WO2023125483A1 - 一种抗tnfr2抗体药物组合物 - Google Patents

一种抗tnfr2抗体药物组合物 Download PDF

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WO2023125483A1
WO2023125483A1 PCT/CN2022/142190 CN2022142190W WO2023125483A1 WO 2023125483 A1 WO2023125483 A1 WO 2023125483A1 CN 2022142190 W CN2022142190 W CN 2022142190W WO 2023125483 A1 WO2023125483 A1 WO 2023125483A1
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pharmaceutical composition
seq
concentration
composition according
antibody
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PCT/CN2022/142190
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English (en)
French (fr)
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李鑫鑫
马飞
胡建中
赵晓峰
唐任宏
任晋生
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山东先声生物制药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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

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  • the invention relates to the field of pharmaceutical preparations, in particular to a pharmaceutical composition comprising TNFR2 antibody or an antigen-binding fragment thereof.
  • the preparations of macromolecular protein drugs are usually injections, which are suitable for parenteral administration, and the administration methods usually include subcutaneous, intravenous, intramuscular, etc. Since proteins are relatively unstable in solution, they are very easy to form particles and aggregates, etc. Stability has become a difficult problem in the development of macromolecular protein drugs. Therefore, the development of liquid formulations of macromolecular protein drugs (such as antibodies, etc.) first needs to solve the problem of protein stability.
  • the present invention provides an anti-TNFR2 antibody pharmaceutical composition.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-TNFR2 antibody or an antigen-binding fragment thereof, a buffer, a protein stabilizer and a surfactant.
  • the anti-TNFR2 antibody or antigen-binding fragment thereof comprises:
  • CDR1-VH is selected from SEQ ID NO.1, 7, or 13;
  • CDR2-VH is selected from SEQ ID NO.2, 8, or 14;
  • CDR3-VH is selected from SEQ ID NO.3, 9, or 15;
  • CDR1-VL is selected from SEQ ID NO. 4, 10, or 16;
  • CDR2-VL is selected from SEQ ID NO. 5, 11, or 17;
  • CDR3-VL is selected from SEQ ID NO. 6, 12, or 18.
  • the anti-TNFR2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region:
  • the heavy chain variable region comprises:
  • the light chain variable region comprises:
  • amino acid insertion, deletion and/or substitution occurs in the heavy chain variable region or/and the FR region of the light chain variable region;
  • the substitution is a conservative amino acid substitution.
  • said heavy chain variable region and light chain variable region are selected from the group consisting of:
  • CDRs are completely consistent with any sequence combination of (1) to (8) above, and FRs have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or 100% sequence identity of VH and VL combinations;
  • amino acid insertion, deletion and/or substitution occurs in the heavy chain variable region or/and the FR region of the light chain variable region;
  • the substitution is a conservative amino acid substitution.
  • the anti-TNFR2 antibody or antigen-binding fragment thereof is selected from monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies) ), monovalent antibody, multivalent antibody, full-length antibody, antibody fragment, Fab, Fab', F(ab')2, Fd, Fv, scFv, or diabody.
  • the concentration of the anti-TNFR2 antibody is 1 mg/mL to 100 mg/mL, preferably 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg /mL, 90mg/mL, more preferably 30mg/mL.
  • the buffer is selected from acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, histidine-histidine hydrochloride buffer or disodium hydrogen phosphate-diphosphate Sodium hydrogen buffer, preferably histidine-histidine hydrochloride buffer.
  • the concentration of the buffer is 10-100 mM, preferably 10-60 mM, more preferably 20 mM.
  • the protein stabilizer is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose or sorbitol, preferably sorbitol.
  • the concentration of the protein stabilizer is 1-10% (w/v), preferably 2-8% (w/v), more preferably 4.7% (w/v).
  • the surfactant is selected from polysorbate 80, poloxamer or glycerin fatty acid ester, preferably polysorbate 80.
  • the concentration of the surfactant is 0.02-0.1% (w/w), preferably 0.02% (w/w), 0.04% (w/w), 0.06% (w/w) w), 0.08% (w/w), 0.1% (w/w), more preferably 0.04% (w/w).
  • the pH value of the pharmaceutical composition is 4.5-7.5, preferably 4.5, 5, 5.5, 6, 6.5, 7, 7.5, more preferably 5.5.
  • the present disclosure provides a pharmaceutical composition comprising:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition is in the form of a liquid preparation.
  • the disclosure of the present invention provides a method for preparing the pharmaceutical composition as described in the first and second aspects, including the step of exchanging the anti-TNFR2 antibody solution with a buffer, and the buffer is preferably histidine Acid-histidine hydrochloride buffer solution, the concentration of the buffer solution is preferably 20 mM, and the pH of the buffer solution is preferably 5.5.
  • the buffer is preferably histidine Acid-histidine hydrochloride buffer solution
  • the concentration of the buffer solution is preferably 20 mM
  • the pH of the buffer solution is preferably 5.5.
  • the present disclosure provides a pharmaceutical composition of an anti-TNFR2 antibody or an antigen-binding fragment thereof, which is prepared by the method described in the third aspect.
  • the present disclosure provides the pharmaceutical composition described in the first to second aspects, the product prepared by the method described in the third aspect, or the pharmaceutical composition described in the fourth aspect in the preparation of therapeutic and /or use in medicines for preventing immune abnormality-related diseases; preferably, the immune abnormality-related diseases are Treg cell and/or MDSC function-related diseases; preferably, the diseases are cancer or autoimmune diseases; more preferably , the cancer is selected from ovarian cancer, advanced epidermal T-cell lymphoma, stage III/IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer, and/or on CTLA-4 and PD-1 Advanced solid tumors resistant to therapy, such as metastatic melanoma; more preferably, the autoimmune disease may be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis optica spectrum disorder, systemic Lupus erythematosus, myasthenia gravis, IgG4-related diseases.
  • the disclosure of the present invention provides a method for treating and/or preventing immune abnormality-related diseases, the method comprising administering to a subject an effective amount of the pharmaceutical composition described in the first to second aspects, the first The product prepared by the method described in the three aspects, or the pharmaceutical composition described in the fourth aspect;
  • the immune abnormality-related disease is a disease related to Treg cell and/or MDSC function;
  • the disease is cancer or Autoimmune disease; more preferably, the cancer is selected from ovarian cancer, advanced epidermal T-cell lymphoma, stage III/IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer, and/or Advanced solid tumors resistant to CTLA-4 and PD-1 therapy, such as metastatic melanoma;
  • the autoimmune disease can be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis , neuromyelitis optica spectrum disease, systemic lupus erythemat
  • the TNFR2 antibody preparation of the present invention can maintain the stability of the anti-TNFR2 antibody during long-term storage and transportation of the product, thereby ensuring the stability, safety and effectiveness of the drug.
  • Figure 1 shows the trend chart of the CEX-HPLC results summary of samples placed at 2-8°C and 25°C in the stability of prescription confirmation
  • Figure 2 shows the trend chart of the SE-HPLC results of the samples placed at 2-8°C and 25°C in the stability of the prescription confirmation
  • Fig. 3 shows the trend chart of the CE-SDS (NR) result summary of samples placed at 2-8°C and 25°C in the stability of prescription confirmation;
  • Fig. 4 shows the trend chart of the CE-SDS(R) result summary of the sample placed at 2-8°C and 25°C in the stability of the formulation confirmation.
  • the invention discloses an anti-TNFR2 antibody pharmaceutical composition, which can be realized by appropriately improving process parameters for those skilled in the art by referring to the contents herein. What needs to be pointed out in particular is that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.
  • the raw materials and reagents used in the anti-TNFR2 antibody pharmaceutical composition provided by the present invention can be purchased from the market.
  • TNFR2 refers to Tumor Necrosis Factor Receptor 2, also known as Tumor Necrosis Factor Receptor Superfamily Member 1B (TNFRSF1B) or CD120b, a membrane-binding tumor necrosis factor- ⁇ (TNF ⁇ ) receptor.
  • TNFRSF1B Tumor Necrosis Factor Receptor Superfamily Member 1B
  • CD120b a membrane-binding tumor necrosis factor- ⁇ receptor.
  • Said TNFR2 is preferably human TNFR2.
  • anti-tumor necrosis factor receptor 2 antibody refers to any immunoglobulin molecule comprising at least a part (such as but not limited to at least one complementarity determining region (CDR) of a heavy chain or a light chain or a ligand-binding portion thereof, a heavy chain or light chain variable region, heavy or light chain constant region, framework region or any portion thereof) protein or peptide-containing molecule.
  • CDR complementarity determining region
  • TNFR2 antibodies also include antibody-like protein scaffolds such as the tenth fibronectin type III domain (10Fn3), which contain BC, DE and FG structural loops similar in structure and solvent accessibility to antibody CDRs.
  • the tertiary structure of the 10Fn3 domain is similar to the tertiary structure of the IgG heavy chain variable region, and by replacing the residues of the BC, DE and FG loops of 10Fn3 with CDR-H1, CDR-H2 or CDR from TNFR2 monoclonal antibody - Residue replacement in the H3 region, those skilled in the art can graft the CDR of the TNFR2 monoclonal antibody onto the fibronectin scaffold.
  • antibody refers to an immunoglobulin molecule that specifically binds to or is immunoreactive with an antigen of interest, including polyclonal, monoclonal, genetically engineered, and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (e.g. bispecific, trispecific and tetraspecific antibodies, diabodies, triabodies and tetrabodies), antibody conjugates) As well as antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments).
