46results about How to "Enhance tumor killing activity" patented technology

The invention discloses a CIK cell, as well as a preparation method and a cell preparation thereof. The method for preparing the CIK cell comprises the following steps: placing a separated mononuclear cell in a culture fluid containing phytohemagglutinin; transplanting the mononuclear cell into a culture flask enveloped by antiCD3 monoclonal antibody and antiCD28 monoclonal antibody after the cell is cultured for 24 to 72 hours; and adding a culture fluid containing IL-1alpha and IL-2 into the culture flask to keep on culturing for 5 to 15 days, and separating the cells into different flasks to be cultured every 2 to 3 days. The CIK cell prepared by the method has the characteristics of obvious improved cell proliferation, great increase of CD8 cell proportion, wide antineoplastic spectrum and strengthened antineoplastic activity. The CIK cell and the cell preparation can effectively prevent the metastasis and the recrudescence for of postoperative patients with tumor, can be combined with chemicotherapy to effectively reduce toxic and side effects of the chemicotherapy, strengthens the survivability tolerance of the patients to improve healing efficacy, and can obviously prolong lifecycle to of end-stage patients so as to improve the life living quality.

This invention relates to interleukin-10 (IL-10) polypeptide conjugates comprising at least one non-naturally-encoded amino acid.

The invention relates to a separation and efficient amplification culture method for an antigen specific T lymphocyte. By means of the immunomagnetic bead technology and the character of an active T cell expression CD137, the antigen specific T lymphocyte is separated from a complex cell mass; then an antigen specific T lymphocyte with high tumor killing activity is obtained through in-vitro efficient amplification culture. CD137L (CD137-ligand), IL-7, IL-15 and IL-2 are added in the culturing process, so that the phenomenon that activated and induced cells are liable to apoptosis in the culturing process is avoided, the amplification time is increased, and the killing activity of the antigen specific T lymphocyte is improved.

The invention relates to an application of a flavonoid compound, the flavonoid compound is 3, 3', 4', 5, 7-pentahydroxyflavone dihydrate, the ELISA and protein fluorescence quenching experiments provethat the compound can effectively inhibit interaction of PD-1 / PD-L1 proteins, and has a strong affinity for PD-L1 protein. Cell clone number formation and ELISA detection of IL-2 secretion assay prove that the compound can effectively enhance the tumor killing activity of T lymphocyte Jurkat cells against MDA-MB-231 and NCI-H460 tumor cells as well as the expression level of IL-2, and then reverse T cellular immunosuppression. The research results of the application provide a drug for tumor immunotherapy targeting the PD-1 / PD-L1 immunological test site.

The invention discloses a method for preparing anti-tumor combined immune cells DC (dendritic cell)-CIKs (cytokine induced killers) and NKs (natural killers) simultaneously and the prepared combined immune cells. Ficoll density gradient centrifugation is used for efficient separation, a mononuclear cell is obtained, sufficient quantities of DCs, CIKs and NKs are obtained through cell culture bags and an immune cell induction culture system, and finally, the induced cells are cultured in a combined manner and applied to clinical treatment, so that a tumor killing effect is realized. The DCs, the CIKs and the NKs are subjected to induction culture respectively with adoption of TexMACS immune cell culture media produced by Miltenyi Biotec, autoserum, various cytokines and a combined culture technology, the cells are mixed for culture and application at certain point in time, application of fetal calf serum is avoided, the pollution rate of an exogenous pyrogen and an exogenous allergen is reduced, and the tumor killing activity of the finally mixed cells is enhanced simultaneously; with adoption of a cell culture bag technology, the cell contamination rate is reduced, and the method is suitable for clinical treatment and application.

The invention discloses a method for preparing high-efficiency tumor-killing DC-CIK immune cells and the prepared DC-CIK immune cells, in particular, adding After Anti-PD-1MAb and Anti-PDL-1MAb, their tumor killing activity was significantly improved. In the present invention, TexMACS immune cell culture medium produced by Miltenyi Company, autologous serum, various cytokines and joint culture technology are used to induce and culture DC and CIK, respectively, and at an appropriate time point, DC and CIK-related PD- 1 site is blocked, and then the cells are mixed cultured and applied at a certain time point, and finally the tumor killing activity of the mixed cells is greatly improved and has higher clinical application value.

