CN116041542A - NK cell preparation method for reversing tumor microenvironment inhibitory signals and application - Google Patents
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Abstract
The invention provides a preparation method and application of immune cells capable of reversing tumor microenvironment immunosuppression signals. The method for reversing the inhibitory signals is to replace an intracellular segment of a TIGIT receptor with a 4-1BB co-stimulatory domain and IL-18R and CD3 intracellular segments, and immune cells expressing the chimeric antigen receptor recognize CD155 in a tumor microenvironment through the TIGIT, so that the immune cells are prevented from losing functions or being exhausted, and the immune cells are stimulated to exert stronger tumor killing activity.
Description
Technical Field
The invention belongs to the field of biological medicine, in particular to preparation of CAR-immune cells and application thereof in tumor treatment, and more particularly relates to a chimeric antigen receptor for reversing tumor microenvironment inhibitory signals, an expression vector, transgenic immune cells, a preparation method of a pharmaceutical composition and application thereof.
Background
In recent years, immunotherapy has been remarkably advanced in the field of tumor treatment, particularly immune checkpoint blocking therapies typified by anti-CTLA-4 and anti-PD-1 or PD-L1 antibodies. The method has the advantages that the combination of the T cell surface inhibitory receptor and the ligand thereof is blocked, the transmission of inhibitory signals is blocked, the immunosuppression mediated by the immunosuppression microenvironment is corrected, the anti-tumor capability of the T cells in the tumor microenvironment is recovered, the higher response rate is obtained in the treatment aspect of various metastatic advanced cancers (including metastatic melanoma, non-small cell lung cancer, renal cancer and the like), and a plurality of advanced cancer patients which have lost the opportunity of treatment and are ineffective in radiotherapy and chemotherapy are expected to be treated again.
However, not all malignant patients are therapeutically effective against PD-1, PD-L1 or CTLA-4 blockade, only 10% -30% of patients who are treated with PD-1 or PD-L1 antibodies show long lasting responses, and most people lack responses, improving clinical response and overcoming resistance are the greatest challenges facing this field. The mechanism of unresponsiveness of tumors to immune card control blocking treatment is explored, other immune control card points affecting immune cell functions are found, and the problem to be solved in the tumor immunotherapy field is urgent. Currently, more and more immune checkpoint molecules are discovered and developed for use.
T cell immunoglobulins and ITIM domain proteins (TIGIT) are primarily expressed on Natural Killer (NK) cells, activated cd8+ T and cd4+ T cells, regulatory T cells (Tregs) and follicular helper T cells (Tfh) surfaces as an important immune checkpoint. TIGIT recognizes ligands CD155 and CD112 that are expressed primarily on monocytes, macrophages, dendritic Cells (DCs), T cells, B cells and many non-hematopoietic cells including tumor cells of different histological types. TIGIT binds CD155 with significantly higher affinity than its competing receptors CD226 and CD96.TIGIT, upon binding to its ligand, transmits an inhibitory signal to T cells or NK cells. TIGIT was found to be highly expressed in T cells or NK cells of various malignant tumors (e.g., non-small cell lung cancer, melanoma, head and neck squamous cell carcinoma, colorectal cancer, glioblastoma, gastric cancer, liver cancer, multiple myeloma, acute myeloid leukemia, and follicular lymphoma). TIGIT ligand CD155 is highly expressed in a variety of solid and hematological tumors including liver, pancreatic, colorectal, gastric, lung, ovarian, head and neck, breast, lymphoma, leukemia, and the like. TIGIT and CD155 expression abundance are closely related to patient prognosis. The current treatment method is to develop targeting TIGIT monoclonal antibody to effectively recover the functions of T cells or NK cells so as to play a role in killing tumors. Of these, some biotechnology/pharmaceutical companies (e.g., rogowski, baji shenzhou, gham, etc.) are working on developing antibodies against TIGIT, and related products are in different stages of clinical development. However, clinical test results show that the therapeutic effect of TIGIT antibody alone is not ideal, and the effect can be improved by combining with PD-1 or PD-L1 antibodies, etc., and the combination of anti-TIGIT monoclonal antibodies developed by Roche and Baiji China and PD-L1 antibodies for treating non-small cell lung cancer has already entered phase III clinical test.
