WO2023051621A1 - 抗lag3抗体、药物组合物及用途 - Google Patents

抗lag3抗体、药物组合物及用途 Download PDF

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WO2023051621A1
WO2023051621A1 PCT/CN2022/122185 CN2022122185W WO2023051621A1 WO 2023051621 A1 WO2023051621 A1 WO 2023051621A1 CN 2022122185 W CN2022122185 W CN 2022122185W WO 2023051621 A1 WO2023051621 A1 WO 2023051621A1
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antibody
seq
antigen
cancer
variable region
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French (fr)
Chinese (zh)
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夏瑜
王忠民
张鹏
李百勇
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Akeso Pharmaceuticals Inc
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Akeso Biopharma Inc
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Priority to IL311738A priority Critical patent/IL311738A/en
Priority to US18/696,559 priority patent/US20250042995A1/en
Priority to AU2022355381A priority patent/AU2022355381A1/en
Priority to MX2024003936A priority patent/MX2024003936A/es
Priority to JP2024519329A priority patent/JP2024536881A/ja
Priority to CA3233205A priority patent/CA3233205A1/en
Application filed by Akeso Biopharma Inc filed Critical Akeso Biopharma Inc
Priority to KR1020247012890A priority patent/KR20240083095A/ko
Priority to EP22875004.8A priority patent/EP4410835A4/en
Publication of WO2023051621A1 publication Critical patent/WO2023051621A1/zh
Anticipated expiration legal-status Critical
Priority to ZA2024/03275A priority patent/ZA202403275B/en
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Definitions

  • the invention belongs to the field of biomedicine, and relates to an anti-LAG3 antibody, a pharmaceutical composition containing the anti-LAG3 antibody and its use.
  • Tumors are diseases that seriously endanger human health in the world today, ranking second among the deaths caused by various diseases. And in recent years, its incidence has shown an obvious upward trend.
  • the curative effect of malignant tumors is poor, the rate of advanced metastasis is high, and the prognosis is often poor.
  • conventional treatment methods such as radiotherapy, chemotherapy and surgery are currently used clinically, although the pain is relieved to a large extent and the survival time is prolonged, these methods have great limitations, and the curative effect is difficult to further improve.
  • Lymphocyte-activation gene 3 (LAG3), or CD223, is a type I transmembrane protein composed of 498 amino acids, which belongs to the immunoglobulin superfamily (IgSF) member.
  • LAG3 is mainly expressed in activated CD4 + T cells and CD8 + T cells, in addition to natural killer (natural killer, NK) cells, B cells, regulatory T cells (regulatory T cells, Treg) and plasmacytoid dendritic cells ( Plasmacytoid dendritic cells, pDC) and other cells also express LAG3.
  • NK natural killer
  • Treg regulatory T cells
  • Plasmacytoid dendritic cells Plasmacytoid dendritic cells, pDC
  • the LAG3 molecular gene is located on human chromosome 12 (20p13.3), adjacent to the CD4 molecular gene, and both have the same exons and introns. Although the amino acid sequence homology between the two is only about 20%, the LAG3 molecule and the CD4 molecule have a high similarity in structure.
  • Major histocompatibility complex class II molecules MHC II
  • LSECtin liver sinusoidal endothelial cell agglutinin
  • galectin-3 galectin-3
  • MHC class II molecules are the main ligands of LAG3, and their affinity with MHC class II molecules (Kd: 60 nmol L-1) is 100 times that of CD4 molecules, indicating that LAG3 molecules can effectively compete for the binding of CD4 to MHC class II molecules and inhibit T cells. activation.
  • the expression of the immunosuppressive molecule LAG3 can be detected 24 hours after T cell activation, leading to T cell incapacity or apoptosis.
  • the LAG3 molecule forms a dimer molecule through its D1 domain (containing a proline-rich ring structure) and specifically binds to the MHCII molecule in the first signaling axis "CD3-TCR-MHCII" of CD4 + T cell activation, On the one hand, it blocks the signal transduction pathway of T cell activation, and on the other hand, the intracellular segment of LAG3 molecule (KIEELE motif) produces an immunosuppressive signal to down-regulate the activity of CD4 + T cells.
