CN116419970A - Low toxicity anti-OX 40 antibodies, pharmaceutical compositions and uses thereof - Google Patents

Abstract

Low toxicity high activity anti-OX 40 antibodies, pharmaceutical compositions and uses thereof are provided. The anti-OX 40 antibodies or antigen-binding fragments thereof and pharmaceutical compositions thereof are useful in treating T cell-related disorders.

Description

Low toxicity anti-OX 40 antibodies, pharmaceutical compositions and uses thereof Technical Field

The present invention relates to anti-OX 40 antibodies, particularly low toxicity anti-OX 40 antibodies, pharmaceutical compositions thereof, and uses thereof.

Background

Breakthroughs and developments in tumour immunotherapy are a great advance in the field of tumour therapy, and are reviewed as "annual breakthroughs" in 2013 by the journal of science. Currently, there are four main approaches that can exploit the power of the immune system to combat tumors: 1) Antibodies against tumor antigens are infused into tumor patients to kill tumor cells, wherein part of the mechanism is that the tumor cells are killed by immune cells expressing Fc receptors after binding the antibodies; 2) The anti-tumor immune cells separated from the tumor patient are amplified in vitro and then are returned to the patient to play an anti-tumor role; 3) Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) expressing recognized tumor antigen after T cell is genetically modified is infused into tumor patient; 4) Monoclonal antibodies (e.g., anti-CTLA-4 and anti-PD-1/PD-L1) directed against targets of immunosuppressive signaling pathways (referred to as "immune checkpoints") that limit immune cell activity, enhance the immune system against the killing of tumors by blocking immunosuppressive signaling against the killer T cell response produced by the tumor cells.

Although the above methods have been used clinically, there is still an urgent need in the field of tumor immunotherapy for new immunotherapies that act through different mechanisms. This is because 1) not all tumor species respond to existing tumor immunotherapy approaches; 2) Even tumor types that respond to existing immunotherapy, not all patients of these tumor types respond to these immunotherapy; 3) Combination therapy is predicted to have a stronger therapeutic effect; 4) Tumors are likely to produce mutations that escape these treatments.

The excited antibody aiming at the immune co-stimulatory molecule can strengthen the anti-tumor immune response to kill tumor cells by combining the target molecule of the immune activation signal transmitted by the immune cell surface and activating the controlled immune activation signal path, and belongs to a treatment method with wide prospect. Of the many immune co-stimulatory molecules, OX40 was one of the earliest molecules found and validated to have an interfering anti-tumor response.

OX40 is a member of the TNF receptor superfamily, expressed predominantly on the surface of activated T cells, including each activated subset of CD4 and CD8T cell functions, e.g., th1, th2, T _FH Th17, and regulatory T cells (Tregs). OX40 has also been reported to have low expression on neutrophils, NK and NKT cells. The feature that OX40 is not expressed in naive T cells, specifically in activated T cells, allows targeting OX40 with a certain selectivity without indiscriminately activating all T cells (e.g. targeting CD28 with more extensive expression). OX40L/CD252 is a natural ligand of OX40 and is mainly expressed in antigen presenting cells such as B cells, dendritic Cells (DCs) and macrophages. Both OX40L and OX40 are antigen-induced expressed, with expression of OX40L being induced by CD40-CD40L signaling, toll-like receptors (TLRs) and inflammatory cytokines. Expression of OX40 is activated by antigen presenting cells to T cells by presenting antigen activated T cell receptor downstream signals, and is positively regulated by CD28-B7.1/2 signals, with peak expression levels occurring between 12 hours and 6 days post T cell activation. Structural analysis of OX40/OX40L supports trimerization of OX40 to OX40L interactions and binding of OX40/OX40L is likely to be directly involved in cellular interactions. Trimerization of OX40 allows its intracellular segments to recruit TRAF2/3/5 and activate NF-kB signaling, up-regulating the expression of anti-apoptotic molecules such as Bcl-2 and Bcl-xL, thereby inhibiting apoptosis and enhancing cell survival. OX40 has also been found to be able to co-act with TCR signals to affect T cell survival and proliferation through PI3K/Akt, and to regulate the expression of cytokines such as IL-2, IL-4, IL-5 and IFN-g through NFAT. Thus, activation of OX40 can inhibit apoptosis, promote proliferation and produce cytokines in activated T cells. These are all agonist anti-OX 40 functions capable of activating OX40 signals.

anti-OX 40 (and OX40L-Fc fusion proteins) has been reported to inhibit tumor growth in a variety of tumor models including melanoma, rectal cancer, fibrosarcoma, B-cell and T-cell lymphomas, and leukemia, including establishing resistance to tumors in some mice. On this basis, a number of anti-human OX40 monoclonal antibodies have entered clinical studies including PF04518600 for pyroxene, BMS-986178 for Shinobody, and a number of anti-OX 40 antibodies for MedImmune. However, development of anti-OX 40 as an anti-tumor drug is not smooth, and since 2013 reports that the drugs of a plurality of companies remain after the first clinical study, no antibodies have been introduced into the clinical third-stage study, and an important reason is that these antibodies have high toxicity to organisms, so that there is a need in the art for low-toxicity and high-efficiency anti-OX 40 antibodies.

Disclosure of Invention

The present invention provides low toxicity and high potency anti-OX 40 antibodies.

The first aspect of the present invention provides an anti-OX 40 antibody or antigen binding fragment thereof, said anti-OX 40 antibody comprising

(1) At least one CDR selected from the following sequences: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, and

(2) A heavy chain constant region comprising a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain connected in sequence from N-terminus to C-terminus, wherein the sequences of the CH1 domain and the hinge region are those derived from a CH1 domain and a hinge region of human IgG2, the sequences of the CH2 domain and the CH3 domain are those derived from a CH2 domain and a CH3 domain of human IgG, and the affinity of the antibody heavy chain constant region to human fcyriib is equal to or higher than the affinity of human IgG1 to human fcyriib, and the I/a ratio of the antibody heavy chain constant region is equal to or higher than the I/a ratio of human IgG 1.

In one or more embodiments, the heavy chain constant region comprises a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain, and the sequences of the CH1 domain and hinge region are those derived from a CH1 domain and hinge region of human IgG2, the sequences of the CH2 domain and CH3 domain being selected from the group consisting of:

a) Sequences derived from the CH2 domain and CH3 domain of human IgG1, and the CH2 domain and CH3 domain comprise the G237D, P238D, P271G and a330R mutations; or alternatively

b) Sequences derived from the CH2 domain and CH3 domain of human IgG1, and the CH2 domain and CH3 domain comprise the G237D, P238D, H268D, P271G and a330R mutations; or alternatively

c) Sequences derived from the CH2 domain and the CH3 domain of human IgG2, and the CH2 domain and the CH3 domain comprise the S267E and L328F mutations; or alternatively

d) Sequences derived from the CH2 domain and the CH3 domain of human IgG2, and the CH2 domain and the CH3 domain comprise the H268D and P271G mutations.

In one or more embodiments, the anti-OX 40 antibody comprises HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 2, and HCDR3 as shown in SEQ ID NO. 3, and/or LCDR1 as shown in SEQ ID NO. 4, LCDR2 as shown in SEQ ID NO. 5, and LCDR3 as shown in SEQ ID NO. 6.

In one or more embodiments, the anti-OX 40 antibody comprises an HCDR1 as set forth in any one of SEQ ID NOS: 7-38, an HCDR2 as set forth in any one of SEQ ID NOS: 39-65, and an HCDR3 as set forth in any one of SEQ ID NOS: 66-114, and/or an LCDR1 as set forth in any one of SEQ ID NOS: 115-145, an LCDR2 as set forth in any one of SEQ ID NOS: 146-159, and an LCDR3 as set forth in any one of SEQ ID NOS: 160-199.

In one or more embodiments, the anti-OX 40 antibody contains HCDR1, HCDR2 and HCDR3 as shown in any one of groups a1 through a71 of table 1, and/or LCDR1, LCDR2 and LCDR3 as shown in any one of groups b1 through b71 of table 2. More preferably, the anti-OX 40 antibody contains HCDR and LCDR of any one of groups c1 through c71 of table 3.

In one or more embodiments, the FR1 of the anti-OX 40 antibody VH may be selected from the FR1 of each antibody-numbered VH in table 4, the FR2 of VH may be selected from the FR2 of each antibody-numbered VH in table 4, the FR3 of VH may be selected from the FR3 of each antibody-numbered VH in table 4, and the FR4 of VH may be selected from the FR4 of each antibody-numbered VH in table 4; and/or FR1 of VL may be selected from FR1 of VL of each antibody number in table 4, FR2 of VL may be selected from FR2 of VL of each antibody number in table 4, FR3 of VL may be selected from FR3 of VL of each antibody number in table 4, and FR4 of VL may be selected from FR4 of VL of each antibody number in table 4.

In one or more embodiments, the FR region of the anti-OX 40 antibody VH is the FR region selected from any one of antibodies SEQ ID NOS: 200-270, and the FR region of the VL is the FR region selected from any one of antibodies SEQ ID NOS: 271-341.

In one or more embodiments, the anti-OX 40 antibody has an amino acid sequence of VH as set forth in any one of SEQ ID NOS: 200-270 and/or has an amino acid sequence of VL as set forth in any one of SEQ ID NOS: 271-341. Preferably, the amino acid sequence of VH and the amino acid sequence of VL of the anti-OX 40 antibody are shown in any one of the rows of table 4.

In some embodiments, the amino acid sequence of the heavy chain constant region of an antibody of the invention has a sequence as set forth in any one of SEQ ID NOS: 342-345, 348-351, and/or the amino acid sequence of the light chain constant region has a sequence as set forth in SEQ ID NOS: 346 or 347. In one or more embodiments, the heavy chain constant region of the anti-OX 40 antibody has a sequence as set forth in any one of SEQ ID NOS: 348-351 and/or the amino acid sequence of the light chain constant region has a sequence as set forth in SEQ ID NO: 347. Preferably, the heavy chain constant region of the anti-OX 40 antibody has a sequence as set forth in SEQ ID NO. 350.

In one or more embodiments, the anti-OX 40 antibody of any one of the embodiments of the invention is a chimeric antibody or a fully human antibody; preferably fully human antibodies.

The invention also provides a pharmaceutical composition comprising an anti-OX 40 antibody or antigen-binding fragment thereof according to any one of the embodiments of the invention, and a pharmaceutically acceptable excipient or carrier.

The invention also provides the use of an anti-OX 40 antibody or antigen-binding fragment thereof of any one of the embodiments of the invention for the preparation of a medicament for the prevention or treatment of a T cell-related disorder; preferably, the T cell associated disease is a T cell associated tumor or OX40 mediated disorder.

In one or more embodiments, the OX40 mediated disorder includes OX40 mediated allergies, asthma, COPD, rheumatoid arthritis, psoriasis, autoimmune diseases and inflammation-related diseases.

The invention also provides a method of treating or preventing a T cell-related disorder, enhancing an endogenous immune response in an animal, comprising administering to a patient in need thereof a therapeutically effective amount of an anti-OX 40 antibody or antigen-binding fragment thereof of any of the embodiments of the invention, or a pharmaceutical composition comprising an anti-OX 40 antibody or antigen-binding fragment thereof of any of the embodiments of the invention. Preferably, the T cell-related disease is a T cell-related tumor or an autoimmune disease.

Drawings

FIG. 1 shows the ability of enzyme-linked immunosorbent assay (ELISA) to determine the binding of single cell sequencing-derived anti-human OX40 JAC3 antibodies to human OX40 extracellular portion antigen. The results showed that the anti-human OX40 JAC3 antibody derived from single cell sequencing, except for a few antibodies, had a certain binding capacity for human OX40 extracellular antigen at a concentration of 3.16. Mu.g/ml for the purified single cell sequencing antibody (A), 0.316. Mu.g/ml for (B), and 3.16. Mu.g/ml-1 ng/ml for gradient dilution.

FIG. 2 shows hFCGR ^Tg hOX40 ^Tg The anti-human OX40 JAC3 antibodies in mouse spleen cells activate T cells and promote their proliferative agonistic activity. The purified anti-human OX40 JAC3 antibody and control antibody were diluted to 3 concentration gradients and cells were cultured with primary cell culture broth containing 0.1 μg/ml anti-murine CD3 for 72 hours before analysis of the murine CD4 by flow-through techniques ⁺ T cells Mean Fluorescence Intensity (MFI) of CFSE at antibody stimulation of 1. Mu.g/ml (A), 0.1. Mu.g/ml (B) and 0.01. Mu.g/ml (C). CFSE is equally diluted in fluorescence intensity with cell division proliferation, so that MFI of CFSE is inversely related to cell proliferation activation, i.e. to the activity of anti-OX 40 antibodies.

FIG. 3 shows the reaction at hFCGR ^Tg hOX40 ^Tg The agonist activity of the anti-human OX40 JAC3 antibody of fig. 2 with certain immune activation ability to activate T cells and promote proliferation was detected in mouse spleen cells. The purified anti-human OX40 PN003, PN013, PN024, PN037 JAC3 antibodies and control antibodies were subjected to gradient dilution (1. Mu.g/ml-1 ng/ml) and cells were incubated with primary cell culture broth containing 0.1. Mu.g/ml anti-murine CD3 for 72 hours, then analyzed for mouse CD4 by flow-through techniques ⁺ Mean Fluorescence Intensity (MFI) of T cell CFSE (A)Each group of antibodies was stimulated with 0.01. Mu.g/ml of CD4 ⁺ Mean Fluorescence Intensity (MFI) of T cell CFSE (B). CFSE is equally diluted in fluorescence intensity with cell division proliferation, so that MFI of CFSE is inversely related to cell proliferation activation, i.e. to the activity of anti-OX 40 antibodies.

FIG. 4 shows that anti-human OX40 JAC3 antibodies have better in vitro activity (vs Pogalizumab and IBI 101). By detection of OVA-specific CD8 ⁺ T cell proliferation, it can be seen that anti-human OX40 JAC3 PN024 antibodies screened by single cell sequencing technique have a stronger stimulation of OVA-specific CD8 than Pogalizumab and IBI101 ⁺ Ability of T cells to secrete IFN-g, A is IFNg ⁺ CD8 ⁺ T is the proportion of CD8+ T cells and B is the absolute number of cells.

Figure 5 shows that anti-human OX40 JAC3 antibodies have good anti-tumor activity (MC 38 system). After different antibody treatments are given to mice inoculated with MC38 tumors, the anti-human OX40 JAC3 PN024 antibody can achieve the effect (A and B) of curing tumors, which is equivalent to the effect of Pogalizumab, and the hepatotoxicity (serum AST level) of the anti-human OX40 JAC3 PN024 antibody is obviously weaker than Pogalizumab (C), which shows that compared with Pogalizumab, the anti-human OX40 JAC3 PN024 antibody can better achieve both curative effect and toxic and side effect.

Figure 6 shows that anti-human OX40 JAC3 antibodies have good anti-tumor activity (MO 4 system). After different antibody treatments of mice vaccinated with MO4 tumor, high dose (5 mg/Kg) and low dose (1.5 mg/Kg) of anti-human OX40 JAC3 PN024 antibody can achieve better tumor curing effect (A and B) than high dose of Pogalizumab and IBI101, and hepatotoxicity (serum AST level) of anti-human OX40 JAC3 PN024 antibody is obviously weaker than Pogalizumab (C), which indicates that the anti-human OX40 JAC3 PN024 antibody has better anti-tumor activity compared with Pogalizumab.

