WO2020043095A1 - 一种tim3抗体药物组合物及其用途 - Google Patents

一种tim3抗体药物组合物及其用途 Download PDF

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Publication number
WO2020043095A1
WO2020043095A1 PCT/CN2019/102839 CN2019102839W WO2020043095A1 WO 2020043095 A1 WO2020043095 A1 WO 2020043095A1 CN 2019102839 W CN2019102839 W CN 2019102839W WO 2020043095 A1 WO2020043095 A1 WO 2020043095A1
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antibody
seq
sequence
variable region
region
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PCT/CN2019/102839
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English (en)
French (fr)
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田晨敏
李皓
刘洵
曹卓晓
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Priority to AU2019330702A priority Critical patent/AU2019330702A1/en
Priority to JP2021508281A priority patent/JP2022513553A/ja
Priority to CA3110002A priority patent/CA3110002A1/en
Priority to KR1020217006876A priority patent/KR20210053294A/ko
Priority to US17/269,456 priority patent/US20210261663A1/en
Priority to CN201980049906.3A priority patent/CN112543647B/zh
Priority to EP19855752.2A priority patent/EP3848049A4/en
Publication of WO2020043095A1 publication Critical patent/WO2020043095A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • C07K16/2803—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
    • 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
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • 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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C07K2317/90—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

Definitions

  • the present disclosure belongs to the field of pharmaceutical preparations, and particularly relates to a pharmaceutical composition containing a TIM3 antibody or an antigen-binding fragment thereof, and its use as an anticancer drug.
  • T cell immunoglobulin and mucin-domain-containing molecule 3 (TIM-3), also known as hepatitis A virus cell receptor 2 (HAVCR-2), is A type I membrane surface protein molecule that belongs to the TIM family.
  • the human TIM-3 molecule consists of 301 amino acids, including a signal peptide, an Ig variable region (IgV region), a serine / threonine-rich mucin region, a transmembrane region, and a cytoplasmic region. Source 63%.
  • TIM-3 is selectively expressed in T helper cells (Th1 and Th17), T regulatory cells (Treg), dendritic cells (DCs), monocytes, mast cells, NK cells, tumor infiltrating cells that secrete IFN- ⁇ Lymphocytes (TILs) (see, eg, Clayton et al., J. Immunol., 192 (2): 782-791 (2014); Jones et al., J. Exp. Med., 205: 2763-2779 (2008)).
  • TILs IFN- ⁇ Lymphocytes
  • Currently known receptors for TIM-3 include phosphatidylserine galectin-9 (Galectin-9, Gal-9), high mobility group protein 1 (HMGB1), and carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) .
  • TIM-3 can regulate the function of the immune system in a variety of ways. Binding to the ligand Gal-9 on the surface of Th1 cells can down-regulate the response of Th1 cells, and can induce the apoptosis of Th1 cells. Lupus, asthma) and immune tolerance play an important role. TIM-3 expressed in monocytes and macrophages interacts with phosphatidylserine to promote phagocytosis of apoptotic cells. In tumor-infiltrating DCs, TIM-3 binds to HMGB1 ligand and inhibits correct nucleic acid transport, thereby inhibiting nucleic acids. Immune response, involved in immune escape.
  • TIM-3 is not only expressed in immune cells, but also overexpressed in tumor cells such as ovarian cancer, meningiomas, and melanoma, and directly promotes tumor growth. Down-regulating the expression of TIM-3 can significantly inhibit the invasion and metastasis of Hela cells. TIM-3 overexpression is closely related to the poor prognosis of lung cancer, gastric cancer, prostate cancer and cervical cancer. In hematological tumors, TIM-3 is overexpressed on leukemia stem cells in acute myeloid leukemia and hematopoietic stem cells in patients with MDS, and TIM-3 + hematopoietic stem cells have malignant biological characteristics of low differentiation, low apoptosis and high proliferation.
  • TIM-3 such as TIM-3 antibodies
  • it is expected to become a new method for treating tumors (see, eg, Ngiow et al1., Cancer Res., 71 (21): 1-5 (2011); Guo et al., Translation alpha medicine, 11: 215 (2013); and Ngiow et al., Cancer Res., 71 (21): 6567-6571 (2011)).
  • the stability of antibody drugs is one of the key factors affecting the drugability of antibodies.
  • the non-enzymatic deamidation of asparagine (Asn) and glutamine (Gln) can cause antibody instability and heterogeneity.
  • Asn asparagine
  • Gln glutamine
  • Huang et al. Found that the deamidation of Asn55 in the heavy chain variable region 2 (CDR2) of the IgG1 monoclonal antibody significantly reduced its binding activity. Therefore, in the process of antibody development, try to avoid or mutate the corresponding amino acids to reduce the occurrence of deamidation of the antibody to increase the stability and bioavailability of the antibody.
  • the present disclosure provides a pharmaceutical composition comprising a TIM3 antibody or an antigen-binding fragment thereof which is more convenient for production and administration and has stable performance.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising a TIM3 antibody or an antigen-binding fragment thereof, and a buffering agent selected from the group consisting of acetate, histidine, succinate, phosphate, and citrate buffers.
  • the agent is preferably an acetate buffer or a histidine buffer.
  • the acetate buffer in the pharmaceutical composition is selected from the group consisting of acetic acid-sodium acetate buffer
  • the histidine buffer is selected from the group consisting of histidine-acetate buffer or histidine- Hydrochloric acid buffer
  • the succinate buffer is selected from succinate-sodium succinate buffer
  • the phosphate buffer is selected from disodium hydrogen phosphate-sodium dihydrogen phosphate buffer
  • the citrate buffer The agent is selected from citrate-sodium citrate; preferably histidine-hydrochloric acid, acetic acid-sodium acetate or a histidine-acetate buffer, most preferably a histidine-acetate buffer.
  • the concentration of the buffer in the pharmaceutical composition is about 5 mM to 30 mM, preferably about 10 mM to 30 mM, preferably about 5 mM to 20 mM, preferably about 10 mM to 20 mM; non-limiting examples include 10 mM, 12 mM , 14 mM, 16 mM, 18 mM, 20 mM, 30 mM, and most preferably about 10 mM.
  • the concentration of the TIM3 antibody or antigen-binding fragment thereof in the pharmaceutical composition is about 1 mg / ml to 100 mg / ml, preferably about 40 mg / ml to 60 mg / ml, preferably 50 mg / ml to 60 mg / ml ;
  • Non-limiting examples include 1mg / ml, 10mg / ml, 20mg / ml, 30mg / ml, 40mg / ml, 41mg / ml, 42mg / ml, 43mg / ml, 44mg / ml, 45mg / ml, 46mg / ml , 47mg / ml, 48mg / ml, 49mg / ml, 50mg / ml, 51mg / ml, 52mg / ml, 53mg / ml, 54mg / /
  • the pH of the buffering agent in the pharmaceutical composition is about 5.0 to 6.5, preferably about 5.0 to 6.0, preferably about 5.2 to 5.8, or preferably about 5.5 to 6.0, most preferably 5.5, non- Restricted examples include 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 about 6.5.
  • the aforementioned pharmaceutical composition further comprises a sugar.
  • sucgar includes conventional composition (CH 2 O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars and the like.
  • the preferred sugar is a non-reducing disaccharide, more preferably trehalose or sucrose, and most preferably sucrose.
  • the sugar concentration in the aforementioned pharmaceutical composition is about 50 mg / ml to about 100 mg / ml, preferably about 60 mg / ml to about 90 mg / ml, preferably about 70 mg / ml to about 90 mg / ml, preferably 70mg / ml to about 80mg / ml, non-limiting examples include 50mg / ml, 60mg / ml, 65mg / ml, 70mg / ml, 75mg / ml, 80mg / ml, 85mg / ml, 90mg / ml, most preferably 80mg / ml.
  • the pharmaceutical composition further comprises a surfactant.
  • a surfactant Can be selected from polysorbate 20, polysorbate 80, polyhydroxyalkylene, Triton, sodium dodecylsulfonate, sodium laurylsulfonate, sodium octylglycoside, lauryl / myristyl / sub Oil-based / stearyl-sulfobetaine, lauryl / myristoyl / linoleyl / stearyl-sarcosine, linoleyl / myristyl / cetyl-betaine, lauryl propyl / Cocaramidopropyl / linoleamidopropyl / myristoylaminopropyl / palmitamidopropyl / isostearylamidopropyl-betaine, myristylpropyl / palmitamide Propyl / isostearylamidopropyl-di
  • the concentration of the surfactant in the pharmaceutical composition is about 0.2 mg / ml to 0.8 mg / ml, preferably 0.2 mg / ml to 0.6 mg / ml, and more preferably 0.4 mg / ml to 0.6 mg / ml.
  • ml non-limiting examples include 0.2mg / ml, 0.3mg / ml, 0.4mg / ml, 0.45mg / ml, 0.5mg / ml, 0.55mg / ml, 0.6mg / ml, 0.7mg / ml, 0.8mg / ml, most preferably 0.4 mg / ml.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • a TIM-3 antibody or antigen-binding fragment described in the pharmaceutical composition of the present disclosure is characterized in that it binds to the extracellular region of human TIM-3, and it is selected from the group consisting of the following (i) Any one of the monoclonal antibodies or antigen-binding fragments to (ii) competes for binding to TIM-3, or is selected from any one of the following (i) to (ii) monoclonal antibodies or antigen-binding fragments thereof:
  • a monoclonal antibody or an antigen-binding fragment thereof comprising one or more CDR region sequences selected from the following or an amino acid sequence having at least 95% sequence identity therewith:
  • Antibody heavy chain variable region HCDR region sequence as shown in SEQ ID NO: 8, 43 and 10 amino acid sequence; and / or antibody light chain variable region LCDR region sequence: as SEQ ID NO: 11, 12 and 13 amino acid sequence Shown
  • a monoclonal antibody or an antigen-binding fragment thereof comprising one or more CDR region sequences selected from the following or an amino acid sequence having at least 95% sequence identity therewith:
  • Antibody heavy chain variable region HCDR region sequence as shown in SEQ ID NO: 14, 15 and 16 amino acid sequence; and / or antibody light chain variable region LCDR region sequence: as SEQ ID NO: 17, 18 and 19 amino acid sequence As shown.
  • TIM-3 antibody or antigen-binding fragment thereof is selected from any one of the monoclonal antibodies or antigen-binding fragments thereof described in (i) or (ii) below:
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of the variable region of the antibody heavy chain as shown in SEQ ID NOs: 8, 43 and 10, respectively, or an amino acid having at least 95% sequence identity therewith.
  • Said SEQ ID NO: 43 is shown in the sequence of DIIPX 1 X 2 X 3 GSKYNQKFKD, wherein X 1 is selected from N, L, V, M or E, and X 2 is selected from N, E, M, H, K , L, A or V, and X 3 is selected from G or A;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of an antibody heavy chain variable region shown in SEQ ID NOs: 14, 15 and 16, respectively, or an amino acid having at least 95% sequence identity therewith; Sequence, and / or the sequence of the antibody light chain variable region LCDR1-3 region shown in SEQ ID NOs: 17, 18, and 19, respectively, or an amino acid sequence having at least 95% sequence identity therewith.
  • the above amino acid sequence having at least 95% sequence identity preferably, it has at least 95%, 96%, 97%, 98%, or 99% sequence identity, and more preferably, it has 97%, 98%, or 99% % Or more, most preferably having at least 99% sequence identity, and the aforementioned amino acid sequence having at least 95% sequence identity includes one or more amino acid deletions, insertions or substitution mutations.
  • the monoclonal antibody or antigen-binding fragment thereof comprises an antibody heavy chain variable region HCDR1-3 region sequence as shown in SEQ ID NO: 8, 43, and 10, respectively, and SEQ ID NO : The sequences of the LCDR1-3 region of the antibody light chain variable region shown in 11, 12 and 13;
  • the TIM-3 antibody or antigen-binding fragment described in the pharmaceutical composition of the present disclosure competes with any of the monoclonal antibodies or antigen-binding fragments of (a) to (m) below for binding to TIM- 3, or any one of the monoclonal antibodies or antigen-binding fragments thereof selected from (a) to (m):
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of the variable region of the antibody heavy chain as shown in SEQ ID NO: 8, 9, and 10, and SEQ ID NO: 11, 12 respectively And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequences of the HCDR1-3 region of the variable region of the antibody heavy chain as shown in SEQ ID NO: 8, 62, and 10, and SEQ ID NO: 11, 12 respectively; And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of the variable region of the antibody heavy chain as shown in SEQ ID NO: 8, 63, and 10, and SEQ ID NO: 11, 12 respectively And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequences of the HCDR1-3 region of the variable region of the antibody heavy chain as shown in SEQ ID NO: 8, 64, and 10, and SEQ ID NO: 11, 12 respectively And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of the variable region of the antibody heavy chain as shown in SEQ ID NO: 8, 65, and 10, and SEQ ID NO: 11, 12 respectively. And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of the variable region of the antibody heavy chain as shown in SEQ ID NO: 8, 66, and 10, and SEQ ID NO: 11, 12 respectively; And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of the variable region of the antibody heavy chain as shown in SEQ ID NO: 8, 67, and 10, and SEQ ID NO: 11, 12 respectively And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of the variable region of the antibody heavy chain as shown in SEQ ID NOs: 8, 68, and 10, and SEQ ID NO: 11, 12 respectively And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of the variable region of the heavy chain of the antibody as shown in SEQ ID NO: 8, 69, and 10, and SEQ ID NO: 11, 12 respectively And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of the variable region of the antibody heavy chain shown in SEQ ID NO: 8, 70, and 10, and SEQ ID NO: 11, 12 respectively And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • (k) A monoclonal antibody or an antigen-binding fragment thereof comprising the sequences of the HCDR1-3 region of the variable region of the antibody heavy chain shown in SEQ ID NOs: 8, 71, and 10, respectively, and SEQ ID NO: 11, 12 respectively And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • a monoclonal antibody or an antigen-binding fragment thereof comprising the sequence of the HCDR1-3 region of the variable region of the antibody heavy chain as shown in SEQ ID NO: 8, 72, and 10, and SEQ ID NO: 11, 12 respectively And the sequence of the LCDR1-3 region of the antibody light chain variable region shown in 13 and 13;
  • the TIM-3 antibody or antigen-binding fragment described in the pharmaceutical composition of the present disclosure is characterized in that it binds to the same epitope as any one of the aforementioned monoclonal antibodies.
  • the pharmaceutical composition of the present disclosure wherein said TIM-3 antibody or antigen-binding fragment, wherein said antibody is a recombinant antibody.
  • the TIM-3 antibody or antigen-binding fragment thereof according to the present disclosure, wherein the monoclonal antibody is selected from the group consisting of a murine antibody, a chimeric antibody, a recombinant antibody of a humanized antibody, or an antigen-binding fragment thereof. .
  • the pharmaceutical composition of the present disclosure wherein the TIM-3 antibody or antigen-binding fragment comprises a heavy chain variable region sequence as shown in SEQ ID NO: 4 and / or as shown in SEQ ID NO: 5
  • the light chain variable region sequence shown in FIG. 1 may include the heavy chain variable region sequence shown in SEQ ID NO: 6 and / or the light chain variable region sequence shown in SEQ ID NO: 7.
  • the pharmaceutical composition of the present disclosure wherein said TIM-3 antibody or antigen-binding fragment, wherein said humanized antibody light chain and heavy chain FR region on the light and heavy chain variable regions
  • the sequences are derived from the human germline light and heavy chains or their mutant sequences, respectively.
  • the pharmaceutical composition of the present disclosure wherein said TIM-3 antibody or antigen-binding fragment, wherein said humanized antibody contains the heavy chain variable region shown in SEQ ID NO: 20 or 31 or A variant thereof; the variant is a sequence having a 1-10 amino acid change on the variable region of the heavy chain shown in SEQ ID NO: 20 or 31.
  • the TIM-3 antibody or antigen-binding fragment thereof wherein the variant has a 1 at the FR region position of the heavy chain variable region shown in SEQ ID NO: 20 or 31 -10 amino acid back mutation; preferably, the back mutation is selected from the group consisting of D89E, R98T, G49A, M48I, M70L, R38K, and V68A in the heavy chain variable region shown in SEQ ID NO: 20 or Multiple amino acid back mutations or Q3K and / or R87K amino acid back mutations in the heavy chain variable region shown in SEQ ID NO: 31.
  • the TIM-3 antibody or antigen-binding fragment thereof according to the present disclosure wherein the CDR region is susceptible to chemical modification sites Amino acid substitutions can be made, preferably NNG in CDR2 is replaced; preferably, the TIM-3 antibody or antigen-binding fragment thereof according to the present disclosure, wherein the humanized antibody comprises a member selected from SEQ ID NO: 45, 45, The heavy chain variable region shown in any one of 46, 47, 48, 49 and 50.
  • the TIM-3 antibody or antigen-binding fragment thereof according to the present disclosure wherein the humanized antibody comprises a heavy chain variable region as shown in SEQ ID NO: 34 or 35.
  • the TIM-3 antibody or antigen-binding fragment thereof according to the present disclosure, wherein the humanized antibody contains a light chain variable region or a variant thereof as shown in SEQ ID NO: 21 or 32;
  • the variant is a sequence having a 1-10 amino acid change on the variable region of the light chain shown in SEQ ID NO: 21 or 32.
  • the TIM-3 antibody or antigen-binding fragment thereof comprises a FR region of a light chain variable region as shown in SEQ ID NO: 21 or 32, or a variant thereof, and the variant is described in SEQ ID
  • the light chain variable region shown by NO: 21 or 32 has a back mutation of 1-10 amino acids; preferably, the back mutation is selected from a back mutation at the A43S site shown in SEQ ID NO: 21, or Back mutation was performed at at least one of Q3K, I48V, K45Q, A43S and T85S shown in SEQ ID NO: 32.
  • the TIM-3 antibody or antigen-binding fragment thereof according to the present disclosure wherein the humanized antibody comprises a light chain variable region selected from SEQ ID NO: 30 or a sequence selected from SEQ ID NO: The light chain variable region shown in any one of 37, 37, 38, 39, and 40.
  • the TIM-3 antibody or antigen-binding fragment thereof described in the present disclosure wherein the site where the CDR region is susceptible to chemical modification may be Amino acid substitution is performed, preferably NNG in CDR2 is substituted to form a CDR2 sequence as shown in SEQ ID NO: 43; in some embodiments, the TIM-3 antibody or antigen-binding fragment thereof according to the present disclosure, wherein
  • the humanized antibody comprises a heavy chain variable region sequence selected from SEQ ID NO: 44, 45, 46, 47, 48, 49 and 50 and a light chain variable selected from SEQ ID NO: 29 or 30 Zone sequence.
  • the TIM-3 antibody or antigen-binding fragment thereof of the present disclosure wherein the humanized antibody comprises any one selected from SEQ ID NOs: 22, 23, 24, 25, 26, 27, and 28 A shown heavy chain variable region sequence and a light chain variable region sequence selected from SEQ ID NO: 29 or 30, preferably selected from the heavy chain variable region sequence shown by SEQ ID NO: 24 and selected from The light chain variable region sequence shown in SEQ ID NO: 29.
  • the TIM-3 antibody or antigen-binding fragment thereof wherein the humanized antibody comprises a heavy chain variable selected from the group consisting of any one of SEQ ID NOs: 33, 34, and 35
  • the region sequence and the light chain variable region sequence selected from any one of SEQ ID NO: 36, 37, 38, 39, and 40 are preferably selected from the heavy chain variable region sequence and selected from SEQ ID NO: 33. From the light chain variable region sequence shown in SEQ ID NO: 36.
  • the TIM-3 antibody or antigen-binding fragment thereof according to the present disclosure wherein the easily deamidated site of the CDR region can be subjected to amino acid Substitution, preferably NNG in CDR2 is replaced; in a preferred embodiment, the TIM-3 antibody or antigen-binding fragment thereof according to the present disclosure, wherein the humanized antibody comprises: selected from SEQ ID NO: 51, The heavy chain variable region sequence shown in any one of 52, 53, 54, 55, 56, 57, 58, 59, 60, and 61 and the light chain variable region sequence selected from SEQ ID NO: 29 or 30 , Preferably selected from the heavy chain variable region sequence shown in SEQ ID NO: 51 and the light chain variable region sequence shown in SEQ ID NO: 29; or selected from the heavy chain shown in SEQ ID NO: 52 A variable region sequence and a light chain variable region sequence selected from the group consisting of SEQ ID NO: 29.
