Ox40l-binding molecule and use thereof

Abstract

The present invention relates to the field of biomedicine. Specifically, the present invention relates to an OX40L-binding molecule (e.g., an anti-OX40L antibody or an antigen-binding fragment thereof according to the present invention), a multi-specific antigen-binding construct, an antibody conjugate, or a pharmaceutical composition, and a use thereof. The present invention further provides a polynucleotide and expression vector encoding the OX40L-binding molecule (e.g., the anti-OX40L antibody or the antigen-binding fragment thereof according to the present invention) or the multi-specific antigen-binding construct, and a method for producing the OX40L-binding molecule (e.g., the anti-OX40L antibody or the antigen-binding fragment thereof according to the present invention) or the multi-specific antigen-binding construct.

Description

OX40L bound molecules and their uses

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202410693961.9, filed on May 30, 2024, which is incorporated herein by reference in its entirety. Technical Field

[0003] This invention belongs to the field of biomedicine. Specifically, this invention relates to an OX40L binding molecule and its uses. Background Technology

[0004] OX40L, also known as CD134L, CD252, and tumor necrosis factor receptor superfamily member 4 (TNFSF4), acts as a ligand for OX40 and participates in enhancing T cell responses triggered by the T cell receptor (TCR). It is a co-stimulatory receptor molecule. Furthermore, OX40L is a type II transmembrane protein with an intracytoplasmic portion, a transmembrane domain, and an extracellular region.

[0005] OX40L is induced to express, primarily on the surface of activated antigen-presenting cells, such as dendritic cells, B cells, and macrophages. The OX40L trimer can bind to three OX40 receptor molecules, promoting the proliferation, survival, and cytokine production of effector T cells; maintaining memory T cells and promoting Th2 cell differentiation; and inhibiting the function of regulatory T cells.

[0006] Studies have found that OX40L and OX40 are highly expressed at inflammatory sites in various diseases, such as multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and asthma. Blocking the OX40L / OX40 signaling pathway can alleviate various inflammatory and autoimmune diseases. The OX40-OX40L interaction is considered a potential therapeutic target for autoimmune diseases, for example, by blocking the OX40-OX40L interaction to improve self-antigen-specific T cell responses. Furthermore, some researchers have found that OX40L blockade may be more effective than OX40 blockade in reducing autoimmunity; for example, OX40L blockade can prevent the proliferation of active CD4+ T cells, alter cytokine production, prevent migration, and affect T cell polarization. Some in vivo mouse experiments have shown that OX40L neutralizing antibodies can inhibit the development of Th1 / Th17 cell-mediated arthritis, experimental autoimmune encephalomyelitis, and inflammatory bowel disease, as well as Th2 cell-mediated asthma and graft-versus-host disease.

[0007] Currently, several molecules targeting OX40L are being tested in clinical trials for inflammatory or autoimmune diseases, and all are antagonistic antibodies. For example, Oxelumab, developed by Roche and Genmab, is undergoing Phase 1 and Phase 2 clinical trials in allergic rhinitis and asthma; Amlitelimab, developed by Sanofi and Kymab, is undergoing Phase 2 or Phase 3 clinical trials in hidradenitis suppurativa, asthma, and atopic dermatitis. Additionally, Amgen, the Baylor Institute, and others are conducting preclinical development of OX40L monoclonal antibodies. However, there is still a need in this field for highly effective drugs that block the action of OX40 / OX40L, such as antibody drugs. Exploring antibody drugs targeting OX40L for the treatment of inflammatory and autoimmune diseases holds promise for providing more treatment options for these conditions.

[0008] This invention provides an OX40L binding molecule, and based on this binding molecule, multispecific antigen-binding constructs, antibody conjugates, pharmaceutical compositions, and their applications in inflammatory and autoimmune diseases. The OX40L binding molecule of this invention has one or more of the following properties: 1) it can bind to OX40L proteins derived from humans and monkeys; 2) it can inhibit the secretion of IL-2 by immune cells; 3) it can inhibit Th2 differentiation. Summary of the Invention

[0009] In a first aspect, the present invention provides an OX40L binding molecule comprising a heavy chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and wherein HCDR1 comprises the sequence shown in SEQ ID NO:3 or 4 or an amino acid sequence differing from the sequence shown in SEQ ID NO:3 or 4 by an amino acid addition, deletion, or substitution of no more than two amino acids; and / or HCDR2 comprises the sequence shown in SEQ ID NO:8 or 9 or an amino acid sequence differing from the sequence shown in SEQ ID NO:8 or 9 by an amino acid addition, deletion, or substitution of no more than two amino acids; and / or HCDR3 comprises the sequence shown in SEQ ID NO:22, 23, or 24 or an amino acid sequence differing from the sequence shown in SEQ ID NO:22, 23, or 24 by an amino acid addition, deletion, or substitution of no more than two amino acids.

[0010] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:3, 8, and 22; or HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:4, 9, and 23; or HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:4, 9, and 24; or HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:3, 10, and 22; or HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:3, 11, and 22; or HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:4, 12, and 23; or HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:4, 14, and 23; or HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:3, 10, and 22. The amino acid sequences shown in SEQ ID NO:4, 15, 23; or HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, 16, 23; or HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, 12, 24; or HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, 14, 24; or HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, 15, 24; or HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, 13, 23; or HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, 17, 24; or HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, 18, 24.

[0011] In one embodiment, the heavy chain variable region comprises 1) an amino acid sequence shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 42, 34, 40, or 41; 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 42, 34, 40, or 41; or 3 ... Compared to the amino acid sequences shown in NO:28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 42, 34, 40 or 41, the amino acid sequences having one or more added, deleted and / or substituted amino acids, preferably, the addition, deletion and / or substitution does not occur in the CDR region.

[0012] In one embodiment, the OX40L binding molecule is a heavy chain antibody and / or a fully humanized antibody.

[0013] In one embodiment, the OX40L binding molecule comprises 1) an amino acid sequence shown in SEQ ID NO:43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 57, 49, 55, or 56; 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 57, 49, 55, or 56; or 3 ... Compared to the amino acid sequences shown in NO:43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 57, 49, 55 or 56, the amino acid sequences have one or more amino acid additions, deletions and / or substitutions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0014] In a second aspect, the present invention provides a multispecific antigen-binding construct comprising a first antigen-binding portion binding OX40L, wherein the first antigen-binding portion comprises the OX40L binding molecule of the first aspect of the present invention. In a preferred embodiment, the multispecific antigen-binding construct comprises a first antigen-binding portion binding OX40L and a second antigen-binding portion binding a second antigen, wherein the first antigen-binding portion comprises the OX40L binding molecule of the first aspect of the present invention.

[0015] In a third aspect, the present invention provides an antibody conjugate comprising an OX40L binding molecule of the first aspect of the present invention or a multispecific antigen binding construct of the second aspect of the present invention conjugated to at least one therapeutic agent.

[0016] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the OX40L binding molecule of the first aspect of the present invention, the multispecific antigen-binding construct of the second aspect of the present invention, or the antibody conjugate of the third aspect of the present invention, and a pharmaceutically acceptable carrier.

[0017] In a fifth aspect, the present invention provides the use of the OX40L binding molecule of the first aspect of the present invention, the multispecific antigen binding construct of the second aspect of the present invention, the antibody conjugate of the third aspect of the present invention, or the pharmaceutical composition of the fourth aspect of the present invention in the preparation of a medicament for treating a disease or condition.

[0018] In a sixth aspect, the present invention provides a polynucleotide encoding an OX40L binding molecule of the first aspect of the present invention or a multispecific antigen binding construct of the second aspect of the present invention.

[0019] In a seventh aspect, the present invention provides an expression vector comprising the polynucleotide of the sixth aspect of the present invention.

[0020] In an eighth aspect, the present invention provides a host cell comprising the polynucleotide of the sixth aspect of the present invention or the expression vector of the seventh aspect of the present invention.

[0021] In a ninth aspect, the present invention provides a method for generating the OX40L binding molecule of the first aspect of the present invention or the multispecific antigen binding construct of the second aspect of the present invention, the method comprising:

[0022] a) Culture the host cells of the eighth aspect of the present invention under suitable conditions to express the OX40L binding molecule of the first aspect of the present invention or the multispecific antigen binding construct of the second aspect of the present invention; and

[0023] b) Isolate the OX40L binding molecule or multispecific antigen binding construct from a host cell or its culture. Attached Figure Description

[0024] Figures 1A-1D show the results of HCAb antibody against OX40L binding to human and monkey OX40L protein.

[0025] Figures 2A-2B show the results of HCAb antibody against OX40L binding to CHO-K1-hOX40L cells.

[0026] Figures 3A-3B show the results of HCAb antibody against OX40L inhibiting the binding of human OX40L to CHO-K1-OX40 cells.

[0027] Figures 4A-4B show the results of the HCAb antibody against OX40L inhibiting the activity of the HEK293T / hOX40L-induced NFKB-luc reporter gene.

[0028] Figures 5A-5B show the results of HCAb antibody against OX40L inhibiting HEK293T / hOX40L-induced secretion of IL-2 and IL-13 by PBMCs.

[0029] Figures 6A-6B show the efficacy study of anti-OX40L HCAb antibody in a GVHD mouse model.

[0030] Figures 7A-7D show the results of HCAb antibody against OX40L and its PTM mutant binding to human and monkey OX40L protein.

[0031] Figures 8A-8C show the results of binding anti-OX40L HCAb antibody and its PTM mutant to CHO-K1-hOX40L cells.

[0032] Figures 9A-9B show the results of HCAb antibody against OX40L and its PTM mutant inhibiting the binding of human OX40L to CHO-K1-OX40 cells.

[0033] Figures 10A-10C show the results of HCAb antibody against OX40L and its PTM mutant inhibiting HEK293T / hOX40L-induced NFKB-luc reporter gene activity.

[0034] Figures 11A-11C show the results of HCAb antibody against OX40L and its PTM mutant inhibiting HEK293T / hOX40L-induced IL-2 secretion by PBMCs. Detailed Implementation

[0035] definition

[0036] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0037] As used herein, “at least one species” or “one or more species” can mean 1, 2, 3, 4, 5, 6, 7, 8, 9 or more species.

[0038] As used herein, the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of” excludes any unspecified elements, steps, or components. The expression “essentially composed of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “essentially composed of” and “composed of” are encompassed within the meaning of the expression “comprising.”

[0039] As used herein, the connecting term “and / or” between multiple referred elements should be understood to include both individual and combined options. In other words, “and / or” includes both “and” and “or”. For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. Further elements qualified by “and / or” are understood in a similar manner and include any one of them and any combination thereof.

[0040] Unless otherwise stated, any numerical value or range, such as concentration or concentration range, shall in any case be understood to be modified by the term “about”. Thus, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions within that range, unless the context clearly indicates otherwise.

[0041] As used in this article, "OX40 (also known as CD134, ACT35, TNFRSF4)" is a protein located on the T cell membrane, belonging to the TNFR superfamily. It plays an important role in the occurrence and development of inflammatory diseases, autoimmune diseases, tumors, and transplant immunity. OX40 contains three whole-cysteine ​​domains (CRDs) and a partial C-terminal CRD1. It is mainly found in activated CD4+ cells. + T cells and CD8 +It is expressed on T cells, but at relatively low levels on NK cells and NKT cells. The exemplary full-length human OX40 protein has the Uniprot accession number P43489; the exemplary full-length cynomolgus monkey OX40 protein has the NCBI accession number XP_005545179.

[0042] As used herein, the term "OX40L (also known as CD252, TNFSF4, CD134L, or gp34)" refers to the ligand of OX40. OX40 regulates cytokine production by T cells, antigen-presenting cells, and natural killer cells by binding to OX40L. Downstream signaling pathways involving the interaction of OX40 and OX40L, including signal transduction involving downstream molecules such as PI3K / PKB, NF-κB, and NFAT, can enhance the intensity of downstream TCR signaling. The Unipol accession number for the exemplary full-length human OX40L protein is P23510; the Unipol accession number for the exemplary full-length cynomolgus monkey OX40L protein is A0A1D5QQH7.

[0043] As used herein, “antibody” refers to an immunoglobulin or a fragment thereof that specifically binds to an antigenic epitope through at least one antigen-binding site. Antibody encompasses antibody fragments. As used herein, the term “antibody” includes synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, fully humanized antibodies, heavy chain antibodies, nanobodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies. The antibodies described herein include members of any immunoglobulin type (e.g., IgG, IgM, IgD, IgE, IgA, and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass (e.g., IgG2a and IgG2b). In a preferred embodiment, the antibodies of the present invention are heavy chain antibodies and / or fully humanized antibodies.

