Search

CN-121991227-A - Anti-PD-1 antibody, CAR-T cell, preparation method and application thereof

CN121991227ACN 121991227 ACN121991227 ACN 121991227ACN-121991227-A

Abstract

Anti-PD-1 antibodies, CAR-T cells, methods of making and uses thereof are provided. The anti-PD-1 antibodies comprise LCDR-1-3 of SEQ ID NOS 1-3, respectively, and HCDR-1-3 of SEQ ID NOS 4-6, respectively. The CAR-T cells herein express a novel element that efficiently blocks PD-1, which is a single chain antibody that cell membrane anchored targeting PD-1. The chimeric antigen receptor and the cell membrane anchored anti-PD-1 scFv are linked by a 2A shear protein. The membrane anchoring type anti-PD-1 scFv expressed by the CAR-T cells can almost completely block the expression of PD-1 on the surface of the T cell membrane, and further block the signal path combined by PD-1/PD-L1, so that the capacity of the CAR-T cells for antagonizing T cell depletion caused by tumors is enhanced, and the aim of enhancing the anti-tumor effect of the CAR-T cells is fulfilled.

Inventors

  • TIAN XIAOLI

Assignees

  • 上海怡豪生物科技有限公司

Dates

Publication Date
20260508
Application Date
20241108

Claims (16)