Therefore, TIGIT and ligand CD155 thereof are expected to become new targets for tumor immunotherapy, block the transmission of the inhibitory signals of the TIGIT on the surface of immune cells, and have great application prospects in tumor immunotherapy.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent.
In order to improve the killing effect of tumors and reduce side effects caused by external medicines, the invention provides a preparation method of immune cells capable of selectively reversing the immune card control point TIGIT inhibitory signals and application of the immune cells in malignant tumor treatment.
The immune cell preparation method for reversing the immune card control point TIGIT inhibitory signal provided by the invention comprises the steps of constructing a chimeric antigen receptor of a targeting TIGIT ligand, wherein the extracellular section is a TIGIT extracellular section, the transmembrane region is a TIGIT transmembrane region, the intracellular section is a 4-1BB co-stimulatory signal domain, and the intracellular section of IL18RB and the intracellular region of CD3 zeta. The structure can convert inhibitory signals of the ligand CD155 of the TIGIT into co-stimulatory activation signals mediated by 4-1BB, IL-18R and CD3, so that inhibitory signals from tumor microenvironment received by NK cells are converted into activation signals, the tumor killing activity of immune cells is effectively enhanced, and the structure can be applied to the treatment of various malignant tumors with high expression of the TIGIT ligand. The inventor finds that when the intracellular segment is the 4-1BB co-stimulatory signal domain, the IL18RB intracellular segment and the CD3 zeta intracellular region, compared with the intracellular segment which is the 4-1BB co-stimulatory signal domain, the IL21R intracellular segment and the CD3 zeta intracellular region, the activated co-stimulatory signal is stronger, and the NK cell can be stimulated to have stronger killing activity on TIGIT ligand positive cells.
Thus, in a first aspect of the invention, the invention provides a chimeric antigen receptor. According to an embodiment of the invention, the chimeric antigen receptor comprises: an extracellular region comprising a TIGIT extracellular segment; a transmembrane region comprising a TIGIT transmembrane region and embedded into the cell membrane of the cell; an intracellular region comprising a 4-1BB costimulatory factor domain, an IL18RB intracellular segment and a CD3 zeta intracellular signal segment, wherein the C-terminal of the extracellular region is connected with the N-terminal of the transmembrane region, and the C-terminal of the transmembrane region is connected with the N-terminal of the intracellular region. The chimeric antigen receptor described in the examples of the present invention is introduced into immune cells for expression. Wherein, the immunosuppression signal received by the original T cells or NK cells is converted into an activation signal, thereby effectively improving the tumor killing effect of the immune cells.
According to embodiments of the present invention, the chimeric antigen receptor described above may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the C-terminal of the 4-1BB costimulatory factor domain in the chimeric antigen receptor is connected with the N-terminal of the intracellular segment of IL18RB, and the C-terminal of the intracellular segment of IL18RB is connected with the N-terminal of the intracellular signal segment of CD3 zeta. Furthermore, the immune cells expressing the chimeric antigen receptor have stronger immune activation effect.
According to an embodiment of the invention, the extracellular region is capable of binding a ligand comprising at least one of the PVR family members.
According to an embodiment of the invention, the PVR family members include CD155 and CD112.
According to an embodiment of the invention, the PVR family member is CD155.
According to an embodiment of the invention, the TIGIT extracellular segment has an amino acid sequence as shown in SEQ ID No. 1.
According to an embodiment of the invention, the TIGIT transmembrane region has the amino acid sequence shown in SEQ ID No. 2.
According to an embodiment of the invention, the 4-1BB co-stimulatory factor domain has an amino acid sequence as shown in SEQ ID NO. 3.
According to an embodiment of the invention, the IL18RB intracellular segment has the amino acid sequence shown in SEQ ID NO. 4.
According to an embodiment of the invention, the intracellular signal segment of CD3 zeta has the amino acid sequence shown in SEQ ID NO. 5.
According to an embodiment of the invention, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO. 6.
MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQV NWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGT YTGRIFLEVLESSVAEHGARFQIPL(SEQ ID NO.1)
LGAMAATLVVICTAVIVVVALTR(SEQ ID NO.2)
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO.3)
SALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEW(SEQ ID NO.4)
RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.5)
MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSALLYRHWIEIVLLYRTYQSKDQTLGDKKDFDAFVSYAKWSSFPSEATSSLSEEHLALSLFPDVLENKYGYSLCLLERDVAPGGVYAEDIVSIIKRSRRGIFILSPNYVNGPSIFELQAAVNLALDDQTLKLILIKFCYFQEPESLPHLVKKALRVLPTVTWRGLKSVPPNSRFWAKMRYHMPVKNSQGFTWNQLRITSRIFQWKGLSRTETTGRSSQPKEWRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO.6)
In a second aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the invention, the nucleic acid molecule encodes the chimeric antigen receptor according to the first aspect of the invention. The nucleic acid molecules according to embodiments of the present invention are expressed in immune cells and convert tumor cell-mediated inhibition signals into activation signals.
According to an embodiment of the present invention, the above-mentioned nucleic acid molecule may further comprise at least one of the following additional technical features:
according to an embodiment of the invention, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO. 7.
ATGCGCTGGTGTCTCCTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATTTCTGCAGAGAAAGGTGGCTCTATCATCTTACAATGTCACCTCTCCTCCACCACGGCACAAGTGACCCAGGTCAACTGGGAGCAGCAGGACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGGTGGCACATCTCCCCATCCTTCAAGGATCGAGTGGCCCCAGGTCCCGGCCTGGGCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTACTTCTGCATCTATCACACCTACCCTGATGGGACGTACACTGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACGGTGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCGCGACGCTGGTGGTCATCTGCACAGCAGTCATCGTGGTGGTCGCGTTGACTAGAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGTGCCCTCCTCTACAGGCACTGGATTGAAATAGTGCTGCTGTACCGGACCTACCAGAGCAAGGATCAGACGCTTGGGGATAAAAAGGATTTTGATGCTTTCGTATCCTATGCAAAATGGAGCTCTTTTCCAAGTGAGGCCACTTCATCTCTGAGTGAAGAACACTTGGCCCTGAGCCTATTTCCTGATGTTTTAGAAAACAAATATGGATATAGCCTGTGTTTGCTTGAAAGAGATGTGGCTCCAGGAGGAGTGTATGCAGAAGACATTGTGAGCATTATTAAGAGAAGCAGAAGAGGAATATTTATCTTGAGCCCCAACTATGTCAATGGACCCAGTATCTTTGAACTACAAGCAGCAGTGAATCTTGCCTTGGATGATCAAACACTGAAACTCATTTTAATTAAGTTCTGTTACTTCCAAGAGCCAGAGTCTCTACCTCATCTCGTGAAAAAAGCTCTCAGGGTTTTGCCCACAGTTACTTGGAGAGGCTTAAAATCAGTTCCTCCCAATTCTAGGTTCTGGGCCAAAATGCGCTACCACATGCCTGTGAAAAACTCTCAGGGATTCACGTGGAACCAGCTCAGAATTACCTCTAGGATTTTTCAGTGGAAAGGACTCAGTAGAACAGAAACCACTGGGAGGAGCTCCCAGCCTAAGGAATGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO:7)
In a third aspect of the invention, the invention provides an expression vector. According to an embodiment of the invention, the expression vector carries a nucleic acid molecule according to the second aspect of the invention. Wherein the expression vector is constructed for the purpose of expressing the gene sequence of interest.
According to an embodiment of the present invention, the above-mentioned expression vector may further include at least one of the following additional technical features:
according to an embodiment of the invention, the expression vector further comprises a promoter.
According to an embodiment of the invention, the promoter is operably linked to a nucleic acid molecule according to the second aspect of the invention.
According to an embodiment of the invention, the promoter is selected from at least one of CMV, EF-1, RSV.
According to an embodiment of the invention, the expression vector is a non-pathogenic viral vector.
According to an embodiment of the invention, the non-pathogenic virus is selected from the group consisting of retrovirus, lentivirus and adenovirus-associated virus, preferably the non-pathogenic virus is a lentivirus.
In a fourth aspect of the invention, the invention provides a lentiviral vector. According to an embodiment of the invention, the lentiviral vector has the sequence of SEQ ID NO:8, and a nucleotide sequence shown in SEQ ID NO. Wherein, after introducing the lentiviral vector into the recipient cell, expression of the helper activation signal in the immune cell is possible.
GGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACTCTAGAATGCGCTGGTGTCTCCTCCTGATCTGGGCCCAGGGGCTGAGGCAGGCTCCCCTCGCCTCAGGAATGATGACAGGCACAATAGAAACAACGGGGAACATTTCTGCAGAGAAAGGTGGCTCTATCATCTTACAATGTCACCTCTCCTCCACCACGGCACAAGTGACCCAGGTCAACTGGGAGCAGCAGGACCAGCTTCTGGCCATTTGTAATGCTGACTTGGGGTGGCACATCTCCCCATCCTTCAAGGATCGAGTGGCCCCAGGTCCCGGCCTGGGCCTCACCCTCCAGTCGCTGACCGTGAACGATACAGGGGAGTACTTCTGCATCTATCACACCTACCCTGATGGGACGTACACTGGGAGAATCTTCCTGGAGGTCCTAGAAAGCTCAGTGGCTGAGCACGGTGCCAGGTTCCAGATTCCATTGCTTGGAGCCATGGCCGCGACGCTGGTGGTCATCTGCACAGCAGTCATCGTGGTGGTCGCGTTGACTAGAAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGTGCCCTCCTCTACAGGCACTGGATTGAAATAGTGCTGCTGTACCGGACCTACCAGAGCAAGGATCAGACGCTTGGGGATAAAAAGGATTTTGATGCTTTCGTATCCTATGCAAAATGGAGCTCTTTTCCAAGTGAGGCCACTTCATCTCTGAGTGAAGAACACTTGGCCCTGAGCCTATTTCCTGATGTTTTAGAAAACAAATATGGATATAGCCTGTGTTTGCTTGAAAGAGATGTGGCTCCAGGAGGAGTGTATGCAGAAGACATTGTGAGCATTATTAAGAGAAGCAGAAGAGGAATATTTATCTTGAGCCCCAACTATGTCAATGGACCCAGTATCTTTGAACTACAAGCAGCAGTGAATCTTGCCTTGGATGATCAAACACTGAAACTCATTTTAATTAAGTTCTGTTACTTCCAAGAGCCAGAGTCTCTACCTCATCTCGTGAAAAAAGCTCTCAGGGTTTTGCCCACAGTTACTTGGAGAGGCTTAAAATCAGTTCCTCCCAATTCTAGGTTCTGGGCCAAAATGCGCTACCACATGCCTGTGAAAAACTCTCAGGGATTCACGTGGAACCAGCTCAGAATTACCTCTAGGATTTTTCAGTGGAAAGGACTCAGTAGAACAGAAACCACTGGGAGGAGCTCCCAGCCTAAGGAATGGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC(SEQ ID NO：8)
In a fifth aspect of the invention, the invention provides a transgenic immune cell. According to an embodiment of the invention, the transgenic immune cell carries the chimeric antigen receptor according to the first aspect of the invention, the nucleic acid molecule according to the second aspect of the invention, the expression vector according to the third aspect of the invention and the lentiviral vector according to the fourth aspect of the invention. Wherein, the expression of the obtained transgenic immune cells can effectively enhance the killing capacity to malignant tumors.
In a sixth aspect of the invention, the invention provides a CAR-immune cell. According to an embodiment of the invention, the CAR-immune cell carries the chimeric antigen receptor according to the first aspect of the invention, the nucleic acid molecule according to the second aspect of the invention, the expression vector according to the third aspect of the invention and the lentiviral vector according to the fourth aspect of the invention.
According to an embodiment of the invention, the CAR-immune cells comprise at least one selected from NK-92 cells, peripheral blood NK cells, umbilical cord blood NK cells, ipscs, CAR-NK cells, CAR-T cells, CAR-NKT cells, CAR- γδ T cells.
In a seventh aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the chimeric antigen receptor according to the first aspect of the invention, the nucleic acid molecule according to the second aspect of the invention, the expression vector according to the third aspect of the invention, the lentiviral vector according to the fourth aspect of the invention, the transgenic immune cell according to the fifth aspect of the invention and the CAR-immune cell according to the sixth aspect of the invention.
According to an embodiment of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable auxiliary materials.
In an eighth aspect of the invention, the invention provides the use of a pharmaceutical composition for the manufacture of a medicament. According to embodiments of the invention, the chimeric antigen receptor, nucleic acid molecule, expression vector, lentiviral vector, transgenic immune cell, CAR-immune cell, and pharmaceutical composition are used for treating or preventing solid tumor or hematological tumor.
