CN115806629A - Autocrine IL-15 and anti-CTLA4 combined fusion protein and application thereof - Google Patents
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Abstract
The invention discloses an autocrine IL-15 and anti-CTLA4 combined fusion protein and application thereof, wherein the fusion protein is constructed by sequentially connecting anti-CTLA4, G4S 4 Linker, IL-15N72D, G4S 4 Linker and IL-15RaSu in series, and the autocrine IL-15 and IL-15RaSu are combined into a superagonist protein. And the membrane containing the immune cells works to express the fusion protein so as to achieve the purpose of enhancing the proliferation capacity, the anti-apoptosis capacity and the killing capacity to tumors of the immune cells.
Description
Technical Field
The invention relates to the technical field of immune cell preparation, and particularly relates to an autocrine IL-15 and anti-CTLA4 combined fusion protein and application thereof.
Background
Tumor (tumor) refers to a new organism (neograwth) formed by local tissue cell proliferation of the body under the action of various tumorigenic factors, because the new organism is mostly in the form of space-occupying block-shaped protrusion, also called neoplasms (neoplasms). Among them, malignant tumors are easy to be metastasized, and they are easy to recur after treatment and are very difficult to cure in some special microenvironments.
IL-15 plays a crucial role in T cells, NK cells and their development, homeostasis and function, and also has various functions on B cells, dendritic Cells (DCs), macrophages and mast cells. IL-15 is a member of the cytokine 4-alpha-helix bundle family, has a molecular weight of 14-15kDa and contains 114 amino acids. IL-15 is of two homogeneous types: (1) SSP: short Signal Peptide (SSP) consisting of 21 amino acids, SSP type IL-15 is sufficiently translated but not secreted, and thus its range of activity is restricted to cytoplasm and nucleus, possibly playing an important role in its transcriptional regulation; (2) and (3) LSP: comprising a Longer Signal Peptide (LSP) of 48 amino acids, LSP-IL-15 is secreted extracellularly as an immunomodulator. IL-15 and IL-15R α are expressed synergistically by antigen presenting cells (monocytes and dendritic cells). IL-15 is widely expressed in a variety of cell types including monocytes, macrophages, DC cells, fibroblasts, epithelial cells and skeletal muscle cells, but does not express IL-15 cytokines in T cells.
The binding mode of IL-15 to antigen receptors is the trans-presentation mode: IL-15 binds to high affinity alpha receptors expressed on antigen presenting cells to form IL-15 Ra; IL-15R α presents IL-15 to IL-2/15R β γ dimer to form a ternary complex. Can activate JAK and STAT type channels and has the functions of promoting the proliferation and the activation of target cells, improving the secretion levels of IFN-gamma and TNF-alpha and the like.
CTLA4 (Cytotoxic T-Lymphocyte Antigen 4), also known as CD152 (Cluster of differentiation 152), is a protein receptor that down-regulates the immune system. CTLA4 is a member of the immunoglobulin superfamily, is expressed on the surface of helper T cells, and transmits inhibitory signals to T cells. The protein comprises an extracellular V domain, a transmembrane domain and a cytoplasmic tail. Alternative splice variants, encoding different isoforms. CTLA4 is similar to the T cell costimulatory protein CD28, with two molecules binding to CD80 and CD86 on antigen presenting cells, also referred to as B7-1 and B7-2, respectively. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also present in regulatory T cells and may be important to their function. Activation of T cells by T cell receptors and CD28 results in increased expression of CTLA4, an inhibitory receptor for B7 molecules. The fusion protein of CTLA4 and antibody (CTLA 4-Ig) has been used in clinical test of rheumatoid arthritis, and CTLA4 Ig can effectively and specifically inhibit cell and humoral immune reaction in vivo and in vitro, has obvious therapeutic action on graft rejection and various autoimmune diseases, has very low toxic and side effects, and is a novel immunosuppressive drug which is considered to be promising at present.