[0044] As used herein, an “antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the antibody’s variable region (e.g., one or more CDRs and / or one or more antibody-binding sites) that binds to an antigen, and thus retains binding specificity as well as at least a portion of the full-length antibody’s specific binding capacity. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as an antibody fragment derived from the antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that, upon insertion into an antibody framework (e.g., by replacing the corresponding region), acquires an antibody that specifically binds to the antigen.

[0045] As used herein, the terms “heavy-chain-only antibody” and “heavy-chain antibody” are used interchangeably and exist in their broadest sense, referring to an antibody that lacks the conventional antibody light chain and contains only a heavy chain variable region and a heavy chain constant region (e.g., Fc fragment) that does not contain CH1.

[0046] "Fc fragment" generally refers to a crystallizable fragment of a conventional antibody or heavy chain antibody after papain digestion. Typically, the Fc fragment of IgG and heavy chain antibodies may contain a partial hinge region, CH2, and CH3. In this article, the Fc fragment may contain at least a partial hinge region (e.g., all or part of the hinge region), CH2, and CH3.

[0047] Variable regions (i.e., “binding domains”) allow binding molecules to selectively recognize and specifically bind to epitopes on antigens. That is, for example, the light chain variable region (VL) and heavy chain variable region (VH) domains of the binding molecule of an antibody, or combinations of these complementarity-determining region (CDR) subgroups, form a variable region that defines a three-dimensional antigen-binding site. More specifically, the antigen-binding site is defined by three CDRs on each VH and VL chain; for example, in the heavy chain antibody of the present invention, the antigen-binding site is defined by three CDRs on the VH chain. These “complementarity-determining regions” or “CDRs” are discontinuous short sequences of amino acids that are specifically localized to form a binding domain as the antibody adopts its three-dimensional conformation in an aqueous environment. The remaining amino acids in the binding domain are called “framework (FR)” regions, exhibiting minor intermolecular differences. The binding domain formed by the localized CDRs defines a surface complementary to the epitope on the immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its complementary epitope. The amino acids constituting the CDRs and framework regions of any given heavy or light chain variable region can be identified by conventional methods (see “Sequences of Proteins of Immunological Interest”, Kabat, E. et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987), which are incorporated herein by reference in their full text). In this paper, the CDRs (CDRL or LCDR) of the light chain variable region may be referred to as LCDR1, LCDR2, and LCDR3, and the CDRs (CDRH or HCDR) of the heavy chain variable region may be referred to as HCDR1, HCDR2, and HCDR3.

[0048] In this invention, the amino acid sequence of CDR is shown according to the Chothia definition rule (the sequence in the claims of this invention is also shown according to the Chothia definition rule). However, it is well known to those skilled in the art that antibody CDRs can be defined in various ways, such as Chothia (see, for example, Chothia, C. et al., Nature, 342, 877-883 (1989); and Al-Lazikani, B. et al., J. Mol. Biol., 273, 927-948 (1997)) based on the antibody's three-dimensional structure and the topology of the CDR ring; Kabat (see, for example, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242) based on antibody sequence variability; and AbM (Martin, ACR and J. Allen (2007) "Bioinformatics tools for antibody engineering," in S. Dübel (ed.), Handbook of Therapeutic Antibodies. Weinheim: Wiley-VCH). Verlag, pp. 95–118), Contact (MacCallum, R.M et al., (1996) J.Mol.Biol. 262: 732-745), IMGT (Lefranc, M.-P., 2011(6), IMGT, the International ImMunoGeneTics Information System Cold Spring Harb Protoc.; and Lefranc, M.-P. et al., Dev.Comp.Immunol., 27, 55-77(2003)), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. In this paper, multiple CDR numbering systems, such as Chothia, AbM, Kabat, Contact, and IMGT, can be used for the same variable region. Those skilled in the art should understand that although CDRs defined by different numbering systems may be different, CDRs corresponding to the same numbering system represent effective antigen-binding sites that can bind antigenic epitopes.Unless otherwise specified, the terms “CDR” and “complementation-determining region” for a given antibody or its region (e.g., variable region) should be understood to encompass complementation-determining regions defined as described in any of the known schemes described herein. While the scope of protection claimed in the claims of this invention is based on the sequence shown in the Chothia definition rule, amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of this invention.

[0049] Therefore, when referring to antibodies defined by a specific CDR sequence as defined in this invention, the scope of said antibody also includes antibodies whose variable region sequence contains the specific CDR sequence, but whose claimed CDR boundaries differ from those defined in this invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0050] As used herein, the terms “frame region” and “architecture region” are used interchangeably. As used herein, the terms “frame region,” “architecture region,” or “FR” residues refer to the amino acid residues in the antibody variable region other than the CDR sequence as defined above.

[0051] As used in this article, the term "disulfide bond" refers to a covalent bond formed between two sulfur atoms. The amino acid cysteine ​​contains a thiol group that can form a disulfide bond or bridge a second thiol group.

[0052] As used herein, the term "humanized antibody" refers to a non-human antibody modified to increase sequence homology with human antibodies. Humanized antibodies typically retain the antigen-binding ability of their derived non-human antibody and exhibit low immunogenicity in humans. Humanized antibodies can be obtained by antibody engineering of any non-human species antibody or antibody containing sequences derived from a non-human species (e.g., chimeric antibodies). Non-human species may include, for example, mice, rats, rabbits, alpacas, sharks, or non-human primates. Techniques for obtaining humanized antibodies from non-human antibodies are well known to those skilled in the art. For example, the CDR sequence of a non-human antibody (e.g., a mouse antibody) can be transplanted into the framework region of a human antibody. In some cases, in order to maintain the antigen-binding ability and / or stability of humanized antibodies, key amino acid residues of the framework sequence of non-human antibodies (e.g., murine antibodies) can be retained in the human antibody framework region, i.e., "reverse mutation" can be performed (see, for example, Morrison et al. (1984) Proc. Natl. Acad. Sci. 81(21):6851-6855; Neuberger et al. (1984) Nature 312:604-608).

[0053] In this invention, the term "fully human antibody" or "fully humanized antibody" generally refers to an antibody expressed by transferring a human antibody-encoding gene into a genetically engineered animal lacking an antibody gene. All parts of the antibody (including the variable and constant regions) are encoded by a human-derived gene. Methods for obtaining fully human antibodies in this art include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology.

[0054] As used herein, the term "percentage (%) sequence identity" or "sequence identity" has a generally accepted definition in the art, referring to the percentage of identical amino acid sequences between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or a known algorithm). This can be determined using methods known to those skilled in the art, such as publicly available computer software like BLAST, BLAST-2, Clustal Omega, and FASTA software.

[0055] In this document, amino acid sequences “derived from” or “from” a reference amino acid sequence are partially or wholly identical or homologous to the reference amino acid sequence. For example, an amino acid sequence derived from the heavy chain constant region of human immunoglobulin may have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the wild-type sequence from which the heavy chain constant region of human immunoglobulin from which it is derived.

[0056] Non-critical regions of peptides (e.g., CDR regions of antibodies, non-critical amino acids in framework regions, amino acids in constant regions) can be modified, for example by adding, deleting, and / or substituting one or more amino acids, without altering the peptide's function. Suitable conserved amino acid substitutions in peptides or proteins are known to those skilled in the art and can generally be performed without changing the biological activity of the resulting molecule. Typically, those skilled in the art recognize that single amino acid substitutions in non-essential regions of a peptide do not substantially alter its biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub.co., p. 224).

[0057] "Affinity" or "binding affinity" measures the strength of the binding between an antibody and an antigen through non-covalent interactions. Affinity can be determined using conventional techniques known in the art, such as biomembrane interference techniques (e.g., using the Octet Fortebio detection system), radioimmunoassay, surface plasmon resonance assay, enzyme-linked immunosorbent assay (ELISA), or flow cytometry (FACS).

[0058] "Specific binding" generally refers to a binding molecule, such as an antibody or its fragments, variants, or derivatives, binding to an epitope through its antigen-binding domain, and this binding requires some complementarity between the antigen-binding domain and the epitope. By this definition, a binding molecule is said to "specifically bind" to an epitope when it binds to it more readily through its antigen-binding domain than to a random, unrelated epitope. In this paper, the term "specificity" is used to qualitatively analyze the relative affinity of an antibody for a given epitope. For example, binding molecule "A" can be considered to have higher specificity for a given epitope than binding molecule "B," or it can be said that binding molecule "A" binds to epitope "C" with higher specificity than its specificity for related epitope "D."

[0059] As used in this article, the term "EC" 50 The "half-maximal effective concentration" (MCI) refers to the concentration of a drug, antibody, or toxicant that induces a 50% response between baseline and maximum after a specific exposure time.

[0060] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to polymers of deoxyribonucleotides (DNA) or polymers of ribonucleotides (RNA). The terms "polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the sequence of nucleotides in a polynucleotide. Those skilled in the art will understand that the DNA coding strand (sense strand) and its encoded RNA can be considered to have the same nucleotide sequence, with the deoxythymidine nucleotide in the DNA coding strand sequence corresponding to the uridine nucleotide in its encoded RNA sequence.

[0061] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided OX40L binding molecule.

[0062] As used herein, the term “expression” includes the transcription and / or translation of nucleotide sequences. Therefore, expression can involve the production of transcripts and / or peptides.

[0063] As used herein, a "vector" is a medium used to introduce exogenous polynucleotides into host cells, whereby the exogenous polynucleotides are amplified or expressed when the vector is transformed into a suitable host cell. Vectors typically remain free, but can be designed to integrate genes or portions thereof into the chromosome of the genome. As used herein, the definition of a vector encompasses plasmids, linearized plasmids, viral vectors, granules, phage vectors, phage particles, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), etc. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, poxvirus vectors, and baculovirus vectors, etc.

[0064] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0065] Terms such as “treatment,” “curing,” “with treatment,” “relief,” or “with relief” refer to therapeutic measures that cure, alleviate, or reduce the symptoms of an existing diagnosed pathological condition or disorder, and / or halt or slow the progression of an existing diagnosed pathological condition or disorder. Terms such as “prevention,” “defense,” “avoidance,” or “containment” refer to preventive or preventative measures that prevent the progression of an undiagnosed target pathological condition or disorder. Therefore, “subjects in need” may include subjects who already have the disease; subjects who are susceptible to the disease; and subjects who need to prevent the disease.

[0066] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0067] The term "therapeutic effective amount" refers to the amount of antibody, polypeptide, polynucleotide, small organic molecule or other drug that is effective for the disease or condition in the "treated" subject or mammal.

[0068] As used in this article, the term "subject" refers to a mammal, such as a human.

[0069] The antibody designations used herein (such as PR002607, PR002608, PR002619, PR004360...PR004370, PR004371, etc., or similar designations) are for the purpose of distinguishing or identifying antibodies or products only, and are not intended to imply that such designations are characteristic of the antibodies or products of this invention. Those skilled in the art will understand that, for example, other antibodies or products may also use such designations for the purpose of distinction or identification, but this does not mean that they are the same or equivalent antibodies or products. Similarly, similar designations used in the examples are merely for illustrative purposes, and the antibodies or products of this invention are defined by the features described in the appended claims.

[0070] OX40L bound molecules

[0071] This invention provides an OX40L-binding molecule. Generally, an OX40L-binding molecule can include any molecule that specifically binds to OX40L.

[0072] In some implementations, the OX40L binding molecule is a peptide or protein, such as an antibody.

[0073] In a preferred embodiment, the OX40L binding molecule is an antibody against OX40L or an antigen-binding fragment thereof.

[0074] OX40L binding molecules include synthetic antibodies, recombinant antibodies, multispecific antibodies (e.g., bispecific antibodies), human antibodies, non-human antibodies, humanized antibodies, fully humanized antibodies, heavy chain antibodies, nanobodies, chimeric antibodies, intracellular antibodies, and antibody fragments, such as, but not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, disulfide-linked Fv (dsFv), Fd fragments, Fd' fragments, single-chain Fv (scFv), single-chain Fab (scFab), biantibodies, anti-idiotypic (anti-Id) antibodies, or antigen-binding fragments of any of the above antibodies.

[0075] In one embodiment, the OX40L binding molecule is an antibody or an antigen-binding fragment thereof containing a heavy chain variable region.

[0076] In one embodiment, the OX40L binding molecule is a heavy chain antibody or its antigen-binding fragment containing a heavy chain variable region.

