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WO2023030393A1 - Récepteur antigénique chimérique et son utilisation - Google Patents

Récepteur antigénique chimérique et son utilisation Download PDF

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WO2023030393A1
WO2023030393A1 PCT/CN2022/116237 CN2022116237W WO2023030393A1 WO 2023030393 A1 WO2023030393 A1 WO 2023030393A1 CN 2022116237 W CN2022116237 W CN 2022116237W WO 2023030393 A1 WO2023030393 A1 WO 2023030393A1
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domain
cells
car
cell
antigen
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Chinese (zh)
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阎锡蕴
段红霞
景林
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Institute of Biophysics of CAS
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Institute of Biophysics of CAS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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 a chimeric antigen receptor (CAR) and its use, especially in the treatment of cancer.
  • CAR chimeric antigen receptor
  • CAR-T chimeric receptor T
  • CAR-T cells are affected by the microenvironment of solid tumors, resulting in reduced killing ability and insufficient persistence, which are the key reasons for their poor therapeutic effect in solid tumors. Therefore, how to further improve the tumor killing ability of CAR-T cells is one of the key issues to solve the inefficiency of CAR-T therapy for solid tumors.
  • CAR-T cells The core of CAR-T cells to achieve their tumor killing function is the CAR molecule, which is designed by simulating the TCR receptor of natural T cells.
  • CAR molecules have a significantly lower ability to activate T cells, which is only about 1/10-1/1000 of TCR activation ability. Therefore, improving the ability of CAR molecules to activate T cells is a key way to improve the tumor killing ability of CAR-T cells.
  • the activation of T cells by CAR molecules mainly depends on the CD3 ⁇ domain of its intracellular segment.
  • the ITAM motif in the CD3 ⁇ domain can be activated by phosphorylation, and the activated CD3 ⁇ activates a series of activation-related signaling pathways by activating downstream ZAP70 and other molecules, thereby activating T cells.
  • How to increase the phosphorylation level of the CD3 ⁇ domain of the CAR molecule is the key to increasing the activation level of CAR-T cells.
  • lymphocyte-specific protein tyrosine kinase (LCK) of the SRC family is a key molecule for phosphorylating the ITAM motif of CD3 ⁇ in CAR-T cells. Therefore, if we can try to recruit more activated forms of LCK kinase to the vicinity of CD3 ⁇ , we can improve the activation efficiency of CD3 ⁇ , and then increase the activation level of CAR-T cells.
  • LCK lymphocyte-specific protein tyrosine kinase
  • the inventors unexpectedly found that the intracellular domain (cytoplasmic region) of the CD146 molecule in T cells can directly interact with the LCK molecule, and can promote the activation of LCK by promoting the autophosphorylation of LCK. This direct interaction is at least partially dependent on the KKGK motif at the membrane-proximal end of the CD146 cytoplasmic domain.
  • the CD146 cytoplasmic region or its near-membrane fragment was integrated into the intracellular domain of the CAR molecule, and its ability to bind and activate LCK was used to integrate more activated forms of LCK molecules are recruited to the intracellular domain of the CAR molecule, thereby better activating the CD3 ⁇ domain of the intracellular domain of the CAR molecule, so as to increase the activation level of CAR-T cells, thereby improving the tumor killing ability of CAR-T cells.
  • the CAR molecule modified by the present invention can improve the activation of CAR-T cells, enhance the tumor killing ability of CAR-T cells, and thus be applied to the immunotherapy of various cancers including solid tumors.
  • the present invention provides a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, an intracellular signaling domain and an intracellular co-stimulatory domain, wherein the The intracellular co-stimulatory domain includes a first co-stimulatory domain capable of binding and activating lymphocyte-specific protein tyrosine kinase (LCK) to enhance immune cell activation.
  • CAR chimeric antigen receptor
  • the intracellular co-stimulatory domain includes a first co-stimulatory domain capable of binding and activating lymphocyte-specific protein tyrosine kinase (LCK) to enhance immune cell activation.
  • LCK lymphocyte-specific protein tyrosine kinase
  • the first co-stimulatory domain may comprise the cytoplasmic region of CD146, or a membrane-proximal fragment thereof.
  • the length of the membrane-proximal fragment of the CD146 cytoplasmic region may be 4-55 amino acids, preferably 10-45 amino acids, more preferably 15-35 amino acids, and even more preferably 15-26 amino acids .
  • the membrane proximal fragment of the CD146 cytoplasmic region comprises amino acids 584-609 of the CD146 molecule.
  • the intracellular co-stimulatory domain may further comprise a second co-stimulatory domain selected from the group consisting of 4-1BB (CD137), OX40 (CD134), ICOS (CD278) , 2B4, HVEM, LAG3, DAP10, DAP12, CD27, CD28, CD30, CD40, glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA-1), MyD88, CD2, Intracellular signaling regions of CD4, CD7, LIGHT, NKG2C and B7-H3 or any combination thereof.
  • the second co-stimulatory domain is the intracellular signaling region of 41BB.
  • the C-terminus of the first costimulatory domain is linked to the N-terminus of the second costimulatory domain, optionally via a linker.
  • the first co-stimulatory domain is the CD146 cytoplasmic region
  • the second co-stimulatory domain is the intracellular signaling region of 41BB
  • the C-terminus of the first co-stimulatory domain is connected to the second co-stimulatory domain. N-terminal linkage of two co-stimulatory domains.
  • the order of the first co-stimulatory domain, the second co-stimulatory domain, and the intracellular signaling domain from the N-terminus to the C-terminus is: first co-stimulatory domain-second Two co-stimulatory domains - intracellular signaling domains.
  • the transmembrane domain may comprise an alpha chain selected from T cell receptor (TCR), beta chain of TCR, zeta chain of TCR, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16 , CD19, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137 (41BB), CD152, CD154 and the transmembrane domain of PD1 or any combination thereof.
  • TCR T cell receptor
  • beta chain of TCR zeta chain of TCR
  • the antigen binding domain can bind one or more tumor-associated antigens (TAAs).
  • TAA tumor-associated antigens
  • the TAA is preferably selected from 5T4, alpha-fetoprotein, BCMA, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, ROR1, GPC3, Tyrp-1, TACI, ALK, C-type lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, ERBB2, FLT3, melanoma-associated antigen, mesothelin, MUC-1, VEGFR2. More preferably, the antigen binding domain specifically binds CD19.
  • the antigen binding domain may be selected from an antigen binding domain derived from an antibody against the antigen and an antigen binding domain derived from a natural ligand of the antigen.
  • said antibody-derived antigen binding domain is in the form of a scFv, Fab or domain antibody (dAb).
  • the invention provides a nucleic acid sequence encoding a CAR of the invention.
  • the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding the CAR of the present invention.
