CN116804063A - Fusion protein combining autocrine IL-15 and anti-TIGIT and application thereof - Google Patents
Fusion protein combining autocrine IL-15 and anti-TIGIT and application thereof Download PDFInfo
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- CN116804063A CN116804063A CN202310887318.5A CN202310887318A CN116804063A CN 116804063 A CN116804063 A CN 116804063A CN 202310887318 A CN202310887318 A CN 202310887318A CN 116804063 A CN116804063 A CN 116804063A
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- fusion protein
- tigit
- cells
- chimeric antigen
- antigen receptor
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Classifications
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
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Abstract
The invention discloses a fusion protein combining autocrine IL-15 and anti-TIGIT and application thereof, wherein the fusion protein is constructed by sequentially and serially connecting the anti-TIGIT, G4S 4Linker, IL-15N72D, G S4 Linker and IL-15RaSu, and the autocrine IL-15 and IL-15RaSu are combined into super-excited protein. And the fusion protein is successfully expressed by the membrane containing the immune cells so as to achieve the effects of enhancing the proliferation capacity, anti-apoptosis capacity and killing capacity to tumors of the immune cells.
Description
Technical Field
The invention relates to the technical field of immune cell preparation, in particular to a fusion protein combining autocrine IL-15 and anti-TIGIT and application thereof.
Background
Tumor (tumor) refers to a new growth (neogram) of a body formed by local tissue cell proliferation under the action of various tumorigenic factors, because the new growth is often in the form of occupied massive protrusions, also called neoplasms (neoplasms). Among them, malignant tumors are easy to metastasize, easy to recur after treatment and extremely difficult to cure under certain special microenvironments.
IL-15 plays a vital 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, with a molecular weight of 14-15kDa and contains 114 amino acids. IL-15 has two homogeneous types: (1) SSP: a shorter signal peptide consisting of 21 amino acids (SSP, short signal peptide), SSP-type IL-15 is fully translated but not secreted, and thus its range of motion is restricted to the cytoplasm and nucleus, possibly playing an important role in its transcriptional regulation; (2) LSP (label switched path): a longer signal peptide consisting of 48 amino acids (LSP, long signal peptide), LSP-IL-15 is secreted extracellular as an immunomodulator. IL-15 and IL-15Rα are expressed synergistically by antigen presenting cells (monocytes and dendritic cells). IL-15 is widely expressed in a variety of cells, 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 receptor is trans-presentation mode: IL-15 binds to a receptor expressed on antigen presenting cells with high affinity to form IL-15Rα; IL-15Rα presents IL-15 to IL-2/15Rβγ dimers to form a ternary complex. Can activate JAK and STAT model channels, and has the functions of promoting proliferation and activation of target cells, improving IFN-gamma and TNF-alpha secretion levels, 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 Inhibitory 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 ligand is CD155 (high affinity), CD112, CD113.
TIGIT can suppress immune cells in multiple steps of tumor immune circulation, specifically as follows:
step1.tigit can inhibit NK cell effects by preventing the initial death of tumor cells and releasing tumor antigens;
step2.TIGIT inhibits dendritic cell co-stimulatory capacity, resulting in decreased 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;
step3.tigit+ Tregs or PVR stimulated myeloid cells can suppress cd8+ T cell effects or skew the polarization of cd4+ T cells;
step4.tigit can directly inhibit cd8+ T cell effect, or tigit+ Treg can inhibit cd8+ T cells, preventing clearance of cancer cells.
Disclosure of Invention
Based on the above problems, the present invention provides a fusion protein for combining autocrine IL-15 and anti-TIGIT, wherein IL-15 and IL-15RaSu are combined to form super-excited protein, the super-excited protein is combined with anti-TIGIT to obtain fusion protein, and immune cells containing the fusion protein successfully secrete protein and express chimeric antigen receptor at the same time, so as to achieve the effects of enhancing immune cell proliferation capability, anti-apoptosis capability and killing capability on tumors.
The technical scheme of the invention is as follows:
a fusion protein of the combination of autocrine IL-15 and anti-TIGIT is embedded in immune cells after gene editing, and can improve the activity of immune cells expressing chimeric antigen receptor and the killing effect on tumor.
An immune cell comprising an auto-secreting IL-15 and anti-TIGIT fusion protein, a fusion protein of an anti-TIGIT and a super-agonistic protein of IL-15 and IL-15RaSu, and successfully obtained a chimeric antigen receptor, e.g., T cell (Chimeric antigen receptor CAR-T), secreting the fusion protein.
