Anti-cd40 antibodies or antigen-binding fragments and uses thereof

Abstract

The present application provides an anti-CD40 antibody or antigen binding fragment, which specifically binds to CD40, can activate antigen presenting cells, stimulate antigen presenting cells to release cytokines, induce tumor cell apoptosis, and inhibit tumor cell proliferation. The application also provides use of the above-mentioned antibody or antigen binding fragment in the preparation of a medicament for treating or improving CD40-related diseases, or in the preparation of a kit for diagnosing CD40-related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and particularly relates to anti-CD40 antibodies or antigen-binding fragments and their applications. Background Technology

[0002] The CD40 receptor and its ligand CD40L (CD154) belong to the TNFR:TNF family. The CD40-CD40L signaling pathway in B cells influences the production and survival of long-lived plasma cells and memory B cells. CD40 is a co-stimulatory receptor expressed on antigen-presenting cells, such as dendritic cells (DCs), B cells, monocytes, and some other non-lymphocytes. The CD40-CD40L interaction (an important signaling pathway) plays a crucial role in regulating the interaction between DCs and T cells (CD4+ T cells and CD8+ T cells), rescuing exhausted CTLs, and reversing DC tolerance. Furthermore, the CD40-CD40L interaction between B cells and T cells affects germinal center responses and induces antibody isotype switching. The interaction between CD40 expressed on T cells and CD154 expressed on APCs leads to DC maturation, increased cytokine production and co-stimulatory factor expression, and enhanced effector T cell differentiation.

[0003] CD40 is a type I transmembrane glycoprotein, while CD40L is a type II transmembrane glycoprotein. Like other members of the TNFR superfamily, CD40 forms a trimer. Although CD40 is considered the primary receptor for CD40L, CD40L can also bind to other receptors in the integrin family, such as αIIbβ3, α5β1 (VLA-5), and αMβ2 (Mac-1).

[0004] CD40 agonist antibodies can not only activate and stimulate innate immunity but also act on adaptive immunity, and can also exert direct cytotoxicity on malignant tumor cells expressing CD40. Therefore, CD40 is a promising therapeutic target for tumor immunotherapy. Summary of the Invention

[0005] This invention provides antibodies or antigen-binding fragments that specifically bind to CD40. In some embodiments, this invention provides antibodies or antigen-binding fragments that specifically bind to human CD40 or monkey CD40. Upon specific binding to CD40, the anti-CD40 antibody of this invention can activate and stimulate innate immunity, producing direct cytotoxicity against malignant tumor cells expressing CD40.

[0006] In some embodiments, the present invention discloses an antibody or antigen-binding fragment that specifically binds to CD40. The antibody or antigen-binding fragment comprises one or more of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 is D, F, G, or Y; X2 is I, N, S, or T; X3 is F; X4 is A, D, F, G, T, S, or Y; X5 is D, G, N, S, or T; X6 is A, F, N, S, or Y; and X7 is A, S, or Y. Furthermore, X1-X2-X3-X4-X5-X6-X7 contains at least three distinct amino acids. CDR2 contains the amino acid sequence Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10, where Z1 is A, G, or S; Z2 is I; Z3 is D, I, K, or S; Z4 is A, G, P, Q, S, T, W, or Y; Z5 is D, G, H, S, or T; Z6 is A, F, G, K, S, T, or W; Z7 is A, D, G, or S; Z8 is D, G, N, S, or T; Z9 is A, H, K, N, P, R, S, or T; and Z10 is A, F, G, H, S, or Y. Furthermore, Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10 contains at least five distinct amino acids. VH CDR3 contains the amino acid sequence shown in SEQ ID NO:79.

[0007] In some embodiments, the present invention discloses an antibody or antigen-binding fragment that specifically binds to CD40, the antibody or antigen-binding fragment comprising one or more of VH CDR1, VH CDR2, and VH CDR3, wherein VH CDR1 comprises the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 is D, F, G, or Y; X2 is I, N, S, or T; X3 is F; X4 is A, D, F, G, T, S, or Y; X5 is D, G, N, S, or T; X6 is A, F, N, S, or Y; and X7 is A, S, or Y, and X1-X2-X3-X4-X5-X6-X7 comprises at least three different amino acids; VH CDR2 comprises the amino acid sequence shown in SEQ ID NO:70 or 74; and VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:79.

[0008] In some implementations, X1 is F, X4 is D or G, X5 is S or T, and X6 is F, N, or Y.

[0009] In some implementations, Z1 is G, Z3 is D or S, Z4 is S or W, Z5 is D or G, Z6 is G or T, Z7 is D or G, Z8 is N, S or T, Z9 is A or P, and Z10 is F, H or S.

[0010] In some embodiments, the VH CDR1 comprises an amino acid sequence as shown in any of SEQ ID NO:4-47.

[0011] In some embodiments, the VH CDR2 comprises an amino acid sequence as shown in any of SEQ ID NO:48-78.

[0012] In some embodiments, the antibody or antigen-binding fragment further comprises one or more of VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:1, VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

[0013] In some embodiments, the antibody or antigen-binding fragment comprises VH CDR1, VH CDR2, VH CDR3, VLCDR1, VL CDR2, and VL CDR3.

[0014] In some embodiments, the antibody or antigen-binding fragment specifically binds to CD40, the antibody or antigen-binding fragment comprising one or more of VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3, wherein VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:1, VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:2, VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:3, VH CDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4-47, VH CDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:48-78, and VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:79.

[0015] In some embodiments, the antibody or antigen-binding fragment comprises VL CDR1, VL CDR2, VL CDR3, VHCDR1, VH CDR2, and VH CDR3, wherein VL CDR1 comprises the amino acid sequence shown in SEQ ID NO:1, VL CDR2 comprises the amino acid sequence shown in SEQ ID NO:2, VL CDR3 comprises the amino acid sequence shown in SEQ ID NO:3, VH CDR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4-47, VH CDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:48-78, and VH CDR3 comprises the amino acid sequence shown in SEQ ID NO:79.

[0016] In some embodiments, the VH CDR1 comprises an amino acid sequence as shown in any one of SEQ ID NO:4-9, and the VH CDR2 comprises an amino acid sequence as shown in any one of SEQ ID NO:48-50.

[0017] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain variable region comprising an amino acid sequence shown in any one of SEQ ID NO:81-126, or an amino acid sequence having at least 90% sequence homology with an amino acid sequence shown in any one of SEQ ID NO:81-126.

[0018] In some embodiments, the antibody or antigen-binding fragment includes a heavy chain variable region comprising an amino acid sequence shown in any one of SEQ ID NO:81-86, or an amino acid sequence having at least 90% sequence homology with an amino acid sequence shown in any one of SEQ ID NO:81-86.

[0019] In some embodiments, the antibody or antigen-binding fragment includes a light chain variable region comprising the amino acid sequence shown in SEQ ID NO:80, or an amino acid sequence having at least 90% sequence homology with the amino acid sequence shown in SEQ ID NO:80.

[0020] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region as shown in any one of SEQ ID NO:81-126 and / or a light chain variable region as shown in SEQ ID NO:80.

[0021] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region as shown in any one of SEQ ID NO:81-86 and / or a light chain variable region as shown in SEQ ID NO:80.

[0022] In some embodiments, the antibody or antigen-binding fragment is an isotype of IgG, IgM, IgA, IgE, or IgD.

[0023] In some embodiments, the antibody or antigen-binding fragment includes a light chain constant region as shown in SEQ ID NO:127, and / or the antibody or antigen-binding fragment includes a heavy chain constant region as shown in SEQ ID NO:128.

[0024] In some implementations, the antibody or antigen-binding fragment is a monoclonal antibody.

[0025] In some implementations, the antibody or antigen-binding fragment is a humanized antibody or a fully human antibody.

[0026] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region as shown in any one of SEQ ID NO:81-86 and a heavy chain constant region as shown in SEQ ID NO:128; the light chain comprising a light chain variable region as shown in SEQ ID NO:80 and a light chain constant region as shown in SEQ ID NO:127.

[0027] In some embodiments, the antibody or antigen-binding fragment comprises a light chain with an amino acid sequence as shown in SEQ ID NO:129, and / or the antibody or antigen-binding fragment comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:130.

[0028] In some embodiments, the affinity of the antibody or antigen-binding fragment to CD40 is KD ≤ 20 nM.

[0029] In some embodiments, the antibody or antigen-binding fragment is a separated antibody or antigen-binding fragment.

[0030] On the other hand, the present invention also provides a nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment. In some embodiments, the nucleic acid molecule is an isolated nucleic acid molecule.

[0031] On the other hand, the present invention also provides a vector or host cell comprising the above-mentioned nucleic acid molecules. In some embodiments, the vector or host cell is an isolated vector or host cell.

[0032] On the other hand, the present invention also provides a composition comprising the above-described antibody or antigen-binding fragment and a pharmaceutically acceptable carrier.

[0033] On the other hand, the present invention also provides the use of the above-mentioned antibody or antigen-binding fragment in the preparation of a medicament for treating or improving CD40-related diseases, or in the preparation of a kit for diagnosing CD40-related diseases.

[0034] On the other hand, the present invention also provides a kit comprising the antibody or antigen-binding fragment.

[0035] In some implementations, the CD40-related diseases include cancer.

[0036] On the other hand, the present invention also provides a method for treating or improving CD40-related diseases in patients in need, the method comprising administering an effective dose of the aforementioned antibody or antigen-binding fragment to the patient.

