CN116535518A - Fusion protein combining IL-15 and DnTRII and application thereof - Google Patents
Fusion protein combining IL-15 and DnTRII and application thereof Download PDFInfo
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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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a fusion protein combining IL-15 and DnTRII and application thereof, wherein the fusion protein comprises cytokines IL-15N72D, G S4 Linker, IL-15RaSu, G4S 4 Linker and DnTRII, the fusion protein is expressed in series according to the sequence of IL-15N72D, G S4 Linker, IL-15RaSu, G4S 4 Linker and DnTRII, and the self-secretion 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, 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, in particular to a fusion protein combining IL-15 and DnTRII 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.
The DnTRII (also known as TGF-. Beta.) signal plays a role in tumor suppression or tumor promotion in a cell and background dependent manner. The tumor suppressive effect of TGF- β signaling derives from its ability to induce the expression of a number of genes involved in inhibiting cell proliferation, inducing apoptosis, activating autophagy, inhibiting growth factor signaling through stromal fibroblasts, inhibiting inflammation, and inhibiting angiogenesis. These effects maintain dynamic balance in normal tissues, preventing early stages of tumor formation. Cancer cells progressively tolerate the inhibition of TGF- β signaling when mutations or epigenetic modifications are introduced during the course of cancer progression. The tumor suppressor arm of the TGF- β signaling pathway is lost and cancer cells utilize this pathway to specifically promote processes that support tumor progression, including cell proliferation, immunosuppression, angiogenesis, cancer stem cell self-renewal, epithelial mesenchymal transition, and metastasis. Understanding the regulatory mechanisms of tumor suppression or tumor promotion of TGF- β signaling may have therapeutic potential, hopefully inhibiting the development of several different types of human cancers.
Transforming growth factor (TGF-beta) is a multifunctional cytokine that acts in a cell-or background-dependent manner to inhibit or promote tumors. TGF-beta signals through a heterotetrameric receptor complex consisting of two type I and two type II transmembrane serine/threonine kinase receptors. Following lipid binding, type II receptors (TGF- βrii) phosphorylate type I receptors (TGF- βri), resulting in recruitment and phosphorylation of Smad2 and Smad3 in most cell types, and Smad1 and Smad5 in some cells that rely on expression of type I receptors. Activated Smad proteins associate with Smad4 and translocate to the nucleus where they recruit more transcriptional regulatory factors, including DNA-binding transcription factors, coactivators, cosuppression factors, and chromosomal remodeling factors, which control the expression of many target genes. Their differential expression may result in a cell-specific response to TGF-beta.
Although studies suggest that many of the tumor-inhibiting and tumor-promoting effects of TGF- β are directly dependent on Smad signaling, TGF- β also activates many other signaling pathways, including Ras/MAPK, par6, rhoA/ROCK1, PI 3-K/Akt, p38, and JNK, which may promote the cancer-related effects of TGF- β signaling in a Smad-dependent or independent manner. Activation of these signaling pathways is cell type specific and context dependent.
Early studies showed that TGF- β could be a potent inhibitor of T cell proliferation. Several mechanisms drive TGF- β mediated T cell proliferation inhibition, including inhibition of IL-2 production, down-regulation of c-myc, and up-regulation of cyclin-dependent kinase inhibitors. However, in some cases TGF- β also plays an important role in promoting cell death to limit T cell expansion after activation. Also, in some cases, TGF- β has been shown to promote survival of activated T cells. Thus, under varying circumstances, TGF- β may inhibit or enhance T cell proliferation, survival and subsequent accumulation at specific tissue sites, and should not be expressed to an excessive or insufficient extent, whereas expression of a partially nonfunctional TGF- β receptor at the surface of a T cell may have the effect of artificially lowering the concentration of TGF- β in the environment.
Disclosure of Invention
Based on the above problems, the present invention is to provide a fusion protein of IL-15 and DnTRII (i.e. intracellular truncated type non-functional TGF-beta receptor), wherein IL-15 and IL-15RaSu are combined into a super-excited fusion protein, the super-excited fusion protein is combined with DnTRII to obtain a fusion protein, and immune cells containing the fusion protein successfully express the fusion protein and express chimeric antigen receptor at the same time, so as to achieve the effects of enhancing immune cell proliferation capacity, anti-apoptosis capacity and killing capacity on tumors.
The technical scheme of the invention is as follows:
a fusion protein of IL-15 and DnTRII, which is edited by genes, can improve the activity of immune cells expressing chimeric antigen receptor and the killing effect on tumor.
