Tumor Epitopes Research Articles

Abstract B024: Using single-cell paired sequencing to isolate cancer-specific T-cell receptors for cancer immunotherapy

Abstract Immunocore’s ImmTAC™ (Immune Mobilising Monoclonal TCR Against Cancer) platform combines affinity-enhanced T-cell receptor (TCR)-based targeting with an anti-CD3 scFv effector function to activate a cytotoxic T-cell response against cancer cells. A key part to this process is the identification of tumour epitope specific TCRs from tumor antigen-reactive T-cells. Here, we describe an integrated in-house process leading to the isolation of TCRs specific for validated cancer epitopes, coupled with rapid identification of TCR chains from individual clones using single cell sequencing. The process involves first strand cDNA generation and universal amplification using SmartSeq2 chemistry, followed by targeted sequencing of the TCR alpha and beta chains using next-generation sequencing (NGS). We have also leveraged the 10x Genomics VDJ/5’ counting platform to label and pool multiple experimental clones for repertoire sequencing within a single run. Together with Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-Seq), we can reliably assign each T-cell clone to its sample of origin paired with transcriptomic information of epitope specific T-cell populations, linking TCR sequences to their functional phenotype. Citation Format: Karolina Lech, Lucia Correia, Max Beckmann, Maria Busz, Sean Collison, Sterenn Davis, Paraskevi Mallini, Sarah Scaife, Joseph Dukes, Bent K. Jakobsen, Luke Williams, Michelle Teng. Using single-cell paired sequencing to isolate cancer-specific T-cell receptors for cancer immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B024.

CD8+ cytotoxic T cell responses to dominant tumor-associated antigens are profoundly weakened by aging yet subdominant responses retain functionality and expand in response to chemotherapy

ABSTRACTIncreasing life expectancy is associated with increased cancer incidence, yet the effect of cancer and anti-cancer treatment on elderly patients and their immune systems is not well understood. Declining T cell function with aging in response to infection and vaccination is well documented, however little is known about aged T cell responses to tumor antigens during cancer progression or how these responses are modulated by standard chemotherapy. We examined T cell responses to cancer in aged mice using AE17sOVA mesothelioma in which ovalbumin (OVA) becomes a ‘spy’ tumor antigen containing one dominant (SIINFEKL) and two subdominant (KVVRFDKL and NAIVFKGL) epitopes. Faster progressing tumors in elderly (22–24 months, cf. 60–70 human years) relative to young (2–3 months, human 15–18 years) mice were associated with increased pro-inflammatory cytokines and worsened cancer cachexia. Pentamer staining and an in-vivo cytotoxic T lymphocyte (CTL) assay showed that whilst elderly mice generated a greater number of CD8+ T cells recognizing all epitopes, they exhibited a profound loss of function in their ability to lyse targets expressing the dominant, but not subdominant, epitopes compared to young mice. Chemotherapy was less effective and more toxic in elderly mice however, similar to young mice, chemotherapy expanded CTLs recognizing at least one subdominant epitope in tumors and draining lymph nodes, yet treatment efficacy still required CD8+ T cells. Given the significant dysfunction associated with elderly CTLs recognizing dominant epitopes, our data suggest that responses to subdominant tumor epitopes may become important when elderly hosts with cancer are treated with chemotherapy.

Neoepitope targets of tumour-infiltrating lymphocytes from patients with pancreatic cancer

BackgroundPancreatic cancer exhibits a poor prognosis and often presents with metastasis at diagnosis. Immunotherapeutic approaches targeting private cancer mutations (neoantigens) are a clinically viable option to improve clinical outcomes.Methods3/40 TIL lines (PanTT26, PanTT39, PanTT77) were more closely examined for neoantigen recognition. Whole-exome sequencing was performed to identify non-synonymous somatic mutations. Mutant peptides were synthesised and assessed for antigen-specific IFN-γ production and specific tumour killing in a standard Cr51 assay. TIL phenotype was tested by flow cytometry. Lymphocytes and HLA molecules in tumour tissue were visualised by immunohistochemistry.ResultsPanTT26 and PanTT39 TILs recognised and killed the autologous tumour cells. PanTT26 TIL recognised the KRASG12v mutation, while a PanTT39 CD4+ TIL clone recognised the neoepitope (GLLRYWRTERLF) from an aquaporin 1-like protein (gene: K7N7A8). Repeated stimulation of TILs with the autologous tumour cells line lead to focused recognition of several mutated targets, based on IFN-γ production. TILs and corresponding PBMCs from PanTT77 showed shared as well as mutually exclusively tumour epitope recognition (TIL-responsive or PBMC-responsive).ConclusionThis study provides methods to robustly screen T-cell targets for pancreatic cancer. Pancreatic cancer is immunogenic and immunotherapeutic approaches can be used to develop improved, targeted therapies.

