Anti-ox40l antibodies and uses thereof

Abstract

The invention relates to a novel antibody and an antibody fragment which specifically bind to OX40L and a composition containing the antibody or the antibody fragment. Furthermore, the invention relates to nucleic acids encoding said antibodies or antibody fragments thereof and to host cells comprising same, as well as to related uses, as well as to therapeutic and diagnostic uses of these antibodies and antibody fragments.

Description

Anti-OX40L antibodies and uses thereof

[0001] The present invention relates to novel antibodies and antibody fragments that specifically bind to OX40L, as well as compositions containing the antibodies or antibody fragments. Furthermore, the present invention relates to nucleic acids encoding the antibodies or antibody fragments thereof, host cells containing the same, and related uses. Furthermore, the present invention relates to therapeutic and diagnostic uses of these antibodies and antibody fragments.

[0002] Background of the Invention

[0003] The T cell co-stimulatory molecule OX40 and its cognate ligand OX40L have attracted extensive research interest as therapeutic targets for T cell-mediated diseases.

[0004] OX40 is a type 1 transmembrane glycoprotein composed of approximately 275 amino acids, with three intact cysteine-rich domains (CRDs) and a partial C-terminal CRD. OX40 has an apparent molecular weight of 50 kDa. OX40, along with other TNFRSF members (such as 4-1BB, CD27, CD30, and CD40), is a T cell co-stimulatory molecule that functions at various stages to regulate and control immune responses. Unlike other constitutive T cell co-stimulatory receptors (such as CD28 and CD27), OX40 is not expressed on naive T cells. OX40 is transiently induced by signals following TCR engagement following antigen (Ag) recognition, peaking 48–72 hours after activation in activated T cells, including activated CD4+ and CD8+ T cells, as well as neutrophils, natural killer (NK) cells, and natural killer T (NKT) cells. The only exception is that OX40 is constitutively expressed in mouse FOXP3+ Treg cells. The dynamics of OX40 expression involve many factors, including the cytokine environment, the persistence of antigen, the inflammatory environment, and the influence of other co-stimulatory pathways. OX40-deficient T cells may normally proliferate and differentiate into effector T cells 2-3 days after TCR recruitment, but they show a significant decrease in survival after 12-13 days. In other words, OX40 signaling may not affect the initial activation stage of T cells, but may play an important role in maintaining the late proliferation and survival of T cells in the effector stage.

[0005] The ligand of OX40 (OX40L, also known as CD252, CD134L, or gp34) is also a member of the TNFR superfamily. OX40L is a type II glycoprotein with a 23-amino acid cytoplasmic tail and a 133-amino acid extracellular domain. Murine OX40L can stimulate both human and mouse T cells; however, human OX40L can only stimulate human T cells. OX40L is primarily expressed on activated antigen-presenting cells (APCs), such as dendritic cells (DCs), activated B cells, and macrophages. The cell types that can be induced to express OX40L are much more diverse. In addition to APCs, OX40L is also expressed on hematopoietic cells, such as activated NK cells, mast cells, or responsive CD4+ T cells, and non-hematopoietic cells, such as endothelial cells or smooth muscle cells. (Yu Fu et al., The significance of OX40 and OX40L to T-cell biology and immune disease, Immunological Reviews 2009, Vol. 229:173–191). OX40L interacts with OX40 and induces receptor trimerization, thereby activating the downstream NfKB signaling pathway, promoting the formation of memory T cells, the secretion of inflammatory factors, and inhibiting the production of regulatory T cells, thereby promoting cellular immune responses.

[0006] Autoimmune diseases are typically characterized by the production of reactive autoantibodies against self-antigens due to the immune system's failure to maintain self-tolerance. The prevalence of some well-known autoimmune diseases is high, as their pathogenesis is poorly understood. These include experimental autoimmune encephalomyelitis (EAE), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), colitis, autoimmune experimental uveitis (AEU), type 1 diabetes, and multiple sclerosis (MS).

[0007] Due to the key role of T cells in controlling immune responses and co-stimulation via OX40-OX40L interactions with T cells, targeting the OX40-OX40L interaction may improve autoantigen-specific T cell responses. Therefore, experiments have been conducted to investigate whether targeting the OX40-OX40L interaction by blocking either OX40 or OX40L has therapeutic benefits in autoimmune diseases, including EAE, SLE, RA, colitis, AEU, type 1 diabetes, MS, graft-versus-host disease (GVHD), and inflammatory bowel disease (IBD). Studies have shown that OX40 is upregulated at autoimmune sites and correlates with disease severity. Therefore, inhibiting this signaling pathway has potential therapeutic benefits for a variety of immune-related diseases.

[0008] Due to the large number of unmet clinical treatment needs, the development of drugs targeting OX40L is very necessary. Although some monoclonal antibodies targeting OX40L have been developed in the existing technology, these antibodies have low affinity, and their biological activity and in vivo efficacy need to be further improved.

[0009] Therefore, there is a need to develop new antibodies against OX40L that have stronger binding affinity to OX40L, higher biological activity, and better in vivo efficacy.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention provides a novel high-affinity antibody for recognizing OX40 ligand (OX40L), which selectively blocks the OX40L-OX40 signaling pathway.

[0012] Thus, the present invention relates to an antibody or fragment, such as an antigen-binding fragment, that specifically binds OX40L, such as human OX40L.

[0013] The antibody or antigen-binding fragment thereof of the present invention can effectively block the interaction between OX40L and its receptor OX40 at both the protein level and the cellular level, and can effectively inhibit the activation, proliferation and / or differentiation of T cells.

[0014] For example, in a mouse model of graft-versus-host disease (GvHD), mice administered with the antibodies or antigen-binding fragments thereof of the present invention can maintain weight gain without clinical symptoms, and / or significantly reduce T cell activation or the production of inflammatory factors.

[0015] The antibody or antigen-binding fragment thereof of the present invention can effectively reduce the probability of chain exchange of the antibody in vivo and improve its stability by mutating the Fc region, such as performing an S228P mutation.

[0016] Therefore, the present invention relates to the following specific aspects.

[0017] In some aspects, the present invention relates to an anti-OX40L antibody or an antigen-binding fragment thereof, the antibody comprising:

[0018] (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 4, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 8; or

[0019] (ii) three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO: 14, and three complementarity determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO: 18;

[0020] For example, wherein the HCDR1 is determined according to the Abm scheme, and HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are determined according to the Kabat scheme.

[0021] In some aspects, the present invention relates to an anti-OX40L antibody or antigen-binding fragment thereof, comprising a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein:

[0022] (i) the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7; or

[0023] (ii) the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or respectively consist of the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17.

[0024] In some embodiments, the anti-OX40L antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region (VH), wherein the heavy chain variable region comprises, consists of, or comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or 14, or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 4 or 14.

[0025] In some embodiments, an anti-OX40L antibody or antigen-binding fragment thereof of the present invention comprises a light chain variable region (VL), wherein the light chain variable region comprises, consists of, or comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 8 or 18, or consists of said sequence.

[0026] In some embodiments, the present invention relates to an anti-OX40L antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein

[0027] (i) the heavy chain variable region comprises, or consists of, an amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable region comprises, or consists of, an amino acid sequence of SEQ ID NO:8 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0028] (ii) the heavy chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 14, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable region comprises, or consists of, the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0029] In some embodiments, the anti-OX40L antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein

[0030] (i) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; or

[0031] (ii) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 18.

[0032] In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention comprise an Fc region. In some specific embodiments, the Fc region is derived from a human IgG Fc, for example, an Fc from human IgG1, an Fc from human IgG2, an Fc from human IgG3, or an Fc from human IgG4. In some specific embodiments, the Fc region comprises a mutation that enhances the stability of the Fc region or reduces chain exchange in the antibody. In some specific embodiments, the mutation is an S228P mutation. In some specific embodiments, wherein the Fc region

[0033] (i) comprising or consisting of the amino acid sequence of SEQ ID NO: 27 or 28;

[0034] (ii) comprises an amino acid sequence that is at least 90% identical, such as 95%, 96%, 97%, 99% or more identical to the amino acid sequence of SEQ ID NO: 28; or

[0035] (iii) comprising an amino acid sequence that is at least 90% identical, such as 95%, 96%, 97%, 99% or more identical to the amino acid sequence of SEQ ID NO: 27, and comprises the S228P mutation.

[0036] In some embodiments, the anti-OX40L antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain constant region, wherein the heavy chain constant region is derived from the constant region of IgG1, IgG2, IgG3 or IgG4. Preferably, the heavy chain constant region is

[0037] (i) comprises or consists of an amino acid sequence selected from SEQ ID NO: 21 or 29;

[0038] (ii) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29; or

[0039] (iii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21 and has an S228P deletion mutation.

[0040] In some embodiments, the anti-OX40L antibody or antigen-binding fragment thereof of the present invention comprises a light chain constant region, wherein the light chain constant region is a lambda or kappa light chain constant region. Preferably, the light chain constant region is

[0041] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; or

[0042] (ii) comprising or consisting of the amino acid sequence of SEQ ID NO: 22.

[0043] In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the invention comprise a heavy chain, wherein the heavy chain

[0044] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 9 or 19; or

[0045] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 9 or 19.

[0046] In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the invention comprise a light chain, wherein the light chain

[0047] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 10 or 20; or

[0048] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 or 20.

[0049] In some embodiments, the present invention relates to an anti-OX40L antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain, wherein

[0050] (i) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9; and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10;

[0051] (ii) the heavy chain comprises, or consists of, an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19; and the light chain comprises, or consists of, an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20.