  • mAb monoclonal antibody
  • mAb monoclonal antibody
  • the term "monoclonal antibody” refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone), without limitation by the method by which the antibody was produced.
  • the terms "antigen-binding fragment” and “antibody fragment” are interchangeable and refer to one or more antibody fragments that retain the ability to specifically bind a target antigen.
  • the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • the antibody fragment may be a Fab, F(ab')2, scFv, SMIP, diabody, triabody, affibody, Nanobody, aptamer or domain antibody.
  • binding fragments encompassing the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab)2 Fragment, a bivalent fragment comprising two Fab fragments connected at the hinge region by disulfide bonds; (iii) Fd fragment consisting of VH and CH1 domains; (iv) VL and VH domains consisting of a single arm of the antibody (V) dAb comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (Ward et al., Nature 341:544-546,1989); (vii) consisting of VH or VL (viii) an isolated complementarity determining region (CDR); and (ix) a combination of two or more isolated CDRs, which CDRs may optionally be joined by a synthetic linker.
  • a Fab fragment a mono
  • the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be joined using recombinant methods through a linker that enables them to be made in which the VL and VH regions pair to form A single protein chain of a monovalent molecule (termed a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 ,1988).
  • scFv single-chain Fv
  • These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as whole antibodies.
  • Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.
  • CDRs complementarity determining regions
  • FR framework regions
  • amino acid positions representing the hypervariable regions of an antibody may vary according to the context and various definitions known in the art. Some positions within variable domains can be considered heterozygous hypervariable positions because these positions can be considered to be within a hypervariable region under one set of criteria (such as IMGT or KABAT) but under a different set Outside the hypervariable region under the standard (such as KABAT or IMGT). One or more of these positions may also be found in extended hypervariable regions.
  • variable domains of the native heavy and light chains each comprise four framework regions predominantly in a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops connecting the sheets , and in some cases form part of the lamellar structure.
  • CDR1, CDR2, and CDR3 CDR1, CDR2, and CDR3 that form loops connecting the sheets , and in some cases form part of the lamellar structure.
  • the CDRs in each chain are held tightly together by the FR regions in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and together with CDRs from other antibody chains contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Protein of Immunological Interest, National Institute of Health, Bethesda, Md. 1987; which is incorporated herein by reference).
  • CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR of the heavy chain variable region (VH), respectively, and these three CDRs constitute the CDR combination (VHCDR combination) of the chain (or its variable region);
  • CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and second CDR of the light chain variable region (VL), respectively
  • Kabat numbering system generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service , National Institutes of Health, Bethesda, Md., 1991).
  • VH refers to the variable region of an immunoglobulin heavy chain of an antibody, including that of an Fv, scFv or Fab.
  • VL refers to the variable region of an immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv or Fab).
  • heavy chain constant region herein refers to the carboxy-terminal portion of the heavy chain of an antibody that is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's available Variable domains have more conserved amino acid sequences.
  • the "heavy chain constant region” at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof.
  • Heavy chain constant region includes "full-length heavy chain constant region” and “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “full-length heavy chain constant region” part".
  • a typical "full-length antibody heavy chain constant region” consists of a CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain In the case of an antibody, it does not include a CH1 domain.
  • a typical "heavy chain constant region segment" can be selected from CH1, Fc or CH3 domains.
  • light chain constant region refers to the carboxy-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and the light chain constant region can be selected from a constant kappa domain or a constant lambda domain.
  • percent (%) sequence identity and “percent (%) sequence identity” are interchangeable and refer to the sequence after aligning sequences and introducing gaps (if necessary) to achieve the maximum percent sequence identity ( For example, for optimal alignment, gaps may be introduced in one or both of the candidate and reference sequences, and non-homologous sequences may be ignored for comparison purposes), after the amino acid (or nucleotide) of the candidate sequence ) residues are identical to the amino acid (or nucleotide) residues of the reference sequence.
  • alignment can be achieved in a number of ways well known to those skilled in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAi) software.
  • a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50% to 100% sequence identity.
  • the length of a candidate sequence aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%) of the length of the reference sequence .
  • amino acid herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:
  • Acidic amino acids Asp(D) and Glu(E);
  • Non-polar uncharged amino acids Cys (C), Met (M) and Pro (P);
  • Aromatic amino acids Phe (F), Tyr (Y) and Trp (W).
  • the term "specific binding” refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, such as those detected by antibodies or their antigens Binding fragment-specific recognition.
  • An antibody or antigen-binding fragment thereof that specifically binds an antigen will bind the antigen with a KD of less than 100 nM.
  • an antibody or antigen-binding fragment thereof that specifically binds an antigen will bind the antigen with a KD of up to 100 nM (eg, between 1 pM and 100 nM).
  • An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a K for that particular antigen or epitope thereof of greater than 100 nM (e.g., greater than 500 nM, 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, or 1 mM).
  • Antibodies that are specifically immunoreactive with a particular protein or carbohydrate can be selected using a variety of immunoassay formats. For example, solid-phase ELISA immunoassays are routinely used to select for antibodies that are specifically immunoreactive with proteins or carbohydrates.
  • pharmaceutical composition means a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components such as physiological/pharmaceutical Pharmaceutically acceptable carriers and excipients.
  • the purpose of the pharmaceutical composition is to maintain the stability of the active ingredient of the antibody, promote the administration to the organism, and facilitate the absorption of the active ingredient to exert biological activity.
  • pharmaceutical composition and “formulation” are not mutually exclusive.
  • CDRs transplantation is used for antibody humanization, that is, based on the sequence selection of the most homologous human antibody to provide antibody framework regions (FRs), the antigen-binding fragment complementarity-determining regions (CDRs) based on the Kabat nomenclature in the target antibody ), transplanted to the former to form the humanized antibody variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • FRs antibody framework regions
  • CDRs antigen-binding fragment complementarity-determining regions
  • CDRs complementarity-determining regions
  • the humanized anti-TNFR2 antibody was expressed using CHO as the host cell, and cultured in Dynamis AGT medium (manufacturer: gibco, article number: A26615-01), the culture period did not exceed 15 days, and the reactor control parameters were pH 6.8-7.6 , dissolved oxygen (DO) is 20% to 80%, the rotation speed is 79RPM to 81RPM, and the initial culture temperature is 36.0°C to 37.0°C; when culturing to the 8th day, reduce the culture temperature to 32.0 ⁇ 0.5°C until harvest.
  • Dynamis AGT medium manufactured by Gibco, article number: A26615-01
  • the purification of humanized anti-TNFR2 antibody adopts multi-step chromatography, concentration and filtration unit operations to purify the antibody sequentially.
  • the supernatant harvest was captured by Protein A affinity chromatography (Mabselect PrismA).
  • the captured antibody solution is incubated with low pH to inactivate potential viruses.
  • the antibody solution was neutralized and the precipitate was removed by depth filtration.
  • the antibody solution was concentrated with an ultrafiltration membrane bag, and the buffer was replaced to complete purification and obtain a humanized anti-TNFR2 antibody protein stock solution.
  • Embodiment 2 buffer system screening
  • the experiment covered 4 kinds of buffer salts (acetate, citrate, histidine salt and phosphate), with pH values ranging from 4.5 to 7.5, and a total of 12 preparation buffer systems were set up for screening (see Table 4).
  • the protein stock solution obtained in Example 1 was subjected to ultrafiltration concentration and liquid replacement treatment, and the stock solution was replaced into the corresponding 12 preparation buffer systems: the protein stock solution was added to the corresponding ultrafiltration tubes in 12 groups, and ultrafiltration concentration was performed using a centrifuge , the protein is intercepted, the buffer solution of the protein stock solution flows through the ultrafiltration membrane to achieve the purpose of protein concentration, and then the corresponding 12 preparation buffer solutions are added respectively, the buffer solution of the original protein stock solution is diluted, and the ultrafiltration concentration is continued, and the concentration is completed Then continue to add the corresponding 12 preparation buffer solutions respectively, continue ultrafiltration and concentration, and repeat the operation until the replacement of the liquid is completed.
  • buffer salts acetate, citrate, histidine salt and phosphate
  • the humanized TNFR2 antibody #2-1 described in Example 1 was used in the above formulations, and the protein concentration was 30 mg/mL.
  • SE-HPLC size exclusion chromatography
  • CE-SDS non-reducing sodium dodecyl sulfate capillary electrophoresis
  • CEX-HPLC charge isomerism
  • DLS dynamic light scattering
  • DFS thermal stability
  • X appearance, pH value, protein concentration, CEX-HPLC, SE-HPLC, CE-SDS(NR), DLS, DSF(T0)
  • Tm protein melting temperature
  • DLS particle size ranking (from small to large): F7, F8, F9>F3, F10, F11, F12>F1>F6, F2>F5, F4;
  • SE-HPLC Purity ranking F7>F8>F2, F9, F3>F1, F5, F6>F10>F11, F4>F12;
  • CE-SDS(NR) Ranking of main peak purity F8, F9, F6, F7, F3>F10>F11, F5>F2>F12, F4>F1;
  • CEX-HPLC main peak purity F8>F7, F9, F6>F3>F2, F1, F5, F10>F4>F11>F12;
  • the acid peak increases, and the order of pros and cons is F7, F1>F8, F2>F3, F4, F5, F6, F9>F10>F11>F12;
  • the pros and cons of each prescription are ranked as follows: F7>F8>F9, F3>F2>F1>F4, F5, F6, F10, F11, F12.
