WO2024027824A1 - 抗cd112r抗体药物组合物及其用途 - Google Patents

抗cd112r抗体药物组合物及其用途 Download PDF

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WO2024027824A1
WO2024027824A1 PCT/CN2023/111228 CN2023111228W WO2024027824A1 WO 2024027824 A1 WO2024027824 A1 WO 2024027824A1 CN 2023111228 W CN2023111228 W CN 2023111228W WO 2024027824 A1 WO2024027824 A1 WO 2024027824A1
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seq
amino acid
concentration
acid sequence
buffer
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PCT/CN2023/111228
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English (en)
French (fr)
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韩秀娟
刘洪川
刘沛想
李宝贤
王静
周岳华
吉丽文
冯辉
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上海君实生物医药科技股份有限公司
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Publication of WO2024027824A1 publication Critical patent/WO2024027824A1/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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to the field of therapeutic pharmaceutical compositions, and in particular to anti-CD112R antibody pharmaceutical compositions and their uses.
  • the immune system is a highly complex system composed of many cell types, including but not limited to T cells, B cells, natural killer cells, antigen-presenting cells, dendritic cells, monocytes, and macrophages. These cells possess complex and subtle systems that control their interactions and responses. Cells utilize activation and inhibitory mechanisms and feedback loops to maintain control of responses and not allow for the negative consequences of uncontrolled immune responses (e.g., autoimmune diseases).
  • CD112R (PVRIG), also known as poliovirus receptor-associated immunoglobulin domain-containing protein, Q6DKI7, or C7orf15, is a 326-amino-acid-long transmembrane domain protein with a signal peptide spanning amino acid 1 to 40), extracellular domain (spanning amino acids 41 to 171), transmembrane domain (spanning amino acids 172 to 190), and cytoplasmic domain (spanning amino acids 191 to 326).
  • CD112R binds to poliovirus receptor-related protein 2 (PVLR2, also known as nectin-2, CD112, or herpesvirus entry mediator B (HVEB), human plasma membrane glycoprotein), the binding partner of CD112R.
  • PVLR2 poliovirus receptor-related protein 2
  • HVEB herpesvirus entry mediator B
  • CD112R is an inhibitory receptor mainly expressed by T cells and NK cells, and competes with the activating receptor CD226 for binding to CD112.
  • CD112 interacts with CD112R with higher affinity than with CD226, thereby effectively regulating CD226-mediated cell activation.
  • Anti-CD112R antibodies that block the interaction with CD112 limit inhibitory signaling directly downstream of CD112R while promoting greater immune cell activation by increasing the interaction of CD226 with CD112.
  • Treatment with anti-CD112R antibodies provides the opportunity to downregulate, and possibly enhance, inhibitory signals that may occur when CD112R-expressing immune cells bind to CD112 on tumor cells and/or myeloid cells within the tumor microenvironment, Increase and maintain anti-tumor immune responses.
  • the pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing an antibody that specifically binds to CD112R.
  • the interaction and synergy between the components of the pharmaceutical composition provide a suitable long-term storage environment for the anti-CD112R monoclonal antibody. It has high stability, strong tolerance to high temperature, shaking and repeated freezing and thawing, and It can block the interaction of CD112/CD112R, improve the activation ability of T cells, mobilize the immune system, and exert a stronger tumor killing effect.
  • the invention provides a pharmaceutical composition, comprising: (1) buffer; (2) anti-CD112R antibody or antigen-binding fragment thereof.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof includes LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3 respectively:
  • LCDR1 KASQSVSNDVA (SEQ ID NO: 1);
  • LCDR2 YVSX 1 RFT, where X 1 is H or N;
  • LCDR3 X 2 QAYRSPWT, where X 2 is H or Q;
  • HCDR1 SYHMS (SEQ ID NO: 6):
  • HCDR2 AIX 3 X 4 X 5 GX 6 X 7 TYYX 8 DTVKG, where X 3 is S or N, X 4 is R or S, X 5 is D or N, or N, X 8 is P or L;
  • HCDR3 HEDYX 9 GFAMDX 10 , X 9 is F or Y, X 10 is S or Y.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof includes:
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 1;
  • amino acid sequence is LCDR2 shown in SEQ ID NO: 2 or 4;
  • amino acid sequence is LCDR3 shown in SEQ ID NO: 3 or 5;
  • amino acid sequence is HCDR1 shown in SEQ ID NO: 6;
  • amino acid sequence is HCDR2 shown in SEQ ID NO: 7 or 9;
  • the amino acid sequence is HCDR3 shown in SEQ ID NO: 8 or 10.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof includes:
  • amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the amino acid sequences are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 3 respectively.
  • amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the amino acid sequences are SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 3 respectively.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof is selected from the group consisting of murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, humanized antibody or antigen-binding fragment thereof, preferably a humanized antibody or Its antigen-binding fragment.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof includes:
  • the anti-CD112R antibodies described above include:
  • amino acid sequence is the light chain amino acid sequence shown in SEQ ID NO: 16, and the amino acid sequence is the heavy chain amino acid sequence shown in SEQ ID NO: 17.
  • amino acid sequence is the light chain amino acid sequence shown in SEQ ID NO: 16, and the amino acid sequence is the heavy chain amino acid sequence shown in SEQ ID NO: 18.
  • amino acid sequence is the light chain amino acid sequence shown in SEQ ID NO: 19, and the amino acid sequence is the heavy chain amino acid sequence shown in SEQ ID NO: 20.
  • the concentration of the anti-CD112R antibody or antigen-binding fragment thereof in the above pharmaceutical composition is about 1 to 100 mg/mL, preferably about 10 to 70 mg/mL, more preferably about 20 to 60 mg/mL; more preferably , the concentration of the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof is about 1 mg/mL, about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 35 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL mL, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL or any two values within these ranges as the range formed by the endpoint, preferably about 35 mg/mL, about 40 mg/mL or about 45 mg/mL. mL.
  • the pH of the above pharmaceutical composition is about 4.5 to 6.5, preferably about 5.5 to 6.5, more preferably about 5.5 to 5.7 or 5.8 to 6.2.
  • Non-limiting examples of the pH of the above pharmaceutical composition are about 4.5. , about 5.0, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5 or a range formed by any two values within these ranges as endpoints, Preferably it is about 5.9, about 6.0 or about 6.1.
  • the pharmaceutical composition has an osmotic pressure of about 250 to 350 mOsm/kg.
  • the above buffer is selected from one or more of acetate buffer, citrate buffer, phosphate buffer and histidine buffer; preferably, the buffer is histidine buffer .
  • the concentration of the above buffer is about 5-100mM, preferably about 10-50mM, preferably about 10 ⁇ 30mM; preferably about 15 ⁇ 25mM, non-limiting examples of the above buffer concentration are about 10mM, about 15mM, about 20mM, about 25mM, about 30mM, about 40mM, about 45mM, about 50mM or any two within these ranges.
  • the range formed by a numerical value as an endpoint is preferably about 15mM, about 20mM or about 25mM.
  • the pH of the above-mentioned buffer is about 4.5-6.5, preferably about 5.5-6.5, more preferably about 5.8-6.2, and non-limiting examples of the pH of the above-mentioned buffer are about 4.5, about 5.0, about 5.5 , about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5 or any two values within these ranges as the range formed by the endpoints, preferably about 5.9, about 6.0 or about 6.1.
  • the above buffer is a histidine buffer; preferably, the histidine buffer is a histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer. liquid, preferably histidine-histidine hydrochloride buffer.
  • the pH of the histidine buffer is 5.5-6.5, preferably 5.8-6.2.
  • the histidine-histidine hydrochloride buffer described above is made from histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made from about 1 to 20 mM L-histidine and about 1 to 20 mM L-histidine monohydrochloride. In some embodiments, the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of about 1:1 to about 1:4. In some embodiments, the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of about 1:1.
  • the histidine buffer is made from histidine and histidine hydrochloride in a molar ratio of about 1:3.
  • the histidine formulation is a histidine buffer with a pH of about 5.5 made from about 4.5 mM L-histidine and about 15.5 mM L-histidine monohydrochloride.
  • the histidine formulation is a histidine buffer with a pH of about 6.0 made from about 10 mM L-histidine and about 10 mM L-histidine hydrochloride.
  • the histidine formulation is a histidine buffer with a pH of about 6.5 made from about 3 mM L-histidine and about 17 mM L-histidine hydrochloride.
  • the pH of the acetate buffer is about 4.5 to 5.7, preferably about 5.3 to 5.7.
  • the acetate buffer is an acetic acid-sodium acetate buffer.
  • the acetic acid-sodium acetate buffer is made from about 1 to 20 mM acetic acid and about 1 to 20 mM sodium acetate.
  • the acetic acid-sodium acetate buffer is made from acetic acid and sodium acetate in a molar ratio of about 1:2 to about 1:3.
  • the acetic acid-sodium acetate buffer is made from acetic acid and sodium acetate in a molar ratio of about 1:5 to about 1:8.
  • the acetic acid-sodium acetate buffer is: an acetate buffer with a pH of about 4.5 made from about 8 mM acetic acid and about 12 mM sodium acetate. In some embodiments, the acetic acid-sodium acetate buffer is: an acetate buffer with a pH of about 5.0 made from about 6.5 mM acetic acid and about 13.5 mM sodium acetate.
  • the acetic acid-sodium acetate buffer is: an acetate buffer with a pH of about 5.0 to 6.0 made from about 2 to 4mM acetic acid and about 16 to 18mM sodium acetate; preferably, it is made from about 3mM acetic acid and about 17mM acetic acid.
  • An acetate buffer made from sodium acetate has a pH of approximately 5.5.
  • the buffer is a citrate buffer.
  • the pH of the citric acid buffer is 5.0-6.0.
  • the citrate buffer is a citric acid-sodium citrate buffer.
  • the citrate buffer is made from about 1 to 20 mM citric acid and about 1 to 20 mM sodium citrate.
  • citrate buffer is determined by the molar ratio Made of citric acid and sodium citrate in a ratio of about 1:1 to 1:4.
  • the citrate buffer is: a citrate buffer with a pH of about 5.0 made from about 15mM citric acid and about 5mM sodium citrate.
  • the citrate buffer is: a citrate buffer with a pH of about 5.5 made from about 12mM citric acid and about 8mM sodium citrate. In some embodiments, the citrate buffer is: a citrate buffer with a pH of about 6.0 made from about 10 mM citric acid and about 10 mM sodium citrate.
  • the above-mentioned buffer is a phosphate buffer.
  • the phosphate buffer is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.
  • the phosphate buffer is made from about 1 to 20 mM sodium hydrogen phosphate disodium and about 1 to 20 mM sodium hydrogen phosphate dibasic.
  • the phosphate buffer is made from disodium hydrogen phosphate and sodium hydrogen phosphate in a molar ratio of about 1:1 to 1:4.
  • the phosphate buffer is: a phosphate buffer with a pH of about 7.0 made from about 10 mM disodium hydrogen phosphate and about 10 mM sodium dihydrogen phosphate.
  • the above pharmaceutical composition also includes a stabilizer, the stabilizer is selected from one or more of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose and trehalose. species;
  • the above-mentioned arginine salt is arginine hydrochloride.
  • the concentration of the above-mentioned stabilizer is about 10 ⁇ 400mM, preferably about 100 ⁇ 300mM, preferably about 150 ⁇ 270mM, preferably about 180 ⁇ 260mM, and the non-limiting example of the above-mentioned stabilizer concentration is about 100mM, About 130mM, about 150mM, about 170mM, about 180mM, about 190mM, about 200mM, about 210mM, about 220mM, about 225mM, about 230mM, about 235mM, about 240mM, about 250mM, about 260mM or any two values within these ranges as The range formed by the endpoint is preferably about 190mM, about 220mM, about 230mM or about 240mM.
  • the above-mentioned stabilizer is trehalose with a concentration of about 150-270mM; or the above-mentioned stabilizer is sucrose with a concentration of about 150-270mM; or the above-mentioned stabilizer is sodium chloride with a concentration of about 20-80mM and a concentration of about 110- A combination of 170mM sucrose; or the above-mentioned stabilizer is a combination of arginine hydrochloride with a concentration of about 20-80mM and sucrose with a concentration of about 110-170mM.
  • the above-mentioned stabilizer is trehalose at a concentration of about 180-260mM, preferably about 220-240mM, sucrose at a concentration of about 180-260mM, preferably about 220-240mM, and sucrose at a concentration of about 30-70mM, preferably about 40-60mM.
  • Sucrose combination is trehalose at a concentration of about 180-260mM, preferably about 220-240mM, sucrose at a concentration of about 180-260mM, preferably about 220-240mM, and sucrose at a concentration of about 30-70mM, preferably about 40-60mM.
  • sucrose combination is trehalose at a concentration of about 180-
  • the above-mentioned stabilizer is trehalose.
  • the above-mentioned stabilizer is mannitol with a concentration of about 100-300mM.
  • the concentration of the above-mentioned mannitol is preferably about 150-270mM, preferably about 180-260mM.
  • a non-limiting example of the above-mentioned mannitol concentration is about 200mM.
  • the stabilizer is sucrose.
  • the above-mentioned stabilizer is sucrose with a concentration of about 100-300mM.
  • the concentration of the above-mentioned sucrose is preferably about 150-270mM, preferably about 180-260mM.
  • the concentration of the above-mentioned sucrose is Non-limiting examples are about 200mM, about 210mM, about 220mM, about 225mM, about 230mM, about 235mM, about 240mM, about 250mM or any two values within these ranges as endpoints, preferably about 225mM, about 230mM. or about 235mM.
  • the stabilizer is a combination of sodium chloride and sucrose.
  • the above-mentioned stabilizer is a combination of about 30-200mM sodium chloride and about 30-200mM sucrose, preferably about 20-80mM sodium chloride and about 110-170mM sucrose, preferably about 30-200mM sucrose.
  • Non-limiting examples of the above-mentioned stabilizer are a combination of about 50mM sodium chloride and about 140mM sucrose, or about 50mM sodium chloride and about 150mM sucrose. Sucrose combination.
  • the above-mentioned stabilizer is a combination of arginine hydrochloride and sucrose.
  • the above-mentioned stabilizer is a combination of about 30-200mM arginine hydrochloride and about 30-200mM sucrose, preferably a combination of about 20-80mM arginine hydrochloride and about 110-170mM sucrose, preferably about A combination of 30-70mM arginine hydrochloride and about 120-160mM sucrose.
  • Non-limiting examples of the above-mentioned stabilizer are a combination of about 50mM arginine hydrochloride and about 140mM sucrose, or about 50mM arginine hydrochloride. Combination of acid with approximately 160mM sucrose.
  • the above pharmaceutical composition further includes a surfactant selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188.
  • the above surfactant is selected from polysorbate 80.
  • the above surfactant concentration is about 0.001% to 0.1%, preferably about 0.01% to 0.1%, preferably about 0.04% to 0.06%; as a non-limiting example, the above The surfactant concentration is about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.1% or any two values within these ranges as The range formed by the endpoint is preferably about 0.02%, about 0.04% or about 0.06%.
  • the pharmaceutical composition includes the components shown in any one of the following (1) to (14), or consists of the components shown in any one of (1) to (14) respectively:
  • the anti-CD112R antibody comprises the light chain amino acid sequence shown in SEQ ID NO: 16 and the heavy chain amino acid sequence shown in SEQ ID NO: 18 Sequence; (b) about 20mM histidine buffer, pH about 6.0, or about 20mM acetate buffer, pH about 5.5; (c) about 230mM trehalose; and (d) about 0.02% (w /v) polysorbate 80; or
  • the anti-CD112R antibody comprises the light chain amino acid sequence shown in SEQ ID NO: 16 and the heavy chain amino acid sequence shown in SEQ ID NO: 18 Sequence; (b) about 20mM histidine buffer, pH about 6.0, or about 20mM acetate buffer, pH about 5.5; (c) about 230mM sucrose; and (d) about 0.02% (w/ v) polysorbate 80; or
  • the anti-CD112R antibody comprises the light chain amino acid sequence shown in SEQ ID NO: 16 and the heavy chain amino acid sequence shown in SEQ ID NO: 18 Sequence; (b) about 20mM histidine buffer, pH about 6.0, or about 20mM acetate buffer, pH about 5.5; (c) about 230mM sucrose; and (d) about 0.04% (w/ v) polysorbate 80; or
  • the anti-CD112R antibody comprises the light chain amino acid sequence shown in SEQ ID NO: 16 and the heavy chain amino acid sequence shown in SEQ ID NO: 18 sequence; (b) about 20mM, Histidine buffer at a pH of about 6.0, or about 20 mM acetate buffer at a pH of about 5.5; (c) about 230 mM sucrose; and (d) about 0.06% (w/v) polysorbate 80; or
  • the anti-CD112R antibody comprises the light chain amino acid sequence shown in SEQ ID NO: 16 and the heavy chain amino acid sequence shown in SEQ ID NO: 18 Sequence; (b) about 20mM histidine buffer with a pH of about 6.0, or about 20mM acetate buffer with a pH of about 5.5; (c) stabilizer, the stabilizer being arginine hydrochloride at a concentration of about 50mM A combination of acid and sucrose at a concentration of about 140 mM; and (d) about 0.02% (w/v) polysorbate 80; or
  • the anti-CD112R antibody comprises the light chain amino acid sequence shown in SEQ ID NO: 16 and the heavy chain amino acid sequence shown in SEQ ID NO: 18 Sequence; (b) about 20mM histidine buffer with a pH of about 6.0, or about 20mM acetate buffer with a pH of about 5.5; (c) stabilizer, the stabilizer being sodium chloride at a concentration of about 50mM In combination with sucrose at a concentration of about 140 mM; and (d) about 0.02% (w/v) polysorbate 80.
  • the pharmaceutical composition described in any of the embodiments herein is a liquid or lyophilized formulation.
  • the pharmaceutical compositions described above are liquid formulations.
  • the above-mentioned liquid preparation or lyophilized preparation is stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.
  • the above-mentioned liquid formulation or lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.
  • the present invention also provides an injection, which contains the pharmaceutical composition described in any scheme herein and sodium chloride solution or glucose solution; preferably, the concentration of the sodium chloride solution is about 0.85 to 0.9% (w /v); Preferably, the concentration of the glucose solution is about 5 to 25% (w/v), more preferably about 5 to 10% (w/v).
  • the above-mentioned pharmaceutical composition or injection is administered via intravenous injection.
  • the present invention also provides the use of the pharmaceutical composition or injection described in any of the aspects herein in the preparation of a medicament for the treatment and/or prevention of CD112R-mediated diseases or conditions.
  • the present invention also provides the pharmaceutical composition or injection described in any of the aspects herein for the treatment and/or prevention of CD112R-mediated diseases or conditions.
  • the present invention also provides a method of treating and/or preventing a CD112R-mediated disease or disorder, comprising administering to a subject in need thereof a pharmaceutical composition or injection as described in any one of the protocols herein.
  • the disease or condition is cancer.
  • Figure 1 Binding and dissociation curves of pharmaceutical compositions and recombinant human PVRIG protein.
  • Figure 2 Binding curves of pharmaceutical compositions and negative controls to human CD112R protein.
  • Figure 3 Ligand competitive inhibition ELISA curve of pharmaceutical composition and negative control competing with CD112 for binding to human CD112R.
  • Figure 4 Binding activity of pharmaceutical compositions to CD112R on the surface of CHO hCD112R cells.
  • Figure 5 Pharmaceutical composition blocks the binding activity of hCD112R protein to CHO CD112 cells.
  • Figure 6 Activity of pharmaceutical compositions in human CD112R/CD112 luciferase reporter gene system.
  • Figure 7 Tumor growth inhibition effects of anti-CD112R humanized antibodies, anti-TIGIT antibodies and their combination.
  • composition means a mixture containing one or more antibodies described herein together with other components, such as physiologically acceptable carriers and excipients.
  • pharmaceutical compositions are to facilitate administration to living organisms and facilitate the absorption of active ingredients to exert biological activity.
  • liquid formulation refers to a formulation in a liquid state and is not intended to refer to a resuspended lyophilized formulation.
  • the liquid formulations of the present invention are stable on storage, and their stability does not depend on lyophilization (or other state-changing methods, such as spray drying).
  • aqueous liquid formulation refers to a liquid formulation using water as a solvent.
  • aqueous liquid formulations are formulations that do not require lyophilization, spray drying, and/or freezing to maintain stability (eg, chemical and/or physical stability and/or biological activity).
  • excipient refers to agents that may be added to a formulation to provide desired properties (eg, consistency, increased stability) and/or to adjust osmotic pressure.
  • desired properties eg, consistency, increased stability
  • excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers. compound.
  • buffer pH of about 4.5 to about 6.5 refers to an agent that renders a solution containing it resistant to changes in pH through the action of its acid/base conjugate component.
  • Buffers used in the formulations of the present invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.
  • buffers that control the pH within this range include acetic acid, acetates (eg, sodium acetate), succinic acid, succinates (eg, sodium succinate), gluconic acid, histidine, histidine, Amine salts (e.g. histidine hydrochloride), methionine, citric acid (citrate), citrate (citrate), phosphates, citrate/phosphate, imidazole, combinations thereof and other organic acid buffers.
  • acetic acid eg, sodium acetate
  • succinic acid eg, sodium succinate
  • succinates eg, sodium succinate
  • gluconic acid histidine
  • histidine histidine
  • Amine salts e.g. histidine hydrochloride
  • methionine e.g. histidine hydrochloride
  • citric acid citrate
  • citrate citrate
  • phosphates citrate/phosphate
  • imidazole imidazole
  • a “histidine buffer” is a buffer containing histidine ions.
  • histidine buffers include histidine and salts of histidine, such as histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, such as histidine containing Histidine buffer with acid and histidine hydrochloride; the histidine buffer of the present invention also includes histidine buffer containing histidine and acetate (such as sodium salt or potassium salt).