[0052] In some embodiments, the anti-OX40L antibody or antigen-binding fragment thereof of the invention comprises a heavy chain and a light chain, wherein

[0053] (i) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 9, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 10;

[0054] (ii) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 19, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0055] In some embodiments, the anti-OX40L antibodies of the invention are monoclonal antibodies.

[0056] In some embodiments, the anti-OX40L antibodies of the invention are humanized antibodies or chimeric antibodies.

[0057] In some embodiments, the antigen-binding fragment of an anti-OX40L antibody of the invention is an antibody fragment selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single-domain antibody such as VHH, dAb (domain antibody), diabody, or linear antibody.

[0058] In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the invention have one or more of the following properties:

[0059] a) binds OX40L (eg, human OX40L) with high affinity;

[0060] b) It can effectively block the interaction between OX40L and its receptor OX40 at both the protein and cellular levels;

[0061] c) inhibiting the OX40 / OX40L signaling pathway;

[0062] d) inhibiting T cell activation, proliferation and / or differentiation;

[0063] e) reducing T cell activation and / or the production of inflammatory factors;

[0064] f) Treatment of graft-versus-host disease.

[0065] In some aspects, the invention relates to an isolated nucleic acid encoding an anti-OX40L antibody or antigen-binding fragment thereof of the invention.

[0066] In some aspects, the present invention relates to a vector comprising the nucleic acid of the present invention, preferably the vector is an expression vector.

[0067] In some aspects, the present invention relates to a host cell comprising a nucleic acid or vector of the present invention, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (such as 293 cells or CHO cells, such as CHO-K cells or HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0068] In some aspects, the present invention relates to a method for preparing an anti-OX40L antibody or an antigen-binding fragment thereof, the method comprising

[0069] a) culturing the host cell of the invention under conditions suitable for expression of a nucleic acid encoding an anti-OX40L antibody or antigen-binding fragment thereof of the invention,

[0070] b) optionally isolating said antibody or antigen-binding fragment thereof,

[0071] c) Optionally, the method further comprises recovering the anti-OX40L antibody or antigen thereof from the host cell

[0072] Binding fragment, optionally, the antibody is purified, for example, by Protein A purification.

[0073] In some aspects, the invention relates to an immunoconjugate comprising an anti-OX40L antibody of the invention or an antigen-binding fragment thereof and other substances, such as toxins, small molecule drugs, cytotoxic agents, apoptotic agents, chelating agents, immunomodulators, such as anti-inflammatory agents or immunosuppressive agents.

[0074] In some aspects, the present invention relates to a pharmaceutical composition comprising an anti-OX40L antibody or antigen-binding fragment or immunoconjugate of the present invention, and optionally a pharmaceutically acceptable excipient.

[0075] In some aspects, the invention relates to a pharmaceutical combination product comprising an anti-OX40L antibody or antigen-binding fragment or immunoconjugate of the invention and one or more other therapeutic agents, e.g., the therapeutic agent is selected from a cytokine, other antibody, small molecule drug, or immunomodulator (e.g., immunosuppressant).

[0076] In some aspects, the present invention relates to a method for preventing or treating a disease or condition associated with inappropriate activation of an OX40L / OX40-mediated pathway in an individual, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, or immunoconjugate, or pharmaceutical composition, or pharmaceutical combination product of the present invention that binds to OX40L. In some embodiments, the subject has abnormal activation of an OX40L / OX40-mediated signaling pathway compared to a healthy individual. In some embodiments, the disease or condition is selected from an autoimmune disease or allogeneic transplantation, for example, an autoimmune disease includes but is not limited to atopic dermatitis, asthma, systemic lupus erythematosus, Sjogren's syndrome, immune thrombocytopenic purpura, multiple sclerosis, lupus nephritis, amyotrophic lateral sclerosis, rheumatoid arthritis, non-rheumatoid arthritis, contact dermatitis, hyper-IgE syndrome, inflammatory bowel disease, myasthenia gravis, Graves' disease, hemolytic anemia, psoriasis, atopic dermatitis, allergic asthma, or idiopathic inflammatory disease. In some embodiments, the method further comprises administering one or more other therapies, such as treatment modalities and / or other therapeutic agents, such as selected from cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants). Description of the drawings:

[0077] FIG1 shows the affinity of anti-OX40L antibodies, their blocking activity on OX40-OX40L, and their effects on the luciferase activity of Jurkat-NFκB cells overexpressing OX40;

[0078] Figure 2 shows the effect of anti-OX40L antibodies on T cell activation;

[0079] Figure 3 shows the changes in IL5 and IL13 secretion during DC-induced Th2 differentiation after anti-OX40L antibody treatment;

[0080] Figure 4 shows the changes in mouse body weight and survival curves in the GvHD mouse model;

[0081] FIG5 shows the proportion of CD4-positive T cells in the blood of mice on day 14 in a GvHD mouse model.

[0082] Detailed Description of the Invention

[0083] I. Definition

[0084] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0085] To interpret this specification, the following definitions will apply, and wherever appropriate, terms used in the singular may also include the plural, and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0086] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0087] As used herein, the term "and / or" means any one of the alternatives or two or more or all of the alternatives.

[0088] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, combinations of the recited elements, integers, or steps are also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0089] An "isolated" antibody or molecule is one that has been separated from a component of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0090] As used herein, "OX40L" refers to any native OX40L polypeptide (e.g., a human OX40L polypeptide) or variant thereof. The term "OX40L" encompasses "full-length" unprocessed OX40L polypeptides as well as any form of OX40L polypeptide produced by processing within a cell. The term also encompasses naturally occurring variants of OX40L, such as those encoded by splice variants and allelic variants. The OX40L polypeptides described herein can be isolated from a variety of sources, such as from human tissue or from another source, such as cynomolgus monkeys, or prepared by recombinant or synthetic methods. In one embodiment of the invention, the human OX40L protein is as shown in (CD252, Uniport: P23510). In one embodiment, the human OX40L gene is as shown in (Uniport: P29279). In one embodiment of the invention, the cynomolgus monkey OX40L protein is as shown in (uniport: A0A1D5QQH7).

[0091] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0092] The term "antigen-binding fragment" of an antibody is a molecule that is different from a full-length antibody and that contains a portion of a full-length antibody, but that can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibodies, single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH, diabodies or fragments thereof, or camelid antibodies, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full antibody below the disulfide bonds in the hinge region with pepsin produces F(ab')2, which is a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by destroying the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. An Fv fragment consists of the VL and VH domains of a single antibody arm. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but recombinant methods can also be used to connect the two domains using a synthetic linker peptide to produce them as a single protein chain, in which the VL and VH regions are paired to form a single-chain Fv (scFv).

[0093] The term "single-chain antibody (scAb)" is used herein in the broadest sense and specifically covers antibodies with monospecificity or multispecificity (e.g., bispecificity) that are initially produced as a single continuous polypeptide chain. Such single-chain antibodies include, but are not limited to, antibodies having two linked VL and VH regions. In one embodiment, the single-chain antibody is an scFv.

[0094] "Diabodies" are small, bivalent antibodies constructed through gene fusion, for example, dimers composed of two polypeptide chains. The VL and VH domains of each polypeptide chain of a diabody are linked by a linker, so that the VL and VH encoded in the same polypeptide chain form a dimer with different single-chain variable region segments. Diabodies generally have two antigen-binding sites.

[0095] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics The IMGT database (http: / / imgt.cines.fr or http: / / imgt.cines.org) and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0096] The following are exemplary schemes for the regional extent of CDRs defined using the Kabat, AbM, Chothia, Contact, and IMGT schemes.

[0097] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention).

[0098] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the position according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0099] In some embodiments, the CDR1 of the heavy chain variable region of the antibody of the present invention is determined according to the AbM scheme, and the CDR2 and CDR3 are determined according to the Kabat scheme. In some embodiments, the CDR of the light chain variable region of the antibody of the present invention is determined according to the Kabat scheme.

[0100] An "antibody that binds to the same or overlapping epitope as a reference antibody" is an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay, whereas conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay.

[0101] An antibody that competes with a reference antibody for binding to its antigen is an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid phase direct or indirect radioimmunoassays (RIAs), solid phase direct or indirect enzyme immunoassays (EIAs), and sandwich competition assays.

[0102] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

[0103] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody is an antibody that has at least 50%, 60%, 70%, 80%, 90% or more than 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0104] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.

[0105] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) while being less immunogenic when administered to humans, for example, as a therapeutic. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing the constant region and portions of the variable region not involved in binding with the corresponding portions of a human antibody).

[0106] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" comprises two or three constant domains, namely a CH2 domain, a CH3 domain, and an optional CH4 domain. For example, in a native antibody, an immunoglobulin Fc domain comprises the second and third constant domains (CH2 domain and CH3 domain) of two heavy chains derived from IgG, IgA, and IgD class antibodies; or the second, third, and fourth constant domains (CH2 domain, CH3 domain, and CH4 domain) of two heavy chains derived from IgM and IgE class antibodies. Unless otherwise indicated herein, amino acid residue numbering in the Fc region or heavy chain constant region is according to the EU numbering system (also called the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0107] As used herein, "conservative changes" include substitutions, deletions, or additions to a polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. In some embodiments, conservative changes are conservative substitutions. A conservative substitution refers to the replacement of one amino acid with another within the same class, such as the replacement of an acidic amino acid with another acidic amino acid, the replacement of a basic amino acid with another basic amino acid, or the replacement of a neutral amino acid with another neutral amino acid. For example, conservative substitutions often result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M). In some embodiments, the term "conservative change" when applied to an antibody molecule amino acid sequence refers to an amino acid modification that does not significantly affect or change the target antigen binding characteristics of the antibody molecule of the present invention containing the amino acid sequence. For example, a conservatively changed variant maintains at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100-110% or higher, binding affinity for the target antigen relative to the parent antibody.

