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WO2019206281A1 - Formulation d'anticorps monoclonal contre la pd-1 humaine, médicament d'association, et utilisation de ces derniers - Google Patents

Formulation d'anticorps monoclonal contre la pd-1 humaine, médicament d'association, et utilisation de ces derniers Download PDF

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Publication number
WO2019206281A1
WO2019206281A1 PCT/CN2019/084558 CN2019084558W WO2019206281A1 WO 2019206281 A1 WO2019206281 A1 WO 2019206281A1 CN 2019084558 W CN2019084558 W CN 2019084558W WO 2019206281 A1 WO2019206281 A1 WO 2019206281A1
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cancer
polysorbate
histidine
sucrose
monoclonal antibody
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English (en)
Chinese (zh)
Inventor
周兵兵
孙丽霞
张乐
王克波
李敏昱
王庆民
吴晓冉
曹传增
郑庆梅
赵春媛
赵洪令
张美娟
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Qilu Pharmaceutical Co Ltd
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Qilu Pharmaceutical Co Ltd
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Priority to CN201980025962.3A priority Critical patent/CN111971062B/zh
Publication of WO2019206281A1 publication Critical patent/WO2019206281A1/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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the invention relates to a pharmaceutical preparation, a combined medicine and a use thereof for a stable monoclonal antibody against human PD-1, and belongs to the field of biotechnology.
  • tumor immunotherapy is to maximize the patient's own immune system response to the tumor. It not only activates the original immune system response in the body, but also maintains the duration of the immune system response and the intensity of the response. The key to cancer.
  • PD-1 Programmed death-1
  • CD279 is essential for regulating the balance between stimulatory and inhibitory signals in the immune system and maintaining peripheral tolerance.
  • the structure of PD-1 is a monomeric type I transmembrane protein that is transformed by an immunoglobulin variable region-like extracellular domain and an immunoreceptor tyrosine inhibition motif (ITIM) and an immunoreceptor tyrosine.
  • ITIM immunoreceptor tyrosine inhibition motif
  • ITM immunoreceptor tyrosine
  • PD-1 is mainly expressed on activated T cells, B cells and myeloid cells.
  • PD-1 has two known ligands: PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273), which are members of the cell surface expression of the B7 family. These two ligands have been identified to specifically interact with PD-1 to induce intracellular signal transduction, which inhibits CD3 and CD28-mediated T cell activation, which in turn impairs T cell activity, for example, Reduce cell proliferation, IL-2 and IFN- ⁇ secretion, and other growth factors and cytokine secretion. Therefore, blocking the binding between PD-L1/PD-1 has become a drug target of great interest in the field of tumor immunotherapy.
  • the anti-PD-1 antibody can specifically bind to PD-1 and block its interaction with the receptor, thereby cutting off the binding inhibition of PD-1 on the surface of tumor cells and PD-1 on T cells for anticancer purposes.
  • the combination of anti-PD-1 antibodies with other therapeutic approaches has also made breakthroughs in clinical practice.
  • Other treatments include radiation therapy, chemotherapy, or other immunological checkpoints other than PD-1 (such as CTLA-4).
  • the inventors obtained a monoclonal antibody (monoclonal antibody) against human PD-1 which is more effective than BMS's Nivolumab (trade name Opdivo) by library screening, and named ZMR01.
  • ZMR01 a monoclonal antibody against human PD-1 which is more effective than BMS's Nivolumab (trade name Opdivo) by library screening.
  • ZMR01 a monoclonal antibody against human PD-1 which is more effective than BMS's Nivolumab (trade name Opdivo) by library screening.
  • ZMR01 monoclonal antibody
  • histidine-acetate buffer had a significant effect on preventing the aggregation and degradation of monoclonal antibody ZMR01.
  • polysorbate was also found.
  • Ester 20 is used as a cosolvent in pharmaceutical preparations, which has a significant benefit in improving the solubility of the drug, enhancing the pharmacological action of the drug or reducing side effects; in addition, it has been unexpectedly found that the pH range of the monoclonal antibody ZMR01 is relatively stable from 4.5 to 5.5.
  • the present invention provides a solution preparation suitable for a specific sequence of a monoclonal antibody against human PD-1 (such as ZMR01) and capable of stably storing the monoclonal antibody, which is capable of sufficiently preventing the monoclonal antibody ZMR01 protein Aggregation, degradation, oxidation or denaturation, etc., in order to maintain the biological activity of its active components, suitable for clinical use.
  • the pharmacological function of the preparation was deeply studied, and the preparation was found to have good antitumor activity, and the preparation was combined with other therapeutic agents, especially with The combination of anti-VEGF monoclonal antibody has a better anti-tumor effect than the use of the preparation alone.
  • a monoclonal antibody e.g., ZMR01
  • the anti-human PD-1 mAb used in the present invention is described herein by taking ZMR01 as an example. Unless otherwise indicated in the examples or otherwise indicated, reference to ZMR01 refers to an anti-human PD-1 monoclonal antibody having a specific sequence used in the present invention.
  • the stable solution formulation of the invention comprises a monoclonal antibody ZMR01 or an antigen binding fragment thereof and a buffer.
  • the solution formulation may also contain stabilizers and/or surfactants.
  • the antibody heavy chain variable region HCDR sequence of the monoclonal antibody ZMR01 or antigen-binding fragment thereof is: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3;
  • the antibody light chain variable region LCDR sequence is: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6.
  • the amino acid sequences are shown in the following table:
  • the monoclonal antibody ZMR01 has the heavy chain variable region amino acid sequence of SEQ ID NO: 7 and the light chain variable region amino acid sequence of SEQ ID NO: 8.
  • the monoclonal antibody ZMR01 has the heavy chain amino acid sequence of SEQ ID NO: 9 and the light chain amino acid sequence of SEQ ID NO: 10.
  • the concentration of the monoclonal antibody ZMR01 in the formulation is preferably 20-30 mg/ml, most preferably 25 mg/ml.
  • the buffering agent of the present invention is selected from the group consisting of a histidine-hydrochloric acid buffer, an acetic acid-sodium acetate buffer, a histidine-acetate buffer; a further preferred pharmaceutically acceptable buffer is a histidine-acetate buffer.
  • concentration of the buffer is 5-20 mM, most preferably 10 mM.
  • the solution formulation of the present invention has a pH in the range of 4.5 to 6.0, preferably a pH in the range of 4.5 to 5.5, most preferably pH 5.2.
  • the stabilizer of the present invention is sucrose or mannitol or trehalose, preferably sucrose, at a concentration of 70-90 mg/ml, most preferably 90 mg/ml.
  • the surfactant of the present invention is polysorbate 20 or polysorbate 80 at a concentration of 0.1-0.5 mg/ml, most preferably polysorbate 20 at a concentration of 0.2 mg/ml.
  • the stable solution formulation of the present invention is an injectable pharmaceutical formulation.
  • the stable solution formulation comprises monoclonal antibody ZMR01 or an antigen binding fragment thereof, a buffer, sucrose, and a surfactant, optionally including water.
