CN110003338B - anti-OX 40 antibodies and uses thereof - Google Patents

Abstract

The present application provides recombinant anti-OX 40(CD134) antibodies and uses thereof. The anti-OX 40(CD134) antibodies of the present application are capable of specifically binding to mammalian OX40, preferably have high affinity for human and/or primate OX40 proteins, block binding of OX40 to its ligands at certain concentrations, modulate receptor activity, stimulate activation and proliferation of T cells, and the like. The antibodies of the present application are useful for treating OX 40-related diseases, such as OX 40-related tumors.

Description

anti-OX 40 antibodies and uses thereof

Technical Field

The present application relates to the field of antibodies, and more specifically, the present application relates to antibodies against OX40 and uses thereof.

Background

The concept of the immune system to recognize and eliminate cancer cells was first proposed over 100 years ago, and the reactivity of T cells to cancer-associated antigens could be detected in the blood of different types of cancer patients. In the process of effective anti-tumor immunity, T cells, which are the core performers, are first activated by T Cell Receptor (TCR) -mediated antigen recognition signals, and simultaneously, a plurality of costimulatory signals and costimulatory signals finely regulate the strength and quality of T cell responses, and these inhibitory signals are immune checkpoints. Under physiological conditions, the co-stimulatory molecules are in equilibrium with immune checkpoint molecules, thereby minimizing damage to surrounding normal tissues, maintaining tolerance to self-tissues, and avoiding autoimmune reactions. Tumor cells can abnormally up-regulate co-suppressor molecules and related ligands through the mechanism, and inhibit T cell activation, thereby avoiding immune killing. Blockade against immune checkpoints is one of the effective strategies to enhance T cell activation and is also a hot target for anti-tumor drug development in recent years. To date, 3 immune checkpoint inhibitors have been approved in the united states for the treatment of a variety of cancer types, targeting CTLA-4 and PD-1/PD-L1, respectively.

Researches show that CTLA-4 and PD-1 play a role in immunosuppression in the process of T cell activation, thereby inhibiting the immune killing function of T cells on tumor cells; the blocking monoclonal antibodies aiming at the two targets can relieve the immunosuppression and restore the anti-tumor immune function of the T cells. In addition to inhibitory immune checkpoints, there is also a class of active immune checkpoints that are becoming new targets for drug development.

Activated immune checkpoint molecules, primarily T-cell activated costimulatory signaling molecules-T-cell costimulatory receptors, which belong to the Tumor Necrosis Factor Receptor (TNFR) family, function to regulate the proliferation, activation and differentiation responses of T-cells, including OX40(TNFRSF4), CD40(TNFRSF5), 4-1BB (T-cell antigen4-1BB homologue, CD137), GITR (glucorticoid-induced TNFR-related protein, GITR), and the like.

OX40, also known as CD134, ACT45 or TNFRSF4, is a member of the TNFR superfamily, whose gene is located on human chromosome 1 and encodes a 50kD type I transmembrane glycoprotein. The extracellular region has 191 amino acids, including three intact and oneA slightly shorter cysteine-rich domain (CRD). OX40 is expressed predominantly on activated effector T cells (Teff) and regulatory T cells (Treg), and is activated CD4⁺T、CD8⁺Activating receptors on the surface of T cells are currently on the forefront of the field known as "T cell co-stimulation" and are also expressed on NKT cells, NK cells and neutrophils. OX40 signal can activate the downstream NF-kappa B, PI3K and PKB pathways, and the continuous activation of the pathways can finally prolong the survival time of T cells, expand T cell memory and promote the cell killing capacity of the T cells; in addition, OX40 can also improve immunosuppressive effects in the tumor microenvironment by inhibiting the differentiation and activity of regulatory T cells (tregs), further enhancing the function of effector T cells.

Therefore, development of an OX40 targeted antibody would be expected to be useful for treating diseases associated with tumors, including advanced malignancies.

Disclosure of Invention

In a first aspect, the present application provides an antibody, or antigen-binding portion thereof, that specifically binds OX40, comprising a heavy chain variable region comprising HCDR1, HCDR2 and/or HCDR 3.

In some embodiments, the HCDR1 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 1-4. In some embodiments, the HCDR2 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 5-8. In some embodiments, the HCDR3 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 9-12. In alternative embodiments, the antigen binding portion is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab')₂A fragment, Fv fragment, scFv fragment, Fd fragment or single domain antibody.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 further comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and/or LCDR3 sequences.

In certain embodiments, the LCDR1 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 13-16. In certain embodiments, the LCDR2 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 17-19. In certain embodiments, the LCDR3 sequence comprises the amino acid sequence set forth in any one of SEQ ID NOs: 20-23.

In some embodiments, the heavy chain variable region of the antibody or antigen-binding portion thereof that specifically binds OX40 comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of SEQ ID NOs:24-30, or an amino acid sequence having at least 80% homology thereto; preferably, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO 24 or 29.

In some embodiments, the light chain variable region of the antibody or antigen-binding portion thereof that specifically binds OX40 comprises an amino acid sequence selected from the group consisting of the amino acid sequences set forth in any one of SEQ ID NOs:31-37, or an amino acid sequence having at least 80% homology thereto; preferably, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO 31 or 36.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 specifically binds primate OX 40.

In some embodiments, the antibody that specifically binds OX40 is a monoclonal antibody.

In some embodiments, the antibody that specifically binds OX40 is a murine antibody.

In some specific embodiments, the heavy chain variable region amino acid sequence of the antibody that specifically binds OX40 is set forth in SEQ ID NO:24 or 29, and the light chain variable region amino acid sequence of the antibody that specifically binds OX40 is set forth in SEQ ID NO:31 or 36.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is at 1 x 10^-8To 1X 10^-7The KD of M binds specifically to the OX40 molecule.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 has OX40 agonist function.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is capable of stimulating the activation and proliferation of T cells.

In a second aspect, the present application provides a nucleotide molecule encoding an antibody or antigen-binding portion thereof of the first aspect that specifically binds OX 40.

In a third aspect, the present application provides an expression vector comprising the nucleotide molecule of the second aspect.

In a fourth aspect, the present application provides a host cell comprising a nucleotide molecule according to the second aspect or an expression vector according to the third aspect.

In a fifth aspect, the present application provides a pharmaceutical composition comprising an antibody or antigen-binding portion thereof that specifically binds OX40 of the first aspect and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition further comprises one or more additional active ingredients. In some embodiments, the active ingredient is a chemotherapeutic agent, a PD-1 binding antagonist, or the like.

In a sixth aspect, the present application provides a vaccine comprising an antibody or antigen-binding portion thereof that specifically binds OX40 as described in the first aspect, and optionally an immunological adjuvant.

In some embodiments, the pharmaceutical composition of the fifth aspect or the vaccine of the sixth aspect is for use in treating an OX 40-related disease, such as a tumor.

In a seventh aspect, the application provides the use of an antibody or antigen-binding portion thereof that specifically binds OX40 of the first aspect, or a pharmaceutical composition of the fifth aspect, in the manufacture of a medicament for inhibiting Treg function, killing OX 40-expressing cells, eliciting a T cell-mediated response, increasing the function of effector T cells, increasing the function of memory T cells, and/or effectively inhibiting tumor growth.

In an eighth aspect, the application provides the use of an antibody or antigen-binding portion thereof that specifically binds OX40 of the first aspect, or a pharmaceutical composition of the fifth aspect, in the manufacture of a medicament for the prevention and/or treatment of an OX 40-related disease, such as a tumor.

In some embodiments, the tumor is selected from the group consisting of colon cancer, melanoma, mesothelioma, renal cell carcinoma, lymphoma, advanced solid tumors, and metastases of the foregoing.

In a ninth aspect, the present application provides a detection reagent or kit comprising an antibody or antigen-binding portion thereof described in the first aspect that specifically binds OX 40.

In other aspects, the application provides methods of preventing and/or treating an OX 40-associated disease, e.g., a tumor, comprising administering to an individual in need thereof an antibody or antigen-binding portion thereof that specifically binds to OX40 as described in the first aspect, or a pharmaceutical composition as described in the fifth aspect, or a vaccine as described in the sixth aspect. Optionally, the method further comprises administering in combination with other therapeutic agents, such as chemotherapeutic agents, PD-1 binding antagonists, and the like.

An antibody or antigen-binding portion thereof of the present application that specifically binds OX40 is capable of specifically binding to OX40 with one or more of the following effects: has OX40 agonist function, stimulates proliferation and activation of T cells, induces OX40 mediated antitumor immune response, and/or inhibits tumor growth.

Brief description of the drawings

FIG. 1 shows the results of validating the functional activity of candidate hybridoma monoclonal antibodies through the NF-. kappa.B signaling pathway: wherein FIG. 1A shows the results of primary screening of 30 monoclonal antibodies, and FIG. 1B shows the results of screening of 7 candidate monoclonal antibodies.