Figure 7 shows that anti-human OX40 JAC3 antibodies are less toxic (MO 4 system). After different antibody treatments were given to mice vaccinated with MO4 tumors, high dose (5 mg/Kg) and low dose (1.5 mg/Kg) of anti-human OX40 JAC3 PN024 antibodies could achieve better tumor curing effect than high dose of Pogalizumab and IBI101 (fig. 6), and the hepatotoxicity (serum AST level) of anti-human OX40 JAC3 PN024 antibodies was significantly weaker than Pogalizumab (a and B) on day 7 blood sampling test, indicating better compromise of therapeutic effect and toxic side effects than Pogalizumab and IBI 101.

Figures 8A-C show the results of detecting anti-human OX40 antibody binding OX40 over-expressing cell surface OX40 using flow cytometry.

FIG. 9 is a result of antigen binding activity assay showing that anti-human OX40 antibody in hybridoma supernatants is capable of specifically binding to human OX40 extracellular portion antigen. Binding of anti-human OX40 antibodies in hybridoma supernatants to human OX40 extracellular antigen was analyzed by ELISA, showing ELISA signals (OD 650) detected by HRP-labeled anti-mouse IgG Fc after binding of various antibodies 1:100 dilutions of supernatants to coated human OX 40.

FIG. 10 shows that the binding capacity of recombinant anti-human OX40 JAC3 antibodies to human OX40 extracellular portion antigen is determined by an enzyme-linked immunosorbent assay (ELISA). The results showed that (A) 3.16 μg/ml of purified recombinant antibody (PN 005/012/017/063/064/065/066/067/071) derived from single cell sequencing and recombinant antibody (PN 101/102/103/104/105/107/108/109/110/111/113/114/115/116/118/119/121/123/125/126/128/129/130) derived from hybridoma technology against human OX40 can bind to human OX40 extracellular antigen, IBI101 and Poga are positive controls, ctrl hIgG and PBS are negative controls; (B) The partial recombinant antibodies are diluted in a gradient (3.16 mu g/ml-1 ng/ml) and all have certain binding capacity to human OX40 extracellular segment antigen, IBI101 and Poga are positive controls, and Ctrl hIgG is a negative control.

FIG. 11 shows the ability of a flow-tested anti-human OX40 recombinant JAC3 antibody to bind human OX40 on OX40 stably transformed cells. The results show that both PN063/064/065/066/067/071, PN101/102/103/104/105/107/108/109/110/113/114/115/116/118/119/121/123/125/126/128/129 bind to the 293T cell line expressing human OX40, wherein PN071/113/115/118 has a relatively weak binding capacity, resulting in the signal strength of the binding antibodies on the human OX 40-expressing cell line. PBS, ctrl hIgG, OX86 (anti-murine OX40hIgG 1) as negative controls, IBI101 and Poga as positive controls, and 293T as negative control cell lines for stably transfected cells.

FIG. 12 shows ELISA detection of cross-reactivity of recombinant JAC3 antibodies with monkey and mouse OX40 proteins. The results show that both recombinant antibodies bind to both human and monkey (cynomolgus and cynomolgus) OX40 proteins and neither recognize mouse OX40 except for PN 116. PBS, ctrl hIgG, OX86 (anti-murine OX40hIgG 1) as negative controls, IBI101 and Poga as positive controls.

FIG. 13 shows the ability of recombinant JAC3 antibodies to compete with human OX40 ligand (OX 40-L) for binding to OX40 protein. The results showed that when 2 μg/ml of the screened antibody competed with 0.2 μg/ml of OX40-L-mFc for binding to OX40 protein, PN012/063/064/065/066/067, PN101/102/103/104/105/107/108/110/114/116/119/121/123/125/128 had a clear epitope competition relationship with OX40-L-mFc, whereas PN005/071/109/111/113/115/118/126/129/130 was less able to compete for the epitope with OX40-L-mFc, wherein the results of PN071/109/115/118/126/129/130 might be related to its relatively weak affinity for binding to OX40 protein. OX40-L-His was used as positive control, ctrl hIgG and PBS was used as negative control.

FIG. 14 shows that recombinant JAC3 antibody activates hFCGR in vitro ^Tg hOX40 ^Tg Experimental results of T cells in mouse spleen cells and promoting proliferation thereof. The results show that (A) at higher concentration (1 mug/ml), PN012/024/037/063/063/065/066/067/071 from single cell has certain capacity of activating T cells and promoting proliferation of the T cells, and positive control Poga and IBI101 also show certain activity; (B) Decreasing the antibody concentration (0.01 μg/ml), single cell derived PN012/024/037/063/064/065/066/067/071 still showed some ability to activate T cells and promote their proliferation, but the positive controls Poga and IBI101 were not apparent in activity; (C) At lower antibody concentrations (0.01 μg/ml), hybridoma-derived PN101/102/103/104/105/107/108/109/110/113/114/116/118/119/121/123/125/126/128/129 had some ability to activate T cells and promote their proliferation. UT is untreated group, CFSE only cells were CFSE stained, CD3only cells were CFSE stained followed by stimulation with anti-mouse CD3 antibody, cd3+cd28 cells were CFSE stained followed by stimulation with anti-mouse CD3 and anti-mouse CD28 antibodies (positive control). (D) PN005/012/024/037/063/071 from single cell sequencing and PN101/102/103/104/105/107/108/109/110/114/116/118 from hybridoma technology under the stimulation of antibodies (3.16 ng/ml-1 mug/ml) at different concentrations 119/121/123/125/126/128/129 all showed a certain activity of promoting T cell proliferation, and the activity was significantly higher than that of the negative control Ctrl hIgG, and positive controls IBI101 and Poga did not show significant activity. The fluorescence intensity of CFSE is equally diluted with cell division proliferation, so that MFI of CFSE is inversely related to cell proliferation activation, i.e. to the activity of anti-OX 40 antibody.

Fig. 15 shows that the recombinant JAC3 antibody PN024 against human OX40 was significantly more active after Fc optimization than before optimization. (A) The T cell in vitro proliferation experimental result shows that under the stimulation of antibodies (1 ng/ml-1 mug/ml) with different concentrations, the Fc segment of the antibody has obviously stronger activity (CFSE reduction) for promoting the proliferation of the T cells after being optimized. (B) The results of the in vivo proliferation model of DEC-OVA T cells show that PN024 after Fc optimization has stronger capacity of promoting T cell proliferation, and the results show that the cells of OVA-specific CD8+ account for the total CD8+T. Ctrl hIgG was used as a negative control.

Detailed Description

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. These techniques are well explained in the literature, such as Molecular Cloning: a Laboratory Manual, second edition (Sambrook et al, 1989); oligonucleotide Synthesis (m.j. Gait edit, 1984); animal Cell Culture (r.i. freshney edit, 1987); methods in Enzymology (Academic Press, inc.); current Protocols in Molecular Biology (F.M. Ausubel et al, 1987 edition and its periodic updates); and (2) PCR: the Polymerase Chain Reaction (Mullis et al, 1994); a Practical Guide to Molecular Cloning (Perbal Bernard v., 1988); phage Display: a Laboratory Manual (Barbas et al, 2001).

OX40 is a cell surface receptor that binds OX40L (CD 252, TNFSF 4) or a receptor complex comprising OX 40. The NCBI accession number for the amino acid sequence of human OX40 (hOX 40) was (NP-003318). OX40 proteins may also include variants and fragments. The fragments include extracellular domains without all or part of the transmembrane, and/or intracellular domains and fragments of extracellular domains. The soluble form of hOX40 includes an extracellular domain or fragment of an extracellular domain that retains the ability to bind BAFF and/or APRIL. "OX40" also includes post-translational modifications of the OX40 amino acid sequence. Post-translational modifications include, but are not limited to, N-and O-linked glycosylation.

OX40 is expressed predominantly on activated effector T cells (Teffs) and regulatory T cells (Tregs), as well as on NKT cells, NK cells and neutrophils. In cancer, OX 40-expressing activated T cells are found in tumor infiltrating lymphocytes. OX40 and its ligand OX40L play a key role in inducing and maintaining T cell responses. Enhancement of anti-tumor T cell function is useful against cancer and other OX40 mediated disorders including, for example, OX40 mediated allergies, asthma, COPD, rheumatoid arthritis, psoriasis, autoimmune diseases and inflammation related diseases.

anti-OX 40 antibodies

The present invention provides antibodies that specifically bind to OX 40.

Herein, the term "antibody" includes monoclonal antibodies (including full length antibodies, which have an immunoglobulin Fc region), antibody compositions having multi-epitope specificity, multi-specific antibodies (e.g., bispecific antibodies), diabodies and single chain molecules, and antibody fragments, particularly antigen binding fragments, e.g., fab, F (ab') 2, and Fv. The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably herein.

The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). IgM antibodies consist of 5 basic heterotetramer units and a further polypeptide called a J chain, comprising 10 antigen binding sites; whereas IgA antibodies comprise 2-5 basic 4-chain units, which can polymerize with J-chains to form multivalent assemblies. In the case of IgG, the 4-chain unit is typically about 150,000 daltons. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the two heavy chains are linked to each other by one or more disulfide bonds, the number of disulfide bonds being dependent on the isotype of the heavy chain. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (CH 1, CH2 and CH3 for each alpha and gamma chain) and four (CH 1, CH2, CH3 and CH 4) constant domains (CH) for the mu and epsilon isoforms and a Hinge region (Hinge) between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus followed by a constant domain (CL) at its other end. VL and VH are aligned together, while CL and the first constant domain of the heavy chain (CH 1) are aligned together. Specific amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The paired VH and VL together form an antigen binding site. For the structure and properties of different classes of antibodies, see e.g. Basic and Clinical Immunology, eighth edition, daniel p.sties, abba i.terr and Tristram g.Parsolw editions, appleton & Lange, norwalk, CT,1994, pages 71 and chapter 6. Light chains from any vertebrate species can be classified, based on their constant domain amino acid sequences, into one of two distinct types called kappa and lambda. Immunoglobulins may be assigned to different classes or isotypes depending on their heavy chain constant domain (CH) amino acid sequence. There are five classes of immunoglobulins: igA, igD, igE, igG and IgM have heavy chains called α, δ, ε, γ and μ, respectively. Based on the relatively small differences in CH sequence and function, the gamma and alpha classes can be further divided into subclasses, e.g., humans express the following subclasses: igG1, igG2A, igG2B, igG3, igG4, igA1 and IgA2.

"variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL", respectively. These domains are typically the most variable parts of an antibody (relative to other antibodies of the same type) and contain antigen binding sites.

The term "variable" refers to the case where certain segments in the variable domain differ widely in antibody sequence. The variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across all amino acids spanned by the variable domains. Instead, it focuses on three segments called hypervariable regions (HVRs), both in the light and heavy chain variable domains, i.e., HCDR1, HCDR2, HCDR3 for the heavy chain variable region and LCDR1, LCDR2 and LCDR3 for the light chain variable region, respectively. The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of the natural heavy and light chains each comprise four FR regions (FR 1, FR2, FR3 and FR 4) that mostly take on a β -sheet conformation, linked by three HVRs that form a loop linkage and in some cases form part of the β -sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and together with the HVRs of the other chain contribute to the formation of the antigen binding site of the antibody (see Kabat et al, sequences of Immunological Interest, fifth edition, national institute of health, bethesda, MD, 1991). Typically, the light chain variable region is of the structure FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4 and the heavy chain variable region is of the structure FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domains are not directly involved in binding of antibodies to antigens, but exhibit a variety of effector functions, such as participation of antibodies in antibody-dependent cell-mediated cytotoxicity.

"Fc region" (crystallizable fragment region) or "Fc domain" or "Fc" refers to the C-terminal region of the antibody heavy chain that mediates binding of immunoglobulins to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (C1 q) of the classical complement system. In IgG, igA and IgD antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 domain and the CH3 domain of the two heavy chains of the antibody; the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, a human IgG heavy chain Fc region is generally defined as the stretch of sequence from the amino acid residue at heavy chain position C226 or P230 to the carboxy-terminus, wherein the numbering is according to the EU index as in Kabat. As used herein, the Fc region may be a native sequence Fc or a variant Fc.

An "Fc receptor" or "FcR" is a receptor that binds an immunoglobulin Fc region. Fcrs that bind IgG antibodies include receptors of the fcγr family, including allelic variants and alternatively spliced forms of these receptors. The human fcγ receptor family includes several members: fcyri (CD 64), fcyriia (CD 32 a), fcyriib (CD 32 b), fcyriiia (CD 16 a), fcyriiib (CD 16 b). Of these, fcyriib is the only inhibitory fcyriib receptor, and others are all activated fcyriib receptors. Most natural effector cell types co-express one or more active fcγr and inhibitory fcγriib, whereas Natural Killer (NK) cells selectively express one active fcγriii (fcγriii in mice, fcγriiia in humans) but not inhibitory fcγriib in mice and humans. These fcγ receptors differ in molecular structure and therefore have different affinities for each IgG antibody subclass. Among these fcγreceptors fcγri is a high affinity receptor, while fcγriia, fcγriib and fcγriiia are low affinity receptors. Genetic polymorphisms are also present in these different fcγ receptors and affect their binding affinity. The most common genetic polymorphisms are R131/H131 of FcgammaRIIA and V158/F158 of FcgammaRIIIA. Some of these polymorphic forms have been found to be associated with a variety of diseases, and the efficacy of some particular therapeutic antibodies is also dependent on whether the patient carries a particular polymorphic form of the fcγ receptor gene.

The "affinity ratio for inhibitory and activating fcγ receptors" or "I/a ratio" as used herein refers to the ratio of the affinity of a protein molecule for an inhibitory Fc receptor to the affinity for an activating Fc receptor, and in the present invention the I/a ratio is calculated as follows: i/a ratio = [ lower KD value of KD (hfcyriia) or KD (hfcyriiia ]/KD (hfcyriib); wherein KD (hFcγRIIA) is the equilibrium dissociation constant of the molecule for the hFcγRIIA receptor (represented by variant hFcγRIIA-R131), KD (hFcγRIIA) is the equilibrium dissociation constant of the molecule for the hFcγRIIA receptor (represented by variant hFcγRIIA-F158), and KD (hFcγRIIB) is the equilibrium dissociation constant of the molecule for the hFcγRIIB receptor; hfcyriia refers to human fcyriia receptor, hfcyriiia refers to human fcyriiia receptor, hfcyriib refers to human fcyriib receptor. By "affinity" is meant the amount of binding capacity between two molecules, which can be measured generally by KD.

"KD" refers to the equilibrium dissociation constant for two molecules (e.g., a particular antibody and antigen or ligand and receptor) to interact. An "antigen binding moiety" refers to a protein that specifically binds an antigen with high affinity, including, but not limited to, antigen binding fragments of antibodies, adnectins, nanobodies (nanobodies), minibodies, affibodies (affibodies), affilins, target binding regions of receptors, cell adhesion molecules, ligands, enzymes, cytokines, chemokines, and the like. Antigens targeted by an antigen binding moiety include, but are not limited to, TNF receptor superfamily members, immunosuppressive receptor molecules, and the like.

In the present invention, a part of the antibodies are agonistic antibodies, and a part of the antibodies are non-agonistic antibodies. An "agonistic antibody" is an antibody that binds to and activates a receptor. Preferably, the CDRs of the agonistic antibody of the present invention can be arbitrarily combined to form an agonistic antibody CDR-set or used to form an agonistic antibody variable region; the CDRs of the non-agonistic antibody of the invention can be combined arbitrarily to form an effective CDR set, thereby forming the non-agonistic antibody CDR set or forming the variable region of the non-agonistic antibody. Herein, "agonistic antibody variable region" refers to a variable region that has agonistic activity to an antigen in the form of an intact antibody, such as an OX40 agonistic antibody variable region or an agonistic OX40 antibody variable region; by "non-agonistic antibody variable region" is meant an antibody variable region that does not have any agonistic activity in the form of an intact antibody, e.g., an OX40 non-agonistic antibody variable region or a non-agonistic OX40 antibody variable region.

Herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal Antibodies to be used according to the invention can be generated by a variety of techniques, including, for example, the Hybridoma method (e.g., kohler and Milstein, nature,256:495-97 (1975); hongo et al, hybrid, 14 (3): 253-260 (1995), harlow et al, antibodies: A Laboratory Manual, cold Spring Harbor Laboratory Press, second edition 1988; hammerling et al, in Monoclonal Antibodies and T-Cell hybrid, 563-681, elsevier, N.Y., 1981), recombinant DNA methods (e.g., U.S. Pat. No. 4,816,567), phage display techniques (e.g., clackson et al, nature,352:624-628 (1991); marks et al, J.mol. Biol.,222:581-597 (1992), sidhu et al, J.mol. Biol.,338 (2): 299-310 (2004), lee et al, J.mol. Biol.,340 (5): 1073-1093 (2004), fellose, proc. Natl. Acad. Sci. USA,101 (34): 12467-12472 (2004), and Lee et al J.immunol. Methods,284 (1-2): 119-132 (2004)), techniques for generating human or human-like Antibodies from animals having a partial or whole human immunoglobulin locus or a gene encoding a human immunoglobulin sequence (e.g., WO1998/24893, WO1996/34096, WO 1991/10741; jakobovits et al, proc. Natl. Acad. Sci. USA,90:2551 (1993)), jabots et al J. Immun. 1993, 255, 1993, and Lee et al J. Immunol. Methods,284 (1-2); 119-132 (2004)), and Brue et al, brBrK.1996/24893; brK 6/340735; brK.1996/1996, WO1996/33735; jakovits, J.Natl. Sci.Sci.USA, 90:25 (1993), nature,368:856-859 (1994); morrison, nature,368:812-813 (1994); fisheld et al, nature biotechnology, 14:845-851 (1996); neuberger, nature biotechnol, 14:826 (1996); and Lonberg and humizar, international rev. Immunol.,13:65-93 (1995), single cell sequencing (Nat Biotechnol.2013Feb;31 (2): 166-9.).

The terms "full length antibody", "whole antibody" or "complete antibody" are used interchangeably to refer to an antibody that is substantially in its whole form (as opposed to an antibody fragment). In particular, complete antibodies include those having heavy and light chains including Fc regions. The constant domain may be a natural sequence constant domain (e.g., a human natural sequence constant domain) or an amino acid sequence variant thereof. In some cases, an intact antibody may have one or more effector functions.

An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and/or variable regions of an intact antibody. The antibody fragment is preferably an antigen binding fragment of an antibody. Examples of antibody fragments include Fab, fab ', F (ab') 2 and Fv fragments; a diabody; linear antibodies (see U.S. Pat. No. 5,641,870, example 2; zapata et al, protein Eng.,8 (10): 1057-1062, 1995); a single chain antibody molecule; an scFv-Fc fragment; multispecific antibodies formed from antibody fragments; and any fragment that should be capable of increasing half-life by chemical modification or by incorporation into liposomes. Digestion of an antibody with papain produces two identical antigen-binding fragments, called "Fab" fragments, and one residual "Fc" fragment, the name of which reflects its ability to crystallize readily. The Fab fragment consists of the complete light chain and heavy chain variable domain (VH) and one heavy chain first constant domain (CH 1). Each Fab fragment is monovalent in terms of antigen binding, i.e. it has a single antigen binding site. Pepsin treatment of antibodies produced a larger F (ab') 2 fragment, roughly equivalent to two Fab fragments linked by disulfide bonds, with different antigen binding activities and still capable of cross-linking the antigen. Fab' fragments differ from Fab fragments by the addition of some additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. F (ab ') 2 antibody fragments were initially generated as pairs of Fab ' fragments with hinge cysteines between the Fab ' fragments. Other chemical couplings of antibody fragments are also known. The Fc fragment comprises the carboxy-terminal portions of two heavy chains held together by disulfide bonds. The effector function of antibodies is determined by sequences in the Fc region, which is also the region recognized by Fc receptors (fcrs) found on certain cell types.

"Fv" is the smallest antibody fragment that contains the complete antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each for heavy and light chains) are highlighted from the fold of these two domains, contributing to the antigen-binding amino acid residues and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with less avidity than the complete binding site.

"Single chain Fv" may also be abbreviated "sFv" or "scFv" and is an antibody fragment comprising the VH and VL domains of an antibody linked into one polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains such that the sFv forms the desired antigen-binding structure. For reviews of sFvs see The Pharmacology of Monoclonal Antibodies, volume 113, rosenburg and Moore editions, springer-Verlag, new York, pp.269-315 (1994).

"chemical modification" of the fragments includes addition of poly (alkylene) glycols such as polyethylene glycol ("pegylation, PEGylation"), including PEGylated fragments of Fv, scFv, fab, F (ab ') 2 and Fab', i.e., fv-PEG, scFv-PEG, fab-PEG, F (ab ') 2-PEG and Fab' -PEG. Such fragments have EGFR binding activity.

Preferably, the antibody fragment, in particular the antigen binding fragment, consists of or comprises a partial sequence of the heavy chain variable region or the light chain variable region of the antibody from which it is derived, said partial sequence being sufficient to retain the same binding specificity and sufficient affinity as the antibody from which it is derived, for OX40 preferably at least equal to 1/100, in a more preferred manner at least equal to 1/10 of the affinity of the antibody from which it is derived. Such antibody fragments will comprise a minimum of 5 amino acids, preferably 10, 15, 25, 50 and 100 consecutive amino acids of the antibody sequence from which they are derived.

Monoclonal antibodies herein also include "chimeric" antibodies (immunoglobulins) 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, and the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; morrison et al, proc.Nat. Acad. Sci. USA,81:6851-6855, 1984).

"humanized" form of a non-human (e.g., murine) antibody refers to a chimeric antibody that minimally comprises sequences derived from a non-human immunoglobulin. Thus, a "humanized antibody" generally refers to a non-human antibody in which the variable domain framework regions are exchanged for sequences found in a human antibody. Typically in humanized antibodies, the entire antibody (except for the CDRs) is encoded by a polynucleotide of human origin or is identical to such an antibody (except for the CDRs). CDRs (some or all of which are encoded by nucleic acids derived from non-human organisms) are grafted into the β -sheet framework of the human antibody variable region to produce antibodies, the specificity of which is determined by the grafted CDRs. The production of such antibodies is described, for example, in WO92/11018; jones,1986, nature,321:522-525; verhoeyen et al, 1988, science,239: 1534-1536. Humanized antibodies can also be generated using mice with a genetically engineered immune system (see Roque et al, 2004, biotechnol. Prog., 20:639-654).

"human antibody" refers to an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or produced using any of the techniques disclosed herein for producing a human antibody. This definition of human antibodies specifically excludes humanized antibodies that comprise non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries. Such techniques can be found in Hoogenboom and Winter, journal of molecular biology, 227:381 (1991); marks et al, journal of molecular biology, 222:581 (1991). Available methods for preparing human monoclonal antibodies are described in Cole et al, monoclonal antibodies and cancer therapies, alan R.List, p.77 (1985); boerner et al, journal of immunology 147 (1): 86-95 (1991). See also van Dijk and van de Winkel, modern pharmaceutical reviews, 5:368-74 (2001). Human antibodies can be prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous locus has been disabled, e.g., by immunized xenograft mice (xenome) (see, e.g., US 6,075,181 and 6,150,584 for XENOMOUSETM technology). See also, for example, li et al, national academy of sciences, 103:3557-3562 (2006) relates to human antibodies produced via human B cell hybridoma technology.

The anti-OX 40 antibodies of the invention can also be minibodies. Minibodies are minimized antibody-like proteins comprising scfvs linked to a CH3 domain (Hu et al, 1996, cancer Res., 56:3055-3061). The anti-OX 40 antibodies of the invention may also be domain antibodies, see for example US 6,248,516. Domain antibodies (dabs) are functional binding domains of antibodies, corresponding to the variable region of the heavy (VH) or light (VL) chain of a human antibody dAb, having a molecular weight of about 13kDa or less than one tenth of the size of an intact antibody. dabs are expressed well in a variety of hosts including bacterial, yeast and mammalian cell systems. In addition, dabs are highly stable and remain active even after being subjected to harsh conditions, such as lyophilization or thermal denaturation. See, for example, US 6,291,158; US 6,582,915; US 6,593,081; US 6,172,197; US 2004/0110841; EP 0368684; U.S. Pat. No. 6,696,245, WO04/058821, WO04/003019 and WO03/002609.

HCDR1 of the anti-OX 40 antibodies of the invention can contain X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ (SEQ ID NO: 1), wherein X ₁ Is G or S, X ₂ A, D, F, G, V or Y, X ₃ S, T or I, X ₄ I, L or F, X ₅ I, S, D, N, T or A, X ₆ N, S, T, D, R or G, X ₇ N, S, T, Y, A, G or F, X ₈ N, Y, A, G, W, S, I, K, F, T or D, X ₉ W, V or none, X ₁₀ N, G or none. In one or more embodiments, X ₁ Is G or S, X ₂ D, F, G or Y, X ₃ Is S or T, X ₄ I, L or F, X ₅ S, D or T, X ₆ N, S, T or D, X ₇ S, T or Y, X ₈ N, Y, A, G or I, X ₉ W, V or none, X ₁₀ N or none. In one or more embodiments, X ₁ Is G or S, X ₂ F, G or Y, X ₃ S, T or I, X ₄ I, L or F, X ₅ S, D or T, X ₆ N, S, T, D, R or G, X ₇ S, Y or A, X ₈ N, G, T, A, Y, W or I, X ₉ W, V or none, X ₁₀ G or none. An exemplary HCDR1 has an amino acid sequence as set forth in any one of SEQ ID NOs 7-38. Preferably, the amino acid sequence of HCDR1 is set forth in any of SEQ ID NOs 9, 10, 12, 13, 19, 21, 23, 33, 34, 38. Also preferably, the amino acid sequence of HCDR1 is set forth in any of SEQ ID NOs 11, 12, 14, 21, 23, 27, 28, 32, 33, 34, 35, 38.

HCDR2 of the anti-OX 40 antibodies of the invention can contain X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ (SEQ ID NO: 2), wherein X ₁ Is M or I; x is X ₂ N, Y, S, K, H or F; x is X ₃ P, W, Q, H, S, G, A or R; x is X ₄ N, S, G, Y, K or D; x is X ₅ S, D, G, N, A or T; x is X ₆ T, N, S, D or G; x is X ₇ N, K, T, S, D, Y, H, G, A or E; x is X ₈ T, I, K, G or none; x is X ₉ T or none; x is X ₁₀ T, I or none. In one or more embodiments, X ₁ Is I; x is X ₂ N, Y, S, H or F; x is X ₃ W, H, S or a; x is X ₄ N, S, G or Y; x is X ₅ S, D, G or N; x is X ₆ T, N, S, D or G; x is X ₇ N, K, T, S or D; x is X ₈ T, I, K or none; x is X ₉ And X ₁₀ Is none. In one or more embodiments, X ₁ Is I; x is X ₂ N, Y, S, K or H; x is X ₃ P, W, H, S or a; x is X ₄ N, S, G, Y or K; x is X ₅ S, D, G, N or a; x is X ₆ N, S, D or G; x is X ₇ T, D, N, S, G or K;X ₈ t, I, K, G or none; x is X ₉ T or none; x is X ₁₀ Is I or none. An exemplary HCDR2 has an amino acid sequence as set forth in any one of SEQ ID NOS.39-65. Preferably, the amino acid sequence of HCDR2 is set forth in any of SEQ ID NOs 42, 43, 46, 47, 50, 58, 59, 63, 64. Also preferably, the amino acid sequence of HCDR2 is set forth in any of SEQ ID NOs 40, 43, 46, 48, 50, 52, 54, 56, 57, 59, 60, 63.

HCDR3 of the anti-OX 40 antibodies of the invention can contain CX ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ (SEQ ID NO: 3), wherein X ₁ V, T, S or A, X ₂ R, K, I, G, A, T, S, H, Q, N or E, X ₃ S, D, W, G, Y, A, E, R, L, V, T, M or K, X ₄ G, Y, F, V, L, A, P, D, S, W, R, Q, E, N or K, X ₅ D, Y, G, S, R, F, T, V, A, P, L, I, E, W or N, X ₆ W, G, E, D, S, R, P, Y, N, L, A, T, I or V, X ₇ H, S, L, A, T, Y, G, F, W, V, E, D or M, X ₈ C, G, L, F, Y, S, W, D, M, T, N, R, H, V or P, X ₉ F, S, D, Y, W, G, T, E, P, L, R, N or none, X ₁₀ D, Y, F, I, N, W, V, P, E, L, G, S, Q or none, X ₁₁ Y, G, D, W, F, A, I, Q, S, N or none, X ₁₂ W, D, Y, F, N, G, R, P or none, X ₁₃ I, W, Y, D, S, F, G, L or none, X ₁₄ Q, W, I, Y, F, D, V, H or none, X ₁₅ H, W, M, D, Y, F or none, X ₁₆ W, D, S, Y or none, X ₁₇ V, W, Y or none, X ₁₈ W or none. At one or more ofIn embodiments, X ₁ Is V or A, X ₂ R, K, S or Q, X ₃ S, D, G, Y, A or E, X ₄ G, Y, D, S, W or N, X ₅ Y, S, F, T, A, L, I or W, X ₆ G, D, P, Y, N, L or T, X ₇ S, L, T, Y, F, W, E or D, X ₈ G, F, Y, S, W, M, T or H, X ₉ F, S, D, Y, E, L or none, X ₁₀ D, Y, V, L, G or none, X ₁₁ Y, G, W, F, Q, N or none, X ₁₂ -X ₁₈ Is none. In one or more embodiments, X ₁ V, T or A, X ₂ S, R, K, G, T or H, X ₃ E, D, S, G, R or A, X ₄ D, W, G, P, Y or L, X ₅ F, A, T, Y, P, G or L, X ₆ D, G, Y or S, X ₇ Y, D, G, A, S, T, E or L, X ₈ W, H, Y, F, G, S, R or T, X ₉ Y, F, D, W, S, G, L, E or none, X ₁₀ Y, D, I, L, S or none, X ₁₁ F, Y, W, G, A, Q, S or none, X ₁₂ W, D, Y, F or none, X ₁₃ I, W, Y, D, F or none, X ₁₄ Q, W, I, Y, F or none, X ₁₅ H, W, M, D or none, X ₁₆ W, D, S or none, X ₁₇ W, V or none, and X ₁₈ W or none. An exemplary HCDR3 has an amino acid sequence as set forth in any one of SEQ ID NOs 66-114. Preferably, the amino acid sequence of HCDR3 is set forth in any one of SEQ ID NOs 67, 78, 79, 80, 86, 93, 96, 99, 100, 107, 108. Also preferably, the amino acid sequence of HCDR3 is set forth in any of SEQ ID NOs 67, 69, 71, 75, 79, 86, 90, 98, 99, 105, 107, 110.