  • the stable solution formulation comprises monoclonal antibody ZMR01 or an antigen binding fragment thereof, a buffer, sucrose, and polysorbate 20, optionally including water.
  • the stable solution formulation comprises monoclonal antibody ZMR01 or an antigen binding fragment thereof, histidine-acetate buffer, sucrose, and polysorbate 20.
  • the stable solution formulation consists of 20-30 mg/ml monoclonal antibody ZMR01 or an antigen binding fragment thereof, 5-20 mM histidine-acetate buffer, 70-90 mg/ml sucrose, 0.1- 0.5 mg/ml polysorbate 20 composition with a pH range of 4.5-5.5.
  • the stable solution formulation comprises:
  • the pH ranges of the above (1) to (10) preparations were all 4.5 to 5.5.
  • the solution formulation of the present invention can effectively inhibit aggregation and deamidation of antibodies, thereby preventing degradation of the antibody product therein, and obtaining a stable injection composition. Moreover, the solution formulation of the present invention has a protective effect on oxidative degradation of proteins, and is also compatible with glass and stainless steel containers, and is also stable in these containers.
  • the present invention also provides a lyophilized preparation obtained by lyophilization of the above solution preparation, or after the lyophilized preparation is reconstituted, the above-mentioned solution preparation is obtained.
  • the invention also provides a method of preparing a solution formulation of the stabilized ZMR01 monoclonal antibody, comprising the steps of:
  • sucrose and polysorbate 20 adding sucrose and polysorbate 20 to the prepared solution, so that the sucrose concentration in the solution reaches 70-90 mg / ml, the concentration of polysorbate 20 reaches 0.1-0.5 mg / ml;
  • the monoclonal antibody ZMR01 was added to the obtained solution to a concentration of 20-30 mg/ml.
  • the invention also provides a combination medicament comprising a solution formulation of a ZMR01 monoclonal antibody and at least one additional therapeutic agent.
  • the combination drug of the present invention, the solution preparation of the ZMR01 monoclonal antibody and the at least one additional therapeutic agent may be mixed together to form a single administration unit, or may be separately used as a administration unit, respectively.
  • Additional therapeutic agents in the combination of the invention may, for example, be selected from inhibitors against the following targets: A2AR, CTLA4, PD-L1, TIGIT, CCR4, CCR8, CSF1R1a, B7H3, B7H4, CD47, CD96, CD73 Claudin 18.2, VEGF, VEGFR, EGFR, FGFR, Her2, IAP, LAG3, STING, TNF- ⁇ , VISTA.
  • Additional therapeutic agents in the combination of the invention may, for example, be selected from agonists directed to the following targets: GITR, 41BB, OX40, CD40, ICOS.
  • Additional therapeutic agents in the combination of the invention may, for example, be selected from the group consisting of IDO inhibitors, TDO inhibitors, and IAP inhibitors.
  • An additional therapeutic agent in the combination of the invention may be, for example, an anti-VEGF antibody, preferably bevacizumab.
  • the invention also provides a kit comprising a solution formulation of a ZMR01 monoclonal antibody or a solution formulation comprising a ZMR01 monoclonal antibody and at least one additional therapeutic agent.
  • Additional therapeutic agents in the kit of the invention may, for example, be selected from inhibitors against the following targets: A2AR, CTLA4, PD-L1, TIGIT, CCR4, CCR8, CSF1R1a, B7H3, B7H4, CD47, CD96, CD73, Claudin 18.2, VEGF, VEGFR, EGFR, FGFR, Her2, IAP, LAG3, STING, TNF- ⁇ , VISTA.
  • Additional therapeutic agents in the kits of the invention may, for example, be selected from agonists directed to the following targets: GITR, 41BB, OX40, CD40, ICOS.
  • Additional therapeutic agents in the kits of the invention may, for example, be selected from the group consisting of IDO inhibitors, TDO inhibitors, and IAP inhibitors.
  • the additional therapeutic agent in the kit of the invention may, for example, be an anti-VEGF antibody, preferably bevacizumab.
  • the present invention also provides the use of the solution preparation or the combination drug or the kit for the preparation of a tumor for preventing and/or treating a PD-1 mediated disease or expressing PD-L1.
  • the tumor may be selected, for example, from lung cancer, gastric cancer, melanoma, renal cancer, breast cancer, intestinal cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, pancreatic cancer, head and neck cancer.
  • the present invention also provides a method of treatment for preventing or treating a PD-1 mediated disease or condition in a subject, preferably a tumor; more preferably a tumor expressing PD-L1;
  • the tumor is preferably lung cancer, gastric cancer, melanoma, kidney cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, pancreatic cancer, head and neck cancer; most preferably non-small cell lung cancer, melanoma and Renal cancer, the method comprising administering to a subject a solution formulation of the invention or the combination drug or kit.
  • a solution preparation of a monoclonal antibody against human PD-1 comprising a monoclonal antibody against human PD-1 or an antigen-binding fragment thereof and a buffer thereof, wherein the monoclonal antibody against human PD-1
  • the amino acid sequences of the three CDRs of the heavy chain variable region of the antigen-binding fragment thereof are: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3
  • the amino acid sequences of the three CDRs of the variable region ie, LCDR1, LCDR2, and LCDR3
  • the buffer is selected from the group consisting of histidine-hydrochloric acid, Acetic acid-acetate (such as sodium acetate) or histidine-acetic acid.
  • Item 5 The solution formulation of any of items 1-4, further comprising a stabilizer.
  • Item 6 The solution formulation of any of items 1-5, further comprising a stabilizer selected from the group consisting of sucrose, mannitol, and trehalose, preferably the stabilizer is sucrose.
  • Item 7 The solution formulation of any of items 1-6, further comprising a surfactant.
  • Item 8 The solution formulation of any of items 1-7, further comprising a surfactant selected from the group consisting of polysorbate 20 and polysorbate 80, preferably polysorbate 20.
  • Item 9 The solution formulation of any of items 1-8, having a pH of from 4.5 to 6.0, preferably from 4.5 to 5.5, such as 5.2.
  • Item 10 The solution formulation of any of items 1-9, which comprises any two or three selected from the group consisting of sucrose, histidine-acetate buffer, and polysorbate 20.
  • Item 11 The solution formulation of any of items 1-10, which comprises 20-30 mg/ml of a monoclonal antibody against human PD-1.
  • Item 12 The solution formulation of any of items 1-11, which comprises 70-90 mg/ml of sucrose.
  • Item 13 The solution formulation of any of items 1-12, comprising 5-20 mM histidine-acetate buffer.
  • Item 14 The solution formulation of any of items 1-13, which comprises 0.1-0.5 mg/ml of polysorbate 20.
  • Item 15 The solution formulation of any of items 1-14, comprising 20-30 mg/ml of monoclonal antibody against human PD-1, 70-90 mg/ml of sucrose, 5-20 mM histidine- Acetate buffer, 0.1-0.5 mg/ml of polysorbate 20, the pH of the solution is 4.5-5.5.
