WO2020094122A1 - 一种TGF-β受体融合蛋白药物组合物及其用途 - Google Patents

一种TGF-β受体融合蛋白药物组合物及其用途 Download PDF

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WO2020094122A1
WO2020094122A1 PCT/CN2019/116593 CN2019116593W WO2020094122A1 WO 2020094122 A1 WO2020094122 A1 WO 2020094122A1 CN 2019116593 W CN2019116593 W CN 2019116593W WO 2020094122 A1 WO2020094122 A1 WO 2020094122A1
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Prior art keywords
tgf
pharmaceutical composition
fusion protein
seq
cancer
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PCT/CN2019/116593
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English (en)
French (fr)
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田晨敏
李皓
刘洵
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to US17/290,707 priority Critical patent/US20220017601A1/en
Priority to CN201980050112.9A priority patent/CN112512550A/zh
Priority to JP2021523471A priority patent/JP7436477B2/ja
Priority to CA3118415A priority patent/CA3118415A1/en
Priority to UAA202102928A priority patent/UA127771C2/uk
Priority to EP19882778.4A priority patent/EP3878461A4/en
Priority to MX2021005018A priority patent/MX2021005018A/es
Priority to BR112021008288-3A priority patent/BR112021008288A2/pt
Priority to KR1020217016571A priority patent/KR20210090643A/ko
Priority to AU2019374363A priority patent/AU2019374363A1/en
Publication of WO2020094122A1 publication Critical patent/WO2020094122A1/zh

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    • A—HUMAN NECESSITIES
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition containing a PD-L1 antibody / TGF- ⁇ RII extracellular domain fusion protein and its use as a medicine.
  • Tumor immunotherapy has attracted much attention in recent years and is the focus of the field of cancer therapy. It is difficult to develop drug resistance is the prominent advantage of this therapy.
  • Tumor immunotherapy mainly uses immunological principles and methods to improve the immunogenicity of tumor cells and the sensitivity to killing effector cells, and stimulate and enhance the body's anti-tumor immune response.
  • immune cells and effector molecules are infused into the host body. The two cooperate with the body's immune system to kill tumors and inhibit tumor growth.
  • Programmed death receptor 1 (programmed death 1, PD-1) is a member of the CD28 superfamily. PD-1 is expressed in activated T cells, B cells and myeloid cells. PD-1 has two ligands, namely, programmed death ligand-1 (PD-L1) and PD-L2. PD-L1 interacts with the PD-1 receptor on T cells and plays an important role in the negative regulation of the immune response. PD-L1 protein expression can be detected in many human tumor tissues. The microenvironment of the tumor site can induce the expression of PD-L1 on the tumor cells. The expressed PD-L1 is conducive to the occurrence and growth of tumors and induces the apoptosis of anti-tumor T cells.
  • PD-1 / PD-L1 pathway inhibitors can block the binding of PD-1 and PD-L1, block negative regulatory signals, and restore the activity of T cells, thereby enhancing the immune response. Therefore, PD-1 / PD- Immunomodulation with L1 as a target has important significance for tumor suppression.
  • Transforming growth factor- ⁇ (transforming growth factor- ⁇ , TGF- ⁇ ) belongs to the TGF- ⁇ superfamily that regulates cell growth and differentiation. TGF- ⁇ transmits signals through a heterotetrameric receptor complex consisting of two type I and two type II transmembrane serine / threonine kinase receptors.
  • TGF- ⁇ is a versatile cytokine that exerts tumor suppression or tumor promotion in a cell- or background-dependent manner.
  • the tumor suppressive effect of TGF- ⁇ stems from its ability to induce the expression of multiple genes.
  • cancer cells gradually tolerate the inhibitory effect of TGF- ⁇ , which ultimately leads to tumor development.
  • TGF- ⁇ signaling pathway can reduce tumor metastasis.
  • Smad2 / 3 negative mutants to inhibit the TGF- ⁇ signaling pathway of breast tumor cell lines, it was found that the metastatic ability of tumor cells was inhibited.
  • Colon cancer microsatellite instability studies have found that inactive mutations of TGF- ⁇ RII reduce metastasis and increase the postoperative survival rate of patients.
  • TGF- ⁇ signaling pathway inhibitors alone in clinical treatment is weak, probably because TGF- ⁇ is abnormally highly expressed in tumor cells, while the inhibition of TGF- ⁇ signaling pathway alone It is difficult for the agent to focus on the tumor, resulting in low drug efficacy or low bioavailability of the inhibitor.
  • inhibition of the PD-1 / PD-L1 pathway can restore the activity of T cells, enhance the immune response, and more effectively improve the effect of inhibiting tumorigenesis and development.
  • Bintrafusp Alfa (WO2015118175, also known as M7824, FP17022).
  • Bintrafusp Alfa has been used in gastric cancer, lung cancer, esophageal cancer, NSCLC, biliary tract cancer and other tumor diseases Start clinical.
  • the antibody drugs in the prior art have large molecular weights, complex structures, and are easily degraded, polymerized, or undesirably chemically modified, etc., and become unstable.
  • the study of stable formulations of antibody drugs is particularly important.
  • the present disclosure provides a pharmaceutical composition containing PD-L1 / TGF- ⁇ RII fusion protein, which is more conducive to production and administration, and has more stable performance, which comprises:
  • the buffer is selected from histidine buffer, succinate buffer, phosphate buffer and citrate buffer.
  • the buffer is a citrate buffer.
  • the histidine buffer is selected from histidine-hydrochloric acid buffer
  • the succinate buffer is selected from succinate-sodium succinate buffer
  • the citrate The buffer is selected from the citrate-sodium citrate buffer; in some embodiments, the buffer is selected from the citrate-sodium citrate buffer.
  • the concentration of the TGF- ⁇ receptor fusion protein in the aforementioned pharmaceutical composition is about 0.5 mg / ml to about 100 mg / ml, preferably about 30 mg / ml to about 70 mg / ml.
  • the concentration of the TGF- ⁇ receptor fusion protein in the pharmaceutical composition is 0.5 mg / ml to 100 mg / ml, preferably 30 mg / ml to 70 mg / ml.
  • TGF- ⁇ receptor fusion protein concentrations include: approximately 30 mg / ml, approximately 35 mg / ml, approximately 40 mg / ml, approximately 45 mg / ml, approximately 50 mg / ml, approximately 55 mg / ml, approximately 60 mg / ml, About 65mg / ml, about 70mg / ml, preferably about 50mg / ml;
  • the concentration of the TGF- ⁇ receptor fusion protein in the pharmaceutical composition is 30 mg / ml, 35 mg / ml, 40 mg / ml, 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, more preferably 50 mg / ml.
  • the pH value of the buffer in the aforementioned pharmaceutical composition is about 5.0 to about 7.5, preferably about 6.0 to about 6.5, and optionally about 6.0, about 6.1, about 6.2, about 63 , About 6.4, about 6.5, more preferably about 6.2;
  • the pH of the buffer is 5.0 to 7.5 or 6.0 to 6.5, preferably 6.0, 6.1, 6.2, 63, 6.4 or 6.5, and more preferably 6.2.
  • the concentration of the buffer is about 5 mM to about 30 mM, preferably about 5 mM to about 20 mM, non-limiting examples include 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 16 mM , 18mM, 20mM, more preferably 10mM.
  • the concentration of the buffer is 5 mM to 30 mM, preferably 5 mM to 20 mM, in some embodiments, the concentration of the buffer is about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, More preferably, it is about 10 mM.
  • the aforementioned pharmaceutical composition further comprises sugar.
  • the “sugar” of the present disclosure includes a conventional composition (CH 2 O) n or derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like.
  • the sugar is selected from the group consisting of glucose, sucrose, trehalose, lactose, fructose, dextran, glycerol, erythritol, glycerin, arabitol, xylitol, Sorbitol, mannitol, melose, melezitose, honey triose, mannose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltone Sugar and so on.
  • the preferred sugar is a non-reducing disaccharide, more preferably trehalose or sucrose, and most preferably sucrose.
  • the concentration of sugar in the aforementioned pharmaceutical composition is about 50 mg / ml to about 100 mg / ml, preferably about 60 mg / ml to about 90 mg / ml, non-limiting examples include 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, most preferably 80 mg / ml.
  • the concentration of sugar is 50 mg / ml to 100 mg / ml, preferably 60 mg / ml to 90 mg / ml, and in some embodiments, the concentration of sugar is about 60 mg / ml, about 65 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 85 mg / ml, or about 90 mg / ml.
  • the aforementioned pharmaceutical composition further comprises a surfactant, which may be selected from polysorbate 20, polysorbate 80, polyhydroxyalkylene, Triton, sodium dodecyl sulfonate, lauryl Sodium sulfonate, sodium octyl glycoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine Acid, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauramide Propyl-betaine, cocaamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitoy
  • the concentration of the surfactant in the aforementioned pharmaceutical composition is from about 0.1 mg / ml to about 0.8 mg / ml, more preferably from about 0.4 mg / ml to about 0.8 mg / ml, in some embodiments
  • the concentration of the surfactant is 0.1 mg / ml to 0.8 mg / ml, preferably 0.4 mg / ml to 0.8 mg / ml, more preferably about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, About 0.55 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml.
  • the concentration of the surfactant is 0.4 mg / ml, 0.45 mg / ml, 0.5 mg / ml, 0.55 mg / ml, 0.6 mg / ml, 0.7 mg / ml or 0.8 mg / ml, more specifically 0.4mg / ml.
  • the aforementioned pharmaceutical composition comprising:
  • the pH of the pharmaceutical composition is about 5.0 to about 7.5, more preferably about 6.0 to about 6.5.
  • the aforementioned pharmaceutical composition comprising:
  • the pH of the pharmaceutical composition is 5.0 to 7.5, more preferably 6.0 to 6.5.
  • the aforementioned pharmaceutical composition comprises:
  • the pH of the pharmaceutical composition is from about 6.0 to about 6.5.
  • the aforementioned pharmaceutical composition comprises:
  • the pH of the pharmaceutical composition is about 6.0 to about 6.5.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pH of the pharmaceutical composition is about 6.2.
  • TGF- ⁇ receptor fusion protein in the aforementioned pharmaceutical composition is represented by the general formula (I):
  • TGF- ⁇ RII ECD is a truncated form of TGF- ⁇ RII extracellular domain
  • Ab is the PD-L1 antibody or antigen-binding fragment thereof
  • L is the connection sequence.
  • the linking sequence described in the aforementioned pharmaceutical composition is (G 4 S) x G, where x is an integer of 3-6. In an alternative embodiment, x is 3, 4, 5 or 6, preferably 4.
  • the truncated form of the aforementioned extracellular domain of TGF- ⁇ RII is the sequence of the extracellular domain of TGF- ⁇ RII (as shown in SEQ ID NO: 14) continuous at its amino terminal (also called N-terminal) Up to 26 amino acid residues are deleted.
  • the truncated form of the extracellular domain of TGF- ⁇ RII is that the sequence of the extracellular domain of TGF- ⁇ RII is continuously deleted at its N-terminus 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 amino acid residues.
  • the sequence of TGF- ⁇ RII ECD described in the aforementioned pharmaceutical composition is shown in SEQ ID NO: 14, 15, 16 or 17; preferably the sequence shown in SEQ ID NO: 15.
  • the PD-L1 antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition comprises:
  • HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and
  • LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the PD-L1 antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition has:
  • HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 10 and SEQ ID NO: 3, and
  • LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
  • the PD-L1 antibody or antigen-binding fragment thereof described in the aforementioned pharmaceutical composition has:
  • the heavy chain amino acid sequence of the PD-L1 antibody described in the prodrug composition is as shown in SEQ ID NO: 12 or has at least 85%, 86 as shown in SEQ ID NO: 12 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity;
  • the light chain amino acid sequence of the PD-L1 antibody is shown in SEQ ID NO: 13 or the sequence shown in SEQ ID NO: 13 has at least 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the aforementioned pharmaceutical composition wherein in the TGF- ⁇ receptor fusion protein, the TGF- ⁇ RII ECD is fused to the carboxy terminus of the PD-L1 antibody heavy chain through a linking sequence.
  • the TGF- ⁇ receptor fusion protein comprises:
  • a fusion peptide fused with PD-L1 antibody heavy chain and TGF- ⁇ RII ECD the amino acid sequence of which is shown in SEQ ID NO: 23 or the amino acid sequence shown in SEQ ID NO: 23 has at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and
  • the light chain of PD-L1 antibody has an amino acid sequence as shown in SEQ ID NO: 13 or at least 85%, 86%, 87%, 88%, 89%, 90%, and SEQ ID NO: 13 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the TGF- ⁇ receptor fusion protein comprises:
  • a fusion peptide fused with PD-L1 antibody heavy chain and TGF- ⁇ RII ECD the amino acid sequence of which is shown in SEQ ID NO: 24 or the amino acid sequence shown in SEQ ID NO: 24 is at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, and
  • the light chain of PD-L1 antibody has an amino acid sequence as shown in SEQ ID NO: 13 or at least 85%, 86%, 87%, 88%, 89%, 90%, and SEQ ID NO: 13 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the present disclosure also provides a method of preparing the aforementioned pharmaceutical composition, which includes the step of contacting the TGF- ⁇ receptor fusion protein with a buffer, for example, replacing the TGF- ⁇ receptor fusion protein stock solution with a buffer, preferably the buffer Citrate buffer, more preferably citrate-sodium citrate buffer, the buffer concentration is preferably about 5mM to about 20mM, non-limiting examples include 5mM, 6mM, 7mM, 8mM, 9mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, more preferably 10 mM; the buffer pH is about 6.0 to about 6.5, non-limiting examples include 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably 6.2.
  • the buffer concentration is 5 mM to 20 mM, non-limiting examples include about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 14 mM, about 16 mM , About 18mM, about 20mM, more preferably about 10mM;
  • the buffer pH is 6.0 to 6.5, non-limiting examples include about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, preferably about 6.2 .
  • the present disclosure also provides a method for preparing the aforementioned pharmaceutical composition.
  • the method further includes: adding sucrose and polysorbitol 80 to the resulting solution (the two do not distinguish the order) And then make up to volume with buffer, wherein the buffer concentration is preferably about 5mM to about 20mM, more preferably 5mM to 20mM, non-limiting examples include 5mM, 8mM, 10mM, 12mM, 14mM, 16mM, 18mM, 20mM
  • the buffer pH is about 6.0 to about 6.5, non-limiting examples include 6.0, 6.1, 6.2, 6.3, 6.4, 6.5.
  • the present disclosure also provides a method for preparing a lyophilized preparation containing a TGF- ⁇ receptor fusion protein, which includes the step of freeze-drying the aforementioned pharmaceutical composition.
  • the aforementioned method for preparing a lyophilized preparation containing a TGF- ⁇ receptor fusion protein wherein the lyophilization is performed according to methods known in the art, such as, but not limited to, including pre-freezing, primary drying, and two The second drying step.
  • methods known in the art such as, but not limited to, including pre-freezing, primary drying, and two The second drying step.
  • the present disclosure also provides a freeze-dried preparation containing TGF- ⁇ receptor fusion protein, which is prepared by the aforementioned method for preparing a freeze-dried preparation.
  • the present disclosure also provides a lyophilized preparation containing the TGF- ⁇ receptor fusion protein.
  • the lyophilized preparation containing the TGF- ⁇ receptor fusion protein can be reconstituted to form the aforementioned pharmaceutical composition.
  • the lyophilized formulation 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. In some embodiments, the lyophilized formulation is stable at 40 ° C for at least 7 days, at least 14 days, or at least 28 days.
  • the present disclosure also provides a reconstituted solution containing the TGF- ⁇ receptor fusion protein, which is obtained by reconstituting the aforementioned freeze-dried preparation containing the TGF- ⁇ receptor fusion protein.
  • the present disclosure also provides a method for preparing a reconstituted solution containing the aforementioned TGF- ⁇ receptor fusion protein, which includes the steps of reconstituting the aforementioned lyophilized preparation.
  • the solution used for reconstitution includes but is not limited to water for injection and physiological saline Or glucose solution, preferably water for injection.
  • the present disclosure further provides an article or kit comprising: a pharmaceutical composition and a container according to the present disclosure.
  • the container is a glass bottle, such as but not limited to a neutral borosilicate glass controlled injection bottle.
  • the present disclosure also provides an article, which includes a container containing the aforementioned pharmaceutical composition, or a lyophilized preparation thereof, or a reconstituted solution of the lyophilized preparation.
  • the present disclosure also provides the use of any one selected from the following in the preparation of a medicament:
  • the aforementioned pharmaceutical composition, or lyophilized preparation, or reconstituted solution, or preparation of the lyophilized preparation; the drug is used to treat or inhibit a disease or condition in which tumor cell proliferation or metastasis is achieved.
  • the disease or disorder is a tumor.
  • the disease or disorder is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, renal cancer, cervical cancer, myeloma cancer, lymphoma, leukemia, thyroid cancer, Endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, Squamous cell skin cancer, protuberant skin fibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.
  • the present disclosure also provides the former method of treating or inhibiting a disease or disorder in which cancer cell proliferation or metastasis includes providing a therapeutically effective amount of the aforementioned pharmaceutical composition, or a lyophilized preparation, or a reconstituted solution to a desired subject, or product.
  • the method comprises administering to the subject a unit dose of the composition containing 0.1-3000 mg of the TGF- ⁇ receptor fusion protein as described above, a pharmaceutical composition, or a lyophilized formulation, or a complex Solution or product.
  • the disease or disorder is a tumor.
  • the disease or disorder is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, renal cancer, cervical cancer, myeloma cancer, lymphoma, leukemia, thyroid cancer, Endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, Squamous cell skin cancer, protuberant skin fibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.
  • the present invention also provides the aforementioned TGF- ⁇ receptor fusion protein, pharmaceutical composition, or lyophilized preparation, or reconstituted solution, or preparation for treating or inhibiting a disease or disorder in which cancer cell proliferation or metastasis.
  • the disease or disorder is a tumor.
  • the disease or disorder is selected from the group consisting of colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, renal cancer, cervical cancer, myeloma cancer, lymphoma, leukemia, thyroid cancer, Endometrial cancer, uterine cancer, bladder cancer, neuroendocrine cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, Squamous cell skin cancer, protuberant skin fibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.