LCDR1 of the anti-OX 40 antibodies of the invention can contain X ₁ X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ X ₁₂ (SEQ ID NO: 4), wherein X ₁ Is H or Q, X ₂ G, D, S or T, X ₃ S, I, L, T or V, X ₄ S, N, R, W, L, V or F, X ₅ T, D, N, S, H, Y or G, X ₆ Y, D, W, S, R, N, G or T, X ₇ N, D, S, Y or none, X ₈ G, N or none, X ₉ Y, N, D, H, S or none, X ₁₀ N, T, R, K or none, X ₁₁ Y, F, S, N or none, X ₁₂ Y or none. In one or more embodiments, X ₁ Is H or Q, X ₂ G, D or S, X ₃ S, I, L or V, X ₄ S, N, W, L or F, X ₅ T, N, S, H, Y or G, X ₆ Y, D, S, R or N, X ₇ N, D, Y or none, X ₈ G or none, X ₉ Y, N, D or none, X ₁₀ N, T or none, X ₁₁ Y or none, X ₁₂ Is none. In one or more embodiments, X ₁ Is Q, X ₂ Is D or S, X ₃ I, L or V, X ₄ S, V or L, X ₅ D, N, S, H, Y or G, X ₆ Y, D, W, S, R or N, X ₇ N, D, S, Y or none, X ₈ G, N or none, X ₉ Y, N, D or none, X ₁₀ N, T, K or none, X ₁₁ Y, F, N or none, X ₁₂ Y or none. An exemplary LCDR1 has an amino acid sequence as set forth in any one of SEQ ID NOS.115-145. Preferably, the amino acid sequence of LCDR1 is set forth in any one of SEQ ID NOs 115, 116, 120, 122, 124, 126, 130, 134, 135, 140, 141. Also preferably, the amino acid sequence of LCDR1 is set forth in any one of SEQ ID NOs 117, 118, 121, 124, 125, 126, 128, 130, 133, 134, 135, 139, 141.

LCDR2 of the anti-OX 40 antibodies of the invention can contain X ₁ X ₂ X ₃ (SEQ ID NO: 5), wherein X ₁ T, K, G, W, E, M, L, D, A or Q; x is X ₂ V, A, T or G; x is X ₃ Is S or A. In one or more embodiments, X ₁ K, G, L, D or a; x is X ₂ V, A or G; x is X ₃ S. In one or more embodiments, X ₁ T, K, G, W, E, M, L or D; x is X ₂ V, A or G; x is X ₃ S. An exemplary LCDR2 has an amino acid sequence as set forth in any one of SEQ ID NOS.146-159. Preferably, the amino acid sequence of LCDR2 is set forth in any one of SEQ ID NOS 147, 148, 153, 154, 155. Also preferably, the amino acid sequence of LCDR2 is set forth in any one of SEQ ID NOs 146, 147, 148, 149, 150, 151, 153, 154.

LCDR3 of the anti-OX 40 antibodies of the invention can contain CX ₁ QX ₂ X ₃ X ₄ X ₅ X ₆ X ₇ X ₈ X ₉ X ₁₀ X ₁₁ (SEQ ID NO: 6), wherein X ₁ M, Q, L or H, X ₂ G, Y, A, V, H, T, F or S, X ₃ T, G, Y, N, L, D or S, X ₄ H, S, T, Q, D, N, G, I or R, X ₅ W, S, T, Y, L, I or N, X ₆ P, S, M, I, W or T, X ₇ W, F, L, P, I, Y, R or T, X ₈ T, L, A, F or W, X ₉ F, L, T or none, X ₁₀ T, F or none, X ₁₁ F or none. In one or more embodiments, X ₁ M, Q or L, X ₂ G, Y, A or S, X ₃ T, G, Y, N, L or D, X ₄ H, S, Q or N, X ₅ W, S, T, Y, L, I or N, X ₆ Is P or I, X ₇ W, F, L, I, Y, R or T, X ₈ Is T or F, X ₉ F or not, X ₁₀ And X ₁₁ Is none. In one or more embodiments, X ₁ Is M or Q, X ₂ G, Y or A, X ₃ T, G, Y, N, L or D, X ₄ H, S, Q, D, N or G, X ₅ W, S, T, Y or L, X ₆ Is P or S, X ₇ W, F, L, P, I or Y, X ₈ Is T or L, X ₉ Is F or L, X ₁₀ T or none, X ₁₁ F or none. An exemplary LCDR3 has an amino acid sequence as set forth in any one of SEQ ID NOS: 160-199. Preferably, the amino acid sequence of LCDR3 is set forth in any one of SEQ ID NOs 160, 161, 166, 168, 172, 182, 192, 193, 195, 198. Also preferably, the amino acid sequence of LCDR3 is set forth in any one of SEQ ID NOs 160, 161, 162, 163, 164, 166, 167, 168, 169, 170.

In some embodiments, the anti-OX 40 antibodies of the invention contain HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 2, and HCDR3 as shown in SEQ ID NO. 3, and/or LCDR1 as shown in SEQ ID NO. 4, LCDR2 as shown in SEQ ID NO. 5, and LCDR3 as shown in SEQ ID NO. 6. Preferably, the anti-OX 40 antibodies of the invention comprise HCDR1 as set forth in any one of SEQ ID NOS.7-38, HCDR2 as set forth in any one of SEQ ID NOS.39-65, and HCDR3 as set forth in any one of SEQ ID NOS.66-114, and/or LCDR1 as set forth in any one of SEQ ID NOS.115-145, LCDR2 as set forth in any one of SEQ ID NOS.146-159, and LCDR3 as set forth in any one of SEQ ID NOS.160-199.

More preferably, the anti-OX 40 antibodies of the invention comprise an HCDR1 as set forth in any one of SEQ ID NOs 9, 10, 12, 13, 19, 21, 23, 33, 34, 38, an HCDR2 as set forth in any one of SEQ ID NOs 42, 43, 46, 47, 50, 58, 59, 63, 64, and an HCDR3 as set forth in any one of SEQ ID NOs 67, 78, 79, 80, 86, 93, 96, 99, 100, 107, 108, and/or an LCDR1 as set forth in any one of SEQ ID NOs 115, 116, 120, 122, 124, 126, 130, 134, 135, 140, 141, an LCDR2 as set forth in any one of SEQ ID NOs 147, 148, 153, 154, 155, and an LCDR3 as set forth in any one of SEQ ID NOs 160, 161, 166, 168, 172, 182, 192, 193, 195, 198.

Or preferably, the anti-OX 40 antibody of the invention comprises an HCDR1 as shown in any of SEQ ID NOs: 11, 12, 14, 21, 23, 27, 28, 32, 33, 34, 35, 38, an HCDR2 as shown in any of SEQ ID NOs: 40, 43, 46, 48, 50, 52, 54, 56, 57, 59, 60, 63, and an HCDR3 as shown in any of SEQ ID NOs: 67, 69, 71, 75, 79, 86, 90, 98, 99, 105, 107, 110, and/or an LCDR1 as shown in any of SEQ ID NOs: 117, 118, 121, 124, 125, 126, 128, 130, 133, 134, 135, 139, 141, an LCDR2 as shown in any of SEQ ID NOs: 146, 147, 148, 149, 150, 151, 153, 154, and an LCDR3 as shown in any of SEQ ID NOs: 160, 161, 162, 163, 164, 166, 167, 168, 169, 170.

Further preferably, the anti-OX 40 antibody of the present invention contains HCDR1, HCDR2 and HCDR3 as shown in any one of the following groups a1 to a 71:

TABLE 1

Group of	HCDR1	HCDR2	HCDR3
a1	23	57	79
a2	23	57	79
a3	23	43	79
a4	23	48	79
a5	34	50	99
a6	35	59	110
a7	27	59	69
a8	28	59	69
a9	32	56	105

a10	23	42	85
a11	12	46	67
a12	14	60	71
a13	34	52	98
a14	11	40	75
a15	23	48	79
a16	21	63	107
a17	23	57	79
a18	23	57	109
a19	34	50	99
a20	33	59	86
a21	38	43	107
a22	32	54	90
a23	33	59	86
a24	31	55	74
a25	24	41	104
a26	36	39	88
a27	32	54	112
a28	31	55	74
a29	30	54	94
a30	12	46	111
a31	32	54	76
a32	21	63	107
a33	21	63	107
a34	21	43	107
a35	21	43	107
a36	38	43	107
a37	14	60	71
a38	37	52	82
a39	23	57	79
a40	18	43	66
a41	23	42	93
a42	25	42	106
a43	25	43	102
a44	23	42	92
a45	22	42	92
a46	23	43	89
a47	23	45	77
a48	23	42	87
a49	23	42	83
a50	26	43	114
a51	17	42	91
a52	23	42	93
a53	37	53	72
a54	22	42	92

a55	7	65	70
a56	32	54	81
a57	13	49	101
a58	23	42	84
a59	10	64	108
a60	14	61	73
a61	20	43	78
a62	15	62	95
a63	9	58	96
a64	25	42	106
a65	13	47	100
a66	16	44	103
a67	32	54	97
a68	19	42	80
a69	23	43	78
a70	29	51	68
a71	8	42	113

Preferably, the composition comprises HCDR1, HCDR2 and HCDR3 selected from any one of the following groups: a3, a5, a11, a16, a19, a20, a21, a41, a59, a63, a65, a68, a69;

and/or LCDR1, LCDR2 and LCDR3 as shown in any one of the following groups b1 to b 71:

TABLE 2

Group of	LCDR1	LCDR2	LCDR3
b1	126	146	160
b2	126	146	160
b3	126	147	160
b4	126	147	160
b5	141	148	161
b6	133	149	162
b7	130	150	163
b8	125	150	163
b9	121	151	164
b10	144	152	165
b11	124	153	166
b12	117	154	167
b13	139	148	168
b14	128	147	160
b15	118	154	169
b16	134	148	168
b17	117	154	167
b18	126	146	160
b19	141	148	168
b20	135	148	168

b21	135	148	168
b22	141	148	170
b23	124	153	171
b24	127	147	172
b25	141	148	173
b26	124	153	174
b27	119	155	175
b28	132	149	176
b29	138	148	177
b30	118	154	178
b31	133	156	179
b32	134	148	168
b33	134	148	168
b34	134	148	168
b35	134	148	168
b36	135	148	168
b37	144	152	165
b38	133	149	180
b39	144	152	165
b40	141	148	181
b41	140	148	182
b42	141	148	183
b43	141	148	168
b44	139	148	184
b45	142	148	182
b46	140	148	182
b47	141	148	168
b48	136	148	185
b49	141	148	186
b50	143	155	187
b51	145	148	188
b52	118	157	189
b53	129	158	190
b54	141	148	183
b55	136	148	191
b56	124	153	174
b57	140	148	182
b58	137	148	168
b59	116	154	192
b60	140	148	182
b61	131	148	193
b62	123	159	194
b63	120	155	195
b64	138	148	196
b65	130	147	172

b66	141	148	197
b67	136	148	185
b68	122	148	198
b69	115	148	193
b70	118	155	199
b71	141	148	182

Preferably selected from the group consisting of LCDR1, LCDR2 and LCDR3 shown in any of the following: b3, b5, b11, b16, b19, b20, b21, b41, b59, b63, b65, b68, b69.

More preferably, the anti-OX 40 antibodies of the present invention contain HCDR and LCDR of any one of groups c1 through c71 of table 3:

TABLE 3 Table 3

In one or more embodiments, the anti-OX 40 antibodies of the present invention contain HCDR and LCDR in table 3 selected from any one of the following groups: c3, c5, c11, c16, c19, c20, c21, c41, c59, c63, c65, c68, c69.

The FR1 of the anti-OX 40 antibody VH of the invention may be selected from the FR1 of each antibody-numbered VH of Table 4, the FR2 may be selected from the FR2 of each antibody-numbered VH of Table 4, the FR3 may be selected from the FR3 of each antibody-numbered VH of Table 4, and the FR4 may be selected from the FR4 of each antibody-numbered VH of Table 4; and/or FR1 of VL may be selected from FR1 of VL of each antibody number in table 4, FR2 may be selected from FR2 of VL of each antibody number in table 4, FR3 may be selected from FR3 of VL of each antibody number in table 4, and FR4 may be selected from FR4 of VL of each antibody number in table 4.

TABLE 4 Table 4

In a preferred embodiment, the FR region of the anti-OX 40 antibody VH of the invention is the FR region of any one of the VH selected from the antibodies SEQ ID NOS: 200-270, and the FR region of the VL is the FR region of any one of the VL selected from the antibodies SEQ ID NOS: 271-341. Further preferably, the HCDR of such antibodies is selected from any of the aforementioned groups a1 to a71, and the LCDR is selected from any of the aforementioned groups b1 to b 71; more preferably, the CDRs of such antibodies are selected from any one of the preceding groups c1 to c 71.

The amino acid sequence of the VH of the anti-OX 40 antibody of the invention may be as shown in any of SEQ ID NOS: 200-270 and/or the amino acid sequence of the VL may be as shown in any of SEQ ID NOS: 271-341. Preferably, the amino acid sequence of VH and the amino acid sequence of VL of the anti-OX 40 antibody of the invention are shown in any one of the rows of table 3.

The amino acid sequence of the heavy chain constant region of an antibody of the invention may have a sequence as set forth in any one of SEQ ID NOS: 342-345, 348-351, and/or the amino acid sequence of the light chain constant region may have a sequence as set forth in SEQ ID NOS: 346 or 347. In one or more embodiments, the heavy chain constant region of the anti-OX 40 antibody has a sequence as set forth in SEQ ID NOS: 348-351 and/or the amino acid sequence of the light chain constant region has a sequence as set forth in SEQ ID NO: 347. Preferably, the heavy chain constant region of the anti-OX 40 antibody has a sequence as set forth in SEQ ID NO. 350. The affinity of the antibody heavy chain constant region of the antibody of the present invention to human fcyriib is equal to or higher than the affinity of human IgG1 to human fcyriib, and the I/a ratio of the antibody heavy chain constant region is equal to or higher than the I/a ratio of human IgG 1. Preferably, the affinity of the antibody heavy chain constant region for human fcyriib and the affinity of human IgG1 for human fcyriib are increased 3.2-fold or more, the I/a ratio of the antibody heavy chain constant region is equal to or higher than 0.32; also preferably, the affinity of the antibody heavy chain constant region to human fcyriib is equal to or higher than the affinity of human IgG1 to human fcyriib, and the I/a ratio of the antibody heavy chain constant region is equal to or higher than 1; more preferably, the affinity of the antibody heavy chain constant region for human fcyriib is increased by 30-fold or more compared to the affinity of human IgG1 for human fcyriib, and the I/a ratio of the antibody heavy chain constant region is equal to or higher than 1; more preferably, the affinity of the antibody heavy chain constant region for human fcyriib is increased by a factor of 60 or more compared to the affinity of human IgG1 for human fcyriib, and the I/a ratio of the antibody heavy chain constant region is equal to or higher than 40; particularly preferably, the affinity of the antibody heavy chain constant region for human fcyriib and the affinity of human IgG1 for human fcyriib are increased by 90-fold or more, and the I/a ratio of the antibody heavy chain constant region is equal to or higher than 100. The heavy chain constant region has higher affinity of the inhibitory Fc receptor, and can obviously enhance the crosslinking of an agonistic antibody or an agonistic molecule (such as an agonistic fusion protein) and the inhibitory Fc receptor, thereby improving the agonistic activity of the agonistic antibody or the agonistic molecule; and at the same time, the antibody has lower affinity of the activated Fc receptor, and can reduce cytotoxicity such as ADCC mediated by the binding of the activated Fc receptor. An agonistic antibody or an agonistic molecule having better activity can be developed based on the heavy chain constant region of the embodiments of the present invention.