  • LAG3 molecules can promote the differentiation of Treg cells, participate in signal transduction and transcription activator 5 downstream signaling, thereby enhancing the inhibitory effect of Treg cells, which is one of the mechanisms for tumors to escape the killing of the immune system (Andrews Lawrence P, Marciscano Ariel E, Drake Charles G et al.LAG3(CD223) as a cancer immunotherapy target.[J].Immunol Rev,2017,276:80-96.).
  • LAG3 is overexpressed in tumor-infiltrating CD8 + T cells in various malignancies.
  • tumor-infiltrating New York esophageal squamous cell carcinoma 1 (NY-ESO-1)-specific CD8 + T cells express high levels of PD-1 and LAG3 and have reduced ability to produce IFN- ⁇ and TNF- ⁇ , resulting in inactivation of lymphocytes.
  • Galectin-3 and LSECtin mainly interact with LAG3 to regulate the activation and function of CD8 + T cells.
  • melanoma antigen-specific T cells isolated from patients with melanoma metastasis were significantly upregulated in the expression of LAG3 and other immune checkpoint molecules CTLA-4 and TIM-3.
  • LAG3 antibody drugs have entered the clinical research stage.
  • Bristol-Myers Squibb's Relatlimab has made the fastest progress.
  • 10 clinical studies have been carried out, and most of them are combined drugs of Relatlimab and Nivolumab. It is used to treat tumors such as hematoma, melanoma, glioblastoma, renal cell carcinoma and non-small cell lung cancer.
  • the present inventor obtained an anti-LAG3 antibody through intensive research and creative work.
  • the inventors have surprisingly found that the anti-LAG3 antibody of the present invention (also referred to as antibody or antibody of the present invention) has superior affinity and/or specificity, and is even superior to a positive control antibody (such as Relatlimab) in one or more aspects .
  • a positive control antibody such as Relatlimab
  • One aspect of the present invention relates to an anti-LAG3 antibody or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region comprises: amino acid sequences shown in SEQ ID NOs: 9-11 respectively HCDR1-HCDR3; and the light chain variable region comprises: amino acid sequences shown in SEQ ID NOs: 12-14 respectively LCDR1-LCDR3;
  • the heavy chain variable region comprises: amino acid sequences such as HCDR1-HCDR3 shown in SEQ ID NOs: 9-11; and the light chain variable region comprises: amino acid sequences such as SEQ ID NO: 12, SEQ ID NO LCDR1-LCDR3 shown in :15 and SEQ ID NO:16;
  • the heavy chain variable region comprises: amino acid sequences such as HCDR1-HCDR3 shown in SEQ ID NOs: 9-11; and the light chain variable region comprises: amino acid sequences such as SEQ ID NO: 17, SEQ ID NO :15 and LCDR1-LCDR3 shown in SEQ ID NO:14.
  • the antibody or antigen-binding fragment thereof wherein,
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:4;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO: 2, and the amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO: 6;
  • amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:2
  • amino acid sequence of the light chain variable region of the antibody is shown in SEQ ID NO:8.
  • the antibody or antigen-binding fragment thereof wherein the antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementary Determining region fragments, single chain antibodies, humanized antibodies or chimeric antibodies.
  • the antibody or antigen-binding fragment thereof wherein, the antibody is less than 0.2nM, such as less than 0.15nM, less than 0.1nM, less than 0.08nM, 0.06nM or less than 0.05nM or A smaller EC50 binding to human LAG3-mFc; preferably, said EC50 is determined by an indirect ELISA method.
  • the antibody or antigen-binding fragment thereof wherein,
  • the antibody includes non-CDR regions, and the non-CDR regions are from a species other than murine, eg, from a human antibody.
  • the antibody or antigen-binding fragment thereof wherein,
  • the antibody whose constant region is derived from a human antibody
  • the constant region of the antibody is selected from the constant region of human IgG1, IgG2, IgG3 or IgG4.
  • the antibody or antigen-binding fragment thereof wherein,
  • the heavy chain constant region of the anti-LAG3 antibody is Ig gamma-1 chain C region (for example, as shown in SEQ ID NO: 18) or Ig gamma-4 chain C region (for example, as shown in SEQ ID NO: 20) and the light chain constant region is an Ig kappa chain C region (eg, as shown in SEQ ID NO: 19).