[0037] In some implementations, the method further includes administering one or more therapeutic agents to the patient.

[0038] In some embodiments, the therapeutic agent includes paclitaxel or a salt thereof, and / or carboplatin or a salt thereof.

[0039] The antibody or antigen-binding fragment provided by this invention can specifically recognize and bind to CD40, blocking the binding of CD40 to CD40L. This antibody or antigen-binding fragment is a CD40 agonist, capable of activating antigen-presenting cells, stimulating the release of cytokines from antigen-presenting cells, inducing tumor cell apoptosis, inhibiting tumor cell proliferation, and inducing the killing of tumor cells through antibody-dependent cytotoxicity, complement-dependent cytotoxicity, and / or antibody-dependent phagocytosis. Attached Figure Description

[0040] Figure 1 The results show the binding of anti-CD40 antibody to Raji cells.

[0041] Figure 2 shows the binding results of anti-CD40 antibody to cCD40-His (cynomolgus monkey CD40) and mCD40-his (mouse CD40), respectively; where Figure 2A The binding result with cCD40-His, Figure 2B The result is for binding with mCD40-His.

[0042] Figure 3 shows the in vitro activation ability of anti-CD40 antibody in the NFκB reporter system; Figure 3A , Figure 3B The results show the in vitro activation of anti-CD40 antibodies that are not crosslinked with Anti-hIgG1-Fc. Figure 3C , Figure 3D The results show the in vitro activation of the anti-CD40 antibody conjugated with Anti-hIgG1-Fc (crosslink).

[0043] Figure 4 shows the effect of anti-CD40 antibody on promoting IFN-γ secretion in MLR experiments. Detailed Implementation

[0044] definition

[0045] Unless otherwise defined, the scientific and technical terms used in this invention have the meanings commonly understood by those skilled in the art. Generally, the nomenclature and techniques used in cell culture, molecular biology, and protein purification described herein are well-known and commonly used in the art. Standard techniques were used for recombinant DNA, oligonucleotide synthesis, and cell culture and transformation (e.g., electroporation, lipid transfection). Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions or those commonly used in the art or described herein. The foregoing techniques and methods are generally used as described in several comprehensive and more specific documents well-known in the art and cited and discussed in this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989)).

[0046] As used herein, the terms “comprising” or “including” mean that compositions and methods include the listed elements, such as components or steps, but do not exclude others. “Substantially composed of” means that compositions and methods exclude other elements that have a fundamental effect on the characterization of the composition, but do not exclude elements that do not substantially affect the composition or method. “Composed of” means excluding elements not specifically listed.

[0047] Where a term used and / or accepted in this field has two or more definitions, the definition of the term used herein includes all of those meanings unless explicitly stated otherwise.

[0048] It should be noted that the term “a” entity refers to one or more of the same entity. For example, “an antibody” should be understood as one or more antibodies. Therefore, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably in this document.

[0049] In this invention, the term "polypeptide" is intended to cover both the singular and plural forms of "polypeptide," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any single or multiple chains of two or more amino acids, and does not imply a specific length of the product. Therefore, the definition of "polypeptide" includes peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to two or more amino acid chains, and the term "polypeptide" can be used in place of any of the foregoing terms, or used interchangeably with any of the foregoing terms. The term "polypeptide" is also intended to refer to products modified after polypeptide expression, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or non-naturally occurring amino acid modifications. Polypeptides can be derived from natural biological sources or produced through recombinant technologies, but they do not necessarily have to be translated from a specified nucleic acid sequence. They can be produced by any means, including chemical synthesis.

[0050] An amino acid is a compound containing both amino and carboxyl functional groups, such as α-amino acids. Two or more amino acids can form a polypeptide through an amide bond (also called a peptide bond). A single amino acid is encoded by a nucleic acid consisting of three nucleotides (so-called a codon or base triplet). Each amino acid is encoded by at least one codon. The fact that the same amino acid is encoded by different codons is called "degeneracy of the genetic code." Amino acids include both natural and non-natural amino acids. Natural amino acids include alanine (three-letter code: Ala, one-letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0051] "Conservative amino acid substitution" refers to the replacement of one amino acid residue with another amino acid residue containing a side chain (R group) with similar chemical properties (such as charge or hydrophobicity). Generally, conservative amino acid substitution does not substantially change the functional properties of a protein. Examples of amino acid classes containing chemically similar side chains include: 1) Aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) Aliphatic hydroxyl side chains: serine and threonine; 3) Amide-containing side chains: asparagine and glutamine; 4) Aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) Basic side chains: lysine, arginine, and histidine; 6) Acidic side chains: aspartic acid and glutamic acid.

[0052] The term "isolated" as used in this invention with respect to cells, nucleic acids, peptides, antibodies, etc., such as "isolated" DNA, RNA, peptides, and antibodies, refers to molecules isolated from one or more other components such as DNA or RNA, respectively, in the cellular natural environment. The term "isolated" as used in this invention also refers to nucleic acids or peptides that, when produced by recombinant DNA technology, are substantially free of cellular material, viral material, or cell culture medium, or chemical precursors or other chemicals used in chemical synthesis. Furthermore, "isolated nucleic acids" is intended to include nucleic acid fragments that are not present in their natural state and will not be present in their natural state. The term "isolated" is also used in this invention to refer to cells or peptides isolated from other cellular proteins or tissues. Isolated peptides are intended to include purified and recombinant peptides. Isolated peptides, antibodies, etc., are typically prepared by at least one purification step. In some embodiments, the purity of the isolated nucleic acids, peptides, antibodies, etc., is at least about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or a range between any two of these values ​​(including the endpoint) or any value therein.

[0053] In this invention, the term "recombination" refers to polypeptides or polynucleotides, meaning forms of polypeptides or polynucleotides that do not exist naturally, which can be combined to produce polynucleotides or polypeptides that do not normally exist.

[0054] "Homology," "identity," or "similarity" refers to the sequence similarity between two polypeptides or two nucleic acid molecules. Homology is determined by comparing comparable positions in each sequence. Molecules are homologous at those positions when the positions in the compared sequences are occupied by the same bases or amino acids. The degree of homology between sequences is a function of the number of shared matching or homologous positions.

[0055] A sequence identity or sequence homology of a polynucleotide or polynucleotide region (or polypeptide or polypeptide region) with another sequence having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) means that, when sequence alignment is performed, that percentage of bases (or amino acids) are identical in the two sequences being compared. This alignment and homology percentage or sequence identity can be determined using software programs known in the art, such as those described in Ausubel et al. eds. (2007) in Current Protocols in Molecular Biology. In some embodiments, alignment is performed using default parameters. One such alignment program is BLAST using default parameters. Biologically equivalent polynucleotides are polynucleotides having the aforementioned specified percentage of homology and encoding polypeptides with the same or similar biological activities.

[0056] Polynucleotides are specific sequences of four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T), or, when the polynucleotide is RNA, thymine replaced by uracil (U). The "polynucleotide sequence" can be represented by the letters of the polynucleotide molecule. This letter representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications, such as functional genomics and homology searches.

[0057] The terms "polynucleotide" and "oligonucleotide" are used interchangeably to refer to a polymeric form of nucleotides of any length, whether deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Examples include: genes or gene fragments (e.g., probes, primers, ESTs, or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribonucleases, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, and isolated RNA of any sequence. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Structural modifications to nucleotides can be made before or after the assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization. This term also refers to double-stranded and single-stranded molecules. Unless otherwise stated or required, any polynucleotide disclosed herein includes both double-stranded forms and each of two complementary single-stranded forms known or predicted to constitute a double-stranded form.

[0058] When the term “encoding” is applied to polynucleotides, it refers to a polynucleotide called the “encoding” polypeptide that, in its natural state or when manipulated by methods known to those skilled in the art, can be transcribed and / or translated to produce the polypeptide and / or fragments thereof.

[0059] In this invention, "antibody" or "antigen-binding fragment" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody or any antigen-binding fragment thereof or a single chain thereof. Therefore, the term "antibody" includes proteins or peptides that contain a portion or the whole of a biologically active immunoglobulin molecule that binds to an antigen. This includes, but is not limited to, the complementarity-determining region (CDR), heavy chain variable region (VH), light chain variable region (VL), heavy chain constant region (CH), light chain constant region (CL), framework region (FR), or any portion thereof of the heavy chain or light chain or its ligand-binding portion. The CDR region includes the light chain CDR region (VL CDR1-3) and the heavy chain CDR region (VH CDR1-3). The variable region may contain the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0060] Antibody heavy chains are classified into γ, μ, α, δ, and ε types, with some subclasses (e.g., γ1-γ4). The properties of this chain determine the "type" of the antibody, namely IgG, IgM, IgA, IgG, or IgE. Some of these can be further divided into immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, etc. Light chains are classified into κ and λ types. Each heavy chain can bind to either a κ or λ light chain. Generally, when immunoglobulins are produced from hybridomas, B cells, or genetically engineered host cells, their light and heavy chains are linked by covalent bonds, and the "tail" portions of the two heavy chains are linked by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the Y-configuration forked end to the C-terminus at the bottom of each chain. The variable region of the immunoglobulin κ light chain is Vκ; the variable region of the immunoglobulin λ light chain is Vλ. The VL commonly used in this invention is Vκ. While some discussions focus on IgG, all immunoglobulin molecules are within the scope of this invention. Regarding IgG, a standard immunoglobulin molecule comprises two identical light-chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy-chain polypeptides with a molecular weight of approximately 53,000-70,000 Daltons. The VL and VH determine antigen recognition and specificity. Antigen-binding sites on the VL and VH recognize antigenic determinants and bind specifically to the antigen. These sites are defined by three CDRs in each VH and VL (i.e., VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3). The CL and CH (CH1, CH2, or CH3) impart important biological properties such as secretion, transplacental migration, Fc receptor binding, complement binding, etc. Conventionally, the numbering of constant regions increases as they move further away from the antibody's antigen-binding site or N-terminus. The N-terminal region is the variable region, and the C-terminal region is the constant region; the CH3 and CL domains actually contain the carboxyl ends of the heavy chain and the light chain, respectively.

