WO2021213479A1 - Shp2特异性失活突变蛋白及其在car-t治疗中应用 - Google Patents

Shp2特异性失活突变蛋白及其在car-t治疗中应用 Download PDF

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WO2021213479A1
WO2021213479A1 PCT/CN2021/089087 CN2021089087W WO2021213479A1 WO 2021213479 A1 WO2021213479 A1 WO 2021213479A1 CN 2021089087 W CN2021089087 W CN 2021089087W WO 2021213479 A1 WO2021213479 A1 WO 2021213479A1
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shp
cells
car
protein
mutant protein
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张雷
高明明
王慧宇
祖睿
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复星凯特生物科技有限公司
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Definitions

  • the present invention relates to the field of medicine, in particular to SHP2 specific inactivating mutant protein and its application in CAR-T therapy.
  • Chimeric Antigen Receptor (“CAR”) is an artificial chimeric protein obtained by fusing a single-chain antibody that recognizes the cell surface antigen of cancer cells with a signal transduction region that induces T cell activation.
  • CAR-T cells T cells capable of expressing CAR molecules.
  • CAR-T cells are tumor-reactive and can kill cancer cells independently of the interaction with the major histocompatibility gene complex (MHC).
  • MHC major histocompatibility gene complex
  • Cancer immunotherapy using CAR-T cells is a therapy in which T cells are collected from patients, the CAR-encoding gene is introduced into the above-mentioned T cells for amplification, and then reinfused into the patient.
  • the current CAR-T technology still faces many problems: for example, the survival efficiency of CAR-T cells in the organism is low, and the proliferation capacity is insufficient; the migration of CAR-T cells to tumor tissues is insufficient; CAR-T cells will be depleted (for example, the activity of CAR-T cells can be inhibited by immunosuppressive checkpoints such as PD-L1/PD-1, TIM-3, LAG-3, etc.); tumor microenvironment and the existing immune system The problem of CAR-T cell inhibition caused by inhibitors such as TGF- ⁇ and IL-10.
  • the purpose of the present invention is to provide a more effective CAR-T cell, which has high survival efficiency, strong proliferation ability, is not prone to exhaustion during the anti-tumor process, and/or is not susceptible to tumor microenvironment Inhibition and other advantages, so that CAR-T functions can be more fully utilized.
  • a SHP-2 mutein in the first aspect of the present invention, has a mutation selected from the group consisting of G60E, D61E, Y62W, R278N, Y279H, K364R, K364Q, Q506K, C459S or a combination thereof, or the SHP-2 mutant protein is a truncated SHP-2 protein; wherein the position of the amino acid is based on SEQ ID No.:1.
  • the mutant protein is a fragment or a full-length mutant of human SHP-2, wherein the fragment has an amino acid sequence corresponding to the N-SH2 domain of the SHP-2 wild-type protein, or The fragment has an amino acid sequence corresponding to the N-SH2 and C-SH2 domains of the SHP-2 wild-type protein; the full-length mutant has an amino acid sequence corresponding to the full-length of the SHP-2 wild-type protein (the fragment Or the full-length mutant has the point mutation at the corresponding site).
  • the mutant protein has an amino acid sequence corresponding to positions Y1 to Y2 of the SHP-2 wild-type protein, wherein Y1 is 1, 2, or 3; and Y2 is 100, 101, 102, 103, 104, or 214, 215, 216, 217, 218.
  • the mutant protein has positions 1-102, 1-104, 2-102, 2-104, 1-216 corresponding to SHP-2 wild-type protein.
  • the mutant protein has an amino acid sequence corresponding to positions 1-593 or 2-593 of the SHP-2 wild-type protein.
  • the mutant protein is inactive.
  • the "inactive type” refers to that, compared with the wild-type SHP-2 protein, these inactive SHP-2 do not have the phosphatase activity of the wild-type SHP-2 protein and have inhibitory properties. The ability of cells to endogenous SHP-2.
  • the mutant protein has an amino acid sequence as shown in SEQ ID No.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. Of peptides.
  • a fusion protein is provided, the fusion protein is artificially constructed, and the fusion protein includes: the SHP-2 mutein of the first aspect of the present invention, and the SHP-2 mutein is a polypeptide element fused together.
  • the polypeptide element includes various elements such as fluorescent protein (such as GFP, RFP) and the like.
  • a polynucleotide which encodes the SHP-2 mutein according to the first aspect of the present invention or the fusion protein according to the second aspect of the present invention.
  • the polynucleotide includes DNA or RNA.
  • an activated SHP-2 mutein expression construct (or expression cassette) is provided.
  • the expression construct includes: an activated promoter and an operative connection with the promoter.
  • the polynucleotide sequence described in the third aspect of the present invention ie, the coding sequence of the SHP-2 mutein described in the first aspect).
  • the activated promoter includes a constitutively activated promoter and a conditionally activated promoter
  • conditionally activated promoter is activated after the CAR-T cell binds to the target molecule through the CAR molecule.
  • the constitutively activated promoter includes but is not limited to CMV promoter, SV40 promoter, PGK promoter, EF1 ⁇ promoter, ⁇ -actin promoter, MSCV Promoter and CAG promoter; conditionally activated promoter is IL2 mini promoter.
  • sequence of the CMV promoter is shown in SEQ ID No: 16.
  • sequence of the IL2 mini promoter is shown in SEQ ID No: 17 or 18.
  • a vector which contains the polynucleotide of the third aspect or the activated SHP-2 mutein expression construct of the fourth aspect.
  • the vector includes an expression vector.
  • the vector includes a viral vector.
  • the expression vector includes: a lentiviral vector, an adenovirus vector, an adeno-associated virus AAV vector, a plasmid, or a combination thereof.
  • a cell which contains the vector of the fifth aspect, or integrates the polynucleotide of the third aspect or the activation of the fourth aspect in the genome.
  • Type SHP-2 mutein expression construct is provided.
  • the expression construct contains a constitutively activated promoter or a conditionally activated promoter.
  • the cells are immune cells.
  • the immune cells express foreign chimeric antigen receptors (CAR).
  • CAR chimeric antigen receptors
  • the immune cells express chimeric antigen receptors for anti-tumor.
  • the chimeric antigen receptor includes: optional leader peptide, extracellular antigen binding domain, hinge region, transmembrane region and intracellular region.
  • the immune cells include: T cells, NK cells, or a combination thereof.
  • the T cells include: CD4 + cells, CD8 + cells, or a combination thereof.
  • the immune cells are autologous or allogeneic.
  • the extracellular binding domain targets CD20, EGFR, FITC, CD19, CD22, CD33, PSMA, GD2, EGFR variant, ROR1, c-Met, HER2, CEA, mesothelin (mesothelin) , GM2, CD7, CD10, CD30, CD34, CD38, CD41, CD44, CD74, CD123, D133, CD171, CD276, CLDN18.2, MUC16, MUC1, CS1 (CD319), IL-13Ra2, BCMA, GPC3, DLL3, LewisY, IgG kappa chain, folate receptor- ⁇ , PSCA, or EpCAM.
  • the immune cell contains an expression construct for expressing the SHP-2 mutein, wherein, in the expression construct, the coding sequence of the SHP-2 mutein and It is operatively connected to constitute an active promoter.
  • the constitutively activated promoter includes but is not limited to CMV promoter, SV40 promoter, PGK promoter, EF1 ⁇ promoter, ⁇ -actin promoter, MSCV Promoter and CAG promoter
  • the immune cell contains an expression construct for expressing the SHP-2 mutein, wherein, in the expression construct, the coding sequence of the SHP-2 mutein and The IL2 mini promoter is operatively linked.
  • the IL2 mini promoter is activated by the binding of activated NFAT.
  • the IL2 mini promoter has the nucleotide sequence described in SEQ ID No: 17 or 18.
  • a pharmaceutical composition which contains:
  • the formulation is a liquid formulation.
  • the cell is an immune cell expressing a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the immune cells include: T cells, NK cells, monocytes, macrophages or a combination thereof.
  • the T cells include: CD4 + cells, CD8 + cells, or a combination thereof.
  • the use of the cell described in the sixth aspect is provided, which is used to prepare drugs for treating tumors.
  • the cell is an immune cell expressing a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • FIG 1 shows the schematic diagrams of the two CAR constructs of the present invention.
  • Figure 2 shows a schematic diagram of the protein molecule working mode of a CAR-T cell of the present invention.
  • Figure 3 shows a schematic diagram of the structure of a lentiviral vector for expressing the mutant SHP-2 of the present invention.
  • Figure 4 shows a schematic diagram of the structure of a SHP-2 mutein CAR expression vector used to express the present invention.
  • Figure 5 shows a schematic structural diagram of a conditionally activated SHP-2 mutein expression construct in an embodiment of the present invention.
  • Figure 6 shows the amino acid sequence of human SHP-2 protein (SEQ ID No.: 1).
  • the underlines are the domains of N-SH2 (positions 2-104) and C-SH2 (positions 112-216). .
  • Figure 7 shows the results of short-term killing experiments of CAR-T cells carrying different SHP-2 mutant protein expression constructs relative to control CAR-T cells (Control-CAR-T) in Example 5 of the present invention.
  • Fig. 8 shows the results of long-term continuous killing experiments of CAR-T cells carrying different SHP-2 mutant protein expression constructs in 60 hours after performing multiple tumor cell killing experiments within 60 hours.
  • Figure 9 shows the flow cytometric detection results of CAR-T cells carrying different SHP-2 mutant protein expression constructs after stimulation with tumor cell specific antigens in Example 6 of the present invention.
  • the inventors unexpectedly discovered for the first time that certain specific types of SHP-2 can significantly improve the efficiency of CAR-T cell therapy.
  • the present inventors used conditional induction technology to improve the treatment efficiency of CAR-T cells by regulating the activity of SHP-2 in CAR-T cells.
  • CAR-T cells carrying an activated SHP-2 mutant protein expression cassette when CAR is activated, the accompanying expression of SHP2 mutant protein effectively inhibits the endogenous SHP-2 in these CAR-Ts, thereby These CAR-T cells showed stronger anti-tumor effects in vitro and in vivo.
  • the term "about” may refer to a value or composition within an acceptable error range of a specific value or composition determined by a person of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • Sequence identity compares two alignments along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein) Sequence, and determine the number of positions where the same residue appears. Normally, this is expressed as a percentage.
  • a predetermined comparison window which can be 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the length of the reference nucleotide sequence or protein Sequence.
  • Src homologous region protein tyrosine phosphatase 2 (SHP-2), also known as PTPN11 (protein tyrosine phosphatase non-receptor type 11), is one of the members of the protein tyrosine phosphatase family.
