TW202128131A - Recombinant anti-programmed cell death protein 1 and anti-cluster of differentiation antigen 137 bispecific antibody preparation and use thereof - Google Patents

Abstract

Disclosed are a recombinant anti-programmed cell death protein 1 (PD-1), an anti-cluster of differentiation antigen 137 (CD137) bispecific antibody preparation and a use thereof.

Description

Recombinant anti-programmed cell death receptor 1 and anti-differentiation antigen cluster 137 bispecific antibody preparation and use thereof

The present invention belongs to the field of biotechnology, and relates to recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody preparations and uses thereof.

Immune checkpoints are a set of membrane proteins expressed on immune cells (such as T cells and dendritic cells), including a variety of co-suppressive and co-stimulatory receptors, which play an important role in regulating the adaptive immune response. The carefully studied checkpoints include PD-1 and CD137. The interaction between PD-1 and its ligands, programmed cell death ligand 1 (PD-L1) and programmed cell death ligand 2 (PD-L2) provides an inhibitory message, which has been shown to be in tumors Immune escape and play an important role in the immunosuppression that occurs in the tumor microenvironment.

Although anti-PD-1 antibody and/or anti-PD-L1 antibody can block PD-1 inhibition message has been clinically verified, and significant clinical progress has been made in the treatment of certain cancers, there are still many patients No response to this, relapse, acquired resistance to PD-1 or PD-L1 antibody therapy, or intolerance to the treatment. CD137, also known as 4-1BB, plays a role in the immune response driven by activated T cells, for example by promoting T cell proliferation and effector functions, enhancing immune memory and inhibiting activation-induced cell death.

Agonistic antibodies that target CD137 have shown promise as monotherapy and combination therapy in murine tumor models. However, due to toxicity and/or lack of efficacy, agonists that target human CD137 are either used as single agents in human cancer patients. Neither drug therapy nor combination therapy showed sufficient response. In fact, no agonistic antibody targeting human CD137 has been approved for human therapeutic use. Therefore, other treatments for the immune checkpoint approach are needed.

Combinations of antibodies that agonize CD137 and antagonize PD-1 have been studied, such as the combination of urelumab (anti-CD137 agonist monoclonal antibody) and nivolumab (anti-PD-1 antagonist monoclonal antibody), which are used in clinical trials for treatment Solid tumors (Tolcher et al., Clin Cancer Res 23 (18) 2017). However, higher potentially effective doses of urelumab have been shown to be associated with elevated transaminitis and other adverse events. Targeting CD137 agonist antibodies specifically targeting those cells expressing PD-1 may limit the adverse events associated with systemic administration of agonistic CD137 antibodies. Therefore, it is necessary to design the bispecific antibody of the present invention to provide an immune-enhancing effect by preferentially binding to cells expressing PD-1, and potentially limit the impact of CD137 agonism on those cells also expressing PD-1.

WO2018/045110 discloses several bispecific antibodies (Fab-scFv-Fc format) that bind to co-inhibitory receptors and co-stimulatory receptors to activate T cells to treat cancer, including [ICOS x PD-1] and [ CD137 x PD-1]. It seems that the CD137 Fab disclosed in WO2018/045110 is derived from BMS20H4.9, which may be related to the development of severe transaminase elevation (Segal et al., Clinical Cancer Research (2016) 1-8). Compared with the bispecific antibodies disclosed in WO2018/045110, IgG-like bispecific antibodies have many advantageous properties related to natural IgG antibodies, such as high stability, long serum half-life and low immunogenicity (Ha et al., Frontiers in Immunology (2016) Article 394). Due to the known toxicity associated with BMS20H4.9 and due to the undesirable structural form of the bispecific antibody described in WO2018/045110, additional bispecific antibodies are needed, which antagonize human PD-1 and agonize CD137 IgG-like bispecific antibodies that promote a powerful anti-cancer immune response and show an acceptable toxicity profile.

At the same time, there is still a need in the art for new pharmaceutical formulations containing double antibodies that antagonize human PD-1 and agonize CD137 that are sufficiently stable and suitable for administration to human subjects. In addition, for such antibody preparations, it is also advantageous to find the simplicity and ease of use of the preparation formulation.

Summary of the invention

In response to the above-mentioned bispecific antibody and corresponding stable antibody preparation needs, the present invention has developed a recombinant anti-programmed cell death receptor 1 (PD-1) (SEQ ID NO: 25) and anti-differentiation antigen cluster 137 (CD137) (SEQ ID NO: 26) bispecific antibody (code IBI319) and stable preparations containing said antibody or functional fragments thereof.

In one aspect, the present invention relates to a liquid antibody preparation comprising a recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof and a buffer, preferably The pH value of the formulation is about 5.0-7.0; more preferably, the pH value of the formulation is about 5.5-6.0; further preferably, the pH value is about 5.7.

In some schemes, the content of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof in the liquid antibody preparation is about 1 mg /mL-150 mg/mL, preferably about 10 mg/mL-100 mg/mL, for example, about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 mg /mL, more preferably about 50.0 mg/ml.

In some aspects, the buffer is selected from histidine, histidine hydrochloride, or a combination thereof, or the buffer is selected from citrate, citrate solvate (e.g., citrate Hydrate) or a combination thereof, for example, sodium citrate, sodium citrate dihydrate or a combination thereof, or the buffer is selected from acetate, acetate solvate (for example, acetate hydrate) or The combination thereof, or the buffer is selected from phosphate, phosphate solvate (for example, phosphate hydrate) or a combination thereof; preferably, the concentration of the buffer is about 5-100 mM, more preferably The ground is about 5-60 mM, for example, about 5, 10, 15, 20, 25, 30, 40, 50, 60 mM; Preferably, the buffering agent is histidine, and the content of histidine is about 0.775 mg/mL-15.5 mg/mL; more preferably, the content of the histidine is about 0.775 mg/mL-9.3 mg/mL, for example about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 mg/mL; further preferably, the group The content of amino acid is about 1.55 mg/ml; Preferably, the buffer is a combination of histidine and histidine hydrochloride, wherein the content of the histidine is about 0.270 mg/mL-5.4 mg/mL, and the content of the histidine hydrochloride is about 0.660 mg/mL-13.33 mg/mL; more preferably, the histidine content is about 0.270 mg/mL-3.24 mg/mL, for example about 0.5, 1.0, 1.5, 2, 2.5, 3 mg/mL, so The content of histidine hydrochloride is about 0.660 mg/mL-8 mg/mL, such as about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 mg/mL; more preferably, the content of the histidine acid is about 0.57 mg/ml, and the content of the histidine hydrochloride is about 1.33 mg/ml.

In some aspects, the formulation further includes a stabilizer; preferably, the formulation further includes a stabilizer, and the stabilizer includes a polyhydric alcohol and/or an amino acid; the polyhydric alcohol is selected from sorbitol, mannitol, Sucrose, trehalose, maltose, and combinations thereof, the amino acid includes arginine, arginine hydrochloride, methionine, glycine, proline or a combination thereof; preferably, the stabilizer is selected from sorbus Alcohol and/or arginine hydrochloride, sorbitol and/or arginine, more preferably, the stabilizer is a combination of sorbitol and arginine hydrochloride, or a combination of sorbitol and arginine, preferably, the spermine The content of arginine hydrochloride or arginine hydrochloride is about 20 mM-200 mM. More preferably, the content of arginine hydrochloride or arginine hydrochloride is about 40 mM-150 mM, such as about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 mM; more preferably, the content of arginine or arginine hydrochloride is about 85 mM; Preferably, the content of the sorbitol is about 10 mg/mL-100 mg/mL, and the content of the arginine hydrochloride is about 4 mg/mL-42.0 mg/mL; further preferably, the content of the sorbitol The content is about 20 mg/mL-60 mg/mL, such as about 30, 40, 50 mg/mL, and the content of arginine hydrochloride is about 8.4 mg/mL-33.7 mg/mL, such as about 15, 20, 25. 30 mg/mL; more preferably, the content of the sorbitol is about 25.00 mg/ml, and the content of the arginine hydrochloride is about 16.85 mg/ml.

In some aspects, the formulation further includes a surfactant; Preferably, the surfactant is selected from non-ionic surfactants, such as one or more of polysorbate 80, polysorbate 20, poloxamer and polyethylene glycol polysorbate 80, more preferably polysorbate Sorbate 80; More preferably, the content of the surfactant is about 0.1 mg/mL-1 mg/mL; further preferably about 0.3 mg/mL-0.7 mg/mL, such as 0.3, 0.4, 0.5, 0.6, 0.7 mg/mL ; It is further preferably about 0.50 mg/ml.

In some aspects, the formulation further includes a chelating agent; Preferably, the chelating agent is selected from edetate disodium, diethyltriaminepentaacetic acid and/or EDTA, more preferably edetate disodium; More preferably, the content of the chelating agent is about 0.005 mg/mL-0.1 mg/mL; further preferably about 0.01 mg/mL-0.05 mg/mL, such as about 0.02, 0.03, 0.04 mg/mL; further preferably It is about 0.01 mg/ml.

In some aspects, a liquid antibody preparation is provided, which contains the following components: about 50.0 mg/ml recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific Antibody, about 0.57 mg/ml histidine, about 1.33 mg/ml histidine hydrochloride, about 25.00 mg/ml sorbitol, about 16.85 mg/ml arginine hydrochloride, about 0.01 mg/ml disodium edetate , About 0.50 mg/ml polysorbate 80, pH about 5.7, the balance is water.

In some aspects, in the liquid antibody preparation, The antibody or antigen-binding fragment thereof includes a first heavy chain (HCl), which includes a first heavy chain variable region (HCVR1) and a constant region; a first light chain (LC1), which includes a first light chain variable region (LCVR1) and the constant region; the second heavy chain (HC2), which includes the second heavy chain variable region (HCVR2) and the constant region; and the second light chain (LC2), which includes the second light chain variable region ( LCVR2) and constant region, where: The HCVR1 includes the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 4, and the LCVR1 includes the light chain variable region shown in SEQ ID NO: 10 LCDR1, LCDR2 and LCDR3 included; and The HCVR2 includes the three complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 16, and the LCVR2 includes the light chain shown in SEQ ID NO: 22 The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the variable region; preferably a) The first heavy chain of the antibody comprises: complementarity determining region 1 (HCDR1) with the amino acid sequence of SEQ ID NO: 1, complementarity determining region 2 (HCDR2) with the amino acid sequence of SEQ ID NO: 2, and having an amino group Complementarity determining region 3 (HCDR3) of the acid sequence SEQ ID NO: 3; b) The first light chain of the antibody comprises: complementarity determining region 1 (LCDR1) with amino acid sequence SEQ ID NO: 7, complementarity determining region 2 (LCDR2) with amino acid sequence SEQ ID NO: 8, and amino acid sequence Complementarity determining region 3 (LCDR3) of the acid sequence SEQ ID NO: 9; c) The second heavy chain of the antibody comprises: complementarity determining region 1 (HCDR1) with the amino acid sequence of SEQ ID NO: 13, and complementarity determining region 2 (HCDR2) with the amino acid sequence of SEQ ID NO: 14, and having an amino group Complementarity determining region 3 (HCDR3) of the acid sequence of SEQ ID NO: 15; d) The second light chain of the antibody comprises: complementarity determining region 1 (LCDR1) with amino acid sequence SEQ ID NO: 19, complementarity determining region 2 (LCDR2) with amino acid sequence SEQ ID NO: 20, and amino acid sequence The complementarity determining region 3 (LCDR3) of the acid sequence SEQ ID NO: 21.

In some aspects, the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody comprises: a first heavy chain, a second heavy chain, and a first light chain. Chain and second light chain, where each chain contains a variable region and a constant region, where: e) The antibody first heavy chain variable region (HCVR) comprises the sequence of SEQ ID NO: 4 or a sequence that has at least 90% 95%, 98% or 99% homology with it; f) The antibody first light chain variable region (LCVR) comprises the sequence of SEQ ID NO: 10 or a sequence that has at least 90%, 95%, 98% or 99% homology with it; g) The antibody second heavy chain variable region (HCVR) comprises the sequence of SEQ ID NO: 16 or a sequence that has at least 90%, 95%, 98% or 99% homology with it; h) The antibody second light chain variable region (LCVR) comprises the sequence of SEQ ID NO: 22 or a sequence with at least 90%, 95%, 98% or 99% homology with it; Preferably, e) The antibody first heavy chain variable region (HCVR) has an amino acid sequence of SEQ ID NO: 4; f) The antibody first light chain variable region (LCVR) has an amino acid sequence of SEQ ID NO: 10; g) The antibody second heavy chain variable region (HCVR) has an amino acid sequence of SEQ ID NO: 16; h) The antibody second light chain variable region (LCVR) has an amino acid sequence of SEQ ID NO:22.

In some aspects, the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody comprises: a first heavy chain, a second heavy chain, and a first light chain. Chain and a second light chain, wherein each chain comprises a variable region and a constant region, wherein: the first heavy chain constant region sequence (HCCR) has an amino acid sequence of SEQ ID NO: 30, and the first light chain constant region sequence ( LCCR) has an amino acid sequence of SEQ ID NO: 31, the second heavy chain constant region (HCCR) sequence has an amino acid sequence of SEQ ID NO: 32, and the second light chain constant region (LCCR) sequence has an amino acid sequence of SEQ ID NO: 33.

In some aspects, the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody comprises: a first heavy chain, a second heavy chain, and a first light chain. Chain and second light chain, where: i) The first heavy chain of the antibody comprises the sequence of SEQ ID NO: 5 or a sequence that has at least 90%, 95%, 98% or 99% homology with it; j) The first light chain of the antibody comprises the sequence of SEQ ID NO: 11 or a sequence that is at least 90%, 95%, 98% or 99% homologous to it; k) The second heavy chain of the antibody comprises the sequence of SEQ ID NO: 17 or a sequence that is at least 90%, 95%, 98% or 99% homologous to it; and 1) The second light chain of the antibody comprises the sequence of SEQ ID NO: 23 or a sequence that has at least 90%, 95%, 98% or 99% homology with it; Preferably, i) The first heavy chain of the antibody has an amino acid sequence of SEQ ID NO: 5; j) The first light chain of the antibody has an amino acid sequence of SEQ ID NO: 11; k) The second heavy chain of the antibody has an amino acid sequence of SEQ ID NO: 17; and 1) The second light chain of the antibody has an amino acid sequence of SEQ ID NO:23.

In some aspects, the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody comprises: a first heavy chain, a second heavy chain, and a first light chain. Chain and second light chain, wherein at least one disulfide bond is formed between the first heavy chain and the first light chain, at least one disulfide bond is formed between the second heavy chain and the second light chain, and the first heavy chain At least one disulfide bond is formed between the second heavy chain; preferably the bispecific antibody is a modified human IgG1.

In certain aspects, the present invention provides a liquid antibody preparation comprising: (i) The recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof of about 1 mg/mL-150 mg/mL; (ii) Histidine of about 5-100 mM; (iii) about 10 mg/mL-100 mg/mL sorbitol and about 20 mM-200 mM arginine or arginine hydrochloride; (iv) about 0.1 mg/mL-1 mg/mL polysorbate 80; and (v) Optionally, about 0.005 mg/mL-0.1 mg/mL disodium edetate, Wherein the pH of the liquid formulation is about 5.0-7.0, for example, about 5.5, 6.0, 6.5; For example, the liquid antibody preparation comprises (i) About 10 mg/mL-100 mg/mL of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof; (ii) Histidine of about 5-60 mM; (iii) about 20 mg/mL-60 mg/mL sorbitol and about 8.4 mg/mL-33.7 mg/mL arginine hydrochloride; (iv) about 0.3 mg/mL-0.7 mg/mL polysorbate 80; and (v) Optionally, about 0.01 mg/mL-0.05 mg/mL disodium edetate, Wherein the pH of the liquid formulation is about 5.5-6.0, for example, about 5.7; Alternatively, the liquid antibody preparation comprises (i) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 1.55 mg/ml histidine, 50.00 mg/ml sorbitol, 0.50 mg/ml polysorbate 80, pH 5.7; or (ii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 1.55 mg/ml histidine, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 1.55 mg/ml histidine, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iv) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 2.94 mg/ml sodium citrate (Dihydrate), 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (v) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 2.94 mg/ml sodium citrate (Dihydrate), 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; or (ii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 0.57 mg/ml histidine, 1.33 mg/ml histidine hydrochloride, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 0.57 mg/ml histidine, 1.33 mg/ml histidine hydrochloride, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iv) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 2.94 mg/ml sodium citrate (Dihydrate), 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; or (v) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 0.57 mg/ml histidine, 1.33 mg/ml histidine hydrochloride, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.01 mg/ml disodium edetate, 0.50 mg/ml polysorbate 80, pH 5.7.

In some aspects, a method for preparing the antibody liquid formulation of the present invention is provided, which includes the following steps: (1) Making a solution with ingredients other than the surfactant; (2) Through ultrafiltration, the solution prepared in step (1) is used to exchange the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibodies or fragments thereof. Liquid, and then concentrated to the target concentration; (3) Add the surfactant to the liquid prepared in step (2).

In some aspects, a solid antibody preparation is provided, which is obtained by curing the liquid antibody preparation of the present invention, and the solidification is performed by, for example, a crystallization method, a spray drying method, or a freeze-drying method. The solid antibody preparation For example, it is in the form of a lyophilized powder injection.

In some aspects, a delivery device is provided, which comprises the liquid antibody preparation or the solid antibody preparation of the present invention.

In some aspects, a pre-filled syringe is provided, which contains the liquid antibody preparation or solid antibody preparation of the present invention for intravenous injection or intramuscular injection.