[0061] In this invention, the heavy chain constant region of an antibody can be derived from different immunoglobulin molecules. For example, the heavy chain constant region of an antibody may include a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In some embodiments, the heavy chain constant region may include a hinge region partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In some embodiments, a portion of the heavy chain may include a chimeric hinge region partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0062] In this invention, the term "hinge region" includes a portion of the heavy chain structure connecting the CH1 and CH2 domains. The hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently.

[0063] In this invention, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. Cysteine ​​contains a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by disulfide bonds, and the two heavy chains are linked by two disulfide bonds.

[0064] In this invention, the terms "fragment," "antibody fragment," or "antigen-binding fragment" refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, etc. Regardless of its structure, an antibody fragment binds to the same antigen recognized by the intact antibody. The term "antigen-binding fragment" includes aptamers, mirror isoforms, and bivalent antibodies, and also includes any synthetic or genetically engineered protein that functions as an antibody by forming a complex with a specific antigen.

[0065] "Single-chain variable fragment" or "scFv" refers to a fusion protein of the VH and VL regions of an immunoglobulin. In some respects, these regions are linked to short linker peptides of about 10 to about 25 amino acids. The linker may be enriched with glycine to increase flexibility, and with serine or threonine to increase solubility, and may link the N-terminus of the VH to the C-terminus of the VL, and vice versa. Although the constant region of the protein has been removed and the linker has been introduced, it retains the specificity of the original immunoglobulin. ScFv molecules are generally known in the art, for example, as described in U.S. Patent 5,892,019.

[0066] The antibodies, antigen-binding fragments, variants, or derivatives disclosed in this invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primate-derived, or chimeric antibodies, single-chain antibodies, and epitope-binding fragments.

[0067] As used in this article, the term "epitope" includes any protein-determining region capable of specifically binding to immunoglobulins or fragments thereof, or T-cell receptors. Epitope-determining regions typically consist of chemically active surface groups of a molecule (such as amino acid or sugar side chains) and usually possess specific three-dimensional structural properties and specific charge properties. When the dissociation constant is less than or equal to 1 μM (e.g., less than or equal to 100 nM, less than or equal to 10 nM, or less than or equal to 1 nM), it can be considered an antibody-specifically bound antigen.

[0068] According to the definitions of Kabat and Chothia, a CDR includes overlaps or subsets of amino acid residues when compared with each other. Nevertheless, the application of either definition to refer to a CDR of an antibody or a variant thereof is within the scope of this invention. The exact residue numbering containing a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can typically determine which specific residues a CDR contains based on the amino acid sequence of the variable region of the antibody.

[0069] Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can apply this "Kabat numbering" system to any variable region sequence without relying on experimental data other than the sequence itself. "Kabat numbering" refers to the numbering system proposed by Kabat et al., USDept. of Health and Human Services in "Sequence of Proteins of Immunological Interest" (1983). Antibodies can also use the EU numbering system.

[0070] The antibodies disclosed in this invention can be derived from any animal, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In some embodiments, the variable region may be of cartilaginous fish origin (e.g., from sharks).

[0071] In this invention, the term "chimeric antibody" is considered to refer to any antibody whose variable region is derived from or derived from a first species, while its constant region (which may be whole, partial, or modified in this invention) is derived from a second species. In some embodiments, the variable region is derived from a non-human source (e.g., mouse or primate), while the constant region is human.

[0072] "Specific binding" or "specific to..." generally refers to the formation of a relatively stable complex by which an antibody or antigen-binding fragment binds to a specific antigen through the complementarity of its antigen-binding domain with the epitope. "Specificity" can be expressed as the relative affinity of the antibody or antigen-binding fragment to a specific antigen or epitope. For example, if antibody "A" has a greater relative affinity to the same antigen than antibody "B," antibody "A" can be considered to have higher specificity for that antigen than antibody "B." Specific binding can be described using the equilibrium dissociation constant (KD), with a smaller KD indicating a tighter binding. Methods for determining whether two molecules bind specifically are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and optical interferometry of biofilm layers. Antibodies that "specifically bind" to the CD40 protein include antibodies with an equilibrium dissociation constant KD of less than or equal to about 100 nM, less than or equal to about 20 nM, less than or equal to about 10 nM, less than or equal to about 5 nM, less than or equal to about 3 nM, or less than or equal to about 1 nM.

[0073] "Treatment" refers to therapeutic treatments and preventative or preventative measures aimed at preventing, mitigating, improving, or stopping adverse physiological changes or disorders, such as disease progression, including but not limited to the following, whether detectable or undetectable: symptom relief, reduction in disease severity, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, mitigation, reduction, or disappearance of the disease state (whether partial or complete), and prolongation of expected survival without treatment. Patients requiring treatment include those already suffering from the condition or disorder, those susceptible to the condition or disorder, or those needing prevention of the condition or disorder, as well as those who can or are expected to benefit from the application of the antibody or pharmaceutical composition disclosed in this invention for detection, diagnostic procedures, and / or treatment.

[0074] "Patient" generally refers to any patient who requires diagnosis, prognosis, or treatment, especially mammalian patients. Mammal patients include humans, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, dairy cows, etc., with humans being the most prominent.

[0075] In this invention, terms such as "patients in need of treatment" include patients who benefit from the use of antibodies or compositions disclosed in this invention for detection, diagnostic procedures, prevention and / or treatment, such as mammalian patients.

[0076] Unless otherwise stated, the term "CD40" refers to any CD40 from any vertebrate source, including mammals such as primates (e.g., humans, rhesus monkeys) and rodents (e.g., mice and rats). The term encompasses "full-length," unprocessed CD40, and any form of CD40 resulting from cellular processing.

[0077] The term "cytokine" is a general term for proteins released by one cell population that act as intercellular mediators to another cell. Examples of such cytokines include lymphokines, monokines, interleukins (ILs) (such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, and IL-15), tumor necrosis factors (such as TNF-α or TNF-β), and other polypeptide factors (including LIF and kit ligand (KL) and gamma interferon). As used herein, the term cytokine includes biologically active equivalents of proteins from natural sources or recombinant cell cultures and of naturally occurring sequence cytokines. Biologically active equivalents include small molecule entities produced through artificial synthesis, and their pharmaceutically acceptable derivatives and salts.

[0078] As used herein, the terms "labeled" or "labeled" refer to polypeptides incorporating a detectable label, for example, by incorporating a radiolabeled amino acid, or by attaching a biotinylated moiety to a labelable avidin (e.g., streptavidin containing a fluorescent label or enzyme-active form detectable by optical or calorimetric methods). In some cases, the label or marker may also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., radioactive isotopes or radionuclides). 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 111 In、 125 I, 131 I) Fluorescent markers (e.g., FITC, Rhodamine, lanthanide phosphors), enzyme markers (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, and predetermined polypeptide epitopes recognized by secondary reporter genes (e.g., leucine zipper pairs, secondary antibody binding sites, metal-binding domains, epitope tags).

[0079] Anti-CD40 antibody

[0080] The antibodies of the present invention have the ability to bind CD40. In some embodiments, the antibodies of the present invention have the ability to bind human CD40.

[0081] In some embodiments, the anti-CD40 antibody or antigen-binding fragment of the present invention comprises a light chain variable region (VL), wherein the VL comprises complementarity-determining regions (CDRs) VL CDR1, VL CDR2, and VL CDR3, wherein VL CDR1 comprises or is composed of an amino acid sequence having at least 50%, 60%, 70%, 80%, or 90% or 100% identity with an amino acid sequence selected from SEQ ID NO:1 (QGISSY), VL CDR2 comprises or is composed of an amino acid sequence having at least 50%, 60%, 70%, 80%, or 90% or 100% identity with an amino acid sequence selected from SEQ ID NO:2 (AASS), and VL CDR3 comprises or is composed of an amino acid sequence having at least 50%, 60%, 70%, 80%, or 90% or 100% identity with an amino acid sequence selected from SEQ ID NO:3 (QQHYTTPP).