  • Src homologous region protein tyrosine phosphatase 2 (SHP-2) is a member of the protein tyrosine phosphatase family. It is a downstream signaling molecule of a variety of cytokines, growth factors and other extracellular stimulating factors. It is widely expressed in Various cells and tissues are closely related to cell proliferation, differentiation and metastasis. Studies have shown that inhibiting the activity of SHP-2 through drugs can increase the cytotoxicity of CD8 + T cells, thereby reducing tumor burden. In addition, the suppression of T cells caused by immunosuppressive molecules such as PD-1 is also closely related to the activation of SHP-2.
  • SHP-2 is encoded by the PTPN11 gene.
  • the human PTPN11 gene is located on chromosome 12 and has 15 exons. Ensembl's registration number is ENST00000635625 .
  • amino acid sequence of human SHP-2 protein is shown in SEQ ID No.: 1 and Figure 6:
  • the N-terminus of SHP-2 contains two SH2 domains: N-SH2 (positions 2-104) and C-SH2 (positions 112-216).
  • the C-terminus contains a catalytically active PTP domain and is rich in proteas.
  • the present invention proves that SHP-2 can exert its catalytic effect through the structural changes of the two SH2 domains at the N-terminal.
  • an SHP2 mutant protein that dominantly acts on SHP-2 endogenous in a cell and can inhibit the activity of endogenous SHP-2 is provided.
  • the mutant contains a partial amino acid sequence of SHP-2, or its amino acid sequence is an amino acid sequence obtained by substituting at least one amino acid with another amino acid compared with the sequence of wild-type SHP-2, and can hinder intracellular intracellular Activation of derived SHP-2.
  • mutant protein of the present invention As used herein, the terms “protein of the present invention”, “mutant protein of the present invention”, “mutant protein of the present invention”, and “mSHP2” are used interchangeably, and all refer to those that have been mutated relative to the wild-type SHP-2 protein. Mutant protein, and the mutant protein can act on endogenous SHP-2 in cells and can inhibit the activity of endogenous SHP-2.
  • the present invention provides 14 mutant proteins, which respectively target specific sites and The C-terminal PTP domain is mutated.
  • FK020-B, FK020-C, and FK020-D mutant proteins all contain 102 amino acids, and they are performed at positions 60, 61, or 62 of the N-SH2 domain, respectively
  • FK020-H mutant protein contains 102 amino acids, which is a truncated mutation of wild-type SHP-2;
  • FK020-E, FK020-F, and FK020-G mutant proteins all contain 216 amino acids.
  • FK020-I mutant protein contains 216 amino acids, which is a truncated mutation of wild-type SHP-2 ;
  • FK020-J, FK020-K, FK020-L, FK020-M, FK020-N, and FK020-O mutant proteins all contain 593 amino acids, and are located at positions 278, 279, and 364 on the PTP domain, respectively.
  • a site-directed mutation of 1 amino acid was carried out at position, 459 or 506.
  • wild-type SHP-2 protein FK020-A is provided, and the present invention also provides 14 mutant SHP-2 proteins: FK020-B, FK020-C, FK020-D, FK020-E , FK020-F, FK020-G, FKC020-H, FKC020-I, FK020-J, FK020-K, FK020-L, FK020-M, FK020-N, FK020-O.
  • amino acid sequences of wild-type SHP-2 protein FK020-A and 14 SHP-2 mutant proteins are shown in Table 2:
  • G60E refers to the mutation of amino acid at position 60 from glycine G to glutamic acid E.
  • D61E refers to the mutation of amino acid at position 61 from aspartic acid D to glutamic acid E.
  • Y62W refers to the mutation of amino acid at position 62 from tyrosine Y to tryptophan W.
  • R278N refers to the mutation of amino acid at position 278 from arginine R to asparagine N.
  • Y279H refers to the mutation of amino acid at position 279 from tyrosine Y to histidine H.
  • K364R refers to the mutation of amino acid at position 364 from arginine R to asparagine N.
  • K364Q refers to the mutation of amino acid at position 364 from arginine R to glutamine Q.
  • C459S refers to the mutation of amino acid at position 459 from Cysteine C to Serine S.
  • Q506K refers to the mutation of amino acid at position 506 from glutamine Q to lysine K.
  • the polynucleotide encoding the mutein of the present invention there is no particular limitation on the polynucleotide encoding the mutein of the present invention, as long as it can encode the mutein of the present invention.
  • the invention also provides a conditionally activated SHP-2 mutant protein expression construct.
  • Immune cells such as CAR-T cells carrying the conditionally activated expression construct will only bind to the expression construct when the immune cells are activated after the CAR molecule binds to the target molecule, and the endogenous NFAT is activated.
  • the IL2 mini promoter before the nucleic acid sequence encoding the SHP-2 mutant protein initiates the expression of the SHP-2 mutant protein.
  • the terms "expression construct”, “expression construct”, and “expression cassette” are used interchangeably and refer to an artificially constructed nucleic acid construct for expressing a target protein.
  • the target protein is a specifically inactivated SHP-2 mutant protein.
  • the expression construct is a constitutive expression construct or a conditional expression construct.
  • the expression construct is a constitutive SHP-2 mutein expression construct.
  • constitutive expression construct As used herein, the terms “constitutive expression construct”, “constitutive expression construct”, and “constitutive expression cassette” are used interchangeably and refer to artificially constructed expression constructs for expression.
  • constitutive expression promoter there is no particular limitation on the constitutive expression promoter.
  • Representative examples include but are not limited to: CMV promoter, SV40 promoter, PGK promoter, EF1 ⁇ promoter, ⁇ -actin promoter, MSCV promoter and CAG promoter.
  • different constitutive expression promoters can be selected according to different coding sequences and different immune cells.
  • the target protein is a specifically inactivated SHP-2 mutant protein.
  • Immune cells (such as CAR-T cells) carrying the constitutive expression construct can start the expression of the SHP-2 mutant protein through the nucleic acid sequence encoding the SHP-2 mutant protein through the constitutive expression promoter while expressing the CAR molecule. And it does not affect the proliferation and activation of CAR-T cells under normal conditions.
  • the expression construct is a conditionally activated SHP-2 mutein expression construct.
  • the present invention uses nuclear factor of activated T cells (NFAT), which is a type of transcription factor family protein.
  • NFAT family proteins have a wide range of physiological functions and play an important role in gene transcription induced by immune response. In addition to T cells, this type of protein can also be expressed on many immune cells, such as B lymphocytes, mast cells, and eosinophils.
  • NFAT can regulate the functions of T cell activation, differentiation and self-tolerance. It also has a multi-directional regulatory function and participates in a variety of biological processes.
  • NFAT is mainly activated through the calcium ion-dependent calmodulin phosphatase C signaling pathway, and its activation and nucleocytoplasmic shuttling are mainly regulated by the dynamic interaction between calmodulin phosphatase C and constitutive kinases.
  • NFAT when NFAT activates the transcription of target genes, it often forms a complex with AP-1 protein or other transcription factors to form a synergistic effect.
  • a special conditionally activated SHP-2 mutein expression construct is designed by using the characteristic that the endogenous NFAT transcription factor in CAR-T cells can bind to the promoter of the IL2 gene.
  • an IL2 mini promoter initiated by activated NFAT binding was constructed before the fragment encoding the SHP-2 mutant protein. Only when CAR-T cells are activated after the CAR molecule binds to the target molecule, the endogenous NFAT is activated and will bind to the IL2mini promoter before the nucleic acid sequence encoding the SHP-2 mutant protein to start the SHP-2 mutant protein. expression. When CAR-T cells are not bound to the target molecule and have not been activated, the SHP-2 mutant protein will not be expressed.
  • the present invention also provides cells expressing the SHP-2 mutant protein of the present invention, especially immune cells.
  • the immune cells of the present invention include (but are not limited to): T cells, NK cells, or a combination thereof.
  • T cells include: CD4 + cells, CD8 + cells, or a combination thereof.
  • immune cells can be autologous or allogeneic.
  • a CAR expression vector is also provided.
  • the expression vector not only contains a nucleic acid sequence encoding a chimeric antigen receptor (CAR), but also contains a nucleic acid sequence encoding a SHP-2 mutein and other related molecules that can improve T cells. .
  • chimeric antigen receptor refers to an artificial chimeric protein molecule obtained by fusing a single-chain antibody that recognizes the cell surface antigen of cancer cells, a signal transduction region that induces T cell activation, and a transcellular membrane region.
  • the nucleic acid encoding the polypeptide constituting the CAR molecule is not particularly limited, including encoding single-chain antibodies that recognize cell surface antigens of cancer cells, transmembrane regions, and those that induce the activation of T cells.
  • the nucleic acid of the polypeptide of the signal transduction region is not particularly limited, including encoding single-chain antibodies that recognize cell surface antigens of cancer cells, transmembrane regions, and those that induce the activation of T cells.
  • the single-chain antibody (scFv) in the CAR can be composed of the light chain variable region (VL) and the heavy chain variable region (VH) from the antigen binding site of the monoclonal antibody, and the light chain variable region An oligopeptide or polypeptide with a linker peptide between the region and the variable region of the heavy chain.
  • the cell surface antigen of the cancer cell recognized by the above-mentioned single-chain antibody may be a biological molecule specifically expressed against cancer cells and their precursor cells, a biological molecule whose expression has been newly confirmed due to cancerization of the cell, Or biological molecules whose expression levels are increased in cancer cells compared to normal cells, including CD20, EGFR, FITC, CD19, CD22, CD33, PSMA, GD2, EGFR variant, ROR1, c-Met, HER2, CEA , Mesothelin, GM2, CD7, CD10, CD30, CD34, CD38, CD41, CD44, CD74, CD123, D133, CD171, CD276, CLDN18.2, MUC16, MUC1, CS1 (CD319), IL-13Ra2 , BCMA, GPC3, DLL3, LewisY, IgG kappa chain, folate receptor- ⁇ , PSCA, EpCAM, CLND, etc.
  • the T cell activation signal transduction region is a region capable of transducing signals into the cell when the single-chain antibody recognizes the cell surface antigen of a cancer cell, and preferably contains a region selected from CD28, 4-1BB (CD137), GITR, and CD27.
  • OX40, ICOS, HVEM, CD3 ⁇ , Fc Receptor-associated ⁇ chain at least one or more of the intracellular region polypeptides, more preferably a polypeptide containing the three intracellular regions of CD28, 4-1BB, and CD3 ⁇ .
  • the polypeptides in each intracellular region can be connected by oligopeptide linkers or polypeptide linkers containing 0-15 (preferably 2-10) amino acids.
