WO2024017362A1 - Récepteur antigénique chimérique ciblant gprc5d et/ou bcma, et son utilisation - Google Patents

Récepteur antigénique chimérique ciblant gprc5d et/ou bcma, et son utilisation Download PDF

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WO2024017362A1
WO2024017362A1 PCT/CN2023/108544 CN2023108544W WO2024017362A1 WO 2024017362 A1 WO2024017362 A1 WO 2024017362A1 CN 2023108544 W CN2023108544 W CN 2023108544W WO 2024017362 A1 WO2024017362 A1 WO 2024017362A1
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cells
seq
chimeric antigen
antigen receptor
car
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PCT/CN2023/108544
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Chinese (zh)
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王义芳
姜福伟
刘婷婷
汪琴
王超
王庆杨
杨翠青
曹卓晓
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上海先博生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of cell therapy, specifically to chimeric antigen receptors targeting GPRC5D and/or BCMA and their applications.
  • GPRC5D is a G protein-coupled receptor, class C, group 5, member D (G protein-coupled receptor, class C, group 5, member D) and is an orphan receptor. Studies have shown that GPRC5D is associated with cancer. It is specifically highly expressed in myeloma but barely expressed in normal tissues. GPRC5D is a potential cancer therapeutic target.
  • BCMA is a B-cell maturation antigen, a member of the tumor necrosis family receptor (TNFR) expressed by cells belonging to the B cell lineage. After binding to a ligand (such as APRIL or BAFF), it can activate B cell proliferation and survival. BCMA is specifically highly expressed on the surface of plasma cells and multiple myeloma cells. Anti-BCMA CAR T has been used to treat multiple myeloma. However, due to the heterogeneity of tumors and the downregulation of BCMA expression during treatment, some patients still have the possibility of recurrence after receiving Anti-BCMA CAR T cell therapy.
  • TNFR tumor necrosis family receptor
  • the present disclosure provides a chimeric antigen receptor targeting GPRC5D or targeting GPRC5D and BCMA, its encoding nucleic acid, and engineered immune effector cells, which have good binding effects on GPRC5D and/or BCMA antigens and are effective against tumors. It has obvious cell killing effect and has broad application prospects.
  • the present disclosure provides a chimeric antigen receptor (CAR), which includes a first polypeptide, from the N-terminus to the C-terminus, the first polypeptide includes: an extracellular antigen-binding domain, a hinge region, a trans- a membrane domain and at least one signal transduction domain, wherein the extracellular antigen-binding region includes a first antigen-binding domain that specifically binds GPRC5D.
  • CAR chimeric antigen receptor
  • the extracellular antigen-binding domain of the first polypeptide also includes a second antigen-binding domain that specifically binds BCMA; optionally, from the N-terminus to the C-terminus, the extracellular antigen-binding structure Domains include first antigen binding domain-linker-second antigen binding domain, or second antigen binding domain-linker-first antigen binding domain.
  • the linker is GGGGSGGGGSGGGGSGGGGS.
  • the chimeric antigen receptor further includes a second polypeptide, from the N-terminus to the C-terminus, the second polypeptide includes: an extracellular antigen-binding domain, a hinge region, a transmembrane domain and at least A signal transduction domain, wherein the extracellular antigen-binding domain includes a second antigen-binding domain that specifically binds BCMA.
  • the first polypeptide and the second polypeptide are operably linked; optionally, the second polypeptide is operably linked to the N-terminus of the first polypeptide, or The first polypeptide is operably linked to the N-terminus of the second polypeptide; preferably, the first polypeptide and the second polypeptide are operably linked through a self-cleaving peptide; more preferably, Upon cleavage of the self-cleaving peptide, the first polypeptide and the second polypeptide form a dimer.
  • the first polypeptide and/or the second polypeptide :
  • the hinge region contains the CD8 hinge region
  • the transmembrane domain contains the NKG2D transmembrane region
  • At least one signaling domain comprises a 2B4 costimulatory domain (natural killer cell receptor 2B4 costimulatory domain) and/or CD3 ⁇ .
  • the hinge region comprises the sequence set forth in SEQ ID NO: 53.
  • the transmembrane domain comprises the sequence set forth in SEQ ID NO: 64.
  • the signaling domain comprises the 2B4 costimulatory domain of the sequence set forth in SEQ ID NO:65 and/or the CD3 ⁇ of the sequence set forth in SEQ ID NO:56.
  • the first polypeptide and/or the second polypeptide :
  • the hinge region contains the CD8 hinge region
  • the transmembrane domain contains the CD8 transmembrane region
  • At least one signaling domain comprises a CD137 costimulatory domain and/or CD3 ⁇ .
  • the hinge region comprises the sequence set forth in SEQ ID NO: 53.
  • the transmembrane domain comprises the sequence set forth in SEQ ID NO: 54.
  • the signaling domain comprises the CD137 costimulatory domain of the sequence set forth in SEQ ID NO:55 and/or the CD3 ⁇ of the sequence set forth in SEQ ID NO:56.
  • the first antigen binding domain comprises LCDR1, LCDR2, and LCDR3 of the light chain variable region (VL) and HCDR1, HCDR2, and HCDR3 of the heavy chain variable region (VH), wherein the light chain variable region
  • the region includes the LCDRs of the light chain variable region of the sequence shown in SEQ ID NO: 9, 11, 13 or 15; and, the heavy chain variable region includes the sequence shown in SEQ ID NO: 10, 12, 14 or 16 HCDRs of the heavy chain variable region of the sequence shown.
  • the first antigen-binding domain includes the following combinations of LCDRs and HCDRs as defined by the Kabat numbering system:
  • the first antigen binding domain comprises a VL as set forth in SEQ ID NO: 9, 11, 13 or 15 and a VH as set forth in SEQ ID NO: 10, 12, 14 or 16;
  • VL and VH combinations there are the following VL and VH combinations:
  • the VL and VH are connected by a glycine- and serine-rich linking peptide GGGGSGGGGSGGGGS.
  • the first antigen binding domain comprises CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 comprise HCDR1, HCDR2 and HCDR3 of the VHH sequence set forth in any one of SEQ ID NOs: 32 to 39;
  • the HCDR1, HCDR2 and HCDR3 are selected from the following combinations:
  • the first antigen-binding domain comprises a sequence set forth in any one of SEQ ID NOs: 32 to 39 or has a 99%, 98, or 98% similarity to any one of SEQ ID NOs: 32 to 39. Sequences that are %, 97%, 96%, 95%, 90%, 85% or 80% identical.
  • the second antigen binding domain comprises CDR1, CDR2 and CDR3, the CDR1, CDR2 and CDR3 comprise the HCDR1, HCDR2 and HCDR3 of the VHH sequence shown in any one of SEQ ID NO: 1-2;
  • the HCDR1, HCDR2 and HCDR3 are selected from the following combinations:
  • the second antigen-binding domain comprises a sequence as set forth in any one of SEQ ID NOs: 1-2 or has a 99%, 98% similarity to any one of SEQ ID NOs: 1-2. Sequences that are %, 97%, 96%, 95%, 90%, 85% or 80% identical.
  • the first and second antigen binding domains are humanized antibodies.
  • the C-terminus of the chimeric antigen receptor also contains an operably linked exogenous cytokine, and the exogenous cytokine is selected from the group consisting of IL2, IL4, IL6, IL7, IL10, IL11, IL12, IL15, IL18 and IL21; preferably, the cytokine is selected from IL-15, and more preferably, the IL15 has the sequence shown in SEQ ID NO: 58.
  • the C-terminus of the chimeric antigen receptor is operably linked to the cytokine through a self-cleaving peptide, the self-cleaving peptide comprising a 2A peptide; more preferably, the self-cleaving peptide comprises a T2A peptide or P2A peptide.
  • the chimeric antigen receptor has the sequence shown in any one of SEQ ID NOs: 59-63 and 66-76, or is identical to any one of SEQ ID NOs: 59-63, 66-76 Sequences shown are sequences with 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity.
  • the present disclosure provides an isolated nucleic acid construct encoding the aforementioned chimeric antigen receptor.
  • the present disclosure provides a vector comprising the aforementioned nucleic acid construct.
  • the present disclosure provides an immune effector cell comprising the expressed chimeric antigen receptor.
  • the immune effector cells are selected from the group consisting of T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, Neutrophils, macrophages and dendritic cells; preferably, the immune effector cells of the present disclosure are NK cells.
  • the present disclosure provides a method for preparing the immune effector cells expressing the chimeric antigen receptor, including introducing the nucleic acid construct or vector encoding the chimeric antigen receptor into the immune effector cells; further, The step of inducing expression of the chimeric antigen receptor may also be included.
  • the present disclosure provides a pharmaceutical composition, which includes the aforementioned chimeric antigen receptor, nucleic acid construct, vector and immune effector cells; further, it also includes a pharmaceutically acceptable carrier.
  • the present disclosure provides a method for treating tumors or cancer, comprising administering to a patient an effective amount of the aforementioned chimeric antigen receptor, nucleic acid construct, vector, immune effector cell and pharmaceutical composition; preferably, the tumor Or the cancer is selected from B-cell lymphoma; more preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.
  • B-cell lymphoma more preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.