[0108] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0109] An "immunoconjugate" is an antibody conjugated to one or more additional substances, including but not limited to a label.

[0110] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating diseases associated with inappropriate activation of OX40 / OX40L-mediated pathways, including cytokines, other antibodies, small molecule drugs, or immunomodulators (eg, immunosuppressants).

[0111] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0112] As used herein, the term "immunomodulator" refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants.

[0113] As used herein, an "immunosuppressant," "immunosuppressive drug," or "immunosuppressant" is a therapeutic agent used in immunosuppressive therapy to suppress or prevent the activity of the immune system.

[0114] The term "effective amount" refers to an amount or dosage of an antibody or fragment or conjugate or composition or combination of the present invention that produces the desired effect in a patient in need of treatment or prevention after administration in single or multiple doses to the patient.

[0115] A "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or its conjugate or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., T cell activation) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70% relative to an untreated subject. In some embodiments, the term "therapeutically effective amount" as used herein is intended to define the amount of treatment necessary to treat a condition (e.g., autoimmune, allergic, inflammatory, GVHD, or anti-transplant, drug, or rejection reaction) or to reduce or eliminate an immune response in a therapeutic regimen.

[0116] A "prophylactically effective amount" refers to an amount effective to achieve the desired prophylactic result at the required dosage and for the required period of time. Typically, a prophylactic effective amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject before or at an earlier stage of the disease. In some embodiments, the term "prophylactically effective amount" as used herein is intended to define the amount necessary to prevent the progression and symptoms of a condition or disease (e.g., autoimmune, allergic, inflammatory, GVHD or anti-transplant, drug or rejection reactions) in a treatment regimen.

[0117] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny screened or selected for the same function or biological activity as the initially transformed cell are included herein.

[0118] The term "label" as used herein refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent to which it is conjugated or fused. The label itself can be detectable (e.g., a radioisotope label or a fluorescent label) or can catalyze a chemical change in a detectable substrate compound or composition in the case of an enzymatic label. The term is intended to encompass direct labeling of a probe or antibody by coupling (i.e., physically connecting) a detectable substance to the probe or antibody and indirect labeling of the probe or antibody by reacting with another reagent of the direct label.

[0119] As used herein, the terms "individual," "subject," or "subject" are used interchangeably and include mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0120] As used herein, the term "subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from fresh, frozen and / or preserved; blood or any blood component; body fluids, such as cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0121] "Nucleic acid encoding an anti-OX40L antibody or fragment thereof" refers to one or more nucleic acid molecules that encode an antibody heavy chain or light chain (or fragment thereof, e.g., a heavy chain variable region or a light chain variable region), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0122] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. Such variants may comprise conservative changes.

[0123] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, etc., which is administered together with the active substance.

[0124] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0125] As used herein, the term "pharmaceutical combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit / test kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the present invention, and (ii) other therapeutic agent) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in separate entities, wherein such administration provides two or more active agents with prophylactic or therapeutically effective levels in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0126] The term "combination therapy" refers to the administration of two or more therapeutic agents or treatment modalities (e.g., radiotherapy or surgery) to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide the beneficial effects of the drug combination in treating disorders or conditions described herein.

[0127] As used herein, "treat," ..."

[0128] As used herein, "prevention" includes the inhibition of the onset or development of a disease or condition or symptoms of a particular disease or condition.

[0129] II Antibodies

[0130] In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention can effectively block the interaction, eg, binding, of OX40L with its receptor OX40 at the protein level and the cellular level, and / or block OX40 signaling activation.

[0131] In some embodiments, the anti-OX40L antibodies of the present invention or antigen-binding fragments thereof can effectively inhibit the activation, proliferation and / or differentiation of T cells, for example, inhibit the secretion level of inflammatory factors such as interleukins such as IL5 and / or IL3 by T cells, or inhibit Th2 inflammatory response.

[0132] In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention can maintain the body weight (or maintain weight gain) of individuals with graft-versus-host disease, render them asymptomatic, reduce T cell activation, and / or inhibit the production of inflammatory factors (e.g., interferons such as IFNγ or interleukins such as IL-6).

[0133] In some embodiments, an anti-OX40L antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0134] In some embodiments, an anti-OX40L antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0135] In some embodiments, an anti-OX40L antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0136] In some aspects, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VH). In some aspects, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region and a light chain variable region (VH). In some embodiments, the heavy chain variable region comprises three complementary determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0137] In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region. In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region. In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region and a light chain constant region.

[0138] In some embodiments, the heavy chain variable region of the present invention:

[0139] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NO: 14; or

[0140] (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 14; or

[0141] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 4 and SEQ ID NO: 14, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0142] In some embodiments, the light chain variable region of the present invention

[0143] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 8 and SEQ ID NO: 18; or

[0144] (ii) comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 8 and SEQ ID NO: 18; or

[0145] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 8 and SEQ ID NO: 18, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0146] In some embodiments, the three complementarity determining regions (HCDRs) of the present invention, HCDR1, HCDR2, and HCDR3, from the heavy chain variable region are selected from

[0147] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 4 or 14;

[0148] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three HCDR regions relative to the sequence of any one of (i),

[0149] For example, wherein the HCDRs can be determined according to any scheme for determining CDRs, such as according to the Kabat, AbM, Chothia, Contact or IMGT schemes, respectively, or a combination thereof;

[0150] For example, the HCDR1 is determined according to the AbM protocol, and the HCDR2 and HCDR3 are each determined according to the Kabat protocol.

[0151] In some embodiments, the three complementarity determining regions (LCDRs) of the present invention, LCDR1, LCDR2, and LCDR3, from the light chain variable region are selected from

[0152] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 8 or 18, or

[0153] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in the three LCDR regions relative to the sequence of any one of (i),

[0154] For example, wherein the LCDR can be determined according to any scheme for determining CDRs, such as according to the Kabat, AbM, Chothia, Contact or IMGT schemes, respectively, or a combination thereof;

[0155] For example, the LCDRs 1, 2 and 3 are each determined according to the Kabat protocol.

[0156] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 1 or 11, or HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 1 or 11.

[0157] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 2 or 12, or HCDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 2 or 12.

[0158] In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 3 or 13, or HCDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 3 or 15.

[0159] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 5 or 15, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 5 or 15.

[0160] In some embodiments, LCDR2 comprises the amino acid sequence of SEQ ID NO: 6 or 16, or consists of the amino acid sequence, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 6 or 16.

[0161] In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 7 or 17, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 7 or 17.

[0162] In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain. In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain. In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain and a light chain. In some embodiments, the heavy chain of the antibodies of the present invention comprises a heavy chain variable region and a heavy chain constant region, or consists of a heavy chain variable region and a heavy chain constant region. In some embodiments, the light chain of the antibodies of the present invention comprises a light chain variable region and a light chain constant region, or consists of a light chain variable region and a light chain constant region. In some embodiments, the antibodies of the present invention comprise two heavy chains and two light chains, or consist of two heavy chains and two light chains.

[0163] In some embodiments, the heavy chain constant region of the antibody of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably the heavy chain constant region of IgG4. In some embodiments, the light chain constant region of the antibody of the present invention is the lambda or kappa light chain constant region, preferably the kappa light chain constant region.

[0164] In some embodiments, the heavy chain constant region of an antibody of the invention comprises an Fc region or a mutated Fc region.

[0165] In some embodiments, the Fc region is a human IgG Fc, e.g., an Fc from human IgG1, an Fc from human IgG2, an Fc from human IgG3, or an Fc from human IgG4. In one embodiment, the Fc region is from human IgG4. In one embodiment, the Fc region comprises or consists of the amino acid sequence of SEQ ID NO: 28, or an amino acid sequence having at least 90% identity thereto, e.g., 95%, 96%, 97%, 99% or more identity thereto.

[0166] In one embodiment, the Fc region is modified with respect to the properties of the effector functions of the Fc region (e.g., the complement activation function of the Fc region). In one embodiment, the effector functions have been reduced or eliminated relative to wild-type Fc regions. In one embodiment, the effector functions are reduced or eliminated by a method selected from the group consisting of: using an Fc isotype that naturally has reduced or eliminated effector functions, and Fc region modifications. The Fc region may also comprise modifications that alter binding affinity for one or more Fc receptors.

[0167] In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region comprises a mutation that reduces binding to the Fcγ receptor. The Fc region also comprises a mutation that improves antibody stability, such as a mutation that eliminates IgG heterogeneity. In some embodiments, the mutation can help reduce the probability of antibody chain exchange in vivo. In some embodiments, the mutation is a mutation that mutates the serine at position 228 to a proline. Therefore, in a preferred embodiment, the Fc region of the present invention has an S228P (EU numbering) mutation. In some embodiments, the Fc region of the antibody or fragment thereof of the present invention is an IgG4 Fc region comprising an S228P mutation.

[0168] Therefore, in a preferred embodiment, the Fc region of the present invention comprises the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 27 and has an S228P mutation.

[0169] In some preferred embodiments, the antibody heavy chain constant region of the present invention is

[0170] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 21 or 29;

[0171] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 21 or 29; or

[0172] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 21 or 29.

[0173] In some preferred embodiments, the heavy chain constant region of an antibody of the present invention comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21, and has an S228P mutation. In some preferred embodiments, the heavy chain constant region of an antibody of the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 21.