According to an embodiment of the present invention, the solid tumor includes at least one selected from the group consisting of tangible tumors occurring in organs, including pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, esophageal cancer, colorectal cancer, lung cancer, head and neck cancer, cervical cancer, glioma, renal cancer, breast cancer, prostate cancer, melanoma, and the like.
According to an embodiment of the invention, the hematological neoplasm comprises at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, multiple myeloma, etc. within the blood cell and hematopoietic system.
Drawings
FIG. 1 is a schematic diagram of a CD 155-targeting CAR activation signal structure according to example 1 of the present invention, wherein the TIGIT extracellular segment represents a nucleotide sequence encoding a receptor binding to CD155, the TIGIT transmembrane segment represents a nucleotide sequence encoding a TIGIT transmembrane segment domain, the 4-1BB intracellular segment represents a nucleotide sequence encoding a 4-1BB costimulatory factor domain, the IL18RB intracellular segment represents a nucleotide sequence encoding an IL18RB domain, and the CD3 zeta intracellular segment represents a nucleotide sequence encoding a CD3 zeta intracellular segment; wherein TIGIT-CAR-2 is a pattern diagram of a CAR activation signal structure targeting CD155 as a control, which differs from the TIGIT-CAR structure of the present example in that the intracellular segments are a 4-1BB intracellular segment, an IL-21 receptor (IL-21R) intracellular segment, and a CD3 ζ intracellular segment;
FIG. 2 is a graph showing the results of detection of the expression level of TIGIT ligand CD155 in tumor cells according to example 2 of the invention, wherein the shaded peaks are isotype antibody staining control groups, and the black solid line is CD155 antibody staining group;
FIG. 3 is a graph showing the results of in vitro killing ability detection of TIGIT-CAR-NK cells according to example 2 of the present invention;
FIG. 4 is a graph showing the detection result of NK cell killing-related degranulation in vitro according to example 2 of the present invention;
FIG. 5 is a graph showing secretion level detection results of IFN-gamma and TNF-alpha of TIGIT-CAR-NK cells according to example 2 of the present invention;
FIG. 6 is a graph showing the results of fluorescence experiments for inhibiting tumor growth in a differentially treated group according to example 3 of the present invention;
FIG. 7 is a graph of the statistics of the fluorescence intensity of the differential treatment group according to example 3 of the present invention;
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. Wherein like or similar reference numerals refer to like or similar elements or elements having like or similar functions throughout. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
In describing the present invention, the terms related thereto are explained and illustrated only for convenience of understanding the scheme and are not to be construed as limiting the protection scheme of the present invention.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
"operably linked" herein refers to the linkage of a foreign gene to a vector such that control elements within the vector, such as transcription control sequences and translation control sequences, and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene. The usual vectors may be, for example, viral vectors, plasmids, phages and the like. After the expression vector according to some embodiments of the present invention is introduced into a suitable recipient cell, the expression of the nucleic acid molecule described above can be effectively achieved under the mediation of a regulatory system, thereby achieving in vitro mass-production of the protein encoded by the nucleic acid molecule.
As used herein, a "chimeric antigen receptor" refers to an artificial receptor fragment expressed on the surface of a cell membrane that includes an extracellular region capable of specifically binding to a corresponding ligand or antigen, a transmembrane region, and an intracellular region that causes activation of an immunostimulatory factor contained in the intracellular region.
The present application constructs a transgenic immune cell expressing a chimeric antigen receptor, wherein the chimeric antigen receptor targets at least one of the members of the ligand PVR family. The chimeric antigen receptor can express an activation signal, enhance the tumor killing activity of CAR immune cells, and is applied to the treatment of solid tumors and hematological tumors.
Embodiments of the present invention will be described in more detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below by referring to the drawings are illustrative and intended to explain the present invention and should not be construed as limiting the invention.
The "plasmid" and "vector" described in the following examples have the same meaning and are used interchangeably.
Example 1: preparation of TIGIT-CAR-NK cells
1.1 construction of pCDH-EF 1-TIGIT-CAR-T2A-copGGFP lentiviral plasmid
The TIGIT-CAR vector sequence designed by the invention comprises an extracellular segment and a transmembrane segment of a TIGIT receptor, a 4-1BB costimulatory signal domain, an IL18RB intracellular segment and a CD3 zeta intracellular region. The structural schematic diagram of the genetic element is shown in figure 1.