[0077] In one embodiment, the OX40L binding molecule is a heavy chain antibody. In another embodiment, the OX40L binding molecule is a fully humanized antibody. In a preferred embodiment, the OX40L binding molecule is both a heavy chain antibody and a fully humanized antibody.

[0078] In one embodiment, the heavy chain variable region contained in OX40L can be fused with another molecule; preferably, the other molecule is the Fc domain of an immunoglobulin; more preferably, the other molecule is the Fc domain of immunoglobulin G1 (IgG1); most preferably, the other molecule is the Fc domain of human immunoglobulin G1 (IgG1).

[0079] In one embodiment, the OX40L binding molecule is a heavy chain antibody containing a heavy chain variable region and an Fc domain of human IgG1.

[0080] In one embodiment, the OX40L binding molecule includes a heavy chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and wherein HCDR1 includes the sequence shown in SEQ ID NO:3 or 4 or an amino acid sequence that differs from the sequence shown in SEQ ID NO:3 or 4 by no more than two amino acid additions, deletions or substitutions.

[0081] In one embodiment, the OX40L binding molecule includes a heavy chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and wherein HCDR2 includes the sequence shown in SEQ ID NO:8 or 9 or an amino acid sequence that differs from the sequence shown in SEQ ID NO:8 or 9 by no more than two amino acid additions, deletions or substitutions.

[0082] In one embodiment, the OX40L binding molecule includes a heavy chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2 and HCDR3, and wherein HCDR3 includes the sequence shown in SEQ ID NO:22, 23 or 24 or an amino acid sequence that differs from the sequence shown in SEQ ID NO:22, 23 or 24 by no more than two amino acid additions, deletions or substitutions.

[0083] In one embodiment, the OX40L binding molecule includes a heavy chain variable region, wherein the heavy chain variable region includes HCDR1, HCDR2, and HCDR3, and wherein HCDR1 includes the sequence shown in SEQ ID NO:3 or 4 or an amino acid sequence that differs from the sequence shown in SEQ ID NO:3 or 4 by no more than two amino acid additions, deletions, or substitutions; HCDR2 includes the sequence shown in SEQ ID NO:8 or 9 or an amino acid sequence that differs from the sequence shown in SEQ ID NO:8 or 9 by no more than two amino acid additions, deletions, or substitutions; and HCDR3 includes the sequence shown in SEQ ID NO:22, 23, or 24 or an amino acid sequence that differs from the sequence shown in SEQ ID NO:22, 23, or 24 by no more than two amino acid additions, deletions, or substitutions.

[0084] As used herein, the term "modification" refers to the presence of a change in a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. Such a change or modification can be obtained by post-synthetic modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide; or by co-translation or post-translational modification of a natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide. The phrase "modified or unmodified" means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide under discussion is optionally modified; that is, the natural amino acid, non-natural amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide under discussion can be modified or unmodified.

[0085] The OX40L binding molecule of the present invention may include modified or unmodified amino acids. For example, during the production, transportation, or storage of the antibody, the amino acid sequence of the antibody may undergo modifications, such as possible post-translational modifications (PTMs), particularly in the CDR region. In the present invention, the PTMs may include modifications currently known to those skilled in the art, including asparagine deamidation (sites NG, NS, NH, etc.), aspartic acid isomerization (sites DG, DP, etc.), N-glycosylation (site NxS / T, where x is any amino acid other than proline), and oxidation, among other related PTMs.

[0086] The presence of PTMs in the variable domains of antibodies, especially antigen-binding regions (such as CDRs), can affect antigen binding and may also alter the physicochemical properties of antibodies. For example, glycosylation, deamidation, isomerization, and oxidation can all increase the instability or heterogeneity of antibody molecules.

[0087] In some implementations, amino acid mutations are used to disrupt the amino acid sequence pattern of PTMs, thereby reducing or eliminating the formation of a specific PTM. Different mutation design methods exist depending on the antibody sequence and the PTM sequence pattern. One method is to replace "hotspot" amino acids (such as N or S in the NS pattern) with amino acids of similar physicochemical properties (such as mutating N to Q). If the PTM sequence pattern originates from a high-frequency mutation in somatic cells and is not present in the germline gene sequence, another method is to replace the sequence pattern with the corresponding germline gene sequence. In practice, multiple mutation removal design methods may be used for the same PTM sequence pattern.

[0088] In some embodiments, the amino acid mutations include mutations at sites of possible post-transcriptional modifications, particularly in the CDR region, including mutations related to antibody aggregation, asparagine deamidation (NG, NS, NH, etc.), aspartic acid isomerization (DG, DP) sensitive sites, N-glycosylation (N-{P}S / T) sensitive sites, and oxidation sensitive sites.

[0089] In some embodiments, the amino acid mutation is based on the amino acid sequences of HCDR1, HCDR2, and HCDR3, where each of HCDR1, HCDR2, and HCDR3 independently introduces no more than two amino acid additions, deletions, or substitutions.

[0090] Exemplary mutations to CDRs (such as HCDR1, HCDR2, or HCDR3) may involve mutations of 2 or 1 amino acid. The same or different numbers of amino acid residues may be selected to mutate among these CDRs (such as HCDR1, HCDR2, or HCDR3). For example, CDR1 may be mutated by 1 amino acid, while CDR2 and CDR3 may not be mutated.

[0091] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:8 (SEQ ID NO:8; NADGSI), wherein the third amino acid is substituted. In another embodiment, the third amino acid in SEQ ID NO:8 is substituted with E (SEQ ID NO:10; NAEGSI).

[0092] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:8 (SEQ ID NO:8; NADGSI), wherein the fourth amino acid is substituted. In another embodiment, the fourth amino acid in SEQ ID NO:8 is substituted with A (SEQ ID NO:11; NADASI).

[0093] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the fourth amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first and fourth amino acids are substituted. In one embodiment, the first amino acid in SEQ ID NO:9 is substituted with A, and the fourth amino acid in SEQ ID NO:9 is substituted with A (SEQ ID NO:12; ASDASS).

[0094] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the fourth amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first and fourth amino acids are substituted. In one embodiment, the first amino acid in SEQ ID NO:9 is substituted with E, and the fourth amino acid in SEQ ID NO:9 is substituted with A (SEQ ID NO:13; ESDASS).

[0095] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the fourth amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first and fourth amino acids are substituted. In one embodiment, the first amino acid in SEQ ID NO:9 is substituted with G, and the fourth amino acid in SEQ ID NO:9 is substituted with A (SEQ ID NO:14; GSDASS).

[0096] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the fourth amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first and fourth amino acids are substituted. In one embodiment, the first amino acid in SEQ ID NO:9 is substituted with S, and the fourth amino acid in SEQ ID NO:9 is substituted with A (SEQ ID NO:15; SSDASS).

[0097] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the fourth amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first and fourth amino acids are substituted. In one embodiment, the first amino acid in SEQ ID NO:9 is substituted with T, and the fourth amino acid in SEQ ID NO:9 is substituted with A (SEQ ID NO:16; TSDASS).

[0098] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the fourth amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first and fourth amino acids are substituted. In one embodiment, the first amino acid in SEQ ID NO:9 is substituted with L, and the fourth amino acid in SEQ ID NO:9 is substituted with A (SEQ ID NO:17; LSDASS).

[0099] In one embodiment, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the fourth amino acid is substituted. In some embodiments, the HCDR2 sequence comprises the amino acid sequence shown in SEQ ID NO:9 (SEQ ID NO:9; NSDGSS), wherein the first and fourth amino acids are substituted. In one embodiment, the first amino acid in SEQ ID NO:9 is substituted with Q, and the fourth amino acid in SEQ ID NO:9 is substituted with A (SEQ ID NO:18; QSDASS).

[0100] In one embodiment, HCDR1 comprises an amino acid sequence as shown in SEQ ID NO:3 or 4.

[0101] In one embodiment, HCDR2 comprises an amino acid sequence as shown in SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.

[0102] In one embodiment, HCDR3 comprises an amino acid sequence as shown in SEQ ID NO:22, 23 or 24.

[0103] In one embodiment, HCDR1 comprises an amino acid sequence as shown in SEQ ID NO:3 or 4; HCDR2 comprises an amino acid sequence as shown in SEQ ID NO:8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18; and HCDR3 comprises an amino acid sequence as shown in SEQ ID NO:22, 23 or 24.

[0104] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 respectively comprise amino acid sequences as shown in SEQ ID NO:3, 8, and 22.

[0105] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 9, and 23, respectively.

[0106] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 9, and 24, respectively.

[0107] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:3, 10, and 22, respectively.

[0108] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:3, 11, and 22, respectively.

[0109] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 12, and 23, respectively.

[0110] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 14, and 23, respectively.

[0111] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 15, and 23, respectively.

[0112] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 16, and 23, respectively.

[0113] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 12, and 24, respectively.

[0114] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 14, and 24, respectively.

[0115] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 15, and 24, respectively.

[0116] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 13, and 23, respectively.

[0117] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 17, and 24, respectively.

[0118] In one embodiment, the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise amino acid sequences as shown in SEQ ID NO:4, 18, and 24, respectively.

[0119] In one embodiment, the heavy chain variable region comprises: 1) an amino acid sequence shown in SEQ ID NO: 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 42, 34, 40, or 41; 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 42, 34, 40, or 41; or 3 ... Compared to the amino acid sequences shown in NO:28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 42, 34, 40 or 41, the amino acid sequences having one or more added, deleted and / or substituted amino acids, preferably, the addition, deletion and / or substitution does not occur in the CDR region.

[0120] In one embodiment, the heavy chain variable region is fused to another molecule. In a preferred embodiment, the other molecule is the Fc domain of an immunoglobulin. In a more preferred embodiment, the other molecule is the Fc domain of human immunoglobulin G1 (IgG1).

[0121] In one embodiment, the OX40L binding molecule comprises 1) an amino acid sequence shown in SEQ ID NO:43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 57, 49, 55, or 56; 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO:43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 57, 49, 55, or 56; or 3 ... Compared to the amino acid sequences shown in NO:43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 57, 49, 55 or 56, the amino acid sequences have one or more amino acid additions, deletions and / or substitutions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

[0122] In one embodiment, the OX40L-binding molecule has at least one of the following characteristics:

[0123] 1) It has affinity activity for OX40L protein;

[0124] 2) It has affinity activity for OX40L positive cells;

[0125] 3) It can inhibit the binding and / or interaction between OX40 and OX40L.

[0126] In one embodiment, the OX40L binding molecule includes antibodies against OX40L PR002607, PR002608, PR002619, PR004360, PR004361, PR004362, PR004363, PR004364, PR004365, PR004366, PR004367, PR004368, PR004369, PR004370, and PR004371, particularly OX40L antibodies PR002607, PR002608, PR002619, PR004360, PR004361, PR004362, PR004364, PR004365, PR004366, PR004367, PR004368, and PR004371.

[0127] Antibody PR002607

[0128] In one aspect, the present invention provides an antibody PR002607 or its antigen-binding fragment targeting OX40L.

[0129] The antibody contains a heavy chain variable region, wherein

[0130] The heavy chain variable region includes:

[0131] HCDR1 contains the amino acid sequence shown in SEQ ID NO:3.

[0132] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:8, and

[0133] HCDR3 contains the amino acid sequence shown in SEQ ID NO:22.

[0134] In one embodiment, the OX40L antibody PR002607 includes a heavy chain variable region (VH).

[0135] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:28 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:28.

[0136] In one embodiment, the OX40L antibody PR002607 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR002607 comprises the amino acid sequence shown in SEQ ID NO:43 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:43.

[0137] Antibody PR002608

[0138] In another aspect, the present invention provides an antibody PR002608 or its antigen-binding fragment targeting OX40L.

[0139] The antibody contains a heavy chain variable region, wherein

[0140] The heavy chain variable region includes:

[0141] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0142] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:9, and

[0143] HCDR3 contains the amino acid sequence shown in SEQ ID NO:23.

[0144] In one embodiment, the OX40L antibody PR002608 includes a heavy chain variable region (VH).

[0145] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:29 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:29.

[0146] In one embodiment, the OX40L antibody PR002608 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR002608 comprises the amino acid sequence shown in SEQ ID NO:44 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:44.

[0147] Antibody PR002619

[0148] In another aspect, the present invention provides an antibody PR002619 or an antigen-binding fragment thereof targeting OX40L.

[0149] The antibody contains a heavy chain variable region, wherein

[0150] The heavy chain variable region includes:

[0151] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0152] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:9, and

[0153] HCDR3 contains the amino acid sequence shown in SEQ ID NO:24.