The fusion protein of IL-15 and DnTRII contains cytokines IL-15N 72N D, G S4 Linker, IL-15RaSu, G4S 4 Linke and DnTRII, and is expressed in series in the sequence of the cytokines IL-15N72D, G S4 Linker, IL-15RaSu, G4S 4 Linke and DnTRII, and the immune cells receiving the gene edits express the fusion protein and are affected by the membrane expression fusion protein.
Immune cells receiving the gene editing, a fusion protein of IL-15 and IL-15RaSu super-agonistic protein and DnTRII were combined and chimeric antigen receptors such as T cells (Chimeric antigen receptor CAR-T) expressing the fusion protein were successfully obtained.
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 protein of membrane-expressed IL-15 and DnTRII 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, and the like.
The invention also provides an application of the immune cells in preparing biological agents for preventing and/or treating cancers or tumors, for example, an application of the biological agents, in particular 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.
Compared with the prior art, the invention has the following beneficial effects:
the immune cells of the membrane expression IL-15 and DnTRII fusion protein provided by the invention express chimeric antigen receptor and can specifically recognize the targeted tumor cell surface antigen; the immune cells combine the IL-15 and IL-15RaSu super-excited protein and DnTRII fusion protein, and enable the immune cells to successfully express the fusion protein through a membrane so as to achieve the purposes 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# -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;
FIGS. 3 and 4 are diagrams of secretory expression of fusion proteins; wherein, FIG. 3 is a bar graph corresponding to the membrane expression DnTRII; FIG. 4 is a bar graph corresponding to IL-15+IL-15RaSu super-agonistic protein secretion;
FIG. 5 is a graph of the amplification growth of each CAR-T;
FIGS. 6, 7, 8, 9 are CAR-T cell surface DnTRII expression flow data; wherein, FIG. 6 is a control T cell phenotype flow chart; FIG. 7 is a phenotypic flow chart of CAR-T CLDN18.2-DnTRII cells; FIG. 8 is a flow chart of the phenotype of CAR-T CLDN18.2-15& DnTRII cells; FIG. 9 is a chart of NC (blank control) phenotype flow; the APC pathway on the abscissa in the figure indicates CD3 expression, positive on the right versus the left; the ordinate PE channel in the graph shows the DnTRII expression condition, and the upper part and the lower part of the cross line are positive;
FIGS. 10 and 11 are graphs showing in vitro tumor killing function evaluation of CAR-T cells; wherein, FIG. 10 is a graph showing the evaluation of the in vitro tumor killing function of HGC-27 target cells by corresponding cells; FIG. 11 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 is specific killing efficiency (%) or killing efficiency (%);
FIG. 12 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 immune cell of membrane expression IL-15 and DnTRII fusion protein, which comprises immune cell membrane expression IL-15 and DnTRII fusion protein edited by genes, wherein the immune cell combines super-excited proteins of IL-15 and IL-15RaSu and DnTRII to obtain fusion protein, the fusion protein can improve the activity of the immune cell and the killing effect on tumors, and the immune cell expresses chimeric antigen receptor.
The fusion protein of the membrane expression IL-15 and DnTRII is sequentially expressed in series according to the sequence of IL-15N72D, G4S 4 Linker, IL-15RaSu, G4S 4 Linker and DnTRII, and the immune cells receiving gene editing express the fusion protein and receive the influence of the membrane expression fusion protein.
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 may be written as IL-15, or IL-15RaSu may be written as IL-15/Ra.
Immune cells do not express the fusion proteins, but in order for immune cell membranes to express the fusion proteins, cells used in tumor therapy, such as CAR-T, CAR-NK, TCR-T, IPS, etc., are subjected to corresponding gene editing, and then the membrane expression fusion proteins are subjected to corresponding gene editing, which are collectively referred to herein as genetically edited immune cells.
The immune cell of the membrane expression IL-15 and DnTRII fusion protein combines the IL-15 and IL-15RaSu super-excited protein and the DnTRII fusion protein, and successfully obtains chimeric antigen receptor of the membrane expression fusion protein, such as T cell (Chimeric antigen receptor CAR-T).
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; target CLDN18.2 is preferred.
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.
In the immune cells, the fusion of the fusion protein and the chimeric antigen receptor gene is realized by constructing expression frames, and the number of the constructed expression frames can be one or more. The vector delivery mode when the expression frame is constructed is slow virus, retrovirus, common plasmid, episome, nanometer delivery system, electric transduction or transposon. That is, vectors in which the genes of immune cells are transferred into chimeric antigen receptors include lentiviruses, retroviruses, common plasmids, episomes, nanodelivery systems, electrotransduction, transposons, or other delivery systems.
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; when the chimeric antigen receptor and the fusion protein are in the same distinct expression cassette, the chimeric antigen receptor and the fusion protein are each expressed or delivered independently, without segmentation.