The invention relates to an isolation culture method for malignant ascites derived TIL cells. At least one of PD-1, LAG-3 and TIM-3 is adopted as a label for isolating tumor-specific T lymphocytes from the TIL cells. The tumor-specific T lymphocytes are separated from the TIL cell mass by combining an immunomagnetic bead technique, and then subjected to in-vitro efficient amplification to obtain the tumor-specific TIL cells having high tumor killing activity. The isolation culture of the tumor-specific TIL cells is simpler in operation procedures, obviously shortened in isolation culture time, low in cost and convenient to popularize and apply. The tumor killing activity of the obtained tumor-specific TIL cells is stronger.

The invention discloses a preparation method for autologous-serum antigen-sensitized DC-CIK cells. The preparation method for the autologous-serum antigen-sensitized DC-CIK cells comprises the steps: A) preparation of an autologous-serum antigen; B) preparation of peripheral blood mononuclear cells; C) separation and culture of DC; D) induction amplification of CIK cells; and E) preparation of the DC-CIK cells through co-culture of DC cells and CIK cells. Compared with the DC-CIK cells prepared by employing a routine culture method, the DC-CIK cells cultured by employing the method are obviously increased in CIK proliferation activity and tumoricidal activity; the average proliferation multiple is 200-500 times and is 2-3 times of that of the DC-CIK cells cultured by employing the routine culture method; and the tumoricidal activity is 70-80% when an in-vitro experiment is performed, and is obviously higher than that of the DC-CIK cells cultured by employing the routine culture method. The method is simple in operation, easily controllable in conditions and relatively low in equipment requirements, and the cell proliferation efficiency of the obtained DC-CIK cells is relatively high.

The invention provides a DC-CIK (dendritic cells-cytokine-induced killers) cell treatment composition derived from umbilical cord blood, wherein DC (dendritic cells) and CIK (cytokine-induced killers) cells are derived from single karyocyte of the same umbilical cord blood. The invention also provides a preparation method of the DC-CIK cell treatment composition. According to the cell treatment composition prepared by adopting the method, CIK cells are high purity and strong in multiplication capacity; the DC is high in purity and strong in antigen presentation capability, so that CIK cell multiplication can be effectively promoted, and the tumor killing activity of the CIK cell can be strengthened; compared with the simple CIK cells, the DC-CIK cells are higher in percentage of CD3<+> CD56<+> cells, and the tumor cell killing activity is remarkably higher.

The invention discloses a preparation method of CIK (cytokine-induced killer) blocked by PD-1 for cancer therapy and belongs to the field of medical treatment. The preparation method includes the coating step, the activating step, the cleaning step, the blocking step and the filtering step. In the coating step, an anti-human CD3 monoclonal antibody is coated in a cell culture flask; in the activating step, the CD3 monoclonal antibody, IFN gamma and IL-2 are utilized to activate and culture a lymphocyte; in the cleaning step, the lymphocyte which is activated and cultured by three times is cleaned; in the blocking step, the cleaned lymphocyte is suspended by normal saline to obtain suspended cell solution, the PD-1 monoclonal antibody is added into the suspended cell solution, and then suspending incubation is carried out; in the filtering step, the cell suspension obtained by incubation of the PD-1 monoclonal antibody is filtered and then is refilled with normal saline to obtain feedback cell suspension. The CIK blocked by PD-1 has the advantages of low consumption of the PD-1 monoclonal antibody, good blocking effect of PD-1 molecules, high killing activity and the like, and also has the advantages of operational simplicity, low preparation cost and convenience in clinical application.

The invention discloses a CIK (Cytokine-Induced Killer Cell) capable of knocking down endogenous PD-1 (programmed death 1) expression and a preparation method and application thereof. The invention provides a method for preparing a CIK preparation capable of knocking down endogenous PD-1 expression, comprising the following steps: A) inducing the differentiation of PBMC to CIK in vitro by using cytokines; B) transferring a vector capable of knocking down endogenous PD-1 expression into the CIK obtained in step A), culturing for 13 to 16 days and harvesting a cell, thereby obtaining the CIK preparation capable of knocking down the endogenous PD-1 expression, wherein the vector capable of knocking down endogenous PD-1 expression contains a plurality of tandem encoded genes targeting miRNAs of PD-1, and the number of the encoded genes is 2 to 30. Experimental results show that the vector can significantly reduce the expression level of endogenous PD 1 in the CIK, thereby significantly enhancing the tumor-killing activity of a CIK cell.