  • citrate buffer also known as “citrate buffer” is a buffer that includes citrate ions.
  • citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.
  • the preferred citrate buffer is citric acid-sodium citrate buffer.
  • Acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like.
  • the preferred acetate buffer is acetic acid-sodium acetate buffer.
  • succinate buffer is a buffer that includes succinate ions.
  • succinic acid buffers include sodium succinate-succinate, potassium succinate-succinate, calcium succinate-succinate, magnesium succinate-succinate, and the like.
  • the preferred succinate buffer is succinate-sodium succinate buffer.
  • Phosphate buffer is a buffer that includes phosphate ions.
  • phosphate buffer include sodium dihydrogen phosphate-disodium hydrogen phosphate, potassium dihydrogen phosphate-dipotassium hydrogen phosphate, and the like.
  • a preferred phosphate buffer is sodium phosphate dibasic-sodium hydrogen phosphate buffer.
  • stabilizer means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation against chemical and/or physical degradation during manufacture, storage and use.
  • Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or their Salt (such as arginine hydrochloride), glycine, alanine (alpha-alanine, beta-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline N-oxidation of acid, 4-hydroxyproline, sarcosine, gamma-aminobutyric acid (GABA), opines, alanopin, octopine, glycine opin (strombine) and trimethylamine (TMAO), human serum albumin (hsa), bovine
  • Some stabilizers such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in controlling osmotic pressure.
  • the stabilizer specifically used in the present invention is selected from one or more types of polyols, amino acids, salts, and sugars.
  • Preferred salts are sodium chloride
  • preferred sugars are sucrose and trehalose
  • preferred polyols are sorbitol and mannitol.
  • Preferred amino acids are arginine, glycine, proline.
  • the amino acid may exist in its D- and/or L-form, but is typically in the L-form.
  • the amino acid may exist in any suitable salt, such as the hydrochloride salt, such as hydrochloric acid.
  • Arginine Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose , sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose.
  • surfactant generally includes agents that protect proteins, such as antibodies, from air/solution interface-induced stress, solution/surface-induced stress to reduce aggregation of antibodies or to minimize the formation of particulate matter in a formulation.
  • exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene- Polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxyethylene Propylene copolymer (poloxamer, Pluronic), sodium dodecyl sulfate (SDS).
  • nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene
  • concentration of polysorbate 20 and “concentration of polysorbate 80” both refer to mass volume concentration (w/v), such as “about 0.02% polysorbate 80" in “ “0.02%” means "100mL of liquid contains 0.02g of polysorbate 80".
  • isotonic means that the preparation has substantially the same osmotic pressure as human blood.
  • Isotonic formulations generally have an osmolality of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression type osmometer.
  • stable formulation is one in which the antibody substantially retains its physical stability and/or chemical stability and/or biological activity during the manufacturing process and/or upon storage. Pharmaceutical preparations can be stable even if the contained antibodies do not retain 100% of their chemical structure or biological function after storage for a certain period of time. In some cases, an antibody that maintains about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of its structure or function after storage for a certain period of time may also be considered " stable”.
  • Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery 247-301, Vincent Lee, Editor-in-Chief, Marcel Dekker, Inc. ., New York, N.Y., Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both incorporated by reference).
  • Stability can be measured by determining the percentage of native antibody remaining in a preparation after it has been stored at a certain temperature for a certain period of time (among other methods).
  • the percentage of native antibodies can be measured by size-exclusion chromatography (eg, size-exclusion high-performance liquid chromatography [SEC-HPLC]), among other methods, with "native" meaning unaggregated and undegraded.
  • protein stability is determined as the percentage of monomeric protein in a solution with a low percentage of degraded (eg, fragmented) and/or aggregated protein.
  • the formulation can be stored stably at room temperature, about 25-30°C, or 40°C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 Months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, up to no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.
  • Stability can be measured by determining (among other methods) the percentage of antibody (“acidic form") that migrates in the more acidic fraction of the main antibody fraction ("mainly charged form") during ion exchange, where stability is related to Inversely proportional to the percentage of acidic form of antibody.
  • the percentage of "acidified” antibody can be measured by ion exchange chromatography (eg, cation exchange high performance liquid chromatography [CEX-HPLC]), among other methods.
  • an acceptable degree of stability means that no more than about 49%, 45%, 40%, 35% of the acidic form of the antibody is detectable in the formulation after storage at a certain temperature for a certain period of time.
  • the period of storage before measuring stability may be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer.
  • a certain temperature at which a pharmaceutical formulation is allowed to be stored may be any temperature in the range of about -80°C to about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C , about 2 ⁇ 8°C, about 5°C, about 25°C, or about 40°C.
  • the antibody shows essentially no signs of, for example, aggregation, precipitation and/or denaturation upon visual inspection of color and/or clarity or when measured by UV light scattering or by pore size exclusion chromatography, the antibody is present in the pharmaceutical combination. "Maintain its physical stability" in the object. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the extent that visible precipitates are formed.
  • Stability such as physical stability
  • Stability can be assessed by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements are related to the turbidity of the formulation.
  • the turbidity of a formulation is partly an intrinsic property of the protein dissolved in solution and is typically measured by turbidimetry and is measured in nephelometric turbidity units (NTU).
  • NTU nephelometric turbidity units
  • the level of turbidity that changes with, for example, the concentration of one or more components in a solution is also referred to as the "opacity" or “opaque appearance" of a formulation.
  • Turbidity levels can be calculated by reference to a standard curve generated using suspensions of known turbidity. Reference standards for determining turbidity levels of pharmaceutical compositions may be based on European Pharmacopoeia standards, 4th edition, "Directorate for the Quality of Medicines" of the Council of Europe (EDQM), France).
  • a clear solution is defined as a solution with a turbidity lower than or equal to that of a reference suspension having a turbidity of about 3 according to the European Pharmacopoeia standards.
  • Nephelometric turbidity measurements can detect Rayleigh scattering in the absence of association or non-ideal effects, which typically varies linearly with concentration. Other methods for assessing physical stability are known in the art.
  • Chemical stability can be assessed, for example, by detecting or quantifying chemically altered forms of the antibody.
  • Chemical alterations may include size changes (eg, clipping), which may be assessed using, for example, size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI/TOF MS).
  • size changes eg, clipping
  • MALDI/TOF MS matrix-assisted laser desorption ionization/time of flight mass spectrometry
  • Other types of chemical changes include charge changes (eg occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.
  • an antibody "retains its biological activity" in a pharmaceutical composition if the antibody is biologically active for its intended purpose. For example, if the preparation is stored for a certain period of time (for example, 1 to 12 months) at temperatures such as 5°C, 25°C, 45°C, etc., the binding affinity of the anti-CD112R antibody contained in the preparation to CD112R is the binding affinity of the antibody before storage. If the ratio is at least 90%, 95% or more, the preparation of the present invention can be considered to be stable. Binding affinity can also be determined using, for example, ELISA or plasmon resonance techniques.
  • a “therapeutically effective amount” or “effective amount” of an antibody means, in the pharmacological sense, an amount that is effective in preventing or treating or alleviating the symptoms of a disorder for which the antibody is effective.
  • a “therapeutically effective amount” or a “therapeutically effective dose” of a drug is any amount of the drug that protects a subject from the onset of a disease or promotes regression of a disease when used alone or in combination with another therapeutic agent, so Regression of said disease is evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods of disease, or the prevention of impairment or disability caused by disease distress.
  • a therapeutically effective amount of a drug includes a "prophylactically effective amount,” that is, any amount that inhibits the progression or recurrence of a disease when administered alone or in combination with other therapeutic agents to a subject at risk for the disease or a subject experiencing recurrence of the disease.
  • Drug is administered alone or in combination with other therapeutic agents to a subject at risk for the disease or a subject experiencing recurrence of the disease.
  • subject or "patient” is intended to include mammalian organisms.
  • subjects/patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human.
  • administer refers to the introduction of a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art.
  • Routes of administration for anti-CD112R antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes, such as injection or infusion.
  • Parenteral administration refers to modes of administration other than enteral or local administration, usually by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, and intracystic , intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intradural and intrasternal injection and infusion as well as in vivo electroporation.
  • antibody as used herein shall be understood to include intact antibody molecules and antigen-binding fragments thereof.
  • antigen-binding portion or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”) refers to an antibody One or more fragments that retain the specific binding ability to human CD112R or its epitope. Therefore, it is used in the broadest sense, specifically including but not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), humanized antibodies, fully human antibodies, Chimeric antibodies and camelized single domain antibodies.
  • isolated antibody refers to the purified state of the binding compound, and in this context means that the molecule is substantially free of other biological molecules, such as nucleic acids, proteins, lipids, sugars, or other materials such as cell debris and growth medium .
  • isolated does not imply the complete absence of such materials or the absence of water, buffers or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the bound compounds described herein.
  • the term "monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, the individual antibodies composing the population are identical except for possible naturally occurring mutations, which may be present in minor amounts. Monoclonal antibodies are highly specific and target a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes.
  • the modifier “monoclonal” indicates the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • murine antibody or “hybridoma antibody” in this disclosure refers to a monoclonal antibody against human CD112R prepared according to the knowledge and skill in the art. The preparation involves injecting a test subject with the CD112R antigen and then isolating hybridomas expressing antibodies with the desired sequence or functional properties.
  • chimeric antibody is an antibody having a variable domain of a first antibody and a constant domain of a second antibody, where the first and second antibodies are from different species.
  • the variable domains are obtained from an antibody such as a rodent (a "parent antibody")
  • the constant domain sequences are obtained from a human antibody such that the resulting chimeric antibody induces in human subjects compared to the parent rodent antibody. There is a lower likelihood of adverse immune responses.
  • humanized antibody refers to antibody forms containing sequences from human and non-human (eg, mouse, rat) antibodies.
  • a humanized antibody contains substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the The framework (FR) region is the framework region of human immunoglobulin sequences.
  • the humanized antibody optionally can comprise at least a portion of a human immunoglobulin constant region (Fc).
  • full-length antibody or "intact antibody molecule” refers to an immunoglobulin molecule that contains four peptide chains, two heavy (H) chains (approximately 50 to 70 kDa in full length) and two light (L) chains (full-length (approximately 25 kDa) are connected to each other through disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conservative regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH or VL region consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of an antibody mediates the response of immunoglobulins to host tissues or factors, including various components of the immune system. Binding of cells (e.g., effector cells) to the first component (Clq) of the classical complement system.
  • CDR refers to the complementarity determining regions within the variable sequences of an antibody. There are 3 CDRs in each variable region of the heavy and light chain, which are named HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each heavy and light chain variable region. The exact boundaries of these CDRs are defined differently by different systems.
  • variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known protocols, including Chothia (Chothia et al. (1989)) based on the three-dimensional structure of the antibody and the topology of the CDR loops. Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997) Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, U.S.
  • the CDRs of the antibodies of the present invention can be determined by those skilled in the art according to any scheme in the art (for example, different (a system or combination of assignments) determines the boundaries.
  • antigen-binding fragment includes fragments of an antibody or derivatives thereof, typically including at least one fragment of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody that retains at least some of the properties of the parent antibody. Binding specificity.
  • antigen-binding fragments include, but are not limited to, Fab, Fab′, F(ab′)2 and Fv fragments; diabodies; linear antibodies; single chain antibody molecules, such as scFv; nanobodies and polypeptides formed from antibody fragments. specific antibodies.
  • the binding fragment or derivative thereof When the binding activity of the antibody is expressed on a molar concentration basis, the binding fragment or derivative thereof generally retains at least 10% of the antigen-binding activity of the parent antibody. Preferably the binding fragment or derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or greater of the antigen binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as “conservative variants" or “functionally conserved variants" of the antibody).
  • the anti-CD112R antibody or antigen-binding fragment thereof of the present invention includes any anti-CD112R antibody or antigen-binding fragment thereof described in the patent application with application number CN 202110178859.1. This article will disclose the patent application with application number CN202110178859.1. All contents are incorporated into this article by introduction.
  • the CDR sequences of the antibodies used in the methods and compositions of the invention include those from the humanized anti-CD112R antibodies Hu16, Hu52 and Hu59 described in CN202110178859.1, their CDRs, variable regions and full-length The amino acid sequence is shown in Table A.
  • the anti-CD112R antibody or antigen-binding fragment thereof used in the methods and compositions of the invention comprises LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively:
  • LCDR1 KASQSVSNDVA (SEQ ID NO: 1);
  • LCDR2 YVSX 1 RFT (SEQ ID NO: 21), where X 1 is H or N;
  • LCDR3 X 2 QAYRSPWT (SEQ ID NO: 22), where X 2 is H or Q;
  • HCDR1 SYHMS (SEQ ID NO: 6);
  • HCDR2 AIX 3 X 4 X 5 GX 6 X 7 TYYX 8 DTVKG (SEQ ID NO: 23), where X 3 is S or N, Or I, X 7 is S or N, X 8 is P or L;
  • HCDR3 HEDYX 9 GFAMDX 10 (SEQ ID NO: 24), X 9 is F or Y, X 10 is S or Y.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof includes:
  • amino acid sequence is LCDR1 shown in SEQ ID NO: 1;
  • amino acid sequence is LCDR2 shown in SEQ ID NO: 2 or 4;
  • amino acid sequence is LCDR3 shown in SEQ ID NO: 3 or 5;
  • amino acid sequence is HCDR1 shown in SEQ ID NO: 6;
  • amino acid sequence is HCDR2 shown in SEQ ID NO: 7 or 9;
  • the amino acid sequence is HCDR3 shown in SEQ ID NO: 8 or 10.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof includes:
  • amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the amino acid sequences are SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 3 respectively.
  • amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the amino acid sequences are SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 9 respectively.
  • amino acid sequences are LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5 respectively, and the amino acid sequences are SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 5 respectively.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof is selected from the group consisting of murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, humanized antibody or antigen-binding fragment thereof, preferably a humanized antibody or Its antigen-binding fragment.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof includes:
  • the anti-CD112R antibodies described above include:
  • amino acid sequence is the light chain amino acid sequence shown in SEQ ID NO: 16, and the amino acid sequence is the heavy chain amino acid sequence shown in SEQ ID NO: 17;
  • amino acid sequence of the light chain amino acid sequence is as shown in SEQ ID NO: 16
  • amino acid sequence of the heavy chain amino acid sequence is as shown in SEQ ID NO: 18;
  • amino acid sequence is the light chain amino acid sequence shown in SEQ ID NO: 19, and the amino acid sequence is the heavy chain amino acid sequence shown in SEQ ID NO: 20.
  • a non-limiting, exemplary antibody used in the embodiments herein is Hu52, which is a humanized antibody comprising the light chain and heavy chain amino acid sequences set forth in SEQ ID NO: 16 and 18.
  • SEQ ID NO:1-20 in this article are as follows:
  • anti-CD112R antibodies or antigen-binding fragments thereof used in the methods and compositions of the invention are humanized antibodies or chimeric antibodies, and may include human constant regions.
  • the constant region is selected from the group consisting of human IgG1, IgG2, IgG3 and IgG4 constant regions; preferably, the anti-CD112R antibody or antigen-binding fragment thereof suitable for use in the methods and compositions of the invention comprises human Heavy chain constant region of IgG1 or IgG4 isotype.
  • the invention provides a method for preparing an anti-CD112R antibody or antigen-binding fragment thereof as described herein, said method comprising expressing the antibody or antigen-binding fragment thereof under conditions suitable for The antibody or antigen-binding fragment thereof is expressed in a host cell described herein, and the expressed antibody or antigen-binding fragment thereof is recovered from the host cell.
  • the invention provides mammalian host cells for expression of the recombinant antibodies of the invention, including a number of immortalized cell lines available from the American Type Culture Collection (ATCC). These include inter alia Chinese hamster ovary (CHO) cells, NS0, SP2/0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many others Cell lines.
  • Mammalian host cells include human, mouse, rat, dog, monkey, porcine, goat, bovine, equine and hamster cells. Particularly preferred cell lines are selected by determining which cell lines have high expression levels.
  • the invention provides a method for preparing an anti-CD112R antibody, wherein the method includes, when introducing the expression vector into a mammalian host cell, by culturing the host cell for a sufficient period of time to allow the antibody to be expressed in the host cell.
  • Antibodies are produced by expressing, or more preferably by secreting, the antibodies into the medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods.
  • antibodies expressed by different cell lines or expressed in transgenic animals have different glycosylation from each other.
  • all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the invention, regardless of glycosylation of the antibody.
  • afucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts and are unlikely to be immunogenic , because their sugar structure is a normal component of natural human serum IgG.
  • the invention provides a pharmaceutical composition, comprising: (1) buffer; (2) anti-CD112R antibody or antigen-binding fragment thereof.
  • anti-CD112R antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is as described in any embodiment of the "anti-CD112R antibody” section of this application.
  • the anti-CD112R antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention includes LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively, and the amino acid sequences are HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 6, SEQ ID NO: 9 and SEQ ID NO: 10 respectively.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof is selected from the group consisting of murine antibody or antigen-binding fragment thereof, chimeric antibody or antigen-binding fragment thereof, humanized antibody or antigen-binding fragment thereof, preferably a humanized antibody or antigen-binding fragment thereof. Combine fragments.
  • the above-mentioned anti-CD112R antibody or antigen-binding fragment thereof includes a light chain variable region as shown in SEQ ID NO: 11, and a heavy chain variable region as shown in SEQ ID NO: 13. More preferably, the above-mentioned anti-CD112R antibody comprises the light chain amino acid sequence shown in SEQ ID NO: 16, and the heavy chain amino acid sequence shown in SEQ ID NO: 18.
  • the concentration of the anti-CD112R antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present invention is about 1 to 100 mg/mL, preferably about 10 to 70 mg/mL, and more preferably about 20 to 60 mg/mL. In some embodiments, the concentration of the anti-CD112R antibody or antigen-binding fragment thereof in the pharmaceutical composition of the invention is 40 ⁇ 10 mg/mL.
