Tumor-specific T-cell Receptor Research Articles

Identification and validation of tumor-specific T cell receptors from tumor infiltrating lymphocytes using tumor organoid co-cultures

T cell receptor-engineered T cells (TCR-Ts) therapy is promising for cancer immunotherapy. Most studies have focused on identifying tumor-specific T cell receptors (TCRs) through predicted tumor neoantigens. However, current algorithms for predicting tumor neoantigens are unreliable and many neoantigens are derived from non-coding regions. Thus, the technological platform for identifying tumor-specific TCRs using natural antigens expressed on tumor cells is urgently needed. In this study, tumor organoids-enriched tumor infiltrating lymphocytes (oeT) were obtained by repeatedly stimulation of autologous patient-derived organoids (PDO) in vitro. The oeT cells specifically responded to autologous tumor PDO by detecting CD137 expression and the secretion of IFN-γ using enzyme-linked immunospot assay. The measurement of oeT cell-mediated killing of three-dimensional organoids was conducted using a caspase3/7 flow cytometry assay kit. Subsequently, tumor-specific T cells were isolated based on CD137 expression and their TCRs were identified through single-cell RT-PCR analysis. The specificity cytotoxicity of TCRs were confirmed by transferring to primary peripheral blood T cells. The co-culture system proved highly effective in generating CD8+ tumor-specific oeT cells. These oeT cells effectively induced IFN-γ secretion and exhibited specificity in killing autologous tumor organoids, while not eliciting a cytotoxic response against normal organoids. The analysis conducted by TCRs revealed a significant expansion of T cells within a specific subset of TCRs. Subsequently, the TCRs were cloned and transferred to peripheral blood T cells generation engineered TCR-Ts, which adequately recognized and killed tumor cell in a patient-specific manner. The co-culture system provided an approach to generate tumor-specific TCRs from tumor-infiltrating lymphocytes of patients with colorectal cancer, and tumor-specific TCRs can potentially be used for personalized TCR-T therapy.

Correlation of peripheral blood monocytic myeloid-derived suppressor cells (M-MDSC) and T-cell receptor (TCR) dynamics with clinical outcomes in patients (pts) with metastatic urothelial carcinoma (mUC) treated with nivolumab (NIVO).

4569 Background: Although PD-1 blockade is active in varied mUC treatment settings, predicting response and overcoming resistance remain unmet needs. We hypothesized that immunosuppressive M-MDSCs (CD14+Lin-/HLA-DRlow/-) and TCR dynamics in tumors and blood may correlate with outcomes from NIVO in pts with platinum-refractory mUC (NCT02553642). Methods: 69 pts with mUC were treated with NIVO 240mg intravenously every 2 weeks (wks). Pre-treatment peripheral M-MDSCs among lineage-negative CD14+ monocytes (%) were estimated by flow cytometry using an algorithm that calculates MDSC frequencies based on HLA-DR mean fluorescence intensity (Kitano et al. 2014). High throughput DNA sequencing of the CDR3 region of the TCR beta chain was performed on baseline tumors (N = 57) and pre and post-treatment (wk 2) peripheral blood mononuclear cells using Adaptive Immunosequencing (Adaptive Biotechnologies). M-MDSC levels and TCR metrics were correlated with clinical outcomes: complete or partial response (CR, PR) vs. stable or progressive disease (SD, PD) by RECIST 1.1, clinical benefit (CR + PR + SD), and progression-free and overall survival (PFS, OS). Groups were compared using Wilcoxon signed rank (paired), Wilcoxon rank sum tests (unpaired), and log-rank tests (time-to-event). Cox proportional hazards and logistic regression models were used to analyze binary and time-to-event outcomes, respectively. Results: Higher pre-treatment M-MDSCs were associated with worse PFS and OS univariably (PFS HR 1.07; 95% CI, 1.01-1.14; p = 0.025; OS HR 1.07; 95% CI, 1.00-1.14; p = 0.038), and after adjustment for liver disease and PD-L1 at baseline (PFS HR 1.12; 95% CI, 1.04-1.21, p = 0.004; OS HR 1.11; 95% CI, 1.03-1.20, p = 0.010). Baseline M-MDSCs did not significantly differ between responders and non-responders, but were significantly lower in patients with clinical benefit (median 12.8 [IQR: 10.7-15.9] vs. median 15.6 [IQR: 13.5-18.3]; p = 0.039), and remained significantly associated after adjustment for PD-L1 score in a multivariable model (OR 0.82; 95% CI, 0.67-0.97; p = 0.037). A higher number of tumor-associated clones in the blood (BTACs) at baseline was associated with response (p = 0.049). Overall, BTACs significantly increased at wk 2 (p < 0.001), and wk 2 values were associated with response (p = 0.003). PFS also significantly differed between BTAC high (high: > median 2,012 clones) and low groups at wk 2, with improved PFS in the high group (log-rank p = 0.026). OS rate for the BTAC high group was 34% vs. 16% for the low group at wk 2, but this did not achieve significance (p = 0.098). Conclusions: Peripheral M-MDSCs may promote resistance to PD-1 blockade in pts with mUC. NIVO stimulated tumor-specific TCR clones in the blood, which correlated with improved response and outcomes. Clinical trial information: NCT02553642 .

