to induce moderate levels of IL-12p40 secretion and upregulation of CD80 and CD86.

[0354] Table 9. Antibody potency in in vitro DC activation assay

[0355]

[0356] 3.11 Antibody-dependent cell-mediated cytotoxicity assay (ADCC)

[0357] To test the ADCC activity of W3525 on CD40-positive hematologic cells, CD40-expressing B cells were used as target cells, and Jurkat-NFAT-CD16.A5 cells were used as effector cells. Human primary B cells were isolated from human PBMCs using human CD19 magnetic beads according to the manufacturer's protocol via magnetic selection. CD40 expression on human primary B cells was tested by FACS. Freshly isolated human B cells were cultured at 4 × 10⁻⁶ cells / cells. 4Cells were seeded at a density of 50 μL per well in 96-well plates. Then, various concentrations of test antibodies (serially diluted 8-fold from 200 nM to 0.0001 nM) were added to the wells in 50 μL volumes. Anti-human CD40 antibodies BMK4 and BMK5 were used as positive controls. Human IgG1 and IgG2 isotype antibodies were used as isotype controls. Next, 50 μL of Jurkat-NFAT-CD16.A5 cells were added to the wells, resulting in an effector cell / target ratio of 2:1. The plates were incubated at 37°C in a 5% CO2 incubator for approximately 5 hours. The bioactivity of the antibodies in ADCC was quantified via luciferase generated by NFAT pathway activation. Luciferase activity in effector cells was quantified using One-Glo and read using a microplate spectrophotometer (Emvision). GraphPad Prism was used to plot the logarithm of antibody concentration (X-axis) relative to the fold change in luciferase intensity (Y-axis). An S-shaped curve was fitted using a four-parameter dose-response model. All samples were tested in duplicate.

[0358] CD40 is expressed not only in tumor cells such as B-cell lymphoma, melanoma, and carcinoma, but also in various normal cell types such as monocytes, dendritic cells (DCs), and B cells. Therefore, we evaluated the ADCC activity of W3525 in human primary B cells. CD40 expression on human B cells was confirmed by FACS. ADCC assay results are shown below. Figure 16 As shown, the results indicate that BMK5 (whose Fc backbone is human IgG1) effectively induces ADCC in a dose-dependent manner on human B cells. However, W3525 and BMK4 (both in human IgG2 form) did not mediate or weakly mediated ADCC activity on human B cells. This suggests that W3525 is unlikely to trigger ADCC on CD40-positive B cells.

[0359] In cancer treatment, the primary mechanism of CD40 agonist antibodies is to initiate APCs to induce anti-tumor T-cell responses, without requiring CD40 expression on tumor cells. However, IgG1 antibodies induce Fc effector function, such as ADCC on CD40-expressing dendritic cells (DCs) and B cells, which may reduce anti-tumor responses. In fact, a decrease in B-cell counts has been observed in clinical trials. [22-23] W3525 is a human IgG2 anti-CD40 antibody that avoids potential damage to CD40-positive normal cells.

[0360] 3.12 Cytokine Release Assay

[0361] As an agonist antibody, the most common reported adverse event for anti-CD40 antibodies in clinical trials is cytokine release syndrome (CRS). The purpose of the cytokine release assay is to assess the cytokine release profile of W3525 under soluble conditions using human PBMCs in the absence of other stimuli.

[0362] Human PBMCs were freshly isolated from healthy donors or purchased from PBMC suppliers using a Ficoll-Paque PLUS gradient centrifugation apparatus. Centrifugation was performed at 1 × 10⁻⁶ centrifugation rates in each well. 5 Isolated PBMCs were added at a density of 100 μl / well. W3525 and other antibodies were added to the plate at a working concentration of 66.7 nM at 100 μl / well. Anti-CD28 antibody TGN1412 was used as a positive control, and human IgG1 and IgG2 isotype antibodies were used as isotype controls. LPS was added at a working concentration of 1 μg / ml. The plate was incubated at 37°C and 5% CO2 for two days. The supernatant was collected and stored at -80°C until ready for testing.

