WO2023040999A1 - 包含pd-l1抗原结合片段的组合物及其用途 - Google Patents

包含pd-l1抗原结合片段的组合物及其用途 Download PDF

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WO2023040999A1
WO2023040999A1 PCT/CN2022/119187 CN2022119187W WO2023040999A1 WO 2023040999 A1 WO2023040999 A1 WO 2023040999A1 CN 2022119187 W CN2022119187 W CN 2022119187W WO 2023040999 A1 WO2023040999 A1 WO 2023040999A1
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concentration
composition
composition according
antigen
binding fragment
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PCT/CN2022/119187
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English (en)
French (fr)
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徐霆
郭康平
恽丽红
吴圆丽
黄艳
胡红琴
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江苏康宁杰瑞生物制药有限公司
苏州康宁杰瑞生物科技有限公司
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Priority to CN202280062580.XA priority Critical patent/CN118215681A/zh
Publication of WO2023040999A1 publication Critical patent/WO2023040999A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • This application relates to the field of biomedicine, in particular to a composition comprising a PD-L1 antigen-binding fragment and its use.
  • PD-L1 expression has been found in several murine and human cancers, including human lung, ovarian, colon, melanoma, and various myelomas.
  • Existing results have shown that PD-L1 highly expressed by tumor cells plays an important role in the immune escape of tumors by increasing the apoptosis of T cells.
  • Antibody drug preparation forms include injection preparations or freeze-dried preparations and the like.
  • the subcutaneous route of administration is preferable. This will enable patients to self-manage, increase the convenience, speed and compliance of treatment, save a lot of money for the healthcare sector, and improve the quality of life of patients.
  • the present application provides a composition comprising an antigen-binding fragment that specifically binds to PD-L1 and auxiliary materials.
  • the composition includes at least one of the following beneficial effects: 1) contains a high concentration (for example, 200 mg/mL) of the antigen-binding fragment; 2) has a suitable viscosity; 3) can be applied to subcutaneous injection, reducing the pain of the subject; 4) good stability (such as accelerated stability and/or long-term stability); 5) avoid side effects caused by infusion; 6) simple components, easy preparation and low cost.
  • the present application provides a composition comprising an antigen-binding fragment and an auxiliary material, wherein the antigen-binding fragment comprises an immunoglobulin single variable domain capable of specifically binding to PD-L1, and the immunoglobulin single variable structure
  • the domain comprises CDR1-3, wherein the CDR3 comprises the amino acid sequence shown in SEQ ID NO:47, wherein the auxiliary material is selected from one or more of the following group: proline, mannitol, sucrose, glycine and L - Arginine hydrochloride.
  • the auxiliary material can also be selected from one or more of the following group: proline, sucrose, mannitol, sorbitol, glycerin and L-arginine hydrochloride.
  • the auxiliary material is preferably selected from one or more of the following group: proline, sucrose, mannitol, sorbitol and glycerin; more preferably proline, sucrose, mannitol and sorbitol one or more.
  • the auxiliary material is L-proline, or the auxiliary material is sucrose, or the auxiliary material is mannitol, or the auxiliary material is sorbitol.
  • the CDR2 comprises the amino acid sequence shown in SEQ ID NO:46.
  • the CDR1 comprises the amino acid sequence shown in SEQ ID NO: 45 or 48.
  • the immunoglobulin single variable domain comprises the amino acid sequence set forth in any one of SEQ ID NO: 1-6.
  • the PD-L1 is human PD-L1.
  • the antigen-binding fragment comprises an Fc region of an immunoglobulin.
  • the N-terminus of the Fc region of the immunoglobulin is directly or indirectly linked to the C-terminus of the immunoglobulin single variable domain.
  • the N-terminal of the Fc region of the immunoglobulin is connected to the C-terminal of the single variable domain of the immunoglobulin through a linker.
  • the linker comprises the amino acid sequence shown in any one of SEQ ID Nos: 42-44.
  • the Fc region of the immunoglobulin comprises an Fc region derived from an immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4.
  • the Fc region of the immunoglobulin does not have ADCC activity; and/or, the Fc region of the immunoglobulin does not have CDC activity.
  • the Fc region of the immunoglobulin is of human origin.
  • the Fc region of the immunoglobulin is derived from human IgG.
  • the Fc region of the immunoglobulin comprises the amino acid sequence shown in any one of SEQ ID NO:7-9.
  • the antigen-binding fragment comprises the amino acid sequence set forth in any one of SEQ ID Nos: 14-31.
  • the concentration of the antigen-binding fragment is from about 1 mg/mL to about 300 mg/mL.
  • the concentration of the antigen-binding fragment is from about 5 mg/mL to about 250 mg/mL.
  • the concentration of the antigen-binding fragment is from about 1 mg/mL to about 220 mg/mL.
  • the concentration of the antigen-binding fragment is from about 20 mg/mL to about 220 mg/mL.
  • the concentration of the antigen-binding fragment is from about 50 mg/mL to about 200 mg/mL.
  • the concentration of the antigen-binding fragment is from about 100 mg/mL to about 280 mg/mL.
  • the concentration of the antigen-binding fragment is from about 150 mg/mL to about 250 mg/mL.
  • the concentration of the antigen-binding fragment is from about 180 mg/mL to about 250 mg/mL.
  • the concentration of the antigen-binding fragment is from about 180 mg/mL to about 220 mg/mL.
  • the concentration of the excipient is about 50 mM to about 500 mM.
  • the excipient comprises proline at a concentration of about 50 mM to about 390 mM.
  • the excipient comprises proline at a concentration of about 150 mM to about 250 mM.
  • the excipient comprises proline at a concentration of about 150 mM to about 220 mM.
  • the excipient comprises L-proline at a concentration of about 150 mM to about 220 mM.
  • the excipient comprises L-proline at a concentration of about 165 mM to about 275 mM.
  • the excipient comprises mannitol at a concentration of about 50 mM to about 500 mM.
  • the excipient comprises sucrose at a concentration of about 50 mM to about 500 mM.
  • the excipient comprises sucrose at a concentration of about 90 mM to about 500 mM.
  • the excipient comprises glycine at a concentration of about 50 mM to about 500 mM.
  • the excipient comprises glycine at a concentration of about 150 mM to about 250 mM.
  • the excipient comprises L-arginine hydrochloride at a concentration of about 50 mM to about 500 mM.
  • the excipient comprises L-arginine hydrochloride at a concentration of about 150 mM to about 500 mM.
  • the excipient comprises sorbitol at a concentration of about 50 mM to about 500 mM.
  • the excipient comprises sorbitol at a concentration of about 100 mM to about 300 mM.
  • the excipient comprises glycerol at a concentration of 50 mM to about 500 mM.
  • the excipient comprises glycerol at a concentration of about 100 mM to about 300 mM.
  • the pH of the composition is from about 5.0 to about 7.5.
  • the pH of the composition is from about 5.9 to about 6.9.
  • the pH of the composition is from about 6.3 to about 6.5.
  • the pH of the composition is about 6.4.
  • the composition comprises a buffering component, wherein the buffering component is selected from one or more of the group consisting of sodium acetate-acetic acid, histidine-acetic acid, L-histidine-L -Histidine hydrochloride, sodium phosphate and citric acid-sodium hydroxide.
  • the buffering component is selected from one or more of the group consisting of sodium acetate-acetic acid, histidine-acetic acid, L-histidine-L -Histidine hydrochloride, sodium phosphate and citric acid-sodium hydroxide.
  • the buffering component is selected from one or more of the group consisting of sodium acetate-acetic acid, L-histidine-L-histidine hydrochloride, and citric acid-sodium hydroxide;
  • the buffer component is sodium acetate-acetic acid or L-histidine-L-histidine hydrochloride.
  • the concentration of the buffer component is from about 5 mM to about 50 mM.
  • the buffer component comprises sodium acetate-acetic acid at a concentration of about 5 mM to about 35 mM.
  • the buffer component comprises sodium acetate-acetic acid at a concentration of about 10 mM to about 30 mM.
  • the buffer component comprises sodium acetate-acetic acid at a concentration of about 20 mM.
  • the buffering 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, even more preferably 20 mM.
  • the composition comprises a surfactant, wherein the surfactant is selected from one or more of the group consisting of polysorbate 20, polysorbate 80, and poloxamer 188.
  • the concentration of the surfactant is from about 0 mg/mL to about 0.8 mg/mL.
  • the surfactant comprises polysorbate 20 at a concentration of about 0 mg/mL to about 0.4 mg/mL.
  • the surfactant comprises polysorbate 20 at a concentration of about 0.2 mg/mL to about 0.4 mg/mL.
  • the surfactant comprises polysorbate 20 at a concentration of about 0.1 mg/mL to about 0.3 mg/mL.
  • the surfactant comprises polysorbate 20 at a concentration of about 0.2 mg/mL.
  • 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-0.3 mg/mL, even more preferably 0.2 mg/mL.
  • 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. mL, more preferably 0.2mg/mL-about 2mg/mL.
  • 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 35 mM about 390 mM proline; 4) about 0 mg/mL to about 0.4 mg/mL polysorbate 20; and a pH of about 5.9 to about 6.9.
  • 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 30 mM sodium acetate-acetic acid; about 250 mM proline; 4) about 0.2 mg/mL to about 0.4 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • 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 30 mM sodium acetate-acetic acid; about 250 mM proline; 4) about 0.2 mg/mL to about 0.4 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • the composition comprises: 1) about 200 mg/mL of the 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 the pH is about 6.4.
  • 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 35 mM sodium acetate-acetic acid; about 500 mM sucrose; 4) about 0.05 mg/mL to about 0.4 mg/mL polysorbate 20; and a pH of about 5.9 to about 6.9.
  • 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 30 mM sodium acetate-acetic acid; about 300 mM sucrose; 4) about 0.1 mg/mL to about 0.3 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • 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 35 mM sodium acetate-acetic acid; about 500 mM mannitol; 4) about 0.05 mg/mL to about 0.4 mg/mL polysorbate 20; and a pH of about 5.9 to about 6.9.
  • 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 30 mM sodium acetate-acetic acid; about 300 mM mannitol; 4) about 0.1 mg/mL to about 0.3 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • 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 35 mM sodium acetate-acetic acid; about 500 mM sorbitol; 4) about 0.05 mg/mL to about 0.4 mg/mL polysorbate 20; and a pH of about 5.9 to about 6.9.
  • 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 30 mM sodium acetate-acetic acid; about 300 mM sorbitol; 4) about 0.1 mg/mL to about 0.3 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • 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 35 mM sodium acetate-acetic acid; about 500 mM glycerol; 4) about 0.05 mg/mL to about 0.4 mg/mL polysorbate 20; and a pH of about 5.9 to about 6.9.
  • 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 30 mM sodium acetate-acetic acid; about 300 mM glycerol; 4) about 0.1 mg/mL to about 0.3 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • 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 3) about 150 mM to about 250 mM proline; 4) about 0.2 mg/mL to about 0.4 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • 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 3) about 50 mM to about 300 mM sucrose; 4) about 0.1 mg/mL to about 0.3 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • 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 3) about 100 mM to about 300 mM mannitol; 4) about 0.1 mg/mL to about 0.3 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • 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 3) about 100 mM to about 300 mM sorbitol; 4) about 0.1 mg/mL to about 0.3 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • 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 3) about 100 mM to about 300 mM glycerol; 4) about 0.1 mg/mL to about 0.3 mg/mL polysorbate 20; and a pH of about 6.3 to about 6.5.
  • the composition is for injection.
  • the composition is for subcutaneous injection.
  • the composition is a formulation.
  • the composition is a liquid formulation.
  • the present application provides a kit comprising the composition described in the present application and a container for containing the composition described in the present application.
  • the container comprises a glass bottle.
  • the volume of the composition in the container is from about 0.5 mL to about 5.0 mL.
  • the volume of the composition in the container is from about 0.5 mL to about 1.5 mL.
  • the present application provides the composition described in the present application; and/or the application of the kit described in the present application in the preparation of medicaments for preventing, alleviating and/or treating tumors.
  • the tumor comprises a PD-L1 positive tumor.
  • the tumor comprises a malignant solid tumor.
  • the present application provides the composition described in the present application; and/or the application of the kit described in the present application in the preparation of medicaments for preventing, alleviating and/or treating infectious diseases.
  • the infectious diseases include diseases caused by viral infections, bacterial infections, fungal infections and/or parasitic infections.
  • Figure 1 shows the Tm value results of formulations comprising the antigen-binding fragments described in the present application.
  • Figure 2 shows the results of detection of aggregation propensity of formulations comprising the antigen-binding fragments described in the present application.
  • Figure 3 shows the detection results of the SEC-HPLC main peak content of the preparation formulation containing the antigen-binding fragment described in the present application.
  • Figure 4 shows the detection results of SEC-HPLC aggregate content of the preparation formulation comprising the antigen-binding fragment described in the present application.
  • Fig. 5 shows the WCX-HPLC detection results of the acidic component of the preparation formulation comprising the antigen-binding fragment described in the present application.
  • Figure 6 shows the results of viscosity testing of formulations containing the antigen-binding fragments described in the present application.
  • Figure 7 shows the results of binding activity of formulations comprising the antigen-binding fragments described herein.
  • Figure 8 shows the results of blocking activity of formulations comprising the antigen-binding fragments described herein.
  • Figure 9 shows the detection results of the SEC-HPLC main peak content of the preparation formulation containing the antigen-binding fragment described in the present application.
  • Fig. 10 shows the detection results of SEC-HPLC aggregate content of the preparation formulation of the antigen-binding fragment described in the present application.
  • Figure 11 shows the WCX-HPLC detection results of the acidic components of the formulation of the antigen-binding fragment described in the present 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 (eg, by binding or steric hindrance, etc.).
  • the antigen-binding function of an antibody can also be achieved by comprising a heavy chain of a fragment of Fv, ScFv, dsFv, Fab, Fab' or F(ab'), or by comprising a Fv, scFv, dsFv, Fab, Fab' or The light chain of a fragment of F(ab')2.
  • Fab fragment that is, a monovalent fragment composed of VL, VH, CL and CH domains
  • F(ab')2 fragment comprising two Fab fragments linked by a disulfide bond at the hinge region Bivalent fragments
  • Fd fragments consisting of VH and CH domains
  • Fv fragments consisting of VL and VH domains of an antibody single arm
  • dAb fragments consisting of VH domains (Ward et al. (1989) Nature 341:544-546);
  • CDR complementarity determining region
  • the monovalent single-chain molecule Fv formed by the pairing of VL and VH (see Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl.Acad.Sci.85 :5879-5883).
  • a type of antibody VHH that lacks the light chain of the antibody and only has the variable region of the heavy chain can also be included (for example, see Kang Xiaozhen et al., Acta Biological Engineering, 2018, 34(12): 1974-1984).
  • the "antigen binding portion” may also include an immunoglobulin fusion protein comprising a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • an immunoglobulin fusion protein comprising a binding domain selected from: (1) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; (2) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide; an immunoglobulin heavy chain CH2 constant region fused to the hinge region; and (3) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region.
  • the term "adjuvant" generally refers to any material selected for whatever type or product form is desired for the composition.
  • the product form may include liquid, granules, powder, paste, spray, tablet or gel.
  • the adjuvant may not affect the form or stability of the antigen-binding fragment, nor may it affect any biological activity of the antigen-binding fragment (such as specific binding to the corresponding antigen).
  • the adjuvant can function as a stabilizer, improve the composition, and/or improve the stability of the antigen-binding fragment in the composition.
  • the term "PD-L1" generally refers to programmed cell death protein 1 ligand 1, which may also be referred to as CD274 or B7H1.
  • the PD-L1 may be PD-L1 derived from mammals, for example, the PD-L1 may be derived from primates or rodents (such as mice or rats).
  • Human PD-L1 may have the amino acid sequence shown in NCBI accession number NP_054862.1.
  • the PD-L1 may comprise natural PD-L1, and may also comprise PD-L1 variants, isoforms, species homologues and analogs having at least one common epitope with PD-L1.
  • immunoglobulin variable domain generally refers to, comprising "framework region 1" or “FR1”, “framework region 2” or “FR2”, “framework region 3” or “FR3”, and the four “framework regions” of “framework region 4" or “FR4" and “complementarity determining region 1" or “CDR1”, “complementarity determining region 2" or “CDR2”, and “complementarity determining region 3” or “CDR3 "Three "complementarity determining regions” or “CDRs” of polypeptide molecules.
  • the immunoglobulin variable domain may have a protein tertiary structure. In this application, the immunoglobulin variable domain may have the function of specifically binding to an antigen (such as PD-L1).
  • the immunoglobulin variable domain may comprise the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the term "immunoglobulin single variable domain” generally refers to the immunoglobulin variable domain that has the independent function of specifically binding to an antigen (such as PD-L1).
  • the immunoglobulin single variable domain can specifically bind an antigen or an epitope thereof without pairing with another immunoglobulin variable domain.
  • the immunoglobulin single variable domain may be a heavy chain variable region VH and/or a light chain variable region VL.
  • the immunoglobulin single variable domain may be VHH.
  • the VHH may be referred to as heavy chain antibody, nano antibody or single domain antibody.
  • the CDR of the VHH can be divided according to the Kabat method (see “Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD): for example, the 1-30th amino acid of the VHH amino acid sequence is FR1, the 31st- Amino acid at position 35 is CDR1, amino acid at position 36-49 is FR2, amino acid at position 50-65 is CDR2, amino acid at position 66-94 is FR3, amino acid at position 95-102 is CDR3, amino acid at position 103-113 is FR4 .
  • the total number of amino acid residues for each CDR may differ 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 not be occupied in the actual sequence , or the actual sequence may contain more amino acid residues than the Kabat numbering allows), so the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
  • composition generally refers to a mixture comprising the antigen-binding fragments described herein.
  • the composition may comprise at least one pharmaceutically acceptable ingredient, such as an excipient as described herein.
  • preparation generally refers to a product comprising the antigen-binding fragment described in the present application in a predetermined amount or ratio, and any preparation directly or indirectly obtained by adding a predetermined amount of the antigen-binding fragment described in the present application. combined products.
  • the meaning of the preparation described in the present application may include pharmaceutical preparations, that is, comprising the antigen-binding fragment described in the present application and the adjuvant, as well as any combination, complex or aggregation of any two or more components directly or indirectly, and produced products.
  • the formulation may be in liquid form.
  • the term “buffering component” generally refers to an agent that has the function of providing a buffering effect, eg resistance to changes in pH.
  • the buffering component can adjust for changes in pH due to the addition and/or release of acidic or basic substances.
  • the buffer component may include a weak acid and its conjugate base; it may also include a weak base and its conjugate acid.
  • surfactant generally refers to an agent that can protect a protein (such as an antigen-binding fragment described herein) from air/solution interface-induced stress, solution/surface-induced stress to reduce the protein. Agents that agglomerate or minimize the formation of particulate matter in the composition.
  • the surfactant may contain both hydrophobic groups and hydrophilic groups.
  • the surfactant may comprise a mixture or combination of one or more surfactants.
  • the surfactant may include a nonionic surfactant.