  • linker sequence include a glycine-serine (G 4 S) continuous sequence.
  • the transmembrane region in the present invention includes ⁇ and ⁇ chains derived from CD8, T cell receptors, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD137L, CD154, GITR transmembrane polypeptides.
  • the CAR molecule is immobilized on the cell membrane of the T cell through this transcellular membrane region.
  • the nucleic acid encoding the chimeric antigen receptor can be based on the nucleic acid sequence encoding the single-chain antibody against the cell surface antigen of the cancer cell, the transmembrane region, and the polypeptide of the T cell activation signal transduction region. It is prepared by chemical synthesis method, PCR amplification method and other techniques. It should be noted that the codons used to encode amino acids can be selected for modification, so as to optimize the expression level of the nucleic acid in the target host cell.
  • the information of the base sequence of the single-chain antibody against the cell surface antigen of the cancer cell, the transmembrane region, and the polypeptide of the T cell activation signal transduction region can be searched through known literature, or N C B I, etc. Appropriately obtained from public databases.
  • information about the base sequence of the polypeptides encoding CD28, 4-1BB, and CD3 ⁇ transmembrane region in the T cell activation signal transduction region can be appropriately obtained by searching databases such as NCBI.
  • Each "-" is independently a connecting peptide or a peptide bond
  • L1 is an optional signal peptide sequence
  • scFv is an antigen binding domain (ie, a binding domain that targets a target antigen (such as CD19));
  • H1 is an optional hinge region
  • TM1 is a transmembrane domain
  • C1 is a costimulatory signal molecule
  • CD3 ⁇ is a cytoplasmic signal transduction sequence derived from CD3 ⁇ .
  • the L1 is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.
  • the H1 is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.
  • the TM1 is a transmembrane region of a protein selected from the group consisting of CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86 , CD134, CD137, CD154, or a combination thereof.
  • the C1 is a costimulatory signal molecule of a protein selected from the group consisting of: OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1 , Dap10, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.
  • the T cells in the present invention may be T cells derived from humans, T cells derived from non-human mammals such as dogs, cats, pigs, and mice.
  • these T cells can be isolated and purified from body fluids such as blood and bone marrow fluid, tissues such as spleen, thymus, and lymph nodes, or immune cells infiltrating cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites.
  • body fluids such as blood and bone marrow fluid, tissues such as spleen, thymus, and lymph nodes, or immune cells infiltrating cancer tissues such as primary tumors, metastatic tumors, and cancerous ascites.
  • T cells include ⁇ T cells, ⁇ T cells, CD8 + T cells, CD4 + T cells, tumor-infiltrating T cells, memory T cells, naive T cells, NKT cells, and the like.
  • the CAR-T cell can recognize the tumor-associated antigen on the surface of the cancer cell through the single-chain antibody on the CAR molecule.
  • the virus vector in the present invention can be a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a transposon vector. If retrovirus, lentiviral vector or transposon vector is used, the original gene encoding the target fragment will be introduced into the genome of the host cell, so the target molecule can be expressed stably for a long time.
  • a spacer composed of any oligopeptides or polypeptides can be provided between the single-chain antibody that recognizes the cell surface antigen of cancer cells and the transmembrane region, the transmembrane region and the T cell activation signal transduction region.
  • the length of the spacer may be 1-100 amino acids, preferably 10-50 amino acids, and the spacer may be a glycine-serine continuous sequence.
  • a CAR construct which comprises: an antigen binding region (scFV region), a transmembrane region, a CD28, or 4-1BB and other co-stimulatory regions, A CD3 ⁇ signal region, and an SHP-2 mutant protein expression construct that constitutes an activated or conditionally activated co-expression.
  • the conditionally activated expression construct uses the NFAT6 IL-2 minimal promotor-SHP2 region.
  • FIG. 1 A typical construct containing the CAR structure and the mSHP2 region is shown in Figure 1.
  • the coding sequence of mSHP2 is operatively linked to a constitutive promoter or IL2 mini promoter.
  • mSHP2 can be expressed constitutively, that is, the expression of mSHP2 and the expression of CAR molecules of immune cells are independent of each other.
  • the mSHP2 coding sequence When the mSHP2 coding sequence is operatively linked to the IL2 mini promoter (or other conditionally activated promoters), once immune cells are activated by the CAR molecule binding to the target molecule, the endogenous NFAT will be activated and will bind to the coding SHP2 The IL2 mini promoter in front of the mutant protein sequence, thereby starting the expression of the SHP2 mutant protein.
  • the working mode of the conditionally activated SHP-2 mutant protein molecule of the present invention is shown in FIG. 2.
  • CAR-T cells do not bind to antigens on the tumor surface, their endogenous SHP-2 is in an inactive state.
  • the N-terminal SH2 domain of SHP-2 and the PTP domain are connected by intermolecular force, and the active site is not exposed and cannot exert phosphatase activity.
  • CAR-T cells When CAR-T cells specifically bind to the target antigen on the tumor surface, the cells are activated, endogenous SHP-2 is activated by immunosuppressive checkpoints and other signals, the PTP domain is exposed, and its phosphatase activity is activated, which can The activation of CAR-T cells conducted by CAR molecules plays a negative feedback regulatory role, which will reduce the degree of cell activation.
  • the endogenous NFAT in the CAR-T cell constructed by the present inventors can bind to the NFAT binding region on the sequence inserted into the host cell genome when the CAR-T cell is activated to initiate the transcription of the mSHP2 mutant protein, and then Translational synthesis inhibitory mutant mSHP2 protein.
  • the mSHP2 can competitively bind to the PTP domain of endogenous SHP-2 in CAR-T cells, and weaken the activation effect of SHP-2 upstream activation signal on endogenous SHP-2, and weaken the activation of endogenous SHP-2 in CAR-T cells.
  • the overall viability of endogenous SHP-2 improves the activation of the intracellular tyrosine kinase pathway, thereby enhancing the anti-tumor effect of CAR-T cells.
  • the constitutively expressed SHP-2 mutant protein molecules of the present invention can be expressed by T cells independently of the CAR molecules to obtain CAR-T cells that constitutively express these mutant SHP-2 protein molecules. These constitutively expressed SHP-2 mutants did not affect the proliferation and activation of CAR-T cells under normal conditions.
  • inhibitory mutant mSHP-2 proteins When encountering cancer cells, these inhibitory mutant mSHP-2 proteins can competitively bind to the PTP domain of endogenous SHP-2 in CAR-T cells, or compete with endogenous SHP-2 for downstream substrates
  • the combination of SHP can weaken the overall viability of the endogenous SHP-2 in CAR-T cells, increase the activation of the intracellular tyrosine kinase pathway, and thereby improve the anti-tumor effect of CAR-T cells.
  • the present invention also provides a corresponding pharmaceutical composition, which contains (a) the SHP-2 mutein of the present invention, a polynucleotide encoding the mutein, a vector, or a cell encoding the mutein, or a combination thereof ; And (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition or preparation of the present invention contains the CAR-T cell of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.
  • the formulation or pharmaceutical composition is a liquid formulation.
  • the preparation is an injection.
  • the concentration of the CAR-T cells in the preparation is 1 ⁇ 10 3 -1 ⁇ 10 8 cells/ml, more preferably 1 ⁇ 10 4 -1 ⁇ 10 7 cells/ml.
  • the formulation may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids such as glycine ; Antioxidants; Chelating agents such as EDTA or glutathione; Adjuvants (for example, aluminum hydroxide); and cryoprotectants such as DMSO, glycerin and the like.
  • the formulations of the invention are preferably formulated for intravenous administration.
  • the invention also provides the therapeutic application of the immune cells of the invention, especially the application in tumor treatment.
  • the present invention includes therapeutic applications of cells (e.g., T cells) transduced with viral vectors (e.g., lentivirus) encoding the expression constructs of the present invention.
  • the transduced T cells can target tumor cells or markers of immune cells that cause autoimmune diseases, and can be used for the treatment of autologous tumors or allogeneic tumors or autoimmune diseases. It can be prepared on a large scale, with uniform and stable quality, and can be used by any patient at any time.
  • the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal, which comprises the following steps: administering the CAR-T cell of the present invention to the mammal.
  • the present invention includes a type of cell therapy in which the modified CAR-T cells of the present invention are directly administered to patients in need.
  • the CAR-T cells of the present invention can undergo stable T cell expansion in vivo and last for an extended amount of time.
  • the CAR-mediated immune response can be part of an adoptive immunotherapy step in which CAR-modified T cells induce an immune response specific to the antigen binding domain in the CAR.
  • anti-CD19 CAR-T cells elicit a specific immune response against CD19-expressing cells.
  • Cancers that can be treated include tumors that have not been vascularized or have not been substantially vascularized, as well as vascularized tumors.
  • the cancer may include non-solid tumors (such as hematological tumors such as leukemia and lymphoma) or may include solid tumors.
  • the types of cancer treated with the CAR of the present invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcoma, carcinoma, and melanoma. It also includes adult tumors/cancers and childhood tumors/cancers.
  • Hematological cancer is cancer of the blood or bone marrow.
  • leukemias include leukemias, including acute leukemias (such as acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic, promyelocytic, myelomonocytic type , Monocytic and erythroleukemia), chronic leukemia (such as chronic myeloid (granulocyte) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, non- Hodgkin's lymphoma (painless and high-grade form), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • acute leukemias such as acute lymphoblastic leukemia, acute myeloid leuk
  • a solid tumor is an abnormal mass of tissue that does not usually contain a cyst or fluid area.
  • Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell type that formed them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcoma and cancer include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, ovarian cancer, and the like.
  • the CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunity and/or in vivo therapy of mammals.
  • the mammal is a human.
  • cells autologous or allogeneic
  • a mammal preferably human
  • genetically modified ie, transduced or transfected in vitro
  • CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits.
  • the mammalian recipient can be a human
  • the CAR-modified cell can be autologous relative to the recipient.
  • the cell may be allogeneic, syngeneic, or xenogeneic relative to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.
  • the present invention provides a method for treating tumors or autoimmune diseases, which comprises: administering to a subject in need thereof a therapeutically effective amount of the CAR-modified T cells of the present invention.
  • the CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and/or other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical composition of the present invention may include the target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelate Mixtures such as EDTA or glutathione; adjuvants (for example, aluminum hydroxide); and cryoprotectants such as DMSO, glycerin, and the like.
  • the composition of the invention is preferably formulated for intravenous administration.
  • the pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented).
  • the number and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease-although the appropriate dosage can be determined by clinical trials.
