CN115806628A - Autocrine IL-15 and anti-TIGIT combined fusion protein and application thereof - Google Patents
Autocrine IL-15 and anti-TIGIT combined fusion protein and application thereof Download PDFInfo
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- CN115806628A CN115806628A CN202210928587.7A CN202210928587A CN115806628A CN 115806628 A CN115806628 A CN 115806628A CN 202210928587 A CN202210928587 A CN 202210928587A CN 115806628 A CN115806628 A CN 115806628A
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an autocrine IL-15 and anti-TIGIT combined fusion protein and application thereof, wherein the fusion protein is sequentially constructed in series according to anti-TIGIT, G4S 4 Linker, IL-15N72D, G S4 Linker and IL-15RaSu, and the autocrine IL-15 and IL-15RaSu are combined into a superagonim 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-TIGIT 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 4-alpha-helix bundle family of cytokines, 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) 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 of IL-15 to antigen receptors is in trans: 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.
TIGIT (T cell Ig and ITIM domain, also known as WUCAM, vstm3, VSIG 9) is a member of the poliovirus receptor (PVR)/Nectin family. It consists of an extracellular immunoglobulin variable region (IgV) domain, a type 1 transmembrane domain, and an intracellular domain with a classical Immunoreceptor Tyrosine Inhibition Motif (ITIM) and an Immunoglobulin Tyrosine Tail (ITT) motif. TIGIT is expressed in lymphocytes, particularly in effector and regulatory CD4+ T cells, follicular helper CD4+ T cells, effector CD8+ T cells and Natural Killer (NK) cells. Its ligands are CD155 (high affinity), CD112, CD113.
TIGIT can suppress immune cells in multiple steps of the tumor immune cycle, as follows:
STEP1. TIGIT can inhibit NK cell effects by preventing initial death of tumor cells and release of tumor antigens;
STEP2. TIGIT inhibits dendritic cell costimulation, leading to reduced cancer antigen presentation and increased anti-inflammatory cytokines such as IL-10, TIGIT can also induce PVR signaling in other cells such as tumor cells;
STEP4. TIGIT can directly inhibit CD8+ T cell effects, or TIGIT + tregs can inhibit CD8+ T cells, preventing the elimination of cancer cells.
Disclosure of Invention
Based on the above problems, the present invention provides a fusion protein combining autocrine IL-15 and anti-TIGIT, wherein IL-15 and IL-15RaSu are combined into a superagonin, the superagonin is combined with anti-TIGIT to obtain a fusion protein, and immune cells containing the fusion protein successfully secrete the 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-TIGIT is embedded into immune cells after being subjected to gene editing, the activity of the immune cells can be improved, the killing effect on tumors can be improved, and the immune cells express chimeric antigen receptors.
The immune cell containing autocrine IL-15 and anti-TIGIT fusion protein, the superagonism protein combining IL-15 and IL-15RaSu and the fusion protein of anti-TIGIT successfully obtain a Chimeric antigen receptor secreting the fusion protein, such as a T cell (Chimeric antigen receptor CAR-T).
The fusion protein combining the autocrine IL-15 and the anti-TIGIT contains cell factors anti-TIGIT, G4S 4 Linker, IL-15N72D, G S4 Linker and IL-15RaSu, and is expressed in series according to the sequence of the cell factors anti-TIGIT, G4S 4 Linker, IL-15N72D, G S4 Linker and IL-15RaSu, and the immune cells receiving gene editing express the fusion protein and receive the influence of the autocrine fusion protein.
The anti-TIGIT in the fusion protein is anti-TIGIT VL, anti-TIGIT VH or the combination of the anti-TIGIT VL and the anti-TIGIT 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, a 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 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 autocrine IL-15 and anti-TIGIT fusion protein are separated by a protein cleavage function; wherein, the protein cleavage functional element is T2A, P2A, E2A, F A 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 autocrine IL-15 and anti-TIGIT fusion protein provided by the invention is embedded into immune cells of the fusion protein to express 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-TIGIT, 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 results of the target cells HGC-27-CLDN18.2 and HGC-27 phenotype flow detection, respectively; wherein HGC-27-CLDN18.2 cells correspond to the FITC-CLDN18.2 flow assay and HGC-27 cells correspond to the FITC-CLDN18.2 flow assay, respectively;
FIG. 3 is a histogram corresponding to IL-15 secreted superagonin/TIGIT 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 CAR-T CLDN18.2-anti TIGIT cell phenotype flow diagram;
FIG. 10 is a flow chart of the CAR-T CLDN18.2-15&TIGIT cell phenotype;
FIG. 11 is a NC (blank control) phenotype flow chart;
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 the 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-TIGIT fusion protein, which comprises an immune cell of the autocrine IL-15 and anti-TIGIT fusion protein edited by a gene, can secrete the fusion protein, can improve the activity of the immune cell and the killing effect on tumors, and expresses a chimeric antigen receptor.