  • the present disclosure provides the use of the aforementioned chimeric antigen receptor, nucleic acid construct, vector, immune effector cell and pharmaceutical composition in the preparation of a drug for treating tumors or cancers, the tumors or cancers being selected from B cells Lymphoma; preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.
  • B cells Lymphoma preferably, the B-cell lymphoma is multiple myeloma (MM); most preferably, the multiple myeloma is refractory or relapsed multiple myeloma.
  • the present disclosure provides immune effector cells and immune checkpoint inhibitors, or immune effector cells and CBL-b inhibitors, or immune effector cells and immune checkpoint inhibitors and CBL-b inhibitors, in preparation Application in medicines for the treatment of cancer or tumors.
  • the immune effector cells are selected from the group consisting of T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, Neutrophils, macrophages and dendritic cells; preferably, the immune effector cells of the present disclosure are NK cells.
  • the immune effector cells express a chimeric antigen receptor, preferably a chimeric antigen receptor targeting BCMA or GPRC5D, more preferably a chimeric antigen receptor targeting BCMA and GPRC5D; more preferably , the immune effector cells express the chimeric antigen receptor described in the first aspect of the present disclosure.
  • the immune checkpoint inhibitor is soluble PD-L1.
  • the immune checkpoint inhibitor is further fused to a cytokine.
  • the cytokine is IL15.
  • the immune checkpoint inhibitor includes a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77.
  • the CBL-b inhibitor is a chemical described in formula (I) or a pharmaceutically acceptable salt of a compound shown in formula (I):
  • the cancer or tumor is selected from solid tumors or hematological tumors.
  • the hematological neoplasm is selected from B-cell lymphoma.
  • the B-cell lymphoma is multiple myeloma.
  • the multiple myeloma is refractory or relapsed multiple myeloma.
  • the present disclosure provides a method of treating cancer or tumors, the method comprising administering an effective amount of immune effector cells to a subject, the method further comprising: (1) before administering the immune effector cells Use a CBL-b inhibitor to stimulate the immune effector cells, and/or, (2) administer an effective amount of an immune checkpoint inhibitor to the subject.
  • the immune effector cells express a chimeric antigen receptor, preferably a chimeric antigen receptor targeting BCMA or GPRC5D, more preferably a chimeric antigen receptor targeting BCMA and GPRC5D; more preferably , the immune effector cells express the chimeric antigen receptor described in the first aspect of the present disclosure.
  • the immune checkpoint inhibitor is soluble PD-L1.
  • the immune checkpoint inhibitor is further fused to a cytokine.
  • the cytokine is IL15.
  • the immune checkpoint inhibitor includes a sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 77.
  • the CBL-b inhibitor is a chemical described in formula (I) or a pharmaceutically acceptable salt of a compound shown in formula (I):
  • the cancer or tumor is selected from solid tumors or hematological tumors.
  • the hematological neoplasm is selected from B-cell lymphoma.
  • the B-cell lymphoma is multiple myeloma.
  • the multiple myeloma is refractory or relapsed multiple myeloma.
  • the present disclosure provides a method for enhancing the expression of chimeric antigen receptors by immune effector cells and/or enhancing the killing ability of immune effector cells.
  • the method includes activating the immune effector cells and/or transfecting the method.
  • the immune effector cells are contacted with the CBL-b inhibitor under the conditions of the chimeric antigen receptor.
  • the CBL-b inhibitor is a chemical described in formula (I) or a pharmaceutically acceptable salt of a compound shown in formula (I):
  • the immune effector cells express a chimeric antigen receptor, preferably a chimeric antigen receptor targeting BCMA or GPRC5D, more preferably a chimeric antigen receptor targeting BCMA and GPRC5D; more preferably , the immune effector cells express the chimeric antigen receptor described in the first aspect of the present disclosure.
  • the immune effector cells are selected from the group consisting of T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, Neutrophils, macrophages and dendritic cells; preferably, the immune effector cells of the present disclosure are NK cells.
  • the present disclosure provides chimeric antigen receptors targeting GPRC5D, which can be used to kill tumor cells expressing GPRC5D antigen and have broad application prospects.
  • the present disclosure provides chimeric antigen receptors targeting GPRC5D and BCMA to enhance the killing ability of the chimeric antigen receptors and improve treatment failure or recurrence of BCMA CART due to tumor heterogeneity or BCMA antigen escape.
  • the present disclosure compares and screens the antibody sequences, CAR elements and/or CAR structures of the aforementioned chimeric antigen receptors to improve the binding, CAR expression, proliferation or killing effects of the chimeric antigen receptors to target antigens (especially long-term lethality).
  • the chimeric antigen receptor described in the present disclosure is particularly suitable for NK cells and is suitable for providing "off-the-shelf" GPRC5D CAR or "off-the-shelf” GPRC5D ⁇ BCMA CAR, which can overcome the high production cost and preparation process of existing autologous CAR T treatments. Complex and potentially toxic or adverse events.
  • the present disclosure combines immune effector cells expressing chimeric antigen receptors with CBL-b inhibitors and/or PDL1-IL15 fusion protein to enhance the killing effect of the immune effector cells on tumor cells.
  • Figure 1 is a schematic structural diagram of the dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D 1A-1D;
  • BI-CAR dual-target chimeric antigen receptor
  • FIG. 2 Schematic structural diagram of the dual-target chimeric antigen receptor (BI-CAR) in the form of NKG2D_TM targeting BCMA and GPRC5D;
  • BI-CAR dual-target chimeric antigen receptor
  • Figure 4A to Figure 4B show the results of multiple rounds of killing experiments by BI-CAR-NK on MOLP8 cells and RPMI 8226 cells;
  • Figure 5 is a schematic structural diagram of the dual-target chimeric antigen receptor (BI-CAR) targeting BCMA and GPRC5D 5A-5B;
  • BI-CAR dual-target chimeric antigen receptor
  • Figure 7A to Figure 7F show the results of multiple rounds of killing experiments by BI-CAR-NK on NCI H929 cells, RPMI 8226 cells, MOLP8 cells and tumor heterogeneous cells;
  • Figure 10A to Figure 10D The positive rate of BI-CAR-NK and the effect of cryopreservation on the positive rate and activity rate of BI-CAR-NK.
  • FIG 11A to Figure 11C The killing activity of BI-CAR-NK and the effect of cryopreservation on the killing activity of BI-CAR-NK.
  • Figure 14 is a schematic diagram of the BCMA and GPRC5D single-targeting CAR structure
  • Figure 16A to Figure 16D Anti-tumor efficacy test of BI-CAR-NK and CBL-b inhibitor and/or PDL1-IL15 in NCI H929 tumor model.
  • compositions comprising, “comprises,” and “having” are used interchangeably herein and are intended to indicate an inclusive nature, meaning that there may be elements other than those listed. At the same time, it should be understood that the descriptions of “including”, “including” and “having” are used in this article, and the solution of “consisting of” is also provided.
  • a composition including A and B should be understood as the following technical solution: a composition composed of A and B, as well as a composition containing other components in addition to A and B, all fall into the category Within the scope of the aforementioned "a composition”.
  • BCMA B cell maturation antigen, which is a member of the tumor necrosis factor receptor family.
  • BCMA is mainly expressed on the surface of late B cells, short-lived proliferating plasmablasts and long-lived plasma cells. It is not expressed on initial B cells, CD34-positive hematopoietic stem cells and other normal tissue cells, but it is highly expressed on MM cells. , by mediating downstream signaling pathways, it plays a key role in the survival, proliferation, metastasis and drug resistance of MM cells, so BCMA is an ideal antigen target for the treatment of MM.
  • An exemplary human BCMA sequence can be found at GenBank Protein Accession No: NP_001183.2.
  • GPRC5D refers to G protein-coupled receptor C5 family subtype D, which is an orphan receptor and is a 7-transmembrane protein with no known ligand. GPRC5D is highly expressed on the surface of primary multiple myeloma cells, while its expression in normal tissues is limited to the hair follicle area. Studies have shown that 65% of multiple myeloma patients have GPRC5D exceeding the 50% expression threshold. With this feature, GPRC5D has become a potential target for the treatment of MM. An exemplary human GPRC5D sequence can be found at GenBank Protein Accession No: NP_061124.1.
  • chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to be expressed on immune effector cells and specifically bind an antigen, which contains at least (1) an extracellular antigen-binding domain, such as an antibody The heavy chain variable region and/or light chain variable region, (2) the transmembrane domain that anchors the CAR into immune effector cells, and (3) the intracellular signaling domain.
  • CARs are able to utilize extracellular antigen-binding domains to redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted manner.
  • signal peptide herein refers to the fragment of a protein or polypeptide used to guide the protein or polypeptide into the secretory pathway and transferred to the cell membrane and/or cell surface.
  • the signal peptide is a CD8 ⁇ signal peptide, optionally, the CD8 ⁇ signal peptide has at least 80% identity (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%): MALPVTALLLPLALLLHAARP.
  • the "hinge region" of a chimeric antigen receptor generally refers to any oligopeptide or polypeptide that serves to connect the transmembrane region and the antigen-binding region. Specifically, the hinge region serves to provide greater flexibility and accessibility to the antigen-binding region.
  • the hinge region may be derived in whole or in part from a natural molecule, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence.