In some aspects, the use of the liquid antibody preparation or solid antibody preparation of the present invention in the preparation of a delivery device or a pre-filled syringe or medicine for the prevention or treatment of cancer is provided. Preferably, the cancer is selected from melanoma, Non-small cell lung cancer, small cell lung cancer, head and neck cancer, liver cancer, colorectal cancer, pancreatic cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, bile duct Cancer, thyroid cancer, hepatocellular carcinoma or mesothelioma; wherein the drug can be administered to cancer patients simultaneously, separately or sequentially with ionizing radiation; or can be administered to cancer patients simultaneously, separately or sequentially with one or more chemotherapeutic drugs; or It can be administered to cancer patients simultaneously, separately or sequentially with ionizing radiation and one or more chemotherapeutic drugs; preferably, the chemotherapeutic drugs are selected from 5-fluorouracil, hydroxyurea, gemcitabine, methotrexate, doxorubicin, etoposide Carboplatin, cisplatin, cyclophosphamide, melphalan, dacarbazine, paclitaxel, camptotheca, FOLFIRI, FOLFOX, docetaxel, daunorubicin, paclitaxel, oxaliplatin, or a combination thereof.

In some aspects, the liquid antibody preparation or solid antibody preparation of the present invention is used to prevent or treat diseases, and the disease is preferably cancer, more preferably selected from melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer , Liver cancer, colorectal cancer, pancreatic cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, cholangiocarcinoma, thyroid cancer, hepatocellular carcinoma, or mesothelial Tumor; wherein the liquid antibody preparation or solid antibody preparation can be administered to cancer patients simultaneously, separately or sequentially with ionizing radiation; or can be administered to cancer patients simultaneously, separately or sequentially with one or more chemotherapeutics; or can be administered to cancer patients simultaneously with ionizing radiation and One or more chemotherapy drugs are administered to cancer patients simultaneously, separately or sequentially; preferably, the chemotherapy drugs are selected from 5-fluorouracil, hydroxyurea, gemcitabine, methotrexate, adriamycin, etoposide carboplatin, cisplatin , Cyclophosphamide, melphalan, dacarbazine, paclitaxel, camptotheca, FOLFIRI, FOLFOX, docetaxel, daunorubicin, paclitaxel, oxaliplatin or a combination thereof.

In some aspects, the present invention provides a method for preventing or treating cancer in a patient, which comprises administering to the patient an effective dose of the liquid antibody preparation or solid antibody preparation of the present invention, or to the patient through the delivery device or pre-filled syringe of the present invention. The patient is administered an effective dose of liquid antibody preparation or solid antibody preparation; the cancer is preferably selected from melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, liver cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, Prostate cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, soft tissue sarcoma, cholangiocarcinoma, thyroid cancer, hepatocellular carcinoma or mesothelioma; wherein the liquid antibody preparation or solid antibody preparation can be combined with ionizing radiation simultaneously, It can be administered to cancer patients separately or sequentially; or can be administered to cancer patients simultaneously, separately or sequentially with one or more chemotherapeutic drugs; or can be administered to cancer patients simultaneously, separately or sequentially with ionizing radiation and one or more chemotherapeutic drugs; preferably The chemotherapy drug is selected from 5-fluorouracil, hydroxyurea, gemcitabine, methotrexate, adriamycin, etoposide carboplatin, cisplatin, cyclophosphamide, melphalan, dacarbazine, paclitaxel, camptotheca Oxaliplatin, FOLFIRI, FOLFOX, docetaxel, daunorubicin, paclitaxel, oxaliplatin, or a combination thereof. It can be administered by parenteral administration, such as injection or infusion.

Detailed description of the invention

Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions in this specification, because the methods and conditions can be changed. In addition, the terms used herein are only for describing specific embodiments, and are not intended to be limiting. definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. For the purpose of the present invention, the following terms are defined below.

The term "about" when used in conjunction with a numerical value means to cover a numerical value within a range that has a lower limit that is 5% smaller than the specified numerical value and an upper limit that is 5% larger than the specified numerical value.

When the term "and/or" is used to connect two or more alternatives, it should be understood to mean any one of the alternatives or any two or more of the alternatives.

As used herein, the term "comprising" or "including" means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps. In this document, when the term "comprises" or "includes" is used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a specific sequence, it is also intended to encompass the antibody variable region composed of the specific sequence.

The term "immune checkpoint molecule" refers to a type of inhibitory message molecule present in the immune system, which prevents tissue damage by regulating the persistence and intensity of immune response in peripheral tissues, and participates in maintaining tolerance to self-antigens (Pardoll DM., The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer, 2012, 12(4): 252-264). Studies have found that one of the reasons why tumor cells can evade the immune system in the body and proliferate out of control is the use of the inhibitory message pathway of immune checkpoint molecules, which inhibits the activity of T lymphocytes, making T lymphocytes unable to effectively kill tumors. Effect (Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2):130-146). Immune checkpoint molecules include, but are not limited to, programmed cell death 1 (PD-1), PD-L1, PD-L2, CD137, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3 and TIM-3.

The terms "whole antibody", "full-length antibody", "full antibody" and "whole antibody" are used interchangeably herein to refer to at least two heavy chains (H) and two light chains interconnected by disulfide bonds. (L) Glycoprotein. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region is composed of three structural domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region consists of a domain CL. The VH and VL regions can be further divided into hypervariable regions [complementarity determining regions (CDR)], with a more conservative region inserted between them [the framework region (FR)]. Each VH and VL consists of three CDRs and 4 FRs, arranged in the following order from the amino end to the carboxyl end: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region does not directly participate in the binding of the antibody to the antigen, but exhibits a variety of effector functions.

The term "antigen-binding fragment" refers to a molecule that is different from an intact antibody, which contains a part of the intact antibody and binds to the antigen to which the intact antibody binds. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies (dAb); linear antibodies; single-chain antibodies (such as scFv); single domains Antibodies (single domain antibodies); antigen-binding fragments of bivalent or bispecific antibodies; camelid antibodies; and other fragments that exhibit the required ability to bind PD-1 and CD137.

As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen binding sites, each of which is associated with a different epitope of the same antigen or is different from Different epitopes of the antigen bind. The antibodies provided herein are generally multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are antibodies that have binding specificities for at least two different epitopes. In one embodiment, provided herein are bispecific antibodies that have binding specificities for a first antigen and a second antigen. For example, the first antigen is PD-1 and the second antigen is CD137.

The term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVR and amino acid residues from human FR. In some embodiments, the humanized antibody comprises all or substantially all of the HVR (eg, CDR) corresponding to those of the non-human antibody and all or substantially all of the FR regions corresponding to those of the human antibody. The humanized antibody optionally can comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain, which contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In certain embodiments, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carbonyl end of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified, the numbering of amino acid residues in the Fc region or the constant region is based on the EU numbering system, which is also called the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service , National Institutes of Health, Bethesda, MD, 1991.

The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies usually have similar structures, where each domain contains four conserved framework regions (FR) and three complementarity determining regions (see, for example, Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co. 91 pages (2007)). A single VH or VL domain may be sufficient to give antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a specific antigen can be used to isolate antibodies that bind to the antigen to screen peptide libraries that complement the VL or VH domains, respectively. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

Variable regions usually exhibit the same general structure of relatively conserved framework regions (FR) connected by three highly variable regions, which are also called complementarity determining regions or CDRs. The CDRs from the two chains of each pair are usually aligned through the framework regions, which CDRs allow binding of specific epitopes. The variable regions of the two light and heavy chains usually comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from N-terminus to C-terminus.

"Complementarity determining region" or "CDR region" or "CDR" or "highly variable region" (herein can be used interchangeably with hypervariable region "HVR") is an antibody variable domain that is highly variable in sequence and forms Structurally defined loops ("hypervariable loops") and/or regions containing antigen contact residues ("antigen contact points"). CDR is mainly responsible for binding to antigen epitopes. The CDRs of the heavy chain and light chain are usually referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundary of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems. Assignment systems include, for example, Chothia (Chothia et al., (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", based on the three-dimensional structure of antibodies and the topology of CDR loops. Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImmunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr/), and affinity propagation clustering based on the use of a large number of crystal structures ) Of the North CDR definition (North et al., "A new clustering of antibody CDR loop confirmations, Journal of Molecular Biology, 406, 228-256 (2011)"). The present invention adopts the North CDR definition.

The term "binding" or "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to bind to a specific antigen can be determined by enzyme-binding immunosorbent assay (ELISA), surface plasmon resonance (SPR) or biofilm optical interference technology (ForteBio) or other conventional binding assays known in the art. As an embodiment of the present invention, it means that the antibody or antigen-binding fragment binds to the epitope in an in vitro assay, preferably in the optical interferometry of a biofilm layer using purified wild-type antigen. In certain embodiments, when an antibody or antigen-binding fragment preferably recognizes its target antigen in a complex mixture of proteins and/or macromolecules, the antibody or antigen-binding fragment is referred to as a specific binding antigen.

The term "antibody preparation" refers to a preparation in a form that allows the biological activity of the antibody as an active ingredient to be effectively exerted, and does not contain other unacceptable toxicity to the subject to which the preparation is administered. Components. Such antibody preparations are usually sterile. Generally, pharmaceutically acceptable excipients are included in antibody preparations. A "pharmaceutically acceptable" excipient is an agent that can be reasonably administered to a tested mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, for example, it may be acceptable for injection.

The term "recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody preparation" is also abbreviated herein as "the antibody preparation of the present invention", meaning that it contains anti-PD -1/CD137 antibody protein as an active ingredient and a preparation containing pharmaceutically acceptable excipients. After the anti-PD-1/CD137 antibody protein is combined with a pharmaceutically acceptable excipient, the anti-PD-1/CD137 antibody protein as the active ingredient is suitable for therapeutic or preventive administration to humans or non-human animals. The antibody preparation of the present invention can be prepared, for example, as an aqueous liquid preparation, for example, a ready-to-use pre-filled syringe, or prepared as a lyophilized preparation, which is carried out by dissolving and/or suspending in a physiologically acceptable solution immediately before use. Reconstitution (ie, reconstitution). In some embodiments, the anti-PD-1/CD137 antibody protein preparation is in the form of a liquid preparation.

A "stable" antibody preparation is when the antibody in the preparation retains an acceptable degree of physical and/or chemical stability after storage under specific conditions. Although the antibody contained in the antibody preparation may not maintain 100% of its chemical structure after storage for a specific time, it usually maintains about 90%, about 95%, about 96%, about 97%, about 98% after storage for a specific time. Or about 99% of the structure or function of the antibody, the antibody preparation is considered "stable." In some specific embodiments, the anti-PD-1/CD137 antibody protein preparation of the present invention exhibits low to undetectable antibody aggregation or degradation or chemical modification during the manufacturing, preparation, transportation and long-term storage, so that there is little or no Even there is no loss of the biological activity of the anti-PD-1/CD137 antibody protein, showing a high degree of stability. In some embodiments, the anti-PD-1/CD137 antibody protein formulation of the present invention substantially retains its physical and chemical stability after storage. Preferably, the liquid formulation of the present invention can be stable at room temperature for at least 6 months or stored at 40°C±2°C for 1 month, and/or stable at 25°C±2°C for at least 3 months, and/or at 2-8 ℃ stable for at least 24 months.

A variety of analytical techniques known in the art can be used to determine protein stability, see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs (1991) and Jones , A. Adv. Drug Delivery Rev. 10: 29-90 (1993). The stability can be measured at a selected temperature and selected storage time. For example, the storage time can be selected based on the expected shelf life of the formulation. Alternatively, an accelerated stability test can be used. In some embodiments, stability testing is performed by performing various stress tests on antibody preparations. These tests can represent extreme conditions that may be encountered during manufacture, storage, or transportation of the formulated antibody preparation, or conditions that may accelerate the instability of the antibody in the antibody preparation during non-manufacture, storage, or transportation. For example, the formulated anti-PD-1/CD137 antibody protein preparation can be filled into a glass vial to test the antibody stability under high temperature stress.

After a period of storage, the preparation does not show aggregation, precipitation, turbidity and/or denaturation; or shows very little aggregation, precipitation, turbidity and/or denaturation, it can be considered that the antibody "maintains its physical stability" in the preparation. The accumulation of antibodies in the preparation can potentially lead to an increased immune response in patients, leading to safety issues. Therefore, there is a need to minimize or prevent aggregation of the antibody in the formulation. The light scattering method can be used to determine the visible aggregates in the formulation. SEC can be used to determine soluble aggregates in the formulation. In addition, the stability of the preparation can be indicated by visually inspecting the appearance, color and/or clarity of the _{preparation, or detecting the turbidity of the preparation by the OD 350 nm} method, or measuring the purity of the preparation by the non-reducing CE-SDS method. In one embodiment, the stability of the formulation is measured by determining the percentage of antibody monomers in the formulation after storage at a specific temperature for a specific time, wherein the higher the percentage of antibody monomers in the formulation, the higher the stability of the formulation .

An "acceptable degree" of physical stability can mean that at least about 90% of the anti-PD-1/CD137 antibody protein monomer is detected in the preparation after storage at a specific temperature for a specific time. In some embodiments, storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months After months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more, an acceptable degree of physical stability indicates At least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/CD137 antibody protein monomer. When evaluating physical stability, the specific temperature at which the pharmaceutical preparation is stored may be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, About 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C. For example, if stored at about 40℃±2℃ for 1 month, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of Anti-PD-1/CD137 antibody protein monomer, the pharmaceutical preparation is considered stable; if stored at about 25°C for 2 months, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/CD137 antibody protein monomer, the pharmaceutical preparation is considered stable; if stored at about 5℃ for 9 months, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/CD137 antibody protein monomer, the pharmaceutical preparation is considered stable.

After a period of storage, if the antibody in the preparation does not show a significant chemical change, it can be considered that the antibody "maintains its chemical stability" in the preparation. Most chemical instabilities result from the formation of covalently modified forms of antibodies (for example, charge variants of antibodies). For example, by aspartic acid isomerization, N and C terminal modification, basic variants can be formed; by deamidation, sialylation and glycation, acidic variants can be produced. Chemical stability can be assessed by detecting and/or quantifying the chemically altered form of the antibody. For example, the charge variant of the antibody in the preparation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (iCIEF). In one embodiment, the stability of the formulation is measured by determining the percentage change in the charge variant of the antibody in the formulation after storage at a specific temperature for a specific time, wherein the smaller the change, the higher the stability of the formulation.

The "acceptable degree" of chemical stability can mean that the percentage change of the charge variant (such as the main component or acidic component or alkaline component) in the formulation after storage at a specific temperature for a specific time does not exceed 50%, for example, does not exceed 30%, not more than 20%. In some embodiments, storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months After months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more, an acceptable degree of chemical stability can be The percentage change value of the main component charge variant does not exceed about 50%, 40%, 30%, 20%, 15%. When evaluating chemical stability, the storage temperature of the pharmaceutical preparation can be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C. For example, if stored at 5℃ for 24 months, the percentage change value of the principal component charge variant is less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17 %, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%, the pharmaceutical preparation can be considered stable; if the percentage change value of the principal component charge variant is less than about 20%, 19%, 18%, 17%, 16% after storage at 25°C for 2 months %, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%, The pharmaceutical preparation can also be regarded as stable; if the percentage change value of the principal component charge variant is less than about 50%, 40%, 30%, 20%, 10%, 5% or 4%, the pharmaceutical preparation can also be considered stable.

The term "lyophilized preparation" refers to a composition obtained or obtainable through a freeze-drying process of a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

The term "reconstituted preparation" refers to a liquid preparation obtained by dissolving and/or suspending a solid preparation (for example, a lyophilized preparation) in a physiologically acceptable solution.

The term "room temperature" as used herein refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, more preferably 25°C.

"Stress conditions" refer to environments that are chemically and/or physically unfavorable to the antibody protein, which can lead to unacceptable instability of the antibody protein, for example, high temperature, shaking, freezing and thawing, and light. "High temperature stress" refers to storing the antibody preparation at room temperature or even at a higher temperature (for example, 40°C±2°C) for a period of time. Through the accelerated test of high temperature stress, the stability of the antibody preparation can be checked.

