Compositions Comprising PD-L1 Antigen-Binding Fragments and Uses Thereof

Abstract

The present invention relates to a composition comprising an antigen-binding fragment comprising an immunoglobulin single variable domain capable of specifically binding to PD-L1, the immunoglobulin single variable domain comprising CDR1-3, and the CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 47, and an excipient which may contain proline. The composition has high stability and suitable viscosity, and can be used for subcutaneous injection.

Description

[Technical field]

[0001] The present application relates to the field of biopharmaceuticals, and in particular to compositions comprising PD-L1 antigen-binding fragments and uses thereof. [Background technology]

[0002] PD-L1 expression has been found in several mouse and human cancers, including human lung, ovarian, and colon cancers, melanoma, and various myelomas. Existing results indicate that PD-L1 highly expressed by tumor cells plays an important role in tumor immune evasion by increasing apoptosis of T cells.

[0003] Antibody drug formulations include injection formulations or freeze-dried formulations, etc. For frequently used products and chronic administration, subcutaneous administration is more preferred, which allows patients to self-manage, improves the convenience, speed and compliance of treatment, saves significant costs for the medical sector and improves the quality of life of patients.

[0004] Therefore, there is a need to develop highly concentrated PD-L1 antibody formulations. Summary of the Invention

[0005] The present application provides a composition comprising an antigen-binding fragment that specifically binds to PD-L1 and an excipient, which has at least one of the following beneficial effects: 1) containing a high concentration (e.g., 200 mg / mL) of the antigen-binding fragment, 2) having an appropriate viscosity, 3) being suitable for subcutaneous injection and capable of reducing the subject's sense of pain, 4) having good stability (e.g., accelerated stability and / or long-term stability), 5) avoiding side effects caused by infusion, and 6) having simple components, being easy to manufacture, and being low cost.

[0006] The present invention provides a composition comprising an antigen-binding fragment comprising an immunoglobulin single variable domain capable of specifically binding to PD-L1, wherein the immunoglobulin single variable domain comprises CDR1-3, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO:47, and one or more excipients selected from the group consisting of proline, mannitol, sucrose, glycine, and L-arginine hydrochloride.

[0007] In another embodiment, the excipient may be one or more selected from the group consisting of proline, sucrose, mannitol, sorbitol, glycerol, and L-arginine hydrochloride.

[0008] In some embodiments, the excipient is preferably one or more selected from the group consisting of proline, sucrose, mannitol, sorbitol and glycerol, more preferably one or more of proline, sucrose, mannitol and sorbitol.

[0009] In some embodiments, the excipient is L-proline, or the excipient is sucrose, or the excipient is mannitol, or the excipient is sorbitol.

[0010] In some embodiments, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO:46.

[0011] In some embodiments, the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:45 or 48.

[0012] In some embodiments, the immunoglobulin single variable domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1-6.

[0013] In some embodiments, the PD-L1 is human PD-L1.

[0014] In some embodiments, the antigen-binding fragment comprises an Fc region of an immunoglobulin.

[0015] In some embodiments, the N-terminus of the Fc region of the immunoglobulin is linked directly or indirectly to the C-terminus of the immunoglobulin single variable domain.

[0016] In some embodiments, the N-terminus of the Fc region of the immunoglobulin is linked to the C-terminus of the immunoglobulin single variable domain by a linker.

[0017] In some embodiments, the linker comprises an amino acid sequence shown in any one of SEQ ID NOs:42-44.

[0018] In some embodiments, the Fc region of the immunoglobulin comprises an Fc region derived from an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

[0019] In some embodiments, the Fc region of the immunoglobulin has no ADCC activity and / or the Fc region of the immunoglobulin has no CDC activity.

[0020] In some embodiments, the Fc region of the immunoglobulin is derived from a human.

[0021] In some embodiments, the Fc region of the immunoglobulin is derived from human IgG.

[0022] In some embodiments, the immunoglobulin Fc region comprises an amino acid sequence shown in any one of SEQ ID NOs: 7-9.

[0023] In some embodiments, the antigen-binding fragment comprises the amino acid sequence set forth in any one of SEQ ID NOs: 14-31.

[0024] In some embodiments, the antigen-binding fragment has a concentration of about 1 mg / mL to about 300 mg / mL.

[0025] In some embodiments, the antigen-binding fragment has a concentration of about 5 mg / mL to about 250 mg / mL.

[0026] In some embodiments, the antigen-binding fragment has a concentration of about 1 mg / mL to about 220 mg / mL.

[0027] In some embodiments, the antigen-binding fragment has a concentration of about 20 mg / mL to about 220 mg / mL.

[0028] In some embodiments, the antigen-binding fragment has a concentration of about 50 mg / mL to about 200 mg / mL.

[0029] In some embodiments, the antigen-binding fragment has a concentration of about 100 mg / mL to about 280 mg / mL.

[0030] In some embodiments, the antigen-binding fragment has a concentration of about 150 mg / mL to about 250 mg / mL.

[0031] In some embodiments, the antigen-binding fragment has a concentration of about 180 mg / mL to about 250 mg / mL.

[0032] In some embodiments, the antigen-binding fragment has a concentration of about 180 mg / mL to about 220 mg / mL.

[0033] In some embodiments, the excipient has a concentration of about 50 mM to about 500 mM.

[0034] In some embodiments, the excipient comprises proline at a concentration of about 50 mM to about 390 mM.

[0035] In some embodiments, the excipient comprises proline at a concentration of about 150 mM to about 250 mM.

[0036] In some embodiments, the excipient comprises proline at a concentration of about 150 mM to about 220 mM.

[0037] In some embodiments, the excipient comprises L-proline at a concentration of about 150 mM to about 220 mM.

[0038] In some embodiments, the excipient comprises L-proline at a concentration of about 165 mM to about 275 mM.

[0039] In some embodiments, the excipient comprises mannitol at a concentration of about 50 mM to about 500 mM.

[0040] In some embodiments, the excipient comprises sucrose at a concentration of about 50 mM to about 500 mM.

[0041] In some embodiments, the excipient comprises sucrose at a concentration of about 90 mM to about 500 mM.

[0042] In some embodiments, the excipient comprises glycine at a concentration of about 50 mM to about 500 mM.

[0043] In some embodiments, the excipient comprises glycine at a concentration of about 150 mM to about 250 mM.

[0044] In some embodiments, the excipient comprises L-arginine hydrochloride at a concentration of about 50 mM to about 500 mM.

[0045] In some embodiments, the excipient comprises L-arginine hydrochloride at a concentration of about 150 mM to about 500 mM.

[0046] In some embodiments, the excipient comprises sorbitol at a concentration of about 50 mM to about 500 mM.

[0047] In some embodiments, the excipient comprises sorbitol at a concentration of about 100 mM to about 300 mM.

[0048] In some embodiments, the excipient comprises glycerol at a concentration of from 50 mM to about 500 mM.

[0049] In some embodiments, the excipient comprises glycerol at a concentration of about 100 mM to about 300 mM.

[0050] In some embodiments, the composition has a pH of about 5.0 to about 7.5.

[0051] In some embodiments, the composition has a pH of about 5.9 to about 6.9.

[0052] In some embodiments, the composition has a pH of about 6.3 to about 6.5.

[0053] In some embodiments, the composition has a pH of about 6.4.

[0054] In some embodiments, the composition comprises one or more buffer components selected from the group consisting of sodium acetate-acetic acid, histidine-acetic acid, L-histidine-L-histidine hydrochloride, sodium phosphate, and citrate-sodium hydroxide.

[0055] In some embodiments, the buffer component is one or more selected from the group consisting of sodium acetate-acetic acid, L-histidine-L-histidine hydrochloride, and citric acid-sodium hydroxide, and preferably, the buffer component is sodium acetate-acetic acid or L-histidine-L-histidine hydrochloride.

[0056] In some embodiments, the buffer components have a concentration of about 5 mM to about 50 mM.

[0057] In some embodiments, the buffer components include sodium acetate-acetic acid at a concentration of about 5 mM to about 35 mM.

[0058] In some embodiments, the buffer components include sodium acetate-acetic acid at a concentration of about 10 mM to about 30 mM.

[0059] In some embodiments, the buffer components include sodium acetate-acetic acid at a concentration of about 20 mM.

[0060] In some embodiments, the buffer component comprises L-histidine-L-histidine hydrochloride at a concentration of about 5 mM to about 35 mM, preferably about 10 mM to about 30 mM, and even more preferably 20 mM. In some embodiments, the composition comprises one or more surfactants selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188.

[0061] In some embodiments, the surfactant has a concentration of about 0 mg / mL to about 0.8 mg / mL.

[0062] In some embodiments, the surfactant comprises polysorbate 20 at a concentration of about 0 mg / mL to about 0.4 mg / mL.

[0063] In some embodiments, the surfactant comprises polysorbate 20 at a concentration of about 0.2 mg / mL to about 0.4 mg / mL. In some embodiments, the surfactant comprises polysorbate 20 at a concentration of about 0.1 mg / mL to about 0.3 mg / mL.

[0064] In some embodiments, the surfactant comprises polysorbate 20 at a concentration of about 0.2 mg / mL.

[0065] In some embodiments, the surfactant comprises polysorbate 80 at a concentration of about 0.05 mg / mL to about 1 mg / mL, preferably 0.1 mg / mL to 0.5 mg / mL, more preferably 0.1 mg / mL to 0.3 mg / mL, and even more preferably 0.2 mg / mL. In some embodiments, the surfactant comprises poloxamer 188 at a concentration of about 0.05 mg / mL to about 5 mg / mL, preferably 0.1 mg / mL to about 3 mg / mL, and more preferably 0.2 mg / mL to about 2 mg / mL.

[0066] In some embodiments, the composition comprises 1) about 20 mg / mL to about 220 mg / mL of the antigen-binding fragment described herein, 2) about 5 mM to about 35 mM sodium acetate-acetic acid, 3) about 50 mM to about 390 mM proline, and 4) about 0 mg / mL to about 0.4 mg / mL of polysorbate 20, and has a pH of about 5.9 to about 6.9.

[0067] In some embodiments, the composition comprises 1) about 50 mg / mL to about 220 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM sodium acetate-acetic acid, 3) about 150 mM to about 250 mM proline, 4) about 0.2 mg / mL to about 0.4 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0068] In some embodiments, the composition comprises 1) about 150 mg / mL to about 250 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM sodium acetate-acetic acid, 3) about 150 mM to about 250 mM proline, 4) about 0.2 mg / mL to about 0.4 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0069] In some embodiments, the composition comprises 1) about 200 mg / mL of an antigen-binding fragment described herein, 2) about 20 mM sodium acetate-acetic acid, 3) about 220 mM proline, 4) about 0.2 mg / mL polysorbate 20, and has a pH of about 6.4.

[0070] In some embodiments, the composition comprises 1) about 100 mg / mL to about 280 mg / mL of the antigen-binding fragment described herein, 2) about 5 mM to about 35 mM sodium acetate-acetic acid, 3) about 50 mM to about 500 mM sucrose, 4) about 0.05 mg / mL to about 0.4 mg / mL of polysorbate 20, and has a pH of about 5.9 to about 6.9.

[0071] In some embodiments, the composition comprises 1) about 150 mg / mL to about 250 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM sodium acetate-acetic acid, 3) about 50 mM to about 300 mM sucrose, 4) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0072] In some embodiments, the composition comprises 1) about 100 mg / mL to about 280 mg / mL of the antigen-binding fragment described herein, 2) about 5 mM to about 35 mM sodium acetate-acetic acid, 3) about 50 mM to about 500 mM mannitol, and 4) about 0.05 mg / mL to about 0.4 mg / mL of polysorbate 20, and has a pH of about 5.9 to about 6.9.