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 expressing the IL-15 and DnTRII fusion protein can be prepared into biological agents which are pharmaceutically acceptable carriers, diluents or excipients.
A biological agent, which comprises an expression cassette, a recombinant vector, a recombinant microorganism or a recombinant cell line, etc. constructed by a nucleic acid sequence or an amino acid sequence encoding a 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 immune cells of the membrane expression IL-15 and DnTRII fusion protein provided by the invention express chimeric antigen receptor and can specifically recognize idiotypes of anti-autoantibodies; the immune cells combine the super-excited protein of IL-15 and IL-15RaSu (also written as IL 15/Ra) and the fusion protein of DnTRII, and enable the immune cells to successfully express the fusion protein through a membrane so as to achieve the effects of enhancing the proliferation capacity, the anti-apoptosis capacity and the killing capacity to tumors of the immune cells; in addition, the immune cells of the invention can specifically kill the membrane to express the fusion protein of IL-15 and DnTRII, and the invention has the advantages of 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 the preparation of CAR-T by T cells in peripheral blood and the membrane expression of IL-15 (also referred to as IL 15) and DnTRII fusion proteins (also referred to as 15& DnTRII), and the preparation method and functional verification of immune cells; the method comprises the following steps:
1. structural design of fusion protein;
2. constructing a membrane expression type CAR-T cell and performing an in vitro function test;
3. membrane-expressed fusion protein CAR-T cell in vivo functional assay.
The specific operation flow of each step is as follows.
1. Structural design of fusion proteins
In this example, the target for the chimeric antigen receptor was chosen to be CLDN18.2.
According to the sequences of IL15, IL15RaSu and DnTRII, according to the structure diagram of IL-15 and DnTRII fusion proteins in A# -D# shown in figure 1, and designing the fusion protein structures into CAR-T-CLDN18.2 cells respectively; wherein, 1# is a control CAR-T immune cell structure diagram, eGFP control, B # is IL15 super-excited protein control, C # is DnTRII control, D # is fusion protein, and 2# to 4# are membrane expression type CAR-T immune cell structure diagrams after IL-15 and DnTRII fusion protein; . Wherein:
the amino acid sequence of IL15 is:
METDTLLLWVLLLWVPGSTGNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS。
the amino acid sequence of IL15RaSu is:
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR。
the DnTRII amino acid sequence is:
MMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVTGISLLPPLGVAISVIIIFYCYRVNRQQKLSSTWETGKTRKLMEFSEHCAIILEDDRSDISSTCANNINHNTELLPIELDTLVGKGRFAEVYKAKLKQNTSEQ。
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 Membrane-expressed CAR-T cells and in vitro functional assays
Constructing membrane-expressed CAR-T cells and performing an in vitro functional assay comprising the steps of:
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 pMD 2. 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 located on the left side of vertical line), and the expression of CLDN18.2 in HGC-27-CLDN18.2 is positive (peak graph is located on the 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;
t cell culture was carried out using TexMACS GMP Medium (Miltenyi Biotec, 170-076-309) medium containing 10% FBS, 2mM L-glutamine and 100IU/ml rhIL2, and the cells were cultured at 37℃and 5% CO 2 Culturing in a constant temperature incubator.
And (2) expressing and purifying the fusion protein in the sequence B# -D# in the fusion protein structure designed in the 1 st item through a CHO fusion protein expression system, then using the fusion protein as ELISA, detecting positive control standard substances of the fusion protein in the sequence 1# -4#, respectively collecting CAR-T cells and culture supernatants, carrying out lysis treatment on the cells to collect cell membrane lysates, detecting the DnTRII expression level by using an ELISA method, wherein the result is shown in figure 3, and detecting IL-15 super-excited protein in the culture supernatant, and the result is shown in figure 4.
FIGS. 3 and 4 are diagrams of secretory expression of fusion proteins; wherein, FIG. 3 is a bar graph corresponding to the membrane expression DnTRII; FIG. 4 is a bar graph of the response of secretor IL15/Ra (i.e., secretion of IL-15+IL-15RaSu super-agonistic protein).
As can be seen from FIGS. 3 and 4, the cell membrane lysates of CART-CLDN18.2-DnTRII and CART-CLDN18.2-15& DnTRII cells can detect DnTRII expression, and CART-CLDN18.2-IL15/Ra can normally express IL15 super-agonistic protein, and the supernatant is not detected because CART-CLDN18.2-15& DnTRII is an immune cell membrane expression protein.
As shown in fig. 5, the proliferation graph of the CAR-T cells prepared by lentiviral packaging, and fig. 5 shows that CART-CLDN18.2-15& dntri has higher cell proliferation multiple than CART-CLDN18.2-IL15/Ra, CART-CLDN18.2-dntri and CART-CLDN18.2, proving that it has more excellent cell proliferation capability.