As used herein, the term "parenteral administration" means administration methods other than enteral and local administration, usually via injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions. In some embodiments, the stabilized anti-PD-1/CD137 antibody protein formulation of the present invention is administered to the subject parenterally. In one embodiment, the anti-PD-1/CD137 antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection. 1. Antibodies

The antibody sequence of the present invention: anti-CD137 arm (7A5*), anti-PD-1 arm (11444*), based on the parental antibody (7A5), parental antibody (11444) and wild-type human IgG1 constant region, wild-type human Lambda constant region and wild-type human Kappa constant region have undergone mutation design. The specific CDR regions, variable regions, constant regions, full-length heavy chains and full-length light chains of the antibody of the present invention, as well as the nucleotide numbers encoding the full-length heavy and light chains, and the parent antibody variable region SEQ ID NOs are shown in Table 1 below ： [01] Table 1 Nucleotide sequence numbers involved in the present invention Anti-CD137 arm (7A5*) Anti-PD-1 arm (11444*) Parent antibody 7A5 Parent antibody 11444 HCDR1 1 13 HCDR2 2 14 HCDR3 3 15 LCDR1 7 19 LCDR2 8 20 LCDR3 9 twenty one HCVR 4 16 34 36 LCVR 10 twenty two 35 37 HCCR 30 32 LCCR 31 33 Heavy chain 5 17 Light chain 11 twenty three Heavy chain DNA 6 18 Light chain DNA 12 twenty four

HCCR: heavy chain constant region; LCCR: light chain constant region

Specific amino acid and nucleotide sequence

7A5* the sequence of

<SEQ ID NO:1; PRT1; Artificial sequence>

KASGGTFSSYAIS

<SEQ ID NO: 2; PRT1; Artificial sequence>

GIIPIFGTANYAQKFQG

<SEQ ID NO: 3; PRT1; Artificial sequence>

ARDLATTAPATYFDL

<SEQ ID NO: 4; PRT1; Artificial sequence>

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRYAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDLATTAPATYFDLWGRGTLVTVSS

<SEQ ID NO: 5; PRT1; Artificial sequence>

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRYAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDLATTAPATYFDLWGRGTLVTVSSASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPDSGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRGDMTKNQVQLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

<SEQ ID NO: 6; DNA; artificial sequence>

CAGGTTCAGTTGGTGCAATCAGGCGCAGAGGTAAAAAAACCAGGGTCCAGCGTGAAAGTCTCATGTAAGGCCTCCGGCGGAACATTCTCCTCCTACGCTATTTCTTGGGTGAGATACGCCCCTGGGCAGGGACTTGAGTGGATGGGAGGCATTATTCCCATATTCGGCACAGCCAATTACGCGCAAAAATTCCAGGGGAGGGTTACTATAACAGCAGATGAGAGTACATCAACTGCGTACATGGAACTGAGCTCCCTGAGGAGCGAAGACACCGCTGTTTACTACTGCGCTAGAGATCTTGCGACGACCGCACCTGCGACGTACTTTGATCTCTGGGGTAGAGGAACCCTCGTAACAGTGTCTTCCGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTGCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCGATTCTGGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTATGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAAGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCC CAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGGGGACATGACCAAGAACCAAGTCCAGCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGCTTCCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGCAAA

<SEQ ID NO: 7; PRT1; artificial sequence>

QASQDIGNSLG

<SEQ ID NO: 8; PRT1; artificial sequence>

FDASDLET

<SEQ ID NO: 9; PRT1; Artificial sequence>

QQGNSFPLT

<SEQ ID NO: 10; PRT1; Artificial sequence>

DIRMTQSPPSLSASVGDRVTITCQASQDIGNSLGWYQRKPGDAPKLVIFDASDLETGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQQGNSFPLTFGQGTRLEIK

<SEQ ID NO: 11; PRT1; Artificial sequence>

DIRMTQSPPSLSASVGDRVTITCQASQDIGNSLGWYQRKPGDAPKLVIFDASDLETGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQQGNSFPLTFGQGTRLEIKRTVAAPSVFIFPTKPSKEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGSSGSLGESFYPREAKVQWKVDNALQSGNSQESVTSGSLGESFYPREAKVQWKVDNALQSGNSQESVTSGSLVTSGSLVTEKPSKESVT

<SEQ ID NO: 12; DNA; artificial sequence>

GACATTAGAATGACACAGTCACCTCCAAGTCTGTCAGCCAGTGTTGGCGACCGGGTGACTATCACCTGCCAGGCTTCCCAAGACATTGGTAATAGTTTGGGTTGGTACCAGCGCAAACCAGGCGATGCTCCGAAACTGGTTATTTTTGACGCCAGTGATTTGGAGACAGGTGTGCCTTCTCGGTTTAGCGGTTCTGGGTCAGGAACTGATTTTTCACTGACAATATCTTCACTGCAGCCGGAAGACTTCGCCACCTATTATTGCCAGCAGGGGAACTCCTTCCCACTCACCTTCGGTCAAGGGACCCGGCTTGAGATTAAGCGGACGGTAGCTGCCCCCTCTGTGTTCATTTTCCCCCCAAGCAAGGAGCAGCTGAAGAGCGGCACGGCCAGCGTGGTATGTCTGCTGAATAACTTTTACCCTCGGGAGGCCAAAGTGCAGTGGAAGGTCGATAATGCTCTTCAATCCGGGAACTCACAGGAATCTGTCACCGAACAAGACAGCAAGGATAGCACGTACAGCCTGTCTAGCACTCTGACCCTTTCCAAAGCAGACTACGAAAAACATAAAGTCTACGCGTGCGAAGTGACCCACCAGGGGCTCAGCTCACCGGTGACGAAATCCTTCAACCGCGGCGAATGC

11444* the sequence of

<SEQ ID NO: 13; PRT1; Artificial sequence>

KASGGTFSSYAIS

<SEQ ID NO: 14; PRT1; Artificial sequence>

LIIPSFDTAGYAQEFQG

<SEQ ID NO: 15; PRT1; Artificial sequence>

ARAEHSSTGTFDY

<SEQ ID NO: 16; PRT1; Artificial sequence>

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRKAPGQGLEWMGLIIPSFDTAGYAQEFQGRVAITVDESTSTAYMELSSLRSEDTAVYYCARAEHSSTGTFDYWGQGTLVTVSS

<SEQ ID NO: 17; PRT1; Artificial sequence>

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRKAPGQGLEWMGLIIPSFDTAGYAQEFQGRVAITVDESTSTAYMELSSLRSEDTAVYYCARAEHSSTGTFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVSTLPPSREEMTKNQVSLMCLVYGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

<SEQ ID NO: 18; DNA; artificial sequence>

CAAGTGCAACTGGTGCAATCAGGGGCTGAGGTGAAGAAGCCTGGAAGTAGCGTTAAAGTCAGTTGCAAAGCGTCCGGTGGGACATTTAGCAGCTATGCCATCAGCTGGGTTCGGAAGGCACCCGGCCAGGGACTGGAGTGGATGGGACTCATAATCCCGAGCTTTGACACTGCTGGTTACGCACAGGAGTTTCAAGGGAGGGTGGCGATCACAGTGGACGAATCAACCAGCACCGCGTATATGGAGCTGTCATCTCTGAGGTCAGAAGACACCGCTGTTTACTATTGTGCCCGCGCTGAGCATTCTTCCACCGGGACCTTCGATTACTGGGGACAAGGAACCCTGGTCACAGTATCATCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGGCCACCGGCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCCGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTATGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACCAGAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAAGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTCCACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAAGTCAGCCTGATGTGCCTGGTCTATGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATTCCGTGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGCAAA

<SEQ ID NO: 19; PRT1; Artificial sequence>

RASQGISSWLA

<SEQ ID NO: 20; PRT1; artificial sequence>

SAASSLQS

<SEQ ID NO: 21; PRT1; Artificial sequence>

QQANHLPFT

<SEQ ID NO: 22; PRT1; Artificial sequence>

RIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQDKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANHLPFTFGGGTKVEIK

<SEQ ID NO: 23; PRT1; Artificial sequence>

RIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQDKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANHLPFTFGGGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCYISDFYPGAVTVAWKADSSPVPEGVAWSTPSKQSNTECTVSYSTVAWKADSSPVPEGVKSTVAWKADSSPVPEQWKSTVAWKADSSPVPEGVAWSTPSKQSNTECV

<SEQ ID NO: 24; DNA; artificial sequence>

CGCATCCAGATGACACAGTCACCTTCAAGCGTCTCCGCCTCCGTGGGAGACAGGGTTACTATTACATGTAGGGCCAGCCAGGGGATCTCTTCATGGCTGGCGTGGTACCAAGACAAGCCAGGCAAAGCCCCCAAGCTCCTTATCTCCGCTGCCTCCTCTCTGCAGTCCGGAGTTCCCTCCCGCTTCAGCGGTAGCGGGTCAGGCACTGACTTCACCCTTACAATCTCTTCTCTGCAACCTGAGGACTTCGCCACATATTATTGCCAGCAGGCAAACCATTTGCCATTTACTTTTGGCGGAGGTACTAAGGTTGAGATTAAAGGCCAGCCTAAAGCTGCCCCTAGCGTTACCCTTTTCCCACCGAGCTCCGAGGAGCTGCAGGCCAATAAAGCAACCTTGGTCTGCTACATATCAGATTTTTACCCTGGCGCCGTGACCGTAGCATGGAAAGCTGATTCATCCCCTGTGAAGGCCGGTGTTGAAACTACAACCCCTTCCAAACAATCTAACAATAAATACGCGGCATGGTCCTACCTGTCCTTGACACCCGAGCAGTGGAAATCTCACAGATCTTACAGCTGCCAGGTCACCCACGAGGGGAGCACTGTGGAGAAGACCGTCGCGCCCACTGAGTGC

people PD-1 and people CD137 sequence

<SEQ ID NO: 25; PRT1; Human>

MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

<SEQ ID NO: 26; PRT1; Human>

MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVISRGVGCELGCFLGCCELGCFLGCCELQDCKQKRGICRPWTNCSLDGKSVLVNGTKERDVISRGEDVGCELGCFLGCCFFKFLG

Wild type IgG1 Constant region sequence

<SEQ ID NO: 27; PRT1; Human>

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Wild type Lambda Constant region sequence

<SEQ ID NO: 28; PRT1; Human>

GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC

Wild type Kappa Constant region sequence

<SEQ ID NO: 29; PRT1; Human>

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Sequence of the constant region of the antibody of the present invention

7A5*

<SEQ ID NO: 30; PRT1; Artificial sequence>

ASTKGPSVFPLAPCSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPDSGDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRGDMTKNQVQLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

<SEQ ID NO: 31; PRT1; Artificial sequence>

RTVAAPSVFIFPPSKEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

11444*

<SEQ ID NO: 32; PRT1; Artificial sequence>

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVATGPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVSTLPPSREEMTKNQVSLMCLVYGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSVLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

<SEQ ID NO: 33; PRT1; Artificial sequence>

GQPKAAPSVTLFPPSSEELQANKATLVCYISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAWSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC

Parent 7A5 Variable region sequence

<SEQ ID NO: 34; PRT1; Artificial sequence>

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDLMTTAPGTYFDLWGRGTLVTVSS

<SEQ ID NO: 35; PRT1; Artificial sequence>

AIRMTQSPPSLSASVGDRVTITCQASQDIGNSLGWYQQKPGKAPKLVIFDASDLETGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQQGNSFPLTFGQGTRLEIK

Parent 11444 Variable region sequence

<SEQ ID NO: 36; PRT1; Artificial sequence>

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGLIIPMFDTAGYAQKFQGRVAITVDESTSTAYMELSSLRSEDTAVYYCARAEHSSTGTFDYWGQGTLVTVSS

<SEQ ID NO: 37; PRT1; Artificial sequence>

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANHLPFTFGGGTKVEIK

＞11444_HC_protein SEQ ID NO: 38

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGLIIPMFDTAGYAQKFQGRVAITVDESTSTAYMELSSLRSEDTAVYYCARAEHSSTGTFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

＞11444_LC_protein SEQ ID NO: 39

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLISAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANHLPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSKDSEKVQWKVDNALQSGNSKDSEKVTEQACEKAVTKDSEKVTEQACE

＞7A5_HC_protein SEQ ID NO: 40

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDLMTTAPGTYFDLWGRGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAEGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

＞7A5_LC_protein SEQ ID NO: 41 IRMTQSPPSLSASVGDRVTITCQASQDIGNSLGWYQQKPGKAPKLVIFDASDLETGVPSRFSGSGSGTDFSLTISSLQPEDFATYYCQQGNSFPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFKVYPREAKVQLKSGTASVVCLLNNFYPREAKVQ

Specifically, the recombinant anti-programmed cell death receptor 1 (PD-1, SEQ ID NO: 25) and anti-differentiation antigen cluster 137 (CD137, SEQ ID NO: 26) bispecific antibody of the present invention comprises: a first heavy chain , The second heavy chain and the first light chain and the second light chain, wherein each chain contains a variable region and a constant region, wherein: a) The first heavy chain of the antibody comprises: complementarity determining region 1 (HCDR1) with the amino acid sequence of SEQ ID NO: 1, complementarity determining region 2 (HCDR2) with the amino acid sequence of SEQ ID NO: 2, and having an amino group Complementarity determining region 3 (HCDR3) of the acid sequence SEQ ID NO: 3; b) The first light chain of the antibody comprises: complementarity determining region 1 (LCDR1) with amino acid sequence SEQ ID NO: 7, complementarity determining region 2 (LCDR2) with amino acid sequence SEQ ID NO: 8, and amino acid sequence Complementarity determining region 3 (LCDR3) of the acid sequence SEQ ID NO: 9; c) The second heavy chain of the antibody comprises: complementarity determining region 1 (HCDR1) with the amino acid sequence of SEQ ID NO: 13, and complementarity determining region 2 (HCDR2) with the amino acid sequence of SEQ ID NO: 14, and having an amino group Complementarity determining region 3 (HCDR3) of the acid sequence of SEQ ID NO: 15; d) The second light chain of the antibody comprises: complementarity determining region 1 (LCDR1) with amino acid sequence SEQ ID NO: 19, complementarity determining region 2 (LCDR2) with amino acid sequence SEQ ID NO: 20, and amino acid sequence The complementarity determining region 3 (LCDR3) of the acid sequence SEQ ID NO: 21.

Preferably, e) the antibody first heavy chain variable region (HCVR) has an amino acid sequence of SEQ ID NO: 4; f) The antibody first light chain variable region (LCVR) has an amino acid sequence of SEQ ID NO: 10; g) The antibody second heavy chain variable region (HCVR) has an amino acid sequence of SEQ ID NO: 16; h) The antibody second light chain variable region (LCVR) has an amino acid sequence of SEQ ID NO:22.

More preferably, the first heavy chain constant region sequence (HCCR) has an amino acid sequence of SEQ ID NO: 30, the first light chain constant region sequence (LCCR) has an amino acid sequence of SEQ ID NO: 31, and the second heavy chain The constant region (HCCR) sequence has an amino acid sequence of SEQ ID NO:32, and the second light chain constant region (LCCR) sequence has an amino acid sequence of SEQ ID NO:33.

More preferably, i) the first heavy chain of the antibody has an amino acid sequence of SEQ ID NO: 5; j) The first light chain of the antibody has an amino acid sequence of SEQ ID NO: 11; k) The second heavy chain of the antibody has an amino acid sequence of SEQ ID NO: 17; and 1) The second light chain of the antibody has an amino acid sequence of SEQ ID NO:23.

Further, at least one disulfide bond is formed between the first heavy chain and the first light chain of the antibody, at least one disulfide bond is formed between the second heavy chain and the second light chain, and the first heavy chain and the second heavy chain At least one disulfide bond is formed between them.

The antibody of the present invention is a modified human IgG1 to reduce the binding of the antibody to the Fc γ receptor.

The binding affinity of the antibody of the present invention to human PD-1 is more than 10 times higher than the binding affinity of the antibody to human CD137, preferably more than 100 times. Therefore, the antibody of the present invention exhibits advantageous pharmaceutical properties. The antibodies of the present invention selectively target cells expressing PD-1 and potentially limit the agonistic effect of CD137 on those cells co-expressing PD-1.

In addition, the present invention also provides nucleotide sequences encoding the first heavy chain, the first light chain, the second heavy chain, and the second light chain of the antibody of the present invention. Preferably, the nucleotide sequences are SEQ ID NO: 6. The nucleotide sequence shown in SEQ ID NO: 12, SEQ ID NO: 18 and SEQ ID NO: 24.

The antibody of the present invention is obtained by culturing mammalian cells capable of expressing the antibody of the present invention (non-limiting examples include CHO, NS0, HEK293 or COS cells) and recovering the antibody. For example, select suitable host cells such as HEK293 or CHO cells, through a preset optimal heavy chain: light chain carrier ratio of secreted antibody expression system, or through a single vector system expressing both heavy and light chains, transient or stable Transfect the host cell. Specifically, for example, encoding the first heavy chain with the amino acid sequence shown in SEQ ID NO: 5, encoding the first light chain with the amino acid sequence shown in SEQ ID NO: 11, and encoding the first heavy chain with SEQ ID NO: The second heavy chain of the amino acid sequence shown in 17, and one or more DNA molecules encoding the second light chain of the amino acid sequence shown in SEQ ID NO: 23 are transiently or stably transformed through the expression system of the secreted protein The host cell is infected to obtain the antibody of the present invention.

Antibody recovery and purification can be achieved by conventional techniques in the art. The application of antibodies as active ingredients of medicines is now widespread. Techniques for purifying therapeutic antibodies to pharmaceutical grade are well known in the art. For example, Tugcu et al. (Maximizing productivity of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering 99 (2008) 599-613.) describe the use of ion exchange chromatography (anionic IEX and/or cationic CEX chromatography after the protein A capture step). ) The monoclonal antibody three-column purification method. Kelley et al. (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies, Biotechnology and Bioengineering 101 (2008) 553–566) describe a two-column purification method in which a weak partitioning anion exchange resin is used after protein A affinity chromatography.

Generally, recombinantly produced monoclonal antibodies can be purified by conventional purification methods to provide pharmaceutical substances with sufficient reproducibility and moderate purity for the preparation of antibody preparations. For example, after the antibody is secreted from the recombinant expression cell into the culture medium, a commercially available protein concentration filter such as Amicon's ultrafiltration device can be used to concentrate the supernatant from the expression system. After that, the antibody can be purified using methods such as chromatography, dialysis, and affinity purification. Protein A is suitable as an affinity ligand for the purification of IgG1, IgG2 and IgG4 type antibodies. Other antibody purification methods, such as ion exchange chromatography, can also be used. After obtaining the antibody of sufficient purity, a preparation containing the antibody can be prepared according to methods known in the art.

For example, the following steps can be used for preparation: (1) After fermentation, the fermentation broth is centrifuged to clarify impurities such as cells to obtain the supernatant; (2) affinity chromatography (for example, specific affinity for IgG1, IgG2, and IgG4 antibodies) Protein A column) to capture antibodies; (3) virus inactivation; (4) purification and purification (usually CEX cation exchange chromatography can be used) to remove impurities in the protein; (5) virus filtration (to make the virus titer) To reduce, for example, 4 log10 or more); (6) Ultrafiltration/diafiltration (which can be used to replace the protein in a formulation buffer that is conducive to its stability and concentrate it to a suitable concentration for injection). See, for example, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57. 2. Antibody preparations

In one aspect, the present invention relates to a liquid antibody preparation comprising a recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof and a buffer, preferably The pH value of the formulation is about 5.0-7.0; more preferably, the pH value of the formulation is about 5.5-6.0; further preferably, the pH value is about 5.7.

In some schemes, the content of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof in the liquid antibody preparation is about 1 mg /mL-150 mg/mL, preferably about 10mg/mL-100 mg/mL, for example, about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90 mg/ mL, more preferably about 50.0 mg/ml.

