WO2021143826A1 - 重组抗程序性死亡受体1和抗分化抗原簇137双特异性抗体制剂及其用途 - Google Patents

重组抗程序性死亡受体1和抗分化抗原簇137双特异性抗体制剂及其用途 Download PDF

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WO2021143826A1
WO2021143826A1 PCT/CN2021/072103 CN2021072103W WO2021143826A1 WO 2021143826 A1 WO2021143826 A1 WO 2021143826A1 CN 2021072103 W CN2021072103 W CN 2021072103W WO 2021143826 A1 WO2021143826 A1 WO 2021143826A1
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antibody
heavy chain
amino acid
seq
histidine
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PCT/CN2021/072103
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English (en)
French (fr)
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张海桃
马丽强
汪音爵
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信达生物制药(苏州)有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the invention belongs to the field of biotechnology, and relates to recombinant anti-programmed death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody preparations and uses thereof.
  • PD-1 anti-programmed death receptor 1
  • CD137 anti-differentiation antigen cluster 137
  • 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 signal, which has been shown to be in tumor Immune escape and play an important role in the immunosuppression that occurs in the tumor microenvironment.
  • CD137 also known as 4-1BB, plays a role in activating T cell-driven immune responses, for example by promoting T cell proliferation and effector functions, enhancing immune memory and inhibiting activation-induced cell death.
  • Agonistic antibodies targeting CD137 have shown promise as monotherapy and combination therapy in murine tumor models. However, due to toxicity and/or lack of efficacy, agonists targeting 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.
  • 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).
  • IgG-like bispecific antibodies 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 Article 394 of Immunology (2016)). 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.
  • the present invention has developed a recombinant anti-programmed 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 a stable preparation containing the antibody or functional fragments thereof.
  • PD-1 anti-programmed death receptor 1
  • CD137 anti-differentiation antigen cluster 137
  • code IBI319 Bispecific antibody
  • the present invention relates to a liquid antibody preparation comprising a recombinant anti-programmed death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof and a buffer, preferably
  • PD-1 recombinant anti-programmed death receptor 1
  • CD137 anti-differentiation antigen cluster 137 bispecific antibody or antigen-binding fragment thereof
  • 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.
  • the content of the recombinant anti-programmed 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 -150mg/mL, preferably about 10mg/mL-100mg/mL, for example, about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90mg/mL, more preferably It is about 50.0mg/ml.
  • 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 a combination thereof, or the buffer is selected from phosphate, a phosphate solvate (for example, phosphate hydrate) or a combination thereof; preferably, the concentration of the buffer is about 5-100 mM, more Preferably, it is about 5-60 mM, for example, about 5, 10, 15, 20, 25, 30, 40, 50, 60 mM;
  • the buffer 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 histamine The acid content is about 1.55mg/ml;
  • 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.
  • the histidine content is about 0.270mg/mL-3.24mg/mL, such as about 0.5, 1.0, 1.5, 2, 2.5, 3mg/mL
  • 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
  • the content of the histidine is about 0.57 mg/ml
  • the content of the histidine hydrochloride is about 1.33 mg/ml.
  • the formulation further includes a stabilizer; preferably, the formulation further includes a stabilizer, and the stabilizer includes a polyol and/or amino acid;
  • the polyol is selected from the group consisting of sorbitol, mannitol, sucrose, Trehalose, maltose and combinations
  • the amino acids include arginine, arginine hydrochloride, methionine, glycine, proline or a combination thereof;
  • the stabilizer is selected from sorbitol and/or arginine hydrochloride Acid, 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 arginine or arginine hydrochloride
  • the content of arginine is about 20mM-200mM, further preferably, the content of arginine or arginine hydrochloride is about
  • 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 is about 20mg/mL-60mg/mL, such as about 30, 40, 50mg/mL, the content of arginine hydrochloride is about 8.4mg/mL-33.7mg/mL, for example, about 15, 20, 25, 30mg/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 formulation further includes a surfactant
  • the surfactant is selected from non-ionic surfactants, such as one or more of polysorbate 80, polysorbate 20, poloxamer and polyethylene glycol polysorbate 80, and more Preferably polysorbate 80;
  • 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; More preferably, it is about 0.50 mg/ml.
  • the formulation further includes a chelating agent
  • the chelating agent is selected from edetate disodium, diethyltriaminepentaacetic acid and/or EDTA, more preferably edetate disodium;
  • 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 0.01mg/ml.
  • a liquid antibody preparation contains the following components: about 50.0 mg/ml recombinant anti-programmed death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific Antibody, about 0.57mg/ml histidine, about 1.33mg/ml histidine hydrochloride, about 25.00mg/ml sorbitol, about 16.85mg/ml arginine hydrochloride, about 0.01mg/ml disodium edetate , About 0.50mg/ml polysorbate 80, pH about 5.7, the balance is water.
  • PD-1 recombinant anti-programmed death receptor 1
  • CD137 anti-differentiation antigen cluster 137 bispecific Antibody
  • liquid antibody preparation 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 first heavy chain of an antibody includes: complementarity determining region 1 (HCDR1) with amino acid sequence SEQ ID NO: 1, complementarity determining region 2 (HCDR2) with amino acid sequence SEQ ID NO: 2, and amino acid sequence SEQ ID NO: Complementarity determining region 3 (HCDR3) of 3;
  • the first light chain of the antibody includes: 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 SEQ ID NO: 9's complementary decision area 3 (LCDR3);
  • LCDR1 complementarity determining region 1
  • LCDR2 complementarity determining region 2
  • LCDR3 complementary decision area 3
  • the second heavy chain of the antibody includes: complementarity determining region 1 (HCDR1) with the amino acid sequence SEQ ID NO: 13, complementarity determining region 2 (HCDR2) with the amino acid sequence SEQ ID NO: 14, and amino acid sequence SEQ ID NO: Complementarity determining region 3 (HCDR3) of 15;
  • the second light chain of the antibody includes: 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 SEQ ID NO: 21's complementary determining region 3 (LCDR3).
  • LCDR1 complementarity determining region 1
  • LCDR2 complementarity determining region 2
  • LCDR3 amino acid sequence SEQ ID NO: 21's complementary determining region 3
  • the recombinant anti-programmed 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 And a second light chain, where each chain contains a variable region and a constant region, where:
  • the antibody first heavy chain variable region contains the sequence of SEQ ID NO: 4 or a sequence that is at least 90%, 95%, 98% or 99% identical to it;
  • the antibody first light chain variable region contains the sequence of SEQ ID NO: 10 or a sequence that is at least 90%, 95%, 98% or 99% identical to it;
  • the antibody second heavy chain variable region contains the sequence of SEQ ID NO: 16 or a sequence that is at least 90%, 95%, 98% or 99% identical to it;
  • the antibody second light chain variable region contains the sequence of SEQ ID NO: 22 or a sequence that is at least 90%, 95%, 98% or 99% identical to it;
  • the antibody first heavy chain variable region has the amino acid sequence SEQ ID NO: 4;
  • the antibody first light chain variable region has the amino acid sequence SEQ ID NO: 10;
  • the antibody second heavy chain variable region has the amino acid sequence SEQ ID NO: 16;
  • the antibody second light chain variable region has the amino acid sequence SEQ ID NO: 22.
  • the recombinant anti-programmed 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 And a second light chain, wherein each chain includes a variable region and a constant region, wherein: the first heavy chain constant region sequence (HCCR) has the amino acid sequence SEQ ID NO: 30, and the first light chain constant region sequence (LCCR) has The amino acid sequence SEQ ID NO: 31, the second heavy chain constant region (HCCR) sequence has the amino acid sequence SEQ ID NO: 32, and the second light chain constant region (LCCR) sequence has the amino acid sequence of SEQ ID NO: 33.
  • HCCR first heavy chain constant region sequence
  • LCCR first light chain constant region sequence
  • LCCR has The amino acid sequence SEQ ID NO: 31
  • the second heavy chain constant region (HCCR) sequence has the amino acid sequence SEQ ID NO: 32
  • the second light chain constant region (LCCR) sequence has the amino acid sequence
  • the recombinant anti-programmed 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 And the second light chain, where:
  • the first heavy chain of the antibody contains the sequence of SEQ ID NO: 5 or a sequence that is at least 90%, 95%, 98% or 99% identical to it;
  • 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% identical to it;
  • 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% identical to it;
  • the second light chain of the antibody comprises the sequence of SEQ ID NO: 23 or a sequence that is at least 90%, 95%, 98% or 99% identical to it;
  • the first heavy chain of the antibody has the amino acid sequence SEQ ID NO: 5;
  • the first light chain of the antibody has the amino acid sequence SEQ ID NO: 11;
  • the second heavy chain of the antibody has the amino acid sequence SEQ ID NO: 17;
  • the second light chain of the antibody has the amino acid sequence SEQ ID NO: 23.
  • the recombinant anti-programmed 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 And a 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 and At least one disulfide bond is formed between the second heavy chains; preferably the bispecific antibody is a modified human IgG1.
  • the present invention provides a liquid antibody preparation comprising:
  • (v) optionally, about 0.005 mg/mL-0.1 mg/mL disodium edetate,
  • pH of the liquid formulation is about 5.0-7.0, for example, about 5.5, 6.0, 6.5;
  • the liquid antibody preparation comprises
  • (v) optionally, about 0.01 mg/mL-0.05 mg/mL disodium edetate,
  • pH of the liquid formulation is about 5.5-6.0, for example, about 5.7;
  • the liquid antibody preparation comprises
  • a method for preparing the antibody liquid formulation of the present invention includes the following steps:
  • the solution prepared in step (1) is used to exchange the recombinant anti-programmed death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibodies or fragments thereof , And then concentrated to the target concentration;
  • PD-1 anti-programmed death receptor 1
  • CD137 anti-differentiation antigen cluster 137
  • 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 The preparation is, for example, in the form of a lyophilized powder injection.
  • a delivery device which comprises the liquid antibody preparation or the solid antibody preparation of the present invention.
  • a pre-filled syringe which contains the liquid antibody preparation or solid antibody preparation of the present invention for intravenous injection or intramuscular injection.
  • 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, Cholangiocarcinoma, 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 Administration; or can be administered to cancer patients simultaneously, separately or sequentially with ionizing radiation and one or more chemotherapeutics; preferably, the chemotherapeutics;
  • 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 chemotherapeutic drugs; or can be administered to cancer patients simultaneously, separately or sequentially with ionizing radiation 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,
  • the present invention provides a method for preventing or treating cancer in a patient, comprising 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 of the present invention
  • An effective dose of liquid antibody preparation or solid antibody preparation is administered 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 simultaneously with ionizing radiation , Separately or sequentially to cancer patients; or can be administered to cancer patients simultaneously, separately or sequentially with one or more chemotherapeutic drugs;
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprises” or “includes” when used, unless otherwise specified, it also encompasses the situation consisting of the stated elements, integers or steps.
  • an antibody variable region that "comprises” a specific sequence 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.
  • immune checkpoint molecule refers to a type of inhibitory signal molecule present in the immune system, which avoids 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).
  • Immune checkpoint molecules include but are not limited to programmed 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 chain (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 more conservative regions (framework regions (FR)) inserted between them.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and 4 FRs, arranged in the following order from the amino terminal to the carboxy terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region does not directly participate in the binding of antibodies to antigens, but exhibits a variety of effector functions.
  • 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.
  • 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.
  • multispecific antibody refers to an antibody having at least two antigen binding sites, each of the at least two antigen binding sites is different from a different epitope of the same antigen or is different from a different epitope of the same antigen. 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.
  • bispecific antibodies that have binding specificities for a first antigen and a second antigen.
  • the first antigen is PD-1 and the second antigen is CD137.
  • humanized antibody refers to a chimeric antibody comprising amino acid residues from non-human HVR and amino acid residues from human FR.
  • the humanized antibody comprises all or substantially all of the HVR (e.g., CDR) corresponding to those of the non-human antibody and all or substantially all of the FR region 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.
  • 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.
  • the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carbonyl end of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • the numbering of amino acid residues in the Fc region or 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.
  • variable region 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., WHFreeman and Co. 91 page (2007)).
  • a single VH or VL domain may be sufficient to give antigen binding specificity.
  • 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 libraries of complementary 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 generally exhibit the same general structure of relatively conserved framework regions (FR) connected by three hypervariable regions, which are also referred to as complementarity determining regions or CDRs.
  • the CDRs from the two chains of each pair are usually aligned by the framework regions, which allow the 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.
  • CDR region or “CDR” or “hypervariable region” (herein can be used interchangeably with hypervariable region “HVR”) is an antibody variable domain that is hypervariable in sequence and A structurally defined loop ("hypervariable loop") and/or a region containing antigen contact residues ("antigen contact point”) is formed.
  • 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 referred to as HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are referred to as LCDR1, LCDR2, and LCDR3.
  • the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, which include For example: Chothia based on the three-dimensional structure of antibodies and the topology of CDR loops (Chothia et al.
  • 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-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) or biofilm optical interference technology (ForteBio) or other conventional binding assays known in the art.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • FormeBio biofilm optical interference technology
  • 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.
  • 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 unacceptable toxicity to the subject to which the preparation is to be administered. Other 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 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-
  • the 1/CD137 antibody protein is a preparation containing pharmaceutically acceptable excipients as the active ingredient. 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 human 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).
  • 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.
  • the antibody preparation is considered “stable.”
  • the anti-PD-1/CD137 antibody protein preparation of the present invention exhibits low to undetectable antibody aggregation or degradation or chemical modification during manufacture, preparation, transportation and long-term storage, so that there is very little Or even there is no loss of the biological activity of the anti-PD-1/CD137 antibody protein, showing a high degree of stability.
  • the anti-PD-1/CD137 antibody protein formulation of the present invention substantially retains its physical and chemical stability after storage.
  • 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°C Stable for at least 24 months.
  • 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, the stability test is performed by performing various stress tests on the antibody preparation.
  • 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.
  • the preparation After a period of storage time, 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. Since the accumulation of antibodies in the preparation can potentially lead to an increased immune response in patients, it leads 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 preparation. SEC can be used to determine soluble aggregates in the formulation.
  • 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 350nm method, or measuring the purity of the preparation by the non-reducing CE-SDS method.
  • 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, where 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.
  • 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.
  • 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.
  • the pharmaceutical preparation is considered stable; if stored at about 40°C ⁇ 2°C 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°C 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.
  • the antibody in the preparation 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).
  • charge variants of antibodies for example, by aspartic acid isomerization, N and C terminal modification, basic variants can be formed; by deamidation, sialylation and saccharification, acidic variants can be generated.
  • Chemical stability can be assessed by detecting and/or quantifying the chemically altered form of the antibody.
  • the charge variant of the antibody in the preparation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (iCIEF).
  • CEX cation exchange chromatography
  • iCIEF imaging capillary isoelectric focusing electrophoresis
  • the stability of the formulation is measured by determining the percentage change in the charge variant of the
  • the "acceptable degree" of chemical stability can mean that the percentage change value of the charge variant (such as the main component or acidic component or alkaline component) in the preparation after storage at a specific temperature for a specific time does not exceed 50%, for example, does not exceed 30%, not more than 20%.
  • 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%.
  • 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.
  • 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 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%, after storage at 40°C for 1 month, 5% or 4%, the pharmaceutical preparation can also
  • lyophilized preparation refers to a composition obtained or obtainable by freeze-drying a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.
  • 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.
  • room temperature 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.
  • parenteral administration means administration methods other than enteral and local administration, usually by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, and intrathecal , Intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspine, epidural and intrasternal injections and infusions .
  • the stabilized anti-PD-1/CD137 antibody protein formulation of the present invention is administered to the subject parenterally.
  • the anti-PD-1/CD137 antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.
  • the antibody sequence of the present invention anti-CD137 arm (7A5*), anti-PD-1 arm (11444*), based on the parent antibody (7A5), parent antibody (11444) and wild-type human IgG1 constant region, wild-type human Lambda constant
  • the mutation design of the wild-type human Kappa constant region is completed.
  • the specific CDR region, variable region, constant region, full-length heavy chain and full-length light chain of the antibody of the present invention, as well as the nucleotide sequence numbers encoding the full-length heavy chain and light chain, and the parent antibody variable region SEQ ID NO are as follows 1 shows:
  • HCCR heavy chain constant region
  • LCCR light chain constant region
  • the recombinant anti-programmed 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 includes: 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 of an antibody includes: complementarity determining region 1 (HCDR1) with amino acid sequence SEQ ID NO: 1, complementarity determining region 2 (HCDR2) with amino acid sequence SEQ ID NO: 2, and amino acid sequence SEQ ID NO: Complementarity determining region 3 (HCDR3) of 3;
  • the first light chain of the antibody includes: 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 SEQ ID NO: 9's complementary decision area 3 (LCDR3);
  • LCDR1 complementarity determining region 1
  • LCDR2 complementarity determining region 2
  • LCDR3 complementary decision area 3
  • the second heavy chain of the antibody includes: complementarity determining region 1 (HCDR1) with the amino acid sequence SEQ ID NO: 13, complementarity determining region 2 (HCDR2) with the amino acid sequence SEQ ID NO: 14, and amino acid sequence SEQ ID NO: Complementarity determining region 3 (HCDR3) of 15;
  • the second light chain of the antibody includes: 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 SEQ ID NO: 21's complementary determining region 3 (LCDR3).
  • LCDR1 complementarity determining region 1
  • LCDR2 complementarity determining region 2
  • LCDR3 amino acid sequence SEQ ID NO: 21's complementary determining region 3
  • the antibody first heavy chain variable region has the amino acid sequence SEQ ID NO: 4;
  • the antibody first light chain variable region has the amino acid sequence SEQ ID NO: 10;
  • the antibody second heavy chain variable region has the amino acid sequence SEQ ID NO: 16;
  • the antibody second light chain variable region has the amino acid sequence SEQ ID NO: 22.
  • the first heavy chain constant region sequence has the amino acid sequence of SEQ ID NO: 30
  • the first light chain constant region sequence has the amino acid sequence of SEQ ID NO: 31
  • the second heavy chain constant region The sequence has the amino acid sequence of SEQ ID NO: 32
  • the second light chain constant region (LCCR) sequence has the amino acid sequence of SEQ ID NO: 33.
  • the first heavy chain of the antibody has the amino acid sequence SEQ ID NO: 5;
  • the first light chain of the antibody has the amino acid sequence SEQ ID NO: 11;
  • the second heavy chain of the antibody has the amino acid sequence SEQ ID NO: 17;
  • the second light chain of the antibody has the amino acid sequence SEQ ID NO: 23.
  • 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 ⁇ 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.
  • the present invention also provides nucleotide sequences encoding the first heavy chain, first light chain, second heavy chain, and second light chain of the antibody of the present invention.
  • the nucleotide sequences are SEQ ID NO: 6.
  • 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, NSO, HEK293 or COS cells) and recovering the antibody. For example, selecting suitable host cells such as HEK293 or CHO cells, by presetting the optimal heavy chain: light chain vector ratio of secreted antibody expression system, or by simultaneously expressing heavy chain and light chain single vector system, transient or stable transfer Infect the host cell. Specifically, for example, encoding the first heavy chain having the amino acid sequence shown in SEQ ID NO: 5, encoding the first light chain having the amino acid sequence shown in SEQ ID NO: 11, and encoding the amino acid sequence shown in SEQ ID NO: 17 can be used.
  • the second heavy chain of the sequence and one or more DNA molecules encoding the second light chain having the amino acid sequence shown in SEQ ID NO: 23 are transiently or stably transfected into host cells through the secreted protein expression system to obtain the antibody of the present invention.
  • Antibody recovery and purification are 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.
  • Tugcu et al. Maximizing productivity of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering 99 (2008) 599-613.
  • ion exchange chromatography anion IEX and/or cation CEX chromatography
  • 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.
  • a commercially available protein concentration filter such as Amicon's ultrafiltration device can be used to concentrate the supernatant from the expression system.
  • 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.
  • a preparation containing the antibody can be prepared according to methods known in the art.
  • the following steps can be used for preparation: (1) After the fermentation, the fermentation broth is centrifuged to clarify impurities such as cells to obtain the supernatant; (2) affinity chromatography (for example, specific for IgG1, IgG2, and IgG4 antibodies) Affinity protein A column) capture antibody; (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) Reduce, for example, 4 log 10 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.