  • the anti-LAG3 antibody is a monoclonal antibody.
  • the anti-LAG3 antibody is in the form of immunoglobulin.
  • the anti-LAG3 antibody is a single chain antibody.
  • an antibody-drug conjugate which includes an antibody or its antigen-binding fragment and a small molecule drug, wherein the antibody or its antigen-binding fragment is any one of the present invention
  • ADC antibody-drug conjugate
  • the small-molecule drug is a small-molecule cytotoxic drug; more preferably, the small-molecule drug is a tumor chemotherapy drug.
  • the chemotherapeutic drugs can be conventional tumor chemotherapeutic drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones, immune preparations and the like.
  • the antibody-drug conjugate wherein the antibody or its antigen-binding fragment is connected to the small molecule drug through a linker;
  • the linker can be a person skilled in the art Known linkers are, for example, hydrazone bonds, disulfide bonds or peptide bonds.
  • the antibody-drug conjugate wherein the molar ratio of the antibody or its antigen-binding fragment to the small-molecule drug is 1: (2-4), for example, 1: 2, 1:3 or 1:4.
  • Another aspect of the present invention relates to an isolated nucleic acid molecule encoding the anti-LAG3 antibody of any one of the present invention.
  • Yet another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.
  • a further aspect of the invention relates to a host cell comprising an isolated nucleic acid molecule of the invention, or a recombinant vector of the invention.
  • Another aspect of the present invention relates to a method for preparing the antibody or antigen-binding fragment thereof according to any one of the present invention, which comprises culturing the host cell of the present invention under suitable conditions, and recovering the obtained antibody from the cell culture. The steps of the antibody or antigen-binding fragment thereof.
  • Another aspect of the present invention relates to a pharmaceutical composition, which comprises the antibody or antigen-binding fragment thereof according to any one of the present invention, and the antibody-drug conjugate according to any one of the present invention; optionally, It also includes pharmaceutically acceptable excipients.
  • Another aspect of the present invention relates to the antibody or its antigen-binding fragment described in any one of the present invention, and the antibody-drug conjugate described in any one of the present invention in the preparation of a drug for treating and/or preventing tumor or anemia use in
  • the tumor is selected from ovarian cancer, esophageal cancer, melanoma, hematoma, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal One or more of cancers of the tract, breast, brain, pancreas, thyroid, head and neck, and kidney;
  • the lung cancer is non-small cell lung cancer
  • the hematological tumor is leukemia
  • the esophageal cancer is esophageal squamous cell carcinoma.
  • the tumor is selected from ovarian cancer, esophageal cancer, melanoma, hematoma, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal One or more of cancers of the tract, breast, brain, pancreas, thyroid, head and neck, and kidney;
  • the lung cancer is non-small cell lung cancer
  • the hematological tumor is leukemia
  • the esophageal cancer is esophageal squamous cell carcinoma.
  • Another aspect of the present invention relates to a method for treating and/or preventing tumor or anemia, comprising administering to a subject in need an effective amount of the antibody or antigen-binding fragment thereof described in any one of the present invention, the present invention The steps of the antibody drug conjugate described in any one of the inventions;
  • the tumor is selected from ovarian cancer, esophageal cancer, melanoma, hematoma, glioblastoma, renal cell carcinoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, gastrointestinal One or more of cancers of the tract, breast, brain, pancreas, thyroid, head and neck, and kidney;
  • the lung cancer is non-small cell lung cancer
  • the hematological tumor is leukemia
  • the esophageal cancer is esophageal squamous cell carcinoma.
  • EC 50 refers to the concentration for 50% of maximal effect, which refers to the concentration that can cause 50% of the maximal effect.
  • antibody refers to an immunoglobulin molecule generally composed of two pairs of polypeptide chains, each pair having a "light” (L) chain and a “heavy” (H) chain.
  • Antibody light chains can be classified as kappa and lambda light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also comprising a "D" region of about 3 or more amino acids.
  • Each heavy chain is composed of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant regions of the antibodies mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4, from amino-terminus to carboxy-terminus.
