WO2024060577A1 - USE OF INTERLEUKIN 15 RECEPTOR α ARMORED CAR-T CELL IN REDUCING INTERLEUKIN 15-INDUCED CYTOTOXICITY - Google Patents

USE OF INTERLEUKIN 15 RECEPTOR α ARMORED CAR-T CELL IN REDUCING INTERLEUKIN 15-INDUCED CYTOTOXICITY 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

The present invention relates to an armored CAR-T cell expressing interleukin 15 receptor α and interleukin 15 and immunotherapeutic use thereof.

Description

白介素15受体α装甲CAR-T细胞在降低白介素15诱导的细胞毒性中的用途Use of interleukin 15 receptor alpha-armored CAR-T cells in reducing interleukin 15-induced cytotoxicity 技术领域Technical field
本发明涉及医学领域，特别是涉及一种表达白介素15受体α和白介素15的装甲CAR-T细胞及其免疫治疗用途。The present invention relates to the medical field, and in particular to an armored CAR-T cell expressing interleukin 15 receptor α and interleukin 15 and its immunotherapy use.
背景技术Background technique
嵌合抗原受体(CAR)-T细胞疗法是一种强大的针对血液癌症的过继性免疫疗法，而白细胞介素(IL)-15是一种重要的免疫刺激剂，具有诱导长期持久性CAR-T细胞的能力(Yang X,et al.Closely related T-memory stem cells correlate with in-vivo expansion of CAR.CD19-T cells in patients and are preserved by IL-7 and IL-15.Blood.(2014),https://doi.org/10.1182/blood-2014-01-552174)。然而，据报道，在CAR-T治疗期间，较高的基线或峰值血清IL-15水平尽管与更好的抗肿瘤反应相关，但也与更高的毒性率相关，所述毒性包括例如细胞因子释放综合征(CRS)、神经毒性和移植物抗宿主病(GVHD)。因此，本领域需要能够降低此类CAR-T细胞的毒性但保持其抗肿瘤效应和在体内的长期持久性的方法。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). However, it has been reported that during CAR-T treatment, higher baseline or peak serum IL-15 levels, although associated with better anti-tumor responses, are also associated with higher rates of toxicity, including, for example, cytokines release syndrome (CRS), neurotoxicity, and graft-versus-host disease (GVHD). Therefore, there is a need in the art for methods that can reduce the toxicity of such CAR-T cells but maintain their anti-tumor effects and long-term persistence in vivo.
发明概述Summary of the invention
经过深入的研究，本发明人应用CD19特异性CAR-T细胞，构建了同时装载了具有特定氨基酸修饰的优化IL-15受体α(IL-15Ra)和IL-I5的装甲CAR-T细胞(armored CAR-T cell)。如本申请实施例所示，在细胞培养条件下，与CAR-IL-15 T细胞相似，装载了优化IL-15Ra的CAR-IL-15 T细胞显示出增强的增殖能力和细胞活力，并保持了Tscm表型；同时与CAR-IL-15 T细胞相比，装载了该IL-15Ra的CAR-IL-15T细胞降低了IL-15向细胞外环境的释放量。进一步，本发明人在小鼠模型中证实，与常规CAR-T细胞和CAR-IL-15 T细胞相比，装载了该IL-15Ra的CAR-IL-15 T细胞延长了小鼠的存活时间，具有优异的抗肿瘤活性；并同时相比于CAR-IL-15 T细胞，在施用后导致降低的血清IL-15水平和相应地的更低毒性。这些结果表明，包含优化IL-15Ra的本发明装甲CAR-T细胞在减少CAR-T治疗过程中的不良事件和增强抗肿瘤能力方面发挥了重要作用。基于这些发现，本发明人由此建立了本发明。After in-depth research, the inventors used CD19-specific CAR-T cells to construct armored CAR-T cells loaded with optimized IL-15 receptor α (IL-15Ra) and IL-I5 with specific amino acid modifications ( armored CAR-T cells). As shown in the examples of this application, under cell culture conditions, similar to CAR-IL-15 T cells, CAR-IL-15 T cells loaded with optimized IL-15Ra showed enhanced proliferation ability and cell viability, and maintained Tscm phenotype; at the same time, compared with CAR-IL-15 T cells, CAR-IL-15T cells loaded with IL-15Ra reduced the release of IL-15 into the extracellular environment. Further, 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.
因此，在第一方面，本发明提供了一种核酸组合，所述组合包含第一核酸分子、第二核酸分子和第三核酸分子，其中所述第一核酸分子包含编码嵌合抗原多肽(CAR)的多核苷酸，第二核酸分子包含编码IL-15的多核苷酸，第三核酸分子包含编码优化IL15Ra的多核苷酸，其中所述优化IL15Ra包含在氨基酸位置202和203的双突变S202R和D203E，其中所述氨基酸位置根据SEQ ID NO:6编号。优选地，所述优化IL-15Ra包含SEQ ID NO:6的氨基酸序列；或与之具有至少90％、92％、95％、96％、97％、98％、99％或99.5％同一性的氨基酸序列。再优选地，所述编码优化IL15Ra的多核苷酸包含SEQ ID NO:3所述序列的多核苷酸、或与之具有至少90％、92％、95％、96％、97％、98％、99％或99.5％同一性的多核苷酸。Therefore, in a first aspect, 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. Preferably, 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.
在一些实施方案中，所述第一、第二和第三核酸分子中的两者或所有三者以功能性连接方式存在单个核酸构建体，例如病毒载体，如慢病毒载体上。在另一些实施方案中，所述第一、第二和第三核酸分子分别存在于不同的核酸构建体，例如病毒载体，如慢病毒载体上。In some embodiments, two or all three of the 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. In other embodiments, 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.
在一个实施方案中，根据本发明的核酸组合为包含第一、第二和第三核酸分子的单个核酸构建体，其中，所述核酸构建体编码从N端到C端具有下式(I)所示结构的融合蛋白：
CAR-(L1)-E1-(L2)-E2   (I)In one embodiment, 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)
其中，in,
CAR表示嵌合抗原受体多肽，CAR stands for Chimeric Antigen Receptor Polypeptide,
L1和L2分别独立地表示连接肽(尤其是包含自剪接位点的连接肽)， L1 and L2 each independently represent a connecting peptide (especially a connecting peptide containing a self-splicing site),
E1和E2互不相同，且彼此独立地选自IL-15和优化IL-15Ra，且E1 and E2 are different from each other and are independently selected from IL-15 and optimized IL-15Ra, and
其中“-”表示式(I)的所述各组分之间功能性连接。Wherein "-" represents the functional connection between the components of formula (I).
在一个实施方案中，E1表示由第二核酸分子编码的IL-15，且E2表示由第三核酸分子编码的优化IL-15Ra。在另一个实施方案中，E2表示由第二核酸分子编码的IL-15，且E1表示由第三核酸分子编码的优化IL-15Ra。In one embodiment, E1 represents IL-15 encoded by the second nucleic acid molecule and E2 represents optimized IL-15Ra encoded by the third nucleic acid molecule. In another embodiment, E2 represents IL-15 encoded by the second nucleic acid molecule and E1 represents optimized IL-15Ra encoded by the third nucleic acid molecule.
在一些实施方案中，连接肽L1和L2相同；在另一些实施方案中，所述连接肽L1和L2不同。在一些实施方案中，L1和L2包含自剪接位点，例如选自：P2A、T2A、E2A或F2A的自剪接位点。在一个实施方案中，L1包含P2A位点(优选地，包含SEQ ID NO:17的氨基酸序列)；L2包含T2A位点(优选地，包含SEQ ID NO:18的氨基酸序列)。In some embodiments, the linking peptides L1 and L2 are the same; in other embodiments, the linking peptides L1 and L2 are different. In some embodiments, L1 and L2 comprise a self-splicing site, eg, a self-splicing site selected from: P2A, T2A, E2A, or F2A. In one embodiment, 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).
在一个实施方案中，第一核酸分子包含编码CAR多肽的多核苷酸，其中所述CAR多肽从N端到C端包含：任选地信号肽(例如GM-CSFRα信号肽)，特异性结合肿瘤抗原的胞外抗原结合结构域、任选地铰链区或间隔区、跨膜结构域、和胞质信号传导结构域，其中所述胞质信号传导结构域包含共刺激结构域和初级信号传导结构域。在一个实施方案中，所述特异性结合肿瘤抗原的抗原结合结构域为抗体或抗体片段，尤其是scFv。在一个实施方案中，所述抗原结合结构域靶向CD19，更优选地包含SEQ ID NO:8的VL氨基酸序列中的LCDR1-3和SEQ ID NO:9的VH氨基酸序列中的HCDR1-3(尤其是Kabat定义的CDR序列)，再优选地，包含SEQ ID NO:8或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上同一性的氨基酸序列的VL和SEQ ID NO:9或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上同一性的氨基酸序列的VH，再优选地，包含SEQ ID NO:11或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上同一性的氨基酸序列的scFv。在一个实施方案中，所述CAR多肽包含铰链区/间隔区，优选地，所述铰链区/间隔区选自：来自IgG的铰链区或来自CD8α或CD28胞外区的间隔区，且优选是人CD8α铰链区或CD28铰链区，例如，包含SEQ ID NO:12所示氨基酸序列或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上同一性的氨基酸序列的CD28铰链区。在一个实施方案中，所述CAR多肽包含跨膜结构域，其选自：CD4,CD8α，CD28和CD3ζ的跨膜结构域，且优选是人CD8跨膜结构域或CD28跨膜结构域，例如，包含SEQ ID NO:13所示氨基酸序列或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上同一性的氨基酸序列的跨膜结构域。在再一实施方案中，所述CAR多肽包含一个或多个(尤其是两个)共刺激结构域，其选自：CD28,CD27,4-1BB,ICOS和OX40的共刺激结构域；且优选包含人CD28共刺激结构域和4-1BB共刺激结构域的组合，例如，包含SEQ ID NO:14和SEQ ID NO:15所示氨基酸序列或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上同一性的氨基酸序列。在一个实施方案中，所述CAR多肽包含初级信号传导结构域，其为CD3ζ初级信号传导结构域，例如，包含SEQ ID NO:16所示氨基酸序列或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上同一性的氨基酸序列。在一个优选实施方案中，所述CAR多肽包含由CD28的共刺激结构域和4-1BB的共刺激结构域以及CD3ζ初级信号传导结构域组成的胞质信号传导结构域，例如，所述胞质信号传导结构域包含SEQ ID NO:21所示的氨基酸序列或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上同一性的氨基酸序列。在再一个实施方案中，所述CAR多肽包含：从N端到C端，抗肿瘤抗原(如CD19)的抗体或抗原结合片段，如scFv，CD28铰链区，CD28跨膜结构域，CD28共刺激结构域，4-1BB共刺激结构域，和CD3ζ初级信号传导结构域。在一个优选实施方案中，所述CAR多肽包含SEQ ID NO:4的氨基酸序列，或与之具有至少90％、92％、95％、96％、97％、98％、99％或以上的同一性的氨基酸序列。In one embodiment, 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 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. In one embodiment, the antigen-binding domain that specifically binds a tumor antigen is an antibody or antibody fragment, especially a scFv. In one embodiment, 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. In one embodiment, 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 CD8α or CD28 extracellular region, and preferably Human CD8α 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. In one embodiment, the CAR polypeptide comprises a transmembrane domain selected from the group consisting of: transmembrane domains of CD4, CD8α, CD28 and CD3ζ, 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. In yet another embodiment, 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. In one embodiment, the CAR polypeptide comprises a primary signaling domain, which is a CD3ζ 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. In a preferred embodiment, the CAR polypeptide comprises a cytoplasmic signaling domain consisting of a costimulatory domain of CD28 and a costimulatory domain of 4-1BB and a CD3ζ 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. In yet another embodiment, 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 CD3ζ primary signaling domain. In a preferred embodiment, 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.
在一个实施方案中，第二核酸分子包含编码IL-15的多核苷酸。在一个实施方案中，第二核酸分子包含编码SEQ ID NO:5或与之具有至少85％、90％、92％、95％、96％、97％、98％、99％或以上同一性的 氨基酸序列的多核苷酸。在一个实施方案中，第二核酸分子包含SEQ ID NO:2的核苷酸序列，或与之具有至少85％、90％、92％、95％、96％、97％、98％、99％或以上的同一性的核苷酸序列。In one embodiment, 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.
在一个实施方案中，第三核酸分子包含编码优化IL-15Ra的多核苷酸。在一个实施方案中，所述优化IL-15Ra包含SEQ ID NO:6的氨基酸序列。In one embodiment, the third nucleic acid molecule comprises a polynucleotide encoding optimized IL-15Ra. In one embodiment, the optimized IL-15Ra comprises the amino acid sequence of SEQ ID NO: 6.
如本申请实施例所证实，本发明的核酸组合，当引入免疫效应细胞如T细胞中后，相比仅引入编码所述CAR的第一核酸分子和/或编码所述IL-15的第二核酸分子的对照免疫效应细胞，导致减少的IL-15在细胞外环境中的释放量，和由此减少的由IL-15诱导的毒性；且优选地，保持了IL-15增加CAR-T细胞的持久性的功能。As demonstrated by the examples of this application, 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.
在第二方面，本发明提供了由本发明的核酸组合编码的多肽，其包含：(i)嵌合抗原受体(CAR)多肽；(ii)IL-15多肽；和(iii)优化IL-15Ra多肽。在一个实施方案中，(i)-(iii)中两者或全部三者彼此功能性连接，尤其是通过连接肽连接为单个多肽链。在另一实施方案中，由本发明的核酸组合编码彼此分离的(i)、(ii)和(iii)多肽。当提及两个或多个多肽彼此分离时，是指所指的多肽之间彼此无(直接或通过连接肽)的共价连接，但所指的分离多肽之间可以存在或不存在非共价的结合，例如，分离的IL-15和优化IL-15Ra之间可以非共价结合为复合体，但与CAR多肽无任何非共价结合。在一个实施方案中，由本发明的核酸组合编码的多肽是包含功能性连接的以下组分的单条融合多肽：(i)嵌合抗原受体(CAR)多肽；(ii)IL-15多肽；和(iii)优化IL-15Ra多肽，更优选地所述融合多肽具有根据本发明的式(I)的结构，再优选地，所述融合多肽在细胞中表达后可以通过位于L1和L2中的自剪接位点而断裂从而产生分离的三个多肽，即，CAR多肽、IL-15多肽和优化IL-15Ra多肽。In a second aspect, 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. In one embodiment, two or all three of (i)-(iii) are functionally linked to each other, especially as a single polypeptide chain via a linking peptide. In another embodiment, the polypeptides (i), (ii) and (iii) are encoded by the nucleic acid combination of the invention and are isolated from each other. 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. For example, 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. In one embodiment, 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. More preferably, the fusion polypeptide has a structure according to formula (I) of the present invention. Even more preferably, 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.
在第三方面，本发明提供包含本发明核酸组合的核酸构建体，尤其是载体，例如病毒载体，如慢病毒载体。在一个实施方案中，包含在本发明核酸组合中的第一、第二和第三核酸分子以多顺反子形式存在于所述载体上。In a third aspect, 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. In one embodiment, 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.
在第四方面，本发明提供了包含本发明核酸组合或核酸构建体或载体的宿主细胞。所述宿主细胞可是免疫效应细胞，例如T细胞或NK细胞。因此，在一个实施方案，本发明也提供了装甲CAR-T细胞及其制备方法，其中所述装甲CAR-T细胞包含根据本发明的核酸组合、或引入了本发明的载体。在一个实施方案中，包含在所述装甲CAR-T细胞中的本发明核酸组合或载体表达产生根据本发明的式(I)融合多肽，任选地，所述融合多肽在细胞中通过包含在式(I)的连接肽中的自剪接位点断裂为三个分离的多肽，即，CAR多肽、IL-15多肽和优化IL-15Ra多肽。在另一个实施方案中，包含在所述装甲CAR-T细胞中的本发明核酸组合或载体表达产生彼此分离的本发明三个多肽，即，CAR多肽、IL-15多肽和优化IL-15Ra多肽。In a fourth aspect, 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. In one embodiment, 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. In another embodiment, 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. .