  • the term "PD-L1-positive" generally means that the proportion of cells expressing PD-L1 in a test tissue sample containing tumor cells and tumor-infiltrating inflammatory cells is higher than that of scoring the sample as expressing cell surface
  • the ratio of cells can be determined by immunohistochemistry (IHC).
  • the PD-L1 positive can 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% in a certain sample.
  • % 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.
  • tumor generally refers to damage caused by abnormal growth of cells.
  • the tumor may form separate masses of tissue through rapid, uncontrolled cell proliferation.
  • the mass of tissue can be benign, pre-malignant or malignant.
  • the tumor may be considered malignant when it has the structure and/or function of unrestricted growth and/or attacks normal tissue.
  • the malignant tumor can be used interchangeably with cancer.
  • malignant solid tumor generally refers to any type of morphological tumor with metastatic potential.
  • infectious disease generally refers to local tissue and systemic inflammatory responses caused by viruses, bacteria, fungi, parasites and other pathogens and specific toxins invading the human body.
  • the present application provides a composition, which may comprise an antigen-binding fragment and an auxiliary material, wherein the antigen-binding fragment may comprise an immunoglobulin single variable domain capable of specifically binding to PD-L1, and the immunoglobulin
  • the protein single variable domain can comprise the amino acid sequence shown in any one of SEQ ID NO:1-6, wherein said auxiliary material is selected from one or more in the following group: proline, mannitol, sucrose, Glycine and L-Arginine HCl.
  • the antigen-binding fragment can specifically bind to PD-L1.
  • the PD-L1 can be human PD-L1.
  • the antigen-binding fragment may comprise the Fc region of an immunoglobulin.
  • the antigen-binding fragment may consist of an amino acid sequence of the formula A-L-B, where A represents an immunoglobulin single variable domain, L represents an amino acid linker or its absence, and B represents a human immunoglobulin Fc region.
  • the N-terminus of the Fc region of the immunoglobulin may be directly or indirectly linked to the C-terminus of the immunoglobulin single variable domain.
  • the N-terminal of the Fc region of the immunoglobulin and the C-terminal of the single variable domain of the immunoglobulin can be linked by a linker.
  • the immunoglobulin single variable domain may comprise complementarity determining regions CDR1-3.
  • the CDR1 may comprise the amino acid sequence shown in SEQ ID NO: 45 or 48.
  • the CDR2 may comprise the amino acid sequence shown in SEQ ID NO:46.
  • the CDR3 may comprise the amino acid sequence shown in SEQ ID NO:47.
  • the linker may have a length of about 1 to about 20 amino acid residues.
  • the linker may have no higher than secondary structure.
  • the linker can be a flexible linker.
  • the linker can be GGGGS, GS or GAP.
  • the linker can comprise the amino acid sequence shown in any one of SEQ ID NOs: 42-44.
  • the Fc region of the immunoglobulin may comprise an Fc region derived from an immunoglobulin selected from the group consisting of IgGl, IgG2, IgG3 and IgG4.
  • the constant regions of human IgG1, IgG2, IgG3 or IgG4 can respectively have the amino acid sequences shown in the entries P01857, P01859, P01860 and P01861 in the www.uniprot.org protein database.
  • the Fc region of the immunoglobulin may be of human origin.
  • the Fc region of the immunoglobulin may be derived from human IgG.
  • the Fc region of the immunoglobulin may be derived from human IgG1.
  • the Fc region of the immunoglobulin may not have ADCC activity; and/or, the Fc region of the immunoglobulin may not have CDC activity.
  • the Fc region of the immunoglobulin can be mutated to lose ADCC activity; and/or, CDC activity.
  • the Fc region of the immunoglobulin may comprise the amino acid sequence shown in any one of SEQ ID NOs: 7-9.
  • the antigen-binding fragment may have a homodimer.
  • the homodimer can be formed by the interaction of the Fc region of the immunoglobulin.
  • the antigen-binding fragment may be in the form of less than 1 ⁇ 10 -7 M, less than 1 ⁇ 10 -8 M, less than 1 ⁇ 10 -9 M, less than 1 ⁇ 10 -10 M, less than 1 ⁇ 10 -11
  • the KD value of M specifically binds to PD-L1.
  • the antigen-binding fragment can specifically bind to human PD-L1 and block the interaction between PD-L1 and PD-1. In this application, the antigen-binding fragment can specifically bind to human PD-L1 and block the interaction between PD-L1 and CD80.
  • 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.
  • the antigen-binding fragment may be resistant to alkaline treatment and/or oxidative treatment.
  • a strong base such as ammonium bicarbonate
  • the antigen-binding fragment still retains the ability to specifically bind PD-L1.
  • an oxidizing agent such as 1% hydrogen peroxide
  • the antigen-binding fragment may comprise the amino acid sequence shown in any one of SEQ ID NO: 14-31.
  • the antigen-binding fragment may comprise functional variants and fragments thereof, derivatives and fragments thereof, analogs and fragments thereof, and/or homologues and fragments thereof.
  • the functional variant may be a polypeptide having substantially the same amino acid sequence or a substantially identical nucleotide sequence as the naturally occurring sequence and capable of having one or more activities of the naturally occurring sequence.
  • the functional variant may be a sequence in which residues are modified and at least one endogenous function is retained on the basis of any of the amino acid sequences shown in SEQ ID NO: 14-31 (for example, specific Binds positively to human PD-L1).
  • the modification may comprise addition, deletion, substitution, modification, replacement and/or variation of at least one amino acid residue.
  • the derivative may comprise any substitution, variation, modification, replacement, deletion and/or addition of one (or more) amino acid residues of the amino acid sequence shown in any one of SEQ ID NO: 14-31, and Sequences that retain at least one endogenous function.
  • the homologue can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5% compared with the amino acid sequence shown in any one of SEQ ID NO: 14-31 %, 99.6%, 99.7%, 99.8% or 99.9% identical amino acid sequences.
  • sequence identity an alignment of sequences can be performed, which can be performed in a variety of ways known to those skilled in the art,
  • the analog may comprise a mimetic of the polypeptide that retains at least one endogenous function for the antigen-binding fragments described herein.
  • the antigen-binding fragments of the present application may have deletions, insertions, or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipathic nature of the residues, so long as endogenous function is preserved.
  • the concentration of the antigen-binding fragment may be from about 1 mg/mL to about 220 mg/mL.
  • the concentration of the antigen-binding fragment can be from about 20 mg/mL to about 220 mg/mL.
  • the concentration of the antigen-binding fragment can be from about 30 mg/mL to about 220 mg/mL, from about 40 mg/mL to about 220 mg/mL, from about 50 mg/mL to about 220 mg/mL, from about 30 mg/mL to about 220 mg/mL. 200 mg/mL, about 40 mg/mL to about 200 mg/mL or about 50 mg/mL to about 200 mg/mL).
  • the concentration of the antigen-binding fragment can be about 1 mg/mL-about 300 mg/mL, for example, the concentration of the antigen-binding fragment can be about 5 mg/mL, 10 mg/mL, 20 mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL mL, 160mg/mL, 170mg/mL, 180mg/mL, 190mg/mL, 200mg/mL, 210mg/mL, 220mg/mL, 230mg/mL, 240mg/mL, 250mg/mL, 260mg/mL, 270mg/mL, 280 mg/mL
  • the concentration of the antigen-binding fragment can be from about 50 mg/mL to about 200 mg/mL.
  • the concentration of the antigen-binding fragment can be about 50 mg/mL; for example, the concentration of the antigen-binding fragment can be about 200 mg/mL.
  • the concentration of the excipient can be from about 50 mM to about 500 mM.
  • the concentration of the excipient can be about 50mM-about 500mM, about 100mM-about 500mM, about 150mM-about 500mM, about 160mM-about 500mM, about 165mM-about 500mM, about 170mM-about 500mM, about 100mM-about 300mM , about 150mM-about 300mM, about 160mM-about 300mM, about 165mM-about 300mM, about 170mM-about 300mM, about 100mM-about 275mM, about 150mM-about 275mM, about 160mM-about 275mM, about 165mM-about 275mM, 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 mM-about
  • the excipient may comprise proline, which may be present at a concentration of about 50 mM to about 500 mM.
  • the concentration of proline can be about 50mM to about 500mM, about 100mM to about 500mM, about 150mM to about 500mM, about 160mM to about 500mM, about 165mM to about 500mM, about 170mM to about 500mM, 50mM to about 390mM, about 100mM-about 390mM, about 150mM-about 390mM, about 160mM-about 390mM, about 165mM-about 390mM, about 170mM-about 390mM, about 100mM-about 300mM, about 150mM-about 300mM, about 160mM-about 300mM, About 165mM to about 300mM, about 170mM to about 300mM, about 100mM to about 275mM, about 150mM to about 275mM, about 160mM to about 275mM.
  • the excipient may comprise proline, which may be present at a concentration of about 165 mM to about 275 mM.
  • the excipient may comprise proline, which may be present at a concentration of about 150 mM to about 250 mM.
  • the excipient may comprise proline, which may be present at a concentration of about 220 mM.
  • the excipient may comprise L-proline, and the concentration of said L-proline may be from about 50 mM to about 500 mM.
  • the concentration of said L-proline can be about 50mM-about 500mM, about 100mM-about 500mM, about 150mM-about 500mM, about 160mM-about 500mM, about 165mM-about 500mM, about 170mM-about 500mM, 50mM -about 390mM, about 100mM-about 390mM, about 150mM-about 390mM, about 160mM-about 390mM, about 165mM-about 390mM, about 170mM-about 390mM, about 100mM-about 300mM, about 150mM-about 300mM, about 160mM-about 300mM, about 165mM-about 300mM, about 170mM-about 300mM, about 100mM-about 275mM, about 150mM-about 275mM, about 160mM-about 275mM, about 165mM-about 275mM, about 170mM-about 275mM, about 100mM-
  • the excipient may comprise L-proline, which may be present at a concentration of about 165 mM to about 275 mM.
  • the excipient may comprise L-proline, and the concentration of said L-proline may be from about 150 mM to about 250 mM.
  • the excipient may comprise L-proline, and the concentration of said L-proline may be about 220 mM.
  • the excipient may comprise mannitol, and the concentration of the mannitol may be from about 50 mM to about 500 mM.
  • the concentration of mannitol can be from about 50 mM to about 500 mM, from about 100 mM to about 500 mM, from about 150 mM to about 500 mM, from about 160 mM to about 500 mM, from about 165 mM to about 500 mM, from about 170 mM to about 500 mM, from 50 mM to about 390 mM , about 100mM-about 390mM, about 150mM-about 390mM, about 160mM-about 390mM, about 165mM-about 390mM, about 170mM-about 390mM, about 100mM-about 300mM, about 150mM-about 300mM, about 160mM-about 300mM, about 165mM-about 300mM, about 170mM-about 300mM, about 100
  • the excipient may comprise sucrose, and the concentration of sucrose may be from about 50 mM to about 500 mM.
  • the concentration of sucrose can be about 50mM to about 500mM, about 90mM to about 500mM, about 100mM to about 500mM, about 150mM to about 500mM, about 160mM to about 500mM, about 165mM to about 500mM, about 170mM to about 500mM , 50mM-about 390mM, about 100mM-about 390mM, about 150mM-about 390mM, about 160mM-about 390mM, about 165mM-about 390mM, about 170mM-about 390mM, about 100mM-about 300mM, about 150mM-about 300mM, about 160mM -about 300mM, about 165mM-about 300mM, about 170mM-about 300mM, about 100mM-about 275mM, about 150mM-about 275
  • the excipient may comprise glycine, which may be present at a concentration of about 50 mM to about 500 mM.
  • the concentration of glycine can be about 50mM to about 500mM, about 100mM to about 500mM, about 150mM to about 500mM, about 160mM to about 500mM, about 165mM to about 500mM, about 170mM to about 500mM, 50mM to about 390mM, About 100mM to about 390mM, about 150mM to about 390mM, about 160mM to about 390mM, about 165mM to about 390mM, about 170mM to about 390mM, about 100mM to about 300mM, about 150mM to about 300mM, about 160mM to about 300mM, about 165mM -about 300mM, about 170mM-about 300mM, about 100mM-about 275mM, about 150mM-about 275mM, about 160mM
  • the excipient may comprise L-arginine hydrochloride, and the concentration of the L-arginine hydrochloride may be from about 50 mM to about 500 mM.
  • the concentration of the L-arginine hydrochloride can be about 50mM to about 500mM, about 100mM to about 500mM, about 150mM to about 500mM, about 160mM to about 500mM, about 165mM to about 500mM, about 170mM to about 500mM, 50mM-about 390mM, about 100mM-about 390mM, about 150mM-about 390mM, about 160mM-about 390mM, about 165mM-about 390mM, about 170mM-about 390mM, about 100mM-about 300mM, about 150mM-about 300mM, about 160mM-about 300mM, about 165mM-about 300mM, about 170mM-about 300mM, about 100mM-about 275
  • the excipient may comprise sorbitol, which may be present in a concentration of about 50 mM to about 500 mM.
  • the concentration of sorbitol can be about 100mM to about 500mM, about 130mM to about 500mM, about 150mM to about 500mM, about 200mM to about 500mM, about 80mM to about 400mM, about 100mM to about 400mM, about 150mM to about 400mM, about 180mM-about 400mM, about 100mM-about 350mM, about 130mM-about 350mM, about 150mM-about 350mM, about 170mM-about 350mM, about 190mM-about 350mM, about 75mM-about 300mM, about 100mM-about 300mM, About 120mM to about 300mM, about 150mM to about 300mM, about 170mM to about 300mM, about 180mM to about 300mM, about 200mM to about 300mM, about 100mM to about
  • the excipient may comprise glycerol, which may be present in a concentration of about 50 mM to about 500 mM.
  • the concentration of sorbitol can be about 100mM to about 500mM, about 130mM to about 500mM, about 150mM to about 500mM, about 200mM to about 500mM, about 80mM to about 400mM, about 100mM to about 400mM, about 150mM to about 400mM, about 180mM-about 400mM, about 100mM-about 350mM, about 130mM-about 350mM, about 150mM-about 350mM, about 170mM-about 350mM, about 190mM-about 350mM, about 75mM-about 300mM, about 100mM-about 300mM, About 120mM to about 300mM, about 150mM to about 300mM, about 170mM to about 300mM, about 180mM to about 300mM, about 200mM to about 300mM, about 100mM to about
  • the excipients may contain stabilizers.
  • the stabilizer may be a component that helps maintain the structural integrity of the composition.
  • the stabilizer can help maintain the structural integrity of the composition during freezing, lyophilization and/or storage.
  • the stabilizer can act as a penetrant that reduces denaturation of a protein (eg, an antigen-binding fragment described herein).
  • the adjuvant described in the present application may further comprise an antioxidant, and the antioxidant may be selected from one of methionine, phenol, cresol, ascorbic acid, glutathione, sodium thiosulfate, citric acid and nicotinamide one or more species.
  • the adjuvant described in the present application may further comprise L-methionine, and the concentration of said L-methionine may be about 0.1 mM-about 100 mM, about 0.5 mM-about 100 mM, about 1 mM-about 100 mM, About 2mM-about 100mM, about 5mM-about 100mM, about 0.2mM-about 50mM, about 1mM-about 50mM, about 3mM-about 50mM, about 4mM-about 50mM, about 5mM-about 50mM, about 0.1mM-about 30mM, About 0.3mM to about 30mM, about 0.6mM to about 30mM, about 1mM to about 30mM, about 2mM to about 30mM, about 5mM to about 30mM, about 0.1mM to about 20mM, about 0.5mM to about 20mM, about 1mM to about 20mM, about 2mM-about 20mM, about 0.1mM-
  • the adjuvant described in the present application can also further comprise a chelating agent, the chelating agent can be EDTA disodium, and the concentration of the EDTA disodium can be about 0.1mM-about 10mM, about 0.5mM-about 10mM, about 1mM-about 10mM , about 0.1mM-about 5mM, about 0.2mM-about 5mM, about 0.3mM-about 5mM, about 0.4mM-about 5mM, about 0.5mM-about 5mM, about 0.1mM-about 3mM, about 0.5mM-about 3mM, about 0.6mM to about 3mM, about 0.7mM to about 3mM, about 0.8mM to about 3mM, about 0.1mM to about 2mM, about 0.2mM to about 2mM, about 0.5mM to about 2mM, about 0.75mM to about 2mM, about 0.9mM to about 2mM, about 1mM to about 2mM
  • the pH of the composition can be from about 5.0 to about 7.5.
  • the pH of the composition can be from about 5.9 to about 6.9.
  • the pH of the composition can be from about 5.9 to about 6.8, from about 5.9 to about 6.7, from about 5.9 to about 6.6, from about 5.9 to about 6.5, from about 5.9 to about 6.4, from about 6.0 to about 6.9, from 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.
  • the pH of the composition can be from about 6.3 to about 6.5.
  • the pH of the composition can be about 6.4.
  • the composition may comprise a buffering component, wherein the buffering component is selected from one or more of the group consisting of sodium acetate-acetic acid, histidine-acetic acid, L-histidine-L-histidine Hydrochloride, Sodium Phosphate and Citric Acid-Sodium Hydroxide.
  • the buffering component is selected from one or more of the group consisting of sodium acetate-acetic acid, histidine-acetic acid, L-histidine-L-histidine Hydrochloride, Sodium Phosphate and Citric Acid-Sodium Hydroxide.
  • the concentration of the buffer component can be from about 5 mM to about 50 mM.
  • the concentration of the buffer component can 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.
  • the buffer component may be present at a concentration of about 10 mM to 50 mM.
  • the concentration of the buffer component can 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.
  • the buffer component can comprise sodium acetate-acetic acid, which can be present at a concentration of about 10 mM to about 50 mM.
  • the sodium acetate-acetic acid concentration can be about 5mM-50mM, about 5mM-about 45mM, about 5mM-about 40mM, about 5mM-about 35mM, about 5mM-about 30mM, about 5mM-about 25mM, about 5mM- about 20 mM, about 10 mM to 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 From about 20 mM to about 30 mM.
  • the buffer component can comprise sodium acetate-acetic acid, which can be present at a concentration of about 5 mM to about 35 mM.
  • the buffer component can comprise sodium acetate-acetic acid, which can be present at a concentration of about 10 mM to about 30 mM.
  • the buffer component can comprise sodium acetate-acetic acid, which can be at a concentration of about 20 mM.
  • the buffer component can comprise L-histidine-L-histidine hydrochloride, and the concentration of the L-histidine-L-histidine hydrochloride can be from about 10 mM to about 50 mM.
  • the concentration of the L-histidine-L-histidine hydrochloride can be about 5mM-50mM, about 5mM-about 45mM, about 5mM-about 40mM, about 5mM-about 35mM, about 5mM-about 30mM , about 5mM-about 25mM, about 5mM-about 20mM, about 10mM-50mM, about 10mM-about 45mM, about 10mM-about 40mM, about 10mM-about 35mM, about 10mM-about 30mM, about 10mM-about 25mM, about 10mM - about 20 mM, about 20 mM to about 50 mM or about 20 mM to about 30 mM.
  • the buffer component can comprise L-histidine-L-histidine hydrochloride, and the concentration of the L-histidine-L-histidine hydrochloride can be from about 5 mM to about 35 mM.
