WO2021143767A1

WO2021143767A1 - Preparation of bispecific antibody combining pd-1 and pd-l1 and use thereof

Info

Publication number: WO2021143767A1
Application number: PCT/CN2021/071757
Authority: WO
Inventors: 谢瑞霞; 马丽强; 汪音爵
Original assignee: 信达生物制药(苏州)有限公司
Priority date: 2020-01-15
Filing date: 2021-01-14
Publication date: 2021-07-22
Also published as: TW202130367A

Abstract

Provided is a pharmaceutical preparation of an anti-PD-1/PD-L1 antibody, a buffer, a stabilizer and a surfactant. In addition, also provided is the use of the preparation for treating or preventing diseases.

Description

Preparation and use of bispecific antibody combining PD-1 and PD-L1

Technical field

The present invention relates to the field of antibody preparations. More specifically, the present invention relates to the stabilization of bispecific antibodies (also known as anti-PD-1/PD-L1 antibodies) that bind human programmed cell death 1 (PD-1) and human PD-1 ligand 1 Preparations, and the therapeutic and/or preventive uses of the preparations.

Background technique

Immune checkpoint pathways are used to maintain self-tolerance and control T cell activation, but cancer cells can use these pathways to inhibit anti-tumor responses and prevent their destruction. The PD-1/PD-L1 pathway is one such immune checkpoint. Human PD-1 is found on T cells, and human PD-L1 is abnormally expressed by a variety of tumor types; the combination of PD-L1 and PD-1 inhibits T cell proliferation and cytokine production. The PD-1/PD-L1 inhibitory axis has been conquered by tumors as part of the natural selection process that shapes tumor evolution in the context of anti-tumor immune responses.

Although therapeutic agents targeting the PD-1/PD-L1 pathway have been clinically validated and have made significant clinical progress in the treatment of cancer, only a portion of patients have benefited from this treatment (see, for example, Sharma, P. and Allison,JP,Immune checkpoint targeting in cancer therapy:toward combination strategies with curative potential.Cell.2015;161:2015-14). For example, only about 20% of patients with non-small cell lung cancer (NSCLC) respond to PD-1 antibody treatment.

Although clinical trials involving the co-administration of PD-L1 antibodies and PD-1 antibodies are currently underway (see, for example, EUROPEAN SOCIETY FOR MEDICAL ONCOLOGY (ESMO), Abstract No. 2130; October 2016), this treatment plan involves transfusion. Note that there are two separate antibody products, and the dose of each antibody is relatively high. In addition, it is not known whether the combination therapy will provide an improvement in efficacy compared to monotherapy without exacerbating the characteristics of adverse events.

Therefore, it is necessary to bind PD-L1 and PD-1 with high affinity, effectively neutralize the activation of PD-L1 and PD-1 by all PDx family ligands, and/or relative to the known target PD-1/PD -Therapeutic agents of the L1 pathway provide bispecific antibodies with excellent activity or even a combination thereof as a more effective pharmacological intervention for certain cancers. For example, PCT application number PCT/US2018/041205 discloses an exemplary therapeutic antibody specific for human PD-1/PD-L1, the disclosure of which is hereby incorporated by reference in its entirety. At the same time, there is a need in the art for anti-PD-1/PD-L1 antibody preparations that can be used to treat, prevent or delay various cancers and immune-related diseases.

Antibodies used in human subjects must be stored and transported to the site of administration before use. Reproducibly obtaining the desired antibody drug level in the subject requires that the drug be stored in a formulation that maintains the biological activity of the drug. At the same time, compared to intravenous infusion, subcutaneous administration of high-concentration preparations is more convenient and safer. It can realize self-administration by patients at home, which is convenient for long-term treatment of chronic diseases. At the same time, high-concentration preparations can meet high-dose administration requirements and reduce Cost of production. The development of high-concentration preparations has become the development trend of monoclonal antibody preparations. At the same time, lyophilized formulations can bring superior formulation stability, and higher concentration liquid formulations can be prepared by adding less liquid during reconstitution.

Therefore, there is still a need in the art for novel pharmaceutical formulations containing anti-PD-1/PD-L1 antibodies that are sufficiently stable and suitable for administration to subjects in various forms. There is a particular need for formulations that can exhibit a long shelf life, are stable during storage and transportation, and are suitable for administration at high concentrations (e.g., for subcutaneous administration) and low concentrations (e.g., for intravenous administration).

Summary of the invention

The present invention meets the above-mentioned needs by providing stable formulations containing antibodies that specifically bind to PD-1/PD-L1.

In the first aspect, the present invention relates to a lyophilized preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which comprises: (i) an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof; ii) a buffer system; (iii) a stabilizer including polyols and/or amino acids; and (iv) a surfactant, wherein the formulation has a pH between 5.5-6.5 when reconstituted, for example, pH Approximately 5.5, 6.0 or 6.5.

In some embodiments, the lyophilized formulation is capable of reconstituted the antibody or antigen-binding fragment thereof at a concentration between about 1 mg/mL to about 200 mg/mL.

In certain embodiments, the surfactant is selected from polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80, or a combination thereof, and is at a rate of about 0.01% (w/v ) Exist in a weight ratio of -1% (w/v).

In some embodiments, the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system, sodium acetate buffer system, phosphate buffer system The system is present in a weight ratio of about 0.01% (w/v) to 1% (w/v).

In certain embodiments, the polyol is selected from sorbitol, mannitol, or a combination thereof, and the amino acid is selected from arginine, arginine hydrochloride, methionine, glycine, proline, or a combination thereof;

Preferably, the stabilizer includes sorbitol and arginine; more preferably, the sorbitol is at about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, The weight ratio of 8, 9, 10% (w/v) is present, and the arginine is present at about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10% (w/v) weight ratio exists;

Preferably, the stabilizer includes sorbitol and arginine hydrochloride; more preferably, the sorbitol is at a concentration of about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10% (w/v) weight ratio exists, about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 The weight ratio of %(w/v) exists.

In certain embodiments, the stabilizer further includes methionine; more preferably, the methionine is present in a weight ratio of about 0.1-10% (w/v).

In the second aspect, the present invention relates to a lyophilized preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which is prepared by lyophilizing an aqueous solution containing: (i) anti-PD-1/PD -L1 antibody or its antigen-binding fragment; (ii) buffer system; (iii) stabilizer, said stabilizer including polyol and/or amino acid; and (iv) surfactant, said liquid formulation has a concentration of about 5.5-6.5 The pH, for example, the pH is about 5.5, 6.0 or 6.5; preferably, the liquid formulation has a pH of about 6.0.

In the third aspect, the present invention relates to a liquid pharmaceutical preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which comprises an aqueous solution comprising: (i) an anti-PD-1/PD-L1 antibody or The antigen-binding fragment thereof; (ii) a buffer system; (iii) a stabilizer, the stabilizer includes a polyol and/or amino acid; and (iv) a surfactant, the liquid formulation has a pH of about 5.5-6.5, for example, The pH is about 5.5, 6.0 or 6.5; preferably, the liquid formulation has a pH of about 6.0.

In certain embodiments, in the above-mentioned lyophilized formulation or liquid formulation, the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof is present in an aqueous solution at a concentration of about 1 mg/mL to about 200 mg/mL, For example, about 1, 5, 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg The concentration of /ml exists in the aqueous solution.

In certain embodiments, in the above-mentioned lyophilized formulation or liquid formulation, the surfactant is selected from polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80, or a combination thereof , And exist at a concentration of about 0.1-10 mg/ml, for example, about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml.

In some embodiments, in the above-mentioned lyophilized preparation or liquid preparation, the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system , Sodium acetate acetate buffer system, phosphate buffer system, and exist in a concentration of about 1-100 mM, for example, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM.

Preferably, the buffer system is a histidine buffer system; more preferably, the histidine is at a concentration of about 0.1-10 mg/mL, such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10mg/ml concentration exists;

Preferably, the buffer system is a histidine and histidine hydrochloride buffer system; more preferably, the histidine and the histidine hydrochloride are respectively about 0.1-10 mg/mL, such as about 0.1, 0.5, Concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml exist.

In some embodiments, in the above-mentioned freeze-dried formulation or liquid formulation, the polyol is selected from sorbitol, mannitol or a combination thereof, and the amino acid is selected from arginine, arginine hydrochloride, and methionine , Glycine, proline or a combination thereof; preferably, the stabilizer includes sorbitol and arginine; more preferably, the sorbitol is about 10-100 mg/mL, such as about 10, 20, 30, 40, The arginine is present at a concentration of 50, 60, 70, 80, 90, 100 mg/mL, and the arginine is present at, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL;

Preferably, the stabilizer includes sorbitol and arginine hydrochloride; more preferably, the sorbitol and the arginine hydrochloride are respectively about 10-100 mg/mL, such as about 10, 20, 30, 40, 50, Concentrations of 60, 70, 80, 90, 100 mg/mL exist.

In some embodiments, in the above-mentioned lyophilized formulation or liquid formulation, the stabilizer further includes methionine; preferably, the methionine is at a concentration of about 1-100 mg/mL, such as about 1, 5 , 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL.

In any of the formulations described herein, the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof binds to human PD-L1 (SEQ ID NO: 1) and human PD-1 (SEQ ID NO: 2). Preferably, the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof comprises: a first heavy chain (HCl), which comprises 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 a constant region; a second heavy chain (HC2), which includes a second heavy chain variable region (HCVR2) and a constant region; and a second light chain (LC2 ), which contains the second light chain variable region (LCVR2) and the 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: 3, and the LCVR1 includes the light chain variable region shown in SEQ ID NO: 4 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: 5, and the LCVR2 includes the light chain shown in SEQ ID NO: 8 The variable region contains three complementary determining regions LCDR1, LCDR2 and LCDR3.

In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention may include: a first heavy chain (HCl), which includes a first heavy chain variable region (HCVR1) and a constant region; The light chain (LC1), which includes the first light chain variable region (LCVR1) and the constant region; the second heavy chain (HC2), which includes the second heavy chain variable region (HCVR2) and the constant region; and the second light chain Chain (LC2), which includes the second light chain variable region (LCVR2) and constant region, where:

a) The HCVR1 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 16, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and the amino acid sequence shown in SEQ ID NO: 18 Sequence complementarity determining region CDR3;

b) The LCVR1 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 19, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 20, and the amino acid sequence shown in SEQ ID NO: 21 Sequence complementarity determining region CDR3;

c) The HCVR2 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 22, and the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24 and having SEQ ID NO : The complementarity determining region CDR3 of the amino acid sequence shown in 25; and

d) The LCVR2 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 26, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 27, and the amino acid sequence shown in SEQ ID NO: 28 The complementarity of the sequence determines the region CDR3.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises HCVR1 that is at least 90%, 95%, 98%, or 99% or more identical to SEQ ID NO: 3; and/or is identical to SEQ ID NO: : 4 LCVR1 with at least 90%, 95%, 98% or 99% or higher identity; and/or at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 5 HCVR2; and/or LCVR2 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 8;

Preferably, in the aforementioned anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof, a) the HCVR1 includes the amino acid sequence shown in SEQ ID NO: 3; b) the LCVR2 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 4; c) the HCVR2 includes the amino acid sequence shown in SEQ ID NO: 5; and d) the LCVR2 includes the amino acid sequence shown in SEQ ID NO: 8.

In certain embodiments, the antibody or antigen-binding fragment thereof comprises HC1 that is at least 90%, 95%, 98%, or 99% or more identical to SEQ ID NO: 49 or SEQ ID NO: 29; and / Or LC1 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 30; and/or with SEQ ID NO: 33 or SEQ ID NO: 31 or SEQ ID NO: 32 HC2 with at least 90%, 95%, 98% or 99% or higher identity; and/or LC2 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 34 ；

Preferably, in the aforementioned anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof, the HC1, the LC1, the HC2 and the LC2 respectively comprise SEQ ID NO: 49, SEQ ID NO: 30, The amino acid sequence shown in SEQ ID NO: 31 and SEQ ID NO: 34; or the amino acid sequence shown in SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34 respectively; or They respectively include the amino acid sequences shown in SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33, and SEQ ID NO: 34.

In certain embodiments, the anti-PD-1/PD-L1 antibody is the anti-PD-1/PD-L1 antibody disclosed in PCT application number PCT/US2018/041205. For the purposes of this application, the entire content of the PCT application is hereby incorporated herein by reference. Preferably, the antibody is selected from: PD-1/PD-L1 bispecific Ab v13844, Ab v3.0 or Ab v3.2.

In certain embodiments, the antibody in the formulation of the present invention exhibits stability for at least about 12 months, preferably, the antibody exhibits stability for at least about 24 months, more preferably The antibody exhibits stability for at least about 36 months.

In certain embodiments, the formulation is stable after storage, for example, after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 6 months, or after storage at 40°C ± 2°C for 1 month. , Preferably has one or more of the following features:

(i) Measured by the SEC-HPLC method, the preparation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, 98%, 99% purity;

(ii) Measured by the reduced or non-reduced CE-SDS method, the preparation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, 98% purity;

(iii) Measured by iCIEF or CEX-HPLC method, the main component is ≥35%, preferably greater than or equal to 35%, 40%, 45% or 50%.

In one aspect, the present invention provides a method for preparing the formulation of the present invention, including the following steps:

(1) Make a solution with ingredients other than surfactants;

(2) Using the solution prepared in step (1) to exchange the PD-1/PD-L1 bispecific antibody or its antigen-binding fragment by ultrafiltration, and then concentrate it to the target concentration;

(3) Add the surfactant to the liquid prepared in step (2).

