CN115873803B - Method and application for improving NK cell survival and anti-tumor activity - Google Patents
Method and application for improving NK cell survival and anti-tumor activity Download PDFInfo
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Abstract
本发明提出一种提高NK细胞存活和抗肿瘤活性的方法及其应用，具体地，提出了一种免疫细胞，所述免疫细胞表达嵌合抗原受体以及融合蛋白，所述融合蛋白包括IL‑15Rα和IL‑15。所述转基因免疫细胞能够同时表达和分泌嵌合抗原受体和融合蛋白，使得免疫细胞能够靶向对应抗原，定位至表达所述抗原的细胞表面，此外，所述融合蛋白进一步促进免疫细胞的活化和增殖，维持免疫细胞在肿瘤局部微环境中的数量和活性，使其保持强大的肿瘤杀伤活性，有效避免全身高剂量或反复多次注射带来的毒副作用。The present invention proposes a method for improving the survival and anti-tumor activity of NK cells and its application. Specifically, an immune cell is proposed, which expresses a chimeric antigen receptor and a fusion protein, wherein the fusion protein includes IL-15Rα and IL-15. The transgenic immune cell can simultaneously express and secrete the chimeric antigen receptor and the fusion protein, so that the immune cell can target the corresponding antigen and localize to the cell surface expressing the antigen. In addition, the fusion protein further promotes the activation and proliferation of immune cells, maintains the number and activity of immune cells in the local microenvironment of the tumor, and maintains a strong tumor killing activity, effectively avoiding the toxic side effects caused by high-dose or repeated injections of the whole body.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a method for improving NK cell survival and anti-tumor activity and application thereof.
Background
In recent years, chimeric antigen receptor T (CHIMERIC ANTIGEN receptorT, CAR-T) cells have achieved dramatic effects in hematological malignancy therapy. However, the CAR-T cells are prone to adverse reactions such as cytokine storm, neurotoxicity and GVHD in clinical application, and the therapeutic effect of the CAR-T cells on solid tumors is not ideal, so that the clinical application of the CAR-T cells still faces challenges.
Compared with CAR-T cells, the CAR-NK cells have the advantages of good safety, generally do not cause side effects such as cytokine storm, GVHD and the like, the NK cells can directly kill tumor cells without antigen presentation and MHC restriction, and the CAR-NK cells can identify and kill tumors by various identification mechanisms such as CAR dependence, NKR dependence and the like, so that the anti-tumor spectrum is wide. Therefore, the CAR-NK cells have wide application prospect in anti-tumor treatment and become hot spots in the field of development of cellular immunotherapy. However, one of the difficulties faced in the development of CAR-NK cells is the short survival time of NK cells in vivo, affecting their in vivo effects.
IL-15 is a cytokine which can promote the survival, proliferation and function of T cells and NK cells, IL-15 shares IL-2/15Rβγc receptor with IL-2, IL-15 and IL-15Rβγc form dimer and then combine with IL-15Rβγc to activate downstream JAK1/JAK3 and STAT3/STAT5 signaling pathway, thereby promoting proliferation, activation and effector functions of NK cells. Thus, IL-15 has become a hot target for drug development to enhance the persistence and proliferative activity of lymphocytes in vivo.
However, the problems associated with the use of IL-15 in vivo are short half-life, limited in vivo efficacy, large doses, and frequent administration, resulting in various side effects, including hypotension, thrombocytopenia, elevated AST and ALT, etc., which may lead to intolerance of such treatment in cancer patients. In clinical application and drug development, the activity of IL-15 for promoting lymphocyte proliferation and persistence and promoting immune response should be maintained as much as possible, and the side effects related to IL-15 should be reduced as much as possible.
Based on the above research and development status, there is a need to further study a safe and effective method for improving the persistence of CAR-NK cells in vivo.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, the invention provides the transgenic immune cell, the proliferation capacity and the in-vivo survival time of which are obviously improved compared with those of natural immune cells, and the transgenic immune cell has higher safety.
Thus, in a first aspect of the invention, the invention provides a transgenic immune cell. According to an embodiment of the invention, the immune cells express chimeric antigen receptor as well as fusion proteins including IL-15Rα and IL-15, hereinafter referred to as super IL-15. The transgenic immune cells according to embodiments of the present invention are capable of simultaneously expressing and secreting chimeric antigen receptor and fusion protein. The chimeric antigen receptor can enable immune cells to target corresponding antigens and locate on the surfaces of cells expressing the antigens, and in addition, the fusion protein further promotes activation and proliferation of the immune cells, maintains the number and activity of the immune cells in a local tumor microenvironment, enables the immune cells to maintain strong tumor killing activity, and can effectively avoid toxic and side effects caused by high-dose whole body or repeated multiple injections.
According to an embodiment of the present invention, the transgenic immune cell may further comprise at least one of the following additional technical features:
According to an embodiment of the application, the chimeric antigen receptor comprises an extracellular region capable of binding specifically to an antigen, a transmembrane region, and an intracellular region comprising an immune co-stimulatory molecule intracellular segment and a signal transduction domain, wherein the extracellular region has a C-terminus linked to the N-terminus of the transmembrane region and a C-terminus linked to the N-terminus of the intracellular region. In the present application, the kind of antigen recognized by the chimeric antigen receptor is not particularly limited, and is suitable for specifically recognizing various antigens.
According to an embodiment of the invention, the antigen is a tumor-associated antigen. According to some embodiments of the invention, the kind of antigen is not particularly limited.
According to an embodiment of the invention, the extracellular region comprises a heavy chain variable region and a light chain variable region of an antibody, which binds to the antigen. It will be appreciated by those skilled in the art that the extracellular region may include a binding region recognizing the antigen, and the extracellular region may include at least one of a full anti-antibody, a Fab 'antibody, a F (ab') 2 antibody, a Fv antibody, a single chain antibody, and a nanobody. According to some preferred embodiments of the invention, the extracellular region comprises a single chain antibody.