  • Figure 1 Schematic diagram of the fusion protein structure.
  • Figure 2 Results of fusion protein binding to human TGF- ⁇ 1 in vitro.
  • Figure 3 Results of fusion protein binding to human TGF- ⁇ 1 in vitro.
  • Figure 4 Results of fusion protein binding to human PD-L1 in vitro.
  • Figure 5 Fusion protein in vitro detection of PD-1 / PD-L1 pathway blocking experiment results.
  • FIG. 6 Fusion protein inhibits TGF ⁇ -induced pSMAD3 reporter activity in a dose-dependent manner.
  • Figure 7 Fusion protein samples are able to enhance the secretion of cytokine IFN- ⁇ by activated T lymphocytes.
  • Figure 8 Effect of fusion protein on tumor weight in tumor-bearing mice.
  • Buffer refers to a solution that is resistant to changes in pH through the action of its acid-base conjugate component.
  • buffers for controlling the pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate Salt, glycylglycine.
  • Hetidine buffer is a buffer containing histidine ions.
  • Histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and other buffers, preferably histidine-hydrochloride Buffer. Histidine-hydrochloride buffer is prepared from histidine and hydrochloric acid.
  • citrate buffer is a buffer that includes citrate ions.
  • the citrate buffer include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like.
  • the preferred citrate buffer is citric acid-sodium citrate.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffers include sodium succinate-succinate, potassium succinate-succinate, calcium succinate-succinate, and the like.
  • the preferred succinate buffer is succinic acid-sodium succinate.
  • Phosphate buffer is a buffer that includes phosphate ions.
  • phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and the like.
  • the preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.
  • Acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetate-sodium acetate, histidine acetate, acetate-potassium acetate, calcium acetate, acetate-magnesium acetate, and the like.
  • the preferred acetate buffer is acetic acid-sodium acetate.
  • “Pharmaceutical composition” means a mixture containing one or more compounds described herein or a physiological / pharmaceutically acceptable salt or prodrug thereof with other chemical components, such as physiological / 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 living body, facilitate the absorption of the active ingredient and exert the biological activity.
  • “pharmaceutical composition” and “formulation” are not mutually exclusive.
  • the solution form of the pharmaceutical composition described in this disclosure is water.
  • “Lyophilized preparation” means a pharmaceutical composition or a pharmaceutical composition obtained after a pharmaceutical composition or a liquid or solution formulation in the form of a liquid or a solution after a freeze-drying step (for example, a vacuum freeze-drying step).
  • the term “about” or “approximately” means that the index value is within an acceptable error range for a specific value determined by one of ordinary skill in the art, and the numerical value depends in part on how to measure or determine (ie, the limit of the measurement system). For example, “about” or “approximately” in the art may mean within 1 or more than 1 standard deviation. Alternatively, “about” or “approximately” or “substantially encompasses a range representing up to 20%. In addition, especially for biological systems or processes, the term may mean up to an order of magnitude or up to 5 times the value.
  • the pharmaceutical composition described in the present disclosure can achieve a stable effect: wherein the TGF- ⁇ receptor fusion protein or its pharmaceutical composition substantially retains its physical stability and / or chemical stability and / or biological properties after storage Chemical activity; preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage.
  • the storage period is generally selected based on the predetermined shelf life of the pharmaceutical composition.
  • analytical techniques for measuring the stability of active ingredients which can measure the stability after storage at a selected temperature for a selected period of time.
  • Stable pharmaceutical antibody or protein preparations are those in which no significant changes are observed when stored at a refrigerated temperature (2-8 ° C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably Up to 2 years.
  • stable liquid preparations include liquid preparations that exhibit desired characteristics after storage at a temperature including 25 ° C for a period of 1 month, 3 months, or 6 months or at 40 ° C for 28 days.
  • Typical acceptable criteria for stability are as follows: as measured by SEC-HPLC, usually no more than about 10%, preferably no more than about 5% of active ingredients (eg, proteins, antibodies) degrade.
  • active ingredients eg, proteins, antibodies
  • the pharmaceutical preparation is light yellow nearly colorless clear liquid or colorless, or clear to slightly milky white, or light yellow nearly colorless clear liquid.
  • concentration, pH and osmolality of the formulation have a variation not exceeding ⁇ 10%. Usually a truncation of no more than about 10%, preferably no more than about 5% is observed. Usually no more than about 10%, preferably no more than about 5%, of aggregates are formed.
  • the antibody If visually inspected for color and / or clarity, or measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), the antibody shows no significant increase in aggregation, precipitation, and / or denaturation , Then the active ingredient "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).
  • the active ingredient eg protein or antibody
  • the active ingredient eg protein or antibody
  • the active ingredient "retains its chemical stability" in the pharmaceutical formulation.
  • the degradation processes that often change the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), and oxidation (by peptide mapping methods such as mass spectrometry or MALDI / TOF / MS) Method to evaluate), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement), and isomerization (by measuring the content of isoaspartic acid, (Peptide mapping method, etc.).
  • the biological activity of the active ingredient eg protein or antibody
  • the biological activity of the active ingredient can be determined, for example, by an antigen binding assay.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through interchain disulfide bonds.
  • the antibody light chain of the present disclosure may comprise a light chain constant region, which comprises human or murine-derived kappa, lambda chains or variants thereof.
  • the antibody heavy chain of the present disclosure may include a heavy chain constant region that contains human or murine IgG1, IgG2, IgG3, IgG4, or a variant thereof.
  • the sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences.
  • the three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each light chain variable region (LCVR or VL) and heavy chain variable region (HCVR or VH) are composed of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1 FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues in the LCVR region and HCVR region of the antibody or antigen-binding fragment described in this disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3), or the numbering positions of kabat and chothia , "Kabat" numbering rules (see Kabat et al.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.
  • the "antibody or antigen-binding" or “functional fragment” described in this disclosure refers to a Fab fragment having an antigen-binding activity, a Fab 'fragment, an F (ab') 2 fragment, and an Fv fragment scFv fragment that binds to an antibody.
  • the Fv fragment contains the variable region of the antibody heavy chain and the variable region of the light chain, but no constant region, and is the smallest antibody fragment with all antigen binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding.
  • Different linkers can also be used to connect the variable regions of two antibodies into a single polypeptide chain, called single chain antibody (single chain antibody) or single chain Fv (sFv).
  • single chain antibody single chain antibody
  • single chain Fv single chain Fv
  • the term "associated with PD-L1" of the present disclosure refers to the ability to interact with human PD-L1.
  • antigen binding site of the present disclosure refers to a discontinuous or continuous three-dimensional space site on an antibody or antigen binding fragment, which can recognize a target antigen and specifically bind to the antigen.
  • murine antibody in this disclosure is a monoclonal antibody against human PD-L1 prepared according to the knowledge and skills in the art. During preparation, the test subjects are injected with PD-L1 antigen, and then hybridomas expressing antibodies with desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody obtained by fusing the variable region of a non-human (eg mouse) antibody with the constant region of a human antibody, which can reduce the immunity induced by the non-human (eg mouse) antibody Response response.
  • a chimeric antibody you must first establish a hybridoma that secretes specific monoclonal antibodies, then clone the variable region gene from the hybridoma cells, and then clone the constant region gene of the human antibody as needed.
  • the variable region gene and the human constant region gene are connected into a chimeric gene and inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the PD-L1 chimeric antibody further comprises a light chain constant region of human ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the PD-L1 chimeric antibody further comprises a heavy chain constant region of human-derived IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the constant region of the human antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof, preferably comprising the human heavy chain constant region of IgG2 or IgG4, or without amino acid mutations without ADCC (antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG4.
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody)
  • CDR-grafted antibody refers to the non-human (eg, mouse) antibody CDR sequence grafted to the human antibody variable region framework, that is different Types of antibodies produced in the framework sequence of human germline antibodies. It can overcome the strong antibody variable antibody reaction induced by the chimeric antibody due to carrying a large number of non-human (eg mouse) protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc. People, 1991 Sequences of Proteins of Immunological Interest, found in the 5th edition.
  • the framework sequence of the variable region of the human antibody may be subjected to the least reverse mutation or back mutation to maintain the activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies obtained after affinity maturation of CDRs by phage display.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the ADCC effect function of the antibody can be reduced or eliminated by modifying the Fc segment on IgG.
  • the modification refers to mutation in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2 / 4 chimera, F234A / L235A mutation of IgG4.
  • sequence identity refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • the sequence identity in this disclosure may be at least 85%, 90% or 95%, preferably at least 95%.
  • Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%.
  • Sequence comparison and percent identity determination between two sequences can be performed by the default settings of the BLASTN / BLASTP algorithm available on the National Center For Biotechnology Institute website.
  • TGF- ⁇ receptor II or “TGF ⁇ RII” or “transforming growth factor ⁇ receptor II” refers to binding ligands (including but not limited to TGF ⁇ 1, TGF ⁇ 2 and TGF ⁇ 3), and thereby triggers intracellular signal transduction Pathway of cell surface receptors.
  • PD-L1 refers to Programmed Death Ligand 1, also known as CD274 and B7H1.
  • PD-L1 is a 290 amino acid protein with extracellular IgV-like and IgC-like domains (amino acids 19-239 of full-length PD-L1), a transmembrane domain, and an intracellular domain of about 30 amino acids.
  • PD-L1 is constitutively expressed on many cells such as antigen-presenting cells (eg, dendritic cells, macrophages, and B cells) as well as hematopoietic cells and non-hematopoietic cells (eg, vascular endothelial cells, islets, and immune pardon sites) .
  • antigen-presenting cells eg, dendritic cells, macrophages, and B cells
  • hematopoietic cells and non-hematopoietic cells eg, vascular endothelial cells, islets, and immune pardon sites
  • PD-L1 is also expressed on various tumor and virus-infected cells and is a member of the immunosuppressive environment (Ribas 2012, NEJM 366: 2517-2519). PD-L1 binds to one of two T cell co-inhibitors PD-1 and B7-1.
  • the "PD-L1 antibody or antigen-binding protein thereof" described in the present disclosure may include any anti-PD-L1 antibody or antigen-binding fragment thereof described in the art.
  • the anti-PD-L1 antibody may be a commercially available PD-L1 antibody that has been disclosed in the literature. Including but not limited to BMS-936559, MPDL3280A, MEDI4736, MSB0010718C (see US2014341917, US20130034559, US8779108), etc.
  • the antibody may be a monoclonal antibody, a chimeric antibody, a humanized antibody, or a human antibody.
  • Antibody fragments include Fab fragments having antigen-binding activity, Fab 'fragments, F (ab') 2 fragments, and Fv fragments and scFv fragments that bind to antibodies.
  • HCDR1 SYWMH SEQ ID NO: 1
  • HCDR2 RIX 1 PNSG X 2 TSYNEKFKN SEQ ID NO: 2
  • HCDR3 GGSSYDYFDY SEQ ID NO: 3
  • X 1 is selected from H or G and X 2 is selected from G or F.
  • the exemplary PD-L1 antibody of the present disclosure further includes the CDR sequence of the light chain variable region as described below:
  • LCDR1 RASESVSIHGTHLMH SEQ ID NO: 4
  • LCDR3 QQSFEDPLT SEQ ID NO: 6;
  • the present disclosure uses the CDR grafting strategy to humanize antibodies to the above CDR regions.
  • the humanized light chain templates of the humanized framework are IGKV7-3 * 01 and hjk2.1, and the humanized heavy
  • the chain templates are IGHV1-46 * 01 and hjh6.1, and the sequence of the humanized variable region is as follows:
  • Humanized PD-L1 antibody heavy chain variable region Humanized PD-L1 antibody heavy chain variable region:
  • Humanized PD-L1 antibody light chain variable region Humanized PD-L1 antibody light chain variable region:
  • the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic in the sequence is the FR sequence, and the underline is the CDR sequence (CDR amino acid residues are determined and annotated by the Kabat numbering system).
  • the back mutation design is shown in Table 1 below:
  • Y91F means to mutate Y back to F at position 91 (natural order number).
  • "Implanted” means that the mouse antibody CDR is implanted into the human germline FR region sequence.
  • New humanized antibodies can be obtained by combining various light and heavy chain mutations in Table 1.
  • Another aspect of the present disclosure provides an embodiment for constructing a humanized clone, as follows:
  • Primer design use online software DNAWorks (v3.2.2) (http://helixweb.nih.gov/dnaworks/) to design multiple primers to synthesize VH / VK containing the required gene fragments for recombination: 5'-30bp signal peptide VH / VK + 30bp CH1 / CL-3 '.
  • Fragment splicing in accordance with the TaKaRa Company Primer STAR GXL DNA polymerase operating instructions, using the multiple primers designed above, two-step PCR amplification to obtain VH / VK gene fragments containing recombination.
  • pHr signal peptide and constant region gene (CH1-Fc / CL) fragment
  • the recognition sequence and the restriction site have different characteristics to design and construct the expression vector pHr (signal peptide and constant region gene (CH1-Fc / CL) fragment).
  • BsmBI digests the carrier, and the gel is recovered for later use.
  • VH / VK contains the required gene fragments for recombination and BsmBI digestion to recover the expression vector pHr (signal peptide and constant region gene (CH1-Fc / CL) fragments) were added to the DH5H competent cells in a 3: 1 ratio, 0 °C ice Bath for 30min, heat shock at 42 ° C for 90s, add 5 volumes of LB medium, incubate at 37 ° C for 45min, coat LB-Amp plate, incubate at 37 ° C overnight, pick single clones and send for sequencing to obtain each clone of interest.
  • pHr signal peptide and constant region gene (CH1-Fc / CL) fragments
  • HCDR2 is SEQ ID NO: 7
  • X 1 is G
  • X 2 is F, that is, the sequence is:
  • the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic in the sequence is the FR sequence; the underline is the CDR sequence (the amino acid residues of the CDR are determined and annotated by the Kabat numbering system).
  • Another aspect of the present disclosure is to provide an example of constructing and expressing anti-PD-L1 human IgG4 type, and further used for fusion protein construction of PD-L1 antibody.
  • This PD-L1 antibody can also be used as a test case control molecule in this disclosure.
  • mutant IgG4 to obtain antibodies without ADCC and CDC.
  • the clones obtained by affinity maturation were converted into IgG4 type, and the core hinge region of IgG4 contained S228P (corresponding to the natural order of sequence SEQ ID NO: 12 position 227) mutation.
  • F234A corresponding to the sequence SEQ ID NO: 12 in the natural order 233
  • L235A corresponding to the sequence SEQ ID NO: 12 in the natural order 234
  • the last K of the PD-L1 antibody heavy chain is mutated to A (corresponding to the sequence SEQ ID NO: 12 The last bit) to increase the stability of the fusion protein.
  • the sequence of the PD-L1 antibody used in the construction of the fusion protein of the present disclosure is as follows:
  • PD-L1 antibody heavy chain sequence IgG4 (AA) (S228P)
  • the underlined part is the heavy chain variable region sequence, and the underlined part is the heavy chain constant region sequence (the italic part is the mutation site);
  • the underlined part is the light chain variable region sequence
  • the ununderlined part is the light chain constant region sequence
  • the fusion protein described in this disclosure is a protein product of two genes co-expressed by DNA recombination.
  • Methods for producing and purifying antibodies and antigen-binding fragments are well known in the prior art (such as Cold Spring Harbor's Antibody Experimental Technology Guide, Chapters 5-8 and 15).
  • mice can be immunized with human PD-L1 or fragments thereof, the obtained antibodies can be renatured and purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the invention is genetically engineered to add one or more human FR regions to a non-human CDR region.
  • Human FR germline sequences can be obtained from the IMmMunoGeneTics (IMGT) website http://imgt.cines.fr by aligning the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351 obtain.
  • IMGT IMmMunoGeneTics
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminal sites in the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human PD-L1. Positive clones were expanded in serum-free medium in the bioreactor to produce antibodies.
  • the antibody-secreted culture fluid can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer is used for purification. Non-specifically bound components are washed away. Then, the bound antibody was eluted by PH gradient method, and the antibody fragments were detected by SDS-PAGE and collected. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as -70 °C, or lyophilized.
  • the "immunomodulatory molecules" described in this disclosure can be used to impair the immune tolerance of cancer cells.
  • the present disclosure adopts a truncated form of the extracellular domain of TGF- ⁇ RII as the immunomodulatory molecular part in the fusion protein.
  • TGF- ⁇ receptor II TGF- ⁇ RII
  • the TGF- ⁇ RII / TGF- ⁇ complex recruits TGF- ⁇ RI to form a signal transduction complex (Won et al. Cancer Res. 1999; 59: 1273-7).
  • the extracellular domain of TGF- ⁇ RII is a stretch of 136 amino acid residues from the N-terminus of TGF- ⁇ RII cells.
  • SEQ ID NO: 14 An exemplary example is shown in SEQ ID NO: 14.
  • Other variants with a length of about 136 amino acids and derived from human having an extracellular region of TGF- ⁇ RII capable of binding to TGF- ⁇ 1 and TGF- ⁇ 3 also belong to the scope of the extracellular domain of TGF- ⁇ RII of the present disclosure .
  • the research in this disclosure found that the structure and function of the continuous truncated form of the N-terminus of the extracellular domain of TGF- ⁇ RII is more stable than that of untruncated molecules.
  • the fusion protein in the form of an untruncated N-terminus of TGF- ⁇ RII extracellular domain (1-136 polypeptide shown in SEQ ID NO: 14) is easily broken.
  • TGF- ⁇ RII extracellular domain is more stable after truncation of consecutive 26 amino acids at the N-terminus, preferably N-terminus is truncated at 14-26 amino acids, and more preferably N-terminus is 14-21 amino acids
  • the truncated form has a higher expression level, and most preferably the N-terminus is truncated with 19 or 21 consecutive amino acids.
  • TGF-beta receptor fusion protein is a fusion protein comprising TGF-beta receptor.
  • the TGF- ⁇ receptor fusion protein of the present disclosure is the TGF- ⁇ receptor fusion protein described in the international patent application PCT / CN2018 / 086451 (WO 2018205985 A1), the entire contents of WO 2018205985 A1 are fully incorporated This disclosure.