[0073] In some embodiments, the composition comprises 1) about 150 mg / mL to about 250 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM sodium acetate-acetic acid, 3) about 100 mM to about 300 mM mannitol, and 4) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0074] In some embodiments, the composition comprises 1) about 100 mg / mL to about 280 mg / mL of the antigen-binding fragment described herein, 2) about 5 mM to about 35 mM sodium acetate-acetic acid, 3) about 50 mM to about 500 mM sorbitol, 4) about 0.05 mg / mL to about 0.4 mg / mL of polysorbate 20, and has a pH of about 5.9 to about 6.9.

[0075] In some embodiments, the composition comprises 1) about 150 mg / mL to about 250 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM sodium acetate-acetic acid, 3) about 100 mM to about 300 mM sorbitol, 4) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0076] In some embodiments, the composition comprises 1) about 100 mg / mL to about 280 mg / mL of the antigen-binding fragment described herein, 2) about 5 mM to about 35 mM sodium acetate-acetic acid, 3) about 50 mM to about 500 mM glycerol, 4) about 0.05 mg / mL to about 0.4 mg / mL of polysorbate 20, and has a pH of about 5.9 to about 6.9.

[0077] In some embodiments, the composition comprises 1) about 150 mg / mL to about 250 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM sodium acetate-acetic acid, 3) about 100 mM to about 300 mM glycerol, 4) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0078] In some embodiments, the composition contains 1) about 150 mg / mL to about 250 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM L-histidine-L-histidine hydrochloride, 3) about 150 mM to about 250 mM proline, 4) about 0.2 mg / mL to about 0.4 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0079] In some embodiments, the composition comprises 1) about 150 mg / mL to about 250 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM L-histidine-L-histidine hydrochloride, 3) about 50 mM to about 300 mM sucrose, and 4) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0080] In some embodiments, the composition comprises 1) about 150 mg / mL to about 250 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM L-histidine-L-histidine hydrochloride, 3) about 100 mM to about 300 mM mannitol, and 4) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0081] In some embodiments, the composition comprises 1) about 150 mg / mL to about 250 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM L-histidine-L-histidine hydrochloride, 3) about 100 mM to about 300 mM sorbitol, and 4) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0082] In some embodiments, the composition comprises 1) about 150 mg / mL to about 250 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM L-histidine-L-histidine hydrochloride, 3) about 100 mM to about 300 mM glycerol, and 4) about 0.1 mg / mL to about 0.3 mg / mL of polysorbate 20, and has a pH of about 6.3 to about 6.5.

[0083] In some embodiments, the composition is for injection.

[0084] In some embodiments, the composition is for subcutaneous injection.

[0085] In some embodiments, the composition is a formulation.

[0086] In some embodiments, the composition is a liquid formulation.

[0087] In another aspect, the present application provides a reagent kit comprising a composition described herein and a container that contains the composition described herein.

[0088] In some embodiments, the container comprises a glass bottle.

[0089] In some embodiments, the composition in the container has a volume of about 0.5 mL to about 5.0 mL.

[0090] In some embodiments, the composition in the container has a volume of about 0.5 mL to about 1.5 mL.

[0091] In another aspect, the present application provides the use of a composition described herein, and / or a reagent kit described herein, in the manufacture of a medicament for preventing, alleviating and / or treating a tumor.

[0092] In some embodiments, the tumor comprises a PD-L1 positive tumor.

[0093] In some embodiments, the tumor comprises a malignant solid tumor.

[0094] In another aspect, the present application provides the use of a composition described herein, and / or a reagent kit described herein, in the manufacture of a medicament for preventing, alleviating, and / or treating an infectious disease.

[0095] In some embodiments, the infectious disease includes diseases resulting from viral, bacterial, fungal, and / or parasitic infections.

[0096] Those skilled in the art can easily discern other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As will be apparent to those skilled in the art, the contents of the present application allow those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention of the present application. Therefore, the drawings and descriptions in the present application are merely illustrative, not limiting.

[0097] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is given below. [Brief description of the drawings]

[0098] [Figure 1] 1 shows the results of Tm values ​​of pharmaceutical formulations containing antigen-binding fragments described herein. [Diagram 2] 1 shows the results of detecting polymer propensity of pharmaceutical formulations containing antigen-binding fragments described herein. [Diagram 3] 1 shows the results of detection of the main peak content by SEC-HPLC of pharmaceutical formulations containing the antigen-binding fragments described herein. [Figure 4] 4 shows the results of detecting polymer content by SEC-HPLC of pharmaceutical formulations containing antigen-binding fragments described herein. [Diagram 5] 1 shows the detection of acidic components by WCX-HPLC of pharmaceutical formulations containing antigen-binding fragments described herein. [Figure 6] 1 shows viscosity profile results of pharmaceutical formulations containing antigen-binding fragments described herein. [Figure 7] 1 shows binding activity results of pharmaceutical formulations containing antigen-binding fragments described herein. [Figure 8] 1 shows blocking activity results of pharmaceutical formulations containing antigen-binding fragments described herein. [Figure 9] 1 shows the results of detection of the main peak content by SEC-HPLC of pharmaceutical formulations containing the antigen-binding fragments described herein. [Figure 10] 4 shows the results of detecting polymer content by SEC-HPLC of pharmaceutical formulations of antigen-binding fragments described herein. [Figure 11] 1 shows the detection of acidic components by WCX-HPLC of pharmaceutical formulations of antigen-binding fragments described herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0099] Hereinafter, the embodiments of the present invention will be described with reference to specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0100] Definition of Terms In this application, the term "antigen-binding fragment" generally refers to one or more portions of an antibody that retain the ability to specifically interact with an epitope of an antigen (e.g., by binding or steric hindrance, etc.). The antigen-binding function of an antibody can also be achieved by a heavy chain, including a fragment of Fv, scFv, dsFv, Fab, Fab', or F(ab')2, or a light chain, including a fragment of Fv, scFv, dsFv, Fab, Fab', or F(ab')2. (1) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH domains; (2) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by disulfide bonds at the hinge region; (3) an Fd fragment consisting of the VH and CH domains; (4) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (5) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); (6) an isolated complementarity determining region (CDR); and (7) a combination of two or more isolated CDRs, optionally linked by a linker. For example, monovalent single chain molecules Fv (scFv) formed from a pair of VL and VH may also be included (see Bird et al., (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. 85:5879-5883). For example, a class of antibody VHHs lacking an antibody light chain and having only a heavy chain variable region may also be included (see, for example, Kang Hsiao-sen et al., Journal of Bioengineering, 2018, 34(12):1974-1984). The above-mentioned "antigen-binding portion" may further include an immunoglobulin fusion protein containing a binding domain selected from (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (2) an immunoglobulin heavy chain CH2 constant region fused to a hinge region, and (3) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region.

[0101] In the present application, the term "excipient" generally refers to any material selected for any type or product form desired for the composition. For example, the product form may include liquid, granule, powder, paste, spray, tablet, or gel. The excipient may not affect the form of existence or stability of the antigen-binding fragment, and may not affect any biological activity of the antigen-binding fragment (e.g., specific binding to the corresponding antigen). In the present application, the excipient may function as a stabilizer and improve the stability of the composition and / or the stability of the antigen-binding fragment in the composition.

[0102] In this application, the term "PD-L1" generally refers to programmed cell death protein 1 ligand 1, which may also be referred to as CD274 or B7H1. In this application, the PD-L1 may be mammalian-derived PD-L1, for example, the PD-L1 may be primate-derived or rodent-derived (e.g., mouse or rat) derived. Human PD-L1 may have the amino acid sequence set forth in NCBI Accession No. NP_054862.1. The PD-L1 may include naturally occurring PD-L1, and may include variants, isotypes, species homologs, and analogs of PD-L1 that share at least one common epitope with PD-L1.

[0103] In the present application, the term "immunoglobulin variable domain" generally refers to a polypeptide molecule comprising four "framework regions", namely, "framework region 1" or "FR1", "framework region 2" or "FR2", "framework region 3" or "FR3", and "framework region 4" or "FR4", and three "complementarity determining regions" or "CDRs", namely, "complementarity determining region 1" or "CDR1", "complementarity determining region 2" or "CDR2", and "complementarity determining region 3" or "CDR3". The immunoglobulin variable domain may have a tertiary structure of a protein. In the present application, the immunoglobulin variable domain may have the function of specifically binding to an antigen (e.g., PD-L1). The immunoglobulin variable domain may comprise the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0104] In the present application, the term "immunoglobulin single variable domain" generally refers to said immunoglobulin variable domain having the independent function of specifically binding to an antigen (e.g., PD-L1). For example, said immunoglobulin single variable domain can specifically bind to an antigen or an epitope thereof without pairing with other immunoglobulin variable domains. For example, said immunoglobulin single variable domain may be a heavy chain variable region VH and / or a light chain variable region VL. In the present application, said immunoglobulin single variable domain may be a VHH. In the present application, said VHH may be referred to as a heavy chain antibody, a nanobody, or a single domain antibody. The CDRs of the above VHHs can be divided according to the Kabat system (see "Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD), e.g., amino acids 1-30 of the VHH amino acid sequence are FR1, amino acids 31-35 are CDR1, amino acids 36-49 are FR2, amino acids 50-65 are CDR2, amino acids 66-94 are FR3, amino acids 95-102 are CDR3, and amino acids 103-113 are FR4. However, it should be noted that the Kabat numbering may or may not correspond to the actual numbering of amino acid residues in the actual sequence, since the total number of amino acid residues in each CDR may be different and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (i.e., one or more positions according to the Kabat numbering may be unoccupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat numbering).

[0105] As used herein, the term "composition" generally refers to a mixture that includes an antigen-binding fragment as described herein. The composition may include at least one pharma- ceutically acceptable ingredient, such as an excipient as described herein.

[0106] In this application, the term "formulation" generally refers to a product that contains a given amount or ratio of an antigen-binding fragment described herein, and any product that is produced by directly or indirectly combining a given amount of an antigen-binding fragment described herein. The meaning of a formulation as used herein includes drug formulations, i.e., any product that results directly or indirectly from the combination, complexation, or aggregation of any two or more components of the antigen-binding fragment described herein and the excipients described above. The formulation may be in liquid form.

[0107] In this application, the term "buffer component" generally refers to a reagent that functions to provide a buffering effect (e.g., resistance to pH changes). For example, the buffer component can adjust the change in pH due to the addition and / or release of an acidic or basic substance. For example, the buffer component may include a weak acid and its conjugate base, or a weak base and its conjugate acid. In this application, the term "surfactant" generally refers to an agent that can protect a protein (e.g., an antigen-binding fragment described herein) from the effects of air / solution interface-induced stress, solution / surface-induced stress, reduce aggregation of the protein, or minimize the formation of particulate matter in the composition. The surfactant may include both hydrophobic and hydrophilic groups. The surfactant may include a mixture or combination of one or more surfactants. The surfactant may include a non-ionic surfactant.

[0108] In the present application, the term "PD-L1 positive" generally refers to a percentage of cells expressing PD-L1 in a test tissue sample, including tumor cells and tumor-infiltrating inflammatory cells, that is higher than the percentage of cells that correspond to the level of cell surface PD-L1 expression assessed for the sample. For example, the percentage of cells can be measured by immunohistochemistry (IHC). In the present application, the PD-L1 positive may be at least about 0.01%, at least about 0.5%, at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% of the total number of cells expressing PD-L1 in a sample.

[0109] In this application, the term "tumor" generally refers to damage caused by abnormal cell growth. For example, the tumor may form a discrete tissue mass due to rapid and uncontrolled cell proliferation. The tissue mass may be benign, pre-malignant, or malignant. The tumor may be considered malignant if it has the structure and / or function of growing uncontrollably and / or attacking normal tissue. The malignant tumor may be used interchangeably with cancer. In this application, the term "malignant solid tumor" generally refers to a tumor of any type and morphology that has metastatic potential.

[0110] In this application, the term "infectious disease" generally refers to local tissue and systemic inflammatory responses resulting from the invasion of the human body by pathogens such as viruses, bacteria, fungi, parasites, and certain toxins.

[0111] In this application, the term "about" generally refers to a normal error range for the corresponding value. Reference herein to "about" a certain value or parameter includes (and describes) the solution to this value or parameter itself. In case of doubt, or if there is a general understanding that the error range for a particular value or parameter is not recognized by the art, "about" refers to ±5% of this value or parameter.