Abstract 2985: Radiotherapy and αCD40 non-redundantly augment immunity to checkpoint blockade in refractory pancreatic ductal adenocarcinoma

Abstract Despite the success of cancer immunotherapy in many disease types, pancreatic ductal adenocarcinoma (PDA) is notably unresponsive to immune checkpoint blockade (ICB) with αPD1 and/or αCTLA4. The mechanism of resistance is poorly understood, but tumor epitopes and the microenvironment, which is immunosuppressive and excludes T cells, are thought to be contributory. To improve response to ICB, we used subcutaneous and orthotopic murine models of PDA to investigate the effect of combination therapy with ICB (CTLA-4 and PD-1 antagonist antibodies), CD40 agonist antibody and radiation therapy (RT). Combination therapy with CD40 agonist antibody, ICB and RT resulted in decrease tumor burden, increase overall survival, and generation of long-term immunity. Response is dependent on T and short-lived myeloid cells, while it is independent of innate activation pathways. Together, these results suggest a dual role for both the innate and adaptive immune response in treating PDA. Citation Format: Hannah Dada, Andrew J. Rech, Christina Twyman-Saint Victor, Andy J. Minn, Robert H. Vonderheide. Radiotherapy and αCD40 non-redundantly augment immunity to checkpoint blockade in refractory pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2985.

Literature Round-Up: Impactful Published Papers

Foiling cleavage (by Marie-Lan Nguyen via Wikimedia Commons)Tumors can avoid immune elimination by NK cells by shedding MICA and MICB, ligands for the activating receptor NKG2D. An antibody that blocks cleavage of these ligands prevents shedding, enhances NKG2D-mediated killing of the tumor cells, and reduces tumor growth in models of lung and melanoma metastasis. The prevalence of MICA and MICB expression on human tumors indicates that such antibodies could have broad therapeutic potential in combination with other approaches.de Andrade LF, …, Wucherpfennig KW. Science 2018 Mar 30;359:1537–42.Multimer peptide exchange (from Fig. 2 of Luimstra et al.)Personalized approaches to antitumor immunity rely upon identification of the tumor epitopes recognized by T cells and quantitation of specific antitumor responses. A simplified, temperature-based method was devised to produce the peptide-MHC multimers needed to identify antigen-specific T cells. Conditional peptides that bind to multimers at 4°C are replaced at higher temperatures by the peptides of interest. Antigen-specific T cells can be detected and immune responses monitored.Luimstra JJ, …, Ovaa H. J Exp Med 2018 Apr 17. DOI: 10.1084/jem.20180156.Malignant melanoma (by Ed Uthman via Flickr)Human CD8+ T cells induce colony-stimulating factor–1 (CSF1) expression by melanoma cells. This attracts immunosuppressive macrophages into the tumor. Treatment of mice with transplanted BrafV600E-driven melanoma tumors with blocking antibodies to both PD-1 and the CFS1 receptor synergistically inhibits tumor growth and increases the survival of tumor-bearing mice.Neubert NJ, …, Speiser DE. Sci Transl Med 2018 Apr 11;10:eaan3311.Discriminating with autophagy (from Amaravadi et al. Clin Cancer Res 2011)Some resistance of melanoma to CTLA-4, but not PD-1, blockade is now linked to expression of the proteins encoded by the MAGE-A supercluster of genes. The MAGE-A proteins interfere with autophagy, a process important to the generation of antitumor responses. Patients whose melanoma expresses these proteins are resistant to anti–CTLA-4, but no association was seen in two cohorts of patients treated with anti–PD-1. This key insight suggests combining autophagy induction with CTLA-4 blockade for patients whose melanomas express the supercluster.Shukla SA, …, Wu CJ. Cell 2018 Apr 19;173:624–33.CLL in bone marrow (from Skarzynski et al. Clin Cancer Res 2016)Only a quarter of chronic lymphocytic leukemia patients respond to CAR T-cell therapy. Complete responders experience expansion and persistence of their CAR T cells, even after 5 years, which are properties found to be intrinsic to the initial transferred T cells. Transcriptome analysis found that the T cells infused into responders are more highly enriched in early memory differentiation genes, and the presence of CD45RO−CD27+CD8+ T cells in peripheral blood prior to CAR transduction is a predictor of response to CAR T-cell therapy.Fraietta JA, …, Melenhorst JJ. Nat Med 2018 Apr 30;24:563–71.Lung tumor (by Lange123 via Wikimedia Commons)A pilot study that treated 20 early-stage lung cancer patients with anti–PD-1 prior to removal of their lung tumors showed that this treatment was safe. Tumor pathology showed responses in 45% of those treated, and responders were more likely to have tumors with greater numbers of mutated sequences. TCR analysis revealed increased expansion of clones specific for tumor neoepitopes.Forde PM, …, Pardoll DM. N Engl J Med 2018 Apr 16. DOI: 10.1056/NEJMoa1716078.

