WO2022022660A1 - 抗pd-1抗体药物组合物及其用途 - Google Patents

抗pd-1抗体药物组合物及其用途 Download PDF

Info

Publication number
WO2022022660A1
WO2022022660A1 PCT/CN2021/109438 CN2021109438W WO2022022660A1 WO 2022022660 A1 WO2022022660 A1 WO 2022022660A1 CN 2021109438 W CN2021109438 W CN 2021109438W WO 2022022660 A1 WO2022022660 A1 WO 2022022660A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
cancer
concentration
heavy chain
Prior art date
Application number
PCT/CN2021/109438
Other languages
English (en)
French (fr)
Inventor
莫希叶乐
颜贞
刘洵
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to US18/018,471 priority Critical patent/US20230295329A1/en
Priority to BR112023001471A priority patent/BR112023001471A2/pt
Priority to CN202180045879.XA priority patent/CN115867316A/zh
Priority to AU2021317805A priority patent/AU2021317805A1/en
Priority to EP21849923.4A priority patent/EP4190353A4/en
Priority to JP2023503241A priority patent/JP2023535384A/ja
Priority to KR1020237006007A priority patent/KR20230044448A/ko
Priority to MX2023001160A priority patent/MX2023001160A/es
Priority to CA3189452A priority patent/CA3189452A1/en
Publication of WO2022022660A1 publication Critical patent/WO2022022660A1/zh

Links

Images

Classifications

    • 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
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K47/06—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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • 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
    • 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
    • C07K16/2818—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 CD28 or CD152
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • 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
    • 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