  • nucleic acid construct has the following structure:
  • BD is the nucleotide sequence encoding the antigen binding domain
  • hinge is the nucleotide sequence encoding the hinge region
  • TM is a nucleotide sequence encoding a transmembrane domain
  • Costi is a nucleotide sequence encoding an intracellular co-stimulatory domain
  • Signal is a nucleotide sequence encoding an intracellular signaling domain.
  • the nucleic acid construct has the following structure:
  • BD is the nucleotide sequence encoding the antigen binding domain
  • hinge is the nucleotide sequence encoding the hinge region
  • TM is a nucleotide sequence encoding a transmembrane domain
  • Costi1 is a nucleotide sequence encoding the first co-stimulatory domain
  • Costi2 is a nucleotide sequence encoding a second co-stimulatory domain
  • Signal is a nucleotide sequence encoding an intracellular signaling domain.
  • the invention provides a vector comprising a nucleic acid sequence or nucleic acid construct of the invention.
  • the vector is selected from lentiviral vectors, retroviral vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors, and artificial chromosomes.
  • the invention provides cells expressing a CAR of the invention.
  • the present invention provides a method of preparing the cells of the present invention, comprising the step of transducing or transfecting the cells with the vector of the present invention.
  • the method may further comprise the step of expanding and/or activating cells before or after said transducing or transfecting.
  • the present invention provides a composition comprising the CAR, nucleic acid sequence, nucleic acid construct, vector, or cell of the present invention, and a pharmaceutically acceptable carrier or excipient.
  • said cells are autologous or allogeneic to said subject.
  • the method can also include administering a second therapeutic agent.
  • the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.
  • the present invention provides the use of the CAR, nucleic acid sequence, nucleic acid construct, vector, cell or composition of the present invention in the manufacture of a medicament for treating cancer in a subject.
  • the invention provides a CAR, nucleic acid sequence, nucleic acid construct, vector, cell or composition of the invention for use in treating cancer in a subject.
  • the cancer may be selected from lymphoma, multiple myeloma, leukemia and solid tumors.
  • Figure 1 shows the interaction assay between CD146 and LCK.
  • A Schematic representation of recombinant CD146 cytoplasmic domain protein.
  • B Pull-down assay of recombinant CD146 cytoplasmic domain protein and LCK-His protein.
  • Figure 3 shows the expression of the CAR molecule of the present invention in T cells.
  • A Structural schematic diagrams of the CAR molecule of the present invention (CD146cyt-4-1BB) and the control CAR molecule (4-1BB).
  • B Expression of the CAR molecule of the present invention (CD146cyt-4-1BB) and the control CAR molecule (4-1BB) in T cells.
  • FIG. 4 shows the activation of T cells expressing the CAR of the present invention measured by flow cytometry.
  • A Activation of T cells after co-culture with target cells.
  • B Activation of T cells after co-culture with CD19 antigen.
  • CTR indicates T cells not transfected with CAR
  • 4-1BB indicates T cells transfected with 41BB-CD3 ⁇ CAR
  • CD146cyt-4-1BB indicates T cells transfected with CD146cyt-41BB-CD3 ⁇ CAR.
  • T cells not transfected with CAR served as a negative control.
  • FIG. 5 shows the cytokine secretion of T cells expressing the CAR of the present invention measured by flow cytometry.
  • 4-1BB indicates T cells transfected with 41BB-CD3 ⁇ CAR
  • CD146cyt-4-1BB indicates T cells transfected with CD146cyt-41BB-CD3 ⁇ CAR.
  • Fig. 6 shows the tumor killing activity of T cells expressing the CAR of the present invention measured by lactate dehydrogenase (LDH) method.
  • CTR indicates T cells not transfected with CAR
  • 4-1BB indicates T cells transfected with 41BB-CD3 ⁇ CAR
  • CD146cyt-4-1BB indicates T cells transfected with CD146cyt-41BB-CD3 ⁇ CAR.
  • T cells not transfected with CAR served as a negative control.
  • Figure 7 shows the in vivo tumor suppressive effect of the CAR T cells of the present invention on a mouse model of hematological tumor observed by in vivo imaging.
  • A When the effect-to-target ratio is 1:1, the CAR T cells of the present invention have an in vivo tumor-inhibiting effect on a mouse model of hematological tumor.
  • B When the effect-to-target ratio is 1:2, the in vivo tumor-suppressive effect of the CAR T cells of the present invention on a mouse model of hematological tumor.
  • CTR indicates the injection of untransfected human primary T cells
  • 4-1BB indicates the injection of 41BB-CD3 ⁇ CAR T cells
  • CD146cyt-4-1BB indicates the injection of CD146cyt-41BB-CD3 ⁇ CAR T cells.
  • FIG. 8 shows the in vivo tumor suppressive effect of the CAR T cells of the present invention on a solid tumor mouse model observed by in vivo imaging.
  • CTR indicates the injection of untransfected human primary T cells
  • 4-1BB indicates the injection of 41BB-CD3 ⁇ CAR T cells
  • CD146cyt-4-1BB indicates the injection of CD146cyt-41BB-CD3 ⁇ CAR T cells.
  • the "membrane-proximal fragment of the CD146 cytoplasmic region” refers to the cytoplasmic region fragment of CD146 close to the transmembrane region, which means the fragment from the 584th amino acid to the 646th amino acid of the CD146 molecule.
  • CD146 plays multiple roles in various cells (Chen J, Luo Y, Hui H, Cai T, Huang H, Yang F, et al. CD146 coordinates brain endothelial cell-pericyte communication for blood-brain barrier development.Proceedings of the National Academy of Sciences of the United States of America.2017;114(36):E7622-E31;Luo Y,Duan H,Qian Y,Feng L,Wu Z,Wang F,et al.Macrophagic CD146 promotes foam cell formation and retention during atherosclerosis. Cell Res.2017; 27(3):352-72; Yan H, Zhang C, Wang Z, Tu T, Duan H, Luo Y, et al.
  • CD146 is required for VEGF- C-induced lymphatic sprouting during lymphangiogenesis.Sci Rep.2017;7(1):7442;Wang Z,and Yan X.CD146,a multi-functional molecule beyond adhesion.Cancer Lett.2013;330(2):150-62 ).
  • LCK also known as lymphocyte-specific protein tyrosine kinase
  • Src kinase is a member of the Src kinase family and is critical in membrane signaling that activates intracellular signaling cascades. LCK deficiency abolishes proximal TCR signaling and blocks T cell development and activation. Accumulating evidence indicates that phosphorylation of LCK at Y394 is critical for its activation (Philipsen L, Reddycherla AV, Hartig R, Gumz J, Kastle M, Credos A, et al. De novo phosphorylation and conformational opening of the tyrosine kinase Lck act in concert to initiate T cell receptor signaling. Sci Signal.2017; 10(462)).