According to an embodiment of the invention, the antigen comprises at least one selected from the group consisting of mesothelin, HER2, EGFR, GPC3, MUC1, CEA, CLDN 18.2, epCAM, GD2, PSCA, CD133, CD19, CD20, CD22, CD30, CD33, BCMA.
According to an embodiment of the invention, the antigen is mesothelin. According to some embodiments of the invention, when the antigen is mesothelin, the transgenic immune cells can effectively target mesothelin-positive tumors, retain higher proliferation activity and have higher anti-tumor capability.
According to an embodiment of the invention, the extracellular region comprises an anti-mesothelin single chain antibody.
According to an embodiment of the invention, the anti-mesothelin single chain antibody comprises a light chain variable region of an anti-mesothelin antibody, a connecting peptide 1 and a heavy chain variable region of an anti-mesothelin antibody.
According to an embodiment of the invention, the connecting peptide 1 has an amino acid sequence shown as (GGGGS) n, wherein n is an integer greater than or equal to 1, preferably 1,2,3, 4, 5,6,7,8, 9 or 10.
According to an embodiment of the invention, the anti-mesothelin single chain antibody comprises the amino acid sequence shown in SEQ ID NO. 14. In some specific embodiments, when the anti-mesothelin single-chain antibody has the above amino acid sequence, the transgenic immune cell can effectively target mesothelin positive tumor, retain higher proliferation activity and have higher anti-tumor capability.
According to an embodiment of the invention, the extracellular region further comprises a hinge region segment, the N-terminus of the hinge region segment being linked to the C-terminus of the single chain antibody.
According to an embodiment of the invention, the hinge region segment comprises at least one of the hinge regions selected from the group consisting of CD8, CD28 and immunoglobulins.
According to an embodiment of the invention, the hinge segment comprises a hinge region of CD 8.
According to an embodiment of the invention, the hinge fragment comprises the amino acid sequence shown in SEQ ID NO. 15.
According to an embodiment of the invention, the transmembrane region comprises at least one selected from the group consisting of CD4, CD8 a, CD28 and CD3 ζ or a fragment thereof.
According to an embodiment of the invention, the transmembrane region comprises a CD8 transmembrane region or fragment thereof.
According to an embodiment of the invention, the transmembrane region has the amino acid sequence shown in SEQ ID NO. 16.
According to an embodiment of the invention, the immune co-stimulatory molecule comprises at least one selected from the group consisting of CD28, ICOS, 4-1BB, OX40 and CD 27.
According to an embodiment of the invention, the intracellular segment of the immune co-stimulatory molecule is an intracellular segment of 4-1BB or CD28 or a fragment thereof.
According to an embodiment of the invention, the intracellular segment of the immune co-stimulatory molecule comprises the amino acid sequence shown in SEQ ID NO. 17.
According to an embodiment of the invention, the C-terminal end of the intracellular segment of the immune co-stimulatory molecule is connected to the N-terminal end of the signaling domain.
According to an embodiment of the invention, the signal transduction domain comprises at least one selected from cd3ζ or fcsriy or a fragment thereof.
It will be appreciated by those skilled in the art that the choice of hinge region, transmembrane region, immune co-stimulatory molecule intracellular segment and signaling domain is not particularly limited and that hinge region, transmembrane region, immune co-stimulatory molecule intracellular segment and signaling domain may be used as are available in chimeric antigen receptors conventional in the art.
According to an embodiment of the invention, the signal transduction domain comprises cd3ζ or fragment thereof.
According to an embodiment of the invention, the signal transduction domain comprises the amino acid sequence shown in SEQ ID NO. 18.
According to an embodiment of the invention, the fusion protein further comprises a connecting peptide 2.
According to an embodiment of the invention, the connecting peptide 2 has the amino acid sequence shown in SEQ ID NO. 19.
According to an embodiment of the application, the immune cells comprise at least one of T cells and NK cells. In the present application, the kind of the immunostimulatory molecule is not particularly limited, and an agent capable of promoting an immune function of at least one of T cells and NK cells may be used, and in some embodiments of the present application, the immunostimulatory molecule is IL-15.
According to an embodiment of the invention, the immune cells are preferably NK cells.
According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells.
According to an embodiment of the invention, the T cells include CD4 + T cells, CD8 + T cells, and γδ T cells.
In a second aspect of the invention, the invention provides an isolated nucleic acid. According to an embodiment of the invention, the isolated nucleic acid comprises 1) a first nucleic acid molecule encoding a chimeric antigen receptor, 2) a second nucleic acid molecule encoding a fusion protein comprising IL-15Rα and IL-15.IL-15 is a pleiotropic cytokine, and IL-15R bind IL-15Rc to activate downstream JAK1/JAK3 and STAT3/STAT5 signaling pathways after forming dimers, thereby promoting proliferation, activation and effector functions of T cells, B cells and NK cells. The isolated nucleic acid according to the embodiments of the present invention can package higher titer viruses after being introduced into recipient cells and achieve specific infection of immune cells, such as NK cells, by the viruses. After the isolated nucleic acid is introduced into immune cells, the immune cells can express and secrete chimeric antigen receptors and fusion proteins simultaneously, so that the immune cells can target corresponding antigens and be positioned on the surfaces of cells expressing the antigens, in addition, the fusion proteins such as IL-15Rα and IL-15 further promote the activation and proliferation of the immune cells, maintain the quantity and activity of the immune cells in the local microenvironment of tumors, ensure that the immune cells keep strong tumor killing activity, effectively avoid toxic and side effects caused by high-dose systemic injection or repeated injection for multiple times, and avoid toxic and side effects caused by systemic application of recombinant IL-15 in high-dose or repeated injection for multiple times.
According to an embodiment of the present invention, the above isolated nucleic acid may further include at least one of the following additional technical features:
According to an embodiment of the invention, the chimeric antigen receptor is as defined in the first aspect.
According to an embodiment of the invention, the second nucleic acid molecule comprises a nucleotide sequence encoding the IL-15Rα as shown in SEQ ID NO. 10 and a nucleotide sequence encoding the IL-15 as shown in SEQ ID NO. 12.
