WO2023000685A1 - Armored car-t cell that increases survivin expression and anti-tumor use thereof - Google Patents

Armored car-t cell that increases survivin expression and anti-tumor use thereof Download PDF

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WO2023000685A1
WO2023000685A1 PCT/CN2022/079966 CN2022079966W WO2023000685A1 WO 2023000685 A1 WO2023000685 A1 WO 2023000685A1 CN 2022079966 W CN2022079966 W CN 2022079966W WO 2023000685 A1 WO2023000685 A1 WO 2023000685A1
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car
cells
nucleic acid
cell
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钟晓松
张莹
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卡瑞济(北京)生命科技有限公司
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Definitions

  • the present invention relates to the medical field, in particular to a CAR-T cell that increases the expression of Survivin and its application alone or in combination with IL-15 in the preparation of anti-tumor drugs.
  • Survivin is an evolutionarily conserved eukaryotic protein that is essential for cell division and acts as an inhibitor of apoptosis by antagonizing caspase activity [1,2]. Therefore, it has received extensive attention as a potential tumor therapeutic target. In addition, Survivin is expressed during development [3] and in proliferating adult cells [4], especially in activated T lymphocytes [5] and self-renewing stem cells [6]. Therefore, Survivin is a marker of actively proliferating cells.
  • Interleukin-15 is a common cytokine involved in T cell differentiation and homeostatic regulation [7]. IL-15 has attracted widespread attention due to its biological similarity to interleukin-2 (IL-2) in cytokine receptors. Both the IL-15 and IL-2 receptors are heterotrimeric complexes composed of the ⁇ and ⁇ c subunits of IL-2 and a unique ⁇ subunit [7]. Due to the shared IL-2 receptor subunit (IL-2/15R ⁇ ), IL-15 and IL-2 share many biological activities, including the growth and migration of activated T cells and NK cells, and the induction of B cell Proliferation and differentiation [8].
  • IL-2 has a strong ability to expand T cells and stimulate T cell functions, and is the earliest cytokine used in clinical cancer trials. However, the strong toxic side effects of IL-2 limit its application. Recently, the clinical use of IL-15 has been further strengthened due to its low toxicity, and it has been reported that overexpression of IL-15 or administration of rIL-15 can protect mice from various infections [9,10].
  • CAR is a synthetic molecule consisting of an extracellular tumor antigen-binding domain, a hinge, a transmembrane and an intracellular signaling domain connected to it, which induces targeting of tumor cells by specifically recognizing surface proteins expressed on tumor cells. T cell responses to eradicate tumors [11].
  • T cells expressing CAR CAR-T cells
  • scFv single chain variable fragment
  • Various cytokines, perforin, granzyme, interferon, etc. induce tumor cell apoptosis[12,13].
  • cytokines perforin, granzyme, interferon, etc.
  • T cell activation is at the heart of the adaptive immune response. T cell expansion, differentiation, and survival depend on the integration of multiple signals from TCR binding, cytokine receptors, and co-stimulatory molecules. These signals lead to activation of the PI3K/AKT pathway signaling network in T cells. PI3K and AKT, key components of the PI3K/AKT pathway, have been shown to be overactivated in most cancers and are therefore the focus of anticancer research. However, there are still many controversies about the role of regulating the PI3K-AKT pathway in CAR-T cells.
  • IL-15 can promote the initial and the survival of memory (Memory) CD8 + T cells[18], and enhance the cross-priming of CD4 + and CD8 + T cells and the activation of TEM cells (effector memory T cells) and CD44 hi or CD122 hi memory CD8 + T cells Proliferation [19].
  • TEM cells effector memory T cells
  • CD44 hi or CD122 hi memory CD8 + T cells Proliferation [19].
  • IL-15 can promote the generation of memory T cells with adoptive immune cell therapy potential, and in animal models, it can cooperate with the anti-tumor effect of CAR-T cells, Effectively enhance the tumor invasiveness and durability of anti-tumor effects of CAR-T cells [20].
  • CAR-T cell therapy is an adoptive immunotherapy technology that has developed rapidly in recent years. How to improve the effectiveness and long-term efficacy of anti-tumor immune cells is crucial to improving the efficacy of cellular immunotherapy. Therefore, there is a continuing need in the art to provide new technological solutions that can be used to generate longer-lasting, more effective CAR-T cell therapy.
  • Uren, A.G., et al., Survivin and the inner centromere protein INCENP show similar cell-cycle localization and gene knockout phenotype. Curr Biol, 2000.10(21):p.1319-28.
  • TSCM Stem like memory T cells
  • IL-15 regulates the expression of survivin through the PI3K/Akt pathway and promotes the expression of CAR-T cells.
  • the formation of a TSCM-like cell population in the middle enhances the persistence of CAR-T cells; and the Armored CAR-T cells (armed CAR-T cells) with increased survivin expression not only promote the survival of the CAR-T cells, but also significantly improve their long-term survival in vivo. Antitumor effect.
  • the present invention aims to provide a CAR-T cell with increased Survivin expression and its application alone or in combination with IL-15 in the preparation of anti-tumor drugs, so as to solve the above-mentioned problems in the prior art.
  • the present invention provides the following scheme:
  • the present invention provides a nucleic acid construct
  • the nucleic acid construct has the following structure: car-(2A)-e, wherein, car represents a chimeric antigen receptor encoding polynucleotide sequence, 2A represents a self-cleaving sequence, e represents the protein coding sequence of functional fusion, and the protein coding gene of described functional fusion is Survivin gene, preferably, the nucleotide sequence of described Survivin gene is as shown in SEQ ID NO:2.
  • the antigen-binding domain of the chimeric antigen receptor specifically targets a tumor antigen
  • the tumor antigen is the membrane antigen CD19
  • the self-cleaving sequence is P2A
  • "-" indicates that adjacent nucleosides are connected Acidic phospholipid bonds or optionally linking peptides.
  • the present invention also provides a recombinant vector, including the nucleic acid construct.
  • the present invention also provides a host cell containing the recombinant vector or the exogenous nucleic acid construct integrated in the chromosome.
  • the present invention also provides a pharmaceutical composition, which includes a pharmaceutically acceptable carrier and the nucleic acid construct, the recombinant vector or the host cell.
  • the pharmaceutical composition further includes cytokine IL-15.
  • the present invention also provides the application of the nucleic acid construct, the recombinant vector or the host cell in the preparation of antitumor drugs or preparations.
  • the tumor comprises a hematological cancer (eg leukemia) or a solid tumor.
  • a hematological cancer eg leukemia
  • a solid tumor e.g., a hematological cancer (eg leukemia) or a solid tumor.
  • said host cells comprise T cells or NK cells.
  • the present invention also provides a method for preparing a modified T cell or NK cell, comprising the following steps: introducing the nucleic acid construct or the recombinant vector into the T cell or NK cell to be modified, constructing a surface expression heterogeneous A modified T cell or NK cell derived from a recipient, wherein the recipient is selected from CAR.
  • the present invention studies the synergistic anti-tumor mechanism of IL-15 and CAR-T from the nuclear molecular level. Through experimental verification, it is found that, first, IL-15 can increase the cytotoxicity of CAR-T cells to various cell lines; 15 enhances the survival of CAR-T cells by increasing the percentage of TSCM. Secondly, it was found that IL-15 can activate an important protein survivin that inhibits apoptosis and promotes cell proliferation. Further analysis showed that IL-15 activated survivin through the PI3K/Akt pathway, and overexpression of survivin confirmed that survivin promoted the survival and anti-tumor effects of CAR-T cells.
  • the molecular mechanism of IL-15 promoting T cell survival and anti-tumor effect is related to the formation of Tscm and the regulation of PI3K/Akt/Survivin signaling pathway activity.
  • the present disclosure may provide new insights into cell survival and Tscm production and lay the foundation for its rapid application in adoptive cell therapy.
  • Figure 1A shows the transfection efficiency of CAR of CD19-CAR-T cells detected by flow cytometry, and PBMC was used as a control;
  • Figure 1B is a schematic diagram of the experimental flow of CAR-T cells cultured with IL-2 or IL-15 for 14 days;
  • Figure 1C shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T was co-cultured with target cell Nalm-6 (10:1) for 6 hours, and the expression of CD107a on the surface of T cells was detected by flow cytometry Express;
  • Figure 1D- Figure 1E shows that after CD19-CAR-T was cultured with IL-15 or IL-2, the expression of IFN ⁇ in T cells was detected with Elispot kit (D), and the culture supernatant was retained, and ELISA kit was used (Thermo) detection of T cell IFN ⁇ release (E);
  • Figure 1F shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T and target cell Nalm-6 were co-cultured (1:1) for 24 hours, the cells were collected, and the remaining tumor cells were detected by flow cytometry ;
  • Figure 1G shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T and target cell Nalm-6 were co-cultured (10:1) for 48 hours or 72 hours, and the cells were collected and detected by Western blot Expression of granzyme A, granzyme B; values below immunoblots indicate expression levels normalized to the addition of antigen alone.
  • Figure 2A shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T was co-cultured with target cell Nalm-6 (10:1), and the number of CD8 + T cells was counted every seven days;
  • Figure 2B shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T was stained with CFSE (10 ⁇ M), co-cultured with target cell Nalm-6 (10:1) for 72 hours, flow cytometry Detection of T cell proliferation;
  • Figure 2C- Figure 2D shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T and target cell Nalm-6 were co-cultured (10:1), cell cycle on day 7 and day 14 detection;
  • Figure 2E shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T was co-cultured with target cell Nalm-6 (10:1) for 24 hours, flow cytometric detection of CD132 on the surface of T cells Express;
  • Figure 2F shows the co-cultivation (10:1) of CD19-CAR-T and target cell Nalm-6 after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, and the Tscm was detected by flow cytometry on the 7th and 14th day Proportion;
  • Figure 3A shows that after EphA2-CAR-T was cultured with IL-15 or IL-2, EphA2-CAR-T was stained with CFSE (10 ⁇ M), co-cultured with target cells U373 cells (10:1) for 72 hours, and flow cytometric detection T cell proliferation;
  • Figure 3B shows the expression of IFN ⁇ in T cells detected by Elispot kit after culturing EphA2-CAR-T with IL-15 or IL-2 respectively;
  • Figure 3C- Figure 3D shows that after EphA2-CAR-T was cultured with IL-15 or IL-2 respectively, EphA2-CAR-T was co-cultured with target cell U373 (2:1), and treated with RTCA (C) and fluorescein (D ) detecting the killing function of T cells on target cells;
  • Figure 4A shows that after CD19-CAR-T was cultured with IL-15 or IL-2, CD19-CAR-T was co-cultured with target cell Nalm-6 (10:1) for 48 hours and 72 hours, RNA was extracted, and survivin was detected by PCR. mRNA expression;
  • Figure 4B shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T was co-cultured with target cell Nalm-6 (10:1) for 72 hours, the protein was collected, and the expression of survivin was detected by Western blot ;
  • Figure 4C- Figure 4D shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T and target cell Nalm-6 were co-cultured (10:1) for 72 hours, and ly294002 (10 ⁇ M) was added at the same time , collect protein, and detect the expression of survivin and P-AKT by Western blot;
  • Figure 5A is a comparison of the structure of Armored CAR (CD19-CAR-Survivin, CD19-CAR-IL-15);
  • Figure 5B shows the transfection efficiency of Armored CAR-T cells detected by flow cytometry.
  • Figure 5C shows that after 24 hours of co-culture (10:1) between Armored CAR-T and target cells Nalm-6, the supernatant was collected and the concentration of IL-15 was detected.
  • Figure 5D shows the co-culture of Armored CAR-T and target cell Nalm-6 (10:1) for 24 hours, the cells were collected, RNA was extracted, reverse transcribed, and the relative expression of survivin was detected by Q-PCR.
  • Figure 6A shows the co-culture of Armored CAR-T and target cells Nalm-6 (10:1), counting the number of cells every 7 days, and calculating the cell proliferation.
  • Figure 6B shows that Armored CAR-T and target cell Nalm-6 were co-cultured (10:1) in medium without IL-2 for 24 hours, the supernatant was collected, and the concentration of IL-2 was detected.
  • Figure 6C and D show the co-culture of Armored CAR-T and target cells Nalm-6 (10:1) for 7 days, the cells were collected, and the expression of CD132 on the cell surface and the ratio of Tscm cells were detected.
  • Figure 6E shows that after 24 hours of co-culture (10:1) of Armored CAR-T and target cell Nalm-6, the supernatant was collected and the concentration of IFN- ⁇ was detected.
  • Figure 6F shows the co-culture of Armored CAR-T and target cell Nalm-6 (10:1) for 7 days, the cells were collected, stained with dyes Annexin V and 7ADD for 15 minutes, and the survival rate and apoptosis of CAR-T cells were detected by flow cytometry death rate.
  • Fig. 7A is a flowchart of animal experiments.
  • Fig. 7B is a fluorescence map of tumor burden in mice.
  • Fig. 7C is a fluorescence statistical graph of tumor burden in mice.
  • Mouse fluorescence was collected with an IVIS instrument (IVIS, Xenogen, Alameda, CA, USA). Each line represents the fluorescence change curve of one mouse.
  • Figure 7D is the survival curve of tumor mice. * indicates p-value ⁇ 0.05.
  • Figure 8A shows that after the experimental mice reached the end point of the experiment, the mice were sacrificed, bone marrow cells were collected, RNA was extracted, and the expression level of the virus vector was detected by Q-PCR. After the RNA was reverse-transcribed into cDNA, Q-PCR primers (F: AGAACCTAGAACCTCGCTGGA, R: CTGCGATGCCGTCTACTTTG) and SYBR green dye were added, and the expression level was detected with a fluorescent quantitative PCR instrument Q6.
  • Q-PCR primers F: AGAACCTAGAACCTCGCTGGA, R: CTGCGATGCCGTCTACTTTG
  • Figure 8B shows that after the experimental mice reached the end point of the experiment, the mice were sacrificed, the plasma was collected, and the concentration of human IL-15 cytokine in the mouse plasma was detected.
  • ELISA kit R&D systeme D1500 was used to detect the concentration of IL-15.
  • Fig. 8C is the result of tumor formation experiment in nude mice.
  • chimeric receptor chimeric antigen receptor
  • CAR chimeric antigen receptor
  • a recombinant protein comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain.
  • peptide a nucleic acid encoding a chimeric antigen receptor is indicated by the lowercase letter "car”.
  • 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.
  • primary signals are elicited, eg, by binding of the TCR/CD3 complex to peptide-loaded MHC molecules and result in mediation of T cell responses including, but not limited to, proliferation, activation, differentiation, and the like.
  • the cytoplasmic domain of any one or more CARs of the invention comprises an intracellular signaling sequence, eg, the signaling sequence of CD3 ⁇ .
  • CD3 ⁇ is defined as the protein provided under accession number UniProtKB-P20963 or an equivalent thereof.
  • a “CD3 ⁇ signaling domain” is defined as a stretch of amino acid residues from the cytoplasmic domain of the CD3 ⁇ chain sufficient to functionally transmit the initial signal necessary for T cell activation.
  • the cytoplasmic domain of CD3 ⁇ comprises residues 52 to 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 equivalent residues for humans, monkeys, apes, etc.).
  • the "CD3 ⁇ signaling domain” is the sequence provided in SEQ ID NO: 15 or a variant thereof.
  • costimulatory molecule refers to a corresponding binding partner on a cell that specifically binds to a costimulatory ligand to mediate a costimulatory response (eg, 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), activating NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28.
  • the "co-stimulatory molecule” is CD28, 4-1BB (ie CD137).
  • co-stimulatory domain refers to the intracellular portion of a co-stimulatory molecule.
  • 4-1BB refers to a member of the TNFR superfamily, also known as CD137, which has the amino acid sequence provided under UniProtKB-Q07011 accession number or is derived from a non-human species (e.g., mouse, rodent, monkey, ape, etc. ) equivalent residues.
  • 4-1BB co-stimulatory domain is defined as the cytoplasmic region from 4-1BB, e.g., amino acid residues 214-255 of UniProtKB-Q07011 or from non-human species (e.g., mouse, rodent , monkey, ape, etc.) equivalent residues.
  • CD28 refers to the amino acid sequence provided under UniProtKB-Q07011 accession number or the equivalent residues from a non-human species (eg, mouse, rodent, monkey, ape, etc.).
  • 4-1BB co-stimulatory domain is defined as amino acid residues 214-255 of UniProtKB-Q07011 or equivalent residues from non-human species (eg, mouse, rodent, monkey, ape, etc.).
  • CD28 co-stimulatory domain is defined as the cytoplasmic region from CD28, e.g., amino acid residues 180-220 of UniProtKB-Q07011 or from non-human species (e.g., mouse, rodent, monkey, etc.) equivalent residues.
  • CD28 transmembrane domain is defined as the transmembrane region from CD28, e.g., amino acid residues 153-179 of UniProtKB-Q07011 or from non-human species (e.g., mouse, rodent, monkey, ape etc.) equivalent residues.
  • CD28 hinge domain is defined as a hinge domain from the extracellular region of CD28, such as amino acid residues 114-152 of UniProtKB-Q07011 or from a non-human species (e.g., mouse, rodent, monkey, Apes, 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 transformed by introducing a heterologous nucleic acid or protein, or by changing itself Modified natural nucleic acid or protein, or refers to a substance derived from a virus or cell thus modified.
  • heterologous nucleic acid sequence also refers to a sequence derived from and introduced into (for example by infection with a viral vector) the same host cell or subject and thus exists in a non-native state, for example, in a different position, in a different The copy number is present, or under the control of different regulatory elements.
  • expression cassette refers to a DNA sequence that encodes and is capable of expressing one or more genes of interest (such as the CAR polypeptide of the present invention, or the Survivin protein, or both).
  • genes of interest such as the CAR polypeptide of the present invention, or the Survivin protein, or both.
  • a heterologous polynucleotide sequence encoding a gene of interest is functionally linked to expression control sequences.
  • linker or “linker” 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).
  • the connecting peptide is 1-50 amino acids in length, eg, 1, 2, 3, 4, 5 amino acids, or 10, 15, 20, 25, 30 amino acids in length.
  • the connecting peptide that can be used between the components of the CAR fusion polypeptide of the present invention is not particularly limited. Suitable linker peptides can be rationally designed using computer programs that model the three-dimensional structures of proteins and peptides.
  • short oligopeptide linkers or polypeptide linkers can form linkages between building block sequences as desired, eg, glycine-serine doublets, or single amino acids, eg, alanine, glycine, can be used as linkers.
  • amino acid change and “amino acid modification” are used interchangeably to refer to additions, deletions, substitutions 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 possesses the desired properties.
  • the amino acid substitutions are non-conservative amino acid substitutions, ie, the substitution of one amino acid with another amino acid having different structural and/or chemical properties.
  • Amino acid substitutions include 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 refers 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.
  • conservative modifications include amino acid substitutions, additions and deletions.
  • Conservative modifications, especially conservative substitutions can be introduced into the CAR fusion polypeptide of the present invention or its constituent elements (such as CAR or Survivin) by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis .
  • 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), nonpolar 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
  • Identity percentage (%) of an amino acid sequence/nucleotide sequence refers to aligning a candidate sequence with a specific amino acid/nucleotide sequence shown in this specification and, if necessary, achieving maximum sequence identity After introducing gaps as percentages, and in the case of amino acid sequences, when not considering any conservative substitutions as part of the sequence identity, the amino acid residues/nuclei of the specific amino acid/nucleotide sequences shown in this specification in the candidate sequence The percentage of amino acid/nucleotide residues with identical nucleotide residues.
  • the invention contemplates variants of the fusion polypeptides or nucleic acid molecules or constituent elements thereof of the invention relative to the fusion polypeptides or nucleic acid molecules or constituent elements thereof specifically disclosed herein (e.g., CAR polypeptide/ Nucleic acid encoding, or Survivin protein/encoding nucleic acid) sequences have a considerable degree of identity, for example, the identity is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or higher. Such variants may contain conservative modifications. For purposes of the present invention, percent identity is determined using the publicly available BLAST tool at https://blast.ncbi.nlm.nih.gov with default parameters.
  • variant or “functional variant” polypeptide or protein means that the polypeptide or protein has substantially the same sequence or significant sequence identity, 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 multiplying another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which 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 inter alia self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009).
  • Other examples of lentiviral vectors that can be used clinically include, for example, but are not limited to, the Lentiviral vector 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 cell refers to a cell that participates in an immune response, eg, participates in promoting an immune effector response.
  • immune effector cells include T cells, eg, ⁇ / ⁇ T cells and ⁇ / ⁇ T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid-derived phagocytes.
  • mammals include, but are not limited to, domesticated animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rodents). 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 rodents.
  • rodents e.g., mice and rodents.
  • an individual or subject is a human.
  • tumor and cancer are used interchangeably herein to encompass both solid and liquid tumors.
  • anti-tumor immunity means that the following immunological effects can be exhibited by various means, including but not limited to, for example, causing a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.
  • the present invention has found through in-depth research that in CAR-based immune cells (for example, CAR-T cells and CAR-NK cells), the persistence and/or Anti-tumor immunity.
  • CAR-based immune cells for example, CAR-T cells and CAR-NK cells
  • the present invention provides a CAR-based immune cell, wherein the immune cell not only comprises a heterologous polynucleotide encoding a CAR polypeptide, but also comprises a heterologous polynucleotide encoding survivin.
  • the immune cells are T cells, such CAR-based immune cells with recombinantly expressed survivin are also referred to as “Armored CAR-T cells” or “Armored CAR-T cells” herein.
  • the heterologous polynucleotide encoding the CAR polypeptide and the heterologous polynucleotide encoding survivin may be located on a single nucleic acid molecule, or located on different nucleic acid molecules that are separated.
  • the invention provides nucleic acid constructs that can be used to form the CAR-based immune cells of the invention.
  • the nucleic acid construct of the present invention comprises a polynucleotide encoding a chimeric antigen receptor (CAR) molecule and a polynucleotide encoding a SURVIVIN protein.
  • the polynucleotide encoding the CAR polypeptide and the polynucleotide encoding the SURVIVIN protein may be located in the same or different expression cassettes, and may be expressed as separate polypeptides, or Expressed as a fusion polypeptide.
  • the expression of the nucleic acid construct produces a fusion polypeptide comprising a CAR polypeptide and a SURVIVIN protein.
  • the CAR polypeptide functions with the SURVIVIN protein through a linker comprising a cleavable site. sexual connection.
  • the nucleic acid construct has the following structure: car-(2A)-e, wherein, car represents the nucleic acid encoding the chimeric antigen receptor, and 2A represents the oligonucleotide encoded from the cleaved 2A peptide , e represents a functionally fused protein-encoding nucleic acid, and "-" represents a phospholipid bond connecting adjacent nucleotides or an oligonucleotide encoding a connecting peptide; wherein, the functionally fused protein-encoding nucleic acid encodes a Survivin gene.