In one aspect, the invention provides a delivery device comprising an effective amount of the liquid antibody preparation or lyophilized preparation of the invention. In one embodiment, the delivery device of the present invention is provided in the form of a pre-filled syringe containing an effective amount of the liquid antibody formulation or lyophilized formulation of the present invention, for example for intravenous, subcutaneous, intradermal or intramuscular injection, or Intravenous infusion.

In yet another aspect, the present invention provides a method for delivering anti-PD-1/PD-L1 antibody protein to a subject, such as a mammal, comprising administering to the subject an effective amount of the liquid antibody preparation or lyophilized preparation of the present invention The delivery is carried out, for example, by a delivery device using a pre-filled syringe.

In yet another aspect, the present invention provides the use of the liquid antibody preparation or lyophilized preparation of the present invention to prepare a delivery device (eg, a pre-filled syringe) or a drug for treating or preventing tumors in a subject. It is, for example, cancer, including but not limited to solid tumors, hematological cancers, soft tissue tumors, and metastatic lesions. In some embodiments, the cancers described herein include, but are not limited to, Hodgkin's or non-Hodgkin's lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, gastric and esophageal cancer, colorectal cancer Cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung (NSCLC), mesothelioma , Squamous cell carcinoma of the head and neck (SCCHN), soft tissue sarcoma or glioblastoma multiforme. Preferably, the lung cancer is NSCLC (squamous and non-squamous), small cell lung cancer or mesothelioma.

The present invention also provides a liquid antibody preparation or lyophilized preparation of the present invention or a delivery device (such as a pre-filled syringe) or a drug containing the liquid antibody preparation or lyophilized preparation of the present invention by administering the liquid antibody preparation or lyophilized preparation of the present invention to a subject. Methods of enhancing immune effector cell response and/or reducing immunosuppression.

The present invention also provides a liquid antibody preparation or lyophilized preparation of the present invention or a delivery device (e.g., pre-filled syringe) or medicine containing the liquid antibody preparation or lyophilized preparation of the present invention to prevent and/ Or a method for treating a subject's disease, such as the above-mentioned tumor.

The beneficial effects of the preparation of the present invention:

1) The freeze-dried and liquid antibody preparations provided by the present invention are stable in nature, and can be stable for at least 24 months at 5°C±3°C.

2) The prescription provided by the present invention can not only ensure the stability of low-concentration liquid preparations, but also can ensure the stability of high-concentration liquid preparations without adding auxiliary materials, providing the possibility of subcutaneous administration of high-concentration liquid preparations. In an implementation case, the high-concentration liquid preparation of the present invention can be directly prepared into a freeze-dried preparation without optimizing the prescription, and can maintain its structural and functional integrity after freeze-drying.

3) The freeze-dried preparation provided by the present invention is easily reconstituted, is within the acceptable injection viscosity range of the liquid preparation (≤10cP), and all the detection indicators have not changed after being placed at room temperature for one day. A higher concentration (for example, 170 mg/ml) liquid preparation can be prepared by reducing the volume of the reconstituted solution. The same amount of administration reduces the volume of administration and improves the compliance of clinical administration.

Other embodiments of the present invention will be made clear by referring to the detailed description hereinafter.

Brief description of the drawings

When read together with the following drawings, the preferred embodiments of the present invention described in detail below will be better understood. For the purpose of illustrating the invention, the figure shows a currently preferred embodiment. However, it should be understood that the present invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1 shows a graph of the protein purity in each sample measured by the SEC-HPLC method over time in the pre-prescription test. Met in the figure represents methionine.

Figure 2 shows the changes in the main components and acidic components of the charge variants in each sample measured by the iCIEF method over time in the pre-prescription test.

Figure 3 shows the variation of protein purity in each sample measured by SEC-HPLC method over time under the condition of 40℃±2℃ in the prescription screening test

Figure 4 shows a graph of the protein purity in each sample measured by SEC-HPLC at 25°C±2°C over time in the prescription screening test.

Figure 5 shows a graph of the protein purity in each sample measured by the non-reduced CE-SDS method over time under the condition of 40°C±2°C in the prescription screening test.

Figure 6 shows a graph of the main components and acidic components of the charge variants in each sample measured by the CEX-HPLC method at 40°C±2°C in the prescription screening test over time.

Figure 7 shows a graph of the main components and acidic components of the charge variants in each sample measured by the CEX-HPLC method under the conditions of 25°C±2°C in the prescription screening test over time.

Detailed description of the invention

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

definition

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

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

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

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

The term "immune checkpoint molecule" refers to a type of inhibitory 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). Studies have found that one of the reasons why tumor cells can escape the immune system in the body and proliferate out of control is the use of the inhibitory signaling pathway of immune checkpoint molecules, which inhibits the activity of T lymphocytes, making T lymphocytes unable to effectively kill tumors Effects (Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling moles for immune modulation. Nat Rev Drug Discov, 2013, 12(2): 130-146). Immune checkpoint molecules include but are not limited to programmed death 1 (PD-1), PD-L1, PD-L2, cytotoxic T lymphocyte antigen 4 (CTLA-4), LAG-3 and TIM-3.

As used herein, the terms "programmed cell death ligand 1", "PD-L1", "programmed cell death ligand 1", "cluster of differentiation 274", "CD274" or "B7 homolog 1" refer to any Any natural PD-L1 of vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The term encompasses "full length", unprocessed PD-L1 and any form of PD-L1 produced by processing in the cell. PD-L1 can exist as a transmembrane protein or as a soluble protein. The term also encompasses naturally occurring variants of PD-L1, such as splice variants or allelic variants. The basic structure of PD-L1 includes 4 domains: extracellular Ig-like V-type domain and Ig-like C2-type domain, transmembrane domain and cytoplasmic domain. Additional information about the human PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 29126. Additional information about mouse PD-L1 gene (including genomic DNA sequence) can be found under NCBI Gene ID No. 60533. The amino acid sequence of an exemplary full-length human PD-L1 protein can be found, for example, under NCBI accession number NP_001254653 or UniProt accession number Q9NZQ7, and an exemplary full-length mouse PD-L1 protein can be found, for example, under NCBI accession number NP_068693 or Uniprot accession number Q9EP73. L1 protein sequence. As used herein, the term "antibody" is used in the broadest sense and refers to a protein containing an antigen-binding site, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to intact antibodies and antigen-binding fragments of antibodies.

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)) interposed between them. Each VH and VL consists of three CDRs and four The FR composition is 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 the antibody to the antigen, but displays a variety of effector functions.

As used herein, the term "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. In one embodiment, provided herein are bispecific antibodies that have binding specificities for a first antigen and a second antigen. For example, the first antigen is PD-L1, and the second antigen is PD-1.

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

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

The term "variable region" or "variable domain" refers to the domain of the heavy or light chain of an antibody that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies usually have similar structures, where each domain contains four conserved framework regions (FR) and three complementarity determining regions (see, for example, Kindt et al. Kuby Immunology, 6th ed., WHFreeman and Co. 91 page (2007)). A single VH or VL domain may be sufficient to give antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a specific antigen can be used to isolate antibodies that bind to the antigen to screen 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.

"Complementarity determining region" or "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 and light chains are usually called CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located in the variable domain of the antibody heavy chain are called HCDR1, HCDR2, and HCDR3, and the CDRs located in the variable domain of the antibody light chain are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence 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. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, USDepartment of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImmunoGeneTics database (IMGT) (World Wide Web imgt.cines.fr/), and North CDR definition based on affinity propagation clustering using a large number of crystal structures (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)).

For example, the different definition ranges of CDR regions numbered by Kabat and Chothia are used.

The CDR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (for example, any of the exemplary CDRs of the present invention).

Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

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

In one embodiment, the CDR of the antibody of the present invention is bounded by North definition rules, for example, as shown in Table 1b and Table 1c below.

However, it should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems are different. Therefore, when it comes to defining antibodies with specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences include the specific CDR sequences, but due to the application of different schemes (for example, Different assignment system rules or combinations) cause the claimed CDR boundary to be different from the specific CDR boundary defined in the present invention.

Antibodies with different specificities (ie, different binding sites for different antigens) have different CDRs. However, although CDRs are different from antibody to antibody, there are only a limited number of amino acid positions within the CDR that directly participate in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact, and North methods, the minimum overlap area can be determined, thereby providing the "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of the CDR. As those skilled in the art know, the structure of the antibody and protein folding can determine the residues of the rest of the CDR sequence. Therefore, the present invention also considers any CDR variants given herein. For example, in a CDR variant, the amino acid residues of the smallest binding unit can remain unchanged, while the remaining CDR residues defined by Kabat or Chothia can be replaced by conserved amino acid residues.

The term "antibody preparation" refers to a preparation in a form that allows the biological activity of the antibody as an active ingredient to be effectively exerted, and does not contain 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 "anti-PD-1/PD-L1 antibody preparation" is also referred to as "antibody preparation of the present invention" herein, meaning that it contains anti-PD-1/PD-L1 antibody protein as an active ingredient and contains pharmaceutically acceptable excipients. Preparation of the agent. After the anti-PD-1/PD-L1 antibody protein is combined with a pharmaceutically acceptable excipient, the anti-PD-1/PD-L1 antibody protein as an 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). In some embodiments, the anti-PD-1/PD-L1 antibody protein preparation is in the form of a liquid preparation.

A "stable" antibody preparation is when the antibody in the preparation retains an acceptable degree of physical and/or chemical stability after storage under specific conditions. Although the antibody contained in the antibody preparation may not maintain 100% of its chemical structure after storage for a specific time, it usually maintains about 90%, about 95%, about 96%, about 97%, about 98% after storage for a specific time. Or about 99% of the structure or function of the antibody, the antibody preparation is considered "stable." In some specific embodiments, the anti-PD-1/PD-L1 antibody protein preparations of the present invention exhibit undetectable antibody aggregation or degradation or chemical modification during manufacturing, preparation, transportation and long-term storage, thereby There is little or no loss of the biological activity of the anti-PD-1/PD-L1 antibody protein, showing a high degree of stability. In some embodiments, the anti-PD-1/PD-L1 antibody protein formulation of the present invention substantially retains its physical and chemical stability after storage. Preferably, the liquid formulation of the present invention can be stable at room temperature for at least 6 months or stored at 40°C±2°C for 1 month, and/or stable at 25°C±2°C for at least 3 months, and/or at 2-8°C Stable for at least 24 months.

A variety of analytical techniques known in the art can be used to determine the stability of proteins, see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs (1991) and Jones , A. Adv. Drug Delivery Rev. 10: 29-90 (1993). The stability can be measured at a selected temperature and selected storage time. For example, the storage time can be selected based on the expected shelf life of the formulation. Alternatively, an accelerated stability test can be used. In some embodiments, the stability test is performed by performing various stress tests on the antibody preparation. These tests can represent extreme conditions that may be encountered during manufacture, storage, or transportation of the formulated antibody preparation, or conditions that may accelerate the instability of the antibody in the antibody preparation during non-manufacture, storage, or transportation. For example, the formulated anti-PD-1/PD-L1 antibody protein preparation can be filled into a glass vial to test the antibody stability under high temperature stress.

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. In addition, the stability of the preparation can be indicated by visually inspecting the appearance, color, and/or clarity of the _{preparation, or detecting the turbidity of the preparation by the OD 350nm} method, or measuring the purity of the preparation by the non-reducing CE-SDS method. In one embodiment, the stability of the formulation is measured by determining the percentage of antibody monomers in the formulation after storage at a specific temperature for a specific time, 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/PD-L1 antibody protein monomer is detected in the preparation after storage at a specific temperature for a specific time. In some embodiments, storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months After months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more, an acceptable degree of physical stability indicates At least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/PD-L1 antibody protein monomer. When evaluating physical stability, the specific temperature at which the pharmaceutical preparation is stored may be any temperature from about -80°C to about 45°C, for example, when stored at about -80°C, about -30°C, about -20°C, about 0°C, About 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C. For example, if stored at about 40℃±2℃ for 1 month, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of Anti-PD-1/PD-L1 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% are detected %, 95%, 96%, 97%, 98%, 99% of the anti-PD-1/PD-L1 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/PD-L1 antibody protein monomer is detected, then the pharmaceutical preparation Considered to be stable.

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

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

The term "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%.

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

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

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

As used herein, the term "parenteral administration" means administration methods other than enteral and local administration, usually 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 . In some embodiments, the stabilized anti-PD-1/PD-L1 antibody protein formulation of the present invention is administered to a subject parenterally. In one embodiment, the anti-PD-1/PD-L1 antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.

I. Antibody preparations

One aspect of the present invention provides a stable freeze-dried formulation, which comprises (i) anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof, (ii) buffer system, (iii) stabilizer, said stabilizer includes multiple An alcohol and/or amino acid, and (iv) a surfactant, wherein the formulation has a pH between 5.5 and 6.5 when reconstituted, for example, the pH is about 5.5, 6.0, or 6.5.

Another aspect of the present invention provides a stable lyophilized preparation, which is prepared by lyophilizing an aqueous solution, the aqueous solution comprising: (i) an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, (ii) a buffer system, (iii) a stabilizer, the stabilizer includes a polyol and/or amino acid, and (iv) a surfactant, the liquid formulation has a pH of about 5.5-6.5, for example, the pH is about 5.5, 6.0 or 6.5; preferably The liquid formulation has a pH of about 6.0.

Another aspect of the present invention provides a stable liquid antibody preparation, which comprises an aqueous solution comprising (i) an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, (ii) a buffer system, and (iii) a stabilizer The stabilizer includes a polyol and/or amino acid, and (iv) a surfactant, and the liquid formulation has a pH of about 5.5-6.5, for example, the pH is about 5.5, 6.0 or 6.5; preferably, the liquid formulation Has a pH of about 6.0.

