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To enable interrogation of tumor HLA LOH as a clinical diagnostic for precision oncology, we developed and validated an assay that detects HLA LOH within the context of an FDA-approved clinical diagnostic test, Tempus xT CDx. Validation was conducted via: (1) analytical evaluation of 17 archival patient samples and 42 cell line admixtures and (2) independent clinical evaluation of LOH prevalence in the HLA-A gene ( HLA-A LOH) across 10,982 patients. To evaluate the prognostic relevance of HLA-A LOH we assessed 256 immunotherapy-treated non-small cell lung cancer (NSCLC) patients. To determine the feasibility of prospectively identifying and enrolling HLA-A LOH patients into a clinical trial, we established BASECAMP-1 (NCT04981119). We observed a positive predictive agreement of 97% and a negative predictive agreement of 100% in samples with ≥ 40% tumor purity. We observed HLA-A LOH in 16.1% of patients (1771/10,982), comparable to previous reports. HLA-A LOH was associated with longer survival among NSCLC adenocarcinoma patients (HR = 0.60, 95% CI [0.37, 0.96], p  = 0.032) with a trend towards shorter survival among squamous cell patients (HR = 1.64, 95% CI [0.80, 3.41], p  = 0.183). In 20 months, we prospectively screened 1720 subjects using the Tempus AWARE program, identifying 26 HLA-A*02 LOH patients at 8 sites, with 14 (54%) enrolled into BASECAMP-1. In conclusion, we developed and validated an investigational assay that detects tumor HLA LOH within an FDA-approved clinical diagnostic test, enabling HLA LOH utilization in diagnostic, prognostic, and therapeutic applications.

BackgroundTCR-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.MethodsTo 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.ResultsAnalysis 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.ConclusionsOverall, 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 ApprovalThe data presented in this abstract does not meet the definition of human subject research and animals were not used in this study.

BackgroundThe AFNT-212 cell therapy consists of autologous CD8+ and CD4+ T cells expressing 1) a T Cell Receptor (TCR) specific for the prevalent oncogenic driver KRAS G12D mutation presented by HLA-A*11:01, 2) a chimeric cytokine receptor, and 3) the CD8α/β coreceptor enabling a coordinated CD4+/CD8+ anti-tumor response to promote T cell activity while minimizing exhaustion. While viral vectors, including lentivirus (LVV), have been a standard modality to deliver transgenes for cell therapies, they are limited by cargo size and manufacturing complexity. In comparison, gene-editing mediated targeted knock-in (KI) of a non-virally delivered transgene cassette overcomes limitations of LVV-mediated delivery and improves function and safety profile of the engineered cells.MethodsHuman CD4+ and CD8+ T cells from healthy volunteers or patients were engineered by a novel CRISPR-Cas nuclease and gRNAs targeting the T-cell receptor α constant (TRAC) and T-cell receptor β constant (TRBC) genes to knock-out the endogenous TCR and simultaneously integrate a non-viral plasmid-based transgene cassette. Human CD4+ and CD8+ T cells were engineered in parallel with lentivirus encoding the same transgene cassette and CRISPR-Cas targeting TRAC and TRBC genes to knock-out endogenous TCR. Engineered T cells were assessed via KRAS G12D peptide stimulation and co-culture with KRAS G12D-expressing tumor cells for in vitro activation and cytotoxicity. In vitro safety studies were performed and in vivo efficacy studies were conducted using human KRAS G12D xenografts in NSG mice.ResultsNon-viral KI generated lower vector copy number per cell than LVV but drove higher transgene expression, suggesting the EF1α promoter within the KI construct outperforms the MSCV promoter of the LVV. T cells engineered with either non-viral KI or LVV demonstrated specific and sensitive recognition of the target KRAS G12D peptide. However, KI-engineered cells demonstrated improved cytotoxicity against endogenously-expressing HLA-A*11–01 and KRAS G12D cell lines in tumor cell rechallenge assays in vitro. KI-engineered cells also showed superior anti-tumor activity in established subcutaneous tumor bearing mice. Off-target assessment was similar for the KI and LVV products, as identified by co-incubation with all possible peptides in the human proteome matching the TCR recognition motif. Optimized KI process generated large number of transgenic cells with naïve and memory phenotype potentially suitable for clinical applications.ConclusionsNon-viral targeted KI engineered AFNT-212 cells drive a robust coordinated CD4/CD8 T cell response against KRAS G12D-harboring tumors and outperform LVV-engineered cells. Our work supports the planned clinical development of this novel TCR-engineered T cell therapy for treating KRAS-mutant solid tumors.Ethics ApprovalThese studies were approved by Affini-T Therapeutics and Explora Biolabs’ Institutional Animal Care and Use Committee, approval number EB17–010-303.

BackgroundAdoptive T cell therapy has demonstrated clinical activity in a subset of patients with solid tumors; however, consistent responses will require further optimization. T cell receptor (TCR)-engineered T cells recognize peptides derived from intracellular and surface proteins presented in the context of MHC class I. Immunologic targeting of recurrently mutated oncogenic drivers, such as KRAS, overcomes many of the major obstacles of this modality because the resulting epitope is: 1) tumor-specific, 2) essential for cancer cell fitness, and 3) derived from a stably expressed non-self protein. AFNT-212 is a next-generation engineered T cell therapy that uses non-viral targeted knock-in (KI) at the TCRα constant (TRAC) locus to express a multi-cistronic cassette that includes 1) a high-affinity TCR specific for KRASG12D mutation, 2) a CD8αβ coreceptor, and 3) a chimeric cytokine receptor.MethodsHuman CD4+ and CD8+ T cells were genetically engineered by a novel CRISPR-Cas nuclease and gRNAs targeting TRAC and the TCRβ constant (TRBC) genes allowing for knock-out of the endogenous TCR loci and simultaneous integration of the non-viral plasmid-based transgene cassette. Engineered T cells were assessed for specificity and potency, including activation, proliferation, and cytotoxicity, against KRAS G12D peptide presented by HLA-A*11:01 and a panel of KRAS G12D-expressing tumor cell lines. In vitro safety studies were performed along with in vivo efficacy studies in multiple human xenograft models.ResultsEngineered primary T cells showed specific recognition of KRAS G12D peptide, demonstrated cytotoxicity against endogenously expressing HLA-A*11:01+/KRAS G12D+ cell lines in tumor cell re-challenge assays in vitro, and mediated robust anti-tumor activity in vivo. Inclusion of the chimeric cytokine receptor allowed for a more potent anti-tumor response stemming from improved T cell expansion and resistance to exhaustion. No off-target liabilities were identified upon co-incubation of AFNT-212 with all possible peptides in the human proteome matching the xScan-defined epitope recognition motif for the TCR, demonstrating specificity. Gene editing safety evaluation did not reveal any off-target activity for the CRISPR-Cas nucleases and engineered T cells did not show cytokine-independent proliferation, collectively supporting a favorable pre-clinical safety profile for AFNT-212.ConclusionsWe report a novel TCR gene therapy approach targeting mutant KRAS G12D-expressing tumors with a coordinated CD4/CD8 T cell response that has a promising efficacy and safety profile. Our work supports the planned clinical development of AFNT-212 as a novel non-viral KI TCR-engineered T cell therapy for KRAS-mutant solid tumors.