  • the buffer component can comprise L-histidine-L-histidine hydrochloride, and the concentration of the L-histidine-L-histidine hydrochloride can be from about 10 mM to about 30 mM.
  • the buffer component may comprise L-histidine-L-histidine hydrochloride, and the concentration of L-histidine-L-histidine hydrochloride may be about 20 mM.
  • the composition may comprise a surfactant, wherein the surfactant may be selected from one or more of the group consisting of polysorbate 20, polysorbate 80 and poloxamer 188.
  • the surfactant may be selected from one or more of the group consisting of polysorbate 20, polysorbate 80 and poloxamer 188.
  • the present application uses mg/mL or % (w/v) to represent the concentration of the surfactant, and those skilled in the art should understand that the two can be converted, for example, 0.02% surfactant can be converted into 0.2 mg/mL.
  • the concentration of the surfactant can be from about 0 mg/mL to about 0.8 mg/mL.
  • the concentration of the surfactant can be from about 0.05 mg/mL to about 0.8 mg/mL.
  • the concentration of the surfactant can 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 mL-about 0.8mg/mL, about 0.1mg/mL-about 0.3mg/mL, about 0.1mg/mL-about 0.2mg/mL, about 0.05mg/mL-about 0.3mg/mL, about 0mg/mL-about 0.4mg/mL, about 0.05mg/mL-about 0.4mg/mL, about 0.05mg/mL-about 0.4mg/mL, about 0.05mg/mL-about 0.4m
  • the surfactant can comprise polysorbate 20, which can be present at a concentration of about 0 mg/mL to about 0.8 mg/mL.
  • the polysorbate 20 may be present at a concentration of about 0.05 mg/mL to about 0.8 mg/mL.
  • the concentration of polysorbate 20 can 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-about 0.8mg/mL, about 0.1mg/mL-about 0.3mg/mL, about 0.1mg/mL-about 0.2mg/mL, about 0.05mg/mL-about 0.3mg/mL, about 0mg/mL- 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.
  • the surfactant can comprise polysorbate 20, which can be present at a concentration of about 0.2 mg/mL to about 0.4 mg/mL.
  • the surfactant can comprise polysorbate 20, which can be present at a concentration of about 0.2 mg/mL.
  • the surfactant can comprise polysorbate 20, which can be present at a concentration of about 0.4 mg/mL.
  • the surfactant can comprise polysorbate 80, which can be present in a concentration of about 0 mg/mL to about 0.8 mg/mL.
  • the polysorbate 80 may be present at a concentration of about 0.05 mg/mL to about 0.8 mg/mL.
  • the concentration of polysorbate 80 can 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-about 0.8mg/mL, about 0.1mg/mL-about 0.3mg/mL, about 0.1mg/mL-about 0.2mg/mL, about 0.05mg/mL-about 0.3mg/mL, about 0mg/mL- 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.
  • the surfactant can comprise polysorbate 80, which can be present in a concentration of about 0.2 mg/mL to about 0.4 mg/mL.
  • the surfactant can comprise polysorbate 80, which can be present at a concentration of about 0.2 mg/mL.
  • the surfactant can comprise polysorbate 80, which can be present at a concentration of about 0.4 mg/mL.
  • the surfactant may comprise Poloxamer 188, and the concentration of Poloxamer 188 may be 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-about 5mg/mL, about 0.2mg/mL-about 5mg/mL, about 0.05mg/mL-about 3mg/mL, about 0.1mg/mL-about 3mg/mL, about 0.15mg/mL-about 3mg /mL, about 0.2mg/mL-about 3mg/mL, about 0.05mg/mL-about 2mg/mL, about 0.075mg/mL-about 2mg/mL, about 0.1mg/mL-about 2mg/mL, about 0.15mg /mL-about 2mg/mL, about 0.2mg/mL-about 2mg/mL, about 0.3mg/mL
  • the composition may comprise the antigen-binding fragment described in the present application, the adjuvant described in the present application, and the pH may be about 5.9 to about 6.9.
  • the composition may comprise an antigen-binding fragment described herein, an adjuvant described herein, and a buffer component described herein, and the pH may be from about 5.9 to about 6.9.
  • the composition may comprise the antigen-binding fragment described herein, the adjuvant described herein, the buffer component described herein, and the surfactant described herein, and the pH may be from about 5.9 to about 6.9 .
  • the composition may comprise the antigen-binding fragment described herein, proline, and the pH may be from about 5.9 to about 6.9.
  • the composition can comprise an antigen-binding fragment described herein, proline, and sodium acetate-acetic acid, and can have a pH of about 5.9 to about 6.9.
  • the composition can comprise an antigen-binding fragment described herein, proline, sodium acetate-acetic acid, and polysorbate 20, and can have a pH of about 5.9 to about 6.9.
  • the composition can comprise an antigen-binding fragment described herein, proline, sodium acetate-acetic acid, and polysorbate 20, and can have a pH of about 6.3 to about 6.5.
  • the composition may comprise: 1) about 20 mg/mL to about 220 mg/mL of an 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 4) about 0.05 mg/mL to about 0.8 mg/mL polysorbate 20; and the pH may be about 5.9 to about 6.9.
  • the composition can be composed 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 0.05mg/mL-about 0.8mg/mL polysorbate 20, and the pH of the composition can be about 5.9-about 6.9.
  • the composition may comprise: 1) about 20 mg/mL to about 220 mg/mL of an 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 4) about 0 mg/mL to about 0.4 mg/mL polysorbate 20; and the pH may be about 5.9 to about 6.9.
  • the composition can 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) from about 0 mg/mL to about 0.4 mg/mL polysorbate 20, and the pH of the composition can be from about 5.9 to about 6.9.
  • the composition may comprise: 1) about 50 mg/mL to about 200 mg/mL of an 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 polysorbate 20; and the pH may be about 6.3 to about 6.5.
  • the composition can 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 0.2mg/mL-about 0.4mg/mL polysorbate 20, and the pH of the composition can be about 6.3-about 6.5.
  • the composition may comprise: 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 ester 20; and the pH may be about 6.4.
  • the composition can be composed 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 composition, and the pH may be about 6.4.
  • the composition can be used for injection.
  • the composition can be used for subcutaneous injection.
  • the composition can also be used for subcutaneous injection, intramuscular injection, intravenous injection, parenteral administration or intravenous infusion.
  • the composition may be a formulation.
  • the composition may be a liquid formulation.
  • the present application provides a kit comprising the composition described in the present application and a container for containing the composition described in the present application.
  • the container can include a vial (eg, vial), an ampoule, a syringe, an injection pen, and/or an IV bag.
  • the container may be sterile.
  • the container may comprise a glass bottle.
  • the kit can include a delivery device that can administer a composition described herein.
  • the delivery device may comprise a vial, ampule, syringe, injection pen and/or IV bag.
  • the volume of the composition in the container may be from about 0.5 mL to about 5.0 mL.
  • the volume of the composition in the container can be from about 0.5 mL to about 1.5 mL.
  • the volume of the composition may be 1.5 mL.
  • the volume of the composition can be 1.0 mL.
  • the concentration of the antigen-binding fragment in the container may be about 200 mg/1.0 mL/bottle; or, it may be about 300 mg/1.5 mL/bottle.
  • the present application provides the composition described in the present application; and/or the application of the kit described in the present application in the preparation of medicaments for preventing, alleviating and/or treating tumors.
  • the present application provides the composition described in the present application; and/or the kit described in the present application, which are used for preventing, alleviating and/or treating tumors.
  • the present application provides a method for preventing, alleviating and/or treating tumors, which includes the following steps: administering the composition described in the present application to a subject in need; and/or the kit described in the present application.
  • the tumor may include a PD-L1 positive tumor.
  • the tumor can include a malignant solid tumor.
  • the tumor may include microsatellite instability (MSI-H)/mismatch repair deficiency (dMMR) tumors (eg, MSI-H/dMMR pan-tumor solid tumors).
  • MSI-H microsatellite instability
  • dMMR microsatellite repair deficiency
  • the tumor may comprise unresectable or metastatic biliary tract carcinoma; and/or, untreated unresectable or metastatic gastric or gastroesophageal junction adenocarcinoma.
  • the present application also provides the composition described in the present application; and/or the application of the kit described in the present application in the preparation of medicaments for preventing, alleviating and/or treating infectious diseases.
  • the present application provides the composition described in the present application; and/or the kit described in the present application, which are used for preventing, alleviating and/or treating infectious diseases.
  • the present application provides a method for preventing, alleviating and/or treating infectious diseases, which comprises the following steps: administering the composition described in the present application to a subject in need; and/or the kit described in the present application.
  • infectious diseases include diseases caused by viral infection, bacterial infection, fungal infection and/or parasitic infection.
  • the present application also provides methods for producing the compositions described herein, and/or the kits described herein.
  • the method may include mixing together any relevant components necessary to prepare the compositions described herein, in any order.
  • the method may comprise preparing a premix (or premix) of the excipient, the buffer component and/or the surfactant.
  • the method may include forming a buffer system.
  • the buffer system may comprise buffer components as described herein.
  • the methods described herein can include removing particulate matter (eg, by filtration).
  • Endogenous fluorescence spectrum Utilize the fluorescence spectrum of endogenous fluorescent amino acids (tryptophan, tyrosine, phenylalanine) in protein molecules in polar and non-polar environments. If the fluorescence spectrum is red-shifted, that is, the maximum emission wavelength changes, and thus indirectly infers the consistency of the higher-order structure of the protein, that is, the mutual transformation of the higher-order structure.
  • the sample was diluted to 40 mg/mL, and the high-throughput endogenous fluorescence spectrometer optim1000 (Pall Company) was used. The test temperature range was 20-95°C, and the test interval was 1°C.
  • the SLS (static scattered light) result at 473nm wavelength is used to judge the polymerization propensity of the sample.
  • the denaturation temperature is the value measured under BCM (centroid wavelength), which is calculated as the Tm value (half denaturation temperature) according to the formula for evaluation.
  • SEC-HPLC Size-Exclusion Chromatography: Refer to "Chinese Pharmacopoeia” 2015 Edition Three General Rules 0514 Size-Exclusion Chromatography, used to detect the monomer content of the product, as well as aggregates and fragments.
  • the chromatographic column is TOSOH G3000 7.8*300mm, 5 ⁇ m; the mobile phase is 20mM Na 2 HPO 4 , 300mMNaCl; the detection wavelength is 280nm.
  • WCX-HPLC Wash Cation Exchange Chromatography: Refer to the "Chinese Pharmacopoeia” 2015 edition three general rules 0512 high performance liquid chromatography, used to detect the main component, acidic component and basic component content of the product. Among them, the chromatographic column is Propac WCX-10 4 ⁇ 250mm, 10 ⁇ m; the mobile phase is 10mM Na 2 HPO 4 , 10mM Na 2 HPO 4 +500mM NaCl; the detection wavelength is 280nm.
  • Binding and blocking activity refer to the test methods described in Examples 5.2 and 5.3 of WO 2017/020802A1, respectively.
  • Viscosity The viscosity is measured in automatic mode by using the American rheosense portable micro viscometer microVISC.
  • Osmotic pressure detected by the osmotic pressure method.
  • CE-SDS purity After the test sample is denatured (non-reducing condition) or denatured and reduced (reducing condition), it is put into a capillary electrophoresis apparatus, analyzed by electrophoresis, and calculated by the area normalization method.
  • Protein content refer to the sixth method of the "Chinese Pharmacopoeia” 2015 edition three general rules 0731 protein content determination method, using a micro-ultraviolet spectrophotometer Nanodrop 2000, according to the light absorption value of the protein at a wavelength of 280nm and the theoretical extinction coefficient of the protein, Calculate protein content.
  • Turbidity measurement Measure the absorbance of the sample at 340 nm on an Agilent 8453 spectrophotometer.
  • Insoluble particles Measure the number of particles ⁇ 10 ⁇ m and ⁇ 25 ⁇ m in the sample on the AccuSizer 780SIS insoluble particle analyzer.
  • DLS Dynamic Light Scattering
  • 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.
  • this antigen-binding protein is simply referred to as "PD-L1 monoclonal antibody".
  • the PD-L1 monoclonal antibody is obtained by artificially synthesizing the amino acid sequence.
  • the PD-L1 monoclonal antibody comprises the amino acid sequence shown in any one of SEQ ID NO: 14-31, especially the amino acid sequence shown in SEQ ID NO: 21.
  • the proline mentioned in the examples includes L-proline.
  • Tm values and aggregation tendency detection results corresponding to prescriptions 1-19 are shown in Figure 1 and Figure 2, respectively.
  • prescription 1 and prescription 3 (pH 6.5 sodium acetate-acetic acid buffer system); prescription 5 and prescription 7 (pH 6.5 sodium phosphate buffer system) were compared respectively. It was found that the inclusion of L-proline was associated with a further increase in stability.
  • the acidic components may include components produced by the decomposition of PD-L1 monoclonal antibody under high temperature conditions.
  • Recipe 1 and Prescription 3 were selected for activity determination of high temperature treatment.
  • FIG. 7 The results of the binding activity and blocking activity of the two are shown in Figure 7, Figure 8 and Table 3, respectively.
  • A-D in Figure 7-8 represent the results of the 0th day of prescription 1, the 0th day of prescription 3, the 28th day of prescription 1, and the 28th day of prescription 3. According to the results of the prescription, there was no significant difference in the activity of prescription 1 and prescription 3 at high temperature for 28 days, and there was no significant difference compared with 0 day.
  • the PD-L1 monoclonal antibody content is 200mg/mL, 20mM sodium acetate-acetic acid buffer solution, 220mM proline (L-proline) stabilizer, and 0.02% polysorbate 20, polysorbate 80, polo Sham P188 is a surfactant, and the target protein is detected by SEC-HPLC and WCX-HPLC on the accelerated 0 day, 14 day and 28 day respectively, and the influence of different surfactant types on the stability of the preparation is investigated. See the preparation prescription 19A and 20A , 21A. The results of the study showed that polysorbate 20, polysorbate 80, and poloxamer P188 can be used as surfactants for PD-L1 monoclonal antibody preparations.
  • PD-L1 monoclonal antibody content 200mg/mL, 20mM sodium acetate-acetic acid buffer, 0.02% polysorbate 20 as surfactant, pH 6.4, respectively choose 220mM L-proline, 90mg/mL sucrose, 250mM glycine , 150mM L-arginine hydrochloride, and 150mM sodium chloride were used as stabilizers, and the target protein was detected by SEC-HPLC and WCX-HPLC on the accelerated 0 day, 14 days, and 28 days, respectively, and the stability of the preparation was investigated by different types of stabilizers. Sexual effects, see formulation prescriptions 19A, 26A, 27A, 28A, 29A. The results of the study showed that L-proline, sucrose, glycine, L-arginine hydrochloride or sodium chloride could be used as stabilizers for PD-L1 monoclonal antibody preparations.
  • the PD-L1 monoclonal antibody content is 200mg/mL, 220mM L-proline is used as a stabilizer, 0.02% polysorbate 20 is used as a surfactant, pH6.4, and 20mM sodium acetate-acetic acid and L-histamine are used respectively Acid-acetic acid, L-histidine-L-histidine hydrochloric acid, sodium phosphate, citric acid-sodium hydroxide buffer, accelerated by SEC-HPLC and WCX-HPLC at 0 days, 14 days and 28 days respectively Detect the target protein, investigate the influence of different buffer components on the stability of the preparation, see preparation prescription 19A, 22A, 23A, 24A, 25A.
  • preparations 33A, 34A and 19A show that preparations containing PD-L1 monoclonal antibody concentrations of 20mg/mL-220mg/mL concentration, under certain buffer, stabilizer, surfactant and/or pH conditions, All meet the requirements of formulation stability.
  • composition described in the present application has a suitable viscosity, which helps to be administered easily (such as subcutaneous injection) and quickly (such as reducing the number of injections and greatly shortening the administration time), while improving the patient's medication compliance and availability. Improving patient quality of life.
  • Viscosity 19A 18.200 20A 18.270 21A 18.470 22A 17.080 23A 17.250 24A 16.390 25A 20.480 26A 22.980 27A 17.440 28A 17.280 29A 19.690 33A 1.239 34A 22.640
  • the prescription formulation is further optimized, and the pH, buffer system, stabilizer and surfactant of the prescription are further screened respectively.
  • Table 9 Summary of test results of pH 4.5-7.5 range investigation 40 ⁇ 2°C high temperature acceleration for 28 days
  • SEC-HPLC test results showed that: under the conditions of pH 5.0 and pH 7.5, the monomers were significantly reduced; WCX-HPLC test results showed that: under the conditions of pH 7.5, the main components were significantly reduced.
  • the detection results of insoluble particles showed that there were fewer insoluble particles under lower pH conditions, and the number of insoluble particles tended to decrease further as the pH increased.
  • the PDI index reflects the uniformity of the solution. The smaller the value, the better the uniformity. The results show that the solution has the best uniformity when the pH is 6.4, and as the pH increases, the PDI value of the solution shows a trend of first decreasing and then increasing.
  • Table 11 Summary of test results of different stabilizers in sodium acetate-acetate buffer solution at 40 ⁇ 2°C for 28 days at high temperature acceleration
  • DLS test results show: average hydration diameter (Z.Av.Diameter) results show: the hydration diameter of the prescription containing sodium chloride is larger, and other prescriptions show a smaller average particle size; distribution coefficient (PDI) results show: Except for the values of sugar, maltose and sodium chloride>0.3, the other prescriptions have no obvious difference, showing good uniformity.
  • Viscosity testing results show that the viscosity of adding 50mg/mL sucrose, 50mg/mL mannitol and 90mg/mL trehalose is >20cp, and other formulations are ⁇ 20cp. Compared with the formulation without adding antioxidant methionine, the viscosity is slightly lower.
  • Table 12 Summary of test results of different stabilizers in L-histidine-L-histidine hydrochloride buffer at 40 ⁇ 2°C for 28 days under high temperature acceleration
  • results of Z.Av.Diameter in the DLS test show that the hydration diameter of the formulation containing sodium chloride is larger, and the average particle size of other formulations is smaller; the results of PDI value show that: except for the formulation containing trehalose and maltose, the PDI value> 0.3 , the other prescriptions had no significant difference, showing better uniformity.
  • Table 13 Summary of test results of different stabilizers in citrate buffer for 28 days accelerated at 40 ⁇ 2°C
  • the SEC-HPLC test results showed that there was no significant difference between different prescriptions; the WCX-HPLC test results showed that only 220mM L-proline was contained, and the main component was significantly reduced compared with other prescriptions. The difference is not obvious.
  • the osmotic pressure of the infusion solution is less than 600mOsmoL/kg is in the safe range, so the osmotic pressure of prescriptions 20, 24, and 28 is in the safe range.
  • buffer system sodium acetate concentration (5-35mM), stabilizer L-proline concentration (50-390mM), polysorbate 20 concentration (0-0.04%) are important for protein detection SEC-HPLC monomer and WCX-
  • the main components of HPLC have no significant effect, and the change is small within this range.
  • Protein content and pH have an impact on protein detection of SEC-HPLC monomers and WCX-HPLC main components, which vary greatly within this range.