  • the "transmembrane (TM) region" of a chimeric antigen receptor refers to the ability of the chimeric antigen receptor to be expressed on the surface of immune cells (such as lymphocytes, NK cells, or NKT cells) and to direct immune cells to target Polypeptide structure of cellular response of target cells.
  • the transmembrane domain may be natural or synthetic and may be derived from any membrane-bound or transmembrane protein.
  • the transmembrane domain enables signaling when the chimeric antigen receptor binds to the target antigen.
  • the hinge region may be a CD8 transmembrane region or an NKG2D transmembrane region.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specified function.
  • the intracellular signaling domain is responsible for primary intracellular signal transmission after the antigen-binding domain binds the antigen, leading to the activation of immune cells and immune responses.
  • the intracellular signaling domain is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is expressed.
  • Exemplary intracellular signaling domains include CD3 ⁇ .
  • host cell herein refers to a cell into which exogenous nucleic acid is introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include primary transformed cells and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical in nucleic acid content to the parent cell but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the originally transformed cells are included herein.
  • immune effector cells refers to cells involved in effector functions during immune responses.
  • Immuno effector cells are preferably capable of binding an antigen, such as an antigen expressed on a cell surface or a cell characterized by the presentation of an antigen or an antigenic peptide derived from the antigen, and mediate an immune response.
  • an antigen such as an antigen expressed on a cell surface or a cell characterized by the presentation of an antigen or an antigenic peptide derived from the antigen, and mediate an immune response.
  • such cells secrete cytokines and/or chemokines, kill microorganisms, secrete antibodies, recognize infected or cancer cells, and optionally eliminate these cells.
  • immunoreactive cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, neutrophils, macrophages Phages and dendritic cells.
  • T cells cytotoxic T cells, helper T cells, tumor-infiltrating T cells
  • B cells natural killer (NK) cells
  • natural killer (NKT) cells natural killer T (NKT) cells
  • neutrophils neutrophils
  • macrophages Phages macrophages Phages and dendritic cells.
  • immunoreactive cells are NK cells.
  • T lymphocyte and "T cell” are used interchangeably herein and refer to the major type of white blood cell that matures in the thymus and has a variety of roles in the immune system, including recognition of specific foreign antigens in the body and Activating and inactivating other immune cells in an MHC class I-restricted manner.
  • the T cell can be any T cell, such as a cultured T cell, such as a primary T cell, or a T cell from a cultured T cell line, such as Jurkat, SupT1, etc., or a T cell obtained from a mammal.
  • T cells can be CD3+ cells.
  • T cells can be any type of T cell and can be at any stage of development, including but not limited to CD4+/CD8+ dual-positive T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells) ), peripheral blood mononuclear cells (PBMC), peripheral blood leukocytes (PBL), tumor-infiltrating lymphocytes (TIL), memory T cells, naive T cells, regulatory T cells, ⁇ T cells (gamma delta T cell/ ⁇ T cell) etc.
  • helper T cells include cells such as Th3 (Treg), Th17, Th9 or Tfh cells.
  • T cells include cells such as central memory T cells (Tcm cells), effector memory T cells (Tem cells and TEMRA cells).
  • T cells may also refer to genetically engineered T cells, such as T cells modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • T cells or T-cell effector cells can also differentiate from stem cells or progenitor cells.
  • T-cell-like derived effector cells can have aspects of the T cell lineage but at the same time possess one or more functional characteristics not present in primary T cells.
  • NK cells or “natural killer cells” herein refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3).
  • adaptive NK cells and “memory NK cells” are interchangeable and refer to a subset of NK cells that are phenotypically CD3- and CD56 + -expressing NKG2C and CD57 and optionally CD16 at least one of, but lacks the expression of one or more of the following: PLZF, SYK, FceR ⁇ , and EAT-2.
  • the isolated CD56 + NK cell subset comprises expression of CD16, NKG2C, CD57, NKG2D, NCR ligand, NKp30, NKp40, NKp46, activating and inhibitory KIR, NKG2A, and/or DNAM-1.
  • CD56 + can be weakly or strongly expressed.
  • NK cells or NK cell-like effector cells can differentiate from stem cells or progenitor cells.
  • NK cell-like derived effector cells can be of the NK cell lineage in some aspects, but at the same time possess one or more functional characteristics not present in primary T cells.
  • NKT cells or “natural killer T cells” refers to CDld-restricted T cells that express the T cell receptor (TCR). Unlike conventional T cells that detect peptide antigens presented by conventional major histocompatibility (MHC) molecules, NKT cells recognize lipid antigens presented by CD1d, a non-classical MHC molecule. Two types of NKT cells are identified. Constant or type I NKT cells express a very limited repertoire of TCRs: a combination of canonical ⁇ chains (V ⁇ 24-J ⁇ 18 in humans) combined with a limited spectrum of ⁇ chains (V ⁇ 11 in humans).
  • TCR T cell receptor
  • a second NKT cell population termed non-classical or non-constant type II NKT cells, shows more uneven TCR ⁇ utilization.
  • Type I NKT cells are considered suitable for immunotherapy.
  • Adaptive or constant (type I) NKT cells can be identified using the expression of at least one or more of the following markers: TCR Va24-Ja18, Vb11, CD1d, CD3, CD4, CD8, aGalCer, CD161, and CD56.
  • autologous herein means any material derived from the same individual to which the material is subsequently reintroduced.
  • Allogeneic refers to a graft derived from a different individual of the same species.
  • KD equilibrium dissociation constant
  • high affinity generally refers to having a KD of about 10 -6 M or lower, 10 -7 M or lower, about 10 -8 M or lower, or about 10 -9 M or lower.
  • the equilibrium dissociation constant KD can be measured using methods well known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis determination.
  • antigen-binding molecule is used herein in its broadest sense and refers to a molecule that specifically binds an antigen.
  • antigen-binding molecules include, but are not limited to, antibodies or antibody mimetics.
  • Antibody mimetic refers to an organic compound or binding domain that can specifically bind to an antigen but has nothing to do with the structure of an antibody.
  • antibody mimetic includes but is not limited to affibody, affitin, affilin, and designed ankyrin repeat proteins. (DARPin), aptamer or Kunitz-type domain peptide.
  • antibody is used herein in its broadest sense and refers to a polypeptide that contains sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen. or peptide combinations.
  • Antibody includes intact antibodies and any antigen-binding fragments (i.e., “antigen-binding portions") or single chains thereof.
  • Antibodies generally comprise a glycoprotein, or an antigen-binding portion thereof, of at least two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (herein abbreviated as VH) and a heavy chain constant region.
  • the heavy chain constant region consists of three domains, CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (herein abbreviated as VL) and a light chain constant region.
  • the light chain constant region consists of one domain, CL.
  • the VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs).
  • CDRs complementarity-determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, which are arranged in the following order from the amino end to the carboxyl end: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of an antibody can mediate 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. Because the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, their antigenicity is also different. Based on this, the "immunoglobulins" in this article can be divided into five categories, or called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ chain and ⁇ chain respectively. , ⁇ chain, ⁇ chain and ⁇ chain.
  • Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • IgA can be divided into IgA1 and IgA2.
  • Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have either a kappa chain or a lambda chain.
  • Antibody herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives.
  • Such scaffolds include antibody-derived scaffolds, which contain mutations introduced to, for example, stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds, which contain, for example, biocompatible polymers. See, e.g., Korndorfer, I.P., Beste, G. & Skerra, A. (2003). Proteins, 53, 121–129.; Roque, A.C.A., Lowe, C.R. & Taipa, M.A. Antibodies and genetically engineered related molecules: production and purification. Biotechnol. Prog. .20,639-654(2004).
  • Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and
  • Antibodies herein also include antibodies that do not contain light chains, for example, those produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and Alpaca ( Heavy-chain antibodies (HCAbs) produced by camelids such as Vicugna pacos and immunoglobulin neoantigen receptors (IgNAR) found in cartilaginous fishes such as sharks.
  • HCAbs Heavy-chain antibodies
  • camelids such as Vicugna pacos and immunoglobulin neoantigen receptors (IgNAR) found in cartilaginous fishes such as sharks.
  • antibody herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, sheep Camel, ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (such as shark).
  • heavy chain antibody herein refers to an antibody lacking the light chain of a conventional antibody.
  • the term specifically includes, but is not limited to, homodimeric antibodies comprising a VH antigen binding domain and CH2 and CH3 constant domains in the absence of a CH1 domain.
  • anobody in this article refers to the natural heavy chain antibody lacking the light chain that exists in camels and other bodies. Cloning its variable region can obtain a single domain antibody consisting only of the heavy chain variable region, also known as VHH (Variable domain of heavy). chain of heavy chain antibody), which is the smallest functional antigen-binding fragment.
  • VHH domain single domain antibody
  • sdAb single domain antibody
  • multispecificity refers to the ability of an antibody or antigen-binding fragment to bind, for example, to different antigens or to at least two different epitopes on the same antigen.
  • terms such as “bispecific,” “trispecific,” “tetraspecific,” etc. refer to the number of different epitopes to which an antibody can bind.
  • conventional monospecific IgG-type antibodies have two identical antigen-binding sites (paratopes) and therefore can only bind to the same epitope (rather than to different epitopes).
  • multispecific antibodies have at least two different types of paratopes/binding sites and therefore can bind to at least two different epitopes.