  • LCK forms asymmetric head-to-tail dimers in which the Y394 activation loop of one monomer is located in the active site of the other monomer. It has been reported that Transautophosphorylation is crucial for the activation of LCK (Eck MJ, Atwell SK, Shoelson SE, and Harrison SC.Structure of the regulatory domains of the Src-family tyrosine kinase Lck.Nature.1994; 368( 6473):764-9; Xu Q, Malecka KL, Fink L, Jordan EJ, Duffy E, Kolander S, et al. Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases. Sci Signal. 2015; 8(405 ):rs13).
  • chimeric antigen receptor refers to an antigen-binding domain engineered to contain an antigen-binding domain that targets a specific antigen and activates immune cells (such as T cells or NK cells) upon binding to that antigen.
  • immune cells such as T cells or NK cells
  • cells such as naive T cells, central memory T cells, effector memory T cells, or combinations thereof
  • CAR-expressing immune cells can target and kill the tumor cells.
  • the classical chimeric antigen receptor is a chimeric type I transmembrane protein that links an extracellular antigen-binding domain to an intracellular signaling domain.
  • the antigen-binding domain is usually a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other forms comprising an antibody-like antigen-binding site or an antigen-binding structure derived from the natural ligand of the antigen area.
  • scFv single-chain variable fragment
  • mAb monoclonal antibody
  • a hinge domain is generally required to separate the antigen-binding domain from the membrane and to allow its proper orientation.
  • a common hinge domain used is the Fc of IgG1.
  • transmembrane domain anchors the protein in the cell membrane and connects the hinge domain to the intracellular domain (intracellular domain).
  • the first generation of CARs there have been at least three "generations" of CAR molecules.
  • these first-generation CARs transmit an immune signal1 that is sufficient to trigger T-cell killing of cognate target cells, but fails to fully activate T-cell proliferation and survival.
  • second-generation CARs have been constructed that possess a composite intracellular domain resulting from the fusion of the intracellular portion of a T-cell co-stimulatory molecule with that of CD3 ⁇ , allowing simultaneous transmission of an activation signal following antigen recognition and costimulatory signals.
  • the most commonly used co-stimulatory domain is that of CD28. This provides the most potent co-stimulatory signal - immune signal 2, which triggers T cell proliferation.
  • Some CARs have also been described that include TNF receptor family intracellular domains such as the closely related OX40 and 41BB that transmit survival signals. Even more potent third-generation CARs have now been described with intracellular domains capable of transmitting activation, proliferation and survival signals.
  • a CAR typically comprises: (i) an antigen-binding domain; (ii) a hinge domain; (iii) a transmembrane domain; and (iv) an intracellular domain comprising a signaling domain and one or more consensus domains. Stimulatory domain.
  • antigen binding domain refers to the portion of a chimeric antigen receptor that recognizes an antigen.
  • the antigen-binding domain comprises: a single-chain variable fragment (scFv) derived from a monoclonal antibody.
  • CARs have also been generated using domain antibodies (dAbs), VHH antigen-binding domains, or antigen-binding domains derived from the natural ligand of the antigen.
  • antigen refers to any molecule that elicits an immune response or is capable of being bound by an antibody or antigen binding molecule.
  • the immune response may involve antibody production or activation of specific immunocompetent cells, or both.
  • any macromolecule including virtually any protein or peptide, can serve as an antigen.
  • Antigens may be expressed endogenously, ie from genomic DNA, or may be expressed recombinantly.
  • An antigen may be specific for certain tissues, such as cancer cells, or it may be expressed broadly.
  • the antigen is a tumor-associated antigen, such as all or a fragment of CD19 or CD20.
  • hinge region refers to the extracellular domain of the CAR molecule that is located between the antigen binding domain and the transmembrane domain and spatially separates the antigen binding domain from the intracellular domain.
  • the hinge region may also be referred to as a "hinge domain” or a "spacer”.
  • a hinge may contribute to receptor expression, activity and/or stability. The hinge can also provide flexibility for binding the target antigen, allowing the antigen-binding domain to be oriented in different orientations to facilitate binding.
  • transmembrane domain refers to a domain that has the property of being present in a membrane when present in a molecule at the cell surface or at the cell membrane (eg, spanning part or all of the cell membrane).
  • a transmembrane domain can be any protein structure that is thermodynamically stable in a membrane. This is usually an alpha helix containing several hydrophobic residues.
  • the transmembrane domain of any transmembrane protein can be used to provide the transmembrane portion of the chimeric receptor.
  • transmembrane domains of proteins can be determined by those skilled in the art using the TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0/). Alternatively, artificially designed TM domains can be used.
  • the intracellular domain is the signaling portion of the chimeric antigen receptor. It contains a signaling domain and one or more co-stimulatory domains. Following antigen recognition, the receptor cluster native CD45 and CD148 are expelled from the synapse and a signal is transmitted to the cell, thereby activating one or more immune cell effector functions (eg, native immune cell effector functions).
  • the most commonly used intracellular domain component is that of CD3 ⁇ containing 3 ITAMs. After antigen binding, it transmits an activation signal to T cells. CD3 ⁇ may not provide a fully sufficient activation signal and additional co-stimulatory signaling may be required. Costimulatory signals promote T cell proliferation and survival. There are two main types of co-stimulatory signals: those belonging to the Ig family (CD28, ICOS) and the TNF family (OX40, 41BB, CD27, GITR, etc.).
  • the signaling domain of the intracellular domain mediates activation of at least one normal effector function of the immune cell.
  • effector functions of T cells may be cytolytic or helper activities involving cytokine secretion.
  • the signaling domain of the intracellular domain mediates T cell activation, proliferation, survival, and/or other T cell functions.
  • the intracellular domain may contain a signaling domain (intracellular signaling domain) as an activation domain.
  • the intracellular domain may also comprise a signaling domain that is a costimulatory signaling domain (intracellular costimulatory domain).
  • CD3 ⁇ as used herein is defined as the protein provided by GenBan Accession No. BAG36664.1, or the equivalent residues from a non-human species such as mouse, rodent, monkey, ape, and the like.
  • a "CD3 ⁇ intracellular signaling region” is defined as the amino acid residues from the cytoplasmic domain of the ⁇ chain that are sufficient to functionally transmit the initial signal required for T cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to 164 of GenBan accession number BAG36664.1, or a functional ortholog thereof, i.e., from a non-human species such as mouse, rodent, monkey, ape, etc. equivalent residues.
  • nucleic acid sequence As used herein, the terms “nucleic acid sequence”, “nucleic acid” and “polynucleotide” are intended to be synonymous with each other. Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides and nucleic acids can encode the same polypeptide. In addition, it should be understood that the skilled artisan can use routine techniques to make nucleotide substitutions that do not affect the sequence of the polypeptide encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptide is to be expressed.