  • the pH of the pharmaceutical composition of the present invention is about 4.5 to 6.5, preferably about 5.5 to 6.5, more preferably about 5.8 to 6.2. In some embodiments, the pH of pharmaceutical compositions described herein is about 5.5 or about 6.0.
  • the buffer in the pharmaceutical composition of the present invention is selected from one or more of acetate buffer, citrate buffer, phosphate buffer and histidine buffer; preferably, the buffer is histamine Acid buffer, preferably, the histidine buffer is histidine-histidine hydrochloride buffer or histidine-histidine acetate buffer, preferably histidine-histidine Hydrochloride buffer.
  • the concentration of the buffer is about 5-100mM, preferably about 10-50mM, preferably about 10-30mM; preferably about 15-25mM.
  • the pH of the buffer is about 4.5 to 6.5, preferably about 5.5 to 6.5, preferably about 5.8 to 6.2. In some embodiments, the pH of the buffers described herein is about 5.5 or about 6.0.
  • the pharmaceutical composition of the present invention may contain: a histidine buffer with a pH of about 5.5 to 6.5, and a concentration of about 10 to 30 mM in the pharmaceutical composition; and any one of the above embodiments of about 10 to 70 mg/mL.
  • a histidine buffer with a pH of about 5.5 to 6.5, and a concentration of about 10 to 30 mM in the pharmaceutical composition may contain any one of the above embodiments of about 10 to 70 mg/mL.
  • the anti-CD112R antibody or its antigen-binding fragment especially the humanized antibody Hu52 or its antigen-binding fragment described herein.
  • the pharmaceutical composition of the present invention further includes a stabilizer selected from one of arginine, arginine salts, sodium chloride, mannitol, sorbitol, sucrose and trehalose. or more; preferably, the above-mentioned arginine salt is arginine hydrochloride.
  • the concentration of the above-mentioned stabilizer is about 10-400mM, preferably about 100-300mM, preferably about 150-270mM, preferably about 180-260mM. It should be understood that when two or more stabilizers are used, the sum of their concentrations is also within the range of 10-400mM, preferably within the range of 150-270mM, and more preferably within the range of 180-260mM.
  • the stabilizer in the pharmaceutical composition of the present invention is: trehalose at a concentration of about 150-270mM; or sucrose at a concentration of about 150-270mM; or sodium chloride at a concentration of about 20-80mM and sodium chloride at a concentration of about 110-170mM.
  • trehalose at a concentration of about 150-270mM
  • sucrose at a concentration of about 150-270mM
  • sodium chloride at a concentration of about 20-80mM and sodium chloride at a concentration of about 110-170mM.
  • the stabilizer in the pharmaceutical composition of the present invention is: trehalose at a concentration of about 180-260mM, preferably about 220-240mM; or sucrose at a concentration of about 180-260mM, preferably about 220-240mM; or
  • the above-mentioned stabilizer is a combination of sodium chloride at a concentration of about 30-70mM, preferably about 40-60mM, and sucrose at a concentration of about 120-160mM, preferably about 130-150mM; or the above-mentioned stabilizer is at a concentration of about 30-70mM, preferably about 40 A combination of ⁇ 60mM arginine hydrochloride and sucrose at a concentration of about 120-160mM, preferably about 130-150mM.
  • the pharmaceutical composition of the present invention may contain: a histidine buffer with a pH of about 5.5 to 6.5, and a concentration of about 10 to 30 mM in the pharmaceutical composition; about 10 to 70 mg/mL
  • the agent is selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose.
  • the above-mentioned arginine salt is arginine hydrochloride.
  • the above-mentioned stabilizer is trehalose with a concentration of about 180-260mM, preferably about 220-240mM; or a concentration of about 180-260mM, preferably about 220-240mM sucrose; or a combination of sodium chloride at a concentration of about 30-70mM, preferably about 40-60mM, and sucrose at a concentration of about 120-160mM, preferably about 130-150mM; or the above stabilizer is A combination of arginine hydrochloride at a concentration of about 30-70mM, preferably about 40-60mM, and sucrose at a concentration of about 120-160mM, preferably about 130-150mM.
  • the above pharmaceutical composition further includes a surfactant selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188.
  • a surfactant selected from one or more of polysorbate 80, polysorbate 20 and poloxamer 188.
  • the surfactant concentration is about 0.001% to 0.1%, preferably about 0.01% to 0.1%, preferably about 0.02% to 0.06%, more preferably about 0.04% to 0.06%.
  • the pharmaceutical composition of the present invention may contain: a histidine buffer with a pH of about 5.5 to 6.5, and a concentration of about 10 to 30mM in the pharmaceutical composition; any one of the above embodiments of about 10 to 70mg/mL.
  • the stabilizer is selected from one or more of arginine, arginine salt, sodium chloride, mannitol, sorbitol, sucrose and trehalose.
  • the above-mentioned arginine salt is hydrochloric acid.
  • Arginine is trehalose with a concentration of about 180-260mM, preferably about 220-240mM; or sucrose with a concentration of about 180-260mM, preferably about 220-240mM; or a concentration of about 30-70mM, preferably about 40-60mM.
  • a combination of sodium chloride and sucrose at a concentration of about 120-160mM, preferably about 130-150mM; or the above-mentioned stabilizer is arginine hydrochloride at a concentration of about 30-70mM, preferably about 40-60mM, and arginine hydrochloride at a concentration of about 120-160mM, preferably A combination of approximately 130 to 150mM sucrose.
  • the pharmaceutical composition of the present invention is a liquid preparation or a freeze-dried preparation.
  • the present invention also provides an injection, which contains the pharmaceutical composition described in any scheme herein and sodium chloride solution or glucose solution; preferably, the concentration of the sodium chloride solution is about 0.85 to 0.9% (w /v); Preferably, the concentration of the glucose solution is about 5 to 25% (w/v), more preferably about 5 to 10% (w/v).
  • the above-mentioned pharmaceutical composition or injection is administered via intravenous injection.
  • the present invention also provides the use of the pharmaceutical composition or injection described in any of the aspects herein in the preparation of a medicament for the treatment and/or prevention of CD112R-mediated diseases or conditions.
  • the present invention also provides the pharmaceutical composition or injection described in any of the aspects herein for the treatment and/or prevention of CD112R-mediated diseases or disorders.
  • the present invention also provides a method of treating and/or preventing a CD112R-mediated disease or disorder, comprising administering to a subject in need thereof a pharmaceutical composition or injection as described in any one of the protocols herein.
  • CD112R-mediated diseases refer to diseases in which CD112R is involved in the occurrence and development of the disease, including but not limited to cancer.
  • Cancer and “cancerous” refer to or describe a physiological disorder in mammals typically characterized by unregulated cell growth. Included in this definition are benign and malignant cancers as well as dormant tumors or micrometastases. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell carcinoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous cell carcinoma of the lung), peritoneal cancer, hepatocellular carcinoma, gastric cancer, or gastric cancer (including gastrointestinal cancer), pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial cancer, or uterine cancer, Salivary gland cancer, kidney cancer or kidney cancer, nasopharyngeal cancer, esophageal squamous cell carcinoma, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer, as well as B-cell lymphoma (including Low-grade/follicular non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, intermediate/follicular NHL
  • compositions or injection may be administered to a subject as described above.
  • administration may be by intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes, such as injection or infusion.
  • Protein concentration was measured using a UV spectrophotometer.
  • the percent extinction coefficient (E1%) was set at 1.389 (mg/m1) - 1 cm -1 .
  • E1% percent extinction coefficient
  • use ultrapure water to dilute the concentration of the test product to about 0.4mg/mL, and try to control the absorbance The reading is between 0.3-0.7.
  • Two solutions were measured in parallel for each sample, and each solution was measured three times. Before measuring each solution, the cuvette must be rinsed twice with 150 ⁇ l of solution, and then 150 ⁇ l of the solution was taken for measurement.
  • C is the protein content in the solution
  • CE-SDS electrophoresis uses high-voltage DC electric field as the driving force and the capillary as the separation channel.
  • the pre-filled gel will form a molecular sieve in the capillary tube.
  • Sodium lauryl sulfate can eliminate the charge effect of different protein molecules.
  • the reducing agent ⁇ -mercaptoethanol can cut off the disulfide bonds in the sample. Samples with different molecular sizes are separated in the capillary tube. Different movement speeds allow separation.
  • the non-reducing CE-SDS electrophoresis purity test of antibody preparations uses a high-voltage DC electric field as the driving force and a capillary tube as the separation channel.
  • the prefilled gel will form a molecular sieve in the capillary tube.
  • the sample is treated with sodium dodecyl sulfate to eliminate the charge effect of different protein molecules, allowing samples with different molecular sizes and different moving speeds in the capillary tube to be separated.
  • Adding alkylating reagents to the test solution can effectively reduce component diffusion, resulting in sharp peak shapes, high separation efficiency, and ensuring that the sample remains in a non-reduced state.
  • sample buffer 1% SDS, 40mM phosphate buffer, pH 6.5
  • sample volume that need to be added when diluting the sample/reference to 1 mg/mL based on the final volume of 100 ⁇ l
  • press the buffer 5.0
  • the prepared samples were centrifuged at 3000 rpm for 30 sec at room temperature, incubated at 70°C for 5 min, cooled to room temperature, centrifuged at 12000 rpm at room temperature for 5 min, and detected using a capillary electrophoresis instrument (Maurice, PA800).
  • CEX-HPLC cation exchange chromatography
  • HPLC Waters e2695 instrument
  • phase B 20mM MES+200mM NaCl (pH6.00 ⁇ 0.02).
  • the peak area normalization method is used to calculate the percentage of the main peak, acidic peak, and basic peak. If automatic integration cannot obtain reasonable integration results, manual integration is used.
  • Table 2 Detailed chromatographic parameters are shown in Table 2 below.
  • Binding ELISA (Binding ELISA) experiment Using the indirect method, HX1 hCD112R Fc is used as the antigen to be coated on a 96-well plate.
  • the antibody preparation can be combined with HX1 hCD112R Fc, and Mouse Anti-Human IgG4 Fc-HRP can be combined with the solid-phase antigen.
  • the antibody preparation specifically binds to (HX1 hCD112R Fc).
  • Mouse Anti-Human IgG4 Fc-HRP can catalyze TMB to turn blue under the action of hydrogen peroxide. The depth of blue is positively correlated with the amount of Mouse Anti-Human IgG4 Fc-HRP bound.
  • the MFI Microfluidic Imaging Particle Analysis System integrates digital microscopy, microfluidics and image processing technology into a fully automatic instrument for analyzing particles.
  • the MFI will capture the mirror image of the sample, and each particle in the mirror image will be analyzed to generate data on the number, size, transparency, morphology, etc. of the particles. It can also be used to analyze the dynamic process of particles in real time. Morphological analysis software can also be used to analyze particles with special morphologies, or to separate some sub-particle populations.
  • iCIEF is detected using a capillary electrophoresis instrument installed with an iCIEF cartridge. After the sample is diluted with the iCIEF mixture, it is detected using a capillary electrophoresis instrument. Components with different isoelectric points are focused at different positions to achieve focusing and separation effects.
  • the emitted light at 330nm of the hydrophobic amino acids inside it is greater than the emitted light at 350nm.
  • the temperature range of protein products is from 35°C to 85°C. If the temperature is increased by 1-2°C and a scan is performed, varying degrees of aggregation will begin to occur at a certain temperature. By monitoring the particle diameter changes and temperature changes in the sample solution, the initial protein aggregation temperature (Tagg) can be obtained.
  • the buffer system closely affects the stability of the antibody.
  • Each antibody with unique physical and chemical properties has the most suitable type of buffer. This example aims to preliminarily screen out the best buffer system and stabilizer so that the anti-CD112R antibody disclosed in the present invention has the best stability and is suitable for clinical application.
  • This example was performed using antibody Hu52.
  • the sample is concentrated by UF/DF ultrafiltration using Millipore Pellicon3 0.11m 2 membrane to a concentration of approximately 52.5 mg/mL.
  • the sample is then dialyzed into the corresponding prescription shown in Table 3, and the final concentration is adjusted to approximately 40 mg/mL.
  • This example was performed using antibody Hu52.
  • the sample is concentrated by UF/DF ultrafiltration using Millipore Pellicon3 0.11m 2 membrane to a concentration of about 55 mg/mL, and then the sample is dialyzed into the corresponding prescription shown in Table 10, and the final concentration is adjusted to about 40 mg/mL, and then added Corresponding concentration of polysorbate 80.
  • the prescriptions FS2-5 (histidine buffer, pH6.0, 230mM sucrose) and FS2-6 (histidine buffer, pH6.0, 230mM trehalose) are more cost-effective. Excellent; judging from the aggregation temperature results, the FS2-5 prescription is better excellent.
  • the pharmaceutical composition of monoclonal antibody with 20mM histidine buffer (pH 6.0) and 230mM sucrose has optimal stability under high temperature conditions.
  • the monoclonal antibody has good stability when combined with 20mM histidine buffer (pH 6.0), 230Mm sucrose and polysorbate 80 (content 0.02% to 0.06%).
  • the prescription samples FS3-1, FS3-2 and FS3-3 of Example 3 were used. Examine the sample's tolerance to shaking conditions: 200 ⁇ 20rpm shaking speed, room temperature, shaking for 2 or 5 days. Examine the sample's tolerance to repeated freezing and thawing: repeated freezing and thawing 3 times and 5 times at -40°C and room temperature. Test items include appearance, SEC-HPLC, SDS-CE-NR/R, CEX and sub-visible particles.
  • Biacore molecular interaction analyzer Biacore was used to couple 40 ⁇ g/mL sheep anti-human Fc fragment antibody (Jackson ImmunoResearch) to the CM5 chip surface, and then capture the pharmaceutical composition (1 ⁇ g/mL, prescription No. FS3-2, the antibody is Hu52), and then inject gradient dilution of human PVRIG protein to detect the binding signal.
  • the gradient dilution concentrations of human PVRIG protein are 16nM, 8nM, 4nM, 2nM, 1nM and 0.5nM, of which 16nM is repeated.
  • the experimental results are shown in Table 30 and Figure 1.
  • the pharmaceutical composition has specific binding activity to human PVRIG protein, with a KD value of 5.77E-11M.
  • Table 30 Table of affinity results between pharmaceutical compositions and recombinant human PVRIG protein
  • the experimental results are shown in Figure 2.
  • the pharmaceutical composition has specific binding activity to human CD112R protein, with an EC 50 value of 1.4ng/mL, and the negative control sample has no binding.
  • Example 7 Competitive inhibitory activity of pharmaceutical composition (BLOCKING ELISA)
  • This experiment was performed at 37°C using a Thermo Scientific microplate reader (Model: Multiskan GO).
  • the plate was coated with 2.0 ⁇ g/mL HX1 hCD112R Fc (Suzhou Junmeng) for 90 min. After washing, the plate was blocked with 2% BSA. 90min, use MX2 hPVRL2 Fc (Suzhou Junmeng) diluted to 4.0 ⁇ g/mL to dilute the pharmaceutical composition (prescription number FS3-2, antibody is Hu52) and negative control (starting at 10 ⁇ g/mL, 2-fold gradient dilution to 4.883ng /mL (12 concentrations in total), add the sample to the blocked plate and incubate for 60 minutes.
  • the experimental results are shown in Figure 3.
  • the pharmaceutical composition can specifically block the binding of human CD112R protein to CD112 protein, with an IC 50 value of 60.0ng/mL.
  • the negative control sample has no blocking effect.
  • the binding of the pharmaceutical composition to CD112R on the surface of CHO hCD112R cells was detected by flow cytometry.
  • Different concentrations of pharmaceutical compositions (prescription number FS3-2, antibody Hu52) and control antibodies (from 0.01ng/ml to 20 ⁇ g/ml) were incubated with CHOHCD112R cells for 30 minutes at 4°C, and then washed away with staining buffer to remove unbound of antibodies.
  • the cells were then mixed with 1% Goat Anti-Human IgG4-PE fluorescent secondary antibody (v/v) prepared in staining buffer at 4°C in the dark. Incubate for 30 minutes.
  • Flow cytometry collects cells and detects fluorescent antibodies bound to the cell surface.
  • FlowJo was used to analyze the raw data to obtain the MFI value (mean fluorescence intensity), and GraphPad Prism software was used to perform a four-parameter curve fitting to obtain the EC 50 value.
  • the experimental results are shown in Figure 4.
  • the pharmaceutical composition and COM701 (Compugen's anti-CD112R antibody, expressed and purified according to the US20180280506A1 sequence) have strong affinity to CHO hCD112R cells, and the EC 50 values of their cell binding abilities are 36.0ng/ml respectively. and 119.2ng/ml, anti-KLH hIgG4 showed negative results, indicating that the pharmaceutical composition can specifically bind to hCD112R on the cell surface with high affinity, and the binding affinity is better than COM701.
  • Example 9 Pharmaceutical composition blocks the binding of HCD112R protein to cell surface CD112R
  • CHO CD112 cells were treated with a fixed concentration of hCD112R protein solution (3 ⁇ g/ml) and different concentrations of the pharmaceutical composition (prescription number FS3-2, The antibody is Hu52) or control antibody (from 0.169ng/ml to 30 ⁇ g/ml, 3-fold dilution) and incubated for 30 minutes at 4°C. Then use staining buffer to wash away unbound antibodies, and then incubate the cells with 1% Goat anti-mouse IgG Fc fluorescent secondary antibody (v/v) prepared in staining buffer for 30 minutes at 4°C in the dark. .
  • the pharmaceutical composition and COM701 can effectively block the binding of hCD112R protein to CHO CD112 cells, while the negative control antibody has no corresponding function.
  • the IC 50 of the pharmaceutical composition for blocking the binding of hCD112R protein to CHO CD112 1D2 cells is 62.2ng/ml, and the activity of COM701 is 84.7ng/ml. The activity levels of the two are equivalent.
  • Example 10 Activity of pharmaceutical composition in CD112R/CD112 reporter gene detection system
  • This experiment used the human CD112R/CD112 luciferase reporter gene system to detect the cellular biology of the pharmaceutical composition and control antibodies COM701 (positive control) and anti-KLH-hIgG4 (negative control) blocking the hCD112R/hCD112 inhibitory pathway and activating NFAT. learning activity.
  • Target cell plating medium use target cell plating medium to plate CHO CD112 1D2 cells expressing human CD112 in a 96-well white flat-bottomed plate at a density of 50,000 cells per well overnight; after draining the medium in the cell wells the next day, add different Concentrated pharmaceutical composition (prescription number FS3-2, antibody Hu52) or control antibody (concentration from 0.15ng/ml to 3 ⁇ g/ml, 3-fold dilution) was added to the target cells and incubated at 37°C for 30 minutes.
  • Concentrated pharmaceutical composition prescription number FS3-2, antibody Hu52
  • control antibody concentration from 0.15ng/ml to 3 ⁇ g/ml, 3-fold dilution
  • the effector cells Jurkat CD112R expressing human CD112R were added to the cell plate at the number of 100,000 cells per well, and incubated in a 37°C incubator for 6 hours; finally, the ONE-Lite Luciferase Assay System was added to the cell antibody mixture system and used with a multifunctional A microplate reader detects the chemiluminescence signal.
  • the four-parameter regression curve was fitted through GraphPad prism software and the EC 50 value was calculated.
  • NFAT is an important transcription factor downstream of T cell receptor activation, which is responsible for IL-2 secretion and T cell proliferation response.
  • Crucial. This experiment used the luciferase reporter gene method to study the ability of the pharmaceutical composition to block the CD112R/CD112 signaling pathway, activate T cells, and activate NFAT signaling. The experimental results are shown in Figure 6. Both the pharmaceutical composition and the positive control COM701 can effectively block the interaction of CD112R/CD112 and promote T cell activation.
  • the EC 50 values are 14.2ng/ml and 104.2ng/ml respectively.
  • the antibody Hu52 The activity was significantly higher than the positive control.
  • Example 11 Pharmacodynamic evaluation of anti-CD112R antibody, anti-TIGIT antibody and their combination in human melanoma A375 mixed PBMC subcutaneous transplant tumor model
  • Anti-TIGIT antibody (JS006): derived from the anti-TIGIT humanized antibody hu20 in PCT application number PCT/CN2020/101883.
  • the anti-CD112R antibody is the humanized antibody hu52 described herein.
  • the anti-CD112R humanized antibody in this example was prepared according to the formula of prescription FS3-2.
  • A375 cells were cultured in DMEM culture medium containing 10% fetal bovine serum (FBS).
  • A375 cells in the logarithmic growth phase were collected and resuspended in HBSS (Hank's balanced salt solution) to a suitable concentration for subcutaneous tumor inoculation in NCG mice.
  • A375 cells used in co-culture experiments were treated with Mitomycin C (mitomycin C) for 2 hours, and then washed three times with PBS.
  • the frozen PBMC were taken, resuscitated and counted, and the obtained PBMC were added to A375 cells treated with Mitomycin C.
  • the PBMC and A375 were co-cultured for 5 days in RPMI 1640 culture medium containing IL-2 and 10% FBS.
  • PBMC and A375 were co-cultured for 5 days, collect PBMC and freshly digested A375 cells, PBMC 4 ⁇ 10 5 , A375 cells 4 ⁇ 10 6 , 0.2ml/cell (containing 50% Matrigel), and inoculate into NCG mouse right side subcutaneously. The day of inoculation was day 0, and the mice were randomly divided into groups for administration. The detailed administration method, dosage and route of administration are shown in Table 31.
  • Dosing volume Adjust the dosing volume (0.1mL/10g) according to the body weight of the tumor-bearing mice.
  • the anti-CD112R antibody hu52 significantly inhibited tumor growth at a dose level of 30mg/kg, with a tumor growth inhibition rate of 37.68% (p ⁇ 0.05); 30mg/ kg anti-CD112R antibody hu52 combined with 30 mg/kg JS006 showed a significant synergistic anti-tumor effect, with a tumor growth inhibition rate of 58.30% (p ⁇ 0.01).