Abstract 13: Discovery and development of T cell receptors targeting MAGE family antigens for adoptive T cell therapy against solid tumors

Abstract Background: T cell receptors (TCRs) recognize epitopes from intracellular and cell surface antigens, enabling targeting of diverse tumor antigen classes including overexpressed, differentiation, cancer-testis antigens, as well as those from driver mutations, frameshift mutations, splice variants, and human endogenous retroviral elements (HERV). Melanoma-associated antigens (MAGE) are highly expressed in various solid tumor types, including melanoma, non-small cell lung cancers (NSCLC), bladder, and head and neck cancers, representing promising targets for TCR-T therapy. However, discovering robust and specific MAGE-reactive TCRs remains challenging due to limited throughput of TCR screening methodologies, and the inherently low frequency of tumor-reactive T cells in tumor samples and peripheral blood. Methods: We employ a proprietary mass-cytometry-based multiplexed tetramer staining approach, TargetScape®, to screen and characterize CD8+ T cells in healthy donors and patients’ samples. Our screening panels contain multiple peptides for MAGE family antigens including A1, C2, and A10. Subsequent single cell sequencing or single cell PCR (scPCR) of identified tumor antigen-specific T cells with high sensitivity enables comprehensive analysis of T cell phenotype, transcriptome, TCR specificity, and paired TCR sequence. TCRs identified through this method are transduced for expression in Jurkat reporter cell lines and their reactivity to corresponding peptides is assessed. Selected TCR candidates are then transduced into primary T cells and evaluated for activation and effector function using a variety of cancer cell lines expressing the corresponding endogenous antigens. Specificity assessment is performed through alanine scans and identification of potential off-target peptides and screening for reactivity. Results: We utilized this approach to discover natural TCRs that target several MAGE family antigens. Through Jurkat reporter screening, we observed specific activation of multiple TCRs in the presence of antigen presenting cells and the peptide initially employed to identify the TCRs. Primary T-cells expressing promising TCR candidates exhibited killing activities and cytokine release when confronted with multiple solid tumor cell lines expressing endogenous levels of the MAGE antigen. Specificity assessment is currently in progress, with preliminary data indicating overall favorable specificity profiles for selected TCRs. Conclusions: Our Deep Immunomics platform enables us to detect, characterize, and validate TCR candidates against highly clinically relevant shared cancer antigens. Leveraging this platform, we identified various natural tumor specific TCRs exhibiting activities against a range of solid tumor cell lines expressing MAGE antigens. Promising candidates are currently undergoing in vivo efficacy studies. Citation Format: Juliana Velez Lujan, Hannah Fields, Kan Xing Wu, Yovita Purwanti, Emily Xin Zi Tang, Michael Cordeau, Kathleen Mendez, Melissa Wirawan, Michael Fehlings, Katja Fink, Zilei Liu, Daniel MacLeod. Discovery and development of T cell receptors targeting MAGE family antigens for adoptive T cell therapy against solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 13.

HLA-class II restricted TCR targeting human papillomavirus type 18 E7 induces solid tumor remission in mice

T cell receptor (TCR)-engineered T cell therapy is a promising potential treatment for solid tumors, with preliminary efficacy demonstrated in clinical trials. However, obtaining clinically effective TCR molecules remains a major challenge. We have developed a strategy for cloning tumor-specific TCRs from long-term surviving patients who have responded to immunotherapy. Here, we report the identification of a TCR (10F04), which is human leukocyte antigen (HLA)-DRA/DRB1*09:01 restricted and human papillomavirus type 18 (HPV18) E784-98 specific, from a multiple antigens stimulating cellular therapy (MASCT) benefited metastatic cervical cancer patient. Upon transduction into human T cells, the 10F04 TCR demonstrated robust antitumor activity in both in vitro and in vivo models. Notably, the TCR effectively redirected both CD4+ and CD8+ T cells to specifically recognize tumor cells and induced multiple cytokine secretion along with durable antitumor activity and outstanding safety profiles. As a result, this TCR is currently being investigated in a phase I clinical trial for treating HPV18-positive cancers. This study provides an approach for developing safe and effective TCR-T therapies, while underscoring the potential of HLA class II-restricted TCR-T therapy as a cancer treatment.