Disclosure of Invention
Based on the above problems, the present invention is to provide a fusion protein combining autocrine IL-15 and anti-CTLA4, wherein IL-15 and IL-15RaSu are combined to form a superagonin, the superagonin is combined with anti-CTLA4 to obtain the fusion protein, and immune cells containing the fusion protein successfully secrete the fusion protein and express a chimeric antigen receptor, so as to enhance the proliferation ability, anti-apoptosis ability and killing ability of the immune cells against tumors.
The technical scheme of the invention is as follows:
the fusion protein combining the autocrine IL-15 and the anti-CTLA4 is embedded into immune cells after being subjected to gene editing, can improve the activity of the immune cells and the killing effect on tumors, and expresses a chimeric antigen receptor by the immune cells.
An immune cell containing autocrine IL-15 and anti-CTLA4 fusion protein, a superagonism protein of IL-15 and IL-15RaSu and the fusion protein of anti-CTLA4 are combined, and a Chimeric antigen receptor secreting the fusion protein, such as a T cell (Chimeric antigen receptor CAR-T), is successfully obtained.
The fusion protein combining the autocrine IL-15 and the anti-CTLA4 contains cell factors of anti-CTLA4, G4S 4 Linker, IL-15N72D, G4S 4 Linker and IL-15RaSu, and is sequentially expressed in series according to the sequence of the cell factors of anti-CTLA4, G4S 4 Linker, IL-15N72D, G4S 4 Linker and IL-15RaSu, and the immune cell receiving gene editing expresses the fusion protein and receives the influence of the autocrine fusion protein.
The anti-CTLA4 in the fusion protein is anti-CTLA4 VL, anti-CTLA4 VH or the combination of the anti-CTLA4 VL and the anti-CTLA4 VH.
The fusion protein constructs an expression cassette through a nucleic acid sequence of a gene code.
In one embodiment, the fusion protein and the gene of the chimeric antigen receptor are realized by constructing an expression cassette; further, the vector delivery means when constructing the expression cassette includes lentivirus, retrovirus, general plasmid, episome, nano delivery system, electric transduction or transposon; wherein, in addition, the vector comprises a nucleic acid sequence or an expression cassette encoding the fusion protein.
In one embodiment, the expression of the chimeric antigen receptor is a chimeric antigen receptor that targets one target or multiple targets.
In one embodiment, the binding region of the chimeric antigen receptor and the target can be scFv, fab, or a combination of scFv and Fab; the scFv region structure can be substituted by one or more of any single-chain antibody, single-chain variable fragment (scFv) and Fab fragment of any target point.
In one embodiment, the target of the chimeric antigen receptor comprises one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA, and EBV; preferably the target is CLDN18.2.
In one embodiment, the chimeric antigen receptor comprises a leader sequence, an scFv that recognizes a tumor associated antigen, a hinge and transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signal CD3Zeta; wherein the scFv is an scFv of an anti-idiotype antibody; the hinge region and transmembrane domain are CD28, or the CD8hinge region and transmembrane domain; the intracellular co-stimulatory domain is CD28, CD137 (4-1 BB), or an ICOS intracellular co-stimulatory domain.
In one embodiment, the binding region between the chimeric antigen receptor and the target can be a single target, a bispecific antibody that binds to two targets, or two or more chimeric antigen receptors that are formed across membranes and that recognize different targets.
In one embodiment, the chimeric antigen receptor comprises a structure comprising one or more of the signal peptide CD8SP, the transmembrane domain CD8Hinger, CD8TM, the intracellular activation element 4-1BB, and CD3Zeta.
In one embodiment, the chimeric antigen receptor and the fusion protein of autocrine IL-15 and anti-CTLA4 are separated by a protein cleavage function; wherein the protein cleavage functional element is T2A, P2A, E2A, F2A or IRES.