In some embodiments, the amino acid sequence of the heavy chain constant region of an antibody of groups d1-d40 in Table 4 has a sequence as set forth in any one of SEQ ID NOS: 345, 348-351, and/or the amino acid sequence of the light chain constant region has a sequence as set forth in SEQ ID NO: 347. In one or more embodiments, the heavy chain constant regions of the antibodies of groups d1-d40 in Table 4 have the sequences shown in SEQ ID NOS: 348-351 and/or the amino acid sequences of the light chain constant regions have the sequences shown in SEQ ID NOS: 347. Preferably, the heavy chain constant region has the sequence shown as SEQ ID NO. 350 and/or the amino acid sequence of the light chain constant region has the sequence shown as SEQ ID NO. 347.

In other embodiments, the heavy chain constant region of an antibody of group d41-d71 of Table 4 has an amino acid sequence as set forth in any one of SEQ ID NOS: 342-344, 348-351, and/or the light chain constant region has an amino acid sequence as set forth in SEQ ID NOS: 346 or 347; preferably, the amino acid sequences of the heavy chain constant regions of the antibodies of groups d41-d71 in Table 4 are shown in the respective corresponding groups in the following table; or the heavy chain constant region of the antibodies of group d41-d71 in Table 4 is shown in any one of SEQ ID NOS: 348-351 and the light chain constant region is shown in SEQ ID NO: 347. Herein, the heavy and light chain constant regions of each antibody can be used with any other heavy and light chain constant region in the art without affecting the binding capacity of the antibody to an antigen.

The antibodies of the invention may be chimeric, humanized or fully human; preferably fully human antibodies. It should be understood that the antibodies provided by the examples of the present invention are fully human antibodies.

One skilled in the art may substitute, add and/or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids from the sequences of the invention to obtain variants of the antibody or functional fragment sequences thereof without substantially affecting the activity of the antibody. They are all considered to be included within the scope of the present invention. Amino acids having similar properties are substituted, for example, in the FR and/or CDR regions of the variable region. Substitutions are preferably conservative substitutions; amino acid residues that can be conservatively substituted are known in the art. In some embodiments, the sequences of the variants of the invention may have at least 95%, 96%, 97%, 98% or 99% identity to the sequence from which they were derived. Sequence identity as described herein can be measured using sequence analysis software. Such as computer programs BLAST, in particular BLASTP or TBLASTN, using default parameters.

The anti-OX 40 antibodies of the invention may be modified to affect function. The invention includes anti-OX 40 antibodies having modified glycosylation patterns. Modifications may be made to remove undesired glycosylation sites, or the absence of fucose moieties on the oligosaccharide chain to enhance Antibody Dependent Cellular Cytotoxicity (ADCC) function, or galactosylation modifications may be made to alter Complement Dependent Cytotoxicity (CDC).

The anti-OX 40 antibodies of the invention can generally have an affinity constant of about 10-9 to about 10-13M.

The anti-OX 40 antibodies of the invention can be prepared using methods conventional in the art, such as hybridoma techniques well known in the art. Alternatively, the anti-OX 40 antibodies of the invention may be expressed in cell lines other than hybridoma cell lines. Suitable mammalian host cells may be transformed with sequences encoding antibodies of the invention. Transformation may be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotides in liposomes, and direct microinjection of DNA into the nucleus, etc. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese Hamster Ovary (CHO) cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hepG 2), and the like. Particularly preferred cell lines are selected by determining which cell lines have high expression levels and produce antibodies with basic OX40 binding properties.

Polynucleotide sequences encoding anti-OX 40 antibodies

The present invention provides nucleic acid molecules comprising a polynucleotide sequence encoding an anti-OX 40 antibody of the present invention. Provided herein are polynucleotide sequences encoding heavy chain variable regions, light chain variable regions, heavy chains, light chains, and CDRs.

Nucleic acid molecules of the invention include DNA and RNA in single and double stranded form, as well as corresponding complementary sequences. DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, PCR amplified DNA, and combinations thereof. Nucleic acid molecules of the invention include combinations of full-length gene or cDNA molecules, and fragments thereof. The nucleic acids of the invention are preferably derived from human sources, but the invention also includes nucleic acids derived from non-human sources.

In the present invention, an isolated nucleic acid molecule refers to a nucleic acid molecule in the form of an independent fragment or as a component of a larger nucleic acid construct. In a preferred embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecule is preferably derived from DNA or RNA isolated at least once in substantially pure form and in an amount or concentration such that its constituent nucleotide sequences can be identified, manipulated and recovered by standard biochemical methods. The sequences are preferably provided and/or constructed in open reading frames uninterrupted by internal untranslated sequences or introns (typically found in eukaryotic genes). The sequence of the untranslated DNA may be present 5 'or 3' to the open reading frame, which also does not affect the manipulation or expression of the coding region.

The invention also includes nucleic acids that hybridize under moderately stringent conditions, preferably under highly stringent conditions, to a nucleic acid encoding an anti-OX 40 antibody as described herein. Basic parameters influencing the choice of hybridization conditions and guidance for the design of appropriate conditions can be found in Sambrook, fritsch and Maniatis (1989,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y, chapters 9 and 11; and Current Protocols in Molecular Biology,1995, ausubel et al, eds., john Wiley & Sons, inc., sections 2.10 and 6.3-6.4).

Variants according to the invention are typically prepared by site-specific mutagenesis of nucleotides in DNA encoding an anti-OX 40 antibody to produce DNA encoding the variant, and thereafter expressing the recombinant DNA in cell culture, as outlined herein, using cassette mutagenesis or PCR mutagenesis or other techniques well known in the art. However, antigen binding fragments comprising fragments having up to about 100-150 residues can be prepared by in vitro synthesis using established techniques.

As will be appreciated by those of skill in the art, due to the degeneracy of the genetic code, a very large number of nucleic acids may be made, all of which encode an anti-OX 40 antibody or antigen binding fragment thereof of the invention. Thus, where a particular amino acid sequence has been identified, one of skill in the art can prepare any number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence encoding the protein.

The invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription cassettes or expression cassettes comprising at least one polynucleotide as described above. In addition, the invention provides host cells comprising the expression system or construct.

The expression vectors used in any host cell typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, a multiple linker region for inserting nucleic acid encoding an antibody to be expressed, and optional marker elements. Each of these sequences is discussed below.

The vector may optionally contain a "tag" coding sequence, i.e., an oligonucleotide molecule located at the 5 'or 3' end of the anti-OX 40 antibody coding sequence; the oligonucleotide sequence encodes polyhistidine (such as 6 His) or another "tag", such as FLAG, HA (hemagglutinin influenza virus) or myc, which are present in commercially available antibodies. This tag is typically fused to the polypeptide when the polypeptide is expressed, and can serve as a means for affinity purification or detection of anti-OX 40 antibodies from host cells. Affinity purification can be accomplished, for example, by column chromatography using antibodies against this tag as an affinity matrix. The tag optionally can then be removed from the purified OX40 anti-OX 40 antibody by various means, such as using certain peptidases for cleavage.

The flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), heterozygous (i.e., a combination of flanking sequences from more than one source), synthetic or natural. Likewise, the source of the flanking sequences may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequences function in and are activatable by the host cell mechanism.

The origin of replication is typically part of those prokaryotic expression vectors purchased on the market, and this origin aids in the amplification of the vector in a host cell. If the vector of choice does not contain an origin of replication site, it can be chemically synthesized based on known sequences and ligated into the vector. For example, the origin of replication from plasmid pBR322 (New England Biolabs, beverly, MA) is suitable for most gram-negative bacteria, and various viral origins (e.g., SV40, polyoma virus, adenovirus, vesicular Stomatitis Virus (VSV) or papillomaviruses, such as HPV or BPV) are suitable for cloning vectors in mammalian cells. Mammalian expression vectors typically do not require an origin of replication component (e.g., often only the SV40 origin is used, as it also contains a viral early promoter).

Transcription termination sequences are typically located 3' to the coding region of the polypeptide to terminate transcription. The transcription termination sequence in a prokaryotic cell is typically a G-C rich fragment followed by a poly-thymidylate sequence.

The selectable marker gene encodes a protein necessary for survival and growth of the host cell grown in the selective medium. Typical selectable marker genes encode (a) conferring resistance to antibiotics or other toxins (e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells); (b) complementing the auxotrophy of the cell; or (c) proteins that provide important nutrients that are not available from the complex or defined medium. Specific selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene. Advantageously, the neomycin resistance gene can also be used for selection in prokaryotic and eukaryotic host cells.

Ribosome binding sites are generally necessary for translation initiation of mRNA and are characterized by Shine-Dalgarno sequences (prokaryotes) or Kozak sequences (eukaryotes). This element is typically located 3 'of the promoter and 5' of the coding sequence of the polypeptide to be expressed.

Expression and cloning vectors of the invention will typically contain a promoter recognized by the host organism and operably linked to a molecule encoding an anti-OX 40 antibody. A promoter is a non-transcribed sequence located upstream of the start codon of a structural gene (typically within about 100 to 1000 bp) that controls transcription of the structural gene.

Suitable promoters for use with yeast hosts are also well known in the art. Yeast enhancers are advantageously used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from viral genomes such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and most preferably simian virus 40 (SV 40). Other suitable mammalian promoters include heterologous mammalian promoters, such as heat shock promoters and actin promoters.

Enhancer sequences may be inserted into the vector to increase transcription of DNA encoding the light or heavy chains comprising the anti-OX 40 antibodies of the invention by higher eukaryotes. Enhancers are cis-acting elements that act on a promoter to increase transcription of DNA, usually about 10-300bp in length. Enhancers have relative directional and positional independence, and have been found at the 5 'and 3' positions of the transcriptional unit. Several enhancer sequences are known which are obtainable from mammalian genes, such as those of globulin, elastase, albumin, alpha-fetoprotein and insulin. However, enhancers from viruses are typically used. The SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma virus enhancers, and adenovirus enhancers known in the art are exemplary enhancing elements for activating eukaryotic promoters.

The expression vectors of the invention may be constructed from a starting vector, such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. If one or more of the flanking sequences described herein are not already present in the vector, they may be obtained separately and ligated to the vector. Methods for obtaining the individual flanking sequences are well known to those skilled in the art.

After constructing the vector and inserting the nucleic acid molecule encoding the light chain, heavy chain, or both the light chain and heavy chain comprising the anti-OX 40 antibody into the appropriate sites of the vector, the completed vector may be inserted into an appropriate host cell for amplification and/or polypeptide expression. The expression vector for the anti-OX 40 antibody can be transformed into the selected host cell by well known methods, including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method portion selected may vary with the type of host cell to be used.

When the host cells are cultured under appropriate conditions to allow synthesis of the anti-OX 40 antibody, the anti-OX 40 antibody can then be collected from the culture medium (if the host cells secrete it into the culture medium) or directly from the host cells producing it (if not secreted). Suitable host cells are as described hereinbefore.

Use of anti-OX 40 antibodies for therapeutic purposes

All aspects of the anti-OX 40 antibodies described herein are useful in the preparation of medicaments to prevent or treat various conditions and diseases described herein, particularly diseases or conditions in which OX 40-expressing immune cells (particularly T cells, NK cells, and neutrophils) are associated. In some embodiments, the conditions and diseases are T cell-related cancers, including but not limited to: bladder cancer, breast cancer, uterine/cervical cancer, ovarian cancer, prostate cancer, testicular cancer, esophageal cancer, gastrointestinal cancer, pancreatic cancer, colorectal cancer, colon cancer, renal cancer, head and neck cancer, lung cancer, gastric cancer, germ cell cancer, bone cancer, liver cancer, thyroid cancer, skin cancer, neoplasms of the central nervous system, lymphoma, leukemia, myeloma, genitourinary system cancer, urothelial cancer, renal cell carcinoma, gastric adenocarcinoma, non-small cell lung cancer, diffuse large B-cell lymphoma, head and neck squamous cell carcinoma, hodgkin lymphoma, gastroesophageal junction adenocarcinoma, melanoma, sarcoma, and virus-associated cancers. In certain embodiments, the cancer is a metastatic cancer, a refractory cancer, or a recurrent cancer. The conditions and diseases may also be other OX40 mediated disorders including, for example, OX40 mediated allergies, asthma, COPD, rheumatoid arthritis, psoriasis, ulcerative colitis, atopic dermatitis, autoimmune diseases and inflammation related diseases.

The anti-OX 40 antibodies described herein may also be used as vaccine adjuvants. The vaccine adjuvant can be used in combination with a vaccine (e.g., OVA) to form a vaccine composition, which can be used for preventing and/or treating tumors, and also can be used for preventing and/or treating infections.

In addition, the anti-OX 40 antibodies described herein may also be used in the preparation of a medicament to enhance an endogenous immune response. As used herein, "enhancing an endogenous immune response" refers to enhancing the effectiveness or strength of an existing immune response in a subject. Such enhancement of efficiency and potential may be achieved, for example, by overcoming mechanisms that suppress the endogenous host immune response, or by stimulating mechanisms that enhance the endogenous host immune response.

Diagnostic uses, assays and kits

The anti-OX 40 antibodies of the invention may be used in diagnostic assays, e.g., binding assays, to detect and/or quantify OX40 expressed in a tissue (such as thymus or spleen) or a cell (such as a T cell). anti-OX 40 antibodies may be used in studies to further investigate the role of OX40 in disease. anti-OX 40 antibodies may be used to further investigate the role of OX40 in the formation of homo-and/or heteromeric receptor complexes and the role of the OX40 receptor complexes in disease.

Serum levels of OX40 may be prognostic. Embodiments of the invention include diagnostic assays and kits to measure that soluble OX40 is a potential replacement for membrane-bound OX40 on tumor cells.

The anti-OX 40 antibodies of the invention can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or conditions associated with OX 40. The present invention provides for detecting the presence of OX40 in a sample using classical immunohistological methods known to those skilled in the art. Detection of OX40 may be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of OX40 include ELISA, FACS, RIA, and the like.

For diagnostic applications, anti-OX 40 antibodies are typically labeled with a detectable label group. Suitable labelling groups include (but are not limited to) the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotin groups, or predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags). In some embodiments, the labeling groups are coupled to the anti-OX 40 antibody through spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used to carry out the present invention.

One aspect of the invention provides for identifying cells that express OX40. In a specific embodiment, the antibody is labeled with a labeling group and binding of the labeled antibody to OX40 is detected. In another specific embodiment, the binding of the antibody to OX40 is detected in vivo. In another specific embodiment, antibody-OX 40 is isolated and measured using techniques known in the art.

Another aspect of the invention provides for detecting the presence of a test molecule that competes with an antibody of the invention for binding to OX40. An example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of OX40 in the presence or absence of a test molecule. An increase in the amount of free antibody (i.e., antibody that does not bind to OX 40) will indicate that the test molecule is able to compete with the antibody for binding to OX40. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.

Pharmaceutical compositions and routes of administration

The present invention provides pharmaceutical compositions comprising a therapeutically effective amount of one or more anti-OX 40 antibodies of the invention in combination with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and/or adjuvant.

In certain embodiments, acceptable diluents, carriers, solubilizers, emulsifiers, preservatives, and/or adjuvants and the like in the pharmaceutical compositions are preferably non-toxic to the recipient at the dosages and concentrations employed. In certain embodiments, the pharmaceutical compositions may contain such materials for improving, maintaining, or retaining, for example, pH, permeability, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption or permeation of the composition. These substances are known from the prior art and can be found, for example, in REMINGTON' S PHARMACEUTICAL SCIENCES, 18 th edition, A.R. Genrmo, code 1990,Mack Publishing Company. The optimal pharmaceutical composition can be determined depending on the intended route of administration, the mode of delivery and the dosage required.