  • the present invention also provides a fusion polypeptide having the following structure: CAR-(2A)-E, wherein, CAR represents a chimeric antigen receptor molecule, 2A represents a self-cleaving 2A peptide, and E represents a functional A fusion protein, and "-" represents a peptide bond or a connecting peptide connecting adjacent amino acids; wherein, the functionally fused protein is Survivin protein.
  • CAR-based immune cells nucleic acid constructs, CAR fusion polypeptides and components thereof of the present invention are described in detail below.
  • the CAR-based immune cells of the present invention may comprise any such combination features, and likewise the nucleic acid constructs and CAR fusion polypeptides of the present invention may also comprise any such combination features.
  • Survivins useful in the present invention include full-length proteins or functional fragments thereof, or variants thereof (including natural allelic variants or species homologues).
  • the amino acid sequence of Survivin from human is given under accession number UniProtKB-015392.
  • Various survivin homologs from other species have also been published, for example, survivin from Felis catus is disclosed under Genbank accession number AB182320.1; survivin from Canis familiaris is disclosed under Genbank accession number AY741504.1, NM_001003348.1, AB180206 .1, AB095108.1, AB095108and NM_001003019; survivin from mice is published under Genbank accession number AAD26200.1. Conway EM et al.
  • CAR-T cells containing survivin or its fragments or variants can be detected and/or screened in the same or similar manner as in the examples, including but not limited to, increasing the persistence of CAR-T cells and improving their resistance to The effect of tumor action.
  • the CAR-T cells recombinantly expressing survivin after co-cultivating with target cells for a period of time, compared with CAR-T cells not expressing survivin, the CAR-T cells recombinantly expressing survivin have an increased survival rate and/or apoptotic ratio.
  • TSCM cells with cell surface markers CD3+CD8+CCR7+CD45RO-CD27+CD95+ in the CAR-T cell population can be identified by flow cytometry.
  • the Survivin protein comprises: i) the amino acid sequence of SEQ ID NO:5; ii) has at least one, two or three modifications to the amino acid sequence of SEQ ID NO:5 but No more than 30, 20 or 10 modified amino acid sequences; or iii) an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO:5.
  • the Survivin-encoding polynucleotide that can be used in the present invention may be any polynucleotide comprising a nucleotide sequence encoding the survivin protein in any of the above-mentioned embodiments of the present invention.
  • the survivin-encoding polynucleotide comprises amino acids encoding SEQ ID NO: 5 or a variant thereof, e.g., at least 95%, 96%, 97%, 98%, or 99% identical thereto sequence.
  • the polynucleotide encoding survivin comprises the nucleotide sequence of SEQ ID NO: 2 or a variant thereof, e.g., at least 95%, 96%, 97%, 98%, or 99% thereof An amino acid sequence of identity; or a nucleotide sequence that hybridizes thereto under stringent hybridization conditions.
  • CAR Chimeric antigen receptor
  • the CAR polypeptides that can be used in the present invention are not particularly limited.
  • the CAR polypeptide of the invention comprises an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic domain.
  • the cytoplasmic domain of a CAR polypeptide of the invention comprises an intracellular signaling domain.
  • the cytoplasmic domain of the CAR polypeptide of the invention comprises a co-stimulatory domain and a cytoplasmic signaling domain.
  • the chimeric antigen receptor (CAR) molecule according to the present invention comprises from N-terminus to C-terminus: composed of (a) an antigen-binding domain specifically binding to a tumor antigen and (b) a hinge or spacer ; (c) a transmembrane domain; and (d) an intracellular signaling domain.
  • the CAR molecule according to the present invention comprises from the N-terminus to the C-terminus: (a) an antigen-binding domain specifically binding to a tumor antigen, (b) a hinge region or a spacer region; (c) a transmembrane domain ; (d) costimulatory domain; and (e) intracellular signaling domain.
  • the target antigen for the CAR polypeptide of the present invention is a membrane antigen expressed on the surface of a target cell, especially a tumor cell, such as a tumor-specific antigen or a tumor-associated antigen.
  • Tumors that may be mentioned include hematological and solid tumors, both primary and metastatic.
  • the target antigen is a tumor cell surface antigen comprising an antigenic cancer epitope immunologically recognized by tumor infiltrating lymphocytes (TILs) derived from a mammal.
  • TILs 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 the group consisting of: CD19, adrenergic A2 receptor (EphA2), folate receptor (FRa) , Mesothelin, EGFRvIII, IL-13Ra, CD123, CD33, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof.
  • the tumor antigen is membrane antigen CD19 or EphA2.
  • 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 confer specific recognition and binding to the CAR molecule and the CAR-T cells containing the CAR molecule ability to target antigens.
  • the extracellular antigen-binding domain of the CAR molecule according to the present invention is a polypeptide molecule having binding affinity for a target antigen.
  • the CAR according to the invention comprises an antigen binding domain derived from an antibody or antibody fragment.
  • the antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL).
  • the antigen-binding domain comprises a scFv formed by linking VL and VH via a linker.
  • scFv can be produced by linking the VH and VL regions together using a flexible polypeptide linker according to methods known in the art.
  • scFv molecules comprise a flexible polypeptide linker 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.
  • Linker sequences can comprise any naturally occurring amino acid.
  • the peptide linker of the scFv consists of amino acid residues such as glycine and/or serine, alone or in combination, to link the variable heavy 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 comprises multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO: 29).
  • the linker comprises the amino acid sequence GSTSGSGKPGSGEGSTKG.
  • the scFv used in the present invention comprises 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 may be derived from natural or synthetic sources.
  • the transmembrane domain may be derived from a membrane-bound protein or a transmembrane protein, such as a transmembrane domain 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 present invention can be linked to the extracellular region of the CAR via a hinge region/spacer.
  • a hinge region/spacer For transmembrane regions and hinge/spacer regions that can be used in CAR polypeptides, see, eg, Kento Fujiwara et al., Cells 2020, 9, 1182; doi:10.3390/cells9051182.
  • the cytoplasmic domain comprised in the CAR polypeptide of the present invention comprises an intracellular signaling domain.
  • the intracellular signaling domain is capable of activating at least one immune effector function of the immune cells into which the CAR of the present 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 helper activity, including secretion of cytokines.
  • cytoplasmic domains for use in CAR polypeptides of the invention include the cytoplasmic domains of T cell receptors (TCRs) and/or co-receptors that can function to initiate signal transduction following binding of the extracellular domain to a target antigen. sequences, as well as any derivatives or variants of these sequences and any recombinant sequences having the same functional capabilities.
  • TCRs T cell receptors
  • 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., the primary intracellular signaling domain) and those that act in an antigen-independent manner to provide co-activation.
  • a CAR polypeptide of the invention comprises a cytoplasmic domain that provides a primary intracellular signaling domain, eg, the intracellular signaling domain of CD3 ⁇ .
  • the cytoplasmic domain of the CAR polypeptide of the invention further comprises a secondary signaling domain, eg, a co-stimulatory domain from a co-stimulatory molecule.
  • the cytoplasmic region of the CAR polypeptide of the invention comprises one or more co-stimulatory domains in tandem with the CD3 ⁇ intracellular signaling domain, such as the co-stimulatory domains of 4-1BB (also known as CD137) and CD28 area.
  • the CAR polypeptide of the present 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.
  • Signal peptides/leaders of any eukaryotic origin eg, signal peptides/leaders of mammalian or human secreted protein origin, may be used.
  • a chimeric antigen receptor (CAR) polypeptide according to the invention comprises an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic domain.
  • the antigen binding domain is an antigen binding domain targeting 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 binding to CD19 comprises: heavy chain complementarity determining region 1 (HC CDR1) of the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 9, 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 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 comprises: i) the amino acid sequence of SEQ ID NO: 9; ii) having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 9 but no more than 30, 20 or 10 A modified amino acid sequence; or iii) an amino acid sequence with 95-99% identity to 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) has at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 8 but no more than 30, 20 or 10 A modified amino acid sequence; or iii) an amino acid sequence having 95-99% identity to 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) having 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 with 95-99% identity to SEQ ID NO: 11.
  • the transmembrane domain comprises a transmembrane domain of a protein selected from the group consisting of: 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 receptors, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors , integrins, and activating NK cell receptors.
  • a protein selected from the group consisting of: 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
  • 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) at least one, two or three modifications but no more than 5 modifications comprising the amino acid sequence of SEQ ID NO: 13 or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO: 13.
  • the cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of: TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b or CD66d.
  • the cytoplasmic domain comprises the functional signaling domain of the CD3 ⁇ protein (also referred to herein, the CD3 ⁇ signaling domain).
  • the cytoplasmic domain comprises: i) the amino acid sequence of SEQ ID NO: 15; ii) at least one, two or three modifications but no more than 20 of the amino acid sequence comprising SEQ ID NO: 15, 10 or 5 modified amino acid sequences; or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO: 15.
  • the cytoplasmic domain further comprises a co-stimulatory domain of a protein selected from the group consisting of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling Lymphocyte activation molecule (SLAM protein), activating NK cell receptor, CD8, ICOS, DAP10, DAP12, OX40, CD40, GITR, 4-1BB (ie CD137), CD27 and CD28.
  • the cytoplasmic domain comprises a costimulatory domain of a protein selected from the group consisting of the costimulatory domains of CD28, CD27, 4-1BB, ICOS and OX40.
  • the cytoplasmic domain comprises a co-stimulatory domain of a protein selected from CD28 and 4-1BB (ie CD137), or a combination thereof. In one embodiment, the cytoplasmic domain comprises a co-stimulatory domain from CD28. In one embodiment, the cytoplasmic domain comprises: i) the amino acid sequence of SEQ ID NO: 14; ii) at least one, two or three modifications but no more than 20 of the amino acid sequence comprising SEQ ID NO: 14, 10 or 5 modified amino acid sequences; or iii) an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 14.
  • the CAR polypeptide comprises a transmembrane domain from CD28 or a cytoplasmic domain comprising a signaling domain from CD3 ⁇ .
  • the cytoplasmic domain of the CAR polypeptide comprises: a co-stimulatory signal from CD28 and a functional signaling domain from CD3 ⁇ .
  • the CAR polypeptide comprises: a transmembrane domain from CD28, and a cytoplasmic domain comprising a co-stimulatory signal from CD28 and a functional signaling domain from CD3 ⁇ .
  • the CAR polypeptide of the invention comprises: (a) antigen binding domain; (b) hinge/spacer; (c) transmembrane domain; (d) costimulatory from 4-1BB or CD28 domain; and (e) a functional signaling domain from CD3 ⁇ .
  • the 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 is selected from 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 comprises: i) the amino acid sequence of SEQ ID NO: 12; ii) at least one, two or three modifications but no more than 5 of the amino acid sequence comprising SEQ ID NO: 12 A modified amino acid sequence; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 12.
  • the expressions "hinge”, “hinge region” and “hinge domain” are used interchangeably.
  • the CAR polypeptide further comprises a leader peptide or 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 the amino acid sequence of SEQ ID NO:7.
  • the 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 to the amino acid sequence of SEQ ID NO:4.
  • the CAR-encoding nucleic acid that can be used in the present invention may be any polynucleotide comprising a nucleotide sequence encoding a CAR polypeptide according to any of the above-mentioned 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., at least 95%, 96%, 97%, 98%, or 99% identical thereto.
  • the CAR-encoding nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 or a variant thereof, e.g., at least 95%, 96%, 97%, 98%, or 99% identical thereto amino acid sequence; or a nucleotide sequence that hybridizes thereto under stringent hybridization conditions.
  • the present invention provides one or more nucleic acid molecules encoding a polynucleotide encoding a CAR polypeptide according to the present invention and a polynucleotide encoding a SURVIVIN protein according to the present invention.
  • the nucleic acid molecule encoding the polynucleotide of the CAR polypeptide according to the present invention is different from the nucleic acid molecule encoding the polynucleotide of the SURVIVIN protein according to the present invention.
  • the polynucleotide encoding the CAR polypeptide according to the present invention and the polynucleotide encoding the SURVIVIN protein according to the present invention are provided in a single nucleic acid molecule.
  • the car-encoding nucleic acid comprises a polynucleotide encoding a CAR polypeptide according to any of the foregoing embodiments of the present invention, especially a polynucleotide encoding a CD19-targeting CAR polypeptide.
  • the car-encoding nucleic acid comprises a polynucleotide encoding the following domains: an extracellular antigen-binding domain specifically binding to a tumor antigen, a hinge or spacer, a transmembrane domain, and a cytoplasmic domain , wherein the cytoplasmic domain comprises a co-stimulatory domain and an intracellular signaling domain.
  • the car-encoding nucleic acid further comprises an oligonucleotide encoding a signal peptide (eg, GM-CSFR ⁇ signal peptide).
  • the car-encoding nucleic acid comprises: i) the nucleotide sequence of SEQ ID NO: 1; ii) a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO: 1 under stringent hybridization conditions or iii) a nucleotide sequence having at least 90-99% identity to the nucleotide sequence of SEQ ID NO:1.
  • the survivin-encoding nucleic acid comprises a polynucleotide encoding survivin according to any of the preceding embodiments of the invention. In one embodiment, the survivin-encoding nucleic acid comprises a polynucleotide encoding human Survivin, eg, the amino acid sequence of human Survivin under accession number UniProtKB-015392. In one embodiment, the survivin-encoding nucleic acid encodes the amino acid sequence of SEQ ID NO:5.
  • the survivin-encoding nucleic acid comprises: i) the nucleotide sequence of SEQ ID NO:2; ii) a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO:2 under stringent hybridization conditions or iii) a nucleotide sequence having at least 90-99% identity with the nucleotide sequence of SEQ ID NO:2.
  • the CAR polypeptide and the Survivin protein are separately expressed from the nucleic acid construct.
  • a fusion polypeptide comprising both the CAR polypeptide and the Survivin protein is produced from the expression of the nucleic acid construct, wherein the fusion polypeptide comprises an optional protein interposed between the CAR polypeptide and the Survivin protein. Cut linker peptide.
  • the gene encoding the Survivin protein is genetically fused to the 3' end of the polynucleotide encoding the CAR polypeptide using a self-cleaving peptide in an in-frame manner.
  • the nucleic acid molecule is a nucleic acid construct having the following structure: car-(2A)-e, wherein car represents a nucleic acid encoding a chimeric antigen receptor (CAR) polypeptide, and 2A represents a nucleic acid encoding a self-splicing
  • the oligonucleotide of the 2A peptide, e represents the coding nucleic acid of the protein of functional fusion, and "-" represents the phospholipid bond connecting adjacent nucleotides or the oligonucleotide encoding the connecting peptide; wherein, the functional fusion
  • the protein-coding nucleic acid comprises a Survivin gene.
  • Self-cleaving peptides that can be used in the present invention include, but are not limited to, P2A, T2A, E2A or F2A peptides.
  • 2A self-cleaving peptide 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 2A peptide is a P2A peptide.
  • the P2A peptide comprises: i) the amino acid sequence of SEQ ID NO: 19; ii) having at least one, two or three modified amino acids but no more than 5 modified amino acids to the amino acid sequence of SEQ ID NO: 19 sequence; or iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 19.
  • a GSG linker can be inserted at the N-terminus of the 2A peptide to further improve its cleavage efficiency.
  • the present invention also provides a CAR-(2A)-SURVIVIN fusion polypeptide, wherein the CAR may comprise a CAR polypeptide according to any of the foregoing embodiments of the present invention; the 2A may comprise any self-splicing 2A according to the foregoing Peptide; said SURVIVIN may comprise a Survivin protein according to any of the preceding embodiments of the present invention.
  • These components can be connected directly or indirectly through linkers (for example, single amino acid residues or short peptides).
  • the invention also provides a vector into which is inserted a nucleic acid molecule(s) of the invention or a nucleic acid construct of the invention.
  • Expression of the nucleic acid encoding the CAR polypeptide and the nucleic acid encoding the SURVIVIN protein can be achieved by operatively linking the nucleic acid encoding the CAR polypeptide and the nucleic acid encoding the SURVIVIN protein to a promoter, and incorporating the construct into an expression vector.
  • the vector may be suitable for replication and integration in eukaryotes. Common cloning vectors contain transcriptional and translational terminators, initiation sequences and promoters for regulating the expression of the desired nucleic acid sequence.
  • retroviruses provide a convenient platform for gene delivery systems.
  • the nucleic acid constructs 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 cells of the subject 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 construct of the present invention is cloned into a lentiviral vector, so that
  • Retroviruses such as 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 added advantage over vectors derived from onco-retroviruses, such as murine leukemia virus, because they can transduce non-proliferative cells, such as hepatocytes. They also have the added advantage of low immunogenicity.
  • a retroviral vector may also be, for example, a gamma retroviral vector.
  • a gamma retroviral vector may, for example, comprise a promoter, a packaging signal ( ⁇ ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, e.g., encoding a CAR gene.
  • Gamma retroviral vectors may lack viral structural genes such as gag, pol, and env.
  • a promoter capable of expressing a CAR transgene 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 the enzymatic delivery of aminoacyl tRNAs to the ribosome.
  • the EF1a promoter has been used extensively in mammalian expression plasmids and has been shown to efficiently drive CAR expression from transgenes cloned into lentiviral vectors. See, eg, Milone et al., Mol. Ther. 17(8):1453-1464 (2009).
  • CMV immediate early cytomegalovirus
  • This promoter sequence is a constitutively strong promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto.
  • other constitutive promoter sequences can 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) promoters, MoMuLV promoters, avian leukemia virus promoters, Epstein-Barr virus immediate early promoters, Rous sarcoma virus promoters, 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 present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of
  • the invention provides methods of expressing a CAR construct of the invention in mammalian immune effector cells (eg, mammalian T cells or mammalian NK cells) and immune effector cells produced thereby.
  • mammalian immune effector cells eg, mammalian T cells or mammalian NK cells
  • a source of cells eg, immune effector cells, eg, T cells or NK cells
  • T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, 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 of skill in the art, such as Ficoll (TM) separation.
  • the cells from the circulating blood of the individual 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 medium for subsequent processing steps.
  • the 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 by positive or negative selection techniques.
  • anti-CD3/anti-CD28 conjugated beads such as M-450CD3/CD28T
  • the period of time is between about 30 minutes and 36 hours or longer. Longer incubation times can be used to isolate T cells wherever 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 can be accomplished through the process of negative selection using a combination of antibodies directed against surface markers unique to the negatively selected cells.
  • One method is the sorting and/or selection of cells by means of negative magnetic immunoadhesion or flow cytometry using the presence of cells on negatively selected cells Monoclonal antibody cocktail for surface markers.
  • the immune effector cells can be allogeneic immune effector cells, eg, T cells or NK cells.
  • the cell can be an allogeneic T cell, e.g., one lacking expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (e.g., HLA class I and/or HLA class II) T cells.
  • TCR T cell receptor
  • HLA human leukocyte antigen
  • the present invention provides a recombinant vector comprising the nucleic acid construct according to any of the preceding embodiments of the present invention, for example, the vector is selected from DNA vectors, RNA vectors, lentiviral vectors, adenoviral vectors or a retroviral vector, preferably a retroviral vector.
  • the present invention provides a host cell, which comprises the recombinant vector according to any of the foregoing embodiments of the present invention, or has integrated the nucleic acid construct according to any of the foregoing embodiments of the present invention in the chromosome,
  • the cells are immune effector cells, such as T cells or NK cells, for example, the T cells are autologous T cells or allogeneic T cells.
  • the present invention provides a CAR-T cell, wherein the cell comprises a heterologous polynucleotide encoding a CAR polypeptide and a heterologous polynucleotide encoding Survivin, preferably, the cell comprises a A nucleic acid construct according to any preceding embodiment of the invention.
  • T-cell therapy was first applied 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.
  • the Armored CAR-T cells of the present invention containing more TCSM cell subpopulations are obtained, so as to facilitate the application of the CAR-T cells in anti-tumor therapy in subjects.
  • immune effector cells such as T cells (such as patient-specific autologous T cells) are engineered to express the nucleic acid construct of the present invention, and the CAR polypeptide and survivin protein of the present invention are heterologously expressed in said cells .
  • T cells such as patient-specific autologous T cells
  • CAR polypeptide and survivin protein of the present invention are heterologously expressed in said cells .
  • After expanding the engineered immune effector cells such as T cells or NK cells), they are used for adoptive cell therapy (ACT).
  • the immune effector cell when treating a patient with an immune effector cell of the invention heterologously expressing survivin, may be an autologous or allogeneic T cell or NK cell.
  • the immune effector cells of the present invention can improve the long-term survival of cells after adoptive transfer and/or the proportion of TSCM subsets.
  • immune effector cells of the invention that heterologously express survivin 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 invention provides methods of 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 construct of the invention.
  • the present invention also provides the use of the aforementioned immune effector cells of the present invention in the preparation of drugs for treating cancer.
  • the cancers include hematological cancers (eg, leukemias) or solid tumors (eg, gliomas), including primary and metastatic cancers.
  • the immune effector cells of the invention are used in combination with IL-15.
  • Human Nalm-6 cell line, U373 cell line and retroviral packaging cell line PG13 were purchased from American Type Culture Collection (ATCC). All these cells were maintained in RPMI-1640 (Lonza) or DMEM (Lonza) with 10% fetal bovine serum (Biosera) and 10,000 IU/ml penicillin/10,00 ⁇ g/ml streptomycin (EallBio Life Sciences). All cells were cultured in 5% CO 2 , 95% air, 37°C humidified incubator.
  • PBMCs were transfected with retrovirus to obtain transfected CD19-CAR-T cells, which were co-cultured with Nalm-6 (CD19 positive) tumor cells.
  • the transfected CAR-T cells were stimulated with IL-2 or IL-15, respectively.
  • the expression of Survivin in CAR-T cells was detected by mRNA and protein levels, and the apoptosis and surface markers of CAR-T cells were detected by flow cytometry. Detect the phosphorylation level of PI3K/Akt in CAR-T cells.
  • CD19-CAR-Survivin T cells and CD19-CAR-IL-15T cells were constructed to investigate the therapeutic effect of Armored CAR-T cells overexpressing survivin or IL-15 on tumors.
  • PBMCs Peripheral blood mononuclear cells
  • T cells in peripheral blood mononuclear cells were stimulated with anti-CD3 and anti-CD28 particles and then infected with retrovirus.
  • retronectin Thermo, 15ug/ml
  • T cells 1.6 ⁇ 10 6 /ml
  • virus liquid 0.5ml
  • seal the well plate with parafilm centrifuge at 700g for 1 hour, and culture in a 37°C incubator to obtain antigen-specific genetically modified T cells.