In some aspects, the buffer is selected from histidine, histidine hydrochloride, or a combination thereof, or the buffer is selected from citrate, citrate solvate (e.g., citrate Hydrate) or a combination thereof, for example, sodium citrate, sodium citrate dihydrate or a combination thereof, or the buffer is selected from acetate, acetate solvate (for example, acetate hydrate) or The combination thereof, or the buffer is selected from phosphate, phosphate solvate (for example, phosphate hydrate) or a combination thereof; preferably, the concentration of the buffer is about 5-100 mM, more preferably The ground is about 5-60 mM, for example, about 5, 10, 15, 20, 25, 30, 40, 50, 60 mM;

Preferably, the buffering agent is histidine, and the content of histidine is about 0.775 mg/mL-15.5 mg/mL; more preferably, the content of the histidine is about 0.775 mg/mL-9.3 mg/mL, for example about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 mg/mL; further preferably, the group The content of amino acid is about 1.55 mg/ml;

Preferably, the buffer is a combination of histidine and histidine hydrochloride, wherein the content of the histidine is about 0.270 mg/mL-5.4 mg/mL, and the content of the histidine hydrochloride is about 0.660 mg/mL-13.33 mg/mL; more preferably, the histidine content is about 0.270 mg/mL-3.24 mg/mL, for example about 0.5, 1.0, 1.5, 2, 2.5, 3 mg/mL, so The content of histidine hydrochloride is about 0.660 mg/mL-8 mg/mL, such as about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 mg/mL; more preferably, the content of the histidine acid is about 0.57 mg/ml, and the content of the histidine hydrochloride is about 1.33 mg/ml.

In some aspects, the formulation further includes a stabilizer;

Preferably, the stabilizer includes a polyhydric alcohol and/or an amino acid; the polyhydric alcohol is selected from sorbitol, mannitol, sucrose, trehalose, maltose, and combinations thereof, and the amino acid includes arginine, spermine hydrochloride Acid, methionine, glycine, proline or a combination thereof; preferably, the stabilizer is selected from sorbitol and/or arginine hydrochloride, sorbitol and/or arginine, more preferably, the The stabilizer is a combination of sorbitol and arginine hydrochloride, or a combination of sorbitol and arginine, preferably, the content of the arginine or arginine hydrochloride is about 20 mM-200 mM, and more preferably, the The content of arginine or arginine hydrochloride is about 40 mM-150 mM, such as about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 mM; more preferably, The content of arginine or arginine hydrochloride is about 85 mM;

Preferably, the content of the sorbitol is about 10 mg/mL-100 mg/mL, and the content of the arginine hydrochloride is about 4 mg/mL-42.0 mg/mL; further preferably, the content of the sorbitol The content is about 20 mg/mL-60 mg/mL, such as about 30, 40, 50 mg/mL, and the content of arginine hydrochloride is about 8.4 mg/mL-33.7 mg/mL, such as about 15, 20, 25. 30 mg/mL; more preferably, the content of the sorbitol is about 25.00 mg/ml, and the content of the arginine hydrochloride is about 16.85 mg/ml.

In some aspects, the formulation further includes a surfactant;

Preferably, the surfactant is selected from non-ionic surfactants, such as one or more of polysorbate 80, polysorbate 20, poloxamer and polyethylene glycol polysorbate 80, more preferably polysorbate Sorbate 80;

More preferably, the content of the surfactant is about 0.1 mg/mL-1 mg/mL; further preferably about 0.3 mg/mL-0.7 mg/mL, such as 0.3, 0.4, 0.5, 0.6, 0.7 mg/mL ; It is further preferably about 0.50 mg/ml.

In some aspects, the formulation further includes a chelating agent;

Preferably, the chelating agent is selected from edetate disodium, diethyltriaminepentaacetic acid and/or EDTA, more preferably edetate disodium;

More preferably, the content of the chelating agent is about 0.005 mg/mL-0.1 mg/mL; further preferably about 0.01 mg/mL-0.05 mg/mL, such as about 0.02, 0.03, 0.04 mg/mL; further preferably It is about 0.01 mg/ml.

In some aspects, a liquid antibody preparation is provided, which contains the following components: about 50.0 mg/ml recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific Antibody, about 0.57 mg/ml histidine, about 1.33 mg/ml histidine hydrochloride, about 25.00 mg/ml sorbitol, about 16.85 mg/ml arginine hydrochloride, about 0.01 mg/ml disodium edetate , About 0.50 mg/ml polysorbate 80, pH about 5.7, the balance is water.

In some aspects, a liquid antibody preparation is provided, which comprises: (i) The recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof of about 1 mg/mL-150 mg/mL; (ii) Histidine of about 5-100 mM; (iii) about 10 mg/mL-100 mg/mL sorbitol and about 20 mM-200 mM arginine or arginine hydrochloride; (iv) about 0.1 mg/mL-1 mg/mL polysorbate 80; and (v) Optionally, about 0.005 mg/mL-0.1 mg/mL disodium edetate, Wherein the pH of the liquid formulation is about 5.0-7.0, for example, about 5.5, 6.0, 6.5; For example, the liquid antibody preparation comprises (i) About 10 mg/mL-100 mg/mL of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof; (ii) Histidine of about 5-60 mM; (iii) about 20 mg/mL-60 mg/mL sorbitol and about 8.4 mg/mL-33.7 mg/mL arginine hydrochloride; (iv) about 0.3 mg/mL-0.7 mg/mL polysorbate 80; and (v) Optionally, about 0.01 mg/mL-0.05 mg/mL disodium edetate, Wherein the pH of the liquid formulation is about 5.5-6.0, for example, about 5.7; Alternatively, the liquid antibody preparation comprises (i) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 1.55 mg/ml histidine, 50.00 mg/ml sorbitol, 0.50 mg/ml polysorbate 80, pH 5.7; or (ii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 1.55 mg/ml histidine, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 1.55 mg/ml histidine, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iv) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 2.94 mg/ml sodium citrate (Dihydrate), 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (v) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 2.94 mg/ml sodium citrate (Dihydrate), 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; or (ii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 0.57 mg/ml histidine, 1.33 mg/ml histidine hydrochloride, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 0.57 mg/ml histidine, 1.33 mg/ml histidine hydrochloride, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iv) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 2.94 mg/ml sodium citrate (Dihydrate), 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; or (v) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 0.57 mg/ml histidine, 1.33 mg/ml histidine hydrochloride, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.01 mg/ml disodium edetate, 0.50 mg/ml polysorbate 80, pH 5.7.

In some aspects, a solid antibody preparation is provided, which is obtained by curing the liquid antibody preparation of the present invention, and the solidification is performed by, for example, a crystallization method, a spray drying method, or a freeze-drying method. The solid antibody preparation For example, it is in the form of a lyophilized powder injection.

The antibody in the formulation has been described in detail above. It should be emphasized here that the antibody and antigen-binding fragments thereof may further cover the following bispecific antibodies or antigen-binding fragments thereof: Contains a first heavy chain variable region (HCVR) having at least 90%, 95%, 98%, or 99% or higher homology with the amino acid sequence SEQ ID NO: 4; and/or with the amino acid sequence SEQ ID NO: 10 has at least 90%, 95%, 98%, or 99% or more homology of the first light chain variable region (LCVR); and/or has at least 90% with the amino acid sequence SEQ ID NO: 16, 95%, 98% or 99% or higher homology of the second heavy chain variable region (HCVR); and/or at least 90%, 95%, 98% or 99% with the amino acid sequence SEQ ID NO: 22 Or higher homology of the second light chain variable region (LCVR). In this context, "sequence homology" refers to the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in the comparison window. The "percentage of sequence homology" can be calculated by the following method: the two optimally aligned sequences are compared in the comparison window to determine the presence of the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) the number of positions to get the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, window size), and multiply the result by 100 to generate the sequence homology percentage. The optimal alignment to determine the percent sequence homology can be achieved in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for sequence alignment, including any algorithm required to achieve maximum alignment within the full-length sequence being compared or within the target sequence region.

In some embodiments, the first heavy chain variable region (HCVR) of the anti-PD-1/CD137 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, compared with SEQ ID NO: 4. 4 or 3 different residues, preferably the different residues are conservative amino acid substitutions. In some embodiments, the first light chain variable region (LCVR) of the anti-PD-1/CD137 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, compared with SEQ ID NO: 10, 4 or 3 different residues, preferably the different residues are conservative amino acid substitutions. In some embodiments, the second heavy chain variable region (HCVR) of the anti-PD-1/CD137 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, compared with SEQ ID NO: 16, 4 or 3 different residues, preferably the different residues are conservative amino acid substitutions. In some embodiments, the second light chain variable region (LCVR) of the anti-PD-1/CD137 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, compared with SEQ ID NO: 22, 4 or 3 different residues, preferably the different residues are conservative amino acid substitutions. "Conservative substitution" refers to an amino acid change that causes a certain amino acid to be replaced with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables for functionally similar amino acids. In any embodiment of the present invention, in a preferred aspect, the conservatively substituted residues are derived from the following conservative substitutions, preferably the preferred substituted residues shown in Table 2. Table 2 Examples of conservative substitutions of amino acids Original residue Exemplary substitution Preferred conservative amino acid substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp; Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe; Leucine Leu Leu (L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Leucine Leu Other excipients

In some embodiments, other excipients are optionally included in the antibody liquid formulation of the present invention. The other excipients include, for example, antimicrobial agents, antistatic agents, antioxidants, gelatin and the like. These and other known pharmaceutical excipients and/or additives suitable for the formulation of the present invention are well known in the art, for example, listed in "The Handbook of Pharmaceutical Excipients, 4th Edition, Rowe et al., eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)". Analysis of preparations

During the storage of antibody preparations, antibodies may undergo aggregation, degradation or chemical modification, resulting in antibody heterogeneity (including size heterogeneity and charge heterogeneity), aggregates and fragments, etc., thereby affecting the quality of antibody preparations. Therefore, it is necessary to monitor the stability of antibody preparations.

Various methods are known in the art that can be used to test the stability of antibody preparations. For example, methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC can be used to analyze the purity of antibody preparations and evaluate the aggregation level of antibodies; it can be through capillary isoelectric focusing electrophoresis (cIEF), imaging capillary, etc. Electrofocusing electrophoresis (iCIEF) and ion exchange chromatography (IEX), etc., analyze charge variants in antibody preparations. In addition, the stability of the preparation can be quickly judged by visually inspecting the appearance of the preparation. The OD350nm method can also be used to detect changes in the turbidity of the preparation, which can give information about the amount of soluble and insoluble aggregates. In addition, ultraviolet spectrophotometry (UV method) can be used to detect changes in protein content in the preparation.

The non-reduced CE-SDS method is a method of antibody purity determination using capillary as a separation channel. In CE-SDS, protein migration is driven by the surface charge caused by SDS binding, and the surface charge is proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar quality-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved in the molecular sieve gel matrix of the capillary. This method has been widely used to monitor the purity of denatured intact antibodies. Generally, in the non-reducing CE-SDS method, the test sample is mixed with SDS sample buffer and iodoacetamide. After that, the mixture can be incubated at 68-72°C for about 10-15 minutes, and the supernatant centrifuged after cooling to room temperature is used for analysis. A UV detector is used to detect the migration of the protein and obtain an electrophoresis spectrum. The purity of the antibody preparation can be calculated as the percentage of the peak area of the main IgG peak to the sum of all peak areas. For a further description of the CE-SDS method, see, for example, Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.

Size exclusion high performance liquid chromatography, namely SEC-HPLC, is another important method for antibody standards and quality control. This method is mainly based on the size of the molecule or the difference in hydrodynamic radius to separate the molecules. Through SEC-HPLC, antibodies can be separated into three main forms: high molecular weight form (HMMS), main peak (mainly antibody monomer), and low molecular weight form (LMMS). Antibody purity can be calculated as the percentage of the main peak area on the chromatogram to the sum of all peak areas. Through the SEC-HPLC method, the percentage of antibody monomers in the preparation product can be measured, and information on the content of soluble aggregates and shears can be given. For a further description of the SEC-HPLC method, see, for example, J. Pharm. Scien., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994); J. Pharm. Bio. Anal. , 15:1928 (1997); J. Pharm. Bio. Anal., 14:1133-1140 (1986). In addition, see, for example, R. Yang et al., High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography (SE-UHPLC), Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx. doi.org/10.1016/j.jpba.2015.02.032; and Alexandre Goyon et al., Protocols for the analytical characterization of therapeutic monoclonal antibodies. I– Non-denaturing chromatographic techniques, Journal of Chromatography, http://dx.doi.org /10.1016/j.jchromb.2017.05.010.

Imaging capillary isoelectric focusing electrophoresis (iCIEF) can be used to analyze the charge heterogeneity of antibodies. This method can provide a quantitative distribution of charge variants. iCIEF achieves molecular separation based on the charge difference (apparent pI value) of molecules in the pH gradient. In iCIEF, the separation column is usually a short capillary (for example, a silica capillary with a length of 5 cm and an inner diameter of 100 μm). The protein is focused in the capillary column under high voltage and passed through the entire tube operated at 280 nM. The column imaging detection system performs real-time online monitoring of the focus. One advantage of this technology is that it can simultaneously record various charge variants of antibody samples through the full-column detection system. Generally speaking, in icIEF, the sample is mixed with urea and icIEF buffer, where the buffer contains methylcellulose, pi molecular weight standards, and amphoteric electrolytes. Then, you can use an iCIEF column such as the iCIEF column assembled by ProtionSimple on an iCIEF analyzer such as iCE280 analyzer (Protein Simple, Santa Clara, CA). After the sample is focused for a certain period of time, the absorbance at 280 nm is measured to obtain the focused antibody Spectra of charge variants. In the iCEIF spectrum, the protein-related peaks eluted before the main peak (ie, the main component) are classified as acidic components; in contrast, the protein-related peaks eluted after the main peak are classified as basic components. The relative amounts of the main components, acidic components, and basic components can be expressed as a percentage of the total peak area. For a further description of iCIEF, see, for example, Salas-Solano O et al., Robustness of iCIEF methodology for the analysis of monoclonal antibodies: an interlaboratory study, J Sep Sci. 2012 Nov;35(22):3124-9. doi: 10.1002 /jssc.201200633. Epub 2012 Oct 15; and Dada OO etc., Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation, Electrophoresis. 2015 Nov;36(21-22):2695-2702. doi: 10.1002/elps.201500219. Epub 2015 Sep 18.

The charge variant of the antibody in the antibody preparation can also be determined by cation exchange high performance liquid chromatography (CEX-HPLC). In this assay, peaks eluted from the CEX-HPLC column earlier than the retention time of the main peak are marked as "acidic peaks", and those eluted from the CEX-HPLC column later than the retention time of the main peak The peaks are labeled as "basic peaks".

Accelerated stability studies can be used to check the stability properties of products, which is conducive to the screening of stable pharmaceutical formulations. For example, a sample of the formulation can be placed at an elevated temperature, such as about 40°C ± 2°C, 25°C ± 2°C, for accelerated stability studies. Detection indicators can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method).

The formulation of the present invention has been subjected to accelerated stability experiments such as forced and 25°C at 40°C to investigate the effects of different pH values and different excipients on protein quality, and to evaluate the formulation of each formulation. The test items during the research process mainly include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method and non-reduced CE-SDS method) and charge variants (iCIEF method), etc. Thus, the stable antibody preparation to be protected by the present invention is obtained. 3. The use of the preparation

The liquid antibody preparation, solid antibody preparation, delivery device or pre-filled syringe of the present invention can prevent or treat cancer in patients, including administering to the patient an effective dose of the liquid antibody preparation or solid antibody preparation of the present invention, or through The delivery device or pre-filled syringe administers an effective dose of liquid antibody preparation or solid antibody preparation to the patient.

The cancer is preferably selected from melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, liver cancer, colorectal cancer, pancreatic cancer, gastric cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, endometrial cancer , Esophageal cancer, soft tissue sarcoma, cholangiocarcinoma, thyroid cancer, hepatocellular carcinoma or mesothelioma.

Wherein the liquid antibody preparation or solid antibody preparation can be administered to cancer patients simultaneously, separately or sequentially with ionizing radiation; or can be administered to cancer patients simultaneously, separately or sequentially with one or more chemotherapeutic drugs; or can be administered to cancer patients simultaneously, separately or sequentially with ionizing radiation; Multiple chemotherapeutic drugs are administered to cancer patients simultaneously, separately or sequentially; preferably, the chemotherapeutic drugs are selected from 5-fluorouracil, hydroxyurea, gemcitabine, methotrexate, doxorubicin, etoposide carboplatin, cisplatin, cyclic Phosphatiamine, melphalan, dacarbazine, paclitaxel, camptothecin, FOLFIRI, FOLFOX, docetaxel, daunorubicin, paclitaxel, oxaliplatin, or a combination thereof.

The therapeutic effect may include reducing physical symptoms. The optimal effective amount and concentration of antibodies for any particular subject will depend on many factors, including the age, weight, health and/or gender of the patient, the nature and extent of the disease, the activity of the particular antibody, and the body’s Clearance rate, and also includes any possible other treatments administered in combination with the antibody formulation. For specific situations, the effective amount delivered can be determined within the judgment of the clinician. In this regard, the application of known antibody-based drugs can provide certain guidance. The dosage can be a single-dose schedule or a multiple-dose schedule.

As used herein, "treatment" refers to slowing, interrupting, blocking, alleviating, stopping, reducing, or reversing the progression or severity of an existing symptom, disorder, condition, or disease. The desired therapeutic effects include, but are not limited to, preventing the appearance or recurrence of the disease, reducing symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving the prognosis. In some embodiments, the antibody molecules of the present invention are used to delay the progression of a disease or to slow the progression of a disease.

As used herein, "prevention" includes the inhibition of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition. In some embodiments, subjects with a family history of cancer are candidates for prophylactic regimens. Generally, in the context of cancer, the term "prevention" refers to the administration of drugs before the onset of signs or symptoms of cancer, especially in subjects at risk of cancer.

"Therapeutically effective amount" refers to the amount that is effective to achieve the desired therapeutic result at the required dose and for the required period of time. The therapeutically effective amount of the antibody or antibody fragment or its conjugate or composition can vary depending on various factors such as disease state, the age, sex and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual. A therapeutically effective amount is also an amount in which any toxic or harmful effects of the antibody or antibody fragment or conjugate or composition thereof are not as good as the therapeutically beneficial effects. Relative to an untreated article, a "therapeutically effective amount" preferably inhibits a measurable parameter (such as tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%. % And still more preferably at least about 80%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors. Alternatively, this property of the composition can be evaluated by testing the compound's ability to inhibit, said inhibition in vitro through assays known to the skilled artisan.