The pharmaceutical compositions of the present invention may be selected for parenteral delivery. Alternatively, the composition may be selected for inhalation or delivery through the digestive tract (such as orally). The preparation of such pharmaceutically acceptable compositions is within the skill of the art.

Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising anti-OX 40 antibodies in sustained or controlled release delivery formulations. Techniques for formulating a variety of other sustained or controlled delivery means, such as liposome carriers, bioerodible particles or porous beads, and depot injections, are also known to those skilled in the art.

Pharmaceutical compositions for in vivo administration are generally provided in the form of sterile formulations. Sterilization is achieved by filtration through sterile filtration membranes. In the case of lyophilization of a composition, this method may be used to sterilize the composition either before or after lyophilization and reconstitution. Compositions for parenteral administration may be stored in lyophilized form or in solution. Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution tape or vial having a stopper pierceable by a hypodermic injection needle.

Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, or as dehydrated or lyophilized powders. The formulation may be stored in a ready-to-use form or reconstituted (e.g., lyophilized) prior to administration. The invention also provides kits for producing single dose administration units. Kits of the invention may each contain a first container having a dried protein and a second container having an aqueous formulation. In certain embodiments of the invention, kits are provided that contain single and multi-chamber prefilled syringes (e.g., liquid syringes and lyophilized syringes).

The invention also provides methods of treating a patient, particularly a T cell-related disease in a patient, such as a T cell-related cancer and an autoimmune disease, by administering an anti-OX 40 antibody or antigen-binding fragment thereof or a pharmaceutical composition thereof according to any of the embodiments of the invention.

The terms "patient," "subject," "individual," "subject" are used interchangeably herein to include any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit, etc.), and most preferably a human. "treating" refers to a subject employing a treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., reduced number of cancer cells, reduced tumor volume, reduced rate of infiltration of cancer cells into peripheral organs, or reduced rate of tumor metastasis or tumor growth). The treatment regimen effective to treat a patient can vary depending on a variety of factors, such as the disease state, age, weight, and ability of the patient to elicit an anti-cancer response in the subject by therapy.

The therapeutically effective amount of the pharmaceutical composition comprising an anti-OX 40 antibody or antigen-binding fragment thereof of the present invention to be employed will depend, for example, on the degree of treatment and the goal. Those skilled in the art will appreciate that the appropriate dosage level for treatment will vary depending in part on the molecule delivered, the indication, the route of administration, and the size (body weight, body surface or organ size) and/or condition (age and general health) of the patient. In certain embodiments, the clinician may titrate the dose and alter the route of administration to obtain the optimal therapeutic effect.

The frequency of administration will depend on the pharmacokinetic parameters of the particular anti-OX 40 antibody in the formulation used. The clinician typically administers the composition until a dose is reached that achieves the desired effect. The composition may thus be administered as a single dose, or over time as two or more doses (which may or may not contain the same amount of the desired molecule), or as a continuous infusion through an implanted device or catheter.

The route of administration of the pharmaceutical composition is according to known methods, for example, by oral, intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, portal or intralesional route injection; either by a sustained release system or by an implanted device.

Some of the embodiments contained herein:

1. an anti-OX 40 antibody or antigen-binding fragment thereof, wherein said anti-OX 40 antibody comprises

(1) HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 2 and HCDR3 as shown in SEQ ID NO. 3, and/or LCDR1 as shown in SEQ ID NO. 4, LCDR2 as shown in SEQ ID NO. 5 and LCDR3 as shown in SEQ ID NO. 6, and

(2) A heavy chain constant region comprising a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain connected in sequence from N-terminus to C-terminus, wherein the sequences of the CH1 domain and the hinge region are those derived from a CH1 domain and a hinge region of human IgG2, the sequences of the CH2 domain and the CH3 domain are those derived from a CH2 domain and a CH3 domain of human IgG, and the affinity of the antibody heavy chain constant region to human fcyriib is equal to or higher than the affinity of human IgG1 to human fcyriib, and the I/a ratio of the antibody heavy chain constant region is equal to or higher than the I/a ratio of human IgG 1.

2. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 1, wherein the anti-OX 40 antibody comprises HCDR1 as set forth in any one of SEQ ID NOs 7-38, HCDR2 as set forth in any one of SEQ ID NOs 39-65, and HCDR3 as set forth in any one of SEQ ID NOs 66-114, and/or LCDR1 as set forth in any one of SEQ ID NOs 115-145, LCDR2 as set forth in any one of SEQ ID NOs 146-159, and LCDR3 as set forth in any one of SEQ ID NOs 160-199.

3. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 1, wherein the anti-OX 40 antibody comprises HCDR1, HCDR2 and HCDR3 as set forth in any one of groups a1 through a71 of table 1, and/or LCDR1, LCDR2 and LCDR3 as set forth in any one of groups b1 through b71 of table 2.

4. The anti-OX 40 antibody or antigen-binding fragment thereof of item 1, which comprises the HCDR and LCDR of any one of groups c 1-c 71 of table 3.

5. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein each FR region of the anti-OX 40 antibody VH is an FR region selected from any one of the VH's set forth in SEQ ID nos. 200-270, and each FR region of the VL is an FR region selected from any one of the VL's set forth in SEQ ID nos. 271-341; more preferably, each FR region of the anti-OX 40 antibody VH and VL is an FR region selected from VH and VL of any one of the antibodies in table 4.

6. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 1, wherein the VH of the anti-OX 40 antibody has an amino acid sequence as set forth in any one of SEQ ID NOs 200-270 and/or the VL has an amino acid sequence as set forth in any one of SEQ ID NOs 271-341; preferably, the amino acid sequence of the VH and the amino acid sequence of the VL of the anti-OX 40 antibody are as shown in any of the antibody numbers of table 4, or

The anti-OX 40 antibody is a chimeric antibody or a fully human antibody; preferably fully human antibodies.

7. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of items 1-6, wherein the sequence of the CH2 domain and the CH3 domain of the heavy chain constant region is selected from the group consisting of:

a) Sequences derived from the CH2 domain and CH3 domain of human IgG1, and the CH2 domain and CH3 domain comprise the G237D, P238D, P271G and a330R mutations; or alternatively

b) Sequences derived from the CH2 domain and CH3 domain of human IgG1, and the CH2 domain and CH3 domain comprise the G237D, P238D, H268D, P271G and a330R mutations; or alternatively

c) Sequences derived from the CH2 domain and the CH3 domain of human IgG2, and the CH2 domain and the CH3 domain comprise the S267E and L328F mutations; or alternatively

d) Sequences derived from the CH2 domain and CH3 domain of human IgG2, and said CH2 domain and CH3 domain comprising the H268D and P271G mutations,

preferably, the amino acid sequence of the heavy chain constant region of the anti-OX 40 antibody has a sequence as set forth in any one of SEQ ID NOS: 348-351 and/or the amino acid sequence of the light chain constant region has a sequence as set forth in SEQ ID NO: 347.

8. A pharmaceutical composition comprising the anti-OX 40 antibody or antigen-binding fragment thereof of any one of items 1-7, and a pharmaceutically acceptable excipient or carrier.

9. A nucleic acid molecule or a vector comprising the nucleic acid molecule selected from the group consisting of:

(1) A polynucleotide sequence encoding an anti-OX 40 antibody or antigen-binding fragment thereof of any one of items 1-7;

(2) The complement of the polynucleotide sequence of (1).

10. Use of an anti-OX 40 antibody or antigen-binding fragment thereof of any one of items 1-7 in the manufacture of a medicament for the treatment of a T cell-related disorder; preferably, the T cell-related disease is a T cell-related tumor or OX 40-mediated disease; more preferably, the OX40 mediated disease comprises OX40 mediated allergy, asthma, COPD, rheumatoid arthritis, psoriasis, autoimmune diseases and inflammation related diseases.

The invention will be illustrated by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. The methods and materials used in the examples are those conventional in the art, unless otherwise indicated.

Examples

Materials and methods

1) Immunoglobulin variable region gene humanization mice acemousebatm were derived from south-hua Kang Hengjian biotechnology limited, lake. The mice are all raised in Hunan Hua Kangheng biological technology limited company, and the development of related animal experiments accords with national laws and regulations of all levels, and the permissions of animal nursing and use committee of Hunan Hua Kang Hengjian biological technology limited company are obtained.

2) OX40 humanized mice (hOX 40) ^Tg Mice): human murine chimeric Tnfrsf4 (OX 40) cDNA-wpre-polyA (wherein the extracellular region (about 2.3 kb) and the front half of the transmembrane region of the OX40 gene are human, including the front half of Exon1-5 and Exon6, the rear half of the transmembrane region and the intracellular region are murine, including the rear half of Exon7 and Exon 6.) this strain of mice was obtained by the Shanghai-south model Biotech Co Ltd using CRISPR/Cas9 technology. Mice of this genotype are suitable for use in studies with murine anti-human OX40 antibodies.

3) Fcγr humanized OX40 humanized mice (hFCGR ^Tg hOX40 ^Tg Mice): i.e., humanized mice with Fc receptor (murine FcgammaRs knockout (FcgammaRα) ^-/- ) Transfer of human Fc gamma Rs (hFc gamma RI) into mice ⁺ /hFcγRIIA ^R131+ /hFcγRIIB ⁺ /hFcγRIIIA ^F158+ /hFcγRIIIB ⁺ ) (the mouse is already at Nat commun.2019 Sep 27;10 (1) 4206.Doi:10.1038/s41467-019-12097-6. Described) and OX40 humanized mice (hOX 40) ^Tg Mice) and mice of this genotype express both human Fc receptors and human OX40 molecules, and are suitable for use in the study of humanized anti-human OX40 antibodies.

4) Bone marrow chimeric mice: considering propagated hFCGR ^Tg hOX40 ^Tg The number of mice is limited and the genotype is hFCGR for obtaining a large number of mice ^Tg hOX40 ^Tg Mice, a large number of mice of interest are obtained in a short period of time by means of bone marrow reconstruction. The specific method for bone marrow reconstruction is as follows: ordering a certain number of C57BL/6 female mice (Ling or Schlemk, china) with a certain size of 8 weeks, feeding with water containing a certain concentration of penicillin for 1-2 weeks, irradiating the mice with X-ray (RS 2000pro X-ray biological Irradiator (Rad Source Technologies, inc., U.S. A.) at a dose of 8Gy (4Gy+4Gy)) to kill the original bone marrow cells, and injecting 2X 10≡6 mice from hFCGR via tail vein after 3 hours ^Tg hOX40 ^Tg Bone marrow cells of mice. Sampling to obtain small part after bone marrow is rebuilt for 2 months The orbital blood of the mice was anticoagulated with heparin and B cells (CD 19 were detected by flow cytometry ⁺ ) And myeloid cell (CD 11 b) ⁺ ) Surface fcyriib expression (human fcyriib vs mouse fcyriib), and the extent of reconstitution is assessed by human fcyriib expression levels.

5)hOX40 ^Tg OT1 mice: i.e. using OT1 mice and OX40 humanized mice (hOX 40) ^Tg Mice) are obtained by hybridization, and the CD8+ T cells of the mice with the genotype have OVA specificity and simultaneously express human OX40 molecules, thereby being suitable for researching human anti-human OX40 antibodies.

All mice were bred in the Shanghai university of traffic medical college animal science laboratory center, and all animal experiments were conducted in compliance with national laws and regulations of all levels, and were licensed by the Shanghai university of traffic medical college animal care and use committee.

Example 1 mice are immunized to produce monoclonal antibodies against human OX40

The first immunization was performed with a commercial recombinant human OX40 antigen protein (6 His tag, cat No. CK60, supplier Novoprotein) and standard freund's complete adjuvant at a dose of 40 μg of human recombinant OX40 protein antigen per 20 g of mouse body weight. The second to fourth immunizations were performed on 6 week old mice using a proprietary expression vector expressing the full length human OX40 protein (sequence reference NCBI database, accession No. nm_ 003327.2). The fifth immunization was performed with commercial recombinant human OX40 antigen protein (6 His tag, cat No. CK60, supplier Novoprotein) and PBS at a dose of 25 μg of human recombinant OX40 protein antigen per mouse.

Example 2 ELISA detection of serum from immunized mice

Human recombinant OX40 antigen protein (Novoprotein, cat. CK 60) was diluted to 0.5 nanograms per microliter with PBS, 100 microliter per well of antigen was added to a 96 well flat bottom ELISA plate of Maxisorp, and the plate was sealed with a preservative film and left overnight at 4 ℃. After antigen removal on every other day, wash with PBS (200. Mu.L/well), remove PBS, beat dry on absorbent paper and add 200. Mu.L blocking solution (PBS containing 10% fetal bovine serum) to each well and block for 2 hours at room temperature. After the end of the sealing, the sealing liquid is poured offAnd (5) drying the absorbent paper. The serum was subjected to concentration gradient dilution by adding 50. Mu.l of a dilution (PBS containing 5% fetal bovine serum) and added to a 96-well plate, and left at room temperature for 1 hour, after which the sample was decanted, washed 3 times with a wash (PBS containing 0.05% Tween-20), and finally the wash was decanted and then dried on absorbent paper. 50 microliter of diluent (PBS containing 5% fetal bovine serum) was added to dilute HRP goat anti-mouse IgG secondary antibody (final concentration 0.4 microgram/milliliter; manufacturer Biolegend, cat. No. 405306) and left at room temperature for 1 hour, after which the liquid was decanted, washed 5 times (200 microliter/well) with wash (PBS containing 0.05% Tween-20) and finally the wash was decanted and then blotted on absorbent paper. 50 μl/well TMB-hydrogen peroxide urea solution (manufacturer Thermo Scientific) ^TM 34029) substrate solution was left at room temperature in the dark for 3-5 minutes, 50. Mu.l/well of 0.25M sulfuric acid was added to terminate the reaction, and then the absorption of light at 450nm wavelength was measured on a multifunctional microplate reader.

Example 3 monoclonal antibody production by Single cell sequencing

After acemousebatm mice were immunized with His-tagged human OX40 extracellular fragment protein (hOX 40-EC-His, novoprotein, cat.No.CK60, lot.No.0331348), mice spleen, inguinal and popliteal lymph node cells were flow sorted and single cell sequencing was performed on the sorted IgD negative B linear cells (10 x Genomics). The sequences of interest (light chain variable region and paired heavy chain variable region sequences) were selected from a number of sequences based on the characteristics of antigen-specific B cell clonal expansion following immunization and constructed as complete antibody constructs (recombinant antibodies) with constant regions (light chain ck, heavy chain constant region is human IgG2 constant region (JAC 3) with mutations that enhance binding to fcyriib). The antibody sequences were inserted into expression vectors, transferred into suspension 293 cells in cotransformation, the supernatant collected and affinity purified with protein G beads. The purified antibody is subjected to molecular sieve (Size-Exclusion Chromatography, SEC) to remove multimers, and the obtained monomer antibody is placed in a refrigerator at 4 ℃ or minus 20 ℃ for later use.

