WO2022111612A1 - Preparation comprising anti-tigit/pd-1 bispecific antibody, method for preparing same, and use thereof - Google Patents

Preparation comprising anti-tigit/pd-1 bispecific antibody, method for preparing same, and use thereof Download PDF

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Publication number
WO2022111612A1
WO2022111612A1 PCT/CN2021/133345 CN2021133345W WO2022111612A1 WO 2022111612 A1 WO2022111612 A1 WO 2022111612A1 CN 2021133345 W CN2021133345 W CN 2021133345W WO 2022111612 A1 WO2022111612 A1 WO 2022111612A1
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antibody
tigit
formulation
liquid
cancer
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PCT/CN2021/133345
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French (fr)
Chinese (zh)
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曹魏
马丽强
汪音爵
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信达生物制药(苏州)有限公司
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Publication of WO2022111612A1 publication Critical patent/WO2022111612A1/en

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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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to the field of antibody formulations. More specifically, the present invention relates to T cell immune receptors (TIGIT) and anti-programmed death receptor 1 (PD- 1) Pharmaceutical formulations, especially stable liquid formulations, of bispecific antibodies (also known as anti-TIGIT/PD-1 bispecific antibodies), and methods for preparing said pharmaceutical formulations, and treatment of said pharmaceutical formulations and/or prophylactic use.
  • T cell immune receptors T cell immune receptors
  • PD- 1 anti-programmed death receptor 1
  • immune checkpoint immune checkpoint
  • the activation of the inhibitory signaling pathway of the immune checkpoint leads to the inability of T lymphocytes to effectively exert the killing effect on tumors (Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2): 130-146), which on the one hand leads to drugs that target only targets on tumor cells (eg trastuzumab) Monoclonal antibody) has poor antitumor effect.
  • Programmed death protein-1 is an important immune checkpoint protein, a 55kDa type I transmembrane protein, mainly inducibly expressed on the surface of activated T cells, and also expressed on B cells and NK cells. , monocytes, DC cells and other cells.
  • Two cell surface glycoprotein ligands for PD-1 have been identified, programmed death protein ligand 1 (PD-L1) and programmed death protein ligand 2 (PD-L2).
  • the ligand of PD-1 is highly expressed on many cancer cells. The binding of PD-1 to PD-1 ligands can lead to T cell apoptosis, immune unresponsiveness, T cell "exhaustion” and secretion of IL-10, etc.
  • Nivolumab (trade name ) is a fully humanized IgG4 antibody molecule
  • Pembrolizumab (trade name ) are humanized IgG4 antibody molecules.
  • the anti-PD-1 monoclonal antibody can inhibit the binding of PD-1 to its ligands PD-L1 and PD-L2 after binding to PD-1 on T lymphocytes, thereby promoting T lymphocyte activation, proliferation and immune generation.
  • TIGIT is a co-inhibitory receptor expressed on activated and depleted T cells.
  • TIGIT binds to the poliovirus receptor (PVR, also known as CD155) on tumor cells and is able to reverse signaling into tumor cells, resulting in the secretion of T-cell inhibitory cytokines.
  • PVR poliovirus receptor
  • CD155 is considered to be the dominant ligand for TIGIT
  • TIGIT can also interact with CD112 and CD113 (Blake et al., Clin CancerRes; 2016; 22(21):5182-5188). The role of TIGIT as an inhibitory immune checkpoint receptor has been investigated.
  • TIGIT is part of the CD226/TIGIT pathway, in which TIGIT not only competes with the co-stimulatory immune receptor CD226 for binding to CD155, but also directly interacts with CD226 in the cell membrane and blocks CD226 homodimerization.
  • Anti-TIGIT antibodies are known in the art and include those disclosed in US 2016/0355589, US 2017/143825, US 2017/088613, US 2016/376365, US 2018/169238, US 2016/176963, and US 2019/100591 .
  • no anti-human TIGIT antibody alone or in combination with an anti-human PD-L1 or anti-human PD-1 antibody, has received regulatory approval for therapeutic use in humans.
  • no bispecific antibodies targeting TIGIT and PD-1 or TIGIT and PD-L1 are approved by regulatory authorities for therapeutic use in humans. Therefore, there is a need for additional treatments that target immune checkpoint pathways.
  • the present invention meets the above needs by providing a pharmaceutical formulation containing an anti-TIGIT/PD-1 bispecific antibody protein that specifically binds TIGIT and PD-1.
  • the antibodies used in the present invention are ineffective in effector function, ie, engineered to minimize Fc receptor binding.
  • the antibodies of the present invention do not contain a native human IgGl framework that can lead to T regulatory cell depletion and adverse events in the immune response.
  • the anti-human TIGIT/anti-human PD-1 bispecific antibodies of the present invention contain different types of light chains, wherein the anti-human TIGIT arm light chain is a kappa light chain, and the anti-human PD-1 light chain is Lambda light chains, which facilitate heteromeric bispecific antibody formation by reducing the likelihood of light chain-light chain dimerization.
  • the present invention provides an anti-human TIGIT/anti-human PD-1 bispecific molecule that minimizes Fc receptor binding, minimizes oxidation, promotes assembly of heteromers, and interacts with human TIGIT/PD-1 and cynomolgus TIGIT /PD-1 cross-reacts and demonstrates in vivo efficacy in established tumor models.
  • the anti-TIGIT/PD-1 bispecific antibodies used in the present invention exhibited synergistic effects in anti-tumor effects.
  • antibody formulations of the invention In addition to enabling the antibody formulations of the invention to be formulated in a manner suitable for administration to a subject, they also maintain their stability during storage and subsequent use.
  • the present invention provides a liquid antibody formulation comprising (i) an anti-TIGIT/PD-1 bispecific antibody protein; (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant , preferably the pH of the antibody formulation is about 4.5-6.8.
  • the anti-TIGIT/PD-1 bispecific antibody protein in the antibody preparation of the present invention comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a specific binding TIGIT (SEQ ID NO: 31) or its extracellular structure
  • the first VH/VL unit of the domain eg, SEQ ID NO:32
  • the second half-antibody comprises a Second VH/VL unit.
  • the anti-TIGIT/PD-1 bispecific antibody protein is capable of acting at less than about 10-7 M, preferably about 10-8 M, and more preferably about 5 nM, 4 nM, 3 nM, 2 nM, 1 nM , KD of 0.5nM or lower binds to PD -1 on the surface of T lymphocytes, and is capable of binding at less than about 10-7 M, preferably about 10-8 M and more preferably about 5nM, 4nM, 3nM, 2nM , 1 nM, 0.5 nM or lower KD binds to TIGIT such that the antibody can be used as a bispecific therapeutic and/or prophylactic targeting PD -1 molecule and TIGIT molecule.
  • the first VH/VL unit comprises the heavy and light chain complementarity determining region (CDR) amino acid sequences SEQ ID NOs: 1-6 or has one or more of the 6 CDRs A sequence of one, two, three, four, five, six or more amino acid changes (eg, amino acid substitutions or deletions); and/or a second VH/VL unit comprising heavy and light chain complementarity determinations Region (CDR) amino acid sequence SEQ ID NOs: 7-12 or one, two, three, four, five, six or more amino acid changes with one or more of the 6 CDRs (eg, amino acid substitutions or deletions).
  • CDR heavy and light chain complementarity determining region
  • the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a first VH/VL unit that specifically binds TIGIT and the second half antibody comprising a second VH/VL unit that specifically binds PD-1, wherein the first VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable sequence of SEQ ID NO: 13/SEQ ID NO: 14 region sequence, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the paired heavy chain variable region sequence/light chain variable region sequence , a sequence of 99% or more sequence identity, and wherein the second VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 17/SEQ ID NO: 18 , or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the paired heavy chain
  • the anti-TIGIT/PD-1 bispecific antibody protein is a heterodimer, wherein the first half antibody and the second half antibody each form two monovalent binding arms. In one embodiment, the two monovalent binding arms are linked by a disulfide bond.
  • the first monovalent binding arm is at a concentration of less than about 10-7 M, preferably about 10-8 M and more preferably about 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM or less KD binds to TIGIT; in a specific embodiment, the second monovalent binding arm is less than about 10-7 M, preferably about 10-8 M and more preferably about 5nM, 4nM, 3nM, 2nM, 1nM, 0.5 A KD of nM or lower binds to PD-1; in another embodiment, the bispecific antibody protein has the same or similar quaternary structure as the common monospecific antibody protein.
  • heavy chain constant regions of the first and second half antibodies in the anti-TIGIT/PD-1 bispecific antibody protein preferably the heavy chain constant regions of IgG1, IgG2 or IgG4 immunoglobulins, or substantially identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to sequences. More preferably, the heavy chain constant region is, or is substantially identical to (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).
  • the heavy chain of the antibody forms at least one disulfide bond with the light chain of the antibody, and the two heavy chains of the antibody form at least one disulfide bond.
  • the antibody is human IgG1 engineered to reduce the binding of the antibody to Fc ⁇ receptors.
  • the anti-TIGIT/PD-1 bispecific antibody protein comprises a heavy chain constant region used in IgG1 (eg, human IgG1). In yet another embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a heavy chain constant region for IgG4 (eg, human IgG4).
  • the Fc domains of the two heavy chains of an anti-TIGIT/PD-1 bispecific antibody comprise hinge regions with "CPPC" amino acid residues, respectively, and/or Y349C and S354C, respectively (according to Kabat's "EU numbering" "), whereby the anti-PD-1 half antibody and the anti-TIGIT half antibody form an interchain disulfide bond in the Fc region, thereby stabilizing the correct pairing of the anti-PD-1 half antibody and the anti-TIGIT half antibody.
  • the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises or has at least 90% of the heavy chain sequence of SEQ ID NO: 21 , a heavy chain sequence that is 95%, 98% or 99% identical, and a light chain sequence that is at least 90%, 95%, 98% or 99% identical to the light chain sequence of SEQ ID NO: 22, and wherein the second half-antibody comprises the heavy chain sequence of SEQ ID NO:23 or a heavy chain sequence at least 90%, 95%, 98% or 99% identical thereto, and the light chain sequence of SEQ ID NO:24 or has A light chain sequence having at least 90%, 95%, 98% or 99% identity.
  • the anti-TIGIT/PD-1 bispecific antibody protein is derived from HEK293 cells or HEK293T, HEK293F, HEK293E cells transformed on the basis of HEK293 cells; CHO cells or transformed on the basis of CHO cells The obtained anti-TIGIT/PD-1 bispecific antibody protein recombinantly expressed in CHO-S, CHO-dhfr-, CHO/DG44, and ExpiCHO.
  • the concentration of anti-TIGIT/PD-1 bispecific antibody protein in the liquid antibody formulation of the present invention is about 1-200 mg/ml, preferably about 1-150 mg/ml, more preferably about 10-100 mg/mL, eg, about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/mL.
  • the buffer in the liquid formulation of the present invention is selected from the group consisting of histidine, histidine hydrochloride, glutamate, phosphate, acetate, citrate and tris and their Combinations, preferably histidine, histidine hydrochloride and combinations thereof.
  • the concentration of buffer in the liquid antibody formulation of the invention is about 0.5-200 mM, about 1-100 mM, about 5-50 mM, about 5-30 mM, or about 5-20 mM.
  • the buffer used in the formulations of the present invention is about 10 mM histidine.
  • the buffer used in the formulation of the present invention is a combination of about 5 mM histidine and about 5 mM histidine hydrochloride.
  • the buffering agents used in the formulations of the present invention can control the pH of the formulations of the present invention to a pH in the range of about 4.5-6.8, preferably about 5.0-6.5, more preferably about 6.0 pH.
  • the antibody formulation of the invention has a pH of about 4.5, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.5, 6.6, 6.7, or 6.8.
  • the stabilizer is selected from saccharides, polyols, amino acids or their salts and combinations thereof, preferably, the saccharides are selected from but not limited to: sucrose, dextrose, lactose, maltose, Trehalose, cyclodextrin, maltodextrin and dextran, the polyol is selected from but not limited to: mannitol, sorbitol and xylitol, the amino acid or its salt is selected from but not limited to arginine , arginine (preferably arginine hydrochloride) and combinations thereof, preferably arginine hydrochloride.
  • the stabilizer is sorbitol.
  • the stabilizer is arginine hydrochloride.
  • the stabilizer is sorbitol, arginine, an arginine salt (preferably arginine hydrochloride), or a combination thereof.
  • the stabilizer of the present invention is present in the liquid formulation of the present invention at about 1-1000 mM, about 10-1000 mM, preferably about 20-800 mM, such as about 50-500, about 100-400, about 100- Present at a concentration of 300 or about 100-200 mM, eg, about 10, 20, 50, 80, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM.
  • the stabilizer of the present invention may also contain antioxidants, EDTA and/or disodium edetate, which are selected from but not limited to: homocysteine, cysteine, cystathionine, methionine, glutathione Cysteine, and peptides comprising any one of homocysteine, cysteine, cystathionine, methionine, and glutathione.
  • antioxidants EDTA and/or disodium edetate
  • the total concentration of stabilizer is as previously described, wherein the concentration of antioxidant is about 1-50 mM, preferably about 5-40 mM, such as about 5, 10, 20, 30, 40 mM; EDTA and/or disodium edetate at a concentration of about 0.001-0.5 mg/ml, eg 0.005-0.02 mg/ml, preferably 0.01 mg/ml.
  • the surfactant in the liquid formulation of the present invention is a nonionic surfactant, eg, an alkyl poly(ethylene oxide).
  • Particular nonionic surfactants that can be included in the formulations of the present invention include, for example, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; Nick et al.
  • polysorbate-80 is included as a surfactant in the liquid formulation of the present invention.
  • the concentration of surfactant in the liquid antibody formulation of the present invention is about 0.01-10 mg/ml, preferably about 0.05-5, about 0.05-2, about 0.1-5, 0.2-2, 0.3-1, 0.4-0.8, 0.5-0.6 mg/ml, eg about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml.
  • the liquid preparation is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection. In one embodiment, the liquid formulation is an intravenous infusion.
  • liquid antibody formulation of the present invention comprises:
  • pH of the liquid formulation is about 5.0-6.5, preferably about 6.0.
  • liquid antibody formulation of the present invention comprises:
  • pH of the liquid formulation is about 5.0-6.5, preferably about 6.0.
  • liquid antibody formulation of the present invention comprises:
  • pH of the liquid formulation is about 5.0-6.5, preferably about 6.0.
  • the present invention provides a solid antibody preparation obtained by subjecting the liquid antibody preparation of the present invention to a solidification treatment.
  • the solidification treatment is carried out by, for example, a crystallization method, a spray-drying method, or a freeze-drying method.
  • the solid antibody formulation is, for example, in the form of a lyophilized powder for injection.
  • Solid antibody formulations can be reconstituted in a suitable vehicle prior to use to form a reconstituted formulation of the invention.
  • the reconstituted formulation is also a liquid antibody formulation of the present invention.
  • the appropriate vehicle is selected from water for injection, organic solvent for injection, including but not limited to oil for injection, ethanol, propylene glycol, etc., or a combination thereof.
  • the liquid formulations of the present invention are stable for long-term storage, eg, at least 24 months or more.
  • the liquid formulation of the present invention may be at about -80°C to about 45°C, eg, -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about Store at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months at 35°C, about 38°C, about 40°C, about 42°C, or about 45°C , at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months , at least 36 months, or longer, and is stable.
  • the liquid formulations of the present invention are storage stable for at least 24 months. In yet another embodiment, the liquid formulations of the present invention are stable at at least 40°C. In yet another embodiment, the liquid formulations of the present invention are stable at about 2°C-8°C for at least 3 months, preferably at least 12 months, more preferably at least 24 months. In one embodiment, the liquid formulations of the present invention are stable at room temperature or, for example, about 25°C for at least 2 months, preferably at least 3 months, more preferably at least 6 months. In yet another embodiment, the liquid formulations of the present invention are stable at about 40°C for at least 2 weeks, preferably at least 1 month.
  • the stability of the formulation after storage can be indicated by detecting changes in the formulation's appearance, visible foreign matter, protein content, purity, and/or charge variants. In one embodiment, it can be in a forced experiment under high temperature stress, eg after storage at 40°C ⁇ 2°C for at least 1 week, 2 weeks or preferably 1 month, or in an accelerated experiment, eg at 25°C ⁇ 2°C
  • the stability of the liquid formulations of the invention is tested after storage for at least 1 month or 2 months, or in long-term experiments, eg, after storage at 5°C ⁇ 3°C for at least 2 months or 3 months.
  • the stability of the liquid formulations of the present invention is visually inspected, wherein the liquid formulations of the present invention remain clear to slightly opalescent in appearance, colorless to pale yellow liquids, and free of foreign matter. In one embodiment, no visible foreign matter is present in the formulation upon visual inspection under a clarity tester. In one embodiment, the stability of the liquid formulations of the invention is checked after storage by measuring the change in protein content, wherein the rate of change in protein content relative to the initial value on day 0 of storage, for example by ultraviolet spectrophotometry (UV) No more than 20%, preferably no more than 10%, eg 7-8%, more preferably no more than 5%.
  • UV ultraviolet spectrophotometry
  • the stability of the liquid formulation of the present invention is examined by determining the change in purity of the liquid formulation of the present invention after storage, wherein by size exclusion high performance liquid chromatography (SEC-HPLC), relative to storage 0 From the initial value of the day, the change in monomer purity is no more than 10%, eg, no more than 5%, 4%, 3%, eg, no more than 1-2%, preferably no more than 1%.
  • SEC-HPLC size exclusion high performance liquid chromatography
  • the stability of the liquid formulation of the present invention is checked by determining the change in purity of the liquid formulation of the present invention after storage, wherein by non-reducing and/or reducing sodium dodecyl sulfate capillary electrophoresis (CE- SDS) method, the change value of monomer purity decreased by no more than 10%, such as no more than 5%, 4%, 3%.
  • CE- SDS non-reducing and/or reducing sodium dodecyl sulfate capillary electrophoresis
  • the stability of the liquid formulations of the invention is tested by imaging capillary isoelectric focusing electrophoresis (iCIEF) after storage, wherein the antibody's charge variant (principal component, acidic The sum of the variation values of components and basic components) does not exceed 50%, for example, does not exceed 40%, 30%, 20%, 10%, 5%.
  • iCIEF capillary isoelectric focusing electrophoresis
  • the formulation is stable after storage, eg, at 2-8°C for at least 24 months, or at room temperature for at least 3 months, or at 40°C ⁇ 2°C for 1 month , preferably with one or more of the following characteristics:
  • the preparation has a purity greater than 90%, preferably greater than 95%, 96%, 97%, 98%, 99%, as measured by the SEC-HPLC method;
  • the preparation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, 98%, as measured by the reduced or non-reduced CE-SDS method;
  • the relative binding activity of the anti-TIGIT/PD-1 bispecific antibody protein in the formulation is 70%-130%, eg, 70%, 80%, relative to the initial value on storage day 0, as measured by ELISA , 90%, 100%, 110%, 120%, 130%.
  • the invention provides a delivery device comprising a liquid antibody formulation or a solid antibody formulation of the invention.
  • the delivery device of the invention is provided in the form of a prefilled syringe containing the liquid antibody formulation or solid antibody formulation of the invention, eg, for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion.
  • the present invention provides a method of delivering an anti-TIGIT/PD-1 bispecific antibody protein to a subject, eg, a mammal, comprising the step of administering to said subject a liquid antibody formulation or solid antibody formulation of the invention , the delivery is carried out, for example, by a delivery device using a prefilled syringe.
  • the present invention provides a liquid antibody formulation or solid antibody formulation of the present invention for use in the manufacture of a delivery device for the treatment, prevention or delay of a disorder associated with the TIGIT signaling pathway and the PD-1 signaling pathway in a subject (such as, prefilled syringes) or drugs, such as various hematological diseases and solid tumors, including but not limited to leukemia, lymphoma, myeloma, brain tumor, head and neck cancer, head and neck squamous cell carcinoma, lung cancer Such as non-small cell lung cancer or small cell lung cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma , prostate cancer, bladder cancer, renal cell carcinoma, melanoma.
  • a subject such as, prefilled syringes
  • Figure 1 Shows the effect of antibody-A on body weight of LOVO mixed PBMC tumor-bearing mice.
  • Figure 2 Shows the effect of antibody-A on tumor tissue growth in LOVO mixed PBMC tumor-bearing mice.
  • Figure 3 Shows the effect of low-dose antibody-A on tumor tissue growth in LOVO mixed PBMC tumor-bearing mice.
  • Figure 4 Shows the effect of high doses of antibody-A on tumor tissue growth in LOVO mixed PBMC tumor-bearing mice.
  • Figure 5 Shows individual tumor profiles of LOVO mixed PBMC tumor-bearing mice.
  • Figure 6 Shows the effect of antibody-A on tumor tissue growth in LOVO mixed PBMC tumor-bearing mice (tumor photos).
  • Figure 7 Shows the effect of antibody-A on tumor tissue weight in LOVO mixed PBMC tumor-bearing mice.
  • Figure 8 Shows the effect of antibody-A on body weight of MC38 tumor-bearing mice.
  • Figure 9 Shows the effect of antibody-A on tumor tissue growth in MC38 tumor-bearing mice.
  • Figure 10 Shows the effect of low doses of antibody-A on tumor tissue growth in MC38 tumor-bearing mice.
  • Figure 11 Shows the effect of high doses of antibody-A on tumor tissue growth in MC38 tumor-bearing mice.
  • Figure 12 Shows individual tumor profiles of MC38 tumor-bearing mice.
  • Figure 13 Shows the effect of antibody-A on tumor tissue growth in MC38 tumor-bearing mice (tumor photos).
  • Figure 14 Shows the effect of antibody-A on tumor tissue weight in MC38 tumor-bearing mice.
  • Figure 15 Shows that antibody-A blocks the activity of PD-1 binding to PD-L1 and TIGIT binding to CD155.
  • FIG. 16 shows T cell activation activity (memory T cell recall method).
  • Figure 17 A graph showing the change trend of protein purity and purity determined by SEC-HPLC method for each sample.
  • Figure 18 A graph showing the change trend of the acidic component of the charge variant in the pH effect experiment (iCIEF method).
  • Figure 19 A graph showing the change trend of the principal components of the charge variants in the pH effect experiment (iCIEF method).
  • Figure 20 A graph showing the change trend of the basic component of the charge variant in the pH effect experiment (iCIEF method).
  • Figure 21 Shows the variation trend of the purity of the formulation screening experiment (SEC-HPLC method).
  • Figure 22 Shows the variation trend of the purity of the formulation screening experiment (non-reducing CE-SDS method).
  • Figure 23 A graph showing the change trend of the acidic components of the charge variant in the formulation screening experiment (iCIEF method).
  • Figure 24 Shows the change trend diagram of the principal components of charge variants in the formulation screening experiment (iCIEF method).
  • FIG. 25 The structure of antibody-A prepared in Example 1 is shown.
  • the term “comprising” or “comprising” means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps.
  • the term “comprising” or “comprising” is used, unless otherwise indicated, it also encompasses situations consisting of the recited elements, integers or steps.
  • reference to an antibody variable region that "comprises” a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.
  • antibody is used in the broadest sense to refer to a protein comprising an antigen-binding site, encompassing natural and artificial antibodies of various structures, including, but not limited to, whole antibodies and antigen-binding fragments of antibodies.
  • the terms “whole antibody”, “full length antibody”, “complete antibody” and “intact antibody” are used interchangeably herein to refer to a composition comprising at least two heavy chains (H) and two Light chain (L) glycoprotein.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions (complementarity determining regions (CDRs), with more conserved regions (framework regions (FR)) interposed therebetween.
  • CDRs complementarity determining regions
  • FR frame regions
  • Each VH and VL consists of three CDRs and four
  • the FRs are composed, from the amino terminus to the carboxy terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions.
  • humanized antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR.
  • the humanized antibody comprises all or substantially all of the HVRs (eg, CDRs) corresponding to those of the non-human antibody and all or substantially all of the FR regions corresponding to those of the human antibody.
  • a humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.
  • half-antibody refers to a monovalent antigen-binding polypeptide.
  • the half-antibody or half-mer comprises a VH/VL unit and optionally at least a portion of an immunoglobulin constant domain.
  • the half-antibody or half-mer comprises one immunoglobulin heavy chain associated with one immunoglobulin light chain, or an antigen-binding fragment thereof.
  • half-antibodies or half-mers are monospecific, ie, bind to a single antigen or epitope.
  • the half-antibody binds to TIGIT and does not bind to PD-1.
  • the half-antibody binds to PD-1 and does not bind to TIGIT.
  • half-antibodies may have antigen-binding domains consisting of a single variable domain (eg, derived from Camelidae).
  • VH/VL unit refers to the antigen-binding region of an antibody comprising at least one VH CDR and at least one VL CDR.
  • the VH/VL unit comprises at least one, at least two, or all three VH CDRs and at least one, at least two, or all three VL CDRs.
  • the VH/VL unit further comprises at least a portion of a framework region (FR).
  • the VH/VL unit comprises three VH CDRs and three VL CDRs.
  • the VH/VL unit comprises at least one, at least two, at least three, or all four VH FRs and at least one, at least two, at least three, or all four VL FRs.
  • bispecific antibody or “bispecific antibody protein” comprises an antigen binding domain that specifically binds to epitopes on two different biomolecules. Unless otherwise stated, the order of the antigens bound by the bispecific antibody in the bispecific antibody designation listed is arbitrary. That is, in some embodiments, the terms “anti-TIGIT/PD-1 bispecific antibody” and “anti-PD-1/TIGIT bispecific antibody” are used interchangeably. Bispecific antibody formats include IgG-like and non-IgG-like antibodies (Fan et al. Journal of Hematology & Oncology. 8:130).
  • IgG-like antibody contains two Fab regions and one Fc region, and the heavy and light chains of each Fab can be derived from separate monoclonal antibodies.
  • Non-IgG-like bispecific antibodies lacking an Fc region, each antigen- or target-binding domain of which can be a Fab, a single-chain variable fragment (scFv), or a fusion of variable domains that mimic two antibodies
  • the different binding domains are linked together by peptide linkers, chemical conjugation, non-covalent linkages, or other means.
  • These formats contain bispecific T-cell adaptors (BiTEs).
  • Bispecific antibodies of the invention can be prepared using any bispecific antibody format or technique.
  • an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (eg, by chemical coupling) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity association, genetic fusion, non-covalent association, or otherwise) to generate bispecific antibodies.
  • bispecific formats that can be used in the context of the present invention include, but are not limited to, the following: scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, tetra-hybrid Quadroma, knobs-into-holes, common light chains (eg, common light chains with knob-in holes, etc.), CrossMab, CrossFab, (SEED)body, Duobody, IgG1/IgG2, dual-acting Fab ( DAF)-IgG and Mab 2 bispecific format.
  • scFv-based or diabody bispecific formats IgG-scFv fusions, dual variable domain (DVD)-Ig, tetra-hybrid Quadroma, knobs-into-holes, common light chains (eg, common light chains with knob-in holes, etc.), CrossMab, CrossFab, (SEED)body, Duobody, IgG1/
  • the bispecific antibody is a heterodimer comprising two monovalent binding arms formed from half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and optionally a heavy chain constant region and at least a portion of a single light chain variable region and optionally at least a portion of the light chain constant region.
  • the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region and does not comprise more than one single heavy chain variable region and does not comprise More than one single light chain variable region.
  • the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half-antibody binds to the first antigen and does not bind to the first antigen.
  • the second antigen binds and the second half-antibody binds to the second antigen and does not bind to the first antigen.
  • the first monovalent binding arm is less than about 10-7 M, preferably about 10-8 M and more preferably about 4nM, 3nM, 2nM, 1nM, or lower K for binding to TIGIT; in a specific embodiment, the second monovalent binding arm is less than about 10-7 M, preferably about 10-8 M and more preferably about 4nM, 3nM, 2nM, 1nM, or lower KD binds to PD -1; in another embodiment, the bispecific antibody protein has the same or similar quaternary structure as the common monospecific antibody protein.
  • antibody preparation refers to a preparation that is in a form that allows the biological activity of the antibody as the active ingredient to be effectively exerted and that does not contain unacceptable toxicity to the subject to which the preparation is to be administered. other components. Such antibody preparations are generally sterile.
  • pharmaceutically acceptable excipients are included in the antibody formulation.
  • a "pharmaceutically acceptable" excipient is an agent that can reasonably be administered to a subject mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, eg, may be acceptable for injection.
  • anti-TIGIT/PD-1 bispecific antibody preparation is also referred to herein simply as "antibody preparation of the present invention", meaning that an anti-TIGIT/PD-1 bispecific antibody protein is contained as an active ingredient and that is pharmaceutically acceptable Preparation of excipients. After combining the anti-TIGIT/PD-1 bispecific antibody protein with a pharmaceutically acceptable excipient, the anti-TIGIT/PD-1 bispecific antibody protein as an active ingredient is suitable for therapeutic or prophylactic administration to humans or non-humans animal.
  • Antibody formulations of the invention can be prepared, for example, as liquid formulations in aqueous form, eg, ready-to-use prefilled syringes, or as lyophilized formulations for reconstitution by dissolving and/or suspending in a physiologically acceptable solution just before use. reconstituted (ie, reconstituted).
  • the anti-TIGIT/PD-1 bispecific antibody protein formulation is in the form of a liquid formulation.
  • a “stable” antibody formulation is one in which the antibody retains an acceptable degree of physical and/or chemical stability after storage under specified conditions. Although antibodies contained in antibody formulations may not retain 100% of their chemical structure after a specified period of storage, they typically maintain about 90%, about 95%, about 96%, about 97%, about 98% after storage for a specified period of time or about 99% of the structure or function of the antibody, an antibody preparation is considered “stable.” In some specific embodiments, the anti-TIGIT/PD-1 bispecific antibody protein formulations of the invention exhibit low to undetectable antibody aggregation or degradation or chemical modification during manufacture, preparation, shipping and long-term storage, There is thus little or even no loss of biological activity of the anti-TIGIT/PD-1 bispecific antibody protein, showing a high degree of stability.
  • the anti-TIGIT/PD-1 bispecific antibody protein formulations of the invention substantially retain their physical and chemical stability upon storage.
  • the liquid formulations of the present invention are stable at room temperature or at 40°C for at least 2 weeks, and/or at 25°C for at least 2 months, and/or at 2-8°C for at least 24 months.
  • Stability can be measured at selected temperatures and selected storage times. For example, the storage time can be selected based on the expected shelf life of the formulation. Alternatively, accelerated stability testing can be used. In some embodiments, stability testing is performed by subjecting antibody formulations to various stress tests.
  • formulated anti-TIGIT/PD-1 bispecific antibody protein preparations can be filled into glass vials to test antibody stability under high temperature stress.
  • the antibody After a period of storage, if the formulation shows no aggregation, precipitation, haze and/or denaturation; or very little aggregation, precipitation, haze and/or denaturation, the antibody may be considered to "retain its physical stability" in the formulation.
  • Safety concerns arise as the aggregation of antibodies in the formulation can potentially lead to an increased immune response in the patient. Therefore, there is a need to minimize or prevent aggregation of antibodies in formulations.
  • Light scattering methods can be used to determine visible aggregates in formulations.
  • SEC can be used to determine soluble aggregates in formulations.
  • the stability of the formulation can be indicated by visual inspection of the formulation's appearance, color and/or clarity, or by measuring the purity of the formulation by non-reducing CE-SDS methods.
  • the stability of the formulation is measured by determining the percentage of antibody monomers in the formulation after storage at a specific temperature for a specific time, wherein the higher the percentage of antibody monomers in the formulation, the higher the stability of the formulation .
  • an "acceptable level" of physical stability may mean that at least about 88%, eg, at least about 92%, of the anti-TIGIT/PD-1 bispecific antibody protein monomers are detected in the formulation after storage at a specific temperature for a specific period of time.
  • An acceptable level of physical stability is expressed after months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more At least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of anti-TIGIT/PD-1 bispecific antibody protein single body.
  • the particular temperature at which the pharmaceutical formulation is stored can be any temperature from about -80°C to about 45°C, eg, at about -80°C, about -30°C, about -20°C, about 0°C, About 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C.
  • the pharmaceutical preparation was considered to be stable. At least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% detected if stored at about 25°C for 2 months
  • the anti-TIGIT/PD-1 bispecific antibody protein monomer is considered stable.
  • An antibody may be considered to "retain its chemical stability" in a formulation if the antibody in the formulation does not exhibit significant chemical changes over a period of storage. Most chemical instability results from the formation of covalently modified forms of the antibody (eg, charge variants of the antibody). For example, by aspartic acid isomerization, N- and C-terminal modifications, basic variants can be formed; by deamidation, sialylation, and glycation, acidic variants can be produced. Chemical stability can be assessed by detecting and/or quantifying chemically altered forms of the antibody. For example, charge variants of the antibody in the preparation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (iCIEF). In one embodiment, the stability of the formulation is measured by determining the percent change in charge variant of the antibody in the formulation after storage at a specific temperature for a specific time, wherein the smaller the change, the more stable the formulation.
  • CEX cation exchange chromatography
  • an "acceptable level" of chemical stability may mean that the percent change in charge variants (eg, principal or acidic or basic components) in the formulation after storage at a specified temperature for a specified period of time does not exceed a value of 50%, such as not more than 50% 30%, no more than 20%.
  • At months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more an acceptable level of chemical stability may be The percent change values expressed as principal component charge variants do not exceed about 50%, 40%, 30%, 20%, 15%.
  • the temperature at which the pharmaceutical formulation is stored can be any temperature from about -80°C to about 45°C, eg, at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C or about 45°C.
  • the pharmaceutical formulation can be considered stable.
  • the pharmaceutical formulation can also be considered stable.
  • a pharmaceutical formulation may also be used if the percent change in the principal component charge variant is less than about 50%, 40%, 30%, 20%, 10%, 5%, or 4% after storage at 40°C for 1 month. considered stable.
  • lyophilized formulation refers to a composition obtained or obtainable by lyophilization of a liquid formulation. Preferably, it is a solid composition with a water content of less than 5%, preferably less than 3%.
  • reconstituted formulation refers to a liquid formulation obtained by dissolving and/or suspending a solid formulation (eg, a lyophilized formulation) in a physiologically acceptable solution.
  • room temperature refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, more preferably 25°C.
  • Stress conditions refers to chemically and/or physically unfavorable environments for an antibody protein that can lead to unacceptable destabilization of the antibody protein.
  • High temperature stress refers to the storage of antibody preparations at room temperature or even at higher temperatures (eg, 40°C ⁇ 2°C) for a period of time. The stability of the antibody preparation can be checked by a high temperature stress accelerated test.
  • parenteral administration means administration other than enteral and topical administration, usually by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal , intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion .
  • the stable anti-TIGIT/PD-1 bispecific antibody protein formulations of the invention are administered parenterally to a subject.
  • the anti-TIGIT/PD-1 bispecific antibody protein formulation of the invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.
  • the present invention provides a liquid antibody formulation comprising (i) an anti-TIGIT/PD-1 bispecific antibody protein, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, preferably the The pH of the antibody formulation is about 4.5-6.8.
  • the liquid antibody formulation of the present invention is in the form of an injectable formulation.
  • the "anti-TIGIT/PD-1 bispecific antibody” in the antibody preparation of the present invention comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a specific binding TIGIT (SEQ ID NO: 31) or its extracellular
  • the first VH/VL unit of the domain (eg, SEQ ID NO:32) and the second half-antibody comprise specific binding to PD-1 (SEQ ID NO:29) or its extracellular domain (eg, SEQ ID NO:30) of the second VH/VL unit.
  • the anti-TIGIT/PD-1 bispecific antibody is a heterodimer, wherein the first half antibody and the second half antibody each form two monovalent binding arms. In one embodiment, the The two monovalent binding arms are connected by a disulfide bond.
  • the bispecific antibody protein has the same or similar quaternary structure as the common monospecific antibody protein.
  • the first monovalent binding arm is below a K of about 10-7 M, preferably about 10-8 M, and more preferably about 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM or less D binds to TIGIT; in a specific embodiment, the second monovalent binding arm is below about 10-7 M, preferably about 10-8 M and more preferably about 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM or lower KD binds to PD -1 on the surface of T lymphocytes.
  • the anti-TIGIT/PD-1 bispecific antibody protein is capable of at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or stronger It binds to PD-1 on the surface of T lymphocytes with an affinity constant of at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or stronger.
  • TIGIT binds so that the antibody can be used as a therapeutic and/or prophylactic agent bispecifically targeting PD-1 molecules and TIGIT molecules.
  • the first VH/VL unit comprises the heavy and light chain complementarity determining region (CDR) amino acid sequences SEQ ID NOs: 1-6 or has one or more of the 6 CDRs A sequence of one, two, three, four, five, six or more amino acid changes (eg, amino acid substitutions or deletions); and/or a second VH/VL unit comprising heavy and light chain complementarity determinations Region (CDR) amino acid sequence SEQ ID NOs: 7-12 or one, two, three, four, five, six or more amino acid changes with one or more of the 6 CDRs (eg, amino acid substitutions or deletions).