[0174] In some embodiments, the antibody light chain constant region of the invention is

[0175] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22;

[0176] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 22; or

[0177] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 22.

[0178] In some specific embodiments, an anti-OX40L antibody of the invention comprises a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3), and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2), and a third light chain complementarity determining region (LCDR3), wherein:

[0179] (i) the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7; or

[0180] (ii) wherein HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17.

[0181] In some embodiments, the VH of the present invention comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, and the HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, or consist of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, and the LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, or consist of the amino acid sequences shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively.

[0182] In some embodiments, the VH of the present invention comprises HCDR1, HCDR2, and HCDR3, and the VL comprises LCDR1, LCDR2, and LCDR3, and the HCDR1, HCDR2, and HCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, or consist of the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13, respectively, and the LCDR1, LCDR2, and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, or consist of the amino acid sequences shown in SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17, respectively.

[0183] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0184] (i) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 4, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 8, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto;

[0185] (ii) a VH comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 14, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and / or a VL comprising, or consisting of, an amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0186] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region and a light chain variable region, wherein

[0187] (i) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 4, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 8; or

[0188] (ii) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 18.

[0189] In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain. In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain. In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain and a light chain.

[0190] In some embodiments, the heavy chain comprises

[0191] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 9 or 19;

[0192] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 9 or 19; or

[0193] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 9 or 19.

[0194] In some embodiments, the light chain comprises

[0195] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 10 or 20;

[0196] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 or 20; or

[0197] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 10 or 20.

[0198] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain, wherein

[0199] (i) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9; and / or the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10;

[0200] (ii) the heavy chain comprises, or consists of, an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19; and / or the light chain comprises, or consists of, an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20.

[0201] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain, wherein

[0202] (i) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 9, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 10; or

[0203] (ii) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 19, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0204] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0205] In a preferred embodiment, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside of the heavy chain variable region and / or outside of the light chain variable region.

[0206] In certain embodiments, the antibodies provided herein are modified to increase or decrease the degree of glycosylation of the antibody. Addition or deletion of the glycosylation site of the antibody can be easily achieved by changing the amino acid sequence to produce or remove one or more glycosylation sites. When the antibody comprises an Fc region, the carbohydrate attached thereto can be changed. In some applications, it can be useful to remove the modification of unwanted glycosylation sites, for example, removing the fucose motif to improve antibody-dependent cellular toxicity (ADCC) function. In other applications, galactosidation can be performed to modify complement-dependent cytotoxicity (CDC).

[0207] In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0208] In certain embodiments, the antibodies provided herein may be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0209] In some embodiments, the anti-OX40L antibodies or antigen-binding fragments thereof of the invention have one or more of the following properties:

[0210] (i) exhibiting the same or similar binding affinity and / or specificity for OX40L as the antibodies of the invention;

[0211] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the invention to OX40L;

[0212] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0213] (iv) competing with the antibodies of the present invention for binding to OX40L;

[0214] (v) possess one or more biological properties of an antibody of the invention.

[0215] In some embodiments, the anti-OX40L antibody of the present invention is an antibody in the form of IgG1, an antibody in the form of IgG2, an antibody in the form of IgG3, or an antibody in the form of IgG4, preferably, an antibody in the form of IgG4.

[0216] In some embodiments, the anti-OX40L antibody is a monoclonal antibody.

[0217] In some embodiments, the anti-OX40L antibody is humanized.

[0218] In some embodiments, the anti-OX40L antibody is a chimeric antibody.

[0219] In one embodiment, the anti-OX40L antibodies of the present invention also encompass antibody fragments thereof (e.g., antigen-binding fragments), preferably antibody fragments selected from the group consisting of: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), diabodies, or linear antibodies.

[0220] In some embodiments, the anti-OX40L antibodies of the invention also encompass multispecific antibodies, such as bispecific antibodies, that specifically bind to OX40L.

[0221] In some embodiments, the anti-OX40L antibodies of the invention are full-length antibodies.

[0222] III. Nucleic acids of the present invention and host cells containing the same

[0223] In one aspect, the present invention provides nucleic acids encoding any chain, monomer, or domain of an antibody or antigen-binding fragment thereof of the present invention. Polynucleotide sequences encoding each chain can be generated using methods well known in the art. For example, when expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human OX40L antigen binding ability. For example, in some embodiments, the nucleic acid encoding the variable region of the heavy chain and / or light chain is operably linked in frame with the nucleic acid encoding the constant region of the heavy chain and / or light chain, thereby producing a nucleic acid encoding the heavy chain and / or light chain of the antibody when expressed from a suitable expression vector.

[0224] In one aspect, the present invention provides a nucleic acid encoding any of the anti-OX40L antibodies or fragments thereof described herein. The nucleic acid may comprise a nucleic acid encoding an amino acid sequence of the light chain variable region and / or heavy chain variable region of the antibody, or a nucleic acid encoding an amino acid sequence of the light chain and / or heavy chain of the antibody.

[0225] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 4, 8-10, 14, and 18-20, or nucleic acids encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from any one of SEQ ID NOs: 4, 8-10, 14, and 18-20. As will be appreciated by those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. Nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, such as de novo solid phase DNA synthesis or PCR amplification. To facilitate production and purification, a secretory signal peptide and / or a tag peptide that facilitates purification can be fused to the N-terminus of the heavy and / or light chains of the antibody.

[0226] The present invention also provides vectors comprising nucleic acids of the present invention. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In preferred embodiments, the expression vector of the present invention is a pcDNA vector, such as the pcDNA3.1 expression vector.

[0227] In one embodiment, a host cell comprising the vector is provided. The present invention also provides a host cell comprising the nucleic acid or the vector. Host cells suitable for replication and support expression of the antibodies of the present invention are well known in the art. Such cells can be transfected or transduced with specific expression vectors, and large quantities of vector-containing cells can be grown for inoculating large-scale fermenters, thereby obtaining sufficient amounts of antibodies for clinical applications. Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. After expression, the antibodies can be separated from the bacterial cell paste in the soluble fraction and can be further purified.

[0228] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells (e.g., CHO-S or CHO-K) or 293 cells (e.g., 293F or HEK293 cells)) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, for example, a bacterium, such as Escherichia coli.

[0229] For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells, CHO-S cells, ExpiCHO, etc.; and myeloma cell lines such as Y0, NS0 and Sp2 / 0. Mammalian host cell lines suitable for producing antibodies are known in the art.

[0230] IV. Production and Purification of Antibody Molecules of the Invention

[0231] In one embodiment, the present invention provides a method for preparing an antibody molecule or fragment thereof (preferably an antigen-binding fragment) of the present invention, wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding an antibody molecule or fragment thereof (preferably an antigen-binding fragment) of the present invention, and optionally isolating the antibody or fragment thereof (e.g., an antigen-binding fragment). In a certain embodiment, the method further comprises recovering the antibody molecule or fragment thereof (e.g., an antigen-binding fragment) of the present invention from the host cell.

[0232] The polynucleotide encoding the polypeptide chain of the antibody of the present invention can be inserted into one or more vectors for further cloning and / or expression in a host cell. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0233] Antibody molecules prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, and the like. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and these will be apparent to those skilled in the art. The purity of the antibody molecules of the invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0234] V. Assay

[0235] The anti-OX40L antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by a variety of assays known in the art.

[0236] The present invention also provides assays for identifying anti-OX40L antibodies with biological activity. Biological activities may include, for example, binding to OX40L (e.g., binding to human OX40L), inhibition of the OX40 / OX40L signaling pathway, blocking the binding of OX40 to OX40L, inhibition of T cell activation, proliferation, and / or differentiation (e.g., differentiation toward Th2), inhibition of inflammatory cytokine production, and therapeutic effects on immune system diseases (e.g., graft-versus-host disease). Antibodies with such biological activities in vivo and / or in vitro are also provided.

[0237] For the determination of the above biological activity, please refer to the exemplary determination methods given in the Examples.

[0238] It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to the anti-OX40L antibody.

[0239] It will be appreciated that any of the above assays can be performed using a combination of an anti-OX40L antibody and an additional therapeutic agent.

[0240] VI. Immunoconjugates

[0241] In some embodiments, the present invention provides immunoconjugates comprising any of the anti-OX40L antibodies provided herein and other substances, such as active agents or labels suitable for forming immunoconjugates with the OX40L antibodies. In some embodiments, the active agent suitable for forming immunoconjugates with the OX40L antibodies can be, for example, a toxin, a small molecule drug, a cytotoxic agent, an apoptotic agent, a chelating agent, an immunomodulatory agent, such as an anti-inflammatory agent or an immunosuppressant.

[0242] In some embodiments, the immunoconjugate is an antibody drug conjugate (ADC).

[0243] VII. Pharmaceutical Compositions and Pharmaceutical Formulations

[0244] In some embodiments, the present invention provides a composition comprising any of the anti-OX40L antibodies or fragments thereof (preferably antigen-binding fragments thereof) or immunoconjugates thereof described herein, preferably a pharmaceutical composition. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an anti-OX40L antibody or fragment thereof or immunoconjugate thereof of the present invention in combination with one or more other therapeutic agents.

[0245] The present invention also includes compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising anti-OX40L antibodies or immunoconjugates thereof, or compositions (including pharmaceutical compositions or pharmaceutical preparations) comprising polynucleotides encoding anti-OX40L antibodies. In certain embodiments, the compositions comprise one or more antibodies or fragments thereof that bind to OX40L, or one or more polynucleotides encoding one or more anti-OX40L antibodies or fragments thereof. These compositions may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0246] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0247] For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0248] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0249] Pharmaceutical formulations comprising the antibodies described herein can be prepared by mixing the antibodies of the invention having the desired degree of purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or an aqueous solution.