[0363] After collecting 10 sets of samples, the cytokines IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A were measured using the Cytometric Bead Array (CBA) Human Th1 / Th2 / Th17 Cytokine Kit according to the manufacturer's protocol. This kit contains pre-captured microbeads of seven cytokines (IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A), premixed standards of the seven cytokines, detection antibodies conjugated with PE, and wash buffer. The CBA kit uses microbead array technology to simultaneously detect multiple cytokine proteins in the study samples. During the assay procedure, the cytokine capture beads and recombinant standards or unknown samples are mixed and incubated with detection antibodies conjugated with PE to form sandwich complexes. The PE fluorescence intensity of each sandwich complex reveals the concentration of that cytokine. After acquiring the samples on a flow cytometer, the concentration of each cytokine can be quantified according to a standard curve.

[0364] We investigated whether an in vitro cytokine release assay could predict the ability of W3525 to activate PBMCs without triggering the release of multiple cytokines. An in vitro cytokine release assay was performed to determine the potential harm of cytokine release. The effect of W3525 on cytokine release in human PBMCs was assessed in soluble form. This assay was performed using PBMCs from 10 donors. Anti-CD28 antibody TGN1412 and LPS were used as positive controls, and human IgG1 and IgG2 isotype antibodies were used as isotype controls. Standard curves were plotted on a log-log plot, with cytokine concentration on the X-axis and MFI on the Y-axis. The best-fit standard curve was plotted using GraphPad Prism via standard points. The concentrations of each cytokine are shown in Table 10. The results showed that treatment with W3525 alone did not stimulate the release of large amounts of cytokines IL-2, IL-4, IL-6, IL-10, TNF, IFN-γ, and IL-17A from human PBMCs. However, in clinical trials, BMK4-induced cytokine release syndrome problems resulted in significantly higher levels of cytokines such as IL-2, IL-4, IL-6, and TNF. All values ​​are shown as mean ± standard error.

[0365] Table 10 Cytokine production in in vitro cytokine release assays

[0366]

[0367]

[0368] Example 4: In vivo characterization of W3525 antibody

[0369] 4.1 In vivo efficacy study of antibody in CD40 humanized mouse MC38 colon cancer model

[0370] To investigate the antitumor activity of W3525 against a CD40-negative tumor model, human CD40 transgenic CD40-Hu mice (Shanghai Model Organisms) were used for tumor cell inoculation. Wild-type MC38 tumor cells (1×10⁻⁶) were used. 5 The tumor cells were resuspended in 0.1 mL of DPBS (Dulbecco phosphate-buffered saline) and subcutaneously injected into the right abdomen of CD40-Hu mice to induce tumor formation. When the average tumor volume reached approximately 80 mm², the tumor was targeted. 3 At that time, tumor-bearing animals were randomly divided into six study groups, each consisting of 8 mice. Table 12 shows the study design. All antibodies were administered intraperitoneally to tumor-bearing mice twice a week. Mouse body weight and tumor volume were measured twice a week. Mice were euthanized according to predefined health criteria, and the study was terminated 25 days after the first administration.

[0371] Table 12 Study Design of In Vivo MC38 Model

[0372]

[0373]

[0374] Tumor growth inhibition (TGI) was calculated and analyzed at the optimal treatment time point (21 days after grouping, with the grouping date being the date of the first dose). Tumor volume results are as follows: Figure 17 As shown, and summarized in Tables 13 and 14.

[0375] Table 13 Summary of Tumor Volume

[0376]

[0377] Note: a, mean ± standard error

[0378] Table 14 Tumor Growth Inhibition

[0379]

[0380]

[0381] Remark:

[0382] a, mean ± standard error.

[0383] b. The mean tumor volume of the treatment group compared to the human IgG2 group on day 21 after grouping was statistically analyzed by independent samples t-test.

[0384] c. The mean tumor volume of G5 compared to G2 on day 21 after grouping, statistically analyzed by independent samples t-test.

[0385] d, the mean tumor volume of G6 compared to G3 on day 21 after grouping, statistically analyzed by independent samples t-test.