In one embodiment, the vector for the transfer of the gene of the immune cell into the chimeric antigen receptor comprises a lentivirus, a retrovirus, a general plasmid, an episome, a nano-delivery system, an electrical transduction, a transposon or other delivery system. In one embodiment, the immune cells comprise T cells, NK cells, NKT cells, macrophages, gamma-delta T cells, TIL cells, TCR-T cells, or other tumor killing cells.
The invention also provides a biological preparation, which comprises an expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell line and the like constructed by the nucleic acid sequence or the amino acid sequence of the coding fusion protein, wherein the recombinant cell line is preferably an immune cell.
The invention also provides the application of the immune cells in preparing biological preparations for preventing and/or treating cancers or tumors, for example, the biological preparations are specifically the application on pharmaceutically acceptable carriers, diluents or excipients; the tumor is selected from a hematologic tumor, a solid tumor, or a combination thereof; the hematological tumor is selected from Acute Myeloid Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphocytic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof; the solid tumor is selected from gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostatic cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal carcinoma, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer or combination thereof.
Compared with the prior art, the invention has the following beneficial effects:
the immune cell of the autocrine IL-15 and anti-CTLA4 fusion protein provided by the invention expresses a chimeric antigen receptor and can specifically recognize a targeted tumor cell surface antigen; the immune cell combines the hyper-agonist protein of IL-15 and IL-15RaSu and the fusion protein of anti-CTLA4, and the immune cell successfully secretes the fusion protein, so as to enhance the proliferation capability, anti-apoptosis capability and killing capability to tumors of the immune cell; the immune cell killing effect of the invention is accurate, the safety is higher, the recurrence is not easy, and the life quality of the patient is improved.
Drawings
FIG. 1 is a structural design drawing of a fusion protein and an amino acid sequence in an immune cell; wherein, the fusion protein structure in A # -D #; 1# -4 # is an amino acid sequence structure design diagram;
FIG. 2 shows the result of the phenotypic flow detection of HGC-27-CLDN18.2 and HGC-27 of target cells;
FIG. 3 is a bar graph corresponding to the secretion of IL-15 superagonin/CTLA 4 SCFV;
FIG. 4 is a bar graph corresponding to IL-15RaSu superagonist protein secreted IL-15+ IL-15;
FIG. 5 is a graph of secretory CAR-T amplification growth;
FIG. 6 is a flow chart of T cell phenotype;
FIG. 7 is a CAR-T CLDN18.2 cell phenotype flow diagram;
FIG. 8 is a flow chart of the CAR-T CLDN18.2-IL-15/Ra cell phenotype;
FIG. 9 is a flow chart of the CAR-T CLDN18.2-anti CTLA4 cell phenotype;
FIG. 10 is a flow chart of the CAR-T CLDN18.2-15&CTLA4 cell phenotype;
FIG. 11 is a phenotype flow chart of NC (blank control);
FIG. 12 is a graph showing the evaluation of the tumoricidal function of corresponding cells in vitro against HGC-27 target cells;
FIG. 13 is a graph showing the in vitro tumoricidal function evaluation of corresponding cells against HGC-27-CLDN18.2 target cells;
FIG. 14 is a CAR-T animal experimental survival curve.
Detailed Description
The preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
The invention provides an autocrine IL-15 and anti-CTLA4 fusion protein, which comprises an immunocyte of the gene-edited autocrine IL-15 and anti-CTLA4 fusion protein, can secrete the fusion protein, can improve the activity of the immunocyte and the killing effect on tumors, and expresses a chimeric antigen receptor.
The autocrine IL-15 and anti-CTLA4 fusion protein is sequentially expressed in series according to the sequence of anti-CTLA4, G4S 4 Linker, IL-15N72D, G4S 4 Linker and IL-15RaSu, and the immune cell receiving gene editing expresses the fusion protein and receives the influence of the autocrine fusion protein. In the present invention, human interleukin is selected, so IL-15 may represent hIL-15, and IL-15RaSu may represent hIL-15RaSu. In the present invention, IL-15 (may be written as IL 15), IL-15RaSu (may be written as IL 15/Ra), and anti-CTLA4 (may be written as CTLA 4).