According to an embodiment of the invention, the first nucleic acid molecule and the second nucleic acid molecule are arranged to express the chimeric antigen receptor and the fusion protein in immune cells, and the fusion protein is in a non-fused form with the chimeric antigen receptor.
According to an embodiment of the invention, the isolated nucleic acid further comprises an internal ribosome entry site sequence which is arranged between the first nucleic acid molecule and the second nucleic acid molecule, the internal ribosome entry site having the nucleotide sequence set forth in SEQ ID NO. 20.
According to an embodiment of the invention, the isolated nucleic acid further comprises a third nucleic acid molecule, which third nucleic acid molecule is arranged between the first nucleic acid molecule and the second nucleic acid molecule, which third nucleic acid molecule encodes a connecting peptide 3, which connecting peptide 3 is capable of being cleaved. The connecting peptide 3 can separate the first nucleic acid molecule from the second nucleic acid molecule, and reduces functional interference of the first nucleic acid molecule and the second nucleic acid molecule.
According to an embodiment of the invention, the connecting peptide 3 comprises a 2A peptide or a fragment thereof. It will be appreciated by those skilled in the art that the connecting peptide 3 is not particularly limited, and conventional peptides having a self-cleavage function may be used.
According to an embodiment of the invention, the connecting peptide 3 comprises at least one of P2A, T2A, E a and F2A or a fragment thereof.
According to an embodiment of the invention, the connecting peptide 3 comprises P2A or a fragment thereof.
According to an embodiment of the invention, the connecting peptide 3 comprises the amino acid sequence shown in SEQ ID NO. 19.
According to an embodiment of the invention, the isolated nucleic acid further comprises a first promoter operably linked to the first nucleic acid molecule and/or a second promoter operably linked to the second nucleic acid molecule.
According to an embodiment of the invention, the first promoter, the second promoter are each independently selected from the group consisting of U6, H1, CMV, EF-1, LTR or RSV promoters.
According to an embodiment of the invention, the isolated nucleic acid further comprises a fourth nucleic acid molecule encoding a signal peptide. According to an embodiment of the present invention, the signal peptide expressed by the gene encoding the signal peptide is located at the amino terminal of the chimeric antigen receptor, is a chimeric antigen receptor membrane localization terminal peptide, and is hydrolyzed and detached after helping the localization of the chimeric antigen receptor to the endoplasmic reticulum after maturation of the protein, so that the chimeric antigen receptor on the viral particle does not contain the signal peptide.
According to an embodiment of the invention, the fourth nucleic acid molecule is operably linked to the first nucleic acid molecule.
According to an embodiment of the invention, the signal peptide comprises at least one selected from CSF2R and CD8 a or a fragment thereof. It will be appreciated by those skilled in the art that the kind of the signal peptide is not particularly limited, and that conventional signal peptides in the art can be used.
According to an embodiment of the invention, the signal peptide comprises CSF2R or a fragment thereof.
According to an embodiment of the invention, the signal peptide comprises the amino acid sequence shown in SEQ ID NO. 13.
According to an embodiment of the invention, the first nucleic acid molecule has at least one of the nucleotide sequences shown in SEQ ID NO. 3, 4, 5, 6 and 7.
According to an embodiment of the invention, the second nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO. 9.
According to an embodiment of the invention, the third nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO. 8.
According to an embodiment of the invention, the fourth nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO. 2.
According to an embodiment of the invention, the isolated nucleic acid has the nucleotide sequence shown in SEQ ID NO. 1.
In a third aspect of the invention, the invention proposes a construct. According to an embodiment of the invention, the construct carries an isolated nucleic acid as described previously. In the case of the above-described isolated nucleic acid being linked to a vector, the isolated nucleic acid may be linked directly or indirectly to control elements on the vector, as long as these control elements are capable of controlling translation and expression of the isolated nucleic acid, etc., i.e., the isolated nucleic acid is operably linked to the control elements. Of course, these control elements may be directly from the carrier itself or may be exogenous, i.e. not from the carrier itself.
According to an embodiment of the present invention, the construct may further comprise at least one of the following additional technical features:
According to an embodiment of the invention, the vector of the construct is a non-pathogenic viral vector. According to some embodiments of the invention, the expression vector has a higher expression efficiency when it is a viral vector.
According to an embodiment of the invention, the viral vector comprises at least one selected from the group consisting of a retroviral vector, a lentiviral vector or an adenovirus-associated viral vector.
In a fourth aspect of the invention, the invention provides a recombinant cell. According to an embodiment of the invention, the recombinant cell carries the isolated nucleic acid or construction vector described previously. Recombinant cells according to embodiments of the invention can be used to express and obtain in vitro under suitable conditions the proteins encoded by the aforementioned isolated nucleic acids, such as chimeric antigen receptor mesothelin and IL-15Rα and IL-15 fusion proteins, in large quantities.
According to an embodiment of the present invention, the recombinant cell may further include at least one of the following additional technical features:
according to an embodiment of the invention, the recombinant cells comprise eukaryotic cells, preferably mammalian cells.
It should be noted that the recombinant cells of the present invention are not particularly limited, and may be prokaryotic cells, eukaryotic cells, or phage. The prokaryotic cells can be escherichia coli, bacillus subtilis, streptomycete, proteus mirabilis and the like, and the eukaryotic cells comprise fungi such as pichia pastoris, saccharomyces cerevisiae, schizosaccharomyces, trichoderma and the like, insect cells such as armyworm and the like, plant cells such as tobacco and the like, and mammalian cells such as BHK cells, CHO cells, COS cells, myeloma cells and the like. In some embodiments, the recombinant cells of the invention are preferably mammalian cells, including T cells, B cells, NK cells, BHK cells, CHO cells, NSO cells, or COS cells, and do not include animal germ cells, fertilized eggs, or embryonic stem cells.