  • CDR heavy and light chain complementarity determining region
  • CDR complementarity determining region
  • CDR region is the amino acid region in the variable region of an antibody that is primarily responsible for binding to an antigenic epitope.
  • the CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus.
  • Various protocols are known in the art for determining the CDR sequences of a given VH or VL or VHH amino acid sequence. For example, Kabat complementarity determining regions (CDRs) or complementarity determining regions (CDRs).
  • Kabat complementarity determining regions are determined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Whereas Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)).
  • AbM HVR is a compromise between Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software.
  • Contact HVR is based on the analysis of available complex crystal structures. For the present invention, the North CDR definitions are used. The North CDR definition (North et al., “A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)) is based on affinity-propagated clustering using bulk crystal structures.
  • amino acid changes are preferably conservative amino acid substitutions.
  • conservative amino acid substitutions refer to amino acid changes that result in the replacement of a certain amino acid with a chemically similar amino acid.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art.
  • the conservatively substituted residues are from the conservative substitutions Table A below, preferably the preferred substitutions shown in Table A.
  • the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a first VH/VL unit that specifically binds TIGIT and the second half antibody comprising a second VH/VL unit that specifically binds PD-1, wherein the first VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable sequence of SEQ ID NO: 13/SEQ ID NO: 14 region sequence, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the paired heavy chain variable region sequence/light chain variable region sequence , a sequence of 99% or more sequence identity, and wherein the second VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 17/SEQ ID NO: 18 , or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the paired heavy chain
  • heavy chain constant regions of the first and second half antibodies in the anti-TIGIT/PD-1 bispecific antibody protein preferably the heavy chain constant regions of IgG1, IgG2 or IgG4 immunoglobulins, or substantially identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to sequences. More preferably, the heavy chain constant region is, or is substantially identical to (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).
  • the heavy chain of the antibody forms at least one disulfide bond with the light chain of the antibody, and the two heavy chains of the antibody form at least one disulfide bond.
  • the antibody is human IgG1 engineered to reduce the binding of the antibody to Fc ⁇ receptors.
  • the anti-TIGIT/PD-1 bispecific antibody protein comprises a heavy chain constant region used in IgG1 (eg, human IgG1). In yet another embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a heavy chain constant region for IgG4 (eg, human IgG4).
  • the Fc domains of the two heavy chains of an anti-TIGIT/PD-1 bispecific antibody comprise hinge regions with "CPPC" amino acid residues, respectively, and/or Y349C and S354C, respectively (according to Kabat's "EU numbering" "), whereby the anti-PD-1 half antibody and the anti-TIGIT half antibody form an interchain disulfide bond in the Fc region, thereby stabilizing the correct pairing of the anti-PD-1 half antibody and the anti-TIGIT half antibody.
  • the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises or has at least 90% of the heavy chain sequence of SEQ ID NO: 21 , a heavy chain sequence that is 95%, 98% or 99% identical, and a light chain sequence that is at least 90%, 95%, 98% or 99% identical to the light chain sequence of SEQ ID NO: 22, and wherein the second half-antibody comprises the heavy chain sequence of SEQ ID NO:23 or a heavy chain sequence at least 90%, 95%, 98% or 99% identical thereto, and the light chain sequence of SEQ ID NO:24 or has A light chain sequence having at least 90%, 95%, 98% or 99% identity.
  • sequence identity refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a window of comparison. "Percent sequence identity” can be calculated by comparing two optimally aligned sequences in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences.
  • the same amino acid residue eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ilc, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met
  • Optimal alignment to determine percent sequence identity can be achieved in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest.
  • the anti-TIGIT/PD-1 bispecific antibody protein in the antibody preparation of the present invention can simultaneously bind to TIGIT and PD-1 protein, and maintain the affinity constant of each parent antibody, thereby blocking the TIGIT signal transduction pathway and blocking Disrupting the PD-1 signaling pathway, thereby being used to treat, prevent or delay various diseases or conditions related to the TIGIT signaling pathway and/or the PD-1 signaling pathway.
  • anti-TIGIT/PD-1 bispecific antibody protein used in the present invention is also described in PCT/US20/34158, the entire contents of which are incorporated by reference.
  • the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the heavy chain sequence of SEQ ID NO: 21, and SEQ ID NO : the light chain sequence of SEQ ID NO:22, and wherein the second half-antibody comprises the heavy chain sequence of SEQ ID NO:23, and the light chain sequence of SEQ ID NO:24.
  • the anti-TIGIT/PD-1 bispecific antibody protein is derived from HEK293 cells or HEK293T, HEK293F, HEK293E cells transformed from HEK293 cells; CHO cells or transformed from CHO cells CHO-S, CHO-dhfr - , CHO/DG44, ExpiCHO were recombinantly expressed and purified.
  • the antibody in the liquid formulation of the present invention exhibits significant anti-tumor activity.
  • anti-TIGIT/PD-1 bispecific antibody administered to LOVO mixed PBMC tumor-bearing mice and MC38 tumor-bearing mice showed that administration of anti-TIGIT compared with administration of anti-PD-1 monoclonal antibody or anti-TIGIT monoclonal antibody
  • the /PD-1 bispecific antibody has significantly improved antitumor activity, which can lead to a significant reduction in tumor volume.
  • the amount of anti-TIGIT/PD-1 bispecific antibody protein included in the antibody formulations of the present invention may vary depending on the specific intended properties of the formulation, the specific environment, and the specific purpose for which the formulation is used.
  • the antibody formulation is a liquid formulation, which may contain about 1-200 mg/ml, preferably about 1-150 mg/ml, more preferably about 10-100 mg/mL, eg, about 5, 10, 15 , 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/ml of anti-TIGIT/PD-1 bispecific antibody protein.
  • a buffer is an agent that can maintain the pH of a solution within an acceptable range.
  • the buffering agents used in the formulations of the present invention can control the pH of the formulations of the present invention to a pH in the range of about 4.5-6.8, preferably about 5.0-6.5, more preferably about 6.0 pH.
  • the antibody formulation of the invention has a pH of about 4.5, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.5, 6.6, 6.7, or 6.8.
  • the buffering agent used in the formulations of the present invention is selected from the group consisting of histidine, histidine hydrochloride, glutamate, phosphate, acetate, citrate and tris and their combination, preferably histidine, histidine hydrochloride and their combination.
  • the concentration of buffer in the liquid antibody formulation of the invention is about 0.5-200 mM, about 1-100 mM, about 5-50 mM, about 5-30 mM, or about 5-20 mM. In one embodiment, the concentration of buffer in the liquid antibody formulation of the invention is about 5-30 mM, eg, about 5, 10, 15, 20, 25 or 30 mM.
  • the buffer used in the formulations of the present invention is about 10 mM histidine.
  • the buffer used in the formulation of the present invention is a combination of about 5 mM histidine and about 5 mM histidine hydrochloride.
  • Suitable stabilizers for use in the present invention may be selected from sugars, polyols, amino acids or salts thereof, and combinations thereof. Further, the stabilizer of the present invention may also contain an antioxidant.
  • the saccharides as stabilizers may be disaccharides, trisaccharides and polysaccharides, and the saccharides may be selected from, but not limited to: sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin and dextran .
  • the saccharides as stabilizers are sucrose and/or trehalose.
  • the polyol as a stabilizer can be selected from, but not limited to: mannitol, sorbitol and xylitol. In one embodiment, the polyol as a stabilizer is sorbitol.
  • the amino acid or its salt as a stabilizer can be selected from, but not limited to, arginine, arginine salt (preferably arginine hydrochloride).
  • the stabilizer is sorbitol. In some embodiments, the stabilizer is arginine hydrochloride. In some embodiments, the stabilizer is sorbitol, arginine, an arginine salt (preferably arginine hydrochloride), or a combination thereof.
  • saccharides and/or polyols as stabilizers are present in the liquid formulations of the present invention at a concentration of about 1-200 mg/ml, preferably about 5-150 mg/ml, for example about 10-100 mg/ml, about 15-80 mg/ml or about 20-50 mg/ml eg, a concentration of about 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 mg/ml exist.
  • the stabilizer of the present invention is present in the liquid formulation of the present invention at about 1-1000 mM, about 10-1000 mM, preferably about 20-800 mM, such as about 50-500, about 100-400, about 100- Present at a concentration of 300 or about 100-200 mM, eg, about 10, 20, 50, 80, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM.
  • Antioxidants that may also be included in the stabilizer of the present invention are selected from, but are not limited to, homocysteine, cysteine, cystathionine, methionine, glutathione, and homocysteine-containing , cysteine, cystathionine, methionine, and glutathione.
  • the total concentration of stabilizers is as previously described, wherein the concentration of antioxidants is about 1-50 mM, preferably about 5-40 mM, eg, about 5, 10, 20, 30, 40 mM.
  • the liquid formulation of the present invention contains a surfactant.
  • the surfactant is a nonionic surfactant, eg, an alkyl poly(ethylene oxide).
  • Particular nonionic surfactants that can be included in the formulations of the present invention include, for example, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; Nick et al.
  • polysorbate-80 is included as a surfactant in the liquid formulation of the present invention.
  • the amount of surfactant contained in the antibody formulation of the invention can vary depending on the specific intended nature of the formulation, the specific environment, and the specific purpose for which the formulation is used.
  • the liquid formulations of the present invention may contain about 0.01-10 mg/ml, preferably about 0.05-5, about 0.05-2, about 0.1-5, 0.2-2, 0.3-1, 0.4-0.8 , 0.5-0.6 mg/ml, eg, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml of a polysorbate-based surfactant (eg, polysorbate-80).
  • the antibody liquid formulation of the present invention further contains a tonicity adjusting agent.
  • the tonicity adjusting agent may be selected from the group consisting of sodium acetate, sodium lactate, sodium chloride, potassium chloride and calcium chloride.
  • liquid formulations of the present invention can be prepared or formulated using pharmaceutically acceptable solvents or solutions known in the art.
  • the pharmaceutically acceptable solvent or solution includes, but is not limited to, for example, water for injection, sterile water, double distilled water, physiological saline, Ringer's solution, dextrose injection and the like.
  • the present invention provides stable formulations comprising anti-TIGIT/PD-1 bispecific antibody proteins.
  • the anti-TIGIT/PD-1 bispecific antibody proteins used in the formulations of the present invention can be prepared using techniques known in the art for producing antibodies.
  • anti-TIGIT/PD-1 bispecific antibody proteins can be produced recombinantly.
  • the anti-TIGIT/PD-1 bispecific antibody protein of the present invention is obtained by transforming HEK293 cells or HEK293 cells based on HEK293T, HEK293F and HEK293E cells; CHO cells or CHO cells as Prepared by recombinant expression in CHO-S, CHO-dhfr-, CHO/DG44, and ExpiCHO obtained by basic transformation, for example, as described in PCT application number PCT/US20/34158, recombinantly prepared anti-TIGIT/PD-1 bispecific Antibody protein.
  • recombinantly produced antibodies can be purified using conventional purification methods to provide drug substance with sufficient reproducibility and modest purity for formulation of antibody preparations.
  • the supernatant from the expression system can be concentrated using a commercially available protein concentration filter such as Amicon's ultrafiltration device.
  • purification of the antibody can be performed using, for example, chromatography, dialysis, and affinity purification.
  • Protein A is suitable as an affinity ligand for the purification of IgG1, IgG2 and IgG4 type antibodies.
  • Other antibody purification methods such as ion exchange chromatography, can also be used.
  • preparations comprising the antibody can be prepared according to methods known in the art.
  • the fermentation broth is centrifuged to remove impurities such as cells to obtain a supernatant;
  • affinity chromatography for example, specific for IgG1, IgG2 and IgG4 antibodies
  • virus inactivation can be used.
  • purification generally, CEX cation exchange chromatography can be used
  • virus filtration to make the virus titer
  • Ultrafiltration/diafiltration can be used to replace the protein in a formulation buffer favorable for its stability and concentrate to a suitable concentration for injection). See, eg, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57.
  • antibodies may aggregate, degrade, or chemically modify, resulting in antibody heterogeneity (including size and charge heterogeneity), as well as aggregates and fragments, which affect the quality of antibody preparations. Therefore, it is necessary to monitor the stability of antibody preparations.
  • the purity of antibody preparations and the level of antibody aggregation can be assessed by methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC; capillary isoelectric focusing electrophoresis (cIEF), imaging capillary isoelectric Analysis of charge variants in antibody preparations by focused electrophoresis (iCIEF) and ion exchange chromatography (IEX), among others.
  • cIEF capillary isoelectric focusing electrophoresis
  • IEX ion exchange chromatography
  • the stability of the formulation can be quickly judged by visually inspecting the appearance of the formulation.
  • the turbidity change of the formulation can also be detected using the OD 350nm method, which can give information on the amount of soluble and insoluble aggregates.
  • ultraviolet spectrophotometry UV method
  • UV method ultraviolet spectrophotometry
  • the non-reducing CE-SDS method is a method for measuring antibody purity with capillary as the separation channel.
  • protein migration is driven by the surface charge caused by the binding of SDS, and SDS binds to the protein to eliminate the difference in the self-charge of the protein. Therefore, in the molecular sieve gel matrix of the capillary, the molecular size or hydrodynamics based on the molecular size can be realized. Electrophoretic separation of radii. This method has been widely used to monitor the purity of denatured intact antibodies.
  • the test sample is mixed with SDS sample buffer and iodoacetamide.
  • the mixture can be incubated at 68-72°C for approximately 10-15 minutes, and the centrifuged supernatant after cooling to room temperature is used for analysis. The migration of the protein was detected by a UV detector, and the electropherogram was obtained. Antibody preparation purity can be calculated as the peak area of the main IgG peak as a percentage of the sum of all peak areas.
  • CE-SDS method see, for example, Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.
  • Size exclusion high performance liquid chromatography is another important method for antibody standardization and quality control. This method is mainly based on the size of the molecules or the difference in the hydrodynamic radius to separate the molecules.
  • SEC-HPLC the antibody can be separated into three main forms: the high molecular weight form (HMMS), the main peak (mainly antibody monomers), and the low molecular weight form (LMMS).
  • HMMS high molecular weight form
  • LMMS low molecular weight form
  • Antibody purity can be calculated as the percentage of the area of the main peak over the sum of all peak areas on the chromatogram.
  • SEC-HPLC the percentage of antibody monomers in the formulated product can be measured, giving information on the content of soluble aggregates and cleavage.
  • Imaging capillary isoelectric focusing electrophoresis can be used to analyze the charge heterogeneity of antibodies. This method can provide a quantitative distribution of charge variants.
  • iCIEF achieves the purpose of molecular separation based on the difference in charge (apparent pi value) of molecules in a pH gradient.
  • the separation column is usually a short capillary (e.g., a 5 cm long, 100 ⁇ m inner diameter silica capillary), the protein is focused in the capillary column at high voltage, and the focusing is performed by a full-column imaging detection system operating at 280 nM Real-time online monitoring.
  • an advantage of this technique is that various charge variants of antibody samples can be simultaneously recorded by this full-column detection system.
  • the sample is mixed with urea and icIEF buffer, wherein the buffer contains methylcellulose, pi molecular weight standards and ampholytes.
  • the focused antibody charge variant can be obtained by measuring the absorbance at 280 nm on an iCIEF analyzer such as an iCE280 analyzer (Protein Simple, Santa Clara, CA) using an iCIEF column such as an iCIEF column assembled by ProtionSimple after focusing the sample for a certain period of time 's spectrum.
  • protein-related peaks eluting before the main peak are classified as acidic components; conversely, protein-related peaks eluting after the main peak are classified as basic components.
  • the relative amounts of principal components, acidic components, and basic components can be expressed as a percentage of the total peak area.
  • Charge variants of antibodies in antibody preparations can also be determined by cation exchange high performance liquid chromatography (CEX-HPLC).
  • CEX-HPLC cation exchange high performance liquid chromatography
  • Accelerated stability studies can be used to examine the stability properties of products and facilitate the screening of stable pharmaceutical formulations.
  • accelerated stability studies can be performed by placing samples of the formulation at elevated temperatures, eg, about 40°C ⁇ 2°C, 25°C ⁇ 2°C.
  • Detection indicators can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reducing CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method).
  • antibodies can be tested for efficacy or biological activity.
  • the ability of an antibody to bind to its antigenic molecules (TIGIT molecule and PD-1 molecule) in the preparation can be tested.
  • TIGIT molecule and PD-1 molecule antigenic molecules
  • a variety of methods are known to those of skill in the art for quantifying specific binding of antibodies to antigens, such as immunoassays, ELISAs, and the like.
  • the anti-TIGIT/PD-1 bispecific antibody protein preparation of the present invention is stable. In one embodiment, after storage at about 5°C, 25°C, 37°C, 40°C, or 45°C for at least 1 month, 2 months, or 3 months, eg, at 5°C ⁇ 3°C for 3 months. Then, the anti-TIGIT/PD-1 bispecific antibody protein purity in the antibody preparation of the present invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99%, as determined by size exclusion chromatography or by non-reducing CS-SDS.
  • At least 60%, preferably at least 65% of the anti-TIGIT/PD-1 bispecific antibody protein in the antibody preparation of the invention is in the non-basic and non-acidic form (ie, the main peak or main charge form), such as by the iCIEF method determined.
  • the antibody preparation of the present invention comprising the anti-TIGIT/PD-1 bispecific antibody protein of the present invention can be used to treat, prevent or delay various diseases related to the TIGIT signaling pathway and/or PD-1 signaling pathway or disease.
  • Diseases or disorders associated with the TIGIT signaling pathway and/or “diseases or disorders associated with the PD-1 signaling pathway” herein refer to treatable with the anti-TIGIT/PD-1 bispecific antibody protein formulations of the invention (eg to ameliorate) or prevent a disease or condition. Any disease or condition that can benefit from the treatment of the antibody formulations of the present invention is suitable for use in the present invention.
  • the formulations of the present invention comprising anti-TIGIT/PD-1 bispecific antibody proteins can be used to prevent or treat various hematological diseases and solid tumors in subjects, including but not limited to leukemia, lymphoma, myeloma, brain tumor, head and neck Cancer, squamous cell carcinoma of head and neck, lung cancer such as non-small cell lung cancer or small cell lung cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer , cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma.
  • lung cancer such as non-small cell lung cancer or small cell lung cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, liver cancer, colorec
  • the present invention also provides the use of the formulation of the present invention in the manufacture of a medicament for delivering an anti-TIGIT/PD-1 bispecific antibody protein to a mammal, or for treating, preventing or ameliorating the above-mentioned diseases and disorders one or more of.
  • the mammal is a human.
  • the antibody formulations of the invention can be administered to a subject or patient in a variety of ways.
  • administration can be by infusion or by syringe.
  • the present invention provides a delivery device (eg, a syringe) comprising an antibody formulation of the present invention (eg, a pre-filled syringe).
  • the patient will receive an effective amount of the anti-TIGIT/PD-1 bispecific antibody protein as the primary active ingredient, ie, an amount sufficient to treat, ameliorate or prevent the disease or disorder of interest.
  • Therapeutic effects may include reduction of physical symptoms.
  • the optimal effective amount and concentration of antibody for use in any particular subject will depend on a variety of factors, including the age, weight, health and/or sex of the patient, the nature and extent of the disease, the activity of the particular antibody, the its clearance, and also any possible other treatments administered in combination with the antibody preparation.
  • the effective amount to be delivered can be determined within the judgment of the clinician.
  • the use of known antibody-based drugs can provide some guidance. Dosage can be a single-dose regimen or a multiple-dose regimen.
  • HCCR heavy chain constant region
  • LCCR light chain constant region
  • HCVR heavy chain variable region
  • LCVR light chain variable region
  • ECD extracellular domain
  • Example 1 Expression and purification of recombinant fully human anti-TIGIT/PD-1 bispecific antibody
  • the antibodies of the present invention can be expressed and purified substantially as follows. Transient or stable transfection of suitable host cells HEK 293 (other hosts may also be possible) with an antibody secreting expression system using an optimal predetermined heavy chain:light chain vector ratio or using a single vector system encoding both heavy and light chains. cells such as CHO cells). Specifically, one or more DNA molecules encoding the following amino acid sequences can be utilized: a first heavy chain having the amino acid sequence SEQ ID NO: 21, a first light chain having the amino acid sequence SEQ ID NO: 22, having the amino acid sequence SEQ ID NO: 22
  • the antibody of the invention also referred to herein as "" antibody-A”.
  • the first heavy chain with the amino acid sequence SEQ ID NO: 21 and the first light chain with SEQ ID NO: 22 together form the first half antibody, and the two respectively constitute its VH/VL unit, and the first half
  • the antibody specifically binds to TIGIT or its extracellular domain
  • the second heavy chain having the amino acid sequence of SEQ ID NO:23 and the second light chain having the amino acid sequence of SEQ ID NO:24 together form a second half-antibody, and
  • Figure 25 shows a schematic diagram of the structure of the above-mentioned antibody.
  • Antibodies can be purified using one of a variety of commonly used techniques.
  • the medium can be conveniently applied to a MabSelect column (GE Healthcare) or a KappaSelect column (GE Healthcare) that has been equilibrated with a compatible buffer such as phosphate buffer (pH 7.4).
  • a compatible buffer such as phosphate buffer (pH 7.4).
  • the column is washed to remove non-specifically bound components.
  • the bound antibody can be eluted, for example, by a pH gradient (eg, 20 mM Tris buffer pH 7.0 to 10 mM sodium citrate buffer pH 3.0, or phosphate buffered saline pH 7.4 to 100 mM glycine buffer pH 3.0).
  • Antibody fractions are detected, eg, by UV absorption or SDS-PAGE, and then pooled. Further purification is optional depending on the intended use. Purified antibodies can be concentrated and/or sterile filtered using conventional techniques. Soluble aggregates and multimers can be efficiently removed by conventional techniques including size exclusion, hydrophobic interactions, ion exchange, multimodal or hydroxyapatite chromatography, and the like. Purified antibodies can be immediately frozen or lyophilized below -30°C.
  • Example 2 Pharmacodynamic study of recombinant fully human anti-TIGIT/PD-1 bispecific antibody (antibody-A) on LOVO mixed PBMC tumor-bearing NOG mice
  • LOVO human colorectal cancer cells
  • PBMC tumor-bearing NOG mice to study the antitumor efficacy of recombinant fully human anti-TIGIT/PD-1 bispecific antibody on LOVO mixed PBMC tumor-bearing NOG mice, and The anti-tumor effects of IBI308(PD-1) and anti-TIGIT alone and in combination were studied.
  • H-IgG injection from Equitech-Bio; product number: SLH56-0001; specification: 7.3mg/ml
  • IBI308 sintilimab injection (trade name From Innovent (Suzhou) Co., Ltd.; batch number: DP1911001; specification: 10mg/ml)
  • anti-TIGIT a monoclonal antibody whose heavy chain is SEQ ID NO: 21; light chain is SEQ ID NO: 22
  • injection Liquid specification: 1.761mg/ml
  • antibody-A injection concentration: 25mg/ml
  • the following test solutions were obtained: 0.6mg/ml H-IgG injection; 0.03mg/ml IBI308 injection; 0.3mg/ml IBI308 injection; 0.03mg/ml anti-TIGIT injection; 0.3mg
  • LOVO cells (source: ATCC; Cat. No.: CCL-229; Lot No.: 60380843) and PBMC cells (Source: AllCells; Cat. No.: LBL-002; Lot No.: 3024811) were dispersed 4:1 in PBS (1 ⁇ ) (source as above). , prepare a cell suspension with a cell concentration of 12.5 ⁇ 10 6 cells/mL: 3.125 ⁇ 10 6 cells/mL.
  • NOG mice 56, female, 14-17g, source: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., certificate number: 1100112011044904
  • LOVO PBMC mixed cell suspension 0.2 mL/mouse
  • the inoculation amount is LOVO 2.5 ⁇ 10 6 cells/mouse: PBMC 0.625 ⁇ 10 6 cells/mouse.
  • mice were randomly divided into 8 groups, with 7 mice in each group, named as H-IgG-6mg/kg group, IBI308-0.3mg/kg group, IBI308- 3mg/kg group, anti-TIGIT-3mg/kg group, IBI308+anti-TIGIT-0.3+0.3mg/kg group, IBI308+ ⁇ TIGIT(321)-3+3mg/kg group, antibody-A-0.6mg/kg group and antibody-A-6mg/kg group; the administration method was intraperitoneal injection, the administration volume was 10ml/kg, and the administration frequency was once every 3 or 4 days, specifically on the 2nd and 6th days after tumor cell inoculation.
  • mice were administered once, and the mice were monitored until day 24.
  • Tumors of mice were collected and weighed 27 days after inoculation.
  • Mice body weight, the largest long axis (L) and the largest wide axis (W) of tumor tissue were monitored twice weekly.
  • the relative tumor inhibition rates of mice in each group were calculated 24 days after inoculation.
  • Tumor volume in mice was determined on days 6, 10, 13, 17, 20, and 24 after tumor inoculation, tumors were measured twice a week, and tumors were weighed at the time of measurement and before each dose. Tumor tissue was weighed and photographed 27 days after tumor inoculation.
  • Tumor inhibition rate TGI 100% ⁇ (Tvol control -Tvol treated )/(Tvol control -Tvol predose )
  • Tvol control -Tvol treated the terminal tumor volume after the administration of the control group - the final volume of the tumor in the administration group after administration;
  • Tvol control -Tvol predose the terminal tumor volume after administration of the control group - the tumor volume of the control group before administration.
  • mice did not change by more than 10% after administration, and it was judged that the drug had no effect on the body weight of the mice.
  • the TGI was calculated compared with the control group to judge the tumor-inhibitory effect of the drug on the tumor-bearing tumor.
  • mice The body weight changes of mice in each group are shown in Figure 1, Table 2 and Table 3. In this experiment, there was no obvious weight loss in each group of mice, and the mice behaved normally. It can be seen that antibody-A has no effect on the body weight of LOVO mixed PBMC tumor-bearing NOG mice.
  • the tumor inhibition rates of the IBI308-0.3mg/kg group and IBI308-3mg/kg group were 11.47% and 57.25%, respectively; the tumor inhibition rate of the anti-TIGIT-3mg/kg group was was 57.25%; the tumor inhibition rates of the IBI308+anti-TIGIT-0.3+0.3mg/kg group and the IBI308+anti-TIGIT-3+3mg/kg group were 65.42% and 35.29%; the antibody-A-0.6mg/kg group and antibody-A-6mg/kg group's tumor inhibition rate was 92.63% and 99.84%.
  • the tumor inhibition rate of antibody-A-0.6mg/kg group and antibody-A-6mg/kg group was significantly better than that of anti-TIGIT-3mg/kg single drug group and IBI308+anti-TIGIT-0.3+0.3mg/kg group,
  • the IBI308+anti-TIGIT-3+3mg/kg group showed that the two antibody parts of the bispecific antibody of the present invention produced a synergistic effect.
  • TGI Tumor Inhibition Rate
  • the tumor inhibitory effect of each dose group of antibody-A was better than that of the IBI308 single-agent group, the anti-TIGIT single-agent group and the combination group, with significant anti-tumor effect, and the anti-TIGIT single-agent group had tumor inhibition.
  • the effect was better than that of IBI308 single drug group.
  • Example 3 Pharmacodynamic study of recombinant fully human anti-TIGIT/PD-1 bispecific antibody (antibody-A) on MC38 tumor-bearing PD-1/TIGIT mice
  • MC38 mouse colon cancer cell tumor-bearing mice were used to study the effect of recombinant fully human anti-TIGIT/PD-1 bispecific antibody on MC38 tumor-bearing PD1/TIGIT mice (ie, PD1/TIGIT double knockout transgenic mice; Source: Biositu Jiangsu Gene Biotechnology Co., Ltd.; Certificate No.: 3207262011001376, the same below) anti-tumor efficacy.
  • test solutions were prepared as described in Example 2: 0.6 mg/ml H-IgG injection; 0.03 mg/ml IBI308 injection; 0.3 mg/ml IBI308 injection; 0.03 mg/ml anti-TIGIT injection; 0.3 mg/ml ml anti-TIGIT injection; 0.06mg/ml antibody-A injection; 0.6mg/ml antibody-A injection.
  • MC38 cells (source: Shanghai Heyuan Biological Co., Ltd.; batch number: HYC0116) were dispersed with PBS (1 ⁇ ) (source as above) to prepare a cell suspension with a cell concentration of 5 ⁇ 10 6 cells/mL.
  • mice 60 mice; female, 16-20 g were shaved on the right back and injected subcutaneously with 0.2 mL/mice of 5 ⁇ 10 6 cells/mL MC38 cell suspension, that is, the inoculation amount was 1 ⁇ 10 6 cells / mouse.
  • mice with tumor volume within a certain range 42.75 mm 3 -98.39 mm 3 .
  • tumor volume they were divided into 8 groups, with 6 mice in each group. They were named as H-IgG-6mg/kg group, IBI308-0.3mg/kg group, IBI308-3mg/kg group, anti-IgG group according to the administration type and dose.
  • TIGIT-3mg/kg group IBI308+anti-TIGIT-0.3+0.3mg/kg group, IBI30+ ⁇ TIGIT(321)-3+3mg/kg group, antibody-A-0.6mg/kg group and antibody-A-6mg group /kg group;
  • the administration method is intraperitoneal injection, the administration volume is 10ml/kg, and the administration frequency is once every 3 or 4 days, that is, specifically on the 7th day, the 10th day, the 14th day, the Each administration was administered once on the 17th day, and the mice were monitored until the 24th day.
  • the tumor of the mice was collected and weighed on the 28th day after the inoculation. Mice body weight, the largest long axis (L) and the largest wide axis (W) of tumor tissue were monitored twice weekly. The relative tumor inhibition rates of mice in each group were calculated 24 days after inoculation.
  • Tumor volume in mice was determined on days 7, 10, 14, 17, 21, and 24 after tumor inoculation. Tumors were measured twice a week, and tumors were weighed at the time of measurement and before each dose. Tumor tissue was weighed and photographed 28 days after tumor inoculation.
  • Tumor inhibition rate TGI 100% ⁇ (Tvol control -Tvol treatment )/(Tvol control -Tvol initial dose )
  • Tvol control -Tvol treated the terminal tumor volume after the administration of the control group - the final volume of the tumor in the administration group after administration;
  • Tvol control -Tvol predose the terminal tumor volume after administration of the control group - the tumor volume of the control group before administration.
  • mice did not change by more than 10% after administration, and it was judged that the drug had no effect on the body weight of the mice.
  • the TGI was calculated compared with the control group to judge the tumor-inhibitory effect of the drug on the tumor-bearing tumor.
  • mice The body weight changes of mice in each group are shown in Figure 8 and Tables 7 and 8. In this experiment, there was no obvious weight loss in each group of mice, and the mice behaved normally. Thus, antibody-A had no effect on the body weight of MC38 tumor-bearing PD1/TIGIT transgenic mice.
  • the tumor inhibition rates of the IBI308-0.3mg/kg group and the IBI308-3mg/kg group were 44.79% and 67.12%, respectively; the tumor inhibition rate of the anti-TIGIT-3mg/kg group was was 13.83%; the tumor inhibition rates of the IBI308+anti-TIGIT-0.3+0.3mg/kg group and the IBI308+anti-TIGIT-3+3mg/kg group were 38.35% and 89.60%; the antibody-A-0.6mg/kg group
  • the tumor inhibition rates of the antibody-A-6 mg/kg and antibody-A-6 mg/kg groups were 68.77% and 95.53%, indicating that the two antibody parts of the bispecific antibody of the present invention produced a synergistic effect.
  • IBI308-0.3mg/kg group, IBI308-3mg/kg group, IBI308+anti-TIGIT-0.3+0.3mg/kg group, IBI308+anti- TIGIT-3+3mg/kg group, antibody-A-0.6mg/kg group and antibody-A-6mg/kg group had certain inhibitory effect on the growth of MC38 tumor bearing.
  • the tumor inhibition rate of the low-dose antibody-A group was better than that of the low-dose IBI308 single-drug group and also better than the combination group of IBI308 and anti-TIGIT low-dose.
  • the drug group was superior to the combination group of IBI308 and high dose of anti-TIGIT.
  • Antibody-A showed a good dose response in MC38 tumor-bearing PD1/TIGIT mice.
  • TGI Tumor Inhibition Rate
  • Example 4 Antibody-A blocking the binding activity of PD-1/PD-L1 and CD155/TIGIT
  • the PD-1-TIGIT-NFAT-Luc reporter system was used to detect the blocking activity of antibody-A on PD-1/PD-L1 and TIGIT/CD155 binding.
  • the assay consists of 2 cell lines: CHO K1 cells (CHOK1-PD-L1-CD155) expressing PD-L1, CD155 and TCR activator on the membrane surface, and Jurkat expressing PD-1, TIGIT and NFAT-Luc reporter genes cells (Jurkat-PD1-TIGIT-NFAT-Luc). After co-incubation of these two cell lines, TCR- and NFAT-mediated luminescence was inhibited by PD-1/PD-L1 and TIGIT/CD155 interactions. This inhibition can be reversed by blocking the PD-1/PD-L1 and TIGIT/CD155 interactions, resulting in TCR activation and luminescence via the NFAT pathway.
  • CHOK1-PD-L1-CD155 cells (Promega, USA, Cat. No. J2102) were cultured to logarithmic growth phase, and the cell density was adjusted to 4 ⁇ 10 5 cells/ml. The cells were plated in a 96-well flat-bottom white plate, 100 ⁇ l/well (4 ⁇ 10 4 cells/well), and cultured at 37° C., 5% CO 2 for 16 h.
  • Antibody-A, IBI308, anti-TIGIT, IBI308+anti-TIGIT, hIgG were prepared using Assay Buffer (RPMI 1640 (Gibco, USA, Cat. No. 22400-089) + 1% FBS (Hyclone, USA, Cat. No. SH30406.05))
  • Assay Buffer RPMI 1640 (Gibco, USA, Cat. No. 22400-089) + 1% FBS (Hyclone, USA, Cat. No. SH30406.05)
  • the configuration concentration is 2000nM
  • the Assay Buffer is used for 4-fold gradient dilution.
  • the 96-well plate cultured with CHOK1-PD-L1-CD155 was removed, and 95 ⁇ l of supernatant was aspirated from each well.
  • a blank control group was set, that is, 40 ⁇ l Assay buffer was added.
  • Jurkat-PD1-TIGIT-NFAT-Luc cells were added, 40 ⁇ l/well (5 ⁇ 10 4 cells/well). Continue to incubate for 6h in a 37°C, 5% CO2 cell incubator.
  • Bio-Glo TM Reagent Bio-Glo TM Luciferase Assay System, Promega, G7940 was added at 80 ⁇ l/well. The reaction was carried out at room temperature for 10 min. Read the fluorescence value using a multi-plate reader.
  • the NFAT-Luc reporter system was used to detect the ability of antibody-A to block the binding of PD-1/PD-L1 and TIGIT/CD155. The results are shown in Figure 15. In the concentration range of 0.015259-1000nM, antibody-A can block PD-1 The combination of -1/PD-L1 and TIGIT/CD155 reactivates T cells, and its blocking activity is significantly stronger than that of IBI308 and anti-TIGIT.
  • Example 5 Study on the effect of antibody-A on the reactivation of memory T cells stimulated by CMV
  • PBMC cell preparation revive a donor's PBMC (stimulator cells), and adjust the cell density to 6 using RPMI 1640 complete medium (Hyclone, USA, catalog number SH30809.01) (containing 10% FBS (Hyclone, USA, catalog number SH30406.05) ⁇ 10 6 cells/ml, 50 ⁇ l cells were added to each well, 300,000 cells/well.
  • CMV cell preparation CMV (Mabtech, Sweden; Cat. No. 3619-1) was lysed with the above-mentioned PBMC-containing medium, 50 ⁇ l/well, at a final concentration of 5 ⁇ g/ml.
  • Antibody preparation use RPMI 1640 complete medium (10% FBS) to prepare hIgG1, antibody-A, IBI308, anti-TIGIT, IBI308+anti-TIGIT (for antibody information, see Example 2), starting at a concentration of 100nM, using Assay Buffer ( For source information, see Example 4) 4-fold serial dilution for a total of 5 concentrations, 100 ⁇ l/well was added to cells, and antibody-A (for antibody sample information, see Example 2), the concentration was always 200 nM, and 4-fold serial dilution with Assay Buffer for a total of 5 Each concentration, 100 ⁇ l/well was added to the cells and incubated in the incubator for 5 days.
  • Assay Buffer For source information, see Example 4) 4-fold serial dilution for a total of 5 concentrations, 100 ⁇ l/well was added to cells, and antibody-A (for antibody sample information, see Example 2), the concentration was always 200 nM, and 4-fold serial dilution with Assay Buffer for
  • Human IFN-gamma DuoSet ELISA Kit (American R&D systems; Cat. No.: DY285B) was used to measure the amount of IFN- ⁇ secretion in the culture supernatant. used in experiments Human T-Activator CD3/ CD28 stimulation served as a positive control.