The immune cells do not express the above-mentioned fusion protein, but the cells for tumor therapy, such as CAR-T, CAR-NK, TCR-T, IPS, etc., which have been edited by the corresponding gene, need to secrete the fusion protein, and the cells are collectively referred to as genetically edited immune cells.
The immune cell capable of autocrine IL-15 and anti-CTLA4 fusion protein provided by the invention combines the superagonism protein of IL-15 and IL-15RaSu and the fusion protein of anti-CTLA4, and successfully obtains a Chimeric antigen receptor secreting the fusion protein, such as a T cell (Chimeric antigen receptor CAR-T).
The anti-CTLA4 in the fusion protein is anti-CTLA4 VL, anti-CTLA4 VH or the combination of the anti-CTLA4 VL and the anti-CTLA4 VH.
In one embodiment, the immune cell expresses a chimeric antigen receptor, e.g., a CAR cell. The chimeric antigen receptor expression may be a chimeric antigen receptor that targets one target or multiple targets, e.g., a CAR cell.
In one embodiment, the chimeric antigen receptor may also be targeted by one or more of the idiotypes CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA and EBV.
The binding region of the chimeric antigen receptor and the target can be scFv, fab or a combination of scFv and Fab; the scFv region structure can be substituted by one or more of any single-chain antibody, single-chain variable fragment (scFv) and Fab fragment of any target point.
The chimeric antigen receptor comprises a leader sequence, a scFv recognizing a tumor-associated antigen, a hinge and transmembrane domain, an intracellular costimulatory domain, and an intracellular activation signal CD3Zeta. Wherein the scFv is an scFv of an anti-idiotype antibody; the hinge region and transmembrane domain are a CD28 or CD8hinge region and transmembrane domain; the intracellular co-stimulatory domain is CD28 or CD137 (4-1 BB) or an ICOS intracellular co-stimulatory domain.
The binding region of the chimeric antigen receptor and the target can be a binding region that binds to one target, or can be a bispecific antibody that binds to two targets, or can be a binding region in which two or more chimeric antigen receptors are formed across membranes and recognize different targets.
In one embodiment, the chimeric antigen receptor comprises a structure comprising one or more of the signal peptide CD8SP, the transmembrane domain CD8Hinger, CD8TM, the intracellular activation element 4-1BB, and CD3Zeta.
The separation mode between the chimeric antigen receptor and the fusion protein of autocrine IL-15 and anti-CTLA4 is a protein cleavage functional element; wherein, the protein cleavage functional element can be T2A, P2A, E2A, F2A or IRES.
Vectors for the transfer of genes into chimeric antigen receptors of immune cells include lentiviruses, retroviruses, common plasmids, episomes, nano-delivery systems, electrical transduction, transposons or other delivery systems.
Immune cells of the invention include T cells, NK cells, NKT cells, macrophages, gamma-delta T cells, TIL cells, TCR-T cells or other tumor killing cells.
The immune cells of the autocrine IL-15 and anti-CTLA4 fusion protein can be prepared into a biological agent which is a pharmaceutically acceptable carrier, diluent or excipient. The biological preparation comprises an expression cassette, a recombinant vector, a recombinant protein, a recombinant microorganism or a recombinant cell line and the like which are constructed by a nucleic acid sequence or an amino acid sequence of a coding fusion protein.
A biological agent, which comprises an expression cassette constructed by a nucleic acid sequence or an amino acid sequence encoding a fusion protein, a recombinant vector, a recombinant microorganism or a recombinant cell line, etc.; the recombinant cell line can be an immune cell, such as a CAR-T cell, CAR-NK, and the like.