Abstract

提供了一种稳定的抗CD112R抗体药物组合物及其用途。该药物组合物含有缓冲液和抗CD112R抗体或其抗原结合片段；其中所述抗CD112R抗体或其抗原结合片段的浓度为约1～100mg/mL。开发得到的抗体制剂各成分之间相互作用、协同配合，为抗CD112R单克隆抗体提供了适宜长期保存的存储环境，具有高稳定性，对于高温、振摇和反复冻融的耐受性强，并且能够阻断CD112/CD112R的相互作用，提高T细胞的激活能力，调动免疫系统，发挥更强的肿瘤杀伤作用。

Description

抗CD112R抗体药物组合物及其用途 技术领域
本发明涉及治疗性药物组合物领域，具体涉及抗CD112R抗体药物组合物及其用途。
背景技术
免疫系统是由许多细胞类型组成的高度复杂的系统，包括但不限于T细胞，B细胞，自然杀伤细胞，抗原呈递细胞，树突状细胞，单核细胞和巨噬细胞。这些细胞拥有复杂而微妙的系统来控制其相互作用和响应。细胞利用激活和抑制机制以及反馈回路来保持反应的控制，并且不允许不受控制的免疫反应(例如，自身免疫疾病)的负面后果。
CD112R(PVRIG)，也称为含有脊髓灰质炎病毒受体相关免疫球蛋白结构域的蛋白、Q6DKI7或C7orf15，是一种长度为326个氨基酸的跨膜结构域蛋白，具有信号肽(跨越氨基酸1至40)、细胞外结构域(跨越氨基酸41至171)、跨膜结构域(跨越氨基酸172至190)和细胞质结构域(跨越氨基酸191至326)。CD112R与脊髓灰质炎病毒受体相关蛋白2(PVLR2，也称为nectin-2、CD112或疱疹病毒侵入介体B(HVEB)、人质膜糖蛋白)，即CD112R的结合配偶体结合。
抗CD112R免疫疗法的施用提供了增加，增强和维持免疫应答的机会。CD112R是主要由T细胞和NK细胞表达的抑制性受体，并与活化受体CD226竞争结合CD112。CD112与CD112R的相互作用比与CD226的相互作用具有更高的亲和力，从而有效调节CD226介导的细胞活化。阻断与CD112相互作用的抗CD112R抗体直接限制了抑制性信号传导CD112R的下游，同时通过增加CD226与CD112的相互作用来促进更大的免疫细胞活化。
用抗CD112R抗体进行的治疗提供了下调抑制信号的机会，该抑制信号可能在表达CD112R的免疫细胞与肿瘤微环境内的肿瘤细胞和/或髓样细胞上的CD112结合时发生，并可能增强，增加并维持抗肿瘤免疫反应。
目前抗CD112R的单抗药物仅有临床前的SRF813(Surface Oncology)，仍需要更多的新型抗CD112R抗体。但是，抗体药物其分子量大，结构复杂，容易降解、聚合或发生不希望发生的化学修饰等而变得不稳定。为了使抗体适合于生产和给药，并且在储存和随后使用过程中能保持稳定性，及发挥更好的效果，抗体药物的稳定制剂研究显得尤为重要。
发明内容
本发明所述的药物组合物是一种含有与CD112R特异性结合的抗体的高稳定性药物组合物。该药物组合物各成分之间相互作用、协同配合，为抗CD112R单克隆抗体提供了适宜长期保存的存储环境，具有高稳定性，对于高温、振摇和反复冻融的耐受性强，并且能够阻断CD112/CD112R的相互作用，提高T细胞的激活能力，调动免疫系统，发挥更强的肿瘤杀伤作用。
本发明提供了一种药物组合物，包含：(1)缓冲液；(2)抗CD112R抗体或其抗原结合片段。
在一些方案中，上述抗CD112R抗体或其抗原结合片段包含的LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3分别为：
LCDR1：KASQSVSNDVA(SEQ ID NO：1)；
LCDR2：YVSX1RFT，其中，X1为H或N；
LCDR3：X2QAYRSPWT，其中，X2为H或Q；
HCDR1：SYHMS(SEQ ID NO：6)：
HCDR2：AIX3X4X5GX6X7TYYX8DTVKG，其中，X3为S或N，X4为R或S，X5为D或N，X6为D或I，X7为S或N，X8为P或L；
HCDR3：HEDYX9GFAMDX10，X9为F或Y，X10为S或Y。
在一些方案中，上述抗CD112R抗体或其抗原结合片段包含：
氨基酸序列如SEQ ID NO：1所示的LCDR1；
氨基酸序列如SEQ ID NO：2或4所示的LCDR2；
氨基酸序列如SEQ ID NO：3或5所示的LCDR3；
氨基酸序列如SEQ ID NO：6所示的HCDR1；
氨基酸序列如SEQ ID NO：7或9所示的HCDR2；
氨基酸序列如SEQ ID NO：8或10所示的HCDR3。
在一些方案中，上述抗CD112R抗体或其抗原结合片段包含：
(1)氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的HCDR1、HCDR2和HCDR3；或
(2)氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3；或
(3)氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：4和SEQ ID NO：5所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：9和SEQ ID NO：10所 示的HCDR1、HCDR2和HCDR3。
在一些方案中，上述抗CD112R抗体或其抗原结合片段选自鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人源化抗体或其抗原结合片段，优选为人源化抗体或其抗原结合片段。
在一些方案中，上述抗CD112R抗体或其抗原结合片段包含：
(1)氨基酸序列如SEQ ID NO：11所示的轻链可变区，和氨基酸序列如SEQ ID NO：12所示的重链可变区；或
(2)氨基酸序列如SEQ ID NO：11所示的轻链可变区，和氨基酸序列如SEQ ID NO：13所示的重链可变区；或
(3)氨基酸序列如SEQ ID NO：14所示的轻链可变区，和氨基酸序列如SEQ ID NO：15所示的重链可变区。
在一些方案中，上述抗CD112R抗体包含：
(1)氨基酸序列如SEQ ID NO：16所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO：17所示的重链氨基酸序列。
(2)氨基酸序列如SEQ ID NO：16所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO：18所示的重链氨基酸序列。
(3)氨基酸序列如SEQ ID NO：19所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO：20所示的重链氨基酸序列。
在一些方案中，上述药物组合物中抗CD112R抗体或其抗原结合片段的浓度为约1～100mg/mL，优选为约10～70mg/mL，更优选为约20～60mg/mL；更优选地，上述抗CD112R抗体或其抗原结合片段浓度为约1mg/mL，约10mg/mL，约20mg/mL，约30mg/mL，约35mg/mL，约40mg/mL，约50mg/mL，约60mg/mL，约70mg/mL，约80mg/mL，约90mg/mL，约100mg/mL或这些范围内任意两个数值作为端点形成的范围，优选为约35mg/mL，约40mg/mL或约45mg/mL。
在一些方案中，上述药物组合物的pH为约4.5～6.5，优选为约5.5～6.5，更优选为约5.5～5.7或5.8～6.2，上述药物组合物的pH非限制性实施例为约4.5，约5.0，约5.5，约5.6，约5.7，约5.8，约5.9，约6.0，约6.1，约6.2，约6.3，约6.4，约6.5或这些范围内任意两个数值作为端点形成的范围，优选为约5.9，约6.0或约6.1。
在一些方案中，上述药物组合物的渗透压为约250～350mOsm/kg。
在一些方案中，上述缓冲液选自醋酸缓冲液、柠檬酸缓冲液、磷酸盐缓冲液和组氨酸缓冲液中的一种或多种；优选地，所述缓冲液为组氨酸缓冲液。
在一些方案中，上述缓冲液的浓度为约5～100mM，优选为约10～50mM，优选为约 10～30mM；优选为约15～25mM，上述缓冲液浓度非限制性实施例为约10mM，约15mM，约20mM，约25mM，约30mM，约40mM，约45mM，约50mM或这些范围内任意两个数值作为端点形成的范围，优选为约15mM，约20mM或约25mM。
在一些方案中，上述缓冲液的pH为约4.5～6.5，优选为约5.5～6.5，更优选为约5.8～6.2，上述缓冲液的pH非限制性实施例为约4.5，约5.0，约5.5，约5.6，约5.7，约5.8，约5.9，约6.0，约6.1，约6.2，约6.3，约6.4，约6.5或这些范围内任意两个数值作为端点形成的范围，优选为约5.9，约6.0或约6.1。
在一些方案中，上述缓冲液为组氨酸缓冲液；优选地，所述组氨酸缓冲液为组氨酸-组氨酸盐酸盐缓冲液或组氨酸-组氨酸醋酸盐缓冲液，优选为组氨酸-组氨酸盐酸盐缓冲液。优选地，所述组氨酸缓冲液的pH为5.5～6.5，优选5.8～6.2。
在一些方案中，上述组氨酸-组氨酸盐酸盐缓冲液由组氨酸和组氨酸盐酸盐制成，优选L-组氨酸和L-组氨酸单盐酸盐。在一些方案中，组氨酸缓冲液由约1～20mM的L-组氨酸和约1～20mM的L-组氨酸单盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为约1∶1到约1∶4的组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为约1∶1组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸缓冲液由摩尔比为约1∶3的组氨酸和组氨酸盐酸盐制成。在一些方案中，组氨酸制剂为：由约4.5mM的L-组氨酸和约15.5mM的L-组氨酸单盐酸盐制成的pH为约5.5的组氨酸缓冲剂。在一些方案中，组氨酸制剂为：由约10mM的L-组氨酸和约10mM的L-组氨酸盐酸盐制成的pH为约6.0的组氨酸缓冲液。在一些方案中，组氨酸制剂为：由约3mM的L-组氨酸和约17mM的L-组氨酸盐酸盐制成的pH为约6.5的组氨酸缓冲液。
在一些方案中，上述醋酸缓冲液的pH为约4.5～5.7，优选约5.3～5.7。优选地，所述醋酸缓冲液为醋酸-醋酸钠缓冲液。在一些方案中，醋酸-醋酸钠缓冲液由约1～20mM的醋酸和约1～20mM的醋酸钠制成。在一些方案中，醋酸-醋酸钠缓冲液由摩尔比为约1∶2到约1∶3的醋酸和醋酸钠制成。在一些方案中，醋酸-醋酸钠缓冲液由摩尔比为约1∶5到约1∶8的醋酸和醋酸钠制成。在一些方案中，醋酸-醋酸钠缓冲液为：由约8mM的醋酸和约12mM的醋酸钠制成的pH为约4.5的醋酸缓冲液。在一些方案中，醋酸-醋酸钠缓冲液为：由约6.5mM的醋酸和约13.5mM的醋酸钠制成的pH为约5.0的醋酸缓冲液。在一些方案中，醋酸-醋酸钠缓冲液为：由约2～4mM的醋酸和约16～18mM的醋酸钠制成的pH为约5.0～6.0的醋酸缓冲液；优选为由约3mM的醋酸和约17mM的醋酸钠制成的pH为约5.5的醋酸缓冲液。
在一些方案中，上述缓冲液为柠檬酸缓冲液。优选地，所述柠檬酸缓冲液的pH为5.0～6.0。优选地，所述柠檬酸缓冲液为柠檬酸-柠檬酸钠缓冲液。在一些方案中，柠檬酸缓冲液由约1～20mM的柠檬酸和约1～20mM的柠檬酸钠制成。在一些方案中，柠檬酸缓冲液由摩尔比 为约1∶1到1∶4的柠檬酸和柠檬酸钠制成。在一些方案中，柠檬酸缓冲液为：由约15mM的柠檬酸和约5mM的柠檬酸钠制成的pH为约5.0的柠檬酸缓冲液。在一些方案中，柠檬酸缓冲液为：由约12mM的柠檬酸和约8mM的柠檬酸钠制成的pH为约5.5的柠檬酸缓冲液。在一些方案中，柠檬酸缓冲液为：由约10mM的柠檬酸和约10mM的柠檬酸钠制成的pH为约6.0的柠檬酸缓冲液。
在一些方案中，上述缓冲液为磷酸盐缓冲液，优选地，所述磷酸盐缓冲液为磷酸氢二钠-磷酸二氢钠缓冲液。在一些方案中，磷酸盐缓冲液由约1～20mM的磷酸氢二钠和约1～20mM的磷酸二氢钠制成。在一些方案中，磷酸盐缓冲液由摩尔比为约1∶1到1∶4的磷酸氢二钠和磷酸二氢钠制成。在一些方案中，磷酸盐缓冲液为：由约10mM的磷酸氢二钠和约10mM的磷酸二氢钠制成的pH为约7.0的磷酸盐缓冲液。
在一些方案中，上述的药物组合物还包括稳定剂，所述稳定剂选自精氨酸、精氨酸盐、氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种；优选地，上述精氨酸盐为盐酸精氨酸。在一些方案中，上述稳定剂的浓度为约10～400mM，优选为约100～300mM，优选为约150～270mM，优选为约180～260mM，上述稳定剂浓度非限制性实施例为约100mM，约130mM，约150mM，约170mM，约180mM，约190mM，约200mM，约210mM，约220mM，约225mM，约230mM，约235mM，约240mM，约250mM，约260mM或这些范围内任意两个数值作为端点形成的范围，优选为约190mM，约220mM，约230mM或约240mM。
在一些方案中，上述稳定剂为浓度约150～270mM的海藻糖；或上述稳定剂为浓度约150～270mM的蔗糖；或上述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的蔗糖的组合；或上述稳定剂为浓度约20～80mM的盐酸精氨酸与浓度约110-170mM的蔗糖的组合。
在一些方案中，上述稳定剂为浓度约180～260mM、优选约220～240mM的海藻糖，浓度约180～260mM、优选约220～240mM的蔗糖，浓度约30～70mM、优选约40～60mM的氯化钠与浓度约120-160mM、优选约130～150mM的蔗糖的组合，或浓度约30～70mM、优选约40～60mM的盐酸精氨酸与浓度约120-160mM、优选约130～150mM的蔗糖的组合。
在一些方案中，上述稳定剂为海藻糖。在一些方案中，上述稳定剂为浓度约100～300mM的甘露醇，上述甘露醇的浓度优选为约150～270mM，优选为约180～260mM，上述甘露醇浓度的非限制性实施例为约200mM，约210mM，约220mM，约225mM，约230mM，约235mM，约240mM，约250mM，约260mM或这些范围内任意两个数值作为端点形成的范围，优选为约225mM，约230mM或约235mM。
在一些方案中，上述稳定剂为蔗糖。在一些方案中，上述稳定剂为浓度约100～300mM的蔗糖，上述蔗糖的浓度优选为约150～270mM，优选为约180～260mM，上述蔗糖浓度的 非限制性实施例为约200mM，约210mM，约220mM，约225mM，约230mM，约235mM，约240mM，约250mM或这些范围内任意两个数值作为端点形成的范围，优选为约225mM，约230mM或约235mM。
在一些方案中，上述稳定剂为氯化钠与蔗糖的组合。在一些方案中，上述稳定剂为约30～200mM的氯化钠与约30～200mM的蔗糖的组合，优选约20～80mM的氯化钠与约110～170mM的蔗糖的组合，优选约30～70mM的氯化钠与约120～160mM的蔗糖的组合，上述稳定剂的非限制性实施例为约50mM的氯化钠与约140mM的蔗糖的组合，或约50mM的氯化钠与约150mM的蔗糖的组合。
在一些方案中，上述稳定剂为盐酸精氨酸与蔗糖的组合。在一些方案中，上述稳定剂为约30～200mM的盐酸精氨酸与约30～200mM的蔗糖的组合，优选约20～80mM的盐酸精氨酸与约110～170mM的蔗糖的组合，优选约30～70mM的盐酸精氨酸与约120～160mM的蔗糖的组合，上述稳定剂的非限制性实施例为约50mM的盐酸精氨酸与约140mM的蔗糖的组合，或约50mM的盐酸精氨酸与约160mM的蔗糖的组合。
在一些方案中，上述药物组合物还包括表面活性剂，所述表面活性剂选自聚山梨酯80、聚山梨酯20和泊洛沙姆188中的一种或多种。
在一些方案中，上述表面活性剂选自聚山梨酯80。
在一些方案中，以w/v计算，上述表面活性剂浓度为约0.001％～0.1％，优选为约0.01％～0.1％，优选为约0.04％～0.06％；作为非限制性实施例，上述表面活性剂的浓度为约0.01％，约0.02％，约0.03％，约0.04％，约0.05％，约0.06％，约0.07％，约0.08％，约0.1％或这些范围内任意两个数值作为端点形成的范围，优选为约0.02％，约0.04％或约0.06％。
在一些方案中，药物组合物包含如下(1)～(14)中任一项所示的组分，或分别由(1)～(14)任一项所示的组分组成：
(1)(a)约10～70mg/mL的上述抗CD112R抗体或其抗原结合片段；(b)约10～30mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5、优选约5.5～6.2；(c)150～270mM的海藻糖；以及(d)约0.01％～0.1％(w/v)的聚山梨酯80；或
(2)(a)约10～70mg/mL的上述抗CD112R抗体或其抗原结合片段；(b)约10～30mM组氨酸缓冲液，pH为约5.5～6.5、优选约5.5～6.2；(c)150～270mM的蔗糖；以及(d)约0.01％～0.1％(w/v)的聚山梨酯80；或
(3)(a)约10～70mg/mL的上述抗CD112R抗体或其抗原结合片段；(b)约10～30mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5、优选约5.5～6.2；(c)稳定剂，所述稳定剂为浓度约20～80mM的盐酸精氨酸与浓度约110-170mM的蔗糖的组合；以及(d)约0.01％～0.1％(w/v)的聚山梨酯80；或
(4)(a)约10～70mg/mL的上述抗CD112R抗体或其抗原结合片段；(b)约10～30mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5、优选约5.5～6.2；(c)稳定剂，所述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的蔗糖的组合；以及(d)约0.01％～0.1％(w/v)的聚山梨酯80；或
(5)(a)约20～60mg/mL的上述抗CD112R抗体或其抗原结合片段；(b)约15～25mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5、优选约5.5～6.2；(c)180～260mM的海藻糖；以及(d)约0.04％～0.06％(w/v)的聚山梨酯80；或
(6)(a)约20～60mg/mL的上述抗CD112R抗体或其抗原结合片段；(b)约15～25mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5、优选约5.5～6.2；(c)180～260mM的蔗糖；以及(d)约0.04％～0.06％(w/v)的聚山梨酯80；或
(7)(a)约20～60mg/mL的上述抗CD112R抗体或其抗原结合片段；(b)约15～25mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5、优选约5.5～6.2；(c)稳定剂，所述稳定剂为浓度约30～70mM的盐酸精氨酸与浓度约120-160mM的蔗糖的组合；以及(d)约0.04％～0.06％(w/v)的聚山梨酯80；或
(8)(a)约20～60mg/mL的上述抗CD112R抗体或其抗原结合片段；(b)约15～25mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5、优选约5.5～6.2；(c)稳定剂，所述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的蔗糖的组合；以及(d)约0.04％～0.06％(w/v)的聚山梨酯80；或
(9)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)约230mM的海藻糖；以及(d)约0.02％(w/v)的聚山梨酯80；或
(10)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)约230mM的蔗糖；以及(d)约0.02％(w/v)的聚山梨酯80；或
(11)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)约230mM的蔗糖；以及(d)约0.04％(w/v)的聚山梨酯80；或
(12)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、 pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)约230mM的蔗糖；以及(d)约0.06％(w/v)的聚山梨酯80；或