[0154] In one embodiment, the OX40L antibody PR002619 includes a heavy chain variable region (VH).

[0155] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:30 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:30.

[0156] In one embodiment, the OX40L antibody PR002619 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR002619 comprises the amino acid sequence shown in SEQ ID NO:45 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:45.

[0157] Antibody PR004360

[0158] In another aspect, the present invention provides an antibody PR004360 or an antigen-binding fragment thereof targeting OX40L.

[0159] The antibody contains a heavy chain variable region, wherein

[0160] The heavy chain variable region includes:

[0161] HCDR1 contains the amino acid sequence shown in SEQ ID NO:3.

[0162] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:10, and

[0163] HCDR3 contains the amino acid sequence shown in SEQ ID NO:22.

[0164] In one embodiment, the OX40L antibody PR004360 includes a heavy chain variable region (VH).

[0165] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:31 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:31.

[0166] In one embodiment, the OX40L antibody PR004360 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004360 comprises the amino acid sequence shown in SEQ ID NO:46 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:46.

[0167] Antibody PR004361

[0168] In another aspect, the present invention provides an antibody PR004361 or its antigen-binding fragment targeting OX40L.

[0169] The antibody contains a heavy chain variable region, wherein

[0170] The heavy chain variable region includes:

[0171] HCDR1 contains the amino acid sequence shown in SEQ ID NO:3.

[0172] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:11, and

[0173] HCDR3 contains the amino acid sequence shown in SEQ ID NO:22.

[0174] In one embodiment, the OX40L antibody PR004361 comprises a heavy chain variable region (VH).

[0175] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:32 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:32.

[0176] In one embodiment, the OX40L antibody PR004361 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004361 comprises the amino acid sequence shown in SEQ ID NO:47 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:47.

[0177] Antibody PR004362

[0178] In another aspect, the present invention provides an antibody PR004362 or its antigen-binding fragment targeting OX40L.

[0179] The antibody contains a heavy chain variable region, wherein

[0180] The heavy chain variable region includes:

[0181] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0182] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:12, and

[0183] HCDR3 contains the amino acid sequence shown in SEQ ID NO:23.

[0184] In one embodiment, the OX40L antibody PR004362 comprises a heavy chain variable region (VH).

[0185] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:33 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:33.

[0186] In one embodiment, the OX40L antibody PR004362 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004362 comprises the amino acid sequence shown in SEQ ID NO:48 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:48.

[0187] Antibody PR004363

[0188] In another aspect, the present invention provides an antibody PR004363 or its antigen-binding fragment targeting OX40L.

[0189] The antibody contains a heavy chain variable region, wherein

[0190] The heavy chain variable region includes:

[0191] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0192] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:13, and

[0193] HCDR3 contains the amino acid sequence shown in SEQ ID NO:23.

[0194] In one embodiment, the OX40L antibody PR004363 contains a heavy chain variable region (VH).

[0195] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:34 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:34.

[0196] In one embodiment, the OX40L antibody PR004363 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004363 comprises the amino acid sequence shown in SEQ ID NO:49 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:49.

[0197] Antibody PR004364

[0198] In another aspect, the present invention provides an antibody PR004364 or its antigen-binding fragment targeting OX40L.

[0199] The antibody contains a heavy chain variable region, wherein

[0200] The heavy chain variable region includes:

[0201] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0202] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:14, and

[0203] HCDR3 contains the amino acid sequence shown in SEQ ID NO:23.

[0204] In one embodiment, the OX40L antibody PR004364 contains a heavy chain variable region (VH).

[0205] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:35 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:35.

[0206] In one embodiment, the OX40L antibody PR004364 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004364 comprises the amino acid sequence shown in SEQ ID NO:50 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:50.

[0207] Antibody PR004365

[0208] In another aspect, the present invention provides an antibody PR004365 or its antigen-binding fragment targeting OX40L.

[0209] The antibody contains a heavy chain variable region, wherein

[0210] The heavy chain variable region includes:

[0211] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0212] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:15, and

[0213] HCDR3 contains the amino acid sequence shown in SEQ ID NO:23.

[0214] In one embodiment, the OX40L antibody PR004365 includes a heavy chain variable region (VH).

[0215] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:36 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:36.

[0216] In one embodiment, the OX40L antibody PR004365 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004365 comprises the amino acid sequence shown in SEQ ID NO:51 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:51.

[0217] Antibody PR004366

[0218] In another aspect, the present invention provides an antibody PR004366 or its antigen-binding fragment targeting OX40L.

[0219] The antibody contains a heavy chain variable region, wherein

[0220] The heavy chain variable region includes:

[0221] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0222] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:16, and

[0223] HCDR3 contains the amino acid sequence shown in SEQ ID NO:23.

[0224] In one embodiment, the OX40L antibody PR004366 comprises a heavy chain variable region (VH).

[0225] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:37 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:37.

[0226] In one embodiment, the OX40L antibody PR004366 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004366 comprises the amino acid sequence shown in SEQ ID NO:52 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:52.

[0227] Antibody PR004367

[0228] In another aspect, the present invention provides an antibody PR004367 or its antigen-binding fragment targeting OX40L.

[0229] The antibody contains a heavy chain variable region, wherein

[0230] The heavy chain variable region includes:

[0231] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0232] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:12, and

[0233] HCDR3 contains the amino acid sequence shown in SEQ ID NO:24.

[0234] In one embodiment, the OX40L antibody PR004367 includes a heavy chain variable region (VH).

[0235] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:38 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:38.

[0236] In one embodiment, the OX40L antibody PR004367 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004367 comprises the amino acid sequence shown in SEQ ID NO:53 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:53.

[0237] Antibody PR004368

[0238] In another aspect, the present invention provides an antibody PR004368 or its antigen-binding fragment targeting OX40L.

[0239] The antibody contains a heavy chain variable region, wherein

[0240] The heavy chain variable region includes:

[0241] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0242] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:14, and

[0243] HCDR3 contains the amino acid sequence shown in SEQ ID NO:24.

[0244] In one embodiment, the OX40L antibody PR004368 includes a heavy chain variable region (VH).

[0245] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:39 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:39.

[0246] In one embodiment, the OX40L antibody PR004368 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004368 comprises the amino acid sequence shown in SEQ ID NO:54 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:54.

[0247] Antibody PR004369

[0248] In another aspect, the present invention provides an antibody PR004369 or an antigen-binding fragment thereof targeting OX40L.

[0249] The antibody contains a heavy chain variable region, wherein

[0250] The heavy chain variable region includes:

[0251] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0252] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:17, and

[0253] HCDR3 contains the amino acid sequence shown in SEQ ID NO:24.

[0254] In one embodiment, the OX40L antibody PR004369 includes a heavy chain variable region (VH).

[0255] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:40 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:40.

[0256] In one embodiment, the OX40L antibody PR004369 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004369 comprises the amino acid sequence shown in SEQ ID NO:55 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:55.

[0257] Antibody PR004370

[0258] In another aspect, the present invention provides an antibody PR004370 or its antigen-binding fragment targeting OX40L.

[0259] The antibody contains a heavy chain variable region, wherein

[0260] The heavy chain variable region includes:

[0261] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0262] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:18, and

[0263] HCDR3 contains the amino acid sequence shown in SEQ ID NO:24.

[0264] In one embodiment, the OX40L antibody PR004370 includes a heavy chain variable region (VH).

[0265] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:41 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:41.

[0266] In one embodiment, the OX40L antibody PR004370 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004370 comprises the amino acid sequence shown in SEQ ID NO:56 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:56.

[0267] Antibody PR004371

[0268] In another aspect, the present invention provides an antibody PR004371 or its antigen-binding fragment targeting OX40L.

[0269] The antibody contains a heavy chain variable region, wherein

[0270] The heavy chain variable region includes:

[0271] HCDR1 contains the amino acid sequence shown in SEQ ID NO:4.

[0272] HCDR2, which contains the amino acid sequence shown in SEQ ID NO:15, and

[0273] HCDR3 contains the amino acid sequence shown in SEQ ID NO:24.

[0274] In one embodiment, the OX40L antibody PR004371 contains a heavy chain variable region (VH).

[0275] The heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:42 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:42.

[0276] In one embodiment, the OX40L antibody PR004371 comprises a heavy chain variable region and an Fc domain of human IgG1, wherein the OX40L antibody PR004371 comprises the amino acid sequence shown in SEQ ID NO:57 or an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95% or higher sequence identity with SEQ ID NO:57.

[0277] Multispecific antigen binding construct

[0278] In a second aspect, the present invention provides a multispecific antigen-binding construct comprising a first antigen-binding portion binding OX40L, wherein the first antigen-binding portion comprises the OX40L binding molecule of the first aspect of the present invention. In a preferred embodiment, the multispecific antigen-binding construct comprises a first antigen-binding portion binding OX40L and a second antigen-binding portion binding a second antigen, wherein the first antigen-binding portion comprises the OX40L binding molecule of the first aspect of the present invention.

[0279] As used herein, a “multispecific antigen-binding construct” or “multispecific molecule” refers to a construct that specifically binds to one or more different antigens or one or more different epitopes within the same antigen. A multispecific antigen-binding construct can be a protein, a protein complex, or an antibody. A multispecific antigen-binding construct comprises an antibody or its antigen-binding fragment, which can associate or link with at least one other functional molecule (e.g., another peptide or protein, such as another antibody or ligand for a receptor) to form a molecule that binds to at least two different binding sites or target molecules. A multispecific molecule may be cross-reactive to other related antigens, for example, cross-reactive to the same antigen from other species (homologous) (such as humans or monkeys, e.g., cynomolgus macaques or chimpanzees), or may bind to epitopes shared between two or more different antigens. Exemplary multispecific molecules include multispecific antibodies, such as bispecific antibodies.

[0280] As used herein, the term "multispecific antibody" refers to an antibody capable of specifically binding to two or more (e.g., 2, 3, 4, 5, or 6) different antigenic epitopes. Multispecific antibodies can be, for example, bispecific, trispecific, or tetraspecific antibodies, capable of specifically binding to 2, 3, or 4 antigenic epitopes, respectively. As used herein, the term "antigenic epitope" or "antigenic determinant" refers to a region of an antigen that specifically binds to an antibody's antigen-binding site. Antigenic epitopes typically consist of chemically active surface groups (such as amino acid or sugar side chains) of the antigen and typically possess specific three-dimensional structural properties and specific charge properties. The second antigen can be an antigen other than OX40L. The second antigen can also be OX40L, which binds to a different antigenic epitope on OX40L with the anti-OX40L antibody of the present invention or its antigen-binding fragment. Whether the antigenic epitopes bound by two antibodies are the same can be determined using methods conventional in the art, such as by measuring the competitive binding of two antibodies to the same antigenic epitope using ELISA, flow cytometry, or surface plasmon resonance.

[0281] Multispecific antibodies can be multivalent (e.g., bivalent, trivalent, tetravalent) antibodies, meaning they have multiple antigen-binding sites. Methods for constructing multispecific antibodies using antibodies or antigen-binding fragments of interest are well known to those skilled in the art (see, for example, WO 93 / 08829; Suresh et al., (1986) Methods in Enzymology, 121:210; and Traunecker et al., (1991) EMBO, 10:3655-3659). Multispecific antibodies can be generated and isolated using various techniques known in the art. For example, a polynucleotide encoding a multispecific antibody can be obtained using recombinant DNA technology, optionally cloned into an expression vector, and then transformed into host cells with the polynucleotide or expression vector. The transformed host cells are then cultured under suitable conditions to allow expression of the polynucleotide or expression vector, and finally the multispecific antibody is isolated and purified from the host cells or culture medium. It is also possible to obtain the various parts of a multispecific antibody separately, such as obtaining the first antigen-binding part and the second antigen-binding part as described herein, and then optionally conjugating the various parts through a linker using enzymatic or chemical conjugation techniques to obtain a multispecific antibody that specifically binds to OX40L and other antigens.

[0282] As used herein, “first antigen-binding part” and “second antigen-binding part” refer to amino acid sequences containing antigen-binding sites that can bind to antigen epitopes, and their definitions fall within the scope of the meaning of antibody or antigen-binding fragment.

[0283] The first antigen-binding moiety can be any form of antibody or antigen-binding fragment, including but not limited to Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', and F(ab')2. In some embodiments, the first antigen-binding moiety comprises the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment). In one embodiment, the first antigen-binding moiety comprises VH, which comprises HCDR1, HCDR2, and HCDR3 of the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment) as described above.