Abstract PR02: Tumor sequestered, neoantigen specific T cells are dynamically increased in circulation following neoadjuvant immunotherapy

Abstract Improved recurrence free survival is observed following neoadjuvant immune checkpoint blockade (ICB), but the mechanisms underlying this clinical benefit are incompletely understood. To gain insights into changes in the tumor microenvironment (TME) that occur following ICB, we performed paired single-cell transcriptomic and T cell receptor (TCR) RNA sequencing on sorted tumor infiltrating T-lymphocytes before and after neoadjuvant treatment of patients with newly diagnosed oral carcinoma with bintrafusp alfa, a bifunctional PD-L1 mAb and TGF-b neutralizing construct. Analysis of pre- and post-treatment TIL with identical TCRs allowed characterization of changes within individual clonotypes after treatment. The addition of deep TCRb complimentary determining region 3 (CDR3) sequencing of peripheral T cells allowed assessment of dynamic changes in individual T cell clonotypes between the tumor and peripheral blood. Within the TME, multiple clearly defined clusters of CD8+ and CD4+ T cells with distinct transcriptional profiles were observed, including T cells expressing activation, exhaustion, and tissue-resident markers (exhausted) as well as T cells expressing progenitor and memory markers (memory). Multiple TCRs from clonotypes observed to be present in the exhausted and memory compartments were cloned for study of their antigen specificity. Putative neoepitopes were synthesized following in silico prediction of putative mutation derived neoantigens. Several TCRs from the exhausted compartment were specific for mutation-derived neoantigens. Conversely, TCRs from the memory compartment were specific for common viral antigens. With treatment, exhausted tumor-specific T cells transitioned between different exhausted clusters and some transitioned into a proliferating cluster and expanded in number. Expression of genes associated with TCR signaling and glycolysis increased in exhausted clonotypes that expanded with treatment. Expression of GLS, a key gene associated with glutaminolysis, associated with clonotypes that did not expand with treatment. Study of the peripheral blood revealed that CDR3 sequences matching exhausted, tumor specific TCRs from the TME were not detected or detected at very low frequencies prior to treatment. However, detected of CDR3 sequences matching exhausted, tumor specific TCRs from the TME significantly increased after treatment. CDR3 sequences matching memory, viral specific TCRs from the TME were detected at high levels in the peripheral blood prior to treatment, did not significantly change with treatment. Thus, neoadjuvant immunotherapy appears to expand exhausted, tumor specific T cells in the TME and results in increased frequency of these T cells in circulation. These data imply that tumor specific T cells may be largely sequestered into tissues, such as the tumor and possibly draining lymph nodes, in patients with newly diagnosed oral carcinoma and that surgical removal of disease without the use of neoadjuvant immunotherapy may remove a significant proportion of the patient’s anti-tumor T cell immunity. Citation Format: Cem Sievers, Marco Craveiro, Jason Redman, Clint T. Allen. Tumor sequestered, neoantigen specific T cells are dynamically increased in circulation following neoadjuvant immunotherapy [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR02.

High-dimensional analysis of the T cell response in multiple myeloma and the potential efficacy of harnessing tumor-reactive CD8 T cells

Abstract Multiple myeloma is an incurable malignancy of plasma cells in the bone marrow increasingly being treated by immunotherapies with improved response and survival over traditional chemotherapy, however an unexplored potential immunotherapy is the adoptive transfer of tumor-specific CD8+ T cells. Here, we investigated the tumor-specific CD8+ T cell response in highly progressed multiple myeloma patients. We predicted neoantigens and tumor-associated antigens for each patient and grew out tumor-specific CD8 T cells from patient TIL’s using autologous B cells loaded with the predicted epitopes. We then utilized TetTCR-SeqHD, a high-throughput tetramer-based workflow, to isolate paired tumor-specific T cell receptor (TCR) sequence and antigen specificities. We subsequently transduced and functionally validated these TCR specificities and their cytotoxicity in vitro. In addition, we performed single-cell transcriptomic and proteomic analyses on TIL’s and peripheral blood T cells before and after immunomodulatory treatment to capture high-dimensional data on the cellular states of the T cell response and their response to stimulation. We observed T cells within the tumor were largely exhausted, but that patients had neoantigen-specific CD8+ T cell populations present responding to a broad array of antigens suggesting adoptive transfer of tumor-specific CD8 T cells is potentially efficacious. Last, we measured TCR repertoire changes and identified T cell clones that had expanded. We also found a number of peripheral blood T cells markers that correlated with treatment response in our patients. Next, we aim to transfer transduced tumor-specific T cells into PDX mouse models to measure preclinical therapeutic potential. Supported by grants from CRI-STAR and NIH R01