  • complementarity determining region refers to the antigen-binding site of an antibody.
  • a single “specificity” may refer to one, two, three or more than three identical CDRs in a single antibody (the actual number of CDRs/binding sites in a single antibody molecule is referred to as " price").
  • a single natural IgG antibody is monospecific and bivalent because it has two identical paratopes.
  • a multispecific antibody contains at least two (different) complementarity determining regions/binding sites.
  • the term “multispecific antibody” refers to an antibody that has more than one paratope and the ability to bind two or more different epitopes.
  • multispecific antibody includes in particular bispecific antibodies as defined above, but generally also includes proteins, such as antibodies, scaffolds that specifically bind three or more different epitopes, i.e. having three or more different epitopes. Antibodies with more than three paratopes/binding sites.
  • valency herein refers to the presence of a specified number of binding sites in the antibody/antigen binding molecule. Therefore, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” refer to one binding site, two binding sites, four binding sites and six binding sites respectively in the antibody/antigen binding molecule. existence of points.
  • Fully-length antibody “intact antibody” and “intact antibody” are used interchangeably herein and refer to having a structure that is substantially similar to the structure of a native antibody.
  • Antigen-binding fragment and “antibody fragment” are used interchangeably herein. They do not have the entire structure of a complete antibody, but only include partial or partial variants of the complete antibody. The partial or partial variants have the ability to bind Antigen capabilities.
  • antigen-binding fragments or “antibody fragments” herein include, but are not limited to, Fab, F(ab')2, Fab', Fab'-SH, Fd, Fv, scFv, diabodies, and single domains Antibody.
  • humanized antibody refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase sequence homology with that of a human antibody.
  • CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) comes from a human source.
  • Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity or the ability to enhance immune response, etc.
  • the term "fully human antibody” herein refers to an antibody having variable regions in which both FRs and CDRs are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences.
  • the fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies” herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.
  • variable region herein refers to the region of the heavy or light chain of an antibody involved in enabling the antibody to bind to the antigen.
  • Heavy chain variable region is used interchangeably with “VH” and “HCVR”
  • light chain variable region is used interchangeably.
  • VL can be used interchangeably with “LCVR”.
  • the variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR). A single VH or VL domain may be sufficient to confer antigen binding specificity.
  • CDR complementarity determining region
  • HVR hypervariable regions
  • FR framework region
  • amino acid positions representing the hypervariable regions of an antibody may vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria (such as IMGT or KABAT) but not considered to be within a different set of criteria (such as KABAT or IMGT). One or more of these locations may also be found in extended hypervariable zones.
  • the invention includes antibodies containing modifications in these hybrid hypervariable positions.
  • the heavy chain variable region CDR can be abbreviated as HCDR, and the light chain variable region can be abbreviated as LCDR.
  • the variable domains of native heavy and light chains each contain four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops that connect the sheet structure , and in some cases form part of the lamellar structure.
  • the CDRs in each chain are closely held together through the FR region in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and contribute to the formation of the antibody's antigen-binding site with CDRs from other antibody chains.
  • CDR herein can be annotated and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but are not limited to AbRSA website (http://cao.labshare. cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign. cgi#results).
  • CDRs herein include overlaps and subsets of differently defined amino acid residues.
  • IMGT numbering system in this article generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., please refer to Lefranc, M.P., Pommié, C., Ruiz, M.,Giudicelli,V.,Foulquier,E.,Truong,L.,...&Lefranc,G.(2003).IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.Developmental&Comparative Immunology ,27(1),55-77.
  • IMGT international ImMunoGeneTics information system
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).
  • Chothia numbering system generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classical rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia, C., & Lesk, A.M. ( 1987). Canonical structures for the hypervariable regions of immunoglobulins. Journal of molecular biology, 196(4),901-917.).
  • amino acids herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity).
  • amino acids in each of the following groups belong to each other's conserved amino acid residues, and the substitution of amino acid residues within the group belongs to the substitution of conservative amino acids:
  • identity herein may be calculated by aligning two amino acid sequences or two nucleic acid sequences for the purpose of optimal comparison (e.g., may be the optimal
  • the alignment introduces gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence.
  • the percent identity between two sequences varies as a function of the identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap.
  • Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences. For example, using the Needlema and Wunsch algorithms already integrated into the GAP program of the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or the PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences. As another example, using the GAP program in the GCG software package (available at www.gcg.com), use the NWSgapdna.CMP matrix with gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between two nucleotide sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. You can also use the PAM120 weighted remainder table, the gap length penalty of 12, the gap penalty of 4, and the E. Meyers and W. Miller algorithms that have been incorporated into the ALIGN program (version 2.0) to determine the difference between two amino acid sequences or nucleotide sequences. The percentage of identity between.
  • nucleic acid herein includes any compound and/or substance that contains a polymer of nucleotides.
  • Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA containing two A polymer that is a mixture of one or more of these molecules.
  • Nucleic acid molecules can be linear or circular.
  • nucleic acid molecule includes both sense and antisense strands, as well as single- and double-stranded forms.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of the antibodies of the invention in vitro and/or in vivo, for example in a host or patient.
  • DNA eg cDNA
  • RNA eg mRNA
  • the mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler, CR, H.,Celik,L.,Hebich,B.,Roth,AS,Roth,RP,...&Sahin,U.(2017).Elimination of large tumors in mice by mRNA-encoded bispecific antibodies.Nature medicine,23( 7), 815-817. or EP2101823B1).
  • control sequences refers to DNA sequences required for expression of an operably linked coding sequence in a particular host organism.
  • Control sequences suitable for use in prokaryotes include, for example, a promoter, optionally an operator sequence and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
  • a nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence.
  • the DNA is operably linked to the DNA of the polypeptide; if the promoter or enhancer affects the transcription of the coding sequence, the promoter or The enhancer is operably linked to the sequence; or, if the ribosome binding site is positioned to promote translation, the ribosome binding site is operably linked to the coding sequence.
  • "operably linked” means that the DNA sequences being linked are contiguous and, with respect to a secretory leader sequence, contiguous and in reading frame. However, enhancers need not be contiguous. Ligation is achieved by ligation at appropriate restriction sites. If these sites are not present, synthetic oligonucleotide adapters or linkers are used according to conventional practice.
  • operably linked refers to the juxtaposition of two or more biological sequences of interest in such a manner that they are placed in a relationship that allows them to function in a purposeful manner, regardless of the presence or absence of a spacer or connector.
  • the term is intended to mean that the polypeptide sequences are linked in such a way that the linked product has the intended biological function.
  • two or more polypeptide sequences can be connected using a self-cleaving peptide that causes the polypeptide sequences to separate from each other after expression rather than fuse together, thereby each performing the function of the polypeptide sequence.
  • the term may also be used for polynucleotides.
  • a polynucleotide encoding a polypeptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), the term is intended to mean that the polynucleotide sequence allows the polypeptide to be produced by The polynucleotides are connected in a manner that regulates expression.
  • a regulatory sequence e.g., a promoter, enhancer, silencer sequence, etc.
  • Isolated nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule but which are present extrachromosomally or at a chromosomal location that is different from its native chromosomal location.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors”.
  • transfection or “transformation” or “transduction” herein refers to the process of transferring or introducing exogenous nucleic acid into a host cell.
  • a “transfected” or “transformed” or “transduced” cell is a cell that has been transfected, transformed or transduced with an exogenous nucleic acid.
  • the cells include primary subject cells and their progeny
  • composition refers to a preparation that is in a form effective to permit the biological activity of the active ingredients contained therein and does not contain unacceptable toxicity to the subject administered the pharmaceutical composition of additional ingredients.
  • pharmaceutically acceptable carrier herein includes any and all solvents, dispersion media, coating materials, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, Agents, salts, preservatives, pharmaceutical stabilizers, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, etc. and combinations thereof are known to those skilled in the art (see For example, Remington's Pharmaceutical Sciences, 18th ed. Mack Printing Company, 1990, pp. 1289-1329). Except where incompatible with the active ingredient, any conventional carrier is contemplated for use in the therapeutic or pharmaceutical compositions.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or lesions in the treated subject, such as cancer and tumors.
  • beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable.
  • Those in need of treatment include those already suffering from the condition or disease as well as those susceptible to the condition or disease or those in whom the condition or disease is intended to be prevented.
  • slow down, alleviation, weakening, alleviation, alleviation their meanings also include elimination, disappearance, non-occurrence, etc.
  • a “subject” herein refers to an organism undergoing treatment for a particular disease or condition as described herein.
  • a “subject” includes a mammal, such as a human, a primate (eg, a monkey), or a non-primate mammal undergoing treatment for a disease or condition.
  • an effective amount herein refers to an amount of a therapeutic agent that is effective to prevent or alleviate the symptoms of a disease or the progression of a disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • Effective amount also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions.
  • the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone.
  • a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.
  • cancer refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes both benign and malignant cancers.
  • tumor or “tumor” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” as used herein are not mutually exclusive.
  • EC50 refers to the half-maximal effective concentration, which includes the antibody concentration that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents 50% of the antibody concentration at which its maximum effect is observed and can be measured by methods known in the art.