[0284] The second antigen-binding moiety can be an antibody or antigen-binding fragment that binds to any antigenic epitope of interest. In one embodiment, the second antigen is an antigen other than OX40L. Antigens that the second antigen-binding moiety can specifically bind to may include: pro-inflammatory cytokines, chemokines, antigens on lymphocytes (such as antigens on T cells or natural killer (NK) cells), immunomodulatory receptors, and immune checkpoint molecules. For example, the second antigen-binding moiety can be an antagonist antibody to an immune-activating receptor or an activator antibody to an immunosuppressive receptor.

[0285] The second antigen-binding part can be any form of antibody or antigen-binding fragment, including but not limited to a single variable domain of an immunoglobulin (e.g., containing the VHH of alpaca or the IgNAR variable domain of shark), Fv, scFv, dsFv, scdsFv, Fab, scFab, Fab', and F(ab')2.

[0286] The first antigen-binding portion and the second antigen-binding portion may optionally be connected via a linker. In some embodiments, the first antigen-binding portion and the second antigen-binding portion are not connected via a linker. In other embodiments, the first antigen-binding portion and the second antigen-binding portion are connected via a linker, such as a peptide linker or a chemical bond. Preferably, the first antigen-binding portion and the second antigen-binding portion are connected via a peptide linker. Exemplary peptide linkers may include, but are not limited to, polyglycine (G), polyalanine (A), polyserine (S), or combinations thereof, such as GGAS, GGGS, GGGSG, or (G4S). n , where n is an integer from 1 to 20.

[0287] Antibody conjugates

[0288] The OX40L binding molecule or multispecific antigen-binding construct of the present invention can be conjugated to at least one therapeutic agent. Conjugates comprising the OX40L binding molecule or multispecific antigen-binding construct of the present invention and a therapeutic agent are also covered within the meaning of the OX40L binding molecule or multispecific antigen-binding construct of the present invention. Therefore, in a third aspect, the present invention also provides an antibody conjugate comprising the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment) or the multispecific antigen-binding construct of the present invention conjugated to at least one therapeutic agent. Antibody-drug conjugates (ADCs) are a typical type of antibody conjugate, wherein the therapeutic agent can be, for example, a hormone molecule, such as a glucocorticoid.

[0289] As used herein, "conjugation" refers to the connection of two or more parts to each other through covalent or non-covalent interactions. In a preferred embodiment, the OX40L binding molecule or multispecific antigen-binding construct of the present invention is conjugated with a therapeutic agent.

[0290] The therapeutic agent may be selected from therapeutic antibodies (e.g., antibodies that specifically bind to another antigen or their antigen-binding fragments), oligonucleotides and their analogues (e.g., interfering RNA), hormonal molecules, immunosuppressants, etc. In one embodiment, the therapeutic agent may include, but is not limited to, hormonal molecules such as prednisone, budesonide, or prednisolone; calcineurin inhibitors such as cyclosporine or tacrolimus; mTOR inhibitors such as sirolimus or everolimus; IMDH inhibitors such as azathioprine, leflunomide, or mycophenolate mofetil; therapeutic antibodies such as abatacept, adalimumab, anakinin, cetuzumab, etanercept, golimumab, infliximab, ixekizumab, nateximab, rituximab, secukinumab, tocilizumab, uterotumab, or vedolizumab; and monoclonal antibodies such as baliximab, dacrolimus, or muromonab.

[0291] The therapeutic agent can be conjugated directly or indirectly (e.g., via a linker) to the OX40L binding molecule or multispecific antigen-binding construct of the present invention. The linker may contain an active group for covalent conjugation, such as an amine, hydroxylamine, maleimide, carboxyl, phenyl, thiol, mercapto, or hydroxyl group. In one embodiment, the linker is a chemical bond. In one embodiment, the linker comprises an amino acid or a peptide consisting of 2-10 amino acids.

[0292] Pharmaceutical Composition

[0293] In a fourth aspect, the present invention also provides a pharmaceutical composition comprising the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment), a multispecific antigen-binding construct, or an antibody conjugate, and a pharmaceutically acceptable carrier.

[0294] Pharmaceutically acceptable carriers may include, but are not limited to: diluents, binders and adhesives, lubricants, disintegrants, preservatives, mediators, dispersants, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, etc. Sugar alcohols, tartaric acid, phosphoric acid, etc.), solubilizers, gelling agents, softeners, solvents (e.g., water, alcohol, acetic acid, and syrup), buffers (e.g., phosphate buffers, histidine buffers, and acetate buffers), surfactants (e.g., nonionic surfactants such as polysorbate 80, polysorbate 20, poloxamer, or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption delay agents, chelating agents, and emulsifiers. For pharmaceutical compositions comprising OX40L binding molecules (e.g., the anti-OX40L antibody or its antigen-binding fragment of the present invention), multispecific antigen-binding constructs, or antibody conjugates, suitable carriers may be selected from buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine buffer, histidine salt buffer), isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamer), or combinations thereof.

[0295] The pharmaceutical compositions provided by this invention can be in various dosage forms, including but not limited to solid, semi-solid, liquid, powder, or lyophilized forms. For pharmaceutical compositions containing OX40L binding molecules (e.g., the anti-OX40L antibody or its antigen-binding fragment of this invention) or multispecific antigen-binding constructs, preferred dosage forms are typically, for example, injections and lyophilized powders.

[0296] The pharmaceutical compositions provided herein can be administered to a subject by any method known in the art, such as systemic or local administration. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, intraperitoneal, intradermal, intramuscular, subcutaneous, or intracavitary), local (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or local). Preferably, the pharmaceutical compositions are suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Administration methods may include, for example, injection or infusion.

[0297] Those skilled in the art will understand that the exact dosage will depend on a variety of factors, such as the pharmacokinetic properties of the pharmaceutical composition, the duration of treatment, the excretion rate of a particular compound, the purpose of treatment, the route of administration, and the condition of the subject, such as the patient’s age, health status, weight, sex, diet, medical history, and other factors known in the medical field.

[0298] As a general guideline, the dosage range for the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody or its antigen-binding fragment of the present invention), multispecific antigen-binding construct, or antibody conjugate may be from about 0.0001 to 100 mg / kg, more typically from 0.01 to 20 mg / kg of subject body weight. Exemplary treatment regimens may require dosing once weekly, twice weekly, three times weekly, every two weeks, every three weeks, every four weeks, once monthly, every three months, every three to six months, or with a slightly shorter initial dosing interval followed by a longer dosing interval.

[0299] treat

[0300] In a fifth aspect, the present invention relates to the use of the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment), multispecific antigen-binding construct, antibody conjugate or pharmaceutical composition in the preparation of a medicament for treating a disease or condition in a subject.

[0301] This invention also relates to the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment), multispecific antigen-binding constructs, antibody conjugates, or pharmaceutical compositions for the treatment of diseases or conditions.

[0302] The present invention also provides a method for treating a disease or condition in a subject, the method comprising administering to the subject a therapeutically effective amount of the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment), a multispecific antigen-binding construct, an antibody conjugate, or a pharmaceutical composition.

[0303] The term "therapeutic effective dose" as used herein refers to a dose that reduces the severity of disease symptoms, increases the frequency and duration of asymptomatic periods, or prevents damage or disability caused by disease-related suffering. Therapeutic effective doses can vary depending on many different factors, including the route of administration, target site, patient's physiological state, whether the patient is human or another animal, other medications administered, and whether the treatment is prophylactic or therapeutic. In some embodiments, the patient is human, but non-human mammals, including transgenic animals, may also be treated. Therapeutic doses can be titrated using conventional methods known to those skilled in the art to optimize safety and efficacy.

[0304] In one embodiment, the disease or condition described above is an inflammatory or autoimmune disease. In one embodiment, the disease or condition includes, but is not limited to, graft-versus-host disease (GVHD), atopic dermatitis, Crohn's disease, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, adolescent / childhood type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, and primary biliary tract infection. Cholangitis, primary biliary cirrhosis, non-alcoholic steatohepatitis (NASH), psoriasis, rheumatoid arthritis, scleroderma, CREST syndrome, Sjögren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, Wegener's granulomatosis, Addison's disease (adrenal insufficiency), Hashimoto's thyroiditis, infertility / sterility, ANCA-associated vasculitis, psoriatic arthritis, celiac disease, ulcerative colitis, lichen sclerosus, Behçet's disease, asthma, allergic rhinitis, and hidradenitis suppurativa.

[0305] The OX40L binding molecules of the present invention (e.g., the anti-OX40L antibody or its antigen-binding fragment of the present invention), multispecific antigen-binding constructs, antibody conjugates, or pharmaceutical compositions may be administered in combination with at least one or more of the therapeutic agents described herein. There are no limitations on the manner of administration. For example, all of the above-described therapeutic agents may be administered at once or separately.

[0306] In one implementation scheme, the combined therapeutic agents may include, but are not limited to, nonsteroidal anti-inflammatory drugs (such as acetylsalicylic acid (aspirin), diflunisal, sulfasalazine, acetaminophen, mefenamic acid, meclofenamic acid, flufenamic acid, ibuprofen, naproxen, fenprofen, tylosin, flurbiprofen, oxapzin, piroxicam, tenoxicam, etc.), and COX-2 inhibitors (such as celecoxib, rofecoxib, vardicoxib, clomecoxib, parecoxib, and etoric acid). oxib), glucocorticoids (such as prednisone / prednisolone, methylprednisolone and fluorinated glucocorticoids (such as dexamethasone and betamethasone)), cDMARDs (common disease-modifying antirheumatic drugs, such as methotrexate, leflunomide, gold compounds, sulfasalazine, azathioprine, cyclophosphamide, antimalarial drugs, d-penicillamine, cyclosporine), and anti-TNF antibodies (such as infliximab, etanercept, adalimumab, golimumab, cetolizumab Pegol)).

[0307] Polynucleotides, vectors and host cells

[0308] In a sixth aspect, the present invention provides a polynucleotide encoding the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment) or a multispecific antigen-binding construct.

[0309] The polynucleotides of the present invention can be obtained using methods known in the art. For example, the polynucleotides of the present invention can be isolated from phage display libraries, yeast display libraries, immunized animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemically synthesized. Polynucleotides encoding binding molecules (e.g., antibodies or their antigen-binding fragments) or multispecific antigen-binding constructs can be prepared from immunized animals or by chemical synthesis, and then expression vectors can be constructed using these polynucleotides. The polynucleotides of the present invention can be codon-optimized for the host cells used for expression.

[0310] In a seventh aspect, the present invention also provides expression vectors comprising the polynucleotides of the present invention. The expression vectors may further comprise additional polynucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The polynucleotides of the present invention may be present in one or more expression vectors. In one embodiment, the polynucleotides of the present invention are prepared as recombinant nucleic acids. Recombinant nucleic acids can be prepared using techniques well known in the art, such as chemical synthesis, DNA recombination techniques (e.g., polymerase chain reaction (PCR) techniques), etc.

[0311] In an eighth aspect, the present invention also provides a host cell comprising the polynucleotide or expression vector of the present invention. The polynucleotide or expression vector of the present invention can be introduced into a suitable host cell using various methods known in the art. Such methods include, but are not limited to, liposome transfection, electroporation, viral transduction, and calcium phosphate transfection.

[0312] In a preferred embodiment, the host cell is used to express the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment) or a multispecific antigen-binding construct. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as *Escherichia coli*) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Suitable mammalian host cells for antibody expression include, but are not limited to, human cervical cancer cells (HeLa cells), human embryonic kidney cells (HEK cells, such as HEK 293 cells), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for antibody expression.

[0313] In a ninth aspect, the present invention also provides a method for generating the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment) or a multispecific antigen-binding construct, the method comprising:

[0314] a) Culture the host cells of the present invention under suitable conditions to express the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment) or a multispecific antigen-binding construct; and

[0315] b) Isolate the OX40L binding molecule (e.g., anti-OX40L antibody or its antigen-binding fragment) or a multispecific antigen-binding construct from a host cell or its culture.