Definitions

  • the present disclosure belongs to the field of pharmaceutical formulations, and particularly relates to pharmaceutical compositions comprising anti-PD-1 antibodies, and their use as medicines.
  • Tumor immunotherapy is a treatment method that fully utilizes and mobilizes killer T cells in tumor patients to kill tumors.
  • tumor cell escape is a huge obstacle for tumor immunotherapy, and tumor cells use their own inhibitory effects on the immune system to promote rapid tumor growth.
  • Tumor-specific killer T cells are biologically active in the early stage of tumor immunotherapy, but lose their killing function in the later stage as the tumor grows.
  • T cells in the human body adopts two signaling pathway systems.
  • a series of co-stimulatory molecules are also required to provide the second signal.
  • T cells produce a normal immune response.
  • This dual signaling pathway system plays a crucial role in the balance of the immune system in the body, and it strictly regulates the body's different immune responses to self and non-self antigens. Absence of the second signal provided by the costimulatory molecule will result in an unresponsive or persistent specific immune response of T cells, resulting in tolerance. Therefore, the second signaling pathway plays a very key regulatory role in the whole process of the body's immune response.
  • PD-1 Programmed death-1
  • CTL-4 cytotoxic T lymphocyte antigen 4
  • PD-1 has two ligands, PD-L1 and PD-L2.
  • PD-L1 is mainly expressed on T cells, B cells, macrophages and dendritic cells (DC), and the expression on cells can be up-regulated after activation.
  • the expression of PD-L2 is relatively limited, mainly expressed on antigen-presenting cells, such as activated macrophages and dendritic cells.
  • Anti-PD-1 antibodies can maximize the patient's own immune system response to tumors by blocking the combination between PD-L1/PD-1, thereby achieving the purpose of killing tumor cells.
  • the present disclosure provides anti-PD-1 antibody pharmaceutical compositions and uses thereof.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-PD-1 antibody and a buffer
  • the buffer is an acetate buffer, a histidine buffer or a succinate buffer.
  • the pH of the buffer is about 4.5 to about 6.0, preferably the pH is about 4.7 to about 5.7, more preferably the pH is about 5.2;
  • the acetate buffer is preferably an acetic acid-sodium acetate buffer, the The histidine buffer is preferably a histidine-acetate buffer, and the succinate buffer is preferably a succinate-sodium succinate buffer;
  • the anti-PD-1 antibody comprises a heavy chain variable region and A light chain variable region comprising: HCDR1 as shown in SEQ ID NO:65, HCDR2 as shown in SEQ ID NO:66, and HCDR3 as shown in SEQ ID NO:67, the The light chain variable region comprises LCDR1 as shown in SEQ ID NO: 68, LCDR2 as shown in SEQ ID NO: 12, and LCD
  • the heavy chain variable region of the anti-PD-1 antibody comprises HCDR1 whose sequence is shown in SEQ ID NO: 8, and whose sequence is shown in SEQ ID NO: 9 HCDR2 shown in SEQ ID NO: 10, and HCDR3 shown in SEQ ID NO: 10;
  • the light chain variable region comprises LCDR2 shown in SEQ ID NO: 12, LCDR3 shown in SEQ ID NO: 13, and LCDR1 whose sequence is shown in the general formula RSSQSX 13 VHSX 14 X 15 X 16 TYLE (SEQ ID NO: 68), wherein X 13 is selected from L, X 14 is selected from N, Q, L, T or D, and X 15 is selected from G, A or V, X 16 is selected from N.
  • the anti-PD-1 antibody is an anti-PD-1 antibody selected from any one of the following (a) to (e):
  • an anti-PD-1 antibody comprising HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NO: 10, respectively; 12 and LCDR2 and LCDR3 shown in SEQ ID NO: 13, and LCDR1 having a sequence shown in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52;
  • an anti-PD-1 antibody comprising HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and HCDR1 whose sequences are shown in SEQ ID NO: 16, respectively: 17.
  • an anti-PD-1 antibody comprising HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively, and HCDR1, HCDR2 and HCDR3 whose sequences are respectively shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23; 24.
  • an anti-PD-1 antibody comprising HCDR1, HCDR2 and HCDR3 whose sequences are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and HCDR1, HCDR2 and HCDR3 whose sequences are respectively shown in SEQ ID NO: 16: 12 and LCDR2 and LCDR3 of SEQ ID NO: 13, and LCDR1 of sequence as set forth in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52; and
  • variable region of the heavy chain comprises HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and the variable region of the light chain comprises the sequence LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO: 17, SEQ ID NO: 12 and SEQ ID NO: 18, respectively.
  • the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises sequences such as SEQ ID NO: 8. HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 9 and SEQ ID NO: 10, and the light chain variable region comprises sequences such as SEQ ID NO: 49, SEQ ID NO: 12 and SEQ ID NO: LCDR1, LCDR2, and LCDR3 shown in 13.
  • the anti-PD-1 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises sequences such as SEQ ID NO: 21. HCDR1, HCDR2 and HCDR3 shown in SEQ ID NO: 22 and SEQ ID NO: 23, and the light chain variable region comprises sequences such as SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: LCDR1, LCDR2, and LCDR3 shown in 26.
  • the anti-PD-1 antibody binds to human PD-1 with a dissociation equilibrium constant (KD value) equal to or less than 10-7 M, in some embodiments , binds to human PD-1 with a dissociation equilibrium constant equal to or less than 10 -8 M, 10 -9 M, 10 -10 M or 10 -11 M.
  • KD value is determined by surface plasmon resonance technology (Biacore); for example, detected by the method described in Test Example 1 of the present disclosure.
  • the pH of the buffer is about 4.5 to about 6.0, preferably 4.5 to 6.0; in some embodiments, the pH of the buffer is about 4.7 to about 5.7, preferably 4.7 to 5.7; in other embodiments, the buffer pH is about 5.2, preferably 5.2; in other embodiments, the buffer is vinegar with a pH of about 4.7 to about 5.7 Acetate buffer; in other embodiments, the buffer is acetate buffer at pH 5.2.
  • non-limiting examples of buffer pH values include about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5 , about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, and any range between these point values.
  • the pH value of the aforementioned pharmaceutical composition is the same or almost the same as the pH value of its buffer (it is well known to those skilled in the art that in the process of preparing pharmaceutical formulations, some pH drifts (the pH of the formulation and the pH of the buffer) There are some differences, called pH drift value), the drift value of pH is usually within the range of ⁇ 0.3, unless otherwise specified, the pH drift value of the pharmaceutical composition of the present disclosure is within the range of ⁇ 0.3, preferably within the range of ⁇ 0.2, More preferably within the range of ⁇ 0.1).
  • the buffer concentration is about 5 mM to about 30 mM, preferably 5 mM to 30 mM; in some embodiments, the buffer concentration is about 5 mM to about 15 mM, preferably 5 mM to 15 mM; in some embodiments, the buffer concentration is about 10 mM, preferably 10 mM.
  • Non-limiting examples of buffer concentrations include about 5 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 18 mM, about 20 mM, about 25 mM, about 30 mM, and any range between these point values.
  • the anti-PD-1 antibody concentration is about 1 mg/mL to about 150 mg/mL, preferably 1 mg/mL to 150 mg/mL;
  • the -1 antibody concentration is about 90 mg/mL to about 150 mg/mL, preferably 90 mg/mL to 150 mg/mL; in some embodiments, the anti-PD-1 antibody concentration is about 100 mg/mL to about 120 mg/mL. In some embodiments, the anti-PD-1 antibody concentration is about 100 mg/mL. In some embodiments, the anti-PD-1 antibody concentration is 100 mg/mL.
  • Non-limiting examples of anti-PD-1 antibody concentrations include: about 1 mg/mL, about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg /mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, about 150 mg/mL, and between these point values any range.
  • the aforementioned pharmaceutical composition further includes an osmotic pressure regulator.
  • the osmotic regulator is a sugar (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.), amino acids (including arginine, glycine, semi- cystine, histidine, etc.) or salts (sodium chloride, potassium chloride, calcium chloride, etc.).
  • the sugar is selected from the group consisting of: glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbose Alcohol (also known as sorbitol), mannitol, mirabiose, melanotriose, raffinose, mannitol, stachyose, maltose, lactulose, maltulose, maltitol, lactitol and iso-maltone sugar.
  • the sugar is a non-reducing disaccharide; in some embodiments, the sugar is preferably trehalose or sucrose, most preferably sucrose.
  • the osmotic pressure regulator is one or more selected from the group consisting of sucrose, trehalose, sorbitol, arginine, glycine, and sodium chloride.
  • the osmolyte concentration is preferably from about 50 mg/mL to about 100 mg/mL, more preferably from about 70 mg/mL to about 90 mg/mL; most preferably about 80 mg/mL.
  • non-limiting examples of osmolyte concentrations include about 50 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg/mL, about 80 mg/mL, about 85 mg/mL mL, about 90 mg/mL, about 95 mg/mL, about 100 mg/mL, and any range between these point values.
  • the aforementioned pharmaceutical composition is an isotonic formulation.
  • the osmotic pressure adjusting agent controls the osmotic pressure of the aforementioned pharmaceutical composition at 280-320 mOsm, preferably at about 300 mOsm.
  • the osmotic pressure regulator is preferably about 70 mg/mL to about 90 mg/mL sucrose, most preferably about 80 mg/mL sucrose.
  • the aforementioned pharmaceutical composition further includes a surfactant
  • the surfactant can be selected from polysorbate 20 (also known as Tween 20), polysorbate 80 (also known as Tween 80), polyhydroxy Alkylene, Triton, Sodium Lauryl Sulfonate, Sodium Lauryl Sulfonate, Sodium Octyl Glycoside, Lauryl-Sulfobetaine, Myristyl-Sulfobetaine, Linoleyl-Sulfobetaine , stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristine Cetyl-betaine, cetyl-betaine, lauramidopropyl-betaine, cocalamidopropyl-betaine, linoleamidopropyl-betaine, my
  • the concentration of the surfactant is about 0.1 mg/mL to about 1.0 mg/mL, preferably about 0.2 mg/mL to about 0.8 mg/mL; in some In embodiments, the concentration of surfactant is about 0.4 mg/mL to about 0.8 mg/mL, preferably about 0.6 mg/mL to about 0.8 mg/mL; in some embodiments, the concentration of surfactant is about 0.6 mg /mL, preferably 0.6 mg/mL.
  • Non-limiting examples include about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, 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 mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1.0 mg/mL, and any range between these point values.
  • the anti-PD-1 antibody in the aforementioned pharmaceutical composition is a murine antibody, a chimeric antibody, a fully human antibody, or a humanized antibody.
  • the anti-PD-1 antibody in the aforementioned pharmaceutical composition is a humanized antibody.
  • the humanized antibody comprises a framework region derived from a human antibody or a framework region variant thereof.
  • the framework region variants are backmutations of up to 11 amino acids, respectively, based on the light chain framework region and/or heavy chain framework region of a human antibody.
  • the framework region variant comprises a mutation selected from any of the following (f) to (h) compared to a framework region derived from a human antibody:
  • (f) comprising a 2G amino acid back mutation in the light chain variable region, and/or one or more amino acid back mutations selected from the group consisting of 27Y, 48I, 67T, 69L, 82F and 93T in the heavy chain variable region;
  • variable region of the light chain comprises a 2V amino acid back mutation
  • variable region of the heavy chain comprises 26D, 27F, 30T, 38K, 43H, 48I, 66K, 67A, 69L, 82F and 93T one or more amino acid backmutations
  • the light chain variable region comprises one or more amino acid backmutations selected from 42G, 44V and 71Y, and/or the heavy chain variable region comprises 1K and/or 94S amino acid backmutations.
  • the anti-PD-1 antibody in the aforementioned pharmaceutical composition comprises an antibody variable region selected from the group consisting of:
  • (a2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10, respectively, and comprising 27Y, 48I, 67T, 69L , a heavy chain framework region backmutated by one or more amino acids in 82F and 93T, and
  • a light chain variable region comprising LCDR2 and LCDR3 as set forth in SEQ ID NO: 12 and SEQ ID NO: 13, respectively, and as set forth in SEQ ID NO: 11, 47, 48, 49, 50, 51 or 52 LCDR1, and a light chain framework region comprising a 2G amino acid back mutation;
  • (b2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively, and comprising 26D, 27F, 30T, 38K , 43H, 48I, 66K, 67A, 69L, 82F and 93T heavy chain framework regions backmutated by one or more amino acids; and
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 17, SEQ ID NO: 12 and SEQ ID NO: 18, respectively, and a light chain framework region comprising a 2V amino acid backmutation;
  • (c2) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having the sequences shown in SEQ ID NO: 21, SEQ ID NO: 22 and SEQ ID NO: 23, respectively, and comprising 1K and/or 94S amino acid back mutations the heavy chain framework region, and
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, respectively, and comprising one or more selected from 42G, 44V and 71Y Multiple amino acid backmutated light chain framework regions.
  • the anti-PD-1 antibody in the aforementioned pharmaceutical composition comprises an antibody variable region selected from any one of (i) to (o) below;
  • a light chain variable region whose sequence is shown in SEQ ID NO:5 or has at least 90% sequence identity with SEQ ID NO:5;
  • a light chain variable region whose sequence is shown in SEQ ID NO:7 or has at least 90% sequence identity with SEQ ID NO:7;
  • (k) a heavy chain variable region, the sequence of which is set forth in SEQ ID NO: 19 or has at least 90% sequence identity to SEQ ID NO: 19, and/or
  • a light chain variable region whose sequence is shown in SEQ ID NO: 20 or has at least 90% sequence identity with SEQ ID NO: 20;
  • a heavy chain variable region the sequence of which is set forth in SEQ ID NO: 27, 30, 31 or 32, or has at least 90% sequence identity with SEQ ID NO: 27, 30, 31 or 32, and/or
  • a light chain variable region whose sequence is shown in SEQ ID NO: 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64, or with SEQ ID NO: 28 , 29, 34, 35, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 have at least 90% sequence identity;
  • a light chain variable region the sequence of which is shown in SEQ ID NO: 34, 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64, or with SEQ ID NO: ID NO: 34, 35, 28, 29, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63 or 64 have at least 90% sequence identity;
  • a light chain variable region the sequence of which is set forth in SEQ ID NO: 42, 43 or 44, or has at least 90% sequence identity with SEQ ID NO: 42, 43 or 44;
  • a light chain variable region whose sequence is set forth in SEQ ID NO:71 or has at least 90% sequence identity to SEQ ID NO:71;
  • a light chain variable region whose sequence is shown in SEQ ID NO: 34 or 35, or has at least 90% sequence identity with SEQ ID NO: 34 or 35;
  • sequence SEQ ID NO:70 and SEQ ID NO:71 are general formulas, and the specific sequence is shown in Table 2:
  • the heavy chain variable region of the anti-PD-1 antibody is shown in SEQ ID NO: 27 or has at least 90% identity with SEQ ID NO: 27
  • the light chain variable region sequence of the anti-PD-1 antibody is shown in SEQ ID NO: 55 or has at least 90% sequence identity with SEQ ID NO: 55.
  • the heavy chain variable region of the anti-PD-1 antibody is as shown in SEQ ID NO:46 or has at least 90% identity with SEQ ID NO:46
  • the light chain variable region sequence of the anti-PD-1 antibody is shown in SEQ ID NO: 43 or has at least 90% sequence identity with SEQ ID NO: 43.
  • the aforementioned "at least 90% identical” includes having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.
  • the anti-PD-1 antibody further comprises an antibody heavy chain constant region and/or an antibody light chain constant region; in other embodiments, the antibody heavy chain The constant regions are selected from the constant regions of human IgGl, IgG2, IgG3 and IgG4 and conventional variants thereof, and the antibody light chain constant regions are selected from the constant regions of human antibody kappa and lambda chains and conventional variants thereof; in other embodiments, the heavy chain constant region of the antibody comprises the heavy chain constant region of IgG4 introduced with one or more mutations in S228P, F234A and L235A, such as three amino acid mutations of S228P, F234A and L235A; in other embodiments , the antibody comprises a heavy chain constant region with a sequence such as SEQ ID NO:72 or a light chain constant region shown in SEQ ID NO:79 and a light chain constant region with a sequence shown in SEQ ID NO:73.
  • the anti-PD-1 antibody comprises or has at least 85%, 86%, 87%, 88% as shown in SEQ ID NO:78 or with SEQ ID NO:78 , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of the light chain and as in SEQ ID NO: 77 or 82 or with SEQ ID NO: 77 or 82 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, A heavy chain of 97%, 98%, 99% or 100% sequence identity; or comprising or having at least 85%, 86%, 87%, 88%, 89 as set forth in SEQ ID NO:75 or with SEQ ID NO:75 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity of the light chain and as SEQ ID NO:78 or with SEQ ID NO:78
  • the anti-PD-1 antibody comprises: a heavy chain as shown in SEQ ID: 74 and a light chain as shown in SEQ ID: 75; or as SEQ ID: The heavy chain shown in 77 and the light chain shown in SEQ ID: 78.
  • the aforementioned pharmaceutical composition comprises:
  • an anti-PD-1 antibody at a concentration of about 1 mg/mL to about 150 mg/mL
  • an osmotic pressure regulator selected from the group consisting of sucrose, trehalose, sorbitol, arginine, glycine, and sodium chloride at a concentration of from about 50 mg/mL to about 100 mg/mL;
  • the aforementioned pharmaceutical composition comprises:
  • A1 an anti-PD-1 antibody at a concentration of about 90 mg/mL to about 150 mg/mL
  • sucrose at a concentration of about 70 mg/mL to about 90 mg/mL
  • polysorbate 80 at a concentration of about 0.6 mg/mL to about 0.8 mg/mL;
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 120 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.0, anti-PD-1 antibody at a concentration of about 100 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 100 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.5, anti-PD-1 antibody at a concentration of about 100 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.7, anti-PD-1 antibody at a concentration of about 100 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM histidine-acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 100 mg/mL, and sucrose at a concentration of about 80 mg/mL , and a concentration of approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 30 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 120 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 100 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.2 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 100 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was about 0.4 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 100 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.8 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 100 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was about 0.6 mg/mL polysorbate 20.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 100 mg/mL, trehalose at a concentration of about 80 mg/mL, and The concentration was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg/mL, and a polysorbate at a concentration of about 0.6 mg/mL Ester 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 100 mg/mL, sorbitol at a concentration of about 50 mg/mL, and The concentration was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg/mL, a concentration of about 100 mM arginine, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg/mL, a concentration of about 100 mM glycine, and a concentration of about 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.2, an anti-PD-1 antibody at a concentration of about 100 mg/mL, a concentration of about 100 mM NaCl, and a concentration of about 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 20 mM acetic acid-sodium acetate buffer at a pH of about 5.7, anti-PD-1 antibody at a concentration of about 150 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 21.9 mM acetic acid-sodium acetate buffer at a pH of about 4.7, anti-PD-1 antibody at a concentration of about 120 mg/mL, sucrose at a concentration of about 80 mg/mL, and The concentration was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 4.7, anti-PD-1 antibody at a concentration of about 90 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 20 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 120 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 20 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 90 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.7, anti-PD-1 antibody at a concentration of about 150 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 30 mM acetic acid-sodium acetate buffer at a pH of about 4.7, anti-PD-1 antibody at a concentration of about 90 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 4.7, anti-PD-1 antibody at a concentration of about 150 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 30 mM acetic acid-sodium acetate buffer at a pH of about 5.2, anti-PD-1 antibody at a concentration of about 150 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 10 mM acetic acid-sodium acetate buffer at a pH of about 5.7, anti-PD-1 antibody at a concentration of about 90 mg/mL, sucrose at a concentration of about 80 mg/mL, and a concentration of was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: about 30 mM acetic acid-sodium acetate buffer at a pH of about 5.7, anti-PD-1 antibody at a concentration of about 107.7 mg/mL, sucrose at a concentration of about 80 mg/mL, and The concentration was approximately 0.6 mg/mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: 10 mM acetic acid-sodium acetate buffer at pH 5.2, anti-PD-1 antibody at a concentration of 120 mg/mL, sucrose at a concentration of 70 mg/mL, and a concentration of 0.6 mg/mL mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: 10 mM acetic acid-sodium acetate buffer at pH 5.2, anti-PD-1 antibody at a concentration of 120 mg/mL, sucrose at a concentration of 90 mg/mL, and a concentration of 0.6 mg/mL mL polysorbate 80.
  • the aforementioned pharmaceutical composition comprises: 10 mM acetic acid-sodium acetate buffer at pH 5.2, anti-PD-1 antibody at a concentration of 120 mg/mL, sucrose at a concentration of 80 mg/mL, and a concentration of 0.6 mg/mL mL polysorbate 80; wherein, the anti-PD-1 antibody has a heavy chain as shown in SEQ ID:74, and a light chain as shown in SEQ ID:75.
  • the present disclosure provides a method of preparing the aforementioned pharmaceutical composition, the method comprising the step of exchanging a stock solution of an anti-PD-1 antibody with a buffer, in some embodiments, the buffer is selected from the group consisting of Acetate buffer, histidine buffer or succinate buffer.
  • the present disclosure also provides a lyophilized formulation, which upon reconstitution can form the pharmaceutical composition of any of the foregoing.
  • the present disclosure provides a lyophilized formulation containing an anti-PD-1 antibody obtained by lyophilizing the aforementioned pharmaceutical composition.
  • the aforementioned preparation wherein the freeze-drying comprises the steps of pre-freezing, primary drying and secondary drying in sequence.
  • the present disclosure provides a lyophilized formulation comprising an anti-PD-1 antibody, which upon reconstitution can form the aforementioned pharmaceutical composition.
  • the present disclosure provides a lyophilized formulation that is a lyophilized form of the pharmaceutical composition of any of the foregoing.
  • the aforementioned pharmaceutical compositions or lyophilized formulations are stable formulations. In some embodiments, the aforementioned pharmaceutical compositions or lyophilized formulations are stored at a refrigerated temperature (2-8° C.) for 6 months, and the SEC The percentage of monomers dropped by no more than 5%. In some embodiments, the aforementioned pharmaceutical compositions or lyophilized formulations are stable at 2-8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months.
  • the percentage of SEC monomer in the formulation decreases by no more than 2% (eg, less than 2%, less than 1%, less than 0.8%, less than 0.5%, or even less). ); in some embodiments, the pharmaceutical composition under the forced degradation conditions (40 ° C M1), compared with the SEC value at D0, the SEC decreases by less than or equal to about 10%; preferably less than or equal to about 9%, About 8%, about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%; more preferably, the SEC reduction is between 1.1% and 2.7%. In some embodiments, the aforementioned pharmaceutical composition is placed at 4°C for 6 months, and the formulation SEC monomer percentage is greater than 94% (eg, greater than 94.5%, 95%, 98%).
  • the present disclosure provides a reconstituted solution containing an anti-PD-1 antibody obtained by reconstituting the aforementioned lyophilized formulation.
  • the solution used for its reconstitution includes but is not limited to water for injection, physiological saline or glucose solution, preferably water for injection.
  • the present disclosure provides a reconstitution solution that is a reconstituted form of the lyophilized formulation of any of the foregoing.
  • the present disclosure provides an article of manufacture comprising a container containing a pharmaceutical composition, lyophilized formulation, or reconstitution solution as previously described.
  • the container is a neutral borosilicate glass vial for injection.
  • the present disclosure also provides use of the aforementioned pharmaceutical compositions, lyophilized formulations, reconstituted solutions or preparations in the manufacture of a medicament for the treatment/prevention of a disease or disorder.
  • the present disclosure also provides the aforementioned pharmaceutical compositions, lyophilized formulations, reconstituted solutions or articles of manufacture as medicaments for the treatment/prevention of a disease or disorder.
  • the present disclosure also provides a method of treating or preventing a disease or disorder, the method comprising administering to a subject a therapeutically effective amount or a prophylactically effective amount of the aforementioned pharmaceutical composition, lyophilized formulation, reconstitution solution or preparation.
  • the disease or disorder of any of the foregoing is a PD-1 related disease or disorder.
  • the disease of any of the foregoing is a tumor.
  • the disease of any preceding item is selected from the group consisting of: head and neck squamous cell carcinoma, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma cell tumor, central nervous system cancer, neuroendocrine tumor, throat cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular tumor, hepatobiliary cancer, pancreatic cancer, gastric cancer, Gastrointestinal cancer, bowel cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell cancer, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma , leukemia, lymphoma, bone cancer, chondrosarcoma, myelo
  • Figure 1 The results of the anti-PD-1 antibody blocking the binding of PD-1 to its ligand
  • Figure 2 The effect of anti-PD-1 antibody on the secretion of IFN ⁇ by PBMC cells
  • Figure 3 The efficacy of anti-PD-1 antibody on mouse colon cancer MC38 xenografts
  • Figure 4 The effect of anti-PD-1 antibody on the tumor volume of mouse colon cancer MC38;
  • Figure 5 DOE fit plot of anti-PD-1 antibody formulations.
  • Buffer refers to a buffer that tolerates changes in pH through the action of its acid-base conjugated component.
  • buffers that control pH in an appropriate range include acetate buffers, succinate buffers, gluconate buffers, histidine buffers, oxalate buffers, lactate buffers, phosphoric acid buffers Salt buffers, citrate buffers, tartrate buffers, fumarate buffers, glycylglycine buffers and other organic acid buffers.
  • Acetate buffer or "acetate buffer”, is a buffer that includes acetate ions.
  • acetate buffers include acetate-sodium acetate, histidine-acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like.
  • the acetate buffer is acetic acid-sodium acetate buffer (also referred to as sodium acetate, abbreviated AA).
  • Hetidine buffer or “histidine buffer” is a buffer containing histidine ions.
  • Histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc. buffers.
  • the buffer is a histidine-acetate buffer (also known as histidine acetate, abbreviated His-AA).
  • succinate buffer or “succinate buffer” is a buffer that includes succinate ions.
  • succinate buffers include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate buffer, and the like.
  • the succinate buffer is a succinate-sodium succinate buffer (also known as sodium succinate, abbreviated SA).
  • citrate buffer or “citrate buffer” is a buffer that includes citrate ions.
  • citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate buffer, and the like.
  • a preferred citrate buffer is citrate-sodium citrate buffer (also known as sodium citrate, abbreviated CA).
  • An osmotic pressure regulator refers to a substance used to adjust the osmotic pressure of a solution.
  • osmotic pressure regulators include, but are not limited to, saccharides (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.), amino acids (including arginine, glycine, cysteine, histidine, etc.), salts (sodium chloride, potassium chloride, calcium chloride, etc.).
  • the osmotic pressure regulator is a sugar selected from the group consisting of: glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol , xylitol, sorbitol (also known as sorbitol), mannitol, miribiose, melanotriose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol , maltitol, lactitol and iso-maltulose; in some embodiments, the sugar is a non-reducing disaccharide; in some embodiments, the sugar is preferably trehalose or sucrose, most preferably sucrose. In some embodiments, the osmotic pressure regulator is an amino acid, preferably arginine, glycine; in some
  • “Isotonic” means that the formulation has substantially the same osmotic pressure as human blood. Isotonicity can be determined by methods known in the art, for example, using a vapor pressure or frozen-type osmometer.
  • the osmotic pressure of the pharmaceutical composition is preferably controlled at 280-320 mOsm, and the osmotic pressure regulator is preferably 70-90 mg/mL sucrose; more preferably, the osmotic pressure of the pharmaceutical preparation is controlled at about 300 mOsm, and the preferred content of the osmotic pressure regulator is 80 mg/mL of sucrose.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, such as a physiologically/pharmaceutically acceptable Carriers and Excipients.
  • the pharmaceutical composition maintains the stability of the active ingredient of the antibody, promotes the administration to the organism, facilitates the absorption of the active ingredient and exerts biological activity.
  • “pharmaceutical composition” and “formulation” are not mutually exclusive.
  • Displacement refers to the displacement of the solvent system that dissolves the antibody protein, eg, the presence of the antibody protein in the stable formulation by physically manipulating a high-salt or hypertonic solvent system containing the antibody protein using the buffer system of the stable formulation.
  • the so-called physical manipulations include, but are not limited to, ultrafiltration, dialysis or reconstitution after centrifugation.
  • the pharmaceutical compositions or formulations of the present disclosure can be prepared by methods well known in the art.
  • preparation of antibody pharmaceutical compositions or preparations the first step: take a certain amount of purified antibody solution, and perform solvent replacement with a buffer without antibody (such as 10mM sodium acetate buffer at pH 5.2) (Ultrafiltration is preferred), the antibody is concentrated to about 130 mg/mL by at least 6 volumes of ultrafiltration membrane displacement.
  • a certain volume of sucrose stock solution was added and mixed to make the final sucrose concentration 80 mg/mL.
  • a certain volume of the polysorbate 80 mother solution was added and mixed to make the final polysorbate 80 concentration 0.6 mg/mL.
  • Step 2 Adjust the filling volume, fill the filtrate in vials, add stoppers, and take samples at the beginning of filling, in the middle of filling, and at the end of filling to detect the difference in filling volume.
  • Step 3 Open the capping machine, add aluminum caps, and perform capping.
  • Step 4 Visual inspection to confirm that the product has no defects such as inaccurate loading. Printing and pasting vial labels; printing carton labels, folding carton boxes, packing boxes, and sticker box labels.
  • the solvent is water.
  • Lyophilized formulation means a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step. Lyophilized formulations can be obtained by freeze-drying pharmaceutical compositions or liquid or solution formulations. Freeze drying is performed by freezing the formulation and then subliming the water at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic or decomposition temperature of the formulation. At suitable pressures, typically in the range of about 50-250 mTorr, the storage temperature for primary drying is typically in the range of about -30 to 25°C (assuming the product remains frozen during the primary drying).
  • the formulation, the size and type of container (eg, glass vial) holding the sample, and the volume of liquid determine the time required for drying, which can range from hours to days (eg, 40-60 hours).
  • the secondary drying stage can be carried out at about 0-40°C, depending mainly on the type and size of the vessel and the type of protein employed.