  • the TIM-3 antibody or antigen-binding fragment thereof according to the present disclosure, wherein the antibody is a full-length antibody, further comprising a human antibody constant region, preferably as shown in SEQ ID NO: 41 or having 85% with it Sequence of the human heavy chain constant region sequence, and preferably the human light chain constant region sequence shown in SEQ ID NO: 42 or having 85% sequence identity therewith.
  • amino acid sequence having at least 85% sequence identity preferably, it has at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, or 99% sequence identity, more preferably, it has 90%, 95%, or 99% or more, most preferably has at least 95% sequence identity, and the above has at least 85% sequence identity
  • Amino acid sequences include those obtained by mutations in one or more amino acid deletions, insertions, or substitutions.
  • the antibody comprises a full-length sequence of a heavy chain as shown in SEQ ID NO: 73 and a full-length sequence of a light chain as shown in SEQ ID NO: 74.
  • the TIM-3 antibody or antigen-binding fragment thereof wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab') 2, and single-chain antibody (scFv) , A dimerized V region (diabody), a disulfide stabilized V region (dsFv), and an antigen-binding fragment of a peptide containing a CDR region.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab ', F (ab') 2, and single-chain antibody (scFv) , A dimerized V region (diabody), a disulfide stabilized V region (dsFv), and an antigen-binding fragment of a peptide containing a CDR region.
  • the present disclosure also provides a method for preparing the aforementioned pharmaceutical composition, which includes the step of replacing the TIM3 antibody stock solution with a buffer solution.
  • the buffer is preferably an acetate buffer or a histidine buffer, more preferably a histidine-hydrochloric acid, an acetic acid-sodium acetate, or a histidine-acetate buffer, and most preferably a histidine -Acetate buffer.
  • the buffer concentration is about 5 mM to 30 mM, preferably about 5 mM to 20 mM, preferably 10 mM to 30 mM, non-limiting examples include 5 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 30 mM , Most preferably about 10 mM.
  • the pH of the buffer is about 5.0 to 6.5, preferably about 5.0 to 6.0, preferably about 5.2 to 5.8, and most preferably 5.5. Non-limiting examples include 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, and about 6.0.
  • the present disclosure also provides a method for preparing the foregoing pharmaceutical composition, and further includes the steps of adding sugar and a surfactant to the solution obtained in the replacement step, and then volume-adjusting with a buffer solution.
  • the preferred sugar is a non-reducing disaccharide, more preferably trehalose or sucrose, most preferably sucrose, and the sugar concentration is from about 50 mg / ml to about 100 mg / ml, more preferably from about 60 mg / ml to About 90 mg / ml, more preferably 70 mg / ml to about 80 mg / ml, non-limiting examples include 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml , Most preferably 80 mg / ml.
  • the surfactant is preferably polysorbate 80 or polysorbate 20, more preferably polysorbate 80; the concentration is about 0.2 mg / ml to 0.8 mg / ml, and more preferably 0.4 mg / ml to 0.6 mg / ml, non-limiting examples include 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.45 mg / ml, 0.5 mg / ml, 0.55 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, and most preferably 0.4 mg / ml.
  • the present disclosure also provides a method for preparing a lyophilized preparation comprising the TIM3 antibody, which comprises a step of freeze-drying the aforementioned pharmaceutical composition.
  • the pharmaceutical composition of the present disclosure comprises: 50 mg / ml of an anti-TIM3 antibody h1799-005, and 10 mM histidine-acetic acid, pH 5.5.
  • the pharmaceutical composition of the present disclosure comprises: 50 mg / ml of an anti-TIM3 antibody h1799-005, and 10 mM histidine-acetic acid, pH 6.0.
  • the pharmaceutical composition of the present disclosure comprises: 50 mg / ml of an anti-TIM3 antibody h1799-005, 10 mM histidine-hydrochloric acid, pH 6.0.
  • the pharmaceutical composition of the present disclosure comprises: 50 mg / ml anti-TIM3 antibody h1799-005, 10 mM acetic acid-sodium acetate, pH 5.5.
  • the pharmaceutical composition of the present disclosure comprises: 50 mg / ml anti-TIM3 antibody h1799-005, 10 mM histidine-hydrochloric acid, pH 5.5.
  • the pharmaceutical composition according to the present disclosure comprises: 50 mg / ml anti-TIM3 antibody h1799-005, 10 mM histidine-acetic acid pH 6.0 and 70 mg / ml sucrose.
  • the pharmaceutical composition according to the present disclosure comprises: 50 mg / ml of an anti-TIM3 antibody h1799-005, 10 mM histidine-acetate pH 6.0 and 70 mg / ml ⁇ , ⁇ -trehalose dihydrate.
  • the pharmaceutical composition of the present disclosure comprises: 50 mg / ml anti-TIM3 antibody h1799-005, 10 mM histidine-acetic acid pH 5.5, 70 mg / ml sucrose, and 0.4 mg / ml polysorbate 80.
  • the pharmaceutical composition of the present disclosure comprises: 50 mg / ml anti-TIM3 antibody h1799-005, 10 mM histidine-acetic acid pH 5.5, 70 mg / ml sucrose, and 0.2 mg / ml polysorbate 80.
  • the pharmaceutical composition of the present disclosure comprises: 50 mg / ml anti-TIM3 antibody h1799-005, 10 mM histidine-acetic acid pH 5.5, 70 mg / ml sucrose, and 0.6 mg / ml polysorbate 80.
  • the pharmaceutical composition of the present disclosure comprises: 50 mg / ml anti-TIM3 antibody h1799-005, 10 mM histidine-acetic acid pH 5.5, 70 mg / ml sucrose, and 0.4 mg / ml polysorbate 20.
  • the pharmaceutical composition described in the present disclosure comprises: 80 mg / ml sucrose, 0.4 mg / ml polysorbate 80, 20 mM histidine-acetic acid pH 5.5 and 60 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose, 0.4 mg / ml polysorbate 80, 10 mM histidine-acetic acid pH 5.5, and 50 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 20 mM histidine-acetic acid pH 5.5 and 50 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 10 mM histidine-acetic acid pH 6.0 and 40 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 30 mM histidine-acetic acid pH 5.5, and 50 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 20 mM histidine-acetic acid pH 6.0 and 50 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 30 mM histidine-acetic acid pH 5.0 and 60 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition described in the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 10 mM histidine-acetic acid pH 6.0 and 60 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition described in the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 20 mM histidine-acetic acid pH 5.0 and 50 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 30 mM histidine-acetic acid pH 6.0, and 60 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 20 mM histidine-acetic acid pH 5.5 and 40 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition described in the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 30 mM histidine-acetic acid pH 6.0 and 40 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 30 mM histidine-acetic acid pH 5.0, and 40 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 10 mM histidine-acetic acid pH 5.0 and 60 mg / ml TIM3 antibody h1799-005.
  • the pharmaceutical composition of the present disclosure comprises: 80 mg / ml sucrose and 0.4 mg / ml polysorbate 80, 10 mM histidine-acetic acid pH 5.0, and 40 mg / ml TIM3 antibody h1799-005.
  • the method of preparing a lyophilized formulation comprising a TIM3 antibody comprises the steps of prefreezing, primary drying, and secondary drying in that order. Freeze-drying is performed by freezing the formulation and subsequently 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 a suitable pressure, typically in the range of about 50-250 millitorr, typically, the storage temperature range for a primary drying is about -30 to 25 ° C (assuming the product remains frozen during the primary drying process).
  • the size and type of the formulation, the container (eg, a glass vial) that holds the sample, and the volume of the liquid determine the time required for drying, which can range from a few hours to a few days (eg, 40-60 hours).
  • the secondary drying stage can be performed at about 0-40 ° C, depending on the type and size of the container and the type of protein used.
  • the secondary drying time is determined by the desired residual humidity level in the product and typically requires at least about 5 hours.
  • the lyophilized formulation has a water content of less than about 5%, preferably less than about 3%.
  • the pressure may be the same as that applied in the primary drying step, and preferably, the secondary drying pressure is lower than the primary drying. Freeze-drying conditions can vary with formulation and vial size.
  • the present disclosure also provides a TIM3 antibody-containing lyophilized preparation prepared by the aforementioned method for preparing a TIM3 antibody-containing lyophilized preparation.
  • the lyophilized formulation is stable at 2-8 ° C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40 ° C for at least 7 days, at least 14 days, or at least 28 days.
  • the present disclosure also provides a method for preparing a reconstituted solution of a lyophilized preparation containing the TIM3 antibody, which comprises a step of reconstituting the aforementioned lyophilized preparation, and the solution used for the reconstitution is selected from, but not limited to, water for injection, physiological saline, or glucose solution .
  • the present disclosure also provides a reconstituted solution of a lyophilized preparation containing a TIM3 antibody prepared by the aforementioned method for reconstituted a lyophilized preparation containing a TIM3 antibody.
  • the present disclosure further provides an article of manufacture or kit comprising a container containing any stable pharmaceutical composition described herein.
  • the container is a neutral borosilicate glass controlled injection vial.
  • the present disclosure also provides an article comprising a container containing the aforementioned pharmaceutical composition or a lyophilized preparation or a reconstituted solution of a lyophilized preparation.
  • the present disclosure also provides the use of the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution or preparation of the lyophilized preparation in the preparation of a diagnostic agent for a disease associated with human TIM-3 positive cells.
  • the present disclosure also provides the use of the aforementioned pharmaceutical composition or lyophilized preparation or reconstituted solution or preparation of the lyophilized preparation for the preparation of a disease associated with human TIM-3 positive cells.
  • the disease related to TIM-3 positive cells is not limited as long as it is a disease related to cells expressing TIM-3, including but not limited to cancer, autoimmune diseases, and allergic diseases.
  • the present disclosure also provides a method for treating or diagnosing cancer, autoimmune diseases, and allergic diseases, comprising administering to a subject a pharmaceutical composition or a lyophilized preparation or a reconstituted solution or preparation of the lyophilized preparation.
  • the cancer includes blood cancer, breast cancer, uterine cancer, colorectal cancer, esophageal cancer, gastric cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small intestine cancer, prostate cancer, and pancreatic cancer.
  • cancer include blood cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer and prostate cancer.
  • Examples of blood cancers include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), multiple myeloma, cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), other lymphocytic leukemia, NK cell lymphoma, Hodgkin's lymphoma, non-Hodgkin Chicking's lymphoma such as Burkitt's lymphoma and the like.
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • MDS myelodysplastic syndrome
  • multiple myeloma cutaneous T-cell lymphoma
  • CTCL cutaneous T-cell lymphoma
  • PTCL peripheral T-cell lymphoma
  • ALCL anaplastic large cell
  • autoimmune diseases include rheumatoid arthritis, psoriasis, Crohn's disease, sclerosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, myocarditis, Sjogren syndrome, autoimmune hemolysis after transplant rejection Anemia, vesicular pemphigoid, Graves' disease, Hashimoto's thyroiditis, systemic lupus erythematosus (SLE), myasthenia gravis, pemphigus, malignant anemia, etc.
  • allergic diseases include acute or chronic reactive airway disease, bronchial asthma, atopic dermatitis, allergic rhinitis, urticaria, PIE syndrome, food allergies, hay fever, allergic nose, bronchial asthma, atopic Dermatitis, anaphylactic shock.
  • the present disclosure also provides the use of the aforementioned pharmaceutical composition or a lyophilized preparation or a reconstituted solution of a lyophilized preparation in the manufacture of a medicament for treating, inhibiting a disease or condition of tumor cell proliferation or metastasis.
  • the present disclosure further provides a method for diagnosing or treating a disease associated with human TIM-3 positive cells, the method comprising using the aforementioned pharmaceutical composition or a lyophilized preparation or a reconstituted solution of a lyophilized preparation.
  • Figure 1 Cell binding experiment curve of candidate TIM-3 antibody. The data show that the candidate antibodies mAb-1701 and mAb-1799 have strong binding activity to cells expressing the antigen;
  • Figures 2A-2C Effects of candidate antibodies and antigen-binding fragments thereof on IFN ⁇ secretion in a mixed lymphocyte reaction experiment;
  • Figure 2B h1701 Effect of -005 and its antigen-binding fragment on IFN ⁇ secretion;
  • the results show that h1701-009 and h1799-005 can effectively stimulate the secretion of IFN ⁇ ;
  • Figures 3A to 3D PBMC activation experiment results;
  • Figures 3A-3D show the FACS analysis of the proportion of IFN ⁇ positive cells, the geometric mean of IFN ⁇ on all cells, the ratio of IFN ⁇ and TIM-3 double positive cells (TIM-3 + IFN ⁇ +%), The geometric mean value of IFN ⁇ on TIM-3 + cells; the results show that h1701-009, h1799-005, and AbTIM-3 all have an effect on the expression of IFN ⁇ in activated PBMC cells, all of which increase the intracellular IFN ⁇ positive to varying degrees Percentage of cells and expression of IFN ⁇ ;
  • Figures 4A to 4B Results of SEB stimulation experiments.
  • Figures 4A and 4B are ELISA results of IL12 and IFN ⁇ , respectively. The results show that after 5 days of SEB stimulation of PBMCs, h1701-009, h1799-005, and AbTIM-3 were effectively increased. SEB-induced secretion of IL12 and IFN ⁇ ;
  • FIG. 5 Fitting results of the software model of the present disclosure by JMP of TIM3 antibody formulation components.
  • Buffering agent refers to a buffering agent that tolerates pH changes through the action of its acid-base conjugate component.
  • Examples of the buffer to control the pH in a suitable range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate Salts, glycylglycine and other organic acid buffers.
  • a “histidine buffer” is a buffer containing histidine ions.
  • histidine buffers include buffers such as histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, and the like, preferably histidine-acetate Buffer or histidine-hydrochloride buffer, histidine-acetate buffer is prepared by histidine and acetic acid, and histidine buffer is made by histidine and hydrochloric acid.
  • a “citrate buffer” is a buffer that includes citrate ions.
  • the citrate buffer include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, citrate-magnesium citrate, and the like.
  • a preferred citrate buffer is citrate-sodium citrate.
  • succinate buffer is a buffer that includes succinate ions.
  • succinate buffer examples include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, and the like.
  • a preferred succinate buffer is succinate-sodium succinate.
  • a “phosphate buffer” is a buffer that includes phosphate ions.
  • the phosphate buffer include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, and the like.
  • a preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.
  • acetate buffer is a buffer that includes acetate ions.
  • examples of the acetate buffer include acetate-sodium acetate, histidine acetate, acetate-potassium acetate, calcium acetate, acetate-magnesium acetate, and the like.
  • a preferred acetate buffer is acetate-sodium acetate.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein or a physiological / pharmaceutically acceptable salt or prodrug thereof with other chemical components, such as physiological / pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to maintain the stability of the active ingredient of the antibody, to promote the administration to the organism, and to facilitate the absorption of the active ingredient and thereby exert the biological activity.
  • composition and “formulation” are not mutually exclusive.
  • the solvents therein are all water.
  • “Lyophilized preparation” means a pharmaceutical composition or a liquid or solution preparation obtained after a vacuum freeze-drying step is performed on the liquid or solution preparation.
  • the terms “about” and “approximately” are index values that are within an acceptable error range for specific values determined by one of ordinary skill in the art, which values depend in part on how to measure or determine (ie, the limits of the measurement system). For example, “about” in each practice in the art may mean within or exceeding a standard deviation of one. Alternatively, “about” or “substantially containing” may mean a range of a specific numerical value ⁇ 20% shown thereafter. Furthermore, particularly for biological systems or processes, the term may mean at most one order of magnitude or up to five times the value. Unless stated otherwise, when a specific value appears in this application and the claims, the meaning of "about” or “substantially containing” should be assumed to be within an acceptable error range for that specific value.
  • the pharmaceutical composition according to the present disclosure can achieve a stable effect: the pharmaceutical composition in which the antibody substantially retains its physical stability and / or chemical stability and / or biological activity after storage, preferably, the drug
  • the composition substantially retains its physical and chemical stability and its biological activity after storage.
  • the shelf life is generally selected based on the intended shelf life of the pharmaceutical composition. There are currently various analytical techniques for measuring protein stability, which can measure the stability after storage at a selected temperature for a selected period of time.
  • Stable pharmaceutical antibody formulations are formulations in which no significant change is observed when stored at refrigerated temperature (2-8 ° C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably as much as 2 years.
  • stable liquid formulations include liquid formulations that exhibit desired characteristics after storage at a temperature of 25 ° C. for a period including 1 month, 3 months, and 6 months.
  • Typical acceptable criteria for stability are as follows: As measured by SEC-HPLC, typically no more than about 10%, preferably no more than about 5%, of the antibody monomer is degraded. By visual analysis, the drug antibody preparation was a pale yellow, nearly colorless, clear liquid or colorless, or clear to slightly milky white.
  • the concentration, pH, and osmolality of the formulations have variations not exceeding ⁇ 10%.
  • Aggregates generally form no more than about 10%, preferably no more than about 5%.
  • the antibody does not show a significant increase in aggregation, precipitation and / or denaturation , Then the antibody "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be evaluated by fluorescence spectroscopy (which determines the tertiary structure of the protein) and by FTIR spectroscopy (which determines the secondary structure of the protein).
  • the antibody If the antibody does not show significant chemical changes, the antibody "retains its chemical stability" in the pharmaceutical formulation. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of proteins include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (by methods such as peptide mapping combined with mass spectrometry or MALDI / TOF / MS, etc.) Method to evaluate), 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, (E.g., peptide mapping).
  • hydrolysis or truncation evaluationated by methods such as size exclusion chromatography and SDS-PAGE
  • oxidation by methods such as peptide mapping combined with mass spectrometry or MALDI
  • An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of the biological activity exhibited in the preparation of the pharmaceutical formulation.
  • the biological activity of an antibody can be determined, for example, by an antigen binding assay.
  • the “antibody” in the present disclosure refers to an immunoglobulin, which is a tetrapeptide chain structure formed by connecting two identical heavy chains and two identical light chains through interchain disulfide bonds.
  • the antibody light chain according to the present disclosure may further include a light chain constant region, and the light chain constant region includes a human or murine ⁇ , ⁇ chain, or a variant thereof.
  • the antibody heavy chain according to the present disclosure may further include a heavy chain constant region, and the heavy chain constant region includes human or murine IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the sequence of about 110 amino acids near the N-terminus of the heavy and light chains of the antibody varies greatly and is a variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four backbone regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of an antibody, also known as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) are composed of three CDR regions and four FR regions.
  • the sequence from the amino terminal to the carboxy terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and HCVR region of the antibody or antigen-binding fragment according to the present disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR1-3) in number and position.
  • the antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, and preferably humanized antibodies.
  • the "antibody or antigen-binding" or “functional fragment” described in the present disclosure refers to a Fab fragment, a Fab 'fragment, an F (ab') 2 fragment, and an Fv fragment scFv fragment that binds an antibody.
  • the Fv fragment contains the variable region of the heavy chain and light chain of the antibody, but has no constant region and has the smallest antibody fragment with all antigen-binding sites.
  • Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding.
  • the variable regions of two antibodies can also be linked into a single polypeptide chain with different linkers, called single chain antibodies (single chain antibodies) or single chain Fv (sFv).
  • antigen-binding site refers to a three-dimensional spatial site on the antigen that is continuous or discontinuous and is recognized by the antibodies or antigen-binding fragments of the present disclosure.
  • murine antibody in this disclosure is a monoclonal antibody to human TIM3 prepared according to the knowledge and skill in the art. Test subjects are injected with TIM3 antigen during preparation, and then hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • chimeric antibody is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, and can reduce the immune response response induced by the murine antibody.