  • the protein concentration is 50-220 mg/mL, and the purity of SEC-HPLC monomers is 95 %, the pH is between 5.9-6.9, and the WCX-HPLC main component is above 74%, which are all within the acceptable range, and it is judged that the change is acceptable.
  • PD-L1 monoclonal antibody protein content is 200 ⁇ 20mg/mL, 20mM sodium acetate-acetic acid, 220mM L-proline, 0.02% polysorbate 20, pH 6.4 ⁇ 0.1, divided into vials, stored at 25 ⁇ 2°C for 9 months, and samples were taken at the end of the 0, 3, 6 and 9 months respectively.
  • the test was carried out according to the key investigation items of stability, and the purity of the sample monomer (SEC-HPLC), the purity of the main component of the charge distribution (WCX-HPLC), the reduction and non-reduction capillary gel electrophoresis (CE-SDS), the protein content, etc. were investigated, and the results were obtained. As shown in Table 20.
  • PD-L1 monoclonal antibody protein content is 200 ⁇ 20mg/mL, 20mM sodium acetate-acetic acid, 220mM L-proline, 0.02% polysorbate 20, pH 6.4 ⁇ 0.1, divided into vials, stored at 5 ⁇ 3°C for 18 months, and samples were taken at the end of the 0, 6, 12 and 18 months respectively.
  • the test was carried out according to the key investigation items of stability, and the purity of the sample monomer (SEC-HPLC), the purity of the main component of the charge distribution (WCX-HPLC), the reduction and non-reduction capillary gel electrophoresis (CE-SDS), the protein content, etc. were investigated, and the results were obtained. As shown in Table 21.

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Abstract

一种组合物，其包含抗原结合片段和辅料，所述抗原结合片段包含能够特异性结合PD-L1的免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含CDR1-3，其中所述CDR3包含SEQ ID NO:47所示的氨基酸序列，所述辅料可以包含脯氨酸。所述组合物稳定性好、粘度适宜，可以用于皮下注射。

Description

包含PD-L1抗原结合片段的组合物及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种包含PD-L1抗原结合片段的组合物及其用途。
背景技术
PD-L1的表达已经在几种鼠和人类癌症中发现，包括人肺癌、卵巢癌、结肠癌、黑色素瘤和各种骨髓瘤。已有的结果显示，肿瘤细胞高表达的PD-L1通过增加T细胞的凋亡从而在肿瘤的免疫逃逸中起着重要的作用。
抗体药物配制剂型包括注射用制剂或冻干制剂等等。对于频繁使用的产品以及慢性给药，皮下给药途径是更可取的。这将使患者能够自我管理，增加治疗的方便性、快捷性和依从性，为医疗保健部门节省大笔开支，提升患者生活质量。
因此需要开发高浓度的PD-L1抗体制剂。
发明内容
本申请提供了一种组合物，其包含特异性结合PD-L1的抗原结合片段和辅料。所述组合物包含以下有益效果中的至少一个：1)包含高浓度(例如200mg/mL)的抗原结合片段；2)粘度适宜；3)能够适用于皮下注射，降低受试者的疼痛感；4)稳定性(例如加速稳定性和/或长期稳定性)好；5)避免输液而引发的副作用；6)组分简单，制备简便且成本低。
本申请提供了一种组合物，其包含抗原结合片段和辅料，其中所述抗原结合片段包含能够特异性结合PD-L1的免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含CDR1-3，其中所述CDR3包含SEQ ID NO:47所示的氨基酸序列，其中所述辅料选自下组中的一种或多种：脯氨酸、甘露醇、蔗糖、甘氨酸和L-精氨酸盐酸盐。
在另一些实施方式中，所述辅料还可以选自下组中的一种或多种：脯氨酸、蔗糖、甘露醇、山梨醇、甘油和L-精氨酸盐酸盐。
在某些实施方式中，所述辅料优选自下组中的一种或多种：脯氨酸、蔗糖、甘露醇、山梨醇和甘油；更优选为脯氨酸、蔗糖、甘露醇和山梨醇中的一种或多种。
在某些实施方式中，所述辅料为L-脯氨酸，或所述辅料为蔗糖，或所述辅料为甘露醇， 或所述辅料为山梨醇。
在某些实施方式中，所述CDR2包含SEQ ID NO:46所示的氨基酸序列。
在某些实施方式中，所述CDR1包含SEQ ID NO:45或48所示的氨基酸序列。
在某些实施方式中，所述免疫球蛋白单一可变结构域包含SEQ ID NO:1-6中任一项所示的氨基酸序列。
在某些实施方式中，所述PD-L1为人PD-L1。
在某些实施方式中，所述抗原结合片段包含免疫球蛋白的Fc区。
在某些实施方式中，所述免疫球蛋白的Fc区的N端与所述免疫球蛋白单一可变结构域的C端直接或间接连接。
在某些实施方式中，所述免疫球蛋白的Fc区的N端与所述免疫球蛋白单一可变结构域的C端通过连接子连接。
在某些实施方式中，所述连接子包含SEQ ID NO:42-44中任一项所示的氨基酸序列。
在某些实施方式中，所述免疫球蛋白的Fc区包含源自选自下组的免疫球蛋白的Fc区：IgG1、IgG2、IgG3和IgG4。
在某些实施方式中，所述免疫球蛋白的Fc区不具有ADCC活性；和/或，所述免疫球蛋白的Fc区不具有CDC活性。
在某些实施方式中，所述免疫球蛋白的Fc区来源于人。
在某些实施方式中，所述免疫球蛋白的Fc区来源于人IgG。
在某些实施方式中，所述免疫球蛋白的Fc区包含SEQ ID NO:7-9中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合片段包含SEQ ID NO:14-31中任一项所示的氨基酸序列。
在某些实施方式中，所述抗原结合片段的浓度为约1mg/mL-约300mg/mL。
在某些实施方式中，所述抗原结合片段的浓度为约5mg/mL-约250mg/mL。
在某些实施方式中，所述抗原结合片段的浓度为约1mg/mL-约220mg/mL。
在某些实施方式中，所述抗原结合片段的浓度为约20mg/mL-约220mg/mL。
在某些实施方式中，所述抗原结合片段的浓度为约50mg/mL-约200mg/mL。
在某些实施方式中，所述抗原结合片段的浓度为约100mg/mL-约280mg/mL。
在某些实施方式中，所述抗原结合片段的浓度为约150mg/mL-约250mg/mL。
在某些实施方式中，所述抗原结合片段的浓度为约180mg/mL-约250mg/mL。
在某些实施方式中，所述抗原结合片段的浓度为约180mg/mL-约220mg/mL。
在某些实施方式中，所述辅料的浓度为约50mM-约500mM。
在某些实施方式中，所述辅料包含脯氨酸，所述脯氨酸的浓度为约50mM-约390mM。
在某些实施方式中，所述辅料包含脯氨酸，所述脯氨酸的浓度为约150mM-约250mM。
在某些实施方式中，所述辅料包含脯氨酸，所述脯氨酸的浓度为约150mM-约220mM。
在某些实施方式中，所述辅料包含L-脯氨酸，所述L-脯氨酸的浓度为约150mM-约220mM。
在某些实施方式中，所述辅料包含L-脯氨酸，所述L-脯氨酸的浓度为约165mM-约275mM。
在某些实施方式中，所述辅料包含甘露醇，所述甘露醇的浓度为约50mM-约500mM。
在某些实施方式中，所述辅料包含蔗糖，所述蔗糖的浓度为约50mM-约500mM。
在某些实施方式中，所述辅料包含蔗糖，所述蔗糖的浓度为约90mM-约500mM。
在某些实施方式中，所述辅料包含甘氨酸，所述甘氨酸的浓度为约50mM-约500mM。
在某些实施方式中，所述辅料包含甘氨酸，所述甘氨酸的浓度为约150mM-约250mM。
在某些实施方式中，所述辅料包含L-精氨酸盐酸盐，所述L-精氨酸盐酸盐的浓度为约50mM-约500mM。
在某些实施方式中，所述辅料包含L-精氨酸盐酸盐，所述L-精氨酸盐酸盐的浓度为约150mM-约500mM。
在某些实施方式中，所述辅料包含山梨醇，所述山梨醇的浓度为约50mM-约500mM。
在某些实施方式中，所述辅料包含山梨醇，所述山梨醇的浓度为约100mM-约300mM。
在某些实施方式中，所述辅料包含甘油，所述甘油的浓度为50mM-约500mM。
在某些实施方式中，所述辅料包含甘油，所述甘油的浓度为约100mM-约300mM。
在某些实施方式中，所述组合物的pH为约5.0-约7.5。
在某些实施方式中，所述组合物的pH为约5.9-约6.9。
在某些实施方式中，所述组合物的pH为约6.3-约6.5。
在某些实施方式中，所述组合物的pH为约6.4。
在某些实施方式中，所述组合物包含缓冲成分，其中所述缓冲成分选自下组中的一种或多种：乙酸钠-乙酸、组氨酸-乙酸、L-组氨酸-L-组氨酸盐酸盐、磷酸钠和柠檬酸-氢氧化钠。
在某些实施方案中，所述缓冲成分选自下组中的一种或多种：乙酸钠-乙酸、L-组氨酸-L-组氨酸盐酸盐和柠檬酸-氢氧化钠；优选地，所述缓冲成分为乙酸钠-乙酸或L-组氨酸-L-组氨 酸盐酸盐。
在某些实施方式中，所述缓冲成分的浓度为约5mM-约50mM。
在某些实施方式中，所述缓冲成分包含乙酸钠-乙酸，所述乙酸钠-乙酸的浓度为约5mM-约35mM。
在某些实施方式中，所述缓冲成分包含乙酸钠-乙酸，所述乙酸钠-乙酸的浓度为约10mM-约30mM。
在某些实施方式中，所述缓冲成分包含乙酸钠-乙酸，所述乙酸钠-乙酸的浓度为约20mM。
在某些实施方式中，所述缓冲成分包含L-组氨酸-L-组氨酸盐酸盐，所述L-组氨酸-L-组氨酸盐酸盐的浓度为约5mM-约35mM，优选为约10mM-约30mM，甚至更优选为20mM。在某些实施方式中，所述组合物包含表面活性剂，其中所述表面活性剂选自下组中的一种或多种：聚山梨酯20、聚山梨酯80和泊洛沙姆188。
在某些实施方式中，所述表面活性剂的浓度为约0mg/mL-约0.8mg/mL。
在某些实施方式中，所述表面活性剂包含聚山梨酯20，所述聚山梨酯20的浓度为约0mg/mL-约0.4mg/mL。
在某些实施方式中，所述表面活性剂包含聚山梨酯20，所述聚山梨酯20的浓度为约0.2mg/mL-约0.4mg/mL。
在某些实施方式中，所述表面活性剂包含聚山梨酯20，所述聚山梨酯20的浓度为约0.1mg/mL-约0.3mg/mL。
在某些实施方式中，所述表面活性剂包含聚山梨酯20，所述聚山梨酯20的浓度为约0.2mg/mL。
在某些实施方式中，所述表面活性剂包含聚山梨酯80，所述聚山梨酯80的浓度为约0.05mg/mL-约1mg/mL，优选为0.1mg/mL-0.5mg/mL，更优选为0.1mg/mL-0.3mg/mL，甚至更优选为0.2mg/mL。
在某些实施方式中，所述表面活性剂包含泊洛沙姆188，所述泊洛沙姆188的浓度为约0.05mg/mL-约5mg/mL，优选为0.1mg/mL-约3mg/mL，更优选为0.2mg/mL-约2mg/mL.
在某些实施方式中，所述组合物包含：1)约20mg/mL-约220mg/mL本申请所述的抗原结合片段；2)约5mM-约35mM乙酸钠-乙酸；3)约50mM-约390mM脯氨酸；4)约0mg/mL-约0.4mg/mL聚山梨酯20；且pH为约5.9-约6.9。
在某些实施方式中，所述组合物包含：1)约50mg/mL-约220mg/mL本申请所述的抗 原结合片段；2)约10mM-约30mM乙酸钠-乙酸；3)约150mM-约250mM脯氨酸；4)约0.2mg/mL-约0.4mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物包含：1)约150mg/mL-约250mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM乙酸钠-乙酸；3)约150mM-约250mM脯氨酸；4)约0.2mg/mL-约0.4mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物包含：1)约200mg/mL本申请所述的抗原结合片段；2)约20mM乙酸钠-乙酸；3)约220mM脯氨酸；4)约0.2mg/mL聚山梨酯20；且pH为约6.4。
在某些实施方式中，所述组合物包含：1)约100mg/mL-约280mg/mL本申请所述的抗原结合片段；2)约5mM-约35mM乙酸钠-乙酸；3)约50mM-约500mM蔗糖；4)约0.05mg/mL-约0.4mg/mL聚山梨酯20；且pH为约5.9-约6.9。
在某些实施方式中，所述组合物包含：1)约150mg/mL-约250mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM乙酸钠-乙酸；3)约50mM-约300mM蔗糖；4)约0.1mg/mL-约0.3mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物包含：1)约100mg/mL-约280mg/mL本申请所述的抗原结合片段；2)约5mM-约35mM乙酸钠-乙酸；3)约50mM-约500mM甘露醇；4)约0.05mg/mL-约0.4mg/mL聚山梨酯20；且pH为约5.9-约6.9。
在某些实施方式中，所述组合物包含：1)约150mg/mL-约250mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM乙酸钠-乙酸；3)约100mM-约300mM甘露醇；4)约0.1mg/mL-约0.3mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物包含：1)约100mg/mL-约280mg/mL本申请所述的抗原结合片段；2)约5mM-约35mM乙酸钠-乙酸；3)约50mM-约500mM山梨醇；4)约0.05mg/mL-约0.4mg/mL聚山梨酯20；且pH为约5.9-约6.9。
在某些实施方式中，所述组合物包含：1)约150mg/mL-约250mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM乙酸钠-乙酸；3)约100mM-约300mM山梨醇；4)约0.1mg/mL-约0.3mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物包含：1)约100mg/mL-约280mg/mL本申请所述的抗原结合片段；2)约5mM-约35mM乙酸钠-乙酸；3)约50mM-约500mM甘油；4)约0.05mg/mL-约0.4mg/mL聚山梨酯20；且pH为约5.9-约6.9。
在某些实施方式中，所述组合物包含：1)约150mg/mL-约250mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM乙酸钠-乙酸；3)约100mM-约300mM甘油；4)约0.1mg/mL-约0.3mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物包含：1)约150mg/mL-约250mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM L-组氨酸-L-组氨酸盐酸盐；3)约150mM-约250mM脯氨酸；4)约0.2mg/mL-约0.4mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物包含：1)约150mg/mL-约250mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM L-组氨酸-L-组氨酸盐酸盐；3)约50mM-约300mM蔗糖；4)约0.1mg/mL-约0.3mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物包含：1)约150mg/mL-约250mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM L-组氨酸-L-组氨酸盐酸盐；3)约100mM-约300mM甘露醇；4)约0.1mg/mL-约0.3mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物包含：1)约150mg/mL-约250mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM L-组氨酸-L-组氨酸盐酸盐；3)约100mM-约300mM山梨醇；4)约0.1mg/mL-约0.3mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物包含：1)约150mg/mL-约250mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM L-组氨酸-L-组氨酸盐酸盐；3)约100mM-约300mM甘油；4)约0.1mg/mL-约0.3mg/mL聚山梨酯20；且pH为约6.3-约6.5。
在某些实施方式中，所述组合物用于注射。
在某些实施方式中，所述组合物用于皮下注射。
在某些实施方式中，所述组合物为制剂。
在某些实施方式中，所述组合物为液体制剂。
另一方面，本申请提供一种试剂盒，其包括本申请所述的组合物和盛放本申请所述的组合物的容器。
在某些实施方式中，所述容器包括玻璃瓶。
在某些实施方式中，所述容器中所述组合物的体积为约0.5mL-约5.0mL。
在某些实施方式中，所述容器中所述组合物的体积为约0.5mL-约1.5mL。
另一方面，本申请提供了本申请所述的组合物；和/或本申请所述的试剂盒在制备预防、缓解和/或治疗肿瘤的药物中的应用。