[0112] In one aspect, the present application provides a composition that can comprise the antigen-binding fragment, which can comprise an immunoglobulin single variable domain capable of specifically binding to PD-L1, wherein the immunoglobulin single variable domain can comprise an amino acid sequence as set forth in any one of SEQ ID NOs: 1-6, and one or more excipients selected from the group consisting of proline, mannitol, sucrose, glycine, and L-arginine hydrochloride.

[0113] antigen binding fragment In the present application, the antigen-binding fragment can specifically bind to PD-L1. For example, the PD-L1 can be human PD-L1. In the present application, the antigen-binding fragment may comprise an immunoglobulin Fc region. For example, the antigen-binding fragment may be composed of an amino acid sequence of the formula ALB, where A represents an immunoglobulin single variable domain, L represents an amino acid linker or is absent, and B represents a human immunoglobulin Fc region.

[0114] For example, the N-terminus of the Fc region of the immunoglobulin may be linked directly or indirectly to the C-terminus of the immunoglobulin single variable domain.

[0115] For example, the N-terminus of the Fc region of the immunoglobulin may be linked by a linker to the C-terminus of the immunoglobulin single variable domain.

[0116] In the present application, the immunoglobulin single variable domain may comprise antigen complementarity determining regions CDR1-3. For example, the CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 45 or 48. For example, the CDR2 may comprise the amino acid sequence shown in SEQ ID NO: 46. For example, the CDR3 may comprise the amino acid sequence shown in SEQ ID NO: 47. For example, the CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 45, the CDR2 may comprise the amino acid sequence shown in SEQ ID NO: 46, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO: 47. For example, the CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 48, the CDR2 may comprise the amino acid sequence shown in SEQ ID NO: 46, and the CDR3 may comprise the amino acid sequence shown in SEQ ID NO: 47.

[0117] In the present application, the linker may have a length of about 1 to about 20 amino acid residues. For example, the linker may not have a secondary or higher structure. For example, the linker may be a flexible linker. For example, the linker may be GGGGS, GS, or GAP. For example, the linker may include an amino acid sequence shown in any one of SEQ ID NOs: 42 to 44.

[0118] For example, the Fc region of the immunoglobulin can comprise an Fc region derived from an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. The human IgG1, IgG2, IgG3, or IgG4 constant region can have the amino acid sequence set forth in entries P01857, P01859, P01860, P01861, respectively, in the www.uniprot.org protein database.

[0119] For example, the Fc region of the immunoglobulin may be derived from a human. For example, the Fc region of the immunoglobulin may be derived from human IgG. For example, the Fc region of the immunoglobulin may be derived from human IgG1.

[0120] In the present application, the Fc region of the immunoglobulin may not have ADCC activity and / or may not have CDC activity. For example, the Fc region of the immunoglobulin may lose ADCC activity and / or CDC activity by mutation. For example, the Fc region of the immunoglobulin may include an amino acid sequence shown in any one of SEQ ID NOs: 7 to 9.

[0121] In the present application, the antigen-binding fragment may comprise a homodimer, which may be formed by interaction of the Fc regions of the immunoglobulins.

[0122] In the present application, the antigen-binding fragment is 1×10 -7 Less than M, 1×10 -8 Less than M, 1×10 -9 Less than M, 1×10 -10 Less than M, 1×10 -11 K less than M D It is capable of specifically binding to PD-L1 at

[0123] In the present application, the antigen-binding fragment is capable of specifically binding to human PD-L1 and blocking the interaction of PD-L1 with PD-1. In the present application, the antigen-binding fragment is capable of specifically binding to human PD-L1 and blocking the interaction of PD-L1 with CD80.

[0124] In the present application, the antigen-binding fragment can inhibit the growth of the tumor by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or more.

[0125] In the present application, the antigen-binding fragment may be resistant to base treatment and / or oxidative treatment. For example, after treating the antigen-binding fragment with a strong base (e.g., ammonium bicarbonate) for at least 8 hours, the antigen-binding fragment still retains the ability to specifically bind to PD-L1. As another example, after treating the antigen-binding fragment with an oxidizing agent (e.g., 1% hydrogen peroxide) for at least 2 hours, the antigen-binding fragment still retains the ability to specifically bind to PD-L1.

[0126] In the present application, the antigen-binding fragment may comprise the amino acid sequence shown in any one of SEQ ID NOs: 14-31.

[0127] In the present application, the antigen-binding fragment may include functional variants and fragments thereof, derivatives and fragments thereof, analogs and fragments thereof, and / or homologs and fragments thereof of the antigen-binding fragment.

[0128] The functional variant may be a polypeptide that has an amino acid sequence substantially identical to the naturally occurring sequence, or is encoded by a nucleotide sequence substantially identical to the naturally occurring sequence, and that can have one or more activities of the naturally occurring sequence. For example, the functional variant may be a sequence based on the amino acid sequence set forth in any one of SEQ ID NOs: 14-31, in which residues therein have been modified and which retains at least one intrinsic function (e.g., the ability to specifically bind to human PD-L1). For example, the modification may include addition, deletion, substitution, modification, exchange, and / or mutation of at least one amino acid residue.

[0129] The above-mentioned derivative may include a sequence that includes any substitution, mutation, modification, exchange, deletion and / or addition of one (or more) amino acid residues in the amino acid sequence shown in any one of SEQ ID NOs: 14 to 31 and retains at least one endogenous function.

[0130] The homologue may be an amino acid sequence that is at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the amino acid sequence shown in any one of SEQ ID NOs: 14 to 31. To determine sequence identity, sequence alignment can be performed, which can be performed by various methods known to those skilled in the art, such as BLAST, BLAST-2, ALIGN, NEEDLE, Megalign (DNASTAR) software, etc. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms necessary to achieve optimal alignment in the full-length sequences being compared. The analogue may include a mimic of a polypeptide that retains at least one endogenous function of the antigen-binding fragment described herein.

[0131] The antigen-binding fragments described herein can have deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent protein. Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphoteric properties of the residues, as long as the endogenous function is retained.

[0132] In the composition described herein, the concentration of the antigen-binding fragment may be about 1 mg / mL to about 220 mg / mL. For example, the concentration of the antigen-binding fragment may be about 20 mg / mL to about 220 mg / mL. For example, the concentration of the antigen-binding fragment may be about 30 mg / mL to about 220 mg / mL, about 40 mg / mL to about 220 mg / mL, about 50 mg / mL to about 220 mg / mL, about 30 mg / mL to about 200 mg / mL, about 40 mg / mL to about 200 mg / mL, or about 50 mg / mL to about 200 mg / mL.For example, the concentration of the antigen-binding fragment may be about 1 mg / mL to about 300 mg / mL. Specifically, for example, the concentration of the antigen-binding fragment is about 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL, 240 mg / mL, 250 mg / mL, 260 mg / mL, 270 mg / mL, 280 mg / mL, 290 mg / mL, 300 mg / mL, 310 mg / mL, 320 mg / mL, 330 mg / mL, 340 mg / mL, 350 mg / mL, 360 mg / mL, 370 mg / mL, 380 mg / mL, 390 mg / mL, 400 mg / mL, 410 mg / mL, 420 mg / mL, 430 mg / mL, 440 mg / mL, 450 mg / mL, 460 mg / mL, 470 mg / mL, 480 mg / mL, 490 mg / mL, 500 mg / mL, 510 mg / mL, 520 mg / mL, 530 mg / mL, 540 mg / mL, 550 mg / mL, 560 mg / mL, 570 mg / mL, 580 mg / mL, 590 mg / For example, the concentration of the antigen-binding fragment may be about 5 mg / mL to about 250 mg / mL, about 10 mg / mL to about 290 mg / mL, about 20 mg / mL to about 290 mg / mL, about 30 mg / mL to about 290 mg / mL, about 50 mg / mL to about 290 mg / mL, about 60 mg / mL to about 280 mg / mL, about 70 mg / mL to about 280 mg / mL, about 80 mg / mL to about 280 mg / mL, about 100 mg / mL to about 280 mg / mL, about 110 mg / mL to about 250 mg / mL, about 130 mg / mL to about 250 mg / mL, about 150 mg / mL to about 250 mg / mL, about 160 mg / mL to about 250 mg / mL, about 170 mg / mL to about 250 mg / mL, about 180 mg / mL to about 250 mg / mL, or about 250 mg / mL to about 250 mg / mL. The amount of the compound may be about 120 mg / mL to about 220 mg / mL, about 140 mg / mL to about 220 mg / mL, about 150 mg / mL to about 220 mg / mL, about 175 mg / mL to about 220 mg / mL, or about 190 mg / mL to about 220 mg / mL.

[0133] For example, the concentration of the antigen-binding fragment may be about 50 mg / mL to about 200 mg / mL. For example, the concentration of the antigen-binding fragment may be about 50 mg / mL, and for example, the concentration of the antigen-binding fragment may be about 200 mg / mL.

[0134] Excipients and other ingredients For example, the concentration of the excipient may be about 50 mM to about 500 mM. For example, the concentrations of the excipients are about 50 mM to about 500 mM, about 100 mM to about 500 mM, about 150 mM to about 500 mM, about 160 mM to about 500 mM, about 165 mM to about 500 mM, about 170 mM to about 500 mM, about 100 mM to about 300 mM, about 150 mM to about 300 mM, about 160 mM to about 300 mM, about 165 mM to about 300 mM, about 170 mM to about 300 mM, about 100 mM to about 275 mM, about 150 mM to about 275 mM, about 160 mM to about 275 mM, about 165 mM to about 275 mM, about 170 mM to about 275 mM, about 100 mM to about 220 mM, about 150 mM to about 220 mM, about 160 mM to about 220 mM, about 165 The concentration may be from about 170 mM to about 220 mM, or from about 170 mM to about 220 mM.

[0135] For example, the excipient may include proline, which may be at a concentration of about 50 mM to about 500 mM. For example, the proline concentration is about 50 mM to about 500 mM, about 100 mM to about 500 mM, about 150 mM to about 500 mM, about 160 mM to about 500 mM, about 165 mM to about 500 mM, about 170 mM to about 500 mM, 50 mM to about 390 mM, about 100 mM to about 390 mM, about 150 mM to about 390 mM, about 160 mM to about 390 mM, about 165 mM to about 390 mM, about 170 mM to about 390 mM, about 100 mM to about 300 mM, about 150 mM to about 300 mM, about 160 mM to about 300 mM, about 165 mM to about 300 mM, about 170 mM to about 300 mM, about 100 mM to about 275 mM, about 150 mM to about 275 mM, about 160 mM to about 275 mM, about 165 mM to about 275 mM, about 170 mM to about 275 mM, about 100 mM to about 250 mM, about 150 mM to about 250 mM, about 160 mM to about 250 mM, about 165 mM to about 250 mM, about 170 mM to about 250 mM, about 100 mM to about 220 mM, about 150 mM to about 220 mM, about 160 mM to about 220 mM, about 165 mM to about 220 mM, about 170 mM to about 220 mM. For example, the excipient may include proline, which may have a concentration of about 165 mM to about 275 mM. For example, the excipient may include proline, which may have a concentration of about 150 mM to about 250 mM. For example, the excipient may include proline, which may have a concentration of about 220 mM. For example, the excipient may include L-proline, which may be at a concentration of about 50 mM to about 500 mM.For example, the L-proline concentration is about 50 mM to about 500 mM, about 100 mM to about 500 mM, about 150 mM to about 500 mM, about 160 mM to about 500 mM, about 165 mM to about 500 mM, about 170 mM to about 500 mM, 50 mM to about 390 mM, about 100 mM to about 390 mM, about 150 mM to about 390 mM, about 160 mM to about 390 mM, about 165 mM to about 390 mM, about 170 mM to about 390 mM, about 100 mM to about 300 mM, about 150 mM to about 300 mM, about 160 mM to about 300 mM, about 165 mM to about 300 mM, about 170 mM to about 300 mM, about 100 mM to about 275 mM, about 150 mM to about 275 mM, about 160 mM to about 275 mM, about 165 mM to about 275 mM, about 170 mM to about 275 mM, about 100 mM to about 250 mM, about 150 mM to about 250 mM, about 160 mM to about 250 mM, about 165 mM to about 250 mM, about 170 mM to about 250 mM, about 100 mM to about 220 mM, about 150 mM to about 220 mM, about 160 mM to about 220 mM, about 165 mM to about 220 mM, about 170 mM to about 220 mM. For example, the excipient may include L-proline, the concentration of which may be about 165 mM to about 275 mM. For example, the excipient may include L-proline, the concentration of which may be about 150 mM to about 250 mM. For example, the excipient may include L-proline, the concentration of which may be about 220 mM.