  1. 1. An anti-PD-1 antibody or antigen-binding fragment thereof comprising light chain complementarity determining regions LCDR 1-3 and heavy chain complementarity determining regions HCDR 1-3, wherein LCDR-1 has an amino acid sequence of SASSSVSYMH (SEQ ID NO: 1), LCDR-2 has an amino acid sequence of GTSNLAS (SEQ ID NO: 2), LCDR-3 has an amino acid sequence of QQWSSYPLT (SEQ ID NO: 3), HCDR-1 has an amino acid sequence of DHIIN (SEQ ID NO: 4), HCDR-2 has an amino acid sequence of RIYPVSGETNYNQKFKG (SEQ ID NO: 5), and HCDR-3 has an amino acid sequence of WDGYYAMDY (SEQ ID NO: 6).
  2. 2. The anti-PD-1 antibody or antigen-binding fragment thereof according to claim 1, which comprises a light chain variable region comprising the amino acid sequence shown in SEQ ID No. 7 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID No. 7 and/or a heavy chain variable region comprising the amino acid sequence shown in SEQ ID No. 8 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID No. 8; Optionally, the light chain variable region comprises the amino acid sequence shown in SEQ ID NO. 7 and/or the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 8.
  3. 3. The anti-PD-1 antibody or an antigen-binding fragment thereof according to claim 1 or 2, wherein the antigen-binding fragment is one or more of a Fab ', fab, F (ab') 2, fd fragment, dAb fragment, camelid antibody, nanobody and single chain Fv, preferably the single chain Fv comprises the amino acid sequence shown in SEQ ID NO. 16 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO. 16.
  4. 4. A fusion comprising a first portion or a first portion and a second portion, wherein: The first moiety comprises, from the N-terminus to the C-terminus, an optional first signal peptide, an anti-PD-1 antibody according to any one of claims 1-3, or an antigen-binding fragment thereof, and a first transmembrane domain; The second portion comprises an optional second signal peptide and a chimeric antigen receptor comprising, from N-terminus to C-terminus, an antigen binding domain, a second transmembrane domain, a co-stimulatory domain, and a signaling domain; when a second portion is present, the first portion is at the C-or N-terminus of the second portion, preferably the second portion is at the N-terminus of the first portion; Preferably, the anti-PD-1 antibody or antigen-binding fragment thereof is in the form of an scFv, Preferably, the anti-PD-1 antibody or antigen-binding fragment thereof is linked to the first transmembrane domain directly or through a linker; preferably, the first transmembrane domain is a CD8 a transmembrane domain; Preferably, the first and second portions are linked by a self-cleaving protein; Preferably, the self-cleaving protein is a 2A shear protein; preferably, the 2A shear protein is selected from the group consisting of P2A, T a and F2A; Preferably, the first signal peptide and the second signal peptide are the same or different, optionally each independently being a CD8 precursor; Preferably, the antigen binding domain binds a cell surface cancer antigen that is one or more of Mesothelin, GD2, CD171, CD19, CD20, BCMA, GPC3, TERT, PTEN, PD-1, PD-L1, NKG 2D-ligand 、CD44v6、FR、CD138、PSMA、NY-ESO、EGFR、CEA、HER2、CD22、CD30、CD123、CD5、CD7、CD33、CEA、EGFR、BRAF、HER-2、MUC1、PSCA、GPC3, or VEGF; Preferably, the co-stimulatory domain is a CD28 co-stimulatory domain, a 4-1BB co-stimulatory domain, or a combination of a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain; Preferably, the signaling domain is a CD3 zeta signaling domain; preferably, the second transmembrane domain is a CD8 a transmembrane domain; preferably, the CD28 co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO. 9 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO. 9; the 4-1BB co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO. 10 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence shown in SEQ ID NO. 10; preferably, the co-stimulatory domain comprises a combination of CD28 and 4-1 BB; preferably, the CD 8. Alpha. Transmembrane domain comprises the amino acid sequence of SEQ ID NO. 11 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO. 11; Preferably, the antigen binding domain of the chimeric antigen receptor comprises HCDR-1 sequence DYAMH (SEQ ID NO: 18), HCDR-2 sequence VISTYNGNINYNQKFKG (SEQ ID NO: 19), HCDR-3 sequence GGYDGTGFDY (SEQ ID NO: 20), LCDR-1 sequence SASSSISYMH (SEQ ID NO: 21), LCDR-2 sequence DTSKLAS (SEQ ID NO: 22), LCDR-3 sequence QQWSSPPT (SEQ ID NO: 23), preferably, the antigen binding domain of the chimeric antigen receptor comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 12; Preferably, the fusion comprises the structure of a CD8 signal peptide-anti-MSLN scFv-CD8 a transmembrane domain-CD 28 co-stimulatory domain-4-1 BB co-stimulatory domain-CD 3 zeta signaling domain-P2A sheaf of proteins-CD 8 signal peptide-anti-PD-1 scFv-CD8 a transmembrane domain.
  5. 5. A composition or conjugate comprising an anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1-3; preferably, the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier; preferably, the conjugate further comprises a cytotoxic agent.
  6. 6. A first nucleic acid encoding an anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1-3, preferably the first nucleic acid comprises the nucleotide sequence set forth in SEQ ID No. 13 or a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID No. 13.
  7. 7. A second nucleic acid encoding the fusion according to claim 4; Preferably, the coding sequence of the first part comprises the nucleotide sequence shown as SEQ ID NO. 14 or a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO. 14; Preferably, the coding sequence of the second part comprises the nucleotide sequence shown as SEQ ID NO. 15 or a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO. 15; Preferably, the second nucleic acid comprises the nucleotide sequence shown as SEQ ID NO.24 or a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO. 24.
  8. 8. An expression cassette comprising a first nucleic acid according to claim 6 or a second nucleic acid according to claim 7 and further comprising a control element operably linked to said nucleic acids, preferably the control element is one or more of a promoter, an enhancer and a terminator, preferably wherein said promoter is selected from one or more of a CMV and an EF1a promoter.
  9. 9. A vector comprising the expression cassette according to claim 8; Preferably, wherein the vector is a viral vector; preferably, wherein the vector is a lentiviral vector or an adeno-associated viral vector.
  10. 10. A virus comprising an expression cassette according to claim 8 or a vector according to claim 9, preferably wherein the virus is a lentivirus or an adeno-associated virus.
  11. 11. Use of an anti-PD-1 antibody or antigen-binding fragment thereof according to any one of claims 1-3 or a composition or conjugate according to claim 5 for the manufacture of a medicament for the treatment of a disease, preferably a cancer, preferably a disease involving the PD-1/PD-L1 signaling pathway, preferably the cancer is one or more of melanoma, non-small cell lung cancer, nasopharyngeal carcinoma, glioblastoma, colon adenocarcinoma, hepatocellular carcinoma, urothelial carcinoma, multiple myeloma, ovarian carcinoma, gastric carcinoma, esophageal carcinoma, pancreatic carcinoma, renal cell carcinoma, breast carcinoma, lymphomas such as hodgkin's lymphoma and leukemia.
  12. 12. A cell comprising a first nucleic acid according to claim 6, a second nucleic acid according to claim 7, an expression cassette according to claim 8, a vector according to claim 9 or a virus according to claim 10, preferably the cell is an immune cell, preferably one or more of a T cell, a macrophage and an NK cell, preferably the T cell is one or more of a Cytotoxic T Lymphocyte (CTL), a regulatory T cell and a natural killer T cell.
  13. 13. An immune cell comprising the first moiety or the first moiety and the second moiety of claim 4, the first moiety being anchored to the cell surface by a first transmembrane domain, or the first moiety and the second moiety being anchored to the cell surface by a first and a second transmembrane domain, respectively, preferably one or more of a T cell, a macrophage and an NK cell, preferably the T cell is one or more of a Cytotoxic T Lymphocyte (CTL), a regulatory T cell and a natural killer T cell.
  14. 14. A method of preparing a cell according to claim 12 or an immune cell according to claim 13, comprising introducing into the cell a first nucleic acid according to claim 6, a second nucleic acid according to claim 7, an expression cassette according to claim 8, a vector according to claim 9 or a virus according to claim 10, thereby blocking expression of PD-1 on the T cell surface and thereby blocking the PD-1/PD-L1 binding signal pathway.
  15. 15. The method of claim 14, comprising one or more of 1) viral vector construction, 2) lentiviral packaging using 293T cells, 3) PBMC isolation and T cell sorting, 4) T cell activation, 5) lentiviral transfection of activated T cells, 6) CAR-T cell culture after infection, detection of CAR positivity, 6) cell killing detection of CAR-T cells, and 7) detection of tumor inhibition of CAR-T cells in a tumor model.
  16. 16. Use of an immune cell according to claim 13 in the manufacture of a medicament for the treatment and/or prophylaxis of cancer, preferably wherein the cancer is selected from liver cancer, bladder cancer, various types of leukemias, multiple myeloma and one or more of malignant lymphoma, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, renal cancer, mesothelioma, head and neck cancer, multiple squamous cell carcinoma, oesophageal cancer, colorectal cancer, melanoma, osteosarcoma, rectal cancer, anal region cancer, cholangiocarcinoma, uterine cancer, ovarian cancer, gastric cancer, prostate cancer, meningioma, pancreatic cancer, breast cancer and medulloblastoma.

