Disease relapse after allogeneic hematopoietic cell transplantation (HCT) affects ∼40% of patients and has high mortality. A potential solution is to target antigens mismatched between transplant recipients and donors. TSC-100 and TSC-101 are allogeneic donor derived T-cell receptor-engineered T cells that target HA-1 and HA-2 hematopoietic cell antigens respectively, both presented on HLA-A*02:01. By choosing donors who are either HLA-A*02:01 or HA-1 negative, TSC-100 or TSC-101 selectively eliminate all residual patient hematopoietic cells post-HCT and prevent relapse (figure 1).TSCAN-001 (NCT05473910) is a multi-center, multi-arm, controlled Phase 1 study evaluating TSC-100 and TSC-101 in adult AML, MDS or ALL eligible for reduced intensity conditioning-based haploidentical donor transplantation. HLA-A*02:01 and HA-1 or HA-2-positive patients receive either TSC-100 or TSC-101 after HCT. HLA-A*02:01-negative control arm patients receive HCT alone. Upon count recovery after HCT, treatment arm patients receive single or repeated doses of TSC-100 or TSC-101.At submission, 11 patients were enrolled, 7 in treatment arms (4 TSC-101, 3 TSC-100) and 4 in the control arm, with a median follow-up of 162 days post-HCT (range 2-339 days). No DLTs occurred, and patients were enrolled at Dose Level 3 in both TSC-100 and TSC-101 arms. Safety was similar in treatment and control arms with expected post-HCT adverse events. Graft-versus-host disease (GvHD) was similar in control (3 events) and treatment arms (4 events) and serious adverse events of GvHD or infections were observed in all arms. No cytokine release syndrome or neurotoxicity occurred after TSC-100/101 with minimal increases in CRP/ ferritin. No clinical relapses or deaths occurred to date.Translational analysis found peak TSC-100/101 expansion and activation 7-14 days post dosing, with ongoing persistence at longest follow-up of 203 days. Chimerism analysis of 8 patients with >100 days follow-up (figure 2) using a high-sensitivity NGS-based AlloHeme assay in whole blood, CD33+ or CD3+ cells found complete donor chimerism (>99.87%) after Day 42 in all cells in all 5 out of 5 TSC-100/101 patients (100%) compared with 1 out of 3 control patients (33%). A control patient required early withdrawal of immunosuppression for increasing mixed chimerism. Post-HCT MRD by NGS (LOD <0.05%) was detected in 1 control and none of the treatment arm patients.The prognostic value of complete donor chimerism by AlloHeme was evaluated in the ACROBAT study (NCT04635384) in which 73 patients achieved >99.9% chimerism in CD33+ cells and, after median follow-up of 9 months/ 270 days (range 72-608 days), 3 relapsed (4%), comparing favorably with 18-20% 6-month relapse rates in CIBMTR data. In summary, TSC-100 and TSC-101 post-HCT induce MRD negativity and complete donor chimerism which may be associated with substantially reduced relapse rates.