[0136] For example, the excipient may include mannitol, which may be at a concentration of about 50 mM to about 500 mM. For example, the concentration of mannitol is about 50 mM to about 500 mM, about 100 mM to about 500 mM, about 150 mM to about 500 mM, about 160 mM to about 500 mM, about 165 mM to about 500 mM, about 170 mM to about 500 mM, 50 mM to about 390 mM, about 100 mM to about 390 mM, about 150 mM to about 390 mM, about 160 mM to about 390 mM, about 165 mM to about 390 mM, about 170 mM to about 390 mM, about 100 mM to about 300 mM, about 150 mM to about 300 mM, about 160 mM to about 300 mM, about 165 mM to about 300 mM, about 170 mM to about 300 mM, about 100 mM to about 275 mM, about 150 mM to about 275 mM, about 160 mM to about 275 The amount of the compound may be about 165 mM to about 275 mM, about 170 mM to about 275 mM, about 100 mM to about 250 mM, about 150 mM to about 250 mM, about 160 mM to about 250 mM, about 165 mM to about 250 mM, about 170 mM to about 250 mM, about 100 mM to about 220 mM, about 150 mM to about 220 mM, about 160 mM to about 220 mM, about 165 mM to about 220 mM, or about 170 mM to about 220 mM.

[0137] For example, the excipient may include sucrose, which may be at a concentration of about 50 mM to about 500 mM. For example, the concentration of sucrose is about 50 mM to about 500 mM, about 90 mM to about 500 mM, about 100 mM to about 500 mM, about 150 mM to about 500 mM, about 160 mM to about 500 mM, about 165 mM to about 500 mM, about 170 mM to about 500 mM, 50 mM to about 390 mM, about 100 mM to about 390 mM, about 150 mM to about 390 mM, about 160 mM to about 390 mM, about 165 mM to about 390 mM, about 170 mM to about 390 mM, about 100 mM to about 300 mM, about 150 mM to about 300 mM, about 160 mM to about 300 mM, about 165 mM to about 300 mM, about 170 mM to about 300 mM, about 100 mM to about 275 mM, about 150 mM to about 275 mM, about 160 mM to about 275 mM, about 165 mM to about 275 mM, about 170 mM to about 275 mM, about 100 mM to about 250 mM, about 150 mM to about 250 mM, about 160 mM to about 250 mM, about 165 mM to about 250 mM, about 170 mM to about 250 mM, about 100 mM to about 220 mM, about 150 mM to about 220 mM, about 160 mM to about 220 mM, about 165 mM to about 220 mM, about 170 mM to about 220 mM, about 30 mM to about 300 mM, about 40 mM to about 300 mM, about 50 mM to about 300 mM, about 60 mM to about 300 mM, about 80 mM to about 300 mM, about 30 mM to about 275 mM, about 40 mM to about 275 mM, about 50 mM to about 275 The amount of the compound may be about 30 mM to about 250 mM, about 40 mM to about 250 mM, about 50 mM to about 250 mM, about 30 mM to about 220 mM, about 40 mM to about 220 mM, or about 50 mM to about 220 mM.

[0138] For example, the excipient may include glycine, where the concentration of glycine may be from about 50 mM to about 500 mM. For example, the concentration of glycine is about 50 mM to about 500 mM, about 100 mM to about 500 mM, about 150 mM to about 500 mM, about 160 mM to about 500 mM, about 165 mM to about 500 mM, about 170 mM to about 500 mM, 50 mM to about 390 mM, about 100 mM to about 390 mM, about 150 mM to about 390 mM, about 160 mM to about 390 mM, about 165 mM to about 390 mM, about 170 mM to about 390 mM, about 100 mM to about 300 mM, about 150 mM to about 300 mM, about 160 mM to about 300 mM, about 165 mM to about 300 mM, about 170 mM to about 300 mM, about 100 mM to about 275 mM, about 150 mM to about 275 mM, about 160 mM to about 275 The amount of the compound may be about 165 mM to about 275 mM, about 170 mM to about 275 mM, about 100 mM to about 250 mM, about 150 mM to about 250 mM, about 160 mM to about 250 mM, about 165 mM to about 250 mM, about 170 mM to about 250 mM, about 100 mM to about 220 mM, about 150 mM to about 220 mM, about 160 mM to about 220 mM, about 165 mM to about 220 mM, or about 170 mM to about 220 mM.

[0139] For example, the excipient may include L-arginine hydrochloride, which may be in a concentration of about 50 mM to about 500 mM. For example, the concentration of L-arginine hydrochloride is about 50 mM to about 500 mM, about 100 mM to about 500 mM, about 150 mM to about 500 mM, about 160 mM to about 500 mM, about 165 mM to about 500 mM, about 170 mM to about 500 mM, 50 mM to about 390 mM, about 100 mM to about 390 mM, about 150 mM to about 390 mM, about 160 mM to about 390 mM, about 165 mM to about 390 mM, about 170 mM to about 390 mM, about 100 mM to about 300 mM, about 150 mM to about 300 mM, about 160 mM to about 300 mM, about 165 mM to about 300 mM, about 170 mM to about 300 mM, about 100 mM to about 275 mM, about 150 mM to about 275 mM, about 160 The amount of the compound may be about 100 mM to about 250 mM, about 150 mM to about 250 mM, about 160 mM to about 250 mM, about 165 mM to about 250 mM, about 170 mM to about 250 mM, about 100 mM to about 220 mM, about 150 mM to about 220 mM, about 160 mM to about 220 mM, about 165 mM to about 220 mM, or about 170 mM to about 220 mM.

[0140] For example, the excipient may include sorbitol, which may be at a concentration of about 50 mM to about 500 mM. For example, the concentration of sorbitol is about 100 mM to about 500 mM, about 130 mM to about 500 mM, about 150 mM to about 500 mM, about 200 mM to about 500 mM, about 80 mM to about 400 mM, about 100 mM to about 400 mM, about 150 mM to about 400 mM, about 180 mM to about 400 mM, about 100 mM to about 350 mM, about 130 mM to about 350 mM, about 150 mM to about 350 mM, about 170 mM to about 350 mM, about 190 mM to about 350 mM, about 75 mM to about 300 mM, about 100 mM to about 300 mM, about 120 mM to about 300 mM, about 150 mM to about 300 mM, about 170 mM to about 300 mM, about 180 mM to about 300 mM, about 200 mM to about 300 The amount of the compound may be about 100 mM to about 275 mM, about 120 mM to about 275 mM, about 150 mM to about 275 mM, about 180 mM to about 275 mM, about 100 mM to about 250 mM, about 130 mM to about 250 mM, about 150 mM to about 250 mM, about 160 mM to about 250 mM, about 170 mM to about 250 mM, about 180 mM to about 250 mM, about 100 mM to about 220 mM, about 120 mM to about 220 mM, about 140 mM to about 220 mM, about 160 mM to about 220 mM, or about 180 mM to about 220 mM.

[0141] For example, the excipient may include glycerol, which may be at a concentration of about 50 mM to about 500 mM. For example, the concentration of sorbitol is about 100 mM to about 500 mM, about 130 mM to about 500 mM, about 150 mM to about 500 mM, about 200 mM to about 500 mM, about 80 mM to about 400 mM, about 100 mM to about 400 mM, about 150 mM to about 400 mM, about 180 mM to about 400 mM, about 100 mM to about 350 mM, about 130 mM to about 350 mM, about 150 mM to about 350 mM, about 170 mM to about 350 mM, about 190 mM to about 350 mM, about 75 mM to about 300 mM, about 100 mM to about 300 mM, about 120 mM to about 300 mM, about 150 mM to about 300 mM, about 170 mM to about 300 mM, about 180 mM to about 300 mM, about 200 mM to about 300 The amount of the compound may be about 100 mM to about 275 mM, about 120 mM to about 275 mM, about 150 mM to about 275 mM, about 180 mM to about 275 mM, about 100 mM to about 250 mM, about 130 mM to about 250 mM, about 150 mM to about 250 mM, about 160 mM to about 250 mM, about 170 mM to about 250 mM, about 180 mM to about 250 mM, about 100 mM to about 220 mM, about 120 mM to about 220 mM, about 140 mM to about 220 mM, about 160 mM to about 220 mM, or about 180 mM to about 220 mM.

[0142] In the present application, the excipient may include a stabilizer. The stabilizer may be a component that helps maintain the structural integrity of the composition. For example, the stabilizer may help maintain the structural integrity of the composition during freezing, lyophilization, and / or storage. For example, the stabilizer may function as a penetrant that reduces denaturation of a protein (e.g., an antigen-binding fragment described in the present application).

[0143] The excipients described herein may further comprise an antioxidant which may be selected from one or more of methionine, phenol, benzophenol, ascorbic acid, glutathione, sodium thiosulfate, citric acid and nicotinamide. For example, the excipients described in the present application may have a concentration of about 0.1 mM to about 100 mM, about 0.5 mM to about 100 mM, about 1 mM to about 100 mM, about 2 mM to about 100 mM, about 5 mM to about 100 mM, about 0.2 mM to about 50 mM, about 1 mM to about 50 mM, about 3 mM to about 50 mM, about 4 mM to about 50 mM, about 5 mM to about 50 mM, about 0.1 mM to about 30 mM, about 0.3 mM to about 30 mM, about 0.6 mM to about 30 mM, about 1 mM to about 30 mM, about 2 mM to about 30 mM, about 5 mM to about 30 mM, about 0.1 mM to about 20 mM, about 0.5 mM to about 20 mM, about 1 mM to about 20 mM, about 2 mM to about 20 mM, about 0.1 mM to about 10 mM, about 0.2 mM to about 10 mM, about 0.5 mM to about 10 The composition may further comprise L-methionine, which may be about 0.5 mM, about 0.8 mM to about 10 mM, about 1 mM to about 10 mM, about 1.5 mM to about 10 mM, about 2 mM to about 10 mM, about 2.5 mM to about 10 mM, about 3 mM to about 10 mM, about 4 mM to about 10 mM, or about 5 mM to about 10 mM.

[0144] The excipient described in the present application may further comprise a chelating agent, and the chelating agent may be disodium EDTA, which may have a concentration of about 0.1 mM to about 10 mM, about 0.5 mM to about 10 mM, about 1 mM to about 10 mM, about 0.1 mM to about 5 mM, about 0.2 mM to about 5 mM, about 0.3 mM to about 5 mM, about 0.4 mM to about 5 mM, about 0.5 mM to about 5 mM, about 0.1 mM to about 3 mM, about 0.5 mM to about 3 mM, about 0.6 mM to about 3 mM, about 0.7 mM to about 3 mM, about 0.8 mM to about 3 mM, about 0.1 mM to about 2 mM, about 0.2 mM to about 2 mM, about 0.5 mM to about 2 mM, about 0.75 mM to about 2 mM, about 0.9 mM to about 2 mM, or about 1 mM to about 2 mM.