  • CBL-b in this article refers to Casitas B-lineage lymphoma proto-oncogene-b (Casitas B-lineage lymphoma proto-oncogene-b) is an E3 ubiquitination ligase.
  • CBL-b inhibitor as used herein refers to compounds that inhibit the activity of CBL-b protein.
  • NCI H929+20% H929-hBCMA-KO heterogeneous tumor cells refers to the mixed cells of NCI H929 cells and H929-hBCMA-KO, NCI H929 cells account for 80%, and NCI H929-hBCMA-KO cells Accounting for 20%.
  • the proportions of various types of cells in the heterogeneous tumor cells of NCI H929+10% H929-hBCMA-KO and NCI H929+20% H929-hBCMA-KO are deduced in turn.
  • Recombinant candidate antibodies were captured using a Protein A chip (GE Healthcare; 29-127-558).
  • Sample and running buffer was HBS-EP+ (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25°C.
  • the sample block was set to 16°C. Both were pretreated with running buffer.
  • the protein A chip is first used to capture the antibody to be tested, then a single concentration of antigen protein is injected, the binding and dissociation process of the antibody and antigen protein is recorded, and finally Glycine pH1.5 (GE Healthcare; BR-1003-54 ) to complete chip regeneration.
  • Binding was measured by injecting different concentrations of human antigen protein in solution for 240 seconds with a flow rate of 30 ⁇ L/min, starting from 200 nM (see detailed results for actual concentrations tested), diluted 1:1, for a total of 5 concentrations.
  • Anti-BCMA nanobodies were prepared by immunizing alpacas. They were first immunized with human BCMA(Met1-Ala54)-hFc protein and then boosted with a mixture of human BCMA(Met1-Ala54)-hFc protein and cynomolgus monkey BCMA(Met1-Ala53)-hFc protein. Immunize alpacas by immunizing them. Blood was collected 1 week after each booster immunization, and serum antibody titer and specificity were detected by ELISA. After verifying that the serum binds to human BCMA-hFc and cynomolgus monkey BCMA-hFc proteins to varying degrees, subsequent phage library construction and panning were carried out.
  • VHH fragments Peripheral blood was collected, PBMC were isolated, total RNA was extracted, cDNA was prepared by reverse transcription, and nested amplification was performed to obtain VHH fragments. After both the VHH fragment and the vector are digested and ligated, the ligation product is electroporated and cultured on a plate. The E. coli library was expanded overnight. Add helper phage M13K07, culture and determine its titer. After identification by ELISA and multiple rounds of panning, clones with a high positive rate of binding to human and monkey BCMA proteins were selected. Positive clones were sent for sequencing and cluster analysis.
  • VHH antibodies that bind well to human and monkey BCMA proteins are screened and identified for humanized design.
  • IMGT http://imgt.cines.fr
  • human antibody heavy and light chain variable region germline gene database we selected heavy chain variable region germline genes with high homology to VHH nanobodies as templates.
  • the CDRs are transplanted into the corresponding human templates respectively.
  • the key amino acids in the backbone sequence are back mutated to the amino acids corresponding to the VHH nanobody to ensure the original affinity.
  • Anti-human GPRC5D monoclonal antibodies were generated by immunizing mice using conventional hybridoma methods.
  • the human GPRC5D overexpression cell line (HEK293T-hGPRC5D, constructed in-house) and the monkey GPRC5D overexpression cell line (HEK293T-cynoGPRC5D, constructed in-house) were alternately immunized, blood was collected and serum was separated, and cell-based enzyme-linked immunosorbent assay (cell-based) was used.
  • ELISA enzyme-linked immunosorbent assay
  • mice with high titers were selected, their spleen cells were fused with sp2/0 myeloma cells, and cell-based ELISA and FACS were used to screen positive clones that combined with human and monkey GPRC5D overexpressing cells and NCI-H929 cells.
  • RNA from hybridoma cells was collected, and the amino acid sequences of the light chain and heavy chain variable regions of the positive clones were determined and clustered. Based on the sequenced light chain and heavy chain variable region sequences, a chimeric antibody containing the human IgG1 constant region was constructed, and human and monkey GPRC5D overexpression cells and NCI-H929 cells were screened using cell-based ELISA and FACS, which are well known to those skilled in the art. All have binding monoclonal antibodies for subsequent humanization design.
  • IMGT http://imgt.cines.fr
  • human antibody heavy and light chain variable region germline gene database we selected heavy chain and light chain variable region germline genes with high homology to mouse antibodies.
  • the CDRs of the mouse antibody are transplanted into the corresponding human template respectively.
  • the key amino acids in the FR region are back mutated to the corresponding amino acids of the mouse antibody to ensure the original affinity.
  • Sites prone to chemical modification have been point mutated to eliminate the risk of modification and obtain humanized monoclonal antibodies.
  • the CDR amino acid residues of the antibody are determined and annotated by the Kabat numbering system.
  • mouse-derived antibodies were finally named GPRC5D-mab03 and GPRC5D-mab06.
  • the humanized antibodies were named GPRC5D-Hab03 and GPRC5D-Hab06.
  • the light chain and heavy chain variable region sequences are as shown in the table. 4, and the CDRs sequences are shown in Table 5.
  • Example 1.1 For the preparation and screening of alpaca-derived VHH antibodies, refer to Example 1.1, in which the immunogen used is CHO-K1-human GPRC5D overexpressing cells. The CDR amino acid residues of the antibody were determined and annotated by the IMGT or Kabat numbering system. Finally, two alpaca sequences were obtained, named GPRC5D-Lab05 and GPRC5D-Lab06; and two humanized sequences were named GPRC5D-Hab05-H2. and GPRC5D-Hab06-H1. The amino acid sequences are shown in Table 6, and the numbered CDRs sequences are shown in Table 7.
  • VHH antibodies Preparation of camel-derived VHH antibodies: Select a camel about 3 weeks old, collect 5 ml of blood before immunization, and reserve it as negative control serum. Human GPRC5D protein was used for the first three immunizations, followed by four immunizations with the CHO-K1-human GPRC5D overexpression cell line. After verifying that the serum binds to both human and monkey GPRC5D proteins, a phage library was constructed and screened. PBMC were isolated from peripheral blood, total RNA was extracted, and cDNA was obtained by reverse transcription. Nested PCR was used to amplify Nanobody (VHH) fragments.
  • VHH Nanobody
  • the vector and target product are digested and ligated, and then the ligation product is electroporated into TG1 competent cells, diluted and plated for culture. After overnight culture, expand the culture, add helper phage M13K07, culture and measure its titer.
  • Human GPRC5D overexpressing cells were used as antigens for phage library panning. After multiple rounds of panning, positive phages were continuously enriched during the panning process, and FACS was used to screen out nanobodies with good specificity and high affinity for overexpressing cells and cells endogenously expressing human GPRC5D. After multiple rounds of optimization and screening, several positive clones capable of recognizing both human and monkey GPRC5D were obtained.
  • sequencing refer to the description in Example 1.1 to construct a humanized antibody, and the CDR of the antibody is annotated by IMGT or Kabat. After verification by FACS, several strains of humanized antibodies with higher affinity were obtained.
  • the camel antibodies were named GPRC5D-Lab03 and GPRC5D-Lab04, and their humanized antibodies were named GPRC5D-Hab03-H10 and GPRC5D-Hab04-H5.
  • the Nanobody sequence is shown in Table 6, and the CDRs sequence is shown in Table 7.
  • the nucleic acid sequence of the chimeric antigen receptor is loaded into the retroviral vector, Construct the target plasmid.
  • the specific steps are as follows: amplify and obtain the CAR nucleotide sequence through PCR, perform a double enzyme digestion reaction between the target gene fragment and the retroviral vector plasmid, and incubate at 37°C for 60 minutes. After the enzyme digestion reaction is completed, the nucleic acid fragment is purified by agarose gel (using a gel purification kit from Taraka Company). The specific operation steps are carried out according to the kit operating instructions.
  • the target nucleic acid molecules after gel purification are incubated at 16°C for 10-12 hours under the action of T4 ligase.
  • the obtained ligation product is transferred into DH5 ⁇ competent cells by heat shock.
  • the obtained competent cells are transferred into cells without added In liquid LB medium with antibiotics, incubate for 2 hours at 37°C with a shaker at 200 rpm, then centrifuge at 4000 rpm for 2 minutes, then centrifuge and resuspend in fresh LB medium, and evenly spread the bacterial liquid into the solid LB medium containing antibiotics. , culture in a 37°C incubator overnight, pick and identify positive clones, and extract the target plasmid of the positive clones for later use.
  • 293T cells purchased from ATCC
  • 293T cells purchased from ATCC
  • ⁇ -MEM medium When transfecting cells, mix the packaging plasmid and the target plasmid, add them to ⁇ -MEM medium, and add them to another centrifuge tube containing ⁇ -MEM medium.
  • Transfection reagent Promega, E2311
  • add the mixture of plasmid and transfection reagent into a 10cm petri dish shake gently 10 times, mix well, and place in the incubator box.
  • NK cells were isolated according to the Human NK Cell isolation kit (Stemcell, 17955).
  • the isolated NK cells were activated with K562 cells.
  • the culture medium is NK cell culture medium (Miltenyi Biotec, 130-114-429) containing 200IU/ml human IL2, placed in an incubator (37°C, 5% CO2); on Day 4, add 3 mL of culture medium to each well. Base; Day 6, cell activation is complete and can be transfected.