在某些实施方式中，所述肿瘤包括PD-L1阳性的肿瘤。
在某些实施方式中，所述肿瘤包括恶性实体瘤。
另一方面，本申请提供了本申请所述的组合物；和/或本申请所述的试剂盒在制备预防、缓解和/或治疗感染性疾病的药物中的应用。
在某些实施方式中，所述感染性疾病包括病毒感染、细菌感染、真菌感染和/或寄生虫感染所引起的疾病。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是包含本申请所述抗原结合片段的制剂处方的Tm值结果。
图2显示的是包含本申请所述抗原结合片段的制剂处方的聚体倾向性检测结果。
图3显示的是包含本申请所述抗原结合片段的制剂处方的SEC-HPLC主峰含量检测结果。
图4显示的是包含本申请所述抗原结合片段的制剂处方的SEC-HPLC聚体含量检测结果。
图5显示的是包含本申请所述抗原结合片段的制剂处方的WCX-HPLC酸性组分检测结果。
图6显示的是包含本申请所述抗原结合片段的制剂处方的粘度检测结果。
图7显示的是包含本申请所述抗原结合片段的制剂处方的结合活性结果。
图8显示的是包含本申请所述抗原结合片段的制剂处方的阻断活性结果。
图9显示的是包含本申请所述抗原结合片段的制剂处方的SEC-HPLC主峰含量检测结果。
图10显示的是本申请所述抗原结合片段的制剂处方的SEC-HPLC聚体含量检测结果。
图11显示的是本申请所述抗原结合片段的制剂处方的WCX-HPLC酸性组分检测结果。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
术语定义
在本申请中，术语“抗原结合片段”通常是指，保留与抗原的表位特异性相互作用的能力(例如通过结合或空间位阻等)的抗体的一个或多个部分。抗体的抗原结合功能也可通过以下来实现：包括Fv、ScFv、dsFv、Fab、Fab’或F(ab’)2的片段的重链，或者，包括Fv、scFv、dsFv、Fab、Fab’或F(ab’)2的片段的轻链。(1)Fab片段，即由VL、VH、CL和CH结构域组成的一价片段；(2)F(ab’)2片段，包含通过铰链区处的二硫键连接的两个Fab片段的二价片段；(3)由VH和CH结构域组成的Fd片段；(4)由抗体单臂的VL和VH结构域组成的Fv片段；(5)由VH结构域组成的dAb片段(Ward等，(1989)Nature 341：544-546)；(6)分离的互补决定区(CDR)和(7)可任选地通过接头连接的两个或以上分离的CDR的组合。例如，还可包括由VL和VH配对形成的一价单链分子Fv(scFv)(参见Bird等(1988)Science 242：423-426；以及Huston等(1988)Proc.Natl.Acad.Sci.85：5879-5883)。例如，还可包括缺失抗体轻链而只有重链可变区的一类抗体VHH(例如，可参见康晓圳等，生物工程学报,2018,34(12):1974-1984)。所述“抗原结合部分”还可包括免疫球蛋白融合蛋白，所述融合蛋白包含选自以下的结合结构域：(1)与免疫球蛋白铰链区多肽融合的结合结构域多肽；(2)与铰链区融合的免疫球蛋白重链CH2恒定区；和(3)与CH2恒定区融合的免疫球蛋白重链CH3恒定区。
在本申请中，术语“辅料”通常是指，针对所述组合物所希望的任何类型或产品形式所需而选择的任何材料。例如，所述产品形式可以包括液体、颗粒、粉末、糊状、喷雾、片剂或凝胶。所述辅料可以不影响所述抗原结合片段的存在形式或稳定性，也可以不影响所述抗原结合片段的任何生物学活性(例如与相应抗原的特异性结合)。在本申请中，所述辅料可以发挥稳定剂的作用，提高所述组合物，和/或提高所述抗原结合片段在所述组合物中的稳定性。
在本申请中，术语“PD-L1”通常是指程序化细胞死亡蛋白1配体1，也可以称为CD274或B7H1。在本申请中，所述PD-L1可以为哺乳动物来源的PD-L1，例如，所述PD-L1可以来源于灵长类，也可以来源于啮齿类(例如小鼠或大鼠)。人PD-L1可以具备NCBI登录号NP_054862.1所示的氨基酸序列。所述PD-L1可以包含天然的PD-L1，也可以包含PD-L1的 变体、同种型、物种同源物以及与PD-L1具有至少一个共同表位的类似物。
在本申请中，术语“免疫球蛋白可变结构域”通常是指，包含“框架区1”或“FR1”、“框架区2”或“FR2”、“框架区3”或“FR3”、及“框架区4”或“FR4”的四个“框架区”以及“互补决定区1”或“CDR1”、“互补决定区2”或“CDR2”、及“互补决定区3”或“CDR3”的三个“互补决定区”或“CDR”的多肽分子。所述免疫球蛋白可变结构域可以具有蛋白质的三级结构。在本申请中，所述免疫球蛋白可变结构域可以具备与抗原(例如PD-L1)特异性结合的功能。所述免疫球蛋白可变结构域可以包含FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的结构。
在本申请中，术语“免疫球蛋白单一可变结构域”通常是指，具有独立与抗原(例如PD-L1)特异性结合的功能的所述免疫球蛋白可变结构域。例如，所述免疫球蛋白单一可变结构域可以在不与其他免疫球蛋白可变结构域配对的情况下特异性结合抗原或其表位。例如，所述免疫球蛋白单一可变结构域可以为重链可变区VH和/或轻链可变区VL。在本申请中，所述免疫球蛋白单一可变结构域可以为VHH。在本申请中，所述VHH，或可称为重链抗体、nano抗体或单域抗体。所述VHH的CDR可以按照Kabat法划分(参见“Sequence of proteins of immunological interest”,US Public Health Services,NIH Bethesda,MD)：例如，VHH氨基酸序列的第1-30位氨基酸为FR1、第31-35位氨基酸为CDR1、第36-49位氨基酸为FR2、第50-65位氨基酸为CDR2、第66-94位氨基酸为FR3、第95-102位氨基酸为CDR3、第103-113位氨基酸为FR4。然而应注意，由于各CDR的氨基酸残基的总数可能不同，且可能不对应于由Kabat编号指示的氨基酸残基的总数(即根据Kabat编号的一个或多个位置可能在实际序列中未被占据，或实际序列可能含有多于Kabat编号所允许数目的氨基酸残基)，因此根据Kabat的编号可能对应也可能不对应于实际序列中氨基酸残基的实际编号。
在本申请中，术语“组合物”通常是指包含本申请所述抗原结合片段的混合物。所述组合物可以包含至少一种可药用的成分，例如本申请所述的辅料。
在本申请中，术语“制剂”通常是指，以预定量或比例包含本申请所述的抗原结合片段的产品，以及任何直接或间接地通过将预定量的本申请所述的抗原结合片段进行组合而产生的产品。本申请所述制剂的含义可以包含药物制剂，即包含了本申请所述的抗原结合片段和所述辅料，以及任何直接或间接地由任意两种或更多种成分组合、复合或聚集，而产生的产品。所述制剂可以以液态的形式存在。
在本申请中，术语“缓冲成分”通常是指具有提供缓冲效应(例如抵抗pH值改变)的功能的试剂。例如，所述缓冲成分可以调节由于酸性或碱性物质的添加和/或释放所导致的pH的 改变。例如，所述缓冲成分可以包括弱酸及其共轭碱；也可以包括弱碱及其共轭酸。
在本申请中，术语“表面活性剂”通常是指可以保护蛋白质(例如本申请所述抗原结合片段)免受空气/溶液界面诱导的应力、溶液/表面诱导的应力的影响以减少该蛋白质的聚集或使所述组合物中颗粒物的形成最小化的试剂。所述表面活性剂可以同时包含疏水性基团和亲水性基团。所述表面活性剂可以包括一种或多种表面活性剂的混合物或组合。所述表面活性剂可以包括非离子型表面活性剂。
在本申请中，术语“PD-L1阳性”通常是指，在包含肿瘤细胞和肿瘤浸润型炎性细胞的供试组织样品中的表达PD-L1细胞比例高于将该样品评为表达细胞表面PD-L1的水平所对应的细胞比例。例如，所述细胞比例可以通过免疫组织化学(IHC)测定。在本申请中，所述PD-L1阳性可以为某一样品中至少约0.01％、至少约0.5％、至少约1％、至少约2％、至少约3％、至少约4％、至少约5％、至少约6％、至少约7％、至少约8％、至少约9％、至少约10％、至少约15％、至少约20％、至少约25％或至少约30％总数的细胞表达PD-L1。
在本申请中，术语“肿瘤”通常是指，细胞不正常生长而导致的损伤。例如，所述肿瘤可以通过快速、不受控的细胞增殖形成单独的组织块。所述组织块可以为良性的、前恶性的或恶性的。所述肿瘤具有无限制生长和/或攻击正常组织的结构和/或功能时，可被认为是恶性的。所述恶性的肿瘤可以与癌症交换使用。
在本申请中，术语“恶性实体瘤”通常是指具有转移潜力的任何类型的具有一定形态的肿瘤。
在本申请中，术语“感染性疾病”通常是指病毒、细菌、真菌、寄生虫等病原体及特定毒素侵入人体所引起的局部组织和全身性炎症反应。
在本申请中，术语“约”通常是指相应值的通常误差范围。在此提及的“约”某一值或参数包括(并描述)了针对该值或参数本身的方案。如果有疑问，或者对于特定值或参数的误差范围没有被该领域承认的通常理解，则“约”意味着该值或参数的±5％。
发明详述
一方面，本申请提供了一种组合物，其可以包含抗原结合片段和辅料，其中所述抗原结合片段可以包含能够特异性结合PD-L1的免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域可以包含SEQ ID NO:1-6中任一项所示的氨基酸序列，其中所述辅料选自下组中的一种或多种：脯氨酸、甘露醇、蔗糖、甘氨酸和L-精氨酸盐酸盐。
抗原结合片段
在本申请中，所述抗原结合片段可以特异性结合PD-L1。例如，所述PD-L1可以为人PD-L1。
在本申请中，所述抗原结合片段可以包含免疫球蛋白的Fc区。例如，所述抗原结合片段可以由式A-L-B的氨基酸序列组成，其中A表示免疫球蛋白单一可变结构域，L表示氨基酸接头或不存在，B表示人免疫球蛋白Fc区。
例如，所述免疫球蛋白的Fc区的N端与所述免疫球蛋白单一可变结构域的C端可以直接或间接连接。
例如，所述免疫球蛋白的Fc区的N端与所述免疫球蛋白单一可变结构域的C端可以通过连接子连接。
在本申请中，所述免疫球蛋白单一可变结构域可以包含抗原互补决定区CDR1-3。例如，所述CDR1可以包含SEQ ID NO:45或48所示的氨基酸序列。例如，所述CDR2可以包含SEQ ID NO:46所示的氨基酸序列。例如，所述CDR3可以包含SEQ ID NO:47所示的氨基酸序列。例如，所述CDR1可以包含SEQ ID NO:45所示的氨基酸序列；所述CDR2可以包含SEQ ID NO:46所示的氨基酸序列；且所述CDR3可以包含SEQ ID NO:47所示的氨基酸序列。例如，所述CDR1可以包含SEQ ID NO:48所示的氨基酸序列；所述CDR2可以包含SEQ ID NO:46所示的氨基酸序列；且所述CDR3可以包含SEQ ID NO:47所示的氨基酸序列。
在本申请中，所述连接子可以具有约1-约20个氨基酸残基的长度。例如，所述连接子可以无二级以上结构。例如，所述连接子可以为柔性连接子。例如，所述连接子可以为GGGGS，GS或GAP。例如，所述连接子可以包含SEQ ID NO:42-44中任一项所示的氨基酸序列。
例如，所述免疫球蛋白的Fc区可以包含源自选自下组的免疫球蛋白的Fc区：IgG1、IgG2、IgG3和IgG4。其中，所述人IgG1、IgG2、IgG3或IgG4的恒定区可以分别据具备参考www.uniprot.org蛋白数据库中的条目P01857、P01859、P01860、P01861所示的氨基酸序列。
例如，所述免疫球蛋白的Fc区可以来源于人。例如，所述免疫球蛋白的Fc区可以来源于人IgG。例如，所述免疫球蛋白的Fc区可以来源于人IgG1。
在本申请中，所述免疫球蛋白的Fc区可以不具有ADCC活性；和/或，所述免疫球蛋白的Fc区可以不具有CDC活性。例如，所述免疫球蛋白的Fc区可以通过突变而丧失ADCC活性；和/或，CDC活性。例如，所述免疫球蛋白的Fc区可以包含SEQ ID NO:7-9中任一项 所示的氨基酸序列。
在本申请中，所述抗原结合片段可以具有同二聚体。所述同二聚体可以通过所述免疫球蛋白的Fc区的相互作用而形成。
在本申请中，所述抗原结合片段可以以小于1×10 -7M，小于1×10 -8M、小于1×10 -9M、小于1×10 -10M、小于1×10 -11M的K D值与PD-L1特异性结合。
在本申请中，所述抗原结合片段可以特异性结合人PD-L1并阻断PD-L1和PD-1的相互作用。在本申请中，所述抗原结合片段可以特异性结合人PD-L1并阻断PD-L1和CD80的相互作用。
在本申请中，所述抗原结合片段可以使所述肿瘤的生长抑制至少约10％，至少约20％，至少约30％，至少约40％，至少约50％，至少约60％，至少约70％，至少约80％或更多。
在本申请中，所述抗原结合片段可以对碱处理和/或氧化处理具有抗性。例如，当强碱(例如碳酸氢铵)处理所述抗原结合片段至少8小时后，所述抗原结合片段仍保留特异性结合PD-L1的能力。又例如，当氧化剂(例如1％双氧水)处理所述抗原结合片段至少2小时后，所述抗原结合片段仍保留特异性结合PD-L1的能力。
在本申请中，所述抗原结合片段可以包含SEQ ID NO:14-31中任一项所示的氨基酸序列。
在本申请中，所述抗原结合片段可以包含所述抗原结合片段的功能性变体及其片段、衍生物及其片段、类似物及其片段和/或同源物及其片段。
所述功能性变体可以为与天然存在序列具有基本上同一的氨基酸序列或由基本上同一的核苷酸序列编码并能够具有天然存在序列的一种或多种活性的多肽。例如，所述功能性变体可以为在SEQ ID NO:14-31中任一项所示的氨基酸序列的基础上，修饰其中残基而且保留至少一种内源功能的序列(例如，可以特异性地结合人PD-L1)。例如，所述修饰可以包含至少一个氨基酸残基的添加、缺失、取代、修饰、替换和/或变异。
所述衍生物可以包含对SEQ ID NO:14-31中任一项所示的氨基酸序列的一个(或多个)氨基酸残基的任何取代、变异、修饰、替换、缺失和/或添加，且保留至少一种内源功能的序列。
所述同源物可以为与SEQ ID NO:14-31中任一项所示的氨基酸序列相比，至少80％、85％、90％、99.1％、99.2％、99.3％、99.4％、99.5％、99.6％、99.7％、99.8％或99.9％相同的氨基酸序列。为了确定序列同一性，可进行序列比对，其可通过本领域技术人员了解的各种方式进行，
例如，使用BLAST、BLAST-2、ALIGN、NEEDLE或Megalign(DNASTAR)软件等。本领域技术人员能够确定用于比对的适当参数，包括在所比较的全长序列中实现最优比对所需要的任何算法。所述类似物可以包含对本申请所述抗原结合片段而言，多肽的模拟物，该模拟物保留至少一种内源功能。
本申请的所述抗原结合片段可以具有氨基酸残基的缺失、插入或取代，所述氨基酸残基产生沉默的变化并导致功能上等同的蛋白质。可以根据残基的极性、电荷、溶解性、疏水性、亲水性和/或两性性质的相似性进行有意的氨基酸取代，只要保留内源性功能即可。
在本申请的所述组合物中，所述抗原结合片段的浓度可以为约1mg/mL-约220mg/mL。例如，所述抗原结合片段的浓度可以为约20mg/mL-约220mg/mL。例如，所述抗原结合片段的浓度可以为约30mg/mL-约220mg/mL、为约40mg/mL-约220mg/mL、为约50mg/mL-约220mg/mL、为约30mg/mL-约200mg/mL、为约40mg/mL-约200mg/mL或者为约50mg/mL-约200mg/mL)。例如，所述所述抗原结合片段的浓度可以为约1mg/mL-约300mg/mL，例如，所述所述抗原结合片段的浓度具体可以为约5mg/mL、10mg/mL、20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL、100mg/mL、110mg/mL、120mg/mL、130mg/mL、140mg/mL、150mg/mL、160mg/mL、170mg/mL、180mg/mL、190mg/mL、200mg/mL、210mg/mL、220mg/mL、230mg/mL、240mg/mL、250mg/mL、260mg/mL、270mg/mL、280mg/mL、290mg/mL、300mg/mL，例如，所述抗原结合片段的浓度可以为约5mg/mL-约250mg/mL、约10mg/mL-约290mg/mL、约20mg/mL-约290mg/mL、约30mg/mL-约290mg/mL、约50mg/mL-约290mg/mL、约60mg/mL-约280mg/mL、约70mg/mL-约280mg/mL、约80mg/mL-约280mg/mL、约100mg/mL-约280mg/mL、约110mg/mL-约250mg/mL、约130mg/mL-约250mg/mL、约150mg/mL-约250mg/mL、约160mg/mL-约250mg/mL、约170mg/mL-约250mg/mL、约180mg/mL-约250mg/mL、约120mg/mL-约220mg/mL、约140mg/mL-约220mg/mL、约150mg/mL-约220mg/mL、约175mg/mL-约220mg/mL、约190mg/mL-约220mg/mL。
例如，所述抗原结合片段的浓度可以为约50mg/mL-约200mg/mL。例如，所述抗原结合片段的浓度可以为约50mg/mL；例如，所述抗原结合片段的浓度可以为约200mg/mL。
辅料及其他组分
例如，所述辅料的浓度可以为约50mM-约500mM。例如，所述辅料的浓度可以为约50mM- 约500mM、约100mM-约500mM、约150mM-约500mM、约160mM-约500mM、约165mM-约500mM、约170mM-约500mM、约100mM-约300mM、约150mM-约300mM、约160mM-约300mM、约165mM-约300mM、约170mM-约300mM、约100mM-约275mM、约150mM-约275mM、约160mM-约275mM、约165mM-约275mM、约170mM-约275mM、约100mM-约220mM、约150mM-约220mM、约160mM-约220mM、约165mM-约220mM、约170mM-约220mM。