[0316] Reagent test kit

[0317] The present invention also provides a kit comprising the OX40L binding molecule of the present invention (e.g., the anti-OX40L antibody of the present invention or its antigen-binding fragment), a multispecific antigen-binding construct, an antibody conjugate, or a pharmaceutical composition, and instructions for use. The kit may also comprise a suitable container. In some embodiments, the kit further comprises a device for administration. The kit generally includes a label indicating the intended use and / or method of use of the kit contents. The term “label” includes any written or recorded material provided on or with the kit or otherwise accompanied by the kit. Beneficial effects

[0318] The OX40L binding molecule of the present invention can exhibit excellent effects, for example, but not limited to: 1) The anti-OX40L antibody of the present invention is a novel fully human antibody containing only the "heavy chain" and has binding activity to human OX40L and cynomolgus monkey OX40L. This heavy chain antibody is only half the size of a conventional IgG antibody. Due to the absence of a light chain, this antibody can be advantageously used as a bispecific antibody and solves the problems of light chain mismatch and heterodimerization; 2) It can bind to OX40L proteins derived from humans and cynomolgus monkeys; 3) It can bind to cells overexpressing OX40L; 4) It can inhibit the binding of OX40L to cells overexpressing OX40; 5) It can inhibit the activity of downstream NF-κB induced by the interaction between OX40 and OX40L; 6) It can inhibit the activity of PBMCs secreting IL-2 and IL-13 induced by the interaction between OX40 and OX40L.

[0319] Example

[0320] A further understanding of the invention can be obtained by referring to some specific embodiments given herein, which are for illustrative purposes only and are not intended to limit the scope of the invention in any way. Obviously, many modifications and variations can be made to the invention without departing from its spirit; therefore, these modifications and variations are also within the scope of protection claimed in this application. Experimental methods in the following embodiments, unless otherwise specified, are performed according to conventional methods and conditions, or as selected in the product manual.

[0321] Example 1. Obtaining fully human HCAb antibodies

[0322] 1.1 Immunization of Harbour HCAb mice

[0323] Harbour HCAb mice (Harbour Antibodies BV, WO 2002 / 085945A3) are transgenic mice carrying a human immunoglobulin library that produce novel, heavy-chain-only antibodies, only half the size of traditional IgG antibodies. These antibodies possess only the variable heavy-chain domain of human antibodies and the constant Fc domain of mice. Due to the absence of light chains, these antibodies virtually eliminate the problems of light chain mismatch and heterodimerization, enabling this technology platform to develop products that are difficult to achieve with traditional technology platforms.

[0324] Harbour H2L2 transgenic mice aged 6-8 weeks were immunized with HEK293-hOX40L (Chempartner, Shanghai), a stable transgenic cell line highly expressing human OX40L, and then housed under specific pathogen-free (SPF) conditions. During the first immunization, each mouse was injected intraperitoneally, axillarily, and in the groin with a total of 2 × 10⁻⁶ cells. 6 Immunogen HEK293-hOX40L cells and 0.2 mL of complete Freund's adjuvant (CFA, Sigma, #F5881). To enhance the immune response, 2 × 10⁻⁶ cells were administered two weeks after the first immunization. 6 Immunogen HEK293-hOX40L cells, along with 200 μL of Ribi (Sigma adjuvant system, Sigma, #S6322), were injected into the intraperitoneal cavity and subcutaneous lymph nodes of each mouse. Subsequently, each mouse received 2 × 10⁻⁶ cells every two weeks. 6 Immunogen and 200 μL Ribi adjuvant were administered, along with the first immunization, for a total of six doses. One week after the fourth and sixth doses during this immunization period, mouse blood was collected and diluted 10-fold to obtain six concentrations (1:100, 1:1000, 1:10000, 1:100000, and 1:1000000). ELISA was performed on ELISA plates coated with human OX40L protein (Acro, Shanghai) to determine the titer of anti-human OX40L in mouse blood. Flow cytometry was used to detect the specific reactivity of two concentrations of mouse blood (1:100 and 1:1000) to OX40L-overexpressing CHO-K1-hOX40L cells (Chempartner, Shanghai) and CHO-K1 blast cells. The blank control group (PB) consisted of serum from mice before immunization.

[0325] 1.2 The HCAb antibody sequence against OX40L was obtained by screening using mammalian cell expression methods.

[0326] Once the titer of OX40L-specific antibodies in mouse serum reached a certain level, mouse spleen cells were removed to isolate B cells. RNA was extracted from the B cells and reverse transcribed into cDNA (SuperScript IV First-Strand synthesis system, Invitrogen, 18091200). Then, the human VH gene was amplified by PCR using specific primers. The amplified VH fragment was constructed into the mammalian cell expression plasmid pCAG vector encoding the Fc domain sequence of the human IgG1 antibody heavy chain.

[0327] The constructed plasmid was transfected into mammalian host cells (such as human embryonic kidney cells HEK293) for expression to obtain HCAb antibodies. The binding of the HCAb-expressing supernatant to human OX40L-His protein was detected using mirrorball screening. The obtained positive monoclonal antibodies were further tested for binding to stable CHO-K1 cell lines overexpressing human OX40L, and screening was performed using flow cytometry.

[0328] 584 monoclonal antibodies specifically binding to CHO-K1-hOX40L and human / monkey OX40L-His proteins were obtained. The nucleotide and amino acid sequences encoding the variable domains of the antibody molecules were obtained using conventional sequencing methods. After removing repetitive sequences, 63 fully human HCAb monoclonal antibodies against OX40L with unique sequences were obtained. Based on the binding ability to human OX40L-overexpressing cells and the ability to inhibit IL-2 secretion by PBMCs, the top 35 antibodies were selected for recombinant expression. The purified monoclonal antibodies were further tested for binding to CHO-K1-hOX40L cells using flow cytometry, and the top 3 antibody sequences were selected as candidate molecules.

[0329] Example 2: Preparation of fully human recombinant antibody against OX40L

[0330] 2.1 Antibody Expression and Purification

[0331] After obtaining the heavy chain variable domain sequence encoding the HCAb antibody molecule, conventional recombinant DNA technology can be used to fuse the heavy chain variable domain sequence with the corresponding human antibody heavy chain constant domain sequence to obtain recombinant HCAb antibody molecules. In this embodiment, the antibody heavy chain variable domain sequence (VH) is synthesized and cloned into a mammalian cell expression plasmid vector encoding the human IgG1 antibody heavy chain constant domain sequence to encode the full-length heavy chain that produces the HCAb antibody.

[0332] Transfecting mammalian host cells (such as human embryonic kidney cells HEK293) with a plasmid encoding the antibody, and then using conventional recombinant and purification techniques, yields purified anti-OX40L fully human recombinant HCAb antibody. The specific steps involve transfecting HEK293 cells into FreeStyle... TM Expand the cell culture in F17 Expression Medium (Thermo, A1383504). Adjust the cell concentration to 6E5 cells / mL and culture at 37°C in an 8% CO2 shaker for 24 h, at which point the cell concentration is approximately 1.2E6 cells / mL. Prepare 30 mL of the above cells. Dissolve the plasmid encoding the fully human recombinant antibody against OX40L from Example 1 in 1.5 mL of Opti-MEM serum-depleted medium (Thermo, 31985088). Add 12 μL of 1 mg / mL PEI (Polysciences, #23966-2) to 1.5 mL of Opti-MEM and let it stand for 5 min. Slowly add the PEI to the plasmid and incubate at room temperature for 10 min. While slowly adding the plasmid-PEI mixture, mix well and culture at 37°C in an 8% CO2 shaker for 5 days. Observe cell viability after 5 days. Collect the culture, centrifuge at 3300G for 10 min, and collect the supernatant. Centrifuge the supernatant at high speed to remove impurities. Equilibrate the culture containing MabSelect with PBS (pH 7.4). TM The sample was washed with 2-5 times the column volume of PBS using a gravity column (Bio-Rad, #7311550) from GE Healthcare Life Science (#71-5020-91AE). The supernatant was then passed through the column, and the column was washed with 5-10 times the column volume of PBS. The target protein was then eluted with 0.1M glycine at pH 3.5, followed by adjustment to neutral with Tris-HCl at pH 8.0. Finally, the solution was concentrated and replaced using an ultrafiltration tube (Millipore, UFC901024) to obtain the purified antibody solution. The antibody concentration was determined by NanoDrop absorbance at 280 nm, and antibody purity was determined using SEC-HPLC and SDS-PAGE.

[0333] 2.2 Protein purity and aggregate composition were analyzed using SEC-HPLC.

[0334] Size exclusion chromatography (SEC) was used to determine and analyze the purity and aggregate form of protein samples. First, an analytical column, TSKgel G3000SWxl (Tosoh Bioscience, 08541, 5 μm, 7.8 mm × 30 cm), was connected to a high-performance liquid chromatograph (HPLC) (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS at room temperature for at least 1 h. The protein concentration was adjusted to 1 mg / mL, and at least 10 μg of protein sample was filtered through a 0.22 μm filter before being injected into the system. The injection method was set as follows: the protein sample was passed through the column with PBS (pH 7.4) at a flow rate of 1 mL / min, the run time was no more than 20 min, and the detection wavelength was 280 nm. After sample collection, the chromatograms were integrated and relevant data were calculated using ChemStation software.

[0335] 2.3 Protein purity and hydrophobicity were analyzed using HIC-HPLC.

[0336] Hydrophobic interaction chromatography (HIC) was used to determine and analyze the purity and hydrophobicity of protein samples. First, an analytical column, TSKgel Butl1-NPR (Tosoh Bioscience, 12947, 4.6 mm × 3.5 cm), was connected to a high-performance liquid chromatograph (HPLC) (Agilent Technologies, Agilent 1260 Infinity II) and equilibrated with PBS at room temperature for at least 1 hour. The protein concentration was adjusted to 1 mg / mL, and the injection volume was 20 μL. The injection method was set as follows: protein sample was injected at a rate of 0.7 mL / min from 100% mobile phase A (20 nM histidine, 1.8 M ammonium sulfate, pH 6.0) to 100% mobile phase B (20 mM histidine, pH 6.0), with a run time not exceeding 16 minutes, and the detection wavelength was 280 nm. After sample collection, the chromatograms were integrated and relevant data were calculated using ChemStation software.

[0337] 2.4 Determination of the thermal stability of antibody molecules using DSF

[0338] The thermal stability of proteins was determined using differential scanning fluorometry (DSF). This method uses a real-time quantitative PCR instrument to monitor changes in the fluorescence intensity of unfolded protein molecules bound to the dye, reflecting the protein denaturation process and thus the thermal stability of the protein, expressed as denaturation temperature (Tm). In this example, 10 μg of protein was added to a 96-well PCR plate (Thermo, AB-0700 / W), followed by 2 μL of a 100-fold diluted SYPROTM dye (Invitrogen, 2008138), and buffer was added to make a total volume of 40 μL per well. The PCR plate was sealed and placed in a real-time quantitative PCR instrument (Bio-Rad CFX96 PCR System), incubated at 25°C for 5 min, and then gradually increased from 25°C to 95°C in 0.2°C / 0.2 min temperature gradients. After the test, the temperature was lowered to 25°C. The data were analyzed using FRET scanning mode and Bio-Rad CFX Maestro software, and the sample Tm was calculated. The expression information and physicochemical properties of the obtained antibodies are shown in Table 1.

[0339] Table 1. Expression and physicochemical properties of anti-OX40L antibody

[0340] 2.5 Sequence of anti-OX40L antibody

[0341] Table 2 lists the heavy chain variable domain (VH) amino acid sequence, the full-length heavy chain (HC) amino acid sequence, and the CDR amino acid sequence as defined by Chothia's rules for candidate anti-OX40L HCAb antibodies. Furthermore, the inventors have also produced and prepared the positive control antibody Oxelumab against OX40L, the amino acid sequence of which is shown in SEQ ID NO:58-59.

[0342] Table 2. Candidate OX40L HCAb antibody sequences (SEQ ID NO:)

[0343] Example 3: Binding of fully human anti-OX40L HCAb antibody to human / monkey OX40L protein

[0344] This embodiment uses ELISA to investigate the ability of fully human anti-OX40L HCAb antibody molecules to bind human and monkey OX40L protein in vitro. 2 mg / mL of human or monkey OX40L protein (Acro) was incubated overnight at 4°C in a high-adsorption 96-well plate (Corning, 9018). After washing three times with PBST, serially diluted anti-OX40L antibody was added and incubated at 37°C for 1.5 h. After washing three times with PBST, peroxidase-conjugated anti-hFc secondary antibody (Jacksonlab) was added and incubated at 37°C for 1 h. After washing three times with PBST, TMB chromogenic substrate was added. After reaching a certain color development level, stop solution (1N HCl) was added and the absorbance value at 450 nm was measured.

[0345] The detection results are shown in Figures 1A-1D and Table 3. All candidate anti-OX40L HCAb antibodies could bind to the human and monkey OX40L protein, and the binding ability of the antibody to the protein was positively correlated with the antibody concentration. The EC50 of the three HCAb antibody molecules was superior to that of the reference antibody Oxelumab.