[0250] The pharmaceutical composition or preparation of the present invention can also include more than one active ingredient, the active ingredient being required for the specific indication being treated, preferably having those active ingredients of complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents, such as cytokines, small molecule drugs, immunomodulators (such as immunosuppressants or anti-inflammatory agents) or other antibodies, etc. The active ingredients are suitably combined in an amount effective for the intended use.

[0251] Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, eg, films, or microcapsules.

[0252] VIII. Pharmaceutical Combinations and Kits

[0253] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-OX40L antibody or fragment thereof (preferably an antigen-binding fragment) of the present invention, or an immunoconjugate thereof, and one or more other therapeutic agents (cytokines, small molecule drugs, immunomodulators (e.g., immunosuppressants or anti-inflammatory agents) or other antibodies, etc.).

[0254] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0255] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0256] - a first container containing a pharmaceutical composition comprising an anti-OX40L antibody or fragment thereof;

[0257] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0258] IX. Use and Method

[0259] In some embodiments, the antibodies of the present invention are used as single agents or in combination to prevent or treat diseases or conditions associated with aberrant activation of OX40L / OX40-mediated pathways in an individual.

[0260] In some embodiments, the antibodies of the present invention can inhibit T cell activation responses by blocking OX40L-OX40 signals to reduce related immune responses and reduce diseases or symptoms associated with excessive autoimmune responses.

[0261] In some embodiments, the patient with the disease or condition has an overstimulation of the immune system mediated by OX40L / OX40 compared to healthy individuals. In some embodiments, the patient with the disease or condition has increased expression of OX40L on cells (e.g., antigen presenting cells) and / or increased expression of OX40 on cells (e.g., T cells, such as activated T cells) in the patient, e.g., compared to corresponding cells in healthy individuals.

[0262] In some embodiments, the disease or illness are selected from autoimmune diseases or graft-versus-host disease.In some embodiments, autoimmune diseases include but are not limited to allergic dermatitis, asthma, systemic lupus erythematosus, Sjogren's syndrome, immune thrombocytopenic purpura, multiple sclerosis, lupus nephritis, amyotrophic lateral sclerosis, rheumatoid arthritis, non-rheumatoid arthritis, contact dermatitis, high IgE syndrome, inflammatory bowel disease, myasthenia gravis, Graves' disease, hemolytic anemia, psoriasis, atopic dermatitis (e.g., moderate to severe atopic dermatitis), allergic asthma or idiopathic inflammatory disease (CN106459196B).

[0263] In some embodiments, the antibodies or antibody fragments or immunoconjugates or compositions or products of the invention delay the onset of a disorder and / or symptoms associated with the disorder.

[0264] In some embodiments, the present invention provides use of an anti-OX40L antibody or fragment thereof, or an immunoconjugate or composition comprising the same, in the production or preparation of a medicament for use as described herein, e.g., for preventing or treating a disease or condition as described herein.

[0265] In some embodiments, the methods of prevention or treatment described herein further comprise administering to the subject or individual an antibody molecule, pharmaceutical composition, or immunoconjugate disclosed herein in combination with one or more other therapies, such as therapeutic modalities and / or other therapeutic agents. In some embodiments, the anti-OX40L antibody or fragment thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as therapeutic modalities and / or other therapeutic agents, for the purposes described herein, e.g., for preventing and / or treating the relevant diseases or conditions mentioned herein.

[0266] In some embodiments, the therapeutic agent is selected from cytokines, small molecule drugs, immunomodulators (such as immunosuppressants or anti-inflammatory agents) or other antibodies, etc.

[0267] In some embodiments, the antibodies described herein can be combined with other antibodies for separate administration, e.g., each as a separate antibody, or when linked (e.g., as a bispecific or multispecific antibody molecule).

[0268] Such combination therapies encompass combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibodies of the invention can occur prior to, concurrently with, and / or after administration of the other therapeutic agents and / or therapies.

[0269] The antibodies or fragments thereof of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, preparations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration and, if desired for local treatment, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion.

[0270] X. Methods and Compositions for Diagnosis and Detection

[0271] In one aspect, the present invention also relates to methods for diagnosis and detection of the antibodies or antigen-binding fragments thereof of the present invention and compositions for diagnosis and detection comprising the same.

[0272] In certain embodiments, any of the anti-OX40L antibodies or fragments thereof (preferably antigen-binding fragments) provided herein can be used to detect the presence of OX40L in a biological sample.

[0273] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid samples of biological origin. In certain embodiments, the biological sample comprises cells or tissues.

[0274] In one embodiment, an anti-OX40L antibody or fragment thereof is provided for use in a method of diagnosis or detection.

[0275] In another aspect, a method for detecting the presence of OX40L in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of OX40L protein in the biological sample. In certain embodiments, OX40L is human OX40L. In certain embodiments, the method comprises contacting the biological sample with an anti-OX40L antibody or fragment thereof as described herein under conditions that allow binding of the anti-OX40L antibody or fragment thereof to OX40L, and detecting whether a complex forms between the anti-OX40L antibody or fragment thereof and OX40L. Formation of a complex indicates the presence of OX40L. The method can be an in vitro or in vivo method. In one embodiment, the anti-OX40L antibody or fragment thereof is used to select a subject suitable for treatment with the anti-OX40L antibody or fragment thereof, e.g., where OX40L is a biomarker used to select the subject.

[0276] In some embodiments, a labeled anti-OX40L antibody or fragment thereof is provided. Labels include, but are not limited to, directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., via an enzymatic reaction or molecular interaction.

[0277] In some embodiments provided herein, the sample is obtained prior to treatment with an anti-OX40L antibody or fragment thereof. In some embodiments, the sample is obtained prior to treatment with other therapies. In some embodiments, the sample is obtained during treatment with other therapies, or after treatment with other therapies.

[0278] In some embodiments, OX40L is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0279] In some embodiments, a method of treating a disease of the present invention is provided, the method comprising: testing a subject (e.g., a sample) (e.g., a subject sample) for the presence of OX40L, thereby determining an OX40L value, comparing the OX40L value to a control value, and if the OX40L value is greater than the control value, administering to the subject a therapeutically effective amount of an anti-OX40L antibody or fragment thereof (e.g., an anti-OX40L antibody or fragment thereof described herein), optionally in combination with one or more other therapies, thereby treating the disease.

[0280] These and other aspects and embodiments of the present invention are described in the accompanying drawings (a brief description of the drawings follows) and the following detailed description of the invention and are exemplified in the following examples. Any or all of the features discussed above and throughout this application may be combined in various embodiments of the present invention. The following examples further illustrate the present invention, however, it should be understood that the examples are described in an illustrative and non-limiting manner, and that various modifications may be made by those skilled in the art. Example

[0281] Equipment: Thermo Fisher MULTISKAN-FC (Examples 1-3)

[0282] Equipment: Fluorescence quantitative microplate reader SPARK (Example 1)

[0283] Equipment: BD FACSymphony A3 Flow Cytometer (Example 4)

[0284] Reagents and raw materials:

[0285] Example 1: Anti-OX40L antibody molecules inhibit the binding of OX40 and OX40L

[0286] Using hybridoma technology, mice were immunized with either a human GS-CHO cell line overexpressing humanOX40L or a human OX40L fusion protein (Human OX40 Ligand / TNFSF4 Protein, His Tag (active trimer) (MALS verified), Acro). The mice were sacrificed, spleens were removed, and digested to a single-cell suspension. The splenocytes were then hybridized with proliferative myeloma cells. Hybridomas expressing positive antibodies were isolated in a selective culture medium. Murine antibodies capable of binding to OX40L and blocking the OX40-OX40L interaction were then screened using in vitro flow cytometry and ELISA.

[0287] CHO-OX40L cell preparation

[0288] The nucleic acid sequence encoding Human OX40L (SEQ ID NO:30) was constructed in the pXC17.4 plasmid and digested with Pvu I (NEB) to obtain a linearized vector. Well-growing GS-CHO cells were electroporated with 40 μg of vector and 1 × 10^7 cells. Following electroporation, the cells were transferred to fresh GS-CHO culture medium without GlutaMax. After the GS-CHO cells grew normally, L-methionine sulfoximine was added to the culture medium to select the cells. After cell viability recovered to above 95%, flow cytometry was performed to isolate the GS-CHO-OX40L (CHO-OX40L).

[0289] immunity

[0290] Balb / c mice (purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were immunized with a human GS-CHO cell line that highly expresses human OX40L (CHO-OX40L) or a human OX40L fusion protein (Human OX40 Ligand / TNFSF4 Protein, His Tag (active trimer) (MALS verified), Acro) once every two weeks for a total of three immunizations.

[0291] Cell fusion

[0292] When the serum titer meets the requirements, the spleen of the mouse is removed to prepare B lymphocyte suspension, which is mixed with SP2 / 0 myeloma cells (ATCC) at a ratio of 1:2 to 1:1 and then electrofused. The fused cells are transferred from the electrode dish into a 50ml centrifuge tube and diluted with HAT-containing culture medium to 1 to 2 × 10 4 Cells were cultured at a concentration of 100 μl per well of a 96-well plate. The selection medium was replaced on the 7th day after fusion. After the 10th day (or longer, depending on the cell growth status), flow cytometry (FACS) was performed to screen for positive clones.