Administration of the biological agent may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The biological agent can be applied to drugs for preventing and/or treating solid tumors, for example, the biological agent is specifically applied to a pharmaceutically acceptable carrier, diluent or excipient; the tumor is selected from a hematologic tumor, a solid tumor or a combination thereof; the hematological tumor is selected from Acute Myeloid Leukemia (AML), multiple Myeloma (MM), chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), diffuse large B-cell lymphoma (DLBCL), or a combination thereof; the solid tumor is selected from gastric cancer, gastric cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostatic cancer, colorectal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal carcinoma, adrenal gland tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer or combination thereof.
The immune cell of the autocrine IL-15 and anti-CTLA4 fusion protein expresses a chimeric antigen receptor and can specifically recognize the idiotype of an anti-autoantibody; the immune cell combines the hyper-agonist protein of IL-15 and IL-15RaSu and the fusion protein of anti-CTLA4, and the immune cell successfully secretes the fusion protein, so as to enhance the proliferation capability, anti-apoptosis capability and killing capability to tumor of the immune cell; in addition, the immune cells can specifically kill and secrete the IL-15 and anti-CTLA4 fusion protein, the killing effect is accurate, the safety is higher, the recurrence is not easy, and the life quality of patients is improved.
The following are descriptions of specific embodiments.
In the following examples, the preparation of immune cells and functional verification thereof are described in detail by taking, as an example, the case where T cells in peripheral blood produce CAR-T and secrete IL-15 and anti-CTLA4 fusion protein (also referred to as 15 &CTLA4).
The preparation method of the immune cell specifically comprises the following steps:
1. structural design of the fusion protein;
2. constructing secretory CAR-T cells and performing in vitro functional tests;
3. secretory fusion protein type CAR-T cell in vivo functional test.
The specific implementation steps are as follows:
1. structural design of fusion proteins
According to the sequences of IL-15 (also written as IL 15), IL-15RaSu (also written as IL 15/Ra) and anti-CTLA4 (also written as CTLA 4), the fusion protein structures in A # -D # are designed according to the structure diagram of the fusion protein shown in figure 1, and the fusion protein structures are designed into CAR-T-CLDN18.2 cells; wherein, 1# is a control CAR-T, and 2# -4 # is a secretory CAR-T; wherein:
the IL-15 amino acid sequence is:
METDTLLLWVLLLWVPGSTGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS；
the amino acid sequence of the IL-15RaSu is as follows:
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR；
the Anti-CTLA4 VH amino acid sequence is as follows:
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSS；
the Anti-CTLA4 VL amino acid sequence is as follows:
EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIK；
the Linker amino acid sequence is as follows: gssssgsssssssgssss.
The structure of the chimeric antigen receptor comprises one or more of a signal peptide CD8SP, a transmembrane domain CD8Hinger, a CD8TM, an intracellular activation element 4-1BB and a CD3Zeta, which are shown in FIG. 1.
2. Construction of secretory CAR-T cells and in vitro functional assays
2.1 cell line culture
Cloning a base sequence expressing CLDN18.2 into a PHBV lentiviral vector skeleton, placing the PHBV lentiviral vector skeleton under a promoter of EF1 alpha (EF-1 alpha) to form PHBVV-EF 1 alpha-CLDN 18.2, and transferring three plasmids such as PHBVV-EF 1 alpha-CLDN 18.2, a lentiviral envelope Plasmid pMD2, G (Addgene, plasmid # 12259) and a lentiviral packaging Plasmid psPAX2 (Addgene Plasmid # 12260) to a lentivirus complete expression vector prepared in 293T cells by using Lipofectamine 3000; viral supernatants were collected at 48h and 72h, respectively, and ultracentrifugation concentration (Merck Millipore) was performed on the collected viral supernatants; the concentrated virus was used to infect HGC-27, resulting in a HGC-27 cell line overexpressing CLDN18.2, designated HGC-27-CLDN18.2.