In a preferred embodiment, the liquid antibody preparation of the present invention is in the form of an injection preparation.

(i) Anti-PD-1/PD-L1 antibody

"Anti-PD-1/PD-L1 antibody" or "bispecific antibody that binds PD-1 and PD-L1" or "antibody that binds PD-1 and PD-L1" refers to an antibody that can It binds to PD-1 and PD-L1 molecules with sufficient affinity so that the antibody can be used as a therapeutic and/or preventive agent that simultaneously targets PD-1 and PD-L1 molecules.

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

As used herein, "binding" or "specific binding" refers to the affinity of an antibody for human PD-L1 (SEQ ID NO: 1), human PD-1 (SEQ ID NO: 2), or both, unless Otherwise, it means that K _{D is} less than about 1×10 ^-6 M, preferably less than about 1×10 ^-9 M, as measured at 37° C. by the above-mentioned common method known in the industry.

The antibody in the present invention is a heterodimeric bispecific antibody that simultaneously targets PD-L1 and PD-1, and is formed by pairing two different heavy chains and two different light chains to form an IgG-like antibody. In addition, the antibody of the present invention provides anti-human PD-L1 and anti-human PD-1 heterodimer bispecific antibodies having one or more of the following characteristics: blocking all three interactions of the PD axis (PD -L1 binds to PD-1, PD-L2 binds to PD-1 and PD-L1 binds to CD80), bridges PD-L1 and PD-1 overexpressing cells, increases T cells due to the proximity of bound T cells and tumor cells Activate and kill tumor cells, exhibit significant anti-tumor activity at low doses in tumor cells with moderate to high PD ligand expression levels, and exhibit physical and chemical stability, including but not limited to in vivo stability and thermal stability , Solubility, low self-association and pharmacokinetic characteristics.

Therefore, in some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention comprises: a) the first heavy chain variable region (HCVR1) comprising the amino acid sequence of SEQ ID NO: 3 Chain (HC1); b) the first light chain (LC1) comprising the light chain variable region (LCVR1) containing the amino acid sequence of SEQ ID NO: 4; c) the heavy chain comprising the amino acid sequence of SEQ ID NO: 5 The second heavy chain (HC2) of the variable region (HCVR2); and d) the second light chain (LC2) including the light chain variable region (LCVR2) containing the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention includes HC1, LC1, HC2, and LC2, wherein: a) the HC1 includes an amino acid sequence of SEQ ID NO: 16 in the HCVR CDR1, CDR1 having the amino acid sequence of SEQ ID NO: 17 and CDR3 having the amino acid sequence of SEQ ID NO: 18; b) the LC1 includes CDR1 having the amino acid sequence of SEQ ID NO: 19 in the LCVR, and having SEQ ID NO CDR2 with the amino acid sequence of: 20 and CDR3 with the amino acid sequence of SEQ ID NO: 21; c) The HC2 includes CDR1 with the amino acid sequence of SEQ ID NO: 22 in the HCVR, and has SEQ ID NO: 23 or SEQ ID NO CDR2 with the amino acid sequence of SEQ ID NO: 25 and CDR3 with the amino acid sequence of SEQ ID NO: 25; d) The LC2 includes CDR1 with the amino acid sequence of SEQ ID NO: 26 in the LCVR, and a CDR1 with the amino acid sequence of SEQ ID NO: 27 CDR2 and CDR3 with the amino acid sequence of SEQ ID NO: 28; e) CH1 domain of HC1 contains amino acid substitutions of S183K; f) the constant region of LC1 contains amino acid substitutions of S176E and Y178E; human lambda variant; g) CH1 of HC2 The domain contains amino acid substitutions of L128E, K147T, and Q175E; h) the constant region of LC2 contains amino acid substitutions of S131R, V133G, and S176R; and i) the CH3 domain of HC1 contains amino acid substitutions of T350V, L351Y, F405A, and Y407V. And the CH3 domain of HC2 contains amino acid substitutions of T350V, T366L, K392L and T394W; or the CH3 domain of HC1 contains amino acid substitutions of T350V, T366L, K392L and T394W, and the CH3 domain of HC2 contains amino acids of T350V, L351Y, F405A and Y407V Substitute; and j) where HC1 and HC2 are human IgG1Fc variants with ineffective immune effector functions, where the numbering is based on the EU index. Preferably, HC1 and HC2 comprise amino acid substitutions of L234A, L235A and D265S.

In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention includes: a) HC1, which sequentially includes HCVR and CH1 containing the amino acid sequence of SEQ ID NO: 3 from N-terminus to C-terminus The amino acid sequence of SEQ ID NO: 9 in the domain, the amino acid sequence of SEQ ID NO: 10 in the CH2 domain, and the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12 in the CH3 domain; b) LC1, which extends from the N-terminus to The C-terminus includes the LCVR containing the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 14 in the constant region in sequence; c) HC2, which sequentially includes the amino acid sequence of SEQ ID NO: 5 from the N-terminus to the C-terminus The amino acid sequence of HCVR, the amino acid sequence of SEQ ID NO: 13 in the CH1 domain, the amino acid sequence of SEQ ID NO: 10 in the CH2 domain, and the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence of SEQ ID NO: 11 in the CH3 domain; And d) LC2, which sequentially includes the LCVR containing the amino acid sequence of SEQ ID NO: 8 and the amino acid sequence of SEQ ID NO: 15 in the constant region from the N-terminus, provided that the amino acid sequence of SEQ ID NO: 11 is stored there. In the CH3 domain of HC1, the amino acid sequence of SEQ ID NO: 12 is stored in the CH3 domain of HC2; or when the amino acid sequence of SEQ ID NO: 12 is stored in the CH3 domain of HC1, the amino acid sequence of SEQ ID NO: 11 The amino acid sequence is stored in the CH3 domain of HC2.

In some embodiments, in the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention: a) The HC1 sequentially includes the HCVR1 and CH1 domains containing the amino acid sequence of SEQ ID NO: 3 from the N-terminus to the C-terminus The amino acid sequence of SEQ ID NO: 9 in the CH2 domain, the amino acid sequence of SEQ ID NO: 10 in the CH2 domain, and the amino acid sequence of SEQ ID NO: 11 in the CH3 domain; b) the LC1, which contains the sequence from the N-terminus to the C-terminus LCVR1 of the amino acid sequence of SEQ ID NO: 4 and the amino acid sequence of SEQ ID NO: 14 in the constant region; and c) the HC2, which sequentially includes HCVR2 containing the amino acid sequence of SEQ ID NO: 6 from the N-terminus to the C-terminus , The amino acid sequence of SEQ ID NO: 13 in the CH1 domain, the amino acid sequence of SEQ ID NO: 10 in the CH2 domain region, and the amino acid sequence of SEQ ID NO: 12 in the CH3 domain; and d) the LC2, which is from the N-terminus The sequence includes LCVR2 containing the amino acid sequence of SEQ ID NO: 8 and the amino acid sequence of SEQ ID NO: 15 in the constant region.

In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention: a) HC1 comprising the amino acid sequence of SEQ ID NO: 49; b) LC1 comprising the amino acid sequence of SEQ ID NO: 30 ; C) HC2 comprising the amino acid sequence of SEQ ID NO: 31; and d) LC2 comprising the amino acid sequence of SEQ ID NO: 34.

The present invention provides antibodies that bind to human PD-L1 (SEQ ID NO: 1) and human PD-1 (SEQ ID NO: 2), which comprise: a) HC1 comprising the amino acid sequence of SEQ ID NO: 49; b) comprising LC1 of the amino acid sequence of SEQ ID NO: 30; c) HC2 of the amino acid sequence of SEQ ID NO: 32; and d) LC2 of the amino acid sequence of SEQ ID NO: 34.

In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention: a) HC1 comprising the amino acid sequence of SEQ ID NO: 29; b) LC1 comprising the amino acid sequence of SEQ ID NO: 30 ; C) HC2 comprising the amino acid sequence of SEQ ID NO: 33; and d) LC2 comprising the amino acid sequence of SEQ ID NO: 34.

In some embodiments, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention may comprise HCVR1 having at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 3; And/or LCVR1 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 4; and/or with SEQ ID NO: 5 (ie SEQ ID NO: 6 or SEQ ID NO :7) HCVR2 with at least 90%, 95%, 98% or 99% or higher identity; and/or at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 8 Sexual LCVR2. As used herein, "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. ) Or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) The number of positions to get the number of matching positions, the number of matching positions is divided by the total number of positions in the comparison window (ie, the window size), and the result is multiplied by 100 to produce the sequence identity percentage. 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.

In some embodiments, the HCVR1 of the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, 4, or 3 different residues compared with SEQ ID NO: 3. Preferably, the different residues are conservative amino acid substitutions. In some embodiments, the LCVR1 of the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, 4, or 3 different residues compared with SEQ ID NO: 4. Preferably, the different residues are conservative amino acid substitutions. In some embodiments, the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10 HCVR2 compared with SEQ ID NO: 5 (ie SEQ ID NO: 6 or SEQ ID NO: 7), Preferably no more than 5, 4 or 3 different residues, preferably said different residues are conservative amino acid substitutions. In some embodiments, the LCVR2 of the anti-PD-1/PD-L1 antibody in the formulation of the present invention has no more than 10, preferably no more than 5, 4, or 3 different residues compared with SEQ ID NO: 8. Preferably, the different residues are conservative amino acid substitutions. "Conservative 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. In any embodiment of the present invention, in a preferred aspect, the conservatively substituted residues are derived from the conservative substitution table A below, preferably the preferred substituted residues shown in Table A.

Table A

原始残基Original residue	示例性取代Exemplary substitution	优选的保守氨基酸取代Preferred conservative amino acid substitution
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Asp；Lys；ArgGln; His; Asp; Lys; Arg	GlnGln
Asp(D)Asp(D)	Glu；AsnGlu; Asn	GluGlu
Cys(C)Cys(C)	Ser；AlaSer; Ala	SerSer
Gln(Q)Gln(Q)	Asn；GluAsn; Glu	AsnAsn
Glu(E)Glu(E)	Asp；GlnAsp; Gln	AspAsp
Gly(G)Gly(G)	AlaAla	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile(I)	Leu；Val；Met；Ala；Phe；正亮氨酸Leu; Val; Met; Ala; Phe; Norleucine	LeuLeu
Leu(L)Leu(L)	正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe(F)	Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr	TyrTyr
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	Val；SerVal; Ser	SerSer
Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; Norleucine	LeuLeu

The antibodies of the present invention are engineered non-naturally occurring polypeptide complexes. The DNA molecule of the present invention is a non-naturally occurring DNA molecule that contains a polynucleotide sequence encoding a polypeptide having the amino acid sequence of a polypeptide in the antibody of the present invention.

The antibody of the present invention is an IgG type antibody, which has a heavy chain and a light chain, which are cross-linked by intra-chain and inter-chain disulfide bonds. Each heavy chain consists of an N-terminal HCVR and a heavy chain constant region ("HCCR"). Each light chain consists of an N-terminal LCVR and a light chain constant region ("LCCR"). The light chains each form a disulfide bond with the heavy chain, and the two heavy chains form two disulfide bonds between each other.

The constant region of the heavy chain contains CH1, CH2 and CH3 domains. CH1 is derived from HCVR; CH1 and HCVR form the heavy chain part of the Fab. CH2 is located after the hinge area and before CH3. CH3 is located after CH2, at the carboxyl end of the heavy chain. The constant region of the light chain contains a domain CL. CL is derived from LCVR; CL and LCVR form the light chain portion of the Fab.

In a preferred embodiment, the anti-PD-1/PD-L1 antibody in the antibody preparation of the present invention is the anti-PD-L1 antibody disclosed in PCT Application No. PCT/US2018/041205 (International Filing Date: July 9, 2018). 1/PD-L1 antibody, the entire content is incorporated herein by reference. The sequence of the anti-PD-1/PD-L1 antibody continued to use the sequence number in the patent application without renumbering. Preferably, the antibody is selected from: PD-1/PD-L1 bispecific Ab v13844, Ab v3.0 or Ab v3.2. More preferably, the antibody is PD-1/PD-L1 bispecific Ab v3.2. In one embodiment, the anti-PD-1/PD-L1 antibody is a purified antibody produced by recombinant expression such as 293 cells or CHO cells, and contains a variant of the Fc portion derived from human IgG1. The antibody in the liquid preparation of the present invention exhibits significant anti-tumor activity. For example, in NSG mice from Jackson Laboratories implanted with a mixture of NCI-H292 tumor cells and PBMC, the antibody of the present invention such as PD-1/PD-L1 bispecific Ab (v13844, v3.0 or v3. 2) Treatment with 10mg/kg qw, complete remission (CR) is 7/8, 5/8 and 5/8, respectively, which is better than combination therapy (parental PD-L1 antibody + parental PD-1 antibody) (CR is 2/ 8) More effective.