  • CMV-stimulated memory T cell recall was used to detect the regulatory activity of antibody-A on human memory T cells.
  • the basic principle is to screen the batches of PBMCs that respond to CMV polypeptides, and after resuscitation, add 5ug/ml of CMV to stimulate the T cells to reactivate them.
  • the drug antibody-A was added to detect the content of IFN- ⁇ in the cell culture supernatant, and the activation activity of antibody-A on memory T cells was reflected according to the secretion of IFN- ⁇ .
  • the results are shown in Figure 16.
  • the experimental results show that in the memory T cell recall experiment, antibody-A can concentration-dependently increase the secretion level of IFN- ⁇ and enhance the function of memory T cells in vitro, and its effect is better than that of anti-PD-1 monoclonal antibody (IBI308) , anti-TIGIT monoclonal antibody and anti-PD-1 monoclonal antibody (IBI308) + anti-TIGIT monoclonal antibody.
  • the following examples relate to the screening and preparation of stable formulations of recombinant anti-TIGIT/PD-1 bispecific antibodies.
  • Drug stability is one of the important indicators to ensure the effectiveness and safety of drugs. Obtaining a formulation that imparts good stability to a drug product is a critical condition for a drug product to remain safe and effective during its shelf life.
  • a formulation screening test was designed to examine different excipients. Effects on the stability of anti-TIGIT/PD-1 bispecific antibody formulations. The materials and methods used in the test are as follows:
  • N/A means "Not applicable”.
  • Antibody preparations were tested for the following items: (1) detection of appearance and presence of visible foreign matter; (2) determination of protein content in preparations by ultraviolet method (UV method); (3) by size exclusion chromatography, for example, size exclusion
  • the purity of antibody preparations was determined by size-exclusion chromatography-HPLC (SEC-HPLC), expressed as the percentage of monomer area to the sum of all peak areas; (4) by non-reducing dodecyl
  • the purity of the antibody preparation was determined by sodium sulfate capillary electrophoresis (non-reducing CE-SDS), expressed as the percentage of the monomer area to the sum of all peak areas; (5)
  • the antibody was determined by imaging capillary isoelectric focusing electrophoresis (iCIEF method) The charge variant in the preparation, expressed as a percentage of the main component, acidic component and basic component; .
  • the protein content in the sample was determined using an ultraviolet spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800).
  • the mobile phase is phosphate buffer (weigh 3.12g of sodium dihydrogen phosphate dihydrate, 8.77g of sodium chloride and 34.84g of arginine, dissolve with 800ml of ultrapure water and adjust the pH with hydrochloric acid. to 6.8 and dilute to 1000ml), the column protection solution is 0.05% (w/v) NaN3, the injection volume is 50 ⁇ l, the flow rate is 0.5ml/min, the collection time is 30 minutes, the column temperature is 25°C, and the detection wavelength is 280nm. Dilute the sample to be tested to 2 mg/ml with preparation buffer as the test solution. The preparation buffer was diluted in the same way as above and used as blank solution. Take 50 ⁇ l of blank solution and 50 ⁇ l of test solution and inject into liquid chromatograph to start detection.
  • the capillary is an uncoated capillary with an inner diameter of 50 ⁇ m, a total length of 30.2 cm, and an effective length of 20.2 cm.
  • the capillary column was washed with 0.1 mol/L sodium hydroxide, 0.1 mol/L hydrochloric acid, ultrapure water, and electrophoresis gel at 70 psi, respectively. Dilute the sample to be tested to 2.0 mg/ml with an appropriate amount of ultrapure water, take 50 ⁇ l of the above diluted sample into a 1.5 ml centrifuge tube, and add 45 ⁇ l of pH 6.5 sample buffer (weigh 0.32 g of citric acid monohydrate to it).
  • Imaging capillary isoelectric focusing electrophoresis was used for detection.
  • the inner diameter of the capillary is 100 ⁇ tm, and the total length is 5 cm.
  • 0.5% methylcellulose solution hereinafter also abbreviated as MC solution
  • ultrapure water should be used to rinse the capillary column.
  • the vacuum sampling method was adopted, the pre-focusing voltage and time were 1.5kV for 1 minute, the focusing voltage and time were 3kV for 8 minutes, the injection time was 55 seconds, the sample pan temperature was 10°C, and the detection wavelength was 280nm.
  • Cathodic Stabilizer is 500 mmol/L arginine solution, 0.5% MC solution reduces the adhesion between protein and capillary.
  • the premix solution is as follows: 70 ⁇ l 3mol urea-0.5% MC solution, 4 ⁇ l ampholyte (pH 3) -10), 2 ⁇ l of cathode stabilizer, 1 ⁇ l of pi 5.85 marker, 1 ⁇ l of pi 9.99 marker), and thoroughly mixed to obtain the sample solution to be tested.
  • the content of main components, acidic components and basic components was calculated according to the area normalization method.
  • This example examines the stability of formulations comprising anti-TIGIT/PD-1 bispecific antibody (antibody-A) at pH 5.0 to 7.0.
  • a buffer containing 10 mM histidine and 5% (w/v) sorbitol was prepared with water for injection, histidine and sorbitol, and the pH was adjusted to 5.0, 5.5, 6.0, 6.5 and 7.0 with hydrochloric acid, respectively.
  • Antibody-A The protein was replaced by ultrafiltration into buffers with different pH values, and the protein content was adjusted to 25 mg/ml; polysorbate 80 was added to make the final concentration 0.2 mg/ml; filtered and dispensed into vials, stoppered, and capped. The above samples were tested for stability under the condition of 40 ⁇ 2°C. The specific scheme is shown in Table 12.
  • the samples were placed at 40 ⁇ 2°C for 2 weeks. Except that the pH 7.0 sample appeared turbid in the second week, no other tests were performed. The appearance and visible foreign matter of the other samples were all qualified.
  • the protein content detection results are shown in Table 14. The results showed that the protein content of samples with different pH values did not change significantly after being placed at 40 ⁇ 2°C for 2 weeks.
  • Embodiment 7 Formulation formula screening experiment
  • This experiment mainly investigated the effects of disodium edetate, sorbitol and arginine hydrochloride on the stability of antibody-A protein.
  • a total of 4 prescriptions were designed.
  • the detailed prescription information is shown in Table 18, and the detailed experimental conditions and sampling plan are shown in Table 19. .
  • % refers to % w/v; adjust pH with hydrochloric acid.
  • the protein content results are shown in Table 20. The results showed that the protein content of each prescription sample did not change after being placed at 40 ⁇ 2°C for 4 weeks.
  • F1 and F2 showed that the addition of disodium edetate in the prescription had limited benefit on the stability of antibody-A protein; (2) Sorbitol performed better in maintaining the purity of antibody-A.
  • the amino acid formulations outperformed the charge variants. Therefore, F4 is the preferred formulation of antibody-A.
  • the buffer system in the formulation was adjusted to histidine and histidine hydrochloride.
  • antibody-A 25.0mg/ml antibody-A bispecific antibody, 0.79mg/ml histidine, 1.03mg/ml histidine hydrochloride, 16.85mg/ml Arginine hydrochloride, 25.00 mg/ml sorbitol and 0.50 mg/ml polysorbate 80, pH 6.0.

Abstract

The present invention relates to a preparation comprising an anti-TIGIT/PD-1 bispecific antibody, and in particular to a pharmaceutical preparation comprising an anti-TIGIT/PD-1 bispecific antibody, a buffering agent, a stabilizing agent, and a surfactant. In addition, the present invention further relates to a use of these preparations in treating or preventing diseases.

Description

包含抗TIGIT/PD-1双特异性抗体的制剂及其制备方法和用途Preparations comprising anti-TIGIT/PD-1 bispecific antibodies, methods for their preparation and use 技术领域technical field
本发明涉及抗体制剂领域。更具体而言，本发明涉及包含重组的抗含免疫球蛋白基序与免疫受体酪氨酸抑制基序结构域的T细胞免疫受体(TIGIT)和抗程序性死亡受体1(PD-1)双特异性抗体(也称为抗TIGIT/PD-1双特异性抗体)的药物制剂，尤其是稳定的液体制剂，以及用于制备所述药物制剂的方法，以及所述药物制剂的治疗和/或预防用途。The present invention relates to the field of antibody formulations. More specifically, the present invention relates to T cell immune receptors (TIGIT) and anti-programmed death receptor 1 (PD- 1) Pharmaceutical formulations, especially stable liquid formulations, of bispecific antibodies (also known as anti-TIGIT/PD-1 bispecific antibodies), and methods for preparing said pharmaceutical formulations, and treatment of said pharmaceutical formulations and/or prophylactic use.
背景技术Background technique
近年来，随着对免疫检查点(immune checkpoint)分子的研究，发现免疫检查点的抑制性信号通路活化导致T淋巴细胞不能有效发挥对肿瘤的杀伤效应(Yao S，Zhu Y和Chen L.，Advances in targeting cell surface signaling molecules for immune modulation.Nat Rev Drug Discov，2013，12(2)：130-146)，这从一个方面导致了仅靶向肿瘤细胞上的靶点的药物(例如曲妥珠单抗)的抗肿瘤效果不佳。In recent years, with the study of immune checkpoint (immune checkpoint) molecules, it was found that the activation of the inhibitory signaling pathway of the immune checkpoint leads to the inability of T lymphocytes to effectively exert the killing effect on tumors (Yao S, Zhu Y and Chen L., Advances in targeting cell surface signaling molecules for immune modulation. Nat Rev Drug Discov, 2013, 12(2): 130-146), which on the one hand leads to drugs that target only targets on tumor cells (eg trastuzumab) Monoclonal antibody) has poor antitumor effect.
程序性死亡蛋白-1(PD-1)是一种重要的免疫检查点蛋白，为55kDa大小的I型跨膜蛋白，主要诱导性表达于活化的T细胞表面，也表达于B细胞、NK细胞、单核细胞、DC细胞等细胞上。已鉴定到PD-1的两种细胞表面糖蛋白配体，分别为程序性死亡蛋白配体1(PD-L1)和程序性死亡蛋白配体2(PD-L2)。PD-1的配体在许多癌细胞上高表达。PD-1与PD-1的配体结合后可导致T细胞凋亡、免疫无应答、T细胞“耗竭”和分泌IL-10等，因此，阻断PD1途径能在癌症患者中恢复T细胞功能(Sheridan，Nature Biotechnology 30(2012)，729-730)。针对PD-1的单克隆抗体已有记载，例如，百时美施贵宝(BMS)公司的纳武单抗(Nivolumab)和默克(Merck)公司的派姆单抗(Pembrolizumab)。纳武单抗(商品名
Figure PCTCN2021133345-appb-000001
)为完全人源化的IgG4抗体分子，派姆单抗(商品名
Figure PCTCN2021133345-appb-000002
)为人源化IgG4抗体分子。所述抗PD-1单克隆抗体与T淋巴细胞上的PD-1结合后能够抑制PD-1与其配体PD-L1和PD-L2的结合，由此促进T淋巴细胞活化、增殖和产生免疫活化型细胞因子如IL-2，并解除PD-1对具有抗肿瘤活性的T淋巴细胞免疫监视的抑制。 Programmed death protein-1 (PD-1) is an important immune checkpoint protein, a 55kDa type I transmembrane protein, mainly inducibly expressed on the surface of activated T cells, and also expressed on B cells and NK cells. , monocytes, DC cells and other cells. Two cell surface glycoprotein ligands for PD-1 have been identified, programmed death protein ligand 1 (PD-L1) and programmed death protein ligand 2 (PD-L2). The ligand of PD-1 is highly expressed on many cancer cells. The binding of PD-1 to PD-1 ligands can lead to T cell apoptosis, immune unresponsiveness, T cell "exhaustion" and secretion of IL-10, etc. Therefore, blocking the PD1 pathway can restore T cell function in cancer patients (Sheridan, Nature Biotechnology 30 (2012), 729-730). Monoclonal antibodies directed against PD-1 have been described, for example, Bristol-Myers Squibb (BMS)'s Nivolumab and Merck's (Merck's) Pembrolizumab. Nivolumab (trade name
Figure PCTCN2021133345-appb-000001
) is a fully humanized IgG4 antibody molecule, Pembrolizumab (trade name
Figure PCTCN2021133345-appb-000002
) are humanized IgG4 antibody molecules. The anti-PD-1 monoclonal antibody can inhibit the binding of PD-1 to its ligands PD-L1 and PD-L2 after binding to PD-1 on T lymphocytes, thereby promoting T lymphocyte activation, proliferation and immune generation. Activating cytokines such as IL-2, and relieve PD-1 inhibition of T lymphocyte immune surveillance with antitumor activity.
虽然抗PD-1抗体对PD-1抑制信号的阻断得到临床证实，并导致某些癌症治疗的显著临床进展，但是有许多患者对PD-1治疗无反应、复发、获得抗性，或对治疗不耐受。Although the blockade of PD-1 inhibitory signaling by anti-PD-1 antibodies is clinically proven and has resulted in significant clinical advances in certain cancer treatments, many patients do not respond to PD-1 therapy, relapse, acquire resistance, or become resistant to PD-1 therapy. Treatment intolerance.
与PD-1类似，TIGIT是在活化和耗尽的T细胞上表达的共抑制受体。TIGIT结合肿瘤细胞上的脊髓灰质炎病毒受体(PVR，也称为CD155)，并能够将信号反向传递到肿瘤细胞中，导致T细胞抑制性细胞因子的分泌。尽管CD155被认为是TIGIT的显性配体，TIGIT也可与CD112和CD113相互作用(Blake et al.，Clin CancerRes；2016；22(21)：5182-5188)。已经研究了TIGIT作为抑制性免疫检查点受体的作用。TIGIT是CD226/TIGIT通路的一部分，其中TIGIT不仅与共刺激免疫受体CD226竞争结合CD155，而且也直接与细胞膜中的CD226相互作用，并阻断CD226同二聚。(Blake et al.，S，Clin Cancer Res；2016；22(21)：5182-5188；Johnston et al.，Cancer Cell 2014；26：923-937；Mahnke et al，Journal of Investigative Dermatology 2016；136： 9-11)。Similar to PD-1, TIGIT is a co-inhibitory receptor expressed on activated and depleted T cells. TIGIT binds to the poliovirus receptor (PVR, also known as CD155) on tumor cells and is able to reverse signaling into tumor cells, resulting in the secretion of T-cell inhibitory cytokines. Although CD155 is considered to be the dominant ligand for TIGIT, TIGIT can also interact with CD112 and CD113 (Blake et al., Clin CancerRes; 2016; 22(21):5182-5188). The role of TIGIT as an inhibitory immune checkpoint receptor has been investigated. TIGIT is part of the CD226/TIGIT pathway, in which TIGIT not only competes with the co-stimulatory immune receptor CD226 for binding to CD155, but also directly interacts with CD226 in the cell membrane and blocks CD226 homodimerization. (Blake et al., S, Clin Cancer Res; 2016; 22(21): 5182-5188; Johnston et al., Cancer Cell 2014; 26: 923-937; Mahnke et al, Journal of Investigative Dermatology 2016; 136: 9-11).
抗TIGIT抗体是本领域已知的，包括US 2016/0355589，US 2017/143825，US 2017/088613，US 2016/376365，US2018/169238，US 2016/176963，和US 2019/100591中公开的那些抗体。然而，没有单独或与抗人PD-L1或抗人PD-1抗体组合的抗人TIGIT抗体已经获得在人中治疗用途的监管批准。此外无靶向TIGIT和PD-1或者TIGIT和PD-L1双特异性抗体被监管部门批准用于人的治疗用途。因此，存在对靶向免疫检查点通路的另外治疗的需要。Anti-TIGIT antibodies are known in the art and include those disclosed in US 2016/0355589, US 2017/143825, US 2017/088613, US 2016/376365, US 2018/169238, US 2016/176963, and US 2019/100591 . However, no anti-human TIGIT antibody, alone or in combination with an anti-human PD-L1 or anti-human PD-1 antibody, has received regulatory approval for therapeutic use in humans. In addition, no bispecific antibodies targeting TIGIT and PD-1 or TIGIT and PD-L1 are approved by regulatory authorities for therapeutic use in humans. Therefore, there is a need for additional treatments that target immune checkpoint pathways.
本领域需要新的能够用来治疗、预防或延缓与免疫检查点通路相关疾病的新特异性抗体及包含这类新抗体的制剂。需要的是所述制剂具有良好稳定性，当在液体中调配时，液体溶液中的抗TIGIT/PD-1双特异性抗体不易分解、聚集或发生不希望的化学修饰等。There is a need in the art for novel specific antibodies and formulations comprising such novel antibodies that can be used to treat, prevent or delay diseases associated with immune checkpoint pathways. What is required is that the formulation has good stability, and when formulated in a liquid, the anti-TIGIT/PD-1 bispecific antibody in the liquid solution is not easily decomposed, aggregated, or undesired chemical modification occurs.
发明概述SUMMARY OF THE INVENTION
本发明通过提供含有特异结合TIGIT和PD-1的抗TIGIT/PD-1双特异性抗体蛋白的药物制剂来满足上述需求。与其它抗人TIGIT抗体不同，本发明中使用的抗体是效应子功能无效的，即，被工程化以最小化Fc受体结合。因此，与其它抗人TIGIT抗体不同，本发明的抗体不包含可导致T调节细胞耗竭和免疫应答不良事件的天然人IgG1框架。此外，本发明的抗人TIGIT/抗人PD-1双特异性抗体含有不同类型的轻链，其中所述抗人TIGIT臂轻链是κ轻链，并且所述抗人PD-1轻链是λ轻链，其通过降低轻链-轻链二聚化的可能性促进异聚体双特异性抗体形成。The present invention meets the above needs by providing a pharmaceutical formulation containing an anti-TIGIT/PD-1 bispecific antibody protein that specifically binds TIGIT and PD-1. Unlike other anti-human TIGIT antibodies, the antibodies used in the present invention are ineffective in effector function, ie, engineered to minimize Fc receptor binding. Thus, unlike other anti-human TIGIT antibodies, the antibodies of the present invention do not contain a native human IgGl framework that can lead to T regulatory cell depletion and adverse events in the immune response. Furthermore, the anti-human TIGIT/anti-human PD-1 bispecific antibodies of the present invention contain different types of light chains, wherein the anti-human TIGIT arm light chain is a kappa light chain, and the anti-human PD-1 light chain is Lambda light chains, which facilitate heteromeric bispecific antibody formation by reducing the likelihood of light chain-light chain dimerization.
本发明提供了抗人TIGIT/抗人PD-1双特异性分子，其最小化Fc受体结合、最小化的氧化、促进性异聚体的组装，并且与人TIGIT/PD-1和猕猴TIGIT/PD-1交叉反应，并且在已建立的肿瘤模型中展示体内功效。本发明中使用的抗TIGIT/PD-1双特异性抗体在抗肿瘤作用方面显示协同作用。The present invention provides an anti-human TIGIT/anti-human PD-1 bispecific molecule that minimizes Fc receptor binding, minimizes oxidation, promotes assembly of heteromers, and interacts with human TIGIT/PD-1 and cynomolgus TIGIT /PD-1 cross-reacts and demonstrates in vivo efficacy in established tumor models. The anti-TIGIT/PD-1 bispecific antibodies used in the present invention exhibited synergistic effects in anti-tumor effects.
本发明的抗体制剂除了能够使抗体以适于施用给受试者的方式调配之外，还能在储存以及后续使用期间维持其稳定性。In addition to enabling the antibody formulations of the invention to be formulated in a manner suitable for administration to a subject, they also maintain their stability during storage and subsequent use.
在一个方面，本发明提供了一种液体抗体制剂，其包含(i)抗TIGIT/PD-1双特异性抗体蛋白；(ii)缓冲剂，(iii)稳定剂，和(iv)表面活性剂，优选地所述抗体制剂的pH为约4.5-6.8。In one aspect, the present invention provides a liquid antibody formulation comprising (i) an anti-TIGIT/PD-1 bispecific antibody protein; (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant , preferably the pH of the antibody formulation is about 4.5-6.8.
本发明抗体制剂中的抗TIGIT/PD-1双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合TIGIT(SEQ ID NO：31)或其胞外结构域(例如SEQ ID NO：32)的第一VH/VL单元并且第二半抗体包含特异性结合PD-1(SEQ ID NO：29)或其胞外结构域(例如SEQ ID NO：30)的第二VH/VL单元。在一些实施方案中，所述抗TIGIT/PD-1双特异性抗体蛋白能够以低于约10 -7M、优选地约10 -8M和更优选地约5nM、4nM、3nM、2nM、1nM、0.5nM或更低的K D与T淋巴细胞表面的PD-1结合，且能够以低于约10 -7M、优选地约10 -8M和更优选地约5nM、4nM、3nM、2nM、1nM、0.5nM或更低的K D与TIGIT结合，以致所述抗体可以用作双特异性靶向PD-1分子和TIGIT分子的治疗剂和/或预防剂。 The anti-TIGIT/PD-1 bispecific antibody protein in the antibody preparation of the present invention comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a specific binding TIGIT (SEQ ID NO: 31) or its extracellular structure The first VH/VL unit of the domain (eg, SEQ ID NO:32) and the second half-antibody comprises a Second VH/VL unit. In some embodiments, the anti-TIGIT/PD-1 bispecific antibody protein is capable of acting at less than about 10-7 M, preferably about 10-8 M, and more preferably about 5 nM, 4 nM, 3 nM, 2 nM, 1 nM , KD of 0.5nM or lower binds to PD -1 on the surface of T lymphocytes, and is capable of binding at less than about 10-7 M, preferably about 10-8 M and more preferably about 5nM, 4nM, 3nM, 2nM , 1 nM, 0.5 nM or lower KD binds to TIGIT such that the antibody can be used as a bispecific therapeutic and/or prophylactic targeting PD -1 molecule and TIGIT molecule.
在一实施方案中，所述第一VH/VL单元包含重链和轻链互补决定区(CDR)氨基酸序列SEQ ID NO：1-6或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；和/或第二VH/VL单元包含 重链和轻链互补决定区(CDR)氨基酸序列SEQ ID NO：7-12或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。In one embodiment, the first VH/VL unit comprises the heavy and light chain complementarity determining region (CDR) amino acid sequences SEQ ID NOs: 1-6 or has one or more of the 6 CDRs A sequence of one, two, three, four, five, six or more amino acid changes (eg, amino acid substitutions or deletions); and/or a second VH/VL unit comprising heavy and light chain complementarity determinations Region (CDR) amino acid sequence SEQ ID NOs: 7-12 or one, two, three, four, five, six or more amino acid changes with one or more of the 6 CDRs (eg, amino acid substitutions or deletions).
在一个实施方案中，抗TIGIT/PD-1双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合TIGIT的第一VH/VL单元并且第二半抗体包含特异性结合PD-1的第二VH/VL单元，其中所述第一VH/VL单元包含SEQ ID NO：13/SEQ ID NO：14的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，并且其中所述第二VH/VL单元包含SEQ ID NO：17/SEQ ID NO：18的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。In one embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a first VH/VL unit that specifically binds TIGIT and the second half antibody comprising a second VH/VL unit that specifically binds PD-1, wherein the first VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable sequence of SEQ ID NO: 13/SEQ ID NO: 14 region sequence, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the paired heavy chain variable region sequence/light chain variable region sequence , a sequence of 99% or more sequence identity, and wherein the second VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 17/SEQ ID NO: 18 , or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the paired heavy chain variable region sequence/light chain variable region sequence Sequences of % or more sequence identity.
在一个实施方案，抗TIGIT/PD-1双特异性抗体蛋白是异源二聚体，其中第一半抗体和第二半抗体各自形成两个单价结合臂。在一个实施方案，所述两个单价结合臂是通过二硫键连接的。In one embodiment, the anti-TIGIT/PD-1 bispecific antibody protein is a heterodimer, wherein the first half antibody and the second half antibody each form two monovalent binding arms. In one embodiment, the two monovalent binding arms are linked by a disulfide bond.
在一个具体的实施方案中，第一单价结合臂以低于约10 -7M、优选地约10 -8M和更优选地约5nM、4nM、3nM、2nM、1nM、0.5nM或更低的KD结合到TIGIT；在一个具体的实施方案中，第二单价结合臂以低于约10 -7M、优选地约10 -8M和更优选地约5nM、4nM、3nM、2nM、1nM、0.5nM或更低的KD结合到PD-1；在另一个实施方案中，与普通的单特异性抗体蛋白相比，双特异性抗体蛋白具有与其相同或相似的四级结构。 In a specific embodiment, the first monovalent binding arm is at a concentration of less than about 10-7 M, preferably about 10-8 M and more preferably about 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM or less KD binds to TIGIT; in a specific embodiment, the second monovalent binding arm is less than about 10-7 M, preferably about 10-8 M and more preferably about 5nM, 4nM, 3nM, 2nM, 1nM, 0.5 A KD of nM or lower binds to PD-1; in another embodiment, the bispecific antibody protein has the same or similar quaternary structure as the common monospecific antibody protein.
不特别地限制抗TIGIT/PD-1双特异性抗体蛋白中第一半抗体和第二半抗体的重链恒定区的类型，优选地是IgG1、IgG2或IgG4免疫球蛋白的重链恒定区，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。更优选地，所述重链恒定区是人IgG1免疫球蛋白的重链恒定区，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。not particularly limiting the type of heavy chain constant regions of the first and second half antibodies in the anti-TIGIT/PD-1 bispecific antibody protein, preferably the heavy chain constant regions of IgG1, IgG2 or IgG4 immunoglobulins, or substantially identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to sequences. More preferably, the heavy chain constant region is, or is substantially identical to (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).
在一个实施方案中，抗体的重链与抗体的轻链形成至少一个二硫键，抗体的两条重链形成至少一个二硫键。In one embodiment, the heavy chain of the antibody forms at least one disulfide bond with the light chain of the antibody, and the two heavy chains of the antibody form at least one disulfide bond.
在另一个实施方案中，抗体是工程化改造以降低抗体与Fcγ受体结合的人IgG1。In another embodiment, the antibody is human IgG1 engineered to reduce the binding of the antibody to Fcγ receptors.
在一个实施方案中，抗TIGIT/PD-1双特异性抗体蛋白包含IgG1(例如，人IgG1)中使用的重链恒定区。在又一个实施方案中，抗TIGIT/PD-1双特异性抗体蛋白包含用于IgG4(例如，人IgG4)的重链恒定区。例如，抗TIGIT/PD-1双特异性抗体的两条重链的Fc结构域中分别包含具有“CPPC”氨基酸残基的铰链区，和/或分别包含Y349C和S354C(根据Kabat的“EU编号”)，由此，抗PD-1半抗体和抗TIGIT半抗体在Fc区形成链间二硫键，由此，稳定抗PD-1半抗体和抗TIGIT半抗体的正确配对。In one embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a heavy chain constant region used in IgG1 (eg, human IgG1). In yet another embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a heavy chain constant region for IgG4 (eg, human IgG4). For example, the Fc domains of the two heavy chains of an anti-TIGIT/PD-1 bispecific antibody comprise hinge regions with "CPPC" amino acid residues, respectively, and/or Y349C and S354C, respectively (according to Kabat's "EU numbering" "), whereby the anti-PD-1 half antibody and the anti-TIGIT half antibody form an interchain disulfide bond in the Fc region, thereby stabilizing the correct pairing of the anti-PD-1 half antibody and the anti-TIGIT half antibody.
在一个实施方案中，抗TIGIT/PD-1双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含SEQ ID NO：21的重链序列或与之具有至少90％，95％，98％或99％同一性 的重链序列，和SEQ ID NO：22的轻链序列或与之具有至少90％，95％，98％或99％同一性的轻链序列，并且其中第二半抗体包含SEQ ID NO：23的重链序列或与之具有至少90％，95％，98％或99％同一性的重链序列，和SEQ ID NO：24的轻链序列或与之具有至少90％，95％，98％或99％同一性的轻链序列。In one embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises or has at least 90% of the heavy chain sequence of SEQ ID NO: 21 , a heavy chain sequence that is 95%, 98% or 99% identical, and a light chain sequence that is at least 90%, 95%, 98% or 99% identical to the light chain sequence of SEQ ID NO: 22, and wherein the second half-antibody comprises the heavy chain sequence of SEQ ID NO:23 or a heavy chain sequence at least 90%, 95%, 98% or 99% identical thereto, and the light chain sequence of SEQ ID NO:24 or has A light chain sequence having at least 90%, 95%, 98% or 99% identity.
在一个实施方案中，所述抗TIGIT/PD-1双特异性抗体蛋白是在HEK293细胞或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293E细胞；CHO细胞或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr-、CHO/DG44、ExpiCHO中重组表达的抗TIGIT/PD-1双特异性抗体蛋白。In one embodiment, the anti-TIGIT/PD-1 bispecific antibody protein is derived from HEK293 cells or HEK293T, HEK293F, HEK293E cells transformed on the basis of HEK293 cells; CHO cells or transformed on the basis of CHO cells The obtained anti-TIGIT/PD-1 bispecific antibody protein recombinantly expressed in CHO-S, CHO-dhfr-, CHO/DG44, and ExpiCHO.
在一个实施方案中，本发明的液体抗体制剂中的抗TIGIT/PD-1双特异性抗体蛋白的浓度为约1-200mg/ml，优选地为约1-150mg/ml，更优选地为约10-100mg/mL，例如约5、10、15、20、25、30、40、50、60、70、80、90或100mg/ml。In one embodiment, the concentration of anti-TIGIT/PD-1 bispecific antibody protein in the liquid antibody formulation of the present invention is about 1-200 mg/ml, preferably about 1-150 mg/ml, more preferably about 10-100 mg/mL, eg, about 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/mL.
在一个实施方案中，本发明液体制剂中的缓冲剂选自组氨酸、盐酸组氨酸、谷氨酸盐、磷酸盐、乙酸盐、柠檬酸盐和三羟甲基氨基甲烷和它们的组合，优选组氨酸、盐酸组氨酸和它们的组合。在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约0.5-200mM、约1-100mM、约5-50mM、约5-30mM或约5-20mM。In one embodiment, the buffer in the liquid formulation of the present invention is selected from the group consisting of histidine, histidine hydrochloride, glutamate, phosphate, acetate, citrate and tris and their Combinations, preferably histidine, histidine hydrochloride and combinations thereof. In one embodiment, the concentration of buffer in the liquid antibody formulation of the invention is about 0.5-200 mM, about 1-100 mM, about 5-50 mM, about 5-30 mM, or about 5-20 mM.
在一个实施方案中，用于本发明制剂中的缓冲剂是约10mM组氨酸。In one embodiment, the buffer used in the formulations of the present invention is about 10 mM histidine.
又在一个实施方案中，用于本发明制剂中的缓冲剂是约5mM组氨酸和约5mM盐酸组氨酸的组合。In yet another embodiment, the buffer used in the formulation of the present invention is a combination of about 5 mM histidine and about 5 mM histidine hydrochloride.
在一些实施方案中，用于本发明制优选剂中的缓冲剂可以将本发明制剂的pH控制在大约4.5-6.8的pH范围，优选大约5.0-6.5，更优选约6.0的pH。在一些具体的实施方案中，本发明的抗体制剂具有约4.5、4.8、5.0、5.2、5.4、5.6、5.8、6.0、6.2、6.4、6.5、6.6、6.7或6.8的pH。In some embodiments, the buffering agents used in the formulations of the present invention can control the pH of the formulations of the present invention to a pH in the range of about 4.5-6.8, preferably about 5.0-6.5, more preferably about 6.0 pH. In some specific embodiments, the antibody formulation of the invention has a pH of about 4.5, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.5, 6.6, 6.7, or 6.8.
在一个实施方案中，所述稳定剂选自糖类、多元醇、氨基酸或其盐及它们的组合，优选地，所述糖类选自但不限于：蔗糖、右旋糖、乳糖、麦芽糖、海藻糖、环糊精、麦芽糖糊精和葡聚糖，所述多元醇选自但不限于：甘露醇、山梨醇和木糖醇，所述氨基酸或其盐选自选自但不限于精氨酸、精氨酸盐(优选精氨酸盐酸盐)和其组合，优选精氨酸盐酸盐。在一些实施方案中，所述稳定剂是山梨醇。在一些实施方案中，所述稳定剂是精氨酸盐酸盐。在一些实施方案中，所述稳定剂是山梨醇、精氨酸、精氨酸盐(优选精氨酸盐酸盐)或其组合。In one embodiment, the stabilizer is selected from saccharides, polyols, amino acids or their salts and combinations thereof, preferably, the saccharides are selected from but not limited to: sucrose, dextrose, lactose, maltose, Trehalose, cyclodextrin, maltodextrin and dextran, the polyol is selected from but not limited to: mannitol, sorbitol and xylitol, the amino acid or its salt is selected from but not limited to arginine , arginine (preferably arginine hydrochloride) and combinations thereof, preferably arginine hydrochloride. In some embodiments, the stabilizer is sorbitol. In some embodiments, the stabilizer is arginine hydrochloride. In some embodiments, the stabilizer is sorbitol, arginine, an arginine salt (preferably arginine hydrochloride), or a combination thereof.
在一些实施方案中，本发明的稳定剂在本发明的液体制剂中以约1-1000mM、约10-1000mM，优选地约20-800mM，例如约50-500、约100-400、约100-300或约100-200mM例如，约10、20、50、80、100、150、200、300、400、500、600、700、800、900、1000mM的浓度存在。In some embodiments, the stabilizer of the present invention is present in the liquid formulation of the present invention at about 1-1000 mM, about 10-1000 mM, preferably about 20-800 mM, such as about 50-500, about 100-400, about 100- Present at a concentration of 300 or about 100-200 mM, eg, about 10, 20, 50, 80, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM.
本发明的稳定剂中还可以包含抗氧化剂、EDTA和/或依地酸二钠，其选自但不限于：同型半胱氨酸、半胱氨酸、胱硫醚、甲硫氨酸、谷胱甘肽、以及包含同型半胱氨酸、半胱氨酸、胱硫醚、甲硫氨酸和谷胱甘肽中任意一种的肽。在包含抗氧化剂、EDTA和/或依地酸二钠的情形下，稳定剂的总浓度如前所述，其中抗氧化剂的浓度为约1-50mM，优选地约5-40mM，例如约5、10、20、30、40mM；EDTA和/或依地酸二钠的浓度为约0.001-0.5mg/ml，例如 0.005-0.02mg/ml，优选0.01mg/ml。The stabilizer of the present invention may also contain antioxidants, EDTA and/or disodium edetate, which are selected from but not limited to: homocysteine, cysteine, cystathionine, methionine, glutathione Cysteine, and peptides comprising any one of homocysteine, cysteine, cystathionine, methionine, and glutathione. In the case of including antioxidant, EDTA and/or disodium edetate, the total concentration of stabilizer is as previously described, wherein the concentration of antioxidant is about 1-50 mM, preferably about 5-40 mM, such as about 5, 10, 20, 30, 40 mM; EDTA and/or disodium edetate at a concentration of about 0.001-0.5 mg/ml, eg 0.005-0.02 mg/ml, preferably 0.01 mg/ml.
在一个实施方案中，本发明的液体制剂中的表面活性剂是非离子型表面活性剂，例如，烷基聚(环氧乙烯)。可包括在本发明制剂中的特定非离子型表面活性剂包括，例如聚山梨酯，诸如聚山梨酯-20、聚山梨酯-80、聚山梨酯-60、或聚山梨酯-40；普洛尼克等。在一个优选实施方案中，本发明的液体制剂中包含聚山梨酯-80作为表面活性剂。In one embodiment, the surfactant in the liquid formulation of the present invention is a nonionic surfactant, eg, an alkyl poly(ethylene oxide). Particular nonionic surfactants that can be included in the formulations of the present invention include, for example, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; Nick et al. In a preferred embodiment, polysorbate-80 is included as a surfactant in the liquid formulation of the present invention.
在优选的一些实施方案中，本发明的液体抗体制剂中的表面活性剂的浓度为约0.01-10mg/ml，优选地约0.05-5、约0.05-2、约0.1-5、0.2-2、0.3-1、0.4-0.8、0.5-0.6mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml。In preferred embodiments, the concentration of surfactant in the liquid antibody formulation of the present invention is about 0.01-10 mg/ml, preferably about 0.05-5, about 0.05-2, about 0.1-5, 0.2-2, 0.3-1, 0.4-0.8, 0.5-0.6 mg/ml, eg about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml.
在一个实施方案中，所述液体制剂为药物制剂，优选为注射剂，更优选为皮下注射剂或静脉内注射剂。在一个实施方案中，所述液体制剂为静脉输注剂。In one embodiment, the liquid preparation is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection. In one embodiment, the liquid formulation is an intravenous infusion.