  • CAR-T cells were incubated in X-VIVO TM 15 serum-free culture system containing 0.5% normal human AB serum (Biosera) for 24h, and then cultured in X-VIVO TM 15 containing 5% GemCell TM normal human AB serum After adding IL-2 (138U/ml) or IL-15 (10ng/ml) and culturing for 7 days, the expression markers of CAR-T cells were detected by flow cytometry to identify the proportion of T cell subsets. This study was approved by the Institutional Review Board of Beijing Shijitan Hospital, and informed consent was obtained from all participants.
  • Flow cytometry was performed on a FACSCanto Plus instrument (BD Biosciences), and data analysis was performed using FlowJo V.10 (FlowJo, LLC).
  • FITC-labeled mouse anti-human CD3 antibody (BD Biosciences), Alexa Fluor 700-labeled mouse anti-human CD8 antibody (BD Biosciences), BV421-labeled mouse anti-human CD4 antibody (BD Biosciences), V450-labeled mouse Anti-human CD107a antibody (BD Biosciences), BV605-labeled mouse anti-human CD45RO (BD Biosciences), PE-Cy7-labeled mouse anti-human CCR7 (BD Biosciences), Alexa Fluo 700-labeled mouse anti-human CD27 (BD Biosciences ), PE-Cy5-labeled mouse anti-human CD95 (BD Biosciences), and flow cytometry to detect the expression of cell surface markers.
  • CD19-CAR-T cells were detected by flow cytometry; Alexa Fluor700-labeled mouse anti-human EphA2 (R&DSystems ) to stain U373 cells, and flow cytometry to detect the expression of EphA2 on the cell surface.
  • CD8 + CD19-CAR-T cells were stimulated with CD19-positive tumor cells (Nalm-6 cells) alone or after co-culture with IL-2 or IL-15. On day 0, day 7, day 14, day 21 and day 28, Vi-CELL cell viability analyzer was used to count viable cells by trypan blue exclusion method.
  • CAR-T cells (1 ⁇ 10 6 ) were resuspended in 300 ⁇ L of PBS, then fixed with 70% ethanol in a volume of 1 ml. After 10 minutes, cells were washed 3 times with PBS, stained with PI/RNase staining buffer (BD Biosciences) for 15 minutes at room temperature, and analyzed by flow cytometry.
  • RNA quantity and purity were measured using a Nanodrop One spectrophotometer (Thermo Fisher Scientific). Only samples with suitable absorbance measurements ( A260 /A280 of ⁇ 2.0 and A260 / A230 of 1.9-2.2 ) were considered in this study.
  • cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).
  • the survivin gene was amplified using 5'-TTGAATCGCGGGACCCGTTGG-3' (forward primer) and 5'-CAGAGGCCTCAATCCATGGCA-3' (reverse primer) primers.
  • GAPDH amplified with primers 5'-GTCATCCCTGAGCTGAACGG-3' (forward primer) and 5'-TGGGTGTCGCTGTTGAAGTC-3' (reverse primer) were used as controls.
  • the membrane was mixed with rabbit anti-human survivin (abcam), rabbit anti-human P-AKT (CST), rabbit anti-human granzyme A (abcam), rabbit anti-human granzyme B (abcam), mouse anti- ⁇ -actin ( abcam) at 4°C overnight, then incubated with HRP-conjugated goat anti-rabbit secondary antibody/mouse secondary antibody (Santa Cruz Biotechnology) for 1 hour at room temperature, and detected the chemiluminescent reaction using an ECL kit (Thermo Fisher Scientific).
  • the inhibitor Ly294002 (Selleck, 10uM) was added to inhibit the PI3K/AKT pathway, and the expression of survivin was detected by western blotting.
  • CD19-CAR gene (nucleotide sequence as shown in SEQ ID NO: 1), survivin gene (nucleotide sequence as shown in SEQ ID NO: 2) and IL-15 gene (nucleotide sequence as shown in SEQ ID NO: 2) respectively cloned NO: 3 shown).
  • CD19-CAR cDNA was synthesized by a biological company, and using this as a template, the CD19-CAR gene was amplified using the primers CD19-CAR-F and CD19-CAR-R shown in Table 1;
  • the cDNA of the Survivin gene purchased in China was used as a template, and the primers Survivin-F and Survivin-R shown in Table 1 were used to amplify the survivin gene;
  • the cDNA of the IL-15 gene purchased by Sino Biological was used as a template, and the primers shown in Table 1 were used IL-15-F and IL-15-R, amplify the IL-15 gene.
  • the amplified product obtained above was connected to the carrier SFG carrier (addgene) through the method of homologous recombination (ClonExpress II One Step Cloning kit) according to the connection system shown in Table 4, and reacted at 37°C for 30 minutes to construct survivin and CAR co- Expression plasmid CD19-CAR-survivin.
  • the CD19-CAR nucleotide sequence (SEQ ID NO: 1) obtained by the above amplification is as follows:
  • amino acid sequence SEQ ID NO: 4 of the protein expressed by the CD19-CAR gene is as follows:
  • Survivin gene nucleotide sequence (SEQ ID NO: 2) is as follows:
  • the protein expressed by Survivin gene has the amino acid sequence (SEQ ID NO: 5) as follows:
  • the IL-15 gene nucleotide sequence (SEQ ID NO: 3) is as follows:
  • amino acid sequence (SEQ ID NO: 6) of the protein expressed by the IL-15 gene is as follows:
  • 6- to 8-week-old NOD-SCID mice were purchased from Victoria Liver. 2 ⁇ 10 6 NALM-6-GFP cells were intravenously injected into NOD-SCID mice to establish 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 a total of 3 days. Tumor development was monitored using IVIS (IVIS, Xenogen, Alameda, CA, USA). With the limb paralysis of the mice as the experimental endpoint, the mice were sacrificed, and the bone marrow cells and plasma of the mice were obtained. All experiments in mice were approved by the Institutional Review Board of Beijing Shijitan Hospital.
  • mice Six to eight-week-old nude mice were purchased from Victoria Liver. 1 ⁇ 107 CD19-CAR-Survivin T cells were injected subcutaneously. Observe and record for tumor formation.
  • Example 1 IL-15 promotes the killing effect of antigen-stimulated CAR-T cells
  • T cells were isolated from healthy donor PBMCs and stimulated with anti-CD3/CD28beads. Two days later, T cells were infected with a retroviral vector encoding a CAR based on a CD19-specific mAb. The transduction efficiency was detected by flow cytometry, and the results showed that 54.5% of the cells expressed CD19-specific CAR (Fig. 1A).
  • CAR-T cells were mixed with IL-2 (138U/ml) (CAR-T/IL-2) or IL- 15 (10ng/ml) (CAR-T/IL-15) was cultured for 14 days (Fig. 1B).
  • the CD19 positive cell line (Nalm-6) was co-cultured with CAR-T cells for 6 hours to study the activation status of CAR-T cells.
  • the expression of CD107a was detected by flow cytometry, and the results showed that IL-15 increased the expression of CD107a on the surface of CD8 + T cells ( FIG. 1C ).
  • Elispot and ELISA detection it was found that compared with CAR-T/IL-2, CAR-T/IL-15 produced more IFN ⁇ expression in cells ( Figure 1D) and in culture medium ( Figure 1E).
  • the anti-tumor activity of CAR-T cells was also tested.
  • FIG. 1F shows that IL-15 promotes the expression of granzyme A and granzyme B with the prolongation of the co-culture time of target cell Nalm-6 antigen and CAR-T cells; CAR-T cells cultured without cytokines, granzyme A and granzyme B expression was the lowest.
  • Example 2 IL-15 promotes T cell proliferation and Tscm formation under antigen stimulation
  • IL-15 and IL-2 are cytokines related to cell proliferation, we directly use cell counting to determine the effect of IL-15 and IL-2 on cell proliferation.
  • CD132 is a co-receptor subunit of IL-2 and IL-15, and its expression was significantly upregulated under the induction of IL-15 (Fig. 2E).
  • Tscm cells (CD45RO - CCR7 + CD27 + CD95 + ), which represent the long-term persistence ability of CAR-T cells, were studied, and the results showed that IL-15 promoted Tscm in CD8 + CAR-T cells compared with IL-2.
  • Example 3 Observation of IL-15-mediated effects in different targeted CAR-T cells
  • EphA2-CAR-T cells which Cells utilize the 4-1BB co-stimulatory domain.
  • sequence information of EphA2-CAR please refer to CN202110919075.X.
  • EphA2-CAR-T cells cultured with IL-15 were less depleted and had higher cell proliferation compared to cells cultured with IL-2 (Fig. 3A).
  • EphA2-CAR-T cells cultured with IL-15 exhibited a more multifunctional phenotype and produced more IFN ⁇ (Fig. 3B).
  • RTCA real-time cell growth monitoring
  • GFP-Luc flow detection results showed that the anti-tumor activity of EphA2-CAR-T/IL-15 cells was enhanced ( Figure 3C and 3D).
  • Example 4 IL-15 up-regulates the expression of Survivin by activating the PI3K/Akt signaling pathway
  • RNA sequencing technology was used to detect differentially expressed genes in CAR-T cells cultured with IL-2 and IL-15.
  • an evolutionarily conserved eukaryotic protein survivin that is critical to cell division and can inhibit cell death was selected for further study.
  • the expression of survivin was detected by PCR and Western blot. The results showed that both IL-2 and IL-15 could up-regulate the mRNA level and protein level of survivin in CAR-T cells under antigen stimulation ( Figure 4A and 4B).
  • FIG. 5A A plasmid vector with CD19-CAR linked to survivin gene or IL-15 gene was constructed (Fig. 5A), and these co-expression retroviral vectors were introduced into T cells.
  • the percentage of CAR-positive T cells was adjusted to be consistent (Fig. 5B).
  • Fig. 5C shows CD19-CAR-IL-15 T cells release IL-15 into the medium.
  • RNA of CAR-T cells was extracted, and the relative expression of survivin was detected by Q-PCR.
  • the results showed that CAR-T cells overexpressing survivin had the highest survivin expression (Fig. 5D).
  • CD19-CAR-IL-15 T cells showed increased expression of survivin compared with CD19-CAR T cells, indicating the induction of survivin expression by IL-15.
  • Example 6 Survivin overexpressed CAR-T cells showed higher proliferation ability and poorly differentiated phenotype in vitro.
  • Vi-CELL cell viability analyzer was used to directly count live cells, showing that Armored CAR-T cells overexpressing survivin and IL-15 exhibited higher proliferation ability (Figure 6A).
  • IL-2 is a growth factor for T cells
  • the concentration of IL-2 in the supernatant of Armored CAR-T was measured, and the results showed that, compared with CD19-CAR T cells, CD19-CAR-survivin and CD19- CAR-IL-15T cells released more IL-2 (Fig. 6B).
  • CD132 is a co-receptor subunit chain of IL-2 and IL-15
  • the expression of CD132 was detected, and the results showed that the expression of CD132 on the surface of CD19-CAR-survivin and CD19-CAR-IL-15 T cells was 22.7%, respectively and 15.0%, while the expression of CD132 on the surface of CD19-CAR T cells was 51.3% (Fig. 6C).
  • CD19-CAR-survivin and CD19-CAR-IL-15T cells showed less IFN ⁇ production, which means that the differentiation phenotype of CD19-CAR-survivin and CD19-CAR-IL-15T cells was less Low.
  • survivin is an important inhibitor of apoptosis
  • Example 7 Armored CAR-T cells exhibit enhanced anti-tumor activity in vivo.
  • NALM-6-GFP cells were intravenously injected into NOD-SCID mice to generate xenograft mouse tumor models.
  • 1 ⁇ 10 7 Armored CAR-T cells were injected intravenously for three consecutive days.
  • Non-transduced T cells (NT) served as a negative control, and tumor burden in mice was monitored for 100 days (Fig. 7A). as the picture shows.
  • the survival time of the mice treated with CD19-CAR-Survivin T cells was prolonged, and two of the mice had no tumor recurrence and survived for more than 100 days (Fig. 7B and 7D).
  • Example 8 The stronger anti-tumor activity of CD19-CAR-Survivin T cells in vivo is positively correlated with its longer survival period
  • Figure 8B shows that mice in the CD19-CAR-Survivin T treatment group contained more human IL-15 in their blood. These results suggest that CD19-CAR-Survivin T cells have longer persistence in mice, thus more IL-15 production, and stronger antitumor activity.
  • CD19-CAR-Survivin T cells were subcutaneously injected into nude mice, and tumor growth was recorded. The results showed that no tumor growth was observed for more than 5 months so far in the experiment ( FIG. 8C ), and the mice were healthy.

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Abstract

Provided are a CAR-T cell that increases Survivin expression and the use thereof, alone or in combination with IL-15, in the preparation of an anti-tumor drug.

Description

增加Survivin表达的Armored CAR-T细胞及其抗肿瘤应用Armored CAR-T cells with increased Survivin expression and their anti-tumor applications 技术领域technical field
本发明涉及医学领域，特别是涉及一种增加Survivin表达的CAR-T细胞及其单独和联合IL-15在制备抗肿瘤药物中的应用。The present invention relates to the medical field, in particular to a CAR-T cell that increases the expression of Survivin and its application alone or in combination with IL-15 in the preparation of anti-tumor drugs.
背景技术Background technique
Survivin是一种进化上保守的真核蛋白质，对细胞分裂至关重要，并通过拮抗半胱天冬酶活性发挥凋亡抑制剂的作用[1,2]。因此，它作为一个潜在的肿瘤治疗靶点受到了广泛的关注。此外，Survivin在发育过程中[3]和增殖的成体细胞中表达[4]，特别是激活的T淋巴细胞[5]和自我更新的干细胞中表达[6]。因此，Survivin是活跃增殖细胞的标志物。Survivin is an evolutionarily conserved eukaryotic protein that is essential for cell division and acts as an inhibitor of apoptosis by antagonizing caspase activity [1,2]. Therefore, it has received extensive attention as a potential tumor therapeutic target. In addition, Survivin is expressed during development [3] and in proliferating adult cells [4], especially in activated T lymphocytes [5] and self-renewing stem cells [6]. Therefore, Survivin is a marker of actively proliferating cells.
白细胞介素-15(IL-15)是参与T细胞分化和稳态调节的常见细胞因子[7]。因与白细胞介素-2(IL-2)在细胞因子受体生物学上的相似性，IL-15引起人们的广泛关注。IL-15和IL-2受体均为异三聚体复合物，这些复合物由IL-2的β和γc亚基以及独特的α亚基组成[7]。由于共享IL-2受体亚单位(IL-2/15Rβγ)，IL-15和IL-2具有许多共同的生物学活性，包括活化的T细胞和NK细胞的生长和迁移，以及诱导B细胞的增殖和分化[8]。IL-2具有很强的扩增T细胞和刺激T细胞功能的能力，是最早用于临床癌症试验的细胞因子。然而，IL-2的较强毒性副作用限制了其应用。最近，IL-15因其低毒性，在临床上的使用得到进一步加强，并且有报道，过表达IL-15或给予rIL-15可以保护小鼠免受各种感染[9,10]。Interleukin-15 (IL-15) is a common cytokine involved in T cell differentiation and homeostatic regulation [7]. IL-15 has attracted widespread attention due to its biological similarity to interleukin-2 (IL-2) in cytokine receptors. Both the IL-15 and IL-2 receptors are heterotrimeric complexes composed of the β and γc subunits of IL-2 and a unique α subunit [7]. Due to the shared IL-2 receptor subunit (IL-2/15Rβγ), IL-15 and IL-2 share many biological activities, including the growth and migration of activated T cells and NK cells, and the induction of B cell Proliferation and differentiation [8]. IL-2 has a strong ability to expand T cells and stimulate T cell functions, and is the earliest cytokine used in clinical cancer trials. However, the strong toxic side effects of IL-2 limit its application. Recently, the clinical use of IL-15 has been further strengthened due to its low toxicity, and it has been reported that overexpression of IL-15 or administration of rIL-15 can protect mice from various infections [9,10].
CAR是一种合成分子，由细胞外肿瘤抗原结合结构域、以及与之相连的铰链、跨膜和细胞内信号结构域组成，其通过特异性识别肿瘤细胞上表达的表面蛋白，诱导针对肿瘤细胞的T细胞反应，以根除肿瘤[11]。表达CAR的T细胞(CAR-T cells)可以通过CAR分子胞外结构域的单链可变片段(single chain variable fragment,scFv)直接识别肿瘤相关抗原,然后被胞内信号转导激活,释放多种细胞因子、穿孔素、颗粒酶、干扰素等,诱导肿瘤细胞凋亡[12,13]。然而，有大量临床前和临床证据表明，CAR-T细胞易衰竭且持久性差，这限制了该免疫疗法的有效性[14]。CAR is a synthetic molecule consisting of an extracellular tumor antigen-binding domain, a hinge, a transmembrane and an intracellular signaling domain connected to it, which induces targeting of tumor cells by specifically recognizing surface proteins expressed on tumor cells. T cell responses to eradicate tumors [11]. T cells expressing CAR (CAR-T cells) can directly recognize tumor-associated antigens through the single chain variable fragment (scFv) of the extracellular domain of the CAR molecule, and then be activated by intracellular signal transduction to release multiple antigens. Various cytokines, perforin, granzyme, interferon, etc., induce tumor cell apoptosis[12,13]. However, there is a large amount of preclinical and clinical evidence that CAR-T cells are prone to exhaustion and poor persistence, which limits the effectiveness of this immunotherapy [14].
T细胞激活是适应性免疫应答的核心。T细胞的扩增、分化和存活，取决于来自TCR结合、细胞因子受体和共刺激分子的多重信号的整合。这些信号在T细胞中导致PI3K/AKT通路信号传导网络的活化。已经显示，PI3K/AKT通路的关键成分PI3K和AKT在大多数癌症中被过度激活，并因此是抗癌研究的焦点。然而，对于调节PI3K-AKT通路在CAR-T细胞中所起的作用，目前仍存在许多争议。Ryan Urak等指出，在离体扩增CAR-T细胞的过程中，阻断CAR T细胞中的AKT信号通路，与产生低分化记忆细胞相关，并且由此可以赋予比未处理细胞更大的生存优势[15]。但其他的数据指出，对于T细胞的发育、分化和激 活机制，PI3K-Akt-mTOR通路是调节的关键[16]。活化的AKT通过抑制Bcl-2家族促凋亡成员而促进细胞存活；而mTOR，作为PI3K/Akt信号转导的重要下游效应子之一，是促进细胞存活和增殖的蛋白质翻译所需的一种丝氨酸/苏氨酸蛋白激酶[17]。因此，PI3K/AKT通路活性在CAR-T细胞过继免疫治疗中的作用，仍有待进一步研究。T cell activation is at the heart of the adaptive immune response. T cell expansion, differentiation, and survival depend on the integration of multiple signals from TCR binding, cytokine receptors, and co-stimulatory molecules. These signals lead to activation of the PI3K/AKT pathway signaling network in T cells. PI3K and AKT, key components of the PI3K/AKT pathway, have been shown to be overactivated in most cancers and are therefore the focus of anticancer research. However, there are still many controversies about the role of regulating the PI3K-AKT pathway in CAR-T cells. Ryan Urak et al pointed out that in the process of ex vivo expansion of CAR-T cells, blocking the AKT signaling pathway in CAR T cells is associated with the generation of poorly differentiated memory cells, and thus can confer greater survival than untreated cells Advantages [15]. However, other data point out that the PI3K-Akt-mTOR pathway is the key to the regulation of T cell development, differentiation and activation mechanisms [16]. Activated AKT promotes cell survival by inhibiting pro-apoptotic members of the Bcl-2 family; mTOR, as one of the important downstream effectors of PI3K/Akt signal transduction, is required for protein translation that promotes cell survival and proliferation Serine/threonine protein kinase [17]. Therefore, the role of PI3K/AKT pathway activity in CAR-T cell adoptive immunotherapy remains to be further studied.
IL-15作为一种重要的免疫刺激因子，与IL-15受体结合后，可以促进初始
Figure PCTCN2022079966-appb-000001
和记忆性(Memory)CD8 +T细胞的存活[18]，并增强CD4 +和CD8 +T细胞的交叉启动以及TEM细胞(效应记忆T细胞)和CD44 hi或CD122 hi记忆性CD8 +T细胞的增殖[19]。此外，已有报道，在CAR-T细胞的体外培养中，IL-15可以促进产生具有过继免疫细胞治疗潜能的记忆T细胞，并且在动物模型中，可配合CAR-T细胞的抗肿瘤作用，有效增强CAR-T细胞的肿瘤侵袭性和抗肿瘤作用的持久性[20]。Valentina Hoyos等[21]也报道了,表达IL-15的CAR-T细胞可通过IL-15信号转导形成记忆干细胞表型，从而形成长期持久性。然而，IL-15促进CAR-T细胞的存活和抗肿瘤作用的具体作用机制尚未明确。 As an important immunostimulatory factor, IL-15 can promote the initial
Figure PCTCN2022079966-appb-000001
and the survival of memory (Memory) CD8 + T cells[18], and enhance the cross-priming of CD4 + and CD8 + T cells and the activation of TEM cells (effector memory T cells) and CD44 hi or CD122 hi memory CD8 + T cells Proliferation [19]. In addition, it has been reported that in the in vitro culture of CAR-T cells, IL-15 can promote the generation of memory T cells with adoptive immune cell therapy potential, and in animal models, it can cooperate with the anti-tumor effect of CAR-T cells, Effectively enhance the tumor invasiveness and durability of anti-tumor effects of CAR-T cells [20]. Valentina Hoyos et al. [21] also reported that CAR-T cells expressing IL-15 can form a memory stem cell phenotype through IL-15 signal transduction, thereby forming long-term persistence. However, the specific mechanism by which IL-15 promotes the survival and anti-tumor effects of CAR-T cells has not yet been clarified.
CAR-T细胞治疗是近年来发展迅速的一种过继免疫治疗技术。如何提高抗肿瘤免疫细胞的有效性和远期疗效，对于提高细胞免疫治疗的疗效至关重要。因此，本领域持续需要提供可以用于产生更持久、更有效CAR-T细胞治疗的新技术方案。CAR-T cell therapy is an adoptive immunotherapy technology that has developed rapidly in recent years. How to improve the effectiveness and long-term efficacy of anti-tumor immune cells is crucial to improving the efficacy of cellular immunotherapy. Therefore, there is a continuing need in the art to provide new technological solutions that can be used to generate longer-lasting, more effective CAR-T cell therapy.