The fusion protein combining the self-secretion IL-15 and the anti-TIGIT contains cytokines of anti-TIGIT, G4S 4Linker, IL-15N72D, G S4 Linker and IL-15RaSu, and is expressed in series according to the sequence of the cytokines of anti-TIGIT, G4S 4Linker, IL-15N72D, G S4 Linker and IL-15RaSu, and the immune cells receiving the gene editing express the fusion protein and receive the influence of the self-secretion fusion protein.
The anti-TIGIT in the fusion protein is an anti-TIGIT VL, an anti-TIGIT VH or a combination of the anti-TIGIT VL and the anti-TIGIT VH.
The fusion protein constructs an expression cassette through a nucleic acid sequence coded by the gene.
In one embodiment, the fusion protein and chimeric antigen receptor genes are achieved by constructing an expression cassette; further, vector delivery means when constructing expression cassettes include lentiviruses, retroviruses, common plasmids, episomes, nanodelivery systems, electrotransduction, or transposons; wherein, in addition, the vector contains 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 a target or targets.
In one embodiment, the binding region of the chimeric antigen receptor and the target can be an scFv, a Fab, or a scFv in combination with a Fab; wherein the scFv region structure can be replaced by one or more of any single-chain antibody, single-chain variable fragment (scFv), fab fragment and the like 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; the preferred 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 region and a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular activation signal CD3Zeta; wherein the scFv is an scFv of an anti-idiotype antibody; the hinge region and the transmembrane domain are CD28, or the CD8hinge region and the 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 of the chimeric antigen receptor and the target may be a bispecific antibody that binds to one target, or may be a bispecific antibody that binds to two targets, or may be a chimeric antigen receptor formed separately across a membrane and recognizing separate targets, respectively.
In one embodiment, the chimeric antigen receptor comprises one or more of the signal peptide CD8SP, the transmembrane domain CD8 ringer, CD8TM, the intracellular activating element 4-1BB and CD3Zeta.
In one embodiment, the split between the chimeric antigen receptor and the fusion proteins of the autocrine IL-15 and anti-TIGIT is a protein cleavage function; wherein the protein cleavage functional element is T2A, P2A, E2A, F A or IRES.
In one embodiment, vectors for gene transfer of immune cells into chimeric antigen receptors include lentiviruses, retroviruses, common plasmids, episomes, nanodelivery systems, electrotransduction, transposons, or other delivery systems.
In one embodiment, the immune cells include 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 agent, which comprises an expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell line constructed by a nucleic acid sequence or an amino acid sequence of the encoding fusion protein, wherein the recombinant cell line is preferably an immune cell.
The invention also provides an application of the immune cells in preparing a medicament for preventing and/or treating cancer or tumor, for example, an application of a biological preparation, in particular a pharmaceutically acceptable carrier, diluent or excipient; the tumor is selected from blood tumor, solid tumor or their combination; the hematological tumor is selected from Acute Myelogenous 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 stomach cancer, stomach cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer or a combination thereof.
Compared with the prior art, the invention has the following beneficial effects:
the self-secretion IL-15 and anti-TIGIT fusion protein provided by the invention can specifically recognize the targeted tumor cell surface antigen through the expression chimeric antigen receptor of immune cells embedded in the fusion protein; the immune cells combine the IL-15 and IL-15RaSu super-excited protein and the anti-TIGIT fusion protein, and enable the immune cells to successfully secrete the fusion protein so as to achieve the effects of enhancing the proliferation capacity, anti-apoptosis capacity and killing capacity on tumors of the immune cells; the invention has the advantages of accurate immune cell killing effect, higher safety, difficult recurrence and improvement of the survival quality of patients.
