Mechanistic Characterization of Cancer-associated Fibroblast Depletion via an Antibody-Drug Conjugate Targeting Fibroblast Activation Protein.

Cancer-associated fibroblasts (CAF) are a prominent cell type within the tumor microenvironment (TME) where they are known to promote cancer cell growth and survival, angiogenesis, drug resistance, and immunosuppression. The transmembrane prolyl protease fibroblast activation protein (FAP) is expressed on the surface of highly protumorigenic CAFs found in the stroma of nearly every cancer of epithelial origin. The widespread expression of FAP has made it an attractive therapeutic target based on the underlying hypothesis that eliminating protumorigenic CAFs will disrupt the cross-talk between components of TME resulting in cancer cell death and immune infiltration. This hypothesis, however, has never been directly proven. To eliminate FAP-expressing CAFs, we developed an antibody-drug conjugate using our anti-FAP antibody, huB12, coupled to a monomethyl auristatin E (huB12-MMAE) payload. After determining that huB12 was an effective targeting vector, we found that huB12-MMAE potently eliminated FAP-expressing cells as monocultures in vitro and significantly prolonged survival in vivo using a xenograft engineered to overexpress FAP. We investigated the effects of selectively eliminating CAFs using a layered, open microfluidic cell coculture platform, known as the Stacks. Analysis of mRNA and protein expression found that treatment with huB12-MMAE resulted in the increased secretion of the proinflammatory cytokines IL6 and IL8 by CAFs and an associated increase in expression of proinflammatory genes in cancer cells. We also detected increased secretion of CSF1, a cytokine involved in myeloid recruitment and differentiation. Our findings suggest that the mechanism of FAP-targeted therapies is through effects on the immune microenvironment and antitumor immune response. SIGNIFICANCE: The direct elimination of FAP-expressing CAFs disrupts the cross-talk with cancer cells leading to a proinflammatory response and alterations in the immune microenvironment and antitumor immune response.

Creating a 'Molecular Band-Aid'; Blocking an Exposed Protease Target Site in Desmoplakin.

Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, a subcellular complex that links the cytoskeleton of one cell to its neighbor. A mutation 'hot-spot' within the NH2-terminal third of the DSP protein (specifically, residues 299-515) is associated with both cardiomyopathies and skin defects. In select DSP variants, disease is linked specifically to the uncovering of a previously-occluded calpain target site (residues 447-451). Here, we partially stabilize these calpain-sensitive DSP clinical variants through the addition of a secondary single point mutation-tyrosine for leucine at amino acid position 518 (L518Y). Molecular dynamic (MD) simulations and enzymatic assays reveal that this stabilizing mutation partially blocks access to the calpain target site, resulting in restored DSP protein levels. This 'molecular band-aid' provides a novel way to maintain DSP protein levels, which may lead to new strategies for treating this subset of DSP-related disorders.