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WO2024125605A1 - Procédé pour améliorer la persistance d'une population de lymphocyte t - Google Patents

Procédé pour améliorer la persistance d'une population de lymphocyte t Download PDF

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WO2024125605A1
WO2024125605A1 PCT/CN2023/138823 CN2023138823W WO2024125605A1 WO 2024125605 A1 WO2024125605 A1 WO 2024125605A1 CN 2023138823 W CN2023138823 W CN 2023138823W WO 2024125605 A1 WO2024125605 A1 WO 2024125605A1
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car
cell population
cell
cells
tscm
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Chinese (zh)
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钟晓松
张莹
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Carriage Pharmaceutic Beijing Co Ltd
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Carriage Pharmaceutic Beijing Co Ltd
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    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
    • A61K39/001195—Prostate specific membrane antigen [PSMA]
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3069—Reproductive system, e.g. ovaria, uterus, testes, prostate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/80—Vaccine for a specifically defined cancer
    • A61K2039/884—Vaccine for a specifically defined cancer prostate
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to the field of medicine, and in particular to a method for improving the persistence of a CAR-T cell population (especially a CAR-T cell population targeting a prostate cancer-specific membrane antigen PSMA) by utilizing a space microgravity environment or a simulated microgravity environment, and the use of the CAR-T cells obtained by the method in the preparation of anti-tumor drugs.
  • Prostate cancer is one of the most common tumors of the male urogenital system.
  • Traditional treatments for prostate cancer include radical prostatectomy, radiotherapy, chemotherapy, and androgen deprivation therapy. Although these therapies have certain efficacy, their effects on alleviating the disease in most patients are still very limited. Therefore, the treatment of prostate cancer patients has long been an unmet medical need.
  • CAR is a synthetic molecule consisting of an extracellular tumor antigen binding domain, a hinge, a transmembrane, and an intracellular signaling domain connected thereto. It specifically recognizes surface proteins expressed on tumor cells and induces T cell responses against tumor cells to eradicate tumors.
  • CAR-expressing T cells i.e., CAR-T cells
  • scFv single chain variable fragment
  • PSMA prostate-specific membrane antigen
  • microgravity environment in space can affect many characteristics of organisms, such as growth rate, cell morphology, and cell metabolism. Therefore, it has been proposed to use the microgravity environment in space or simulated microgravity environment to develop new species and new pharmaceutical products. However, there has been no report on the application of the microgravity environment in space or simulated microgravity environment on CAR-T cells and the related application effects.
  • Tscm Stem like memory T cells
  • Tcm central memory T cells
  • Tscm and Tcm memory cell subsets in the CAR-T cell population are associated with the in vivo persistence of CAR-T cells, and it is proposed that increasing the proportion of Tscm and Tcm memory cell subsets is valuable in the clinical application of CAR-T cells. See, Chimeric antigen receptor T cell persistence and memory cell formation, Immunology & Cell Biology 2019; 97: 664–674.
  • the inventors surprisingly found that the space microgravity environment can effectively promote the proportion of Tscm/Tcm-like cell subsets in the CAR-T cell population targeting the prostate cancer-specific membrane antigen PSM, and enhance the persistence of CAR-T cells.
  • the space environment interferes with/reduces the immune function of astronauts or the immune function of lymphocytes therefrom, the above findings of the inventors are particularly surprising.
  • the present invention provides a method for improving the persistence of a CAR-T cell group (especially a CAR-T cell group targeting a prostate cancer-specific membrane antigen (PSMA)), the method comprising: maintaining the CAR-T cell group in a space microgravity environment or a simulated microgravity environment, thereby obtaining a CAR-T cell group (CAR-T-S cell group) with increased persistence compared to a control CAR-T cell group that has not received the treatment.
  • a CAR-T cell group especially a CAR-T cell group targeting a prostate cancer-specific membrane antigen (PSMA)
  • PSMA prostate cancer-specific membrane antigen
  • CAR-T-S cell a CAR-T cell group treated in a space microgravity environment or a simulated microgravity environment
  • PSMA-CAR-T-S cell group a CAR-T cell group treated in a space microgravity environment or a simulated microgravity environment
  • PSMA-CAR-T-S cell group a CAR-T cell group treated in a space microgravity environment or a simulated microgravity environment
  • the present invention provides a method for increasing the ratio of Tscm and/or Tcm or the sum of the ratios of the two in a CAR-T cell population, comprising:
  • a CAR-T cell group (CAR-T-S cell group) with increased Tscm and/or Tcm ratio is obtained.
  • the remaining steps of the method are performed in a normal gravity environment (i.e., an environment with the same gravity as the earth).
  • the CAR-T cell population is maintained in a space microgravity environment by orbital space flight.
  • the present invention provides a CAR-TS cell population (especially, a PSMA-CAR-TS cell population) obtained by the method of the present invention.
  • the CAR-TS cell population has increased persistence compared to a control CAR-T cell population that has not been treated in a space microgravity environment or a simulated microgravity environment.
  • the CAR-TS cell population has an increased Tscm ratio and/or Tcm ratio or both ratios compared to the control CAR-T cell population. Examples and.
  • the CAR-TS cell group produces a reduced amount of IFN ⁇ under stimulation with the target antigen PSMA compared to the control CAR-T cell group.
