AbstractDisorganized intercellular junctions are critical for maintaining the integrity of solid epithelial tumors and prevent the infiltration of oncological therapies into the bulk of the malignancy. We have developed small, recombinant proteins which bind a critical junction protein, desmoglein 2, triggering the transient and specific opening of tumor tight junctions allowing for infiltration of the tumor with immune cells, oncolytic viruses, drugs, and other therapeutics. Our new molecule, JOC-x, is a promising candidate for a new class of tumor-targeting agents that accumulate both around and within tumors and remodel the tumor microenvironment. Native cysteines were removed from the parental protein, JO-4, followed by addition of a single cysteine to allow for convenient attachment of various payloads that can be targeted directly to the tumor. Our tumor-targeting protein exhibits high avidity, minimal aggregation, and is easily purified at good yields from E. coli. For proof of concept, we demonstrate effective conjugation to biotin as a model for flexible co-targeting, addition of metal ion chelators as models for imaging and radiotherapy, and linkage of the TLR3 agonist poly(I:C) as a model immune-oncologic agent. This second-generation cancer co-therapeutic protein is optimized for activity and primed for cGMP manufacture in preparation for upcoming clinical studies.