  • the F7 formulation is superior to other formulations in terms of purity, degradation, aggregation, and charge heterogeneity in this 40°C accelerated experiment, showing good stability. Therefore, the F7 prescription (20mM histidine-histidine hydrochloride, pH5.5) was preliminarily selected as the optimal prescription as the preparation buffer system of this product for the next step of excipient screening research.
  • the optimal buffer system including sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol, and sorbitol, a total of 7 prescriptions,
  • the protein concentration was 30 mg/mL.
  • X appearance, pH value, protein concentration, CEX-HPLC, SE-HPLC, CE-SDS(NR), DLS, DSF(T0), osmotic pressure(T0)
  • the sample concentration was 30mg/mL, and five freeze-thaw experiments from -20°C to 25°C and excipient screening at 40°C for 4 weeks were carried out.
  • Appearance is yellowish, micro-opalescent liquid, pH and concentration are within the target value, no significant change;
  • DSF Thermal stability test
  • Tm1 and Tm2 protein melting temperature: F7-4, F7-5>F7-2, F7-6, F7-7, without excipients (F7)>F7-1, F7 -3;
  • DLS 4 weeks at 40°C, the particle size of F7-1 increased significantly compared to T0; the particle size of F7-2 and F7-6 increased significantly compared to T0 after freezing and thawing for 5 times;
  • SE-HPLC 4 weeks at 40°C, purity F7-2, F7-3, F7-4, F7-5, F7-6, F7-7>F7-1; freeze-thaw 5 times, purity F7-1, F7 -3, F7-4, F7-5, F7-7>F7-2>F7-6;
  • CE-SDS(NR) 4 weeks at 40°C, CE(NR) main peak purity F7-7, F7-5, F7-6, F7-4, F7-2>F7-1, F7-3; freeze-thaw 5 no significant change;
  • CEX-HPLC investigation at 40°C for 4 weeks, main peak purity F7-3>F7-1, F7-6, F7-7>F7-2, F7-4, F7-5; acidic peak increased, ranking F7-1 , F7-3>F7-6, F7-7>F7-4, F7-5, F7-2; there was no significant difference among the prescriptions after freezing and thawing for 5 times.
  • the F7-7 prescription has the highest performance in terms of purity, degradation, aggregation, charge heterogeneity, etc. Aspect synthesis is superior to other prescriptions, showing good stability. Therefore, the formulation F7-7 (4.7% sorbitol (W/V), 20mM histidine-histidine hydrochloride, pH5.5) was initially selected as the optimal formulation for further surfactant screening research.
  • Embodiment 4 surfactant screening
  • X appearance, pH value, protein concentration, CEX-HPLC, SE-HPLC, CE-SDS (NR), DLS, insoluble particles (microfluidic imaging (MFI), T0 and investigation endpoint)
  • the sample concentration was 30mg/mL, and the investigation was carried out at 40°C for 4 weeks. Appearance is colorless micro-opalescent liquid; pH, concentration are within the target value, no significant change;
  • SE-HPLC pros and cons of each prescription, F7-7-3, F7-7-5>F7-7-2, F7-7-6>F7-7-4>F7-7-1;
  • CE-SDS(NR) the advantages and disadvantages of each prescription, F7-7-6, F7-7-2>F7-7-4, F7-7-3>F7-7-5, F7-7-1;
  • DLS the advantages and disadvantages of each formulation of particle size, F7-7-1, F7-7-6>F7-7-5>F7-7-2>F7-7-4>F7-7-3;
  • Insoluble particles the advantages and disadvantages of each prescription, F7-7-1 prescription ⁇ 10 ⁇ m particles are always significantly more than other prescriptions; there is no significant difference in other prescriptions;
  • CEX-HPLC The F7-7-3 formulation was slightly better than the other formulations.
  • the screening of excipients passed the evaluation of each stability index: F7-7-3, F7-7-6>F7-7-2, F7-7-4>F7-7-5>F7-7-1, That is, the optimal prescription is F7-7-3 or F7-7-6.
  • the content of polysorbate 80 in prescription F7-7-2 and F7-7-4 is at the upper and lower edges of the content of polysorbate 80 in prescription F7-7-3, and the content of polysorbate 80 in prescription F7-7-5
  • the content of polysorbate 80 in prescription F7-7-6 was on the edge of the content of polysorbate 80 in prescription F7-7-6, while prescriptions F7-7-2 and F7-7-4 were better than prescription F7-7-5.
  • F7-7-3 prescription 0.04% polysorbate 80 (W/W), 20mM histidine-histidine hydrochloride, 4.7% sorbitol, pH5.5
  • the F7-7-3 prescription 0.04% polysorbate 80 (W/W), 20mM histidine-histidine hydrochloride, 4.7% sorbitol (W/V), pH5.5
  • the optimal prescription is prepared, and the next step of the prescription confirmation stability study is prepared, and the final confirmation of this prescription is carried out.
  • Embodiment 5 prescription confirms stability
  • this prescription confirms the stability and packaging material selection research experiment, and determines 1 prescription: 30mg/mL TNFR2 monoclonal antibody (#2-1), 20mM histidine-hydrochloric acid group Amino acid, 4.7% sorbitol (W/V), 0.04% polysorbate 80 (W/W), pH5.5, 5mL/bottle, divided into vials, using rubber stoppers and aluminum-plastic caps to form airtight
  • the packaging system was upright and inverted respectively, and the real-time stability and accelerated stability investigations were carried out for 6 months, and the stability confirmation study was carried out on the final formulation.
  • X appearance, pH value, protein concentration, CEX-HPLC, SE-HPLC, CE-SDS(NR), CE-SDS(R), osmotic pressure (T0), activity, insoluble particles (microfluidic imaging method, MFI)
  • the activity detection method adopts Elisa and Cell assay blocking methods, as follows:
  • Elisa method use the anti-TNFR2 monoclonal antibody to bind to human TNFR2, and detect the half-maximal concentration of the anti-TNFR2 monoclonal antibody on 50ng of TNFR2.
  • Antigen human TNFR2-his
  • Stino Biological, 10417-H08H was pre-coated on the 96-well plate, the anti-TNFR2 monoclonal antibody combined with the antigen on the microtiter plate, the plate was washed to remove various components that did not bind, and then Peroxidase was added -conjugated Mouse anti Human IgG Fc ⁇ (Jackson Immuno, 209-035-098), wash the plate after incubation to remove excess enzyme-labeled antibody; finally add the HRP substrate TMB (Thermo, 34029) to produce a blue product with light and dark colors Proportional to the concentration of anti-TNFR2 monoclonal antibody. After the stop solution is terminated, the blue product turns to yellow, and the OD value
  • Humanized anti-TNFR2 monoclonal antibody is a monoclonal antibody that specifically binds to Human TNFR2, and can inhibit the binding of Human TNF ⁇ to the receptor Human TNFR2.
  • the monoclonal cell line CT26-2A03 overexpressing TNFR2 obtained through self-screening is used as the detection cell.
  • TNF ⁇ -biotin biotin-labeled TNF ⁇
  • AcroBiosystems, TNA-H82E3 Biotin-labeled TNF ⁇
  • the ligand TNF ⁇ interacts with receptor TNFR2
  • PE-labeled Streptavidin Biolegend , 405204
  • the cells are indirectly labeled with PE dye, and a clear positive signal can be seen by microplate reader detection.
  • humanized anti-TNFR2 monoclonal antibody and Human TNFR2 conflict with the binding site of Human TNF ⁇ , it will affect the binding of Human TNF ⁇ and Human TNFR2, and finally affect the intensity of PE signal. For the weakening of PE signal.
  • the function of humanized anti-TNFR2 monoclonal antibody to inhibit the combination of Human TNFR2 and Human TNF ⁇ at the cellular level can be detected.
  • the upright and inverted samples After being stored at low temperature at 2-8°C for 6 months, the upright and inverted samples had no significant changes in routine detection indicators, insoluble particles, purity, charge heterogeneity, activity, etc., and there was no significant difference between the upright and inverted samples.