  • RetroNectin reagent (Takara, T202) at a concentration of 7 ⁇ g/mL, 500 ⁇ L per well, and incubate at 4°C overnight.
  • RetroNectin reagent (Takara, T202) at a concentration of 7 ⁇ g/mL, 500 ⁇ L per well, and incubate at 4°C overnight.
  • To count NK cells add 3 ⁇ 10 5 cells/well to a 24-well plate and centrifuge at 400g for 5 minutes at room temperature. Place the 24-well plate into an incubator (37°C, 5% CO 2 ).
  • Day 3 replace the transfected
  • the BCMA-Hab02-VHH7 antibody and GPRC5D-scFv (GPRC5D-Hab06) antibody screened in Example 1 were used to design and construct a dual-target chimeric antigen receptor targeting BCMA and GPRC5D as shown in 1A-1D of Figure 1 ( BI-CAR) were named BI-CAR01 ⁇ BI-CAR04 respectively; BCMA-Hab02-VHH7 and GPRC5D-scFv (GPRC5D-Hab03) antibodies were used to design and construct the BI-CAR05 whose structure is shown in 1A of Figure 1; the scFv structures are all according to The N-terminal to C-terminal is a VL-VH structural design.
  • Example 2 The molecular biology method shown in Example 2 was used to load the nucleic acid sequence of BI-CAR into a retroviral vector, construct the target plasmid and viral vector, induce NK cells and prepare CAR-NK cells.
  • the amino acid sequences of each domain of BI-CAR and exemplary BI-CAR01 to 05 sequences are shown in Table 8.
  • the method for detecting BCMA-CAR NK surface molecule expression in BI-CAR is as follows: Take 2 ⁇ 10 5 cells into a 96-well U-shaped plate, centrifuge, discard the supernatant, and after washing with buffer, add 100 ⁇ l of a final concentration of 2 ⁇ g/ml.
  • FITC-hBCMA-Fc ACRO Biosystems, BCA-HF254.
  • the method for detecting surface molecule expression of GPRC5D-CAR NK cells in BI-CAR is as follows: Take 2 ⁇ 10 5 cells into a 96-well U-shaped plate, centrifuge, discard the supernatant, and after washing with buffer, add 100 ⁇ l to a final concentration of 10 ⁇ g/ml.
  • the FACS expression detection results of BI-CAR are shown in Table 9. The results show that in the 1A form of Figure 1 and the 1B form of Figure 1, the expression of anti-BCMA and anti-GPRC5D is overall higher and has no position dependence; in the 1C form of Figure 1 and the 1D form of Figure 1 The expression of BCMA-CAR or GPRC5D-CAR is affected by its location.
  • NCI H929, RPMI8226, MOLP8 and NCI H929-hBCMA-KO were selected as target cells. All cells contained luciferase reporter genes through conventional gene manipulation methods.
  • NCI H929-hBCMA-KO is an NCI H929 cell line that uses conventional gene manipulation methods to knock out BCMA. Add target cells diluted in 1640 culture medium to a white opaque 96-well plate at 2 ⁇ 10 4 cells/50 ⁇ l/well.
  • NK cells to the above target cells at an effective-to-target ratio of 5:1, 2.5:1, and 1.25:1, and Place the 96-well plate in a 37°C, 5% CO2 incubator; after 4 hours, add 30 ⁇ l FIREFLYGLO luciferase reporter gene detection reagent (Meilun Biotech, MA0519-1), and incubate at room temperature in the dark for 10 minutes. Measure with a microplate reader and calculate the killing rate.
  • FIREFLYGLO luciferase reporter gene detection reagent Meilun Biotech, MA0519-1
  • BI-CAR01 ⁇ BI CAR05 had specific killing effects on both NCI H929-Lu and NCI H929-hBCMA-KO-Lu.
  • BI-CAR01 ⁇ BI-CAR03 and BI-CAR05 have good specific killing effects on the four tumor cells as a whole.
  • BI-CAR01, BI-CAR-03 and BI-CAR05 are effective against NCI H929-hBCMA- KO-Lu tumor cells showed stronger killing function, indicating that BI-CAR01, BI-CAR03 and BI-CAR05 forms have better killing effects on tumor cells with down-regulated or deleted BCMA expression.
  • the antibodies BCMA-Hab02-VHH7 and GPRC5D-Hab06 corresponding to BI-CAR01 and BI-CAR03 screened in Example 3 were used.
  • the design and construction are shown in Figure 2
  • the structure of a dual-target chimeric antigen receptor targeting BCMA and GPRC5D was named BI-CAR06.
  • the dual-target chimeric antigen receptor targeting BCMA and GPRC5D includes CD8 ⁇ signal peptide (SP), anti-GPRC5D-scFv and anti-BCMA-VHH, CD8 hinge region, NKG2D transmembrane region, 2B4 costimulatory domain and CD3 ⁇ , and IL15 linked via the self-cleaving peptide P2A. See Table 11 for specific sequences.
  • the molecular biology method in Example 2 was used to load the nucleic acid sequence into the retroviral vector to construct the target plasmid.
  • Example 2 use the same MOI to infect NK cells to prepare BI-CAR-NK cells with different transmembrane regions and intracellular stimulating domain structures.
  • Example 3.2 to detect the expression of CAR.
  • the results are as shown in the table 12 shown.
  • the results show that the CAR expression shown in 1A of Figure 1 is better than the CAR expression shown in 1C of Figure 1 .
  • BI-CAR cells The proliferation of BI-CAR cells was detected by AO/PI counting, and the results are shown in Figure 3. After 14 days of NK activation, the proliferation rates of BI-CAR cells from high to low were: BI-CAR06 (5566 times)>NK (3124 times)>BI-CAR03 (1705 times)>BI-CAR05 (1245.2 times)>BCMA -CAR01 (1012 times). Based on the above results, it can be seen that the proliferation rate of BI-CAR cells loaded with NKG2D TM elements after optimization is significantly higher than that of BI-CAR cells using other transmembrane elements.
  • target cells NCI H929, RPMI 8226, MOLP8 and NCI H929-hBCMA-KO diluted in 1640 medium were added at 2 ⁇ 10 4 cells/50 ⁇ l/well.
  • NK cells In a white opaque 96-well plate, add NK cells to the above target cells at an effective-to-target ratio of 9:1 and 3:1, and place the 96-well plate in a 37°C, 5% CO2 incubator for culture; after 4 hours, add 30 ⁇ l FIREFLYGLO luciferase reporter gene detection reagent (Meilun Biotech, MA0519-1), incubate at room temperature in the dark for 10 minutes, then use a microplate reader to measure and calculate the killing rate.
  • FIREFLYGLO luciferase reporter gene detection reagent Meilun Biotech, MA0519-1
  • NK cells On the 6th day after retrovirus infection of NK cells, the in vitro multiple rounds of killing effects of BI-CAR01, BI-CAR-03, BI-CAR-05, and BI-CAR-06 on RPMI 8226 cells and MOLP8 cells were detected: (1) First round of killing: Place target cells RPMI8226 or MOLP8 diluted in 1640 culture medium into a 12-well plate at a rate of 2.5 ⁇ 10 5 cells/500 ⁇ l/well. Add NK cells to a 12-well plate at an effective-to-target ratio of 1:1 and culture them in a 37°C, 5% CO2 incubator for 24 hours.
  • Next round of killing Take the cells from the previous round of 12-well plate, count the NK cells, and add the previous round of NK cells to the 12-well plate seeded with new target cells at an effective-to-target ratio of 1:1 , repeat step (1), measure the NK cell killing rate and continue the next round of killing test.
  • FIG. 4 The results of multiple rounds of killing tests of BI-CAR NK cells are shown in Figure 4.
  • the killing effect of BI-CAR06 loaded with NKG2D TM elements is better than other forms of BI-CAR; BI-CAR06 can still maintain 50% cell killing in the third round of killing MOLP8 cells (R3-1d) rate, the third round of killing (R3-1d) of RPMI 8226 cells can still maintain a cell killing rate of 40%.
  • BCMA-Hab02-VHH7 and GPRC5D-VHH antibodies were designed Construct the 1A form of BI-CAR in Figure 1.
  • the dual-target chimeric antigen receptor targeting BCMA and GPRC5D includes CD8 ⁇ signal peptide (SP), anti-GPRC5D-VHH and anti-BCMA, CD8 hinge region, and CD8 transmembrane region, CD137 costimulatory domain, and CD3 ⁇ .
  • the BI-CAR obtained by screening in Example 5.1 was further constructed into a BI-CAR NK with a CD8 transmembrane region (the structure is shown in 1A of Figure 1) to verify its expression.
  • the BI-CAR sequence is shown in Table 14.
  • the BI-CAR NK expression detection method is the same as in Example 3.2.
  • the expression of BCMA CAR could reach more than 80%, and the expression of GPRC5D CAR could reach more than 50%.
  • a 4-hour in vitro killing experiment was performed on the BI-CAR NK cells prepared in Example 5.2: target cells NCI H929, RPMI 8226, MOLP8 and NCI H929-hBCMA-KO diluted in 1640 medium Add 2 ⁇ 10 4 cells/50 ⁇ l/well to a white opaque 96-well plate, add NK cells to the above target cells according to the effect-to-target ratio of 5:1, 2.5:1, and 1.25:1, and place the 96-well plate at 37°C.