(13)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)稳定剂，所述稳定剂为浓度约50mM的盐酸精氨酸与浓度约140mM的蔗糖的组合；以及(d)约0.02％(w/v)的聚山梨酯80；或
(14)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)稳定剂，所述稳定剂为浓度约50mM的氯化钠与浓度约140mM的蔗糖的组合；以及(d)约0.02％(w/v)的聚山梨酯80。
在一些方案中，本文任一项方案中所述的药物组合物为液体制剂或冻干制剂。
在一些方案中，上述药物组合物为液体制剂。
在一些方案中，上述液体制剂或冻干制剂于2～8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。
在一些方案中，上述液体制剂或冻干制剂于40℃稳定至少7天，至少14天或至少28天。
本发明还提供了一种注射剂，其含有本文任一项方案中所述的药物组合物与氯化钠溶液或葡萄糖溶液；优选地，所述氯化钠溶液浓度为约0.85～0.9％(w/v)；优选地，所述葡萄糖溶液浓度为约5～25％(w/v)，更优选为约5～10％(w/v)。
在一些方案中，上述药物组合物或注射剂，其经静脉注射施用。
本发明还提供了本文任一项方案中所述的药物组合物或注射剂在制备用于治疗和/或预防CD112R介导的疾病或病症的药物中的用途。
本发明还提供了本文任一项方案中所述的药物组合物或注射剂，其用于治疗和/或预防CD112R介导的疾病或病症。
本发明还提供了一种治疗和/或预防CD112R介导的疾病或病症的方法，其包括向有需要的受试者施用如本文任一项方案中所述的药物组合物或注射剂。
在一些方案中，上述疾病或病症为癌症。
附图说明
图1：药物组合物与重组人PVRIG蛋白的结合解离曲线。
图2：药物组合物及阴性对照与人CD112R蛋白的结合曲线。
图3：药物组合物及阴性对照与CD112竞争结合人CD112R的配体竞争抑制ELISA曲线。
图4：药物组合物与CHO hCD112R细胞表面CD112R的结合活性。
图5：药物组合物阻断hCD112R蛋白与CHO CD112细胞的结合活性。
图6：药物组合物在人CD112R/CD112荧光素酶报告基因系统中的活性。
图7：抗CD112R人源化抗体、抗TIGIT抗体及其联用的肿瘤生长抑制作用。
具体实施方式
定义和说明
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。应理解本发明不限于具体的方法、试剂、化合物、组合物或生物系统，当然可以对以上进行变化。还应理解本申请所用术语仅为了描述具体的实施方式，并不旨在进行限制。本文所引用的所有参考文献，包括专利、专利申请、论文、教科书诸如此类，以及其中所引用的参考文献，就其尚未被引用的程度而言，在此其全文通过引用并入。如果所并入的文献和类似材料中的一个或多个与本申请不同或矛盾，包括但不限于所定义的术语、术语用法、所描述的技术诸如此类，则以本申请为准。
除非该内容被另外明确说明，否则本说明书以及所附权利要求中所用的单数形式“一个”、“一种”和“该”包括复数指代。因此，例如，提及“一种多肽”包括了两种或更多种多肽等的组合。
术语“药物组合物”或“制剂”表示含有一种或多种本文所述抗体与其他组分的混合物，所述其他组分例如生理学可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“液体制剂”是指处于液体状态下的制剂，且不意图指称重悬浮的冻干制剂。本发明的液体制剂在储存时稳定，并且其稳定性不依赖于冻干(或其他状态改变方法，例如喷雾干燥)。
术语“水性液体制剂”是指使用水作为溶剂的液体制剂。在一些方案中，水性液体制剂是不需冻干、喷雾干燥和/或冷冻来维持稳定性(例如化学和/或物理稳定性和/或生物活性)的制剂。
术语“赋形剂”是指可以向制剂添加以提供所需特性(例如稠度、提高的稳定性)和/或调节渗透压的试剂。常用赋形剂的实例包括但不限于糖类、多元醇、氨基酸、表面活性剂和聚 合物。
本申请所用的“约”在指代可测量数值(如量、持续时间等)时意在涵盖相对于具体数值±20％或±10％的变化，包括±5％、±1％和±0.1％，因为这些变化适于进行所公开的方法。
术语“缓冲液pH为约4.5～6.5”是指这样的试剂，通过其酸/碱共轭组分的作用使得包含该试剂的溶液能抵抗pH变化。本发明的制剂中使用的缓冲液可具有约5.0至约6.5范围内的pH、或约5.5至约6.5范围内的pH、或约5.0至约6.0范围内的pH。
在本文中，将pH控制在该范围内的“缓冲液”实例包括醋酸、醋酸盐(例如醋酸钠)、琥珀酸、琥珀酸盐(例如琥珀酸钠)、葡萄糖酸、组氨酸、组胺酸盐(例如组氨酸盐酸盐)、甲硫氨酸、柠檬酸(枸橼酸)、柠檬酸盐(枸橼酸盐)、磷酸盐、柠檬酸盐/磷酸盐、咪唑、其组合和其他有机酸缓冲剂。
“组氨酸缓冲液”为包含组氨酸离子的缓冲液。组氨酸缓冲液的实例包括组氨酸和组氨酸的盐，如组氨酸盐酸盐、组氨酸乙酸盐、组氨酸磷酸盐和组氨酸硫酸盐等，如含有组氨酸与组氨酸盐酸盐的组氨酸缓冲液；本发明的组氨酸缓冲液也包括含有组氨酸和醋酸盐(如钠盐或钾盐)的组氨酸缓冲液。
“柠檬酸缓冲液”，又称“枸橼酸缓冲液”，是包括柠檬酸根离子的缓冲液。柠檬酸盐缓冲液的实例包括柠檬酸-柠檬酸钠、柠檬酸-柠檬酸钾、柠檬酸-柠檬酸钙、柠檬酸-柠檬酸镁等。优选的柠檬酸盐缓冲液为柠檬酸-柠檬酸钠缓冲液。
“醋酸缓冲液”是包括醋酸根离子的缓冲液。醋酸盐缓冲液的实例包括醋酸-醋酸钠、醋酸-醋酸钾、醋酸-醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲液为醋酸-醋酸钠缓冲液。
“琥珀酸缓冲液”是包括琥珀酸根离子的缓冲液。琥珀酸缓冲液的实例包括琥珀酸-琥珀酸钠、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙、琥珀酸-琥珀酸镁等。优选的琥珀酸盐缓冲液为琥珀酸-琥珀酸钠缓冲液。
“磷酸盐缓冲液”是包括磷酸根离子的缓冲液。磷酸盐缓冲液的实例包括磷酸二氢钠-磷酸氢二钠、磷酸二氢钾-磷酸氢二钾等。优选的磷酸盐缓冲液为磷酸二氢钠-磷酸氢二钠缓冲液。
术语“稳定剂”表示药学上可接受的赋形剂，其在制造、储存和应用过程中保护活性药物成分和/或制剂免受化学和/或物理降解。稳定剂包括但不限于如以下定义的糖，氨基酸，盐，多元醇和他们的代谢产物，例如氯化钠、氯化钙、氯化镁、甘露醇、山梨醇、蔗糖、海藻糖、精氨酸或其盐(如盐酸精氨酸)、甘氨酸、丙氨酸(α-丙氨酸、β-丙氨酸)、甜菜碱、亮氨酸、赖氨酸、谷氨酸、天冬氨酸、脯氨酸、4-羟基脯氨酸、肌氨酸、γ-氨基丁酸(GABA)、奥品类(opines)、丙氨奥品、章鱼碱、甘氨奥品(strombine)和三甲胺的N-氧化物(TMAO)、人血清白蛋白(hsa)、牛血清白蛋白(BSA)、α-酪蛋白、球蛋白、α-乳白蛋白、LDH、溶 菌酶、肌红蛋白、卵清蛋白和RNAaseA。部分稳定剂，如氯化钠、氯化钙、氯化镁、甘露醇、山梨醇、蔗糖等也可起到控制渗透压的作用。在本发明中具体地使用的稳定剂选自多元醇、氨基酸、盐、糖中的一种或一种以上。优选的盐为氯化钠，优选的糖为蔗糖和海藻糖，优选的多元醇为山梨醇和甘露醇。优选的氨基酸为精氨酸、甘氨酸、脯氨酸，氨基酸可以以其D-和/或L-型存在，但典型是L-型，氨基酸可以任何合适的盐存在，例如盐酸盐，如盐酸精氨酸。优选的稳定剂为氯化钠、甘露醇、山梨醇、蔗糖、海藻糖、盐酸精氨酸、甘氨酸、脯氨酸、氯化钠-山梨醇、氯化钠-甘露醇、氯化钠-蔗糖、氯化钠-海藻糖、盐酸精氨酸-甘露醇、盐酸精氨酸-蔗糖。
术语“表面活性剂”一般包括保护蛋白质例如抗体免受空气/溶液界面诱导的应力、溶液/表面诱导的应力的影响以减少抗体的聚集或使制剂中颗粒物的形成最小化的试剂。示例性的表面活性剂包括但不限于非离子型表面活性剂例如聚氧乙烯脱水山梨醇脂肪酸酯(如聚山梨酯20和聚山梨酯80)、聚乙烯-聚丙烯共聚物、聚乙烯-聚丙烯二醇、聚氧乙烯-硬脂酸酯、聚氧乙烯烷基醚、例如聚氧乙烯单月桂基醚、烷基苯基聚氧乙烯醚(Triton-X)、聚氧乙烯-聚氧丙烯共聚物(泊洛沙姆，Pluronic)、十二烷基硫酸钠(SDS)。本文中，如无特别说明，术语“聚山梨酯20的浓度”和“聚山梨酯80的浓度”均是指质量体积浓度(w/v)，如“约0.02％聚山梨酯80”中“0.02％”即指“100mL液体中含有0.02g的聚山梨酯80”。
术语“等渗”是指该制剂具有与人血液基本相同的渗透压。等渗制剂一般具有约250至350mOsm的渗透压。可使用蒸汽压或冰点下降式的渗透压计测量等渗性。
术语“稳定的”制剂是其中的抗体在制造过程期间和/或储存时基本上保持其物理稳定性和/或化学稳定性和/或生物活性的制剂。即使所含的抗体在经过一定时间储存之后未能保持其100％的化学结构或生物功能，医药制剂也可以是稳定的。在某些情况下，在经过一定时间储存之后，能维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，也可被认为是“稳定的”。用于测量蛋白质稳定性的各种分析技术在本技术领域中是可得的，并综述在《肽和蛋白质药物递送》(Peptide and Protein Drug Delivery)247～301，Vincent Lee主编，Marcel Dekker，Inc.，New York，N.Y.，Pubs.(1991)，和Jones，A.(1993)Adv.Drug Delivery Rev.10：29～90中(二者引入作为参考)。
制剂在一定温度下经过一定时间的储存之后，通过测定其中剩余的天然抗体的百分比(及其它方法)，可以测量其稳定性。除其它方法外，天然抗体的百分比可以通过尺寸排阻色谱法(例如尺寸排阻高效液相色谱法[SEC-HPLC])来测量，“天然的”指未聚集的和未降解的。在一些方案中，蛋白质的稳定性按照具有低百分比的降解(例如片段化)和/或聚集蛋白质的溶液中单体蛋白质的百分数来确定。在一些方案中，制剂可以在室温、约25～30℃或40℃下稳定储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4 个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，或更长，最多不超过约6％、5％、4％、3％、2％、1％、0.5％，或0.1％聚集形式的抗体。
通过测定在离子交换期间在此抗体主馏分(“主要荷电形式”)较为酸性的馏分中迁移的抗体(“酸性形式”)的百分比(及其它方法)，可以测量稳定性，其中稳定性与酸性形式抗体的百分比成反比。除其它方法外，“酸化”抗体的百分比可以通过离子交换色谱法(例如阳离子交换高效液相色谱法[CEX-HPLC])来测量。在一些实施方式中，可接受程度的稳定性意为当制剂在一定温度下经过一定时间的储存之后，其中可检测出的酸性形式的抗体最多不超过约49％、45％、40％、35％、30％、25％、20％、15％、10％、5％、4％、3％、2％、1％、0.5％或0.1％。在测量稳定性之前储存的一定时间可以是至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，或更长。当评估稳定性时，容许储存医药制剂的一定温度可以是约-80℃至约45℃范围内的任何温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约2～8℃、约5℃、约25℃，或约40℃。
如果抗体在颜色和/或澄清度目测检查时或通过UV光散射或通过孔径排阻层析测量时基本上不显示出例如聚集、沉淀和/或变性的迹象，则所述抗体在该药物组合物中“保持其物理稳定性”。聚集是单个分子或复合物共价或非共价缔合以形成聚集体的过程。聚集可以进行到形成可见沉淀物的程度。
制剂的稳定性例如物理稳定性可以通过本技术领域中公知的方法来评估，包括测量样品的表观消光度(吸光度或光密度)。这样的消光测量与制剂的浊度相关。制剂的浊度部分地是溶解在溶液中的蛋白质的固有性质，并且通常通过比浊法来测量，并用比浊法浊度单位(NTU)来量度。
随着例如溶液中一种或多种组分的浓度(例如蛋白质和/或盐浓度)而变化的浊度水平也被称为制剂的“乳浊”或“乳浊外观”。浊度水平可以参照使用已知浊度的悬液产生的标准曲线来计算。用于测定药物组合物的浊度水平的参比标准品可以基于《欧洲药典》标准(《欧洲药典》(European Pharmacopoeia)，第四版，“欧洲药品质量委员会指令”(Directorate for the Quality of Medicine of the Council of Europe)(EDQM)，Strasbourg，France)。根据《欧洲药典》标准，澄清溶液被定义为浊度低于或等于按照《欧洲药典》标准具有约3的参比悬液的浊度的溶液。比浊法的浊度测量可以检测在不存在缔合或非理想效应的情况下的瑞利散射，其通常随浓度线性变化。用于评估物理稳定性的其他方法在本技术领域中是公知的。
如果抗体在给定时间点的化学稳定性使得抗体被认为仍保持如下文中所定义的其生物 活性，则所述抗体在药物组合物中“保持其化学稳定性”。可以通过例如检测或定量抗体的化学改变的形式来评估化学稳定性。化学改变可以包括尺寸改变(例如剪短)，其可以使用例如孔径排阻层析、SDS-PAGE和/或基质辅助的激光解吸电离/飞行时间质谱(MALDI/TOF MS)来评估。其他类型的化学改变包括电荷改变(例如作为脱酰胺或氧化的结果而发生)，其可以通过例如离子交换层析来评估。
如果药物组合物中的抗体对于其预期目的来说是生物活性的，则所述抗体在药物组合物中“保持其生物活性”。例如，如果制剂于例如5℃、25℃、45℃等温度下储存一定时间(例如1至12个月)之后，该制剂所含抗CD112R抗体与CD112R结合的亲和力为所述储存之前抗体结合亲和力的至少90％、95％或以上，则可认为本发明之制剂是稳定的。结合亲和力也可用例如ELISA或等离子共振技术测定。
在本发明的情形中，在药理学意义上，抗体的“治疗有效量”或“有效量”是指在抗体可以有效治疗的障碍的症状的预防或治疗或减轻方面有效的量。本发明中，药物的“治疗有效量”或“治疗有效剂量”是当单独使用或与另一种治疗剂组合使用时保护受试者免于疾病发作或促进疾病消退的任何量的药物，所述疾病消退通过疾病症状的严重性的降低，疾病无症状期的频率和持续时间的增加，或由疾病痛苦引起的损伤或失能的预防来证明。药物促进疾病消退的能力可以使用本领域技术人员已知的多种方法来评价，比如在临床试验期间的人受试者中，在预测人类功效的动物模型系统中，或通过在体外测定法中测定所述药剂的活性。药物治疗有效量包括“预防有效量”，即当单独或如与其它治疗药物组合给与处于患病风险的受试者或患病复发的受试者时，抑制疾病的发展或复发的任何量的药物。
术语“受试者”或“患者”意图包括哺乳动物生物体。受试者/患者的实例包括人类和非人类哺乳动物，例如非人类灵长动物、狗、奶牛、马、猪、绵羊、山羊、猫、小鼠、兔、大鼠和转基因非人类动物。在本发明的特定实施方式中，受试者是人类。
术语“施用”、“给与”及“处理”是指采用本领域技术人员已知的各种方法或递送系统中的任意一种将包含治疗剂的组合物引入受试者。抗CD112R抗体的给药途径包括静脉内、肌内、皮下、腹膜、脊髓或其他胃肠外给药途径，比如注射或输注。“胃肠外给药”是指除了肠内或局部给药以外的通常通过注射的给药方式，包括但不限于静脉内、肌内、动脉内、鞘内、淋巴内、损伤内、囊内、框内、心内、皮内、腹膜内、经气管、皮下、表皮下、关节内、囊下、蛛网膜下、脊柱内、硬膜内和胸骨内注射和输注以及经体内电穿孔。
抗CD112R抗体
本文所用的术语“抗体”应被理解为包括完整抗体分子及其抗原结合片段。本文所用的术语抗体的“抗原结合部分”或“抗原结合片段”(或简称为“抗体部分”或“抗体片段”)是指抗体 中保持了与人CD112R或其表位特异性结合能力的一个或多个片段。因此，其以最广义使用，具体包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、人源化抗体、全人抗体、嵌合抗体和骆驼源化单结构域抗体。
术语“分离的抗体”是指结合化合物的纯化状态，且在这种情况下意指该分子基本不含其它生物分子，例如核酸、蛋白质、脂质、糖或其它物质例如细胞碎片和生长培养基。术语“分离(的)”并非意指完全不存在这类物质或不存在水、缓冲液或盐，除非它们以明显干扰本文所述结合化合物的实验或治疗应用的量存在。
术语“单克隆抗体”是指获自基本均质抗体群的抗体，即组成该群的各个抗体除可少量存在的可能天然存在的突变之外是相同的。单克隆抗体是高度特异性的，针对单一抗原表位。相比之下，常规(多克隆)抗体制备物通常包括大量针对不同表位(或对不同表位有特异性)的抗体。修饰语“单克隆”表明获自基本均质抗体群的抗体的特征，且不得解释为需要通过任何特定方法产生抗体。
术语“鼠源抗体”或“杂交瘤抗体”在本公开中为根据本领域知识和技能制备的抗人CD112R的单克隆抗体。制备时用CD112R抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体”是具有第一抗体的可变结构域和第二抗体的恒定结构域的抗体，其中第一抗体和第二抗体来自不同物种。通常，可变结构域获自啮齿动物等的抗体(“亲代抗体”)，而恒定结构域序列获自人抗体，使得与亲代啮齿动物抗体相比，所得嵌合抗体在人受试者中诱导不良免疫应答的可能性较低。
术语“人源化抗体”是指含有来自人和非人(例如小鼠、大鼠)抗体的序列的抗体形式。一般而言，人源化抗体包含基本所有的至少一个、通常两个可变结构域，其中所有或基本所有的超变环相当于非人免疫球蛋白的超变环，而所有或基本所有的构架(FR)区是人免疫球蛋白序列的构架区。人源化抗体任选可包含至少一部分的人免疫球蛋白恒定区(Fc)。
术语“全长抗体”或“完整抗体分子”指包含四条肽链的免疫球蛋白分子，两条重(H)链(全长时约50～70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种 细胞(例如，效应细胞)和经典补体系统的第一组分(Clq)的结合。
术语“CDR”是指抗体可变序列内的互补决定区。在重链和轻链的各个可变区中存在3个CDR，其对于各个重链和轻链可变区被命名为HCDR1、HCDR2和HCDR3或LCDR1、LCDR2和LCDR3。这些CDR的准确边界按照不同的系统有不同的定义。
本发明的所述抗体的可变区CDR的精确氨基酸序列边界可使用许多公知的方案的任何方案来确定，包括基于抗体的三维结构和CDR环的拓扑学的Chothia(Chothia等人.(1989)Nature 342：877-883，A1-Lazikani等人，“Standard conformations for the canonical structures of immunoglobulins”，Journal of Molecular Biology，273，927-948(1997)基于抗体序列可变性的Kabat(Kabat等人，Sequences of Proteins of Immunological Interest，第4版，U.S.Department of Health and Human Services，National Institutes of Health(1987)，AbM(University of Bath)，Contact(University College London)，国际ImMunoGeneTics database(IMGT)(1999 Nucleic Acids Research，27，209-212)，以及基于利用大量晶体结构的近邻传播聚类(affinity propagation clustering)的North CDR定义。本发明抗体的CDR可以由本领域的技术人员根据本领域的任何方案(例如不同的指派系统或组合)确定边界。
本文所使用的“抗原结合片段”包括抗体的片段或其衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗原结合片段的实例包括但不限于Fab，Fab′，F(ab′)2和Fv片段；双抗体；线性抗体；单链抗体分子，例如scFv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗体的结合活性在摩尔浓度基础上表示时，结合片段或其衍生物通常保持亲代抗体抗原结合活性的至少10％。优选结合片段或其衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。
本发明所述的抗CD112R抗体或其抗原结合片段包括申请号为CN 202110178859.1的专利申请中描述的任意一个抗CD112R抗体或其抗原结合片段，本文将申请号为CN202110178859.1的专利申请所公开的全部内容以引入的方式纳入本文。在一些方案中，在本发明的方法和组合物中使用的抗体的CDR序列包括来自于CN202110178859.1中描述的人源化抗CD112R抗体Hu16、Hu52和Hu59，其CDR、可变区和全长氨基酸序列如表A所示。
表A：人源化抗CD112R抗体氨基酸序列(KABAT方案)

