Determination of Slow-binding HDAC Inhibitor Potency and Subclass Selectivity

Histone deacetylases (HDACs) 1-3 regulate chromatin structure and gene expression. These three enzymes are targets for cancer chemotherapy and are studied for the treatment of immune disorders and neurodegeneration, but there is a lack of selective pharmacological tool compounds to unravel their individual roles. Potent inhibitors of HDACs 1-3 often display slow-binding kinetics, which causes a delay in inhibitor-enzyme equilibration and may affect assay readout. Here, we compare the potency and selectivity of slow-binding inhibitors measured by discontinuous and continuous assays. We find that entinostat, a clinical candidate, inhibits HDACs 1-3 by a two-step, slow-binding mechanism with lower potencies than previously reported. In addition, we show that RGFP966, commercialized as HDAC3-selective probe, is a slow-binding inhibitor with inhibitor constants of 57 nM, 31 nM, and 13 nM against HDACs 1-3, respectively. These data highlight a need for thorough kinetic investigation in the development of selective HDAC probes.

Discovery of TSC-101: A First-in-Class Natural HA-2-Specific TCR to Treat Leukemia Following Hematopoietic Stem Cell Transplant Therapy

Background Approximately 50% of AML patients relapse following allogeneic hematopoietic stem cell transplant therapy, leaving them with very few treatment options (Rautenberg et al. (2019) Int. J. Mol. Sci. 20:228). Rare patients who naturally develop a minor antigen-specific graft-versus-leukemia T cell response show substantially lower relapse rates (Marijt et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2742-2747; Spierings et al. (2013) Biol. Blood Marrow Transplant. 19:1244-1253). HA-2 (YIGEVLVSV, genotype RS_61739531 C/C or T/C) is an HLA-A*02:01- and haematopoietically-restricted minor histocompatibility antigen derived from the class I myosin protein, MYO1G (Pierce et al. (2001) J. Immunol. 167:3223-3230). Patients receiving donor lymphocyte infusion from HA-2-mismatched donors who develop HA-2-specific T cells show a graft vs leukemia response and often experience long-term remission (Marijt et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100:2742-2747), making HA-2 an ideal candidate for TCR-engineered T cell immunotherapy of liquid tumors. Methods Using TScan's proprietary ReceptorScan platform, we discovered 1,302 HA-2-specific TCRs by screening 237 million naïve CD8 + T cells from 5 healthy HA-2-negative donors. We evaluated these TCRs using our proprietary DexScan platform to select the 15 TCRs with the highest surface expression and greatest affinity for the HA-2 peptide when transferred into primary human T cells. We further tested each TCR individually in our clinical vector backbone for surface expression, selective cytotoxicity, cytokine production, and proliferation using a panel of cell lines that express varying levels of HLA-A*02:01 and MYO1G. Finally, the top 5 TCRs were evaluated for alloreactivity using an array-based screen assessing 108 MHC-I molecules individually, and for off-target cross-reactivity using our proprietary genome-wide TargetScan platform. A lead TCR with limited alloreactivity and a narrow off-target profile was selected as our lead TSC-101 TCR. The avidity of TSC-101 for its putative off-targets was further measured in peptide-pulsed experiments to better appreciate the toxicity risks associated with our lead clinical candidate. Results and Conclusion Of the 1,302 HA-2-specific TCRs identified by our ReceptorScan platform, we identified TSC-101 as the most active TCR. TSC-101 displayed no alloreactivity to 107/108 HLAs tested and limited off-target risks in a genome-wide screens. Potential off-target peptides identified for TSC-101 displayed extremely weak avidities, predicting an absence of toxicity risks for our clinical candidate. Based on these results, TSC-101 has been advanced to IND-enabling activities to prepare for first-in-human testing in 2022. To our knowledge, this is the first clinical grade HA-2-specifc TCR being developed for immunotherapy for liquid tumors. Disclosures Macbeath: TScan Therapeutics: Current Employment, Current equity holder in publicly-traded company.