  • the TGF- ⁇ receptor fusion protein is a PD-L1 antibody / TGF- ⁇ RII extracellular domain fusion protein (PD-L1 / TGF- ⁇ trap), which uses the TGF- ⁇ RII extracellular domain as a fusion
  • the immunomodulatory molecular part of the protein, the PD-L1 antibody serves as the targeting part of the fusion protein, and the extracellular domain of TGF- ⁇ RII (such as shown in SEQ ID NO: 14, 15, 16, or 17) is connected by a connecting sequence (such as (G 4 S) x G, x is 3-6) connected to the C-terminus (also called carboxy terminus) of the heavy chain of the PD-L1 antibody to form a fusion sequence, and the fusion sequence and the light chain of the PD-L1 antibody are connected by an interchain disulfide bond to form a final
  • the structure of PD-L1 / TGF- ⁇ trap fusion protein is shown in Figure 1.
  • the TGF- ⁇ receptor fusion protein is shown in
  • Linker refers to a connecting polypeptide sequence used to connect protein domains, usually with a certain degree of flexibility. Lost.
  • the linking sequence is (G 4 S) x G, where x is 3-6, for example, the linking sequence is: (G 4 S) 3 G, (G 4 S) 4 G, (G 4 S) 5 G or (G 4 S) 6 G and other polypeptides.
  • Constant modification or “conservative substitution or substitution” means that other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) replace amino acids in the protein so that frequent Make changes without changing the biological activity of the protein.
  • Those skilled in the art know that, generally speaking, a single amino acid substitution in a non-essential region of a polypeptide does not substantially change biological activity (see, for example, Watson et al. (1987) Molecular Biology of The Gene, The Benjamin / Cummings Pub. Co., Page 224, (4th edition)).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions and animals , Humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids contact cells.
  • administering “administering,” and “treating” also mean in vitro and ex vivo treatment of, for example, cells by an agent, diagnosis, binding composition, or by another cell.
  • administering or “treatment” when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
  • Treatment means administration of a therapeutic agent for internal or external use to a subject, for example, comprising a composition of the present disclosure, the subject has one or more disease symptoms, and the therapeutic agent is known to have a treatment for these symptoms effect.
  • the therapeutic agent is administered to the treated subject or population in an amount effective to relieve the symptoms of one or more diseases to induce the regression of such symptoms or inhibit the development of such symptoms to any clinically measurable extent.
  • the amount of therapeutic agent effective to relieve the symptoms of any specific disease may vary according to various factors, such as the disease state, age and weight of the subject, and the effect of the drug in the subject ability.
  • embodiments of the present disclosure may be ineffective in relieving the symptoms of each target disease, according to any statistical test methods known in the art such as Student's test, chi-square test, according to Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should alleviate the target disease symptoms in a statistically significant number of subjects.
  • Effective amount includes an amount sufficient to improve or prevent the symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject or veterinary subject can vary depending on factors such as the condition to be treated, the general health of the subject, the route and dose of administration, and the severity of side effects.
  • the effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Tm value refers to the temperature of protein thermal denaturation, that is, the temperature when half of the protein is unfolded. At this time, the spatial structure of the protein is destroyed, so the higher the Tm value, the higher the thermal stability of the protein.
  • Replacement refers to the replacement of a solvent system that dissolves antibody proteins.
  • a high-salt or hypertonic solvent system containing antibody proteins is physically replaced with a buffer system of a stable preparation, so that the antibody protein is present in the stable preparation.
  • the so-called physical operation methods include but are not limited to ultrafiltration, dialysis or reconstitution after centrifugation.
  • test examples or preparation examples are for illustrative purposes only and are not intended to limit the scope of the present disclosure.
  • the experimental methods in the examples, test examples or preparation examples of the present disclosure without specifying specific conditions are generally in accordance with conventional conditions; or in accordance with the conditions recommended by the manufacturers of raw materials or commodities.
  • Reagents that do not indicate a specific source are conventional reagents purchased on the market.
  • the extracellular domain of TGF- ⁇ RII (full-length or truncated form of SEQ ID NO: 14) is used as the immunomodulatory molecular part in the fusion protein, and the PD-L1 antibody is used as the target part of the fusion protein to form the PD-L1 antibody / TGF- ⁇ RII extracellular domain fusion protein (PD-L1 / TGF- ⁇ trap).
  • the truncated form of the extracellular domain of TGF- ⁇ RII is more stable in structure and function, especially after truncating the N-terminus of less than 26 amino acids, it is more stable, preferably 14-26 amino acids.
  • Non-limiting example sequences of the TGF- ⁇ RII extracellular domain and truncated forms of the present disclosure are as follows:
  • TGF- ⁇ RII extracellular domain sequence ECD (1-136)
  • TGF- ⁇ RII extracellular domain sequence has a truncation or deletion of 19 amino acids at the N-terminus: ECD (20-136)
  • TGF- ⁇ RII extracellular domain sequence has 21 amino acids truncated or deleted at the N-terminus: ECD (22-136)
  • TGF- ⁇ RII extracellular domain sequence has 14 amino acids truncated or deleted at the N-terminus: ECD (15-136)
  • the C-terminus of the heavy chain of the PD-L1 antibody of the present disclosure (such as the heavy chain shown in SEQ ID NO: 12 and the light chain shown in SEQ ID NO: 13) using homologous recombination technology
  • Amino acids are connected to the extracellular regions of TGF- ⁇ RII of different lengths through a linker ((G 4 S) x G, x is 3-6), and together with the PD-L1 antibody light chain, are routinely expressed by the 293 expression system to obtain
  • Ab is the PD-L1 antibody shown in SEQ ID NO: 12 for the heavy chain sequence and SEQ ID NO: 13 for the light chain sequence.
  • the ECD (n-136) in the sequence description is TGF- ⁇ RII
  • the full-length or truncated form of the extracellular domain, n is the starting number of amino acids after truncation of the extracellular domain of TGF- ⁇ RII.
  • the structure of the fusion protein of the present disclosure is shown in FIG. 1; N19A is the mutation of amino acid 19 from N to A in the extracellular region of the full-length TGF- ⁇ RII (as shown in SEQ ID NO: 14).
  • the nucleotide sequence encoding the PD-L1 antibody, the nucleotide sequence encoding the extracellular region of TGF- ⁇ RII, and the nucleotide sequence of the linker protein fragment ((G 4 S) x G) are obtained by conventional technical means in the art.
  • the C-terminal nucleotide of the PD-L1 antibody was connected to the N-terminal nucleotide of the extracellular region of TGF- ⁇ RII of different lengths through a linker protein, and cloned into the Phr-BsmbI vector.
  • Recombinant PD-L1 / TGF- ⁇ trap was expressed in 293 cells and purified by Example 2. The purified protein can be used in the experiments in the following examples.
  • the supernatant was collected after centrifugation of the cell culture solution at high speed, and the first step of purification was performed by affinity chromatography.
  • the chromatographic medium is Protein A that interacts with Fc or a derivative filler, such as GE's Mabselect.
  • the equilibration buffer is 1 ⁇ PBS (137 mmol / L NaCl, 2.7 mmol / L KCl, 10 mmol / L Na 2 HPO 4 , 2 mmol / L KH 2 PO 4 , pH7.4), after equilibrating 5 times the column volume, place the cells on Clear the sample and combine, the flow rate is controlled to the sample retention time on the column ⁇ 1min.
  • the column was rinsed with 1 ⁇ PBS (pH7.4) until the UV absorption of A280 dropped to the baseline. Then, the column was washed with elution buffer of 0.1 M glycine (pH 3.0), the elution peak was collected according to the A280 ultraviolet absorption peak, and the collected elution sample was neutralized with 1 M Tris (pH 8.5).
  • the neutralized eluted sample was ultrafiltered and concentrated, and then subjected to size exclusion chromatography.
  • the buffer solution was 1 ⁇ PBS
  • the chromatography column was XK26 / 60 Superdex200 (GE)
  • the flow rate was controlled at 4 ml / min
  • the loading volume was 5ml
  • the collected protein was identified by SEC-HPLC with a purity greater than 95%, and was identified as correct by LC-MS and then used for distribution.
  • PD-L1 / TGF- ⁇ trap was obtained.
  • the following uses biochemical test methods to verify the performance and beneficial effects of the PD-L1 antibody / TGF- ⁇ fusion protein in the present disclosure.
  • Test cases biological evaluation of in vivo and in vitro activities
  • Test Example 1 ELISA detection of PD-L1 / TGF- ⁇ trap in vitro binding TGF- ⁇ 1 experiment
  • the detection process is described as follows:
  • a 96-well plate was coated with 100 ⁇ l / well of human-derived TGF- ⁇ 1 (8915LC, CST) at a concentration of 1 ⁇ g / ml at 4 ° C overnight.
  • TGF- ⁇ trap is a positive control, and incubate at 37 °C for 1 hour.
  • Test Example 2 ELISA detection of PD-L1 / TGF- ⁇ trap binding to PD-L1 in vitro
  • the detection process is described as follows:
  • a 96-well plate was coated with 100 ⁇ l / well of human-derived PD-L1-His (SEQ ID NO: 18) at a concentration of 5 ⁇ g / ml at 4 ° C overnight.
  • Test Example 3 In vitro detection of PD-1 / PD-L1 pathway blocking experiment
  • 1PD-L1 antibody heavy chain sequence is shown in SEQ ID NO: 12, light chain sequence is shown in SEQ ID NO: 13;
  • Control 1 (20T-Fc): ECD (20-136) -Fc, TGF- ⁇ RII extracellular region truncated fragment ECD (20-136) and Fc fusion protein, the sequence is as follows:
  • TGF- ⁇ receptor fusion protein prepared in Example 1 of the present disclosure: fusion protein 9, fusion protein 15:
  • the fusion peptide sequence of fusion protein 9 fused PD-L1 antibody heavy chain- (G 4 S) 4 G-TGF- ⁇ RII ECD (20-136) is as follows:
  • the normal type is the PD-L1 antibody heavy chain sequence
  • the italic type is the linking sequence
  • the underlined is the TCD- ⁇ RII extracellular region truncated fragment ECD (20-136) sequence.
  • the light chain sequence of the PD-L1 antibody in fusion protein 9 is as follows:
  • fusion peptide sequence of fusion PD-L1 antibody heavy chain- (G 4 S) 5 G-TGF- ⁇ RII ECD (22-136) in fusion protein 15 is as follows:
  • the normal type is the PD-L1 antibody heavy chain sequence
  • the italic type is the linking sequence
  • the underline is the TGF- ⁇ RII extracellular region truncated fragment ECD (22-136) sequence
  • the light chain sequence of the PD-L1 antibody in fusion protein 15 is as follows:
  • 5Human IgG blank control, human immunoglobulin purified from mixed normal human sera using traditional affinity chromatography methods such as ProteinA;
  • Positive control PD-L1 antibody 2 / TGF- ⁇ RII extracellular domain fusion protein
  • CHO / PD-L1 cells (CS187108, Promega) were taken, digested and resuspended in F-12 Nutrient Mixture (Ham) complete medium. According to the results of cell counting, adjust the cell density to 4 ⁇ 10 5 / mL using complete medium, transfer the cell suspension to the sample loading tank, add it to a 96-well plate at 100 ⁇ L / well using a multi-channel pipette, and place it in 37 °C, 5% CO 2 incubator for 20 to 24 hours; prepare Jurkat / PD-1 (CS187102, Promega) cell suspension the next day, resuspend the cells using the analysis medium according to the results of cell counting, and adjust the cell density to 1.25 ⁇ 10 6 / mL; remove the cell culture plate added with CHO / PD-L1 cells from the incubator, use a multichannel pipette to take out 95 ⁇ L of culture medium per well, and add the gradient diluted fusion protein according to 40 ⁇ L / well
  • the Bio-Glo TM Reagent was removed and its temperature returned to room temperature. Take out the cell culture plate, put it at room temperature for 5-10min, then add 40 ⁇ L Bio-Glo TM Reagent to each well, incubate in a safe cabinet for 5-10min, and read the chemiluminescence signal value using a multifunctional microplate reader.
  • the fusion protein 9 of the present disclosure can effectively block the binding of Jurkat cells expressing PD-1 molecules to CHO / PD-L1 cells like positive molecules, and has a dose-dependent effect on drug concentration.
  • Fusion protein 15 has the same level of blocking ability as fusion protein 9.
  • Protein A chip is used to capture a certain amount of PD-L1 / TGF- ⁇ trap, and then flow through human or mouse TGF- ⁇ 1 (8915LC, CST) or human PD-L1 (Sino Biological) on the surface of the chip, using Biacore real-time
  • the reaction signal was detected to obtain binding and dissociation curves, and then the biochip was washed and regenerated with glycine-hydrochloric acid (pH 1.5, GE).
  • the buffer solution used in the experiment was HBS-EP Buffer (GE).
  • the data obtained by the experiment was fitted with the BIAevaluation version 4.1 software (GE) using the (1: 1) Langmuir model, and the affinity values shown in Table 3 were obtained.
  • the fusion protein binding activity is shown in Table 3. The results show that the fusion protein 9 and fusion protein 15 of the present disclosure have extremely high affinity for human and mouse TGF- ⁇ 1 and human PD-L1.
  • HepG2 cells expressed Smad3 binding original (SBE) with luciferase reporter gene to study the inhibitory effect of PD-L1 / TGF- ⁇ trap on TGF- ⁇ 1-induced Smad3 activation, and evaluate PD-L1 / TGF- ⁇ trap based on IC50 size In vitro activity.
  • SBE Smad3 binding original
  • Test sample fusion protein 9, positive control (FP17022)
  • HepG2 cells were cultured using MEM complete medium (GE, SH30243.01) containing 10% FBS, and passaged every 3 days.
  • MEM complete medium GE, SH30243.01
  • a 96-well plate (Corning, 3903) was seeded at a density of 25,000 cells per well, and cultured at 37 ° C and 5% CO 2 for 24 hours.
  • the medium in the cell culture plate was discarded and 100 ng of 3TP-Lux plasmid was transfected per well.
  • the cells were continuously cultured at 37 ° C and 5% CO 2 for 24 hours.
  • the complete medium in the 96-well plate was discarded, and 80 ⁇ L of incomplete medium (MEM + 0.5% FBS) was added to each well.
  • IC50 value of the sample to be tested is calculated using the data processing software Graphpad Prism5.0.
  • Figure 6 shows that fusion protein 9 inhibits TGF ⁇ -induced pSMAD3 reporter activity in a dose-dependent manner and has comparable efficacy and IC 50 (concentration required to inhibit 50% of maximum activity) as the positive control FP17022.
  • IC 50 concentration required to inhibit 50% of maximum activity
  • the test result of PD-L1 antibody shows that it has no inhibitory effect (IC 50 > 500 nM).
  • Test Example 6 In vitro detection of tuberculin (TB) to stimulate PBMC to release IFN ⁇
  • PBMC peripheral blood mononuclear cells
  • the cell culture plate was placed in a 5% CO 2 incubator at 37 ° C for 3 days. Remove the cell culture plate, centrifuge (4000rpm, 10min) to take the supernatant from each well, after 10-fold dilution, use ELISA method (human IFN- ⁇ detection kit, Xin Bo Sheng, EHC102g.96) to detect the level of IFN- ⁇ .
  • ELISA method human IFN- ⁇ detection kit, Xin Bo Sheng, EHC102g.96
  • fusion protein 9 can dose-dependently enhance the secretion of cytokine IFN- ⁇ by activated T lymphocytes, and has a stronger activation effect than PD-L1 antibody, 20T-FC.
  • SD rats Three SD rats were used in the experiment, female, with light / dark adjustment for 12/12 hours, constant temperature of 24 ⁇ 3 °C, humidity of 50-60%, and free access to drinking and drinking water. Purchased from Jiesijie Experimental Animal Co., Ltd. On the day of the experiment, SD rats were separately injected with fusion protein via the tail vein at a dose of 6 mg / kg and an injection volume of 5 ml / kg.
  • the blood sampling time points are: 15min, 7h, 24h (day 2), day 3, day 4, day 6, day 8, day 10, day 15, day 15 after administration on the first day Blood was taken from the fundus vein, 200 ⁇ l each time (equivalent to taking 100 ⁇ l of serum); the collected blood samples were placed at room temperature for half an hour to agglomerate, and then centrifuged at 10000xg for 10 minutes at 4 ° C. Collect the supernatant and store it immediately at -80 ° C. The concentration of fusion protein in serum was detected by ELISA.
  • the detection process is described as follows:
  • a 96-well plate was coated with 100 ⁇ l / well of human PD-L1-His at a concentration of 2 ⁇ g / ml as an antigen, and overnight at 4 ° C.
  • the PK analysis results indicate that the fusion protein 9 of the present disclosure has a half-life of about 236h (9.8 days) in rats.
  • Test Example 8 Effect of PD-L1 / TGF- ⁇ trap on subcutaneously transplanted tumors of human breast cancer MDA-MB-231 mice
  • the mouse strain used in this experiment was a NOD / SCID female mouse (Cavens).
  • the human peripheral blood mononuclear cells used in the experiment were obtained from freshly collected blood.
  • the extraction method is as follows: heparin anticoagulated venous blood Mix with the same volume of PBS containing 2% FBS. After mixing, slowly add 25ml of diluted blood to a centrifuge tube containing 15ml of lymphocyte separation solution. Centrifuge at 1200g for 10 minutes at room temperature.
  • Tube add PBS to wash cells, centrifuge at 300g for 8 minutes at room temperature, repeat once, resuspend cells in RPMI-1640 medium containing 10% FBS, add cells to CD3 antibody (OKT3, 40ng / ml) coated in advance
  • CD3 antibody OKT3, 40ng / ml coated in advance
  • 2 ⁇ 10 6 cells (2 ml) per well were placed in a 37 ° C incubator for 4 days.
  • 4PD-L1 antibody 4mpk
  • 5PD-L1 antibody 20mpk
  • MDA-MB-231 cells were resuspended in serum-free RPMI-1640 medium, mixed with an equal volume of Matrigel, and 100 ⁇ l (2.3 ⁇ 10 6 ) were inoculated subcutaneously in the right rib of NOD / SCID mice, and the tumor volume was removed after 11 days. After being oversized and small animals, the mice were randomly divided into groups of 9 animals. 5 ⁇ 10 5 stimulated PBMCs (60 ⁇ l) were injected into the tumor tissue, and the remaining PBMCs stopped stimulation and continued to be cultured. One week later, 5 ⁇ 10 6 PBMCs (100 ⁇ l) were intraperitoneally injected into tumor-bearing mice. Treated as the first round of injection.