在一些方案中，在本发明的方法和组合物中使用的抗CD112R抗体或其抗原结合片段包含的LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3分别为：
LCDR1：KASQSVSNDVA(SEQ ID NO：1)；
LCDR2：YVSX1RFT(SEQ ID NO：21)，其中，X1为H或N；
LCDR3：X2QAYRSPWT(SEQ ID NO：22)，其中，X2为H或Q；
HCDR1：SYHMS(SEQ ID NO：6)；
HCDR2：AIX3X4X5GX6X7TYYX8DTVKG(SEQ ID NO：23)，其中，X3为S或N，X4为R或S，X5为D或N，X6为D或I，X7为S或N，X8为P或L；
HCDR3：HEDYX9GFAMDX10(SEQ ID NO：24)，X9为F或Y，X10为S或Y。
在一些方案中，上述抗CD112R抗体或其抗原结合片段包含：
氨基酸序列如SEQ ID NO：1所示的LCDR1；
氨基酸序列如SEQ ID NO：2或4所示的LCDR2；
氨基酸序列如SEQ ID NO：3或5所示的LCDR3；
氨基酸序列如SEQ ID NO：6所示的HCDR1；
氨基酸序列如SEQ ID NO：7或9所示的HCDR2；
氨基酸序列如SEQ ID NO：8或10所示的HCDR3。
在一些方案中，上述抗CD112R抗体或其抗原结合片段包含：
(1)氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的HCDR1、HCDR2和HCDR3；或
(2)氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3；或
(3)氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：4和SEQ ID NO：5所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3。
在一些方案中，上述抗CD112R抗体或其抗原结合片段选自鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人源化抗体或其抗原结合片段，优选为人源化抗体或其抗原结合片段。
在一些方案中，上述抗CD112R抗体或其抗原结合片段包含：
(1)氨基酸序列如SEQ ID NO：11所示的轻链可变区，和氨基酸序列如SEQ ID NO：12所示的重链可变区；或
(2)氨基酸序列如SEQ ID NO：11所示的轻链可变区，和氨基酸序列如SEQ ID NO：13所示的重链可变区；或
(3)氨基酸序列如SEQ ID NO：14所示的轻链可变区，和氨基酸序列如SEQ ID NO：15所示的重链可变区。
在一些方案中，上述抗CD112R抗体包含：
(1)氨基酸序列如SEQ ID NO：16所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO：17所示的重链氨基酸序列；
(2)氨基酸序列如SEQ ID NO：16所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO：18所示的重链氨基酸序列；或
(3)氨基酸序列如SEQ ID NO：19所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO：20所示的重链氨基酸序列。
在一些方案中，在本文实施例中所用的非限制性、示范性抗体为Hu52，其为包含序列SEQ ID NO：16和18所示的轻链及重链氨基酸序列的人源化抗体。
本文中的SEQ ID NO：1-20如下所示：


在一些实施方式中，在本发明的方法和组合物中使用的抗CD112R抗体或其抗原结合片段为人源化抗体或嵌合抗体，且可包括人恒定区。在一些实施方式中，恒定区是选自人IgG1、IgG2、IgG3及IgG4恒定区组成的组；优选地，适用于本发明所述的方法和组合物的抗CD112R抗体或其抗原结合片段包含人IgG1或IgG4同种型的重链恒定区。
在一些实施方式中，本发明提供了一种用于制备如本文所述的抗CD112R抗体或其抗原结合片段的方法，所述方法包括在适合于所述抗体或其抗原结合片段表达的条件下在本文所述的宿主细胞中表达所述抗体或其抗原结合片段，并从所述宿主细胞回收所表达的抗体或其抗原结合片段。
本发明提供用于表达本发明的重组抗体的哺乳动物宿主细胞，包括可获自美国典型培养物保藏中心(ATCC)的许多永生化细胞系。这些尤其包括中国仓鼠卵巢(CHO)细胞、NS0、SP2/0细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞、A549细胞、293T细胞和许多其它细胞系。哺乳动物宿主细胞包括人、小鼠、大鼠、狗、猴、猪、山羊、牛、马和仓鼠细胞。通过测定哪种细胞系具有高表达水平来选择特别优选的细胞系。
在一个实施方式中，本发明提供制备抗CD112R抗体的方法，其中所述方法包括，将表达载体导入哺乳动物宿主细胞中时，通过将宿主细胞培养足够的一段时间，以允许抗体在宿主细胞中表达，或者更优选抗体分泌到宿主细胞生长的培养基中，来产生抗体。可采用标准蛋白质纯化方法从培养基中回收抗体。
很可能由不同细胞系表达或在转基因动物中表达的抗体彼此具有不同的糖基化。然而，由本文提供的核酸分子编码的或包含本文提供的氨基酸序列的所有抗体是本发明的组成部分，而不论抗体的糖基化如何。同样，在某些实施方式中，非岩藻糖基化抗体是有利的，因为它们通常在体外和体内具有比其岩藻糖基化对应物更强力的功效，并且不可能是免疫原性的，因为它们的糖结构是天然人血清IgG的正常组分。
医药制剂
本发明提供了一种药物组合物，包含：(1)缓冲液；(2)抗CD112R抗体或其抗原结合片段。
本发明所述药物组合物中的抗CD112R抗体或其抗原结合片段如本申请“抗CD112R抗体”部分任一实施方式所述。
例如，本发明所述药物组合物中的抗CD112R抗体或其抗原结合片段包含氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3。优选地，上述抗CD112R抗体或其抗原结合片段选自鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人源化抗体或其抗原结合片段，优选为人源化抗体或其抗原结合片段。优选地，上述抗CD112R抗体或其抗原结合片段包含如SEQ ID NO：11所示的轻链可变区，和如SEQ ID NO：13所示的重链可变区。更优选地，上述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列，和如SEQ ID NO：18所示的重链氨基酸序列。
本发明所述药物组合物中的抗CD112R抗体或其抗原结合片段的浓度为约1～100mg/mL，优选为约10～70mg/mL，更优选为约20～60mg/mL。在一些实施方案中，本发明所述药物组合物中的抗CD112R抗体或其抗原结合片段的浓度为40±10mg/mL。
本发明所述药物组合物的pH为约4.5～6.5，优选为约5.5～6.5，更优选为约5.8～6.2。在一些实施方案中，本发明所述药物组合物的pH为约5.5或约6.0。
本发明所述药物组合物中的缓冲液选自醋酸缓冲液、柠檬酸缓冲液、磷酸盐缓冲液和组氨酸缓冲液中的一种或多种；优选地，所述缓冲液为组氨酸缓冲液，优选地，所述组氨酸缓冲液为组氨酸-组氨酸盐酸盐缓冲液或组氨酸-组氨酸醋酸盐缓冲液，优选为组氨酸-组氨酸盐酸盐缓冲液。
优选地，缓冲液的浓度为约5～100mM，优选为约10～50mM，优选为约10～30mM；优选为约15～25mM。优选地，缓冲液的pH为约4.5～6.5，优选为约5.5～6.5，优选为约5.8～6.2。在一些实施方案中，本发明所述缓冲液的pH为约5.5或约6.0。
因此，本发明的药物组合物可含有：pH约为5.5～6.5的组氨酸缓冲液，其在药物组合物中的浓度约为10～30mM；和约10～70mg/mL的前文任一实施方式所述的抗CD112R抗体或其抗原结合片段，尤其是本文所述的人源化抗体Hu52或其抗原结合片段。
在一些方案中，本发明所述药物组合物还包括稳定剂，所述稳定剂选自精氨酸、精氨酸盐、氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种；优选地，上述精氨酸盐为盐酸精氨酸。优选地，上述稳定剂的浓度为约10～400mM，优选为约100～300mM，优选为约150～270mM，优选为约180～260mM。应理解，当使用两种或两种以上稳定剂时，其浓度之和也在所述10～400mM的范围内，优选在150～270mM的范围内，更优选在180～260mM的范围内。
优选地，本发明所述药物组合物中的稳定剂为：浓度约150～270mM的海藻糖；或浓度约150～270mM的蔗糖；或浓度约20～80mM的氯化钠与浓度约110-170mM的蔗糖的组合；或浓度约20～80mM的盐酸精氨酸与浓度约110-170mM的蔗糖的组合。在一些实施方案中，本发明所述药物组合物中的稳定剂为：浓度约180～260mM、优选约220～240mM的海藻糖；或浓度约180～260mM、优选约220～240mM的蔗糖；或上述稳定剂为浓度约30～70mM、优选约40～60mM的氯化钠与浓度约120-160mM、优选约130～150mM的蔗糖的组合；或上述稳定剂为浓度约30～70mM、优选约40～60mM的盐酸精氨酸与浓度约120-160mM、优选约130～150mM的蔗糖的组合。
因此，在一些实施方案中，本发明的药物组合物可含有：pH约为5.5～6.5的组氨酸缓冲液，其在药物组合物中的浓度约为10～30mM；约10～70mg/mL的前文任一实施方式所述的抗CD112R抗体或其抗原结合片段，尤其是本文所述的人源化抗体Hu52或其抗原结合片段；以及约150～270mM的稳定剂，优选地，所述稳定剂选自精氨酸、精氨酸盐、氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种，优选地，上述精氨酸盐为盐酸精氨酸。优选地，上述稳定剂为浓度约180～260mM、优选约220～240mM的海藻糖；或浓度约 180～260mM、优选约220～240mM的蔗糖；或浓度约30～70mM、优选约40～60mM的氯化钠与浓度约120-160mM、优选约130～150mM的蔗糖的组合；或上述稳定剂为浓度约30～70mM、优选约40～60mM的盐酸精氨酸与浓度约120-160mM、优选约130～150mM的蔗糖的组合。
在一些方案中，上述药物组合物还包括表面活性剂，所述表面活性剂选自聚山梨酯80、聚山梨酯20和泊洛沙姆188中的一种或多种。优选地，以w/v计算，上述表面活性剂浓度为约0.001％～0.1％，优选为约0.01％～0.1％，优选为约0.02～0.06％，更优选为约0.04％～0.06％。
因此，本发明的药物组合物可含有：pH约为5.5～6.5的组氨酸缓冲液，其在药物组合物中的浓度约为10～30mM；约10～70mg/mL的前文任一实施方式所述的抗CD112R抗体或其抗原结合片段，尤其是本文所述的人源化抗体Hu52或其抗原结合片段；约150～270mM的稳定剂；以及以w/v计，约0.01％～0.1％的聚山梨酯80。优选地，所述稳定剂选自精氨酸、精氨酸盐、氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种，优选地，上述精氨酸盐为盐酸精氨酸。优选地，所述稳定剂为浓度约180～260mM、优选约220～240mM的海藻糖；或浓度约180～260mM、优选约220～240mM的蔗糖；或浓度约30～70mM、优选约40～60mM的氯化钠与浓度约120-160mM、优选约130～150mM的蔗糖的组合；或上述稳定剂为浓度约30～70mM、优选约40～60mM的盐酸精氨酸与浓度约120-160mM、优选约130～150mM的蔗糖的组合。
本发明的药物组合物为液体制剂或冻干制剂。
本发明还提供了一种注射剂，其含有本文任一项方案中所述的药物组合物与氯化钠溶液或葡萄糖溶液；优选地，所述氯化钠溶液浓度为约0.85～0.9％(w/v)；优选地，所述葡萄糖溶液浓度为约5～25％(w/v)，更优选为约5～10％(w/v)。
在一些方案中，上述药物组合物或注射剂，其经静脉注射施用。
医药用途和方法
本发明还提供了本文任一项方案中所述的药物组合物或注射剂在制备用于治疗和/或预防CD112R介导的疾病或病症的药物中的用途。
本发明还提供了用于治疗和/或预防CD112R介导的疾病或病症的本文任一项方案中所述的药物组合物或注射剂。
本发明还提供了一种治疗和/或预防CD112R介导的疾病或病症的方法，其包括向有需要的受试者施用如本文任一项方案中所述的药物组合物或注射剂。
本发明中，CD112R介导的疾病指在CD112R参与了疾病的发生和发展的疾病，包括但不限于癌症。
“癌症”和“癌性”指或描述哺乳动物中特征通常为细胞生长不受调控的生理疾患。此定义中包括良性和恶性癌症以及休眠肿瘤或微转移。癌症的例子包括但不限于癌，淋巴瘤，母细胞瘤，肉瘤，和白血病。此类癌症的更具体例子包括鳞状细胞癌，肺癌(包括小细胞肺癌，非小细胞肺癌，肺的腺癌，和肺的鳞癌)，腹膜癌，肝细胞癌，胃的癌或胃癌(包括胃肠癌)，胰腺癌，成胶质细胞瘤，宫颈癌，卵巢癌，肝癌，膀胱癌，肝瘤(hepatoma)，乳腺癌，结肠癌，结肠直肠癌，子宫内膜癌或子宫癌，唾液腺癌，肾癌或肾的癌，鼻咽癌，食管鳞状细胞癌，肝癌，前列腺癌，外阴癌，甲状腺癌，肝的癌，及各种类型的头颈癌，以及B细胞淋巴瘤(包括低级/滤泡性非何杰金氏淋巴瘤(NHL)，小淋巴细胞性(SL)NHL，中级/滤泡性NHL，中级弥漫性NHL，高级成免疫细胞性NHL，高级成淋巴细胞性NHL，高级小无核裂细胞性NHL，贮积病(bulky disease)NHL，套细胞淋巴瘤，AIDS相关淋巴瘤，和瓦尔登斯特伦氏(Waldenstrom)巨球蛋白血症)，慢性淋巴细胞性白血病(CLL)，急性成淋巴细胞性白血病(ALL)，毛细胞性白血病，慢性成髓细胞性白血病，和移植后淋巴增殖性病症(PTLD)，以及与瘢痣病(phakomatoses)，水肿(诸如与脑瘤有关的)和梅格斯氏(Meigs)综合征有关的异常血管增殖。
可如前文所述给予受试者施用所述药物组合物或注射剂。例如，可通过静脉内、肌内、皮下、腹膜、脊髓或其他胃肠外给药途径，比如注射或输注。
实施例
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并非意图限制本发明的范围。本文已经详细描述了本发明，其中也公开了其具体实施方式。对本领域的技术人员而言，在不脱离本发明精神和范围的情况下，针对本发明具体实施方式进行各种变化和改进将是显而易见的的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。实施例中所用到的方法和材料，除非另有说明，否则为本领域的常规方法和材料。
实施例中所用的检测方法
(1)外观
采用目检法来检测外观。确保澄明度检测仪的光照强度保持在10001x～15001x之间。将样品保持在眼睛的同一水平面，轻轻摇晃或颠倒以避免产生气泡。分别在黑色背景和白色背景前进行目检。从颜色，乳光和可见异物三个方面记录结果。
(2)蛋白含量
蛋白浓度使用紫外分光光度计来检测。将百分比消光系数(E1％)设定在1.389(mg/m1)- 1cm-1。根据检品蛋白质浓度预测值，用超纯水稀释检品浓度至约0.4mg/mL，尽量控制吸光 读数在0.3-0.7之间。每个样品平行测定2份溶液，每份溶液重复测定3次；每份溶液测定之前均需先用150μl溶液润洗比色皿2次，然后取150μl溶液进行测定。
浓度计算公式：
C(mg/mL)＝A/ε×DF；样品蛋白质含量(mg/mL)＝(C1+C2)/2
C为溶液中蛋白质含量；
A为280nm吸光度值，A＝(A1+A2+A3)/3；
ε：消光系数(1.389(mg/mL)-1cm-1)；
DF：稀释倍数。
(3)SEC-HPLC纯度
SEC-HPLC纯度采用安装了SEC色谱柱(WatersXbrigeBEH200A，7.8*300mm，3.5μm)的HPLC(Waters e2695仪器)进行检测。流动相组成为50mM磷酸盐，300mM NaCl，pH6.8±0.2。采用峰面积归一化对结果进行定量分析。分别计算单体、聚体和片段的峰面积百分比，以单体的峰面积百分比作为样品的纯度，聚体和片段的峰面积百分比作为聚体和片段的含量。色谱参数见下表1。
表1：SEC-HPLC色谱参数
(4)R-CE-SDS纯度
抗体制剂还原CE-SDS电泳法纯度检测以高压直流电场为驱动力，以毛细管为分离通道。预先填充的凝胶会在毛细管内形成分子筛，十二烷基硫酸钠可消除不同蛋白质分子的电荷效应，还原剂β-巯基乙醇可切除样品中的二硫键，分子大小不同的样品在毛细管内移动速度不同，因而可进行分离。用1％SDS，40mM磷酸盐缓冲液，pH6.5的溶液将样品稀释至1mg/mL，然后取95μl，加入β-巯基乙醇5.0μl混合均匀；同时将超纯水以与参比品相同稀释倍数稀释，取95μl，加入β-巯基乙醇5.0μl混合均匀，作为空白对照。制备的样品3000rpm室温离心30sec，70℃孵育15min，冷却至室温，12000rpm室温离心5min，分离时间30min， 使用毛细管电泳仪(Maurice、PA800)检测。计算重链(HC)，非糖基化重链(NGHC)和轻链(LC)的纯度，三者之和即为样品的纯度。
(5)NR-CE-SDS纯度
抗体制剂非还原CE-SDS电泳法纯度检测以高压直流电场为驱动力，以毛细管为分离通道。预先填充的凝胶会在毛细管内形成分子筛，用十二烷基硫酸钠处理样品以消除不同蛋白质分子的电荷效应，使分子大小不同而在毛细管内移动速度不同的样品进行分离。供试品溶液中加入烷基化试剂，能够有效减小组分扩散，使所得峰型尖锐，分离效率高，且可以保证样品保持非还原状态。按终体积100μl计算好将样品/参比品稀释至1mg/mL时需要加入的样品缓冲液(1％SDS，40mM磷酸盐缓冲液，pH6.5)及样品量，然后依次按缓冲液、5.0μl0.25MNEM、样品的顺序加入1.5mLEP管中混匀；以超纯水作为空白对照，稀释比例和参比品保持一致。制备的样品3000rpm室温离心30sec，70℃孵育5min，冷却至室温，12000rpm室温离心5min，使用毛细管电泳仪(Maurice、PA800)检测。
(6)CEX-HPLC纯度
电荷异质性采用阳离子交换色谱法(CEX-HPLC)进行检测。CEX-HPLC纯度采用安装了色谱柱(ProPac WCX-10，4*250mm)的HPLC(Waters e2695仪器)进行检测。流动相组成为A相：20mM MES(pH6.00±0.02)；B相：20mM MES+200mM NaCl(pH6.00±0.02)。采用峰面积归一化法计算主峰、酸性峰、碱性峰百分比，如果采用自动积分无法获得合理的积分结果，则采用手动积分。详细色谱参数见下表2。
表2：CEX-HPLC色谱参数