  • affinity chromatography for example, specific for IgG1, IgG2, and IgG4 antibodies
  • Affinity protein A column capture antibody
  • the present invention relates to a liquid antibody preparation comprising a recombinant anti-programmed death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific antibody or antigen-binding fragment thereof and a buffer, preferably
  • PD-1 recombinant anti-programmed death receptor 1
  • CD137 anti-differentiation antigen cluster 137 bispecific antibody or antigen-binding fragment thereof
  • 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.
  • the content of the recombinant anti-programmed 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 -150mg/mL, preferably about 10mg/mL-100mg/mL, for example, about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90mg/mL, more preferably It is about 50.0mg/ml.
  • 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 a combination thereof, or the buffer is selected from phosphate, a phosphate solvate (for example, phosphate hydrate) or a combination thereof; preferably, the concentration of the buffer is about 5-100 mM, more Preferably, it is about 5-60 mM, for example, about 5, 10, 15, 20, 25, 30, 40, 50, 60 mM;
  • the buffer 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 histamine The acid content is about 1.55mg/ml;
  • 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.
  • the histidine content is about 0.270mg/mL-3.24mg/mL, such as about 0.5, 1.0, 1.5, 2, 2.5, 3mg/mL
  • 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
  • the content of the histidine is about 0.57 mg/ml
  • the content of the histidine hydrochloride is about 1.33 mg/ml.
  • the formulation further includes a stabilizer
  • 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, arginine hydrochloride, and methionine.
  • the stabilizer is selected from sorbitol and/or arginine hydrochloride, sorbitol and/or arginine, more preferably, the stabilizer is sorbitol and hydrochloric acid
  • the combination of arginine, or the combination of sorbitol and arginine, preferably, the content of the arginine or arginine hydrochloride is about 20mM-200mM, further preferably, the arginine or arginine hydrochloride
  • the content of is about 40mM-150mM, such as about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150mM; more preferably, the content of arginine or arginine hydrochloride Is about 85mM;
  • 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 is about 20mg/mL-60mg/mL, such as about 30, 40, 50mg/mL, the content of arginine hydrochloride is about 8.4mg/mL-33.7mg/mL, for example, about 15, 20, 25, 30mg/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 formulation further includes a surfactant
  • the surfactant is selected from non-ionic surfactants, such as one or more of polysorbate 80, polysorbate 20, poloxamer and polyethylene glycol polysorbate 80, and more Preferably polysorbate 80;
  • 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; More preferably, it is about 0.50 mg/ml.
  • the formulation further includes a chelating agent
  • the chelating agent is selected from edetate disodium, diethyltriaminepentaacetic acid and/or EDTA, more preferably edetate disodium;
  • 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 0.01mg/ml.
  • a liquid antibody preparation contains the following components: about 50.0 mg/ml recombinant anti-programmed death receptor 1 (PD-1) and anti-differentiation antigen cluster 137 (CD137) bispecific Antibody, about 0.57mg/ml histidine, about 1.33mg/ml histidine hydrochloride, about 25.00mg/ml sorbitol, about 16.85mg/ml arginine hydrochloride, about 0.01mg/ml disodium edetate , About 0.50mg/ml polysorbate 80, pH about 5.7, the balance is water.
  • PD-1 recombinant anti-programmed death receptor 1
  • CD137 anti-differentiation antigen cluster 137 bispecific Antibody
  • a liquid antibody preparation which comprises:
  • (v) optionally, about 0.005 mg/mL-0.1 mg/mL disodium edetate,
  • pH of the liquid formulation is about 5.0-7.0, for example, about 5.5, 6.0, 6.5;
  • the liquid antibody preparation comprises
  • (v) optionally, about 0.01 mg/mL-0.05 mg/mL disodium edetate,
  • pH of the liquid formulation is about 5.5-6.0, for example, about 5.7;
  • the liquid antibody preparation comprises
  • 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 The preparation is, for example, in the form of a lyophilized powder injection.
  • antibody and antigen-binding fragments thereof may further cover the following bispecific antibodies or antigen-binding fragments thereof:
  • HCVR first heavy chain variable region having at least 90%, 95%, 98%, or 99% or more identity with the amino acid sequence SEQ ID NO: 4; and/or with the amino acid sequence SEQ ID NO: 10
  • sequence identity refers to the degree of sequence identity on a nucleotide-by-nucleotide or amino acid-by-amino-acid basis in the comparison window.
  • the “percent sequence identity” can be calculated in the following way: the two best aligned sequences are compared in the comparison window, and the same nucleic acid bases (for example, A, T, C, G, I, etc.) are present in the two sequences.
  • sequence identity percentage e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met
  • the optimal alignment to determine the percent sequence identity 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 algorithms required to achieve the maximum alignment within the full-length sequence being compared or within the target sequence region.
  • 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.
  • 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, 4 or 3 different residues, preferably the different residues are conservative amino acid substitutions.
  • 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.
  • 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, 4 or 3 different residues, preferably the different residues are conservative amino acid substitutions.
  • Constant substitution refers to an amino acid change that results in the substitution of a certain amino acid with a chemically similar amino acid. It is well known in the art to provide conservative substitution tables of functionally similar amino acids.
  • the conservatively substituted residues are derived from the following conservative substitutions, preferably the preferred substituted residues shown in Table 2.
  • 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, edited by Rowe et al., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005)".
  • 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.
  • 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; capillary isoelectric focusing (cIEF), imaging capillary, etc. Focusing electrophoresis (iCIEF) and ion exchange chromatography (IEX), etc., analyze charge variants in antibody preparations.
  • 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.
  • ultraviolet spectrophotometry UV method
  • UV method ultraviolet spectrophotometry
  • the non-reduced CE-SDS method is a method of antibody purity determination using capillary as a separation channel.
  • 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 mass-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.
  • the test sample is mixed with SDS sample buffer and iodoacetamide.
  • 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.
  • Size exclusion high performance liquid chromatography 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.
  • 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).
  • HMMS high molecular weight form
  • LMMS low molecular weight form
  • Antibody purity can be calculated as the percentage of the main peak area on the chromatogram to the sum of all peak areas.
  • the SEC-HPLC method the percentage of antibody monomers in the preparation product can be measured, and the content information of soluble aggregates and shears can be given.
  • Imaging capillary isoelectric focusing electrophoresis 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.
  • 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 ⁇ m).
  • the protein is focused in the capillary column under high voltage, and the focusing is performed by a whole column imaging detection system operating at 280 nM. Real-time online monitoring.
  • One advantage of this technology is that the whole column detection system can simultaneously record various charge variants of antibody samples.
  • icIEF the sample is mixed with urea and icIEF buffer, where the buffer contains methyl cellulose, pi molecular weight standards and amphoteric electrolytes.
  • iCIEF column such as an iCIEF column assembled by ProtionSimple on an iCIEF analyzer such as iCE280 analyzer (Protein Simple, Santa Clara, CA).
  • iCE280 analyzer Protein Simple, Santa Clara, CA.
  • the protein-related peaks eluted before the main peak 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.
  • the charge variant of the antibody in the antibody preparation can also be determined by cation exchange high performance liquid chromatography (CEX-HPLC).
  • CEX-HPLC cation exchange high performance liquid chromatography
  • Accelerated stability studies can be used to check the stability properties of products, which is conducive to the screening of stable pharmaceutical formulations.
  • 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 preparation of the present invention passes 40°C forced and 25°C accelerated stability experiments to investigate the influence of different pH values and different excipients on protein quality, and evaluate the formulation of each preparation.
  • 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.
  • the stable antibody preparation to be protected by the present invention is obtained.
  • 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.
  • 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 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, 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 Its clearance rate, and also includes any possible other treatments administered in combination with the antibody formulation.
  • the effective amount delivered can be determined within the judgment of the clinician.
  • the application of known antibody-based drugs can provide certain guidance.
  • the dosage can be a single-dose schedule or a multiple-dose schedule.
  • 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.
  • the antibody molecules of the present invention are used to delay the progression of a disease or to slow the progression of a disease.
  • prevention includes the inhibition of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.
  • subjects with a family history of cancer are candidates for prophylactic regimens.
  • 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 according to 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 deleterious effects of the antibody or antibody fragment or its conjugate or composition are not as good as the therapeutically beneficial effects.
  • 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%.
  • a compound to inhibit a measurable parameter e.g., cancer
  • this property of the composition can be evaluated by testing the compound's ability to inhibit, said inhibition in vitro by an assay 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 subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • 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).
  • T0 means 0 days
  • 1W means 1 week
  • 2W means 2 weeks
  • 4W means 4 weeks.
  • T0 means 0 days
  • 1M means January
  • 2M means February.
  • F1, F2, F3, F4, and F5 represent prescription 1, prescription 2, prescription 3, prescription 4, and prescription 5, respectively.
  • Figure 13 shows the Jurkat/CD137-NFKB-luc reporter system to verify the activation activity of IBI319 on CD137 binding: co-incubation 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: no CHO-S/PD-1 co-incubation.
  • 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.
  • 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):
  • the chromatographic column is Knitted Reactor Coil 5m x 0.50mm ID, SUPELCO, mobile phase is 0.15mol/L sodium chloride, 0.05mol/L Tris, pH 8.0, 5 % Acetonitrile, 5.0umol/L NPN (N-phenyl-1-naphthylamine), 15ppm Brij, column protection solution is 0.05% (w/v) NaN 3 , injection volume 50 ⁇ l, flow rate 0.5ml/min, collection The time is 30 minutes, the column temperature is 25°C, and the detection wavelength is 280nm.
  • Turbidity (OD350nm method): Use an ultraviolet spectrophotometer (produced by Shimadzu, Japan, model UV-1800) to measure the absorbance of the sample at 350nm to determine the turbidity of the sample.
  • the mobile phase is phosphate buffer (weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, after dissolving in ultrapure water, adjust the pH to 6.8 with hydrochloric acid And dilute to 1000ml), the column protection solution is 0.05% (w/v) NaN 3 , the injection volume is 50 ⁇ l, the flow rate is 0.5ml/min, the collection time is 30 minutes, the column temperature is 25°C, and the detection wavelength is 280nm. Take the sample to be tested and dilute it to 2mg/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 ⁇ l each of the blank solution and the test solution into the liquid chromatograph to start the test.
  • phosphate buffer weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, after dissolving in ultrapure water
  • the capillary is an uncoated capillary with an inner diameter of 50 ⁇ m, a total length of 30.2cm, and an effective length of 20.2cm. Wash the capillary column with 0.1mol/L sodium hydroxide, 0.1mol/L hydrochloric acid, ultrapure water, and 70psi electrophoresis gel before electrophoresis.
  • Sample injection conditions -5kV for 20 seconds; separation voltage: -15kV for 35 minutes.
  • the capillary column temperature is controlled at 25°C, and the detection wavelength is 220nm.
  • the capillary has an inner diameter of 100 ⁇ m and a total length of 5cm.
  • 0.5% methyl cellulose solution hereinafter also abbreviated as MC solution
  • ultrapure water should be used to rinse the capillary column respectively.
  • the vacuum sampling method is adopted.
  • the pre-focusing voltage and time are 1.5kV for 1 minute, the focusing voltage and time are 3kV for 8 minutes, the sampling time is 55 seconds, the sample tray temperature is 10°C, and the detection wavelength is 280nm.
  • Cathodic Stabilizer is 500mmol/L arginine solution, 0.5% MC solution reduces the adhesion between protein and capillary.
  • the test product Dilute the test product to 1.0 mg/ml with water, take 20 ⁇ l of the diluted test product solution, and add 78 ⁇ l of the pre-mixed solution to it (the pre-mixed solution is as follows: 70 ⁇ l pI 0.5% MC solution, 4 ⁇ l amphoteric electrolyte (pH 3-10) ), 2 ⁇ l cathode stabilizer, 1 ⁇ l pI 5.85 marker, 1 ⁇ l 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.
  • the antibody of the present invention was obtained, the code is IBI319, and the antibody has SEQ ID NO as described in Table 1. :
  • the heavy chain sequence of 5 and 17 and the light chain sequence of SEQ ID NO: 11 and 23 are humanized antibodies.
  • 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.
  • the antibody A of the present invention can be transiently or stably transfected by an expression system that secretes the antibody.
  • the DNA molecule encodes the first heavy chain with 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, and 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 frozen immediately at -70°C or can be lyophilized.
  • One of many common techniques can be used to purify antibodies.
  • the medium can be conveniently applied to MabSelect chromatographic columns (GE Healthcare) or KappaSelect chromatographic columns (GE Healthcare) that have been equilibrated with compatible buffers (such as phosphate buffer (pH 7.4)).
  • the column can be washed to remove non-specifically bound components.
  • the bound antibody can be eluted, for example, by a pH gradient (for example, 20 mM Tris buffer from pH 7 to 10 mM to 10 mM sodium citrate at pH 3.0, glycine buffer from pH 7.4 to 100 mM phosphate buffered saline at pH 7.4).
  • the antibody fractions can be detected, for example, by UV absorption or SDS-PAGE, and then they can be combined.
  • further purification is optional.
  • the purified antibody can be concentrated and/or sterile filtered using conventional techniques.
  • Soluble aggregates and multimers 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).
  • LONZA basic medium Dynamis AGT Medium
  • Cell Boost 7a Cell Boost 7b
  • HyClone reagents
  • cells were inoculated into a 2L cell reactor at a density of 1.0 ⁇ 10 6 cells/ml, the DO was set at 40%, the pH was controlled at 7.05 ⁇ 0.20, and the pre-culture temperature was 36.5 °C, cultivate to 33.0°C on the 6th day.
  • the gravity column used for purification was treated with 0.5M NaOH overnight, and the glass bottles were washed with distilled water and then dried at 180°C for 4 hours to obtain a purification column. Before purification, the collected medium was centrifuged at 4500 rpm for 30 min, and the cells were discarded. Filter the supernatant with a 0.22 ⁇ l filter. Each tube is filled with 1ml Protein A and equilibrated with 10ml binding buffer (sodium phosphate 20mM.NaCl 150mM, pH7.0). Add the filtered supernatant to the purification column and re-equilibrate with 15ml of binding buffer.
  • 10ml binding buffer sodium phosphate 20mM.NaCl 150mM, pH7.0
  • elution buffer citric acid+sodium citrate 0.1M, pH3.5
  • Tris-HCl 80 ⁇ l Tris-HCl to every 1ml of eluate to adjust the pH to 7.0.
  • the collected antibody is purified by 1ml S-type strong cation exchange chromatography.
  • the column is first equilibrated with 10ml equilibration buffer (20mM citric acid+sodium citrate, pH5.0), and the affinity collected sample is added to the purification column and used 10ml
  • the equilibration buffer is re-equilibrated, and then the elution buffer (20mM citric acid+sodium citrate+1M sodium chloride, pH5.0) is used for gradient elution, the elution volume is 20ml, the collected sample is ultrafiltration concentrated and exchanged to PBS (Gibco , 70011-044), and detect the concentration.
  • Example 2 The above-mentioned prepared and purified antibody of the present invention was tested for the following functions
  • the biofilm layer interference (BLI) assay was used to determine the antibody IBI319 of the present invention and the proteins of different species: 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 parent antibody 11444 antibody and 4-1BB.
  • human 4-1BB Human 4-1BB
  • Cyno 4-1BB human cynomolgus 4-1BB
  • human PD-1 Human PD-1
  • cyno PD-1 Cyno PD-1
  • each sample prepared above is the maximum concentration sample tested.
  • the antigen is diluted by 2 times to the required concentration gradient for detection.
  • Biotin-labeled antibody used to detect the binding of the antibody to the Fc-tagged antigen
  • the experimental grouping information is shown in Table 6. Add 200 ⁇ L of the corresponding sample to each well.
  • Detection and detection are divided into 5 steps. Each cycle is in sequence: Baseline ⁇ Loadings ⁇ Baseline ⁇ Binding to the diluted protein (Association) ⁇ Dissociation.
  • Judgment criteria ForteBio Octet instrument analysis software (Fortebio data analysis 11.0), using 1:1 binding (global fitting) to fit the curve. The reliability of the result is judged by analyzing the degree of fit of the fitted curve and R2 value. The fitting result accords with R2>0.95, and the KD value is calculated by the combination and dissociation constant.
  • 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 IBI319 and human PD-1, cynomolgus PD-1, human 4-1BB (CD137), and crab-eating The affinity of monkey 4-1BB.
  • the coupled anti-human Fc antibody captures IBI319, gradiently diluted human PD-1, cynomolgus PD-1, human 4-1BB, Cynomolgus 4-1BB combined with captured IBI319 respectively.
  • IBI319 parent antibody 11444 (anti-PD-1 antibody) and parent antibody 7A5 (anti-4-1BB antibody or anti-CD137 antibody) were used as controls, respectively.
  • the affinity value is calculated by fitting the binding and dissociation curve.
  • the buffer used in the experiment was HBS-EP+(10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% P20) solution with pH 7.4, and the detection temperature was 25°C.
  • the experiment included: 1) coupling 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.
  • the experimental results show that the affinity of IBI319 to Human PD-1 and Cyno PD-1 is about 0.1 nM and 0.04 nM, respectively, and the affinity to 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.
  • Antibody antigen K D average ⁇ 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
  • 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 The strong affinity of IBI319 with PD-1 is conducive to a stronger PD-1/PD-L1 blocking effect.
  • the 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 T cell activation function.
  • 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 determination
  • 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, the parent antibody 11444, the parent antibody 7A5 and hIgG1 are measured to reflect the blocking activity of the drug on PD-1/PD-L1 by measuring the luciferase activity in the system.
  • Use Assay buffer (RPMI 1640+1% FBS) to prepare IBI319, parent antibody 11444, parent antibody 7A5, parent antibody 11444 + parent antibody 7A5, IgG1, starting with a concentration of 2000 nM, 4-fold dilution, and a total of 11 gradient points. See Table 9 for details.
  • the anti-PD-1 end of IBI319 showed the activity of blocking PD-1/PD-L1 binding.
  • IBI319 in vitro efficacy 2 IBI319 CD137 agonistic activity determination
  • Co-culture Jurkat cells Jurkat/CD137-NF ⁇ B-luc expressing CD137 and NF ⁇ B-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, parent antibody 11444, parent antibody 7A5 and hIgG1, and measure the luciferase activity in the system to reflect the drug's agonistic activity on the CD137 pathway and the cross-linking effect with CD137-PD-1.
  • Jurkat cell culture medium expressing CD137 and NF ⁇ B-luc reporter 90% RPMI 1640 with 1-glutamine, 10% FBS, 200 ⁇ g/ml hygromycin B, 500 ⁇ g/ml Geneticin, 1 mM sodium pyruvate, 0.1 mM MEM NEAA;
  • CHO-S cell culture medium expressing hPD-1 CD Forti CHO+1mM MTX+1%ACA+1%GlutaMAX.
  • IBI319 ⁇ PD1/CD137
  • parent antibody 11444 ⁇ PD-1
  • parent antibody 7A5 pool3
  • final concentration of IgG1 400, 80, 16, 3.2, 0.64, 0.128, 0.0256, 0.00512, 0.001024nM (5 times serial dilution)
  • IBI319 showed the activity of activating the NFKB_Luciferase signal. This activation activity was very weak under the condition of incubating with Jurkat/CD137-NFKB-luc cells alone ( Figure 14), while the addition of CHO -S/PD-1 cells were significantly enhanced when incubated together ( Figure 13). The degree of enhancement is positively correlated with the ratio of CHO-S/PD-1 cells ( Figure 15).
  • the anti-CD137 end of IBI319 shows activating activity, which depends on the expression of PD-molecules.
  • IBI319 in mixed lymphocyte reaction (MLR) can induce T cell activation
  • PBMC Human peripheral blood mononuclear cells
  • AllCells frozen state
  • Ficoll-Paque PLUS GE Healthcare density gradient centrifugation.
  • hGM-CSF R&D Systems, 215-GM-050 or Sanofi, NDC 0024-5843-01
  • 500IU/mL hIL-4 R&D Systems, 204-IL-050
  • DC Immature dendritic cells
  • the two types of cells from different donors were mixed in a 96-well V-shaped plate, and each well contained 100 ⁇ L of AIM-V medium (Thermo Fisher Scientific), 5 ⁇ 10 4 -1 ⁇ 10 5 CD4+ T cells and 5 ⁇ 10 3 immature DC cells.
  • AIM-V medium Thermo Fisher Scientific
  • 5 ⁇ 10 4 -1 ⁇ 10 5 CD4+ T 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 ⁇ L/well (total 8 replicate wells), 37°C, 5% CO 2 Cultivate for 67 hours.
  • 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.