In a fifth aspect of the invention, the invention provides a CAR-NK or CAR-T cell. According to an embodiment of the invention, the CAR-NK cells carry the isolated nucleic acid or construct described previously. The CAR-NK cells according to embodiments of the present invention are capable of simultaneously expressing and secreting chimeric antigen receptors and fusion proteins. The inventor finds in experiments that the fusion protein modification strategy provided by the invention can enable NK cells or T cells to locally secrete super IL-15, namely IL-15Rα and IL-15 fusion protein in tumors, obviously improve the in-vivo and in-vitro proliferation capacity of the CAR-NK cells or the T cells, enhance the in-vivo survival time of the NK cells or the T cells and improve the in-vivo anti-tumor function of the NK cells or the T cells. And the IL-15 released slowly and continuously in local can avoid toxic and side effects caused by systemic application of recombinant IL-15 or repeated administration.
According to some embodiments of the invention, the NK cells comprise at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells.
According to an embodiment of the invention, the T cells include CD4 + T cells, CD8 + T cells, and γδ T cells.
In a sixth aspect of the invention, the invention provides a method of obtaining a virus. According to an embodiment of the invention, the construct as described above is introduced into a first recipient cell, and the first recipient cell into which the construct is introduced is cultured to obtain the virus. Methods according to some preferred embodiments of the invention can achieve higher titers of virus.
According to an embodiment of the invention, the virus comprises a lentivirus.
According to an embodiment of the invention, the first recipient cell is 293T.
In a seventh aspect of the invention, the invention provides a virus. According to an embodiment of the invention, obtained by the method for obtaining viruses described above.
In an eighth aspect of the invention, the invention provides a virus. According to an embodiment of the invention, the virus comprises a nucleotide sequence as shown in SEQ ID NO. 1.
According to an embodiment of the invention, the virus comprises at least one of a retrovirus, a lentivirus and an adenovirus.
According to an embodiment of the invention, the virus comprises a lentivirus.
In an eighth aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises an isolated transgenic immune cell, nucleic acid, construct, recombinant cell, CAR-NK or CAR-T cell or virus as described previously. As described above, the isolated nucleic acid, expression vector or cell or virus carrying the isolated nucleic acid or expression vector can simultaneously express and secrete chimeric antigen receptor and fusion protein, so that immune cells can target corresponding antigens and be positioned on the surface of the cell expressing the antigens, and in addition, the fusion protein, such as super IL-15, namely IL-15Rα and IL-15 fusion protein, further promotes the activation and proliferation of immune cells, maintains the quantity and activity of immune cells in the local microenvironment of tumor, so that the immune cells maintain strong tumor killing activity, effectively avoids toxic and side effects caused by high dosage or repeated multiple injections of whole body, and also avoids toxic and side effects caused by high dosage or repeated multiple injections of recombinant IL-15. Therefore, the pharmaceutical composition comprising the above-mentioned substances also has the above-mentioned functions, and will not be described here again.
According to an embodiment of the present invention, the above pharmaceutical composition may further include at least one of the following additional technical features:
According to an embodiment of the invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, except insofar as any conventional excipients are incompatible with the compounds of the invention, such as any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, the use of which is also contemplated by the present invention.
For example, the isolated nucleic acids, expression vectors, or cells carrying the isolated nucleic acids or expression vectors of the invention may be incorporated into a medicament suitable for parenteral administration (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). These drugs can be prepared in various forms. Such as liquid, semi-solid, and solid dosage forms, and the like, including but not limited to liquid solutions (e.g., injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The drug is typically in the form of an injection solution or infusion solution. The isolated nucleic acid, expression vector, or cell carrying the isolated nucleic acid or expression vector may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
The effective amount of the isolated nucleic acid, expression vector or cell carrying the isolated nucleic acid or expression vector of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc., the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times per day, or the dose may be proportionally reduced, depending on the urgent requirements of the treatment situation.
In a tenth aspect of the invention, the invention provides a kit. According to an embodiment of the invention, the kit comprises an isolated nucleic acid, construct or virus as described previously. The isolated nucleic acid, construct or virus can significantly promote the activation or proliferation of NK cells or T cells, and thus, the kit comprising the same also has the function of promoting the activation or proliferation of NK cells or T cells. The kit can be used for scientific research, such as reversing NK cells or T cells with low proliferation activity, so that the proliferation activity of the NK cells or the T cells is increased from low to obtain a biological sample meeting expectations.
In an eleventh aspect of the invention, the invention provides a method of introducing a virus into an activated immune cell. According to an embodiment of the invention, the activated immune cells are electrotransfected or transfected with the construct described above or infected with the virus described above.
According to an embodiment of the invention, the immune cells comprise at least one of T cells and NK cells.
According to an embodiment of the invention, the immune cells are preferably NK cells.
According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells.
According to an embodiment of the invention, the T cells include CD4 + T cells, CD8 + T cells, and γδ T cells.
In a twelfth aspect of the invention, the invention provides a method of obtaining chimeric antigen receptors and fusion proteins. According to an embodiment of the invention, the method comprises introducing the construct or virus described previously into a second recipient cell, and culturing the second recipient cell into which the construct or virus was introduced, so as to obtain the chimeric antigen receptor and fusion protein. As previously described, the construct or virus is capable of simultaneously expressing the chimeric antigen receptor and the fusion protein under suitable conditions, and thus the chimeric antigen receptor and the fusion protein can be obtained in large amounts according to the method of the embodiment of the present invention.
According to an embodiment of the invention, the introduction of the second recipient cell is by electrotransfection, transfection or infection. The "electrotransformation" or "transfection" is a method of introducing a viral vector into a recipient cell, and the "infection" refers to a process in which a virus actively binds to and fuses a cell membrane and enters the cell. Wherein "electrotransfection" refers to a method of introducing a viral packaging vector into a recipient cell by means of electrical stimulation, and "transfection" refers to a method of introducing a viral packaging vector into a recipient cell by means of a chemical mediator, such as a liposome.
According to an embodiment of the invention, the second recipient cell is at least one of a T cell and an NK cell.
According to an embodiment of the invention, the second recipient cell is an NK cell.