[0293] Hybridoma positive clone screening (FACS)

[0294] Hybridoma cells that specifically express anti-OX40L antibodies were screened by flow cytometry (FACS). The cells to be tested (GS-CHO humanOX40L) were counted and diluted to 1×10 6 For each cell / ml, add 100 μl / well of a U-bottom 96-well plate. Centrifuge at 500 g for 5 minutes and remove the cell culture medium. Add the supernatant from the hybridoma 96-well plate to the U-bottom plate and resuspend the cells. Add 100 μl to each well and incubate on ice for 30 minutes. Centrifuge at 500 g for 5 minutes and remove the supernatant. Wash the cells once with PBS. Centrifuge at 500 g for 5 minutes and remove the PBS. Add 100 μl of FITC-conjugated secondary antibody (1:500 dilution in PBS) against mouse Fab (Jackson Immunoresearch, Cat#115-545-006) to each well. For the positive control antibody, add 100 μl of PE-conjugated secondary antibody against human Fc (Biolegend, Cat#409304). Incubate on ice for 30 minutes in the dark. Remove the supernatant at 500 g for 5 minutes and wash the cells once with PBS. Resuspend the cells in 50 μl of 1× PBS and analyze on a FACS machine.

[0295] Screening of hybridoma cells blocking OX40 / OX40L function

[0296] Positive hybridoma clones were selected and further tested for their activity in blocking the binding of OX40 and OX40L. Based on the blocking results, subclones of the positive clones were selected.

[0297] Positive hybridoma cell subcloning

[0298] Prepare a 96-well plate and add 200 μl of culture medium to each well. This medium is the same as the screening medium except that HAT is replaced with HT (Gibco, Cat#11067-030). Prepare a cell suspension from the cells in the positive wells screened for fusion. Add 100 μl of the cell suspension from the first row to each well and mix thoroughly. Then, add 100 μl of the cell suspension from the first row to the second row, mix thoroughly, and add 100 μl to the next row. Repeat this process. Let the 96-well plate stand for 30 minutes and count under a microscope. Add 20 ml of culture medium corresponding to 100 cells, mix thoroughly, and plate 200 μl per well. After one week, observe under a microscope to identify and mark the wells with single clones. When the confluence of each well reaches 50% or higher, perform the same FACS screening and blocking assay as described above. Select the positive wells for in vitro screening. The resulting hybridoma supernatant is then assayed for antigen affinity using thin-layer biofilm interferometry (ForteBio).

[0299] Hybridoma sequencing

[0300] The positive clones obtained by screening were subjected to hybridoma sequencing to obtain the antibody variable region sequence.

[0301] RNA extraction: RNA extraction kit (Macherey-Nagel, Cat#740984.250) was used to extract hybridoma cell RNA. Approximately 5×10 freshly cultured cells were taken. 6 Centrifuge at 300g for 5 minutes, remove the supernatant, add 500μl of LBP buffer to the pellet, and mix until clear. Add to a DNA removal tube, centrifuge at 11,000rpm for 1 minute, and collect the flow-through. Add 100μl of LBS to the flow-through and mix. Add the clarified solution to an RNA collection tube, centrifuge at 11,000rpm for 1 minute to remove the liquid, add 200μl of WB1, and centrifuge at 11,000rpm for 1 minute to remove the liquid. Add 600μl of WB2 to the RNA collection tube, centrifuge at 11,000rpm for 1 minute to remove the liquid. Add 250μl of WB2 to the RNA collection tube, centrifuge at 11,000rpm for 2 minutes, discard the liquid, and spin the RNA collection tube for 1 minute to completely remove the liquid. Uncap the RNA column and transfer it to a new 1.5mL EP tube. Evaporate the ethanol for 2 minutes, add 50μl of DEPC-treated ddH2O, let it stand for 2 minutes, and centrifuge at 11,000g for 1 minute to collect the eluate. Determine RNA concentration.

[0302] Reverse transcription PCR: using PrimeScript TMcDNA was obtained by reverse transcription using the RT reagent kit (Takara). The reaction system was as follows: 4 μl of 5× PrimeScript Buffer, 1 μl of 1× PrimeScript RT Enzyme Mix, 1 μl of Oligo dT Primer (50 μM), 8 μl of Total RNA, and 6 μl of ddH₂O. The reaction procedure was as follows: 37°C for 15 min, 85°C for 5 s, and a 4°C hold. RT-PCR product cDNA was obtained.

[0303] PCR amplification of VH / VL sequences: Two cDNAs were used to amplify the heavy and light chain variable regions, respectively. The reaction system was as follows: 0.5 μl of ExTag (ExTaq Hot Start Version, Takara), 3 μl of 10× ExTaq buffer, 4 μl of dNTPs, 2 μl of forward primer, 2 μl of reverse primer, 1 μl of template (cDNA), and 17.5 μl of ddH2O. The reaction procedure was as follows: Step 1: 94°C for 5 min; Step 2: 94°C for 30 s; Step 3: 55°C for 30 s; Step 4: 72°C for 40 s; Step 5: 72°C for 10 min. Steps 2 and 4 were cycled 30 times. The PCR products were ligated into a T-vector using the Mighty TA-cloning Kit (Takara).

[0304] Transformation: Remove TOP10 competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd.) at -80°C and thaw on ice. Add 5 μl of the ligation product obtained above to the thawed TOP10 competent cells, mix well, and incubate on ice for 30 minutes. Heat shock at 42°C for 90 seconds, then quickly cool on ice for 2 minutes. Add 900 μl of LB medium (Sangon Biotech (Shanghai) Co., Ltd.) to an EP tube and incubate at 37°C with a shaker at 220 rpm for 1 hour. Centrifuge at 3000 g for 2 minutes, remove 800 μl of the supernatant, resuspend the cells in the remaining medium, and plate on ampicillin-resistant plates. Incubate overnight at 37°C. Select clones for sequencing, and analyze and compare the results using MEGA7 software.

[0305] Construction of chimeric antibodies

[0306] Using molecular biology techniques, the heavy and light chain sequences of the anti-OX40L antibody produced by the hybridoma cells were obtained. The variable region gene fragments of each light and heavy chain were ligated into the pcDNA3.1 vector using homologous recombinases. The constant regions were selected to be of the IgG4 subtype (heavy chain constant region: containing the S228P mutation, SEQ ID NO:21; light chain constant region: SEQ ID NO:22), generating expression plasmids for both the light and heavy chains. The light and heavy chain plasmids of the same antibody were then mixed at a 1:1 molar ratio and transfected into 293F cells using polyethyleneimine (PEI) (Polysciences, Cat#23966). After 5-7 days of culture, when cell viability dropped below 60%, the cell culture supernatant was collected and the monoclonal antibody was purified using a Protein A affinity column to obtain the human-mouse chimeric antibody.

[0307] Chimeric antibody screening: Flow cytometry and luciferase reporter gene assays were used for in vitro screening to obtain anti-OX40L antibodies that could both bind to OX40L and block the OX40-OX40L interaction.

[0308] Antibody humanization

[0309] The chimeric antibody obtained in the above example was humanized according to conventional methods. The CDR sequences, light chain variable region and heavy chain variable region sequences, and amino acid sequences of the light chain and heavy chain of the humanized antibody are shown in the table below.

[0310] Expression and purification of humanized antibodies

[0311] HEK293 cells (Invitrogen) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5 × 10 6 cells / ml. The cell density on the day of transfection was approximately 3×10 6cells / ml. Take Opti-MEM medium (Gibco catalog number: 31985-070) at a final volume of 1 / 10 (v / v) as transfection buffer, add the expression plasmid pcDNA3.1 vector containing the heavy and light chains of the humanized antibodies (which contains nucleic acids encoding the heavy and light chains of the HZ18 and HZ22 antibodies), mix thoroughly, and filter through a 0.22μm filter until ready for use. Add the appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid from the previous step (plasmid to PEI mass ratio of 1:3), mix thoroughly, and incubate at room temperature for 10 minutes to obtain a DNA / PEI mixture. The DNA / PEI mixture was gently poured into HEK293 cells and mixed thoroughly. After incubation at 37°C, 8% CO2 for 24 h, VPA (Sigma, Catalog No.: P4543-100G) was added to a final concentration of 2 mM and 2% (v / v) Feed solution (1 g / L Phytone Peptone + 1 g / L Difco Select Phytone) was added. Culture was continued for 6 days.

[0312] After cell culture, the cell culture medium was centrifuged at 13,000 rpm for 20 minutes, and the supernatant was collected and purified using a prepacked Hitrap Mabselect Sure column (GE, 11-0034-95) according to the manufacturer's instructions, and the concentration was determined. 100 μg of the purified protein was adjusted to a concentration of 1 mg / mL and the protein purity was determined using a gel filtration column SW3000 (TOSOH Catalog No. 18675). The results showed that a highly pure chimeric antibody was obtained.

[0313] Humanized antibody affinity determination

[0314] The affinity of the humanized antibodies for binding to antigens (human and cynomolgus monkey OX40L) was determined using the ForteBio assay and expressed as the equilibrium dissociation constant (KD). The results are shown in Table 1.

[0315] Table 1. Affinity constants (M) for antigen-antibody binding assays performed by ForteBio.

[0316] In this application, the inventors screened for antibodies with higher affinity for OX40L and humanized them to obtain two humanized antibodies, HZ18 and HZ22. In this example, their blocking effects on OX40L at the molecular and cellular levels were tested. Furthermore, in a co-culture system of CHO-OX40L-overexpressing cells and Jurkat-OX40-NFκB-Luc reporter cells, the addition of the anti-OX40L antibodies of the present invention blocked the binding of OX40L on the surface of CHO cells and OX40 on the surface of Jurkat-OX40-NFκB-Luc reporter cells, thereby inhibiting the activation of NFκB and luciferase production downstream of Jurkat cells, and reducing the fluorescence signal of the system.