[0082] In some embodiments, the antibody or antigen-binding fragment of the present invention comprises one or more of VH CDR1, VH CDR2, and VHCDR3, wherein VH CDR1 comprises the amino acid sequence X1-X2-X3-X4-X5-X6-X7, where X1 is D, F, G, or Y; X2 is I, N, S, or T; X3 is F; X4 is A, D, F, G, T, S, or Y; X5 is D, G, N, S, or T; X6 is A, F, N, S, or Y; and X7 is A, S, or Y, and X1-X2-X3-X4-X5-X6-X7 comprises at least three different amino acids, VH CDR2 contains the amino acid sequence Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10, where Z1 is A, G, or S; Z2 is I; Z3 is D, I, K, or S; Z4 is A, G, P, Q, S, T, W, or Y; Z5 is D, G, H, S, or T; Z6 is A, F, G, K, S, T, or W; Z7 is A, D, G, or S; Z8 is D, G, N, S, or T; Z9 is A, H, K, N, P, R, S, or T; and Z10 is A, F, G, H, S, or Y. Furthermore, Z1-Z2-Z3-Z4-Z5-Z6-Z7-Z8-Z9-Z10 contains at least five distinct amino acids. VH CDR3 contains the amino acid sequence shown in SEQ ID NO:79. In some embodiments, the antibody or antigen-binding fragment of the present invention has X1 as F, X4 as D or G, X5 as S or T, and X6 as F, N, or Y. In some embodiments, the antibody or antigen-binding fragment of the present invention has Z1 as G, Z3 as D or S, Z4 as S or W, Z5 as D or G, Z6 as G or T, Z7 as D or G, Z8 as N, S, or T, Z9 as A or P, and Z10 as F, H, or S.

[0083] In some embodiments, the anti-CD40 antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region (VH), wherein said VH comprises complementarity-determining regions VH CDR1, VH CDR2, and VH CDR3. VH CDR1 comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 80%, or 90% or 100% identity with, or is composed of, an amino acid sequence selected from SEQ ID NO:4-47; VH CDR2 comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 80%, or 90% or 100% identity with, or is composed of, an amino acid sequence selected from SEQ ID NO:48-78; and VH CDR3 comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 80%, or 90% or 100% identity with, or is composed of, an amino acid sequence selected from SEQ ID NO:79.

[0084] In some embodiments, the anti-CD40 antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region (VH), wherein the VH comprises complementarity-determining regions VH CDR1, VH CDR2, and VH CDR3. VH CDR1 comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 80%, or 90% or 100% identity with an amino acid sequence selected from SEQ ID NO:4-9; VH CDR2 comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 80%, or 90% or 100% identity with an amino acid sequence selected from SEQ ID NO:48-50; and VH CDR3 comprises or consists of an amino acid sequence having at least 50%, 60%, 70%, 80%, or 90% or 100% identity with an amino acid sequence selected from SEQ ID NO:79.

[0085] In some embodiments, the VH CDR1 of the anti-CD40 antibody or antigen-binding fragment of the present invention contains the amino acid sequence shown in SEQ ID NO:4, the VH CDR2 contains the amino acid sequence shown in SEQ ID NO:48, and the VH CDR3 contains the amino acid sequence shown in SEQ ID NO:79; the VL CDR1 contains the amino acid sequence shown in SEQ ID NO:1, the VLCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and the VL CDR3 contains the amino acid sequence shown in SEQ ID NO:3.

[0086] In some embodiments, the VH CDR1 of the anti-CD40 antibody or antigen-binding fragment of the present invention contains the amino acid sequence shown in SEQ ID NO:5, the VH CDR2 contains the amino acid sequence shown in SEQ ID NO:48, and the VH CDR3 contains the amino acid sequence shown in SEQ ID NO:79; the VL CDR1 contains the amino acid sequence shown in SEQ ID NO:1, the VLCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and the VL CDR3 contains the amino acid sequence shown in SEQ ID NO:3.

[0087] In some embodiments, the VH CDR1 of the anti-CD40 antibody or antigen-binding fragment of the present invention contains the amino acid sequence shown in SEQ ID NO:6, the VH CDR2 contains the amino acid sequence shown in SEQ ID NO:49, and the VH CDR3 contains the amino acid sequence shown in SEQ ID NO:79; the VL CDR1 contains the amino acid sequence shown in SEQ ID NO:1, the VLCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and the VL CDR3 contains the amino acid sequence shown in SEQ ID NO:3.

[0088] In some embodiments, the VH CDR1 of the anti-CD40 antibody or antigen-binding fragment of the present invention contains the amino acid sequence shown in SEQ ID NO:7, the VH CDR2 contains the amino acid sequence shown in SEQ ID NO:49, and the VH CDR3 contains the amino acid sequence shown in SEQ ID NO:79; the VL CDR1 contains the amino acid sequence shown in SEQ ID NO:1, the VLCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and the VL CDR3 contains the amino acid sequence shown in SEQ ID NO:3.

[0089] In some embodiments, the VH CDR1 of the anti-CD40 antibody or antigen-binding fragment of the present invention contains the amino acid sequence shown in SEQ ID NO:8, the VH CDR2 contains the amino acid sequence shown in SEQ ID NO:50, and the VH CDR3 contains the amino acid sequence shown in SEQ ID NO:79; the VL CDR1 contains the amino acid sequence shown in SEQ ID NO:1, the VLCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and the VL CDR3 contains the amino acid sequence shown in SEQ ID NO:3.

[0090] In some embodiments, the VH CDR1 of the anti-CD40 antibody or antigen-binding fragment of the present invention contains the amino acid sequence shown in SEQ ID NO:9, the VH CDR2 contains the amino acid sequence shown in SEQ ID NO:48, and the VH CDR3 contains the amino acid sequence shown in SEQ ID NO:79; the VL CDR1 contains the amino acid sequence shown in SEQ ID NO:1, the VLCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and the VL CDR3 contains the amino acid sequence shown in SEQ ID NO:3.

[0091] In some embodiments, the VH CDR1 of the anti-CD40 antibody or antigen-binding fragment of the present invention contains the amino acid sequence shown in SEQ ID NO:18, the VH CDR2 contains the amino acid sequence shown in SEQ ID NO:60, and the VH CDR3 contains the amino acid sequence shown in SEQ ID NO:79; the VL CDR1 contains the amino acid sequence shown in SEQ ID NO:1, the VLCDR2 contains the amino acid sequence shown in SEQ ID NO:2, and the VL CDR3 contains the amino acid sequence shown in SEQ ID NO:3.

[0092] In the antibody or its antigen-binding fragment of the present invention, VH CDR1, VH CDR2, and VH CDR3 can be any combination of the amino acid sequences corresponding to each CDR in Table 1.

[0093] Table 1 CDR Sequence

[0094]

[0095]

[0096] In some embodiments, the anti-CD40 antibody of the present invention or its antigen-binding fragment comprises a light chain variable region VL, which comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with an amino acid sequence selected from SEQ ID NO:80.

[0097] In some embodiments, the anti-CD40 antibody or fragment thereof of the present invention comprises a heavy chain variable region VH, which comprises or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or 100% identity with an amino acid sequence selected from SEQ ID NO: 81-126.

[0098] In some embodiments, the anti-CD40 antibody or fragment thereof of the present invention comprises VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO:81, and VL comprises the amino acid sequence shown in SEQ ID NO:80.

[0099] In some embodiments, the anti-CD40 antibody or fragment thereof of the present invention comprises VH and VL, wherein VH comprises an amino acid sequence as shown in SEQ ID NO:82, and VL comprises an amino acid sequence as shown in SEQ ID NO:80.

[0100] In some embodiments, the anti-CD40 antibody or fragment thereof of the present invention comprises VH and VL, wherein VH comprises an amino acid sequence as shown in SEQ ID NO:83, and VL comprises an amino acid sequence as shown in SEQ ID NO:80.

[0101] In some embodiments, the anti-CD40 antibody or fragment thereof of the present invention comprises VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO:84, and VL comprises the amino acid sequence shown in SEQ ID NO:80.

[0102] In some embodiments, the anti-CD40 antibody or fragment thereof of the present invention comprises VH and VL, wherein VH comprises the amino acid sequence shown in SEQ ID NO:85, and VL comprises the amino acid sequence shown in SEQ ID NO:80.

[0103] In some embodiments, the anti-CD40 antibody or fragment thereof of the present invention comprises VH and VL, wherein VH comprises an amino acid sequence as shown in SEQ ID NO:86, and VL comprises an amino acid sequence as shown in SEQ ID NO:80.

[0104] In some embodiments, the anti-CD40 antibody or fragment thereof of the present invention comprises VH and VL, wherein VH comprises an amino acid sequence as shown in SEQ ID NO:96, and VL comprises an amino acid sequence as shown in SEQ ID NO:80.

[0105] In some embodiments, the antibody or fragment thereof of the present invention further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

[0106] In some embodiments, the antibody of the present invention comprises a light chain constant region (CL) as shown in SEQ ID NO:127 and a heavy chain constant region (CH) as shown in SEQ ID NO:128.

[0107] In some embodiments, the antibody of the present invention comprises a heavy chain composed of VH and CH, and a light chain composed of VL and CL. The sequence of VH is shown in SEQ ID NO:81, the sequence of CH is shown in SEQ ID NO:128, the sequence of VL is shown in SEQ ID NO:80, and the sequence of CL is shown in SEQ ID NO:127.

[0108] In some embodiments, the antibody of the present invention comprises a heavy chain composed of VH and CH, and a light chain composed of VL and CL. The sequence of VH is shown in SEQ ID NO:82, the sequence of CH is shown in SEQ ID NO:128, the sequence of VL is shown in SEQ ID NO:80, and the sequence of CL is shown in SEQ ID NO:127.

[0109] In some embodiments, the antibody of the present invention comprises a heavy chain composed of VH and CH, and a light chain composed of VL and CL. The sequence of VH is shown in SEQ ID NO:83, the sequence of CH is shown in SEQ ID NO:128, the sequence of VL is shown in SEQ ID NO:80, and the sequence of CL is shown in SEQ ID NO:127.