  • a nucleic acid according to the invention may comprise DNA or RNA. They can be single-stranded or double-stranded.
  • polynucleotides may also be polynucleotides which include synthetic or modified nucleotides. Many different types of modifications to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones with the addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For purposes of the uses described herein, it is understood that polynucleotides may be modified by any method available in the art. Such modifications can be made to enhance the in vivo activity or longevity of the polynucleotide of interest.
  • cell refers to any type of cell, such as eukaryotic or prokaryotic cells, capable of expressing the CAR of the present invention.
  • transfection is the process of introducing nucleic acid molecules or polynucleotides, including vectors, into target cells.
  • transduction is generally used to describe the virus-mediated transfer of nucleic acid molecules or polynucleotides.
  • Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of material. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with the material to generate liposomes that fuse with cell membranes and deposit their cargo inside.
  • Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation.
  • treatment includes therapeutic or prophylactic treatment in a subject in need thereof.
  • “Therapeutic or prophylactic treatment” includes prophylactic treatment aimed at the complete prevention of clinical and/or pathological manifestations or therapeutic treatment aimed at amelioration or alleviation of clinical and/or pathological manifestations.
  • the term “treating” also includes ameliorating or preventing a disease.
  • treatment may include: (i) preventing the disease, disorder and/or condition in a patient who may be predisposed to the disease, disorder and/or condition but has not been diagnosed with the disease, disorder and/or condition; (ii ) inhibiting said disease, disorder and/or condition, ie arresting its development; or (iii) alleviating said disease, disorder and/or condition, ie causing regression of said disease, disorder and/or condition.
  • an effective amount means an amount of a therapeutic agent which, when administered to a subject for the treatment or prevention of a disease, is sufficient to effect such treatment or prevention.
  • the “effective amount” may vary depending on the compound, the disease and its severity, and the age, weight, etc. of the subject to be treated.
  • “Therapeutically effective amount” refers to an effective amount for therapeutic treatment.
  • “Prophylactically effective amount” refers to an effective amount for prophylactic treatment.
  • the terms "subject,” “individual,” and “patient” are art-recognized and are used interchangeably herein to refer to any subject, particularly a mammal, in need of treatment subject. Examples include, but are not limited to, humans and other primates, including non-human primates such as chimpanzees and other ape and monkey species. The terms individual, subject, and patient per se do not denote a specific age, sex, race, or the like.
  • autologous refers to any substance derived from the same individual into which it is later reintroduced.
  • the treatment methods herein involve collecting lymphocytes from a patient, then engineering them to express, for example, a CAR of the invention, and then administering them back to the same patient.
  • allogeneic refers to any substance derived from one individual and then introduced into another individual of the same species, such as an allogeneic T cell transplant.
  • the present invention provides a chimeric antigen receptor (CAR) comprising an antigen binding domain, a transmembrane domain, an intracellular signaling domain and an intracellular co-stimulatory domain, wherein the intracellular
  • the costimulatory domain comprises a first costimulatory domain capable of binding and activating lymphocyte-specific protein tyrosine kinase (LCK) to enhance immune cell activation.
  • LCK lymphocyte-specific protein tyrosine kinase
  • a CAR may contain a co-stimulatory domain, for example, to increase signaling potency.
  • a co-stimulatory domain for example, to increase signaling potency.
  • a CAR of the invention comprises one or more co-stimulatory domains that activate one or more immune cell effector functions, such as the innate immune cell effector functions described herein.
  • a portion of such co-stimulatory domains may be used as long as the portion transduces an effector function signal.
  • the use of Its ability to bind and activate LCK recruits more activated LCK molecules to the intracellular domain of the CAR molecule, thereby increasing the activation level of CAR-T cells, thereby improving the tumor-killing ability of CAR-T cells.
  • the first co-stimulatory domain may comprise a sequence derived from the cytoplasmic region of the CD146 molecule.
  • the first co-stimulatory domain may comprise a KKGK motif at the membrane-proximal end of the cytoplasmic region of CD146. In some embodiments, the first co-stimulatory domain may comprise the cytoplasmic region of CD146, or a membrane-proximal fragment thereof. In some embodiments, the first co-stimulatory domain may comprise a functional variant of the CD146 cytoplasmic region or its proximal membrane fragment, and the functional variant may comprise one or more amino acid insertions or deletions relative to the wild-type sequence , substitution or addition, so long as the variant retains the ability to bind and activate LCK to enhance immune cell activation.
  • the functional variant of the CD146 cytoplasmic region or its membrane-near end fragment may retain the KKGK motif and contain one or more amino acid insertions, deletions, or substitutions at other positions except the KKGK motif in the CD146 cytoplasmic region or added peptides.
  • the term "functional variant” refers to a polypeptide that has significant sequence identity to a parent polypeptide and that retains the biological activity of the parent polypeptide. Functional variants encompass, for example, a CD146 cytoplasmic region or its vicinity described herein that retains the ability to bind and activate LCK to a similar extent, to the same extent, or to a greater extent than the parent polypeptide. Variant of the membrane end fragment.
  • the amino acid sequence of the functional variant may, for example, have at least about 30%, 50%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the amino acid sequence of the parent polypeptide. identity.
  • a functional variant may be a functional variant formed by insertion, deletion or substitution of one or more amino acids in a parent polypeptide.
  • a functional variant may comprise the amino acid sequence of a parent polypeptide with at least one conservative amino acid substitution.
  • Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and/or chemical properties is exchanged for another amino acid having the same chemical or physical properties.
  • a conservative amino acid substitution can be an acidic amino acid for another acidic amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly , Val, Ile, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid is replaced by another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain is replaced by a polar side chain Another amino acid (Asn, Cys, Gln, Ser, Thr, Tyr, etc.) etc.
  • the membrane-proximal segment of the CD146 cytoplasmic region refers to the cytoplasmic region fragment of CD146 near the transmembrane region, which means the fragment from the 584th amino acid to the 646th amino acid of the CD146 molecule.
  • the membrane-proximal segment of the cytoplasmic region of CD146 may be 4-55 amino acids in length, preferably 10-45 amino acids in length, more preferably 15-35 amino acids in length, and even more preferably 15-26 amino acids in length.
  • the membrane-proximal segment of the cytoplasmic region of CD146 comprises amino acids 584-609 of the CD146 molecule.
  • the first co-stimulatory domain may comprise the amino acid sequence shown in SEQ ID NO: 7 or a functional variant thereof formed by insertion, deletion or substitution of one or more amino acids.
  • the intracellular co-stimulatory domain can also comprise a second co-stimulatory domain.