[0346] Table 3. Antibody binding to human or monkey OX40L protein

[0347] Example 4: Binding of fully human anti-OX40L HCAb antibody to cells overexpressing human OX40L

[0348] CHO-K1-hOX40L cells digested with trypsin were resuspended in PBS to a concentration of 1×10⁻⁶ cells / mL. 6 Add 100 mL of cell suspension to each well of a 96-well V-plate (Corning, 3894). Centrifuge the 6-well V-plates at 1000 rpm for 5 minutes, and wash once with PBS + 0.5% BSA (VWR, #0332-100G). Resuspend the cells in serially diluted antibody and incubate at 4°C for 1 hour. After washing twice with PBS + 0.5% BSA, incubate the cells with 100 μL of Alexa Fluor 488 goat anti-human IgG (Life Technologies, 1:1000, #A-11013) in the dark at 4°C for 1 hour, and wash twice with PBS + 0.5% BSA. Resuspend the cells in 200 μL of PBS + 0.5% BSA. Analyze the fluorescence intensity of the cells using FACS flow cytometry.

[0349] The detection results are shown in Figures 2A-2B and Table 4. All three candidate HCAb antibody molecules specifically bound to CHO-K1-hOX40L cells, and the detected antibody binding ability was positively correlated with the antibody concentration. The EC50 of the three HCAb antibody molecules was comparable to that of the reference antibody Oxelumab.

[0350] Table 4. Antibody binding to CHO-K1-hOX40L cells

[0351] Example 5: Affinity determination of fully human anti-OX40L HCAb antibody to human OX40L protein

[0352] The KD value of HCAb antibodies was determined using the conventional Octet method in this art. Candidate antibodies were immobilized onto the AHC sensor to a height of 0.2 nm. They were bound to diluted human OX40L protein for 200 seconds, dissociated for 600 seconds, and then regenerated for 15 seconds with 10 mM glycine hydrochloride buffer (pH 1.5). The binding rate (kon) and dissociation rate (kdis) (also known as koff) were calculated using a simple one-to-one Languir binding model (Octet Red96 software). The equilibrium dissociation constant (KD) was calculated as the ratio kdis(koff) / kon. The results are shown in Table 5. The affinity of the candidate antibody was comparable to or better than that of the reference antibody Oxelumab.

[0353] Table 5. Affinity assay of antibody binding to human OX40L protein

[0354] Example 6: Antibody inhibits the binding of human OX40L to CHO-K1-OX40 cells.

[0355] This embodiment uses FACS to investigate the ability of antibody molecules to block the binding of human OX40L protein to CHO-K1-OX40. The concentration of trypsin-digested CHO-K1-OX40 cells (Beijing Kangyuan Bochuang, KYinno) was adjusted to 1E6 / mL. Serially diluted antibody and 0.25 mg / mL hOX40L-mFc (Sino Bio, 13127-H04H) were incubated at room temperature for 15 min. 100 mL of the cell suspension was added to a 96-well V plate. After centrifugation at 500g for 5 min, the supernatant was discarded. The mixture of antibody and hOX40L-mFc was added to the cells and incubated at 4°C for 1 h. After washing twice with FACS buffer (PBS + 2% FBS), 100 μL of anti-mFc-APC was added, and the cells were incubated at 4°C in the dark for 0.5 h. After washing twice more with FACS buffer, the fluorescence intensity of the cells was analyzed using FACS flow cytometry.

[0356] The test results are shown in Figures 3A-3B and Table 6. The IC50 values ​​of the three HCAb antibody molecules were all superior to those of the reference antibody Oxelumab.

[0357] Table 6. Antibody inhibition of binding between human OX40L and CHO-K1-OX40 cells

[0358] Example 7: Antibody inhibits HEK293T / hOX40L-activated NFKB-luc reporter gene activity

[0359] It has been reported that the OX40-OX40L interaction can directly activate the NF-κB signaling pathway and enhance downstream signaling pathways of the TCR, such as PI3K / PKB and NFAT (see, for example, Willoughby J, et al. OX40: Structure and function - What questions remain? Mol Immunol. 2017 Mar; 83:13-22.). This example investigated the effect of anti-OX40L antibody on the activity of fluorescent reporter genes regulated by the NFκB gene promoter. Two E4 HEK293T / hOX40L cells (Beijing Kangyuan Bochuang, KYinno, HEK293T cells overexpressing human OX40L) were seeded in each well and incubated overnight at 37°C in black 96-well plates. The next day, 50 μL of culture medium was removed, and 20 μL of serially diluted antibody was added. After incubating at 37°C for 30 min, add 20 μL of 293T-OX40-NF-κB cells (HEK293 reporter cells that express the luciferase reporter gene under the control of OX40 and NF-κB response elements, BPS Biosciences, #60482), 5E4 cells per well. After incubating at 37°C for 5 / 6 h, add 50 μL of L Bio-glo reagent, mix thoroughly, and read the fluorescence value using Envision.

[0360] The detection results are shown in Figures 4A-4B and Table 7. All three HCAb antibody molecules can inhibit the reporter gene activity of NFKB-luc, and the inhibition effect is significantly better than that of Oxelumab.

[0361] Table 7. Antibody Inhibition of Human NFKB-luc Reporter Gene Activity

[0362] Example 8: Antibody inhibits the activity of HEK293T / hOX40L-stimulated PBMCs in secreting IL-2 and IL-13.

[0363] This study investigated the inhibitory effect of candidate anti-OX40L antibodies on the secretion of IL-2 and IL-13 by HEK293T / hOX40L-stimulated PBMCs. 8E3 HEK293T / hOX40L cells per well were seeded in 96-well plates (Corning, 3599) overnight. The next day, 50 μL of serially diluted antibody was added, followed by 100 μL of PBMCs (1.5E5 per well) and phytohemagglutinin (PHA) to a final concentration of 0.25 μg / mL. After incubation at 37°C for 48 h, the supernatant was collected, and the levels of IL-2 (Thermo, 88-7025-88) and IL-13 (R&D, DY213-05) were detected by ELISA.

[0364] The test results are shown in Figures 5A-5B and Table 8. All three HCAb antibody molecules could inhibit the secretion of IL-2 and IL-13. Among them, PR002608 and PR002619 showed comparable or better inhibitory effects compared to Oxelumab.

[0365] Table 8. Antibody inhibition of IL-2 and IL-13 secretion by human OX40L-induced PBMCs

[0366] Example 9: Therapeutic effect of antibody in GVHD mouse disease model

[0367] NCG mice (Gempharmatech) were injected with 1E7 human PBMCs via the tail vein. Starting from day one of injection, NCG mice were injected with 200 μg of candidate anti-OX40L antibody or PBS as a control three times a week for a total of 3 weeks.

[0368] Survival curve analysis showed that the candidate anti-OX40L antibody or the positive control Oxelumab could inhibit mouse mortality by 100% (Figure 6A). Using FACS to determine the proportion of human CD45 in all CD45 (human and mouse CD45) positive lymphocytes in whole mouse blood, it was found that the candidate anti-OX40L antibody or the positive control Oxelumab significantly reduced the proportion of human CD45 positive lymphocytes, demonstrating that the proliferation of human CD45 positive lymphocytes was inhibited (Figure 6B).

[0369] Furthermore, according to Figures 6A-6B, the PR002608 molecule showed comparable therapeutic effects to the control molecule Oxelumab.

[0370] Example 10 Optimization of antibody post-translational modification (PTM) sites

[0371] Post-translational modifications (PTMs) sometimes introduce chemical modifications into the amino acid chains of proteins or peptides after translation and synthesis in cells. For antibodies, some PTM sites are highly conserved. For example, the conserved amino acid asparagine (Asn) at position 297 (EU number) of the constant domain of human IgG1 antibody typically undergoes glycosylation to form a glycan, which is crucial for antibody structure and related effector function. However, if PTMs are present in the variable domains of antibodies, especially antigen-binding regions (such as CDRs), their presence can affect antigen binding and alter the physicochemical properties of the antibody. For example, glycosylation, deamidation, isomerization, and oxidation can increase the instability or heterogeneity of antibody molecules, thereby increasing the difficulty and risk of antibody development. Therefore, avoiding potential PTMs is crucial for the development of therapeutic antibodies. Currently, some PTMs have been found to be highly correlated with the composition of the amino acid sequence, especially the "pattern" of adjacent amino acid sequences, allowing potential PTMs to be predicted from the primary amino acid sequence of the protein. For example, the NxS / T sequence pattern (where the first position is asparagine, the second position is any amino acid other than proline, and the third position is serine or threonine) predicts an N-linked glycosylation site. The amino acid sequence pattern causing PTM may originate from germline gene sequences, such as the human germline gene fragment IGHV3-33, which naturally possesses the glycosylation pattern NST in the FR3 region; it may also originate from high-frequency mutations in somatic cells.

[0372] Amino acid mutations can disrupt the amino acid sequence pattern of PTMs, thereby reducing or eliminating the formation of specific PTMs. Different mutation design methods exist depending on the antibody sequence and the PTM sequence pattern. One method is to replace "hotspot" amino acids (such as N or S in the NS pattern) with amino acids of similar physicochemical properties (such as mutating N to Q). If the PTM sequence pattern originates from a high-frequency mutation in somatic cells and does not exist in the germline gene sequence, another method is to replace the sequence pattern with the corresponding germline gene sequence. In practice, multiple mutation removal design methods may be used for the same PTM sequence pattern. In this example, new PTM mutant antibodies were obtained by amino acid mutation of the sequences of three HCAb antibody molecules PR002607, PR002608, and PR002619, and their amino acid sequences are shown in Table 9.

[0373] Table 9. Amino acid sequences of PTM mutants (SEQ ID NO:)

[0374] Example 11 Affinity determination of the parent molecule and its PTM mutant molecule

[0375] The KD value of candidate HCAb antibodies was determined using the Octet method, a standard technique in the art. Candidate antibodies were immobilized onto the AHC sensor to a height of 0.2 nm. They were bound to diluted human OX40L protein for 200 seconds, dissociated for 600 seconds, and then regenerated for 15 seconds with 10 mM glycine hydrochloride buffer (pH 1.5). The binding rate (kon) and dissociation rate (kdis) were calculated using a simple one-to-one Langmuir binding model (Octet Red96 software). The equilibrium dissociation constant (KD) was calculated as the ratio kdis / kon.

[0376] The test results are shown in Table 10. The antigen affinity of PTM mutants PR004362, PR004369, PR004368, PR004370, PR004367, PR004371, PR004360, and PR004361 is comparable to that of the parent molecule.

[0377] Table 10. Antigen affinity determination of HCAb parent molecules and their PTM mutants

[0378] Example 12: Binding of the parent molecule and its PTM mutant molecule to human / monkey OX40L protein

[0379] This embodiment uses ELISA to investigate the ability of the fully human anti-OX40L HCAb parent molecule and its PTM mutant molecule to bind human and monkey OX40L protein in vitro. 2 μg / mL of human or monkey OX40L (Acro) protein was incubated overnight at 4°C in a highly absorbent 96-well plate (Corning, 9018). After washing three times with PBST, serially diluted anti-OX40L antibody was added and incubated at 37°C for 1.5 h. After washing three times with PBST, peroxidase-conjugated anti-hFc secondary antibody was added and incubated at 37°C for 1 h. After washing three times with PBST, TMB chromogenic substrate was added. After reaching a certain color development level, stop solution (1N HCl) was added and the absorbance value at 450 nm was measured.

[0380] The test results are shown in Figures 7A-7D and Table 11. All candidate anti-OX40L HCAb antibodies could bind to the human and monkey OX40L protein, and the binding ability of the antibody to the protein was positively correlated with the antibody concentration. All PTM mutants showed better antigen-binding ability (lower EC50 value) compared to the control antibody Oxelumab.

[0381] Table 11. Binding of HCAb parent molecules and their PTM mutants to human or monkey OX40L protein

[0382] Example 13: Binding of the parent molecule and its PTNM mutant molecule to overexpressed human OX40L cells

[0383] CHO-K1-hOX40L cells digested with trypsin were resuspended in PBS to a concentration of 1×10⁻⁶ cells / mL. 6 Add 100 μL of cell suspension to each well of a 96-well V-plate (Corning, 3894). Centrifuge the 96-well V-plate at 1000 rpm for 5 min, and wash once with PBS + 0.5% BSA (VWR, #0332-100G). Resuspend the cells in serially diluted antibody and incubate at 4°C for 1 h. Wash twice with PBS + 0.5% BSA, then incubate the cells with 100 μL of Alexa Fluor 488 goat anti-human IgG (Life Technologies, 1:1000, #A-11013) in the dark at 4°C for 1 h, and wash twice with PBS + 0.5% BSA. Resuspend the cells in 200 μL of PBS + 0.5% BSA. Analyze the fluorescence intensity of the cells using FACS flow cytometry.