The fusion protein of the autocrine IL-15 and anti-TIGIT is sequentially expressed in series according to the sequence of anti-TIGIT, G4S 4 Linker, IL-15N72D, G S4 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, since human interleukins are selected, IL-15 can also represent hIL-15, and IL-15RaSu can also represent hIL-15RaSu. In the present invention, IL-15 (may be written as IL 15), IL-15RaSu (may be written as IL 15/Ra), anti-TIGIT (may be written as TIGIT) are exemplified.
The immune cells do not express the above-mentioned fusion protein, but the cells for tumor therapy, such as CAR-T, CAR-NK and TCR-T, IPS, which are edited by the corresponding gene and secrete the fusion protein, need to be edited by the corresponding gene in order to make the immune cells secrete the fusion protein, and such cells are collectively referred to as the immune cells subjected to gene editing.
The immune cell capable of autocrine IL-15 and anti-TIGIT fusion protein provided by the invention combines IL-15 and IL-15RaSu superagonist protein and anti-TIGIT fusion protein, and successfully obtains a Chimeric antigen receptor secreting the fusion protein, such as a T cell (Chimeric anti receptor CAR-T).
The anti-TIGIT in the fusion protein is anti-TIGIT VL, anti-TIGIT VH or the combination of the anti-TIGIT VL and the anti-TIGIT 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 for the chimeric antigen receptor and the target may 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 division mode between the chimeric antigen receptor and the fusion protein of autocrine IL-15 and anti-TIGIT is a protein cutting functional element; wherein, the protein cutting functional element can be T2A, P2A, E2A, F A 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-TIGIT 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 provided comprises an expression cassette, a recombinant vector, 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; 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 autocrine IL-15 and anti-TIGIT fusion protein provided by the invention has the advantages that immune cells embedded into the fusion protein express chimeric antigen receptors, and can specifically recognize idiotypes of anti-autoantibodies; the immune cell combines the hyper-agonist protein of IL-15 and IL-15RaSu and the fusion protein of anti-TIGIT, and the immune cell successfully secretes the fusion protein, so as to achieve the purpose of enhancing the proliferation capability, anti-apoptosis capability and killing capability to tumors of the immune cell; in addition, the immune cell can specifically kill and secrete IL-15 and anti-TIGIT fusion protein, has accurate killing effect and higher safety, is not easy to relapse, and improves the life quality of patients.
The following are descriptions of specific embodiments.
In the following examples, the preparation method of immune cells, functional verification, and the like will be described in detail, taking as an example the case where T cells in peripheral blood produce CAR-T and secrete IL-15 and anti-TIGIT fusion protein (also referred to as 15& -TIGIT).
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-TIGIT (also written as TIGIT), 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-TIGIT VH amino acid sequence is as follows:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYHMYWVRQAPGKGLEWVAYISKGGISTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQSSYDFAMDYWGRGTLVTVSS；
the Anti-TIGIT VL amino acid sequence is as follows:
DIQMTQSPSFLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWASARHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPLTFGQGTKLEIK；
the Linker amino acid sequence is as follows: GSSSSGSSSSGSSSSGSSSS.
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 lentiviral complete expression vector prepared in 293T cells by using Lipofectamine 3000; virus supernatants were collected at 48h and 72h, respectively, and ultracentrifugation concentration (Merck Millipore) was performed on the collected virus 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 (the peak pattern is located on the left side of the vertical line) and CLDN18.2 expression in HGC-27-CLDN18.2 is positive (the peak pattern is located on the right side of the vertical line). Negative means no expression of CLDN18.2, and positive means expression of CLDN18.2.
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 containing 10% FBS, 2mM L-glutamine and 100IU/ml rhIL2 was used, and the cells were cultured at 37 ℃ in a 5-vol% CO2 incubator.
The fusion 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 then used as ELISA to detect a positive control standard substance secreted by the protein in item 1# -4 #, CAR-T culture supernatant is collected, and the protein secretion in cell culture supernatant is detected, wherein the detection data are shown in figures 3 and 4.