Figure 2 shows the results of validating the functional activity of candidate hybridoma monoclonal antibodies through the NFAT signaling pathway.

FIG. 3 shows the KD values of the Fortebio assay anti-OX 40 monoclonal antibodies: wherein FIGS. 3A-3G correspond to KD values for anti-OX 40 monoclonal antibodies 18B10, 4F2, 6H12, 6A6, 4B9, 6C1, and 13F7, respectively.

FIG. 4 shows the results of an experiment in which anti-OX 40 monoclonal antibodies bound to human OX40 and monkey OX 40.

FIG. 5 shows the blocking effect of anti-OX 40 monoclonal antibodies on human OX40 binding to its ligand OX 40L.

FIG. 6 shows the results of an assay of the EC50 value functional activity of an anti-OX 40 monoclonal antibody.

FIG. 7 shows that anti-OX 40 monoclonal antibodies stimulated human CD4⁺Node for T cell proliferation and IL-2 and IFN-gamma secretionAnd (4) fruit: wherein FIG. 7A is a CFSE flow detection CD4⁺T cell proliferation results, FIG. 7B shows the results of ELISA detection of IL-2 secretion, and FIG. 7C shows the results of ELISA detection of IFN-. gamma.secretion.

FIG. 8 shows in vivo efficacy and safety studies of anti-OX 40 monoclonal antibodies in MC38 tumor-bearing mice of humanized OX 40: wherein fig. 8A is a graph of tumor volume change trend of the mice, fig. 8B is a graph of body weight change trend of the mice, fig. 8C is a graph of ALT detection results, fig. 8D is a graph of AST detection results, fig. 8E is a photograph of tumors obtained after dissection, and fig. 8F is a graph of tumor weight as a percentage of the total body weight of the mice.

Detailed Description

The present application provides novel anti-OX 40 antibodies, or antigen-binding portions thereof, that specifically bind to OX 40. In preferred embodiments, the antibodies or antigen-binding portions thereof of the present application bind to OX40 molecules on the surface of target cells and have OX40 agonist function. Also provided are nucleotide molecules encoding the antibodies or antigen-binding fragments thereof, expression vectors comprising the nucleotide molecules, host cells comprising the nucleotide molecules or expression vectors, methods of making and purifying the antibodies, and medical and biological applications of the antibodies or antigen-binding fragments thereof, such as preventing or treating OX 40-related diseases or disorders. Methods of using the antibodies or antigen binding fragments thereof to detect OX40 and modulate OX40 activity are also encompassed by the present application, as well as related detection reagents or kits.

To facilitate understanding of the present application, certain terms used herein are first defined.

As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) and two light (L) chains interconnected by a disulfide bond, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated VH) and a heavy chain constant region (abbreviated CH). The heavy chain constant region comprises three domains, CH1, CH2, and CH 3. Each light chain comprises a light chain variable region (abbreviated VL) and a light chain constant region (abbreviated CL). The light chain constant region comprises a domain (CL 1). The VH and VL regions can be further subdivided into hypervariable regions known as Complementarity Determining Regions (CDRs) into which conserved regions known as Framework Regions (FRs) are interspersed. In some embodiments, both the light and heavy chain variable domains comprise, from N-terminus to C-terminus, FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4.

As used herein, the term "antigen-binding portion" of an antibody refers to a portion or segment of an intact antibody molecule that is responsible for binding to an antigen. The antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Antigen-binding fragments of antibodies can be prepared from intact antibody molecules using any suitable standard technique, including proteolytic digestion or recombinant genetic engineering techniques, among others. Non-limiting examples of antigen-binding moieties include: fab fragment, F (ab')₂Fragments, Fd fragments, Fv fragments, single chain Fv (scfv) molecules, single domain antibodies, dAb fragments, and minimal recognition units (e.g., isolated CDRs) consisting of amino acid residues that mimic a hypervariable region of an antibody. The term "antigen-binding portion" also includes other engineered molecules, such as diabodies, triabodies, tetrabodies, minibodies, and the like. For example, the Fd fragment herein refers to an antibody fragment consisting of VH and CH1 domains; the Fv fragment consists of the VL and VH domains in a single arm of the antibody; dAb fragments (Ward et al, Nature 1989; 341: 544-546) consist of VH domains.

It is well known to those skilled in the art that the complementarity determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. There are two common definitions of CDR Sequences for VH or VL, namely Kabat definition and Chothia definition, see, for example, Kabat et al, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et Al, J Mol Biol 273:927-948 (1997); and Martin et al, Proc.Natl.Acad.Sci.USA 86: 9268-. For a given antibody variable region sequence, can according to Kabat definition or Chothia definition to determine VH and VL sequence in CDR region sequence. In embodiments of the present application, the CDR sequences are defined using Kabat. Herein, CDR1, CDR2 and CDR3 of the heavy chain variable region are abbreviated as HCDR1, HCDR2 and HCDR3, respectively; CDR1, CDR2, and CDR3 of the light chain variable region are abbreviated as LCDR1, LCDR2, and LCDR3, respectively.

The CDR region sequences in the variable region sequences can be analyzed in a variety of ways for the variable region sequences of a given antibody, such as can be determined using the online software Abysis (http:// www.abysis.org /).

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, e.g., binding of an antibody to an epitope of an antigen, e.g., the ability of an antibody to bind to a specific antigen with at least two times greater affinity than its affinity for a non-specific antigen. It will be appreciated, however, that an antibody is capable of specifically binding to two or more antigens associated with its sequence. For example, an antibody of the invention can specifically bind to OX40 in humans and non-humans (e.g., mice or non-human primates).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations in a small number of individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies, so long as they exhibit the desired biological activity (see, U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

As used herein, the term "murine antibody" refers to any antibody in which all constant domain sequences are mouse sequences. Such antibodies can be produced by hybridomas.

As used herein, the term "chimeric antibody" refers to an antibody comprising segments from two or more different antibodies. In some embodiments, one or more CDRs are derived from a mouse anti-OX 40 antibody. In other embodiments, all of the CDRs are derived from a mouse anti-OX 40 antibody. In some embodiments, the CDRs from more than one mouse anti-OX 40 antibody are combined in a chimeric antibody. For example, a chimeric antibody can comprise a CDR1 from the light chain in a first mouse anti-OX 40 antibody, a CDR2 from the light chain in a second mouse anti-OX 40 antibody, and a CDR3 from the light chain in a third mouse anti-OX 40 antibody, and the CDRs from the heavy chain can be derived from one or more other anti-OX 40 antibodies. In addition, the framework regions may be from the same anti-OX 40 antibody or from one or more different individuals.

Suitable techniques that may additionally be used in the antibody method include OX 40-based affinity purification, non-denaturing gel purification, HPLC or RP-HPLC, size exclusion, purification on a protein a column, or any combination of these techniques. The OX40 antibody isotype can be determined using an ELISA assay, e.g., human Ig can be identified using mouse Ig-adsorbed anti-human Ig.

OX40 suitable for antibody production can be produced by any of a variety of standard protein purification or recombinant expression techniques known in the art. Forms of OX40 suitable for generating an immune response include OX40 subsequences (e.g., immunogenic fragments). Additional forms of OX40 include OX40 expressing cells, OX40 containing preparations or cell extracts or fractions, partially purified OX 40.

As used herein, the term "homology" is defined as the percentage of residues in an amino acid or nucleotide sequence variant that are identical, if necessary to the maximum percentage, after alignment and the introduction of gaps in the sequence. Methods and computer programs for alignment are well known in the art. As used herein, "at least 80% homology" means any value of 80% to 100% homology, e.g., 85%, 90%, 95%, 99%, etc.

As used herein, the term "OX 40-associated disease" includes diseases and/or symptoms associated with the OX40 signaling pathway. Exemplary OX 40-related diseases or disorders include tumors, such as colon cancer, melanoma, mesothelioma, renal cell carcinoma, lymphoma, advanced solid tumors, and metastases of the foregoing.

As used herein, the term "EC 50" refers to the concentration of the half maximal effect (concentration for 50% of the maximum effect, EC50), and refers to the concentration that causes 50% of the maximal effect.

In a first aspect, the present application provides an antibody, or antigen-binding portion thereof, that specifically binds OX40, comprising HCDR1, HCDR2 and/or HCDR3 of the heavy chain variable region. The CDR, heavy chain variable region and light chain variable region amino acid sequences suitable for use in the antibodies disclosed herein are exemplified in tables 1-4 below. In certain embodiments, the anti-OX 40 antibody or antigen-binding portion thereof comprises HCDR1, HCDR2, and/or HCDR3 sequences independently selected from any one of the HCDR1, HCDR2, and/or HCDR3 sequences shown in table 1. In certain embodiments, the anti-OX 40 antibodies of the present application may further comprise LCDR1, LCDR2, and/or LCDR3 independently selected from any one of the LCDR1, LCDR2, and/or LCDR3 sequences shown in table 2. For example, an anti-OX 40 antibody of the present application can comprise any of the heavy chain variable regions shown in table 3, optionally in combination or pairing with any of the light chain variable regions shown in table 4.