[0384] The test results are shown in Figures 8A-8C and Tables 12A-12C. All PTM mutants could specifically bind to CHO-K1-hOX40L cells. The antibody binding ability was positively correlated with the antibody concentration. Moreover, the detected binding ability was comparable to that of their respective HCAb parent molecules and superior to that of the control antibody Oxelumab.

[0385] Table 12A. Binding of PR002607 and its PTM mutant with CHO-K1-hOX40L cells

[0386] Table 12B. Binding of PR002608 and its PTM mutant with CHO-K1-hOX40L cells

[0387] Table 12 shows the binding of C.PR002619 and its PTM mutant to CHO-K1-hOX40L cells.

[0388] Example 14: Parent molecule and its PTM mutant molecule inhibit the binding of human OX40L to CHO-K1-OX40 cells.

[0389] This embodiment uses FACS to investigate the ability of antibody molecules to block the binding of human OX40L protein to CHO-K1-OX40. The concentration of trypsin-digested CHO-K1-OX40 cells (Beijing Kangyuan Bochuang, KYinno) was adjusted to 1E6 / mL. Serially diluted antibody was incubated with 0.25 μg / mL hOX40L-mFc (Sino Bio, 13127-H04H) at room temperature for 15 min. 100 μL of the cell suspension was added to a 96-well V plate. After centrifugation at 500g for 5 min, the supernatant was discarded. The antibody and hOX40L-mFc mixture was added to the cells and incubated at 4°C for 1 h. After washing twice with FACS buffer (PBS + 2% FBS), 100 μL of anti-mFc-APC was added, and the cells were incubated at 4°C in the dark for 0.5 h. After washing twice more with FACS buffer, the fluorescence intensity of the cells was analyzed using FACS flow cytometry.

[0390] The test results are shown in Figures 9A-9B and Tables 13A-13B. All PTM mutants were able to inhibit the binding of human OX40L to CHO-K1-OX40 cells, and their inhibitory ability was superior to that of the reference antibody Oxelumab.

[0391] Table 13A. Antibody inhibition of binding of human OX40L to CHO-K1-OX40 cells

[0392] Table 13B. Antibody inhibition of binding of human OX40L to CHO-K1-OX40 cells

[0393] Example 15: The maternal molecule and its PTM mutant molecule inhibit the activity of HEK293T / hOX40L in NFKB-luc reporter gene activation.

[0394] This study investigated the effect of candidate anti-OX40L antibodies on the activity of fluorescent reporter genes regulated by the NFKB gene promoter. Two E4 HEK293T / hOX40L cells (Beijing Kangyuan Bochuang, KYinno, HEK293T cells overexpressing human OX40L) were seeded in black 96-well plates and incubated overnight at 37°C. The next day, 50 μL of culture medium was removed, and 20 μL of serially diluted antibody was added. After incubation at 37°C for 30 min, 20 μL of 293T-OX40-NF-KB cells (HEK293 reporter cells expressing luciferase reporter genes under the control of OX40 and NF-κB response elements, BPS Biosciences, #60482) were added, resulting in 5 E4 cells per well. After incubation at 37°C for 5 / 6 h, 50 μL of L'O-glo reagent was added, and after thorough mixing, fluorescence values ​​were read using Envision.

[0395] The test results are shown in Figures 10A-10C and Table 14. All PTM mutants can inhibit the activity of HEK293T / hOX40L in NFKB-luc reporter gene activation, and the effect is comparable to that of the reference antibody Oxelumab.

[0396] Table 14. Maternal molecule and its PTM mutant inhibit HEK293T / hOX40L activation of NFKB-luc reporter gene activity

[0397] Example 16: The parent molecule and its PTM mutant molecule inhibit the activity of HEK293T / hOX40L-stimulated PBMCs to secrete IL-2.

[0398] This study investigated the inhibitory effect of candidate anti-OX40L antibodies on IL-2 secretion by HEK293T / hOX40L-stimulated PBMCs. Eight E3 HEK293T / hOX40L cells were seeded in 96-well plates (Corning, 3599) overnight. The next day, 50 μL of serially diluted antibody was added, followed by 100 μL of PBMCs (1.5 E5 per well) and PHA to a final concentration of 0.25 μg / mL. After incubation at 37°C for 48 h, the supernatant was collected, and IL-2 levels were detected by ELISA (Thermo, 88-7025-88).

[0399] The test results are shown in Figures 11A-11C and Table 15. All tested PTM mutants were able to inhibit the ability of HEK293T / hOX40L to stimulate PBMCs to secrete IL-2, and the effect was comparable to that of their parent molecules, and comparable to or better than that of the reference antibody Oxelumab.

[0400] Table 15. Inhibition of HEK293T-hOX40L-stimulated PBMC secretion of IL-2 by maternal molecules and their PTM mutants

[0401] sequence list

[0402] PR002607

[0403] PR002619

[0404] PR004360

[0405] PR004361

[0406] PR004362

[0407] PR004363

[0408] PR004364

[0409] PR004365

[0410] PR004366

[0411] PR004367

[0412] PR004368

[0413] PR004369

[0414] PR004370

[0415] PR004371

[0416] Oxelumab

Claims

1. An OX40L binding molecule comprising a heavy chain variable region, wherein the heavy chain variable region comprises a HCDR1, a HCDR2, and a HCDR3, and wherein the HCDR1 comprises the sequence set forth in SEQ ID NO: 3 or 4 or an amino acid sequence that differs from the sequence set forth in SEQ ID NO: 3 or 4 by no more than 2 amino acid additions, deletions, or substitutions of amino acids; and / or the HCDR2 comprises the sequence set forth in SEQ ID NO: 8 or 9 or an amino acid sequence that differs from the sequence set forth in SEQ ID NO: 8 or 9 by no more than 2 amino acid additions, deletions, or substitutions of amino acids; and / or the HCDR3 comprises the sequence set forth in SEQ ID NO: 22, 23, or 24 or an amino acid sequence that differs from the sequence set forth in SEQ ID NO: 22, 23, or 24 by no more than 2 amino acid additions, deletions, or substitutions of amino acids.

2. The OX40L binding molecule of claim 1, wherein the OX40L binding molecule is an antibody or antigen-binding fragment thereof to OX40L.

3. The OX40L binding molecule of claim 1 or 2, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 3 or 4; the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; and / or the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 22, 23, or 24.

4. The OX40L binding molecule of any one of claims 1-3, wherein the heavy chain variable region comprises a HCDR1, a HCDR2, and a HCDR3, and wherein the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 3, 8, 22, respectively; or the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 4, 9, 23, respectively; or the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 4, 9, 24, respectively; or the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 3, 10, 22, respectively; or the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 3, 11, 22, respectively; or the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 4, 12, 23, respectively; or the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 4, 14, 23, respectively; or the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 4, 15, 23, respectively; or the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 4, 16, 23, respectively; or the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 4, 17, 23, respectively; or the HCDR1, the HCDR2, and the HCDR3 comprise the amino acid sequences set forth in SEQ ID NO: 4, 18, 23, respectively. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ HCDR1, HCDR2, and HCDR3 contain the amino acid sequences shown in SEQ ID NO:4, 16, and 23, respectively; or HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, 12, and 24; or HCDR1, HCDR2, and HCDR3 contain the amino acid sequences shown in SEQ ID NO:4, 14, and 24, respectively; or HCDR1, HCDR2, and HCDR3 respectively contain the amino acid sequences shown in SEQ ID NO:4, 15, and 24; or HCDR1, HCDR2, and HCDR3 contain the amino acid sequences shown in SEQ ID NO:4, 13, and 23, respectively; or HCDR1, HCDR2, and HCDR3 contain the amino acid sequences shown in SEQ ID NO:4, 17, and 24, respectively; or HCDR1, HCDR2 and HCDR3 contain amino acid sequences as shown in SEQ ID NO:4, 18 and 24, respectively.

5. The OX40L-bound molecule according to any one of claims 1-4, wherein The heavy chain variable region comprises: 1) an amino acid sequence shown in SEQ ID NO: 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 42, 34, 40, or 41; 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 42, 34, 40, or 41; or 3 ... Compared to the amino acid sequences shown in NO:28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 42, 34, 40 or 41, the amino acid sequences having one or more added, deleted and / or substituted amino acids, preferably, the addition, deletion and / or substitution does not occur in the CDR region.

6. The OX40L binding molecule of any one of claims 1-5, wherein it is a heavy chain antibody and / or a fully humanized antibody.

7. The OX40L-bound molecule of any one of claims 1-5, wherein the heavy chain variable region can be fused with another molecule; Preferably, the other molecule is the Fc domain of an immunoglobulin; More preferably, the other molecule is the Fc domain of immunoglobulin G1 (IgG1).

8. The OX40L binding molecule of any one of claims 1-7, comprising: 1) an amino acid sequence shown in SEQ ID NO: 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 57, 49, 55 or 56; 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 57, 49, 55 or 56; or 3 ... Compared to the amino acid sequences shown in NO:43, 44, 45, 46, 47, 48, 50, 51, 52, 53, 54, 57, 49, 55 or 56, the amino acid sequences have one or more amino acid additions, deletions and / or substitutions, preferably, the additions, deletions and / or substitutions do not occur in the CDR region.

9. The OX40L-bound molecule of any one of claims 1-8, having at least one of the following characteristics: 1) It has affinity activity for OX40L protein; 2) It has affinity activity for OX40L positive cells; 3) It can inhibit the binding and / or interaction between OX40 and OX40L.

10. A multispecific antigen-binding construct comprising a first antigen-binding portion that binds OX40L, wherein the first antigen-binding portion comprises the OX40L binding molecule of any one of claims 1-9; Preferably, the multispecific antigen-binding construct comprises a first antigen-binding portion that binds to OX40L and a second antigen-binding portion that binds to a second antigen; Preferably, the second antigen-binding portion specifically binds to pro-inflammatory cytokines, chemokines, antigens on lymphocytes, immunomodulatory receptors, and immune checkpoint molecules.

11. An antibody conjugate comprising an OX40L binding molecule of any one of claims 1-9 or a multispecific antigen-binding construct of claim 10 conjugated to at least one therapeutic agent.

12. A pharmaceutical composition comprising the OX40L binding molecule of any one of claims 1-9, the multispecific antigen-binding construct of claim 10, the antibody conjugate of claim 11, and a pharmaceutically acceptable carrier.

13. Use of the OX40L binding molecule of any one of claims 1-9, the multispecific antigen-binding construct of claim 10, the antibody conjugate of claim 11, or the pharmaceutical composition of claim 12 in the preparation of a medicament for treating a disease or condition; Preferably, the disease or condition is an inflammatory or autoimmune disease; Preferably, the disease or condition is selected from graft-versus-host disease (GVHD), atopic dermatitis, Crohn's disease, alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, type 1 diabetes, adolescent / childhood type 1 diabetes, juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary idiopathic arthritis, etc. Bile cholangitis, primary biliary cirrhosis, nonalcoholic steatohepatitis (NASH), psoriasis, rheumatoid arthritis, scleroderma, CREST syndrome, Sjögren's syndrome, systemic lupus erythematosus, thyroiditis, uveitis, vitiligo, Wegener's granulomatosis, Addison's disease, Hashimoto's thyroiditis, infertility / sterility, ANCA-associated vasculitis, psoriatic arthritis, celiac disease, ulcerative colitis, lichen sclerosus, Behçet's disease, asthma, allergic rhinitis, and hidradenitis suppurativa.

14. A polynucleotide encoding the OX40L binding molecule of any one of claims 1-9 or the multispecific antigen binding construct of claim 10.

15. An expression vector comprising the polynucleotide of claim 14.

16. A host cell comprising the polynucleotide of claim 14 or the expression vector of claim 15.

17. A method for generating the OX40L binding molecule of any one of claims 1-9 or the multispecific antigen-binding construct of claim 10, the method comprising: a) Culturing the host cells of claim 16 under suitable conditions to express the OX40L binding molecule of any one of claims 1-9 or the multispecific antigen-binding construct of claim 10; and b) Isolate the OX40L binding molecule or multispecific antigen binding construct from a host cell or its culture.

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