  • To establish 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 according to the need, The gene is linked to the human constant region gene to form a chimeric gene and inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the TIM3 chimeric antibody further comprises a light chain constant region of a human-derived ⁇ , ⁇ chain, or a variant thereof.
  • the heavy chain of the TIM3 chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof.
  • the constant region of a human antibody may be selected from the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4, or variants thereof, and preferably contains human IgG2 or IgG4 heavy chain constant regions, or there is no ADCC (antibody-dependent) after amino acid mutation is used. cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG4.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline Antibodies produced in antibody framework sequences. It can overcome the strong heterogeneous response induced by the chimeric antibody because it carries a large amount of mouse protein components.
  • framework sequences can be obtained from a public DNA database including germline antibody gene sequences or published references.
  • 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 www.mrccpe.com.ac.uk/vbase ), and in Kabat, EA, etc.
  • human antibody variable region framework sequence may 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 further undergo affinity affinity CDRs by phage display.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the "ADCC” described in the present disclosure refers to that the cells expressing Fc receptors are directly killed and coated with antibodies by recognizing the Fc segment of the antibody. Target cells.
  • ADCC effector function of the antibody can be reduced or eliminated.
  • the modification refers to mutation in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A selected from IgG1; IgG2 / 4 chimera, and F234A / L235A mutation of IgG4.
  • “Mutations” in the mutated sequences described in this disclosure include, but are not limited to, “back mutations”, “conservative modifications” or “conservative substitutions or substitutions.”
  • “Conservative modification” or “conservative substitution or substitution” as described in this disclosure refers to other amino acid replacement proteins with similar characteristics (such as charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.) Amino acid, which allows frequent changes to be made without altering the biological activity of the protein.
  • Those skilled in the art know that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co. ,, P. 224, (4th edition)).
  • structural or functionally similar amino acid substitutions are unlikely to disrupt biological activity.
  • the “mutated sequence” described in the present disclosure refers to the nucleotide sequence and / or the nucleotide sequence and / or obtained in the present disclosure when mutations such as substitution, insertion, or deletion of the nucleotide sequence and / or amino acid sequence are appropriately performed.
  • Amino acid sequences have nucleotide sequences and / or amino acid sequences with varying percentages of sequence identity.
  • the sequence identity described in this disclosure may be at least 85%, 90%, or 95%, and preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%. Sequence comparisons and percent identity determinations between two sequences can be performed using the default settings of the BLASTN / BLASTP algorithm available on the National Center for Biotechnology Institute website.
  • TIM3 is a widely expressed cell membrane surface immunoglobulin, also known as integrin-related protein.
  • Singal regulatory protein SIRP
  • SIRP Singal regulatory protein
  • TIM3 is an extracellular ligand of human signal-regulating protein ⁇ (SIRP ⁇ ). It binds SIRP ⁇ on the surface of macrophages to conduct inhibitory signals to reduce phagocytic activity, and then suppresses the innate immune system. This signal is described as " don't eat me “signal.
  • binding to TIM3 refers to the ability to interact with human TIM3.
  • anti-TIM3 antibody and "TIM3 antibody” agree, and both are antibodies that bind to TIM3.
  • the fusion protein described in the present disclosure is a protein product in which the two genes obtained are co-expressed by DNA recombination.
  • Recombinant TIM3 extracellular domain Fc fusion protein Through DNA recombination, a fusion protein that coexpresses the extracellular domain of TIM3 and human antibody Fc fragments.
  • the TIM3 extracellular region refers to a part of the TIM3 protein expressed outside the cell membrane, and the sequence is shown in SEQ ID NO: 1.
  • 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 expression systems cause glycosylation of antibodies, especially the highly conserved N-terminal site in the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human TIM3. Positive clones were expanded in serum-free medium in the bioreactor to produce antibodies.
  • the culture medium in which the antibody is secreted can be purified by conventional techniques.
  • a or G Sepharose FF columns with adjusted buffers for purification. Non-specifically bound components are washed away. Then bound antibody was eluted by pH gradient method, and antibody fragments were detected by SDS-PAGE and collected. The antibody can be concentrated by filtration using a conventional method. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange. The resulting product needs to be immediately frozen, such as -70 ° C, or lyophilized.
  • administering and “treatment” when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids refer to exogenous drugs, therapeutic agents, diagnostic agents or compositions and animals, humans, recipients Contact with a subject, cell, tissue, organ, or biological fluid.
  • administering and “treating” may refer to, for example, treatment, pharmacokinetics, diagnostics, research, and experimental methods.
  • Treatment of a cell includes contact of a reagent with a cell, and contact of a reagent with a fluid, wherein the fluid is in contact with the cell.
  • administering and “treating” also mean in vitro and ex vivo treatment of cells, such as cells, by an agent, diagnostic, binding composition, or by another cell.
  • Treatment when applied to a human, veterinary or research subject refers to therapeutic treatment, preventive or prophylactic measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent to a patient, such as a composition comprising any of the binding compounds of the present disclosure, said patient has one or more symptoms of a disease, and said therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a treated patient or population in an amount effective to alleviate the symptoms of one or more diseases to induce the deterioration of such symptoms or inhibit the development of such symptoms to any clinically measurable degree.
  • the amount of therapeutic agent also known as a "therapeutically effective amount" effective to alleviate the symptoms of any particular disease can vary depending on a variety of 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.
  • the formulations of the present disclosure can be used to treat diseases associated with TIM-3 positive cells.
  • diseases associated with TIM-3 positive cells There is no limitation on a disease associated with TIM-3 positive cells as long as it is a disease associated with cells expressing TIM-3, such as cancer, autoimmune diseases, and allergic diseases.
  • Cancers include blood cancer, breast cancer, uterine cancer, colorectal cancer, esophageal cancer, gastric cancer, ovarian cancer, lung cancer, kidney cancer, rectal cancer, thyroid cancer, cervical cancer, small bowel cancer, prostate cancer, and pancreatic cancer.
  • Preferable examples of cancer include blood cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer and prostate cancer.
  • Examples of blood cancers include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), multiple myeloma, cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), other lymphocytic leukemia, NK cell lymphoma, Hodgkin's lymphoma, non-Hodgkin Chicking's lymphoma such as Burkitt's lymphoma and the like.
  • AML acute myeloid leukemia
  • CML chronic myeloid leukemia
  • MDS myelodysplastic syndrome
  • multiple myeloma cutaneous T-cell lymphoma
  • CTCL cutaneous T-cell lymphoma
  • PTCL peripheral T-cell lymphoma
  • ALCL anaplastic large cell
  • autoimmune diseases include rheumatoid arthritis, psoriasis, Crohn's disease, sclerosing spondylitis, multiple sclerosis, type I diabetes, hepatitis, myocarditis, Sjogren syndrome, autoimmune hemolysis after transplant rejection Anemia, vesicular pemphigoid, Graves' disease, Hashimoto's thyroiditis, systemic lupus erythematosus (SLE), myasthenia gravis, pemphigus, malignant anemia, etc.
  • allergic diseases include acute or chronic reactive airway disease, bronchial asthma, atopic dermatitis, allergic rhinitis, urticaria, PIE syndrome, food allergies, hay fever, allergic nose, bronchial asthma, atopic Dermatitis, anaphylactic shock, etc.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disease.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the patient's overall health, the route and dose of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosage regimen to avoid significant side effects or toxic effects.
  • Replacement refers to the replacement of the solvent system that dissolves the antibody protein.
  • a buffer system using a stable formulation replaces a high-salt or hypertonic solvent system containing the antibody protein by a physical operation, so that the antibody protein exists in the stable formulation.
  • the so-called physical operation methods include, but are not limited to, ultrafiltration, dialysis, or reconstitution after centrifugation.
  • “Pharmaceutical composition” means a mixture containing one or more of the compounds described herein or a physiological / pharmaceutically acceptable salt or prodrug thereof with other chemical components, such as physiological / pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, which is beneficial to the absorption of the active ingredient and then exerts the biological activity.
  • Exemplary antibody pharmaceutical composition (formulation) preparation process :
  • the first step take a certain amount of purified TIM3 antibody solution, solvent replacement (preferably ultrafiltration) with an antibody-free buffer (such as 10mM, pH 5.5 histidine-acetate buffer), and pass the ultrafiltration membrane at least 6 Volume replacement, protein was concentrated to about 70 mg / mL.
  • solvent replacement preferably ultrafiltration
  • an antibody-free buffer such as 10mM, pH 5.5 histidine-acetate buffer
  • protein was concentrated to about 70 mg / mL.
  • a certain volume of polysorbate 80 mother liquor was added and mixed until the final polysorbate 80 concentration was 0.4 mg / mL.
  • Add 10 mM pH 5.5 histidine-acetic acid buffer to make up the volume so that the protein concentration is 50 mg / mL (other preparations to be tested or stable preparations are prepared by referring to similar steps).
  • the product is filtered and tested for sterility after being filtered.
  • the stock solution was passed through a 0.22 ⁇ m PVDF filter element, and the filtrate was collected.
  • the second step adjust the filling volume to 2.15ml, fill the filtrate in a 2ml vial, stopper, and sample the difference between the filling control at the beginning of filling, middle of filling and end of filling.
  • Step 3 Turn on 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.
  • TIM-3 UniProt Hepatitis A virus virus cellular receptor 2 (human HAVCR2, human TIM-3, Uniprot number: Q8TDQ0) was used as a template for the TIM-3 of the present disclosure to design the amino acid sequence of the antigen and detection protein involved in the present disclosure, optionally in TIM -3 protein is fused with different tags, cloned into pHr vector (self-produced) or pTargeT vector (promega, A1410), and transiently expressed or purified by CHO-S in 293 cells. Detection protein.
  • the following TIM-3 antigens refer to human TIM-3 unless otherwise specified.
  • TIM-3 extracellular region and hIgG1Fc fusion protein TIM-3-Fc (SEQ ID NO: 1), for mouse immunization:
  • the underlined part is the signal peptide, and the italic part is Fc.
  • TIM-3 extracellular region with Flag tag and His tag TIM-3-Flag-His (SEQ ID NO: 2), used to detect:
  • the underlined part is the signal peptide, and the italic part is the Flag-His tag.
  • TIM-3 full-length amino acid sequence used to construct TIM-3 overexpressing cell lines, TIM-3- full length (SEQ ID NO: 3):
  • the single horizontal line is the signal peptide
  • the double horizontal line is the transmembrane region
  • the wavy line is the intracellular region
  • the unlined part is the TIM-3 extracellular region.
  • NI-NTA affinity column (QIAGEN, Cat. No. 30721) was equilibrated with PBS, and the column volume was washed 2-5 times. The dedifferentiated cell expression supernatant sample was applied to the column. The column was rinsed with PBS until the A280 reading dropped to baseline. Rinse the column with PBS, rinse the protein, and collect. The target protein was eluted with a washing buffer (imidazole 20 mM) and an elution buffer (imidazole 300 mM) in this order, and the elution peak was collected.
  • a washing buffer imidazole 20 mM
  • an elution buffer imidazole 300 mM
  • the collected eluate was further purified by ion exchange (Hiload 16/600 superdex 200 column). Equilibrate about 2 column volumes with PBS to ensure pH 7.4. The elution buffer containing the protein of interest was concentrated and loaded, and the samples were collected. The samples were identified by SDS-PAGE and LC-MS as correct and divided into equipment.
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities.
  • the hybridoma expression supernatant was purified using a Protein G column, and the recombinant antibody and Fc fusion protein expression supernatant were purified using a Protein A column.
  • the column was rinsed with PBS until the A280 reading dropped to baseline.
  • the target protein was eluted with 100 mM acetic acid, pH 3.0, and neutralized with 1 M Tris-HCl, pH 8.0.
  • the eluted samples were appropriately concentrated and further purified using Superdex 200 (GE) gel chromatography equilibrated with PBS. The depolymerized peaks were collected and used for equipment.
  • GE Superdex 200
  • Anti-human TIM-3 monoclonal antibodies are produced by immunizing mice.
  • SJL white mice for experiments, female, 6-8 weeks of age (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Rearing environment SPF level. After the mice were purchased, they were reared in a laboratory environment for 1 week, with a 12/12 hour light / dark cycle adjustment, a temperature of 20-25 ° C, and a humidity of 40-60%. Environment-adapted mice were immunized according to the following protocol.
  • the immunization antigen was the extracellular region of human TIM-3 with an Fc tag (SEQ ID NO: 1).
  • Immunization protocol Mice were immunized with QuickAntibody-Mouse5W (KX0210041). The ratio of antigen to adjuvant is 1: 1, 10 ⁇ g / piece / time (first immunization / boosting). The antigen and adjuvant were quickly and thoroughly mixed and inoculated. The inoculation time was 21 days between the first and second immunizations, and 14 days after each immunization. Blood was collected 7 days after each immunization, and the antibody titer in mouse serum was determined by ELISA method. Mice with high antibody titers and plateaus in the serum were selected for splenocyte fusion. Immunization was strengthened 3 days before splenocyte fusion, and an antigen solution prepared by physiological saline (20 ⁇ g / head) was injected intraperitoneally (IP).
  • IP intraperitoneally
  • the spleen lymphocytes and myeloma cells Sp2 / 0 cells ( CRL-8287 TM ) to obtain hybridoma cells.
  • the fused hybridoma cells were resuspended in complete medium (containing DMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 4-5 ⁇ 10 5 / ml, and 100 ⁇ l / well were seeded in a 96-well plate. After incubation at 37 ° C, 5% CO 2 for 3-4 days, 100 ⁇ l / well of HAT complete medium was supplemented, and culture was continued for 3-4 days until needle-like clones were formed.
  • HT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HT, and 1 ⁇ OPI
  • the hybridoma culture supernatant was detected by a combined ELISA method (see Test Example 1).
  • the TIM-3 overexpressing cell binding experiment was performed on the supernatant of the positive well cells combined with ELISA detection (see Test Example 2).
  • Wells that are positive for both protein binding and cell binding are expanded in time for cryopreservation and two to three subclones until a single cell clone is obtained.
  • the hybridoma clones were obtained through the above experiments and the antibodies mAb-1701 and mAb-1799 secreted by the hybridomas were obtained.
  • the antibodies were further prepared by a serum-free cell culture method, and the antibodies were purified according to the purification examples for use in the detection examples.
  • the process of cloning sequences from positive hybridomas is as follows.
  • the logarithmic growth phase hybridoma cells were collected, and RNA was extracted using Trizol (Invitrogen, Cat No. 15596-018) according to the instructions of the kit, and reverse transcription was performed using a PrimeScript TM Reverse Transcriptase kit (Takara, Cat No. 2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sent to a sequencing company for sequencing. Get the amino acid sequences corresponding to the heavy and light chain variable region DNA sequences of mAb-1701 and mAb-1799:
  • the underlined portions are CDR regions, and each sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in that order.
  • the CDR sequences in the light and heavy chains of each antibody are shown in Table 1.
  • the heavy chain and light chain variable region germline genes with high homology to mAb-1701 antibody were selected as templates, respectively.
  • the CDRs were transplanted into the corresponding human-derived templates to form variable region sequences in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the amino acid residues are identified and annotated by the Kabat numbering system.
  • the humanized light chain template of the mouse antibody mAb-1701 is IGKV1-33 * 01 and hjk4.1, and the humanized heavy chain template is IGHV1-18 * 01 and hjh4.1.
  • the sequence of the humanized variable region is as follows :
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR sequences in italics, and CDR sequences underlined.
  • A43S means numbering according to the natural order, the 43 A is mutated back to S. Grafted represents the human antibody CDR sequence inserted into the human germline FR region.
  • This table shows the sequences obtained from various combinations of mutations. As indicated by h1701-007, there are two mutations in the light chain h1701_VL.1A and the heavy chain h1701_VH.1A on the humanized murine antibody h1701-007. Other analogies.
  • the heavy chain and light chain variable region germline genes with high homology to mAb-1799 antibody were selected as templates, respectively, and the mouse
  • the CDRs of the antibodies were transplanted into corresponding human-derived templates to form variable region sequences in the sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the amino acid residues are identified and annotated by the Kabat numbering system.
  • the humanized light chain template for mouse antibody 1799 is IGKV1-39 * 01 and hjk2.1, and the humanized heavy chain template is IGHV3-7 * 01 and hjh4.1.
  • the sequence of the humanized variable region is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, FR sequences in italics, and CDR sequences underlined.
  • This table shows the sequences obtained from various combinations of mutations. As shown by h1799-005, there are two mutations on the humanized murine antibody h1799-005, the light chain h1799_VL.1A and the heavy chain h1799_VH.1. Other analogies.
  • the antibody uses the constant region of human heavy chain IgG4 / light chain kappa in combination with various variable regions, and a S228P mutation is made in the Fc segment to increase the stability of the IgG4 antibody.
  • Other known mutations in the art can also be used to increase its performance.
  • the heavy chain constant region sequence is shown in SEQ ID NO: 41:
  • the light chain constant region sequence is shown in SEQ ID NO: 42:
  • the italic sequence is the FR region sequence
  • the underlined is the CDR region sequence
  • the wavy line is the constant region sequence.
  • variable region coding gene sequence After sequencing the positive antibody molecule obtained by hybridoma screening, the variable region coding gene sequence is obtained. Primer and tail primers were designed based on the sequence obtained. Using the sequenced gene as a template, the VH / VK gene fragments of each antibody were constructed by PCR, and then combined with the expression vector pHr (with signal peptide and hIgG4 / hkappa constant region gene (CH1-FC / CL) fragment). Homologous recombination was performed to construct the full-length expression plasmid VH-CH1-FC-pHr / VL-CL-pHr of the recombinant chimeric antibody to form two chimeric antibodies, Ch1701 and Ch1799.
  • the human-designed antibody sequence was codon-optimized to generate the human codon-preferred coding gene sequence.
  • Primer PCR was designed to construct each antibody VH / VK gene fragment, and then combined with the expression vector pHr (with signal peptide and hIgG4 / hkappa constant region Gene (CH1-FC / CL) fragment) was subjected to homologous recombination to construct a full-length humanized antibody expression plasmid VH-CH1-FC-pHr / VL-CL-pHr.
  • the plasmids expressing the light and heavy chains of the antibodies were respectively transfected into HEK293E cells at a ratio of 1: 1.2. After 6 days, the expression supernatants were collected, the impurities were removed by high-speed centrifugation, and purification was performed using a Protein A column. The column was rinsed with PBS until the A280 reading dropped to baseline. The target protein was eluted with an acidic eluent of pH 3.0-pH 3.5, and neutralized with 1 M Tris-HCl, pH 8.0-9.0. The eluted sample was appropriately concentrated, and further purified using Superdex 200 (GE) gel chromatography equilibrated to remove the polymer, and the monomer peaks were collected and used for equipment.
  • GE Superdex 200
  • Deamidation modification is a common chemical modification in antibodies that may affect the later stability.
  • some amino acids in the CDR region are highly deamidated, oxidized or isomerized. Generally, they are chosen to avoid or reduce mutations as much as possible.
  • the NNG in the heavy chain CDR2 of h1701 antibody is prone to deamidation.
  • the NNG is located at positions 54-56 of the variable region of the heavy chain of h1701 antibody, respectively. Natural sequence number), according to amino acid properties and computer antibody structure simulation technology, the amino acids at the above positions can be substituted with any amino acid.
  • the CDR2 mutant of h1701 is: DIIPX 1 X 2 X 3 GSKYNQKFKD (SEQ ID NO: 43 ), Where X 1 X 2 X 3 are amino acid residues 54 to 56 of the heavy chain variable region of h1701 antibody, X 1 is selected from Asn, Leu, Val, Met or Glu, and X 2 is selected from Asn, Glu, Met , His, Lys, Leu, Ala or Val, and X 3 is selected from Gly or Ala.
  • the CDR2 containing the 54th to 56th mutations described above and the FR region containing different back mutations can form the following heavy chain variable region:> h1701_VH.1-CDR2 mutant (SEQ ID NO: 44)
  • Exemplary sequences related to the HCDR2 mutant of h1701, and the humanized sequence including the corresponding CDR2 mutant h1701_VH.1B-CDR2 mutant (SEQ ID NO: 46) are shown in the following mutants and Table 6.