Abstract Checkpoint immunotherapies have revolutionized solid tumor treatment yet durably benefit a minority of patients, as they rely on endogenous anti-tumor T cells. A potential solution for patients lacking functional endogenous anti-tumor T cells is engineering their T cells with exogenous T cell receptors (TCRs) to target and kill tumor cells. Initial clinical trials with TCR engineered T cell therapies (TCR-Ts) targeted single tumor antigens on single HLA and produced partial, short-lasting responses. Solid tumors are notoriously heterogenous with highly variable antigen expression. Recent discoveries also identified HLA loss of heterozygosity in up to 40% of solid tumors, allowing tumor cells to evade T cell attack. To overcome this heterogeneity, TScan has developed T-Plex, a multiplexed cell therapy comprising 2-3 different TCR-Ts, chosen from a collection of TCR-Ts called the ImmunoBank, to target different tumor antigens on different HLA types with confirmed tumor expression. To deepen clinical responses, TCR-T cells are engineered to express CD8α/β co-receptors that, in preclinical experiments, enable CD4+ helper T cells to have >100-fold improved cytotoxicity and cytokine secretion over CD4+ cells expressing the TCR alone. Finally, to allow T cell persistence despite immunosuppressive TGF-β in the solid tumor microenvironment, TCR-T cells also express the dominant negative TGF-βreceptor, enabling ~10-fold improved proliferation in the presence of TGF- β compared to T cells expressing the TCR and CD8α/β co-receptors alone. A proprietary transposon vector with larger cargo limit enables the inclusion of these additional genes. The Phase 1 study utilizes a separate screening protocol to pre-identify patients with head and neck, cervical, anogenital cancers, NSCLC, and melanoma, any time during standard clinical care, enabling rapid enrollment into the treatment protocol upon disease progression. Screening comprises germline HLA testing, and archival tumor testing for antigen expression and exclusion of HLA loss. Treatment includes standard non-myeloablative lymphodepletion followed by one or 2 doses of T-Plex infused 28 days apart. Dose escalation under the interval 3+3 design starts with testing single TCR-Ts in dose levels 1 and 2. Thereafter, TCR-Ts are combined and escalated in dose levels 3 and 4. TCR-Ts currently in the master protocol target PRAME, MAGE-A1 or HPV16 on HLA-A*02:01 or MAGE-A1 on HLA-C*07:02. Additional TCR-Ts added to the ImmunoBank and master protocol go through dose levels 1 and 2 as single therapies before becoming available for multiplexed dose levels 3 and 4. Primary endpoints include safety, feasibility and identifying the recommended Phase 2 dose. Secondary endpoints are rates and durations of response and exploratory endpoints measure T cell activation and persistence. Two additional TCR-Ts are on track to be added to the ImmunoBank, which could allow 50-80% of common solid tumor patients to qualify for multiplexed TCR-T therapy. Citation Format: Justin Moser, Brian Pico, Brian Henick, Rom Leidner, Jared Weiss, James Isaacs, Jaspreet Grewal, Michael Hurwitz, Jim Murray, Marlyane Motta, Yun Wang, Shrikanta Chattopadhyay, Debora Barton, Gavin MacBeath, Sajeve Thomas. Trial in progress: A phase 1, first in human clinical trial for T-Plex, a multiplexed, enhanced T cell receptor-engineered T cell therapy (TCR-T) for solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT170.

Current regulation of T cell receptor (TCR)-based therapeutics may require repeated testing of patients for specific HLA alleles as well as companion diagnostics development, despite the invariant nature of the HLA genotype and availability of robust clinical HLA tests. This increases the burden on patients and the organizations developing these products. We propose regulatory flexibility to facilitate the development of and access to TCR-based therapeutics.