在一个实施方案中，本发明的液体抗体制剂包含：In one embodiment, the liquid antibody formulation of the present invention comprises:
(i)约1-150mg/ml的抗TIGIT/PD-1双特异性抗体蛋白；(i) about 1-150 mg/ml of anti-TIGIT/PD-1 bispecific antibody protein;
(ii)约5-50mM的组氨酸和/或盐酸组氨酸；(ii) about 5-50 mM histidine and/or histidine hydrochloride;
(iii)约50-500mM的山梨醇；和/或约1-600mM的精氨酸或盐酸精氨酸；和(iii) about 50-500 mM sorbitol; and/or about 1-600 mM arginine or arginine hydrochloride; and
(iv)约0.05-2mg/ml聚山梨醇酯80；(iv) about 0.05-2 mg/ml polysorbate 80;
其中所述液体制剂的pH为约5.0-6.5，优选地约6.0。wherein the pH of the liquid formulation is about 5.0-6.5, preferably about 6.0.
在一个实施方案中，本发明的液体抗体制剂包含：In one embodiment, the liquid antibody formulation of the present invention comprises:
(i)约10-100mg/ml的抗TIGIT/PD-1双特异性抗体蛋白；(i) about 10-100 mg/ml of anti-TIGIT/PD-1 bispecific antibody protein;
(ii)约5-20mM的组氨酸和/或盐酸组氨酸；(ii) about 5-20 mM histidine and/or histidine hydrochloride;
(iii)约100-300mM的山梨醇；和/或约50-200mM的精氨酸或盐酸精氨酸；和(iii) about 100-300 mM sorbitol; and/or about 50-200 mM arginine or arginine hydrochloride; and
(iv)约0.2-2mg/ml聚山梨醇酯80；(iv) about 0.2-2 mg/ml polysorbate 80;
其中所述液体制剂的pH为约5.0-6.5，优选地约6.0。wherein the pH of the liquid formulation is about 5.0-6.5, preferably about 6.0.
在一个实施方案中，本发明的液体抗体制剂包含：In one embodiment, the liquid antibody formulation of the present invention comprises:
(i)约25mg/ml的抗TIGIT/PD-1双特异性抗体蛋白；(i) about 25 mg/ml of anti-TIGIT/PD-1 bispecific antibody protein;
(ii)约5mM的组氨酸和约5mM盐酸组氨酸；或者约10mM的组氨酸；(ii) about 5 mM histidine and about 5 mM histidine hydrochloride; or about 10 mM histidine;
(iii)约25mg/ml的山梨醇；和约80mM的精氨酸或盐酸精氨酸；和(iii) about 25 mg/ml of sorbitol; and about 80 mM of arginine or arginine hydrochloride; and
(iv)约0.5mg/ml聚山梨醇酯80；(iv) about 0.5 mg/ml polysorbate 80;
其中所述液体制剂的pH为约5.0-6.5，优选地约6.0。wherein the pH of the liquid formulation is about 5.0-6.5, preferably about 6.0.
另一方面，本发明提供了一种固体抗体制剂，其是通过将本发明的液体抗体制剂经固化处理而获得的。所述固化处理是通过例如结晶法、喷雾干燥法、冷冻干燥法实施的。在一个优选的实施方案中，所述固体抗体制剂例如是冻干粉针剂形式。固体抗体制剂可在使用前，通过重构于适当的溶媒中，形成本发明的重构制剂。所述重构制剂也是一种本发明的液体抗体制剂。在一个实施方案中，所述适当的溶媒选自注射用水、注射用有机溶剂，包括但不限于注射用油、乙醇、丙二醇等，或其组合。In another aspect, the present invention provides a solid antibody preparation obtained by subjecting the liquid antibody preparation of the present invention to a solidification treatment. The solidification treatment is carried out by, for example, a crystallization method, a spray-drying method, or a freeze-drying method. In a preferred embodiment, the solid antibody formulation is, for example, in the form of a lyophilized powder for injection. Solid antibody formulations can be reconstituted in a suitable vehicle prior to use to form a reconstituted formulation of the invention. The reconstituted formulation is also a liquid antibody formulation of the present invention. In one embodiment, the appropriate vehicle is selected from water for injection, organic solvent for injection, including but not limited to oil for injection, ethanol, propylene glycol, etc., or a combination thereof.
本发明的液体制剂可以长期稳定储存，例如至少24个月或更长时间。在一个实施方案中，本发明的液体制剂可以在约-80℃至约45℃，例如-80℃、约-30℃、约-20℃、约0℃、约5℃、 约25℃、约35℃、约38℃、约40℃、约42℃或约45℃的条件下，储存至少10天、至少20天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，至少36个月，或更长时间，且是稳定的。The liquid formulations of the present invention are stable for long-term storage, eg, at least 24 months or more. In one embodiment, the liquid formulation of the present invention may be at about -80°C to about 45°C, eg, -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about Store at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months at 35°C, about 38°C, about 40°C, about 42°C, or about 45°C , at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months , at least 36 months, or longer, and is stable.
在一个实施方案中，本发明的液体制剂可以稳定储存至少24个月。在再一实施方案中，本发明的液体制剂在至少40℃是稳定的。在再一实施方案中，本发明的液体制剂在约2℃-8℃保持稳定至少3个月，优选至少12个月，更优选至少24个月。在一个实施方案中，本发明的液体制剂在室温或例如约25℃保持稳定至少2个月，优选至少3个月，更优选至少6个月。在再一实施方案中，本发明的液体制剂在约40℃保持稳定至少2周、优选至少1个月。In one embodiment, the liquid formulations of the present invention are storage stable for at least 24 months. In yet another embodiment, the liquid formulations of the present invention are stable at at least 40°C. In yet another embodiment, the liquid formulations of the present invention are stable at about 2°C-8°C for at least 3 months, preferably at least 12 months, more preferably at least 24 months. In one embodiment, the liquid formulations of the present invention are stable at room temperature or, for example, about 25°C for at least 2 months, preferably at least 3 months, more preferably at least 6 months. In yet another embodiment, the liquid formulations of the present invention are stable at about 40°C for at least 2 weeks, preferably at least 1 month.
在一个实施方案中，可以通过检测制剂的外观、可见异物、蛋白含量、纯度、和/或电荷变异体的变化，来指示储存后制剂的稳定性。在一个实施方案中，可以在高温胁迫的强制实验中，例如在40℃±2℃储存至少1周、2周或优选地1个月后，或在加速实验中，例如在25℃±2℃储存至少1个月或2个月后，或在长期实验中，例如在5℃±3℃储存至少2个月或3个月后，检测本发明液体制剂的稳定性。In one embodiment, the stability of the formulation after storage can be indicated by detecting changes in the formulation's appearance, visible foreign matter, protein content, purity, and/or charge variants. In one embodiment, it can be in a forced experiment under high temperature stress, eg after storage at 40°C ± 2°C for at least 1 week, 2 weeks or preferably 1 month, or in an accelerated experiment, eg at 25°C ± 2°C The stability of the liquid formulations of the invention is tested after storage for at least 1 month or 2 months, or in long-term experiments, eg, after storage at 5°C ± 3°C for at least 2 months or 3 months.
在一个实施方案中，在储存后，通过目视检查本发明液体制剂的稳定性，其中本发明液体制剂在外观上保持为澄明至微乳光，为无色至淡黄色液体，且无异物。在一个实施方案中，在澄明度检测仪下目视检查，制剂中无可见异物存在。在一个实施方案中，在储存后，通过测定蛋白含量变化，检查本发明液体制剂的稳定性，其中例如通过紫外分光光度(UV)法，相对于储存第0天的初始值，蛋白含量变化率不超过20％，优选不超过10％，例如7-8％，更优选不超过5％。在一个实施方案中，在储存后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过体积排阻高效液相色谱法(SEC-HPLC)，相对于储存第0天的初始值，单体纯度的变化值不超过10％，例如不超过5％、4％、3％、例如变化值不超过1-2％，优选不超过1％。在一个实施方案中，在储存后，通过测定本发明液体制剂的纯度变化，检查本发明液体制剂的稳定性，其中通过非还原型和/或还原型十二烷基硫酸钠毛细管电泳(CE-SDS)法，单体纯度的变化值下降不超过10％，例如不超过5％、4％、3％。在一个实施方案中，在储存后，通过成像毛细管等电聚焦电泳(iCIEF)检测本发明液体制剂的稳定性，其中相对于储存第0天的初始值，抗体的电荷变异体(主成分、酸性组分和碱性组分)的变化值总和不超过50％，例如不超过40％、30％、20％、10％、5％。In one embodiment, after storage, the stability of the liquid formulations of the present invention is visually inspected, wherein the liquid formulations of the present invention remain clear to slightly opalescent in appearance, colorless to pale yellow liquids, and free of foreign matter. In one embodiment, no visible foreign matter is present in the formulation upon visual inspection under a clarity tester. In one embodiment, the stability of the liquid formulations of the invention is checked after storage by measuring the change in protein content, wherein the rate of change in protein content relative to the initial value on day 0 of storage, for example by ultraviolet spectrophotometry (UV) No more than 20%, preferably no more than 10%, eg 7-8%, more preferably no more than 5%. In one embodiment, the stability of the liquid formulation of the present invention is examined by determining the change in purity of the liquid formulation of the present invention after storage, wherein by size exclusion high performance liquid chromatography (SEC-HPLC), relative to storage 0 From the initial value of the day, the change in monomer purity is no more than 10%, eg, no more than 5%, 4%, 3%, eg, no more than 1-2%, preferably no more than 1%. In one embodiment, the stability of the liquid formulation of the present invention is checked by determining the change in purity of the liquid formulation of the present invention after storage, wherein by non-reducing and/or reducing sodium dodecyl sulfate capillary electrophoresis (CE- SDS) method, the change value of monomer purity decreased by no more than 10%, such as no more than 5%, 4%, 3%. In one embodiment, the stability of the liquid formulations of the invention is tested by imaging capillary isoelectric focusing electrophoresis (iCIEF) after storage, wherein the antibody's charge variant (principal component, acidic The sum of the variation values of components and basic components) does not exceed 50%, for example, does not exceed 40%, 30%, 20%, 10%, 5%.
在一个实施方案中，制剂在储存后，例如在2-8℃储存至少24个月后，或在室温储存至少3个月后，或在40℃±2℃储存1个月后，是稳定的，优选地具有如下特征之一或多项：In one embodiment, the formulation is stable after storage, eg, at 2-8°C for at least 24 months, or at room temperature for at least 3 months, or at 40°C ± 2°C for 1 month , preferably with one or more of the following characteristics:
(i)通过SEC-HPLC法测量，制剂具有大于90％的纯度，优选大于95％、96％、97％、98％、99％的纯度；(i) the preparation has a purity greater than 90%, preferably greater than 95%, 96%, 97%, 98%, 99%, as measured by the SEC-HPLC method;
(ii)通过还原型或非还原型CE-SDS法测量，制剂具有大于90％的纯度，优选大于92％、94％、96％、98％的纯度；(ii) the preparation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, 98%, as measured by the reduced or non-reduced CE-SDS method;
(iii)通过iCIEF法测量，相对于储存第0天的初始值，制剂中抗TIGIT/PD-1双特异性抗体蛋白的各组分(主成分、酸性组分和碱性组分)的变化值总和不超过50％，例如不超过40％、 30％、20％、10％、5％；(iii) Changes in each component (principal component, acidic component and basic component) of the anti-TIGIT/PD-1 bispecific antibody protein in the preparation relative to the initial value on day 0 of storage, measured by the iCIEF method The sum of the values does not exceed 50%, such as 40%, 30%, 20%, 10%, 5%;
(iv)通过ELISA法测量，相对于储存第0天的初始值，制剂中抗TIGIT/PD-1双特异性抗体蛋白的相对结合活性为70％-130％，例如，为70％、80％、90％、100％、110％、120％、130％。(iv) The relative binding activity of the anti-TIGIT/PD-1 bispecific antibody protein in the formulation is 70%-130%, eg, 70%, 80%, relative to the initial value on storage day 0, as measured by ELISA , 90%, 100%, 110%, 120%, 130%.
在一个方面，本发明提供了一种递送装置，其包含本发明的液体抗体制剂或固体抗体制剂。在一个实施方案中，本发明的递送装置以包含本发明的液体抗体制剂或固体抗体制剂的预填充注射器形式提供，例如用于静脉内、皮下、皮内或者肌内注射、静脉内输注。In one aspect, the invention provides a delivery device comprising a liquid antibody formulation or a solid antibody formulation of the invention. In one embodiment, the delivery device of the invention is provided in the form of a prefilled syringe containing the liquid antibody formulation or solid antibody formulation of the invention, eg, for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion.
在又一方面，本发明提供向受试者，例如哺乳动物递送抗TIGIT/PD-1双特异性抗体蛋白的方法，包括给予所述受试者本发明的液体抗体制剂或固体抗体制剂的步骤，所述递送是例如通过使用预填充注射器的递送装置实施的。In yet another aspect, the present invention provides a method of delivering an anti-TIGIT/PD-1 bispecific antibody protein to a subject, eg, a mammal, comprising the step of administering to said subject a liquid antibody formulation or solid antibody formulation of the invention , the delivery is carried out, for example, by a delivery device using a prefilled syringe.
在又一方面，本发明提供本发明的液体抗体制剂或固体抗体制剂用于制备在受试者中治疗、预防或延缓与TIGIT信号传导通路和PD-1信号传导通路相关的病症的递送装置(如，预填充注射器)或药物的用途，所述病症例如各种血液病和实体瘤，包括但不限于白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈癌、头颈部鳞状细胞癌、肺癌例如非小细胞肺癌或小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、胆管癌、肝癌、结直肠癌、乳腺癌、卵巢癌、***、子宫内膜癌、子宫肉瘤、***癌、膀胱癌、肾细胞癌、黑色素瘤。In yet another aspect, the present invention provides a liquid antibody formulation or solid antibody formulation of the present invention for use in the manufacture of a delivery device for the treatment, prevention or delay of a disorder associated with the TIGIT signaling pathway and the PD-1 signaling pathway in a subject ( such as, prefilled syringes) or drugs, such as various hematological diseases and solid tumors, including but not limited to leukemia, lymphoma, myeloma, brain tumor, head and neck cancer, head and neck squamous cell carcinoma, lung cancer Such as non-small cell lung cancer or small cell lung cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma , prostate cancer, bladder cancer, renal cell carcinoma, melanoma.
本发明的其它实施方案将通过参阅此后的详细说明而清楚明了。Other embodiments of the present invention will become apparent by reference to the detailed description which follows.
附图说明Description of drawings
图1.显示了antibody-A对LOVO混合PBMC荷瘤小鼠体重的影响。Figure 1. Shows the effect of antibody-A on body weight of LOVO mixed PBMC tumor-bearing mice.
图2.显示了antibody-A对LOVO混合PBMC荷瘤小鼠肿瘤组织生长的影响。Figure 2. Shows the effect of antibody-A on tumor tissue growth in LOVO mixed PBMC tumor-bearing mice.
图3.显示了低剂量antibody-A对LOVO混合PBMC荷瘤小鼠瘤组织生长的影响。Figure 3. Shows the effect of low-dose antibody-A on tumor tissue growth in LOVO mixed PBMC tumor-bearing mice.
图4.显示了高剂量antibody-A对LOVO混合PBMC荷瘤小鼠瘤组织生长的影响。Figure 4. Shows the effect of high doses of antibody-A on tumor tissue growth in LOVO mixed PBMC tumor-bearing mice.
图5.显示了LOVO混合PBMC荷瘤小鼠个体肿瘤曲线图。Figure 5. Shows individual tumor profiles of LOVO mixed PBMC tumor-bearing mice.
图6.显示了antibody-A对LOVO混合PBMC荷瘤小鼠瘤组织生长的影响(肿瘤照片)。Figure 6. Shows the effect of antibody-A on tumor tissue growth in LOVO mixed PBMC tumor-bearing mice (tumor photos).
图7.显示了antibody-A对LOVO混合PBMC荷瘤小鼠瘤组织重量的影响。Figure 7. Shows the effect of antibody-A on tumor tissue weight in LOVO mixed PBMC tumor-bearing mice.
图8.显示了antibody-A对MC38荷瘤小鼠体重的影响。Figure 8. Shows the effect of antibody-A on body weight of MC38 tumor-bearing mice.
图9.显示了antibody-A对MC38荷瘤小鼠瘤组织生长的影响。Figure 9. Shows the effect of antibody-A on tumor tissue growth in MC38 tumor-bearing mice.
图10.显示了低剂量antibody-A对MC38荷瘤小鼠瘤组织生长的影响。Figure 10. Shows the effect of low doses of antibody-A on tumor tissue growth in MC38 tumor-bearing mice.
图11.显示了高剂量antibody-A对MC38荷瘤小鼠瘤组织生长的影响。Figure 11. Shows the effect of high doses of antibody-A on tumor tissue growth in MC38 tumor-bearing mice.
图12.显示了MC38荷瘤小鼠个体肿瘤曲线图。Figure 12. Shows individual tumor profiles of MC38 tumor-bearing mice.
图13.显示了antibody-A对MC38荷瘤小鼠瘤组织生长的影响(肿瘤照片)。Figure 13. Shows the effect of antibody-A on tumor tissue growth in MC38 tumor-bearing mice (tumor photos).
图14.显示了antibody-A对MC38荷瘤小鼠瘤组织重量的影响。Figure 14. Shows the effect of antibody-A on tumor tissue weight in MC38 tumor-bearing mice.
图15.显示了antibody-A阻断PD-1结合PD-L1及TIGIT结合CD155的活性。Figure 15. Shows that antibody-A blocks the activity of PD-1 binding to PD-L1 and TIGIT binding to CD155.
图16：显示了T细胞激活活性(memory T cell recall法)。Figure 16: shows T cell activation activity (memory T cell recall method).
图17.显示了通过SEC-HPLC法对各样品测定的蛋白纯度纯度变化趋势图。Figure 17. A graph showing the change trend of protein purity and purity determined by SEC-HPLC method for each sample.
图18.显示了pH影响实验中电荷变异体酸性组分变化趋势图(iCIEF法)。Figure 18. A graph showing the change trend of the acidic component of the charge variant in the pH effect experiment (iCIEF method).
图19.显示了pH影响实验中电荷变异体主成分变化趋势图(iCIEF法)。Figure 19. A graph showing the change trend of the principal components of the charge variants in the pH effect experiment (iCIEF method).
图20.显示了pH影响实验中电荷变异体碱性组分变化趋势图(iCIEF法)。Figure 20. A graph showing the change trend of the basic component of the charge variant in the pH effect experiment (iCIEF method).
图21.显示了处方筛选实验纯度变化趋势图(SEC-HPLC法)。Figure 21. Shows the variation trend of the purity of the formulation screening experiment (SEC-HPLC method).
图22.显示了处方筛选实验纯度变化趋势图(非还原型CE-SDS法)。Figure 22. Shows the variation trend of the purity of the formulation screening experiment (non-reducing CE-SDS method).
图23.显示了处方筛选实验电荷变异体酸性组分变化趋势图(iCIEF法)。Figure 23. A graph showing the change trend of the acidic components of the charge variant in the formulation screening experiment (iCIEF method).
图24.显示了处方筛选实验电荷变异体主成分变化趋势图(iCIEF法)。Figure 24. Shows the change trend diagram of the principal components of charge variants in the formulation screening experiment (iCIEF method).
图25.显示了实施例1制备的antibody-A的结构。Figure 25. The structure of antibody-A prepared in Example 1 is shown.
发明详述Detailed description of the invention
在详细描述本发明之前，应了解，本发明不受限于本说明书中的特定方法及实验条件，因为所述方法以及条件是可以改变的。另外，本文所用术语仅是供说明特定实施方案之用，而不意欲为限制性的。Before the present invention is described in detail, it is to be understood that this invention is not limited to the particular methods and experimental conditions set forth in this specification, as such methods and conditions may vary. Also, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
定义definition
除非另有定义，否则本文中使用的所有技术和科学术语均具有与本领域一般技术人员通常所理解的含义相同的含义。为了本发明的目的，下文定义了以下术语。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。The term "about" when used in conjunction with a numerical value is intended to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.
术语“和/或”当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或多项。The term "and/or" when used in conjunction with two or more alternatives should be understood to mean any one of the alternatives or any two or more of the alternatives.
术语“任选的”或“任选地”意指其后的成分可以存在或不存在，或着其后的事件可以发生或不发生。The term "optional" or "optionally" means that a subsequent ingredient may or may not be present, or that a subsequent event may or may not occur.
如本文中所用，术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其他要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。As used herein, the term "comprising" or "comprising" means the inclusion of stated elements, integers or steps, but not the exclusion of any other elements, integers or steps. Herein, when the term "comprising" or "comprising" is used, unless otherwise indicated, it also encompasses situations consisting of the recited elements, integers or steps. For example, reference to an antibody variable region that "comprises" a particular sequence is also intended to encompass antibody variable regions that consist of that particular sequence.
在本文中，术语“抗体”以最广意义使用，指包含抗原结合位点的蛋白质，涵盖各种结构的天然抗体和人工抗体，包括但不限于完整抗体和抗体的抗原结合片段。As used herein, the term "antibody" is used in the broadest sense to refer to a protein comprising an antigen-binding site, encompassing natural and artificial antibodies of various structures, including, but not limited to, whole antibodies and antigen-binding fragments of antibodies.
术语“全抗体”、“全长抗体”、“完全抗体”和“完整抗体”在本文中可互换地用来指包含由二硫键相互连接的至少两条重链(H)和两条轻链(L)的糖蛋白。每条重链由重链可变区(本文中缩写为VH)和重链恒定区组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每条轻链由轻链可变区(本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH区和VL区可以进一步再划分为超变区(为互补决定区(CDR)，其间插有较保守的区域(为构架区(FR))。每个VH和VL由三个CDR和4个FR组成，从氨基端到羧基端以如下顺序排列：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。恒定区不直接参与抗体与抗原的结合，但是显示出多种效应子功能。The terms "whole antibody", "full length antibody", "complete antibody" and "intact antibody" are used interchangeably herein to refer to a composition comprising at least two heavy chains (H) and two Light chain (L) glycoprotein. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions (complementarity determining regions (CDRs), with more conserved regions (framework regions (FR)) interposed therebetween. Each VH and VL consists of three CDRs and four The FRs are composed, from the amino terminus to the carboxy terminus, in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant region is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions.
术语“人源化”抗体指包含来自非人类HVR的氨基酸残基和来自人FR的氨基酸残基的嵌合抗体。在一些实施方案中，人源化抗体包含全部或基本上全部的HVR(例如，CDR)与非人抗体的那些HVR对应并且全部或基本上全部的FR区与人抗体的那些FR对应。人源化抗体任选地可以包含从人抗体衍生的抗体恒定区的至少一部分。抗体(例如非人抗体)的“人源化形式”指已经历过人源化的抗体。The term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from a non-human HVR and amino acid residues from a human FR. In some embodiments, the humanized antibody comprises all or substantially all of the HVRs (eg, CDRs) corresponding to those of the non-human antibody and all or substantially all of the FR regions corresponding to those of the human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (eg, a non-human antibody) refers to an antibody that has undergone humanization.
术语“半抗体”或“半聚物”指单价抗原结合多肽。在一些实施方案中，半抗体或半聚物包含VH/VL单元和任选地免疫球蛋白恒定结构域的至少一部分。在一些实施方案中，半抗体或半聚物包含与一条免疫球蛋白轻链缔合的一条免疫球蛋白重链，或其抗原结合片段。在一些实施方案中，半抗体或半聚物是单特异的，即，与单个抗原或表位结合。在一些具体的实施方案中，半抗体与TIGIT结合并且不与PD-1结合。在一些具体的实施方案中，半抗体与PD-1结合并且不与TIGIT结合。本领域技术人员将会轻易地理解，半抗体可以具有由单一可变结构域组成(例如，源自骆驼属(Camelidae))的抗原结合结构域。The term "half-antibody" or "hemimer" refers to a monovalent antigen-binding polypeptide. In some embodiments, the half-antibody or half-mer comprises a VH/VL unit and optionally at least a portion of an immunoglobulin constant domain. In some embodiments, the half-antibody or half-mer comprises one immunoglobulin heavy chain associated with one immunoglobulin light chain, or an antigen-binding fragment thereof. In some embodiments, half-antibodies or half-mers are monospecific, ie, bind to a single antigen or epitope. In some specific embodiments, the half-antibody binds to TIGIT and does not bind to PD-1. In some specific embodiments, the half-antibody binds to PD-1 and does not bind to TIGIT. Those of skill in the art will readily appreciate that half-antibodies may have antigen-binding domains consisting of a single variable domain (eg, derived from Camelidae).
术语“VH/VL单元”指抗体中包含至少一个VH CDR和至少一个VL CDR的抗原结合区。在一些实施方案中，VH/VL单元包含至少一个、至少两个或全部三个VH CDR和至少一个、至少两个或全部三个VL CDR。在某些实施方案中，VH/VL单元还包含构架区(FR)的至少一部分。在一些实施方案中，VH/VL单元包含三个VH CDR和三个VL CDR。在一些实施方案中，VH/VL单元包含至少一个、至少两个、至少三个或全部四个VH FR和至少一个、至少两个、至少三个或全部四个VL FR。The term "VH/VL unit" refers to the antigen-binding region of an antibody comprising at least one VH CDR and at least one VL CDR. In some embodiments, the VH/VL unit comprises at least one, at least two, or all three VH CDRs and at least one, at least two, or all three VL CDRs. In certain embodiments, the VH/VL unit further comprises at least a portion of a framework region (FR). In some embodiments, the VH/VL unit comprises three VH CDRs and three VL CDRs. In some embodiments, the VH/VL unit comprises at least one, at least two, at least three, or all four VH FRs and at least one, at least two, at least three, or all four VL FRs.
在本文中，术语“双特异性抗体”或“双特异性抗体蛋白”包含与两种不同生物分子上的表位特异性结合的抗原结合结构域。除非另外说明，否则列出的双特异性抗体名称中双特异性抗体结合的抗原的顺序是任意的。即，在一些实施方案中，术语“抗TIGIT/PD-1双特异性抗体”和“抗PD-1/TIGIT双特异性抗体”可以互换使用。双特异性抗体形式包含IgG样和非IgG样抗体(Fan等人
Figure PCTCN2021133345-appb-000003
Journal of Hematology&Oncology.8：130)。最常见的IgG样抗体类型包含两个Fab区域和一个Fc区域，每个Fab的重链和轻链可以来自单独的单克隆抗体。非IgG样双特异性抗体缺乏Fc区域，其每一个抗原或靶标结合结构域可以是Fab，也可以是单链可变片段(scFv)，还可以是模拟两种抗体的可变结构域的融合蛋白，不同的结合结构域通过肽接头、化学偶联、非共价键连接或者其他方式连接到一起。这些形式包含双特异性T-细胞衔接子(BiTE)。 As used herein, the term "bispecific antibody" or "bispecific antibody protein" comprises an antigen binding domain that specifically binds to epitopes on two different biomolecules. Unless otherwise stated, the order of the antigens bound by the bispecific antibody in the bispecific antibody designation listed is arbitrary. That is, in some embodiments, the terms "anti-TIGIT/PD-1 bispecific antibody" and "anti-PD-1/TIGIT bispecific antibody" are used interchangeably. Bispecific antibody formats include IgG-like and non-IgG-like antibodies (Fan et al.
Figure PCTCN2021133345-appb-000003
Journal of Hematology & Oncology. 8:130). The most common type of IgG-like antibody contains two Fab regions and one Fc region, and the heavy and light chains of each Fab can be derived from separate monoclonal antibodies. Non-IgG-like bispecific antibodies lacking an Fc region, each antigen- or target-binding domain of which can be a Fab, a single-chain variable fragment (scFv), or a fusion of variable domains that mimic two antibodies In proteins, the different binding domains are linked together by peptide linkers, chemical conjugation, non-covalent linkages, or other means. These formats contain bispecific T-cell adaptors (BiTEs).
可以使用任何双特异性抗体形式或技术来制备本发明的双特异性抗体。例如，具有第一抗原结合特异性的抗体或其片段可以与如具有第二抗原结合特异性的另一种抗体或抗体片段等一种或多种其它分子实体功能性连结(例如，通过化学偶联、遗传融合、非共价缔合或以其它方式)，以产生双特异性抗体。可以在本发明的背景下使用的具体示例性双特异性形式包含但不限于以下：基于scFv的或双抗体双特异性形式、IgG-scFv融合、双可变域(DVD)-Ig、四杂交瘤(Quadroma)、杵臼(knobs-into-holes)、普通轻链(例如，具有旋钮入孔的 普通轻链等)、CrossMab、CrossFab，(SEED)body、Duobody、IgG1/IgG2、双作用Fab(DAF)-IgG以及Mab 2双特异性形式。 Bispecific antibodies of the invention can be prepared using any bispecific antibody format or technique. For example, an antibody or fragment thereof having a first antigen-binding specificity can be functionally linked (eg, by chemical coupling) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity association, genetic fusion, non-covalent association, or otherwise) to generate bispecific antibodies. Specific exemplary bispecific formats that can be used in the context of the present invention include, but are not limited to, the following: scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, tetra-hybrid Quadroma, knobs-into-holes, common light chains (eg, common light chains with knob-in holes, etc.), CrossMab, CrossFab, (SEED)body, Duobody, IgG1/IgG2, dual-acting Fab ( DAF)-IgG and Mab 2 bispecific format.
在一些实施方案中，双特异性抗体是异源二聚体，其包含两个由半抗体形成的单价结合臂，其中每个半抗体包含单个重链可变区和任选地重链恒定区的至少一部分以及单个轻链可变区和任选地轻链恒定区的至少一部分。在一些实施方案中，双特异性抗体包含两个半抗体，其中每个半抗体包含单个重链可变区和单个轻链可变区并且不包含多于一个单个重链可变区且不包含多于一个单个轻链可变区。在一些实施方案中，双特异性抗体包含两个半抗体，其中每个半抗体包含单个重链可变区和单个轻链可变区，并且其中第一半抗体与第一抗原结合且不与第二抗原结合并且第二半抗体与第二抗原结合且不与第一抗原结合。在一个具体的实施方案中，第一单价结合臂以低于约10 -7M、优选地约10 -8M和更优选地约
Figure PCTCN2021133345-appb-000004
4nM、3nM、2nM、1nM、
Figure PCTCN2021133345-appb-000005
或更低的K D结合到TIGIT；在一个具体的实施方案中，第二单价结合臂低于约10 -7M、优选地约10 -8M和更优选地约
Figure PCTCN2021133345-appb-000006
4nM、3nM、2nM、1nM、
Figure PCTCN2021133345-appb-000007
或更低的K D结合到PD-1；在另一个实施方案中，与普通的单特异性抗体蛋白相比，双特异性抗体蛋白具有与其相同或相似的四级结构。 In some embodiments, the bispecific antibody is a heterodimer comprising two monovalent binding arms formed from half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and optionally a heavy chain constant region and at least a portion of a single light chain variable region and optionally at least a portion of the light chain constant region. In some embodiments, the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region and does not comprise more than one single heavy chain variable region and does not comprise More than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half-antibodies, wherein each half-antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half-antibody binds to the first antigen and does not bind to the first antigen. The second antigen binds and the second half-antibody binds to the second antigen and does not bind to the first antigen. In a specific embodiment, the first monovalent binding arm is less than about 10-7 M, preferably about 10-8 M and more preferably about
Figure PCTCN2021133345-appb-000004
4nM, 3nM, 2nM, 1nM,
Figure PCTCN2021133345-appb-000005
or lower K for binding to TIGIT; in a specific embodiment, the second monovalent binding arm is less than about 10-7 M, preferably about 10-8 M and more preferably about
Figure PCTCN2021133345-appb-000006
4nM, 3nM, 2nM, 1nM,
Figure PCTCN2021133345-appb-000007
or lower KD binds to PD -1; in another embodiment, the bispecific antibody protein has the same or similar quaternary structure as the common monospecific antibody protein.
术语“抗体制剂”指一种制备物，所述制备物处于允许作为活性成分的抗体的生物活性可以有效发挥的形式，并且不含有对于待施用该制剂的受试者而言具有不可接受毒性的其它组分。这类抗体制剂通常是无菌的。通常，抗体制剂中包含可药用赋形剂。“可药用”赋形剂是可以合理地施用至受试哺乳动物以便制剂中所用活性成分的有效剂量可以递送至受试者的试剂。赋形剂的浓度与施用模式相适应，例如可以是注射可接受的。The term "antibody preparation" refers to a preparation that is in a form that allows the biological activity of the antibody as the active ingredient to be effectively exerted and that does not contain unacceptable toxicity to the subject to which the preparation is to be administered. other components. Such antibody preparations are generally sterile. Typically, pharmaceutically acceptable excipients are included in the antibody formulation. A "pharmaceutically acceptable" excipient is an agent that can reasonably be administered to a subject mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, eg, may be acceptable for injection.
术语“抗TIGIT/PD-1双特异性抗体制剂”在本文中也简称为“本发明的抗体制剂”，意指包含抗TIGIT/PD-1双特异性抗体蛋白作为活性成分并包含可药用赋形剂的制备物。将抗TIGIT/PD-1双特异性抗体蛋白与可药用赋形剂组合后，作为活性成分的抗TIGIT/PD-1双特异性抗体蛋白适于治疗性或预防性施与人类或非人类动物。本发明的抗体制剂可以例如制备成水性形式的液体制剂，例如，即用式预填充注射器，或者制备成冻干制剂，在即将使用前通过溶解和/或悬浮于生理可接受的溶液中进行重构(即，复溶)。在一些实施方案中，抗TIGIT/PD-1双特异性抗体蛋白制剂是液体制剂形式。The term "anti-TIGIT/PD-1 bispecific antibody preparation" is also referred to herein simply as "antibody preparation of the present invention", meaning that an anti-TIGIT/PD-1 bispecific antibody protein is contained as an active ingredient and that is pharmaceutically acceptable Preparation of excipients. After combining the anti-TIGIT/PD-1 bispecific antibody protein with a pharmaceutically acceptable excipient, the anti-TIGIT/PD-1 bispecific antibody protein as an active ingredient is suitable for therapeutic or prophylactic administration to humans or non-humans animal. Antibody formulations of the invention can be prepared, for example, as liquid formulations in aqueous form, eg, ready-to-use prefilled syringes, or as lyophilized formulations for reconstitution by dissolving and/or suspending in a physiologically acceptable solution just before use. reconstituted (ie, reconstituted). In some embodiments, the anti-TIGIT/PD-1 bispecific antibody protein formulation is in the form of a liquid formulation.
“稳定的”抗体制剂是制剂中的抗体在储存于特定条件下之后保有可接受程度的物理稳定性和/或化学稳定性。尽管抗体制剂中所含的抗体在储存特定时间之后可能不会100％维持其化学结构，但通常在储存特定时间之后维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，则认为抗体制剂是“稳定的”。在一些具体的实施方案中，本发明的抗TIGIT/PD-1双特异性抗体蛋白制剂在制造、制备、运输和长期储存过程中表现出低至检测不到的抗体聚集或降解或化学修饰，从而极少或甚至是没有抗TIGIT/PD-1双特异性抗体蛋白的生物活性损失，表现出高度稳定性。在一些实施方案中，本发明的抗TIGIT/PD-1双特异性抗体蛋白制剂在储存后，基本上保留其物理和化学稳定性。优选地，本发明液体制剂可以在室温或在40℃稳定至少2周，和/或在25℃稳定至少2个月，和/或在2-8℃稳定至少24个月。A "stable" antibody formulation is one in which the antibody retains an acceptable degree of physical and/or chemical stability after storage under specified conditions. Although antibodies contained in antibody formulations may not retain 100% of their chemical structure after a specified period of storage, they typically maintain about 90%, about 95%, about 96%, about 97%, about 98% after storage for a specified period of time or about 99% of the structure or function of the antibody, an antibody preparation is considered "stable." In some specific embodiments, the anti-TIGIT/PD-1 bispecific antibody protein formulations of the invention exhibit low to undetectable antibody aggregation or degradation or chemical modification during manufacture, preparation, shipping and long-term storage, There is thus little or even no loss of biological activity of the anti-TIGIT/PD-1 bispecific antibody protein, showing a high degree of stability. In some embodiments, the anti-TIGIT/PD-1 bispecific antibody protein formulations of the invention substantially retain their physical and chemical stability upon storage. Preferably, the liquid formulations of the present invention are stable at room temperature or at 40°C for at least 2 weeks, and/or at 25°C for at least 2 months, and/or at 2-8°C for at least 24 months.