  • IBI319 can induce a unique immune gene expression profile in the H292 NSCLC Winn tumor model
  • mice are injected intraperitoneally with a mixture of H292 tumor cells and cryopreserved 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.
  • 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 to analyze the gene expression level of total RNA (500ng).
  • 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 one-way analysis of variance.
  • 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- ⁇ , GZMB), multiple cytokines and chemotaxis Factors, as well as MHC class I and II antigens (such as HLA-B, HLA-DRA).
  • T cell infiltration and activation related genes such as CD3E, CD4, CD8B, IFN- ⁇ , GZMB
  • multiple cytokines and chemotaxis Factors such as CDHC class I and II antigens (such as HLA-B, HLA-DRA).
  • MHC class I and II antigens such as HLA-B, HLA-DRA
  • 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 10x10 6 cells/ml and the density of PBMC to 2.5x10 6 cells/ml.
  • mice 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) according to the formula ⁇ / 6 * length * width Calculated,% T / C according to the formula 100x ⁇ T / ⁇ C (eg ⁇ T> 0) is calculated. Use the MIXED program of SAS software for statistical analysis.
  • the combination of p-11444+p-7A5 and monoclonal antibodies p-7A5 and p-11444 did not show efficacy.
  • 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 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.
  • 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.
  • Preparation of prescription 1 weigh 1.55 g histidine, 50.00 g sorbitol and purified water to make the volume up to 980 mL, adjust the pH to 5.7 with HCl, and make the volume up to 1L.
  • the IBI319 protein was replaced by ultrafiltration into the above solution. After the replacement is completed, adjust the protein content to about 50mg/ml, and finally add 0.50g polysorbate 80 to obtain prescription 1.
  • the composition of prescription 1 is: 50.0mg/ml IBI319, 1.55mg/ml histidine, 50.00mg/ml Sorbitol, 0.50mg/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 50mg/ml, and finally 0.50g polysorbate 80 is added to obtain prescription 2.
  • composition of prescription 2 is: 50.0mg/ml IBI319, 1.55mg/ml histidine, 25.00mg/ml sorbitol, 16.85mg /ml arginine hydrochloride, 0.50mg/ml polysorbate 80, pH 5.7, the balance is water.
  • Preparation of prescription 3 weigh 1.55 g histidine, 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. After the replacement, adjust the protein content to about 50mg/ml, and finally add 0.50g polysorbate 80 to obtain prescription 3.
  • the composition of prescription 3 is: 50.0mg/ml IBI319, 1.55mg/ml histidine, 16.85mg/ml Arginine hydrochloride, 0.50mg/ml polysorbate 80, pH 5.7, the balance is water.
  • Preparation of prescription 4 weigh 2.94g sodium citrate (dihydrate), 16.85g arginine hydrochloride and purified water to make the volume to 980mL 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 50mg/ml, and finally add 0.50g polysorbate 80 to obtain prescription 4.
  • the composition of prescription 4 is: 50.0mg/ml IBI319, 2.94mg/ml sodium citrate (dihydrate) , 16.85mg/ml arginine hydrochloride, 0.50mg/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 disodium edetate and dilute to 980 mL with purified water, adjust the pH to 5.7 with HCl, and dilute 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 0.50g polysorbate 80 to obtain prescription 5.
  • composition of prescription 5 is: 50.0mg/ml IBI319, 1.55mg/ml histidine, 25.00mg/ ml sorbitol, 16.85mg/ml arginine hydrochloride, 0.01mg/ml disodium edetate, 0.50mg/ml polysorbate 80, pH 5.7, the balance is water.
  • Prescription 1 used hydrochloric acid to adjust pH
  • Prescription 2 used hydrochloric acid to adjust pH
  • prescription 4 used citric acid to adjust pH
  • the samples of prescription 1 to prescription 5 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.
  • Table 19 shows that the protein content of each prescription did not change significantly when placed at 40°C for 4 weeks and at 25°C for 2 months.
  • 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°C 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.
  • 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, and the polysorbate 80 of prescription 4 and prescription 5 did not decrease.
  • Table 23 shows the results of the shaking experiment of the formulation 5 samples.
  • 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.
  • 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.
  • formulation 5 was finally selected as the IBI319 formulation formulation.
  • fixed ratios of histidine hydrochloride and histidine are selected to achieve pH 5.7. Its composition: 50.0mg/ml IBI319, 0.57mg/ml histidine, 1.33mg/ml histidine hydrochloride, 25.00mg/ml sorbitol, 16.85mg/ml arginine hydrochloride, 0.01mg/ml edetic acid Sodium, 0.50mg/ml polysorbate 80, pH 5.7.

Abstract

本发明公开了一种重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体制剂及其用途。

Description

重组抗程序性死亡受体1和抗分化抗原簇137双特异性抗体制剂及其用途 技术领域
本发明属于生物技术领域，涉及重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体制剂及其用途。
背景技术
免疫检查点是在免疫细胞(例如T细胞和树突状细胞)上表达的一组膜蛋白，包括多种共抑制和共刺激受体，它们在调节适应性免疫应答中起重要作用。精心研究的检查点包括PD-1和CD137。PD-1及其配体，程序性细胞死亡配体1(PD-L1)和程序性细胞死亡配体2(PD-L2)之间的相互作用提供了抑制信号，该信号已被证明在肿瘤免疫逃逸和在肿瘤微环境中发生的免疫抑制中起到重要作用。
虽然抗PD-1抗体和/或抗PD-L1抗体对PD-1抑制信号的阻断已在临床上得到验证，并已在治疗某些癌症方面取得了重大的临床进展，但仍有许多患者对此无反应，复发，获得对PD-1或PD-L1抗体治疗的耐药性，或者对治疗不耐受。CD137，也称为4-1BB，在激活T细胞驱动的免疫反应中起作用，例如通过促进T细胞增殖和效应子功能，增强免疫记忆并抑制激活诱导的细胞死亡。
靶向CD137的激动性抗体已在鼠肿瘤模型中显示出作为单药治疗和联合治疗的前景，但是，由于毒性和/或缺乏疗效，靶向人CD137的激动剂在人癌症患者中无论作为单药治疗还是联合治疗均未显示出足够的反应。实际上，还没有批准靶向人CD137的激动性抗体用于人的治疗用途。因此，需要针对免疫检查点途径的其他治疗方法。
已经研究了激动CD137和拮抗PD-1的抗体的组合，例如urelumab(即抗CD137激动剂单克隆抗体)和nivolumab(即抗PD-1拮抗剂单克隆抗体)的组合，在临床试验用于治疗实体瘤(Tolcher等，Clin Cancer Res 23(18)2017)。然而，较高的潜在有效剂量的urelumab显示与转氨酶升高(transaminitis)和其他不良事件有关。将CD137激动抗体特异性靶向表达PD-1的那些细胞可能 会限制与激动CD137抗体全身给药相关的不良事件。因此，需要设计本发明的双特异性抗体，以通过优先结合表达PD-1的细胞来提供免疫增强作用，且潜在地限制CD137激动作用对那些也表达PD-1的细胞的影响。
WO2018/045110公开了几种双特异性抗体(Fab-scFv-Fc形式)，其与共抑制受体和共刺激受体结合以激活T细胞来治疗癌症，包括[ICOS x PD-1]和[CD137 x PD-1]。似乎在WO2018/045110中公开的CD137 Fab衍生自BMS20H4.9，其可能与严重的转氨酶升高的发展有关(Segal等人，Clinical Cancer Research(2016)1-8)。与WO2018/045110中公开的双特异性抗体相比，IgG样双特异性抗体具有许多与天然IgG抗体相关的有利特性，例如高稳定性，长血清半衰期和低免疫原性(Ha等人，Frontiers in Immunology(2016)第394条)。由于与BMS20H4.9相关的已知毒性以及由于WO2018/045110中描述的双特异性抗体的不合需要的结构形式，因此需要另外的双特异性抗体，它们是拮抗人PD-1且激动人CD137的IgG样双特异性抗体，其促进强大的抗癌免疫反应，并显示出可接受的毒性谱。
同时，在本领域中对于含有足够稳定且适于施用给人受试者的拮抗人PD-1且激动人CD137的双抗的新药物制剂也仍存在需要。此外，对于这样的抗体制剂，寻找制剂处方的简单和易用性，也是有利的。
发明内容
发明简述
针对上述所述双特异性抗体及相应的稳定的抗体制剂的需求，本发明开发了一种重组抗程序性死亡受体1(PD-1)(SEQ ID NO：25)和抗分化抗原簇137(CD137)(SEQ ID NO：26)双特异性抗体(代码IBI319)以及含有所述抗体或其功能性片段的稳定的制剂。
一方面，本发明涉及一种液体抗体制剂，包含重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段以及缓冲剂，优选地所述制剂的pH值为约5.0-7.0；更优选，所述制剂的pH值为约5.5-6.0；进一步优选地，所述pH值为约5.7。
在某些方案中，液体抗体制剂中所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段的含量为约 1mg/mL-150mg/mL，优选为约10mg/mL-100mg/mL，例如，约15、20、25、30、35、40、45、50、55、60、70、80、90mg/mL，更优选地为约50.0mg/ml。
在某些方案中，所述缓冲剂选自组氨酸、盐酸组氨酸或它们的组合，或者所述缓冲剂选自枸橼酸盐、枸橼酸盐溶剂合物(例如，柠檬酸盐水合物)或它们的组合，例如，枸橼酸钠、二水枸橼酸钠或它们的组合，或者所述缓冲剂选自醋酸盐、醋酸盐溶剂合物(例如，醋酸盐水合物)或它们的组合，或者所述缓冲剂选自磷酸盐、磷酸盐溶剂合物(例如，磷酸盐水合物)或它们的组合；优选地，所述缓冲剂的浓度为约5-100mM，更优选地为约5-60mM，例如，约5、10、15、20、25、30、40、50、60mM；
优选地，所述缓冲剂为组氨酸，所述组氨酸的含量为约0.775mg/mL-15.5mg/mL；更优选地，所述组氨酸的含量为约0.775mg/mL-9.3mg/mL，例如约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、9mg/mL；进一步优选地，所述组氨酸的含量为约1.55mg/ml；
优选地，所述缓冲剂为组氨酸和盐酸组氨酸的组合，其中所述组氨酸的含量为约0.270mg/mL-5.4mg/mL，所述盐酸组氨酸的含量为约0.660mg/mL-13.33mg/mL；更优选地，所述组氨酸的含量为约0.270mg/mL-3.24mg/mL，例如约0.5、1.0、1.5、2、2.5、3mg/mL，所述盐酸组氨酸的含量为约0.660mg/mL-8mg/mL，例如约1.0、1.5、2.0、2.5、3.0、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8mg/mL；更优选地，所述组氨酸的含量为约0.57mg/ml，所述盐酸组氨酸的含量为约1.33mg/ml。
在某些方案中，所述制剂还包含稳定剂；优选地，所述制剂还包含稳定剂，所述稳定剂包括多元醇和/或氨基酸；所述多元醇选自山梨醇、甘露醇、蔗糖、海藻糖、麦芽糖及组合，所述氨基酸包括精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸或其组合；优选地，所述稳定剂选自山梨醇和/或盐酸精氨酸、山梨醇和/或精氨酸，更优选地，所述稳定剂为山梨醇和盐酸精氨酸的组合、或山梨醇和精氨酸的组合，优选地，所述精氨酸或盐酸精氨酸的含量为约20mM-200mM，进一步优选地，所述精氨酸或盐酸精氨酸的含量为约40mM-150mM，例如约40、50、60、70、80、90、100、110、120、130、140、150mM；更优选地，所述精氨酸或盐酸精氨酸的含量为约85mM；
优选地，所述山梨醇的含量为约10mg/mL-100mg/mL，所述盐酸精氨酸的含量为约4mg/mL-42.0mg/mL；进一步优选地，所述山梨醇的含量为约20mg/mL-60mg/mL，例如约30、40、50mg/mL，所述盐酸精氨酸的含量为约8.4mg/mL-33.7mg/mL，例如约15、20、25、30mg/mL；更优选地，所述山梨醇的含量为约25.00mg/ml，所述盐酸精氨酸的含量为约16.85mg/ml。
在某些方案中，所述制剂还包含表面活性剂；
优选地，所述表面活性剂选自非离子型表面活性剂，例如聚山梨酯80、聚山梨酯20、泊洛沙姆和聚乙二醇聚山梨酯80中的一种或多种，更优选聚山梨酯80；
更优选地，所述表面活性剂的含量为约0.1mg/mL-1mg/mL；进一步优选为约0.3mg/mL-0.7mg/mL，例如0.3、0.4、0.5、0.6、0.7mg/mL；进一步优选地为约0.50mg/ml。
在某些方案中，所述制剂还包含螯合剂；
优选地，所述螯合剂选自依地酸二钠、二乙基三胺五乙酸和/或EDTA，更优选依地酸二钠；
更优选地，所述螯合剂的含量为约0.005mg/mL-0.1mg/mL；进一步优选为约0.01mg/mL-0.05mg/mL，例如约0.02、0.03、0.04mg/mL；进一步优选地为约0.01mg/ml。
在某些方案中，提供一种液体抗体制剂，所述制剂含有以下组分：约50.0mg/ml重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体、约0.57mg/ml组氨酸、约1.33mg/ml盐酸组氨酸、约25.00mg/ml山梨醇、约16.85mg/ml盐酸精氨酸、约0.01mg/ml依地酸二钠、约0.50mg/ml聚山梨酯80，pH约5.7，余量为水。
在某些方案中，所述液体抗体制剂中，
所述抗体或其抗原结合片段包含第一重链(HCl)，其包含第一重链可变区(HCVR1)和恒定区；第一轻链(LC1)，其包含第一轻链可变区(LCVR1)和恒定区；第二重链(HC2)，其包含第二重链可变区(HCVR2)和恒定区；以及第二轻链(LC2)，其包含第二轻链可变区(LCVR2)和恒定区，其中：
所述HCVR1包含SEQ ID NO：4所示的重链可变区所含的三个互补决定区域HCDR1、HCDR2和HCDR3，并且所述LCVR1包含SEQ ID NO：10所示的 轻链可变区所含的LCDR1、LCDR2和LCDR3；以及
所述HCVR2包含SEQ ID NO：16所示的重链可变区中所含的三个互补决定区域(CDR)HCDR1、HCDR2和HCDR3，并且所述LCVR2包含SEQ ID NO：22所示的轻链可变区所含的三个互补决定区域LCDR1、LCDR2和LCDR3；优选地
a)抗体第一重链包含：具有氨基酸序列SEQ ID NO：1的互补决定区1(HCDR1)、具有氨基酸序列SEQ ID NO：2的互补决定区2(HCDR2)、具有氨基酸序列SEQ ID NO：3的互补决定区3(HCDR3)；
b)抗体第一轻链包含：具有氨基酸序列SEQ ID NO：7的互补决定区1(LCDR1)、具有氨基酸序列SEQ ID NO：8的互补决定区2(LCDR2)、具有氨基酸序列SEQ ID NO：9的互补决定区3(LCDR3)；
c)抗体第二重链包含：具有氨基酸序列SEQ ID NO：13的互补决定区1(HCDR1)、具有氨基酸序列SEQ ID NO：14的互补决定区2(HCDR2)、具有氨基酸序列SEQ ID NO：15的互补决定区3(HCDR3)；
d)抗体第二轻链包含：具有氨基酸序列SEQ ID NO：19的互补决定区1(LCDR1)、具有氨基酸序列SEQ ID NO：20的互补决定区2(LCDR2)、具有氨基酸序列SEQ ID NO：21的互补决定区3(LCDR3)。
在某些方案中，所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体包含：第一重链、第二重链以及第一轻链和第二轻链，其中每条链包含可变区和恒定区，其中：
e)抗体第一重链可变区(HCVR)包含SEQ ID NO：4的序列或与其具有至少90％，95％，98％或99％同一性的序列；
f)抗体第一轻链可变区(LCVR)包含SEQ ID NO：10的序列或与其具有至少90％，95％，98％或99％同一性的序列；
g)抗体第二重链可变区(HCVR)包含SEQ ID NO：16的序列或与其具有至少90％，95％，98％或99％同一性的序列；
h)抗体第二轻链可变区(LCVR)包含SEQ ID NO：22的序列或与其具有至少90％，95％，98％或99％同一性的序列；
优选地，