  • the CAR-TS cell group has more cells in the cell division phase (S phase) compared to the control CAR-T cell group by cell cycle detection. In some embodiments, the CAR-TS cell group has an increased survival rate compared to the control CAR-T cell group; and/or delayed or reversed T cell exhaustion.
  • the present invention also provides a use of a CAR-T-S cell population obtained by the method of the present invention in the preparation of a drug for treating a tumor.
  • the CAR-T cell population is a PSMA-targeted CAR-T cell population
  • the tumor is prostate cancer.
  • Figure 1 shows the cell sorting gating step for detecting T cell subpopulations after PSMA-CAR-T cells and PSMA-CAR-T-S cells were cultured with target cells for seven days.
  • the horizontal black arrows shown in each sub-graph of Figures a-f indicate the cell subpopulations in the corresponding sorting boxes (a-d: black boxes; e: Q4-1 boxes; f: Q1-6 boxes) for the next sorting step; the sub-graph of Figure g shows the sorting box corresponding to Tscm cells.
  • Figure 2 shows the proportion of T cell subsets in the PSMA-CAR-T cell population analyzed by flow cytometry.
  • the upper figure shows that in the ground control PSMA-CAR-T cell population that has not been treated in space, the proportion of CD3 + Tcm cell subsets (CD45RO + CCR7 + ) is 1.4%; and the proportion of CD3 + Tscm cell subsets (CD45RO - CCR7 + CD95 + CD27 + ) is 0.2%.
  • the lower figure shows that in this control PSMA-CAR-T cell population, the proportion of CD3 + CD8 + Tcm cell subsets is 1.0%; and the proportion of CD3 + CD8 + Tscm cell subsets is 0.3%.
  • Figure 3 shows the proportion of T cell subsets in the PSMA-CAR-TS cell population analyzed by flow cytometry.
  • the upper figure shows that in the space-treated PSMA-CAR-TS cell population, the proportion of CD3 + Tcm cell subsets is 3.3%; and the proportion of CD3 + Tscm cell subsets is 0.5%.
  • the lower figure shows that in this PSMA-CAR-TS cell population, the proportion of CD3 + CD8 + Tcm cell subsets is 2.8%; and the proportion of CD3 + CD8 + Tscm cell subsets is 1.1%.
  • FIG4 shows that the PSMA-CAR-TS cell population has increased sum of Tscm and Tcm ratios in both the CD3 + T cell subset and in the CD3 + CD8 + T cell subset compared to the PSMA-CAR-T cell population.
  • FIG5 shows the proportion of cells in the S phase in the PSMA-CAR-TS cell population and the PSMA-CAR-T cell population as determined by cell cycle after contact with the target antigen PSMA.
  • FIG6 shows the secretion of cytokine IFN ⁇ by the PSMA-CAR-T-S cell population and the PSMA-CAR-T cell population after contact with the target antigen (K562 cells expressing PSMA), compared with the negative control.
  • space microgravity environment is also referred to as “space environment” or “space environment” and refers to the space microgravity environment provided by orbital space flight.
  • microgravity environment is also called “zero gravity environment”, which refers to an environment where the apparent weight of a system under the action of gravity is much less than its actual weight.
  • Ground facilities that simulate microgravity have been developed, including devices such as the fast rotating cyclotron (FRC), rotating wall container (RWV) or random positioning machine (RPM).
  • FRC fast rotating cyclotron
  • RWV rotating wall container
  • RPM random positioning machine
  • chimeric receptor refers to a recombinant polypeptide comprising at least an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain.
  • the lowercase letter “car” represents a nucleic acid encoding a chimeric antigen receptor.
  • CAR-T cell refers to a T cell that contains and expresses CAR on the cell surface.
  • PSMA-CAR-T-S cells or cell groups refer to CAR-T cells or cell groups targeting prostate cancer-specific membrane antigen PSMA that have been treated in a space microgravity environment or a simulated microgravity environment.
  • CAR-T cells and CAR-T cell groups are used interchangeably, and “CAR-T-S cells” and “CAR-T-S cell groups” are used interchangeably, referring to CAR-T cell groups containing multiple T cell subsets (for example, but not limited to Tscm and Tcm).
  • the term "stimulatory molecule” refers to a molecule expressed by a T cell that provides a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex in a stimulatory manner in at least some aspect of the T cell signaling pathway.
  • the primary signal is initiated, for example, by the binding of the TCR/CD3 complex to the MHC molecule loaded with a peptide and results in mediating a T cell response, including but not limited to proliferation, activation, differentiation, etc.
  • the cytoplasmic domain of any one or more CARs of the present invention comprises an intracellular signaling sequence, for example, a signaling sequence of CD3 ⁇ .
  • CD3 ⁇ is defined as a protein or its equivalent provided under UniProtKB-P20963 accession number.
  • a "CD3 ⁇ signaling domain” is defined as an amino acid residue segment from the cytoplasmic domain of the CD3 ⁇ chain, which is sufficient to functionally propagate the initial signal necessary for T cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to residue 164 of the amino acid sequence under UniProtKB-P20963 accession number or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.) as their functional homologs.
  • costimulatory molecule refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand to mediate a costimulatory response of the cell (e.g., but not limited to, proliferation).
  • Costimulatory molecules are cell surface molecules that contribute to an effective immune response other than antigen receptors or their ligands.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD28.
  • costimulatory molecules are CD28, 4-1BB (i.e., CD137).
  • costimulatory domain refers to the intracellular portion of a costimulatory molecule.