Drawings
FIG. 1 is a diagram showing the structural design of fusion proteins and amino acid sequences in immune cells; wherein, the fusion protein structure in A# to D#; 1# to 4# are amino acid sequence structural design diagrams;
FIG. 2 shows the results of the phenotypic flow assays of target cells HGC-27-CLDN18.2 and HGC-27, respectively; wherein, HGC-27-CLDN18.2 cells correspond to FITC-CLDN18.2 flow assay, and correspondingly, HGC-27 cells also correspond to FITC-CLDN18.2 flow assay;
FIG. 3 is a bar graph corresponding to IL-15 secretion of super-agonistic protein/TIGIT SCFV;
FIG. 4 is a bar graph corresponding to IL-15+IL-15RaSu super-agonistic protein secretion;
FIG. 5 is a graph of secretory CAR-T amplification growth;
FIG. 6 is a flow chart of T cell phenotypes;
FIG. 7 is a CAR-T CLDN18.2 cell phenotype flow chart;
FIG. 8 is a phenotype flow chart of CAR-T CLDN18.2-il-15/Ra cells;
FIG. 9 is a flow chart of the phenotype of CAR-T CLDN18.2-anti TIGIT cells;
FIG. 10 is a phenotype flow chart of CAR-T CLDN18.2-15& TIGIT cells;
FIG. 11 is a chart of NC (blank control) phenotype flow;
FIG. 12 is a graph of in vitro tumoricidal function evaluation of corresponding cells against HGC-27 target cells;
FIG. 13 is a graph of in vitro tumoricidal function evaluation of corresponding cells against HGC-27-CLDN18.2 target cells;
FIG. 14 is a graph of experimental survival of CAR-T animals.
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 immune cells of the autocrine IL-15 and anti-TIGIT fusion protein edited by genes, wherein the immune cells can secrete the fusion 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 proteins of the autocrine IL-15 and the anti-TIGIT are sequentially expressed in series according to the sequence of the anti-TIGIT, the G4S 4Linker, the IL-15N72D, G S4 Linker and the IL-15RaSu, and the immune cells receiving the gene editing express the fusion proteins and receive the influence of the autocrine fusion proteins. In the present invention, IL-15 may be expressed as hIL-15, and IL-15RaSu may be expressed as hIL-15RaSu, since human interleukin is selected. In the present invention, IL-15 (IL-15 may be written), IL-15RaSu (IL-15/Ra may be written), and anti-TIGIT (TIGIT may be written).
Immune cells do not express the fusion proteins, but in order for immune cells to secrete the fusion proteins, the cells need to be subjected to corresponding gene editing, such as CAR-T, CAR-NK, TCR-T, IPS and the like, which are used for tumor treatment, are subjected to corresponding gene editing, and then the cells secrete the fusion proteins to be subjected to corresponding gene editing, and the cells are collectively called immune cells subjected to gene editing.
The immune cell of the self-secreting IL-15 and anti-TIGIT fusion protein combines the IL-15 and IL-15RaSu super-excited protein and the anti-TIGIT fusion protein, and successfully obtains chimeric antigen receptor, such as T cell (Chimeric antigen receptor CAR-T), secreting the fusion protein.
The anti-TIGIT in the fusion protein is an anti-TIGIT VL, an anti-TIGIT VH or a combination of the anti-TIGIT VL and the anti-TIGIT VH.
In one embodiment, the immune cells express a chimeric antigen receptor, e.g., a CAR cell. Chimeric antigen receptor expression can be chimeric antigen receptor that targets a target or targets, e.g., CAR cells.
In one embodiment, the chimeric antigen receptor can 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 may be scFv, fab or a combination of scFv and Fab; wherein the scFv region structure can be replaced by one or more of any single-chain antibody, single-chain variable fragment (scFv), fab fragment and the like of any target point.
The chimeric antigen receptor comprises a leader sequence, an scFv that recognizes a tumor associated antigen, a hinge region and a transmembrane domain, an intracellular co-stimulatory domain, and an intracellular activation signal CD3Zeta. Wherein the scFv is an scFv of an anti-idiotype antibody; the hinge and transmembrane domains are CD28 or CD8hinge and transmembrane domains; the intracellular co-stimulatory domain is CD28 or CD137 (4-1 BB) or ICOS intracellular co-stimulatory domain.
The binding region of the chimeric antigen receptor and the target may be a bispecific antibody that binds to one target, or to two targets, or may be formed by the respective transmembrane formation of two or more chimeric antigen receptors and recognizing the respective different targets.
In one embodiment, the chimeric antigen receptor comprises one or more of the signal peptide CD8SP, the transmembrane domain CD8 ringer, CD8TM, the intracellular activating element 4-1BB and CD3Zeta.
The division mode between the chimeric antigen receptor and the fusion protein of the autocrine IL-15 and the anti-TIGIT is a protein cleavage functional element; wherein the protein cleavage functional element may be T2A, P2A, E2A, F a or IRES.
Vectors for gene transfer of immune cells into chimeric antigen receptors include lentiviruses, retroviruses, common plasmids, episomes, nanodelivery systems, electrotransduction, transposons, or other delivery systems.
