Neoantigen-specific T-cell Research Articles

Abstract B092: Tumor infiltrating T-cells from renal cell carcinoma patients recognize neoantigens derived from point and frameshift mutations

Abstract Mutation-derived neoantigens are important targets of T-cell mediated reactivity towards tumors. Their unique tumor-restriction poses an advantage compared to shared tumor antigens in that they are in principle both foreign and tumor specific, hence presumably less impacted by T-cell tolerance and for therapeutic applications less prone to mediate immune-related destruction of noncancerous tissue. Moreover, the mutational burden and predicted number of neoantigens correlate to favorable clinical outcome and benefit from immune checkpoint therapy. Neoantigen-reactive T-cells have been detected across a number of solid cancers, ranging from immunogenic tumors such as melanoma and non-small cell lung cancer to less immunogenic tumors such as breast cancer. Renal cell carcinomas (RCCs) are among medium-range mutational burden tumors and present with the highest pan-cancer number and proportion of frameshift mutations, a mutation type considered to be highly immunogenic. However, to our knowledge, yet no reports have described neoantigen-specific T-cells in this malignancy. In this study, the mutational landscape and HLA (human leukocyte antigen) profile of tumors from six renal cell carcinoma patients were analyzed by whole-exome sequencing (WXS) of DNA from tumor fragments (TFs), autologous tumor cell lines (TCLs) and tumor-infiltrating lymphocytes (TILs, germline reference). Hereafter the online MuPeXi tool was used to predict binding of mutated peptide sequences of 9-11mer length to the HLAs of each patient, using a rank score < 2 for selection of peptide binding, hereby creating patient-specific libraries of putative neo-peptides. TILs extracted from the patients tumors were screened for T-cell recognition of the peptide libraries by use of a novel high-throughput platform based on DNA barcode labeled peptide-MHC multimers, and responses were verified by conventional fluorochrome labeled MHC multimers. In four of six patients WXS was performed on both TF and TCL, in two of six patients only on TF. The average mutational burden of the six patients was 271 for TF (range 146–381, n=6) and 289 for TCL (range 182-404, n=4). Prediction of HLA-restricted peptides within the mutated sequences resulted in patient specific libraries of average 269 peptides for TF and TCL combined (range 126-443, n=6). Half of the peptides were predicted from both sources (52%, range 28-74%, n=4) compared to 20% (range 8-31%, n=4) predicted solely from TF and 29% (range 18-41%, n=4) predicted solely from TCL. The proportion of predicted peptides derived from frameshift mutations out of total mutations was 16% (range 7-24%, n=6). A total of 67 neoantigen-specific T-cell responses were detected across all patients by use of a novel high-throughput DNA barcode screening platform, with the number of detected responses ranging from 4-30 and spanning 3-5 HLA restrictions per patient. Of note, we detected a number of T-cell responses towards HLA-C restricted peptides, which have previously been poorly described. For several patients, the number of HLA-C restricted T-cell responses observed was substantially higher than for both HLA-A and -B, highlighting the importance of including this HLA type for neoepitope analyses. In the four patients in whom peptides were predicted from both TF and TCL, the distribution of responses was 37% on TF (range 14-60%, n=4), 36% on TCL (range 29-43%, n=4) and 27% (range 0-43%) on TF+TCL combined. The proportion of responses towards frameshift mutations was 17% (range 0-24%, n=6) of total responses. The identification of neoantigen-specific T-cells within tumors from RCC patients is an important step towards the use of neoantigens as therapeutic targets and predictors of response to immunotherapy in this cancer subtype. Moreover, our study points toward the importance of broad peptide prediction platforms covering multiple sources for WXS and mutational analyses covering both point and frameshift mutations. Citation Format: Sofie Ramskov, Ulla Kring Hansen, Anne-Mette Bjerregaard, Amalie Kai Bentzen, Marco Donia, Rikke Andersen, Zoltan Szallasi, Inge Marie Stentoft Svane, Aron Charles Eklund, Sine Reker Hadrup. Tumor infiltrating T-cells from renal cell carcinoma patients recognize neoantigens derived from point and frameshift mutations [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 B092.