"Prophylactically effective amount" refers to an amount that effectively achieves the desired preventive result at the required dose and for the required period of time. Generally, since the prophylactic dose is used in the article before or in the early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount. Example embodiments of the detailed description of the preferred embodiments

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention and not to limit the scope of the present invention.

Histidine is a product of Shanghai Ajinomoto Amino Acid Co., Ltd., pharmaceutical grade.

Histidine hydrochloride is a product of Shanghai Ajinomoto Amino Acid Co., Ltd., pharmaceutical grade.

Sorbitol is a French Roquette product, the article number is H20110265, pharmaceutical grade.

Arginine hydrochloride is a product of Shanghai Ajinomoto Amino Acid Co., Ltd., pharmaceutical grade.

Sodium citrate (dihydrate) is a product of Merck from Germany, the product number is 1.37042.5000, pharmaceutical grade.

Sodium citrate (monohydrate) is a product of German Merck, the article number is 1.00242.5000, pharmaceutical grade.

Edetate disodium is a product of Nanjing Chemical Reagent Co., Ltd., Su Yao Zhunzi F15431201, pharmaceutical grade.

Polysorbate 80 is a product of Nanjing Weir Chemical Co., Ltd., Suyao Zhunzi F15423203, pharmaceutical grade.

The hydrochloric acid is a product of Merck, Germany, the product number is 1.00314.2508, pharmaceutical grade.

Protein content (UV method): Use an ultraviolet spectrophotometer (produced by Shimadzu, Japan, model UV-1800) to determine the protein content in the sample.

Polysorbate 80 content high performance liquid chromatography-fluorescence detection method (HPLC-FLD method): Use high performance liquid chromatography (Agilent, 1260 or equivalent) to separate, the chromatography column is Knitted Reactor Coil 5 mx 0.50mm ID, SUPELCO, mobile phase is 0.15 mol/L sodium chloride, 0.05 mol/L Tris, pH 8.0, 5% acetonitrile, 5.0 umol/L NPN (N-phenyl-1-naphthylamine), 15 ppm Brij, layer The column protection solution is 0.05% (w/v) NaN ₃ , the injection volume is 50 μl, the flow rate is 0.5 ml/min, the collection time is 30 minutes, the column temperature is 25°C, and the detection wavelength is 280 nm. Take the sample to be tested and dilute it to 2 mg/ml with ultrapure water as the test solution. Take the preparation buffer solution and dilute it with the same treatment method as above and use it as a blank solution. Take 10 μl each of the blank solution and the test solution into the liquid chromatograph to start the test.

Turbidity (OD350 nm method): Use an ultraviolet spectrophotometer (produced by Shimadzu, Japan, model UV-1800) to measure the absorbance of the sample at 350 nm to determine the turbidity of the sample.

Purity (SEC-HPLC method): Use size exclusion chromatography column to separate, the mobile phase is phosphate buffer solution (weigh 3.12 g sodium dihydrogen phosphate dihydrate, 8.77 g sodium chloride and 34.84 g arginine, ultra After the pure water is dissolved, adjust the pH to 6.8 with hydrochloric acid and make the volume to 1000 ml), the column protection solution is 0.05% (w/v) NaN ₃ , the injection volume is 50 μl, the flow rate is 0.5 ml/min, and the acquisition time is 30 Minutes, the column temperature is 25°C, and the detection wavelength is 280 nm. Take the sample to be tested and dilute it to 2 mg/ml with ultrapure water as the test solution. Take the preparation buffer solution and dilute it with the same treatment method as above and use it as a blank solution. Take 50 μl each of the blank solution and the test solution into the liquid chromatograph to start the test.

Purity (non-reduced CE-SDS method): Detected by capillary gel electrophoresis. The capillary is an uncoated capillary with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20.2 cm. Wash the capillary column with 0.1 mol/L sodium hydroxide, 0.1 mol/L hydrochloric acid, ultrapure water, and 70 psi electrophoresis gel before electrophoresis. Dilute the sample to be tested with an appropriate amount of ultrapure water to 2.0 mg/ml, take 50 μl of the above diluted sample into a 1.5 ml centrifuge tube, and add 45 μl of pH 6.5 sample buffer to it (weigh out one water of citric acid 0.32 g, 2.45 g of disodium hydrogen phosphate dodecahydrate, dissolved in 45 ml of ultrapure water, and dilute to 50 ml to prepare a citric acid-phosphate buffer solution. Accurately measure 200 μl of the buffer solution and add 10% (w/v) 80 μl of sodium lauryl sulfate solution, add water to 1 ml, mix well, ready to get), 1 μl internal standard (10 kDa protein, 5 mg/mL) (Beckman Coulter, catalog number: 390953) and 5 μl 250 mmol/L NEM solution (weigh 62 mg of N-ethylmaleimide and dissolve in 2 ml of ultrapure water), mix well and heat at 70±2℃ for 10±2 minutes, and cool After reaching room temperature, transfer to the sample bottle as the test solution. Take the same volume of preparation buffer as the test product, and perform the same operation as described above to prepare a blank solution. Sample injection conditions: -5 kV for 20 seconds; separation voltage: -15 kV for 35 minutes. The capillary column temperature is controlled at 25°C, and the detection wavelength is 220 nm.

Charge variants (iCIEF method): Use imaging capillary isoelectric focusing electrophoresis (iCIEF method) to detect. The capillary has an inner diameter of 100 μm and a total length of 5 cm. Before sample electrophoresis, 0.5% methyl cellulose solution (hereinafter also abbreviated as MC solution) and ultrapure water should be used to rinse the capillary column. The vacuum sampling method is adopted. The pre-focusing voltage and time are 1.5 kV for 1 minute, the focusing voltage and time are 3 kV for 8 minutes, the sampling time is 55 seconds, the sample tray temperature is 10°C, and the detection wavelength is 280 nm. Cathodic Stabilizer is 500 mmol/L arginine solution, 0.5% MC solution reduces the adhesion between protein and capillary. Dilute the test product to 1.0 mg/ml with water, take 20 μl of the diluted test product solution, and add 78 μl of the pre-mixed solution to it (the pre-mixed solution is as follows: 70 µl pI 0.5% MC solution, 4 µl amphoteric electrolyte ( pH 3-10), 2 µl cathode stabilizer, 1 µl pI 5.85 marker, 1 µl pI 9.99 marker), mix well to prepare the sample solution to be tested. Sample injection analysis, according to the area normalization method, calculate the main component, acidic component and alkaline component content. Example 1. Preparation of IBI319 protein

According to U.S. Provisional Application Nos. US62/700525 and US62/799274 and its follow-up PCT Application No. PCT/US2019/041517, the antibody of the present invention was obtained, code-named IBI319, and the antibody has the SEQ ID NO as described in Table 1. : The heavy chain sequences of 5 and 17 and the light chain sequences of SEQ ID NOs: 11 and 23 are humanized antibodies.

For example: The antibody of the present invention can be expressed and purified basically as follows. A suitable predetermined heavy chain: light chain vector ratio or a single vector system encoding heavy and light chains can be used to transiently or stably transfect an appropriate host cell, such as HEK 293 or CHO, with an antibody-secreting expression system. Through the use of one or more DNA molecules, the antibody A of the present invention can be transiently or stably transfected by the expression system of secreting antibodies, the DNA molecule encoding the first heavy chain having the amino acid sequence of SEQ ID NO: 5, The first light chain having the amino acid sequence of SEQ ID NO: 11, the second heavy chain having the amino acid sequence of SEQ ID NO: 17, the second light chain having the amino acid sequence of SEQ ID NO: 23 . The antibody fragments can be detected, for example, by UV absorption or SDS-PAGE, and can then be pooled. Common techniques can be used to concentrate and/or sterile filter the antibody. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, multimodal or hydroxyapatite chromatography. The purified product can be immediately frozen at -70°C or can be lyophilized. One of many common techniques can be used to purify antibodies. For example, the medium can be conveniently applied to a MabSelect chromatography column (GE Healthcare) or a KappaSelect chromatography column (GE Healthcare) that has been equilibrated with a compatible buffer [e.g., phosphate buffer (pH 7.4)]. The column can be washed to remove non-specifically bound components. The bound antibody can be eluted, for example, through a pH gradient (for example, 20 mM Tris buffer at pH 7 to 10 mM to 10 mM sodium citrate at pH 3.0, glycine buffer at pH 7.4 to 100 mM phosphate buffered saline at pH 7.4). The antibody fractions can be detected by UV absorption or SDS-PAGE, for example, and then they can be combined. Depending on the intended use, further purification is optional. The purified antibody can be concentrated and/or sterile filtered using conventional techniques. Soluble aggregates and polymers can be effectively removed by conventional techniques, including size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, multimodal chromatography or hydroxyapatite chromatography. The purified antibody can be immediately frozen at -70°C or lyophilized.

The following exemplarily shows the culture process of the antibody of the present invention, code-named IBI319, using CHO Gs cells (LONZA) transfection, basic medium Dynamis AGT Medium (Gibco); feeding reagents Cell Boost 7a, Cell Boost 7b ( HyClone). In the cell culture experiment, in the cell culture experiment on day 0, ^{inoculate cells at a density of 1.0x10 6} cells/ml into a 2 L cell reactor, with DO set to 40%, pH control at 7.05±0.20, and pre-culture temperature at 36.5 ℃, cultivate to 33.0℃ on the 6th day. Incubate to the 3rd, 5th, 7th, 9th, and 11th day, respectively, add 6.0% (w/w) of the initial culture weight of Fed Reagent A (purchased from HyClone), and 0.6% (w/w) of the initial culture weight of Fed Reagent B (purchased from HyClone). According to the results of the glucose concentration test every day, when the glucose is lower than 3.0 g/L, supplement the glucose concentrate to increase the glucose concentration in the cell sap to 4.0 g/L. According to the foam produced by ventilation, add 10% ADCF Antifoam Solution manually when the foam is 5 cm above the liquid level, and the total amount should not exceed 50 ppm. Monitor cell density, viability and cell biochemical metabolism every day. When the culture is continued to the 15th day or when the cell viability is ≤ 60%, the cell supernatant is collected for purification.

The gravity column used for purification was treated with 0.5 M NaOH overnight, and the glass bottles were washed with distilled water and then dry-baked at 180°C for 4 hours to obtain a purified column. Before purification, centrifuge the collected medium at 4500 rpm for 30 min, and discard the cells. Filter the supernatant with a 0.22 μl filter. Each tube is filled with 1 ml of Protein A and equilibrated with 10 ml of binding buffer (sodium phosphate 20mM NaCl 150mM, pH7.0). Add the filtered supernatant to the purification column and re-equilibrate with 15 ml of binding buffer. Add 5 ml of elution buffer (citric acid + sodium citrate 0.1 M, pH 3.5), collect the eluate, add 80 μl Tris-HCl for every 1 ml of eluate to adjust the pH to 7.0. The collected antibody is purified by 1 ml S-type strong cation exchange chromatography. The column is first equilibrated with 10 ml equilibration buffer (20 mM citric acid + sodium citrate, pH 5.0), and the affinity collected sample is added to the purification tube After the column, re-equilibrate with 10 ml of equilibration buffer, and then perform gradient elution with elution buffer (20 mM citric acid + sodium citrate + 1 M sodium chloride, pH 5.0), with an elution volume of 20 ml, and collect the sample Ultrafiltration was concentrated and exchanged into PBS (Gibco, 70011-044), and the concentration was detected. Example 2. The antibody of the present invention prepared and purified above was subjected to the following functional verification IBI319 affinity specificity and affinity determination (BLI assay )

Using the Fortebio Octet platform (Fortebio, USA), using the biofilm layer interference (BLI) assay method to determine the exemplary antibody IBI319 of the present invention and different species of proteins: human 4-1BB (Human 4-1BB), cynomolgus 4-1BB (Cyno) 4-1BB), human PD-1 (Human PD-1), Cyno PD-1 (Cyno PD-1) protein affinity, judge the affinity specificity of IBI319, and compare IBI319 and parent antibody 7A5 antibody with 4 The difference in affinity between -1BB and the affinity between IBI319 and the parental antibody 11444 antibody and 4-1BB. Table 3 Experimental samples name batch number source concentration IBI319 GG20191001 Homemade, Cinda Bio 50 mg/ml Parent antibody 7A5 20190806B Homemade, Cinda Bio 12.8 mg/ml Parent antibody 11444 DP1901002 Homemade, Cinda Bio 10 mg/ml For the antibody sequence of parent antibody 7A5 and parent antibody 11444, please refer to the sequence section 38-41 above. Other key materials Table 4 Reagent information name Origin and brand Item No. batch number PBS Shanghai Biotech E607016-0500 F517FA0002 BSA Jackson Immuno, USA 001-000162 144028 EZ-Link Sulfo-NHS-LC-Biotin American Thermo Scientific 21327 S1254537 Human 4-1BB U.S. R&D Systems 9220-4B DGCI0318011 Cyno 4-1BB Chinese novoprotein CB18 0331280 Human PD-1 ACRO Biosystems, USA PD1-H5221 C42bp1-958F1-P1 Cyno PD-1 ACROBiosystems, USA PD1-C5223 643-7CTF1-HZ Table 5 Solution preparation method name Preparation method SD buffer Add 0.05 g of BSA IgG-Free to 50 mL PBS (1x) solution, then add 25 µL of Tween-20 100 nM IBI319 solution Take 100 µL of 50 mg/mL IBI319, add 900 µL of PBS buffer, and mix to get 5 mg/mL, then take 200 µL of 5 mg/mL IBI319, add 800 µL of PBS buffer, and mix to get 1 mg/mL, then Take another 30 µL and add it to 1970 µL SD buffer, mix well, and the final concentration is 100 nM. 100 nM parent antibody 7A5 antibody solution Take 78 µL of the 12.8 mg/mL parent antibody 7A5 antibody, add 922 µL of PBS buffer, and mix to obtain 1 mg/mL, then add 30 µL to 1970 µL of SD buffer to a final concentration of 100 nM. 100 nM parent antibody 11444 solution Take 100 µL of 10 mg/mL parent antibody 11444 antibody, add 900 µL of PBS buffer, and mix to obtain 1 mg/mL, then take 30 µL and add 1970 µL of SD buffer to a final concentration of 100 nM. 1600 nM Human 4-1BB solution After dissolving the protein powder to 0.5 mg/mL, take 115 µL of Human 4-1BB and add it to 1885 µL of SD buffer to a final concentration of 1600 nM. 200 nM Cyno 4-1BB solution After dissolving the protein powder to 0.5 mg/mL, take 20 µL 0.5 mg/mL Cyno 4-1BB, add 180 µL PBS buffer, and mix to get 0.05 mg/mL, then take 145 µL and add 1855 µL SD buffer , The final concentration is 200 nM. 50 nM Human PD-1 solution After dissolving the protein powder to 0.5 mg/mL, take 20 µL of 0.5 mg/mL Human PD-1, add 180 µL of PBS buffer, and mix to obtain 0.05 mg/mL, then take 33.6 µL of 0.05 mg/mL Human PD -1, add 1966 µL SD buffer to a final concentration of 50 nM. 100 nM Cyno PD-1 solution After dissolving the protein powder to 0.5 mg/mL, take 20 µL 0.5 mg/mL Cyno PD-1, add 180 µL PBS buffer, and mix to get 0.05 mg/mL, then take 67 µL and add 1933 µL SD buffer , The final concentration is 100 nM.

Note: Each sample prepared above is the maximum concentration sample tested. According to the experimental design, the antigen is diluted by 2 times to the required concentration gradient for detection. 1. Biotin-labeled antibodies (used to detect the binding of antibodies to Fc-labeled antigens)

Take 180 µL of ultrapure water and add it to 1 mg EZ-Link Sulfo-NHS-LC-Biotin, dissolve it into a 10 mM solution, and then dilute it 100 times to 100 µM. According to the molar concentration of 1:1, mix the calculated amount of EZ-Link Sulfo-NHS-LC-Biotin with the protein to be labeled. After incubating for 1 hour at room temperature, use a desalting column to remove the unlabeled Biotin, and recover the Biotin label in an equal volume. The protein should be stored at 4℃ for later use. 2. Grouping of experimental samples

Place the sensor in 200 μL SD buffer and soak for 30 minutes before use.