Introduction: To date, engineered T cell therapies have not proven effective for non-B cell hematologic malignancies due to a lack of antigens that spare healthy myeloid cells. Allogeneic hematopoietic cell transplantation (HCT) remains the best curative option for these malignancies, yet ~40% of patients relapse post-HCT with high mortality after relapse. A potential solution to prevent relapse is to target hematopoietic lineage-specific minor histocompatibility antigens (MiHAs) that are genetically mismatched between HCT patients and donors. These mismatches enable engineered T cells to selectively eliminate residual patient hematopoietic cells, normal or malignant, and spare donor cells. TSC-100 and TSC-101 are allogeneic, donor derived T-cell receptor-engineered T (TCR-T) cells that target MiHAs HA-1 and HA-2 respectively, both presented on HLA-A*02:01. By choosing HCT patients who are HLA-A*02:01 positive and donors who are either HLA-A*02:01 or MiHA negative, TSC-100 or TSC-101 are designed to eliminate residual patient hematopoietic cells post-HCT and thus prevent relapse (figure). Methods: Study TSCAN-001 (NCT05473910) is a multi-center, multi-arm non-randomized, controlled Phase 1 study evaluating the feasibility, safety and preliminary efficacy of TSC-100 and TSC-101. Adults with AML, MDS or ALL eligible for reduced intensity conditioning (RIC)-based haploidentical donor transplantation from HLA or MiHA mismatched donors are enrolled. HLA-A*02:01 and HA-1 or HA-2-positive patients receive either TSC-100 or TSC-101 after HCT. HLA-A*02:01-negative control arm patients receive HCT alone. Upon count recovery after HCT, patients in treatment arms receive a single dose of TSC-100 or TSC-101 at Dose Level 1 or repeat doses at Dose Levels 2 and 3. Dose escalation follows interval 3+3 rules. Primary endpoints include adverse event profiles and dose-limiting toxicities (DLTs). Secondary endpoints are relapse rates, disease-free and overall survival. Exploratory endpoints include biomarkers of efficacy such as minimal residual disease (MRD) by deep next-generation sequencing (NGS), and donor chimerism using novel high-sensitivity and standard assays. Results: At submission time, 8 patients were enrolled, 5 in treatment arms (3 TSC-101, 2 TSC-100) and 3 in the control arm, with a median of 111 days post-HCT follow-up (range 33-266 days). No DLTs occurred, and patients were enrolled at Dose Level 2 in both TSC-100 and TSC-101 arms. Safety analysis found expected post-HCT adverse events similar in treatment and control arms. Incidence of graft-versus-host disease (GvHD) was similar in control (3 events) and treatment arms (4 events). Serious adverse events of grade 3 GvHD and infections were observed in TSC-101 and control arms (1 event each). No cytokine release syndrome or neurotoxicity occurred after TSC-100/101 and minimal changes in CRP/ ferritin occurred, consistent with general safety of TCR-Ts with low target cell burden. No clinical relapses occurred to date. Translational analysis found peak TSC-100/101 expansion and activation 7-14 days post dosing, with ongoing persistence at longest follow-up of 138 days. In repeat dose cohorts, substantial increases of early expansion were noted after dose 2 of TSC-101/100, supporting the benefit of repeat dosing. Chimerism analysis (table) using a novel high-sensitivity NGS-based AlloHeme assay (limit of detection (LOD) 0.13%) in whole blood, CD33+ or CD3+ subsets found mixed chimerism in 2 out of 2 control patients but complete donor chimerism in 4 out of 4 TSC-100/101 patients after Day 42. Both control patients had declining donor chimerism after Day 100 by AlloHeme and standard STR-based chimerism assays (LOD 1-2%) prompting early withdrawal of immunosuppression in one patient. Marrow MRD analysis by NGS (LOD <0.05%) found 1 control patient was MRD positive pre-HCT and remained MRD positive post-HCT. In contrast, 2 TSC-100/101 patients who were MRD positive pre-HCT turned MRD negative after HCT and TSC-100/101 therapy including a TP53 mutated MDS patient confirmed MRD negative by droplet digital PCR (LOD 0.01%). Conclusions: Targeting MiHAs HA-1 or HA-2 with TSC-100/ 101 following HCT shows early safety and biomarker evidence of efficacy by completing elimination of all detectable patient hematopoietic cells, normal or malignant, thereby reducing relapse risk. Updated results will be presented at the meeting.

TPS2678 Background: Engineered T cell therapies such as CAR-T cell therapies have transformed the treatment of B-cell but not non-B cell hematologic malignancies. Allogeneic hematopoietic cell transplantation (HCT) remains the best curative option for hematologic malignancies but ~40% of patients relapse post-HCT with up to 90% mortality due to residual disease post-HCT. A potential solution is targeting minor histocompatibility antigens (MiHAs) that are homogenously expressed on all hematopoietic cells and are genetically mismatched between donors and patients undergoing HCT. These mismatches enable engineered T cells to selectively eliminate residual patient hematopoietic cells, normal or malignant, leaving donor cells untouched. TScan has developed allogeneic donor derived T-cell products TSC-100 and TSC-101, targeting MiHAs HA-1 and HA-2 respectively, both presented on HLA-A*02:01. By choosing HCT patients who are HLA-A*02:01 positive (>98% of whom are either HA-1 or HA-2 positive) and donors who are either HLA-A*02:01 or MiHA negative, TSC-100 or TSC-101 can potentially eliminate all residual patient-derived hematopoietic cells after HCT, to prevent disease relapse. Methods: Study NCT05473910 is a multi-center, multi-arm, non-randomized controlled Phase 1 umbrella study evaluating the feasibility, safety and preliminary efficacy of TSC-100 and TSC-101. Inclusion criteria include adults with AML, MDS or ALL eligible for reduced intensity conditioning-based haploidentical donor transplantation from HLA or MiHA mismatched donors. HLA-A*02:01-positive patients undergo HA-1/ HA-2 testing and are assigned to either TSC-100 or TSC-101 treatment arms in addition to HCT. HLA-A*02:01-negative patients in the control arm receive HCT alone. Upon count recovery after HCT, patients in treatment arms receive either TSC-100 or TSC-101, administered as single or two doses. Primary endpoints include adverse event profiles and dose limiting toxicities. Secondary endpoints include relapse rates, disease-free survival and overall survival. Exploratory endpoints include surrogates of efficacy such as donor chimerism rates and kinetics and minimal residual disease (MRD) rates. Donor chimerism is measured by standard STR-based and novel high-sensitivity NGS-based assays to quantify residual patient-derived hematopoietic cells. MRD is measured before and after HCT using flow cytometry and NGS. Together, these assays measure elimination of residual patient hematopoietic cells, malignant or normal, and could provide early evidence of biological activity. Clinical trial information: NCT05473910 .