[0386] Following the fourth injection, animals in the WBP352-BMK4.hIgG4K, 10 mg / kg group experienced a weight loss exceeding 10%, and all mice in this group were found to be lethargic and hypothermic, a condition that persisted for over a week until the end of the experiment. The average body weight results are shown in Table 15 and... Figure 18 middle.

[0387] Table 15 Weight Changes

[0388]

[0389] Note: a, mean ± standard error.

[0390] On day 21 after grouping, the TGI values ​​of W3525 at doses of 10 mg / kg and 1 mg / kg were 101.01% and 51.24%, respectively. The mean tumor volume in these two groups was statistically significantly different from that in the human IgG2 group (P<0.05). The TGI value of W3525 at 0.1 mg / kg was 26.06%, but this was not statistically different from that in the human IgG2 group (p>0.05). These results indicate that W3525 has significant antitumor activity at dose levels of 10 mg / kg and 1 mg / kg, while its tumor growth inhibitory activity is limited at a dose level of 0.1 mg / kg.

[0391] The TGI values ​​of BMK4 at 10 mg / kg and 1 mg / kg were 102.75% and 54.79%, respectively. Meanwhile, the mean tumor volume in these groups was statistically significantly different from that in the human IgG2 group (P<0.05). These results indicate that the BMK4 reference antibody exhibits significant antitumor activity at both 10 mg / kg and 1 mg / kg.

[0392] However, following the fourth dose, over 10% weight loss, lethargy, and hypothermia were observed in the BMK4 10 mg / kg group, and these signs persisted for more than a week, suggesting potential toxicity of the reference antibody at this dose level. In contrast, all dose levels of W3525 were well tolerated in treated mice.

[0393] In summary, W3525 demonstrated significant inhibitory effects on tumor growth at both 1 mg / kg and 10 mg / kg dose levels, and the inhibitory effect was dose-dependent. More importantly, mice in all groups treated with W3525 tolerated the treatment well, while mice in the high-dose BMK4 group showed intolerance to BMK4 treatment. W3525 mediates antitumor activity comparable to BMK4 (i.e., Pfizer's CP-870, 893) but with lower toxicity, demonstrating the possibility and potential therapeutic utility of W3525 in separating agonist effects from toxicity in clinical trials.

[0394] 4.2 In vivo efficacy study of antibody in humanized CD40 mouse B16F10 melanoma model This study aimed to evaluate the antitumor activity of W3525 in a B16F10 melanoma model (typically a "cold tumor" unresponsive to immunotherapy). B16F10 tumor cells (5 × 10⁻⁶) were used. 6 The sample was resuspended in 0.1 mL of DPBS and subcutaneously injected into the right abdomen of human CD40 transgenic CD40-Hu mice to induce tumor formation. The tumor volume was approximately 60-80 mm². 3At that time, tumor-bearing animals were randomly divided into 7 study groups, each consisting of 7 mice. The study design is shown in Table 16. All antibodies were administered to tumor-bearing mice intraperitoneally every three days (Q3d). Mouse body weight and tumor volume were measured every three days. Mice were euthanized according to predefined health criteria, and the study was terminated 24 days after the first administration.

[0395] Table 16 Study Design of In Vivo B16F10 Model

[0396] Group test product Animal number dose route of administration Dosage frequency G1 DPBS 7 / IP Q3d×6 G2 W3525 7 1mg / kg IP Q3d×6 G3 W3525 7 3mg / kg IP Q3d×6 G4 W3525 7 10mg / kg IP Q3d×6 G5 APX005M 7 1mg / kg IP Q3d×6 G6 APX005M 7 3mg / kg IP Q3d×6 G7 APX005M 7 10mg / kg IP Q3d×6

[0397] Throughout the experiment, tumor growth and body weight were closely monitored in all mice, with tumor size measured and recorded every three days. Tumor growth inhibition (TGI) was calculated and analyzed at the optimal treatment time point (12 days after grouping, with the grouping date being the first administration date). Tumor volume results are as follows: Figure 19 The results are shown and summarized in Tables 17 and 18.

[0398] Table 17 Summary of Tumor Volume

[0399]

[0400] Note: a, mean ± standard error.