Abstract B022: Properties of T-cell-recognized neoantigens

Abstract Over the past years we have learned that the T-cell-based immune system frequently responds to the neoantigens that arise as a consequence of the accumulated DNA damage causing the malignant transformation. Furthermore, recognition of neoantigens appears an important driver of the clinical activity of both T-cell checkpoint blockade and adoptive T-cell therapy as cancer immunotherapies. From the efforts dissecting the neoantigen-specific T-cell response it has become clear that only a very minor fraction of the accumulated mutations is recognized by the immune system, and the challenge to unravel the neoantigen-specific T-cell response lies in identifying which neoantigens are more likely to be true T-cell epitopes. We have analyzed neoantigen-specific T-cell reactivity in 12 melanoma patients using an in silico epitope prediction pipeline based on RNA expression, predicted HLA binding affinity, proteasomal processing and self-similarity to predict potential neoepitopes. We screened for T-cell recognition of 7000 epitopes from these 12 patients (average ~550 epitopes per patient, range: 96-1902) using our pMHC multimer combinatorial encoding technology and found 19 epitopes to be recognized by T-cells (hits) and 6981 to be “non-hits.” Based on these data we have examined the properties of T-cell recognized neoantigens. An intriguing observation is an enrichment within T-cell recognized epitopes of epitopes with the mutation positioned within the last 4 amino acids (C-terminal end of the peptide) compared to the screened set of epitopes. Fifteen out of 19 hits (approximately 80%) harbored a mutation within the last 4 amino acids of the peptide, whereas within the full set of screen epitopes it is 43%. While it is currently unclear what the reason is for this, this could reflect a biologic importance in T-cell recognition of the C-terminal part of the epitope. Furthermore, RNA expression and predicted binding affinity to HLA are important informative parameters for selecting T-cell recognized epitopes. A striking observation is that predicted binding affinity not only correlates with likelihood of observing a T-cell response but also the magnitude of this T-cell response, suggesting a hierarchy within neoantigens, and that not all neoantigens are of equal immunologic quality. In summary, our findings indicate that T-cell recognized neoantigens may differ from the neoantigen pool not recognized. In particular regarding position of the mutation with the epitope, RNA abundance and predicted HLA binding affinity. Importantly, our data reveal a hierarchy within neoantigens comparable to immunodominance known from viral infections. This hierarchy appears to depend mostly on binding affinity. These observations are likely to be highly relevant when selecting neoantigens for therapeutic manipulation such as vaccines. Citation Format: Pia Kvistborg, Marit M. van Buuren, Daisy Philips, Nienke van Rooij, Arno Velds, Sam Behjati, Marlous van den Braber, Mireille Toebes, Lorenzo Fanchi, Maarten Slagter, Marie Stentoft Svane, Patrick Hwu, Joost van den Berg, Michael Stratton, Christian Blank, John B.A.G. Haanen, Can Kesmir, Ton N.M. Schumacher. Properties of T-cell-recognized neoantigens [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 B022.

Abstract B094: Successful identification of neoantigen-specific T-cell responses in low mutation burden colorectal cancers for personalized cancer vaccine development

Abstract Innovative treatment options are required to improve cure rates in advanced colorectal cancer patients. Immune checkpoint blockade therapy (anti-PD-1) was shown to be effective in colorectal cancers with high mutation burden (e.g., mismatch repair deficient) as antitumor reactivity is largely explained by the recognition of somatically mutated antigens (neoantigens). No immunotherapeutic strategies are currently available for patients diagnosed with low mutation burden colorectal cancer. We hypothesized that if neoantigen-reactive T-cells are present in low mutation burden patients, the latter could benefit from immunotherapeutic interventions that stimulate neoantigen recognition and the onset of a robust antitumor immune response.In order to detect neoantigens, whole exome and RNA next-generation sequencing analyses were performed in cancer and healthy tissues from five colorectal cancer patients. Corresponding neoepitopes were synthesized and tested for their ability to induce immune cell activation in T-cells isolated from the tumor tissue (TIL) and from peripheral blood. Neoantigen-specific T-cell responses were identified in the majority of patients that presented with tumors carrying 25 to 36 transcribed, non-synonymous variants. Up to six different neoantigens were recognized per tumor, which resulted in a higher detection rate than anticipated based on published data. Moreover, we discovered the merits of isolating CD39+CD103+CD8+ T-cells for detection of a broad recognition of HLA class I-restricted neoantigens. This CD39+CD103+CD8+ T-cell subset comprises the majority and a broader repertoire of neoantigen-specific T-cells compared to bulk TIL populations or lymphocytes derived from peripheral blood. In conclusion, we developed a neoantigen screening pipeline to unlock the immunogenic potential of colorectal cancers with low mutation burden. We have detected a relatively high number of neoantigens that are recognized by tumor- and/or PBMC-derived T-cells in mismatch repair proficient, low mutation burden colorectal cancer patients, and show the importance of the CD39+CD103+CD8+ T-cell subset for neoantigen-based immunotherapies. These findings warrant the further exploration of the potential to employ neoantigen-targeted therapies to improve clinical outcomes of colorectal cancer patients. Citation Format: Jitske van den Bulk, Dina Ruano, Marieke E. Ijsselsteijn, Marten Visser, Ruud van der Breggen, Koen C.M.J. Peeters, Thomas Duhen, Rebekka Duhen, Andrew D Weinberg, Sjoerd S.H. van der Burg, Els M.E. Verdegaal, Noel F. de Miranda. Successful identification of neoantigen-specific T-cell responses in low mutation burden colorectal cancers for personalized cancer vaccine development [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 B094.