  • the NNG design mutation in HCDR2 of h1701-009 is mutated to NLG, NVG, NNA, NMA, NEA, NHA, NMG, NEG, NKG, NAG, NHG (the CDR2 amino acid mutant of the above heavy chain variable region)
  • the sequences are shown in SEQ ID NOs: 51-61 respectively).
  • Expression plasmid construction and 293E expression were performed by molecular cloning. After purification, the affinity and stability of the mutant antibodies were further tested.
  • a series of amino acid mutations to h1701-009. Specific related sequences include but are not limited to those described in Table 6.
  • Test example 1 Anti-TIM-3 antibody binding to human TIM-3 overexpressing CHO-s cells
  • the binding ability of the anti-TIM-3 antibody was detected by binding experiments of the antibody to CHO-S cells overexpressing the TIM-3 protein.
  • the full-length TIM-3 plasmid (internally produced, SEQ ID NO: 3) was transfected into CHO-S cells by electrotransfection and screened under pressure for two weeks, and the expression of TIM-3 was detected. After the over-expressing cells were fixed on the bottom of a 96-well plate, the strength of the signal after antibody addition was used to judge the binding activity of the antibody to Tim-3 over-expressing CHO-S cells.
  • the specific experimental method is as follows.
  • the positive control antibody used was AbTim-3, and the sequence was derived from Table 2 of US20150218274A1, which was prepared by our company.
  • PBST buffer pH 7.4PBS containing 0.05% tweeen-20
  • test antibody hybrida purified antibody
  • sample dilution solution or humanized antibody
  • incubate for 1 hour in a 37 ° C incubator.
  • wash the plate 5 times with PBST and add 100 ⁇ l / well HRP-labeled goat anti-mouse secondary antibody (Jackson Immuno Research, Cat No. 115-035-003) or goat anti-human secondary antibody (Jackson Immuno Research, Cat No. 109-035-003), and incubated at 37 ° C for 1 hour.
  • HRP-labeled goat anti-mouse secondary antibody Jackson Immuno Research, Cat No. 115-035-003
  • goat anti-human secondary antibody Jackson Immuno Research, Cat No. 109-035-003
  • TMB chromogenic substrate KPL, Cat No. 52-00-03
  • 1M H 2 SO 4 50 ⁇ l / well of 1M H 2 SO 4 to stop the reaction.
  • the NOVOStar microplate reader reads the absorption value at a wavelength of 450nm, and calculates the EC50 value of TIM-3 antibody binding to Tim-3 overexpressing CHO-S cells. The test results are shown in Figure 1 and Table 7.
  • TIM-3 antibodies 1701 and 1799 and their humanized antibodies had good binding activity to CHO-s cells overexpressing human TIM-3 full-length protein.
  • Test example 2 Anti-TIM-3 antibody group competition test
  • Antibody A was diluted to a concentration of 2 ⁇ g / ml with PBS (Sigma, Cat No. P4417-100TAB) buffer at pH 7.4, and added to a 96-well microtiter plate (Corning, Cat No. CLS3590-100EA) in a volume of 50 ⁇ l / well. In a 37 ° C incubator for 2 hours. After discarding the liquid, 200 ⁇ l / well of 5% skimmed milk (bright skimmed milk) blocking solution diluted with PBS was added, and incubated at 37 ° C for 2.5 hours or at 4 ° C overnight (16-18 hours) for blocking.
  • PBS Sigma, Cat No. P4417-100TAB
  • CLS3590-100EA 96-well microtiter plate
  • the mouse anti-capture antibody was covalently coupled to the CM5 biosensor chip (Cat. # BR-1000-12, GE) according to the method described in the mouse anti-capture kit (Cat. # BR-1008-38, GE) instructions. ), So that the antibody to be detected is affinity captured, and then the TIM-3-His (Sino. Biol, Cat. 10390-H03H) antigen is flowed on the chip surface, and the reaction signal is detected in real time using a Biacore instrument to obtain the binding and dissociation curve. Affinity is obtained by fitting. After the dissociation of each cycle was completed in the experiment, the biochip was washed and regenerated with the regeneration solution provided in the mouse anti-capture kit, and the results are shown in Table 10.
  • TIM-3 antibodies mAb-1701 and mAb-1799 have strong binding activity and affinity for human TIM-3 protein.
  • the human anti-capture antibody was covalently coupled to a CM5 biosensor chip (Cat. # BR-1000-12, GE) according to the method described in the human anti-capture kit (Cat. # BR-1008-39, GE) instructions. ), So that the antibody to be detected is affinity captured, and then the TIM-3-Flag-His (in-house production, SEQ ID NO: 2) antigen is passed on the surface of the chip, and the reaction signal is detected in real time using a Biacore instrument to obtain the binding and dissociation curve. Affinity values were obtained by fitting, see Table 11 below. After each cycle of dissociation in the experiment was completed, the biochip was washed and regenerated with the regeneration solution provided in the human anti-capture kit.
  • Test Example 4 In vitro immune cell function test
  • MLR assay mixed lymphocyte response assay
  • PBMC activation assay PBMC activation assay
  • SEB superantigen Staphylococcus aureus enterotoxin B
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • Test Example 5 Rat PK determination of anti-TIM-3 humanized antibodies, h1701-009 and h1799-005
  • the two groups of rats were injected intravenously with the test drug h1701-009 at a dose of 3mg / kg and 10mg / kg; the two groups of rats were intravenously injected with the test drug h1799-005 at a dose of 3mg / kg. And 10mg / kg.
  • the intravenous injection volume was 5 ml / kg.
  • Blood was collected before and at 5min, 8h, 1d, 2d, 4d, 7d, 10d, 14d, 21d, and 28d. 0.2ml of whole blood was taken from each animal without anticoagulant. After the blood was collected, it was placed at 4 ° C for 30min, centrifuged at 1000g for 15min, and the supernatant was placed in an EP tube and stored at 80 ° C.
  • SD rats were administered intravenous injection of anti-TIM-3 antibody h1701-009, h1799-005 at a dose of 10mg / kg, and the exposure in rats was similar; the increase in the exposure and dosage of antibody h1799-005 at a dose of 3,10mg / kg
  • the growth was basically linear.
  • the exposure of antibody h1701-009 at a dose of 3,10 mg / kg increased slightly higher than the dose increase; the elimination half-lives of antibodies h1701-009 and h1799-005 were similar.
  • Test Example 6 Detection of the thermal stability of h1701-009, h1799-005 antibodies by DSC
  • Exemplary buffer systems corresponding to different pHs were 10 mM PB (pH 7), 15 mM His (pH 6.0), and 10 mM acetate (pH 5.2).
  • the sample was replaced in the corresponding buffer, and the concentration of the sample was controlled at about 1 mg / ml, and the detection was performed using MicroCal * VP-Capillary DSC (Malvern).
  • degas each sample and blank buffer with a vacuum degasser for 1-2 minutes. Add 400 ⁇ l of sample or blank buffer to each well of the sample plate (300 ⁇ l on the instrument). Add 14% Decon 90 and ddH 2 O to the last two pairs of well plates for cleaning.
  • the scanning temperature is from 25 ° C to 100 ° C, and the scanning rate is 60 ° C / h.
  • Table 14 The specific results are shown in Table 14. In several test systems, h1701-009 and h1799-005 all showed good thermal stability.
  • Test Example 7 Monitoring the purity of samples by SEC-HPLC to investigate the cyclic stability under a certain concentration
  • Exemplary conditions such as controlling the sample concentration to about 50 mg / ml, comparing the stability of different antibodies in PBS (pH 7.4) system, such as repeated freeze-thaw cycles at -80 ° C for 5 times and storage at 4 ° C and 40 ° C for one month .
  • the Xbridge protein BEH 200A (Waters) HPLC column was used to detect the purity of the antibody. After a month's investigation, h1701-009 and h1799-005 showed good stability, and there was no significant change in the purity of SEC at 40 ° C in one month.
  • H1701-009, h1799-005, and h1701-009 mutants shown in SEQ ID NO: 51-55 were tested for chemical stability.
  • an anti-TIM3 antibody (h1799-005, prepared in Example 1, the same below) formulation with an antibody concentration of 50 mg / ml:
  • D means day, for example, D7 means day 7.
  • An anti-TIM3 antibody (h1799-005) preparation with an antibody concentration of 50 mg / ml was prepared with the following buffers:
  • D is the day, for example, D21 is the 21st day.
  • an anti-TIM3 antibody (h1799-005) preparation with an antibody concentration of 50 mg / ml:
  • the prepared formulation samples were taken for high-temperature (40 ° C) and low-temperature light (5 ⁇ 3 ° C, 4500lx) stability studies. The results are shown in Table 18. The results show that the stability of sucrose and trehalose against TIM3 antibodies is similar. When the sucrose concentration is 80 mg / ml, the osmotic pressure is about 300 mosm / kg, which is close to isotonicity. Therefore, the sucrose concentration is preferably 80 mg / ml.
  • D indicates the day, for example, D21 indicates the 21st day.
  • Anti-TIM3 antibody (h1799-005) preparation with sucrose concentration of 70mg / ml and antibody concentration of 50mg / ml was prepared with the following different concentrations and types of surfactant buffers:
  • the samples of the 1-5 groups of anti-TIM3 antibody preparations prepared according to the above method were diluted to 0.9% sodium chloride injection, respectively, and the protein concentration was diluted to 0.5 mg / ml, and the insoluble particles of each group of samples were observed after dilution.
  • the test results are shown in Table 19. The results show that no less than 0.4 mg / ml polysorbate 80 has better stability against TIM3 antibodies, so 0.4-0.6 mg / ml polysorbate 80 is selected, and 0.4 mg / ml polysorbate is preferred. Ester 80 is shown as an active agent for anti-TIM3 antibodies.
  • the samples of 1-15 groups of anti-TIM3 antibody preparations prepared according to the above method were stored at 40 ° C for stability analysis.
  • the difference between the neutral peak value and the acidic peak value of the IEC was used as the evaluation index, and the results were analyzed by the least square method.
  • Statistical analysis The test results are shown in Figure 5 and Table 20.
  • the results show that the ionic strength is 10mM and the pH 5.5 histidine-acetic acid system is more conducive to the stability of the anti-TIM3 antibody.
  • the protein concentration is relatively stable between 40-60 mg / ml. Take the median value of 50 mg / ml as the final protein concentration of the anti-TIM3 antibody.
  • M means month, for example M1 means 1 month.
  • anti-TIM3 antibody pharmaceutical formulations of other formulations including but not limited to:

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Abstract

本发明公开了一种TIM3抗体药物组合物及其用途。具体而言，所述药物组合物包含在缓冲液中的TIM3抗体或其抗原结合片段。除此之外，该药物组合物还包含糖和非离子型表面活性剂。本发明中的药物组合物具备良好的稳定性。

Description

一种TIM3抗体药物组合物及其用途 技术领域
本公开属于药物制剂领域，具体涉及一种包含TIM3抗体或其抗原结合片段的药物组合物，以及其作为抗癌药物的用途。
背景技术
这里的陈述仅提供与本发明有关的背景信息，而不必然地构成现有技术。
T细胞免疫球蛋白黏蛋白分子3(T cell immunoglobulinand mucin-domain-containing molecule 3，TIM-3)，又称甲型肝炎病毒细胞受体2(hepatitis A virus cellular receptor 2，HAVCR-2)，是一种I型膜表面蛋白分子，属TIM家族成员。人类TIM-3分子由301个氨基酸组成，包含信号肽、Ig可变区(IgV区)、富含丝氨酸/苏氨酸的黏蛋白区、跨膜区和胞质区，人鼠之间的同源性63％。
TIM-3选择性地表达在分泌IFN-γ的T辅助细胞(Th1和Th17)、T调控细胞(Treg)、树突状细胞(DCs)、单核细胞、肥大细胞、NK细胞、肿瘤浸润性淋巴细胞(TILs)上(see,e.g.,Clayton et al.,J.Immunol.,192(2):782-791(2014)；Jones et al.,J.Exp.Med.,205:2763-2779(2008))。目前已知的TIM-3的受体包括磷脂酰丝氨酸半乳糖凝集素9(Galectin-9，Gal-9)、高迁移率族蛋白1(HMGB1)和癌胚抗原细胞粘附分子1(CEACAM1)。