[0401] Table 18 Tumor Growth Inhibition

[0402]

[0403] Remark:

[0404] a, mean ± standard error.

[0405] b. Statistical analysis of the mean tumor volume of the treatment group compared to the DPBS group on day 12 after grouping, using an independent samples t-test.

[0406] No significant weight loss was observed in this study. The results for average weight are shown in Table 19 and... Figure 20 middle.

[0407] Table 19 Weight Changes

[0408]

[0409]

[0410] Note: a, mean ± standard error.

[0411] The survival rate of the mice was shown Figure 21 In the study, W3525 prolonged the survival rate of mice inoculated with B16F10 tumors at doses of 3 mg / kg and 10 mg / kg.

[0412] Twelve days after grouping, the TGI values ​​of W3525 at 3 mg / kg and 10 mg / kg were 71.45% and 76.43%, respectively. The mean tumor volume in both groups was statistically significantly different from the DPBS group (P<0.05). The TGI value of W3525 at 1 mg / kg was 25.61%, but there was no statistically significant difference compared to the DPBS group (P>0.05). These results indicate that W3525 has significant antitumor activity at doses of 10 mg / kg and 3 mg / kg, but its tumor growth inhibitory activity is limited at the 1 mg / kg dose level.

[0413] The TGI values ​​of APX005M at 1 mg / kg, 3 mg / kg, and 10 mg / kg were 16.05%, 23.14%, and 32.25%, respectively. However, the mean tumor volume in these groups was not statistically significantly different from that in the DPBS group (P<0.05). These results indicate that the reference antibody has little or weak antitumor activity at these three dose levels.

[0414] The above results indicate that W3525 is more effective than APX005M in the B16F10 tumor model. W3525 not only inhibits tumor growth but also improves the survival rate of tumor-bearing mice.

[0415] Besides CP-870, 893, and CDX-1140, other CD40 agonist antibodies are in human IgG1 form (wild-type or with modified Fc to enhance specific FcγR binding) and typically require FCγR cross-linking to achieve their agonist activity. CP-870 and 893 are fully human IgG2 isotype antibodies with potent agonist activity and have shown promising therapeutic effects in early clinical trials. [15-16] However, CP-870,893 has a limited therapeutic window, with a maximum tolerated dose of 0.2 mg / kg. Three dose-limiting toxicities were observed, including venous thromboembolism at 0.3 mg / kg, grade 3 headache at 0.3 mg / kg, and transient elevation of serum transaminases at 0.2 mg / kg. The most common adverse events associated with CP-870,893 treatment were cytokine release syndromes (grades 1 and 2), which included chills, shivering, and fever (NCT02225002). Although intratumoral injection of CP-870,893 can modulate the reach of the drug to tumors (NCT02665416), this route of administration largely limits its clinical application. Conversely, CDX-1140 (another CD40 agonist antibody in the form of human IgG2) has a low affinity for human CD40, despite a favorable safety profile in cynomolgus monkeys. Furthermore, its agonist activity in activating dendritic cells (DCs) is limited. ADC-1013 also showed a favorable safety profile, but its therapeutic efficacy in clinical studies as a single agent was limited (NCT02379741). APX-005M is a human IgG1 antibody with enhanced CD32b binding; although promising efficacy was observed in a phase 2 clinical trial in patients with untreated metastatic pancreatic adenocarcinoma, its safety remains a significant concern.

[0416] W3525-1.9.16-P5-uIgG2K is a fully human agonist antibody possessing the wild-type human IgG2 constant region. This antibody blocks the binding of CD40L to CD40, while CP-870,893 failed to prevent the binding of CD40L to CD40. It has been shown that CD40L-blocking antibodies tend to have more potent CD40 agonist activity than CD40L-non-blocking antibodies.

[21] .

[0417] Those skilled in the art will further recognize that the invention can be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the invention discloses only exemplary embodiments thereunder, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims to indicate the scope and content of the invention.