例如，所述辅料可以包含脯氨酸，所述脯氨酸的浓度可以为约50mM-约500mM。例如，所述脯氨酸的浓度可以为约50mM-约500mM、约100mM-约500mM、约150mM-约500mM、约160mM-约500mM、约165mM-约500mM、约170mM-约500mM、50mM-约390mM、约100mM-约390mM、约150mM-约390mM、约160mM-约390mM、约165mM-约390mM、约170mM-约390mM、约100mM-约300mM、约150mM-约300mM、约160mM-约300mM、约165mM-约300mM、约170mM-约300mM、约100mM-约275mM、约150mM-约275mM、约160mM-约275mM、约165mM-约275mM、约170mM-约275mM、约100mM-约250mM、约150mM-约250mM、约160mM-约250mM、约165mM-约250mM、约170mM-约250mM、约100mM-约220mM、约150mM-约220mM、约160mM-约220mM、约165mM-约220mM、约170mM-约220mM。例如，所述辅料可以包含脯氨酸，所述脯氨酸的浓度可以为约165mM-约275mM。例如，所述辅料可以包含脯氨酸，所述脯氨酸的浓度可以为约150mM-约250mM。例如，所述辅料可以包含脯氨酸，所述脯氨酸的浓度可以为约220mM。例如，所述辅料可以包含L-脯氨酸，所述L-脯氨酸的浓度可以为约50mM-约500mM。例如，所述L-脯氨酸的浓度可以为约50mM-约500mM、约100mM-约500mM、约150mM-约500mM、约160mM-约500mM、约165mM-约500mM、约170mM-约500mM、50mM-约390mM、约100mM-约390mM、约150mM-约390mM、约160mM-约390mM、约165mM-约390mM、约170mM-约390mM、约100mM-约300mM、约150mM-约300mM、约160mM-约300mM、约165mM-约300mM、约170mM-约300mM、约100mM-约275mM、约150mM-约275mM、约160mM-约275mM、约165mM-约275mM、约170mM-约275mM、约100mM-约250mM、约150mM-约250mM、约160mM-约250mM、约165mM-约250mM、约170mM-约250mM、约100mM-约220mM、约150mM-约220mM、约160mM-约220mM、约165mM-约220mM、约170mM-约220mM。例如，所述辅料可以包含L-脯氨酸，所述L-脯氨酸的浓度可以为约165mM-约275mM。例如，所述辅料可以包含L-脯氨酸，所述L-脯氨酸的浓度可以为约150mM-约250mM。例如， 所述辅料可以包含L-脯氨酸，所述L-脯氨酸的浓度可以为约220mM。
例如，所述辅料可以包含甘露醇，所述甘露醇的浓度可以为约50mM-约500mM。例如，所述甘露醇的浓度可以为约50mM-约500mM、约100mM-约500mM、约150mM-约500mM、约160mM-约500mM、约165mM-约500mM、约170mM-约500mM、50mM-约390mM、约100mM-约390mM、约150mM-约390mM、约160mM-约390mM、约165mM-约390mM、约170mM-约390mM、约100mM-约300mM、约150mM-约300mM、约160mM-约300mM、约165mM-约300mM、约170mM-约300mM、约100mM-约275mM、约150mM-约275mM、约160mM-约275mM、约165mM-约275mM、约170mM-约275mM、约100mM-约250mM、约150mM-约250mM、约160mM-约250mM、约165mM-约250mM、约170mM-约250mM、约100mM-约220mM、约150mM-约220mM、约160mM-约220mM、约165mM-约220mM、约170mM-约220mM。
例如，所述辅料可以包含蔗糖，所述蔗糖的浓度可以为约50mM-约500mM。例如，所述蔗糖的浓度可以为约50mM-约500mM、约90mM-约500mM、约100mM-约500mM、约150mM-约500mM、约160mM-约500mM、约165mM-约500mM、约170mM-约500mM、50mM-约390mM、约100mM-约390mM、约150mM-约390mM、约160mM-约390mM、约165mM-约390mM、约170mM-约390mM、约100mM-约300mM、约150mM-约300mM、约160mM-约300mM、约165mM-约300mM、约170mM-约300mM、约100mM-约275mM、约150mM-约275mM、约160mM-约275mM、约165mM-约275mM、约170mM-约275mM、约100mM-约250mM、约150mM-约250mM、约160mM-约250mM、约165mM-约250mM、约170mM-约250mM、约100mM-约220mM、约150mM-约220mM、约160mM-约220mM、约165mM-约220mM、约170mM-约220mM、约30mM-约300mM、约40mM-约300mM、约50mM-300mM、约60mM-300mM、约80mM-300mM、约30mM-约275mM、约40mM-约275mM、约50mM-约275mM、约30mM-约250mM、约40mM-约250mM、约50mM-约250mM、约30mM-约220mM、约40mM-约220mM、约50mM-约220mM。
例如，所述辅料可以包含甘氨酸，所述甘氨酸的浓度可以为约50mM-约500mM。例如，所述甘氨酸的浓度可以为约50mM-约500mM、约100mM-约500mM、约150mM-约500mM、约160mM-约500mM、约165mM-约500mM、约170mM-约500mM、50mM-约390mM、约100mM-约390mM、约150mM-约390mM、约160mM-约390mM、约165mM-约390mM、约170mM-约390mM、约100mM-约300mM、约150mM-约300mM、约160mM-约300mM、约 165mM-约300mM、约170mM-约300mM、约100mM-约275mM、约150mM-约275mM、约160mM-约275mM、约165mM-约275mM、约170mM-约275mM、约100mM-约250mM、约150mM-约250mM、约160mM-约250mM、约165mM-约250mM、约170mM-约250mM、约100mM-约220mM、约150mM-约220mM、约160mM-约220mM、约165mM-约220mM、约170mM-约220mM。
例如，所述辅料可以包含L-精氨酸盐酸盐，所述L-精氨酸盐酸盐的浓度可以为约50mM-约500mM。例如，所述L-精氨酸盐酸盐的浓度可以为约50mM-约500mM、约100mM-约500mM、约150mM-约500mM、约160mM-约500mM、约165mM-约500mM、约170mM-约500mM、50mM-约390mM、约100mM-约390mM、约150mM-约390mM、约160mM-约390mM、约165mM-约390mM、约170mM-约390mM、约100mM-约300mM、约150mM-约300mM、约160mM-约300mM、约165mM-约300mM、约170mM-约300mM、约100mM-约275mM、约150mM-约275mM、约160mM-约275mM、约165mM-约275mM、约170mM-约275mM、约100mM-约250mM、约150mM-约250mM、约160mM-约250mM、约165mM-约250mM、约170mM-约250mM、约100mM-约220mM、约150mM-约220mM、约160mM-约220mM、约165mM-约220mM、约170mM-约220mM。
例如，所述辅料可以包含山梨醇，所述山梨醇的浓度可以为约50mM-约500mM。例如，所述山梨醇的浓度可以为约100mM-约500mM、约130mM-约500mM、约150mM-约500mM、约200mM-约500mM、约80mM-约400mM、约100mM-约400mM、约150mM-约400mM、约180mM-约400mM、约100mM-约350mM、约130mM-约350mM、约150mM-约350mM、约170mM-约350mM、约190mM-约350mM、约75mM-约300mM、约100mM-约300mM、约120mM-约300mM、约150mM-约300mM、约170mM-约300mM、约180mM-约300mM、约200mM-约300mM、约100mM-约275mM、约120mM-约275mM、约150mM-约275mM、约180mM-约275mM、约100mM-约250mM、约130mM-约250mM、约150mM-约250mM、约160mM-约250mM、约170mM-约250mM、约180mM-约250mM、约100mM-约220mM、约120mM-约220mM、约140mM-约220mM、约160mM-约220mM、约180mM-约220mM。
例如，所述辅料可以包含甘油，所述甘油的浓度可以约为50mM-约500mM。例如，所述山梨醇的浓度可以为约100mM-约500mM、约130mM-约500mM、约150mM-约500mM、约200mM-约500mM、约80mM-约400mM、约100mM-约400mM、约150mM-约400mM、约180mM-约400mM、约100mM-约350mM、约130mM-约350mM、约150mM-约350mM、约 170mM-约350mM、约190mM-约350mM、约75mM-约300mM、约100mM-约300mM、约120mM-约300mM、约150mM-约300mM、约170mM-约300mM、约180mM-约300mM、约200mM-约300mM、约100mM-约275mM、约120mM-约275mM、约150mM-约275mM、约180mM-约275mM、约100mM-约250mM、约130mM-约250mM、约150mM-约250mM、约160mM-约250mM、约170mM-约250mM、约180mM-约250mM、约100mM-约220mM、约120mM-约220mM、约140mM-约220mM、约160mM-约220mM、约180mM-约220mM。
在本申请中，所述辅料可以包含稳定剂。所述稳定剂可以为有助于维持所述组合物的结构完整性的组分。例如，所述稳定剂可以在冷冻、冻干和/或储存期间帮助维持所述组合物的结构完整性。例如，所述稳定剂可以起到减轻蛋白质(例如本申请所述的抗原结合片段)变性的渗透剂的作用。
本申请所述辅料还可以进一步包含抗氧化剂，所述抗氧化剂可以选自甲硫氨酸、苯酚、苯甲酚、抗坏血酸、谷胱甘肽、硫代硫酸钠、柠檬酸和烟酰胺中的一种或多种。例如，本申请所述辅料还可以进一步包含L-甲硫氨酸，所述L-甲硫氨酸的浓度可以为约0.1mM-约100mM、约0.5mM-约100mM、约1mM-约100mM、约2mM-约100mM、约5mM-约100mM、约0.2mM-约50mM、约1mM-约50mM、约3mM-约50mM、约4mM-约50mM、约5mM-约50mM、约0.1mM-约30mM、约0.3mM-约30mM、约0.6mM-约30mM、约1mM-约30mM、约2mM-约30mM、约5mM-约30mM、约0.1mM-约20mM、约0.5mM-约20mM、约1mM-约20mM、约2mM-约20mM、约0.1mM-约10mM、约0.2mM-约10mM、约0.5mM-约10mM、约0.8mM-约10mM、约1mM-约10mM、约1.5mM-约10mM、约2mM-约10mM、约2.5mM-约10mM、约3mM-约10mM、约4mM-约10mM、约5mM-约10mM。
本申请所述辅料还可以进一步包含螯合剂，所述螯合剂可以为EDTA二钠，所述EDTA二钠的浓度可以为约0.1mM-约10mM、约0.5mM-约10mM、约1mM-约10mM、约0.1mM-约5mM、约0.2mM-约5mM、约0.3mM-约5mM、约0.4mM-约5mM、约0.5mM-约5mM、约0.1mM-约3mM、约0.5mM-约3mM、约0.6mM-约3mM、约0.7mM-约3mM、约0.8mM-约3mM、约0.1mM-约2mM、约0.2mM-约2mM、约0.5mM-约2mM、约0.75mM-约2mM、约0.9mM-约2mM、约1mM-约2mM。
例如，所述组合物的pH可以为约5.0-约7.5。例如，所述组合物的pH可以为约5.9-约6.9。例如，所述组合物的pH可以为约5.9-约6.8、约5.9-约6.7、约5.9-约6.6、约5.9-约6.5、约5.9-约6.4、约6.0-约6.9、约6.1-约6.9、约6.2-约6.9、约6.3-约6.9、约6.4-约6.9、约6.0- 约6.5、约6.1-约6.5、约6.2-约6.5、约6.3-约6.5或者约6.4-约6.5。例如，所述组合物的pH可以为约6.3-约6.5。例如，所述组合物的pH可以为约6.4。
例如，所述组合物可以包含缓冲成分，其中所述缓冲成分选自下组中的一种或多种：乙酸钠-乙酸、组氨酸-乙酸、L-组氨酸-L-组氨酸盐酸盐、磷酸钠和柠檬酸-氢氧化钠。
例如，所述缓冲成分的浓度可以为约5mM-约50mM。例如，所述缓冲成分的浓度可以为约5mM-约50mM、约5mM-约45mM、约5mM-约40mM、约5mM-约35mM、约5mM-约30mM、约5mM-约25mM、约5mM-约20mM、约5mM-约50mM或者约5mM-约30mM。例如，所述缓冲成分的浓度可以为约10mM-50mM。例如，所述缓冲成分的浓度可以为约10mM-约50mM、约10mM-约45mM、约10mM-约40mM、约10mM-约35mM、约10mM-约30mM、约10mM-约25mM、约10mM-约20mM、约20mM-约50mM或者约20mM-约30mM。
例如，所述缓冲成分可以包含乙酸钠-乙酸，所述乙酸钠-乙酸的浓度可以为约10mM-约50mM。例如，所述乙酸钠-乙酸的浓度可以为约5mM-50mM、约5mM-约45mM、约5mM-约40mM、约5mM-约35mM、约5mM-约30mM、约5mM-约25mM、约5mM-约20mM、约10mM-50mM、约10mM-约45mM、约10mM-约40mM、约10mM-约35mM、约10mM-约30mM、约10mM-约25mM、约10mM-约20mM、约20mM-约50mM或者约20mM-约30mM。例如，所述缓冲成分可以包含乙酸钠-乙酸，所述乙酸钠-乙酸的浓度可以为约5mM-约35mM。例如，所述缓冲成分可以包含乙酸钠-乙酸，所述乙酸钠-乙酸的浓度可以为约10mM-约30mM。例如，所述缓冲成分可以包含乙酸钠-乙酸，所述乙酸钠-乙酸的浓度可以为约20mM。
例如，所述缓冲成分可以包含L-组氨酸-L-组氨酸盐酸盐，所述L-组氨酸-L-组氨酸盐酸盐的浓度可以为约10mM-约50mM。例如，所述L-组氨酸-L-组氨酸盐酸盐的浓度可以为约5mM-50mM、约5mM-约45mM、约5mM-约40mM、约5mM-约35mM、约5mM-约30mM、约5mM-约25mM、约5mM-约20mM、约10mM-50mM、约10mM-约45mM、约10mM-约40mM、约10mM-约35mM、约10mM-约30mM、约10mM-约25mM、约10mM-约20mM、约20mM-约50mM或者约20mM-约30mM。例如，所述缓冲成分可以包含L-组氨酸-L-组氨酸盐酸盐，所述L-组氨酸-L-组氨酸盐酸盐的浓度可以为约5mM-约35mM。例如，所述缓冲成分可以包含L-组氨酸-L-组氨酸盐酸盐，所述L-组氨酸-L-组氨酸盐酸盐的浓度可以为约10mM-约30mM。例如，所述缓冲成分可以包含L-组氨酸-L-组氨酸盐酸盐，所述L-组氨酸-L-组氨酸盐酸盐的浓度可以为约20mM。
例如，所述组合物可以包含表面活性剂，其中所述表面活性剂可以选自下组中的一种或 多种：聚山梨酯20、聚山梨酯80和泊洛沙姆188。本申请使用mg/mL或％(w/v)来表示表面活性剂的浓度，本领域技术人员应当理解两者可以进行换算，例如，0.02％表面活性剂可换算为0.2mg/mL。
例如，所述表面活性剂的浓度可以为约0mg/mL-约0.8mg/mL。例如，所述表面活性剂的浓度可以为约0.05mg/mL-约0.8mg/mL。例如，所述表面活性剂的浓度可以为约0mg/mL-约0.8mg/mL、约0.1mg/mL-约0.8mg/mL、约0.2mg/mL-约0.8mg/mL、约0.3mg/mL-约0.8mg/mL、约0.1mg/mL-约0.3mg/mL、约0.1mg/mL-约0.2mg/mL、约0.05mg/mL-约0.3mg/mL、约0mg/mL-约0.4mg/mL、约0.05mg/mL-约0.4mg/mL、约0.05mg/mL-约0.2mg/mL、约0.2mg/mL-约0.4mg/mL或者约0mg/mL-约0.2mg/mL。
例如，所述表面活性剂可以包含聚山梨酯20，所述聚山梨酯20的浓度可以为约0mg/mL-约0.8mg/mL。例如，所述聚山梨酯20的浓度可以为约0.05mg/mL-约0.8mg/mL。例如，所述聚山梨酯20的浓度可以为约0mg/mL-约0.8mg/mL、约0.1mg/mL-约0.8mg/mL、约0.2mg/mL-约0.8mg/mL、约0.3mg/mL-约0.8mg/mL、约0.1mg/mL-约0.3mg/mL、约0.1mg/mL-约0.2mg/mL、约0.05mg/mL-约0.3mg/mL、约0mg/mL-约0.4mg/mL、约0.05mg/mL-约0.4mg/mL、约0.05mg/mL-约0.2mg/mL、约0.2mg/mL-约0.4mg/mL或者约0mg/mL-约0.2mg/mL。例如，所述表面活性剂可以包含聚山梨酯20，所述聚山梨酯20的浓度可以为约0.2mg/mL-约0.4mg/mL。例如，所述表面活性剂可以包含聚山梨酯20，所述聚山梨酯20的浓度可以为约0.2mg/mL。例如，所述表面活性剂可以包含聚山梨酯20，所述聚山梨酯20的浓度可以为约0.4mg/mL。
例如，所述表面活性剂可以包含聚山梨酯80，所述聚山梨酯80的浓度可以为约0mg/mL-约0.8mg/mL。例如，所述聚山梨酯80的浓度可以为约0.05mg/mL-约0.8mg/mL。例如，所述聚山梨酯80的浓度可以为约0mg/mL-约0.8mg/mL、约0.1mg/mL-约0.8mg/mL、约0.2mg/mL-约0.8mg/mL、约0.3mg/mL-约0.8mg/mL、约0.1mg/mL-约0.3mg/mL、约0.1mg/mL-约0.2mg/mL、约0.05mg/mL-约0.3mg/mL、约0mg/mL-约0.4mg/mL、约0.05mg/mL-约0.4mg/mL、约0.05mg/mL-约0.2mg/mL、约0.2mg/mL-约0.4mg/mL或者约0mg/mL-约0.2mg/mL。例如，所述表面活性剂可以包含聚山梨酯80，所述聚山梨酯80的浓度可以为约0.2mg/mL-约0.4mg/mL。例如，所述表面活性剂可以包含聚山梨酯80，所述聚山梨酯80的浓度可以为约0.2mg/mL。例如，所述表面活性剂可以包含聚山梨酯80，所述聚山梨酯80的浓度可以为约0.4mg/mL。
例如，所述表面活性剂可以包含泊洛沙姆188，所述泊洛沙姆188的浓度可以为约0.05mg/mL-约5mg/mL、约0.08mg/mL-约5mg/mL、约0.1mg/mL-约5mg/mL、约0.2mg/mL-约5mg/mL、约0.05mg/mL-约3mg/mL、约0.1mg/mL-约3mg/mL、约0.15mg/mL-约3mg/mL、约0.2mg/mL-约3mg/mL、约0.05mg/mL-约2mg/mL、约0.075mg/mL-约2mg/mL、约0.1mg/mL-约2mg/mL、约0.15mg/mL-约2mg/mL、约0.2mg/mL-约2mg/mL、约0.3mg/mL-约2mg/mL、约0.5mg/mL-约2mg/mL。
在本申请中，所述组合物可以包含本申请所述的抗原结合片段、本申请所述的辅料，并且pH可以为约5.9-约6.9。例如，所述组合物可以包含本申请所述的抗原结合片段、本申请所述的辅料和本申请所述的缓冲成分，并且pH可以为约5.9-约6.9。例如，所述组合物可以包含本申请所述的抗原结合片段、本申请所述的辅料、本申请所述的缓冲成分和本申请所述的表面活性剂，并且pH可以为约5.9-约6.9。
在本申请中，所述组合物可以包含本申请所述的抗原结合片段、脯氨酸，并且pH可以为约5.9-约6.9。例如，所述组合物可以包含本申请所述的抗原结合片段、脯氨酸和乙酸钠-乙酸，并且pH可以为约5.9-约6.9。例如，所述组合物可以包含本申请所述的抗原结合片段、脯氨酸、乙酸钠-乙酸和聚山梨酯20，并且pH可以为约5.9-约6.9。例如，所述组合物可以包含本申请所述的抗原结合片段、脯氨酸、乙酸钠-乙酸和聚山梨酯20，并且pH可以为约6.3-约6.5。
