WO2024060577A1 - UTILISATION D'UNE CELLULE CAR-T RENFORCÉE EXPRIMANT LE RÉCEPTEUR α DE L'INTERLEUKINE 15 DANS LA RÉDUCTION DE LA CYTOTOXICITÉ INDUITE PAR L'INTERLEUKINE 15 - Google Patents

UTILISATION D'UNE CELLULE CAR-T RENFORCÉE EXPRIMANT LE RÉCEPTEUR α DE L'INTERLEUKINE 15 DANS LA RÉDUCTION DE LA CYTOTOXICITÉ INDUITE PAR L'INTERLEUKINE 15 Download PDF

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WO2024060577A1
WO2024060577A1 PCT/CN2023/086217 CN2023086217W WO2024060577A1 WO 2024060577 A1 WO2024060577 A1 WO 2024060577A1 CN 2023086217 W CN2023086217 W CN 2023086217W WO 2024060577 A1 WO2024060577 A1 WO 2024060577A1
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car
nucleic acid
cells
polypeptide
amino acid
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Chinese (zh)
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钟晓松
白玥
张莹
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卡瑞济(北京)生命科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the medical field, and in particular to an armored CAR-T cell expressing interleukin 15 receptor ⁇ and interleukin 15 and its immunotherapy use.
  • Chimeric antigen receptor (CAR)-T cell therapy is a powerful adoptive immunotherapy against hematological cancers, and interleukin (IL)-15 is an important immune stimulator with the ability to induce long-lasting CAR -The ability of T cells (Yang ), https://doi.org/10.1182/blood-2014-01-552174).
  • IL-15 interleukin-15
  • CRS cytokines release syndrome
  • GVHD graft-versus-host disease
  • CAR-IL-15T cells loaded with optimized IL-15 receptor ⁇ (IL-15Ra) and IL-I5 with specific amino acid modifications ( armored CAR-T cells).
  • IL-15Ra IL-15 receptor ⁇
  • IL-I5 IL-I5 with specific amino acid modifications
  • the inventors confirmed in a mouse model that compared with conventional CAR-T cells and CAR-IL-15 T cells, CAR-IL-15 T cells loaded with IL-15Ra prolonged the survival time of mice. , has excellent anti-tumor activity; and at the same time results in reduced serum IL-15 levels and correspondingly lower toxicity after administration compared to CAR-IL-15 T cells.
  • These results indicate that the armored CAR-T cells of the present invention containing optimized IL-15Ra play an important role in reducing adverse events during CAR-T treatment and enhancing anti-tumor ability. Based on these findings, the present inventors thus established the present invention.
  • the invention provides a nucleic acid combination comprising a first nucleic acid molecule, a second nucleic acid molecule and a third nucleic acid molecule, wherein the first nucleic acid molecule comprises a chimeric antigen polypeptide (CAR ), the second nucleic acid molecule comprises a polynucleotide encoding IL-15, and the third nucleic acid molecule comprises a polynucleotide encoding an optimized IL15Ra, wherein the optimized IL15Ra comprises the double mutations S202R and S202R at amino acid positions 202 and 203. D203E, wherein the amino acid position is numbered according to SEQ ID NO:6.
  • CAR chimeric antigen polypeptide
  • the second nucleic acid molecule comprises a polynucleotide encoding IL-15
  • the third nucleic acid molecule comprises a polynucleotide encoding an optimized IL15Ra, wherein the optimized IL15Ra
  • the optimized IL-15Ra comprises the amino acid sequence of SEQ ID NO: 6; or has at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 99.5% identity thereto. Amino acid sequence. More preferably, the polynucleotide encoding optimized IL15Ra comprises the polynucleotide of the sequence described in SEQ ID NO: 3, or has at least 90%, 92%, 95%, 96%, 97%, 98%, A polynucleotide that is 99% or 99.5% identical.
  • first, second and third nucleic acid molecules are present in a functionally linked manner on a single nucleic acid construct, such as a viral vector, such as a lentiviral vector.
  • the first, second and third nucleic acid molecules are each present on a different nucleic acid construct, such as a viral vector, such as a lentiviral vector.
  • the nucleic acid combination according to the present invention is a single nucleic acid construct comprising a first, a second and a third nucleic acid molecule, wherein the nucleic acid construct encodes from the N-terminus to the C-terminus the following formula (I) Fusion protein with the structure shown: CAR-(L1)-E1-(L2)-E2 (I)
  • CAR stands for Chimeric Antigen Receptor Polypeptide
  • L1 and L2 each independently represent a connecting peptide (especially a connecting peptide containing a self-splicing site),
  • E1 and E2 are different from each other and are independently selected from IL-15 and optimized IL-15Ra, and
  • E1 represents IL-15 encoded by the second nucleic acid molecule and E2 represents optimized IL-15Ra encoded by the third nucleic acid molecule.
  • E2 represents IL-15 encoded by the second nucleic acid molecule and E1 represents optimized IL-15Ra encoded by the third nucleic acid molecule.
  • the linking peptides L1 and L2 are the same; in other embodiments, the linking peptides L1 and L2 are different.
  • L1 and L2 comprise a self-splicing site, eg, a self-splicing site selected from: P2A, T2A, E2A, or F2A.
  • L1 comprises a P2A site (preferably, comprising the amino acid sequence of SEQ ID NO: 17);
  • L2 comprises a T2A site (preferably, comprising the amino acid sequence of SEQ ID NO: 18).
  • the first nucleic acid molecule comprises a polynucleotide encoding a CAR polypeptide, wherein said CAR polypeptide comprises from N-terminus to C-terminus: optionally a signal peptide (e.g., GM-CSFRa signal peptide) that specifically binds to a tumor
  • a signal peptide e.g., GM-CSFRa signal peptide
  • the extracellular antigen-binding domain optionally a hinge or spacer region, a transmembrane domain, and a cytoplasmic signaling domain of an antigen, wherein the cytoplasmic signaling domain comprises a costimulatory domain and a primary signaling structure area.
  • the antigen-binding domain that specifically binds a tumor antigen is an antibody or antibody fragment, especially a scFv.
  • the antigen binding domain targets CD19, more preferably comprising LCDR1-3 in the VL amino acid sequence of SEQ ID NO:8 and HCDR1-3 in the VH amino acid sequence of SEQ ID NO:9 ( Especially the CDR sequence defined by Kabat), further preferably, comprising SEQ ID NO: 8 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity therewith
  • the VL and SEQ ID NO: 9 or a VH having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical thereto further preferably, comprise SEQ ID NO :11 or a scFv having an amino acid sequence at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical thereto.
  • the CAR polypeptide comprises a hinge region/spacer region, preferably the hinge region/spacer region is selected from: a hinge region from IgG or a spacer region from CD8 ⁇ or CD28 extracellular region, and preferably Human CD8 ⁇ hinge region or CD28 hinge region, for example, includes the amino acid sequence shown in SEQ ID NO: 12 or has at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity therewith. Amino acid sequence of the CD28 hinge region.
  • the CAR polypeptide comprises a transmembrane domain selected from the group consisting of: transmembrane domains of CD4, CD8 ⁇ , CD28 and CD3 ⁇ , and preferably a human CD8 transmembrane domain or a CD28 transmembrane domain, e.g. , a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity with it.
  • the CAR polypeptide comprises one or more (especially two) costimulatory domains selected from: the costimulatory domains of CD28, CD27, 4-1BB, ICOS and OX40; and preferably A combination comprising a human CD28 costimulatory domain and a 4-1BB costimulatory domain, for example, comprising the amino acid sequence shown in SEQ ID NO: 14 and SEQ ID NO: 15 or having at least 90%, 92%, 95%, 96 %, 97%, 98%, 99% or more identical amino acid sequences.
  • the CAR polypeptide comprises a primary signaling domain, which is a CD3 ⁇ primary signaling domain, for example, comprising or at least 90%, 92%, 95% the amino acid sequence set forth in SEQ ID NO: 16 , 96%, 97%, 98%, 99% or more identical amino acid sequences.
  • the CAR polypeptide comprises a cytoplasmic signaling domain consisting of a costimulatory domain of CD28 and a costimulatory domain of 4-1BB and a CD3 ⁇ primary signaling domain, for example, the cytoplasmic signaling domain
  • the signaling domain includes the amino acid sequence shown in SEQ ID NO: 21 or an amino acid sequence having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity therewith.
  • the CAR polypeptide comprises: from N-terminus to C-terminus, an antibody or antigen-binding fragment, such as scFv, against a tumor antigen (eg, CD19), CD28 hinge region, CD28 transmembrane domain, CD28 costimulatory domain, the 4-1BB costimulatory domain, and the CD3 ⁇ primary signaling domain.