e)抗体第一重链可变区(HCVR)具有氨基酸序列SEQ ID NO：4；
f)抗体第一轻链可变区(LCVR)具有氨基酸序列SEQ ID NO：10；
g)抗体第二重链可变区(HCVR)具有氨基酸序列SEQ ID NO：16；
h)抗体第二轻链可变区(LCVR)具有氨基酸序列SEQ ID NO：22。
在某些方案中，所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体包含：第一重链、第二重链以及第一轻链和第二轻链，其中每条链包含可变区和恒定区，其中：第一重链恒定区序列(HCCR)具有氨基酸序列SEQ ID NO：30，第一轻链恒定区序(LCCR)具有氨基酸序列SEQ ID NO：31，第二重链恒定区(HCCR)序列具有氨基酸序列SEQ ID NO：32，第二轻链恒定区(LCCR)序列具有氨基酸序列SEQ ID NO：33。
在某些方案中，所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体包含：第一重链、第二重链以及第一轻链和第二轻链，其中：
i)抗体第一重链包含SEQ ID NO：5的序列或与其具有至少90％，95％，98％或99％同一性的序列；
j)抗体第一轻链包含SEQ ID NO：11的序列或与其具有至少90％，95％，98％或99％同一性的序列；
k)抗体第二重链包含SEQ ID NO：17的序列或与其具有至少90％，95％，98％或99％同一性的序列；以及
l)抗体第二轻链包含SEQ ID NO：23的序列或与其具有至少90％，95％，98％或99％同一性的序列；
优选地，
i)抗体第一重链具有氨基酸序列SEQ ID NO：5；
j)抗体第一轻链具有氨基酸序列SEQ ID NO：11；
k)抗体第二重链具有氨基酸序列SEQ ID NO：17；以及
l)抗体第二轻链具有氨基酸序列SEQ ID NO：23。
在某些方案中，所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体包含：第一重链、第二重链以及第一轻链和第二轻链，其中，第一重链与第一轻链之间形成至少一个二硫键，第二重链与第二轻链之间形成至少一个二硫键，以及第一重链和第二重链之间形成至少一个二硫键；优选地所述双特异性抗体是改性的人IgG1。
在某些方案中，本发明提供一种液体抗体制剂，其包含：
(i)约1mg/mL-150mg/mL的所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段；
(ii)约5-100mM的组氨酸；
(iii)约10mg/mL-100mg/mL的山梨醇以及约20mM-200mM精氨酸或盐酸精氨酸；
(iv)约0.1mg/mL-1mg/mL聚山梨酯80；和
(v)任选地，约0.005mg/mL-0.1mg/mL的依地酸二钠，
其中所述液体制剂的pH为约5.0-7.0，例如，约5.5、6.0、6.5；
例如，所述液体抗体制剂包含
(i)约10mg/mL-100mg/mL的所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段；
(ii)约5-60mM的组氨酸；
(iii)约20mg/mL-60mg/mL的山梨醇以及约8.4mg/mL-33.7mg/mL盐酸精氨酸；
(iv)约0.3mg/mL-0.7mg/mL聚山梨酯80；和
(v)任选地，约0.01mg/mL-0.05mg/mL的依地酸二钠，
其中所述液体制剂的pH为约5.5-6.0，例如，约5.7；
或者，所述液体抗体制剂包含
(i)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、1.55mg/ml组氨酸，50.00mg/ml山梨醇，0.50mg/ml聚山梨酯80，pH 5.7；或
(ii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、1.55mg/ml组氨酸，25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
(iii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、1.55mg/ml组氨酸，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
(iv)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、2.94mg/ml枸橼酸钠(二水)， 16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
(v)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、2.94mg/ml枸橼酸钠(二水)，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；或
(ii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、0.57mg/ml组氨酸、1.33mg/ml盐酸组氨酸，25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
(iii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、0.57mg/ml组氨酸、1.33mg/ml盐酸组氨酸，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
(iv)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、2.94mg/ml枸橼酸钠(二水)，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；或
(v)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、0.57mg/ml组氨酸、1.33mg/ml盐酸组氨酸、25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.01mg/ml依地酸二钠，0.50mg/ml聚山梨酯80，pH 5.7
在某些方案中，提供一种制备本发明所述抗体液体制剂的方法，包括以下步骤：
(1)将除所述表面活性剂之外的成分配制成溶液；
(2)通过超滤，采用步骤(1)制备的溶液对所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其片段进行换液，然后浓缩至目标浓度；
(3)添加所述表面活性剂到步骤(2)制备的液体中。
在某些方案中，提供一种固体抗体制剂，其通过固化本发明所述的液体抗体制剂而获得，所述固化是通过例如结晶法、喷雾干燥法、冷冻干燥法实施的，所述固体抗体制剂例如是冻干粉针剂形式。
在某些方案中，提供一种递送装置，其包含本发明所述的液体抗体制剂或固体抗体制剂。
在某些方案中，提供一种预填装注射器，其包含本发明所述的液体抗体制剂或固体抗体制剂，用于静脉内注射或者肌内注射。
在某些方案中，提供本发明所述液体抗体制剂或固体抗体制剂在制备用于预防或治疗癌症的递送装置或预填装注射器或药物药物中的用途，优选的所述癌症选自黑色素瘤、非小细胞肺癌、小细胞肺癌、头颈癌、肝癌、结直肠癌、胰腺癌、胃癌、肾癌、膀胱癌、前列腺癌、乳腺癌、卵巢癌、子宫内膜癌、食管癌、软组织肉瘤、胆管癌、甲状腺癌、肝细胞癌或间皮瘤；其中所述药物可以与电离辐射同时、分开或者依次向癌症患者施用；或者可以与一种或多种化疗药物同时、分开或者依次向癌症患者施用；或者可以与电离辐射以及一种或多种化疗药物同时、分开或者依次向癌症患者施用；优选地所述化疗药物选自5-氟尿嘧啶、羟基脲、吉西他滨、甲氨蝶呤、阿霉素、足叶乙甙卡铂、顺铂、环磷酰胺、美法仑、达卡巴嗪、紫杉醇、喜树碱、FOLFIRI、FOLFOX、多西紫杉醇、柔红霉素、紫杉酚、奥沙利铂或其组合。
在某些方案中，本发明所述液体抗体制剂或固体抗体制剂用于预防或治疗疾病，所述疾病优选为癌症，更优选为选自黑色素瘤、非小细胞肺癌、小细胞肺癌、头颈癌、肝癌、结直肠癌、胰腺癌、胃癌、肾癌、膀胱癌、前列腺癌、乳腺癌、卵巢癌、子宫内膜癌、食管癌、软组织肉瘤、胆管癌、甲状腺癌、肝细胞癌或间皮瘤；其中所述液体抗体制剂或固体抗体制剂可以与电离辐射同时、分开或者依次向癌症患者施用；或者可以与一种或多种化疗药物同时、分开或者依次向癌症患者施用；或者可以与电离辐射以及一种或多种化疗药物同时、分开或者依次向癌症患者施用；优选地所述化疗药物选自5-氟尿嘧啶、羟基脲、吉西他滨、甲氨蝶呤、阿霉素、足叶乙甙卡铂、顺铂、环磷酰胺、美法仑、达卡巴嗪、紫杉醇、喜树碱、FOLFIRI、FOLFOX、多西紫杉醇、柔红霉素、紫杉酚、奥沙利铂或其组合。
在某些方案中，本发明提供一种预防或治疗患者癌症的方法，包括向患者施用有效剂量的本发明所述液体抗体制剂或固体抗体制剂，或者通过本发明的递送装置或预填装注射器向患者施用有效剂量的液体抗体制剂或固体抗体制剂；癌症优选为选自黑色素瘤、非小细胞肺癌、小细胞肺癌、头颈癌、肝癌、结直肠癌、胰腺癌、胃癌、肾癌、膀胱癌、前列腺癌、乳腺癌、卵巢癌、子宫内膜癌、食管癌、软组织肉瘤、胆管癌、甲状腺癌、肝细胞癌或 间皮瘤；其中所述液体抗体制剂或固体抗体制剂可以与电离辐射同时、分开或者依次向癌症患者施用；或者可以与一种或多种化疗药物同时、分开或者依次向癌症患者施用；或者可以与电离辐射以及一种或多种化疗药物同时、分开或者依次向癌症患者施用；优选地所述化疗药物选自5-氟尿嘧啶、羟基脲、吉西他滨、甲氨蝶呤、阿霉素、足叶乙甙卡铂、顺铂、环磷酰胺、美法仑、达卡巴嗪、紫杉醇、喜树碱、FOLFIRI、FOLFOX、多西紫杉醇、柔红霉素、紫杉酚、奥沙利铂或其组合。可以通过肠胃外施用给药，例如注射或输注方式。
发明详述
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。
定义
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或多项。
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“免疫检查点分子”意指免疫系统中存在的一类抑制性信号分子，通过调节外周组织中免疫反应的持续性和强度避免组织损伤，并参与维持对于自身抗原的耐受(Pardoll DM.，The blockade of immune checkpoints in cancer immunotherapy.Nat Rev Cancer，2012，12(4)：252-264)。研究发现，肿瘤细胞能够逃避体内免疫系统而失控增殖的原因之一是利用了免疫检查点分子的 抑制性信号通路，由此抑制了T淋巴细胞活性，使得T淋巴细胞不能有效发挥对肿瘤的杀伤效应(Yao S，Zhu Y和Chen L，Advances in targeting cell surface signaling molecules for immune modulation.Nat Rev Drug Discov，2013，12(2)：130-146)。免疫检查点分子包括但不限于程序性死亡1(PD-1)、PD-L1、PD-L2、CD137、细胞毒T淋巴细胞抗原4(CTLA-4)、LAG-3和TIM-3。
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步再划分为超变区(为互补决定区(CDR))，其间插有较保守的区域(为构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。
术语“抗原结合片段”指与完整抗体不同的分子，其包含完整抗体的一部分且结合完整抗体所结合的抗原。抗原结合片段的例子包括但不限于Fv，Fab，Fab’，Fab’-SH，F(ab’) 2；双抗体(diabodies，dAb)；线性抗体；单链抗体(例如scFv)；单结构域抗体(单域抗体)；双价或双特异性抗体的抗原结合片段；骆驼科抗体；和表现出所需的结合PD-1以及CD137能力的其它片段。
如本文所用，术语“多特异性”抗体指具有至少两个抗原结合位点的抗体，所述至少两个抗原结合位点中的每一个抗原结合位点与相同抗原的不同表位或与不同抗原的不同表位结合。本文提供的抗体通常是多特异性抗体，例如双特异性抗体。多特异性抗体是对至少两个不同抗原表位具有结合特异性的抗体。在一个实施方案中，本文提供了这样的双特异性抗体，其具有针对第一抗原和第二抗原的结合特异性。例如第一抗原为PD-1，第二抗原为CD137。
术语“人源化”抗体指包含来自非人类HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在一些实施方案中，人源化抗体包含全部或基本上全部的HVR(例如，CDR)与非人抗体的那些HVR对应并且全部或基本上全 部的FR区与人抗体的那些FR对应。人源化抗体任选地可以包含从人抗体衍生的抗体恒定区的至少一部分。抗体(例如非人抗体)的“人源化形式”指已经历过人源化的抗体。
术语“Fc区”在本文中用于定义免疫球蛋白重链的C端区域，所述区域包含至少一部分的恒定区。该术语包括天然序列Fc区和变体Fc区。在某些实施方案中，人IgG重链Fc区从Cys226或Pro230延伸至重链的羰基端。然而，Fc区的C端赖氨酸(Lys447)可以存在或者可以不存在。除非另外说明，Fc区或恒定区中的氨基酸残基的编号是根据EU编号系统，其也被称为EU索引，如在Kabat等，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，MD，1991中所述。
术语“可变区”或“可变结构域”是指参与抗体与抗原结合的抗体重或轻链的结构域。天然抗体的重链和轻链的可变结构域通常具有相似的结构，其中每个结构域包含四个保守的框架区(FR)和三个互补决定区(参见，例如，Kindt等Kuby Immunology，6th ed.，W.H.Freeman and Co.91页(2007))。单个VH或VL结构域可以足以给予抗原结合特异性。此外，可以使用来自与特定抗原结合的抗体的VH或VL结构域来分离结合所述抗原的抗体，以分别筛选互补VL或VH结构域的文库。参见，例如，Portolano等，J.Immunol.150：880-887(1993)；Clarkson等，Nature 352：624-628(1991)。
可变区通常表现出由三个高变区连接的相对保守的构架区(FR)的相同的一般结构，所述高变区也被称为互补决定区或CDR。通常通过构架区定位(align)来自每对的两条链的CDR，所述CDR使得可结合特异性表位。两条轻链和重链可变区从N-末端到C-末端通常包含结构域FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。
“互补决定区”或“CDR区”或“CDR”或“高变区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变结构域中在序列上高变并且形成在结构上确定的环(“超变环”)和/或含有抗原接触残基(“抗原接触点”)的区域。CDR主要负责与抗原表位结合。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。位于抗体重链可变结构域内的CDR被称作HCDR1、HCDR2和HCDR3，而位于抗体轻链可变结构域内的CDR被称 作LCDR1、LCDR2和LCDR3。在一个给定的轻链可变区或重链可变区氨基酸序列中，各CDR的精确氨基酸序列边界可以使用许多公知的抗体CDR指派系统的任一种或其组合确定，所述指派系统包括例如：基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342：877-883，Al-Lazikani等人，“Standard conformations for the canonical structures of immunoglobulins”，Journal of Molecular Biology，273，927-948(1997))，基于抗体序列可变性的Kabat(Kabat等人，Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services，National Institutes of Health(1987))，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(万维网imgt.cines.fr/)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义(North等，“A new clustering of antibody CDR loop confirmations，Journal of Molecular Biology，406，228-256(2011)”)。本发明采用North CDR定义。
术语“结合”或“特异性结合”意指结合作用对抗原是选择性的，并且可以与不想要的或非特异的相互作用区别开。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、表面等离子共振法(SPR)或生物膜层光学干涉技术(ForteBio)或本领域已知的其它常规结合测定法测定。作为本发明的实施例，指抗体或抗原结合片段在体外测定法中，优选地在采用纯化的野生型抗原的生物膜层光学干涉测量中与抗原表位结合。在某些实施方案中，在抗体或抗原结合片段优选地识别蛋白质和/或大分子的复杂混合物中其靶抗原时，将抗体或抗原结合片段称作特异性结合抗原。
术语“抗体制剂”指一种制备物，所述制备物处于允许作为活性成分的抗体的生物活性可以有效发挥的形式，并且不含有对于待施用该制剂的受试者而言具有不可接受毒性的其它组分。这类抗体制剂通常是无菌的。通常，抗体制剂中包含可药用赋形剂。“可药用”赋形剂是可以合理地施用至受试哺乳动物以便制剂中所用活性成分的有效剂量可以递送至受试者的试剂。赋形剂的浓度与施用模式相适应，例如可以是注射可接受的。
术语“重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体制剂”在本文中也简称为“本发明的抗体制剂”，意指包含抗 PD-1/CD137抗体蛋白作为活性成分并包含可药用赋形剂的制备物。将抗PD-1/CD137抗体蛋白与可药用赋形剂组合后，作为活性成分的抗PD-1/CD137抗体蛋白适于治疗性或预防性施与人类或非人类动物。本发明的抗体制剂可以例如制备成水性形式的液体制剂，例如，即用式预填装注射器，或者制备成冻干制剂，在即将使用前通过溶解和/或悬浮于生理可接受的溶液中进行重构(即，复溶)。在一些实施方案中，抗PD-1/CD137抗体蛋白制剂是液体制剂形式。
“稳定的”抗体制剂是制剂中的抗体在储存于特定条件下之后保有可接受程度的物理稳定性和/或化学稳定性。尽管抗体制剂中所含的抗体在储存特定时间之后可能不会100％维持其化学结构，但通常在储存特定时间之后维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，则认为抗体制剂是“稳定的”。在一些具体的实施方案中，本发明的抗PD-1/CD137抗体蛋白制剂在制造、制备、运输和长期储存过程中表现出低至检测不到的抗体聚集或降解或化学修饰，从而极少或甚至是没有抗PD-1/CD137抗体蛋白的生物活性损失，表现出高度稳定性。在一些实施方案中，本发明的抗PD-1/CD137抗体蛋白制剂在储存后，基本上保留其物理和化学稳定性。优选地，本发明液体制剂可以在室温稳定至少6个月或在40℃±2℃储存1个月，和/或在25℃±2℃稳定至少3个月，和/或在2-8℃稳定至少24个月。
本领域已知多种分析技术可以用于测定蛋白质的稳定性，参见例如Peptide and Protein Drug Delivery，247-301，Vincent Lee Ed.，Marcel Dekker，Inc.，New York，N.Y.，Pubs(1991)and Jones，A.Adv.Drug Delivery Rev.10：29-90(1993)。可以在选定的温度和选定的储存时间测量稳定性。例如，可以基于预期的制剂货架期来选择储存时间。备选地，可以使用加速稳定性试验。在一些实施方案中，通过对抗体制剂进行各种胁迫测试来进行稳定性测试。这些测试可以代表调配的抗体制剂在制造、储存或运输期间可能遭遇到的极端条件，也可以代表在非制造、储存或运输期间可能使抗体制剂中的抗体的不稳定性加速的条件。例如，可以将经调配的抗PD-1/CD137抗体蛋白制剂充填至玻璃小瓶中以检验在高温胁迫下的抗体稳定性。
经一段储存时间后，制剂不显示聚集、沉淀、混浊和/或变性；或显示 非常少的聚集、沉淀、混浊和/或变性，则可以认为抗体在制剂中“保持其物理稳定性”。由于制剂中抗体的聚集可以潜在地导致患者增加的免疫反应，从而导致安全性问题。因此，需要使在制剂中的抗体聚集最小化或防止聚集。光散射法可以用于测定制剂中的可见聚集物。SEC可以用于测定制剂中的可溶性聚集物。此外，可以通过目视检查制剂的外观、颜色和/或澄清度、或者通过OD 350nm法检测制剂的浊度、或者通过非还原型CE-SDS法测定制剂的纯度，来指示制剂的稳定性。在一个实施方案中，通过测定在特定温度下储存特定时间之后制剂中的抗体单体的百分比来测量制剂的稳定性，其中制剂中的抗体单体的百分比越高，则制剂的稳定性越高。
“可接受程度的”物理稳定性可以表示于特定温度下储存特定时间之后，在制剂中检测到至少约90％的抗PD-1/CD137抗体蛋白单体。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的物理稳定性表示至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/CD137抗体蛋白单体。当评估物理稳定性时，药物制剂储存的特定温度可为约-80℃至约45℃的任一温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃、约35℃、约37℃、约40℃、约42℃或约45℃。例如，若储存于约40℃±2℃1个月之后，检测到至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/CD137抗体蛋白单体，则药物制剂视为是稳定的；若储存于约25℃2个月之后，检测到至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/CD137抗体蛋白单体，则药物制剂视为是稳定的；若储存于约5℃9个月之后，检测到至少约90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗PD-1/CD137抗体蛋白单体，则药物制剂视为是稳定的。
经一段储存时间后，如果制剂中的抗体不显示显著的化学改变，则可以认为抗体在制剂中“保持其化学稳定性”。大多数化学不稳定性源自于形成了抗体的共价修饰形式(例如，抗体的电荷变异体)。例如由天冬氨酸异构化、 N和C末端修饰，可以形成碱性变异体；由脱酰胺化、唾液酸化和糖化，可以产生酸性变异体。化学稳定性可以通过检测和/或定量抗体的化学改变形式来评估。例如，可以通过阳离子交换色谱(CEX)或成像毛细管等电聚焦电泳(iCIEF)检测制剂中抗体的电荷变异体。在一个实施方案中，通过测定在特定温度下储存特定时间之后制剂中抗体的电荷变异体百分比变化值来测量制剂的稳定性，其中该变化值越小，则制剂的稳定性越高。