  • Item 16 The solution formulation of item 1-15, which comprises a monoclonal antibody against human PD-1 and
  • the pH of the formulation ranges from 4.5 to 5.5.
  • Item 17 The solution formulation of item 16, wherein the concentration of the monoclonal antibody against human PD-1 is 20-30 mg/ml.
  • Item 18 A lyophilized preparation obtained by lyophilizing the solution preparation according to any one of Items 1 to 17, or the solution preparation of any one of Items 1 to 17 after the lyophilized preparation is reconstituted.
  • Item 19 The method of preparing a solution formulation of a monoclonal antibody against human PD-1 according to any one of items 1-17, comprising:
  • sucrose and polysorbate 20 added to the prepared buffer to make the sucrose concentration in the solution reach 70-90 mg / ml, the concentration of polysorbate 20 reaches 0.1-0.5 mg / ml;
  • Item 20 A combination medicament comprising the formulation of any of items 1-18 and at least one additional therapeutic agent.
  • Item 21 The combination drug of item 20, wherein the additional therapeutic agent is an inhibitor against a target selected from the group consisting of: A2AR, CTLA4, PD-L1, TIGIT, CCR4, CCR8 CSF1R1a, B7H3, B7H4, CD47, CD96, CD73, Claudin 18.2, VEGF, VEGFR, EGFR, FGFR, Her2, IAP, LAG3, STING, TNF- ⁇ and VISTA.
  • a target selected from the group consisting of: A2AR, CTLA4, PD-L1, TIGIT, CCR4, CCR8 CSF1R1a, B7H3, B7H4, CD47, CD96, CD73, Claudin 18.2, VEGF, VEGFR, EGFR, FGFR, Her2, IAP, LAG3, STING, TNF- ⁇ and VISTA.
  • Item 22 The combination of claim 20, wherein the additional therapeutic agent is an agonist directed to: GITR, 41BB, OX40, CD40 and ICOS.
  • Item 23 The combination of claim 20, wherein the additional therapeutic agent is selected from the group consisting of an IDO inhibitor, a TDO inhibitor, an IAP inhibitor, and an anti-VEGF antibody, for example, the anti-VEGF antibody is preferably bevacizumab .
  • Item 24 A kit comprising the formulation of any of items 1-18 or the combination of any of items 20-23.
  • Item 25 The kit of item 24, further comprising an additional therapeutic agent.
  • Item 26 The kit of item 25, wherein the additional therapeutic agent is an inhibitor against a target selected from the group consisting of: A2AR, CTLA4, PD-L1, TIGIT, CCR4, CCR8, CSF1R1a, B7H3, B7H4, CD47, CD96, CD73, Claudin 18.2, VEGF, VEGFR, EGFR, FGFR, Her2, IAP, LAG3, STING, TNF- ⁇ and VISTA.
  • a target selected from the group consisting of: A2AR, CTLA4, PD-L1, TIGIT, CCR4, CCR8, CSF1R1a, B7H3, B7H4, CD47, CD96, CD73, Claudin 18.2, VEGF, VEGFR, EGFR, FGFR, Her2, IAP, LAG3, STING, TNF- ⁇ and VISTA.
  • Item 27 The kit of item 25, wherein the additional therapeutic agent is an agonist directed to a target selected from the group consisting of: GITR, 41BB, OX40, CD40, and ICOS.
  • Item 28 The kit of item 25, wherein the additional therapeutic agent is selected from the group consisting of an IDO inhibitor, a TDO inhibitor, an IAP inhibitor, and an anti-VEGF antibody, for example, the anti-VEGF antibody is bevacizumab.
  • Item 29 The preparation of the monoclonal antibody against human PD-1 according to any one of Items 1 to 18, or the combination drug according to any one of Items 20 to 23 or the kit according to any one of Items 24-28, Use in the preparation of a medicament for the prevention and/or treatment of a PD-1 mediated disease or a tumor expressing PD-L1.
  • Item 30 The use of item 29, wherein the tumor is selected from the group consisting of lung cancer, gastric cancer, melanoma, kidney cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, pancreatic cancer, and Head and neck tumor; preferably selected from the group consisting of non-small cell lung cancer, melanoma, and kidney cancer.
  • Item 31 A method for preventing and/or treating a PD-1 mediated disease or condition in a subject, comprising administering to the subject a formulation of the invention or the combination or the drug box.
  • the disease or condition is preferably a tumor; more preferably a tumor expressing PD-L1; the tumor is preferably selected from the group consisting of lung cancer, gastric cancer, melanoma, kidney cancer, breast cancer, colon cancer, liver cancer, ovarian cancer, cervix Cancer, bladder cancer, esophageal cancer, pancreatic cancer, and head and neck cancer; most preferably selected from the group consisting of non-small cell lung cancer, melanoma, and kidney cancer.
  • Figure 1 Affinity determination of ZMR01 and PD-1 antigen combined with dissociation map.
  • Figure 2 Affinity determination of Nivolumab with PD-1 antigen in combination with dissociation map.
  • Figure 4 Effect of ZMR01 formulation on SEB-activated PBMC cytokine IL-2 release.
  • Figure 7 shows the MC38 tumor growth curve.
  • Figure 8 A431 tumor growth curve.
  • anti-human PD-1 refers to an antibody that recognizes and binds to a PD-1 molecule derived from a human.
  • antibody generally refers to a Y-type tetramer comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by a covalent disulfide bond and a non-covalent interaction. protein.
  • Natural IgG antibodies have such a structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH) and a constant region.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin G
  • IgG immunoglobulin G
  • IgM immunoglobulin M
  • the corresponding heavy chain constant domains are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively, and IgG and IgA can be further divided into different Subclasses such as IgG can be classified into IgG1, IgG2, IgG3, IgG4, and IgA can be classified into IgA1 and IgA2.
  • the light chain of an antibody from any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of its constant domain.
  • this constant region comprises three domains called CH1, CH2 and CH3 (IgM and IgE have a fourth domain CH4).
  • the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable length proline- and cysteine-rich segment.
  • Each type of antibody further comprises an interchain and intrachain disulfide bond formed by a paired cysteine residue.
  • variable region or “variable domain” shows a significant change in the amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding.
  • the variable region of each light/heavy chain pair forms an antibody binding site such that the intact IgG antibody has two binding sites (ie, it is bivalent).
  • the variable region (VH) of the heavy chain and the variable region (VL) domain of the light chain each comprise three regions of extreme variability, referred to as hypervariable regions (HVRs), or more commonly, referred to as The complementarity determining region (CDR), VH and VL each have four skeleton regions FR, which are represented by FR1, FR2, FR3, and FR4, respectively.
  • CDR and FR sequences typically appear in the following sequences of the heavy chain variable domain (or light chain variable domain): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (LCDR2)-FR3-HCDR3 (LCDR3)- FR4.
  • Fc is used to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • antibody can include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies (CDR- Grafted antibody), human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies Including mutant proteins and their variants) and so on.