Abstract B124: Personalized peptide vaccination based on patient-individual tumor-specific variants induces T-cell responses in pediatric patients

Abstract Background: We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. Accumulation of somatic mutations is one characteristic feature of malignanT-cells. These single-nucleotide variants (SNVs) can lead to altered amino acid sequences of the translated proteins, which in turn can be presented by malignant cells as antigenic peptides on HLA molecules. A peptide vaccination to induce neoantigen-specific T-cell responses, therefore, is a promising and versatile immunotherapeutic approach for the treatment of malignant diseases. Such approaches were predominantly applied in malignancies with high mutational load in adult patients until now. We developed a vaccine design platform based on sequencing data generated from a cohort of acute lymphoblastic leukemia (ALL) patients and tested individualized peptide vaccines in pediatric patients suffering from a variety of tumors with low mutational profiles. Methods: Nonsynonymous mutations were identified by whole-exome and transcriptome sequencing of patient leukemic blasts and healthy reference tissue. HLA binding peptides harboring the altered amino acids were subsequently predicted in silico by algorithms SYFPEITHI, NetMHC and NetMHCpan for the patients’ individual HLA type. Individual peptides for treatment attempts were produced by chemical synthesis and vaccination cocktails were formulated. The vaccination schedule was 16 vaccinations over 33 weeks using GM-CSF and Imiquimod as adjuvants. Some patients received simultaneous checkpoint blockade treatment with pembrolizumab or nivolumab. Response to the vaccination was monitored by detection of T-cells recognizing the vaccinated peptides occurring over time in peripheral blood of the patients. Monitoring was performed for each vaccination time point by pre-stimulation with the peptides and subsequent intracellular cytokine staining (ICS) of T-cells and FACS analysis. Results: Whole-exome sequencing was performed for 25 patients to identify ALL-specific SNVs using a comparative bioinformatics pipeline. We found an average of 39.2 mutations per patient on DNA level, with an average validation rate of 47% by RNA sequencing. Based on these data, an average of 35.1 HLA binders could be predicted per patient. We applied our platform for 6 patients with various malignancies based on compassionate need and designed individual peptide vaccines. In all cases validated mutations could be identified and epitope prediction was performed for HLA Class I and II binders. In 6/6 patients a de novo induced T-cell response against the vaccinated mutated HLA-binding peptides was detectable. Combination therapy with PD-1 blockade and peptide vaccination was well tolerated. T-cell responses were predominantly, but not exclusively, CD4+-restricted. Encouraged by these findings, we started a phase I/II clinical trial in patients with primary/relapsed ALL with the aim to prevent relapse and show safety and immunogenicity of the personalized peptide vaccine. Conclusions: We established a platform for the design of patient-individual peptide vaccination cocktails by combination of whole exome sequencing of tumor and normal tissue with in silico epitope prediction algorithms for individual patient HLA types. Whole-exome sequencing of pediatric ALL patients is feasible and yields a small amount of tumor-specific mutations per patient. However, these few mutations are sufficient to predict HLA-binding peptides that are immunogenic when vaccinated and elicit specific T-cell responses in patients. The universal character of individualized peptide vaccination allows for application in virtually any malignant disease, as well as combination therapy approaches. The concept is now translated to clinical application in a phase I/II clinical trial in ALL, started in 2016 (NCT03559413). Citation Format: Armin Rabsteyn, Christina Kyzirakos, Christopher Schroeder, Marc Sturm, Christopher Mohr, Jakob Matthes, Magdalena Feldhahn, Nicolas Casadei, Martin Ebinger, Stefan Stevanovic, Peter Bauer, Oliver Kohlbacher, Cecile Gouttefangeas, Juergen Schaefer, Hans-Georg Rammensee, Rupert Handgretinger, Peter Lang. Personalized peptide vaccination based on patient-individual tumor-specific variants induces T-cell responses in pediatric patients [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 B124.