The total gene synthesized TIGIT-CAR fragment was inserted into the lentiviral vector pCDH-EF 1-MSC-T2A-copGGFP vector through the cleavage sites XbaI and BamHI. After colony PCR identification and sequencing verification, the sequence is correct, the construction of pCDH-EF 1-TIGIT-CAR-T2A-copGGFP plasmid is successful.
1.2 packaging of lentiviruses and concentration of viral fluids
Taking 293T cells in logarithmic growth phase 5X 10 6 Inoculating into 10cm culture dish, adding 10ml of LDMEM culture medium, 37 deg.C, and 5% CO 2 Culturing overnight in an incubator. Waiting for cell DensityWhen 80% was reached, 10mL of fresh DMEM medium was replaced and the culture was continued in an incubator.
Preparing a slow virus packaging system: adding 6 mu g of psPAX2 plasmid, 3 mu g of pMD2.G plasmid and 6 mu g of pCDH-EF1-TIGIT-CAR-T2A-copGFP plasmid into 250 mu L serum-free DMEM medium, uniformly mixing to prepare a DNA mixture; mu.L of PEIpro was added to 235. Mu.L of serum-free DMEM medium and mixed to prepare a PEIpro mixture. And adding the PEIpro mixed solution into the DNA mixed solution at one time, standing and mixing uniformly, and incubating for 15min at room temperature. The mixture was added to 293T cell culture dishes. Culturing for 24 hr, changing liquid, and placing the culture dish back to 37deg.C, 5% CO 2 In an incubator. After 48h, the cell supernatant was collected, centrifuged at 400 Xg for 5min, the cell debris removed and the supernatant filtered with a 0.45 μm filter head into a new 50ml centrifuge tube. Add 5 XPEG 8000 solution, invert the tube upside down, mix well and place in a refrigerator at 4deg.C overnight. Centrifuging at 4deg.C at 4000 Xg for 20min, discarding supernatant, adding appropriate amount of serum-free DMEM medium to resuspend virus precipitate, packaging into EP tube, and storing in refrigerator at-80deg.C.
1.3 lentivirus infection of human NK cells
NK-92 cells (purchased from ATCC) in logarithmic phase were taken, and 2mL of alpha-MEM medium was added to resuspend the cells to adjust the cell density to 5X 10 5 And each mL. 5X 10 was inoculated into 24-well plate 5 NK-92 cells, 1mL virus concentrate, 1. Mu.L protamine (from Soy pal, final concentration 8. Mu.g/mL). Placing at 37deg.C and 5% CO 2 Culturing in an incubator. After 24h, the cell status was observed, the liquid was changed, the infected cells were transferred into EP tube, centrifuged at 100 Xg for 5min, the cells were resuspended in a small amount of fresh alpha-MEM medium, transferred into cell culture flasks, and continued culture by adding 10mL of fresh alpha-MEM medium and IL-2 (final concentration of 200 IU/mL). After cell expansion, cells were transferred into a flow tube, resuspended in 3mL of 1 XPBS solution, centrifuged at 100 XP for 5min, the supernatant discarded, and the cell pellet vortexed and repeated once. The infected NK-92 cells were flow-sorted GFP-positive, flow-sorted GFP-positive TIGIT-CAR-NK cells by flow meter for the subsequent experiments.
Example 2: biological function identification of TIGIT-CAR-NK cells
2.1 expression of TIGIT ligand CD155 in tumor cells
Studies have shown that TIGIT ligand CD155 is highly expressed on the surface of ovarian cancer tissues and a variety of ovarian cancer cells. The inventors examined the expression of CD155 on the cell surface of human ovarian cancer cell lines HO8910 and SKOV-3 by flow cytometry. The results showed that both HO8910 and SKOV-3 cells highly expressed CD155 molecules (FIG. 2).