本领域已知多种分析技术可以用于测定蛋白质的稳定性，参见例如Peptide and Protein Drug Delivery，247-301，Vincent Lee Ed.，Marcel Dekker，Inc.，New York，N.Y.，Pubs(1991)and Jones，A.Adv.Drug Delivery Rev.10：29-90(1993)。可以在选定的温度和选定的储存时间测量稳定性。例如，可以基于预期的制剂货架期来选择储存时间。备选地，可以使用加速稳定性试验。在一些实施方案中，通过对抗体制剂进行各种胁迫测试来进行稳定性测试。这些测试可以代表调配的抗体制剂在制造、储存或运输期间可能遭遇到的极端条件，也可以代表在非制造、储存或运输期间可能使抗体制剂中的抗体的不稳定性加速的条件。例如，可以将经调配的抗TIGIT/PD-1双特异性抗体蛋白制剂充填至玻璃小瓶中以检验在高温胁迫下的抗体稳定性。Various analytical techniques are known in the art that can be used to determine the stability of proteins, see eg Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs (1991) and Jones , A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at selected temperatures and selected storage times. For example, the storage time can be selected based on the expected shelf life of the formulation. Alternatively, accelerated stability testing can be used. In some embodiments, stability testing is performed by subjecting antibody formulations to various stress tests. These tests can represent extreme conditions that may be encountered during manufacture, storage, or transportation of the formulated antibody formulation, as well as conditions that may accelerate instability of the antibody in the antibody formulation during non-manufacturing, storage, or transportation. For example, formulated anti-TIGIT/PD-1 bispecific antibody protein preparations can be filled into glass vials to test antibody stability under high temperature stress.
经一段储存时间后，制剂不显示聚集、沉淀、混浊和/或变性；或显示非常少的聚集、沉淀、混浊和/或变性，则可以认为抗体在制剂中“保持其物理稳定性”。由于制剂中抗体的聚集可以潜在地导致患者增加的免疫反应，从而导致安全性问题。因此，需要使在制剂中的抗体聚集最小化或防止聚集。光散射法可以用于测定制剂中的可见聚集物。SEC可以用于测定制剂中的可溶性聚集物。此外，可以通过目视检查制剂的外观、颜色和/或澄清度、或者通过非还原型CE-SDS法测定制剂的纯度，来指示制剂的稳定性。在一个实施方案中，通过测定在特定温度下储存特定时间之后制剂中的抗体单体的百分比来测量制剂的稳定性，其中制剂中的抗体单体的百分比越高，则制剂的稳定性越高。After a period of storage, if the formulation shows no aggregation, precipitation, haze and/or denaturation; or very little aggregation, precipitation, haze and/or denaturation, the antibody may be considered to "retain its physical stability" in the formulation. Safety concerns arise as the aggregation of antibodies in the formulation can potentially lead to an increased immune response in the patient. Therefore, there is a need to minimize or prevent aggregation of antibodies in formulations. Light scattering methods can be used to determine visible aggregates in formulations. SEC can be used to determine soluble aggregates in formulations. In addition, the stability of the formulation can be indicated by visual inspection of the formulation's appearance, color and/or clarity, or by measuring the purity of the formulation by non-reducing CE-SDS methods. In one embodiment, the stability of the formulation is measured by determining the percentage of antibody monomers in the formulation after storage at a specific temperature for a specific time, wherein the higher the percentage of antibody monomers in the formulation, the higher the stability of the formulation .
“可接受程度的”物理稳定性可以表示于特定温度下储存特定时间之后，在制剂中检测到至少约88％例如至少约92％的抗TIGIT/PD-1双特异性抗体蛋白单体。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的物理稳定性表示至少约88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗TIGIT/PD-1双特异性抗体蛋白单体。当评估物理稳定性时，药物制剂储存的特定温度可为约-80℃至约45℃的任一温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃、约35℃、约37℃、约40℃、约42℃或约45℃。例如，若储存于约40℃±2℃1个月或4周之后，检测到至少约88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗TIGIT/PD-1双特异性抗体蛋白单体，则药物制剂视为是稳定的。若储存于约25℃2个月之后，检测到至少约88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗TIGIT/PD-1双特异性抗体蛋白单体，则药物制剂视为是稳定的。若储存于约5℃9个月之后，检测到至少约88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的抗TIGIT/PD-1双特异性抗体蛋白单体，则药物制剂视为是稳定的。An "acceptable level" of physical stability may mean that at least about 88%, eg, at least about 92%, of the anti-TIGIT/PD-1 bispecific antibody protein monomers are detected in the formulation after storage at a specific temperature for a specific period of time. In some embodiments, at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at the specified temperature An acceptable level of physical stability is expressed after months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more At least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of anti-TIGIT/PD-1 bispecific antibody protein single body. When assessing physical stability, the particular temperature at which the pharmaceutical formulation is stored can be any temperature from about -80°C to about 45°C, eg, at about -80°C, about -30°C, about -20°C, about 0°C, About 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C. For example, if stored at about 40°C ± 2°C for 1 month or 4 weeks, at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the anti-TIGIT/PD-1 bispecific antibody protein monomer, the pharmaceutical preparation was considered to be stable. At least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% detected if stored at about 25°C for 2 months The anti-TIGIT/PD-1 bispecific antibody protein monomer is considered stable. At least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% detected if stored at about 5°C for 9 months The anti-TIGIT/PD-1 bispecific antibody protein monomer is considered stable.
经一段储存时间后，如果制剂中的抗体不显示显著的化学改变，则可以认为抗体在制剂中“保持其化学稳定性”。大多数化学不稳定性源自于形成了抗体的共价修饰形式(例如，抗体的电荷变异体)。例如由天冬氨酸异构化、N和C末端修饰，可以形成碱性变异体；由脱酰胺化、唾液酸化和糖化，可以产生酸性变异体。化学稳定性可以通过检测和/或定量抗体的化学 改变形式来评估。例如，可以通过阳离子交换色谱(CEX)或成像毛细管等电聚焦电泳(iCIEF)检测制剂中抗体的电荷变异体。在一个实施方案中，通过测定在特定温度下储存特定时间之后制剂中抗体的电荷变异体百分比变化值来测量制剂的稳定性，其中该变化值越小，则制剂的稳定性越高。An antibody may be considered to "retain its chemical stability" in a formulation if the antibody in the formulation does not exhibit significant chemical changes over a period of storage. Most chemical instability results from the formation of covalently modified forms of the antibody (eg, charge variants of the antibody). For example, by aspartic acid isomerization, N- and C-terminal modifications, basic variants can be formed; by deamidation, sialylation, and glycation, acidic variants can be produced. Chemical stability can be assessed by detecting and/or quantifying chemically altered forms of the antibody. For example, charge variants of the antibody in the preparation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (iCIEF). In one embodiment, the stability of the formulation is measured by determining the percent change in charge variant of the antibody in the formulation after storage at a specific temperature for a specific time, wherein the smaller the change, the more stable the formulation.
“可接受程度”的化学稳定性可以表示于特定温度下储存特定时间之后制剂中电荷变异体(例如主成分或酸性组分或碱性组分)的百分比变化值不超过50％，例如不超过30％、不超过20％。在一些实施方案中，在特定温度储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月或更久后，可接受程度的化学稳定性可以表现为主成分电荷变异体的百分比变化值不超过约50％、40％、30％、20％、15％。当评估化学稳定性时，储存药物制剂的温度可为约-80℃至约45℃的任一温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约4℃-8℃、约5℃、约25℃或约45℃。例如，若在储存于5℃24个月之后，主成分电荷变异体的百分比变化值少于约25％、24％、23％、22％、21％、20％、19％、18％、17％、16％、15％、14％、13％、12％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.1％，则药物制剂可被视为是稳定的。若在储存于25℃2个月后，主成分电荷变异体的百分比变化值少于约20％、19％、18％、17％、16％、15％、14％、13％、12％、10％、9％、8％、7％、6％、5％、4％、3％、2％、1％、0.5％或0.1％，则药物制剂亦可被视为是稳定的。若在储存于40℃1个月之后，主成分电荷变异体的百分比变化值少于约50％、40％、30％、20％、10％、5％或4％，则药物制剂亦可被视为是稳定的。An "acceptable level" of chemical stability may mean that the percent change in charge variants (eg, principal or acidic or basic components) in the formulation after storage at a specified temperature for a specified period of time does not exceed a value of 50%, such as not more than 50% 30%, no more than 20%. In some embodiments, at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at the specified temperature After months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more, an acceptable level of chemical stability may The percent change values expressed as principal component charge variants do not exceed about 50%, 40%, 30%, 20%, 15%. When assessing chemical stability, the temperature at which the pharmaceutical formulation is stored can be any temperature from about -80°C to about 45°C, eg, at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C or about 45°C. For example, if after 24 months of storage at 5°C, the percent change in principal component charge variants is less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17 %, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%, the pharmaceutical formulation can be considered stable. If after 2 months of storage at 25°C, the percent change in the charge variant of the principal component is less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%, the pharmaceutical formulation can also be considered stable. A pharmaceutical formulation may also be used if the percent change in the principal component charge variant is less than about 50%, 40%, 30%, 20%, 10%, 5%, or 4% after storage at 40°C for 1 month. considered stable.
术语“冻干制剂”是指通过液体制剂的冷冻干燥处理得到或能够得到的组合物。优选地，其为具有少于5％、优选少于3％水含量的固体组合物。The term "lyophilized formulation" refers to a composition obtained or obtainable by lyophilization of a liquid formulation. Preferably, it is a solid composition with a water content of less than 5%, preferably less than 3%.
术语“重构制剂”是指将固体制剂(例如冻干制剂)溶解和/或悬浮于生理可接受的溶液中得到的液体制剂。The term "reconstituted formulation" refers to a liquid formulation obtained by dissolving and/or suspending a solid formulation (eg, a lyophilized formulation) in a physiologically acceptable solution.
文中使用的术语“室温”是指15℃至30℃、优选20℃至27℃、更优选25℃的温度。The term "room temperature" as used herein refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, more preferably 25°C.
“胁迫条件”是指在化学和/或物理上不利于抗体蛋白的环境，所述环境可以导致不可接受的抗体蛋白失稳定。“高温胁迫”是指，将抗体制剂置于室温或甚至于更高温度(例如40℃±2℃)储存一段时间。通过高温胁迫加速试验，可以检查抗体制剂的稳定性。"Stress conditions" refers to chemically and/or physically unfavorable environments for an antibody protein that can lead to unacceptable destabilization of the antibody protein. "High temperature stress" refers to the storage of antibody preparations at room temperature or even at higher temperatures (eg, 40°C ± 2°C) for a period of time. The stability of the antibody preparation can be checked by a high temperature stress accelerated test.
如本文所使用，术语“肠胃外施用”意指肠内和局部给药以外的给药方式，通常通过注射或输注方式，并且包括但不限于，静脉内、肌内、动脉内、鞘内、囊内、眶内、心内、皮内、腹膜内、经气管、皮下、表皮下(subcuticular)、关节内、囊下、蛛网膜下、脊柱内、硬膜外和胸骨内注射以及输注。在一些实施方案中，本发明的稳定抗TIGIT/PD-1双特异性抗体蛋白制剂肠胃外施用于受试者。在一个实施方案中，本发明的抗TIGIT/PD-1双特异性抗体蛋白制剂以皮下、皮内、肌内或静脉内注射方式施用于受试者。As used herein, the term "parenteral administration" means administration other than enteral and topical administration, usually by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal , intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion . In some embodiments, the stable anti-TIGIT/PD-1 bispecific antibody protein formulations of the invention are administered parenterally to a subject. In one embodiment, the anti-TIGIT/PD-1 bispecific antibody protein formulation of the invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.
I.抗体制剂I. Antibody Preparations
本发明提供一种液体抗体制剂，其包含(i)抗TIGIT/PD-1双特异性抗体蛋白，(ii)缓冲剂，(iii)稳定剂，和(iv)表面活性剂，优选地所述抗体制剂的pH为约4.5-6.8。在一个优选方案中， 本发明的液体抗体制剂是注射制剂形式。The present invention provides a liquid antibody formulation comprising (i) an anti-TIGIT/PD-1 bispecific antibody protein, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, preferably the The pH of the antibody formulation is about 4.5-6.8. In a preferred embodiment, the liquid antibody formulation of the present invention is in the form of an injectable formulation.
(i)抗TIGIT/PD-1双特异性抗体蛋白(i) Anti-TIGIT/PD-1 bispecific antibody protein
本发明抗体制剂中的“抗TIGIT/PD-1双特异性抗体”包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合TIGIT(SEQ ID NO：31)或其胞外结构域(例如SEQ ID NO：32)的第一VH/VL单元并且第二半抗体包含特异性结合PD-1(SEQ ID NO：29)或其胞外结构域(例如SEQ ID NO：30)的第二VH/VL单元。The "anti-TIGIT/PD-1 bispecific antibody" in the antibody preparation of the present invention comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a specific binding TIGIT (SEQ ID NO: 31) or its extracellular The first VH/VL unit of the domain (eg, SEQ ID NO:32) and the second half-antibody comprise specific binding to PD-1 (SEQ ID NO:29) or its extracellular domain (eg, SEQ ID NO:30) of the second VH/VL unit.
在一个实施方案中，所述抗TIGIT/PD-1双特异性抗体”是异源二聚体，其中第一半抗体和第二半抗体各自形成两个单价结合臂。在一个实施方案，所述两个单价结合臂是通过二硫键连接的。In one embodiment, the anti-TIGIT/PD-1 bispecific antibody" is a heterodimer, wherein the first half antibody and the second half antibody each form two monovalent binding arms. In one embodiment, the The two monovalent binding arms are connected by a disulfide bond.
在另一个实施方案中，与普通的单特异性抗体蛋白相比，双特异性抗体蛋白具有与其相同或相似的四级结构。In another embodiment, the bispecific antibody protein has the same or similar quaternary structure as the common monospecific antibody protein.
在一个具体的实施方案中，第一单价结合臂低于约10 -7M、优选地约10 -8M和更优选地约5nM、4nM、3nM、2nM、1nM、0.5nM或更低的K D与TIGIT结合；在一个具体的实施方案中，第二单价结合臂低于约10 -7M、优选地约10 -8M和更优选地约5nM、4nM、3nM、2nM、1nM、0.5nM或更低的K D与T淋巴细胞表面的PD-1结合。 In a specific embodiment, the first monovalent binding arm is below a K of about 10-7 M, preferably about 10-8 M, and more preferably about 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM or less D binds to TIGIT; in a specific embodiment, the second monovalent binding arm is below about 10-7 M, preferably about 10-8 M and more preferably about 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM or lower KD binds to PD -1 on the surface of T lymphocytes.
在一些实施方案中，所述抗TIGIT/PD-1双特异性抗体蛋白能够以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与T淋巴细胞表面的PD-1结合，且能够以至少约10 7M -1、优选地约10 8M -1和更优选地约10 9M -1或更强的亲和力常数与TIGIT结合，以致所述抗体可以用作双特异性靶向PD-1分子和TIGIT分子的治疗剂和/或预防剂。 In some embodiments, the anti-TIGIT/PD-1 bispecific antibody protein is capable of at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or stronger It binds to PD-1 on the surface of T lymphocytes with an affinity constant of at least about 10 7 M -1 , preferably about 10 8 M -1 and more preferably about 10 9 M -1 or stronger. TIGIT binds so that the antibody can be used as a therapeutic and/or prophylactic agent bispecifically targeting PD-1 molecules and TIGIT molecules.
在一实施方案中，所述第一VH/VL单元包含重链和轻链互补决定区(CDR)氨基酸序列SEQ ID NO：1-6或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列；和/或第二VH/VL单元包含重链和轻链互补决定区(CDR)氨基酸序列SEQ ID NO：7-12或与所述6个CDR中的一个或多个CDR具有一个、两个、三个、四个、五个、六个或更多个氨基酸变化(例如，氨基酸置换或缺失)的序列。In one embodiment, the first VH/VL unit comprises the heavy and light chain complementarity determining region (CDR) amino acid sequences SEQ ID NOs: 1-6 or has one or more of the 6 CDRs A sequence of one, two, three, four, five, six or more amino acid changes (eg, amino acid substitutions or deletions); and/or a second VH/VL unit comprising heavy and light chain complementarity determinations Region (CDR) amino acid sequence SEQ ID NOs: 7-12 or one, two, three, four, five, six or more amino acid changes with one or more of the 6 CDRs (eg, amino acid substitutions or deletions).
术语“CDR”或“互补决定区”或“CDR区”(在本文中与超变区“HVR”可以互换使用)，是抗体可变区中主要负责与抗原表位结合的氨基酸区域。重链和轻链的CDR通常被称作CDR1、CDR2和CDR3，从N-端开始顺序编号。本领域公知多种用于在一个给定的VH或VL或VHH氨基酸序列中确定其CDR序列的方案。例如，Kabat互补决定区(CDR)或互补决定区(CDR)。Kabat互补决定区(CDR)是基于序列变异性确定的(Kabat等人，Sequences of Proteins of Immunological Interest，5th Ed.Public Health Service，National Institutes of Health，Bethesda，Md.(1991))。而Chothia指的是结构环的位置(Chothia和Lesk，J.Mol.Biol.196：901-917(1987))。AbM HVR是Kabat HVR和Chothia结构环之间的折中，并且由Oxford Molecular的AbM抗体建模软件使用。“接触性”(Contact)HVR基于对可获得的复杂晶体结构的分析。对于本发明，使用North CDR定义。North CDR定义(North etal.，“A New Clustering of Antibody CDR Loop  Conformations”，Journal of Molecular Biology，406，228-256(2011))是基于利用大量晶体结构的亲和力传播聚类。The term "CDR" or "complementarity determining region" or "CDR region" (used interchangeably herein with the hypervariable region "HVR"), is the amino acid region in the variable region of an antibody that is primarily responsible for binding to an antigenic epitope. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. Various protocols are known in the art for determining the CDR sequences of a given VH or VL or VHH amino acid sequence. For example, Kabat complementarity determining regions (CDRs) or complementarity determining regions (CDRs). Kabat complementarity determining regions (CDRs) are determined based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Whereas Chothia refers to the position of the structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). AbM HVR is a compromise between Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. "Contact" HVR is based on the analysis of available complex crystal structures. For the present invention, the North CDR definitions are used. The North CDR definition (North et al., "A New Clustering of Antibody CDR Loop Conformations", Journal of Molecular Biology, 406, 228-256 (2011)) is based on affinity-propagated clustering using bulk crystal structures.
所述氨基酸变化，例如，氨基酸置换优选地是保守氨基酸取代。“保守氨基酸取代”是指导致某个氨基酸置换为化学上相似的氨基酸的氨基酸改变。提供功能上相似氨基酸的保守性置换表是本领域熟知的。在本发明任一实施方案中，在一个优选的方面，保守取代残基来自以下的保守替代表A，优选地为表A中所示优选置换残基。The amino acid changes, eg, amino acid substitutions, are preferably conservative amino acid substitutions. "Conservative amino acid substitutions" refer to amino acid changes that result in the replacement of a certain amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. In any embodiment of the invention, in a preferred aspect, the conservatively substituted residues are from the conservative substitutions Table A below, preferably the preferred substitutions shown in Table A.
表ATable A
原始残基original residue 示例性取代Exemplary substitution 优选的保守氨基酸取代Preferred conservative amino acid substitutions
Ala(A)Ala(A) Val；Leu；IleVal; Leu; Ile ValVal
Arg(R)Arg(R) Lys；Gln；AsnLys; Gln; Asn LysLys
Asn(N)Asn(N) Gln；His；Asp；Lys；ArgGln; His; Asp; Lys; Arg GlnGln
Asp(D)Asp(D) Glu；AsnGlu; Asn GluGlu
Cys(C)Cys(C) Ser；AlaSer; Ala SerSer
Gln(Q)Gln(Q) Asn；GluAsn;Glu AsnAsn
Glu(E)Glu(E) Asp；GlnAsp;Gln AspAsp
Gly(G)Gly(G) AlaAla AlaAla
His(H)His(H) Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg ArgArg
Ile(I)Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸Leu; Val; Met; Ala; Phe; Norleucine LeuLeu
Leu(L)Leu(L) 正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; Phe IleIle
Lys(K)Lys(K) Arg；Gln；AsnArg; Gln; Asn ArgArg
Met(M)Met(M) Leu；Phe；IleLeu; Phe; Ile LeuLeu
Phe(F)Phe(F) Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr TyrTyr
Pro(P)Pro(P) AlaAla AlaAla
Ser(S)Ser(S) ThrThr ThrThr
Thr(T)Thr(T) Val；SerVal; Ser SerSer
Trp(W)Trp(W) Tyr；PheTyr; Phe TyrTyr
Tyr(Y)Tyr(Y) Trp；Phe；Thr；SerTrp; Phe; Thr; Ser PhePhe
Val(V)Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; Norleucine LeuLeu
在一个实施方案中，抗TIGIT/PD-1双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合TIGIT的第一VH/VL单元并且第二半抗体包含特异性结合PD-1的第二VH/VL单元，其中所述第一VH/VL单元包含SEQ ID NO：13/SEQ ID NO：14的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，并且其中所述第二VH/VL单元包含SEQ ID NO：17/SEQ ID NO：18的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列。In one embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises a first VH/VL unit that specifically binds TIGIT and the second half antibody comprising a second VH/VL unit that specifically binds PD-1, wherein the first VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable sequence of SEQ ID NO: 13/SEQ ID NO: 14 region sequence, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% with the paired heavy chain variable region sequence/light chain variable region sequence , a sequence of 99% or more sequence identity, and wherein the second VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 17/SEQ ID NO: 18 , or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% with the paired heavy chain variable region sequence/light chain variable region sequence Sequences of % or more sequence identity.
不特别地限制抗TIGIT/PD-1双特异性抗体蛋白中第一半抗体和第二半抗体的重链恒定区的类型，优选地是IgG1、IgG2或IgG4免疫球蛋白的重链恒定区，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。更优选地，所述重链恒定区是人IgG1免疫球蛋白的重链恒定区，或与之基本上同一(例如，至少80％、85％、90％、92％、95％、97％、98％、99％或更多同一)的序列。not particularly limiting the type of heavy chain constant regions of the first and second half antibodies in the anti-TIGIT/PD-1 bispecific antibody protein, preferably the heavy chain constant regions of IgG1, IgG2 or IgG4 immunoglobulins, or substantially identical (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical) to sequences. More preferably, the heavy chain constant region is, or is substantially identical to (eg, at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more identical).
在一个实施方案中，抗体的重链与抗体的轻链形成至少一个二硫键，抗体的两条重链形成至少一个二硫键。In one embodiment, the heavy chain of the antibody forms at least one disulfide bond with the light chain of the antibody, and the two heavy chains of the antibody form at least one disulfide bond.
在另一个实施方案中，抗体是工程化改造以降低抗体与Fcγ受体结合的人IgG1。In another embodiment, the antibody is human IgG1 engineered to reduce the binding of the antibody to Fcγ receptors.
在一个实施方案中，抗TIGIT/PD-1双特异性抗体蛋白包含IgG1(例如，人IgG1)中使用的重链恒定区。在又一个实施方案中，抗TIGIT/PD-1双特异性抗体蛋白包含用于IgG4(例如，人IgG4)的重链恒定区。例如，抗TIGIT/PD-1双特异性抗体的两条重链的Fc结构域中分别包含具有“CPPC”氨基酸残基的铰链区，和/或分别包含Y349C和S354C(根据Kabat的“EU编号”)，由此，抗PD-1半抗体和抗TIGIT半抗体在Fc区形成链间二硫键，由此，稳定抗PD-1半抗体和抗TIGIT半抗体的正确配对。In one embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a heavy chain constant region used in IgG1 (eg, human IgG1). In yet another embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a heavy chain constant region for IgG4 (eg, human IgG4). For example, the Fc domains of the two heavy chains of an anti-TIGIT/PD-1 bispecific antibody comprise hinge regions with "CPPC" amino acid residues, respectively, and/or Y349C and S354C, respectively (according to Kabat's "EU numbering" "), whereby the anti-PD-1 half antibody and the anti-TIGIT half antibody form an interchain disulfide bond in the Fc region, thereby stabilizing the correct pairing of the anti-PD-1 half antibody and the anti-TIGIT half antibody.
在一个实施方案中，抗TIGIT/PD-1双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含SEQ ID NO：21的重链序列或与之具有至少90％，95％，98％或99％同一性的重链序列，和SEQ ID NO：22的轻链序列或与之具有至少90％，95％，98％或99％同一性的轻链序列，并且其中第二半抗体包含SEQ ID NO：23的重链序列或与之具有至少90％，95％，98％或99％同一性的重链序列，和SEQ ID NO：24的轻链序列或与之具有至少90％，95％，98％或99％同一性的轻链序列。In one embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises or has at least 90% of the heavy chain sequence of SEQ ID NO: 21 , a heavy chain sequence that is 95%, 98% or 99% identical, and a light chain sequence that is at least 90%, 95%, 98% or 99% identical to the light chain sequence of SEQ ID NO: 22, and wherein the second half-antibody comprises the heavy chain sequence of SEQ ID NO:23 or a heavy chain sequence at least 90%, 95%, 98% or 99% identical thereto, and the light chain sequence of SEQ ID NO:24 or has A light chain sequence having at least 90%, 95%, 98% or 99% identity.
在本文中，“序列同一性”是指在比较窗中以逐个核苷酸或逐个氨基酸为基础的序列相同的程度。可以通过以下方式计算“序列同一性百分比”：将两条最佳比对的序列在比较窗中进行比较，确定两条序列中存在相同核酸碱基(例如，A、T、C、G、I)或相同氨基酸残基(例如，Ala、Pro、Ser、Thr、Gly、Val、Leu、Ilc、Phe、Tyr、Trp、Lys、Arg、His、Asp、Glu、Asn、Gln、Cys和Met)的位置的数目以得到匹配位置的数目，将匹配位置的数目除以比较窗中的总位置数(即，窗大小)，并且将结果乘以100，以产生序列同一性百分比。为了确定序列同一性百分数而进行的最佳比对，可以按本领域已知的多种方式实现，例如，使用可公开获得的计算机软件如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的适宜参数，包括为实现正在比较的全长序列范围内或目标序列区域内最大比对所需要的任何算法。As used herein, "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis over a window of comparison. "Percent sequence identity" can be calculated by comparing two optimally aligned sequences in a comparison window to determine the presence of identical nucleic acid bases (e.g., A, T, C, G, I) in the two sequences. ) or the same amino acid residue (eg, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ilc, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) To obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window (ie, the window size), and multiply the result by 100 to yield the percent sequence identity. Optimal alignment to determine percent sequence identity can be achieved in a variety of ways known in the art, eg, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length sequences being compared or within the region of the sequence of interest.
本发明抗体制剂中的抗TIGIT/PD-1双特异性抗体蛋白能够同时与TIGIT和PD-1蛋白结合，且维持了各亲本抗体的亲和力常数，由此，能够阻断TIGIT信号传导通路和阻断PD-1信号传导通路，从而用于治疗、预防或延缓各种与TIGIT信号传导通路和/或与PD-1信号传导通路相关的疾病或病症。The anti-TIGIT/PD-1 bispecific antibody protein in the antibody preparation of the present invention can simultaneously bind to TIGIT and PD-1 protein, and maintain the affinity constant of each parent antibody, thereby blocking the TIGIT signal transduction pathway and blocking Disrupting the PD-1 signaling pathway, thereby being used to treat, prevent or delay various diseases or conditions related to the TIGIT signaling pathway and/or the PD-1 signaling pathway.
本发明使用的抗TIGIT/PD-1双特异性抗体蛋白还记载于PCT/US20/34158，将其全部内容引入作为参考。The anti-TIGIT/PD-1 bispecific antibody protein used in the present invention is also described in PCT/US20/34158, the entire contents of which are incorporated by reference.
在一个优选的实施方案中，抗TIGIT/PD-1双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含SEQ ID NO：21的重链序列，和SEQ ID NO：22的轻链序列，并且其中第二半抗体包含SEQ ID NO：23的重链序列，和SEQ ID NO：24的轻链序列。In a preferred embodiment, the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the heavy chain sequence of SEQ ID NO: 21, and SEQ ID NO : the light chain sequence of SEQ ID NO:22, and wherein the second half-antibody comprises the heavy chain sequence of SEQ ID NO:23, and the light chain sequence of SEQ ID NO:24.
在一个实施方案中，该抗TIGIT/PD-1双特异性抗体蛋白由HEK293细胞或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293E细胞；CHO细胞或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr -、CHO/DG44、ExpiCHO重组表达产生并经纯化。优选地，在本发明液体制剂中的所述抗体表现出显著的抗肿瘤活性。对LOVO混合PBMC荷瘤小鼠和MC38荷瘤小鼠施用抗TIGIT/PD-1双特异性抗体，结果表明，与施用抗PD-1单克隆抗体或抗TIGIT单克隆抗体相比较，施用抗TIGIT/PD-1双特异性抗体具有显著提高的抗肿瘤活性，可以导致肿瘤体积的显著缩小。 In one embodiment, the anti-TIGIT/PD-1 bispecific antibody protein is derived from HEK293 cells or HEK293T, HEK293F, HEK293E cells transformed from HEK293 cells; CHO cells or transformed from CHO cells CHO-S, CHO-dhfr - , CHO/DG44, ExpiCHO were recombinantly expressed and purified. Preferably, the antibody in the liquid formulation of the present invention exhibits significant anti-tumor activity. Administration of anti-TIGIT/PD-1 bispecific antibody to LOVO mixed PBMC tumor-bearing mice and MC38 tumor-bearing mice showed that administration of anti-TIGIT compared with administration of anti-PD-1 monoclonal antibody or anti-TIGIT monoclonal antibody The /PD-1 bispecific antibody has significantly improved antitumor activity, which can lead to a significant reduction in tumor volume.
本发明的抗体制剂中所包含的抗TIGIT/PD-1双特异性抗体蛋白的量可随着制剂的特定目的特性、特定环境、和使用制剂的特定目的而改变。在一些实施方案中，抗体制剂为液体制剂，其可含有约1-200mg/ml，优选地为约1-150mg/ml，更优选地为约10-100mg/mL，例如约5、10、15、20、25、30、40、50、60、70、80、90或100mg/ml的抗TIGIT/PD-1双特异性抗体蛋白。The amount of anti-TIGIT/PD-1 bispecific antibody protein included in the antibody formulations of the present invention may vary depending on the specific intended properties of the formulation, the specific environment, and the specific purpose for which the formulation is used. In some embodiments, the antibody formulation is a liquid formulation, which may contain about 1-200 mg/ml, preferably about 1-150 mg/ml, more preferably about 10-100 mg/mL, eg, about 5, 10, 15 , 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 mg/ml of anti-TIGIT/PD-1 bispecific antibody protein.
(ii)缓冲剂(ii) Buffers
缓冲剂是可以将溶液的pH维持在可接受范围的试剂。在一些实施方案中，用于本发明制优选剂中的缓冲剂可以将本发明制剂的pH控制在大约4.5-6.8的pH范围，优选大约5.0-6.5，更优选约6.0的pH。在一些具体的实施方案中，本发明的抗体制剂具有约4.5、4.8、5.0、5.2、5.4、5.6、5.8、6.0、6.2、6.4、6.5、6.6、6.7或6.8的pH。A buffer is an agent that can maintain the pH of a solution within an acceptable range. In some embodiments, the buffering agents used in the formulations of the present invention can control the pH of the formulations of the present invention to a pH in the range of about 4.5-6.8, preferably about 5.0-6.5, more preferably about 6.0 pH. In some specific embodiments, the antibody formulation of the invention has a pH of about 4.5, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.5, 6.6, 6.7, or 6.8.
在一些实施方案中，用于本发明制剂中的缓冲剂选自组氨酸、盐酸组氨酸、谷氨酸盐、磷酸盐、乙酸盐、柠檬酸盐和三羟甲基氨基甲烷和它们的组合，优选组氨酸、盐酸组氨酸和它们的组合。在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约0.5-200mM、约1-100mM、约5-50mM、约5-30mM或约5-20mM。在一个实施方案中，本发明的液体抗体制剂中的缓冲剂的浓度为约5-30mM，例如，约5、10、15、20、25或30mM。In some embodiments, the buffering agent used in the formulations of the present invention is selected from the group consisting of histidine, histidine hydrochloride, glutamate, phosphate, acetate, citrate and tris and their combination, preferably histidine, histidine hydrochloride and their combination. In one embodiment, the concentration of buffer in the liquid antibody formulation of the invention is about 0.5-200 mM, about 1-100 mM, about 5-50 mM, about 5-30 mM, or about 5-20 mM. In one embodiment, the concentration of buffer in the liquid antibody formulation of the invention is about 5-30 mM, eg, about 5, 10, 15, 20, 25 or 30 mM.
在一个实施方案中，用于本发明制剂中的缓冲剂是约10mM组氨酸。In one embodiment, the buffer used in the formulations of the present invention is about 10 mM histidine.
又在一个实施方案中，用于本发明制剂中的缓冲剂是约5mM组氨酸和约5mM盐酸组氨酸的组合。In yet another embodiment, the buffer used in the formulation of the present invention is a combination of about 5 mM histidine and about 5 mM histidine hydrochloride.
(iii)稳定剂(iii) Stabilizers
用于本发明的合适的稳定剂可以选自糖类、多元醇、氨基酸或其盐及它们的组合。进一步地，本发明的稳定剂还可以包含抗氧化剂。Suitable stabilizers for use in the present invention may be selected from sugars, polyols, amino acids or salts thereof, and combinations thereof. Further, the stabilizer of the present invention may also contain an antioxidant.
作为稳定剂的糖类可以是二糖、三糖和多糖，而且糖类可以选自但不限于：蔗糖、右旋糖、乳糖、麦芽糖、海藻糖、环糊精、麦芽糖糊精和葡聚糖。在一个实施方案中，作为稳定剂的糖类是蔗糖和/或海藻糖。The saccharides as stabilizers may be disaccharides, trisaccharides and polysaccharides, and the saccharides may be selected from, but not limited to: sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin and dextran . In one embodiment, the saccharides as stabilizers are sucrose and/or trehalose.
作为稳定剂的多元醇可以选自但不限于：甘露醇、山梨醇和木糖醇。在一个实施方案中， 作为稳定剂的多元醇是山梨醇。The polyol as a stabilizer can be selected from, but not limited to: mannitol, sorbitol and xylitol. In one embodiment, the polyol as a stabilizer is sorbitol.
作为稳定剂的氨基酸或其盐可以选自但不限于精氨酸、精氨酸盐(优选精氨酸盐酸盐)。The amino acid or its salt as a stabilizer can be selected from, but not limited to, arginine, arginine salt (preferably arginine hydrochloride).
在一些实施方案中，所述稳定剂是山梨醇。在一些实施方案中，所述稳定剂是精氨酸盐酸盐。在一些实施方案中，所述稳定剂是山梨醇、精氨酸、精氨酸盐(优选精氨酸盐酸盐)或其组合。In some embodiments, the stabilizer is sorbitol. In some embodiments, the stabilizer is arginine hydrochloride. In some embodiments, the stabilizer is sorbitol, arginine, an arginine salt (preferably arginine hydrochloride), or a combination thereof.
在一些实施方案中，作为稳定剂的糖类和/或多元醇在本发明的液体制剂中以约1-200mg/ml，优选地约5-150mg/ml，例如约10-100mg/ml、约15-80mg/ml或约20-50mg/ml例如，约1、5、10、15、20、25、30、40、50、60、70、80、90、100、150、200mg/ml的浓度存在。In some embodiments, saccharides and/or polyols as stabilizers are present in the liquid formulations of the present invention at a concentration of about 1-200 mg/ml, preferably about 5-150 mg/ml, for example about 10-100 mg/ml, about 15-80 mg/ml or about 20-50 mg/ml eg, a concentration of about 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 mg/ml exist.
在一些实施方案中，本发明的稳定剂在本发明的液体制剂中以约1-1000mM、约10-1000mM，优选地约20-800mM，例如约50-500、约100-400、约100-300或约100-200mM例如，约10、20、50、80、100、150、200、300、400、500、600、700、800、900、1000mM的浓度存在。In some embodiments, the stabilizer of the present invention is present in the liquid formulation of the present invention at about 1-1000 mM, about 10-1000 mM, preferably about 20-800 mM, such as about 50-500, about 100-400, about 100- Present at a concentration of 300 or about 100-200 mM, eg, about 10, 20, 50, 80, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mM.
本发明的稳定剂中还可以包含的抗氧化剂选自但不限于：同型半胱氨酸、半胱氨酸、胱硫醚、甲硫氨酸、谷胱甘肽、以及包含同型半胱氨酸、半胱氨酸、胱硫醚、甲硫氨酸和谷胱甘肽中任意一种的肽。在包含抗氧化剂的情形下，稳定剂的总浓度如前所述，其中抗氧化剂的浓度为约1-50mM，优选地约5-40mM，例如约5、10、20、30、40mM。Antioxidants that may also be included in the stabilizer of the present invention are selected from, but are not limited to, homocysteine, cysteine, cystathionine, methionine, glutathione, and homocysteine-containing , cysteine, cystathionine, methionine, and glutathione. Where antioxidants are included, the total concentration of stabilizers is as previously described, wherein the concentration of antioxidants is about 1-50 mM, preferably about 5-40 mM, eg, about 5, 10, 20, 30, 40 mM.