Table 1: sequence numbers of the heavy chain CDR amino acid sequences of exemplary anti-OX 40 antibodies

Table 2: sequence numbers of light chain CDR amino acid sequences of exemplary anti-OX 40 antibodies

Table 3: sequence numbers of amino acid sequences of heavy chain variable regions of exemplary anti-OX 40 antibodies

Table 4: sequence number of the light chain variable region amino acid sequence of an exemplary anti-OX 40 antibody

In some embodiments, the amino acid sequence of HCDR1 of an antibody or antigen-binding portion thereof disclosed herein is as set forth in any one of SEQ ID NOs 1-4. In some embodiments, the amino acid sequence of HCDR2 is as set forth in any one of SEQ ID NOs: 5-8. In some embodiments, the amino acid sequence of HCDR3 is as set forth in any one of SEQ ID NOs: 9-12.

In alternative embodiments, the antigen binding portion is selected from the group consisting of a Fab fragment, a Fab 'fragment, a F (ab')₂A fragment, Fv fragment, scFv fragment, Fd fragment or single domain antibody.

The antibodies or antigen-binding portions thereof disclosed herein may further comprise a light chain variable region in addition to the heavy chain variable region.

In some embodiments, the amino acid sequence of LCDR1 of an antibody or antigen-binding portion thereof disclosed herein is as set forth in any one of SEQ ID NOs 13-16. In some embodiments, the amino acid sequence of LCDR2 is as set forth in any one of SEQ ID NOs: 17-19. In some embodiments, the amino acid sequence of LCDR3 is as set forth in any one of SEQ ID NOs: 20-23.

In some specific embodiments, the heavy chain variable region of an antibody, or antigen-binding portion thereof, disclosed herein is at least 80% homologous, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to an amino acid sequence selected from the group consisting of SEQ ID NOs: 24-30. In some specific embodiments, the antibody heavy chain variable region consists of an amino acid sequence selected from any one of SEQ ID NOs: 24-30. In some more specific embodiments, the amino acid sequence of the variable region of the antibody heavy chain is set forth in SEQ ID NO:24 or 29.

In some specific embodiments, the light chain variable region of an antibody, or antigen-binding portion thereof, disclosed herein is at least 80% homologous, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous, to a sequence selected from SEQ ID NOs: 31-37. In some specific embodiments, the antibody light chain variable region consists of an amino acid sequence selected from any one of SEQ ID NOs: 31-37. In some more specific embodiments, the amino acid sequence of the variable region of the light chain of the above antibody is as set forth in SEQ ID NO 31 or 36.

In some embodiments, the heavy chain variable region or the light chain variable region of the antibodies disclosed herein can be substituted, deleted, or added with at least one amino acid based on the respective corresponding specific amino acid sequences listed above, and the resulting variants still retain the activity of binding to OX 40.

In certain embodiments, the number of amino acid substitutions, deletions or additions described above is any number between 1 and 30 or between 1 and 30, preferably between 1 and 20, more preferably between 1 and 10. In preferred embodiments, the sequence variant differs from the original amino acid sequence by substitutions, deletions and/or additions of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In more preferred embodiments, the sequence variant differs from the original amino acid sequence by a substitution, deletion or addition of about 1, 2, 3, 4 or5 amino acids. In particular embodiments, the amino acid substitution is a conservative substitution.

In a preferred embodiment, the antibody disclosed herein is antibody 6A6 or 4F2, wherein the heavy chain variable region amino acid sequence of antibody 6A6 is set forth in SEQ ID NO:29 and the light chain variable region amino acid sequence is set forth in SEQ ID NO: 36; the amino acid sequence of the heavy chain variable region of the antibody 4F2 is shown in SEQ ID NO. 24, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 31.

The antibodies, or antigen binding portions thereof, disclosed herein are capable of specifically binding OX 40. In some embodiments, the OX40 antibody exhibits species and molecular selectivity. In some specific embodiments, the antibody, or antigen-binding portion thereof, specifically binds to primate OX40, or OX40 of a species having high homology to primate OX 40. In some specific embodiments, the antibody, or antigen binding portion thereof, specifically binds to human OX40 and monkey OX 40. In some more specific embodiments, the anti-OX 40 antibody binds to human, cynomolgus or rhesus OX 40. In some other embodiments, the anti-OX 40 antibody does not bind to mouse, rat, dog, or rabbit OX 40.

As used herein, the term "KD" is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of KD to ka (i.e., KD/ka) and expressed in molar concentration (M). The KD values of antibodies can be determined using well established methods in the art. Preferred methods for determining the KD of an antibody are by using surface plasmon resonance, preferably using biosensor systems (e.g. SPR systems) or flow cytometry and Scatchard analysis.

As used herein, the term "high affinity" for an IgG antibody means that the antibody has 10 to the target antigen^-8M or less, preferably 10^-9M is less, and more preferably 10^-10M or less KD. However, for other antibody isotypes, "high affinity" binding may vary. For example, a "high affinity" binding for an IgM isotype refers to an antibody having 10^-7M or less, preferably 10^-8M or less KD.

As used herein, the term "antibody that inhibits the binding of OX40-L to OX 40" refers to an antibody that inhibits the binding of OX40-L to OX40, e.g., inhibits the binding of OX40-L to OX40 in a binding assay using HEK:: OX 40100B 5 cells, at an antibody concentration that partially or fully blocks the binding of 0.5 μ g of OX40-L to OX40 in art-recognized methods (e.g., FACS-based binding assays described herein).

As used herein, the terms "inhibit" or "block" (e.g., in reference to inhibition/blocking of binding of OX40-L to OX40 on a cell) are used interchangeably and encompass partial and complete inhibition/blocking. In certain embodiments, the anti-OX 40 antibody inhibits binding of OX40-L to OX40 by at least about 50%, e.g., about 60%, 70%, 80%, 90%, 95%, 99%, or 100%. In certain embodiments, the anti-OX 40 antibody inhibits binding of OX40-L to OX40 by no more than 50%, e.g., by about 40%, 30%, 20%, 10%, 5%, or 1%.

As used herein, the term "nucleotide molecule" is intended to include DNA molecules as well as RNA molecules. The nucleotide molecule may be single-stranded or double-stranded, and may be a cDNA.

In some more specific embodiments, the antibodies disclosed herein are anti-human OX40 monoclonal antibodies. The type and subtype of OX40 antibody can be determined by any means known in the art. Generally, antibody types and subtypes can be determined using antibodies specific for a particular antibody type and subtype.

As used herein, the term "agonist antibody" refers to an antibody that, when added to a cell, tissue, or organism that expresses OX40, increases one or more OX40 activities by at least about 20%. In certain embodiments, an antibody with agonist function increases OX40 activity by at least 40%, 50%, or 60%. The activated antibody may augment or replace the effect of OX40L on OX 40. In some embodiments, the activatable antibody is substantially a mimetic of OX40L and competes with OX40L for binding to OX 40. The terms "agonist antibody", "agonistic antibody" and "activating antibody" are used interchangeably herein.

In some embodiments, an anti-OX 40 activating monoclonal antibody can inhibit or eradicate a tumor by at least one of the following mechanisms: enhanced activation of tumor-specific CD4⁺And CD8⁺Lymphocytes inhibit Treg cells in tumors, and enhance the memory cells of tumor killing, etc. In some embodiments, other anti-tumor activities may be mediated by other immune enhancing effects of OX40 signaling (e.g., production of chemokines and cytokines, and enhancement of CTL and NK cytolytic activity, etc.), as well as direct elimination of tumors by inducing apoptosis or stimulating a humoral response to ADCC, etc.

In some embodiments, the antibodies disclosed herein are anti-OX 40 agonist antibodies that are active in cell function and stimulate human CD4 compared to the existing Genentech IgG1 subtype OX40 antibody (Genentech accession number: MOXR0916)⁺The method has certain advantages in the aspect of T cell proliferation, and can quickly stimulate the proliferation and activation of specific T cells and inhibit the growth of tumor cells in a mouse body.

In some specific embodiments, the antibodies disclosed herein are capable of blocking the binding of human OX40 protein to its ligand OX40L, in a dose-effect relationship.