  • the secondary drying time is determined by the desired residual moisture level in the product, and typically requires at least about 5 hours.
  • the moisture content of the lyophilized formulation is less than about 5%, preferably less than about 3%.
  • the pressure may be the same as the pressure applied in the primary drying step, preferably, the pressure for the secondary drying is lower than that for the primary drying. Freeze drying conditions can vary with formulation and vial size.
  • the surfactant of the present disclosure may be selected from polysorbate 20, polysorbate 80, polyhydroxyalkene, Triton, sodium lauryl sulfonate, sodium lauryl sulfonate, sodium octyl glycoside, lauryl-sulfonic acid Betaine, Myristyl-Sulfobetaine, Linoleyl-Sulfobetaine, Stearyl-Sulfobetaine, Lauryl-sarcosine, Myristyl-sarcosine, Linole- sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocalamidopropyl-beet Alkali, Linoleamidopropyl-Betaine, Myristamidopropyl-Betaine, Palmamidopropyl-Betaine, Isostearamidoprop
  • the term “about” or “approximately” refers to an index value within an acceptable error range of the particular value determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (ie, the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation in every practice in the art. Alternatively, “about” or “approximately” can mean a range of up to 20%.
  • the pharmaceutical composition described in the present disclosure can achieve a stable effect, wherein the antibody substantially retains its physical stability and/or chemical stability and/or biological activity after storage.
  • Pharmaceutical composition preferably, pharmaceutical composition Its physical and chemical stability and its biological activity are substantially retained after storage.
  • the shelf life is generally selected based on the intended shelf life of the pharmaceutical composition.
  • analytical techniques for measuring protein stability which can measure stability after storage at a selected temperature for a selected period of time.
  • a stable drug antibody formulation is one in which no significant change is observed when stored at refrigerated temperatures (2-8°C), for example, for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably Up to 2 years.
  • Stable formulations e.g., drug antibody formulations are colorless, or clear to slightly opalescent, by visual analysis; the formulations have no more than ⁇ 10% variation in concentration, pH, and osmolality; typically no more than ⁇ 10% is observed About 10%, preferably no more than about 5% truncation; generally no more than about 10%, preferably no more than about 5% aggregates are formed, and the like.
  • the pharmaceutical compositions or lyophilized formulations of the present disclosure are 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.
  • formulations can be observed by observing forced degradation (eg, 40°C M1), accelerated conditions (eg, 25°C M6), shaking D7 (eg, 25°C, 300 rpm, shaking for 10 days), multiple freeze-thaw cycles
  • forced degradation eg, 40°C M1
  • accelerated conditions eg, 25°C M6
  • shaking D7 eg, 25°C, 300 rpm, shaking for 10 days
  • multiple freeze-thaw cycles The relative stability of the preparation was detected by the appearance of the preparation under the same conditions, SEC, non-reduced CE-SDS and iCIEF and other indicators.
  • Antibodies show no significant increase in aggregation, precipitation and/or denaturation if after visual inspection for color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS) , then the antibody "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).
  • An antibody "retains its chemical stability” in a pharmaceutical formulation if it does not exhibit significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of proteins include hydrolysis or truncation (as assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (by peptide mapping such as combined with mass spectrometry or MALDI/TOF/MS, etc.) methods), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.), and isomerization (by measuring isoaspartic acid content, Peptide Mapping, etc.).
  • An antibody "retains its biological activity" in a pharmaceutical formulation if its biological activity at a given time is within a predetermined range of the biological activity exhibited at the time of preparation of the pharmaceutical formulation.
  • the biological activity of an antibody can be determined, for example, by antigen binding assays.
  • programmed cell death 1 means programmed cell death 1
  • protein PD-1 protein PD-1
  • PD-1 protein PD-1
  • PDCD1 protein PD-1
  • hPD-1 hPD-1
  • the terms “programmed cell death 1”, “programmed cell death 1”, “protein PD-1”, “PD-1”, “PDCD1” and “hPD-1” are used interchangeably and include human PD-1 Variants, isoforms, species homologues, and analogs that share at least one epitope with PD-1.
  • the complete PD-1 sequence can be found at GenBank Accession No. U64863.
  • P-L1 programmed death ligand-1
  • PD-L1 is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2), which downregulates T when bound to PD-1 Cell activation and cytokine secretion.
  • the term "PD-L1” as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and interspecies homologues of hPD-L1, and 5 species having at least one of hPD-L1 Analogues of common epitopes. The complete hPD-L1 sequence can be found using GenBank accession number Q9NZQ7.
  • cytokine is a general term for proteins released by a population of cells that act as intercellular mediators on other cells.
  • cytokines include lymphokines, monokines, chemokines and traditional polypeptide hormones.
  • exemplary cytokines include: human IL-2, IFN- ⁇ , IL-6, TNF ⁇ , IL-17, and IL-5.
  • Antibody as used in this disclosure is used herein in the broadest sense and includes various antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, murine antibodies, chimeric antibodies, humanized antibodies , multispecific antibodies (eg, bispecific antibodies) and antibody fragments so long as they exhibit the desired antigen-binding activity and specificity.
  • Antibody as used in the present disclosure includes “full-length antibody” and "antigen-binding fragments” thereof.
  • the anti-PD-1 antibody is the anti-PD-1 antibody described in international patent application PCT/CN2020/074098, such as "Hu23-11.IgG4AA” antibody. This disclosure incorporates the entire contents of International Patent Application PCT/CN2020/074098 into the present application.
  • full-length antibody intact antibody
  • complete antibody complete antibody
  • whole antibody whole antibody
  • antigen-binding fragment or “functional fragment” is one or more fragments of an intact antibody that retain the ability to specifically bind an antigen (eg, PD-1).
  • antigen-binding fragments include, but are not limited to (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F(ab') 2 fragments, comprising disulfide bridges across the hinge region Bivalent fragment of two Fab fragments linked, (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VH and VL domains of one-armed antibody; (v) single domain or dAb fragments (Ward et al.
  • Antigen binding moieties can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • antigen-binding fragments of the present disclosure include Fab, F(ab')2, Fab', single chain antibody (scFv), dimerized V region (diabody), disulfide stabilized V Region (dsFv), etc.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • Each light chain variable region (VL) and heavy chain variable region (VH) are composed of 3 CDR regions and 4 FR regions. The order from the amino terminus to the carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • LCDR1, LCDR2, LCDR3 three CDRs in each light chain variable region.
  • the amino acid sequence boundaries of CDRs can be determined using any of a variety of well-known schemes, including the "Kabat” numbering convention (see Kabat et al.
  • VH variable domain
  • VL variable domain
  • CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50 -56 (LCDR2) and 89-97 (LCDR3).
  • CDR amino acids in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and amino acids in VL Residue numbers are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).
  • the CDRs are defined by amino acid residues 26-35 in human VH (HCDR1 ), 50-65 (HCDR2) and 95-102 (HCDR3) and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3) in human VL.
  • VH The CDR amino acid residue numbers in VL are approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are approximately 27-32 (CDR1), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR regions of the antibody can be determined using the program IMGT/DomainGap Align.
  • the CDR amino acids in VH are numbered 26-32 (HCDR1), 50-58 ( HCDR2) and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2 ) and 89-97 (LCDR3).
  • the "Kabat" numbering convention applies to the antibody variable regions and CDR sequences involved in the examples of the present disclosure.
  • inventions of the human antibody heavy chain constant region and the human antibody light chain constant region mentioned in the present disclosure refer to the human-derived heavy chain constant regions disclosed in the prior art that do not alter the structure and function of the antibody variable region or variants of the light chain constant region
  • exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed reengineering and amino acid substitutions of the heavy chain constant region, specifically replacing YTE as known in the art Mutations, L234A and/or L235A mutations, S228P mutations, and/or mutations that acquire a knob-into-hole structure (giving the antibody heavy chain a knob-Fc and hole-Fc combination), which have been shown to confer antibodies with novel performance, but does not alter the function of the variable region of the antibody.
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, preferably humanized antibodies.
  • murine antibody in this disclosure is a monoclonal antibody directed against human PD-1 prepared according to the knowledge and skill in the art. In preparation, test subjects are injected with the PD-1 antigen, and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine anti-PD-1 antibody may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise murine IgG1, IgG2, IgG3 or the heavy chain constant region of a variant thereof.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can alleviate the immune response induced by the murine antibody.
  • a chimeric antibody usually, to build a chimeric antibody, first establish a hybridoma that secretes a mouse-specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as needed, and then clone the mouse variable region gene from the mouse hybridoma cell.
  • the gene is linked with the human constant region gene to form a chimeric gene and inserted into an expression vector, and finally the chimeric antibody is expressed in a eukaryotic system or a prokaryotic system.
  • the antibody light chain of the PD-1 chimeric antibody further comprises a light chain constant region of a human ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the PD-1 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably a human IgG1, IgG2 or IgG4 heavy chain constant region, or using IgG1, IgG2 or IgG4 variants with amino acid mutations (eg L234A and/or L235A mutations, and/or S228P mutations).
  • humanized antibody also known as CDR-grafted antibody, refers to the grafting of murine CDR sequences into a human antibody variable region framework, i.e. a different type of human germline antibody antibodies produced in framework sequences.
  • the heterologous reaction induced by chimeric antibodies can be overcome because they carry a large amount of murine 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), and in Kabat, EA, et al.
  • human antibody variable region framework sequence can be subjected to minimal reverse mutation or back mutation to maintain the activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies that have been further subjected to affinity maturation mutation of the CDRs by yeast display.
  • human antibody (HuMAb), “human antibody”, “fully human antibody”, “fully human antibody” are used interchangeably, the amino acid sequence of which corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from amino acid sequences of non-human origin using human antibody repertoires or other human antibody coding sequences.
  • This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen-binding residues.
  • fully human antibodies can be constructed by gene or chromosomal transfection methods and phage display techniques, or by in vitro activated B cells, all of which are known in the art.
  • amino acid difference or “amino acid mutation” refers to the presence of amino acid changes or mutations in a variant protein or polypeptide compared to the original protein or polypeptide, including 1, 2, 3, or Insertion, deletion or substitution of more amino acids.
  • antibody framework or "FR region” refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • epitopes refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (eg, a specific site on a PD-1 molecule).
  • Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, eg, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (KD) of less than about 10-7 M, eg, less than about 10-9 M, 10-10 M, 10-11 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.
  • the antibodies of the present disclosure bind PD-1 with a dissociation equilibrium constant (KD) of less than about 10-7 M, eg, less than about 10-8 M or 10-9 M, eg, as using surface plasmon resonance (SPR) Techniques are measured in a BIACORE instrument.
  • SPR surface plasmon resonance
  • the term “compete” in the context of antigen-binding proteins competing for the same epitope (eg, neutralizing antigen-binding proteins or neutralizing antibodies), it means competition between antigen-binding proteins, as determined by the following assay:
  • the antigen-binding protein eg, antibody or immunologically functional fragment thereof
  • the antigen-binding protein prevents or inhibits (eg, reduces) the interaction of a reference antigen-binding protein (eg, ligand or reference antibody) with a common antigen (eg, PD-1 antigen or fragment thereof).
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA solid-phase direct or indirect enzyme immunoassay
  • Sandwich competition assay see, eg, Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid-phase direct biotin-avidin EIA see, eg, Kirkland et al., 1986, J. Immunol. 137:3614-3619
  • solid-phase Phase Direct Labeling Assay Solid Phase Direct Labeling Sandwich Assay (see e.g.
  • Solid Phase Direct Labeling with I-125 Label RIA see, eg, Morel et al., 1988, Molec. Immunol. 25:7-15
  • solid-phase direct biotin-avidin EIA see, eg, Cheung, et al., 1990, Virology 176:546-552
  • directly labeled RIA Methyl et al., 1990, Scand. J. Immunol. 32:77-82
  • the assay involves the use of purified antigen bound to a solid surface or cell bearing either an unlabeled test antigen binding protein and a labeled reference antigen binding protein.
  • Antigen-binding proteins identified by competitive assays include: antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein; and antigen-binding proteins that bind to adjacent epitopes sufficiently close to the binding epitope of the reference antigen-binding protein protein, the two epitopes sterically prevent each other from binding. Additional details regarding methods for determining competitive binding are provided in the Examples herein.
  • a competing antigen binding protein when present in excess, it will inhibit (eg decrease) by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70% -75% or 75% or more specific binding of the reference antigen binding protein to a common antigen. In certain instances, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • nucleic acid molecule refers to DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, preferably double-stranded DNA or single-stranded mRNA or modified mRNA. A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors with a bacterial origin of replication) or may integrate into the host cell's genome after introduction into the host cell, thereby following The host genome replicates together (eg, a non-episomal mammalian vector).
  • mice can be immunized with human PD-1 or fragments thereof, and the resulting antibodies can be renatured, purified, and amino acid sequenced using conventional methods.
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the antibody or antigen-binding fragment of the present invention uses genetic engineering to add one or more human FR regions to the non-human CDR regions.
  • Human FR germline sequences can be obtained by aligning the IMGT human antibody variable region germline gene database with MOE software, from the website of ImMunoGeneTics (IMGT) at http://imgt.cines.fr, or from the Journal of Immunoglobulins, 2001 ISBN012441351 get.
  • IMGT ImMunoGeneTics
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • the engineered antibodies or antigen-binding fragments of the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian-like expression systems lead to glycosylation of the antibody, especially at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human PD-1. Positive clones were expanded in serum-free medium in bioreactors for antibody production.
  • the antibody-secreted culture medium can be purified by conventional techniques.
  • a or G Sepharose FF column with adjusted buffer. Non-specifically bound components are washed away. 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, ion exchange. The obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean in vitro and ex vivo treatment of, eg, cells by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, eg, a composition comprising any of the binding compounds of the present disclosure, to a patient having one or more disease symptoms for which the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a patient or population to be treated in an amount effective to alleviate one or more symptoms of a disease, to induce regression of such symptoms or to inhibit progression of such symptoms to any clinically measured degree.
  • the amount of a therapeutic agent effective to relieve symptoms of any particular disease can vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient.
  • Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (eg, methods of treatment or articles of manufacture) may be ineffective in alleviating symptoms of each target disease, the method of The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • H test Kruskal-Wallis test
  • Jonckheere-Terpstra test Jonckheere-Terpstra test
  • Wilcoxon test determine that it should reduce symptoms of the target disease in a statistically significant number of patients.
  • Constant modification or “conservative substitution or substitution” refers to the replacement of amino acids in a protein by other amino acids with similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., 224, (4th ed.).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity. Exemplary conservative substitutions are set forth in the table "Exemplary Conservative Amino Acid Substitutions" below.
  • an “effective amount” or “effective dose” refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction in severity, or delay in onset of disorders, including biochemical, tissue academic and/or behavioral symptoms.
  • beneficial or desired outcomes include clinical outcomes, such as reducing the incidence or amelioration of one or more symptoms of the various target antigen-related disorders of the present disclosure, reducing the amount of other agents required to treat the disorder dose, enhances the efficacy of another agent, and/or delays the progression of a disorder associated with the target antigen of the present disclosure in a patient.
  • Exogenous refers to a substance produced outside an organism, cell, or human body, as the case may be.
  • Endogenous refers to a substance produced in a cell, organism, or human body as the case may be.
  • Homology refers to the sequence similarity between two polynucleotide sequences or between two polypeptides. Two DNA molecules are homologous when a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position is occupied by an adenine, then the molecules are homologous at that position . The percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100.
  • sequences when sequences are optimally aligned, two sequences are 60% homologous if 6 matches or homology at 10 positions in the two sequences; if 95 matches at 100 positions in the two sequences or homologous, then the two sequences are 95% homologous.
  • comparisons are made when aligning two sequences to give the greatest percent homology.
  • the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are chosen to give the maximum match between the respective sequences over the entire length of the respective reference sequences.
  • the following references refer to the BLAST algorithm frequently used in sequence analysis: BLAST Algorithm (BLAST ALGORITHMS): Altschul, SF et al., (1990) J. Mol. Biol.
  • the expressions "cell”, “cell line” and “cell culture” are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of transfers. It should also be understood that, due to deliberate or unintentional mutations, all progeny may not be exactly the same in terms of DNA content. Mutant progeny that have the same function or biological activity as screened in the original transformed cell are included. Where a different name is meant, it is clear from the context.
  • Polymerase chain reaction or "PCR” as used herein refers to a procedure or technique in which traces of a specified portion of nucleic acid, RNA and/or DNA are amplified as described in, eg, US Pat. No. 4,683,195. Generally, sequence information from the end of the target region or beyond is required to allow the design of oligonucleotide primers; these primers are identical or similar in sequence to the corresponding strand of the template to be amplified. The 5'-terminal nucleotides of the two primers can be identical to the ends of the material to be amplified.
  • PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA and cDNA, phage or plasmid sequences transcribed from total cellular RNA, and the like. See generally Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263; Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.).
  • PCR as used herein is considered to be one example, but not the only example, of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, which method includes the use of known nucleic acids and nucleic acid polymerases as primers to amplify or Generate a specific portion of nucleic acid.
  • isolated refers to a purified state, and in this case means that the named molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates, or other materials, such as cell debris and growth media. Generally, the term “isolated” is not intended to mean the complete absence of these materials or the absence of water, buffers, or salts, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds as described herein.
  • the present disclosure includes an agent for treating a disease associated with a target antigen (eg, PD-1) positive cell, the agent comprising the anti-PD-1 antibody of the present disclosure as an active ingredient.
  • a target antigen eg, PD-1 positive cell
  • the disease associated with PD-1 in the present disclosure is not limited as long as it is a disease associated with PD-1, eg, a therapeutic response induced using the molecules of the present disclosure can be achieved by binding to human PD-1 and then repressing PD-1 and its associated Binding of ligands PD-L1 and PD-L2, or killing tumor cells overexpressing PD-1.
  • the molecules of the present disclosure are very useful to persons suffering from tumors or cancers, preferably melanoma, colon cancer, breast cancer, lung cancer, stomach cancer, Colon cancer, kidney cancer, non-small cell lung cancer, bladder cancer, etc.
  • the present disclosure relates to methods for immunodetection or assay of target antigen (eg PD-1), reagents for immunodetection or assay of target antigen (eg PD-1), for immunodetection or assay of expression of target antigen (eg PD-1) PD-1) cells and diagnostic agents for diagnosing diseases associated with target antigen (eg, PD-1) positive cells, comprising the present disclosure that specifically recognizes a target antigen (eg, human PD-1) and interacts with cells
  • An antibody or antibody fragment to which the amino acid sequence of the outer region or its three-dimensional structure is bound serves as an active ingredient.
  • the method for detecting or determining the amount of the target antigen can be any known method.
  • it includes immunodetection or assay methods.
  • An immunodetection or assay method is a method for detecting or measuring the amount of antibody or antigen using labeled antigen or antibody.
  • immunodetection or assay methods include radioactive substance-labeled immunoantibody methods (RIA), enzyme immunoassays (EIA or ELISA), fluorescent immunoassays (FIA), luminescence immunoassays, Western blotting, physicochemical methods Wait.
  • the above-mentioned diseases associated with PD-1 positive cells can be diagnosed by detecting or assaying cells expressing PD-1 with the antibodies or antibody fragments of the present disclosure.
  • immunodetection methods In order to detect the cells expressing the polypeptide, known immunodetection methods can be used, and preferably, immunoprecipitation methods, fluorescent cell staining methods, immunohistochemical staining methods, and the like are used. In addition, a fluorescent antibody staining method using the FMAT8100HTS system (Applied Biosystem) and the like can be used.
  • the living sample for detecting or measuring the target antigen eg PD-1
  • the target antigen eg PD-1
  • cells expressing the target antigen such as tissue cells , blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody or antibody fragment thereof of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting a reaction.
  • Reagents for performing antigen-antibody reactions include buffers, salts, and the like.
  • Reagents for detection include those commonly used in immunodetection or assay methods, such as a labeled secondary antibody that recognizes the monoclonal antibody, its antibody fragment or its conjugate, and a substrate corresponding to the label, and the like.
  • the antigen is prepared as follows:
  • the human PD-1-IgG1 Fc fusion protein was designed and synthesized.
  • the N-terminal is 150 amino acids in the extracellular domain of human PD-1, and the C-terminal is the Fc segment of human IgG1 (hIgG1Fc).
  • high-purity recombinant PD-1-Fc protein can be obtained, which can be used to detect the binding of anti-PD-1 antibody to antigen.
  • the underlined part is the signal peptide
  • the normal part is the extracellular region of human PD-1
  • the italicized part is hIgG1Fc ( signal peptide +extracellular region+hIgG1Fc).
  • the PD-1 antigen (SEQ ID NO: 3) encoded by the nucleic acid of the transfected cells:
  • anti-human PD-1 antibodies can be produced by immunizing mice, and can also be obtained by anti-human PD-1 phage mouse immunization library.
  • the method for preparing anti-human PD-1 antibody by immunizing mice is as follows:
  • mice SJL white mice, female, 6-8 weeks old and Balb/c white mice, female, 6-8 weeks old, were used for the experiment. Breeding environment: SPF grade. After the mice were purchased, they were reared in a laboratory environment for 1 week, regulated by a 12/12 hour light/dark cycle, with a temperature of 20-25°C and a humidity of 40-60%. The acclimated mice were immunized according to different protocols, 6-10 mice in each group.
  • the immunizing antigen can be purified recombinant protein PD-1-IgG1Fc (see SEQ ID NO: 1), PD-1-his (see SEQ ID NO: 2), or PD-1 as an antigen (see SEQ ID NO: 3)
  • Transfected Jurkat/CHO-PD-1 cells can be cross-immunized with a single antigen with different immune adjuvants or different types of immunogens.
  • the immunization site can be the abdominal cavity or the back subcutaneous, or alternate immunization between the two positions.
  • immune adjuvant Gold Adjuvant (hereinafter referred to as Titermax, purchased from Sigma product number T2684) was cross-immunized with Imject Alum Adjuvant (hereinafter referred to as Alum, purchased from Pierce product number 77161).
  • Titermax was 1:1
  • Alum is 3:1.
  • Jurkat/CHO-PD-1 cells/only On the 0th day, intraperitoneal injection of 25-50 ⁇ g/mouse emulsified antigen, once a week or once every two weeks after the first immunization, alternately used Titermax and Alum, a total of 5-8 times.
  • mice with high antibody titers in serum were selected for splenocyte fusion, and the mice after sprint immunization for 72 h (“h” is the abbreviation of “hour”, the same below) were bled from the eyeballs, killed by pulling their necks, and released. Sterilize in 75% ethanol.
  • Hybridoma cells were obtained by fusing spleen lymphocytes with myeloma cells Sp2/0 cells (Chinese Academy of Sciences) using an optimized PEG-mediated fusion procedure.
  • HAT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • 96-well cell culture plates (1 ⁇ 105/150 ⁇ L/ well)
  • HAT complete medium 50 ⁇ L/well
  • incubate at 37°C, 5% CO2.
  • the medium was completely changed, 200 ⁇ L/well, and incubated at 37°C with 5% CO2.
  • Hybridoma cell screening On the 7th to 9th day after fusion, according to the cell growth density, the ELISA method of antibody binding to PD-1 was carried out, and the detected positive well cells were subjected to PD-1/PDL1 binding blocking ELISA After detection, the positive wells were replaced with medium and expanded to 24-well plates in time according to the cell density. The cell lines transferred into the 24-well plate were retested for seed conservation and the first subcloning. The first subcloning screen is positive for conservation, and the second or third subcloning is performed until a single-cell clone is obtained. The hybridoma cells with the effect of blocking the binding of PD-1 to PDL1 were obtained by multiple fusions.
  • the method for obtaining anti-human PD-1 antibody from the anti-human PD-1 phage mouse immune library is as follows:
  • the single-chain antibody fragment and the transformed library building vector pCantab5E were digested with Sfi1 (NEB Cat No.#R0123L), and electrophoresed with Gel Extraction Kit (Omega Cat No. D2500-02) was used for purification and recovery. Then use T4 DNA ligase (NEB Cat No.#M0202L) to ligate at 16°C for 16-18 hours, then use the above kit for purification and recovery, and finally eluate with deionized water. 1 ⁇ g of the ligation product was mixed with one electrotransformation competent TG1 (Lucigen Cat No.
  • the supernatant was added with 10 ⁇ g/mL biotinylated human PD-1-ECD-his protein (purchased from Sino Biological) and incubated at room temperature for 1 hour, followed by the addition of 100 ⁇ L streptavidin-coated magnetic beads (1 mL MPBS pre- Incubation) for 1 hour at room temperature. It was loaded on a magnetic rack system for sorting, and the supernatant was aspirated.
  • PBST phosphate buffered saline containing 0.1% Tween-20
  • elution solution 50 ⁇ L 10 mg/mL trypsin stock solution (stock solution) in 450 [mu]L PBS
  • min room temperature
  • TG1 eluted phage
  • the phage mouse immune library utilizes biotinylated human PD-1-ECD-his antigen, and after 2-3 rounds of MACS screening (streptomycin magnetic beads, Invitrogen), the final product with binding to PD-1 and blocking PD-1 is obtained. The monoclonal binding to PD-L1 was verified by sequencing, and the variable region sequence of the antibody was obtained.
  • the purification method of antibody or antigenic protein is as follows:
  • ProteinG is preferred for affinity chromatography.
  • the cultured hybridomas are centrifuged to take the supernatant, and 10-15% volume of 1M Tris-HCl (pH8.0-8.5) is added according to the supernatant volume. Clear pH.
  • the ProteinG column is washed with 6M guanidine hydrochloride for 3-5 times the column volume, and then washed with pure water for 3-5 times the column volume; use a buffer system such as 1 ⁇ PBS (pH7.4) as the equilibration buffer to equilibrate the column for 3-5 times.
  • the column is washed with 1 ⁇ PBS (pH 7.4) for 3-5 times the column volume until the UV absorption falls back to Baseline; use 0.1M acetic acid/sodium acetate (pH3.0) buffer for sample elution, collect elution peaks according to UV detection, and use 1M Tris-HCl (pH8.0) to quickly adjust the pH of the eluted product to 5-6 temporarily live.
  • the eluted product can be replaced by solution using methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution replacement to the desired buffer system, or using molecular exclusion such as G-25 desalting to replace the desired buffer system Buffer system, or use a high-resolution size exclusion column such as Superdex 200 to remove the polymer component in the eluted product to improve the purity of the sample.
  • methods well known to those skilled in the art such as ultrafiltration concentration using an ultrafiltration tube and solution replacement to the desired buffer system, or using molecular exclusion such as G-25 desalting to replace the desired buffer system Buffer system, or use a high-resolution size exclusion column such as Superdex 200 to remove the polymer component in the eluted product to improve the purity of the sample.
  • the cell culture supernatant expressing the antigen protein or antibody is subjected to high-speed centrifugation to collect the supernatant.
  • the ProteinA affinity column was washed with 6M guanidine hydrochloride for 3-5 column volumes, and then washed with pure water for 3-5 column volumes.
  • the column is equilibrated for 3-5 column volumes using a buffer system such as 1 ⁇ PBS (pH 7.4) as the equilibration buffer.
  • the cell supernatant is loaded and combined with low flow rate, and the flow rate is controlled so that the retention time is about 1min or longer.
  • the column is washed with 1 ⁇ PBS (pH7.4) for 3-5 times the column volume until the UV absorption falls back to the baseline. .
  • 0.1M acetic acid/sodium acetate (pH3.0-3.5) buffer for sample elution, collect elution peaks according to UV detection, and use 1M Tris-HCl (pH8.0) to quickly adjust the pH of the eluted product to 5-6 temporarily live.
  • the eluted product can be replaced by solution using methods well known to those skilled in the art, such as ultrafiltration concentration using an ultrafiltration tube and solution replacement to the desired buffer system, or using molecular exclusion such as G-25 desalting to replace the desired buffer system Buffer system, or use a high-resolution size exclusion column such as Superdex 200 to remove the polymer component in the eluted product to improve the purity of the sample.