  • Antibody fusion protein 9 (4.8 mg / kg, 24 mg / kg) can significantly inhibit the growth of human breast cancer MDA-MB-231 mice subcutaneously transplanted tumors, showing a dose-dependent relationship between high and low doses, and excellent For each equivalent molar dose of the reference drug PD-L1 antibody (4mg / kg, 20mg / kg), TGF- ⁇ RII control molecule 20T-FC (2.14mg / kg) and combination group (PD-L1 antibody-4mg / kg + 20T-FC-2.14mg / kg).
  • Each dose of fusion protein 9 has maintained the ideal tumor suppressing effect since 14 days after administration, and its high dose has obvious advantages compared with PD-L1 antibody-20mpk (p ⁇ 0.05); 25 after administration
  • the anti-tumor effect of each antibody was the best.
  • the anti-tumor rates of the fusion protein 9 and PDL-1 antibodies were 37.24%, 52.38%, 30.24%, 28.01%, and 31.38% respectively in the high-dose and combination groups.
  • The% TGI of the low-dose group was 36.68% and 50.76%, respectively, and the tumor volume was statistically different from that of the control group (p ⁇ 0.05).
  • Test Example 9 Physical stability of PD-L1 / TGF- ⁇ trap
  • This test case is used to detect the stability of the fusion proteins 9 and 15.
  • DSC Different Scanning Calorimetry
  • Fusion protein 9 ( ⁇ %) Fusion protein 15 ( ⁇ %) 40 °C 3.39% 1.8% -80 °C freeze-thaw 1.44% 1.39%
  • ⁇ % represents the rate of change.
  • Deamidation modification is a common chemical modification in antibodies that may affect the stability in the later stage.
  • the highly deamidation modification of some amino acids in the CDR region is generally avoided as much as possible or the mutation is reduced.
  • N represents the modified asparagine, and the number represents the position where the N-terminus of the light or heavy chain begins to count.
  • the percentage represents the ratio of the deamidation modification detected by LC-MS to the total peptide signal at the site.
  • the first step take a certain amount of purified TGF- ⁇ receptor fusion protein stock solution, and use a protein-free buffer (such as 10mM, pH6.2 citrate-sodium citrate buffer) for solvent replacement (preferably ultrafiltration ), At least 6 times volume replacement by ultrafiltration membrane, protein concentration to about 70mg / mL.
  • a protein-free buffer such as 10mM, pH6.2 citrate-sodium citrate buffer
  • solvent replacement preferably ultrafiltration
  • protein concentration to about 70mg / mL.
  • Add 10 mM pH 6.2 citrate buffer to bring the volume to 50 mg / mL (other preparations to be tested or stability preparations should be prepared according to similar procedures).
  • the product is filtered and tested for sterility after controlled sampling. Pass the original solution through a 0.22 ⁇ m PVDF filter element and collect the filtrate.
  • the second step adjust the filling volume to 6.3ml, fill the filtrate in a 6ml vial, stopper and take samples at the beginning of filling, the middle of filling, and the end of filling to control the difference in volume.
  • Step 3 Turn on the capping machine, add aluminum caps, and perform capping.
  • the fourth step visual inspection to confirm that the product has no defects such as inaccurate loading.
  • Print and paste the vial label print the carton label, folding carton, boxing, sticker box label.
  • TGF- ⁇ receptor fusion protein (fusion protein 9) preparation with a protein concentration of 50 mg / ml:
  • the shaking conditions are: D1: 130rpm, D2: 200rpm, D3-D7: 300rpm; D means day, T means time, M means month.
  • TGF- ⁇ receptor fusion protein (fusion protein 9) preparation with a protein concentration of 50 mg / ml:
  • Each formulation was filtered and filled into a 2mL neutral borosilicate glass controlled injection bottle with 1.2mL / bottle, and a water needle was pressed to stopper and the lid was sealed. Store the samples at 25 ° C for stability analysis, 6 months of SEC or non-reduced CE-SDS testing.
  • T means time
  • D means day
  • M means month
  • TGF- ⁇ receptor fusion protein preparation with a protein concentration of 50 mg / ml using the following buffers of different sugar species:
  • Each formulation was filtered and filled into a 2mL neutral borosilicate glass controlled injection bottle with 1.2mL / bottle, and a water needle was pressed to stopper and the lid was sealed. Take samples for 25 °C normal temperature and 2-8 °C low temperature long-term storage experiments.
  • sucrose and trehalose have similar stability effects on the TGF- ⁇ receptor fusion protein (fusion protein 9), and sucrose was selected as the stabilizer of the TGF- ⁇ receptor fusion protein (fusion protein 9).
  • sucrose concentration is 80 mg / ml
  • the osmotic pressure is about 300 mosm / kg, which is close to isotonicity, so the sucrose concentration is 80 mg / ml.
  • TGF- ⁇ receptor fusion protein preparation with a protein concentration of 50 mg / ml using the following buffer solutions of different concentrations and types of surfactants:
  • TGF- ⁇ receptor fusion protein (fusion protein 9) with a protein concentration of 50 mg / ml using the following buffers of different types of surfactants:
  • Each formulation was filtered and filled into a 2mL neutral borosilicate glass controlled injection bottle with 1.2mL / bottle, and a water needle was pressed to stopper and the lid was sealed. Take samples for 2-8 °C low temperature long-term storage experiment.
  • T time
  • D day
  • M month
  • the TGF- ⁇ receptor fusion protein (fusion protein 9) was formulated at 50 mg / ml in 10 mM citrate-sodium citrate buffer, 80 mg / ml sucrose, 0.4 mg / ml polysorbate 80, pH 6.2. The preparation was passed through 0.22 ⁇ m PES filter membrane and PVDF filter membrane and sampled at the beginning, middle and last.
  • TGF- ⁇ receptor fusion protein (fusion protein 9) is stable during the time of contact with the filter.
  • the preparation is compatible with PES and PVDF filters. Content.
  • T means time
  • TGF- ⁇ receptor fusion protein (fusion protein 9) with a concentration of 50 mg / ml, containing 80 mg / ml sucrose, 0.4 mg / ml polysorbate Alcohol ester 80 TGF- ⁇ receptor fusion protein (fusion protein 9) preparation.
  • the antibody was filled into a 20 mL vial at 6.3 mL / bottle, placed in a lyophilizer, and lyophilized.
  • the freeze-drying procedure is pre-freezing, primary drying and secondary drying. After the freeze-drying procedure is completed, the plug is vacuumed. The reconstituted samples were compared before and after lyophilization. The results show that the reconstituted solution can maintain the good performance of the liquid formulation.
  • TGF- ⁇ receptor fusion protein fusion protein 9

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Abstract

本公开公开了一种TGF-β受体融合蛋白药物组合物及其用途。具体而言，该药物组合物包含在枸橼酸钠缓冲液中的TGF-β受体融合蛋白，所述的TGF-β受体融合蛋白包含PD-L1抗体靶向部分和TGF-βRII胞外区。除此之外，该药物组合物还可含有糖和非离子型表面活性剂。

Description

一种TGF-β受体融合蛋白药物组合物及其用途
本申请要求2018年11月09日提交的专利申请201811328326.1的优先权，其通过引用整体并入此处。
技术领域
本公开属于药物制剂领域，具体涉及一种包含PD-L1抗体/TGF-βRII胞外区融合蛋白的药物组合物，以及其作为药物的用途。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然地构成现有技术。
在肿瘤治疗中，人们早已认识到化疗所带来的高毒性，以及化疗可导致耐药性癌细胞产生。即使是使用靶向性的治疗手段，来针对过度表达或过度激活的与肿瘤生存生长相关的蛋白，仍会有癌细胞通过变异来减少或逃脱对靶向性治疗所针对通路的依赖，并利用其它的通路继续生存。
肿瘤免疫治疗近年来备受关注，是肿瘤治疗领域的焦点，较难产生耐药性是该疗法的突出优势。肿瘤免疫治疗主要通过免疫学原理和方法，提高肿瘤细胞的免疫原性和对效应细胞杀伤的敏感性，激发和增强机体抗肿瘤免疫应答。肿瘤免疫治疗将免疫细胞和效应分子输注宿主体内，两者协同机体免疫系统杀伤肿瘤、抑制肿瘤生长。
程序性死亡受体1(programmed death 1,PD-1)为CD28超家族成员。PD-1表达于活化的T细胞、B细胞及髓系细胞。PD-1有两个配体，即程序性死亡配体-1(programmed death ligand 1,PD-L1)和PD-L2。PD-L1与T细胞上的受体PD-1相互作用，在免疫应答的负调控方面发挥着重要作用。在许多人类肿瘤组织中均可检测到PD-L1蛋白的表达。肿瘤部位的微环境可诱导肿瘤细胞上的PD-L1的表达，表达的PD-L1有利于肿瘤的发生和生长，诱导抗肿瘤T细胞的凋亡。PD-1/PD-L1通路抑制剂可以阻断PD-1与PD-L1的结合，阻断负向调控信号，使T细胞恢复活性，从而增强免疫应答，因此，以PD-1/PD-L1为靶点的免疫调节对肿瘤抑制有重要的意义。
转化生长因子-β(transforming growth factor-β,TGF-β)属于调节细胞生长和分化的TGF-β超家族。TGF-β通过异源四聚体受体复合物传递信号，这个受体复合物是由两个I型和两个II型的跨膜丝氨酸/苏氨酸激酶受体组成。
TGF-β是一种多功能的细胞因子，以细胞或背景依赖的方式发挥肿瘤抑制或肿瘤促进的作用。TGF-β的肿瘤抑制作用源于其诱导多个基因表达的能力。当肿瘤发展过程中引入突变或表观遗传修饰时，癌细胞逐渐耐受TGF-β的抑制作用，最终导致肿瘤的发展。
研究发现阻断TGF-β信号传导通路能够减少肿瘤的转移。运用截短的Smad2/3显负性突变体抑制乳腺肿瘤细胞系的TGF-β信号通路，结果发现肿瘤细胞的转移能力被抑制。结肠癌的微卫星不稳定性研究发现，TGF-βRII无活性的突变，使转移减少，增加了患者术后的存活率。但总体而言，在临床治疗中单独施用TGF-β信号通路的抑制剂，其效果微弱，可能是因为TGF-β主要在肿瘤细胞内异常性高表达，而单独的TGF-β信号通路的抑制剂很难集中靶向肿瘤，而导致药效不高或抑制剂的生物利用度不高。
因此，在肿瘤微环境中靶向并中和TGF-β基础上，抑制PD-1/PD-L1通路可以使T细胞恢复活性，增强免疫应答，更有效地提高抑制肿瘤发生和发展的效果。
本申请人在先的PCT申请PCT/CN2016/104320(公开号WO2017084495)提供了一种PD-L1抗体。目前已有抗体/TGF-β受体融合蛋白公开，如WO2006074451A2、WO2009152610A1、WO2011109789A2、WO2013164694A1、WO2014164427A1、WO2015077540A2、WO9309228A1、WO9409815A1、WO2015077540A2、WO2015118175A2等。其中，默克公开一种PD-L1/TGF-β双功能融合蛋白Bintrafusp Alfa(WO2015118175，也称M7824、FP17022)，目前Bintrafusp Alfa已经在胃癌、肺癌、食管癌、NSCLC、胆道癌等肿瘤疾病中开展临床。但是，现有技术中的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。为了使抗体适合于给药，并且在储存及随后使用过程中能保持稳定性，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
本公开提供一种更利于生产和给药，性能更稳定的包含PD-L1/TGF-βRII融合蛋白的药物组合物，其包含：
-TGF-β受体融合蛋白，以及
-缓冲液，
所述缓冲液选自组氨酸盐缓冲液、琥珀酸盐缓冲液、磷酸盐缓冲液和枸橼酸盐缓冲液。