The immune cells of the present 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 autotaxin-15 and anti-TIGIT fusion protein can be prepared into biological agents which are pharmaceutically acceptable carriers, diluents or excipients. The biological agent comprises an expression cassette, a recombinant vector, a recombinant protein, a recombinant microorganism or a recombinant cell line and the like constructed by a nucleic acid sequence or an amino acid sequence of the encoding fusion protein.
A biological agent is provided, which comprises an expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell line constructed by a nucleic acid sequence or an amino acid sequence of a coding fusion protein; the recombinant cell line may be an immune cell, such as a CAR-T cell, CAR-NK, or the like.
Administration of the biologic may be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The biological preparation can be applied to medicines for preventing and/or treating solid tumors, for example, the biological preparation is particularly applied to pharmaceutically acceptable carriers, diluents or excipients; the tumor is selected from blood tumor, solid tumor or their combination; the hematological tumor is selected from Acute Myelogenous 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 stomach cancer, stomach cancer peritoneal metastasis, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, cervical cancer, ovarian cancer, lymph cancer, nasopharyngeal cancer, adrenal tumor, bladder tumor, non-small cell lung cancer (NSCLC), brain glioma, endometrial cancer or a combination thereof.
The self-secretion IL-15 and anti-TIGIT fusion protein provided by the invention is embedded into immune cells of the fusion protein to express chimeric antigen receptor, so that idiotypes of anti-autoantibodies can be specifically identified; the immune cell combines the IL-15 and IL-15RaSu super-excited protein and the anti-TIGIT fusion protein, and enables the immune cell to successfully secrete the fusion protein so as to achieve the effects of enhancing the proliferation capacity, anti-apoptosis capacity and killing capacity on tumors of the immune cell; in addition, the immune cells of the invention can specifically kill and secrete the fusion protein of IL-15 and anti-TIGIT, and have accurate killing effect, higher safety, difficult recurrence and improved survival quality of patients.
The following is a description of specific embodiments.
The following examples illustrate methods for preparing immune cells and verifying functions, taking as an example the preparation of CAR-T by T cells in peripheral blood and secretion of IL-15 and anti-TIGIT fusion protein (which may also be written as 15& TIGIT).
The preparation method of the immune cell specifically comprises the following steps:
1. structural design of fusion protein;
2. constructing a secretory CAR-T cell and performing an in vitro function test;
3. in vivo functional assay of secreted fusion protein type CAR-T cells.
The specific implementation steps are as follows:
1. structural design of fusion proteins
Based on the sequence of IL-15 (IL 15 may be written), IL-15RaSu (IL 15/Ra may be written), anti-TIGIT (TIGIT may be written), the structure of the fusion protein in A# to D# is designed according to the structure diagram of the fusion protein shown in FIG. 1, and the fusion protein is designed into CAR-T-CLDN18.2 cells; wherein #1 is control CAR-T, and # 2-4 is secretory CAR-T; wherein:
the amino acid sequence of IL-15 is:
METDTLLLWVLLLWVPGSTGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS；
the amino acid sequence of IL-15RaSu is:
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR；
the amino acid sequence of the Anti-TIGIT VH is:
EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYHMYWVRQAPGKGLEWVAYISKGGISTYYPDTVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARQSSYDFAMDYWGRGTLVTVSS；
the amino acid sequence of the Anti-TIGIT VL is:
DIQMTQSPSFLSASVGDRVTITCKASQDVGTSVAWYQQKPGKAPKLLIYWASARHTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSSYPLTFGQGTKLEIK；
the Linker amino acid sequence was: GSSSSGSSSSGSSSSGSSSS.
The chimeric antigen receptor comprises one or more of signal peptide CD8SP, transmembrane domain CD8 ringer, CD8TM, intracellular activating element 4-1BB and CD3Zeta, and is shown in figure 1.
2. Construction of secreted CAR-T cells and in vitro functional assays
2.1 cell line culture
Cloning a base sequence for expressing CLDN18.2 into a PHBLV lentiviral vector skeleton, placing under a promoter of EF1 alpha (EF-1 alpha) to form PHBLV-EF1 alpha-CLDN 18.2, and transferring three plasmids, namely PHBLV-EF1 alpha-CLDN 18.2, lentiviral envelope Plasmid pMD2.G (Addgene, plasmid # 12259), lentiviral packaging Plasmid psPAX2 (Addgene Plasmid # 12260) and the like, onto a lentiviral complete expression vector prepared in 293T cells by using Lipofectamine 3000; collecting virus supernatant at 48h and 72h, respectively, and subjecting the collected virus supernatant to ultracentrifugation concentration (Merck Millipore); the concentrated virus can be used for infecting HGC-27, and finally the HGC-27 cell line which over-expresses the CLDN18.2 is obtained and named HGC-27-CLDN18.2.