  • a tumor antigen eg, CD19
  • CD28 hinge region CD28 transmembrane domain
  • CD28 costimulatory domain CD28 costimulatory domain
  • 4-1BB costimulatory domain the CD3 ⁇ primary signaling domain.
  • the CAR polypeptide comprises the amino acid sequence of SEQ ID NO: 4, or is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical thereto. sexual amino acid sequence.
  • the second nucleic acid molecule comprises a polynucleotide encoding IL-15. In one embodiment, the second nucleic acid molecule comprises SEQ ID NO: 5 or is at least 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical thereto. Amino acid sequence of a polynucleotide. In one embodiment, the second nucleic acid molecule comprises, or is at least 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% identical to, the nucleotide sequence of SEQ ID NO: 2 or more identical nucleotide sequences.
  • the third nucleic acid molecule comprises a polynucleotide encoding optimized IL-15Ra.
  • the optimized IL-15Ra comprises the amino acid sequence of SEQ ID NO: 6.
  • the nucleic acid combination of the present invention when introduced into immune effector cells such as T cells, is more effective than only introducing the first nucleic acid molecule encoding the CAR and/or the second nucleic acid molecule encoding the IL-15.
  • Control immune effector cells of nucleic acid molecules resulting in reduced release of IL-15 in the extracellular environment, and thereby reduced toxicity induced by IL-15; and preferably, maintaining IL-15 increases CAR-T cells The persistence function.
  • the invention provides a polypeptide encoded by the nucleic acid combination of the invention, comprising: (i) a chimeric antigen receptor (CAR) polypeptide; (ii) an IL-15 polypeptide; and (iii) optimized IL-15Ra Peptides.
  • CAR chimeric antigen receptor
  • IL-15 polypeptide IL-15 polypeptide
  • optimized IL-15Ra Peptides two or all three of (i)-(iii) are functionally linked to each other, especially as a single polypeptide chain via a linking peptide.
  • the polypeptides (i), (ii) and (iii) are encoded by the nucleic acid combination of the invention and are isolated from each other.
  • polypeptides When two or more polypeptides are referred to as being isolated from each other, it is meant that the polypeptides are not covalently linked to each other (either directly or through a linking peptide), but that there may or may be no non-covalent linkage between the isolated polypeptides.
  • isolated IL-15 and optimized IL-15Ra can be non-covalently bound to each other as a complex, but there is no non-covalent binding to the CAR polypeptide.
  • the polypeptide encoded by the nucleic acid combination of the invention is a single fusion polypeptide comprising the following components functionally linked: (i) a chimeric antigen receptor (CAR) polypeptide; (ii) an IL-15 polypeptide; and (iii) Optimize the IL-15Ra polypeptide.
  • the fusion polypeptide has a structure according to formula (I) of the present invention.
  • the fusion polypeptide can be expressed through the self-containing polypeptides located in L1 and L2 after being expressed in cells. The splice site is cleaved to produce three isolated polypeptides, namely, a CAR polypeptide, an IL-15 polypeptide and an optimized IL-15Ra polypeptide.
  • the invention provides nucleic acid constructs, especially vectors, such as viral vectors, such as lentiviral vectors, comprising a combination of nucleic acids according to the invention.
  • vectors such as viral vectors, such as lentiviral vectors
  • the first, second and third nucleic acid molecules comprised in the nucleic acid combination of the invention are present on said vector in polycistronic form.
  • the invention provides a host cell comprising a nucleic acid combination or nucleic acid construct or vector of the invention.
  • the host cells may be immune effector cells, such as T cells or NK cells. Therefore, in one embodiment, the present invention also provides armored CAR-T cells and preparation methods thereof, wherein the armored CAR-T cells comprise the nucleic acid combination according to the present invention, or are introduced with the vector of the present invention.
  • the nucleic acid combination or vector expression of the invention contained in the armored CAR-T cells produces a fusion polypeptide of formula (I) according to the invention, optionally, the fusion polypeptide is expressed in the cell by being contained in The self-splicing site in the linker peptide of formula (I) is cleaved into three separate polypeptides, namely, a CAR polypeptide, an IL-15 polypeptide and an optimized IL-15Ra polypeptide.
  • expression of the nucleic acid combination or vector of the invention contained in said armored CAR-T cells produces three polypeptides of the invention that are separated from each other, namely, a CAR polypeptide, an IL-15 polypeptide and an optimized IL-15Ra polypeptide. .
  • armored CAR-T cells of the invention expressing IL-15 and optimized IL-15Ra exhibit enhanced proliferation compared to CAR-T cells expressing only the CAR molecule, as determined as described in the Examples competence and cell viability, as well as an increased proportion of T cell subsets with Tscm phenotype.
  • the armored CAR-T cells of the invention expressing IL-15 and optimized IL-15Ra reduce the amount of IL-15 released in the extracellular environment compared to CAR-T cells expressing only IL-15, and thereby have reduced toxicity induced by IL-15.
  • the invention provides a pharmaceutical composition comprising the armored CAR-T cells of the invention.
  • the present invention provides the use of the armored CAR-T cells of the present invention in the preparation of drugs for preventing or treating cancer or providing anti-tumor immunity, and the use of the armored CAR-T cells of the present invention in subjects.
  • Methods for preventing or treating cancer or providing anti-tumor immunity are administered systemically (eg, intravenously) or used locally (eg, intratumorally).
  • the tumor is a hematological tumor or a solid tumor.
  • the present invention provides optimization of the IL15Ra polypeptide and its encoding nucleic acid molecule, and its application in reducing the toxicity of CAR-T cells recombinantly expressing IL15.
  • the application includes in the CAR-T cells
  • the nucleic acid molecule encoding the optimized IL-15Ra polypeptide is introduced and expressed in the cell.
  • the CAR-T cell contains a nucleic acid molecule encoding the fusion protein according to the structure shown in Formula (I) of the present invention.
  • the present invention provides a method for increasing the persistence of CAR-T cells and reducing their toxicity, which includes introducing and expressing the nucleic acid combination or vector according to the present invention in the CAR-T cells.
  • FIG. 1 schematically shows the structure of armored CAR-T cells according to the present invention.
  • CD19-CAR represents a chimeric antigen receptor polypeptide targeting CD19, which contains anti-CD19scFv, CD28 spacer/transmembrane region, CD28 costimulatory domain, 4-1BB costimulatory domain and CD3 ⁇ from N-terminus to C-terminus.
  • Signaling domain SD represents splice donor site
  • SA represents splice acceptor site
  • LTR represents long terminal repeat sequence
  • P2A and T2A represent self-splicing peptides P2A and T2A respectively
  • IL-15 represents IL-15 protein
  • Figure 2 shows the characterization of CAR, CAR-IL15, and CAR-IL15-IL15Ra T cells produced after T cells were transduced with retroviral vectors encoding CAR.
  • a and B Transduction efficiency was determined by flow cytometry to quantify CD4+ and CD8+ T cell numbers and CD19+CD8+CAR T cell numbers.
  • C Confirmation of IL-15 and IL-15Ra expression levels in three CAR-T cells by PCR. As a control, the housekeeping gene GAPDH in the cells was also amplified.
  • Figure 3 shows the detection of in vitro proliferation (A) and cytokine IL-2 production (B) of armored CAR-T cells overexpressing IL-15 and/or IL-15Ra when stimulated by cytokines or target tumor cells.
  • FIG. 4 shows that after 7 days of stimulation of target tumor cells with NAML-6-eGFP, the differentiation phenotype of armored CAR-T cells overexpressing IL-15 and/or IL-15Ra was detected, where flow cytometry was used to detect the proportion of Tscm (CD8 + CD45RO - CCR7 + CD27 + CD95 + ) in the cell population.
  • Tscm CD8 + CD45RO - CCR7 + CD27 + CD95 +
  • Figure 5 shows that after incubation with target tumor cells NAML-6-eGFP for a period of time, cytokine IFN ⁇ secretion (A), as well as cell apoptosis percentage (B) and Cell viability (C).
  • Figure 6 shows IL-15 secretion (A) and CD132 cell surface expression (B) of CAR, CAR-IL15 and CAR-IL15-IL15Ra T cells under in vitro culture conditions.
  • Figure 7 shows that after CAR-T cells were co-cultured with target cells NAML-6-eGFP cells (2:1) for 24 hours, the cells were collected.
  • A GFP signal was detected by flow cytometry, indicating surviving NAML-6-eGFP cells. number; and
  • B the corresponding histogram of statistical results.
  • Figure 8 shows the xenograft mouse tumor model experiment.
  • A Animal experiment flow chart;
  • B Fluorescence chart of mouse tumor burden.
  • Figure 9 shows the changes in tumor burden of individual mice in each group over time in the xenograft mouse tumor model experiment.