例如，所述组合物可以包含：1)约20mg/mL-约220mg/mL本申请所述的抗原结合片段；2)约10mM-约50mM乙酸钠-乙酸；3)约50mM-约500mM脯氨酸；4)约0.05mg/mL-约0.8mg/mL聚山梨酯20；且pH可以为约5.9-约6.9。
例如，所述组合物可以由1)约20mg/mL-约220mg/mL本申请所述的抗原结合片段；2)约10mM-约50mM乙酸钠-乙酸；3)约50mM-约500mM脯氨酸和4)约0.05mg/mL-约0.8mg/mL聚山梨酯20组成，且所述组合物的pH可以为约5.9-约6.9。
例如，所述组合物可以包含：1)约20mg/mL-约220mg/mL本申请所述的抗原结合片段；2)约5mM-约35mM乙酸钠-乙酸；3)约50mM-约390mM脯氨酸；4)约0mg/mL-约0.4mg/mL聚山梨酯20；且pH可以为约5.9-约6.9。
例如，所述组合物可以由1)约20mg/mL-约220mg/mL本申请所述的抗原结合片段；2)约5mM-约35mM乙酸钠-乙酸；3)约50mM-约390mM脯氨酸和4)约0mg/mL-约0.4mg/mL聚山梨酯20组成，且所述组合物的pH可以为约5.9-约6.9。
例如，所述组合物可以包含：1)约50mg/mL-约200mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM乙酸钠-乙酸；3)约150mM-约250mM脯氨酸；4)约0.2mg/mL-约0.4mg/mL聚山梨酯20；且pH可以为约6.3-约6.5。
例如，所述组合物可以由1)约50mg/mL-约200mg/mL本申请所述的抗原结合片段；2)约10mM-约30mM乙酸钠-乙酸；3)约150mM-约250mM脯氨酸和4)约0.2mg/mL-约0.4mg/mL聚山梨酯20组成，且所述组合物的pH可以为约6.3-约6.5。
例如，所述组合物可以包含：1)约200mg/mL本申请所述的抗原结合片段；2)约20mM乙酸钠-乙酸；3)约220mM脯氨酸；4)约0.2mg/mL聚山梨酯20；且pH可以为约6.4。
例如，所述组合物可以由1)约200mg/mL本申请所述的抗原结合片段；2)约20mM乙酸钠-乙酸；3)约220mM脯氨酸和4)约0.2mg/mL聚山梨酯20组成，且pH可以为约6.4。
在本申请中，所述组合物可以用于注射。在本申请中，所述组合物可以用于皮下注射。例如，所述组合物也可以用于皮下注射、肌内注射、静脉注射、经肠胃施用或静脉输注。
在本申请中，所述组合物可以为制剂。例如，所述组合物可以为液体制剂。
另一方面，本申请提供一种试剂盒，其包括本申请所述的组合物和盛放本申请所述的组合物的容器。
例如，所述容器可以包括小瓶(例如，玻璃瓶)、安瓿、注射器、注射笔和/或静脉袋。在本申请中，所述容器可以为无菌。例如，所述容器可以包括玻璃瓶。
例如，所述试剂盒可以包括一个递送装置，所述递送装置可以施用本申请所述的组合物。例如，所述递送装置可以包括小瓶、安瓿、注射器、注射笔和/或静脉袋。
在本申请中，所述容器中所述组合物的体积可以为约0.5mL-约5.0mL。例如，所述容器中所述组合物的体积可以为约0.5mL-约1.5mL。例如，所述组合物的体积可以为1.5mL。例如，所述组合物的体积可以为1.0mL。在本申请中，其中所述容器中，所述抗原结合片段的浓度可以为约200mg/1.0mL/瓶；或者，可以为约300mg/1.5mL/瓶。
另一方面，本申请提供了本申请所述的组合物；和/或本申请所述的试剂盒在制备预防、缓解和/或治疗肿瘤的药物中的应用。
本申请提供了本申请提供了本申请所述的组合物；和/或本申请所述的试剂盒，其用于预防、缓解和/或治疗肿瘤。
本申请提供了预防、缓解和/或治疗肿瘤的方法，其包括以下步骤：向有需要的受试者施用本申请所述的组合物；和/或本申请所述的试剂盒。
在本申请中，所述肿瘤可以包括PD-L1阳性的肿瘤。例如，所述肿瘤可以包括恶性实体瘤。
在本申请中，所述肿瘤可以包括微卫星不稳定(MSI-H)/错配修复功能缺陷(dMMR)的肿瘤(例如MSI-H/dMMR泛瘤种实体瘤)。例如可以包括微卫星不稳定(MSI-H)/错配修复功能缺陷(dMMR)的晚期结直肠癌或胃癌。例如，所述肿瘤可以包括不能手术切除或转移性的胆道癌；和/或，未治疗的不能切除或转移性胃或胃食管结合部腺癌。
另一方面，本申请还提供了本申请所述的组合物；和/或本申请所述的试剂盒在制备预防、缓解和/或治疗感染性疾病的药物中的应用。
本申请提供了本申请提供了本申请所述的组合物；和/或本申请所述的试剂盒，其用于预防、缓解和/或治疗感染性疾病。
本申请提供了预防、缓解和/或治疗感染性疾病的方法，其包括以下步骤：向有需要的受试者施用本申请所述的组合物；和/或本申请所述的试剂盒。
在本申请中，所述感染性疾病包括病毒感染、细菌感染、真菌感染和/或寄生虫感染所引起的疾病。
本申请还提供了生产本申请所述组合物，和/或本申请所述试剂盒的方法。所述方法可以包括以任何顺序将制备本申请所述组合物所必需的任何相关组分混合在一起。例如，所述方法可以包括制备所述辅料、所述缓冲成分和/或所述表面活性剂的预混物(或预混液)。例如，所述方法可以包括形成缓冲系统。所述缓冲系统可以包含本申请所述的缓冲成分。本申请所述的方法可以包括去除颗粒物质(例如通过过滤)。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的制剂、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
检测项目及方法：
(1)内源荧光光谱：利用蛋白质分子中内源能发荧光的氨基酸(色氨酸、酪氨酸、苯丙氨酸)在极性和非极性环境中的荧光光谱。若荧光光谱发生红移时，即最大发射波长改变，由此间接推断蛋白质高级结构的一致性，即高级结构的相互转变。样品稀释至40mg/mL，采用高通量的内源荧光光谱仪optim1000(pall公司)，测试温度范围为20-95℃，测试间隔为1℃。采用473nm波长的SLS(静态散射光)结果来判断样品的聚合倾向性，变性温度是BCM (质心波长)下测定的值，根据公式计算成Tm值(变性一半的温度)来评估。
(2)SEC-HPLC(分子排阻色谱法)：参考《中国药典》2015版三部通则0514分子排阻色谱法，用于检测产品的单体含量，以及聚体和片段。其中，色谱柱为TOSOH G3000 7.8*300mm，5μm；流动相为20mM Na 2HPO 4，300mMNaCl；检测波长280nm。
(3)WCX-HPLC(弱阳离子交换色谱法)：参考《中国药典》2015版三部通则0512高效液相色谱法，用于检测产品的主成分、酸性成分及碱性成分含量。其中，色谱柱为Propac WCX-10 4×250mm,10μm；流动相为10mM Na 2HPO 4，10mM Na 2HPO 4+500mM NaCl；检测波长280nm。
(4)结合和阻断活性：分别参考WO 2017/020802A1的实施例5.2和5.3所记载的试验方法。
(5)粘度：采用美国rheosense便携式微量粘度计microVISC，自动模式下测量其粘度。
(6)渗透压：利用摩尔渗透压法进行检测。
仪器：渗透压仪；试剂：300mosm/Kg校准液、900mosm/Kg校准液、氯化钠
耗材：离心管、无尘纸等；
实验步骤：取100μL加入样品管中，确保样品中管中无气泡产生，用无尘纸擦拭样品探针及二级针后，将样品管放到检测探针上，降低测量探针，样品管进入冷阱中，按“Enter”键进行测试。供试品平行测定2次。零点校准：在±2之间；300点校准，在±4之间；900点校准：在±8之间。
(7)CE-SDS纯度：供试品经变性(非还原条件)或变性还原(还原条件)后，放入毛细管电泳仪，电泳分析，按面积归一化法计算。
(8)蛋白质含量：参考《中国药典》2015版三部通则0731蛋白质含量测定法第六法，采用微量紫外分光光度计Nanodrop 2000，根据蛋白质在280nm波长下的吸光值和蛋白质的理论消光系数，计算蛋白质含量。
(9)浊度测量：在Agilent 8453分光光度计上测量样品于340nm的吸光值。
(10)不溶性颗粒：在AccuSizer 780SIS不溶性微粒分析仪上测量样品≥10μm和≥25μm颗粒的数量。
(11)动态光散色(DLS)：在PUNK型粒度仪上测量样品平均粒子直径(Z.average.Diameter)和分布系数(PDI)。
抗原结合蛋白的制备：
抗原结合蛋白包含能够特异性结合PD-L1的免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域可以包含SEQ ID NO:2所示的氨基酸序列。以下将该抗原结合蛋白简称为“PD-L1单克隆抗体”。通过氨基酸序列人工合成的方法获得所述PD-L1单克隆抗体。所述PD-L1单克隆抗体包含SEQ ID NO:14-31中任一项所示的氨基酸序列，特别是SEQ ID NO：21所示的氨基酸序列。
实施例中涉及的脯氨酸包括L-脯氨酸。
实施例1
按照表1获得如下的制剂处方：
表1制剂处方筛选实验不同组成
Figure PCTCN2022119187-appb-000001
注：--表示未添加
按照表2对表1中制剂处方进行筛选检测。
表2制剂处方筛选检测项目
Figure PCTCN2022119187-appb-000002
1)内源荧光光谱检测
处方1-19对应的Tm值及聚体倾向性检测结果分别如图1和图2所示。
图1的结果显示，处方1-4和12的结构更为稳定；图2的结果显示，处方1-4和10更不易形成聚体。可见在该项检测中，处方1-4的稳定性非常好，即使用乙酸钠-乙酸缓冲体系与稳定性的进一步提高相关。
分别比较处方1-2和处方3-4的结果，可见较低的pH与稳定性的进一步提高相关。
2)SEC-HPLC检测主峰含量
处方1-19对应的SEC-HPLC检测主峰含量和聚体含量的检测结果分别如图3和图4所示。
结果显示，高温28天条件下，除处方11以外，其他处方中PD-L1单克隆抗体的纯度在97％以上，可见缓冲体系是必要的。
分别比较处方1、3、5、7和处方2、4、6、8的结果，可见较低的pH与处方稳定性的进一步提高相关。
分别比较处方1和处方3(pH 6.5乙酸钠-乙酸缓冲体系)；处方5和处方7(pH 6.5磷酸钠缓冲体系)的稳定性。结果发现包含L-脯氨酸与稳定性的进一步提高相关。
分别比较处方6和8、13和14的结果，结果发现包含L-精氨酸或者包含L-脯氨酸与稳定性的进一步提高相关。
分别比较处方2、6和10，结果发现使用乙酸钠-乙酸缓冲体系与稳定性的进一步提高相关。
分别比较处方14和16、处方13和17、处方6和18的结果，发现PD-L1单克隆抗体含 量50mg/mL和200mg/mL，似乎都不影响处方的稳定性。
3)WCX-HPLC检测酸性组分含量
WCX-HPLC检测酸性组分含量结果参见图5，其可以评价处方中酸性组分(该酸性组分可包括在高温条件下PD-L1单克隆抗体分解而产生的成分)的变化趋势。
分别比较处方1、3、5、7和处方2、4、6、8的结果，可见较低的pH与处方酸性组分的进一步减少相关。
分别比较处方1和处方5、处方3和处方7以及处方2、6和10的酸性组分，结果发现乙酸钠-乙酸缓冲体系与处方酸性组分的进一步减少相关。
比较处方6、8、13-14的酸性组分，结果发现包含L-脯氨酸与处方酸性组分的进一步减少相关。
分别比较处方14和16、处方13和17以及处方6和18的结果，可见PD-L1单克隆抗体含量50mg/mL和200mg/mL，似乎都不影响处方酸性含量的水平。
4)粘度检测
粘度检测的结果如图6所示。除处方8以外，其余处方的粘度均符合皮下注射对粘度的要求。
分别比较处方14和16、处方13和17的结果，可见PD-L1单克隆抗体含量200mg/mL与含量50mg/mL相比，虽粘度有所上升，但仍不影响皮下注射施用。
5)活性检测
选择处方1和处方3分别进行高温处理的活性测定。
两者的结合活性和阻断活性的结果分别如图7、图8和表3所示。其中图7-8中A-D分别表示处方1第0天、处方3第0天、处方1第28天、处方3第28天的结果。处方结果发现，处方1和处方3高温28天活性无明显差异，和0天相比无明显差异。
表3处方1和处方3的结合活性和阻断活性检测结果
Figure PCTCN2022119187-appb-000003
实施例2
在实施例1处方筛选的基础上，进一步考察各因素对处方性能的影响，研究制剂处方如 表4所示。
表4制剂处方优化不同组成
Figure PCTCN2022119187-appb-000004
注：NA表示未添加
经高温(40±2℃)加速14天、28天后，评估PD-L1单克隆抗体SEC-HPLC纯度和WCX-HPLC酸碱组分变化情况，并检测了0天样品的粘度。制剂组成其他补充实验考察及评价项目如表5所示。
表5制剂处方优化检测项目
Figure PCTCN2022119187-appb-000005
Figure PCTCN2022119187-appb-000006
SEC-HPLC检测结果如表6所示。
表6制剂处方组成研究SEC-HPLC检测结果
Figure PCTCN2022119187-appb-000007
WCX-HPLC检测结果如表7所示。
表7制剂处方组成研究WCX-HPLC检测结果
Figure PCTCN2022119187-appb-000008
Figure PCTCN2022119187-appb-000009
(1)表面活性剂种类对制剂稳定性的影响研究
PD-L1单克隆抗体含量200mg/mL，20mM乙酸钠-乙酸缓冲液，220mM脯氨酸(L-脯氨酸)稳定剂，分别选用0.02％的聚山梨酯20、聚山梨酯80、泊洛沙姆P188为表面活性剂，加速0天、14天、28天分别用SEC-HPLC、WCX-HPLC法检测目的蛋白，考察不同表面活性剂种类对制剂稳定性的影响，见制剂处方19A、20A、21A。研究结果显示，聚山梨酯20、聚山梨酯80、泊洛沙姆P188可作为PD-L1单克隆抗体制剂的表面活性剂。
(2)稳定剂种类对制剂稳定性影响研究
PD-L1单克隆抗体含量200mg/mL，20mM乙酸钠-乙酸缓冲液，0.02％聚山梨酯20为表面活性剂，pH6.4，分别选用220mM L-脯氨酸、90mg/mL蔗糖、250mM甘氨酸、150mM L-精氨酸盐酸盐、150mM氯化钠为稳定剂，加速0天、14天、28天分别用SEC-HPLC、WCX-HPLC法检测目的蛋白，考察不同稳定剂种类对制剂稳定性的影响，见制剂处方19A、26A、27A、28A、29A。研究结果显示，L-脯氨酸、蔗糖、甘氨酸、L-精氨酸盐酸盐或氯化钠可作为PD-L1单克隆抗体制剂的稳定剂。
(3)缓冲成分种类对对制剂稳定性的影响
PD-L1单克隆抗体含量200mg/mL，220mM的L-脯氨酸为稳定剂，0.02％聚山梨酯20为表面活性剂，pH6.4，分别选用20mM的乙酸钠-乙酸、L-组氨酸-乙酸、L-组氨酸-L-组氨酸盐酸、磷酸钠、柠檬酸-氢氧化钠的缓冲液，加速0天、14天、28天分别用SEC-HPLC、WCX-HPLC法检测目的蛋白，考察不同缓冲液成分对制剂稳定性的影响，见制剂处方19A、22A、23A、24A、25A。研究结果显示，乙酸钠-乙酸、L-组氨酸-乙酸、L-组氨酸-L-组氨酸盐酸、磷酸钠、柠檬酸-氢氧化钠的缓冲液等均可作为PD-L1单克隆抗体制剂的缓冲成分。具体来说，各缓冲系统配方的SEC-HPLC检测结果无显著差异；WCX-HPLC结果显示，高温加速28天后，磷酸钠与柠檬酸-氢氧化钠体系的配方主成分含量稍低，其余几种缓冲体系的主成分含量 接近。
(4)PD-L1单克隆抗体浓度对制剂稳定性的影响
制剂处方33A、34A及19A研究结果显示，包含20mg/mL-220mg/mL浓度的PD-L1单克隆抗体浓度的制剂，在一定的缓冲液、稳定剂、表面活性剂和/或pH条件下，均符合制剂稳定性的要求。
第0天粘度检测结果如表8所示。结果显示：除了低PD-L1单克隆抗体浓度的处方33A粘度较低，其余制剂处方的粘度差异不明显，总体均在可接受范围内。这就意味着包含较高浓度(例如220mg/mL)的PD-L1单克隆抗体的制剂具有适于成药(例如适于用于注射，例如适于用于皮下注射)的粘度特性。可见本申请所述的组合物具有合适的粘度，从而有助于简便(例如皮下注射)、快捷(例如减少注射次数，大大缩短给药时间)地被施用，同时提升病人用药的依从性和可及性，提升患者生活质量。
表8制剂组成研究粘度检测结果
处方 粘度(mPa.s)
19A 18.200
20A 18.270
21A 18.470
22A 17.080
23A 17.250
24A 16.390
25A 20.480
26A 22.980
27A 17.440
28A 17.280
29A 19.690
33A 1.239
34A 22.640
实施例3
根据实施例1和2的结果进一步优化处方配方，对处方的pH、缓冲系统、稳定剂和表面活性剂分别做进一步筛选。
1、pH的筛选
考察含有200±12mg/mLPD-L1单克隆抗体的不同处方在pH 5.0-7.5范围内，放置于40±2℃高温条件下的稳定性。通过SEC-HPLC和WCX-HPLC检测蛋白的纯度差异，通过不溶性微粒和DLS检测蛋白的颗粒及均一性差异。具体处方组成及检测结果汇总见表9。
表9：pH 4.5-7.5范围考察40±2℃高温加速28天检测结果汇总
Figure PCTCN2022119187-appb-000010
“NA”表示未检测
SEC-HPLC检测结果显示：pH 5.0和pH 7.5条件下，单体明显降低；WCX-HPLC检测结果显示：pH 7.5条件下，主成分降低明显。不溶性微粒检测结果显示，较低pH条件下，不溶性微粒较少，且随着pH升高，不溶性微粒数量有进一步减少的趋势。PDI指标反映溶液的均一性，该数值越小均一性越好，结果显示pH 6.4时溶液均一性最佳，且随着pH的上升，溶液的PDI值呈现出先降低后升高的趋势。
根据上述结果，进一步考察PD-L1单克隆抗体在pH 6.1-6.7范围内的稳定性。将含有200±12mg/mLPD-L1单克隆抗体的不同处方置于40±2℃高温条件下，通过SEC-HPLC和WCX-HPLC检测蛋白的纯度差异，比较340nm处OD值检测浊度差异。具体处方组成及检测结果汇总见表10。
表10：pH 6.1-6.7范围考察40±2℃高温加速28天检测结果汇总
Figure PCTCN2022119187-appb-000011
在pH 6.1-6.7范围，SEC-HPLC结果显示不同处方之间差异不明显；WCX-HPLC结果显 示随着pH值升高，主成分含量有稍微降低的趋势；OD 340nm检测的浊度结果随pH升高有先下降后升高的趋势。
2、稳定剂与缓冲系统的选择
选择醋酸钠-醋酸、L-组氨酸-L-组氨酸盐酸盐和柠檬酸盐缓冲体系结合不同稳定剂进一步考察稳定剂的作用。评估的稳定剂包括糖类的蔗糖、海藻糖和麦芽糖；氨基酸类的L-脯氨酸、L-精氨酸盐酸盐和甲硫氨酸；醇类的甘露醇、山梨醇和甘油；氯化钠作为对照同步进行比较。
通过SEC-HPLC和WCX-HPLC检测蛋白的纯度差异，通过不溶性微粒和DLS检测不同配方中的颗粒及均一性差异。具体处方组成及检测结果见表11-13。
表11：醋酸钠-醋酸缓冲液中考察不同稳定剂40±2℃高温加速28天检测结果汇总
Figure PCTCN2022119187-appb-000012
高温加速28天后，目测外观显示，除含有麦芽糖组成的处方颜色发黄，其它处方外观差异不明显。