“可接受程度”的化学稳定性可以表示于特定温度下储存特定时间之后制剂中电荷变异体(例如主成分或酸性组分或碱性组分)的百分比变化值不超过50％，例如不超过30％、不超过20％。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的化学稳定性可以表现为主成分电荷变异体的百分比变化值不超过约50％、40％、30％、20％、15％。当评估化学稳定性时，储存药物制剂的温度可为约-80℃至约45℃的任一温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃或约45℃。例如，若在储存于5℃24个月之后，主成分电荷变异体的百分比变化值少于约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％或0.1％，则药物制剂可被视为是稳定的；若在储存于25℃2个月后，主成分电荷变异体的百分比变化值少于约20％、19％、18％、17％、16％、15％、14％、13％、12％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.1％，则药物制剂亦可被视为是稳定的；若在储存于40℃1个月之后，主成分电荷变异体的百分比变化值少于约50％、40％、30％、20％、10％、5％或4％，则药物制剂亦可被视为是稳定的。
术语“冻干制剂”是指通过液体制剂的冷冻干燥处理得到或能够得到的组合物。优选地，其为具有少于5％、优选少于3％水含量的固体组合物。
术语“重构制剂”是指将固体制剂(例如冻干制剂)溶解和/或悬浮于生理可接受的溶液中得到的液体制剂。
文中使用的术语“室温”是指15℃至30℃、优选20℃至27℃、更优选 25℃的温度。
“胁迫条件”是指在化学和/或物理上不利于抗体蛋白的环境，所述环境可以导致不可接受的抗体蛋白失稳定，例如，高温、振荡、冻融、光照。“高温胁迫”是指，将抗体制剂置于室温或甚至于更高温度(例如40℃±2℃)储存一段时间。通过高温胁迫加速试验，可以检查抗体制剂的稳定性。
如本文所使用，术语“肠胃外施用”意指肠内和局部给药以外的给药方式，通常通过注射或输注方式，并且包括但不限于，静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下(subcuticular)、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射以及输注。在一些实施方案中，本发明的稳定抗PD-1/CD137抗体蛋白制剂肠胃外施用于受试者。在一个实施方案中，本发明的抗PD-1/CD137抗体蛋白制剂以皮下、皮内、肌内或静脉内注射方式施用于受试者。
1.抗体
本发明抗体序列：抗CD137臂(7A5*)、抗PD-1臂(11444*)，是分别基于亲本抗体(7A5)、亲本抗体(11444)以及野生型人IgG1恒定区、野生型人Lambda恒定区和野生型人Kappa恒定区进行突变设计完成。具体的本发明抗体CDR区、可变区、恒定区、全长重链和全长轻链以及编码全长重链和轻链的核苷酸序列号以及亲本抗体可变区SEQ ID NO如下表1所示：
表1本发明涉及的核苷酸序列编号
Figure PCTCN2021072103-appb-000001
HCCR：重链恒定区；LCCR：轻链恒定区
具体氨基酸和核苷酸序列
7A5＊的序列
<SEQ ID NO：1；PRT1；人工序列>
Figure PCTCN2021072103-appb-000002
<SEQ ID NO：2；PRT1；人工序列>
Figure PCTCN2021072103-appb-000003
<SEQ ID NO：3；PRT1；人工序列>
Figure PCTCN2021072103-appb-000004
<SEQ ID NO：4；PRT1；人工序列>
Figure PCTCN2021072103-appb-000005
<SEQ ID NO：5；PRT1；人工序列>
Figure PCTCN2021072103-appb-000006
<SEQ ID NO：6；DNA；人工序列>
Figure PCTCN2021072103-appb-000007
Figure PCTCN2021072103-appb-000008
<SEQ ID NO：7；PRT1；人工序列>
Figure PCTCN2021072103-appb-000009
<SEQ ID NO：8；PRT1；人工序列>
Figure PCTCN2021072103-appb-000010
<SEQ ID NO：9；PRT1；人工序列>
Figure PCTCN2021072103-appb-000011
<SEQ ID NO：10；PRT1；人工序列>
Figure PCTCN2021072103-appb-000012
<SEQ ID NO：11；PRT1；人工序列>
Figure PCTCN2021072103-appb-000013
<SEQ ID NO：12；DNA；人工序列>
Figure PCTCN2021072103-appb-000014
11444＊的序列
<SEQ ID NO：13；PRT1；人工序列>
Figure PCTCN2021072103-appb-000015
<SEQ ID NO：14；PRT1；人工序列>
Figure PCTCN2021072103-appb-000016
<SEQ ID NO：15；PRT1；人工序列>
Figure PCTCN2021072103-appb-000017
<SEQ ID NO：16；PRT1；人工序列>
Figure PCTCN2021072103-appb-000018
<SEQ ID NO：17；PRT1；人工序列>
Figure PCTCN2021072103-appb-000019
<SEQ ID NO：18；DNA；人工序列>
Figure PCTCN2021072103-appb-000020
Figure PCTCN2021072103-appb-000021
<SEQ ID NO：19；PRT1；人工序列>
Figure PCTCN2021072103-appb-000022
<SEQ ID NO：20；PRT1；人工序列>
Figure PCTCN2021072103-appb-000023
<SEQ ID NO：21；PRT1；人工序列>
Figure PCTCN2021072103-appb-000024
<SEQ ID NO：22；PRT1；人工序列>
Figure PCTCN2021072103-appb-000025
<SEQ ID NO：23；PRT1；人工序列>
Figure PCTCN2021072103-appb-000026
Figure PCTCN2021072103-appb-000027
<SEQ ID NO：24；DNA；人工序列>
Figure PCTCN2021072103-appb-000028
人PD-1和人CD137序列
<SEQ ID NO：25；PRT1；人类>
Figure PCTCN2021072103-appb-000029
<SEQ ID NO：26；PRT1；人类>
Figure PCTCN2021072103-appb-000030
Figure PCTCN2021072103-appb-000031
野生型人IgG1恒定区序列
<SEQ ID NO：27；PRT1；人类>
Figure PCTCN2021072103-appb-000032
野生型人Lambda恒定区序列
<SEQ ID NO：28；PRT1；人类>
Figure PCTCN2021072103-appb-000033
野生型人Kappa恒定区序列
<SEQ ID NO：29；PRT1；人类>
Figure PCTCN2021072103-appb-000034
本发明抗体恒定区序列
7A5*
<SEQ ID NO：30；PRT1；人工序列>
Figure PCTCN2021072103-appb-000035
Figure PCTCN2021072103-appb-000036
<SEQ ID NO：31；PRT1；人工序列>
Figure PCTCN2021072103-appb-000037
11444*
<SEQ ID NO：32；PRT1；人工序列>
Figure PCTCN2021072103-appb-000038
<SEQ ID NO：33；PRT1；人工序列>
Figure PCTCN2021072103-appb-000039
亲本7A5可变区序列
<SEQ ID NO：34；PRT1；人工序列>
Figure PCTCN2021072103-appb-000040
<SEQ ID NO：35；PRT1；人工序列>
Figure PCTCN2021072103-appb-000041
亲本11444可变区序列
<SEQ ID NO：36；PRT1；人工序列>
Figure PCTCN2021072103-appb-000042
<SEQ ID NO：37；PRT1；人工序列>
Figure PCTCN2021072103-appb-000043
＞11444_HC_protein SEQ ID NO：38
Figure PCTCN2021072103-appb-000044
＞11444_LC_protein SEQ ID NO：39
Figure PCTCN2021072103-appb-000045
＞7A5_HC_protein SEQ ID NO：40
Figure PCTCN2021072103-appb-000046
Figure PCTCN2021072103-appb-000047
＞7A5_LC_protein SEQ ID NO：41
Figure PCTCN2021072103-appb-000048
具体地，本发明重组抗程序性死亡受体1(PD-1，SEQ ID NO：25)和抗分化抗原簇137(CD137，SEQ ID NO：26)双特异性抗体包含：第一重链、第二重链以及第一轻链和第二轻链，其中每条链包含可变区和恒定区，其中：
a)抗体第一重链包含：具有氨基酸序列SEQ ID NO：1的互补决定区1(HCDR1)、具有氨基酸序列SEQ ID NO：2的互补决定区2(HCDR2)、具有氨基酸序列SEQ ID NO：3的互补决定区3(HCDR3)；
b)抗体第一轻链包含：具有氨基酸序列SEQ ID NO：7的互补决定区1(LCDR1)、具有氨基酸序列SEQ ID NO：8的互补决定区2(LCDR2)、具有氨基酸序列SEQ ID NO：9的互补决定区3(LCDR3)；
c)抗体第二重链包含：具有氨基酸序列SEQ ID NO：13的互补决定区1(HCDR1)、具有氨基酸序列SEQ ID NO：14的互补决定区2(HCDR2)、具有氨基酸序列SEQ ID NO：15的互补决定区3(HCDR3)；
d)抗体第二轻链包含：具有氨基酸序列SEQ ID NO：19的互补决定区1(LCDR1)、具有氨基酸序列SEQ ID NO：20的互补决定区2(LCDR2)、具有氨基酸序列SEQ ID NO：21的互补决定区3(LCDR3)。
优选地，e)抗体第一重链可变区(HCVR)具有氨基酸序列SEQ ID NO：4；
f)抗体第一轻链可变区(LCVR)具有氨基酸序列SEQ ID NO：10；
g)抗体第二重链可变区(HCVR)具有氨基酸序列SEQ ID NO：16；
h)抗体第二轻链可变区(LCVR)具有氨基酸序列SEQ ID NO：22。
更优选地，第一重链恒定区序列(HCCR)具有氨基酸序列SEQ ID NO：30，第一轻链恒定区序(LCCR)具有氨基酸序列SEQ ID NO：31，第二重链 恒定区(HCCR)序列具有氨基酸序列SEQ ID NO：32，第二轻链恒定区(LCCR)序列具有氨基酸序列SEQ ID NO：33。
更优选地，i)抗体第一重链具有氨基酸序列SEQ ID NO：5；
j)抗体第一轻链具有氨基酸序列SEQ ID NO：11；
k)抗体第二重链具有氨基酸序列SEQ ID NO：17；以及
l)抗体第二轻链具有氨基酸序列SEQ ID NO：23。
进一步地，抗体第一重链与第一轻链之间形成至少一个二硫键，第二重链与第二轻链之间形成至少一个二硫键，以及第一重链和第二重链之间形成至少一个二硫键。
本发明抗体是改性的人IgG1以减少抗体与Fcγ受体的结合。
本发明抗体与人PD-1的结合亲和力比抗体与人CD137的结合亲和力高10倍以上，优选高100倍以上，因此本发明抗体展现出有利的药学特性。本发明抗体选择性地靶向表达PD-1的细胞并潜在地限制CD137对那些共表达PD-1的细胞的激动作用。
另外，本发明还提供编码本发明抗体第一重链、第一轻链、第二重链以及第二轻链的核苷酸序列，优选地，所述核苷酸序列分别为SEQ ID NO：6、SEQ ID NO：12、SEQ ID NO：18和SEQ ID NO：24所示的核苷酸序列。
本发明的抗体通过培养能够表达本发明抗体的哺乳动物细胞(非限制性的例子包括CHO、NS0、HEK293或COS细胞)并回收所述抗体获得。例如，选择合适的宿主细胞如HEK293或CHO细胞，通过预设最佳重链：轻链载体比例的分泌抗体的表达系统，或者通过同时表达重链和轻链的单载体系统，瞬时或稳定转染所述宿主细胞。具体的，例如可以使用编码具有SEQ ID NO：5所示氨基酸序列的第一重链、编码具有SEQ ID NO：11所示氨基酸序列的第一轻链、编码具有SEQ ID NO：17所示氨基酸序列的第二重链、编码具有SEQ ID NO：23所示氨基酸序列的第二轻链的一个或多个DNA分子，通过分泌蛋白的表达系统瞬时或者稳定转染宿主细胞获得本发明的抗体。
抗体回收和纯化通过本领域常规技术实现。抗体作为药物的活性成分的应用现在已经很广泛。用于将治疗性抗体纯化至药用级的技术是本领域公知的。例如，Tugcu等(Maximizing productivity of chromatography steps for purification of monoclonal antibodies，Biotechnology and Bioengineering 99 (2008)599-613.)描述在蛋白A捕获步骤后使用离子交换色谱(阴离子IEX和/或阳离子CEX色谱)的单克隆抗体三柱纯化方法。Kelley等(Weak partitioning chromatography for anion exchange purification of monoclonal antibodies，Biotechnology and Bioengineering 101(2008)553-566)描述了两柱纯化法，其中在蛋白A亲和色谱后使用弱分配阴离子交换树脂。
一般，重组产生的单克隆抗体可以利用常规的纯化方法纯化，以提供具有足够的可重复性和适度纯度的药物物质用于抗体制剂的配制。例如，在抗体从重组表达细胞分泌至培养基中后，可以使用商业可得的蛋白浓缩过滤器例如Amicon的超滤装置，浓缩来自该表达系统的上清液。之后，可以使用例如色谱、透析和亲和纯化等方式进行抗体的纯化。蛋白A适应于作为亲和配体用于纯化IgG1、IgG2和IgG4型抗体。也可以使用其它抗体纯化方法，例如离子交换色谱。在获得足够纯度的抗体后，可以按照本领域已知的方法，制备包含抗体的制剂。
例如，可以采用如下步骤进行制备：(1)在发酵结束后将发酵液离心澄清去除细胞等杂质以获得上清；(2)使用亲和层析(例如对IgG1、IgG2和IgG4型抗体具有特异亲和力的蛋白A柱)捕获抗体；(3)进行病毒灭活；(4)精制纯化(一般可以采用CEX阳离子交换层析)，以去除蛋白中的杂质；(5)病毒过滤(使病毒滴度降低例如4 log 10以上)；(6)超滤/渗滤(可以用于将蛋白置换于利于其稳定的制剂缓冲液中并浓缩至合适的浓度供注射用)。参见例如，B.Minow，P.Rogge，K.Thompson，BioProcess International，Vol.10，No.6，2012，pp.48-57。
2.抗体制剂
一方面，本发明涉及一种液体抗体制剂，包含重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段以及缓冲剂，优选地所述制剂的pH值为约5.0-7.0；更优选，所述制剂的pH值为约5.5-6.0；进一步优选地，所述pH值为约5.7。
在某些方案中，液体抗体制剂中所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段的含量为约1mg/mL-150mg/mL，优选为约10mg/mL-100mg/mL，例如，约15、20、25、30、35、40、45、50、55、60、70、80、90mg/mL，更优选地为约50.0mg/ml。
在某些方案中，所述缓冲剂选自组氨酸、盐酸组氨酸或它们的组合，或者所述缓冲剂选自枸橼酸盐、枸橼酸盐溶剂合物(例如，柠檬酸盐水合物)或它们的组合，例如，枸橼酸钠、二水枸橼酸钠或它们的组合，或者所述缓冲剂选自醋酸盐、醋酸盐溶剂合物(例如，醋酸盐水合物)或它们的组合，或者所述缓冲剂选自磷酸盐、磷酸盐溶剂合物(例如，磷酸盐水合物)或它们的组合；优选地，所述缓冲剂的浓度为约5-100mM，更优选地为约5-60mM，例如，约5、10、15、20、25、30、40、50、60mM；
优选地，所述缓冲剂为组氨酸，所述组氨酸的含量为约0.775mg/mL-15.5mg/mL；更优选地，所述组氨酸的含量为约0.775mg/mL-9.3mg/mL，例如约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、9mg/mL；进一步优选地，所述组氨酸的含量为约1.55mg/ml；
优选地，所述缓冲剂为组氨酸和盐酸组氨酸的组合，其中所述组氨酸的含量为约0.270mg/mL-5.4mg/mL，所述盐酸组氨酸的含量为约0.660mg/mL-13.33mg/mL；更优选地，所述组氨酸的含量为约0.270mg/mL-3.24mg/mL，例如约0.5、1.0、1.5、2、2.5、3mg/mL，所述盐酸组氨酸的含量为约0.660mg/mL-8mg/mL，例如约1.0、1.5、2.0、2.5、3.0、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8mg/mL；更优选地，所述组氨酸的含量为约0.57mg/ml，所述盐酸组氨酸的含量为约1.33mg/ml。
在某些方案中，所述制剂还包含稳定剂；
优选地，所述稳定剂包括多元醇和/或氨基酸；所述多元醇选自山梨醇、甘露醇、蔗糖、海藻糖、麦芽糖及组合，所述氨基酸包括精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸或其组合；优选地，所述稳定剂选自山梨醇和/或盐酸精氨酸、山梨醇和/或精氨酸，更优选地，所述稳定剂为山梨醇和盐酸精氨酸的组合、或山梨醇和精氨酸的组合，优选地，所述精氨酸或盐酸精氨酸的含量为约20mM-200mM，进一步优选地，所述精氨酸或盐酸精氨酸的含量为约40mM-150mM，例如约40、50、60、70、80、90、100、110、120、130、140、150mM；更优选地，所述精氨酸或盐酸精氨酸的含量为约85mM；
优选地，所述山梨醇的含量为约10mg/mL-100mg/mL，所述盐酸精氨酸的含量为约4mg/mL-42.0mg/mL；进一步优选地，所述山梨醇的含量为约 20mg/mL-60mg/mL，例如约30、40、50mg/mL，所述盐酸精氨酸的含量为约8.4mg/mL-33.7mg/mL，例如约15、20、25、30mg/mL；更优选地，所述山梨醇的含量为约25.00mg/ml，所述盐酸精氨酸的含量为约16.85mg/ml。
在某些方案中，所述制剂还包含表面活性剂；
优选地，所述表面活性剂选自非离子型表面活性剂，例如聚山梨酯80、聚山梨酯20、泊洛沙姆和聚乙二醇聚山梨酯80中的一种或多种，更优选聚山梨酯80；
更优选地，所述表面活性剂的含量为约0.1mg/mL-1mg/mL；进一步优选为约0.3mg/mL-0.7mg/mL，例如0.3、0.4、0.5、0.6、0.7mg/mL；进一步优选地为约0.50mg/ml。
在某些方案中，所述制剂还包含螯合剂；
优选地，所述螯合剂选自依地酸二钠、二乙基三胺五乙酸和/或EDTA，更优选依地酸二钠；
更优选地，所述螯合剂的含量为约0.005mg/mL-0.1mg/mL；进一步优选为约0.01mg/mL-0.05mg/mL，例如约0.02、0.03、0.04mg/mL；进一步优选地为约0.01mg/ml。
在某些方案中，提供一种液体抗体制剂，所述制剂含有以下组分：约50.0mg/ml重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体、约0.57mg/ml组氨酸、约1.33mg/ml盐酸组氨酸、约25.00mg/ml山梨醇、约16.85mg/ml盐酸精氨酸、约0.01mg/ml依地酸二钠、约0.50mg/ml聚山梨酯80，pH约5.7，余量为水。
在某些方案中，提供一种所述的液体抗体制剂，其包含：
(i)约1mg/mL-150mg/mL的所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段；
(ii)约5-100mM的组氨酸；
(iii)约10mg/mL-100mg/mL的山梨醇以及约20mM-200mM精氨酸或盐酸精氨酸；
(iv)约0.1mg/mL-1mg/mL聚山梨酯80；和
(v)任选地，约0.005mg/mL-0.1mg/mL的依地酸二钠，
其中所述液体制剂的pH为约5.0-7.0，例如，约5.5、6.0、6.5；
例如，所述液体抗体制剂包含
(i)约10mg/mL-100mg/mL的所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段；
(ii)约5-60mM的组氨酸；
(iii)约20mg/mL-60mg/mL的山梨醇以及约8.4mg/mL-33.7mg/mL盐酸精氨酸；
(iv)约0.3mg/mL-0.7mg/mL聚山梨酯80；和
(v)任选地，约0.01mg/mL-0.05mg/mL的依地酸二钠，
其中所述液体制剂的pH为约5.5-6.0，例如，约5.7；
或者，所述液体抗体制剂包含
(i)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、1.55mg/ml组氨酸，50.00mg/ml山梨醇，0.50mg/ml聚山梨酯80，pH 5.7；或
(ii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、1.55mg/ml组氨酸，25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
(iii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、1.55mg/ml组氨酸，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