  • the IVIS imaging system was used to obtain quantitative bioluminescence imaging data of all mice (i.e., the absolute number of photons emitted from the animal's body surface per unit time, unit area, and unit arc (photons/sec/cm 2 /sr)). Higher values indicate greater tumor burden.
  • Figure 10 shows the overall survival and serum IL-15 concentration of xenograft tumor-bearing mice.
  • A Overall survival of xenograft tumor-bearing mice was measured using the Kaplan-Meier method.
  • B On the 50th day, the blood of mice in each group was collected, and the concentration of human IL-15 was detected in the serum.
  • IL-15 refers to the interleukin 15 cytokine.
  • An example of IL-15 is an interleukin IL-15 of human origin, such as the protein under UniProtKB-accession number P40933, or a homologue thereof, such as an interleukin IL-15 of non-human mammalian origin, such as a non-human mammal. Human primates, rodents, domestic animals, sporting animals, etc.
  • IL-15Ra or "IL-15R ⁇ ” refers to the interleukin 15 receptor alpha protein.
  • An example of IL-15Ra is the interleukin IL-15 receptor alpha of human origin, such as the protein under UniProtKB-accession number Q13261, or a homologue, or variant thereof.
  • the IL-15Ra of the invention is an interleukin 15 receptor alpha protein in which modifications (eg, double mutations S202R and D203E) were introduced into the parent IL-15Ra receptor protein, also referred to as "optimized IL-15Ra.”
  • the parent IL-15Ra receptor protein may be of mammalian origin, such as human origin, or native or wild-type IL-15Ra of a non-human mammalian animal.
  • the parent IL-15Ra receptor protein comprises the motif YPQGHRET at positions 197-204.
  • chimeric receptor chimeric antigen receptor
  • CAR chimeric antigen receptor
  • the cytoplasmic signaling domain comprises a primary signaling domain from a stimulatory molecule as described below, such as that of CD3- ⁇ .
  • the intracellular signaling domain further comprises one or more functional signaling domains from at least one, preferably two costimulatory molecules, such as CD28 and 4-1BB.
  • CAR polypeptides can be expressed on any cells, such as immune effector cells such as T cells or NK cells.
  • a primary cytoplasmic signaling sequence that modulates the TCR in a stimulatory manner in at least some aspect of the T cell signaling pathway Primary activation of the complex.
  • a primary signal can be initiated (e.g., via binding of a TCR/CD3 complex to a peptide-loaded MHC molecule) and subsequently mediate a T cell response, including but not limited to proliferation, activation, differentiation, and the like.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may include immunoreceptor tyrosine activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine activation motifs
  • ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those from TCR zeta and CD3 zeta intracellular signaling domain.
  • the cytoplasmic domain of the CAR polypeptide of the invention comprises at least one functional cytoplasmic signaling sequence from a stimulatory molecule, for example, the cytoplasmic signaling sequence of CD3 ⁇ .
  • CD3 ⁇ is defined as the protein provided under UniProtKB-P20963 accession number or its equivalent.
  • a “CD3 ⁇ signaling domain” is defined as a segment of amino acid residues from the cytoplasmic domain of the CD3 ⁇ chain that 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 as a functional ortholog thereof from a non-human species (e.g., mouse, rodent species, monkeys, apes, etc.).
  • the "CD3 ⁇ signaling domain” is the sequence provided in SEQ ID NO: 16, or a variant thereof.
  • costimulatory molecule refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand thereby mediating a costimulatory response (such as, but not limited to, proliferation) of the cell.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an effective immune response.
  • 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), activated NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28.
  • the "costimulatory molecule” is CD28, 4-1BB (ie, CD137).
  • costimulatory domain refers to the intracellular portion of the costimulatory molecule.
  • 4-1BB refers to a TNFR superfamily member, also known as CD137, having the amino acid sequence provided under UniProtKB-Q07011 accession number or from a non-human species (e.g., mouse, rodent, monkey, ape, etc. ) equivalent residues.
  • 4-1BB costimulatory domain is defined as derived from the cytoplasmic region of 4-1BB, e.g., amino acid residues 214-255 of UniProtKB-Q07011 or from a non-human species (e.g., mouse, rodent , monkey, ape, etc.) equivalent residues.
  • CD28 refers to the amino acid sequence provided under UniProtKB-P10747 accession number or equivalent residues from a non-human species (eg, mouse, rodent, monkey, ape, etc.).
  • CD28 costimulatory domain is defined as derived from the cytoplasmic region of CD28, e.g., amino acid residues 180-220 of UniProtKB-P10747 or from a non-human species (e.g., mouse, rodent, monkey, ape etc.) equivalent residues.
  • CD28 transmembrane domain is defined as the transmembrane region from CD28, e.g., amino acid residues 153-179 of UniProtKB-P10747 or from a non-human species (e.g., mouse, rodent, monkey, ape etc.) equivalent residues.
  • the term "recombinant" when referring to, for example, a virus or a cell or a nucleic acid or a protein or a vector, means that the virus, cell, nucleic acid, protein or vector has been modified by introducing a heterologous nucleic acid or protein, or by altering its own existing A natural nucleic acid or protein that has been modified, or a substance derived from a virus or cell that has been modified thereby.
  • 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, for example, the sequence is located in 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 encoding and capable of expressing one or more genes of interest (such as the CAR polypeptide of the present invention, or IL-15 protein, or optimized IL-15Ra, or two or three thereof).
  • a heterologous polynucleotide sequence encoding a gene of interest is functionally linked to expression control sequences.
  • the expression cassette may contain two or more genes of interest in a polycistronic form under the control of the same promoter, thereby encoding and expressing a single polypeptide chain in which the two Two or more target proteins encoded by one or more target genes are functionally connected to each other.
  • linker or “linker peptide” or “linker” are used interchangeably and refer to a short amino acid sequence consisting of amino acids, such as alanine (A), glycine (G) alone or in combination and/or serine (S) and/or threonine residues (T), or a self-splicing peptide comprising a self-splicing site.
  • the linking peptide is 1-50 amino acids in length, 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 components of the CAR fusion polypeptide of the present invention are not particularly limited.
  • Computer programs can be used to model the three-dimensional structures of proteins and peptides to rationally design suitable linker peptides.
  • short oligopeptide linkers or polypeptide linkers can be used to form linkages between component sequences as desired, e.g., glycine-serine doublets, or single amino acids, e.g., alanine, glycine, can be used as linkers.
  • 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 additions, deletions, substitutions, and other modifications can be made, provided that the final polypeptide sequence has the desired properties.
  • the substitution of amino acids is a non-conservative amino acid substitution, ie, one amino acid is replaced by another amino acid with different structural and/or chemical properties.
  • Amino acid substitutions include substitutions with non-naturally occurring amino acids or naturally occurring amino acid derivatives of the twenty standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxy Lysine) substitution.
  • conservative sequence modification and “conservative sequence change” refer to amino acid modifications or changes that do not significantly affect or change the characteristics of the parent polypeptide containing the amino acid sequence or its constituent elements. Such conservative modifications include amino acid substitutions, additions and deletions. Conservative modifications can be introduced into the CAR fusion polypeptides of the invention or component elements thereof (e.g., CAR or IL-15 or IL-15Ra) by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. , especially conservative substitutions. A conservative substitution is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those 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), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), ⁇ -side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenyl Alanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • Percent identity (%) of an amino acid sequence/nucleotide sequence means that the candidate sequence is compared to the specific amino acid/nucleotide sequence shown in this specification and, if necessary, to achieve maximum sequence identity. After introducing gaps, and in the case of amino acid sequences, without considering any conservative substitutions as part of the sequence identity, the number of amino acid residues/core in the candidate sequence is identical to the specific amino acid/nucleotide sequence shown in this specification. Percentage of amino acid/nucleotide residues whose nucleotide residues are identical.
  • the present invention contemplates variants of the fusion polypeptides or nucleic acid molecules of the invention, or constituent elements thereof, that are relative to the fusion polypeptides or nucleic acid molecules, or constituent elements thereof, specifically disclosed herein (e.g., CAR polypeptides/ coding nucleic acid, or IL-15 protein/encoding nucleic acid, or optimized IL-15Ra protein/nucleic acid) has a substantial degree of identity, for example, the identity is at least 80%, 85%, 90%, 95%, 97 %, 98% or 99% or higher.
  • the variants may contain conservative modifications. For the purposes of this invention, percent identity is determined using the publicly available BLAST tool at https://blast.ncbi.nlm.nih.gov, using default parameters.
  • variants or functional variants means that the polypeptide or protein has substantially the same sequence or significant sequence identity as compared with the reference polypeptide or protein. and maintain 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 linked.
  • 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 they have been introduced. Some vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • lentivirus refers to a genus of the family Retroviridae. 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 examples of lentiviruses.