在一些实施方案中，表达IL-15和优化IL-15Ra的本发明装甲CAR-T细胞，相比仅表达CAR分子的CAR-T细胞，如按实施例中所述测定，显示出增强的增殖能力和细胞活力，以及增加的Tscm表型T细胞亚群比例。在一些实施方案中，与仅表达IL-15的CAR-T细胞相比，表达IL-15和优化IL-15Ra的本发明装甲CAR-T细胞减少IL-15在细胞外环境中的释放量，和由此具有降低的由IL-15诱导的毒性。在再一些实施方案中，表达IL-15和优化IL-15Ra的本发明装甲CAR-T细胞，如按实施例中所述测定，显示出体内抗肿瘤功效和减少的CAR-T治疗中与IL-15相关的不良事件。In some embodiments, 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. In some embodiments, 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. In further embodiments, armored CAR-T cells of the invention expressing IL-15 and optimized IL-15Ra, as determined as described in the Examples, exhibit in vivo anti-tumor efficacy and reduced CAR-T treatment with IL -15 related adverse events.
在第五方面，本发明提供了包含本发明的装甲CAR-T细胞的药物组合物。In a fifth aspect, the invention provides a pharmaceutical composition comprising the armored CAR-T cells of the invention.
在第六方面，本发明提供了本发明的装甲CAR-T细胞在制备用于预防或治疗癌症或提供抗肿瘤免疫的药物中的用途、以及使用本发明的装甲CAR-T细胞在受试者中预防或治疗癌症或提供抗肿瘤免疫的方法。在一些实施方案中，所述CAR-T细胞全身(例如静脉内)施用或局部(例如瘤内)使用。在一些实施方案中，所述肿瘤是血液肿瘤或实体瘤。 In a sixth aspect, 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. In some embodiments, the CAR-T cells are administered systemically (eg, intravenously) or used locally (eg, intratumorally). In some embodiments, the tumor is a hematological tumor or a solid tumor.
在第七方面，本发明提供了优化IL15Ra多肽及其编码核酸分子、以及其在降低重组表达IL15的CAR-T细胞的毒性中的应用，优选地，所述应用包括在所述CAR-T细胞中引入并表达编码所述优化IL-15Ra多肽的核酸分子，更优选地，所述CAR-T细胞包含编码根据本发明式(I)所示结构的融合蛋白的核酸分子。In a seventh aspect, 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. Preferably, 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. More preferably, 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.
在第八方面，本发明提供了一种用于增加CAR-T细胞的持久性并降低其毒性的方法，其包括在所述CAR-T细胞中引入并表达根据本发明的核酸组合或载体。In an eighth aspect, 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.
附图说明Description of the drawings
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.
图1示意性展示根据本发明的装甲CAR-T细胞的结构。其中：CD19-CAR表示靶向CD19的嵌合抗原受体多肽，从N端到C端包含抗CD19scFv、CD28间隔区/跨膜区、CD28共刺激结构域、4-1BB共刺激结构域和CD3ζ信号传导结构域；SD表示剪接供体位点；SA表示剪接受体位点；LTR表示长末端重复序列；P2A和T2A分别表示自剪接肽P2A和T2A；IL-15表示IL-15蛋白；IL-15Ra表示修饰的IL-15Ra蛋白。Figure 1 schematically shows the structure of armored CAR-T cells according to the present invention. Among them: 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 CD3ζ 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; IL-15Ra Represents modified IL-15Ra protein.
图2显示，以编码CAR的逆转录病毒载体转导T细胞后，对产生的CAR、CAR-IL15和CAR-IL15-IL15Ra T细胞进行表征。(A和B)通过流式细胞术，定量CD4+和CD8+T细胞数和CD19+CD8+CAR T细胞数，确定转导效率。(C)通过PCR确认三种CAR-T细胞中的IL-15和IL-15Ra表达水平。作为对照，同时扩增细胞中的管家基因GAPDH。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.
图3显示，检测IL-15和/或IL-15Ra过表达的装甲CAR-T细胞在细胞因子或靶肿瘤细胞刺激下的体外增殖(A)和细胞因子IL-2产生(B)。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.
图4显示，在靶肿瘤细胞NAML-6-eGFP刺激7天后，检测IL-15和/或IL-15Ra过表达的装甲CAR-T细胞的分化表型，其中流式细胞术检测细胞群体中Tscm(CD8+CD45RO-CCR7+CD27+CD95+)的比例。(A)最高测量的Tscm细胞水平，三组分别为1.67％，9.23％和4.84％。(B)测量结果的柱状统计图Figure 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. (A) The highest measured Tscm cell levels were 1.67%, 9.23% and 4.84% in the three groups, respectively. (B) Bar graph of the measurement results
图5显示，在与靶肿瘤细胞NAML-6-eGFP孵育一段时间后，CAR、CAR-IL15和CAR-IL15-IL15Ra T细胞的细胞因子IFNγ分泌(A)、以及细胞凋亡百分数(B)和细胞生存率(C)。Figure 5 shows that after incubation with target tumor cells NAML-6-eGFP for a period of time, cytokine IFNγ secretion (A), as well as cell apoptosis percentage (B) and Cell viability (C).
图6显示，CAR、CAR-IL15和CAR-IL15-IL15Ra T细胞在体外培养条件下的IL-15分泌(A)和CD132细胞表面表达(B)。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.
图7显示，CAR-T细胞与靶细胞NAML-6-eGFP细胞(2：1)共培养24小时后，收集细胞，(A)通过流式检测GFP信号，表示存活的NAML-6-eGFP细胞数；以及(B)相应的统计结果柱状图。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.
图8显示，异种移植小鼠肿瘤模型实验。(A)动物实验流程图；(B)小鼠肿瘤负荷荧光图。Figure 8 shows the xenograft mouse tumor model experiment. (A) Animal experiment flow chart; (B) Fluorescence chart of mouse tumor burden.
图9显示，异种移植小鼠肿瘤模型实验中各组小鼠个体随时间的肿瘤负荷变化。应用IVIS成像系统获取所有小鼠的定量生物发光成像数据(即，单位时间、单位面积、单位弧度从动物体表发出的绝对光子数(photons/sec/cm2/sr))。该数值越高指示肿瘤负荷越大。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.
图10显示，异种移植物荷瘤小鼠的总体存活率和血清IL-15浓度。(A)使用Kaplan-Meier方法，测量异种移植物荷瘤小鼠的总体存活率。(B)在第50天，采集各组小鼠的血液，用血清检测人IL-15的浓度。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.
具体实施方式Detailed ways
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。 Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that every intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range and any other stated value or value intermediate within a stated range is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。It will be apparent to those skilled in the art that various modifications and changes can be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to the skilled person from the description of the invention. The specification and examples are intended to be illustrative only.
I.定义I.Definition
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。For the purpose of interpreting this specification, the following definitions will be used and, wherever appropriate, terms used in the singular may also include the plural and vice versa. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组成的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。The words "includes", "includes", "has", "contains", etc. used in this article are all open terms, which mean including but not limited to. When the term "comprises" or "includes" is used herein, it also encompasses a combination of the stated elements, integers, or steps unless otherwise indicated. For example, when reference is made to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass antibody variable regions that consist of that particular sequence.
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。The term "about" when used in conjunction with a numerical value is intended to encompass a range of numerical values having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.
如本文所用，术语“和/或”意指可选项中的任一项或可选项的两项或多项。As used herein, the term "and/or" means any one of the options or two or more of the options.
术语“IL-15”是指白介素15细胞因子。IL-15的一个实例是人来源的白介素IL-15，例如UniProtKB-登录号P40933下的蛋白质，或其同源物，例如非人哺乳动物来源的白介素IL-15，所述哺乳动物为例如非人灵长类动物、啮齿类动物、家畜、运动动物等。The term "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.
术语“IL-15Ra”或“IL-15Rα”是指白介素15受体α蛋白。IL-15Ra的一个实例是人来源的白介素IL-15受体α，例如UniProtKB-登录号Q13261下的蛋白质、或其同源物、或变体。在一个实施方案中，本发明的IL-15Ra是在亲本IL-15Ra受体蛋白中引入修饰(例如双突变S202R和D203E)的白介素15受体α蛋白，也称作“优化IL-15Ra”。在一些实施方案中，所述亲本IL-15Ra受体蛋白可以是哺乳动物来源，例如人来源的、或非人哺乳动物动物的天然或野生型IL-15Ra。在一些实施方案中，所述亲本IL-15Ra受体蛋白包含在位置197-204的基序YPQGHRET。The term "IL-15Ra" or "IL-15Rα" 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. In one embodiment, 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." In some embodiments, 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. In some embodiments, the parent IL-15Ra receptor protein comprises the motif YPQGHRET at positions 197-204.
术语“嵌合受体”、“嵌合抗原受体”或“CAR”在本文中可互换使用，是指至少包含胞外抗原结合结构域、跨膜结构域及胞质信号传导结构域的重组多肽。在一个方面，所述胞质信号传导结构域包含来自如下所述刺激分子的初级信号传导结构域，例如CD3-ζ的初级信号传导结构域。在另一方面，所述胞内信号传导结构域还包含来自至少一种，优选两种共刺激分子，例如CD28和4-1BB的一个或多个功能性信号结构域。CAR多肽可以表达在任何细胞，例如免疫效应细胞如T细胞或NK细胞上。The terms "chimeric receptor", "chimeric antigen receptor" or "CAR" are used interchangeably herein to refer to a receptor that includes at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. Recombinant peptides. In one aspect, the cytoplasmic signaling domain comprises a primary signaling domain from a stimulatory molecule as described below, such as that of CD3-ζ. In another aspect, 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.
术语“刺激分子”指由T细胞表达的分子，所述分子提供初级胞质信号传导序列，所述的初级胞质信号传导序列在T细胞信号传导途径的至少某个方面以刺激性方式调节TCR复合体的初级活化。在一个方面，初级信号可以(例如通过TCR/CD3复合体与载有肽的MHC分子的结合)被引发，并随后介导T细胞反应，包括但不限于增殖、活化、分化等。以刺激方式起作用的初级胞质信号传导序列可以包含免疫受体酪氨酸激活基序(ITAM)。含有ITAM的初级胞质信号传导序列的例子包括，但不限于，来自TCR zeta和CD3 zeta 的胞内信号传导结构域。在本发明中，本发明CAR多肽的胞质结构域包含来自刺激分子的至少一个功能性胞质信号传导序列，例如，CD3ζ的胞质信号传导序列。The term "stimulatory molecule" refers to a molecule expressed by a T cell that provides 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. In one aspect, 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). Examples of ITAM-containing primary cytoplasmic signaling sequences include, but are not limited to, those from TCR zeta and CD3 zeta intracellular signaling domain. In the present invention, 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 CD3ζ.
术语“CD3ζ”定义为UniProtKB-P20963登录号下提供的蛋白质或其等同物。在本文中，“CD3ζ信号传导结构域”定义为来自CD3ζ链胞质结构域的氨基酸残基区段，所述氨基酸残基区段足以在功能上传播T细胞活化必需的初始信号。在一个实施方案中，CD3ζ的胞质结构域包含UniProtKB-P20963登录号下氨基酸序列的残基52至残基164或作为其功能直向同源物的来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在一个实施方案中，“CD3ζ信号传导结构域”是在SEQ ID NO:16中提供的序列或其变体。The term "CD3ζ" is defined as the protein provided under UniProtKB-P20963 accession number or its equivalent. As used herein, a "CD3ζ signaling domain" is defined as a segment of amino acid residues from the cytoplasmic domain of the CD3ζ chain that is sufficient to functionally propagate the initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of CD3ζ 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.). In one embodiment, the "CD3ζ signaling domain" is the sequence provided in SEQ ID NO: 16, or a variant thereof.
术语“共刺激分子”是指细胞上的与共刺激配体特异性结合从而介导细胞的共刺激反应(例如但不限于增殖)的相应结合配偶体。共刺激分子是除抗原受体或其配体之外的有助于有效免疫应答的细胞表面分子。共刺激分子包括但不限于MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号传导淋巴细胞活化分子(SLAM蛋白)、激活NK细胞受体、OX40、CD40、GITR、4-1BB(即CD137)、CD27和CD28。在一些实施方案中，“共刺激分子”是CD28、4-1BB(即CD137)。在本文中，“共刺激结构域”是指共刺激分子的胞内部分。The term "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. In some embodiments, the "costimulatory molecule" is CD28, 4-1BB (ie, CD137). As used herein, "costimulatory domain" refers to the intracellular portion of the costimulatory molecule.
术语“4-1BB”指TNFR超家族成员，也称作CD137,所述成员具有在UniProtKB-Q07011登录号下提供的氨基酸序列或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在本文中，术语“4-1BB共刺激结构域”定义为来自4-1BB的胞质区，例如，UniProtKB-Q07011的氨基酸残基214-255或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。The term "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. As used herein, the term "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.
术语“CD28”是指在UniProtKB-P10747登录号下提供的氨基酸序列或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在本文中，术语“CD28共刺激结构域”定义为来自CD28的胞质区，例如，UniProtKB-P10747的氨基酸残基180-220或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在本文中，术语“CD28跨膜结构域”定义为来自CD28的跨膜区，例如，UniProtKB-P10747的氨基酸残基153-179或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。在本文中，术语“CD28铰链结构域”与“CD28间隔区”可互换使用，定义为来自CD28胞外区的铰链结构域，例如UniProtKB-P10747的氨基酸残基114-152或来自非人类物种(例如，小鼠、啮齿类、猴、猿等)的等同残基。The term "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.). As used herein, the term "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. As used herein, the term "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. In this article, the terms "CD28 hinge domain" and "CD28 spacer" are used interchangeably and are defined as the hinge domain from the extracellular region of CD28, such as amino acid residues 114-152 of UniProtKB-P10747 or from non-human species. (e.g., mouse, rodent, monkey, ape, etc.) equivalent residues.
在本文中，术语“重组”，当涉及例如病毒或细胞或核酸或蛋白或载体时，指所述病毒、细胞、核酸、蛋白或载体已经通过引入异源核酸或蛋白、或通过改变自身已有的天然核酸或蛋白而被修饰、或指来自于由此修饰的病毒或细胞的物质。As used herein, 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.
描述核酸或蛋白质时所用的术语“外源的”或“异源的”可互换使用，是指相对于包含或待包含所述核酸或蛋白质的宿主细胞而言，该核酸或蛋白质是外来的，即其在所述宿主细胞中的存在位置并不是其在自然情况下的天然存在位置。异源核酸序列也指衍生自并引入(例如通过病毒载体感染而引入)相同宿主细胞或受试者而由此以非天然状态存在的序列，例如，所述序列位于不同的位置、以不同的拷贝数存在，或处于不同调控元件的控制下。The terms "exogenous" or "heterologous" used to describe a nucleic acid or protein are used interchangeably and refer to a nucleic acid or protein that is foreign to a host cell that contains or is to contain the nucleic acid or protein, i.e., that is, that is present in a location in the host cell that is not its natural location under natural conditions. A heterologous nucleic acid sequence also 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.
术语“表达盒”是指，编码并能够表达一个或多个目的基因(例如本发明CAR多肽、或IL-15蛋白、或优化IL-15Ra、或其中两者或三者)的DNA序列。在表达盒中，通常，编码目的基因的异源多核苷酸序列与表达控制序列功能性连接。例如，表达盒可以包含在同一启动子控制下、以多顺反子形式存在的两个或两个以上目的基因，并由此编码并表达单条多肽链，在该单条多肽链中，所述两个或两个以上目的基因编码的两个或两个以上目的蛋白彼此功能性连接。The term "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). In an expression cassette, typically, a heterologous polynucleotide sequence encoding a gene of interest is functionally linked to expression control sequences. For example, 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.