[0110] In some embodiments, the antibody of the present invention comprises a heavy chain composed of VH and CH, and a light chain composed of VL and CL. The sequence of VH is shown in SEQ ID NO:84, the sequence of CH is shown in SEQ ID NO:128, the sequence of VL is shown in SEQ ID NO:80, and the sequence of CL is shown in SEQ ID NO:127.

[0111] In some embodiments, the antibody of the present invention comprises a heavy chain composed of VH and CH, and a light chain composed of VL and CL. The sequence of VH is shown in SEQ ID NO:85, the sequence of CH is shown in SEQ ID NO:128, the sequence of VL is shown in SEQ ID NO:80, and the sequence of CL is shown in SEQ ID NO:127.

[0112] In some embodiments, the antibody of the present invention comprises a heavy chain composed of VH and CH, and a light chain composed of VL and CL. The sequence of VH is shown in SEQ ID NO:86, the sequence of CH is shown in SEQ ID NO:128, the sequence of VL is shown in SEQ ID NO:80, and the sequence of CL is shown in SEQ ID NO:127.

[0113] In some embodiments, the antibody of the present invention comprises a heavy chain composed of VH and CH, and a light chain composed of VL and CL. The sequence of VH is shown in SEQ ID NO:96, the sequence of CH is shown in SEQ ID NO:128, the sequence of VL is shown in SEQ ID NO:80, and the sequence of CL is shown in SEQ ID NO:127.

[0114] In some embodiments, the antibody of the present invention comprises a heavy chain as shown in SEQ ID NO:130 and a light chain as shown in SEQ ID NO:129.

[0115] In some embodiments, the regulation of the immune response by the anti-CD40 antibody and fragment provided herein can be measured in a mixed lymphocyte (MLR) reaction. In one embodiment, the anti-CD40 antibody provided herein increases the level of cytokine production by lymphocytes in the MLR. In one embodiment, the anti-CD40 antibody increases the level of IFNγ production in the MLR.

[0116] The Fc region of an anti-CD40 antibody can interact with most Fc receptors to deliver a range of important functional capabilities, known as effector functions. In some embodiments, the present invention provides a conjugate comprising the anti-CD40 antibody of the present invention conjugated to an Fc receptor. In some embodiments, in vitro experiments use an anti-human IgG1-Fc antibody to mimic the Fc receptor expressed in vivo by cells, reflecting the effect of conjugation of the anti-CD40 antibody to the Fc receptor on the activity of the anti-CD40 antibody. In some embodiments, the anti-CD40 antibody of the present invention can be conjugated to an anti-human IgG1-Fc antibody to enhance the activity of the anti-CD40 antibody. In some embodiments, the anti-CD40 antibody of the present invention can be conjugated to an Fc receptor to enhance the activity of the anti-CD40 antibody. In some embodiments, the anti-CD40 antibody of the present invention can bind to the Fc receptor on the surface of cells expressing the Fc receptor to enhance the activity of the anti-CD40 antibody. In some embodiments, the anti-CD40 antibody of the present invention can be conjugated in vivo through cells expressing the Fc receptor to enhance the activity of the anti-CD40 antibody. Fc receptors are expressed in a variety of immune cells, including monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells, and T cells.

[0117] In some embodiments, anti-CD40 antibodies, antigen-binding fragments, variants, or derivatives are disclosed. A variant is an antibody or antigen-binding fragment obtained by deleting and / or replacing, or inserting, one or more amino acid residues in the antibody or antigen-binding fragment. Derivatives include modified derivatives, i.e., modified by covalently linking the antibody to any type of molecule, wherein the covalent link does not prevent the antibody from binding to the epitope. Examples of modifications include, but are not limited to, glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins. Any of the numerous chemical modifications can be performed using existing techniques, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin. Furthermore, antibodies may contain one or more unnatural amino acids.

[0118] In some implementations, antibodies may be conjugated with therapeutic agents, drug precursors, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceuticals, or PEG.

[0119] Antibodies can be conjugated or fused with therapeutic agents, which may include detectable markers such as radioactive markers, immunomodulators, hormones, enzymes, oligonucleotides, photosensitizing therapeutic agents or diagnostic agents, cytotoxic agents that may be drugs or toxins, ultrasound enhancers, non-radioactive markers and combinations thereof, and other such agents known in the art.

[0120] Antibodies can be detectably labeled by conjugating them to chemiluminescent compounds. The presence of the chemiluminescently labeled antigen-binding fragment is then determined by detecting the luminescence that occurs during the chemical reaction. Examples of particularly useful chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridine esters, imidazoles, acridine salts, and oxalates.

[0121] In some embodiments, the antibodies of the present invention also cover variants of the amino acid sequence of anti-CD40 antibodies, as well as antibodies that bind to the same epitope as any of the antibodies described above.

[0122] In some embodiments, the anti-CD40 antibody of the present invention also encompasses its antibody fragments; in some embodiments, the antibody fragments are selected from the following: Fab, Fab'-SH, Fv, scFv or (Fab')2 fragments.

[0123] In some embodiments, the present invention provides a nucleic acid encoding any of the above-described anti-CD40 antibodies or fragments. In one embodiment, a vector comprising said nucleic acid is provided. In one embodiment, the vector is an expression vector. Expression vectors include plasmids, retroviruses, YACs, EBV-derived episomes, etc. In one embodiment, a host cell comprising said vector is provided. In one embodiment, said host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293F cells), or other cells suitable for preparing antibodies or antigen-binding fragments.

[0124] In some embodiments, the nucleic acid sequence encoding an anti-CD40 antibody or fragment can be obtained from the amino acid sequence of the anti-CD40 antibody or fragment using methods conventional in the art. In some embodiments, the nucleic acid encoding the antibody light chain as shown in SEQ ID NO:129 is shown in SEQ ID NO:131, wherein the underlined portion may encode an antibody CDR region. In some embodiments, the nucleic acid encoding the antibody heavy chain as shown in SEQ ID NO:130 is shown in SEQ ID NO:132, wherein the underlined portion may encode an antibody CDR region.

[0125] Table 2. Amino acid and nucleic acid sequences of anti-CD40 antibody (antibody 25)

[0126]

[0127]

[0128] In some embodiments, the antibody is chimeric, humanized, or fully human. In some embodiments, the antibody or fragment can activate T cells, promoting their proliferation or secretion of inflammatory factors. In some embodiments, the antibody or fragment is an IgG isotype selected from the group consisting of IgG1, IgG2, IgG3, and / or IgG4 isotypes. In some embodiments, the antibody or antigen-binding fragment is an IgG isotype selected from IgG1.

[0129] This invention also includes antibodies that bind to the same epitope as the anti-CD40 antibody described herein. For example, the antibodies of this invention specifically bind to epitopes comprising one or more amino acid residues on human CD40 (the sequence of human CD40 is shown at P25942 on Uniprot).

[0130] Those skilled in the art will recognize that it is only necessary to determine whether an antibody binds to the same epitope as the antibody described herein by examining whether the test antibody prevents a known antibody from binding to CD40, without requiring extensive experiments. If the test antibody competes with the antibody disclosed herein, the two antibodies may bind to the same or similar epitopes.

[0131] An alternative method for determining whether an antibody has the specificity of the antibody described herein is to pre-incubate the antibody described herein with a soluble CD40 protein to which the antibody typically reacts, and then add the test antibody to determine whether the ability of the test antibody to bind to CD40 is inhibited. If the test antibody is inhibited, it has the same or functional epitope specificity as the antibody of this disclosure.

[0132] In some embodiments, the antibodies of the present invention can be prepared using methods described, for example, in the examples provided below. In some embodiments, they can also be prepared using Trioma technology, human B-cell hybridoma technology (see Kozbor et al., 1983, Immunol Today 4:72), and EBV hybridoma technology (see Cole et al., 1985, In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

[0133] Using conventional recombinant DNA techniques, one or more CDRs of the antibodies of this invention can be inserted into a frame region, for example, into a human frame region, to construct a humanized non-fully human antibody. The frame region can be a naturally occurring or common frame region, preferably a human frame region (see Chothia et al., J. Mol. Biol. 278:457-479 (1998), which lists a series of human frame regions). Some polynucleotides can encode antibodies that specifically bind to at least one epitope of a target antigen, containing a combination of frame region and CDR. One or more amino acid substitutions can be made within the frame region, selectively choosing amino acid substitutions that improve antibody binding to its antigen. Additionally, this method can be used to substitute or delete cysteine ​​residues in one or more variable regions involved in interchain disulfide bond formation, thereby producing antibody molecules lacking one or more interchain disulfide bonds. Other modifications to polynucleotides within the scope of the art are also covered in this invention.

[0134] Furthermore, antibodies can be prepared using conventional recombinant DNA techniques. Vectors and cell lines for antibody production can be selected, constructed, and cultured using recombinant DNA techniques well-known to those skilled in the art. These techniques are described in various laboratory manuals and major publications, such as *Recombinant DNA Technology for Production of Protein Therapeutics in Cultured Mammalian Cells*, DL Hacker, FMWurm, in *Reference Module in Life Sciences*, 2017, the entire contents of which, including supplementary information, are incorporated herein by reference.