Abstract

提供了一种TNFR2抗体药物组合物，所述的组合物包括TNFR2抗体或其抗原结合片段、缓冲液、蛋白稳定剂和表面活性剂。该药物组合物能够在产品长期贮存和运输过程中维持TNFR2抗体的稳定性，进而保证药品的稳定性、安全性和有效性。

Description

一种抗TNFR2抗体药物组合物
本申请要求于2021年12月28日提交中国专利局、申请号为202111624712.7、发明名称为“一种抗TNFR2抗体药物组合物”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及药物制剂领域，特别涉及一种包含TNFR2抗体或其抗原结合片段的药物组合物。
背景技术
大分子蛋白类药物(如抗体等)的制剂通常为注射剂，适合于非经肠道给药，其给药方式通常包括皮下、静脉、肌内等。由于蛋白质在溶液中较不稳定，非常容易形成颗粒和聚集等，稳定性成为大分子蛋白类药物开发的一个难题。因此，开发大分子蛋白类药物(如抗体等)的液体制剂首先需要解决蛋白的稳定性问题。
发明内容
有鉴于此，本发明提供一种抗TNFR2抗体药物组合物。
为了实现上述发明目的，本发明提供以下技术方案：
在第一个方面，本发明公开提供了一种药物组合物，所述的药物组合物包含抗TNFR2抗体或其抗原结合片段、缓冲液、蛋白稳定剂和表面活性剂。
在一个实施方案中，所述抗TNFR2抗体或其抗原结合片段包含：
CDR1-VH选自SEQ ID NO.1、7、或13；
CDR2-VH选自SEQ ID NO.2、8、或14；
CDR3-VH选自SEQ ID NO.3、9、或15；
CDR1-VL选自SEQ ID NO.4、10、或16；
CDR2-VL选自SEQ ID NO.5、11、或17；和
CDR3-VL选自SEQ ID NO.6、12、或18。
在一个具体的实施方案中，所述的抗TNFR2抗体或其抗原结合片段包含重链可变区和轻链可变区：
(1)所述重链可变区包含：
a.选自SEQ ID NO：19-23任一所示的VH序列，
b.选自与SEQ ID NO：19-23任一序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH序列，或，
c.选自与SEQ ID NO：19-23任一序列相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸***、缺失和/或替换的VH序列；
(2)所述轻链可变区包含：
a.选自SEQ ID NO：24-28任一所示的VL序列，
b.选自与SEQ ID NO：24-28任一序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VL序列，或，
c.选自与SEQ ID NO：24-28任一序列相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸***、缺失和/或替换的VL序列；
可选地，所述氨基酸***、缺失和/或替换发生在重链可变区或/和轻链可变区的FR区；
可选地，所述替换为保守氨基酸的替换。
在另一个具体的实施方案中，所述重链可变区和轻链可变区选自如下组：
(1)具有SEQ ID NO.19所示的VH和SEQ ID NO.24所示的VL；
(2)具有SEQ ID NO.19所示的VH和SEQ ID NO.25所示的VL；
(3)具有SEQ ID NO.20所示的VH和SEQ ID NO.26所示的VL；
(4)具有SEQ ID NO.21所示的VH和SEQ ID NO.26所示的VL；
(5)具有SEQ ID NO.22所示的VH和SEQ ID NO.27所示的VL；
(6)具有SEQ ID NO.23所示的VH和SEQ ID NO.28所示的VL；
(7)具有与上述(1)～(6)任一序列组合相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合；
(8)具有与上述(1)～(7)任一项序列组合相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸***、缺失和/或替换的VH和VL组合；或
(9)具有与上述(1)～(8)任一项序列组合相比CDR完全一致，FR具有至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合；
可选地，所述氨基酸***、缺失和/或替换发生在重链可变区或/和轻链可变区的FR区；
可选地，所述替换为保守氨基酸的替换。
在一个具体的实施方案中，所述抗TNFR2抗体或其抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、或双抗体(diabody)。
在另一个实施方案中，所述抗TNFR2抗体的浓度为1mg/mL至100mg/mL，优选为20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL，更优选为30mg/mL。
在另一个实施方案中，所述的缓冲液选自醋酸-醋酸钠缓冲液、枸橼酸-枸橼酸钠缓冲液、组氨酸-盐酸组氨酸缓冲液或磷酸氢二钠-磷酸二氢钠缓冲液，优选为组氨酸-盐酸组氨酸缓冲液。
在一个具体的实施方案中，所述的缓冲液浓度为10-100mM，优选为10-60mM，更优选为20mM。
在另一个实施方案中，所述的蛋白稳定剂选自氯化钠、甘氨酸、盐酸精氨酸、蔗糖、海藻糖或山梨醇，优选山梨醇。
在一个具体的实施方案中，所述的蛋白稳定剂的浓度为1-10％(w/v)，优选为2-8％(w/v)，更优选为4.7％(w/v)。
在另一个实施方案中，所述的表面活性剂选自聚山梨酯80、泊洛沙姆或甘油脂肪酸酯，优选为聚山梨酯80。
在一个具体的实施方案中，所述的表面活性剂的浓度为0.02～0.1％(w/w)，优选为0.02％(w/w)、0.04％(w/w)、0.06％(w/w)、0.08％(w/w)、0.1％(w/w)，更优选为0.04％(w/w)。
在另一个实施方案中，所述的药物组合物的pH值为4.5-7.5，优选为4.5、5、5.5、6、6.5、7、7.5，更优选为5.5。
在第二个方面，本发明公开提供了一种药物组合物，所述的药物组合物包含：
a)浓度为1mg/mL至100mg/mL的抗TNFR2抗体，b)浓度为10-100mM的组氨酸-盐酸组氨酸缓冲液，pH值为4.5-7.5，c)浓度为1-10％(w/v)的山梨醇，和d)浓度为0.02～0.1％(w/w)的聚山梨酯80；
优选的，所述的药物组合物包含：
a)浓度为10mg/mL至60mg/mL的抗TNFR2抗体，b)浓度为10-60mM的组氨酸- 盐酸组氨酸缓冲液，pH值为5.0-6.0，c)浓度为2-8％(w/v)的山梨醇，和d)浓度为0.04％(w/w)的聚山梨酯80；
更优选的，所述药物组合物包含：
a)浓度为30mg/mL的抗TNFR2抗体，b)浓度为20mM的组氨酸-盐酸组氨酸缓冲液，pH值为5.5，c)浓度为4.7％(w/v)的山梨醇，和d)浓度为0.04％(w/w)的聚山梨酯80。
在一个具体的实施方案中，所述的药物组合物的形式为液体制剂。
在第三个方面，本发明公开提供了一种制备如第一、二方面所述的药物组合物的方法，包括将抗TNFR2抗体溶液经过缓冲液置换的步骤，所述的缓冲液优选组氨酸-盐酸组氨酸缓冲液，所述缓冲液的浓度优选20mM，所述缓冲液的pH优选为5.5。
在第四个方面，本发明公开提供了一种抗TNFR2抗体或其抗原结合片段的药物组合物，所述药物组合物通过第三个方面所述的方法制备获得。
在第五个方面，本发明公开提供了第一至二个方面所述的药物组合物，第三个方面所述方法制备的产品，或第四个方面所述的药物组合物在制备治疗和/或预防免疫异常相关疾病的药物中的用途；优选地，所述免疫异常相关疾病是Treg细胞和/或MDSC功能相关疾病；优选地，所述疾病是癌症或自身免疫性疾病；更优选地，所述癌症选自卵巢癌、晚期表皮T细胞淋巴瘤、III/IV期转移性结直肠癌、三阴乳腺癌、胰腺癌、非小细胞肺癌、和/或对CTLA-4和PD-1疗法耐药的晚期实体瘤，如转移性黑色素瘤；更优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、***性硬化症、视神经脊髓炎谱系病、***性红斑狼疮、重症肌无力、IgG4相关性疾病。
在第六个方面，本发明公开提供了一种治疗和/或预防免疫异常相关疾病的方法，所述方法包括向受试者施用有效量的第一至二个方面所述药物组合物，第三个方面所述方法制备的产品，或第四个方面所述药物组合物；优选地，所述免疫异常相关疾病是Treg细胞和/或MDSC功能相关疾病；优选地，所述疾病是癌症或自身免疫性疾病；更优选地，所述癌症选自卵巢癌、晚期表皮T细胞淋巴瘤、III/IV期转移性结直肠癌、三阴乳腺癌、胰腺癌、非小细胞肺癌、和/或对CTLA-4和PD-1疗法耐药的晚期实体瘤，如转移性黑色素瘤；更优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、***性硬化症、视神经脊髓炎谱系病、***性红斑狼疮、重症肌无力、IgG4相关性疾病。
有益效果：本发明所述TNFR2抗体制剂可以在产品长期贮存和运输过程中维持抗TNFR2抗体的稳定性，进而保证药品的稳定性、安全性和有效性。
附图说明
图1示处方确认稳定性中样品放置2-8℃和25℃考察CEX-HPLC结果汇总的趋势图；
图2示处方确认稳定性中样品放置2-8℃和25℃考察SE-HPLC结果汇总的趋势图；
图3示处方确认稳定性中样品放置2-8℃和25℃考察CE-SDS(NR)结果汇总的趋势图；
图4示处方确认稳定性中样品放置2-8℃和25℃考察CE-SDS(R)结果汇总的趋势图。
具体实施方式
