152 results about "Cell kill" patented technology

Methods and compositions for the development of effective cancer therapies using mitotic inhibitors which have limited general toxicity to normal, non-cancerous cells and tissues are provided. The methods and compositions utilize cytotoxic compounds comprised of a cell-binding agent (e.g., antibodies) conjugated to an anti-mitotic compound (e.g., maytansinoids). The invention further provides antibodies which are substantially incapable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC), thereby ensuring that the therapeutic effect is mediated primarily by the anti-mitotic component of the cytotoxic compound, rather than by indirect cell killing via ADCC and/or CDC. The antibodies of the invention further are capable of differentiating between polypeptide antigens which are more highly expressed on proliferating cancer cells as compared to proliferating non-cancer cells.

This invention provides a non-radioactive assay to monitor and quantify the target-cell killing activities mediated by cytotoxic T lymphocytes (CTLs). This assay is predicated on the discovery that apoptosis pathway activation and, in particular, granzyme B activity, provides a measure of cytotoxic effector cell activity. In one embodiment, measurement of CTL-induced granzyme B activation in target cells is achieved through detection of the specific cleavage of fluorogenic granzyme B substrates. This assay reliably detects antigen-specific CTL killing of target cells, and provides a more sensitive, more informative and safer alternative to the standard ⁵¹Cr-release assay most often used to quantify CTL responses. The assay can be used to study CTL-mediated killing of primary host target cells of different cell lineages, and enables the study of antigen-specific cellular immune responses in real time at the single-cell level. As such, the assay can provide a valuable tool for studies of infectious disease pathogenesis and development of new vaccines and immunotherapies.

The invention relates to the field of tumor cellular immunotherapy, and in particular relates to a recombinant lentivirus and application thereof. The recombinant lentivirus comprises a chimeric antigen receptor, wherein the chimeric antigen receptor mainly comprises signal peptide, an antigen recognition domain, a transmembrane domain, an intracellular co-stimulation signal transduction domain and a CD3 zeta signal transduction domain which are serially connected; the intracellular co-stimulation signal transduction domain mainly comprises a human TLR2 (Toll Like Receptor 2) intracellular domain. A GPC3 CAT T (Glypican 3 CAT T) cell prepared from the recombinant lentivirus has an intense cell killing effect on liver cancer cells, a Th1 cell factor can be highly expressed, a tumor killing effect caused by non-CAR T (Chimeric Antigen Receptor T) cell can be stimulated to the maximum extent, escape and potential reoccurrence risk of GPC 3-tumor cells can be effectively prevented, the tumor cells can be killed by T cells expressing the chimeric antigen receptor, normal tissue can be slightly damaged, a tumor immunosuppression micro environment can be broken through, and thus a relatively good treatment effect on solid tumor can be achieved.

The present invention includes a method of measuring cytolytic activity including providing a device capable of monitoring cell-substrate impedance operably connected to an impedance analyzer, adding target cells to at least one well of the device, adding effector cells to the at least one well, monitoring impedance of the at least one well and optionally determining a cell index from the impedance, wherein monitoring impedance includes measuring impedance during at least one time point before and at least one time point after adding effector cells, and determining viability of said target cells after adding effector cells by comparing the impedance or optionally the cell index at the at least one time point after adding effector cells to the impedance or optionally the cell index at the at least one time point before adding effector cells

Cell-targeted serine protease constructs are provided. Such constructs can be used in methods for targeted cell killing such as for treatment cell of proliferative diseases (e.g., cancer). In some aspects, recombinant serine proteases, such as Granzyme B polypeptides, are provided that exhibit improved stability and cell toxicity. Methods and compositions for treating lapatinib or trastuzumab-resistant cancers are also provided.