  • lentiviral vector refers to a vector derived from at least a portion of a lentiviral genome, including in particular 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 e.g. Including, but not limited to, 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.
  • immune effector cells refers to cells involved in an immune response, such as in promoting an immune effector response.
  • immune effector cells include T cells, eg, alpha/beta T cells and gamma/delta 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). mouse).
  • 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 being.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • anti-tumor immunity refers to immunological effects that can be demonstrated through various means, including but not limited to, for example, causing a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation or a reduction in tumor cell survival.
  • the present invention found in in-depth research that in CAR-based immune cells (for example, CAR-T cells and CAR-NK cells), the immunity can be promoted by increasing IL-15 and optimizing the expression of IL-15Ra gene. Cell persistence and/or anti-tumor immunity while limiting toxicity induced by IL-15 released from the environment (e.g., serum).
  • CAR-based immune cells for example, CAR-T cells and CAR-NK cells
  • the invention provides CAR-based immune cells, wherein the immune cells comprise not only heterologous polynucleotides encoding CAR polypeptides, but also heterologous polynucleotides encoding IL-15 and optimized IL-15Ra .
  • the immune cells are T cells, in this article, such CAR-based immune cells with recombinantly expressed IL15 and optimized IL-15Ra are also called “armored CAR-T cells" or “Armored CAR-T cells. ".
  • the heterologous polynucleotide encoding the CAR polypeptide and the heterologous polynucleotide encoding IL-15 and optimized IL-15Ra may be located on a single nucleic acid molecule, or on separate different nucleic acid molecules.
  • the invention provides combinations of nucleic acids useful in forming CAR immune cells according to the invention.
  • the nucleic acid combination of the invention comprises a first nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, a second nucleic acid molecule encoding an IL-15 protein, and a third nucleic acid molecule encoding an optimized IL-15Ra .
  • CAR chimeric antigen receptor
  • the armored CAR-T cells of the present invention containing optimized IL-15Ra exhibit excellent performance in reducing adverse events during CAR-T treatment and enhancing anti-tumor effects.
  • the CAR polypeptide encoding polynucleotide, the IL-15 protein encoding polynucleotide, and the optimized IL-15Ra encoding nucleic acid can be used as all three or any two. Located in the same expression cassette or in different expression cassettes, and can be expressed as separate polypeptides, or any two or all three of them can be expressed as fusion polypeptides.
  • the nucleic acid combination is in the form of a single nucleic acid molecule encoding and expressing a single fusion polypeptide comprising the CAR polypeptide, IL-15 and optimized IL15Ra, preferably in the In the fusion polypeptide, IL-15 and IL15Ra proteins are functionally linked together, and the CAR polypeptide is functionally linked to one of IL-15 and IL15Ra proteins through a linker peptide containing a cleavable site.
  • the fusion polypeptide has the structure of formula (I): CAR-(L1)-E1-(L2)-E2 (I)
  • CAR means a chimeric antigen receptor polypeptide encoded by the first nucleic acid molecule
  • L1 and L2 independently represent connecting peptides (especially self-splicing peptides),
  • E1 and E2 are different from each other and independently represent IL-15 or optimized IL-15Ra encoded by the second or third nucleic acid molecule, respectively, and
  • the present invention also provides a fusion polypeptide having the structure of the above formula (I).
  • CAR-based immune cells nucleic acid combinations, polypeptides and components thereof of the present invention will be described in detail below.
  • any technical features mentioned in describing components and any combination thereof are within the scope of consideration of the present invention; and, a person skilled in the art can understand , unless the context clearly indicates otherwise, the CAR-based immune cells of the invention may comprise any such combination of features, and similarly the nucleic acid constructs and CAR fusion polypeptides of the invention may also comprise any such combination of features.
  • IL-15Ra of the present invention is a membrane-bound protein with characteristic double mutations S202R and D203E.
  • the IL-15Ra of the invention can be derived from any functional single-spanning native full-length IL-15Ra protein or a variant thereof (including natural allelic variants or species homologues), wherein, in Double mutations S202R and D203E were introduced at amino acid positions 202 and 203, which are numbered according to SEQ ID NO:6.
  • Double mutations S202R and D203E were introduced at amino acid positions 202 and 203, which are numbered according to SEQ ID NO:6.
  • "numbered according to SEQ ID NO:6" means that it is determined by reference to the amino acid sequence of SEQ ID NO:6.
  • amino acid sequence alignment can be performed with SEQ ID NO:6 (e.g., using BLAST; Basic Local Alignment Search available at http://blast.ncbi.nlm.nih.gov Tool, using default parameters, performing the alignment), identifies the corresponding amino acid position on the given IL-15Ra polypeptide.
  • mutation S202R refers to the mutation from serine (S) to arginine (R) at amino acid position 202
  • mutation D203E refers to the mutation from aspartic acid (D) to glutamic acid (E) at amino acid position 203.
  • the IL-15Ra polypeptide comprises: i) the amino acid sequence of SEQ ID NO: 6; ii) having at least An amino acid sequence with one, two or three modifications but no more than 30, 20 or 10 modifications; or iii) an amino acid sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 6.
  • IL-15Ra of the invention comprises the motif YPQGHSDT at positions 197-204.
  • the IL-15Ra polypeptides of the invention retain the signal peptide of the native IL-15 parent polypeptide from which they are derived.
  • the IL-15Ra polypeptide of the invention has a heterologous signal peptide from another transmembrane eukaryotic protein, such as a mammalian protein, to direct its expression in the cell and subsequent integration into the cell membrane after processing.
  • the IL-15Ra polypeptide of the invention forms a non-covalent complex with an IL-15 polypeptide expressed in the same cell and is transported to the cell membrane surface.
  • a polynucleotide encoding an optimized IL-15Ra polypeptide useful in the present invention may be any polynucleotide comprising a nucleotide sequence encoding an optimized IL-15Ra protein according to any of the above embodiments of the present invention.
  • the optimized IL-15Ra encoding polynucleotide comprises encoding SEQ ID NO: 6 or a variant thereof, e.g., is at least 95%, 96%, 97%, 98%, or 99% identical thereto. sexual amino acid sequence.
  • the IL-15Ra encoding polynucleotide comprises the nucleotide sequence of SEQ ID NO: 3 or a variant thereof, e.g., is at least 95%, 96%, 97%, 98%, or An amino acid sequence that is 99% identical; or a nucleotide sequence that hybridizes to it under stringent hybridization conditions.
  • IL-15 polypeptides useful in the present invention include, but are not limited to, full-length native IL-15 protein or functional fragments thereof, or variants thereof (including native allelic variants or species homologs).
  • the amino acid sequence of IL-15 from human is given under UniProtKB-P40933 accession number.
  • the IL-15 polypeptide comprises: i) the amino acid sequence of SEQ ID NO: 5; ii) at least one, two or three of the amino acid sequence of SEQ ID NO: 5 An amino acid sequence that is modified but not more than 30, 20 or 10 modified; or iii) an amino acid sequence that is at least 95-99% identical to the amino acid sequence of SEQ ID NO: 5.
  • the IL-15 encoding polynucleotide useful in the present invention may be any polynucleotide comprising a nucleotide sequence encoding an IL-15 polypeptide according to any of the above embodiments of the invention.
  • the IL-15 encoding polynucleotide comprises encoding SEQ ID NO:5 or a variant thereof, for example, a nucleotide sequence having at least 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence thereof.
  • the IL-15 encoding polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2 or a variant thereof, e.g., is at least 95%, 96%, 97%, 98%, or An amino acid sequence that is 99% identical; or a nucleotide sequence that hybridizes to it under stringent hybridization conditions.
  • the amount of IL-15 released from the cell is reduced.
  • the released IL-15 may include the IL-15 polypeptide itself, or a heterodimer formed with soluble IL-15Ra (eg, soluble IL-15Ra shed from the cell membrane).
  • the reduction is relative to control cells expressing only IL-15 and not the optimized IL-15Ra polypeptide of the invention.
  • the control cells express wild-type full-length IL-15Ra polypeptide without mutations S202R and D203E.
  • CAR Chimeric antigen receptor
  • a CAR polypeptide of the invention includes an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain.
  • the cytoplasmic signaling domain of a CAR polypeptide of the invention comprises a primary signaling domain.
  • the cytoplasmic signaling domain of a CAR polypeptide of the invention comprises a costimulatory domain and a primary signaling domain.
  • a chimeric antigen receptor (CAR) molecule comprises from N-terminus to C-terminus: (a) an antigen-binding domain that specifically binds a tumor antigen and (b) a hinge region or spacer region ; (c) transmembrane domain; and (d) cytoplasmic signaling domain.