Example 4 ELISA determination of the ability of Single cell sequencing antibodies to bind to hOX40-EC

1) 100 μl of hOX40-EC-His (Novoprotein) was coated in a 96-well high-adsorption microplate (nunc) at a concentration of 2 μg/ml, and incubated overnight in a four-degree refrigerator;

2) The plate was discarded, the plate was washed 2 times with PBST (PBS with 0.05% Tween 20), 200. Mu.l of 1% BSA (PBS formulation) was added, and the plate was blocked at room temperature for 2 hours;

3) Purified single cell sequencing antibody, negative control antibody Ctrl hIgG and positive control antibody IBI101 (Xinda), pogalizumab (Roche) were diluted appropriately (3.16. Mu.g/ml-1 ng/ml), 100. Mu.l each was taken into the wells after blocking (PBST washed 3 times, liquid was discarded), and incubated at room temperature for 1 hour;

4) PBST plates were washed 4 times, the liquid was patted dry, 100. Mu.l of detection antibody goat anti-human IgG Fc HRP (1:100000, bethy) was added to each well and incubated for 1 hour at room temperature;

5) The plates were washed 5 times with PBST, the liquid was dried by beating, 100. Mu.l of HRP substrate TMB (equal volume mixing of liquid A and liquid B, KPL) was added to each well, and after 10-30 minutes of development, the signal value of OD650 was read with an ELISA reader.

As shown in fig. 1, the anti-human OX40 JAC3 antibody has OX40 binding capacity.

Example 5 in vitro immune activation Activity assay of anti-human OX40 JAC3 antibodies

1) Preparation of hFCGR ^Tg hOX40 ^Tg Mouse spleen leukocyte single cell suspension: taking hFCGR under aseptic conditions ^Tg hOX40 ^Tg Spleen of the mouse is ground, then is lysed on ACK ice for 5 minutes by using 5ml of red blood cell lysate, 9ml of PBS is added to terminate the lysis and clean spleen cells, 400g is centrifuged for 5 minutes, and the spleen cells are resuspended by using 2ml of PBS, thus obtaining spleen single cell suspension. Diluting a small amount of single cell suspension by 1:100, and counting cells by using a modified Newpall counting plate;

2) CFSE marks spleen cells: according to the experimental requirement, a part of cells are taken out, diluted with PBS to make the cell density be (2-4) 10-7/ml, the volume is smaller than 5ml, and CFSE is added to make the final concentration be 5 mu M. Mixing, and incubating in a 37 ℃ incubator for 15min. The cells were then washed 2 times with a wash containing 9ml of 5% FBS, centrifuged at 400g for 5min and the supernatant discarded. Cells were resuspended in 2ml of primary cell culture medium (RPMI+10% FBS+1% Pen/strep+1% HEPES+1% sodium pyruvate+0.1% 2ME (final concentration 50 uM) +1% L-glutamine+1% optional amino acids) and counted;

3) In addition to the controls (untreated, CFSE only, CD3 only, CD3+CD28), anti-mouse CD3e (clone: 145-2C11,BD Pharmingen) containing 0.2. Mu.g/ml was used ^TM ) The CFSE labeled cells were resuspended in primary cell culture medium to a cell density of 2 x 10≡6/ml. 100 μl of the above cells were added to a 96-well cell culture flat bottom plate according to the experimental design so that the number of cells per well was 2 x 10≡5. Untreated groups were supplemented with cells not labeled with CFSE, and no anti-murine CD3 and other antibodies were added. Only CFSE groups were added with CFSE-labeled cells, but no anti-murine CD3 and other antibodies. Only CD3 group with CFSE labeled cells and anti-murine CD3, but no other antibodies. CFSE-labeled cells and anti-murine CD3 were added to the CD3+CD28 group, and anti-murine CD28 antibody (clone 37.51 (RUO), BD Pharmingen) was added at a concentration of 2. Mu.g/ml ^TM ) (final concentrations of anti-murine CD3 and CD28 after isovolumetric dilution in step 4 were 0.1. Mu.g/ml and 1. Mu.g/ml, respectively);

4) Different concentrations of antibodies were formulated: the primary medium was used to gradient dilute the anti-hOX 40 JAC3 antibody, ctrl hIgG (Jackson ImmunoResearch) and the positive control antibody IBI101 (Xinda), pogalizumab (Roche) to a final concentration of 2-fold, and 100. Mu.l each was added to the above-described well plate with anti-mouse CD3 antibody and CFSE labeled spleen cells. 100 μl of medium was added to each control group;

5) Culturing the cells in a 37 ℃ incubator for 3 days;

6) Flow cytometry detects proliferation of T cells: the cultured cells were transferred to a 96-well U-bottom plate, washed with PBS for 2 times, centrifuged at 500g for 5 minutes, and the supernatant was spun off to resuspend the cells in a cell line containing PE anti-mouse CD4 (clone: GK1.5,1:500, BD) and APC anti-mouse CD8a (clone: 53-6.7,1:500, biolegend)In 50. Mu.l FACS buffer (PBS containing 0.5% FBS,2mM EDTA) on ice incubated for 15 min in the absence of light, then the cells were washed twice with PBS buffer, resuspended in a buffer containing DAPI (0.5. Mu.g/ml, invitrogen) and CountBIght ^TM Absolute Counting Beads (Life Technologies, 2. Mu.l/sample) in 200. Mu.l FACS buffer and analyzed by flow cytometry.

As shown in fig. 2 and 3, the anti-human OX40 JAC3 antibodies had in vitro activity, with the anti-human OX40 JAC3 PN024 and PN037 antibodies having superior in vitro activity.

Example 6 in vivo immune activation Activity assay of anti-human OX40 JAC3 antibodies

The OVA-specific cd8+ T cell proliferation model was used to detect the in vivo immune activation activity of anti-human OX40 JAC3 antibodies. The specific process is as follows:

1) Immunization of hFCGR by intraperitoneal injection with 5. Mu.g/DEC-OVA protein alone and a dose of antibody at day0 ^Tg hOX40 ^Tg And (3) a mouse. Grouping and antibody doses were as follows:

group 1 ctrl higg,100 μg/n=4 only

Group 2 ibi101,100 μg/n=5 only

Group 3 pn037,100 μg/n=3 only

Group 4 Pogalizumab,100 μg/n=5 only

Group 5 Pogalizumab,30 μg/n=4 only

Group 6 pn024,100 μg/n=4 only

Group 7 pn024,30 μg/n=5 only

Group 8 pn024,10 μg/n=4 only

2) Mice were sacrificed at day7, spleens of mice were aseptically removed, single cell suspensions were prepared, 1/10 spleen cells were resuspended in 150. Mu.l of medium (RPMI with 10%FBS,1%Pen Strep,10mM HEPES,50. Mu.M 2-mercaptoethanol) containing 1. Mu.g/ml of anti-mouse CD28 antibody and 1. Mu.g/ml of OVA peptide SIINFEKL, and after 1 hour incubation in 96-well flat bottom plates, brefeldin A (BFA) was diluted with the above medium containing 1. Mu.g/ml of anti-mouse CD28 antibody and 1. Mu.g/ml of OVA peptide SIINFEKL, and 50. Mu.l was added to each well to give a final concentration of 10. Mu.g/ml. Placing in a cell incubator at 37 ℃ for continuous culture for 5 hours;

3) After centrifugation (400 g 5 min) of the cultured cells, the supernatant was discarded, washed twice with 200. Mu.l of PBS, and 50. Mu.l of Fixable Viability Stain (BD) diluted with PBS was added to each well, and the cells were stained at room temperature for 15 minutes.

4) Washing twice with PBS, and ice-staining with 50. Mu.l of the DNA containing PE-labeled anti-murine CD4 (clone: GK1.5,1:500, BD) and APC-labeled anti-murine CD 8. Alpha. (clone: 53-6.7,1:500, biolegend) for 15 minutes;

5) Washing twice with PBS, discarding the supernatant, resuspending the cells with 100. Mu.l BD cell fixing/membrane rupture fluid (BD), and placing on ice for 20 minutes;

6) With Perm/Wash ^TM Washing (BD) was performed 2 times, and stained with anti-murine IFN-gamma (clone: XMG1.2 (RUO), 1:200, BD) containing PECy7 markers on ice for 30 minutes in the absence of light;

7) With Perm/Wash ^TM Washing with washing solution (BD) containing CountB right for 2 times ^TM Absolute Counting Beads (Life Technologies, 2. Mu.l/sample) of 200. Mu.l PBS was used to re-suspend the cells and analyzed by flow cytometry.

As shown in FIG. 4, the anti-human OX40 JAC3 PN024 antibody had a greater ability to stimulate IFN-g secretion from OVA-specific CD8+ T cells than Pogalizumab and IBI 101.

Example 7 anti-human OX40 JAC3 antibody has good anti-tumor Activity (MC 38)

MC38 is a mouse colon cancer cell line given hFCGR at day-8 ^Tg hOX40 ^Tg Mice were inoculated subcutaneously with 2 x 10 x 6 MC38 cells, one week later (day-1) with a calipers measuring the long side (L1) and short side (L2) of the tumor, and (L1 x L2)/2 was used to calculate tumor size. Mice were divided into groups according to tumor size ordering, mice in each group were given 63 μg Ctrl hIgG, pogalizumab, anti-human OX40 IAC3 PN024 antibody and PN003 antibody by intraperitoneal injection, respectively, and serum AST levels were measured at day3 and day6 orbital blood collection to measure the hepatotoxicity of the antibodies, and tumor sizes were measured at day-1, 3, 6, 10, 13, 16, 20, 23 to evaluate the anti-tumor activity of the antibodies. As shown in FIG. 5, the anti-human OX40 JAC3 PN024 antibody was better able to achieve both therapeutic effect and toxic and side effects than Pogalizumab.

Example 8 anti-human OX40 JAC3 antibody has good anti-tumor Activity (MO 4)

MO4 was an OVA transfected mouse melanoma cell line given hFCGR at day-10 ^Tg hOX40 ^Tg Mice were inoculated subcutaneously with 2 x 10≡6 MO4 cells, and after 9 days (day-1) the long side (L1) and short side (L2) of the tumor were measured with vernier calipers and the tumor size was calculated using (L1 x L2≡2)/2. Mice were divided into groups according to tumor size order, mice of each group were given 2 μg of DEC-OVA antigen and DEC-OVA solutions of different concentrations of antibodies by intraperitoneal injection at day0, respectively, including Ctrl hIgG (5 mg/kg), pogalizumab (5 mg/kg), IBI101 (5 mg/kg), anti-human OX40 JAC3 PN024 antibody (5 mg/kg and 1.5 mg/kg) and anti-human OX40 JAC3 PN037 antibody (5 mg/kg), and serum AST levels were measured by intraperitoneal injection at day4 and day7 eyesockets to measure the hepatotoxicity of the antibodies, and tumor sizes were measured at day-1, 4, 7, 11, 14, 18, 21 to evaluate the anti-tumor activity of the antibodies. As shown in fig. 6, the anti-human OX40 JAC3 PN024 antibody had better anti-tumor activity than Pogalizumab. As shown in fig. 7, the anti-human OX40 JAC3 PN024 antibody was significantly less hepatotoxic (serum AST levels) than ponglizumab. Therefore, compared with Pogalizumab and IBI101, the anti-human OX40 JAC3 PN024 antibody can better achieve both curative effect and toxic and side effects.

Example 9 obtaining hybridoma cells from immunized mouse spleen cells Using electrofusion technique

Resuscitates SP2/0 mouse myeloma cells in advance; taking SP2/0 mouse myeloma cell count on the fusion day; the spleen of the OX40 mice after successful immunization was placed in transport medium HB medium (containing 1% p/S) and immediately the cell count in the spleen was extracted and isolated for use; take 1.5x10 ⁸ Spleen cells and 1.5X10 ⁸ Mixing ratio of SP2/0 mouse myeloma; centrifuging at 200G for 5 minutes after mixing, discarding the supernatant, and washing twice with 20 ml of fusion solution for standby; centrifuging at 200G at room temperature for 5 minutes, discarding the supernatant and adding 6.4 ml of electrofusion buffer to resuspend the precipitate; a 50 ml centrifuge tube was prepared and 13 ml of pre-heated HB medium was added; adding the mixed cell suspension to CUY497X which has been sterilized with 75% alcohol and air-dried ₁₀ The electrodes were combined using an ECFG21 electrofusion machine (manufacturer NEPAGENE,model ECFG 21) for cell electrofusion; after fusion, the cells were aspirated and placed in 13 ml HB media pre-warmed in an incubator; the cells were resuspended in HAT medium according to 5X 10 ⁵ Cell/well plating 96-well flat bottom plate; the 96-well plate was placed in an incubator at 37℃for stationary culture, cells were observed every day, and the supernatant was subjected to ELISA primary screening on day 10.

Example 10 selection of hybridoma anti-human OX40 monoclonal antibodies Using an ELISA reaction

OX40 antigen (Novoprotein cat: CK 60) was diluted to 0.5 nanograms/microliter with PBS; 100 microliters/well antigen was added to a 96-well flat-bottom ELISA plate of Maxisorp, and the plate was sealed with a preservative film and left at 4℃overnight; after antigen is removed every other day, PBS (200 microliters/hole) is used for washing once, after PBS is removed, the mixture is dried by beating on absorbent paper, and 200 microliters of sealing liquid is added into each hole and is placed at room temperature for sealing for 2 hours; pouring off the sealing liquid after sealing, and then beating the sealing liquid on the absorbent paper; 50. Mu.l of the sample (OX 40 hybridoma cell culture supernatant obtained in example 9) was added, left at room temperature for 1 hour, after which the sample was poured off, washed 3 times with a washing liquid, and finally poured off and then patted dry on a piece of absorbent paper; adding 50 microliters of diluent (PBS containing 5% fetal bovine serum) to dilute HRP goat anti-mouse IgG secondary antibody, standing at room temperature for 1 hour, pouring off the liquid, washing with washing liquid for 5 times (200 microliters/hole), and finally pouring off the washing liquid and then beating on water-absorbing paper to dry; the reaction was terminated by adding 50. Mu.l/well TMB-hydrogen peroxide urea solution substrate solution, standing at room temperature in the dark for 3-5 minutes, adding 50. Mu.l/well 0.25M sulfuric acid, and then detecting the light absorption at 450nm wavelength on a multifunctional microplate reader. The results are shown in the following table:

Example 11 detection of anti-human OX40 antibody binding to OX40 overexpressing cell surface OX40 assay Using flow cytometry

After 200G centrifugation to collect OX40 overexpressing cells, the cells were washed once with 3% FCS in PBS and resuspended in 2.5 ml 3% FCS in PBS and plated in 96-well plates, 25 wells eachMicroliter of cells (2.5X10) ⁵ ) 75 microliters of each sample (OX 40 hybridoma cell culture supernatant obtained in example 9) was added, and a positive reference well was set with 75 microliters of anti-OX 40 antibody (clone ACT35, manufacturer Biolegend final concentration 1 μg/ml); female ginseng well 1 was added with 75. Mu.l IgG2a isotype control (clone MG2a-53, manufacturer Biolegend, final concentration 1. Mu.g/ml); the female ginseng hole 2 is added with 75 microliters of PBS containing 3% FCS, incubated for 1 hour at 4 ℃ in a refrigerator, and then washed twice with PBS containing 3% FCS; after decanting the supernatant, 50 μl of 500 ng/ml secondary antibody (manufacturer Ebioscience, sheep anti-mouse IgG-PE, cat# 12-4010-82) was added to sample wells, yang Cankong, wenyujin 1 and Wenyujin 2; the plates were then incubated at 4℃in the absence of light for 30 minutes, washed twice with 3% FCS in PBS, and the cells were finally resuspended in 50. Mu.l of 3% FCS in PBS and examined by flow cytometry. The results are shown in the following table and in FIGS. 8A-C.