  • Example 1 Obtaining anti-human PD-1 murine antibody
  • the anti-human PD-1 mouse-derived antibody obtained by the aforementioned method was subjected to an antigen binding experiment, and multiple strains of antibodies with good activity were screened: including M23, M32 and M33, the single-cell clone was expanded and cultured, RNA was extracted, and mouse-Ig was used.
  • the degenerate primers were used for reverse transcription amplification (RT-PCR) to obtain the variable region sequence of the antibody.
  • the murine antibody variable region sequence is linked with the human antibody constant region sequence, the chimeric antibody expressing the murine monoclonal antibody is cloned and recombinantly expressed, and the in vitro activity experiment is performed to confirm that the obtained monoclonal antibody variable region sequence is correct.
  • variable region sequences of murine antibodies M23, M32 and M33 were determined as follows:
  • Heavy chain variable region of murine antibody M23 (SEQ ID NO: 4):
  • Heavy chain variable region of murine antibody M32 (SEQ ID NO: 6):
  • Heavy chain variable region of murine antibody M33 (SEQ ID NO: 19)
  • the human germline heavy and light chain variable region germline genes with high sequence identity to the light and heavy chain sequences of M23, M32, and M33 were selected as the template, the CDRs of the three murine antibodies were transplanted into the corresponding human antibody templates, respectively, to construct their corresponding humanized antibodies.
  • the humanized light chain templates of murine antibody M23 are IGKV2-40*01 and IGKJ4*01, and the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01.
  • the variable region sequences after humanization are as follows ( The underscore is the CDR sequence):
  • Hu23VL-CDR grafting (SEQ ID NO:28)
  • Grafted means that the mouse antibody CDR is implanted into the human germline FR region sequence.
  • the amino acid residues are determined and annotated by the Kabat numbering system.
  • I2G means that according to the Kabat numbering system, the 2nd I of the Kabat numbering is mutated back to G.
  • the humanized antibody light/heavy chain variable region sequences of M23 are as follows:
  • the antibody obtained after humanization of murine antibody M23 and its variable region combination are shown in the following table.
  • Hu23-1 refers to the antibody whose light chain variable region is Hu23VL1 and the heavy chain variable region is Hu23VH1, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu23-1) referred to in the table above can be linked with antibody light/heavy chain constant regions, respectively, to form full-length antibodies;
  • the light chain variable region is connected with the Kappa chain constant region shown in SEQ ID NO: 73 to form an antibody light chain
  • the heavy chain variable region is connected with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO: 72.
  • the IgG4- ⁇ heavy chain constant region shown in ID NO: 79 is linked to form an antibody heavy chain, and is represented by the name of the antibody light/heavy chain variable region combination in the table (eg Hu23-1) plus the suffix ".IgG4AA".
  • a full-length antibody formed by linking the constant region of an IgG4-AA heavy chain, adding the suffix ".IgG4P” indicates a full-length antibody formed by linking the constant region of an IgG4-P heavy chain
  • "Hu23-1.IgG4AA” indicates a heavy chain formed by Hu23VH1
  • SEQ ID NO: 72 the variable region and the constant region of the IgG4-AA heavy chain shown in SEQ ID NO: 72 are connected, and the variable region of the light chain of Hu23VL1 and the constant region of the Kappa chain shown in SEQ ID NO: 73 are connected
  • Human23-1.IgG4P means a heavy chain formed by linking the variable region of the heavy chain of Hu23VH1 and the constant region of the heavy chain of IgG4-P shown in SEQ ID NO: 79, and the variable region of the light chain of Hu23VL1 and the variable region of the light chain of Hu23VL1 shown in SEQ ID NO: 79.
  • the humanized light chain templates of murine antibody M32 are IGKV2-40*01 and IGKJ4*01, the humanized heavy chain templates are IGHV1-69*02 and IGHJ6*01, and the humanized variable region sequences are as follows (underlined as CDR sequence):
  • Hu32 VH-CDR grafts (SEQ ID NO: 33) IGHV1-69*02 and IGHJ6*01
  • Hu32VL-CDR grafting (SEQ ID NO:34)
  • Grafted means that the mouse antibody CDR is implanted into the human germline FR region sequence. Amino acid residues are identified and annotated by the Kabat numbering system, eg, I2V represents a mutation of I back to V at position 2 of the Kabat numbering according to the Kabat numbering system.
  • variable region sequence of the humanized antibody light and heavy chain of the murine antibody M32 is as follows:
  • Antibody obtained after humanization of murine antibody M32 and its variable region combination.
  • Hu32-1 in the table refers to the antibody light/heavy chain variable region combination whose light chain variable region is Hu32VL1 and the heavy chain variable region is Hu32VH1, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu32-1) referred to in the above table can be linked with antibody light/heavy chain constant regions, respectively, to form full-length antibodies;
  • the light chain variable region is linked with the Kappa chain constant region shown in SEQ ID NO: 73 to form an antibody light chain
  • the heavy chain variable region is connected with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO: 72.
  • the IgG4- ⁇ heavy chain constant region shown in ID NO: 79 is linked to form an antibody heavy chain, and is represented by the name of the antibody light/heavy chain variable region combination (eg Hu32-1) in the table with the suffix ".IgG4AA".
  • a full-length antibody formed by linking the constant region of an IgG4-AA heavy chain, adding the suffix ".IgG4P" indicates a full-length antibody formed by linking the constant region of an IgG4-P heavy chain, e.g., "Hu32-1.IgG4AA” indicates a heavy chain formed by Hu32VH1 A heavy chain formed by linking the variable region and the constant region of the IgG4-AA heavy chain as shown in SEQ ID NO:72, linked to the variable region of the Hu32VL1 light chain and the constant region of the Kappa chain as shown in SEQ ID NO:73 A full-length antibody formed from the resulting light chain.
  • Hu32-1.IgG4P means a heavy chain formed by linking the variable region of the heavy chain of Hu32VH1 and the constant region of the heavy chain of IgG4-P shown in SEQ ID NO: 79, with the variable region of the light chain of Hu32VL1 and the variable region of the light chain of Hu32VL1 shown in SEQ ID NO: 79.
  • the humanized light chain templates of murine antibody M33 are IGKV1-39*01 and IGKJ4*01, the humanized heavy chain templates are IGHV3-7 and IGHJ6*01, and the humanized variable region sequences are as follows:
  • Hu33VL-CDR grafting (SEQ ID NO:42):
  • Grafted means that the mouse antibody CDR is implanted into the human germline FR region sequence. Amino acid residues are identified and annotated by the Kabat numbering system, eg, F71Y indicates that F at position 71 of the Kabat numbering is mutated back to Y according to the Kabat numbering system.
  • the humanized antibody light chain variable region and heavy chain variable region sequences of murine antibody M33 are as follows:
  • “Hu33-6” in the table refers to the antibody light/heavy chain variable region combination in which the antibody light chain variable region is Hu33VL2 and the heavy chain variable region is Hu33VH3, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu33-6) referred to in the table above can be linked with antibody light/heavy chain constant regions, respectively, to form full-length antibodies;
  • the light chain variable region is linked with the Kappa chain constant region shown in SEQ ID NO: 73 to form an antibody light chain
  • the heavy chain variable region is connected with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO: 72.
  • the IgG4- ⁇ heavy chain constant region shown in ID NO: 79 is linked to form an antibody heavy chain, and is represented by the name of the antibody light/heavy chain variable region combination (eg Hu33-6) in the table with the suffix ".IgG4AA".
  • a full-length antibody formed by linking the constant region of an IgG4-AA heavy chain, adding the suffix ".IgG4P” indicates a full-length antibody formed by linking the constant region of an IgG4-P heavy chain, e.g., "Hu33-6.IgG4AA”, indicating a heavy chain derived from Hu33VH3 A heavy chain in which the chain variable region and the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 are linked, with the Hu33VL2 light chain variable region and the Kappa chain constant region shown in SEQ ID NO: 73 A full-length antibody formed from linked light chains.
  • Hu33-6.IgG4P means a heavy chain formed by linking the variable region of the heavy chain of Hu33VH3 and the constant region of the heavy chain of IgG4-P shown in SEQ ID NO: 79, with the variable region of the light chain of Hu33VL2 and the variable region of the light chain of Hu33VL2 as shown in SEQ ID NO: 79.
  • Hu23LCDR1(N28Q) represents the mutated sequence of LCDR1 in which the variable region of the light chain variable region of Hu23 humanized antibody Hu23VL1 or Hu23VL2 is mutated to Q at position 28 of the Kabat numbering rule
  • Hu23LCDR1(G29A) represents the light chain of Hu23 humanized antibody light chain
  • the mutated sequence of LCDR1 in which the G at position 29 of the Kabat numbering system of the variable region Hu23VL1 or Hu23VL2 is mutated to A (CDRs are determined by the Kabat numbering system).
  • the light chain variable region sequence of the Hu23 humanized antibody mutated by LCDR1 is as follows:
  • “Hu23-11” in the table refers to the antibody light/heavy chain variable region combination of which the variable region of the antibody light chain is Hu23VL1 (N28T) and the variable region of the heavy chain is Hu23VH1, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu23-11) referred to in the table above can be linked with antibody light/heavy chain constant regions, respectively, to form full-length antibodies;
  • the light chain variable region is connected with the Kappa chain constant region shown in SEQ ID NO: 73 to form an antibody light chain
  • the heavy chain variable region is connected with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO: 72.
  • the IgG4- ⁇ heavy chain constant region shown in ID NO: 79 is linked to form an antibody heavy chain, and is represented by the name of the antibody light/heavy chain variable region combination in the table (eg Hu23-11) plus the suffix ".IgG4AA".
  • a full-length antibody formed by linking the constant region of an IgG4-AA heavy chain, adding the suffix ".IgG4P” indicates a full-length antibody formed by linking the constant region of an IgG4-P heavy chain, e.g., "Hu23-11.IgG4AA”, indicating a heavy chain derived from Hu23VH1 A heavy chain in which the chain variable region and the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 are linked, with the light chain variable region of Hu23VL1 (N28T) and Kappa shown in SEQ ID NO: 73
  • Human23-11.IgG4P means the heavy chain composed of the variable region of the heavy chain of Hu23VH1 and the constant region of the heavy chain of IgG4-P shown in SEQ ID NO: 79, and the variable region of the light chain of Hu23VL1 (N28T) A full-length antibody formed by a light chain linked to the Kappa chain constant region shown in SEQ ID NO: 73.
  • “Hu32a-85” in the table refers to the antibody light/heavy chain variable region combination with the antibody light chain variable region being Hu23VL1 (N28T) and the heavy chain variable region being Hu32VH6, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu32a-85) referred to in the table above can be linked to antibody light/heavy chain constant regions, respectively, to form full-length antibodies;
  • the light chain variable region is connected with the Kappa chain constant region shown in SEQ ID NO: 73 to form an antibody light chain
  • the heavy chain variable region is connected with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO: 72.
  • the IgG4- ⁇ heavy chain constant region shown in ID NO: 79 is linked to form an antibody heavy chain, and is represented by the name of the antibody light/heavy chain variable region combination in the table (eg Hu32a-85) plus the suffix ".IgG4AA".
  • a full-length antibody formed by linking the constant region of an IgG4-AA heavy chain with the suffix ".IgG4P” indicating a full-length antibody formed by linking the constant region of an IgG4-P heavy chain, e.g., "Hu32a-85.IgG4AA”, indicating that the heavy chain is composed of Hu32VH6 A heavy chain in which the chain variable region and the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 are linked, with the light chain variable region of Hu23VL1 (N28T) and Kappa shown in SEQ ID NO: 73 A full-length antibody formed from a light chain formed by linking the constant regions of the chains.
  • “Hu32a-85.IgG4P” means the heavy chain formed by linking the variable region of the heavy chain of Hu32VH6 and the constant region of the heavy chain of IgG4-P shown in SEQ ID NO: 79, and the variable region of the light chain of Hu23VL1 (N28T) A full-length antibody formed by a light chain linked to the Kappa chain constant region shown in SEQ ID NO: 73.
  • Hu23a-57 in the table refers to the antibody light/heavy chain variable region combination whose light chain variable region is Hu32VL1 and the heavy chain variable region is Hu23VH1, and so on.
  • the antibody light/heavy chain variable region combinations (such as Hu23a-57) referred to in the above table can be linked with antibody light/heavy chain constant regions, respectively, to form full-length antibodies;
  • the light chain variable region is connected with the Kappa chain constant region shown in SEQ ID NO: 73 to form an antibody light chain
  • the heavy chain variable region is connected with the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72 or SEQ ID NO: 72.
  • the IgG4- ⁇ heavy chain constant region shown in ID NO: 79 is linked to form an antibody heavy chain, and is represented by the name of the antibody light/heavy chain variable region combination in the table (eg Hu32a-85) plus the suffix ".IgG4AA".
  • a full-length antibody formed by linking the constant region of an IgG4-AA heavy chain with the suffix ".IgG4P” indicating a full-length antibody formed by linking the constant region of an IgG4-P heavy chain, e.g., "Hu23a-57.IgG4AA”, indicating a heavy chain derived from Hu23VH1
  • a heavy chain formed by linking the chain variable region and the IgG4-AA heavy chain constant region shown in SEQ ID NO: 72, with the Hu32VL1 light chain variable region and the Kappa chain constant region shown in SEQ ID NO: 73 A full-length antibody formed from linked light chains.
  • Human23a-57.IgG4P means a heavy chain formed by linking the variable region of the heavy chain of Hu23VH1 and the constant region of the heavy chain of IgG4-P shown in SEQ ID NO: 79, with the variable region of the light chain of Hu32VL1 and A full-length antibody formed by a light chain formed by linking the Kappa chain constant regions shown in SEQ ID NO: 73.
  • Design primers PCR to build each humanized antibody VH/VK gene fragment, and then carry out homologous recombination with the expression vector pHr (with signal peptide and constant region gene (CH1-Fc/CL) fragment) to construct the full-length antibody expression vector VH- CH1-Fc-pHr/VK-CL-pHr.
  • IgG4-P represents the S228P (corresponding to sequence SEQ ID NO:72 or position 108 of SEQ ID NO:79) mutation
  • IgG4-AA represents F234A (corresponding to sequence SEQ ID NO:72 or position 114 of SEQ ID NO:79) position)
  • L235A corresponding to sequence SEQ ID NO: 72 or position 115 of SEQ ID NO: 79
  • S228P corresponding to sequence SEQ ID NO: 72 or position 108 of SEQ ID NO: 79 mutations
  • IgG4- AA and IgG4-P antibody formats can be obtained by simple point mutation of the IgG4 antibody format.
  • the IgG4-AA heavy chain constant region sequence is as follows (SEQ ID NO: 72):
  • the light chain (Kappa chain) constant region sequence of the antibody is as follows (SEQ ID NO: 73):
  • the constructed IgG4AA format full-length antibody sequence is exemplified as follows:
  • Hu23-11.IgG4AA antibody heavy chain (SEQ ID NO:74):
  • Hu23-11.IgG4AA light chain (SEQ ID NO:75):
  • Hu32a-85.IgG4AA heavy chain (SEQ ID NO:76):
  • Hu33-6.IgG4AA heavy chain (SEQ ID NO:77):
  • Hu33-6.IgG4AA light chain (SEQ ID NO:78):
  • the heavy chain constant region sequence of IgG4-P is as follows (SEQ ID NO: 79):
  • the constructed IgG4-P format full-length antibody sequence is exemplified as follows:
  • Hu23-11.IgG4P antibody heavy chain (SEQ ID NO:80):
  • Hu23-11.IgG4P light chain (same as the light chain of Hu23-11.IgG4AA, SEQ ID NO: 75):
  • Hu32a-85.IgG4P heavy chain (SEQ ID NO: 81):
  • Hu33-6.IgG4P heavy chain (SEQ ID NO: 82):
  • Hu33-6.IgG4P light chain (same as Hu33-6.IgG4AA light chain, SEQ ID NO: 78):
  • Test Example 1 Binding of anti-PD-1 antibody to PD-1 ligand in vitro and binding blocking ELISA experiment
  • PD-L1 on the surface of tumor cells can inhibit the proliferation of T cells by binding to PD-1 on the surface of T cells.
  • Antibodies to PD-1 can block the PD-L1/PD-1 signaling pathway by binding to PD-1, thereby stimulating the proliferation of T cells.
  • the PD-1/PD-L1 binding blocking assay was used to detect the blocking activity of anti-PD-1 antibodies on the signaling pathway.
  • the anti-PD-1 antibodies to be tested including antibodies: Hu23-11.IgG4AA, Hu32a-85 .IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1 (see H005-1 antibody in WO2015085847), for incubation reaction; HRP-labeled goat anti-human IgG (H+L) antibody (Cat. #109-035-003, Jackson ImmunoResearch), incubate the reaction. After washing the plate, detect the binding amount of HRP-labeled goat anti-human IgG (H+L), and calculate the amount of anti-PD-1 antibody bound to ligand PD-1. EC 50 value.
  • the anti-PD-1 antibody to be tested including Antibodies: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody: H005-1 (see H005-1 antibody in WO2015085847), carry out incubation reaction; add biotin-labeled PD later -L1/PD-L2, incubation reaction. After washing the plate, detect the binding amount of biotin-labeled PD-L1/PD-L2, and calculate the amount of anti-PD-1 antibody that blocks the binding of ligand PD-L1/PD-L2. IC50 value.
  • PD-1-Fc was diluted to 1 ⁇ g/mL with pH 9.6 CB buffer (1.59 g Na 2 CO 3 and 2.93 g NaHCO 3 dissolved in 1 L of distilled water), added to 96-well plate at a volume of 100 ⁇ L/well, at 4 °C for 16h-20h.
  • PBST pH 7.4 PBS containing 0.05% tween20
  • the exemplary anti-PD-1 antibodies of the present disclosure can effectively block the binding of PD-1 to PD-L1/PD-L2, and their blocking activity is similar to that of PD-L1/PD-L2. Positive control antibodies were similar.
  • CHOK1/PD-L1 cells (Promega) were digested, added to a 96-well plate at 100 ⁇ L/well, and placed in a 37° C., 5% CO 2 incubator for 24 hours. Controls and samples were diluted to desired concentrations in PBS. Count Jurkat/PD-1 cells (Jurkat cells stably transfected with PD-1), and add 10 ⁇ L/well of diluted antibody (antibody) to the cell culture plate (90 ⁇ L/well) of CHOK1/PD-L1 cells in a certain proportion.
  • Test Example 4 The secretion effect of antibody on cellular IFN ⁇ in PBMC-T lymphocyte activation experiment
  • PBMC peripheral blood mononuclear cells
  • Fresh blood was obtained by Ficoll-Hypaque (17-5442-02, GE), density gradient centrifugation (Stem Cell Technologies) to obtain PBMC, and cultured in RPMI 1640 (SH30809.01, GE) medium supplemented with 10% ( v/v) FBS (10099-141, Gibco), cultured at 37°C, 5% CO 2 .
  • Freshly isolated and purified PBMCs were adjusted to a density of 2 ⁇ 106 cells/mL in RPMI 1640 medium, and 40 ⁇ L of tuberculin ( 97-8800 , Synbiotics) was added to 20 mL of cell suspension. sky. On the 5th day, the cultured cells were collected and centrifuged, resuspended in fresh RPMI 1640 medium, adjusted to a density of 1.1 ⁇ 10 6 cells/mL, and seeded into a 96-well cell culture plate with 90 ⁇ L per well.
  • tuberculin 97-8800 , Synbiotics
  • serially diluted antibody samples including the antibodies of the present disclosure: Hu23-11.IgG4AA, Hu32a-85.IgG4AA and Hu33-6.IgG4AA, positive control antibody H005-1, and negative control IgG4 protein were added at the same time, and the antibody gradient concentration was 0.3mg/mL, 3mg/mL, 30mg/mL), diluted with PBS (B320, Shanghai Yuanpei Biotechnology Co., Ltd.), 10 ⁇ L per well. Place the cell culture plate in a 37°C, 5% CO2 incubator for 3 days.
  • the cell culture plate was taken out, and the cell culture supernatant was collected by centrifugation (4000 rpm, 10 min), and the level of IFN- ⁇ was detected by ELISA (human IFN- ⁇ detection kit (EHC102g.96, Xinbosheng)).
  • ELISA human IFN- ⁇ detection kit (EHC102g.96, Xinbosheng)
  • the test results are shown in Fig. 2, and the results show that the anti-PD-1 antibodies Hu23-11.IgG4AA, Hu32a-85.IgG4AA, and Hu33-6.IgG4AA of the present disclosure can effectively activate the secretion of IFN- ⁇ .
  • Test Example 5 The effect of anti-PD-1 antibody in transgenic PD-1 mouse colon cancer model MC38
  • mice 5 ⁇ 10* 5 cells/mouse/100 ⁇ L of MC38 cells were inoculated subcutaneously in the right flank of 90 hPD-1TG mice (Biocytometer). After 10 days, the animals with too large or too small tumors were removed, and the average tumor volume was approximately 120mm ⁇ 3 mice were randomly divided into: blank control Vehicle (PBS), positive control H005-1 3mpk, Hu32a-85.IgG4AA 1mpk, Hu32a-85.IgG4AA 3mpk, Hu23-11.IgG4AA 1mpk, Hu23-11.IgG4AA 3mpk, Hu33-6.IgG4AA 3mpk, a total of 7 groups, 8 mice in each group.
  • PBS blank control Vehicle
  • H005-1 3mpk Hu32a-85.IgG4AA 1mpk
  • Hu32a-85.IgG4AA 3mpk Hu23-11.IgG4AA 1mpk
  • Hu23-11.IgG4AA 3mpk Hu33-6.
  • each group of antibodies was injected intraperitoneally three times a week. After the first week of administration, it was found that the tumor was significantly inhibited. The second and third weeks adjusted the administration frequency to once a week, with a total of 5 administrations. Tumor volumes, animal weights were monitored and data recorded twice weekly. Tumor-bearing animals were euthanized as experimental endpoints when tumor volume exceeded 2000 mm3 or when most tumors ruptured or lost 20% of body weight.
  • Tumor volume (TV) 1/2 ⁇ L long ⁇ L short 2
  • Tumor proliferation rate (T/C%) (T-T0)/(C-C0) ⁇ 100%
  • TGI% 1-T/C%
  • T and T0 represent the tumor volume at the end of the experiment and the beginning of the experiment in the antibody administration group, respectively
  • C and C0 represent the tumor volume at the end of the experiment and the beginning of the experiment in the blank control group, respectively.
  • test results are shown in Table 19 and accompanying drawing 3.
  • the test results show that, compared with the blank control, the antibodies of the present disclosure can significantly inhibit the growth of mouse colon cancer MC38 xenograft tumors, and Hu32a-85.IgG4AA has the highest tumor inhibition rate.
  • the tumor inhibition rate at the last measurement was 77.64%.
  • Test Example 6 The effect of anti-PD-1 antibody in transgenic PD-1 mouse colon cancer model MC38
  • Transgenic PD-1 mice were derived from purchased transgenic PD-1 mice (ISIS INNOVATION LIMITED, University Offices, Wellington Square, Oxford OX1 2JD, England) of fifth generation mice bred at Cephrim Biosciences, Inc. MC38 cells were inoculated into hPD-1 transgenic mice (half males and females) subcutaneously at the back of the right flank at 5x105 cells/100 ⁇ L/mice. When the average tumor volume of the mice reached 80-100mm3, the body weight, tumor size and tumor size were removed.
  • mice For animals that are too small, the tumor-bearing mice were randomly divided into 5 groups (8 mice in each group) according to the tumor volume: negative control hIgG control 30mpk, H005-1 10mpk, H005-1 30mpk, Hu33-6.IgG4AA 10mpk, Hu33 -6. IgG4AA 30mpk.
  • the day of group administration was set as Day 0.
  • each drug was administered intraperitoneally for a period of 22 days, once every two days, for a total of 11 times.
  • the tumor volume was measured twice a week, the body weight was weighed, and the data were recorded.
  • the body weight and tumor volume of animals in each group were expressed as mean ⁇ standard deviation (Mean ⁇ SEM), and were plotted using Graphpad Prism 5 and Excel software, and statistical analysis was performed using student t test.
  • Tumor volume (TV) 0.5236 ⁇ L long ⁇ L short 2
  • Tumor proliferation rate T/C% (T-T0)/(C-C0) ⁇ 100%
  • T and T0 represent the tumor volume at the end of the experiment and the beginning of the experiment in the antibody administration group, respectively
  • C and C0 represent the tumor volume at the end of the experiment and the beginning of the experiment in the blank control group, respectively.
  • the test results are shown in Table 20 and accompanying drawing 4.
  • the test results show that, compared with the control group, the antibody of the present disclosure can significantly inhibit the growth of mouse colon cancer MC38 transplanted tumor, and the tumor inhibition rate is the highest Hu33-6.
  • the tumor inhibition rate was 80.4% when measured on the 20th day.
  • the efficacy of Hu33-6.IgG4AA-10mpk was better than that of the positive control H005-1-10mpk.
  • the unit of the average tumor volume of each group in the table is: mm 3 .
  • Test Example 7 Pharmacokinetic test of anti-PD-1 antibody in cynomolgus monkeys
  • the room temperature was controlled at 18°C-26°C, the relative humidity was 40%-70%, and the light was alternated between light and dark for 12 hours. Unlimited access to feed and water, except where fasting is required.
  • mice were weighed before dosing and weighed between 2.81-3.52 kg.
  • 5min, 0.25h, 0.5h after the start of intravenous infusion (immediately after the end of administration), 1h, 2h, 4h, 8h, 1d, 2d, 3d, 4d, 5d, 7d, 10d, 13d, 14d , 21d and 28d whole blood was collected from the hindlimb vein, and serum was separated.
  • An anti-PD-1 antibody stable preparation is prepared by an exemplary test below.
  • the anti-PD-1 antibody in the implementation of the following preparation is the aforementioned Hu23-11.IgG4AA.
  • the equipment used in the preparation process and the calculation method of the results are as follows:
  • SEC Size Exclusion Chromatography An analytical method for the separation of solutes based on the relative relationship between the pore size of the gel pores and the coil size of the polymer sample molecules.
  • SEC monomer content percentage A monomer / A total * 100% (A monomer is the peak area of the main peak monomer in the sample, A total is the sum of all peak areas).
  • Instrument for SEC measurement Agilent 1260; Column: waters, XBrige SEC (300 x 7.8 mm 3.5 ⁇ m).
  • CE capillary gel electrophoresis a method in which the gel is moved into a capillary as a support medium for electrophoresis, and is separated according to the molecular weight of the sample under a certain voltage.
  • Non-reduced CE (NR-CE) purity percentage A main peak/A total * 100% (A main peak is the peak area of the main peak in the sample, A total is the sum of all peak areas.
  • CE determination instrument Beckman, model plus800.
  • iCIEF Imaging Capillary Isofocus Electrophoresis A technique for separating proteins based on their isoelectric point pI.
  • iCIEF neutral peak content percentage neutral peak area/total area*100% (total area is the sum of acidic peak, neutral peak and basic peak area).
  • Instrument used for iCIEF determination simple protein, model muarice.
  • Osmotic pressure The freezing point method is used to measure the osmotic pressure. Based on the proportional relationship between the freezing point drop value and the molar concentration of the solution, a high-sensitivity temperature sensing element is used to measure the freezing point of the solution and convert it into osmotic pressure through electricity. Instrument manufacturer Roser Loser, model OM815.
  • Test Example 8 Anti-PD-1 Antibody Preparation Buffer System and pH Screening
  • An anti-PD-1 antibody (Hu23-11.IgG4AA) formulation containing 80 mg/mL sucrose and 0.6 mg/mL polysorbate 80 (PS80) at a protein concentration of 100 mg/mL was prepared using the following buffers:
  • the finished formulation is filtered, filled, stoppered, and capped.
  • the stability of the samples under forced degradation conditions 40°CM1, that is, placed at a high temperature of 40°C for 1 month
  • accelerated conditions 25°CM6, that is, placed at a temperature of 25°C for 6 months
  • Appearance, SEC and non-reduced CE-SDS were used as evaluation indicators to investigate the stability of the formulation.
  • the experimental results are shown in Table 22.
  • D0 means the start of the experiment
  • M means month
  • M1 means one month.
  • the finished formulation is filtered, filled, stoppered, and capped.
  • the stability of the samples under accelerated conditions 25°C M6, that is, 6 months at 25°C was investigated, and the SEC and iCIEF indexes of the formulations were investigated to investigate the stability of the formulations.
  • the experimental results are shown in Table 23.
  • the preparation buffer system is preferably an acetate and histidine buffer system, preferably an AA or His-AA buffer system.
  • Test Example 9 Ionic Strength Screening of Anti-PD-1 Antibody Preparation Buffer System
  • Anti-PD-1 antibody (Hu23- 11. IgG4AA) formulations. Ingredients:
  • the finished formulation is filtered, filled, stoppered, and capped.
  • the stability of the sample under forced degradation conditions (40°CM1) was investigated, and the SEC was used as the evaluation index to investigate the stability of the formulation.
  • the experimental results are shown in Table 24.
  • the experimental data show that under the forced degradation condition of M1 at 40°C, the SEC data of the 10mM ionic strength group is slightly higher than that of the 30mM ionic strength group, so the ionic strength of the pH5.2 sodium acetate buffer system is preferably 10mM.
  • Test Example 10 Surfactant Screening in Anti-PD-1 Antibody Preparations
  • Anti-PD-1 antibody (Hu23-11.IgG4AA) formulations containing various concentrations of Tween 80 below 1)-5) were prepared in 10 mM sodium acetate buffer at pH 5.2. The protein concentration is 100mg/mL, and other excipients are as follows:
  • the finished formulation is filtered, filled, stoppered, and capped.
  • the samples were shaken (25°C, 300rpm, 10 days) and long-term stability (2-8°C for 6 months) experiments, and the stability of the formulation was investigated with appearance, SEC, and non-reduced CE-SDS as evaluation indicators.
  • the experimental results are shown in Table 25.
  • polysorbate 80 is selected as the surfactant, and the concentration is preferably 0.6 mg/mL.
  • D in the table means days, such as D10 means 10 days; D0 means the beginning of the experiment, "M” means months, such as M6 means six months.
  • Test Example 11 Screening of osmotic pressure regulators in anti-PD-1 antibody preparations
  • Anti-PD-1 antibody (Hu23-11.IgG4AA) preparations with a protein concentration of 100 mg/mL were prepared in 10 mM sodium acetate buffer containing the following 1)-7) different kinds of excipients and pH 5.2.
  • the specific accessories are as follows:
  • the finished formulation is filtered, filled, stoppered, and capped.
  • the stability of the samples under forced degradation conditions 40°C D22 (22 days at 40°C high temperature) was investigated, and the stability of the formulation was investigated with appearance, SEC and non-reducing CE-SDS as evaluation indicators.
  • the experimental results are shown in Table 26.
  • the SEC and non-reduced CE-SDS of the preparations in the 80mg/mL sucrose, 80mg/mL trehalose and 50mg/mL sorbitol groups were slightly lower. higher than other groups, and there was no significant difference in the purity item among the three groups of preparations.
  • the osmotic pressure of the preparation is controlled at 280-320 mOsm, therefore, the sucrose content is preferably 70-90 mg/mL.
  • the osmotic pressure of the preparation containing 80 mg/mL sucrose is about 300 mOsm, and the sucrose content is preferably 80 mg/mL.
  • D in the table means days, for example, D22 means 22 days; D0 means the beginning of the experiment.
  • Test Example 12 Screening of protein concentration of anti-PD-1 antibody preparations
  • Anti-PD-1 antibody (Hu23) containing 80 mg/mL sucrose, 0.6 mg/mL polysorbate 80 (PS80), and protein concentration of 100 mg/mL or 120 mg/mL was prepared using 10 mM sodium acetate with a buffer system of pH 5.2. -11. IgG4AA) formulation.
  • the experimental data showed that under the forced degradation condition of M1 at 40°C, the appearance and purity items of the 100 mg/mL and 120 mg/mL formulation groups were not significantly different.
  • the prepared formulation is filtered, filled, stoppered, and capped.
  • the stability of the samples under forced degradation conditions 40°C M1, freezing and thawing 5 times and shaking D7 (25°C, 300rpm) was investigated, and appearance, SEC, non-reducing CE-SDS and IEC were used as evaluation indicators to investigate the formulations. stability.
  • the experimental results are shown in Table 28.
  • Test Example 14 Prescription optimization experiment of anti-PD-1 antibody preparations
  • the DOE experimental design was carried out with the ionic strength, pH value and protein concentration of sodium acetate as variables.
  • the DOE experimental factors and levels were set as ionic strength 10-30 mM, pH 4.7-5.7, anti-PD-1 antibody (Hu23-11.IgG4AA) ) concentration of 90-150mg/mL, a series of prescriptions (see Table 30) were designed, and the forced degradation experiment was placed under the high temperature condition of 40°C for one month. Analysis, the results are shown in Table 31 and Figure 5.
  • anti-PD-1 antibody samples have good stability in the range of protein concentration of 90-150 mg/mL, ionic strength of 10-30 Mm and pH of 4.7-5.7.
  • Test Example 15 Lyophilization of anti-PD-1 antibody preparation
  • Anti-PD-1 antibody (Hu23-11.IgG4AA) formulations containing 100 mg/mL antibody, 80 mg/mL sucrose, 0.6 mg/mL PS80 were prepared in 10 mM AA pH 5.2 buffer, samples of the formulation were lyophilized, lyophilized The drying procedure was pre-freezing, primary drying and secondary drying (see Table 32 for parameters). After the lyophilization procedure is complete, vacuum stopper. The reconstituted samples were compared before and after lyophilization. The results show that the reconstituted solution can maintain the good properties of the liquid formulation.