  • CAR chimeric antigen receptor
  • the CAR molecule according to the present invention includes from N-terminus to C-terminus: (a) an antigen-binding domain that specifically binds a tumor antigen, (b) a hinge region or a spacer region; (c) a transmembrane domain ; (d) two costimulatory domains from CD28 and 4-1BB; and (e) primary signaling domain from CD3.
  • the target antigen for the CAR polypeptides of the invention is a membrane antigen expressed on the surface of target cells, especially tumor cells, such as a tumor-specific antigen or a tumor-associated antigen.
  • Tumors that may be mentioned include hematological tumors and solid tumors, both primary and metastatic.
  • the target antigen is a tumor cell surface antigen comprising an antigenic cancer epitope that is immunologically recognized by tumor-infiltrating lymphocytes (TIL) derived from mammals.
  • TIL tumor-infiltrating lymphocytes
  • the target antigen is a tumor cell surface antigen comprising one or more antigenic cancer epitopes associated with malignancy.
  • the extracellular antigen-binding domain of the CAR molecule of the present invention targets a tumor antigen.
  • the tumor antigen is selected from: CD19, epinephrine 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 the membrane antigen CD19.
  • 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.
  • scFv can be generated by linking the VH and VL regions together using flexible polypeptide linkers according to methods known in the art.
  • scFv molecules comprise flexible polypeptide linkers of 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 contain any naturally occurring amino acid.
  • the peptide linker of the scFv consists of amino acids such as glycine and/or serine residues used alone or in combination to link the variable heavy chain and variable light chain regions together.
  • flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 (SEQ ID NO:27) or (Gly4Ser)3 (SEQ ID NO:28).
  • the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO:29).
  • the linker comprises the GSTGSSGKPGSGEGSTKG amino acid sequence.
  • the scFv used in the present invention contains from N-terminus to C-terminus: VL-linker-VH; or VH-linker-VL.
  • the CAR polypeptides of the invention comprise at least one transmembrane domain, which can be derived from natural or synthetic sources.
  • the transmembrane domain may be derived from a membrane-binding or transmembrane protein, such as that from CD3 ⁇ , CD4, CD28, CD8 (eg, CD8 ⁇ , CD8 ⁇ ).
  • the transmembrane domain confers membrane attachment to the CAR polypeptide of the invention.
  • the transmembrane domain in the CAR of the invention can be connected to the extracellular region of the CAR via a hinge region/spacer region.
  • transmembrane region and hinge region/spacer region that can be used in CAR polypeptides, see, for example, Kento Fujiwara et al., Cells 2020, 9, 1182; doi:10.3390/cells9051182.
  • the cytoplasmic signaling domain included in the CAR polypeptide of the present invention at least includes a primary signaling domain.
  • the primary signaling domain is capable of activating at least one immune effector function of the immune cell into which the CAR of the invention has been introduced.
  • the immune effector function includes, but is not limited to, for example, enhancing or promoting the function or response of immune attack target cells.
  • the effector function of T cells may be, for example, cytolytic activity or auxiliary activity, including secretion of cytokines.
  • cytoplasmic signaling domains for use in CAR polypeptides of the invention include T cell receptors (TCRs) and/or coreceptors that function to initiate signal transduction upon binding of the extracellular domain to a target antigen.
  • TCRs T cell receptors
  • coreceptors that function to initiate signal transduction upon binding of the extracellular domain to a target antigen.
  • cytoplasmic signaling sequences those that initiate antigen-dependent primary activation through the TCR (i.e., primary signaling domains) and those that act in an antigen-independent manner to provide costimulatory signals those sequences (i.e., secondary cytoplasmic domains, e.g., costimulatory domains).
  • a CAR polypeptide of the invention comprises a cytoplasmic domain that provides a primary signaling domain, e.g., the intracellular primary signaling domain of CD3 ⁇ .
  • the cytoplasmic domain of a CAR polypeptide of the invention further comprises a secondary signaling domain, e.g., a costimulatory domain from a costimulatory molecule.
  • the cytoplasmic region of the CAR polypeptide of the invention comprises one or more (especially two) costimulatory domains in tandem with the CD3 ⁇ intracellular signaling domain, such as 4-1BB (also known as CD137) and the costimulatory domain of CD28.
  • the CAR polypeptide of the invention may comprise a signal peptide or leader sequence located at the N-terminus of the extracellular antigen-binding domain. Through the signal peptide/leader sequence, the nascent CAR polypeptide can be guided to the endoplasmic reticulum of the cell and then anchored on the cell membrane.
  • a signal peptide/leader sequence of any eukaryotic origin may be used, such as a signal peptide/leader sequence of mammalian or human secretory protein origin.
  • chimeric antigen receptor (CAR) polypeptides according to the invention comprise an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain.
  • the antigen binding domain is one that targets a tumor antigen.
  • the tumor antigen is a membrane antigen, such as CD19 or EphA2, and preferably CD19.
  • the extracellular antigen binding domain is an antigen binding domain that binds CD19.
  • the extracellular antigen binding domain comprises a murine, human or humanized antigen binding domain that binds CD19.
  • the antigen-binding domain that binds CD19 comprises: the heavy chain complementarity determining region 1 (HC CDR1) of the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 9, the heavy chain complementarity determining region 2 ( HC CDR2) and heavy chain complementarity determining region 3 (HC CDR3); and/or light chain complementarity determining region 1 (LC CDR1) of the light chain variable region (VL) amino acid sequence of SEQ ID NO:8, light chain complementarity determining region 1 Region 2 (LC CDR2) and light chain complementarity determining region 3 (LC CDR3).
  • the antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region includes: i) the amino acid sequence of SEQ ID NO: 9; ii) having at least one, two or three modifications but no more than 30, 20 or 10 modifications to the amino acid sequence of SEQ ID NO: 9 Modified amino acid sequence; or iii) an amino acid sequence having 95-99% identity with the heavy chain variable region amino acid sequence of SEQ ID NO: 9; and/or
  • the light chain variable region comprises: i) the amino acid sequence of SEQ ID NO:8; ii) having at least one, two or three modifications but no more than 30, 20 or 10 modifications to the amino acid sequence of SEQ ID NO:8 A modified amino acid sequence; or iii) an amino acid sequence having 95-99% identity with the heavy chain variable region amino acid sequence of SEQ ID NO:8.
  • the antigen binding domain comprises: i) the amino acid sequence of SEQ ID NO: 11; ii) at least one, two or three modifications to SEQ ID NO: 11 but no more than 30, 20 or 10 modified amino acid sequences; or iii) an amino acid sequence that is 95-99% identical to SEQ ID NO: 11.
  • the transmembrane domain comprises a transmembrane domain of a protein selected from: CD4, CD8 ⁇ , CD28, CD3 ⁇ , TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD9, CD16, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), Fc ⁇ RI, CD66d, alpha, beta or zeta chain of T cell receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor , integrins, and activating NK cell receptors.
  • a protein selected from: CD4, CD8 ⁇ , CD28, CD3 ⁇ , TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD9, CD16, CD22, CD79a, CD79b, CD278 (also known as "ICOS”), Fc ⁇ RI, CD66d, alpha, beta or zeta chain of T cell
  • the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD4, CD8 ⁇ , CD28 and CD3 ⁇ .
  • the transmembrane domain comprises: i) the amino acid sequence of SEQ ID NO: 13; ii) comprising at least one, two or three modifications but no more than 5 modifications of the amino acid sequence of SEQ ID NO: 13 an amino acid sequence; or iii) an amino acid sequence having 95-99% sequence identity with SEQ ID NO: 13.
  • the cytoplasmic signaling domain comprises a functional signaling domain of a protein selected from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b, or CD66d.
  • the cytoplasmic signaling domain comprises the functional signaling domain of the CD3 ⁇ protein (also referred to herein as the CD3 ⁇ primary signaling domain).
  • the cytoplasmic signaling domain comprises: i) the amino acid sequence of SEQ ID NO: 16; ii) comprising at least one, two or three modifications but no more than 20 of the amino acid sequence of SEQ ID NO: 16 , 10 or 5 modified amino acid sequences; or iii) an amino acid sequence having 95-99% sequence identity with SEQ ID NO: 15.
  • the cytoplasmic signaling domain further comprises a co-stimulatory domain of one or more proteins selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, CD8, ICOS, DAP10, DAP12, OX40, CD40, GITR, 4-1BB (i.e., CD137), CD27, and CD28.
  • the cytoplasmic signaling domain comprises a co-stimulatory domain of one or two proteins selected from the group consisting of: CD28, CD27, 4-1BB, ICOS, and OX40.
  • the cytoplasmic signaling domain comprises a co-stimulatory domain of a protein selected from the group consisting of: CD28 and 4-1BB (i.e., CD137), or a combination thereof.