  • the second co-stimulatory domain may be selected from 4-1BB (CD137), OX40 (CD134), ICOS (CD278), 2B4, HVEM, LAG3, DAP10, DAP12, CD27, CD28, CD30, CD40, glucocorticoid-induced tumors Necrosis factor receptor (GITR), lymphocyte function-associated antigen-1 (LFA-1), MyD88, CD2, CD4, CD7, LIGHT, NKG2C, and the intracellular signaling region of B7-H3, or any combination thereof.
  • the second co-stimulatory domain is the intracellular signaling region of 41BB.
  • the amino acid sequences of the second co-stimulatory domains provided herein are known in the art.
  • the second co-stimulatory domain can comprise the amino acid sequence set forth in SEQ ID NO:9.
  • the first costimulatory domain is the cytoplasmic region of CD146
  • the second costimulatory domain is the intracellular signaling region of 41BB
  • the C-terminus of the first costimulatory domain is connected to the N-terminal connection.
  • Intracellular signaling domains that can transduce signals upon antigen binding to immune cells are known, any of which can be included in the CARs of the present disclosure.
  • the cytoplasmic sequence of the T cell receptor (TCR) is known to initiate signal transduction upon binding of the TCR to an antigen (see, e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).
  • an intracellular signaling domain of the invention may comprise a signaling domain selected from the group consisting of TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, ICOS (CD278), and CD66d. area or any combination thereof.
  • the intracellular signaling domain is preferably the signaling region of CD3 ⁇ .
  • the amino acid sequences of the intracellular signaling domains provided herein are known in the art.
  • the intracellular signaling domain can comprise the amino acid sequence set forth in SEQ ID NO: 11.
  • transmembrane domain contained in the CAR of the present invention is not limited to any type.
  • a transmembrane domain is selected that is naturally associated with the antigen binding domain and/or the intracellular domain.
  • transmembrane domains comprise modifications (e.g., deletions, insertions, and/or substitutions) of one or more amino acids, e.g., to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins to minimize interaction with other members of the receptor complex.
  • Transmembrane domains can be derived from natural sources or from synthetic sources. When the source is a natural source, the domains may be derived from any membrane-bound or transmembrane protein.
  • Exemplary transmembrane domains can be derived from T cell receptor (TCR) alpha chain, TCR beta chain, TCR zeta chain, CD28, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD7, CD8, CD8 ⁇ , CD8 ⁇ , CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB/CD137, activated NK cell receptor , BAFFR, BLAME(SLAMF8), BTLA, CD100(SEMA4D), CD103, CD160(BY55), CD18, CD19, CD19a, CD2, CD247, CD276
  • the transmembrane domain may comprise an alpha chain selected from T cell receptor (TCR), beta chain of TCR, zeta chain of TCR, CD3 ⁇ , CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD19 , CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137(41BB), CD152, CD154 and the transmembrane domain of PD1 or any combination thereof.
  • TCR T cell receptor
  • beta chain of TCR zeta chain of TCR
  • the transmembrane domain is that of CD8.
  • the transmembrane domain may be synthetic (and may, for example, comprise predominantly hydrophobic residues such as leucine and valine). In some embodiments, a triplet of phenylalanine, tryptophan, and valine is included at each end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly linked to the intracellular domain. In some embodiments, short oligopeptide or polypeptide linkers (eg, between 2 and 10 amino acids in length) can form a link between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine doublet.
  • transmembrane domains provided herein are known in the art.
  • the transmembrane domain can comprise the amino acid sequence set forth in SEQ ID NO:5.
  • the antigen-binding domain refers to the portion of the chimeric antigen receptor that recognizes an antigen.
  • an antigen binding domain of the invention can bind one or more tumor-associated antigens (TAAs).
  • TAA is preferably selected from 5T4, alpha-fetoprotein, BCMA, CA-125, carcinoembryonic antigen, CD19, CD20, CD22, CD23, CD30, CD33, CD40, CD56, CD79, CD78, CD123, CD138, c-Met, CSPG4, ROR1, GPC3, Tyrp-1, TACI, ALK, C-type lectin-like molecule 1 (CLL-1), EGFR, EGFRvIII, ERBB2, FLT3, melanoma-associated antigen, mesothelin, MUC-1, VEGFR2. More preferably, the antigen binding domain specifically binds CD19.
  • the antigen binding domain may be selected from an antigen binding domain derived from an antibody against the antigen and an antigen binding domain derived from a natural ligand of the antigen.
  • the antigen binding domain derived from an antibody may be in the form of a scFv, Fab or domain antibody (dAb).
  • the antigen binding domain may be in the form of a Fab fragment of a monoclonal antibody.
  • a Fab CAR comprises two chains: one with an antibody light chain variable region (VL) and constant region (CL); and one with a heavy chain variable region (VH) and constant region (CH).
  • VL antibody light chain variable region
  • CL constant region
  • CH constant region
  • One chain also contains a transmembrane domain and an intracellular domain. The association between CL and CH leads to the assembly of the receptor.
  • VH-CH-hinge region-transmembrane domain-intracellular domain VL-CL
  • the antigen binding domain consists of a VH from one polypeptide chain and a VL from the other polypeptide chain.
  • a polypeptide chain may comprise a linker between VH/VL and CH/CL.
  • Linkers can be flexible and serve to spatially separate VH/VL from CH/CL.
  • Flexible linkers can consist of small non-polar residues such as glycine, threonine and serine.
  • a linker may comprise one or more repeats of a glycine-serine linker, such as a ( Gly4Ser ) n linker, where n is the number of repeats.
  • the or each linker may be less than 50, 40, 30, 20 or 10 amino acids in length.
  • the CAR of the present invention may also comprise a hinge region.
  • the hinge region can be located between the antigen binding domain and the transmembrane domain.
  • the hinge region may comprise a hinge or fragment thereof selected from the group consisting of CD8, IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM.
  • the hinge region may be a CD8 hinge.
  • the hinge region can comprise the amino acid sequence set forth in SEQ ID NO:3.
  • nucleic acid construct has the following structure:
  • BD is a nucleotide sequence encoding an antigen-binding domain
  • hinge is the nucleotide sequence encoding the hinge region
  • TM is a nucleotide sequence encoding a transmembrane domain
  • Costi is a nucleotide sequence encoding an intracellular co-stimulatory domain
  • Signal is a nucleotide sequence encoding an intracellular signaling domain.
  • the nucleic acid construct has the following structure:
  • BD is a nucleotide sequence encoding an antigen-binding domain
  • hinge is the nucleotide sequence encoding the hinge region
  • TM is a nucleotide sequence encoding a transmembrane domain
  • Costi1 is a nucleotide sequence encoding the first co-stimulatory domain
  • Costi2 is a nucleotide sequence encoding a second co-stimulatory domain
  • Signal is a nucleotide sequence encoding an intracellular signaling domain.