术语“功能性连接”也称作“有效连接”，意指指定的各组分处于一种允许它们以预期的方式起作用的关系中。 The term "functionally linked", also referred to as "effectively linked," means that the specified components are in a relationship that allows them to function in an intended manner.
在本文中，术语“连接子”或“连接肽”或“接头”可互换使用，是指由氨基酸组成的短氨基酸序列，例如单独或组合使用的丙氨酸(A)、甘氨酸(G)和/或丝氨酸(S)和/或苏氨酸残基(T)、或包含自剪接位点的自剪接肽。在一个实施方案中，连接肽具有1-50个氨基酸长度，例如，1,2,3,4,5个氨基酸，或10,15,20,25,30个氨基酸长度。可以用于本发明CAR融合多肽的各组件之间的连接肽并不特定限制。可以使用计算机程序模拟蛋白和肽的三维结构来合理地设计合适的连接肽。例如，短寡肽接头或多肽接头可以根据需要，在组件序列之间形成键接，例如，甘氨酸-丝氨酸双联体，或单个氨基酸，例如，丙氨酸、甘氨酸可以用作接头。In this article, the terms "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. In one embodiment, 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. For example, 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.
术语“氨基酸变化”和“氨基酸修饰”可互换地使用，是指氨基酸的添加、缺失、取代和其他修饰。可以进行氨基酸的添加、缺失、取代和其他修饰的任意组合，条件是最终的多肽序列具有所需的特性。在一些实施方案中，氨基酸的取代是非保守氨基酸取代，即用具有不同结构和/或化学性质的另一种氨基酸取代一种氨基酸。氨基酸取代包括用非天然存在的氨基酸或二十种标准氨基酸的天然存在的氨基酸衍生物(例如、4-羟基脯氨酸、3-甲基组氨酸、鸟氨酸、高丝氨酸、5-羟基赖氨酸)的取代。The terms "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. In some embodiments, 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.
术语“保守序列修饰”、“保守序列变化”指未显著影响或改变含有氨基酸序列的亲本多肽或其组成元件的特征的氨基酸修饰或变化。这类保守修饰包括氨基酸取代、添加和缺失。可以通过本领域已知的标准技术，如位点定向诱变和PCR介导的诱变向本发明的CAR融合多肽或其组成元件(例如CAR或IL-15或IL-15Ra)中引入保守修饰，尤其是保守性取代。保守性取代是氨基酸残基由具有相似侧链的氨基酸残基替换的氨基酸取代。已经在本领域中定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、蛋氨酸)、β-侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。The terms "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), β-side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenyl Alanine, tryptophan, histidine) amino acids.
氨基酸序列/核苷酸序列的“同一性百分数(％)”是指，将候选序列与本说明书中所示的具体氨基酸/核苷酸序列进行比对并且如有必要的话为达到最大序列同一性百分数而引入空位后，且在氨基酸序列的情况下，不考虑任何保守置换作为序列同一性的一部分时，候选序列中与本说明书中所示的具体氨基酸/核苷酸序列的氨基酸残基/核苷酸残基相同的氨基酸/核苷酸残基百分数。在一些实施方案中，本发明考虑本发明融合多肽或核酸分子或其组成元件的变体，所述变体相对于在本文中具体公开的融合多肽或核酸分子或其组成元件(例如CAR多肽/编码核酸，或IL-15蛋白/编码核酸,或优化IL-15Ra蛋白/核酸)的序列而言具有相当程度的同一性，例如同一性为至少80％,85％,90％,95％,97％,98％或99％或更高。所述变体可以包含保守性修饰。根据本发明的目的，同一性百分数应用https://blast.ncbi.nlm.nih.gov上公众可得的 BLAST工具，采用默认参数进行确定。"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. In some embodiments, 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.
在本文中，表述“变体”或“功能性变体”多肽或蛋白是指，所述的多肽或蛋白，与参照多肽或蛋白相比，具有实质上相同的序列或显著的序列同一性、并保持参照多肽或蛋白的期望生物学活性。As used herein, the expression "variant" or "functional variant" polypeptide or protein 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.
在本文中当谈及核酸时使用的术语“载体(vector)”是指能够增殖与其相连的另一个核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及结合到已经引入其的宿主细胞的基因组中的载体。一些载体能够指导与其有效相连的核酸的表达。这样的载体在本文中被称为“表达载体”。The term "vector" as used herein when referring to nucleic acids 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."
术语“慢病毒”指逆转录病毒科(Retroviridae)的一个属。慢病毒在逆转录病毒当中的独特之处在于能够感染非分裂性细胞；它们可以递送显著量的遗传信息至宿主细胞，从而它们是基因递送载体的最高效方法之一。HIV、SIV和FIV均是慢病毒的例子。The term "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.
术语“慢病毒载体”指从慢病毒基因组的至少一部分衍生的载体，尤其包括如Milone等人,Mol.Ther.17(8):1453–1464(2009)中提供的自我失活慢病毒载体。可以在临床使用的慢病毒载体的其他例子，例如， 包括但不限于，来自Oxford BioMedica的基因递送技术、来自Lentigen的LENTIMAXTM载体系统等。非临床类型的慢病毒载体也是可获得的并且是本领域技术人员已知的。The term "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.
术语“免疫效应细胞”指参与免疫应答，例如参与促进免疫效应反应的细胞。免疫效应细胞的例子包括T细胞，例如，α/βT细胞和γ/δT细胞、B细胞、天然杀伤(NK)细胞、天然杀伤T(NKT)细胞、肥大细胞、和髓细胞衍生的吞噬细胞。The term "immune effector cells" refers to cells involved in an immune response, such as in promoting an immune effector response. Examples of 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.
术语“个体”或“受试者”可互换地使用，包括哺乳动物。哺乳动物包括但不限于驯化动物(例如，牛、羊、猫、犬和马)、灵长类(例如，人和非人灵长类如猴)、兔和啮齿类(例如，小鼠和大鼠)。特别地，个体或受试者是人。The terms "individual" or "subject" are used interchangeably and include mammals. 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). In particular, the individual or subject is a human being.
术语“肿瘤”和“癌症”在本文中互换地使用，涵盖实体瘤和液体肿瘤。The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid and liquid tumors.
术语“抗肿瘤免疫”是指，可以通过多种手段展示如下免疫学效果，包括但不限于例如，引起肿瘤体积减少、肿瘤细胞数目减少、肿瘤细胞增殖减少或肿瘤细胞存活减少。The term "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.
II.本发明的核酸组合、多肽和武装CAR-T细胞II. Nucleic acid combinations, polypeptides and armed CAR-T cells of the invention
本发明在深入的研究中发现，在基于CAR的免疫细胞(例如，CAR-T细胞和CAR-NK细胞)中，可以通过增加IL-15和优化IL-15Ra基因的表达，来促进所述免疫细胞的持久性和/或抗肿瘤免疫作用，同时限制由释放自环境(例如血清)中的IL-15诱导的毒性。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).
因此，在一个方面，本发明提供了基于CAR的免疫细胞，其中所述免疫细胞不仅包含编码CAR多肽的异源多核苷酸，还包含编码IL-15和优化IL-15Ra的异源多核苷酸。在所述免疫细胞为T细胞时，在本文中，具有重组表达的IL15和优化IL-15Ra的这种基于CAR的免疫细胞，也称作“装甲CAR-T细胞”或“Armored CAR-T细胞”。在所述免疫细胞中，所述编码CAR多肽的异源多核苷酸以及编码IL-15和优化IL-15Ra的异源多核苷酸可以位于单个核酸分子上，或位于分开的不同核酸分子上。Accordingly, in one aspect, 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 . When 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. ". In the immune cell, 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.
在再一个方面中，本发明提供了可用于形成根据本发明的CAR免疫细胞的核酸组合。在一个实施方案中，本发明的核酸组合包含编码嵌合抗原受体(CAR)分子的第一核酸分子、编码IL-15蛋白的第二核酸分子、和编码优化IL-15Ra的第三核酸分子。尽管不受任何理论的约束，但认为：本发明的优化IL-15Ra由于特别设计的双突变，当与IL-15在相同细胞中表达时，可以与IL-15结合形成稳定的异二聚体结构并展示在细胞膜上，从而在保证IL-15以稳定的功能性构象发挥其功能的同时，限制IL-15释放到环境(例如动物血清)中的量，从而降低由血清高IL-15水平诱导的毒性。如本申请实施例所证实，包含优化IL-15Ra的本发明装甲CAR-T细胞在减少CAR-T治疗过程的不良事件和增强抗肿瘤方面展示了优异的性能。In yet another aspect, the invention provides combinations of nucleic acids useful in forming CAR immune cells according to the invention. In one embodiment, 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 . Although not bound by any theory, it is believed that the optimized IL-15Ra of the present invention, due to the specially designed double mutations, can combine with IL-15 to form a stable heterodimer when expressed in the same cell as IL-15. Structure and displayed on the cell membrane, thereby ensuring that IL-15 performs its function in a stable functional conformation while limiting the amount of IL-15 released into the environment (such as animal serum), thereby reducing high serum IL-15 levels induced toxicity. As demonstrated in the examples of this application, 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.
在本发明的核酸组合中，所述的CAR多肽编码多核苷酸，所述的IL-15蛋白编码多核苷酸、与所述的优化IL-15Ra编码核酸，可以以全部三者或任意两者位于相同的表达盒中或位于不同的表达盒中，并可以分别表达为单独的多肽，或以任意两者或全部三者表达为融合多肽。在一个实施方案中，所述核酸组合为单个核酸分子的形式，所述单个核酸分子编码并表达产生包含所述CAR多肽、IL-15和优化IL15Ra的单个融合多肽，优选地，在所述的融合多肽中，IL-15和IL15Ra蛋白功能性连接在一起，且CAR多肽通过包含可剪切位点的连接肽与IL-15和IL15Ra蛋白之一功能性连接。在一些优选实施方案中，从N端到C端，所述融合多肽具有式(I)结构：
CAR-(L1)-E1-(L2)-E2   (I)In the nucleic acid combination of the present invention, 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. In one embodiment, 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. In some preferred embodiments, from N-terminus to C-terminus, the fusion polypeptide has the structure of formula (I):
CAR-(L1)-E1-(L2)-E2 (I)
其中，in,
CAR表示由第一核酸分子编码的嵌合抗原受体多肽，CAR means a chimeric antigen receptor polypeptide encoded by the first nucleic acid molecule,
L1和L2分别独立地表示连接肽(尤其是自剪接肽)，L1 and L2 independently represent connecting peptides (especially self-splicing peptides),
E1和E2互不相同，且彼此独立地表示分别由第二或第三核酸分子编码的IL-15或优化IL-15Ra，且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
其中式(I)的所述各组分之间功能性连接。 The components of formula (I) are functionally connected.
在再一方面，本发明也提供了一种具有上述式(I)结构的融合多肽。In yet another aspect, the present invention also provides a fusion polypeptide having the structure of the above formula (I).
以下就本发明的基于CAR的免疫细胞、核酸组合、多肽及其组件，分别进行详述描述。本领域技术人员可以理解，除非上下文有明确相反指示，否则，在对组件进行描述时提及的任何技术特征及其任何组合，均在本发明考虑范畴之中；并且，本领域技术人员可以理解，除非上下文有明确相反指示，否则本发明的基于CAR的免疫细胞可以包含任何这样的组合特征，同样地本发明核酸构建体和CAR融合多肽也可以包含任何这样的组合特征。The CAR-based immune cells, nucleic acid combinations, polypeptides and components thereof of the present invention will be described in detail below. A person skilled in the art can understand that, unless the context clearly indicates otherwise, 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.
优化IL-15Ra多肽及其编码核酸Optimizing IL-15Ra polypeptides and their encoding nucleic acids
本发明的IL-15Ra是具有特征性双突变S202R和D203E的膜结合型蛋白。在一个实施方案中，本发明的IL-15Ra可来自任何功能性的单跨膜天然全长IL-15Ra蛋白或其变体(包括天然等位基因变体或物种同源物)，其中，在氨基酸位置202和203引入了双突变S202R和D203E，其中所述氨基酸位置根据SEQ ID NO:6编号。在本文中，当提及IL-15Ra蛋白的氨基酸位置时，“根据SEQ ID NO:6编号”是指，通过参考SEQ ID NO:6的氨基酸序列予以确定。对于任何一个给定的IL-15Ra多肽，可以通过与SEQ ID NO:6进行氨基酸序列比对(例如使用BLAST；可从http://blast.ncbi.nlm.nih.gov获得的Basic Local Alignment Search Tool，使用默认参数，进行比对)，鉴定在该给定的IL-15Ra多肽上的对应氨基酸位置。因此，例如，当提及一个给定IL-15Ra多肽的氨基酸位置202和203时，是指SEQ ID NO:6的第202位和第203位(当所述IL-15Ra包含SEQ ID NO:6时)，或经比对在该给定多肽序列上的对应氨基酸位置(当所述IL-15Ra包含与SEQ ID NO:6具有一定百分比，例如90-99.5％同一性的氨基酸序列时)。在本文中，突变S202R是指氨基酸位置202具有丝氨酸(S)到精氨酸(R)的突变；突变D203E是指氨基酸位置203具有天冬氨酸(D)到谷氨酸(E)的突变。在根据本发明的任何实施方案中，优选地，除所述双突变外，IL-15Ra多肽包含:i)SEQ ID NO:6的氨基酸序列；ii)对SEQ ID NO:6的氨基酸序列具有至少一个，两个或三个修饰但不超过30,20或10个修饰的氨基酸序列；或iii)与SEQ ID NO:6的氨基酸序列具有至少95-99％同一性的氨基酸序列。优选地，本发明的IL-15Ra包含位置197-204的基序YPQGHSDT。IL-15Ra of the present invention is a membrane-bound protein with characteristic double mutations S202R and D203E. In one embodiment, 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. In this article, when referring to the amino acid position of the IL-15Ra protein, "numbered according to SEQ ID NO:6" means that it is determined by reference to the amino acid sequence of SEQ ID NO:6. For any given IL-15Ra polypeptide, 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. Thus, for example, when referring to amino acid positions 202 and 203 of a given IL-15Ra polypeptide, reference is made to positions 202 and 203 of SEQ ID NO:6 (when said IL-15Ra contains SEQ ID NO:6 when), or when aligned to the corresponding amino acid position on the given polypeptide sequence (when the IL-15Ra includes an amino acid sequence that has a certain percentage, for example, 90-99.5% identity to SEQ ID NO: 6). In this article, 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. . In any embodiment according to the invention, preferably, in addition to said double mutation, 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. Preferably, IL-15Ra of the invention comprises the motif YPQGHSDT at positions 197-204.
在一个优选的实施方案中，本发明的IL-15Ra多肽保留其来源天然IL-15亲本多肽的信号肽。在另一些实施方案中，本发明的IL-15Ra多肽具有来自另一跨膜真核蛋白如哺乳动物蛋白的异源信号肽，以指导其在细胞中表达和加工后整合在细胞膜上。在一个实施方案中，本发明的IL-15Ra多肽在细胞表达后，与在相同细胞中表达的IL-15多肽，形成非共价复合物并转运到细胞膜表面。In a preferred embodiment, the IL-15Ra polypeptides of the invention retain the signal peptide of the native IL-15 parent polypeptide from which they are derived. In other embodiments, 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. In one embodiment, after expression in cells, 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.
可用于本发明的优化IL-15Ra多肽编码多核苷酸，可以是包含编码根据本发明的上述任何实施方案中的优化IL-15Ra蛋白的核苷酸序列的任何多核苷酸。在一个实施方案中，所述的优化IL-15Ra编码多核苷酸包含编码SEQ ID NO:6或其变体，例如，与其具有至少95％,96％,97％,98％,或99％同一性的氨基酸序列。在一个实施方案中，所述的IL-15Ra编码多核苷酸包含SEQ ID NO:3的核苷酸序列或其变体，例如，与其具有至少95％,96％,97％,98％,或99％同一性的氨基酸序列；或与其在严谨杂交条件杂交的核苷酸序列。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. In one embodiment, 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. In one embodiment, 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多肽及其编码核酸IL-15 polypeptide and its encoding nucleic acid
可用于本发明的IL-15多肽包括但不限于，全长天然IL-15蛋白或其功能性片段，或其变体(包括天然等位基因变体或物种同源物)。在UniProtKB-P40933登录号下给出了来自人的IL-15氨基酸序列。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.