According to an embodiment of the present invention, the NK cells include at least one selected from peripheral blood NK cells, umbilical cord blood NK cells, induced pluripotent cell (iPSC) -derived NK cells, and NK-92 cells.
According to an embodiment of the invention, the T cells include CD4 + T cells, CD8 + T cells, and γδ T cells.
According to an embodiment of the invention, the virus comprises at least one selected from the group consisting of retrovirus, lentivirus and adenovirus.
According to an embodiment of the invention, the virus comprises a lentivirus.
In a thirteenth aspect of the invention, the invention proposes a method of obtaining CAR-NK or CAR-T cells of chimeric antigen receptor and fusion protein. According to an embodiment of the invention, comprising introducing the construct or virus described previously into NK cells or T cells, culturing the NK cells or T cells into which the construct or virus was introduced so as to obtain said CAR-NK or CAR-T cells. According to some specific embodiments of the invention, a lentiviral expression vector is constructed, wherein the lentiviral expression vector is used for targeting mesothelin and simultaneously expressing super IL-15 (namely IL-15 and IL-15Rα fusion protein), virus particles are packaged by lentivirus to infect NK cells or T cells, so that high infection efficiency and CAR positive NK cells or T cells are obtained, and the CAR-NK or CAR-T cells not only can target malignant tumors positive with killing mesothelin, but also have higher proliferation capacity and killing activity than unmodified NK cells or T cells, especially can maintain long-term survival of NK cells or T cells in vivo, can maintain higher proliferation activity and killing activity and exert stronger capacity of continuously killing tumors due to the fact that the CAR-NK or CAR-T cells can locally and continuously secrete super IL-15. More importantly, the local secreted IL-15 plays an effective biological function in the tumor part, and can effectively avoid toxic and side effects caused by systemic application of high dose or repeated injection of recombinant IL-15.
According to embodiments of the invention, the introduction of NK cells or T cells is by electrotransfection, transfection or infection.
In a fourteenth aspect of the invention, the invention proposes the use of an isolated nucleic acid, construct, recombinant cell, CAR-NK or CAR-T cell or virus as described hereinbefore for the preparation of a pharmaceutical composition for the treatment or prevention of a tumor. As described above, the isolated nucleic acid, construct, or cell carrying the above can simultaneously express and secrete chimeric antigen receptor and fusion protein, such as IL-15Rα and IL-15 fusion protein, under appropriate conditions, so that the cell can target the corresponding antigen and locate on the surface of the cell expressing the antigen, in addition, the IL-15Rα and IL-15 fusion protein further promotes the activation and proliferation of immune cells, maintains the number and activity of immune cells in the local microenvironment of tumor, so as to maintain strong tumor killing activity, effectively avoid the toxic and side effects caused by high dosage or repeated multiple injections of whole body, and avoid the toxic and side effects caused by high dosage or repeated multiple injections of recombinant IL-15.
According to an embodiment of the invention, the tumor is at least one of a mesothelin-positive tumor, a HER 2-positive tumor, an EGFR-positive tumor, a GPC 3-positive tumor, a MUC 1-positive tumor, a CEA-positive tumor, a CLDN 18.2-positive tumor, an EpCAM-positive tumor, a GD 2-positive tumor, a PSCA-positive tumor, a CD 133-positive tumor, a CD 19-positive tumor, a CD 20-positive tumor, a CD 22-positive tumor, a CD 30-positive tumor, a CD 33-positive tumor, and a BCMA-positive tumor.
According to an embodiment of the invention, the mesothelin-positive tumor comprises at least one of pancreatic cancer, ovarian cancer, mesothelioma, cholangiocarcinoma and lung cancer.
In a fifteenth aspect of the invention, the invention provides the use of an isolated nucleic acid, construct or virus as hereinbefore described in the preparation of a kit for promoting NK cell or T cell activation or proliferation. According to some embodiments of the invention, the isolated nucleic acid, construct or virus is capable of significantly promoting the activation or proliferation of NK cells or T cells, and therefore, a kit comprising the same also has the function of promoting the activation or proliferation of NK cells or T cells. The kit can be used for scientific research, such as reversing NK cells or T cells with low proliferation activity, so that the proliferation activity of the NK cells or the T cells is increased from low to obtain a biological sample meeting expectations.
Drawings
FIG. 1 is a schematic diagram of a CAR targeting MSLN and modified with a fusion protein (RIL) according to example 1 of the present invention, wherein SP represents a nucleotide sequence encoding a signal peptide, α -MSLN-scFv represents a nucleotide sequence encoding an anti-MSLN single chain antibody, CD8hinge +TM represents a nucleotide sequence encoding a CD8hinge region and a transmembrane region, 4-1BB represents a nucleotide sequence encoding a 4-1BB costimulatory signal domain, CD3Z represents a nucleotide sequence encoding a CD3Z intracellular region, P2A represents a nucleotide sequence encoding a P2A self-cleaving region, IL15Rα represents a nucleotide sequence encoding the full length of IL15Rα, linker represents a nucleotide sequence encoding a Linker peptide, and IL15 represents a nucleotide sequence encoding the full length of IL 15;
FIG. 2 is a graph showing the results of detection of secretion levels of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-RIL-NK-92 cell IL-15 according to example 2 of the present invention;
FIG. 3 is a graph of results of detection of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-RIL-NK-92 cell STAT5 phosphorylation levels according to example 2 of the invention;
FIG. 4 is a graph of the results of in vitro killing ability assays of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-RIL-NK-92 cells according to example 2 of the present invention;
FIG. 5 is a graph showing the results of in vitro proliferation potency detection of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-RIL-NK-92 cells according to example 2 of the present invention, wherein the abscissa (Days) represents Days and the ordinate (Cell number) represents Cell number;
FIG. 6 is a flow chart of the operation of RIL-expressing CAR-NK cells for the treatment of pancreatic cancer Aspc-1 cell tumor-bearing mice according to example 3 of the present invention;
FIG. 7 is a graph of the viability assay of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-RIL-NK-92 cells in pancreatic cancer Aspc-1 cell tumor-bearing mice according to example 3 of the present invention;
FIG. 8 is a graph showing the results of detection of antitumor ability of NK-92, α -MSLN-CAR-NK-92 and α -MSLN-CAR-RIL-NK-92 cells against pancreatic cancer Aspc-1 cell Tumor-bearing mice according to example 3 of the present invention, wherein the abscissa (DAYS AFTERNK CELL TREATMENT) represents days after treatment with NK cells and the ordinate (Tumor) represents the volume of Tumor cells.