本发明公开了一种抗TNFR2抗体药物组合物，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技 术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的一种抗TNFR2抗体药物组合物中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
术语解释:
除非另外说明，本文所用术语具有所属技术领域普通技术人员通常理解的含义。对于本文中明确定义的术语，则该术语的含义以所述定义为准。
如本文所用，术语“TNFR2”是指肿瘤坏死因子受体2，也称为肿瘤坏死因子受体超家族成员1B(TNFRSF1B)或CD120b，这是一种结合肿瘤坏死因子-α(TNFα)的膜受体。所述TNFR2优选地是人TNFR2。
如本文所用，术语“抗-肿瘤坏死因子受体2抗体”、“肿瘤坏死因子受体2抗体”、“抗TNFR2抗体”、“TNFR2抗体”、“抗-TNFR2抗体部分”和/或“抗-TNFR2抗体片段”等是指任何包含能够特异性结合TNFR2的免疫球蛋白分子的至少一部分(例如但不限于重链或轻链的至少一个互补决定区(CDR)或其配体结合部分、重链或轻链可变区、重链或轻链恒定区、框架区或其任何部分)的含蛋白质或肽的分子。TNFR2抗体还包括抗体样蛋白支架(如第十纤连蛋白III型结构域(10Fn3))，其含有与抗体CDR在结构和溶剂可及性上相似的BC、DE和FG结构环。10Fn3结构域的三级结构类似于IgG重链可变区的三级结构，并且通过将10Fn3的BC、DE和FG环的残基用来自TNFR2单克隆抗体的CDR-H1、CDR-H2或CDR-H3区的残基替换，本领域技术人员可以将例如TNFR2单克隆抗体的CDR接枝到纤连蛋白支架上。
如本文所用，术语“抗体”(Ab)是指与目标抗原特异性结合或具有免疫反应性的免疫球蛋白分子，包括抗体的多克隆、单克隆、基因工程化和其他修饰形式(包括但不限于嵌合抗体，人源化抗体，全人源抗体，异源偶联抗体(例如双特异性、三特异性和四特异性抗体，双抗体，三抗体和四抗体)，抗体缀合物)以及抗体的抗原结合片段(包括例如Fab’、F(ab’)2、Fab、Fv、rIgG和scFv片段)。此外，除非另有说明，否则术语“单克隆抗体”(mAb)意指包括能够特异性结合靶蛋白的完整抗体分子以及不完整的抗体片段(例如Fab和F(ab’)2片段，它们缺少完整抗体的Fc片段(从动物循环中更快地清除)，因此缺乏Fc介导的效应功能(effector function)(参见Wahl等人，J.Nucl.Med.24:316,1983；其内容援引加入本文)。
如本文所用，术语“单克隆抗体”是指来源于单个克隆(包括任何真核、原核、或噬菌体克隆)的抗体，而不限于该抗体的产生方法。
如本文所用，术语“抗原结合片段”和“抗体片段”可互换，是指保留特异性结合靶抗原的能力的一个或更多个抗体片段。抗体的抗原结合功能可以由全长抗体的片段执行。抗体片段可以是Fab、F(ab’)2、scFv、SMIP、双抗体、三抗体、亲和体(affibody)、纳米抗体、适体或结构域抗体。涵盖术语抗体的“抗原结合片段”的结合片段的实例包括但不限于：(i)Fab片段，一种由VL、VH、CL和CHl结构域组成的单价片段；(ii)F(ab)2片段，一种包含由二硫键在铰链区连接的两个Fab片段的双价片段；(iii)由VH和CHl结构域组成的Fd片段；(iv)由抗体单臂的VL和VH结构域组成的Fv片段；(V)包含VH和VL结构域的dAb；(vi)由VH结构域组成的dAb片段(Ward等人，Nature 341:544-546,1989)；(vii)由VH或VL结构域组成的dAb；(viii)分离的互补决定区(CDR)；以及(ix)两个或更多个分离的CDR的组合，所述CDR可以任选地由合成接头连接。此外，虽然Fv片段的两个结构域VL和VH是通过独立的基因编码的，但是这两个结构域可以使用重组方法通过接头接合，该接 头能够使其制成其中VL和VH区配对以形成单价分子的单蛋白质链(称为单链Fv(scFv)；参见例如，Bird等人，Science 242:423-426,1988以及Huston等人，Proc.Natl.Acad.Sci.USA 85:5879-5883,1988)。这些抗体片段可以使用本领域技术人员已知的常规技术获得，并且这些片段被筛选用于与完整抗体相同的方式使用。可以通过重组DNA技术、完整免疫球蛋白的酶促或化学裂解、或在一些实施方式中通过本领域已知的化学肽合成程序来产生抗原结合片段。
如本文所用，术语“互补决定区”(CDR)指在轻链和重链可变结构域中均发现的高变区。可变结构域中更高保守性的部分称为框架区(FR)。如本领域所理解的，表示抗体的高变区的氨基酸位置可以根据上下文和本领域已知的各种定义而变化。可变结构域内的一些位置可以被视为杂合高变位置，因为这些位置可以被认为是在一组标准(如IMGT或KABAT)下的可高变区之内，而被认为在不同组的标准(如KABAT或IMGT)下的可高变区之外。这些位置中的一个或更多个也可以在延伸的可高变区中找到。本发明包括在这些杂合可高变的位置中包含修饰的抗体。天然重链和轻链的可变结构域各自包含主要采用片层构型的四个框架区，其通过三个CDR(CDR1、CDR2和CDR3)连接，这三个CDR形成连接片层结构的环，并且在一些情况下形成片层结构的一部分。每条链中的CDR通过FR区按顺序FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4紧密保持在一起，并且与来自其他抗体链的CDR促成了抗体的抗原结合位点的形成(参见Kabat等人，Sequences of Protein sofImmunological Interest,National Institute of Health,Bethesda,Md.1987；其通过援引加入并入本文)。例如在本文中，CDR1-VH、CDR2-VH和CDR3-VH分别是指重链可变区(VH)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了重链(或其可变区)的CDR组合(VHCDR组合)；CDR1-VL、CDR2-VL和CDR3-VL分别是指轻链可变区(VL)的第一个CDR、第二个CDR和第三个CDR，这三个CDR构成了轻链(或其可变区)的CDR组合(VLCDR组合)。
如本文所用，术语“Kabat编号***”通常是指由Elvin A.Kabat提出的免疫球蛋白比对及编号***(参见，例如Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)。
如本文所用，术语“VH”是指抗体的免疫球蛋白重链(包括Fv、scFv或Fab的重链)的可变区。术语“VL”是指免疫球蛋白轻链(包括Fv、scFv、dsFv或Fab的轻链)的可变区。
本文术语“重链恒定区”是指抗体重链的羧基端部分，其不直接参与抗体与抗原的结合，但是表现出效应子功能，诸如与Fc受体的相互作用，其相对于抗体的可变结构域具有更保守的氨基酸序列。“重链恒定区”至少包含：CH1结构域，铰链区，CH2结构域，CH3结构域，或其变体或片段。“重链恒定区”包括“全长重链恒定区”和“重链恒定区片段”，前者具有基本上与天然抗体恒定区基本相似的结构，而后者仅包括“全长重链恒定区的一部分”。示例性地，典型的“全长抗体重链恒定区”由CH1结构域-铰链区-CH2结构域-CH3结构域组成；当抗体为IgE时，其还包括CH4结构域；当抗体为重链抗体时，则其不包括CH1结构域。示例性地，典型的“重链恒定区片段”可选自CH1、Fc或CH3结构域。
本文术语“轻链恒定区”是指抗体轻链的羧基端部分，其不直接参与抗体与抗原的结合，所述轻链恒定区可选自恒定κ结构域或恒定λ结构域。
如本文所用，术语“百分比(％)序列一致性”和“百分比(％)序列同一性”可互换，是指在为达到最大百分比序列一致性而比对序列和引入空位(如果需要)(例如，为了最佳比对，可以在候选和参比序列中的一个或两个中引入空位，并且出于比 较的目的，可以忽略非同源序列)之后，候选序列的氨基酸(或核苷酸)残基与参比序列的氨基酸(或核苷酸)残基相同的百分比。出于确定百分比序列一致性的目的，可以用本领域技术人员熟知的多种方式来实现比对，例如使用公众可得的计算机软件，如BLAST、ALIGN或Megalign(DNASTAIi)软件。本领域技术人员可以确定用于测量比对的适当参数，包括需要在被比较序列的全长范围实现最大比对的任何算法。例如，用于与候选序列进行比较而比对的参比序列可以显示候选序列在候选序列的全长或候选序列的连续氨基酸(或核苷酸)残基的选定部分上表现出从50％至100％的序列同一性。出于比较目的而比对的候选序列的长度可以是例如参比序列的长度的至少30％(例如30％、40％、50％、60％、70％、80％、90％或100％)。当候选序列中的位置被与在参比序列中的相应位置相同的氨基酸(或核苷酸)残基占据时，则这些分子在那个位置是相同的。
本文术语“保守氨基酸”通常是指属于同一类或具有类似特征(例如电荷、侧链大小、疏水性、亲水性、主链构象和刚性)的氨基酸。示例性地，下述每组内的氨基酸属于彼此的保守氨基酸残基，组内氨基酸残基的替换属于保守氨基酸的替换：
(1)酸性氨基酸：Asp(D)和Glu(E)；
(2)碱性氨基酸：Lys(K)、Arg(R)和His(H)；
(3)亲水性不带电荷氨基酸：Ser(S)、Thr(T)、Asn(N)和Gln(Q)；
(4)脂肪族不带电荷氨基酸：Gly(G)、Ala(A)、Val(V)、Leu(L)和Ile(I)；
(5)非极性不带电荷的氨基酸：Cys(C)、Met(M)和Pro(P)；
(6)芳香族氨基酸：Phe(F)、Tyr(Y)和Trp(W)。