在根据本发明的任何实施方案中，优选地，IL-15多肽包含:i)SEQ ID NO:5的氨基酸序列；ii)对SEQ ID NO:5的氨基酸序列具有至少一个，两个或三个修饰但不超过30,20或10个修饰的氨基酸序列；或iii)与SEQ ID NO:5的氨基酸序列具有至少95-99％同一性的氨基酸序列。In any embodiment according to the invention, preferably, 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.
可用于本发明的IL-15编码多核苷酸，可以是包含编码根据本发明的上述任何实施方案中的IL-15多肽的核苷酸序列的任何多核苷酸。在一个实施方案中，所述的IL-15编码多核苷酸包含编码SEQ ID NO:5 或与其变体，例如，与其具有至少95％,96％,97％,98％,或99％同一性的氨基酸序列的核苷酸序列。在一个实施方案中，所述的IL-15编码多核苷酸包含SEQ ID NO:2的核苷酸序列或其变体，例如，与其具有至少95％,96％,97％,98％,或99％同一性的氨基酸序列；或与其在严谨杂交条件杂交的核苷酸序列。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. In one embodiment, 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. In one embodiment, 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.
在一些实施方案中，当在IL-15多肽的相同细胞中表达本发明的优化IL15Ra多肽时，减少自细胞释放的IL-15量。所述释放的IL-15可以包括IL-15多肽本身、或其与可溶性IL-15Ra(例如，自细胞膜上脱落的可溶性IL-15Ra)形成的异二聚体。在一个实施方案中，所述减少相对于仅表达IL-15而不表达本发明优化IL-15Ra多肽的对照细胞而言。在再一实施方案中，所述对照细胞表达不具有突变S202R和D203E的野生型全长IL-15Ra多肽。In some embodiments, when an optimized IL15Ra polypeptide of the invention is expressed in the same cells as the IL-15 polypeptide, 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). In one embodiment, the reduction is relative to control cells expressing only IL-15 and not the optimized IL-15Ra polypeptide of the invention. In yet another embodiment, the control cells express wild-type full-length IL-15Ra polypeptide without mutations S202R and D203E.
嵌合抗原受体(CAR)多肽及其编码多核苷酸Chimeric antigen receptor (CAR) polypeptides and encoding polynucleotides thereof
可用于本发明的CAR多肽并无特别限制。在一方面，本发明的CAR多肽包含细胞外抗原结合结构域，跨膜结构域，和胞质信号传导结构域。在一个实施方案中，本发明CAR多肽的胞质信号结构域包含初级信号传导结构域。在一个实施方案中，本发明CAR多肽的胞质信号传导结构域包含共刺激结构域和初级信号传导结构域。在一个实施方案中，根据本发明的嵌合抗原受体(CAR)分子从N端到C端包含：由(a)特异性结合肿瘤抗原的抗原结合结构域和(b)铰链区或间隔区；(c)跨膜结构域；和(d)胞质信号传导结构域。在一个实施方案中，根据本发明的CAR分子从N端到C端包含：(a)特异性结合肿瘤抗原的抗原结合结构域，(b)铰链区或间隔区；(c)跨膜结构域；(d)来自CD28和4-1BB的两个共刺激结构域；和(e)来自CD3的初级信号传导结构域。The CAR polypeptides that can be used in the present invention are not particularly limited. In one aspect, a CAR polypeptide of the invention includes an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain. In one embodiment, the cytoplasmic signaling domain of a CAR polypeptide of the invention comprises a primary signaling domain. In one embodiment, the cytoplasmic signaling domain of a CAR polypeptide of the invention comprises a costimulatory domain and a primary signaling domain. In one embodiment, a chimeric antigen receptor (CAR) molecule according to the invention 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. In one embodiment, 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.
在一些实施方案中，用于本发明CAR多肽的靶抗原是靶细胞，尤其是肿瘤细胞上表面表达的膜抗原，例如肿瘤特异性抗原或肿瘤相关抗原。可以提及的肿瘤包括血液性肿瘤和实体肿瘤，包括原发性和转移性肿瘤。在一些实施方案中，靶抗原是包含可以被源自哺乳动物的肿瘤浸润性淋巴细胞(TIL)免疫识别的抗原性癌表位的肿瘤细胞表面抗原。在另一些实施方案中，靶抗原是包含一个或多个与恶性肿瘤相关的抗原性癌表位的肿瘤细胞表面抗原。在优选的实施方案中，本发明CAR分子的胞外抗原结合结构域靶向肿瘤抗原，优选地，所述肿瘤抗原选自：CD19,肾上腺素A2受体(EphA2),叶酸受体(FRa),间皮素，EGFRvIII,IL-13Ra,CD123,CD33,BCMA,GD2,CLL-1,CA-IX,MUC1,HER2，及其任何组合。更优选地，所述肿瘤抗原为膜抗原CD19。In some embodiments, 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. In some embodiments, 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. In other embodiments, the target antigen is a tumor cell surface antigen comprising one or more antigenic cancer epitopes associated with malignancy. In a preferred embodiment, the extracellular antigen-binding domain of the CAR molecule of the present invention targets a tumor antigen. Preferably, 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.
根据所要靶向的抗原，本发明的CAR可被构建以包括对所需抗原靶标特异的适当的抗原结合结构域，以赋予CAR分子以及包含所述CAR分子的CAR-T细胞特异性识别并结合靶抗原的能力。在一个实施方案中，根据本发明的CAR分子的胞外抗原结合结构域是，对靶抗原具有结合亲和力的多肽分子。在一个实施方案中，根据本发明的CAR包含来源于抗体或抗体片段的抗原结合结构域。在再一实施方案中，所述抗原结合结构域包含重链可变区(VH)和轻链可变区(VL)。在优选的实施方案中，所述抗原结合结构域包含由VL和VH经由接头连接而成的scFv。According to the antigen to be targeted, 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. In one embodiment, 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. In one embodiment, the CAR according to the present invention includes an antigen binding domain derived from an antibody or antibody fragment. In another embodiment, the antigen binding domain includes a heavy chain variable region (VH) and a light chain variable region (VL). In a preferred embodiment, the antigen binding domain includes a scFv formed by connecting VL and VH via a joint.
scFv可以根据本领域已知的方法，通过使用柔性多肽接头将VH和VL区连接在一起而产生。在一些实施方案中，scFv分子包含具有优化的长度和/或氨基酸组成的柔性多肽接头。在一些实施方案中，scFv包含位于其VL和VH区之间的接头，其中所述接头包含至少5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40,45,50个或更多个氨基酸残基。接头序列可以包含任何天然存在的氨基酸。在一个实施方案中，scFv的肽接头由单独或组合使用的氨基酸如甘氨酸和/或丝氨酸残基组成，以将可变重链和可变轻链区连接在一起。在一个实施方案中，柔性多肽接头是Gly/Ser接头，并且例如包含氨基酸序列(Gly-Gly-Gly-Ser)n，其中n是等于或大于1的正整数(SEQ ID NO:40)。例如，n＝1,n＝2,n＝3.n＝4,n＝5和n＝6,n＝7,n＝8,n＝9和n＝10。在一个实施方案中，柔性多肽接头包括但不限于(Gly4Ser)4(SEQ ID NO:27)或(Gly4Ser)3(SEQ ID NO:28)。在另一个实施方案中，接头包括(Gly2Ser),(GlySer)或(Gly3Ser)的多个重复 (SEQ ID NO:29)。在再一实施方案中，接头包含GSTSGSGKPGSGEGSTKG氨基酸序列。在一个实施方案中，用于本发明的scFv从N端到C端包含：VL-接头-VH；或VH-接头-VL。scFv can be generated by linking the VH and VL regions together using flexible polypeptide linkers according to methods known in the art. In some embodiments, scFv molecules comprise flexible polypeptide linkers of optimized length and/or amino acid composition. In some embodiments, 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. In one embodiment, 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. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and, for example, comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1 (SEQ ID NO: 40). For example, n=1, n=2, n=3. n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In one embodiment, flexible polypeptide linkers include, but are not limited to, (Gly4Ser)4 (SEQ ID NO:27) or (Gly4Ser)3 (SEQ ID NO:28). In another embodiment, the linker includes multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO:29). In yet another embodiment, the linker comprises the GSTGSSGKPGSGEGSTKG amino acid sequence. In one embodiment, the scFv used in the present invention contains from N-terminus to C-terminus: VL-linker-VH; or VH-linker-VL.
本发明的CAR多肽包含至少一个跨膜结构域，其可以衍生自天然来源或合成来源。例如，跨膜结构域可以衍生自膜结合蛋白或跨膜蛋白，例如来自CD3ζ、CD4、CD28、CD8(例如，CD8α，CD8β)的跨膜结构域。在本发明的嵌合抗原受体(CAR)多肽中，跨膜结构域赋予本发明的CAR多肽的膜附着。在一些实施方案中，本发明的CAR中的跨膜结构域可以借助铰链区/间隔区与CAR的胞外区连接。关于可用于CAR多肽中的跨膜区和铰链区/间隔区，可以参见例如，Kento Fujiwara等，Cells 2020,9,1182；doi:10.3390/cells9051182。The CAR polypeptides of the invention comprise at least one transmembrane domain, which can be derived from natural or synthetic sources. For example, the transmembrane domain may be derived from a membrane-binding or transmembrane protein, such as that from CD3ζ, CD4, CD28, CD8 (eg, CD8α, CD8β). In the chimeric antigen receptor (CAR) polypeptides of the invention, the transmembrane domain confers membrane attachment to the CAR polypeptide of the invention. In some embodiments, 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. Regarding the 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.
本发明的CAR多肽中包含的胞质信号传导结构域至少包含初级信号结构域。初级信号结构域能够活化引入了本发明CAR的免疫细胞的至少一个免疫效应功能。所述的免疫效应功能包括但不限于，例如增强或促进免疫攻击靶细胞的功能或应答。T细胞的效应功能例如可以是溶细胞活性或辅助活性，包括分泌细胞因子。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.
用于本发明CAR多肽中的胞质信号传导结构域的例子包括，可以发挥作用以在胞外结构域结合靶抗原后启动信号转导的T细胞受体(TCR)和/或共受体的胞质序列，以及这些序列的任何衍生物或变体和具有相同功能性能力的任何重组序列。T细胞的活化由两类不同的胞质信号传导序列介导：通过TCR启动抗原依赖性初级活化的那些序列(即，初级信号结构域)和以抗原非依赖性方式发挥作用以提供共刺激信号的那些序列(即，次级胞质结构域，例如，共刺激结构域)。在一个实施方案中，本发明的CAR多肽包含提供初级信号结构域的胞质结构域，例如，CD3ζ的胞内初级信号结构域。在另一实施方案中，本发明CAR多肽的胞质结构域还包含次级信号结构域，例如，来自共刺激分子的共刺激结构域。在一个实施方案中，本发明CAR多肽的胞质区包含与CD3ζ胞内信号传导结构域串联的一个或多个(尤其是两个)共刺激结构域，如4-1BB(也称为CD137)和CD28的共刺激结构域的组合。Examples of 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. Cytoplasmic sequences, as well as any derivatives or variants of these sequences and any recombinant sequences having the same functional capabilities. Activation of T cells is mediated by two distinct classes of 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). In one embodiment, a CAR polypeptide of the invention comprises a cytoplasmic domain that provides a primary signaling domain, e.g., the intracellular primary signaling domain of CD3ζ. In another embodiment, 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. In one embodiment, the cytoplasmic region of the CAR polypeptide of the invention comprises one or more (especially two) costimulatory domains in tandem with the CD3ζ intracellular signaling domain, such as 4-1BB (also known as CD137) and the costimulatory domain of CD28.
在一些实施方案中，本发明的CAR多肽可以包含位于胞外抗原结合结构域N端的信号肽或前导序列。通过信号肽/前导序列，新生的CAR多肽可以被引导到细胞的内质网，并之后锚定在细胞膜上。可以使用任何真核来源的信号肽/前导序列，例如哺乳动物或人分泌蛋白来源的信号肽/前导序列。In some embodiments, 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.
在一些实施方案中，根据本发明的嵌合抗原受体(CAR)多肽包含细胞外抗原结合结构域，跨膜结构域，和胞质信号传导结构域。In some embodiments, chimeric antigen receptor (CAR) polypeptides according to the invention comprise an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain.
在一个实施方案中，抗原结合结构域是靶向肿瘤抗原的抗原结合结构域。在一个实施方案中，所述肿瘤抗原为膜抗原，例如CD19或EphA2，且优选CD19。在一个实施方案中，细胞外抗原结合结构域是结合CD19的抗原结合结构域。在一个实施方案中，细胞外抗原结合结构域包含结合CD19的鼠、人或人源化抗原结合结构域。在一个实施方案中，结合CD19的抗原结合结构域包含:SEQ ID NO:9的重链可变区(VH)氨基酸序列的重链互补决定区1(HC CDR1)，重链互补决定区2(HC CDR2)和重链互补决定区3(HC CDR3)；和/或SEQ ID NO:8的轻链可变区(VL)氨基酸序列的轻链互补决定区1(LC CDR1)，轻链互补决定区2(LC CDR2)和轻链互补决定区3(LC CDR3)。在一个实施方案中，所述抗原结合结构域包含重链可变区和轻链可变区，其中，In one embodiment, the antigen binding domain is one that targets a tumor antigen. In one embodiment, the tumor antigen is a membrane antigen, such as CD19 or EphA2, and preferably CD19. In one embodiment, the extracellular antigen binding domain is an antigen binding domain that binds CD19. In one embodiment, the extracellular antigen binding domain comprises a murine, human or humanized antigen binding domain that binds CD19. In one embodiment, 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). In one embodiment, the antigen binding domain comprises a heavy chain variable region and a light chain variable region, wherein,
所述重链可变区包含：i)SEQ ID NO:9的氨基酸序列；ii)对SEQ ID NO:9的氨基酸序列具有至少一个，两个或三个修饰但不超过30,20或10个修饰的氨基酸序列；或iii)与SEQ ID NO:9的重链可变区氨基酸序列具有95-99％同一性的氨基酸序列；和/或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
所述轻链可变区包含:i)SEQ ID NO:8的氨基酸序列；ii)对SEQ ID NO:8的氨基酸序列具有至少一个，两个或三个修饰但不超过30,20或10个修饰的氨基酸序列；或iii)与SEQ ID NO:8的重链可变区氨基酸序列具有95-99％同一性的氨基酸序列。 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.
在一个实施方案中，所述抗原结合结构域包含:i)SEQ ID NO:11的氨基酸序列；ii)对SEQ ID NO:11具有至少一个，两个或三个修饰但不超过30,20或10个修饰的氨基酸序列；或iii)与SEQ ID NO：11具有95-99％同一性的氨基酸序列。In one embodiment, 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.
在一个实施方案中，跨膜结构域包含选自以下的蛋白质的跨膜结构域:CD4,CD8α，CD28，CD3ζ，TCRζ，FcRγ，FcRβ，CD3γ，CD3δ，CD3ε，CD5，CD9,CD16，CD22，CD79a，CD79b，CD278(也称为“ICOS”)，FcεRI，CD66d，T细胞受体的α，β或ζ链，MHC I类分子，TNF受体蛋白，免疫球蛋白样蛋白，细胞因子受体，整联蛋白，和激活NK细胞受体。在一个实施方案中，跨膜结构域包含选自以下的蛋白质的跨膜结构域:CD4,CD8α，CD28和CD3ζ。在一个实施方案中，跨膜结构域包含:i)SEQ ID NO:13的氨基酸序列；ii)包含SEQ ID NO:13的氨基酸序列的至少一个，两个或三个修饰但不超过5个修饰的氨基酸序列；或iii)与SEQ ID NO:13具有95-99％序列同一性的氨基酸序列。In one embodiment, the transmembrane domain comprises a transmembrane domain of a protein selected from: CD4, CD8α, CD28, CD3ζ, TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD9, CD16, CD22, CD79a, CD79b, CD278 (also known as "ICOS"), FcεRI, 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. In one embodiment, the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of CD4, CD8α, CD28 and CD3ζ. In one embodiment, 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.