[0135] In some embodiments, antibody-encoding DNA can be designed and synthesized according to the antibody amino acid sequence described herein using conventional methods, placed into an expression vector, and then transfected into host cells. The transfected host cells are then cultured in a culture medium to produce monoclonal antibodies. In some embodiments, the antibody expression vector includes at least one promoter element, an antibody-encoding sequence, a transcription termination signal, and a polyA tail. Other elements include an enhancer, a Kozak sequence, and donor and acceptor sites for RNA splicing flanking the insert sequence. Efficient transcription can be achieved using early and late promoters of SV40, early promoters of long terminal repeat sequences from retroviruses such as RSV, HTLV1, HIV, and cytomegalovirus, or other cellular promoters such as actin promoters. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or Plncx, pcDNA3.1(+ / -), pcDNA / Zeo(+ / -), pcDNA3.1 / Hygro(+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI, and pCS2, etc. Commonly used mammalian cell lines include 293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells, and CHO cells, etc.

[0136] In some implementations, the inserted gene fragment needs to contain selection markers. Common selection markers include dihydrofolate reductase, glutamine synthase, neomycin resistance, and hygromycin resistance selection genes to facilitate the selection and isolation of successfully transfected cells. The constructed plasmid is transfected into host cells, cultured in a selective medium, and the successfully transfected cells grow in large numbers, producing the desired target protein.

[0137] Antibodies can be purified using well-known techniques, such as affinity chromatography using protein A or protein G, immunoaffinity chromatography, etc. For example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (2000), pp. 25-28) discusses the purification of immunoglobulins.

[0138] Fc modification

[0139] The effector functions of the antibodies described in this article can be modified to enhance, for example, the effectiveness of antibodies in treating diseases and disorders related to CD40 signaling. For instance, because regulatory T cells (Tregs) in tumor infiltration are ubiquitously expressed, one or more mutations can be introduced into the Fc region of the antibody to enhance ADCC function, thereby more effectively killing Tregs. Also, for example, full activation of intracellular CD40 signaling requires the aggregation of multiple CD40 receptors, or even their oligomerization; therefore, one or more mutations can be introduced into the Fc region of the antibody to enhance the antibody's own aggregation ability or its binding affinity to Fc receptors to promote antibody aggregation, thereby more fully activating intracellular CD40 signaling.

[0140] In some embodiments, the antibody described herein is an IgG isotype. In some embodiments, the constant region of the antibody is a human IgG1 isotype. In some embodiments, specific amino acids on the human IgG1 constant region are modified to alter the antibody's glycosylation, for example, by defucosylating N297. In some embodiments, the antibody or antigen-binding fragment contains a small amount of fucose modification, or almost no fucose modification, or no fucose modification, resulting in significantly improved ADCC efficacy. In some embodiments, the antibody or fragment has at most one (or at most two, or three) amino acid residues that are fucose modified. In some embodiments, the protein molecule comprising the antibody or fragment has at most less than 0.01%, 0.1%, 1%, 2%, 3%, 4%, or 5% fucose modification.

[0141] In some implementations, specific amino acids on the constant region of the antibody are modified to alter Fc receptor interactions, such as mutations in N297A, L234A, and / or L235A.

[0142] In some embodiments, the constant region of the antibody is the human IgG2 isotype. In some embodiments, the constant region of the antibody is the human IgG4 isotype.

[0143] For the use of anti-CD40 antibodies or antigen-binding fragments

[0144] Those skilled in the art will understand that the antibodies of the present invention have a variety of uses. For example, the antibodies of the present invention can be used as therapeutic agents, as reagents in diagnostic kits or as diagnostic tools, or as reagents in competitive experiments to generate therapeutic agents.

[0145] In some embodiments, a method is provided for preventing, treating, or improving CD40-related diseases in patients in need, the method comprising administering to the patient an effective dose of the antibody or antigen-binding fragment described herein. In some embodiments, the use of the antibody or antigen-binding fragment described herein is provided in the preparation of a medicament for the prevention, treatment, or improvement of CD40-related diseases. Diseases or conditions that can be treated by the anti-CD40 antibodies described herein include hematologic malignancies and / or solid tumors.

[0146] Blood cancers include, for example, leukemia, lymphoma, and myeloma. In some implementations, leukemia includes acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); chronic lymphocytic leukemia (CLL); chronic myeloid leukemia (CML); and myeloproliferative disorders / tumors (MPDS). Lymphomas include Hodgkin's lymphoma, painless and aggressive non-Hodgkin's lymphoma, Burkitt's lymphoma, and follicular lymphoma (small cell and large cell). Myeloma includes multiple myeloma (MM), giant cell myeloma, heavy chain myeloma, and light chain or Bens-Jones myeloma. Solid tumors include, for example, breast cancer, ovarian cancer, lung cancer, pancreatic cancer, prostate cancer, melanoma, colorectal cancer, lung cancer, head and neck cancer, bladder cancer, esophageal cancer, liver cancer, and kidney cancer.

[0147] In some embodiments, the antibodies of the present invention can activate an immune response, thereby being used to treat infections.

[0148] Infection is the process by which pathogenic agents invade the tissues of an organism, multiply, and the host tissues respond to these organisms and the toxins they produce. Infections can be caused by infectious agents such as viruses, viroids, prions, bacteria, nematodes such as parasitic roundworms and pinworms, arthropods such as ticks, mites, fleas and lice, fungi such as tinea, and other large parasites such as tapeworms and other worms. In some cases, infectious agents are bacteria, such as Gram-negative bacteria. In others, infectious agents are viruses, such as DNA viruses, RNA viruses, and retroviruses. Non-limiting examples of viruses include adenoviruses, Coxsackieviruses, Epstein-Barr virus (EBV), hepatitis A virus, hepatitis B virus, hepatitis C virus, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, human herpesvirus type 8, HIV, influenza virus, measles virus, mumps virus, human papillomavirus, parainfluenza virus, poliovirus, rabies virus, respiratory syncytial virus, rubella virus, and varicella-zoster virus.

[0149] The antibodies of this invention can also be used to treat infectious diseases caused by microorganisms, or to eliminate microorganisms by targeting and binding to microorganisms and killing them with immune cells. In one respect, microorganisms include viruses (RNA and DNA viruses), Gram-positive bacteria, Gram-negative bacteria, protozoa, or fungi.

[0150] The therapeutically effective dose of the antibody of the present invention relates to the amount required to achieve the therapeutic goal. The amount required for administration depends on the antibody's binding affinity to its specific antigen, the severity of the disease, disorder, or condition, the route of administration, the rate at which the antibody is depleted from its free volume in the recipient, etc. In some embodiments, the therapeutically effective dose of the antibody or antibody fragment of the present invention ranges from about 0.01 mg / kg to about 100 mg / kg. In some embodiments, the therapeutically effective dose of the antibody or antibody fragment of the present invention ranges from about 0.1 mg / kg to about 30 mg / kg. The dosing frequency range may be, for example, once every two weeks or once every three weeks.

[0151] In some embodiments, the antibody of the present invention may be used in combination with a therapeutic agent. The therapeutic agent is any substance that inhibits or prevents cellular function and / or causes cell damage. In some embodiments, the therapeutic agent may be one or more of maytansine or a derivative thereof, camptothecin or a derivative thereof, paclitaxel or a derivative thereof, carboplatin or a derivative thereof, cisplatin or a derivative thereof, gemcitabine or a derivative thereof. In some embodiments, the therapeutic agent may be paclitaxel or a salt thereof. In some embodiments, the therapeutic agent may be carboplatin or a salt thereof. In some embodiments, the therapeutic agent may be paclitaxel and carboplatin.

[0152] In some implementations, anti-CD40 antibodies are given to patients diagnosed with clinical symptoms associated with one or more of the aforementioned diseases (including, but not limited to, cancer or other neoplastic conditions). Following diagnosis, the anti-CD40 antibody is administered to alleviate or reverse the clinical symptoms associated with one or more of the aforementioned diseases.

[0153] CD40 overexpression has been observed in certain tumor samples, and patients with CD40-overexpressing cells may respond to treatment using the anti-CD40 antibody of the present invention. Therefore, the antibody of the present invention can also be used for diagnosis and prognosis.

[0154] In some embodiments, the cell-containing sample can be obtained from a patient, who may be a cancer patient or a patient awaiting diagnosis. The cells are cells from tumor tissue or tumor masses, blood samples, urine samples, or any sample from the patient. After selectively pretreating the sample, it can be incubated with the antibody of the present invention under conditions that allow the antibody to interact with the CD40 protein that may be present in the sample, using the anti-CD40 antibody to detect the presence of the CD40 protein in the sample.

[0155] The antibodies of the present invention are also used to detect CD40 in patient samples and are therefore useful for diagnosis. For example, the anti-CD40 antibody of the present invention is used in in vitro assays (such as ELISA) to detect CD40 levels in patient samples.

[0156] In one embodiment, the anti-CD40 antibody of the present invention is immobilized on a solid support (such as the wells of a microtiter plate). The immobilized antibody acts as a capture antibody, capturing any CD40 that may be present in the test sample. Before exposing the immobilized antibody to the patient sample, the solid support is washed and treated with a blocking agent (such as milk protein or albumin) to avoid non-specific adsorption of the analyte. The wells are then treated with a test sample that may contain the antigen or with a solution containing a standard amount of the antigen. Such samples are, for example, serum samples from an individual who may have circulating antigen levels considered diagnostic of a particular lesion. After washing away the test sample or standard, the solid support is treated with a detectable labeled secondary antibody. The labeled secondary antibody serves as the detection antibody. The level of the detectable label is measured, and the concentration of CD40 in the test sample is determined by comparing it with a standard curve established using standard samples.