The invention belongs to the immunobiology and biological medicine fields, and relates to a polypeptide for enclosing a TGF-beta acceptor or an IL-10 acceptor, a pharmaceutical composition and an application. Concretely, the invention relates to the polypeptide or a polypeptide combination, an amino acid sequence is one or more shown as the following (1)-(4), 1) the amino acid sequence shown in a SEQ ID NO: 6, 2) the amino acid sequence shown in a SEQ ID NO: 14, 3) the amino acid sequence shown in a SEQ ID NO: 25, and 4) the amino acid sequence shown in a SEQ ID NO: 37. The polypeptide can effectively enclose the TGF-beta acceptor or the IL-10 acceptor, thereby the tumor killing activity of the T cell (especially the antigenic specificity T cell) is enhanced, and the polypeptide has potential as a medicine for treating or assistantly treating prostate.

The invention discloses a peripheral blood NKT cell culture solution and a culture method. The NKT cell culture method comprises the following steps of adding peripheral blood mononuclear cells into a lymphocyte serum-free culture medium of autoserum containing cytokines, and culturing for about 14 days; in the culture period, a culture solution containing cell factors needing to be added every 2-3 days; after culture is finished, performing centrifugal treatment and cleaning with a 0.9% sodium chloride solution to obtain peripheral blood NKT cells; adding human serum albumin into the peripheral blood NKT cells, and fixing the volume by using a 0.9% sodium chloride solution to obtain the peripheral blood NKT cells. According to the culture method, a peripheral blood single core has no immunological rejection reaction on autoserum, cells are purer, and the safety is higher; the NKT cells are fast in growth and high in amplification rate; and the NKT cell killing activity is high, and a relatively strong tumor cell killing effect is achieved.

The invention relates to an isolated culture method of malignant pleural effusion-sourced TIL cells. The isolated culture method comprises the following steps: using at least one of PD-1, LAG-3 and TIM-3 as a marker for isolating tumor-specific T lymphocytes from TIL cells, isolating the tumor-specific T lymphocytes from a TIL cell population in combination with an immunomagnetic bead technology, and performing in-vitro efficient amplification to obtain tumor-specific TIL cells with high tumor-killing activities. By the isolated culture method, the isolated culture of the tumor-specific TIL cells is simpler in operational process and the isolated culture time is significantly shortened; the isolated culture method is low in cost and convenient to popularize and apply; in addition, the tumor-killing activities of the isolated tumor-specific TIL cells are stronger.

The invention discloses a quasi-chimeric antigen receptor-modified CIK (cytokine induced killer) as well as a preparation method and the application thereof. The method for preparing a quasi-chimericantigen receptor-modified CIK cell preparation comprises the following steps: silencing 5'-UTR, 3'-UTR and / or an intracellular region of a CTLA4 mRNA in the CIK by using multiple serially-connected miRNAs, and expressing a CTLA4 extracellular region-CD28-41BB-CD3 fusion protein, so as to prepare the quasi-chimeric antigen receptor-modified CIK cell preparation. An experiment shows that the carriercan greatly reduce the expression of endogenesis CTLA4 in a CIK cell, and after the expressed CTLA4 extracellular region-CD28-41BB-CD3 fusion protein is combined with B7 on the surface of a cell T, the CIK cell is activated, thereby obviously improving the tumoricidal activity of the CIK cell.

The invention relates to application of a polypeptide XZZ-5 to induction and enhancement of tumor killing activity of CIK (Cytokine Induced Killer) cells. A research shows that the polypeptide XZZ-5 is used for inducing CIK or DC-CIK cells, and the multiplication and differentiation of the cells can be effectively enhanced, so that the tumor killing activity of the cells is improved; the application is hopefully developed to a brand new biological therapy manner.

The invention provides a gingseng extract product FQR1 as well as a preparation and an application thereof, and provides a FQR1 induced culture method for DC-CIK cell. The method comprises the following steps: a collection of peripheral blood, an acquisition of tumor antigens, a separation of mononuclear cells, a washing, an induction of DC-CIK cells, a culture, etc. The obtained gingseng extract product FQR1 can effectively increase propagation of DC-CIK cells, improve tumor killing activity of DC-CIK cells, and prolong the survival time with tumor of tumor-bearing mice with the DC-CIK cell therapy.