在一个实施方案中，胞质信号传导结构域包含选自以下的蛋白质的功能性信号传导结构域:TCRζ,FcRγ，FcRβ,CD3γ,CD3δ,CD3ε,CD5，CD22,CD79a,CD79b或CD66d。在一个实施方案中，胞质信号传导结构域包含CD3ζ蛋白的功能信号传导结构域(在本文中也称作，CD3ζ初级信号传导结构域)。在一个实施方案中，胞质信号传导结构域包含:i)SEQ ID NO:16的氨基酸序列；ii)包含SEQ ID NO:16的氨基酸序列的至少一个，两个或三个修饰但不超过20个，10个或5个修饰的氨基酸序列；或iii)与SEQ ID NO:15具有95-99％序列同一性的氨基酸序列。In one embodiment, the cytoplasmic signaling domain comprises a functional signaling domain of a protein selected from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, or CD66d. In one embodiment, the cytoplasmic signaling domain comprises the functional signaling domain of the CD3ζ protein (also referred to herein as the CD3ζ primary signaling domain). In one embodiment, 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.
在一个实施方案中，胞质信号传导结构域进一步包含选自以下的一个或多个蛋白质的共刺激结构域：MHC I类分子、TNF受体蛋白、免疫球蛋白样蛋白、细胞因子受体、整联蛋白、信号传导淋巴细胞活化分子(SLAM蛋白)、激活NK细胞受体、CD8、ICOS，DAP10,DAP12,OX40、CD40、GITR、4-1BB(即CD137)、CD27和CD28。在一个实施方案中，胞质信号传导结构域包含选自以下的一个或两个蛋白质的共刺激结构域：CD28,CD27,4-1BB,ICOS和OX40的共刺激结构域。在一个实施方案中，胞质信号传导结构域包含选自以下的蛋白质的共刺激结构域：CD28和4-1BB(即CD137)，或其组合。在一个实施方案中，胞质信号传导结构域包含CD28共刺激结构域和4-1BB共刺激结构域，其中优选地，所述CD28共刺激结构域包含:i)SEQ ID NO:14的氨基酸序列；ii)包含SEQ ID NO:14的氨基酸序列的至少一个，两个或三个修饰但不超过20个，10个或5个修饰的氨基酸序列；或iii)与SEQ ID NO:14的氨基酸序列具有95-99％同一性的氨基酸序列；且优选地，所述4-1BB共刺激结构域包含:i)SEQ ID NO:15的氨基酸序列；ii)包含SEQ ID NO:15的氨基酸序列的至少一个，两个或三个修饰但不超过20个，10个或5个修饰的氨基酸序列；或iii)与SEQ ID NO:15的氨基酸序列具有95-99％同一性的氨基酸序列。In one embodiment, 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. In one embodiment, 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. In one embodiment, 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. In one embodiment, 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 ID NO: 15; or iii) an amino acid sequence with 95-99% identity with the amino acid sequence of SEQ ID NO: 15.
在一个优选的实施方案中，CAR多肽的胞质信号传导结构域包含：包含来自CD28和4-1BB的共刺激信号和来自CD3ζ的初级信号传导结构域。在一个实施方案中，所述胞质信号传导结构域包含：i)SEQ ID NO:1的氨基酸序列；ii)包含SEQ ID NO:1的氨基酸序列的至少一个，两个或三个修饰但不超过20个，10个或5个修饰的氨基酸序列；或iii)与SEQ ID NO:1的氨基酸序列具有95-99％同一性的氨基酸序列。In a preferred embodiment, the cytoplasmic signaling domain of the CAR polypeptide comprises costimulatory signals from CD28 and 4-1BB and a primary signaling domain from CD3ζ. In one embodiment, 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.
在一个实施方案中，CAR多肽包含跨膜结构域和细胞外抗原结合结构域，并且还包含置于所述跨膜结构域和所述细胞外抗原结合结构域之间的铰链区或间隔区。在一个实施方案中，铰链区/间隔区选自GS铰链，CD8铰链，IgG4铰链，IgD铰链，CD16铰链，和CD64铰链。在一个实施方案中，CAR多肽包含来自CD28胞外区的铰链区。在一个实施方案中，铰链区/间隔区包含:i)SEQ ID NO:12的氨基酸序列；ii)包含SEQ ID NO:12的氨基酸序列的至少一个，两个或三个修饰但不超过5个修饰的氨基酸序列；或iii)与SEQ ID NO:12的氨基酸序列具有95-99％同一性的氨基酸序列。在本文中，表述“铰链”、“铰链区”和“铰链结构域”可互换使用。 In one embodiment, 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. In one embodiment, 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. In one embodiment, the CAR polypeptide comprises a hinge region from the extracellular region of CD28. In one embodiment, 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. In this document, the expressions "hinge", "hinge region" and "hinge domain" are used interchangeably.
在一个优选的实施方案中，本发明的CAR多肽包含：(a)抗原结合结构域；(b)铰链区/间隔区；(c)跨膜结构域；(d)来自CD28和4-1BB的共刺激结构域；和(e)来自CD3ζ的初级信号传导结构域。In a preferred embodiment, 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 CD3ζ.
在一个实施方案中，CAR多肽还包含前导肽或信号肽，例如来自人粒细胞-巨噬细胞集落刺激因子受体α链(GM-CSFRα)的信号肽。在一个实施方案中，CAR多肽包含具有SEQ ID NO:7的氨基酸序列的信号肽。In one embodiment, 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-CSFRα). In one embodiment, the CAR polypeptide comprises a signal peptide having an amino acid sequence of SEQ ID NO:7.
在一个实施方案中，根据本发明的CAR多肽包含:i)SEQ ID NO:4的氨基酸序列；ii)对SEQ ID NO:4的氨基酸序列具有至少一个，两个或三个修饰但不超过30,20或10个修饰的氨基酸序列；或iii)与SEQ ID NO:4的氨基酸序列具有至少95-99％同一性的氨基酸序列。In one embodiment, 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.
可用于本发明的CAR编码核酸，可以是包含编码根据本发明的上述任何实施方案的CAR多肽的核苷酸序列的任何多核苷酸。在一个实施方案中，所述的CAR编码核酸包含编码SEQ ID NO:4或其变体，例如，与其具有至少95％,96％,97％,98％,或99％同一性的氨基酸序列的核苷酸序列。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. In one embodiment, 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.
本发明的核酸构建体Nucleic acid construct of the invention
在一个方面，本发明提供了包含编码根据本发明的CAR多肽的多核苷酸、编码根据本发明的IL-15多肽的多核苷酸、和编码根据本发明的IL-15Ra多肽的多核苷酸中一者、两者或全部三者的一个或多个核酸构建体。在一个实施方案中，编码根据本发明的CAR多肽的多核苷酸，编码根据本发明的IL-15多肽的多核苷酸、与编码根据本发明的优化IL-15Ra多肽的多核苷酸，分别位于三个不同的核酸构建体上。在一个实施方案中，编码根据本发明的CAR多肽的多核苷酸，编码根据本发明的IL-15多肽的多核苷酸、与编码根据本发明的优化IL-15Ra多肽的多核苷酸，在单一一个核酸构建体中提供。In one aspect, 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. One or more nucleic acid constructs of one, two, or all three. In one embodiment, 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. In one embodiment, 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.
在一个实施方案中，根据本发明的核酸构建体是包含表达盒的质粒或病毒载体。在一个实施方案中，编码根据本发明的CAR多肽的多核苷酸、编码根据本发明的IL-15多肽的多核苷酸、和编码根据本发明的IL-15Ra多肽的多核苷酸，存在于单一一个核酸构建体上，并分别位于不同的表达盒中置于相同或不同的启动子控制下。在一个实施方案中，编码根据本发明的CAR多肽的多核苷酸、编码根据本发明的IL-15多肽的多核苷酸、和编码根据本发明的IL-15Ra多肽的多核苷酸，以多顺反子形式，存在于单一一个表达盒中。In one embodiment, the nucleic acid construct according to the invention is a plasmid or viral vector comprising an expression cassette. In one embodiment, 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. In one embodiment, 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. Antisubforms, existing in a single expression box.
在一个实施方案中，编码根据本发明的CAR多肽的多核苷酸包含编码根据本发明前述任何实施方案的CAR多肽的多核苷酸，尤其是编码靶向CD19的CAR多肽的多核苷酸。In one embodiment, 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.
在一个实施方案中，编码根据本发明的IL-15多肽的多核苷酸包含编码根据本发明前述任何实施方案的IL-15的多核苷酸。在一个实施方案中，所述IL-15编码多核苷酸编码SEQ ID NO:5的氨基酸序列。在一个实施方案中，所述IL-15编码多核苷酸包含：i)SEQ ID NO:2的核苷酸序列；ii)与SEQ ID NO:2的核苷酸序列在严谨杂交条件下杂交的核苷酸序列；或iii)与SEQ ID NO:2的核苷酸序列具有至少90-99％同一性的核苷酸序列。In one embodiment, 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. In one embodiment, the IL-15 encoding polynucleotide encodes the amino acid sequence of SEQ ID NO:5. In one embodiment, 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.
在一个实施方案中，编码根据本发明的IL-15Ra多肽的多核苷酸包含编码根据本发明前述任何实施方案的IL-15Ra的多核苷酸。在一个实施方案中，所述IL-15Ra编码多核苷酸编码SEQ ID NO:6的氨基酸序列。在一个实施方案中，所述IL-15Ra编码多核苷酸包含：i)SEQ ID NO:3的核苷酸序列；ii)与SEQ ID NO:3的核苷酸序列在严谨杂交条件下杂交的核苷酸序列；或iii)与SEQ ID NO:2的核苷酸序列具有至少90-99％同一性的核苷酸序列。In one embodiment, 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. In one embodiment, the IL-15Ra encoding polynucleotide encodes the amino acid sequence of SEQ ID NO: 6. In one embodiment, 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.
在一个实施方案中，所述的CAR多肽、IL-15及IL-15Ra多肽分别单独地自根据本发明的核酸构建体表达。在另一实施方案中，自根据本发明的核酸构建体表达产生包含所述CAR多肽、IL-15及IL-15Ra多肽的融合多肽，其中，所述融合多肽包含置于表达的所述CAR多肽、IL-15及IL-15Ra多肽之间的可剪切连接肽，由此在细胞中表达后所述融合多肽可以被断裂以产生单独的所述CAR多肽、IL-15及IL-15Ra多 肽。在一些实施方案中，编码IL-15或IL-15Ra多肽的多核苷酸，在其一端，使用自剪切肽，以符合读框的方式，与编码CAR多肽的多核苷酸遗传融合；且在另一端，使用自剪切肽，以符合读框的方式，与编码IL-15Ra或IL-15多肽的多核苷酸遗传融合。In one embodiment, the CAR polypeptide, IL-15 and IL-15Ra polypeptide are each individually expressed from the nucleic acid construct according to the invention. In another embodiment, 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. Peptides. In some embodiments, 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.
在一个实施方案中，根据本发明的核酸构建体包含编码具有式(I)结构的融合多肽的多核苷酸：
CAR-(L1)-E1-(L2)-E2   (I)In one embodiment, 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)
其中，CAR，L1，L2，E1和E2如上所定义。Among them, CAR, L1, L2, E1 and E2 are as defined above.
优选地，L1和L2包含自剪切位点。可以用于本发明的自剪切位点包括，但不限于，包含P2A,T2A，E2A或F2A自剪接位点。关于2A自剪切位点的序列和应用，可以参见Jin Hee Kim等,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。Preferably, 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.
在根据本发明的一些实施方案中，优选地，所述L1包含P2A位点且所述L2包含T2A位点。在一个实施方案中，P2A位点包含:i)SEQ ID NO:17的氨基酸序列；ii)对SEQ ID NO:17的氨基酸序列具有至少一个，两个或三个修饰但不超过5个修饰的氨基酸序列；或iii)与SEQ ID NO:17的氨基酸序列具有至少95％,96％,97％,98％,或99％同一性的氨基酸序列。在一个实施方案中，T2A位点包含:i)SEQ ID NO:18的氨基酸序列；ii)对SEQ ID NO:18的氨基酸序列具有至少一个，两个或三个修饰但不超过5个修饰的氨基酸序列；或iii)与SEQ ID NO:18的氨基酸序列具有至少95％,96％,97％,98％,或99％同一性的氨基酸序列。在一个实施方案中，在2A肽的N端可以插入GSG接头以进一步改善其切割效率。In some embodiments according to the present invention, preferably, the L1 comprises a P2A site and the L2 comprises a T2A site. In one embodiment, 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. In one embodiment, 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. In one embodiment, a GSG linker may be inserted at the N-terminus of the 2A peptide to further improve its cleavage efficiency.
因此，再一方面，本发明也提供了根据式(I)的融合多肽，优选地所述L1和L2可以包含根据前述的任何自剪接2A位点。在所述式(I)结构的各组件之间可以直接地，或间接地通过接头(例如，单氨基酸残基或短肽)功能性连接。Therefore, in yet another aspect, 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).
在一些实施方案中，为实现本发明核酸组合在转移到细胞中后的表达，本发明核酸构建体包含与编码根据本发明的CAR多肽的多核苷酸、编码根据本发明的IL-15多肽的多核苷酸、和编码根据本发明的IL-15Ra多肽的多核苷酸有效连接的启动子。在一些实施方案中，所述核酸构建体是载体。可适用于本发明的载体包括任何适合在真核生物中复制和整合的载体；且含有用于调节所需核酸序列的表达的转录和翻译终止子、起始序列和启动子。In some embodiments, in order to realize the expression of the nucleic acid combination of the present invention after being transferred into cells, 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. In some embodiments, 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.
已经开发了众多基于病毒的系统用于转移基因至哺乳动物细胞中。例如，逆转录病毒载体提供了用于基因递送系统的便利平台。可以使用本领域已知的技术，将本发明核酸组合插入载体并且包装在逆转录病毒粒子中。随后可以分离重组病毒并将其在体内或离体递送至受试者的细胞。众多逆转录病毒系统是本领域已知的。在一些实施方案中，使用慢病毒载体。例如，将本发明核酸组合的核酸序列克隆至慢病毒载体中，以便Numerous virus-based systems have been developed for gene transfer into mammalian cells. For example, 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. In some embodiments, lentiviral vectors are used. For example, the nucleic acid sequence of the nucleic acid combination of the invention is cloned into a lentiviral vector, so that
(i)在单个编码框中产生全长CAR多肽、在单个编码框中产生IL-15多肽、和在单个编码框中产生优化IL-15Ra多肽，或(i) producing a full-length CAR polypeptide in a single coding frame, producing an IL-15 polypeptide in a single coding frame, and producing an optimized IL-15Ra polypeptide in a single coding frame, or
(ii)在单个编码框中产生全长CAR多肽和在单个编码框中产生通过自剪接肽连接的IL-15多肽和IL-15Ra多肽；或(ii) producing a full-length CAR polypeptide in a single coding frame and producing an IL-15 polypeptide and an IL-15Ra polypeptide linked by a self-splicing peptide in a single coding frame; or
(ii)在单个编码框中产生包含通过自剪接肽连接的全长CAR多肽、IL-15多肽和IL-15Ra多肽的融合多肽。(ii) Generating a fusion polypeptide comprising a full-length CAR polypeptide, an IL-15 polypeptide and an IL-15Ra polypeptide linked by a self-splicing peptide in a single coding frame.
