358 T cell receptor engineering targeting EGFR-mutant non-small cell lung cancer

<h3>Background</h3> Epidermal Growth Factor Receptor (EGFR) is mutated in 30% of metastatic non-small cell lung cancer (NSCLC). Tyrosine kinase inhibitors (TKIs) have been developed to target key mutations in EGFR, leading to improved survival. Despite this success, resistance inevitably occurs. Therefore, alternative therapeutic strategies are needed to overcome this resistance and provide durable responses. Cellular therapies have resulted in impressive durable clinical responses. Among cellular therapies, T cell receptor (TCR) engineering allows focused targeting of a tumor antigen of interest. Here, we assess the immunogenicity of EGFR mutations and investigate the feasibility of targeting them using TCR engineering. <h3>Methods</h3> We focused on the prevalent L858R, T790M, and C797S mutations of EGFR and predicted high affinity neoantigens binding to the 10 most prevalent HLA alleles in the United States population. Peptides were synthesized and pulsed onto dendritic cells to stimulate blood from healthy donors and lung cancer patients. Antigen-specific T cell populations were isolated by tetramer sorting, underwent rapid expansion and were exposed to cancer cells endogenously expressing mutated EGFR to ensure immunogenic epitopes were naturally processed and presented. Reactive T cells then underwent single cell TCR/RNA sequencing to identify full abTCR sequences. TCRs were synthesized and transduced into HLA-matched PBMCs via retroviral vector. Antigen-specific T cell activation and cytotoxicity was assessed by ELISpot and chromium-51 release. Peptide dose-responses were performed to determine TCR specificity and sensitivity. <h3>Results</h3> Epitopes derived from the L858R, T790M, and C797S mutations were confirmed to be immunogenic on HLA-A*03:01, HLA-A*02:01, and HLA-A*02:01, respectively, by antigen-specific T cell stimulation and tetramer staining. Antigen-specific T cells lysed H1975 cells expressing endogenous EGFR mutations at a 20:1 effector:target ratio confirming antigens were truly endogenously-processed and presented (L858R: peptide-pulsed, 97%; endogenous, 26%; parental, 0%/T790M: peptide-pulsed, 48%; endogenous, 15%; parental, 3%/<i>p</i><i>&lt;0.0001</i>). TCRs were successfully expressed in healthy donor PBMCs, purified, and expanded for functional assessment. Engineered TCR-T cells were capable of IFN-g secretion as detected by ELISpot (37 spots vs. 0.33 spots, <i>p=0.0019</i>). TCR-T cells also demonstrated cytotoxic potential against HLA-matched cell lines, as indicated by Chromium-51 release at a 20:1 effector:target ratio (L858R: 63% vs. 23%, <i>p&lt;0.0001</i>/T790M: 40% vs. 8%, <i>p&lt;0.0001</i>/C797S: 24% vs. 11%, <i>p&lt;0.001</i>). Peptide dose-responses revealed TCR-T cells were specific and sensitive. <h3>Conclusions</h3> Our studies confirm the immunogenicity of EGFR mutations and feasibility of targeting these mutations using TCR-engineering <i>in vitro. In vivo</i> efficacy studies are underway.