The experiment grouping information is shown in Table 6. Add 200 μL of the corresponding sample to each well. Table 6 Experiment grouping information Cured Analyte Analyte concentration (nM) IBI319 Human 4-1BB 0, 100, 200, 400, 800, 1600 nM IBI319 Cyno 4-1BB 0, 100, 200, 400, 800, 1600 nM Parent antibody 7A5 Human 4-1BB 0, 12.5, 25, 50, 100, 200 nM Parent antibody 7A5 Cyno 4-1BB 0, 6.25, 12.5, 25, 50, 100 nM IBI319 Human PD-1 0, 3.125, 6.25, 12.5, 25, 50 nM IBI319 Cyno PD-1 0, 3.125, 6.25, 12.5, 25, 50 nM Parent antibody 11444 Human PD-1 0, 6.25, 12.5, 25, 50, 100 nM Parent antibody 11444 Cyno PD-1 0, 6.25, 12.5, 25, 50, 100 nM 3. Detection

The detection is divided into 5 steps, and each loop is as follows: Baseline→Loadings→Baseline→Binding to the serially diluted protein (Association)→Dissociation. Judgment criteria:

The curve was fitted with the ForteBio Octet instrument analysis software (Fortebio data analysis 11.0) using 1:1 binding (global fitting). The reliability of the result is judged by analyzing the degree of fit of the fitted curve and the R2 value. The fitting result accords with R2>0.95, and the KD value is calculated by the combination and dissociation constant. Experimental results:

Affinity test results of IBI319 with PD-1 of different species and 4-1BB of different species Table 7 Affinity test results of IBI319 with PD-1 of different species and 4-1BB of different species Human PD-1 KD (nM) Cyno PD-1 K _D average (M) ± SD Human CD137 K _D average (M) ± SD Cyno 4-1BB K _D average (M) ± SD IBI319 6.32E-10± 5.94E-11 7.64E-10± 1.25E-10 7.19E-07± 2.14E-07 2.08E-08± 5.04E-09 Parent antibody 11444 1.14E-09± 8.96E-11 1.66E-09± 1.48E-10 Not detected Not detected Parent antibody 7A5 Not detected Not detected 4.62E-07± 1.30E-07 6.58E-09± 4.19E-10 in conclusion:

IBI319 has a strong affinity with human PD-1 and cynomolgus PD-1, and IBI319 has a weak affinity with human 4-1BB and cynomolgus 4-1BB. IBI319 affinity study determination ( surface plasmon resonance , SPR method ) :

Using the Biacore platform (GE Healthcare, Sweden), the SPR method was used to detect the affinity of IBI319 with human PD-1, cynomolgus PD-1, human 4-1BB (CD137), and cynomolgus 4-1BB. method:

Using the SPR method, after coupling the anti-human Fc antibody to the surface of the CM5 chip, the coupled anti-human Fc antibody was used to capture IBI319, gradiently diluted human PD-1, cynomolgus PD-1, human 4-1BB, and crab-eating Monkey 4-1BB binds to captured IBI319 respectively. IBI319's parent antibody 11444 (anti-PD-1 antibody) and parent antibody 7A5 (anti-4-1BB antibody or anti-CD137 antibody) were used as controls, respectively. Finally, the affinity value is calculated by fitting the binding and dissociation curve. Affinity test:

The buffer used in the experiment was HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20) solution with pH 7.4, and the detection temperature was 25°C. The experiment included: 1) coupling the anti-human Fc antibody to the surface of the CM5 chip; 2) detecting the affinity of IBI319 and control antibodies with Human PD-1, Cyno PD-1, Human 4-1BB, and Cyno 4-1BB. Experimental results:

Experimental results show that the affinities of IBI319 with Human PD-1 and Cyno PD-1 are about 0.1 nM and 0.04 nM, respectively, and the affinities with Human 4-1BB and Cyno 4-1BB are about 394 nM and 68.5 nM, respectively. The affinity data is shown in Table 8 below. Table 8 Test results of affinity between IBI319 and Human PD-1, Cyno PD-1, human 4-1BB, and cynomolgus 4-1BB Antibody antigen K _D mean ±SD (M) IBI319 Human PD-1 1.02E-10±8.31E-12 Parent antibody 11444 Human PD-1 1.37E-10±2.89E-11 IBI319 Cyno PD-1 4.09E-11±8.95E-12 Parent antibody 11444 Cyno PD-1 1.51E-10±1.99E-11 IBI319 Human 4-1BB 3.94E-07±2.30E-08 Parent antibody 7A5 Human 4-1BB 2.19E-07±1.87E-08 IBI319 Cyno 4-1BB 6.85E-08±1.44E-09 Parent antibody 7A5 Cyno 4-1BB 5.25E-08±2.99E-09 in conclusion:

IBI319 has a strong affinity with human PD-1 and cynomolgus PD-1, and IBI319 has a weak affinity with human 4-1BB and cynomolgus 4-1BB. discuss:

The strong affinity of IBI319 with PD-1 is conducive to a stronger PD-1/PD-L1 blocking effect. Anti-4-1BB antibody has a balance between T cell activation ability and liver toxicity, and the two have a great correlation with their affinity. IBI319 has a weak affinity with 4-1BB, and it is more conducive to obtaining a larger function and safety window while retaining the activation function of T cells. The bispecific antibody design of IBI319 can play a better synergistic effect on the immune suppression and T cell activation in the tumor microenvironment. IBI319 in vitro efficacy 1 : IBI319 PD-L1 binding blocking activity test protocol:

Co-culture CHO cells (CHO-K1/PD-L1) expressing PD-L1 and TCR activator on the membrane surface and Jurkat cells (Jurkat/PD1-NFAT-luc) expressing PD-1 and NFAT-luc reporter, adding different concentrations The IBI319, parental antibody 11444, parental antibody 7A5 and hIgG1 can reflect the blocking activity of the drug on PD-1/PD-L1 by measuring the luciferase enzyme activity in the system. Experimental steps: (1) CHO-K1/PD-L1 cell plating (2) antibody preparation

Use Assay buffer (RPMI 1640+1% FBS) to prepare IBI319, parental antibody 11444, parental antibody 7A5, parental antibody 11444 + parental antibody 7A5, IgG1, starting from a constant concentration of 2000 nM, 4-fold dilution, total 11 gradient points, see Table 9 for details. Table 9 Abs The initial concentration Preparation concentration 200 µl added volume (μl) IBI319 50 mg/ml 4000 nM (0.6 mg/ml) 2.4 Parent antibody 11444 10 mg/ml 12 Parent antibody 7A5 12.8 mg/ml 9.4 Parental antibody 11444 + Parental antibody 7A5 Same as above 6, 4.7 IgG1 7.3 mg/ml 16.4 (3) Preparation of Jurkat cells (4) Loading 1. Take out the 96-well plate for culturing CHO-K1/PD-L1, and remove 95 µl of supernatant from each well. 2. Quickly add 40 µl/well of the prepared antibody according to the plate layout, set the medium control group, that is, add 40 µl assay buffer. 3. Add 40 µl/well of Jurkat/PD1-NFAT-luc cells (5x10 ⁴ cells/well). Incubate at 37°C, 5% CO _{2 for} 6 h. (5) The test results of luciferase enzyme activity measured by the multifunctional enzyme label reader (MOLECULAR DEVICES):

Through Jurkat / NFAT-luc reporter system experiments, IBI319 NFAT_Luciferase message display activation activity, its activity EC ₅₀ of 8.14 nM. _{The EC 50} of the control antibody parent antibody 11444 to activate NFAT_Luciferase was 1.08 nM; the CD137 monoclonal antibody showed no activity. See Figure 12 for details. Experimental results:

The anti-PD-1 end of IBI319 showed the activity of blocking PD-1/PD-L1 binding. IBI319 in vitro efficacy 2 : Experimental protocol for determining the agonistic activity of IBI319 CD137:

Co-culture Jurkat cells (Jurkat/CD137-NFκB-luc) expressing CD137 and NFκB-luc reporter with CHO-S/hPD-1 cells (CHO-S expressing hPD-1), or without CHO-S/hPD- 1 cell, then add different concentrations of IBI319, parental antibody 11444, parental antibody 7A5 and hIgG1, by measuring the luciferase enzyme activity in the system to reflect the drug's agonistic activity on the CD137 pathway and cross-linking with CD137-PD-1 effect. Experimental steps:

Jurkat cell culture medium for CD137 and NFκB-luc reporter: 90% RPMI 1640 with l-glutamine, 10% FBS, 200µg/ml hygromycin B, 500µg/ml Geneticin, 1mM sodium pyruvate, 0.1mM MEM NEAA;

CHO-S cell culture medium expressing hPD-1: CD Forti CHO+1mM MTX+1%ACA+1%GlutaMAX. (1) Antibody preparation

Use Assay buffer (RPMI 1640+1% FBS) to prepare IBI319 (αPD1/CD137), parent antibody 11444 (αPD-1), parent antibody 7A5 (pool3), final concentration of IgG1: 400, 80, 16, 3.2, 0.64, 0.128, 0.0256, 0.00512, 0.001024 nM (5-fold serial dilution). (2) Cell preparation 1. Cultivate Jurkat/CD137-NFκB-luc until the doubling time is stable, and the cell density is 3.5x10 ⁵ ~ ^{2.2x10 6} cells/ml. After the cell count, use Assay buffer to adjust the cell density to 1.0x10 ⁶ cells/ml for use. 2. Cultivate CHO-S/ hPD-1 cells to the logarithmic phase. After cell counting, adjust the cell density to 2.0x10 ⁶ cells/ml with Assay buffer for use. (3) Loading and plating 1. Add the prepared antibody to a 96-well flat-bottom white plate according to the plate layout, 25 µl/well, set the medium control group, that is, add 25 µl assay buffer. 2. After mixing the adjusted density of CHOS/hPD-1 and Jurkat/CD137-NFkB-luc in equal volumes, add 50 µl/well to the cell dish (5.0x10 ⁴ cells/well and 2.5×104 cells/well, respectively ).

Set the no CHO-S group, that is, add 25 µl assay buffer and 25 µl Jurkat/CD137-NFkB-luc.

Set the backgroud group and add only 75 µl assay buffer. 3. Place in a 37℃±2℃, 5%±1% CO ₂ incubator and incubate for 6 hours. (4) The test results of luciferase enzyme activity determined by the multifunctional enzyme labeling reader:

Through the experiment of the Jurkat/CD137-NFKB-luc reporter system, IBI319 showed the activity of activating the NFKB_Luciferase message. This activation activity is very weak under the condition of independent culture with Jurkat/CD137-NFKB-luc cells (Figure 14), and after adding CHO -S/PD-1 cells were significantly enhanced when they were co-cultured (Figure 13). The degree of enhancement is positively correlated with the ratio of CHO-S/PD-1 cells (Figure 15). Experimental results:

The anti-CD137 end of IBI319 shows activation activity, which depends on the performance of PD-molecules. Mixed lymphocyte reaction (MLR) can be induced in T cell activation IBI319

The activity of blocking PD-1/PD-L1 binding at the PD1 end of IBI319 was detected by human allogeneic mixed lymphocyte reaction. The specific method is as follows: Human peripheral blood mononuclear cells (PBMC) are derived from frozen state (AllCells), or obtained from fresh whole blood by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation. Use human monocyte separation kit II or CD14 magnetic beads (Miltenyi Biotec) and AutoMACS Pro separator (Miltenyi Biotec) to separate CD14+ monocytes from PBMC. In the presence of 1,000 IU/mL hGM-CSF (R&D Systems, 215-GM-050 or Sanofi, NDC 0024-5843-01) and 500 IU/mL hIL-4 (R&D Systems, 204-IL-050), Monocytes were cultured in RPMI-1640 complete medium containing 10% FBS for 2 days (Table 14) or 5 days (Table 16, 17) to obtain immature dendritic cells (DC). The human CD4+ T cell isolation kit (Miltenyi Biotec) was used to purify CD4+ T cells from human PBMC (AllCells or Indiana Blood Center) from different healthy donors. After that, the two types of cells from different donors were mixed in a 96-well V-plate, each well containing 100 µL AIM-V medium (Thermo Fisher Scientific), 5x10 ⁴ -1x10 ⁵ CD4+ T cells and 5x10 ³ immature DC cells. Dilute the antibody to be tested (human IgG1-EN, p-11444, p-7A5, IBI319) and the control antibody, add them to the reaction plate, 100 µL/well (a total of 8 replicate wells), 37°C, 5% CO ₂ training 67 hours. Or add the antibody to be tested to the reaction plate, 200 µL/well (3 replicate wells in total), and incubate for 4 days. The supernatant was collected, and the human IFN-γ ELISA kit (R&D Systems; SIF50 or DY285) and the human IL-2 ELISA kit (R&D Systems; S2050) were used to detect the contents of IFN-γ and IL-2, respectively, according to the instructions. Nine different donor pairs were used for testing. GraphPad Prism (GraphPad Software) was used to calculate the EC ₅₀ values of three pairs of different donor cells.

In the MLR experiment, using allogeneic human DC cells and CD4+ T cells, adding p-11444, p-11444+p-7A5, and IBI319, compared with IgG1, enhanced T cell activation and showed a dose-dependent manner. Table 10 shows _{the EC 50 of the} combined amount of IFN-γ and IL-2. The experiment did not detect the activity of CD137 monoclonal antibody.

In summary, the results of the allogeneic mixed lymphocyte reaction show that for multiple donor pairs, IBI319 retains the PD-1 blocking activity similar to that of p-11444, and increases the release of cytokines. Table 10 PD1 blocking activity of IBI319 in MLR experiment [cytokine mean EC ₅₀ (nM) values across 3 donors] Abs Control IgG1 p-11444 p-7A5 p-11444+ p-7A5 IBI319 IFN-γ ＞32 0.088± 0.071 ＞32 0.038± 0.013 0.091± 0.074 IL-2 ＞32 0.238± 0.209 ＞32 0.158± 0.063 0.297± 0.305 IBI319 can induce a unique immune gene expression profile in the H292 NSCLC Winn tumor model

The expression of human immune-related genes in tumor tissues of H292 Winn mouse model treated with IBI319, p-7A5, p-11444 and/or control human IgG1 was tested. The general method is as follows: On day 0, mice are injected intraperitoneally with a mixture of H292 tumor cells and frozen human peripheral blood mononuclear cells. The test antibody and the control human IgG1 were administered sequentially at the dose of 10 mg/kg on the first, eighth, and fifteenth days. On the 16th day, the tumor tissues were collected and quickly frozen in liquid nitrogen. The tumor tissue was lysed using MagMAX-96 total RNA extraction kit (Life Technologies), and homogenized with a tissue grinder (Qiagen). MagMAX Express-96 Deep Well Magnetic Particle Processor (Life Technologies) was used to extract total RNA, and OD _{260 and OD 280} were measured with a spectrophotometer for quantification. QuantiGene 2.0 plex assay (Affymetrix) and FLEXMAP 3D Luminex instrument (ThermoFisher) were used for gene expression level analysis of total RNA (500 ng). Use a quality control script to convert the MFI value into relative gene expression (normalized net MFI value). The fold change of the expression of each gene compared with the control group is calculated by the formula (normalized net MFI value/average normalized net MFI value). The average fold change of each gene expression compared with the control group was calculated by single factor analysis of variance.

Gene expression analysis showed that IBI319 induced a special gene expression profile in the in vivo model H292 tumor tissue, including T cell infiltration and activation related genes (such as CD3E, CD4, CD8B, IFN-γ, GZMB), multiple cytokines and chemotaxis Hormones, and MHC class I and II antigens (such as HLA-B, HLA-DRA). As shown in Table 11 below, these gene changes induced by IBI319 are different from the two affinity antibodies and combination antibodies. Table 11 Gene Fold Change P-value IBI319 p-7A5 p-11444 p-7A5 + p-11444 IBI319 p-7A5 p-11444 p-7A5 + p-11444 CD3E 7.26 2.11 0.81 1.76 0.00 0.20 0.72 0.36 CD4 5.12 1.73 0.7 1.07 0.01 0.34 0.53 0.91 CD8B 9.04 3.48 1.3 3.89 0.00 0.03 0.64 0.03 IFNγ 8.27 2.62 0.87 1.30 0.00 0.08 0.80 0.64 HLA-B 2.23 1.38 0.73 1.08 0.02 0.27 0.29 0.79 HLA-DRA 4.81 1.86 0.52 1.24 0.02 0.30 0.28 0.73 CCL5 9.56 2.39 1.06 3.30 0.00 0.16 0.92 0.07 CXCL10 11.05 2.95 0.54 1.58 0.01 0.20 0.46 0.60 GZMB 8.22 3.06 0.53 2.28 0.01 0.10 0.35 0.25 PRF1 11.59 3.45 0.65 2.48 0.00 0.08 0.52 0.22 IBI319 shows anti-tumor activity in H292 NSCLC Winn tumor model

The tumor growth inhibition rate of the H292 tumor xenograft mouse model treated with IBI319, p-11444, p-7A5, p-11444+p-7A5 was tested. Female NOD/SCID Gamma (NSG) mice (Jackson Laboratories) were used for the experiment. The human NSCLC cell line NCIH292 (ATCC; CRL-1848) and human PBMC (Stem Cell Technologies) were mixed at a ratio of 4:1. After centrifugation, resuspend in HBSS, adjust the cell density of NCI-H292 to ¹⁰ ×10 6 cells/ml and the density of PBMC to 2.5× ^{10 6} cells/ml. On day 0, each mouse was injected subcutaneously with 0.2 ml of cell mixture on the right side, and one control group was injected with tumor cells only. On the first day, the mice were randomly divided into groups of 8 mice/group, and the administration was started. Antibodies to be tested include control IgG, p-7A5, p-11444, combination p-11444 + p-7A5 and IBI319. It was injected intraperitoneally at a dose of 10 mg/kg, once a week for 4 weeks. Body weight and tumor volume are measured twice a week. Tumor volume (mm ³ ) is calculated according to the formula π/6 * length * width 2, and %T/C is calculated according to the formula 100 x △T /△C (such as △T ＞ 0). Use the MIXED program of SAS software for statistical analysis.

As shown in Table 12 below, the IBI319 (10 mg/kg) treatment group inhibited tumor growth compared with the control group IgG (%T/C=~2%, p＜.001), and 6 out of 8 mice reached A complete remission. The combination of p-11444+p-7A5 and monoclonal antibodies p-7A5 and p-11444 did not show efficacy. Table 12 Treatment/ Comparison Xenograft p-value for tumor volume T/C% CR Control IgG 10 mg/kg NCI-H292 p=.695 115.8 0/8 Control IgG 10 mg/kg NCI-H292 + hPBMC NA NA 0/8 p-11444 10 mg/kg NCI-H292 + hPBMC p=.377 71.5 1/8 p-7A5 10 mg/kg NCI-H292 + hPBMC p=.702 115.4 1/8 p-11444 + p-7A5 NCI-H292 + hPBMC p=.144 57.0 1/8 IBI319 10 mg/kg NCI-H292 + hPBMC p＜.001 1.9 6/8 Example 3. pH Screening Experiment

This experiment mainly investigates the influence of different pH values on the stability of IBI319 protein. A total of 5 pH values were designed, namely pH 5.0, 5.5, 6.0, 6.5 and 7.0. Through pH screening experiments, a better pH range was obtained.

The specific experimental steps are as follows: 1. Prepare a buffer solution containing 10 mM histidine and 5% (w/v) sorbitol, adjust the pH to 5.0, 5.5, 6.0, 6.5 and 7.0 with hydrochloric acid, and replace the IBI319 protein by ultrafiltration to the above different pH values Adjust the protein content to 30 mg/ml in the buffer solution, and add polysorbate 80 to 0.2 mg/ml to obtain pH 5.0, pH 5.5, pH 6.0, pH 6.5 and pH 7.0 samples, which are filtered and aliquoted into vials. Stoppered, capped. 2. Take samples of the above samples on the 0th day, 1st week, 2nd week and 4th week respectively, and conduct stability investigation under the condition of 40℃±2℃, observe the appearance and visible foreign matter, and detect the protein content and turbidity , Purity [size exclusion high performance liquid chromatography (SEC-HPLC method) and non-reduced sodium dodecyl sulfate capillary electrophoresis (non-reduced CE-SDS method)] and charge variants [imaging capillary isoelectric focusing Electrophoresis (iCIEF method)].