[0317] ELISA detection of antibody blocking OX40 / OX40L protein binding level

[0318] Experimental procedures: Dilute OX40L recombinant protein to a concentration of 1μg / ml using citrate-buffered saline, add 100μl per well to a 96-well microtiter plate, and incubate overnight at 4°C. The next day, remove the coated microtiter plate, wash three times with PBST buffer (1×PBS, 0.05% Tween), pat dry, and add blocking buffer (1×PBS, 0.05% Tween, 1% BSA) to the 96-well microtiter plate and incubate at room temperature for 2 hours. Wash three times with PBST buffer and pat dry. Dilute OX40-Avi to 1μg / ml in blocking buffer and dilute the antibody to be tested in blocking buffer. Add 50μl of the diluted OX40-Avi and 50μl of the diluted antibody to be tested to each 96-well microtiter plate and incubate at room temperature for 2 hours. Wash three times with PBST buffer and pat dry. Dilute Streptavidin-HRP (1:3000) in blocking buffer and add 100 μl per well to a 96-well microtiter plate. Incubate at room temperature in the dark for 1 hour. Wash three times with PBST buffer and pat dry. Develop the plate with 100 μl of TMB colorimetric solution and stop the color development with stop buffer. Read the OD450 absorbance in a microtiter plate reader.

[0319] Reporter gene system detection of antibody blocking OX40 signaling activation

[0320] Experimental Procedure: Prepare well-grown OX40L-overexpressing cells (CHO-OX40L) and OX40-overexpressing reporter cells (Jurkat-OX40-NFκB-Luc). Count the cells, adjust the CHO-OX40L to 5.5×10^4 / ml, and the Jurkat-OX40-NFκB-Luc to 2.2×10^6 / ml using 1640 complete medium. Also dilute the antibody using 1640 complete medium. Add 45μl of CHO-OX40L and 45μl of Jurkat-OX40-NFκB-Luc cells to a 96-well white-bottom plate, along with 10μl of the antibody. Incubate the 96-well white-bottom plate in a 37°C cell culture incubator for 6 hours. After incubation, add 100μl of Bio-Glo Reagent to each well. Incubate at room temperature in the dark for 10 minutes, then read the fluorescence on a multi-function microplate reader. Figure 1 shows the experimental results. Among them, antibody KY1005 (US10654935B2).

[0321] Example 2: Inhibition of primary T cell activation by anti-OX40L antibodies

[0322] In a co-culture system of CHO-OX40L-overexpressing cells and primary T cells, the effect of anti-OX40L antibodies on T cell activation was detected, and the inhibitory effect was reflected by measuring the secretion level of IL-2 in the culture system.

[0323] The experimental steps are as follows: OKT3 was diluted to 1 μg / ml, 100 μl was added to each well of a 96-well U-bottom plate, and the plate was coated overnight at 4°C. The next day, the liquid in the 96-well U-bottom plate was removed, the plate was washed three times with 1× PBS, and patted dry. Pan T cells were isolated from PBMC using the Pan-T Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions, and the cell density was adjusted to 1×10 cells using RPMI medium 1640 + 5% FBS. 6 / ml, and add anti-human CD28. Use 1640 complete culture medium to dilute CHO-OX40L cells to 0.8×105 / ml, and use 1640 complete culture medium to dilute the antibody to be tested. Add 100μl Pan T cells, 50μl CHO-OX40L and 50μl of the antibody to be tested to the patted dry 96-well U-bottom plate. Place the 96-well U-bottom plate in a 37°C cell culture incubator and incubate for 3 days. After three days, remove the 96-well U-bottom plate, centrifuge at 500×g for 10 minutes, and aspirate the culture supernatant. Use IL-2 ELISA (Human IL-2 DuoSet ELISA, R&D) to detect the IL-2 content in the supernatant to indicate the activation of T cells.

[0324] The experimental results are shown in Figure 2. As shown in Figure 2, it can be seen that the blocking antibody against OX40L can significantly inhibit the activation level of T cells in vitro.

[0325] Example 3: Effect of anti-OX40L antibodies on DCs-induced T cell differentiation into Th2

[0326] Existing studies have shown that thymic stromal lymphopoietin (TSLP) can induce the expression of OX40L on dendritic cells, thereby promoting T cell differentiation into Th2 during antigen presentation and secreting Th2 cell-related factors IL-4, IL-5 and IL-13.

[0327] In this experiment, isolated primary DCs were stimulated with recombinant hTSLP (Sino Biological), and then DCs were isolated CD4 T cells were co-cultured for 6 days, and then anti-CD3 / anti-CD28 antibodies were added to further activate the T cells. Anti-OX40L antibodies or isotype controls were added to the entire culture system, and the inhibitory effect of the antibodies was evaluated by measuring the secretion levels of IL-5 and IL-13.

[0328] Experimental steps:

[0329] Isolate mDCs using the EasySep Human Myeloid DC Enrichment Kit (Stemcell):

[0330] Remove PBMCs, thaw quickly, and wash twice with 1× PBS; resuspend PBMCs to 5×107 cells / ml in MACS Buffer; add FcR blocker 15μl / ml, Myeloid DC Enrichment Cocktail Component A 50μl / ml, and DC Enrichment Cocktail Component B to the system, mix well, and incubate at room temperature in the dark for 30 minutes; remove the separated magnetic beads, vortex to mix, and add them to the system at 100μl / ml, mix well, and incubate at room temperature in the dark for 10 minutes; add MACS Buffer to the volume of the system to 2.5ml and transfer to a flow tube; place the flow tube on a magnetic stand and wait at room temperature for 5 minutes; aspirate the liquid into a new flow tube, place the new flow tube in the magnetic stand again, and wait at room temperature for 5 minutes; aspirate the liquid into a 15ml tube and centrifuge at 500×g for 10 minutes; resuspend mDCs using X-VIVO 15 and centrifuge at 500×g for 10 minutes. Resuspend mDCs using an X-VIVO 15 (Lonza) and adjust the cell density to 1×10^6 cells / ml. Add recombinant hTSLP to the mDC suspension at a final concentration of 20 ng / ml, and seed the cells into ultra-low-attachment 96-well U-bottom plates with 200 μl per well. Culture the mDCs in the ultra-low-attachment 96-well U-bottom plates at 37°C for 2 days.

[0331] Remove the mDC cells from the incubator and centrifuge at 500×g for 10 minutes; remove the supernatant, resuspend the cells in fresh X-VIVO 15 culture medium, and centrifuge at 500×g for 10 minutes; resuspend the cells in fresh X-VIVO 15 culture medium again, centrifuge at 500×g for 10 minutes, and remove the supernatant; adjust the mDC concentration to 160,000 cells / ml using X-VIVO 15.

[0332] Naive CD4+ T cells were isolated using the EasySep Human Naive CD4+ T Cell Isolation Kit (Stemcell): PBMCs were removed, thawed quickly, and washed twice with 1× PBS; PBMCs were resuspended in MACS buffer to 5×107 cells / ml; 50μl / ml of Biotinylated Anti-CD45RO Antibody and 50μl / ml of Isolation Cocktail were added, mixed, and incubated at room temperature for 5 min; the separated magnetic beads were removed, vortexed to mix, and then added to the system at 50μl / ml, mixed, and incubated at room temperature for 5 min; MACS buffer was added to the system to a volume of 2.5ml and transferred to a flow cytometry tube; the flow cytometry tube was placed on a magnetic stand and waited at room temperature for 5 min; the liquid was pipetted into a new flow cytometry tube, which was placed in the magnetic stand again and waited at room temperature for 5 min; the liquid was pipetted into a 15ml tube and centrifuged at 500×g for 10 min; T cells were resuspended using an X-VIVO 15 and centrifuged at 500×g for 10 min. Resuspend naive CD4+ T cells in X-VIVO 15 and adjust the cell density to 400,000 cells / ml.

[0333] Add 50 μl of diluted mDCs, 100 μl of naive CD4+ T cells, and 50 μl of antibody to an ultra-low-adhesion 96-well U-bottom plate and incubate the plate in a 37°C, 5% CO2 cell culture incubator for 4 days. After 4 days of incubation, remove the plate and centrifuge at 500 × g for 10 minutes. Discard 140 μl of supernatant. Dilute the 4× antibody as above and add 120 μl of fresh X-VIVO 15 culture medium and 40 μl of 4× antibody to the plate. Homogenize the cells in the plate by air-drying. Incubate the plate in a 37°C, 5% CO2 cell culture incubator for another 3 days. Remove the ultra-low-adsorption 96-well U-bottom plate and centrifuge at 500×g for 10 minutes. Discard 150μl of the supernatant. Dilute Anti-Human CD28 to 3.33μg / ml (1.67×) using X-VIVO 15 and add 120μl per well to the U-bottom plate coated with OKT3 the day before. Dilute the test antibody 4× as above and add 40μl per well to the U-bottom plate coated the day before. Sweep the cells that have been centrifuged to the bottom of the ultra-low-adsorption 96-well U-bottom plate and transfer them all to the 96-well U-bottom plate coated the day before. Incubate in a 37°C incubator for 1 day. The 96-well U-bottom plate was removed from the incubator and centrifuged at 500 × g for 10 min. 140 μl of the supernatant was transferred to a new V-bottom plate and placed at -80°C for ELISA experiments using Human IL-5 DuoSet ELISA and Human IL-13 ELISA BASIC kit (HRP) to detect cytokines.

