CN115806627A - Autocrine IL-15 and anti-LAG3 combined fusion protein and application thereof - Google Patents
Autocrine IL-15 and anti-LAG3 combined fusion protein and application thereof Download PDFInfo
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- CN115806627A CN115806627A CN202210928582.4A CN202210928582A CN115806627A CN 115806627 A CN115806627 A CN 115806627A CN 202210928582 A CN202210928582 A CN 202210928582A CN 115806627 A CN115806627 A CN 115806627A
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a fusion protein combining autocrine IL-15 and anti-LAG3 and application thereof, the fusion protein is sequentially constructed in series according to anti-LAG3, G4S 4 Linker, IL-15N72D, G4S 4 Linker and IL-15RaSu, 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 field of cell biological agents, in particular to an autocrine IL-15 and anti-LAG3 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 proliferation and activation of target cells, increasing IFN-gamma and TNF-alpha secretion levels and the like.
LAG-3 (lymphocyte-activation gene 3) is an immune checkpoint receptor protein expressed on the surface of effector T lymphocytes and regulatory T cells. LAG-3 has functions of regulating T cell immune response, activation and growth. Preclinical studies have found that LAG-3 inhibition can cause T cells to regain cytotoxicity, thereby enhancing the killing effect on tumors. Simultaneous inhibition of LAG-3 also reduces the ability of regulatory T cells to suppress immune responses, and thus LAG-3 is considered to be a more attractive target than other immune checkpoint proteins.
LAG-3 not only inhibits the proliferation of CD8+ T cells with antitumor activity, but also directly affects the immune function, and meanwhile, LAG-3 can also enhance the inhibitory activity of regulatory T cells to further inhibit immune response. While low expression of LAG-3 is one of the characteristics of memory T cells. Clinical data also show that in a variety of cancer types, such as melanoma, colon cancer, breast cancer, etc., tumor Infiltrating Lymphocytes (TILs) express LAG-3, which correlates with a clinical feature of cancer cell aggressiveness. Blocking LAG-3 can reverse the above inhibitory effect, restore CD8+ T cell proliferation and activity, reduce the number of regulatory T cells, and improve the sensitivity of T cell immune response. Simultaneously, PD-1 is blocked, the immune response is enhanced in a synergistic manner, and tumors are inhibited.
Since the presence of FGL1 was not known at that time due to earlier development, previous antibodies may not block the binding of LAG-3 and FGL 1. That is, the inability to completely block the LAG-3 pathway may be a cause of poor clinical data.
Disclosure of Invention
Based on the above problems, the present invention provides a fusion protein combining autocrine IL-15 and anti-LAG3, wherein a superagonin of IL-15 and IL-15RaSu is then combined with anti-LAG3 to obtain the 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:
an autocrine IL-15 and anti-LAG3 fusion protein is embedded into immune cells after gene editing, the fusion protein can improve the activity of the immune cells and the killing effect on tumors, and the immune cells express chimeric antigen receptors.
An immune cell comprising a fusion protein of autocrine IL-15 and anti-LAG3, a superagonin of IL-15 and IL-15RaSu, and a fusion protein of anti-LAG3 were combined and successfully obtained a Chimeric antigen receptor, such as a T cell (Chimeric anti receptor CAR-T), secreting the fusion protein.
The fusion protein of autocrine IL-15 and anti-LAG3 contains cytokines anti-LAG3, G4S 4 Linker, IL-15N72D, G4S 4 Linker and IL-15RaSu, and is expressed in series according to the sequence of anti-LAG3, 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 autocrine fusion protein.
The anti-LAG3 in the fusion protein is anti-LAG3 VL, anti-LAG3 VH or the combination of anti-LAG3 VL and anti-LAG3 VH.
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, electrical transduction, or transposon.
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 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; preferably the target is CLDN18.2.
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 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 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-LAG3 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 transferring the gene of the immune cell into the chimeric antigen receptor comprises a lentivirus, retrovirus, general plasmid, episome, nano-delivery system, electrical transduction, 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 encoding 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 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.
Compared with the prior art, the invention has the following beneficial effects:
the autocrine IL-15 and anti-LAG3 fusion protein provided by the invention is embedded into immune cells of the fusion protein to express a chimeric antigen receptor, and can specifically identify 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-LAG3, 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; wherein HGC-27-CLDN18.2 cells correspond to a FITC-CLDN18.2 flow assay and HGC-27 cells correspond to a FITC-CLDN18.2 flow assay;
FIGS. 3, 4 are histograms of secretory CAR-T fusion protein secretion; wherein, FIG. 3 is a histogram corresponding to secretion of IL-15 super agonist protein/LAG 3 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;
FIGS. 6, 7, 8, 9, 10, 11 are CAR-T cell phenotype flow data, respectively; wherein, 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 LAG3 cell phenotype; FIG. 10 is a flow chart of the CAR-T CLDN18.2-15 and LAG3 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 LAG3 expression, and the "ten" word line is positive above and below relative to each other;
FIGS. 12 and 13 are graphs for evaluating the in vitro tumoricidal function of CAR-T cells, respectively; 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 showing the in vitro tumoricidal function evaluation of corresponding cells against HGC-27-CLDN18.2 target cells; in the abscissa, E: T represents the effective target ratio; the ordinate is specific killing efficiency (%) or killing efficiency (%);
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-LAG3 fusion protein, which comprises immune cells of IL-15 and anti-LAG3 fusion protein edited by genes, wherein the immune cells can be fused with the protein, the fusion protein can improve the activity of the immune cells and the killing effect on tumors, and the immune cells express chimeric antigen receptors.