  • monoclonal antibody refers to an antibody that is substantially homogeneous, produced by a single cell clone, directed only to a particular antigenic epitope.
  • Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant techniques, phage display technology, transgenic animals, synthetic techniques, or combinations of the above.
  • antibody fragment encompasses at least a portion of an intact antibody.
  • a “fragment” of an antibody molecule includes an "antigen-binding fragment” of an antibody, and the term “antigen-binding fragment” refers to an immunoglobulin or antibody that specifically binds to a selected antigen or an immunogenic determining portion thereof. Or a polypeptide fragment of the reaction, or a fusion protein product further derived therefrom, such as a single chain antibody, an extracellular binding region in a chimeric antigen receptor, and the like.
  • Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to, variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single domain antibodies, linear An antibody, a single-chain antibody (scFv), a bispecific antibody or a multispecific antibody formed from an antibody fragment, and the like.
  • an antigen refers to a substance that is recognized and specifically bound by an antibody or antibody-binding fragment.
  • an antigen can include any immunogenic fragment or determinant of a selected target, including single-epitope, multi-epitope, single-structure. Domain, multidomain, intact extracellular domain (ECD) or protein. Peptides, proteins, glycoproteins, polysaccharides and lipids, part of which and combinations thereof, can constitute an antigen.
  • Non-limiting exemplary antigens include tumor antigens or pathogen antigens and the like.
  • Antigen can also refer to a molecule that elicits an immune response.
  • antigen or cell or preparation containing the antigen can be used to generate antibodies specific for the antigenic determinant.
  • the antigen may be an isolated full length protein, a cell surface protein (eg, immunized with cells expressing at least a portion of the antigen on its surface), or a soluble protein (eg, immunized only with the ECD portion of the protein) or protein. Construct (eg, Fc antigen).
  • the antigen can be produced in genetically modified cells. Any of the foregoing antigens may be used alone or in combination with one or more immunogenic enhancing adjuvants known in the art.
  • the DNA encoding the antigen can be genomic or non-genomic (eg, cDNA) and can encode at least a portion of the ECD sufficient to elicit an immunogenic response.
  • Any vector can be used to transform a cell in which the antigen is expressed, including but not limited to an adenoviral vector, a lentiviral vector, a plasmid, and a non-viral vector such as a cationic lipid.
  • epitope refers to a site on an antigen that specifically binds to an immunoglobulin or antibody.
  • An epitope can be formed by an adjacent amino acid, or a non-adjacent amino acid juxtaposed by a tertiary folding of a protein. Epitopes formed from adjacent amino acids are typically retained after exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost after treatment with denaturing solvents. Epitopes usually exist in a unique spatial conformation and include at least 3-15 amino acids. Methods for determining the epitope to which it binds from a given antibody are well known in the art, including immunoblotting and immunoprecipitation assays, and the like. Methods for determining the spatial conformation of an epitope include techniques in the art, such as X-ray crystallography and two-dimensional nuclear magnetic resonance.
  • binding affinity refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen).
  • KD refers to the dissociation constant of a particular antibody-antigen interaction. Binding affinities can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical overspeed Centrifugation and flow cytometry.
  • biological activity refers to the ability of an antibody to bind to an antigen and result in a measurable biological response, which can be measured in vitro or in vivo.
  • pharmaceutical formulation or “formulation” or “prescription formulation” means an article that is present in a form that allows the biological activity of the active ingredient to be effective and does not contain other components that are toxic to the subject to which the formulation is to be administered. .
  • solution formulation means a formulation that is liquid at a temperature of at least about 2 ° C to about 8 ° C at atmospheric pressure.
  • deamidation means that one or more asparagine residues in an antibody have been derivatized, for example, aspartic acid or iso-aspartic acid.
  • aggregated antibody is an antibody that has been found to aggregate with other antibody molecules, particularly after freezing and/or agitation.
  • stable formulation is a formulation in which the protein retains substantially its physical stability and/or chemical stability and/or biological activity upon storage. Preferably, the formulation substantially retains its physical and chemical stability, as well as its biological activity, after storage.
  • the shelf life is generally chosen based on the shelf life of the formulation.
  • Various analytical techniques for measuring protein stability are known in the art. The stability can be measured for a selected time at the selected temperature.
  • Stability can be assessed qualitatively and/or quantitatively in a number of different ways, including assessment of aggregate formation (eg, by size exclusion chromatography, by measurement of turbidity, and/or by visual inspection); by using cation exchange chromatography Or capillary partition electrophoresis to assess charge heterogeneity; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibodies; peptide mapping; assessment of biological activity or antigen binding function of antibodies; and many more.
  • Instabilities may include any one or more of the following: aggregation, deamidation (eg, Asn deamidation), oxidation (eg, Met oxidation), isomerization (eg, Asp isomerization), shear Cut/hydrolysis/fragmentation (eg, hinge region fragmentation), succinimide formation, unpaired cysteine, N-terminal extension, C-terminal processing, differences in glycosylation, and the like.
  • deamidation eg, Asn deamidation
  • oxidation eg, Met oxidation
  • isomerization eg, Asp isomerization
  • shear Cut/hydrolysis/fragmentation eg, hinge region fragmentation
  • succinimide formation unpaired cysteine, N-terminal extension, C-terminal processing, differences in glycosylation, and the like.
  • buffer refers to a pharmaceutically acceptable excipient that stabilizes the pH of the pharmaceutical formulation.
  • Suitable buffering agents are well known in the art and can be found in the literature.
  • Preferred pharmaceutically acceptable buffers include, but are not limited to, histidine buffer, citrate buffer, succinate buffer, acetate buffer, arginine buffer, phosphate buffer or Mixtures and so on.
  • the pH of the buffer is adjusted with an acid or base known in the art, and the pH can be adjusted to a value in the range of 4.5 to 6.0, in particular to a value in the range of 4.5 to 5.5, most particularly to pH 5. 2.
  • stabilizer means a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and application.
  • Stabilizers include, but are not limited to, sugars, amino acids, polyols, cyclodextrins, and the like.
  • surfactant means a pharmaceutically acceptable excipient for protecting a protein preparation against physical stress such as agitation and shear.
  • Pharmaceutically acceptable surfactants include: polyoxyethylene sorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (such as those sold under the trademark BrijTM), and polyoxyethylene-polyoxypropylene copolymers. (Polosham, Pluronic).
  • Polyoxyethylene sorbitan-fatty acid esters include polysorbate 20 (sold under the trademark Tween 20TM) and polysorbate 80 (sold under the trademark Tween 80TM).
  • combination drug refers to a combination comprising two or more pharmaceutical preparations each having an active ingredient, which are required to be used in combination when administered to a subject.
  • the active ingredients may be mixed together to form a single administration unit, or may be separately used as a administration unit, respectively.
  • an effective amount refers to a dose of a pharmaceutical formulation of an antibody or fragment of the invention which, after administration to a patient in a single or multiple doses, produces the desired effect in the patient being treated.
