Myosin binding protein C (MyBP-C) is expressed in striated muscles, where it plays key roles in the modulation of actomyosin cross-bridges. Slow MyBP-C (sMyBP-C) consists of multiple variants sharing common domains but also containing unique segments within the NH2 and COOH termini. Two missense mutations in the NH2 terminus (W236R) and COOH terminus (Y856H) of sMyBP-C have been causally linked to the development of distal arthrogryposis-1 (DA-1), a severe skeletal muscle disorder. Using a combination of in vitro binding and motility assays, we show that the COOH terminus mediates binding of sMyBP-C to thick filaments, while the NH2 terminus modulates the formation of actomyosin cross-bridges in a variant-specific manner. Consistent with this, a recombinant NH2-terminal peptide that excludes residues 34-59 reduces the sliding velocity of actin filaments past myosin heads from 9.0 ± 1.3 to 5.7 ± 1.0 μm/s at 0.1 μM, while a recombinant peptide that excludes residues 21-59 fails to do so. Notably, the actomyosin regulatory properties of sMyBP-C are completely abolished by the presence of the DA-1 mutations. In summary, our studies are the first to show that the NH2 and COOH termini of sMyBP-C have distinct functions, which are regulated by differential splicing, and are compromized by the presence of missense point mutations linked to muscle disease.

An artificial cell-signaling pathway is developed that capitalizes on the T-cell’s innate extravasation ability and transforms it into a vector (T-cell Biofactory) for synthesizing calibrated amounts of engineered proteins in vivo. The modularity of this pathway enables reprogramming of the T-cell Biofactory to target biomarkers on different disease cells, e.g. cancer, viral infections, autoimmune disorders. It can be expected that the T-cell Biofactory leads to a “living drug” that extravasates to the disease sites, assesses the disease burden, synthesizes the calibrated amount of engineered therapeutic proteins upon stimulation by the diseased cells, and reduces targeting of normal cells.

We have developed a set of genetically engineered isogenic cell-lines to express either Folate Receptor alpha (FRa) or Mesothelin (MSLN), and a control cell-line negative for both antigens. These cell lines also expressed fluorescent and bioluminescent reporter transgenes. The cell lines were used to authenticate specificity and function of a T-cell Biofactory, a living vector that we developed to express proportionate amounts of engineered proteins upon engaging with disease cells through their specific antigenic biomarkers. The engineered cell lines were also used to assess the cytolytic function and specificity of primary T cells engineered with chimeric antigen receptors (CAR T cells); and the specificity of monoclonal antibodies. The strategy described can be used to generate other cell lines to present different disease-specific biomarkers for use as quality control tools.

Current protein-based cancer therapies have several disadvantages, which include general toxicity, lack of response at the administered dose and differing responses from patients to patient. To circumvent these issues, we propose a platform in which engineered T-cells specifically recognize tumors and secrete therapeutic peptides directly at the disease site leading to targeted cancer cell killing. This approach will increase the specificity of the therapy and decrease toxicity to healthy cells. We transformed acute T-cell lymphoma cells into biofactories for site-specific synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. The effector T-cell line was engineered by stably introducing a chimeric T-cell receptor to recognize Folate-Receptor alpha (FRα) or Mesothelin (MSLN) protein. Upon binding of the effector T-cells to the receptor, an intracellular cascade directed expression of non-human proteins is induced. Specific T-cell binding to human ovarian cancer cell lines and signaling was measured by in vitro co-culture using luciferase production as a surrogate for therapeutic peptide secretion. We demonstrated that T-cells can be genetically programed to synthesize and secrete proportionate amounts of engineered proteins upon engaging the tumor-associated antigens (FRα or MSLN) on a human ovarian cancer cell line, OVCAR3 (FRα+MSLN+). A FRα-MSLN- ovarian cancer cell line, A2780cis, was used as the non-targeted negative control. The difference in protein secretion following stimulation by the two cell lines, as measured by luciferase activity, was statistically significant within 1 hour. It reached ~35-fold within 1 to 3 days, and we observed stable expression for at least 10 days. The luminescent signal was proportionate to the number of OVCAR3 cells. To further validate the specificity of target engagement, we generated A2780cis-FRα positive and A2780cis-MSLN positive cell lines and demonstrated selective binding and activation of the corresponding effector cells in co-culture assays. No binding was detected to the A2780cis-vector control cells. In vivo results for T-cell biofactories targeting OVCAR3 tumors 24 hours post-stimulation validated the in vitro results. Currently, we are engineering T-cell biofactories to release cytotoxic peptides and are assessing their therapeutic efficacy against cancer cells in vitro using co-culture assays and supernatant transfer. Our results show that T-cells can be genetically reprogrammed to serve as biofactories for the synthesis of therapeutic proteins upon stimulation by antigen-presenting disease cells. Importantly, these studies demonstrate the feasibility of developing the next generation of adoptively transferred T-cell therapies to target tumors that express FRα (e.g., ovarian, breast, lung) and/or MSLN (e.g. ovarian, lung, pancreatic) on their cell surfaces for cancer therapy. Citation Format: Claire E. Repellin, Puja Patel, Lucia Beviglia, Harold Javitz, Lidia C. Sambucetti, Parijat Bhatnagar. Assessing the therapeutic efficacy of disease-specific T-cell biofactories [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2557.