Background Engineered T cell therapy holds great promise for treating solid tumors. To date, clinical investigations of TCR-T cell therapies have targeted one antigen/HLA at a time and have produced encouraging but partial response rates with limited durations. While heterogeneity of antigen expression is appreciated as a likely driver of patient relapse, the contribution of HLA loss of heterozygosity (LOH), occurring in up to 40% of tumors, is only now gaining attention. To address both antigen heterogeneity and HLA LOH requires a collection of TCRs recognizing multiple targets presented on multiple HLAs. MAGE-A1 is a cancer-testes antigen previously identified as the target of expanded tumor infiltrating T-cells using TScan's screening technology. Currently, TScan has two MAGE-A1-TCR-T products, recognizing epitopes on A*02:01 and C*07:02 approved for clinical development. Here we report discovery and lead selection of a MAGE-A1 TCR recognizing an epitope on A*01:01 (~24% population frequency). Methods We discovered TCRs specific for an A*01:01- restricted MAGE-A1-derived epitope using TScan's proprietary ReceptorScan platform. Using an activation-based screening technology termed ActivScan, we screened a library of MAGE-A1-specific TCRs to select for greatest avidity and expression. These TCRs were functionally characterized using a panel of MAGE-A1 expressing A*01:01-positive cell lines and a xenograft mouse model. Lead TCRs were assessed for potential off-target reactivity using our proprietary SafetyScan platform, which evaluates recognition of antigens from all proteins that comprise the human proteome. Safety was further evaluated by examining alloreactivity to high-frequency Class I HLAs and by testing TCR reactivity to normal primary human cells and cell lines. Results ReceptorScan identified 1181 TCRs specific for the MAGE-A1 A*01:01 epitope. Following selection of high-expressing and high avidity MAGE-A1-specific TCRs in ActivScan, 14 TCRs were evaluated for their cytotoxic function, and 5 TCRs compared favorably to a clinical-stage benchmark TCR for cytotoxicity and cytokine release. Safety assessment demonstrated that few putative off-target peptides were recognized, minimal alloreactivity was observed to 110 allotypes tested, and no reactivity to target-negative cell lines were observed. Conclusions A novel HLA-A*01:01 restricted TCR-T cell therapy candidate has advanced to pre-clinical studies. Addition of this product to TScan's ImmunoBank (collection of TCRs) would extend MAGE-A1 TCR-T therapy for solid tumors on three different HLA alleles, potentially expanding the addressable patient population. Importantly, this creates a unique opportunity to simultaneously target tumor antigens presented on HLA alleles on different chromosomes, thus circumventing tumor evasion by HLA LOH with the goal of improving patient responses. Ethics Approval The Testing facility specifically 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. The animal care and use program at Explora, now CR Discovery Services, is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), which assures compliance with accepted standards for the care and use of laboratory animals. IACUC number: EB17–010-301.

