WO2021213479A1

WO2021213479A1 - Shp2 specific inactivating mutant protein and application thereof in car-t therapy

Info

Publication number: WO2021213479A1
Application number: PCT/CN2021/089087
Authority: WO
Inventors: 张雷; 高明明; 王慧宇; 祖睿
Original assignee: 复星凯特生物科技有限公司
Priority date: 2020-04-22
Filing date: 2021-04-22
Publication date: 2021-10-28
Also published as: CN113528483A

Abstract

A SHP2 specific inactivating mutant protein and an application thereof in CAR-T therapy are provided. Specifically, the SHP2 specific inactivating mutant protein, a corresponding conditionally activated SHP-2 mutant protein expression cassette, and CAR-T cells carrying the conditionally activated SHP-2 mutant protein expression cassette are provided. When the CAR of the CAR-T cells is activated, the accompanying expression of the SHP2 mutant protein can effectively inhibit the endogenous SHP-2 in the CAR-T cells, thereby having a stronger in vitro and in vivo anti-tumor effects.

Description

SHP2 specific inactivating mutant protein and its application in CAR-T therapy

Technical field

The present invention relates to the field of medicine, in particular to SHP2 specific inactivating mutant protein and its application in CAR-T therapy.

Background technique

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. By introducing the gene encoding CAR into the patient's peripheral blood T cells, it is possible to produce a large number of T cells capable of expressing CAR molecules (referred to as "CAR-T cells"). CAR-T cells are tumor-reactive and can kill cancer cells independently of the interaction with the major histocompatibility gene complex (MHC). 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.

However, 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-β and IL-10.

Therefore, there is an urgent need in this field to develop more effective CAR-T cells to give full play to the functions of CAR-T.

Summary of the invention

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.

In the first aspect of the present invention, a SHP-2 mutein is provided, the SHP-2 mutein 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.

In another preferred example, 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).

In another preferred example, 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.

In another preferred example, the mutant protein has positions 1-102, 1-104, 2-102, 2-104, 1-216 corresponding to SHP-2 wild-type protein. The amino acid sequence of positions 2 to 216.

In another preferred example, the mutant protein has an amino acid sequence corresponding to positions 1-593 or 2-593 of the SHP-2 wild-type protein.

In another preferred embodiment, the mutant protein is inactive.

In another preferred example, 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.

In another preferred embodiment, 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.

In the second aspect of the present invention, 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.

In another preferred embodiment, the polypeptide element includes various elements such as fluorescent protein (such as GFP, RFP) and the like.

In the third aspect of the present invention, a polynucleotide is provided, 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.

In another preferred embodiment, the polynucleotide includes DNA or RNA.

In the fourth aspect of the present invention, 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).

In another preferred example, the activated promoter includes a constitutively activated promoter and a conditionally activated promoter

In another preferred example, the conditionally activated promoter is activated after the CAR-T cell binds to the target molecule through the CAR molecule.

In another preferred example, the constitutively activated promoter (or constitutive expression promoter) includes but is not limited to CMV promoter, SV40 promoter, PGK promoter, EF1α promoter, β-actin promoter, MSCV Promoter and CAG promoter; conditionally activated promoter is IL2 mini promoter.

In another preferred example, the sequence of the CMV promoter is shown in SEQ ID No: 16.

In another preferred embodiment, the sequence of the IL2 mini promoter is shown in SEQ ID No: 17 or 18.

In the fifth aspect of the present invention, a vector is provided, which contains the polynucleotide of the third aspect or the activated SHP-2 mutein expression construct of the fourth aspect.

In another preferred embodiment, the vector includes an expression vector.

In another preferred embodiment, the vector includes a viral vector.

In another preferred embodiment, the expression vector includes: a lentiviral vector, an adenovirus vector, an adeno-associated virus AAV vector, a plasmid, or a combination thereof.

In the sixth aspect of the present invention, there is provided 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.

In another preferred example, the expression construct contains a constitutively activated promoter or a conditionally activated promoter.

In another preferred embodiment, the cells are immune cells.

In another preferred embodiment, the immune cells express foreign chimeric antigen receptors (CAR).

In another preferred embodiment, the immune cells express chimeric antigen receptors for anti-tumor.