在一些实施方案中，所述缓冲液为枸橼酸盐缓冲液。在一些实施方案中，所述的组氨酸盐缓冲液选自组氨酸-盐酸缓冲液，所述的琥珀酸盐缓冲液选自琥珀酸-琥珀酸钠缓冲液，所述枸橼酸盐缓冲液选自枸橼酸-枸橼酸钠缓冲液；在一些实施方案中，所述缓冲液选自枸橼酸-枸橼酸钠缓冲液。
在可选的实施方案中，前述药物组合物中所述TGF-β受体融合蛋白浓度为大约0.5mg/ml至大约100mg/ml，优选为大约30mg/ml至大约70mg/ml。
在一些实施方案中，药物组合物中所述TGF-β受体融合蛋白浓度为0.5mg/ml至100mg/ml，优选为30mg/ml至70mg/ml。TGF-β受体融合蛋白浓度非限制性实 施例包括：大约30mg/ml、大约35mg/ml、大约40mg/ml、大约45mg/ml、大约50mg/ml、大约55mg/ml、大约60mg/ml、大约65mg/ml、大约70mg/ml，优选大约50mg/ml；
在一些实施方案中，药物组合物中所述TGF-β受体融合蛋白浓度为30mg/ml、35mg/ml、40mg/ml、45mg/ml、50mg/ml、55mg/ml、60mg/ml、65mg/ml、70mg/ml，更优选50mg/ml。
在可选的实施方案中，前述药物组合物中所述缓冲液的pH值为大约5.0到大约7.5，优选为大约6.0至大约6.5，还可选为约6.0、约6.1、约6.2、约63、约6.4、约6.5，更优选为大约6.2；
在一些实施方案中，所述缓冲液的pH值为5.0至7.5或6.0至6.5，优选为6.0、6.1、6.2、63、6.4或6.5，更优选为6.2。
在可选的实施方案中，缓冲液的浓度为约5mM至约30mM，优选为大约5mM至大约20mM，非限制性实施例包括5mM、6mM、7mM、8mM、9mM、10mM、12mM、14mM、16mM、18mM、20mM，更优选为10mM。
在一些实施方案中，缓冲液的浓度为5mM至30mM，优选为5mM至20mM，在一些实施方案中，缓冲液的浓度为约10mM、约12mM、约14mM、约16mM、约18mM、约20mM，更优选为约10mM。
在可选的实施方案中，前述药物组合物还包含糖。本公开的“糖”包含常规组合物(CH 2O) n或其衍生物，包括单糖、二糖、三糖、多糖、糖醇、还原性糖、非还原性糖等等。在一些实施方案中，所述糖选自：葡萄糖、蔗糖、海藻糖、乳糖、果糖、右旋糖苷、甘油、赤藻糖醇、丙三醇、阿拉伯糖醇、木糖醇(X ylitol)、山梨糖醇、甘露醇、密里二糖、松三糖、蜜三糖、甘露三糖、水苏糖、麦芽糖、乳果糖、麦芽酮糖、山梨醇、麦芽糖醇、乳糖醇、异-麦芽酮糖等等。优选的糖是非还原性二糖，更优选为海藻糖或蔗糖，最优选为蔗糖。
在可选的实施方案中，前述药物组合物中糖的浓度为约50mg/ml至约100mg/ml，优选为约60mg/ml至约90mg/ml，非限制性实施例包括60mg/ml、65mg/ml、70mg/ml、75mg/ml、80mg/ml、85mg/ml、90mg/ml，最优选为80mg/ml。
在一些实施方案中，糖的浓度为50mg/ml至100mg/ml，优选为60mg/ml至90mg/ml，在一些实施方案中，糖的浓度为约60mg/ml、约65mg/ml、约70mg/ml、约75mg/ml、约80mg/ml、约85mg/ml或约90mg/ml。
在可选的实施方案中，前述药物组合物还包含表面活性剂，可选自聚山梨醇酯20、聚山梨醇酯80、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜 菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等等。优选的表面活性剂是聚山梨醇酯80或聚山梨醇酯20，更优选为聚山梨醇酯80。
在可选的实施方案中，前述药物组合物中表面活性剂的浓度为约0.1mg/ml至约0.8mg/ml，更优选为约0.4mg/ml至约0.8mg/ml，在一些实施方案中，表面活性剂的浓度为0.1mg/ml至0.8mg/ml，优选为0.4mg/ml至0.8mg/ml，更优选约0.4mg/ml、约0.45mg/ml、约0.5mg/ml、约0.55mg/ml、约0.6mg/ml、约0.7mg/ml、约0.8mg/ml。
在一些实施方案中，表面活性剂的浓度为0.4mg/ml、0.45mg/ml、0.5mg/ml、0.55mg/ml、0.6mg/ml、0.7mg/ml或0.8mg/ml，更具体为0.4mg/ml。
在可选的实施方案中，前述药物组合物，其包含：
(a)大约0.5mg/ml至大约100mg/ml的TGF-β受体融合蛋白，(b)大约5mM至大约30mM的枸橼酸盐缓冲液，(c)大约50mg/ml至大约100mg/ml的蔗糖，和(d)大约0.1mg/ml至大约0.8mg/ml的聚山梨醇酯80，优选所述药物组合物的pH为大约5.0至大约7.5，更优选为大约6.0至大约6.5。
在可选的实施方案中，前述药物组合物，其包含：
Figure PCTCN2019116593-appb-000001
优选所述药物组合物的pH为5.0至7.5，更优选为6.0至6.5。
在可选的实施方案中，前述药物组合物包含：
(a)大约30mg/ml至大约70mg/ml的TGF-β受体融合蛋白，(b)大约5mM至大约20mM的枸橼酸-枸橼酸钠缓冲液，(c)大约60mg/ml至大约90mg/ml的蔗糖，和(d)大约0.4mg/ml至大约0.8mg/ml的聚山梨醇酯80，优选地，所述药物组合物的pH为大约6.0至大约6.5。
在可选的实施方案中，前述药物组合物包含：
Figure PCTCN2019116593-appb-000002
所述药物组合物的pH为大约6.0至大约6.5。
在可选的实施方案中，所述药物组合物包含：
(a)大约50mg/ml的TGF-β受体融合蛋白，(b)大约10mM的枸橼酸-枸橼酸钠缓冲液，(c)大约80mg/ml的蔗糖，和(d)大约0.4mg/ml的聚山梨醇酯80， 优选所述药物组合物的pH约为6.2。
在可选的实施方案中，所述药物组合物包含：
Figure PCTCN2019116593-appb-000003
优选所述药物组合物的pH约6.2。
在可选的实施方案中，前述药物组合物中所述的TGF-β受体融合蛋白如通式(I)所示：
Ab-L-TGF-βRII ECD      (I)
其中TGF-βRII ECD为TGF-βRII胞外区的截短形式；
Ab为PD-L1抗体或其抗原结合片段；
L为连接序列。
在可选的实施方案中，前述药物组合物中所述的连接序列为(G 4S) xG，其中x为3-6的整数。在可选的实施方案中，x为3、4、5或6，优选为4。
在可选的实施方案中，前述TGF-βRII胞外区的的截短形式是TGF-βRII胞外结构域序列(如SEQ ID NO：14所示)在其氨基端(也称N端)连续缺失至多26个氨基酸残基。在一些实施方案中，TGF-βRII胞外区的的截短形式是TGF-βRII胞外结构域序列在其N端连续缺失14、15、16、17、18、19、20、21、22、23、24、25或26个氨基酸残基。在一些实施方案中，前述药物组合物中所述的TGF-βRII ECD的序列如SEQ ID NO：14、15、16或17所示；优选如SEQ ID NO：15所示的序列。
在可选的实施方案中，前述药物组合物中所述的PD-L1抗体或其抗原结合片段包含：
分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和
分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在可选的实施方案中，前述药物组合物中所述的PD-L1抗体或其抗原结合片段具有：
分别如SEQ ID NO:1、SEQ ID NO:10和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和
分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
在可选的实施方案中，前述药物组合物中所述的PD-L1抗体或其抗原结合片段具有：
如SEQ ID NO:7所示的重链可变区，和
如SEQ ID NO:8所示的轻链可变区；
或，具有：
如SEQ ID NO:9所示的重链可变区，和
如SEQ ID NO:11所示的轻链可变区。
在可选的实施方案中，前药物组合物中所述的PD-L1抗体的重链氨基酸序列如SEQ ID NO：12所示或与SEQ ID NO：12所示的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性；所述PD-L1抗体的轻链氨基酸序列如SEQ ID NO：13所示或与SEQ ID NO：13所示的序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性。
在可选的实施方案中，前述的药物组合物，其中所述TGF-β受体融合蛋白中，所述TGF-βRII ECD通过连接序列融合至PD-L1抗体重链羧基末端。
在一些实施方案中，所述TGF-β受体融合蛋白包含：
· 融合有PD-L1抗体重链和TGF-βRII ECD的融合肽，其氨基酸序列如SEQ ID NO：23所示或与SEQ ID NO：23所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性，以及
· PD-L1抗体轻链，其氨基酸序列如SEQ ID NO：13所示或与SEQ ID NO：13所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性。
在另一些实施方案中，所述TGF-β受体融合蛋白包含：
· 融合有PD-L1抗体重链和TGF-βRII ECD的融合肽，其氨基酸序列如SEQ ID NO：24所示或与SEQ ID NO：24所示的氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性，以及
· PD-L1抗体轻链，其氨基酸序列如SEQ ID NO：13所示或与SEQ ID NO：13所示氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性。
本公开还提供制备前述药物组合物的方法，其中包括：使TGF-β受体融合蛋白和缓冲液接触的步骤，例如将TGF-β受体融合蛋白原液经缓冲液置换，所述缓冲液优选枸橼酸盐缓冲液，更优选为枸橼酸-枸橼酸钠缓冲液，所述缓冲液浓度优选为大约5mM至大约20mM，非限制性实施例包括5mM、6mM、7mM、8mM、9mM、10mM、12mM、14mM、16mM、18mM、20mM，更优选为10mM；所述缓冲液pH为约6.0至约6.5，非限制性实施例包括6.0、6.1、6.2、6.3、6.4、6.5，优选6.2。在可选的实施方案中，所述缓冲液浓度为5mM至20mM，非限制性实 施例包括大约5mM、大约6mM、大约7mM、大约8mM、大约9mM、大约10mM、大约12mM、大约14mM、大约16mM、大约18mM、大约20mM，更优选为大约10mM；所述缓冲液pH为6.0至6.5，非限制性实施例包括大约6.0、大约6.1、大约6.2、大约6.3、大约6.4、大约6.5，优选大约6.2。
本公开还提供制备前述药物组合物的方法，在TGF-β受体融合蛋白和缓冲液接触之后，进一步地还包括：向所得溶液中加入蔗糖和聚山梨醇80(两者不区分先后次序)，再经缓冲液定容，其中缓冲液浓度优选为大约5mM至大约20mM，更优选为5mM至20mM，非限制性实施例包括5mM、8mM、10mM、12mM、14mM、16mM、18mM、20mM；所述缓冲液pH为约6.0至约6.5，非限制性实施例包括6.0、6.1、6.2、6.3、6.4、6.5。
本公开还提供一种制备包含TGF-β受体融合蛋白的冻干制剂的方法，其中包括将前述药物组合物经冷冻干燥的步骤。
在可选的实施方案中，前述制备包含TGF-β受体融合蛋白的冻干制剂的方法，其中所述冷冻干燥按照本领域公知的方法进行，例如但不限于包括预冻、一次干燥和二次干燥的步骤。技术人员理解，任何将水从本公开所述药物组合物中移除的方法均适用于本公开。
本公开还提供一种含有TGF-β受体融合蛋白冻干制剂，其是经前述的制备冻干制剂的方法制备所得。
本公开还提供一种含有TGF-β受体融合蛋白的冻干制剂，所述含有TGF-β受体融合蛋白的冻干制剂经复溶后可形成前述的药物组合物。
在一些实施方案中，该冻干制剂于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。在一些实施方案中，该冻干制剂于40℃稳定至少7天，至少14天或至少28天。
本公开还提供一种含有TGF-β受体融合蛋白的复溶溶液，其是通过将前述含有TGF-β受体融合蛋白的冻干制剂复溶获得。
本公开还提供一种前述含有TGF-β受体融合蛋白的复溶溶液的制备方法，其中包括：将前述冻干制剂经复溶的步骤，复溶所用溶液包括但不限于注射用水、生理盐水或葡萄糖溶液，优选注射用水。
本公开进一步提供一种制品或试剂盒，其包含：根据本公开的药物组合物和容器。
在一些实施方案中，容器是玻璃瓶，例如但不限于中性硼硅玻璃管制注射剂瓶。
本公开还提供一种制品，其包括容器，该容器中装有前述的药物组合物、或其冻干制剂、或冻干制剂的复溶溶液。
本公开还提供选自以下的任一项在制备药物中的用途：
前述的药物组合物、或冻干制剂、或冻干制剂的复溶溶液、或制品；所述药 物用于治疗或抑制肿瘤细胞增殖或转移的疾病或病症。
在一些实施方案中，所述疾病或病症为肿瘤。
在一些实施方案中，所述疾病或病症选自：结直肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、前列腺癌、肾癌、宫颈癌、骨髓瘤癌、淋巴瘤、白血病、甲状腺癌、子宫内膜癌、子宫癌、膀胱癌、神经内分泌癌、头部颈部癌、肝癌、鼻咽癌、睾丸癌、肺癌、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合征。
本公开还提供前一种治疗或抑制癌细胞增殖或转移的疾病或病症的方法，包括向所需受试者提供治疗有效量的前述药物组合物、或冻干制剂、或复溶溶液、或制品。在一些实施方案中，所述方法包括向受试者施用单位剂量的组合物中含有0.1-3000mg的如前所述的TGF-β受体融合蛋白、药物组合物、或冻干制剂、或复溶溶液、或制品。在一些实施方案中，所述疾病或病症为肿瘤。在一些实施方案中，所述疾病或病症选自：结直肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、前列腺癌、肾癌、宫颈癌、骨髓瘤癌、淋巴瘤、白血病、甲状腺癌、子宫内膜癌、子宫癌、膀胱癌、神经内分泌癌、头部颈部癌、肝癌、鼻咽癌、睾丸癌、肺癌、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合征。
本发明还提供一种用于治疗或抑制癌细胞增殖或转移的疾病或病症的前述TGF-β受体融合蛋白、药物组合物、或冻干制剂、或复溶溶液、或制品。在一些实施方案中，所述疾病或病症为肿瘤。在一些实施方案中，所述疾病或病症选自：结直肠癌、乳腺癌、卵巢癌、胰腺癌、胃癌、前列腺癌、肾癌、宫颈癌、骨髓瘤癌、淋巴瘤、白血病、甲状腺癌、子宫内膜癌、子宫癌、膀胱癌、神经内分泌癌、头部颈部癌、肝癌、鼻咽癌、睾丸癌、肺癌、小细胞肺癌、非小细胞肺癌、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、成胶质细胞瘤、胶质瘤、肉瘤、间皮瘤，和骨髓增生异常综合征。
如本领域技术人员所熟知的，本公开中所述各个实施方案的一项、一些或所有特性可以进一步组合以形成本公开的其它实施方案。本公开的以上实施方案和通过组合得到的其他实施方案通过下面的详述进一步说明。
附图说明
图1：融合蛋白结构示意图。
图2：融合蛋白体外结合人源TGF-β1的结果。
图3：融合蛋白体外结合人源TGF-β1的结果。
图4：融合蛋白体外结合人源PD-L1的结果。
图5：融合蛋白体外检测PD-1/PD-L1通路阻断实验结果。
图6：融合蛋白以剂量依赖性形式抑制TGFβ诱导的pSMAD3报告物活性。
图7：融合蛋白样品均能够增强激活的T淋巴细胞分泌细胞因子IFN-γ。
图8：融合蛋白对荷瘤小鼠瘤重的影响。
具体实施方式
术语
为了更容易理解本公开，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公开所属领域的一般技术人员通常理解的含义。
“缓冲液”指通过其酸-碱共轭组分的作用而耐受pH变化的溶液。将pH控制在适当范围中的缓冲液的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸盐、草酸盐、乳酸盐、磷酸盐、枸橼酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸。
“组氨酸盐缓冲液”是包含组氨酸根离子的缓冲液。组氨酸盐缓冲液的示例包括组氨酸-盐酸盐，组氨酸-醋酸盐，组氨酸-磷酸盐，组氨酸-硫酸盐等缓冲液，优选组氨酸-盐酸盐缓冲液。组氨酸-盐酸盐缓冲液是组氨酸与盐酸配制而成。
“枸橼酸盐缓冲液”是包括枸橼酸根离子的缓冲液。枸橼酸盐缓冲液的示例包括枸橼酸-枸橼酸钠、枸橼酸-枸橼酸钾、枸橼酸-枸橼酸钙、枸橼酸-枸橼酸镁等。优选的枸橼酸盐缓冲液是枸橼酸-枸橼酸钠。
“琥珀酸盐缓冲液”是包括琥珀酸根离子的缓冲液。琥珀酸盐缓冲液的示例包括琥珀酸-琥珀酸钠、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选的琥珀酸盐缓冲液是琥珀酸-琥珀酸钠。
“磷酸盐缓冲液”是包括磷酸根离子的缓冲液。磷酸盐缓冲液的示例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾等。优选的磷酸盐缓冲液是磷酸氢二钠-磷酸二氢钠。
“醋酸盐缓冲液”是包括醋酸根离子的缓冲液。醋酸盐缓冲液的示例包括醋酸-醋酸钠、醋酸组氨酸盐、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是醋酸-醋酸钠。
“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。
本公开中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂均为水。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经冷冻干燥步骤(例如真空冷冻干燥步骤)之后获得的制剂或药物组合物。
本文所用术语“约”或“大约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域中“约”或“大约”可意味着在1内或超过1的标准差。或者，“约”或“大约”或“基本上包含表示至多20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。本公开中，除非另外说明，否则“约XX”或“大约XX”或“基本上包含XX”的含义是该具体值“XX”的可接受误差范围内的数值(包括数值“XX”本身，以及本领域一般技术人员测定该数值的可接受误差范围内的数值)。
本公开所述的药物组合物能够达到一种稳定的效果：其中TGF-β受体融合蛋白或其药物组合物在贮藏后，基本上保留其物理稳定性和/或化学稳定性和/或生物学活性；优选地，药物组合物在贮藏后基本上保留其物理和化学稳定性以及其生物学活性。贮藏期一般基于药物组合物的预定保存期来选择。目前有多种测量活性成分稳定性的分析技术，可测量在选定温度贮藏选定时间段后的稳定性。
稳定的药物抗体或蛋白制剂是在下述情况下没有观察到显著变化的制剂：在冷藏温度(2-8℃)保存至少3个月、优选6个月、更优选1年，且甚至更优选地多达2年。另外，稳定的液体制剂包括这样的液体制剂：其在包括25℃的温度保存包括1个月、3个月、6个月在内或者在40℃保存28天的时段后表现出期望的特征。
稳定性的典型的可接受标准如下：通过SEC-HPLC测得，通常不超过约10％、优选不超过约5％的活性成分(例如蛋白、抗体)发生降解。通过视觉分析，药物制剂是淡黄色近无色澄明液体或者无色，或澄清至稍微乳白色，或淡黄色近无色澄明液体。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、优选不超过约5％的截短。通常形成不超过约10％、优选不超过约5％的聚集。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述活性成分在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果活性成分(例如蛋白或抗体)没有显示出显著的化学改变，那么所述活性成分(例如蛋白或抗体)在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白或抗体，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和SDS-PAGE等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