FIGS. 3, 4 are histograms of secreted CAR-T fusion protein secretion; wherein, FIG. 3 is a histogram corresponding to IL-15 secreted superagonin/TIGIT SCFV; FIG. 4 is a bar chart corresponding to IL-15+ IL-15RaSu superagonin secretion.
As can be seen from FIGS. 3 and 4, the proteins can be normally secreted by CART-CLDN18.2-IL-15/Ra, CART-CLDN18.2-anti TIGIT, CART-CLDN18.2-15&TIGIT, and have substantially the same protein secretion efficiency.
As shown in FIG. 5, the obtained CAR-T proliferated by using the CAR-T prepared by lentivirus packaging, wherein the CART-CLDN18.2-15 and TIGIT have higher cell proliferation fold than the CART-CLDN18.2-IL-15/Ra, the CART-CLDN18.2-anti TIGIT and the CART-CLDN18.2, which proves that the CAR-T has more excellent cell proliferation capability.
The positive rate and phenotype results of CAR-T prepared by lentivirus infection are shown in Table 1, FIGS. 6, 7, 8, 9, 10 and 11.
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 CART-CLDN18.2, CART-CLDN18.2-IL-15/Ra, CART-CLDN18.2-anti TIGIT and CART-CLDN18.2-15&TIGIT cells.
FIGS. 6, 7, 8, 9, 10, 11 are CAR-T cell phenotype flow data, respectively; wherein, FIG. 6 is a T cell phenotype flow chart; 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 TIGIT cell phenotype; FIG. 10 is a flow chart of the CAR-T CLDN18.2-15&TIGIT cell phenotype; FIG. 11 is a NC (blank control) phenotype flow chart; in each figure, the APC channel on the abscissa indicates CD3 expression, the right side is positive relative to the left side, the PE channel on the ordinate indicates TIGIT expression, and the "ten" word line is positive above and below relative to each other.
In FIGS. 6 to 11, the negative regions were divided with NC as a control ("lower left portion ratio in cross quadrant graph), and TIGIT expression levels of both CART-CLDN18.2-anti TIGIT and CART-CLDN18.2-15 and TIGIT cells secreting anti-TIGIT antibody and IL-15 and TIGIT fusion protein (" upper right portion ratio in cross quadrant graph) were significantly lower than those of T cells, CART-CLDN18.2 and CART-CLDN18.2-IL-15/Ra, demonstrating that IL-15 and TIGIT fusion protein can effectively inhibit TIGIT expression on the surface of CAR-T cells.
2.3 cell in vitro killing experiment
FIGS. 12 and 13 are graphs showing the tumor killing function evaluation of CAR-T cells in vitro; wherein, FIG. 12 is a graph showing the in vitro tumoricidal function evaluation of corresponding cells against HGC-27 target cells; FIG. 13 is a graph of the in vitro tumor killing function evaluation of corresponding cells against HGC-27-CLDN18.2 target cells; in the abscissa, E: T represents the effective target ratio; the ordinate represents specific killing efficiency (%) or killing efficiency (%).
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. As shown in FIGS. 12 and 13, the results of the assay showed that, in contrast, the CART-CLDN18.2-15&TIGIT secreting the fusion protein had the strongest killing effect on HGC-27-CLDN18.2 positive target cells.
3. CAR-T cell in vivo functional evaluation
Selecting 6-8 weeks old NSG mice (weight 18-22 g) 24 mice, adaptively feeding for one week, subcutaneously inoculating HGC-27-CLDN18.2 positive tumor cell strain, and inoculating 5 × 10 for each mouse 6 Closely observing the state of the animal, measuring the tumor volume of the mouse every three days by using a vernier caliper, and when the tumor volume reaches 100mm 3 CAR-T cells or control T cells were infused via tail vein after randomized grouping according to mouse body 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&TIGIT secretory CAR-T can greatly prolong the survival time of mice.
The above examples demonstrate that: CAR-T secreting IL-15 and anti-TIGIT fusion protein has stronger proliferation capacity and in-vivo 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 (12)
1. The fusion protein is characterized by comprising cytokines anti-TIGIT, G4S 4 Linker, IL-15N72D, G S4 Linker and IL-15RaSu, wherein the fusion protein is sequentially constructed in series according to the sequence of anti-TIGIT, G4S 4 Linker, IL-15N72D, G S4 Linker and IL-15RaSu, and the autocrine IL-15 is firstly combined with the IL-15RaSu to form the superagonist protein and then is combined with the anti-TIGIT to obtain the fusion protein.