[0157] In some embodiments, when using the anti-CD40 antibody or antigen-binding fragment described herein, the antibody or fragment is present in the form of a pharmaceutical composition. This pharmaceutical composition may consist of an anti-CD40 antibody or antigen-binding fragment and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delayers compatible with drug administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences. Such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and / or 5% human serum albumin.

[0158] In some embodiments, the pharmaceutical composition containing the anti-CD40 antibody is compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., local), transmucosal, and rectal administration. The pharmaceutical composition may include one or more of the following components: a sterile diluent for injection, such as water, saline solution, fixative oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; a bacteriostatic agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid (EDTA); a buffer, such as histidine hydrochloride, acetate, citrate, or phosphate; an osmolarity regulator, such as sodium chloride or dextran; a stabilizer, such as arginine, methionine, trehalose, sucrose, or sorbitol; and a surfactant, such as Tween 20 or Tween 80. The pH may be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Pharmaceutical compositions can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic. In some embodiments, pharmaceutical compositions suitable for injection include sterile aqueous solutions (in this case, water-soluble) or dispersions, as well as sterile powders for the immediate preparation of sterile injectable solutions or dispersions. When used, the composition must be sterile and should be fluid enough for easy injection. It must be stable under manufacturing and storage conditions and must be resistant to contamination by microorganisms such as bacteria and fungi. Prolonged absorption of the injectable composition can be achieved by including an absorption-delaying agent, such as aluminum monostearate and gelatin, in the composition. If necessary, a sterile injectable solution can be prepared by incorporating an antibody in the desired amount into a suitable solvent having one or more of the components listed above (as needed), followed by filtration sterilization. Alternatively, the aforementioned sterile solution can be freeze-dried to obtain a powder for use in preparing a sterile injectable solution at the time of administration.

[0159] In some embodiments, the anti-CD40 antibody and other therapeutic agents are prepared as a single therapeutic composition and administered simultaneously. Alternatively, the anti-CD40 antibody and other therapeutic agents are independent of each other, for example, prepared as separate therapeutic compositions and administered simultaneously, or administered at different times during a treatment regimen. For example, the anti-CD40 antibody may be administered before or after the other therapeutic agents, or administered in an alternating manner. In this document, the anti-CD40 antibody and other therapeutic agents are administered in a single dose or multiple doses.

[0160] Example

[0161] Unless otherwise specified, the materials and reagents used in the following examples can be obtained commercially or by known methods.

[0162] Example 1: Preparation of positive control antibodies CP870893 and APX005.

[0163] The heavy and light chain sequences of CP870893 are as follows:

[0164] CP870893 heavy chain amino acid sequence:

[0165] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFD YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKC CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTIS KTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:133);

[0166] CP870893 light chain amino acid sequence:

[0167] DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:134).

[0168] The heavy and light chain sequences of APX005 are shown in patent US9676861.

[0169] The nucleotides encoding the heavy and light chains of the antibodies described above were ligated into the pcDNA3.0 vector (purchased from Invitrogen) via enzyme digestion and ligation to obtain recombinant plasmids for expressing the total antibody. The corresponding light chain recombinant plasmid and heavy chain plasmid were transiently expressed by co-transfection of HEK293 cells with PEI. After 7 days of culture, the supernatant was collected, and finally purified to obtain CP870893 and APX005 anti-CD40 antibodies, which were used in the various examples below.

[0170] Example 2: Preparation of anti-CD40 antibody

[0171] The variable and constant regions of the 46 antibodies are shown in Table 3. The heavy chain of each antibody consists of VH and CH, and the light chain consists of VL and CL. The numbers "1-VH" refer to the VH sequence of antibody 1, and so on; the VL, CH, and CL are the same for all 46 antibodies. Using molecular cloning technology, the nucleic acid sequences encoding the above antibodies were inserted into the pcDNA3.0 vector (purchased from Invitrogen) to obtain recombinant plasmids for expressing the complete antibodies. The corresponding light chain recombinant plasmids and heavy chain plasmids were co-transfected into HEK293 cells via PEI for transient expression. The recombinant plasmids were then transformed into the 293F cell line, cultured for 7 days, and the supernatant was collected. Finally, the 46 antibodies were purified, and their sequences were confirmed by sequencing for use in the following examples.

[0172] Table 3. VH, VL, CH, and CL sequences of each antibody

[0173]

[0174]

[0175]

[0176]

[0177] Example 3: Identification of the binding ability of anti-human CD40 antibody

[0178] 3.1 Affinity of anti-CD40 antibody

[0179] The affinity of antibodies 1, 7, 15, 25, 28, 35, 41, and the positive control antibody CP870893 was determined using the BIACORE method, a conventional method in this field. The results are shown in Table 4.

[0180] Table 4. Affinity (KD) of anti-CD40 antibody determined by the BIACORE method.

[0181]

[0182]

[0183] 3.2 Binding ability of anti-CD40 antibody to Daudi cell surface antigen

[0184] The binding ability of anti-CD40 antibody to CD40 molecules on the cell surface was detected using Daudi cells expressing CD40.

[0185] Antibodies 1, 7, 25, 28, 35, 41, and the positive control antibody APX005 were used to incubate Daudi cells with different concentrations of antibodies. Antibody 1 and Antibody 7 were tested at ten concentration points starting from 10 μg / ml. Antibody 25, Antibody 28, Antibody 35, and Antibody 41 were tested at nine concentration points starting from 6 μg / ml and diluted 2.5-fold downwards. The mean fractional oxidative stress (MFI) was calculated, and the data were processed using SoftMax Pro. The EC50 values ​​of Antibody 1, Antibody 7, Antibody 25, Antibody 28, Antibody 35, Antibody 41, and Antibody APX005 bound to Daudi cells were 961.6 ng / ml, 620.7 ng / ml, 413.8 ng / ml, 651.3 ng / ml, 223.3 ng / ml, 765.2 ng / ml, and 859.7 ng / ml, respectively. Using the same detection method, the EC50 values ​​of Antibody 27, 29, 36, and 45 bound to Daudi cells were 92.53 ng / ml, 323.3 ng / ml, 185.4 ng / ml, and 125.9 ng / ml, respectively.

[0186] 3.3 Binding ability of anti-CD40 antibody to HT1080-hCD40 cell surface antigen

[0187] HT1080-hCD40 cells (HT1080 cells expressing human CD40, purchased from Kangyuan Bochuang Biotechnology (Beijing) Co., Ltd., KC-0142) were incubated with a series of antibody concentrations: antibody 1, antibody 7, antibody 25, antibody 28, antibody 35, antibody 41, and control antibody APX005. The antibody concentrations were started at 15 μg / ml and diluted 2.5-fold downwards for a total of ten concentration points. MFI was calculated, and the data were processed using SoftMax Pro. The EC50 values ​​of antibody 1, antibody 7, antibody 25, antibody 28, antibody 35, antibody 41, and APX005 bound to HT1080-hCD40 cells were 2154 ng / ml, 2191 ng / ml, 593.3 ng / ml, 944.4 ng / ml, 4698 ng / ml, 3517 ng / ml, and 995.1 ng / ml, respectively.

[0188] 3.4 Cell binding specificity of anti-CD40 antibody

[0189] Jurkat cells, CHO cells, 293F cells, and Raji cells were incubated with 100 nM anti-CD40 antibodies (antibody 1, antibody 7, antibody 25, antibody 28, antibody 34, and antibody 41) or human IgG1 (negative control) for 1 h. Anti-hIgG-FC-PE (Thermofisher, Ref 12-4998-82) was then added, and the cells were analyzed using an Accuri C6 flow cytometer with signal acquisition via the FL2 channel. No CD40 antigen was expressed on Jurkat, CHO, or 293F cells, but CD40 antigen was expressed on Raji cells. The anti-CD40 antibodies showed no significant binding to Jurkat, CHO, or 293F cells, but exhibited strong binding to Raji cells, demonstrating the good cell-specific binding of the anti-CD40 antibodies. (See [link to relevant documentation]). Figure 1 .

[0190] 3.5 Species specificity of anti-CD40 antibodies

[0191] Using the ELISA method, mouse CD40 (mCD40-his, purchased from Nearshore Biotech, Catalog #CS43) and monkey CD40 (cCD40-his, purchased from Nearshore Biotech, Catalog #CA90) were first coated at 2 μg / ml and incubated overnight at 4°C. After washing, the mixture was incubated with anti-CD40 antibodies (antibody 1, antibody 7, antibody 25, antibody 28, antibody 34, antibody 41), a positive control antibody (APX005), and a negative control antibody (IgG1). Finally, the mixture was labeled and developed using HRP-labeled secondary antibody. The anti-CD40 antibodies bound to monkey cCD40-His in a gradient-dependent manner but did not bind to mouse mCD40-his. The obtained OD values ​​are shown below. Figure 2A and Figure 2B As shown.

[0192] Example 4: Detection of the bioactivity of anti-CD40 antibody

[0193] 4.1 The in vitro activation activity of anti-CD40 antibody was detected using the NFκB reporter gene system.

