The present invention relates to methods for developing engineered T-cells for immunotherapy that are non-alloreactive. The present invention relates to methods for modifying T-cells by inactivating both genes encoding T-cell receptor and an immune checkpoint gene to unleash the potential of the immune response. This method involves the use of specific rare cutting endonucleases, in particular TALE-nucleases (TAL effector endonuclease) and polynucleotides encoding such polypeptides, to precisely target a selection of key genes in T-cells, which are available from donors or from culture of primary cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.

A method of expanding TCRalpha deficient T-cells by expressing pTalpha or functional variants thereof into said cells, thereby restoring a functional CD3 complex. This method is particularly useful to enhance the efficiency of immunotherapy using primary T-cells from donors. This method involves the use of pTalpha or functional variants thereof and polynucleotides encoding such polypeptides to expand TCRalpha deficient T-cells. Such engineered cells can be obtained by using specific rare-cutting endonuclease, preferably TALE-nucleases. The use of Chimeric Antigen Receptor (CAR), especially multi-chain CAR, in such engineered cells to target malignant or infected cells. The invention opens the way to standard and affordable adoptive immunotherapy strategies for treating cancer and viral infections.

The present invention includes compositions and methods for modifying a T cell with a nucleic acid encoding a switch molecule comprising an extracellular domain comprising a membrane receptor or fragment thereof and an intracellular domain comprising a signaling receptor or fragment thereof. In one aspect, a method comprises introducing a nucleic acid encoding a switch molecule and a nucleic acid encoding a soluble fusion protein and/or a nucleic acid encoding a bispecific antibody into a population of cells comprising T cells, wherein the T cells transiently expresses the switch molecule and soluble fusion protein or bispecific antibody. In other aspect, compositions of T cells and methods of treating a disease or condition, such as cancer or an autoimmune disease, are also included.

The present disclosure relates in some aspects to methods, cells, and compositions for preparing cells and compositions for genetic engineering and cell therapy. Provided in some embodiments are streamlined cell preparation methods, e.g., for isolation, processing, incubation, and genetic engineering of cells and populations of cells. Also provided are cells and compositions produced by the methods and methods of their use. The cells can include immune cells, such as T cells, and generally include a plurality of isolated T cell populations or types. In some aspects, the methods are capable of preparing of a plurality of different cell populations for adoptive therapy using fewer steps and/or resources and/or reduced handling compared with other methods.

The invention provides improved T cell compositions and methods for manufacturing T cells. More particularly, the invention provides methods of T cell manufacturing that result in adoptive T cell immunotherapies with improved survival, expansion, and persistence in vivo.

The present invention provides compositions and methods for immunotherapy in human. The invention includes a B cell receptor like complex expressed in T cells and comprising an extracellular antigen recognition domain, a trans-membrane domain, a CD79αβ heterodimer, and a signaling region that controls T cell activation. The extracellular antigen recognition domain and trans-membrane are derived from the same human or humanized B cell receptor and form a single unit in the complex. The signaling region comprises a T cell signaling domain in combination with a co-stimulatory signaling domain. The signaling region is fused to the CD79αβ heterodimer. Furthermore, the B cell receptor like complex of the present invention can use a targeting molecule as a bridge between cytotoxic T cells and targeted cells.

FOXP3.sup.+ regulatory T cells (Tregs) can represent powerful adoptive immunotherapies for autoimmune diseases, metabolic diseases, and other chronic inflammatory diseases. The present invention is related to the ability to maintain and expand stable Treg lines and can provide insight into FOXP3.sup.+ Treg physiology and can enable feasible strategies of Treg-based immunotherapy.

The present invention relates to compositions and methods comprising cell surface markers for pluripotent-derived cells, in particular, pancreatic endoderm-type cells, derived from pluripotent stem cells.

A system that easily and stably separates various living cells from a living body-derived tissue, the system including: a mincing unit that minces the living body-derived tissue based on a parameter; a measurement unit that acquires information regarding the living body-derived tissue being minced; and an analysis unit that calculates a ratio of impurities to the living body-derived tissue being minced from the information acquired by the measurement unit. Methods for separating various living cells from a living body-derived tissue are also disclosed.

The present invention provides a cell which co-expresses a first chimeric antigen receptor (CAR) and second CAR at the cell surface, each CAR comprising an antigen-binding domain, wherein the antigen-binding domain of the first CAR binds to CD19 and the antigen-binding domain of the second CAR binds to CD22.