[0145] For example, the pH of the composition may be about 5.0 to about 7.5. For example, the pH of the composition may be about 5.9 to about 6.9. For example, the pH of the composition may be about 5.9 to about 6.8, about 5.9 to about 6.7, about 5.9 to about 6.6, about 5.9 to about 6.5, about 5.9 to about 6.4, about 6.0 to about 6.9, about 6.1 to about 6.9, about 6.2 to about 6.9, about 6.3 to about 6.9, about 6.4 to about 6.9, about 6.0 to about 6.5, about 6.1 to about 6.5, about 6.2 to about 6.5, about 6.3 to about 6.5, or about 6.4 to about 6.5. For example, the pH of the composition may be about 6.3 to about 6.5. For example, the pH of the composition may be about 6.4.

[0146] For example, the composition may include one or more buffer components selected from the group consisting of sodium acetate-acetic acid, histidine-acetic acid, L-histidine-L-histidine hydrochloride, sodium phosphate, and citrate-sodium hydroxide.

[0147] For example, the concentration of the buffer component may be about 5 mM to about 50 mM. For example, the concentration of the buffer component may be about 5 mM to about 50 mM, about 5 mM to about 45 mM, about 5 mM to about 40 mM, about 5 mM to about 35 mM, about 5 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 5 mM to about 50 mM, or about 5 mM to about 30 mM. For example, the concentration of the buffer component may be about 10 mM to about 50 mM. For example, the concentration of the buffer component may be about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 20 mM to about 50 mM, or about 20 mM to about 30 mM.

[0148] For example, the buffer component may contain sodium acetate-acetic acid, which may have a concentration of about 10 mM to about 50 mM. For example, the concentration of the sodium acetate-acetic acid may be about 5 mM to about 50 mM, about 5 mM to about 45 mM, about 5 mM to about 40 mM, about 5 mM to about 35 mM, about 5 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 20 mM to about 50 mM, or about 20 mM to about 30 mM. For example, the buffer component may contain sodium acetate-acetic acid, which may have a concentration of about 5 mM to about 35 mM. For example, the buffer component may contain sodium acetate-acetic acid, which may have a concentration of about 10 mM to about 30 mM. For example, the buffer components may include sodium acetate-acetic acid, which may be at a concentration of about 20 mM.

[0149] For example, the buffer component may contain L-histidine-L-histidine hydrochloride, which may have a concentration of about 10 mM to about 50 mM. For example, the concentration of the L-histidine-L-histidine hydrochloride may be about 5 mM to about 50 mM, about 5 mM to about 45 mM, about 5 mM to about 40 mM, about 5 mM to about 35 mM, about 5 mM to about 30 mM, about 5 mM to about 25 mM, about 5 mM to about 20 mM, about 10 mM to about 50 mM, about 10 mM to about 45 mM, about 10 mM to about 40 mM, about 10 mM to about 35 mM, about 10 mM to about 30 mM, about 10 mM to about 25 mM, about 10 mM to about 20 mM, about 20 mM to about 50 mM, or about 20 mM to about 30 mM. For example, the buffer components may include L-histidine-L-histidine hydrochloride, which may have a concentration of about 5 mM to about 35 mM. For example, the buffer components may include L-histidine-L-histidine hydrochloride, which may have a concentration of about 10 mM to about 30 mM. For example, the buffer components may include L-histidine-L-histidine hydrochloride, which may have a concentration of about 20 mM.

[0150] For example, the composition may include one or more surfactants, which may be selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188. The present application uses mg / mL or % (w / v) to express surfactant concentrations, and it should be understood that one of skill in the art can convert between the two, e.g., converting 0.02% surfactant to 0.2 mg / mL.

[0151] For example, the concentration of the surfactant may be about 0 mg / mL to about 0.8 mg / mL.For example, the concentration of the surfactant may be about 0.05 mg / mL to about 0.8 mg / mL. For example, the concentration of the surfactant may be about 0 mg / mL to about 0.8 mg / mL, about 0.1 mg / mL to about 0.8 mg / mL, about 0.2 mg / mL to about 0.8 mg / mL, about 0.3 mg / mL to about 0.8 mg / mL, about 0.1 mg / mL to about 0.3 mg / mL, about 0.1 mg / mL to about 0.2 mg / mL, about 0.05 mg / mL to about 0.3 mg / mL, about 0 mg / mL to about 0.4 mg / mL, about 0.05 mg / mL to about 0.4 mg / mL, about 0.05 mg / mL to about 0.2 mg / mL, about 0.2 mg / mL to about 0.4 mg / mL, or about 0 mg / mL to about 0.2 mg / mL.

[0152] For example, the surfactant may include polysorbate 20, which may have a concentration of about 0 mg / mL to about 0.8 mg / mL. For example, the concentration of the polysorbate 20 may be about 0.05 mg / mL to about 0.8 mg / mL. For example, the concentration of the polysorbate 20 may be about 0 mg / mL to about 0.8 mg / mL, about 0.1 mg / mL to about 0.8 mg / mL, about 0.2 mg / mL to about 0.8 mg / mL, about 0.3 mg / mL to about 0.8 mg / mL, about 0.1 mg / mL to about 0.3 mg / mL, about 0.1 mg / mL to about 0.2 mg / mL, about 0.05 mg / mL to about 0.3 mg / mL, about 0 mg / mL to about 0.4 mg / mL, about 0.05 mg / mL to about 0.4 mg / mL, about 0.05 mg / mL to about 0.2 mg / mL, about 0.2 mg / mL to about 0.4 mg / mL, or about 0 mg / mL to about 0.2 mg / mL. For example, the surfactant may contain polysorbate 20, which may have a concentration of about 0.2 mg / mL to about 0.4 mg / mL. For example, the surfactant may include polysorbate 20, which may have a concentration of about 0.2 mg / mL. For example, the surfactant may include polysorbate 20, which may have a concentration of about 0.4 mg / mL.

[0153] For example, the surfactant may include polysorbate 80, which may have a concentration of about 0 mg / mL to about 0.8 mg / mL. For example, the concentration of the polysorbate 80 may be about 0.05 mg / mL to about 0.8 mg / mL. For example, the concentration of the polysorbate 80 may be about 0 mg / mL to about 0.8 mg / mL, about 0.1 mg / mL to about 0.8 mg / mL, about 0.2 mg / mL to about 0.8 mg / mL, about 0.3 mg / mL to about 0.8 mg / mL, about 0.1 mg / mL to about 0.3 mg / mL, about 0.1 mg / mL to about 0.2 mg / mL, about 0.05 mg / mL to about 0.3 mg / mL, about 0 mg / mL to about 0.4 mg / mL, about 0.05 mg / mL to about 0.4 mg / mL, about 0.05 mg / mL to about 0.2 mg / mL, about 0.2 mg / mL to about 0.4 mg / mL, or about 0 mg / mL to about 0.2 mg / mL. For example, the surfactant may include polysorbate 80, which may have a concentration of about 0.2 mg / mL to about 0.4 mg / mL. For example, the surfactant may include polysorbate 80, which may have a concentration of about 0.2 mg / mL. For example, the surfactant may include polysorbate 80, which may have a concentration of about 0.4 mg / mL.

[0154] For example, the surfactant may comprise poloxamer 188, which may have a concentration of about 0.05 mg / mL to about 5 mg / mL, about 0.08 mg / mL to about 5 mg / mL, about 0.1 mg / mL to about 5 mg / mL, about 0.2 mg / mL to about 5 mg / mL, about 0.05 mg / mL to about 3 mg / mL, about 0.1 mg / mL to about 3 mg / mL, about 0.15 mg / mL to about 3 mg / mL, about 0.2 mg / mL to about 3 mg / mL, about 0.05 mg / mL to about 2 mg / mL, about 0.075 mg / mL to about 2 mg / mL, about 0.1 mg / mL to about 2 mg / mL, about 0.15 mg / mL to about 2 mg / mL, about 0.2 mg / mL to about 2 mg / mL, about 0.3 mg / mL to about 2 mg / mL, or about 0.5 mg / mL to about 2 mg / mL.

[0155] In the present application, the composition may comprise the antigen-binding fragment described in the present application, the excipient described in the present application, and may have a pH of about 5.9 to about 6.9. For example, the composition may comprise the antigen-binding fragment described in the present application, the excipient described in the present application, and the buffer component described in the present application, and may have a pH of about 5.9 to about 6.9. For example, the composition may comprise the antigen-binding fragment described in the present application, the excipient described in the present application, the buffer component described in the present application, and the surfactant described in the present application, and may have a pH of about 5.9 to about 6.9.

[0156] In the present application, the composition may comprise the antigen-binding fragment described herein, proline, and may have a pH of about 5.9 to about 6.9. For example, the composition may comprise the antigen-binding fragment described herein, proline, and sodium acetate-acetic acid, and may have a pH of about 5.9 to about 6.9. For example, the composition may comprise the antigen-binding fragment described herein, proline, sodium acetate-acetic acid, and polysorbate 20, and may have a pH of about 5.9 to about 6.9. For example, the composition may comprise the antigen-binding fragment described herein, proline, sodium acetate-acetic acid, and polysorbate 20, and may have a pH of about 6.3 to about 6.5.

[0157] For example, the composition may contain 1) about 20 mg / mL to about 220 mg / mL of the antigen-binding fragment described in the present application, 2) about 10 mM to about 50 mM sodium acetate-acetic acid, 3) about 50 mM to about 500 mM proline, and 4) about 0.05 mg / mL to about 0.8 mg / mL of polysorbate 20, and may have a pH of about 5.9 to about 6.9.

[0158] For example, the composition may be comprised of 1) about 20 mg / mL to about 220 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 50 mM sodium acetate-acetic acid, 3) about 50 mM to about 500 mM proline, and 4) about 0.05 mg / mL to about 0.8 mg / mL of polysorbate 20, and the pH of the composition may be about 5.9 to about 6.9.

[0159] For example, the composition may contain 1) about 20 mg / mL to about 220 mg / mL of the antigen-binding fragment described in the present application, 2) about 5 mM to about 35 mM sodium acetate-acetic acid, 3) about 50 mM to about 390 mM proline, and 4) about 0 mg / mL to about 0.4 mg / mL of polysorbate 20, and may have a pH of about 5.9 to about 6.9.

[0160] For example, the composition may be composed of 1) about 20 mg / mL to about 220 mg / mL of the antigen-binding fragment described herein, 2) about 5 mM to about 35 mM sodium acetate-acetic acid, 3) about 50 mM to about 390 mM proline, and 4) about 0 mg / mL to about 0.4 mg / mL of polysorbate 20, and the pH of the composition may be about 5.9 to about 6.9.

[0161] For example, the composition may contain 1) about 50 mg / mL to about 200 mg / mL of the antigen-binding fragment described in the present application, 2) about 10 mM to about 30 mM sodium acetate-acetic acid, 3) about 150 mM to about 250 mM proline, and 4) about 0.2 mg / mL to about 0.4 mg / mL of polysorbate 20, and may have a pH of about 6.3 to about 6.5.

[0162] For example, the composition may be composed of 1) about 50 mg / mL to about 200 mg / mL of the antigen-binding fragment described herein, 2) about 10 mM to about 30 mM sodium acetate-acetic acid, 3) about 150 mM to about 250 mM proline, and 4) about 0.2 mg / mL to about 0.4 mg / mL of polysorbate 20, and the pH of the composition may be about 6.3 to about 6.5.

[0163] For example, the composition may include 1) about 200 mg / mL of an antigen-binding fragment described herein, 2) about 20 mM sodium acetate-acetic acid, 3) about 220 mM proline, 4) about 0.2 mg / mL polysorbate 20, and may have a pH of about 6.4.

[0164] For example, the composition may be comprised of 1) about 200 mg / mL of an antigen-binding fragment described herein, 2) about 20 mM sodium acetate-acetic acid, 3) about 220 mM proline, and 4) about 0.2 mg / mL polysorbate 20, and may have a pH of about 6.4.

[0165] In the present application, the composition can be used for injection. In the present application, the composition can be used for subcutaneous injection. For example, the composition can be used for subcutaneous injection, intramuscular injection, intravenous injection, parenteral administration or intravenous infusion.

[0166] In the present application, the composition may be a formulation. For example, the composition may be a liquid formulation.

[0167] In another aspect, the present application provides a reagent kit comprising a composition described herein and a container that contains the composition described herein.