(iv)表面活性剂(iv) Surfactant
在一个实施方案中，本发明的液体制剂中含有表面活性剂。在一实施方案中，所述表面活性剂是非离子型表面活性剂，例如，烷基聚(环氧乙烯)。可包括在本发明制剂中的特定非离子型表面活性剂包括，例如聚山梨酯，诸如聚山梨酯-20、聚山梨酯-80、聚山梨酯-60、或聚山梨酯-40；普洛尼克等。在一个优选实施方案中，本发明的液体制剂中包含聚山梨酯-80作为表面活性剂。In one embodiment, the liquid formulation of the present invention contains a surfactant. In one embodiment, the surfactant is a nonionic surfactant, eg, an alkyl poly(ethylene oxide). Particular nonionic surfactants that can be included in the formulations of the present invention include, for example, polysorbates such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; Nick et al. In a preferred embodiment, polysorbate-80 is included as a surfactant in the liquid formulation of the present invention.
本发明抗体制剂中所含的表面活性剂的量可随制剂的特定目的特性、特定环境、和使用制剂的特定目的而改变。在优选的一些实施方案中，本发明的液体制剂可含有约0.01-10mg/ml，优选地约0.05-5、约0.05-2、约0.1-5、0.2-2、0.3-1、0.4-0.8、0.5-0.6mg/ml，例如约0.2、0.3、0.4、0.5、0.6、0.7、0.8mg/ml的聚山梨酯类表面活性剂(例如，聚山梨酯-80)。The amount of surfactant contained in the antibody formulation of the invention can vary depending on the specific intended nature of the formulation, the specific environment, and the specific purpose for which the formulation is used. In preferred embodiments, the liquid formulations of the present invention may contain about 0.01-10 mg/ml, preferably about 0.05-5, about 0.05-2, about 0.1-5, 0.2-2, 0.3-1, 0.4-0.8 , 0.5-0.6 mg/ml, eg, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml of a polysorbate-based surfactant (eg, polysorbate-80).
(v)其它赋形剂(v) Other excipients
本发明的抗体液体制剂中可以包含或不包含其它赋形剂。例如，本发明的抗体液体制剂还包含张力调节剂。张力调节剂可以选自下组：醋酸钠、乳酸钠、氯化钠、氯化钾和氯化钙。Other excipients may or may not be included in the antibody liquid formulation of the present invention. For example, the antibody liquid formulation of the present invention further contains a tonicity adjusting agent. The tonicity adjusting agent may be selected from the group consisting of sodium acetate, sodium lactate, sodium chloride, potassium chloride and calcium chloride.
这些和另外已知的药物赋形剂和/或适用于本发明制剂的添加剂是本领域公知的，例如，列出于“The Handbook of Pharmaceutical Excipients，第4版，Rowe等人编，American Pharmaceuticals Association(2003)；和Remington：the Science and Practice of Pharmacy，第21版，Gennaro编，Lippincott Williams&Wilkins(2005)”。These and other known pharmaceutical excipients and/or additives suitable for use in the formulations of the present invention are well known in the art, and are listed, for example, in "The Handbook of Pharmaceutical Excipients, 4th Ed., Rowe et al., American Pharmaceuticals Association (2003); and Remington: The Science and Practice of Pharmacy, 21st Ed. Gennaro ed. Lippincott Williams & Wilkins (2005)”.
应当理解，如无特别说明，本发明的液体制剂可使用本领域已知的可药用的溶剂或溶液 来制备或配制。所述可药用的溶剂或溶液包括但不限于例如注射用水、无菌水、双蒸水、生理盐水、林格氏溶液、葡萄糖注射液等。It should be understood that, unless otherwise specified, the liquid formulations of the present invention can be prepared or formulated using pharmaceutically acceptable solvents or solutions known in the art. The pharmaceutically acceptable solvent or solution includes, but is not limited to, for example, water for injection, sterile water, double distilled water, physiological saline, Ringer's solution, dextrose injection and the like.
II.制剂的制备II. Preparation of Formulations
本发明提供了包含抗TIGIT/PD-1双特异性抗体蛋白的稳定制剂。在本发明制剂中使用的抗TIGIT/PD-1双特异性抗体蛋白可以使用本领域已知的用于生产抗体的技术进行制备。例如，可以重组制备抗TIGIT/PD-1双特异性抗体蛋白。在一个优选的实施方案中，本发明的抗TIGIT/PD-1双特异性抗体蛋白通过在HEK293细胞或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293E细胞；CHO细胞或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr-、CHO/DG44、ExpiCHO中重组表达而制备，例如，如PCT申请号PCT/US20/34158中所述，重组制备抗TIGIT/PD-1双特异性抗体蛋白。The present invention provides stable formulations comprising anti-TIGIT/PD-1 bispecific antibody proteins. The anti-TIGIT/PD-1 bispecific antibody proteins used in the formulations of the present invention can be prepared using techniques known in the art for producing antibodies. For example, anti-TIGIT/PD-1 bispecific antibody proteins can be produced recombinantly. In a preferred embodiment, the anti-TIGIT/PD-1 bispecific antibody protein of the present invention is obtained by transforming HEK293 cells or HEK293 cells based on HEK293T, HEK293F and HEK293E cells; CHO cells or CHO cells as Prepared by recombinant expression in CHO-S, CHO-dhfr-, CHO/DG44, and ExpiCHO obtained by basic transformation, for example, as described in PCT application number PCT/US20/34158, recombinantly prepared anti-TIGIT/PD-1 bispecific Antibody protein.
抗体作为药物的活性成分的应用现在已经很广泛。用于将治疗性抗体纯化至药用级的技术是本领域公知的。例如，Tugcu等(Maximizing productivity of chromatography steps for purification of monoclonal antibodies，Biotechnology and Bioengineering 99(2008)599-613)描述了在蛋白A捕获步骤后使用离子交换色谱(阴离子IEX和/或阳离子CEX色谱)的抗体三柱纯化方法。Kelley等(Weak partitioning chromatography for anion exchange purification of monoclonal antibodies，Biotechnology and Bioengineering 101(2008)553-566)描述了两柱纯化法，其中在蛋白A亲和色谱后使用弱分配阴离子交换树脂。The use of antibodies as active ingredients of drugs is now widespread. Techniques for purifying therapeutic antibodies to pharmaceutical grades are well known in the art. For example, Tugcu et al. (Maximizing productivity of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering 99 (2008) 599-613) describe the use of ion-exchange chromatography (anionic IEX and/or cationic CEX chromatography) following a protein A capture step Antibody three-column purification method. Kelley et al. (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies, Biotechnology and Bioengineering 101 (2008) 553-566) describe a two-column purification method in which a weak partitioning anion exchange resin is used after protein A affinity chromatography.
一般地，重组产生的抗体可以利用常规的纯化方法纯化，以提供具有足够的可重复性和适度纯度的药物物质用于抗体制剂的配制。例如，在抗体从重组表达细胞分泌至培养基中后，可以使用商业可得的蛋白浓缩过滤器例如Amicon的超滤装置，浓缩来自该表达***的上清液。之后，可以使用例如色谱、透析和亲和纯化等方式进行抗体的纯化。蛋白A适应于作为亲和配体用于纯化IgG1、IgG2和IgG4型抗体。也可以使用其它抗体纯化方法，例如离子交换色谱。在获得足够纯度的抗体后，可以按照本领域已知的方法，制备包含抗体的制剂。Generally, recombinantly produced antibodies can be purified using conventional purification methods to provide drug substance with sufficient reproducibility and modest purity for formulation of antibody preparations. For example, after the antibody is secreted from the recombinant expression cells into the culture medium, the supernatant from the expression system can be concentrated using a commercially available protein concentration filter such as Amicon's ultrafiltration device. Afterwards, purification of the antibody can be performed using, for example, chromatography, dialysis, and affinity purification. Protein A is suitable as an affinity ligand for the purification of IgG1, IgG2 and IgG4 type antibodies. Other antibody purification methods, such as ion exchange chromatography, can also be used. After obtaining the antibody of sufficient purity, preparations comprising the antibody can be prepared according to methods known in the art.
例如，可以采用如下步骤进行制备：(1)在发酵结束后将发酵液离心澄清去除细胞等杂质以获得上清；(2)使用亲和层析(例如对IgG1、IgG2和IgG4型抗体具有特异亲和力的蛋白A柱)捕获抗体；(3)进行病毒灭活；(4)精制纯化(一般可以采用CEX阳离子交换层析)，以去除蛋白中的杂质；(4)病毒过滤(使病毒滴度降低例如4log10以上)；(5)超滤/渗滤(可以用于将蛋白置换于利于其稳定的制剂缓冲液中并浓缩至合适的浓度供注射用)。参见例如，B.Minow，P.Rogge，K.Thompson，BioProcess International，Vol.10，No.6，2012，pp.48-57。For example, it can be prepared by the following steps: (1) after the fermentation, the fermentation broth is centrifuged to remove impurities such as cells to obtain a supernatant; (2) affinity chromatography (for example, specific for IgG1, IgG2 and IgG4 antibodies) can be used. (3) virus inactivation; (4) purification (generally, CEX cation exchange chromatography can be used) to remove impurities in the protein; (4) virus filtration (to make the virus titer) (5) Ultrafiltration/diafiltration (can be used to replace the protein in a formulation buffer favorable for its stability and concentrate to a suitable concentration for injection). See, eg, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57.
III.制剂的分析方法III. METHODS OF ANALYSIS OF PREPARATIONS
在抗体制剂的储存过程中，抗体可能会发生聚集、降解或化学修饰，导致抗体异质性(包括大小异质性和电荷异质性)以及聚集物和片段等，从而影响抗体制剂的质量。因此，有必要进行抗体制剂稳定性的监测。During storage of antibody preparations, antibodies may aggregate, degrade, or chemically modify, resulting in antibody heterogeneity (including size and charge heterogeneity), as well as aggregates and fragments, which affect the quality of antibody preparations. Therefore, it is necessary to monitor the stability of antibody preparations.
在本领域中已知多种方法可以用于检测抗体制剂的稳定性。例如，可以通过还原型 CE-SDS、非还原型CE-SDS和SEC-HPLC等方法，分析抗体制剂的纯度和评估抗体的聚集水平；可以通过毛细管等电聚焦电泳(cIEF)、成像毛细管等电聚焦电泳(iCIEF)和离子交换色谱(IEX)等，分析抗体制剂中的电荷变异体。此外，可以通过目视检测制剂外观，快速地判断制剂的稳定性。也可以使用OD 350nm法检测制剂的浊度改变，该方法可以给出有关可溶性和不溶性聚集物量的信息。此外，可以使用紫外分光光度法(UV法)检测制剂中的蛋白质含量变化。 Various methods are known in the art for testing the stability of antibody formulations. For example, the purity of antibody preparations and the level of antibody aggregation can be assessed by methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC; capillary isoelectric focusing electrophoresis (cIEF), imaging capillary isoelectric Analysis of charge variants in antibody preparations by focused electrophoresis (iCIEF) and ion exchange chromatography (IEX), among others. In addition, the stability of the formulation can be quickly judged by visually inspecting the appearance of the formulation. The turbidity change of the formulation can also be detected using the OD 350nm method, which can give information on the amount of soluble and insoluble aggregates. In addition, ultraviolet spectrophotometry (UV method) can be used to detect changes in protein content in the formulation.
非还原型CE-SDS法是一种以毛细管为分离通道进行的抗体纯度测定方法。在CE-SDS中，蛋白迁移由SDS结合引起的表面电荷来驱动，SDS与蛋白结合，消除蛋白自身电荷的差异，故可以在毛细管的分子筛凝胶基质中，实现基于分子的大小或流体动力学半径的电泳分离。该方法已经被广泛地用于监测变性的完整抗体的纯度。一般，在非还原型CE-SDS法中，供试样品与SDS样品缓冲液和碘乙酰胺混合。之后，混合物可以于68-72℃孵育约10-15分钟，冷却至室温后离心的上清液用于分析。采用紫外检测器检测蛋白的迁移，获得电泳谱图。抗体制剂纯度可以计算为IgG主峰的峰面积占所有峰面积之和的百分比。关于CE-SDS法的进一步描述，可以参见例如Richard R.等，Application of CE SDS gel in development of biopharmaceutical antibody-based products，Electrophoresis，2008，29，3612-3620。The non-reducing CE-SDS method is a method for measuring antibody purity with capillary as the separation channel. In CE-SDS, protein migration is driven by the surface charge caused by the binding of SDS, and SDS binds to the protein to eliminate the difference in the self-charge of the protein. Therefore, in the molecular sieve gel matrix of the capillary, the molecular size or hydrodynamics based on the molecular size can be realized. Electrophoretic separation of radii. This method has been widely used to monitor the purity of denatured intact antibodies. Generally, in the non-reducing CE-SDS method, the test sample is mixed with SDS sample buffer and iodoacetamide. Afterwards, the mixture can be incubated at 68-72°C for approximately 10-15 minutes, and the centrifuged supernatant after cooling to room temperature is used for analysis. The migration of the protein was detected by a UV detector, and the electropherogram was obtained. Antibody preparation purity can be calculated as the peak area of the main IgG peak as a percentage of the sum of all peak areas. For a further description of the CE-SDS method, see, for example, Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.
体积排阻高效液相色谱法，即SEC-HPLC法，是用于抗体标准和质控的另一重要方法。该方法主要依据分子的尺寸大小或流体动力学半径差异来进行分子的分离。通过SEC-HPLC，抗体可以分离出三种主要形式：高分子量形式(HMMS)、主峰(主要是抗体单体)、和低分子量形式(LMMS)。抗体纯度可以计算为色谱图上主峰面积占所有峰面积之和的百分比。通过SEC-HPLC法，可以测量制剂产品中抗体单体的百分数，给出可溶性聚集物和剪切物的含量信息。关于SEC-HPLC法的进一步描述，可以参见例如，J.Pharm.Scien.，83：1645-1650，(1994)；Pharm.Res.，11：485(1994)；J.Pharm.Bio.Anal.，15：1928(1997)；J.Pharm.Bio.Anal.，14：1133-1140(1986)。此外，也可以参见例如，R.Yang等，High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography(SE-UHPLC)，Journal of Pharmaceutical and Biomedical Analysis(2015)，http：//dx.doi.org/10.1016/j.jpba.2015.02.032；和Alexandre Goyon等，Protocols for the analytical characterization of therapeutic monoclonal antibodies.I-Non-denaturing chromatographic techniques，Journal of Chromatography，http：//dx.doi.org/10.1016/j.jchromb.2017.05.010。Size exclusion high performance liquid chromatography, or SEC-HPLC, is another important method for antibody standardization and quality control. This method is mainly based on the size of the molecules or the difference in the hydrodynamic radius to separate the molecules. By SEC-HPLC, the antibody can be separated into three main forms: the high molecular weight form (HMMS), the main peak (mainly antibody monomers), and the low molecular weight form (LMMS). Antibody purity can be calculated as the percentage of the area of the main peak over the sum of all peak areas on the chromatogram. By SEC-HPLC, the percentage of antibody monomers in the formulated product can be measured, giving information on the content of soluble aggregates and cleavage. For a further description of the SEC-HPLC method, see, eg, J. Pharm. Scien., 83: 1645-1650, (1994); Pharm. Res., 11: 485 (1994); , 15: 1928 (1997); J. Pharm. Bio. Anal., 14: 1133-1140 (1986). Also see, e.g., R. Yang et al., High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography (SE-UHPLC), Journal of Pharmaceutical and Biomedical Analysis (2015), http://dx. doi.org/10.1016/j.jpba.2015.02.032; and Alexandre Goyon et al., Protocols for the analytical characterization of therapeutic monoclonal antibodies. I-Non-denaturing chromatographic techniques, Journal of Chromatography, http://dx.doi.org /10.1016/j.jchromb.2017.05.010.
成像毛细管等电聚焦电泳(iCIEF)可以用于分析抗体的电荷异质性。该方法可以提供电荷变异体的定量分布情况。iCIEF基于分子在pH梯度中的电荷差异(表观pI值)来实现分子分离的目的。在iCIEF中，分离柱通常是短毛细管(例如，5cm长，100μm内径的二氧化硅毛细管)，蛋白质在高电压下在毛细管柱中聚焦，并通过在280nM操作的全柱成像检测***对聚焦进行实时在线监测。该技术的一个优点是，可以通过该全柱检测***同时记录抗体样品的各种电荷变异体。一般而言，在icIEF中，将样品与尿素和icIEF缓冲液混合，其中所述缓冲液含有甲基纤维素、pI分子量标准和两性电解质。然后，可以在iCIEF分析仪例如iCE280分析仪(Protein Simple，Santa Clara，CA)上，使用iCIEF柱例如ProtionSimple组装的iCIEF柱，在样 品聚焦一定时间后，测定280nm的吸光度，获得聚焦抗体电荷变异体的谱图。在iCEIF谱图中，在主峰(即主成分)之前洗脱的蛋白相关峰被分类为酸性组分；相对地，在主峰之后洗脱的蛋白相关峰被分类为碱性组分。主成分、酸性组分和碱性组分的相对量可以表示为占总峰面积的百分数。关于iCIEF的进一步描述，可以参见例如，Salas-Solano O等，Robustness of iCIEF methodology for the analysis of monoclonal antibodies：an interlaboratory study，J Sep Sci.2012 Nov；35(22)：3124-9.doi：10.1002/jssc.201200633.Epub 2012 Oct 15；和Dada OO等，Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation，Electrophoresis.2015 Nov；36(21-22)：2695-2702.doi：10.1002/elps.201500219.Epub 2015 Sep 18.Imaging capillary isoelectric focusing electrophoresis (iCIEF) can be used to analyze the charge heterogeneity of antibodies. This method can provide a quantitative distribution of charge variants. iCIEF achieves the purpose of molecular separation based on the difference in charge (apparent pi value) of molecules in a pH gradient. In iCIEF, the separation column is usually a short capillary (e.g., a 5 cm long, 100 μm inner diameter silica capillary), the protein is focused in the capillary column at high voltage, and the focusing is performed by a full-column imaging detection system operating at 280 nM Real-time online monitoring. An advantage of this technique is that various charge variants of antibody samples can be simultaneously recorded by this full-column detection system. In general, in icIEF, the sample is mixed with urea and icIEF buffer, wherein the buffer contains methylcellulose, pi molecular weight standards and ampholytes. Then, the focused antibody charge variant can be obtained by measuring the absorbance at 280 nm on an iCIEF analyzer such as an iCE280 analyzer (Protein Simple, Santa Clara, CA) using an iCIEF column such as an iCIEF column assembled by ProtionSimple after focusing the sample for a certain period of time 's spectrum. In the iCEIF spectrum, protein-related peaks eluting before the main peak (ie, principal component) are classified as acidic components; conversely, protein-related peaks eluting after the main peak are classified as basic components. The relative amounts of principal components, acidic components, and basic components can be expressed as a percentage of the total peak area. For a further description of iCIEF, see, eg, Salas-Solano O et al., Robustness of iCIEF methodology for the analysis of monoclonal antibodies: an interlaboratory study, J Sep Sci. 2012 Nov;35(22):3124-9.doi:10.1002 /jssc.201200633.Epub 2012 Oct 15; and Dada OO et al., Characterization of acidic and basic variants of IgG1 therapeutic monoclonal antibodies based on non-denaturing IEF fractionation, Electrophoresis. 2015 Nov;36(21-22):2695-2702. doi: 10.1002/elps.201500219.Epub 2015 Sep 18.
也可以通过阳离子交换高效液相色谱法(CEX-HPLC)测定抗体制剂中抗体的电荷变异体。在该测定法中，以比主峰的保留时间更早从CEX-HPLC柱洗脱出的峰被标记为“酸性峰”，而那些以比主峰的保留时间更晚从CEX-HPLC柱洗脱出的峰被标记为“碱性峰”。Charge variants of antibodies in antibody preparations can also be determined by cation exchange high performance liquid chromatography (CEX-HPLC). In this assay, peaks eluting from the CEX-HPLC column with an earlier retention time than the main peak are labeled as "acidic peaks", while those eluting from the CEX-HPLC column with a later retention time than the main peak The peaks are marked as "Basic Peaks".
加速稳定性研究可以用于检查产品的稳定性性质，有利于筛选稳定药物制剂形式。例如，可以将制剂样品放置于升高的温度，例如约40℃±2℃、25℃±2℃条件下进行加速稳定性研究。检测指标可以包括外观、可见异物、蛋白含量、浊度、纯度(SEC-HPLC法、非还原型CE-SDS法)和电荷变异体(iCIEF法、CEX-HPLC法)。Accelerated stability studies can be used to examine the stability properties of products and facilitate the screening of stable pharmaceutical formulations. For example, accelerated stability studies can be performed by placing samples of the formulation at elevated temperatures, eg, about 40°C ± 2°C, 25°C ± 2°C. Detection indicators can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reducing CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method).
此外，可以检测抗体的功效或生物活性。例如，可以检测制剂中抗体与其抗原分子(TIGIT分子和PD-1分子)的结合能力。本领域技术人员已知多种方法可以用于定量抗体与抗原的特异性结合，例如免疫测定试验，ELISA等。In addition, antibodies can be tested for efficacy or biological activity. For example, the ability of an antibody to bind to its antigenic molecules (TIGIT molecule and PD-1 molecule) in the preparation can be tested. A variety of methods are known to those of skill in the art for quantifying specific binding of antibodies to antigens, such as immunoassays, ELISAs, and the like.
本发明的抗TIGIT/PD-1双特异性抗体蛋白制剂是稳定的。在一个实施方案中，于约5℃、25℃、37℃、40℃、或45℃储存至少1个月、2个月或3个月后，例如，在5℃±3℃储存3个月后，本发明的抗体制剂中的抗TIGIT/PD-1双特异性抗体蛋白纯度是至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％以上，如通过体积排阻色谱法或通过非还原型CS-SDS所测定。在一个实施方案中，于约5℃、25℃、37℃、40℃、或45℃储存至少1个月、2个月或3个月后，例如，在5℃±3℃储存3个月后，本发明的抗体制剂中抗TIGIT/PD-1双特异性抗体蛋白的至少60％，优选至少65％是非碱性及非酸性形式(亦即，主峰或主要电荷形式)，如通过iCIEF法所测定。The anti-TIGIT/PD-1 bispecific antibody protein preparation of the present invention is stable. In one embodiment, after storage at about 5°C, 25°C, 37°C, 40°C, or 45°C for at least 1 month, 2 months, or 3 months, eg, at 5°C ± 3°C for 3 months Then, the anti-TIGIT/PD-1 bispecific antibody protein purity in the antibody preparation of the present invention is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99%, as determined by size exclusion chromatography or by non-reducing CS-SDS. In one embodiment, after storage at about 5°C, 25°C, 37°C, 40°C, or 45°C for at least 1 month, 2 months, or 3 months, eg, at 5°C ± 3°C for 3 months Then, at least 60%, preferably at least 65% of the anti-TIGIT/PD-1 bispecific antibody protein in the antibody preparation of the invention is in the non-basic and non-acidic form (ie, the main peak or main charge form), such as by the iCIEF method determined.
IV.制剂的用途IV. USE OF THE FORMULATION
本发明的包含抗TIGIT/PD-1双特异性抗体蛋白的本发明的抗体制剂可以用于治疗、预防或延缓各种与TIGIT信号传导通路和/或与PD-1信号传导通路相关的疾病或病症。“与TIGIT信号传导通路相关的疾病或病症”和/或“与PD-1信号传导通路相关的疾病或病症”在本文中指可以用本发明抗TIGIT/PD-1双特异性抗体蛋白制剂进行治疗(例如改善)或预防的疾病或病症。任何可以得益于本发明抗体制剂治疗的疾病或病症都适用于本发明。The antibody preparation of the present invention comprising the anti-TIGIT/PD-1 bispecific antibody protein of the present invention can be used to treat, prevent or delay various diseases related to the TIGIT signaling pathway and/or PD-1 signaling pathway or disease. "Diseases or disorders associated with the TIGIT signaling pathway" and/or "diseases or disorders associated with the PD-1 signaling pathway" herein refer to treatable with the anti-TIGIT/PD-1 bispecific antibody protein formulations of the invention (eg to ameliorate) or prevent a disease or condition. Any disease or condition that can benefit from the treatment of the antibody formulations of the present invention is suitable for use in the present invention.
包含抗TIGIT/PD-1双特异性抗体蛋白的本发明制剂能够用于预防或治疗受试者的各种血液病和实体瘤，包括但不限于白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈癌、头颈部鳞状细胞癌、肺癌例如非小细胞肺癌或小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、胆 管癌、肝癌、结直肠癌、乳腺癌、卵巢癌、***、子宫内膜癌、子宫肉瘤、***癌、膀胱癌、肾细胞癌、黑色素瘤。The formulations of the present invention comprising anti-TIGIT/PD-1 bispecific antibody proteins can be used to prevent or treat various hematological diseases and solid tumors in subjects, including but not limited to leukemia, lymphoma, myeloma, brain tumor, head and neck Cancer, squamous cell carcinoma of head and neck, lung cancer such as non-small cell lung cancer or small cell lung cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer , cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma.
本发明也提供本发明的制剂在制备药物中的用途，其中所述药物用于向哺乳动物递送抗TIGIT/PD-1双特异性抗体蛋白，或用于治疗、预防或改善上述疾病和病症中的一种或多种。优选地，哺乳动物是人。The present invention also provides the use of the formulation of the present invention in the manufacture of a medicament for delivering an anti-TIGIT/PD-1 bispecific antibody protein to a mammal, or for treating, preventing or ameliorating the above-mentioned diseases and disorders one or more of. Preferably, the mammal is a human.
可以以多种途径将本发明的抗体制剂施用于受试者或患者。例如，施用可以通过输注或通过注射器进行。因此，在一个方面，本发明提供了一种递送装置(例如注射器)，其包含本发明的抗体制剂(例如，预填充注射器)。患者将接受有效量的抗TIGIT/PD-1双特异性抗体蛋白作为主要活性成分，即足以治疗、改善或预防目的疾病或病症的量。The antibody formulations of the invention can be administered to a subject or patient in a variety of ways. For example, administration can be by infusion or by syringe. Accordingly, in one aspect, the present invention provides a delivery device (eg, a syringe) comprising an antibody formulation of the present invention (eg, a pre-filled syringe). The patient will receive an effective amount of the anti-TIGIT/PD-1 bispecific antibody protein as the primary active ingredient, ie, an amount sufficient to treat, ameliorate or prevent the disease or disorder of interest.
治疗效果可包括减少生理症状。用于任何特定受试者的抗体的最佳有效量和浓度将取决于多种因素，包括患者的年龄、体重、健康状况和/或性别、疾病的性质和程度、特定抗体的活性，身体对其清除率，并且也包括与所述抗体制剂组合施用的任何可能的其它治疗。对于具体的情况，所递送的有效量可以在临床医师的判断范围内来确定。在这方面，已知的基于抗体的药物的应用可以提供一定的指导。剂量可以是单剂量方案或多剂量方案。Therapeutic effects may include reduction of physical symptoms. The optimal effective amount and concentration of antibody for use in any particular subject will depend on a variety of factors, including the age, weight, health and/or sex of the patient, the nature and extent of the disease, the activity of the particular antibody, the its clearance, and also any possible other treatments administered in combination with the antibody preparation. For a particular case, the effective amount to be delivered can be determined within the judgment of the clinician. In this regard, the use of known antibody-based drugs can provide some guidance. Dosage can be a single-dose regimen or a multiple-dose regimen.
文中所提到的序列编号对应于下面表1中所列的SEQ ID NO编号The sequence numbers mentioned in the text correspond to the SEQ ID NO numbers listed in Table 1 below
Figure PCTCN2021133345-appb-000008
Figure PCTCN2021133345-appb-000008
HCCR：重链恒定区；LCCR：轻链恒定区；HCVR：重链可变区；LCVR：轻链可变区；ECD：胞外结构域HCCR: heavy chain constant region; LCCR: light chain constant region; HCVR: heavy chain variable region; LCVR: light chain variable region; ECD: extracellular domain
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成限 制本发明的保护范围。The following examples are described to assist in the understanding of the present invention. The examples are not intended and should not be construed in any way to limit the scope of protection of the present invention.
缩略词描述Abbreviation Description
Figure PCTCN2021133345-appb-000009
Figure PCTCN2021133345-appb-000009
具体实施方式Detailed ways
实施例1：重组全人源抗TIGIT/PD-1双特异性抗体的表达与纯化Example 1: Expression and purification of recombinant fully human anti-TIGIT/PD-1 bispecific antibody
本发明的抗体可以基本上如下表达和纯化。可以采用最佳的预定的重链：轻链载体比率或采用同时编码重链和轻链的单一载体***，用分泌抗体的表达***瞬时或稳定转染合适的宿主细胞HEK 293(也可是其他宿主细胞例如CHO细胞)。具体而言，可以利用编码以下氨基酸序列的一种或多种DNA分子：具有氨基酸序列SEQ ID NO：21的第一重链，具有SEQ ID NO：22的第一轻链，具有氨基酸序列SEQ ID NO：23的第二重链和具有氨基酸序列SEQ ID NO：24的第二轻链，并利用分泌抗体的表达***瞬时或稳定地转染，来制备本发明的抗体(在文中也称为“antibody-A”)。其中，具有氨基酸序列SEQ ID NO：21的第一重链和具有SEQ ID NO：22的第一轻链共同形成第一半抗体，并且两者分别构成其VH/VL单元，并且该第一半抗体与TIGIT或其胞外结构域特异性结合；以及，具有氨基酸序列SEQ ID NO：23的第二重链和具有氨基酸序列SEQ ID NO：24的第二轻链共同形成第二半抗体，并且两者分别构成其VH/VL单元，并且该第二半抗体与PD-1或其胞外结构域特异性结合；以及，第一半抗体 和第二半抗体共同形成本发明的完整的双特异性抗体。图25展示了上述抗体的结构示意图。可以使用多种常用技术之一来纯化抗体。例如，可将培养基方便地应用于已用兼容的缓冲液例如磷酸盐缓冲液(pH 7.4)平衡的MabSelect柱(GE Healthcare)或KappaSelect柱(GE Healthcare)。洗涤柱以去除非特异性结合组分。结合的抗体可以例如通过pH梯度洗脱(例如20mM Tris缓冲液pH 7.0至10mM柠檬酸钠缓冲液pH 3.0，或磷酸盐缓冲盐水pH 7.4至100mM甘氨酸缓冲液pH 3.0)。例如通过紫外线吸收或SDS-PAGE检测抗体级分，然后合并抗体级分。根据预期用途，任选进一步纯化。可以使用常规技术浓缩和/或无菌过滤纯化的抗体。可以通过常规技术有效去除可溶性聚集体和多聚体，所述常规技术包括体积排阻、疏水相互作用、离子交换、多模式或羟磷灰石色谱等。纯化的抗体可立即在-30℃以下冷冻或冻干。The antibodies of the present invention can be expressed and purified substantially as follows. Transient or stable transfection of suitable host cells HEK 293 (other hosts may also be possible) with an antibody secreting expression system using an optimal predetermined heavy chain:light chain vector ratio or using a single vector system encoding both heavy and light chains. cells such as CHO cells). Specifically, one or more DNA molecules encoding the following amino acid sequences can be utilized: a first heavy chain having the amino acid sequence SEQ ID NO: 21, a first light chain having the amino acid sequence SEQ ID NO: 22, having the amino acid sequence SEQ ID NO: 22 The antibody of the invention (also referred to herein as "" antibody-A”). Wherein, the first heavy chain with the amino acid sequence SEQ ID NO: 21 and the first light chain with SEQ ID NO: 22 together form the first half antibody, and the two respectively constitute its VH/VL unit, and the first half The antibody specifically binds to TIGIT or its extracellular domain; and, the second heavy chain having the amino acid sequence of SEQ ID NO:23 and the second light chain having the amino acid sequence of SEQ ID NO:24 together form a second half-antibody, and The two respectively constitute its VH/VL unit, and the second half antibody specifically binds to PD-1 or its extracellular domain; and, the first half antibody and the second half antibody together form the complete bispecific of the present invention Sexual antibodies. Figure 25 shows a schematic diagram of the structure of the above-mentioned antibody. Antibodies can be purified using one of a variety of commonly used techniques. For example, the medium can be conveniently applied to a MabSelect column (GE Healthcare) or a KappaSelect column (GE Healthcare) that has been equilibrated with a compatible buffer such as phosphate buffer (pH 7.4). The column is washed to remove non-specifically bound components. The bound antibody can be eluted, for example, by a pH gradient (eg, 20 mM Tris buffer pH 7.0 to 10 mM sodium citrate buffer pH 3.0, or phosphate buffered saline pH 7.4 to 100 mM glycine buffer pH 3.0). Antibody fractions are detected, eg, by UV absorption or SDS-PAGE, and then pooled. Further purification is optional depending on the intended use. Purified antibodies can be concentrated and/or sterile filtered using conventional techniques. Soluble aggregates and multimers can be efficiently removed by conventional techniques including size exclusion, hydrophobic interactions, ion exchange, multimodal or hydroxyapatite chromatography, and the like. Purified antibodies can be immediately frozen or lyophilized below -30°C.
实施例2：重组全人源抗TIGIT/PD-1双特异性抗体(antibody-A)对LOVO混合PBMC荷瘤NOG小鼠的药效研究Example 2: Pharmacodynamic study of recombinant fully human anti-TIGIT/PD-1 bispecific antibody (antibody-A) on LOVO mixed PBMC tumor-bearing NOG mice
采用LOVO(人结/直肠癌细胞)混合PBMC荷瘤的NOG小鼠，研究重组全人源抗TIGIT/PD-1双特异性抗体对LOVO混合PBMC荷瘤NOG小鼠的抗肿瘤药效，并研究了IBI308(PD-1)与anti-TIGIT单药及联用的抗肿瘤效应进行对比。Using LOVO (human colorectal cancer cells) mixed with PBMC tumor-bearing NOG mice to study the antitumor efficacy of recombinant fully human anti-TIGIT/PD-1 bispecific antibody on LOVO mixed PBMC tumor-bearing NOG mice, and The anti-tumor effects of IBI308(PD-1) and anti-TIGIT alone and in combination were studied.
1.体内药效学研究1. In vivo pharmacodynamic studies
1.1测试溶液配制1.1 Test solution preparation
将H-IgG注射液(来自Equitech-Bio；货号：SLH56-0001；规格：7.3mg/ml)、IBI308(信迪利单抗)注射液(商品名
Figure PCTCN2021133345-appb-000010
来自信达生物(苏州)有限公司；批号：DP1911001；规格：10mg/ml)、anti-TIGIT(一种单抗，其重链为SEQ ID NO：21；轻链为SEQ ID NO：22)注射液(规格：1.761mg/ml)和根据实施例7处方F5制备的antibody-A注射液(浓度：25mg/ml)用PBS(1×)(中国上海生工，货号：F607008-0500)稀释，得到以下测试溶液：0.6mg/ml H-IgG注射液；0.03mg/ml IBI308注射液；0.3mg/ml IBI308注射液；0.03mg/ml anti-TIGIT注射液；0.3mg/ml anti-TIGIT注射液；0.06mg/ml antibody-A注射液；0.6mg/ml antibody-A注射液。 H-IgG injection (from Equitech-Bio; product number: SLH56-0001; specification: 7.3mg/ml), IBI308 (sintilimab) injection (trade name
Figure PCTCN2021133345-appb-000010
From Innovent (Suzhou) Co., Ltd.; batch number: DP1911001; specification: 10mg/ml), anti-TIGIT (a monoclonal antibody whose heavy chain is SEQ ID NO: 21; light chain is SEQ ID NO: 22) injection Liquid (specification: 1.761mg/ml) and antibody-A injection (concentration: 25mg/ml) prepared according to recipe F5 in Example 7 were diluted with PBS (1×) (Shanghai Sangong, China, product number: F607008-0500), The following test solutions were obtained: 0.6mg/ml H-IgG injection; 0.03mg/ml IBI308 injection; 0.3mg/ml IBI308 injection; 0.03mg/ml anti-TIGIT injection; 0.3mg/ml anti-TIGIT injection ; 0.06mg/ml antibody-A injection; 0.6mg/ml antibody-A injection.
1.2.实验操作1.2. Experimental operation
以PBS(1×)(来源同上)按4∶1分散LOVO细胞(来源：ATCC；货号：CCL-229；批号：60380843)和PBMC细胞(来源：AllCells；货号：LBL-002；批号：3024811)，制备成细胞浓度为12.5×10 6个/mL：3.125×10 6个/mL的细胞悬液。 LOVO cells (source: ATCC; Cat. No.: CCL-229; Lot No.: 60380843) and PBMC cells (Source: AllCells; Cat. No.: LBL-002; Lot No.: 3024811) were dispersed 4:1 in PBS (1×) (source as above). , prepare a cell suspension with a cell concentration of 12.5×10 6 cells/mL: 3.125×10 6 cells/mL.