  • the variable regions (VH and VL) of each heavy chain/light chain pair form the antibody binding site, respectively. Assignment of amino acids to regions or domains follows Bethesda M.d., Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, (1987 and 1991)), or Chothia & Lesk J. Mol. Biol. 1987; 196:901-917; Chothia et al.
  • antibody is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. Antibodies can be of different isotypes, eg, IgG (eg, IgGl, IgG2, IgG3, or IgG4 subtype), IgAl, IgA2, IgD, IgE, or IgM antibodies.
  • IgG eg, IgGl, IgG2, IgG3, or IgG4 subtype
  • IgAl IgA2, IgD, IgE, or IgM antibodies.
  • the terms “monoclonal antibody” and “monoclonal antibody” refer to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, except for natural mutations that may occur spontaneously, A population of identical antibody molecules.
  • mAbs are highly specific for a single epitope on an antigen.
  • polyclonal antibodies usually contain at least two or more different antibodies, and these different antibodies usually recognize different epitopes on antigens.
  • Monoclonal antibodies can usually be obtained using hybridoma technology first reported by Kohler et al. ( G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but it can also be obtained by recombinant DNA technology (see US Patent 4,816,567).
  • humanized antibody refers to the replacement of all or part of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody).
  • Antibodies or antibody fragments, wherein the donor antibody can be a non-human (eg, mouse, rat or rabbit) antibody with the desired specificity, affinity or reactivity.
  • some amino acid residues in the framework region (FR) of the acceptor antibody can also be replaced by amino acid residues of corresponding non-human antibodies, or by amino acid residues of other antibodies, so as to further improve or optimize the performance of the antibody.
  • isolated means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated or “isolated” do not exclude the admixture of artificial or synthetic substances, nor the presence of other impurities which do not affect the activity of the substance.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector is called an expression vector.
  • a vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phage such as lambda phage or M13 phage and animal viruses.
  • artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC)
  • Phage such as lambda phage or M13 phage and animal viruses.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, papillomaviruses, Polyoma vacuolar virus (eg SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • baculoviruses such as herpes simplex virus
  • papillomaviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyoma vacuolar virus eg
  • the term "host cell” refers to cells that can be used to introduce vectors, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as Escherichia coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • an antibody that specifically binds to an antigen refers to an antibody that is less than about 10 -5 M, such as less than about 10 -6 M, 10 -7 M, Binds the antigen with an affinity (K D ) of 10 ⁇ 8 M, 10 ⁇ 9 M or 10 ⁇ 10 M or less.
  • KD refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and antigen.
  • the antibody has a dissociation equilibrium constant (K D ) of less than about 10 ⁇ 5 M, such as less than about 10 ⁇ 6 M, 10 ⁇ 7 M, 10 ⁇ 8 M, 10 ⁇ 9 M, or 10 ⁇ 10 M or less.
  • Binds antigen eg, PD-1 protein). KD can be determined using methods known to those skilled in the art, for example using a Fortebio Molecular Interaction Instrument.
  • amino acids are generally represented by single-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” refers to a carrier and/or excipient compatible with the subject and the active ingredient pharmacologically and/or physiologically, These are well known in the art (see e.g. Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers agent.
  • pH regulators include but not limited to phosphate buffer
  • surfactants include but not limited to cationic, anionic or nonionic surfactants such as Tween-80
  • ionic strength enhancers include but not limited to sodium chloride.
  • an effective amount refers to an amount sufficient to achieve, or at least partially achieve, the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, arrest, or delay the occurrence of a disease (such as a tumor);
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the occurrence of a disease in a patient Amount of disease and its complications. Determining such an effective amount is well within the capability of those skilled in the art.
  • amounts effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and sex, the mode of administration of the drug, and other treatments administered concomitantly etc.
  • lymphocyte-activation gene 3 when referring to the amino acid sequence of lymphocyte activation gene 3 (lymphocyte-activation gene 3, LAG3), it includes the full length of the LAG3 protein, or the extracellular fragment LAG3 ECD of LAG3 or a protein comprising LAG3 ECD Fragments; also include full-length fusion proteins of LAG3 protein or fusion proteins of LAG3 ECD, for example, fragments fused with Fc protein fragments (mFc or hFc) of mouse or human IgG.