  • An effective amount can be readily determined by an attending physician as a person skilled in the art by considering various factors such as ethnic differences; body weight, age, and health status; specific diseases involved; severity of the disease; response of the individual patient; Specific antibodies administered; mode of administration; bioavailability characteristics of the administered formulation; selected dosing regimen; and use of any concomitant therapy.
  • kit includes an effective amount of a pharmaceutical formulation or a combination of the invention in one or more unit dosage forms.
  • the kit may contain a sterile container of a therapeutic or prophylactic composition; such a container may be a cartridge, ampule, bottle, vial, tube, bag, blister pack, or other suitable as is known in the art.
  • Container form Such containers may be made of plastic, glass, laminated paper, metal foil or other materials suitable for holding the drug.
  • the kit further includes instructions for administering the pharmaceutical preparation or the combination medicament of the present invention to an individual.
  • the specification generally includes a method of using the pharmaceutical preparation of the present invention or a combination of drugs to treat or prevent a disease.
  • the engineered antibodies or antigen-binding fragments thereof of the invention can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified and collected by a conventional technique.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the term "individual” or “subject” as used herein refers to any animal, such as a mammal or marsupial. Individuals of the invention include, but are not limited to, humans, non-human primates (eg, cynomolgus or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any species. Poultry.
  • non-human primates eg, cynomolgus or rhesus monkeys or other types of macaques
  • mice pigs, horses, donkeys, cows, sheep, rats, and any species. Poultry.
  • tumor refers to a disease characterized by pathological hyperplasia of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation.
  • a tumor can affect a variety of cells, tissues or organs including, but not limited to, selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, Nerve tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organs, or tissue or corresponding cells.
  • Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).
  • the tumor of the present invention may include, but is not limited to, leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute granulocyte-monocytic leukemia, Acute monocytic leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia, weight Chain disease, solid tumors such as sarcoma and cancer (eg fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphangisarcoma, angiosarcoma, lymphatic endothelial sarcoma, mesotheli
  • the "tumor” includes, but is not limited to, pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, Kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.
  • disease refers to any alteration or disorder that impairs or interferes with the normal function of a cell, tissue or organ.
  • disease includes, but is not limited to, a tumor, a pathogen infection, an autoimmune disease, a T cell dysfunction disease, or a defect in immune tolerance (eg, transplant rejection).
  • treatment refers to a clinical intervention in an attempt to alter a disease caused by an individual or a cell, both prophylactically and in a clinical pathological process.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing the direct or indirect pathological consequences of any disease, preventing metastasis, slowing the progression of the disease, improving or ameliorating the condition, alleviating or improving the prognosis.
  • the invention is further illustrated in detail by the following examples. It is still considered to be part of the present invention to alter the concentration of the components of the formulation or to add other substances on the basis of the present invention, but to have no significant effect on improving the protein stability of the ZMR01 monoclonal antibody.
  • This assay utilizes Waters Xbridge BEH SEC A 7.8 x 300 mm column was run on a Waters e2695-2489 HPLC system.
  • the mobile phase was 100 mM sodium phosphate, 150 mM sodium chloride, pH 6.8.
  • the sample was diluted with mobile phase to 1 mg/mL with an injection volume of 25 ⁇ L.
  • the protein was eluted isocratically for 30 min at a flow rate of 0.5 mL/min, and the absorbance of the eluate was measured at 215 nm. Integrate with Empower 3 software.
  • CE-SDS Capillary Electrophoresis
  • the % main peak and % (LC + HC) purity were determined by non-reducing CE-SDS (nrCE) and reducing CE-SDS (rCE), respectively. This measurement was performed on a BECKMAN COULTER PA800 plus capillary electrophoresis system with 50 ⁇ m ID uncoated quartz. The capillary was carried out, and the capillary was effectively separated by a length of 20 cm, a total length of 30.2 cm, and a PDA 220 nm bandwidth of 10 nm.
  • anti-PD-1 mAb to compete with PD-L1 for binding to PD-1 was tested by ELISA.
  • This product competitively blocks the binding of PD-L1 to PD-1. Therefore, competitive ELISA was used to determine the ability of this product to compete with PD-L1 for binding to PD-1 antigen.
  • Human PD-L1 was first coated on a 96-well plate and then competed with the gradient-diluted product for binding to Biotin-PD-1. The higher the concentration of this product, the less Biotin-PD-1 binds to human PD-L1. After the substrate was added for color development, the absorbance was read to calculate the ability of the product to compete with human PD-L1 for binding to PD-1 antigen.
  • test data is analyzed by four-parameter fitting using SoftMax Pro or other similar software, and the results are automatically analyzed as follows: the reference value (home-made) concentration is the X-axis and the absorbance is the Y-axis.
  • the software will give the test sample.
  • the half effective concentration (EC50) of the reference product and draw an "S" curve.
  • the biological activity of anti-PD-1 mAb was detected by reporter gene method.
  • the reporter gene method comprises two kinds of cells: CHO-PD-L1-CD3L cells as target cells, which stably express PD-L1 and anti-CD3-single-chain antibody fragment (scFv) immobilized on the cell membrane; -PD-1-NFAT cells act as effector cells, which stably express PD-1 and luciferase, and the luciferase gene is regulated by NFAT elements.
  • the anti-CD3-scFv on the CHO cell membrane binds to CD3 on the surface of Jurkat cells, and then expresses an activation signal to Jurkat cells to express luciferase; PD-L1 on the surface of CHO cells binds to PD-1 on the surface of Jurkat cells.
  • the inhibition signal is delivered to Jurkat cells, inhibiting the expression of luciferase; and the anti-PD-1/anti-PD-L1 antibody can block the binding of PD-1 to PD-L1, thereby releasing the delivery of the inhibitory signal. Therefore, the reporter gene method can be used to determine the biological activities of different kinds of anti-PD-1/anti-PD-L1 antibodies.
  • test data is analyzed by four-parameter fitting using SoftMax Pro or other similar software, and the results are automatically analyzed as follows: the reference value (home-made) concentration is the X-axis and the absorbance is the Y-axis.
  • the software will give the test sample. And the half effective concentration (EC 50 ) of the reference product, and draw an "S" curve.
  • Tm midpoint temperature
  • the stability of the biomolecule can be visually explained, and the higher the Tm value, the more stable the biomolecule.
  • Direct, in situ characterization of protein stability by measuring the amount of heat absorbed or released by proteins and other biomolecules during controlled heating or cooling.
  • two peaks appear during the assay, representing the different domains of the antibody, with the main peak representing the Fab region of the antibody and the other peak being the CH2 or CH3 of the antibody.
  • CH2 and CH3 are relatively conserved, while Fab is a variable region whose sequence is not fixed between different antibodies, so the peak of the Fab region may cover CH2 or CH3.
  • the screening range is wide, and some conditions may occur such that the antibody configuration changes drastically, and the corresponding domain is unfolded without corresponding Tm value.
  • the monoclonal antibody ZMR01 against human PD-1 was obtained according to the screening method described in PCT/US2017/060122 (see Example 2 of the literature).