[0418] References

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[0442]

[24] An HJ, Kim YJ, Song DH, et al. Crystalographic and mutational analysis of the CD40-CD154 complex and its implications for receptor activation[J]. J Biol Chem. 2011, 286(13):11226-11235. sequence list <110> InnoLake Pharmaceutical (Hangzhou) Co., Ltd. <120> CD40 agonist antibodies and their usage <130> IEC206026PCT <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Artificial sequence <220> <223> HCDR1 <400> 1 Gly Asp Thr Val Ser Ser Tyr Gly Ile Ser 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial sequence <220> <223> HCDR2 <400> 2 Gly Ser Ile Pro Phe Phe Gly Thr Pro Asn Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210> 3 <211> 17 <212> PRT <213> Artificial sequence <220> <223> HCDR3 <400> 3 Ala Arg Arg Gly Tyr Ser Tyr Gly His Tyr His Tyr Tyr Ala Met Asp 1 5 10 15 Val <210> 4 <211> 16 <212> PRT <213> Artificial sequence <220> <223> LCDR1,p5 <400> 4 Arg Ser Gly Gln Ser Leu Leu His Asn Asn Ala Tyr Asn Tyr Leu Asp 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Artificial sequence <220> <223> LCDR2 <400> 5 Leu Gly Ser Asn Arg Ala Ser 1 5 <210> 6 <211> 9 <212> PRT <213> artificial sequence <220> <223> LCDR3 <400> 6 Met Gln Ala Leu Gln Thr Pro Leu Ser 1 5 <210> 7 <211> 16 <212> PRT <213> artificial sequence <220> <223> LCDR1 <400> 7 Arg Ser Gly Gln Ser Leu Leu His Asn Asn Gly Tyr Asn Tyr Leu Asp 1 5 10 15 <210> 8 <211> 126 <212> PRT <213> artificial sequence <220> <223> VH <400> 8 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Thr Val Ser Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ser Ile Pro Phe Phe Gly Thr Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Phe Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Arg Arg Gly Tyr Ser Tyr Gly His Tyr His Tyr Tyr Ala 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ala Ser 115 120 125 <210> 9 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> VL, p5 <(400)> 9 Asp Thr Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Gly Gln Ser Leu Leu His Asn 20 25 30 Asn Ala Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Note: For the tag <(400)>, it's not clear if it's a correct tag format in the original. If it's a misprint and should be something else, this translation might need adjustment accordingly. Also, the "p5" in <223> VL, p5 is left as is without further context for a more accurate translation of what it might represent. 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Leu Ser Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 10 <211> 112 <212> PRT <213> Synthetic sequence <220> <223> VL, parental antibody <400> 10 Asp Thr Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Gly Gln Ser Leu Leu His Asn 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 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 Met Gln Ala 85 90 95 Leu Gln Thr Pro Leu Ser Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 11 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> VH <400> 11 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60 tcctgcaagg cttctggaga caccgtcagc agctatggta tcagctgggt gcgacaggcc 120 cctggacaag ggcttgagtg gatgggaggg agcatccctt tctttggtac accaaactac 180 gcacagaagt tccagggcag agtcacgttt accgcggacg aatccacgag cacagcctac 240 atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagcgaga 300 cgtggataca gctatggtca ctaccactac tacgctatgg acgtctgggg ccaagggacc 360 acggtcaccg tcgcctca 378 <210> 12 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> VL, p5 <400> 12 gatactgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctggtca gagcctcctg cataataatg cgtacaacta tttggattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactcct 300 ctcagtttcg gcggagggac caaggtggag atcaaa 336 <210> 13 <211> 336 <212> DNA <213> Artificial sequence <220> <223> VL, parental antibody <400> 13 gatactgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 atctcctgca ggtctggtca gagcctcctg cataataatg gatacaacta tttggattgg 120 tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc 180 tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240 agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct acaaactcct 300 ctcagtttcg gcggagggac caaggtggag atcaaa 336 <210> 14 <211> 452 <212> PRT <213> Artificial sequence <220> <223> Heavy chain, p5 <400> 14 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Thr Val Ser Ser Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ser Ile Pro Phe Phe Gly Thr Pro Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Phe Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Arg Arg Gly Tyr Ser Tyr Gly His Tyr His Tyr Tyr Ala 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ala Ser Ala Ser 115 120 125 Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 130 135 140 Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165 170 175 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 180 185 190 Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr 195 200 205 Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val 210 215 220 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val 225 230 235 240 Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300 Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Pro Gly Lys 450 <210> 15 <211> 219 <212> PRT <213> Artificial sequence <220> <223> p5, light chain <400> 15 Asp Thr Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 2]1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Gly Gln Ser Leu Leu His Asn 20 25 30 Asn Ala Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala 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 Met Gln Ala 85 90 95 Leu Gln Thr Pro Leu Ser Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 +125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215