The invention provides an astragalus extract FQR-8 and a preparation method and application thereof and further provides a method for using the FQR-8 to induce and culture CIK or DC-CIK cells. The preparation method comprises the steps of peripheral blood collection, tumor antigen obtaining, mononuclear cell separation, washing, induction and culture of the DC-CIK cells and the like. The astragalus extract FQR-8 prepared by means of the preparation method can effectively promote proliferation of the CIK cells, improve the tumor killing activity of the DC-CIK cells and prolong the survival time of a tumor-bearing mouse treated by using the DC-CIK cells.

The invention relates to the technical field of biological cells, in particular to an immune cell capable of secreting a bispecific antibody and application of the immune cell; the immune cell capableof secreting the bispecific antibody comprises an immune cell expression chimeric antigen receptor and expresses a secreted bispecific antibody. By secreted bispecific antibodies, two negative feedback pathways of PD-1 and CTLA-4 are blocked, so that negative regulation and control of a tumor microenvironment on immune cells are weakened, depletion of the immune cells is reduced, the tumor killing function of the immune cells is enhanced, and inhibition of the tumor microenvironment on the immune cells is reduced.

The invention relates to HPK1-targeted gRNA and an editing method aiming at an HPK1 gene. According to HPK1gRNA and the editing method of the HPK1 gene, the HPK1 gene of the T cell can be knocked out,the killing activity of the T cell can be improved, the Th1 cell factor level of a peripheral blood monouclear cell can be increased; and by knocking out the HPK1 gene of the T cell, the expression of PD-1 and TIM3 on the surface of the T cell can be decreased, and the failure of the T cell can be inhibited. Therefore, HPK1-targeted gRNA can be used for preparing tumor treatment drug and can be particularly applied to targeting therapy methods (CRA-T, CAR-NK, CAR-NKT and CAR-gammadelta) for chimeric antigen receptor cell tumors.

The invention relates to the technical field of biological cells, in particular to an immune cell and application thereof. A chimeric antigen receptor is expressed on the cytomembrane of the immune cell, and in addition, an immune checkpoint antibody protein fused with a cytokine receptor is also expressed on the cytomembrane. The expression CAR cell of the immune checkpoint antibody protein fused with the cytokine receptor is also expressed on the cytomembrane; through the immune checkpoint antibody protein which is fused with the cytokine receptor and is expressed on the immune cytomembrane, two negative feedback approaches PD-1 and CTLA-4 are blocked so as to weaken negative regulation and control of a tumor microenvironment for the immune cell, reduce exhaustion of the immune cell, enhance the tumor killing function of the immune cell and reduce inhibition of the immune cell by the tumor microenvironment; and in addition, through binding with the cytokine receptor, corresponding JAK and STAT signal channels are activated, and the cells are induced to secrete various active substances, including IFN-[gamma], perforin, granzyme and the like so as to inhibit growth of tumor cells, enhance an anti-tumor capacity, reduce the exhaustion and reduce the inhibition of the immune cell by the tumor microenvironment.

The invention relates to the technical field of cell culture, and in particular to a cell separation culture solution and a T cell separation culture method. The cell separation culture solution includes a culture solution A, a culture solution B, and a culture solution C, and the three culture solutions cooperate in synergy and work together, and are conducive to stimulating transformation of monocytes to CIK cells. According to the cell separation culture solution and the T cell separation culture method, by adding neutrophil separation fluid to blood cells, content of neutrophils in the cells is reduced; after mononuclear cells are extracted by using the neutrophil separation liquid, most of red blood cells in mononuclear cell layer are removed by using a red blood cell lysis liquid, content of the red blood cells in the mononuclear cells is obviously reduced, and number of lymphocytes is increased; after the final culture, effective cell proportion in the cells is increased, and tumoricidal activity of cell population is increased.

The invention discloses a method for preparing a high-toxicity CIK (cytokine-induced killer) cell by combining a novel TLR7 agonist GD. The method includes the steps of 1) collecting peripheral venous blood to obtain mononuclear cells; 2) adding the novel TLR7 agonist, a small molecule compound GD to perform CIK cell induced culture; 3) performing CIK cell multiplication culture to obtain a novel heterogeneous cell population, namely a GD-CIK cell. The method has the advantages that compared with a CIK cell cultured by a conventional method, the GD-CIK cell prepared by the method is obviously higher in tumor killing cytotoxic activity and is up to above 95% in tumor killing rate (effector-target ratio: 20:1); the method is low in preparation cost, simple in process, easily controllable in condition, low in requirement on equipment, easy for scale production and the like; the GD-CIK cell prepared by the method is mainly applied to cancer patient treatment or prevention of high-risk groups of cancers.