Oncolytic vaccines increase the response to PD-L1 blockade in immunogenic and poorly immunogenic tumors

ABSTRACTActivation of immune checkpoint pathways and limited T- cell infiltration result in immunological escape of tumors. Although immune checkpoint inhibitors are currently approved for several types of cancers, the response rate is often limited by the lack of tumor specific T-cells within the malignant tissue. Therefore, new combinatorial strategies are needed to enhance the clinical benefit of immune checkpoint inhibitors.We have previously developed PeptiCRAd, an oncolytic vaccine platform capable of directing the immune response toward tumor epitopes. In this study, we evaluated whether the platform could be used to increase the response rate to checkpoint inhibitors in both highly immunogenic and poorly immunogenic tumors, such as melanoma and triple negative breast cancer (TNBC).We report here that anti-PD-L1 therapy in combination with PeptiCRAd significantly reduced the growth of melanomas and increased the response rate to checkpoint inhibition. In fact, we registered a higher rate of complete responses among mice treated with the combination. This approach promoted the presence of non-exhausted antigen-specific T-cells within the tumor in comparison to anti-PD-L1 monotherapy. Furthermore, we found that targeting both MHC-I and II restricted tumor epitopes was necessary to decrease the growth of the poorly immunogenic TNBC model 4T1 and that combination with PD-L1 blockade increased the number of responders to checkpoint inhibition. Finally, the described strategy was validated in a translational in vitro model using HLA matched human PBMCs and tumor cell lines. Consistent to our previous results, improved cytotoxicity was observed with combination of PeptiCRAd and anti-PD-L1.These results demonstrate that oncolytic virus based cancer vaccine can significantly improve the response rate to checkpoint blocking antibodies in the context of immunogenic and non-immunogenic tumors.

Divergent T-cell receptor recognition modes of a HLA-I restricted extended tumour-associated peptide

Human leukocyte antigen (HLA)-I molecules generally bind short peptides (8–10 amino acids), although extended HLA-I restricted peptides (>10 amino acids) can be presented to T cells. However, the function of such extended HLA-I epitopes in tumour immunity, and how they would be recognised by T-cell receptors (TCR) remains unclear. Here we show that the structures of two distinct TCRs (TRAV4+TRAJ21+-TRBV28+TRBJ2-3+ and TRAV4+TRAJ8+-TRBV9+TRBJ2-1+), originating from a polyclonal T-cell repertoire, bind to HLA-B*07:02, presenting a 13-amino-acid-long tumour-associated peptide, NY-ESO-160–72. Comparison of the structures reveals that the two TCRs differentially binds NY-ESO-160–72–HLA-B*07:02 complex, and induces differing extent of conformational change of the NY-ESO-160–72 epitope. Accordingly, polyclonal TCR usage towards an extended HLA-I restricted tumour epitope translates to differing TCR recognition modes, whereby extensive flexibility at the TCR–pHLA-I interface engenders recognition.

Adoptive cell therapy with CD4+ T helper 1 cells and CD8+ cytotoxic T cells enhances complete rejection of an established tumour, leading to generation of endogenous memory responses to non-targeted tumour epitopes.