Abstract 605: Single cell RNA sequencing reveals tumor immune microenvironment and T cell receptor diversity in epidermal growth factor receptor (EGFR) mutated lung cancer

Abstract Background: Somatic alterations in the Epidermal Growth Factor Receptor (EGFR) gene represent a common subset of oncogene-driven lung cancer. Patients who receive EGFR-targeted tyrosine kinase inhibitor (TKI) therapy inevitably develop treatment resistance with limited options after disease progression, with immune checkpoint inhibitor therapy (ICI) typically ineffective. Tumor Immune Microenvironment (TME) characteristics, including T-cell receptor (TCR) sequencing and clonality have been incompletely explored in this population. Methods: We further analyzed our group’s available data from a previous study on therapy-induced evolution of human lung cancer (Maynard et. al, PMID: 32822576). Tumor samples were sorted and subjected to smartseq-based single-cell RNA sequencing. A total of 7495 cells were derived from 17 samples from 10 patients with tumors that harbored EGFR driver gene mutations. This included 1322 gene marker-annotated T-cells. Samples were grouped into three treatment conditions: treatment naïve (TN), residual disease (RD), and progressive disease (PD). We then used the TraCeR tool to infer the TCR α, β, and the complementarity-determining region 3 (CDR3) sequences. Results: T-cells were enriched in RD (55.2%) relative to TN (12.8%) and PD (31.9%) (p<0.0001, Chi-square test) with cytotoxic T-cells as the predominant phenotype across treatment conditions. Increased expression of CTLA4, and LAG3 occurred in PD (p<0.0001, Kruskal-Wallis test). Single-cell TCR analysis revealed increased TCR clonal diversity in PD (Shannon Diversity Index: 0.261 PD > 0.255 TN > 0.205 RD) while increased clonal expansion occurred in RD (32 clones from 4 patients) as compared to TN (17 clones from 1 patient) and PD (14 clones from 1 patient) (p<0.0001, Chi-square test). Conclusion: T-cell infiltration is greatest in RD, while immunosuppressive genes were significantly expressed in PD. Increased TCR clonal expansion with less clonal diversity in RD suggests the presence of tumor-specific TCR clones and that this may be the optimal time to initiate ICI treatment. Future directions will include the identification of clonally expanded TCR and matched neoantigens to provide potential tailored immunotherapies for patients with TKI-resistant tumors. Citation Format: Turja Chakrabarti, Wei Wu, Daniel L. Kerr, Paul Allegakoen, Trever G. Bivona, Collin M. Blakely. Single cell RNA sequencing reveals tumor immune microenvironment and T cell receptor diversity in epidermal growth factor receptor (EGFR) mutated lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 605.

TCR sequencing and cloning methods for repertoire analysis and isolation of tumor-reactive TCRs

T cell receptor (TCR) technologies, including repertoire analyses and Tcell engineering, are increasingly important in the clinical management of cellular immunity in cancer, transplantation, and other immune diseases. However, sensitive and reliable methods for repertoire analyses and TCR cloning are still lacking. Here, we report on SEQTR, a high-throughput approach to analyze human and mouse repertoires that is more sensitive, reproducible, and accurate as compared with commonly used assays, and thus more reliably captures the complexity of blood and tumor TCR repertoires. We also present a TCR cloning strategy to specifically amplify TCRs from Tcell populations. Positioned downstream of single-cell or bulk TCR sequencing, it allows time- and cost-effective discovery, cloning, screening, and engineering of tumor-specific TCRs. Together, these methods will accelerate TCR repertoire analyses in discovery, translational, and clinical settings and permit fast TCR engineering for cellular therapies.

Primary Mouse Invariant Natural Killer T (iNKT) Cell Purification and Transduction.

