Brad Nelson

Current cancer immunotherapies primarily leverage prognostically favourable tumour-infiltrating T cells (TIL-Ts); however, optimal anti-tumour immunity additionally requires tumour-infiltrating B cells (TIL-Bs). Many solid cancers such as high-grade serous ovarian cancer (HGSC) remain resistant to immunotherapies despite harbouring TIL-Ts and TIL-Bs, suggesting that further research is needed to devise optimal immunotherapeutic strategies for these cancers. One of the most successful T cell-based immunotherapies, chimeric antigen receptor (CAR)-T cell therapy, ideally targets a surface antigen that is exclusively and uniformly expressed by tumours; however, no such antigen is known for HGSC. Therefore, we investigated the potential for multi-antigenic targeting with antigens commonly over-expressed in HGSC: CA125, mesothelin, and folate receptor alpha. We used immunofluorescence to quantify co-expression patterns of these antigens in HGSC. We identified that a CAR-T cell strategy targeting CA125 and/or mesothelin was the most promising approach, yet no approach would target 100% of tumour cells in all cases, prompting further research into immunotherapeutic strategies for HGSC. While TIL-Ts are positively prognostic, the best prognosis is observed with additional presence of TIL-Bs; however, the target antigens and mechanisms through which TIL-Bs confer this beneficial effect remain poorly understood. We endeavoured to decipher the role of TIL-Bs in anti-tumour immunity to guide the development of better immunotherapies that leverage the prognostic benefit associated with TIL-Bs. We performed single-cell RNA sequencing using TIL-Bs from patient-derived HGSC tumours. We identified 1,379 TIL-B B cell receptors (BCRs) and produced 51 of the most expanded, somatically hypermutated, class-switched BCRs as recombinant antibodies (rAbs). Flow cytometry, western blotting, and immunohistochemistry revealed that TIL-B-derived rAbs frequently recognize intracellular antigens across normal, embryonic, and cancerous cells/tissues. By phage-based cDNA library immunoscreening and immunoprecipitation-mass spectrometry, we identified three TIL-B target antigens, which are all intracellular self-antigens over-expressed in cancer. By ELISA, a substantial proportion of TIL-B-derived rAbs were polyreactive, yet matched patient serum did not display elevated levels of autoreactivity. Overall, my results highlight the opportunities and shortcomings of existing CAR-T cell strategies while providing critical insights into TIL-B-mediated immunity in HGSC, suggesting multiple avenues through which to develop future immunotherapies that harness the positive prognosis associated with TIL-Bs.

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A hallmark of cancer is the accumulation of mutations, and a small proportion of these give rise to mutant neoantigens – mutated peptides bound to Major Histocompatibility Complex (MHC) and recognized by T cells. Accumulating evidence suggests that mutant neoantigens (hereafter referred to as “neoantigens”) underlie successful immune therapies in cancers with high mutation loads, such as melanoma. Moreover, neoantigen-specific vaccines have successfully targeted highly mutated murine tumor models. However, less is known about neoantigen-specific T cell responses in cancers with moderate mutation loads, such as ovarian cancer. I hypothesized that (1) modified peptide-based vaccination schedules can lead to enhanced antigen-specific T cell responses; (2) neoantigen-specific vaccines can elicit T cell responses that eradicate murine ovarian tumors; and (3) neoantigen-reactive T cells are detectable in human ovarian tumors and peripheral blood. To activate high frequencies of antigen-specific T cells, I developed a vaccination method involving repeated, daily immunizations with long peptides and adjuvant. This method elicited robust T cell responses that eliminated established murine tumors. I used these enhanced vaccination methods to target tumor-specific mutations identified by exome- and RNA-sequencing of the ovarian tumor model ID8-G7. Prophylactic and therapeutic vaccinations were performed targeting all expressed mutations that had a predicted MHCI binding affinity 1500 nM (n=17 mutations). Though the vaccines elicited robust T cell responses to most mutations, activated T cells failed to recognize ID8-G7 tumors in vitro and failed to control tumor growth in vivo, indicating that none of the evaluated mutations represented authentic neoantigens. I also investigated the neoantigen-specific T cell repertoire in blood and tumor samples donated by an ovarian cancer patient. A neoantigen-specific T cell response was identified in the first recurrence tumor sample, and a derived T cell clone recognized tumor from each of three time points. Furthermore, peripheral blood samples donated prior disease recurrence harbored T cells recognizing the same mutation. The results presented here show that neoantigen-specific T cells can arise spontaneously in ovarian tumors and are detectable in peripheral blood; however, low mutation burdens in ovarian cancer may limit the utility of neoantigen-targeted vaccines to a minority of patients with this disease.

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