The antibody of the present invention is a heterodimer because each arm of the antibody exhibits selective monovalent binding to its homologous antigen due to two different heavy chains and two different light chains that form the antibody. In the present invention, one arm of the antibody binds human PD-L1 (SEQ ID NO: 1), and the other arm binds human PD-1 (SEQ ID NO: 2). The CH2 and/or CH3 domains of these polypeptide chains do not need to be identical in sequence, and are advantageously modified to promote the complexation between the two polypeptide chains. For example, amino acid substitutions (preferably with amino acid substitutions containing bulky side groups forming a "knob", such as tryptophan) can be introduced into the CH2 or CH3 domain so that steric interference will prevent interaction with similar mutations. The domain will force the mutation domain to pair with the domain for which complementary or regulatory mutations have been designed, that is, "holes" (for example, replacement with glycine). These mutant sets can be engineered into any polypeptide pair comprising the CH2-CH3 domain that forms the Fc region. Protein engineering methods that favor heterodimerization rather than homodimerization are well known in the art, especially regarding the engineering of immunoglobulin-like molecules (see, for example, WO96/27011, WO98/050431, EP1870459, WO2007 /110205, WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO 2011/143545, WO2012/058768, WO2013/157954, WO2013/096291, EP1870459A1, and Ridgway etc. (1996)" 'Knobs-Into-Holes' engineered antibody CH3 heavy chain domain heterodimerization, "Protein Engineering 9: 617-621, Atwell et al. (1997)" Use phage display library to modify the stable heterodimerization of the homodimer domain Biol. 270: 26-35, and Xie et al. (2005) "A new form of bispecific antibodies: high-efficiency heterodimerization, expression and tumor cell lysis", J. Immunol. Methods 296: 95-101). Generally, in methods known in the art, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are both engineered in a complementary manner, so that the heavy chain containing an engineered CH3 domain is no longer It can be homodimerized with another heavy chain of the same structure (for example, the first heavy chain of CH3-engineering can no longer be homodimerized with the first heavy chain of another CH3 engineering; and CH3 -The second engineered heavy chain can no longer be homodimerized with another CH3-engineered second heavy chain. This forces the heavy chain containing an engineered CH3 domain and the heavy chain containing the CH3 domain The other heavy chain is heterodimerized and the CH3 domain is engineered in a complementary manner. For this embodiment of the invention, the CH3 domain of the first heavy chain and the CH3 domain of the second heavy chain are complementary by amino acid substitutions The method is engineered so that the first heavy chain and the second heavy chain are forced to heterodimerize. The first heavy chain and the second heavy chain can no longer homodimerize (for example, for spatial reasons).

The different methods known in the art for supporting heavy chain heterodimerization are supported as different alternatives used in the bispecific antibodies according to the present invention. In some embodiments of the invention, mutations are incorporated into the heavy chain sequence in the CH1 and CH3 regions and the light chain sequence in the light chain constant region. CH1 and LC mutations facilitate the natural pairing of the necessary light and heavy chain pairs and are not conducive to light chain mismatches. Mutation of CH3 is conducive to the pairing of heterodimers of two different heavy chains and is not conducive to the formation of homodimers.

In some embodiments, the antibodies of the invention contain variants of the Fc portion derived from human IgG1. It is well known that IgG1 binds to the Fc-γ receptor family (FcγR) and C1q. Interactions with these receptors can induce antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Therefore, for the antibodies of the present invention, certain amino acid substitutions are introduced into the human IgG1 Fc region to eliminate immune effector functions. Mutations in the CH2 region of the antibody heavy chain can include positions 234, 235, and 265 in the EU numbering to reduce or eliminate immune effector functions.

By operably linking the DNA encoding HCVR to another DNA molecule encoding the heavy chain constant region or a variant thereof, the isolated DNA encoding the HCVR region can be converted into a full-length heavy chain gene. The sequences of human and other mammalian heavy chain constant region genes are known in the art. DNA fragments containing these regions can be obtained, for example, by standard PCR amplification. Preferably, for the antibody of the present invention, the heavy chain constant region of the heavy chain is a variant of human IgG1.

By operably linking the DNA encoding the LCVR to another DNA molecule encoding the light chain constant region or a variant thereof, the isolated DNA encoding the LCVR region can be converted into a full-length light chain gene. The sequences of human and other mammalian light chain constant region genes are known in the art. DNA fragments containing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region. Preferably, for the antibody of the present invention, the light chain constant region of the anti-PD-L1 portion of the antibody is a variant of the lambda light chain, and the light chain constant region of the anti-PD-1 portion of the antibody is a variant of the kappa light chain.

After the sequence is operably linked to the expression control sequence, the polynucleotide of the present invention can be expressed in the host cell. An expression vector can usually be used as an episome or as an attachment to replicate a component of the host chromosomal DNA in the host organism. Generally, expression vectors contain selectable markers such as tetracycline, neomycin, glutamine synthetase and dihydrofolate reductase to allow detection of those cells transformed with the desired DNA sequence.

The amount of the antibody or antigen-binding fragment thereof contained in the antibody preparation of the present invention may vary according to the specific purpose characteristics of the preparation, the specific environment, and the specific purpose for which the preparation is used. In some embodiments, the antibody preparation is a liquid preparation, which may contain about 1-200 mg/ml, such as about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/ml, preferably about 10-100 mg/mL, for example about 10, 15, 20, 25, 30, 35, 40, 50, 60 , 70, 80, 90 or 100mg/ml of anti-PD-1/PD-L1 antibody. In some embodiments, the antibody formulation is a lyophilized formulation that can be at a concentration between about 1 mg/mL to about 200 mg/mL, such as about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/ml, preferably about 10-100 mg/mL, for example about 10, 15, 20 , 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 mg/ml concentration to reconstitute the antibody or antigen-binding fragment thereof.

(ii) Buffer system

The buffer system is a system that can maintain the pH of the solution in an acceptable range. In some embodiments, the buffer system used in the formulation of the present invention can control the pH of the formulation of the present invention in a pH range of about 5.5-6.5, such as a pH of about 5.5, 6.0, or 6.5. In some specific embodiments, the antibody preparations of the invention have a pH of about 6.

In some embodiments, in the lyophilized formulation of the present invention, the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system , Sodium acetate acetate buffer system, phosphate buffer system, and the weight ratio of about 0.01% (w/v)-1% (w/v), such as about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v) by weight ratio; preferably, the buffer system is present in a weight ratio of about 0.01% (w/v)-0.2% (w/v),

In some embodiments, the buffer system is a histidine buffer system. Preferably, the histidine content is about 0.01-1% (w/v), such as 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v) The weight ratio exists. In some embodiments, the histidine is present in a weight ratio of about 0.155% (w/v);

In some embodiments, the buffer system is a histidine and histidine hydrochloride buffer system. Preferably, the histidine and the histidine hydrochloride are respectively about 0.01-1% (w/v), such as about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, It exists in a weight ratio of 0.9 and 1% (w/v). In some embodiments, the histidine and histidine hydrochloride are present in a weight ratio of about 0.085% (w/v) and 0.1% (w/v), respectively.

In some embodiments, the buffer system in the liquid antibody preparation of the present invention is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system, sodium acetate acetate Buffer system, phosphate buffer system, and exist in a concentration of about 1-100 mM, for example, 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 mM. Preferably, the buffer system is present at a concentration of about 1-20 mM, for example, 1, 5, 10, 15, 20 mM; more preferably, it is present at a concentration of about 10 mM.

In one embodiment, the buffer system in the liquid antibody preparation of the present invention is a histidine buffer system. Optionally, the histidine is present at a concentration of about 0.1-10 mg/mL, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml. In a more preferred embodiment, the histidine is present at a concentration of about 1.55 mg/mL;

The buffer system in the liquid antibody preparation of the present invention is a histidine and histidine hydrochloride buffer system. Preferably, the histidine and the histidine hydrochloride are respectively about 0.1-10 mg/mL, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/mL. The concentration of ml exists. In a more preferred embodiment, the buffering agent used in the formulation of the present invention is histidine and histidine hydrochloride at a total concentration of about 10 mM, for example, the histidine and the histidine hydrochloride are respectively about 0.85 There are concentrations of mg/mL and about 1.00 mg/mL.

(iii) Stabilizer

Whether used in lyophilized formulations or liquid formulations, suitable stabilizers used in the present invention may be selected from polyols, amino acids, and any combination thereof.

The polyol as a stabilizer can be selected from but not limited to: mannitol, sorbitol or a combination thereof. In one embodiment, the polyol used as a stabilizer is sorbitol.

The amino acid as a stabilizer can be selected from but not limited to: arginine, arginine hydrochloride, methionine, glycine, proline, or a combination thereof. In some embodiments, the amino acid used as a stabilizer is arginine and/or arginine hydrochloride.

In some embodiments, in the lyophilized formulation of the present invention, the stabilizer includes sorbitol and arginine. Preferably, the sorbitol is present in a weight ratio of about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v) The arginine is present in a weight ratio of about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v). In a more preferred embodiment, the sorbitol and the arginine are present in a weight ratio of about 3% (w/v) and about 1.742% (w/v), respectively.

In some embodiments, in the lyophilized formulation of the present invention, the stabilizer includes sorbitol and arginine hydrochloride. Preferably, the sorbitol is present in a weight ratio of about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v) , Present in a weight ratio of about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v). In a more preferred embodiment, the sorbitol and the arginine hydrochloride are present in a weight ratio of about 3% (w/v) and about 2.107% (w/v), respectively.

In some embodiments, in the lyophilized formulation of the present invention, the stabilizer further includes methionine. In the liquid formulation of the present invention, the methionine may be present in a weight ratio of about 0.1-10% (w/v), for example, in a weight ratio of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10% (w/v) weight ratio exists. In one embodiment, the methionine is present in a weight ratio of about 0.749% (w/v).

In some embodiments, the liquid formulation of the present invention contains sorbitol and arginine as stabilizers. The concentration of the sorbitol and the arginine in the liquid preparation of the present invention may be about 10-100 mg/mL, for example about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL, respectively. mL. Preferably, the sorbitol and the arginine in the liquid formulation of the present invention may be present at a concentration of about 10-50 mg/mL, for example, about 10, 20, 30, 40, 50 mg/mL, respectively. In one embodiment, the sorbitol and the arginine are present at a concentration of about 30 mg/mL and about 17.42 mg/mL, respectively;

In some embodiments, the liquid formulation of the present invention contains sorbitol and arginine hydrochloride as stabilizers. The sorbitol and the arginine hydrochloride in the liquid preparation of the present invention may be about 10-100 mg/mL, for example about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL, respectively. The concentration exists. Preferably, the sorbitol and the arginine hydrochloride in the liquid formulation of the present invention may be present at a concentration of about 10-50 mg/mL, for example, about 10, 20, 30, 40, 50 mg/mL, respectively. In one embodiment, sorbitol and arginine hydrochloride are present at a concentration of about 30 mg/mL and about 21.07 mg/mL, respectively.

In some embodiments, the stabilizer in the liquid formulation of the present invention further includes methionine. In the liquid formulation of the present invention, the methionine may be about 1-100 mg/mL, for example, about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL The concentration exists. More preferably, the methionine is at about 1-50 mg/mL. In one embodiment, the methionine is present at a concentration of about 7.49 mg/mL.

(iv) Surfactant

As used herein, the term "surfactant" refers to an organic substance with an amphiphilic structure; that is, they are composed of groups with opposite solubility tendencies, usually oil-soluble hydrocarbon chains and water-soluble ionic groups. group.

Whether used in lyophilized formulations or liquid formulations, suitable stabilizers for use in the present invention may be non-ionic surfactants, for example, alkyl poly(ethylene oxide). Specific nonionic surfactants that can be included in the formulation of the present invention include, for example, polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80, and the like. In a preferred embodiment, the formulation of the present invention contains polysorbate-80 as a surfactant.

The amount of the surfactant contained in the antibody preparation of the present invention may vary depending on the specific purpose characteristics of the preparation, the specific environment, and the specific purpose for which the preparation is used.

In some embodiments, in the lyophilized formulation of the present invention, the surfactant is at a concentration of about 0.01% (w/v) to 1% (w/v), such as 0.01, 0.05, 0.1, 0.2, There are weight ratios of 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v). Preferably, it is present in a weight ratio of about 0.01% (w/v)-0.5% (w/v), for example 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5% (w/v).

In some embodiments, in the lyophilized formulation of the present invention, the surfactant is polysorbate 80, more preferably, the polysorbate 80 is about 0.02% (w/v) by weight Than exist.

In some embodiments, in the liquid formulation of the present invention, the surfactant is about 0.1-10 mg/ml, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, Exist at concentrations of 8, 9, 10 mg/ml. Preferably, it is present at a concentration of about 0.1-5 mg/ml, for example about 0.1, 0.5, 1, 2, 3, 4, 5 mg/ml.

In some preferred embodiments, the formulation may contain about 0.1-10 mg/ml, for example about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml of polysorbates Surfactant (for example, polysorbate-80). More preferably, the polysorbate 80 is present at a concentration of about 0.2 mg/ml.

(v) Other excipients

In some embodiments, other excipients are optionally included in the antibody formulations of the present invention. The other excipients include, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, 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)".

In some embodiments, the antibody lyophilized preparation of the present invention contains: about 2-17% (w/v) recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 ( PD-L1) bispecific antibody, about 0.294% (w/v), about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 5.5.

In some preferred embodiments, the antibody lyophilized preparation of the present invention contains: about 2-17% (w/v) recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, about 0.155% (w/v) histidine, about 3% (w/v) sorbitol, about 0.749% (w/v) methionine, about 1.742% (w/v) Arginine, about 0.20 mg/ml polysorbate 80, the formulation has a pH of 6.0 when reconstituted.

In some preferred embodiments, the antibody lyophilized preparation of the present invention contains: about 2-17% (w/v) recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, about 0.085% (w/v) histidine, about 0.1% (w/v) histidine hydrochloride, about 3.00% (w/v) sorbitol, about 0.749% (w/v) methionine, about 2.107% (w/v) arginine hydrochloride, about 0.02% (w/v) polysorbate 80; the formulation has a pH of 6.0 when reconstituted.