In some embodiments, the antibodies disclosed herein can stimulate CD4⁺T cellsProliferate and increase the secretion of IL-2 and IFN-gamma. For example, in some specific embodiments, the antibodies disclosed herein can be effective in promoting CD4⁺T cell proliferation and cytokine secretion, including but not limited to IL-2, IFN-gamma, etc..

In some embodiments, the antibodies disclosed herein are effective in activating tumor-killing T cells (e.g., activating specific T cells in hOX40 KI mice) and inhibiting intratumoral Treg cells.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is capable of killing cells that express OX40, such as cells that express high levels of OX 40.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is capable of eliciting a T cell-mediated response.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is capable of increasing the function of effector T cells.

In some embodiments, the antibody or antigen-binding portion thereof that specifically binds OX40 is capable of increasing the function of memory T cells.

In some embodiments, the antibodies disclosed herein can inhibit the growth of a tumor. For example, in some specific embodiments, the antibodies disclosed herein can inhibit the growth of MC38 subcutaneous transplants of hOX40 KI mice. In some specific embodiments, the above antibody inhibits tumor growth by at least 80%, 85%, or 90%. In some embodiments, inhibition of tumor growth is detectable 14 days after a tumor-bearing individual is treated with an antibody disclosed herein. In other embodiments, inhibition of tumor growth is detected 6 days after initial antibody treatment.

The present application also provides nucleotide molecules encoding the antibodies disclosed herein, or antigen-binding portions thereof, expression vectors comprising the nucleotide molecules, host cells comprising the nucleotide molecules or expression vectors, and methods of making and purifying the antibodies.

In some embodiments, the nucleotide molecule encoding the antibody, or antigen-binding portion thereof, is operably linked to a control sequence that is recognized by a host cell transformed with the expression vector.

In some embodiments, any suitable expression vector may be used in the present application. For example, the expression vector may be any one of pQK1, pTT5, pUC57, pDR1, pcDNA3.1(+), pDFHF, and pCHO1.0. Expression vectors may include fusion DNA sequences with appropriate transcriptional and translational regulatory sequences attached.

In some embodiments, useful host cells are cells containing the above-described expression vectors, which may be eukaryotic cells, such as mammalian or insect host cell culture systems, can be used for expression of the antibodies or antigen-binding portions thereof of the present application. For example, HEK 293 cells, COS cells, CHO cells, NS0 cells, sf9 cells, sf21 cells, and the like can be used in the present invention. The host cell may be a prokaryotic cell containing the above expression vector, and may be, for example, DH5 α, BL21(DE3), TG1 or the like.

In some embodiments, methods of making anti-OX 40 monoclonal antibodies disclosed herein comprise: culturing the host cell under expression conditions such that the anti-OX 40 monoclonal antibody is expressed; isolation and purification of the expressed anti-OX 40 monoclonal antibody. Using the above method, the recombinant protein can be purified as a substantially homogeneous substance, for example, as a single band on SDS-PAGE electrophoresis.

In some embodiments, the anti-OX 40 antibodies disclosed herein can be isolated and purified using methods of affinity chromatography, and the anti-OX 40 antibody bound to the affinity column can be eluted using conventional methods, such as high salt buffers, varying pH, and the like, depending on the characteristics of the affinity column being utilized.

In some embodiments, antibodies and/or antibody-producing cells can be obtained from within an animal after the animal is inoculated with an OX40 antigen. Antibody-producing immortalized cell lines can be prepared from cells isolated from immunized animals. Following immunization, the animal is killed, lymph node and/or spleen B cells are immortalized, treated with an oncogenic compound and a mutagenic compound, and fused with immortalized cells (e.g., myeloma cells) to inactivate tumor suppressor genes. When myeloma cells are used for fusion, the myeloma cells preferably do not secrete immunoglobulin polypeptides (non-secreting cell lines). Screening for immortalized cells uses OX40, a portion thereof, or cells expressing OX 40. In a preferred embodiment, the primary screening is performed using enzyme-linked immunosorbent assay (ELISA). Cells, such as hybridomas, that produce anti-OX 40 antibodies are selected for cloning and further screened for desired characteristics, including good growth, high antibody production, and desirable antibody characteristics. Methods for screening, cloning and amplifying hybridomas are well known to those of ordinary skill in the art. In some embodiments, the immunized animal is a non-human animal, wherein the splenic B cells are fused with a myeloma cell line from the same species as the non-human animal. In some embodiments, the immunized animal is a Balb/c mouse and the myeloma cell line is the non-secreting mouse myeloma cell SP 2/0.

The present application provides pharmaceutical compositions comprising an antibody, or antigen-binding portion thereof, disclosed herein and a pharmaceutically acceptable carrier. The above-described anti-OX 40 antibodies (e.g., anti-human OX40 monoclonal antibodies) disclosed herein can be formulated into pharmaceutical preparations together with pharmaceutically acceptable carriers, thereby exerting the therapeutic effects more stably. In some embodiments, these formulations can ensure the conformational integrity of the amino acid core sequence of an anti-OX 40 antibody disclosed herein (e.g., an anti-human OX40 monoclonal antibody), while also protecting the multifunctional groups of the protein from degradation (including but not limited to aggregation, deamidation, or oxidation). In some embodiments, for liquid formulations, it may be generally shelf stable for at least one year at 2 ℃ to 8 ℃. In some embodiments, for a lyophilized formulation, it remains stable at 30 ℃ for at least six months. In some embodiments, the pharmaceutical composition further comprises one or more additional active ingredients. In some embodiments, the active ingredient is an anti-tumor drug.

The present application provides a vaccine comprising an antibody or antigen-binding portion thereof that specifically binds OX40 as described in the first aspect, and optionally an immunological adjuvant. Such immunological adjuvants include, but are not limited to: 1) biological adjuvants such as bacteria or products thereof (e.g., mycobacteria (tuberculosis, bacille calmette-guerin), corynebacterium parvum, bordetella pertussis, gram-negative bacillus endotoxins), granulocyte-macrophage colony stimulating factor, interleukin-l, interleukin-2, interferon- γ, etc.; 2) inorganic adjuvants such as aluminum hydroxide, alum, aluminum phosphate, and the like; 3) artificially synthesized adjuvants such as double-stranded polyinosinic acid, cytidylic acid, double-stranded polyadenylic acid; 4) oils such as peanut oil emulsion adjuvant, mineral oil, vegetable oil, lanolin, etc.; 5) freund's adjuvant, such as Freund's incomplete adjuvant and Freund's complete adjuvant.

The present application also provides methods of preventing and/or treating OX 40-related diseases, such as tumors, in the field of tumor immunization comprising administering to an individual an anti-OX 40 antibody, or a pharmaceutical composition or vaccine comprising an anti-OX 40 antibody (e.g., an anti-human OX40 monoclonal antibody). In some embodiments, the anti-tumor effect is evident upon administration to animals, including humans. Specifically, the anti-OX 40 antibodies disclosed herein are effective in preventing and/or treating tumors, and can be used as antitumor agents.

In some embodiments, an anti-OX 40 antibody, or a pharmaceutical composition or vaccine comprising an anti-OX 40 antibody (e.g., an anti-human OX40 monoclonal antibody), of the present application can be administered in combination with other therapeutic agents (e.g., chemotherapeutic agents, PD-1 binding antagonists, etc.).

The application also provides for the use of an anti-OX 40 antibody or antigen-binding portion thereof, or a pharmaceutical composition comprising the anti-OX 40 antibody or antigen-binding portion thereof, in the manufacture of a medicament for the prevention and/or treatment of an OX 40-associated disease, such as a tumor.

In some embodiments, the tumor is colon cancer, melanoma, mesothelioma, renal cell carcinoma, lymphoma, advanced solid tumors, or metastases thereof, and the like.

The anti-human OX40 antibody and the pharmaceutical composition comprising the anti-human OX40 antibody disclosed herein, when administered to animals including humans, are administered at doses varying depending on the age and body weight of the individual, the nature and severity of the disease and the route of administration, and the total dose cannot exceed a certain range with reference to the results and general conditions of animal experiments.

The dosage and frequency of administration of the antibody or the pharmaceutical composition comprising the antibody may vary depending on the disease to be prevented or treated. In prophylactic applications, a composition comprising an antibody of the present application or a mixture thereof is administered to a patient who is not already in a disease state to enhance the patient's resistance, this amount being defined as a "prophylactically effective dose". In this use, the specific dosage will again depend on the health of the patient and the systemic immunity. Relatively low doses are typically administered at relatively infrequent intervals for extended periods of time. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until disease progression is slowed or terminated, and preferably until the patient shows partial or complete improvement in disease symptoms. Thereafter, a prophylactic regimen may be administered to the patient. The specific dosage and frequency can be readily determined by one of ordinary skill in the art based on the actual need.

The present application also provides a detection reagent or kit comprising an antibody or antigen-binding portion thereof disclosed herein.