The results of adoptive T-cell therapies (ACTs) are very encouraging and show clinical evidence that ACT can provide a cure for patients with metastatic disease. However, various response rates and long-term cancer remission have been observed in different ACT trials. The types of T cells, prior treatment with chemotherapy and co-administration of other immune-target therapies have been found to influence the efficacy of ACT. In this study, we investigate the ability of ACT using CD4+ T helper 1 (Th1) cells and CD8+ cytotoxic T lymphocytes (CTLs) to reject the growth of established B16-ovalbumin (OVA) melanoma. CD8+ CTLs were found to be the main effector T cells that mediated tumour regression. However, low tumour-free survival rates were observed in ACT with CD8+ CTLs only. Co-transferring CD4+ Th1 cells and CD8+ CTLs has been observed to induce a synergistic antitumour response, resulting in complete regression in 80% of the tumour-bearing mice. We also examined a prior Dacarbazine (DTIC) and after virus-like particle (VLP)-OVA vaccine treatment to enhance ACT, but no therapeutic benefit was observed during primary B16-OVA tumour growth. Nevertheless, the ACT-mediated antitumour response was able to generate memory responses to both B16-OVA and B16-gp33 tumours. VLP-OVA vaccination following ACT enhances the memory responses to tumours that express a heterogenic population of both B16-OVA and B16-gp33 cells; however, it abolished the memory response to tumours consisting of only gp33-expressing cells. These findings provide important information for designing therapeutic treatments for patients with metastatic disease and cancer relapse to achieve durable cancer remission.

Peritumoral IL-15 Promotes Regional NK Activation and DC Licensing to Augment Radiation-Induced Anti-tumor Immunity

Radiotherapy (RT) can elicit anti-tumor T cells by multiple mechanisms but the induced immune response is limited by the immunosuppression in the tumor microenvironment. The common gamma-chain cytokines IL-15 promotes the activation of natural killer (NK) cells and the proliferation of activated CD8+ T cells and is under investigation for its ability to induce T cell response in cancer patients. Here we tested the hypothesis that IL-15 strengthens the pro-immunogenic effect of local RT to potentiate durable tumor-specific immunity. The poorly immunogenic mouse TSA breast cancer cells were implanted s.c. in syngeneic wild type (WT) or batf3-deficient BALB/c mice and when tumors reached 5mm in average diameter, were randomly assigned to one of 4 treatment groups: control, RT, IL-15 or RT+IL-15. Local RT was delivered consecutively in 8 Gy fractions on days 13, 14, and 15 post tumor implantation. IL-15 (2 μg/mouse) was administered s.c. peritumorally daily for 10 days starting on the first day of RT. Mice were followed for tumor growth and surviving mice subsequently re-challenged. Some animals were depleted of CD8 and NK cells (anti-asialo-GM1 Ab) using standard protocols. A parallel experiment was done to characterize tumor-infiltrating lymphocytes (TILs) and measure ex vivo IFNg production by tumor-specific CD8 T-cells in the draining lymph nodes at the end of treatment (day 22). IL-15 as monotherapy had no effect on tumor control but significantly improved the ability of RT to delay tumor growth in WT mice (P < 0.01, RT vs RT+IL-15). Combination treatment led to durable and robust memory responses in 50% of animals compared to 0% of mice treated with RT alone. RT enhanced intratumoral infiltration of DX5+NKp46+ NK cells and was further increased by the addition of IL-15 (22.86% of CD45 TILs in RT vs 32% in RT+IL-15, P<0.05). CD8+ T cells expressing the activation marker CD137 were significantly increased in tumors of RT+IL-15-treated mice while the increase was modest with each monotherapy (35.3% in RT+IL-15 vs 18.8% in RT, 20.7% in IL-15, 5.90% in control, P < 0.05). Systemic depletion of either CD8 T-cells or NK cells abrogated the therapeutic effect of the combination. RT+IL-15 elicited more priming of tumor-specific T-cells in response to tumor epitope AH-1-A5 (1238 pg/mL in RT+IL-15 vs 3.3 pg/mL in control vs 28.7 pg/mL in IL-15 RT vs 2.63 pg/mL in RT; P<0.05). Batf3-/- mice that CD103+ DCs showed reduce response to RT+IL-15 compared to WT mice. Here we show that local IL-15 administration improves priming of anti-tumor CD8+ T cell and leads to long-term memory responses by a mechanism that is dependent on NK cells and Batf3-dependent DCs. Data suggest that IL-15 activated NK cells kill tumor cells and license DCs leading to a long-term T cell-mediated memory response. Overall these results support the testing of IL-15 in combination with tumor-targeted radiotherapy.