2.2 in vitro killing ability of TIGIT-CAR-NK cells
NK-92, TIGIT-CAR-NK-92 and TIGIT-CAR2-NK-92 are taken as effector cells, an ovarian cancer cell line HO8910 is taken as target cells, and the effective target ratio is set to be 5:1, 2.5:1 and 1.25:1. The effector cells were incubated with the target cells for 4 hours, and the killing efficiency of the effector cells against the target cells was examined by ldh (lactate dehydrogenase) release method. The results show that the killing efficiency of the TIGIT-CAR cells on H08910 cells is obviously higher than that of NK-92 and TIGIT-CAR2 (the amino acid sequence is shown as SEQ ID NO: 10) cell groups (figure 3). The results show that the killing capacity of NK cells to CD155 positive tumor cells can be obviously improved through TIGIT-CAR gene modification, and the TIGIT-CAR structure with the intracellular sections of 4-1BB and CD3 zeta is superior to the CAR structure with the intracellular sections of 4-1BB and IL-21 receptor (IL-21R, the amino acid sequence of which is shown as SEQ ID NO: 9) and CD3 zeta, so that the stronger killing capacity of NK cells to CD155 positive tumor cells can be stimulated.
SLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQAS(SEQ ID NO:9)
MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGADFQIPLLGAMAATLVVICTAVIVVVALTRKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELSLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDFKKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQEPAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDAEGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVSAGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGSPLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPPPLSSPGPQASRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:10)
In addition, NK cell killing-related degranulation was examined, different effector cells were incubated with HO8910 cells for 4h, and NK cells were collected in flow tubes, and expression of CD107a, granzyme (granzyme b) and Perforin (Perforin) on NK cells were examined by flow cytometry, respectively. As a result, after co-incubation with HO8910 cells, the expression level of CD107a on TIGIT-CAR cells was significantly higher than that of NK-92 group, and there was no statistical difference in the expression level of Perforin (FIG. 4). It was further verified that TIGIT-CAR modification significantly improved the degranulation level (expression level of CD107a, granzyme and perforin) and killing function of NK cells on CD155 positive tumor cells.
2.3 secretion levels of IFN-gamma and TNF-alpha by TIGIT-CAR-NK cells
The change in the secretion capacity of IFN-gamma and TNF-alpha from TIGIT-CAR-NK-92 cells was examined by flow cytometry. After incubating NK cells with ovarian cancer HO8910 cells for 4 hours, collecting the NK cells in a flow tube, and detecting secretion levels of NK cells IFN-gamma and TNF-alpha by flow cytometry through fixation membrane rupture treatment. The results showed that TIGIT-CAR-NK-92 cells secreted IFN- γ and TNF- α levels that were significantly higher than NK-92 group (fig. 5). The TIGIT-CAR can obviously improve the secretion capacity of IFN-gamma and TNF-alpha when NK cells are contacted with CD155 positive tumor cells.
Example 3: TIGIT-CAR-NK cells anti-tumor ability in vivo
And carrying out abdominal cavity tumor loading by using the luciferase-marked ovarian cancer HO8910 cells, and establishing an ovarian cancer abdominal cavity metastasis model. 5-week-old female hyperimmune NCG mice were selected for intraperitoneal tumor-bearing, each 2X 10 mice by intraperitoneal injection 5 Luciferase-labeled HO8910 cells. NK cell reinfusion therapy was performed on day 2 post tumor-bearing. Mice were randomized into control, NK-92 treatment and TIGIT-CAR-NK-92 treatment groups. Treatment group mice were injected intraperitoneally with NK cells 5X 10 6 Individual/individual, controlGroups were injected with equal volumes of 1 XPBS solution, once every other week for a total of 3 treatments. IL-2 (50000 IU/dose) was intraperitoneally injected every 3 days. The tumor size is observed by a small animal living body imaging technology, and then a tumor growth curve is drawn.
The results show that compared with the tumor-bearing control group, the NK-92 and TIGIT-CAR-NK-92 treatment groups can obviously inhibit the growth of tumors, and the effect of taking the TIGIT-CAR-NK-92 as the treatment group is optimal, and the intensity of tumor fluorescence signals observed by the in-vivo imaging technology is obviously smaller than that of the NK-92 treatment group (figures 6 and 7). Indicating that the TIGIT-CAR gene expresses an activating signal after modification and activates the tumor killing effect of NK cells.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the present invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (17)
1. A chimeric antigen receptor comprising:
an extracellular region comprising a TIGIT extracellular segment;
a transmembrane region comprising a TIGIT transmembrane region and embedded into the cell membrane of the cell; and
an intracellular region comprising a 4-1BB costimulatory factor domain, an IL18RB intracellular segment, and a cd3ζ intracellular signaling segment;
wherein the C end of the extracellular region is connected with the N end of the transmembrane region, and the C end of the transmembrane region is connected with the N end of the intracellular region.