As shown in fig. 2, HGC-27-CLDN18.2 cells expressed the detection result of CLDN 18.2; wherein, the detection map corresponding to the HGC-27 is a comparison map; from the results of detection of HGC-27-CLDN18.2 corresponding to HGC-27, it can be seen from FIG. 2 that the results of detection of the expression level of CLDN18.2 antigen at the FITC channel show that CLDN18.2 expression in HGC-27 is negative (peak pattern located at left peak of vertical line), and CLDN18.2 expression in HGC-27-CLDN18.2 is positive (peak pattern located at right peak of vertical line).
2.2 isolation of peripheral blood PBMC and expansion of T cells
Separating mononuclear cells from donor peripheral blood, performing density gradient centrifugation using a ficol method, and enriching T cells using a T cell sorting kit, such as CD3 MicroBeads, human-lysoinvented or 130-097-043, and activating cultured and expanded T cells using magnetic beads coupled with anti-CD3/anti-CD 28;
for T cell culture, texMACS GMP Medium (Miltenyi Biotec, 170-076-309) Medium was used, and the Medium was usedContaining 10% of FBS, 2mM L-glutamine and 100IU/ml rhIL2, the cell culture is carried out at 37 ℃,5% of CO 2 Culturing in a constant temperature incubator.
As shown in fig. 1, the fusion degree protein in the B # -D # sequence in the structural design of the fusion protein of item 1 is expressed and purified by a CHO fusion protein expression system and used as ELISA to detect the positive control standard for protein secretion in 1# -4 #, CAR-T culture supernatant is collected and protein secretion in cell culture supernatant is detected, and the detection data are shown in fig. 3 and 4. As can be seen from FIGS. 3 and 4, CART-CLDN18.2-IL-15/Ra, CART-CLDN18.2-anti CTLA4, CART-CLDN18.2-15&CTLA4 can normally secrete proteins and have substantially the same protein secretion efficiency.
As shown in FIG. 5, the obtained CAR-T proliferated by lentiviral packaging of the obtained CAR-T, wherein CART-CLDN18.2-15 and CTLA4 had higher cell proliferation fold than CART-CLDN18.2-IL-15/Ra, CART-CLDN 18.2-anticlast CTLA4 and CART-CLDN18.2, demonstrating that it has more excellent cell proliferation ability.
The positive rate and the phenotype result of CAR-T prepared by lentivirus infection are shown in table 1 and figures 6 to 11; in each figure, the APC channel on the abscissa indicates the expression of CD3, the positive channels on the right side relative to the left side, the PE channel on the ordinate indicates the expression of CTLA4, and the positive channels above and below the "ten" word line are indicated.
TABLE 1 CAR-T cell positivity and phenotypic flow assay results
The results in Table 1 show that CAR-T positive cells can be efficiently produced by the lentivirus infection method (the positive rate is more than 50%), and that there is no significant difference between the phenotypes of the CART-CLDN18.2, CART-CLDN18.2-IL-15/Ra, CART-CLDN 18.2-anticCTLA 4 and CART-CLDN18.2-15&CTLA4 cells. In FIGS. 6 to 11, the negative region was divided with NC as a control ("lower left portion ratio in cross-quadrant graph), and the CTLA4 expression levels of both the CART-CLDN18.2-anti CTLA4 and the CART-CLDN18.2-15 and the CTLA4 cells secreting anti-CTLA4 antibody and IL-15 and CTLA4 fusion protein (" upper right portion ratio in cross-quadrant graph) were significantly lower than those of the T cells, the CART-CLDN18.2 and the CART-CLDN18.2-IL-15/Ra, demonstrating that the IL-15 and CTLA4 fusion protein could effectively inhibit the CTLA4 expression on the surface of the CAR-T cells.