衍生自逆转录病毒(如慢病毒)的载体是实现长期基因转移的合适工具，因为它们允许转基因的长期、稳定整合和其在子代细胞中增殖。慢病毒载体具有胜过衍生自癌-逆转录病毒(如鼠白血病病毒)的载体的额外优点，因为它们可以转导非增殖性细胞，如肝细胞。它们还具有额外的低免疫原性优点。逆转录病 毒载体也可以例如是γ逆转录病毒载体。γ逆转录病毒载体可以例如包含启动子、包装信号(ψ)、引物结合位点(PBS)、一个或多个(例如，两个)长末端重复序列(LTR)和目的转基因，例如，编码CAR的基因。γ逆转录病毒载体可以缺少病毒结构性基因如gag、pol和env。Vectors derived from retroviruses (e.g., lentiviruses) are suitable tools for long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in progeny cells. 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 (ψ), 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.
能够在哺乳动物T细胞中表达转基因的启动子的例子是EF1a启动子。天然EF1a启动子驱动延伸因子-1复合体的α亚基表达，所述α亚基负责酶促递送氨酰基tRNA至核糖体。已经在哺乳动物表达质粒中广泛使用了EF1a启动子并且已经显示有效驱动从克隆至慢病毒载体中的转基因表达。参见，例如，Milone等人,Mol.Ther.17(8):1453–1464(2009)。An example of 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).
启动子的另一个例子是立即早期巨细胞病毒(CMV)启动子序列。这个启动子序列是能够驱动与之有效连接的任何多核苷酸序列高水平表达的组成型强启动子序列。但是，也可以使用其他组成型启动子序列，所述其他组成型启动子序列包括但不限于猴病毒40(SV40)早期启动子、小鼠乳腺瘤病毒(MMTV)、人类免疫缺陷病毒(HIV)长末端重复序列(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、埃巴病毒立即早期启动子、劳斯肉瘤病毒启动子以及人基因启动子，如但不限于肌动蛋白启动子、肌球蛋白启动子、延伸因子-1α启动子、血红蛋白启动子和肌酸激酶启动子。另外，本发明不应当限于使用组成型启动子。还构思了诱导型启动子作为本发明的部分。Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a constitutively strong promoter sequence capable of driving high-level expression of any polynucleotide sequence to which it is operably linked. However, 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 promoters, such as but not limited to the actin promoter , myosin promoter, elongation factor-1α 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.
III.包含本发明核酸组合的细胞III. Cells containing nucleic acid combinations of the invention
本发明还提供了引入了本发明核酸组合或本发明的核酸构建体的细胞。可以通过本领域任何已知的核酸转移方法，将本发明的核酸组合或本发明的核酸构建体引入细胞。在一个实施方案中，所述细胞是哺乳动物细胞，例如免疫效应细胞。在一个实施方案中，所述细胞是装甲CAR-T细胞，其包含根据本发明的IL-15和优化IL-15Ra多肽以及CAR多肽。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. In one embodiment, the cells are mammalian cells, such as immune effector cells. In one embodiment, 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.
在一些实施方案中，因此，本发明也提供了在哺乳动物免疫效应细胞(例如哺乳动物T细胞或哺乳动物NK细胞)中引入并表达本发明的核酸组合和由此产生免疫效应细胞，尤其是根据本发明的装甲CAR-T细胞的方法。在一些实施方案中，本发明也提供了通过所述方法可获得的免疫效应细胞，尤其是根据本发明的装甲CAR-T细胞。In some embodiments, therefore, 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. In some embodiments, the invention also provides immune effector cells obtainable by said method, especially armored CAR-T cells according to the invention.
在一些实施方案中，为产生根据本发明的免疫效应细胞，从受试者获得细胞来源，例如，免疫效应细胞，例如，T细胞或NK细胞。术语“受试者”意在包括可以激发免疫应答的活生物(例如，哺乳动物)。可以从众多来源获得T细胞，包括外周血单个核细胞、骨髓、淋巴结组织、脐带血、胸腺组织、来自感染部位的组织、腹水、胸腔积液、脾组织和肿瘤。In some embodiments, to produce immune effector cells according to the present invention, a cell source, e.g., immune effector cells, e.g., T cells or NK cells, is obtained from a subject. The term "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.
可以使用本领域技术人员已知的任何技术(如FicollTM分离法)，从采集自受试者的血液成分中获得T细胞。在一个优选的方面，通过单采血液成分术获得来自个体循环血液的细胞。单采产物一般含有淋巴细胞，包括T细胞、单核细胞、粒细胞、B细胞、其他有核的白细胞、红细胞和血小板。在一个实施方案中，可以洗涤通过单采血液成分术采集的细胞，以除去血浆级分并且以在用于后续加工步骤的适宜缓冲液或培养基中放置细胞。在本发明的一个方面，用磷酸盐缓冲盐水(PBS)洗涤细胞。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 ™ isolation. In a preferred aspect, 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. In one embodiment, 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. In one aspect of the invention, cells are washed with phosphate buffered saline (PBS).
可以通过正向或负向选择技术进一步分离特定的T细胞亚群，如CD3+、CD28+、CD4+、CD8+、CD45RA+和CD45RO+T细胞。例如，在一个实施方案中，通过与抗CD3/抗CD28缀合的珠(如M-450CD3/CD28T)温育一段足够正向选择所需T细胞的时间，分离T细胞。在一些实施方案中，该时间段是约30分钟至36小时之间或更长时间。较长的温育时间可以用来在存在少量T细胞的任何情况下分离T细胞，如用于从肿瘤组织或从免疫受损个体分离肿瘤浸润型淋巴细胞(TIL)。另外，使用较长的温育时间可以增加捕获CD8+T细胞的效率。因此，通过简单地缩短或延长该时间，允许T细胞与CD3/CD28珠结合和/或通过增加或减少珠对T细胞的比率，可以在培养伊始或在培养过程期间的其他时间点偏好地选择T细胞亚群。 Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated through positive or negative selection techniques. For example, in one embodiment, by conjugating beads with anti-CD3/anti-CD28 (e.g. M-450CD3/CD28T) was incubated for a period of time sufficient to positively select the desired T cells, and the T cells were isolated. In some embodiments, 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. Additionally, using longer incubation times can increase the efficiency of capturing CD8+ T cells. Thus, by simply shortening or extending this time, allowing T cells to bind to CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells, one can preferentially select at the beginning of the culture or at other time points during the culture process T cell subsets.
可以用抗体的组合，通过负选择过程完成T细胞群体的富集，其中所述抗体针对负向选择的细胞独有的表面标志物。一种方法是借助负向磁力免疫粘附法或流式细胞术分选和/或选择细胞，所述负向磁力免疫粘附法或流式细胞术使用针对负向选择的细胞上存在的细胞表面标志物的单克隆抗体混合物。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.
在一些实施方案中，免疫效应细胞可以是同种异体免疫效应细胞，例如，T细胞或NK细胞。例如，细胞可以是同种异体T细胞，例如，缺少功能性T细胞受体(TCR)和/或人白细胞抗原(HLA)(例如，HLA I类和/或HLA II类)表达的同种异体T细胞。In some embodiments, the immune effector cells may be allogeneic immune effector cells, such as T cells or NK cells. For example, 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. T cells.
在一些实施方案中，所述免疫效应细胞是根据本发明的装甲CAR-T细胞。在一个实施方案中，所述装甲CAR-T细胞表达根据式(I)的融合多肽，且任选地所述融合多肽在表达后自剪接产生彼此分离的本发明CAR多肽、IL-15多肽和优化IL-15Ra多肽。在一个实施方案中，根据本发明的装甲CAR-T细胞具有至少一项如下性质：In some embodiments, the immune effector cells are armored CAR-T cells according to the invention. In one embodiment, 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. In one embodiment, the armored CAR-T cells according to the invention have at least one of the following properties:
-相比仅表达所述CAR多肽的对照CAR-T细胞，具有增强的增殖能力和细胞活力，以及增加的Tscm表型T细胞亚群比例；-Have enhanced proliferation ability and cell viability, as well as an increased proportion of Tscm phenotype T cell subsets compared to control CAR-T cells that only express the CAR polypeptide;
-相比仅表达所述CAR多肽和所述IL-15的对照CAR-T细胞，减少IL-15在细胞外环境中的释放量，和由此具有降低的由IL-15诱导的毒性；和- reduce the amount of IL-15 released in the extracellular environment, and thereby have reduced toxicity induced by IL-15, compared to control CAR-T cells expressing only the CAR polypeptide and the IL-15; and
-体内抗肿瘤功效和减少的CAR-T治疗中与IL-15相关的不良事件。- In vivo anti-tumor efficacy and reduced IL-15-related adverse events in CAR-T therapy.
IV.本发明免疫效应细胞的用途和使用本发明免疫效应细胞的治疗方法IV. Uses of the immune effector cells of the present invention and therapeutic methods using the immune effector cells of the present invention
T细胞治疗首次应用于血液学B细胞恶性肿瘤的治疗，并显示出有效和令人鼓舞的结果。然而，CAR-T细胞治疗的抗肿瘤活性受限于CAR-T细胞的有限持久性。为了满足CAR T细胞在多种癌症中的应用，急需能有效调整CAR-T细胞的体外和体内持久性的技术手段。为此提出了在CAR-T细胞中重组表达IL-15的方式。然而，释放的血清中的活性IL-15具有诱导毒性的可能性。为了保证CAR-T细胞的持久性并最小化IL-15相关毒性或副作用，通过深入研究，本发明人提出，通过在CAR-T细胞中同时表达IL-15以及本发明的优化IL-15Ra，由此获得了包含更多TCSM细胞亚群且具有降低的毒性的本发明装甲CAR-T细胞，以利于所述CAR-T细胞在受试者中的抗肿瘤治疗应用。T-cell therapy was applied for the first time in the treatment of hematological B-cell malignancies and showed effective and encouraging results. However, the antitumor activity of CAR-T cell therapy is limited by the limited persistence of CAR-T cells. In order to meet the application of CAR T cells in various cancers, technical means that can effectively adjust the persistence of CAR-T cells in vitro and in vivo are urgently needed. To this end, a method of recombinantly expressing IL-15 in CAR-T cells has been proposed. However, the released active IL-15 in serum has the potential to induce toxicity. In order to ensure the persistence of CAR-T cells and minimize IL-15-related toxicity or side effects, through in-depth research, the inventors proposed that by simultaneously expressing IL-15 and the optimized IL-15Ra of the present invention in CAR-T cells, Thus, armored CAR-T cells of the present invention containing more TCSM cell subpopulations and with reduced toxicity are obtained, so as to facilitate the anti-tumor therapeutic application of the CAR-T cells in subjects.
在一些实施方案中，工程化改造免疫效应细胞，例如T细胞(例如患者特异的自体T细胞)以引入本发明的核酸组合或载体，由此在所述细胞中异源共表达本发明的CAR多肽、IL-15和优化IL-15Ra多肽。通过扩增所述工程化改造的免疫效应细胞(例如T细胞或NK细胞)后，可将其用于过继细胞治疗(ACT)。In some embodiments, 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).
在一些实施方案中，在利用本发明免疫效应细胞治疗患者时，所述免疫效应细胞可以是自体或同种异体T细胞或NK细胞。在一些实施方案中，相比于使用无异源IL-15和IL-15Ra表达的对照CAR-T或CAR-NK细胞，本发明免疫效应细胞可以提高过继性转移后细胞的长期存活和/或TSCM亚群占比。In some embodiments, when treating a patient with immune effector cells of the invention, the immune effector cells 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.
在一个实施方案中，本发明免疫效应细胞用于在受试者中治疗癌症，并且能够减轻癌症的至少一种症状或指征的严重性或抑制癌细胞生长。In one embodiment, 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.
本发明提供了在受试者中治疗癌症的方法，其包括向有需要的个体施用治疗有效量的表达本发明核酸组合的免疫效应细胞。本发明也提供了前述的本发明免疫效应细胞在制备用于治疗癌症的药物中的用途。所述癌症包括血液学癌症(例如白血病)或者实体瘤(例如，胶质瘤)，包括原发性和转移性癌症。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.
本发明所述的各个实施方案/技术方案以及各个实施方案/技术方案中的特征应当被理解为可以任意进行相互组合，这些相互组合得到的各个方案均包括在本发明的范围内，就如同在本文中具体地且逐一地列出了这些相互组合而得到的方案一样，除非上下文清楚地显示并非如此。It should be understood that the various embodiments/technical solutions and the features of each embodiment/technical solution described in the present invention can be combined with each other arbitrarily, and the various solutions obtained by these mutual combinations are included in the scope of the present invention, just as in These are specifically and individually listed herein as the same as the resulting combinations, unless the context clearly indicates otherwise.
描述以下实施例以辅助对本发明的理解。不意在且不应当以任何方式将实施例解释成对本发明的保护范围的限制。The following examples are described to aid understanding of the invention. The examples are not intended and should not be construed in any way as limiting the scope of the invention.
实施例 Example
材料和方法Materials and methods
细胞系cell lines
人NALM-6细胞系和逆转录病毒包装细胞系PG13购自美国典型培养物保藏中心(ATCC)。由逆转录病毒感染产生表达增强型GFP的NAML-6-eGFP细胞。NAML-6-eGFP细胞维持在含有10％胎牛血清(Biosera)和10,000IU/mL青霉素/10,000μg/mL链霉素(EallBio Life Sciences)的RPMI-1640(Lonza)中。所有细胞均在37℃、5％CO2、95％空气中在加湿培养箱中培养。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 μg/mL streptomycin (EallBio Life Sciences). All cells were cultured in a humidified incubator at 37°C, 5% CO2 , 95% air.
生成编码CD19特异性CAR的逆转录病毒载体Generation of retroviral vector encoding CD19-specific CAR
由生物公司合成三代CD19-CAR基因，其中IL-15基因的核苷酸序列如SEQ ID NO：2所示和优化的IL-15Ra基因的核苷酸序列如SEQ ID NO：3所示。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.
将合成的上述核苷酸序列，通过同源重组(ClonExpress II One Step Cloning kit)的方法，按照图1所示，亚克隆到载体SFG载体(addgene)上，构建产生构建体CD19-CAR、CD19-CAR-IL15、和CD19-CAR-IL15-IL15Ra。The above synthesized nucleotide sequence was subcloned into the vector SFG vector (addgene) through the method of homologous recombination (ClonExpress II One Step Cloning kit) as shown in Figure 1 to construct the constructs CD19-CAR, CD19 -CAR-IL15, and CD19-CAR-IL15-IL15Ra.
三代CD19-CAR基因表达的蛋白，其氨基酸序列(SEQ ID NO：4)如下所示，其从N端到C端包含，信号肽(粗体下划线)、CD19scFv、短连接肽(粗体下划线)、CD28间隔区/跨膜区(斜体)、CD28共刺激结构域(下划线)、4-1BB共刺激结构域(加框粗斜体)、和CD3ζ信号传导结构域：
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 CD3ζ signaling domain:
IL-15基因的核苷酸序列(SEQ ID NO：2)如下：
The nucleotide sequence of the IL-15 gene (SEQ ID NO: 2) is as follows:
IL-15基因表达的蛋白，其氨基酸序列(SEQ ID NO：5)如下所示：
The protein expressed by the IL-15 gene has the amino acid sequence (SEQ ID NO: 5) as follows:
优化IL-15Ra基因的核苷酸序列(SEQ ID NO：3)如下：

The optimized nucleotide sequence of the IL-15Ra gene (SEQ ID NO: 3) is as follows:

优化IL-15Ra基因表达的蛋白(SEQ ID NO：6)如下：
The protein (SEQ ID NO: 6) optimized for IL-15Ra gene expression is as follows:
构建的所有CAR构建体均通过测序验证。在瞬时转染后，应用PG13细胞产生逆转录病毒颗粒。All CAR constructs constructed were verified by sequencing.After transient transfection, PG13 cells were used to produce retroviral particles.
