Zheng Pan

. Here we developed a clinical-grade approach based on CRISPR-Cas9 non-viral precision genome-editing to simultaneously knockout the two endogenous TCR genes TRAC (which encodes TCRα) and TRBC (which encodes TCRβ). We also inserted into the TRAC locus two chains of a neoantigen-specific TCR (neoTCR) isolated from circulating T cells of patients. The neoTCRs were isolated using a personalized library of soluble predicted neoantigen-HLA capture reagents. Sixteen patients with different refractory solid cancers received up to three distinct neoTCR transgenic cell products. Each product expressed a patient-specific neoTCR and was administered in a cell-dose-escalation, first-in-human phase I clinical trial ( NCT03970382 ). One patient had grade 1 cytokine release syndrome and one patient had grade 3 encephalitis. All participants had the expected side effects from the lymphodepleting chemotherapy. Five patients had stable disease and the other eleven had disease progression as the best response on the therapy. neoTCR transgenic T cells were detected in tumour biopsy samples after infusion at frequencies higher than the native TCRs before infusion. This study demonstrates the feasibility of isolating and cloning multiple TCRs that recognize mutational neoantigens. Moreover, simultaneous knockout of the endogenous TCR and knock-in of neoTCRs using single-step, non-viral precision genome-editing are achieved. The manufacture of neoTCR engineered T cells at clinical grade, the safety of infusing up to three gene-edited neoTCR T cell products and the ability of the transgenic T cells to traffic to the tumours of patients are also demonstrated.

Nanoparticle-based cancer immunotherapy has shown promising therapeutic potential in clinical settings. However, current research mainly uses nanoparticles as delivery vehicles but overlooks their potential to directly modulate immune responses. Inspired by the endogenous endoplasmic reticulum (ER) stress caused by unfolded/misfolded proteins, we present a rationally designed immunogenic cell death (ICD) inducer named NanoICD, which is a nanoparticle engineered for ER targeting and retention. By carefully controlling surface composition and properties, we have obtained NanoICD that can effectively accumulate in the ER, induce ER stress, and activate ICD-associated immune responses. In addition, NanoICD is generally applicable to various proteins and enzymes to further enhance the immunomodulatory capacity, exemplified by encapsulating catalase (CAT) to obtain NanoICD/CAT, effectively alleviated immunosuppressive tumor microenvironment and induced robust antitumor immune responses in 4T1-bearing mice. This work demonstrates engineered nanostructures' potential to autonomously regulate biological processes and provides insights into the development of advanced nanomedicines for cancer treatment.

Abstract Introduction: PACT Pharma is a clinical-stage adoptive cell therapy company that develops personalized neoTCR-T cells using a state-of-the-art approach to discover and validate predicted neoepitopes and their cognate T cell receptors (clinical trial NCT03970382). An important mechanism of resistance to adoptive cell therapy targeting tumor-specific neoantigens is the genetic loss of human leukocyte antigen (HLA) alleles or alterations in HLA expression in tumor cells. Therapies targeting neoantigens presented by HLA alleles that have either been deleted, mutated or are strongly downregulated are likely to lack efficacy due to the absence or reduction of epitope presentation. Surveying neoepitope presentation escape mechanisms is key for personalized immunotherapy. Methods: PACT Pharma has developed PACT-ESCAPE, a method that integrates DNA and RNA sequencing data to quantify allelic imbalance at HLA loci, including the most extreme case of loss of heterozygosity (LOH). Allelic imbalance is first determined at the DNA level by determining the patient’s maternal and paternal haplotypes across chromosome 6p to infer their copy number states. Twenty-six HLA Class I and II loci from chromosome 6 are then genotyped at high resolution, enabling accurate assignment of copy number states to the patient’s alleles, including any alleles that have been deleted in the tumor. Unlike other currently available methods, PACT-ESCAPE also measures the relative RNA expression of HLA alleles to provide an orthogonal estimate of allelic imbalance across chromosome 6 and support loss of heterozygosity calls made at the DNA level. Presentation machinery genes are also surveyed for loss of function mutations. To benchmark PACT-ESCAPE, we compared loss of heterozygosity calls at HLA-A, HLA-B, and HLA-C between our method and a previously published DNA-based HLA LOH classifier for 17 samples for which we had matched tumor/normal whole exome sequencing. Results: We found 100% concordance between PACT-ESCAPE and the benchmark, with four samples showing LOH at HLA-A, HLA-B, and HLA-C at the DNA level. Next, we evaluated RNA sequencing from tumor samples and confirmed that the relative expression of the lost allele compared to the kept allele was lower when LOH was called. High levels of allelic imbalance in gene expression, however, were not unique to samples with LOH, suggesting that differential expression of HLA alleles might also be an important contributor to immune escape in cancer. Conclusions: Evaluation of allelic imbalance in DNA copy number and RNA expression in PACT-ESCAPE provides new insights into immune escape in cancer. Adoptive cell therapy targeting neoantigens will benefit by enabling the accurate selection of targets most likely to be presented by the tumor. Citation Format: Chad C. Smith, Yan Ma, Katie Campbell, Zheng Pan, Eric Stawiski. Uncovering HLA loss of heterozygosity and allelic imbalance in cancer for the improvement of personalized neoTCR immunotherapy with PACT-ESCAPE [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1213.

Abstract BACKGROUND Low-grade gliomas (LGGs) exhibit low overall mutational burden and an immunosuppressive microenvironment, contributing to immunotherapy resistance. Intratumoral heterogeneity (ITH) further complicates the targeting of tumor-specific antigens (TSAs), yet its influence on the antigenic and immune landscapes in LGG remains understudied, particularly due to reliance on single-biopsy analyses. METHODS We performed exome and transcriptome sequencing on 70 spatially mapped biopsies from seven IDH-mutant Grade II astrocytoma patients. Tumor purity, immune deconvolution, and neoantigen prediction were conducted to assess regional immune variation and antigenic burden. In one patient (P375), neoantigen-specific CD8+ T cells were isolated using barcoded peptide-HLA multimers. Reactive T-cell receptor (TCR) clonotypes were identified via single-cell TCR sequencing and functionally validated in Jurkat76 and CD8+ T cells. RESULTS Spatial profiling of whole tumors revealed most mutations were biopsy-specific, with a steep drop in mutations shared across multiple regions. RNA-seq–based unsupervised clustering with xCell and DESeq2 revealed two distinct immune microenvironments: immune-hot regions with elevated immune infiltration, and immune-cold regions with minimal immune presence. Notably, mutation-derived n-mer peptides from immune-hot regions showed enhanced predicted immunogenicity relative to those from immune-cold areas. In one patient (P375), neoantigen-specific CD8⁺ TCR clonotypes were identified targeting PRMT5 mutations found in immune-hot regions. A mutant PRMT5-specific TCR demonstrated robust, antigen-dependent activation and dose-dependent cytotoxicity in vitro. This TCR showed high specificity toward the mutant peptide with minimal cross-reactivity to the wild-type sequence. Furthermore, engineered CD8⁺ T cells effectively killed glioma cells endogenously expressing the full-length mutant PRMT5, supporting its candidacy as a viable immunotherapeutic target. CONCLUSIONS Our findings reveal significant 3D spatial and immunologic heterogeneity in low-grade gliomas and demonstrate that neoantigens such as mutant PRMT5 can be selectively targeted by highly specific, cytotoxic TCR-engineered CD8⁺ T cells. These results support the potential of personalized TCR-based immunotherapy for low-grade glioma.