In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 5.5.

In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.

In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 2.94mg/ml sodium citrate, about 50mg/ml sorbitol, about 2.94mg/ml methionine, about 0.20mg/ml polysorbate 80, the aqueous solution has about The pH of 6.0.

In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 2.94 mg/ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.5.

In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 1.55 mg/ml histidine, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.

In some embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD -L1) Bispecific antibody, about 1.55 mg/ml histidine, about 50 mg/ml sorbitol, about 7.49 mg/ml methionine, about 0.20 mg/ml polysorbate 80, the aqueous solution has about 6.0的pH.

In some preferred embodiments, the aqueous solution in the antibody liquid formulation of the present invention contains: 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 ( PD-L1) bispecific antibody, about 1.55mg/ml histidine, about 30mg/ml sorbitol, about 7.49mg/ml methionine, about 17.42mg/ml arginine, about 0.20mg/ml poly Sorbate 80, the aqueous solution has a pH of about 6.0.

In some preferred embodiments, in the antibody liquid preparation of the present invention, the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibody, about 0.85mg/ml histidine, about 1.00mg/ml histidine hydrochloride, about 30.00mg/ml sorbitol, about 7.49mg/ml methionine, about 21.07 mg/ml arginine hydrochloride, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0.

II. Preparation of the formulation

The present invention provides a stable formulation containing anti-PD-1/PD-L1 antibody protein. The anti-PD-1/PD-L1 antibody protein used in the formulation of the present invention can be prepared using techniques known in the art for antibody production. For example, antibodies can be produced recombinantly. In some embodiments, the antibodies of the present invention can be produced in CHO, NSO, HEK293 or COS cells. Preferably, the antibody of the present invention can be prepared in CHO or HEK293 cells. The vector containing the polynucleotide sequence of interest (for example, the polynucleotide encoding the polypeptide of the antibody and the expression control sequence) can be transferred to the host cell by a well-known method, and the method varies according to the type of the host cell.

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

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

For example, the following steps can be used for preparation: (1) After 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; (4) virus filtration (to make the virus titer Reduce, for example, 4 log10 or more); (5) 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.

III. Analytical method of preparation

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

Various methods are known in the art that can be used to test the stability of antibody preparations. For example, methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC can be used to analyze the purity of antibody preparations and evaluate the aggregation level of antibodies; capillary isoelectric focusing (cIEF), imaging capillary, etc. Focused electrophoresis (iCIEF) and ion exchange chromatography (IEX) are used to analyze charge variants in antibody preparations. In addition, the stability of the preparation can be quickly judged by visually inspecting the appearance of the preparation. _{The OD 350nm} method can also be used to detect changes in the turbidity of the preparation, which can give information about the amount of soluble and insoluble aggregates. In addition, ultraviolet spectrophotometry (UV method) can be used to detect changes in the protein content of the preparation.

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

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

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

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

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

The anti-PD-1/PD-L1 antibody protein preparation of the present invention is stable. In some embodiments, the anti-PD-1/PD-L1 antibody protein formulation of the present invention is stored, for example, after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 6 months, or after 40 It is stable after storage at ℃±2℃ for 1 month. In some embodiments, after storage at about 5°C, 25°C, 37°C, 40°C, or 45°C for at least 1 month, 2 months, or 3 months, for example, storage at 5°C ± 3°C for 3 months Afterwards, the anti-PD-1/PD-L1 antibody protein purity in the antibody preparation of the present invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more, as determined by size exclusion chromatography or by non-reduced CE-SDS. In one embodiment, after storage at about 5°C, 25°C, 37°C, 40°C, or 45°C for at least 1 month, 2 months or 3 months, for example, storage at 5°C ± 3°C for 3 months Later, at least 55%, preferably at least 60% of the anti-PD-1/PD-L1 antibody protein in the antibody preparation of the present invention is in the non-basic and non-acidic form (that is, the main peak or the main charge form), such as by CEX-HPLC Measured by law.

IV. Use of the preparation

The antibody preparation of the present invention containing the anti-PD-1/PD-L1 antibody protein of the present invention can be used to treat or prevent tumors and the like.

The present invention further provides the use of the antibody preparation of the present invention for the manufacture of a drug for the treatment or prevention of cancer, wherein the drug can be simultaneously, separately or sequentially with one or more anti-tumor agents selected from the group consisting of Administration: Cisplatin, carboplatin, dacarbazine, liposomal doxorubicin, docetaxel, cyclophosphamide and more Rubicin, navelbine, eribulin, paclitaxel, paclitaxel protein-binding particles for injectable suspension, ixabepilone, capecitabine (capecitabine), FOLFOX (leucovorin, fluorouracil and oxaliplatin), FOLFIRI (calcium folinate, fluorouracil and irinotecan), gemcitabine, Topo Topotecan, liposomal irinotecan, pemetrexed, cetuximab, nivolumab, ipilimumab, pilizumab Mab (pidilizumab), pembrolizumab, tremelimumab, urelumab, lirilumab, atezolizumab, love Epacadostat and durvalumab.

The present invention provides the use of the antibody preparation of the present invention for the manufacture of drugs for the treatment of cancer, wherein the drugs can be administered simultaneously, separately or sequentially with ionizing radiation.

In some embodiments, cancers to be prevented or treated include, but are not limited to, Hodgkin's or non-Hodgkin's lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, stomach and esophageal cancer , Colorectal cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), Mesothelioma, squamous cell carcinoma of the head and neck (SCCHN), soft tissue sarcoma, or glioblastoma multiforme. In some embodiments, the cancer is a gastrointestinal cancer, such as gastric cancer, rectal cancer, colon cancer, colorectal cancer, and the like.

The present invention also provides the use of the preparation of the present invention in the preparation of medicines, wherein the medicines are used to deliver anti-PD-1/PD-L1 antibody proteins to mammals. The present invention also provides a method for the preparation of the present invention to treat or prevent one or more of the above-mentioned diseases and disorders. Preferably, the mammal is a human.

The antibody formulation of the present invention can be administered to a subject or patient in a variety of ways. For example, administration can be by infusion or by syringe. Therefore, in one aspect, the present invention provides a delivery device (e.g., a syringe) comprising the antibody preparation of the present invention (e.g., a pre-filled syringe). The patient will receive an effective amount of anti-PD-1/PD-L1 antibody protein as the main active ingredient, that is, an amount sufficient to treat, ameliorate or prevent the target disease or condition.

The present invention further provides a method for preventing and/or treating tumors, which comprises administering to a subject an effective amount of the formulation of the present invention. For example, the tumor is cancer. Preferably, the cancer is Hodgkin or non-Hodgkin lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, gastric and esophageal cancer, colorectal cancer, Liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), mesothelioma, head and neck Squamous cell carcinoma (SCCHN), soft tissue sarcoma or glioblastoma multiforme; more preferably, the lung cancer is NSCLC, small cell lung cancer or mesothelioma.

In some embodiments, the method of the present invention further includes simultaneous, separate or sequential administration of one or more anti-tumor agents selected from the group consisting of cisplatin (cisplatin), carboplatin (carboplatin), and Carbachol (dacarbazine), liposomal doxorubicin (liposomal doxorubicin), docetaxel (docetaxel), cyclophosphamide (cyclophosphamide) and doxorubicin, navelbine (navelbine), eribulin ( eribulin, paclitaxel, paclitaxel protein-binding particles for injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin), fluorouracil ) And oxaliplatin), FOLFIRI (leucovorin, fluorouracil and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed Pemetrexed, cetuximab, nivolumab, ipilimumab, pidilizumab, pembrolizumab, trime Tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat and durvalumab.

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. For specific situations, the effective amount delivered can be determined within the judgment of the clinician. In this regard, the application of known antibody-based drugs can provide certain guidance. The dosage can be a single-dose schedule or a multiple-dose schedule.

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

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

"Therapeutically effective amount" refers to the amount that is effective to achieve the desired therapeutic result at the required dose and for the required period of time. The therapeutically effective amount of the antibody or antibody fragment or its conjugate or composition can vary 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. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits a measurable parameter (such as tumor growth rate) by at least about 20%, more preferably at least about 40%, even more preferably at least about 50%, 60%, or 70%. % And still more preferably at least about 80%. The ability of a compound to inhibit a measurable parameter (e.g., cancer) can be evaluated in an animal model system that predicts efficacy in human tumors. Alternatively, this property of the composition can be evaluated by testing the compound's ability to inhibit, said inhibition in vitro 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.

V. Exemplary bispecific antibodies of the invention

Table 1a The amino acid sequence numbers of the light chain, heavy chain, light chain variable region and heavy chain variable region of antibodies v3.2, v3.0 and v13844

To	抗体v3.2Antibody v3.2	抗体v3.0Antibody v3.0	抗体v13844Antibody v13844
第一重链可变区HCVR1-抗PD-L1First heavy chain variable region HCVR1-anti-PD-L1	33	33	33
第二重链可变区HCVR2-抗PD-1The second heavy chain variable region HCVR2-anti-PD-1	66	77	77
第一轻链可变区LCVR1-抗PD-L1First light chain variable region LCVR1-anti-PD-L1	44	44	44
第二轻链可变区LCVR2-抗PD-1Second light chain variable region LCVR2-anti-PD-1	88	88	88
第一重链HC1-抗PD-L1First heavy chain HC1-anti-PD-L1	4949	4949	2929
第二重链HC2-抗PD-1Second heavy chain HC2-anti-PD-1	3131	3232	3333
第一轻链LC1-抗PD-L1First light chain LC1-anti-PD-L1	3030	3030	3030
第二轻链L2-抗PD-1Second light chain L2-anti-PD-1	3434	3434	3434

Table 1b The amino acid sequence numbers of the CDRs of the PD-L1 binding variable regions of antibodies v3.2, v3.0 and v13844 (determined according to the North rule)

Table 1c The amino acid sequence numbers of the CDRs of the PD-1 binding variable regions of antibodies v3.2, v3.0 and v13844 (determined according to the North rule)

The following examples are described to assist the understanding of the present invention. It is not intended and should not be construed in any way to limit the scope of protection of the present invention.

Abbreviation description

CEX-HPLC: Cation Exchange High Performance Liquid Chromatography

CE-SDS: Sodium Lauryl Sulfate Capillary Gel Electrophoresis

iCIEF: Imaging Capillary Isoelectric Focusing Electrophoresis

SEC-HPLC: size exclusion high performance liquid chromatography

Example

In order to develop formulations for anti-PD-1/PD-L1 antibody injections and freeze-dried formulations for long-term stable storage, and to ensure that the product quality is controllable within the validity period (at least 24 months), a prescription screening test was designed and different The effect of excipients on the stability of anti-PD-1/PD-L1 antibody preparations. The materials and methods used in the test are as follows:

Materials and Method

1.1. Materials used in the formulation of the present invention

1.2. The equipment used in the formulation research of the present invention

1.3. Test items and test methods for the stability of the preparation

The following items were tested for antibody preparations: (1) Check the appearance and presence of visible foreign matter; (2) Determine the protein content in the preparation by ultraviolet method (UV method); (3) Determine the turbidity by detecting the absorbance at 350nm (4) Determine the purity of the antibody preparation by size exclusion chromatography, for example, size-exclusion high performance liquid chromatography (size-exclusion chromatography-HPLC; SEC-HPLC), expressed as the area of the monomer in the area of all peaks (5) Determination of antibody preparations by reduced sodium dodecyl sulfate capillary electrophoresis (reduced CE-SDS) and/or non-reduced sodium dodecyl sulfate capillary electrophoresis (non-reduced CE-SDS) The purity is expressed as the percentage of the area of the monomer to the sum of all peak areas; (6) Determination of antibody preparations by imaging capillary isoelectric focusing electrophoresis (iCIEF method) and cation exchange high performance liquid chromatography (CEX-HPLC) Charge variant, expressed as the percentage of the main component, acidic component and alkaline component.

Visible foreign body detection

According to the method described in the National Pharmacopoeia Commission, the Pharmacopoeia of the People’s Republic of China (2015 edition, Four General Principles 0904 "Visible Foreign Body Inspection Method", Beijing: China Medical Science and Technology Press. 2015), the clarity detector (Tianjin Tianda Tianfa) Production, model YB-2), check for visible foreign objects in the sample.

Protein content determination

An ultraviolet spectrophotometer (produced by Shimadzu, Japan, model UV-1800) was used to determine the protein content in the sample.

Turbidity measurement

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

Purity (SEC-HPLC method)

Use size exclusion chromatography column to separate, the mobile phase is phosphate buffer (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 50μl, 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 50μl each of the blank solution and the test solution into the liquid chromatograph to start the test.

Purity (reduced CE-SDS method)

Detected by capillary gel electrophoresis. The capillary is an uncoated capillary with an inner diameter of 50μm, 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. Dilute the sample to be tested with an appropriate amount of ultrapure water to 2.0mg/ml, take 50μl of the above diluted sample into a 1.5ml centrifuge tube, and add 45μl pH 6.5 sample buffer to it (weigh 0.32g monohydrate citric acid , 2.45g disodium hydrogen phosphate dodecahydrate, dissolve in 45ml ultrapure water, dilute to 50ml to prepare citric acid-phosphate buffer solution, accurately measure 200μl of the buffer solution, add 10%(w/v) Sodium lauryl sulfate solution 80μl, add water to 1ml, mix well, ready to get), 1μl internal standard (10kDa protein, 5mg/mL) (Beckman Coulter, catalog number: 390953) and 5μl β-mercaptoethanol, mix well for 70 Heat at ±2°C for 10±2 minutes, cool to room temperature and transfer to a sample bottle as the test solution. Take the same volume of preparation buffer as the test product, and perform the same operation as described above to prepare a blank solution. Sample injection conditions: -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.