在给定时间内，如果活性成分(例如蛋白或抗体)的生物活性在制备药物制剂时所表现出的生物活性的预定范围内，那么所述活性成分(例如蛋白或抗体)在药物制剂中“保留它的生物活性”。活性成分(例如蛋白或抗体)的生物活性可以例如通过抗原结合测定来确定。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem，243，p3558(1968)中所述。
本公开所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。
在本公开中，本公开所述的抗体轻链可包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本公开中，本公开所述的抗体重链可包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR或VL)和重链可变区(HCVR或VH)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)，或者符合kabat和chothia的编号规则，“Kabat”编号规则(参见Kabat等(1991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)，“Chothia”编号规则(参见Al-Lazikani等人，(1997)JMB 273：927-948)。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
本公开中所述的“抗体或其抗原结合”或“功能片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，以及与抗体结合的Fv片段scFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，是具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。本公开的术语“与PD-L1结合”，指能与人PD-L1相互作用。本公开的术语“抗原结合位点”指抗体或抗原结合片段上不连续或连续的三维空间位点，其能识别靶抗原并与抗原特异性结合。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人PD-L1的单 克隆抗体。制备时用PD-L1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(chimeric antibody)”，是将非人(例如小鼠)抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻非人(例如小鼠)抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌特异性单抗的杂交瘤，然后从杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将非人(例如小鼠)抗体可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的PD-L1嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的PD-L1嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG4。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人(例如小鼠)抗体CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量非人(例如小鼠)蛋白成分，从而诱导的强烈的抗体可变抗体反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后获得的人源化抗体。
本公开中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A、L234A、L235A；IgG2/4嵌合体，IgG4的F234A/L235A突变。
本公开中所述“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。本公开中的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。
术语“TGF-β受体II”或“TGFβRII”或“转化生长因子β受体II”是指结合配体(包括但不限于TGFβ1、TGFβ2和TGFβ3)，并且由此引发细胞内的信号转导途径的细胞表面受体。
术语“PD-L1”是指程序性死亡配体1，也称为CD274和B7H1。PD-L1是具有胞外IgV样和IgC样结构域(全长PD-L1的氨基酸19-239)、跨膜结构域和约30个氨基酸的胞内结构域的290个氨基酸的蛋白质。PD-L1在许多细胞例如抗原呈递细胞(例如，树突细胞、巨噬细胞和B细胞)上以及造血细胞和非造血细胞(例如，血管内皮细胞、胰岛、和免疫赦免部位)上组成型表达。PD-L1也在多种肿瘤和病毒感染的细胞上表达，并且是免疫抑制环境(immunosuppressive milieu)的成员(Ribas 2012，NEJM 366：2517-2519)。PD-L1与两种T细胞共抑制剂PD-1和B7-1之一结合。
本公开所述的“PD-L1抗体或其抗原结合蛋白”可包括本领域中所述的任何抗PD-L1抗体或其抗原结合片段。抗PD-L1抗体可以是市售可得的或已在文献中公开的PD-L1抗体。包括但不限于BMS-936559，MPDL3280A，MEDI4736，MSB0010718C(参见US2014341917、US20130034559、US8779108)等。抗体可以是单克隆抗体、嵌合抗体、人源化抗体，或人抗体。抗体片段包括具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，以及与抗体结合的Fv片段和scFv片段。
本公开示例性的PD-L1抗体的制备过程已在PCT申请PCT/CN2016/104320(公开号WO2017084495)中公开，包括如下所述的重链可变区的CDR序列：
HCDR1：SYWMH                      SEQ ID NO：1
HCDR2：RIX 1PNSG X 2TSYNEKFKN       SEQ ID NO：2
HCDR3：GGSSYDYFDY                 SEQ ID NO：3
在可选的实施方案中，X 1选自H或G，X 2选自G或F。
在另一个实施方案中，本公开示例性的PD-L1抗体进一步包括如下所述的轻链可变区的CDR序列：
LCDR1：RASESVSIHGTHLMH          SEQ ID NO：4
LCDR2：AASNLES                  SEQ ID NO：5
LCDR3：QQSFEDPLT                SEQ ID NO：6；
在另一个实施方案中，本公开对上述的CDR区采用CDR移植策略进行抗体人源化，人源化构架的人源化轻链模板为IGKV7-3*01和hjk2.1，人源化重链模板为IGHV1-46*01和hjh6.1，人源化可变区序列如下：
人源化PD-L1抗体重链可变区：
Figure PCTCN2019116593-appb-000004
SEQ ID NO：7，其中X 1选自H或G，X 2选自G或F。
人源化PD-L1抗体轻链可变区：
Figure PCTCN2019116593-appb-000005
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列(CDR的氨基酸残基由Kabat编号系统确定并注释)。
在另一个实施方案中，对本公开人源化抗体的回复突变设计，回复突变设计见下表1：
表1.回复突变设计
Figure PCTCN2019116593-appb-000006
注：如Y91F表示将第91位(自然顺序编号)Y突变回F。“植入”代表鼠抗体CDR植入人种系FR区序列。
将表1中各种轻重链的突变组合可得到新的人源化抗体。
本公开的另一方面，提供一种构建人源化克隆的实施例，如下：
设计引物，PCR搭建各人源化抗体VH/VK基因片段，再与表达载体pHr(带信号肽及恒定区基因(CH1-Fc/CL)片段)进行同源重组，构建抗体全长表达载体VH-CH1-Fc-pHr/VK-CL-pHr。
1.引物设计：利用在线软件DNAWorks(v3.2.2)(http://helixweb.nih.gov/dnaworks/)设计多条引物合成VH/VK含重组所需基因片段：5’-30bp信号肽+VH/VK+30bp CH1/CL-3’。
2.片段拼接：按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。
3.表达载体pHr(带信号肽及恒定区基因(CH1-Fc/CL)片段)构建及酶切
利用一些特殊的限制性内切酶，如BsmBI，识别序列与酶切位点不同的特性设计构建表达载体pHr(带信号肽及恒定区基因(CH1-Fc/CL)片段)。BsmBI酶切载体，切胶回收备用。
4.重组构建表达载体VH-CH1-FC-pHr/VK-CL-pHr
VH/VK含重组所需基因片段与BsmBI酶切回收表达载体pHr(带信号肽及恒定区基因(CH1-Fc/CL)片段)按3:1比例分别加入DH5H感受态细胞中，0℃冰浴30min，42℃热击90s，加入5倍体积LB介质，37℃孵育45min，涂布LB-Amp 平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
5.根据本实施例的设计方案构建质粒，然后表达纯化蛋白，用检测例SPR测定所得蛋白亲和力。
6.最终用BIACORE测试人源化回复突变体与人源PD-L1-his或杂交瘤抗体的亲和力，筛选得到的人源化回复突变位点的选择及序列组合如下：
PD-L1抗体重链可变区：
Figure PCTCN2019116593-appb-000007
其中，HCDR2为SEQ ID NO：7的X 1为G，X 2为F，即序列为：
Figure PCTCN2019116593-appb-000008
PD-L1抗体轻链可变区：
Figure PCTCN2019116593-appb-000009
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列；下划线为CDR序列(CDR的氨基酸残基由Kabat编号系统确定并注释)。
本公开的另一方面，提供一种构建和表达抗PD-L1人源IgG4类型的实施例，并进一步用于融合蛋白构建的PD-L1抗体。该PD-L1抗体也可用作本公开中的测试例对照分子。
由于PD-L1在活化T细胞中也有表达，如果采用野生型IgG1恒定区会引起Fc介导的效应作用，比如ADCC和CDC，从而导致活化T细胞的消减。本公开选择突变IgG4以得到无ADCC和CDC的抗体。将亲合力成熟得到的克隆转换成IgG4类型，IgG4的核心铰链区包含S228P(对应于序列SEQ ID NO：12的自然顺序第227位)突变。并进一步引入F234A(对应于序列SEQ ID NO：12的自然顺序第233位)和L235A(对应于序列SEQ ID NO：12的自然顺序第234位)突变(mAbs 4:3，310-318；2012五月/六月)。同时，为防止抗体重链C末端在引入连接肽连接TGF-βRII胞外区时发生断裂，又将PD-L1抗体重链的最后一位K突变成A(对应于序列SEQ ID NO：12的最后一位)，以增加融合蛋白的稳定性。本公开用于融合蛋白构建的PD-L1抗体序列如下：
PD-L1抗体重链序列：Ig G4(AA)(S228P)
Figure PCTCN2019116593-appb-000010
Figure PCTCN2019116593-appb-000011
备注：下划线部分为重链可变区序列，无下划线部分为重链恒定区序列(斜体部分为突变位点)；
PD-L1抗体轻链序列：
Figure PCTCN2019116593-appb-000012
备注：下划线部分为轻链可变区序列，无下划线部分为轻链恒定区序列。
本公开中所述的融合蛋白是一种通过DNA重组得到的两个基因共表达的蛋白产物。现有技术中熟知生产和纯化抗体和抗原结合片段的方法(如冷泉港的抗体实验技术指南，5-8章和15章)。例如，小鼠可以用人PD-L1或其片段免疫，所得到的抗体能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人PD-L1特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用PH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
本公开所述的“免疫调节分子”可用于削弱癌细胞的免疫耐受性。本公开采用TGF-βRII胞外结构域的截短形式作为融合蛋白中免疫调节分子部分。“TGF-β受体II(TGF-βRII)”以高亲和力结合配体TGF-β1和TGF-β3。TGF-βRII/TGF-β复合物招募TGF-βRI以形成信号转导复合物(Won等，Cancer Res.1999；59:1273-7)。TGF-βRII的胞外结构域是TGF-βRII细胞外自N端开始的一段长136个氨基酸残基的肽段，其中一个示例性的例子如SEQ ID NO：14所示。其他长度约为136个 氨基酸，并且来源于人的具有TGF-βRII的胞外区的能够与TGF-β1和TGF-β3相结合的变体同样属于本公开TGF-βRII的胞外结构域的范围。本公开研究发现，TGF-βRII胞外结构域N端连续截短形式的结构和功能较未截短分子稳定。含TGF-βRII胞外结构域N端未截短(SEQ ID NO：14所示的1-136多肽)形式的融合蛋白容易断裂。尤其是在TGF-βRII胞外结构域N端作连续26个以下氨基酸的截短后更为稳定，优选N端作连续14-26个氨基酸的截短，更优选N端作14-21个氨基酸截短形式，具有更高的表达量，最优选N端作连续19或21个氨基酸的截短。
术语“TGF-β受体融合蛋白”是包含TGF-β受体的融合蛋白。在一些实施方案中，本公开的TGF-β受体融合蛋白为描述于国际专利申请PCT/CN2018/086451(WO 2018205985 A1)中的TGF-β受体融合蛋白，WO 2018205985 A1的全文内容全部引入本公开。在一些实施方案中，所述TGF-β受体融合蛋白为PD-L1抗体/TGF-βRII胞外区融合蛋白(PD-L1/TGF-βtrap)，其以TGF-βRII胞外结构域作为融合蛋白的免疫调节分子部分，PD-L1抗体作为融合蛋白的靶向部分，TGF-βRII胞外结构域(例如SEQ ID NO：14、15、16或17所示)通过连接序列(例如(G 4S) xG，x为3-6)与PD-L1抗体的重链C末端(也称羧基末端)连接形成融合序列，融合序列与PD-L1抗体轻链通过链间二硫键连接最终形成PD-L1/TGF-βtrap融合蛋白，其结构如图1所示。在一些实施方案中，所述TGF-β受体融合蛋白为本公开实施例1的表2所述融合蛋白。
术语“Linker”或“接头”或“连接子”或“连接序列”指用于连接蛋白质结构域的连接性多肽序列，通常具有一定的柔性，接头的使用不会使蛋白质结构域原有的功能丧失。本公开的一些实施方案中，连接序列为(G 4S) xG，其中x为3-6，例如连接序列为:(G 4S) 3G、(G 4S) 4G、(G 4S) 5G或(G 4S) 6G等多肽。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着特定序列的抗体重链可变区可以但不必须存在。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的 接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“施用”或“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，例如包含本公开的的组合物，所述受试者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“Tm值”是指蛋白质热变性温度，即一半蛋白去折叠时的温度，此时蛋白的空间结构被破坏，所以Tm值越高，蛋白热稳定性越高。
“置换”是指溶解抗体蛋白的溶剂体系的置换，例如，使用稳定制剂的缓冲体系经物理操作方式将含抗体蛋白的高盐或高渗溶剂体系置换，从而使抗体蛋白存在于稳定制剂中。所称物理操作方式包括但不限于超滤、透析或离心后复溶。
具体实施方式
通过以下实施例、测试例或制备例进一步详细说明本公开。这些实施例、测试例或制备例仅用于说明性目的，而并不用于限制本公开的范围。
本公开实施例、测试例或制备例中未注明具体条件的实验方法，通常按照常规条件；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
实施例1.融合蛋白PD-L1/TGF-βtrap克隆和表达
采用TGF-βRII胞外结构域(SEQ ID NO：14的全长或截短形式)作为融合蛋 白中免疫调节分子部分，将PD-L1抗体作为融合蛋白的靶向部分，形成PD-L1抗体/TGF-βRII胞外区融合蛋白(PD-L1/TGF-βtrap)。
出乎意料地发现，TGF-βRII胞外结构域截短形式的结构和功能较为稳定，尤其是在其N端作26个以下氨基酸的截短后更为稳定，优选14-26个氨基酸的截短，更优选N端截短14-21个连续氨基酸的形式，具有更高的表达量和稳定的结构，更优选N端截短14、19或21个连续氨基酸的形式。
本公开TGF-βRII胞外结构域及其截短形式的非限制性实施例序列如下：
TGF-βRII胞外结构域序列：ECD(1-136)
Figure PCTCN2019116593-appb-000013
TGF-βRII胞外结构域序列在N端有19个氨基酸的截短或缺失：ECD(20-136)
Figure PCTCN2019116593-appb-000014
TGF-βRII胞外结构域序列在N端有21个氨基酸的截短或缺失：ECD(22-136)
Figure PCTCN2019116593-appb-000015
TGF-βRII胞外结构域序列在N端有14个氨基酸的截短或缺失：ECD(15-136)
Figure PCTCN2019116593-appb-000016
示例性地，利用同源重组技术将本公开PD-L1抗体(如重链如SEQ ID NO：12所示，轻链如SEQ ID NO：13所示的PD-L1抗体)的重链C末端氨基酸通过连接子((G 4S) xG，x为3-6)连接不同长度的TGF-βRII胞外区，并与PD-L1抗体轻链一起，通过293表达系统进行常规表达，得到如表2所示的融合蛋白：
表2.PD-L1抗体/TGF-βRII胞外区融合蛋白
融合蛋白例 序列描述 N端连续氨基酸缺失数
融合蛋白1 Ab-(G4S)4G-ECD(1-136) 未缺失
融合蛋白2 Ab-(G 4S) 3G-ECD(15-136) 14
融合蛋白3 Ab-(G 4S) 3G-ECD(15-136,N19A) 14
融合蛋白4 Ab-(G 4S) 3G-ECD(20-136) 19
融合蛋白5 Ab-(G 4S) 3G-ECD(22-136) 21
融合蛋白6 Ab-(G 4S) 3G-ECD(27-136) 26
融合蛋白7 Ab-(G4S) 4G-ECD(15-136) 14
融合蛋白8 Ab-(G4S) 4G-ECD(15-136,N19A) 14
融合蛋白9 Ab-(G4S) 4G-ECD(20-136) 19
融合蛋白10 Ab-(G4S) 4G-ECD(22-136) 21
融合蛋白11 Ab-(G4S) 4G-ECD(27-136) 26
融合蛋白12 Ab-(G 4S) 5G-ECD(15-136) 14
融合蛋白13 Ab-(G 4S) 5G-ECD(15-136,N19A) 14
融合蛋白14 Ab-(G 4S) 5G-ECD(20-136) 19
融合蛋白15 Ab-(G 4S) 5G-ECD(22-136) 21
融合蛋白16 Ab-(G 4S) 5G-ECD(27-136) 26
融合蛋白17 Ab-(G 4S) 6G-ECD(27-136) 26
