Abstract
Disorganized 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.
Highlights
Junction Opener 1 (JO-1) is a protein that binds to DSG2 and preferentially opens tumor tight junctions[7] (Fig. 1)
We reintroduced a single cysteine to act as a designer sulfhydryl-based site for targeted conjugation at various locations within the proteins as follows: in GB3 the cysteine was added at the C-terminus following a flexible glycine serine linker [(G3S)3]; in GB4 and GB10, the single cysteinyl residue was added at the N-terminus either before (GB4) or immediately following (GB10) the His tag; and for GB6, the cysteine was introduced proximal to the glutamic acid residue at position aa 299 within the H-I loop (PECTT, underlined in Fig. 1a), a key region known to stabilize DSG2 binding
We have described a new approach to exposing tumors to the action of the immune system and oncological therapy using designer proteins that bind to DSG2 and open tumor tight junctions
Summary
Junction Opener 1 (JO-1) is a protein that binds to DSG2 and preferentially opens tumor tight junctions[7] (Fig. 1). These proteins did not produce the desired tight-junction opening effects possibly due to inability of the knob protein to form active higher-order quaternary structures[10,11]. To overcome this obstacle, a dimerization domain (DD) was added to facilitate multimerization[10]. Depolarization of cells during EMT as www.nature.com/scientificreports well as upregulation in DSG2 expression in tumors relative to normal tissues makes for an attractive target for our molecules
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