Background T-Plex is an autologous TCR-T cell therapy product comprising customized combinations of 2–3 TCR-T cell components that recognize different tumor antigens presented on specific HLA class I molecules. Each component of T-Plex is engineered using a transposon-based vector encoding the therapeutic TCR, CD8α and CD8β co-receptors, a CD34 epitope tag, a dominant-negative TGFβRII (DN-TGFβRII), and a mutated form of dihydrofolate reductase (DHFRdm). TSC-200-A0201 is intended for the treatment of HPV16+ HLA-A*02:01+ cancers. HPV16 is an oncogenic virus responsible for ~57% of cervical cancers and ~21% of head and neck squamous cell carcinomas. HPV16 E7 oncoprotein drives oncogenic transformation of infected cells and is not expressed by healthy tissues, making it a compelling immunotherapeutic target. Methods The TSC-200-A0201 TCR is a naturally occurring TCR discovered using TScan's proprietary ReceptorScan platform. TSC-200-A0201 TCR-T cells engineered using a full-scale representative workflow for the planned manufacturing process were used to investigate the in-vitro pharmacology and toxicology of TSC-200-A0201. TSC-200-A0201 was evaluated for avidity and target-dependent cytotoxicity, proliferation, and cytokine secretion in vitro as well as for anti-tumor efficacy in vivo. The contribution of DN-TGFβRII was assessed by testing the ability of TSC-200-A0201 TCR-T cells to resist the immuno-suppressive effects of TGFβ. Further, TSC-200-A0201 was assessed for risk of alloreactivity and off-target recognition using TScan's SafetyScan screen. Finally, to assess the risk of off-target/off-tumor activity, TSC-200-A0201 TCR-T cells were tested for their reactivity to an extensive panel of 74 healthy human primary and iPSC-derived cells from tissues that are traditionally assessed in toxicology studies, including those expressing high levels of the putative off-targets of TSC-200-A0201. Results TSC-200-A0201 displayed high avidity for the cognate peptide (EC50 of ~4.2 pg/mL) and target-dependent secretion of inflammatory cytokines, killing of target cells, and proliferation of both engineered CD4+ and CD8+ T cells. Moreover, TSC-200-A0201 successfully controlled the growth of HLA-A*02:01+ HPV16+ tumors in mice. Target-dependent IFN-γ production and proliferation of TSC-200-A0201 was maintained in the presence of physiological levels of TGFβ. Further, TSC-200-A0201 displayed no alloreactivity to the 110 most common class I HLAs in the US population. Although a few putative off-targets were identified in the SafetyScan screen, TSC-200-A0201 showed no reactivity to normal primary or iPSC-derived cells. Conclusions TSC-200-A0201 exhibits high specificity and potency. Based on these results, TSC-200-A0201 has been cleared by the U.S. FDA for clinical development and has been incorporated in the T-Plex Phase 1 clinical trial master protocol. Ethics Approval Animal studies were performed at CR Discovery Services, a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). This facility complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care. IACUC number: EB17–010-301.

Background The cancer/testis antigen PRAME exemplifies an ideal TCR-T cell therapy target due to its high expression in multiple malignancies and its absence in normal tissues. Initially identified in metastatic cutaneous melanoma,1 PRAME is highly expressed in various additional solid tumors including lung, head & neck, and ovarian cancers. PRAME plays a pivotal role in multiple cellular processes and has been demonstrated to exhibit protumorigenic function primarily through inhibition of retinoic acid receptor signaling.2 Targeting of PRAME in solid tumors, particularly when performed as part of a TCR-T multiplexing strategy, represents a promising therapeutic approach in the treatment of many cancer indications Methods We discovered TCRs specific for 5 different A*02:01-restricted PRAME-derived epitopes using TScan's proprietary ReceptorScan platform. Using an activation-based screening technology termed ActivScan, we identified the most functional TCRs from a library of 1300 PRAME-specific TCRs to select for TCRs with greatest avidity and expression. These highly active TCRs were examined for their cytotoxic function using a panel of PRAME-expressing A*02:01-positive cell lines. Lead TCRs were assessed for potential off-target reactivity using our proprietary SafetyScan platform, in which off-target recognition of antigens derived from all proteins that comprise the human proteome is evaluated. Safety was further confirmed by examining alloreactivity to high-frequency class I HLAs and by testing TCR reactivity to a panel of normal primary human cells. Lastly, we tested TCR-T cells for their ability to control tumor growth in vivo using PRAME-expressing xenograft mouse models. Results We screened 871 million naïve CD8+ T cells from 16 unique healthy donors in ReceptorScan to identify 5706 TCRs specific for 5 PRAME epitopes. PRAME425–434-specific TCRs demonstrated superior recognition of a PRAME-expressing cell line compared to all other PRAME epitopes tested. Following selection of high-expressing and high avidity PRAME425–434-specific TCRs in ActivScan, TCRs were evaluated for their cytotoxic function, and two TCRs compared favorably to a clinical-stage benchmark TCR with respect to cytotoxicity, cytokine release, and T cell proliferation. Safety assessment demonstrated that few off-target peptides were recognized by lead TCRs, minimal alloreactivity was observed to 110 allotypes tested, and no reactivity to normal primary human cells was found. PRAME425–434-specific TCR-T cells were also able to control tumor growth in vivo following infusion into immunodeficient mice implanted with PRAME-expressing xenografts. Conclusions Based on its demonstrated activity in vitro and in vivo, this autologous TCR-T cell therapy candidate, TSC-203-A02, has been advanced to IND-enabling studies. References Ikeda H, Lethe B, Lehmann F, van Baren N, Baurain JF, de Smet C, et al. Characterization of an antigen that is recognized on a melanoma showing partial HLA loss by CTL expressing an NK inhibitory receptor. Immunity. 1997;6(2):199–208. Epping MT, Wang L, Edel MJ, Carlee L, Hernandez M, Bernards R. The human tumor antigen PRAME is a dominant repressor of retinoic acid receptor signaling. Cell. 2005;122(6):835–47.