产生CAR-T细胞Generate CAR-T cells
通过Lymphoprep(MP Biomedicals)梯度离心，自健康供体的外周血单个核细胞(PBMC)分离原代T细胞。为了产生抗原特异性的转基因T细胞，PBMC中的T细胞用抗CD3和抗CD28珠刺激，然后用逆转录病毒感染。感染7天后，对CAR-T细胞进行CAR表达检测，然后在含有5％GemCellTM人血清AB和IL-2(138U/ml)的X-VIVOTM15无血清培养基中扩增。使用CD8阳性分离试剂盒(Thermo Fisher Scientific)分离CD8+T细胞。本研究经北京世纪坛医院机构审查委员会批准，并获得所有参与者的知情同意。Primary T cells were isolated from peripheral blood mononuclear cells (PBMC) of healthy donors by Lymphoprep (MP Biomedicals) gradient centrifugation. To generate antigen-specific transgenic T cells, T cells in PBMCs were stimulated with anti-CD3 and anti-CD28 beads and then infected with retroviruses. Seven days after infection, CAR-T cells were assayed for CAR expression and then expanded in X-VIVO ™ 15 serum-free medium containing 5% GemCell ™ 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.
流式细胞术Flow Cytometry
流式细胞术在FACSCanto Plus仪器(BD Biosciences)上进行。FlowJo v.10(FlowJo,LLC)用于数据分析。用APC-cy7标记的小鼠抗人CD3抗体(BD Biosciences)、FITC标记的小鼠抗人CD8抗体(BD Biosciences)、Alexa Fluor 700标记的小鼠抗人CD8抗体(BD Biosciences)、BV421标记的小鼠抗人CD4抗体(BD Biosciences)、V450标记的小鼠抗人CD107a(BD Biosciences)、BV605标记的小鼠抗人CD45RO(BD Biosciences)、PE-cy7标记的小鼠抗人CCR7(BD Biosciences)、Alexa Fluor 700标记的小鼠抗人CD27(BD Biosciences)、PE-cy5标记的小鼠抗人CD95(BD Biosciences)和Alexa Fluor 647标记的山羊抗小鼠IgG(Fab特异性)F(ab')2抗体(Jackson ImmunoResearch)染色，检测CAR-T细胞。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-mouse IgG (Fab specific) F(ab')2 antibody labeled with Alexa Fluor 647 (Jackson ImmunoResearch).
细胞毒性测定Cytotoxicity assay
CAR-T细胞在24孔板中，以不同的效靶比(E:T)，与表达荧光蛋白GFP的靶肿瘤细胞NAML-6-EGFP-eGFP共培养。24小时后，收集细胞并使用流式细胞仪(BD FacsCanto II Plus)通过表面标志物检测肿瘤细胞。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).
增殖测定Proliferation assay
CD19-CAR-T细胞、CD19-CAR-IL-15 T细胞和CD19-CAR-IL15-IL15Ra T细胞与IL-2一起培养，并使用NALM-6刺激(E:T＝10:1)。第0天、第7天、第14天和第21天，使用Vi-CELL细胞活力分析仪，通过台盼蓝排除法，计数活细胞数。 CD19-CAR-T cells, CD19-CAR-IL-15 T cells and CD19-CAR-IL15-IL15Ra T cells were cultured with IL-2 and stimulated with NALM-6 (E:T=10:1). On days 0, 7, 14 and 21, use the Vi-CELL cell viability analyzer and count the number of viable cells by the trypan blue exclusion method.
细胞因子产生分析Cytokine production analysis
将CAR-T细胞与靶细胞(NAML-6-eGFP)，以2:1的E:T比，共培养24小时。收集上清液，并根据制造商的说明，通过ELISA试剂盒(DY285B、D1500、DY202、R&D systems)进行IFN-γ、IL-15和IL-2检测。CAR-T cells and target cells (NAML-6-eGFP) were co-cultured at an E:T ratio of 2:1 for 24 hours. The supernatant was collected and tested for IFN-γ, IL-15 and IL-2 by ELISA kits (DY285B, D1500, DY202, R&D systems) according to the manufacturer's instructions.
PCRPCR
通过PCR证实抗原刺激的CAR-T细胞中的IL-15和IL-15Ra表达。按照制造商的说明，使用TRIzol试剂(Invitrogen)从CAR-T细胞中提取总RNA。使用Nanodrop One分光光度计(Thermo Fisher Scientific)测量RNA的量和纯度。只有具有合适的吸光度测量值的样品(A260/A280为～2.0，且A260/A230为1.9-2.2)才被考虑纳入本研究。使用High Capacity cDNA逆转录试剂盒(Thermo Fisher Scientific)合成cDNA。IL-15 and IL-15Ra expression in antigen-stimulated CAR-T cells was confirmed by PCR. Total RNA was extracted from CAR-T cells using TRIzol reagent (Invitrogen) 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).
使用引物5'-ATGGATGCAATGAAGAGAGGG-3'(有义)和5'-CGACGTGTTCATGAACATCTGGA-3'(反义)扩增IL-15；使用引物5'-ATGGCCCCGAGGCGGGCGCGAGG-3'(有义)和5'-TAGGTGGTGCGAGCAGT-3'(反义)扩增IL-15Ra；使用引物5'-TGACCACAGTCCATGCCATC-3'(有义)和5'-GTGAGCTTCCCGTTCAGCTC-3'(反义)扩增GAPDH作为对照。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).
异种移植小鼠肿瘤模型Xenograft mouse tumor model
6至8周龄的NOD-SCID小鼠购自Charles River Laboratories。将1×106NAML-6-eGFP细胞静脉注射到NOD-SCID小鼠体内，构建异种移植小鼠模型。肿瘤细胞注射1天后，尾静脉注射1×107CAR-T细胞，每天一次，持续3天。使用IVIS(IVIS,Xenogen,Alameda,CA,USA)监测肿瘤发展。包括小鼠在内的所有实验均经北京世纪坛医院机构审查委员会批准。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.
统计分析Statistical Analysis
使用Graphpad Prism 8.0.2进行图表和统计分析。使用student t检验分析数据，p值<0.05被认为是显著；*p<0.05；**p<0.01；***p<0.001；****p<0.0001；ns，不显著。使用Kaplan-Meier方法，测量具有NAML-6-eGFP异种移植物的小鼠的总体存活率，并使用Cox比例风险回归分析进行组间比较。所有实验至少重复3次。Charts and statistical analyzes were performed using Graphpad Prism 8.0.2. Data were analyzed using student t test, p value <0.05 was considered significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant. Overall survival of mice with NAML-6-eGFP xenografts was measured using the Kaplan-Meier method and compared between groups using Cox proportional hazards regression analysis. All experiments were repeated at least three times.
实施例1构建并表征装甲CD19特异性CAR-T细胞Example 1 Construction and Characterization of Armored CD19-Specific CAR-T Cells
如材料和方法中所述，构建与CD19-CAR基因相连的IL-15基因和IL-15Ra基因(图1)，并将共表达这些基因的逆转录病毒载体转导到T细胞中，用流式细胞术检测转导效率。图2A显示了三种CAR构建体获得了相似的CAR转导效率，分别具有35.4％、26.1％和17.4％CD8+T细胞表达CD19特异性CAR。图2B显示，三组之间具有相似的CD4+/CD8+T细胞比率。As described in Materials and Methods, the 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.
接下来，使用PCR确认IL-15和IL-15Ra在装甲CAR-T细胞中的成功表达。提取CAR-T细胞的总RNA，并进行PCR扩增。结果(图2C)表明，CD19-CAR-IL-15 T细胞过表达IL-15，CD19-CAR-IL-15-IL-15Ra T细胞过表达IL-15以及IL-15Ra。Next, 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.
实施例2检测IL-15装甲CAR-T细胞的体外增殖和分化表型 Example 2 Detection of in vitro proliferation and differentiation phenotypes of IL-15 armored CAR-T cells
通过直接细胞计数，检测IL-15和/或IL-15Ra过表达的CAR-T细胞的体外增殖。简言之，CD19-CAR-T细胞、CD19-CAR-IL-15 T细胞和CD19-CAR-IL15-IL15Ra T细胞与IL-2一起培养。第0天、第3天、第7天和第14天，使用Vi-CELL细胞活力分析仪，通过台盼蓝排除法，计数活细胞数。结果显示，IL-15和/或IL-15Ra过表达的两种装甲CAR-T细胞均表现出了比CAR-T细胞更高的增殖能力(图3A)。In vitro proliferation of CAR-T cells overexpressing IL-15 and/or IL-15Ra was detected by direct cell counting. Briefly, CD19-CAR-T cells, CD19-CAR-IL-15 T cells, and CD19-CAR-IL15-IL15Ra T cells were cultured with IL-2. On days 0, 3, 7 and 14, use the Vi-CELL cell viability analyzer and count the number of viable cells by the trypan blue exclusion method. The results showed that both armored CAR-T cells overexpressing IL-15 and/or IL-15Ra showed higher proliferation ability than CAR-T cells (Figure 3A).
此外，由于IL-2是T细胞的生长因子，因此测量了上清液中IL-2的浓度。简言之，将CAR-T细胞与靶细胞(NAML-6-eGFP)，以2:1的E:T比，共培养24小时，培养基中不含外加的IL-2。之后，收集上清液，并通过ELISA试剂盒进行IL-2检测。结果表明，CD19-CAR-IL-15和CD19-CAR-IL-15-IL15Ra T细胞与CD19-CAR T细胞相比，释放更多的IL-2(图3B)。In addition, since IL-2 is a growth factor for T cells, the 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).
NAML-6-eGFP细胞刺激7天后，通过流式细胞术，检测CAR-T细胞的分化表型。对代表CAR-T细胞长期持久性能力的Tscm细胞(CD8+CD45RO-CCR7+CD27+CD95+)进行研究。结果显示，相对于CD8+CD19-CAR T细胞，CD19-CAR-IL-15和CD19-CAR-IL-15-IL-15Ra T细胞有显著更多的Tscm细胞(图4A最高测量的Tscm细胞水平，三组分别为1.67％，9.23％和4.84％；图4B显示了测量结果的柱状统计图)。After 7 days of stimulation with NAML-6-eGFP cells, the differentiation phenotype of CAR-T cells was detected by flow cytometry. 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).
此外，已经报道，分化程度较低的T细胞在抗原刺激时产生较少的IFNγ。因此，用NAML-6-eGFP细胞，以2:1的E:T比，刺激CD19-CAR、CD19-CAR-IL-15和CD19-CAR-IL15-IL15Ra T细胞24小时，并通过ELISA测量IFNγ的浓度。如图5A所示，表达IL-15和IL-15Ra的T细胞产生较少的IFNγ，这意味着CD19-CAR-IL-15和CD19-CAR-IL-15-IL-15Ra T细胞的分化程度较低。Furthermore, it has been reported that less differentiated T cells produce less IFNγ upon antigen stimulation. Therefore, 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 IFNγ was measured by ELISA concentration. As shown in Figure 5A, T cells expressing IL-15 and IL-15Ra produced less IFNγ, implying the degree of differentiation of CD19-CAR-IL-15 and CD19-CAR-IL-15-IL-15Ra T cells. lower.
随后，考虑到IL-15对T细胞增殖的作用，分析了装甲CAR-T细胞在与靶细胞共孵育一定时间后的凋亡细胞百分比和细胞存活率。简言之，两种装甲CAR-T和对照CAR-T细胞，分别与靶细胞NAML-6-eGFP共培养(10:1)7天，之后收集细胞，用染料Annexin V和7ADD染色15分钟，流式细胞仪检测CAR-T细胞存活率和细胞凋亡比例。结果显示，IL-15和IL-15Ra抑制了CAR-T细胞凋亡并增强了细胞活力(图5B和5C)。Subsequently, considering the effect of IL-15 on T cell proliferation, the percentage of apoptotic cells and cell survival rate of armored CAR-T cells after co-incubation with target cells for a certain period of time were analyzed. Briefly, two armored CAR-T and control CAR-T cells were co-cultured with target cells NAML-6-eGFP (10:1) for 7 days, and then the cells were collected and stained with dyes Annexin V and 7ADD for 15 minutes. Flow cytometry was used to detect the survival rate and apoptosis ratio of CAR-T cells. The results showed that IL-15 and IL-15Ra inhibited CAR-T cell apoptosis and enhanced cell viability (Figures 5B and 5C).
实施例3.IL-15Ra在体外降低IL-15诱导的毒性Example 3. IL-15Ra reduces IL-15-induced toxicity in vitro
为研究细胞培养条件下IL-15Ra对IL-15的影响，CAR-T细胞与NALM-6-eGFP共培养24小时(E:T＝2：1)。之后，收集装甲CAR-T细胞上清液，采用ELISA检测IL-15浓度。结果表明(图6A)，CD19-CAR-IL-15 T细胞具有最高的IL-15释放，而CD19-CAR-IL-15-IL15Ra T细胞与CD19-CAR-T细胞两者相当，具有显著较低的IL-15释放水平。图2B已经证实CAR-IL-15-IL-15Ra T细胞可以成功表达IL-15和IL-15Ra两者。因此，尽管不受任何理论约束，但认为CAR-IL-15-IL-15Ra T细胞可以通过使表达的IL-15与细胞膜表面表达的修饰IL-15Ra结合，减少释放到培养基中的IL-15量，并由此降低由释放的IL-15诱导的细胞毒性。To study the effect of IL-15Ra on IL-15 under cell culture conditions, CAR-T cells were co-cultured with NALM-6-eGFP for 24 hours (E:T=2:1). Afterwards, the armored CAR-T cell supernatant was collected, and IL-15 concentration was detected using ELISA. The results showed (Figure 6A) that CD19-CAR-IL-15 T cells had the highest IL-15 release, while CD19-CAR-IL-15-IL15Ra T cells were comparable to CD19-CAR-T cells, with significantly higher Low IL-15 release levels. Figure 2B has confirmed that CAR-IL-15-IL-15Ra T cells can successfully express both IL-15 and IL-15Ra. Therefore, while not being bound by any theory, it is thought that 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.
此外，鉴于CD132是IL-2和IL-15的共同受体亚基链并且其高表达与GVHD(移植物抗宿主病)有关，检测并比较了两种装甲CAR-T细胞的细胞表面CD132表达。简言之，CAR-T细胞与NALM-6-eGFP共培养7天(E:T＝10：1)后，收集细胞，CD132抗体染色。通过流式细胞术，检测细胞表面CD132的表达。结果(图6B)显示，CAR-IL-15-IL-15Ra T细胞具有最低的CD132表达(CAR-IL-15-IL-15Ra T细胞60.8％vs.CAR-IL-15 T细胞65.5％vs.CAR-T细胞93.2％)。Furthermore, given that 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 . Briefly, after CAR-T cells were co-cultured with NALM-6-eGFP for 7 days (E:T=10:1), the cells were collected and stained with CD132 antibody. The expression of CD132 on the cell surface was detected by flow cytometry. The results (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%).
此外，将CAR-T细胞与靶细胞NALM-6-eGFP细胞(E:T＝2：1)共培养24小时。收集细胞，通过流式检测GFP信号，以确定存活的NALM-6细胞数。结果显示在图7A和7B中。 In addition, CAR-T cells were co-cultured with target cells NALM-6-eGFP cells (E:T=2:1) for 24 hours. Cells were collected and the GFP signal was detected by flow cytometry to determine the number of surviving NALM-6 cells. The results are shown in Figures 7A and 7B.
实施例4.表达IL-15Ra的IL-15装甲CAR-T细胞在体内表现出增强的抗肿瘤活性和降低的毒性Example 4. IL-15Ra-expressing IL-15-armored CAR-T cells exhibit enhanced anti-tumor activity and reduced toxicity in vivo
为了检查装甲T细胞的体内抗肿瘤活性，将NAML-6-eGFP细胞静脉内注射到NOD-SCID小鼠中以产生异种移植小鼠模型。一天后，静脉内注射CAR-T细胞，并将未转导T细胞(NT)作为阴性对照。对小鼠进行三个月以上的监测(图8A)。To examine the in vivo antitumor activity of armored T cells, NAML-6-eGFP cells were intravenously injected into NOD-SCID mice to generate a xenograft mouse model. One day later, 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).