  • the precise amount of the composition of the present invention to be administered can be determined by the physician, who considers the patient (subject ) Individual differences in age, weight, tumor size, degree of infection or metastasis, and disease. May generally indicated: including those described herein, the pharmaceutical compositions of T cells may be 104 to 109 doses cells / kg body weight, preferably 105 to 106 cells / kg body weight doses (including all integers within that range Value) application. The T cell composition can also be administered multiple times at these doses.
  • the cells can be administered by using injection techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988).
  • the optimal dosage and treatment regimen for a specific patient can be easily determined by those skilled in the medical field by monitoring the patient's signs of disease and adjusting the treatment accordingly.
  • administration can refer to treatment, pharmacokinetics, diagnosis, research and experimental methods.
  • the treatment of cells includes contact between reagents and cells, contact between reagents and fluids, and contact between fluids and cells.
  • administering and “treatment” also mean treatment by reagents, diagnostics, binding compositions, or by another cell in vitro and ex vivo.
  • Treatment when applied to humans, animals, or research subjects, refers to treatment, preventive or preventive measures, research, and diagnosis.
  • the administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation.
  • the compositions described herein can be administered to patients subcutaneously, intracutaneously, intratumorally, intranodal, intraspinal, intramuscular, by intravenous (i.v.) injection, or intraperitoneally.
  • the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell composition of the present invention is preferably administered by i.v. injection.
  • the composition of T cells can be injected directly into tumors, lymph nodes or sites of infection.
  • cells activated and expanded using the methods described herein or other methods known in the art to expand T cells to therapeutic levels are combined with any number of relevant treatment modalities (e.g., previous , At the same time or after) administration to the patient.
  • relevant treatment modalities e.g., previous , At the same time or after
  • the subject receives an infusion of immune cells of the invention.
  • the expanded cells are administered before or after surgery.
  • the dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the dosage ratio for human administration can be implemented according to the practice accepted in the art.
  • 1 ⁇ 10 6 to 1 ⁇ 10 10 modified T cells (for example, CAR-T cells) of the present invention can be administered to the patient by, for example, intravenous infusion, per treatment or per course of treatment. .
  • the SHP-2 mutant protein of the present invention has good specificity, can specifically regulate the viability of endogenous SHP-2, reduce the exhaustion of CAR-T cells, and enhance its anti-tumor effect.
  • the CAR-T cell of the present invention contains a constitutively activated or conditionally activated SHP-2 mutant protein expression construct.
  • the coding sequence of the SHP-2 mutant protein is operatively linked to the constitutive promoter, which can produce a specific mutant SHP-2 protein and regulate the activity of the endogenous SHP-2 phosphatase in the cell .
  • the coding sequence of the SHP-2 mutant protein is operatively linked to IL2 minimal promotor, so that the expression of the SHP-2 mutant protein is downstream of the activation signal mediated by the CAR molecule.
  • the constitutively activated or conditionally activated SHP-2 mutant protein of the present invention does not affect or basically does not affect the proliferation and activation of CAR-T cells under normal conditions, and can reduce the tumor cells and tumor microenvironment caused CAR-T cell exhaustion.
  • IL2 mini promoter see Table 1, SEQ ID No: 17 or 18;
  • the six mutant proteins were respectively subjected to point mutations to the two SH2 domains at the N-terminus of the SHP-2 protein, among which FK020-B, FK020- C, FK020-D mutant protein contains 102 amino acids, and site-directed mutation of 1 amino acid was carried out at position 60, 61, or 62 of the N-SH2 domain; while FK020-H mutant protein contains 102 amino acids.
  • Amino acids which are truncated mutations of wild-type SHP-2; FK020-E, FK020-F, and FK020-G mutant proteins all contain 216 amino acids, and are located at positions 60 and 61 of the N-SH2 domain, respectively Or a site-directed mutation of 1 amino acid was carried out at position 62; and the FK020-I mutant protein contains 216 amino acids, which is a truncated mutation of wild-type SHP-2; FK020-J, FK020-K, FK020-L, FK020- M, FK020-N, and FK020-O mutant proteins all contain 593 amino acids, and have 1 amino acid at positions 278, 279, 364, 459, or 506 on the PTP domain, respectively. Site-directed mutation.
  • a lentiviral vector expressing the mutant protein was designed and synthesized. After 48 hours and 72 hours, the virus was collected and concentrated for virus MOI determination.
  • the vector nucleic acid sequence pattern is shown in Figure 3. Show.
  • the virus obtained by the above method was transduced into Jurkat cells growing in suspension, and the expression of HA tag was detected by western immunoblotting after 48 hours. It can be found that the expression levels of these different SHP-2 mutants in cells are consistent.
  • M-PER TM Mammalian Protein Extraction Reagent and 5 ⁇ 10 7 Jurkat cells were mixed on ice. Add 10-50ug protein supernatant, add an appropriate amount of SHP2 antibody (1-5ug), mix and incubate overnight at 4°C. Add dextran microbeads (Protein G sepharose), mix well at 4°C and incubate for 4-6h. Centrifuge at high speed to collect the beads.
  • the cell lysate treated with NaF, Na 3 VO 4 was used as the sample, and the PTPase activity was detected using the Non-Radioactive Phosphatase detection system (Promega).
  • Western blot method was used to detect the activation of downstream signaling pathways, such as SHP-2 phosphorylation, MARP/ERK expression and phosphorylation.
  • SHP-2 mutants with obvious inhibitory ability were selected to construct a CAR that expresses the constitutively activated SHP-2 conditional activation type.
  • Expression vector, or CAR expression vector pWPXLd-ScFV-NFAT6-mSHP2-GFP
  • the structural model of the conditionally activated plasmid is shown in FIG. 4.
  • a lentiviral vector for transfecting HEK293T cells was prepared. Use these lentiviral vectors to prepare CAR-T cells expressing SHP-2 mutant protein.
  • CAR-T cells are briefly described as follows: PBMC or T cells are cultured for 48 hours in culture medium and monoclonal antibodies against T cell activation signals such as CD3 and CD28 or similar activation reagents; activated PMBC or T cells are collected and used Resuspend in fresh medium, add the required amount of virus according to the MOI, and conduct transduction during culture; after the end of transduction, centrifuge the cells, discard the supernatant, resuspend the cells in fresh medium, and continue to expand the culture until harvest cell.
  • T cell activation signals such as CD3 and CD28 or similar activation reagents
  • the structure of the NFAT6-binding IL2 minimal promotor (6 NFAT binding sites with IL2 minimal promoter) structure is shown in FIG. 5.
  • the CAR-T cells prepared in this example contain a NFAT6-binding IL2 minimal promotor-SHP2 construct (that is, the expression cassette of the SHP-2 mutein), which makes the SHP-2 mutein
  • the expression is downstream of the activation signal mediated by the CAR molecule.
  • the CMV constitutive promoter (SEQ ID No: 16) was used to prepare a constitutively activated CAR expression vector (pWPXLd-ScFV-CMV-mSHP2-GFP) for expressing the SHP-2 mutant protein.
  • the structural pattern of the constitutively activated plasmid is shown in Figure 1 (a).
  • CAR-T cells and target cells were co-cultured. Add the sample to the 96-well culture plate and set up 3 multiple wells. Mix well and incubate in a carbon dioxide incubator for 24 hours to detect the killing effect on target cells.
  • the constitutively expressed SHP-2 mutein also continues to inhibit the viability of the endogenous SHP-2 in these CAR-Ts, thereby This makes these CAR-T cells show a stronger killing effect on target cells.
  • CAR-T cells and target cells were co-cultured. Add the sample to the 96-well culture plate and set up 3 multiple wells. After mixing and incubating in a carbon dioxide incubator for 12 hours, observe under a microscope that after the target cells are completely killed, add them according to the initial number of target cells, and mix and place in a carbon dioxide incubator to continue incubating. After multiple killings, observe the remaining target cells under a microscope, and take CAR-T cells for flow cytometric analysis of the proliferation of CAR-positive cells.
  • the SHP-2 mutant protein (FK020-J, K, L, M, N and O, especially FK020-N and FK020-O) inhibit the activity of endogenous SHP-2 in CAR-T, so that these CAR-T cells show better expansion ability and stronger sustained killing effect on target cells.