The invention relates to a preparation method of a CAR-T cell targeting B7H3. The preparation method includes first preparing a PBMC cell; then co-transfecting a 293T cell with a shuttle plasmid LV-B7H3 containing the CAR structure, a helper plasmid psPAX2 and an envelope plasmid VSV-G to obtain a packaged B7H3-CAR virus; then taking a PBMC cell, using anti-human CD3 and anti-human CD28 as activators, culturing and activating for 48 hours and adding the B7H3-CAR virus for infection. By means of the preparation scheme, the expression of IFN-gamma in the CAR-T cell is increased, and the cell killing activity is high. The CAR-T cell targeting B7H3 has a killing effect on various solid tumor cells, has high killing activity, is safe and effective, and can be used for immunotherapy of kidney cancer, lung cancer, liver cancer, glioma, ovarian cancer, breast cancer and the like.

The invention discloses an immunity enhancing reagent comprising AAVs carrying molecule encoding genes of antibodies at immunodetection points, wherein the immunodetection points are one or several from PD-1, PD-L1 and CTLA-4. The AAVs carrying the molecule encoding genes of the antibodies at different immunodetection points are combined with each other, T cells, natural killer cells, cytotoxic T lymphocytes or cell factor induced killer cells and other immune cells are infected in vitro, and the antibodies at the immunodetection points are expressed and secreted after the infected immune cells are fed back to bodies of patients suffering from tumors, so that the combination of receptors at the immunodetection points and ligands of the receptors is blocked, the transfer of an immunosuppression signal is effectively blocked, the immune tumor suppression microenviroment is destroyed, the immune cell killing capacity is improved, and furthermore the curative effect of adoptive immune therapy is improved.

The invention provides a method for evaluating cell killing efficacy. The evaluation method comprises the following steps: (1) co-culturing a target cell carrying a fluorescence label A and an effector cell, and dyeing and labeling dead cells generated after co-culture by using a fluorescence label B, wherein the excitation light wavelengths corresponding to the fluorescence label A and the fluorescence label B are different; (2) respectively using a bright field, a fluorescence channel matched with the fluorescence label A and a fluorescence channel matched with the fluorescence label B to perform microscopic imaging on the dyed and labeled cells to obtain a microscopic image; and (3) carrying out superposition synthesis analysis on the obtained microscopic image, and evaluating the cellkilling efficacy of the effector cells according to an analysis result. According to the invention, the fluorescent reagent A and the fluorescent label B are respectively used for staining and markingthe target cells and dead cells, and then fluorescence microscopic imaging and image synthesis analysis are carried out, so that the evaluation result of the cell killing efficacy can be quickly, intuitively and accurately obtained.

Nucleic acid compositions encoding a pro-apoptotic protein, Bok (Bcl-2-related ovarian killer) are identified. Bok has conserved Bcl-2 homology domains 1, 2 and 3 and a C-terminal transmembrane region present in other Bcl-2 related proteins, but lacks the BH4 domain found only in anti-apoptotic Bcl-2 proteins. Over-expression of Bok induces apoptosis. Cell killing induced by Bok is suppressed by co-expression with selective anti-apoptotic Bcl-2 proteins. Bok is highly expressed in the ovary, testis and uterus, particularly in granulosa cells, the cell type that undergoes apoptosis during follicle atresia. Identification of Bok as a new pro-apoptotic protein with wide tissue distribution and hetero-dimerization properties facilitates elucidation of apoptosis mechanisms in reproductive and other tissues, and provides a means for manipulating apoptosis.

The invention discloses a construction method and application of an NKG2D-ACE2 CAR-NK cell secreting super IL 15. An expression vector mainly comprises an NKG2D extracellular segment, an ACE2 extracellular segment, 4-1BB, CD3[zeta] and IL15R[alpha]-IL15 thawing protein. NKG2D is an activation receptor of the NK cell and is used for identifying ligands (MICA, MICB, ULBP1-6) on the surfaces of virusinfected cells or tumor cells; ACE2 is a receptor of SARS-CoV-2 and is combined with S protein of a virus envelope; the NK cell modified by CAR is proved to be capable of breaking through the limitation of an inhibitory receptor to activate the NK cell, so that the specific killing effect of the NK cell on target cells is enhanced, and no serious toxic or side effect exists; and the activity of an IL15 super agonist is increased by 20 times compared with that of natural IL15 in the body, and the pharmacokinetic property is improved, so that the CAR-NK cell has enhanced durability and target cell killing activity. Therefore, the ACE2 and the NKG2D respectively target the S protein of the SARS-CoV-2 virus and NKG2DL on the surfaces of the infected cells, and the powerful synergistic effectof the super IL15 is achieved, so that the NKG2D-ACE2 CAR-NK cell can specifically remove SARS-CoV-2 virus particles and the infected cells, and a safe and effective cell therapy is provided for COVID-19.