CD40 Signaling Drives Potent Cellular Immune Responses in Heterologous Cancer Vaccinations.

Antagonistic antibodies targeting coinhibitory receptors have revolutionized the treatment of cancer by inducing durable immune responses and clinical remissions in patients. In contrast, success of agonistic costimulatory antibodies has thus far been limited because of the insufficient induction of adaptive immune responses. Here, we describe a novel vaccination method consisting of a primary dendritic cell (DC) immunization followed by a composite vaccination, including an agonistic CD40 antibody, soluble antigen, and a TLR3 agonist, referred to as CoAT. In mice, DC/CoAT prime-boost vaccinations targeting either MHC class I or II neoantigens or tumor-associated antigens rendered up to 60% of the total T-cell population specific for a single tumor epitope. DC/CoAT induced durable and complete remissions of large subcutaneous tumors without detectable side effects. Thus, booster vaccinations with agonistic costimulatory antibodies represent an ideal means to amplify DC vaccinations and induce robust T-cell immune responses while providing maximum flexibility regarding the choice of antigen. Cancer Res; 77(8); 1918-26. ©2017 AACR.

TANTIGEN: a comprehensive database of tumor T cell antigens.

Tumor T cell antigens are both diagnostically and therapeutically valuable molecules. A large number of new peptides are examined as potential tumor epitopes each year, yet there is no infrastructure for storing and accessing the results of these experiments. We have retroactively cataloged more than 1000 tumor peptides from 368 different proteins, and implemented a web-accessible infrastructure for storing and accessing these experimental results. All peptides in TANTIGEN are labeled as one of the four categories: (1) peptides measured in vitro to bind the HLA, but not reported to elicit either in vivo or in vitro T cell response, (2) peptides found to bind the HLA and to elicit an in vitro T cell response, (3) peptides shown to elicit in vivo tumor rejection, and (4) peptides processed and naturally presented as defined by physical detection. In addition to T cell response, we also annotate peptides that are naturally processed HLA binders, e.g., peptides eluted from HLA in mass spectrometry studies. TANTIGEN provides a rich data resource for tumor-associated epitope and neoepitope discovery studies and is freely available at http://cvc.dfci.harvard.edu/tantigen/ or http://projects.met-hilab.org/tadb (mirror).

Understanding CD8+ T-cell responses toward the native and alternate HLA-A*02:01-restricted WT1 epitope.

The Wilms' tumor 1 (WT1) antigen is expressed in solid and hematological malignancies, but not healthy tissues, making it a promising target for cancer immunotherapies. Immunodominant WT1 epitopes, the native HLA-A2/WT1126-134 (RMFPNAPYL) (HLA-A2/RMFPNAPYL epitope (WT1A)) and its modified variant YMFPNAPYL (HLA-A2/YMFPNAPYL epitope (WT1B)), can induce WT1-specific CD8+ T cells, although WT1B is more stably bound to HLA-A*02:01. Here, to further determine the benefits of those two targets, we assessed the naive precursor frequencies; immunogenicity and cross-reactivity of CD8+ T cells directed toward these two WT1 epitopes. Ex vivo naive WT1A- and WT1B-specific CD8+ T cells were detected in healthy HLA-A*02:01+ individuals with comparable precursor frequencies (1 in 105–106) to other naive CD8+ T-cell pools (for example, A2/HIV-Gag77-85), but as expected, ~100 × lower than those found in memory populations (influenza, A2/M158-66; EBV, A2/BMLF1280-288). Importantly, only WT1A-specific naive precursors were detected in HLA-A2.1 mice. To further assess the immunogenicity and recruitment of CD8+ T cells responding to WT1A and WT1B, we immunized HLA-A2.1 mice with either peptide. WT1A immunization elicited numerically higher CD8+ T-cell responses to the native tumor epitope following re-stimulation, although both regimens produced functionally similar responses toward WT1A via cytokine analysis and CD107a expression. Interestingly, however, WT1B immunization generated cross-reactive CD8+ T-cell responses to WT1A and could be further expanded by WT1A peptide revealing two distinct populations of single- and cross-reactive WT1A+CD8+ T cells with unique T-cell receptor-αβ gene signatures. Therefore, although both epitopes are immunogenic, the clinical benefits of WT1B vaccination remains debatable and perhaps both peptides may have separate clinical benefits as treatment targets.