(iv)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、2.94mg/ml枸橼酸钠(二水)，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
(v)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、2.94mg/ml枸橼酸钠(二水)，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；或
(ii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、0.57mg/ml组氨酸、1.33mg/ml盐酸组氨酸，25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
(iii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗 原簇137(CD137)双特异性抗体或其抗原结合片段、0.57mg/ml组氨酸、1.33mg/ml盐酸组氨酸，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
(iv)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、2.94mg/ml枸橼酸钠(二水)，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；或
(v)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、0.57mg/ml组氨酸、1.33mg/ml盐酸组氨酸、25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.01mg/ml依地酸二钠，0.50mg/ml聚山梨酯80，pH 5.7。
在某些方案中，提供一种固体抗体制剂，其通过固化本发明所述的液体抗体制剂而获得，所述固化是通过例如结晶法、喷雾干燥法、冷冻干燥法实施的，所述固体抗体制剂例如是冻干粉针剂形式。
对于制剂中抗体，上文已经进行了详细描述，在此需要强调的是所述抗体及其抗原结合片段可以进一步涵盖如下双特异性抗体或其抗原结合片段：
包含与氨基酸序列SEQ ID NO：4具有至少90％，95％，98％或99％或更高同一性的第一重链可变区(HCVR)；和/或与氨基酸序列SEQ ID NO：10具有至少90％，95％，98％或99％或更高同一性的第一轻链可变区(LCVR)；和/或与氨基酸序列SEQ ID NO：16具有至少90％，95％，98％或99％或更高同一性的第二重链可变区(HCVR)；和/或与氨基酸序列SEQ ID NO：22具有至少90％，95％，98％或99％或更高同一性的第二轻链可变区(LCVR)。在本文中，“序列同一性”是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ile、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign (DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。
在一些实施方案中，本发明制剂中的抗PD-1/CD137抗体的第一重链可变区(HCVR)与SEQ ID NO：4相比具有不超过10个，优选地不超过5个、4个或3个不同残基，优选地所述不同残基为保守氨基酸替代。在一些实施方案中，本发明制剂中的抗PD-1/CD137抗体的第一轻链可变区(LCVR)与SEQ ID NO：10相比具有不超过10个，优选地不超过5个、4个或3个不同残基，优选地所述不同残基为保守氨基酸替代。在一些实施方案中，本发明制剂中的抗PD-1/CD137抗体的第二重链可变区(HCVR)与SEQ ID NO：16相比具有不超过10个，优选地不超过5个、4个或3个不同残基，优选地所述不同残基为保守氨基酸替代。在一些实施方案中，本发明制剂中的抗PD-1/CD137抗体的第二轻链可变区(LCVR)与SEQ ID NO：22相比具有不超过10个，优选地不超过5个、4个或3个不同残基，优选地所述不同残基为保守氨基酸替代。“保守性取代”是指导致某个氨基酸置换为化学上相似的氨基酸的氨基酸改变。提供功能上相似氨基酸的保守性置换表是本领域熟知的。在本发明任一实施方案中，在一个优选的方面，保守取代残基来自以下的保守替代，优选地为表2中所示优选置换残基。
表2氨基酸保守取代示例表
原始残基 示例性取代 优选的保守氨基酸取代
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；正亮氨酸 Leu
Leu(L) 正亮氨酸；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；正亮氨酸 Leu
其它赋形剂
在一些实施方案中，本发明的抗体液体制剂中任选地包含其它赋形剂。所述其它赋形剂包括，例如，抗微生物剂、抗静电剂、抗氧化剂、明胶等等。这些和另外已知的药物赋形剂和/或适用于本发明制剂的添加剂是本领域公知的，例如，列出于“The Handbook of Pharmaceutical Excipients，第4版，Rowe等人编，American Pharmaceuticals Association(2003)；和Remington：the Science and Practice of Pharmacy，第21版，Gennaro编，Lippincott Williams&Wilkins(2005)”。
制剂的分析
在抗体制剂的储存过程中，抗体可能会发生聚集、降解或化学修饰，导致抗体异质性(包括大小异质性和电荷异质性)以及聚集物和片段等，从而影响抗体制剂的质量。因此，有必要进行抗体制剂稳定性的监测。
在本领域中已知多种方法可以用于检测抗体制剂的稳定性。例如，可 以通过还原型CE-SDS、非还原型CE-SDS和SEC-HPLC等方法，分析抗体制剂的纯度和评估抗体的聚集水平；可以通过毛细管等电聚焦电泳(cIEF)、成像毛细管等电聚焦电泳(iCIEF)和离子交换色谱(IEX)等，分析抗体制剂中的电荷变异体。此外，可以通过目视检测制剂外观，快速地判断制剂的稳定性。也可以使用OD350nm法检测制剂的浊度改变，该方法可以给出有关可溶性和不溶性聚集物量的信息。此外，可以使用紫外分光光度法(UV法)检测制剂中的蛋白质含量变化。
非还原型CE-SDS法是一种以毛细管为分离通道进行的抗体纯度测定方法。在CE-SDS中，蛋白迁移由SDS结合引起的表面电荷来驱动，而该表面电荷与蛋白质的分子量成正比。由于所有的SDS-蛋白质复合物都具有相似的质量-电荷比，故可以在毛细管的分子筛凝胶基质中，实现基于分子的大小或流体动力学半径的电泳分离。该方法已经被广泛地用于监测变性的完整抗体的纯度。一般，在非还原型CE-SDS法中，供试样品与SDS样品缓冲液和碘乙酰胺混合。之后，混合物可以于68-72℃孵育约10-15分钟，冷却至室温后离心的上清液用于分析。采用紫外检测器检测蛋白的迁移，获得电泳谱图。抗体制剂纯度可以计算为IgG主峰的峰面积占所有峰面积之和的百分比。关于CE-SDS法的进一步描述，可以参见例如Richard R.等，Application of CE SDS gel in development of biopharmaceutical antibody-based products，Electrophoresis，2008，29，3612-3620。
尺寸排阻高效液相色谱法，即SEC-HPLC法，是用于抗体标准和质控的另一重要方法。该方法主要依据分子的尺寸大小或流体动力学半径差异来进行分子的分离。通过SEC-HPLC，抗体可以分离出三种主要形式：高分子量形式(HMMS)、主峰(主要是抗体单体)、和低分子量形式(LMMS)。抗体纯度可以计算为色谱图上主峰面积占所有峰面积之和的百分比。通过SEC-HPLC法，可以测量制剂产品中抗体单体的百分数，给出可溶性聚集物和剪切物的含量信息。关于SEC-HPLC法的进一步描述，可以参见例如，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)。此外，也可以参见例如，R.Yang等，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；和Alexandre Goyon等，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。
成像毛细管等电聚焦电泳(iCIEF)可以用于分析抗体的电荷异质性。该方法可以提供电荷变异体的定量分布情况。iCIEF基于分子在pH梯度中的电荷差异(表观pI值)来实现分子分离的目的。在iCIEF中，分离柱通常是短毛细管(例如，5cm长，100μm内径的二氧化硅毛细管)，蛋白质在高电压下在毛细管柱中聚焦，并通过在280nM操作的全柱成像检测系统对聚焦进行实时在线监测。该技术的一个优点是，可以通过该全柱检测系统同时记录抗体样品的各种电荷变异体。一般而言，在icIEF中，将样品与尿素和icIEF缓冲液混合，其中所述缓冲液含有甲基纤维素、pI分子量标准和两性电解质。然后，可以在iCIEF分析仪例如iCE280分析仪(Protein Simple，Santa Clara，CA)上，使用iCIEF柱例如ProtionSimple组装的iCIEF柱，在样品聚焦一定时间后，测定280nm的吸光度，获得聚焦抗体电荷变异体的谱图。在iCEIF谱图中，在主峰(即主成分)之前洗脱的蛋白相关峰被分类为酸性组分；相对地，在主峰之后洗脱的蛋白相关峰被分类为碱性组分。主成分、酸性组分和碱性组分的相对量可以表示为占总峰面积的百分数。关于iCIEF的进一步描述，可以参见例如，Salas-Solano O等，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；和Dada OO等，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。
也可以通过阳离子交换高效液相色谱法(CEX-HPLC)测定抗体制剂中抗体的电荷变异体。在该测定法中，以比主峰的保留时间更早从CEX-HPLC柱洗脱出的峰被标记为“酸性峰”，而那些以比主峰的保留时间更晚从CEX-HPLC柱洗脱出的峰被标记为“碱性峰”。
加速稳定性研究可以用于检查产品的稳定性性质，有利于筛选稳定药物制剂形式。例如，可以将制剂样品放置于升高的温度，例如约40℃±2℃、25℃±2℃条件下进行加速稳定性研究。检测指标可以包括外观、可见异物、蛋白含量、浊度、纯度(SEC-HPLC法、非还原型CE-SDS法)和电荷变异体(iCIEF法、CEX-HPLC法)。
本发明的制剂通过40℃强制和25℃等加速稳定性实验，考察不同pH值和不同辅料对蛋白质量的影响，对各制剂处方进行评估。研究过程中检测项目主要包括外观、可见异物、蛋白含量、浊度、纯度(SEC-HPLC法和非还原型CE-SDS法)和电荷变异体(iCIEF法)等。由此获得了本发明要保护的稳定的抗体制剂。
3.制剂的用途
本发明所述液体抗体制剂、固体抗体制剂、递送装置或预填装注射器可以预防或治疗患者癌症，包括向患者施用有效剂量的本发明所述液体抗体制剂或固体抗体制剂，或者通过本发明的递送装置或预填装注射器向患者施用有效剂量的液体抗体制剂或固体抗体制剂。
癌症优选为选自黑色素瘤、非小细胞肺癌、小细胞肺癌、头颈癌、肝癌、结直肠癌、胰腺癌、胃癌、肾癌、膀胱癌、前列腺癌、乳腺癌、卵巢癌、子宫内膜癌、食管癌、软组织肉瘤、胆管癌、甲状腺癌、肝细胞癌或间皮瘤。
其中所述液体抗体制剂或固体抗体制剂可以与电离辐射同时、分开或者依次向癌症患者施用；或者可以与一种或多种化疗药物同时、分开或者依次向癌症患者施用；或者可以与电离辐射以及一种或多种化疗药物同时、分开或者依次向癌症患者施用；优选地所述化疗药物选自5-氟尿嘧啶、羟基脲、吉西他滨、甲氨蝶呤、阿霉素、足叶乙甙卡铂、顺铂、环磷酰胺、美法仑、达卡巴嗪、紫杉醇、喜树碱、FOLFIRI、FOLFOX、多西紫杉醇、柔红霉素、紫杉酚、奥沙利铂或其组合。
治疗效果可包括减少生理症状。用于任何特定受试者的抗体的最佳有效量和浓度将取决于多种因素，包括患者的年龄、体重、健康状况和/或性别、疾病的性质和程度、特定抗体的活性，身体对其清除率，并且也包括与所述抗体制剂组合施用的任何可能的其它治疗。对于具体的情况，所递送的有效 量可以在临床医师的判断范围内来确定。在这方面，已知的基于抗体的药物的应用可以提供一定的指导。剂量可以是单剂量方案或多剂量方案。
用于本文时，“治疗”指减缓、中断、阻滞、缓解、停止、降低、或逆转已存在的症状、病症、病况或疾病的进展或严重性。想要的治疗效果包括但不限于防止疾病出现或复发、减轻症状、减小疾病的任何直接或间接病理学后果、防止转移、降低病情进展速率、改善或缓和疾病状态，以及缓解或改善预后。在一些实施方案中，本发明的抗体分子用来延缓疾病发展或用来减慢疾病的进展。
用于本文时，“预防”包括对疾病或病症或特定疾病或病症的症状的发生或发展的抑制。在一些实施方式中，具有癌症家族病史的受试者是预防性方案的候选。通常，在癌症的背景中，术语“预防”是指在癌症的病征或症状发生前，特别是在具有癌症风险的受试者中发生前的药物施用。
“治疗有效量”指以需要的剂量并持续需要的时间段，有效实现所需治疗结果的量。抗体或抗体片段或其缀合物或组合物的治疗有效量可以根据多种因素如疾病状态、个体的年龄、性别和重量和抗体或抗体部分在个体中激发所需反应的能力而变动。治疗有效量也是这样的一个量，其中抗体或抗体片段或其缀合物或组合物的任何有毒或有害作用不及治疗有益作用。相对于未治疗的对象，“治疗有效量”优选地抑制可度量参数(例如肿瘤生长率)至少约20％、更优选地至少约40％、甚至更优选地至少约50％、60％或70％和仍更优选地至少约80％。可以在预示人肿瘤中的功效的动物模型系统中评价化合物抑制可度量参数(例如，癌症)的能力。可选地，可以通过检验化合物抑制的能力评价组合物的这种特性，所述抑制在体外通过熟练技术人员已知的测定法。
“预防有效量”指以需要的剂量并持续需要的时间段，有效实现所需预防结果的量。通常，由于预防性剂量在对象中在疾病较早阶段之前或在疾病较早阶段使用，故预防有效量将小于治疗有效量。
附图说明
图1为IBI319分子结构示意图。
图2为pH筛选浊度变化趋势图。
图3为pH筛选纯度(SEC-HPLC法)变化趋势图。
图4为pH筛选纯度(非还原型CE-SDS法)变化趋势图。
图5为pH筛选电荷变异体-主成分变化趋势图。
图6为pH筛选电荷变异体-酸性组分变化趋势图。
图7为处方筛选纯度变化趋势图。
图8为处方筛选电荷变异体-主成分变化趋势图(iCIEF法，40℃)。
图9为处方筛选电荷变异体-酸性组分变化趋势图(iCIEF法，40℃)。
图10为处方筛选电荷变异体-主成分变化趋势图(iCIEF法，25℃)。
图11为处方筛选电荷变异体-酸性组分变化趋势图(iCIEF法，25℃)。
图2-9中，T0表示0天，1W表示1周，2W表示2周，4W表示4周。图10和11中，T0表示0天，1M表示1月，2M表示2月。图7-图11中，F1、F2、F3、F4和F5分别表示处方1、处方2、处方3、处方4和处方5。
图12为通过NFAT-luc报告系统验证IBI319对PD-1/PD-L1结合的阻断活性；注：诱导倍数＝RLU(induced-background)/RLU(no antibody control-background)。
图13为通过Jurkat/CD137-NFKB-luc报告系统验证IBI319对CD137结合的激活活性：与CHO-S/PD-1共孵育。
图14为通过Jurkat-CD137-NFKB-luc报告系统验证IBI319对CD137结合的激活活性：不加CHO-S/PD-1共孵育。
图15通过Jurkat-CD137-NFKB-luc报告系统验证IBI319对CD137结合的激活活性：与不同比例的CHO-S/PD-1共孵育；注：诱导倍数＝RLU(induced-background)/RLU(no antibody control-background)。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
以下结合具体实施例，对本发明作进一步说明。应理解，以下实施例仅用于说明本发明而非用于限定本发明的范围。
组氨酸为上海味之素氨基酸有限公司产品，药用级。
盐酸组氨酸为上海味之素氨基酸有限公司产品，药用级。
山梨醇为法国罗盖特产品，货号为H20110265，药用级。
盐酸精氨酸为上海味之素氨基酸有限公司产品，药用级。
枸橼酸钠(二水)为德国Merck产品，货号为1.37042.5000，药用级。
枸橼酸钠(一水)为德国Merck产品，货号为1.00242.5000，药用级。
依地酸二钠为南京化学试剂股份有限公司产品，苏药准字F15431201，药用级。
聚山梨酯80为南京威尔化工有限公司产品，苏药准字F15423203，药用级。
盐酸为德国Merck产品，货号为1.00314.2508，药用级。
蛋白含量(UV法)：使用紫外分光光度计(日本岛津生产，型号UV-1800)测定样品中的蛋白质含量。
聚山梨酯80含量(高效液相色谱-荧光检测法(HPLC-FLD法)：
使用高效液相色谱(Agilent，1260或等效)分离，色谱柱为Knitted Reactor Coil 5m x 0.50mm ID，SUPELCO，流动相为0.15mol/L氯化钠，0.05mol/L Tris，pH 8.0，5％乙腈，5.0umol/L NPN(N-苯基-1-萘胺)，15ppm Brij，色谱柱保护液为0.05％(w/v)NaN 3，进样量50μl，流速0.5ml/分钟，采集时间30分钟，柱温25℃，检测波长280nm。取待测样品用超纯水稀释至2mg/ml，作为供试品溶液。取制剂缓冲液用上述相同处理方式稀释后做为空白溶液。取空白溶液、供试品溶液各10μl注入液相色谱仪，开始检测。
浊度(OD350nm法)：使用紫外分光光度计(日本岛津生产，型号UV-1800)，测定样品在350nm的吸光度，确定样品浊度。
纯度(SEC-HPLC法)：
使用体积排阻色谱柱分离，流动相为磷酸盐缓冲液(称取3.12g二水合磷酸二氢钠，8.77g氯化钠和34.84g精氨酸，超纯水溶解后用盐酸调节pH至6.8并定容至1000ml)，色谱柱保护液为0.05％(w/v)NaN 3，进样量50μl，流速0.5ml/分钟，采集时间30分钟，柱温25℃，检测波长280nm。取待测样品用超纯水稀释至2mg/ml，作为供试品溶液。取制剂缓冲液用上述相同处理方式稀释后做为空白溶液。取空白溶液、供试品溶液各50μl注入液相色谱仪，开始检测。
纯度(非还原型CE-SDS法)：
采用毛细管凝胶电泳法检测。毛细管为无涂层毛细管，内径50μm，总长30.2cm，有效长度20.2cm。电泳前分别使用0.1mol/L氢氧化钠、0.1mol/L盐酸、超纯水、电泳胶70psi冲洗毛细管柱。将待测样品用适量超纯水稀释至2.0mg/ml，取以上稀释后的样品50μl于1.5ml离心管中，分别向其中加入45μl pH 6.5的样品缓冲液(称取一水柠檬酸0.32g，十二水合磷酸氢二钠2.45g，溶于45ml超纯水中，定容至50ml，制得柠檬酸-磷酸盐缓冲液，精密量取该缓冲液200μl，加10％(w/v)十二烷基硫酸钠溶液80μl，加水至1ml，混匀，即得)、1μl内标(10kDa蛋白质，5mg/mL)(Beckman Coulter，货号：390953)和5μl 250mmol/L NEM溶液(称取N-乙基顺丁稀二酰亚胺62mg，溶于2ml超纯水中)，充分混匀后70±2℃加热10±2分钟，冷却至室温后转移至样品瓶作为供试品溶液。取与供试品相同体积的制剂缓冲液，按上述方法同样操作，制得空白溶液。样品进样条件：-5kV 20秒；分离电压：-15kV 35分钟。毛细管柱温控制在25℃，检测波长为220nm。
电荷变异体(iCIEF法)：
采用成像毛细管等电聚焦电泳(iCIEF法)检测。毛细管内径100μm，总长5cm。样品电泳前需分别使用0.5％甲基纤维素溶液(下文中也缩写为MC溶液)、超纯水冲洗毛细管柱。采用真空进样方式，预聚焦电压及时间为1.5kV 1分钟，聚焦电压及时间为3kV 8分钟，进样时间55秒，样品盘温度为10℃，检测波长为280nm。阴极稳定剂(Cathodic Stabilizer)为500mmol/L精氨酸溶液，0.5％MC溶液降低蛋白与毛细管之间的粘附。将供试品用水稀释至1.0mg/ml，取稀释后的供试品溶液20μl，向其中加入78μl预混液(预混液配比如下：70μl pI 0.5％MC溶液，4μl两性电解质(pH 3-10)，2μl阴极稳定剂，1μl pI 5.85标志物，1μl pI 9.99标志物)，充分混匀制得待测样品溶液。进样分析，根据面积归一化法，计算主成分、酸性组分及碱性组分含量。
实施例1、IBI319蛋白的制备
根据美国临时申请号US62/700525和US62/799274以及其后续PCT申请号PCT/US2019/041517所述，获得本发明所述的抗体，代号为IBI319，该抗体具有如表1种记载的SEQ ID NO：5和17的重链序列和SEQ ID NO：11和23的轻链序列，为人源化抗体。
举例：
本发明的抗体可以基本上如下表达和纯化。可以使用合适的预定重链：轻链载体比例或编码重链和轻链的单个载体系统，用分泌抗体的表达系统瞬时或稳定转染适当的宿主细胞，例如HEK 293或CHO。通过使用一种或多种DNA分子，本发明的抗体A可以被分泌抗体的表达系统瞬时或稳定地转染，所述DNA分子编码具有SEQ ID NO：5的氨基酸序列的第一重链，具有SEQ ID NO：11的氨基酸序列的第一轻链，具有SEQ ID NO：17的氨基酸序列的第二重链，具有SEQ ID NO：23的氨基酸序列的第二轻链。可例如借由UV吸收或SDS-PAGE检测抗体片段，且随后可汇集。可使用常见技术浓缩及/或无菌过滤抗体。可溶性聚集体及多聚体可借由常见技术有效移除，该等技术包括尺寸排除、疏水相互作用、离子交换、多峰或羟磷灰石色谱。可将纯化的产物立即于-70℃下冷冻或可经冻干。可以使用许多常用技术之一来纯化抗体。例如，可将介质方便地应用于已用兼容缓冲液(例如磷酸盐缓冲液(pH 7.4))平衡的MabSelect色谱柱(GE Healthcare)或KappaSelect色谱柱(GE Healthcare)。可以洗涤柱以去除非特异性结合组分。结合的抗体可以例如通过pH梯度(例如pH 7至10mM的20mM Tris缓冲液至pH 3.0的10mM柠檬酸钠，pH 7.4至100mM的甘氨酸缓冲液pH 7.4的磷酸盐缓冲盐水)洗脱。可以例如通过UV吸收或SDS-PAGE检测抗体级分，然后可以将其合并。根据预期用途，进一步纯化是可选的。纯化的抗体可以使用常规技术浓缩和/或无菌过滤。可溶性聚集体和多聚体可通过常规技术进行有效去除，包括尺寸排阻色谱，疏水相互作用色谱，离子交换色谱，多峰(multimodal)色谱或羟磷灰石色谱。纯化的抗体可立即冷冻在-70℃或冻干。