如本文所用，术语“特异性结合”是指一种结合反应，其决定抗原在蛋白质和其他生物分子的一个异质性群体中的存在状况，所述蛋白质和其他生物分子例如被抗体或其抗原结合片段特异性识别。与抗原特异性结合的抗体或其抗原结合片段将以小于100nM的KD与抗原结合。例如，与抗原特异性结合的抗体或其抗原结合片段将以高达100nM(例如，1pM至100nM之间)的KD与抗原结合。不显示与特定抗原或其表位特异性结合的抗体或其抗原结合片段将显示对该特定抗原或其表位的大于100nM(例如，大于500nM、1μM、100μΜ、500μΜ或1mM)的KD。可以使用多种免疫测定方式来选择与特定蛋白或碳水化合物进行特异性免疫反应的抗体。例如，常规地使用固相ELISA免疫测定法来选择与蛋白质或碳水化合物进行特异性免疫反应的抗体。参见，Harlow&Lane，Antibodies,ALaboratory Manual,Cold Spring Harbor Press,NewYork(1988)以及Harlow&Lane,Using Antibodies，A Laboratory Manual,Cold Spring Harbor Press,NewYork(1999)，其描述了可以用于确定特异免疫反应性的免疫测定方式和条件。
如本文所用，“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，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保持抗体活性成分的稳定性，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。本文中，“药物组合物”和“制剂”并不互相排斥。
下面结合实施例，进一步阐述本发明：
实施例1抗TNFR2单克隆抗体人源化和表达纯化
1.1抗TNFR2单克隆抗体人源化
采用“CDRs移植”方法进行抗体人源化，即基于序列挑选同源性最高的人源性抗体提供抗体骨架区(FRs)，把目标抗体中基于Kabat命名方法的抗原结合片段互补 决定区(CDRs)，移植到前者形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的人源化抗体可变区序列。其中，TNFR2抗体Kabat命名方法的抗原结合片段互补决定区(CDRs)详见表1，表2示人源化抗体分子的VH和VL序列，表3示人源化抗TNFR2抗体的VH和VL序列配对情况。
表1.候选抗体CDRs的KABAT分析结果
Figure PCTCN2022142190-appb-000001
表2.人源化抗TNFR2抗体的VH和VL序列
Figure PCTCN2022142190-appb-000002
Figure PCTCN2022142190-appb-000003
表3.人源化抗TNFR2抗体的VH和VL序列配对情况
抗体编号 VH VL
#1-1 VH1(SEQ ID NO.19) VL1(SEQ ID NO.24)
#1-2 VH1(SEQ ID NO.19) VL2(SEQ ID NO.25)
#2-1 VH2(SEQ ID NO.20) VL3(SEQ ID NO.26)
#2-2 VH3(SEQ ID NO.21) VL3(SEQ ID NO.26)
#3-1 VH4(SEQ ID NO.22) VL4(SEQ ID NO.27)
#3-2 VH5(SEQ ID NO.23) VL5(SEQ ID NO.28)
1.2人源化抗TNFR2抗体的表达
人源化抗TNFR2抗体利用CHO作为宿主细胞进行表达，在Dynamis AGT培养基(厂家：gibco,货号：A26615-01)中进行培养，培养周期不超过15天，反应器控制参数为pH 6.8～7.6，溶解氧(DO)为20％～80％，转速79RPM～81RPM，初始培养温度36.0℃～37.0℃；当培养至第8天时，降低培养温度至32.0±0.5℃直到收获。
1.3人源化抗TNFR2抗体的纯化
人源化抗TNFR2抗体的纯化采用多步骤层析、浓缩和过滤单元操作依次对抗体进行纯化。上清收获液经过Protein A亲和层析(Mabselect PrismA)进行捕获。捕获后的抗体溶液采用低pH孵育处理以灭活潜在的病毒。抗体溶液中和后通过深层过滤除去沉淀。然后依次进行阴离子交换层析(Capto Q)以去除HCD,HCP,脱落Protein A等杂质，以及阳离子交换(Capto S Impact)层析以去除HCP和聚集体等杂质。通过纳滤膜包过滤以去除潜在的内外源病毒。之后用超滤膜包对抗体溶液进行浓缩，缓冲液置换，从而完成纯化，获得人源化抗TNFR2抗体蛋白原液。
实施例2缓冲体系筛选
2.1缓冲体系筛选方案
实验涵盖了4种缓冲盐(醋酸盐、枸橼酸盐、组氨酸盐和磷酸盐)，pH值从4.5至7.5，共设置12个制剂缓冲体系进行筛选(参见表4)。将实施例1获得的蛋白原液进行超滤浓缩换液处理，原液被置换至对应的12个制剂缓冲体系中：分12组将蛋白原液加入到对应超滤管中，使用离心机进行超滤浓缩，蛋白被截留，蛋白原液的缓冲液从超滤膜流穿，达到蛋白浓缩的目的，然后分别加入对应的12个制剂缓冲液，原蛋白原液的缓冲液被稀释，继续超滤浓缩，浓缩完毕后继续分别加入对应的12个制剂缓冲液，继续超滤浓缩，重复操作，直到换液完毕。以上各处方中采用实施例1中所述的人源化TNFR2抗体#2-1，蛋白浓度为30mg/mL。通过40℃高温加速试验，以外观、pH值、浓度、纯度(分子排阻色谱法(SE-HPLC)、非还原十二烷基硫酸 钠毛细管电泳(CE-SDS(NR))、电荷异构(离子交换色谱法(CEX-HPLC))、动态光散射(DLS)、热稳定性(差示扫描荧光法(DSF))为指标，综合考察比较此蛋白在12种缓冲体系中的稳定性，以筛选出最优的缓冲体系。具体处方组成和考察方案见下表4。
表4制剂缓冲体系筛选实验方案
Figure PCTCN2022142190-appb-000004
备注：X＝外观、pH值、蛋白浓度、CEX-HPLC、SE-HPLC、CE-SDS(NR)、DLS、DSF(T0)
T0＝考察起始点；W＝周；DSF(T0)表示只在T0时考察DSF参数即可。
2.2缓冲体系筛选结果
缓冲体系筛选主要结果汇总见下表5-表6。
表5制剂缓冲体系筛选40℃加速试验结果
Figure PCTCN2022142190-appb-000005
Figure PCTCN2022142190-appb-000006
Figure PCTCN2022142190-appb-000007
备注：NA＝不适用。
表6制剂缓冲体系筛选40℃加速试验结果
Figure PCTCN2022142190-appb-000008
Figure PCTCN2022142190-appb-000009
备注：NA＝不适用。
2.3缓冲体系筛选结论
12个缓冲体系内，40℃考察4周，外观均为微黄色，微乳光液体，pH、浓度均在目标值之内，无明显变化；
热稳定性检测，蛋白的熔融温度(Tm)：F4处方Tm1为68.46℃，明显低于其他处方，其他处方Tm1均大于69℃，无明显差异；
DLS：粒径优劣排序(从小到大排序)：F7、F8、F9>F3、F10、F11、F12>F1>F6、F2>F5、F4；
SE-HPLC：纯度优劣排序F7>F8>F2、F9、F3>F1、F5、F6>F10>F11、F4>F12；
CE-SDS(NR)：主峰纯度优劣排序F8、F9、F6、F7、F3>F10>F11、F5>F2>F12、F4>F1；
CEX-HPLC：主峰纯度F8>F7、F9、F6>F3>F2、F1、F5、F10>F4>F11>F12；
酸性峰增加，优劣排序F7、F1>F8、F2>F3、F4、F5、F6、F9>F10>F11>F12；
综上述，经过缓冲体系筛选，各个处方优劣排序如下：F7>F8>F9、F3>F2>F1>F4、F5、F6、F10、F11、F12。F7处方在本次40℃加速实验考察中在纯度、降解、聚集、电荷异质体等方面综合优于其他处方，表现出良好的稳定性。因此，初步选定F7处方(20mM组氨酸-盐酸组氨酸，pH5.5)为最优处方作为本产品制剂缓冲体系进行下一步的辅料筛选研究。
实施例3辅料筛选
3.1辅料筛选方案
结合缓冲体系筛选结果以及开发冻干制剂的目标，向最优缓冲体系中加入相应辅料，包括氯化钠、甘氨酸、盐酸精氨酸、蔗糖、海藻糖、甘露醇、山梨醇共计7个处方，蛋白浓度为30mg/mL。通过冻融试验(-20℃到25℃为一轮冻融，连续冻融3-5轮)、40℃高温加速试验，以外观、pH值、浓度、纯度(分子排阻色谱法(SE-HPLC)、非还原十二烷基硫酸钠毛细管电泳分子排阻色谱法(CE-SDS(NR))、电荷异构(离子交换色谱法(CEX-HPLC))、动态光散射(DLS)、热稳定性(差示扫描荧光法(DSF))为指标考察各处方的稳定性，筛选出1个最优制剂处方进行下一轮的表面活性剂筛选。具体处方组成和考察方案见下表7。
表7辅料筛选方案
Figure PCTCN2022142190-appb-000010
Figure PCTCN2022142190-appb-000011
备注：X＝外观、pH值、蛋白浓度、CEX-HPLC、SE-HPLC、CE-SDS(NR)、DLS、DSF(T0)、渗透压(T0)
T0＝考察起始点；W＝周；DSF(T0)表示只在T0时考察DSF参数即可；渗透压(T0)表示只在T0时考察渗透压参数即可。
3.2辅料筛选结果
辅料筛选主要结果汇总见下表8-表11。
表8辅料筛选40℃加速试验结果
Figure PCTCN2022142190-appb-000012
备注：NA＝不适用。
表9辅料筛选40℃加速试验结果
Figure PCTCN2022142190-appb-000013
备注：NA＝不适用。
表10辅料筛选-20℃到25℃冻融试验结果
Figure PCTCN2022142190-appb-000014
Figure PCTCN2022142190-appb-000015
备注：NA＝不适用。
表11辅料筛选-20℃到25℃冻融试验结果
Figure PCTCN2022142190-appb-000016
备注：NA＝不适用。
3.3辅料筛选结论
样品浓度为30mg/mL，分别进行-20℃到25℃的5次冻融实验和40℃下放置4周的辅料筛选。