The positive rate and the phenotype result of the CAR-T cells prepared by lentivirus infection are shown in Table 1, and FIGS. 6, 7, 8 and 9.
TABLE 1 CAR-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, and the phenotypes of CART-CLDN18.2, CART-CLDN18.2-IL15/Ra, CART-CLDN18.2-DnTRII and CART-CLDN18.2-15& DnTRII have no obvious difference.
FIGS. 6, 7, 8, 9 are CAR-T cell surface DnTRII expression flow data, respectively; wherein, FIG. 6 is a control T cell phenotype flow chart; FIG. 7 is a phenotypic flow chart of CAR-T CLDN18.2-DnTRII cells; FIG. 8 is a flow chart of the phenotype of CAR-T CLDN18.2-15& DnTRII cells; FIG. 9 is a chart of NC (blank control) phenotype flow; the APC pathway on the abscissa in the figure indicates CD3 expression, positive on the right versus the left; the ordinate PE channel in the graph shows that DnTRII is expressed, and the upper part and the lower part of the cross line are positive.
In FIGS. 6 to 9, the NC was used as a control to divide the negative area (the ratio of the lower left part of the cross-shaped quadrant), and the expression levels of the CART-CLDN18.2-DnTRII and CART-CLDN18.2-15 cells of the membrane expression DnTRII and IL15& DnTRII fusion proteins (the ratio of the upper right part of the cross-shaped quadrant, 39.90% and 42.40% respectively) were significantly higher than the expression level of the CART-CLDN18.2 cells (0.5%), which demonstrated that the expression levels of the DnTRII protein and IL15& DnTRII fusion proteins were normally expressed on the surfaces of CAR-T cells.
2.3 in vitro cell killing experiments
FIGS. 10 and 11 are graphs showing in vitro tumor killing function evaluation of CAR-T cells; wherein, FIG. 10 is a graph showing the evaluation of the in vitro tumor killing function of HGC-27 target cells by corresponding cells; FIG. 11 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 is 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 10 and 11, and in contrast, the CART-CLDN18.2-15& DnTRII of the membrane expression 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 the results of FIG. 12, CART-CLDN18.2-15& DnTRII membrane-expressed CAR-T can greatly prolong the survival of mice.
The above examples demonstrate that: the CAR-T with the membrane expressing the fusion protein of IL-15 and dntri has stronger proliferation capacity and in vitro and in vivo tumoricidal activity on tumors than CAR-T with no other cytokines or CAR-T with only 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 (12)
1. A fusion protein of IL-15 and dntri, wherein the fusion protein comprises cytokines IL-15N72D, G S4 Linker, IL-15RaSu, G4S 4 Linke and dntri, and the fusion protein is expressed in tandem in the order IL-15N72D, G S4 Linker, IL-15RaSu, G4S 4 Linke and dntri, and the autocrine IL-15 and IL-15RaSu bind as a super-agonistic protein.
2. An expression cassette comprising the nucleic acid sequence encoding a fusion protein of claim 1.
3. A vector comprising the fusion protein of claim 1 or the expression cassette of claim 2.
4. A recombinant microorganism comprising the fusion protein of claim 1, or comprising the expression cassette of claim 2, or comprising the vector of claim 3.
5. An immune cell comprising the fusion protein of claim 1, or comprising the expression cassette of claim 2, or comprising the vector of claim 3.
6. The immune cell of claim 5, wherein the immune cell is formed by fusion of a fusion protein with a chimeric antigen receptor gene.
7. The immune cell of claim 6, wherein fusion of the fusion protein to a chimeric antigen receptor gene is achieved by constructing an expression cassette from the vector; wherein, 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; or alternatively
When the chimeric antigen receptor and the fusion protein are in the same distinct expression cassette, the chimeric antigen receptor and the fusion protein are each expressed or delivered independently, without segmentation.
8. The immune cell of claim 6 or 7, wherein when the chimeric antigen receptor is targeted to a target, the binding region of the chimeric antigen receptor to the target is scFv, fab, or a combination of scFv and Fab.
9. The immune cell of claim 6 or 7, wherein 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.
10. The immune cell of claim 9, wherein the chimeric antigen receptor-targeting target is CLDN18.2.
11. A biological agent comprising the fusion protein of claim 1, or comprising the expression cassette of claim 2, or comprising the vector of claim 3, or comprising the immune cell of any one of claims 5 to 9.
12. Use of a biological agent according to claim 11 in a medicament for the treatment and/or therapy of cancer or tumour.
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