  • the three CDRs of the heavy chain variable region of ZMR01 were determined by amino acid sequence: HCDR1, SEQ ID NO: 1; HCDR2, SEQ ID NO: 2; HCDR3, SEQ ID NO: 3; 3 CDRs of the light chain variable region are: LCDR1, SEQ ID NO: 4; LCDR2, SEQ ID NO: 5; LCDR3, SEQ ID NO: 6.
  • the heavy chain variable region is the sequence shown in SEQ ID NO: 7, and the light chain variable region is the sequence shown in SEQ ID NO: 8.
  • the heavy chain is the sequence shown in SEQ ID NO: 9, and the light chain is the sequence shown in SEQ ID NO: 10.
  • Anti-human Fc antibody protein (GE Healthcare, Cat. No. BR-1008-39) was diluted to 25 ⁇ g/mL with pH 5.0 sodium acetate solution (GE Healthcare, Cat. No. BR-1003-51) and coupled with amino (GE Healthcare, article number The BR-1000-50) was immobilized on two channels (channel 1, channel 2) of the CM5 chip (GE Healthcare, Cat. No. BR-1005-30) with a coupling level of approximately 8000 RU. The sample was captured on channel 2 at a concentration of 2 ⁇ g/mL, 30 ⁇ L/min for 35 seconds, and PD-1 antigen (Sino Biological, Cat. No. 10377-H02H) was diluted to 100 nM with running buffer (GE Healthcare, Cat. No.
  • the affinity experiments of ZMR01 and PD-1 antigen were repeated twice before and after the determination of affinity of all samples (ZMR01-1 and ZMR01-2), and the affinity KD was 1.91nM and 1.82nM (average 1.87nM), respectively.
  • the results of the repeated results were basically the same, indicating that the experimental method was reproducible, and the average of the two replicates was taken as the final affinity of the sample to PD-1, that is, the affinity of ZMR01 to PD-1 antigen was 1.87 nM.
  • Nivolumab (Opdivo, Britol-Myers Squibb, lot AAL9430) to the PD-1 antigen was 8.06 nM; the results showed that ZMR01 had a higher affinity for PD-1 antigen than Nivolumab.
  • Table 1 The specific data is shown in Table 1.
  • the sensor map and the fitting curve are shown in Figure 1 and Figure 2.
  • CHO-PD-L1-CD3L cells purchased from China Food and Drug Administration
  • CHO-PD-L1-CD3L cells purchased from China Food and Drug Administration
  • adjusted cell density to 5 ⁇ 10 5 /mL, 80 ⁇ L / well plating, 37 ° C, 5% CO 2 culture
  • dilute ZMR01 and Nivolumab Nivolumab (Britol-Myers Squibb, batch AAL9430) to 1 mg/mL with sterile water, then dilute to 80 ⁇ g/ml with viable medium from 80 ⁇ g/mL Start 5 times more than 7 times, a total of 8 gradients.
  • the biological activity of ZMR01 was significantly higher than that of Nivolumab in the comparison of the biological activity of ZMR01 and Nivolumab.
  • N.D stands for no detection. Since the buffer affects the antibody configuration, the corresponding Tm value is not detected.
  • ZMR01 antibody was added with polysorbate 20 and polysorbate 80 respectively in histidine-hydrochloric acid and histidine-acetic acid solution.
  • the stability of the formulation was investigated, and a relatively stable formulation was selected.
  • the composition is as follows:
  • R1 R2 R3 R4 antibody 20mg/mL 20mg/mL 20mg/mL 20mg/mL 20mg/mL sucrose 70mg/mL 70mg/mL 70mg/mL 70mg/mL Polysorbate 20 0.2mg/mL / 0.2mg/mL / Polysorbate 80 / 0.2mg/mL / 0.2mg/mL Histidine hydrochloride 10mM 10mM / / Histidine acetate / / 10mM 10mM pH value 5.5 5.5 5.5 5.5 5.5 5.5
  • N/A means the item has not been tested.
  • the histidine-acetic acid buffer is slightly better than the histidine-hydrochloric acid buffer, although the difference is not very obvious.
  • the histidine-hydrochloride buffer has a corrosive effect on the stainless steel product, and the antibody solution has an opportunity to come into contact with the stainless steel container during preparation, circulation and storage. Therefore, a histidine-acetate buffer is preferably selected so as not only to protect the antibody, but also to protect the storage container. From the above test results, it was also unexpectedly found that the polysorbate 20 was more stable to the ZMR01 antibody than the polysorbate 80.
  • Sucrose was used as a stabilizer at a concentration of 70 mg/mL, and polysorbate 20 was selected as a surfactant at a concentration of 0.2 mg/mL; histidine-acetic acid was used as a buffer solution at a concentration of 10 mM.
  • the formulation of the preparation is as follows:
  • N.D stands for no detection.
  • the pH change affects the antibody configuration and the corresponding Tm value is not detected.
  • N/A means the item has not been tested
  • the inventors have unexpectedly found that the ZMR01 antibody formulation is stable in the range of pH 4.5-6.0 with an optimum pH range of 4.5-5.5.
  • the concentration of the ZMR01 antibody was 25 mg/mL, and the pH was 5.2 ⁇ 0.3.
  • the composition and content of the other preparations were as shown in Table 9, and the osmotic pressure of the preparation was simultaneously measured.
  • N/A means the item has not been tested.
  • the ZMR01 antibody was stable in all 10 formulation formulations.
  • the human osmotic pressure ranged from 280 to 320 mOsmol/kg, and according to the values of the osmotic pressure test in Table 9, the osmotic pressure of the R1, R2, and R3 formulations was slightly smaller. Therefore, combining the results of Tables 9 and 10, the most preferred formulation composition is: 25 mg/ml ZMR01 antibody, 90 mg/ml sucrose, 10 mM histidine-acetate buffer, 0.2 mg/ml polysorbate 20, pH 5.2. .
  • ZMR01 antibody 25mg/ml, prepared in 10mM histidine-acetate buffer, 90mg/ml sucrose, 0.2mg/ml polysorbate 20, pH 5.2, the preparation was added to glass bottles, stainless steel and silicone tubes, room temperature Leave for 24 hours. Detection of SEC, nrCE, rCE, binding activity:
  • ZMR01 antibody 25 mg/ml was prepared in 10 mM histidine-acetate buffer, 90 mg/ml sucrose, 0.2 mg/ml polysorbate 20, pH 5.2, and the preparation was filtered through a 0.22 ⁇ m PVDF, PES filter. The protein content and polysorbate content of the filtered sample were examined.
  • N/A means the item has not been tested.
  • the stable ZMR01 antibody formulation has no significant change in the protein concentration after filtration and the concentration of polysorbate 20 in the PVDF, PES filter.
  • Formulation composition ZMR01 antibody 25 mg/ml, 90 mg/ml sucrose, 10 mM histidine-acetate buffer, 0.2 mg/ml polysorbate 20.
