Provided is a ROBO1 CAR-NK cell carrying a suicide gene, and a preparation method and application thereof. In order to increase the safety and controllability of a CAR-NK therapy, on the basis of a present ROBO1 CAR-NK cell, a suicide gene switch element is integrated into a genome by means of a lentiviral transfection technology to form a CAR-NK carrying a suicide gene. By adding the suicide gene, the CAR-NK cell can be better controlled, and the clinical safety is further improved.

An object of the invention is to provide a novel pharmaceutical composition. The pharmaceutical composition of the disclosure contains a DNA encoding a suicide gene having at least one intron sequence. The intron sequence has a donor sequence or an acceptor sequence to be used in a tumor cell with abnormal splicing not in a normal cell. In a transcript of the DNA, the suicide gene is expressed when the intron is abnormally spliced and the suicide gene is not expressed when the intron is not abnormally spliced.

Provided herein are immune cells expressing antigenic receptors, such as a chimeric antigen receptor and a T cell receptor. Further provided herein are methods of treating immune-related disorder by administering the antigen-specific immune cells.

Provided herein are methods for cell therapy by modifying transfused cells to express an inducible caspase 9 protein, so that the cells may be selectively killed if the patient experiences dangerous side effects. Provided also within relates in part to methods for preventing or treating Graft versus Host Disease by modifying T cells before administration to a patient, so that they may be selectively killed if GvHD develops in the patient.

Provided herein are modified caspase-9 polypeptides, and chimeric caspase-9 proteins containing the modified caspase-9 polypeptides. The disclosure further provides polynucleotides encoding these proteins, engineered host cells containing these polynucleotides and proteins, including host cells that co-express a chimeric antigen receptor, and methods of making and using the same.

The technology relates in part to methods for controlling elimination of therapeutic cells, for example, cells that express a chimeric antigen receptor. The technology further relates to a two-step method of controlling destruction of therapeutic cells in a patient following an adverse event. The two-step system may include a rapamycin or rapamycin analog-based level of control and a second, rimiducid, level of control. The technology also relates in part to methods for cell therapy using cells that express the inducible caspase polypeptide and the rapamycin-sensitive polypeptide, where the proportion of therapeutic cells eliminated by apoptosis is related to the choice and amount of the administered ligand.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome of a cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing enzymes or enzyme domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 and nucleic acid editing enzymes or domains, are provided.

Disclosed are a gene expression cassette for expressing an N-terminal-methionine-truncated target protein comprising a nucleic acid encoding a target protein and a nucleic acid encoding a cysteine protease, in which a cysteine protease recognition sequence is inserted between methionine (Met), which is the first amino acid at the N-terminus of the target protein, and the second amino acid and N-terminal methionine is cleaved with the cysteine protease, and a method of producing an N-terminal-methionine-truncated target protein using the same.

The technology relates generally to the field of immunology and relates in part to methods for activating T cells and other cells resulting in an immune response against a target antigen. The technology also relates to costimulation of therapeutic cells that express chimeric antigen receptors that recognize target antigens using chimeric MyD88- and CD40-derived polypeptides. The technology further relates in part to therapeutic cells that express chimeric antigen receptors, wherein the chimeric antigen receptors have an endodomain that includes MyD88- and CD40-derived polypeptides, and methods for treating patients using the modified therapeutic cells.

We disclose various improvements for compositions of genetically-modified T cells which include a suicide switch. For instance, the composition may comprise CD4+ T cells and CD8+ T cells, wherein the ratio of CD4+ T cells to CD8+ T cells is less than 2.