The fusion protein of the autocrine IL-15 and the anti-LAG3 is expressed in a tandem manner according to the sequence of anti-LAG3, G4S 4 Linker, IL-15N72D, G4S 4 Linker and IL-15RaSu, and the immune cells receiving gene editing express the fusion protein and are influenced by the autocrine fusion protein.
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-LAG3 fusion protein provided by the invention combines IL-15 and IL-15RaSu superagonin and anti-LAG3 fusion protein, and successfully obtains a Chimeric antigen receptor, such as a T cell (Chimeric anti receptor CAR-T), which secretes the fusion protein.
The anti-LAG3 in the fusion protein is anti-LAG3 VL, anti-LAG3 VH or the combination of anti-LAG3 VL and anti-LAG3 VH.
In one embodiment, the immune cell expresses a chimeric antigen receptor, e.g., a CAR cell. Chimeric antigen receptor expression can 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-LAG3 is a protein cleavage functional element; wherein, the protein cleavage function 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-LAG3 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.
The biological preparation provided by the invention 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.
The autocrine IL-15 and anti-LAG3 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-LAG3, and enables the immune cell to successfully secrete the fusion protein, so as to achieve the purpose of enhancing the proliferation capacity, the anti-apoptosis capacity and the killing capacity on tumors of the immune cell; in addition, the immune cell can specifically kill and secrete IL-15 and anti-LAG3 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 of immune cells and functional verification thereof 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-LAG3 fusion proteins.
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-LAG3 (also written as LAG 3), the fusion protein structures in A # -D # are designed according to the structure diagram of the fusion protein shown in figure 1, and 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-LAG3 VH amino acid sequence is as follows:
QVQLVQSGAEVKKPGASVKVSCKASGFTLTNYGMNWVRQARGQRLEWIGWINTDTGEPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARNPPYYYGTNNAEAMDYWGQGTTVTVSS；
the Anti-LAG3 VL amino acid sequence is as follows:
DIQMTQSPSSLSASVGDRVTITCSSSQDISNYLNWYLQKPGQSPQLLIYYTSTLHLGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYYNLPWTFGQGTKVEIK；
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
Constructing secretory CAR-T cells and performing in vitro functional tests, comprising the following steps:
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), a lentiviral packaging Plasmid psPAX2 (Addgene Plasmid # 12260) and the like 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 the detection of HGC-27-CLDN18.2 corresponding to HGC-27, as can be seen from the vertical two graphs of the dotted line frame a portion in FIG. 2, the results of the detection of the expression level of CLDN18.2 antigen in the FITC channel show that CLDN18.2 expression in HGC-27 is negative (the peak graph is located on the left side of the vertical line I) and CLDN18.2 expression in HGC-27-CLDN18.2 is positive (the peak graph is located on the right side of the vertical line I).
2.2 isolation of peripheral blood PBMC and expansion of T cells
Separating mononuclear cells from peripheral blood of a donor, performing density gradient centrifugation using a ficol method, and enriching T cells using a T cell sorting kit, such as CD3 MicroBeads, human-lysophilized, or 130-097-043, and activating cultured and expanded T cells using anti-CD3/anti-CD28 coupled magnetic beads;
for T cell culture, texMACS GMP Medium (Miltenyi Biotec, 170-076-309) Medium containing 10% of FBS, 2mM L-glutamine and 100IU/ml rhIL2 was used, and the cells were cultured at 37 ℃ and 5% CO 2 Culturing in a constant temperature incubator.
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 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 secretory CAR-T fusion protein secretion; wherein, FIG. 3 is a histogram corresponding to secretion of IL-15 super agonist protein/LAG 3 SCFV; FIG. 4 is a bar chart corresponding to IL-15+ IL-15RaSu superagonin secretion.
As can be seen from FIGS. 3 and 4, CART-CLDN18.2-IL-15/Ra, CART-CLDN 18.2-antaLAG 3, CART-CLDN18.2-15&LAG3 can normally secrete proteins and have substantially the same protein secretion efficiency.
As shown in FIG. 5, the obtained CAR-T proliferated by packaging a lentivirus-derived CAR-T, which had a higher cell proliferation factor than CART-CLDN18.2-IL-15/Ra, CART-CLDN18.2-anti LAG3 and CART-CLDN18.2, demonstrated a more excellent cell proliferation ability with respect to CART-CLDN18.2-15 and LAG 3.