Claims

1. An antibody or antigen-binding moiety thereof isolated from CD40, wherein the isolated antibody or antigen-binding moiety thereof comprises: HCDR1 as shown in SEQ ID NO:1; HCDR2 as shown in SEQ ID NO:2; HCDR3 as shown in SEQ ID NO:3; LCDR1 as shown in SEQ ID NO:4 or 7; LCDR2 as shown in SEQ ID NO:5; and LCDR3 as shown in SEQ ID NO:

6.

2. The isolated antibody or its antigen-binding portion according to claim 1, wherein the isolated antibody or its antigen-binding portion comprises: (a) Heavy chain variable region (VH); (i) Its amino acid sequence is shown in SEQ ID NO:8; (ii) Contains an amino acid sequence having at least 85%, 90%, or 95% identity with the amino acid sequence of SEQ ID NO:8; or (iii) An amino acid sequence comprising one or more added, deleted, and / or substituted amino acids compared to the amino acid sequence of SEQ ID NO:8; and / or (b) Variable region (VL) of light chain; (i) Its amino acid sequence is shown in SEQ ID NO:9 or 10; (ii) Contains an amino acid sequence having at least 85%, at least 90%, or at least 95% identity with the amino acid sequence of SEQ ID NO: 9 or 10; or (iii) An amino acid sequence comprising one or more added, deleted and / or substituted amino acids compared to the amino acid sequence of SEQ ID NO:9 or 10.

3. The isolated antibody or its antigen-binding portion according to claim 2, wherein the isolated antibody or its antigen-binding portion comprises: The heavy chain variable region of the amino acid sequence shown in SEQ ID NO:8 and the light chain variable region of the amino acid sequence shown in SEQ ID NO:9 or 10.

4. The isolated antibody or its antigen-binding portion thereof of any of the preceding claims, wherein the isolated antibody further comprises a human IgG constant domain.

5. The isolated antibody or its antigen-binding portion of claim 4, wherein the human IgG constant domain is a human IgG1 or IgG2 constant domain.

6. The isolated antibody or its antigen-binding portion of claim 5, wherein the human IgG constant domain is the human IgG2 constant domain.

7. The isolated antibody or its antigen-binding portion according to claim 1, wherein the antibody is a chimeric antibody, a humanized antibody, or a fully human antibody.

8. The isolated antibody or its antigen-binding portion of claim 7, wherein the antibody is a fully human monoclonal antibody.

9. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a heavy chain variable region of an isolated antibody as defined in any one of claims 1-8 and a nucleic acid sequence encoding a light chain variable region.

10. A vector comprising the nucleic acid molecule of claim 9.

11. A host cell comprising the vector of claim 10.

12. A pharmaceutical composition comprising at least one antibody or antigen-binding portion thereof as defined in any one of claims 1-8 and a pharmaceutically acceptable carrier.

13. A method for preparing an antibody or its antigen-binding portion as defined in any one of claims 1-8, comprising the following steps: - Culture host cells containing one or more expression vectors encoding the antibody or a portion thereof under suitable conditions; and - Isolate the antibody or its antigen-binding portion from the cell culture.

14. Use of the antibody or its antigen-binding portion as defined in any one of claims 1-8 in the preparation of a medicament for treating or preventing cancer, wherein the cancer is selected from breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, bladder cancer, renal cell carcinoma, liver cancer, prostate cancer, gastric cancer, pancreatic cancer, chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and multiple myeloma.

15. The use of claim 14, wherein the cancer is colon cancer or melanoma.

16. A kit comprising a container containing at least one antibody or antigen-binding portion thereof as defined in any one of claims 1-8.

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