  • Example 7 In vivo verification of specific inactivation SHP-2 CAR-T in vivo activity, proliferation and anti-tumor effect

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Abstract

提供了SHP2特异性失活突变蛋白及其在CAR-T治疗中应用。具体地，提供了SHP2特异性失活突变蛋白以及相应的条件激活型SHP-2突变蛋白表达盒，以及携带该条件激活型SHP-2突变蛋白表达盒的CAR-T细胞。当该CAR-T细胞的CAR被激活时，伴随表达的SHP2突变蛋白可有效地抑制了这些CAR-T中内源性的SHP-2，从而具有更强的体外和体内抑瘤效果。

Description

SHP2特异性失活突变蛋白及其在CAR-T治疗中应用 技术领域
本发明涉及医药领域，具体地涉及SHP2特异性失活突变蛋白及其在CAR-T治疗中应用。
背景技术
嵌合抗原受体(Chimeric Antigen Receptor，“CAR”)是将识别癌细胞的细胞表面抗原的单链抗体与诱导T细胞活化的信号转导区融合而得到的人工嵌合蛋白质。通过向患者的外周血T细胞导入编码CAR的基因，从而得以能够大量制备能够表达CAR分子的T细胞(简称为“CAR-T细胞”)。CAR-T细胞具有肿瘤反应性，能够不依赖与主要组织相容性基因复合体(MHC)的相互作用而杀伤癌细胞。利用CAR-T细胞的癌症免疫疗法，是从患者采集T细胞、将编码CAR的基因导入上述T细胞进行扩增、然后再次回输患者的疗法。
然而，当前CAR-T技术依然面临很多问题：例如，CAR-T细胞在生物体内的存活效率较低，增殖能力不足；由CAR-T细胞向肿瘤组织的迁移不足；在抗肿瘤免疫过程中的CAR-T细胞会出现耗竭现象(如CAR-T细胞的活性可以被经由PD-L1/PD-1、TIM-3、LAG-3等免疫抑制检查点抑制)；肿瘤微环境及存在其中的免疫抑制因子TGF-β、IL-10等引起的CAR-T细胞抑制的问题。
因此，本领域迫切需要开发更有效的CAR-T细胞，以充分发挥CAR-T的功能。
发明内容
本发明的目的是提供一种更为有效的CAR-T细胞，所述的CAR-T细胞具有存活效率高、增殖能力强、在抗肿瘤过程中不易出现耗竭、和/或不易受肿瘤微环境抑制等优点，从而可以更充分地发挥CAR-T的功能。
在本发明的第一方面，提供了一种SHP-2突变蛋白，所述的SHP-2突变蛋白具有选自下组的突变：G60E、D61E、Y62W、R278N、Y279H、K364R、K364Q、Q506K、C459S或其组合，或者所述的SHP-2突变蛋白为截断型SHP-2蛋白；其中，氨基酸的位置基于SEQ ID No.:1。
在另一优选例中，所述的突变蛋白为人SHP-2的片段或全长突变体，其中，所述片段具有对应于SHP-2野生型蛋白的N-SH2结构域的氨基酸序列，或者所述片段具有对应于SHP-2野生型蛋白的N-SH2和C-SH2结构域的氨基酸序列；所述的全长突变体具有对应于SHP-2野生型蛋白全长的氨基酸序列(所述片段或全长突变体在相应的位点具有所述的点突变)。
在另一优选例中，所述的突变蛋白具有对应于SHP-2野生型蛋白的第Y1至Y2 位的氨基酸序列，其中，Y1为1,2或3；而Y2为100、101、102、103、104，或214、215、216、217、218。
在另一优选例中，所述的突变蛋白具有对应于SHP-2野生型蛋白的第1-102位、第1-104位，第2-102位、第2-104位、第1-216位、第2-216位的氨基酸序列。
在另一优选例中，所述的突变蛋白具有对应于SHP-2野生型蛋白第1-593位或第2-593位的氨基酸序列。
在另一优选例中，所述的突变蛋白为失活型。
在另一优选例中，所述的“失活型”指，与野生型SHP-2蛋白相比，这些失活型SHP-2不具备野生型SHP-2蛋白的磷酸酶活性，并且有抑制细胞内源性的SHP-2的能力。
在另一优选例中，所述的突变蛋白为氨基酸序列如SEQ ID No.:2、3、4、5、6、7、8、9、10、11、12、13、14或15所示的多肽。
在本发明的第二方面，提供了一种融合蛋白，所述的融合蛋白是人工构建的，并且所述融合蛋白包括：本发明第一方面所述的SHP-2突变蛋白，以及与所述SHP-2突变蛋白融合在一起的多肽元件。
在另一优选例中，该多肽元件包括如荧光蛋白(如GFP、RFP)等的各类元件。
在本发明的第三方面，提供了一种多核苷酸，所述的多核苷酸编码本发明第一方面所述的SHP-2突变蛋白或第二方面所述的融合蛋白。
在另一优选例中，所述的多核苷酸包括DNA、或RNA。
在本发明的第四方面，提供了一种激活型SHP-2突变蛋白表达构建体(或表达盒)，所述的表达构建体包括：激活型启动子、以及与所述启动子操作性连接的本发明第三方面所述的多核苷酸序列(即第一方面所述的SHP-2突变蛋白的编码序列)。
在另一优选例中，所述的激活型启动子包括组成激活型启动子和条件激活型启动子
在另一优选例中，所述的条件激活型启动子在CAR-T细胞通过CAR分子结合靶分子后才被激活。
在另一优选例中，所述的组成激活型启动子(或组成型表达启动子)包括但不限于CMV启动子、SV40启动子、PGK启动子、EF1α启动子、β-actin启动子、MSCV启动子和CAG启动子；条件激活型启动子为IL2 mini启动子。
在另一优选例中，所述的CMV启动子的序列如SEQ ID No:16所示。
在另一优选例中，所述的IL2 mini启动子的序列如SEQ ID No:17或18所示。
在本发明的第五方面，提供了一种载体，所述的载体含有第三方面所述的多核苷酸或第四方面所述的激活型SHP-2突变蛋白表达构建体。
在另一优选例中，所述的载体包括表达载体。
在另一优选例中，所述的载体包括病毒载体。
在另一优选例中，所述的表达载体包括：慢病毒载体、腺病毒载体、腺相关病毒AAV载体、质粒、或其组合。
在本发明的第六方面，提供了一种细胞，所述的细胞含有第五方面所述的载体，或者在基因组中整合有第三方面所述的多核苷酸或第四方面所述的激活型SHP-2突变蛋白表达构建体。
在另一优选例中，所述的表达构建体含有组成激活型启动子或条件激活型启动子。
在另一优选例中，所述的细胞为免疫细胞。
在另一优选例中，所述的免疫细胞表达外源的嵌合抗原受体(CAR)。
在另一优选例中，所述的免疫细胞表达用于抗肿瘤的嵌合抗原受体。
在另一优选例中，所述的嵌合抗原受体包括：任选的前导肽、胞外抗原结合域、铰链区、跨膜区和胞内区。
在另一优选例中，所述的免疫细胞包括：T细胞、NK细胞、或其组合。
在另一优选例中，所述的T细胞包括：CD4 +细胞、CD8 +细胞、或其组合。
在另一优选例中，所述的免疫细胞为自体的或异体的。
在另一优选例中，所述的胞外结合域针对CD20、EGFR、FITC、CD19、CD22、CD33、PSMA、GD2、EGFR variant、ROR1、c-Met、HER2、CEA、间皮素(mesothelin)、GM2、CD7、CD10、CD30、CD34、CD38、CD41、CD44、CD74、CD123、D133、CD171、CD276、CLDN18.2、MUC16、MUC1、CS1(CD319)、IL-13Ra2、BCMA、GPC3、DLL3、LewisY、IgG kappa链、叶酸受体-α、PSCA、或EpCAM。
在另一优选例中，所述的免疫细胞中，含有用于表达所述SHP-2突变蛋白的表达构建体，其中，在所述的表达构建体中，SHP-2突变蛋白的编码序列与组成激活型启动子操作性连接。
在另一优选例中，所述的组成激活型启动子(或组成型表达启动子)包括但不限于CMV启动子、SV40启动子、PGK启动子、EF1α启动子、β-actin启动子、MSCV启动子和CAG启动子
在另一优选例中，所述的免疫细胞中，含有用于表达所述SHP-2突变蛋白的表达构建体，其中，在所述的表达构建体中，SHP-2突变蛋白的编码序列与IL2 mini启动子操作性连接。
在另一优选例中，所述的IL2 mini启动子通过活化的NFAT的结合而被启动。
在另一优选例中，所述的IL2 mini启动子具有SEQ ID No:17或18所述的核苷酸序列。
表1 IL2 mini启动子
Figure PCTCN2021089087-appb-000001
在本发明的第七方面，提供了一种药物组合物，所述的药物组合物含有：
(a)第一方面所述的SHP-2突变蛋白、第三方面所述的多核苷酸、第五方面所述的载体、第六方面所述的细胞、或其组合；和
(b)药学上可接受的载体。
在另一优选例中，所述的制剂为液态制剂。
在另一优选例中，所述的细胞为表达嵌合抗原受体(CAR)的免疫细胞。
在另一优选例中，所述的免疫细胞包括：T细胞、NK细胞、单核细胞、巨噬细胞或其组合。
在另一优选例中，所述的T细胞包括：CD4 +细胞、CD8 +细胞、或其组合。
在本发明的第八方面，提供了第六方面所述的细胞的用途，它被用于制备***的药物。
在另一优选例中，所述的细胞为为表达嵌合抗原受体(CAR)的免疫细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明的两种CAR构建物的结构示意图。
图2显示了本发明的一种CAR-T细胞的蛋白分子工作模式的示意图。
图3显示了用于表达本发明的突变型SHP-2的一种慢病毒载体结构示意图。
图4显示了用于表达本发明的一种SHP-2突变蛋白CAR表达载体的结构示意图。
图5显示了本发明一个实施例中，条件激活型SHP-2突变蛋白表达构建体的结构示意图。
图6显示了人SHP-2蛋白的氨基酸序列(SEQ ID No.:1)，图中，下划线分别为N-SH2(第2-104位)和C-SH2(第112-216位)结构域。
图7显示了本发明实施例5中，携带不同SHP-2突变蛋白表达构建体的CAR-T细胞，相对于对照组CAR-T细胞(Control-CAR-T)的短期杀伤实验结果。
图8显示了本发明实施例6中，携带不同SHP-2突变蛋白表达构建体的CAR-T细胞，在60h内进行多次肿瘤细胞杀伤实验后的长期持续杀伤实验结果。
图9显示了本发明实施例6中，携带不同SHP-2突变蛋白表达构建体的CAR-T细胞，在肿瘤细胞特异性抗原刺激后的CAR阳性细胞流式检测结果。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现，某些特定类型的SHP-2可显著提高CAR-T细胞治疗效率。具体地，本发明人利用条件诱导技术，通过调节CAR-T细胞中的SHP-2的活性来提高CAR-T细胞治疗效率。实验表明，对于携带激活型SHP-2突变蛋白表达盒的CAR-T细胞，当CAR被激活时，伴随表达的SHP2突变蛋白有效地抑制了这些CAR-T中内源性的SHP-2，从而使得这些CAR-T细胞表现出了更强的体外和体内抑瘤效果。在此基础上，本发明人完成了本发明。
术语
为了可以更容易地理解本公开，首先定义某些术语。如本申请中所使用的，除非本文另有明确规定，否则以下术语中的每一个应具有下面给出的含义。在整个申请中阐述了其它定义。
术语“约”可以是指在本领域普通技术人员确定的特定值或组成的可接受误差范围内的值或组成，其将部分地取决于如何测量或测定值或组成。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