2.3 cell in vitro killing experiment
The in vitro tumor killing function of the CAR-T is verified by a flow detection method by using HGC-27-CLDN18.2 cells and HGC-27 cells as positive target cells and negative target cells respectively. The results of the tests are shown in FIGS. 12 and 13 (in the figures, E: T represents the effective target ratio), and the results of the tests show that, in contrast, the CART-CLDN18.2-15&CTLA4 secreting the fusion protein has the strongest killing effect on HGC-27-CLDN18.2 positive target cells.
3. CAR-T cell in vivo functional evaluation
24 NSG mice (18-22 g in weight) 6-8 weeks old are taken, after adaptive feeding for one week, the mice are inoculated with HGC-27-CLDN18.2 positive tumor cell strains subcutaneously, and each mouse is inoculated with 5 x 10 6 Closely observing the state of each tumor cell, measuring the tumor volume of the mice by using a vernier caliper every three days until the tumor volume reaches 100mm 3 CAR-T cells or control T cells were infused via tail vein after randomized grouping according to mouse weight and tumor size. The detailed administration method, administration dose and administration route are shown in table 2.
TABLE 2 animal protocol
As shown in FIG. 14, the CART-CLDN18.2-15&CTLA4 secretory CAR-T can greatly prolong the survival of mice.
The above examples demonstrate that: CAR-T secreting IL-15 and anti-CTLA4 fusion protein has stronger proliferation capacity and in-vitro tumor killing activity on tumors compared with CAR-T not secreting other cytokines or CAR-T secreting only one cytokine.
It should be understood that the above description is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting the scope of the invention, which is defined by the appended claims.
Claims (13)
1. The fusion protein combining the autocrine IL-15 and the anti-CTLA4 is characterized by comprising cytokines anti-CTLA4, G4S 4 Linker, IL-15N72D, G4S 4 Linker and IL-15RaSu, wherein the fusion protein is sequentially constructed in series according to the sequence of the anti-CTLA4, the G4S 4 Linker, the IL-15N72D, the G4S 4 Linker and the IL-15RaSu, and the autocrine IL-15 and the IL-15RaSu are combined into a superagonist protein.
2. The fusion protein of claim 1, wherein the anti-CTLA4 is anti-CTLA4 VL, anti-CTLA4 VH, or any combination of anti-CTLA4 VL and anti-CTLA4 VH.
3. An expression cassette comprising the nucleic acid sequence of claim 1 or 2 encoding a fusion protein.
4. A vector comprising the fusion protein of claim 1 or 2 or comprising the expression cassette of claim 3.
5. A recombinant microorganism comprising the fusion protein of claim 1 or 2, or comprising the expression cassette of claim 3, or comprising the vector of claim 4.
6. An immune cell comprising the fusion protein of claim 1 or 2, or comprising the expression cassette of claim 3, or the vector of claim 4.
7. The immune cell of claim 6, wherein the immune cell is formed from a fusion protein genetically fused to a chimeric antigen receptor.
8. The immune cell of claim 7, wherein the fusion protein and the chimeric antigen receptor are achieved by constructing an expression cassette through the vector; when the chimeric antigen receptor and the fusion protein are positioned in the same expression frame, a protein segmentation functional element is arranged between the chimeric antigen receptor and the fusion protein; the protein dividing functional element is T2A, P2A, E2A, F2A or IRES; alternatively, when the chimeric antigen receptor and the fusion protein are in different expression cassettes, the chimeric antigen receptor and the fusion protein are each expressed or delivered independently without segmentation.
9. The immune cell of claim 7 or 8, wherein the chimeric antigen receptor targets one or more targets comprising one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA, and EBV.
10. The immune cell of claim 9, wherein when the chimeric antigen receptor is targeted to a target, the binding region of the chimeric antigen receptor to the target is an scFv, a Fab, or a combination of an scFv and a Fab.
11. The immune cell of claim 9, wherein the chimeric antigen receptor targeting target is CLDN18.2.
12. A biological agent comprising the fusion protein of claim 1 or 2.
13. Use of a biological agent according to claim 12 in a medicament for the treatment and/or prophylaxis of cancer or tumour.
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