  • the preparation was filled in a 4 ml vial at 4 ml/vial and the cap was capped.
  • the samples were placed in 4500 ⁇ 500 Lx intense light, 40 ⁇ 2 ° C high temperature, 25 ° C, 150 rpm shaking for 5 days, low temperature (2-8 ° C and 40 ° C respectively for two days for one cycle) cycle 3 times and repeated freezing
  • the fusion two cycles at -20 ° C and 25 ° C for two days was cycled three times, and SEC, rCE, nrCE, and binding activity were measured. The results are shown in Table 13.
  • the ZMR01 antibody preparation was accelerated at 25 °C ⁇ 2 °C for 1 month (1M), 2 months (2M), 3 months (3M), 6 months (6M), and 2-8 °C for 3 months. (3M), 6 months (6M), 12 months (12M) stability investigation, detection of biological activity, SEC, nrCE, rCE, the results are shown in Table 14.
  • N/A means the item has not been tested.
  • composition of the ZMR01 preparation On the basis of obtaining the composition of the ZMR01 preparation, the following examples will use the preparation to study the pharmacological function of the monoclonal antibody, which will have a more guiding significance for future clinical applications.
  • PBMC Peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • MEM NEAA Inactivated fetal bovine serum FBS + non-essential amino acids (MEM NEAA, Gibco, Cat. No. 11140-050) + sodium pyruvate (SP, Gibco, Cat. No. 11360-070) + 1 ⁇ penicillin streptomycin (Gibco, Cat. No.
  • the PBMC cell density was adjusted to 1 ⁇ 10 5 /well, and a 96-well transparent round bottom plate was inoculated with 150 ⁇ L per well.
  • 50 ⁇ L of ZMR01 or Nivolumab (Britol-Myers Squibb, batch number AAS1144) were added to the corresponding wells of the 96-well plate inoculated with cells to a final concentration of 10, 3.33, 1.11, 0.370, 0.123, 0.0412, 0.0137, 0.00457, 0.00152, respectively.
  • DC dendritic cells
  • PBMC peripheral blood mononuclear cells
  • CD4 + T cells from different volunteers.
  • Peripheral blood of volunteer A was taken, and PBMC was isolated by density gradient centrifugation using Ficoll-PaqueTM Plus reagent (GE Healthcare, Cat. No. 17-1440-02), and then CD14 + monocytes were obtained by magnetic bead sorting (Human).
  • Monocyte Isolation Kit STEMCELL, Catalog No. 19359
  • RPMI 1640 complete medium containing 10% fetal calf serum, 1% non-essential amino acid (Gibco, Cat. No. 11140-050), 1% sodium pyruvate (Gibco, Cat.
  • cytokine GM-CSF 100ng /mL, PEPROTECH, Cat. No. 300-03-100UG
  • IL-4 50ng/mL, PEPROTECH, Cat. No. 200-04-100UG
  • imDC was further added to RPMI1640 complete medium containing 10% fetal calf serum, 1% non-essential amino acid, 1% sodium pyruvate by adding cytokine IL-1 ⁇ (10 ng/mL, PEPROTECH, Cat. No. 200-01B-50UG).
  • TNF- ⁇ (10 ng/mL, PEPROTECH, Cat. No. 300-01A-50UG)
  • IL-6 (10 ng/mL, PEPROTECH, Cat. No. 200-06-50 UG)
  • PGE2 (1 ⁇ g/mL, Sigma, Cat. No. P6532-1MG)
  • the culture was induced for 24 hours or 48 hours in a 37 ° C, 5% CO 2 cell incubator to become mature dendritic cells (mDC).
  • PBMC peripheral blood of volunteer B was extracted, and PBMC was isolated by density gradient centrifugation, and then sorted by magnetic beads (EasySepTM Human CD4 + T Cell Isolation Kit, STEMCELL, Cat. No. 17952) to obtain CD4 + T lymphocytes.
  • the isolated T lymphocytes (inoculation density 1 ⁇ 10 5 /well) and the induced mature DC cells (inoculation density 2 ⁇ 10 4 /well) were co-inoculated into 96-well round bottom culture plates, and different concentration gradients were added.
  • the mixed cell culture system was cultured for 5 days at 37 ° C in a 5% CO 2 cell incubator, and the cell culture supernatant was collected using Human IFN- ⁇ ELISA Ready-SET-Go!
  • the T cell IFN- ⁇ secretion level was measured by a kit (invitrogen, Cat. No. 88-7316-88).
  • the concentration of the added antibody was plotted on the abscissa and the IFN- ⁇ content was plotted on the ordinate.
  • the histogram was performed using the GRAPHPAD PRISM software.
  • the results are shown in Fig. 5.
  • Four times of MLR experiments were performed using peripheral blood of four different volunteers, and the paired t-test was performed using the GRAPHPAD PRISM software.
  • the results are shown in Fig. 6.
  • the above experimental results show that the ZMR01 preparation can effectively enhance the secreted cytokine IFN- ⁇ function of T cells in the mixed lymphocyte reaction system; when the antibody concentration is 0.1 ⁇ g/mL and 10 ⁇ g/mL, the ZMR01 preparation enhances the secretion of IFN- ⁇ by T cells.
  • the ability is better than BMS's Nivolumab mAb (*p ⁇ 0.05).
  • MC38 tumor cells were inoculated into the right flank of female B-hPD-1 humanized mice (Bai Sai Tu Jiangsu Genetic Biotechnology Co., Ltd.) at a concentration of 5 ⁇ 10 5 / 0.1 mL, 0.1 mL / volume.
  • the tumor volume is randomly divided into groups, 10 in each group, which are control hIgG 4 (Beijing Yiqiao Shenzhou Technology Co., Ltd., batch number MA09JL0905) group, Nivolumab group (3mg/kg, Bristol- Myers Squibb, batch number AAS1144), ZMR01 (0.3 mg/kg), ZMR01 (1.0 mg/kg) and ZMR01 (3.0 mg/kg) groups.
  • the drug was administered twice a week by intraperitoneal injection for a total of 6 times, and the experiment was terminated on the second day after the last administration.
  • tumor volume 0.5 ⁇ long diameter ⁇ short diameter 2.
  • TGI% tumor growth inhibition rate
  • the tumor growth inhibition rates of the Nivolumab group (3 mg/kg), ZMR01 (0.3 mg/kg), ZMR01 (1 mg/kg), and ZMR01 (3 mg/kg) groups were 27.5, respectively, compared with the hIgG 4 group. %, 60.9% (p ⁇ 0.01), 67.4% (p ⁇ 0.001), and 76.6% (p ⁇ 0.001), ZMR01 (0.3 mg/kg), ZMR01 (1 mg/kg), and ZMR01 (3 mg/kg) with a negative control There was a significant difference in mean tumor volume between the hIgG 4 groups.
  • ZMR01 has a significant inhibitory effect on tumor growth at doses of 0.3, 1, and 3 mg/kg; ZMR01 has a significant dose-effect relationship in this experimental model, and the antitumor activity increases with the dose administered. Enhanced, and the anti-tumor effect of ZMR01 is superior to BMS's Nivolumab mAb (p ⁇ 0.001).