[0194] The hCD40 gene sequence was constructed into the pCDNA3.1 vector to obtain the pCDNA3.1-hCD40 plasmid. The pCDNA3.1-hCD40 plasmid and pGL4.32[luc2P-NF-κB-RE-Hygro] Vector (purchased from Promega) were co-transfected into 293T cells via PEI to construct the 293T-CD40-NFkb-luciferase-reporter cell line, which was used to detect the cell activity of anti-CD40 antibody in vitro.

[0195] The method for detecting anti-CD40 antibody cell activity (reporting system) is as follows: 293T-CD40-NFkb-luciferase-reporter cells in good condition are used at 5 × 10⁻⁶ cells / day. 4 Cells were plated overnight. The next day, serially diluted anti-CD40 antibodies (antibody 1, antibody 7, antibody 25, antibody 28, antibody 34, antibody 41) and positive antibody APX005 were added, starting at 5 μg / ml and 3-fold diluted in 8 concentrations. The cells were incubated in a cell culture incubator for 6 hours. Then, 100 μl of ONE-Glo reagent was added to each well. TM The Luciferase Assay System (purchased from Promega) was used. Fluorescence values ​​were read within 10 minutes of reagent addition. The anti-CD40 antibody and the positive antibody APX005 activated the reporter system in a gradient-dependent manner. Results are as follows... Figure 3A , Figure 3B As shown.

[0196] In the cell viability assay (reporter system) for anti-CD40 antibody, the above method involved adding 5 μg / ml of anti-human IgG1-Fc antibody (Anti-hIgG1-Fc, purchased from Sigma, catalog number I2136) conjugated with anti-CD40 antibody simultaneously with the antibody, while other procedures remained the same. The results showed that ( Figure 3C , Figure 3D Anti-hIgG1-Fc conjugation significantly enhances the activity of anti-CD40 antibodies. The anti-CD40 antibody of this invention can be conjugated in vivo with cells expressing the Fc receptor to enhance its activity.

[0197] 4.2 The promoting effect of anti-CD40 antibody on IFN-γ secretion in MLR (mixed lymphocyte reaction) assay

[0198] Peripheral blood-derived dendritic cells (Mo-DCs) from volunteer A (age: 20, male, in good health) were induced to mature using conventional methods in the art. Specifically, after PBMCs adhered to the culture vessel, IL4 and GM-CSF were added for 5 days of induction culture, followed by TNF-α, IL-β, IL6, and PEG2 for 2 days of maturation culture to obtain mature Mo-DCs. The Mo-DCs from volunteer A were co-cultured with PBMCs from volunteer B (age: 29, male, in good health) in 96-well plates. Serially diluted anti-CD40 antibodies (antibody 1, antibody 7, antibody 25, antibody 28, antibody 34, antibody 41) and positive antibody APX005 were added simultaneously. Antibody concentrations started at 166 ng / ml, were 3-fold diluted, and five concentration points were established. After 72 hours, IFN-γ in the supernatant of the co-culture system was detected using an IFN-γ ELISA kit (Sinbosheng Biotech, EHC 102g). Anti-CD40 antibody and positive antibody APX005 can increase IFN-γ secretion in MLR assays in a gradient-dependent manner (IFN-γ difference is expressed as OD450). The results are shown in Figure 4.

[0199] All publications and patent documents cited herein are incorporated herein by reference as if each of these publications or documents were specifically and individually indicated to be incorporated herein by reference. Reference to the foregoing publications and patent documents does not imply an admission that any of the foregoing content is applicable prior art, nor does it imply an admission of its content or date. Now that the invention has been described in writing, those skilled in the art will recognize that the invention can be practiced in various embodiments, and that the foregoing description and examples are intended to illustrate, not limit, the claims of the invention.

Claims

1. An anti-CD40 antibody or its antigen-binding fragment, characterized in that, The anti-CD40 antibody or its antigen-binding fragment comprises VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, and VH CDR3, wherein the amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:4, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:48, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:5, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:48, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:18, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:60, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:6, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:49, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:28, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:65, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:7, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:49, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:29, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:66, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:32, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:48, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:8, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:50, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:33, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:69, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:9, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:48, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:79; or, The amino acid sequence of VL CDR1 is shown in SEQ ID NO:1, the amino acid sequence of VL CDR2 is shown in SEQ ID NO:2, the amino acid sequence of VL CDR3 is shown in SEQ ID NO:3, the amino acid sequence of VH CDR1 is shown in SEQ ID NO:40, the amino acid sequence of VH CDR2 is shown in SEQ ID NO:49, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO:

79.

2. The anti-CD40 antibody or its antigen-binding fragment as described in claim 1, characterized in that, The anti-CD40 antibody or its antigen-binding fragment includes a heavy chain variable region, which contains an amino acid sequence represented by any one of SEQ ID NO: 81-86, 96, 106, 107, 110, 111 or 118.

3. The anti-CD40 antibody or its antigen-binding fragment as described in claim 1, characterized in that, The anti-CD40 antibody or its antigen-binding fragment includes a light chain variable region, which contains the amino acid sequence shown in SEQ ID NO:

80.

4. The anti-CD40 antibody or its antigen-binding fragment as described in claim 2, characterized in that, The anti-CD40 antibody or its antigen-binding fragment includes a light chain variable region, which contains the amino acid sequence shown in SEQ ID NO:

80.

5. The anti-CD40 antibody or its antigen-binding fragment as described in claim 1, characterized in that, The anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in any one of SEQ ID NO:81-86, 96, 106, 107, 110, 111 or 118 and a light chain variable region as shown in SEQ ID NO:

80.

6. The anti-CD40 antibody or its antigen-binding fragment as described in claim 2, characterized in that, The anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in any one of SEQ ID NO:81-86, 96, 106, 107, 110, 111 or 118 and a light chain variable region as shown in SEQ ID NO:

80.

7. The anti-CD40 antibody or its antigen-binding fragment as described in claim 3, characterized in that, The anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in any one of SEQ ID NO:81-86, 96, 106, 107, 110, 111 or 118 and a light chain variable region as shown in SEQ ID NO:

80.

8. The anti-CD40 antibody or its antigen-binding fragment as described in claim 4, characterized in that, The anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain variable region as shown in any one of SEQ ID NO:81-86, 96, 106, 107, 110, 111 or 118 and a light chain variable region as shown in SEQ ID NO:

80.

9. The anti-CD40 antibody or its antigen-binding fragment as described in any one of claims 1-8, characterized in that, The anti-CD40 antibody or its antigen-binding fragment is an isotype of IgG, IgM, IgA, IgE or IgD.

10. The anti-CD40 antibody or its antigen-binding fragment according to any one of claims 1-8, characterized in that, The anti-CD40 antibody or its antigen-binding fragment includes a light chain constant region as shown in SEQ ID NO:127, and the anti-CD40 antibody or its antigen-binding fragment includes a heavy chain constant region as shown in SEQ ID NO:

128.

11. The anti-CD40 antibody or its antigen-binding fragment as described in claim 9, characterized in that, The anti-CD40 antibody or its antigen-binding fragment includes a light chain constant region as shown in SEQ ID NO:127, and the anti-CD40 antibody or its antigen-binding fragment includes a heavy chain constant region as shown in SEQ ID NO:

128.

12. The anti-CD40 antibody or its antigen-binding fragment according to any one of claims 1-8, characterized in that, The anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region as shown in any one of SEQ ID NO:81-86, 96, 106, 107, 110, 111 or 118, and a heavy chain constant region as shown in SEQ ID NO:128; and the light chain comprises a light chain variable region as shown in SEQ ID NO:80, and a light chain constant region as shown in SEQ ID NO:

127.

13. The anti-CD40 antibody or its antigen-binding fragment as described in claim 9, characterized in that, The anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region as shown in any one of SEQ ID NO:81-86, 96, 106, 107, 110, 111 or 118, and a heavy chain constant region as shown in SEQ ID NO:128; and the light chain comprises a light chain variable region as shown in SEQ ID NO:80, and a light chain constant region as shown in SEQ ID NO:

127.

14. The anti-CD40 antibody or its antigen-binding fragment as described in claim 10, characterized in that, The anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region as shown in any one of SEQ ID NO:81-86, 96, 106, 107, 110, 111 or 118, and a heavy chain constant region as shown in SEQ ID NO:128; and the light chain comprises a light chain variable region as shown in SEQ ID NO:80, and a light chain constant region as shown in SEQ ID NO:

127.

15. The anti-CD40 antibody or its antigen-binding fragment as described in claim 11, characterized in that, The anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain variable region as shown in any one of SEQ ID NO:81-86, 96, 106, 107, 110, 111 or 118, and a heavy chain constant region as shown in SEQ ID NO:128; and the light chain comprises a light chain variable region as shown in SEQ ID NO:80, and a light chain constant region as shown in SEQ ID NO:

127.

16. An anti-CD40 antibody or its antigen-binding fragment, characterized in that, The anti-CD40 antibody or its antigen-binding fragment comprises a light chain with an amino acid sequence as shown in SEQ ID NO:129, and the anti-CD40 antibody or its antigen-binding fragment comprises a heavy chain with an amino acid sequence as shown in SEQ ID NO:

130.

17. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the anti-CD40 antibody or its antigen-binding fragment as described in any one of claims 1 to 16.

18. A carrier, characterized in that, The carrier comprises the nucleic acid molecule as described in claim 17.

19. A host cell, characterized in that, The host cell contains the nucleic acid molecules as described in claim 17.

20. A reagent kit, characterized in that, The kit contains an anti-CD40 antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 16.

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