[0168] For example, the container may include a vial (e.g., a glass bottle), an ampoule, a syringe, an injection pen, and / or an intravenous bag. In the present application, the container may be sterile. For example, the container may include a glass bottle.

[0169] For example, the reagent kit may include a delivery device capable of administering the compositions described herein. For example, the delivery device may include a vial, an ampoule, a syringe, an injection pen, and / or an intravenous bag.

[0170] In the present application, the volume of the composition in the container may be about 0.5 mL to about 5.0 mL. For example, the volume of the composition in the container may be about 0.5 mL to about 1.5 mL. For example, the volume of the composition may be 1.5 mL. For example, the volume of the composition may be 1.0 mL. In the present application, the concentration of the antigen-binding fragment in the container may be about 200 mg / 1.0 mL / vial, or about 300 mg / 1.5 mL / vial.

[0171] In another aspect, the present application provides the use of a composition described herein, and / or a reagent kit described herein, in the manufacture of a medicament for preventing, alleviating and / or treating a tumor.

[0172] The present application provides a composition as described herein, and / or a reagent kit as described herein, for preventing, alleviating and / or treating tumors.

[0173] The present application provides a method for preventing, alleviating, and / or treating a tumor, comprising administering to a subject in need thereof a composition described herein, and / or a reagent kit described herein.

[0174] In the present application, the tumor may include a PD-L1 positive tumor. For example, the tumor may include a malignant solid tumor.

[0175] In the present application, the tumor may include a microsatellite unstable (MSI-H) / mismatch repair deficient (dMMR) tumor (e.g., a MSI-H / dMMR pan-tumor solid tumor). For example, the tumor may include advanced microsatellite unstable (MSI-H) / mismatch repair deficient (dMMR) colorectal cancer or gastric cancer. For example, the tumor may include biliary tract cancer that cannot be surgically resected or metastasized, and / or untreated unresectable or metastatic gastric or gastroesophageal junction adenocarcinoma.

[0176] In another aspect, the present application further provides the use of a composition described herein, and / or a reagent kit described herein, in the manufacture of a medicament for preventing, alleviating and / or treating an infectious disease. The present application provides a composition as described herein, and / or a reagent kit as described herein, for preventing, alleviating and / or treating an infectious disease.

[0177] The present application provides a method for preventing, alleviating and / or treating an infectious disease, comprising administering to a subject in need thereof a composition described herein, and / or a reagent kit described herein.

[0178] In the present application, the infectious disease includes diseases resulting from viral, bacterial, fungal and / or parasitic infections.

[0179] The present application further provides a method for producing the compositions described herein and / or the reagent kits described herein. The method can include mixing any relevant components necessary to produce the compositions described herein in any order. For example, the method can include producing a premix (or premixed solution) of the excipients, the buffer components, and / or the surfactants. For example, the method can include forming a buffer system. The buffer system can include a buffer component described herein. The method can include removing particulate matter (e.g., by filtration).

[0180] Without being limited to any theory, the following examples are merely intended to illustrate the formulations, manufacturing methods, and uses of the present application, and are not intended to limit the scope of the present invention. EXAMPLES

[0181] Detection items and methods: (1) Intrinsic fluorescence spectrum: Fluorescence spectra of endogenous fluorescent amino acids (tryptophan, tyrosine, phenylalanine) in polar and non-polar environments in protein molecules are used. When the fluorescence spectrum is red-shifted, i.e., when the maximum emission wavelength changes, the conformation of the higher-order structure of the protein, i.e., the interconversion of the higher-order structure, is indirectly estimated. The sample is diluted to 40 mg / mL and a high-throughput intrinsic fluorescence spectrometer optim1000 (Pall) is used, the test temperature range is 20 to 95 °C, and the test interval is 1 °C. The polymerization tendency of the sample is judged using the SLS (static scattered light) result at a wavelength of 473 nm, and the denaturation temperature is the value measured at BCM (centroid wavelength) and is calculated and evaluated as the Tm value (half denaturation temperature) from the formula.

[0182] (2) SEC-HPLC (molecular exclusion chromatography): Refer to the 2015 edition of the Chinese Pharmacopoeia, Part III, General Provisions 0514 Molecular Exclusion Chromatography, used to detect the monomer content of the product, and polymers and fragments. The column is 7.8*300 mM, 5 μm TOSOH G3000, the mobile phase is 20 mM Na2HPO4, 300 mM NaCl, and the detection wavelength is 280 nm.

[0183] (3) WCX-HPLC (Weak Cation Exchange Chromatography): Refer to the 2015 edition of the Chinese Pharmacopoeia, Part III, General Rules 0512 High Performance Liquid Chromatography, used to detect the content of main components, acidic components and basic components of the product. The column is 4×250 mM, 10 μm Propac WCX-10, the mobile phase is 10 mM Na2HPO4, 10 mM Na2HPO4+500 mM NaCl, and the detection wavelength is 280 nm.

[0184] (4) Binding and blocking activity: refer to the test methods described in Examples 5.2 and 5.3 of WO2017 / 020802A1, respectively.

[0185] (5) Viscosity: The viscosity is measured using the American Rheosense portable microviscometer microVISC in automatic mode.

[0186] (6) Osmotic pressure: Measured by molar osmometry.

[0187] Equipment: Osmometer, Reagents: 300 mosm / kg calibration solution, 900 mosm / kg calibration solution, Sodium chloride Consumables: Centrifugal separator tubes, dust-free paper, etc.

[0188] Experimental procedure: take 100 μL and add it to the sample tube, ensure that no air bubbles are generated in the sample tube, wipe the sample probe and secondary needle with dust-free paper, then place the sample tube on the detection probe, lower the measurement probe, place the sample tube in the cold trap, press the "Enter" key to measure. Measure the sample twice in parallel. Zero calibration: between ±2 points, 300-point calibration: between ±4 points, 900-point calibration: between ±8 points.

[0189] (7) CE-SDS purity: After a sample is denatured (non-reducing conditions) or denatured and reduced (reducing conditions), it is placed in a capillary electrophoresis device and subjected to electrophoretic analysis, and calculated by the area normalization method.

[0190] (8) Protein content: Refer to Method 6 of General Provisions 0731, Determination of Protein Content, Part III of the 2015 Edition of the Chinese Pharmacopoeia, and use the Nanodrop 2000 microultraviolet spectrophotometer to calculate the protein content based on the absorbance of protein at a wavelength of 280 nm and the theoretical extinction coefficient of protein.

[0191] (9) Turbidity measurement: Measure the absorbance of the sample at 340 nm using an Agilent 8453 spectrophotometer.

[0192] (10) Insoluble Particles: Measure the number of particles ≧10 μm and ≧25 μm in the sample using an AccuSizer 780SIS Insoluble Particle Analyzer.

[0193] (11) Dynamic Light Scattering (DLS): The average particle size (Z.average.Diameter) and particle distribution index (PDI) of the sample are measured using a PUNK type particle size analyzer.

[0194] Production of antigen-binding proteins: The antigen-binding protein comprises an immunoglobulin single variable domain capable of specifically binding to PD-L1, and the immunoglobulin single variable domain may comprise the amino acid sequence shown in SEQ ID NO:2. Hereinafter, this antigen-binding protein is simply referred to as "PD-L1 monoclonal antibody". The PD-L1 monoclonal antibody is obtained by an artificial synthesis method of an amino acid sequence. The PD-L1 monoclonal antibody comprises the amino acid sequence shown in any one of SEQ ID NOs:14 to 31, in particular the amino acid sequence shown in SEQ ID NO:21.

[0195] Proline in the embodiments includes L-proline.

[0196] Example 1 The following formulations were obtained according to Table 1:

[0197] [Table 1]

[0198] For the formulations in Table 1, screening detection was carried out according to Table 2.

[0199] [Table 2]

[0200] 1) Detection by intrinsic fluorescence spectrum The results of detecting the Tm values ​​and polymerization tendencies corresponding to formulations 1 to 19 are shown in FIG. 1 and FIG. 2, respectively.

[0201] The results in Figure 1 show that the structures of formulations 1-4 and 12 are more stable, and the results in Figure 2 show that formulations 1-4 and 10 are less likely to form polymers. In this detection, the stability of formulations 1-4 is very good, that is, the use of a sodium acetate-acetic acid buffer system leads to further improvement of stability.

[0202] Comparing Formulations 1-2 and Formulations 3-4, it was found that a relatively low pH leads to further improvement in stability.

[0203] 2) Detection of main peak content by SEC-HPLC The results of detecting the main peak content and polymer content by SEC-HPLC corresponding to Formulations 1 to 19 are shown in FIG. 3 and FIG. 4, respectively.

[0204] The results showed that under high temperature conditions for 28 days, apart from formulation 11, the purity of PD-L1 monoclonal antibody was above 97% in the other formulations, and a buffer system was required.

[0205] Comparing formulations 1, 3, 5, 7 and formulations 2, 4, 6, 8, respectively, it was found that a lower pH was associated with further improved formulation stability.

[0206] The stability of Formulation 1 and Formulation 3 (pH 6.5 sodium acetate-acetic acid buffer system) and Formulation 5 and Formulation 7 (pH 6.5 sodium phosphate buffer system) were compared. As a result, it was found that the inclusion of L-proline leads to further improvement in stability.

[0207] As a result of comparing formulations 6 and 8, and 13 and 14, respectively, it was found that the inclusion of L-arginine or L-proline led to further improvement in stability.

[0208] By comparing formulations 2, 6 and 10, it was found that the use of a sodium acetate-acetic acid buffer system further improved stability.

[0209] Comparing the results for formulations 14 and 16, formulations 13 and 17, and formulations 6 and 18, respectively, it was found that the PD-L1 monoclonal antibody contents of 50 mg / mL and 200 mg / mL did not appear to affect the stability of the formulations.

[0210] 3) Detection of acidic component content by WCX-HPLC The detection results of the acidic component content by WCX-HPLC are shown in Figure 5, which can evaluate the change trend of the acidic components in the formulations (the acidic components may include components resulting from the degradation of PD-L1 monoclonal antibody under high temperature conditions).

[0211] Comparing formulations 1, 3, 5, 7 and formulations 2, 4, 6, 8, respectively, it was found that a lower pH led to a further reduction in the acidic components of the formulations.

[0212] As a result of comparing the acidic components of Formulations 1 and 5, Formulations 3 and 7, and Formulations 2, 6, and 10, respectively, it was found that the sodium acetate-acetic acid buffer system led to a further reduction in the acidic components of the formulations.

[0213] By comparing the acidic components of formulations 6, 8, and 13-14, it was found that the inclusion of L-proline led to a further reduction in the acidic components of the formulations.

[0214] Comparing formulations 14 and 16, formulations 13 and 17, and formulations 6 and 18, respectively, it was found that the PD-L1 monoclonal antibody contents of 50 mg / mL and 200 mg / mL did not appear to affect the level of acidic content of the formulations.

[0215] 4) Viscosity detection The viscosity detection results are shown in Figure 6. In addition to formulation 8, the viscosities of the other formulations all met the viscosity requirements for subcutaneous injection.

[0216] Comparing formulations 14 and 16, and formulations 13 and 17, respectively, it was found that the PD-L1 monoclonal antibody content of 200 mg / mL increased viscosity compared to the 50 mg / mL content, but this did not affect subcutaneous injection administration.

[0217] 5) Liveness detection Formulations 1 and 3 were selected and activity measurements were carried out at high temperatures.

[0218] The results of the binding activity and blocking activity of both are shown in Figure 7, Figure 8 and Table 3, respectively. A to D in Figures 7 to 8 show the results of Formulation 1 on day 0, Formulation 3 on day 0, Formulation 1 on day 28, and Formulation 3 on day 28. As a result of the formulation, it was found that Formulation 1 and Formulation 3 had no significant difference in activity at high temperature for 28 days, and no significant difference compared to day 0.

[0219] [Table 3]

[0220] Example 2 Based on the screening of the formulation in Example 1, the influence of each factor on the formulation performance was further considered, and the formulation study results are shown in Table 4.