The judging criteria for unchanged quality are shown in Table 13. Table 13 Judgment criteria for unchanged quality Test items Judgment criteria unchanged Appearance (observation method) Clear to slightly opalescent, colorless to light yellow liquid, no foreign matter Visible foreign body (Visible foreign body inspection method) Comply with the general rule 0904 of "The Pharmacopoeia of the People's Republic of China" (2015 edition, four parts) Protein content (UV method) Change rate ≤10% Polysorbate 80 content (HPLC-FLD method) 0.3~0.7mg/ml Turbidity (OD350 nm method) Change value ≤0.02 Purity (SEC-HPLC method) Main peak change value ≤ 1% Purity (non-reduced CE-SDS method) Main peak change value ≤ 2% Charge variant (iCIEF method) The change value of the main component and the acidic acid component ≤ 2%

The results are as follows: 1. Appearance and visible foreign matter

The pH 5.0, pH 5.5, pH 6.0, pH 6.5, and pH 7.0 samples were placed at 40°C±2°C for 4 weeks, and the appearance and visible foreign matter were all qualified. 2. Protein content

The test results of protein content are shown in Table 14. Table 14 Results of pH screening protein content (UV method, mg/ml) sample name time 0 days 1 week Two weeks 4 weeks pH 5.0 29.5 29.0 29.0 29.3 pH 5.5 29.7 29.6 29.4 29.4 pH 6.0 29.4 29.3 29.4 29.2 pH 6.5 30.0 29.9 29.9 29.6 pH 7.0 28.6 28.4 28.3 28.2

Table 14 shows that the protein content of each sample did not change significantly after being placed at 40°C±2°C for 4 weeks. 3. Turbidity

The turbidity results are shown in Table 15, and the trend of change is shown in Figure 2. Table 15 pH screening turbidity results (OD350 nm method) sample name time 0 days 1 week Two weeks 4 weeks pH 5.0 0.040 0.049 0.051 0.054 pH 5.5 0.054 0.062 0.061 0.060 pH 6.0 0.056 0.062 0.060 0.068 pH 6.5 0.060 0.065 0.070 0.090 pH 7.0 0.069 0.081 0.085 0.108

The results showed that the turbidity of the samples at pH 5.0, pH 5.5, pH 6.0, pH 6.5 and pH 7.0 increased by 0.014, 0.006, 0.012, 0.03 and 0.039 after being placed at 40℃±2℃ for 4 weeks. The samples were at pH 5.0~pH The change in turbidity is small between 6.0. 4. Purity

The results of purity (SEC-HPLC method and non-reducing CE-SDS method) are shown in Table 16, and the trend of change is shown in Figs. 3 and 4. Table 16 Purity results of pH screening Detection item sample name time 0 days 1 week Two weeks 4 weeks Purity (SEC-HPLC method) pH 5.0 99.9 96.5 95.6 94.8 pH 5.5 99.9 99.5 99.3 98.9 pH 6.0 99.8 99.5 99.3 98.9 pH 6.5 99.8 99.3 99.0 98.5 pH 7.0 99.8 99.1 98.9 98.4 Purity (non-reduced CE-SDS method) pH 5.0 92.9 92.7 92.3 90.4 pH 5.5 92.8 92.5 92.6 91.7 pH 6.0 92.9 92.6 92.6 91.3 pH 6.5 92.7 92.6 92.5 90.8 pH 7.0 93.0 92.6 92.2 90.2

The results showed that the sample purity of pH 5.0, pH 5.5, pH 6.0, pH 6.5, and pH 7.0 (SEC-HPLC method) decreased by 5.1%, 1.0%, 0.9%, 1.3%, respectively, after being placed at 40℃±2℃ for 4 weeks. And 1.4%. The sample purity of pH 5.0, pH 5.5, pH 6.0, pH 6.5 and pH 7.0 (non-reducing CE-SDS method) decreased by 2.5%, 1.1%, 1.6%, 1.9% and 2.8%, respectively. IBI319 protein is relatively stable under the conditions of pH 5.5 and pH 6.0. 5. Charge variants

The results of the charge variant (iCIEF method) are shown in Table 17, and the trend of its change is shown in Figure 5 and Figure 6. Table 17 Results of pH screening of charge variants (iCIEF method, %) sample name time 0 days 1 week Two weeks 4 weeks pH 5.0 Acidic component 17.9 20.8 25.2 34.0 main ingredient 76.3 72.3 67.4 59.4 Alkaline component 5.9 6.9 7.5 6.6 pH 5.5 Acidic component 17.4 20.4 23.8 32.8 main ingredient 77.4 72.7 69.3 60.7 Alkaline component 5.1 6.8 6.9 6.5 pH 6.0 Acidic component 17.6 20.9 24.6 32.5 main ingredient 76.9 72.7 68.6 61.3 Alkaline component 5.5 6.5 6.8 6.3 pH 6.5 Acidic component 17.7 22.5 27.1 38.1 main ingredient 76.8 71.6 67.3 57.3 Alkaline component 5.5 5.9 5.6 4.6 pH 7.0 Acidic component 18.2 26.9 34.0 47.4 main ingredient 76.5 67.8 60.9 48.4 Alkaline component 5.4 5.3 5.1 4.2

The results showed that the main components and acidic components of the charge variants of all samples changed significantly after being placed for 4 weeks under the condition of 40℃±2℃. Compared with day 0, the main components of charge variants of pH 5.0, pH 5.5, pH 6.0, pH 6.5, and pH 7.0 samples decreased by 16.9%, 16.7%, 15.6%, 19.5%, and 28.1%, respectively, in the fourth week. They increased by 16.1%, 15.4%, 14.9%, 20.4% and 29.2% respectively. From the analysis of the test results, IBI319 protein is more stable under the conditions of pH 5.5 and pH 6.0.

In summary, the results of pH screening experiments show that IBI319 protein is relatively stable at pH 5.5-6.0. Take pH 5.7 as an example for subsequent prescription screening experiments. Example 4. Prescription screening experiment

This experiment investigates the effect of buffer system (histidine and citric acid) and stabilizers (sorbitol and arginine hydrochloride) on the stability of IBI319 protein.

1. Design the following 5 prescriptions: Preparation of prescription 1: weigh 1.55 g of histidine, 50.00 g of sorbitol and purified water to make the volume to 980 mL, adjust the pH to 5.7 with HCl, and make the volume to 1 L. The IBI319 protein was replaced by ultrafiltration into the above solution. After the replacement, adjust the protein content to about 50 mg/ml, and finally add 0.50 g of polysorbate 80 to obtain prescription 1. The composition of prescription 1 is: 50.0 mg/ml IBI319, 1.55 mg/ml histidine, 50.00 mg/ml ml sorbitol, 0.50 mg/ml polysorbate 80, pH 5.7, the balance is water.

Preparation of prescription 2: weigh 1.55 g histidine, 25.00 g sorbitol, 16.85 g arginine hydrochloride and purified water to make the volume to 980 mL, adjust the pH to 5.7 with HCl, and make the volume to 1L. The IBI319 protein was replaced by ultrafiltration into the above solution. The protein content is about 50 mg/ml, and finally 0.50 g of polysorbate 80 is added to obtain prescription 2. The composition of prescription 2 is: 50.0 mg/ml IBI319, 1.55 mg/ml histidine, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7, the balance is water.

Preparation of prescription 3: weigh 1.55 g of histidine, 16.85 g of arginine hydrochloride and purified water to make the volume to 980 mL, adjust the pH to 5.7 with HCl, and make the volume to 1L. The IBI319 protein was replaced by ultrafiltration into the above solution. After the replacement, adjust the protein content to about 50 mg/ml, and finally add 0.50 g of polysorbate 80 to obtain prescription 3. The composition of prescription 3 is: 50.0 mg/ml IBI319, 1.55 mg/ml histidine, 16.85 mg/ml ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7, the balance is water.

Preparation of prescription 4: weigh 2.94 g of sodium citrate (dihydrate), 16.85 g of arginine hydrochloride and purified water to make the volume to 980 mL of citric acid, adjust the pH to 5.7, and make the volume to 1L. The IBI319 protein was replaced by ultrafiltration into the above solution. After the replacement, adjust the protein content to about 50 mg/ml, and finally add 0.50 g of polysorbate 80 to obtain prescription 4. The composition of prescription 4 is: 50.0 mg/ml IBI319, 2.94 mg/ml sodium citrate (dihydrate ), 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7, the balance is water.

Preparation of prescription 5: weigh 1.55 g histidine, 25.00 g sorbitol, 16.85 g arginine hydrochloride, 0.01 g edetate disodium and purified water to make the volume to 980 mL, adjust the pH to 5.7 with HCl, and make the volume to 1 L. The IBI319 protein was replaced by ultrafiltration into the above solution. After the replacement, adjust the protein content to about 50 mg/ml, and finally add 0.50 g of polysorbate 80 to obtain prescription 5. The composition of prescription 5 is: 50.0 mg/ml IBI319, 1.55 mg/ml histidine, 25.00 mg/ml ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.01 mg/ml disodium edetate, 0.50 mg/ml polysorbate 80, pH 5.7, the balance is water.

Prescription 1, Prescription 2, Prescription 3, and Prescription 5 used hydrochloric acid to adjust pH, and prescription 4 used citric acid to adjust pH.

Filter each prescription sample into a vial, stopper and cap.

2. The above-mentioned prescription samples were subjected to 40°C, 25°C, shaking, and freeze-thaw stability investigations. The specific plan is shown in Table 18. Table 18 Experimental conditions and sampling Experiment name prescription Experimental conditions and sampling plan Test items 40℃ stability test Prescription 1~Prescription 5 Placed at 40℃, sampling at 0 days, 1 week, 2 weeks and 4 weeks Appearance, visible foreign matter, protein content, purity (SEC-HPLC method and non-reduced CE-SDS method), charge variant (iCIEF method) 25℃ stability test Prescription 1~Prescription 5 Placed at 25℃, sampling at 0 days, 1 month and 2 months Oscillation experiment Prescription 5 650 r/min, room temperature, protected from light, sampling on the 0th and 5th days Freeze-thaw experiment Prescription 5 Freeze and store below -30℃ for 1 day, thawing at room temperature as a loop, sampling at the 0th and 6th loops

The judging criteria for unchanged quality are shown in Table 13.

The results are as follows: 1. Appearance and visible foreign matter

Prescription 1 to prescription 5 samples were placed at 40°C for 4 weeks and at 25°C for 2 months, and the appearance and visible foreign matter were all qualified. 2. Protein content

The test results of protein content are shown in Table 19. Table 19 Prescription screening results of protein content (UV method, mg/ml) sample name 0 days 40℃ 25℃ 1 week Two weeks 4 weeks January February Prescription 1 51.8 51.5 51.4 51.5 50.8 51.7 Prescription 2 52.5 52.7 52.7 52.3 52.5 52.1 Prescription 3 52.3 52.3 52.0 51.8 52.7 52.3 Prescription 4 52.3 52.3 52.0 51.8 52.7 52.3 Prescription 5 53.4 53.2 52.8 52.5 52.7 52.1

Table 19 shows that the protein content of each prescription did not change significantly after being placed at 40°C for 4 weeks and at 25°C for 2 months. 3. Purity

The results of purity (SEC-HPLC method and non-reducing CE-SDS method) are shown in Table 20. Table 20 Prescription screening purity results Detection item sample name 0 days 40℃ 25℃ 1 week Two weeks 4 weeks January February Purity (SEC-HPLC method) Prescription 1 99.6 99.1 98.8 98.2 99.3 98.9 Prescription 2 99.6 98.7 98.4 97.2 99.1 98.7 Prescription 3 99.6 98.5 98.0 96.6 99.0 98.4 Prescription 4 99.6 98.5 98.0 96.8 99.0 98.5 Prescription 5 99.5 98.9 98.6 97.9 99.2 98.9 Purity (non-reduced CE-SDS method) Prescription 1 94.5 94.5 93.9 93.0 94.3 93.6 Prescription 2 94.4 94.4 93.7 92.8 94.0 93.3 Prescription 3 94.5 94.3 93.7 92.6 94.1 93.1 Prescription 4 94.3 94.3 94.0 92.9 93.9 93.4 Prescription 5 94.4 94.4 94.0 93.0 94.3 93.8

Table 20 shows that when placed at 40°C for 4 weeks and at 25°C for 2 months, the purity of the samples (non-reduced CE-SDS method) did not change significantly. After being placed at 40℃ for 4 weeks, the purity (SEC-HPLC method) of the samples from prescription 1 to prescription 5 all changed significantly. Compared with day 0, the purity (SEC-HPLC method) decreased by 1.4%, 2.4%, 3.0%, respectively. 2.8% and 1.6%. The change trend is shown in Figure 7. When placed at 25°C for 2 months, the purity of prescription 3 and prescription 4 (SEC-HPLC method) decreased by 1.2% and 1.1%, which exceeded the judgment standard. In summary, prescription 1 and prescription 5 are better than other prescriptions. 4. Charge variant

The results of the charge variant (iCIEF method) are shown in Table 21, and the trend of change is shown in Figs. 8-11. Table 21 Results of prescription screening of charge variants sample name 0 days 40℃ 25℃ 1 week Two weeks 4 weeks January February Prescription 1 Acidic component 20.9 24.2 29.2 35.8 23.4 27.7 main ingredient 77.8 73.6 68.1 61.3 74.8 70.4 Alkaline component 1.3 2.2 2.7 3.0 1.9 2.0 Prescription 2 Acidic component 21.6 24.4 28.2 34.8 22.9 27.2 main ingredient 77.4 73.7 69.2 62.3 74.9 70.8 Alkaline component 1.0 1.9 2.6 2.9 2.2 2.0 Prescription 3 Acidic component 21.8 23.9 27.6 34.2 22.9 26.5 main ingredient 77.1 74.3 69.6 63.1 75.0 71.4 Alkaline component 1.1 1.8 2.8 2.7 2.1 2.0 Prescription 4 Acidic component 21.8 24.3 29.1 35.4 23.3 27.9 main ingredient 77.2 73.9 68.3 61.9 74.5 69.9 Alkaline component 1.0 1.7 2.6 2.7 2.2 2.2 Prescription 5 Acidic component 21.6 24.0 26.9 32.4 22.3 26.1 main ingredient 77.3 74.2 70.5 64.6 75.5 71.9 Alkaline component 1.1 1.9 2.6 3.0 2.2 2.1

The results showed that the main components and acidic components of the charge variants of each square sample changed significantly after being placed at 40°C for 4 weeks. Compared with day 0, the main components of prescription 1, prescription 2, prescription 3, prescription 4, and prescription 5 decreased by 16.5%, 15.1%, 14%, 15.3%, and 12.7%, respectively, and the acidic component increased by 14.9%, 13.2%, and 12.7%, respectively. 12.4%, 13.6% and 10.8%. After being placed at 25℃ for 2 months, the main components and acidic components of the samples of various prescriptions changed significantly. Compared with day 0, the main components of samples of various prescriptions decreased by 7.4%, 6.6%, 5.7%, 7.3% and 5.4%, respectively. The acid component increased by 6.8%, 5.6%, 4.7%, 6.1% and 4.5% respectively. In summary, from the analysis of the detection results of charge variants, prescription 5 is better than other prescriptions. 5. Polysorbate

The polysorbate results are shown in Table 22. Table 22 Prescription screening polysorbate 80 (HPLC-FLD method) results sample name time 0 days Two weeks 4 weeks Prescription 1 0.51 0.50 0.31 Prescription 2 0.50 0.50 0.31 Prescription 3 0.50 0.50 0.31 Prescription 4 0.50 0.53 0.49 Prescription 5 0.52 0.54 0.51

Table 22 shows that when placed at 40°C for 4 weeks, the polysorbate 80 of prescription 1, prescription 2, and prescription 3 all decreased, while the polysorbate 80 of prescription 4 and prescription 5 did not decrease.

The above experimental results show that from the analysis of purity (SEC-HPLC method) results, prescription 2, prescription 3 and prescription 4 can be excluded; from the analysis of charge variant results, prescription 5 is better than other prescriptions. Select prescription 5 to carry out subsequent shaking and freeze-thaw experiments. 6. Oscillation experiment

Table 23 shows the results of the shaking experiment of the formulation 5 samples. Table 23 Prescription Screening and Shaking Results sample name Detection Indicator Time (days) 0 5 days Prescription 5 Appearance (observation method) qualified qualified Visible foreign body (Visible foreign body inspection method) qualified qualified Protein content (UV method, mg/ml) 50.8 50.7 Charge variant (iCIEF method, %) Acidic component 16.5 16.8 main ingredient 82.2 82.0 Alkaline component 1.3 1.2 Purity (SEC-HPLC method, %) 99.6 99.6 Purity (non-reduced CE-SDS method, %) 96.3 96.3

Table 23 shows that, after shaking for 5 days at room temperature and 650 r/min in the dark, the appearance and visible foreign matter of prescription 5 are all qualified; the protein content, purity and charge variants have not changed significantly. 7. Freeze-thaw experiment

The results of the freeze-thaw experiment are shown in Table 24. Table 24 Prescription screening freeze-thaw results sample name Detection Indicator Freeze-thaw cycles (times) 0 6 Prescription 5 Appearance (observation method) qualified qualified Visible foreign body (Visible foreign body inspection method) qualified qualified Protein content (UV method, mg/ml) 50.8 50.7 Charge variant (iCIEF method, %) Acidic component 16.5 16.0 main ingredient 82.2 83.0 Alkaline component 1.3 1.0 Purity (SEC-HPLC method, %) 99.6 99.7 Purity (non-reduced CE-SDS method, %) 96.3 96.3

Table 24 shows that after repeated freezing and thawing 6 times, the appearance and visible foreign matter of the prescription 5 samples are all qualified; the protein content, purity and charge variants have not changed significantly.