  • BI-CAR07 shows a tumor cell killing effect comparable to that of BI-CAR01, and the tumor killing effects of humanized BI-CAR14 and BI-CAR15 are lower than those of unhumanized BI-CAR16 and BI-CAR17.
  • Example 3.2 to detect the expression of BI CAR18-21.
  • the results are shown in Table 18.
  • the expression of BCMA CAR and GPRC5D CAR can be detected after infecting NK cells.
  • the positive rate of BCMA CAR is high and can reach more than 60%; the positive rate of GPRC5D CAR is low, and the expression rates from high to low are BI-CAR06 (69.60%)>BI-CAR21 (54.20%)>BI-CAR20 (54.00%) )>BI-CAR18 (32.30%)>BI-CAR19 (13.80%).
  • BI-CAR NK cells proliferated about 1473 to 2166 times on the 11th day after activation.
  • NK cells to a 12-well plate at an effective-to-target ratio of 1:1 and culture them in a 37°C, 5% CO2 incubator for 24 hours. After culturing for 24 hours, add 100 ⁇ l of the above-mentioned uniformly mixed cells into a 96-well plate with a white bottom and an opaque bottom, add 30 ⁇ l of FIREFLYGLO luciferase reporter gene detection reagent (Meilun Biotech, MA0519-1), and incubate at room temperature in the dark for 10 minutes. Use a microplate reader to measure fluorescence intensity and calculate NK cell killing efficiency. After that, enter the next round of killing tests.
  • Next round of killing Take the cells from the previous round of 12-well plate, count the NK cells, and add the previous round of NK cells to the 12-well plate seeded with new target cells at an effective-to-target ratio of 1:1 , repeat step (1), measure the NK cell killing rate and continue the next round of killing test.
  • BI-CAR21 has the best killing effect on NCI H929 cells, and can still maintain a cell killing rate of 30% in the fourth round of killing (R4-1d) experiments; BI-CAR19, BI-CAR20 and BI-CAR21 are effective against NCI H929+20 % (or 10% or 5%) H929-hBCMA-KO has the best killing effect on heterogeneous tumor cells, RPMI 8226 cells and MOLP8 cells, and has the best killing effect on NCI H929+20% (or 10% or 5%) H929-hBCMA- KO's heterogeneous tumor cells can still maintain a cell killing rate of more than 30% in the fourth round of killing (R4-1d) experiments, and can still maintain a cell killing rate of more than 30% in the fifth round of killing (R5-1d) experiments on RPMI 8226 cells and MOLP8 cells. Cell killing rate of about 10%.
  • Example 2 Prepare BI-CAR21 with reference to Example 2, transfect NK cells, and add the CBL-b inhibitor prepared in Example 7.1 (final concentration 1 ⁇ M) during the activation and transfection of NK cells.
  • the CAR positive rate of the NK cells was detected with reference to Example 3.2.
  • the results showed that after adding CBL-b inhibitor, the CAR positive rate of NK cells increased.
  • the expression rate of BI-CAR21 was detected by BCMA, wherein, compared with the control without adding CBL-B inhibitor, after adding BL-b inhibitor, the expression rate of BI-CAR was determined by 80% rose to 92.5%.
  • Example 2 use retrovirus to transfect NK cells and culture them for 6 days.
  • Example 6.4 to detect the combination of CAR-NK (BI-CAR21) targeting GPRC5D and BCMA and CBL-b inhibitor and/or PDL1-IL15. Multiple rounds of killing effect on NCI H929+10% NCI H929-hBCMA-KO cells after use. See Table 20 for the experimental design plan, where the CBL-b inhibitor and its addition method are shown in 7.2, and the PDL1-IL15 is added when the target cells and NK cells are incubated. The results of multiple rounds of killing are shown in Figure 9.
  • CBL-b inhibitors to stimulate NK cells during the culture process or adding PDL1-IL15 fusion protein when killing CAR-NK cells can enhance the killing effect of BCMA-GPRC5D CAR-NK.
  • the combination of CBL-b inhibitors and PDL1-IL15 can further enhance the killing effect of BCMA-GPRC5D CAR-NK.
  • the G5 group still maintains a high target cell killing rate on R5-1d, which is significantly higher than the G3 group and G4 Group.
  • sequence information of PDL1-IL15 is as follows:
  • Example 2 to prepare BI-CAR06, BI-CAR19, BI-CAR20, and BI-CAR21 for in vivo efficacy verification, and transfect NK cells.
  • NK cells Six days after retroviral transfection of NK cells, NK cells were detected according to Example 3.2. CAR positive rate.
  • FIG 10A through BCMA detection, the CAR positive rate of NK cells was above 80%.
  • GPRC5D detection the CAR positive rate of NK cells was above 70%.
  • Figure 10B and Figure 10C 0 hours after cryopreservation and recovery, the CAR positive rate of NK cells decreased by approximately 10%.
  • the viability of BI-CAR cells after recovery was detected by AO/PI counting.
  • NCI H929 was selected as the target cells, and the cells all contained luciferase reporter genes through conventional gene manipulation methods.
  • NK cells to the above target cells at an effective-to-target ratio of 5:1, 2.5:1, and 1.25:1, and Place the 96-well plate in a 37°C, 5% CO2 incubator; after 4 hours, add 30 ⁇ l FIREFLYGLO luciferase reporter gene detection reagent (Meilun Biotech, MA0519-1), and incubate at room temperature in the dark for 10 minutes. Measure with a microplate reader and calculate the killing rate.
  • FIREFLYGLO luciferase reporter gene detection reagent Meilun Biotech, MA0519-1
  • a myeloma vein transplant tumor model was used to conduct an anti-tumor efficacy test. details as follows:
  • NCI H929-Luc cells in the logarithmic growth phase and in good growth status were collected, and 2 ⁇ 10 6 cells were inoculated into the tail vein of each NPG mouse (combined immunodeficient mouse).
  • the weight of the mice was measured 1 day after tumor inoculation, and mice with a weight of about 19.76-25.52g were selected based on the random number principle, with an average value of 21.66g for random grouping.
  • freeze-resuscitated CAR-NK cells (8 ⁇ 10 6 /animal) and CAR-T cells (3 ⁇ 10 6 /animal) were injected into the tail vein.
  • the injection volume was 200 ⁇ l/animal.
  • CAR-NK/T cells were injected The diary is Day0.
  • the grouping of mice and the injection of CAR-NK cells are shown in Table 21. Use an IVIS intravital imager to monitor tumor growth (fluorescence signal ROI value), and weigh the mice twice a week.
  • BCMA-CAR-T preparation The nucleic acid encoding the chimeric antigen receptor for T cells is cloned into the retroviral shuttle plasmid through conventional molecular cloning technology, and the virus is packaged through the packaging plasmid.
  • T cells were isolated from fresh PBMC using Stemcell Easy Sep Kit (Stemcell, Cat#19055). The isolated T cells were transferred to culture dishes pre-coated with 1 ⁇ g/ml CD3/CD28 antibody (MY-ebioscience-16-0289-85, MY-ebioscience-16-0037-85).
  • the medium components are X-VIVO15 (Lonza, Cat#BEBP02-054Q), 5% human AB serum (Gemini, Cat#100-512), 100U/ml penicillin-streptomycin (Gibco, Cat#15140-122) and 200IU/ml human IL2 (Beijing Shuanglu, Cat#S19991007).
  • Cell counting was performed twice a week, and when the cell density reached 2.5 ⁇ 10 6 cells/ml, the cells were subcultured and expanded.
  • T cells Take the above-mentioned T cells in good condition and inoculate them into a 12-well cell culture plate pre-coated with 5 ⁇ g/ml recombinant human fibrin (Takara, Cat#T100B) at 1 ⁇ 10 6 cells/well. Add 50 ⁇ l of concentrated lentivirus, 4 Centrifuge at 1000 g for 1 hour at °C. After centrifugation, culture at 37°C and 5% CO2 . Count twice a week. When the cell density reaches 2.5 ⁇ 10 6 cells/ml, the cells are passaged and expanded. The prepared CAR-T cells are cryopreserved for later use.
  • BCMA-CAR-T sequence information is as follows:
  • the results of fluorescence signal detection of mouse tumor growth are shown in Figure 12A.
  • the results show that on the 22nd day after injection of CAR-NK cells, the three groups BI-CAR06, BI-CAR19 and BI-CAR20 significantly inhibited tumor burden and effectively controlled As for tumor progression, the tumors of 4, 2 and 3 animals in the three groups were completely eliminated respectively.
  • the BI-CAR21 group had the most recurrences as the detection time was extended.
  • the BI-CAR21 treatment group could slow down the growth rate of the tumors, but could not eliminate the tumors.
  • the Molp8 myeloma vein transplant tumor model was used to conduct an anti-tumor efficacy test. details as follows:
  • Molp8-Luc cells in the logarithmic growth phase and in good growth status were collected, and 5 ⁇ 10 5 cells were inoculated into the tail vein of each NPG mouse (combined immunodeficient mouse).
  • the mouse body weight and tumor fluorescence signal value were measured. Mice with a weight of about 21.81 to 27.25g were selected based on the random number principle, and the average value was 23.98g for random grouping.