[0221] [Table 4]

[0222] After 14 and 28 days of accelerated incubation at elevated temperature (40±2°C), the purity of the PD-L1 monoclonal antibody was evaluated by SEC-HPLC and the changes in its acid-base components by WCX-HPLC, and the viscosity of the sample at day 0 was measured. Other additional experimental considerations and evaluation items of the formulation composition are listed in Table 5.

[0223] [Table 5]

[0224] The detection results by SEC-HPLC are shown in Table 6.

[0225] [Table 6]

[0226] The detection results by WCX-HPLC are shown in Table 7.

[0227] [Table 7]

[0228] (1) Study on the effect of surfactant type on formulation stability At a PD-L1 monoclonal antibody content of 200 mg / mL, 20 mM sodium acetate-acetic acid was used as the buffer, 220 mM proline (L-proline) was used as the stabilizer, and 0.02% polysorbate 20, polysorbate 80, and poloxamer P188 were selected as surfactants. After 0 days, 14 days, and 28 days of accelerated incubation, the target protein was detected by SEC-HPLC and WCX-HPLC methods, respectively, and the effects of different types of surfactants on the formulation stability were discussed, as shown in formulations 19A, 20A, and 21A. The study results showed that polysorbate 20, polysorbate 80, and poloxamer P188 could be used as surfactants for PD-L1 monoclonal antibody formulations.

[0229] (2) Examination of the effect of stabilizer type on formulation stability The content of PD-L1 monoclonal antibody was 200 mg / mL, 20 mM sodium acetate-acetic acid was the buffer, 0.02% polysorbate 20 was the surfactant, the pH was 6.4, and 220 mM L-proline, 90 mg / mL sucrose, 250 mM glycine, 150 mM L-arginine hydrochloride, and 150 mM sodium chloride were selected as stabilizers, respectively, and the target protein was detected by SEC-HPLC and WCX-HPLC methods after 0 days, 14 days, and 28 days of accelerated incubation, respectively, and the effects of different types of stabilizers on the formulation stability were investigated, as shown in formulation formulations 19A, 26A, 27A, 28A, and 29A. The results showed that L-proline, sucrose, glycine, L-arginine hydrochloride, and sodium chloride could be used as stabilizers for PD-L1 monoclonal antibody formulations.

[0230] (3) Effect of the type of buffer ingredient on formulation stability The PD-L1 monoclonal antibody content was 200 mg / mL, and 220 mM L-proline was used as the stabilizer, 0.02% polysorbate 20 was used as the surfactant, pH was 6.4, and 20 mM sodium acetate-acetic acid, L-histidine-L-histidine hydrochloride, sodium phosphate, and citric acid-sodium hydroxide buffer solutions were selected, respectively, and the target protein was detected by SEC-HPLC and WCX-HPLC methods after 0 days, 14 days, and 28 days of accelerated incubation, respectively, and the effects of different buffer components on the formulation stability were examined, as shown in formulations 19A, 22A, 23A, 24A, and 25A. The results showed that sodium acetate-acetic acid, L-histidine-acetic acid, L-histidine-L-histidine hydrochloride, sodium phosphate, and citric acid-sodium hydroxide buffer solutions could all be used as buffer components for PD-L1 monoclonal antibody formulations. Specifically, there was no significant difference in the detection results of each buffer system formulation by SEC-HPLC, and the results of WCX-HPLC showed that after 28 days of accelerated incubation at high temperature, the main component contents of sodium phosphate and citric acid-sodium hydroxide system formulations were slightly lower, while the main component contents of several other buffer systems were close.

[0231] (4) Effect of PD-L1 monoclonal antibody concentration on formulation stability The results of the study of formulations 33A, 34A, and 19A showed that formulations containing PD-L1 monoclonal antibody at concentrations ranging from 20 mg / mL to 220 mg / mL met the formulation stability requirements under certain buffer, stabilizer, surfactant, and / or pH conditions.

[0232] The viscosity detection results on day 0 are shown in Table 8. As can be seen from the results, except for formulation 33A with a low concentration of PD-L1 monoclonal antibody, which has a relatively low viscosity, the difference in viscosity of the other formulations was not obvious and was generally within the acceptable range. This means that the formulation containing PD-L1 monoclonal antibody with a relatively high concentration (e.g., 220 mg / mL) has viscosity properties suitable for drug formation (e.g., suitable for use in injection, e.g., suitable for use in subcutaneous injection). The composition described herein has a suitable viscosity, which is conducive to convenient (e.g., subcutaneous injection) and rapid administration (e.g., reducing the number of injections and greatly shortening the administration time), while at the same time improving patient compliance and accessibility, and improving the quality of life of patients.

[0233] [Table 8]

[0234] Example 3 The formulation of the formulation was further optimized according to the results of Examples 1 and 2, and the pH, buffer system, stabilizer and surfactant of the formulation were further screened, respectively.

[0235] 1. pH screening The stability of different formulations containing 200±12 mg / mL PD-L1 monoclonal antibody was investigated at high temperatures of 40±2℃ in the pH range of 5.0-7.5. Differences in protein purity were detected by SEC-HPLC and WCX-HPLC, and differences in protein particle size and homogeneity were detected by insoluble microparticles and DLS. The specific formulation compositions and detection results are summarized in Table 9.

[0236] [Table 9]

[0237] The results of the detection by SEC-HPLC showed that the monomers were obviously decreased under the conditions of pH 5.0 and pH 7.5, and the results of the detection by WCX-HPLC showed that the main components were obviously decreased under the condition of pH 7.5. The results of the detection of insoluble particles showed that there were few insoluble particles under the condition of relatively low pH, and the number of insoluble particles tended to decrease further as the pH increased. The PDI index reflects the homogeneity of the solution, and the smaller this value, the higher the homogeneity. The results showed that the homogeneity of the solution was most favorable when the pH was 6.4, and as the pH increased, the PDI value of the solution tended to decrease first and then increase.

[0238] Based on the above results, the stability of PD-L1 monoclonal antibody in the pH range of 6.1-6.7 was further investigated. Different formulations containing 200±12 mg / mL PD-L1 monoclonal antibody were placed under high temperature conditions at 40±2℃, and the differences in protein purity were detected by SEC-HPLC and WCX-HPLC, and the turbidity differences were detected by comparing the OD values ​​at 340 nm. The specific formulation compositions and detection results are summarized in Table 10.

[0239] [Table 10]

[0240] In the range of pH 6.1-6.7, the SEC-HPLC results showed that the difference between different formulations was not obvious, and the WCX-HPLC results showed that as the pH value increased, the content of the main components tended to decrease slightly, and the OD 340 nm The turbidity measured by the method tended to decrease first and then increase as the pH increased.

[0241] 2. Selection of stabilizers and buffer systems Sodium acetate-acetic acid, L-histidine-L-histidine hydrochloride and citrate buffer systems were selected to be combined with different stabilizers and to further study the effects of the stabilizers.The stabilizers evaluated included the sugars sucrose, trehalose and maltose, the amino acids L-proline, L-arginine hydrochloride and methionine, and the alcohols mannitol, sorbitol and glycerol, and were simultaneously compared with sodium chloride as a control.

[0242] The difference in protein purity was detected by SEC-HPLC and WCX-HPLC, and the difference in particle size and uniformity in different formulations was detected by insoluble microparticles and DLS. The specific formulation composition and detection results are shown in Tables 11 to 13.

[0243] [Table 11]

[0244] After 28 days of accelerated heating at high temperature, visual observation of the appearance showed that except for the color of the formulation containing the maltose composition turning yellow, no obvious difference was observed in the appearance of the other formulations.

[0245] The results of detection by SEC-HPLC showed that the content of monomer detected in the formulation containing maltose was relatively low, and the difference between the monomer detected in the other formulations was not obvious, 98.3%-98.6%; the results of detection by WCX-HPLC showed that the peak shape detected in the formulation containing maltose was deformed, and the difference between the main components detected in the other formulations was not obvious, 83.3%-85.5%. The results of detection of insoluble fine particles showed that there was no obvious difference between different stabilizers. As can be seen from the DLS detection results, the results of the average hydration diameter (Z.Av.Diameter) showed that the hydration diameter of the formulation containing sodium chloride was relatively large, and the average particle size of the other formulations was relatively small; the results of the distribution coefficient (PDI) showed that there was no obvious difference between the other formulations, except for the formulation containing trehalose, maltose and sodium chloride, whose values ​​were >0.3, and showed high uniformity. The viscosity detection results showed that the formulations containing 50 mg / mL sucrose, 50 mg / mL mannitol, and 90 mg / mL trehalose had a viscosity of >20 cp, while the other formulations all had a viscosity of <20 cp, and the formulation containing methionine as an antioxidant had a slightly lower viscosity than the formulation without it.

[0246] [Table 12]

[0247] After 28 days of accelerated high temperature, visual observation of the appearance showed that except for the color of the formulation containing the maltose composition turning yellow, no obvious difference was observed in the appearance of the other sets.

[0248] The results of detection by SEC-HPLC showed that the content of monomer detected in the formulation containing maltose was relatively low, and the difference between the detected monomers in other formulations was not obvious, being 98.6%-98.9%. The results of detection by WCX-HPLC showed that the peak shape detected in the formulation containing maltose was deformed, and the difference between the detected main components in other formulations was not obvious, being 83.2%-84.9%. The results of detection of insoluble fine particles showed that there was no obvious difference between different stabilizers. The results of Z.Av.Diameter in DLS detection showed that the hydrated diameter of the formulation containing sodium chloride was relatively large, and the average particle size of the other formulations was small. The results of PDI value showed that the difference between other formulations was not obvious, except for the PDI value of the formulation containing trehalose and maltose, which was >0.3, and the homogeneity was high.

[0249] [Table 13]

[0250] The detection results of insoluble microparticles show that there is no obvious difference between different stabilizers. The detection results of DLS show that, except for the formulation containing L-arginine hydrochloride and other buffer systems, the formulations using other stabilizers have larger hydration diameters in citrate buffer system compared with the other two buffer systems mentioned above, according to the Z.Av.Diameter value, which is unfavorable to the structural stability of protein. The PDI values ​​detected by different stabilizers are different in each formulation, and the PDI values ​​of the formulation containing L-arginine hydrochloride and mannitol are relatively small and highly uniform.

[0251] 3. Screening of surfactants The stability of PD-L1 monoclonal antibody in different formulations was investigated, especially the effect of different surfactants on protein stability. The purity difference of protein SEC-HPLC and WCX-HPLC was detected by high temperature 40±2℃ accelerated experiment, insoluble particles were detected by shaking at 25±2℃, 150 rpm for 7 days, and the particle and uniformity difference in different formulations was detected by DLS. The specific formulation composition and detection results are shown in Table 14.

[0252] [Table 14]

[0253] After accelerated incubation at 40±2℃ for 28 days, the detection results by SEC-HPLC showed that there was no obvious difference between different formulations, and the detection results by WCX-HPLC showed that only L-proline at 220 mM was contained, and the reduction of the main components was more obvious than that of other formulations, and the comparative difference between other formulations was not obvious.

[0254] After shaking at 25±2℃ and 150 rpm for 7 days, the detection results of insoluble particles showed that the formulations with added surfactants had low insoluble particles, while the other formulations had relatively high insoluble particles. The detection results by DLS showed that the PDI value could be improved by adding different surfactants, polysorbate 20, polysorbate 80, or poloxamer 188, and the difference between the formulations with different surfactants was not obvious.

[0255] Example 4 According to the results of the previous example, the excipients and pH of the formulation were further optimized, and the following pharmaceutical formulation was obtained according to Table 15:

[0256] [Table 15]

[0257] For the formulations in Table 15, screening detection was performed according to Table 16.

[0258] [Table 16]

[0259] 1) Detection of main peak content by SEC-HPLC The results of detecting the main peak content and polymer content by SEC-HPLC corresponding to formulations 20 to 31 are shown in Figures 9 and 10, respectively.