TIM-3可从多方面调节免疫系统的功能，在Th1细胞表面与配体Gal-9结合可下调Th1细胞应答，并能诱导Th1细胞凋亡，在自身和异体免疫性疾病(如系统性红斑狼疮、哮喘)以及免疫耐受中起着重要作用。在单核-巨噬细胞表达的TIM-3与磷脂酰丝氨酸相互作用，促进凋亡细胞的吞噬；在肿瘤浸润DC中，TIM-3则与HMGB1配体结合，抑制核酸正确转运，从而抑制核酸免疫反应，参与免疫逃逸。TIM-3不仅表达于免疫细胞，在卵巢癌、脑膜瘤、黑色素瘤等肿瘤细胞中也有过表达，并直接促进肿瘤的增长。下调TIM-3的表达可以明显抑制Hela细胞的侵袭和转移。TIM-3的过表达均与肺癌、胃癌、前列腺癌和宫颈癌的不良预后密切相关。在血液系统肿瘤中，TIM-3过表达于急性髓系白血病的白血病干细胞、MDS患者的造血干细胞上，且TIM-3+造血干细胞具有低分化、低凋亡及高增殖的恶性生物学特征。因此，通过抑制TIM-3的活性(如TIM-3抗体)以提高固有免疫系统的功能，有望成为治疗肿瘤的新方法(see,e.g.,Ngiow et a1.,Cancer Res.,71(21):1-5(2011)；Guo et al.,Journαl of Translationαl Medicine,11:215(2013)；and Ngiow et al.,Cancer Res.,71(21):6567-6571(2011))。
抗体药物的稳定性是影响抗体成药性的关键因素之一，其中天冬酰胺(Asn)和谷氨酰胺(Gln)的非酶促脱酰胺作用，能够引起抗体的不稳定和异质性，是抗体 分子常见化学降解途径之一。已有文献报道，多个IgGl亚型抗体因为其CDR区氨基酸的脱酰胺和异构化作用，而失去了其生物学活性。Huang等人发现，位于IgG1单克隆抗体重链可变区2(CDR2)的Asn55的脱酰胺作用，明显降低了其结合活性。因此，在抗体的研发过程中应尽量避免或者突变相应氨基酸以降低抗体脱酰胺情况的发生，以增加抗体的稳定性和生物利用率。
尽管目前已有专利报道了有关TIM-3的抗体，如WO2011159877、WO2013006490、WO2015117002、WO2016144803、WO2016161270、US20150218274，但就国内外而言，仅有两个TIM-3抗体处于临床研究阶段，其他抗体药物仍处于发现和研究阶段，尚没有TIM-3抗体进入临床应用。因此，有必要进一步开发具有更高活性、高亲和力和高稳定性的TIM-3的抗体，以进行TIM-3相关疾病的治疗研究和应用。
本公开提供一种更利于生产和给药，性能稳定的包含TIM3抗体或其抗原结合片段的药物组合物。
发明内容
本公开提供一种药物组合物，其包含TIM3抗体或其抗原结合片段，以及缓冲剂，所述缓冲剂选自醋酸盐、组氨酸盐、琥珀酸盐、磷酸盐、枸橼酸盐缓冲剂，优选醋酸盐缓冲剂或组氨酸盐缓冲液。
在一些实施方案中，药物组合物中所述的醋酸盐缓冲剂选自醋酸-醋酸钠缓冲剂，所述的组氨酸盐缓冲剂选自组氨酸-醋酸缓冲剂或组氨酸-盐酸缓冲剂；所述的琥珀酸盐缓冲剂选自琥珀酸-琥珀酸钠缓冲剂；所述磷酸盐缓冲剂选自磷酸氢二钠-磷酸二氢钠缓冲剂；所述枸橼酸盐缓冲剂选自枸橼酸-枸橼酸钠；优选组氨酸-盐酸、醋酸-醋酸钠或组氨酸-醋酸缓冲剂，最优选组氨酸-醋酸缓冲剂。
在一些实施方案中，药物组合物中所述缓冲剂浓度为大约5mM至30mM，优选为大约10mM-30mM，优选为大约5mM至20mM，优选大约10mM至20mM；非限制性实施例包括10mM、12mM、14mM、16mM、18mM、20mM、30mM，最优选为大约10mM。
在一些实施方案中，药物组合物中所述TIM3抗体或其抗原结合片段浓度为大约1mg/ml至100mg/ml，优选为大约40mg/ml至60mg/ml，优选为50mg/ml至60mg/ml；非限制性实施例包1mg/ml、10mg/ml、20mg/ml、30mg/ml、40mg/ml、41mg/ml、42mg/ml、43mg/ml、44mg/ml、45mg/ml、46mg/ml、47mg/ml、48mg/ml、49mg/ml、50mg/ml、51mg/ml、52mg/ml、53mg/ml、54mg/ml、55mg/ml、56mg/ml、57mg/ml、58mg/ml、59mg/ml、60mg/m、70mg/m、80mg/m、90mg/m、100mg/ml，最优选为50mg/ml。
在一些实施方案中，药物组合物中所述缓冲剂的pH值约为5.0到6.5，优选为大约5.0至6.0，优选为大约5.2至5.8，或优选为大约5.5至6.0，最优选5.5， 非限制的实施例包括约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、约6.5。
进一步的，在一些实施方案中，前述药物组合物还包含糖。本公开的“糖”包含常规组合物(CH 2O) n及其衍生物，包括单糖，二糖，三糖，多糖，糖醇，还原性糖，非还原性糖等等。可选自葡萄糖，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，甘露醇，密里二糖，松三糖，蜜三糖，甘露三糖，水苏糖，麦芽糖，乳果糖，麦芽酮糖，山梨醇，麦芽糖醇，乳糖醇，异-麦芽酮糖等等。优选的糖是非还原性二糖，更优选为海藻糖或蔗糖，最优选为蔗糖。
在一些实施方案中，前述药物组合物中糖的浓度为约50mg/ml至约100mg/ml，优选为约60mg/ml至约90mg/ml，优选为约70mg/ml至约90mg/ml，优选为70mg/ml至约80mg/ml，非限制性实施例包括50mg/ml、60mg/ml、65mg/ml、70mg/ml、75mg/ml、80mg/ml、85mg/ml、90mg/ml，最优选为80mg/ml。
进一步地，在一些实施方案中，药物组合物还包含表面活性剂。可选自聚山梨醇酯20、聚山梨醇酯80、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基/肉豆蔻基/亚油基/硬脂基-磺基甜菜碱、月桂基/肉豆蔻基/亚油基/硬脂基-肌氨酸、亚油基/肉豆蔻基/鲸蜡基-甜菜碱、月桂酰胺基丙基/柯卡酰胺基丙基/亚油酰胺基丙基/肉豆蔻酰胺基丙基/棕榈酰胺基丙基/异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基/棕榈酰胺基丙基/异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等等。优选的表面活性剂是聚山梨醇酯80或聚山梨醇酯20，更优选为聚山梨醇酯80。
在一些实施方案中，药物组合物中表面活性剂的浓度为约0.2mg/ml至0.8mg/ml，优选为0.2mg/ml至0.6mg/ml，更优选为0.4mg/ml至0.6mg/ml，非限制性实施例包括0.2mg/ml、0.3mg/ml、0.4mg/ml、0.45mg/ml、0.5mg/ml、0.55mg/ml、0.6mg/ml、0.7mg/ml、0.8mg/ml，最优选为0.4mg/ml。
在可选的实施方案中，所述药物组合物包含：
(a)大约1mg/ml-100mg/ml的TIM3抗体或其抗原结合片段，
(b)大约5mM-30mM的醋酸盐缓冲剂或组氨酸盐缓冲液，
(c)大约50mg/ml-100mg/ml的糖，和
(d)大约0.2mg/ml-0.8mg/ml的聚山梨醇酯。
在可选的实施方案中，所述药物组合物包含：
(a)1mg/ml-100mg/ml的TIM3抗体或其抗原结合片段，
(b)5mM-30mM的组氨酸-醋酸缓冲剂或醋酸-醋酸钠缓冲剂，pH约为5.0至6.5，
(c)50mg/ml-100mg/ml的糖，和
(d)0.2mg/ml-0.8mg/ml的聚山梨醇酯80。
在可选的实施方案中，所述药物组合物包含：
(a)40mg/ml-60mg/ml的TIM3抗体或其抗原结合片段，
(b)10mM-30mM的组氨酸-醋酸缓冲剂或醋酸-醋酸钠缓冲剂，pH约为5.0至6.0，
(c)70mg/ml-90mg/ml的蔗糖或海藻糖，和
(d)0.4mg/ml-0.6mg/ml的聚山梨醇酯80。
在可选的实施方案中，所述药物组合物包含：
(a)约50mg/ml的TIM3抗体或其抗原结合片段，
(b)约10mM的组氨酸-醋酸缓冲剂，pH约5.5，
(c)约80mg/ml的蔗糖，和
(d)约0.4mg/ml的聚山梨醇酯80。
在一些实施方案中，本公开药物组合物中所述的一种TIM-3抗体或抗原结合片段，其特征在于，其与人TIM-3胞外区结合，且其与选自下面(i)至(ii)中的任一种单克隆抗体或抗原结合片段竞争结合TIM-3，或是选自下面(i)至(ii)中的任一种单克隆抗体或其抗原结合片段：
(i)单克隆抗体或其抗原结合片段，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：
抗体重链可变区HCDR区序列：如SEQ ID NO：8、43和10氨基酸序列所示；和/或抗体轻链可变区LCDR区序列：如SEQ ID NO：11、12和13氨基酸序列所示；
(ii)单克隆抗体或其抗原结合片段，其包含1个或多个选自以下的CDR区序列或与其具有至少95％序列同一性的氨基酸序列：
抗体重链可变区HCDR区序列：如SEQ ID NO：14、15和16氨基酸序列所示；和/或抗体轻链可变区LCDR区序列：如SEQ ID NO：17、18和19氨基酸序列所示。
在一些实施方案中，其中所述的TIM-3抗体或其抗原结合片段选自下面(i)或(ii)所述的任一种单克隆抗体或其抗原结合片段：
(i)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、43和10所示的抗体重链可变区HCDR1-3区序列或与其具有至少95％序列同一性的氨基酸序列，和/或分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列或与其具有至少95％序列同一性的氨基酸序列；
其中所述的SEQ ID NO:43如DIIPX 1X 2X 3GSKYNQKFKD序列所示，其中，X 1选自N、L、V、M或E，X 2选自N、E、M、H、K、L、A或V，X 3选自G或A；
(ii)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：14、15和 16所示的抗体重链可变区HCDR1-3区序列或与其具有至少95％序列同一性的氨基酸序列，和/或分别如SEQ ID NO：17、18和19所示的抗体轻链可变区LCDR1-3区序列或与其具有至少95％序列同一性的氨基酸序列。
上述具有至少95％序列同一性的氨基酸序列，优选地，其具有至少95％、96％、97％、98％、或99％序列一致性，更优选地，其具有97％、98％或99％以上，最优选的具有至少99％以上序列一致性，上述具有至少95％序列同一性的氨基酸序列包括通过一个或者多个氨基酸缺失、插入或替换突变获得。
在一些实施方案中，所述单克隆抗体或其抗原结合片段其包含分别如SEQ ID NO：8、43和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
或包含分别如SEQ ID NO：14、15和16所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：17、18和19所示的抗体轻链可变区LCDR1-3区序列。
在一些实施方案中，本公开药物组合物中所述的TIM-3抗体或抗原结合片段，其与如下(a)至(m)中的任一种单克隆抗体或抗原结合片段竞争结合TIM-3，或是选自(a)至(m)中的任一种单克隆抗体或其抗原结合片段：
(a)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、9和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(b)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、62和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(c)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、63和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(d)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、64和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(e)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、65和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(f)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、66和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(g)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、67和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所 示的抗体轻链可变区LCDR1-3区序列；
(h)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、68和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(i)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、69和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(g)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、70和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(k)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、71和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(l)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、72和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
(m)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：14、15和16所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：17、18和19所示的抗体轻链可变区LCDR1-3区序列。
在一些实施方案中，本公开药物组合物中所述的TIM-3抗体或抗原结合片段，其特征在于，其与选自前述任何一种单克隆抗体结合相同的表位。
在一些实施方案中，本公开药物组合物，其中所述的TIM-3抗体或抗原结合片段，其中所述抗体是重组抗体。
在一些的实施方式中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述单克隆抗体选自鼠源抗体、嵌合抗体、人源化抗体的重组抗体或其抗原结合片段。
在一些实施方案中，本公开药物组合物，其中所述的TIM-3抗体或抗原结合片段包含如SEQ ID NO:4所示的重链可变区序列和/或如SEQ ID NO:5所示的轻链可变区序列，或包含如SEQ ID NO:6所示的重链可变区序列和/或如SEQ ID NO:7所示的轻链可变区序列。
在一些实施方案中，本公开药物组合物，其中所述的TIM-3抗体或抗原结合片段，其中所述的人源化抗体轻链和重链可变区上的轻链和重链FR区序列分别来源于人种系轻链和重链或其突变序列。
在一些实施方案中，本公开药物组合物，其中所述的TIM-3抗体或抗原结合片段，其中所述的人源化抗体含有SEQ ID NO:20或31所示的重链可变区或其变体；所述变体是在SEQ ID NO:20或31所示的重链可变区上具有1-10个氨基酸变 化的序列。
在一些实施方式中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述变体是在SEQ ID NO:20或31所示的重链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自在SEQ ID NO:20所示的重链可变区上进行D89E、R98T、G49A、M48I、M70L、R38K和V68A中的一个或多个氨基酸回复突变或在SEQ ID NO:31所示的重链可变区上进行Q3K和/或R87K的氨基酸回复突变。
在一些实施方式中，为消除抗体CDR区的乙酰化、脱酰胺化等化学修饰，本公开所述的TIM-3抗体或其抗原结合片段，其中所述CDR区的易发生化学修饰的位点可以进行氨基酸的置换，优选CDR2中的NNG被置换；优选地，本公开所述的TIM-3抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:45、45、46、47、48、49和50中任一个所示的重链可变区。
在一些实施方式中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述人源化抗体包含SEQ ID NO:34或35所示的重链可变区。
在一些实施方式中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述的人源化抗体含有SEQ ID NO:21或32所示的轻链可变区或其变体；所述变体是在SEQ ID NO:21或32所示的轻链可变区上具有1-10个氨基酸变化的序列。
在一些方案中，所述的TIM-3抗体或其抗原结合片段含有如SEQ ID NO:21或32所示的轻链可变区的FR区或其变体，所述变体是在SEQ ID NO:21或32所示的轻链可变区上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自在SEQ ID NO:21所示的A43S位点进行回复突变，或在SEQ ID NO:32所示的Q3K、I48V、K45Q、A43S和T85S中的至少一个位点进行回复突变。
在一些的实施方式中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:30所示的轻链可变区或包含选自SEQ ID NO:37、37、38、39和40中任一个所示的轻链可变区。
在一些实施方案中，为消除抗体CDR区的乙酰化、脱酰胺化等化学修饰，本公开所述的TIM-3抗体或其抗原结合片段，其中所述CDR区易发生化学修饰的位点可以进行氨基酸的置换，优选CDR2中的NNG被置换，形成如SEQ ID NO：43所示的CDR2序列；在一些实施方案中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:44、45、46、47、48、49和50所示的重链可变区序列和选自SEQ ID NO:29或30所示的轻链可变区序列。
在一些实施方案中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述人源化抗体包含选自SEQ ID NO:22、23、24、25、26、27和28中任一个所示的重链可变区序列和选自SEQ ID NO:29或30所示的轻链可变区序列，优选选自SEQ ID NO:24所示的重链可变区序列和选自SEQ ID NO:29所示的轻链可变区序列。
在一些实施方案中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述 人源化抗体包含选自SEQ ID NO:33、34和35中任一个所示的重链可变区序列和选自SEQ ID NO:36、37、38、39和40中任一个所示的轻链可变区序列，优选选自SEQ ID NO:33所示的重链可变区序列和选自SEQ ID NO:36所示的轻链可变区序列。
在一些实施方案中，为消除抗体CDR区氨基酸位点的脱酰胺，本公开所述的TIM-3抗体或其抗原结合片段，其中所述CDR区的易脱酰胺化的位点可以进行氨基酸的置换，优选CDR2中的NNG被置换；一种优选的实施方式中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述人源化抗体包含：选自SEQ ID NO:51、52、53、54、55、56、57、58、59、60和61中任一个所示的重链可变区序列和选自SEQ ID NO:29或30所示的轻链可变区序列，优选选自SEQ ID NO:51所示的重链可变区序列和选自SEQ ID NO:29所示的轻链可变区序列；或选自SEQ ID NO:52所示的重链可变区序列和选自SEQ ID NO:29所示的轻链可变区序列。
在一些实施方式中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述抗体为全长抗体，进一步包括人抗体恒定区，优选SEQ ID NO:41所示或与其具有85％序列同一性的人重链恒定区序列，和优选SEQ ID NO:42所示或与其具有85％序列同一性的人轻链恒定区序列。上述具有至少85％序列同一性的氨基酸序列，优选地，其具有至少86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列一致性，更优选地，其具有90％、95％或99％以上，最优选的具有至少95％以上序列一致性，上述具有至少85％序列同一性的氨基酸序列包括通过一个或者多个氨基酸缺失、插入或替换突变获得。最优选的，所述抗体包含如SEQ ID NO:73所示的重链全长序列和如SEQ ID NO:74所示的轻链全长序列。
在一种些实施方式中，本公开所述的TIM-3抗体或其抗原结合片段，其中所述抗原结合片段是选自Fab、Fab'、F(ab')2、单链抗体(scFv)、二聚化的V区(双抗体)、二硫键稳定化的V区(dsFv)和包含CDR区的肽的抗原结合片段。
本公开还提供制备前述药物组合物的方法，其中包括：TIM3抗体原液经缓冲液置换的步骤。在一些实施方案中，所述缓冲液优选醋酸盐缓冲剂或组氨酸盐缓冲剂，更优选组氨酸-盐酸、醋酸-醋酸钠或组氨酸-醋酸缓冲剂，最优选组氨酸-醋酸缓冲剂。在一些实施方案中，所述缓冲液浓度为大约5mM至30mM，优选为大约5mM至20mM，优选10mM至30mM，非限制性实施例包括5mM、10mM、12mM、14mM、16mM、18mM、20mM、30mM，最优选为大约10mM。在一些实施方案中，所述缓冲剂的pH值约为5.0到6.5，优选为大约5.0至6.0，优选为大约5.2至5.8，最优选5.5，非限制的实施例包括约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。
本公开还提供制备前述药物组合物的方法，进一步地还包括向置换步骤所得溶液中加入糖和表面活性剂，再经缓冲液定容的步骤。在一些实施方案中，优选 的糖是非还原性二糖，更优选为海藻糖或蔗糖，最优选为蔗糖，糖的浓度为约50mg/ml至约100mg/ml，更优选为约60mg/ml至约90mg/ml，更优选为70mg/ml至约80mg/ml，非限制性实施例包括60mg/ml、65mg/ml、70mg/ml、75mg/ml、80mg/ml、85mg/ml、90mg/ml，最优选为80mg/ml。在一些实施方案中，表面活性剂优选聚山梨醇酯80或聚山梨醇酯20，更优选为聚山梨醇酯80；浓度为约0.2mg/ml至0.8mg/ml，更优选为0.4mg/ml至0.6mg/ml，非限制性实施例包括0.2mg/ml、0.3mg/ml、0.4mg/ml、0.45mg/ml、0.5mg/ml、0.55mg/ml、0.6mg/ml、0.7mg/ml、0.8mg/ml，最优选为0.4mg/ml。
本公开还提供一种制备包含TIM3抗体的冻干制剂的方法，其中包括将前述药物组合物经冷冻干燥的步骤。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，和10mM组氨酸-醋酸，pH 5.5。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，和10mM组氨酸-醋酸，pH 6.0。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，10mM组氨酸-盐酸，pH 6.0。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，10mM醋酸-醋酸钠，pH 5.5。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，10mM组氨酸-盐酸，pH 5.5。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，10mM组氨酸-醋酸pH 6.0和70mg/ml蔗糖。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，10mM组氨酸-醋酸pH 6.0和70mg/mlα,α-二水合海藻糖。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，10mM组氨酸-醋酸pH 5.5，70mg/ml蔗糖和0.4mg/ml聚山梨酯80。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，10mM组氨酸-醋酸pH 5.5，70mg/ml蔗糖和0.2mg/ml聚山梨酯80。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，10mM组氨酸-醋酸pH 5.5，70mg/ml蔗糖和0.6mg/ml聚山梨酯80。