  • the cytoplasmic signaling domain comprises a CD28 co-stimulatory domain and a 4-1BB co-stimulatory domain, wherein preferably, the CD28 co-stimulatory domain comprises: i) an amino acid sequence of SEQ ID NO: 14; ii) an amino acid sequence comprising at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ ID NO: 14; or iii) an amino acid sequence with 95-99% identity with the amino acid sequence of SEQ ID NO: 14; and preferably, the 4-1BB co-stimulatory domain comprises: i) an amino acid sequence of SEQ ID NO: 15; ii) an amino acid sequence comprising at least one, two or three modifications but not more than 20, 10 or 5 modifications of the amino acid sequence of SEQ
  • the cytoplasmic signaling domain of the CAR polypeptide comprises costimulatory signals from CD28 and 4-1BB and a primary signaling domain from CD3 ⁇ .
  • the cytoplasmic signaling domain comprises: i) the amino acid sequence of SEQ ID NO: 1; ii) comprising at least one, two or three modifications of the amino acid sequence of SEQ ID NO: 1 but not More than 20, 10 or 5 modified amino acid sequences; or iii) an amino acid sequence having 95-99% identity with the amino acid sequence of SEQ ID NO:1.
  • a CAR polypeptide comprises a transmembrane domain and an extracellular antigen-binding domain, and further comprises a hinge or spacer region disposed between said transmembrane domain and said extracellular antigen-binding domain.
  • the hinge/spacer region is selected from the group consisting of a GS hinge, a CD8 hinge, an IgG4 hinge, an IgD hinge, a CD16 hinge, and a CD64 hinge.
  • the CAR polypeptide comprises a hinge region from the extracellular region of CD28.
  • the hinge region/spacer region comprises: i) the amino acid sequence of SEQ ID NO: 12; ii) comprising at least one, two or three modifications but no more than 5 of the amino acid sequence of SEQ ID NO: 12 A modified amino acid sequence; or iii) an amino acid sequence having 95-99% identity with the amino acid sequence of SEQ ID NO: 12.
  • the expressions "hinge”, “hinge region” and “hinge domain” are used interchangeably.
  • the CAR polypeptide of the invention comprises: (a) antigen-binding domain; (b) hinge region/spacer region; (c) transmembrane domain; (d) from CD28 and 4-1BB A costimulatory domain; and (e) a primary signaling domain from CD3 ⁇ .
  • the CAR polypeptide further comprises a leader peptide or a signal peptide, such as a signal peptide from human granulocyte-macrophage colony stimulating factor receptor alpha chain (GM-CSFR ⁇ ).
  • the CAR polypeptide comprises a signal peptide having an amino acid sequence of SEQ ID NO:7.
  • a CAR polypeptide according to the present invention comprises: i) the amino acid sequence of SEQ ID NO: 4; ii) having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 4 but no more than 30 , 20 or 10 modified amino acid sequences; or iii) an amino acid sequence having at least 95-99% identity with the amino acid sequence of SEQ ID NO: 4.
  • the CAR-encoding nucleic acid useful in the present invention can be any polynucleotide comprising a nucleotide sequence encoding a CAR polypeptide according to any of the above embodiments of the present invention.
  • the CAR-encoding nucleic acid comprises an amino acid sequence encoding SEQ ID NO: 4 or a variant thereof, e.g., an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99% identical thereto. Nucleotide sequence.
  • the invention provides a polynucleotide comprising a polynucleotide encoding a CAR polypeptide according to the invention, a polynucleotide encoding an IL-15 polypeptide according to the invention, and a polynucleotide encoding an IL-15Ra polypeptide according to the invention.
  • a polynucleotide comprising a polynucleotide encoding a CAR polypeptide according to the invention, a polynucleotide encoding an IL-15 polypeptide according to the invention, and a polynucleotide encoding an IL-15Ra polypeptide according to the invention.
  • the polynucleotide encoding the CAR polypeptide according to the invention, the polynucleotide encoding the IL-15 polypeptide according to the invention, and the polynucleotide encoding the optimized IL-15Ra polypeptide according to the invention are located respectively in on three different nucleic acid constructs.
  • a polynucleotide encoding a CAR polypeptide according to the invention, a polynucleotide encoding an IL-15 polypeptide according to the invention, and a polynucleotide encoding an optimized IL-15Ra polypeptide according to the invention are present in a single Provided in a nucleic acid construct.
  • the nucleic acid construct according to the invention is a plasmid or viral vector comprising an expression cassette.
  • a polynucleotide encoding a CAR polypeptide according to the invention, a polynucleotide encoding an IL-15 polypeptide according to the invention, and a polynucleotide encoding an IL-15Ra polypeptide according to the invention are present in a single on a nucleic acid construct and located in different expression cassettes under the control of the same or different promoters.
  • a polynucleotide encoding a CAR polypeptide according to the invention, a polynucleotide encoding an IL-15 polypeptide according to the invention, and a polynucleotide encoding an IL-15Ra polypeptide according to the invention are expressed as polypeptides.
  • the polynucleotide encoding a CAR polypeptide according to the invention comprises a polynucleotide encoding a CAR polypeptide according to any of the preceding embodiments of the invention, in particular a polynucleotide encoding a CAR polypeptide targeting CD19.
  • a polynucleotide encoding an IL-15 polypeptide according to the invention comprises a polynucleotide encoding IL-15 according to any of the preceding embodiments of the invention.
  • the IL-15 encoding polynucleotide encodes the amino acid sequence of SEQ ID NO:5.
  • the IL-15 encoding polynucleotide comprises: i) the nucleotide sequence of SEQ ID NO:2; ii) hybridizes to the nucleotide sequence of SEQ ID NO:2 under stringent hybridization conditions. A nucleotide sequence; or iii) a nucleotide sequence that is at least 90-99% identical to the nucleotide sequence of SEQ ID NO:2.
  • a polynucleotide encoding an IL-15Ra polypeptide according to the invention comprises a polynucleotide encoding an IL-15Ra according to any of the preceding embodiments of the invention.
  • the IL-15Ra encoding polynucleotide encodes the amino acid sequence of SEQ ID NO: 6.
  • the IL-15Ra encoding polynucleotide comprises: i) the nucleotide sequence of SEQ ID NO:3; ii) hybridizes to the nucleotide sequence of SEQ ID NO:3 under stringent hybridization conditions. A nucleotide sequence; or iii) a nucleotide sequence that is at least 90-99% identical to the nucleotide sequence of SEQ ID NO:2.
  • the CAR polypeptide, IL-15 and IL-15Ra polypeptide are each individually expressed from the nucleic acid construct according to the invention.
  • a fusion polypeptide comprising the CAR polypeptide, IL-15 and IL-15Ra polypeptide is produced from expression of a nucleic acid construct according to the invention, wherein the fusion polypeptide comprises the CAR polypeptide placed for expression , a cleavable linker peptide between IL-15 and IL-15Ra polypeptides, whereby the fusion polypeptide can be cleaved after expression in cells to produce separate CAR polypeptides, IL-15 and IL-15Ra polypeptides.
  • a polynucleotide encoding an IL-15 or IL-15Ra polypeptide is genetically fused at one end thereof to a polynucleotide encoding a CAR polypeptide using a self-cleaving peptide in an in-frame manner; and On the other end, a self-cleaving peptide is used to genetically fuse in frame with a polynucleotide encoding IL-15Ra or IL-15 polypeptide.
  • the nucleic acid construct according to the invention comprises a polynucleotide encoding a fusion polypeptide having the structure of formula (I): CAR-(L1)-E1-(L2)-E2 (I)
  • CAR, L1, L2, E1 and E2 are as defined above.
  • L1 and L2 contain self-cleaving sites.
  • Self-splicing sites that may be used in the present invention include, but are not limited to, self-splicing sites including P2A, T2A, E2A or F2A.
  • For the sequence and application of the 2A self-cleavage site please refer to Jin Hee Kim et al., High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice, PLoS ONE ⁇ April 2011, DOI: 10.1371 /journal.pone.0018556.
  • the L1 comprises a P2A site and the L2 comprises a T2A site.
  • the P2A site comprises: i) an amino acid sequence of SEQ ID NO: 17; ii) an amino acid sequence having at least one, two or three modifications but not more than 5 modifications to the amino acid sequence of SEQ ID NO: 17; or iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 17.
  • the T2A site comprises: i) an amino acid sequence of SEQ ID NO: 18; ii) an amino acid sequence having at least one, two or three modifications but not more than 5 modifications to the amino acid sequence of SEQ ID NO: 18; or iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 18.
  • a GSG linker may be inserted at the N-terminus of the 2A peptide to further improve its cleavage efficiency.
  • the present invention also provides a fusion polypeptide according to formula (I), preferably said L1 and L2 may comprise any self-splicing 2A site according to the foregoing.