Abstract 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.

Background Approximately 30–40% of AML patients relapse following allogeneic hematopoietic stem cell transplant therapy, leaving them with very few treatment options.1 2 Rare patients that naturally develop an HA-1-specific graft-versus-leukemia T cell response, however, show substantially lower relapse rates.3 4 HA-1 (VLHDDLLEA, genotype RS_1801284 A/G or A/A) is an HLA-A*02:01-and hematopoietically restricted minor histocompatibility antigen, making it an ideal candidate for TCR immunotherapy for liquid tumors.5 Methods We developed a high-throughput TCR discovery platform that enables rapid cloning of antigen-specific TCRs from healthy donors. We then used this platform to screen 178.3 million naïve CD8+ T cells from six unique HA-1- (VLRDDLLEA, genotype RS_1801284 G/G) donors, identifying 329 HA-1-specific TCRs. We tested each TCR for expression and the ability to kill HA-1+ target cells, using a previously published, clinical-stage HA-1-specific TCR as a benchmark for these studies.6 In parallel, we tested TCR constant region modifications to promote expression and proper pairing of exogenous TCR alpha and beta chains and designed a lentiviral vector to co-deliver CD8 coreceptors as well as a CD34 enrichment tag to enable purification of engineered T cells. The top 11 candidates were cloned into our optimized backbone and evaluated for cytotoxicity, cytokine production, and T cell proliferation using a panel of HLA-A*02:01+ HA-1+ cell lines. Finally, the top two TCRs were evaluated for allo-reactivity and off-target cross-reactivity using our proprietary genome-wide T-Scan platform. Results The TCR discovery and evaluation platform described here identified 329 HA-1-specific TCRs from a total of 178.3 million naïve T cells, and TSC-100 as the most active TCR. Defined mutations in the constant region of TSC-100 enhanced its surface expression while decreasing expression of endogenous TCRs, and co-introduction of CD8 enabled efficient engagement and function of engineered CD4 cells. Overall, TSC-100 exhibited comparable activity to a clinical-stage benchmark TCR when challenged with cell lines expressing moderate to high levels of HA-1, and superior activity when incubated with cell lines expressing low levels of both HA-1 and MHC-I.6 In addition, TSC-100 exhibited no detectable allo-reactivity to 108 different HLA types tested, and minimal off-target effects when challenged with a genome-wide library expressing peptides derived from human proteins. Conclusions TSC-100 exhibits comparable or superior activity to a clinical-stage therapeutic TCR, with minimal allo-reactivity or off-target effects. Based on these results, TSC-100 has been advanced to IND-enabling activities to prepare for first-in-human testing in 2021. Ethics Approval All clinical samples used in the study were collected by STEMCELL Technologies, StemExpress and HemaCare using their IRB approved protocols. References Pavletic SZ, Kumar S, Mohty M, et al. NCI First International Workshop on the Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation: Report from The Committee on the Epidemiology and Natural History of Relapse following Allogeneic Cell Transplantation. Biol Blood Marrow Transplant. 2010;16(7):871–890. Miller JS, Warren EH, van den Brink MR, et al. NCI First International Workshop on The Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation: Report from the Committee on the Biology Underlying Recurrence of Malignant Disease following Allogeneic HSCT: Graft-versus-Tumor/Leukemia Reaction. Biol Blood Marrow Transplant 2010;16(5):565–586. Marijt WAE, Heemskerk MHM, Kloosterboer FM, et al. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia. PNAS 2003;100:2742–2747. Spierings E, Kim Y, Hendriks M, et al. Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-Matched related and unrelated hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2013; 19: 1244–1253. Bleakley M, Riddell SR. Exploiting T cells specific for human minor histocompatibility antigens for therapy of leukemia. Immunol Cell Biol 2011;89(3):396–407. Dossa RG, Cunningham T, Sommermeyer D, et al. Development of T-cell immunotherapy for hematopoietic stem cell transplantation recipients at risk of leukemia relapse. Blood 2018;131(1):108–120.