  • 4-1BB refers to a member of the TNFR superfamily, also known as CD137, which has a The amino acid sequence provided under accession number or the equivalent residue from non-human species (e.g., mouse, rodent, monkey, ape, etc.).
  • 4-1BB costimulatory domain is defined as the cytoplasmic region from 4-1BB, for example, the amino acid residue 214-255 of UniProtKB-Q07011 or the equivalent residue from non-human species (e.g., mouse, rodent, monkey, ape, etc.).
  • CD28 refers to the amino acid sequence provided under the accession number of UniProtKB-P10747 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).
  • CD28 co-stimulatory domain is defined as the cytoplasmic region from CD28, for example, amino acid residues 180-220 of UniProtKB-P10747 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).
  • CD28 transmembrane domain is defined as the transmembrane region from CD28, for example, amino acid residues 153-179 of UniProtKB-P10747 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).
  • CD8 refers to the amino acid sequence provided under UniProtKB-P01732 accession number or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).
  • CD8 hinge region is defined as a transmembrane region from CD8, e.g., amino acid residues 117-178 of UniProtKB-P01732 or equivalent residues from non-human species (e.g., mice, rodents, monkeys, apes, etc.).
  • the CD8 hinge region that can be used for CAR is also described in EP3115373A1, which is incorporated herein by reference in its entirety.
  • the term "recombinant" when referring to, for example, a virus or cell or nucleic acid or protein or vector, means that the virus, cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein, or by changing the existing natural nucleic acid or protein, or refers to a substance from a virus or cell modified thereby. Therefore, in the present invention, the exogenous CAR encoding nucleic acid introduced into a mammalian cell is a recombinant nucleic acid, and the host cell into which the nucleic acid is introduced is a recombinant cell.
  • heterologous nucleic acid sequence refers to a sequence that is derived from and introduced (e.g., by infection with a viral vector) into the same host cell or subject and thereby exists in a non-natural state, e.g., the sequence is located at a different location, exists in a different copy number, or is under the control of a different regulatory element.
  • expression cassette refers to a DNA sequence that encodes and is capable of expressing one or more target genes (e.g., CAR polypeptides of the present invention).
  • a heterologous polynucleotide sequence encoding a target gene is functionally connected to an expression control sequence.
  • a nucleic acid encoding CAR can be placed in a suitable expression cassette and functionally connected to an expression control sequence.
  • connecting peptide refers to a short amino acid sequence consisting of amino acids, such as alanine (A), glycine (G) and/or serine (S) and/or threonine residues (T) used alone or in combination.
  • the connecting peptide has a length of 1-50 amino acids, for example, 1,2,3,4,5 amino acids, or 10,15,20,25,30 amino acids in length.
  • the connecting peptides that can be used between the components of the CAR fusion polypeptide of the present invention are not specifically limited. Computer programs can be used to simulate the three-dimensional structure of proteins and peptides to rationally design suitable connecting peptides.
  • a short oligopeptide linker or a polypeptide linker can form a bond between component sequences as needed, for example, a glycine-serine doublet, or a single amino acid, for example, alanine, glycine can be used as a linker.
  • amino acid change and “amino acid modification” are used interchangeably and refer to the addition, deletion, substitution and other modifications of amino acids. Any combination of amino acid addition, deletion, substitution and other modifications may be performed, provided that the final polypeptide sequence has the desired properties.
  • the amino acid substitution is a non-conservative amino acid substitution, i.e., replacing an amino acid with another amino acid having a different structure and/or chemical property.
  • Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine).
  • amino acid changes or amino acid modifications may be non-conservative amino acid substitutions, but in some cases, conservative amino acid modifications or conservative amino acid changes are preferred.
  • conservative sequence modifications and “conservative sequence changes” refer to amino acid modifications or changes that do not significantly affect or change the characteristics of a parent polypeptide containing an amino acid sequence or its constituent elements. Such conservative modifications include amino acid substitutions, additions and deletions. Conservative modifications, especially conservative substitutions, can be introduced into the CAR polypeptide of the present invention or its constituent elements by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative substitutions are amino acid substitutions in which amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta-side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • the "percentage (%) identity" of an amino acid sequence/nucleotide sequence refers to the percentage of amino acid/nucleotide residues in the candidate sequence that are identical to the amino acid residues/nucleotide residues of the specific amino acid/nucleotide sequence described, after aligning the candidate sequence with the specific amino acid/nucleotide sequence described and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity, and in the case of amino acid sequences, not considering any conservative substitutions as part of the sequence identity.
  • the present invention contemplates variants of the CAR polypeptide or nucleic acid molecule of the present invention or its constituent elements, the variants relative to The specific CAR polypeptide or nucleic acid molecule or its constituent element sequence has a considerable degree of identity, for example, the identity is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or higher.
  • the variant may include conservative modifications.
  • the identity percentage is determined using the BLAST tool available to the public at https://blast.ncbi.nlm.nih.gov using default parameters.
  • variants or functional variants refers to a polypeptide or protein that has substantially the same sequence or significant sequence identity as a reference polypeptide or protein and retains the desired biological activity of the reference polypeptide or protein.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which it has been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operatively attached. Such vectors are referred to herein as "expression vectors.”
  • lentivirus refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in their ability to infect non-dividing cells; they can deliver significant amounts of genetic information to host cells, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentiviral genome, and particularly includes self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8): 1453–1464 (2009).