The invention relates to a method for evaluating CAR-T killing activity in vitro. IN the prior art, the existing CAR-T cell in-vitro killing activity detection method relates to isotope safety, operation is tedious and other problems exist. Based on the problems in the prior art, in order to conveniently and simply realize the detection of in vitro killing activity of CAR-T cells, a cell strain capable of evaluating the CAR-T killing function in vitro is constructed, and a method for detecting the CAR-T in-vitro killing activity through RTCA is provided, wherein the CAR-T cell killing activitycan be efficiently, sensitively and continuously detected in vitro through the method so as to provide the guarantee for the subsequent treatment effect of CAR-T cells.

The invention provides a construction method of an immune cell tumor killing capacity detection model. The construction method comprises the following steps: (1) 24 after egg membranes of embryos of zebrafish fertilized for 24-96 hours are removed, injecting tumor cells labeled by a first living cell staining agent into zebrafish bodies, then executing culturing in culture water containing a melanin inhibitor, and screening out the zebrafish with the tail parts labeled by the first living cell staining agent; (2) after the collected zebrafish is anesthetized, the zebrafish is divided into an immune cell injection group and a control group, injecting immune cells labeled by a second living cell staining agent into the immune cell injection group in a microinjection mode, injecting a carriersolution into the control group, and then putting the zebrafish in the culture water containing the melanin inhibitor for continuous culture. Short period, time saving, labor saving and low modelingcost are achieved by adopting the construction method, and a constructed detection model can accurately and objectively evaluate the effect of tumor immunotherapy. The invention also provides a detection model prepared based on the method and application thereof.

The invention provides a cell killing efficacy detection method and application thereof. The detection method comprises the following steps: carrying out mixed co-culture on target cells carrying a fluorescence label A and effector cells, then adding a fluorescence label B to label the target cells and the effector cells, and adding a fluorescence label C to carry out dyeing labeling on dead cellsgenerated after co-culture; and then respectively carrying out microscopic imaging on the cells by using different fluorescence channels, identifying and analyzing the cells in the same region in a microscopic image by combining an image synthesis analysis method, and obtaining the cell killing efficacy of effector cells according to an analysis result. Therefore, the detection method can effectively eliminate the interference of impurities and cell debris, can directly obtain a plurality of data such as cell death rate, cell self-injury rate and cell specific killing rate from the image, andhas the advantages of intuitive and visible detection result, high accuracy and high sensitivity.

The invention discloses a method for efficient in-vitro amplification and culture of natural killer T cells with high killing capacity. The method comprises the following steps: a, taking fresh human peripheral blood and obtaining mononuclear cells with a gradient centrifugation method; b, pre-stimulating mononuclear cells in an in vitro medium; c, expanding culture of peripheral blood derived mononuclear cells, detecting the proportion of various cells, and judging amplification efficiency and activity of the target NK/NKT cells; d, detecting the proportion of NK/NKT cells to the cell population after culture for 3-4 weeks, and detecting cell killing efficiency; e, further purifying the NK/NKT cells. The method is simple to operate, the NK cell population rich in NKT cells with high killing activity is obtained and contains no impurity cells, immunogenicity caused by the impurity cells is avoided, and side effects in clinical application are lower. Besides, the method has the advantages that materials are easily available, the cells are suitable for large-scale culture, damage to the body is small and the like, and is more suitable for adjuvant treatment of tumor patients.

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.