The present disclosure describes engineering of cells to co-express TNF-Related Apoptosis-Inducing Ligand (TRAIL) and Decoy Receptor 1 (DcR1). The expression of DcR1 results in competition for TRAIL binding to Death Receptors 4 and 5, thereby protecting the engineered cells from TRAIL-induced apoptosis. Such cells will exhibit longer survival such as when used in cell-based therapies for cancer.

Disclosed herein is a method for enhancing antitumor T cell responses in subjects. The method involves administering to the subject in need thereof a composition comprising a demethylating agent in an amount effective to demethylate STING proteins in the tumor cells. This method is particularly useful in subjects with deficient STING expression in the tumor cells. Therefore, also disclosed is a method for treating a tumor in a subject that involves detecting in a biopsy sample from the subject reduced STING expression, reduced cGAS expression, or a combination thereof; and then administering to the subject a demethylating agent in an amount effective to demethylate STING proteins in the tumor cells. The method can further involve administering to the subject a therapeutically effective amount of a STING agonist. The method can further involve administering to the subject tumor infiltrating lymphocytes (TILs), such as HLA-matched TILs.

Provided are methods for isolating T-cells with T cell receptors (TCRs) optimized for reactivity to specific peptides and decreased cross-reactivity to non-target peptides. Advantageously, TCRs of the invention can be optimized to target cancer antigens and peptides while having reducing reactivity to healthy cells. Methods of the invention utilize a novel combination of culturing conditions that increase T-cell activation and allow for validation of TCR activity. Culturing conditions of the invention further reduce culturing times generally needed to achieve expanded reactive T-cells. Because of the robust nature of the activation and validation conditions of the present invention, variants of identified TCRs can also be optimized and validated for their response to peptides, including cancer peptides.

The present disclosure describes, in part, a Micro-organosphere immune-oncology assay and methods of making and using same. The assay quickly measures the potency of effector immune cells, such as tumor infiltrating lymphocytes, at killing a patient's tumor cells. Understanding the potency of effector immune cells is critical for adoptive T cell therapy.

The disclosure provides immune cells comprising a first activator receptor specific to mesothelin and a second inhibitory receptor specific to a ligand that has been lost in a mesothelin-positive cancer cell, and methods of making and using same for the treatment of cancer.

A method for producing microvesicles ex vivo for use in systemic treatment of cancer. The method comprises isolating patient cancerous tumor primary cells, culturing isolated patient cancerous tumor primary cells in appropriate culture media, treating cultured patient cancerous tumor primary cells non cold atmospheric plasma, after apoptosis of cultured patient cancerous tumor primary cells occurs, collecting apoptotic cell-derived extracellular microvesicles from the culture media by differential centrifugation, directly applying apoptotic cell-derived extracellular microvesicles to one of a naïve T cell culture or a dendritic cell culture, isolating antigen specific T cells from said one of a T cell culture and a dendritic cell culture, and storing said isolated antigen specific T cells.

A method of treating a metastatic lesion that presents a peptide containing SLLQHLIGL (SEQ ID NO: 310) on a cell surface, including selecting a patient having a metastatic lesion and administering to the patient a composition containing recombinant T lymphocytes or activated T lymphocytes that express a T cell receptor, or a functional fragment thereof, that is reactive with, or binds to, an MHC ligand containing SLLQHLIGL (SEQ ID NO: 310).

Expansion of cytotoxic T lymphocytes (CTLs) is a crucial step in almost all cancer immunotherapeutic methods. Current techniques for expansion of tumor-reactive CTLs present major limitations. The present invention comprises a novel method to effectively produce and expand tumor-activated CTLs using high-voltage pulsed electric fields. Tumor cells were subjected to high-frequency irreversible electroporation (HFIRE) with various electric field magnitudes and pulse widths, or irreversible electroporation (IRE). The treated tumor cells were subsequently cocultured with CD8+ cytotoxic T cells along with antigen-presenting cells. Tumor-activated CTLs can be produced and expanded when exposed to treated tumor cells. The activated CTLs produced with the present invention could be used for clinical applications with the goal of targeting and eliminating tumors.

Disclosed herein, are methods for identifying a drug candidate for treating cancer metastasis. The method comprising culturing an embedded organoid/natural killer (NK) cell co-culture with a drug candidate; and measuring one or more metastatic properties of the embedded organoid or tumor-killing potential or tumor-promoting potential of the NK cells in the co-culture.

Composition and methods for ex vivo expansion of natural killer (NK) cells, and methods for cell-based cancer immunotherapy are disclosed. Leukemic cell-derived dendritic cells and anti-PD-L1 antibodies, and certain embodiments with addition of PBMCs are used for in vivo administration for cancer treatment. Leukemic cell-derived dendritic cells and anti-PD-L1 antibodies are also used for ex vivo expansion of NK cells.