  • Fc protein fragments mFc or hFc
  • the anti-LAG3 antibody of the present invention has superior affinity and specificity
  • the anti-LAG3 antibody of the present invention can effectively block the interaction between LAG3 and MHC-II, and specifically relieve the immune suppression of the body by LAG3.
  • Figure 1 The detection results of the binding activity between H7L8(hG1WT) and antigen LAG3-mFc determined by indirect ELISA method.
  • Figure 2 ELISA method to determine the binding activity of H7L8(hG4WT), H7L9(hG4WT), H7L10(hG4WT) to antigen human LAG3-mFc.
  • FIG. 3 FACS detection of the binding activity of H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT) to the surface antigen LAG3 of 293T-LAG3 cells.
  • Figure 4 The results of competitive flow cytometry determination of H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT) and LAG3-mFc for binding to 293T-LAG3 cell membrane surface antigen MHC II.
  • Figure 5 The results of the mixed lymphocyte reaction MLR detection of the biological activity of anti-LAG3 antibody to promote IFN- ⁇ secretion.
  • Figure 6 The results of the mixed lymphocyte reaction MLR detection of the biological activity of the anti-LAG3 antibody in promoting IL-2 secretion.
  • Figure 7 Detection results of biological activity of anti-LAG antibody blocking the interaction between LAG-3 and MHC-II.
  • Relatlimab refers to the US Patent Publication: US20160326248A1 for its sequence.
  • amino acid sequence of the heavy chain refers to SEQ ID NO:1 in the patent disclosure
  • amino acid sequence of the light chain refers to SEQ ID NO:2 in the patent disclosure.
  • Relatlimab is an anti-LAG-3 antibody.
  • control antibody 14C12H1L1 is an anti-PD-1 antibody, prepared by Akeso Bio, batch number B105Y2080601.
  • the 293T-LAG3 cell line was constructed by Zhongshan Akefang Biopharmaceutical Co., Ltd.
  • the 293T-LAG3 cell line was obtained by virus infection of HEK293T cells.
  • the virus was prepared using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector used in it is plenti6.3/V5-huLAG3FL-BSD (where LAG3, Genebank ID: NM_002277.4; vector plenti6.3/V5-BSD, purchased from Invitrogen, product number: K5315-20).
  • the Raji-PDL1 cell line was constructed by Zhongshan Akefang Biopharmaceutical Co., Ltd.
  • the Raji-PDL1 cell line was obtained by virus infection of Raji cells using 3rd Generation Lentiviral Systems, see, for example, A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector used in it is plenti6.3/V5-PDL1 (wherein PDL1, Genebank ID: NP_054862.1; vector plenti6. 3/V5, purchased from Invitrogen, catalog number: K5315-20).
  • the Jurkat-NFAT-PD1-LAG3 cell line was constructed by Zhongshan Akeso Biopharmaceutical Co., Ltd.
  • the Jurkat-NFAT-PD1-LAG3 cell line is obtained from PD-1 Effector cells (CPM), manufacturer: Promega, product number: J112A) cells infected by virus, the virus is prepared using 3rd Generation Lentiviral Systems, See, for example A Third Generation Lentivirus Vector with a Conditional Packaging System. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, and Naldini L.J Virol.1998.72(11):8463-8471.
  • the lentiviral expression vector is pCDH-huLAG3FL-RFP-NEO (wherein LAG3, Genebank ID: NM_002277.4; vector pCDH-CMV-MCS-EF1-RFP+Neo, purchased from Youbao Biology, product number: VT9005).
  • the nucleotide sequence of the heavy chain variable region H7v of H7L9 is identical to the nucleotide sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:1.
  • the amino acid sequence of the heavy chain variable region H7v of H7L9 is identical to the amino acid sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:2.
  • the nucleotide sequence of the heavy chain variable region H7v of H7L10 is identical to the nucleotide sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:1.
  • the amino acid sequence of the heavy chain variable region H7v of H7L10 is identical to the amino acid sequence of the heavy chain variable region H7v of H7L8, namely SEQ ID NO:2.