As used herein, the term "immune response" refers to a biological response in a vertebrate against an external agent that protects an organism against such agent and the disease caused by it. The immune response is mediated by the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, Natural Killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and any of these cells or soluble macromolecules produced by the liver, including antibodies, cytokines, and complements, which results in the selective targeting, binding, damage, destruction, and/or elimination from the vertebrate body of invading pathogens, pathogen-infected cells or tissues, cancerous or other abnormal cells, or normal human cells or tissues in the case of autoimmune or pathological inflammation. The immune response includes T cells (e.g., effector T cells) or Th cells (e.g., CD 4)⁺Or CD8⁺T cells) or suppression of Treg cells.

As used herein, the term "T cell-mediated response" refers to a response mediated by T cells (including effector T cells (e.g., CD 8)⁺Cells) and helper T cells (e.g., CD 4)⁺Cell)) mediated response. T cell mediated responses include T cell cytotoxicity and proliferation.

As used herein, the term "cancer" refers to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in vivo. Dysregulated cell division can form malignant tumors or cells that invade adjacent tissues and can metastasize to distant parts of the body via the lymphatic system or the bloodstream.

As used herein, the term "inhibiting the growth of a tumor" includes any measurable reduction in tumor growth, such as at least about 10%, e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100% inhibition of tumor growth.

As used herein, the term "treatment" refers to any type of intervention or method performed on a subject or administration of an active agent thereto, wherein the objective is to reverse, alleviate, ameliorate, inhibit or ameliorate or prevent the progression, development, severity or recurrence of a symptom, complication, condition, or biochemical marker associated with the disease. Prevention refers to administration to a subject not suffering from a disease, to prevent the disease from occurring or to minimize its effects (if any).

In the present description and claims, the words "comprise", "comprises" and "comprising" mean "including but not limited to", and are not intended to exclude other moieties, additives, components or steps.

It should be understood that features, characteristics, components or steps described in a particular aspect, embodiment or example of the present application may be applied to any other aspect, embodiment or example described herein unless incompatible therewith.

The above disclosure generally describes the present application and the following examples are presented to further illustrate the present application and should not be construed as limiting the present application. The examples do not include detailed descriptions of conventional methods such as those for constructing vectors and plasmids, methods for inserting genes encoding proteins into vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those of ordinary skill in the art and are described in numerous publications, see, for example, Sambrook, j., Fritsch, ef.and maniis, T. (1989) Molecular Cloning: a Laboratory Manual, 2nd edition, Cold spring Harbor Laboratory Press.

Antibodies that specifically bind to mammalian (human, primate, etc.) OX40 are disclosed, and the antibodies or antibody portions are useful for modulating receptor activity, stimulating activation and proliferation of T cells, and the like. The invention provides applications of the protein in treatment, screening, detection and the like, such as application in cancer treatment. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. Those skilled in the art can implement and use the invention by making modifications, or appropriate alterations and combinations, of the methods and applications described herein without departing from the spirit, scope, and content of the invention.

In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to the embodiments.

Examples

Example 1 preparation of hybridomas producing anti-OX 40 antibodies

1. Balb/c mice, 8-10 weeks old, were inoculated intraperitoneally with OX40 protein (10. mu.g/dose/mouse). Human OX40(TNFRSF4) protein was ordered from Cassia, cat No.10481-H08H, DNA sequence NP-003318.1, protein sequence extracellular Met1-Ala 216, and C-terminal His-tag, and its molecular weight was confirmed to be 40-45kDa by SDS-PAGE. This dose is repeated 5-7 times within 3-8 weeks. Mice were injected with the last dose of OX40 protein 4 days prior to fusion.

2. 1 day before fusion, ordinary Balb/c mouse intraperitoneal macrophages are taken as trophoblasts and inoculated in a 96-well plate.

3. Lymphocytes from the spleen and lymph nodes of immunized mice were fused with a non-secretory myeloma SP2/0 cell line, and the fused cells were HAT-selected by adding the cells to a 96-well plate previously plated with a trophoblast (Galfre and Milstein, Methods Enzymol 1981; 73: 3-46).

4. A panel of hybridoma cells that all secrete anti-OX 40-specific antibodies was recovered. The primary screen was performed by using enzyme-linked immunosorbent assay (ELISA) to determine the titer of anti-OX 40 antibody secreted by the hybridomas.

Example 2 detection of antibody Activity by NF-. kappa.B Signal pathway

1. OX 40100B 5 cells were transferred to a 15mL centrifuge tube, washed 1 time with 5mL DMEM medium (Gibco) containing 10% FBS (Gemini), and centrifuged at 1000rpm for5 min; the supernatant was discarded and 5mL of PBS containing 2% FBS was added to resuspend the cell counts.

2. Cells were diluted to 2X 10⁵cells/mL, 100. mu.L/well into 96-well plates, 5% CO₂At 37 ℃ for 30 min.

3. The monoclonal antibodies to be detected and the control antibody (humanized antibody of the subclass of antibody MOXR0916, IgG1, manufactured by Roche Co.), the human 4-1BB ligand (h-OX40L, Popsece), the antibody of the same subclass (mouse IgG1 antibody (mIgG1), the human IgG1 antibody (hIgG1), Biolegend) were diluted at the following concentrations: 4. mu.g/mL, 0.4. mu.g/mL and 0.04. mu.g/mL were added to a 96-well plate at 50. mu.L/well, and the multiple wells were set so that the final concentrations of the monoclonal antibody in the reaction system were 1. mu.g/mL, 0.1. mu.g/mL and 0.01. mu.g/mL, respectively. Meanwhile, a DMEM medium group without the antibody at all is added, and an anti-mIgG-Fc group with the secondary antibody alone is used as a background value control.

4. Anti-mouse IgG Fc (Biolegend) and anti-human IgG Fc (Biolegend) were added as secondary antibodies to mimic Fc receptor cross-linking in vivo, and the final concentration of the secondary antibody was 3 times that of the primary antibody, and the secondary antibodies were prepared in a 50 μ L/well system and added to a 96-well plate.

5. Place 96-well plate in 5% CO₂Incubating for 20h in a cell culture box at 37 ℃; then, the 96-well plate to be tested was centrifuged at 1000rpm at room temperature for 5min, 160. mu.L of Quanti Blue (Invivo Gen) which had been incubated in advance at 37 ℃ for 30min was added to each well of the new 96-well plate, and 40. mu.L of cell supernatant was added and incubated at 37 ℃ for 1 h.

6. OD was measured at 620nm using a microplate reader and GraphPad Prism was selected for data mapping.

Partial results are shown in FIG. 1A, and 30 monoclonal antibodies were screened; dominant clone validation was further selected and 7 candidate monoclonal antibodies were obtained, labeled 4F2, 4B9, 6C1, 6H12, 13F7, 6a6 and 18B10, respectively, as shown in fig. 1B.

Example 3 detection of antibody Activity by NFAT Signaling pathway

1. The collected 1 vial (T75) of Jurkat OX40 cells were transferred to a 15mL centrifuge tube, washed 1 time with 5mL 1640 medium (Gibco) containing 10% FBS, centrifuged 5min at 850rpm, the supernatant discarded, 5mL of RPMI 1640 medium containing 1% FBS was added to resuspend the cells and counted.

2. Cells were diluted to 1.0X 10⁶cells/mL, 25. mu.L/well into 96-well plates, 5% CO₂At 37 ℃ for 30 min.

3. Control antibodies MOXR0916 of the same subtype (mIgG1 and hIgG1, Biolegend) were added as in the control design of example 2 and each antibody to be detected was diluted at the following concentrations: 3. mu.g/ml, 0.3. mu.g/ml and 0.03. mu.g/ml were added to a 96-well plate at 25. mu.L/well, and multiple wells were set. A group of RPMI 1640 medium (labeled as 1640 in the figure) containing no antibody at all was added at the same time as a background control.

4. The final concentration of the anti-mouse IgG Fc (Biolegend) and anti-human IgG Fc (Biolegend) as secondary antibodies was 3 times that of the primary antibody, and they were prepared in a system of 25. mu.L/well and added to a 96-well plate.

5. Place 96-well plate in 5% CO₂At 37 ℃ for 6 h.

6. Bio-glo (Promega) equilibrated to room temperature was added to the 96-well plate at 75. mu.L/well, and luminescence detection was performed.

The results are shown in fig. 2, and 7 candidate monoclonal antibodies were obtained, labeled 4F2, 4B9, 6C1, 6H12, 13F7, 6a6, and 18B10, respectively.

Example 4 determination of kinetic parameters of antibodies

1. Kinetic parameters of human OX40 binding to anti-OX 40 monoclonal antibodies (4F2, 4B9, 6C1, 6H12, 13F7, 6a6, and 18B10) were determined using a Fortebio molecular interaction instrument.