序列同一性通过沿着预定的比较窗(其可以是参考核苷酸序列或蛋白的长度的 50％、60％、70％、80％、90％、95％或100％)比较两个对齐的序列，并且确定出现相同的残基的位置的数目来确定。通常地，这表示为百分比。核苷酸序列的序列同一性的测量是本领域技术人员熟知的方法。
SHP-2及其突变蛋白
Src同源区蛋白络氨酸磷酸酶2(SHP-2)，也称为PTPN11(protein tyrosine phosphatase non-receptor type 11)，属于蛋白酪氨酸磷酸酶家族成员之一。
Src同源区蛋白络氨酸磷酸酶2(SHP-2)属于蛋白酪氨酸磷酸酶家族成员之一，是多种细胞因子、生长因子及其他胞外刺激因素的下游信号分子，广泛表达于各种细胞和组织，与细胞的增殖、分化、转移密切相关。研究表明，通过药物抑制SHP-2的活性可以提高CD8 +T细胞的细胞毒作用，进而降低肿瘤负荷。此外，PD-1等免疫抑制性分子引起的T细胞抑制也和SHP-2的活化密切相关。
SHP-2由PTPN11基因所编码。人源PTPN11基因位于12号染色体，有15个外显子。Ensembl登录序号为 ENST00000635625。
人SHP-2蛋白氨基酸序列如SEQ ID No.:1和图6所示：
Figure PCTCN2021089087-appb-000002
SHP-2的N末端含有2个SH2结构域：N-SH2(第2-104位)和C-SH2(第112-216位),C末端含有一个具有催化活性的PTP结构域及富含脯氨酸基团和酪氨酸磷酸化位点的C端结构域(C terminal tail)。本发明证实，SHP-2可通过N末端的2个SH2结构域的结构变化而发挥其催化作用。
已有研究表明，通过药物抑制SHP-2的活性可以提高CD8 +T细胞的细胞毒作用，进而降低肿瘤负荷。在小鼠的T细胞中特异性敲除SHP-2可以延缓T细胞耗竭，增强T细胞介导的抗肿瘤作用。在人源CAR-T细胞中共表达SHP-2显性失活突变体增强了CAR-T细胞在体外对表达PD-L1肿瘤细胞的杀伤。当T细胞激活后，免疫抑制性调控信号激活了SHP-2，则CAR-T细胞的杀伤，增殖以及细胞因子的分泌都是减弱的。如果选择性地抑制SHP-2，则CAR-T细胞的肿瘤杀伤能力会有所提高。
SHP-2突变蛋白
在本发明中，提供了显性作用于细胞内源性的SHP-2且能够抑制内源性SHP-2的活性的SHP2突变蛋白(mutant)。所述突变体含有SHP-2的部分氨基酸序列，或其氨基酸序列与野生型SHP-2的序列相比有至少1个氨基酸被其他的氨基酸取代而得到的氨基酸序列，且能够阻碍细胞内的内源性SHP-2的活化。
如本文所用，术语“本发明蛋白”、“本发明的突变蛋白”、“本发明的突变型蛋白”、“mSHP2”可互换使用，均指相对于野生型SHP-2蛋白发生了突变的突变蛋白，并且该突变蛋白可作用于细胞内源性的SHP-2且能够抑制内源性SHP-2的活性。
优选地，相对于野生型SHP-2蛋白(FK020-A)的氨基酸序列，本发明提供了14种突变蛋白，它们分别针对SHP-2蛋白N末端的2个SH2结构域中的特定位点和C末端的PTP结构域进行突变，其中FK020-B、FK020-C、FK020-D突变蛋白均含有102个氨基酸，并且分别在N-SH2结构域的第60位、第61位或第62位进行了1个氨基酸的定点突变；而FK020-H突变蛋白含有102个氨基酸，是野生型SHP-2的截短体突变；FK020-E、FK020-F、FK020-G突变蛋白均含有216个氨基酸，并且分别在N-SH2结构域的第60位、第61位或第62位进行了1个氨基酸的定点突变；FK020-I突变蛋白含有216个氨基酸，是野生型SHP-2的截短体突变；另外，FK020-J、FK020-K、FK020-L、FK020-M、FK020-N、FK020-O突变蛋白均含有593个氨基酸，并且分别在PTP结构域上的第278位、279位、364位、459位或506位进行了1个氨基酸的定点突变。
在本发明的一个实施例中，提供了野生型SHP-2蛋白FK020-A，本发明还提供了14种突变型SHP-2蛋白：FK020-B、FK020-C、FK020-D、FK020-E、FK020-F、FK020-G、FKC020-H、FKC020-I、FK020-J、FK020-K、FK020-L、FK020-M、FK020-N、FK020-O。
野生型SHP-2蛋白FK020-A和14种SHP-2突变蛋白的氨基酸序列如表2所示：
表2野生型SHP-2蛋白和SHP-2突变蛋白
Figure PCTCN2021089087-appb-000003
Figure PCTCN2021089087-appb-000004
Figure PCTCN2021089087-appb-000005
Figure PCTCN2021089087-appb-000006
Figure PCTCN2021089087-appb-000007
如本文所用，术语“G60E”是指第60位氨基酸由甘氨酸G突变为谷氨酸E。
如本文所用，术语“D61E”指第61位氨基酸由天冬氨酸D突变为谷氨酸E。
如本文所用，术语“Y62W”指第62位氨基酸由酪氨酸Y突变为色氨酸W。
如本文所用，术语“R278N”指第278位氨基酸由精氨酸R突变为天冬酰胺N。
如本文所用，术语“Y279H”指第279位氨基酸由酪氨酸Y突变为组氨酸H。
如本文所用，术语“K364R”指第364位氨基酸由精氨酸R突变为天冬酰胺N。
如本文所用，术语“K364Q”指第364位氨基酸由精氨酸R突变为谷氨酰胺Q。
如本文所用，术语“C459S”指第459位氨基酸由半胱氨酸C突变为丝氨酸S。
如本文所用，术语“Q506K”指第506位氨基酸由谷氨酰胺Q突变为赖氨酸K。
在本发明中，对于编码本发明突变蛋白的多核苷酸没有特别限制，只要其能够编码本发明的突变蛋白即可。
组成激活型或条件激活型SHP-2突变蛋白表达构建体
本发明还提供了一种条件激活型SHP-2突变蛋白表达构建体。携带该条件激活型表达构建体的免疫细胞(如CAR-T细胞)，只有当免疫细胞通过CAR分子结合靶分子后被激活时，内源性NFAT被活化，才会结合到该表达构建体上编码SHP-2突变蛋白的核酸序列前的IL2 mini启动子上，启动SHP-2突变蛋白的表达。
如本文所用，术语“表达构建体(expression construct)”、“表达构建物”、“表达盒(expression cassette)”可互换使用，指人工构建的用于表达一目标蛋白的核酸构建物。在本发明中，所述的目标蛋白为特异性失活的SHP-2突变蛋白。优选地，所述的表达构建体为组成激活型表达构建体(constitutive expression construct)或条件激活型表达构建体(conditional expression construct)。
优选地，所述所述的表达构建体是组成型SHP-2突变蛋白表达构建体。如本文所用，术语“组成型表达构建体(constitutive expression construct)”、“组成激活型表达构建物”、“组成型表达盒(constitutive expression cassette)”可互换使用，指人工构建的用于表达一目标蛋白的核酸构建物，其中包含组成型表达启动子。
在本发明中，对于组成型表达启动子没有特别限制，代表性的例子包括但不限于：CMV启动子、SV40启动子、PGK启动子、EF1α启动子、β-actin启动子、MSCV启动子和CAG启动子。具体地，可根据不同的编码序列和不同的免疫细胞选择不同的组成型表达启动子。在本发明中，所述的目标蛋白为特异性失活的SHP-2突变蛋白。
携带该组成型表达构建体的免疫细胞(如CAR-T细胞)，可在表达CAR分子的同时通过组成型表达启动子编码SHP-2突变蛋白的核酸序列，启动SHP-2突变蛋白的表达，并且不影响正常条件下CAR-T细胞的增殖和活化。
在另一优选例中，所述所述的表达构建体是条件激活型SHP-2突变蛋白表达构建体。
优选地，本发明使用活化T细胞核因子(Nuclear factor of activated T cells，NFAT)，它是一类转录因子家族蛋白。NFAT家族蛋白具有广泛生理功能，对由免疫反应诱导的基因转录起重要作用。除T细胞外，这类蛋白质还可以在许多免疫细胞上进行表达，如B淋巴细胞、肥大细胞、嗜酸性粒细胞等。NFAT可以调节T细胞的活化,分化及自身耐受性等功能，同时具有多向调节功能，参与多种生物学过程。
NFAT主要通过钙离子依赖性的钙调蛋白磷酸酯酶C信号路径被激活，它的激活与核质穿梭主要是通过钙调蛋白磷酸酯酶C与组成型激酶的动力学相互作用来调控的。同时NFAT激活靶基因转录时常常与AP-1蛋白或其他转录因子形成复合物发生协同作用。
本发明的优选例中，利用CAR-T细胞中内源性的NFAT转录因子可以结合于IL2基因的启动子的特性，设计了特殊的条件激活型SHP-2突变蛋白表达构建体。在该条件激活型表达构建体中，在编码SHP-2突变蛋白的片段前构建了由活化的NFAT结合启动的IL2 mini启动子。只有当CAR-T细胞通过CAR分子结合靶分子后被激活时，内源性NFAT被活化，才会结合到编码SHP-2突变蛋白的核酸序列前的IL2mini启动子上，启动SHP-2突变蛋白的表达。而当CAR-T细胞未结合靶分子尚未活化时，SHP-2突变蛋白则不会被表达。
在本发明中，通过这种优选的条件激活型的分子调控方式，可以减少在未活化的CAR-T细胞中，因SHP-2活力被意外干扰产生的不利影响，从而提高了CAR-T细胞的安全性。
CAR与CAR-T细胞
本发明还提供了表达本发明的SHP-2突变蛋白的细胞，尤其是免疫细胞。
典型地，本发明的免疫细胞包括(但并不限于)：T细胞、NK细胞、或其组合。优选地，所述的T细胞包括：CD4 +细胞、CD8 +细胞、或其组合。
在本发明中，免疫细胞可以为自体的或异体的。
在本发明中，还提供了CAR表达载体，该表达载体不仅含有编码嵌合抗原受体(CAR)的核酸序列，还含有编码能改善T细胞的SHP-2突变蛋白和其他相关分子的核酸序列。
所谓嵌合抗原受体，是指将识别癌细胞的细胞表面抗原的单链抗体和诱导T细胞活化的信号转导区，及跨细胞膜区融合而得到的人工嵌合蛋白分子。
本发明中，作为编码CAR分子的核酸序列，编码构成CAR分子的多肽的核酸 则没有特别限制，包括编码识别癌细胞的细胞表面抗原的单链抗体、跨细胞膜区、及诱导T细胞的活化的信号转导区的多肽的核酸。
本发明中作为CAR中的单链抗体(scFv)，可由来自单克隆抗体的抗原结合位点的轻链可变区(VL)及重链可变区(VH)组成、且在轻链可变区与重链可变区之间具有接头(linker)肽的寡肽或多肽。
本发明中作为上述单链抗体识别的癌细胞的细胞表面抗原，可以是针对癌细胞及其前体细胞特异性表达的生物体分子、由于细胞的癌化而新确认到表达的生物体分子、或在癌细胞中与正常细胞相比表达水平升高的生物体分子，可举出CD20、EGFR、FITC、CD19、CD22、CD33、PSMA、GD2、EGFR variant、ROR1、c-Met、HER2、CEA、间皮素(mesothelin)、GM2、CD7、CD10、CD30、CD34、CD38、CD41、CD44、CD74、CD123、D133、CD171、CD276、CLDN18.2、MUC16、MUC1、CS1(CD319)、IL-13Ra2、BCMA、GPC3、DLL3、LewisY、IgG kappa链、叶酸受体-α、PSCA、EpCAM、CLND等。