(7)结合活性
结合ELISA(Binding ELISA)实验：采用间接法，用HX1 hCD112R Fc作为抗原包被到96孔板上，抗体制剂可与HX1 hCD112R Fc结合，Mouse Anti-Human IgG4 Fc-HRP可与结合到固相抗原(HX1 hCD112R Fc)上的抗体制剂特异性结合，Mouse Anti-Human IgG4 Fc-HRP在过氧化氢作用下可催化TMB显蓝色，蓝色深浅与Mouse Anti-Human IgG4 Fc-HRP结合量呈正相关，用2M盐酸终止后变为黄色。用酶标仪在450nm/620nm波长下测定吸光度(OD值)，通过标准曲线的有效结合活性(EC50)来考察抗体制剂结合CD112R的能力。
(8)亚可见颗粒(MFI)检测
MFI微流成像颗粒分析系统将数字显微技术、微流体和图像处理技术整合成一个全自动分析颗粒的仪器。当样品流过检测区时，MFI会捕捉样品的镜像，镜像中的每个颗粒将被分析，生成关于颗粒的数量、尺寸、透明度、形态等方面的数据。也能用于实时分析颗粒的动态过程。形态分析软件还可用于分析特殊形态的颗粒，或者用于分离一些亚颗粒群体。
(9)iCIEF纯度
iCIEF采用安装了iCIEF卡盒的毛细管电泳仪进行检测。样品经iCIEF混合液稀释处理后，使用毛细管电泳仪检测。不同等电点组分被聚焦在不同位置而达到聚焦和分离的效果。
(10)热稳定性(Tm)
蛋白处于折叠状态时，其内部的疏水氨基酸在330nm处的发射光是要大于在其350nm处的发射光。当我们对蛋白进行加热或者化学变性剂处理时，蛋白会发生去折叠并且当氨基酸暴露于液相环境中，此时最大发射光会发生位移，最终在350nm处的发射光会大于在330nm处的发射光，因此我们可以通过20℃～95℃升温，1℃/min升温速率利用荧光值的变化来检测温度或者化学变性剂所引起的蛋白质去折叠的过程，从而计算出半数解链温度，即Tm值。
(11)聚集温度(Tagg)
蛋白类产品升温范围由35℃到85℃，升温1-2℃进行一次扫描，在某一温度开始会出现不同程度的聚集。通过对样品溶液中颗粒直径变化和温度变化的监测，可得到蛋白初始聚集温度(Tagg)。
以下实施例中的缩写说明：“h”表示小时，“W”表示周，“M”表示月，“C”表示冻融循环的次数，“FT”表示冻融循环，“SH”表示振摇，“ST”表示搅拌，“RT”表示室温，“rpm”表示转/分钟，“T0”表示处方样品经放样处理前的起始测试。
实施例1：缓冲液体系筛选实验
液体型药物组合物中，缓冲液体系密切影响抗体的稳定性，每种具有独特理化性质的抗体都具有最适宜的缓冲液的种类。本实施例旨在初步筛选出最佳缓冲液体系和稳定剂，使本发明公开的抗CD112R抗体具有最佳的稳定性以适宜临床应用。
1.1实验步骤
本实施例以抗体Hu52进行。样品使用Millipore Pellicon3 0.11m2膜进行UF/DF超滤浓缩至浓度约52.5mg/mL，再将样品透析至对应的如表3所示的处方中，将最终浓度调至约40mg/mL，再加入对应浓度的聚山梨酯80。在超净台无菌灌装到2R西林瓶，2.0mL/瓶，进行稳定性放样和检测。
表3：缓冲液体系筛选实验中的处方信息
1.2实验结果
1.2.1外观
根据表4的结果，经高温或长期条件放置4周，所有样品的外观均未出现明显变化；经反复冻融5次，所有样品的外观均未出现显著变化。
表4：外观测试结果

1.2.2 SEC-HPLC纯度
根据表5中的SEC纯度结果，经高温条件下放置4周，所有处方聚体和片段均增加，处方FS1-1和FS1-4的单体纯度明显下降，处方FS1-8表现相对较优；经长期条件放置4周，所有处方SEC纯度均未出现明显变化。经反复冻融5次，所有样品SEC纯度均未出现明显变化。
表5：SEC-HPLC测试结果

1.2.3 R-CE-SDS纯度
根据表6中的R-CE-SDS纯度结果，经高温条件下放置4周，处方FS1-4和FS1-5纯度下降明显，其他组间差异不大。经长期条件放置4周及冻融五次后，所有样品R-CE-SDS纯度均未出现显著变化。
表6：R-CE-SDS测试结果

1.2.4 NR-CE-SDS纯度
根据表7中的NR-CE-SDS纯度结果，经高温条件下放置4周，所有样品NR-CE-SDS纯度均出现下降，其中处方FS1-2和FS1-4纯度降低相对较快；所有样品经长期放置4周及冻融五次后NR-CE-SDS纯度均未出现显著变化。
表7：NR-CE-SDS测试结果
1.2.5 iCIEF纯度
根据表8中的iCIEF纯度结果，经高温条件下放置4周，所有样品均出现酸峰明显增加和主峰纯度降低的现象，其中处方FS1-2、FS1-3、FS1-7和FS1-8主峰纯度降低较慢，处方FS1-4稳定性表现最差；经长期条件下放置4周以及反复冻融五次后，所有样品主峰纯度无明显变化。
表8：iCIEF测试结果

1.2.6亚可见颗粒
根据表9中的亚可见颗粒结果，经高温条件放置4周和反复冻融5次条件下，处方FS1-3、FS1-6、FS1-7和FS1-8亚可见颗粒数量增加相对缓慢。
表9：亚可见颗粒测试结果
1.3缓冲液体系筛选结论
综上所述，在高温条件下放置4周，处方FS1-3(醋酸缓冲液，pH5.5)和FS1-8(组氨酸缓冲液，pH6.0)的主峰纯度明显高于其它处方，亚可见颗粒较少。此外，所有处方的外观均没有显著性变化。因此，单克隆抗体与20mM醋酸缓冲液(pH5.5)和0.02％聚山梨酯80(II)、20mM组氨酸缓冲液(pH6.0)和0.02％聚山梨酯80(II)的药物组合物在高温条件下具有最优的稳定性。
实施例2：稳定剂筛选实验
为了进一步探究不同辅料对抗体稳定性影响，我们选取不同的稳定剂进行了比较测试。考察pH5.5、20mM醋酸缓冲体系和pH6.0、20mM组氨酸缓冲体系，抗体Hu52浓度40mg/mL条件下，上述不同辅料对稳定性的影响。
2.1实验步骤
本实施例以抗体Hu52进行。样品使用Millipore Pellicon3 0.11m2膜进行UF/DF超滤浓缩至浓度约55mg/mL，再将样品透析至对应的如表10所示的处方中，将最终浓度调至约40mg/mL，再加入对应浓度的聚山梨酯80。在超净台无菌灌装到2R西林瓶，2.0mL/瓶，进行稳定性放样和检测。
表10：稳定剂筛选实验中的处方信息
注“/”表示无。
2.2实验结果
2.2.1热稳定性(Tm)
根据表11中的热稳定性(Tm)检测结果，处方FS2-5和FS2-6的Tm值相对较高。
表11：热稳定性(Tm)测试结果
2.2.2聚集温度(Tagg)
根据表12中聚集温度(Tagg)检测结果，处方FS2-5聚集温度较高。
表12：聚集温度(Tagg)测试结果

2.2.3外观
根据表13中的外观结果，经高温、加速或长期条件下放置4周，所有处方外观均正常。
表13：外观测试结果
2.2.4 SEC-HPLC纯度
根据表14中的SEC-HPLC纯度结果，所有样品经高温、加速条件或长期条下放置4周，SEC纯度均有下降，样品无组间显著差异。
表14：SEC-HPLC纯度测试结果

2.2.5 R-CE-SDS纯度
根据表15中的R-CE-SDS纯度结果，经高温、加速及长期条件下放置4周，样品纯度均无明显变化。
表15：R-CE-SDS纯度测试结果

2.2.6 NR-CE-SDS纯度
根据表16中的NR-CE-SDS纯度结果，经高温条件下放置4周，样品纯度均有下降，样品组间无显著差异。经加速和长期条件下放置4周样品纯度无明显变化。
表16：NR-CE-SDS测试结果
2.2.7 CEX-HPLC纯度结果
根据表17中的CEX-HPLC纯度结果，经高温条件下放置4周，所有样品CEX-HPLC纯度均有下降，醋酸缓冲液pH5.5(FS2-1～FS2-4)酸性峰增加速率优于组氨酸缓冲液pH6.0(FS2-5～FS2-8)，酸性峰和pH有一定相关性；经加速条件下放置4周，所有样品CEX纯度均有所下降但无明显组间差异；长期条件下放置4周，所有样品CEX-HPLC纯度均无显著变化。
表17：CEX-HPLC测试结果

2.2.8结合活性
根据表18中的结合活性结果，经高温、加速条件和长期条件下放置4周，所有样品结合活性均未出现显著变化的情况。
表18：结合活性测试结果
2.2.9亚可见颗粒
根据表19中的亚可见颗粒检测结果，经高温、加速条件或长期条件下放置4周，所有样品亚可见颗粒均未出现显著变化。
表19：亚可见颗粒测试结果
2.3稳定剂筛选结论
综上所述，从溶解温度结果来看，FS2-5(组氨酸缓冲液，pH6.0，230mM蔗糖)和FS2-6(组氨酸缓冲液，pH6.0，230mM海藻糖)处方较优；从聚集温度结果来看，FS2-5处方较 优。此外，所有处方的外观、SEC纯度、R-CE-SDS纯度、NR-CE-SDS纯度、CEX-HPLC纯度、结合活性和亚可见颗粒均没有显著性变化。因此，单克隆抗体与20mM组氨酸缓冲液(pH6.0)和230mM蔗糖的药物组合物在高温条件下具有最优的稳定性。
实施例3：表面活性剂筛选实验
3.1实验步骤
将样品Hu52单抗使用Millipore Pellicon3 0.11m2进行UF/DF超滤浓缩换液约8倍体积至含230mM蔗糖的20mM组氨酸盐酸盐缓冲液(pH6.0)中，将最终浓度调至约40mg/mL，再加入对应浓度的聚山梨酯80(如表20)，进行稳定性实验。在超净台无菌灌装到2R西林瓶，2.0mL/瓶，进行稳定性放样和检测。
表20：表面活性剂筛选实验方案
3.2实验结果
3.2.1外观
根据表21中的外观结果，经高温、加速或长期条件放置4周，所有样品的外观均未出现明显变化。
表21：外观测试结果
3.2.2 SEC-HPLC纯度
根据表22中的SEC-HPLC纯度结果，经高温条件下放置4周，所有处方有轻微聚体 增加，在加速和长期条件下，所有样品的纯度均未出现显著变化，未见明显组间差异。
表22：SEC-HPLC纯度测试结果
3.2.3 R-CE-SDS纯度
根据表23中的R-CE-SDS纯度结果，经高温条件下放置4周，所有处方纯度均有下降，未出现显著组间差异。在加速和长期条件下，所有样品纯度均未发现明显变化。
表23：R-CE-SDS纯度测试结果
3.2.4 NR-CE-SDS纯度
根据表24中的NR-CE-SDS纯度结果，经高温条件下放置4周，所有处方纯度均有下降，在加速和长期条件下，所有样品纯度均未发现明显变化，未出现显著组间差异。
表24：NR-CE-SDS纯度测试结果

3.2.5 CEX-HPLC纯度
根据表25中的CEX-HPLC纯度结果及图3-2中的CEX-HPLC纯度趋势图，所有纯度变化未出现显著组间差异。
表25：CEX-HPLC纯度测试结果
3.2.6结合活性
根据表26中的结合活性结果，经高温、加速条件和长期条件下放置4周，所有样品结合活性均未出现显著变化的情况。
表26：结合活性测试结果

3.2.7亚可见颗粒
根据表27中的亚可见颗粒检测结果，经高温、加速条件或长期条件下放置4周，所有样品亚可见颗粒均未出现显著变化。
表27：亚可见颗粒测试结果
3.3表面活性剂筛选实验结论
综上所述，经高温、加速和长期条件下所有样品外观、纯度、活性及亚可见颗粒均未出现显著差异，三个处方表现出较好的稳定性。因此单克隆抗体与20mM组氨酸缓冲液(pH6.0)、230Mm蔗糖和聚山梨酯80(含量0.02％～0.06％)处方均具有较好的稳定性。
实施例4：强制降解实验
4.1实验步骤
使用实施例3的处方样品FS3-1、FS3-2和FS3-3。考察样品对于振摇条件的耐受性：200±20rpm振摇速度，室温条件，振摇2、5天。考察样品对于反复冻融的耐受性：-40℃与室温条件下反复冻融3次和5次。检测项目包括外观、SEC-HPLC、SDS-CE-NR/R、CEX和亚可见微粒。
4.2实验结果
4.2.1振摇实验
根据表28中结果，FS3-1处方样品经振摇实验考察后，乳光明显加重、亚可见颗粒数量明显增加，且FS3-1处方的R-CE-SDS纯度下降速度相对较快。FS3-2和FS3-3样品的外观、SEC-HPLC纯度、R-CE-SDS纯度、NR-CE-SDS纯度、CEX-HPLC纯度、亚可见颗粒数量均未发现显著变化。
表28：振摇实验测试结果

4.2.2冻融实验
根据表29中结果，经过反复冻融5次所有样品的外观、SEC-HPLC纯度、R-CE-SDS纯度、NR-CE-SDS纯度、CEX-HPLC纯度、亚可见颗粒数量均未发现显著变化。
表29：冻融实验测试结果