2554 Background: Checkpoint immunotherapies have revolutionized the treatment of solid tumors yet durably benefit a minority of patients because they rely on endogenous anti-tumor T cells. In patients lacking functional anti-tumor T cells, a solution is engineering their T cells with exogenous T cell receptors (TCRs) to efficiently target and kill tumor cells. Initial clinical trials with TCR engineered T cell therapies (TCR-Ts) only produced partial, short-lasting responses because they targeted single tumor antigens. Solid tumors are notoriously heterogenous not only with highly variable antigen expression levels but also with recently identified HLA gene losses occurring in up to 40% of solid tumors, enabling tumor cells to evade T cell attack. Methods: To overcome this heterogeneity, TScan has developed T-Plex, a multiplexed therapy comprising 2-3 different TCR-Ts, chosen from a collection of TCR-Ts called the ImmunoBank, to target different tumor antigens presented on different HLA types with confirmed tumor expression. Results: To deepen clinical responses, TCR-T cells are engineered to express the CD8α/β co-receptors that, in preclinical experiments, enable CD4+ helper T cells to become >100-fold more cytotoxic and secrete cytokines at >100-fold lower target antigen levels. Finally, to enable T cell persistence despite immunosuppressive TGF-β in the tumor microenvironment, engineered TCR-T cells express the dominant negative TGF-β receptor allowing ~10-fold improved proliferation and ~2-fold improved cytokine production in the presence of TGF-β. The inclusion of these additional genes is enabled by a transposon manufacturing system with no cargo limit. Patient selection for the Phase 1 study uses a separate screening protocol to identify patients any time during standard clinical care so they can rapidly enroll into the treatment protocol upon progression. Screening comprises germline HLA testing, then archival tumor testing for antigen expression and HLA loss. Treatment includes standard lymphodepletion followed by 2 doses of T-Plex infused 28 days apart. T-Plex initially starts with 2 TCR-Ts targeting different MAGE-A1 antigens presented either on HLA-A*02:01 or HLA-C*07:02. Dose escalation starts with testing the single TCR-Ts individually in dose levels 1 and 2. The two TCR-Ts will then be combined and escalated in dose levels 3 and 4. Additional TCR-Ts added to the ImmunoBank will go through dose levels 1 and 2 as single therapies before becoming available for multiplexed dose levels 3 and 4. Primary endpoints include safety and feasibility, secondary endpoints are response rates and duration of response and exploratory endpoints measure T cell persistence. Conclusions: Four additional TCR-Ts are on track to be added to the ImmunoBank in 2023 which makes 50-80% of patients with common solid tumors eligible for multiplexed TCR-T therapy.

Heregulin is expressed in MCF-7Ca and LTLT-Ca cells

Quantified target bands from Figure 2

Brief descriptions of the supplementary figure data provided

Background TCR-engineered T cell therapy has shown encouraging response rates in solid tumors, but complete responses are rare and partial responses are often short-lived. We submit that the primary reason underlying these results is that solid tumors exhibit heterogeneous target expression and HLA loss is common. Consequently, tumor cells that lack or lose the targeted antigen are resistant to single-targeted TCR-T therapies and drive relapse. To address these challenges, TScan has developed clinical trial assays to assess target expression and HLA loss in patient tumors. These assays enable prospective patient selection and assignment of treatment with multi-targeted TCR-T therapy. T-Plex is a multiplexed TCR-T cell product consisting of customized combinations of 2–3 TCR-T cell components selected from a pre-existing collection of TCR-Ts. Methods To enable T-Plex, TScan is developing an ImmunoBank of TCRs targeting MAGE-A1, HPV16, PRAME, and two additional undisclosed targets across multiple HLAs. TScan and Neogenomics have developed IHC and RNA-ISH assays to assess target expression in FFPE tumor samples. In addition, TScan and Tempus have developed a novel NGS-based pan-HLA-A/B/C Loss of Heterozygosity (LOH) algorithm to assess partial or clonal loss of HLA class I alleles in solid tumors. Results Analysis of >150 tumor samples revealed the prevalence of MAGE-A1, HPV16, and PRAME across various solid tumor types. For example, PRAME expression was observed in 95% of melanoma samples, but only in 55% of NSCLC and HNSCC. Furthermore, the intensity and uniformity of expression varied considerably. H-scores for PRAME ranged from 66–300 (melanoma), 5–170 (NSCLC) and 2–135 (HNSCC). Similarly, MAGE-A1 expression was observed in 40% of melanomas and 20% of NSCLC and HNSCC. H-scores for MAGE-A1 varied considerably, ranging from 1–200 (melanoma), 1–50 (NSCLC) and 3–180 (HNSCC). Notably, co-expression of PRAME and MAGE-A1 was observed in ~31%, ~10% and ~9% of melanomas, NSCLC, and HNSCC, respectively. Heterogeneity of HLA expression was also observed. Data collected at Tempus showed that clonal and subclonal loss of HLA occurs in approximately 14% and 29% of melanomas, 23% and 16% of NSCLC, and 27% and 14% of HNSCC. Importantly, HLA-A/B/C alleles were almost always lost together, indicating that HLA loss most frequently occurs through haplotype loss, informing a strategy to direct multiplexed TCR-T to the remaining HLA haplotype. Conclusions Overall, these data highlight the importance of a multiplexed TCR-T cell therapy targeting various intact tumor antigens presented on intact HLA alleles in order to effectively address solid tumors. Ethics Approval The data presented in this abstract does not meet the definition of human subject research and animals were not used in this study.