2. Each monoclonal antibody was captured and immobilized with a ProA sensor and, after equilibration in PBS, bound to the antigen human OX 40.

3. Human OX40 was diluted to 2 concentrations, 400nM and 200nM respectively, with PBS and dissociated in PBS.

The results of the parametric measurements are shown in Table 1, and the kinetic binding dissociation pattern is shown in FIG. 3, with all candidate antibodies having affinity at sub-nM level.

TABLE 1 kinetic parameters for binding of human OX40 to anti-OX 40 monoclonal antibodies

Example 5 binding experiments of candidate anti-OX 40 monoclonal antibodies to human OX40 and monkey OX40

1. Human OX40 and monkey OX40 membrane expression plasmids were transiently transfected in 293T cells in advance.

2. Untransfected 293T cells were selected as control cells, 293T cells expressing human OX40 and 293T cells expressing monkey OX40 were taken as experimental groups, three cells were digested with pancreatin, washed twice with PBS containing 2% FBS, centrifuged at 850rpm for 5min, the supernatant was discarded, and resuspended cells were added in PBS containing 2% FBS and counted.

3. Cells were diluted to a concentration of 1.0X 10⁷cells/mL, 50. mu.L/well into the flow tube.

4. Each monoclonal antibody (4F2, 4B9, 6C1, 6H12, 13F7, 6A6 and 18B10) was diluted to 2. mu.g/mL with PBS containing 2% FBS, and added at 50. mu.L/well to a flow tube containing 50. mu.L of cells.

Incubate at 5.4 ℃ for 30min, wash 2 times with 3mL PBS containing 2% FBS, and centrifuge at 1000rpm for5 min.

6. The supernatant was discarded, the cells were resuspended in 100. mu.L of 1 XPBS containing 2% FBS, and APC anti-mouse IgG Fc (Biolegend) was added as a secondary antibody.

Incubate at 7.4 ℃ for 30min, wash 1 time with 3mL PBS containing 2% FBS, and centrifuge at 1000rpm for5 min.

8. The supernatant was discarded, and the cells were resuspended in 100. mu.L of PBS containing 2% FBS, and the positive rate was obtained by flow machine detection.

The results are shown in fig. 4, candidate anti-OX 40 monoclonal antibodies 18B10, 4F2, 6C1, 6H12 and 6a6 have relatively high affinity binding to both human OX40 and monkey OX40, 4B9 and 13F7 show weaker affinity to monkey OX40, and both candidate antibodies 4B9 and 13F7 were discarded in view of facilitating the safety evaluation of the subsequent drug in monkeys.

Example 6 blocking of anti-OX 40 monoclonal antibodies against human OX40L (hFc) binding to human OX40

1. OX 40100B 5 cells were transferred to a 15mL centrifuge tube in 1 vial (T75) of HEK, washed 1 time with 8mL PBS containing 2% FBS, and centrifuged at 1000rpm for5 min; the supernatant was discarded, and 1mL of PBS containing 2% FBS was added to resuspend the cells and counted.

2. Experimental sample set-up: blank, 0 μ g human OX40L (speed biosystems) group, 0.5 μ g human OX40L group, and four concentration gradient groups for each monoclonal antibody (4F2, 6C1, 6H12, 6a6, and 18B 10).

3. Cells were diluted to 1X 10⁷cells/mL were added to the flow tube at 10. mu.L/well, and the monoclonal antibody was diluted with PBS containing 2% FBS to give final concentrations of 0.01. mu.g/mL, 0.1. mu.g/mL, 1. mu.g/mL and 10. mu.g/mL, respectively.

4. mu.L of each of the diluted antibodies corresponding to the four concentration gradients was added to the monoclonal antibody sample groups, and the amounts of the corresponding monoclonal antibodies were 1ng, 10ng, 100ng and 1000ng, respectively. To the remaining groups, 100. mu.L of PBS containing 2% FBS was added, ensuring that all reaction systems were 110. mu.L.

Binding was performed at 5.4 ℃ for 30min, washed 2 times with 3mL of PBS containing 2% FBS, and centrifuged at 1000rpm for5 min.

6. The supernatant was discarded, and human OX40L 100. mu.L at a concentration of 5. mu.g/mL was added to each sample tube except for the blank and 0. mu.g of human OX 40L.

Binding was performed at 7.4 ℃ for 30min, washed 2 times with 3mL of PBS containing 2% FBS, and centrifuged at 1000rpm for5 min.

8. Cells were resuspended in 100. mu.L of 1 XPBS containing 2% FBS, and APC anti-human IgG Fc (Biolegend) was added as a secondary antibody to each of the groups except the blank group.

Binding was performed at 9.4 ℃ for 30min, washed 1 time with 3mL of PBS 2% FBS, and centrifuged at 1000rpm for5 min.

10. The supernatant was discarded, and the cells were resuspended in 100. mu.L of 2% FBS-containing 1 XPBS and tested by flow machine to obtain a positive rate.

The results are shown in FIG. 5, where the candidate antibody was able to partially or completely block the binding of 0.5. mu.g of OX40L to OX40 protein at concentrations of 100ng and above. Among them, candidate antibodies 18B10 and 6C1 blocked slightly less effectively.

Example 7 EC50 value detection of anti-OX 40 monoclonal antibodies

EC50 value detection experiment for detecting antibody activity by NF-kB signal channel

1. OX 40100B 5 cells were transferred to a 15mL centrifuge tube, washed 1 time with 5mL DMEM medium (Gibco) containing 10% FBS (Gemini), and centrifuged at 1000rpm for5 min; the supernatant was discarded, 5mL of PBS containing 2% FBS was added to resuspend the cells and counted.

2. Cells were diluted to 2X 10⁵cells/mL, 100. mu.L/well into 96-well plates, 5% CO₂At 37 ℃ for 30 min.

3. The formulated concentrations of the candidate 5 monoclonal antibodies (4F2, 6C1, 6H12, 6a6, and 18B10) and the control antibody MOXR0916 were diluted down in A3-fold gradient at an initial concentration of 200nM for 10 concentration points. Multiple wells were set at 50 μ L/well in 96-well plates, i.e., the final concentration of monoclonal antibody was 50nM starting concentration, decreasing in a 3-fold gradient.

4. The final concentration of anti-mouse IgG Fc (Biolegend) as a secondary antibody was 3 times that of the primary antibody, and the secondary antibody was prepared in a 50. mu.L/well system and added to a 96-well plate.

5. Place 96-well plate in 5% CO₂At 37 ℃ for 20 h.

6. The 96-well plate to be tested was centrifuged at 1000rpm at room temperature for 5min, 160. mu.L of Quanti Blue (Invivo Gen) which had been incubated in advance at 37 ℃ for 30min was added to each well of the new 96-well plate, and 40. mu.L of cell supernatant was added and incubated at 37 ℃ for 1 h.

7. OD was measured at 620nm using a microplate reader and GraphPad Prism was selected for data mapping.

The corresponding EC50 values for the anti-OX 40 monoclonal antibody are shown in table 2 and the EC50 values are fitted to the graph shown in figure 6. The binding force between the antibody and the cell level is detected in the early-stage flow mode, and the functional activity of the antibody in the play of the test is investigated. The results show that the candidate antibodies 6a6 and 4F2 have superior functional activity to the control antibody; 6C1 was relatively poor, so the antibody was discarded in subsequent experiments. Further selected candidate monoclonal antibodies were 4F2, 6H12, 6a6 and 18B 10.

TABLE 2 summary of EC50 values for candidate anti-OX 40 monoclonal antibodies

Example 8 anti-OX 40 monoclonal antibodies stimulated human CD4⁺Experiment on T cell proliferation

CD3 coated 96-well plates: 100 μ L/well, 0.05 μ g/mL or 0.01 μ g/mL of CD3(OKT3), incubated overnight at 4 ℃.

2. Sorting cells: using CD4⁺Negative selection magnetic bead sorting kit (Miltenyi Biotec) for obtaining CD4 from PBMC⁺Cells were counted.

CFSE (MCE) marker: leaving a suitable amount of cells, the remaining cells (10)⁷Individually) in 1mL of 5% FBS-containing PBS, CFSE labeling was performed by adding CFSE to a final concentration of 2. mu.M, incubation at room temperature for 10min, 3 washes with 5mL of 5% FBS-containing PBS to stop staining, and resuspension in a 10% FBS-containing 1640 medium.

4. 96-well plates coated with different CD3 concentrations were selected: 100 μ L/well, 3X 10⁵Individual cell/well CD4⁺T cells, 2 replicates.