4.3强制降解实验结论
药物组合物经过反复冻融和振摇后，20mM组氨酸缓冲液(pH6.0)、230mM蔗糖和聚山梨酯80(含量0.04％～0.06％)处方外观、纯度和亚可见微粒均无明显变化，证明本发明的药物组合物对于振摇和反复冻融的耐受性良好。
综上所述，我们通过对不同缓冲体系，不同pH条件和不同辅料组成进行考察，目标pH范围控制在5.5～6.5，渗透压范围在250～350mOsm/kg，选择了最优制剂配方：约20mM组氨酸缓冲液(pH约为6.0)，约230mM蔗糖和约0.04～0.06％聚山梨酯80。
实施例5：药物组合物与CD112R的结合解离特性(BIACORE)
本实验使用GE医疗生命科学公司的T200型分子相互作用分析仪Biacore，将40μg/mL羊抗人Fc片断抗体(Jackson ImmunoResearch)偶联在CM5芯片表面，然后捕获药物组合物(1μg/mL，处方编号FS3-2，抗体为Hu52)，再进样梯度稀释的人PVRIG蛋白，检测结合信号。人PVRIG蛋白梯度稀释浓度为16nM、8nM、4nM、2nM、1nM和0.5nM，其中16nM为重复。使用Biacore分析软件Biacore T200 Evaluation Software 3.0的动力学模型分析，拟合得到亲和力KD值。
实验结果如表30和图1所示，药物组合物与人PVRIG蛋白有特异性结合活性，KD数值为5.77E-11M。
表30：药物组合物与重组人PVRIG蛋白亲和力结果表
实施例6：药物组合物结合活性(BINDING ELISA)
本实验均在37℃条件下进行，使用Thermo Scientific的酶标仪(型号：Multiskan GO)，用1.0μg/mL的HX1 hCD112R Fc(苏州君盟)包板90min，洗板后用2％BSA封闭90min，加入梯度稀释的药物组合物(处方编号FS3-2，抗体为Hu52)及阴性对照(1μg/mL起始，3倍梯度稀释至0.0056ng/mL，共12个浓度)孵育60min，洗板后加入稀释5000倍的Mouse Anti-Human IgG4 Fc-HRP(Southern Biotech，9200-05)作为检测抗体孵育60min进行检测，然后用0.1mg/mL TMB显色15min，最后用2M HCl终止反应，在450nm/620nm下读板。使用四参数对数回归(4PL)模型拟合EC50。
实验结果如图2所示，药物组合物与人CD112R蛋白有特异性结合活性，EC50值为1.4ng/mL，阴性对照样品无结合。
实施例7：药物组合物的竞争抑制活性(BLOCKING ELISA)
本实验均在37℃条件下进行，使用Thermo Scientific的酶标仪(型号：Multiskan GO)，用2.0μg/mL的HX1 hCD112R Fc(苏州君盟)包板90min，洗板后用2％BSA封闭90min，用稀释至4.0μg/mL的MX2 hPVRL2 Fc(苏州君盟)稀释药物组合物(处方编号FS3-2，抗体为Hu52)及阴性对照(10μg/mL起始，2倍梯度稀释至4.883ng/mL共12个浓度)，样品加至封闭后的板中孵育60min，洗板后加入稀释5000倍的Anti Mouse IgG(Fc Specific)-Peroxidase antibody produced in goat(Sigma，A2554)作为检测抗体孵育60min进行检测，然后用0.1mg/mL TMB显色15min，最后用2M HCl终止反应，在450nm/620nm下读板。使用四参数对数回归(4PL)模型拟合IC50。
实验结果如图3所示，药物组合物可特异性阻断人CD112R蛋白与CD112蛋白的结合，IC50值为60.0ng/mL，阴性对照样品无阻断作用。
实施例8：药物组合物与细胞表面CD112R的结合
通过流式细胞术检测药物组合物与CHO hCD112R细胞表面CD112R的结合。将不同浓度药物组合物(处方编号FS3-2，抗体为Hu52)和对照抗体(从0.01ng/ml到20μg/ml)与CHOhCD112R细胞4℃共孵育30分钟，然后用染色缓冲液洗去未结合的抗体。再将细胞与用染色缓冲液配置的含1％的Goat Anti-Human IgG4-PE荧光二抗(v/v)在4℃条件下避光 孵育30分钟。流式细胞仪收集细胞，检测细胞表面结合的荧光抗体。最后用FlowJo分析原始数据得到MFI值(平均荧光强度)，并用GraphPad Prism软件进行四参数拟合曲线得出EC50值。
实验结果如图4显示，药物组合物和COM701(Compugen的抗CD112R抗体，根据US20180280506A1序列表达纯化得到)与CHO hCD112R细胞有很强的亲和力，其细胞结合能力的EC50值分别为36.0ng/ml和119.2ng/ml，anti-KLH hIgG4为阴性结果，表明药物组合物能以高亲和力特异性地与细胞表面的hCD112R结合，结合亲和力优于COM701。
实施例9：药物组合物阻断HCD112R蛋白与细胞表面CD112R的结合
为了检测药物组合物竞争抑制对CD112R蛋白与细胞表面的CD112结合的活性，将CHO CD112细胞与固定浓度的hCD112R蛋白溶液(3μg/ml)，以及不同浓度的药物组合物(处方编号FS3-2，抗体为Hu52)或对照抗体(从0.169ng/ml到30μg/ml，3倍稀释)在4℃条件下共孵育30min。然后使用染色缓冲液洗去未结合的抗体，再将细胞与用染色缓冲液配置的含1％的Goat anti-mouse IgG Fc荧光二抗(v/v)在4℃条件下避光孵育30分钟。最后用流式细胞仪收集细胞，检测细胞表面结合的荧光抗体。最后用FlowJo分析原始数据得到MFI值，并用GraphPad Prism软件进行四参数拟合曲线得出IC50值。
如图5所示，药物组合物和COM701能有效阻断hCD112R蛋白与CHO CD112细胞的结合，而阴性对照抗体则无相应功能。药物组合物阻断hCD112R蛋白与CHO CD112 1D2细胞结合的IC50为62.2ng/ml，COM701活性是84.7ng/ml，二者活性水平相当。
实施例10：药物组合物在CD112R/CD112报告基因检测系统中的活性
本实验通过人CD112R/CD112荧光素酶报告基因系统，检测了药物组合物和对照抗体COM701(阳性对照)以及anti-KLH-hIgG4(阴性对照)阻断hCD112R/hCD112抑制通路并激活NFAT的细胞生物学活性。首先用靶细胞铺板培养基将表达人CD112靶细胞CHO CD112 1D2细胞在96孔白色平底板中按每个孔50000个细胞数铺板过夜；第二天吸尽细胞孔中的培养基后，将不同浓度的药物组合物(处方编号FS3-2，抗体为Hu52)或者对照抗体(浓度从0.15ng/ml到3μg/ml，3倍稀释)加入靶细胞中并在37℃孵育30分钟。然后将表达人CD112R的效应细胞Jurkat CD112R按照每孔100000个细胞数加入细胞板中，并在37℃培养箱中共孵育6小时；最后在细胞抗体混合体系中加入ONE-Lite Luciferase Assay System并用多功能酶标仪检测化学发光信号。通过GraphPad prism软件拟合四参数回归曲线，计算EC50值。
NFAT是T细胞受体激活下游的一个重要转录因子，对于IL-2分泌及T细胞增殖应答 至关重要。本实验利用荧光素酶报告基因法研究了药物组合物阻断CD112R/CD112信号通路，活化T细胞并激活NFAT信号的能力。实验结果如图6所示，药物组合物及阳性对照COM701均能够有效阻断CD112R/CD112的相互作用，促进T细胞激活，EC50值为分别为14.2ng/ml和104.2ng/ml，抗体Hu52活性显著高于阳性对照。
实施例11：抗CD112R抗体、抗TIGIT抗体及其联用在人黑色素瘤A375混合PBMC皮下移植瘤模型上的药效学评价
实验动物：48只NCG小鼠，雌性，6-8周龄，体重约18-22g。购自江苏集萃药康生物科技有限公司。
抗TIGIT抗体(JS006)：来源于PCT申请号PCT/CN2020/101883中抗TIGIT人源化抗体hu20。
抗CD112R抗体为本文所述的人源化抗体hu52。本实施例抗CD112R人源化抗体按处方FS3-2的配方配制。
A375细胞培养在含10％胎牛血清(FBS)的DMEM培养液中。收集对数生长期的A375细胞，HBSS(Hank′s平衡盐溶液)重悬至适合浓度用于NCG小鼠皮下肿瘤接种。共培养实验中用的A375细胞用Mitomycin C(丝裂霉素C)处理2h，然后PBS洗三次。取冻存的PBMC，复苏计数，将得到的PBMC加入经Mitomycin C处理的A375细胞中，PBMC与A375共培养5天，培养液为含IL-2和10％FBS的RPMI 1640培养液。PBMC与A375共培养5天后，收取PBMC与新鲜消化下来的A375细胞，PBMC 4×105个，A375细胞4×106个，0.2ml/只(含50％Matrigel(基质胶))，接种于NCG小鼠右侧皮下。接种当天为第0天，根据小鼠体重随机进行分组给药，详细的给药方法、给药剂量和给药途径见表31。
表31.给药方案
注：N：使用动物数量；i.p.：腹腔注射；Q3D：每三天给药一次。给药体积：根据荷瘤鼠体重调整给药体积(0.1mL/10g)。
实验开始后，每周测量2次肿瘤体积和小鼠体重，实验结果见表32和图7。
表32.各组小鼠肿瘤平均体积(Mean±SEM)
注：p＜0.05即认为具有显著性差异；T/C(％)为相对肿瘤增殖率，即在某一时间点，给药组相对于对照组的肿瘤体积的百分比值，T和C分别为给药组和对照组在某一特定时间点的肿瘤体积(TV)；肿瘤生长抑制率TGI(％)＝(1-T/C)×100％。
实验结束时(给药第25天后)，与对照组相比，抗CD112R抗体hu52在30mg/kg的剂量水平，显著抑制了肿瘤生长，肿瘤生长抑制率为37.68％(p＜0.05)；30mg/kg的抗CD112R抗体hu52联合30mg/kg的JS006表现显著的协同抗肿瘤作用，肿瘤生长抑制率为58.30％(p＜0.01)。

Claims (10)

  1. 一种药物组合物，包含：
    (1)缓冲液；和
    (2)抗CD112R抗体或其抗原结合片段；
    其中，所述抗CD112R抗体或其抗原结合片段包含的LCDR1、LCDR2、LCDR3、HCDR1、HCDR2和HCDR3分别为：
    LCDR1：KASQSVSNDVA(SEQ ID NO：1)；
    LCDR2：YVSX1RFT，其中，X1为H或N；
    LCDR3：X2QAYRSPWT，其中，X2为H或Q；
    HCDR1：SYHMS(SEQ ID NO：6)；
    HCDR2：AIX3X4X5GX6X7TYYX8DTVKG，其中，X3为S或N，X4为R或S，X5为D或N，X6为D或I，X7为S或N，X8为P或L；
    HCDR3：HEDYX9GFAMDX10，X9为F或Y，X10为S或Y；
    优选地，所述抗CD112R抗体或其抗原结合片段的浓度为约1～100mg/mL，优选为约10～70mg/mL，更优选为约20～60mg/mL；
    优选地，所述药物组合物的pH为约4.5～6.5，优选为约5.5～6.5；
    优选地，所述药物组合物的渗透压为约250～350mOsm/kg；
    优选地，所述药物组合物经静脉注射施用。
  2. 如权利要求1所述的药物组合物，其中所述抗CD112R抗体或其抗原结合片段包含：
    氨基酸序列如SEQ ID NO：1所示的LCDR1；
    氨基酸序列如SEQ ID NO：2或4所示的LCDR2；
    氨基酸序列如SEQ ID NO：3或5所示的LCDR3；
    氨基酸序列如SEQ ID NO：6所示的HCDR1；
    氨基酸序列如SEQ ID NO：7或9所示的HCDR2；
    氨基酸序列如SEQ ID NO：8或10所示的HCDR3；
    优选地，所述抗CD112R抗体或其抗原结合片段包含：
    (1)氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的HCDR1、HCDR2和HCDR3；或
    (2)氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：2和SEQ ID NO：3所示的LCDR1、 LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3；或
    (3)氨基酸序列分别如SEQ ID NO：1、SEQ ID NO：4和SEQ ID NO：5所示的LCDR1、LCDR2和LCDR3，和氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3；
    优选地，所述抗CD112R抗体或其抗原结合片段选自鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、人源化抗体或其抗原结合片段，优选为人源化抗体或其抗原结合片段。
  3. 如权利要求1或2所述的药物组合物，其中所述抗CD112R抗体或其抗原结合片段包含：
    (1)氨基酸序列如SEQ ID NO：11所示的轻链可变区，和氨基酸序列如SEQ ID NO：12所示的重链可变区；或
    (2)氨基酸序列如SEQ ID NO：11所示的轻链可变区，和氨基酸序列如SEQ ID NO：13所示的重链可变区；或
    (3)氨基酸序列如SEQ ID NO：14所示的轻链可变区，和氨基酸序列如SEQ ID NO：15所示的重链可变区；
    优选地，所述抗CD112R抗体包含：
    (1)氨基酸序列如SEQ ID NO：16所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO：17所示的重链氨基酸序列；
    (2)氨基酸序列如SEQ ID NO：16所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO：18所示的重链氨基酸序列；
    (3)氨基酸序列如SEQ ID NO：19所示的轻链氨基酸序列，和氨基酸序列如SEQ ID NO：20所示的重链氨基酸序列。
  4. 如权利要求1-3中任一项所述的药物组合物，其中所述缓冲液选自醋酸缓冲液、柠檬酸缓冲液、磷酸盐缓冲液和组氨酸缓冲液中的一种或多种；优选地，所述缓冲液为组氨酸缓冲液，优选地，所述组氨酸缓冲液为组氨酸-组氨酸盐酸盐缓冲液或组氨酸-组氨酸醋酸盐缓冲液，优选为组氨酸-组氨酸盐酸盐缓冲液；优选地，所述缓冲液的浓度为约5～100mM，优选为约10～50mM，优选为约10～30mM；优选地，所述缓冲液的pH为约4.5～6.5，优选为约5.5～6.5。
  5. 如权利要求1-4中任一项所述的药物组合物，其中所述药物组合物还包括稳定剂，所述稳定剂选自精氨酸、精氨酸盐、氯化钠、甘露醇、山梨醇、蔗糖和海藻糖中的一种或多种；优选地，所述精氨酸盐为盐酸精氨酸；优选地，所述稳定剂的浓度为约100～300mM， 优选为约150～270mM，更优选为约180～260mM。
  6. 如权利要求5所述的药物组合物，其中，所述稳定剂为浓度约150～270mM的海藻糖、浓度约150～270mM的蔗糖、浓度约20～80mM的氯化钠与浓度约110-170mM的蔗糖的组合、或、浓度约20～80mM的盐酸精氨酸与浓度约110-170mM的蔗糖的组合；
    优选地，所述稳定剂为浓度约180～260mM、优选约220～240mM的海藻糖，浓度约180～260mM、优选约220～240mM的蔗糖，浓度约30～70mM、优选约40～60mM的氯化钠与浓度约120-160mM、优选约130～150mM的蔗糖的组合，或浓度约30～70mM、优选约40～60mM的盐酸精氨酸与浓度约120-160mM、优选约130～150mM的蔗糖的组合。
  7. 如权利要求1-6中任一项所述的药物组合物，其中所述药物组合物还包括表面活性剂，所述表面活性剂选自聚山梨酯80、聚山梨酯20和泊洛沙姆188中的一种或多种；优选地，以w/v计算，所述表面活性剂浓度为约0.01％～0.1％，优选为约0.02～0.06％，更优选为约0.04％～0.06％。
  8. 如权利要求1-7中任一项所述的药物组合物，其分别包含如下(1)～(14)中任一项所示的组分：
    (1)(a)约10～70mg/mL的所述抗CD112R抗体或其抗原结合片段；(b)约10～30mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5；(c)150～270mM的海藻糖；以及(d)约0.01％～0.1％(w/v)的聚山梨酯80；或
    (2)(a)约10～70mg/mL的所述抗CD112R抗体或其抗原结合片段；(b)约10～30mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5；(c)150～270mM的蔗糖；以及(d)约0.01％～0.1％(w/v)的聚山梨酯80；或
    (3)(a)约10～70mg/mL的所述抗CD112R抗体或其抗原结合片段；(b)约10～30mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5；(c)稳定剂，所述稳定剂为浓度约20～80mM的盐酸精氨酸与浓度约110-170mM的蔗糖的组合；以及(d)约0.01％～0.1％(w/v)的聚山梨酯80；或
    (4)(a)约10～70mg/mL的所述抗CD112R抗体或其抗原结合片段；(b)约10～30mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5；(c)稳定剂，所述稳定剂为浓度约20～80mM的氯化钠与浓度约110-170mM的蔗糖的组合；以及(d)约0.01％～0.1％(w/v)的聚山梨酯80；或
    (5)(a)约20～60mg/mL的所述抗CD112R抗体或其抗原结合片段；(b)约15～25mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5；(c)180～260mM的海藻糖；以及(d)约0.04％～0.06％(w/v)的聚山梨酯80；或
    (6)(a)约20～60mg/mL的所述抗CD112R抗体或其抗原结合片段；(b)约15～25mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5；(c)180～260mM的蔗糖；以及(d)约0.04％～0.06％(w/v)的聚山梨酯80；或
    (7)(a)约20～60mg/mL的所述抗CD112R抗体或其抗原结合片段；(b)约15～25mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5；(c)稳定剂，所述稳定剂为浓度约30～70mM的盐酸精氨酸与浓度约120-160mM的蔗糖的组合；以及(d)约0.04％～0.06％(w/v)的聚山梨酯80；或
    (8)(a)约20～60mg/mL的所述抗CD112R抗体或其抗原结合片段；(b)约15～25mM组氨酸缓冲液或醋酸缓冲液，pH为约5.5～6.5；(c)稳定剂，所述稳定剂为浓度约30～70mM的氯化钠与浓度约120-160mM的蔗糖的组合；以及(d)约0.04％～0.06％(w/v)的聚山梨酯80；或
    (9)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)约230mM的海藻糖；以及(d)约0.02％(w/v)的聚山梨酯80；或
    (10)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)约230mM的蔗糖；以及(d)约0.02％(w/v)的聚山梨酯80；或
    (11)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)约230mM的蔗糖；以及(d)约0.04％(w/v)的聚山梨酯80；或
    (12)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)约230mM的蔗糖；以及(d)约0.06％(w/v)的聚山梨酯80；或
    (13)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)稳定剂，所述稳定剂为浓度约50mM的盐酸精氨酸与浓度约140mM的蔗糖的组合；以及(d)约0.02％(w/v)的聚山梨酯80；或
    (14)(a)约40mg/mL的抗CD112R抗体，优选地，所述抗CD112R抗体包含如SEQ ID NO：16所示的轻链氨基酸序列和如SEQ ID NO：18所示的重链氨基酸序列；(b)约20mM、pH为约6.0的组氨酸缓冲液，或约20mM、pH约为5.5的醋酸缓冲液；(c)稳定剂，所述稳定剂为浓度约50mM的氯化钠与浓度约140mM的蔗糖的组合；以及(d)约0.02％(w/v)的聚山梨酯80。
  9. 一种注射剂，其含有如权利要求1-8中任一项所述的药物组合物与氯化钠溶液或葡萄糖溶液；优选地，所述氯化钠溶液浓度为约0.85～0.9％(w/v)；优选地，所述葡萄糖溶液浓度为约5～25％(w/v)，优选为约5～10％(w/v)。
  10. 如权利要求1-8中任一项所述的药物组合物或如权利要求9所述的注射剂在制备用于治疗和/或预防CD112R介导的疾病或病症的药物中的用途；优选地，所述疾病或病症为癌症。
PCT/CN2023/111228 2022-08-05 2023-08-04 抗cd112r抗体药物组合物及其用途 WO2024027824A1 (zh)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200040081A1 (en) * 2018-07-20 2020-02-06 Surface Oncology, Inc. Anti-CD112R Compositions and Methods
WO2021097294A1 (en) * 2019-11-15 2021-05-20 Surface Oncology, Inc. Compositions and methods for immunotherapy
WO2021180205A1 (zh) * 2020-03-13 2021-09-16 江苏恒瑞医药股份有限公司 Pvrig结合蛋白及其医药用途
CN114414808A (zh) * 2021-12-07 2022-04-29 上海药明生物医药有限公司 一种检测tigit抗体和pvrig抗体的协同生物学活性的方法
CN114615993A (zh) * 2019-07-29 2022-06-10 康姆普根有限公司 抗pvrig抗体制剂及其用途
CN114644711A (zh) * 2022-03-07 2022-06-21 南京诺艾新生物技术有限公司 重组抗人pvrig抗体及应用
CN114907479A (zh) * 2021-02-09 2022-08-16 上海君实生物医药科技股份有限公司 抗cd112r抗体及其用途

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200040081A1 (en) * 2018-07-20 2020-02-06 Surface Oncology, Inc. Anti-CD112R Compositions and Methods
CN114615993A (zh) * 2019-07-29 2022-06-10 康姆普根有限公司 抗pvrig抗体制剂及其用途
WO2021097294A1 (en) * 2019-11-15 2021-05-20 Surface Oncology, Inc. Compositions and methods for immunotherapy
WO2021180205A1 (zh) * 2020-03-13 2021-09-16 江苏恒瑞医药股份有限公司 Pvrig结合蛋白及其医药用途
CN114907479A (zh) * 2021-02-09 2022-08-16 上海君实生物医药科技股份有限公司 抗cd112r抗体及其用途
CN114414808A (zh) * 2021-12-07 2022-04-29 上海药明生物医药有限公司 一种检测tigit抗体和pvrig抗体的协同生物学活性的方法
CN114644711A (zh) * 2022-03-07 2022-06-21 南京诺艾新生物技术有限公司 重组抗人pvrig抗体及应用

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