Abstract

本披露涉及抗PD-1抗体药物组合物及其用途。具体地，本披露提供一种药物组合物，其包含抗PD-1抗体以及缓冲液。

Description

抗PD-1抗体药物组合物及其用途 技术领域
本披露属于药物制剂领域，具体涉及包含抗PD-1抗体的药物组合物，以及其作为药物的用途。
背景技术
这里的陈述仅是提供与本披露有关的背景信息，而不必然地构成现有技术。
肿瘤免疫治疗是充分利用、调动肿瘤患者体内的杀伤性T细胞，对肿瘤进行杀伤的治疗方法。与此同时，肿瘤细胞逃逸是肿瘤免疫治疗面临的一个巨大障碍，肿瘤细胞利用其自身对免疫系统的抑制作用促进了肿瘤的快速生长。肿瘤的免疫逃逸机制与机体对肿瘤的免疫应答之间存在着极为复杂的关系。在肿瘤免疫治疗早期肿瘤特异性的杀伤性T细胞是有其生物活性的，但随着肿瘤生长在后期失去了杀伤的功能。
人体内T细胞的活化采取了两条信号通路系统，除了需要通过抗原呈递细胞递呈MHC-抗原肽给T细胞提供第一信号外，还需要一系列协同刺激分子提供第二信号，进而才能使T细胞产生正常的免疫应答。这个双信号通路系统对体内免疫系统的平衡起着至关重要的作用，它严格调控机体对自身和非自身抗原产生不同的免疫应答。如果缺少协同刺激分子提供的第二信号，将会导致T细胞的无应答或持续特异性免疫应答，从而产生耐受。因此，第二信号通路在机体免疫应答的整个过程中起着非常关键的调节作用。
程序性死亡分子1(programmed death-l，PD-l)是1992年发现的表达在T细胞表面的一个蛋白受体，参与到细胞的凋亡过程之中。PD-l属于CD28家族，与细胞毒性T淋巴细胞抗原4(cytotoxic T Iymphocyte antigen 4，CTLA-4)具有23％的氨基酸同源性，但其表达却与CTLA不同，主要表达在活化的T细胞、B细胞和髓系细胞上。PD-1有两个配体，分别为PD-L1和PD-L2。PD-L1主要表达于T细胞、B细胞、巨噬细胞和树突状细胞(dendritic cell，DC)上，在活化后细胞上的表达能够进行上调。而PD-L2的表达相对较局限，主要表达在抗原呈递细胞上，如活化的巨噬细胞和树突状细胞。
抗PD-1抗体可通过阻断PD-L1/PD-1之间结合，最大限度的提高患者自身对肿瘤的免疫系统反应，从而达到对肿瘤细胞进行杀伤的目的。
发明内容
本披露提供抗PD-1抗体药物组合物及其用途。
一方面，本披露提供一种药物组合物，其包含抗PD-1抗体以及缓冲液，其中， 所述缓冲液为醋酸盐缓冲液、组氨酸盐缓冲液或琥珀酸盐缓冲液。优选地，所述缓冲液的pH为约4.5至约6.0，优选pH为约4.7至约5.7，更优选pH为约5.2；所述醋酸盐缓冲液优选为醋酸-醋酸钠缓冲液，所述组氨酸盐缓冲液优选为组氨酸-醋酸盐缓冲液，所述琥珀酸盐缓冲液优选为琥珀酸-琥珀酸钠缓冲液；所述抗PD-1抗体包含重链可变区和轻链可变区，所述重链可变区包含：如SEQ ID NO：65所示的HCDR1，如SEQ ID NO：66所示的HCDR2，和如SEQ ID NO：67所示的HCDR3，所述轻链可变区包含如SEQ ID NO：68所示的LCDR1，如SEQ ID NO：12所示的LCDR2，和如SEQ ID NO：69所示的LCDR3；优选地，其中所述抗PD-1抗体的重链可变区包含：如SEQ ID NO：8所示的HCDR1，如SEQ ID NO：9所示的HCDR2，和如SEQ ID NO：10所示的HCDR3，轻链可变区包含：如SEQ ID NO：49所示的LCDR1，如SEQ ID NO：12所示的LCDR2，和如SEQ ID NO：13所示的LCDR3。具体序列见下表1：
表1.抗体CDR序列
Figure PCTCN2021109438-appb-000001
在一些可选的实施方案中，在前述药物组合物中，所述抗PD-1抗体的重链可变区包含序列如SEQ ID NO：8所示的HCDR1，序列如SEQ ID NO：9所示的HCDR2，和序列如SEQ ID NO：10所示的HCDR3；所述轻链可变区包含序列如SEQ ID NO：12所示的LCDR2，序列如SEQ ID NO：13所示的LCDR3，和序列如通式RSSQSX 13VHSX 14X 15X 16TYLE(SEQ ID NO：68)所示的LCDR1，其中X 13选自L，X 14选自N、Q、L、T或D，X 15选自G、A或V，X 16选自N。
在一些可选的实施方案中，在前述药物组合物中，所述抗PD-1抗体是选自如下(a)至(e)任一项所述的抗PD-1抗体：
(a)一种抗PD-1抗体，其包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，和序列分别如SEQ ID NO： 12和SEQ ID NO：13所示的LCDR2和LCDR3，和序列如SEQ ID NO：11、47、48、49、50、51或52所示的LCDR1；
(b)一种抗PD-1抗体，其包含序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，和序列分别如SEQ ID NO：17、SEQ ID NO：12和SEQ ID NO：18所示的LCDR1、LCDR2和LCDR3；
(c)一种抗PD-1抗体，其包含序列分别如SEQ ID NO：21、SEQ ID NO：22和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，和序列分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3；
(d)一种抗PD-1抗体，其包含序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，和序列分别如SEQ ID NO：12和SEQ ID NO：13所示的LCDR2和LCDR3，以及序列如SEQ ID NO：11、47、48、49、50、51或52所示的LCDR1；和
(e)所述重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含序列分别如SEQ ID NO：17、SEQ ID NO：12和SEQ ID NO：18所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，在前述药物组合物中，所述抗PD-1抗体包含重链可变区和轻链可变区，其中所述的重链可变区包含序列分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含序列分别如SEQ ID NO：49、SEQ ID NO：12和SEQ ID NO：13所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，在前述药物组合物中，所述抗PD-1抗体包含重链可变区和轻链可变区，其中所述的重链可变区包含序列分别如SEQ ID NO：21、SEQ ID NO：22和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，和所述轻链可变区包含序列分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，在本披露的前述药物组合物中，所述抗PD-1抗体以等于或小于10 -7M解离平衡常数(KD值)与人PD-1结合，在一些实施方案中，以等于或小于10 -8M、10 -9M、10 -10M或10 -11M解离平衡常数与人PD-1结合。所述KD值通过表面等离子共振技术(Biacore)所测定；例如，通过本披露的测试例1所述方法进行检测。
在一些实施方案中，在前述药物组合物中，所述缓冲液的pH值为约4.5至约6.0，优选为4.5至6.0；在一些实施方案中，所述缓冲液pH值为约4.7至约5.7，优选为4.7至5.7；在另一些实施方案中，所述缓冲液pH值为约5.2，优选为5.2；在另一些实施方案中，所述缓冲液为pH为约4.7至约5.7的醋酸盐缓冲液；在另一些实施方案中，所述缓冲液为pH为5.2的醋酸盐缓冲液。在另一些实施方案中， 缓冲液的pH值非限制的实施例包括约4.5、约4.6、约4.7、约4.8、约4.9、约5.0、约5.1、约5.2、约5.3、约5.4、约5.5、约5.6、约5.7、约5.8、约5.9、约6.0，以及这些点值之间的任一范围。在一些实施方案中，前述药物组合物的pH值与其缓冲液pH值一致或几乎一致(本领域技术人员公知，在制备药物制剂的过程中，有的会存在pH漂移(制剂pH与缓冲液pH存在一些差值，称为pH漂移值)，pH的漂移值通常在±0.3范围内，如无特别说明，本披露药物组合物的pH漂移值在±0.3范围内，优选在±0.2范围内，更优选在±0.1范围内)。
在一些实施方案中，在前述的药物组合物中，所述缓冲液浓度为大约5mM至大约30mM，优选为5mM至30mM；在一些实施方案中，缓冲液浓度为大约5mM至大约15mM，优选5mM至15mM；在一些实施方案中，缓冲液浓度为大约10mM，优选10mM。缓冲液浓度的非限制性实施例包括约5mM、约7mM、约8mM、约9mM、约10mM、约11mM、约12mM、约13mM、约14mM、约15mM、约16mM、约18mM、约20mM、约25mM、约30mM，以及这些点值之间的任一范围。
在一些实施方案中，在前述的药物组合物中，所述抗PD-1抗体浓度为大约1mg/mL至大约150mg/mL，优选1mg/mL至150mg/mL；在一些实施方案中，抗PD-1抗体浓度为大约90mg/mL至大约150mg/mL，优选90mg/mL至150mg/mL；在一些实施方案中，抗PD-1抗体浓度为大约100mg/mL至大约120mg/mL。在一些实施方案中，抗PD-1抗体浓度为大约100mg/mL。在一些实施方案中，抗PD-1抗体浓度为100mg/mL。抗PD-1抗体浓度非限制性实施例包括：大约1mg/mL、大约10mg/mL、大约20mg/mL、大约30mg/mL、大约40mg/mL、大约50mg/mL、大约60mg/mL、大约70mg/mL、大约80mg/mL、大约90mg/mL、大约100mg/mL、大约110mg/mL、大约120mg/mL、大约130mg/mL、大约140mg/mL、大约150mg/mL，以及这些点值之间的任一范围。
在一些实施方案中，前述的药物组合物还包括渗透压调节剂。在一些实施方案中，渗透压调节剂为糖(包括单糖，二糖，三糖，多糖，糖醇，还原性糖，非还原性糖等等)、氨基酸(包括精氨酸、甘氨酸、半胱氨酸、组氨酸等等)或盐类(氯化钠、氯化钾、氯化钙等)。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，麦芽糖，右旋糖苷，甘油，赤藻糖醇，丙三醇，阿拉伯糖醇，木糖醇，山梨糖醇(也称山梨醇)，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，麦芽糖醇，乳糖醇和异-麦芽酮糖。在一些实施方案中，所述的糖是非还原性二糖；在一些实施方案中，所述的糖优选为海藻糖或蔗糖，最优选为蔗糖。在一些实施方案中，所述渗透压调节剂选自由蔗糖、海藻糖、山梨糖醇、精氨酸、甘氨酸和氯化钠组成的组中的一种或多种。在一些实施方案中，渗透压调节剂浓度优选为大约50mg/mL至大约100mg/mL，更优选为大约70mg/mL至大约90mg/mL；最优选为大约80mg/mL。在一些实施方案中，渗透压调节剂浓度非限制性实施例包括约50mg/mL、约60mg/mL、约 65mg/mL、约70mg/mL、约75mg/mL、约80mg/mL、约85mg/mL、约90mg/mL、约95mg/mL、约100mg/mL，以及这些点值之间的任一范围。在一些实施方案中，前述药物组合物为等渗制剂。在一些实施方案中，所述渗透压调节剂使前述药物组合物渗透压控制在280-320mOsm，优选渗透压控制在约300mOsm。在一些实施方案中，所述渗透压调节剂优选大约70mg/mL至大约90mg/mL蔗糖，最优选为大约80mg/mL蔗糖。
在一些实施方案中，前述药物组合物还包括表面活性剂，所述表面活性剂可选自聚山梨酯20(也称吐温20)、聚山梨酯80(也称吐温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、聚丙二醇和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酯80或聚山梨酯20，更优选为聚山梨酯80。
在一些实施方案中，在前述的药物组合物中，所述表面活性剂的浓度为大约0.1mg/mL至大约1.0mg/mL，优选为大约0.2mg/mL至大约0.8mg/mL；在一些实施方案中，表面活性剂的浓度为大约0.4mg/mL至大约0.8mg/mL，优选大约0.6mg/mL至大约0.8mg/mL；在一些实施方案中，表面活性剂的浓度为大约0.6mg/mL，优选0.6mg/mL。非限制性实施例包括约0.1mg/mL、约0.2mg/mL、约0.3mg/mL、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、约0.9mg/mL、约1.0mg/mL，以及这些点值之间的任一范围。
在一些实施方案中，前述药物组合物中的抗PD-1抗体是鼠源抗体、嵌合抗体、全人抗体，或人源化抗体。
在一些实施方案中，前述药物组合物中的抗PD-1抗体为人源化抗体。在一些实施方案中，所述人源化抗体包含来源自人抗体的框架区或其框架区变体。在一些实施方案中，所述框架区变体为在人抗体的轻链框架区和/或重链框架区基础上分别具有至多11个氨基酸的回复突变。在一些实施方案中，所述框架区变体与来源自人抗体的框架区相比，包含选自以下(f)至(h)任一所述的突变：
(f)轻链可变区中包含2G氨基酸回复突变，和/或重链可变区中包含选自27Y、48I、67T、69L、82F和93T中的一个或更多个氨基酸回复突变；
(g)轻链可变区中包含2V氨基酸回复突变，和/或重链可变区中包含选自26D、27F、30T、38K、43H、48I、66K、67A、69L、82F和93T中的一个或更多个氨 基酸回复突变；和
(h)轻链可变区中包含选自42G、44V和71Y中的一个或更多个氨基酸回复突变，和/或重链可变区中包含1K和/或94S氨基酸回复突变。
在一些实施方案中，前述药物组合物中的抗PD-1抗体包含选自如下所述的抗体可变区：
(a2)重链可变区，其包含分别如SEQ ID NO：8、SEQ ID NO：9和SEQ ID NO：10所示的HCDR1、HCDR2和HCDR3，和包含选自27Y、48I、67T、69L、82F和93T中的一个或更多个氨基酸回复突变的重链框架区，和
轻链可变区，其包含分别如SEQ ID NO：12和SEQ ID NO：13所示的LCDR2和LCDR3，和如SEQ ID NO：11、47、48、49、50、51或52所示的LCDR1，和包含2G氨基酸回复突变的轻链框架区；
(b2)重链可变区，其包含分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的HCDR1、HCDR2和HCDR3，和包含选自26D、27F、30T、38K、43H、48I、66K、67A、69L、82F和93T中的一个或更多个氨基酸回复突变的重链框架区；和
轻链可变区，其包含分别如SEQ ID NO：17、SEQ ID NO：12和SEQ ID NO：18所示的LCDR1、LCDR2和LCDR3，和包含2V氨基酸回复突变的轻链框架区；
(c2)重链可变区，其包含序列分别如SEQ ID NO：21、SEQ ID NO：22和SEQ ID NO：23所示的HCDR1、HCDR2和HCDR3，和包含1K和/或94S氨基酸回复突变的重链框架区，和
轻链可变区，其包含分别如SEQ ID NO：24、SEQ ID NO：25和SEQ ID NO：26所示的LCDR1、LCDR2和LCDR3，和包含选自42G、44V和71Y中的一个或更多个氨基酸回复突变的轻链框架区。
在一些实施方案中，前述药物组合物中的抗PD-1抗体包含选自如下(i)至(o)任一所述的抗体可变区；
(i)重链可变区，其序列如SEQ ID NO:4所示或与SEQ ID NO：4具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：5所示或与SEQ ID NO：5具有至少90％序列同一性；
(j)重链可变区，其序列如SEQ ID NO:6所示或与SEQ ID NO：6具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：7所示或与SEQ ID NO：7具有至少90％序列同一性；
(k)重链可变区，其序列如SEQ ID NO:19所示或与SEQ ID NO：19具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：20所示或与SEQ ID NO：20具有至少 90％序列同一性；
(l)重链可变区，其序列如SEQ ID NO:27、30、31或32所示，或与SEQ ID NO：27、30、31或32具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：28、29、53、54、55、56、57、58、59、60、61、62、63或64所示，或与SEQ ID NO：28、29、34、35、53、54、55、56、57、58、59、60、61、62、63或64具有至少90％序列同一性；
(m)重链可变区，其序列如SEQ ID NO:33、36、37、38、39或40所示，或与SEQ ID NO:33、36、37、38、39或40具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：34、35、28、29、53、54、55、56、57、58、59、60、61、62、63或64所示，或与SEQ ID NO：34、35、28、29、53、54、55、56、57、58、59、60、61、62、63或64具有至少90％序列同一性；
(n)重链可变区，其序列如SEQ ID NO:41、45或46所示，或与SEQ ID NO:41、45或46具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：42、43或44所示，或与SEQ ID NO：42、43或44具有至少90％序列同一性；
(o)重链可变区，其序列如SEQ ID NO:70所示或与SEQ ID NO：70具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：71所示或与SEQ ID NO：71具有至少90％序列同一性；和
(p)重链可变区，其序列如SEQ ID NO:27、30、31或32所示，或与SEQ ID NO:27、30、31或32具有至少90％序列同一性，和/或
轻链可变区，其序列如SEQ ID NO：34或35所示，或与SEQ ID NO：34或35具有至少90％序列同一性；
其中，序列SEQ ID NO：70和SEQ ID NO：71为通式，具体序列见表2：
表2.抗体可变区序列
Figure PCTCN2021109438-appb-000002
在一些实施方案中，在前述药物组合物中，所述的抗PD-1抗体的重链可变区如SEQ ID NO：27所示或与SEQ ID NO：27具有至少90％的同一性，且所述抗 PD-1抗体的轻链可变区序列如SEQ ID NO：55所示或与SEQ ID NO：55具有至少90％的序列同一性。
在一些实施方案中，在前述药物组合物中，所述的抗PD-1抗体的重链可变区如SEQ ID NO：46所示或与SEQ ID NO：46具有至少90％的同一性，且所述抗PD-1抗体的轻链可变区序列如SEQ ID NO：43所示或与SEQ ID NO：43具有至少90％的序列同一性。
前述的“至少90％同一性”包含具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性。
在另外一些实施方案中，在前述药物组合物中，所述的抗PD-1抗体进一步包含抗体重链恒定区和/或抗体轻链恒定区；在另外一些实施方案中，所述抗体重链恒定区选自人IgG1、IgG2、IgG3和IgG4的恒定区及其常规变体，所述抗体轻链恒定区选自人抗体κ和λ链的恒定区及其常规变体；在另外一些实施方案中，所述抗体重链恒定区包含引入S228P、F234A和L235A中的一个或更多个突变的IgG4的重链恒定区，例如含S228P、F234A和L235A三个氨基酸突变；在另外一些实施方案中，所述抗体包含序列如SEQ ID NO:72或如SEQ ID NO:79所示的重链恒定区和序列如SEQ ID NO:73所示的轻链恒定区。
在一些实施方案中，在前述药物组合物中，所述的抗PD-1抗体包含如SEQ ID NO：78所示或与SEQ ID NO：78具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％序列同一性的轻链和如SEQ ID NO：77或82所示或与SEQ ID NO：77或82具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％序列同一性的重链；或包含如SEQ ID NO：75所示或与SEQ ID NO：75具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％序列同一性的轻链和如SEQ ID NO：74、76、80或81所示或与SEQ ID NO：74、76、80或81具有至少85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％或100％序列同一性的重链。
在一些实施方案中，在前述药物组合物中，所述的抗PD-1抗体包含：如SEQ ID：74所示的重链和如SEQ ID：75所示的轻链；或如SEQ ID：77所示的重链和如SEQ ID：78所示的轻链。
在一些实施方案中，前述药物组合物包含：
a)浓度为大约1mg/mL至大约150mg/mL的抗PD-1抗体，
b)浓度为大约5mM至大约30mM，pH为大约4.5至约6.0的醋酸盐缓冲液，
c)浓度为大约50mg/mL至大约100mg/mL的渗透压调节剂，所述渗透压调节剂选自蔗糖、海藻糖、山梨糖醇、精氨酸、甘氨酸和氯化钠；
d)浓度为大约0.2mg/mL至大约0.8mg/mL的聚山梨酯；
在一些实施方案中，前述药物组合物包含：
A1)浓度为大约90mg/mL至大约150mg/mL的抗PD-1抗体，
B1)浓度为大约10mM至大约30mM，pH为大约4.7至约5.7的醋酸盐缓冲液，
C1)浓度为大约70mg/mL至大约90mg/mL的蔗糖，和
D1)浓度为大约0.6mg/mL至大约0.8mg/mL的聚山梨酯80；
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约120mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.0的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.5的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.7的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的组氨酸-醋酸盐缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约30mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约120mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.2mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.4mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.8mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋 酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯20。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约80mg/mL海藻糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约50mg/mL山梨醇，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约100mM精氨酸，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约100mM甘氨酸，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约100mg/mL的抗PD-1抗体，浓度为大约100mM NaCl，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约20mM的pH为大约5.7的醋酸-醋酸钠缓冲液，浓度为大约150mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约21.9mM的pH为大约4.7的醋酸-醋酸钠缓冲液，浓度为大约120mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约4.7的醋酸-醋酸钠缓冲液，浓度为大约90mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约20mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约120mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约20mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约90mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.7的醋 酸-醋酸钠缓冲液，浓度为大约150mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约30mM的pH为大约4.7的醋酸-醋酸钠缓冲液，浓度为大约90mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约4.7的醋酸-醋酸钠缓冲液，浓度为大约150mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约30mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约150mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约10mM的pH为大约5.7的醋酸-醋酸钠缓冲液，浓度为大约90mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：大约30mM的pH为大约5.7的醋酸-醋酸钠缓冲液，浓度为大约107.7mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：pH为5.2的10mM的醋酸-醋酸钠缓冲液，浓度为120mg/mL的抗PD-1抗体，浓度为70mg/mL蔗糖，和浓度为0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：pH为5.2的10mM的醋酸-醋酸钠缓冲液，浓度为120mg/mL的抗PD-1抗体，浓度为90mg/mL蔗糖，和浓度为0.6mg/mL聚山梨酯80。
在一些实施方案中，前述药物组合物包含：pH为5.2的10mM的醋酸-醋酸钠缓冲液，浓度为120mg/mL的抗PD-1抗体，浓度为80mg/mL蔗糖，和浓度为0.6mg/mL聚山梨酯80；其中，所述抗PD-1抗体具有如SEQ ID：74所示的重链，和如SEQ ID：75所示的轻链。在一些实施方案中，本披露提供一种制备前述的药物组合物的方法，所述方法包括将抗PD-1抗体原液经缓冲液置换的步骤，在一些实施方案中，所述缓冲液选自醋酸盐缓冲液、组氨酸缓冲液或琥珀酸盐缓冲液。
本披露还提供一种冻干制剂，其复溶后可形成前述任一项所述的药物组合物。
在一些实施方案中，本披露提供一种含抗PD-1抗体的冻干制剂，所述冻干制剂通过将前述的药物组合物经冷冻干燥获得。在可选的实施方案中，前述制备其中所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。
在一些实施方案中，本披露提供一种含抗PD-1抗体的冻干制剂，所述冻干制剂经复溶可形成前述的药物组合物。
在一些实施方案中，本披露提供一种冻干制剂，所述冻干制剂为前面任一项 所述的药物组合物的冻干形式。
在一些实施方案中，前述药物组合物或冻干制剂为稳定制剂，在一些实施方案中，前述药物组合物或冻干制剂在冷藏温度(2-8℃)条件下保存6个月时，SEC单体百分比下降不超过5％。在一些实施方案中，前述药物组合物或冻干制剂于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。在一些实施方案中，前述药物组合物在4℃条件下放置6个月，制剂SEC单体百分比下降不超过2％(例如小于2％，小于1％、小于0.8％、小于0.5％甚至更小)；在一些实施方案中，所述药物组合物在强制降解条件(40℃M1)下，相比D0时的SEC值，SEC下降幅度小于或等于约10％；优选小于或等于约9％、约8％、约7％、约6％、约5％、约4％、约3％、约2％或约1％；更优选SEC下降幅度为1.1％-2.7％之间。在一些实施方案中，前述药物组合物在4℃条件下放置6个月,制剂SEC单体百分比大于94％(例如大于94.5％、95％、98％)。
在一些实施方案中，本披露提供一种含抗PD-1抗体的复溶溶液，所述复溶溶液通过将前述的冻干制剂经复溶获得。其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。
在一些实施方案中，本披露提供一种复溶溶液，所述复溶溶液为前面任一项所述的冻干制剂的复溶形式。
在一些实施方案中，本披露提供一种制品，其包括容器，该容器中装有如前所述的药物组合物、冻干制剂或复溶溶液。在一些实施方案中，该容器为中性硼硅玻璃管制注射剂瓶。
本披露还提供前述的药物组合物、冻干制剂、复溶溶液或制品在制备用于治疗/预防疾病或病症的药物中的用途。
本披露还提供作为治疗/预防疾病或病症的药物的前述药物组合物、冻干制剂、复溶溶液或制品。
本披露还提供一种治疗或预防疾病或病症的方法，所述方法包括向受试者施用治疗有效量或预防有效量的前述药物组合物、冻干制剂、复溶溶液或制品。
在一些实施方案中，前面任一项述疾病或病症是PD-1相关的疾病或病症。
在一些实施方案中，前面任一项述所述疾病为肿瘤。在另一些实施方案中，前面任一项述所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；在其中一些实施方案中，所述淋巴瘤选自：何杰金 淋巴瘤、非何杰金淋巴瘤、弥漫性大B-细胞淋巴瘤、滤泡性淋巴瘤、原发性纵隔大B-细胞淋巴瘤、套细胞淋巴瘤、小淋巴细胞性淋巴瘤、富含T-细胞/组织细胞的大B-细胞淋巴瘤和淋巴浆细胞性淋巴瘤，所述肺癌选自：非小细胞肺癌和小细胞肺癌，所述白血病选自：慢性髓细胞样白血病、急性髓细胞样白血病、淋巴细胞白血病、成淋巴细胞性白血病、急性成淋巴细胞性白血病、慢性淋巴细胞性白血病和髓样细胞白血病；在另一些实施方案中，所述疾病选自：PD-L1阳性的黑色素瘤、肺癌、非小细胞肺癌、乳腺癌、胃癌、肾癌、膀胱癌、肠癌和结肠癌；在另一些实施方案中，所述疾病选自：黑色素瘤、肺癌、非小细胞肺癌、乳腺癌、胃癌、肾癌、膀胱癌、肠癌和结肠癌。
附图说明
图1：抗PD-1抗体阻断PD-1与其配体的结合测试结果；
图2：抗PD-1抗体对PBMC细胞分泌IFNγ的影响；
图3：抗PD-1抗体对小鼠结肠癌MC38移植瘤的疗效；
图4：抗PD-1抗体对小鼠结肠癌MC38肿瘤体积的影响；
图5：抗PD-1抗体制剂的DOE拟合图。
具体实施方式
术语
为了更容易理解本披露，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披露所属领域的一般技术人员通常理解的含义。
“缓冲液”或“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲液。将pH控制在适当范围中的缓冲液的例子包括醋酸盐缓冲液、琥珀酸盐缓冲液、葡萄糖酸盐缓冲液、组氨酸缓冲液、草酸盐缓冲液、乳酸盐缓冲液、磷酸盐缓冲液、柠檬酸盐缓冲液、酒石酸盐缓冲液、延胡索酸盐缓冲液、甘氨酰甘氨酸缓冲液和其它有机酸缓冲液。
“醋酸盐缓冲剂”或“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、组氨酸-醋酸盐、醋酸-醋酸钾、醋酸-醋酸钙、醋酸-醋酸镁等。本披露在一些实施方案中，醋酸盐缓冲液是醋酸-醋酸钠缓冲液(也称醋酸钠盐，简写AA)。
“组氨酸盐缓冲剂”或“组氨酸缓冲液”，是包含组氨酸离子的缓冲液。组氨酸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。本披露在一些实施方案中，缓冲液是组氨酸-醋酸盐缓冲液(也称组氨酸醋酸盐，简写His-AA)。
“琥珀酸盐缓冲剂”或“琥珀酸盐缓冲液”，是包括琥珀酸离子的缓冲液。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-琥珀酸钙缓冲液等。本披露在一些实施方案中，琥珀酸盐缓冲液是琥珀酸-琥珀酸钠缓冲液(也称琥珀酸钠盐，简写SA)。
“枸橼酸盐缓冲剂”或“枸橼酸盐缓冲液”是包括枸橼酸根离子的缓冲剂。枸橼酸盐缓冲剂的实例包括枸橼酸-枸橼酸钠、枸橼酸-枸橼酸钾、枸橼酸-枸橼酸钙、枸橼酸-枸橼酸镁缓冲液等。优选的枸橼酸盐缓冲剂是枸橼酸-枸橼酸钠缓冲液(也称枸橼酸钠盐，简写CA)。
渗透压调节剂是指用于调节溶液渗透压的物质。渗透压调节剂的实例包括但不限于，糖类(包括单糖，二糖，三糖，多糖，糖醇，还原性糖，非还原性糖等等)、氨基酸(包括精氨酸、甘氨酸、半胱氨酸、组氨酸等等)、盐类(氯化钠、氯化钾、氯化钙等)。在一些实施方案中，渗透压调节剂为糖，所述糖选自：葡萄糖，蔗糖，海藻糖，乳糖，果糖，麦芽糖，右旋糖苷，甘油，赤藻糖醇，丙三醇，阿拉伯糖醇，木糖醇，山梨糖醇(也称山梨醇)，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，乳糖醇和异-麦芽酮糖；在一些实施方案中，糖是非还原性二糖；在一些实施方案中，所述的糖优选为海藻糖或蔗糖，最优选为蔗糖。在一些实施方案中，所述渗透压调节剂为氨基酸，优选精氨酸、甘氨酸；在一些实施方案中，所述渗透压调节剂为盐类，优选氯化钠。
“等渗”表示制剂具有与人血液基本上相同的渗透压。等渗可以通过本领域公知方法测定，例如可以采用蒸汽压力或冰冻型渗压计测定。当给药途径为皮下注射时，药物组合物渗透压优选控制在280-320mOsm，渗透压调节剂优选70-90mg/mL蔗糖；更优选药物制剂渗透压控制在300mOsm左右，渗透压调节剂优选含量80mg/mL的蔗糖。
“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。
“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。
本披露的药物组合物或制剂可通过本领域公知的方法制备。示例性的，抗体药物组合物或制剂的制备：第一步：取一定量的纯化的抗体溶液，用不含抗体的缓冲剂(如pH5.2的10mM的醋酸钠盐缓冲液)进行溶剂置换(优选超滤)，经超滤膜至少6倍体积置换，抗体浓缩到约130mg/mL。加入一定体积的蔗糖母液，混匀，使最终蔗糖浓度为80mg/mL。加入一定体积的聚山梨酯80母液，混匀，使最终聚山梨酯80浓度为0.6mg/mL。加pH5.2的10mM醋酸钠盐缓冲液定容，使 抗体浓度为120mg/mL(其他待测试制剂或稳定性制剂可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滤芯，收集滤液。第二步：调节装量，将滤液灌装于西林瓶中，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。
本披露中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂为水。
“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真空冷冻干燥步骤之后获得的制剂或药物组合物。冻干制剂可通过将药物组合物或液体或溶液制剂经冷冻干燥获得。通过冷冻制剂和随后在适于一次干燥的温度使水升华，进行冷冻干燥。在此条件下，产物温度低于制剂的低共熔点或分解温度。在通常约50-250毫托范围的合适压力下，通常，一次干燥的存放温度范围为约-30至25℃(假设产物在一次干燥过程中保持冷冻)。制剂、容纳样品的容器(例如，玻璃小瓶)的大小和类型以及液体的体积决定了干燥所需的时间，所述时间的范围可为几小时至几天(例如40-60小时)。二次干燥阶段可在约0-40℃进行，这主要取决于容器的类型和大小以及采用的蛋白的类型。二次干燥时间由产物中的期望残余湿度水平决定，通常需要至少约5小时。通常，低压冻干的制剂的含水量小于约5％，优选小于约3％。压力可与在一次干燥步骤中应用的压力相同，优选的，二次干燥的压力低于一次干燥。冷冻干燥条件可以随制剂和小瓶大小而变化。
本披露的表面活性剂可选自聚山梨酯20、聚山梨酯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、聚丙二醇和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酯80或聚山梨酯20，更优选为聚山梨酯80。
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，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“大约”可意味着至多20％的范围。例如在“约”或“大约”后具体数值的基础上±20％、±19％、±18％、±17％、±16％、±15％、±14％、±13％、±12％、±11％、±10％、±9％、±8％、±7％、±6％、±5％、±4％、±3％、±2％、±1％或更小，该范围取决于本领域技术人员通常采用的测定方法、技术条件、检测试剂和/或检测仪器，这样 的范围是本领域公知的。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。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“大约”的含义应该假定为在该具体值的可接受误差范围内。
本披露所述的药物组合物能够达到稳定的效果，其中的抗体在贮藏后基本上保留其物理稳定性和/或化学稳定性和/或生物学活性的药物组合物，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。目前有多种测量蛋白质稳定性的分析技术，可测量在选定温度贮藏选定时间段后的稳定性。例如，稳定药物抗体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℃)保存例如至少3个月、优选6个月、更优选1年，且甚至更优选地最多达2年。稳定制剂，例如，通过视觉分析，药物抗体制剂是无色的，或澄清至稍微乳白色；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％的聚集等。在一些实施方案中，本披露中的药物组合物或冻干制剂于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。在一些实施方案中，可通过观察制剂在强制降解(例如40℃M1)、加速条件下(例如25℃M6)，振摇D7(例如25℃，300rpm，振摇10天)、多次冻融等条件下的制剂外观、SEC、非还原CE-SDS和iCIEF等指标来检测制剂的相对稳定性。