  • Other examples of lentiviral vectors that can be used clinically include, but are not limited to, lentiviral vectors from Oxford BioMedica. Gene delivery technology, LENTIMAX TM vector system from Lentigen, etc.
  • Non-clinical types of lentiviral vectors are also available and known to those skilled in the art.
  • a lentiviral expression vector comprising a CAR encoding nucleic acid of the present invention, and introducing it into a host immune effector cell (e.g., T cell, NK cell), etc., to produce the CAR-T cell of the present invention.
  • a host immune effector cell e.g., T cell, NK cell
  • immune effector cell refers to a cell that participates in an immune response, e.g., participates in promoting an immune effector reaction.
  • immune effector cells include T cells, e.g., ⁇ / ⁇ T cells and ⁇ / ⁇ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid derived phagocytes.
  • mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • tumor and cancer are used interchangeably herein and encompass both solid tumors and liquid tumors.
  • anti-tumor immunity refers to an immunological effect that can be exhibited by various means, including but not limited to, for example, causing a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.
  • PSMA-CAR-T-S After culturing with target antigen-positive tumor cells (K562-PSMA), both PSMA-CAR-T-S and PSMA-CAR-T cells produced more IFN ⁇ release under stimulation, but PSMA-CAR-T-S produced less IFN ⁇ .
  • Effector CD8+T cells are the main effector cells of anti-tumor immunity and anti-infection immunity in vivo.
  • tumor-infiltrating effector CD8+T cells are prone to apoptosis, and tumor is a chronic disease. Therefore, relying solely on effector CD8+T cells is often not enough to induce tumor elimination, and long-term surviving memory CD8+T cells are needed to maintain sustained anti-tumor immunity.
  • T cm Central memory T cells
  • T scm stem cell memory T cells
  • TEM effector memory T cells
  • TEFF effector T cells
  • Tscm stem cell memory T cells
  • T cm central memory T cells
  • the expression "persistence" related to the CAR-T cell population can be characterized by the ratio of memory cells Tscm and/or Tcm subpopulations in the CAR-T cell population, or by the sum of the ratio of Tscm subpopulations and Tcm subpopulations.
  • the PSMA-CAR-TS cell population according to the present invention has a higher ratio of memory cells Tscm and/or Tcm subpopulations, or a higher sum of the ratio of Tscm subpopulations and Tcm subpopulations.
  • the ratio of Tscm and Tcm subpopulations in the CD3 and/or CD8 subpopulations of the CAR-T cell population after contact with tumor cells expressing the PSMA target antigen can be determined according to the flow cytometry assay method described in the examples, and the "persistence" of the CAR-T cell population is thereby determined.
  • the cell cycle determination method described in the examples can be further used to determine the proportion of cells in the S phase of the CAR-T cell population; and/or the cytokine determination method described in the examples can be further used to determine the proportion of cells in the S phase of the CAR-T cell population. IFN ⁇ secretion was measured to further characterize the persistence of the CAR-T cells.
  • the present invention provides a method for improving the persistence of a CAR-T cell group (especially a CAR-T cell group targeting a prostate cancer-specific membrane antigen (PSMA)), the method comprising: maintaining the CAR-T cell group in a space microgravity environment or a simulated microgravity environment, thereby obtaining a CAR-T cell group (CAR-T-S cell group) with increased persistence compared to a control CAR-T cell group that has not received the treatment.
  • a CAR-T cell group especially a CAR-T cell group targeting a prostate cancer-specific membrane antigen (PSMA)
  • PSMA prostate cancer-specific membrane antigen
  • CAR-T-S cell a CAR-T cell group treated in a space microgravity environment or a simulated microgravity environment
  • PSMA-CAR-T-S cell a CAR-T cell group treated in a space microgravity environment or a simulated microgravity environment
  • PSMA-CAR-T-S cell a CAR-T cell group treated in a space microgravity environment or a simulated microgravity environment
  • PSMA-CAR-T-S cell PSMA-CAR-T-S cell
  • the method includes: placing the CAR-T cell group in a space microgravity environment or a simulated microgravity environment, and maintaining a time sufficient to increase the Tscm and/or Tcm ratio in the CAR-T cell group, thereby increasing the persistence of the CAR-T cells.
  • the method includes: maintaining the CAR-T cell group in a space microgravity environment or a simulated microgravity environment for at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours, for example, about 1-5 days, for example, about 3 days.
  • the CAR-TS cell population obtained by the method of the present invention has an increased Tscm ratio and/or Tcm ratio after contact with the target antigen compared to the control CAR-T cell population, preferably, an increased sum of Tscm and Tcm ratios, wherein preferably, the sum of the Tscm and T CM ratios is increased by at least 0.5 times, 0.8 times, 1 times, 1.2 times, 1.5 times, 1.8 times, 2.0 times, 2.2 times, 2.5 times, 2.8 times, or 3 times compared to the control CAR-T cell population.
  • the Tscm and the Tcm are CD3 + T cells.
  • the T SCM and the Tcm are CD3 + CD8 + T cells.
  • the CAR-T cells of the CAR-T cell population comprise a chimeric antigen receptor (CAR) polypeptide, wherein the chimeric antigen receptor polypeptide comprises from N-terminus to C-terminus: an extracellular antigen binding domain, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain, preferably, wherein the chimeric antigen receptor polypeptide comprises from N-terminus to C-terminus: optionally a signal peptide, a single-chain scFv antibody that specifically binds to prostate cancer-specific membrane antigen (PSMA), a CD28 transmembrane domain, a CD28 and/or 4-1BB co-stimulatory domain, and a CD3 ⁇ signaling domain.