  • NOG mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into groups according to body weight, 8 rats in each group, A431 cells + 0.9% NaCl injection group, A431 cells + PBMC + hIgG 4 + hIgG group 1 (Beijing Yiqiao Shenzhou Technology Co., Ltd., hIgG 4 batch number MA09JL0905, hIgG 1 batch number MB11MA1306), A431 cells + PBMC+ZMR01 (1mg/kg) group, A431 cells + PBMC + anti-human VEGF humanized antibody (QL1101, Qilu Pharmaceutical Co., Ltd.
  • the negative control group was inoculated with A431 cells (6 ⁇ 106 cells/case, sc, purchased from ATCC), and the other experiments were inoculated with A431 cells (6 ⁇ 10 6 cells/sc, sc) and PBMC (4 ⁇ 10 6 cells/only). , iv, purchased from Stetexpress, lot number 1702170123), the day of vaccination is recorded as day 0.
  • the administration was started on the third day, twice a week for a total of 7 times, and the second day of the last administration was the end point of the test, and the animals were sacrificed by the method of euthanasia.
  • TGI% tumor growth inhibition rate
  • the tumor growth inhibition rate of ZMR01 (1 mg/kg), QL1101 (0.5 mg/kg), and ZMR01 (1 mg/kg) + QL1101 (0.5 mg/kg) group was compared with hIgG 4 + hIgG 1 group.
  • the heavy chain sequence of the QL1101 antibody is set forth in SEQ ID NO: 11
  • the light chain sequence of the QL1101 antibody is set forth in SEQ ID NO: 12.

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Abstract

La présente invention concerne une formulation pharmaceutique stable d'un anticorps contre la PD-1 humaine, un médicament d'association, et l'utilisation de ces derniers. Ladite formulation pharmaceutique comprend un anticorps contre la PD-1 humaine, un stabilisant, un tampon et un tensioactif. La formulation pharmaceutique de l'anticorps contre la PD-1 humaine de la présente invention peut inhiber efficacement l'agrégation et la désamidation de l'anticorps, permettant ainsi d'empêcher la dégradation d'une protéine d'anticorps et d'obtenir une formulation pharmaceutique stable. En ce qui concerne le médicament d'association de la présente invention, la formulation pharmaceutique de l'anticorps contre la PD-1 humaine est utilisée en association avec d'autres agents thérapeutiques supplémentaires. L'invention concerne en outre l'utilisation de la formulation pharmaceutique ou du médicament d'association dans la préparation d'un médicament antitumoral.
PCT/CN2019/084558 2018-04-28 2019-04-26 Formulation d'anticorps monoclonal contre la pd-1 humaine, médicament d'association, et utilisation de ces derniers Ceased WO2019206281A1 (fr)

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CN115666649A (zh) * 2020-06-12 2023-01-31 上海君实生物医药科技股份有限公司 一种新型冠状病毒抗体的药物组合物及其用途
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WO2023217294A1 (fr) * 2022-05-09 2023-11-16 浙江特瑞思药业股份有限公司 Formulation de nano-anticorps anti-pd-1 et son utilisation
WO2025011439A1 (fr) * 2023-07-07 2025-01-16 齐鲁制药有限公司 Préparation pharmaceutique d'anticorps monoclonal anti-ccr8 humain et son utilisation
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CN113842456B (zh) * 2020-06-28 2022-07-26 上海齐鲁制药研究中心有限公司 一种抗人4-1bb的单克隆抗体制剂及其用途
CN114712495B (zh) * 2021-01-06 2024-08-20 盛禾(中国)生物制药有限公司 一种多功能抗体的组合物
CN113925963B (zh) * 2021-10-15 2023-08-11 江苏太平洋美诺克生物药业股份有限公司 一种稳定的包含抗cd147单克隆抗体的药物制剂
CN118660720A (zh) * 2022-02-22 2024-09-17 齐鲁制药有限公司 包含抗ctla4和抗pd1的混合抗体的药物组合物及其治疗用途
CN116688115B (zh) * 2022-03-18 2024-02-06 上海齐鲁制药研究中心有限公司 一种PD-L1/TGF-β双功能融合蛋白制剂及其用途
CN119300862A (zh) * 2022-06-28 2025-01-10 齐鲁制药有限公司 包含抗ctla4和抗pd1的混合抗体的药物组合物及其治疗用途
CN119300863A (zh) * 2022-06-30 2025-01-10 齐鲁制药有限公司 包含抗ctla4和抗pd1的混合抗体的药物组合物及其治疗用途
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WO2021228069A1 (fr) * 2020-05-12 2021-11-18 信达生物制药(苏州)有限公司 Utilisation d'une combinaison d'anticorps anti-vegf et d'anticorps anti-pd-1 pour prévenir ou traiter une maladie
CN115803055A (zh) * 2020-06-11 2023-03-14 三生国健药业（上海）股份有限公司 一种抗pd-1单克隆抗体液体制剂
WO2021249551A1 (fr) * 2020-06-11 2021-12-16 三生国健药业（上海）股份有限公司 Préparation liquide d'anticorps monoclonaux anti-pd-1
CN115666649A (zh) * 2020-06-12 2023-01-31 上海君实生物医药科技股份有限公司 一种新型冠状病毒抗体的药物组合物及其用途
US20230295329A1 (en) * 2020-07-31 2023-09-21 Jiangsu Hengrui Pharmaceuticals Co., Ltd. Anti-pd-1 antibody pharmaceutical composition and use thereof
WO2022063193A1 (fr) * 2020-09-24 2022-03-31 上海齐鲁制药研究中心有限公司 MOLÉCULE BIFONCTIONNELLE CIBLANT SIMULTANÉMENT PD-L1 ET TGFβ ET SON UTILISATION MÉDICALE
WO2022178319A1 (fr) * 2021-02-18 2022-08-25 Qilu Puget Sound Biotherapeutics Corporation Combinaisons d'anticorps anti-pd1 et anti-ctla4
EP4294531A4 (fr) * 2021-02-18 2025-07-16 Qilu Puget Sound Biotherapeutics Corp Combinaisons d'anticorps anti-pd1 et anti-ctla4
US20230025464A1 (en) * 2021-07-09 2023-01-26 Macrogenics, Inc. Pharmaceutical compositions of a pd-1 antibody and use of the same
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WO2023217294A1 (fr) * 2022-05-09 2023-11-16 浙江特瑞思药业股份有限公司 Formulation de nano-anticorps anti-pd-1 et son utilisation
WO2025011439A1 (fr) * 2023-07-07 2025-01-16 齐鲁制药有限公司 Préparation pharmaceutique d'anticorps monoclonal anti-ccr8 humain et son utilisation
WO2025140495A1 (fr) * 2023-12-28 2025-07-03 齐鲁制药有限公司 Composition pharmaceutique stable d'anticorps anti-pd-1

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