在一些实施方案中，本公开所述的药物组合物包含：50mg/ml的抗TIM3抗体h1799-005，10mM组氨酸-醋酸pH 5.5，70mg/ml蔗糖和0.4mg/ml聚山梨酯20。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖，0.4mg/ml聚山梨酯80，20mM组氨酸-醋酸pH 5.5和60mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖，0.4mg/ml聚山梨酯80，10mM组氨酸-醋酸pH 5.5和50mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，20mM组氨酸-醋酸pH 5.5和50mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，10mM组氨酸-醋酸pH 6.0和40mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，30mM组氨酸-醋酸pH 5.5和50mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，20mM组氨酸-醋酸pH 6.0和50mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，30mM组氨酸-醋酸pH 5.0和60mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，10mM组氨酸-醋酸pH 6.0和60mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，20mM组氨酸-醋酸pH 5.0和50mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，30mM组氨酸-醋酸pH 6.0和60mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，20mM组氨酸-醋酸pH 5.5和40mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，30mM组氨酸-醋酸pH 6.0和40mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，30mM组氨酸-醋酸pH 5.0和40mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，10mM组氨酸-醋酸pH 5.0和60mg/ml TIM3抗体h1799-005。
在一些实施方案中，本公开所述的药物组合物包含：80mg/ml蔗糖和0.4mg/ml聚山梨酯80，10mM组氨酸-醋酸pH 5.0和40mg/ml TIM3抗体h1799-005。
在一些实施方案中，制备包含TIM3抗体的冻干制剂的方法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。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。
本公开还提供一种经前述制备包含TIM3抗体的冻干制剂的方法制备的含TIM3抗体的冻干制剂。
在一些实施方案中，该冻干制剂于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。在一些实施方案中，该冻干制剂于40℃稳定至少7天，至少14天或至少28天。
本公开还提供制备含有TIM3抗体的冻干制剂的复溶溶液的方法，其中包括将前述冻干制剂经复溶的步骤，其复溶所用溶液选自但不限于注射用水、生理盐水或葡萄糖溶液。
本公开还提供一种经前述含有TIM3抗体的冻干制剂的复溶溶液的方法制备的含有TIM3抗体的冻干制剂的复溶溶液。
本公开进一步提供一种制品或试剂盒，其包含装有本文所述任何稳定的药物组合物的容器。在一些实施方案中，该容器为中性硼硅玻璃管制注射剂瓶。
本公开还提供一种制品，其包括容器，该容器中装有前述的药物组合物或冻干制剂或冻干制剂的复溶溶液。
本公开还提供前述的药物组合物或冻干制剂或冻干制剂的复溶溶液或制品在制备与人TIM-3阳性细胞相关的疾病的诊断剂中的应用。
本公开还提供前述的药物组合物或冻干制剂或冻干制剂的复溶溶液或制品在制备与人TIM-3阳性细胞相关的疾病的治疗中的应用。所述与TIM-3阳性细胞相关的疾病没有限制，只要它是与表达TIM-3的细胞相关的疾病即可，包括但不限于癌症、自体免疫性疾病和过敏性疾病。
本公开还提供一种治疗或诊断癌症、自体免疫性疾病和过敏性疾病方法，包括给予对象施用前述的药物组合物或冻干制剂或冻干制剂的复溶溶液或制品。优选地，癌症包括血液癌、乳腺癌、子宫癌、结直肠癌、食道癌、胃癌、卵巢癌、肺癌、肾癌、直肠癌、甲状腺癌、宫颈癌、小肠癌、前列腺癌和胰腺癌。癌症的优选实例包括血液癌、食道癌、胃癌、结直肠癌、肝癌和前列腺癌。血液癌的实例包括急性髓性白血病(AML)、慢性髓性白血病(CML)、骨髓增生异常综合征(MDS)、多发性骨髓瘤、皮肤T细胞淋巴瘤(CTCL)、外周T细胞淋巴瘤(PTCL)、间变性大细胞淋巴瘤(ALCL)、急性淋巴细胞性白血病(ALL)、慢性淋巴细胞性白血病(CLL)、其他淋巴性白血病、NK细胞淋巴瘤、霍奇金氏淋巴瘤、非霍奇金氏淋巴瘤例如Burkitt淋巴瘤等等。自体免疫性疾病的具体实例包括类风湿关节炎、牛皮癣、克罗恩病、强硬性脊柱炎、多发性硬化症、I型糖尿病、肝炎、心肌炎、Sjogren综合征、移植排斥后的自体免疫性溶血性贫血、水疱性类天疱疮、格雷夫氏病、桥本甲状腺炎、系统性红斑狼疮(SLE)、重症肌无力、天疱疮、恶性贫血等。过敏性疾病的实例包括急性或慢性反应性气道疾病、支气 管哮喘、特应性皮炎、过敏性鼻炎、荨麻疹、PIE综合征、食物过敏、花粉热、过敏性鼻、支气管哮喘、特应性皮炎、过敏性休克。
本公开还提供前述的药物组合物或冻干制剂或冻干制剂的复溶溶液在制备用于治疗、抑制肿瘤细胞增殖或转移的疾病或病症的药物中的用途。
本公开进一步提供一种用于诊断或治疗与人TIM-3阳性细胞相关的疾病的方法，所述方法包括使用前述的药物组合物或冻干制剂或冻干制剂的复溶溶液。
如本领域技术人员所熟知的，本公开中所述各个实施方案的一项、一些或所有特性可以进一步组合以形成本公开的其它实施方案。本公开的以上实施方案和通过组合得到的其他实施方案通过下面的详述进一步说明。
附图说明
图1：候选TIM-3抗体的细胞结合实验曲线，数据显示候选抗体mAb-1701和mAb-1799对表达抗原的细胞均具有很强的结合活性；
图2A-图2C：在混合淋巴细胞反应实验中本公开的候选抗体和其抗原结合片段对IFNγ分泌的影响；图2A，h1701-009及其抗原结合片段对IFNγ分泌的影响；图2B，h1701-005及其抗原结合片段对IFNγ分泌的影响；图2C，h1701-009,h1799-005对IFNγ分泌的影响。结果显示h1701-009、h1799-005均能够有效刺激IFNγ的分泌；
图3A至图3D：PBMC活化实验结果图；图3A-3D分别展示FACS分析IFNγ阳性细胞比例、在所有细胞上IFNγ的几何平均值，IFNγ和TIM-3双阳性细胞比例(TIM-3+IFNγ+％)，TIM-3+细胞上IFNγ的几何平均值；结果显示h1701-009,h1799-005，AbTIM-3对活化后PBMC细胞内IFNγ表达均有影响，均不同程度提高了细胞内IFNγ阳性细胞的百分比以及IFNγ的表达；
图4A至图4B：SEB刺激实验结果图，图4A和图4B分别是IL12和IFNγ的ELISA检测结果，结果显示SEB刺激PBMC5天后，h1701-009,h1799-005以及AbTIM-3均有效升高了SEB诱导的IL12和IFNγ的分泌；
图5：TIM3抗体制剂成分JMP本公开软件模型拟合结果。
具体实施方式
术语的详细说明
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
本公开将申请PCT/CN2018/077190中的全部内容引入本申请。
“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲剂。将pH控制在适当范围中的缓冲剂的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸盐、 草酸盐、乳酸盐、磷酸盐、枸橼酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲剂。
“组氨酸盐缓冲剂”是包含组氨酸根离子的缓冲剂。组氨酸盐缓冲剂的实例包括组氨酸-盐酸盐，组氨酸-醋酸盐，组氨酸-磷酸盐，组氨酸-硫酸盐等缓冲剂，优选组氨酸-醋酸盐缓冲剂或组氨酸-盐酸缓冲剂，组氨酸-醋酸盐缓冲剂是组氨酸与醋酸配制而成，组氨酸盐缓冲剂是组氨酸与盐酸配制而成。
“枸橼酸盐缓冲剂”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-枸橼酸镁等。优选的枸橼酸盐缓冲剂是枸橼酸-枸橼酸钠。
“琥珀酸盐缓冲剂”是包括琥珀酸根离子的缓冲剂。琥珀酸盐缓冲剂的实例包括琥珀酸-琥珀酸钠、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选的琥珀酸盐缓冲剂是琥珀酸-琥珀酸钠。
“磷酸盐缓冲剂”是包括磷酸根离子的缓冲剂。磷酸盐缓冲剂的实例包括磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾、磷酸氢二钠-枸橼酸等。优选的磷酸盐缓冲剂是磷酸氢二钠-磷酸二氢钠。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠、醋酸组氨酸盐、醋酸-醋酸钾、醋酸醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲剂是醋酸-醋酸钠。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是保持抗体活性成分的稳定性，促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本文中，“药物组合物”和“制剂”并不互相排斥。
本公开中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂均为水。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经真空冷冻干燥步骤之后获得的制剂或药物组合物。
本文所用术语“约”、“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“基本上包含”可意味着其后所示的具体数值±20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。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“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本公开所述的药物组合物能够达到一种稳定的效果：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。
稳定的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℃)保存至少3个月、优选6个月、更优选1年，且甚至更优选地多达2年。另外，稳定的液体制剂包括这样的液体制剂：其在包括25℃的温度保存包括1个月、3个月、6个月在内的时段后表现出期望的特征。稳定性的典型的可接受的标准如下：通过SEC-HPLC测得，通常不超过约10％、优选不超过约5％的抗体单体发生降解。通过视觉分析，药物抗体制剂是淡黄色近无色澄明液体或者无色，或澄清至稍微乳白色。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％的减少。通常形成不超过约10％、优选不超过约5％的聚集。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体在药物制剂中“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。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本公开所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。
在本公开中，本公开所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本公开中，本公开所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR) 和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR1-3)。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
本公开中所述的“抗体或其抗原结合”或“功能片段”，指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，以及与抗体结合的Fv片段scFv片段。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。
本公开的术语“抗原结合位点”指抗原上连续或不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
术语“鼠源抗体”在本公开中为根据本领域知识和技能制备的对人TIM3的单克隆抗体。制备时用TIM3抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体(chimeric antibody)”，是将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。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小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，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的TIM3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TIM3嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG4。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的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构架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的异源性反应。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。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。
本公开中所述的“ADCC”，即antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A、L234A、L235A；IgG2/4嵌合体，IgG4的F234A/L235A突变。
本公开中所述的突变序列中的“突变”包括但不限于“回复突变”、“保守修饰”或“保守置换或取代”。本公开中所述的“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。
本公开所述的“突变序列”是指对本公开的核苷酸序列和/或氨基酸序列进行适当的替换、插入或缺失等突变修饰情况下，得到的与本公开的核苷酸序列和/或氨基酸序列具有不同百分比序列同一性程度的核苷酸序列和/或氨基酸序列。本公开中所述的序列同一性可以至少为85％、90％或95％，优选至少为95％。非限制性实施例包括85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。
术语“TIM3”是一种广泛表达的细胞膜表面的免疫球蛋白，又称整合素相关蛋白。信号调节蛋白(singal regulatory protein，SIRP)是抑制性受体超家族的一个成员，属于免疫球蛋白超家族，主要表达于巨噬细胞、树突状细胞和神经细胞表面，通过细胞表面受配体接触调节细胞的迁移和吞噬活性、免疫自稳及神经元网络。TIM3是人信号调节蛋白α(SIRPα)的胞外配体，通过与巨噬细胞表面SIRPα结合传导抑制性信号而降低吞噬活性，进而对固有免疫系统产生抑制作用，该信号被形象的描述为“don't eat me(别吃我)”信号。
术语“与TIM3结合”，指能与人TIM3相互作用。
术语“抗TIM3抗体”与“TIM3抗体”同意，均为与TIM3结合的抗体。
本公开中所述的融合蛋白是一种通过DNA重组，将得到的两个基因共表达的 蛋白产物。重组TIM3胞外区Fc融合蛋白通过DNA重组，把TIM3胞外区和人抗体Fc片段共表达的融合蛋白。所述的TIM3胞外区，是指TIM3蛋白表达在细胞膜以外的部分，序列见SEQID NO:1。
生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港实验室出版的《抗体实验技术指南》第5-8章和15章。小鼠可以用人TIM3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。本公开所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR区。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人TIM3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，用含调整过的缓冲剂的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。
“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。
“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。“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。“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，例如包含本公开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可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据 多种因素变化，例如患者的疾病状态、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。尽管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
本公开制剂可用于治疗与TIM-3阳性细胞相关的疾病。对与TIM-3阳性细胞相关的疾病没有限制，只要它是与表达TIM-3的细胞相关的疾病即可，例如癌症、自体免疫性疾病和过敏性疾病。癌症包括血液癌、乳腺癌、子宫癌、结直肠癌、食道癌、胃癌、卵巢癌、肺癌、肾癌、直肠癌、甲状腺癌、宫颈癌、小肠癌、前列腺癌和胰腺癌。癌症的优选实例包括血液癌、食道癌、胃癌、结直肠癌、肝癌和前列腺癌。血液癌的实例包括急性髓性白血病(AML)、慢性髓性白血病(CML)、骨髓增生异常综合征(MDS)、多发性骨髓瘤、皮肤T细胞淋巴瘤(CTCL)、外周T细胞淋巴瘤(PTCL)、间变性大细胞淋巴瘤(ALCL)、急性淋巴细胞性白血病(ALL)、慢性淋巴细胞性白血病(CLL)、其他淋巴性白血病、NK细胞淋巴瘤、霍奇金氏淋巴瘤、非霍奇金氏淋巴瘤例如Burkitt淋巴瘤等等。自体免疫性疾病的具体实例包括类风湿关节炎、牛皮癣、克罗恩病、强硬性脊柱炎、多发性硬化症、I型糖尿病、肝炎、心肌炎、Sjogren综合征、移植排斥后的自体免疫性溶血性贫血、水疱性类天疱疮、格雷夫氏病、桥本甲状腺炎、系统性红斑狼疮(SLE)、重症肌无力、天疱疮、恶性贫血等。过敏性疾病的实例包括急性或慢性反应性气道疾病、支气管哮喘、特应性皮炎、过敏性鼻炎、荨麻疹、PIE综合征、食物过敏、花粉热、过敏性鼻、支气管哮喘、特应性皮炎、过敏性休克等。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“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。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是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。
示例性抗体药物组合物(制剂)制备工艺：
第一步：取一定量的纯化的TIM3抗体溶液，用不含抗体的缓冲剂(如10mM，pH 5.5组氨酸-醋酸缓冲剂)进行溶剂置换(优选超滤)，经超滤膜至少6倍体积置换，蛋白浓缩到约70mg/mL。加入一定体积的蔗糖母液，混匀，使最终蔗糖浓度为80mg/mL。加入一定体积的聚山梨酯80母液，混匀，使最终聚山梨酯80浓度为0.4mg/mL。加10mM pH 5.5组氨酸-醋酸缓冲剂定容，使蛋白浓度为50mg/mL(其他待测试制剂或稳定性制剂参照相似步骤进行配制)。
产品经过滤后中控取样检测无菌。将原液过0.22μm PVDF滤芯，收集滤液。
第二步：调节装量至2.15ml，将滤液灌装于2ml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。
以下结合实施例进一步描述本公开，但这些实施例并非是对本公开范围的限制。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如参照冷泉港实验室出版的《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.TIM3抗体的制备
一、TIM-3抗原及检测用蛋白的制备
1.TIM-3抗原设计及表达
以UniProt Hepatitis A virus cellular receptor 2(人HAVCR2，人TIM-3，Uniprot号：Q8TDQ0)作为本公开TIM-3的模板，设计本公开涉及的抗原及检测用蛋白的氨基酸序列，可选的在TIM-3蛋白基础上融合不同的标签，分别克隆到pHr载体上(自产)或pTargeT载体上(promega，A1410)，在293细胞瞬转表达或CHO-S稳定表达纯化，获得编码本公开抗原及检测用蛋白。以下TIM-3抗原未特殊说明的均指人TIM-3。
TIM-3胞外区和hIgG1Fc的融合蛋白：TIM-3-Fc(SEQ ID NO：1)，用于小鼠免疫：
Figure PCTCN2019102839-appb-000001
注释：划横线部分为信号肽，斜体部分为Fc。
带Flag标签和His标签的TIM-3胞外区：TIM-3-Flag-His(SEQ ID NO：2)，用于检测：
Figure PCTCN2019102839-appb-000002
注释：划横线部分为信号肽，斜体部分为Flag-His标签。
人TIM-3全长氨基酸序列：用于构建TIM-3过表达细胞株，TIM-3-全长(SEQ ID NO：3)：
Figure PCTCN2019102839-appb-000003
注释：单横线部分为信号肽，双横线为跨膜区，波浪线为胞内区，没有划线部分为TIM-3胞外区。
2.TIM-3相关重组蛋白的纯化，以及杂交瘤抗体、重组抗体的纯化
2.1 TIM-3-Flag-His重组蛋白的纯化步骤：
将样品高速离心去除杂质，并浓缩至适当体积。利用PBS平衡NI-NTA亲和柱(QIAGEN，Cat No.30721)，冲洗2-5倍柱体积。将除杂后的细胞表达上清样品上柱。用PBS冲洗柱子，至A280读数降至基线。用PBS冲洗柱子，冲洗杂蛋白，并收集。依次用洗涤缓冲液(咪唑20mM)和洗脱缓冲液(咪唑300mM)洗脱目的蛋白，并收集洗脱峰。
收集的洗脱液用离子交换(Hiload 16/600 superdex 200柱)进一步纯化。用PBS平衡2个柱体积左右，保证pH7.4。将鉴定含目的蛋白的洗脱缓冲液(elution buffer)浓缩后上样，收集样品，SDS-PAGE和LC-MS鉴定为正确后分装备用。
2.2杂交瘤，重组抗体，Fc融合蛋白的纯化
将细胞表达上清样品高速离心去除杂质，杂交瘤表达上清用Protein G柱，重组抗体、Fc融合蛋白表达上清用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用100mM乙酸pH3.0洗脱目的蛋白，用1M Tris-HCl，pH8.0中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，去聚体的峰收集好后分装备用。
二、抗人TIM-3单克隆抗体的制备
1.动物免疫
抗人TIM-3单克隆抗体通过免疫小鼠产生。实验用SJL白小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。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 按以下方案免疫。免疫抗原为带Fc标签的人TIM-3胞外区(SEQ ID NO:1)。
免疫方案：用QuickAntibody-Mouse5W(KX0210041)对小鼠进行免疫。抗原与佐剂比例为1:1，10μg/只/次(首免/加强免疫)。抗原与佐剂迅速充分混匀后接种，接种时间为第1和第2次免疫间隔21天，之后每次免疫间隔14天。于每次免疫后7天取血，用ELISA方法确定小鼠血清中的抗体滴度。选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射20μg/只的生理盐水配制的抗原溶液。
2.脾细胞融合
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(
Figure PCTCN2019102839-appb-000004
CRL-8287 TM)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞以4-5×10 5/ml的密度用完全培养基(含20％FBS、1×HAT、1×OPI的DMEM培养基)重悬，100μl/孔种于96孔板中，37℃，5％CO 2孵育3-4天后，补充HAT完全培养基100μl/孔，继续培养3-4天至形成针尖般克隆。去除上清，加入200μl/孔的HT完全培养基(含20％FBS、1×HT和1×OPI的RPMI-1640培养基)，37℃，5％CO 2培养3天后进行ELISA检测。
3.杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用结合ELISA方法进行杂交瘤培养上清检测(见测试例1)。并将结合ELISA检测的阳性孔细胞上清进行TIM-3过表达细胞结合实验(见测试例2)。蛋白结合和细胞结合均为阳性的孔细胞及时进行扩增冻存保种和二到三次亚克隆直至获得单细胞克隆。
每次亚克隆细胞均需进行TIM-3结合ELISA、细胞结合实验检测。通过以上实验筛选得到杂交瘤克隆，并获得其分泌的抗体mAb-1701和mAb-1799，用无血清细胞培养法进一步制备抗体，按纯化实例纯化抗体，供在检测例中使用。
4.杂交瘤阳性克隆序列测定