Neoantigen Landscape of Relapsed Acute Leukemia Following Allogeneic Stem Cell Transplantation

INTRODUCTION: The potent graft versus leukemia (GVL) effect of allogeneic stem cell transplantation (allo-SCT) is considered as a blueprint for cellular immunotherapy. However, failure of GVL leads to relapse of underlying leukemia, the major cause of death after allo-SCT. In solid tumors, higher tumor mutation burdens are associated with better response to check point inhibitors which implies the importance of neoantigen specific T-cell functions in cancer immunity. In contrast, the frequencies of somatic mutations in acute leukemia are generally low, therefore the role of neoantigens in GVL remains undetermined. Here, we developed a platform to screen for potential neoantigens by performing whole exome sequencing (WES) and RNA sequencing (RNAseq) in matched samples: leukemic blasts at relapse after allo-SCT, recipient T cell controls, and donor cells.METHODS: Leukemic blasts from patients in relapse were enriched by flow sorting from bone marrow aspirate or peripheral blood samples. Recipient T cells were isolated from pre-transplant peripheral blood as germline controls, and donor monocytes or CD34-positive cells were used as hematopoietic-lineage cell controls. WES was performed to 100X coverage, paired with RNAseq 40M reads per sample. Somatic mutations were detected with mutect and mpileup, followed by annotation with SnpEff. High confidence somatic mutations were subjected to pVAC-seq for neoantigen predictions.RESULTS: Six patients with relapsed acute leukemia (AML 5, ALL 1) after allo-SCT and their transplant donors (matched sibling 3, haplo-identical 3) had suitable samples available for analysis. On average, somatic mutations were identified in 297 genes (range 108- 609) by comparing leukemic blasts and germline control T cells. Among those mutations, potential candidates of neoantigen were identified in five out of six subjects. Allele frequencies of mutant genes varied. Most of neoantigens were predicted to bind HLA of both class I (median 5, range 0-15) and class II (median 6, range 0-12). One subject had only HLA class II restricted peptides as predicted neoantigens. Of interest majority of antigens were derived from molecules known to play important roles in leukemia or tumor biology which include ETV6, CCNY, IDH2, PTPN11, SF3B1, and TP53. Evolution analysis of neoantigen showed an emergence of new antigens in relapsed leukemia while a few driver gene mutations persisted after allo-SCT (Figure).CONCLUSION: Our in-silico analysis demonstrated the possibility that somatic mutation in acute leukemia could serve as putative neoantigens applicable for novel immunotherapy after allo-SCT. The binding capacity of mutant peptides to class I and II HLA implies the importance of both CD4 and CD8 contributions to anti-neoantigen immunity. Next, we will search for neoantigen specific T cells exerting an anti-leukemia effect to validate the GVL potential of these mutations in allo-SCT. DisclosuresNo relevant conflicts of interest to declare.

T-cell Responses to TP53 "Hotspot" Mutations and Unique Neoantigens Expressed by Human Ovarian Cancers.

Purpose: This was a study prospectively evaluating intratumoral T-cell responses to autologous somatic mutated neoepitopes expressed by human metastatic ovarian cancers.Patients and Methods: Tumor-infiltrating lymphocytes (TIL) were expanded from resected ovarian cancer metastases, which were analyzed by whole-exome and transcriptome sequencing to identify autologous somatic mutations. All mutated neoepitopes, independent of prediction algorithms, were expressed in autologous antigen-presenting cells and then cocultured with TIL fragment cultures. Secretion of IFNγ or upregulation of 41BB indicated a T-cell response.Results: Seven women with metastatic ovarian cancer were evaluated, and 5 patients had clear, dominant T-cell responses to mutated neoantigens, which were corroborated by comparison with the wild-type sequence, identification of the minimal epitope, human leukocyte antigen (HLA) restriction element(s), and neoantigen-specific T-cell receptor(s). Mutated neoantigens were restricted by HLA-B, -C, -DP, -DQ, and/or -DR alleles and appeared to principally arise from random, somatic mutations unique to each patient. We established that TP53 "hotspot" mutations (c.659A>G; p.Y220C and c.733G>A; p.G245S) expressed by two different patients' tumors were both immunogenic in the context of HLA-DRB3*02:02.Conclusions: Mutation-reactive T cells infiltrated ovarian cancer metastases at sufficient frequencies to warrant their investigation as adoptive cell therapy. In addition, transfer of TP53 "hotspot" mutation-reactive T-cell receptors into peripheral blood T cells could be evaluated as a gene therapy for a diverse range of tumor histologies. Clin Cancer Res; 24(22); 5562-73. ©2018 AACR See related commentary by McNeish, p. 5493.

Two Weeks' Notice from Allogeneic Sources

A novel pipeline for neoantigen-specific T-cell receptor (TCR) identification has been validated in ovarian cancer, making use of HLA-matched allogeneic healthy donor T cells. This workflow allows for the identification of tumor-specific TCRs 2 weeks after antigen-specific stimulation and eliminates problematic patient-to-patient variation in the selection of neoantigen-specific TCRs. Clin Cancer Res; 24(21); 5195-7. ©2018 AACR See related article by Matsuda et al., p. 5357.

Induction of Neoantigen-Specific Cytotoxic T Cells and Construction of T-cell Receptor-Engineered T Cells for Ovarian Cancer.