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发明内容Contents of the invention
干细胞样记忆性T细胞(stem like memory T cells,TSCM)具有抗原特异性记忆、初始T细胞的干细胞样特征和体内长期存活等特点，具有潜在的临床应用价值。本发明人在研究IL-15对CAR-T细胞的细胞存活的影响及其作用机制的过程中，令人惊奇地发现，IL-15通过PI3K/Akt通路来调节survivin表达，促进CAR-T细胞中TSCM样细胞群体形成，增强CAR-T细胞的持久性；而增加survivin表达的Armored CAR-T细胞(武装CAR-T细胞)不仅促进该CAR-T细胞存活，也显著提升其在体内的长期抗肿瘤作用。Stem like memory T cells (TSCM) have the characteristics of antigen-specific memory, stem cell-like characteristics of naive T cells and long-term survival in vivo, and have potential clinical application value. In the process of studying the effect of IL-15 on the cell survival of CAR-T cells and its mechanism of action, the inventors surprisingly found that IL-15 regulates the expression of survivin through the PI3K/Akt pathway and promotes the expression of CAR-T cells. The formation of a TSCM-like cell population in the middle enhances the persistence of CAR-T cells; and the Armored CAR-T cells (armed CAR-T cells) with increased survivin expression not only promote the survival of the CAR-T cells, but also significantly improve their long-term survival in vivo. Antitumor effect.
因此，本发明旨在提供一种增加Survivin表达的CAR-T细胞及其单独和联合IL-15在制备抗肿瘤药物中的应用，以解决上述现有技术存在的问题。Therefore, the present invention aims to provide a CAR-T cell with increased Survivin expression and its application alone or in combination with IL-15 in the preparation of anti-tumor drugs, so as to solve the above-mentioned problems in the prior art.
为实现上述目的，本发明提供了如下方案：To achieve the above object, the present invention provides the following scheme:
本发明提供一种核酸构建体，所述核酸构建体具有如下所示结构：car-(2A)-e，其中，car表示嵌合抗原受体编码多核苷酸序列，2A表示自剪切序列，e表示功能性融合的蛋白编码序列，所述功能性融合的蛋白编码基因为Survivin基因，优选地，所述Survivin基因的核苷酸序列如SEQ ID NO：2所示。The present invention provides a nucleic acid construct, the nucleic acid construct has the following structure: car-(2A)-e, wherein, car represents a chimeric antigen receptor encoding polynucleotide sequence, 2A represents a self-cleaving sequence, e represents the protein coding sequence of functional fusion, and the protein coding gene of described functional fusion is Survivin gene, preferably, the nucleotide sequence of described Survivin gene is as shown in SEQ ID NO:2.
优选的是，所述嵌合抗原受体的抗原结合结构域特异性靶向肿瘤抗原，所述肿瘤抗原为膜抗原CD19，所述自剪切序列为P2A，“-”表示连接相邻核苷酸的磷脂键或任选地连接肽。Preferably, the antigen-binding domain of the chimeric antigen receptor specifically targets a tumor antigen, the tumor antigen is the membrane antigen CD19, the self-cleaving sequence is P2A, and "-" indicates that adjacent nucleosides are connected Acidic phospholipid bonds or optionally linking peptides.
本发明还提供一种重组载体，包括所述的核酸构建体。The present invention also provides a recombinant vector, including the nucleic acid construct.
本发明还提供一种宿主细胞，其含有所述的重组载体或者染色体中整合有外源的所述的核酸构建体。The present invention also provides a host cell containing the recombinant vector or the exogenous nucleic acid construct integrated in the chromosome.
本发明还提供一种药物组合物，其包括药学上可接受的载体以及所述的核酸构建体、所述的重组载体或所述的宿主细胞。The present invention also provides a pharmaceutical composition, which includes a pharmaceutically acceptable carrier and the nucleic acid construct, the recombinant vector or the host cell.
优选的是，所述药物组合物还包括细胞因子IL-15。Preferably, the pharmaceutical composition further includes cytokine IL-15.
本发明还提供如所述的核酸构建体、所述的重组载体或所述的宿主细胞的应用，应用于制备抗肿瘤药物或者制剂中。The present invention also provides the application of the nucleic acid construct, the recombinant vector or the host cell in the preparation of antitumor drugs or preparations.
优选的是，所述肿瘤包括血液学癌症(例如白血病)或者实体瘤。Preferably, the tumor comprises a hematological cancer (eg leukemia) or a solid tumor.
优选的是，所述宿主细胞包括T细胞或NK细胞。Preferably, said host cells comprise T cells or NK cells.
本发明还提供一种制备修饰的T细胞或NK细胞的方法，包括以下步骤：将所述的核酸构建体、或所述的重组载体导入待修饰的T细胞或NK细胞内，构建表面表达异源受体的修饰的T细胞或NK细胞，其中，所述受体选自CAR。The present invention also provides a method for preparing a modified T cell or NK cell, comprising the following steps: introducing the nucleic acid construct or the recombinant vector into the T cell or NK cell to be modified, constructing a surface expression heterogeneous A modified T cell or NK cell derived from a recipient, wherein the recipient is selected from CAR.
本发明公开了以下技术效果：The invention discloses the following technical effects:
本发明从细胞核分子水平上研究了IL-15和CAR-T协同抗肿瘤的作用机制，通过实验验证发现，首 先，IL-15可以增加CAR-T细胞对多种细胞系的细胞毒性；IL-15通过增加TSCM的百分率而增强CAR-T细胞的存活。其次，发现IL-15可激活一种重要的抑制细胞凋亡、促进细胞增殖的蛋白survivin。进一步分析表明，IL-15通过PI3K/Akt途径激活survivin，过表达survivin证实survivin促进CAR-T细胞的存活和抗肿瘤效应。上述结果表明，IL-15促进T细胞存活和抗肿瘤作用的分子机制与Tscm的形成和PI3K/Akt/Survivin信号通路活性的调节有关。因此，本发明公开的内容可为细胞存活和Tscm的产生提供新的见解，并为其在过继细胞治疗中的快速应用奠定基础。The present invention studies the synergistic anti-tumor mechanism of IL-15 and CAR-T from the nuclear molecular level. Through experimental verification, it is found that, first, IL-15 can increase the cytotoxicity of CAR-T cells to various cell lines; 15 enhances the survival of CAR-T cells by increasing the percentage of TSCM. Secondly, it was found that IL-15 can activate an important protein survivin that inhibits apoptosis and promotes cell proliferation. Further analysis showed that IL-15 activated survivin through the PI3K/Akt pathway, and overexpression of survivin confirmed that survivin promoted the survival and anti-tumor effects of CAR-T cells. The above results indicate that the molecular mechanism of IL-15 promoting T cell survival and anti-tumor effect is related to the formation of Tscm and the regulation of PI3K/Akt/Survivin signaling pathway activity. Thus, the present disclosure may provide new insights into cell survival and Tscm production and lay the foundation for its rapid application in adoptive cell therapy.
附图说明Description of 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 technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some of the present invention. Embodiments, for those of ordinary skill in the art, other drawings can also be obtained based on these drawings without any creative effort.
图1A为流式检测CD19-CAR-T细胞的CAR的转染效率，PBMC作为对照；Figure 1A shows the transfection efficiency of CAR of CD19-CAR-T cells detected by flow cytometry, and PBMC was used as a control;
图1B为CAR-T细胞分别与IL-2或IL-15培养14天的实验流程简图；Figure 1B is a schematic diagram of the experimental flow of CAR-T cells cultured with IL-2 or IL-15 for 14 days;
图1C为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T与靶细胞Nalm-6共培养(10:1)6小时，流式检测CD107a在T细胞表面的表达；Figure 1C shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T was co-cultured with target cell Nalm-6 (10:1) for 6 hours, and the expression of CD107a on the surface of T cells was detected by flow cytometry Express;
图1D-图1E为分别用IL-15或IL-2培养CD19-CAR-T后，用Elispot试剂盒检测T细胞中的IFNγ的表达(D)，并且培养物上清保留，用ELISA试剂盒(Thermo)检测T细胞IFNγ的释放(E)；Figure 1D-Figure 1E shows that after CD19-CAR-T was cultured with IL-15 or IL-2, the expression of IFNγ in T cells was detected with Elispot kit (D), and the culture supernatant was retained, and ELISA kit was used (Thermo) detection of T cell IFNγ release (E);
图1F为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T与靶细胞Nalm-6共培养(1:1)24小时，收集细胞，流式检测肿瘤细胞残留；Figure 1F shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T and target cell Nalm-6 were co-cultured (1:1) for 24 hours, the cells were collected, and the remaining tumor cells were detected by flow cytometry ;
图1G为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T与靶细胞Nalm-6共培养(10:1)48小时或72小时，收集细胞，Western blot检测颗粒酶A、颗粒酶B的表达；免疫印迹下方数值指示，相对于仅加入抗原进行标化的表达水平。Figure 1G shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T and target cell Nalm-6 were co-cultured (10:1) for 48 hours or 72 hours, and the cells were collected and detected by Western blot Expression of granzyme A, granzyme B; values below immunoblots indicate expression levels normalized to the addition of antigen alone.
图2A为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T与靶细胞Nalm-6共培养(10:1)，每七天计数CD8 +T细胞数； Figure 2A shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T was co-cultured with target cell Nalm-6 (10:1), and the number of CD8 + T cells was counted every seven days;
图2B为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T用CFSE(10μM)染色,与靶细胞Nalm-6共培养(10:1)72小时，流式检测T细胞增殖；Figure 2B shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T was stained with CFSE (10 μM), co-cultured with target cell Nalm-6 (10:1) for 72 hours, flow cytometry Detection of T cell proliferation;
图2C-图2D为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T与靶细胞Nalm-6共培养(10:1)，第7天和14天细胞周期检测；Figure 2C-Figure 2D shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T and target cell Nalm-6 were co-cultured (10:1), cell cycle on day 7 and day 14 detection;
图2E为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T与靶细胞Nalm-6共培养(10:1)24 小时，流式检测CD132在T细胞表面的表达；Figure 2E shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T was co-cultured with target cell Nalm-6 (10:1) for 24 hours, flow cytometric detection of CD132 on the surface of T cells Express;
图2F为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T与靶细胞Nalm-6共培养(10:1)，第7天和14天流式检测Tscm的比例；Figure 2F shows the co-cultivation (10:1) of CD19-CAR-T and target cell Nalm-6 after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, and the Tscm was detected by flow cytometry on the 7th and 14th day Proportion;
图3A为分别用IL-15或IL-2培养EphA2-CAR-T后，EphA2-CAR-T用CFSE(10μM)染色,与靶细胞U373细胞共培养(10:1)72小时，流式检测T细胞增殖；Figure 3A shows that after EphA2-CAR-T was cultured with IL-15 or IL-2, EphA2-CAR-T was stained with CFSE (10 μM), co-cultured with target cells U373 cells (10:1) for 72 hours, and flow cytometric detection T cell proliferation;
图3B为分别用IL-15或IL-2培养EphA2-CAR-T后，用Elispot试剂盒检测T细胞中的IFNγ的表达；Figure 3B shows the expression of IFNγ in T cells detected by Elispot kit after culturing EphA2-CAR-T with IL-15 or IL-2 respectively;
图3C-图3D为分别用IL-15或IL-2培养EphA2-CAR-T后，EphA2-CAR-T与靶细胞U373共培养(2:1)，用RTCA(C)和荧光素(D)检测T细胞对靶细胞的杀伤功能；Figure 3C-Figure 3D shows that after EphA2-CAR-T was cultured with IL-15 or IL-2 respectively, EphA2-CAR-T was co-cultured with target cell U373 (2:1), and treated with RTCA (C) and fluorescein (D ) detecting the killing function of T cells on target cells;
图4A为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T与靶细胞Nalm-6共培养(10:1)48小时和72小时，提取RNA，PCR检测survivin mRNA的表达；Figure 4A shows that after CD19-CAR-T was cultured with IL-15 or IL-2, CD19-CAR-T was co-cultured with target cell Nalm-6 (10:1) for 48 hours and 72 hours, RNA was extracted, and survivin was detected by PCR. mRNA expression;
图4B为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T与靶细胞Nalm-6共培养(10:1)72小时，收集蛋白，Western blot检测survivin的表达；Figure 4B shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T was co-cultured with target cell Nalm-6 (10:1) for 72 hours, the protein was collected, and the expression of survivin was detected by Western blot ;
图4C-图4D为分别用IL-15或IL-2培养CD19-CAR-T后，CD19-CAR-T与靶细胞Nalm-6共培养(10:1)72小时，同时加入ly294002(10μM)，收集蛋白，Western blot检测survivin和P-AKT的表达；Figure 4C-Figure 4D shows that after CD19-CAR-T was cultured with IL-15 or IL-2 respectively, CD19-CAR-T and target cell Nalm-6 were co-cultured (10:1) for 72 hours, and ly294002 (10 μM) was added at the same time , collect protein, and detect the expression of survivin and P-AKT by Western blot;
图5A为Armored CAR的结构示意图对比(CD19-CAR-Survivin，CD19-CAR-IL-15)；Figure 5A is a comparison of the structure of Armored CAR (CD19-CAR-Survivin, CD19-CAR-IL-15);
图5B为流式检测Armored CAR-T细胞的转染效率。Figure 5B shows the transfection efficiency of Armored CAR-T cells detected by flow cytometry.
图5C为Armored CAR-T与靶细胞Nalm-6共培养(10:1)24小时后，收集上清，检测IL-15的浓度。Figure 5C shows that after 24 hours of co-culture (10:1) between Armored CAR-T and target cells Nalm-6, the supernatant was collected and the concentration of IL-15 was detected.
图5D为Armored CAR-T与靶细胞Nalm-6共培养(10:1)24小时后，收集细胞，提取RNA，逆转录，Q-PCR检测survivin的相对表达量。Figure 5D shows the co-culture of Armored CAR-T and target cell Nalm-6 (10:1) for 24 hours, the cells were collected, RNA was extracted, reverse transcribed, and the relative expression of survivin was detected by Q-PCR.
图6A为Armored CAR-T与靶细胞Nalm-6共培养(10:1)，每7天计数细胞量，计算细胞增殖情况。Figure 6A shows the co-culture of Armored CAR-T and target cells Nalm-6 (10:1), counting the number of cells every 7 days, and calculating the cell proliferation.
图6B为Armored CAR-T与靶细胞Nalm-6在不含有IL-2的培养基中共培养(10:1)24小时，收集上清，检测IL-2的浓度。Figure 6B shows that Armored CAR-T and target cell Nalm-6 were co-cultured (10:1) in medium without IL-2 for 24 hours, the supernatant was collected, and the concentration of IL-2 was detected.
图6C和D为Armored CAR-T与靶细胞Nalm-6共培养(10:1)7天，收集细胞，检测细胞表面CD132的表达以及Tscm细胞的比例。Figure 6C and D show the co-culture of Armored CAR-T and target cells Nalm-6 (10:1) for 7 days, the cells were collected, and the expression of CD132 on the cell surface and the ratio of Tscm cells were detected.
图6E为Armored CAR-T与靶细胞Nalm-6共培养(10:1)24小时后，收集上清，检测IFN-γ的浓度。Figure 6E shows that after 24 hours of co-culture (10:1) of Armored CAR-T and target cell Nalm-6, the supernatant was collected and the concentration of IFN-γ was detected.
图6F为Armored CAR-T与靶细胞Nalm-6共培养(10:1)7天，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。Figure 6F shows the co-culture of Armored CAR-T and target cell Nalm-6 (10:1) for 7 days, the cells were collected, stained with dyes Annexin V and 7ADD for 15 minutes, and the survival rate and apoptosis of CAR-T cells were detected by flow cytometry death rate.
图7A为动物实验流程图。Fig. 7A is a flowchart of animal experiments.
图7B为小鼠肿瘤负荷荧光图。Fig. 7B is a fluorescence map of tumor burden in mice.
图7C为小鼠肿瘤负荷荧光统计图。用IVIS仪器(IVIS,Xenogen,Alameda,CA,USA)采集小鼠荧光。每条线代表一只小鼠的荧光变化曲线。Fig. 7C is a fluorescence statistical graph of tumor burden in mice. Mouse fluorescence was collected with an IVIS instrument (IVIS, Xenogen, Alameda, CA, USA). Each line represents the fluorescence change curve of one mouse.
图7D为肿瘤小鼠生存曲线。*表示p值<0.05。Figure 7D is the survival curve of tumor mice. * indicates p-value <0.05.
图8A为实验小鼠到达实验终点后，处死小鼠，收集骨髓细胞，提取RNA，Q-PCR方法检测病毒载体的表达量。RNA逆转录成cDNA后,加入Q-PCR引物(F:AGAACCTAGAACCTCGCTGGA,R:CTGCGATGCCGTCTACTTTG)与SYBR green染料，用荧光定量PCR仪Q6，检测所述表达量。Figure 8A shows that after the experimental mice reached the end point of the experiment, the mice were sacrificed, bone marrow cells were collected, RNA was extracted, and the expression level of the virus vector was detected by Q-PCR. After the RNA was reverse-transcribed into cDNA, Q-PCR primers (F: AGAACCTAGAACCTCGCTGGA, R: CTGCGATGCCGTCTACTTTG) and SYBR green dye were added, and the expression level was detected with a fluorescent quantitative PCR instrument Q6.
图8B为实验小鼠到达实验终点后，处死小鼠，收集血浆，检测小鼠血浆中人IL-15细胞因子的浓度。采用ELISA试剂盒(R&D systeme D1500)，检测IL-15浓度。Figure 8B shows that after the experimental mice reached the end point of the experiment, the mice were sacrificed, the plasma was collected, and the concentration of human IL-15 cytokine in the mouse plasma was detected. ELISA kit (R&D systeme D1500) was used to detect the concentration of IL-15.
图8C为裸鼠成瘤实验结果。Fig. 8C is the result of tumor formation experiment in nude mice.
具体实施方式detailed description
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 present invention will now be described in detail. The detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features and embodiments of the present 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 terminology described in the present invention is only used to describe specific embodiments, and is not used to limit the present invention. In addition, regarding the numerical ranges in the present invention, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in a stated range is encompassed 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 present 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 are described. In case of conflict with any incorporated document, the contents of this specification control.
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 in the specific embodiments of the present invention described herein without departing from the scope or spirit of the present invention. Other embodiments will be apparent to the skilled person from the description of the present invention. The specification and examples in this application are exemplary 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。In order to explain this specification, the following definitions will be used, and whenever 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。As used herein, "comprising", "comprising", "having", "comprising" and so on are all open terms, meaning including but not limited to. Herein, when the term "comprising" or "comprises" is used, unless otherwise specified, it also covers the situation consisting of the mentioned elements, integers or steps. For example, when referring to an antibody variable region that "comprises" a particular sequence, it is also intended to encompass an antibody variable region that consists 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 meant to encompass a numerical value within a range having a lower limit of 5% less and an upper limit of 5% greater than the stated 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 alternatives or two or more of the alternatives.
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、跨膜结构域及胞内信号结构域的重组多肽。在本文中，以小写字母“car”表示编码嵌合抗原受体的核酸。The terms "chimeric receptor", "chimeric antigen receptor" or "CAR" are used interchangeably herein to refer to a recombinant protein comprising at least an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. peptide. Herein, a nucleic acid encoding a chimeric antigen receptor is indicated by the lowercase letter "car".
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。在一个实施方案中，初级信号例如通过TCR/CD3复合体与载有肽的MHC分子的结合引发并且导致介导T细胞反应，包括但不限于增殖、活化、分化等。在本发明的具体CAR中，本发明的任一种或多种CAR的胞质结构域包含胞内信号传导序列，例如，CD3ζ的信号传导序列。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 embodiment, primary signals are elicited, eg, by binding of the TCR/CD3 complex to peptide-loaded MHC molecules and result in mediation of T cell responses including, but not limited to, proliferation, activation, differentiation, and the like. In particular CARs of the invention, the cytoplasmic domain of any one or more CARs of the invention comprises an intracellular signaling sequence, eg, the 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:15中提供的序列或其变体。The term "CD3ζ" is defined as the protein provided under accession number UniProtKB-P20963 or an equivalent thereof. Herein, a "CD3ζ signaling domain" is defined as a stretch of amino acid residues from the cytoplasmic domain of the CD3ζ chain sufficient to functionally transmit the initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of CD3ζ comprises residues 52 to 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 equivalent residues for humans, monkeys, apes, etc.). In one embodiment, the "CD3ζ signaling domain" is the sequence provided in SEQ ID NO: 15 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 to mediate a costimulatory response (eg, 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), activating NK cell receptors, OX40 , CD40, GITR, 4-1BB (ie CD137), CD27 and CD28. In some embodiments, the "co-stimulatory molecule" is CD28, 4-1BB (ie CD137). As used herein, "co-stimulatory domain" refers to the intracellular portion of a co-stimulatory 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 member of the TNFR superfamily, also known as CD137, which has the amino acid sequence provided under UniProtKB-Q07011 accession number or is derived from a non-human species (e.g., mouse, rodent, monkey, ape, etc. ) equivalent residues. Herein, the term "4-1BB co-stimulatory domain" is defined as the cytoplasmic region from 4-1BB, e.g., amino acid residues 214-255 of UniProtKB-Q07011 or from non-human species (e.g., mouse, rodent , monkey, ape, etc.) equivalent residues.
术语“CD28”是指在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。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-Q07011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-Q07011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”定义为来自CD28胞外区的铰链结构域，例如UniProtKB-Q07011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-Q07011 accession number or the equivalent residues from a non-human species (eg, mouse, rodent, monkey, ape, etc.). Herein, the term "4-1BB co-stimulatory domain" is defined as amino acid residues 214-255 of UniProtKB-Q07011 or equivalent residues from non-human species (eg, mouse, rodent, monkey, ape, etc.). Herein, the term "CD28 co-stimulatory domain" is defined as the cytoplasmic region from CD28, e.g., amino acid residues 180-220 of UniProtKB-Q07011 or from non-human species (e.g., mouse, rodent, monkey, etc.) equivalent residues. Herein, the term "CD28 transmembrane domain" is defined as the transmembrane region from CD28, e.g., amino acid residues 153-179 of UniProtKB-Q07011 or from non-human species (e.g., mouse, rodent, monkey, ape etc.) equivalent residues. Herein, the term "CD28 hinge domain" is defined as a hinge domain from the extracellular region of CD28, such as amino acid residues 114-152 of UniProtKB-Q07011 or from a non-human species (e.g., mouse, rodent, monkey, Apes, 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 transformed by introducing a heterologous nucleic acid or protein, or by changing itself Modified natural nucleic acid or protein, or refers to a substance derived from a virus or cell thus modified.
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" are used interchangeably when describing a nucleic acid or protein to mean that the nucleic acid or protein is foreign to the host cell that contains or is to contain the nucleic acid or protein , that is, its location in the host cell is not its natural location in nature. Heterologous nucleic acid sequence also refers to a sequence derived from and introduced into (for example by infection with a viral vector) the same host cell or subject and thus exists in a non-native state, for example, in a different position, in a different The copy number is present, or under the control of different regulatory elements.
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Survivin蛋白、或两者)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。The term "expression cassette" refers to a DNA sequence that encodes and is capable of expressing one or more genes of interest (such as the CAR polypeptide of the present invention, or the Survivin protein, or both). In an expression cassette, typically, a heterologous polynucleotide sequence encoding a gene of interest is functionally linked to expression control sequences.