Tumor antigen-specific CD8+ T cells are negatively regulated by PD-1 and Tim-3 in human gastric cancer

Cytotoxic CD8 T lymphocytes that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally important in eliciting tumor rejection. NY-ESO-1 is a major target of CD8+ T cell recognition in gastric cancer (GC) and is among the most immunogenic tumor antigens defined to date. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8+ T cell dysfunction may reveal effective strategies for immunotherapy. In an effort to understand in vivo tolerance mechanisms, we assessed the phenotype and function of NY-ESO-1-specific CD8+ T cells derived from peripheral blood lymphocytes (PBLs) and tumor-associated lymphocytes (TALs) of GC patients. Here, we report that Tim-3 expression defines a subpopulation of PD-1+ exhausted NY-ESO-1-specific CD8+ T cell and PD-1+Tim-3+ CD8+ T cells represented the largest subset of NY-ESO-1-specific CD8+ T cells in GC patients. Functionally, CD8+PD-1+Tim-3+ T cells were more impaired in IFN-γ, TNF-α and IL-2 production compared with PD-1+Tim-3− or PD-1−Tim-3− subsets. Dual blockade of Tim-3 and PD-1 during T-cell priming efficiently augmented proliferation and cytokine production by NY-ESO-1-specific CD8+ T cells could potentially be improved by therapeutic targeting of these inhibitory receptors, indicating that antitumor function of NY-ESO-1-specific CD8+ T cells could potentially be improved by therapeutic targeting of these inhibitory receptors.

Epitope-Specific Antitumor Immunity Suppresses Tumor Spread in Papillary Thyroid Cancer.

Papillary thyroid cancer (PTC) is characterized by a lymphocytic infiltration. PTC patients with lymphocytic infiltration may have a better clinical outcome. Characterization of tumor epitope-specific immunity and correlation analyses with the clinical outcome. 150 PTC patients; 40 Hashimoto thyroiditis (HT) patients; 21 healthy controls; 27,239 healthy whites (for HLA typing). HLA class I restricted thyroperoxidase (TPO) and thyroglobulin (Tg) epitope-specific T cells (tetramer analyses), correlation analyses between HLA class II phenotypes, T cell immunity, and the clinical course. The frequency of TPO- and Tg-specific CD8+ T cells in PTC patients was largely increased compared with healthy controls (TPO and Tg, P < 0.005 and P < 0.005) and was similar to those in HT patients. HLA-DQB1*03-positive PTC patients had a significantly lower risk [risk ratio (RR), 0.170; 95% confidence interval (CI), 0.037 to 0.755; P < 0.05] and HLA-DRB1*03-positive and HLA-DQB1*02-positive PTC patients a significantly higher risk (HLA-DRB1*03: RR, 4.400; 95% CI, 1.378 to 14.05; P < 0.05; HLA-DQB1*02: RR, 3.692; 95% CI, 1.102 to 12.38; P < 0.05) for distant metastases, compared with patients with other haplotypes. HLA-DQB1*03-positive PTC patients revealed an increased responsiveness of tumor epitopes in vitro. These tumor epitope-specific CD8+ T cells were also found in lymph node metastases of HLA-DQB1*03-positive PTC patients. We demonstrate a tumor epitope-specific immunity in PTC patients and the protective role of HLA-DQB1*03 against metastatic spread. These results have direct implications for new treatment options with immune checkpoint inhibitors.

Abstract PR02: A personalized neoantigen vaccine in patients with high risk melanoma