Purpose: Current evolution of cancer immunotherapies, such as immune checkpoint blockade, has implicated neoantigens as major targets of anticancer cytotoxic T cells. Adoptive T-cell therapy with neoantigen-specific T-cell receptor (TCR)-engineered T cells would be an attractive therapeutic option for advanced cancers where the host antitumor immune function is strongly inhibited. We previously developed a rapid and efficient pipeline for production of neoantigen-specific TCR-engineered T cells using peripheral blood from an HLA-matched healthy donor. Our protocol required only 2 weeks from stimulation of T cells with neoantigen-loaded dendritic cells to the identification of neoantigen-specific TCRs. We conducted the pilot study to validate our protocol.Experimental Design: We used tumors from 7 ovarian cancer patients to validate our protocol.Results: We chose 14 candidate neoantigens from 7 ovarian tumors (1-3 candidates for each patient) and then successfully induced three neoantigen-specific T cells from 1 healthy donor and identified their TCR sequences. Moreover, we validated functional activity of the three identified TCRs by generating TCR-engineered T cells that recognized the corresponding neoantigens and showed cytotoxic activity in an antigen dose-dependent manner. However, one case of neoantigen-specific TCR-engineered T cells showed cross-reactivity against the corresponding wild-type peptide.Conclusions: This pilot study demonstrated the feasibility of our efficient process from identification of neoantigen to production of the neoantigen-targeting cytotoxic TCR-engineered T cells for ovarian cancer and revealed the importance of careful validation of neoantigen-specific TCR-engineered T cells to avoid severe immune-related adverse events. Clin Cancer Res; 24(21); 5357-67. ©2018 AACR See related commentary by Anczurowski and Hirano, p. 5195.

Abstract CT079: Neoadjuvant PD-1 blockade in resectable lung cancer

Abstract Background: Programmed death-1 (PD-1) blocking antibodies improve survival in patients with advanced non-small-cell lung cancer (NSCLC) but have not been tested in resectable NSCLC, where little progress has been made over the last decade. Methods: Adults with untreated surgically resectable stage I-IIIA NSCLC received two doses of nivolumab (anti-PD-1) preoperatively. The primary endpoints of the study were safety and feasibility. Tumor pathologic response, PD-L1 expression, mutation burden and mutation-associated neoantigen-specific T-cell responses were evaluated. Results: Neoadjuvant nivolumab was had an acceptable side effect profile without surgical delays, and 20 of 21 tumors were completely resected. Major pathologic response occurred in 45% (9/20) of resected tumors. Responses occurred in both PD-L1 positive and negative tumors. Pathologic response significantly correlated with pre-treatment tumor somatic mutation burden. T-cell clones shared between the tumor and peripheral blood increased systemically upon anti-PD-1 treatment in 8 of 9 patients analyzed. Mutation-associated neoantigen-specific T-cell clones, from a primary tumor that underwent pathologic complete response, rapidly expanded in peripheral blood at 2-4 weeks post-treatment, some of these clones were not detected before anti-PD-1. Conclusions: Neoadjuvant nivolumab was associated with few side effects, did not delay surgery, and induced major pathologic responses in 45% of resected tumors. Tumor mutation burden is predictive of pathologic response to anti-PD-1. Anti-PD-1 can induce expansion of mutation-associated neoantigen-specific T-cell clones in peripheral blood. [P.M.F., J.E.C., and K.N.S. contributed equally to this work.] Citation Format: Patrick M. Forde, Jamie E. Chaft, Kellie N. Smith, Valsamo Anagnostou, Tricia R. Cottrell, Matthew D. Hellmann, Marianna Zahurak, Stephen C. Yang, David R. Jones, Stephen Broderick, Richard J. Battafarano, Moises J. Velez, Natasha Rekhtman, Zachary Olah, Jarushka Naidoo, Kristen A. Marrone, Franco Verde, Haidan Guo, Jiajia Zhang, Justina X. Caushi, Hok Yee Chan, John-William Sidhom, Robert B. Scharpf, James White, Edward Gabrielson, Hao Wang, Gary L. Rosner, Valerie Rusch, Jedd D. Wolchok, Taha Merghoub, Janis M. Taube, Victor E. Velculescu, Suzanne L. Topalian, Julie R. Brahmer, Drew M. Pardoll. Neoadjuvant PD-1 blockade in resectable lung cancer [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 CT079.