  • PSMA prostate cancer-specific membrane antigen
  • the method further comprises: after the CAR-T-S cell population contacts the target antigen, detecting the proportion of Tscm and Tcm cell subpopulations in the CAR-T-S cell population by flow cytometry.
  • the flow cytometry detection is performed according to the cell gating step shown in FIG. 1 .
  • the present invention provides a method for increasing the ratio of Tscm and/or Tcm or the sum of the ratios of the two in a CAR-T cell population, comprising:
  • a CAR-T cell group (CAR-T-S cell group) with an increased Tscm and/or Tcm ratio is obtained.
  • the remaining steps of the method are performed in a normal gravity environment (i.e., an environment with the same gravity as the earth).
  • the method includes: placing the CAR-T cell group in a space microgravity environment or a simulated microgravity environment, and maintaining a time sufficient to increase the Tscm and/or Tcm ratio in the CAR-T cell group, thereby obtaining a CAR-T-S cell group with increased persistence.
  • the method includes: maintaining the CAR-T cell group in a space microgravity environment or a simulated microgravity environment for at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours, for example, about 1-5 days, for example, about 3 days.
  • the CAR-TS cell population obtained by the method of the present invention has an increased Tscm ratio and/or Tcm ratio after contact with the target antigen compared to the control CAR-T cell population, preferably, an increased sum of Tscm and Tcm ratios, wherein preferably, the sum of the Tscm and T CM ratios is increased by at least 0.5 times, 0.8 times, 1 times, 1.2 times, 1.5 times, 1.8 times, 2.0 times, 2.2 times, 2.5 times, 2.8 times, or 3 times compared to the control CAR-T cell population.
  • the Tscm and the Tcm are CD3 + T cells.
  • the T SCM and the Tcm are CD3 + CD8 + T cells.
  • the CAR-T cells of the CAR-T cell population comprise a chimeric antigen receptor (CAR) polypeptide, wherein the chimeric antigen receptor polypeptide comprises from N-terminus to C-terminus: an extracellular antigen binding domain, a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular signaling domain, preferably, wherein the chimeric antigen receptor polypeptide comprises from N-terminus to C-terminus: optionally a signal peptide, a single-chain scFv antibody that specifically binds to prostate cancer-specific membrane antigen (PSMA), a CD28 transmembrane domain, a CD28 and/or 4-1BB co-stimulatory domain, and a CD3 ⁇ signaling domain.
  • PSMA prostate cancer-specific membrane antigen
  • the method further comprises: contacting the CAR-T-S cell population from step (b) with the target antigen, and determining the ratio of Tscm and Tcm subpopulations in the CAR-T-S cell population.
  • the ratio of Tscm and Tcm cell subpopulations in the CAR-T-S cell population is detected by flow cytometry.
  • the flow cytometry detection is performed according to the cell gating step shown in Figure 1.
  • the CAR-TS cell group produces a reduced amount of IFN ⁇ under target antigen stimulation compared to the control CAR-T cell group.
  • the CAR-TS cell group compared to the control CAR-T cell group, has a higher cell proliferation capacity after contact with the target antigen, preferably, by cell cycle determination, the proportion of cells in the cell division phase (S phase) is determined in the CAR-T cell group to determine the cell proliferation capacity.
  • the CAR-TS cell group compared to the control CAR-T cell group, has an increased survival rate; and/or a delayed or reversed T cell exhaustion phenotype.
  • the method of the present invention further comprises one or more steps selected from the following to further characterize the persistence of the obtained CAR-T-S cell population:
  • the present invention also provides the use of a space microgravity environment or simulated microgravity for improving the persistence of a CAR-T cell population, or for increasing the Tscm and/or Tcm ratio or the sum of the two ratios of a CAR-T cell population, or for increasing the survival rate of a CAR-T cell population and/or reversing or delaying T cell exhaustion.
  • the step of maintaining the CAR-T cell population in a space microgravity environment is implemented by orbital space flight.
  • the present invention provides a CAR-T-S cell group (especially, a PSMA-CAR-T-S cell group) obtained by the method of the present invention.
  • the CAR-T-S cell group has increased persistence compared to a control CAR-T cell group that has not been treated in a space microgravity environment or a simulated microgravity environment.
  • the CAR-T-S cell group has an increased Tscm ratio and/or Tcm ratio or the sum of the two ratios compared to the control CAR-T cell group.
  • the CAR-T-S cell group produces a reduced amount of IFN ⁇ under the stimulation of the target antigen PSMA compared to the control CAR-T cell group.
  • the CAR-T-S cell group has more cells in the cell division phase (S phase) compared to the control CAR-T cell group.
  • the PSMA-CAR-T-S cell group has an increased survival rate compared to the control CAR-T cell group; and/or delayed or reversed T cell exhaustion.
  • the CAR polypeptides of the present invention include an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic domain.
  • the cytoplasmic domain of the CAR polypeptide of the present invention includes an intracellular signaling domain.
  • the cytoplasmic domain of the CAR polypeptide of the present invention includes a co-stimulatory domain and a cytoplasmic signaling domain.