本发明中T细胞活化信号转导区是在上述单链抗体识别癌细胞的细胞表面抗原时能够向细胞内转导信号的区域，优选含有选自CD28、4-1BB(CD137)、GITR、CD27、OX40、ICOS、HVEM、CD3ζ、Fc Receptor-associatedγchain的细胞内区的多肽中的至少1种或2种以上，更优选含有CD28、4-1BB、及CD3ζ这3种的细胞内区的多肽。
本发明中各细胞内区的多肽可以由含有0～15个(较佳地2-10)氨基酸的寡肽接头或多肽接头被连接。作为所述接头序列，可举出甘氨酸-丝氨酸(G 4S)连续序列。
本发明中的跨细胞膜区，可举出来自CD8、T细胞受体的α、β链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD137L、CD154、GITR的跨细胞膜区的多肽。通过该跨细胞膜区将CAR分子固定于T细胞的细胞膜上。
本发明中对于编码嵌合抗原受体的核酸而言，可基于编码针对癌细胞的细胞表面抗原的单链抗体、跨细胞膜区、及T细胞激活信号转导区的多肽的核酸序列，可以利用化学合成的方法、通过PCR进行扩增的方法等技术进行制备。需要说明的是，可选择用于编码氨基酸的密码子进行改造，从而优化核酸在目标宿主细胞中的表达水平。
本发明中编码针对癌细胞的细胞表面抗原的单链抗体、跨细胞膜区、及T细胞激活信号转导区的多肽的碱基序列的信息可通过检索已知的文献、或N C B I等公共数据库而适当获得。
本发明中编码T细胞激活信号转导区中的CD28、4-1BB、及CD3ζ的跨细胞膜区的多肽的碱基序列的信息可通过检索NCBI等数据库而适当获得。
在本发明中，典型的CAR的结构如下式I所示：
L1-scFv-H1-TM1-C1-CD3ζ    (I)
式中，
各“-”独立地为连接肽或肽键；
L1为任选的信号肽序列；
scFv为抗原结合结构域(即靶向目标抗原(如CD19)的结合域)；
H1为任选的铰链区；
TM1为跨膜结构域；
C1为共刺激信号分子；
CD3ζ为源于CD3ζ的胞浆信号传导序列。
在另一优选例中，所述的L1为选自下组的蛋白的信号肽：CD8、CD28、GM-CSF、CD4、CD137、或其组合。
在另一优选例中，所述的H1为选自下组的蛋白的铰链区：CD8、CD28、CD137、或其组合。
在另一优选例中，所述的TM1为选自下组的蛋白的跨膜区：CD28、CD3epsilon、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154、或其组合。
在另一优选例中，所述的C1为选自下组的蛋白的共刺激信号分子：OX40、CD2、CD7、CD27、CD28、CD30、CD40、CD70、CD134、4-1BB(CD137)、PD1、Dap10、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)、NKG2D、GITR、TLR2、或其组合。
本发明中T细胞，可为来自人的T细胞、来自狗、猫、猪、小鼠等非人哺乳动物的T细胞。另外，这些T细胞可从血液、骨髓液等体液、脾脏、胸腺、***等组织、或浸润原发肿瘤、转移性肿瘤、癌性腹水等癌症组织的免疫细胞中分离、纯化而得到。这些T细胞，可举出αβT细胞、γδT细胞、CD8 +T细胞、CD4 +T细胞、肿瘤浸润T细胞、记忆T细胞、幼稚T细胞、NKT细胞等。
通过本发明设计的CAR-T细胞，通过CAR分子上的单链抗体，CAR-T细胞能够识别在癌细胞的表面上的肿瘤相关抗原。
本发明中病毒载体，可为逆转录病毒载体、慢病毒载体、腺病毒载体、腺相关病毒载体、转座子载体。若使用逆转录病毒、慢病毒载体或转座子载体，则会将编码目的片段的基因原件导入宿主细胞的基因组中，因此能够长期且稳定地表达目的分子。
本发明中在识别癌细胞的细胞表面抗原的单链抗体与跨细胞膜区、跨细胞膜区与T细胞活化信号转导区之间，可设置由任意的寡肽或多肽组成的间隔区。作为间隔区的长度，可为1～100个氨基酸，优选为10～50个氨基酸，作为该间隔区，可为甘氨酸-丝氨酸连续序列。
本发明的一个实施例中，提供了一种CAR构建物，该构建物包含：一个抗原结合区(scFV区)、一个跨膜区、一个CD28，或4-1BB等其他形式的共刺激区，一 个CD3ζ信号区，以及一个组成激活型或条件激活型共表达的SHP-2突变蛋白表达构建体。典型地，所述条件激活型表达构建体采用NFAT6 IL-2 minimal promotor-SHP2区域。
一种典型的包含CAR结构和mSHP2区域的构建物示于图1。图中，mSHP2的编码序列与组成型启动子或IL2 mini启动子操作性连接。当mSHP2编码序列与组成型启动子操作性连接时，可组成型表达mSHP2，即mSHP2的表达与免疫细胞的CAR分子的表达是各自独立的。当mSHP2编码序列与IL2 mini启动子(或其他条件激活型启动子)操作性连接时，一旦免疫细胞通过CAR分子结合靶分子后被激活时，内源性NFAT被活化，才会结合到编码SHP2突变蛋白的序列前的IL2 mini启动子上，从而启动SHP2突变蛋白的表达。
本发明的条件激活性SHP-2突变蛋白分子工作模式如图2所示。当CAR-T细胞未与肿瘤表面的抗原相结合时，其内源性的SHP-2处于未活化状态。SHP-2的N端的SH2结构域与PTP结构域以分子间作用力连接，活性位点未暴露，不能发挥磷酸酶活性。当CAR-T细胞与肿瘤表面的靶抗原特异性结合时，细胞活化，内源性SHP-2被免疫抑制检查点等信号激活，PTP结构域得以暴露出来，其磷酸酶活力被激活，能对CAR分子传导的CAR-T细胞活化起到负反馈调节作用，会降低细胞活化的程度。本发明人构建的该CAR-T细胞中内源性的NFAT在CAR-T细胞活化时，可以与***到宿主细胞基因组中的序列上的NFAT结合区结合，启动mSHP2突变蛋白的转录，并进而翻译合成抑制性突变mSHP2蛋白。该mSHP2可以竞争性结合到CAR-T细胞中内源性的SHP-2的PTP结构域，并削弱SHP-2上游活化信号对内源性SHP-2的活化效果，减弱CAR-T细胞中的内源性SHP-2的整体活力，提高细胞内酪氨酸激酶通路的激活程度，进而提高CAR-T细胞的抗肿瘤效果。
本发明的组成型表达的SHP-2突变蛋白分子可以独立于CAR分子被T细胞表达，获得组成性表达这些突变体SHP-2蛋白分子的CAR-T细胞。这些组成性表达的SHP-2突变体不影响正常条件下CAR-T细胞的增殖和活化。在遇到癌细胞时，这些抑制性突变的mSHP-2蛋白，可以竞争性结合到CAR-T细胞中内源性SHP-2的PTP结构域，或与内源性SHP-2竞争下游底物的结合，能够减弱CAR-T细胞中的内源性SHP-2的整体活力，提高细胞内酪氨酸激酶通路的激活程度，进而提高CAR-T细胞的抗肿瘤效果。
药物组合物
本发明还提供了相应的药物组合物，该药物组合物含有(a)本发明的SHP-2突变蛋白、编码该突变蛋白的多核苷酸、载体、或编码该突变蛋白的细胞、或其组合；和(b)药学上可接受的载体。
优选地，本发明的药物组合物或制剂含有本发明所述的CAR-T细胞，以及药学上可接受的载体、稀释剂或赋形剂。
在一个实施方式中，所述制剂或药物组合物为液态制剂。优选地，所述制剂为注射剂。优选地，所述制剂中所述CAR-T细胞的浓度为1×10 3-1×10 8个细胞/ml，更优地1×10 4-1×10 7个细胞/ml。
在一个实施方式中，所述制剂可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和冷冻保护剂如DMSO，甘油等。本发明的制剂优选配制用于静脉内施用。
治疗性应用
本发明还提供了本发明的免疫细胞在治疗上的应用，尤其是在肿瘤治疗方面的应用。
本发明包括用编码本发明表达构建体的病毒载体(如慢病毒)转导的细胞(例如，T细胞)进行的治疗性应用。转导的T细胞可靶向肿瘤细胞或引起自免疫疾病的免疫细胞的标志物，并且可以用于自体肿瘤或异体肿瘤或自身免疫疾病治疗。可以大规模制备，质量均一稳定，随时可调用给任何患者使用。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法，其包括以下步骤：给哺乳动物施用本发明的CAR-T细胞。
在一个实施方式中，本发明包括一类细胞疗法，给需要的患者直接施用本发明改造的CAR-T细胞。
在一个实施方式中，本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，抗CD19CAR-T细胞引起抗表达CD19的细胞的特异性免疫应答。
可治疗的癌症包括没有被血管化或基本上还没有被血管化的肿瘤，以及血管化的肿瘤。癌症可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性 的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌、卵巢癌等。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩增细胞(自体或异体的)，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞(自体或异体的)从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即，体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
本发明提供了***或自身免疫疾病的方法，其包括：施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如本文所述的靶细胞群，与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和和冷冻保护剂如DMSO，甘油等。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度—尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂 量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
本发明中“给予”和“处理”可以指治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触、以及试剂与流体的接触、流体与细胞的接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理。“处理”当应用于人、动物或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，***或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者。在一些实施方式中，在移植后，对象接受本发明的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6个至1×10 10个本发明经修饰的T细胞(如，CAR-T细胞)，通过例如静脉输注的方式，施用于患者。