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，那么所述抗体在药物制剂中“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。
如果抗体没有显示出显著的化学改变，那么所述抗体在药物制剂中“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，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)。
如果抗体在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围内，那么所述抗体在药物制剂中“保留它的生物活性”。抗体的生物活性可以例如通过抗原结合测定来确定。
术语“程序性死亡1”、“细胞程序性死亡1”、“蛋白PD-1”、“PD-1”、“PDCD1”和“hPD-1”可互换使用，且包括人PD-1的变体、同种型、物种同源物、以及与PD-1 具有至少一个共同表位的类似物。完整的PD-1序列可以GenBank登录号U64863找到。
术语“程序性死亡配体-1(PD-L1)”是PD-1的两种细胞表面糖蛋白配体之一(另一种为PD-L2)，它在与PD-1结合时下调T细胞活化和细胞因子分泌。如本文中使用的术语“PD-L1”包括人PD-L1(hPD-L1)，hPD-L1的变体、同种型、和种间同源物，以及5种与hPD-L1具有至少一个共同表位的类似物。完整的hPD-L1序列可以用GenBank登录号Q9NZQ7查到。
术语“细胞因子”是由一个细胞群体释放的、作为细胞间介质作用于其它细胞的蛋白质的一般术语。这样的细胞因子的例子包括淋巴因子、单核因子、趋化因子和传统的多肽激素。示例性的细胞因子包括：人IL-2、IFN-γ、IL-6、TNFα、IL-17和IL-5。
本披露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。
本披露所述的“抗体”在本文中以最广意义使用，并且包括不同的抗体结构，包括，但不限于，单克隆抗体，多克隆抗体，鼠源抗体、嵌合抗体、人源化抗体、多特异性抗体(例如，双特异性抗体)和抗体片段，只要它们显示所需的抗原结合活性和特异性即可。本披露所述“抗体”包含“全长抗体”及其“抗原结合片段”。本披露的一些实施例中所述抗PD-1抗体为国际专利申请PCT/CN2020/074098所述的抗PD-1抗体，例如“Hu23-11.IgG4AA”抗体。本披露将国际专利申请PCT/CN2020/074098的全部内容引入本申请。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。
术语“抗原结合片段”或“功能片段”是完整抗体的保持特异性结合抗原(例如，PD-1)的能力的一个或更多个片段。抗原结合片段的实例包括但不限于(i)Fab片段，由VL、VH、CL和CH1结构域组成的单价片段；(ii)F(ab') 2片段，包含通过铰链区上的二硫桥连接的两个Fab片段的二价片段，(iii)由VH和CH1结构域组成的Fd片段；(iv)由抗体的单臂的VH和VL结构域组成的Fv片段；(v)单结构域或dAb片段(Ward等人，(1989)Nature341：544-546)，其由VH结构域组成。此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。在一些实施方案中，本披露的抗原结合片段包 括Fab、F(ab')2、Fab'、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)等。
抗体重链和轻链中靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C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(VL)和重链可变区(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。
术语“互补决定区”、“CDR”或“高变区”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)，每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“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)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist，7，132-136(1999)；Lefranc，M.P.等，Dev.Comp.Immunol.，27，55-77(2003)等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。遵循AbM规则，VH中的CDR氨基酸编号为26-32(HCDR1)、50-58(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。除非另有说明，本披露实施例涉及的抗体可变区和CDR序列均适用“Kabat”编号规则。
本披露中所述人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变， L234A和/或L235A突变，S228P突变，和/或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
本披露的抗体包括鼠源抗体、嵌合抗体、人源化抗体和全人抗体，优选人源化抗体。
术语“鼠源抗体”在本披露中为根据本领域知识和技能制备的针对人PD-1的单克隆抗体。制备时用PD-1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。在本披露一个优选的实施方案中，所述的鼠源抗PD-1抗体，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1、IgG2、IgG3或其变体的重链恒定区。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。通常，建立嵌合抗体，先建立分泌鼠源性特异性单抗的杂交瘤，然后从鼠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，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体。在本披露一个优选的实施方案中，所述的PD-1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-1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，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(例如L234A和/或L235A突变，和/或S228P突变)的IgG1、IgG2或IgG4变体。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的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框架序列中产生的抗体。可以克服嵌合抗体由于携带大量鼠蛋白成分，从而诱导的异源性反应。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。本披露的人源化抗体也包括进一步由酵母菌展示对CDR进行亲和力成熟突变后的人源化抗体。
本披露中“人抗体”(HuMAb)、“人源抗体”、“全人抗体”、“完全人抗体”可以互换使用，其氨基酸序列对应于由人或人细胞产生的抗体的氨基酸序列、或衍生自利用人抗体组库或其它人抗体编码序列的非人来源的氨基酸序列。该人抗体的定义明确排除了包含非人抗原结合残基的人源化抗体。在一些技术方案中，完全人抗体可以通过基因或染色体转染方法以及噬菌体展示技术来构建，或者由体外活化的B细胞构建，所有的这些都是本领域已知的。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个、2个、3个或更多个氨基酸的插入、缺失或替换。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位(例如，PD-1分子上的特定部位)。表位通常以独特的空间构象包括至少3，4，5，6，7，8，9，10，11，12，13，14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular B iology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -7M，例如大约小于10 -9M、10 -10M、10 -11M或更小的亲和力(KD)结合。
术语“KD”或“Kd”是指特定抗体-抗原相互作用的解离平衡常数。通常，本披露的抗体以小于大约10 -7M，例如小于大约10 -8M或10 -9M的解离平衡常数(KD)结合PD-1，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其免疫学功能片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原(例如PD-1抗原或其片段)的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用结合荷有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白任一种的固态表面或细胞的纯化的抗原。通过测量在所测抗原结合蛋白存在下结合固态表面或细胞的标记的量来测量竞争性抑制。通常所测抗原结合蛋白过量存在。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的 邻近表位的抗原结合蛋白，所述两个表位在空间上互相妨碍发生结合。在本文实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
本文中使用的术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，优选是双链DNA或单链mRNA或修饰的mRNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语“载体”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
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，鼠可以用人PD-1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区加上一个或更多个人源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。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
本披露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-1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。
“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。“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”意指给予患者内用或外用治疗剂，例如包含本披露的任一种结合化合物的组合物，所述患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，在受治疗患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右测量的程度。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，例如患者的疾病状态、年龄和体重，以及药物在患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。尽管本披露的实施方案(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患者中应当减轻目标疾病症状。
“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。示例性保守取代于下表“示例性氨基酸保守取代”中陈述。
表3.示例性氨基酸保守取代
原始残基 保守取代
Ala(A) Gly；Ser
Arg(R) Lys；His
Asn(N) Gln；His；Asp
Asp(D) Glu；Asn
Cys(C) Ser；Ala；Val
Gln(Q) Asn；Glu
Glu(E) Asp；Gln
Gly(G) Ala
His(H) Asn；Gln
Ile(I) Leu；Val
Leu(L) Ile；Val
Lys(K) Arg；His
Met(M) Leu；Ile；Tyr
Phe(F) Tyr；Met；Leu
Pro(P) Ala
Ser(S) Thr
Thr(T) Ser
Trp(W) Tyr；Phe
Tyr(Y) Trp；Phe
Val(V) Ile；Leu
“有效量”或“有效剂量”指指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，诸如减少各种本披露靶抗原相关病症的发病率或改善所述病症的一个或更多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓患者的本披露靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”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。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。通常，当比对两个序列时进行比较以给出最大百分比同源性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS):Altschul，S.F.等人，(1990)J.Mol.Biol.215:403-410；Gish，W.等人，(1993)Nature Genet.3:266-272；Madden，T.L.等人，(1996)Meth.Enzymol.266:131-141；Altschul，S.F.等人，(1997)Nucleic Acids Res.25:3389-3402；Zhang，J.等人，(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也 为本领域技术人员所熟知。
本文使用的表述“细胞”、“细胞系”和“细胞培养物”可互换使用，并且所有这类名称都包括后代。因此，单词“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑转移数目。还应当理解的是，由于故意或非有意的突变，所有后代在DNA含量方面不可能精确相同。包括具有与最初转化细胞中筛选的相同的功能或生物学活性的突变后代。在意指不同名称的情况下，其由上下文清楚可见。
本文使用的“聚合酶链式反应”或“PCR”是指其中微量的特定部分的核酸、RNA和/或DNA如在例如美国专利号4，683，195中所述扩增的程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物；这些引物在序列方面与待扩增模板的对应链相同或相似。2个引物的5’末端核苷酸可以与待扩增材料的末端一致。PCR可用于扩增特定的RNA序列、来自总基因组DNA的特定DNA序列和由总细胞RNA转录的cDNA、噬菌体或质粒序列等。一般参见Mullis等(1987)Cold Spring Harbor Symp.Ouant.Biol.51:263；Erlich编辑，(1989)PCR TECHNOLOGY(Stockton Press，N.Y.)。本文使用的PCR被视为用于扩增核酸测试样品的核酸聚合酶反应法的一个实例，但不是唯一的实例，所述方法包括使用作为引物的已知核酸和核酸聚合酶，以扩增或产生核酸的特定部分。
“分离的”指纯化状态，并且在这种情况下意味着在指定的分子基本上不含其他生物分子，例如核酸、蛋白质、脂质、碳水化合物或其他材料，例如细胞碎片和生长培养基。通常，术语“分离的”并不意图指完全不存在这些材料或不存在水、缓冲液或盐，除非它们以显著干扰如本文所述的化合物的实验或治疗用途的量存在。
“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。
此外，本披露包括用于治疗与目标抗原(例如PD-1)阳性细胞相关的疾病的药剂，所述药剂包含本披露的抗PD-1抗体作为活性成分。
本披露中与PD-1相关的疾病没有限制，只要它是与PD-1相关的疾病即可，例如利用本披露的分子诱导的治疗反应可通过结合人类PD-1，然后阻遏PD-1与其配体PD-L1、PD-L2的结合，或杀伤过表达PD-1的肿瘤细胞。因此，当处于适于治疗应用的制备物和制剂中时，本披露的分子对这样一些人是非常有用的，他们患有肿瘤或癌症，优选黑色素瘤、结肠癌、乳腺癌、肺癌、胃癌、肠癌、肾癌、非小细胞肺癌、膀胱癌等。
此外，本披露涉及用于免疫检测或测定目标抗原(例如PD-1)的方法、用于免疫检测或测定目标抗原(例如PD-1)的试剂、用于免疫检测或测定表达目标抗 原(例如PD-1)的细胞的方法和用于诊断与目标抗原(例如PD-1)阳性细胞相关的疾病的诊断剂，其包含本披露的特异性识别目标抗原(例如人PD-1)并与胞外区的氨基酸序列或其三维结构结合的抗体或抗体片段作为活性成分。
在本披露中，用于检测或测定目标抗原(例如PD-1)的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的实例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与PD-1阳性细胞相关的疾病可以通过用本披露的抗体或抗体片段检测或测定表达PD-1的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本披露中，对用于检测或测定目标抗原(例如PD-1)的活体样品没有特别限制，只要它具有包含表达目标抗原(例如PD-1)的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本披露的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
在一些技术方案中，抗原的制备如下：
设计并合成人PD-1-IgG1Fc融合蛋白，N端为人PD-1胞外区150个氨基酸，C端为人IgG1的Fc段(hIgG1Fc)。经Protein A的亲和柱纯化，可获得高纯度的重组PD-1-Fc蛋白，用于检测抗PD-1抗体与抗原的结合。
人PD-1-IgG1Fc(SEQ ID NO：1)：
Figure PCTCN2021109438-appb-000003
注释：下划线部分为信号肽，正体部分为人PD-1胞外区，斜体部分为hIgG1Fc ( 信号肽+胞外区+hIgG1Fc)。
人PD-1-his(SEQ ID NO：2)：
Figure PCTCN2021109438-appb-000004
转染细胞核酸编码的PD-1抗原(SEQ ID NO：3)：
Figure PCTCN2021109438-appb-000005
在一些技术方案中，抗人PD-1抗体可通过免疫小鼠产生，也可通过抗人PD-1噬菌体小鼠免疫文库获得。
通过免疫小鼠制备抗人PD-1抗体的方法如下：
1.免疫：实验用SJL白小鼠，雌性，6-8周龄和Balb/c白小鼠，雌性，6-8周龄。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按不同方案免疫，每组6-10只。免疫抗原可以是纯化的重组蛋白PD-1-IgG1Fc(见SEQ ID NO：1)、PD-1-his(见SEQ ID NO：2)、或PD-1作为抗原(见SEQ ID NO：3)转染的Jurkat/CHO-PD-1细胞，可以使用单独一种抗原配合不同的免疫佐剂或者不同类型免疫原交叉免疫。免疫部位可以是腹腔或者背部皮下，或者两种位置交替免疫。免疫佐剂
Figure PCTCN2021109438-appb-000006
Gold Adjuvant(以下简称Titermax，购自Sigma货号T2684)与Imject Alum Adjuvant(以下简称Alum，购自Pierce货号77161)交叉免疫。抗原与佐剂(Titermax)比例为1:1，抗原与佐剂(Alum)比例为3:1，25-50μg/只(首免)，50μg/只(加强免疫)，或是1×107个Jurkat/CHO-PD-1细胞/只。第0天腹膜内注射25-50μg/只的乳化后抗原，首免后每周一次或是每两周一次，Titermax和Alum交替使用，共5-8次。
2.细胞融合：选择血清中抗体滴度高的小鼠进行脾细胞融合，将冲刺免疫72h(“h”是“小时”的缩写，下同)后的小鼠眼球放血，拉颈处死，放入75％乙醇中消毒。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(中国科学院)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的RPMI-1640培养基)重悬，分装于96孔细胞培养板中(1×105/150μL/孔)，37℃，5％CO2孵育，种板10-30块左右。融合后的第5天加入HAT完全培养基，50μL/孔，37℃，5％CO2孵育。融合后 第7天至8天，根据细胞生长密度，全换液，200μL/孔，37℃，5％CO2孵育。
3.杂交瘤细胞筛选：融合后第7-9天，根据细胞生长密度，进行抗体与PD-1结合的ELISA方法检测，并将检测的阳性孔细胞进行PD-1/PDL1结合的阻断ELISA检测，阳性孔换液，并根据细胞密度及时扩大至24孔板中。移入24孔板的细胞株经过复测后进行保种和第一次亚克隆。第一次亚克隆筛选为阳性的进行保种，并进行第二次或第三次亚克隆，直至获得单细胞克隆。多次融合获得有阻断PD-1与PDL1结合效果的杂交瘤细胞。
通过抗人PD-1噬菌体小鼠免疫文库获得抗人PD-1抗体的方法如下：
1.构建抗人PD-1噬菌体小鼠免疫文库：选择血清中抗体滴度高的小鼠的脾脏，用Trizol(Invitrogen Cat No.15596-018)提取组织总RNA。使用PrimeScript TMII 1st Strand cDNA Synthesis Kit试剂盒(Takara Cat No.6210A)进行反转录获得cDNA。根据IMGT数据库设计并合成构建文库的引物。通过三轮PCR反应，获得单链抗体片段。将单链抗体片段和经过改造的建库载体pCantab5E(Amersham Biosciences/GE Cat No.27-9400-01)用Sfi1(NEB Cat No.#R0123L)进行酶切，电泳后用
Figure PCTCN2021109438-appb-000007
Gel Extraction Kit(Omega Cat No.D2500-02)进行纯化回收。然后用T4DNA连接酶(NEB Cat No.#M0202L)16℃连接16-18小时，再用上述试剂盒进行纯化回收，最后用去离子水洗脱。取1μg连接产物与1支电转化感受态TG1(Lucigen Cat No.60502-2)混合，电转化仪(Bio Rad Micropulser)参数设至2.5kV，200Ω，25uF，进行电转化。重复转化10次，涂平板，37℃倒置培养16-18小时。将所有菌落刮洗下来混和在一起，加入终浓度为15％的甘油，-80℃保存备用。
2.抗人PD-1噬菌体小鼠免疫文库的筛选：将包装好的抗人PD-1噬菌体免疫文库(1×10 12-1×10 13)与100μL链菌素微珠(Mi1envi Biotec，Auburn，CA)加入1mL含2％脱脂牛奶-磷酸盐缓冲液(缩写MPBS)中于室温下孵育1小时，放置在磁力架上，取上清。上清加入10μg/mL生物素化的人PD-1-ECD-his蛋白(购自Sino Biological)中于室温下孵育1小时，再加入100μL链霉亲和素包被的磁珠(1mL MPBS预孵育)于室温下孵育1小时。并使其负载于磁力架系统上用于分选，吸去上清。加入1mL PBST(含0.1％Tween-20的磷酸盐缓冲液)，翻转多次，吸尽后再加入新鲜洗液，重复11次，以去除未结合的抗体片段，加入0.5mL洗脱液(50μL 10mg/mL trypsin stock solution(存储液)加入450μL PBS中)。室温下摇晃15min(“min”是“分钟”的缩写，下同)。放置在磁力架上，吸出上清至一新EP管中。TG1接入2YT培养基中扩增至培养细菌密度OD600＝0.4时。每管加入1.75mL TG1(OD600＝0.4)，并加入250μL洗脱后噬菌体(phage)，37℃水浴中静置孵育30min，梯度稀释涂板，用于测试滴度。其余TG1溶液离心，涂板，37℃过夜孵育。
噬菌体小鼠免疫文库利用生物素化的人PD-1-ECD-his抗原，经过2-3轮 MACS筛选(链霉素磁珠，Invitrogen)，最终获得具有结合PD-1和阻断PD-1与PD-L1结合的单克隆，测序验证，得到抗体的可变区序列。
在一些技术方案中，抗体或抗原蛋白的纯化方法如下：
1.杂交瘤上清分离纯化/ProteinG亲和层析：
对于小鼠杂交瘤上清纯化首选ProteinG进行亲和层析，将培养所得杂交瘤离心取上清，根据上清体积加入10-15％体积的1M Tris-HCl(pH8.0-8.5)调节上清pH。ProteinG柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积；利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积；细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间；利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线；利用0.1M醋酸/醋酸钠(pH3.0)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。
2.Protein A亲和层析纯化蛋白或抗体：
首先将表达抗原蛋白或者抗体的细胞培养上清进行高速离心收取上清。ProteinA亲和柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积。利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积。细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线。利用0.1M醋酸/醋酸钠(pH3.0-3.5)缓冲液进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样品纯度。
以下结合实施例用于进一步描述本披露，但这些实施例并非限制着本披露的范围。本披露实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；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1.抗人PD-1鼠源抗体获得
将经前述方法获得的抗人PD-1鼠源抗体进行抗原结合实验，筛选得到多株活性良好的抗体：其中包括M23、M32和M33，将单细胞克隆扩培养，提取RNA， 利用mouse-Ig的简并引物进行反转录扩增(RT-PCR)，得到抗体的可变区序列。将该鼠抗体可变区序列与人抗体恒定区序列连接，克隆并重组表达出该鼠单克隆抗体的嵌合抗体，进行体外活性实验，确认所得到的单克隆抗体可变区序列正确。
测得鼠源抗体M23、M32和M33的可变区序列如下：
鼠源抗体M23的重链可变区(SEQ ID NO：4)：
Figure PCTCN2021109438-appb-000008
鼠源抗体M23的轻链可变区(SEQ ID NO：5)：
Figure PCTCN2021109438-appb-000009
鼠源抗体M32的重链可变区(SEQ ID NO：6)：
Figure PCTCN2021109438-appb-000010
鼠源抗体M32的轻链可变区(SEQ ID NO：7)：
Figure PCTCN2021109438-appb-000011
鼠源抗体M33的重链可变区：(SEQ ID NO：19)
Figure PCTCN2021109438-appb-000012
鼠源抗体M33的轻链可变区：(SEQ ID NO：20)
Figure PCTCN2021109438-appb-000013
备注：上述抗体的重链可变区和轻链可变区序列中，下划线为Kabat编号系统确定的CDR序列，依次为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。
表4.鼠源抗体M23、M32和M33重链及轻链CDR区序列
Figure PCTCN2021109438-appb-000014
Figure PCTCN2021109438-appb-000015
备注：表中的抗体CDR序列是根据Kabat编号系统确定。
实施例2.抗人PD-1单克隆抗体的人源化
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件分析，分别挑选与M23，M32，M33的轻重链序列同一性高的人种系重轻链可变区种系基因作为模板，将这3个鼠源抗体的CDR分别移植到相应的人源抗体模板中，分别构建其对应的人源化抗体。
1.鼠源抗体M23人源化
1.1鼠源抗体M23人源化构架选择
鼠源抗体M23的人源化轻链模板为IGKV2-40*01和IGKJ4*01，人源化重链模板为IGHV1-69*02和IGHJ6*01，人源化改造后可变区序列如下(下划线为CDR序列)：
Hu23VH-CDR嫁接：(SEQ ID NO：27)
Figure PCTCN2021109438-appb-000016
Hu23VL-CDR嫁接：(SEQ ID NO：28)
Figure PCTCN2021109438-appb-000017
1.2鼠源抗体M23的人源化模板选择和回复突变设计
表5.鼠源抗体M23的人源化抗体的回复突变
Figure PCTCN2021109438-appb-000018
Figure PCTCN2021109438-appb-000019
注：嫁接(Grafted)代表鼠抗体CDR植入人种系FR区序列，氨基酸残基由Kabat编号系统确定并注释，如I2G表示依照Kabat编号系统，将Kabat编号的第2位I突变回G。
M23的人源化抗体轻/重链可变区序列如下：
>Hu23VL1(同Hu23VL-CDR grafted)：(SEQ ID NO：28)
Figure PCTCN2021109438-appb-000020
>Hu23VL2(SEQ ID NO：29)
Figure PCTCN2021109438-appb-000021
>Hu23VH1(同Hu23VH-CDR grafted)：(SEQ ID NO：27)
Figure PCTCN2021109438-appb-000022
>Hu23VH2(SEQ ID NO：30)
Figure PCTCN2021109438-appb-000023
>Hu23VH3(SEQ ID NO：31)
Figure PCTCN2021109438-appb-000024
>Hu23VH4(SEQ ID NO：32)
Figure PCTCN2021109438-appb-000025
1.3鼠源抗体M23的人源化序列组合
鼠源抗体M23的人源化后获得的抗体及其可变区组合见下表。
表6.人源化Hu23抗体可变区组合
Figure PCTCN2021109438-appb-000026
备注：“Hu23-1”指代抗体轻链可变区为Hu23VL1且重链可变区为Hu23VH1的抗体，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu23-1)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本披露中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu23-1)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu23-1.IgG4AA”表示由Hu23VH1重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由Hu23VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu23-1.IgG4P”表示由Hu23VH1重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由Hu23VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
2.鼠源抗体M32人源化
2.1鼠源抗体M32人源化构架选择
鼠源抗体M32的人源化轻链模板为IGKV2-40*01和IGKJ4*01，人源化重链模板为IGHV1-69*02和IGHJ6*01，人源化可变区序列如下(下划线为CDR序列)：
Hu32VH-CDR嫁接：(SEQ ID NO：33)IGHV1-69*02和IGHJ6*01
Figure PCTCN2021109438-appb-000027
Hu32VL-CDR嫁接：(SEQ ID NO：34)
Figure PCTCN2021109438-appb-000028
2.2鼠源抗体M32的人源化模板选择和回复突变设计
表7.鼠源抗体M32的人源化抗体的回复突变
Figure PCTCN2021109438-appb-000029
Figure PCTCN2021109438-appb-000030
注：嫁接(Grafted)代表鼠抗体CDR植入人种系FR区序列。氨基酸残基由Kabat编号系统确定并注释，如I2V表示依照Kabat编号系统，将Kabat编号的第2位I突变回V。
鼠源抗体M32的人源化抗体轻重链可变区序列如下：
>Hu32VL1(同Hu32VL-CDR嫁接)：(SEQ ID NO：34)
Figure PCTCN2021109438-appb-000031
>Hu32VL2(SEQ ID NO：35)
Figure PCTCN2021109438-appb-000032
>Hu32VH1(同Hu32VH-CDR grafted)：(SEQ ID NO：33)
Figure PCTCN2021109438-appb-000033
>Hu32VH2(SEQ ID NO：36)
Figure PCTCN2021109438-appb-000034
>Hu32VH3(SEQ ID NO：37)
Figure PCTCN2021109438-appb-000035
>Hu32VH4(SEQ ID NO：38)
Figure PCTCN2021109438-appb-000036
>Hu32VH5(SEQ ID NO：39)
Figure PCTCN2021109438-appb-000037
>Hu32VH6(SEQ ID NO：40)
Figure PCTCN2021109438-appb-000038
2.3鼠源抗体M32的人源化序列组合
鼠源抗体M32的人源化后获得的抗体及其可变区组合。
表8.人源化抗体Hu32轻/重链可变区组合
Figure PCTCN2021109438-appb-000039
备注：表中例如“Hu32-1”指代抗体轻链可变区为Hu32VL1且重链可变区为Hu32VH1的抗体轻/重链可变区组合，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu32-1)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本披露中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu32-1)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu32-1.IgG4AA”表示由 Hu32VH1重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由 Hu32VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu32-1.IgG4P”表示由 Hu32VH1重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由 Hu32VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
3.鼠源抗体M33人源化
3.1鼠源抗体M33人源化构架选择
鼠源抗体M33的人源化轻链模板为IGKV1-39*01和IGKJ4*01，人源化重链模板为IGHV3-7和IGHJ6*01，人源化可变区序列如下：
Hu33VH-CDR嫁接(SEQ ID NO：41)：
Figure PCTCN2021109438-appb-000040
Hu33VL-CDR嫁接(SEQ ID NO：42)：
Figure PCTCN2021109438-appb-000041
3.2鼠源抗体M33的人源化模板选择和回复突变设计
表9.鼠源抗体M33人源化抗体的回复突变
Figure PCTCN2021109438-appb-000042
注：嫁接(Grafted)代表鼠抗体CDR植入人种系FR区序列。氨基酸残基由Kabat编号系统确定并注释，如F71Y表示依照Kabat编号系统，将Kabat编号的第71位F突变回Y。
鼠源抗体M33的人源化抗体轻链可变区和重链可变区序列如下：
>Hu33VL1(同Hu33VL-CDR grafted)：(SEQ ID NO：42)
Figure PCTCN2021109438-appb-000043
>Hu33VL2(SEQ ID NO：43)
Figure PCTCN2021109438-appb-000044
>Hu33VL3(SEQ ID NO：44)
Figure PCTCN2021109438-appb-000045
>Hu33VH1(同Hu33VH-CDR grafted)：(SEQ ID NO：41)
Figure PCTCN2021109438-appb-000046
>Hu33VH2(SEQ ID NO：45)
Figure PCTCN2021109438-appb-000047
>Hu33VH3(SEQ ID NO：46)
Figure PCTCN2021109438-appb-000048
Figure PCTCN2021109438-appb-000049
3.3鼠源抗体M33的人源化序列组合
表10.人源化抗体轻重链可变区组合
Figure PCTCN2021109438-appb-000050