将NOG小鼠(56只，雌性，14-17g，来源：北京维通利华实验动物技术有限公司，合格证号：1100112011044904)右侧背部剃毛，皮下注射12.5×10 6个/mL：3.125×10 6个/mL的LOVO：PBMC混合细胞悬液0.2mL/只，即接种量为LOVO 2.5×10 6个/小鼠：PBMC 0.625×10 6个/小鼠。 NOG mice (56, female, 14-17g, source: Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., certificate number: 1100112011044904) were shaved on the right back and injected subcutaneously at 12.5×10 6 /mL: 3.125 ×10 6 cells/mL of LOVO:PBMC mixed cell suspension 0.2 mL/mouse, that is, the inoculation amount is LOVO 2.5×10 6 cells/mouse: PBMC 0.625×10 6 cells/mouse.
肿瘤细胞接种2天将56只小鼠随机分为8组，每组7只小鼠，按照给药类型、剂量命名 为H-IgG-6mg/kg组、IBI308-0.3mg/kg组、IBI308-3mg/kg组、anti-TIGIT-3mg/kg组、IBI308+anti-TIGIT-0.3+0.3mg/kg组、IBI308+αTIGIT(321)-3+3mg/kg组、antibody-A-0.6mg/kg组和antibody-A-6mg/kg组；给药方式为腹腔注射，给药体积10ml/kg，给药频率为每3或4天一次，即具体在肿瘤细胞接种后的第2天、第6天、第10天、第13天各给药1次，监测至24天，于接种后的27天取小鼠肿瘤，并称重。每周监测小鼠体重、肿瘤组织最大长轴(L)和最大宽轴(W)两次。在接种后24天计算各组小鼠的相对肿瘤抑制率。2 days after tumor cell inoculation, 56 mice were randomly divided into 8 groups, with 7 mice in each group, named as H-IgG-6mg/kg group, IBI308-0.3mg/kg group, IBI308- 3mg/kg group, anti-TIGIT-3mg/kg group, IBI308+anti-TIGIT-0.3+0.3mg/kg group, IBI308+αTIGIT(321)-3+3mg/kg group, antibody-A-0.6mg/kg group and antibody-A-6mg/kg group; the administration method was intraperitoneal injection, the administration volume was 10ml/kg, and the administration frequency was once every 3 or 4 days, specifically on the 2nd and 6th days after tumor cell inoculation. Day 10, day 13, each administration was administered once, and the mice were monitored until day 24. Tumors of mice were collected and weighed 27 days after inoculation. Mice body weight, the largest long axis (L) and the largest wide axis (W) of tumor tissue were monitored twice weekly. The relative tumor inhibition rates of mice in each group were calculated 24 days after inoculation.
肿瘤接种后第6、10、13、17、20、24天测定小鼠肿瘤体积，肿瘤测量每周两次，肿瘤测量时称重，以及每一次给药前称重。肿瘤接种27天后取瘤体组织称重并拍照。Tumor volume in mice was determined on days 6, 10, 13, 17, 20, and 24 after tumor inoculation, tumors were measured twice a week, and tumors were weighed at the time of measurement and before each dose. Tumor tissue was weighed and photographed 27 days after tumor inoculation.
1.3.肿瘤体积测定1.3. Determination of tumor volume
采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积按如下公式计算：V＝L×W 2/2。 Vernier calipers were used to measure the maximum long axis (L) and maximum wide axis (W) of the tumor, and the tumor volume was calculated according to the following formula: V=L×W 2 /2.
1.4.相对肿瘤抑制率计算1.4. Calculation of relative tumor inhibition rate
肿瘤抑制率TGI(％)：100％×(Tvol control-Tvol treated)/(Tvol control-Tvol predose) Tumor inhibition rate TGI (%): 100%×(Tvol control -Tvol treated )/(Tvol control -Tvol predose )
Tvol control-Tvol treated：对照组给药后肿瘤终末体积-给药组给药后肿瘤终末体积； Tvol control -Tvol treated : the terminal tumor volume after the administration of the control group - the final volume of the tumor in the administration group after administration;
Tvol control-Tvol predose：对照组给药后肿瘤终末体积-对照组给药前肿瘤体积。 Tvol control -Tvol predose : the terminal tumor volume after administration of the control group - the tumor volume of the control group before administration.
2.统计分析2. Statistical analysis
实验结果采用Two-wayANOVA分析比较各给药组肿瘤体积大小之间的差异，P＜0.05认为具有统计学差异。The experimental results were analyzed by Two-way ANOVA to compare the differences in tumor volume between each administration group, and P<0.05 was considered to be statistically significant.
给药后小鼠体重变化不超过10％，判断该药物对小鼠体重无影响。给药后，与对照组相比计算TGI，判断该药物对荷瘤的抑瘤效果。The body weight of the mice did not change by more than 10% after administration, and it was judged that the drug had no effect on the body weight of the mice. After administration, the TGI was calculated compared with the control group to judge the tumor-inhibitory effect of the drug on the tumor-bearing tumor.
3.实验结果3. Experimental results
3.1 antibody-A对荷瘤小鼠体重的影响3.1 The effect of antibody-A on the body weight of tumor-bearing mice
各组小鼠体重变化见图1、表2和表3。本实验中，各组小鼠未出现明显的体重下降情况，小鼠表现正常。由此可见，antibody-A对LOVO混合PBMC荷瘤NOG小鼠的体重没有影响。The body weight changes of mice in each group are shown in Figure 1, Table 2 and Table 3. In this experiment, there was no obvious weight loss in each group of mice, and the mice behaved normally. It can be seen that antibody-A has no effect on the body weight of LOVO mixed PBMC tumor-bearing NOG mice.
Figure PCTCN2021133345-appb-000011
Figure PCTCN2021133345-appb-000011
Figure PCTCN2021133345-appb-000012
Figure PCTCN2021133345-appb-000012
3.2 antibody-A对LOVO混合PBMC荷瘤小鼠肿瘤生长的影响3.2 The effect of antibody-A on tumor growth in LOVO mixed PBMC tumor-bearing mice
本实验研究了antibody-A对LOVO混合PBMC荷瘤NOG小鼠的抗肿瘤药效。各组小鼠在给药后不同时间的肿瘤抑制率及肿瘤体积如表4-表6和图2-图7所示。本药效学实验基于接种后第24天的肿瘤体积数据计算相对肿瘤抑制率(如表4)。与H-IgG-6mg/kg组相比，IBI308-0.3mg/kg组、IBI308-3mg/kg组的肿瘤抑制率分别是11.47％、57.25％；anti-TIGIT-3mg/kg组的肿瘤抑制率为57.25％；IBI308+anti-TIGIT-0.3+0.3mg/kg组和IBI308+anti-TIGIT-3+3mg/kg组的肿瘤抑制率是65.42％和35.29％；antibody-A-0.6mg/kg组和antibody-A-6mg/kg组的肿瘤抑制率为92.63％与99.84％。antibody-A-0.6mg/kg组、antibody-A-6mg/kg组的肿瘤抑制率显著优于anti-TIGIT-3mg/kg单药组及IBI308+anti-TIGIT-0.3+0.3mg/kg组、IBI308+anti-TIGIT-3+3mg/kg组，显示本发明的双特异性抗体的两抗体部分产生协同作用。In this experiment, the antitumor efficacy of antibody-A on LOVO mixed PBMC tumor-bearing NOG mice was investigated. The tumor inhibition rate and tumor volume of each group of mice at different times after administration are shown in Table 4-Table 6 and Figure 2-Figure 7. In this pharmacodynamic experiment, the relative tumor inhibition rate was calculated based on the tumor volume data on the 24th day after inoculation (as shown in Table 4). Compared with the H-IgG-6mg/kg group, the tumor inhibition rates of the IBI308-0.3mg/kg group and IBI308-3mg/kg group were 11.47% and 57.25%, respectively; the tumor inhibition rate of the anti-TIGIT-3mg/kg group was was 57.25%; the tumor inhibition rates of the IBI308+anti-TIGIT-0.3+0.3mg/kg group and the IBI308+anti-TIGIT-3+3mg/kg group were 65.42% and 35.29%; the antibody-A-0.6mg/kg group and antibody-A-6mg/kg group's tumor inhibition rate was 92.63% and 99.84%. The tumor inhibition rate of antibody-A-0.6mg/kg group and antibody-A-6mg/kg group was significantly better than that of anti-TIGIT-3mg/kg single drug group and IBI308+anti-TIGIT-0.3+0.3mg/kg group, The IBI308+anti-TIGIT-3+3mg/kg group showed that the two antibody parts of the bispecific antibody of the present invention produced a synergistic effect.
表4.肿瘤抑制率(TGI)和肿瘤消失数目Table 4. Tumor Inhibition Rate (TGI) and Number of Tumor Disappearances
Figure PCTCN2021133345-appb-000013
Figure PCTCN2021133345-appb-000013
Figure PCTCN2021133345-appb-000014
Figure PCTCN2021133345-appb-000014
表6.antibody-A对荷瘤小鼠瘤组织重量的影响(g，Mean±SD，n＝7)Table 6. Effect of antibody-A on tumor tissue weight in tumor-bearing mice (g, Mean±SD, n=7)
Figure PCTCN2021133345-appb-000015
Figure PCTCN2021133345-appb-000015
本实验中，antibody-A各剂量组的肿瘤抑制效果均优于IBI308单药组、anti-TIGIT单药组及联合用药组，具有显著的抗肿瘤效果，且anti-TIGIT单药组的肿瘤抑制效果均优于IBI308单药组。In this experiment, the tumor inhibitory effect of each dose group of antibody-A was better than that of the IBI308 single-agent group, the anti-TIGIT single-agent group and the combination group, with significant anti-tumor effect, and the anti-TIGIT single-agent group had tumor inhibition. The effect was better than that of IBI308 single drug group.
实施例3：重组全人源抗TIGIT/PD-1双特异性抗体(antibody-A)对MC38荷瘤PD-1/TIGIT小鼠的药效研究Example 3: Pharmacodynamic study of recombinant fully human anti-TIGIT/PD-1 bispecific antibody (antibody-A) on MC38 tumor-bearing PD-1/TIGIT mice
采用MC38(鼠结肠癌细胞)荷瘤的小鼠，研究重组全人源抗TIGIT/PD-1双特异性抗体对MC38荷瘤PD1/TIGIT小鼠(即PD1/TIGIT双敲的转基因小鼠；来源：百奥赛图江苏基因生物技术有限公司；合格证号：3207262011001376，下同)的抗肿瘤药效。MC38 (mouse colon cancer cell) tumor-bearing mice were used to study the effect of recombinant fully human anti-TIGIT/PD-1 bispecific antibody on MC38 tumor-bearing PD1/TIGIT mice (ie, PD1/TIGIT double knockout transgenic mice; Source: Biositu Jiangsu Gene Biotechnology Co., Ltd.; Certificate No.: 3207262011001376, the same below) anti-tumor efficacy.
1.体内药效学研究1. In vivo pharmacodynamic studies
1.1测试溶液配制1.1 Test solution preparation
如实施例2所述配制如下测试溶液：0.6mg/ml H-IgG注射液；0.03mg/ml IBI308注射液；0.3mg/ml IBI308注射液；0.03mg/ml anti-TIGIT注射液；0.3mg/ml anti-TIGIT注射液；0.06mg/ml antibody-A注射液；0.6mg/ml antibody-A注射液。The following test solutions were prepared as described in Example 2: 0.6 mg/ml H-IgG injection; 0.03 mg/ml IBI308 injection; 0.3 mg/ml IBI308 injection; 0.03 mg/ml anti-TIGIT injection; 0.3 mg/ml ml anti-TIGIT injection; 0.06mg/ml antibody-A injection; 0.6mg/ml antibody-A injection.
1.2.实验操作1.2. Experimental operation
以PBS(1×)(来源同上)分散MC38细胞(来源：上海和元生物有限公司；批号：HYC0116)，制备成细胞浓度为5×10 6个/mL细胞悬液。 MC38 cells (source: Shanghai Heyuan Biological Co., Ltd.; batch number: HYC0116) were dispersed with PBS (1×) (source as above) to prepare a cell suspension with a cell concentration of 5×10 6 cells/mL.
将PD1/TIGIT小鼠(60只；雌性，16-20g)右侧背部剃毛，皮下注射5×10 6个/mL的MC38细胞悬液0.2mL/只，即接种量为1×10 6个/只小鼠。 PD1/TIGIT mice (60 mice; female, 16-20 g) were shaved on the right back and injected subcutaneously with 0.2 mL/mice of 5×10 6 cells/mL MC38 cell suspension, that is, the inoculation amount was 1×10 6 cells / mouse.
肿瘤细胞接种7天后测量每只小鼠瘤体积，挑选出瘤体积在一定范围内 (42.75mm 3～98.39mm 3)的小鼠，共48只小鼠。按瘤体积平均分为8组，每组6只小鼠，按照给药类型、剂量命名为H-IgG-6mg/kg组、IBI308-0.3mg/kg组、IBI308-3mg/kg组、anti-TIGIT-3mg/kg组、IBI308+anti-TIGIT-0.3+0.3mg/kg组、IBI30+αTIGIT(321)-3+3mg/kg组、antibody-A-0.6mg/kg组和antibody-A-6mg/kg组；给药方式为腹腔注射，给药体积10ml/kg，给药频率为每3或4天一次，即具体地在肿瘤细胞接种后的第7天、第10天、第14天、第17天各给药1次，监测至24天，于接种后的28天取小鼠肿瘤，并称重。每周监测小鼠体重、肿瘤组织最大长轴(L)和最大宽轴(W)两次。在接种后24天计算各组小鼠的相对肿瘤抑制率。 Seven days after tumor cell inoculation, the tumor volume of each mouse was measured, and the mice with tumor volume within a certain range (42.75 mm 3 -98.39 mm 3 ) were selected, totaling 48 mice. According to tumor volume, they were divided into 8 groups, with 6 mice in each group. They were named as H-IgG-6mg/kg group, IBI308-0.3mg/kg group, IBI308-3mg/kg group, anti-IgG group according to the administration type and dose. TIGIT-3mg/kg group, IBI308+anti-TIGIT-0.3+0.3mg/kg group, IBI30+αTIGIT(321)-3+3mg/kg group, antibody-A-0.6mg/kg group and antibody-A-6mg group /kg group; the administration method is intraperitoneal injection, the administration volume is 10ml/kg, and the administration frequency is once every 3 or 4 days, that is, specifically on the 7th day, the 10th day, the 14th day, the Each administration was administered once on the 17th day, and the mice were monitored until the 24th day. The tumor of the mice was collected and weighed on the 28th day after the inoculation. Mice body weight, the largest long axis (L) and the largest wide axis (W) of tumor tissue were monitored twice weekly. The relative tumor inhibition rates of mice in each group were calculated 24 days after inoculation.
肿瘤接种后第7、10、14、17、21、24天测定小鼠肿瘤体积，肿瘤测量每周两次，肿瘤测量时称重，以及每一次给药前称重。肿瘤接种28天后取瘤体组织称重并拍照。Tumor volume in mice was determined on days 7, 10, 14, 17, 21, and 24 after tumor inoculation. Tumors were measured twice a week, and tumors were weighed at the time of measurement and before each dose. Tumor tissue was weighed and photographed 28 days after tumor inoculation.
1.3肿瘤体积测定1.3 Determination of tumor volume
采用游标卡尺测定肿瘤的最大长轴(L)和最大宽轴(W)，肿瘤体积(Tvol)按如下公式计算：V＝L×W 2/2。 Vernier calipers were used to measure the maximum long axis (L) and the maximum wide axis (W) of the tumor, and the tumor volume (Tvol) was calculated according to the following formula: V=L×W 2 /2.
1.4相对肿瘤抑制率计算1.4 Calculation of relative tumor inhibition rate
肿瘤抑制率TGI(％)：100％×(Tvol 对照-Tvol 治疗)/(Tvol 对照-Tvol 初始剂量) Tumor inhibition rate TGI (%): 100%×(Tvol control -Tvol treatment )/(Tvol control -Tvol initial dose )
Tvol control-Tvol treated：对照组给药后肿瘤终末体积-给药组给药后肿瘤终末体积； Tvol control -Tvol treated : the terminal tumor volume after the administration of the control group - the final volume of the tumor in the administration group after administration;
Tvol control-Tvol predose：对照组给药后肿瘤终末体积-对照组给药前肿瘤体积。 Tvol control -Tvol predose : the terminal tumor volume after administration of the control group - the tumor volume of the control group before administration.
2.统计分析2. Statistical analysis
实验结果采用Two-way ANOVA分析比较各给药组肿瘤体积大小之间的差异，P＜0.05认为具有统计学差异。The experimental results were analyzed by Two-way ANOVA to compare the differences in tumor volume between each administration group, and P<0.05 was considered statistically significant.
给药后小鼠体重变化不超过10％，判断该药物对小鼠体重无影响。给药后，与对照组相比计算TGI，判断该药物对荷瘤的抑瘤效果。The body weight of the mice did not change by more than 10% after administration, and it was judged that the drug had no effect on the body weight of the mice. After administration, the TGI was calculated compared with the control group to judge the tumor-inhibitory effect of the drug on the tumor-bearing tumor.
3.实验结果3. Experimental results
3.1 antibody-A对MC38荷瘤小鼠体重的影响3.1 The effect of antibody-A on body weight of MC38 tumor-bearing mice
各组小鼠体重变化见图8和表7、8。本实验中，各组小鼠未出现明显的体重下降情况，小鼠表现正常。由此可见，antibody-A对MC38荷瘤PD1/TIGIT转基因小鼠的体重没有影响。The body weight changes of mice in each group are shown in Figure 8 and Tables 7 and 8. In this experiment, there was no obvious weight loss in each group of mice, and the mice behaved normally. Thus, antibody-A had no effect on the body weight of MC38 tumor-bearing PD1/TIGIT transgenic mice.
Figure PCTCN2021133345-appb-000016
Figure PCTCN2021133345-appb-000016
Figure PCTCN2021133345-appb-000017
Figure PCTCN2021133345-appb-000017
3.2 antibody-A对MC38荷瘤小鼠肿瘤生长的影响3.2 The effect of antibody-A on tumor growth in MC38 tumor-bearing mice
本实验研究了antibody-A对MC38荷瘤PD1/TIGIT转基因小鼠的抗肿瘤药效。各组小鼠在给药后不同时间的肿瘤抑制率及肿瘤体积如表9-表11和图9-图14所示。本药效学实验基于接种后第24天的肿瘤体积数据计算相对肿瘤抑制率(如表9)。与H-IgG-6mg/kg组相比，IBI308-0.3mg/kg组、IBI308-3mg/kg组的肿瘤抑制率分别是44.79％、67.12％；anti-TIGIT-3mg/kg组的肿瘤抑制率为13.83％；IBI308+anti-TIGIT-0.3+0.3mg/kg组和IBI308+anti-TIGIT-3+3mg/kg组的肿瘤抑制率是38.35％和89.60％；antibody-A-0.6mg/kg组和antibody-A-6mg/kg组的肿瘤抑制率为68.77％与95.53％，显示本发明的双特异性抗体的两抗体部分产生协同作用。In this experiment, the antitumor efficacy of antibody-A on MC38 tumor-bearing PD1/TIGIT transgenic mice was investigated. The tumor inhibition rate and tumor volume of each group of mice at different times after administration are shown in Table 9-Table 11 and Figure 9-Figure 14. In this pharmacodynamic experiment, the relative tumor inhibition rate was calculated based on the tumor volume data on the 24th day after inoculation (as shown in Table 9). Compared with the H-IgG-6mg/kg group, the tumor inhibition rates of the IBI308-0.3mg/kg group and the IBI308-3mg/kg group were 44.79% and 67.12%, respectively; the tumor inhibition rate of the anti-TIGIT-3mg/kg group was was 13.83%; the tumor inhibition rates of the IBI308+anti-TIGIT-0.3+0.3mg/kg group and the IBI308+anti-TIGIT-3+3mg/kg group were 38.35% and 89.60%; the antibody-A-0.6mg/kg group The tumor inhibition rates of the antibody-A-6 mg/kg and antibody-A-6 mg/kg groups were 68.77% and 95.53%, indicating that the two antibody parts of the bispecific antibody of the present invention produced a synergistic effect.
本实验中，相对于H-IgG-6mg/kg组而言，IBI308-0.3mg/kg组、IBI308-3mg/kg组、IBI308+anti-TIGIT-0.3+0.3mg/kg组、IBI308+anti-TIGIT-3+3mg/kg组、antibody-A-0.6mg/kg组和antibody-A-6mg/kg组对MC38荷瘤生长均有一定的抑制效果。antibody-A低剂量组的肿瘤抑制率优于IBI308低剂量的单药组也胜于IBI308和anti-TIGIT低剂量的联合组，antibody-A高剂量组的肿瘤抑制作用也要强过IBI308高剂量单药组且优于IBI308和anti-TIGIT高剂量的联合组。antibody-A在MC38荷瘤的PD1/TIGIT小鼠中显示很好的剂量效应。In this experiment, compared with H-IgG-6mg/kg group, IBI308-0.3mg/kg group, IBI308-3mg/kg group, IBI308+anti-TIGIT-0.3+0.3mg/kg group, IBI308+anti- TIGIT-3+3mg/kg group, antibody-A-0.6mg/kg group and antibody-A-6mg/kg group had certain inhibitory effect on the growth of MC38 tumor bearing. The tumor inhibition rate of the low-dose antibody-A group was better than that of the low-dose IBI308 single-drug group and also better than the combination group of IBI308 and anti-TIGIT low-dose. The drug group was superior to the combination group of IBI308 and high dose of anti-TIGIT. Antibody-A showed a good dose response in MC38 tumor-bearing PD1/TIGIT mice.
表9.肿瘤抑制率(TGI)和肿瘤消失数目Table 9. Tumor Inhibition Rate (TGI) and Number of Tumor Disappearances
Figure PCTCN2021133345-appb-000018
Figure PCTCN2021133345-appb-000018
Figure PCTCN2021133345-appb-000019
Figure PCTCN2021133345-appb-000019
表11.antibody-A对MC38荷瘤小鼠瘤组织重量的影响(g，Mean±SD，n＝6)Table 11. Effect of antibody-A on tumor tissue weight in MC38 tumor-bearing mice (g, Mean±SD, n=6)
Figure PCTCN2021133345-appb-000020
Figure PCTCN2021133345-appb-000020
实施例4：antibody-A阻断PD-1/PD-L1及CD155/TIGIT结合活性研究Example 4: Antibody-A blocking the binding activity of PD-1/PD-L1 and CD155/TIGIT
本研究通过PD-1-TIGIT-NFAT-Luc报告***检测antibody-A对PD-1/PD-L1及TIGIT/CD155结合的阻断活性。该检测方法由2个细胞系组成：膜表面表达PD-L1、CD155和TCR activator的CHO K1细胞(CHOK1-PD-L1-CD155)，以及表达PD-1、TIGIT和NFAT-Luc报告基因的Jurkat细胞(Jurkat-PD1-TIGIT-NFAT-Luc)。这两种细胞系共孵育后，TCR和NFAT介导的发光可被PD-1/PD-L1及TIGIT/CD155相互作用抑制。通过阻断PD-1/PD-L1及TIGIT/CD155相互作用，则可逆转这种抑制作用，从而使TCR活化并通过NFAT途径产生发光。In this study, the PD-1-TIGIT-NFAT-Luc reporter system was used to detect the blocking activity of antibody-A on PD-1/PD-L1 and TIGIT/CD155 binding. The assay consists of 2 cell lines: CHO K1 cells (CHOK1-PD-L1-CD155) expressing PD-L1, CD155 and TCR activator on the membrane surface, and Jurkat expressing PD-1, TIGIT and NFAT-Luc reporter genes cells (Jurkat-PD1-TIGIT-NFAT-Luc). After co-incubation of these two cell lines, TCR- and NFAT-mediated luminescence was inhibited by PD-1/PD-L1 and TIGIT/CD155 interactions. This inhibition can be reversed by blocking the PD-1/PD-L1 and TIGIT/CD155 interactions, resulting in TCR activation and luminescence via the NFAT pathway.
实验步骤Experimental procedure
培养CHOK1-PD-L1-CD155细胞(美国Promega，货号J2102)至对数生长期，调整细胞密度至4x10 5个/ml。将细胞铺于96孔平底白板中，100μl/孔(4×10 4个/孔)，37℃，5％CO 2培养16h。 CHOK1-PD-L1-CD155 cells (Promega, USA, Cat. No. J2102) were cultured to logarithmic growth phase, and the cell density was adjusted to 4×10 5 cells/ml. The cells were plated in a 96-well flat-bottom white plate, 100 μl/well (4×10 4 cells/well), and cultured at 37° C., 5% CO 2 for 16 h.
利用Assay Buffer(RPMI 1640(美国Gibco，货号22400-089)+1％FBS(美国Hyclone，货号SH30406.05))配制antibody-A、IBI308、anti-TIGIT、IBI308+anti-TIGIT、hIgG(这些抗体的信息参见实施例2)，配置浓度为2000nM，用Assay Buffer4倍梯度稀释。Antibody-A, IBI308, anti-TIGIT, IBI308+anti-TIGIT, hIgG (these antibodies) were prepared using Assay Buffer (RPMI 1640 (Gibco, USA, Cat. No. 22400-089) + 1% FBS (Hyclone, USA, Cat. No. SH30406.05)) For information, see Example 2), the configuration concentration is 2000nM, and the Assay Buffer is used for 4-fold gradient dilution.
培养Jurkat-PD1-TIGIT-NFAT-Luc(美国Promega，货号J2102)至对数生长期，利用Assay Buffer(RPMI 1640+1％FBS(来源信息同上))调整细胞密度为1.25×10 6cells/ml。 Cultivate Jurkat-PD1-TIGIT-NFAT-Luc (Promega, USA, product number J2102) to logarithmic growth phase, and adjust the cell density to 1.25×10 6 cells/ml with Assay Buffer (RPMI 1640+1% FBS (same source information)) .
将培养CHOK1-PD-L1-CD155的96孔板取出，每孔吸去95μl上清。加入配制好的antibody-A、IBI308、anti-TIGIT、IBI308+anti-TIGIT、hIgG抗体稀释液，40μl/孔。设置空白对照组，即加入40μl Assay buffer。再加入Jurkat-PD1-TIGIT-NFAT-Luc细胞，40μl/孔(5×10 4 个/孔)。37℃，5％CO 2细胞培养箱中继续孵育6h。 The 96-well plate cultured with CHOK1-PD-L1-CD155 was removed, and 95 μl of supernatant was aspirated from each well. Add the prepared antibody-A, IBI308, anti-TIGIT, IBI308+anti-TIGIT, hIgG antibody dilutions, 40 μl/well. A blank control group was set, that is, 40 μl Assay buffer was added. Then Jurkat-PD1-TIGIT-NFAT-Luc cells were added, 40 μl/well (5×10 4 cells/well). Continue to incubate for 6h in a 37°C, 5% CO2 cell incubator.
将96孔板取出，平衡至室温。加入Bio-Glo TM Reagent(Bio-Glo TM Luciferase Assay System，Promega，G7940)，80μl/孔。室温反应10min。利用多功能酶标仪读取荧光值。 Remove the 96-well plate and equilibrate to room temperature. Bio-Glo ™ Reagent (Bio-Glo ™ Luciferase Assay System, Promega, G7940) was added at 80 μl/well. The reaction was carried out at room temperature for 10 min. Read the fluorescence value using a multi-plate reader.
利用GraphPad Prism 8.0拟合曲线。根据公式倍数诱导(Fold induction)＝RLU(抗体组)/RLU(空白对照组)计算阻断活性倍数，拟合S型曲线，计算并比较EC 50值。平行重复三次。 Curves were fitted using GraphPad Prism 8.0. The fold of blocking activity was calculated according to the formula Fold induction=RLU (antibody group)/RLU (blank control group), sigmoidal curve was fitted, and EC 50 values were calculated and compared. Repeat three times in parallel.
实验结果Experimental results
利用NFAT-Luc报告***检测antibody-A阻断PD-1/PD-L1及TIGIT/CD155结合的能力，结果如图15所示，在0.015259～1000nM的浓度范围内，antibody-A能够阻断PD-1/PD-L1及TIGIT/CD155的结合，从而重新激活T细胞，其阻断活性明显强于IBI308和anti-TIGIT。The NFAT-Luc reporter system was used to detect the ability of antibody-A to block the binding of PD-1/PD-L1 and TIGIT/CD155. The results are shown in Figure 15. In the concentration range of 0.015259-1000nM, antibody-A can block PD-1 The combination of -1/PD-L1 and TIGIT/CD155 reactivates T cells, and its blocking activity is significantly stronger than that of IBI308 and anti-TIGIT.
实施例5：antibody-A对CMV刺激记忆性T细胞再次活化的作用研究Example 5: Study on the effect of antibody-A on the reactivation of memory T cells stimulated by CMV
PBMC样本信息PBMC Sample Information
名称name 货号article number 批号batch number 公司company
Donor1Donor1 pb004F-Cpb004F-C LP200622LP200622 All cellsAll cells
Donor2Donor2 pb004F-Cpb004F-C LP200715BLP200715B All cellsAll cells
Donor3Donor3 SLB-HP100BSLB-HP100B 190077190077 赛笠生物科技Saili Biotechnology
T细胞激活活性(memory T cell recall法)T cell activation activity (memory T cell recall method)
PBMC细胞准备：复苏一个供体的PBMC(stimulator cells)，利用RPMI 1640完全培养基(美国Hyclone，货号SH30809.01)(含10％FBS(美国Hyclone，货号SH30406.05))调整细胞密度为6×10 6个/ml，每孔加入50μl细胞，30万/孔。 PBMC cell preparation: revive a donor's PBMC (stimulator cells), and adjust the cell density to 6 using RPMI 1640 complete medium (Hyclone, USA, catalog number SH30809.01) (containing 10% FBS (Hyclone, USA, catalog number SH30406.05) ×10 6 cells/ml, 50 μl cells were added to each well, 300,000 cells/well.
CMV细胞准备：用上述的含PBMC的培养基将CMV(瑞典Mabtech；货号3619-1)溶解，50μl/孔，终浓度为5μg/ml。CMV cell preparation: CMV (Mabtech, Sweden; Cat. No. 3619-1) was lysed with the above-mentioned PBMC-containing medium, 50 μl/well, at a final concentration of 5 μg/ml.
抗体准备：利用RPMI 1640完全培养基(10％FBS)配制hIgG1、antibody-A、IBI308、anti-TIGIT、IBI308+anti-TIGIT(抗体信息参见实施例2)，起始终浓度100nM，用Assay Buffer(来源信息参见实施例4)4倍梯度稀释共5个浓度，100μl/孔加到细胞中，antibody-A(抗体样品信息参见实施例2)起始终浓度200nM，用Assay Buffer 4倍梯度稀释共5个浓度，100μl/孔加到细胞中，于培养箱中孵育5天。利用Human IFN-gamma DuoSet ELISA Kit(美国R&D systems；货号：DY285B)测定培养上清中IFN-γ分泌量。实验中使用
Figure PCTCN2021133345-appb-000021
Human  T-Activator CD3/CD 28刺激作为阳性对照。 Antibody preparation: use RPMI 1640 complete medium (10% FBS) to prepare hIgG1, antibody-A, IBI308, anti-TIGIT, IBI308+anti-TIGIT (for antibody information, see Example 2), starting at a concentration of 100nM, using Assay Buffer ( For source information, see Example 4) 4-fold serial dilution for a total of 5 concentrations, 100 μl/well was added to cells, and antibody-A (for antibody sample information, see Example 2), the concentration was always 200 nM, and 4-fold serial dilution with Assay Buffer for a total of 5 Each concentration, 100 μl/well was added to the cells and incubated in the incubator for 5 days. Human IFN-gamma DuoSet ELISA Kit (American R&D systems; Cat. No.: DY285B) was used to measure the amount of IFN-γ secretion in the culture supernatant. used in experiments
Figure PCTCN2021133345-appb-000021
Human T-Activator CD3/ CD28 stimulation served as a positive control.
实验结果及结论Experimental results and conclusions
T细胞激活活性T cell activation activity
本实验利用CMV刺激记忆性T细胞再次活化(memory T cell recall)检测antibody-A对人记忆性T细胞的调控活性。基本原理为筛选对CMV多肽有反应的PBMC批号，复苏后加入5ug/ml CMV刺激记性T细胞将其再次活化。加入药物antibody-A，检测细胞培养上清中IFN-γ的含量，根据IFN-γ的分泌量，来反映antibody-A对记忆性T细胞的激活活性。In this experiment, CMV-stimulated memory T cell recall was used to detect the regulatory activity of antibody-A on human memory T cells. The basic principle is to screen the batches of PBMCs that respond to CMV polypeptides, and after resuscitation, add 5ug/ml of CMV to stimulate the T cells to reactivate them. The drug antibody-A was added to detect the content of IFN-γ in the cell culture supernatant, and the activation activity of antibody-A on memory T cells was reflected according to the secretion of IFN-γ.
结果见图16。实验结果表明，在memory T cell recall实验中，antibody-A在体外能浓度依赖地提高IFN-γ分泌水平，增强记忆性T细胞的功能，且其作用优于抗PD-1单抗(IBI308)、anti-TIGIT单抗以及抗PD-1单抗(IBI308)+anti-TIGIT单抗。The results are shown in Figure 16. The experimental results show that in the memory T cell recall experiment, antibody-A can concentration-dependently increase the secretion level of IFN-γ and enhance the function of memory T cells in vitro, and its effect is better than that of anti-PD-1 monoclonal antibody (IBI308) , anti-TIGIT monoclonal antibody and anti-PD-1 monoclonal antibody (IBI308) + anti-TIGIT monoclonal antibody.
以下实施例涉及重组抗TIGIT/PD-1双特异性抗体的稳定制剂的筛选和制备。The following examples relate to the screening and preparation of stable formulations of recombinant anti-TIGIT/PD-1 bispecific antibodies.
药品稳定性是保证药品有效性和安全性的重要指标之一。获得赋予药品良好稳定性的制剂处方是药品在货架期内保持其安全有效的关键条件。为了开发出重组抗TIGIT/PD-1双特异性抗体注射液长期稳定储存的制剂处方，确保产品在有效期内(例如至少24个月)的质量可控，设计了处方筛选试验，考察了不同辅料对于抗TIGIT/PD-1双特异性抗体制剂稳定性的影响。试验所用材料和方法如下：Drug stability is one of the important indicators to ensure the effectiveness and safety of drugs. Obtaining a formulation that imparts good stability to a drug product is a critical condition for a drug product to remain safe and effective during its shelf life. In order to develop a formulation for long-term stable storage of recombinant anti-TIGIT/PD-1 bispecific antibody injection, and to ensure the quality of the product is controllable within the validity period (for example, at least 24 months), a formulation screening test was designed to examine different excipients. Effects on the stability of anti-TIGIT/PD-1 bispecific antibody formulations. The materials and methods used in the test are as follows:
材料和方法Materials and methods
1.1.本发明的制剂研究中使用的材料1.1. Materials used in formulation studies of the present invention
名称name 级别level 产地及品牌Origin and brand 货号article number 符合标准Standards compliant
组氨酸Histidine 药用级Pharmaceutical grade 德国MerckMerck, Germany 1.04352.10001.04352.1000 EP，USP，JPEP, USP, JP
组氨酸Histidine 药用级Pharmaceutical grade 上海味之素Shanghai Ajinomoto N/AN/A ChP，USP，JPChP, USP, JP
盐酸精氨酸Arginine hydrochloride 药用级Pharmaceutical grade 上海味之素Shanghai Ajinomoto N/AN/A ChP，USP，JPChP, USP, JP
山梨醇Sorbitol 药用级Pharmaceutical grade 法国罗盖特Roquette, France H20110265H20110265 EP，BP，NF，USPEP, BP, NF, USP
依地酸二钠Disodium Edetate 药用级Pharmaceutical grade 南京化学试剂Nanjing Chemical Reagent N/AN/A ChPChP
聚山梨酯80Polysorbate 80 药用级Pharmaceutical grade 南京威尔Nanjing Well N/AN/A ChP，USPChP, USP
盐酸hydrochloric acid 药用级Pharmaceutical grade 南京化学试剂Nanjing Chemical Reagent N/AN/A ChPChP
注：N/A表示“不适用”(Not applicable)。Note: N/A means "Not applicable".