In another preferred embodiment, the chimeric antigen receptor includes: optional leader peptide, extracellular antigen binding domain, hinge region, transmembrane region and intracellular region.

In another preferred embodiment, the immune cells include: T cells, NK cells, or a combination thereof.

In another preferred embodiment, the T cells include: CD4 ⁺ cells, CD8 ⁺ cells, or a combination thereof.

In another preferred embodiment, the immune cells are autologous or allogeneic.

In another preferred embodiment, 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-α, PSCA, or EpCAM.

In another preferred example, 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.

In another preferred example, the constitutively activated promoter (or constitutive expression promoter) includes but is not limited to CMV promoter, SV40 promoter, PGK promoter, EF1α promoter, β-actin promoter, MSCV Promoter and CAG promoter

In another preferred example, 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.

In another preferred embodiment, the IL2 mini promoter is activated by the binding of activated NFAT.

In another preferred embodiment, the IL2 mini promoter has the nucleotide sequence described in SEQ ID No: 17 or 18.

Table 1 IL2 mini promoter

In the seventh aspect of the present invention, there is provided a pharmaceutical composition, which contains:

(a) The SHP-2 mutein of the first aspect, the polynucleotide of the third aspect, the vector of the fifth aspect, the cell of the sixth aspect, or a combination thereof; and

(b) A pharmaceutically acceptable carrier.

In another preferred embodiment, the formulation is a liquid formulation.

In another preferred embodiment, the cell is an immune cell expressing a chimeric antigen receptor (CAR).

In another preferred embodiment, the immune cells include: T cells, NK cells, monocytes, macrophages or a combination thereof.

In the eighth aspect of the present invention, the use of the cell described in the sixth aspect is provided, which is used to prepare drugs for treating tumors.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, I will not repeat them one by one here.

Description of the drawings

Figure 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). In the figure, 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.

Detailed ways

After extensive and in-depth research, 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. Specifically, 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. Experiments have shown that for 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. On this basis, the inventor completed the present invention.

the term

In order to make the present disclosure easier to understand, first define certain terms. As used in this application, unless expressly stated otherwise herein, each of the following terms shall have the meaning given below. Other definitions are stated throughout the application.

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. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, 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. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

SHP-2 and its mutant protein

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 .

The 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 C terminal tail of the amino acid group and tyrosine phosphorylation site. 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.

Studies have shown that inhibiting the activity of SHP-2 by drugs can increase the ^{cytotoxicity of CD8 +} T cells, thereby reducing tumor burden. Knockout of SHP-2 specifically in mouse T cells can delay T cell exhaustion and enhance T cell-mediated anti-tumor effects. Co-expression of SHP-2 dominant negative mutants in human CAR-T cells enhanced the killing of PD-L1 tumor cells by CAR-T cells in vitro. When T cells are activated, SHP-2 is activated by immunosuppressive regulatory signals, and the killing, proliferation and secretion of cytokines of CAR-T cells are weakened. If SHP-2 is selectively inhibited, the tumor-killing ability of CAR-T cells will be improved.

SHP-2 mutant protein

In the present invention, an SHP2 mutant protein (mutant) 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.

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.

Preferably, relative to the amino acid sequence of the wild-type SHP-2 protein (FK020-A), 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. And a site-directed mutation of 1 amino acid was carried out at position 60, 61, or 62 of the N-SH2 domain; FK020-I mutant protein contains 216 amino acids, which is a truncated mutation of wild-type SHP-2 ; In addition, 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.

In an embodiment of the present invention, 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.

The amino acid sequences of wild-type SHP-2 protein FK020-A and 14 SHP-2 mutant proteins are shown in Table 2:

Table 2 Wild-type SHP-2 protein and SHP-2 mutant protein

As used herein, the term "G60E" refers to the mutation of amino acid at position 60 from glycine G to glutamic acid E.

As used herein, the term "D61E" refers to the mutation of amino acid at position 61 from aspartic acid D to glutamic acid E.

As used herein, the term "Y62W" refers to the mutation of amino acid at position 62 from tyrosine Y to tryptophan W.

As used herein, the term "R278N" refers to the mutation of amino acid at position 278 from arginine R to asparagine N.