Invariant natural killer T (iNKT) cells are a non-conventional T-cell population expressing a conserved semi-invariant T-cell receptor (TCR) that reacts to lipid antigens, such as α-galactosyl ceramide (α-GalCer), presented by the monomorphic molecule CD1d. iNKT cells play a central role in tumor immunosurveillance and represent a powerful tool for anti-cancer treatment, notably because they can be efficiently redirected against hematological or solid malignancies by engineering with tumor-specific chimeric antigen receptors (CARs) or TCRs. However, iNKT cells are rare and require specific ex vivo pre-selection and substantial in vitro expansion to be exploited for adoptive cell therapy (ACT). This protocol describes a robust method to obtain a large number of mouse iNKT cells that can be effectually engineered by retroviral (RV) transduction. A major advantage of this protocol is that it requires neither particular instrumentation nor a high number of mice. iNKT cells are enriched from the spleens of iVα14-Jα18 transgenic mice; the rapid purification protocol yields a highly enriched iNKT cell population that is activated by anti-CD3/CD28 beads, which is more reproducible and less time consuming than using bone marrow-derived dendritic cells loaded with α-GalCer, without risks of expanding contaminant T cells. Forty-eight hours after activation, iNKT cells are transduced with the selected RV by spin inoculation. This protocol allows to obtain, in 15 days, millions of ready-to-use, highly pure, and stably transduced iNKT cells that might be exploited for in vitro assays and ACT experiments in preclinical studies.

Deep learning reveals predictive sequence concepts within immune repertoires to immunotherapy.

T cell receptor (TCR) sequencing has been used to characterize the immune response to cancer. However, most analyses have been restricted to quantitative measures such as clonality that do not leverage the complementarity-determining region 3 (CDR3) sequence. We use DeepTCR, a framework of deep learning algorithms, to reveal sequence concepts that are predictive of response to immunotherapy. We demonstrate that DeepTCR can predict response and use the model to infer the antigenic specificities of the predictive signature and their unique dynamics during therapy. The predictive signature of nonresponse is associated with high frequencies of TCRs predicted to recognize tumor-specific antigens, and these tumor-specific TCRs undergo a higher degree of dynamic changes on therapy in nonresponders versus responders. These results are consistent with a biological model where the hallmark of nonresponders is an accumulation of tumor-specific T cells that undergo turnover on therapy, possibly because of the dysfunctional state of these T cells in nonresponders.

TCR-engineered iNKT cells induce robust antitumor response by dual targeting cancer and suppressive myeloid cells.

Adoptive immunotherapy with T cells engineered with tumor-specific T cell receptors (TCRs) holds promise for cancer treatment. However, suppressive cues generated in the tumor microenvironment (TME) can hinder the efficacy of these therapies, prompting the search for strategies to overcome these detrimental conditions and improve cellular therapeutic approaches. CD1d-restricted invariant natural killer T (iNKT) cells actively participate in tumor immunosurveillance by restricting suppressive myeloid populations in the TME. Here, we showed that harnessing iNKT cells with a second TCR specific for a tumor-associated peptide generated bispecific effectors for CD1d- and major histocompatibility complex (MHC)-restricted antigens in vitro. Upon in vivo transfer, TCR-engineered iNKT (TCR-iNKT) cells showed the highest efficacy in restraining the progression of multiple tumors that expressed the cognate antigen compared with nontransduced iNKT cells or CD8+ T cells engineered with the same TCR. TCR-iNKT cells achieved robust cancer control by simultaneously modulating intratumoral suppressive myeloid populations and killing malignant cells. This dual antitumor function was further enhanced when the iNKT cell agonist α-galactosyl ceramide (α-GalCer) was administered as a therapeutic booster through a platform that ensured controlled delivery at the tumor site, named multistage vector (MSV). These preclinical results support the combination of tumor-redirected TCR-iNKT cells and local α-GalCer boosting as a potential therapy for patients with cancer.

Indomethacin-induced oxidative stress enhances death receptor 5 signaling and sensitizes tumor cells to adoptive T-cell therapy

BackgroundAdoptive cell therapy (ACT) using genetically modified T cells has evolved into a promising treatment option for patients with cancer. However, even for the best-studied and clinically validated CD19-targeted chimeric...

Pinpointing the tumor-specific T cells via TCR clusters.

Adoptive cell transfer (ACT) is a promising approach to cancer immunotherapy, but its efficiency fundamentally depends on the extent of tumor-specific T cell enrichment within the graft. This can be estimated via activation with identifiable neoantigens, tumor-associated antigens (TAAs), or living or lysed tumor cells, but these approaches remain laborious, time-consuming, and functionally limited, hampering clinical development of ACT. Here, we demonstrate that homology cluster analysis of T cell receptor (TCR) repertoires efficiently identifies tumor-reactive TCRs allowing to: (1) detect their presence within the pool of tumor-infiltrating lymphocytes (TILs); (2) optimize TIL culturing conditions, with IL-2low/IL-21/anti-PD-1 combination showing increased efficiency; (3) investigate surface marker-based enrichment for tumor-targeting T cells in freshly isolated TILs (enrichment confirmed for CD4+ and CD8+ PD-1+/CD39+ subsets), or re-stimulated TILs (informs on enrichment in 4-1BB-sorted cells). We believe that this approach to the rapid assessment of tumor-specific TCR enrichment should accelerate T cell therapy development.

