T cell receptor engineered T cell (TCR T) therapy has emerged as a promising therapeutic modality for solid cancer following recent trials demonstrating the safety and efficacy of TCR T therapies against some types of metastatic solid cancers. However, the broader application of TCR T towards many solid tumors, including metastatic breast cancer (MBC), has been limited by several factors, chiefly among them the current scarcity of tumor selective target antigens. Neoantigens, which are expressed exclusively in cancer cells, are currently underrepresented in TCR T development, being targeted in only about 7% of trials conducted to date, and thus represent a relatively untapped source of potentially safe and effective novel targets. Driver mutations in AKT1, ESR1, PIK3CA, and TP53 are common in patients with MBC, and could serve as ideal neoantigen targets for TCR T therapies. We hypothesized that we could generate MBC driver mutation-specific T cells from which we could isolate and clone neoantigen-specific TCRs to generate TCR T products for MBC. We identified 13 driver missense mutations that are among the most frequent in patients with MBC, which included AKT1 (E17K), ESR1 (K303R, Y537S, D538G), PIK3CA (E542K, E545K, H1047L, H1047R), and TP53 (R175H, R248Q, R248W, R273C, R273H), then designed peptide libraries consisting of 15-mer overlapping peptides that contain these mutations. To determine if these neopeptides could elicit T cell responses, we isolated T cells from 15 healthy donors and 11 MBC patients who expressed at least one of the targeted mutations and performed successive stimulations with neopeptide pulsed dendritic cells, then screened the resulting T cell lines for neoantigen specificity using an IFN-γ ELISpot assay. We observed neopeptide T cell responses in 8/16 lines generated from healthy donors and 7/11 lines generated from MBC patients, which were collectively directed against 11/13 of the targeted driver mutations. To isolate neoantigen-specific TCRs from one of these lines, we performed IFN-γ capture, limiting dilution, and 5’ RACE, and isolated an HLA-B*35 restricted TP53 R248W-specifc TCR. Gene transfer of this TCR conferred edited T cells with potent activity towards the TP53 R248W and not the TP53 WT peptide as assessed by ELISpot (1036 vs 46 SFU/1 × 105 cells, respectively) and chromium release cytoxicity assay targeting peptide pulsed autologous PHA blasts (37.5% vs 0% lysis at E:T 40:1, respectively). To increase the throughput of TCR discovery, we next used a single cell RNA sequencing based TCR discovery approach whereby we stimulated T cells from one of the generated lines with ESR1 WT or neopeptide and identified responsive T cell clones through upregulation of IFN-γ and/or TNF-α. This strategy has so far enabled us to identify and validate two ESR1 mutant-specific TCRs. This includes an HLA-C*01 restricted TCR that confers edited T cells with dual activity towards both ESR1 Y537S and D538G, but not WT peptide as determined by both ELISpot (2094, 3194, and 79 SFU, respectively) and chromium release cytotoxicity (31.3%, 77.8%, and 9.1% lysis, respectively), as well as an HLA-B*40 restricted TCR that confers high ESR1 Y537S specificity (5039 vs 138 SFU in response to ESR1 Y537S vs WT peptide, respectively). In summary, we have demonstrated responses of T cells derived from both healthy donors and MBC patients towards neopeptides derived from common MBC driver mutations. We have so far isolated neoantigen specific TCRs from two of the neoantigen-specific T cells lines, including TCRs specific towards TP53 R248W, ESR1 Y537S, dual ESR1 Y537S+D538G that are restricted to three different HLA alleles, and have successfully used these TCRs to generate TCR T products with high neoantigen activity. These results encourage further efforts to identify TCRs recognizing these MBC driver mutations, with our ultimate aim to translate neoantigen-targeted TCR T therapies to clinical trials of MBC.

Citation Format: Paul Shafer, Wingchi K. Leung, Mae L. Woods, Carlos Rodriguez-Plata, Arushana Ali, Saisha Nalawade, Lauren M. Kelley, Jarrett Joubert, Anthony Manliguez, Spyridoula Vasileiou, Suzanne A. Fuqua, Premal Lulla, Cliona Rooney, Ann Leen, Valentina Hoyos. Engineered neoantigen-specific T cell receptors to treat metastatic breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-10-11.