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.

Pharmacodynamic analyses of HRG and ERBB3 expression in tumors harvested post-treatment

Background Engineered T cell therapies such as CAR-T cell therapies have transformed the treatment of B-cell but not non-B cell hematologic malignancies. Allogeneic hematopoietic cell transplantation (HCT) remains the best curative option for hematologic malignancies but ~40% of patients relapse post-HCT with up to 90% mortality due to residual disease post-HCT. A key challenge in non-B cell malignancies is identifying the right antigens for T cell targeting. Cancer-associated antigens are heterogenous, enabling rapid escape of malignant cells with low antigens, while targeting lineage-specific antigens without distinct expression in malignant versus normal myeloid cells can cause prolonged neutropenia. A potential solution is targeting minor histocompatibility antigens (MiHAs) that are homogenously expressed on all hematopoietic cells and are genetically mismatched between donors and patients undergoing HCT. These mismatches enable T cells to selectively eliminate residual patient hematopoietic cells, normal or malignant, leaving donor cells untouched. TScan has developed allogeneic donor derived T-cell products TSC-100 and TSC-101, targeting MiHAs HA-1 and HA-2 respectively, both presented on HLA-A*02:01. By choosing HCT patients who are HLA-A*02:01 positive (>98% of whom are either HA-1 or HA-2 positive) and donors who are either HLA-A*02:01 or MiHA negative, TSC-100 or TSC-101 can potentially eliminate all residual patient-derived hematopoietic cells after HCT, sparing donor cells, to prevent disease relapse. Methods Study NCT05473910 is a multi-center, multi-arm, non-randomized controlled Phase 1 umbrella study evaluating the feasibility, safety and preliminary efficacy of TSC-100 and TSC-101. Inclusion criteria (figure 1) include adults with AML, MDS or ALL eligible for reduced intensity conditioning-based haploidentical donor transplantation from HLA or MiHA mismatched donors. HLA-A*02:01-positive patients undergo HA-1/HA-2 testing and are assigned to either TSC-100 or TSC-101 treatment arms in addition to HCT. HLA-A*02:01-negative patients in the control arm receive HCT alone. Upon count recovery after HCT, patients in treatment arms receive either TSC-100 or TSC-101, administered as single or two doses. Primary endpoints include adverse event profiles and dose limiting toxicities. Secondary endpoints include relapse rates, disease-free survival and overall survival. Exploratory endpoints include surrogates of efficacy such as minimal residual disease (MRD) rates and donor chimerism rates and kinetics. MRD is measured before and after HCT using flow cytometry, NGS and ddPCR. Donor chimerism is measured by standard STR-based and novel high-sensitivity NGS-based assays to quantify residual patient-derived hematopoietic cells. Together, these assays measure elimination of residual patient hematopoietic cells, malignant or normal, and could provide early evidence of biological activity. Trial Registration NCT05473910 Ethics Approval The study obtained ethics approval from WCG-IRB (20220488). Participants will give/have given informed consent before taking part.

Dosing everolimus in combination with letrozole and seribantumab causes weight loss in mice