Combined IL-2, agonistic CD3 and 4-1BB stimulation preserve clonotype hierarchy in propagated non-small cell lung cancer tumor-infiltrating lymphocytes

BackgroundAdoptive cell transfer (ACT) of tumor-infiltrating lymphocytes (TIL) yielded clinical benefit in patients with checkpoint blockade immunotherapy-refractory non-small cell lung cancer (NSCLC) prompting a renewed interest in TIL-ACT. This preclinical...

Adoptive cell therapy with tumor-specific Th9 cells induces viral mimicry to eliminate antigen-loss-variant tumor cells.

Resistance can occur in patients receiving adoptive cell therapy (ACT) due to antigen-loss-variant (ALV) cancer cell outgrowth. Here we demonstrate that murine and human T helper (Th) 9 cells, but not Th1/Tc1 or Th17 cells, expressing tumor-specific Tcell receptors (TCRs) or chimeric antigen receptors (CARs), eradicate advanced tumors that contain ALVs. This unprecedented antitumor capacity of Th9 cells is attributed to both enhanced direct tumor cell killing and bystander antitumor effects promoted by intratumor release of interferon (IFN) α/β. Mechanistically, tumor-specific Th9 cells increase the intratumor accumulation of extracellular ATP (eATP; released from dying tumor cells), because of a unique feature of Th9 cells that lack the expression of ATP degrading ectoenzyme cluster of differentiation (CD) 39. Intratumor enrichment of eATP promotes the monocyte infiltration and stimulates their production of IFNα/β by inducing eATP-endogenous retrovirus-Toll-like receptor 3 (TLR3)/mitochondrial antiviral signaling (MAVS) pathway activation. These results identify tumor-specific Th9 cells as a unique Tcell subset endowed with the unprecedented capacity to eliminate ALVs for curative responses.

A Novel Strategy for Off-the-Shelf T Cell Therapy Which Evades Allogeneic T Cell and NK Cell Rejection

Introduction. Despite the success of autologous chimeric antigen receptor (CAR)-T cells, barriers to a more widespread use of this potentially curative therapy include manufacturing failures and the high cost of individualized production. There is a strong desire for an immediately available cell therapy option; however, development of “off-the-shelf” T cells is challenging. Alloreactive T cells from unrelated donors can cause graft versus host disease (GvHD) for which researchers have successfully used nucleases to reduce expression of the endogenous T cell receptor (TCR) in the allogeneic product. The recognition of allogeneic cells by the host is a complex issue that has not been fully solved to date. Some approaches utilize prolonged immune suppression to avoid immune rejection and increase persistence. Although showing responses in the clinic, this approach carries the risk of infections and the durability of the adoptive T cells is uncertain. Other strategies include deletion of the B2M gene to remove HLA class I molecules and avoid recognition by host CD8 T cells. However, loss of HLA class I sends a “missing-self” signal to natural killer (NK) cells, which readily eliminate B2Mnull T cells. To overcome this, researchers are exploring insertion of the non-polymorphic HLA-E gene, which can provide partial but not full protection from NK cell-mediated lysis. Because activated T cells upregulate HLA class II, rejection by alloreactive CD4 T cells should also be addressed.Methods. Here, we developed an immunologically stealth “off-the-shelf” T cell strategy by leveraging our CRISPR/Cas9 platform and proprietary sequential editing process. To solve the issue of rejection by alloreactive CD4 and CD8 T cells, we knocked out (KO) select HLA class I and class II expression with a sequential editing process. Additionally, we utilize potent TCR-α and -β constant chain (TRAC, TRBC) gRNAs that achieve >99% KO of the endogenous TCR, addressing the risk of GvHD. An AAV-mediated insertion of a CAR or TCR into the TRAC locus is used in parallel with the TRAC KO step to redirect the T cells to tumor targets of interest. Alloreactivity by CD4 and CD8 T cells, NK killing, GvHD induction and T cell function was assessed in vitro and/or in vivo.Results. By knocking out select HLA class I and class II proteins, we were able to avoid host CD4- and CD8-T cell-mediated recognition. Edited T cells were protected from host NK cells, both in vitro and in an in vivo model engrafted with functional human NK cells. TRAC edited donor T cells did not induce GvHD in an immune compromised mouse model over the 90-day evaluation period. Using our proprietary T cell engineering process, we successfully generated allogeneic T cells with sequential KOs and insertion of a tumor-specific TCR or CAR with high yield. Importantly, these allogeneic T cells had comparable functional activity to their autologous T cell counterparts in in vitro assays (tumor cell killing and cytokine release) as well as in vivo tumor models. With a relatively small bank of donors, we can provide an “off-the-shelf” CAR or TCR-T cell solution for a large proportion of the population.Conclusions. We have successfully developed a differentiated “off-the-shelf” approach, which is expected to be safe and cost-effective. It is designed to provide long-term persistence without the need for an immune suppressive regimen. This promising strategy is being applied to our T cell immuno-oncology and autoimmune research candidates. DisclosuresZhang: Intellia Therapeutics: Current Employment. Goel: Intellia Therapeutics: Current Employment. Prodeus: Intellia Therapeutics: Current Employment. Jetley: Intellia Therapeutics: Current Employment. Tan: Intellia Therapeutics: Current Employment. Averill: Intellia Therapeutics: Current Employment. Ranade: Intellia Therapeutics: Current Employment. Balwani: Intellia Therapeutics: Current Employment. Dutta: Intellia Therapeutics: Current Employment. Sharma: Intellia Therapeutics: Current Employment. Venkatesan: Intellia Therapeutics: Current Employment. Liu: Intellia Therapeutics: Current Employment. Roy: Intellia Therapeutics: Current Employment. O′Connell: Intellia Therapeutics: Current Employment. Arredouani: Intellia Therapeutics: Current Employment. Keenan: Intellia Therapeutics: Current Employment. Lescarbeau: Intellia Therapeutics: Current Employment. Schultes: Intellia Therapeutics: Current Employment.