备注：表中例如“Hu33-6”指代抗体轻链可变区为Hu33VL2且重链可变区为Hu33VH3的抗体轻/重链可变区组合，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu33-6)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本披露中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu33-6)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu33-6.IgG4AA”，表示由 Hu33VH3重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由 Hu33VL2轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu33-6.IgG4P”，表示由 Hu33VH3重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由 Hu33VL2轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
4.人源化抗体的突变体
4.1 Hu23人源化抗体的突变抗体
通过计算机模拟，对Hu23人源化抗体的轻链LCDR1(SEQ ID NO：11)的特定位点氨基酸进行定点突变，具体突变见表11：
表11.Hu23轻链LCDR1的突变序列:
Figure PCTCN2021109438-appb-000051
Figure PCTCN2021109438-appb-000052
注：Hu23LCDR1(N28Q)表示对Hu23人源化抗体轻链可变区Hu23VL1或Hu23VL2的Kabat编号规则第28位N突变为Q的LCDR1突变序列，Hu23LCDR1(G29A)表示对Hu23人源化抗体轻链可变区Hu23VL1或Hu23VL2的Kabat编号规则第29位G突变为A的LCDR1突变序列(由Kabat编号系统确定CDR)。
LCDR1突变后的Hu23人源化抗体轻链可变区序列如下：
>Hu23VL1(N28Q)序列为：
Figure PCTCN2021109438-appb-000053
>Hu23VL1(N28L)序列为：
Figure PCTCN2021109438-appb-000054
>Hu23VL1(N28T)序列为：
Figure PCTCN2021109438-appb-000055
>Hu23VL1(N28D)序列为：
Figure PCTCN2021109438-appb-000056
>Hu23VL1(G29A)序列为：
Figure PCTCN2021109438-appb-000057
>Hu23VL1(G29V)序列为：
Figure PCTCN2021109438-appb-000058
>Hu23VL2(N28Q)序列为：
Figure PCTCN2021109438-appb-000059
>Hu23VL2(N28L)序列为：
Figure PCTCN2021109438-appb-000060
>Hu23VL2(N28T)序列为：
Figure PCTCN2021109438-appb-000061
>Hu23VL2(N28D)序列为：
Figure PCTCN2021109438-appb-000062
>Hu23VL2(G29A)序列为：
Figure PCTCN2021109438-appb-000063
>Hu23VL2(G29V)序列为：
Figure PCTCN2021109438-appb-000064
表12.Hu23人源化抗体轻/重链可变区组合
Figure PCTCN2021109438-appb-000065
备注：表中例如“Hu23-11”指代抗体轻链可变区为Hu23VL1(N28T)且重链可变区为Hu23VH1的抗体轻/重链可变区组合，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu23-11)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本披露中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu23-11)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu23-11.IgG4AA”，表示由Hu23VH1重链可变区和如SEQ ID NO：72所示的 IgG4-AA重链恒定区连接而成的重链，与由Hu23VL1(N28T)轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu23-11.IgG4P”，表示由Hu23VH1重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由Hu23VL1(N28T)轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
实验结果显示，Hu23LCDR1(N28Q)、Hu23LCDR1(N28L)、Hu23LCDR1(N28T)、Hu23LCDR1(N28D)、Hu23LCDR1(G29A)、Hu23LCDR1(G29V)位点突变后的人源化抗体均保持与PD-1的结合能力(表16)。
4.2 Hu32人源化抗体的突变抗体
经序列分析，M23来源的系列人源化抗体Hu23和M32来源的系列人源化抗体Hu32的序列同一性较高，将Hu23轻链可变区和Hu32的重链可变区组合成新的轻重链可变区组合。实验结果显示，包含新组合轻重链可变区的人源化抗体均保持与PD-1抗原的结合能力(表16)。
表13.Hu32和Hu23抗体可变区共有序列通式
Figure PCTCN2021109438-appb-000066
Figure PCTCN2021109438-appb-000067
表14.Hu32重链可变区与Hu23轻链可变区的组合
Figure PCTCN2021109438-appb-000068
备注：表中例如“Hu32a-85”指代抗体轻链可变区为Hu23VL1(N28T)且重链可变区为Hu32VH6的抗体轻/重链可变区组合，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu32a-85)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本披露中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu32a-85)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu32a-85.IgG4AA”，表示由Hu32VH6重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由Hu23VL1(N28T)轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu32a-85.IgG4P”，表示由Hu32VH6重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由Hu23VL1(N28T)轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
表15.Hu23重链可变区与Hu32轻链可变区的组合
Figure PCTCN2021109438-appb-000069
Figure PCTCN2021109438-appb-000070
备注：表中例如“Hu23a-57”指代抗体轻链可变区为Hu32VL1且重链可变区为Hu23VH1的抗体轻/重链可变区组合，其它依此类推。
上表中所指代的抗体轻/重链可变区组合(例如Hu23a-57)可以分别与抗体轻/重链恒定区连接形成全长抗体；在本披露中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA重链恒定区或SEQ ID NO：79所示的IgG4-P重链恒定区连接形成抗体重链，并以表中指代抗体轻/重链可变区组合的名称(例如Hu32a-85)加后缀“.IgG4AA”表示与IgG4-AA重链恒定区连接形成的全长抗体，加后缀“.IgG4P”表示与IgG4-P重链恒定区连接形成的全长抗体，例如，“Hu23a-57.IgG4AA”，表示由Hu23VH1重链可变区和如SEQ ID NO：72所示的IgG4-AA重链恒定区连接而成的重链，与由Hu32VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。“Hu23a-57.IgG4P”，表示由Hu23VH1重链可变区和如SEQ ID NO：79所示的IgG4-P重链恒定区连接而成的重链，与由Hu32VL1轻链可变区和如SEQ ID NO：73所示的Kappa链恒定区连接而成的轻链形成的全长抗体。
5.人源化抗体的筛选
通过Biacore进行不同人源化抗体的亲和力检测(方法参见测试例3)，结果见表16，结果显示不同的人源化抗体保持了对于PD-1的结合能力，部分人源化抗体的亲和力甚至和其鼠源抗体基本接近。
表16.Hu23人源化抗体与人PD-1的亲和力
Figure PCTCN2021109438-appb-000071
Figure PCTCN2021109438-appb-000072
实施例3.构建和表达PD-1人源化抗体
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。IgG4-P代表S228P(对应于序列SEQ ID NO：72或SEQ ID NO：79的第108位)突变，IgG4-AA代表F234A(对应于序列SEQ ID NO：72或SEQ ID NO：79的第114位)、L235A(对应于序列SEQ ID NO：72或SEQ ID NO：79的第115位)和S228P(对应于序列SEQ ID NO：72或SEQ ID NO：79的第108位)突变，IgG4-AA和IgG4-P抗体形式可以通过IgG4抗体形式简单点突变获得。
IgG4-AA重链恒定区序列如下(SEQ ID NO：72)：
Figure PCTCN2021109438-appb-000073
抗体的轻链(Kappa链)恒定区序列如下(SEQ ID NO：73)：
Figure PCTCN2021109438-appb-000074
构建的IgG4AA形式全长抗体序列示例性列举如下：
Hu23-11.IgG4AA抗体重链(SEQ ID NO：74)：
Figure PCTCN2021109438-appb-000075
Figure PCTCN2021109438-appb-000076
Hu23-11.IgG4AA轻链(SEQ ID NO：75):
Figure PCTCN2021109438-appb-000077
Hu32a-85.IgG4AA重链(SEQ ID NO：76)：
Figure PCTCN2021109438-appb-000078
Hu32a-85.IgG4AA的轻链(同Hu23-11.IgG4AA的轻链，SEQ ID NO：75)：
Figure PCTCN2021109438-appb-000079
Hu33-6.IgG4AA重链(SEQ ID NO：77)：
Figure PCTCN2021109438-appb-000080
Hu33-6.IgG4AA轻链(SEQ ID NO：78)：
Figure PCTCN2021109438-appb-000081
Figure PCTCN2021109438-appb-000082
IgG4-P的重链恒定区序列如下(SEQ ID NO：79)：
Figure PCTCN2021109438-appb-000083
构建的IgG4-P形式全长抗体序列示例性列举如下：
Hu23-11.IgG4P抗体重链(SEQ ID NO：80)：
Figure PCTCN2021109438-appb-000084
Hu23-11.IgG4P轻链(同Hu23-11.IgG4AA的轻链，SEQ ID NO：75):
Figure PCTCN2021109438-appb-000085
Hu32a-85.IgG4P重链(SEQ ID NO：81)：
Figure PCTCN2021109438-appb-000086
Hu32a-85.IgG4P的轻链(同Hu23-11.IgG4AA的轻链，SEQ ID NO：75)：
Figure PCTCN2021109438-appb-000087
Hu33-6.IgG4P重链(SEQ ID NO：82)：
Figure PCTCN2021109438-appb-000088
Hu33-6.IgG4P轻链(同Hu33-6.IgG4AA轻链，SEQ ID NO：78)：
Figure PCTCN2021109438-appb-000089
测试例
测试例1.抗PD-1抗体在体外与PD-1配体的结合和结合阻断ELISA实验
肿瘤细胞表面的PD-L1通过和T细胞表面的PD-1的结合，从而对T细胞的增殖起到抑制的效果。PD-1的抗体能通过和PD-1的结合，而阻断PD-L1/PD-1的信号通路，进而刺激T细胞的增殖。PD-1/PD-L1的结合阻断实验用于检测抗PD-1抗体对于信号通路的阻断活性。
本实验中，将PD-1-His蛋白(Cat.#10377H08H，Sino Biological)包被96孔板后，分别加入待测的抗PD-1抗体(包括抗体：Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA，阳性对照抗体：H005-1(参见WO2015085847中H005-1抗体)，进行孵育反应；稍后再HRP标记的goat anti-human IgG(H+L)抗体(Cat.#109-035-003，Jackson ImmunoResearch)，孵育反应。洗板后，检测HRP标记的goat anti-human IgG(H+L)结合量，计算得到抗PD-1抗体对配体PD-1结合的EC 50值。
本实验中，将胞外区与Fc融合的PD-1蛋白(PD-1-Fc，序列见SEQ ID NO：1)包被96孔板后，分别加入待测的抗PD-1抗体(包括抗体：Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA，阳性对照抗体：H005-1(参见WO2015085847 中H005-1抗体)，进行孵育反应；稍后再加入生物素标记的PD-L1/PD-L2，孵育反应。洗板后，检测生物素标记的PD-L1/PD-L2结合量，计算得到抗PD-1抗体对配体PD-L1/PD-L2结合阻断的IC 50值。
用pH 9.6CB缓冲液(1.59g Na 2CO 3和2.93g NaHCO 3溶于1L蒸馏水)将PD-1-Fc稀释至1μg/mL，以100μL/孔的体积加于96孔板中，于4℃放置16h-20h。将96孔板中PBS缓冲液吸掉，用PBST(pH7.4PBS含0.05％tween20)缓冲液洗板1次后，加入120μL/孔PBST/1％milk，室温孵育1h进行封闭。移去封闭液，用PBST缓冲液洗板1次后，加入90μL用样品稀释液(pH7.4PBS含5％BSA，0.05％Tween20)稀释至合适浓度的待测抗PD-1抗体，置4℃预孵育1h。以10μL/孔的体积加入10×浓度的生物素标记PD-L1/PD-L2(北京义翘神州生物技术有限公司)(10μg/mL)，在振荡器上振荡、混匀后，置37℃孵育1h。移去反应体系，用PBST洗板6次后，加入100μL/孔用PBST缓冲液1:400稀释的Streptavidin–Peroxidase Polymer(链霉亲和素-过氧化物酶聚合物)，室温振荡孵育50分钟。用PBST洗板6次后，加入100μL/孔TMB，于室温孵育5-10min。加入100μL/孔1M H 2SO 4终止反应。用酶标仪在450nm处读取吸收值，计算抗PD-1抗体对配体PD-L1/PD-L2结合阻断的IC 50值。数据详见下表17。
表17.本披露的抗PD-1抗体和PD-1结合及对配体PD-L1/PD-L2结合阻断ELISA
Figure PCTCN2021109438-appb-000090
本披露示例性抗PD-1抗体Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA都能够有效阻断PD-1与PD-L1/PD-L2的结合，其阻断活性与阳性对照抗体相似。
测试例2.示例性抗体的配体阻断试验
研究抗体对PD-1与PD-L1结合的阻断作用。实验过程简单描述如下：
消化CHOK1/PD-L1细胞(Promega)，按照100μL/孔加入到96孔板中，放置于37℃，5％CO 2培养箱培养24小时。使用PBS稀释对照品和样品至所需浓度。计数Jurkat/PD-1细胞(稳转PD-1的Jurkat细胞)，按一定比例种CHOK1/PD-L1细胞的细胞培养板中(90μL/孔)同时加入10μL/孔加入稀释后的抗体(抗体：Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA，阳性对照抗体：H005-1)，阴性对照IgG4蛋白，抗体梯度稀释浓度为0.3mg/mL、3mg/mL、30mg/mL)，置 于37℃，5％CO 2培养箱培养5小时。取出细胞培养板，置于室温放置5分钟，然后每孔加入50μL Bio-Glo TMReagent，室温孵育5分钟，读板。实验结果见附图1。
结果表明，本披露中示例性的抗PD-1抗体Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA能够有效阻断PD-1与PD-L1的结合。
测试例3.示例性抗体的BIAcore抗体亲和力实验
用Protein A生物传感芯片(Cat.#29127556，GE)亲和捕获IgG，human PD-1抗原(Cat.#10377H08H，Sino Biological)、Cyno PD-1抗原(购自Sino Biological)流过芯片表面，Biacore T200仪器实时检测PD-1抗体和抗原PD-1反应信号获得结合和解离曲线。在每个实验循环解离完成后，用10mM Glycine-HCl pH1.5的缓冲液将生物传感芯片洗净再生。实验缓冲体系为1×HBS-EP缓冲溶液(Cat#BR-1001-88，GE)。实验结束后用GE Biacore T200 Evaluation version 3.0软件以(1:1)Langmuir模型拟合数据，得出亲和力数值，结果见表18。
表18.抗PD-1抗体与人PD-1和猴PD-1的亲和力
Figure PCTCN2021109438-appb-000091
结果显示，本披露示例性的抗PD-1抗体Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA均能够与人PD-1和猴PD-1结合。
测试例4.抗体在PBMC-T淋巴细胞激活实验中对细胞IFNγ的分泌作用
为了研究抗PD-1抗体对人原代T淋巴细胞功能的影响，收集和纯化人外周血单核细胞(PBMC)，采用结核菌素(TB)体外刺激5天后，检测细胞因子IFNγ分泌水平。实验过程简单描述如下：
新鲜血液利用Ficoll-Hypaque(17-5442-02，GE)，密度梯度离心(Stem Cell Technologies)得到PBMC，于RPMI 1640(SH30809.01，GE)培养基中培养，该培养基中添加10％(v/v)FBS(10099-141，Gibco)，37℃，5％CO 2条件下培养。
新鲜分离纯化的PBMC以RPMI 1640培养基调整密度为2×10 6个/mL，20mL细胞悬液中加入40μL结核菌素(97-8800，Synbiotics)，37℃，5％CO 2培养箱培养5天。第5天，收集上述培养的细胞离心，重悬至新鲜的RPMI 1640培养基中，调整密度为1.1×10 6个/mL，接种至96孔细胞培养板，每孔90μL。同时加入梯度稀释的抗体样品(包括本披露的抗体：Hu23-11.IgG4AA、Hu32a-85.IgG4AA和Hu33-6.IgG4AA，阳性对照抗体H005-1，和阴性对照IgG4蛋白，抗体梯度稀释浓度为0.3mg/mL、3mg/mL、30mg/mL)，用PBS(B320，上海源培生物科 技股份有限公司)稀释，每孔10μL。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)收集细胞培养上清，采用ELISA的方法(人IFN-γ检测试剂盒(EHC102g.96，欣博盛))，检测IFN-γ的水平。具体操作参考试剂说明书。
试验结果见图2，结果表明本披露的抗PD-1抗体Hu23-11.IgG4AA、Hu32a-85.IgG4AA、和Hu33-6.IgG4AA均能有效激活IFN-γ的分泌。
测试例5.抗PD-1抗体在转基因PD-1小鼠结肠癌模型MC38中的作用
将MC38细胞5×10* 5细胞/小鼠/100μL接种于90只hPD-1TG小鼠(百奥赛图)右肋部皮下，10天后去除肿瘤体积过大过小的动物，按平均肿瘤体积约120mm^3将小鼠随机分为：空白对照Vehicle(PBS)、阳性对照H005-1 3mpk、Hu32a-85.IgG4AA 1mpk、Hu32a-85.IgG4AA 3mpk、Hu23-11.IgG4AA 1mpk、Hu23-11.IgG4AA 3mpk、Hu33-6.IgG4AA 3mpk共7组，每组8只。Day0(第0天)起每周三次腹腔注射各组抗体，第一周给药结束后发现肿瘤被明显抑制，第二、三周调整给药频率为每周一次，共给药5次。每周2次监测肿瘤体积、动物重量并记录数据。当肿瘤体积超过2000mm 3或多数肿瘤出现破溃或体重下降20％时，将荷瘤动物进行安乐死作为实验终点。
肿瘤体积(TV)＝1/2×L 长×L 短 2
肿瘤增殖率(T/C％)＝(T-T0)/(C-C0)×100％
抑瘤率(TGI％)＝1-T/C％
其中，T、T0分别表示抗体给药组试验结束和试验开始时的肿瘤体积，C、C0分别表示空白对照组试验结束和试验开始时的肿瘤体积。
试验结果见表19和附图3，试验结果表明，与空白对照相比，本披露的抗体均能显著抑制小鼠结肠癌MC38移植瘤的生长，其中抑瘤率最高的是Hu32a-85.IgG4AA-3mpk组，末次测量时抑瘤率为77.64％。当给药频率为一周三次给药3次，在第七天检测时，结果显示本披露的抗体的抑瘤率均明显优于阳性对照抗体H005-1；其后给药频率降为一周一次，给药2次后(Day21)，本披露的抗体间药效逐渐拉开差距，且表现出剂量依赖性，其中Hu32a-85.IgG4AA明显优于同等剂量的H005-1(p＜0.05)。而且荷瘤小鼠对抗PD-1抗体均能很好耐受，在整个给药过程中体重平稳上升，无明显药物致体重减轻等症状发生。
表19.抗PD-1抗体对小鼠结肠癌MC38的抑瘤率影响(mm 3)
Figure PCTCN2021109438-appb-000092
测试例6.抗PD-1抗体在转基因PD-1小鼠结肠癌模型MC38中的作用
转基因PD-1小鼠来源于购买的转基因PD-1小鼠(ISIS INNOVATION LIMITED，University Offices，Wellington Square，Oxford OX1 2JD，England)在Cephrim Biosciences，Inc.培育的第五代小鼠。将MC38细胞以5x10 5个/100μL/只接种到hPD-1转基因小鼠(雌雄各半)右肋后部皮下，待小鼠平均肿瘤体积达到80-100mm3之间时，去除体重、肿瘤过大和过小的动物，按照肿瘤体积大小将荷瘤小鼠随机分为5组(每组8只)：阴性对照hIgG control 30mpk、H005-1 10mpk、H005-1 30mpk、Hu33-6.IgG4AA 10mpk、Hu33-6.IgG4AA 30mpk。分组给药日期设定为Day 0。分组后腹腔给予各药物，给药周期22天，每两天给药一次，共11次。每周测2次瘤体积，称体重，记录数据。各组动物体重、肿瘤体积均用平均值±标准差(Mean±SEM)表示，并用Graphpad Prism 5和Excel软件作图，使用student t test统计分析。
肿瘤体积(TV)＝0.5236×L 长×L 短 2
肿瘤增殖率T/C％＝(T-T0)/(C-C0)×100％
抑瘤率％TGI＝1-T/C％
其中，T、T0分别表示抗体给药组试验结束和试验开始时的肿瘤体积，C、C0分别表示空白对照组试验结束和试验开始时的肿瘤体积。
试验结果见表20和附图4所示，试验结果表明，与对照组相比，本披露的抗体能显著抑制小鼠结肠癌MC38移植瘤的生长，其中抑瘤率最高的是Hu33-6.IgG4AA 30mpk组，第20天测量时抑瘤率为80.4％。在低剂量组(10mpk)，Hu33-6.IgG4AA-10mpk的药效好于阳性对照H005-1-10mpk。
表20.抗PD-1抗体对小鼠结肠癌MC38肿瘤体积影响
Figure PCTCN2021109438-appb-000093
备注：表中各组肿瘤平均体积的单位为：mm 3。
测试例7.抗PD-1抗体食蟹猴的药代动力学试验
实验用食蟹猕猴，雄性，6只，2-5岁，2-5公斤。购于于广东前沿生物科技有限公司，许可证号：SCXK(粤)2015-0037，动物合格证号：44613900000219。
饲养环境：室温控制在18℃-26℃，相对湿度在40％-70％，光照12小时明暗交替。除了需要禁食的情况外，无限量获取饲料和水。
动物给药前称重，体重介于2.81-3.52kg之间。使用注射泵于前肢或后肢皮下静脉输注给药，各组给药剂量均为1mg/kg(1mpk)，单次静脉注射给药，给药速度0.1mL/kg/min，给药时间约30min。动物于给药前，静脉输注开始后5min、0.25h、 0.5h(给药结束即刻)，1h、2h、4h、8h，1d、2d、3d、4d、5d、7d、10d、13d、14d、21d和28d，于后肢静脉采集全血，分离血清。其中给药前，静脉输注开始后14d，21d，28d采集全血约2mL，其余采血点采集全血约1mL。用ELISA检测血清中的血药浓度，进行PK分析，结果见表21。
表21.人源化抗PD-1抗体食蟹猕猴药代动力学
  Hu23-11.IgG4AA Hu33-6.IgG4AA
t1/2(day) 5.5±0.7 4.6±1.3
Cmax(μg/mL) 23.75±2.29 21.47±2.13
AUC(h*μg/mL) 2775±241 2319±518
CL(mL/day/kg) 8.7±0.7 10.7±2.3
Vz(mL/kg) 69±3.6 67.9±3.4
结果显示，Hu23-11.IgG4AA和Hu33-6.IgG4AA的药代动力学活性良好。
以下通过示例性试验配制抗PD-1抗体稳定制剂，以下制剂实施中的抗PD-1抗体为前述的Hu23-11.IgG4AA，制备过程中使用的设备及结果计算方法如下：
SEC分子排阻色谱法：根据凝胶孔隙的孔径大小与高分子样品分子的线团尺寸间的相对关系而对溶质进行分离的分析的方法。SEC单体含量百分比＝A单体/A总*100％(A单体为样品中主峰单体的峰面积，A总为所有峰面积之和)。SEC测定用仪器：安捷伦1260；柱子：waters，XBrige
Figure PCTCN2021109438-appb-000094
SEC(300×7.8mm 3.5μm)。
CE毛细管凝胶电泳：将凝胶移到毛细管中作为支持介质进行的一种电泳，并在一定的电压下根据样品分子量的大小进行分离的方法。非还原CE(NR-CE)纯度百分比＝A主峰/A总*100％(A主峰为样品中主峰的峰面积，A总为所有峰面积之和。CE测定用仪器：Beckman，型号plus800。
iCIEF成像毛细管等点聚焦电泳(简称iCE)：根据蛋白质等电点pI不同进行分离的技术。iCIEF中性峰含量百分比＝中性峰面积/总面积*100％(总面积为酸性峰、中性峰和碱性峰面积之和)。iCIEF测定所用仪器：simple protein，型号muarice。
渗透压：冰点法测定渗透压，以冰点下降值与溶液的摩尔浓度成正比例关系为基础，采用高灵敏度感温元件，测定溶液结冰点，通过电量转化为渗透压。仪器厂家罗泽Loser，型号OM815。
测试例8：抗PD-1抗体制剂缓冲体系和pH筛选
使用下列缓冲液，配制含80mg/mL蔗糖和0.6mg/mL聚山梨酯80(PS80)，蛋白浓度为100mg/mL的抗PD-1抗体(Hu23-11.IgG4AA)制剂，其中缓冲液如下：
1)10mM醋酸钠盐(简称：AA)，pH 5.0；
2)10mM醋酸钠盐，pH 5.2；
3)10mM醋酸钠盐，pH 5.5；
4)10mM醋酸钠盐，pH5.7；
5)10mM琥珀酸钠盐(简称：SA)，pH5.2；
6)10mM枸橼酸钠盐(简称：CA)，pH5.2；
制备完成的制剂过滤，灌装，加塞，轧盖。考察样品在强制降解条件下(40℃M1，也即40℃高温条件下放置1个月)和加速条件下(25℃M6，也即25℃温度条件下放置6个月)的稳定性，并以外观、SEC和非还原CE-SDS为评价指标，考察制剂稳定性。实验结果见表22。
40℃M1强制降解条件下，AA和SA缓冲体系的蛋白制剂外观优于CA体系；pH5.2-5.7的AA缓冲体系的蛋白制剂纯度项优于pH5.0AA缓冲体系和pH5.2 SA缓冲体系；25℃M6加速条件下，外观组间无显著性差异。
表22.pH和缓冲体系筛选实验结果
Figure PCTCN2021109438-appb-000095
备注：D0表示实验开始时，“M”表示月，例如M1表示一个月。
另外，对另一批次抗PD-1抗体(Hu23-11.IgG4AA)，使用下列缓冲液，配制含80mg/mL蔗糖和0.6mg/mL聚山梨酯80(PS80)，蛋白浓度为100mg/mL的抗PD-1抗体(Hu23-11.IgG4AA)制剂，其中缓冲液如下：
1)10mM醋酸钠盐，pH 5.2；
2)10mM琥珀酸钠盐，pH5.2；
3)10mM组氨酸醋酸盐(简称：His-AA)，pH5.2；
制备完成的制剂过滤，灌装，加塞，轧盖。考察样品在加速条件下(25℃M6，也即25℃温度条件下放置6个月)的稳定性，考察制剂SEC和iCIEF指标，考察制剂稳定性。实验结果见表23。
25℃M6加速条件下，pH5.2 AA和pH5.2 His-AA的SEC/iCIEF数据高于pH 5.2 SA组，pH5.2 AA缓冲体系的iCIEF略高于pH5.2 His-AA缓冲体系。综合考虑，制剂缓冲体系优选醋酸盐和组氨酸盐缓冲体系，优选AA或His-AA缓冲体系。
表23.pH和缓冲体系筛选实验结果
Figure PCTCN2021109438-appb-000096
备注：表中“D”表示天，D0表示实验开始时,“M”表示月，例如M1表示一个月。
测试例9：抗PD-1抗体制剂缓冲体系离子强度筛选
使用缓冲体系离子强度分别为10mM和30mM的pH5.2的醋酸钠盐，配制含80mg/mL蔗糖，0.6mg/mL聚山梨酯80，蛋白浓度为120mg/mL的抗PD-1抗体(Hu23-11.IgG4AA)制剂。辅料组方：
1)10mM pH5.2 AA，0.6mg/mL PS80和80mg/mL蔗糖；
2)30mM pH5.2 AA，0.6mg/mL PS80和80mg/mL蔗糖；
制备完成的制剂过滤，灌装，加塞，轧盖。考察样品在强制降解条件下(40℃M1)的稳定性，并以SEC为评价指标，考察制剂稳定性。实验结果见表24。
实验数据显示，在40℃M1强制降解条件下，10mM离子强度组的SEC数据略高于30mM离子强度组，因此pH5.2醋酸钠盐缓冲体系的离子强度优选10mM。
表24.离子强度筛选实验结果
Figure PCTCN2021109438-appb-000097
备注：表中“D”表示天，D0表示实验开始时，“M”表示月，例如M1表示一个 月。
测试例10：抗PD-1抗体制剂中表面活性剂筛选
在pH5.2的10mM醋酸钠盐缓冲液中制备含下列1)-5)不同浓度吐温80的抗PD-1抗体(Hu23-11.IgG4AA)制剂。其中蛋白浓度为100mg/mL，其它辅料如下：
1)80mg/mL蔗糖，0.2mg/mL聚山梨酯80(PS80)；
2)80mg/mL蔗糖，0.4mg/mL PS80；
3)80mg/mL蔗糖，0.6mg/mL PS80；
4)80mg/mL蔗糖，0.8mg/mL PS80；
5)80mg/mL蔗糖，0.6mg/mL聚山梨酯20(PS20)。
制备完成的制剂过滤，灌装，加塞，轧盖。将样品进行振摇(25℃，300rpm，10天)和长期稳定性(2-8℃6个月)实验，以外观、SEC、非还原CE-SDS为评价指标，考察制剂稳定性。实验结果见表25。
实验结果显示，在振摇D10条件下，0.6mg/mL PS80、0.8mg/mL PS80和0.6mg/mL PS20组的外观优于出现大量颗粒的0.2mg/mL PS80和0.4mg/mL PS80组；在2-8℃条件下放置6个月时，从外观看0.6mg/mL PS80、0.8mg/mL PS80优于出现浑浊的0.6mg/mL PS20组，纯度项组间无显著差异。因此表面活性剂选择聚山梨酯80，浓度优选0.6mg/mL。
表25.制剂中表面活性剂筛选实验结果
Figure PCTCN2021109438-appb-000098
备注：表中“D”表示天，例如D10表示10天；D0表示实验开始时,“M”表示月，例如M6表示六个月。
测试例11：抗PD-1抗体制剂中渗透压调节剂筛选
在含下列1)-7)不同种类辅料及pH5.2的10mM醋酸钠盐缓冲液中，制备蛋白浓度为100mg/mL的抗PD-1抗体(Hu23-11.IgG4AA)制剂。具体辅料如下：
1)0.6mg/mL聚山梨酯80(PS80)；
2)80mg/mL蔗糖，0.6mg/mL PS80；
3)80mg/mL海藻糖，0.6mg/mL PS80；
4)50mg/mL山梨醇，0.6mg/mL PS80；
5)100mM精氨酸，0.6mg/mL PS80；
6)100mM甘氨酸，0.6mg/mL PS80；
7)100mM NaCl，0.6mg/mL PS80；
制备完成的制剂过滤，灌装，加塞，轧盖。考察样品在强制降解条件下(40℃D22(40℃高温条件下放置22天))的稳定性，并以外观、SEC和非还原CE-SDS为评价指标，考察制剂稳定性。实验结果见表26。
40℃D22强制降解条件下，各组间外观无显著性差异；从纯度项数据看，80mg/mL蔗糖、80mg/mL海藻糖和50mg/mL山梨醇组制剂的SEC和非还原CE-SDS略高于其他组别，而这三组制剂间纯度项无显著性差异。当给药途径为皮下注射，制剂渗透压控制在280-320mOsm，因此，蔗糖含量优选70-90mg/mL。含80mg/mL蔗糖制剂的渗透压为300mOsm左右，蔗糖含量优选80mg/mL。
表26.制剂中渗透压筛选实验结果
Figure PCTCN2021109438-appb-000099
备注：表中“D”表示天，例如D22表示22天；D0表示实验开始时。
测试例12：抗PD-1抗体制剂蛋白浓度筛选
使用缓冲体系为pH5.2的10mM醋酸钠盐，配制含80mg/mL蔗糖，0.6mg/mL聚山梨酯80(PS80)，蛋白浓度为100mg/mL或120mg/mL的抗PD-1抗体(Hu23-11.IgG4AA)制剂。
1)100mg/mL蛋白浓度，0.6mg/mL PS80和80mg/mL蔗糖；
2)120mg/mL蛋白浓度，0.6mg/mL PS80和80mg/mL蔗糖；
将每种制剂过滤，灌装，加塞，轧盖。考察样品在强制降解条件下(40℃M1)的稳定性，并以外观、SEC、非还原CE-SDS和ICE为评价指标，考察制剂稳定性。实验结果见表27。
实验数据显示，在40℃M1强制降解条件下，100mg/mL和120mg/mL的制剂组的外观和纯度项无显著差异。
表27.不同浓度制剂对比实验结果
Figure PCTCN2021109438-appb-000100
备注：表中“D”表示天，D0表示实验开始时,“M”表示月，例如M1表示一个月。
测试例13：抗PD-1抗体制剂稳定性实验
制备含蛋白浓度120mg/mL，10mM醋酸钠盐pH5.2，80mg/mL蔗糖，0.6mg/mL PS80的抗PD-1抗体(Hu23-11.IgG4AA)制剂。
将制备好的制剂过滤，灌装，加塞，轧盖。考察样品在强制降解条件(40℃M1)、冻融5次和振摇D7(25℃，300rpm)下的稳定性，并以外观、SEC、非还原CE-SDS和IEC为评价指标，考察制剂稳定性。实验结果见表28。
实验结果显示，最终处方制剂从外观看在各强制降解条件下保持澄明；从纯度项数据看，在冻融五次或进行振摇7天(25℃，300rpm)下无显著变化，较D0 40℃M1强制降解条件下SEC下降1.9％，NR-CE下降2.5％和IEC下降14.8％，具有较好的稳定性。
表28.制剂稳定性实验结果
Figure PCTCN2021109438-appb-000101
备注：表中“D”表示天，D0表示实验开始时,“M”表示月，例如M1表示一个月。
另外，还观察了制剂在25℃M6(25℃温度条件下放置6个月)加速降解条 件下以及4℃M6(4℃温度条件下放置6个月)条件下的制剂的稳定性，实验结果见表29，实验结果显示，4℃M6长期条件下本披露的制剂具有良好的稳定性，即使在25℃M6加速降解条件下本披露的制剂仍有良好的稳定性，相比D0，制剂的SEC仅下降2.2％，非还原CE-SDS仅降2.7％，IEC下降9.3％。
表29.制剂稳定性实验结果
Figure PCTCN2021109438-appb-000102
测试例14：抗PD-1抗体制剂处方优化实验
以醋酸钠盐的离子强度、pH值和蛋白浓度为变量进行DOE实验设计，DOE实验因子及水平设为离子强度10-30mM、pH4.7-5.7、抗PD-1抗体(Hu23-11.IgG4AA)浓度90-150mg/mL，设计一系列处方(见表30)，通过40℃高温条件下放置一个月的强制降解实验，以外观、SEC、IEC为评价指标，采用最小二乘法对结果进行统计分析，结果见表31和图5。
结果显示，40℃高温条件下放置一个月，各处方外观均澄明，纯度项SEC下降幅度为1.1％-2.7％之间、iCIEF下降幅度为5.6％-8.5％之间，在强制降解条件下的下降幅度可接受范围之内，并且组间差异在仪器的检测误差范围内。因此抗PD-1抗体样品在蛋白浓度90-150mg/mL，离子强度10-30Mm和pH4.7-5.7，范围内均具有良好的稳定性。将数据进行拟合，观察到低离子强度时纯度项处于更安全的范围，因此可优选：蛋白浓度120mg/mL，离子强度10mM，pH5.2。
表30.DOE处方筛选实验处方设计
组别 pH 蛋白浓度(mg/mL) 离子强度(mM)
1 5.7 150 20
2 4.7 120 21.9
3 5.2 120 10
4 4.7 90 10
5 5.2 120 20
6 5.2 90 20
7 5.7 150 10
8 4.7 90 30
9 4.7 150 10
10 5.2 150 30
11 5.7 90 10
12 5.7 107.7 30
注：所有处方均加入80mg/mL蔗糖和0.6mg/mL PS80。
表31.DOE筛选实验结果
Figure PCTCN2021109438-appb-000103
备注：表中“D”表示天，D0表示实验开始时,“M”表示月，例如M1表示一个月；N表示本表不适用；SEC单体下降幅度(％)＝40℃M1时制剂SEC单体(％)与D0时制剂SEC单体(％)的差值；iCIEF中性峰下降幅度(％)＝40℃M1时制剂iCIEF中性峰(％)与D0时制剂iCIEF中性峰(％)的差值。
测试例15：抗PD-1抗体制剂的冻干
在10mM AA pH5.2的缓冲液中制备包含100mg/mL抗体，80mg/mL蔗糖，0.6mg/mL PS80的抗PD-1抗体(Hu23-11.IgG4AA)制剂，将制剂样品进行冻干，冻干程序为预冻、一次干燥和二次干燥(参数见表32)。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32.冻干程序
Figure PCTCN2021109438-appb-000104
Figure PCTCN2021109438-appb-000105
虽然为了清楚的理解，已经借助于附图和实例详细描述了上述发明，但是描述和实例不应当解释为限制本披露的范围。本文中引用的所有专利和科学文献的公开内容通过引用完整地清楚结合。