Based on the above experimental results and the experience of the formulation formulation development platform, formulation 5 was finally selected as the IBI319 formulation formulation. In order to obtain a robust and easy-to-implement production process, a fixed ratio of histidine hydrochloride and histidine acid was selected to achieve a pH of 5.7. Its composition: 50.0 mg/ml IBI319, 0.57 mg/ml histidine, 1.33 mg/ml histidine hydrochloride, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.01 mg/ml edetate two Sodium, 0.50 mg/ml polysorbate 80, pH 5.7.

Figure 1 is a schematic diagram of the molecular structure of IBI319.

Figure 2 is a graph showing the change trend of turbidity in pH screening.

Figure 3 is a graph showing the change trend of pH screening purity (SEC-HPLC method).

Figure 4 is a graph showing the change trend of pH screening purity (non-reduced CE-SDS method).

Figure 5 is a graph showing the change trend of pH screening charge variants-principal components.

Figure 6 is a graph showing the change trend of pH screening charge variants-acidic components.

Figure 7 is a graph showing the change trend of prescription screening purity.

Figure 8 is a graph showing the change trend of prescription screening charge variant-principal component (iCIEF method, 40°C).

Figure 9 is a graph showing the change trend of the charge variant-acidic component of the prescription screening (iCIEF method, 40°C).

Figure 10 is a graph showing the change trend of prescription screening charge variant-principal component (iCIEF method, 25°C).

Figure 11 is a graph showing the change trend of charge variants-acidic components in prescription screening (iCIEF method, 25°C).

In Figure 2-9, T0 means 0 days, 1W means 1 week, 2W means 2 weeks, and 4W means 4 weeks. In Figures 10 and 11, T0 means 0 days, 1M means January, and 2M means February. In Figures 7-11, F1, F2, F3, F4, and F5 represent prescription 1, prescription 2, prescription 3, prescription 4, and prescription 5, respectively.

Figure 12 shows the verification of the blocking activity of IBI319 on PD-1/PD-L1 binding through the NFAT-luc reporting system; Note: Induction factor = RLU (induced–background) /RLU (no antibody control–background).

Figure 13 shows the verification of the activation activity of IBI319 on CD137 binding through the Jurkat/CD137-NFKB-luc reporter system: co-cultivation with CHO-S/PD-1.

Figure 14 shows the verification of the activation activity of IBI319 on CD137 binding through the Jurkat-CD137-NFKB-luc reporter system: without CHO-S/PD-1 co-cultivation.

Figure 15 Verification of the activation activity of IBI319 on CD137 binding through the Jurkat-CD137-NFKB-luc reporting system: co-cultivation with different ratios of CHO-S/PD-1; Note: Induction factor = RLU (induced–background) /RLU (no antibody control–background).

Claims (19)

A liquid antibody preparation comprising recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibodies or antigen-binding fragments thereof and a buffer, preferably the pH of the preparation It is about 5.0-7.0; more preferably, the pH value of the formulation is about 5.5-6.0; further preferably, the pH value is about 5.7. The liquid antibody preparation according to claim 1, wherein the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof The content is about 1 mg/mL-150 mg/mL, preferably about 10 mg/mL-100 mg/mL, for example, about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70 , 80, 90 mg/mL, more preferably about 50.0 mg/ml. The liquid antibody preparation according to any one of claims 1-2, wherein the buffer is selected from histidine, histidine hydrochloride or a combination thereof, or the buffer is selected from citrate , Citrate solvate (for example, citrate hydrate) or a combination thereof, for example, sodium citrate, sodium citrate dihydrate or a combination thereof, or the buffer is selected from acetate, Acetate solvate (for example, acetate hydrate) or a combination thereof, or the buffer is selected from phosphate, phosphate solvate (for example, phosphate hydrate) or a combination thereof; preferably, the The concentration of the buffer is about 5-100 mM, more preferably about 5-60 mM, for example, about 5, 10, 15, 20, 25, 30, 40, 50, 60 mM; preferably, the buffer Is histidine, the content of the histidine is about 0.775 mg/mL-15.5 mg/mL; more preferably, the content of the histidine is about 0.775 mg/mL-9.3 mg/mL, for example, about 1.0 , 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9 mg/mL; further preferably, the content of the histidine is about 1.55 mg/ml; Preferably, the buffer is a combination of histidine and histidine hydrochloride, wherein the content of histidine is about 0.270 mg/mL-5.4 mg/mL, and the content of histidine hydrochloride is 0.660 mg/mL. mL-13.33 mg/mL; more preferably, the content of the histidine is about 0.270 mg/mL-3.24 mg/mL, for example about 0.5, 1.0, 1.5, 2, 2.5, 3 mg/mL, the hydrochloric acid The content of histidine is about 0.660 mg/mL-8 mg/mL, for example about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8 mg/mL mL; More preferably, the content of the histidine acid is about 0.57 mg/ml, and the content of the histidine hydrochloride is about 1.33 mg/ml. The liquid antibody preparation according to any one of claims 1-3, characterized in that: the preparation further comprises a stabilizer, and the stabilizer comprises a polyol and/or an amino acid; the polyol is selected from sorbitol, Mannitol, sucrose, trehalose, maltose and combinations thereof, the amino acid includes arginine, arginine hydrochloride, methionine, glycine, proline or a combination thereof; preferably, the stabilizer Selected from sorbitol and/or arginine hydrochloride, sorbitol and/or arginine, more preferably, the stabilizer is a combination of sorbitol and arginine hydrochloride, or a combination of sorbitol and arginine, preferably, The content of arginine or arginine hydrochloride is about 20 mM to 200 mM, and more preferably, the content of arginine or arginine hydrochloride is about 40 mM to 150 mM, for example, about 40, 50, 60. , 70, 80, 90, 100, 110, 120, 130, 140, 150 mM; more preferably, the content of arginine or arginine hydrochloride is about 85 mM; Preferably, the content of the sorbitol is about 10 mg/mL-100 mg/mL, and the content of the arginine hydrochloride is about 4 mg/mL-42.0 mg/mL; further preferably, the content of the sorbitol The content is about 20 mg/mL-60 mg/mL, such as about 30, 40, 50 mg/mL, and the content of arginine hydrochloride is about 8.4 mg/mL-33.7 mg/mL, such as about 15, 20, 25. 30 mg/mL; more preferably, the content of the sorbitol is about 25.00 mg/ml, and the content of the arginine hydrochloride is about 16.85 mg/ml. The liquid antibody preparation according to any one of claims 1-4, characterized in that: the preparation further comprises a surfactant; Preferably, the surfactant is selected from non-ionic surfactants, such as one or more of polysorbate 80, polysorbate 20, poloxamer and polyethylene glycol polysorbate 80, more preferably polysorbate Sorbate 80; More preferably, the content of the surfactant is about 0.1 mg/mL-1 mg/mL; further preferably about 0.3 mg/mL-0.7 mg/mL, such as 0.3, 0.4, 0.5, 0.6, 0.7 mg/mL ; It is further preferably about 0.50 mg/ml. The liquid antibody preparation according to any one of claims 1-5, characterized in that: the preparation further comprises a chelating agent; Preferably, the chelating agent is selected from edetate disodium, diethyltriaminepentaacetic acid and/or EDTA, more preferably edetate disodium; More preferably, the content of the chelating agent is about 0.005 mg/mL-0.1 mg/mL; further preferably about 0.01 mg/mL-0.05 mg/mL, such as about 0.02, 0.03, 0.04 mg/mL; further preferably It is about 0.01 mg/ml. The liquid antibody preparation according to any one of claims 1-6, characterized in that: the preparation contains the following components: about 50.0 mg/ml recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation Antigen cluster 137 (CD137) bispecific antibody, about 0.57 mg/ml histidine, about 1.33 mg/ml histidine hydrochloride, about 25.00 mg/ml sorbitol, about 16.85 mg/ml arginine hydrochloride, about 0.01 mg/ml disodium edetate, about 0.50 mg/ml polysorbate 80, pH about 5.7, the balance is water. The liquid antibody preparation according to any one of claims 1-7, wherein the antibody or antigen-binding fragment thereof comprises a first heavy chain (HCl), which comprises a first heavy chain variable region (HCVR1) and Constant region; the first light chain (LC1), which includes the first light chain variable region (LCVR1) and the constant region; the second heavy chain (HC2), which includes the second heavy chain variable region (HCVR2) and the constant region ; And the second light chain (LC2), which includes the second light chain variable region (LCVR2) and constant region, wherein: The HCVR1 includes the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 4, and the LCVR1 includes the light chain variable region shown in SEQ ID NO: 10 LCDR1, LCDR2 and LCDR3 included; and The HCVR2 includes the three complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 16, and the LCVR2 includes the light chain shown in SEQ ID NO: 22 The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the variable region; preferably a) The first heavy chain of the antibody comprises: complementarity determining region 1 (HCDR1) with the amino acid sequence of SEQ ID NO: 1, complementarity determining region 2 (HCDR2) with the amino acid sequence of SEQ ID NO: 2, and having an amino group Complementarity determining region 3 (HCDR3) of the acid sequence SEQ ID NO: 3; b) The first light chain of the antibody comprises: complementarity determining region 1 (LCDR1) with amino acid sequence SEQ ID NO: 7, complementarity determining region 2 (LCDR2) with amino acid sequence SEQ ID NO: 8, and amino acid sequence Complementarity determining region 3 (LCDR3) of the acid sequence SEQ ID NO: 9; c) The second heavy chain of the antibody comprises: complementarity determining region 1 (HCDR1) with the amino acid sequence of SEQ ID NO: 13, and complementarity determining region 2 (HCDR2) with the amino acid sequence of SEQ ID NO: 14, and having an amino group Complementarity determining region 3 (HCDR3) of the acid sequence of SEQ ID NO: 15; d) The second light chain of the antibody comprises: complementarity determining region 1 (LCDR1) with amino acid sequence SEQ ID NO: 19, complementarity determining region 2 (LCDR2) with amino acid sequence SEQ ID NO: 20, and amino acid sequence The complementarity determining region 3 (LCDR3) of the acid sequence SEQ ID NO: 21. The liquid antibody preparation according to any one of claims 1-8, wherein the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibodies comprise : The first heavy chain, the second heavy chain, and the first light chain and the second light chain, where each chain includes a variable region and a constant region, where: e) The antibody first heavy chain variable region (HCVR) comprises the sequence of SEQ ID NO: 4 or a sequence with at least 90%, 95%, 98% or 99% homology with it; f) The antibody first light chain variable region (LCVR) comprises the sequence of SEQ ID NO: 10 or a sequence that has at least 90%, 95%, 98% or 99% homology with it; g) The antibody second heavy chain variable region (HCVR) comprises the sequence of SEQ ID NO: 16 or a sequence that has at least 90%, 95%, 98% or 99% homology with it; h) The antibody second light chain variable region (LCVR) comprises the sequence of SEQ ID NO: 22 or a sequence that has at least 90%, 95%, 98% or 99% homology with it; Preferably, e) The antibody first heavy chain variable region (HCVR) has an amino acid sequence of SEQ ID NO: 4; f) The antibody first light chain variable region (LCVR) has an amino acid sequence of SEQ ID NO: 10; g) The antibody second heavy chain variable region (HCVR) has an amino acid sequence of SEQ ID NO: 16; h) The antibody second light chain variable region (LCVR) has an amino acid sequence of SEQ ID NO:22. The liquid antibody preparation according to any one of claims 1-9, wherein the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibodies comprise : The first heavy chain, the second heavy chain, and the first light chain and the second light chain, where each chain includes a variable region and a constant region, wherein: the first heavy chain constant region sequence (HCCR) has an amino acid sequence SEQ ID NO: 30, the first light chain constant region sequence (LCCR) has an amino acid sequence of SEQ ID NO: 31, the second heavy chain constant region (HCCR) sequence has an amino acid sequence of SEQ ID NO: 32, the second The light chain constant region (LCCR) sequence has an amino acid sequence of SEQ ID NO:33. The liquid antibody preparation according to any one of claims 1-10, wherein the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibodies comprise ：The first heavy chain, the second heavy chain, the first light chain and the second light chain, where: i) The first heavy chain of the antibody comprises the sequence of SEQ ID NO: 5 or a sequence that has at least 90%, 95%, 98% or 99% homology with it; j) The first light chain of the antibody comprises the sequence of SEQ ID NO: 11 or a sequence that is at least 90%, 95%, 98% or 99% homologous to it; k) The second heavy chain of the antibody comprises the sequence of SEQ ID NO: 17 or a sequence that is at least 90%, 95%, 98% or 99% homologous to it; and 1) The second light chain of the antibody comprises the sequence of SEQ ID NO: 23 or a sequence that has at least 90%, 95%, 98% or 99% homology with it; Preferably, i) The first heavy chain of the antibody has an amino acid sequence of SEQ ID NO: 5; j) The first light chain of the antibody has an amino acid sequence of SEQ ID NO: 11; k) The second heavy chain of the antibody has an amino acid sequence of SEQ ID NO: 17; and 1) The second light chain of the antibody has an amino acid sequence of SEQ ID NO:23. The liquid antibody preparation according to any one of claims 1-11, wherein the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody comprises ：The first heavy chain, the second heavy chain, and the first light chain and the second light chain, wherein at least one disulfide bond is formed between the first heavy chain and the first light chain, and the second heavy chain and the second light chain At least one disulfide bond is formed between, and at least one disulfide bond is formed between the first heavy chain and the second heavy chain; preferably, the bispecific antibody is a modified human IgG1. The liquid antibody preparation according to any one of claims 1-12, which comprises: (i) The recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof of about 1 mg/mL-150 mg/mL; (ii) Histidine of about 5-100 mM; (iii) about 10 mg/mL-100 mg/mL sorbitol and about 20 mM-200 mM arginine or arginine hydrochloride; (iv) about 0.1 mg/mL-1 mg/mL polysorbate 80; and (v) Optionally, about 0.005 mg/mL-0.1 mg/mL disodium edetate, Wherein the pH of the liquid antibody preparation is about 5.0-7.0, for example, about 5.5, 6.0, 6.5; For example, the liquid antibody preparation comprises (i) About 10 mg/mL-100 mg/mL of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof; (ii) Histidine of about 5-60 mM; (iii) about 20 mg/mL-60 mg/mL sorbitol and about 8.4 mg/mL-33.7 mg/mL arginine hydrochloride; (iv) about 0.3 mg/mL-0.7 mg/mL polysorbate 80; and (v) Optionally, about 0.01 mg/mL-0.05 mg/mL disodium edetate, Wherein the pH of the liquid antibody preparation is about 5.5-6.0, for example, about 5.7; Alternatively, the liquid antibody preparation comprises (i) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 1.55 mg/ml histidine, 50.00 mg/ml sorbitol, 0.50 mg/ml polysorbate 80, pH 5.7; or (ii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 1.55 mg/ml histidine, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 1.55 mg/ml histidine, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iv) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 2.94 mg/ml sodium citrate (Dihydrate), 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (v) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 2.94 mg/ml sodium citrate (Dihydrate), 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; or (ii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 0.57 mg/ml histidine, 1.33 mg/ml histidine hydrochloride, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iii) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 0.57 mg/ml histidine, 1.33 mg/ml histidine hydrochloride, 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; (iv) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 2.94 mg/ml sodium citrate (Dihydrate), 16.85 mg/ml arginine hydrochloride, 0.50 mg/ml polysorbate 80, pH 5.7; or (v) 50.0 mg/ml of the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or its antigen-binding fragment, 0.57 mg/ml histidine, 1.33 mg/ml histidine hydrochloride, 25.00 mg/ml sorbitol, 16.85 mg/ml arginine hydrochloride, 0.01 mg/ml disodium edetate, 0.50 mg/ml polysorbate 80, pH 5.7. A method for preparing the preparation according to any one of claim items 1-13, comprising the following steps: (1) Making a solution with ingredients other than the surfactant; (2) Through ultrafiltration, the solution prepared in step (1) is used to exchange the recombinant anti-programmed cell death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibodies or fragments thereof. Liquid, and then concentrated to the target concentration; (3) Add the surfactant to the liquid prepared in step (2). A solid antibody preparation obtained by curing the liquid antibody preparation described in any one of Claims 1-13, the solidification being carried out by, for example, a crystallization method, a spray drying method, or a freeze-drying method. The solid antibody The preparation is, for example, in the form of a lyophilized powder injection. A delivery device comprising the liquid antibody preparation of any one of claims 1-13 or the solid antibody preparation of claim 15. A pre-filled syringe containing the liquid antibody preparation of any one of claims 1-13 or the solid antibody preparation of claim 15 for intravenous injection or intramuscular injection. The use of the liquid antibody preparation according to any one of claims 1-13 or the solid antibody preparation according to claim 15 in the preparation of a delivery device or a pre-filled syringe or medicine for the prevention or treatment of cancer, preferably The cancer is selected from melanoma, non-small cell lung cancer, small cell lung cancer, head and neck cancer, liver cancer, colorectal cancer, pancreatic cancer, stomach cancer, kidney cancer, bladder cancer, prostate cancer, breast cancer, ovarian cancer, endometrial cancer, esophagus Carcinoma, soft tissue sarcoma, cholangiocarcinoma, thyroid cancer, hepatocellular carcinoma or mesothelioma. The use according to claim 18, wherein the drug is administered to cancer patients simultaneously, separately or sequentially with ionizing radiation; or administered to cancer patients simultaneously, separately or sequentially with one or more chemotherapeutic drugs; or administered to cancer patients simultaneously, separately or sequentially with ionizing radiation; or Multiple chemotherapeutic drugs are administered to cancer patients simultaneously, separately or sequentially. Preferably, the chemotherapeutic drugs are selected from 5-fluorouracil, hydroxyurea, gemcitabine, methotrexate, doxorubicin, etoposide carboplatin, cisplatin, cyclic Phosphatiamine, melphalan, dacarbazine, paclitaxel, camptothecin, FOLFIRI, FOLFOX, docetaxel, daunorubicin, paclitaxel, oxaliplatin, or a combination thereof.

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