  • freeze-resuscitated CAR-NK cells (8 ⁇ 10 6 /animal) and CAR-T cells (3 ⁇ 10 6 /animal) were injected into the tail vein. The injection volume was 200 ⁇ l/animal.
  • CAR-NK/T cells were injected The diary is Day0.
  • the grouping of mice and the injection of CAR-NK cells are shown in Table 22.
  • Tumor growth was monitored using an IVIS intravital imager. The body weight of mice was measured and recorded twice a week and the survival rate of mice was observed.
  • the fluorescence signal detection results of mouse tumor growth are shown in Figure 13A.
  • the results show that on the 15th day after injection of CAR-NK cells, the BI-CAR21+CBL/B inhibitor treatment group significantly inhibited tumor growth, with 3 animals The tumors were completely eliminated; in the BI-CAR06 group, the tumors of 2 animals were completely eliminated on Day 15; the three treatment groups of BI-CAR19, BI-CAR20 and BI-CAR21 could significantly inhibit the growth rate of mouse tumors, but could not completely eliminate them. Tumor, manifested as the recurrence and growth of all animal tumors. After administration, the photon value of the mouse tumor was continuously measured. Compared with the Parental NK group, each CAR NK treatment group showed a significantly lower trend (as shown in Figure 13B).
  • the mortality rates of the BI-CAR06, BI-CAR19, BI-CAR21 and Parental NK groups were 80%, 20%, 60% and 100% respectively. As mentioned above, they were all due to the rapid expansion of NK. The heavy load on major organs caused death; no animal death occurred in the other treatment groups (as shown in Figure 13C); at the same time, the body weight of mice in the three groups of BI-CAR06, BI-CAR19 and BI-CAR21 treatment showed a decrease in the experiment. The downward trend is due to the poor condition of some animals in the group due to high NK cell expansion, while the Parental NK group has lost weight due to model disease (as shown in Figure 13D).
  • BCMA single-targeting CAR-NK was constructed using the sequence of BCMA-Hab02-VHH7, named mono-B-CAR; GPRC5D-Hab06 human The humanized antibody was used to construct a GPRC5D single-targeting CAR-NK based on the N-terminal-C-terminal VL-VH scFv, named mono-G-CAR.
  • Single-targeting CARs all adopt the structure of the NKG2D transmembrane region and the 2B4 intracellular region. The specific structure is shown in Figure 14, and the sequence is shown in Table 23.
  • NK cells On the 6th day after retrovirus infection of NK cells, a 4-h in vitro killing experiment was performed on the prepared mono-B-CAR, mono-G-CAR and BI-CAR06NK cells: targets carrying luciferase reporter genes diluted in 1640 medium Cells NCI H929-Lu and NCI H929-hBCMA-KO-Lu were added to a white opaque 96-well plate at 2 ⁇ 10 4 cells/50 ⁇ l/well, and NK cells were added according to the effect-to-target ratio of 5:1, 2.5:1, and 1.25:1.
  • the 4-hour in vitro cell killing effects of mono-B-CAR, mono-G-CAR and BI-CAR06NK cells on the above target cells are detailed in Table 25. According to Table 25, mono-B-CAR, mono-G-CAR and BI-CAR06NK cells all have good specific killing effect on NCI H929 tumor cells, and mono-G-CAR and BI-CAR06NK cells have good specific killing effect on NCI H929-hBCMA. -KO-Lu tumor cells all have good specific killing effect. The killing effect of mono-B-CAR on NCI H929-hBCMA-KO-Lu tumor cells is only equivalent to that of Parental NK.
  • mice Collect 50% H929-Luc cells and 50% H929-Luc (BCMA-KO) cells that are in logarithmic growth phase and in good growth status, mix them, and inoculate 2 cells into the tail vein of each NPG mouse (combined immunodeficient mouse). ⁇ 10 6 cells.
  • the weight of the mice was measured 1 day after tumor inoculation, and mice with a weight of about 21.57-27.47g were selected based on the random number principle, with an average value of 24.37g for random grouping.
  • freshly prepared CAR-NK cells (5 ⁇ 10 6 cells/animal) were injected into the tail vein, with an injection volume of 200 ⁇ l/animal.
  • the CAR-NK cell injection diary was Day 0.
  • the grouping of mice and the injection of CAR-NK cells are shown in Table 26.
  • the ROI value of tumor growth fluorescence signal was monitored using IVIS intravital imager. Weigh and record the body weight of mice twice a week.
  • Example 2 the same MOI was used to infect NK cells to prepare BCMA single-targeting mono-B-CAR and GPRC5D single-targeting mono-G-CAR and BCMA and GPRC5D dual-targeting BI-CAR06 cells.
  • Example 3.2 the method in Example 3.2 to detect the expression of CAR, and the results are shown in Table 27.
  • a 4-h in vitro killing experiment was performed on the prepared mono-B-CAR, mono-G-CAR and BI-CAR06NK cells: target cells NCI H929 and NCI H929- diluted in 1640 medium hBCMA-KO was added to a white opaque 96-well plate at 2 ⁇ 10 4 cells/50 ⁇ l/well, and NK cells were added to the above target cells at an effective-to-target ratio of 10:1, 5:1, 2.5:1, and 1.25:1, and The 96-well plate was cultured in a 37°C, 5% CO2 incubator; after 4 hours, add 30 ⁇ l FIREFLYGLO luciferase reporter gene detection reagent (Meilun Biotech, MA0519-1), and incubate at room temperature in the dark for 10 minutes before use. A microplate reader takes the measurement and calculates the kill rate.
  • the 4-hour in vitro cell killing effects of mono-B-CAR, mono-G-CAR and BI-CAR06NK cells on the above target cells are detailed in Table 28. According to Table 28, mono-B-CAR, mono-G-CAR and BI-CAR06NK cells all have good specific killing effect on NCI H929 tumor cells, and mono-B-CAR, mono-G-CAR is slightly better than BI -CAR06; both mono-G-CAR and BI-CAR06NK cells have good specific killing effect on NCI H929-hBCMA-KO tumor cells. The killing effect of mono-G-CAR on NCI H929-hBCMA-KO tumor cells is only the same as that of Parental NK is quite good.
  • the fluorescence signal detection results of mouse tumor growth are shown in Figure 15A.
  • the results show that on the 60th day after injection of CAR-NK cells, mono-B-CAR and mono-G-CAR treatment groups can inhibit mouse tumor growth. However, none of them could eliminate the tumors, which was manifested as the recurrence of all animal tumors.
  • the tumor growth was significantly inhibited and the tumor progression was effectively controlled. Only 2 animals had tumor recurrence in 60 days, and the remaining 3 were completely eliminated.
  • the survival rate was continuously monitored until 96 days.
  • the PBS group one animal died on day 53, and the mortality rate was 100% on day 96; in the mono-B-CAR group, one animal died on day 61, and on day 96, one animal died.
  • the mortality rate was 40%; one animal died in the mono-G-CAR group on day 69, and the mortality rate was 20% on day 96; no animal death occurred in the BI-CAR06 group (as shown in Figure 15C).
  • none of the three groups of mice treated with CARNK showed weight loss after administration, and showed an overall increasing trend. Only the PBS group continued to lose weight after 40 days due to the occurrence of model disease (as shown in Figure 15D).
  • H929-Luc cells in the logarithmic growth phase and in good growth status were collected, and 2 ⁇ 10 6 cells were inoculated into the tail vein of each NPG mouse (combined immunodeficient mouse).
  • the weight of the mice was measured 1 day after tumor inoculation, and mice with a weight of about 21.03-27.65g were selected based on the random number principle, with an average value of 24.42g for random grouping.
  • freeze-resuscitated CAR-NK cells (6 ⁇ 10 6 /animal) and CAR-T cells (3 ⁇ 10 6 /animal) were injected into the tail vein.
  • the injection volume was 200 ⁇ l/animal.
  • CAR-NK/T cells were injected The diary is Day0.
  • the grouping of mice and the injection of CAR-NK/T cells are shown in Table 29. Tumor growth was monitored using an IVIS intravital imager (fluorescence signal ROI value).
  • the mortality rates of the BI-CAR06 and BI-CAR06+PD-L1/IL-15+CBL/B inhibitor combination treatment groups were 75% and 25% respectively. According to analysis, this was due to the rapid decline of NK. Amplification accumulates in major organs with higher burden and leads to death; the mortality rates of the Parental NK group, the PD-L1/IL-15 group and the CBL/B inhibitor group were 25%, 25% and 75% respectively.

Abstract

L'invention concerne un récepteur antigénique chimérique ciblant GPRC5D et BCMA, et son utilisation. En particulier, l'invention concerne des acides nucléiques codant pour le récepteur antigénique chimérique ciblant GPRC5D et BCMA, un récepteur antigénique chimérique correspondant, un vecteur, une cellule effectrice immunitaire, un procédé de préparation et un produit associé, une composition pharmaceutique, ainsi qu'une utilisation pharmaceutique et une méthode de traitement de tumeurs ou du cancer.
PCT/CN2023/108544 2022-07-22 2023-07-21 Récepteur antigénique chimérique ciblant gprc5d et/ou bcma, et son utilisation WO2024017362A1 (fr)

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