As used herein, the term "Y279H" refers to the mutation of amino acid at position 279 from tyrosine Y to histidine H.

As used herein, the term "K364R" refers to the mutation of amino acid at position 364 from arginine R to asparagine N.

As used herein, the term "K364Q" refers to the mutation of amino acid at position 364 from arginine R to glutamine Q.

As used herein, the term "C459S" refers to the mutation of amino acid at position 459 from Cysteine C to Serine S.

As used herein, the term "Q506K" refers to the mutation of amino acid at position 506 from glutamine Q to lysine K.

In 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.

Constitutively activated or conditionally activated SHP-2 mutant protein expression construct

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.

As used herein, 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. In the present invention, the target protein is a specifically inactivated SHP-2 mutant protein. Preferably, the expression construct is a constitutive expression construct or a conditional expression construct.

Preferably, the expression construct is a constitutive SHP-2 mutein 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. A nucleic acid construct of the target protein, which contains a constitutive expression promoter.

In the present invention, there is no particular limitation on the constitutive expression promoter. Representative examples include but are not limited to: CMV promoter, SV40 promoter, PGK promoter, EF1α promoter, β-actin promoter, MSCV promoter and CAG promoter. Specifically, different constitutive expression promoters can be selected according to different coding sequences and different immune cells. In the present invention, 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.

In another preferred embodiment, the expression construct is a conditionally activated SHP-2 mutein expression construct.

Preferably, 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. At the same time, 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.

In a preferred example of the present invention, 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. In this conditionally activated expression construct, 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.

In the present invention, through this preferred conditionally activated molecular control method, the adverse effects of accidental interference with SHP-2 activity in unactivated CAR-T cells can be reduced, thereby increasing CAR-T cells. Security.

CAR and CAR-T cells

The present invention also provides cells expressing the SHP-2 mutant protein of the present invention, especially immune cells.

Typically, the immune cells of the present invention include (but are not limited to): T cells, NK cells, or a combination thereof. Preferably, the T cells include: CD4 ⁺ cells, CD8 ⁺ cells, or a combination thereof.

In the present invention, immune cells can be autologous or allogeneic.

In the present invention, 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. .

The so-called 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.

In the present invention, as the nucleic acid sequence encoding the CAR molecule, 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.

In the present invention, 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.

In the present invention, 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-α, PSCA, EpCAM, CLND, etc.

In the present invention, 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, CD3ζ, Fc Receptor-associated γ 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 CD3ζ.

In the present invention, the polypeptides in each intracellular region can be connected by oligopeptide linkers or polypeptide linkers containing 0-15 (preferably 2-10) amino acids. Examples of the linker sequence include a glycine-serine (G ₄ S) continuous sequence.

The transmembrane region in the present invention includes α and β chains derived from CD8, T cell receptors, CD28, CD3ε, 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.

In the present invention, 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.

In the present invention, 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.

In the present invention, information about the base sequence of the polypeptides encoding CD28, 4-1BB, and CD3ζ transmembrane region in the T cell activation signal transduction region can be appropriately obtained by searching databases such as NCBI.

In the present invention, the structure of a typical CAR is shown in the following formula I:

L1-scFv-H1-TM1-C1-CD3ζ (I)

Where

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;

CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

In another preferred example, 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.

In another preferred example, the H1 is the hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

In another preferred embodiment, 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.

In another preferred example, 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. In addition, 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. Examples of these T cells include αβ T cells, γδ T cells, CD8 ⁺ T cells, CD4 ⁺ T cells, tumor-infiltrating T cells, memory T cells, naive T cells, NKT cells, and the like.

Through the CAR-T cell designed in the present invention, 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.

In the present invention, 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.

In one embodiment of the present invention, a CAR construct is provided, which comprises: an antigen binding region (scFV region), a transmembrane region, a CD28, or 4-1BB and other co-stimulatory regions, A CD3ζ signal region, and an SHP-2 mutant protein expression construct that constitutes an activated or conditionally activated co-expression. Typically, the conditionally activated expression construct uses the NFAT6 IL-2 minimal promotor-SHP2 region.