Clone name	28E3-4	21F3-11	29B8-3	24F10-1	55H6-6
Y586 channel PE fluorescence value	195411	170723	388242	274205	479073
Clone name	24E10-4	30B1-3	15A7-6	22D6-3	23D1-8
Y586 channel PE fluorescence value	343778	208478	180959	461976	166107
Clone name	24B9-10	24A12-12	29A9-5	26G9-9	35B8-6
Y586 channel PE fluorescence value	313454	343068	220618	221755	243286
Clone name	38D9-12	4D9-12	48D10-5	18F11-4	18F5-2
Y586 channel PE fluorescence value	243343	186812	358123	402043	431911
Clone name	26C3-4	25F4-10	25F4-7	16G2-10	22E6-11
Y586 channel PE fluorescence value	416371	612396	616613	475068	549525
Clone name	17D5-11	27C8-12	21F9-12	23C4-1-5	16E4-10-5
Y586 channel PE fluorescence value	475068	400528	423614	63905	376890
Clone name	1F10-3-1	14D3-7-3	1F10-12-3	20G6-7-1	19C12-9-6
Y586 channel PE fluorescence value	172510	273719	169911	351786	315714
Clone name	20D2-12	24F8-7	21D2-3	anti-OX 40Ab	IgG2a
Y586 channel PE fluorescence value	540481	437030	201913	209583	1828
Clone name	PBS
Y586 channel PE fluorescence value	1388

Example 12 ELISA detection of the ability of anti-hOX40 murine antibodies in hybridoma supernatants to bind hOX40-EC

1) 100 μl of hOX40-EC-His (Novoprotein) was coated in a 96-well high-adsorption microplate (nunc) at a concentration of 1 μg/ml, and incubated overnight in a four-degree refrigerator;

2) The plate was discarded, the plate was washed 2 times with PBST (PBS containing 0.05% Tween 20), 200. Mu.l of 1% BSA (PBS formulation) was added, and the plate was blocked at room temperature for 2 hours;

3) The hybridoma supernatants were serially diluted (1:10/1:100/1:1000/1:10000), 100. Mu.l each was taken into the above-mentioned blocked wells (PBST washed 2 times, liquid discarded), and incubated for 1 hour at room temperature;

4) PBST plates were washed 4 times, the liquid was patted dry, 100. Mu.l of detection antibody goat anti-mouse IgG Fc HRP (1:5000,Jackson ImmunoResearch) was added to each well and incubated for 1 hour at room temperature;

5) Washing the PBST plate for 5 times, beating the liquid, adding 100 mu l of HRP substrate TMB (equal volume mixing of liquid A and liquid B, KPL) into each hole, and reading the OD650 signal value by an enzyme-labeling instrument after a period of time;

6) And selecting signal values of the samples at the dilution of 1:100 for result statistics.

The results are shown in the following table and fig. 9, demonstrating that hybridoma antibody clones have the ability to bind OX 40.

Example 13 construction of antibodies Using the variable regions of hybridoma antibodies and determination of the ability of the antibodies to bind to hOX40-EC

The hybridoma antibodies obtained according to examples 10-12 obtained their light and heavy chain variable region sequences and constructed them as complete antibody structures (recombinant antibodies) with constant regions (light chain ck, heavy chain constant region is human IgG2 constant region (JAC 3) with mutations that enhance binding to fcyriib). The antibody sequences were inserted into expression vectors, transferred into suspension 293 cells in cotransformation, the supernatant collected and affinity purified with protein G beads. The purified antibody is subjected to molecular sieve (Size-Exclusion Chromatography, SEC) to remove multimers, and the obtained monomer antibody is placed in a refrigerator at 4 ℃ or minus 20 ℃ for later use. The ability of the antibodies to bind hOX40-EC was determined by ELISA as described in example 4, and the results indicated that the reconstituted hybridoma anti-human OX40 JAC3 antibodies had OX40 binding ability.

Example 14 ability of anti-human OX40 antibodies to bind to hOX40 to stably transduce OX40 on 293T cells

1) hOX40 stably transformed 293T cells constitutively expressed human OX40 extracellular portion protein. Mu.l of hOX40 stably transformed 293T cells or 293T cells not expressing OX40 (PBS formulated) were added to 96-well U-plates according to the experimental design such that the number of cells per well was 1X 10≡6, 400g was centrifuged for 5min, and the supernatant was discarded. 2) Dilution of antibodies: anti-human OX40 JAC3 antibody, ctrl igg (Jackson ImmunoResearch) and positive control antibody IBI101 (belief), pogalizumab (roche) and anti-murine OX40 antibody OX86 (igg 1) were diluted to 1 μg/ml with PBS.

3) The antibody was aspirated at 50. Mu.l each, the cells were resuspended and incubated on ice for 15 min.

4) Wash 2 times with PBS, centrifuge 400g for 5min, discard supernatant.

5) Binding of flow cytometry detection reporter cells: cells were resuspended in 50. Mu.l FACS buffer (PBS containing 0.5% FBS,2mM EDTA) containing anti-human IgG Fab-PE (clone: M1310G05,1:200, bioLegend), incubated for 15 min on ice in the dark, then washed twice with PBS buffer, resuspended in 200. Mu.l FACS buffer containing DAPI (0.5. Mu.g/ml, invitrogen), and analyzed by flow cytometry.

As shown in fig. 11, the anti-human OX40 antibody has the ability to bind OX40 on hOX40 stably transformed 293T cells).

Example 15 ELISA determination of the ability of anti-human OX40 antibodies to bind human, murine, monkey OX40-EC protein

1) 100. Mu.l of hOX40-EC-His (Novoprotein, CK 60), mouse OX40-EC (CK 52), rhesus OX40-EC (sinobioglogic, 90846-C08H), cynomomolgus OX40-EC (Novoprotein, CB 17) were each coated in 96-well high-adsorptivity assay plates (nunc) at a concentration of 2. Mu.g/ml, and incubated overnight in a four-degree refrigerator;

2) The plate was discarded, the plate was washed 2 times with PBST (PBS with 0.05% Tween 20), 200. Mu.l of 1% BSA (PBS formulation) was added, and the plate was blocked at room temperature for 2 hours;

3) Purified anti-human OX40 antibody, negative control antibody Ctrl hIgG and positive control antibody IBI101 (belief), pogalizumab (rogowski) were diluted appropriately (3.16 μg/ml), 100 μl each was taken into the above-mentioned blocked wells (PBST washed 3 times, liquid was discarded), and incubated for 1 hour at room temperature;

4) PBST plates were washed 4 times, the liquid was patted dry, 100. Mu.l of detection antibody goat anti-human IgG Fc HRP (1:50000, bethy, A80-104 p) was added to each well and incubated for 1 hour at room temperature;

5) The plates were washed 5 times with PBST, the liquid was dried by beating, 100. Mu.l of HRP substrate TMB (equal volume mixing of liquid A and liquid B, KPL) was added to each well, and after 10-30 minutes of development, the signal value of OD650 was read with an ELISA reader.

As shown in fig. 12, recombinant anti-human OX40 antibodies have the ability to specifically bind to human and monkey OX40 proteins, typically not to mouse OX40.

Example 16 ELISA determination of the ability of anti-human OX40 antibodies to competitively bind to hOX40-EC

1) 100. Mu.l of hOX40-EC-His (Novoprotein, CK 60) was coated in 96-well high-adsorptivity microplate (nunc) at a concentration of 2. Mu.g/ml, and incubated overnight in a four-degree refrigerator;

2) The plate was discarded, the plate was washed 2 times with PBST (PBS with 0.05% Tween 20), 200. Mu.l of 1% BSA (PBS formulation) was added, and the plate was blocked at room temperature for 2 hours;

3) Purified anti-human OX40 antibody, negative control antibody Ctrl hIgG and positive control antibody IBI101 (Xinda), pogalizumab (Roche), human OX40-L-6His (Novoprotein, CJ 45) were diluted to 2. Mu.g/ml with a solution containing 0.2. Mu.g/ml human OX40-L-mFc (Novoprotein, CW 25) (antibody to ligand ratio 10:1), 100. Mu.l each were taken into the wells after blocking (PBST wash 3 times, discard the liquid), incubated at room temperature for 1 hour;

4) PBST plates were washed 4 times, the liquid was patted dry, 100. Mu.l of detection antibody goat anti-mouse IgG1 HRP (1:5000,Jackson ImmunoResearch Laboratories,115-035-205) was added to each well and incubated for 1 hour at room temperature;

5) The plates were washed 5 times with PBST, the liquid was dried by beating, 100. Mu.l of HRP substrate TMB (equal volume mixing of liquid A and liquid B, KPL) was added to each well, and after 10-30 minutes of development, the signal value of OD650 was read with an ELISA reader.

As shown in fig. 13, the partially anti-human OX40 antibodies have the ability to compete for binding epitopes of the ligand.

Example 17 in vivo immune activation Activity assay of anti-human OX40 antibodies

The OVA-specific cd8+ T cell proliferation model was used to detect the in vivo immune activation activity of anti-human OX40 JAC3 antibodies. The specific process is as follows:

1) hFCGR at day-1 ^Tg hOX40 ^Tg Mice were injected via tail vein with OT-IhOX40 ^Tg Spleen cells of mice (2X 10≡6/mouse).

2) Immunization of hFCGR by intraperitoneal injection with 5. Mu.g/DEC-OVA protein alone and a dose of anti-human OX40 antibody and Ctrl hIgG (Jackson ImmunoResearch) at day0 ^Tg hOX40 ^Tg And (3) a mouse. Grouping and antibody doses were as follows:

set 1 ctrl higg,50 μg/n=5 only

Group 2 pn024 pre-optimization (heavy chain constant region is human IgG constant region as shown in SEQ ID NO: 345), 50 μg/n=5 only

Group 3 pn024,50 μg/n=5 only

3) Mice were sacrificed at day6, spleens of the mice were taken under aseptic conditions, and after grinding, the spleens were lysed on ice with 5ml of red blood cell lysate ACK for 5 minutes, the lysis was terminated by adding 9ml of PBS and washing the spleen cells, centrifugation was performed for 5 minutes at 400g, and the spleen cells were resuspended with 2ml of PBS to obtain a spleen single cell suspension.

4) Flow cytometry detects proliferation of T cells: 200 μl of the above spleen cells were transferred to 96-well U bottom plate, centrifuged at 500g for 5min, the supernatant was removed, the cells were resuspended in 50 μl of FACS buffer (PBS containing 0.5% FBS,2mM EDTA) containing PE anti-mouse CD4 (clone: GK1.5,1:500, BD) and APC anti-mouse CD8a (clone: 53-6.7,1:500, biolegend), the cells were washed twice with PBS buffer and resuspended in PBS containing 0.5% FBS,2mM EDTA Containing DAPI (0.5. Mu.g/ml, invitrogen) and CountBIght ^TM Absolute Counting Beads (Life Technologies, 2. Mu.l/sample) in 200. Mu.l FACS buffer and analyzed by flow cytometry.

As shown in fig. 15, the optimized recombinant anti-human OX40 antibodies significantly enhanced immune activation activity over the optimized precursors.

Claims (10)

1. An anti-OX 40 antibody or antigen-binding fragment thereof, wherein said anti-OX 40 antibody comprises

   (1) HCDR1 as shown in SEQ ID NO. 1, HCDR2 as shown in SEQ ID NO. 2 and HCDR3 as shown in SEQ ID NO. 3, and/or LCDR1 as shown in SEQ ID NO. 4, LCDR2 as shown in SEQ ID NO. 5 and LCDR3 as shown in SEQ ID NO. 6, and

   (2) A heavy chain constant region comprising a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain connected in sequence from N-terminus to C-terminus, wherein the sequences of the CH1 domain and the hinge region are those derived from a CH1 domain and a hinge region of human IgG2, the sequences of the CH2 domain and the CH3 domain are those derived from a CH2 domain and a CH3 domain of human IgG, and the affinity of the antibody heavy chain constant region to human fcyriib is equal to or higher than the affinity of human IgG1 to human fcyriib, and the I/a ratio of the antibody heavy chain constant region is equal to or higher than the I/a ratio of human IgG 1.
2. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 1, wherein the anti-OX 40 antibody comprises HCDR1 as set forth in any one of SEQ ID NOs 7-38, HCDR2 as set forth in any one of SEQ ID NOs 39-65, and HCDR3 as set forth in any one of SEQ ID NOs 66-114, and/or LCDR1 as set forth in any one of SEQ ID NOs 115-145, LCDR2 as set forth in any one of SEQ ID NOs 146-159, and LCDR3 as set forth in any one of SEQ ID NOs 160-199.
3. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 1, wherein the anti-OX 40 antibody comprises HCDR1, HCDR2 and HCDR3 as set forth in any one of groups a1 through a71 of table 1, and/or LCDR1, LCDR2 and LCDR3 as set forth in any one of groups b1 through b71 of table 2.
4. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 1, wherein the anti-OX 40 antibody comprises an HCDR and an LCDR as set forth in any one of groups c 1-c 71 of table 3.
5. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein each FR region of the anti-OX 40 antibody VH is an FR region selected from any one of the VH's set forth in SEQ ID nos. 200-270, and each FR region of the VL is an FR region selected from any one of the VL's set forth in SEQ ID nos. 271-341; more preferably, each FR region of the anti-OX 40 antibody VH and VL is an FR region selected from VH and VL of any one of the antibodies in table 4.
6. The anti-OX 40 antibody or antigen-binding fragment thereof of claim 1, wherein the VH of the anti-OX 40 antibody has an amino acid sequence as set forth in any one of SEQ ID NOs 200-270 and/or the VL has an amino acid sequence as set forth in any one of SEQ ID NOs 271-341; preferably, the amino acid sequence of the VH and the amino acid sequence of the VL of the anti-OX 40 antibody are as shown in any of the antibody numbers of table 4, or

   The anti-OX 40 antibody is a chimeric antibody or a fully human antibody; preferably fully human antibodies.
7. The anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-6, wherein the sequence of the CH2 domain and the CH3 domain of the heavy chain constant region is selected from the group consisting of:

   a) Sequences derived from the CH2 domain and CH3 domain of human IgG1, and the CH2 domain and CH3 domain comprise the G237D, P238D, P271G and a330R mutations; or alternatively

   b) Sequences derived from the CH2 domain and CH3 domain of human IgG1, and the CH2 domain and CH3 domain comprise the G237D, P238D, H268D, P271G and a330R mutations; or alternatively

   c) Sequences derived from the CH2 domain and the CH3 domain of human IgG2, and the CH2 domain and the CH3 domain comprise the S267E and L328F mutations; or alternatively

   d) Sequences derived from the CH2 domain and CH3 domain of human IgG2, and said CH2 domain and CH3 domain comprising the H268D and P271G mutations,

   preferably, the amino acid sequence of the heavy chain constant region of the anti-OX 40 antibody has a sequence as set forth in any one of SEQ ID NOS: 348-351 and/or the amino acid sequence of the light chain constant region has a sequence as set forth in SEQ ID NO: 347.
8. A pharmaceutical composition comprising an anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-7, and a pharmaceutically acceptable excipient or carrier.
9. A nucleic acid molecule or a vector comprising the nucleic acid molecule selected from the group consisting of:

   (1) A polynucleotide sequence encoding an anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-7;

   (2) The complement of the polynucleotide sequence of (1).
10. Use of an anti-OX 40 antibody or antigen-binding fragment thereof of any one of claims 1-7 in the manufacture of a medicament for the treatment of a T cell-related disorder; preferably, the T cell-related disease is a T cell-related tumor or OX 40-mediated disease; more preferably, the OX40 mediated disease comprises OX40 mediated allergy, asthma, COPD, rheumatoid arthritis, psoriasis, autoimmune diseases and inflammation related diseases.

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