注：Ab为本公开所述重链序列如SEQ ID NO：12所示，轻链序列如SEQ ID NO：13所示的PD-L1抗体，序列描述中ECD(n-136)为TGF-βRII胞外区的全长或截短形式，n为TGF-βRII胞外区截短后的氨基酸起始位数。本公开融合蛋白结构如图1所示；N19A是全长TGF-βRII胞外区(如SEQ ID NO：14所示)的第19位氨基酸由N突变为A。
编码PD-L1抗体的核苷酸序列、编码TGF-βRII胞外区的核苷酸序列、接头蛋白片段((G 4S) xG)的核苷酸序列通过所属领域常规技术手段获得。利用同源重组技术将PD-L1抗体的C末端核苷酸通过接头蛋白连接不同长度TGF-βRII胞外区的N末端核苷酸，克隆到Phr-BsmbI载体上。重组的PD-L1/TGF-βtrap在293细胞表达，通过实施例2进行纯化。纯化的蛋白可用于下述各实施例实验中。
实施例2.PD-L1/TGF-βtrap融合蛋白纯化
细胞培养液高速离心后收集上清，利用亲合层析进行第一步纯化。层析介质为与Fc相互作用的Protein A或者衍生填料，如GE的Mabselect。平衡缓冲液为1×PBS(137mmol/L NaCl，2.7mmol/L KCl，10mmol/L Na 2HPO 4，2mmol/L KH 2PO 4，pH7.4)，平衡5倍柱体积后，将细胞上清上样结合，流速控制为样品在柱上保留时间≧1min。上样结束后，用1×PBS(pH7.4)冲洗柱子，直至A280紫外吸收降至基线。然后用0.1M甘氨酸(pH3.0)的洗脱缓冲液冲洗层析柱，根据A280紫外吸收峰收集洗脱峰，收集的洗脱样品用1M Tris(pH8.5)中和。
将上述中和后的洗脱样品超滤浓缩后进行体积排阻层析，缓冲液为1×PBS，层析柱为XK26/60 Superdex200(GE)，流速控制在4ml/min，上样体积小于5ml，根据A280紫外吸收合并目的蛋白峰。收集的蛋白经SEC-HPLC鉴定纯度大于95％，经LC-MS鉴定为正确后分装备用。得到PD-L1/TGF-βtrap。
以下用生化测试方法验证本公开中PD-L1抗体/TGF-β融合蛋白性能及有益效果。
测试例(体内、外活性生物学评价)
测试例1.ELISA检测PD-L1/TGF-βtrap体外结合TGF-β1实验
检测流程描述如下：
a.以1μg/ml浓度的人源TGF-β1(8915LC，CST)为抗原按100μl/孔包被96孔板，4℃过夜。
b.250μl 1×PBST洗涤3次，加入250μl 5％牛奶PBS，37℃封闭2小时。
c.250μl 1×PBST洗涤3次，加入梯度稀释的PD-L1/TGF-βtrap，TGF-βtrap为阳性对照，37℃孵育1小时。
d.250μl 1×PBST洗涤3次。
e.每孔加入100μl抗-人FC抗体-HRP(1:4000)，37℃孵育40分钟。
f.每孔加入100μl TMB，室温孵育10分钟后加入100μl 1M H 2SO 4终止反应。
g.酶标仪上检测450nm测吸收值，Graphpad Prism5分析数据。
融合蛋白体外结合人源TGF-β1的结果如图2、图3所示。ELISA显示表2中融合蛋白1未保留对人源TGF-β1的结合活性。质谱分析结果显示融合蛋白1(即TGF-βRII胞外区非截短形式(1-136))不稳定，容易在重链TGF-βRII部分断裂，阳性对照同样存在相同情况，而融合蛋白7、9、10、12-15等TGFβRII胞外区N端截短形式的融合蛋白对于结合至人源TGF-β1是具有特异性的。
测试例2.ELISA检测PD-L1/TGF-βtrap体外结合PD-L1实验
检测用抗原：PD-L1-His
Figure PCTCN2019116593-appb-000017
检测流程描述如下：
a.以5μg/ml浓度的人源PD-L1-His(SEQ ID NO：18)为抗原按100μl/孔包被96孔板，4℃过夜。
b.250μl 1×PBST洗涤3次，加入250μl 5％牛奶PBS 37℃封闭2小时。
c.250μl 1×PBST洗涤3次，加入梯度稀释的PD-L1/TGF-βtrap，PD-L1抗体为阳性对照，37℃孵育1小时。
d.250μl 1×PBST洗涤3次。
e.每孔加入100μl抗人FC抗体-HRP(1:4000)，37℃孵育40分钟。
f.每孔加入100μl TMB，室温孵育10分钟后加入100μl 1M H2SO4终止反应。
g.酶标仪上检测450nm测吸收值，Graphpad Prism5分析数据。
本公开融合蛋白体外结合人源PD-L1的结果如图4所示。ELISA结果显示融合蛋白均保留了对人源PD-L1的结合活性。
测试例3.体外检测PD-1/PD-L1通路阻断实验
1.测试目的：
为了研究PD-L1/TGF-βtrap对PD-1/PD-L1信号通路的阻断作用，采用来自Promaga公司构建的分别带有人源PD-1和PD-L1受体分子的细胞，进行基于细胞水平上的抗体阻断实验。
2.测试样品：
①PD-L1抗体：重链序列如SEQ ID NO：12所示，轻链序列如SEQ ID NO：13所示；
②对照1(20T-Fc)：ECD(20-136)-Fc，TGF-βRII胞外区截短片段ECD(20-136)与Fc的融合蛋白，序列如下：
Figure PCTCN2019116593-appb-000018
③对照2(22T-Fc)：ECD(22-136)-Fc，TGF-βRII胞外区截短片段ECD(22-136)与Fc的融合蛋白，序列如下：
Figure PCTCN2019116593-appb-000019
④本公开实施例1制备的TGF-β受体融合蛋白：融合蛋白9、融合蛋白15：
融合蛋白9中融合PD-L1抗体重链-(G 4S) 4G-TGF-βRII ECD(20-136)的融合肽序列如下：
Figure PCTCN2019116593-appb-000020
Figure PCTCN2019116593-appb-000021
备注：正体字为PD-L1抗体重链序列，斜体为连接序列，下划线为TGF-βRII胞外区截短片段ECD(20-136)序列。
融合蛋白9中的PD-L1抗体轻链序列如下：
Figure PCTCN2019116593-appb-000022
融合蛋白15中的融合PD-L1抗体重链-(G 4S) 5G-TGF-βRII ECD(22-136)的融合肽序列如下：
Figure PCTCN2019116593-appb-000023
备注：正体字为PD-L1抗体重链序列，斜体为连接序列，下划线为TGF-βRII胞外区截短片段ECD(22-136)序列；
融合蛋白15中的PD-L1抗体轻链序列如下：
Figure PCTCN2019116593-appb-000024
Figure PCTCN2019116593-appb-000025
⑤人IgG：空白对照，从混合的正常人血清中，利用传统的亲和层析方法如ProteinA纯化获得的人免疫球蛋白；
⑥阳性对照(FP17022)：PD-L1抗体2/TGF-βRII胞外区融合蛋白
FP17022融合蛋白中的PD-L1抗体2轻链的氨基酸序列
Figure PCTCN2019116593-appb-000026
FP17022融合蛋白中的PD-L1抗体2重链/TGF-βRII胞外区(1-136)的融合肽的氨基酸序列：
Figure PCTCN2019116593-appb-000027
3.测试过程
取CHO/PD-L1细胞(CS187108，Promega)，消化并用F-12Nutrient Mixture(Ham)完全培养基重悬细胞。根据细胞计数结果使用完全培养基调整细胞密度至4×10 5/mL，将细胞悬液转移至加样槽中，使用多道移液器以100μL/孔加入到96孔板中，放置于37℃，5％CO 2培养箱培养20～24h；第二天制备Jurkat/PD-1(CS187102，Promega)细胞悬液，根据细胞计数结果使用分析培养基重悬细胞，并调整细胞密度至1.25×10 6/mL；将加入CHO/PD-L1细胞的细胞培养板从培养箱中取出，使用多道移液器每孔取出95μL培养液，按照40μL/孔加入梯度稀释的融合蛋白，以PD-L1抗体及阳性对照(FP17022)，然后将Jurkat/PD-1细胞悬液转移至加样槽中，以40μL/孔加入到细胞培养板中，置于37℃，5％CO 2培养箱培养5～6h。在蛋白孵育期间，将Bio-Glo TM Reagent取出使其温度恢复至室温。取出细 胞培养板，置于室温放置5～10min，然后每孔加入40μL Bio-Glo TM Reagent，置于安全柜中孵育5～10min，使用多功能酶标仪读取化学发光信号值。
4.结果
如图5所示，本公开融合蛋白9同阳性分子一样均能够有效地阻断表达有PD-1分子的Jurkat细胞同CHO/PD-L1细胞结合，并且有药物浓度剂量依赖效应。融合蛋白15与融合蛋白9有相同水平的阻断能力。
测试例4.Biacore检测体外结合亲和力和动力学实验
通过Biacore T200(GE)测定待测分子与人或鼠源TGF-β1或人源PD-L1蛋白的亲和力，实验过程描述如下：
用Protein A芯片亲和捕获一定量的PD-L1/TGF-βtrap，然后于芯片表面流经人或鼠源TGF-β1(8915LC，CST)或人源PD-L1(Sino Biological)，利用Biacore实时检测反应信号，从而获得结合和解离曲线，然后用甘氨酸-盐酸(pH 1.5，GE)将生物芯片洗净再生。实验中用到的缓冲溶液为HBS-EP Buffer(GE)。实验得到的数据用BIAevaluation version 4.1软件(GE)以(1:1)Langmuir模型进行拟合，得出如表3所示的亲和力数值。
表3.本公开融合蛋白与TGF-β1或人源PD-L1的体外亲和力
Figure PCTCN2019116593-appb-000028
*对应序号的融合蛋白形式见表2。
融合蛋白结合活性见表3，结果表明，本公开融合蛋白9和融合蛋白15均对人、小鼠TGF-β1以及人PD-L1具极高的亲和力。
测试例5.SMAD3报告基因抑制实验
1、测试目的
该实验通过HepG2细胞表达带荧光素酶报告基因的Smad3结合原件(SBE)来研究PD-L1/TGF-βtrap对TGF-β1诱导Smad3活化的抑制作用，根据IC50大小评价PD-L1/TGF-βtrap的体外活性。
2、测试样品：融合蛋白9、阳性对照(FP17022)
3、测试过程
HepG2细胞使用含有10％FBS的MEM完全培养基(GE，SH30243.01)培养，每3天传代一次。实验第一天以每孔25,000个细胞的密度接种于96孔板(Corning，3903)，在37℃、5％CO 2条件下培养24小时。第二天，弃去细胞培养板中的培 养基，每孔转染100ng 3TP-Lux质粒。细胞在37℃、5％CO 2条件下继续培养24小时。加入待测样品前6小时，弃去96孔板中完全培养基，每孔加入80μL不完全培养基(MEM+0.5％FBS)。6小时后再加入10μL使用不完全培养基配制的人TGF-β1(R&D，240-B-010)溶液，终浓度为2ng/mL和10μL待测样品，终浓度为500、50、5、0.5、0.05、0.005、0.0005和0nM，以人TGF-β1溶剂为对照，细胞在37℃、5％CO 2条件下继续培养18h。然后每孔加入100μL配制好的萤光素酶底物ONE-GloTM Luciferase Assay system(promega，E6110)，室温避光放置10分钟，然后使用Victor3多功能酶标仪(Perkin Elmer)读取发光信号值。待测样品的IC50值使用数据处理软件Graphpad Prism5.0计算得到。
图6显示融合蛋白9以剂量依赖性形式抑制TGFβ诱导的pSMAD3报告物活性，且与阳性对照FP17022具有相当的功效和IC 50(抑制最大活性的50％所需的浓度)。PD-L1抗体的测试结果显示其不具有抑制作用(IC 50＞500nM)。
测试例6.体外检测结核杆菌素(TB)刺激PBMC释放IFNγ实验
1、测试目的
为了研究PD-L1/TGF-βtrap对T淋巴细胞的激活作用，收集和纯化人外周血单核细胞(PBMC)，采用结核杆菌素(TB)体外刺激5天，检测IFNγ细胞因子的分泌水平。
2、测试样品：
①人IgG；
②PD-L1抗体；
③融合蛋白9；
④对照1(20T-Fc)：ECD(20-136)-Fc；
⑤PD-L1抗体+对照1(20T-Fc)。
3、测试过程
新鲜分离纯化的PBMC，15mL约3×10 7个，加入20μL结核菌素，37℃、5％CO 2培养箱培养5天。第6天，收集上述培养的细胞离心，用PBS洗一次，重悬至新鲜的培养基中，调整密度为1×10 6个每毫升，接种至96孔细胞培养板，每孔90μL。将不同浓度的抗体分别加入上述96孔细胞培养板的对应孔中，每孔10μL，对照组和空白组分别加入10μL PBS。细胞培养板置于37℃，5％CO 2培养箱孵育3天。取出细胞培养板，离心(4000rpm，10min)每孔取上清，10倍稀释后，采用ELISA的方法(人IFN-γ检测试剂盒，欣博盛，EHC102g.96)检测IFN-γ的水平。具体操作参考试剂说明书。结果如下表4所示，PD-L1/TGF-βtrap融合蛋白样品均能够增强激活的T淋巴细胞分泌细胞因子IFN-γ，并且有药物浓度剂量效应。
表4.细胞因子IFN-γ的分泌结果
Figure PCTCN2019116593-appb-000029
4、结果
如图7、表4所示，融合蛋白9能够剂量依赖地增强激活的T淋巴细胞分泌细胞因子IFN-γ，并且具有比PD-L1抗体，20T-FC更强的激活作用。
测试例7.药代动力学评价
实验用SD大鼠3只，雌性，12/12小时光/暗调节，温度24±3℃恒温，湿度50-60％，自由进食饮水。购自杰思捷实验动物有限公司。实验当天对SD大鼠分别尾静脉注射融合蛋白，给药剂量为6mg/kg，注射体积为5ml/kg。
取血时间点为：第1天给药后15min、7h、24h(第2天)，第3天，第4天，第6天，第8天，第10天，第15天，于大鼠眼底静脉取血，每次200μl(相当于取血清100μl)；收集的血样在室温下置放半小时至凝集，然后4℃下10000x g离心10分钟。收集上清，立即放置-80℃贮存。用ELISA检测血清中的融合蛋白浓度。
检测流程描述如下：
a.以2μg/ml浓度的人源PD-L1-His为抗原按100μl/孔包被96孔板，4℃过夜。
b.250μl 1×PBST洗涤4次，加入250μl 5％牛奶PBS，37℃封闭3小时。
c.250μl 1×PBST洗涤4次，加入100μl梯度稀释的待测血清，以融合蛋白9为阳性对照，37℃孵育1小时。
d.250μl 1×PBST洗涤5次。
e.每孔加入100μl生物素化的抗人源TGF-βRII的抗体(R&D)，37℃孵育1小时。
f.250μl 1×PBST洗涤5次。
g.每孔加入100μl TMB，室温孵育10分钟后加入100μl 1M H 2SO 4终止反应。
h.酶标仪上检测450nm测吸收值，Graphpad Prism5分析数据。
表5.融合蛋白在SD大鼠中的T1/2
受试药物 给药方式 T1/2
    (平均值±SD，h)
融合蛋白9 IV(6mg/kg) 236±10
参见表5，PK分析结果表明，本公开的融合蛋白9在大鼠体内的半衰期约为236h(9.8天)。
测试例8.PD-L1/TGF-βtrap对人乳腺癌MDA-MB-231小鼠皮下移植瘤的疗效
本实验应用的小鼠品系为NOD/SCID雌性小鼠(卡文斯)，实验使用的人外周血单个核细胞从新鲜采集的血液中提取获得，提取方法如下：将肝素抗凝处理的静脉血与同体积含2％FBS的PBS混合，混匀后将25ml稀释后的血液缓慢加入到含15ml淋巴细胞分离液的离心管中，室温下1200g离心10分钟，吸取淋巴细胞层转移到另一个离心管，加入PBS清洗细胞，室温下300g离心8分钟，重复一次后，用含10％FBS的RPMI-1640培养基重悬细胞，将细胞加入到事先包被好CD3抗体(OKT3，40ng/ml)的6孔板中，每孔2×10 6个细胞(2ml)，置于37℃培养箱中培养4天。
实验样品：
①空白对照：PBS；
②融合蛋白9：4.8mpk；
③融合蛋白9：24mpk；
④PD-L1抗体：4mpk；
⑤PD-L1抗体：20mpk；
⑥PD-L1抗体4mpk+对照1(20T-Fc)2.14mpk；
⑦对照1(20T-Fc)：2.14mpk。
将MDA-MB-231细胞重悬于无血清RPMI-1640培养基中，与等体积基质胶混合后100μl(2.3×10 6)接种于NOD/SCID小鼠右肋部皮下，11天后去除肿瘤体积过大过小动物后，将小鼠随机分组，每组9只。将5×10 5个刺激后的PBMC(60μl)注射到肿瘤组织中，剩余的PBMC停止刺激并继续培养，1周后将5×10 6个PBMC(100μl)腹腔注射到荷瘤小鼠体内，视为第1轮注射。整个实验周期，进行2轮半、共5次PBMC注射。首次瘤内注射当日开始腹腔给药，一周三次，共给药14次，给药方式见表6。每周2次测量瘤体积，称体重。实验结果见表7。实验结束后将荷瘤小鼠安乐死并剥瘤称重。
表6.试验分组及给药情况
Figure PCTCN2019116593-appb-000030
Figure PCTCN2019116593-appb-000031
表7.融合蛋白9对MDA-MB-231小鼠皮下移植瘤的疗效
Figure PCTCN2019116593-appb-000032
第0天:第一次给药时间；*p＜0.05**p＜0.01***p＜0.001通过student t检验与PBS对比。
实验结果如图8显示，抗体融合蛋白9(4.8mg/kg、24mg/kg)能明显抑制人乳腺癌MDA-MB-231小鼠皮下移植瘤的生长，高低剂量间呈现剂量依赖关系，且优于各自等同摩尔剂量的参比药物PD-L1抗体(4mg/kg、20mg/kg)、TGF-βRII对照分子20T-FC(2.14mg/kg)和联用组(PD-L1抗体-4mg/kg+20T-FC-2.14mg/kg)。各剂量的融合蛋白9从给药后14天开始，就一直保持理想的抑瘤效果，且其高剂量与PD-L1抗体-20mpk相比，优势非常明显(p＜0.05)；给药后25天，各抗体抑瘤效果最好，融合蛋白9和PDL-1抗体低、高剂量与联用组的抑瘤率分别为37.24％、52.38％、30.24％、28.01％、31.38％；给药后32天，融合蛋白9的抑瘤作用仍十分明显，低高剂量组％TGI分别为36.68％和50.76％，且瘤体积与对照组相比都存在统计学差异(p＜0.05)。
测试例9.PD-L1/TGF-βtrap的物理稳定性
本测试例用于检测融合蛋白融合蛋白9和融合蛋白15的稳定性。
利用DSC(Differential scanning calorimetry，差示扫描量热法)检测不同抗体的热稳定性，比较了不同的缓冲体系下的稳定性情况，不同缓冲体系如10mM醋 酸盐/135mM NaCl(pH 5.5)和10mM醋酸盐/9％海藻糖(pH 5.5)。
将样品置换到对应缓冲液中，控制样品浓度在50mg/ml左右，利用MicroCal*VP-Capillary DSC(Malvern)进行检测。检测前，将各个样品及空白缓冲液用真空脱气器脱气1～2min。样品板每个孔加入400μl样品或空白缓冲液(仪器上样量为300μl)。最后两对孔板分别加入14％Decon 90和ddH 2O，以备清洗用，样品板加样完毕后，套上塑料软盖板。扫描温度从25℃开始到100℃结束，扫描速率60℃/h。具体结果如下表8所示，在两个测试体系中融合蛋白9、融合蛋白15均表现了良好的热稳定性。
表8.热稳定性测试
Figure PCTCN2019116593-appb-000033
通过SEC-HPLC监测样品纯度考察一定浓度条件下周期性稳定性，示例性的条件比如将样品浓度控制在约50mg/ml，在10mM醋酸盐/135mM NaCl(pH 5.5)比较在比如-80℃反复冻融5次及40℃保存一个月的稳定性情况。利用Xbridge protein BEH SEC 200A(Waters)HPLC柱子检测。检测结果如下表9所示，两个融合蛋白均表现出良好的稳定性。
表9.稳定性
  融合蛋白9(△％) 融合蛋白15(△％)
40℃ 3.39％ 1.8％
-80℃冻融 1.44％ 1.39％
注：△％表示变化率。
测试例10.融合蛋白的化学稳定性
脱酰胺修饰是抗体中可能影响后期稳定性的一种常见的化学修饰，尤其是CDR区域的部分氨基酸高度脱酰胺修饰一般选择尽量避免或者突变降低。取1600μg待测抗体溶于200μl 10mM醋酸盐/135mM NaCl(pH 5.5)中，40℃恒温箱存放；分别于0、14、28天取样，用于酶解实验。将100μg不同时间点取样的样品溶于100μl 0.2M His-HCl，8M Gμa-HCl，pH 6.0溶液中，加3μl 0.1g/mL DTT,50℃水浴1小时，后用0.02M His-HCl，pH 6.0的溶液超滤两次，加入3μL 0.25mg/mL的胰蛋白酶，37℃水浴酶解过夜。Agilent 6530 Q-TOF进行LC-MS检测脱酰胺修饰情况，结果如下表10所示。