Abstract Neoantigens arise from somatic tumor mutations and can encode amino acid changes, generating peptides with the potential to bind to HLA molecules. Since these neoepitopes provide exquisite tumor specificity and are expected to be highly immunogenic as they are not subject to central tolerance, they were long-envisioned as highly valuable tumor antigens. Growing evidence has supported tumor neoepitopes as key antigens triggering immune-mediated tumor killing in vivo across settings of effective cancer immunotherapy, including checkpoint blockade and adoptive transfer with tumor-infiltrating lymphocytes. Vaccination is a potentially effective approach for inducing tumor-specific immune responses, but has been limited by the lack of a sufficiently potent vaccine immunogen. By leveraging next-generation sequencing technologies and new analytical pipelines to systematically identify personal tumor neoantigens, in a phase 1 trial we tested the feasibility, safety, and immunogenicity of a multi-epitope cancer vaccine targeting personal tumor neoantigens in patients with high-risk melanoma. Mutations were identified by DNA and RNA sequencing; epitopes were selected based on class I HLA binding prediction algorithms. The vaccines, comprised of up to 20 long peptides spanning mutated tumor epitopes and admixed with the Toll-Like Receptor 3 agonist poly-ICLC, were administered using a prime-boost approach. Of 12 patients enrolled, the initial 6 patients who received the full set of priming and booster vaccinations experienced only minimal adverse events, consisting of mild and transient injection site reaction, flu-like symptoms, and rash. We identified strong, polyfunctional CD4+ and CD8+ T cell responses against multiple neoepitopes in all dosed patients. Processing and presentation of neoantigens on autologous tumors was confirmed by mass spectrometry and recognition of autologous tumor by vaccine-induced neoantigen-specific T cells was verified. These studies show that a personalized neoantigen vaccine is safe, feasible, and strongly immunogenic in cancer patients. The data should reinvigorate the field of cancer vaccines, providing a rational path for the building of combinatorial approaches to extend their efficacy. Citation Format: Patrick A. Ott, Zhuting Hu, Derin B. Keskin, Sachet A. Shukla, Jing Sun, Ed Fritsch, Nir Hacohen, Catherine J. Wu. A personalized neoantigen vaccine in patients with high risk melanoma [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr PR02.

Suppression of inducible CD4 regulatory cells by MHC class I-restricted human tumor epitope specific TCR engineered multifunctional CD4 T cells

Regulatory T cells (Treg) can interfere with the generation and function of anti-tumor immune effectors. Accordingly, ways that could block Treg function would be useful in cancer immunotherapy. We have previously shown that incorporation of CD4+CD25-ve T cells in an in vitro cytolytic T lymphocyte (CTL) generation assay leads to generation of induced regulatory T cells (iTregs), and that these iTreg block the generation of productive CTL response (Chattopadhyay et al., 2006). We here show that human CD4 T cells engineered to express MHC class I-restricted human melanoma associated epitope, MART-127–35, specific T cell receptor (TCR), that can simultaneously exhibit helper as well as cytolytic effector functions (Chhabra et al., 2008, Ray et al., 2010), can interfere with the generation of inducible Treg, block iTreg-mediated suppression, and allow the activation and expansion of MART-127–35 specific CTL responses, in vitro. We also show that mitigation of Treg generation by TCR engineered CD4 T cells is not mediated by a soluble factor and may involve “licensing/conditioning” of the dendritic cells (DC). Our data offer novel insights on the biology of MHC class I restricted TCReng CD4 T cells and have translational implications.

Exposure to Melan-A/MART-126-35 tumor epitope specific CD8(+)T cells reveals immune escape by affecting the ubiquitin-proteasome system (UPS).

Efficient processing of target antigens by the ubiquitin-proteasome-system (UPS) is essential for treatment of cancers by T cell therapies. However, immune escape due to altered expression of IFN-γ-inducible components of the antigen presentation machinery and consequent inefficient processing of HLA-dependent tumor epitopes can be one important reason for failure of such therapies. Here, we show that short-term co-culture of Melan-A/MART-1 tumor antigen-expressing melanoma cells with Melan-A/MART-126-35-specific cytotoxic T lymphocytes (CTL) led to resistance against CTL-induced lysis because of impaired Melan-A/MART-126-35 epitope processing. Interestingly, deregulation of p97/VCP expression, which is an IFN-γ-independent component of the UPS and part of the ER-dependent protein degradation pathway (ERAD), was found to be essentially involved in the observed immune escape. In support, our data demonstrate that re-expression of p97/VCP in Melan-A/MART-126-35 CTL-resistant melanoma cells completely restored immune recognition by Melan-A/MART-126-35 CTL. In conclusion, our experiments show that impaired expression of IFN-γ-independent components of the UPS can exert rapid immune evasion of tumor cells and suggest that tumor antigens processed by distinct UPS degradation pathways should be simultaneously targeted in T cell therapies to restrict the likelihood of immune evasion due to impaired antigen processing.