  • the components of the structure of formula (I) may be functionally connected directly or indirectly through a linker (eg, a single amino acid residue or a short peptide).
  • the nucleic acid construct of the present invention includes a polynucleotide encoding a CAR polypeptide according to the present invention, a polynucleotide encoding an IL-15 polypeptide according to the present invention. polynucleotide, and a promoter operably linked to the polynucleotide encoding an IL-15Ra polypeptide according to the invention.
  • the nucleic acid construct is a vector.
  • Vectors suitable for use in the present invention include any vector suitable for replication and integration in eukaryotes; and containing transcriptional and translational terminators, initiation sequences and promoters for regulating expression of the desired nucleic acid sequence.
  • retroviral vectors provide a convenient platform for gene delivery systems.
  • the nucleic acid combinations of the invention can be inserted into vectors and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to the subject's cells in vivo or ex vivo.
  • Numerous retroviral systems are known in the art.
  • lentiviral vectors are used.
  • the nucleic acid sequence of the nucleic acid combination of the invention is cloned into a lentiviral vector, so that
  • Retroviruses e.g., lentiviruses
  • Lentiviral vectors have the additional advantage over vectors derived from onco-retroviruses (such as murine leukemia virus) in that they can transduce non-proliferating cells, such as hepatocytes. They also have the additional advantage of being low immunogenicity.
  • retropathies Viral vectors may also be, for example, gamma retroviral vectors.
  • a gamma retroviral vector may, for example, comprise a promoter, a packaging signal ( ⁇ ), a primer binding site (PBS), one or more (eg, two) long terminal repeats (LTR), and a transgene of interest, e.g., encoding a CAR genes.
  • Gamma retroviral vectors can lack viral structural genes such as gag, pol and env.
  • a promoter capable of expressing transgenes in mammalian T cells is the EF1a promoter.
  • the native EF1a promoter drives expression of the alpha subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl tRNA to ribosomes.
  • the EF1a promoter has been widely used in mammalian expression plasmids and has been shown to efficiently drive transgene expression from cloning into lentiviral vectors. See, eg, Milone et al., Mol. Ther. 17(8):1453–1464 (2009).
  • CMV immediate early cytomegalovirus
  • 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 promoters, such as but not limited to the actin promoter , myosin promoter, elongation factor-1 ⁇ promoter, hemoglobin promoter and creatine kinase promoter. Additionally, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.
  • the invention also provides cells into which a nucleic acid combination of the invention or a nucleic acid construct of the invention has been introduced.
  • the nucleic acid combination of the invention or the nucleic acid construct of the invention can be introduced into a cell by any nucleic acid transfer method known in the art.
  • the cells are mammalian cells, such as immune effector cells.
  • the cell is an armored CAR-T cell comprising IL-15 and an optimized IL-15Ra polypeptide according to the invention and a CAR polypeptide.
  • the invention also provides for the introduction and expression of the nucleic acid combinations of the invention in mammalian immune effector cells (eg, mammalian T cells or mammalian NK cells) and the generation of immune effector cells therefrom, in particular Methods of armoring CAR-T cells according to the invention.
  • the invention also provides immune effector cells obtainable by said method, especially armored CAR-T cells according to the invention.
  • a cell source e.g., immune effector cells, e.g., T cells or NK cells
  • T cells e.g., T cells or NK cells
  • 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 can be obtained from blood components collected from a subject using any technique known to those skilled in the art, such as Ficoll TM isolation.
  • cells from the individual's circulating blood are obtained by apheresis.
  • Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in a suitable buffer or culture medium for subsequent processing steps.
  • cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • T cell subsets such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated through positive or negative selection techniques.
  • anti-CD3/anti-CD28 e.g. M-450CD3/CD28T
  • the time period is between about 30 minutes and 36 hours or longer. Longer incubation times can be used to isolate T cells in any situation where small numbers of T cells are present, such as for isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals.
  • TILs tumor-infiltrating lymphocytes
  • Enrichment of T cell populations through a negative selection process can be accomplished using a combination of antibodies directed against surface markers unique to the negatively selected cells.
  • One method is to sort and/or select cells by means of negative magnetic immunoadhesion or flow cytometry, which uses cells present on the negatively selected cells. Mixture of monoclonal antibodies to surface markers.
  • the immune effector cells may be allogeneic immune effector cells, such as T cells or NK cells.
  • the cells may be allogeneic T cells, e.g., allogeneic that lack functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (e.g., HLA class I and/or HLA class II) expression.
  • TCR T cell receptor
  • HLA human leukocyte antigen
  • the immune effector cells are armored CAR-T cells according to the invention.
  • the armored CAR-T cells express a fusion polypeptide according to formula (I), and optionally the fusion polypeptide auto-splices after expression to produce the CAR polypeptide of the invention, the IL-15 polypeptide and the IL-15 polypeptide of the invention that are separated from each other. Optimization of IL-15Ra peptides.
  • the armored CAR-T cells according to the invention have at least one of the following properties:
  • T-cell therapy was applied for the first time in the treatment of hematological B-cell malignancies and showed effective and encouraging results.
  • the antitumor activity of CAR-T cell therapy is limited by the limited persistence of CAR-T cells.
  • technical means that can effectively adjust the persistence of CAR-T cells in vitro and in vivo are urgently needed.
  • a method of recombinantly expressing IL-15 in CAR-T cells has been proposed.
  • the released active IL-15 in serum has the potential to induce toxicity.
  • immune effector cells such as T cells (e.g., patient-specific autologous T cells) are engineered to incorporate nucleic acid combinations or vectors of the invention, thereby heterologously co-expressing the CAR of the invention in the cells.
  • Peptides, IL-15 and optimized IL-15Ra peptides After expanding the engineered immune effector cells (such as T cells or NK cells), they can be used for adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • the immune effector cells when treating a patient with immune effector cells of the invention, may be autologous or allogeneic T cells or NK cells. In some embodiments, the immune effector cells of the invention can improve the long-term survival of the cells after adoptive transfer and/or compared to the use of control CAR-T or CAR-NK cells without heterologous IL-15 and IL-15Ra expression. Proportion of TSCM subgroups.
  • the immune effector cells of the invention are used to treat cancer in a subject and are capable of reducing the severity of at least one symptom or indication of cancer or inhibiting cancer cell growth.
  • the present invention provides a method for treating cancer in a subject, comprising administering to an individual in need thereof a therapeutically effective amount of an immune effector cell expressing a nucleic acid combination of the present invention.
  • the present invention also provides the use of the aforementioned immune effector cell 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 human NALM-6 cell line and the retrovirus packaging cell line PG13 were purchased from the American Type Culture Collection (ATCC).
  • NAML-6-eGFP cells expressing enhanced GFP were generated by retroviral infection.
  • NAML-6-eGFP cells were maintained in RPMI-1640 (Lonza) containing 10% fetal calf serum (Biosera) and 10,000 IU/mL penicillin/10,000 ⁇ g/mL streptomycin (EallBio Life Sciences). All cells were cultured in a humidified incubator at 37°C, 5% CO2 , 95% air.
  • the third generation CD19-CAR gene was synthesized by a biological company.
  • the nucleotide sequence of the IL-15 gene is shown in SEQ ID NO: 2 and the nucleotide sequence of the optimized IL-15Ra gene is shown in SEQ ID NO: 3.
  • the protein expressed by the third generation CD19-CAR gene has an amino acid sequence (SEQ ID NO: 4) as shown below, which includes from N-terminus to C-terminus, signal peptide (bold underline), CD19scFv, and short connecting peptide (bold underline) , CD28 spacer/transmembrane region (italics), CD28 costimulatory domain (underlined), 4-1BB costimulatory domain (boxed bold italics), and CD3 ⁇ signaling domain:
  • the nucleotide sequence of the IL-15 gene (SEQ ID NO: 2) is as follows:
  • the protein expressed by the IL-15 gene has the amino acid sequence (SEQ ID NO: 5) as follows:
  • the optimized nucleotide sequence of the IL-15Ra gene (SEQ ID NO: 3) is as follows:
  • the protein (SEQ ID NO: 6) optimized for IL-15Ra gene expression is as follows:
  • T cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy donors by Lymphoprep (MP Biomedicals) gradient centrifugation.
  • PBMC peripheral blood mononuclear cells
  • T cells in PBMCs were stimulated with anti-CD3 and anti-CD28 beads and then infected with retroviruses.
  • CAR-T cells were assayed for CAR expression and then expanded in X-VIVO TM 15 serum-free medium containing 5% GemCell TM human serum AB and IL-2 (138 U/ml).
  • CD8 + T cells were isolated using the CD8 Positive Isolation Kit (Thermo Fisher Scientific). This study was approved by the Institutional Review Board of Beijing Millennium Hospital, and informed consent was obtained from all participants.