  • the antigen binding domain, hinge region, transmembrane domain, intracellular costimulatory domain, first costimulatory domain and second costimulatory domain are as defined above.
  • the antigen binding domain specifically binds to CD19; the hinge region is the CD8 hinge region; the transmembrane domain is the CD8 transmembrane domain; the first co-stimulatory domain is the cytoplasmic region of the CD146 molecule; the second co-stimulatory domain is the 41BB intracellular signaling domain; and the intracellular signaling domain is the CD3 ⁇ intracellular signaling domain.
  • the nucleotide sequence encoding the antigen binding domain can be a nucleotide sequence as shown in SEQ ID NO: 2, or at least 80%, at least 85%, at least 90% identical to SEQ ID NO: 2 %, at least 95% or 99% sequence identity of nucleotide sequences.
  • the nucleotide sequence encoding the hinge region can be a nucleotide sequence as shown in SEQ ID NO: 4, or have at least 80%, at least 85%, at least 90%, at least 90%, Nucleotide sequences with at least 95% or 99% sequence identity.
  • the nucleotide sequence encoding the transmembrane domain can be a nucleotide sequence as shown in SEQ ID NO:6, or have at least 80%, at least 85%, at least 90% of SEQ ID NO:6 %, at least 95% or 99% sequence identity of nucleotide sequences.
  • the nucleotide sequence encoding the first co-stimulatory domain can be a nucleotide sequence as shown in SEQ ID NO: 8, or at least 80%, at least 85%, Nucleotide sequences having at least 90%, at least 95%, or 99% sequence identity.
  • the nucleotide sequence encoding the second co-stimulatory domain can be a nucleotide sequence as shown in SEQ ID NO: 10, or at least 80%, at least 85%, Nucleotide sequences having at least 90%, at least 95%, or 99% sequence identity.
  • the nucleotide sequence encoding the intracellular signaling domain can be a nucleotide sequence as shown in SEQ ID NO: 12, or have at least 80%, at least 85%, Nucleotide sequences having at least 90%, at least 95%, or 99% sequence identity.
  • the invention provides a vector comprising a nucleic acid sequence or nucleic acid construct of the invention.
  • Non-limiting examples of vectors include, but are not limited to, plasmids, viral vectors (including retroviral vectors, lentiviral vectors, adenoviral vectors, vaccinia viral vectors, polyoma viral vectors, and adeno-associated vectors (AAV)), bacteriophage, phage Bacteroids, cosmids and artificial chromosomes (including BAC and YAC).
  • the vector itself is usually a sequence of nucleotides, usually a DNA sequence containing the insert (transgene) and a larger sequence that acts as the "backbone" of the vector.
  • Engineered vectors typically contain an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS).
  • a vector may additionally comprise a promoter, genetic marker, reporter gene, targeting sequence and/or protein purification tag.
  • suitable vectors are provided in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, New York (2012), which is hereby incorporated by reference in its entirety.
  • the vector is preferably selected from lentiviral vectors, retroviral vectors, plasmids, DNA vectors, mRNA vectors, transposon-based vectors and artificial chromosomes.
  • the invention provides cells expressing a CAR of the invention.
  • the T cells are T cells isolated from humans.
  • T cells can be of any type and at any stage of development, including but not limited to CD4+/CD8+ double positive T cells, CD4+ helper T cells such as Th1 and Th2 cells, CD4+ T cells, CD8+ T cells (eg, cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T cells (eg, central memory T cells and effector memory T cells), naive T cells, and the like.
  • the cells are immune cells, preferably T cells, NK cells or macrophages.
  • the cells are stem cells, preferably pluripotent stem cells, induced pluripotent stem cells (iPSC), mesenchymal stem cells, hematopoietic stem cells or lymphoid progenitor cells.
  • iPSC induced pluripotent stem cells
  • mesenchymal stem cells hematopoietic stem cells or lymphoid progenitor cells.
  • the present invention provides a method of preparing the cells of the present invention, comprising the step of transducing or transfecting the cells with the vector of the present invention.
  • the method may further comprise the step of expanding and/or activating cells before or after said transducing or transfecting.
  • the present invention provides a composition comprising the CAR, nucleic acid sequence, nucleic acid construct, vector, or cell of the present invention, and a pharmaceutically acceptable carrier or excipient.
  • compositions according to the invention are in particular compositions suitable for administration to humans. However, it generally also encompasses compositions suitable for administration to non-human animals.
  • the composition and its components i.e. the active agent and optionally the carrier or excipient
  • a pharmaceutically acceptable composition of the invention may, for example, be sterile.
  • the term "pharmaceutically acceptable” may mean approval by a regulatory agency or other recognized pharmacopoeia for use in animals, more particularly in humans.
  • excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesive agents, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweeteners Agents, solvents, co-solvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers, and tonicity regulators.
  • suitable excipients for the preparation of the compositions of the invention is known to those skilled in the art.
  • Exemplary carriers for use in compositions of the invention include saline, buffered saline, dextrose and water.
  • suitable excipients depends inter alia on the active agent used, the disease to be treated and the desired formulation of the composition.
  • the composition may further comprise a second therapeutic agent, preferably, the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs.
  • the second therapeutic agent include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, Gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E, vincristine, and doxorubicin; and peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase, and RNase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs such as antibody-directed enzyme prodrugs; immunostimulants such as IL-2,
  • the invention provides a method of treating cancer in a subject comprising administering to said subject an effective amount of a cell of the invention.
  • cells may be administered to a subject already suffering from a disease or condition to alleviate, reduce or ameliorate at least one symptom associated with the disease and/or to slow, reduce or block the progression of the disease.
  • the effective amount of cells administered can be determined by one skilled in the art using known techniques.
  • a suitable dosage provides a sufficient amount of the active agent of the invention, and preferably is therapeutically effective, ie sufficient to elicit eg a therapeutic or prophylactic response in a subject or animal within a reasonable time frame.
  • the dose of cells of the invention should be within a period of about 2 hours or more, such as 12 hours to 24 hours or more (e.g., 6 months, 12 months, 24 months, etc.) from the time of administration Sufficient to bind a cancer antigen or to detect, treat or prevent cancer. In certain embodiments, the period of time can be even longer.
  • an assay can be used to determine the starting dose to be administered to a mammal, the assay comprising administering a given dose of T cells expressing a CAR of the invention to mammals in a group of mammals (each determined by After administration of different doses of T cells), the extent to which target cell lysis or IFN- ⁇ secretion by such T cells is compared is compared.
  • the extent to which target cell lysis or IFN- ⁇ secretion is achieved following administration of a certain dose can be determined by methods known in the art. Dosages of the cells of the invention are also determined by the presence, nature and extent of any adverse side effects that may accompany the administration of the cells of the invention.