Neoadjuvant PD-1 Blockade in Resectable Lung Cancer

BackgroundAntibodies that block programmed death 1 (PD-1) protein improve survival in patients with advanced non–small-cell lung cancer (NSCLC) but have not been tested in resectable NSCLC, a condition in which little progress has been made during the past decade.MethodsIn this pilot study, we administered two preoperative doses of PD-1 inhibitor nivolumab in adults with untreated, surgically resectable early (stage I, II, or IIIA) NSCLC. Nivolumab (at a dose of 3 mg per kilogram of body weight) was administered intravenously every 2 weeks, with surgery planned approximately 4 weeks after the first dose. The primary end points of the study were safety and feasibility. We also evaluated the tumor pathological response, expression of programmed death ligand 1 (PD-L1), mutational burden, and mutation-associated, neoantigen-specific T-cell responses.ResultsNeoadjuvant nivolumab had an acceptable side-effect profile and was not associated with delays in surgery. Of the 21 tumors that were removed, 20 were completely resected. A major pathological response occurred in 9 of 20 resected tumors (45%). Responses occurred in both PD-L1–positive and PD-L1–negative tumors. There was a significant correlation between the pathological response and the pretreatment tumor mutational burden. The number of T-cell clones that were found in both the tumor and peripheral blood increased systemically after PD-1 blockade in eight of nine patients who were evaluated. Mutation-associated, neoantigen-specific T-cell clones from a primary tumor with a complete response on pathological assessment rapidly expanded in peripheral blood at 2 to 4 weeks after treatment; some of these clones were not detected before the administration of nivolumab.ConclusionsNeoadjuvant nivolumab was associated with few side effects, did not delay surgery, and induced a major pathological response in 45% of resected tumors. The tumor mutational burden was predictive of the pathological response to PD-1 blockade. Treatment induced expansion of mutation-associated, neoantigen-specific T-cell clones in peripheral blood. (Funded by Cancer Research Institute–Stand Up 2 Cancer and others; ClinicalTrials.gov number, NCT02259621.)

Intratumoral injection of IFN-β induces chemokine production in melanoma and augments the therapeutic efficacy of anti-PD-L1 mAb

Despite recent advances in treatment for melanoma patients through using immune checkpoint inhibitors, these monotherapies have limitations and additional treatments have been explored. Type I IFNs have been used to treat melanoma and possess immunomodulatory effects including enhancement of T-cell infiltration. T-cell plays a critical role in immune checkpoint therapies via restoration of effector functions and tumor infiltration by T-cells predicts longer survival in a variety of cancer types. Moreover, tumor-infiltrating T-cells are associated with the expression of chemokines such as CCL5 and CXCR3 ligands in tumor tissues. We therefore investigated whether intratumoral injection of IFN-β induces the expression of CCL5 and CXCR3 ligands in melanoma cells and has additional antitumor effects when combined with anti-PD-L1 mAb treatment. IFN-β treatment enhanced CD8+ T-cell infiltration into tumors and CCL5 and CXCR3 ligand expression. In vivo studies using a mouse model showed that monotherapy with IFN-β, but not with anti-PD-L1 mAb, inhibited tumor growth in comparison to control. However, the therapeutic efficacy of IFN-β was significantly enhanced by the addition of anti-PD-L1 mAb. This antitumor response of combination therapy was abrogated by anti-CD8 mAb and IFN-β augmented the neoantigen-specific T-cell response of anti-PD-L1 mAb. Our findings suggest that IFN-β induces the expression of CCL5 and CXCR3 ligands in melanoma, which could play a role in T-cell recruitment, and enhances the efficacy of anti-PD-L1 mAb treatment in a CD8-dependent manner.

A rapid single-cell RNA-seq approach to identify neoantigen-specific T-cell receptors targeting tumor-specific mutations for use in gene-engineered T-cell immunotherapy

Abstract Accumulated evidence suggested that effective cancer immunotherapies were associated with neoantigen-reactive T cells, and adoptive transfer of neoantigen-reactive tumor infiltrating lymphocytes (TILs) could result in tumor regressions. To improve the efficacy of adoptive T-cell therapy targeting tumor-specific mutations, we have proposed a new therapeutic approach, which involves the genetic modification of autologous T cells with neoantigen-specific TCRs and the transfer of these T cells back to cancer patients. However, the current techniques to isolate neoantigen-specific TCRs are labor-intensive, time-consuming and technically challenging, not suitable for clinical applications. To facilitate this process, a new approach was developed, which included the co-culture of TILs with tandem minigene (TMG)-pulsed antigen-presenting cells, single-cell RNA-seq analysis of T cells and the identification of paired TCR sequences associated with cells expressing high levels of IFN-γ and IL-2 mRNA. In this study, TIL fragment cultures were grown from tumors resected from three patients with metastatic gastrointestinal cancers, and then were screened against TMG libraries encoding tumor-specific mutations identified by whole-exome sequencing. Following this new approach, three TCRs were identified, synthesized and cloned into a retroviral vector. These TCRs were then transduced into donor T cells, and these transduced T cells were shown to specifically recognize the following neoantigens encoded by the TMGs: USP8 (R667H), KRAS (G12D) and NBAS (C144S). In conclusion, this approach provides a rapid procedure to isolate neoantigen-specific TCRs for basic and translational research, as well as clinical applications.