本发明的主要优点包括：
1.本发明的SHP-2突变蛋白具有很好的特异性，能够特异性调节内源性SHP-2的活力，减少CAR-T细胞的耗竭，提升其抗肿瘤效果。
2.本发明的CAR-T细胞中，含有组成激活型或条件激活型SHP-2突变蛋白表达构建体。在组成型激活构建体，SHP-2突变蛋白的编码序列与组成型启动子操作性连接，从而可产生特定的突变型SHP-2蛋白，调节细胞中内源性的SHP-2磷酸酶的活力。在条件激活型表达构建体中，SHP-2突变蛋白的编码序列与IL2 minimal promotor操作性连接，使SHP-2突变蛋白的表达处于CAR分子介导的活化信号的下游。实验表明，组成激活型或条件激活型表达本发明的SHP-2突变蛋白，不影响或基本不影响正常条件下CAR-T细胞的增殖和活化，同时能减少有肿瘤细胞和肿瘤微环境引起的CAR-T细胞耗竭。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明 本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非特别说明，否则本发明实施例中所用材料和试剂均为市售产品。
材料和序列
IL2 mini启动子：见表1,SEQ ID No:17或18；
实施例1 SHP-2突变蛋白
通过对SHP-2结构的分析，设计并通过重组制备14种SHP-2的突变蛋白。野生型SHP-2和14种SHP-2突变蛋白的编号和氨基酸序列如表2和3所示：
表3野生型SHP-2和8种SHP-2突变蛋白
Figure PCTCN2021089087-appb-000008
具体地，相对于野生型SHP-2蛋白(FK020-A)的氨基酸序列，其中6种突变蛋白分别针对SHP-2蛋白N末端的2个SH2结构域进行点突变，其中FK020-B、FK020-C、FK020-D突变蛋白均含有102个氨基酸，并且分别在N-SH2结构域的第60位、第61位或第62位进行了1个氨基酸的定点突变；而FK020-H突变蛋白含有102个氨基酸，是野生型SHP-2的截短体突变；FK020-E、FK020-F、FK020-G突变蛋白均含有216个氨基酸，并且分别在N-SH2结构域的第60位、第61位或第62位进行了1个氨基酸的定点突变；而FK020-I突变蛋白含有216个氨基酸，是野生型SHP-2的截短体突变；FK020-J、FK020-K、FK020-L、FK020-M、FK020-N、 FK020-O突变蛋白均含有593个氨基酸，并且分别在PTP结构域上的第278位、第279位、第364位、第459位或第506位进行了1个氨基酸的定点突变。
实施例2构建表达SHP-2突变蛋白的Jurkat细胞
将实施例1中SHP-2突变蛋白作为目标片段，设计并合成表达突变蛋白的病毒的慢病毒载体，48小时和72小时后收集病毒并浓缩进行病毒MOI测定，载体核酸序列模式如图3所示。
用上述方法获得的病毒转导至悬浮生长的Jurkat细胞中，48小时后采用western免疫印迹法检测HA标签的表达量。可以发现这些不同的SHP-2突变体在细胞中的表达水平是一致的。
实施例3表达SHP-2突变蛋白的Jurkat细胞中SHP-2酶活力的评价
在本实施例中，对表达SHP-2突变蛋白的Jurkat细胞中SHP-2酶活力进行评价，方法如下：
M-PER TM Mammalian Protein Extraction Reagent与5×10 7Jurkat细胞(ATCC)(未处理组及100ng/ml PMA处理组)冰上混匀。加入10～50ug蛋白上清，加入适量SHP2抗体(1-5ug)，4℃混匀孵育过夜。加入葡聚糖微珠(Protein G sepharose)，4℃混匀孵育4-6h。高速离心收集微珠。
以NaF,Na 3VO 4处理过的细胞裂解液作为样品，使用Non-Radioactive Phosphatase检测***(promega公司)检测PTPase活性。
用Western blot的方法检测细胞下游信号通路的激活情况，如SHP-2的磷酸化，MARP/ERK的表达及磷酸化。
结果表明，这些SHP-2突变蛋白，在细胞内可抑制细胞中内源性SHP-2酶活力。多数突变体SHP-2表现出了对内源性SHP-2活力的抑制效果，其中FKC020-E，FKC020-N和FKC020-O的效果尤为明显。
实施例4制备表达SHP-2突变蛋白的CAR-T细胞
根据实施例2和3的结果，选择具有明显抑制能力的SHP-2突变体(如FKC020-E，FKC020-N和FKC020-O)，构建了表达组成激活性的SHP-2条件激活型的CAR表达载体，或表达SHP-2突变蛋白的CAR表达载体(pWPXLd-ScFV-NFAT6-mSHP2-GFP)。所述条件激活型质粒的结构模式如图4所示。使用得到的CAR表达载体，制备转染HEK293T细胞的慢病毒载体。使用这些慢病毒载体制备表达SHP-2突变蛋白的CAR-T细胞。
CAR-T细胞的制备步骤简述如下：PBMC或T细胞使用培养基和抗CD3、CD28等T细胞激活信号的单克隆抗体或类似的激活试剂培养48小时；收集激活的PMBC或T细胞，用新鲜的培养基重悬，根据MOI加入所需的病毒量，在培养进行转导； 结束转导后，细胞离心，弃上清，用新鲜的培养基重悬细胞,继续进行扩大培养，直到收获细胞。
所述NFAT6结合的IL2 minimal promotor(6 NFAT binding sites with IL2 minimal promoter)的构建物结构如图5所示。
相比传统CAR-T细胞，本实施例中制备的CAR-T细胞中含有一个NFAT6结合的IL2 minimal promotor-SHP2构建物(即SHP-2突变蛋白的表达盒)，使SHP-2突变蛋白的表达处于CAR分子介导的活化信号的下游。
类似地，采用CMV组成型启动子(SEQ ID No:16)，制得组成激活型表达SHP-2突变蛋白的CAR表达载体(pWPXLd-ScFV-CMV-mSHP2-GFP)。所述组成激活型质粒的结构模式如图1的(a)所示。
结果同样显示，采用组成激活型表达载体时，SHP-2突变蛋白的表达独立于CAR分子的表达。
实施例5 CAR-T细胞短期毒性评价
按照E:T比例(2:1)将CAR-T细胞和靶细胞共培养。加样于96孔培养板中，设3个复孔。混匀置二氧化碳培养箱中孵育24h，检测对靶细胞的杀伤效果。
结果表明，如图7所示，对于携带条件激活型SHP-2突变蛋白表达构建体的CAR-T细胞，当CAR被激活时，伴随表达的SHP-2突变蛋白(FK020-J、K、L、M、N和O)确实抑制了这些CAR-T中内源性的SHP-2的活力，从而使得这些CAR-T细胞表现出了对靶细胞更强的杀伤效果。
类似地，对于携带组成激活型SHP-2突变蛋白表达构建体的CAR-T细胞，组成表达的SHP-2突变蛋白也持续抑制了这些CAR-T中内源性的SHP-2的活力，从而使得这些CAR-T细胞表现出了对靶细胞更强的杀伤效果。
实施例6 CAR-T细胞长期杀伤能力评价
按照E:T比例(2:1)将CAR-T细胞和靶细胞共培养。加样于96孔培养板中，设3个复孔。混匀置二氧化碳培养箱中孵育12h后，显微镜下观察，靶细胞完全杀伤后，按照初始靶细胞数量补加，混匀置二氧化碳培养箱中继续孵育培养。多次杀伤后，显微镜下观察靶细胞剩余情况，并取CAR-T细胞进行流式分析CAR阳性细胞增殖情况。
结果表明，如图8和图9所示，对于携带SHP-2突变蛋白表达构建体的CAR-T细胞，SHP-2突变蛋白(FK020-J、K、L、M、N和O,尤其是FK020-N和FK020-O)抑制了CAR-T中内源性SHP-2的活力，从而使得这些CAR-T细胞表现出更优的扩增能力和对靶细胞更强的持续杀伤效果。
实施例7体内验证特异性失活SHP-2 CAR-T的体内活性，增殖及抗肿瘤效果
构建人源肿瘤异种移植模型(Patient-Derived tumor Xenograft，PDX)模型，制备得到的CAR-T细胞注射至小鼠体内，每两天检测如下指标：
a)小鼠的体重；
b)小鼠的生存率；
c)肿瘤的大小；
d)流式细胞术检测CAR-T细胞的体内增殖；
e)流式细胞术检测CAR-T细胞因子的分泌情况。
结果表明，对于携带条件激活型SHP-2突变蛋白表达构建体的CAR-T细胞，当CAR被激活时，伴随表达的SHP2突变蛋白确实抑制了这些CAR-T中内源性的SHP-2，从而使得这些CAR-T细胞表现出了更强的抑制小鼠体内肿瘤生长的效果。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (10)

  1. 一种SHP-2突变蛋白，其特征在于，所述的SHP-2突变蛋白具有选自下组的突变：G60E、D61E、Y62W、R278N、Y279H、K364R、K364Q、Q506K、C459S或其组合，或者所述的SHP-2突变蛋白为截断型SHP-2蛋白；其中，氨基酸的位置基于SEQ ID No.:1。
  2. 如权利要求1所述的突变蛋白，其特征在于，所述的突变蛋白为人SHP-2的片段或全长突变体，其中，所述片段具有对应于SHP-2野生型蛋白的N-SH2结构域的氨基酸序列，或者所述片段具有对应于SHP-2野生型蛋白的N-SH2和C-SH2结构域的氨基酸序列；所述的全长突变体具有对应于SHP-2野生型蛋白全长的氨基酸序列。
  3. 如权利要求1所述的突变蛋白，其特征在于，所述的突变蛋白具有对应于SHP-2野生型蛋白的第1-102位、第1-104位，第2-102位、第2-104位、第1-216位、第2-216位的氨基酸序列；或者所述的突变蛋白具有对应于SHP-2野生型蛋白第1-593位或第2-593位的氨基酸序列。
  4. 如权利要求1所述的突变蛋白，其特征在于，所述的突变蛋白为氨基酸序列如SEQ ID No.:2、3、4、5、6、7、8、9、10、11、12、13、14或15所示的多肽。
  5. 一种融合蛋白，其特征在于，所述的融合蛋白是人工构建的，并且所述融合蛋白包括：权利要求1所述的SHP-2突变蛋白，以及与所述SHP-2突变蛋白融合在一起的多肽元件。
  6. 一种多核苷酸，其特征在于，所述的多核苷酸编码权利要求1-4中任一所述的SHP-2突变蛋白或权利要求5所述的融合蛋白。
  7. 一种激活型SHP-2突变蛋白表达构建体，其特征在于，所述的表达构建体包括：激活型启动子、以及与所述启动子操作性连接的权利要求6所述的多核苷酸序列。
    7a.如权利要求7所述的表达构建体，其特征在于，所述的激活型启动子包括组成激活型启动子和条件激活型启动子。
  8. 一种载体，其特征在于，所述的载体含有权利要求6所述的多核苷酸或权利要求7所述的激活型SHP-2突变蛋白表达构建体。
  9. 一种细胞，其特征在于，所述的细胞含有权利要求8所述的载体，或者在基因组中整合有权利要求6所述的多核苷酸或权利要求7所述的激活型SHP-2突变蛋白表达构建体。
  10. 一种药物组合物，其特征在于，所述的药物组合物含有：
    (a)权利要求1所述的SHP-2突变蛋白、权利要求6所述的多核苷酸、权利要求8所述的载体、权利要求9所述的细胞、或其组合；和
    (b)药学上可接受的载体。
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