  • amino acid sequence of the CDR of antibody H7L8 is as follows (according to the IMGT numbering system):
  • HCDR1 GGSISDYY (SEQ ID NO: 9);
  • HCDR2 INHRGTT (SEQ ID NO: 10);
  • HCDR3 AFGYSDYEYDWFDP (SEQ ID NO: 11);
  • LCDR1 QTISSY (SEQ ID NO: 12);
  • LCDR2 DAS (SEQ ID NO: 13);
  • LCDR3 QQRSNWPIT (SEQ ID NO: 14).
  • amino acid sequence of the CDR of antibody H7L9 is as follows (according to the IMGT numbering system):
  • HCDR1 GGSISDYY (SEQ ID NO: 9);
  • HCDR2 INHRGTT (SEQ ID NO: 10);
  • HCDR3 AFGYSDYEYDWFDP (SEQ ID NO: 11);
  • LCDR1 QTISSY (SEQ ID NO: 12);
  • LCDR2 DGS (SEQ ID NO: 15);
  • LCDR3 QQRSNWPLT (SEQ ID NO: 16).
  • amino acid sequence of the CDR of antibody H7L10 is as follows (according to the IMGT numbering system):
  • HCDR1 GGSISDYY (SEQ ID NO: 9);
  • HCDR2 INHRGTT (SEQ ID NO: 10);
  • HCDR3 AFGYSDYEYDWFDP (SEQ ID NO: 11);
  • LCDR1 QSISSY (SEQ ID NO: 17);
  • LCDR2 DGS (SEQ ID NO: 15);
  • LCDR3 QQRSNWPIT (SEQ ID NO: 14).
  • the heavy chain cDNA sequence of H7L8 (hG1WT) (the variable region coding sequence is shown in SEQ ID NO: 1; the constant region is the Ig gamma-1 chain C region) and the cDNA sequence of the light chain (the variable region coding sequence is shown in SEQ ID NO: ID NO: 3; the constant region is human Ig kappa chain C region) were cloned into pUC57simple (provided by GenScript) vectors to obtain pUC57simple-H7 and pUC57simple-L8 plasmids, respectively.
  • pUC57simple provided by GenScript
  • Plasmids pUC57simple-H7 and pUC57simple-L8 were digested (HindIII & EcoRI), and the heavy and light chains recovered by electrophoresis were subcloned into pcDNA3.1 vectors, and the recombinant plasmids were extracted and co-transfected into 293F cells. After the cells were cultured for 7 days, the culture medium was concentrated by high-speed centrifugation and the supernatant was concentrated and loaded onto the HiTrap MabSelect SuRe column. The protein was eluted with Elution Buffer in one step, and the target sample was recovered and replaced with PBS.
  • the heavy chain cDNA sequences of H7L8 (hG4WT), H7L9 (hG4WT) and H7L10 (hG4WT) (the variable region coding sequence is shown in SEQ ID NO: 1; the constant region is Ig gamma-4 chain C region) and H7L8 (hG4WT ) light chain cDNA sequence (the variable region coding sequence is shown in SEQ ID NO: 3; the constant region is human Ig kappa chain C region), the cDNA sequence of the H7L9 (hG4WT) light chain (the variable region coding sequence is shown in SEQ ID Shown in NO: 5; the constant region is human Ig kappa chain C region), the cDNA sequence of H7L10 (hG4WT) light chain (the variable region coding sequence is shown in SEQ ID NO: 7; the constant region is human Ig kappa chain C region ) were respectively cloned into pUC57simple (provided by GenScript
  • the plasmids pUC57simple-H7 and pUC57simple-L8, pUC57simple-L9, pUC57simple-L10 were digested with restriction enzymes (HindIII & EcoRI), and the heavy and light chains recovered by electrophoresis were respectively subcloned into pcDNA3.1 vectors, and the recombinant plasmids were extracted and co-transfected into 293F cell. After the cells were cultured for 7 days, the culture medium was concentrated by high-speed centrifugation and the supernatant was concentrated and loaded onto the HiTrap MabSelect SuRe column. The protein was eluted in one step with Elution Buffer, and the target sample was recovered and replaced with PBS.