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 "operably linked", means that the named components are in a relationship permitting them to function in their 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)。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。As used herein, the terms "linker" or "linker" 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). In one embodiment, the connecting peptide is 1-50 amino acids in length, eg, 1, 2, 3, 4, 5 amino acids, or 10, 15, 20, 25, 30 amino acids in length. The connecting peptide that can be used between the components of the CAR fusion polypeptide of the present invention is not particularly limited. Suitable linker peptides can be rationally designed using computer programs that model the three-dimensional structures of proteins and peptides. For example, short oligopeptide linkers or polypeptide linkers can form linkages between building block sequences as desired, eg, glycine-serine doublets, or single amino acids, eg, 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 to refer to additions, deletions, substitutions 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 possesses the desired properties. In some embodiments, the amino acid substitutions are non-conservative amino acid substitutions, ie, the substitution of one amino acid with another amino acid having different structural and/or chemical properties. Amino acid substitutions include 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Survivin)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", "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, especially conservative substitutions, can be introduced into the CAR fusion polypeptide of the present invention or its constituent elements (such as CAR or Survivin) by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis . 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), nonpolar 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Survivin蛋白/编码核酸)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。"Identity percentage (%)" of an amino acid sequence/nucleotide sequence refers to aligning a candidate sequence with a specific amino acid/nucleotide sequence shown in this specification and, if necessary, achieving maximum sequence identity After introducing gaps as percentages, and in the case of amino acid sequences, when not considering any conservative substitutions as part of the sequence identity, the amino acid residues/nuclei of the specific amino acid/nucleotide sequences shown in this specification in the candidate sequence The percentage of amino acid/nucleotide residues with identical nucleotide residues. In some embodiments, the invention contemplates variants of the fusion polypeptides or nucleic acid molecules or constituent elements thereof of the invention relative to the fusion polypeptides or nucleic acid molecules or constituent elements thereof specifically disclosed herein (e.g., CAR polypeptide/ Nucleic acid encoding, or Survivin protein/encoding nucleic acid) sequences have a considerable degree of identity, for example, the identity is at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or higher. Such variants may contain conservative modifications. For purposes of the present invention, percent identity is determined using the publicly available BLAST tool at https://blast.ncbi.nlm.nih.gov with 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。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, 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 a nucleic acid refers to a nucleic acid molecule capable of multiplying another nucleic acid to which it has been linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which 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
Figure PCTCN2022079966-appb-000002
基因递送技术、来自Lentigen的LENTIMAX TM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 inter alia self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that can be used clinically include, for example, but are not limited to, the Lentiviral vector from Oxford BioMedica
Figure PCTCN2022079966-appb-000002
Gene delivery technology, LENTIMAX ™ 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 cell" refers to a cell that participates in an immune response, eg, participates in promoting an immune effector response. Examples of immune effector cells include T cells, eg, α/β T cells and γ/δ 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 rodents). mouse). In particular, an individual or subject is a human.
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" means that the following immunological effects can be exhibited by various means, including but not limited to, for example, causing a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.
II..本发明的核酸分子和多肽II.. Nucleic acid molecules and polypeptides of the present 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survivin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。The present invention has found through in-depth research that in CAR-based immune cells (for example, CAR-T cells and CAR-NK cells), the persistence and/or Anti-tumor immunity.
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survivin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survivin的这种基于CAR的免疫细胞，也称作“Armored 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survivin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。Therefore, in one aspect, the present invention provides a CAR-based immune cell, wherein the immune cell not only comprises a heterologous polynucleotide encoding a CAR polypeptide, but also comprises a heterologous polynucleotide encoding survivin. When the immune cells are T cells, such CAR-based immune cells with recombinantly expressed survivin are also referred to as "Armored CAR-T cells" or "Armored CAR-T cells" herein. In the immune cell, the heterologous polynucleotide encoding the CAR polypeptide and the heterologous polynucleotide encoding survivin may be located on a single nucleic acid molecule, or located on different nucleic acid molecules that are separated.
在再一个方面中，本发明提供了可用于形成本发明的基于CAR的免疫细胞的核酸构建体。在一个实施方案中，本发明的核酸构建体包含编码嵌合抗原受体(CAR)分子的多核苷酸以及编码SURVIVIN蛋白的多核苷酸的核酸构建体。在本发明的核酸构建体中，所述的CAR多肽编码多核苷酸，与所述的SURVIVIN蛋白编码多核苷酸，可以位于相同或不同的表达盒中，并可以分别表达为单独的多肽，或表达为融合多肽。在一个实施方案中，所述核酸构建体表达产生包含CAR多肽和SURVIVIN蛋白的融合多肽，优选地，在所述的融合多肽中，CAR多肽通过包含可剪切位点的接头，与SURVIVIN蛋白功能性连接。在一些优选实施方案中，所述核酸构建体具有如下结构：car-(2A)-e，其中，car表示嵌合抗原受体的编码核酸，2A表示编码自剪切2A肽的寡核苷酸，e表示功能性融合的蛋白编码核酸，且“-”表示连接相邻核苷酸的磷脂键或编码连接肽的寡核苷酸；其中，所述功能性融合的蛋白编码核酸编码Survivin基因。In yet another aspect, the invention provides nucleic acid constructs that can be used to form the CAR-based immune cells of the invention. In one embodiment, the nucleic acid construct of the present invention comprises a polynucleotide encoding a chimeric antigen receptor (CAR) molecule and a polynucleotide encoding a SURVIVIN protein. In the nucleic acid construct of the present invention, the polynucleotide encoding the CAR polypeptide and the polynucleotide encoding the SURVIVIN protein may be located in the same or different expression cassettes, and may be expressed as separate polypeptides, or Expressed as a fusion polypeptide. In one embodiment, the expression of the nucleic acid construct produces a fusion polypeptide comprising a CAR polypeptide and a SURVIVIN protein. Preferably, in the fusion polypeptide, the CAR polypeptide functions with the SURVIVIN protein through a linker comprising a cleavable site. sexual connection. In some preferred embodiments, the nucleic acid construct has the following structure: car-(2A)-e, wherein, car represents the nucleic acid encoding the chimeric antigen receptor, and 2A represents the oligonucleotide encoded from the cleaved 2A peptide , e represents a functionally fused protein-encoding nucleic acid, and "-" represents a phospholipid bond connecting adjacent nucleotides or an oligonucleotide encoding a connecting peptide; wherein, the functionally fused protein-encoding nucleic acid encodes a Survivin gene.
在再一方面，本发明也提供了一种具有如下结构的融合多肽：CAR-(2A)-E，其中，CAR表示嵌合抗原受体分子，2A表示自剪切2A肽，E表示功能性融合的蛋白，且“-”表示连接相邻氨基酸的肽键或连接肽；其中，所述功能性融合的蛋白为Survivin蛋白。In yet another aspect, the present invention also provides a fusion polypeptide having the following structure: CAR-(2A)-E, wherein, CAR represents a chimeric antigen receptor molecule, 2A represents a self-cleaving 2A peptide, and E represents a functional A fusion protein, and "-" represents a peptide bond or a connecting peptide connecting adjacent amino acids; wherein, the functionally fused protein is Survivin protein.
以下就本发明的基于CAR的免疫细胞、核酸构建体、CAR融合多肽及其组件，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 constructs, CAR fusion polypeptides and components thereof of the present invention are described in detail below. Those skilled in the art can understand that, unless the context clearly indicates otherwise, any technical feature and any combination thereof mentioned when describing components are within the scope of the present invention; and those skilled in the art can understand , unless the context clearly indicates otherwise, the CAR-based immune cells of the present invention may comprise any such combination features, and likewise the nucleic acid constructs and CAR fusion polypeptides of the present invention may also comprise any such combination features.
SURVIVIN多肽及其编码核酸SURVIVIN polypeptide and its encoding nucleic acid
可用于本发明的Survivin包括全长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-O15392登录号下给出了来自人的Survivin氨基酸序列。来自其它物种的多种survivin同源物也已经公开，例如，来自Felis catus的survivin公开在Genbank登录号AB182320.1下；来自Canis familiaris的survivin公开在Genbank登录号AY741504.1,NM_001003348.1,AB180206.1, AB095108.1,AB095108and NM_001003019下；来自小鼠的survivin公开在Genbank登录号AAD26200.1下。Conway EM等(2000)Blood 95,1435-42；Mahotka C等(1999)Cancer Res.59,6097-102和Caldas H等(2005)MoI.Cancer 4,11，描述了survivin的功能性变体。可以按照实施例中相同或相似的方式，检测和/或筛选包含survivin或其片段或变体的基于CAR-T细胞的效应，包括但不限于，增加CAR-T细胞的持久性和改善其抗肿瘤作用的效应。Survivins useful in the present invention include full-length proteins or functional fragments thereof, or variants thereof (including natural allelic variants or species homologues). The amino acid sequence of Survivin from human is given under accession number UniProtKB-015392. Various survivin homologs from other species have also been published, for example, survivin from Felis catus is disclosed under Genbank accession number AB182320.1; survivin from Canis familiaris is disclosed under Genbank accession number AY741504.1, NM_001003348.1, AB180206 .1, AB095108.1, AB095108and NM_001003019; survivin from mice is published under Genbank accession number AAD26200.1. Conway EM et al. (2000) Blood 95, 1435-42; Mahotka C et al. (1999) Cancer Res. 59, 6097-102 and Caldas H et al. (2005) MoI. Cancer 4, 11, describe functional variants of survivin. The effects of CAR-T cells containing survivin or its fragments or variants can be detected and/or screened in the same or similar manner as in the examples, including but not limited to, increasing the persistence of CAR-T cells and improving their resistance to The effect of tumor action.
在一个实施方案中，在与靶细胞共培养一段时间后，相比于未表达survivin的CAR-T细胞，重组表达survivin的CAR-T细胞具有增加的存活率和/或细胞凋亡比例。在另一实施方案中，在与靶细胞共培养一段时间后，相比于未表达survivin的CAR-T细胞，重组表达survivin的CAR-T细胞具有增加的TSCM细胞比例。可以通过流式检测，鉴定CAR-T细胞群中具有细胞表面标志物CD3+CD8+CCR7+CD45RO-CD27+CD95+的TSCM细胞。In one embodiment, after co-cultivating with target cells for a period of time, compared with CAR-T cells not expressing survivin, the CAR-T cells recombinantly expressing survivin have an increased survival rate and/or apoptotic ratio. In another embodiment, after a period of co-culture with the target cells, the CAR-T cells expressing survivin recombinantly have an increased proportion of TSCM cells compared to CAR-T cells not expressing survivin. TSCM cells with cell surface markers CD3+CD8+CCR7+CD45RO-CD27+CD95+ in the CAR-T cell population can be identified by flow cytometry.
在根据本发明的任何实施方案中，优选地，Survivin蛋白包含: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 present invention, preferably, the Survivin protein comprises: i) the amino acid sequence of SEQ ID NO:5; ii) has at least one, two or three modifications to the amino acid sequence of SEQ ID NO:5 but No more than 30, 20 or 10 modified amino acid sequences; or iii) an amino acid sequence having at least 95-99% identity to the amino acid sequence of SEQ ID NO:5.
可用于本发明的Survivin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survivin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survivin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。在一个实施方案中，所述的survivin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 Survivin-encoding polynucleotide that can be used in the present invention may be any polynucleotide comprising a nucleotide sequence encoding the survivin protein in any of the above-mentioned embodiments of the present invention. In one embodiment, the survivin-encoding polynucleotide comprises amino acids encoding SEQ ID NO: 5 or a variant thereof, e.g., at least 95%, 96%, 97%, 98%, or 99% identical thereto sequence. In one embodiment, the polynucleotide encoding survivin comprises the nucleotide sequence of SEQ ID NO: 2 or a variant thereof, e.g., at least 95%, 96%, 97%, 98%, or 99% thereof An amino acid sequence of identity; or a nucleotide sequence that hybridizes thereto under stringent hybridization conditions.
嵌合抗原受体(CAR)多肽及其编码多核苷酸Chimeric antigen receptor (CAR) polypeptide and its encoding polynucleotide
可用于本发明的CAR多肽并无特别限制。在第一方面，本发明的CAR多肽包含细胞外抗原结合结构域，跨膜结构域，和胞质结构域。在一个实施方案中，本发明CAR多肽的胞质结构域包含胞内信号传导结构域。在一个实施方案中，本发明CAR多肽的胞质结构域包含共刺激结构域和胞质信号传导结构域。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)胞内信号传导结构域。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)共刺激结构域；和(e)胞内信号传导结构域。The CAR polypeptides that can be used in the present invention are not particularly limited. In a first aspect, the CAR polypeptide of the invention comprises an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic domain. In one embodiment, the cytoplasmic domain of a CAR polypeptide of the invention comprises an intracellular signaling domain. In one embodiment, the cytoplasmic domain of the CAR polypeptide of the invention comprises a co-stimulatory domain and a cytoplasmic signaling domain. In one embodiment, the chimeric antigen receptor (CAR) molecule according to the present invention comprises from N-terminus to C-terminus: composed of (a) an antigen-binding domain specifically binding to a tumor antigen and (b) a hinge or spacer ; (c) a transmembrane domain; and (d) an intracellular signaling domain. In one embodiment, the CAR molecule according to the present invention comprises from the N-terminus to the C-terminus: (a) an antigen-binding domain specifically binding to a tumor antigen, (b) a hinge region or a spacer region; (c) a transmembrane domain ; (d) costimulatory domain; and (e) intracellular signaling domain.
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或EphA2。In some embodiments, the target antigen for the CAR polypeptide of the present invention is a membrane antigen expressed on the surface of a target cell, especially a tumor cell, such as a tumor-specific antigen or a tumor-associated antigen. Tumors that may be mentioned include hematological and solid tumors, both primary and metastatic. In some embodiments, the target antigen is a tumor cell surface antigen comprising an antigenic cancer epitope immunologically recognized by tumor infiltrating lymphocytes (TILs) derived from a mammal. 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 the group consisting of: CD19, adrenergic A2 receptor (EphA2), folate receptor (FRa) , Mesothelin, EGFRvIII, IL-13Ra, CD123, CD33, BCMA, GD2, CLL-1, CA-IX, MUC1, HER2, and any combination thereof. More preferably, the tumor antigen is membrane antigen CD19 or EphA2.
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 confer specific recognition and binding to the CAR molecule and the CAR-T cells containing the CAR molecule ability to target antigens. In one embodiment, the extracellular antigen-binding domain of the CAR molecule according to the present invention is a polypeptide molecule having binding affinity for a target antigen. In one embodiment, the CAR according to the invention comprises an antigen binding domain derived from an antibody or antibody fragment. In yet another embodiment, the antigen binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL). In a preferred embodiment, the antigen-binding domain comprises a scFv formed by linking VL and VH via a linker.
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 produced by linking the VH and VL regions together using a flexible polypeptide linker according to methods known in the art. In some embodiments, scFv molecules comprise a flexible polypeptide linker 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. Linker sequences can comprise any naturally occurring amino acid. In one embodiment, the peptide linker of the scFv consists of amino acid residues such as glycine and/or serine, alone or in combination, to link the variable heavy 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 comprises multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO: 29). In yet another embodiment, the linker comprises the amino acid sequence GSTSGSGKPGSGEGSTKG. In one embodiment, the scFv used in the present invention comprises 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 may be derived from natural or synthetic sources. For example, the transmembrane domain may be derived from a membrane-bound protein or a transmembrane protein, such as a transmembrane domain from CD3ζ, CD4, CD28, CD8 (eg, CD8α, CD8β). In 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 present invention can be linked to the extracellular region of the CAR via a hinge region/spacer. For transmembrane regions and hinge/spacer regions that can be used in CAR polypeptides, see, eg, Kento Fujiwara et al., Cells 2020, 9, 1182; doi:10.3390/cells9051182.
本发明的CAR多肽中包含的胞质结构域包含胞内信号结构域。胞内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 domain comprised in the CAR polypeptide of the present invention comprises an intracellular signaling domain. The intracellular signaling domain is capable of activating at least one immune effector function of the immune cells into which the CAR of the present 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 helper activity, including secretion of cytokines.
用于本发明CAR多肽中的胞质结构域的例子包括，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，初级胞内信号结构域)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多肽包含提供初级胞内信号结构域的胞质结构域，例如，CD3ζ的胞内信号结构域。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共刺激结构域，如4-1BB(也称为CD137)和CD28的共刺激结构域。Examples of cytoplasmic domains for use in CAR polypeptides of the invention include the cytoplasmic domains of T cell receptors (TCRs) and/or co-receptors that can function to initiate signal transduction following binding of the extracellular domain to a target antigen. 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., the primary intracellular signaling domain) and those that act in an antigen-independent manner to provide co-activation. Those sequences that stimulate the signal (ie, the secondary cytoplasmic domain, eg, costimulatory domain). In one embodiment, a CAR polypeptide of the invention comprises a cytoplasmic domain that provides a primary intracellular signaling domain, eg, the intracellular signaling domain of CD3ζ. In another embodiment, the cytoplasmic domain of the CAR polypeptide of the invention further comprises a secondary signaling domain, eg, a co-stimulatory domain from a co-stimulatory molecule. In one embodiment, the cytoplasmic region of the CAR polypeptide of the invention comprises one or more co-stimulatory domains in tandem with the CD3ζ intracellular signaling domain, such as the co-stimulatory domains of 4-1BB (also known as CD137) and CD28 area.
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 present 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. Signal peptides/leaders of any eukaryotic origin, eg, signal peptides/leaders of mammalian or human secreted protein origin, may be used.
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，和胞质结构域。In some embodiments, a chimeric antigen receptor (CAR) polypeptide according to the invention comprises an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic 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。In one embodiment, the antigen binding domain is an antigen binding domain targeting 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.
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 binding to CD19 comprises: heavy chain complementarity determining region 1 (HC CDR1) of the heavy chain variable region (VH) amino acid sequence of SEQ ID NO: 9, 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 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 comprises: i) the amino acid sequence of SEQ ID NO: 9; ii) having at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 9 but no more than 30, 20 or 10 A modified amino acid sequence; or iii) an amino acid sequence with 95-99% identity to 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) has at least one, two or three modifications to the amino acid sequence of SEQ ID NO: 8 but no more than 30, 20 or 10 A modified amino acid sequence; or iii) an amino acid sequence having 95-99% identity to 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) having 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 with 95-99% identity 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 the group consisting of: 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 receptors, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors , 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) at least one, two or three modifications but no more than 5 modifications comprising the amino acid sequence of SEQ ID NO: 13 or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO: 13.
在一个实施方案中，胞质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信号传导结构域)。在一个实施方案中，胞质结构域包含: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具有95-99％序列同一性的氨基酸序列。In one embodiment, the cytoplasmic domain comprises a functional signaling domain of a protein selected from the group consisting of: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b or CD66d. In one embodiment, the cytoplasmic domain comprises the functional signaling domain of the CD3ζ protein (also referred to herein, the CD3ζ signaling domain). In one embodiment, the cytoplasmic domain comprises: i) the amino acid sequence of SEQ ID NO: 15; ii) at least one, two or three modifications but no more than 20 of the amino acid sequence comprising SEQ ID NO: 15, 10 or 5 modified amino acid sequences; or iii) an amino acid sequence having 95-99% sequence identity to SEQ ID NO: 15.
在一个实施方案中，胞质结构域进一步包含选自以下的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，胞质结构域包含选自以下的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，胞质结构域包含来自CD28的共刺激结构域。在一个实施方案中，胞质结构域包含: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。In one embodiment, the cytoplasmic domain further comprises a co-stimulatory domain of a protein selected from the group consisting of MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling Lymphocyte activation molecule (SLAM protein), activating NK cell receptor, CD8, ICOS, DAP10, DAP12, OX40, CD40, GITR, 4-1BB (ie CD137), CD27 and CD28. In one embodiment, the cytoplasmic domain comprises a costimulatory domain of a protein selected from the group consisting of the costimulatory domains of CD28, CD27, 4-1BB, ICOS and OX40. In one embodiment, the cytoplasmic domain comprises a co-stimulatory domain of a protein selected from CD28 and 4-1BB (ie CD137), or a combination thereof. In one embodiment, the cytoplasmic domain comprises a co-stimulatory domain from CD28. In one embodiment, the cytoplasmic domain comprises: i) the amino acid sequence of SEQ ID NO: 14; ii) at least one, two or three modifications but no more than 20 of the amino acid sequence comprising SEQ ID NO: 14, 10 or 5 modified amino acid sequences; or iii) an amino acid sequence with 95-99% identity to the amino acid sequence of SEQ ID NO: 14.
在一个实施方案中，CAR多肽包含来自CD28的跨膜结构域或包含来自CD3ζ的信号传导结构域的胞质结构域。在一个实施方案中，所述CAR多肽的胞质结构域包含：包含来自CD28的共刺激信号和来自CD3ζ的功能信号传导结构域。在一个实施方案中，所述CAR多肽包含：来自CD28的跨膜结构域、和包含来自CD28的共刺激信号和来自CD3ζ的功能信号传导结构域的胞质结构域。In one embodiment, the CAR polypeptide comprises a transmembrane domain from CD28 or a cytoplasmic domain comprising a signaling domain from CD3ζ. In one embodiment, the cytoplasmic domain of the CAR polypeptide comprises: a co-stimulatory signal from CD28 and a functional signaling domain from CD3ζ. In one embodiment, the CAR polypeptide comprises: a transmembrane domain from CD28, and a cytoplasmic domain comprising a co-stimulatory signal from CD28 and a functional signaling domain from CD3ζ.
在一个实施方案中，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4-1BB或CD28的共刺激结构域；和(e)来自CD3ζ的功能信号传导结构域。In one embodiment, the CAR polypeptide of the invention comprises: (a) antigen binding domain; (b) hinge/spacer; (c) transmembrane domain; (d) costimulatory from 4-1BB or CD28 domain; and (e) a functional signaling domain from CD3ζ.
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, the 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 is selected from 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 comprises: i) the amino acid sequence of SEQ ID NO: 12; ii) at least one, two or three modifications but no more than 5 of the amino acid sequence comprising SEQ ID NO: 12 A modified amino acid sequence; or iii) an amino acid sequence having 95-99% identity to the amino acid sequence of SEQ ID NO: 12. Herein, the expressions "hinge", "hinge region" and "hinge domain" are used interchangeably.
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 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 the 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, the 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 to 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。在一个实施方案中，所述的CAR编码核酸包含SEQ ID NO:1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 CAR-encoding nucleic acid that can be used in the present invention may be any polynucleotide comprising a nucleotide sequence encoding a CAR polypeptide according to any of the above-mentioned 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., at least 95%, 96%, 97%, 98%, or 99% identical thereto. In one embodiment, the CAR-encoding nucleic acid comprises the nucleotide sequence of SEQ ID NO: 1 or a variant thereof, e.g., at least 95%, 96%, 97%, 98%, or 99% identical thereto amino acid sequence; or a nucleotide sequence that hybridizes thereto under stringent hybridization conditions.