[0334] The experimental results are shown in Figure 3. As shown in Figure 3, it can be seen that the blocking antibody against OX40L can significantly inhibit the Th2 inflammatory response.

[0335] Example 4: Effect of anti-OX40L antibodies in a graft-versus-host disease (GvHD) mouse model

[0336] Part I: GvHD animal experimental process and disease scoring

[0337] NOG mice were intravenously injected with PBMC (1*10^7 / mouse) to establish a GVHD model. The mice were immediately randomized into 4 groups of 9 mice each. The drug was administered once a week for a total of four times, namely D0, D7, D14, and D21. The body weight of the mice was recorded twice a week, and their survival was observed. A weight change chart and survival curve were prepared, as shown in Figure 4. Blood was collected on D14 for flow cytometry. In this experiment, HZ22 effectively reduced the weight loss caused by GVHD in mice and significantly reduced the mortality rate of mice; HZ18 significantly reduced the mortality rate of mice.

[0338] PBMC information

[0339] Manufacturer: AllCellS Item No.: FPB005F-C Lot No.: 3024702

[0340] Mouse information

[0341] Strain: NOG Gender: Female Source: Weitonglihua Laboratory Animal Technology Co., Ltd. Certificate No.: 110011211111216336

[0342] Antibody information

[0343] Part 2: Streaming detection related markers

[0344] In this experiment, blood was collected on day 14 of modeling and analyzed by flow cytometry for CD4 T cell counts. The proportion of Granzyme B-positive CD4 T cells was also measured. Serum IFN-γ and IL-6 levels were also measured by ELISA. As shown in Figure 5, the use of an OX40L blocking antibody inhibited IFN-γ and IL-6 secretion in the peripheral blood of GvHD mice, as well as T cell activation.

[0345] Sequence summary:

Claims

1. An anti-OX40L antibody or an antigen-binding fragment thereof, the antibody comprising: (i) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO:4, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO:8; or (ii) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:14, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:

18.

2. An anti-OX40L antibody or antigen-binding fragment thereof, comprising a first heavy chain complementarity determining region (HCDR1), a second heavy chain complementarity determining region (HCDR2), a third heavy chain complementarity determining region (HCDR3) and a first light chain complementarity determining region (LCDR1), a second light chain complementarity determining region (LCDR2) and a third light chain complementarity determining region (LCDR3), wherein: (i) the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7; or (ii) the HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 or respectively consist of the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:

17.

3. The anti-OX40L antibody or antigen-binding fragment thereof of claim 1 or 2, comprising a heavy chain variable region (VH), wherein the heavy chain variable region comprises, consists of, or comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or 14 having at least 90% identity, or consists of, or comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4 or 14 having at least 90% identity; or comprising a light chain variable region (VL), wherein the light chain variable region comprises, consists of, or comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8 or 18 having at least 90% identity.

4. An anti-OX40L antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises or consists of an amino acid sequence as set forth in SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable region comprises or consists of an amino acid sequence as set forth in SEQ ID NO:8 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; (ii) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:8; (iii) the heavy chain variable region comprises, or consists of, an amino acid sequence of SEQ ID NO:14, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and the light chain variable region comprises, or consists of, an amino acid sequence of SEQ ID NO:18 that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto; or (iv) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:

18.

5. The anti-OX40L antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:4, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:8; or (ii) the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 14, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:

18.

6. The anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 1 to 5, comprising an Fc region.

7. The anti-OX40L antibody or antigen-binding fragment thereof of claim 6, wherein the Fc region is Fc from human IgG, for example, Fc from human IgG1, Fc from human IgG2, Fc from human IgG3, or Fc from human IgG4.

8. The anti-OX40L antibody or antigen-binding fragment thereof of claim 7, wherein the Fc region comprises a mutation that enhances the stability of the Fc region or reduces chain exchange in the antibody.

9. The anti-OX40L antibody or antigen-binding fragment thereof of claim 8, wherein the mutation is an S228P mutation.

10. The anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 6 to 9, wherein the Fc region (i) comprising or consisting of the amino acid sequence of SEQ ID NO: 27 or 28; (ii) comprises an amino acid sequence that is at least 90% identical, such as 95%, 96%, 97%, 99% or more identical to the amino acid sequence of SEQ ID NO: 28; or (iii) comprising an amino acid sequence having at least 90% identity, such as 95%, 96%, 97%, 99% or more identity to the amino acid sequence of SEQ ID NO: 27, and comprising the S228P mutation.

11. The anti-OX40L antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, comprising a heavy chain constant region, wherein the heavy chain constant region is derived from the constant region of IgG1, IgG2, IgG3 or IgG4, preferably, the heavy chain constant region (i) comprises or consists of an amino acid sequence selected from SEQ ID NO: 21 or 29; (ii) comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29; or (iii) comprising an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21, and having an S228P deletion mutation.

12. The anti-OX40L antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, comprising a light chain constant region, wherein the light chain constant region is a lambda or kappa light chain constant region, preferably, the light chain constant region (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 22; or (ii) comprises the amino acid sequence of SEQ ID NO: 22 or consists of the amino acid sequence.

13. The anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 1-12, comprising a heavy chain, wherein the heavy chain (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 9 or 19; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 9 or 19; or It comprises a light chain, wherein the light chain (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 10 or 20; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 or 20.

14. An anti-OX40L antibody or antigen-binding fragment thereof, comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9; and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10; (ii) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 9, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 10; (iii) the heavy chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 19; and the light chain comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20; or (iv) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 19, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:

20.

15. The anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 1-14, comprising a heavy chain and a light chain, wherein (i) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 9, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO: 10; (ii) the heavy chain comprises or consists of the amino acid sequence of SEQ ID NO: 19, and the light chain comprises or consists of the amino acid sequence of SEQ ID NO:

20.

16. The anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 1-15, wherein the antibody is a monoclonal antibody.

17. The anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein the antibody is a humanized antibody or a chimeric antibody.

18. The anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 1 to 17, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody (e.g., scFv), (Fab')2, single domain antibody such as VHH, dAb (domain antibody), diabody, or linear antibody.

19. The anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 1-18, wherein the antibody or antigen-binding fragment thereof has one or more of the following properties: a) binds OX40L (eg, human OX40L) with high affinity; b) It can effectively block the interaction between OX40L and its receptor OX40 at both the protein and cellular levels; c) inhibiting the OX40 / OX40L signaling pathway; d) inhibiting T cell activation, proliferation and / or differentiation; e) reducing T cell activation and / or the production of inflammatory factors; f) Treatment of graft-versus-host disease.

20. An isolated nucleic acid encoding the anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 1-19.

21. A vector comprising the nucleic acid of claim 20, preferably said vector is an expression vector.

22. A host cell comprising the nucleic acid of claim 20 or the vector of claim 21, preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells (e.g., 293 cells or CHO cells, such as CHO-K cells or HEK293 cells) or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

23. A method for preparing an anti-OX40L antibody or an antigen-binding fragment thereof, the method comprising a) culturing the host cell of claim 22 under conditions suitable for expressing a nucleic acid encoding an anti-OX40L antibody or antigen-binding fragment thereof according to any one of claims 1 to 19, b) optionally isolating the antibody or antigen-binding fragment thereof, c) Optionally, the method further comprises recovering the anti-OX40L antibody or antigen-binding fragment thereof from the host cell. Optionally, the antibody is purified, for example, purified by Protein A.

24. An immunoconjugate comprising the anti-OX40L antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 and other substances, such as toxins, small molecule drugs, cytotoxic agents, apoptotic agents, chelating agents, immunomodulators, such as anti-inflammatory agents or immunosuppressants.

25. A pharmaceutical composition comprising the anti-OX40L antibody or antigen-binding fragment thereof according to any one of claims 1 to 19 or the immunoconjugate according to claim 24, and optionally a pharmaceutically acceptable excipient.

26. A pharmaceutical combination comprising the anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 1 to 19 or the immunoconjugate of claim 24, and one or more other therapeutic agents, for example, the therapeutic agent is selected from cytokines, other antibodies, small molecule drugs or immunomodulators (e.g., immunosuppressants).

27. A method for preventing or treating a disease or condition associated with inappropriate activation of an OX40L / OX40-mediated pathway in an individual, the method comprising administering to the subject an effective amount of the anti-OX40L antibody or antigen-binding fragment thereof of any one of claims 1 to 19, or the immunoconjugate of claim 24, or the pharmaceutical composition of claim 25, or the pharmaceutical combination product of claim 26.

28. The method of claim 27, wherein the subject has abnormal activation of an OX40L / OX40-mediated signaling pathway compared to healthy individuals.

29. The method of claim 27 or 28, wherein the disease or disorder is selected from an autoimmune disease or an allogeneic transplant, for example, an autoimmune disease includes but is not limited to atopic dermatitis, asthma, systemic lupus erythematosus, Sjogren's syndrome, immune thrombocytopenic purpura, multiple sclerosis, lupus nephritis, amyotrophic lateral sclerosis, rheumatoid arthritis, non-rheumatoid arthritis, contact dermatitis, hyper-IgE syndrome, inflammatory bowel disease, myasthenia gravis, Graves' disease, hemolytic anemia, psoriasis, atopic dermatitis, allergic asthma or idiopathic inflammatory disease.

30. The method of any one of claims 27-29, wherein the method further comprises administering one or more other therapies, e.g., treatment modalities and / or other therapeutic agents, e.g., the therapeutic agent is selected from cytokines, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

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