图8B和图9显示，与对照组相比，用CD19-CAR-IL-15 T细胞和CD19-CAR-IL-15-IL-15Ra T细胞治疗的小鼠没有肿瘤复发，这说明IL-15诱导了增强的抗肿瘤活性。然而，尽管没有肿瘤复发，但与CD19-CAR和CD19-CAR-IL-15-IL-15Ra T细胞治疗组相比，CD19-CAR-IL-15 T细胞治疗组的所有小鼠70天内死亡，存活率最低(图8B)。这说明IL-15对动物具有毒性。Figure 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.
使用Kaplan-Meier方法，测量具有NAML-6-eGFP异种移植物的小鼠的总体存活率，并使用Cox比例风险回归分析进行组间比较。如图10A所示，与CD19-CAR组只有20％的小鼠存活超过90天相比，CD19-CAR-IL-15-IL-15Ra的大约40％小鼠存活超过90天。Overall survival of mice with NAML-6-eGFP xenografts was 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.
在第50天，采集各组小鼠的血液，用血清检测人IL-15的浓度。图10B表明，CD19-CAR-IL-15 T治疗的小鼠在血液中具有最多的人IL-15，而CD19-CAR-IL-15-IL-15Ra T细胞处理组小鼠与对照CAR-T细胞处理小鼠相当，具有显著减少的血液IL-15水平。这表明，CD19-CAR-IL-15-IL-15Ra T细胞中IL-15Ra的共表达阻断了IL-15的血液释放以及由此阻断了IL-15在血清中的毒性，延长了所治疗的荷瘤小鼠的存活时间。 On day 50, blood was collected from each group of mice, and the concentration of human IL-15 was detected in serum. Figure 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.
序列表

sequence list

Claims (23)

  1. 一种核酸组合，所述组合包含第一核酸分子、第二核酸分子和第三核酸分子，其中：A nucleic acid combination comprising a first nucleic acid molecule, a second nucleic acid molecule and a third nucleic acid molecule, wherein:
    -所述第一核酸分子包含编码嵌合抗原多肽(CAR)的多核苷酸，- the first nucleic acid molecule comprises a polynucleotide encoding a chimeric antigen polypeptide (CAR),
    -所述第二核酸分子包含编码IL-15的多核苷酸，- the second nucleic acid molecule comprises a polynucleotide encoding IL-15,
    -所述第三核酸分子包含编码优化IL15Ra的多核苷酸，- the third nucleic acid molecule comprises a polynucleotide encoding optimized IL15Ra,
    其中所述优化IL15Ra包含在氨基酸位置202和203的双突变S202R和D203E，其中所述氨基酸位置根据SEQ ID NO:6编号。The optimized IL15Ra comprises double mutations S202R and D203E at amino acid positions 202 and 203, wherein the amino acid positions are numbered according to SEQ ID NO:6.
  2. 根据权利要求1的核酸组合，其中所述优化IL-15Ra包含双突变S202R和D203E，优选包含位置197-204的基序YPQGHSDT，且包含SEQ ID NO:6的氨基酸序列；或与之具有至少90％、92％、95％、96％、97％、98％、99％或99.5％同一性的氨基酸序列；优选地，所述编码优化IL15Ra的多核苷酸包含SEQ ID NO:3所述序列的多核苷酸、或与之具有至少90％、92％、95％、96％、97％、98％、99％或99.5％同一性的多核苷酸。The nucleic acid combination according to claim 1, wherein the optimized IL-15Ra comprises double mutations S202R and D203E, preferably comprising the motif YPQGHSDT at positions 197-204, and comprising the amino acid sequence of SEQ ID NO: 6; or having at least 90 %, 92%, 95%, 96%, 97%, 98%, 99% or 99.5% identity of the amino acid sequence; Preferably, the polynucleotide encoding optimized IL15Ra includes the sequence described in SEQ ID NO:3 polynucleotide, or a polynucleotide having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 99.5% identity thereto.
  3. 根据权利要求1-2的核酸组合，其中，第一核酸分子包含编码CAR多肽的多核苷酸，其中所述CAR多肽从N端到C端包含：任选地信号肽，特异性结合肿瘤抗原的胞外抗原结合结构域，任选地铰链区或间隔区、跨膜结构域、和胞质信号传导结构域，且其中所述胞质信号传导结构域包含CD28的共刺激结构域和4-1BB的共刺激结构域的组合以及CD3ζ初级信号传导结构域。The nucleic acid combination according to claims 1-2, wherein the first nucleic acid molecule comprises a polynucleotide encoding a CAR polypeptide, wherein the CAR polypeptide comprises from the N-terminus to the C-terminus: optionally a signal peptide specifically binding to a tumor antigen. An extracellular antigen binding domain, optionally a hinge or spacer region, a transmembrane domain, and a cytoplasmic signaling domain, and wherein the cytoplasmic signaling domain comprises the costimulatory domain of CD28 and 4-1BB A combination of costimulatory domains as well as the CD3ζ primary signaling domain.
  4. 根据权利要求1-3的核酸组合，其中，所述CAR多肽包含：从N端到C端，The nucleic acid combination according to claims 1-3, wherein the CAR polypeptide includes: from the N terminus to the C terminus,
    抗肿瘤抗原(如CD19)的抗体或抗原结合片段，如scFv，Antibodies or antigen-binding fragments, such as scFv, against tumor antigens (such as CD19),
    CD28铰链区，CD28 hinge region,
    CD28跨膜结构域，CD28 transmembrane domain,
    CD28共刺激结构域，CD28 co-stimulatory domain,
    4-1BB共刺激结构域，和4-1BB costimulatory domain, and
    CD3ζ初级信号传导结构域，CD3ζ primary signaling domain,
    优选地，所述CAR多肽包含SEQ ID NO:4的氨基酸序列，或与之具有至少90％、92％、95％、96％、97％、98％、99％或以上的同一性的氨基酸序列。Preferably, the CAR polypeptide comprises the amino acid sequence of SEQ ID NO: 4, or an amino acid sequence that is at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identical thereto. .
  5. 根据权利要求1-4的核酸组合，其中，第二核酸分子包含编码IL-15的多核苷酸，且优选地，第二核酸分子包含编码SEQ ID NO:5或与之具有至少85％、90％、92％、95％、96％、97％、98％、99％或以上同一性的氨基酸序列的多核苷酸，且更优选地，第二核酸分子包含SEQ ID NO:2的核苷酸序列，或与之具有至少85％、90％、92％、95％、96％、97％、98％、99％或以上的同一性的核苷酸序列。According to the nucleic acid combination of claims 1-4, wherein the second nucleic acid molecule comprises a polynucleotide encoding IL-15, and preferably, the second nucleic acid molecule comprises a polynucleotide encoding SEQ ID NO:5 or an amino acid sequence having at least 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto, and more preferably, the second nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto.
  6. 根据权利要求1-5的核酸组合，其中所述第一、第二和第三核酸分子中的两者或所有三者以功能性连接的方式存在于单个核酸构建体，例如病毒载体，如慢病毒载体上。The nucleic acid combination according to claims 1-5, wherein two or all three of the 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.
  7. 根据权利要求1-6的核酸组合，其中所述核酸组合为包含第一、第二和第三核酸分子的单个核酸构建体，其中，所述核酸构建体编码从N端到C端具有下式(I)所示结构的融合蛋白：
    CAR-(L1)-E1-(L2)-E2   (I)    The nucleic acid combination according to claims 1-6, wherein the nucleic acid combination is a single nucleic acid construct comprising first, second and third nucleic acid molecules, wherein the nucleic acid construct encodes from the N-terminus to the C-terminus the following formula (I) Fusion protein of the structure shown:
    CAR-(L1)-E1-(L2)-E2 (I)
    其中，in,
    CAR表示嵌合抗原受体多肽，CAR stands for Chimeric Antigen Receptor Polypeptide,
    L1和L2分别独立地表示包含自剪接位点的连接肽， L1 and L2 each independently represent a connecting peptide containing a self-splicing site,
    E1和E2互不相同，且独立地选自IL-15和优化IL-15Ra，E1 and E2 are different from each other and are independently selected from IL-15 and optimized IL-15Ra,
    其中，“-”表示式(I)的所述各组分之间功能性连接。Wherein, "-" represents the functional connection between the components of formula (I).
  8. 根据权利要求7的核酸组合，其中E1表示由第二核酸分子编码的IL-15，且E2表示由第三核酸分子编码的优化IL-15Ra。The nucleic acid combination according to claim 7, wherein E1 represents IL-15 encoded by the second nucleic acid molecule, and E2 represents optimized IL-15Ra encoded by the third nucleic acid molecule.
  9. 根据权利要求7-8的核酸组合，其中L1和L2互不相同，彼此独立地包含选自P2A、T2A、E2A或F2A的自剪接位点；优选地，L1包含P2A位点(优选地，包含SEQ ID NO:17的氨基酸序列)；且L2包含T2A位点(优选地，包含SEQ ID NO:18的氨基酸序列)。The nucleic acid combination according to claims 7-8, wherein L1 and L2 are different from each other and independently comprise a self-splicing site selected from P2A, T2A, E2A or F2A; preferably, L1 comprises a P2A site (preferably, comprising The amino acid sequence of SEQ ID NO:17); and L2 includes the T2A site (preferably, the amino acid sequence of SEQ ID NO:18).
  10. 根据权利要求1-9的核酸组合，其中所述核酸组合，当引入免疫效应细胞如T细胞中后，相比仅引入编码所述CAR的第一核酸分子和编码所述IL-15的第二核酸分子的对照免疫效应细胞，导致减少的IL-15在细胞外环境中的释放量，和由此减少的由IL-15诱导的毒性。The nucleic acid combination according to claims 1-9, wherein the nucleic acid combination, 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 the second nucleic acid molecule encoding the IL-15. Control of immune effector cells with nucleic acid molecules results in reduced release of IL-15 in the extracellular environment, and thus reduced toxicity induced by IL-15.
  11. 由权利要求1-10的核酸组合编码的多肽，优选地，所述多肽为包含功能性连接的以下组分的单条融合多肽：The polypeptide encoded by the nucleic acid combination of claims 1-10, preferably, the polypeptide is a single fusion polypeptide comprising the following functionally linked components:
    (i)嵌合抗原受体(CAR)多肽；(i) Chimeric antigen receptor (CAR) polypeptide;
    (ii)IL-15多肽；和(ii) IL-15 polypeptide; and
    (iii)优化IL-15Ra多肽。(iii) Optimizing IL-15Ra polypeptide.
    更优选地，所述融合多肽具有根据权利要求7-9中描述的式(I)结构。More preferably, the fusion polypeptide has a structure according to formula (I) as described in claims 7-9.
  12. 包含权利要求1-11的核酸组合的载体，如慢病毒载体。Vectors comprising the nucleic acid combination of claims 1-11, such as lentiviral vectors.
  13. 权利要求12的载体，其中所述第一、第二和第三核酸分子以多顺反子形式存在于所述载体上。The vector of claim 12, wherein said first, second and third nucleic acid molecules are present on said vector in a polycistronic form.
  14. 装甲CAR-T细胞，其中所述装甲CAR-T细胞包含根据权利要求1-10的核酸分子组合、或引入权利要求13的载体。Armored CAR-T cells, wherein the armored CAR-T cells comprise a combination of nucleic acid molecules according to claims 1-10, or a vector incorporating claim 13.
  15. 根据权利要求14的装甲CAR-T细胞，其包含权利要求7-9中描述的式(I)融合多肽。The armored CAR-T cell according to claim 14, comprising the fusion polypeptide of formula (I) described in claims 7-9.
  16. 根据权利要求14-15的装甲CAR-T细胞，其包含彼此分离的所述CAR多肽、IL-15多肽和优化IL-15Ra多肽。The armored CAR-T cell according to claims 14-15, comprising said CAR polypeptide, IL-15 polypeptide and optimized IL-15Ra polypeptide separated from each other.
  17. 根据权利要求14-16的装甲CAR-T细胞，其具有至少一项如下性质：The armored CAR-T cell according to claims 14-16, which has at least one of the following properties:
    -相比仅表达所述CAR多肽的对照CAR-T细胞，具有增强的增殖能力和细胞活力，以及增加的Tscm表型T细胞亚群比例；-Have enhanced proliferation ability and cell viability, as well as an increased proportion of Tscm phenotype T cell subsets compared to control CAR-T cells that only express the CAR polypeptide;
    -相比仅表达所述CAR多肽和所述IL-15的对照CAR-T细胞，减少IL-15在细胞外环境中的释放量，和由此具有降低的由IL-15诱导的毒性；和- reduce the amount of IL-15 released in the extracellular environment, and thereby have reduced toxicity induced by IL-15, compared to control CAR-T cells expressing only the CAR polypeptide and the IL-15; and
    -体内抗肿瘤功效和减少的CAR-T治疗中与IL-15相关的不良事件。- In vivo anti-tumor efficacy and reduced IL-15-related adverse events in CAR-T therapy.
  18. 包含权利要求14-17的装甲CAR-T细胞的药物组合物。Pharmaceutical composition comprising the armored CAR-T cells of claims 14-17.
  19. 权利要求14-17的装甲CAR-T细胞在制备预防或治疗癌症或提供抗肿瘤免疫的药物中的用途，优选地，所述肿瘤是血液肿瘤或实体瘤，再优选地，所述CAR-T细胞全身(例如静脉内)施用或局部(例如瘤内)使用。The use of the armored CAR-T cells of claims 14-17 in the preparation of drugs for preventing or treating cancer or providing anti-tumor immunity. Preferably, the tumor is a hematological tumor or a solid tumor, and further preferably, the CAR-T The cells are administered systemically (eg intravenously) or used locally (eg intratumorally).
  20. 优化IL15Ra多肽，其包含在氨基酸位置201和202的双突变S202R和D203E，其中所述氨基酸位置根据SEQ ID NO:6确定，且优选地，还包含SEQ ID NO:6的氨基酸序列；或与之具有至少90％、92％、95％、96％、97％、98％、99％或99.5％同一性的氨基酸序列。An optimized IL15Ra polypeptide comprising double mutations S202R and D203E at amino acid positions 201 and 202, wherein the amino acid positions are determined according to SEQ ID NO: 6, and preferably, further comprising the amino acid sequence of SEQ ID NO: 6; or together with Amino acid sequences having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or 99.5% identity.
  21. 编码权利要求20的优化IL15Ra的多核苷酸，优选地，其包含SEQ ID NO:3所述序列的多核苷酸、或与之具有至少90％、92％、95％、96％、97％、98％、99％或99.5％同一性的多核苷酸。 The polynucleotide encoding the optimized IL15Ra of claim 20, preferably, it comprises the polynucleotide of the sequence described in SEQ ID NO: 3, or has at least 90%, 92%, 95%, 96%, 97%, Polynucleotides that are 98%, 99% or 99.5% identical.
  22. 一种用于增加CAR-T细胞的持久性并降低其毒性的方法，其包括在所述CAR-T细胞中引入并表达根据权利要求1-10的核酸组合。A method for increasing the persistence of CAR-T cells and reducing their toxicity, which includes introducing and expressing a nucleic acid combination according to claims 1-10 in the CAR-T cells.
  23. 权利要求22的方法，其中编码所述优化IL-15Ra多肽的核酸分子与编码所述CAR多肽的核酸分子和编码IL-15多肽的核酸分子彼此功能性连接在单个核酸构建体中，并表达产生具有根据权利要求7-9中描述的式(I)结构的融合多肽。 The method of claim 22, wherein the nucleic acid molecule encoding the optimized IL-15Ra polypeptide and the nucleic acid molecule encoding the CAR polypeptide and the nucleic acid molecule encoding the IL-15 polypeptide are functionally linked to each other in a single nucleic acid construct and expressed to produce Fusion polypeptide having a structure according to formula (I) as described in claims 7-9.
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