  • the chimeric antigen receptor (CAR) molecule according to the present invention includes from N-terminus to C-terminus: (a) an antigen binding domain that specifically binds to a tumor antigen; and (b) a hinge region or a spacer; (c) a transmembrane domain; and (d) an intracellular signaling domain.
  • the CAR molecule according to the present invention includes from N-terminus to C-terminus: (a) an antigen binding domain that specifically binds to a tumor antigen, (b) a hinge region or a spacer; (c) a transmembrane domain; (d) a co-stimulatory domain; and (e) an intracellular signaling domain.
  • the target antigen for the CAR polypeptide of the present invention is a membrane antigen expressed on the surface of a target cell, especially a tumor cell, such as a tumor-specific antigen or a tumor-associated antigen.
  • Tumors that may be mentioned include hematological tumors and solid tumors, including primary and metastatic tumors.
  • the target antigen is a protein that can be derived from a mammal. Tumor cell surface antigens of antigenic cancer epitopes immune-recognized by tumor infiltrating lymphocytes (TIL).
  • TIL tumor infiltrating lymphocytes
  • the target antigen is a tumor cell surface antigen comprising one or more antigenic cancer epitopes associated with malignant tumors.
  • the extracellular antigen binding domain of the CAR molecule of the present invention targets tumor antigens, preferably, the tumor antigen is selected from: CD19, adrenergic A2 receptor (EphA2), folate receptor (FRa), mesothelin, EGFRvIII, IL-13Ra, CD123, CD33, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof. More preferably, the tumor antigen is a prostate cancer-specific membrane antigen PSMA.
  • the CAR of the present invention can be constructed to include an appropriate antigen binding domain specific to the desired antigen target, so as to give the CAR molecule and the CAR-T cell comprising the CAR molecule the ability to specifically recognize and bind to the target antigen.
  • the extracellular antigen binding domain of the CAR molecule according to the present invention is a polypeptide molecule with binding affinity to the target antigen.
  • the CAR according to the present invention includes an antigen binding domain derived from an antibody or antibody fragment.
  • the antigen binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL).
  • the antigen binding domain includes a scFv formed by connecting VL and VH via a joint.
  • the scFv can be produced by connecting VH and VL regions together using a flexible polypeptide linker according to methods known in the art.
  • the scFv molecule comprises a flexible polypeptide linker with an optimized length and/or amino acid composition.
  • the scFv comprises a linker between its VL and VH regions, wherein the linker comprises at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more amino acid residues.
  • the linker sequence may comprise any naturally occurring amino acid.
  • the peptide linker of the scFv is composed of amino acids such as glycine and/or serine residues used alone or in combination to connect the variable heavy chain and variable light chain regions together.
  • the flexible polypeptide linker includes but is not limited to (Gly4Ser)4 or (Gly4Ser)3.
  • the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser).
  • the linker comprises the GSTSGSGKPGSGEGSTKG amino acid sequence.
  • the scFv used in the present invention comprises from N-terminus to C-terminus: VL-linker-VH; or VH-linker-VL.
  • the CAR polypeptide of the present invention comprises at least one transmembrane domain, which can be derived from a natural source or a synthetic source.
  • the transmembrane domain can be derived from a membrane-bound protein or a transmembrane protein, such as a transmembrane domain from CD3 ⁇ , CD4, CD28, CD8 (e.g., CD8 ⁇ , CD8 ⁇ ).
  • the transmembrane domain confers membrane attachment properties to the CAR polypeptide of the present invention.
  • the transmembrane domain in the CAR polypeptide of the present invention can be connected to the extracellular region of the CAR by means of a hinge region/spacer.
  • a hinge region/spacer Regarding the transmembrane region and hinge region/spacer that can be used in the CAR polypeptide
  • For the partition region see, for example, Kento Fujiwara et al., Cells 2020, 9, 1182; doi: 10.3390/cells9051182.
  • the cytoplasmic domain included in the CAR polypeptide of the present invention includes an intracellular signaling domain.
  • the intracellular signaling domain can activate at least one immune effector function of the immune cell into which the CAR of the present invention is introduced.
  • the immune effector function includes, but is not limited to, for example, enhancing or promoting the function or response of immune cells attacking target cells.
  • the immune effector cell is a T cell
  • such an immune effector function can be, for example, cytolytic activity or auxiliary activity, including secretion of cytokines.
  • cytoplasmic domains used in the CAR polypeptides of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and/or co-receptors that can play a role in initiating signal transduction after the extracellular domain binds to the target antigen, and any derivatives or variants of these sequences and any recombinant sequences with the same function or property.
  • TCRs T cell receptors
  • co-receptors that can play a role in initiating signal transduction after the extracellular domain binds to the target antigen
  • the activation of T cells is mediated by two different types of cytoplasmic signaling sequences: those sequences that initiate antigen-dependent primary activation by TCR (i.e., primary intracellular signaling domains) and those sequences that act in an antigen-independent manner to provide costimulatory signals (i.e., secondary cytoplasmic domains, e.g., costimulatory domains).
  • the CAR polypeptide of the present invention includes a cytoplasmic domain that provides a primary intracellular signaling domain, e.g., an intracellular signaling domain of CD3 ⁇ .
  • the cytoplasmic domain of the CAR polypeptide of the present invention also includes a secondary signaling domain, e.g., a costimulatory domain from a costimulatory molecule.