Direct identification of clinically relevant neoepitopes presented on native human melanoma tissue by mass spectrometry.

Although mutations may represent attractive targets for immunotherapy, direct identification of mutated peptide ligands isolated from human leucocyte antigens (HLA) on the surface of native tumour tissue has so far not been successful. Using advanced mass spectrometry (MS) analysis, we survey the melanoma-associated immunopeptidome to a depth of 95,500 patient-presented peptides. We thereby discover a large spectrum of attractive target antigen candidates including cancer testis antigens and phosphopeptides. Most importantly, we identify peptide ligands presented on native tumour tissue samples harbouring somatic mutations. Four of eleven mutated ligands prove to be immunogenic by neoantigen-specific T-cell responses. Moreover, tumour-reactive T cells with specificity for selected neoantigens identified by MS are detected in the patient's tumour and peripheral blood. We conclude that direct identification of mutated peptide ligands from primary tumour material by MS is possible and yields true neoepitopes with high relevance for immunotherapeutic strategies in cancer.

Abstract CT022: IVAC® MUTANOME - A first-in-human phase I clinical trial targeting individual mutant neoantigens for the treatment of melanoma

Abstract One of the hallmarks of cancer is the inherent instability of the genome leading to multiple genomic alterations and epigenetic changes that ultimately drive carcinogenesis. These processes lead to a unique molecular profile of every given tumor and to substantial intratumoral heterogeneity of cancer tissues. Recently, a series of independent reports revealed that pre-formed neoantigen specific T-cell responses are of crucial relevance for the clinical efficacy of immune checkpoint inhibitors. However, spontaneous immune recognition of neoantigens seems to be a rare event with only less than 1% of mutations inducing a T-cell response in the tumor-bearing patient. Accordingly, only patients with a high burden of mutations profit from currently approved therapies. To overcome this restriction, the IVAC® MUTANOME-project harnesses the individual patient's mutation profile by manufacturing highly potent neoantigen-coding RNA vaccines. To this end, the individual mutation repertoire is identified by next-generation-sequencing, potentially immunogenic mutations are selected and incorporated into a poly-epitopic RNA vaccine that is tailored to activate and expand the individual patient's neoantigen-specific CD4+ and CD8+ T cells. A phase I study to test this novel concept of an active individualized cancer vaccine for the treatment of malignant melanoma was initiated in 2013 (NCT02035956). Notably, BioNTech RNA Pharmaceutical's IVAC® MUTANOME trial is the first-in-human trial that introduces a fully personalized RNA vaccine for the treatment of malignant melanoma. The objective of this clinical trial is to study the feasibility, safety, tolerability, immunogenicity and the potential clinical activity of the IVAC® MUTANOME approach. The recruitment of a patient into the trial triggers the multi-step IVAC® MUTANOME process covering (i) the receipt and processing of tumor and blood sample specimens, (ii) the identification, prioritization and confirmation of mutations, (iii) testing of pre-existing immunity against identified tumor mutations, (iv) the selection of mutant neoantigen sequences as vaccine targets, (v) design, production of a DNA lead structure, (vi) GMP manufacturing and release of the patient-specific mRNA, (vii) shipment to the clinical trial site and (viii) the administration of the IMP to patients. Detailed information on the trial, the recruitment and treatment status as well as data on the assessment of vaccine induced immune responses will be presented. Citation Format: Matthias Miller, Carmen Loquai, Björn-Philipp Kloke, Sebastian Attig, Nicole Bidmon, Stefanie Bolte, Valesca Bukur, Evelyna Derhovanessian, Jan Diekmann, Angela Filbry, Sandra Heesch, Christoph Hoeller, Klaus Kuehlcke, David Langer, Martin Loewer, Felicitas Mueller, Inga Ortseifer, Burkhard Otte, Anna Paruzynski, Richard Rae, Barbara Schroers, Christine Seck, Kristina Spiess, Arbel D. Tadmor, Isabel Vogler, Mathias Vormehr, Alexandra Kemmer-Brueck, Andreas N. Kuhn, Ulrich Luxemburger, Sebastian Kreiter, Jochen Utikal, Stephan Grabbe, Oezlem Tuereci, Ugur Sahin. IVAC® MUTANOME - A first-in-human phase I clinical trial targeting individual mutant neoantigens for the treatment of melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT022.

Neoantigen landscape dynamics during human melanoma-T cell interactions.