[0260] Comparison of formulations 20–23, 24–27, and 28–31 revealed that there was no clear difference in the effect of pH 6.1–6.7 on stability.

[0261] As a result of comparing the polymer contents of Formulations 20 to 23 and Formulations 28 to 31, it was found that the inclusion of L-proline led to further improvement in stability.

[0262] By comparing formulations 24-27 and formulations 28-31, respectively, it was found that there was no significant difference in the effect of 220 mM L-proline and 150 mM L-proline on stability.

[0263] 2) Detection of acidic component content by WCX-HPLC After high-temperature (40±2°C) accelerated experiments, the detection results of the acidic component contents of formulations 20 to 31 by WCX-HPLC are shown in Figure 11.

[0264] When the acidic components of Formulations 20 to 23, Formulations 24 to 27, and Formulations 28 to 31 were compared, it was found that the content of acidic components at pH 6.1 to 6.7 tended to increase as the pH value increased, but all were within the acceptable range.

[0265] As a result of comparing the acidic components of Formulations 20 to 23 and Formulations 28 to 31, it was found that the inclusion of L-proline led to further improvement in stability.

[0266] When the acidic components of formulations 24-27 and 28-31 were compared, respectively, it was found that there was no obvious difference in the effects of 220 mM L-proline and 150 mM L-proline on stability.

[0267] 3) Detection of osmotic pressure The detection results of osmotic pressure are shown in Table 17.

[0268] [Table 17]

[0269] According to the standards of the INS (infusion nurses society), if the infusion osmolality is less than 600 mOsmoL / kg, it is within the safe range, so the osmolality of prescriptions 20, 24, and 28 is within the safe range.

[0270] Example 5 Through a series of experiments on protein content, pH range, buffer screening, and stabilizer screening, an optimal combination of sodium acetate-acetic acid as the buffer system, L-proline as the stabilizer, and polysorbate 20 as the surfactant was selected. The DoE experimental design further considered the effects of sodium acetate-acetic acid concentration, L-proline concentration, polysorbate 20 concentration, PD-L1 monoclonal antibody protein content and pH on protein stability. A classic two-level screening design in JMP15.0 software was adopted to consider these five factors, with one iteration of two levels and one center point, for a total of 18 combinations. The experimental design is shown in Table 18 below.

[0271] [Table 18]

[0272] The above formulations were accelerated at high temperature (40±2°C) for 14 days and 28 days, and then the monomer content was detected by SEC-HPLC and the main component content was detected by WCX-HPLC. The results are summarized in Table 19.

[0273] [Table 19]

[0274] The results showed that the concentration of sodium acetate as a buffer system (5-35 mM), the concentration of L-proline as a stabilizer (50-390 mM), and the concentration of polysorbate 20 (0-0.04%) had no obvious effect on the SEC-HPLC monomer and WCX-HPLC main component for protein detection, and the fluctuation was small within this range. The protein content and pH affected the SEC-HPLC monomer and WCX-HPLC main component for protein detection, and changed significantly within this range. At high temperature for 28 days, the protein concentration was 50-220 mg / mL, the purity of the SEC-HPLC monomer was above 95%, the pH was between 5.9-6.9, and the WCX-HPLC main component was above 74%, all of which were within the acceptable range, and the changes were deemed acceptable.

[0275] Example 6 An accelerated stability study was conducted on the preferred formulation: the formulation used in the sample was PD-L1 monoclonal antibody with a protein content of 200±20 mg / mL, 20 mM sodium acetate-acetic acid, 220 mM L-proline, 0.02% polysorbate 20, pH 6.4±0.1, dispensed into vials, and stored at 25±2°C for 9 months, with samples taken at the end of 0, 3, 6, and 9 months, respectively. The stability was examined according to the key items for consideration, including sample monomer purity (SEC-HPLC), charge distribution main component purity (WCX-HPLC), reduced and non-reduced capillary gel electrophoresis (CE-SDS), protein content, etc., and the results are shown in Table 20.

[0276] [Table 20]

[0277] As a result, it was shown that after being left at 25±2° C. for 9 months, all of the indexes of the sample were within the acceptable range compared to day 0. It was shown that the formulation of the present invention maintains stability even at room temperature for 9 months.

[0278] Example 7 A long-term stability study was conducted on the preferred formulation: the formulation used in the sample was PD-L1 monoclonal antibody with a protein content of 200±20 mg / mL, 20 mM sodium acetate-acetic acid, 220 mM L-proline, 0.02% polysorbate 20, pH 6.4±0.1, dispensed into vials and stored at 5±3℃ for 18 months, with samples taken at the end of 0, 6, 12, and 18 months, respectively. The stability was examined according to the key items for consideration, including sample monomer purity (SEC-HPLC), charge distribution main component purity (WCX-HPLC), reduced and non-reduced capillary gel electrophoresis (CE-SDS), protein content, etc., and the results are shown in Table 21.

[0279] [Table 21]

[0280] As a result, after leaving the sample for 18 months at 5±3°C, all of the relevant indicators of sample detection were shown to be within the acceptable range compared to day 0. The present formulation was shown to maintain stability for 18 months at 5±3°C.

[0281] The above detailed description is provided for purposes of explanation and illustration, and is not intended to limit the scope of the appended claims. Various modifications to the embodiments set forth herein will be apparent to those skilled in the art and remain within the scope of the appended claims and their equivalents.

Claims

1. an antigen-binding fragment comprising an immunoglobulin single variable domain capable of specifically binding to PD-L1, wherein the immunoglobulin single variable domain comprises CDR1-3, wherein the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 47, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 46, and the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 45 or 48; and one or more selected from the group consisting of proline, mannitol, sucrose, glycine, and L-arginine hydrochloride; or one or more excipients selected from the group consisting of proline, sucrose, mannitol, sorbitol and glycerol, composition.

2. The immunoglobulin single variable domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6.

10. The composition of claim 1.

3. the antigen-binding fragment comprises an Fc region of an immunoglobulin; Preferably, the N-terminus of the Fc region of said immunoglobulin is linked directly or indirectly to the C-terminus of said immunoglobulin single variable domain, More preferably, the N-terminus of the Fc region of the immunoglobulin is linked to the C-terminus of the immunoglobulin single variable domain by a linker.

10. The composition of claim 1.

4. The linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 42 to 44.

4. The composition of claim 3.

5. the Fc region of the immunoglobulin (a) comprises an Fc region derived from an immunoglobulin selected from the group consisting of IgG1, IgG2, IgG3, and IgG4; (b) does not have ADCC activity, and / or the Fc region of the immunoglobulin does not have CDC activity; (c) is derived from a human; and / or (d) is derived from a human IgG; Preferably, the immunoglobulin Fc region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 7 to 9.

4. The composition of claim 3.

6. The antigen-binding fragment comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14 to 31.

10. The composition of claim 1.

7. the antigen-binding fragment has a concentration of about 1 mg / mL to about 220 mg / mL; Preferably, it has a concentration of about 20 mg / mL to about 220 mg / mL; More preferably, it has a concentration of about 50 mg / mL to about 200 mg / mL, More preferably, it has a concentration of about 180 mg / mL to about 220 mg / mL, 10. The composition of claim 1.

8. the antigen-binding fragment has a concentration of about 100 mg / mL to about 280 mg / mL, or has a concentration of about 150 mg / mL to about 250 mg / mL; 10. The composition of claim 1.

9. the excipient has a concentration of about 50 mM to about 500 mM; 10. The composition of claim 1.

10. The excipient is (a) containing proline at a concentration of about 50 mM to about 390 mM; Preferably, the concentration of proline is about 150 mM to about 250 mM; More preferably, the concentration of proline is about 150 mM to about 220 mM; More preferably, the concentration of L-proline is about 150 mM to about 220 mM; or L-proline at a concentration of about 165 mM to about 275 mM; (b) mannitol at a concentration of about 50 mM to about 500 mM; (c) containing sucrose at a concentration of about 50 mM to about 500 mM; Preferably, the concentration of sucrose is about 90 mM to about 500 mM; (d) glycine at a concentration of about 50 mM to about 500 mM; Preferably, the concentration of glycine is about 150 mM to about 250 mM; (e) L-arginine hydrochloride at a concentration of about 50 mM to about 500 mM; Preferably, the concentration of L-arginine hydrochloride is about 150 mM to about 500 mM; (f) sorbitol at a concentration of about 50 mM to about 500 mM; Preferably, the concentration of sorbitol is about 100 mM to about 300 mM; or (g) glycerol at a concentration of 50 mM to about 500 mM; Preferably, the concentration of glycerol is between 100 mM and about 300 mM.

10. The composition of claim 9.

11. a pH of about 5.0 to about 7.5; Preferably, the pH is about 5.9 to about 6.9; More preferably, the pH is from about 6.3 to about 6.5; More preferably, the pH is about 6.

4.

10. The composition of claim 1.

12. one or more buffer components selected from the group consisting of sodium acetate-acetic acid, histidine-acetic acid, L-histidine-L-histidine hydrochloride, sodium phosphate, and citric acid-sodium hydroxide; Preferably, the buffer components have a concentration of about 5 mM to about 50 mM.

10. The composition of claim 1.

13. the buffer components include sodium acetate-acetic acid at a concentration of about 5 mM to about 35 mM; Preferably, the sodium acetate-acetic acid has a concentration of about 10 mM to about 30 mM; More preferably, the concentration of sodium acetate-acetic acid is about 20 mM.

13. The composition of claim 12.

14. comprising one or more surfactants selected from the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188; Preferably, the surfactant has a concentration of about 0 mg / mL to about 0.8 mg / mL.

10. The composition of claim 1.

15. the surfactant comprises polysorbate 20 at a concentration of about 0 mg / mL to about 0.4 mg / mL; Preferably, the concentration of polysorbate 20 is about 0.2 mg / mL to about 0.4 mg / mL; More preferably, the concentration of polysorbate 20 is about 0.1 mg / mL to about 0.3 mg / mL, More preferably, the concentration of polysorbate 20 is about 0.2 mg / mL.

15. The composition of claim 14.

16. 1) about 20 mg / mL to about 220 mg / mL of the antigen-binding fragment of claim 1; 2) about 5 mM to about 35 mM sodium acetate-acetic acid; 3) about 50 mM to about 390 mM proline; and 4) about 0 mg / mL to about 0.4 mg / mL of polysorbate 20; and a pH of about 5.9 to about 6.9; Preferably, 1) about 50 mg / mL to about 220 mg / mL of the antigen-binding fragment of claim 1; 2) about 10 mM to about 30 mM sodium acetate-acetic acid; 3) about 150 mM to about 250 mM proline; 4) about 0.2 mg / mL to about 0.4 mg / mL of polysorbate 20; and a pH of about 6.3 to about 6.5; More preferably, 1) about 200 mg / mL of the antigen-binding fragment of claim 1; 2) about 20 mM sodium acetate-acetic acid; 3) about 220 mM L-proline; 4) about 0.2 mg / mL of polysorbate 20; and the pH is about 6.

4.

10. The composition of claim 1.

17. It is used for injection, preferably for subcutaneous injection.

10. The composition of claim 1.

18. formulation, preferably a liquid formulation; 10. The composition of claim 1.

19. A composition according to any one of claims 1 to 18 and a container containing the composition according to any one of claims 1 to 18, Preferably, the container comprises a glass bottle; Reagent kit.

20. the composition in the container has a volume of about 0.5 mL to about 5.0 mL; Preferably, it has a volume of about 0.5 mL to about 1.5 mL.

20. The reagent kit according to claim 19.

21. A composition according to any one of claims 1 to 18 in the manufacture of a medicament for preventing, alleviating and / or treating tumors, use.

22. The tumor comprises a PD-L1 positive tumor and / or the tumor comprises a malignant solid tumor.

22. The use according to claim 21.

23. A composition according to any one of claims 1 to 18 in the manufacture of a medicament for preventing, alleviating and / or treating an infectious disease. use.

24. The infectious disease includes diseases caused by viral infection, bacterial infection, fungal infection, and / or parasitic infection.

24. The use according to claim 23.

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