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WO2022111612A1 - Préparation comprenant un anticorps bispécifique anti-tigit/pd-1, son procédé de préparation et son utilisation - Google Patents

Préparation comprenant un anticorps bispécifique anti-tigit/pd-1, son procédé de préparation et son utilisation 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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Prior art keywords
antibody
tigit
formulation
liquid
cancer
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PCT/CN2021/133345
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English (en)
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/fr

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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.

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Abstract

La présente invention concerne une préparation comprenant un anticorps bispécifique anti-TIGIT/PD-1, et en particulier une préparation pharmaceutique comprenant un anticorps bispécifique anti-TIGIT/PD-1, un agent tampon, un agent stabilisant et un tensioactif. La présente invention se rapporte en outre à une utilisation de ces préparations dans le traitement ou la prévention de maladies.
PCT/CN2021/133345 2020-11-26 2021-11-26 Préparation comprenant un anticorps bispécifique anti-tigit/pd-1, son procédé de préparation et son utilisation WO2022111612A1 (fr)

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