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 isolated nucleic acid 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 isolated nucleic acid.
As used herein, the term "suitable conditions" refers to conditions suitable for expression of the proteins encoded by the isolated nucleic acids of the application. Those skilled in the art will readily appreciate that conditions suitable for expression of the protein encoded by the isolated nucleic acid include, but are not limited to, suitable transformation or transfection means, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, suitable cell culture time. The "suitable conditions" are not particularly limited, and those skilled in the art can optimize the conditions for optimal expression of the protein encoded by the isolated nucleic acid according to the specific environment of the laboratory.
The application constructs a transgenic immune cell simultaneously expressing a chimeric antigen receptor and an immune stimulation molecule, wherein the chimeric antigen receptor can target a plurality of antigens, so that the immune cell can target the corresponding antigens and be positioned on the surface of the cell expressing the antigens, and the immune stimulation molecule can further promote the activation and proliferation of the immune cell, such as IL-15 used in the application, after experimental verification, the proliferation activity and tumor killing capability of the immune cell simultaneously expressing the chimeric antigen receptor and the IL-15 are obviously improved, and the toxic and side effects caused by systemic high dose or repeated multiple injections are effectively avoided.
The amino acid or nucleic acid sequences referred to herein are shown below.
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro Ala Phe Leu Leu Ile Pro(SEQ ID NO:13)
Asp Ile Gln Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala Ser Val Gln Val Ser Cys Arg Ala Ser Gly Tyr Ser Ile Asn Thr Tyr Tyr Met Gln Trp Val Arg Gln Ala Pro Gly Ala Gly Leu Glu Trp Met Gly Val Ile Asn Pro Ser Gly Val Thr Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Leu Thr Asn Asp Thr Ser Thr Asn Thr Val Tyr Met Gln Leu Asn Ser Leu Thr Ser Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Ala Leu Trp Gly Asp Phe Gly MetAsp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Ile Gly Asp Arg Val Thr Ile Thr Cys ArgAla Ser Glu Gly Ile Tyr His Trp LeuAla Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Ser LeuAla Ser GlyAla Pro SerArg Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile LysArg(SEQ ID NO:14)
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro AlaAla Gly GlyAla Val His ThrArg Gly LeuAsp PheAla Cys Asp(SEQ ID NO:15)
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys(SEQ ID NO:16)
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe MetArg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu(SEQ ID NO:17)
Arg Val Lys Phe SerArg SerAlaAsp Ala Pro Ala Tyr Gln Gln Gly GlnAsn Gln Leu TyrAsn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu TyrAsn Glu Leu Gln Lys Asp Lys MetAla Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu ArgArg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser ThrAla Thr Lys Asp Thr Tyr Asp Ala Leu His Met GlnAla Leu Pro ProArg(SEQ ID NO:18)
Ala ThrAsn Phe Ser Leu Leu Lys GlnAla GlyAsp Val Glu GluAsn Pro Gly Pro(SEQ ID NO:19)
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 CAR-NK cells
1.1 Construction of CAR expression plasmids
The invention designs a CAR vector (anti-MSLN-CAR-RIL) sequence which targets Mesothelin (MSLN) and expresses super IL-15, and comprises a Signal Peptide (SP), an extracellular region (anti-MSLN single-chain antibody, anti-MSLN scFv) which targets and recognizes MSLN, a CD8a Hinge region (Hinge) and a transmembrane region (TM), a 4-1BB intracellular co-stimulatory signal domain and intracellular signal transduction molecule CD3 zeta and an IL-15 Ralpha-linker-IL-15 (RIL) gene fragment connected by P2A. The structure of each genetic element in the CAR vector is shown in figure 1. Wherein:
the full-length sequence of the gene of the CAR vector formed by the elements is shown as SEQ ID NO. 1;
The signal peptide is CSF2R, and the nucleotide sequence of the signal peptide is shown as SEQ ID NO. 2;
the nucleotide sequence of the anti-MSLN scFv is shown as SEQ ID NO. 3;
the nucleotide sequence of the CD8 hinge region is shown as SEQ ID NO. 4;
The nucleotide sequence of the CD8 transmembrane region is shown as SEQ ID NO. 5;
the nucleotide sequence of the 4-1BB co-stimulatory signal domain is shown as SEQ ID NO. 6;
The nucleotide sequence of the CD3 zeta intracellular area is shown as SEQ ID NO. 7;
the nucleotide sequence of the self-shearing region P2A is shown as SEQ ID NO. 8;
the nucleotide sequence of the RIL is shown as SEQ ID NO. 9;
The nucleotide sequence of the IL15Rα is shown as SEQ ID NO.10 in a sequence table;
the nucleotide sequence of the Linker is shown as SEQ ID NO.11 in the sequence table;
the nucleotide sequence of the IL15 is shown as SEQ ID NO.12 in a sequence table.
Firstly, inserting the anti-MSLN-CAR fragment into a lentiviral vector pLent-EF1 alpha-P2A-CMV-GP to construct a pLent-anti-MSLN-P2A-CMV-GP vector. A total gene synthesis IL-15R-IL-15 (RIL) gene fragment was inserted into pLent-anti-MSLN-P2A-CMV-GP vector through cleavage site Not I. The correct sequence is verified by PCR identification and sequencing, which shows that pLent-anti-MSLN-P2A-RIL15-CMV-GP vector is successfully constructed.