2. The fusion protein of claim 1, wherein the anti-TIGIT is any one of an anti-TIGIT VL, an anti-TIGIT VH, or an anti-TIGIT VL and an anti-TIGIT VH.
3. An expression cassette comprising the nucleic acid sequence encoding the fusion protein of claim 1 or 2.
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 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 chimeric antigen receptor are achieved by constructing an expression cassette from 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 segmentation functional element is T2A, P2A, E2A, F A 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 6, wherein the chimeric antigen receptor is expressed as a target or targets and the binding region of the chimeric antigen receptor and the target is scFv, fab or a combination of scFv and Fab.
10. The immune cell of claim 6, wherein the target comprises one or more of CLDN18.2, GPC3, HER2, TAA, GD2, MSLN, EGFR, NY-ESO-1, MUC1, PSMA, and EBV.
11. A biological agent comprising the fusion protein according to claim 1 or 2, or comprising the expression cassette according to claim 3, or comprising the vector according to claim 4, or comprising the immune cell according to any one of claims 6 to 10.
12. Use of a biological agent according to claim 11 in a medicament for the treatment and/or prophylaxis of cancer or tumour.
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| CN202310887318.5A Pending CN116804063A (en) | 2022-08-03 | 2023-07-18 | Fusion protein combining autocrine IL-15 and anti-TIGIT and application thereof |
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| CN202310887318.5A Pending CN116804063A (en) | 2022-08-03 | 2023-07-18 | Fusion protein combining autocrine IL-15 and anti-TIGIT and application thereof |
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| CN (2) | CN115806628A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110799528A (en) * | 2016-10-21 | 2020-02-14 | 阿尔托生物科学有限公司 | Multimeric IL-15-based molecules |
| US20200140512A1 (en) * | 2018-10-12 | 2020-05-07 | Xencor, Inc. | Pd-1 targeted il-15/il-15ralpha fc fusion proteins and uses in combination therapies thereof |
| CN111744007A (en) * | 2019-03-29 | 2020-10-09 | 江苏恒瑞医药股份有限公司 | anti-TIGIT antibody pharmaceutical composition and application thereof |
| CN113106068A (en) * | 2021-03-26 | 2021-07-13 | 深圳市先康达生命科学有限公司 | Immune cell of autocrine IL-15 and anti-PD1 fusion protein |
| WO2021244626A1 (en) * | 2020-06-05 | 2021-12-09 | 上海交通大学 | Chimeric antigen receptor targeting cldn18.2 and use thereof |
-
2022
- 2022-08-03 CN CN202210928587.7A patent/CN115806628A/en not_active Withdrawn
-
2023
- 2023-07-18 CN CN202310887318.5A patent/CN116804063A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110799528A (en) * | 2016-10-21 | 2020-02-14 | 阿尔托生物科学有限公司 | Multimeric IL-15-based molecules |
| US20200140512A1 (en) * | 2018-10-12 | 2020-05-07 | Xencor, Inc. | Pd-1 targeted il-15/il-15ralpha fc fusion proteins and uses in combination therapies thereof |
| CN111744007A (en) * | 2019-03-29 | 2020-10-09 | 江苏恒瑞医药股份有限公司 | anti-TIGIT antibody pharmaceutical composition and application thereof |
| WO2021244626A1 (en) * | 2020-06-05 | 2021-12-09 | 上海交通大学 | Chimeric antigen receptor targeting cldn18.2 and use thereof |
| CN113106068A (en) * | 2021-03-26 | 2021-07-13 | 深圳市先康达生命科学有限公司 | Immune cell of autocrine IL-15 and anti-PD1 fusion protein |
Non-Patent Citations (3)
| Title |
|---|
| JOE-MARC CHAUVIN ET AL.: "IL-15 stimulation with TIGIT blockade reverses CD155-mediated NK cell dysfunction in melanoma", 《CLIN CANCER RES》, vol. 26, no. 20, pages 5520 * |
| MARIELLA DELLA CHIESA ET AL.: "NK Cell-Based Immunotherapy in Colorectal Cancer", 《VACCINES》, vol. 10, no. 1033, pages 1 - 20 * |
| 王蓉 等: "免疫检查点TIGIT 在肿瘤治疗中的 最新研究进展", 《河北医学》, vol. 29, no. 1, pages 172 - 176 * |
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| Publication number | Publication date |
|---|---|
| CN116804063A (en) | 2023-09-26 |
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