The invention discloses non-cell-derived polypeptide-sensitized DC-CIK cells, and a construction method and application thereof. In a plurality of typical embodiments, the construction method comprises the following steps: acquiring peripheral blood, and carrying out isolating to obtain mononuclear cells; isolating mononuclear cells to obtain monocytes and lymphocytes; inducing the differentiationof the monocytes into mature polypeptide-sensitized DC; inducing the differentiation of the lymphocytes into CIK cells; and subjecting the DC and CIK cells to co-culture so as to obtain the polypeptide-sensitized DC-CIK cells. The DC-CIK cells prepared in the invention have high proliferation activity, can specifically kill tumors and are high in tumoricidal activity; and the construction methodhas low requirements on equipment, and is simple in process, easily controllable in conditions and suitable for large-scale implementation.

The invention discloses an immune cell for expressing a cytokine receptor fusion type chimeric antigen receptor and application of the immune cell. A cytokine receptor fusion type chimeric antigen receptor is expressed on a cell membrane of the immune cell, and a cytokine receptor intracellular domain is fused in an intracellular domain structure of the cytokine receptor fusion type chimeric antigen receptor. The immune cell can enhance the proliferation capacity of the cell, reduce the influence of a tumor microenvironment and enhance the tumor killing activity of the immune cell under the condition of receiving antigen stimulation, so that the negative regulation of the tumor microenvironment on the immune cell is weakened, the exhaustion of the immune cell is reduced, and corresponding JAK and STAT signal channels are activated by combining with a cytokine receptor, the cells are induced to secrete various active substances such as IFN-gamma, perforin and granzyme, so that the growth of tumor cells is inhibited, the anti-tumor capability is enhanced, and the inhibition of a tumor microenvironment on immune cells is reduced.

The invention relates to the technical field of biological medicine and oncology, in particular to a composite exosome loaded with membrane-bound tumor necrosis factor-related apoptosis-inducing ligand and small-molecule antitumor drug as well as a preparation method and application of the composite exosome. The membrane surface of the composite exosome carries TRAIL protein, and the small-molecule antitumor drug is entrapped in a membrane. A preparation process of the composite exosome is mature, efficient, good in reproducibility and low in cost. The composite exosome has the advantages of obvious in-vitro and in-vivo anti-tumor effects, low drug dosage, no toxic or side effect and good biological safety, and provides a new strategy for clinical treatment of tumors.

The invention discloses a CIK modified by similar chimeric antigen receptor, as well as a preparation method and application thereof. The method comprises the following steps: transferring a recombinant vector which can knock down endogenous PD-1 expression and can further express PD1 extracellular region-CD28-41BB-CD3 fusion protein into CIK, and harvesting cells after culturing is performed, soas to obtain a CIK cell preparation; ensuring that the recombinant vector contains 5'-UTR and 3'-UTR of a plurality of targeted PD-1mRNA in series connection and / or encoding gene of intracellular region miRNA. Experiments prove that the vector can greatly reduce the endogenous PD-1 expression in a CIK cell, and the expressed PD1 extracellular region-CD28-41BB-CD3 fusion protein activates the CIK cell after being combined with PD-L1 / L2 on the surface of tumor cell, so that the tumoricidal activity of the CIK cell is obviously improved.

The invention provides a DC-CIK (dendritic cells-cytokine-induced killers) cell treatment composition derived from umbilical cord blood, wherein DC (dendritic cells) and CIK (cytokine-induced killers) cells are derived from single karyocyte of the same umbilical cord blood. The invention also provides a preparation method of the DC-CIK cell treatment composition. According to the cell treatment composition prepared by adopting the method, CIK cells are high purity and strong in multiplication capacity; the DC is high in purity and strong in antigen presentation capability, so that CIK cell multiplication can be effectively promoted, and the tumor killing activity of the CIK cell can be strengthened; compared with the simple CIK cells, the DC-CIK cells are higher in percentage of CD3<+> CD56<+> cells, and the tumor cell killing activity is remarkably higher.