Longitudinal and spatial heterogeneity of intra-tumoral TCR repertoires during immunotherapy in metastatic cancers

Introduction: Spatial and temporal heterogeneity and evolution of the T cell receptor (TCR) landscape may influence immunotherapy outcomes. Studying TCR repertoires and their antigen specificities, together with T cell dynamics, could provide important insights into the quality of anti-tumor immune responses and their impact on cancer immunotherapy effectiveness. Methods: We performed serial and multi-region sampling at critical timepoints (n=52 samples) during the clinical course of 7 patients with metastatic lung and head and neck cancer, who received immunotherapy-containing regimens. To study the TCR repertoire, we performed bulk TCR sequencing of TCR-beta CDR3 regions and recovered 69,135 unique productive clones (ImmunoSeq assay, Adaptive Biotechnologies). To determine differential responses to immunotherapy across tumor sites and patients, we evaluated TCR repertoire similarity using Morisita's overlap index (MI) based on clonotype frequency and identity. Next, we clustered TCR clones of similar antigen specificities using GIANA (15,476 clusters) and performed differential T cell abundance analyses at the clone- and cluster-level to assess T cell dynamics. Statistically significant TCR expansions and regressions were determined using Fisher's exact test (p≤0.05). We used the VDJDB and CEDAR databases to de-orphanize 2,019 TCR clusters, mapping to TCRs with known antigen specificities. Results: We found significant TCR repertoire heterogeneity between patients (MI between patients ≈ 0), suggesting private TCR landscapes at a patient level. In individual patients, a higher heterogeneity was noted between primary and metastatic sites (MI baseline-metastatic = 0.01 - 0.34) while TCR repertoires of metastatic sites from proximal anatomic locations shared similarities (MI proximal metastasis = 0.47 - 0.96). Despite the largely private expanded clusters and clones in each patient, similar dynamics were observed in regressing compared to progressing tumors in the context of immunotherapy, while at the autopsy sites, significantly expanded clusters dominated their respective TCR repertoires (14-28% abundance). TCR sequences recognizing viral antigen epitopes, such as GLCTLVAML (EBV), KLGGALQAK (CMV) and RAKFKQLL (EBV) were identified across patients and were part of the significantly expanded clusters in progressing metastatic sites. Several significantly expanding TCR clusters (FDR p≤0.05) were detected; while these had unmapped antigen specificity, they could recognize tumor antigens and mutation-associated neoantigens relevant for mounting anti-tumor immune responses. Conclusions: Spatial and temporal TCR repertoire analyses provided a better understanding of the patients' immune landscape during therapy and offered insights that can be used to further dissect the heterogeneity of adaptive immune responses in the context of immunotherapy.