  • restriction enzymes HindIII & EcoRI
  • H7L8 H7L9
  • H7L10 H7L10
  • Human anti-Hen Egg Lysozyme IgG human anti-Hen Egg Lysozyme IgG, anti-HEL, namely human IgG, referred to as hIgG
  • its sequence comes from Affinity maturation increases the stability and plasticity of the Fv domain of published by Acierno et al.
  • Variable region sequence of Fab F10.6.6 sequence in anti-protein antibodies study (Acierno et al. J Mol Biol. 2007; 374(1):130-46.). The preparation method is as follows:
  • Experimental example 1 Determination of the binding activity of anti-LAG3 antibody and antigen by ELISA method
  • Relatlimab and H7L8 can effectively bind to the antigen human LAG3-mFc, and the binding efficiency is dose-dependent.
  • the absorbance intensity of each dose is shown in Table 1.
  • Embodiment 2 ELISA method measures the binding activity of anti-LAG3 antibody and antigen
  • Example 3 Detection of binding activity of anti-LAG3 antibody to cell surface antigen LAG3 by flow cytometry
  • the host cell 293T-LAG3 expressing LAG3 antigen obtained in the above steps was digested with conventional trypsin, and the number of cells in each collection tube was 3 ⁇ 10 5 , and 1% PBSA (containing 1 %BSA in PBS) to prepare LAG3 antibody dilutions with final concentrations of 0.0123nM, 0.123nM, 1.23nM, 3.7nM, 11.1nM, 33.3nM, 100nM, and 300nM, and incubate with 293T cells expressing LAG3 on ice for 1 hour.
  • PBSA containing 1 %BSA in PBS
  • FITC goat anti-human IgG purchased from Jackson, catalog number: 109-095-098
  • 100 ⁇ L FITC goat anti-human IgG purchased from Jackson, catalog number: 109-095-098
  • 200 ⁇ L of 1% PBSA was added to resuspend the cells, and the fluorescent signal was detected with the FITC channel on the flow cytometer.
  • Table 3 shows the EC 50 of the binding efficiency of each anti-LAG3 antibody to the 293T-LAG3 surface antigen.
  • Table 3 The binding activity of anti-LAG3 antibody to 293T-LAG3 surface antigen detected by flow cytometry
  • the anti-LAG3 antibody can effectively bind the target LAG3 protein on the surface of host cell 293T-LAG3, and the binding activity of anti-LAG3 antibodies H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT) to the surface antigen of 293T-LAG3 Comparable to the positive control antibody Relatlimab.
  • hIgG1 manufactured by Akeso, batch number: 20190410
  • a final concentration of 300 nM added to each tube according to the experimental design, and incubate 100 ⁇ L of each tube on ice for 1 h; add 200 ⁇ L of 1% PBSA to the incubated Raji cells, centrifuge at 600 ⁇ g for 5 min, and remove supernatant.
  • Raji-PDL1 cells were conventionally subcultured; revived PBMCs were cultured with 10 mL of 1640 complete medium, and stimulated with 0.5 ⁇ g/mL SEB (Staphylococcal enterotoxin B) (Denotec, product number: S010201) for two days.
  • Raji-PDL1 cells were treated with 25 ⁇ g/mL MMC (Stressmarq, product number: SIH-246-10MG), and placed in a 37°C, 5% CO 2 carbon dioxide incubator for 1 hour; PBMCs were collected after being stimulated by SEB for 2 days and treated with MMC.
  • the 1-hour Raji-PDL1 cells were washed twice with PBS and resuspended in complete medium (ie RPMI1640+10% FBS) for counting.
  • PBMC and Raji-PDL1 were each added to a U-shaped 96-well plate (Corning, model: 3799) at 10 ⁇ 10 4 cells/well for co-cultivation.
  • antibodies according to the experimental design the final concentration of each antibody is 300nM, 30nM, 3nM when used alone or in combination
  • co-cultivate in the incubator for 3 days; after 3 days, centrifuge at 1200rpm for 5min, collect the cell culture supernatant, and perform ELISA.
  • IFN- ⁇ detection the final concentration of each antibody is 300nM, 30nM, 3nM when used alone or in combination
  • the mixed culture of human PBMC and Raji-PDL1 cells can promote the secretion of IFN- ⁇ in PBMC, and the addition of antibodies in the mixed culture system can significantly induce PBMC to further secrete IFN- ⁇ .