外观均为微黄色，微乳光液体，pH、浓度均在目标值之内，无明显变化；
热稳定性检测(DSF)，蛋白的熔融温度(Tm1和Tm2)：F7-4、F7-5>F7-2、F7-6、F7-7、不加辅料(F7)>F7-1、F7-3；
DLS：40℃考察4周，F7-1粒径相对T0有明显增加；冻融5次F7-2、F7-6粒径相对于T0有明显增加；
SE-HPLC：40℃考察4周，纯度F7-2、F7-3、F7-4、F7-5、F7-6、F7-7>F7-1；冻融5次，纯度F7-1、F7-3、F7-4、F7-5、F7-7>F7-2>F7-6；
CE-SDS(NR)：40℃考察4周，CE(NR)主峰纯度F7-7、F7-5、F7-6、F7-4、F7-2>F7-1、F7-3；冻融5次无明显变化；
CEX-HPLC：40℃考察4周，主峰纯度F7-3>F7-1、F7-6、F7-7>F7-2、F7-4、F7-5；酸性峰增加，优劣排序F7-1、F7-3>F7-6、F7-7>F7-4、F7-5、F7-2；冻融5次各处方无明显差异。
综上述，辅料筛选通过对各个稳定性指标的评估，F7-7处方在本次40℃加速实验、-20到25℃的5次冻融考察中在纯度、降解、聚集、电荷异质体等方面综合优于 其他处方，表现出良好的稳定性。因此，初步选定F7-7处方(4.7％山梨醇(W/V)，20mM组氨酸-盐酸组氨酸，pH5.5)为最优处方进行下一步的表面活性剂筛选研究。
实施例4表面活性剂筛选
4.1表面活性剂筛选方案
向最优缓冲体系、辅料中加入不同浓度的表面活性剂，包括不含聚山梨酯80、0.02％、0.04％、0.06％、0.08％、0.1％的聚山梨酯80(W/W)共计6个处方，蛋白浓度为30mg/mL，通过40℃加速试验、300rpm振摇试验，以外观、pH值、浓度、纯度(分子排阻色谱法(SE-HPLC)、非还原十二烷基硫酸钠毛细管电泳分子排阻色谱法(CE-SDS(NR))、电荷异构(离子交换色谱法(CEX-HPLC))、动态光散射(DLS)、不溶性微粒为指标考察各处方的稳定性，筛选出1个最优制剂处方进行处方确认稳定性实验，对最终的处方进行了确认。具体处方组成和考察方案见下表12。
表12表面活性剂筛选方案
Figure PCTCN2022142190-appb-000017
备注：X＝外观、pH值、蛋白浓度、CEX-HPLC、SE-HPLC、CE-SDS(NR)、DLS、不溶性微粒(微流成像法(MFI)，T0和考察终点)
T0＝考察起始点；W＝周；D＝天。
4.2表面活性剂筛选结果
表面活性剂筛选主要结果汇总见下表13-表14。
表13表面活性剂筛选40℃加速试验结果
Figure PCTCN2022142190-appb-000018
Figure PCTCN2022142190-appb-000019
备注：NA＝不适用。
表14表面活性剂筛选振摇试验结果
Figure PCTCN2022142190-appb-000020
备注：NA＝不适用。
4.3表面活性剂筛选结论
样品浓度为30mg/mL，40℃考察4周。外观均为无色微乳光液体；pH、浓度均在目标值之内，无明显变化；
SE-HPLC：各个处方优劣，F7-7-3、F7-7-5>F7-7-2、F7-7-6>F7-7-4>F7-7-1；
CE-SDS(NR)：各个处方优劣，F7-7-6、F7-7-2>F7-7-4、F7-7-3>F7-7-5、F7-7-1；
DLS：粒径各个处方优劣，F7-7-1、F7-7-6>F7-7-5>F7-7-2>F7-7-4>F7-7-3；
不溶性微粒：各个处方优劣，F7-7-1处方≥10μm的颗粒始终明显多于其他处方；其他处方均无明显差异；
CEX-HPLC：F7-7-3处方略优于其他处方。
25℃,300rpm振摇7天。SE-HPLC、DLS、CEX-HPLC：各个处方无明显差异；不溶性微粒：F7-7-1处方≥10μm的颗粒始终明显多于其他处方；其他处方均无明显差异CE-SDS(NR)：主峰含量F7-7-3、F7-7-6>F7-7-4>F7-7-1>F7-7-2、F7-7-5。
综上述，辅料筛选通过对各个稳定性指标的评估：F7-7-3、F7-7-6>F7-7-2、F7-7-4>F7-7-5>F7-7-1，即最优的处方为F7-7-3或F7-7-6。
然而，处方F7-7-2、F7-7-4中的聚山梨脂80含量处于处方F7-7-3中的聚山梨脂80含量的上下两个边缘，处方F7-7-5中的聚山梨脂80含量处于处方F7-7-6中的聚山梨脂80含量的边缘，而处方F7-7-2、F7-7-4均优于F7-7-5处方。
所以，从生产的可操作空间考虑，选择F7-7-3处方(0.04％聚山梨酯80(W/W),20mM组氨酸-盐酸组氨酸,4.7％山梨醇,pH5.5)更为合适。因此，F7-7-3处方(0.04％聚山梨酯80(W/W),20mM组氨酸-盐酸组氨酸,4.7％山梨醇(W/V),pH5.5)为本次筛选的最优处方，并准备进行下一步的处方确认稳定性研究，对本处方进行最终的确认。
实施例5处方确认稳定性
5.1处方确认稳定性方案
本处方确认稳定性和包材选型研究实验基于缓冲体系、辅料和表面活性剂筛选的结果，确定1个处方：30mg/mLTNFR2单克隆抗体(#2-1)，20mM组氨酸-盐酸组氨酸，4.7％山梨醇(W/V)，0.04％聚山梨酯80(W/W)，pH5.5，5mL/瓶，分装至西林瓶中，使用胶塞和铝塑盖组成密闭的包装***，分别正置和倒置，进行6个月的实时稳定性和加速稳定性考察，对最终的处方进行稳定性确认研究。
具体考察方案见下表15。
表15处方确认稳定性方案
Figure PCTCN2022142190-appb-000021
备注：X＝外观、pH值、蛋白浓度、CEX-HPLC、SE-HPLC、CE-SDS(NR)、CE-SDS(R)、渗透压(T0)、活性、不溶性微粒(微流成像法，MFI)
T0＝考察起始点；M＝月；渗透压(T0)表示只在T0时考察渗透压参数即可。
在本方案中活性检测方法采用Elisa和Cell assay blocking方法，具体如下：
Elisa方法：利用抗TNFR2单克隆抗体与人TNFR2结合作用，检测抗TNFR2单克隆抗体对于50ng的TNFR2的半数作用浓度。96孔板上预先包被抗原(human  TNFR2-his)(Sino Biological,10417-H08H)，抗TNFR2单克隆抗体与酶标板上的抗原结合，洗板去除不结合的各种成分，再加入Peroxidase-conjugated Mouse anti Human IgG Fcγ(Jackson Immuno,209-035-098)，孵育后洗板去除多余的酶标抗体；最后加入HRP的底物TMB(Thermo,34029)，产生蓝色的产物，颜色深浅与抗TNFR2单克隆抗体浓度成正比。终止液终止后蓝色产物转变成黄色，用酶标仪读取OD值。
Cell assay blocking方法：人源化抗TNFR2单克隆抗体是一种特异性结合Human TNFR2的单克隆抗体，可以抑制Human TNFα与受体Human TNFR2的结合。本方法采用经过自行筛选得到的过表达TNFR2的单克隆细胞株CT26-2A03作为检测用细胞。先加入人源化抗TNFR2单克隆抗体，然后加入生物素标记的TNFα(TNFα-biotin)(AcroBiosystems,TNA-H82E3)，配体TNFα与受体TNFR2相互作用,之后再加入PE标记的Streptavidin(Biolegend,405204)进行孵育，形成CHO-TNFR2～TNFα-biotin～Streptavidin-PE复合物，此时细胞间接被标记上PE染料，经酶标仪检测可看到明显的阳性信号。人源化抗TNFR2单克隆抗体与Human TNFR2的结合位点若与Human TNFα结合位点有冲突，则会影响Human TNFα与Human TNFR2的结合，最终影响PE信号的强度，在酶标仪上检测表现为PE信号的减弱。利用此原理，可以检测人源化抗TNFR2单克隆抗体在细胞水平上抑制HumanTNFR2与HumanTNFα结合的功能。
5.2处方确认稳定性结果
处方确认稳定性主要结果汇总见下表16-表17，图1-图4。
表16处方确认稳定性结果
Figure PCTCN2022142190-appb-000022
Figure PCTCN2022142190-appb-000023
表17处方确认稳定性结果
Figure PCTCN2022142190-appb-000024
Figure PCTCN2022142190-appb-000025
5.3处方确认稳定性研究结论
2～8℃低温放置6个月，正置和倒置样品在常规检测指标、不溶性微粒、纯度、电荷异质体、活性等方面均没有显著改变，且正置和倒置样品之间无明显差异。
25℃加速稳定性考察6个月，正置和倒置样品在常规检测指标、不溶性微粒、纯度、电荷异质体、活性等方面均在质量标准范围内，且正置和倒置样品之间无明显差异。
40℃加速稳定性考察3个月，正置和倒置样品仅纯度指标中的CE-SDS(NR)、CEX-HPLC超出了质量标准，其他的纯度指标、常规检测指标、不溶性微粒、电荷异质体、活性等均在质量标准范围内，且正置和倒置样品之间无明显差异。
综上所述，在低温(2～8℃)实时稳定性考察过程中，样品的常规检测指标、不溶性微粒、纯度、电荷异质体、活性等均没有显著改变，在25℃加速实验中，各指标均在质量标准范围内，说明本处方中蛋白具有较好的稳定性，达到了本发明的目的。在高温40℃加速稳定性考察实验中，纯度、电荷异质体方面有下降趋势，这与处方筛选阶段该处方的质量指标趋势一致。在高温条件下，一些关键指标呈下降趋势，也说明本产品对高温敏感，建议本产品低温(2～8℃)保存和运输。
以上对本发明所提供的一种抗TNFR2抗体药物组合物进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
序列表
Figure PCTCN2022142190-appb-000026
Figure PCTCN2022142190-appb-000027