Claims (15)

  1. 一种药物组合物，其包含抗PD-1抗体以及缓冲液，其中，
    所述缓冲液为醋酸盐缓冲液、组氨酸盐缓冲液或琥珀酸盐缓冲液，优选地，所述缓冲液的pH为约4.5至约6.0，优选pH为约4.7至约5.7，更优选pH为约5.2；所述醋酸盐缓冲液优选为醋酸-醋酸钠缓冲液，所述组氨酸盐缓冲液优选为组氨酸-醋酸盐缓冲液，所述琥珀酸盐缓冲液优选为琥珀酸-琥珀酸钠缓冲液；
    所述抗PD-1抗体包含重链可变区和轻链可变区，所述重链可变区包含：如SEQ ID NO：65所示的HCDR1，如SEQ ID NO：66所示的HCDR2，和如SEQ ID NO：67所示的HCDR3，所述轻链可变区包含如SEQ ID NO：68所示的LCDR1，如SEQ ID NO：12所示的LCDR2，和如SEQ ID NO：69所示的LCDR3；优选地，其中所述抗PD-1抗体的重链可变区包含：如SEQ ID NO：8所示的HCDR1，如SEQ ID NO：9所示的HCDR2，和如SEQ ID NO：10所示的HCDR3，轻链可变区包含：如SEQ ID NO：49所示的LCDR1，如SEQ ID NO：12所示的LCDR2，和如SEQ ID NO：13所示的LCDR3。
  2. 根据权利要求1所述的药物组合物，其中所述缓冲液浓度为大约5mM至大约30mM，优选为大约10mM至大约30mM，更优选为大约10mM。
  3. 根据权利要求1或2所述的药物组合物，其中所述抗PD-1抗体浓度为大约1mg/mL至大约150mg/mL，优选为大约90mg/mL至大约150mg/mL，更优选为大约120mg/mL或大约100mg/mL。
  4. 根据权利要求1至3中任一项所述的药物组合物，其还包括表面活性剂，所述表面活性剂优选为聚山梨酯，更优选为聚山梨酯80；表面活性剂的浓度优选为大约0.2mg/mL至大约0.8mg/mL，更优选为大约0.6mg/mL至大约0.8mg/mL，最优选为大约0.6mg/mL。
  5. 根据权利要求1至4中任一项所述的药物组合物，其还包括渗透压调节剂，优选地，所述渗透压调节剂为糖、氨基酸和/或盐；更优选地，所述渗透压调节剂选自由蔗糖、海藻糖、山梨糖醇、精氨酸、甘氨酸和氯化钠组成的组中的一种或更多种；所述渗透压调节剂浓度优选为大约50mg/mL至大约100mg/mL，更优选为大约70mg/mL至大约90mg/mL；最优选为大约80mg/mL。
  6. 根据权利要求1至5中任一项所述的药物组合物，其中所述抗PD-1抗体的重链可变区如SEQ ID NO：27所示或与SEQ ID NO：27具有至少90％序列同一性，和轻链可变区如SEQ ID NO：55所示或与SEQ ID NO：55具有至少90％序列同一 性；优选地，所述抗PD-1抗体具有如SEQ ID NO：27所示的重链可变区，和如SEQ ID NO：55所示的轻链可变区。
  7. 根据权利要求1至6中任一项所述的药物组合物，其中所述抗PD-1抗体包含重链恒定区和/或轻链恒定区；优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体；更优选地，所述抗PD-1抗体包含如SEQ ID NO：72所示的重链恒定区和如SEQ ID NO：73所示的轻链恒定区；最优选地，所述抗PD-1抗体包含：如SEQ ID：74所示或与SEQ ID NO：74具有至少85％序列同一性的重链，和如SEQ ID：75所示或与SEQ ID NO：75具有至少85％序列同一性的轻链。
  8. 根据权利要求1至7中任一项所述的药物组合物，其包含：
    a)浓度为大约1mg/mL至大约150mg/mL的抗PD-1抗体，
    b)浓度为大约5mM至大约30mM，pH为大约4.5至约6.0的醋酸盐缓冲液，
    c)浓度为大约50mg/mL至大约100mg/mL的渗透压调节剂，所述渗透压调节剂选自蔗糖、海藻糖、山梨糖醇、精氨酸、甘氨酸和氯化钠；
    d)浓度为大约0.2mg/mL至大约0.8mg/mL的聚山梨酯；
    优选地，所述的药物组合物包含：
    A1)浓度为大约90mg/mL至大约150mg/mL的抗PD-1抗体，
    B1)浓度为大约10mM至大约30mM，pH为大约4.7至约5.7的醋酸盐缓冲液，
    C1)浓度为大约70mg/mL至大约90mg/mL的蔗糖，和
    D1)浓度为大约0.6mg/mL至大约0.8mg/mL的聚山梨酯80；
    更优选地，所述的药物组合物包含：大约10mM的pH为大约5.2的醋酸-醋酸钠缓冲液，浓度为大约120mg/mL的抗PD-1抗体，浓度为大约80mg/mL蔗糖，和浓度为大约0.6mg/mL聚山梨酯80。
  9. 一种药物组合物，其包含：pH为5.2的10mM的醋酸-醋酸钠缓冲液，浓度为120mg/mL的抗PD-1抗体，浓度为80mg/mL蔗糖，和浓度为0.6mg/mL聚山梨酯80；其中，所述抗PD-1抗体具有如SEQ ID：74所示的重链，和如SEQ ID：75所示的轻链。
  10. 制备如权利要求1至9中任一项所述的药物组合物的方法，所述方法包括将抗PD-1抗体原液经缓冲液置换的步骤。
  11. 一种含抗PD-1抗体的冻干制剂，所述冻干制剂通过将权利要求1至9中任一项所述的药物组合物经冷冻干燥获得。
  12. 一种含抗PD-1抗体的复溶溶液，所述复溶溶液通过将权利要求11所述的冻干制剂经复溶获得。
  13. 一种含抗PD-1抗体的冻干制剂，所述冻干制剂经复溶可形成如权利要求1至9中任一项所述的药物组合物。
  14. 一种制品，其包括容器，该容器中装有如权利要求1至9中任一项所述的药物组合物、如权利要求11或13所述的冻干制剂或如权利要求12所述的复溶溶液。
  15. 一种治疗或预防疾病或病症的方法，所述方法包括向受试者施用有效量的如权利要求1至9中任一项所述的药物组合物，或如权利要求11或13所述的冻干制剂，或如权利要求12所述的复溶溶液，或如权利要求14所述的制品；优选地，其中所述疾病或病症为肿瘤；更优选地，所述疾病选自：头和颈鳞状细胞癌、头和颈癌、脑癌、神经胶质瘤、多形性成胶质细胞瘤、神经母细胞瘤、中枢神经系统癌、神经内分泌肿瘤、咽喉癌、鼻咽癌、食管癌、甲状腺癌、恶性胸膜间皮瘤、肺癌、乳腺癌、肝癌、肝细胞瘤、肝胆癌、胰腺癌、胃癌、胃肠道癌、肠癌、结肠癌、结肠直肠癌、肾癌、透明细胞肾细胞癌、卵巢癌、子宫内膜癌、子宫颈癌、膀胱癌、前列腺癌、睾丸癌、皮肤癌、黑色素瘤、白血病、淋巴瘤、骨癌、软骨肉瘤、骨髓瘤、多发性骨髓瘤、骨髓异常增生综合征、骨髓增生性肿瘤、鳞状细胞癌、尤因氏肉瘤、全身性轻链淀粉样变性和梅克尔细胞癌；最优选地，所述疾病选自：PD-L1阳性的黑色素瘤、肺癌、非小细胞肺癌、乳腺癌、胃癌、肾癌、膀胱癌、肠癌和结肠癌；可选的，所述疾病为与PD-1相关的疾病。
PCT/CN2021/109438 2020-07-31 2021-07-30 抗pd-1抗体药物组合物及其用途 WO2022022660A1 (zh)

Priority Applications (9)

Application Number Priority Date Filing Date Title
US18/018,471 US20230295329A1 (en) 2020-07-31 2021-07-30 Anti-pd-1 antibody pharmaceutical composition and use thereof
BR112023001471A BR112023001471A2 (pt) 2020-07-31 2021-07-30 Composição farmacêutica de anticorpo anti-pd-1 e uso da mesma
CN202180045879.XA CN115867316A (zh) 2020-07-31 2021-07-30 抗pd-1抗体药物组合物及其用途
AU2021317805A AU2021317805A1 (en) 2020-07-31 2021-07-30 Anti-PD-1 antibody pharmaceutical composition and use thereof
EP21849923.4A EP4190353A4 (en) 2020-07-31 2021-07-30 PHARMACEUTICAL COMPOSITION OF ANTI-PD -1 ANTIBODY AND ITS USE
JP2023503241A JP2023535384A (ja) 2020-07-31 2021-07-30 抗ｐｄ－１抗体医薬組成物及びその用途
KR1020237006007A KR20230044448A (ko) 2020-07-31 2021-07-30 항-pd-1 항체 약학적 조성물 및 이의 용도
MX2023001160A MX2023001160A (es) 2020-07-31 2021-07-30 Composicion farmaceutica del anticuerpo anti-pd-1 y uso del mismo.
CA3189452A CA3189452A1 (en) 2020-07-31 2021-07-30 Anti-pd-1 antibody pharmaceutical composition and use thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202010755954 2020-07-31
CN202010755954.9 2020-07-31
CN202110831575.8 2021-07-22
CN202110831575 2021-07-22

Publications (1)

Publication Number Publication Date
WO2022022660A1 true WO2022022660A1 (zh) 2022-02-03

Family

ID=80037648

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/109438 WO2022022660A1 (zh) 2020-07-31 2021-07-30 抗pd-1抗体药物组合物及其用途

Country Status (11)

Country Link
US (1) US20230295329A1 (zh)
EP (1) EP4190353A4 (zh)
JP (1) JP2023535384A (zh)
KR (1) KR20230044448A (zh)
CN (1) CN115867316A (zh)
AU (1) AU2021317805A1 (zh)
BR (1) BR112023001471A2 (zh)
CA (1) CA3189452A1 (zh)
MX (1) MX2023001160A (zh)
TW (1) TW202214298A (zh)
WO (1) WO2022022660A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024058289A1 (ko) * 2022-09-15 2024-03-21 주식회사 와이바이오로직스 Pd-1에 특이적으로 결합하는 항체를 유효성분으로 포함하는 신경내분비 신생물의 예방 또는 치료용 약학 조성물

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20210097882A (ko) * 2020-01-30 2021-08-10 삼성바이오에피스 주식회사 안정한 항-pd-1 항체 약제학적 제제

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
WO2015085847A1 (zh) 2013-12-12 2015-06-18 上海恒瑞医药有限公司 Pd-1抗体、其抗原结合片段及其医药用途
CN106390115A (zh) * 2015-07-29 2017-02-15 上海君实生物医药科技股份有限公司 一种人源化单克隆抗体的稳定制剂
WO2017054646A1 (zh) * 2015-09-28 2017-04-06 江苏恒瑞医药股份有限公司 一种抗pd-1抗体制剂及其在医药上的应用
CN109966487A (zh) * 2017-12-28 2019-07-05 上海复宏汉霖生物制药有限公司 一种包含抗pd-l1单克隆抗体的药物配制剂
WO2019206987A1 (en) * 2018-04-25 2019-10-31 Medimmune Limited Formulations of human anti-pd-l1 antibodies
CN110787292A (zh) * 2020-01-06 2020-02-14 上海复宏汉霖生物技术股份有限公司 一种细胞程序性死亡受体1抗体制剂及其用途
CN110869002A (zh) * 2017-05-02 2020-03-06 默沙东公司 程序性死亡受体1(pd-1)抗体的稳定制剂及其使用方法
CN110974958A (zh) * 2019-12-25 2020-04-10 北京东方百泰生物科技有限公司 一种抗pd-l1单克隆抗体的注射制剂
WO2020097139A1 (en) * 2018-11-07 2020-05-14 Merck Sharp & Dohme Corp. Co-formulations of anti-lag3 antibodies and anti-pd-1 antibodies

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106699889A (zh) * 2015-11-18 2017-05-24 礼进生物医药科技（上海）有限公司 抗pd-1抗体及其治疗用途

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (zh) 1986-01-30 1990-11-27 Cetus Corp
WO2015085847A1 (zh) 2013-12-12 2015-06-18 上海恒瑞医药有限公司 Pd-1抗体、其抗原结合片段及其医药用途
CN106390115A (zh) * 2015-07-29 2017-02-15 上海君实生物医药科技股份有限公司 一种人源化单克隆抗体的稳定制剂
WO2017054646A1 (zh) * 2015-09-28 2017-04-06 江苏恒瑞医药股份有限公司 一种抗pd-1抗体制剂及其在医药上的应用
CN110869002A (zh) * 2017-05-02 2020-03-06 默沙东公司 程序性死亡受体1(pd-1)抗体的稳定制剂及其使用方法
CN109966487A (zh) * 2017-12-28 2019-07-05 上海复宏汉霖生物制药有限公司 一种包含抗pd-l1单克隆抗体的药物配制剂
WO2019206987A1 (en) * 2018-04-25 2019-10-31 Medimmune Limited Formulations of human anti-pd-l1 antibodies
WO2020097139A1 (en) * 2018-11-07 2020-05-14 Merck Sharp & Dohme Corp. Co-formulations of anti-lag3 antibodies and anti-pd-1 antibodies
CN110974958A (zh) * 2019-12-25 2020-04-10 北京东方百泰生物科技有限公司 一种抗pd-l1单克隆抗体的注射制剂
CN110787292A (zh) * 2020-01-06 2020-02-14 上海复宏汉霖生物技术股份有限公司 一种细胞程序性死亡受体1抗体制剂及其用途

Non-Patent Citations (21)

* Cited by examiner, † Cited by third party
Title
"Antibodies: A Laboratory Manual and Molecular Cloning: A Laboratory Manual", COLD SPRING HARBOR LABORATORY
AL-LAZIKANI ET AL., JMB, vol. 273, 1997, pages 927 - 948
ALTSCHUL, S.F. ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410
ALTSCHUL, S.F. ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
CHEUNG ET AL., VIROLOGY, vol. 176, 1990, pages 546 - 552
EPITOPE MAPPING PROTOCOLS IN METHODS IN MOLECULAR BIOLOGY, vol. 66, 1996
GISH, W. ET AL., NATURE GENET., vol. 3, 1993, pages 266 - 272
HUSTON ET AL., PROC. NATL. ACAD. SCI USA, vol. 85, 1988, pages 5879 - 5883
KABAT, E. A. ET AL., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 1991
KIRKLAND ET AL., J. IMMUNOL., vol. 137, 1986, pages 3614 - 3619
LEFRANC M. P., IMMUNOLOGIST, vol. 7, 1999, pages 132 - 136
LEFRANC, M. P. ET AL., DEV. COMP. IMMUNOL., vol. 27, 2003, pages 55 - 77
MADDEN, T.L. ET AL., METH. ENZYMOL., vol. 266, 1996, pages 131 - 141
MOLDENHAUER ET AL., SCAND. J. IMMUNOL., vol. 32, 1990, pages 77 - 82
MOREL ET AL., MOLEC. IMMUNOL., vol. 25, 1988, pages 7 - 15
See also references of EP4190353A4
STAHLI ET AL., METHODS IN ENZYMOLOGY, vol. 9, 1983, pages 242 - 253
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546
WATSON ET AL.: "Molecular Biology of the Gene", 1987, THE BENJAMIN/CUMMINGS PUB. CO., pages: 224
ZHANG, J. ET AL., GENOME RES., vol. 7, 1997, pages 649 - 656

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024058289A1 (ko) * 2022-09-15 2024-03-21 주식회사 와이바이오로직스 Pd-1에 특이적으로 결합하는 항체를 유효성분으로 포함하는 신경내분비 신생물의 예방 또는 치료용 약학 조성물

Also Published As

Publication number Publication date
CA3189452A1 (en) 2022-02-03
KR20230044448A (ko) 2023-04-04
CN115867316A (zh) 2023-03-28
MX2023001160A (es) 2023-02-22
TW202214298A (zh) 2022-04-16
JP2023535384A (ja) 2023-08-17
EP4190353A4 (en) 2024-02-14
US20230295329A1 (en) 2023-09-21
EP4190353A1 (en) 2023-06-07
AU2021317805A1 (en) 2023-03-16
BR112023001471A2 (pt) 2023-03-14

Similar Documents

Publication Publication Date Title
CN111744007B (zh) 一种抗tigit抗体药物组合物及其用途
TWI829799B (zh) 一種TGF-β受體融合蛋白醫藥組成物及其用途
CN110538321B (zh) 一种cd47抗体药物组合物及其用途
WO2020156509A1 (zh) 抗pd-1抗体、其抗原结合片段及医药用途
CN111356476B (zh) Lag-3抗体药物组合物及其用途
WO2022022660A1 (zh) 抗pd-1抗体药物组合物及其用途
WO2022117060A1 (zh) 一种含抗结缔组织生长因子抗体的药物组合物
WO2021190582A1 (zh) 一种抗ox40抗体药物组合物及其用途
JP2022513671A (ja) Ｃｄ４０抗体医薬品組成物およびその使用
CN113507938A (zh) 包含抗il-5抗体的药物组合物及其用途
WO2023040935A1 (zh) 一种含抗pvrig/tigit双特异性抗体的药物组合物
RU2778572C1 (ru) Фармацевтическая композиция на основе антител к cd40 и ее применение
US20230088052A1 (en) Pharmaceutical composition containing anti-il-4r antibody and use thereof
BR112021015168A2 (pt) Anticorpo anti-pd-1, fragmento de ligação ao antígeno do mesmo e uso farmacêutico do mesmo
TW202320847A (zh) 一種抗ａｎｇｐｔｌ３抗體或其抗原結合片段的醫藥組成物及其用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21849923

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3189452

Country of ref document: CA

ENP Entry into the national phase

Ref document number: 2023503241

Country of ref document: JP

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112023001471

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20237006007

Country of ref document: KR

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 2023100354

Country of ref document: RU

Ref document number: 2021849923

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2021849923

Country of ref document: EP

Effective date: 20230228

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 112023001471

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20230126

ENP Entry into the national phase

Ref document number: 2021317805

Country of ref document: AU

Date of ref document: 20210730

Kind code of ref document: A