  • the attending physician determines the dosage of the cells of the invention to be administered to each individual patient, taking into account factors such as age, body weight, general health, diet, sex, active agent to be administered, route of administration, and severity of the condition being treated degree.
  • the number of cells administered per infusion can vary, for example, from about 1 ⁇ 10 6 to about 1 ⁇ 10 12 cells or more. In certain embodiments, less than 1 x 106 cells may be administered.
  • treatment may require a single administration of a therapeutically effective dose or multiple administrations of a therapeutically effective dose of an agent of the invention.
  • some compositions may be administered every 3 to 4 days, weekly, or once every two weeks, or once in a month, depending on the formulation, half-life, and clearance rate of the particular composition.
  • the cells of the invention are suitable for a variety of routes of administration. Typically, administration is accomplished parenterally.
  • Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.
  • a subject refers to any subject in need of treatment, especially a mammalian subject.
  • mammalian subjects include humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, and the like.
  • the cells and pharmaceutical compositions provided herein are specifically envisioned for use in the treatment of human subjects.
  • said cells are autologous or allogeneic to said subject.
  • the cells are CAR-T cells or CAR-NK cells.
  • the method comprises the steps of: (i) isolating a sample containing cells from the subject; (ii) transducing or transfecting the cells with a vector of the invention; and (iii) converting The cells obtained in step (ii) are administered to a subject.
  • a sample containing, for example, T cells can be isolated from the subject or from other sources, for example, from the patient's own peripheral blood (first party), or in a hematopoietic stem cell transplant from donor peripheral blood (second party). environment, or isolate cells from peripheral blood (third party) from an unrelated donor.
  • the method can also include administering a second therapeutic agent.
  • the second therapeutic agent is selected from antibodies, chemotherapeutic agents and small molecule drugs. Preferred examples of the second therapeutic agent are described above.
  • the cancer may be selected from lymphoma, multiple myeloma, leukemia, and solid tumors.
  • the cancer may be acute lymphoblastic leukemia (ALL) (including non-T-cell ALL), acute myeloid leukemia, B-cell prolymphocytic leukemia, B-cell acute lymphoblastic leukemia (“BALL”) ), blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloid leukemia, chronic or acute leukemia, diffuse Large B-cell Lymphoma (DLBCL), Follicular Lymphoma (FL), Hairy Cell Leukemia, Hodgkin's Disease, Malignant Lymphoproliferative Conditions, MALT Lymphoma, Mantle Cell Lymphoma, Marginal Zone Lymphoma, Significance Monoclonal gammopathy of unspecified (MGUS), multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hod
  • the cancer may be alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal or rectoanal cancer, eye cancer, intrahepatic cholangiocarcinoma, joint cancer, neck cancer, Cancer of the gallbladder or pleura, cancer of the nose, nasal cavity or middle ear, oral cavity, vagina, vulva, chronic lymphocytic leukemia, chronic myeloid, colon, esophagus, cervix, gastrointestinal carcinoid tumors , glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, Oropharyngeal, ovarian, penile, pancreatic, peritoneal,
  • the present invention provides a method for enhancing the activation of immune cells, which comprises introducing the CAR of the present invention into the immune cells.
  • the immune cells are T cells or NK cells.
  • the method includes the step of transducing or transfecting immune cells with a vector of the invention.
  • the present invention provides the use of the CAR, nucleic acid sequence, nucleic acid construct, vector, cell or composition of the present invention in the manufacture of a medicament for treating cancer in a subject.
  • the invention provides a CAR, nucleic acid sequence, nucleic acid construct, vector, cell or composition of the invention for use in treating cancer in a subject.
  • the cancer may be selected from lymphoma, multiple myeloma, leukemia and solid tumors.
  • Non-limiting examples of cancer are as described above.
  • IP immunoprecipitation
  • CD146 truncated parts of CD146 were constructed, namely C19 (627 amino acids, deletion of amino acids 628-646), C37 (609 amino acids, deletion of 610 -646 amino acids) and C63 (583 amino acids, deletion of 584-646 amino acids) ( Figure 2A). Plasmids encoding these CD146 truncations were transfected into LCK stably transfected 293T cells. Cells were lysed with cell lysate, cell lysates were pre-cleared with protein A/G Sepharose beads, and supernatants were immunoprecipitated overnight at 4°C with anti-CD146 antibody AA1.
  • a plasmid encoding this mutant was transfected into 293T cells that had been stably transfected with a plasmid encoding LCK, and immunoprecipitation experiments were performed as described above. The results showed that the KKGK mutant significantly weakened the interaction with LCK, suggesting that KKGK is indeed required for LCK interaction.
  • another mutant RRS-AAS with a positively charged amino acid cluster RRS motif (which is similar to the motif RRR interacting with LCK in CD4 or CD44) was constructed, and similar experiments were carried out. The results showed that the mutation The body did not lead to weakened interaction with LCK (Fig. 2B-C). Therefore, the interaction of CD146 with LCK is mainly dependent on the KKGK motif.
  • the second-generation CAR was engineered so that its intracellular domain (eg, at the membrane-proximal end) contained the cytoplasmic region of CD146.
  • a vector encoding an anti-CD19 CAR was constructed using the Lenti-EF1a-CD19(FMC63)-2nd-CAR(4-1BB)-EGFRt vector (Aikond Biomedical Technology (Suzhou) Co., Ltd.).
  • the vector is designed to contain anti-CD19 scFv (FMC63), CD8 hinge region, CD8 transmembrane domain, CD146 cytoplasmic region and 41BB-CD3 ⁇ intracellular signal region.
  • the CAR molecule is connected to the EGFRt fragment through the P2A linker to realize the simultaneous transcription and translation of the inserted gene fragment and the EGFRt marker molecule, so that subsequent experiments can detect the lentiviral transfection efficiency through the EGFRt marker molecule.
  • Vectors were subjected to lentiviral packaging for subsequent transduction of T cells according to the manufacturer's (and Metabiota's) instructions.