本发明的核酸构建体Nucleic acid constructs of the invention
在一个方面，本发明提供了编码根据本发明的CAR多肽的多核苷酸和编码根据本发明的SURVIVIN蛋白的多核苷酸的一个或多个核酸分子。在一个实施方案中，编码根据本发明的CAR多肽的多核苷酸的核酸分子，与编码根据本发明的SURVIVIN蛋白的多核苷酸的核酸分子不同。在一个实施方案中，编码根据本发明的CAR多肽的多核苷酸，与编码根据本发明的SURVIVIN蛋白的多核苷酸，在单一一个核酸分子中提供。In one aspect, the present invention provides one or more nucleic acid molecules encoding a polynucleotide encoding a CAR polypeptide according to the present invention and a polynucleotide encoding a SURVIVIN protein according to the present invention. In one embodiment, the nucleic acid molecule encoding the polynucleotide of the CAR polypeptide according to the present invention is different from the nucleic acid molecule encoding the polynucleotide of the SURVIVIN protein according to the present invention. In one embodiment, the polynucleotide encoding the CAR polypeptide according to the present invention and the polynucleotide encoding the SURVIVIN protein according to the present invention are provided in a single nucleic acid molecule.
在一个实施方案中，car编码核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。在一个实施方案中，所述car编码核酸包含编码以下结构域的多核苷酸：特异性结合肿瘤抗原的胞外抗原结合结构域，铰链区或间隔区、跨膜结构域、和胞质结构域，其中所述胞质结构域包含共刺激结构域和胞内信号传导结构域。在一个实施方案中，所述car编码核酸还包含编码信号肽(例如GM-CSFRα信号肽)的寡核苷酸。在一个实施方案中，所述car编码核酸包含：i)SEQ ID NO:1的核苷酸序列；ii)与SEQ ID NO:1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:1的核苷酸序列具有至少90-99％同一性的核苷酸序列。In one embodiment, the car-encoding nucleic acid comprises a polynucleotide encoding a CAR polypeptide according to any of the foregoing embodiments of the present invention, especially a polynucleotide encoding a CD19-targeting CAR polypeptide. In one embodiment, the car-encoding nucleic acid comprises a polynucleotide encoding the following domains: an extracellular antigen-binding domain specifically binding to a tumor antigen, a hinge or spacer, a transmembrane domain, and a cytoplasmic domain , wherein the cytoplasmic domain comprises a co-stimulatory domain and an intracellular signaling domain. In one embodiment, the car-encoding nucleic acid further comprises an oligonucleotide encoding a signal peptide (eg, GM-CSFRα signal peptide). In one embodiment, the car-encoding nucleic acid comprises: i) the nucleotide sequence of SEQ ID NO: 1; ii) a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO: 1 under stringent hybridization conditions or iii) a nucleotide sequence having at least 90-99% identity to the nucleotide sequence of SEQ ID NO:1.
在一个实施方案中，survivin编码核酸包含编码根据本发明前述任何实施方案的survivin多核苷酸。在一个实施方案中，survivin编码核酸包含编码人Survivin的多核苷酸，例如，UniProtKB-O15392登录号下的人Survivin氨基酸序列。在一个实施方案中，所述survivin编码核酸编码SEQ ID NO:5的氨基酸序列。 在一个实施方案中，所述survivin编码核酸包含：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, the survivin-encoding nucleic acid comprises a polynucleotide encoding survivin according to any of the preceding embodiments of the invention. In one embodiment, the survivin-encoding nucleic acid comprises a polynucleotide encoding human Survivin, eg, the amino acid sequence of human Survivin under accession number UniProtKB-015392. In one embodiment, the survivin-encoding nucleic acid encodes the amino acid sequence of SEQ ID NO:5. In one embodiment, the survivin-encoding nucleic acid comprises: i) the nucleotide sequence of SEQ ID NO:2; ii) a nucleotide sequence that hybridizes with the nucleotide sequence of SEQ ID NO:2 under stringent hybridization conditions or iii) a nucleotide sequence having at least 90-99% identity with the nucleotide sequence of SEQ ID NO:2.
在一个实施方案中，所述的CAR多肽和Survivin蛋白分别单独地自所述核酸构建体表达。在另一实施方案中，自所述的核酸构建体表达产生包含所述CAR多肽和Survivin蛋白两者的融合多肽，其中，所述融合多肽包含置于所述CAR多肽和Survivin蛋白之间的可剪切连接肽。在一些实施方案中，编码Survivin蛋白的基因，使用自剪切肽，以符合读框的方式，遗传融合在编码CAR多肽的多核苷酸的3’端。In one embodiment, the CAR polypeptide and the Survivin protein are separately expressed from the nucleic acid construct. In another embodiment, a fusion polypeptide comprising both the CAR polypeptide and the Survivin protein is produced from the expression of the nucleic acid construct, wherein the fusion polypeptide comprises an optional protein interposed between the CAR polypeptide and the Survivin protein. Cut linker peptide. In some embodiments, the gene encoding the Survivin protein is genetically fused to the 3' end of the polynucleotide encoding the CAR polypeptide using a self-cleaving peptide in an in-frame manner.
在一个实施方案中，所述核酸分子是具有如下结构的核酸构建体：car-(2A)-e，其中，car表示嵌合抗原受体(CAR)多肽的编码核酸，2A表示编码自剪切2A肽的寡核苷酸，e表示功能性融合的蛋白的编码核酸，且“-”表示连接相邻核苷酸的磷脂键或编码连接肽的寡核苷酸；其中，所述功能性融合的蛋白编码核酸包含Survivin基因。In one embodiment, the nucleic acid molecule is a nucleic acid construct having the following structure: car-(2A)-e, wherein car represents a nucleic acid encoding a chimeric antigen receptor (CAR) polypeptide, and 2A represents a nucleic acid encoding a self-splicing The oligonucleotide of the 2A peptide, e represents the coding nucleic acid of the protein of functional fusion, and "-" represents the phospholipid bond connecting adjacent nucleotides or the oligonucleotide encoding the connecting peptide; wherein, the functional fusion The protein-coding nucleic acid comprises a Survivin gene.
可以用于本发明的自剪切肽包括，但不限于，P2A,T2A，E2A或F2A肽。关于2A自剪切肽的序列和应用，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。Self-cleaving peptides that can be used in the present invention include, but are not limited to, P2A, T2A, E2A or F2A peptides. For the sequence and application of 2A self-cleaving peptide, 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2A肽为P2A肽。在一个实施方案中，P2A肽包含:i)SEQ ID NO:19的氨基酸序列；ii)对SEQ ID NO:19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:19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 2A peptide is a P2A peptide. In one embodiment, the P2A peptide comprises: i) the amino acid sequence of SEQ ID NO: 19; ii) having at least one, two or three modified amino acids but no more than 5 modified amino acids to the amino acid sequence of SEQ ID NO: 19 sequence; or iii) an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 19. In one embodiment, a GSG linker can 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CAR-(2A)-SURVIVIN融合多肽，其中所述CAR可以包含根据本发明前述任何实施方案的CAR多肽；所述2A可以包含根据前述的任何自剪接2A肽；所述SURVIVIN可以包含根据本发明前述任何实施方案的Survivin蛋白。在所述的这些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)连接。Therefore, in another aspect, the present invention also provides a CAR-(2A)-SURVIVIN fusion polypeptide, wherein the CAR may comprise a CAR polypeptide according to any of the foregoing embodiments of the present invention; the 2A may comprise any self-splicing 2A according to the foregoing Peptide; said SURVIVIN may comprise a Survivin protein according to any of the preceding embodiments of the present invention. These components can be connected directly or indirectly through linkers (for example, single amino acid residues or short peptides).
III.包含本发明核酸分子的载体和细胞III. Vectors and Cells Comprising Nucleic Acid Molecules of the Invention
本发明还提供了插入有(一个或多个)本发明核酸分子或本发明的核酸构建体的载体。可以通过将编码CAR多肽的核酸和编码SURVIVIN蛋白的核酸有效连接至启动子，并将构建体并入表达载体中，实现编码CAR的核酸和编码SURVIVIN蛋白的核酸的表达。载体可以适合在真核生物中复制和整合。常见的克隆载体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。The invention also provides a vector into which is inserted a nucleic acid molecule(s) of the invention or a nucleic acid construct of the invention. Expression of the nucleic acid encoding the CAR polypeptide and the nucleic acid encoding the SURVIVIN protein can be achieved by operatively linking the nucleic acid encoding the CAR polypeptide and the nucleic acid encoding the SURVIVIN protein to a promoter, and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Common cloning vectors contain transcriptional and translational terminators, initiation sequences and promoters for regulating the 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，将本发明核酸构建体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，将本发明核酸构建体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, retroviruses provide a convenient platform for gene delivery systems. The nucleic acid constructs 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 cells of the subject 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 construct of the present invention is cloned into a lentiviral vector, so that
(i)在单个编码框中产生全长CAR多肽和在单个编码框中产生Survivin多肽，或(i) producing a full-length CAR polypeptide in a single coding frame and producing a Survivin polypeptide in a single coding frame, or
(ii)在单个编码框中产生包含CAR多肽和Survivin多肽的CAR融合多肽。(ii) producing a CAR fusion polypeptide comprising a CAR polypeptide and a Survivin polypeptide 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 such as 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 added advantage over vectors derived from onco-retroviruses, such as murine leukemia virus, because they can transduce non-proliferative cells, such as hepatocytes. They also have the added advantage of low immunogenicity. A retroviral vector may also be, for example, a gamma retroviral vector. A gamma retroviral vector may, for example, comprise a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTRs), and a transgene of interest, e.g., encoding a CAR gene. Gamma retroviral vectors may lack viral structural genes such as gag, pol, and env.
能够在哺乳动物T细胞中表达CAR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CAR。参见，例如，Milone等人,Mol.Ther.17(8):1453–1464(2009)。An example of a promoter capable of expressing a CAR transgene 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 the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EF1a promoter has been used extensively in mammalian expression plasmids and has been shown to efficiently drive CAR expression from transgenes cloned 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 operatively linked thereto. However, other constitutive promoter sequences can 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) promoters, MoMuLV promoters, avian leukemia virus promoters, Epstein-Barr virus immediate early promoters, Rous sarcoma virus promoters, 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 present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention.
在一些实施方案中，本发明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)中表达本发明的CAR构建体的方法和由此产生的免疫效应细胞。In some embodiments, the invention provides methods of expressing a CAR construct of the invention in mammalian immune effector cells (eg, mammalian T cells or mammalian NK cells) and immune effector cells produced thereby.
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。A source of cells (eg, immune effector cells, eg, T cells or NK cells) is obtained from a subject. The term "subject" is intended to include living organisms (eg, mammals) that can elicit an immune response. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, 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Ficoll TM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 of skill in the art, such as Ficoll (TM) separation. In a preferred aspect, the cells from the circulating blood of the individual 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 medium for subsequent processing steps. In one aspect of the invention, the 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
Figure PCTCN2022079966-appb-000003
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 by positive or negative selection techniques. For example, in one embodiment, anti-CD3/anti-CD28 conjugated beads (such as
Figure PCTCN2022079966-appb-000003
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 period of time is between about 30 minutes and 36 hours or longer. Longer incubation times can be used to isolate T cells wherever 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 selectively 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 can be accomplished through the process of negative selection using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method is the sorting and/or selection of cells by means of negative magnetic immunoadhesion or flow cytometry using the presence of cells on negatively selected cells Monoclonal antibody cocktail for 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 can be allogeneic immune effector cells, eg, T cells or NK cells. For example, the cell can be an allogeneic T cell, e.g., one lacking expression of a functional T cell receptor (TCR) and/or human leukocyte antigen (HLA) (e.g., HLA class I and/or HLA class II) T cells.
在一个实施方案中，本发明提供了重组载体，其包含根据本发明的前述任何实施方案所述的核酸构建体，例如，所述载体选自DNA载体、RNA载体、慢病毒载体、腺病毒载体或逆转录病毒载体，优选地，逆转录病毒载体。In one embodiment, the present invention provides a recombinant vector comprising the nucleic acid construct according to any of the preceding embodiments of the present invention, for example, the vector is selected from DNA vectors, RNA vectors, lentiviral vectors, adenoviral vectors or a retroviral vector, preferably a retroviral vector.
在一个实施方案中，本发明提供了宿主细胞，其包含根据本发明的前述任何实施方案所述的重组载体，或者在染色体中整合有根据本发明的前述任何实施方案所述的核酸构建体，其中所述细胞是免疫效应细胞，例如T细胞或NK细胞，例如，所述T细胞是自体T细胞或同种异体T细胞。In one embodiment, the present invention provides a host cell, which comprises the recombinant vector according to any of the foregoing embodiments of the present invention, or has integrated the nucleic acid construct according to any of the foregoing embodiments of the present invention in the chromosome, Wherein the cells are immune effector cells, such as T cells or NK cells, for example, the T cells are autologous T cells or allogeneic T cells.
在一个实施方案中，本发明提供了一种CAR-T细胞，其中所述细胞包含编码CAR多肽的异源多核苷酸和编码Survivin的异源多核苷酸，优选地，所述细胞包含根据本发明的前述任何实施方案所述的核酸构建体。In one embodiment, the present invention provides a CAR-T cell, wherein the cell comprises a heterologous polynucleotide encoding a CAR polypeptide and a heterologous polynucleotide encoding Survivin, preferably, the cell comprises a A nucleic acid construct according to any preceding embodiment of the invention.
IV.表达SURVIVIN的本发明免疫效应细胞的用途和使用表达SURVIVIN的本发明免疫效应细胞的治疗方法IV. Use of the Immune Effector Cells of the Invention Expressing SURVIVIN and Therapeutic Methods Using the Immune Effector Cells of the Invention Expressing SURVIVIN
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。本发明通过在CAR-T细胞中增加survivin表达，获得了包含更多TCSM细胞亚群的本发明Armored 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 first applied 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, there is an urgent need for technical means that can effectively adjust the in vitro and in vivo persistence of CAR-T cells. In the present invention, by increasing the expression of survivin in CAR-T cells, the Armored CAR-T cells of the present invention containing more TCSM cell subpopulations are obtained, so as to facilitate the application of the CAR-T cells in anti-tumor therapy 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)以表达本发明的核酸构建体，在所述细胞中异源表达本发明的CAR多肽和survivin蛋白。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，将其用于过继细胞治疗(ACT)。In some embodiments, immune effector cells, such as T cells (such as patient-specific autologous T cells) are engineered to express the nucleic acid construct of the present invention, and the CAR polypeptide and survivin protein of the present invention are heterologously expressed in said cells . After expanding the engineered immune effector cells (such as T cells or NK cells), they are used for adoptive cell therapy (ACT).
在一些实施方案中，在利用异源表达survivin的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survivin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 an immune effector cell of the invention heterologously expressing survivin, the immune effector cell may be an autologous or allogeneic T cell or NK cell. In some embodiments, compared with control CAR-T or CAR-NK cells without heterologous survivin expression, the immune effector cells of the present invention can improve the long-term survival of cells after adoptive transfer and/or the proportion of TSCM subsets.
在一个实施方案中，异源表达survivin的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, immune effector cells of the invention that heterologously express survivin 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构建体的免疫效应细胞。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 invention provides methods of 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 construct of the invention. The present invention also provides the use of the aforementioned immune effector cells of the present invention in the preparation of drugs for treating cancer. The cancers include hematological cancers (eg, leukemias) or solid tumors (eg, gliomas), including primary and metastatic cancers.
在一些实施方案中，本发明的免疫效应细胞与IL-15联合应用。In some embodiments, the immune effector cells of the invention are used in combination with IL-15.
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。The various embodiments/technical solutions described in the present invention and the features in each embodiment/technical solution should be understood as being able to be combined arbitrarily with each other, and each solution obtained by these mutual combinations is included in the scope of the present invention, just as in These solutions obtained in combination are specifically and individually listed herein 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 in the understanding of the present invention. The examples are not intended and should not be construed as limiting the scope of the invention in any way.
实施例Example
一、材料和方法1. Materials and methods
1、实验材料1. Experimental materials
细胞系：人Nalm-6细胞系，U373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)。所有这些细胞均保存在含有10％胎牛血清(Biosera)和10,000IU/ml青霉素/10,00μg/ml链霉素(EallBio Life Sciences)的RPMI-1640(Lonza)或DMEM(Lonza)中。所有细胞在5％CO 2,95％空气,37℃加湿培养箱中培养。 Cell lines: Human Nalm-6 cell line, U373 cell line and retroviral packaging cell line PG13 were purchased from American Type Culture Collection (ATCC). All these cells were maintained in RPMI-1640 (Lonza) or DMEM (Lonza) with 10% fetal bovine serum (Biosera) and 10,000 IU/ml penicillin/10,00 μg/ml streptomycin (EallBio Life Sciences). All cells were cultured in 5% CO 2 , 95% air, 37°C humidified incubator.
2、实验方法2. Experimental method
用逆转录病毒转染PBMCs得到转染的CD19-CAR-T细胞，与Nalm-6(CD19阳性)肿瘤细胞共培养。分别用IL-2或IL-15刺激转染的CAR-T细胞。通过mRNA和蛋白水平检测CAR-T细胞的Survivin表达，流式细胞术检测CAR-T细胞的凋亡和表面标志物。检测CAR-T细胞中PI3K/Akt磷酸化水平。最后构建CD19-CAR-Survivin T细胞和CD19-CAR-IL-15T细胞，考察过表达survivin或IL-15的Armored CAR-T细胞对肿瘤的治疗作用。PBMCs were transfected with retrovirus to obtain transfected CD19-CAR-T cells, which were co-cultured with Nalm-6 (CD19 positive) tumor cells. The transfected CAR-T cells were stimulated with IL-2 or IL-15, respectively. The expression of Survivin in CAR-T cells was detected by mRNA and protein levels, and the apoptosis and surface markers of CAR-T cells were detected by flow cytometry. Detect the phosphorylation level of PI3K/Akt in CAR-T cells. Finally, CD19-CAR-Survivin T cells and CD19-CAR-IL-15T cells were constructed to investigate the therapeutic effect of Armored CAR-T cells overexpressing survivin or IL-15 on tumors.
(1)CAR-T细胞的产生(1) Generation of CAR-T cells
用梯度离心法分离健康供者外周血单个核细胞(PBMCs)。用抗CD3和抗CD28颗粒刺激外周血单个核细胞中的T细胞，然后用逆转录病毒感染。具体为：0.5ml Retronectin(Thermo,15ug/ml)加入12孔板，室温避光孵育2h。弃掉上清，加入0.5％human AB血清(Biosera)(PBS配制)，温育30min,弃掉上清。加入0.5ml T细胞(1.6×10 6/ml)与0.5ml病毒液体，用封口膜封闭孔板，700g，离心1h后，放入37℃培养箱培养，获得抗原特异性的基因修饰T细胞。7天后，CAR-T细胞在含0.5％正常人AB血清(Biosera)的X-VIVO TM15无血清培养系统中孵育24h，然后在含5％GemCell TM正常人AB血清的X-VIVO TM15培养基中扩增，加入IL-2(138U/ml)或IL-15(10ng/ml)培养7天后，对CAR-T细胞表达标志物进行流式检测，鉴定T细胞亚群的比例。本研究获得了北京世纪坛医院机构评审委员会的批准，并获得了所有参与者的知情同意。 Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated by gradient centrifugation. T cells in peripheral blood mononuclear cells were stimulated with anti-CD3 and anti-CD28 particles and then infected with retrovirus. Specifically: add 0.5ml Retronectin (Thermo, 15ug/ml) into a 12-well plate, and incubate at room temperature for 2 hours in the dark. The supernatant was discarded, 0.5% human AB serum (Biosera) (prepared in PBS) was added, incubated for 30 min, and the supernatant was discarded. Add 0.5ml of T cells (1.6×10 6 /ml) and 0.5ml of virus liquid, seal the well plate with parafilm, centrifuge at 700g for 1 hour, and culture in a 37°C incubator to obtain antigen-specific genetically modified T cells. After 7 days, CAR-T cells were incubated in X-VIVO TM 15 serum-free culture system containing 0.5% normal human AB serum (Biosera) for 24h, and then cultured in X-VIVO TM 15 containing 5% GemCell TM normal human AB serum After adding IL-2 (138U/ml) or IL-15 (10ng/ml) and culturing for 7 days, the expression markers of CAR-T cells were detected by flow cytometry to identify the proportion of T cell subsets. This study was approved by the Institutional Review Board of Beijing Shijitan Hospital, and informed consent was obtained from all participants.
(2)流式细胞术(2) Flow cytometry
流式细胞术在FACSCanto Plus仪器(BD Biosciences)上进行，数据分析使用FlowJo V.10(FlowJo，LLC)。用FITC标记的小鼠抗人CD3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抗体(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 Fluo 700标记的小鼠抗人CD27(BD Biosciences),PE-Cy5标记的小鼠抗人CD95 (BD Biosciences)，流式细胞术检测细胞表面标志物表达。用Alexa Fluor 647标记的山羊抗小鼠IgG(Fab特异性)F(ab')2(Invitrogen)，流式细胞术检测CD19-CAR-T细胞；用Alexa Fluor700标记的小鼠抗人EphA2(R&DSystems)染色U373细胞，流式细胞术检测细胞表面EphA2的表达。Flow cytometry was performed on a FACSCanto Plus instrument (BD Biosciences), and data analysis was performed using FlowJo V.10 (FlowJo, LLC). FITC-labeled mouse anti-human CD3 antibody (BD Biosciences), Alexa Fluor 700-labeled mouse anti-human CD8 antibody (BD Biosciences), BV421-labeled mouse anti-human CD4 antibody (BD Biosciences), V450-labeled mouse Anti-human CD107a antibody (BD Biosciences), BV605-labeled mouse anti-human CD45RO (BD Biosciences), PE-Cy7-labeled mouse anti-human CCR7 (BD Biosciences), Alexa Fluo 700-labeled mouse anti-human CD27 (BD Biosciences ), PE-Cy5-labeled mouse anti-human CD95 (BD Biosciences), and flow cytometry to detect the expression of cell surface markers. Alexa Fluor 647-labeled goat anti-mouse IgG (Fab-specific) F(ab')2 (Invitrogen), CD19-CAR-T cells were detected by flow cytometry; Alexa Fluor700-labeled mouse anti-human EphA2 (R&DSystems ) to stain U373 cells, and flow cytometry to detect the expression of EphA2 on the cell surface.
(3)细胞毒性测定(3) Cytotoxicity assay
在96孔板中，CAR-T细胞以不同的效靶比(E:T)与靶细胞共培养6或24小时后，收集细胞，并使用流式细胞仪(BD FacsCanto II Plus)通过表面标记检测肿瘤细胞残留。In 96-well plates, after CAR-T cells were co-cultured with target cells at different effector-to-target ratios (E:T) for 6 or 24 hours, the cells were collected and detected by surface markers using a flow cytometer (BD FacsCanto II Plus). Detection of tumor cell residues.