As shown in FIG. 2, HGC-27-CLDN18.2 cells express the detection result of CLDN 18.2; wherein, the detection graph corresponding to HGC-27 is a comparison graph; from the results of the detection of HGC-27-CLDN18.2 corresponding to HGC-27, it can be seen from FIG. 2 that the result of detecting the expression level of CLDN18.2 antigen in FITC channel shows that the expression of CLDN18.2 in HGC-27 is negative (peak graph is left side of vertical line) and the expression of CLDN18.2 in HGC-27-CLDN18.2 is positive (peak graph is right side of vertical line). Negative is CLDN18.2 not expressed, positive is CLDN18.2 expressed.
2.2 isolation of peripheral blood PBMC and expansion of T cells
Isolation of mononuclear cells from donor peripheral blood, density gradient centrifugation using ficol method, and enrichment of T cells with T cell sorting kit, e.g., CD3 MicroBeads, human-lyophilized or 130-097-043, and activation of 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% FBS, 2mM L-glutamine and 100IU/ml rhIL2 was used, and the cells were cultured in a constant temperature incubator at 37℃and 5% CO 2.
And (3) expressing and purifying the fusion protein in the sequence B# -D# in the structural design of the fusion protein in the 1 st item by using a CHO fusion protein expression system as ELISA, detecting positive control standard substances of protein secretion in the 1# -4 # and collecting CAR-T culture supernatant, and detecting protein secretion in cell culture supernatant, wherein the detection data are shown in figures 3 and 4.
FIGS. 3, 4 are secretory histograms of secreted CAR-T fusion proteins; wherein, FIG. 3 is a bar graph corresponding to IL-15 secretion super-agonistic protein/TIGIT SCFV; FIG. 4 is a bar graph of IL-15+ IL-15RaSu super-agonistic protein secretion.
From FIGS. 3 and 4, it is understood that CART-CLDN18.2-IL-15/Ra, CART-CLDN18.2-antiTIGIT, CART-CLDN18.2-15& TIGIT can normally secrete proteins and have substantially the same protein secretion efficiency.
As shown in figure 5, the CAR-T obtained by lentiviral packaging has higher cell proliferation factor than CART-CLDN18.2-IL-15/Ra, CART-CLDN 18.2-anti-TIGIT and CART-CLDN18.2, and the obtained CART-CLDN18.2 has more excellent cell proliferation capacity.
The positive rate and the phenotypic results of the CAR-T prepared by lentiviral infection are shown in Table 1, FIGS. 6, 7, 8, 9, 10 and 11.
TABLE 1CAR-T cell Positive Rate and phenotypic flow assay results
The results in Table 1 show that the lentivirus infection method can effectively prepare CAR-T positive cells (the positive rate is more than 50%), and the phenotypes of CART-CLDN18.2, CART-CLDN18.2-IL-15/Ra, CART-CLDN 18.2-anti-TIGIT and CART-CLDN18.2-15& TIGIT cells have no obvious difference.
FIGS. 6, 7, 8, 9, 10, 11 are CAR-T cell phenotype flow data, respectively; wherein, FIG. 6 is a T cell phenotype flow diagram; FIG. 7 is a CAR-T CLDN18.2 cell phenotype flow chart; FIG. 8 is a flow chart of the CAR-TCLDN18.2-il-15/Ra cell phenotype; FIG. 9 is a flow chart of the phenotype of CAR-T CLDN18.2-anti TIGIT cells; FIG. 10 is a phenotype flow chart of CAR-T CLDN18.2-15& TIGIT cells; FIG. 11 is a chart of NC (blank control) phenotype flow; in each figure, the FITC channel on the abscissa indicates CD3 expression, the right side is positive relative to the left side, the APC channel on the ordinate indicates TIGIT expression, and the upper side of the "ten" word line is positive relative to the lower side.
In FIGS. 6 to 11, negative areas are divided by NC as a control (the ratio of the lower left part of the cross quadrant), and the TIGIT expression levels of the anti-TIGIT antibody and the CART-CLDN 18.2-anti-TIGIT and the CART-CLDN 18.2-15-TIGIT cells secreted by the anti-TIGIT antibody and the IL-15& TIGIT fusion protein (the ratio of the upper right part of the cross quadrant) are obviously lower than those of the T cells, the CART-CLDN18.2 and the CART-CLDN18.2-IL-15/Ra, so that the IL-15& TIGIT fusion protein can effectively inhibit the TIGIT expression on the surface of the CAR-T cells.