Description

Anti-PD-1 antibody, CAR-T cell, preparation method and application thereof Technical Field The invention belongs to the field of biological medicine, and particularly relates to an anti-PD-1 antibody, a CAR-T cell, a preparation method and application thereof. Background T cell adoptive immunotherapy is a promising approach for cancer treatment. Immunotherapy treatment approaches utilize genetically modified isolated human T cells to enhance their specificity for specific tumor-associated antigens. Genetic modification may involve expression of chimeric antigen receptors or exogenous T cell receptors to specifically transplant antigens onto T cells. In contrast to exogenous T cell receptors, the specificity of chimeric antigen receptors is derived from the variable domains of monoclonal antibodies. Thus, T cells expressing chimeric antigen receptors (CAR-T cells) induce tumor immunoreactivity in a non-limiting manner by the major histocompatibility complex. T cell adoptive immunotherapy has been used as a clinical therapy for many cancers, including B cell malignancy, multiple myeloma, neuroblastoma, glioblastoma, advanced glioma, ovarian cancer, mesothelioma, melanoma, prostate cancer, pancreatic cancer, and the like. Despite the potential usefulness of CAR-T cells as cancer treatments, adoptive immunotherapy with CAR-T cells is also a great challenge in the treatment of solid tumors. The key problems with CAR-T cell treatment of solid tumors are the presence of tumor heterogeneity and tumor immunosuppressive microenvironment. The tumor microenvironment, which consists of immune checkpoint mediated immunosuppression signals, plays an important role in promoting tumor immune escape. CAR-T cells, upon activation by antigen, inhibit their proliferation and cytokine secretion functions by binding to the relevant ligand. In particular on tumor infiltrating CAR-T cells, these inhibitory molecules are expressed further up-regulated, thereby greatly limiting the anti-tumor immune effect of CAR-T cells at the site of solid tumors. PD-1 is one of the representative inhibitors expressed on the surface of T cells, and binds to PD-L1 of tumor cells to mediate the depletion of T cells. In view of this, drugs directed to the PD-1/PD-L1 binding signaling pathway have been marketed in various forms, such as PD-1 and PD-L1 mab drugs, CAR-T cells that secrete PD-1 or PD-L1 mab, and the like. However, currently, there is a need for a novel drug capable of blocking the PD-1/PD-L1 signaling pathway, which is capable of solving the above problems, because of the short half-life of the drug requiring multiple injections (antibody drug), or because of uncontrollable secretion and high safety risk (CAR-T secreting antibody). Disclosure of Invention CAR-T currently faces many challenges in the treatment of solid tumors, the key reasons among which are tumor heterogeneity and tumor immunosuppressive microenvironment. When CAR-T cells enter the tumor, the ligand expressed on the tumor surface will bind to the inhibitor expressed on the T cell surface, thereby causing T cell depletion. Therefore, to solve the problem of poor treatment of CAR-T cells in solid tumors, the problem of T cell depletion by the tumor must be solved. PD-1 is one of the putative T cell inhibitors, and monoclonal anti-tumor drugs aiming at PD-1 are already marketed. Furthermore, CAR-T cells secreting PD-1 antibodies have been reported to enter the clinic. However, drugs directed against the PD-1/PD-L1 signaling pathway have been marketed, including those in the clinical research stage, either with short half-life and large molecular weight (e.g., antibody drugs) or with uncontrollable secretion (e.g., antibody-secreting CAR-T cells). It is an object of the present invention to provide anti-PD-1 antibodies which are particularly suitable for use with chimeric antibody receptors, which can completely block the expression of cell surface PD-1. It is another object of the invention to synchronize the expression of anti-PD-1 scFv with a CAR by means of an initial cell membrane anchoring, which anchoring is non-secretory to anti-PD-1 scFv and can be synchronously and continuously expressed on the surface of T cells together with the CAR. The data of the examples show that the anchored anti-PD-1 scFv almost completely blocks the expression of the cell surface PD-1, solving the problems existing in the prior art. The invention also provides a preparation method and application of the CAR-T cell for synchronously expressing the novel element capable of effectively blocking PD-1. In a first aspect, provided herein is an anti-PD-1 antibody or antigen-binding fragment thereof comprising light chain complementarity determining regions (LCDRs) 1-3 and heavy chain complementarity determining regions (HCDRs) 1-3, wherein LCDR-1 has an amino acid sequence of SASSSVSYMH (SEQ ID NO: 1), LCDR-2 has an amino acid sequence of GTSNLAS (SEQ ID NO: 2), LCDR-3 has an amino acid sequence