(4)增殖测定(4) Proliferation assay
单独或与IL-2或IL-15共培养后，用CD19阳性肿瘤细胞(Nalm-6细胞)刺激CD8 +CD19-CAR-T细胞。第0天、第7天、第14天、第21天和第28天，采用Vi-CELL细胞活力分析仪，通过台盼蓝排除法进行活细胞计数。 CD8 + CD19-CAR-T cells were stimulated with CD19-positive tumor cells (Nalm-6 cells) alone or after co-culture with IL-2 or IL-15. On day 0, day 7, day 14, day 21 and day 28, Vi-CELL cell viability analyzer was used to count viable cells by trypan blue exclusion method.
(5)细胞周期测定(5) Cell cycle assay
将CAR-T细胞(1×10 6)重新悬浮于300μL PBS中，然后用体积为1ml的70％乙醇固定。10分钟后，用PBS洗涤细胞3次，室温下用PI/RNase染色缓冲液(BD Biosciences)染色15分钟，再用流式细胞仪分析。 CAR-T cells (1×10 6 ) were resuspended in 300 μL of PBS, then fixed with 70% ethanol in a volume of 1 ml. After 10 minutes, cells were washed 3 times with PBS, stained with PI/RNase staining buffer (BD Biosciences) for 15 minutes at room temperature, and analyzed by flow cytometry.
(6)PCR检测(6) PCR detection
PCR检测通过IL-2和IL-15刺激后CAR-T细胞中Survivin的表达。按照制造商的说明，使用TRIzol试剂(Invitrogen)从CD8 +CAR-T细胞中提取总RNA。使用Nanodrop One分光光度计(Thermo Fisher Scientific)测量RNA的数量和纯度。本研究只考虑吸光度测量值合适的样品(A 260/A2 80为～2.0,A 260/A 230为1.9-2.2)。利用High Capacity cDNA逆转录试剂盒(Thermo Fisher Scientific)合成cDNA。 The expression of Survivin in CAR-T cells stimulated by IL-2 and IL-15 was detected by PCR. Total RNA was extracted from CD8 + CAR-T cells using TRIzol reagent (Invitrogen) following the manufacturer's instructions. RNA quantity and purity were measured using a Nanodrop One spectrophotometer (Thermo Fisher Scientific). Only samples with suitable absorbance measurements ( A260 /A280 of ~2.0 and A260 / A230 of 1.9-2.2 ) were considered in this study. cDNA was synthesized using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).
利用5'-TTGAATCGCGGGACCCGTTGG-3'(正向引物)和5'-CAGAGGCCTCAATCCATGGCA-3'(反向引物)引物扩增survivin基因。用引物5'-GTCATCCCTGAGCTGAACGG-3'(正向引物)和5'-TGGGTGTCGCTGTTGAAGTC-3'(反向引物)扩增的GAPDH作为对照。The survivin gene was amplified using 5'-TTGAATCGCGGGACCCGTTGG-3' (forward primer) and 5'-CAGAGGCCTCAATCCATGGCA-3' (reverse primer) primers. GAPDH amplified with primers 5'-GTCATCCCTGAGCTGAACGG-3' (forward primer) and 5'-TGGGTGTCGCTGTTGAAGTC-3' (reverse primer) were used as controls.
(7)免疫印迹检测(7) Western blot detection
细胞用PBS洗涤三次，然后用RIPA缓冲液提取蛋白质。蛋白质样品用Pierce BCA蛋白测定试剂盒(Thermo Fisher Scientific)定量，然后在十二烷基硫酸钠(SDS)/巯基乙醇样品缓冲液中变性。样品(10微克)在15％的SDS-聚丙烯酰胺凝胶上分离,电泳转印到聚偏氟乙烯膜(Millipore)上。将该膜与兔抗人survivin(abcam)、兔抗人P-AKT(CST)、兔抗人颗粒酶A(abcam)、兔抗人颗粒酶B(abcam)、小鼠抗β肌动蛋白(abcam)在4℃下孵育过夜，然后用HRP结合的山羊抗兔二抗/鼠二抗(Santa Cruz Biotechnology)室温孵育1小时，使用ECL试剂盒(Thermo Fisher Scientific)进行化学发光反应的检测。加入抑制剂 Ly294002(Selleck，10uM)抑制PI3K/AKT通路，免疫印迹检测survivin的表达。Cells were washed three times with PBS, and then proteins were extracted with RIPA buffer. Protein samples were quantified with the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) and then denatured in sodium dodecyl sulfate (SDS)/mercaptoethanol sample buffer. Samples (10 micrograms) were separated on 15% SDS-polyacrylamide gel, electrophoretically transferred to polyvinylidene fluoride membrane (Millipore). The membrane was mixed with rabbit anti-human survivin (abcam), rabbit anti-human P-AKT (CST), rabbit anti-human granzyme A (abcam), rabbit anti-human granzyme B (abcam), mouse anti-β-actin ( abcam) at 4°C overnight, then incubated with HRP-conjugated goat anti-rabbit secondary antibody/mouse secondary antibody (Santa Cruz Biotechnology) for 1 hour at room temperature, and detected the chemiluminescent reaction using an ECL kit (Thermo Fisher Scientific). The inhibitor Ly294002 (Selleck, 10uM) was added to inhibit the PI3K/AKT pathway, and the expression of survivin was detected by western blotting.
(8)Armored CAR-T细胞的构建方法(8) Construction method of Armored CAR-T cells
分别克隆CD19-CAR基因(核苷酸序列如SEQ ID NO：1所示)，survivin基因(核苷酸序列如SEQ ID NO：2所示)和IL-15基因(核苷酸序列如SEQ ID NO：3所示)。Cloning CD19-CAR gene (nucleotide sequence as shown in SEQ ID NO: 1), survivin gene (nucleotide sequence as shown in SEQ ID NO: 2) and IL-15 gene (nucleotide sequence as shown in SEQ ID NO: 2) respectively cloned NO: 3 shown).
根据CD19-CAR的碱基序列，由生物公司合成cDNA，并以此为模板，利用表1所示的引物CD19-CAR-F和CD19-CAR-R，扩增CD19-CAR基因；以义翘神州购买的Survivin基因的cDNA为模板，利用表1所示引物Survivin-F和Survivin-R，扩增survivin基因；以义翘神州购买的IL-15基因的cDNA为模板，利用表1所示引物IL-15-F和IL-15-R，扩增IL-15基因。According to the base sequence of CD19-CAR, cDNA was synthesized by a biological company, and using this as a template, the CD19-CAR gene was amplified using the primers CD19-CAR-F and CD19-CAR-R shown in Table 1; The cDNA of the Survivin gene purchased in China was used as a template, and the primers Survivin-F and Survivin-R shown in Table 1 were used to amplify the survivin gene; the cDNA of the IL-15 gene purchased by Sino Biological was used as a template, and the primers shown in Table 1 were used IL-15-F and IL-15-R, amplify the IL-15 gene.
表1 扩增引物Table 1 Amplification primers
Figure PCTCN2022079966-appb-000004
Figure PCTCN2022079966-appb-000004
扩增反应体系如表2所示：The amplification reaction system is shown in Table 2:
表2 反应体系Table 2 Reaction system
试剂Reagent 体积(μl)Volume (μl)
10×KOD buffer10×KOD buffer 55
2mM dNTPs 2mM dNTPs 55
25mM MgSO4 25mM MgSO4 33
Primer 1(10uM)Primer 1(10uM) 0.750.75
Primer 2(10uM)Primer 2(10uM) 0.750.75
模板 template 11
KOD-plus-NeuKOD-plus-Neu 11
ddH 2O ddH 2 O 33.533.5
扩增反应程序如表3所示：The amplification reaction program is shown in Table 3:
表3 反应程序Table 3 Reaction program
Figure PCTCN2022079966-appb-000005
Figure PCTCN2022079966-appb-000005
上述得到的扩增产物，通过同源重组(ClonExpress II One Step Cloning kit)的方法，按照表4所示连接体系连接到载体SFG载体(addgene)上，37℃，反应30min，构建survivin和CAR共表达质粒CD19-CAR-survivin。The amplified product obtained above was connected to the carrier SFG carrier (addgene) through the method of homologous recombination (ClonExpress II One Step Cloning kit) according to the connection system shown in Table 4, and reacted at 37°C for 30 minutes to construct survivin and CAR co- Expression plasmid CD19-CAR-survivin.
表4 连接体系Table 4 connection system
试剂Reagent 体积(μl)Volume (μl)
PCR片段 PCR fragment 44
载体(SFG)Carrier (SFG) 11
连接酶 Ligase 11
ddH 2O ddH 2 O 1414
上述扩增得到的CD19-CAR核苷酸序列(SEQ ID NO：1)如下：The CD19-CAR nucleotide sequence (SEQ ID NO: 1) obtained by the above amplification is as follows:
Figure PCTCN2022079966-appb-000006
Figure PCTCN2022079966-appb-000006
Figure PCTCN2022079966-appb-000007
Figure PCTCN2022079966-appb-000007
CD19-CAR基因表达的蛋白，其氨基酸序列(SEQ ID NO：4)如下所示：The amino acid sequence (SEQ ID NO: 4) of the protein expressed by the CD19-CAR gene is as follows:
Figure PCTCN2022079966-appb-000008
Figure PCTCN2022079966-appb-000008
Survivin基因核苷酸序列(SEQ ID NO：2)如下：Survivin gene nucleotide sequence (SEQ ID NO: 2) is as follows:
Figure PCTCN2022079966-appb-000009
Figure PCTCN2022079966-appb-000009
Figure PCTCN2022079966-appb-000010
Figure PCTCN2022079966-appb-000010
Survivin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 Survivin gene has the amino acid sequence (SEQ ID NO: 5) as follows:
Figure PCTCN2022079966-appb-000011
Figure PCTCN2022079966-appb-000011
IL-15基因核苷酸序列(SEQ ID NO：3)如下：The IL-15 gene nucleotide sequence (SEQ ID NO: 3) is as follows:
Figure PCTCN2022079966-appb-000012
Figure PCTCN2022079966-appb-000012
IL-15基因表达的蛋白，其氨基酸序列(SEQ ID NO：6)如下所示：The amino acid sequence (SEQ ID NO: 6) of the protein expressed by the IL-15 gene is as follows:
Figure PCTCN2022079966-appb-000013
Figure PCTCN2022079966-appb-000013
(9)异种移植小鼠肿瘤模型(9) Xenograft mouse tumor model
从维通利华购买了6至8周龄的NOD-SCID小鼠。将2×10 6个NALM-6-GFP细胞静脉注射到NOD-SCID小鼠体内，建立异种移植小鼠模型。肿瘤细胞注射后1d，尾静脉注射1×10 7个CAR-T细胞，每日1次，共3d。使用IVIS(IVIS，Xenogen，Alameda，CA，USA)监测肿瘤的发展。以小鼠肢体瘫痪为实验终点，处死小鼠，获得小鼠骨髓细胞与血浆。小鼠的所有实验均由北京世纪坛医院机构审查委员会批准。 6- to 8-week-old NOD-SCID mice were purchased from Victoria Liver. 2×10 6 NALM-6-GFP cells were intravenously injected into NOD-SCID mice to establish 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 a total of 3 days. Tumor development was monitored using IVIS (IVIS, Xenogen, Alameda, CA, USA). With the limb paralysis of the mice as the experimental endpoint, the mice were sacrificed, and the bone marrow cells and plasma of the mice were obtained. All experiments in mice were approved by the Institutional Review Board of Beijing Shijitan Hospital.
(10)裸鼠成瘤实验(10) Tumor formation experiment in nude mice
从维通利华购买了6至8周龄的裸鼠。皮下注射1×10 7个CD19-CAR-Survivin T细胞。观察并记录是否有肿瘤形成。 Six to eight-week-old nude mice were purchased from Victoria Liver. 1 ×107 CD19-CAR-Survivin T cells were injected subcutaneously. Observe and record for tumor formation.
(11)统计分析(11) Statistical analysis
使用Graphpad Prism 8.0.2进行图形和统计分析。数据采用t检验分析，以p值<0.05为显著性： *P<0.05； **P<0.01； ***P<0.001； ****P<0.0001；NS(不显著)。所有实验至少重复三次。 Graphical and statistical analyzes were performed using Graphpad Prism 8.0.2. The data were analyzed by t-test, and p-value<0.05 was considered significant: * P<0.05; ** P<0.01; *** P<0.001; **** P<0.0001; NS (not significant). All experiments were repeated at least three times.
实施例1：IL-15促进抗原刺激的CAR-T细胞的杀伤作用Example 1: IL-15 promotes the killing effect of antigen-stimulated CAR-T cells
从健康供体PBMCs中分离原代T细胞，并用anti-CD3/CD28beads刺激。2天后，用基于CD19特异性单抗的编码CAR的逆转录病毒载体感染T细胞。用流式细胞术检测转导效率，结果显示54.5％的细胞表达CD19特异性CAR(图1A)。为了评估IL-2和IL-15对T细胞功能的不同影响，分离CAR-T细胞后，CAR-T细胞分别与IL-2(138U/ml)(CAR-T/IL-2)或IL-15(10ng/ml)(CAR-T/IL-15)培养14天(图1B)。之后用CD19阳性细胞系(Nalm-6)与CAR-T细胞共培养6小时，研究CAR-T细胞的激活状况。用流式细胞术检测CD107a的表达，结果显示：IL-15增加CD8 +T细胞表面的CD107a表达(图1C)。通过Elispot和ELISA检测，发现与CAR-T/IL-2相比，CAR-T/IL-15在细胞内(图1D)和在培养基中(图1E)产生更多的IFNγ表达。此外，还检测了CAR-T细胞的抗肿瘤活性。经流式检测，CAR-T/IL-15细胞对肿瘤细胞表现出较强的细胞毒性(2.2％肿瘤细胞残留)，而CAR-T/IL-2的抗肿瘤活性较弱(53.5％肿瘤细胞残留)(图1F)。随后检测代表T细胞活化水平的颗粒酶A和颗粒酶B的蛋白表达。图1G显示，IL-15随靶细胞Nalm-6抗原与CAR-T细胞共培养时间的延长，促进颗粒酶A和颗粒酶B的表达；未加细胞因子培养的CAR-T细胞，颗粒酶A和颗粒酶B表达最低。 Primary T cells were isolated from healthy donor PBMCs and stimulated with anti-CD3/CD28beads. Two days later, T cells were infected with a retroviral vector encoding a CAR based on a CD19-specific mAb. The transduction efficiency was detected by flow cytometry, and the results showed that 54.5% of the cells expressed CD19-specific CAR (Fig. 1A). In order to evaluate the different effects of IL-2 and IL-15 on T cell function, after isolation of CAR-T cells, CAR-T cells were mixed with IL-2 (138U/ml) (CAR-T/IL-2) or IL- 15 (10ng/ml) (CAR-T/IL-15) was cultured for 14 days (Fig. 1B). Afterwards, the CD19 positive cell line (Nalm-6) was co-cultured with CAR-T cells for 6 hours to study the activation status of CAR-T cells. The expression of CD107a was detected by flow cytometry, and the results showed that IL-15 increased the expression of CD107a on the surface of CD8 + T cells ( FIG. 1C ). Through Elispot and ELISA detection, it was found that compared with CAR-T/IL-2, CAR-T/IL-15 produced more IFNγ expression in cells (Figure 1D) and in culture medium (Figure 1E). In addition, the anti-tumor activity of CAR-T cells was also tested. Through flow cytometry, CAR-T/IL-15 cells showed strong cytotoxicity to tumor cells (2.2% tumor cells remained), while CAR-T/IL-2 had weak anti-tumor activity (53.5% tumor cells residual) (Fig. 1F). The protein expression of granzyme A and granzyme B, which represent the activation level of T cells, was then detected. Figure 1G shows that IL-15 promotes the expression of granzyme A and granzyme B with the prolongation of the co-culture time of target cell Nalm-6 antigen and CAR-T cells; CAR-T cells cultured without cytokines, granzyme A and granzyme B expression was the lowest.
实施例2：IL-15在抗原刺激下促进T细胞增殖及Tscm的形成Example 2: IL-15 promotes T cell proliferation and Tscm formation under antigen stimulation
由于IL-15和IL-2是细胞增殖相关的细胞因子，我们直接用细胞计数来测定IL-15和IL-2对细胞增殖的影响。Since IL-15 and IL-2 are cytokines related to cell proliferation, we directly use cell counting to determine the effect of IL-15 and IL-2 on cell proliferation.
用Nalm-6每隔7天刺激CAR-T细胞，结果显示CAR-T/IL-15细胞具有较高的生长速率(图2A和2B)。CAR-T cells were stimulated with Nalm-6 every 7 days, and the results showed that CAR-T/IL-15 cells had a higher growth rate (Figure 2A and 2B).
细胞周期的流式细胞术分析显示，在第7天，约21.67％的CAR-T/IL-15细胞处于细胞分裂S期，而约16.07％的CAR-T/IL-2细胞处于细胞分裂S期(图2C)。随着时间的延长，细胞增殖能力略有下降，分别有15.3％和9.7％的细胞细胞分裂S期阶段(图2D)。Flow cytometry analysis of the cell cycle showed that at day 7, about 21.67% of CAR-T/IL-15 cells were in the S phase of cell division, while about 16.07% of CAR-T/IL-2 cells were in the S phase of cell division period (Figure 2C). With the prolongation of time, the cell proliferation ability decreased slightly, with 15.3% and 9.7% of the cells in the S phase of cell division, respectively (Fig. 2D).
接下来检测CAR-T细胞的表型变化。CD132是IL-2和IL-15的共同受体亚基，在IL-15的诱导下表达显著上调(图2E)。对代表CAR-T细胞长期持久性能力的Tscm细胞(CD45RO -CCR7 +CD27 +CD95 +)进行了研究,结果表明,与IL-2相比,IL-15促进了CD8 +CAR-T细胞中Tscm细胞的形成(分别为1.08％和0.31％)(图2F),表明CAR-T/IL-15细胞具有向多亚群分化和自我更新的能力。 Next, the phenotypic changes of CAR-T cells were detected. CD132 is a co-receptor subunit of IL-2 and IL-15, and its expression was significantly upregulated under the induction of IL-15 (Fig. 2E). Tscm cells (CD45RO - CCR7 + CD27 + CD95 + ), which represent the long-term persistence ability of CAR-T cells, were studied, and the results showed that IL-15 promoted Tscm in CD8 + CAR-T cells compared with IL-2. The cells formed (1.08% and 0.31%, respectively) (Figure 2F), indicating that CAR-T/IL-15 cells have the ability to differentiate into multiple subpopulations and self-renew.
实施例3：在不同靶向的CAR-T细胞中观察IL-15介导的效应Example 3: Observation of IL-15-mediated effects in different targeted CAR-T cells
为了确定在CD19-CAR-T/IL-15细胞上观察到的表型和功能特征是否可推广到其他CAR分子，使用胶质瘤靶向的EphA2-CAR-T细胞进行了类似的研究，该细胞利用4-1BB共刺激结构域。EphA2-CAR序列信息参见CN202110919075.X。与IL-2培养的细胞相比，用IL-15培养的EphA2-CAR-T细胞消耗较少，细胞增殖更高(图3A)。与CD19-CAR-T细胞相似，用IL-15培养的EphA2-CAR-T细胞表现出更多的多功能表型，产生更多的IFNγ(图3B)。同时，实时细胞生长监测(RTCA)系统和GFP-Luc流式检测结果显示，EphA2-CAR-T/IL-15细胞抗肿瘤活性增强(图3C和3D)。To determine whether the phenotypic and functional features observed on CD19-CAR-T/IL-15 cells could be generalized to other CAR molecules, a similar study was performed using glioma-targeted EphA2-CAR-T cells, which Cells utilize the 4-1BB co-stimulatory domain. For the sequence information of EphA2-CAR, please refer to CN202110919075.X. EphA2-CAR-T cells cultured with IL-15 were less depleted and had higher cell proliferation compared to cells cultured with IL-2 (Fig. 3A). Similar to CD19-CAR-T cells, EphA2-CAR-T cells cultured with IL-15 exhibited a more multifunctional phenotype and produced more IFNγ (Fig. 3B). At the same time, the real-time cell growth monitoring (RTCA) system and GFP-Luc flow detection results showed that the anti-tumor activity of EphA2-CAR-T/IL-15 cells was enhanced (Figure 3C and 3D).
实施例4：IL-15通过激活PI3K/Akt信号通路上调Survivin的表达Example 4: IL-15 up-regulates the expression of Survivin by activating the PI3K/Akt signaling pathway
为探讨IL-15对CAR-T细胞有较好的增殖作用的机制，采用高通量RNA测序技术检测IL-2和IL-15培养的CAR-T细胞差异表达基因。在研究发现的上调基因中，选择了一种进化上保守的、对细胞分裂至关重要的、能抑制细胞死亡的真核蛋白survivin，进行进一步研究。在分别用IL-15和IL-2培养CAR-T细胞后，应用PCR和Western blot检测survivin的表达。结果表明，IL-2和IL-15在抗原刺激下均可上调CAR-T细胞中survivin的mRNA水平和蛋白的水平(图4A和4B)。In order to explore the mechanism of IL-15's better proliferation effect on CAR-T cells, high-throughput RNA sequencing technology was used to detect differentially expressed genes in CAR-T cells cultured with IL-2 and IL-15. Among the up-regulated genes found in the study, an evolutionarily conserved eukaryotic protein survivin that is critical to cell division and can inhibit cell death was selected for further study. After the CAR-T cells were cultured with IL-15 and IL-2, the expression of survivin was detected by PCR and Western blot. The results showed that both IL-2 and IL-15 could up-regulate the mRNA level and protein level of survivin in CAR-T cells under antigen stimulation (Figure 4A and 4B).
由于PI3K/Akt通路与刺激细胞增殖和生长有关，本研究探讨了IL-15诱导survivin的作用机制。结果显示，IL-2或IL-15激活了CAR-T细胞的PI3K/Akt通路，IL-15具有更强作用(图4C)。为了证实IL-15诱导的survivin表达增加是通过PI3K/Akt途径，使用PI3K/Akt途径抑制剂ly194002，结果表明ly294002抑制survivin的表达(图4D)。Since the PI3K/Akt pathway is related to the stimulation of cell proliferation and growth, this study explored the mechanism of IL-15-induced survivin. The results showed that IL-2 or IL-15 activated the PI3K/Akt pathway of CAR-T cells, and IL-15 had a stronger effect (Figure 4C). In order to confirm that the IL-15-induced increase in survivin expression is through the PI3K/Akt pathway, the PI3K/Akt pathway inhibitor ly194002 was used, and the results showed that ly294002 inhibited the expression of survivin (Fig. 4D).