Purity (non-reduced CE-SDS method)

Detected by capillary gel electrophoresis. The capillary is an uncoated capillary with an inner diameter of 50μm, 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. Dilute the sample to be tested with an appropriate amount of ultrapure water to 2.0mg/ml, take 50μl of the above diluted sample into a 1.5ml centrifuge tube, and add 45μl pH 6.5 sample buffer to it (weigh 0.32g monohydrate citric acid , 2.45g disodium hydrogen phosphate dodecahydrate, dissolve in 45ml ultrapure water, dilute to 50ml to prepare citric acid-phosphate buffer solution, accurately measure 200μl of the buffer solution, add 10%(w/v) Sodium lauryl sulfate solution 80μl, add water to 1ml, mix well, ready to get), 1μl internal standard (10kDa protein, 5mg/mL) (Beckman Coulter, catalog number: 390953) and 5μl 250mmol/L NEM solution (weigh N -62mg of ethyl maleimide, dissolved in 2ml of ultrapure water), fully mixed and heated at 70±2°C for 10±2 minutes, cooled to room temperature and transferred to the sample bottle as the test solution. Take the same volume of preparation buffer as the test product, and perform the same operation as described above to prepare a blank solution. Sample injection conditions: -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.

Charge variant (iCIEF)

Using imaging capillary isoelectric focusing electrophoresis (iCIEF method) detection. The capillary has an inner diameter of 100μm and a total length of 5cm. Before sample electrophoresis, 0.5% methyl cellulose solution (hereinafter also abbreviated as MC solution) and ultrapure water should be used to rinse the capillary column 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. Dilute the test product to 1.0 mg/ml with water, take 20 μl of the diluted test product solution, and add 78 μl of the pre-mixed solution to it (the pre-mixed solution is as follows: 70 μl pI 0.5% MC solution, 4 μl amphoteric electrolyte (pH 3-10) ), 2μl cathode stabilizer, 1μl pI 5.85 marker, 1μl 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.

Charge variant (CEX-HPLC method)

It is detected by ion exchange chromatography (CEX-HPLC method). Use ion exchange chromatography column to separate, the mobile phase is 10mmol/L phosphate buffer (weigh 1.15g sodium dihydrogen phosphate dihydrate, 0.95g disodium hydrogen phosphate dodecahydrate, dissolve in 800ml ultrapure water and dilute to 1000ml ) And 10mmol/L phosphate+200mmol/L sodium chloride buffer (weigh 1.15g sodium dihydrogen phosphate dihydrate, 0.95g disodium hydrogen phosphate dodecahydrate, 11.69g sodium chloride dissolved in 800ml ultrapure water Constant volume to 1000ml), flow rate 0.5ml/min, detection wavelength 280nm, column temperature 25℃. Dilute the sample to 2mg/ml with mobile phase as the test solution; dilute the working reference product to 2mg/ml with mobile phase as the system suitability solution. Take the preparation buffer and dilute the same multiple with the mobile phase as a blank solution. Take 50μl each of the blank solution, system suitability solution, and test solution into the liquid chromatograph, the mobile phase flow rate is 1.0ml/min, the collection time is 35 minutes, the column temperature is 35°C, and the detection wavelength is 280nm.

1.4 Judgment criteria

According to the knowledge of the product and the precision of the instrument and method, the judgment standard of the quality of the sample detection index value and the initial value has not changed to determine whether the sample has changed. See the table below for details.

Judgment criteria for unchanged quality

检测项目Test items	未发生变化的判定标准Judgment criteria unchanged
外观(液体制剂及冻干制剂复溶后)Appearance (after reconstitution of liquid preparations and freeze-dried preparations)	澄明至微乳光，无色至微黄色液体，无异物Clear to slightly opalescent, colorless to slightly yellow liquid, no foreign matter
外观(冻干制剂)Appearance (lyophilized preparation)	白色至淡黄色块状疏松体White to light yellow lumpy loose body
可见异物(液体制剂及冻干制剂复溶Visible foreign bodies (liquid preparations and lyophilized preparations reconstituted	符合《中华人民共和国药典》(2015年版，四部)Comply with the "Pharmacopeia of the People's Republic of China" (2015 edition, four)

检测项目Test items	未发生变化的判定标准Judgment criteria unchanged
后)Rear)	通则0904General Rules 0904
蛋白含量(UV法)Protein content (UV method)	变化率≤10％Change rate ≤10%
纯度(SEC-HPLC法)Purity (SEC-HPLC method)	主峰纯度≥95％Main peak purity ≥95%
纯度(还原型CE-SDS法)Purity (reduced CE-SDS method)	主峰纯度≥90％Main peak purity ≥90%
纯度(非还原型CE-SDS法)Purity (non-reduced CE-SDS method)	主峰纯度≥90％Main peak purity ≥90%
电荷变异体(iCIEF法/CEX-HPLC法)Charge variants (iCIEF method/CEX-HPLC method)	主成分≥35％Main component ≥ 35%

Example 1. Preparation and purification of anti-PD-1/PD-L1 antibodies

According to the PCT application number PCT/US2018/041205, the light chain of new antibodies v3.2, v3.0 and v13844, and antibodies v3.2, v3.0 and v13844 that specifically bind to PD-1 and PD-L1 were obtained The SEQ ID NOs of the amino acid sequences of the heavy chain, light chain variable region, and heavy chain variable region are shown in Table 1(a) above. In addition, the SEQ ID NOs of the amino acid sequences of the CDRs of the PD-L1 and PD-1 binding variable regions of antibodies v3.2, v3.0, and v13844 are shown in Table 1(b) and Table 1(c), respectively.

The antibodies of the present invention include but are not limited to antibodies v3.2, v3.0 and v13844, and are basically prepared and purified as follows. A quadruple vector (ie, a single vector encoding the expression of two light chains and two heavy chains), a dual vector (ie, two vectors encoding two different light chains and two different heavy chains together), or a quadruple vector can be used. A single vector (two of which code for different light chains and two of which code for different heavy chains) is transiently or stably transfected into an appropriate host cell (such as CHO) with an antibody-secreting expression system. The medium in which the purified antibody is secreted can be used by any of many commonly used techniques. For example, the culture medium can be applied to a MabSelect column (GE Healthcare) or a KappaSelect column (GE Healthcare) for Fab fragments, and the column has been equilibrated with a compatible buffer (for example, phosphate buffered saline (pH 7.4)). The column can be washed to remove non-specific binding components. For example, the bound can be eluted by a pH gradient (for example, 20mM Tris buffer (pH 7) to 10mM citrate buffer (pH 3.0), or physiological saline (pH 7.4) to 100mM glycine buffer (pH 3.0)). Antibody. The antibody antibody fragments can be detected, for example, by SDS-PAGE, and can then be pooled. Soluble aggregates and multimers can be effectively removed by common techniques, including size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, multimodal chromatography, or hydroxyapatite chromatography. Common techniques can be used to concentrate and/or sterile filter the antibody. The product can be frozen immediately at -70°C or can be lyophilized.

An exemplary preparation method of v3.2 is described below.

Specifically, synthesize the nucleotide sequence encoding the heavy chain and light chain nucleotide sequence of the humanized antibody PD-1; synthesize the nucleotide sequence encoding the heavy chain and light chain core of the humanized antibody PD-L1 Nucleotide sequence. The heavy chain of PD-1 and the heavy chain of PD-L1 are respectively connected to the eukaryotic plasmid expression vector PEE6.4, and the light chain of PD-1 and the light chain of PD-L1 are respectively connected to the eukaryotic plasmid expression vector PEE12.4. The intermediate expression vector containing the heavy chain of PD-1 and the intermediate expression vector containing the light chain of PD-1 are combined to obtain a recombinant expression vector containing the nucleotide sequences of the heavy chain and light chain of PD-1. The intermediate expression vector containing the heavy chain of PD-L1 and the intermediate expression vector containing the light chain of PD-L1 are combined to obtain a recombinant expression vector containing the nucleotide sequences of the heavy chain and light chain of PD-L1. After culturing, two recombinant expression vectors were finally obtained for cell transfection to 269M cells (Lonza Biologics), and the selection pressure was LM7300 medium without glutamine (from the main medium). Use a bioreactor for cultivation with an inoculation density of not less than 1.0×10 ⁶ cells/ml; when it is cultivated to the 12th to 16th day or the cell viability is less than 60%, harvest, and the cell harvest liquid is used for downstream purification.

The cell harvest liquid is filtered with an adsorption depth filter membrane bag to filter the harvest liquid. During the filtration process, keep the aeration and stirring parameter settings of the bioreactor unchanged, and then rinse the membrane package with the affinity balance solution, combine the rinse solution and the filtrate, and name the clarified collection solution. Pack Mabselect Sure LX chromatography column, balance with affinity balance solution, after balance, add the clear collection solution to the column, and then rinse with balance solution, then rinse with affinity washing solution, and then affinity balance solution. Then start elution, collect the eluate, and name it as the affinity collection solution. The pH of the affinity collection solution was adjusted to about 6.0, and it was named the virus inactivation collection solution. The virus inactivation collection solution uses a depth filtration membrane package to perform adsorption and deep filtration of the virus inactivation collection solution, and collects the filtrate and the top washing solution, which is named as the absorption depth filtration collection solution. Pack the Poros XQ column, balance the column with an anion balance solution, then start to load the sample, collect the flow-through solution, and name it as the anion collection solution. Pack the Poros HS column, equilibrate with the cation balance solution, load the sample, rinse with the balance solution after the sample is loaded, eluate with the eluent, collect the eluent, and name it as the cation collector. The sample has an anti-PD-1/PD-L1 antibody v3.2 protein content of about 5.0-25.0 mg/ml.

The following examples take antibody v3.2 as an example, and its product code is IBI318.

Example 2. Test of the influence of pH on the stability of the formulation

This example examines the stability of the formulation containing the anti-PD-1/PD-L1 antibody at pH 5.5 to 6.5. A total of 3 pH values were designed, namely 5.5, 6.0 and 6.5.

2.1 Experimental steps

Prepare the buffer solution of each prescription according to Table 5, adjust the pH to 5.5, 6.0 and 6.5 with dilute hydrochloric acid, and replace the purified anti-PD-1/PD-L1 antibody of Example 1 by ultrafiltration to the different pH values In the solution. After the replacement, adjust the anti-PD-1/PD-L1 antibody protein content in the sample to about 20mg/ml; then add polysorbate 80 to make the final concentration of polysorbate 80 0.20mg/ml; filter and aliquot to The vials were stoppered and capped and studied according to the experimental scheme shown in Table 6. The detection indicators are appearance, protein content, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method).

Table 5. Pre-prescription test prescription form

Table 6. Pre-prescription test protocol

2.2 Experimental results

(1) Appearance

Placed for 2 weeks under the condition of 40℃±2℃, the appearance of each group of samples is qualified.

(2) Protein content

Under the condition of 40℃±2℃, the protein content of each group of samples did not change (see Table 7).

Table 7. Test protein content results before prescription (UV method, mg/ml)

(3) Purity

Purity (SEC-HPLC method): Accelerating for 2 weeks at 40℃±2℃, the purity of samples in each group changed significantly. As the pH increases, the purity of the samples decreases faster, which is manifested as an increase in aggregates; Thionine can slow down the rate of purity decline. (See Table 8, Figure 1).

Purity (non-reduced CE-SDS method): Accelerated for 2 weeks at 40°C±2°C, and the purity of the sample under each pH condition did not change significantly. (See Table 9).

Table 8. Pre-prescription test purity results (SEC-HPLC method, %)

Table 9. Test purity results before prescription (non-reduced CE-SDS method, %)

(4) Charge variant

Under the condition of 40°C±2°C, the charge variant-acid component and main component of each square have obvious changes, and the trend is the same among all squares, and there is no obvious difference. (See Table 10 and Figure 2)

Table 10. Test results of charge variants before prescription (iCIEF method)

Pre-prescription test results show that the protein is stable at pH 5.5-6.5, especially at pH 6.0. Based on the above results, the pH was selected as 6.0 for the next round of prescription screening test.

Example 3. Prescription determination experiment (1)

3.1 Experimental steps

This example examines the effects of different buffer systems and stabilizers on the stability of anti-PD-1/PD-L1 antibody formulations.

A total of 4 prescriptions were designed, and the detailed prescription information is shown in Table 11.

Prepare the buffer solution of each prescription according to Table 11, and replace the IBI318 protein into the respective prescription solution by ultrafiltration. After the replacement, the protein concentration of each prescription was diluted to about 20mg/ml, and polysorbate 80 was added to make the final concentration 0.2mg/ml. After being filtered and dispensed into vials, stoppered and capped, the accelerated stability was investigated under the conditions of 40℃±2℃ and 25℃±2℃, and its shaking and freeze-thaw stability were also investigated. The detection indicators are appearance, visible foreign matter, protein content, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variant (CEX-HPLC method).

Table 11. Alternative prescription information form for prescription screening test

Note: Prescription 1 uses citric acid to adjust pH, and prescriptions 2 to 4 use hydrochloric acid to adjust pH.

The detailed test conditions and sampling plan are shown in Table 12.

Table 12. Test conditions and sampling table

3.2 Test results

(1) Appearance, visible foreign matter

Observed at 40℃±2℃ for 1 month, 25℃±2℃ for 3 months, shaking for 5 days, freezing and thawing 6 times, the appearance of the four prescriptions and visible foreign bodies were all qualified.