1.2 Packaging of lentiviruses and concentration of viral fluids
293T cells in the logarithmic growth phase were inoculated in 5X 10 6 cells in a10 cm dish, 10mLDMEM medium was added and cultured overnight in a 37℃5% CO 2 incubator. When the cell density reached 80%, 10mL of fresh DMEM medium was replaced for virus packaging and the cell culture dishes were kept in an incubator for further use. Preparing a slow virus packaging system, adding psPAX mu g of slow virus packaging auxiliary plasmid and 6 mu g of target gene vector plasmid into 250 mu L of serum-free DMEM culture medium to prepare plasmid mixed solution, and uniformly mixing. 15 mu L PEIpro was added to a volume of 235. Mu.L of serum-free DMEM medium and mixed well. Will beThe mixed solution is added into the plasmid mixed solution at one time, evenly mixed, incubated for 15min at room temperature, and added into a 293T cell culture dish after the incubation is finished. After 24h, the culture dish was replaced, placed back into 37℃in a 5% CO 2 incubator, after 48h of culture, the cell supernatant was collected, centrifuged at 400 Xg for 5min, the cell debris was removed, and the supernatant was filtered into a 50mL centrifuge tube with a 0.45 μm filter head. 5 XPEG 8000 solution was added to concentrate the virus solution, the centrifuge tube was turned upside down and mixed well, and the mixture was placed in a 4℃refrigerator overnight. Centrifuging at 4deg.C and 4000 Xg for 20min, discarding supernatant, adding appropriate amount of serum-free DMEM to resuspend virus precipitate, transferring into EP tube, and storing in-80deg.C refrigerator.
1.3 Lentiviral titer assay
293T cells in the logarithmic growth phase were collected and the concentration was adjusted to 1X 10 5/mL. A24-well plate was used, and 1mL of the cell suspension (1X 10 5/well) was added to each well, and 3 added virus volume gradients were set. Incubated overnight in a 37℃5% CO 2 incubator. The concentrated virus solution is diluted 10 times, namely a 1mLEP tube is taken, 60 mu L of the virus concentrate is sucked into an EP tube, and diluted with 540 mu LDMEM culture medium and uniformly mixed. 293T cells were replaced with fresh DMEM medium, 5. Mu.L, 50. Mu.L and 500. Mu.L of diluted virus solution were added to the corresponding wells, labeled, and the plates were returned to 37℃in a 5% CO 2 incubator. After 24h, the well plates were blotted for virus and 1mL fresh DMEM medium was added. After 72h, cells were harvested by pancreatin digestion, 293T cells GFP expression was measured using a flow meter and viral titer was converted according to the following formula:
Titer (TU/mL) = (c×n×d×1000)/V
Wherein C=GFP positive rate of flow assay
N=number of cells at infection (about 1×10 5)
D = dilution factor of viral vector
V = volume of diluted virus added.
1.4 Lentivirus infection of human NK cells
NK-92 cells (purchased from ATCC) in the logarithmic phase of growth were harvested by centrifugation at 100 Xg for 5min, and the cells were resuspended in an appropriate amount of alpha-MEM medium to adjust the cell density to 5X 10 5 cells/mL. 5X 10 5 NK-92 cells were inoculated into a 24-well plate, respectively, 1mL of the virus concentrate and protamine (purchased from Soxhibao, final concentration 8. Mu.g/mL) were mixed uniformly, and cultured in a 37℃and 5% CO 2 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 cultured for a further 48h with 10mL of fresh alpha-MEM medium and IL-2 (final concentration of 200 IU/mL). Cells were transferred into a inflow tube, 3mL of 1 XPBS solution was added, 100 XPS was centrifuged for 5min, the supernatant was discarded, the cell pellet was sprung, and washed once again with 1 XPBS solution. The expression rate of GFP was measured using a flow meter. And continuing to expand culture, and adjusting the state of NK-92 cells after infection to expand. The infected NK-92 cells were sorted by flow meter for GFP-positive CAR-NK-92 cells for later experiments.
Example 2 measurement of IL-15 secretion level and cell proliferation ability of CAR-NK cells
This example uses the CAR-NK-92 (referred to as CAR-NK in short) cells obtained in example 1 to measure IL-15 secretion level and cell proliferation ability
2.1ELISA detection of secretion level of IL-15 by CAR-NK-92 cells
NK-92, alpha-MSLN-CAR-NK-92 (carrying MSLN-targeted CAR) and alpha-MSLN-CAR-RIL-NK-92 (carrying MSLN-targeted CAR and expressing IL-15 and IL15Rα) cells were cultured, respectively, and supernatants were collected for 24 hours. ELISA detects the IL-15 content in supernatants of different groups. The results of the experiment are shown in FIG. 2, in which IL-15 was hardly detected in NK-92 and alpha-MSLN-CAR-NK-92 cell supernatants. However, significant IL-15 was detected in the supernatant of α -CAR-IL15-NK-92 cells at a level of 67.48.+ -. 5.96pg/mL. Demonstrating the ability of the engineered modified alpha-MSLN-CAR-RIL-NK cells of the invention to secrete IL-15.
2.2 Detection of STAT5 phosphorylation level of CAR-NK cells
IL-15 acts by first binding to the IL-15Rα chain to form a dimer, and then, after IL-15Rα -IL-15 and IL-15Rβγc chains are bound, downstream STAT5 phosphorylation (pSTAT 5) is activated, and pSTAT5 enters the nucleus to promote activation, proliferation and expression of genes associated with apoptosis. Thus, the inventors further observed whether the IL-15 secreted by the modified CAR-NK cells of the invention is biologically active, phosphorylating downstream STAT 5. The NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-RIL-NK-92 cells were cultured in serum-free RPMI 1640 medium for 12 hours, respectively, to perform starvation treatment for reducing the phosphorylation level of autologous STAT 5. Cells were harvested and the level of pSTAT5 was detected by flow-through. The results are shown in FIG. 3, where the level of STAT5 phosphorylation was lower in the NK-92 cell group and the α -MSLN-CAR-NK-92 cell group, and significantly higher in the α -MSLN-CAR-RIL-NK-92 cell group than in the control group.