Pulsed Radiation Therapy to Improve Systemic Control of Metastatic Cancer.

Radiation therapy (RT) is emerging as an interventional modality in the cancer-immunity cycle, augmenting the activation of an adaptive immune response against tumors. RT, particularly in combination with immunotherapy, can enhance immune memory effects and shape the tumor-directed T-cell populations. However, a single cycle of RT delivered to a limited number of polymetastatic lesions is rarely sufficient to achieve systemic control. We hypothesize that several rounds of RT, akin to several rounds of immunotherapeutic drugs, is likely to provide greater clinical benefit to patients with metastatic disease. We propose that the repeated exposure to tumor antigens released by “pulsed-RT” (i.e., treating 2-4 tumor lesions with 3 irradiation cycles given one month apart) may amplify the adaptive immune response by expanding the tumor-specific T-cell receptor repertoire, the production of high-affinity tumor antibodies, and the generation of memory lymphocytes and thereby improve immune control of systemic disease.

Engineered T Cell Therapy for Gynecologic Malignancies: Challenges and Opportunities.

Gynecologic malignancies, mainly including ovarian cancer, cervical cancer and endometrial cancer, are leading causes of death among women worldwide with high incidence and mortality rate. Recently, adoptive T cell therapy (ACT) using engineered T cells redirected by genes which encode for tumor-specific T cell receptors (TCRs) or chimeric antigen receptors (CARs) has demonstrated a delightful potency in B cell lymphoma treatment. Researches impelling ACT to be applied in treating solid tumors like gynecologic tumors are ongoing. This review summarizes the preclinical research and clinical application of engineered T cells therapy for gynecologic cancer in order to arouse new thoughts for remedies of this disease.