  • the cytoplasmic region of the CAR polypeptide of the present invention includes one or more costimulatory domains in series with the CD3 ⁇ intracellular signaling domain, such as 4-1BB (also referred to as CD137) and CD28 costimulatory domains.
  • the CAR polypeptide of the present invention may include a signal peptide or leader sequence located at the N-terminus of the extracellular antigen binding domain.
  • the signal peptide/leader sequence Through the signal peptide/leader sequence, the nascent CAR polypeptide can be directed to the endoplasmic reticulum of the cell and then anchored to the cell membrane.
  • Any signal peptide/leader sequence of eukaryotic origin can be used, such as a signal peptide/leader sequence of a mammalian or human secretory protein origin.
  • the CAR-T cells used in the present invention can be produced by any CAR-T preparation method known in the art.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the nucleic acid construct encoding the CAR of the present invention can be inserted into a viral vector and packaged in a retroviral particle using techniques known in the art.
  • the recombinant virus can then be separated and delivered in vitro to T cells (e.g., T cells from a subject).
  • T cells e.g., T cells from a subject.
  • retroviral systems are known in the art.
  • Retroviral vectors can be, for example, lentiviral vectors.
  • Vectors derived from lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of transgenes and their proliferation in daughter cells.
  • Lentiviral vectors have additional advantages over vectors derived from onco-retroviruses (such as murine leukemia viruses) because they can transduce non-proliferative cells, such as hepatocytes. They also have the additional advantage of low immunogenicity.
  • Retroviral vectors can also be, for example, gamma-retroviral vectors. Viral vector.
  • a gamma retroviral vector may, for example, comprise a promoter, a packaging signal ( ⁇ ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, e.g., a gene encoding a CAR.
  • a gamma retroviral vector may lack viral structural genes such as gag, pol, and env.
  • the promoter capable of expressing the CAR transgene in mammalian T cells is not particularly limited.
  • a promoter can be an EF1a promoter.
  • the natural EF1a promoter drives the expression of the ⁇ subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl tRNA to the ribosome.
  • the EF1a promoter has been widely used in mammalian expression plasmids and has been shown to effectively drive the expression of CAR from a transgene cloned into a lentiviral vector. See, for example, Milone et al., Mol. Ther. 17 (8): 1453–1464 (2009).
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a constitutive strong promoter sequence that can drive any polynucleotide sequence effectively connected thereto to express at a high level.
  • other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter and human gene promoter, such as but not limited to actin promoter, myosin promoter, elongation factor-1 ⁇ promoter, hemoglobin promoter and creatine kinase promoter.
  • the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention.
  • the cell source for constructing CAR-T cells can be separated or preserved T cells or T cells obtained from a subject.
  • subject is intended to include living organisms (e.g., mammals) that can stimulate an immune response.
  • T cells can be obtained from numerous sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells from blood components collected from a subject can be obtained by apheresis.
  • Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes and platelets.
  • the cells collected by apheresis can be washed to remove the plasma fraction and to place cells in a suitable buffer or culture medium for subsequent processing steps.
  • cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the antibody is expressed by anti-CD3/anti-CD28 conjugated beads (e.g. T cells were isolated from peripheral blood mononuclear cells from the subject by incubation with M-450 CD3/CD28T) for a period of time.
  • anti-CD3/anti-CD28 conjugated beads e.g. T cells were isolated from peripheral blood mononuclear cells from the subject by incubation with M-450 CD3/CD28T
  • the CAR-T cells can be maintained in a space microgravity environment or a simulated microgravity environment for a time sufficient to enhance the ratio of Tscm and Tcm subpopulations according to the method of the present invention as described above. To obtain the CAR-TS cell population according to the present invention.
  • the present invention also provides the use of the CAR-T-S cell population obtained by the method of the present invention in the preparation of a drug for treating tumors.
  • the present invention also provides a method for treating cancer in a subject, comprising administering a therapeutically effective amount of a CAR-T-S cell group of the present invention to an individual in need thereof.
  • the present invention also provides the use of the aforementioned CAR-T-S cell group of the present invention in the preparation of a medicament for treating cancer.
  • the cancer includes hematological cancers (e.g., leukemia) or solid tumors (e.g., gliomas), including primary and metastatic cancers.
  • the CAR-T-S cell population of the present invention is used for adoptive cell therapy (ACT), or for the preparation of a cell therapy product for the ACT treatment.
  • the CAR-T-cell population can be a T cell population from an autologous or allogeneic source.
  • the cell population of the present invention is used to treat cancer in a subject, and can reduce the severity of at least one symptom or indication of cancer or inhibit cancer cell growth.
  • the CAR-T cell population is a CAR-T cell population targeting PSMA, and the tumor is prostate cancer.
  • K562-PSMA tumor cells are K562 cells that stably express human PSMA by retroviral infection. All these cells were maintained in RPMI-1640 (Lonza) or DMEM (Lonza) containing 10% fetal bovine serum (Biosera) and 10,000 IU/ml penicillin/10,00 micrograms/ml streptomycin (EallBio Life Sciences). All cells were cultured in a humidified incubator at 5% CO 2 , 95% air, 37°C.
  • Retroviral vector encoding PSMA-specific CAR basically as described previously (MC Gong et al., Cancer patient T cells genetically targeted to prostate-specific membrane antigen specifically lyse prostate cancer cells and release cytokines in response to prostate-specific membrane antigen, Neoplasia v Vol.1, No.2, June 1999, pp.123–127 123)
  • the retroviral vector was constructed, which contained a CAR polypeptide having a single-chain antibody fragment (scFv) that specifically binds to PSMA, a CD8 hinge region and a transmembrane region, and a CD3 ⁇ cytoplasmic domain.