5. Adding monoclonal antibodies: 100 μ L/well, 5 μ g/mL (final concentration of 2.5 μ g/mL) of antibody (4F2, 6H12, 6A6, 18B10 and control antibody MOXR0916), negative antibody controls (mIgG1 and hIgG1, Biolegend) and blank well controls were set and incubated at 37 ℃.

6. After 20h incubation, 60. mu.L of the supernatant was centrifuged and assayed for IL-2 by ELISA, as shown in FIG. 7B.

7. The 96-well plate was further incubated for 5-7 days, and 100. mu.L of the supernatant was subjected to IFN-. gamma.ELISA, and the results are shown in FIG. 7C.

8. Cells were collected for CFSE flow detection of CD4, and the results are shown in fig. 7A.

The results show that 4 candidate monoclonal antibodies are all effective in stimulating the proliferation of human CD4+ T cells and increasing the secretion of IL-2 and IFN-gamma, and under the experimental conditions, the candidate antibodies are all superior to the control antibody. Combining the results of the previous experiments, especially paying attention to the detection of the biological activity of the EC50 value, comprehensively considering, selecting 6A6 and 4F2 to carry out the research of the drug effect tumor inhibition experiment in the body of the mouse.

Example 9 anti-human OX40 monoclonal antibodies in OX40 humanized mice in vivo tumor suppression experiments

1. Cell culture: mouse colorectal cancer MC38 cells at 37 ℃ and 5% CO₂The culture medium is DMEM medium containing 10% FBS, and the cells are subjected to flask culture for 1 time every 2 days.

2. Mouse inoculation of cells: resuspended MC38 tumor cells in PBS at 5X 10⁶The cells/mL concentration, 0.1 mL/volume, were inoculated subcutaneously into the right flank of OX40 humanized mice (Beijing Baioectozei Gene Biotechnology Co., Ltd.) and 26 mice were co-transfected. When the mean tumor volume reached about 100mm³When mice with moderate individual tumor volumes were selected for inclusion in groups, animals were randomly assigned to 4 groups based on tumor volume, including control group (PBS group) and three experimental groups (MOXR0916 control antibody group, 6a6 group and 4F2 group), with 6 animals per group, dosing started on the day of the group, with the specific dosing schedule shown in table 3:

TABLE 3 Experimental dosing regimen for tumor inhibition

Note:^ameans 1 dose every 3 days, 4 doses in total.

3. After administration, the body weight and tumor growth status of the experimental animals were continuously observed. Tumor volume and animal body weight were measured twice weekly and measurements were recorded. The results of tumor volume measurements are shown in FIG. 8A, where the MOXR0916 control antibody group when compared to the control,^*P<0.05，^**P<0.01; when 6A6 group was compared to control，^#P<0.05，^##P<0.01; when the 4F2 group was compared to the control,^&P<0.05. the results of the animal body weight measurements are shown in figure 8B.

4. After administration, observation and recording are carried out for 14 days, the mice are dissected, serum is taken for ALT and AST biochemical detection, the detection data are respectively shown as 8C and 8D, and the results show that the data are all in a normal range and no obvious hepatotoxicity is found. Tumor, liver and spleen tissues were dissected, weighed and subjected to data statistics, and the tumor anatomy is shown in fig. 8E. The percentage of the anatomical tumor weight in the total body weight of the mice is calculated, the difference between groups is more intuitively examined, the data statistical result is shown in figure 8F, the experimental group has very obvious statistical difference compared with the control group,^***P<0.001。

5. tumor inhibition rate (TGI) data were analyzed simultaneously and the results are shown in table 4. After administration, the candidate antibodies (6a6 and 4F2) showed comparable tumor suppression effects to the control antibody (MOXR0916), and clone 6a6 was slightly superior to 4F 2.

TABLE 4 Experimental group TGI statistics

It will be understood that, although the invention herein has been described in the foregoing specific forms, these inventions are not to be limited to the particulars described in these specific forms. It will be obvious to those skilled in the art that various equivalent changes may be made in the technical features of the invention involved therein without departing from the spirit of the invention described in the present application, and these changes should be construed as being within the scope of the invention.

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<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Trp Ala Ser Ile Arg His Thr

1 5

<210> 18

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Ala Ala Ser Thr Leu Asp Ser

1 5

<210> 19

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Tyr Thr Ser Arg Leu His Ser

1 5

<210> 20

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Gln Gln His Tyr Ser Thr Pro Trp Thr

1 5

<210> 21

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Gln Pro His Tyr Ser Thr Pro Trp Thr

1 5

<210> 22

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Leu Gln Tyr Ala Ser Tyr Pro Leu Thr

1 5

<210> 23

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Gln Gln Leu Asn Thr Leu Pro Trp Thr

1 5

<210> 24

<211> 140

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile

1 5 10 15

Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30

Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

35 40 45

Ser Gly Tyr Asn Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu

50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn Pro

65 70 75 80

Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln

85 90 95

Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr

100 105 110

Tyr Cys Ala Arg Gln Gly Ser Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 25

<211> 140

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile

1 5 10 15

Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30

Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

35 40 45

Ser Gly Tyr Asn Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu

50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro

65 70 75 80

Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln

85 90 95

Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr

100 105 110

Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 26

<211> 141

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Thr Ala Gly

1 5 10 15

Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Thr Ala Ser Gly His Ala Phe

35 40 45

Ser Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr

50 55 60

Leu Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asn Asn Tyr Asn

65 70 75 80

Pro Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn

85 90 95

Gln Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr

100 105 110

Tyr Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 27

<211> 140

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile

1 5 10 15

Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30

Phe Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

35 40 45

Ser Gly Tyr Tyr Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu

50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro

65 70 75 80

Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln

85 90 95

Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr

100 105 110

Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 28

<211> 140

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Met Lys Val Leu Ser Leu Leu Tyr Leu Leu Thr Ala Ile Pro Gly Ile

1 5 10 15

Leu Ser Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro

20 25 30

Ser Gln Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr

35 40 45

Ser Gly Tyr Asn Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu

50 55 60

Glu Trp Met Gly Tyr Ile Ser Tyr Asp Gly Ser Asp Asn Tyr Asn Pro

65 70 75 80

Ser Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln

85 90 95

Phe Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr

100 105 110

Tyr Cys Ala Arg Glu Gly Arg Ser Gly His Phe Tyr Tyr Ala Met Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

130 135 140

<210> 29

<211> 138

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Met Gly Trp Asn Trp Ile Phe Ile Leu Ile Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45

Thr Gly Tyr Tyr Met Asn Trp Val Lys Gln Ser Pro Glu Lys Ser Leu

50 55 60

Glu Trp Ile Gly Glu Ile Asn Pro Ser Thr Gly Gly Thr Thr Tyr Asn

65 70 75 80

Gln Lys Phe Lys Ala Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Ser Gly Leu Pro Tyr Tyr Asp Val Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210> 30

<211> 134

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Met Gly Trp Ser Arg Ile Phe Leu Phe Leu Leu Ser Ile Thr Ala Gly

1 5 10 15

Val His Cys Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Ile Ser Cys Thr Ala Ser Gly His Ala Phe

35 40 45

Ser Asn Ser Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Glu Asn Thr Asn Tyr Asn

65 70 75 80

Gly Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val

100 105 110

Tyr Phe Cys Ala Arg Gly Leu Tyr Ser Val Gln Trp Gly Gln Gly Thr

115 120 125

Thr Leu Thr Val Ser Ser

130

<210> 31

<211> 131

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 31

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala His

20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45

Ala Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95

Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys

100 105 110

Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

<210> 32

<211> 131

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 32

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala His

20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45

Ala Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95

Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys

100 105 110

Leu Gln Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Asn Leu

115 120 125

Glu Leu Lys

130

<210> 33

<211> 131

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 33

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ala His

20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45

Ala Ser Gln Asp Val Ser Ser Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95

Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Leu Tyr Tyr Cys

100 105 110

Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

<210> 34

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 34

Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe Leu Trp Leu Ser

1 5 10 15

Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser

20 25 30

Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asp

35 40 45

Val Asn Thr Ala Val Ala Trp Tyr Gln His Lys Pro Gly Gln Ser Pro

50 55 60

Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr Gly Val Pro Asp

65 70 75 80

Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser

85 90 95

Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Pro His Tyr

100 105 110

Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

<210> 35

<211> 131

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Ala Phe Val Phe Val Phe

1 5 10 15

Leu Trp Leu Ser Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser His

20 25 30

Lys Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Ile Thr Cys Lys

35 40 45

Ala Ser Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Ile Arg His Thr

65 70 75 80

Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Tyr Thr

85 90 95

Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys

100 105 110

Gln Pro His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu

115 120 125

Glu Ile Lys

130

<210> 36

<211> 129

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Met Asp Met Arg Val Pro Ala His Val Phe Gly Phe Leu Leu Leu Trp