从阳性杂交瘤中克隆序列过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,Cat No.15596-018)按照试剂盒说明书步骤提取RNA，用PrimeScript TM Reverse Transcriptase试剂盒反转录(Takara,Cat No.2680A)。将反转录得到的cDNA采用小鼠Ig-Primer Set(Novagen,TB326 Rev.B 0503)进行PCR扩增后送测序公司测序。得到mAb-1701和mAb-1799的重链和轻链可变区DNA序列对应的氨基酸序列：
mAb-1701重链可变区(SEQ ID NO:4)
Figure PCTCN2019102839-appb-000005
mAb-1701轻链可变区(SEQ ID NO:5)
Figure PCTCN2019102839-appb-000006
mAb-1799重链可变区(SEQ ID NO:6)
Figure PCTCN2019102839-appb-000007
mAb-1799轻链可变区(SEQ ID NO:7)
Figure PCTCN2019102839-appb-000008
上述mAb-1701和mAb-1799轻重链可变区序列中，下划线部分为CDR区，各序列依次顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4。其中各抗体轻重链中CDR序列如表1所示。
表1各重链及轻链CDR区序列
Figure PCTCN2019102839-appb-000009
三、抗人TIM-3鼠杂交瘤单克隆抗体的人源化
1.抗TIM-3抗体mAb-1701号的人源化
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，分别挑选与mAb-1701抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。其中氨基酸残基由Kabat编号系统确定并注释。
1.1杂交瘤克隆mAb-1701号人源化构架选择
鼠源抗体mAb-1701号的人源化轻链模板为IGKV1-33*01和hjk4.1，人源化重链模板为IGHV1-18*01和hjh4.1，人源化可变区序列如下：
h1701VH-CDR graft(SEQ ID NO：20)
Figure PCTCN2019102839-appb-000010
h1701VL-CDR graft(SEQ ID NO：21)
Figure PCTCN2019102839-appb-000011
Figure PCTCN2019102839-appb-000012
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
1.2 h1701号的模板选择和回复突变设计
具体突变设计见下表2：
表2 h1701的模板选择和回复突变设计
Figure PCTCN2019102839-appb-000013
注：如A43S表示依照自然顺序编号，将43位A突变回S。Grafted代表鼠抗体CDR植入人种系FR区序列。
表3 h1701人源化抗体重轻链可变区相互组合表
  h1701_VL.1 h1701_VL.1A
h1701_VH.1 h1701-005 h1701-006
h1701_VH.1A h1701-007 h1701-008
h1701_VH.1B h1701-009 h1701-010
h1701_VH.1C h1701-011 h1701-012
h1701_VH.1D h1701-013 h1701-014
h1701_VH.1E h1701-015 h1701-016
h1701_VH.1F h1701-017 h1701-018
注：该表表示各种突变组合所得的序列。如h1701-007表示，在人源化的鼠抗体h1701-007上的有轻链h1701_VL.1A、重链h1701_VH.1A两种突变。其它类推。
1701号人源化具体序列如下：
>h1701_VH.1(同h1701VH-CDR graft，SEQ ID NO：22)
Figure PCTCN2019102839-appb-000014
>h1701h1701_VH.1A(SEQ ID NO：23)
Figure PCTCN2019102839-appb-000015
>h1701_VH.1B(SEQ ID NO：24)
Figure PCTCN2019102839-appb-000016
>h1701_VH.1C(SEQ ID NO：25)
Figure PCTCN2019102839-appb-000017
>h1701_VH.1D(SEQ ID NO：26)
Figure PCTCN2019102839-appb-000018
>h1701_VH.1E(SEQ ID NO：27)
Figure PCTCN2019102839-appb-000019
>h1701_VH.1F(SEQ ID NO：28)
Figure PCTCN2019102839-appb-000020
>h1701_VL.1(同h1701VL-CDR graft，SEQ ID NO：29)
Figure PCTCN2019102839-appb-000021
>h1701_VL.1A(SEQ ID NO：30)
Figure PCTCN2019102839-appb-000022
2.抗TIM-3抗体mAb-1799号的人源化
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，分别挑选与mAb-1799号抗体同源性高的重链和轻链可变区种系基因作为模板，将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。其中氨基酸残基由Kabat编号系统确定并注释。
2.1杂交瘤克隆1799号人源化构架选择
鼠源抗体1799号的人源化轻链模板为IGKV1-39*01和hjk2.1，人源化重链模板为IGHV3-7*01和hjh4.1，人源化可变区序列如下：
h1799VH-CDR graft(SEQ ID NO：31)
Figure PCTCN2019102839-appb-000023
h1799VL-CDR graft(SEQ ID NO：32)
Figure PCTCN2019102839-appb-000024
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
2.2杂交瘤克隆1799号的模板选择和回复突变设计，见下表4：
表4 h1799的模板选择和回复突变设计
Figure PCTCN2019102839-appb-000025
注：如I48V表示依照自然顺序编号，将48位I突变回V。Grafted代表鼠抗体CDR植入人种系FR区序列。
表5鼠抗1799号人源化抗体重轻链可变区序列组合
Figure PCTCN2019102839-appb-000026
注：该表表示各种突变组合所得的序列。如h1799-005表示，在人源化的鼠抗体h1799-005上的有轻链h1799_VL.1A、重链h1799_VH.1两种突变。其它类推。
1799号人源化具体序列如下：
>h1799_VH.1(同h1799VH-CDR graft，SEQ ID NO：33)
Figure PCTCN2019102839-appb-000027
>h1799_VH.1A(SEQ ID NO：34)
Figure PCTCN2019102839-appb-000028
>h1799_VH.1B(SEQ ID NO：35)
Figure PCTCN2019102839-appb-000029
>h1799_VL.1(同h1799VL-CDR graft，SEQ ID NO：36)
Figure PCTCN2019102839-appb-000030
>h1799_VL.1A(SEQ ID NO：37)
Figure PCTCN2019102839-appb-000031
>h1799_VL.1B(SEQ ID NO：38)
Figure PCTCN2019102839-appb-000032
>h1799_VL.1C(SEQ ID NO：39)
Figure PCTCN2019102839-appb-000033
>h1799_VL.1D(SEQ ID NO：40)
Figure PCTCN2019102839-appb-000034
四、重组嵌合抗体以及人源化抗体的制备
抗体选用人重链IgG4/轻链kappa的恒定区与各可变区组合，在Fc段做了S228P突变来增加IgG4抗体的稳定性，也可选用本领域其它已知的突变来增加其性能。
重链恒定区序列如SEQ ID NO：41所示：
Figure PCTCN2019102839-appb-000035
轻链恒定区序列如SEQ ID NO：42所示：
Figure PCTCN2019102839-appb-000036
其中，h1799-005抗体重链全长序列如SEQ ID NO：73所示：
Figure PCTCN2019102839-appb-000037
h1799-005抗体轻链全长序列如SEQ ID NO：74所示：
Figure PCTCN2019102839-appb-000038
备注：上述h1799-005抗体全长序列中，斜体为FR区序列，下划线为CDR区序列，波浪线为恒定区序列。
1.重组嵌合抗体的分子克隆
杂交瘤筛选所获得的阳性抗体分子经过测序后，得到可变区编码基因序列。以测序所得序列设计首尾引物，以测序基因为模板，经过PCR搭建各抗体VH/VK基因片段，再与表达载体pHr(带信号肽及hIgG4/hkappa恒定区基因(CH1-FC/CL)片段)进行同源重组,构建重组嵌合抗体全长表达质粒VH-CH1-FC-pHr/VL-CL-pHr，形成Ch1701和Ch1799两个嵌合抗体。
2.人源化抗体的分子克隆
人源设计之后的抗体序列，经过密码子优化后产生人密码子偏好的编码基因序列，设计引物PCR搭建各抗体VH/VK基因片段，再与表达载体pHr(带信号肽及hIgG4/hkappa恒定区基因(CH1-FC/CL)片段)进行同源重组，构建人源化抗体全长表达质粒VH-CH1-FC-pHr/VL-CL-pHr。
3.重组嵌合抗体以及人源化抗体的表达与纯化
分别表达抗体轻重链的质粒以1：1.2的比例转染HEK293E细胞，6天后收集表达上清，高速离心去除杂质，用Protein A柱进行纯化。用PBS冲洗柱子，至A280读数降至基线。用pH3.0-pH3.5的酸性洗脱液洗脱目的蛋白，用1M Tris-HCl,pH8.0-9.0中和。洗脱样品适当浓缩后，利用PBS平衡好的凝胶层析Superdex200(GE)进一步纯化，以去除聚体，收集单体峰，分装备用。
五、h1701抗体的点突变
脱酰胺修饰是抗体中可能影响后期稳定性的一种常见的化学修饰，尤其是 CDR区域的部分氨基酸高度脱酰胺、氧化或者异构化修饰，一般选择尽量避免或者突变降低。根据加速稳定性实验和计算机模拟抗体结构和热点预测，h1701抗体重链CDR2中的NNG是易于发生脱酰胺化的位点，上述NNG分别位于h1701抗体重链可变区的第54-56位(自然顺序编号)，根据氨基酸性质和计算机抗体结构模拟技术，上述位点的氨基酸可以进行任意的氨基酸取代，优选地，h1701的CDR2突变体为：DIIPX 1X 2X 3GSKYNQKFKD(SEQ ID NO：43)，其中X 1X 2X 3分别为h1701抗体重链可变区第54-56位氨基酸残基，X 1选自Asn、Leu、Val、Met或Glu，X 2选自Asn、Glu、Met、His、Lys、Leu、Ala或Val，X 3选自Gly或Ala。
进一步地，含上述第54-56位突变的CDR2与含不同回复突变的FR区可形成如下的重链可变区：>h1701_VH.1-CDR2突变体(SEQ ID NO：44)
Figure PCTCN2019102839-appb-000039
>h1701_VH.1A-CDR2突变体(SEQ ID NO：45)
Figure PCTCN2019102839-appb-000040
>h1701_VH.1B-CDR2突变体(SEQ ID NO：46)
Figure PCTCN2019102839-appb-000041
>h1701_VH.1C-CDR2突变体(SEQ ID NO：47)
Figure PCTCN2019102839-appb-000042
>h1701_VH.1D-CDR2突变体(SEQ ID NO：48)
Figure PCTCN2019102839-appb-000043
>h1701_VH.1E-CDR2突变体(SEQ ID NO：49)
Figure PCTCN2019102839-appb-000044
>h1701_VH.1F-CDR2突变体(SEQ ID NO：50)
Figure PCTCN2019102839-appb-000045
h1701的HCDR2突变体相关的示例性序列，及包含相应CDR2突变体的人源化序列h1701_VH.1B-CDR2突变体(SEQ ID NO：46)见以下突变体及表6。
示例性地，把h1701-009的HCDR2中的NNG设计突变为NLG、NVG、NNA、NMA、NEA、NHA、NMG、NEG、NKG、NAG、NHG(上述重链可变区的CDR2氨基酸突变体的序列分别如SEQ ID NO：51-61所示)，通过分子克隆的方法进行表达质粒构建和293E表达，纯化后进一步检测突变体抗体的亲和力和稳定性。对h1701-009进行系列氨基酸突变具体相关序列包括但不限于表6所述
表6 h1701-009防脱酰胺化修饰突变体重链可变区序列
重链可变区 VH序列代号 对应的HCDR2序列
h1701-009 SEQ ID NO：24 DIIPNNGGSKYNQKFKD(SEQ ID NO：9)
h1701-009NLG SEQ ID NO：51 DIIPNLGGSKYNQKFKD(SEQ ID NO：62)
h1701-009NVG SEQ ID NO：52 DIIPNVGGSKYNQKFKD(SEQ ID NO：63)
h1701-009NNA SEQ ID NO：53 DIIPNNAGSKYNQKFKD(SEQ ID NO：64)
h1701-009NMA SEQ ID NO：54 DIIPNMAGSKYNQKFKD(SEQ ID NO：65)
h1701-009NEA SEQ ID NO：55 DIIPNEAGSKYNQKFKD(SEQ ID NO：66)
h1701-009NHA SEQ ID NO：56 DIIPNHAGSKYNQKFKD(SEQ ID NO：67)
h1701-009NMG SEQ ID NO：57 DIIPNMGGSKYNQKFKD(SEQ ID NO：68)
h1701-009NEG SEQ ID NO：58 DIIPNEGGSKYNQKFKD(SEQ ID NO：69)
h1701-009NKG SEQ ID NO：59 DIIPNKGGSKYNQKFKD(SEQ ID NO：70)
h1701-009NAG SEQ ID NO：60 DIIPNAGGSKYNQKFKD(SEQ ID NO：71)
h1701-009NHG SEQ ID NO：61 DIIPNHGGSKYNQKFKD(SEQ ID NO：72)
六、抗体性能及效果检测
以下用测试方法验证本公开抗体性能及有益效果：
测试例1：抗TIM-3抗体与人TIM-3过表达CHO-s细胞的结合实验
抗TIM-3抗体的结合力通过抗体与过表达TIM-3蛋白的CHO-S细胞的结合实验来检测。通过电转染的方法将TIM-3全长质粒(内部生产，SEQ ID NO:3)转染进CHO-S细胞中后加压筛选两周后，检测TIM-3的表达量。将过表达细胞固定于96孔板底后，抗体加入后信号的强弱被用于判断抗体和Tim-3过表达CHO-S细胞的结合活性，具体实验方法如下。所使用的阳性对照抗体为AbTim-3，序列来源自US20150218274A1表2，为我公司自行制备。
将细胞以4×10 5/ml密度，100μl/孔接种于96孔板中过夜培养。弃上清，用PBS洗三遍后，加入100μl/孔4％PFA室温固定半小时，PBS洗三遍。弃去液体后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μl/孔，37℃孵育箱孵育2.5小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(PH7.4PBS含0.05％tweeen-20)洗板5次后，加入50μl/孔用样品稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体或人源化抗体)，放于37℃孵育箱孵育1小时。孵育结束后用PBST洗板5次，加入100μl/孔用样品稀释液稀释的HRP标记的羊抗鼠二抗(Jackson Immuno Research，Cat No.115-035-003)或羊抗人二抗(Jackson Immuno Research，Cat No.109-035-003)，37℃孵育1小时。用PBST洗板6次后，加入50μl/ 孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-15min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在波长450nm处读取吸收值，计算TIM-3抗体对Tim-3过表达CHO-S细胞的结合EC50值。试验结果见图1及表7。
表7候选抗体在细胞结合实验中的EC50的测定
Figure PCTCN2019102839-appb-000046
结果表明，TIM-3抗体1701号和1799号及其人源化抗体与过表达人TIM-3全长蛋白的CHO-s细胞均有很好的结合活性。
对h1701-009抗体的HCDR2点突变抗体进行的人TIM-3过表达细胞结合活性检测，结果见表8。
表8候选抗体在细胞结合实验中的EC50的测定
候选抗体 结合ELISA EC50(nM)
h1701-009 0.503
h1701-009 NNA 0.515
h1701-009 NHA 0.530
h1701-009 NEA 0.667
h1701-009 NMA 0.629
h1701-009 NMG 0.603
h1701-009 NEG 0.673
h1701-009 NVG 0.717
h1701-009 NLG 0.637
h1701-009 NKG 0.438
h1701-009 NAG 0.512
h1701-009 NHG 0.317
结果显示，h1701-009抗体的HCDR2点突变抗体对过表达人TIM-3的CHO-s细胞仍显示出很强的结合活性。
测试例2：抗TIM-3抗体分组竞争实验
用pH7.4的PBS(Sigma，Cat No.P4417-100TAB)缓冲液将抗体A稀释至2μg/ml浓度，以50μl/孔的体积加入96孔酶标板(Corning，Cat No.CLS3590-100EA)中，于37℃孵育箱中放置2小时。弃去液体后，加入用PBS稀释的5％脱脂牛奶(光明脱脂奶粉)封闭液200μl/孔，37℃孵育箱孵育2.5小时或4℃放置过夜(16-18小时)进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(PH7.4PBS含0.05％tweeen-20)洗板5次后，加入50μl/孔用样品稀释液(PH7.4PBS含1％BSA)稀释至20μg/ml和4μg/ml的抗体B及终浓度为0.4μg/ml的生物素标记的TIM-3-Fc蛋白(内部生产，SEQ ID NO:1)的预混液，置37℃孵育箱孵育1小时。孵育结束后，弃去酶标板中的反应液，用PBST洗板5次后，加入100μl/孔用样品稀释液稀释的HRP标记的streptavidin(Sigma，Cat No.S2438)，37℃孵育1小时。用PBST洗板5次后，加入50μl/孔TMB显色底物(KPL，Cat No.52-00-03)，于室温孵育5-10min，加入50μl/孔1M H 2SO 4终止反应，用NOVOStar酶标仪在波长450nm处读取吸收值，计算不同抗体对Tim-3结合的竞争作用。
表9 mAb-1701和mAb-1799与TIM-3结合的竞争试验
Figure PCTCN2019102839-appb-000047
结果表明，在结合TIM-3时，mAb-1701与mAb-1799两者之间没有竞争关系。
测试例3：BIAcore检测抗TIM-3抗体亲和力实验
1、鼠源抗体BIAcore亲和力检测
按照鼠抗捕获试剂盒(Cat.#BR-1008-38，GE)说明书中所述的方法将鼠抗捕获抗体共价偶联于CM5生物传感芯片(Cat.#BR-1000-12，GE)上，从而亲和捕获待测抗体，然后于芯片表面流经TIM-3-His(Sino.Biol，Cat.10390-H03H)抗原，利用Biacore仪器实时检测反应信号从而获得结合和解离曲线，通过拟合得到亲和力数值。在实验中每个循环解离完成后，用鼠抗捕获试剂盒里配置的再生溶液将生物芯片洗净再生，结果如表10所示。
表10候选抗体的亲和力测定
Figure PCTCN2019102839-appb-000048
结果表明，TIM-3抗体mAb-1701号和mAb-1799号对人TIM-3蛋白有较强的 结合活性和亲和力。
2、嵌合抗体和人源化抗体BIAcore亲和力检测
按照人抗捕获试剂盒(Cat.#BR-1008-39，GE)说明书中所述的方法将人抗捕获抗体共价偶联于CM5生物传感芯片(Cat.#BR-1000-12，GE)上，从而亲和捕获待测抗体，然后于芯片表面流经TIM-3-Flag-His(内部生产，SEQ ID NO:2)抗原，利用Biacore仪器实时检测反应信号从而获得结合和解离曲线，通过拟合得到亲和力数值，见下表11。在实验中每个循环解离完成后，用人抗捕获试剂盒里配置的再生溶液将生物芯片洗净再生。
表11抗TIM-3抗体的亲和力
Figure PCTCN2019102839-appb-000049
结果表明，本公开筛选得到的人源化抗体对人TIM-3蛋白有较强的结合活性和亲和力。
对h1701-009抗体的HCDR2点突变抗体进行的BIAcore亲和力检测，结果见表12。
表12抗TIM-3抗体的亲和力
Figure PCTCN2019102839-appb-000050
Figure PCTCN2019102839-appb-000051
结果显示，上述h1701-009突变体仍具有很强的TIM-3亲和力。在NNG位点的突变并不影响抗体与抗原的亲和力。
测试例4：体外免疫细胞功能实验
为研究抗TIM-3抗体对T淋巴细胞激活效果，我们建立了3个体外功能实验：混合淋巴细胞反应实验(MLR assay)，PBMC活化实验以及超抗原金黄色葡萄球菌肠毒素B(SEB)刺激实验。
1.混合淋巴细胞反应实验
实验过程简单描述如下：
1)收集和纯化人外周血单核细胞(PBMC)；
2)将PBMC以2×10 6cell/ml密度种于6孔NUNC板中培养2小时；
3)去除非贴壁细胞，使用GM-CSF(100ng/ml)和IL-4(100ng/ml)刺激贴壁细胞5天，然后再和TNFα(100ng/ml)孵育2天诱导DC细胞成熟；
4)将成熟的DC细胞和不同献血者的PBMC以1:5的比例混合培养在预先包被10ng/ml CD3抗体的96孔板中，同时加入不同浓度的h1701-009，h1799-005，AbTIM-3以及阴性对照Fc-IgG；
5)5天后使用ELISA的方法检测上清中的IFNγ浓度(结果如图2A至图2C所示)。
结果显示，h1701-009，h1799-005以及AbTIM-3均有效刺激了IFNγ的分泌，h1701-009，h1799-005则显示出更强的刺激作用。
2.PBMC活化实验
实验过程简单描述如下：
1)收集和纯化人外周血单核细胞(PBMC)，并用CD3和CD28抗体包被96孔板，浓度均为10ng/ml；
2)将PBMC以1×10 5cell/孔的密度种于包被好的96孔板中。同时加入不同浓度的TIM-3抗体以及阴性对照Fc-IgG；
3)6天后收集细胞进行细胞内IFNγ和TIM-3染色，利用FACS分析IFNγ阳性细胞比例(IFNγ+％)，在所有细胞上IFNγGeo mean值，IFNγ和TIM-3双阳性细胞比例(TIM-3+IFNγ+％)，在TIM-3+细胞上IFNγGeo mean值(图3A至图3D)。
结果显示，h1701-009，h1799-005以及AbTIM-3不同程度提高了细胞内IFNγ阳性细胞的百分比以及IFNγ的表达，h1701-009，h1799-005的作用更强。
3.SEB刺激实验
实验过程简单描述如下：
1)收集和纯化人外周血单核细胞(PBMC)；
2)将PBMC以1×10 5cell/孔的密度种于96孔板中。同时加入1ng/ml SEB和不同浓度的TIM-3抗体以及阴性对照Fc-IgG；
3)5天后收集上清用ELISA检测IL12和IFNγ(分别见图4A和图4B)；
结果显示，h1701-009，h1799-005以及AbTIM-3均有效升高了SEB诱导的IL12和IFNγ的分泌，h1701-009，h1799-005在低剂量时显示出更高的优势。
测试例5：抗TIM-3人源化抗体，h1701-009和h1799-005的大鼠PK测定
SD大鼠12只，体重180-240g，购自西普尔-必凯实验动物有限公司。饲养期间自由摄取饲料和水，实验室环境适应性饲养不小于3天，12/12小时光/暗周期调节，温度16-26℃，相对湿度40-70％。实验开始前一天，对SD大鼠进行编号，随机分组，每组3只(2雄1雌)。实验当天，两组大鼠分别静脉注射受试药物h1701-009，给药剂量为3mg/kg和10mg/kg；两组大鼠分别静脉注射受试药物h1799-005，给药剂量为3mg/kg和10mg/kg。静脉注射体积为5ml/kg。
于给药前及给药后5min,8h,1d,2d,4d,7d,10d,14d,21d,28d各时间点采血。每只动物取全血0.2ml，不加抗凝剂，取血后在4℃放置30min，1000g离心15min，取上清置于EP管中，80℃保存。
采用ELISA方法检测血清中的抗体浓度，采用Winnolin软件计算受试药物的药动学参数。所得主要药动学参数见表13。
表13 h1799-005,h1701-009在大鼠体内的半衰期
Figure PCTCN2019102839-appb-000052
SD大鼠静脉注射给予10mg/kg抗TIM-3抗体h1701-009,h1799-005后，在大鼠体内的暴露量相近；抗体h1799-005在3,10mg/kg剂量下的暴露量增长与剂量增长基本呈线性关系，抗体h1701-009在3,10mg/kg剂量下的暴露量增长略高于剂量增长倍数；抗体h1701-009，h1799-005的消除半衰期相近。
测试例6：利用DSC检测h1701-009,h1799-005抗体的热稳定性