(2) Protein content

Under the conditions of 40℃±2℃ and 25℃±2℃, as well as shaking and freeze-thaw conditions, the protein content of the four groups of prescriptions did not change (see Table 13).

Table 13. Prescription screening test protein content results (UV method, mg/ml)

(3) Purity

Purity (SEC-HPL C method): 1 month at 40°C±2°C and 3 months at 25°C±2°C, the purity of prescription 1 (citrate buffer system) decreased, mainly manifested by increased aggregates , The purity of other prescriptions has not changed significantly. The purity of each cube remained unchanged under shaking and freezing and thawing conditions. See Table 14, Figure 3 and Figure 4 for details.

Purity (non-reduced CE-SDS method): Under the condition of 40℃±2℃, the purity of each prescription has a downward trend, and there is no difference between the prescriptions; under the condition of 25℃±2℃, the purity of each prescription does not change significantly. See Table 15 and Figure 5 for details.

Table 14. Purity results of prescription screening test (SEC-HPLC method, %)

Table 15. Purity results of prescription screening test (non-reduced CE-SDS method, %)

(4) Charge variant

Under the conditions of 40°C±2°C and 25°C±2°C, the charge variant-acidic components and principal components of each prescription changed significantly. The rate of change is prescription 1> prescription 2> prescription 3> prescription 4, indicating the histidine system It is better than citric acid and sodium citrate system, and IBI318 protein has the best stability under the combined action of sorbitol, methionine and arginine (see Table 16, Figure 6-7). Under the conditions of oscillation and freezing and thawing, there is no obvious difference in charge variants between each square.

Table 16. Results of prescription screening test charge variants (CEX-HPLC method, %)

Based on the above test results, prescription 4 was selected as the IBI318 formulation prescription. In order to avoid the use of concentrated hydrochloric acid to adjust pH during production, the buffer system was adjusted to 10mmol/L histidine and histidine hydrochloride, that is, the prescription of IBI318: 20mg/ml recombinant fully human anti-programmed death receptor 1 (PD- 1) And anti-programmed death ligand 1 (PD-L1) bispecific antibody, 0.85mg/ml histidine, 1.00mg/ml histidine hydrochloride, 30.00mg/ml sorbitol, 7.49mg/ml methyl sulfide Acid, 21.07mg/ml arginine hydrochloride, 0.20mg/ml polysorbate 80, pH 6.0.

Example 4: Stability test of IBI318 high-concentration liquid preparation and its freeze-dried preparation

This experiment is used to investigate the stability of IBI318 high-concentration liquid preparation and its freeze-dried preparation under this prescription. The inventors unexpectedly discovered that through this prescription, not only a stable low concentration, for example, 20 mg/ml liquid antibody preparation, but also a stable high concentration, such as 100 mg/ml, liquid preparation and its lyophilized preparation can be prepared.

4.1 Test procedure

Change the antibody by ultrafiltration to the buffer in Example 3 (20mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific Antibody, 0.85mg/ml histidine, 1.00mg/ml histidine hydrochloride, 30.00mg/ml sorbitol, 7.49mg/ml methionine, 21.07mg/ml arginine hydrochloride, pH 6.0), After being concentrated to about 100 mg/ml, polysorbate 80 was added to make the final concentration 0.2 mg/ml. Filter and dispense into vials, 0.5ml each, 22 bottles in total. Take 11 bottles, stopper and cap and label them as PD20191109-1. Take another 11 bottles, freeze-dry them, stopper and cap and mark them as PD20191109-2. See Table 17 for stability investigation according to the experimental scheme. The detection indicators are appearance, protein content, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (CEX-HPLC method).

Table 17. Stability study protocol

4.2 Test results

(1) Appearance, visible foreign matter

When inspected at 40℃±2℃ for 1 month, and at 25℃±2℃ for 1 month, the appearance of high-concentration preparations and visible foreign bodies are all qualified; the appearance of freeze-dried preparations is qualified, and the appearance after reconstitution is visible. All foreign objects are qualified.

(2) Protein content

Under the conditions of 40℃±2℃ and 25℃±2℃ for 1 month, the protein content of high-concentration liquid preparations did not change; the protein content of the lyophilized preparations was reconstituted, and there was no change.

Table 18. Protein content results (UV method, mg/ml)

Note: ND means that the check item is not set, - means that the test is in progress, the same below.

K20180504 is a 20mg/ml liquid preparation; PD20191109-1 is a 100mg/ml high-concentration liquid preparation; PD20191109-2 is a 100mg/ml high-concentration lyophilized preparation.

(3) Purity

Purity (SEC-HPL C method): The purity of high-concentration liquid preparations decreases at 40°C and 25°C, which is mainly manifested by the increase in aggregates, and the decline rate is slightly faster than that of 20mg/ml liquid preparations, but at 40°C 1 The monthly purity is still greater than 95%, and the stability is acceptable. Compared with high-concentration liquid formulations, lyophilized formulations show better stability. See Table 19 for details.

Purity (non-reduced CE-SDS method): The purity of high-concentration liquid preparations decreases at 40°C, and the rate of decrease is equivalent to that of 20mg/ml liquid preparations. At 40°C, the purity is still greater than 90% for 1 month, and the stability is good. accept. Compared with high-concentration liquid preparations, freeze-dried preparations show better stability, and the purity (non-reduced CE-SDS method) does not change significantly after being placed at 40°C for 1 month. See Table 20 for details.

Table 19. Purity results of stability study (SEC-HPLC method, %)

Table 20. Purity results of stability study (non-reduced CE-SDS method, %)

(4) Charge variant

Under the condition of 40℃, the acidic components of high-concentration liquid preparations increased, and the main components decreased. The rate of change was equivalent to that of 20mg/ml liquid preparations. The charge variants of freeze-dried preparations did not change significantly. The charge variants of high-concentration liquid preparations and freeze-dried preparations did not change significantly at 25°C. See Table 21 for details.

Table 21. Results of stability study charge variants (CEX-HPLC method, %)

Test Conclusions

Based on the existing results of the above various experiments, it can be known that the high-concentration preparation prescription (100mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies , 0.85mg/ml histidine, 1.00mg/ml histidine hydrochloride, 30.00mg/ml sorbitol, 7.49mg/ml methionine, 21.07mg/ml arginine hydrochloride, 0.20mg/ml polysorbate 80, pH 6.0) The stability of the prepared liquid preparations and freeze-dried preparations all meet the requirements.

Example 5: Reconstitution test and viscosity test of IBI318 freeze-dried preparation

The lyophilized preparation can be reconstituted to prepare a higher concentration liquid preparation by adding less water. The IBI318 freeze-dried preparation PD20191109-2 can be reconstituted to form a liquid preparation with a protein concentration as high as 170mg/ml, with a viscosity lower than 10cP, and within the acceptable injection viscosity range of the liquid preparation. And when placed at room temperature (25°C±2°C, 60%RH±5%RH, 500Lux±50Lux) for one day, there is no change in all the detection indicators. The above results indicate that it is feasible to reconstitute a lyophilized preparation to prepare a high-concentration liquid preparation for subcutaneous injection.

Table 22 Stability research results of the reconstitution experiment of IBI318 freeze-dried preparation

The exemplary embodiments of the present invention have been described above, and those skilled in the art should understand that these disclosures are only exemplary, and various other substitutions, adaptations and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed in the text.

Claims

A freeze-dried preparation of an anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof, which comprises:

(i) Anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof;

(ii) Buffer system;

(iii) a stabilizer, which includes a polyol and/or amino acid; and

(iv) Surfactants,

Wherein the formulation has a pH between 5.5-6.5 when reconstituted, for example, the pH is about 5.5, 6.0 or 6.5.
The lyophilized formulation of claim 1, wherein the formulation has a pH of about 6.0 when reconstituted.
The freeze-dried preparation according to any one of claims 1-2, which can reconstitute the antibody or antigen-binding fragment thereof at a concentration between about 1 mg/mL and about 150 mg/mL, for example, the preparation can At a concentration of about 1 mg/mL to about 200 mg/mL, for example, about 1, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140 The antibody or antigen-binding fragment thereof is reconstituted at a concentration of 150, 160, 170, 180, 190, or 200 mg/ml; preferably, the preparation can be reconstituted at a concentration between about 20 mg/mL and about 170 mg/mL The antibody or antigen-binding fragment thereof.
The freeze-dried formulation according to any one of claims 1 to 3, wherein the surfactant is selected from polysorbate 80, polysorbate 20, poloxamer, polyethylene glycol polysorbate 80 or a combination thereof , And at about 0.01%(w/v)-1%(w/v), such as 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1%(w/v) Preferably, the surfactant is polysorbate 80, and more preferably, the polysorbate 80 is present at a weight ratio of about 0.02% (w/v).
The lyophilized preparation according to any one of claims 1 to 4, wherein the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, citric acid and sodium citrate buffer system , Sodium acetate acetate buffer system, phosphate buffer system, and exist in a weight ratio of about 0.01% (w/v) to 1% (w/v), such as about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5 , 0.6, 0.7, 0.8, 0.9, 1% (w/v) by weight ratio; preferably, the buffer system is present in a weight ratio of about 0.01% (w/v)-0.2% (w/v),

Preferably, the buffer system is a histidine buffer system; more preferably, the histidine content is about 0.01-1% (w/v), such as 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v) by weight ratio; more preferably, the histidine is present at a weight ratio of about 0.155% (w/v);

Preferably, the buffer system is a histidine and histidine hydrochloride buffer system; more preferably, the histidine and the histidine hydrochloride are respectively about 0.01-1% (w/v), such as 0.01 , 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1% (w/v) in weight ratio; more preferably, the histidine and histidine hydrochloride are respectively about It is present in a weight ratio of 0.085% (w/v) and about 0.1% (w/v).
The freeze-dried preparation according to any one of claims 1 to 5, wherein the polyol is selected from sorbitol, mannitol or a combination thereof, and the amino acid includes arginine, arginine hydrochloride, methionine, Glycine, proline or a combination thereof;

Preferably, the stabilizer includes sorbitol and arginine; more preferably, the sorbitol is at about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7, The weight ratio of 8, 9, 10% (w/v) is present, and the arginine is present at about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10% (w/v) weight ratio; more preferably, the sorbitol and the arginine are present in a weight ratio of about 3% (w/v) and about 1.742% (w/v) ；

Preferably, the stabilizer includes sorbitol and arginine hydrochloride; more preferably, the sorbitol is at a concentration of about 1-10% (w/v), such as about 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10% (w/v) weight ratio exists, about 1-10% (w/v), for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 % (W/v); more preferably, the sorbitol and the arginine hydrochloride are present in a weight ratio of about 3% (w/v) and about 2.107% (w/v), respectively.
The freeze-dried formulation according to any one of claims 1 to 6, wherein the stabilizer further comprises methionine; preferably, the methionine is about 0.1-10% (w/v) by weight Ratio exists, for example in a weight ratio of about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% (w/v); more preferably, the methionine It is present in a weight ratio of about 0.749% (w/v).
A freeze-dried preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof is prepared by freeze-drying an aqueous solution, the aqueous solution comprising:

(i) Anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof;

(ii) Buffer system;

(iii) a stabilizer, which includes a polyol and/or amino acid; and

(iv) Surfactants,

The liquid formulation has a pH of about 5.5-6.5, for example, the pH is about 5.5, 6.0, or 6.5; preferably, the liquid formulation has a pH of about 6.0.
A liquid preparation of an anti-PD-1/PD-L1 antibody or an antigen-binding fragment thereof, which comprises an aqueous solution, and the aqueous solution comprises:

(i) Anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof;

(ii) Buffer system;

(iii) a stabilizer, which includes a polyol and/or amino acid; and

(iv) Surfactants,

The liquid formulation has a pH of about 5.5-6.5, for example, the pH is about 5.5, 6.0, or 6.5; preferably, the liquid formulation has a pH of about 6.0.
The lyophilized formulation of claim 8 or the liquid formulation of claim 9, wherein the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof is at a concentration of about 1 mg/mL to about 200 mg/mL Exist in an aqueous solution, such as about 1, 5, 10, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, The concentration of 180, 190 or 200 mg/ml is present in the aqueous solution; preferably, the anti-PD-1/PD-L1 antibody or antigen-binding fragment thereof is present in the aqueous solution at a concentration between about 20 mg/mL and about 170 mg/mL middle.
The freeze-dried formulation according to claim 8 or the liquid formulation according to any one of claims 9-10, wherein the surfactant is selected from the group consisting of polysorbate 80, polysorbate 20, poloxamer, polysorbate Ethylene glycol polysorbate 80 or a combination thereof, and is present at a concentration of about 0.1-10 mg/ml, such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml ; Preferably, the surfactant is polysorbate 80, more preferably, the polysorbate 80 is present at a concentration of about 0.2 mg/ml.
The lyophilized preparation according to claim 8 or the liquid preparation according to any one of claims 9-11, wherein the buffer system is selected from the group consisting of histidine buffer system, histidine and histidine hydrochloride buffer system, Citrate and sodium citrate buffer system, sodium acetate buffer system, phosphate buffer system, and the amount of about 1-100mM, for example, about 1, 5, 10, 15, 20, 30, 40, 50, 60, It is present at a concentration of 70, 80, 90, 100 mM; preferably, the buffer system is present at a concentration of about 1-20 mM, for example, 1, 5, 10, 15, 20 mM;

Preferably, the buffer system is a histidine buffer system; more preferably, the histidine is at a concentration of about 0.1-10 mg/mL, such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10 mg/ml; more preferably, the histidine is present at a concentration of about 1.55 mg/mL;