2. The chimeric antigen receptor according to claim 1, wherein the C-terminus of the 4-1BB co-stimulatory factor domain is linked to the N-terminus of the IL18RB intracellular segment, and wherein the C-terminus of the IL18RB intracellular segment is linked to the N-terminus of the cd3ζ intracellular signaling segment.
3. The chimeric antigen receptor according to claim 1, wherein the extracellular region is capable of binding a ligand comprising at least one member of the PVR family;
optionally, the PVR family member comprises CD155 and CD112;
preferably, the PVR family member is CD155.
4. The chimeric antigen receptor according to claim 1, wherein the TIGIT extracellular segment has an amino acid sequence shown in SEQ ID No. 1;
the TIGIT transmembrane region has an amino acid sequence shown as SEQ ID NO. 2;
the 4-1BB co-stimulatory factor domain has an amino acid sequence shown as SEQ ID NO. 3;
the IL18RB intracellular segment has an amino acid sequence shown in SEQ ID NO. 4;
the intracellular signal segment of the CD3 zeta has an amino acid sequence shown in SEQ ID NO. 5;
the chimeric antigen receptor has an amino acid sequence shown in SEQ ID NO. 6.
5. A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1 to 4.
6. The nucleic acid molecule of claim 5, wherein said nucleic acid molecule has the nucleotide sequence set forth in SEQ ID NO. 7.
7. An expression vector carrying the nucleic acid molecule of claim 5 or 6.
8. The expression vector of claim 7, further comprising: a promoter operably linked to the nucleic acid molecule of claim 5 or 6.
9. The expression vector of claim 8, wherein the promoter is selected from at least one of CMV, EF-1, rsv.
10. The expression vector of claim 7, wherein the expression vector is a non-pathogenic viral vector.
11. The expression vector of claim 10, wherein the non-pathogenic virus is selected from the group consisting of a retrovirus, a lentivirus, and an adenovirus-associated virus, preferably wherein the non-pathogenic virus is a lentivirus.
12. A lentiviral vector having the sequence set forth in SEQ ID NO:8, and a nucleotide sequence shown in SEQ ID NO.
13. A transgenic immune cell expressing the chimeric antigen receptor of any one of claims 1 to 4, carrying the nucleic acid molecule of any one of claims 5 to 6, the expression vector of any one of claims 7 to 11, or the lentiviral vector of claim 12.
14. A CAR-immune cell expressing the chimeric antigen receptor of any one of claims 1-4, carrying the nucleic acid molecule of any one of claims 5-6, the expression vector of any one of claims 7-11, or the lentiviral vector of claim 12;
optionally, the CAR-immune cells comprise at least one selected from NK-92 cells, peripheral blood NK cells, umbilical cord blood NK cells, ipscs, CAR-NK cells, CAR-T cells, CAR-NKT cells, CAR- γδ T cells.
15. A pharmaceutical composition comprising:
expressing the chimeric antigen receptor of any one of claims 1 to 4, carrying the nucleic acid molecule of any one of claims 5 to 6, the expression vector of any one of claims 7 to 11 or the lentiviral vector of claim 12, the transgenic immune cell of claim 13 or the CAR-immune cell of claim 14;
optionally, further comprising: pharmaceutically acceptable auxiliary materials.
16. Use of a chimeric antigen receptor according to any one of claims 1 to 4, carrying a nucleic acid molecule according to any one of claims 5 to 6, an expression vector according to any one of claims 7 to 11 or a lentiviral vector according to claim 12, a transgenic immune cell according to claim 13 or a CAR-immune cell according to claim 14 in the manufacture of a medicament for the treatment or prophylaxis of a solid or hematological tumor.
17. The use according to claim 16, wherein the solid tumor comprises a tangible tumor selected from the group consisting of pancreatic cancer, ovarian cancer, mesothelioma, liver cancer, cholangiocarcinoma, gastric cancer, colorectal cancer, esophageal cancer, lung cancer, head and neck cancer, cervical cancer, brain glioma, renal cancer, breast cancer, prostate cancer, melanoma;
optionally, the hematological neoplasm comprises at least one selected from acute myeloid leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, hodgkin's lymphoma, non-hodgkin's lymphoma, multiple myeloma within the blood cell and hematopoietic system.
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