The positive rate and phenotype results of CAR-T prepared by lentivirus infection are shown in Table 1, and FIG. 6, FIG. 7, FIG. 8, FIG. 9, FIG. 10 and FIG. 11.
TABLE 1 CAR-T cell positivity and phenotypic flow assay results
The results in Table 1 show that CAR-T positive cells (positive rate greater than 50%) could be efficiently produced by the lentivirus infection method and that there was no significant difference between the phenotypes of the CART-CLDN18.2, CART-CLDN18.2-IL-15/Ra, CART-CLDN 18.2-antisense LAG3 and CART-CLDN18.2-15 and LAG3 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 LAG3 cell phenotype; FIG. 10 is a flow chart of the CAR-T CLDN18.2-15&LAG3 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 LAG3 expression, and the "ten" word line is positive above and below relative to each other.
In FIGS. 6 to 11, negative regions were divided with NC as a control ("lower left proportion in cross quadrant), and LAG3 expression levels of both CART-CLDN18.2-anti LAG3 and CART-CLDN18.2-15 and LAG3 cells secreting anti-LAG3 antibody and IL-15 and LAG3 fusion protein (" upper right proportion in cross quadrant), were significantly lower than those of T cells, CART-CLDN18.2 and CART-CLDN18.2-IL-15/Ra, demonstrating that IL-15 and LAG3 fusion protein can effectively inhibit LAG3 expression on the surface of CAR-T cells.
2.3 cell in vitro killing experiment
The in vitro tumor killing function of 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 tests showed that, in contrast, the fusion protein-secreting CART-CLDN18.2-15&LAG3 had 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&LAG3 secreted CAR-T could greatly prolong the survival of mice.
The above examples demonstrate that: CAR-T secreting IL-15 and anti-LAG3 fusion protein has stronger proliferation capacity and in-vitro tumor killing activity on tumors compared with CAR-T secreting no 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-LAG3, G4S 4 Linker, IL-15N72D, G4S 4 Linker and IL-15RaSu, wherein the fusion protein is constructed by sequentially connecting anti-LAG3, G4S 4 Linker, IL-15N72D, G4S 4 Linker and IL-15RaSu in series, and the autocrine IL-15 is firstly combined with IL-15RaSu to form superagonin and then combined with anti-LAG3 to obtain the fusion protein.
2. The fusion protein of claim 1, wherein the anti-LAG3 is anti-LAG3 VL, anti-LAG3 VH, or any one of anti-LAG3 VL and anti-LAG3 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 the expression cassette of claim 3, or 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 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 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 being split.
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 of claim 1 or 2, or comprising the expression cassette of claim 3, or comprising the vector of claim 4, or comprising the immune cell of 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20190365861A1 (en) * | 2018-04-18 | 2019-12-05 | Xencor, Inc. | Il-15/il-15ra heterodimeric fc fusion proteins and uses thereof |
| CN110894241A (en) * | 2018-09-13 | 2020-03-20 | 中国科学院生物物理研究所 | Fusion protein for inducing differentiation of target antigen specific T cells to memory stem cells |
| 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 |
| CN113195534A (en) * | 2018-04-18 | 2021-07-30 | Xencor股份有限公司 | LAG-3 targeting heterodimeric fusion proteins comprising an IL-15/IL-15RA FC fusion protein and a LAG-3 antigen binding domain |
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| CN114525260A (en) * | 2022-02-24 | 2022-05-24 | 深圳市先康达生命科学有限公司 | Immune cell for simultaneously expressing fusion protein and chimeric antigen receptor and application thereof |
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2022
- 2022-08-03 CN CN202210928582.4A patent/CN115806627A/en not_active Withdrawn
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2023
- 2023-07-18 CN CN202310885980.7A patent/CN116891535A/en active Pending
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| US20190365861A1 (en) * | 2018-04-18 | 2019-12-05 | Xencor, Inc. | Il-15/il-15ra heterodimeric fc fusion proteins and uses thereof |
| CN113195534A (en) * | 2018-04-18 | 2021-07-30 | Xencor股份有限公司 | LAG-3 targeting heterodimeric fusion proteins comprising an IL-15/IL-15RA FC fusion protein and a LAG-3 antigen binding domain |
| CN110894241A (en) * | 2018-09-13 | 2020-03-20 | 中国科学院生物物理研究所 | Fusion protein for inducing differentiation of target antigen specific T cells to memory stem cells |
| CN113913379A (en) * | 2020-07-07 | 2022-01-11 | 深圳市菲鹏生物治疗股份有限公司 | T lymphocyte and application thereof |
| WO2022046755A1 (en) * | 2020-08-28 | 2022-03-03 | Innovative Cellular Therapeutics Holdings, Ltd. | Fusion protein enhancing cell therapy |
| 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 |
| CN114525260A (en) * | 2022-02-24 | 2022-05-24 | 深圳市先康达生命科学有限公司 | Immune cell for simultaneously expressing fusion protein and chimeric antigen receptor and application thereof |
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| CN116891535A (en) | 2023-10-17 |
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