2.3 Detection of in vitro killing Capacity of CAR-NK cells
The inventors incubated the above NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-RIL-NK-92 cells with pancreatic cancer cell line Aspc-1 for 5 hours, respectively, and examined the killing efficiency. As can be seen from FIG. 4, the killing efficiency of alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-RIL-NK-92 on Aspc-1 cells at an effective target ratio of 5:1 is 48.01 + -2.00% and 58.42% + -2.46, respectively, which is significantly higher than that of NK-92 cells (35.59 + -2.46%), while the killing efficiency of alpha-MSLN-CAR-RIL-NK-92 cells is also significantly higher than that of alpha-MSLN-CAR-NK-92 group, indicating that the insertion of RIL fragment not only can promote secretion of IL-15 by NK cells, but also can enhance the killing function of alpha-MSLN-CAR-NK-92 cells.
2.4 Detection of in vitro proliferation Capacity of CAR-NK cells
The inventors further validated the pro-survival effect of autocrine RIL on NK-92 cells. Cell proliferation curves were drawn by plating the NK-92, alpha-MSLN-CAR-NK-92 and alpha-MSLN-CAR-RIL-NK-92 cells of the same cell number into 96-well plates, counting cells every 3 days, culturing for 24 days, respectively. As can be seen from fig. 5, the number of α -MSLN-CAR-RIL-NK-92 cells started to be different from the two groups of NK-92, α -MSLN-CAR-NK-92 cells by day 12 of culture. By day 21, there was a significant difference starting. The modified NK cells of the present invention have a stronger proliferation capacity probably due to the fact that RIL is IL-15 in the form of a super agonist, which is able to bind more effectively to the IL-15βγc chain in a trans-binding manner, activating the downstream signaling pathway.
Example 3 in vivo anti-tumor Capacity of CAR-NK cells and in vivo viability assay of CAR-NK cells
This example establishes a pancreatic cancer Aspc-1 cell tumor-bearing mouse transplantation model to observe the therapeutic effect of CAR-NK-92 cells on pancreatic cancer. The specific experimental procedure is as follows:
BALB/c-nu nude mice at 6 weeks of age were selected for underarm subcutaneous tumor-bearing at a dose of 2X 10 6 cells/mouse. After about 4 days, cell therapy was started after one week. Tumor volume size was measured prior to treatment and randomly divided into PBS group, NK-92 cell treatment group, alpha-MSLN-CAR-NK-92 cell treatment group and alpha-MSLN-CAR-RIL-NK-92 cell treatment group according to tumor volume size. Mice in the treatment group were given 1×10 7 effector cells/mouse tail intravenous, untreated groups were given an equal volume of 1×pbs, once every other week for 5 total treatments, and 3 tail intravenous IL-2 (5×10 4 IU/mouse) was given every 3 days, and specific experimental setup and operational flow are described in fig. 6. To study the pro-survival effect of RIL on CAR-NK-92 cells, peripheral blood was collected from mice on the first, third and seventh days after treatment, perCP/cyanine5.5anti-human CD56 antibody was labeled after erythrocyte lysis, and the proportion of CD56 cell population in peripheral blood lymphocytes, i.e. NK-92 cells, was flow-detected. Tumor volume was measured every 3 days and tumor growth curves were drawn.
As a result, as shown in FIG. 7, the NK-92 cells, the. Alpha. -MSLN-CAR-NK-92 cells and the. Alpha. -MSLN-CAR-RIL-NK-92 cells in mice were 27.3%, 29.6% and 29.3% respectively on the peripheral blood lymphocytes, and the NK-92 cells in each group were similar in the body at the time of NK cell treatment for one day. On the third day, the in vivo NK-92 cells, alpha-MSLN-CAR-NK-92 cells and alpha-MSLN-CAR-RIL-NK-92 cells in mice accounted for 8.32%, 9.83% and 16.8% of peripheral blood lymphocytes, respectively. It can be seen that the proportion of NK-92 cells was decreased in each group at the third day compared to the first day, the NK-92 group was decreased from 27.3% to 8.32%, the α -MSLN-CAR-NK-92 group was decreased from 29.6% to 9.83%, and the α -MSLN-CAR-RIL-NK-92 group was decreased from 29.3% to 16.8%. However, it can be seen that the cells of the α -MSLN-CAR-RIL-NK-92 group had the highest proportion in vivo and the persistence in vivo was more prominent. On day seven, the in vivo proportions of NK-92 cells, alpha-MSLN-CAR-NK-92 cells and alpha-MSLN-CAR-RIL-NK-92 cells were 0.13%, 3.32%, and 14.7%, respectively. It can be seen that RIL-genetically modified α -MSLN-CAR-RIL-NK-92 cells have a higher in vivo proportion and a greater in vivo persistence than the NK-92 and α -MSLN-CAR-NK-92 groups. Therefore, expression of RIL can improve the viability of NK cells in vivo, and the locally secreted super IL-15 (RIL) has obvious effect of maintaining the survival of NK cells in vivo.
By measuring tumor size and plotting tumor growth curves, it can be seen from fig. 8 that compared with the PBS group and NK-92 cell treatment group of the control group, the α -MSLN-CAR-NK92 cell and the α -MSLN-CAR-RIL-NK-92 cell treatment can significantly inhibit tumor growth, and the α -MSLN-CAR-RIL-NK-92 cell shows better antitumor effect than the α -MSLN-CAR-NK-92 cell. The results show that the mesothelin-targeted CAR-NK-92 cells can inhibit the growth of pancreatic cancer and have good treatment effect. The RIL gene modification can improve the persistence of the CAR-NK-92 cells in vivo, improve the viability of the CAR-NK-92 cells in vivo and improve the anti-tumor effect of the CAR-NK-92 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.
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| CN116410336B (en) * | 2023-06-02 | 2023-09-22 | 云南赛元生物技术有限公司 | Chimeric antigen receptor encoding nucleotide, CAR-NK cell, construction method and application thereof |
| CN117467022B (en) * | 2023-09-28 | 2024-10-22 | 上海恩凯细胞技术有限公司 | Chimeric antigen receptor and uses thereof |
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