  • Coding sequence SEQ ID NO: 2 of anti-CD19 antigen-binding domain (FMC63):
  • CD146 cytoplasmic region SEQ ID NO:8
  • Amino acid sequence (SEQ ID NO: 9) of the 41BB intracellular signal region :
  • Coding sequence (SEQ ID NO: 12) of CD3 ⁇ intracellular signal region:
  • T cells that can stably express the CAR molecule of the present invention are constructed by the following procedure:
  • Petri dish coating Coat the cell culture plate overnight at 4°C in advance with anti-human CD3 antibody (1 ⁇ g/ml), anti-human CD28 antibody (1 ⁇ g/ml) and recombinant human fibrin fragment (5 ⁇ g/ml);
  • PBMC Peripheral blood mononuclear cells
  • CB6100 human peripheral blood lymphocyte isolation medium
  • MojoSort TM Human CD3 T Cell Isolation Kit Cat. No. 480021
  • T cell activation culture T cell proliferation medium (recipe: RPMI 1640 medium + 10% heat-inactivated fetal bovine serum + sodium pyruvate + non-essential amino acids + penicillin/streptomycin + 1000IU/ml human IL2) Resuspend T cells to a concentration of 1 ⁇ 10 6 cells/ml. 500 ⁇ l per well was inoculated into a 48-well cell culture plate coated with anti-human CD3 antibody, anti-human CD28 antibody and recombinant human fibrin fragment at 4°C overnight, and activated in a 37°C, 5% CO 2 incubator. After 24-36 hours of activation, the T cells are attached to the bottom of the cell culture plate under the action of antibodies and fibronectin. The cell volume becomes larger, and the shape gradually changes from round to polarized. At this time, the cells are about to start to proliferate rapidly. in lentiviral infection.
  • T cell proliferation medium for T cells the formula is RPMI 1640 medium + 10% heat-inactivated fetal bovine serum + sodium pyruvate + non-essential amino acids + penicillin/streptomycin + 1000IU /ml human IL2
  • T cells transfected with 41BB-CD3 ⁇ second-generation CAR molecules T cells transfected with CD146 cytoplasmic region (CD146cyt)-41BB-CD3 ⁇ CAR molecules were compared with Raji cells highly expressing CD19 molecules.
  • Raji cells highly expressing CD19 molecules Human Burkitt lymphoma cell line
  • E:T ratio effector cell-target cell ratio
  • the CD19 antigen was coated on a 48-well plate according to the specified concentration (1 ⁇ g/ml, 2 ⁇ g/ml, 4 ⁇ g/ml), and then the above-mentioned T cells transfected with 41BB-CD3 ⁇ second-generation CAR molecule, transfected with CD146cyt T cells with -41BB-CD3 ⁇ CAR molecules were added into the well plate, and the CD69 + cell ratios of the two T cells were detected by flow cytometry after 5 hours, and the results are shown in Figure 4B.
  • T cells transfected with 41BB-CD3 ⁇ second-generation CAR molecules and T cells transfected with CD146cyt-41BB-CD3 ⁇ CAR molecules were co-cultured with Raji cells highly expressing CD19 molecules at an E:T ratio of 1:1 for 48 h. Then, the production of TNF- ⁇ , IL-2 and IFN- ⁇ in T cells was detected by flow cytometry, and the results are shown in FIG. 5 .
  • T cells transfected with 41BB-CD3 ⁇ second-generation CAR molecules T cells transfected with CD146cyt-41BB-CD3 ⁇ CAR molecules were compared with Raji cells highly expressing CD19 molecules at a ratio of 1:1 and 2 After 36 hours of co-cultivation at an E:T ratio of 1, the supernatant and cells were collected by centrifugation.
  • Lactate dehydrogenase in the culture supernatant was detected by using a non-isotope-labeled cell death kit (CytoTox96 Non-radioactive Cytotoxicity Assay) (the detection principle is that after cell death, intracellular lactate dehydrogenase (LDH ) is released into the medium, so the level of LDH in the medium is detected by enzymatic discoloration reaction, and the number of cell death can be converted).
  • the target cell maximum release group deducted the volume to correct the control group, and after the experimental group and the self-release group deducted the medium blank control group, the killing rate was calculated according to the following formula:
  • Killing rate (%) (experimental group release-spontaneous release of effector cells-spontaneous release of target cells/maximum release of target cells-spontaneous release of target cells) ⁇ 100%.
  • Example 7 In vivo killing activity of CAR-T cells against hematological tumors
  • NSG immunodeficient mice were used as a model. NSG mice lack T cells, B cells, NK cells, and the complement system, and their macrophages and dendritic cells are also defective. NSG mice have severe immunodeficiency and are widely used in preclinical research on T cell therapy because they do not have rejection reactions to transplanted tumors and T cells.
  • 6-week-old female NSG mice were used, and the difference in body weight of each batch of experiments was controlled within 2 g. Mice were housed in individually ventilated cages within specific pathogen-free (SPF) clean-grade barriers, provided with a normal diet and drinking water with an acidic pH to prevent pathogen contamination.
  • SPPF pathogen-free
  • mice were numbered and randomly divided into 3 groups, with three mice in each group.
  • the three groups of mice were: human T cell group (injection of untransfected human primary T cells), 41BB CAR-T group (injection of FMC63-4-1BB-2 nd -CAR T cells), CD146cyt-41BB-CAR -T group (injection of FMC63-CD8TM-CD146cyt-4-1BB-CD3-CAR T cells);
  • Example 8 In vivo killing activity of CAR-T cells against solid tumors
  • mice were used in this example to subcutaneously inoculate the aforementioned Raji-luc cells with tumors, and each mouse was inoculated with 3* 106 cells.
  • the tumor-bearing mice were numbered and randomized. Divide into 3 groups with two mice in each group. The three groups of mice were: human T cell group (injection of untransfected human primary T cells), 41BB CAR-T group (injection of FMC63-4-1BB-2 nd -CAR T cells), CD146cyt-41BB-CAR -T group (injection of FMC63-CD8TM-CD146cyt-4-1BB-CD3-CAR T cells).
  • Each mouse in each group was injected with 4*10 6 corresponding T cells, and the images were detected on the 13th day and the 18th day after inoculation respectively, and the results are shown in FIG. 8 .

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  • Epidemiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne un récepteur antigénique chimérique (CAR) et son utilisation. Le CAR comprend un premier domaine de co-stimulation capable de se lier à la protéine tyrosine kinase spécifique des lymphocytes et d'activer la protéine tyrosine kinase spécifique des lymphocytes (LCK) pour améliorer l'activation des cellules immunitaires. L'invention concerne également une séquence d'acide nucléique codant pour le CAR, une construction d'acide nucléique comprenant la séquence d'acide nucléique codant pour le CAR, un vecteur comprenant la séquence d'acide nucléique ou la construction d'acide nucléique, une cellule exprimant le CAR, et un procédé de préparation de la cellule.
PCT/CN2022/116237 2021-09-01 2022-08-31 Récepteur antigénique chimérique et son utilisation Ceased WO2023030393A1 (fr)

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US20210206826A1 (en) * 2015-11-19 2021-07-08 The Regents Of The University Of California Conditionally repressible immune cell receptors and methods of use thereof

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US20210206826A1 (en) * 2015-11-19 2021-07-08 The Regents Of The University Of California Conditionally repressible immune cell receptors and methods of use thereof
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