表10.脱酰胺修饰
Figure PCTCN2019116593-appb-000034
Figure PCTCN2019116593-appb-000035
备注：N代表检测到修饰的天冬酰胺，数字代表所处轻链或者重链N端开始计数所处的位置。百分含量代表LC-MS检测到的脱酰胺修饰占该位点所处全部肽段信号的比例。
质谱检测结果显示两个融合蛋白都没有明显的脱酰胺修饰位点，提示其后期化学稳定性良好。
制备例
示例性融合蛋白药物组合物(制剂)制备工艺
第一步：取一定量的纯化的TGF-β受体融合蛋白原液，用不含蛋白的缓冲液(如10mM，pH 6.2枸橼酸-枸橼酸钠缓冲液)进行溶剂置换(优选超滤)，经超滤膜至少6倍体积置换，蛋白浓缩到约70mg/mL。加入一定体积的蔗糖母液，混匀，使最终蔗糖浓度为80mg/mL。加入一定体积的吐温-80母液，混匀，使最终吐温-80浓度为0.4mg/mL。加10mM pH 6.2枸橼酸盐缓冲液定容，使蛋白浓度为50mg/mL(其他待测试制剂或稳定性制剂参照相似步骤进行配制)。
产品经过滤后中控取样检测无菌。将原液过0.22μm PVDF滤芯，收集滤液。
第二步：调节装量至6.3ml，将滤液灌装于6ml西林瓶中，加塞，分别于灌装开始、灌装中间、灌装结束时取样中控检测装量差异。
第三步：开启轧盖机，加铝盖，进行轧盖。
第四步：目检，确认产品无装量不准等缺陷。打印、粘贴西林瓶标签；打印纸盒标签，折叠纸盒，装盒，贴纸盒标签。
制备例1.TGF-β受体融合蛋白制剂缓冲体系pH值的筛选
用下列缓冲液，配制蛋白浓度为50mg/ml的TGF-β受体融合蛋白(融合蛋白9)制剂：
1)10mM组氨酸-醋酸，pH 5.0
2)10mM组氨酸-醋酸，pH 6.0
3)10mM组氨酸-醋酸，pH 6.5
4)10mM磷酸二氢钠-磷酸氢二钠，pH 7.0
5)10mM磷酸二氢钠-磷酸氢二钠，pH 7.5
过滤每种制剂并以1.2mL/瓶填充入2mL中性硼硅玻璃管制注射剂瓶中，水针压塞轧盖封口。取样品分别进行40℃高温和振摇实验，实验结果如表11所示，结果表明TGF-β受体融合蛋白在pH 6.0-6.5时稳定性较好。
表11.强制降解实验筛选结果
Figure PCTCN2019116593-appb-000036
Figure PCTCN2019116593-appb-000037
注：振摇条件为：D1:130rpm，D2:200rpm，D3-D7:300rpm；D表示天，T表示时间，M表示月。
制备例2.TGF-β受体融合蛋白制剂缓冲体系的筛选
用下列缓冲液，配制蛋白浓度为50mg/ml的TGF-β受体融合蛋白(融合蛋白9)制剂：
1)10mM琥珀酸-琥珀酸钠，pH 6.0
2)10mM枸橼酸-枸橼酸钠，pH 6.0
3)10mM枸橼酸-枸橼酸钠，pH 6.5
4)10mM磷酸二氢钠-磷酸氢二钠，pH 6.5
5)10mM组氨酸-盐酸盐，pH 6.5
过滤每种制剂并以1.2mL/瓶填充入2mL中性硼硅玻璃管制注射剂瓶中，水针压塞轧盖封口。取样品进行振摇(25℃，300rpm)实验，实验结果见表12，结果表明，磷酸二氢钠-磷酸氢二钠组在振摇第6天见大量小颗粒，SEC检测聚体达1.8％，而其它组仅偶见小颗粒，可见TGF-β受体融合蛋白在枸橼酸、组氨酸和琥珀酸盐缓冲体系中的稳定性优于磷酸盐缓冲体系。
表12.缓冲体系及pH值筛选实验结果
Figure PCTCN2019116593-appb-000038
Figure PCTCN2019116593-appb-000039
注：D表示天。
制备例3.TGF-β受体融合蛋白制剂缓冲体系的进一步筛选
用pH 6.2的含10mM组氨酸-盐酸盐或10mM枸橼酸-枸橼酸钠的缓冲液制备含80mg/ml蔗糖，0.4mg/ml聚山梨醇酯80，TGF-β受体融合蛋白(融合蛋白9)浓度为50mg/ml的制剂。
过滤每种制剂并以1.2mL/瓶填充入2mL中性硼硅玻璃管制注射剂瓶中，水针压塞轧盖封口。将样品保存于25℃，进行稳定性分析，6个月的SEC或非还原CE-SDS检测。
实验结果见表13，结果表明，枸橼酸-枸橼酸钠体系优于组氨酸-盐酸盐体系(M6的SEC聚体：1.8％V 2.2％；非还原CE-SDS：94.5％V 92.2％)，因此可选择枸橼酸体系作为TGF-β受体融合蛋白的缓冲体系。
表13.缓冲体系筛选25℃加速稳定性实验结果
Figure PCTCN2019116593-appb-000040
注：T表示时间；D表示天；M表示月。
制备例4.TGF-β受体融合蛋白制剂中稳定剂的筛选
用下列不同糖种类的缓冲液，制备蛋白浓度为50mg/ml的TGF-β受体融合蛋白(融合蛋白9)制剂：
1)10mM枸橼酸-枸橼酸钠，80mg/ml蔗糖，pH 6.2
2)10mM枸橼酸-枸橼酸钠，80mg/mlα,α-二水合海藻糖，pH 6.2
过滤每种制剂并以1.2mL/瓶填充入2mL中性硼硅玻璃管制注射剂瓶中，水针压塞轧盖封口。取样品分别进行25℃常温和2-8℃低温长期保存实验。
实验结果见表14，结果表明蔗糖与海藻糖对TGF-β受体融合蛋白(融合蛋白9)的稳定性作用相似，选择蔗糖作为TGF-β受体融合蛋白(融合蛋白9)的稳定剂。当蔗糖浓度为80mg/ml时，渗透压约为300mosm/kg，接近等渗，因此可选蔗糖浓度为80mg/ml。
表14.糖种类筛选实验结果
Figure PCTCN2019116593-appb-000041
注：T表示时间，M表示月。
制备例5.TGF-β受体融合蛋白制剂中表面活性剂的筛选
用下列不同浓度及种类表面活性剂的缓冲液，制备蛋白浓度为50mg/ml的TGF-β受体融合蛋白(融合蛋白9)制剂：
1)10mM组氨酸-盐酸盐，0.1mg/ml聚山梨醇酯20，pH 6.2
2)10mM组氨酸-盐酸盐，0.2mg/ml聚山梨醇酯20，pH 6.2
3)10mM组氨酸-盐酸盐，0.4mg/ml聚山梨醇酯20，pH 6.2
4)10mM组氨酸-盐酸盐，0.6mg/ml聚山梨醇酯20，pH 6.2
5)10mM组氨酸-盐酸盐，0.8mg/ml聚山梨醇酯20，pH 6.2
6)10mM组氨酸-盐酸盐，0.1mg/ml聚山梨醇酯80，pH 6.2
7)10mM组氨酸-盐酸盐，0.2mg/ml聚山梨醇酯80，pH 6.2
8)10mM组氨酸-盐酸盐，0.4mg/ml聚山梨醇酯80，pH 6.2
9)10mM组氨酸-盐酸盐，0.6mg/ml聚山梨醇酯80，pH 6.2
10)10mM组氨酸-盐酸盐，0.8mg/ml聚山梨醇酯80，pH 6.2
过滤每种制剂并取0.5mL注射入50mL生理盐水注射液或5％葡萄糖注射液，使稀释后蛋白浓度为0.5mg/mL。观察样品的稀释稳定性。实验结果见表15，结果表明，处方中聚山梨酯20浓度达到0.2mg/ml以上时，稀释后不溶性微粒有显著下降；聚山梨酯80，氯化钠稀释产生的不溶性微粒随着其浓度增加而减少，当达 到0.4mg/ml以上时，10μm以上颗粒可降低到10个/ml以内。
表15.聚山梨酯筛选-稀释振摇实验结果
Figure PCTCN2019116593-appb-000042
制备例6.TGF-β受体融合蛋白制剂中表面活性剂的进一步筛选
用下列不同种类表面活性剂的缓冲液，制备蛋白浓度为50mg/ml的TGF-β受体融合蛋白(融合蛋白9)：
1)10mM枸橼酸-枸橼酸钠，0.4mg/ml聚山梨醇酯80，pH 6.2
2)10mM枸橼酸-枸橼酸钠，0.6mg/ml聚山梨醇酯20，pH 6.2
过滤每种制剂并以1.2mL/瓶填充入2mL中性硼硅玻璃管制注射剂瓶中，水针压塞轧盖封口。取样品进行2-8℃低温长期保存实验。
实验结果见表16，结果表明聚山梨醇酯80对TGF-β受体融合蛋白(融合蛋白9)的稳定性作用更好，因此选择聚山梨醇酯80作为TGF-β受体融合蛋白(融合蛋白9)的表明活性剂。
表16.聚山梨酯筛选2-8℃长期稳定性实验结果
Figure PCTCN2019116593-appb-000043
Figure PCTCN2019116593-appb-000044
注：T表示时间，D表示天，M表示月。
制备例7.TGF-β受体融合蛋白制剂滤膜相容性实验
TGF-β受体融合蛋白(融合蛋白9)以50mg/ml配制在10mM枸橼酸-枸橼酸钠缓冲液，80mg/ml蔗糖，0.4mg/ml聚山梨醇酯80，pH 6.2中。将制剂分别过0.22μm PES滤膜和PVDF滤膜并于开始、中间和最后取样检测。
实验结果见表17，蛋白含量、外观和纯度分析表明，TGF-β受体融合蛋白(融合蛋白9)在与滤膜接触的时间内是稳定的，该制剂与PES和PVDF滤膜均可以相容。
表17.滤膜相容性实验结果
Figure PCTCN2019116593-appb-000045
注：T表示时间。
制备例8.TGF-β受体融合蛋白制剂的冻干
用pH6.2的含10mM枸橼酸-枸橼酸钠的缓冲剂，制备TGF-β受体融合蛋白(融合蛋白9)浓度为50mg/ml，含80mg/ml蔗糖，0.4mg/ml聚山梨醇酯80的TGF-β受体融合蛋白(融合蛋白9)制剂。将抗体以6.3mL/瓶填充入20mL西林瓶中，装入冻干箱中，冻干。
冻干程序为预冻、一次干燥和二次干燥。冻干程序结束后，真空加塞。复溶样品进行冻干前后对比。结果表明，复溶溶液可保持液体制剂良好的性能。
表18.制剂的冻干步骤
Figure PCTCN2019116593-appb-000046
Figure PCTCN2019116593-appb-000047
制备例9.其它可选择制剂组成
此外，本公开还提供其它制剂配方的TGF-β受体融合蛋白(融合蛋白9)药物制剂：
(1)70mg/ml融合蛋白9，75mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和20mM枸橼酸-枸橼酸钠缓冲液，最终pH为6.4；
(2)80mg/ml融合蛋白9，85mg/ml蔗糖，0.5mg/ml的聚山梨醇酯80，和15mM枸橼酸-枸橼酸钠缓冲液，最终pH为6.2；
(3)60mg/ml融合蛋白9，90mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80，和5mM枸橼酸-枸橼酸钠缓冲液，最终pH为6.2；
(4)30mg/ml融合蛋白9，60mg/ml蔗糖，0.3mg/ml的聚山梨醇酯80，和30mM枸橼酸-枸橼酸钠缓冲液，最终pH为6.3；
(5)90mg/ml融合蛋白9，95mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和10mM枸橼酸-枸橼酸钠缓冲液，最终pH为6.0；
(6)100mg/ml融合蛋白9，70mg/ml蔗糖，0.1mg/ml的聚山梨醇酯80，和25mM枸橼酸-枸橼酸钠缓冲液，最终pH为6.5；
(7)50mg/ml融合蛋白9，80mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和10mM枸橼酸-枸橼酸钠缓冲液，最终pH为7.0；
(8)50mg/ml融合蛋白9，80mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和10mM枸橼酸-枸橼酸钠缓冲液，最终pH为7.5；
(9)50mg/ml融合蛋白9，80mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和10mM枸橼酸-枸橼酸钠缓冲液，最终pH为5.0；
(10)60mg/ml融合蛋白9，70mg/ml蔗糖，0.5mg/ml的聚山梨醇酯80，和15mM枸橼酸-枸橼酸钠缓冲液，最终pH为5.5；
(11)40mg/ml融合蛋白9，80mg/ml蔗糖，0.5mg/ml的聚山梨醇酯80，和10mM枸橼酸-枸橼酸钠缓冲液，最终pH为6.2；
(12)55mg/ml融合蛋白9，75mg/ml蔗糖，0.3mg/ml的聚山梨醇酯80，和5mM枸橼酸-枸橼酸钠缓冲液，最终pH为6.0；
(13)65mg/ml融合蛋白9，90mg/ml蔗糖，0.7mg/ml的聚山梨醇酯80，和30mM枸橼酸-枸橼酸钠缓冲液，最终pH为7.5；
(14)70mg/ml融合蛋白9，75mg/ml蔗糖，0.8mg/ml的聚山梨醇酯80，和30mM枸橼酸-枸橼酸钠缓冲液，最终pH为7.0；
(15)50mg/ml融合蛋白9，80mg/ml蔗糖，0.8mg/ml的聚山梨醇酯80，和 10mM枸橼酸-枸橼酸钠缓冲液，最终pH为7.0。

Claims (23)

  1. 一种药物组合物，其包含：
    TGF-β受体融合蛋白，以及
    缓冲液；
    其中，所述缓冲液选自：组氨酸盐缓冲液、琥珀酸盐缓冲液、枸橼酸盐缓冲液。
  2. 根据权利要求1所述的药物组合物，其中：
    所述的组氨酸盐缓冲液是组氨酸-盐酸缓冲液，
    所述的琥珀酸盐缓冲液是琥珀酸-琥珀酸钠缓冲液，
    所述枸橼酸盐缓冲液是枸橼酸-枸橼酸钠缓冲液；
    优选地，所述缓冲液是枸橼酸-枸橼酸钠缓冲液。
  3. 根据权利要求1或2所述的药物组合物，其中所述缓冲液的浓度为大约5mM至大约30mM，优选为大约5mM至大约20mM，最优选为大约10mM。
  4. 根据权利要求1至3任一项所述的药物组合物，其中所述TGF-β受体融合蛋白的浓度为大约0.5mg/ml至大约100mg/ml，优选为大约30mg/ml至大约70mg/ml，最优选为大约50mg/ml。
  5. 根据权利要求1至4任一项所述的药物组合物，其中所述药物组合物的pH为大约5.0至大约7.5，优选为大约6.0至大约6.5，最优选为大约6.2。
  6. 根据权利要求1至5任一项所述的药物组合物，其中：
    所述药物组合物还包含糖，
    优选地，所述糖选自：海藻糖和蔗糖，
    最优选地，所述糖为蔗糖。
  7. 根据权利要求6所述的药物组合物，其中所述糖的浓度为大约50mg/ml至大约100mg/ml，优选为大约60mg/ml至大约90mg/ml，最优选为大约80mg/ml。
  8. 根据权利要求1至7任一项所述的药物组合物，其中：
    所述药物组合物还包含表面活性剂，
    优选，所述表面活性剂为聚山梨醇酯，
    更优选，所述表面活性剂为聚山梨醇酯80。
  9. 根据权利要求8所述的药物组合物，其中所述表面活性剂的浓度为大约0.1mg/ml至大约0.8mg/ml，优选为大约0.4mg/ml至大约0.8mg/ml，更优选为大约0.4mg/ml。
  10. 根据权利要求1至9任一项所述的药物组合物，其包含：
    Figure PCTCN2019116593-appb-100001
    优选，所述药物组合物的pH为大约5.0至大约7.5；
    更优选，所述药物组合物的pH为大约6.0至大约6.5；
    优选地，所述药物组合物包含：
    Figure PCTCN2019116593-appb-100002
    优选地，所述药物组合物的pH为大约6.0至大约6.5；
    更优选地，所述药物组合物包含：
    Figure PCTCN2019116593-appb-100003
    优选地，所述药物组合物的pH为约6.2。
  11. 根据权利要求1至10任一项所述的药物组合物，其中所述的TGF-β受体融合蛋白如通式(I)所示：
    Ab-L-TGF-βRII ECD    (I)
    其中TGF-βRII ECD为TGF-βRII胞外区的截短形式；
    Ab为PD-L1抗体或其抗原结合片段；
    L为连接序列。
  12. 根据权利要求11所述的药物组合物，其中所述的连接序列为(G 4S) xG，其中x为3、4、5或6，优选x为4。
  13. 根据权利要求11或12所述的药物组合物，其中，所述TGF-βRII胞外区的截短形式是TGF-βRII胞外结构域序列在氨基端连续缺失至多26个氨基酸残基；优选TGF-βRII胞外结构域序列在氨基端连续缺失14、15、16、17、18、19、20、 21、22、23、24、25或26个氨基酸残基；
    更优选地，所述TGF-βRII ECD的序列如SEQ ID NO：14、15、16或17所示；优选如SEQ ID NO：15所示。
  14. 根据权利要求11至13任一项所述的药物组合物，其中所述的PD-L1抗体或其抗原结合片段包含：
    (A)分别如SEQ ID NO:1、SEQ ID NO:2和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和
    分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3；或
    (B)分别如SEQ ID NO:1、SEQ ID NO:10和SEQ ID NO:3所示的HCDR1、HCDR2和HCDR3，和
    分别如SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6所示的LCDR1、LCDR2和LCDR3。
  15. 根据权利要求11至14任一项所述的药物组合物，其中所述的PD-L1抗体或其抗原结合片段包含：
    (C)如SEQ ID NO:7所示的重链可变区，和如SEQ ID NO:8所示的轻链可变区；或
    (D)如SEQ ID NO:9所示的重链可变区，和如SEQ ID NO:11所示的轻链可变区。
  16. 根据权利要求11至15任一项所述的药物组合物，其中：
    所述PD-L1抗体的重链氨基酸序列如SEQ ID NO：12所示或与SEQ ID NO：12所示的氨基酸序列具有至少85％的同一性，
    所述PD-L1抗体的轻链氨基酸序列如SEQ ID NO：13所示或与SEQ ID NO：13所示氨基酸序列具有至少85％的同一性。
  17. 根据权利要求11至16任一项所述的药物组合物，其中所述TGF-βRII ECD通过连接序列融合至PD-L1抗体重链羧基末端；
    优选地，所述TGF-β受体融合蛋白包含：
    (E)PD-L1抗体重链和TGF-βRII ECD形成的融合肽，其序列如SEQ ID NO：23所示或与SEQ ID NO：23所示序列具有至少85％同一性，和
    PD-L1抗体轻链，其序列如SEQ ID NO：13所示或与SEQ ID NO：13所示序列具有至少85％的同一性；或
    (F)PD-L1抗体重链和TGF-βRII ECD形成的融合肽，其序列如SEQ ID NO：24所示或与SEQ ID NO：24所示序列具有至少85％的同一性，和
    PD-L1抗体轻链，其序列如SEQ ID NO：13所示或与SEQ ID NO：13所示序列具有至少85％的同一性。
  18. 一种制备如权利要求1至17任一项所述的药物组合物的方法，所述方法包括：使TGF-β受体融合蛋白和缓冲液接触的步骤；
    优选，所述缓冲液枸橼酸-枸橼酸钠缓冲液，
    优选，所述缓冲液的浓度为大约5mM至大约20mM，所述缓冲液的pH为约6.0至约6.5。
  19. 一种含有TGF-β受体融合蛋白的冻干制剂，所述含有TGF-β受体融合蛋白的冻干制剂通过将权利要求1至17任一项所述的药物组合物经冷冻干燥获得。
  20. 一种含有TGF-β受体融合蛋白的冻干制剂，所述含有TGF-β受体融合蛋白的冻干制剂经复溶后可形成如权利要求1至17中任一项所述的药物组合物。
  21. 一种含有TGF-β受体融合蛋白的复溶溶液，所述含有TGF-β受体融合蛋白的复溶溶液是通过将权利要求19或20所述的冻干制剂经复溶获得。
  22. 一种制品，其包括容器，所述容器中包含：
    如权利要求1至17任一项所述的药物组合物、或
    如权利要求19或20所述的含有TGF-β受体融合蛋白的冻干制剂、或
    如权利要求21所述的含有TGF-β受体融合蛋白的复溶溶液。
  23. 选自以下的任一项在制备药物中的用途：
    如权利要求1至17任一项所述的药物组合物、或如权利要求19或20所述的含有TGF-β受体融合蛋白的冻干制剂、或如权利要求21所述的含有TGF-β受体融合蛋白的复溶溶液、或如权利要求22所述的制品；
    优先地，所述药物用于治疗或抑制与肿瘤细胞增殖或肿瘤细胞转移相关的疾病或病症；
    更优选地，所述疾病或病症为肿瘤；
    更优选地，所述疾病或病症选自：头部颈部癌、成胶质细胞瘤、胶质瘤、鼻咽癌、甲状腺癌、肺癌、骨髓瘤癌、骨髓增生异常综合征、神经内分泌癌、淋巴瘤、白血病、黑素瘤、基底细胞皮肤癌、鳞状细胞皮肤癌、隆突性皮肤纤维肉瘤、梅克尔细胞癌、肉瘤、间皮瘤、胃癌、肝癌、胰腺癌、肾癌、膀胱癌、结直肠癌、乳腺癌、子宫内膜癌、子宫癌、宫颈癌、卵巢癌、前列腺癌、睾丸癌；所述肺癌选自：小细胞肺癌、非小细胞肺癌。
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