Background TCR-T adoptive cell therapy is a promising approach to treating solid tumors, but the heterogeneous expression of TCR targets by the tumor and T cell evasion mechanisms are barriers to durable responses. HLA heterogeneity further limits the addressable population. Multiplexing TCR-T products offers a unique strategy to address the heterogenous landscape of targets presented by a diverse array of HLAs but requires the identification of novel epitopes presented on unaddressed HLAs. TScan's proprietary platform, TargetScan, is an unbiased method to discover the natural targets of T cell clones responding to tumors. Methods The target landscape recognized by the most expanded T cell clones derived from clinical melanoma TIL therapy products was evaluated using TScan's TargetScan platform. TCR Targets were identified using a strategy of screening TCRs from the most expanded T cell clones against a peptidome wide library, and the next most frequent T cell clones against a focused cancer testis antigen (CTA) library. Using this approach, TCRs recognizing targets with a favorable tissue expression profile for targeting with a TCR-T therapy were identified. Individual TCRs were cloned and evaluated for cytotoxicity, cytokine release, and T cell proliferation in response to co-culture with cancer cell lines expressing their cognate antigens. Results Peptidome wide screens of the10 most expanded TCRs revealed several known targets including the A*02:01 presented MART126–35 epitope as well as previously unknown cancer associated targets with limited tissue specific off tumor expression including brain tissue. CTA focused screens identified novel targets including a novel clinically relevant B*07:02 presented epitope of the cancer testis antigen MAGEC2. The reactive TCR was identified and exhibited cytotoxicity, cytokine release, and T cell proliferation when co-cultured with B*07:02 expressing cells including a thyroid cancer cell line FTC133 and melanoma cell lines A101D and SKLMS1 with the degree of the response corresponding to the level of MAGEC2 expression in the cell lines. Conclusions Using our TargetScan platform, we have shown that expanded T cell clones from clinical TIL products express TCRs that recognize tumor associated targets; the novel B*07:02 restricted epitope of MAGEC2 is a promising target for TCR-T therapy potentially enabling us to target 20% of the US patient population. TargetScan mediated discovery of novel epitopes across a diverse set of HLAs will further enable a multiplexing approach to TCR-T therapy. Ethics Approval Data presented in this abstract used deidentified human materials and do not meet the definition for human subjects research

ERBB3 levels are significantly higher following co-treatment with letrozole and everolimus

Background Adoptive cell transfer with genetically engineered T cells holds great promise for treating solid tumors. To date, clinical investigations of TCR-engineered T cell therapies (TCR-T) have targeted one antigen at a time and have produced response rates ranging from 30-50%. Unfortunately, complete responses have been rare, and responses are often short-lived. One possible reason why patients rapidly relapse is that their tumors exhibit substantial heterogeneity of antigen expression: not every cancer cell within a tumor expresses the target of a mono TCR therapy and, even when they do, the target is expressed at variable levels among the individual tumor cells. This suggests that TCR-T targeting one antigen could allow the cells lacking the treated antigen to escape and drive relapse. Methods To address antigen heterogeneity, we are developing multiplexed TCR-T cell therapy in which a patient is treated with multiple TCR-T cell products, chosen from a collection of pre-vetted TCRs matched to the patient's tumor antigens and HLA type. As proof-of-concept, we selected two different cancer/testis antigens targeted by two different TCRs. One of these antigens, MAGEA1, was identified as the target of expanded tumor infiltrating T-cells from a head & neck cancer patient using TScan's screening technology.1 The other one, PRAME, is highly expressed in a variety of cancers. Using our ReceptorScan platform, we developed two high affinity TCRs that recognize HLA-A*02:01-restricted epitopes from MAGEA1 and PRAME, and assessed the benefits of combining these two TCR-T cell products using a variety of pre-clinical models. Results Individually, both TCRs show strong cytotoxic activity in vitro when co-cultured with HLA-matched cancer cell lines expressing endogenous MAGEA1 and PRAME. Additionally, in xenograft mouse models, each TCR was able to control the growth of tumors expressing their cognate antigens. To test whether the two TCRs exhibit additive or synergistic activity, we developed two different tumor models. In one model, we used a cancer cell line that expresses both MAGEA1 and PRAME. In the other model, a mixture of two different cell lines expressing either MAGEA1 or PRAME were grown as xenograft tumors in mice. Notably, when treated with multiplexed MAGEA1/PRAME TCR-T, the mice achieved longer lasting tumor control compared to TCR-T targeting a single antigen. Conclusions These findings support the hypothesis that multiplexed TCR-T mimics the natural oligoclonal T-cell response to cancer and has the potential to overcome antigen heterogeneity, which may contribute to the observed lack of durability in monotherapy TCR-T clinical trials. Reference Luoma AM, Suo S, Wang Y, Gunasti L, Porter CBM, Nabilsi N, Tadros J, Ferretti AP, Liao S, Gurer C, Chen YH, Criscitiello S, Ricker CA, Dionne D, Rozenblatt-Rosen O, Uppaluri R, Haddad RI, Ashenberg O, Regev A, Van Allen EM, MacBeath G, Schoenfeld JD, Wucherpfennig KW. Tissue-resident memory and circulating T cells are early responders to pre-surgical cancer immunotherapy. Cell. 2022;185:2918-2935.