Preferably, the buffer system is a histidine and histidine hydrochloride buffer system; more preferably, the histidine and the histidine hydrochloride are respectively about 0.1-10 mg/mL, such as about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/ml; more preferably, the histidine and the histidine hydrochloride are present at a concentration of about 0.85 mg/mL and about 1.00 There is a concentration of mg/mL.
The freeze-dried formulation according to claim 8 or the liquid formulation according to any one of claims 9-12, wherein the polyol is selected from sorbitol, mannitol or a combination thereof, and the amino acid includes arginine, Arginine hydrochloride, methionine, glycine, proline or a combination thereof;

Preferably, the stabilizer includes sorbitol and arginine; more preferably, the sorbitol is at a concentration of about 10-100 mg/mL, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, The arginine is present at a concentration of 100 mg/mL, and the arginine is present at, for example, about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 mg/mL; more preferably, the sorbitol and the arginine The acid exists at a concentration of about 30 mg/mL and about 17.42 mg/mL, respectively;

Preferably, the stabilizer includes sorbitol and arginine hydrochloride; more preferably, the sorbitol and the arginine hydrochloride are respectively about 10-100 mg/mL, such as about 10, 20, 30, 40, 50, It is present at a concentration of 60, 70, 80, 90, 100 mg/mL; more preferably, the sorbitol and the arginine hydrochloride are present at a concentration of about 30 mg/mL and about 21.07 mg/mL, respectively.
The lyophilized formulation according to claim 8 or the liquid formulation according to any one of claims 9-13, wherein the stabilizer further comprises methionine; preferably, the methionine is about 1 -100mg/mL, such as about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100mg/mL; more preferably, the methionine is present at a concentration of about 1- 50 mg/mL; more preferably, the methionine is present at a concentration of about 7.49 mg/mL.
The freeze-dried preparation according to claim 8 or the liquid preparation according to any one of claims 9-14, characterized in that:

The aqueous solution contains: about 20-170 mg/ml of recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 2.94 mg/ml Sodium citrate, about 50mg/ml sorbitol, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 5.5;

Or the aqueous solution contains: about 20-170 mg/ml of recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 2.94 mg/ml ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0;

Or the aqueous solution contains: about 20-170 mg/ml of recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 2.94 mg/ml ml sodium citrate, about 50 mg/ml sorbitol, about 2.94 mg/ml methionine, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0;

Or the aqueous solution contains: about 20-170 mg/ml of recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 2.94 mg/ml ml sodium citrate, about 50mg/ml sorbitol, about 0.20mg/ml polysorbate 80, the aqueous solution has a pH of about 6.5;

Or the aqueous solution contains: about 20-170 mg/ml of recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 2.94 mg/ml ml sodium citrate, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0;

Or the aqueous solution contains: about 20-170mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 1.55 mg/ml ml histidine, about 50 mg/ml sorbitol, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0;

Or the aqueous solution contains: about 20-170mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 1.55 mg/ml ml histidine, about 50 mg/ml sorbitol, about 7.49 mg/ml methionine, about 0.20 mg/ml polysorbate 80, the aqueous solution has a pH of about 6.0;

More preferably, the aqueous solution contains: 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 1.55 mg/ml histidine, about 30 mg/ml sorbitol, about 7.49 mg/ml methionine, about 17.42 mg/ml arginine, about 0.20 mg/ml polysorbate 80, the aqueous solution has about 6.0 pH;

More preferably, the aqueous solution contains: about 20-170 mg/ml recombinant fully human anti-programmed death receptor 1 (PD-1) and anti-programmed death ligand 1 (PD-L1) bispecific antibodies, about 0.85mg/ml histidine, about 1.00mg/ml histidine hydrochloride, about 30.00mg/ml sorbitol, about 7.49mg/ml methionine, about 21.07mg/ml arginine hydrochloride, about 0.20mg/ ml polysorbate 80, the aqueous solution has a pH of about 6.0.
The lyophilized preparation according to any one of claims 1-8 or the liquid preparation according to any one of claims 9-15, wherein the antibody or antigen-binding fragment thereof binds to human PD-L1 (SEQ ID NO: 1) And human PD-1 (SEQ ID NO: 2);

Preferably, the antibody or antigen-binding fragment thereof comprises a first heavy chain (HCl), which comprises a first heavy chain variable region (HCVR1) and a constant region; a first light chain (LC1), which comprises a first light chain The 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:

1) The HCVR1 includes the three complementarity determining regions HCDR1, HCDR2, and HCDR3 contained in the heavy chain variable region shown in SEQ ID NO: 3, and the LCVR1 includes the light chain variable shown in SEQ ID NO: 4 LCDR1, LCDR2 and LCDR3 contained in the area; and

2) 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: 5, and the LCVR2 includes the sequence shown in SEQ ID NO: 8. The three complementary determining regions LCDR1, LCDR2 and LCDR3 contained in the light chain variable region.
The lyophilized preparation according to any one of claims 1-8 or the liquid preparation according to any one of claims 9-15, wherein the antibody or antigen-binding fragment thereof comprises a first heavy chain (HCl), which comprises a One heavy chain variable region (HCVR1) and constant region; the first light chain (LC1), which contains the first light chain variable region (LCVR1) and constant region; the second heavy chain (HC2), which contains the second heavy chain Chain variable region (HCVR2) and constant region; and second light chain (LC2), which includes the second light chain variable region (LCVR2) and constant region, wherein:

a) The HCVR1 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 16, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 17, and the amino acid sequence shown in SEQ ID NO: 18 Sequence complementarity determining region CDR3;

b) The LCVR1 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 19, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 20, and the amino acid sequence shown in SEQ ID NO: 21 Sequence complementarity determining region CDR3;

c) The HCVR2 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 22, and the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 23 or SEQ ID NO: 24 and having SEQ ID NO : The complementarity determining region CDR3 of the amino acid sequence shown in 25; and

d) The LCVR2 includes the complementarity determining region CDR1 having the amino acid sequence shown in SEQ ID NO: 26, the complementarity determining region CDR2 having the amino acid sequence shown in SEQ ID NO: 27, and the amino acid sequence shown in SEQ ID NO: 28 The complementarity of the sequence determines the region CDR3.
The lyophilized preparation according to any one of claims 1-8 or the liquid preparation according to any one of claims 9-15, wherein the antibody or antigen-binding fragment thereof comprises a first heavy chain (HCl), which comprises a One heavy chain variable region (HCVR1) and constant region; the first light chain (LC1), which contains the first light chain variable region (LCVR1) and constant region; the second heavy chain (HC2), which contains the second heavy chain Chain variable region (HCVR2) and constant region; and second light chain (LC2), which includes the second light chain variable region (LCVR2) and constant region, wherein:

The antibody or antigen-binding fragment thereof comprises HCVR1 having at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 3; and/or having at least 90% with SEQ ID NO: 4, LCVR1 with 95%, 98% or 99% or higher identity; and/or HCVR2 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 5; and/or with SEQ ID NO: 8 LCVR2 with at least 90%, 95%, 98% or 99% or higher identity;

Preferably, a) the HCVR1 includes the amino acid sequence shown in SEQ ID NO: 3; b) the LCVR1 includes the amino acid sequence shown in SEQ ID NO: 4; c) the HCVR2 includes the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in 5; and d) the LCVR2 includes the amino acid sequence shown in SEQ ID NO: 8.
The lyophilized preparation according to any one of claims 1-8 or the liquid preparation according to any one of claims 9-15, wherein the antibody or antigen-binding fragment thereof comprises a first heavy chain (HCl), which comprises a One heavy chain variable region (HCVR1) and constant region; the first light chain (LC1), which contains the first light chain variable region (LCVR1) and constant region; the second heavy chain (HC2), which contains the second heavy chain Chain variable region (HCVR2) and constant region; and second light chain (LC2), which includes the second light chain variable region (LCVR2) and constant region, wherein:

The antibody or antigen-binding fragment thereof comprises HC1 which is at least 90%, 95%, 98% or 99% or more identical to SEQ ID NO: 49 or SEQ ID NO: 29; and/or SEQ ID NO: 30 LC1 with at least 90%, 95%, 98% or 99% or higher identity; and/or at least 90%, 95% with SEQ ID NO: 33 or SEQ ID NO: 31 or SEQ ID NO: 32 , HC2 with 98% or 99% or higher identity; and/or LC2 with at least 90%, 95%, 98% or 99% or higher identity with SEQ ID NO: 34;

Preferably, the HC1, the LC1, the HC2 and the LC2 respectively comprise the amino acid sequences shown in SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 31 and SEQ ID NO: 34; or Respectively include the amino acid sequences shown in SEQ ID NO: 49, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34; or respectively include SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 33 and the amino acid sequence shown in SEQ ID NO: 34;

More preferably, the antibody or antigen-binding fragment thereof contains a variant of the Fc portion derived from human IgG1.
The lyophilized preparation according to any one of claims 1-8 or the liquid preparation according to any one of claims 9-15, wherein the antibody is selected from: PD-1/PD-L1 bispecific Ab v13844, Ab v3.0 or Ab v3.2; preferably, the antibody is PD-1/PD-L1 bispecific Ab v3.2.
The lyophilized formulation according to any one of claims 1-8 or the liquid formulation according to any one of claims 9-15, wherein the antibody or antigen-binding fragment thereof exhibits stability for at least about 12 months, Preferably, the antibody or antigen-binding fragment thereof exhibits stability for at least about 24 months, and more preferably, the antibody or antigen-binding fragment thereof exhibits stability for at least about 36 months.
The lyophilized formulation according to any one of claims 1-8 or the liquid formulation according to any one of claims 9-15, wherein the formulation is stored after storage, for example, after storage at 2-8°C for at least 24 months, It is stable after storage at room temperature for at least 6 months, or storage at 40°C±2°C for 1 month, and preferably has one or more of the following characteristics:

(i) Measured by the SEC-HPLC method, the preparation has a purity greater than or equal to 90%, preferably greater than or equal to 92%, 94%, 95%, 96%, 98% or 99% purity;

(ii) Measured by the reduced or non-reduced CE-SDS method, the preparation has a purity greater than or equal to 90%, preferably greater than or equal to 92%, 94%, 96%, 98% purity;

(iii) Measured by iCIEF or CEX-HPLC method, the main component is ≥35%, preferably greater than or equal to 35%, 40%, 45% or 50%.
A delivery device comprising the freeze-dried formulation according to any one of claims 1-8 or the liquid formulation according to any one of claims 9-15, for intravenous, subcutaneous, intradermal or intramuscular injection, or intravenous Infusion.
A pre-filled syringe containing the freeze-dried formulation according to any one of claims 1-8 or the liquid formulation according to any one of claims 9-15 for intravenous, subcutaneous, intradermal or intramuscular injection, Or intravenous infusion.
The freeze-dried formulation according to any one of claims 1-8 or the use of the liquid formulation according to any one of claims 9-15 for preparing a delivery device or a pretreatment device for preventing and/or treating tumors in a subject Fill a syringe or medicine; for example, the tumor is cancer, preferably, the cancer is Hodgkin's or non-Hodgkin's lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, stomach and esophagus Cancer, colorectal cancer, liver cancer, hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC) , Mesothelioma, squamous cell carcinoma of the head and neck (SCCHN), soft tissue sarcoma or glioblastoma multiforme; more preferably, the lung cancer is NSCLC, small cell lung cancer or mesothelioma; preferably, wherein said The delivery device or pre-filled syringe or drug can be administered simultaneously, separately or sequentially with one or more anti-tumor agents selected from the group consisting of: cisplatin, carboplatin, dacarbazine (dacarbazine), liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin , Paclitaxel, paclitaxel protein-binding particles for injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin), fluorouracil (fluorouracil) and Oxaliplatin), FOLFIRI (leucovorin, fluorouracil and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed ( pemetrexed), cetuximab, nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab Anti-tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat and durvalumab; more preferred In particular, the delivery device or pre-filled syringe or drug can be administered simultaneously, separately or sequentially with ionizing radiation.
A method for preventing and/or treating tumors, comprising administering to a subject an effective amount of the freeze-dried formulation according to any one of claims 1-8 or the liquid formulation according to any one of claims 9-15; for example , The tumor is cancer, preferably, the cancer is Hodgkin's or non-Hodgkin's lymphoma, melanoma, renal cell carcinoma, kidney cancer, lung cancer, bladder cancer, stomach and esophageal cancer, colorectal cancer, liver cancer , Hepatocellular carcinoma, cholangiocarcinoma, pancreatic cancer, breast cancer, triple-negative breast cancer, ovarian cancer, endometrial cancer, prostate cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), mesothelioma, head and neck cancer Squamous cell carcinoma (SCCHN), soft tissue sarcoma or glioblastoma multiforme; more preferably, the lung cancer is NSCLC, small cell lung cancer or mesothelioma.
The method according to claim 26, further comprising simultaneously, separately or sequentially administering one or more anti-tumor agents selected from the group consisting of cisplatin, carboplatin, dacarbazine ), liposomal doxorubicin, docetaxel, cyclophosphamide and doxorubicin, navelbine, eribulin, Pacific Paclitaxel, paclitaxel protein-binding particles for injectable suspension, ixabepilone, capecitabine, FOLFOX (leucovorin), fluorouracil, and oxabe Oxaliplatin), FOLFIRI (leucovorin, fluorouracil and irinotecan), gemcitabine, topotecan, liposomal irinotecan, pemetrexed , Cetuximab, nivolumab, ipilimumab, pidilizumab, pembrolizumab, tremelimumab ( tremelimumab, urelumab, lirilumab, atezolizumab, epacadostat and durvalumab.