实施例5：Armored CAR-T细胞的构建Example 5: Construction of Armored CAR-T cells
构建了CD19-CAR连接survivin基因或IL-15基因的质粒载体(图5A)，并将这些共表达逆转录病毒载体导入T细胞。为了更好地评估CAR-T细胞的不同功能并排除转染效率的干扰，基于测定的Armored CAR-T细胞的转染效率，将CAR阳性T细胞的百分比调整为一致(图5B)。接下来进一步证实IL-15和survivin的成功表达。收集CAR-T细胞上清液，用ELISA试剂盒测定IL-15浓度。图5C显示CD19-CAR-IL-15T细胞释放IL-15到培养基中。此外，提取CAR-T细胞总RNA，用Q-PCR检测survivin的相对表达量。结果表明，过表达survivin的CAR-T细胞具有最高的survivin表达(图5D)。此外，受自分泌IL-15的影响，与CD19-CAR T细胞相比，CD19-CAR-IL-15T细胞的survivin表达增加，表明IL-15对survivin表达的诱导作用。A plasmid vector with CD19-CAR linked to survivin gene or IL-15 gene was constructed (Fig. 5A), and these co-expression retroviral vectors were introduced into T cells. In order to better evaluate the different functions of CAR-T cells and exclude the interference of transfection efficiency, based on the measured transfection efficiency of Armored CAR-T cells, the percentage of CAR-positive T cells was adjusted to be consistent (Fig. 5B). Next, we further confirmed the successful expression of IL-15 and survivin. The CAR-T cell supernatant was collected, and the IL-15 concentration was determined with an ELISA kit. Figure 5C shows CD19-CAR-IL-15 T cells release IL-15 into the medium. In addition, the total RNA of CAR-T cells was extracted, and the relative expression of survivin was detected by Q-PCR. The results showed that CAR-T cells overexpressing survivin had the highest survivin expression (Fig. 5D). Furthermore, under the influence of autocrine IL-15, CD19-CAR-IL-15 T cells showed increased expression of survivin compared with CD19-CAR T cells, indicating the induction of survivin expression by IL-15.
实施例6：Survivin过表达的CAR-T细胞在体外表现出较高的增殖能力和低分化表型。Example 6: Survivin overexpressed CAR-T cells showed higher proliferation ability and poorly differentiated phenotype in vitro.
采用Vi-CELL细胞活力分析仪，通过直接活细胞计数法，显示survivin和IL-15过表达的Armored CAR-T细胞表现出更高的增殖能力(图6A)。此外，由于IL-2是T细胞的生长因子，因此测量了Armored CAR-T上清液中的IL-2浓度，结果表明，与CD19-CAR T细胞相比，CD19-CAR-survivin和CD19-CAR-IL-15T细胞释放更多的IL-2(图6B)。此外，鉴于CD132是IL-2和IL-15的共同受体亚基链，检测了CD132的表达，结果显示CD19-CAR-survivin和CD19-CAR-IL-15T细胞表面CD132的表达分别为22.7％和15.0％，而CD19-CAR T细胞表面CD132的表达为51.3％(图6C)。Vi-CELL cell viability analyzer was used to directly count live cells, showing that Armored CAR-T cells overexpressing survivin and IL-15 exhibited higher proliferation ability (Figure 6A). In addition, since IL-2 is a growth factor for T cells, the concentration of IL-2 in the supernatant of Armored CAR-T was measured, and the results showed that, compared with CD19-CAR T cells, CD19-CAR-survivin and CD19- CAR-IL-15T cells released more IL-2 (Fig. 6B). In addition, given that CD132 is a co-receptor subunit chain of IL-2 and IL-15, the expression of CD132 was detected, and the results showed that the expression of CD132 on the surface of CD19-CAR-survivin and CD19-CAR-IL-15 T cells was 22.7%, respectively and 15.0%, while the expression of CD132 on the surface of CD19-CAR T cells was 51.3% (Fig. 6C).
接下来分析了Armored CAR-T细胞的分化表型。用NALM-6细胞刺激7天后，结果显示只有1.67％的CD8 +CD19-CAR T细胞为Tscm，而CD19-CAR-survivin和CD19-CAR-IL-15T细胞具有更多的Tscm细胞(分别为4.06％和9.23％)(图6D)。此外，据报道，分化程度较低的T细胞在抗原刺激下产生较少的IFNγ。因此，用NALM-6细胞刺激CD19-CAR、CD19-CAR-survivin和CD19-CAR-IL-15T细胞24小时，并通过ELISA测定IFNγ的浓度。如图6E所示，CD19-CAR-survivin和CD19-CAR-IL-15T细胞显示出较少的IFNγ产生，这意味着CD19-CAR-survivin和CD19-CAR-IL-15T细胞的分化表型较低。随后，由于survivin是一种重要的凋亡抑制剂，我们分析了凋亡细胞的百分比和细胞存活率。结果显示，survivin和IL-15降低了CAR-T细胞凋亡并提高了细胞活力(图6F)。 Next, the differentiation phenotype of Armored CAR-T cells was analyzed. After 7 days of stimulation with NALM-6 cells, the results showed that only 1.67% of CD8 + CD19-CAR T cells were Tscm, while CD19-CAR-survivin and CD19-CAR-IL-15 T cells had more Tscm cells (4.06 % and 9.23%) (Fig. 6D). In addition, it has been reported that less differentiated T cells produce less IFNγ upon antigen stimulation. Therefore, CD19-CAR, CD19-CAR-survivin and CD19-CAR-IL-15T cells were stimulated with NALM-6 cells for 24 hours, and the concentration of IFNγ was determined by ELISA. As shown in Figure 6E, CD19-CAR-survivin and CD19-CAR-IL-15T cells showed less IFNγ production, which means that the differentiation phenotype of CD19-CAR-survivin and CD19-CAR-IL-15T cells was less Low. Subsequently, since survivin is an important inhibitor of apoptosis, we analyzed the percentage of apoptotic cells and cell viability. The results showed that survivin and IL-15 decreased CAR-T cell apoptosis and increased cell viability (Fig. 6F).
实施例7：Armored CAR-T细胞在体内表现出增强的抗肿瘤活性。Example 7: Armored CAR-T cells exhibit enhanced anti-tumor activity in vivo.
进一步检测Armored CAR-T细胞在体内的抗肿瘤活性，将NALM-6-GFP细胞静脉注射到NOD-SCID小鼠体内，以生成异种移植小鼠肿瘤模型。一天后，静脉注射1×10 7个Armored CAR-T细胞，连续三天。非转导T细胞(NT)作为阴性对照，并对小鼠的肿瘤负荷进行100天的监测(图7A)。如图所示。与NT细胞和CD19-CAR T细胞治疗组小鼠相比，CD19-CAR-Survivin T细胞治疗组的小鼠存活时间延长，其中有两只小鼠肿瘤无复发，并且存活时间超过100天(图7B和图7D)。此外，CD19-CAR和CD19-CAR-Survivin T组之间的肿瘤负担不同，CD19-CAR-Survivin T治疗组的小鼠肿瘤发展较慢(图7B和7C)。证明过表达survivin的Armored CAR-T细胞在体内具有更强的抗肿瘤活性。 To further test the anti-tumor activity of Armored CAR-T cells in vivo, NALM-6-GFP cells were intravenously injected into NOD-SCID mice to generate xenograft mouse tumor models. One day later, 1×10 7 Armored CAR-T cells were injected intravenously for three consecutive days. Non-transduced T cells (NT) served as a negative control, and tumor burden in mice was monitored for 100 days (Fig. 7A). as the picture shows. Compared with the mice treated with NT cells and CD19-CAR T cells, the survival time of the mice treated with CD19-CAR-Survivin T cells was prolonged, and two of the mice had no tumor recurrence and survived for more than 100 days (Fig. 7B and 7D). In addition, the tumor burden was different between the CD19-CAR and CD19-CAR-Survivin T groups, and the mice in the CD19-CAR-Survivin T-treated group developed slower tumors (Figure 7B and 7C). It is proved that the Armored CAR-T cells overexpressing survivin have stronger anti-tumor activity in vivo.
实施例8：CD19-CAR-Survivin T细胞在体内更强的抗肿瘤活性与其较长的存活周期成正相关Example 8: The stronger anti-tumor activity of CD19-CAR-Survivin T cells in vivo is positively correlated with its longer survival period
为了研究CAR-T细胞在荷瘤小鼠体内的持久性，在实施例7的动物实验中，处死小鼠时收集骨髓细胞并提取总RNA。用Q-PCR检测SFG逆转录病毒载体的表达。结果显示，与不存在SFG逆转录病毒载体表达的NT组和CD19-CAR组相比，CD19-CAR-Survivin组具有更多的SFG逆转录病毒载体表达(图 8A)，这意味着Survivin促进了小鼠体内CAR-T细胞的存活。除此之外，第50天，收集每只小鼠的血液，分离血清并检测人IL-15的浓度。图8B显示CD19-CAR-Survivin T治疗组的小鼠血液中含有更多的人IL-15。这些结果表明，CD19-CAR-Survivin T细胞在小鼠体内具有更长的持续性，因此有更多的IL-15产生，并且具有较强的抗肿瘤活性。In order to study the persistence of CAR-T cells in tumor-bearing mice, in the animal experiment of Example 7, bone marrow cells were collected and total RNA was extracted when the mice were sacrificed. The expression of SFG retroviral vector was detected by Q-PCR. The results showed that compared with the NT group and CD19-CAR group without SFG retroviral vector expression, the CD19-CAR-Survivin group had more SFG retroviral vector expression (Figure 8A), which meant that Survivin promoted Survival of CAR-T cells in mice. Besides, on the 50th day, the blood of each mouse was collected, the serum was separated and the concentration of human IL-15 was detected. Figure 8B shows that mice in the CD19-CAR-Survivin T treatment group contained more human IL-15 in their blood. These results suggest that CD19-CAR-Survivin T cells have longer persistence in mice, thus more IL-15 production, and stronger antitumor activity.
尽管如此，Survivin作为一种癌基因，被认为具有致瘤作用。因此，我们利用裸鼠研究了CD19-CAR-Survivin T细胞的致瘤性。将CD19-CAR-Survivin T细胞皮下注射到裸鼠体内，并记录肿瘤生长情况。结果表明，至实验目前为止5个月以上，并没有观察到肿瘤生长(图8C)，小鼠健康。Nevertheless, Survivin, as an oncogene, is considered to have tumorigenic effects. Therefore, we studied the tumorigenicity of CD19-CAR-Survivin T cells using nude mice. CD19-CAR-Survivin T cells were subcutaneously injected into nude mice, and tumor growth was recorded. The results showed that no tumor growth was observed for more than 5 months so far in the experiment ( FIG. 8C ), and the mice were healthy.
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。The above-mentioned embodiments are only to describe the preferred mode of the present invention, not to limit the scope of the present invention. Without departing from the design spirit of the present invention, those skilled in the art may make various Variations and improvements should fall within the scope of protection defined by the claims of the present invention.
序列表sequence listing
Figure PCTCN2022079966-appb-000014
Figure PCTCN2022079966-appb-000014
Figure PCTCN2022079966-appb-000015
Figure PCTCN2022079966-appb-000015
Figure PCTCN2022079966-appb-000016
Figure PCTCN2022079966-appb-000016

Claims (21)

  1. 一种核酸构建体，其中，所述核酸构建体具有如下所示结构：car-(2A)-e，其中，car表示嵌合抗原受体多肽的编码核酸，2A表示编码自剪切2A肽的寡核苷酸，e表示功能性融合的蛋白的编码核酸，且“-”表示连接相邻核苷酸的磷脂键或编码连接肽的寡核苷酸；其中，所述功能性融合的蛋白编码核酸包含Survivin基因。A nucleic acid construct, wherein the nucleic acid construct has the following structure: car-(2A)-e, wherein car represents the nucleic acid encoding the chimeric antigen receptor polypeptide, and 2A represents the nucleic acid encoded from the cleaved 2A peptide Oligonucleotide, e represents the coding nucleic acid of the protein of functional fusion, and "-" represents the phospholipid bond connecting adjacent nucleotides or the oligonucleotide encoding the connecting peptide; wherein, the protein coding of the functional fusion The nucleic acid comprises a Survivin gene.
  2. 权利要求1的核酸构建体，其中，所述嵌合抗原受体的抗原结合结构域特异性靶向肿瘤抗原，优选地所述肿瘤抗原为膜抗原，例如CD19或EphA2，且优选CD19。The nucleic acid construct of claim 1, wherein the antigen binding domain of the chimeric antigen receptor specifically targets a tumor antigen, preferably the tumor antigen is a membrane antigen, such as CD19 or EphA2, and preferably CD19.
  3. 权利要求1-2任一项的核酸构建体，其中，所述自剪切2A肽选自：P2A,T2A，E2A或F2A肽，且优选地为P2A肽。The nucleic acid construct according to any one of claims 1-2, wherein the self-cleaving 2A peptide is selected from: P2A, T2A, E2A or F2A peptide, and is preferably a P2A peptide.
  4. 权利要求1-3任一项的核酸构建体，其中，所述car编码核酸编码嵌合抗原受体(CAR)多肽，所述嵌合抗原受体多肽从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，铰链区或间隔区、跨膜结构域、和胞质结构域，其中所述胞质结构域包含共刺激结构域和胞内信号传导结构域。The nucleic acid construct according to any one of claims 1-3, wherein the car-encoding nucleic acid encodes a chimeric antigen receptor (CAR) polypeptide, and the chimeric antigen receptor polypeptide comprises from the N-terminus to the C-terminus: optionally Signal peptide (such as GM-CSFRα signal peptide), extracellular antigen binding domain specifically binding tumor antigen, hinge region or spacer, transmembrane domain, and cytoplasmic domain, wherein said cytoplasmic domain comprises co- Stimulatory domain and intracellular signaling domain.
  5. 权利要求1-4任一项的核酸构建体，其中，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，The nucleic acid construct according to any one of claims 1-4, wherein the antigen-binding domain specifically binding to a tumor antigen is an antibody or an antibody fragment, especially scFv,
    优选地，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的VL和SEQ ID NO:9的VH，再优选地，包含SEQ ID NO:11所示的氨基酸序列的scFv。Preferably, the antigen binding domain targets CD19, more preferably comprises 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 Kabat defined CDR sequence), more preferably, comprising the VL of SEQ ID NO:8 and the VH of SEQ ID NO:9, more preferably, comprising the scFv of the amino acid sequence shown in SEQ ID NO:11.
  6. 权利要求1-5任一项的核酸构建体，其中，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所示氨基酸序列的CD28铰链区。The nucleic acid construct according to any one of claims 1-5, wherein the hinge region/spacer is selected from: a hinge region from IgG or a spacer from CD8α or CD28 extracellular region, and is preferably a human CD8α hinge region or A CD28 hinge region, for example, a CD28 hinge region comprising the amino acid sequence shown in SEQ ID NO: 12.
  7. 权利要求1-6任一项的核酸构建体，其中，所述跨膜结构域选自：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所示氨基酸序列的跨膜结构域。The nucleic acid construct according to any one of claims 1-6, wherein the transmembrane domain is selected from the group consisting of CD4, CD8α, CD28 and CD3ζ transmembrane domains, and is preferably human CD8 transmembrane domain or CD28 transmembrane A structural domain, for example, a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 13.
  8. 权利要求1-7任一项的核酸构建体，其中，所述共刺激结构域选自：CD28,CD27,4-1BB,ICOS和OX40的共刺激结构域；且优选是人CD28和4-1BB的共刺激结构域，例如，包含SEQ ID NO:14所示氨基酸序列的共刺激结构域。The nucleic acid construct according to any one of claims 1-7, wherein the co-stimulatory domain is selected from the group consisting of: CD28, CD27, 4-1BB, ICOS and OX40 co-stimulatory domains; and preferably human CD28 and 4-1BB A co-stimulatory domain, for example, a co-stimulatory domain comprising the amino acid sequence shown in SEQ ID NO: 14.
  9. 权利要求1-8任一项的核酸构建体，其中，所述胞内信号传导结构域为CD3ζ信号传导结构域，例如，包含SEQ ID NO:15所示氨基酸序列的CD3ζ信号传导结构域。The nucleic acid construct according to any one of claims 1-8, wherein the intracellular signaling domain is a CD3ζ signaling domain, for example, comprising the CD3ζ signaling domain of the amino acid sequence shown in SEQ ID NO:15.
  10. 权利要求1-9任一项的核酸构建体，其中所述survivin基因包含编码SEQ ID NO:5所示的氨基酸序列或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上的同一性的氨基酸序列的多核苷酸；优选地，所述survivin基因包含SEQ ID NO:4所示的多核苷酸，或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上的同一性的核苷酸序列的多核苷酸。The nucleic acid construct of any one of claims 1-9, wherein said survivin gene comprises encoding the amino acid sequence shown in SEQ ID NO: 5 or has at least 90%, 92%, 95%, 96%, 97%, 98% thereof %, 99% or more identical amino acid sequence polynucleotides; preferably, the survivin gene comprises the polynucleotide shown in SEQ ID NO: 4, or has at least 90%, 92%, 95%, A polynucleotide having a nucleotide sequence of 96%, 97%, 98%, 99% or more identity.
  11. 权利要求1-10任一项的核酸构建体，其中,所述嵌合抗原受体编码核酸包含：The nucleic acid construct according to any one of claims 1-10, wherein said chimeric antigen receptor encoding nucleic acid comprises:
    编码抗人CD19的单链抗体的核苷酸序列、Nucleotide sequence encoding a single-chain antibody against human CD19,
    编码CD28铰链区的核苷酸序列、The nucleotide sequence encoding the hinge region of CD28,
    编码CD28跨膜结构域的核苷酸序列、Nucleotide sequence encoding CD28 transmembrane domain,
    编码CD28共刺激结构域的核苷酸序列、和a nucleotide sequence encoding a CD28 co-stimulatory domain, and
    编码CD3ζ信号传导结构域的核苷酸序列，a nucleotide sequence encoding the CD3ζ signaling domain,
    优选地，所述嵌合抗原受体编码核酸包含编码SEQ ID NO:2或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上的同一性的氨基酸序列的多核苷酸；优选地，所述嵌合抗原受体编码核酸包含SEQ ID NO:1或与其具有至少90％、92％、95％、96％、97％、98％、99％或以上的同一性的核苷酸序列的多核苷酸。Preferably, the chimeric antigen receptor-encoding nucleic acid comprises an amino acid encoding SEQ ID NO: 2 or having at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more identity thereto sequence; preferably, said chimeric antigen receptor encoding nucleic acid comprises SEQ ID NO: 1 or has at least 90%, 92%, 95%, 96%, 97%, 98%, 99% or more thereof The identity of a nucleotide sequence to a polynucleotide.
  12. 一种重组载体，其包含权利要求1-11任一项所述的核酸构建体，例如，所述载体选自DNA载体、RNA载体、慢病毒载体、腺病毒载体或逆转录病毒载体，优选地，逆转录病毒载体。A recombinant vector, which comprises the nucleic acid construct described in any one of claims 1-11, for example, the carrier is selected from DNA vectors, RNA vectors, lentiviral vectors, adenoviral vectors or retroviral vectors, preferably , a retroviral vector.
  13. 一种宿主细胞，其包含权利要求12所述的重组载体，或者在染色体中整合有权利要求1-11任一项的核酸构建体，其中所述细胞是免疫效应细胞，例如T细胞或NK细胞，例如，所述T细胞是自体T细胞或同种异体T细胞。A host cell comprising the recombinant vector of claim 12, or the nucleic acid construct of any one of claims 1-11 integrated in the chromosome, wherein the cell is an immune effector cell, such as T cell or NK cell , for example, the T cells are autologous T cells or allogeneic T cells.
  14. 一种CAR-T细胞，其中所述细胞包含编码CAR多肽的异源多核苷酸和编码Survivin的异源多核苷酸，优选地，所述细胞包含权利要求1-11任一项的核酸构建体。A CAR-T cell, wherein the cell comprises a heterologous polynucleotide encoding a CAR polypeptide and a heterologous polynucleotide encoding Survivin, preferably, the cell comprises the nucleic acid construct according to any one of claims 1-11 .
  15. 制备权利要求13或14的细胞的方法，包括用权利要求12的载体转导所述细胞。A method of producing a cell according to claim 13 or 14, comprising transducing said cell with the vector of claim 12.
  16. 一种药物组合物，其包括药学上可接受的载体以及权利要求1-11任一项所述的核酸构建体、权利要求12所述的重组载体、权利要求13所述的宿主细胞、或权利要求14所述的CAR-T细胞。A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the nucleic acid construct according to any one of claims 1-11, the recombinant vector according to claim 12, the host cell according to claim 13, or the The CAR-T cells described in 14 are required.
  17. 权利要求16的药物组合物，所述药物组合物还包括细胞因子IL-15。The pharmaceutical composition of claim 16, further comprising the cytokine IL-15.
  18. 权利要求1-11任一项所述的核酸构建体、权利要求12所述的重组载体、权利要求13所述的宿主细胞、权利要求14所述的CAR-T细胞、或权利要求16-17任一项的药物组合物的应用，用于在受试者中预防或治疗肿瘤或提供抗肿瘤免疫，或用于制备抗肿瘤药物或者制剂。The nucleic acid construct according to any one of claims 1-11, the recombinant vector according to claim 12, the host cell according to claim 13, the CAR-T cell according to claim 14, or the recombinant vector according to claim 16-17 The application of any one of the pharmaceutical compositions is used to prevent or treat tumors or provide anti-tumor immunity in a subject, or to prepare anti-tumor drugs or preparations.
  19. 一种在受试者中预防或治疗癌症或提供抗肿瘤免疫的方法，包括向有需要的受试者施用有效量的 权利要求1-11任一项所述的核酸构建体、权利要求12所述的重组载体、权利要求13所述的宿主细胞、权利要求14所述的CAR-T细胞、或权利要求16-17任一项的药物组合物。A method for preventing or treating cancer or providing anti-tumor immunity in a subject, comprising administering to a subject in need an effective amount of the nucleic acid construct according to any one of claims 1-11, or the nucleic acid construct described in claim 12. The recombinant vector described in claim 13, the CAR-T cell described in claim 14, or the pharmaceutical composition of any one of claims 16-17.
  20. 权利要求18的用途或权利要求19的方法，其中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)。The use of claim 18 or the method of claim 19, wherein the cancer comprises a hematological cancer (eg leukemia) or a solid tumor (eg glioma).
  21. 一种用于增加CAR-T细胞的持久性的方法，包括向所述CAR-T细胞中引入包含编码SURVIVIN蛋白的异源多核苷酸的核酸构建体。A method for increasing the persistence of CAR-T cells, comprising introducing into the CAR-T cells a nucleic acid construct comprising a heterologous polynucleotide encoding a SURVIVIN protein.
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