Figure PCTCN2022142190-appb-000028
Figure PCTCN2022142190-appb-000029
Figure PCTCN2022142190-appb-000030
Figure PCTCN2022142190-appb-000031
Figure PCTCN2022142190-appb-000032
Figure PCTCN2022142190-appb-000033

Claims (19)

  1. 一种药物组合物，其特征在于，所述的药物组合物包含抗TNFR2抗体或其抗原结合片段、缓冲液、蛋白稳定剂和表面活性剂。
  2. 根据权利要求1所述的药物组合物，其特征在于，所述抗TNFR2抗体或其抗原结合片段包含：
    CDR1-VH选自SEQ ID NO.1、7、或13；
    CDR2-VH选自SEQ ID NO.2、8、或14；
    CDR3-VH选自SEQ ID NO.3、9、或15；
    CDR1-VL选自SEQ ID NO.4、10、或16；
    CDR2-VL选自SEQ ID NO.5、11、或17；和
    CDR3-VL选自SEQ ID NO.6、12、或18。
  3. 根据权利要求1或2所述的药物组合物，其特征在于，所述的抗TNFR2抗体或其抗原结合片段包含重链可变区和轻链可变区：
    (1)所述重链可变区包含：
    a.选自SEQ ID NO：19-23任一所示的VH序列，
    b.选自与SEQ ID NO：19-23任一序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH序列，或，
    c.选自与SEQ ID NO：19-23任一序列相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸***、缺失和/或替换的VH序列；
    (2)所述轻链可变区包含：
    a.选自SEQ ID NO：24-28任一所示的VL序列，
    b.选自与SEQ ID NO：24-28任一序列相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VL序列，或，
    c.选自与SEQ ID NO：24-28任一序列相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸***、缺失和/或替换的VL序列；
    可选地，所述氨基酸***、缺失和/或替换发生在重链可变区或/和轻链可变区的FR区；
    可选地，所述替换为保守氨基酸的替换。
  4. 根据权利要求3所述的药物组合物，其特征在于，所述重链可变区和轻链可变区选自如下组：
    (1)具有SEQ ID NO.19所示的VH和SEQ ID NO.24所示的VL；
    (2)具有SEQ ID NO.19所示的VH和SEQ ID NO.25所示的VL；
    (3)具有SEQ ID NO.20所示的VH和SEQ ID NO.26所示的VL；
    (4)具有SEQ ID NO.21所示的VH和SEQ ID NO.26所示的VL；
    (5)具有SEQ ID NO.22所示的VH和SEQ ID NO.27所示的VL；
    (6)具有SEQ ID NO.23所示的VH和SEQ ID NO.28所示的VL；
    (7)具有与上述(1)～(6)任一序列组合相比具有至少80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合；
    (8)具有与上述(1)～(7)任一项序列组合相比具有1、2、3、4、5、6、7、8、9、10或更多个氨基酸***、缺失和/或替换的VH和VL组合；或
    (9)具有与上述(1)～(8)任一项序列组合相比CDR完全一致，FR具有 至少85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列一致性的VH和VL组合；
    可选地，所述氨基酸***、缺失和/或替换发生在重链可变区或/和轻链可变区的FR区；
    可选地，所述替换为保守氨基酸的替换。
  5. 根据权利要求1-4任一项所述的药物组合物，其特征在于，所述的抗TNFR2抗体或其抗原结合片段选自单克隆抗体、多克隆抗体、天然抗体、工程化抗体、单特异性抗体、多特异性抗体(例如双特异性抗体)、单价抗体、多价抗体、全长抗体、抗体片段、Fab、Fab’、F(ab’)2、Fd、Fv、scFv、或双抗体(diabody)。
  6. 根据权利要求1-5任一项所述的药物组合物，其特征在于，所述抗TNFR2抗体的浓度为1mg/mL至100mg/mL，优选为20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL，更优选为30mg/mL。
  7. 根据权利要求1-6任一项所述的药物组合物，其特征在于，所述的缓冲液选自醋酸-醋酸钠缓冲液、枸橼酸-枸橼酸钠缓冲液、组氨酸-盐酸组氨酸缓冲液或磷酸氢二钠-磷酸二氢钠缓冲液，优选为组氨酸-盐酸组氨酸缓冲液。
  8. 根据权利要求7所述的药物组合物，其特征在于，所述的缓冲液浓度为10-100mM，优选为10-60mM，更优选为20mM。
  9. 根据权利要求1-8任一项所述的药物组合物，其特征在于，所述的蛋白稳定剂选自氯化钠、甘氨酸、盐酸精氨酸、蔗糖、海藻糖或山梨醇，优选山梨醇。
  10. 根据权利要求9所述的药物组合物，其特征在于，所述蛋白稳定剂的浓度为1-10％(w/v)，优选为2-8％(w/v)，更优选为4.7％(w/v)。
  11. 根据权利要求1-10任一项所述的药物组合物，其特征在于，所述的表面活性剂选自聚山梨酯80、泊洛沙姆或甘油脂肪酸酯，优选为聚山梨酯80。
  12. 根据权利要求11所述的药物组合物，其特征在于，所述的表面活性剂的浓度为0.02～0.1％(w/w)，优选为0.02％(w/w)、0.04％(w/w)、0.06％(w/w)、0.08％(w/w)、0.1％(w/w)，更优选为0.04％(w/w)。
  13. 根据权利要求1-12任一项所述的药物组合物，其特征在于，所述的药物组合物的pH值为4.5-7.5，优选为4.5、5、5.5、6、6.5、7、7.5，更优选为5.5。
  14. 根据权利要求1-13任一项所述的药物组合物，其特征在于，其中包含：
    a)浓度为1mg/mL至100mg/mL的抗TNFR2抗体，
    b)浓度为10-100mM的组氨酸-盐酸组氨酸缓冲液，pH值为4.5-7.5，
    c)浓度为1-10％(w/v)的山梨醇，和
    d)浓度为0.02～0.1％(w/w)的聚山梨酯80；
    优选的，所述药物组合物包含：
    a)浓度为10mg/mL至60mg/mL的抗TNFR2抗体，
    b)浓度为10-60mM的组氨酸-盐酸组氨酸缓冲液，pH值为5.0-6.0，
    c)浓度为2-8％(w/v)的山梨醇，和
    d)浓度为0.04％(w/w)的聚山梨酯80；
    更优选的，所述药物组合物包含：
    a)浓度为30mg/mL的抗TNFR2抗体，
    b)浓度为20mM的组氨酸-盐酸组氨酸缓冲液，pH值为5.5，
    c)浓度为4.7％(w/v)的山梨醇，和
    d)浓度为0.04％(w/w)的聚山梨酯80。
  15. 根据权利要求1-14任一项所述的药物组合物，其特征在于，所述的药物组合 物的形式为液体制剂。
  16. 制备如权利要求1-15任一项所述的药物组合物的方法，其特征在于，包括将所述抗TNFR2抗体溶液经过缓冲液置换的步骤，所述的缓冲液优选组氨酸-盐酸组氨酸缓冲液，所述缓冲液的浓度优选20mM，所述缓冲液的pH优选为5.5。
  17. 一种含抗TNFR2抗体或其抗原结合片段的药物组合物，其特征在于，所述药物组合物通过权利要求16所述方法制备获得。
  18. 如权利要求1-15任一项所述的药物组合物、权利要求16方法制备的产品、或权利要求17所述的药物组合物，在制备治疗和/或预防免疫异常相关疾病的药物中的用途；
    优选地，所述免疫异常相关疾病是Treg细胞和/或MDSC功能相关疾病；
    优选地，所述疾病是癌症或自身免疫性疾病；
    更优选地，所述癌症选自卵巢癌、晚期表皮T细胞淋巴瘤、III/IV期转移性结直肠癌、三阴乳腺癌、胰腺癌、非小细胞肺癌、和/或对CTLA-4和PD-1疗法耐药的晚期实体瘤，如转移性黑色素瘤；
    更优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、***性硬化症、视神经脊髓炎谱系病、***性红斑狼疮、重症肌无力、IgG4相关性疾病。
  19. 一种治疗和/或预防免疫异常相关疾病的方法，其特征在于，所述方法包括向受试者施用有效量的权利要求1-15任一项所述的药物组合物、权利要求16方法制备的产品、或权利要求17所述的药物组合物；
    优选地，所述免疫异常相关疾病是Treg细胞和/或MDSC功能相关疾病；
    优选地，所述疾病是癌症或自身免疫性疾病；
    更优选地，所述癌症选自卵巢癌、晚期表皮T细胞淋巴瘤、III/IV期转移性结直肠癌、三阴乳腺癌、胰腺癌、非小细胞肺癌、和/或对CTLA-4和PD-1疗法耐药的晚期实体瘤，如转移性黑色素瘤；
    更优选地，所述自身免疫性疾病可选自类风湿性关节炎、多发性硬化症、***性硬化症、视神经脊髓炎谱系病、***性红斑狼疮、重症肌无力、IgG4相关性疾病。
PCT/CN2022/142190 2021-12-28 2022-12-27 一种抗tnfr2抗体药物组合物 WO2023125483A1 (zh)

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