以下示例性地示出本发明所述的抗体，代号为IBI319的培养过程，采用CHO Gs细胞(LONZA)转染，基础培养基Dynamis AGT Medium(Gibco)；流加试剂Cell Boost 7a、Cell Boost 7b(HyClone)。在细胞培养试验中，第0天在细胞培养实验中，按密度1.0×10 6个/ml接种细胞至2L细胞反应器中，DO设置为40％，pH控制为7.05±0.20，前期培养温度36.5℃，培养至第6天降温到33.0℃。培养至第3、5、7、9、11天分别流加6.0％(w/w)初始培养重量的流加试剂A(购自HyClone)，0.6％(w/w)初始培养重量的流加试剂B(购自HyClone)。每天根据葡萄糖浓度检测结果，在葡萄糖低于3.0g/L时，补加 葡萄糖浓缩液使细胞液中葡萄糖浓度提高到4.0g/L。根据通气产生的泡沫情况，泡沫高于液面5cm时手动加入10％ADCF Antifoam Solution，总量不能超过50ppm。每天监测细胞密度、活率以及细胞生化代谢情况。连续培养至第15天或者细胞活力≤60％时，收集细胞上清进行纯化。
将纯化使用的重力柱使用0.5M NaOH过夜处理，玻璃瓶等用蒸馏水洗净后在180℃干烤4h，获得纯化柱。纯化前将收集的培养基4500rpm离心30min，弃掉细胞。再将上清使用0.22μl的滤器过滤。每管装填1ml Protein A，并使用10ml结合缓冲液(磷酸钠20mM.NaCl 150mM，PH7.0)平衡。将过滤后的上清加入纯化柱后使用15ml结合缓冲液再平衡。加5ml洗脱缓冲液(柠檬酸+柠檬酸钠0.1M，PH3.5)，收集洗脱液，每1ml的洗脱液加入80μl Tris-HCl将pH调至7.0。收集的抗体用1ml S型强阳离子交换层析纯化，层析柱先用10ml平衡缓冲液(20mM柠檬酸+柠檬酸钠，pH5.0)平衡，将亲和收集的样品加入纯化柱后使用10ml平衡缓冲液再平衡，之后用洗脱缓冲液(20mM柠檬酸+柠檬酸钠+1M氯化钠，pH5.0)进行梯度洗脱，洗脱体积20ml，收集样品超滤浓缩交换到PBS(Gibco，70011-044)中，并检测浓度。
实施例2.上述制备并纯化的本发明抗体进行如下功能验证
IBI319亲和特异性和亲和力测定(BLI测定法)
运用Fortebio Octet平台(美国Fortebio)，采用生物膜层干涉(BLI)测定法测定本发明示例抗体IBI319与不同种属蛋白：人4-1BB(Human 4-1BB)、食蟹猴4-1BB(Cyno 4-1BB)、人PD-1(Human PD-1)、食蟹猴PD-1(Cyno PD-1)蛋白的亲和力，判断IBI319的亲和特异性，同时比较IBI319和亲本抗体7A5抗体与4-1BB的亲和力差异以及IBI319和亲本抗体11444抗体与4-1BB的亲和力差异。
表3实验样品
Figure PCTCN2021072103-appb-000049
亲本抗体7A5以及亲本抗体11444抗体序列请见上文序列部分38-41
其他关键材料
表4试剂信息
Figure PCTCN2021072103-appb-000050
表5溶液配制方法
Figure PCTCN2021072103-appb-000051
注：以上每个配制的样品为检测的最大浓度样品，根据实验设计，抗原按2倍稀释至所需检测浓度梯度。
1.生物素标记抗体(用于检测抗体与带Fc标签抗原的结合)
取180μL超纯水加入1mg EZ-Link Sulfo-NHS-LC-Biotin中，溶解成10 mM的溶液，再稀释100倍至100μM。按摩尔浓度1∶1，将计算量的EZ-Link Sulfo-NHS-LC-Biotin与待标记蛋白混匀，室温孵育1小时后使用脱盐柱去除未标记的Biotin，并等体积回收Biotin标记的蛋白，保存于4℃备用。
2.实验样品分组
将传感器置于200μL SD缓冲液中，浸泡30min后使用。
实验分组信息如表6所示，每孔加入200μL对应样品。
表6实验分组信息
Figure PCTCN2021072103-appb-000052
3.检测检测分为5个步骤，每个循环依次为：平衡(Baseline)→固化抗体(Loadings)→平衡(Baseline)→结合梯度稀释的蛋白(Association)→解离(Dissociation)。
判断标准：通过ForteBio Octet仪器分析软件(Fortebio data analysis 11.0)，采用1∶1 binding(global fitting)对曲线进行拟合。通过分析拟合曲线拟合程度、R2值进行判断结果的可靠性。拟合结果符合R2＞0.95，通过结合和解离常数计算出KD值。
实验结果IBI319与不同种属PD-1、不同种属4-1BB的亲和力检测结果
表7 IBI319与不同种属PD-1、不同种属4-1BB的亲和力检测结果
Figure PCTCN2021072103-appb-000053
结论：IBI319与人PD-1、食蟹猴PD-1有很强的亲和力，IBI319与人4-1BB、食蟹猴4-1BB的亲和力较弱。
IBI319亲和力研究测定(表面等离子共振，SPR法)：运用Biacore平台(瑞典GE Healthcare)，采用SPR法检测IBI319与人PD-1、食蟹猴PD-1、人4-1BB(CD137)、食蟹猴4-1BB的亲和力。
方法：采用SPR法，将抗人Fc抗体偶联在CM5芯片表面之后，通过偶联的抗人Fc抗体捕获IBI319，梯度稀释的人PD-1、食蟹猴PD-1、人4-1BB、食蟹猴4-1BB分别与被捕获的IBI319结合。IBI319的亲本抗体11444(抗PD-1抗体)和亲本抗体7A5(抗4-1BB抗体或抗CD137抗体)分别作为对照。最后通过对结合解离曲线的拟合，计算出亲和力的数值。
亲和力检测
实验所用缓冲液为pH7.4的HBS-EP+(10mM HEPES，150mM NaCl，3mM EDTA，0.05％P20)溶液，检测温度25℃。实验包括：1)将抗人Fc抗体偶联到CM5芯片表面；2)IBI319及对照抗体与Human PD-1、Cyno PD-1、Human 4-1BB、Cyno 4-1BB亲和力检测。
实验结果
实验结果表明IBI319与Human PD-1，Cyno PD-1亲和力分别约为0.1nM和0.04nM，与Human 4-1BB，Cyno 4-1BB的亲和力分别约为394nM和68.5nM。亲和力数据见下表8。
表8 IBI319与Human PD-1，Cyno PD-1，人4-1BB、食蟹猴4-1BB亲和力检测结果
抗体 抗原 K D平均值±SD(M)
IBI319 Human PD-1 1.02E-10±8.31E-12
亲本抗体11444 Human PD-1 1.37E-10±2.89E-11
IBI319 Cyno PD-1 4.09E-11±8.95E-12
亲本抗体11444 Cyno PD-1 1.51E-10±1.99E-11
IBI319 Human 4-1BB 3.94E-07±2.30E-08
亲本抗体7A5 Human 4-1BB 2.19E-07±1.87E-08
IBI319 Cyno 4-1BB 6.85E-08±1.44E-09
亲本抗体7A5 Cyno 4-1BB 5.25E-08±2.99E-09
结论：IBI319与人PD-1、食蟹猴PD-1有很强的亲和力，IBI319与人4-1BB、食蟹猴4-1BB的亲和力较弱。
讨论：
IBI319与PD-1的强亲和力，有利于发挥更强的PD-1/PD-L1阻断作用。抗4-1BB的抗体存在T细胞激活能力与肝毒性的平衡，且两者与其亲和力有很大的相关性。IBI319与4-1BB的亲和力较弱，在保留T细胞激活功能的情况下，更有利于获得更大的功能与安全窗口。IBI319的双特异性抗体设计，能将解除免疫抑制与T细胞激活功能在肿瘤微环境中发挥更好的协同作用。
IBI319体外药效1：IBI319 PD-L1结合的阻断活性测定
实验方案
将膜表面表达PD-L1和TCR activator的CHO细胞(CHO-K1/PD-L1)与表达PD-1和NFAT-luc reporter的Jurkat细胞(Jurkat/PD1-NFAT-luc)共培养，加入不同浓度的IBI319、亲本抗体11444、亲本抗体7A5和hIgG1，通过测定体系中的luciferase酶活来反应药物对PD-1/PD-L 1的阻断活性。
实验步骤
(一)CHO-K1/PD-L1细胞铺板
(二)抗体准备
利用Assay缓冲液(RPMI 1640+1％FBS)配制IBI319、亲本抗体11444、 亲本抗体7A5、亲本抗体11444+亲本抗体7A5、IgG1，起始终浓度为2000nM，4倍梯度稀释，共11个梯度点，具体参见表9。
表9
Figure PCTCN2021072103-appb-000054
(三)Jurkat细胞准备
(四)加样
1.将培养CHO-K1/PD-L1的96孔板取出，每孔吸去95μl上清。
2.按照板布局迅速加入40μl/孔配制好的抗体，设置medium control组，即加入40μl assay buffer。
3.再加入40μl/孔Jurkat/PD1-NFAT-luc细胞(5×10 4个/孔)。37℃，5％CO 2孵育6h。
(五)利用多功能酶标仪(MOLECULAR DEVICES)测定luciferase酶活
实验结果
通过Jurkat/NFAT-luc reporter system实验，IBI319显示了激活NFAT_Luciferase信号的活性，其活性的EC 50为8.14nM。对照抗体亲本抗体11444激活NFAT_Luciferase的EC 50为1.08nM；CD137单抗未显示出活性。具体见图12。
实验结论：
IBI319的抗PD-1端显示了阻断PD-1/PD-L1结合的活性。
IBI319体外药效2：IBI319 CD137端的激动活性测定
实验方案
将表达CD137和NFκB-luc reporter的Jurkat细胞(Jurkat/CD137-NFκB-luc)与CHO-S/hPD-1细胞(CHO-S表达hPD-1)共培养，或不加CHO-S/hPD-1细胞，再加入不同浓度的IBI319、亲本抗体11444、 亲本抗体7A5和hIgG1，通过测定体系中的luciferase酶活来反应药物对CD137通路的激动活性以及与CD137-PD-1的cross-linking作用。
实验步骤
表达CD137和NFκB-luc reporter的Jurkat细胞培养基：90％RPMI 1640 with 1-glutamine，10％FBS，200μg/ml hygromycin B，500μg/ml Geneticin，1mM sodium pyruvate，0.1mM MEM NEAA；
表达hPD-1的CHO-S细胞培养基：CD Forti CHO+1mM MTX+1％ACA+1％GlutaMAX。
(一)抗体准备
利用Assay缓冲液(RPMI 1640+1％FBS)配制IBI319(αPD1/CD137)、亲本抗体11444(αPD-1)、亲本抗体7A5(pool3)、IgG1终浓度：400，80，16，3.2，0.64，0.128，0.0256，0.00512，0.001024nM(5倍梯度稀释)
(二)细胞准备
1.培养Jurkat/CD137-NFκB-luc至倍增时间稳定，细胞密度在3.5×10 5～2.2×10 6cells/ml。细胞计数后，利用Assay缓冲液调整细胞密度为1.0×10 6cells/ml，备用。
2.培养CHO-S/hPD-1细胞至对数期，细胞计数后，利用Assay缓冲液调整细胞密度为2.0×10 6cells/ml，备用。
(三)加样、铺板
1.按照板布局将配制好的抗体加入96孔平底白板中，25μl/孔，设置medium control组，即加入25μl assay buffer。
2.将调整好密度的CHOS/hPD-1和Jurkat/CD137-NFkB-luc等体积混合后，50μl/孔加入到细胞板中(分别为5.0×10 4个/孔和2.5×10 4个/孔)。
设置no CHO-S组，即加入25μl assay buffer和25μl Jurkat/CD137-NFkB-luc。
设置backgroud组，只加入75μl assay buffer。
3.置于37℃±2℃，5％±1％CO 2培养箱中孵育6h。
(四)多功能酶标仪测定luciferase酶活
实验结果：
通过Jurkat/CD137-NFKB-luc reporter system实验，IBI319显示了激活 NFKB_Luciferase信号的活性，此激活活性在与Jurkat/CD137-NFKB-luc细胞单独孵育的条件下很弱(图14)，而在加入CHO-S/PD-1细胞共同孵育时显著增强(图13)。其增强的程度与CHO-S/PD-1细胞的比例成正相关(图15)。
实验结论：
IBI319的抗CD137端显示了激活活性，此激活活性依赖于PD-分子的表达。
混合淋巴细胞反应(MLR)中IBI319可诱导T细胞激活
通过人异体混合淋巴细胞反应检测IBI319中PD1端阻断PD-1/PD-L1结合的活性。具体方法如下：人外周血单个核细胞(PBMC)来源于冻存状态(AllCells)，或从新鲜全血中利用Ficoll-Paque PLUS(GE Healthcare)密度梯度离心分离获得。利用人单核细胞分离试剂盒II或CD14磁珠(Miltenyi Biotec)及AutoMACS Pro separator(Miltenyi Biotec)从PBMC中分选出CD14+单核细胞。在1,000IU/mL hGM-CSF(R&D Systems，215-GM-050或Sanofi，NDC 0024-5843-01)和500IU/mL hIL-4(R&D Systems，204-IL-050)存在情况下，在含10％FBS的RPMI-1640完全培养基中培养单核细胞2天(Table 14)或5天(Table 16、17)获得未成熟树突细胞(DC)。使用人CD4+T细胞分离试剂盒(Miltenyi Biotec)从不同健康供体的人PBMC(AllCells或Indiana Blood Center))中纯化得到CD4+T细胞。之后在96孔V型板中混合不同供体的两种类型细胞，每孔含100μL AIM-V培养基(Thermo Fisher Scientific)，5x10 4-1x10 5CD4+T细胞及5x10 3未成熟DC细胞。将待测抗体(人IgG1-EN、p-11444、p-7A5、IBI319)及对照抗体梯度稀释，加至反应板中，100μL/孔(共8个复孔)，37℃，5％CO 2培养67小时。或将待测抗体加至反应板中，200μL/孔(共3个复孔)，培养4天。收集上清液，并根据说明书使用人IFN-γELISA试剂盒(R&D Systems；SIF50或DY285)和人IL-2 ELISA试剂盒(R&D Systems；S2050)分别检测IFN-γ和IL-2含量。利用9个不同的供体对进行测试。使用GraphPad Prism(GraphPad Software)计算三对不同的供体细胞的EC 50值。
在MLR实验中，利用同种异体人DC细胞和CD4+T细胞，添加p-11444，p-11444+p-7A5、IBI319，与IgG1相比，增强了T细胞激活，并呈 现剂量依赖性。IFN-γ和IL-2合成量的EC 50如表10所示。该实验未检测到CD137单抗的活性。
综上，同种异体混合淋巴细胞反应结果显示，对于多组供体对，IBI319保留了与p-11444类似的PD-1端的阻断活性，增加了细胞因子的释放量。
表10 MLR实验中IBI319的PD1阻断活性(cytokine mean EC50(nM)values across 3 donors)
Figure PCTCN2021072103-appb-000055
IBI319在H292 NSCLC Winn肿瘤模型中可诱导独特的免疫基因表达谱
检测了H292 Winn小鼠模型经IBI319、p-7A5、p-11444和/或对照人IgG1给药处理后的肿瘤组织中人免疫相关基因的表达情况。大致方法为：在第0天，小鼠经腹腔注射H292肿瘤细胞和冻存的人外周血单个核细胞的混合液。待测抗体和对照人IgG1以10mg/kg的剂量在第1、8、15天依次给药。第16天收集肿瘤组织，速冻于液氮。利用MagMAX-96总RNA提取试剂盒(Life Technologies)裂解肿瘤组织，并用组织研磨机(Qiagen)将其匀浆化。使用MagMAX Express-96 Deep Well Magnetic Particle Processor(Life Technologies)提取总RNA，用分光光度计测定OD 260和OD 280用以定量。利用QuantiGene 2.0 plex assay(Affymetrix)和FLEXMAP 3D Luminex仪器(ThermoFisher)对总RNA(500ng)进行基因表达水平分析。利用一个质控脚本将MFI值转化为相对基因表达量(归一化净MFI值)。各基因表达量与对照组相比的倍数变化通过公式计算得到(归一化净MFI值/平均归一化净MFI值)。各基因表达量与对照组相比的平均倍数变化通过单因子方差分析计算。
基因表达分析显示IBI319在体内模型H292肿瘤组织中诱导了特殊的基因表达谱，包括T细胞浸润和激活相关基因(如CD3E，CD4，CD8B，IFN-γ，GZMB)、多个细胞因子和趋化因子，以及MHC I类和II类抗原(如HLA-B，HLA-DRA)。如下表11所示，IBI319诱导的这些基因变化与两个亲和抗体、 组合抗体有所不同。
表11
Figure PCTCN2021072103-appb-000056
IBI319在H292 NSCLC Winn肿瘤模型显示出抗肿瘤活性
检测了H292肿瘤异种移植小鼠模型经IBI319、p-11444、p-7A5、p-11444+p-7A5给药处理后的肿瘤生长抑制率。使用雌性NOD/SCID Gamma(NSG)小鼠(Jackson Laboratories)用于该实验。将人NSCLC细胞株NCIH292(ATCC；CRL-1848)和人PBMC(Stem Cell Technologies)以4∶1比例混合。离心后重悬于HBSS，调整NCI-H292细胞密度为10x10 6个/ml，PBMC密度为2.5x10 6个/ml。在第0天，每只小鼠在右侧皮下注射0.2ml细胞混合液，其中一个对照组只注射肿瘤细胞。第1天，将小鼠进行随机分组，8只/组，开始给药。待测抗体包括对照IgG、p-7A5、p-11444、组合p-11444+p-7A5和IBI319。以10mg/kg的剂量腹腔注射，每周给药一次，共4周。体重和肿瘤体积每周测量两次。肿瘤体积(mm 3)根据公式π/6*长度*宽度 2计算，％T/C根据公式100xΔT/ΔC(如ΔT＞0)计算。利用SAS软件的MIXED程序进行统计分析。
如下表12所示，IBI319(10mg/kg)处理组与对照组IgG相比抑制了肿瘤生长(％T/C＝～2％，p＜.001)，8只小鼠中的6只达到了完全缓解。组合p-11444+p-7A5和单抗p-7A5、p-11444未显示出药效。
表12
Figure PCTCN2021072103-appb-000057
实施例3、pH筛选实验
本实验主要考察不同pH值对IBI319蛋白稳定性的影响，共设计了5个pH值，分别为pH 5.0、5.5、6.0、6.5和7.0，通过pH筛选实验，获得较优的pH值范围。
具体实验步骤如下：
一、配制含有10mM组氨酸、5％(w/v)山梨醇缓冲液，用盐酸分别将pH调节为5.0、5.5、6.0、6.5和7.0，将IBI319蛋白超滤置换到上述不同pH值的缓冲液中，调节蛋白含量至30mg/ml，加入聚山梨酯80至0.2mg/ml，得到pH 5.0、pH 5.5、pH 6.0、pH 6.5和pH 7.0样品，分别过滤分装至西林瓶，加塞，轧盖。
二、将上述样品在第0天、第1周、第2周和第4周分别取样，于40℃±2℃条件下进行稳定性考察，观察外观和可见异物，并检测蛋白含量、浊度、纯度(体积排阻高效液相色谱法(SEC-HPLC法)和非还原型十二烷基硫酸钠毛细管电泳(非还原型CE-SDS法))和电荷变异体(成像毛细管等电聚焦电泳(iCIEF法))。
质量未发生变化的判断标准如表13所示。
表13质量未发生变化的判断标准
Figure PCTCN2021072103-appb-000058
结果如下：
1、外观和可见异物
pH 5.0、pH 5.5、pH 6.0、pH 6.5和pH 7.0样品在40℃±2℃条件下放置4周，外观、可见异物均合格。
2、蛋白含量
蛋白含量检测结果如表14所示。
表14 pH筛选蛋白含量结果(UV法，mg/ml)
Figure PCTCN2021072103-appb-000059
表14表明，在40℃±2℃条件下放置4周，各样品蛋白含量均未发生显著变化。
3、浊度
浊度结果如表15所示，其变化趋势如图2所示。
表15 pH筛选浊度结果(OD350nm法)
Figure PCTCN2021072103-appb-000060
结果表明，在40℃±2℃条件下放置4周，pH 5.0、pH 5.5、pH 6.0、pH 6.5和pH 7.0样品浊度分别上升0.014、0.006、0.012、0.03和0.039，样品在pH 5.0～pH 6.0之间浊度变化较小。
4、纯度
纯度(SEC-HPLC法和非还原型CE-SDS法)结果如表16所示，其变化趋势如图3和图4所示。
表16 pH筛选纯度结果
Figure PCTCN2021072103-appb-000061
结果表明，在40℃±2℃条件下放置4周，pH 5.0、pH 5.5、pH 6.0、pH 6.5和pH 7.0样品纯度(SEC-HPLC法)分别下降5.1％、1.0％、0.9％、 1.3％和1.4％。pH 5.0、pH 5.5、pH 6.0、pH 6.5和pH 7.0样品纯度(非还原型CE-SDS法)分别下降2.5％、1.1％、1.6％、1.9％和2.8％。IBI319蛋白在pH 5.5和pH 6.0条件下较为稳定。
5、电荷变异体
电荷变异体(iCIEF法)结果如表17所示，其变化趋势如图5和图6所示。
表17 pH筛选电荷变异体结果(iCIEF法，％)
Figure PCTCN2021072103-appb-000062
结果表明，在40℃±2℃条件下放置4周，所有样品电荷变异体主成分和酸性组分均发生明显变化。与第0天比较，第4周的pH 5.0、pH 5.5、pH 6.0、pH 6.5和pH 7.0样品电荷变异体主成分分别下降16.9％、16.7％、15.6％、19.5％和28.1％，酸性组分分别上升16.1％、15.4％、14.9％、20.4％和29.2％。从该检测结果分析，IBI319蛋白在pH 5.5和pH 6.0条件下更为稳定。
综上所述，pH筛选实验结果表明，IBI319蛋白在pH 5.5～6.0时较为 稳定，以pH 5.7为例进行后续处方筛选实验。
实施例4、处方筛选实验
本实验考察缓冲体系(组氨酸和枸橼酸)和稳定剂(山梨醇和盐酸精氨酸)对IBI319蛋白稳定性的影响。
一、设计如下5个处方：
处方1的制备：称取1.55克组氨酸，50.00g山梨醇加纯化水定容至980mL，HCl调pH至5.7，定容至1L。将IBI319蛋白超滤置换至上述溶液中。置换完成后，调节蛋白含量至约50mg/ml，最后加入0.50g聚山梨酯80，得到处方1，处方1的组成为：50.0mg/ml IBI319、1.55mg/ml组氨酸，50.00mg/ml山梨醇，0.50mg/ml聚山梨酯80，pH 5.7，余量为水。