  • scFv single-chain antibody fragment
  • a polynucleotide containing the CAR coding sequence was synthesized and then subcloned into the SFG retroviral vector (addgene). The cloning of CAR was verified by sequencing. After 48 hours of transient transfection, the retroviral packaging cell line PG13 was used to produce retroviral particles.
  • PBMCs Peripheral blood mononuclear cells
  • T cells in peripheral blood mononuclear cells were stimulated with anti-CD3 and anti-CD28 particles, and then infected with retrovirus.
  • retronectin 15ug/ml was added to a 12-well plate and incubated at room temperature in the dark for 2h. The supernatant was discarded, 0.5% human AB serum (prepared in PBS) was added, cultured for 30min, and the supernatant was discarded.
  • T cells 1.6 ⁇ 10 6 /ml
  • virus liquid 0.5ml T cells (1.6 ⁇ 10 6 /ml) and 0.5ml virus liquid were added, the well plate was sealed with a sealing film, 700g, centrifuged for 1h, and then placed in a 37°C incubator for culture to obtain antigen-specific gene-modified T cells.
  • CAR-T cells were incubated in X-VIVO TM 15 serum-free medium (Lonza) containing 0.5% normal human AB serum for 24 h, and then expanded in X-VIVO TM 15 medium containing 5% GemCell TM human serum AB, and IL-2 (138 U/ml) or IL-15 (10 ng/ml) and target tumor cells were added.
  • X-VIVO TM 15 serum-free medium Licosarcomase
  • IL-2 138 U/ml
  • IL-15 10 ng/ml
  • the space CAR-T cell treatment group and the ground CAR-T cell treatment group performed T cell extraction, activation, and transduction in the same manner and procedure as above.
  • the space CAR-T cell treatment group was cultured in the microgravity environment of orbital flight for another three days (i.e., the fourth, fifth, and sixth days of the experiment), and stayed for one day after returning to the ground (i.e., the seventh day of the experiment).
  • the space CAR-T cell treatment group and the ground CAR-T control treatment group performed the above-mentioned culture, amplification, and flow cytometry in parallel.
  • Flow cytometry was performed on a FACSCanto Plus instrument (BD Biosciences), and data analysis was performed using FlowJo V.10 (FlowJo, LLC).
  • the fluorescent labeled antibodies used included: APC-Cy7 labeled mouse anti-human CD3 antibody (BD Biosciences), FITC labeled mouse anti-human CD8 antibody (BD Biosciences), BV421 labeled mouse anti-human CD4 antibody (BD Biosciences), BV605 labeled mouse anti-human CD45RO (BD Biosciences), PE-Cy7 labeled mouse anti-human CCR7 (BD Biosciences), Alexa Fluo 700 labeled mouse anti-human CD27 (BD Biosciences), and PE-Cy5 labeled mouse anti-human CD95 (BD Biosciences).
  • CAR-T cells (1 ⁇ 10 6 ) were resuspended in 300 ⁇ L PBS and then fixed with 1 ml of 70% ethanol. After 10 minutes, the cells were washed three times with PBS, stained with PI/RNase staining buffer (BD Biosciences) at room temperature for 15 minutes, and analyzed by flow cytometry.
  • PI/RNase staining buffer (BD Biosciences)
  • PSMA-CAR-T cells were generated by transfecting PBMCs with retrovirus.
  • PSMA-CAR-T-S cell populations treated with space flight for 3 days or ground-based PSMA-CAR-T cell populations that were not treated with space flight were co-cultured with K562-PSMA tumor cells for 24 hours, and the supernatant was obtained to detect cytokine concentrations; and after 7 days of co-culture, cells were obtained to detect T subsets and cell cycles.
  • T cell subsets were detected according to the cell sorting and gating steps shown in Figure 1.
  • PSMA-CAR-T-S cells had more Tscm and Tcm ( Figures 2 to 4), including a larger proportion of Tscm and Tcm in the CD3 cell subset and in the CD8 cell subset, indicating that PSMA-CAR-T-S cells have a younger cell phenotype.

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Abstract

La présente invention concerne : le domaine des produits pharmaceutiques, et plus particulièrement un procédé permettant d'améliorer la persistance de la population de lymphocytes CAR-T (en particulier la population de lymphocytes CAR-T ciblant l'antigène membranaire spécifique de la prostate, ou PSMA) au moyen de l'utilisation d'un environnement spatial ou d'un environnement de microgravité simulé; un lymphocyte CAR-T obtenu par le procédé; et son utilisation dans la préparation d'un médicament antitumoral.
PCT/CN2023/138823 2022-12-14 2023-12-14 Procédé pour améliorer la persistance d'une population de lymphocyte t Ceased WO2024125605A1 (fr)

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WANG CHONGZHEN, ZHAO YONG, LUO HAIYING, FENG MEIFU: "Effects of microgravity on immune cells", SCIENCE BULLETIN, KEXUE CHUBANSHE, BEIJING, CN, vol. 58, no. 26, 20 September 2013 (2013-09-20), CN , pages 2679 - 2689, XP093180871, ISSN: 0023-074X, DOI: 10.1360/972013-191 *

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