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Developing effective strategies to prevent or treat coronavirus disease 2019 (COVID-19) requires understanding the natural immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8+ T cells of COVID-19 patients. In total, we identified 3-8 epitopes for each of the 6 most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8+ T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8+ T cell immunity to SARS-CoV-2.

Background Approximately 30–40% of AML patients relapse following allogeneic hematopoietic stem cell transplant therapy, leaving them with very few treatment options.1 2 Rare patients that naturally develop an HA-1-specific graft-versus-leukemia T cell response, however, show substantially lower relapse rates.3 4 HA-1 (VLHDDLLEA, genotype RS_1801284 A/G or A/A) is an HLA-A*02:01-and hematopoietically restricted minor histocompatibility antigen, making it an ideal candidate for TCR immunotherapy for liquid tumors.5 Methods We developed a high-throughput TCR discovery platform that enables rapid cloning of antigen-specific TCRs from healthy donors. We then used this platform to screen 178.3 million naive CD8+ T cells from six unique HA-1- (VLRDDLLEA, genotype RS_1801284 G/G) donors, identifying 329 HA-1-specific TCRs. We tested each TCR for expression and the ability to kill HA-1+ target cells, using a previously published, clinical-stage HA-1-specific TCR as a benchmark for these studies.6 In parallel, we tested TCR constant region modifications to promote expression and proper pairing of exogenous TCR alpha and beta chains and designed a lentiviral vector to co-deliver CD8 coreceptors as well as a CD34 enrichment tag to enable purification of engineered T cells. The top 11 candidates were cloned into our optimized backbone and evaluated for cytotoxicity, cytokine production, and T cell proliferation using a panel of HLA-A*02:01+ HA-1+ cell lines. Finally, the top two TCRs were evaluated for allo-reactivity and off-target cross-reactivity using our proprietary genome-wide T-Scan platform. Results The TCR discovery and evaluation platform described here identified 329 HA-1-specific TCRs from a total of 178.3 million naive T cells, and TSC-100 as the most active TCR. Defined mutations in the constant region of TSC-100 enhanced its surface expression while decreasing expression of endogenous TCRs, and co-introduction of CD8 enabled efficient engagement and function of engineered CD4 cells. Overall, TSC-100 exhibited comparable activity to a clinical-stage benchmark TCR when challenged with cell lines expressing moderate to high levels of HA-1, and superior activity when incubated with cell lines expressing low levels of both HA-1 and MHC-I.6 In addition, TSC-100 exhibited no detectable allo-reactivity to 108 different HLA types tested, and minimal off-target effects when challenged with a genome-wide library expressing peptides derived from human proteins. Conclusions TSC-100 exhibits comparable or superior activity to a clinical-stage therapeutic TCR, with minimal allo-reactivity or off-target effects. Based on these results, TSC-100 has been advanced to IND-enabling activities to prepare for first-in-human testing in 2021. Ethics Approval All clinical samples used in the study were collected by STEMCELL Technologies, StemExpress and HemaCare using their IRB approved protocols. References Pavletic SZ, Kumar S, Mohty M, et al. NCI First International Workshop on the Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation: Report from The Committee on the Epidemiology and Natural History of Relapse following Allogeneic Cell Transplantation. Biol Blood Marrow Transplant. 2010;16(7):871–890. Miller JS, Warren EH, van den Brink MR, et al. NCI First International Workshop on The Biology, Prevention, and Treatment of Relapse After Allogeneic Hematopoietic Stem Cell Transplantation: Report from the Committee on the Biology Underlying Recurrence of Malignant Disease following Allogeneic HSCT: Graft-versus-Tumor/Leukemia Reaction. Biol Blood Marrow Transplant 2010;16(5):565–586. Marijt WAE, Heemskerk MHM, Kloosterboer FM, et al. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia. PNAS 2003;100:2742–2747. Spierings E, Kim Y, Hendriks M, et al. Multicenter analyses demonstrate significant clinical effects of minor histocompatibility antigens on GvHD and GvL after HLA-Matched related and unrelated hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2013; 19: 1244–1253. Bleakley M, Riddell SR. Exploiting T cells specific for human minor histocompatibility antigens for therapy of leukemia. Immunol Cell Biol 2011;89(3):396–407. Dossa RG, Cunningham T, Sommermeyer D, et al. Development of T-cell immunotherapy for hematopoietic stem cell transplantation recipients at risk of leukemia relapse. Blood 2018;131(1):108–120.

Developing effective strategies to prevent or treat coronavirus disease 2019 (COVID-19) requires understanding the natural immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used an unbiased, genome-wide screening technology to determine the precise peptide sequences in SARS-CoV-2 that are recognized by the memory CD8+ T cells of COVID-19 patients. In total, we identified 3–8 epitopes for each of the 6 most prevalent human leukocyte antigen (HLA) types. These epitopes were broadly shared across patients and located in regions of the virus that are not subject to mutational variation. Notably, only 3 of the 29 shared epitopes were located in the spike protein, whereas most epitopes were located in ORF1ab or the nucleocapsid protein. We also found that CD8+ T cells generally do not cross-react with epitopes in the four seasonal coronaviruses that cause the common cold. Overall, these findings can inform development of next-generation vaccines that better recapitulate natural CD8+ T cell immunity to SARS-CoV-2., Graphical Abstract, Highlights • Unbiased screens identified SARS-CoV-2 targets of CD8+ T cells in COVID-19 patients • CD8+ T cells predominantly recognize 3–8 shared epitopes for each HLA type studied • ∼90% of shared epitopes are not located in the spike protein • CD8+ T cells show almost no cross-reactivity with epitopes in seasonal coronaviruses, Ferretti et al. reveal specific SARS-CoV-2 epitopes that are broadly shared by CD8+ T cells of COVID-19 patients but exhibit limited cross-reactivity with seasonal coronaviruses. Most epitopes are located outside of the spike protein, suggesting that next-generation vaccines incorporating these epitopes might be needed to generate more robust and durable immunity.

Development of effective strategies to detect, treat, or prevent COVID-19 requires a robust understanding of the natural immune response to SARS-CoV-2, including the cellular response mediated by T cells. We used an unbiased, genome-wide screening technology, termed T-Scan, to identify specific epitopes in SARS-CoV-2 that are recognized by the memory CD8+ T cells of 25 COVID-19 convalescent patients, focusing on epitopes presented by the six most prevalent HLA types: A*02:01, A*01:01, A*03:01, A*11:01, A*24:02, and B*07:02. For each HLA type, the patients’ T cells recognized 3–8 immunodominant epitopes that are broadly shared among patients. Remarkably, 94% of screened patients had T cells that recognized at least one of the three most dominant epitopes for a given HLA, and 53% of patients had T cells that recognized all three. Subsequent validation studies in 18 additional A*02:01 patients confirmed the presence of memory CD8+ T cells specific for the top six A*02:01 epitopes, and single-cell sequencing revealed that patients often have many different T cell clones targeting each epitope, but that the same T cell receptor Vα regions are predominantly used to recognize these epitopes, even across patients. In total, we identified 29 shared epitopes across the six HLA types studied. T cells that target most of these epitopes (27 of 29) do not cross-react with the endemic coronaviruses that cause the common cold, and the epitopes do not occur in regions with high mutational variation. Notably, only 3 of the 29 epitopes reside in the spike protein, highlighting the need to design new classes of vaccines that recapitulate natural CD8+ T cell responses to SARS-CoV-2.