比较了不同的缓冲体系不同pH条件下的热稳定性情况，不同pH对应的示例性缓冲体系如10mM PB(pH7)，15mM His(pH6.0)，10mM醋酸盐(pH5.2)。将样品置换到对应缓冲液中，控制样品浓度在1mg/ml左右，利用MicroCal*VP-Capillary DSC(Malvern)进行检测。检测前，将各个样品及空白缓冲液用真空脱气器脱气1～2min。样品板每个孔加入400μl样品或空白缓冲液(仪器上样量为300μl)。最后两对孔板分别加入14％Decon 90和ddH 2O，以备清洗用，样品板加样完毕后，套上塑料软盖板。扫描温度从25℃开始到100℃结束，扫描速率60℃/h。具体结果如表14所示，在几个测试体系中h1701-009、h1799-005均表现了较好的热稳定性。
表14 h1701-009和h1799-005抗体不同pH热稳定性监测表
Figure PCTCN2019102839-appb-000053
测试例7：通过SEC-HPLC监测样品纯度考察一定浓度条件下周期性稳定性
示例性的条件比如将样品浓度控制在约50mg/ml，在PBS(pH7.4)体系中比较不同抗体在比如-80℃反复冻融5次及4℃和40℃保存一个月的稳定性情况。利用Xbridge protein BEH SEC 200A(Waters)HPLC柱子检测抗体纯度，通过一个月的考察，h1701-009、h1799-005均表现了良好的稳定性，一个月40℃加速SEC纯度无明显变化。
测试例8：抗体的化学稳定性
对h1701-009、h1799-005以及SEQ ID NO：51-55所示的h1701-009突变体进行化学稳定性检测。
取500μg待测抗体溶于500μl pH 7.4的PBS中，40℃水浴；分别于0、14、28天取样，用于酶解实验。将100μg不同时间点取样的样品溶于100μl 0.2M His-HCl，8M Gua-HCl，pH 6.0溶液中，加3μl 0.1g/mL DTT，50℃水浴1小时，后用0.02M His-HCl，pH 6.0的溶液超滤两次，加入3μL 0.25mg/mL的胰蛋白酶，37℃水浴酶解过夜。Agilent 6530 Q-TOF进行LC-MS检测分析，结果显示h1799-005在40度条件加速一个月均未发现明显的异构化、氧化及脱酰胺修饰，提示分子良好的化学稳定性。h1701-009检测中发现无明显的氧化及异构化修饰，但存在N54N55G56位点的强脱酰胺修饰，尝试对其进行系列氨基酸突变。其中示例性突变如NLG、NVG、NMA、NEA、NNA等，体外活性验证这些突变均不影响其生物活性，加速实验后进行LC-MS分析显示这些突变体均能够有效降低脱酰胺修饰，表15中所示为加速后不同突变体脱酰胺修饰比例。
表15 h1701-009抗体CDR2突变体化学稳定性监测表
  0天 一周 两周
h1701-009 11.16％ 17.55％ 34.62％
h1701-009NLG 0 0 0
h1701-009NVG 0 0 0
h1701-009NMA 0 0 0
h1701-009NEA 0 0 0
h1701-009NNA 0 0 13.58％
实施例2.抗TIM3抗体制剂缓冲体系pH值的筛选
用下列缓冲剂，配制抗体浓度为50mg/ml的抗TIM3抗体(h1799-005，实施例1制备获得，以下同)制剂：
1)10mM组氨酸-醋酸，pH 4.5
2)10mM组氨酸-醋酸，pH 5.0
3)10mM组氨酸-醋酸，pH 5.5
4)10mM组氨酸-醋酸，pH 6.0
取样品进行高温(40℃)稳定性研究，结果表明(见表16)，抗TIM3抗体较适pH范围为5.5～6.0。
表16.抗TIM3抗体pH值筛选结果
Figure PCTCN2019102839-appb-000054
注：D表示天，例如D7表示第7天。
实施例3.抗TIM3抗体制剂缓冲体系筛选
用下列缓冲剂，配制抗体浓度为50mg/ml的抗TIM3抗体(h1799-005)制剂：
1)10mM组氨酸-醋酸，pH 6.0
2)10mM枸橼酸-枸橼酸钠，pH 6.0
3)10mM组氨酸-盐酸，pH 6.0
4)10mM磷酸二氢钠-磷酸氢二钠，pH 6.0
5)10mM组氨酸-醋酸，pH 5.5
6)10mM醋酸-醋酸钠，pH 5.5
7)10mM琥珀酸-琥珀酸钠，pH 5.5
8)10mM琥珀酸-琥珀酸钠，pH 6.0
9)10mM组氨酸-盐酸，pH 5.5
10)10mM枸橼酸-枸橼酸钠，pH 5.5
取样品进行高温(40℃)和低温光照(4℃/4500lx)稳定性研究。结果表明(见表17)，抗TIM3抗体较适缓冲体系为组氨酸-盐酸(His-HCl)、醋酸-醋酸钠(AA)和组氨酸-醋酸(His-AA)缓冲液。考虑实际生产样品实际pH可能与目标pH发生一点漂移，故TIM3稳定性最优的缓冲体为His-AA体系，其次可选His-HCl和AA体系。
表17.抗TIM3抗体不同pH缓冲体系筛选试验结果
Figure PCTCN2019102839-appb-000055
Figure PCTCN2019102839-appb-000056
注：D表示天，例如D21表示第21天。
实施例4.抗TIM3抗体制剂中稳定剂的筛选
用不同糖及缓冲液，制备抗体浓度为50mg/ml的抗TIM3抗体(h1799-005)制剂：
1)10mM组氨酸-醋酸pH 6.0，70mg/ml蔗糖；
2)10mM组氨酸-醋酸pH 6.0，70mg/mlα,α-二水合海藻糖；
取配制好的上述制剂样品分别进行高温(40℃)和低温光照(5±3℃，4500lx)稳定性研究，结果见表18，结果表明蔗糖与海藻糖对抗TIM3抗体的稳定性作用相似。当蔗糖浓度为80mg/ml时，渗透压约为300mosm/kg，接近等渗，因此优选蔗糖浓度为80mg/ml。
表18.稳定剂筛选实验结果
Figure PCTCN2019102839-appb-000057
Figure PCTCN2019102839-appb-000058
注：D表示日，例如D21表示第21天。
实施例5.抗TIM3抗体制剂中表面活性剂的筛选
用下列不同浓度及种类表面活性剂的缓冲剂，制备蔗糖浓度为70mg/ml，抗体浓度为50mg/ml的抗TIM3抗体(h1799-005)制剂：
1)10mM组氨酸-醋酸pH 5.5；
2)10mM组氨酸-醋酸pH 5.5，0.2mg/ml聚山梨酯80；
3)10mM组氨酸-醋酸pH 5.5，0.4mg/ml聚山梨酯80；
4)10mM组氨酸-醋酸pH 5.5，0.6mg/ml聚山梨酯80；
5)10mM组氨酸-醋酸pH 5.5，0.4mg/ml聚山梨酯20；
将依照上述方法配制的1-5组抗TIM3抗体制剂样品分别稀释至0.9％氯化钠注射液中，蛋白浓度稀释为0.5mg/ml，观察各组样品稀释后不溶性微粒。试验结果见表19，结果表明，不低于0.4mg/ml聚山梨酯80对抗TIM3抗体的稳定性作用更好，因此选择0.4-0.6mg/ml聚山梨酯80，优选0.4mg/ml聚山梨酯80作为抗TIM3抗体的表明活性剂。
表19抗TIM3抗体聚山梨酯筛选-稀释结果
Figure PCTCN2019102839-appb-000059
Figure PCTCN2019102839-appb-000060
实施例6.制剂成分的综合筛选
为了进一步对蛋白浓度、缓冲体系种类、pH进行优化，运用JMP软件进行DOE实验设计，应用RSM模型得到一系列处方(如下)。在含下列不同离子强度的缓冲体系、不同pH的缓冲液中，制备不同抗体浓度，含80mg/ml蔗糖和0.4mg/ml聚山梨酯80的抗TIM3抗体(h1799-005)制剂：
1)20mM组氨酸-醋酸pH 5.5，60mg/ml TIM3抗体；
2)10mM组氨酸-醋酸pH 5.5，50mg/ml TIM3抗体；
3)20mM组氨酸-醋酸pH 5.5，50mg/ml TIM3抗体；
4)10mM组氨酸-醋酸pH 6.0，40mg/ml TIM3抗体；
5)30mM组氨酸-醋酸pH 5.5，50mg/ml TIM3抗体；
6)20mM组氨酸-醋酸pH 6.0，50mg/ml TIM3抗体；
7)30mM组氨酸-醋酸pH 5.0，60mg/ml TIM3抗体；
8)10mM组氨酸-醋酸pH 6.0，60mg/ml TIM3抗体；
9)20mM组氨酸-醋酸pH 5.0，50mg/ml TIM3抗体；
10)30mM组氨酸-醋酸pH 6.0，60mg/ml TIM3抗体；
11)20mM组氨酸-醋酸pH 5.5，40mg/ml TIM3抗体；
12)30mM组氨酸-醋酸pH 6.0，40mg/ml TIM3抗体；
13)30mM组氨酸-醋酸pH 5.0，40mg/ml TIM3抗体；
14)10mM组氨酸-醋酸pH 5.0，60mg/ml TIM3抗体；
15)10mM组氨酸-醋酸pH 5.0，40mg/ml TIM3抗体；
将依照上述方法配制的1-15组抗TIM3抗体制剂样品保存于40℃条件下进行稳定性分析，以IEC中性峰的差值和酸性峰差值为评价指标，采用最小二乘法对结果进行统计分析，试验结果见图5和表20，结果表明，离子强度为10mM，pH 5.5组氨酸-醋酸体系更利于抗TIM3抗体的稳定性，蛋白浓度在40～60mg/ml之间均较稳定，取中值50mg/ml作为抗TIM3抗体的最终蛋白浓度。
表20 TIM3抗体高温保存稳定性实验结果
Figure PCTCN2019102839-appb-000061
Figure PCTCN2019102839-appb-000062
注：M表示月，例如M1表示1个月。
实施例7.其它可选择制剂配方
此外，本公开还提供其它制剂配方的抗TIM3抗体药物制剂，包含但不限于：
(1)50mg/ml TIM3抗体(h1799-005)，80mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80和pH 6.0的10mM醋酸-醋酸钠缓冲剂；
(2)50mg/ml TIM3抗体(h1799-005)，80mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80和pH为5.5的20mM醋酸-醋酸钠缓冲剂；
(3)50mg/ml TIM3抗体(h1799-005)，80mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80和pH为6.5的10mM醋酸-醋酸钠缓冲剂；
(4)100mg/ml TIM3抗体(h1799-005)，80mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80和pH为5.4的10mM醋酸-醋酸钠缓冲剂；
(5)100mg/ml TIM3抗体(h1799-005)，80mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80和pH为5.4的20mM醋酸-醋酸钠缓冲剂；
(6)50mg/ml TIM3抗体(h1799-005)，90mg/ml海藻糖，0.6mg/ml的聚山梨醇酯80和pH为5.2的10mM醋酸-醋酸钠缓冲剂；
(7)60mg/ml TIM3抗体(h1799-005)，70mg/ml蔗糖，0.8mg/ml的聚山梨 醇酯80和pH为5.2的10mM醋酸-醋酸钠缓冲剂；
(8)40mg/ml TIM3抗体(h1799-005)，70mg/ml蔗糖，0.8mg/ml的聚山梨醇酯80和pH为5.4的10mM醋酸-醋酸钠缓冲剂；
(9)50mg/ml TIM3抗体(h1799-005)，90mg/ml蔗糖，0.5mg/ml的聚山梨醇酯80和pH为5.5的30mM醋酸-醋酸钠缓冲剂；
(10)40mg/ml TIM3抗体(h1799-005)，90mg/ml海藻糖，0.5mg/ml的聚山梨醇酯80和pH为6.0的20mM醋酸-醋酸钠缓冲剂；
(11)50mg/ml TIM3抗体(h1799-005)，70mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80和pH为5.2的10mM醋酸-醋酸钠缓冲剂；
(12)60mg/ml TIM3抗体(h1799-005)，75mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80和pH为5.2的10mM醋酸-醋酸钠缓冲剂；
(13)55mg/ml TIM3抗体(h1799-005)，75mg/ml蔗糖，0.5mg/ml的聚山梨醇酯80和pH为5.8的10mM醋酸-醋酸钠缓冲剂；
(14)45mg/ml TIM3抗体(h1799-005)，85mg/ml蔗糖，0.7mg/ml的聚山梨醇酯80和pH为5.8的10mM醋酸-醋酸钠缓冲剂；
(15)50mg/ml TIM3抗体(h1799-005)，80mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80和pH为5.9的10mM醋酸-醋酸钠缓冲剂；
(16)100mg/ml TIM3抗体(h1799-005)，80mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80和pH为5.5的10mM组氨酸-醋酸缓冲剂；
(17)70mg/ml TIM3抗体(h1799-005)，90mg/ml蔗糖，0.8mg/ml的聚山梨醇酯80和pH为5.5的5mM组氨酸-醋酸缓冲剂；
(18)1mg/ml TIM3抗体(h1799-005)，60mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80和pH为5.8的20mM组氨酸-醋酸缓冲剂；
(19)90mg/ml TIM3抗体，60mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80，和pH为5.8的20mM组氨酸-醋酸缓冲剂。

Claims (25)

  1. 一种药物组合物，其包含TIM3抗体或其抗原结合片段，以及缓冲剂，所述缓冲剂选自醋酸盐、组氨酸盐、琥珀酸盐、磷酸盐、枸橼酸盐缓冲剂，所述缓冲剂优选是醋酸盐缓冲剂或组氨酸盐缓冲剂，所述缓冲剂的pH为5.0-6.5，优选为5.5-6.0，最优选为大约5.5。
  2. 根据权利要求1所述的药物组合物，其中所述的醋酸盐缓冲剂选自醋酸-醋酸钠缓冲剂；所述的组氨酸盐缓冲剂选自组氨酸-醋酸缓冲剂或组氨酸-盐酸缓冲剂；所述的琥珀酸盐缓冲剂选自琥珀酸-琥珀酸钠缓冲剂；所述磷酸盐缓冲剂选自磷酸氢二钠-磷酸二氢钠缓冲剂；所述缓冲剂优选为组氨酸-盐酸、醋酸-醋酸钠或组氨酸-醋酸缓冲剂，最优选为组氨酸-醋酸缓冲剂。
  3. 根据权利要求1或2所述的药物组合物，其中所述缓冲液浓度为5mM至30mM，优选为10mM至20mM，最优选为大约10mM。
  4. 根据权利要求1至3任一所述的药物组合物，其中所述TIM3抗体或其抗原结合片段浓度为1mg/ml至100mg/ml，优选为40mg/ml至60mg/ml，最优选为约50mg/ml。
  5. 根据权利要求1至4任一所述的药物组合物，其还包括糖，所述糖优选为海藻糖或蔗糖，最优选为蔗糖。
  6. 根据权利要求5所述的药物组合物，其中所述糖浓度为50mg/ml至100mg/ml，选为70mg/ml至90mg/ml，最优选为约80mg/ml。
  7. 根据权利要求1至6任一所述的药物组合物，其还包括表面活性剂，所述表面活性剂优选为聚山梨醇酯，更优选为聚山梨醇酯80。
  8. 根据权利要求7所述的药物组合物，其中所述的表面活性剂的浓度为0.2mg/ml至0.8mg/ml，优选为0.4mg/ml至0.6mg/ml，最优选为约0.4mg/ml。
  9. 根据权利要求1至8任一所述的药物组合物，其包含：
    (a)大约1mg/ml-100mg/ml的TIM3抗体或其抗原结合片段，
    (b)大约5mM-30mM的醋酸盐缓冲剂或组氨酸盐缓冲剂，
    (c)大约50mg/ml-100mg/ml的糖，和
    (d)大约0.2mg/ml-0.8mg/ml的表面活性剂；
    所述药物组合物优选包含：(a)40mg/ml-60mg/ml的TIM3抗体或其抗原结合片段，(b)10mM-30mM的组氨酸-醋酸缓冲剂或醋酸-醋酸钠缓冲剂，pH约为5.0至6.0，(c)70mg/ml-90mg/ml的蔗糖或海藻糖，和(d)0.4mg/ml-0.6mg/ml的聚山梨醇酯；
    所述药物组合物最优选包含：(a)约50mg/ml的TIM3抗体或其抗原结合片段，(b)约10mM的组氨酸-醋酸缓冲剂或醋酸-醋酸钠缓冲剂，pH约为5.5，(c)约80mg/ml的蔗糖，和(d)约0.4mg/ml的聚山梨醇酯80。
  10. 根据权利要求1至9任一项所述的药物组合物，其中所述的TIM-3抗体或其抗原结合片段选自下面(i)或(ii)所述的任一种单克隆抗体或其抗原结合片段：
    (i)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、43和10所示的抗体重链可变区HCDR1-3区序列或与其具有至少95％序列同一性的氨基酸序列，和/或分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列或与其具有至少95％序列同一性的氨基酸序列；
    其中所述的SEQ ID NO:43如DIIPX 1X 2X 3GSKYNQKFKD序列所示，其中，X 1选自N、L、V、M或E，X 2选自N、E、M、H、K、L、A或V，X 3选自G或A；或
    (ii)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：14、15和16所示的抗体重链可变区HCDR1-3区序列或与其具有至少95％序列同一性的氨基酸序列，和/或分别如SEQ ID NO：17、18和19所示的抗体轻链可变区LCDR1-3区序列或与其具有至少95％序列同一性的氨基酸序列。
  11. 根据权利要求10所述的药物组合物，其中所述的TIM-3抗体或其抗原结合片段选自下面(a)至(m)中的任一种单克隆抗体或其抗原结合片段：
    (a)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、9和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (b)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、62和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (c)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、63和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (d)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、64和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (e)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、65和10 所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (f)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、66和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (g)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、67和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (h)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、68和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (i)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、69和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (g)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、70和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (k)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、71和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；
    (l)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：8、72和10所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：11、12和13所示的抗体轻链可变区LCDR1-3区序列；和
    (m)单克隆抗体或其抗原结合片段，其包含分别如SEQ ID NO：14、15和16所示的抗体重链可变区HCDR1-3区序列，和分别如SEQ ID NO：17、18和19所示的抗体轻链可变区LCDR1-3区序列。
  12. 根据权利要求10或11所述的药物组合物，其中所述的TIM-3抗体或其抗原结合片段选自鼠源抗体、嵌合抗体和人源化抗体。
  13. 根据权利要求12所述的药物组合物，其中所述的TIM-3抗体或其抗原结合片段为人源化抗体，其中所述的人源化抗体轻链和重链FR区序列分别来源于人种系轻链和重链或其突变序列。
  14. 根据权利要求13所述的药物组合物，其中所述的TIM-3抗体或其抗原结合片段含有如SEQ ID NO:20或31所示的重链可变区或其变体，所述变体是在SEQ  ID NO:20或31所示的重链可变区的FR区位置上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自在SEQ ID NO:20所示的D89E、R98T、G49A、M48I、M70L、R38K和V68A中的至少一个位点进行回复突变，或在SEQ ID NO:31中所示的Q3K和/或R87K位点进行回复突变。
  15. 根据权利要求13或14所述的药物组合物，所述的TIM-3抗体或其抗原结合片段，还含有如SEQ ID NO:21或32所示的轻链可变区或其变体，所述变体是在SEQ ID NO:21或32所示的轻链可变区上具有1-10个氨基酸的回复突变；优选的，所述回复突变选自在SEQ ID NO:21所示的A43S位点进行回复突变，或在SEQ ID NO:32所示的Q3K、I48V、K45Q、A43S和T85S中的至少一个位点进行回复突变。
  16. 根据权利要求1至15任一项所述的药物组合物，其中所述的TIM-3抗体或其抗原结合片段包含选自如SEQ ID NO:44、45、46、47、48、49和50中任一个所示的重链可变区序列和/或选自如SEQ ID NO:29或30所示的轻链可变区序列，或包含选自如SEQ ID NO:22、23、24、25、26、27和28中任一个所示的重链可变区序列和/或选自如SEQ ID NO:29或30所示的轻链可变区序列，或包含选自如SEQ ID NO:33、34和35中任一个所示的重链可变区序列和/或选自如SEQ ID NO:36、37、38、39和40中任一个所示的轻链可变区序列，或包含选自SEQ ID NO:51、52、53、54、55、56、57、58、59、60和61中任一个所示的重链可变区序列和/或选自SEQ ID NO:29或30所示的轻链可变区序列，或包含如SEQ ID NO:4所示的重链可变区序列和/或如SEQ ID NO:5所示的轻链可变区序列，或包含如SEQ ID NO：6所示的重链可变区序列和/或如SEQ ID NO:7所示的轻链可变区序列；
    优选地包含：
    如SEQ ID NO:24所示的重链可变区序列和如SEQ ID NO:29所示的轻链可变区序列；或
    包含如SEQ ID NO:33所示的重链可变区序列和如SEQ ID NO:36所示的轻链可变区序列；或
    包含如SEQ ID NO:51所示的重链可变区序列和如SEQ ID NO:29所示的轻链可变区序列；或
    或如SEQ ID NO:52所示的重链可变区序列和如SEQ ID NO:29所示的轻链可变区序列。
  17. 根据权利要求1至16任一项所述的药物组合物，其中所述抗体进一步包括人抗体恒定区；优选包含如SEQ ID NO:41所示的人重链恒定区序列和如SEQ ID NO:42所示的人轻链恒定区序列；最优选的，所述抗体包含如SEQ ID NO:73所示 的重链和如SEQ ID NO:74所示的轻链。
  18. 根据权利要求1至17任一项所述的药物组合物，其包含TIM3抗体或其抗原结合片段，其中所述抗体与权利要求10至17任一项所述的抗体或其抗原结合片段竞争结合TIM-3。
  19. 制备权利要求1至18任一项所述的药物组合物的方法，包括将TIM3抗体原液经缓冲液置换的步骤。
  20. 一种含TIM3抗体的冻干制剂，其中所述的冻干制剂通过将权利要求1至18任一项所述的药物组合物经冷冻干燥获得。
  21. 一种含有TIM3抗体的复溶溶液，其特征在于所述复溶溶液是通过将权利要求20所述的冻干制剂经复溶制备获得。
  22. 一种制品，其包括容器，该容器中装有如权利要求1至18任一项所述的药物组合物或权利要求20所述的冻干制剂或权利要求21所述的复溶溶液。
  23. 根据权利要求1至18任一项所述的药物组合物或权利要求20所述的冻干制剂或权利要求21所述的复溶溶液或权利要求22所述的制品在制备用于治疗、诊断与人TIM-3阳性细胞相关的疾病的药物中的应用。
  24. 一种治疗癌症、自体免疫性疾病和过敏性疾病方法，包括将治疗有效量的权利要求1至18任一项所述的药物组合物或权利要求20所述的冻干制剂或权利要求21所述的复溶溶液或权利要求22所述制品给予所述疾病患者。
  25. 根据权利要求24所述的方法，其中所述的癌症选自肺癌、鳞状细胞肺癌、黑素瘤、肾癌、乳腺癌、IM-TN乳腺癌、结肠直肠癌、白血病、膀胱癌、宫颈癌、结肠癌、胆囊癌、喉癌、肝癌、甲状腺癌、胃癌、唾液腺癌、前列腺癌，胰腺癌和默克尔细胞癌；其中所述的自体免疫性疾病选自多发性硬化症、I型糖尿病、类风湿性关节炎、硬皮病、克隆氏病、牛皮癣、系统性红斑狼疮(SLE)和溃疡性结肠炎。
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