A typical construct containing the CAR structure and the mSHP2 region is shown in Figure 1. In the figure, the coding sequence of mSHP2 is operatively linked to a constitutive promoter or IL2 mini promoter. When the mSHP2 coding sequence is operatively linked with a constitutive 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. 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. When 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. 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. 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.

Pharmaceutical composition

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.

Preferably, 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.

In one embodiment, the formulation or pharmaceutical composition is a liquid formulation. Preferably, the preparation is an injection. Preferably, 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.

In one embodiment, 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.

Therapeutic application

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.

Therefore, 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.

In one embodiment, 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.

In one embodiment, the CAR-T cells of the present invention can undergo stable T cell expansion in vivo and last for an extended amount of time. In addition, 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. For example, 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. Examples of hematological (or hematogenic) cancers 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.

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. Preferably, the mammal is a human.

For ex vivo immunization, at least one of the following occurs in vitro before the cells are administered into the mammal: i) expansion of the cells (autologous or allogeneic), ii) introduction of nucleic acid encoding CAR into the cells, and/or iii) Store cells frozen.

In vitro procedures are well known in the art and are discussed more fully below. Briefly, cells (autologous or allogeneic) are isolated from a mammal (preferably human) and genetically modified (ie, transduced or transfected in vitro) with a vector expressing the CAR disclosed herein. CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits. The mammalian recipient can be a human, and the CAR-modified cell can be autologous relative to the recipient. Alternatively, the cell may be allogeneic, syngeneic, or xenogeneic relative to the recipient.

In addition to the use of cell-based vaccines for ex vivo immunization, 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. Briefly, 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. Such 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.

When referring to "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount", 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.

In the present invention, "administration" and "treatment" 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. "Administration" 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. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, 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.

In certain embodiments of the present invention, 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. In some embodiments, after transplantation, the subject receives an infusion of immune cells of the invention. In an additional embodiment, 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. ^{Generally, 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 main advantages of the present invention include:

1. 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.

2. The CAR-T cell of the present invention contains a constitutively activated or conditionally activated SHP-2 mutant protein expression construct. In the constitutive activation 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 . In the conditionally activated expression construct, 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. Experiments have shown that 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.

The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples usually follow conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing The conditions suggested by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and parts by weight.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

Material and sequence

IL2 mini promoter: see Table 1, SEQ ID No: 17 or 18;

Example 1 SHP-2 mutant protein

By analyzing the structure of SHP-2, 14 kinds of mutant proteins of SHP-2 were designed and recombined. The numbers and amino acid sequences of wild-type SHP-2 and 14 SHP-2 mutant proteins are shown in Tables 2 and 3:

Table 3 Wild-type SHP-2 and 8 SHP-2 mutant proteins

Specifically, relative to the amino acid sequence of the wild-type SHP-2 protein (FK020-A), 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.

Example 2 Construction of Jurkat cells expressing SHP-2 mutant protein

Using the SHP-2 mutant protein in Example 1 as the target fragment, 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.

Example 3 Evaluation of SHP-2 enzyme activity in Jurkat cells expressing SHP-2 mutant protein

In this example, the SHP-2 enzyme activity in Jurkat cells expressing SHP-2 mutant protein was evaluated, and the method was as follows:

M-PER ^TM Mammalian Protein Extraction Reagent and 5×10 ⁷ Jurkat cells (ATCC) (untreated group and 100ng/ml PMA treated group) 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℃ 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.

The results show that these SHP-2 mutant proteins can inhibit the endogenous SHP-2 enzyme activity in the cell. Most of the mutant SHP-2 showed an inhibitory effect on the endogenous SHP-2 activity, among which FKC020-E, FKC020-N and FKC020-O were particularly effective.

Example 4 Preparation of CAR-T cells expressing SHP-2 mutant protein

According to the results of Examples 2 and 3, SHP-2 mutants with obvious inhibitory ability (such as FKC020-E, FKC020-N and FKC020-O) 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) expressing SHP-2 mutant protein. The structural model of the conditionally activated plasmid is shown in FIG. 4. Using the obtained CAR expression vector, a lentiviral vector for transfecting HEK293T cells was prepared. Use these lentiviral vectors to prepare CAR-T cells expressing SHP-2 mutant protein.

The preparation steps of 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.