1.2.制剂稳定性的检测项目和检测方法1.2. Testing items and testing methods for formulation stability
对抗体制剂检测了以下项目：(1)检测外观以及是否存在可见异物；(2)通过紫外法(UV法)测定制剂中的蛋白质含量；(3)通过体积排阻色谱法，例如，体积排阻高效液相色谱法 (size-exclusion chromatography-HPLC；SEC-HPLC)测定抗体制剂的纯度，表示为单体的面积占所有峰面积之和的百分数；(4)通过非还原型十二烷基硫酸钠毛细管电泳(非还原型CE-SDS)测定抗体制剂的纯度，表示为单体的面积占所有峰面积之和的百分数；(5)通过成像毛细管等电聚焦电泳法(iCIEF法)测定抗体制剂中电荷变异体，表示为主成分、酸性组分和碱性组分的百分数；。Antibody preparations were tested for the following items: (1) detection of appearance and presence of visible foreign matter; (2) determination of protein content in preparations by ultraviolet method (UV method); (3) by size exclusion chromatography, for example, size exclusion The purity of antibody preparations was determined by size-exclusion chromatography-HPLC (SEC-HPLC), expressed as the percentage of monomer area to the sum of all peak areas; (4) by non-reducing dodecyl The purity of the antibody preparation was determined by sodium sulfate capillary electrophoresis (non-reducing CE-SDS), expressed as the percentage of the monomer area to the sum of all peak areas; (5) The antibody was determined by imaging capillary isoelectric focusing electrophoresis (iCIEF method) The charge variant in the preparation, expressed as a percentage of the main component, acidic component and basic component; .
可见异物检测Visible foreign body detection
按照国家药典委员会，中华人民共和国药典(2020年版，四部通则0904“可见异物检查法”，北京：中国医药科技出版社.2020)中所记载的方法，采用澄明度检测仪(天津天大天发生产，型号YB-2)，检查样品中的可见异物。According to the method recorded in the State Pharmacopoeia Commission, the Pharmacopoeia of the People's Republic of China (2020 edition, the four general rules 0904 "Visible foreign body inspection method", Beijing: China Pharmaceutical Science and Technology Press. 2020), the clarity detector (Tianjin Tianda Tianfa) was used. production, model YB-2), inspect the sample for visible foreign matter.
蛋白含量测定Determination of protein content
使用紫外分光光度计(日本岛津生产，型号UV-1800)测定样品中的蛋白质含量。The protein content in the sample was determined using an ultraviolet spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800).
纯度(SEC-HPLC法)Purity (SEC-HPLC method)
使用体积排阻色谱柱分离，流动相为磷酸盐缓冲液(称取3.12g二水合磷酸二氢钠，8.77g氯化钠和34.84g精氨酸，用800ml超纯水溶解后用盐酸调节pH至6.8并定容至1000ml)，色谱柱保护液为0.05％(w/v)NaN3，进样量50μl，流速0.5ml/分钟，采集时间30分钟，柱温25℃，检测波长280nm。取待测样品用制剂缓冲液稀释至2mg/ml，作为供试品溶液。取制剂缓冲液用上述相同处理方式稀释后做为空白溶液。取空白溶液、供试品溶液各50μl注入液相色谱仪，开始检测。Use size exclusion chromatography column for separation, the mobile phase is phosphate buffer (weigh 3.12g of sodium dihydrogen phosphate dihydrate, 8.77g of sodium chloride and 34.84g of arginine, dissolve with 800ml of ultrapure water and adjust the pH with hydrochloric acid. to 6.8 and dilute to 1000ml), the column protection solution is 0.05% (w/v) NaN3, the injection volume is 50μl, the flow rate is 0.5ml/min, the collection time is 30 minutes, the column temperature is 25°C, and the detection wavelength is 280nm. Dilute the sample to be tested to 2 mg/ml with preparation buffer as the test solution. The preparation buffer was diluted in the same way as above and used as blank solution. Take 50 μl of blank solution and 50 μl of test solution and inject into liquid chromatograph to start detection.
纯度(非还原型CE-SDS法)Purity (non-reducing CE-SDS method)
采用毛细管凝胶电泳法检测。毛细管为无涂层毛细管，内径50μm，总长30.2cm，有效长度20.2cm。电泳前分别使用0.1mol/L氢氧化钠、0.1mol/L盐酸、超纯水、电泳胶70psi冲洗毛细管柱。将待测样品用适量超纯水稀释至2.0mg/ml，取以上稀释后的样品50μl于1.5ml离心管中，分别向其中加入45μl pH 6.5的样品缓冲液(称取一水柠檬酸0.32g，十二水合磷酸氢二钠2.45g，溶于45ml超纯水中，定容至50ml，制得柠檬酸-磷酸盐缓冲液，精密量取该缓冲液200μl，加10％(w/v)十二烷基硫酸钠溶液80μl，加水至1ml，混匀，即得)和5μl 250mmol/L NEM溶液(称取N-乙基顺丁稀二酰亚胺62mg，溶于2ml超纯水中)，充分混匀后70±2℃加热10±2分钟，冷却至室温后转移至样品瓶作为供试品溶液。取与供试品相同体积的制剂缓冲液，按上述方法同样操作，制得空白溶液。样品进样条件：-5kV 20秒；分离电压：-15kV 35分钟。毛细管柱温控制在25℃，检测波长为220nm。detected by capillary gel electrophoresis. The capillary is an uncoated capillary with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20.2 cm. Before electrophoresis, the capillary column was washed with 0.1 mol/L sodium hydroxide, 0.1 mol/L hydrochloric acid, ultrapure water, and electrophoresis gel at 70 psi, respectively. Dilute the sample to be tested to 2.0 mg/ml with an appropriate amount of ultrapure water, take 50 μl of the above diluted sample into a 1.5 ml centrifuge tube, and add 45 μl of pH 6.5 sample buffer (weigh 0.32 g of citric acid monohydrate to it). , 2.45g of disodium hydrogen phosphate dodecahydrate, dissolved in 45ml of ultrapure water, the volume was adjusted to 50ml to prepare a citric acid-phosphate buffer, 200μl of the buffer was accurately measured, and 10% (w/v) 80μl of sodium dodecyl sulfate solution, add water to 1ml, mix well, and 5μl of 250mmol/L NEM solution (weigh 62mg of N-ethyl maleimide, dissolve in 2ml of ultrapure water) , fully mixed and heated at 70±2°C for 10±2 minutes, cooled to room temperature and transferred to a sample bottle as the test solution. Take the same volume of preparation buffer as the test sample, and operate in the same way as above to prepare a blank solution. Sample injection conditions: -5kV for 20 seconds; separation voltage: -15kV for 35 minutes. The capillary column temperature was controlled at 25 °C, and the detection wavelength was 220 nm.
电荷变异体(iCIEF法)Charge variant (iCIEF method)
采用成像毛细管等电聚焦电泳(iCIEF法)检测。毛细管内径100μtm，总长5cm。样品电泳前需分别使用0.5％甲基纤维素溶液(下文中也缩写为MC溶液)、超纯水冲洗毛细管柱。采用真空进样方式，预聚焦电压及时间为1.5kV 1分钟，聚焦电压及时间为3kV 8分钟，进样时间55秒，样品盘温度为10℃，检测波长为280nm。阴极稳定剂(Cathodic Stabilizer)为500mmol/L精氨酸溶液，0.5％MC溶液降低蛋白与毛细管之间的粘附。将供试品用水稀释至1.0mg/ml，取稀释后的供试品溶液20μl，向其中加入78μl预混液(预混液配比如下：70μl 3mol尿素-0.5％ MC溶液，4μl两性电解质(pH 3-10)，2μl阴极稳定剂，1μl pI 5.85标志物，1μl pI 9.99标志物)，充分混匀制得待测样品溶液。进样分析，根据面积归一化法，计算主成分、酸性组分及碱性组分含量。Imaging capillary isoelectric focusing electrophoresis (iCIEF method) was used for detection. The inner diameter of the capillary is 100 μtm, and the total length is 5 cm. Before sample electrophoresis, 0.5% methylcellulose solution (hereinafter also abbreviated as MC solution) and ultrapure water should be used to rinse the capillary column. The vacuum sampling method was adopted, the pre-focusing voltage and time were 1.5kV for 1 minute, the focusing voltage and time were 3kV for 8 minutes, the injection time was 55 seconds, the sample pan temperature was 10°C, and the detection wavelength was 280nm. Cathodic Stabilizer is 500 mmol/L arginine solution, 0.5% MC solution reduces the adhesion between protein and capillary. Dilute the test product to 1.0 mg/ml with water, take 20 μl of the diluted test solution, add 78 μl of the premix solution (the premix solution is as follows: 70 μl 3mol urea-0.5% MC solution, 4 μl ampholyte (pH 3) -10), 2 μl of cathode stabilizer, 1 μl of pi 5.85 marker, 1 μl of pi 9.99 marker), and thoroughly mixed to obtain the sample solution to be tested. In the analysis of sample injection, the content of main components, acidic components and basic components was calculated according to the area normalization method.
实施例6.pH对制剂的稳定性影响试验Example 6. pH effect test on formulation stability
本实施例考察了包含抗TIGIT/PD-1双特异性抗体(antibody-A)的制剂在pH 5.0至7.0的稳定性。This example examines the stability of formulations comprising anti-TIGIT/PD-1 bispecific antibody (antibody-A) at pH 5.0 to 7.0.
6.1实验步骤6.1 Experimental steps
用注射用水、组氨酸和山梨醇配制含10mM组氨酸、5％(w/v)山梨醇的缓冲液，分别用盐酸调节pH至5.0、5.5、6.0、6.5和7.0，将antibody-A蛋白超滤置换至不同pH值的缓冲液中，调节蛋白含量至25mg/ml；加入聚山梨酯80，使其终浓度为0.2mg/ml；过滤分装至西林瓶，加塞、轧盖。上述样品于40±2℃条件下进行稳定性考察，具体方案见表12。A buffer containing 10 mM histidine and 5% (w/v) sorbitol was prepared with water for injection, histidine and sorbitol, and the pH was adjusted to 5.0, 5.5, 6.0, 6.5 and 7.0 with hydrochloric acid, respectively. Antibody-A The protein was replaced by ultrafiltration into buffers with different pH values, and the protein content was adjusted to 25 mg/ml; polysorbate 80 was added to make the final concentration 0.2 mg/ml; filtered and dispensed into vials, stoppered, and capped. The above samples were tested for stability under the condition of 40±2°C. The specific scheme is shown in Table 12.
表12.实验方案Table 12. Experimental protocol
Figure PCTCN2021133345-appb-000022
Figure PCTCN2021133345-appb-000022
注：时间点取样后均先放入超低温冰箱中冻存待检，按需化冻送检。Note: After sampling at the time point, the samples were placed in an ultra-low temperature freezer and stored for inspection, and then thawed and sent for inspection as needed.
6.2判断标准6.2 Judgment Criteria
根据对产品的认识以及仪器和方法的精密度，设定了样品检测指标数值与初始值相比质量未发生变化的判断标准，具体见表13。According to the knowledge of the product and the precision of the instrument and method, the standard for judging that the quality of the sample detection index value has not changed compared with the initial value is set, as shown in Table 13.
表13.质量未发生变化的判断标准Table 13. Judgment Criteria for No Change in Quality
检测项目Test items 未发生变化的判断标准Judgment Criteria That Have Not Changed
外观Exterior 澄明至微乳光，无色至淡黄色液体，无异物Clear to slightly opalescent, colorless to pale yellow liquid, no foreign matter
可见异物visible foreign body 符合《中华人民共和国药典》(2020版，四部)0904 [1] Comply with "Pharmacopoeia of the People's Republic of China" (2020 edition, four volumes) 0904 [1]
蛋白含量(UV法)Protein content (UV method) 变化率≤10％Change rate≤10%
纯度(SEC-HPLC法)Purity (SEC-HPLC method) 主峰纯度变化值≤1％Main peak purity change value ≤ 1%
纯度(非还原型CE-SDS法)Purity (non-reducing CE-SDS method) 主峰纯度变化值≤2％Main peak purity change value ≤ 2%
电荷变异体(iCIEF法)Charge variant (iCIEF method) 主成分及酸碱各组分变化值≤2％The main component and the change value of each component of acid and alkali are less than or equal to 2%
6.3实验结果6.3 Experimental results
(1)外观和可见异物(1) Appearance and visible foreign matter
在40±2℃条件下放置2周，除pH 7.0样品第2周出现浑浊，不进行其他检测，其他各组样品外观和可见异物均合格。The samples were placed at 40±2°C for 2 weeks. Except that the pH 7.0 sample appeared turbid in the second week, no other tests were performed. The appearance and visible foreign matter of the other samples were all qualified.
(2)蛋白含量(2) protein content
蛋白含量检测结果见表14。结果表明，在40±2℃条件下放置2周，不同pH值样品蛋白含量均未发生显著变化。The protein content detection results are shown in Table 14. The results showed that the protein content of samples with different pH values did not change significantly after being placed at 40±2℃ for 2 weeks.
表14.蛋白含量结果(UV法，mg/ml)Table 14. Protein content results (UV method, mg/ml)
Figure PCTCN2021133345-appb-000023
Figure PCTCN2021133345-appb-000023
注：“-”表示未进行该项检测，下同。Note: "-" indicates that this test has not been carried out, the same below.
(3)纯度(3) Purity
纯度(SEC-HPLC法)结果见表15。结果表明，在40±2℃条件下考察2周，不同pH值样品纯度均下降，pH 5.0、pH 5.5、pH 6.0、pH 6.5样品与0天比较纯度分别下降1.1％、1.2％、1.4％和1.9％。The results of the purity (SEC-HPLC method) are shown in Table 15. The results showed that after 2 weeks of investigation at 40±2°C, the purity of samples with different pH values decreased, and the purity of samples at pH 5.0, pH 5.5, pH 6.0, and pH 6.5 decreased by 1.1%, 1.2%, 1.4% and 1.4%, respectively, compared with 0 days. 1.9%.
纯度(非还原型CE-SDS法)结果见表16，结果表明，在40℃条件下考察2周，各组样品纯度(非还原型CE-SDS法)均未发生变化。The results of purity (non-reducing CE-SDS method) are shown in Table 16. The results show that the purity of each group of samples (non-reducing CE-SDS method) did not change after 2 weeks of investigation at 40°C.
表15.通过SEC-HPLC法对各样品测定的蛋白纯度结果(％)Table 15. Results (%) of protein purity by SEC-HPLC method for each sample
Figure PCTCN2021133345-appb-000024
Figure PCTCN2021133345-appb-000024
表16.通过非还原型CE-SDS法对各样品测定的纯度结果(％)Table 16. Purity results (%) of each sample measured by non-reducing CE-SDS method
Figure PCTCN2021133345-appb-000025
Figure PCTCN2021133345-appb-000025
(4)电荷变异体(4) Charge variants
电荷变异体(iCIEF法)结果见表17，其变化趋势见图18～20。结果表明，在40±2℃条件下考察2周，pH 5.0、pH 5.5、pH 6.0、pH 6.5样品与0天比较主成分分别下降9.5％、9.1％、8.7％和9.2％。The results of the charge variant (iCIEF method) are shown in Table 17, and the change trend is shown in Figures 18-20. The results showed that the main components of pH 5.0, pH 5.5, pH 6.0, and pH 6.5 samples decreased by 9.5%, 9.1%, 8.7% and 9.2%, respectively, compared with 0 days under the condition of 40±2℃ for 2 weeks.
表17通过iCIEF法测定的各样品的电荷变异体结果(％)Table 17 Charge variant results (%) of each sample determined by the iCIEF method
Figure PCTCN2021133345-appb-000026
Figure PCTCN2021133345-appb-000026
研究结果表明，antibody-A蛋白在pH 6.0较稳定，选定pH 6.0进行处方筛选实验。The results showed that the antibody-A protein was relatively stable at pH 6.0, and pH 6.0 was selected for the formulation screening experiment.
实施例7.制剂处方筛选实验Embodiment 7. Formulation formula screening experiment
1.实验步骤1. Experimental procedure
本实验主要考察依地酸二钠、山梨醇、盐酸精氨酸对antibody-A蛋白稳定性的影响，共设计了4个处方，详细处方信息见表18，详细实验条件及取样计划见表19。This experiment mainly investigated the effects of disodium edetate, sorbitol and arginine hydrochloride on the stability of antibody-A protein. A total of 4 prescriptions were designed. The detailed prescription information is shown in Table 18, and the detailed experimental conditions and sampling plan are shown in Table 19. .
按照表18配制各个处方的缓冲液，将antibody-A蛋白超滤置换至各自的处方溶液中。置 换完成后，调节各处方蛋白含量至25mg/ml；加入聚山梨酯80，使其终浓度为0.5mg/ml；过滤分装至西林瓶，加塞、轧盖。上述样品于40±2℃条件下进行稳定性考察。Prepare the buffers of each recipe according to Table 18, and replace the antibody-A protein into the respective recipe solutions by ultrafiltration. After the replacement is completed, adjust the protein content of each recipe to 25 mg/ml; add polysorbate 80 to make the final concentration 0.5 mg/ml; filter and dispense into vials, stopper and cap. The above samples were tested for stability at 40±2°C.
表18.备选处方信息表Table 18. Alternative Prescribing Information Sheet
Figure PCTCN2021133345-appb-000027
Figure PCTCN2021133345-appb-000027
注：％是指％w/v；用盐酸调节pH。Note: % refers to % w/v; adjust pH with hydrochloric acid.
表19.处方筛选实验方案Table 19. Prescription Screening Protocol
Figure PCTCN2021133345-appb-000028
Figure PCTCN2021133345-appb-000028
2.实验结果2. Experimental results
(1)外观和可见异物(1) Appearance and visible foreign matter
在40±2℃条件下放置4周，各处方样品外观、可见异物均合格。After being placed at 40±2℃ for 4 weeks, the appearance and visible foreign matter of all prescription samples were qualified.
(2)蛋白含量(2) protein content
蛋白含量结果见表20。结果表明，在40±2℃条件下放置4周，各处方样品蛋白含量均未发生变化。The protein content results are shown in Table 20. The results showed that the protein content of each prescription sample did not change after being placed at 40±2℃ for 4 weeks.
表20.处方筛选实验蛋白含量结果(UV法，mg/ml)Table 20. Results of protein content of prescription screening experiments (UV method, mg/ml)
Figure PCTCN2021133345-appb-000029
Figure PCTCN2021133345-appb-000029
(3)纯度(3) Purity
纯度(SEC-HPLC法)结果见表21。结果表明，在40±2℃条件下放置4周，制剂F1～F4纯度分别下降2.2％、2.3％、4.8％、3.2％。The purity (SEC-HPLC method) results are shown in Table 21. The results showed that the purity of preparations F1 to F4 decreased by 2.2%, 2.3%, 4.8% and 3.2% respectively after being placed at 40±2°C for 4 weeks.
纯度(非还原型CE-SDS法)结果见表22。结果表明，在40±2℃条件下放置4周，各处方下降趋势一致，制剂F2～F4发生明显变化。The results of the purity (non-reducing CE-SDS method) are shown in Table 22. The results showed that when placed at 40±2℃ for 4 weeks, the decreasing trend of all prescriptions was consistent, and the preparations F2-F4 changed significantly.
表21.处方筛选实验纯度结果(SEC-HPLC法，％)Table 21. Purity results of formulation screening experiments (SEC-HPLC method, %)
Figure PCTCN2021133345-appb-000030
Figure PCTCN2021133345-appb-000030
表22.处方筛选实验纯度结果(非还原型CE-SDS法，％)Table 22. Purity results of formulation screening experiments (non-reduced CE-SDS method, %)
Figure PCTCN2021133345-appb-000031
Figure PCTCN2021133345-appb-000031
(4)电荷变异体(4) Charge variants
电荷变异体(iCIEF法)结果见表23。电荷变异体变化趋势见图23和图24。结果表明，40±2℃条件下考察4周，各处方电荷变异体均发生明显变化。F1～F4酸性组分别增加9.9％、10.4％、8.8％和8.2％，主成分分别下降10.7％、11.3％、9.8％和9.8％。Charge variant (iCIEF method) results are shown in Table 23. Figure 23 and Figure 24 show the changing trends of charge variants. The results showed that after 4 weeks of investigation at 40±2℃, the charge variants of each recipe changed significantly. The F1-F4 acid groups increased by 9.9%, 10.4%, 8.8% and 8.2%, respectively, and the principal components decreased by 10.7%, 11.3%, 9.8% and 9.8%, respectively.
表23.处方筛选实验电荷变异体结果(iCIEF法，％)Table 23. Formulation Screening Experiment Charge Variant Results (iCIEF Method, %)
Figure PCTCN2021133345-appb-000032
Figure PCTCN2021133345-appb-000032
Figure PCTCN2021133345-appb-000033
Figure PCTCN2021133345-appb-000033
(1)F1与F2各项检测结果表明：处方中添加依地酸二钠对antibody-A蛋白稳定性获益有限；(2)山梨醇在antibody-A纯度维持上表现较优，添加盐酸精氨酸的处方在电荷变异体方面表现较优。因此，F4为antibody-A的较优处方。此外，为避免在生产过程中使用盐酸调节pH，将制剂处方中缓冲体系调整为组氨酸和盐酸组氨酸。(1) The test results of F1 and F2 showed that the addition of disodium edetate in the prescription had limited benefit on the stability of antibody-A protein; (2) Sorbitol performed better in maintaining the purity of antibody-A. The amino acid formulations outperformed the charge variants. Therefore, F4 is the preferred formulation of antibody-A. In addition, in order to avoid using hydrochloric acid to adjust pH in the production process, the buffer system in the formulation was adjusted to histidine and histidine hydrochloride.
通过上述研究结果，确定antibody-A的优选制剂处方(F5)：25.0mg/ml antibody-A双特异性抗体、0.79mg/ml组氨酸、1.03mg/ml盐酸组氨酸、16.85mg/ml盐酸精氨酸、25.00mg/ml山梨醇和0.50mg/ml聚山梨酯80，pH 6.0。Based on the above research results, the preferred formulation of antibody-A was determined (F5): 25.0mg/ml antibody-A bispecific antibody, 0.79mg/ml histidine, 1.03mg/ml histidine hydrochloride, 16.85mg/ml Arginine hydrochloride, 25.00 mg/ml sorbitol and 0.50 mg/ml polysorbate 80, pH 6.0.
以上描述了本发明的示例性实施方案，本领域技术人员应当理解的是，这些公开内容仅是示例性的，在本发明的范围内可以进行各种其它替换、适应和修改。因此，本发明不限于文中列举的具体实施方案。Exemplary embodiments of the present invention have been described above, and it should be understood by those skilled in the art that these disclosures are merely exemplary and various other substitutions, adaptations and modifications may be made within the scope of the present invention. Therefore, the invention is not limited to the specific embodiments recited herein.

Claims (15)

  1. 一种液体抗体制剂，其包含A liquid antibody preparation comprising
    (i)抗TIGIT/PD-1双特异性抗体蛋白；(i) anti-TIGIT/PD-1 bispecific antibody protein;
    (ii)缓冲剂，(ii) buffers,
    (iii)稳定剂，和(iii) stabilizers, and
    (iv)表面活性剂，(iv) surfactants,
    其中所述抗TIGIT/PD-1双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含特异性结合TIGIT(SEQ ID NO：31)或其胞外结构域(例如SEQ ID NO：32)的第一VH/VL单元并且第二半抗体包含特异性结合PD-1(SEQ ID NO：29)或其胞外结构域(例如SEQ ID NO：30)的第二VH/VL单元，其中所述第一VH/VL单元包含重链和轻链互补决定区(CDR)氨基酸序列SEQ ID NO：1-6；且第二VH/VL单元包含重链和轻链互补决定区(CDR)氨基酸序列SEQ ID NO：7-12，wherein the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half-antibody and a second half-antibody, wherein the first half-antibody comprises specifically binding TIGIT (SEQ ID NO: 31) or its extracellular domain (e.g. The first VH/VL unit of SEQ ID NO:32) and the second half antibody comprises a second VH that specifically binds PD-1 (SEQ ID NO:29) or its extracellular domain (eg, SEQ ID NO:30) /VL unit, wherein the first VH/VL unit comprises heavy and light chain complementarity determining region (CDR) amino acid sequences SEQ ID NOs: 1-6; and the second VH/VL unit comprises heavy and light chain complementarity determining regions Region (CDR) amino acid sequences SEQ ID NOs: 7-12,
    优选地所述抗体制剂的pH为约4.5-6.8，优选大约5.0-6.5，更优选约6.0的pH。Preferably the pH of the antibody formulation is about 4.5-6.8, preferably about 5.0-6.5, more preferably about 6.0 pH.
  2. 权利要求1所述的液体抗体制剂，其中所述第一VH/VL单元包含SEQ ID NO：13/SEQ ID NO：14的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列，并且其中所述第二VH/VL单元包含SEQ ID NO：17/SEQ ID NO：18的成对重链可变区序列/轻链可变区序列，或与所述成对重链可变区序列/轻链可变区序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更多序列同一性的序列；The liquid antibody formulation of claim 1, wherein the first VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 13/SEQ ID NO: 14, or with The paired heavy chain variable region sequence/light chain variable region sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more A sequence of multiple sequence identity, and wherein said second VH/VL unit comprises the paired heavy chain variable region sequence/light chain variable region sequence of SEQ ID NO: 17/SEQ ID NO: 18, or the Paired heavy chain variable region sequence/light chain variable region sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence sequence of identity;
    优选地，抗体的重链与抗体的轻链形成至少一个二硫键，抗体的两条重链形成至少一个二硫键；Preferably, the heavy chain of the antibody forms at least one disulfide bond with the light chain of the antibody, and the two heavy chains of the antibody form at least one disulfide bond;
    更优选地，抗体是工程化改造以降低抗体与Fcγ受体结合的人IgG1。More preferably, the antibody is human IgG1 engineered to reduce the binding of the antibody to Fcγ receptors.
  3. 权利要求1或2所述的液体抗体制剂，其中所述抗TIGIT/PD-1双特异性抗体蛋白包含第一半抗体和第二半抗体，其中第一半抗体包含SEQ ID NO：21的重链序列或与之具有至少90％，95％，98％或99％同一性的重链序列，和SEQ ID NO：22的轻链序列或与之具有至少90％，95％，98％或99％同一性的轻链序列，并且其中第二半抗体包含SEQ ID NO：23的重链序列或与之具有至少90％，95％，98％或99％同一性的重链序列，和SEQ ID NO：24的轻链序列或与之具有至少90％，95％，98％或99％同一性的轻链序列。The liquid antibody formulation of claim 1 or 2, wherein the anti-TIGIT/PD-1 bispecific antibody protein comprises a first half antibody and a second half antibody, wherein the first half antibody comprises the heavyweight of SEQ ID NO:21. chain sequence or a heavy chain sequence at least 90%, 95%, 98% or 99% identical to it, and a light chain sequence of SEQ ID NO: 22 or at least 90%, 95%, 98% or 99% identical thereto % identical light chain sequence, and wherein the second half antibody comprises or has a heavy chain sequence at least 90%, 95%, 98% or 99% identical to the heavy chain sequence of SEQ ID NO: 23, and SEQ ID NO: 23 The light chain sequence of NO: 24 or a light chain sequence having at least 90%, 95%, 98% or 99% identity therewith.
  4. 权利要求1-3中任何一项所述的液体抗体制剂，所述抗TIGIT/PD-1双特异性抗体蛋白是在HEK293细胞或以HEK293细胞为基础改造而得到的HEK293T、HEK293F、HEK293E细胞；CHO细胞或以CHO细胞为基础改造而得到的CHO-S、CHO-dhfr-、CHO/DG44、ExpiCHO中重组表达的抗TIGIT/PD-1双特异性抗体蛋白。The liquid antibody preparation according to any one of claims 1-3, wherein the anti-TIGIT/PD-1 bispecific antibody protein is HEK293T, HEK293F, and HEK293E cells transformed from HEK293 cells or based on HEK293 cells; Anti-TIGIT/PD-1 bispecific antibody protein recombinantly expressed in CHO cells or CHO-S, CHO-dhfr-, CHO/DG44, and ExpiCHO transformed from CHO cells.
  5. 权利要求1-4中任何一项所述的液体抗体制剂，其中所述的抗TIGIT/PD-1双特异性抗体蛋白的浓度为约1-200mg/ml，优选地为约1-150mg/ml，更优选地为约10-100mg/mL。The liquid antibody formulation of any one of claims 1-4, wherein the anti-TIGIT/PD-1 bispecific antibody protein has a concentration of about 1-200 mg/ml, preferably about 1-150 mg/ml , more preferably about 10-100 mg/mL.
  6. 权利要求1-5中任何一项所述的液体抗体制剂，其中所述液体制剂中的缓冲剂选自组 氨酸、盐酸组氨酸、谷氨酸盐、磷酸盐、乙酸盐、柠檬酸盐和三羟甲基氨基甲烷和它们的组合，优选组氨酸、盐酸组氨酸和它们的组合；优选地，所述液体抗体制剂中的缓冲剂的浓度为约0.5-200mM、约5-50mM或约5-20mM。The liquid antibody formulation of any one of claims 1-5, wherein the buffer in the liquid formulation is selected from the group consisting of histidine, histidine hydrochloride, glutamate, phosphate, acetate, citric acid salts and tris and combinations thereof, preferably histidine, histidine hydrochloride and combinations thereof; preferably, the concentration of the buffer in the liquid antibody formulation is about 0.5-200 mM, about 5- 50mM or about 5-20mM.
  7. 权利要求1-6中任何一项所述的液体抗体制剂，其中所述稳定剂选自糖类、多元醇、氨基酸或其盐及它们的组合，优选地，所述稳定剂选自：蔗糖、右旋糖、乳糖、麦芽糖、海藻糖、环糊精、麦芽糖糊精、葡聚糖、甘露醇、山梨醇、木糖醇、精氨酸、精氨酸盐(优选精氨酸盐酸盐)，更优选地，所述稳定剂是山梨醇、精氨酸、精氨酸盐或其组合；The liquid antibody preparation of any one of claims 1-6, wherein the stabilizer is selected from carbohydrates, polyols, amino acids or their salts and combinations thereof, preferably, the stabilizer is selected from: sucrose, Dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin, dextran, mannitol, sorbitol, xylitol, arginine, arginine salt (preferably arginine hydrochloride) , more preferably, the stabilizer is sorbitol, arginine, arginine salt or a combination thereof;
    优选地，所述稳定剂在本发明的液体制剂中以约1-1000mM、或约50-500的浓度存在。Preferably, the stabilizer is present in the liquid formulation of the present invention at a concentration of about 1-1000 mM, or about 50-500.
  8. 权利要求1-7中任何一项所述的液体抗体制剂，其中所述稳定剂是山梨醇、精氨酸盐酸盐或者山梨醇和精氨酸盐酸盐的组合。7. The liquid antibody formulation of any one of claims 1-7, wherein the stabilizer is sorbitol, arginine hydrochloride, or a combination of sorbitol and arginine hydrochloride.
  9. 权利要求1-8中任何一项所述的液体抗体制剂，其中所述表面活性剂选自聚山梨酯-20、聚山梨酯-80、聚山梨酯-60、或聚山梨酯-40；普洛尼克，优选为聚山梨酯-80；The liquid antibody formulation of any one of claims 1-8, wherein the surfactant is selected from polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; Lonic, preferably polysorbate-80;
    优选地，所述液体抗体制剂中的表面活性剂的浓度为约0.01-10mg/ml、约0.05-5、约0.1-5、或0.2-2mg/ml。Preferably, the concentration of surfactant in the liquid antibody formulation is about 0.01-10 mg/ml, about 0.05-5, about 0.1-5, or 0.2-2 mg/ml.
  10. 权利要求1-9中任何一项所述的液体抗体制剂，其中所述液体抗体制剂注射剂，更优选为皮下注射剂、静脉内注射剂或静脉输注剂。The liquid antibody preparation according to any one of claims 1 to 9, wherein the liquid antibody preparation for injection is more preferably a subcutaneous injection, an intravenous injection or an intravenous infusion.
  11. 权利要求1-10中任何一项所述的液体抗体制剂，其中所述液体抗体制剂包含：The liquid antibody formulation of any one of claims 1-10, wherein the liquid antibody formulation comprises:
    (i)约1-150mg/ml的抗TIGIT/PD-1双特异性抗体蛋白；(i) about 1-150 mg/ml of anti-TIGIT/PD-1 bispecific antibody protein;
    (ii)约5-50mM的组氨酸和/或盐酸组氨酸；(ii) about 5-50 mM histidine and/or histidine hydrochloride;
    (iii)约50-500mM的山梨醇；和/或约1-600mM的精氨酸或盐酸精氨酸；和(iii) about 50-500 mM sorbitol; and/or about 1-600 mM arginine or arginine hydrochloride; and
    (iv)约0.05-2mg/ml聚山梨醇酯80；(iv) about 0.05-2 mg/ml polysorbate 80;
    其中所述液体制剂的pH为约5.0-6.5，优选地约6.0；或者wherein the pH of the liquid formulation is about 5.0-6.5, preferably about 6.0; or
    所述液体抗体制剂包含：The liquid antibody formulation comprises:
    (i)约10-100mg/ml的抗TIGIT/PD-1双特异性抗体蛋白；(i) about 10-100 mg/ml of anti-TIGIT/PD-1 bispecific antibody protein;
    (ii)约5-20mM的组氨酸和/或盐酸组氨酸；(ii) about 5-20 mM histidine and/or histidine hydrochloride;
    (iii)约100-300mM的山梨醇；和/或约50-200mM的精氨酸或盐酸精氨酸；和(iii) about 100-300 mM sorbitol; and/or about 50-200 mM arginine or arginine hydrochloride; and
    (iv)约0.2-2mg/ml聚山梨醇酯80；(iv) about 0.2-2 mg/ml polysorbate 80;
    其中所述液体制剂的pH为约5.0-6.5，优选地约6.0；或者wherein the pH of the liquid formulation is about 5.0-6.5, preferably about 6.0; or
    所述液体抗体制剂包含：The liquid antibody formulation comprises:
    (i)约25mg/ml的抗TIGIT/PD-1双特异性抗体蛋白；(i) about 25 mg/ml of anti-TIGIT/PD-1 bispecific antibody protein;
    (ii)约5mM的组氨酸和约5mM盐酸组氨酸；或者约10mM的组氨酸；(ii) about 5 mM histidine and about 5 mM histidine hydrochloride; or about 10 mM histidine;
    (iii)约25mg/ml的山梨醇；和约80mM的精氨酸或盐酸精氨酸；和(iii) about 25 mg/ml of sorbitol; and about 80 mM of arginine or arginine hydrochloride; and
    (iv)约0.5mg/ml聚山梨醇酯80；(iv) about 0.5 mg/ml polysorbate 80;
    其中所述液体制剂的pH为约5.0-6.5，优选地约6.0。wherein the pH of the liquid formulation is about 5.0-6.5, preferably about 6.0.
  12. 一种固体抗体制剂，其是通过将权利要求1-11中任何一项所述的液体抗体制剂经固化处理而获得的；所述固体抗体制剂例如是冻干粉针剂形式。A solid antibody preparation obtained by subjecting the liquid antibody preparation of any one of claims 1 to 11 to solidification treatment; the solid antibody preparation is, for example, in the form of a lyophilized powder for injection.
  13. 一种递送装置，其包含权利要求1-11中任何一项所述的液体抗体制剂或权利要求12所述的固体抗体制剂。A delivery device comprising the liquid antibody formulation of any one of claims 1-11 or the solid antibody formulation of claim 12.
  14. 权利要求13的递送装置，其是预填充注射器形式的，用于静脉内、皮下、皮内或者肌内注射、静脉内输注。14. The delivery device of claim 13 in the form of a prefilled syringe for intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion.
  15. 根据权利要求1-11中任何一项的液体抗体制剂或权利要求12的固体抗体制剂用于制备在受试者中治疗、预防或延缓与TIGIT信号传导通路和PD-1信号传导通路相关的病症的递送装置(如，预填充注射器)或药物的用途，所述病症例如各种血液病和实体瘤，包括但不限于白血病、淋巴瘤、骨髓瘤、脑肿瘤、头颈癌、头颈部鳞状细胞癌、肺癌例如非小细胞肺癌或小细胞肺癌、鼻咽癌、食道癌、胃癌、胰腺癌、胆囊癌、胆管癌、肝癌、结直肠癌、乳腺癌、卵巢癌、***、子宫内膜癌、子宫肉瘤、***癌、膀胱癌、肾细胞癌、黑色素瘤。A liquid antibody formulation according to any one of claims 1-11 or a solid antibody formulation according to claim 12 for the preparation of a treatment, prevention or delay in a subject of a disorder associated with the TIGIT signaling pathway and the PD-1 signaling pathway Use of a delivery device (eg, a prefilled syringe) or a drug for conditions such as various hematological diseases and solid tumors, including but not limited to leukemia, lymphoma, myeloma, brain tumor, head and neck cancer, head and neck squamous Cell cancer, lung cancer such as non-small cell lung cancer or small cell lung cancer, nasopharyngeal cancer, esophagus cancer, stomach cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, liver cancer, colorectal cancer, breast cancer, ovarian cancer, cervical cancer, endometrium carcinoma, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma, melanoma.
PCT/CN2021/133345 2020-11-26 2021-11-26 Preparation comprising anti-tigit/pd-1 bispecific antibody, method for preparing same, and use thereof WO2022111612A1 (en)

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