194. Adenoviruses Armed with TNFα and IL-2 Successfully Enable Adoptive T-Cell Therapy

The field of T-cell therapy of cancer holds great promise, as demonstrated by the results obtained in Chimeric Antigen Receptor (CAR) modified T-cell therapy of CD19+ leukemia. However, the approach has not yielded similar results in the treatment of solid tumors with the exception of tumor infiltrating lymphocyte (TIL) therapy of melanoma. Recent work has identified key obstacles which has limited the efficacy of the approach: i) lack of recruitment of transferred T-cells to the tumor due to lack of trafficking signals ii) anergy of transferred cells at the tumor due to tumor immunosuppression iii) lack of propagation of transferred T-cells at the tumor. Previously, we have identified interleukin 2 (IL-2) and Tumor Necrosis Factor alpha (TNFα) as the most promising factors to stimulate the graft used in adoptive T-cell therapy. Both cytokines are important activators of immune cells and also known for their direct anti-tumor properties. Notably, these cytokines can cause severe side effects when administered systemically. Previously, we and others have achieved long lasting, high level cytokine expression locally but low level systemically when using armed oncolytic viruses in vivo. Oncolytic viruses armed with immunostimulatory molecules constitute a potent form of immunotherapy. In essence, the danger signal caused by virus replication, coupled with the actions of the transgene, and effective presentation of tumor epitopes by lysis of the cells, results in a personalized cancer vaccine. One attractive aspect of this approach is the ease of combinations with standard therapies including chemotherapy, radiation and immune checkpoint inhibiting antibodies for example. Therefore, we developed oncolytic adenoviruses expressing one or both above mentioned human cytokines. For more detailed immunological studies in immune competent mouse models, we constructed non-replicative adenoviruses with murine cytokines (Ad5-CMV-mIL2 and Ad5-CMV-mTNFα). These viruses were studied in combination with adoptive transfer of OT-1 TCR transgenic T-cells to treat C57BL/6 mice bearing B16-OVA melanoma tumors. The animals were administered intraperitoneally with CD8+-enriched OT-1 cells with or without intratumoral injections of cytokine-coding viruses. Combination treatment with Ad5-CMV-mIL2 and OT-1 resulted in statistically significant antitumor efficacy when compared with either monotherapy or untreated control. In further experiments a triple combination of Ad5-CMV-mIL2 + Ad5-CMV-mTNFa and OT-1 T-cells improved antitumor efficacy was observed over dual agent therapies. Furthermore, in Syrian hamster model, oncolytic virus successfully improved the efficacy of TIL therapy. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes.

Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes.

Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.

Oncolytic adenoviruses coated with MHC-I tumor epitopes increase the antitumor immunity and efficacy against melanoma

ABSTRACT The stimulation of the immune system using oncolytic adenoviruses (OAds) has attracted significant interest and several studies suggested that OAds immunogenicity might be important for their efficacy. Therefore, we developed a versatile and rapid system to adsorb tumor-specific major histocompatibility complex class I (MHC-I) peptides onto the viral surface to drive the immune response toward the tumor epitopes. By studying the model epitope SIINFEKL, we demonstrated that the peptide-coated OAd (PeptiCRAd) retains its infectivity and the cross presentation of the modified-exogenous epitope on MHC-I is not hindered. We then showed that the SIINFEKL-targeting PeptiCRAd achieves a superior antitumor efficacy and increases the percentage of antitumor CD8+ T cells and mature epitope-specific dendritic cells in vivo. PeptiCRAds loaded with clinically relevant tumor epitopes derived from tyrosinase-related protein 2 (TRP-2) and human gp100 could reduce the growth of primary-treated tumors and secondary-untreated melanomas, promoting the expansion of antigen-specific T-cell populations. Finally, we tested PeptiCRAd in humanized mice bearing human melanomas. In this model, a PeptiCRAd targeting the human melanoma-associated antigen A1 (MAGE-A1) and expressing granulocyte and macrophage colony-stimulating factor (GM-CSF) was able to eradicate established tumors and increased the human MAGE-A1-specific CD8+ T cell population. Herein, we show that the immunogenicity of OAds plays a key role in their efficacy and it can be exploited to direct the immune response system toward exogenous tumor epitopes. This versatile and rapid system overcomes the immunodominance of the virus and elicits a tumor-specific immune response, making PeptiCRAd a promising approach for clinical testing.