Abstract 127: Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer

Abstract The adoptive transfer of tumor-infiltrating lymphocytes (TILs) expanded in vitro can mediate regressions of established tumors, yet only a minority of intratumoral T cells are reactive to cancer antigens while the majority represent bystander cells. This complexity underpins the need to phenotypically characterize T cells infiltrating the tumor microenvironment. In this study, we performed single-cell RNA and T cell receptor (TCR) sequencing (scRNA/TCR-seq) on over 45,000 T cells isolated from ten archival metastatic tumor samples whose primary histologies included colon, rectal, breast, and melanoma. By transcriptomic clustering of the T cells and mapping TCR identity data onto these blueprints, we observed a diversity of T cell phenotypes spanning from memory-like to dysfunctional effector-like, with clonal expansion occurring more widely in the effector-like cells. Through sequencing of peripheral blood lymphocytes (PBL) obtained at the time of tumor resection, we found T cell clusters that were either tumor enriched or distinctly PBL enriched, potentially representing recent tumor immigrants. Prior knowledge of 30 CD4 and CD8 tumor neoantigen-reactive TCRs (NeoTCRs) from eight patients allowed us to project these NeoTCRs onto the TIL transcriptomic map where we observed 380 total T cells bearing these NeoTCRs. The majority (>85%) of NeoTCRs were expressed by T cells within 2 clusters, one CD4+ and one CD8+, marked by expression of CXCL13, ENTPD1 (CD39), TOX, and PDCD1 (PD-1), indicating a dysfunctional state. We reasoned that other dominant T cell clonotypes within the NeoTCR clusters could contain additional novel NeoTCR clonotypes. We expressed these predicted NeoTCRs in donor PBL and screened them in vitro with antigen presenting cells (APCs) pulsed with peptides or tandem minigenes encoded by patient-specific tumor mutations, yielding 24 new NeoTCR clones (251 cells), which included neoantigen reactivities undetected in the same patients' fragment TIL screens. Using a NeoTCR signature score derived from the archival samples, three prospective patients' TIL were analyzed by scRNA/TCRseq and clusters scored according to their NeoTCR signature. TCRs reconstructed from these clusters and tested against patients' tumor-specific reagents yielded novel CD4 and CD8 NeoTCRs. Altogether, this study enabled successful enrichment and detection of tumor-specific NeoTCRs in the sequenced TIL of 12/12 patients for whom reactivity was either previously assessed or prospectively identified. Deconvolution of NeoTCRs from bystander TCRs within the tumor-immune microenvironment represents an important step in the development of personalized immunotherapy, as prospective NeoTCR isolation based on transcriptional phenotype of TIL will allow for rapid therapeutic development in the form of T cells expressing these tumor-specific TCRs. Citation Format: Frank J. Lowery, Sri Krishna, Rami Yossef, Praveen D. Chatani, Neilesh B. Parikh, Yong-Chen Lu, Nikolaos Zacharakis, Noam Levin, Ken-ichi Hanada, James C. Yang, Paul F. Robbins, Maria R. Parkhurst, Steven A. Rosenberg. Single cell mapping of tumor infiltrating lymphocytes enables neoantigen-reactive T cell identification in metastatic human cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 127.

Abstract PO-015: Systematic discovery of KRAS neoantigens

Abstract Response rates to immunotherapies in pancreatic ductal adenocarcinoma (PDAC) patients are low. However, cases with long-term survival have been correlated with both T cell infiltration and quality of neoantigens (i.e., epitopes from mutations presented by Human Leukocyte Antigen (HLA) molecules). It is known that mutations in the KRAS oncogene, which occur in the majority of PDAC patients, can be recognized by T cells as neoantigens on specific HLA molecules and lead to tumor control. KRAS neoantigens may thus be an attractive therapeutic target in a substantial fraction of PDAC patients. In this study, we systematically evaluated a broad range of KRAS neoantigens using an integrated discovery and validation pipeline. To date, the discovery and validation of KRAS neoantigens has mainly relied on the detection of stochastic T cell responses in patient samples or the use of transgenic mouse models. As such, the reported coverage of common HLA alleles is relatively limited. To overcome these issues, we established a systematic pipeline that addresses two fundamental principles: (1) the presentation of the neoantigen on cells with the mutation and (2) ability of the neoantigen to elicit T cell responses. The presentation of potential neoantigens was evaluated by applying a targeted mass spectrometry approach to detect peptides eluted from specific class I HLA molecules isolated from cells harboring a KRAS mutation of interest. T cell responses were elicited to MS-observed peptides using a proprietary in vitro stimulation protocol, after which the T cells can further be used in cytotoxicity assays to orthogonally confirm neoantigen presentation. We substantiated our pipeline using KRAS codon 12 neoantigens reported in the literature (e.g., G12D on HLA-C*08:02), then evaluated novel neoantigens predicted to be presented by common class I HLA molecules in the U.S. population. Through this pipeline, we have increased the number of validated KRAS neoantigens and thus the targetable patient population expected to harbor at least one KRAS neoantigen. A more comprehensive understanding of KRAS neoantigens will help drive focused therapies, such as the development of tumor-specific T cell receptor (TCR) therapies or neoantigen vaccines in appropriate patient populations in PDAC or other indications. Citation Format: Jaewon Choi, Scott P Goulding, Brandon P. Conn, Christopher D. McGann, Jared L. Dietze, Jessica Kohler, Divya R. Lenkala, Antoine Boudot, Daniel A. Rothenberg, Paul J. Turcott, John S. Srouji, Michael S. Rooney, Marit M. van Buuren, Richard B. Gaynor, Jennifer G. Abelin, Terri A. Addona, Vikram R. Juneja. Systematic discovery of KRAS neoantigens [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-015.