  • anti-LAG3 antibodies H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) combined with 14C12H1L1 (hG1TM) and the positive control antibody Relatlimab combined with 14C12H1L1 (hG1TM) could promote IFN- ⁇ secretion. And its activity is quite.
  • Raji-PDL1 cells were conventionally subcultured; revived PBMCs were cultured with 10 mL of 1640 complete medium, and 0.5 ⁇ g/mL of SEB (Staphylococcal enterotoxin B, purchased from Denotec, Cat. No.: S010201) was added to stimulate for two days.
  • Raji-PDL1 cells were treated with 25 ⁇ g/mL MMC (Stressmarq, product number: SIH-246-10MG), and placed in a 37° C., 5% CO 2 carbon dioxide incubator for 1 hour.
  • PBMC and Raji-PDL1 were each added to a U-shaped 96-well plate (Corning, model: 3799) at 10 ⁇ 10 4 cells/well for co-culture.
  • add antibodies according to the experimental design the final concentration of each antibody is 300nM, 30nM, 3nM when used alone or in combination), and co-culture for 3 days; after 3 days, centrifuge at 1200rpm for 5min, collect the cell culture supernatant, and use ELISA method to detect IL-2 detection.
  • the mixed culture of human PBMCs (from healthy donors) and Raji-PDL1 cells can promote the secretion of IL-2 in PBMCs, and adding antibodies to the mixed culture system can significantly induce PBMCs to further increase
  • the secretion of IL-2 has a significant dose-dependent relationship.
  • anti-LAG3 antibodies H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT) combined with 14C12H1L1 (hG1TM), positive control Relatlimab Both 14C12H1L1 (hG1TM) can promote IL-2 secretion, and their activities are equivalent.
  • Jurkat-NFAT-PD1-LAG3 cells and Raji cells were used as reporter gene system. After adding SEE superantigen, the TCR-NFAT signaling pathway was activated to induce the expression of luciferase.
  • LAG-3 on Jurkat cells binds to MHC-II on Raji cells, inhibits the NFAT signaling pathway, and down-regulates the expression of luciferase. The antibody releases inhibition and upregulates luciferase expression by specifically binding to LAG-3.
  • Jurkat-NFAT-PD1-LAG3 cells were inoculated into black-bottom 96-well plates (Corning, model: 3916) at 105 cells/well, 30 ⁇ L/well; antibodies were added according to the experimental design (final concentrations were 0.3nM, 3nM, 300nM) , 10 ⁇ L/well, and placed in a 37°C, 5% CO 2 incubator for pre-incubation for 30 min.
  • SEE Staphylococcal Enterotoxins E, purchased from Toxin Technology, product number: ET404
  • the SEE-treated Raji cells at 2 ⁇ 104 cells/well (40 ⁇ L/well) to the above-mentioned 96-well plate containing Jurkat-NFAT-PD1-LAG3 cells, the final volume of the system is 80 ⁇ L, mix well Afterwards, they were placed in a 37°C, 5% CO 2 incubator and incubated for 6h. After the incubation, the culture plate was taken out, equilibrated to room temperature, 80 ⁇ L/well of Bright-Glo TM Luciferase Assay System (purchased from Promega, product number: E2650) was added, and the RLU value was read after incubation in the dark for 2 minutes.
  • the isotype control hIgG1DM is made by Akeso Bio, batch number: 20181107
  • the isotype control hG4WT is made by Akeso Bio, batch number: 20190910.
  • anti-LAG antibodies H7L8 (hG4WT), H7L9 (hG4WT), H7L10 (hG4WT), and the positive control antibody Relatlimab can block the interaction between LAG-3 and MHC-II, and up-regulate the expression of luciferase , and the activities of anti-LAG antibodies H7L8 (hG4WT), H7L9 (hG4WT), and H7L10 (hG4WT) were better than the control antibody Relatlimab.

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PCT/CN2022/122185 2021-09-29 2022-09-28 抗lag3抗体、药物组合物及用途 Ceased WO2023051621A1 (zh)

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