Abstract: 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.

Abstract: 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.

Abstract: Described herein are single chain trimer (SCT) polypeptides comprising or consisting essentially of a target peptide, a first linker, at least a portion of a beta-2 microglobulin domain, a second linker, and at least a portion of a major histocompatibility complex (MHC) I alpha chain, or pharmaceutically acceptable derivatives thereof. The SCT polypeptides may further include a leader peptide, e.g., a PHO5, SUC2, app8, or HLA A2 leader sequence at the N-terminus of the target peptide. Further described herein are polypeptide compositions comprising or consisting essentially of a first polypeptide comprising a target peptide, and a second polypeptide comprising at least a portion of a beta-2 microglobulin domain, a second linker, and at least a portion of a major histocompatibility complex (MHC) I alpha chain, a third linker, and a tether peptide, or pharmaceutically acceptable derivatives thereof. The first polypeptide and/or the second polypeptide may further include a leader peptide, e.g.

Abstract: The present invention features compositions and methods for enhancing endosomal escape of therapeutic agents in the cytoplasm of a cell to thereby provide more effective therapy. The endocytic pathway is the major uptake mechanism of cells, and biological agents such as proteins, DNA, and siRNA become entrapped in endosomes and subsequently degraded by lysosomal enzymes. The present invention facilitates endosomal escape by providing fusion proteins with membrane disruptive activity. In some embodiments, the fusion protein has endosomolytic activity that is activated at endosomal or lysosomal pH. In other embodiments, the fusion protein has reversible and pH sensitive membrane disruptive activity so that once internalized into the endosomal or lysosomal compartments, the resulting reduction in pH causes the pH sensitive fusion protein to activate and disrupt the endosomal or lysosomal membrane.

Abstract: The present invention features compositions and methods for delivering a therapeutic agent to the cytoplasm of a cell. We have developed, inter alia, a system in which two or more distinct moieties—at least one therapeutic moiety and at least one potentiating moiety—selectively target and specifically bind cell surface molecules that are then internalized to an intracellular, membrane-bound compartment, such as an endosome. In some embodiments, as discussed further below, a third moiety that induces clustering of the targeted cell surface molecule can also be employed. Regardless of whether the compositions and methods include two or three moieties, the therapeutic agent can be any agent one wishes to deliver to the cytoplasm of a cell, and the potentiating agent can be any agent that destabilizes the intracellular, sub-cellular compartment in which the therapeutic agent is sequestered. The potentiating moiety can include, for example, a lytic agent (i.e.