  • Flow cytometry was performed on a FACSCanto Plus instrument (BD Biosciences).
  • FlowJo v.10 (FlowJo, LLC) was used for data analysis.
  • CAR-T cells were detected by staining with mouse anti-human CD3 antibody labeled with APC-cy7 (BD Biosciences), mouse anti-human CD8 antibody labeled with FITC (BD Biosciences), mouse anti-human CD8 antibody labeled with Alexa Fluor 700 (BD Biosciences), mouse anti-human CD4 antibody labeled with BV421 (BD Biosciences), mouse anti-human CD107a labeled with V450 (BD Biosciences), mouse anti-human CD45RO labeled with BV605 (BD Biosciences), mouse anti-human CCR7 labeled with PE-cy7 (BD Biosciences), mouse anti-human CD27 labeled with Alexa Fluor 700 (BD Biosciences), mouse anti-human CD95 labeled with PE-cy5 (BD Biosciences), and goat anti-m
  • CAR-T cells were co-cultured in 24-well plates with target tumor cells NAML-6-EGFP-eGFP expressing the fluorescent protein GFP at different efficacy-to-target ratios (E:T). After 24 hours, cells were collected and tumor cells were detected by surface markers using flow cytometry (BD FacsCanto II Plus).
  • CAR-T cells and target cells were co-cultured at an E:T ratio of 2:1 for 24 hours.
  • the supernatant was collected and tested for IFN- ⁇ , IL-15 and IL-2 by ELISA kits (DY285B, D1500, DY202, R&D systems) according to the manufacturer's instructions.
  • RNA quantity and purity were measured using a Nanodrop One spectrophotometer (Thermo Fisher Scientific). Only samples with suitable absorbance measurements ( ⁇ 2.0 for A260/A280 and 1.9-2.2 for A260/A230) were considered for inclusion in this study.
  • cDNA was synthesized using High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).
  • IL-15 was amplified using primers 5'-ATGGATGCAATGAAGAGAGGG-3' (sense) and 5'-CGACGTGTTCATGAACATCTGGA-3' (antisense); IL-15Ra was amplified using primers 5'-ATGGCCCCGAGGCGGGCGCGAGG-3' (sense) and 5'-TAGGTGGTGCGAGCAGT-3' (antisense); GAPDH was amplified as a control using primers 5'-TGACCACAGTCCATGCCATC-3' (sense) and 5'-GTGAGCTTCCCGTTCAGCTC-3' (antisense).
  • NOD-SCID mice aged 6 to 8 weeks were purchased from Charles River Laboratories. 1 ⁇ 10 6 NAML-6-eGFP cells were intravenously injected into NOD-SCID mice to construct a xenograft mouse model. One day after tumor cell injection, 1 ⁇ 10 7 CAR-T cells were injected into the tail vein once a day for 3 days. Tumor development was monitored using IVIS (IVIS, Xenogen, Alameda, CA, USA). All experiments, including mice, were approved by the Institutional Review Board of Beijing Millennium Hospital.
  • mice with NAML-6-eGFP xenografts were measured using the Kaplan-Meier method and compared between groups using Cox proportional hazards regression analysis. All experiments were repeated at least three times.
  • IL-15 gene and IL-15Ra gene linked to the CD19-CAR gene were constructed (Fig. 1), and retroviral vectors co-expressing these genes were transduced into T cells using flow. Transduction efficiency was measured by cytometry.
  • Figure 2A shows that the three CAR constructs achieved similar CAR transduction efficiencies, with 35.4%, 26.1%, and 17.4% of CD8 + T cells expressing CD19-specific CAR, respectively.
  • Figure 2B shows similar CD4+/CD8+ T cell ratios among the three groups.
  • PCR was used to confirm the successful expression of IL-15 and IL-15Ra in armored CAR-T cells.
  • Total RNA from CAR-T cells was extracted and amplified by PCR.
  • the results ( Figure 2C) showed that CD19-CAR-IL-15 T cells overexpressed IL-15, and CD19-CAR-IL-15-IL-15Ra T cells overexpressed IL-15 and IL-15Ra.
  • IL-2 is a growth factor for T cells
  • concentration of IL-2 in the supernatant was measured. Briefly, CAR-T cells and target cells (NAML-6-eGFP) were co-cultured at an E:T ratio of 2:1 for 24 hours without additional IL-2 in the culture medium. Afterwards, the supernatant was collected and tested for IL-2 by ELISA kit. The results showed that CD19-CAR-IL-15 and CD19-CAR-IL-15-IL15Ra T cells released more IL-2 compared with CD19-CAR T cells ( Figure 3B).
  • Tscm cells (CD8 + CD45RO - CCR7 + CD27 + CD95 + ) representing the long-term persistence of CAR-T cells were studied.
  • the results showed that CD19-CAR-IL-15 and CD19-CAR-IL-15-IL-15Ra T cells had significantly more Tscm cells compared to CD8+CD19-CAR T cells (Figure 4A The highest measured Tscm cell levels were 1.67%, 9.23% and 4.84% in the three groups, respectively; Figure 4B shows a bar chart of the measurement results).
  • CD19-CAR, CD19-CAR-IL-15, and CD19-CAR-IL15-IL15Ra T cells were stimulated with NAML-6-eGFP cells at an E:T ratio of 2:1 for 24 hours, and IFN ⁇ was measured by ELISA concentration.
  • T cells expressing IL-15 and IL-15Ra produced less IFN ⁇ , implying the degree of differentiation of CD19-CAR-IL-15 and CD19-CAR-IL-15-IL-15Ra T cells. lower.
  • CAR-IL-15-IL-15Ra T cells may reduce the amount of IL-15 released into the culture medium by binding the expressed IL-15 to modified IL-15Ra expressed on the cell membrane surface. 15 amount and thereby reduce the cytotoxicity induced by released IL-15.
  • CD132 is a common receptor subunit chain for IL-2 and IL-15 and its high expression is associated with GVHD (graft versus host disease)
  • the cell surface CD132 expression of the two armored CAR-T cells was detected and compared .
  • the expression of CD132 on the cell surface was detected by flow cytometry.
  • Figure 6B showed that CAR-IL-15-IL-15Ra T cells had the lowest CD132 expression (CAR-IL-15-IL-15Ra T cells 60.8% vs. CAR-IL-15 T cells 65.5% vs. CAR-T cells 93.2%).
  • Example 4 IL-15Ra-expressing IL-15-armored CAR-T cells exhibit enhanced anti-tumor activity and reduced toxicity in vivo
  • NAML-6-eGFP cells were intravenously injected into NOD-SCID mice to generate a xenograft mouse model.
  • CAR-T cells were injected intravenously, and non-transduced T cells (NT) were used as negative controls. Mice were monitored over three months (Fig. 8A).
  • FIG. 8B and Figure 9 show that compared with the control group, mice treated with CD19-CAR-IL-15 T cells and CD19-CAR-IL-15-IL-15Ra T cells had no tumor recurrence, indicating that IL-15 Induced enhanced antitumor activity. However, despite no tumor recurrence, all mice in the CD19-CAR-IL-15 T cell treatment group died within 70 days compared with the CD19-CAR and CD19-CAR-IL-15-IL-15Ra T cell treatment groups. The survival rate was the lowest (Fig. 8B). This shows that IL-15 is toxic to animals.
  • mice with NAML-6-eGFP xenografts were measured using the Kaplan-Meier method and compared between groups using Cox proportional hazards regression analysis. As shown in Figure 10A, approximately 40% of mice in the CD19-CAR-IL-15-IL-15Ra survived more than 90 days compared with only 20% of mice in the CD19-CAR group.
  • FIG. 10B shows that mice treated with CD19-CAR-IL-15 T cells had the most human IL-15 in the blood, while mice in the CD19-CAR-IL-15-IL-15Ra T cell treatment group had significantly reduced blood IL-15 levels comparable to control CAR-T cell treated mice. This suggests that the co-expression of IL-15Ra in CD19-CAR-IL-15-IL-15Ra T cells blocks the blood release of IL-15 and thereby blocks the toxicity of IL-15 in serum, prolonging the survival of the treated tumor-bearing mice.

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Abstract

La présente invention concerne une cellule CAR-T renforcée exprimant le récepteur α de l'interleukine 15 et l'interleukine 15, ainsi qu'une utilisation immunothérapeutique associée.
PCT/CN2023/086217 2022-09-23 2023-04-04 UTILISATION D'UNE CELLULE CAR-T RENFORCÉE EXPRIMANT LE RÉCEPTEUR α DE L'INTERLEUKINE 15 DANS LA RÉDUCTION DE LA CYTOTOXICITÉ INDUITE PAR L'INTERLEUKINE 15 WO2024060577A1 (fr)

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