处方2的制备：称取1.55克组氨酸，25.00g山梨醇，16.85g盐酸精氨酸加纯化水定容至980mL，HCl调pH至5.7，定容至1L。将IBI319蛋白超滤置换至上述溶液中。蛋白含量至约50mg/ml，最后加入0.50g聚山梨酯80，得到处方2，处方2的组成为：50.0mg/ml IBI319、1.55mg/ml组氨酸，25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7，余量为水。
处方3的制备：称取1.55克组氨酸，16.85g盐酸精氨酸加纯化水定容至980mL，HCl调pH至5.7，定容至1L。将IBI319蛋白超滤置换至上述溶液中。置换完成后，调节蛋白含量至约50mg/ml，最后加入0.50g聚山梨酯80，得到处方3，处方3的组成为：50.0mg/ml IBI319、1.55mg/ml组氨酸，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7，余量为水。
处方4的制备：称取2.94g枸橼酸钠(二水)，16.85g盐酸精氨酸加纯化水定容至980mL枸橼酸调pH至5.7，定容至1L。将IBI319蛋白超滤置换至上述溶液中。置换完成后，调节蛋白含量至约50mg/ml，最后加入0.50g聚山梨酯80，得到处方4，处方4的组成为：50.0mg/ml IBI319、2.94mg/ml枸橼酸钠(二水)，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7，余量为水。
处方5的制备：称取1.55克组氨酸，25.00g山梨醇、16.85g盐酸精氨酸、0.01g依地酸二钠加纯化水定容至980mL，HCl调pH至5.7，定容至1L。将IBI319蛋白超滤置换至上述溶液中。置换完成后，调节蛋白含量至约50 mg/ml，最后加入0.50g聚山梨酯80，得到处方5，处方5的组成为：50.0mg/ml IBI319、1.55mg/ml组氨酸，25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.01mg/ml依地酸二钠，0.50mg/ml聚山梨酯80，pH 5.7，余量为水。
处方1、处方2、处方3和处方5用盐酸调节pH，处方4用枸橼酸调节pH。
将各个处方样品过滤分装至西林瓶中，加塞，轧盖。
二、将上述处方样品进行40℃、25℃、振荡和冻融稳定性考察。具体方案如表18所示。
表18实验条件及取样
Figure PCTCN2021072103-appb-000063
质量未发生变化的判断标准如表13所示。
结果如下：
1、外观和可见异物
处方1～处方5样品在40℃条件下放置4周和在25℃条件下放置2月，外观、可见异物均合格。
2、蛋白含量
蛋白含量检测结果如表19所示。
表19处方筛选蛋白含量结果(UV法，mg/ml)
Figure PCTCN2021072103-appb-000064
表19表明，在40℃条件下放置4周和在25℃条件下放置2月，各处方样品蛋白含量均未发生显著变化。
3、纯度
纯度(SEC-HPLC法和非还原型CE-SDS法)结果如表20所示。
表20处方筛选纯度结果
Figure PCTCN2021072103-appb-000065
表20表明，在40℃条件下放置4周和在25℃条件下放置2月，各处方样品纯度(非还原型CE-SDS法)均未发生显著变化。在40℃条件下放置4周，处方1～处方5样品纯度(SEC-HPLC法)均发生显著变化，与0天比较，纯度(SEC-HPLC法)分别下降1.4％、2.4％、3.0％、2.8％和1.6％。其变化趋势见图7。在25℃条件下放置2月，处方3和处方4纯度(SEC-HPLC 法)下降1.2％和1.1％，超出判断标准。综上所述，处方1和处方5优于其他处方。
4、电荷变异体
电荷变异体(iCIEF法)结果如表21所示，其变化趋势如图8至图11所示。
表21处方筛选电荷变异体结果
Figure PCTCN2021072103-appb-000066
结果表明，在40℃条件下放置4周，各处方样品电荷变异体主成分和酸性组分均发生明显变化。与0天比较，处方1、处方2、处方3、处方4和处方5样品主成分分别下降16.5％、15.1％、14％、15.3％和12.7％，酸性组分分别上升14.9％、13.2％、12.4％、13.6％和10.8％。在25℃条件下放置2月，各处方样品主成分和酸性组分均发生明显变化，与0天比较，各处方样品主成分分别下降7.4％、6.6％、5.7％、7.3％和5.4％，酸性组分分别上升6.8％、5.6％、4.7％、6.1％和4.5％。综上所述，从电荷变异体检测结果分析， 处方5优于其他处方。
5、聚山梨酯
聚山梨酯结果如表22所示。
表22处方筛选聚山梨酯80(HPLC-FLD法)结果
Figure PCTCN2021072103-appb-000067
表22表明，在40℃条件下放置4周，处方1、处方2和处方3聚山梨酯80均下降，处方4和处方5聚山梨酯80未发生下降。
上述实验结果表明，从纯度(SEC-HPLC法)结果分析，可以排除处方2、处方3和处方4；从电荷变异体结果分析，处方5优于其他处方。选择处方5开展后续振荡和冻融实验。
6、振荡实验
处方5样品的振荡实验结果如表23所示。
表23处方筛选振荡结果
Figure PCTCN2021072103-appb-000068
表23表明，在室温、避光650r/min条件下振荡5天，处方5外观、 可见异物均合格；蛋白含量、纯度和电荷变异体均未发生明显变化。
7、冻融实验
冻融实验结果如表24所示。
表24处方筛选冻融结果
Figure PCTCN2021072103-appb-000069
表24表明，反复冻融6次后，处方5样品外观、可见异物均合格；蛋白含量、纯度和电荷变异体均未发生明显变化。
综合上述实验结果及制剂处方开发平台经验，最终选定处方5为IBI319制剂处方。为获得稳健并易于实现的生产工艺，选用固定配比的盐酸组氨酸和组氨酸来实现pH 5.7。其组成：50.0mg/ml IBI319、0.57mg/ml组氨酸、1.33mg/ml盐酸组氨酸、25.00mg/ml山梨醇、16.85mg/ml盐酸精氨酸、0.01mg/ml依地酸二钠、0.50mg/ml聚山梨酯80，pH 5.7。

Claims (19)

  1. 一种液体抗体制剂，包含重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段以及缓冲剂，优选地所述制剂的pH值为约5.0-7.0；更优选，所述制剂的pH值为约5.5-6.0；进一步优选地，所述pH值为约5.7。
  2. 根据权利要求1所述的液体抗体制剂，其特征在于：所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段的含量为约1mg/mL-150mg/mL，优选为约10mg/mL-100mg/mL，例如，约15、20、25、30、35、40、45、50、55、60、70、80、90mg/mL，更优选地为约50.0mg/ml。
  3. 根据权利要求1-2任一项所述的液体抗体制剂，其特征在于：所述缓冲剂选自组氨酸、盐酸组氨酸或它们的组合，或者所述缓冲剂选自枸橼酸盐、枸橼酸盐溶剂合物(例如，柠檬酸盐水合物)或它们的组合，例如，枸橼酸钠、二水枸橼酸钠或它们的组合，或者所述缓冲剂选自醋酸盐、醋酸盐溶剂合物(例如，醋酸盐水合物)或它们的组合，或者所述缓冲剂选自磷酸盐、磷酸盐溶剂合物(例如，磷酸盐水合物)或它们的组合；优选地，所述缓冲剂的浓度为约5-100mM，更优选地为约5-60mM，例如，约5、10、15、20、25、30、40、50、60mM；
    优选地，所述缓冲剂为组氨酸，所述组氨酸的含量为约0.775mg/mL-15.5mg/mL；更优选地，所述组氨酸的含量为约0.775mg/mL-9.3mg/mL，例如约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、9mg/mL；进一步优选地，所述组氨酸的含量为约1.55mg/ml；
    优选地，所述缓冲剂为组氨酸和盐酸组氨酸的组合，其中所述组氨酸的含量为约0.270mg/mL-5.4mg/mL，所述盐酸组氨酸的含量为约0.660mg/mL-13.33mg/mL；更优选地，所述组氨酸的含量为约0.270mg/mL-3.24mg/mL，例如约0.5、1.0、1.5、2、2.5、3mg/mL，所述盐酸组氨酸的含量为约0.660mg/mL-8mg/mL，例如约1.0、1.5、2.0、2.5、3.0、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8mg/mL；更优选地，所述组氨酸的含量为约0.57mg/ml，所述盐酸组氨酸的含量为约1.33mg/ml。
  4. 根据权利要求1-3任一项所述的液体抗体制剂，其特征在于：所述制 剂还包含稳定剂，所述稳定剂包括多元醇和/或氨基酸；所述多元醇选自山梨醇、甘露醇、蔗糖、海藻糖、麦芽糖及组合，所述氨基酸包括精氨酸、盐酸精氨酸、甲硫氨酸、甘氨酸、脯氨酸或其组合；优选地，所述稳定剂选自山梨醇和/或盐酸精氨酸、山梨醇和/或精氨酸，更优选地，所述稳定剂为山梨醇和盐酸精氨酸的组合、或山梨醇和精氨酸的组合，优选地，所述精氨酸或盐酸精氨酸的含量为约20mM-200mM，进一步优选地，所述精氨酸或盐酸精氨酸的含量为约40mM-150mM，例如约40、50、60、70、80、90、100、110、120、130、140、150mM；更优选地，所述精氨酸或盐酸精氨酸的含量为约85mM；
    优选地，所述山梨醇的含量为约10mg/mL-100mg/mL，所述盐酸精氨酸的含量为约4mg/mL-42.0mg/mL；进一步优选地，所述山梨醇的含量为约20mg/mL-60mg/mL，例如约30、40、50mg/mL，所述盐酸精氨酸的含量为约8.4mg/mL-33.7mg/mL，例如约15、20、25、30mg/mL；更优选地，所述山梨醇的含量为约25.00mg/ml，所述盐酸精氨酸的含量为约16.85mg/ml。
  5. 根据权利要求1-4任一项所述的液体抗体制剂，其特征在于：所述制剂还包含表面活性剂；
    优选地，所述表面活性剂选自非离子型表面活性剂，例如聚山梨酯80、聚山梨酯20、泊洛沙姆和聚乙二醇聚山梨酯80中的一种或多种，更优选聚山梨酯80；
    更优选地，所述表面活性剂的含量为约0.1mg/mL-1mg/mL；进一步优选为约0.3mg/mL-0.7mg/mL，例如0.3、0.4、0.5、0.6、0.7mg/mL；进一步优选地为约0.50mg/ml。
  6. 根据权利要求1-5任一项所述的液体抗体制剂，其特征在于：所述制剂还包含螯合剂；
    优选地，所述螯合剂选自依地酸二钠、二乙基三胺五乙酸和/或EDTA，更优选依地酸二钠；
    更优选地，所述螯合剂的含量为约0.005mg/mL-0.1mg/mL；进一步优选为约0.01mg/mL-0.05mg/mL，例如约0.02、0.03、0.04mg/mL；进一步优选地为约0.01mg/ml。
  7. 根据权利要求1-6任一项所述的液体抗体制剂，其特征在于：所述制 剂含有以下组分：约50.0mg/ml重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体、约0.57mg/ml组氨酸、约1.33mg/ml盐酸组氨酸、约25.00mg/ml山梨醇、约16.85mg/ml盐酸精氨酸、约0.01mg/ml依地酸二钠、约0.50mg/ml聚山梨酯80，pH约5.7，余量为水。
  8. 根据权利要求1-7任一项所述的液体抗体制剂，其特征在于，
    所述抗体或其抗原结合片段包含第一重链(HCl)，其包含第一重链可变区(HCVR1)和恒定区；第一轻链(LC1)，其包含第一轻链可变区(LCVR1)和恒定区；第二重链(HC2)，其包含第二重链可变区(HCVR2)和恒定区；以及第二轻链(LC2)，其包含第二轻链可变区(LCVR2)和恒定区，其中：
    所述HCVR1包含SEQ ID NO：4所示的重链可变区所含的三个互补决定区域HCDR1、HCDR2和HCDR3，并且所述LCVR1包含SEQ ID NO：10所示的轻链可变区所含的LCDR1、LCDR2和LCDR3；以及
    所述HCVR2包含SEQ ID NO：16所示的重链可变区中所含的三个互补决定区域(CDR)HCDR1、HCDR2和HCDR3，并且所述LCVR2包含SEQ ID NO：22所示的轻链可变区所含的三个互补决定区域LCDR1、LCDR2和LCDR3；优选地
    a)抗体第一重链包含：具有氨基酸序列SEQ ID NO：1的互补决定区1(HCDR1)、具有氨基酸序列SEQ ID NO：2的互补决定区2(HCDR2)、具有氨基酸序列SEQ ID NO：3的互补决定区3(HCDR3)；
    b)抗体第一轻链包含：具有氨基酸序列SEQ ID NO：7的互补决定区1(LCDR1)、具有氨基酸序列SEQ ID NO：8的互补决定区2(LCDR2)、具有氨基酸序列SEQ ID NO：9的互补决定区3(LCDR3)；
    c)抗体第二重链包含：具有氨基酸序列SEQ ID NO：13的互补决定区1(HCDR1)、具有氨基酸序列SEQ ID NO：14的互补决定区2(HCDR2)、具有氨基酸序列SEQ ID NO：15的互补决定区3(HCDR3)；
    d)抗体第二轻链包含：具有氨基酸序列SEQ ID NO：19的互补决定区1(LCDR1)、具有氨基酸序列SEQ ID NO：20的互补决定区2(LCDR2)、具有氨基酸序列SEQ ID NO：21的互补决定区3(LCDR3)。
  9. 根据权利要求1-8任一项所述的液体抗体制剂，其特征在于，所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体包 含：第一重链、第二重链以及第一轻链和第二轻链，其中每条链包含可变区和恒定区，其中：
    e)抗体第一重链可变区(HCVR)包含SEQ ID NO：4的序列或与其具有至少90％，95％，98％或99％同一性的序列；
    f)抗体第一轻链可变区(LCVR)包含SEQ ID NO：10的序列或与其具有至少90％，95％，98％或99％同一性的序列；
    g)抗体第二重链可变区(HCVR)包含SEQ ID NO：16的序列或与其具有至少90％，95％，98％或99％同一性的序列；
    h)抗体第二轻链可变区(LCVR)包含SEQ ID NO：22的序列或与其具有至少90％，95％，98％或99％同一性的序列；
    优选地，
    e)抗体第一重链可变区(HCVR)具有氨基酸序列SEQ ID NO：4；
    f)抗体第一轻链可变区(LCVR)具有氨基酸序列SEQ ID NO：10；
    g)抗体第二重链可变区(HCVR)具有氨基酸序列SEQ ID NO：16；
    h)抗体第二轻链可变区(LCVR)具有氨基酸序列SEQ ID NO：22。
  10. 根据权利要求1-9任一项所述的液体抗体制剂，其特征在于，所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体包含：第一重链、第二重链以及第一轻链和第二轻链，其中每条链包含可变区和恒定区，其中：第一重链恒定区序列(HCCR)具有氨基酸序列SEQ ID NO：30，第一轻链恒定区序(LCCR)具有氨基酸序列SEQ ID NO：31，第二重链恒定区(HCCR)序列具有氨基酸序列SEQ ID NO：32，第二轻链恒定区(LCCR)序列具有氨基酸序列SEQ ID NO：33。
  11. 根据权利要求1-10任一项所述的液体抗体制剂，其特征在于，所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体包含：第一重链、第二重链以及第一轻链和第二轻链，其中：
    i)抗体第一重链包含SEQ ID NO：5的序列或与其具有至少90％，95％，98％或99％同一性的序列；
    j)抗体第一轻链包含SEQ ID NO：11的序列或与其具有至少90％，95％，98％或99％同一性的序列；
    k)抗体第二重链包含SEQ ID NO：17的序列或与其具有至少90％，95％， 98％或99％同一性的序列；以及
    l)抗体第二轻链包含SEQ ID NO：23的序列或与其具有至少90％，95％，98％或99％同一性的序列；
    优选地，
    i)抗体第一重链具有氨基酸序列SEQ ID NO：5；
    j)抗体第一轻链具有氨基酸序列SEQ ID NO：11；
    k)抗体第二重链具有氨基酸序列SEQ ID NO：17；以及
    l)抗体第二轻链具有氨基酸序列SEQ ID NO：23。
  12. 根据权利要求1-11任一项所述的液体抗体制剂，其特征在于，所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体包含：第一重链、第二重链以及第一轻链和第二轻链，其中，第一重链与第一轻链之间形成至少一个二硫键，第二重链与第二轻链之间形成至少一个二硫键，以及第一重链和第二重链之间形成至少一个二硫键；优选地所述双特异性抗体是改性的人IgG1。
  13. 根据权利要求1-12中任一项所述的液体抗体制剂，其包含：
    (i)约1mg/mL-150mg/mL的所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段；
    (ii)约5-100mM的组氨酸；
    (iii)约10mg/mL-100mg/mL的山梨醇以及约20mM-200mM精氨酸或盐酸精氨酸；
    (iv)约0.1mg/mL-1mg/mL聚山梨酯80；和
    (v)任选地，约0.005mg/mL-0.1mg/mL的依地酸二钠，
    其中所述液体制剂的pH为约5.0-7.0，例如，约5.5、6.0、6.5；
    例如，所述液体抗体制剂包含
    (i)约10mg/mL-100mg/mL的所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段；
    (ii)约5-60mM的组氨酸；
    (iii)约20mg/mL-60mg/mL的山梨醇以及约8.4mg/mL-33.7mg/mL盐酸精氨酸；
    (iv)约0.3mg/mL-0.7mg/mL聚山梨酯80；和
    (v)任选地，约0.01mg/mL-0.05mg/mL的依地酸二钠，
    其中所述液体制剂的pH为约5.5-6.0，例如，约5.7；
    或者，所述液体抗体制剂包含
    (i)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、1.55mg/ml组氨酸，50.00mg/ml山梨醇，0.50mg/ml聚山梨酯80，pH 5.7；或
    (ii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、1.55mg/ml组氨酸，25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
    (iii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、1.55mg/ml组氨酸，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
    (iv)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、2.94mg/ml枸橼酸钠(二水)，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
    (v)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、2.94mg/ml枸橼酸钠(二水)，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；或
    (ii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、0.57mg/ml组氨酸、1.33mg/ml盐酸组氨酸，25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
    (iii)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、0.57mg/ml组氨酸、1.33mg/ml盐酸组氨酸，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；
    (iv)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、2.94mg/ml枸橼酸钠(二水)，16.85mg/ml盐酸精氨酸，0.50mg/ml聚山梨酯80，pH 5.7；或
    (v)50.0mg/ml所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其抗原结合片段、0.57mg/ml组氨酸、1.33mg/ml 盐酸组氨酸、25.00mg/ml山梨醇，16.85mg/ml盐酸精氨酸，0.01mg/ml依地酸二钠，0.50mg/ml聚山梨酯80，pH 5.7。
  14. 一种制备权利要求1-13任一项所述的制剂的方法，包括以下步骤：
    (1)将除所述表面活性剂之外的成分配制成溶液；
    (2)通过超滤，采用步骤(1)制备的溶液对所述重组抗程序性死亡受体1(PD-1)和抗分化抗原簇137(CD137)双特异性抗体或其片段进行换液，然后浓缩至目标浓度；
    (3)添加所述表面活性剂到步骤(2)制备的液体中。
  15. 一种固体抗体制剂，其通过固化权利要求1-13中任何一项所述的液体抗体制剂而获得，所述固化是通过例如结晶法、喷雾干燥法、冷冻干燥法实施的，所述固体抗体制剂例如是冻干粉针剂形式。
  16. 递送装置，其包含权利要求1-13中任何一项的液体抗体制剂或权利要求15的固体抗体制剂。
  17. 预填装注射器，其包含权利要求1-13中任何一项的液体抗体制剂或权利要求15的固体抗体制剂，用于静脉内注射或者肌内注射。
  18. 权利要求1-13任一项所述的液体抗体制剂或权利要求15所述的固体抗体制剂在制备用于预防或治疗癌症的递送装置或预填装注射器或药物药物中的用途，优选的所述癌症选自黑色素瘤、非小细胞肺癌、小细胞肺癌、头颈癌、肝癌、结直肠癌、胰腺癌、胃癌、肾癌、膀胱癌、前列腺癌、乳腺癌、卵巢癌、子宫内膜癌、食管癌、软组织肉瘤、胆管癌、甲状腺癌、肝细胞癌或间皮瘤。
  19. 根据权利要求18所述的用途，其中所述药物与电离辐射同时、分开或者依次向癌症患者施用；或者与一种或多种化疗药物同时、分开或者依次向癌症患者施用；或者与电离辐射以及一种或多种化疗药物同时、分开或者依次向癌症患者施用，优选地所述化疗药物选自5-氟尿嘧啶、羟基脲、吉西他滨、甲氨蝶呤、阿霉素、足叶乙甙卡铂、顺铂、环磷酰胺、美法仑、达卡巴嗪、紫杉醇、喜树碱、FOLFIRI、FOLFOX、多西紫杉醇、柔红霉素、紫杉酚、奥沙利铂或其组合。
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