SEC-HPLC检测结果显示：除麦芽糖检测单体含量较低，其它处方检测单体差异不明显，在98.3％～98.6％之间；WCX-HPLC检测结果显示：除麦芽糖检测峰型有变形，其它配方检测 主成分差异不明显，在83.3％～85.5％之间。不溶性微粒检测结果显示：不同稳定剂之间差异不明显。DLS检测结果显示：平均水合直径(Z.Av.Diameter)结果显示：含有氯化钠的处方的水合直径较大，其它处方显示均粒径较小；分布系数(PDI)结果显示：除含有海藻糖、麦芽糖和氯化钠值＞0.3外，其它处方差异不明显，显示均一性较好。粘度检测结果显示：添加50mg/mL蔗糖、50mg/mL甘露醇和90mg/mL海藻糖的粘度＞20cp，其他配方都＜20cp，添加抗氧化剂甲硫氨酸与未添加的处方比较，粘度稍降低。
表12：L-组氨酸-L-组氨酸盐酸盐缓冲液中考察不同稳定剂40±2℃高温加速28天检测结果汇总
Figure PCTCN2022119187-appb-000013
高温加速28天后，目测外观显示，除含有麦芽糖组成的处方颜色发黄，其他各组外观差异不明显。
SEC-HPLC检测结果显示：除麦芽糖检测单体含量较低，其它处方检测单体差异不明显，在98.6％～98.9％之间；WCX-HPLC检测结果显示：除麦芽糖检测峰型有变形，其它处方检测主成分差异不明显，在83.2％～84.9％之间。不溶性微粒检测结果显示：不同稳定剂之间差异不明显。DLS检测中Z.Av.Diameter结果显示：含有氯化钠的处方的水合直径较大，其它处方显示均粒径较小；PDI值结果显示：除含有海藻糖和麦芽糖的配方PDI值＞0.3外，其它处方差异不明显，显示均一性较好。
表13：柠檬酸盐缓冲液中考察不同稳定剂40±2℃高温加速28天检测结果汇总
Figure PCTCN2022119187-appb-000014
不溶性微粒检测结果显示：不同稳定剂之间差异不明显。DLS检测结果显示：除含有L-精氨酸盐酸盐处方与其他缓冲体系中差异不大，采用其他稳定剂的处方在柠檬酸盐缓冲体系中，较上述其他两个缓冲体系，Z.Av.Diameter值显示其水合直径都较大，不利于蛋白的结构稳定；不同稳定剂检测PDI值各处方大小不一，其中含有L-精氨酸盐酸盐和甘露醇的处方PDI值较小，显示均一性较好。
3、表面活性剂的筛选
考察PD-L1单克隆抗体在不同处方中的稳定性，特别是不同表面活性剂对蛋白稳定性的影响。通过高温40±2℃加速实验检测蛋白SEC-HPLC和WCX-HPLC的纯度差异，通过25±2℃150rpm振荡7天检测不溶性微粒和DLS检测不同处方中的颗粒及均一性差异。具体处方组成及检测结果如表14所示。
表14：不同表面活性剂种类筛选考察检测结果汇总
Figure PCTCN2022119187-appb-000015
“-”表示配方中未添加，“+”表示配方中有添加
40±2℃加速28天，SEC-HPLC检测结果显示：不同处方差异不明显；WCX-HPLC检测结果显示：只含有220mM L-脯氨酸，主成分降低较其他处方明显，其他处方之间比较差异不明显。
25±2℃150rpm振荡7天，不溶性微粒检测结果显示，添加表面活性剂处方，其不溶性微粒低，而其他处方都较高。DLS检测结果显示，添加不同表面活性剂聚山梨酯20、聚山梨酯80或泊洛沙姆188，可改善PDI值，不同表面活性剂的处方差异不明显。
实施例4
根据之前实施例的结果，进一步对处方的辅料及pH进行优化，按照表15获得如下的制剂处方：
表15制剂处方确认实验不同组成
Figure PCTCN2022119187-appb-000016
按照表16对表15中制剂处方进行筛选检测。
表16制剂处方确认检测项目
Figure PCTCN2022119187-appb-000017
1)SEC-HPLC检测主峰含量
处方20-31对应的SEC-HPLC检测主峰含量和聚体含量的检测结果分别如图9和图10所示。
分别比较处方20-23、处方24-27和处方28-31的结果，可见pH 6.1-6.7对稳定性的影响差异不显著。
分别比较处方20-23、处方28-31的聚体含量，结果发现包含L-脯氨酸与稳定性的进一步提高相关。
分别比较处方24-27和处方28-31的结果，发现220mM L-脯氨酸和150mM L-脯氨酸对稳定性的影响无显著差异。
2)WCX-HPLC检测酸性组分含量
经过高温(40±2℃)加速实验，处方20-31的WCX检测酸性组分含量结果如图11所示。
分别比较处方20-23、处方24-27和处方28-31的酸性组分，可见pH 6.1-6.7酸性组分含量随pH值升高有上升的趋势，但是都在可接受范围内。
分别比较处方20-23、处方28-31的酸性组分，结果发现包含L-脯氨酸与稳定性的进一步提高相关。
分别比较处方24-27和处方28-31的酸性组分，发现220mM L-脯氨酸和150mM L-脯氨酸对稳定性的影响差异不显著。
3)渗透压检测
渗透压检测的结果如表17所示。
表17渗透压检测结果
处方 辅料 渗透压(mOsmoL/kg)
20 150mM甘氨酸 306
24 220mM L-脯氨酸 373
28 150mM L-脯氨酸 296
根据INS(infusion nurses society)的标准，输液渗透压小于600mOsmoL/kg都属于安全范围，因此处方20、24、28的渗透压属于安全范围。
实施例5
通过对蛋白质含量、pH范围、缓冲液筛选和稳定剂筛选的系列实验，选择较优的组合，醋酸钠-醋酸缓冲体系，L-脯氨酸作为稳定剂，聚山梨酯20作为表面活性剂，通过DoE实验 设计进一步考察醋酸钠-醋酸浓度、L-脯氨酸浓度、聚山梨酯20浓度、PD-L1单克隆抗体蛋白含量和pH对蛋白稳定性的影响，采用JMP15.0软件经典两水平筛选设计，考察了这5个因素，2个水平，加上1个中心点的1次重复，共18个组合，实验设计见下表18。
表18制剂处方DOE实验不同组成
Figure PCTCN2022119187-appb-000018
以上处方经高温(40±2℃)加速14天、28天后，使用SEC-HPLC检测单体含量，WCX-HPLC检测主成分含量，结果汇总见表19。
表19：制剂组成DOE实验设计不同处方检测结果汇总
Figure PCTCN2022119187-appb-000019
Figure PCTCN2022119187-appb-000020
结果显示：缓冲系统乙酸钠浓度(5-35mM)，稳定剂L-脯氨酸浓度(50-390mM)，聚山梨酯20浓度(0-0.04％)对于蛋白检测SEC-HPLC单体和WCX-HPLC主成分无显著影响，在该范围内变化较小。蛋白质含量及pH对于蛋白检测SEC-HPLC单体和WCX-HPLC主成分有影响，在该范围内变化较大，高温28天，蛋白浓度在50-220mg/mL，SEC-HPLC单体纯度在95％以上，pH在5.9-6.9之间，WCX-HPLC主成分在74％以上，皆在可接受范围内，判断该变化属于可接受的。
实施例6
对优选处方进行加速稳定性研究：样品采用处方组成为：PD-L1单克隆抗体蛋白含量为200±20mg/mL，20mM醋酸钠-醋酸，220mM L-脯氨酸，0.02％聚山梨酯20，pH6.4±0.1，分装在西林瓶中，在25±2℃的条件下放置9个月，分别于第0、3、6、9个月末取样。按照稳定性重点考察项目进行检测，考察样品单体纯度(SEC-HPLC)、电荷分布主成分纯度(WCX-HPLC)、还原和非还原毛细管凝胶电泳(CE-SDS)、蛋白质含量等，结果如表20所示。
表20：加速(25±2℃，9个月)稳定性研究数据
Figure PCTCN2022119187-appb-000021
Figure PCTCN2022119187-appb-000022
结果显示，在25±2℃条件下放置9个月，同第0天比较，样品各项指标都在可接受范围内。表明本发明的制剂处方在室温9个月仍能保持稳定性。
实施例7
对优选处方进行长期稳定性研究：样品采用处方组成为：PD-L1单克隆抗体蛋白含量为200±20mg/mL，20mM醋酸钠-醋酸，220mM L-脯氨酸，0.02％聚山梨酯20，pH6.4±0.1，分装在西林瓶中，在5±3℃的条件下放置18个月，分别于第0、6、12、18个月末取样。按照稳定性重点考察项目进行检测，考察样品单体纯度(SEC-HPLC)、电荷分布主成分纯度(WCX-HPLC)、还原和非还原毛细管凝胶电泳(CE-SDS)、蛋白质含量等，结果如表21所示。
表21：长期(5±3℃，18个月)稳定性研究数据
Figure PCTCN2022119187-appb-000023
结果显示，在5±3℃条件下放置18个月，同第0天比较，样品检测的相关指标都在可接受范围内。表明本发明的制剂处方在5±3℃下，18个月仍能保持稳定性。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (69)

  1. 组合物，其包含抗原结合片段和辅料，其中所述抗原结合片段包含能够特异性结合PD-L1的免疫球蛋白单一可变结构域，所述免疫球蛋白单一可变结构域包含CDR1-3，其中所述CDR3包含SEQ ID NO:47所示的氨基酸序列，
    其中所述辅料选自下组中的一种或多种：脯氨酸、甘露醇、蔗糖、甘氨酸和L-精氨酸盐酸盐，
    或其中所述辅料选自下组中的一种或多种：脯氨酸、蔗糖、甘露醇、山梨醇和甘油。
  2. 根据权利要求1所述的组合物，其中所述CDR2包含SEQ ID NO:46所示的氨基酸序列。
  3. 根据权利要求1-2中任一项所述的组合物，其中所述CDR1包含SEQ ID NO:45或48所示的氨基酸序列。
  4. 根据权利要求1-3中任一项所述的组合物，所述免疫球蛋白单一可变结构域包含SEQ ID NO:1-6中任一项所示的氨基酸序列。
  5. 根据权利要求1-4中任一项所述的组合物，其中所述PD-L1为人PD-L1。
  6. 根据权利要求1-5中任一项所述的组合物，其中所述抗原结合片段包含免疫球蛋白的Fc区。
  7. 根据权利要求6所述的组合物，其中所述免疫球蛋白的Fc区的N端与所述免疫球蛋白单一可变结构域的C端直接或间接连接。
  8. 根据权利要求6-7中任一项所述的组合物，其中所述免疫球蛋白的Fc区的N端与所述免疫球蛋白单一可变结构域的C端通过连接子连接。
  9. 根据权利要求8所述的组合物，其中所述连接子包含SEQ ID NO:42-44中任一项所示的氨基酸序列。
  10. 根据权利要求6-9中任一项所述的组合物，其中所述免疫球蛋白的Fc区包含源自选自下组的免疫球蛋白的Fc区：IgG1、IgG2、IgG3和IgG4。
  11. 根据权利要求6-10中任一项所述的组合物，其中所述免疫球蛋白的Fc区不具有ADCC活性；和/或，所述免疫球蛋白的Fc区不具有CDC活性。
  12. 根据权利要求6-11中任一项所述的组合物，其中所述免疫球蛋白的Fc区来源于人。
  13. 根据权利要求6-12中任一项所述的组合物，其中所述免疫球蛋白的Fc区来源于人IgG。
  14. 根据权利要求6-13中任一项所述的组合物，其中所述免疫球蛋白的Fc区包含SEQ ID NO:7-9中任一项所示的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的组合物，其中所述抗原结合片段包含SEQ ID NO:14-31中任一项所示的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的组合物，其中所述抗原结合片段的浓度为约1mg/mL- 约220mg/mL。
  17. 根据权利要求1-16中任一项所述的组合物，其中所述抗原结合片段的浓度为约20mg/mL-约220mg/mL。
  18. 根据权利要求1-17中任一项所述的组合物，其中所述抗原结合片段的浓度为约50mg/mL-约200mg/mL。
  19. 根据权利要求1-17中任一项所述的组合物，其中所述抗原结合片段的浓度为约180mg/mL-约220mg/mL。
  20. 根据权利要求1-15任一项所述的组合物，其中抗原结合片段的浓度为约100mg/mL-约280mg/mL。
  21. 根据权利要求1-15任一项所述的组合物，其中抗原结合片段的浓度为约150mg/mL-约250mg/mL。
  22. 根据权利要求1-21中任一项所述的组合物，其中所述辅料的浓度为约50mM-约500mM。
  23. 根据权利要求1-22中任一项所述的组合物，其中所述辅料包含脯氨酸，所述脯氨酸的浓度为约50mM-约390mM。
  24. 根据权利要求1-23中任一项所述的组合物，其中所述辅料包含脯氨酸，所述脯氨酸的浓度为约150mM-约250mM。
  25. 根据权利要求1-24中任一项所述的组合物，其中所述辅料包含脯氨酸，所述脯氨酸的浓度为约150mM-约220mM。
  26. 根据权利要求1-25中任一项所述的组合物，其中所述辅料包含L-脯氨酸，所述L-脯氨酸的浓度为约150mM-约220mM。
  27. 根据权利要求1-26中任一项所述的组合物，其中所述辅料包含L-脯氨酸，所述L-脯氨酸的浓度为约165mM-约275mM。
  28. 根据权利要求1-27中任一项所述的组合物，其中所述辅料包含甘露醇，所述甘露醇的浓度为约50mM-约500mM。
  29. 根据权利要求1-28中任一项所述的组合物，其中所述辅料包含蔗糖，所述蔗糖的浓度为约50mM-约500mM。
  30. 根据权利要求1-29中任一项所述的组合物，其中所述辅料包含蔗糖，所述蔗糖的浓度为约90mM-约500mM。
  31. 根据权利要求1-30中任一项所述的组合物，其中所述辅料包含甘氨酸，所述甘氨酸的浓度为约50mM-约500mM。
  32. 根据权利要求1-31中任一项所述的组合物，其中所述辅料包含甘氨酸，所述甘氨酸的浓 度为约150mM-约250mM。
  33. 根据权利要求1-32中任一项所述的组合物，其中所述辅料包含L-精氨酸盐酸盐，所述L-精氨酸盐酸盐的浓度为约50mM-约500mM。
  34. 根据权利要求1-33中任一项所述的组合物，其中所述辅料包含L-精氨酸盐酸盐，所述L-精氨酸盐酸盐的浓度为约150mM-约500mM。
  35. 根据权利要求1-34中任一所述的组合物，其中所述辅料包含山梨醇，所述山梨醇的浓度为约50mM-约500mM。
  36. 根据权利要求1-35中任一所述的组合物，其中所述辅料包含山梨醇，所述山梨醇的浓度为约100mM-约300mM。
  37. 根据权利要求1-36中任一所述的组合物，其中所述辅料包含甘油，所述甘油的浓度为50mM-约500mM。
  38. 根据权利要求1-37中任一所述的组合物，其中所述辅料包含甘油，所述甘油的浓度为100mM-约300mM。
  39. 根据权利要求1-38中任一项所述的组合物，其pH为约5.0-约7.5。
  40. 根据权利要求1-39中任一项所述的组合物，其pH为约5.9-约6.9。
  41. 根据权利要求1-40中任一项所述的组合物，其pH为约6.3-约6.5。
  42. 根据权利要求1-41中任一项所述的组合物，其pH为约6.4。
  43. 根据权利要求1-42中任一项所述的组合物，其包含缓冲成分，其中所述缓冲成分选自下组中的一种或多种：乙酸钠-乙酸、组氨酸-乙酸、L-组氨酸-L-组氨酸盐酸盐、磷酸钠和柠檬酸-氢氧化钠。
  44. 根据权利要求43所述的组合物，其中所述缓冲成分的浓度为约5mM-约50mM。
  45. 根据权利要求43-44中任一项所述的组合物，其中所述缓冲成分包含乙酸钠-乙酸，所述乙酸钠-乙酸的浓度为约5mM-约35mM。
  46. 根据权利要求43-45中任一项所述的组合物，其中所述缓冲成分包含乙酸钠-乙酸，所述乙酸钠-乙酸的浓度为约10mM-约30mM。
  47. 根据权利要求43-46中任一项所述的组合物，其中所述缓冲成分包含乙酸钠-乙酸，所述乙酸钠-乙酸的浓度为约20mM。
  48. 根据权利要求1-47中任一项所述的组合物，其包含表面活性剂，其中所述表面活性剂选自下组中的一种或多种：聚山梨酯20、聚山梨酯80和泊洛沙姆188。
  49. 根据权利要求48所述的组合物，其中所述表面活性剂的浓度为约0mg/mL-约0.8mg/mL。
  50. 根据权利要求48-49中任一项所述的组合物，其中所述表面活性剂包含聚山梨酯20，所述 聚山梨酯20的浓度为约0mg/mL-约0.4mg/mL。
  51. 根据权利要求48-50中任一项所述的组合物，其中所述表面活性剂包含聚山梨酯20，所述聚山梨酯20的浓度为约0.2mg/mL-约0.4mg/mL。
  52. 根据权利要求48-50中任一项所述的组合物，其中所述表面活性剂包含聚山梨酯20，所述聚山梨酯20的浓度为约0.1mg/mL-约0.3mg/mL。
  53. 根据权利要求48-52中任一项所述的组合物，其中所述表面活性剂包含聚山梨酯20，所述聚山梨酯20的浓度为约0.2mg/mL。
  54. 根据权利要求1-53中任一项所述的组合物，其包含：
    1)约20mg/mL-约220mg/mL权利要求1-15中任一项所述的抗原结合片段；
    2)约5mM-约35mM乙酸钠-乙酸；
    3)约50mM-约390mM脯氨酸；
    4)约0mg/mL-约0.4mg/mL聚山梨酯20；
    且pH为约5.9-约6.9。
  55. 根据权利要求1-54中任一项所述的组合物，其包含：
    1)约50mg/mL-约220mg/mL权利要求1-15中任一项所述的抗原结合片段；
    2)约10mM-约30mM乙酸钠-乙酸；
    3)约150mM-约250mM脯氨酸；
    4)约0.2mg/mL-约0.4mg/mL聚山梨酯20；
    且pH为约6.3-约6.5。
  56. 根据权利要求1-55中任一项所述的组合物，其包含：
    1)约200mg/mL权利要求1-15中任一项所述的抗原结合片段；
    2)约20mM乙酸钠-乙酸；
    3)约220mML-脯氨酸；
    4)约0.2mg/mL聚山梨酯20；
    且pH为约6.4。
  57. 根据权利要求1-56中任一项所述的组合物，其用于注射。
  58. 根据权利要求1-57中任一项所述的组合物，其用于皮下注射。
  59. 根据权利要求1-58中任一项所述的组合物，其为制剂。
  60. 根据权利要求1-59中任一项所述的组合物，其为液体制剂。
  61. 试剂盒，其包括权利要求1-60中任一项所述的组合物和盛放权利要求1-60中任一项所述的组合物的容器。
  62. 根据权利要求61所述的试剂盒，其中所述容器包括玻璃瓶。
  63. 根据权利要求61-62中任一项所述的试剂盒，其中所述容器中所述组合物的体积为约0.5mL-约5.0mL。
  64. 根据权利要求61-63中任一项所述的试剂盒，其中所述容器中所述组合物的体积为约0.5mL-约1.5mL。
  65. 权利要求1-60中任一项所述的组合物；和/或权利要求61-64中任一项所述的试剂盒在制备预防、缓解和/或治疗肿瘤的药物中的应用。
  66. 根据权利要求65所述的应用，其中所述肿瘤包括PD-L1阳性的肿瘤。
  67. 根据权利要求65-66中任一项所述的应用，其中所述肿瘤包括恶性实体瘤。
  68. 权利根据要求1-60中任一项所述的组合物；和/或权利要求61-64中任一项所述的试剂盒在制备预防、缓解和/或治疗感染性疾病的药物中的应用。
  69. 根据权利要求68所述的应用，其中所述感染性疾病包括病毒感染、细菌感染、真菌感染和/或寄生虫感染所引起的疾病。
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