The structure of the NFAT6-binding IL2 minimal promotor (6 NFAT binding sites with IL2 minimal promoter) structure is shown in FIG. 5.

Compared with traditional CAR-T cells, 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.

Similarly, 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).

The results also showed that when using a constitutively activated expression vector, the expression of SHP-2 mutein was independent of the expression of CAR molecules.

Example 5 Short-term toxicity evaluation of CAR-T cells

According to the E:T ratio (2:1), 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 results show that, as shown in Figure 7, for CAR-T cells carrying the conditionally activated SHP-2 mutein expression construct, when CAR is activated, the expression of SHP-2 mutein (FK020-J, K, L , M, N and O) indeed inhibited the viability of the endogenous SHP-2 in these CAR-Ts, so that these CAR-T cells showed a stronger killing effect on target cells.

Similarly, for CAR-T cells carrying a construct that constitutes an activated SHP-2 mutein expression construct, 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.

Example 6 Evaluation of the long-term killing ability of CAR-T cells

According to the E:T ratio (2:1), 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 results show that, as shown in Figure 8 and Figure 9, for CAR-T cells carrying the SHP-2 mutant protein expression construct, 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

Construct a human-derived tumor xenograft (Patient-Derived tumor Xenograft, PDX) model, inject the prepared CAR-T cells into mice, and detect the following indicators every two days:

a) The weight of the mouse;

b) Survival rate of mice;

c) The size of the tumor;

d) Flow cytometry to detect the proliferation of CAR-T cells in vivo;

e) Flow cytometry to detect the secretion of CAR-T cytokines.

The results show that for CAR-T cells carrying the conditionally activated SHP-2 mutant protein expression construct, when CAR is activated, the accompanying expression of SHP2 mutant protein does indeed inhibit the endogenous SHP-2 in these CAR-Ts. As a result, these CAR-T cells showed a stronger effect of inhibiting tumor growth in mice.

All documents mentioned in the present invention are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

A SHP-2 mutant protein, characterized in that the SHP-2 mutant protein 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 of claim 1, wherein the mutant protein is a fragment or a full-length mutant of human SHP-2, wherein the fragment has an N-SH2 structure corresponding to the SHP-2 wild-type protein The amino acid sequence of the domain, or the fragment has the amino acid sequence corresponding to the N-SH2 and C-SH2 domains of the SHP-2 wild-type protein; the full-length mutant has the full-length corresponding to the SHP-2 wild-type protein The amino acid sequence.
The mutant protein of claim 1, wherein the mutant protein has positions 1-102, 1-104, 2-102, and 2-102 corresponding to SHP-2 wild-type protein. The amino acid sequence at position 104, position 1-216, and position 2-216; or the mutant protein has an amino acid sequence corresponding to position 1-593 or position 2-593 of the SHP-2 wild-type protein.
The mutant protein of claim 1, wherein the mutant protein has an amino acid sequence such as SEQ ID No.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, The polypeptide represented by 13, 14 or 15.
A fusion protein, characterized in that the fusion protein is artificially constructed, and the fusion protein comprises: the SHP-2 mutein of claim 1, and fused with the SHP-2 mutein Of peptide elements.
A polynucleotide, characterized in that the polynucleotide encodes the SHP-2 mutein of any one of claims 1-4 or the fusion protein of claim 5.
An activated SHP-2 mutein expression construct, wherein the expression construct comprises: an activated promoter and the polynucleotide sequence of claim 6 operably linked to the promoter .

7a. The expression construct of claim 7, wherein the activated promoter comprises a constitutively activated promoter and a conditionally activated promoter.
A vector, characterized in that the vector contains the polynucleotide of claim 6 or the activated SHP-2 mutein expression construct of claim 7.
A cell, characterized in that the cell contains the vector according to claim 8, or integrates the polynucleotide according to claim 6 or the activated SHP-2 mutein according to claim 7 into the genome Expression construct.
A pharmaceutical composition, characterized in that the pharmaceutical composition contains:

(a) The SHP-2 mutein of claim 1, the polynucleotide of claim 6, the vector of claim 8, the cell of claim 9, or a combination thereof; and

(b) A pharmaceutically acceptable carrier.