2.3 in vitro cell killing experiments
FIGS. 12 and 13 are graphs showing in vitro tumor killing function evaluation of CAR-T cells, respectively; wherein, FIG. 12 is a graph of in vitro tumoricidal function evaluation of the corresponding cells against HGC-27 target cells; FIG. 13 is a graph of 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 indicates specific killing efficiency (%) or killing efficiency (%).
The in vitro tumoricidal function of CAR-T was verified using a flow assay using HGC-27-CLDN18.2 and HGC-27 cells as positive and negative target cells, respectively. The detection results are shown in figures 12 and 13, and the detection results show that the CART-CLDN18.2-15& TIGIT secreting the fusion protein has the strongest killing effect on HGC-27-CLDN18.2 positive target cells.
3. In vivo functional assessment of CAR-T cells
24 NSG mice (weight 18-22 g) with age of 6-8 weeks are taken, after being adapted to feed for one week, HGC-27-CLDN18.2 positive tumor cell strains are inoculated subcutaneously, and each mouse is inoculated with 5X 10 6 Tumor cells are used for closely observing animal states, the tumor volume of the mice is measured every three days by using a vernier caliper, and when the tumor volume reaches 100mm 3 After random grouping according to mouse body weight and tumor size, CAR-T cells or control T cells were infused via the tail vein. The detailed methods of administration, dosages and routes of administration are shown in Table 2.
Table 2 animal protocol
As shown in fig. 14, CART-CLDN18.2-15& tigit secretory CAR-T can greatly extend the survival of mice.
The above examples demonstrate that: the CAR-T of the self-secreting IL-15 and anti-TIGIT fusion protein has stronger proliferation capacity and in-vitro and in-vivo tumor killing activity on tumors compared with the CAR-T which does not secrete other cytokines or the CAR-T which only secretes one cytokine.
It is to be understood that the foregoing description of the preferred embodiments of the invention is not to be considered as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. A fusion protein combining autocrine IL-15 and anti-TIGIT, which is characterized by comprising cytokines of anti-TIGIT, G4S 4Linker, IL-15N72D, G S4 Linker and IL-15RaSu, wherein the fusion protein is constructed according to the sequence of anti-TIGIT, G4S 4Linker, IL-15N72D, G S4 Linker and IL-15RaSu in series, and the autocrine IL-15 is combined with IL-15RaSu to be super-excited protein and then combined with anti-TIGIT to obtain the fusion protein.
2. The fusion protein of claim 1, wherein the anti-TIGIT is any of an anti-TIGIT VL, an anti-TIGIT VH, or an anti-TIGIT VL and an anti-TIGIT VH.
3. An expression cassette comprising a nucleic acid sequence encoding a fusion protein according to claim 1 or 2.
4. A vector comprising the fusion protein of claim 1 or 2 or 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 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 by gene fusion of a fusion protein and 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, F A or IRES; alternatively, when the chimeric antigen receptor and the fusion protein are in separate expression cassettes, the chimeric antigen receptor and the fusion protein are each expressed or delivered independently, without segmentation.
9. 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 8.
10. Use of a biological agent according to claim 9 for the preparation of a medicament for the prevention, treatment of cancer or tumour.
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| CN202210928587.7A Withdrawn CN115806628A (en) | 2022-08-03 | 2022-08-03 | Autocrine IL-15 and anti-TIGIT combined fusion protein and application thereof |
| 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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| CN118562016A (en) * | 2016-10-21 | 2024-08-30 | 艾尔特生物科技公司 | Multimeric IL-15 based molecules |
| PE20211055A1 (en) * | 2018-10-12 | 2021-06-07 | Xencor Inc | IL-15 / IL-15 RALPHA F C FUSION PROTEINS TARGETING PD-1 AND USES IN COMBINATION THERAPIES OF THE SAME |
| CN111744007B (en) * | 2019-03-29 | 2023-05-12 | 江苏恒瑞医药股份有限公司 | anti-TIGIT antibody pharmaceutical composition and application thereof |
| CN113754778A (en) * | 2020-06-05 | 2021-12-07 | 上海交通大学 | Chimeric antigen receptor targeting CLDN18.2 and uses 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 |
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