1 5 10 15

Phe Pro Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser

20 25 30

Leu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser

35 40 45

Gln Glu Ile Ser Gly Tyr Leu Ser Trp Leu His Gln Lys Pro Asp Gly

50 55 60

Thr Ile Lys Arg Leu Ile Tyr Ala Ala Ser Thr Leu Asp Ser Gly Val

65 70 75 80

Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr

85 90 95

Ile Ser Ser Leu Glu Ser Glu Asp Phe Ala Asp Tyr Tyr Cys Leu Gln

100 105 110

Tyr Ala Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Asn Leu Glu Leu

115 120 125

Lys

<210> 37

<211> 127

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 37

Met Met Ser Ser Ala Gln Phe Leu Gly Leu Leu Leu Leu Cys Phe Gln

1 5 10 15

Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Thr Ser Ser Ser Leu Ser

20 25 30

Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp

35 40 45

Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Ile Val

50 55 60

Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser

85 90 95

Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Leu Asn

100 105 110

Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

115 120 125

Claims (30)

1. An antibody or antigen-binding portion thereof that specifically binds OX40, comprising a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 sequences and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 sequences, wherein

The sequence of the HCDR1 is SGYWN (SEQ ID NO:1), the sequence of the HCDR2 is YISYDGNNYNPSLRN (SEQ ID NO:5), the sequence of the HCDR3 is QGSSGHFYYAMDY (SEQ ID NO:9), the sequence of the LCDR1 is KASQDVSSAVA (SEQ ID NO:13), the sequence of the LCDR2 is WASIRHT (SEQ ID NO:17) and the sequence of the LCDR3 is QQHYSTPWT (SEQ ID NO: 20); or

The sequence of the HCDR1 is SGYWN (SEQ ID NO:1), the sequence of the HCDR2 is YISYDGSDNYNPSLKN (SEQ ID NO:6), the sequence of the HCDR3 is EGRSGHFYYAMDY (SEQ ID NO:10), the sequence of the LCDR1 is KASQDVSSAVA (SEQ ID NO:13), the sequence of the LCDR2 is WASIRHT (SEQ ID NO:17) and the sequence of the LCDR3 is LQYASYPLT (SEQ ID NO: 22); or

The sequence of the HCDR1 is SGYYWN (SEQ ID NO:2), the sequence of the HCDR2 is YISYDGNNYNPSLRN (SEQ ID NO:5), the sequence of the HCDR3 is EGRSGHFYYAMDY (SEQ ID NO:10), the sequence of the LCDR1 is KASQDVSSAVA (SEQ ID NO:13), the sequence of the LCDR2 is WAHT SIRHT (SEQ ID NO:17) and the sequence of the LCDR3 is QQHYSTPWT (SEQ ID NO: 20); or

The sequence of the HCDR1 is SGYYWN (SEQ ID NO:2), the sequence of the HCDR2 is YISYDGSDNYNPSLKN (SEQ ID NO:6), the sequence of the HCDR3 is EGRSGHFYYAMDY (SEQ ID NO:10), the sequence of the LCDR1 is KASQDVNTAVA (SEQ ID NO:14), the sequence of the LCDR2 is WASIRHT (SEQ ID NO:17) and the sequence of the LCDR3 is QPHYSTPWT (SEQ ID NO: 21); or

The sequence of the HCDR1 is SGYWN (SEQ ID NO:1), the sequence of the HCDR2 is YISYDGSDNYNPSLKN (SEQ ID NO:6), the sequence of the HCDR3 is EGRSGHFYYAMDY (SEQ ID NO:10), the sequence of the LCDR1 is KASQDVNTAVA (SEQ ID NO:14), the sequence of the LCDR2 is WASIRHT (SEQ ID NO:17) and the sequence of the LCDR3 is QPHYSTPWT (SEQ ID NO: 21); or

The sequence of the HCDR1 is GYYMN (SEQ ID NO:3), the sequence of the HCDR2 is EINPSTGGTTYNQKFKA (SEQ ID NO:7), the sequence of the HCDR3 is SGLPYYDVDY (SEQ ID NO:11), the sequence of the LCDR1 is RASQEISGYLS (SEQ ID NO:15), the sequence of the LCDR2 is AASTLDS (SEQ ID NO:18) and the sequence of the LCDR3 is LQYASYPLT (SEQ ID NO: 22); or

The sequence of the HCDR1 is NSWMN (SEQ ID NO:4), the sequence of the HCDR2 is RIYPGDENTNYNGKFKG (SEQ ID NO:8), the sequence of the HCDR3 is GLYSVQ (SEQ ID NO:12), the sequence of the LCDR1 is RASQDISNYLN (SEQ ID NO:16), the sequence of the LCDR2 is YTSRLHS (SEQ ID NO:19) and the sequence of the LCDR3 is QQLNTLPWT (SEQ ID NO: 23);

wherein the antigen binding moiety is selected from: fab fragment, Fab 'fragment, F (ab')₂Fragments, Fv fragments and scFv fragments.

2. The antibody or antigen binding portion thereof of claim 1, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of those set forth in any one of SEQ ID NOs: 24-30.

3. The antibody or antigen binding portion thereof of claim 2, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO 24 or 29.

4. The antibody or antigen binding portion thereof of claim 1, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of those set forth in any one of SEQ ID NOs: 31-37.

5. The antibody or antigen binding portion thereof of claim 4, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO 31 or 36.

6. The antibody or antigen-binding portion thereof of any one of claims 1-5, wherein the antibody or antigen-binding portion thereof specifically binds to primate OX 40.

7. The antibody or antigen binding portion thereof of claim 6, wherein the primate OX40 is selected from human OX40 or monkey OX 40.

8. The antibody, or antigen-binding portion thereof, of any one of claims 1-5 and 7, wherein the antibody is a murine antibody.

9. The antibody or antigen binding portion thereof of claim 6, wherein the antibody is a murine antibody.

10. The antibody or antigen binding portion thereof of claim 8, wherein

The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 24, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 31; or

The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 29, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 36.

11. The antibody or antigen binding portion thereof of claim 9, wherein

The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 24, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 31; or

The heavy chain variable region amino acid sequence of the antibody is shown as SEQ ID NO. 29, and the light chain variable region amino acid sequence is shown as SEQ ID NO. 36.

12. The antibody or antigen binding portion thereof of any one of claims 1-5, 7, and 9-11, wherein the antibody or antigen binding portion thereof is at 1 x 10^-8To 1X 10^-7 The KD of M binds specifically to the OX40 molecule.

13. The antibody or antigen binding portion thereof of claim 6, wherein the antibody or antigen binding portion thereof is at 1 x 10^-8To 1X 10^-7 The KD of M binds specifically to the OX40 molecule.

14. The antibody or antigen binding portion thereof of claim 8, wherein the antibody or antigen binding portion thereof is at 1 x 10^-8To 1X 10^-7 The KD of M binds specifically to the OX40 molecule.

15. The antibody, or antigen binding portion thereof, of claim 12, wherein the antibody has OX40 agonist function, stimulating activation and proliferation of T cells.

16. The antibody, or antigen binding portion thereof, of claim 13 or 14, wherein the antibody has OX40 agonist function, stimulating activation and proliferation of T cells.

17. A pharmaceutical composition comprising the antibody or antigen-binding portion thereof of any one of claims 1-16 and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition further comprises one or more additional active ingredients.

19. The pharmaceutical composition of claim 18, wherein the active ingredient is selected from the group consisting of a chemotherapeutic agent and a PD-1 binding antagonist.

20. A vaccine comprising the antibody or antigen-binding portion thereof of any one of claims 1-16.

21. The vaccine of claim 20, further comprising an immunological adjuvant.

22. A nucleotide molecule encoding the antibody or antigen-binding portion thereof of any one of claims 1-16.

23. An expression vector comprising the nucleotide molecule of claim 22.

24. A host cell comprising the nucleotide molecule of claim 22 or the expression vector of claim 23.

25. Use of the antibody or antigen-binding portion thereof of any one of claims 1-16, or the pharmaceutical composition of any one of claims 17-19, in the manufacture of a medicament for eliciting a T cell-mediated response, increasing the function of effector T cells, increasing the function of memory T cells, and/or effectively inhibiting tumor growth.

26. Use of the antibody or antigen-binding portion thereof of any one of claims 1-16, or the pharmaceutical composition of any one of claims 17-19, in the manufacture of a medicament for the prevention and/or treatment of a tumor.

27. The use of claim 26, wherein the tumor is selected from one or more of: colon cancer, melanoma, mesothelioma, renal cell carcinoma, lymphoma, and metastases of the foregoing.

28. The use of claim 26, wherein the tumor is an advanced solid tumor.

29. A detection reagent comprising the antibody or antigen-binding portion thereof of any one of claims 1-16.

30. A kit comprising the antibody or antigen-binding portion thereof of any one of claims 1-16.

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