Recognition of neoantigens that are formed as a consequence of DNA damage is likely to form a major driving force behind the clinical activity of cancer immunotherapies such as T-cell checkpoint blockade and adoptive T-cell therapy. Therefore, strategies to selectively enhance T-cell reactivity against genetically defined neoantigens are currently under development. In mouse models, T-cell pressure can sculpt the antigenicity of tumours, resulting in the emergence of tumours that lack defined mutant antigens. However, whether the T-cell-recognized neoantigen repertoire in human cancers is constant over time is unclear. Here we analyse the stability of neoantigen-specific T-cell responses and the antigens they recognize in two patients with stage IV melanoma treated by adoptive T-cell transfer. The T-cell-recognized neoantigens can be selectively lost from the tumour cell population, either by overall reduced expression of the genes or loss of the mutant alleles. Notably, loss of expression of T-cell-recognized neoantigens was accompanied by development of neoantigen-specific T-cell reactivity in tumour-infiltrating lymphocytes. These data demonstrate the dynamic interactions between cancer cells and T cells, which suggest that T cells mediate neoantigen immunoediting, and indicate that the therapeutic induction of broad neoantigen-specific T-cell responses should be used to avoid tumour resistance.

Viral Infection of Tumors Overcomes Resistance to PD-1-immunotherapy by Broadening Neoantigenome-directed T-cell Responses.

There is evidence that viral oncolysis is synergistic with immune checkpoint inhibition in cancer therapy but the underlying mechanisms are unclear. Here, we investigated whether local viral infection of malignant tumors is capable of overcoming systemic resistance to PD-1-immunotherapy by modulating the spectrum of tumor-directed CD8 T-cells. To focus on neoantigen-specific CD8 T-cell responses, we performed transcriptomic sequencing of PD-1-resistant CMT64 lung adenocarcinoma cells followed by algorithm-based neoepitope prediction. Investigations on neoepitope-specific T-cell responses in tumor-bearing mice demonstrated that PD-1 immunotherapy was insufficient whereas viral oncolysis elicited cytotoxic T-cell responses to a conserved panel of neoepitopes. After combined treatment, we observed that PD-1-blockade did not affect the magnitude of oncolysis-mediated antitumoral immune responses but a broader spectrum of T-cell responses including additional neoepitopes was observed. Oncolysis of the primary tumor significantly abrogated systemic resistance to PD-1-immunotherapy leading to improved elimination of disseminated lung tumors. Our observations were confirmed in a transgenic murine model of liver cancer where viral oncolysis strongly induced PD-L1 expression in primary liver tumors and lung metastasis. Furthermore, we demonstrated that combined treatment completely inhibited dissemination in a CD8 T-cell-dependent manner. Therefore, our results strongly recommend further evaluation of virotherapy and concomitant PD-1 immunotherapy in clinical studies.

Abstract 4704: Neo-antigen enriched TIL therapy mediates superior tumor eradication in a patient-derived xenograft model of human melanoma

Abstract The mutational load in melanoma is high relative to that in most other human malignancies, resulting in the possible expression of large numbers of patient-specific mutated antigens. This may, in part, explain the immunogenicity of this disease and the high rate of responsiveness to immunotherapeutic strategies such as tumor-infiltrating lymphocyte (TIL) therapy. Indeed, recent data have shown that cytotoxic T-cell reactivity targeting neo antigens may be common in human melanoma TILs. Importantly though, the clinical relevance of neo-antigen specific T cell populations remains uncertain. To directly address the tumoricidal potential of defined neo-antigen specific T cell populations, we first identified two neo-antigen specific T-cell populations within a bulk melanoma TIL culture by the combination of exome sequencing and MHC multimer-based T cell screens. Subsequently, we generated TIL products that are highly enriched for these neo-antigen reactivities and we compared the anti-tumor activity of these neo-antigen enriched TIL with that of ‘standard’ bulk TIL in NSG mice bearing the autologous tumor. We observed outgrowth of the tumors in mice treated with standard TIL. In contrast, tumors in mice treated with enriched TILs were controlled long-term. Additional experiments showed that this superior activity of neo-antigen enriched TIL was caused by the increased numbers of T cells with a high anti-tumor activity, rather than the depletion of cell populations with inhibitory activity. Furthermore, once tumors eventually recurred these were still recognized in vitro by cells from the initially infused TIL culture. Together, these preclinical data demonstrate that neo-antigen reactive T cells within bulk TIL cultures form a critical component of anti-tumor reactivity and provide a further basis to target mutated antigens with cancer immunotherapy. Citation Format: Sander Kelderman, Laura Bies, Marit M. Van Buuren, Nienke Van Rooij, John Haanen, Pia Kvistborg, Ton Schumacher. Neo-antigen enriched TIL therapy mediates superior tumor eradication in a patient-derived xenograft model of human melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4704. doi:10.1158/1538-7445.AM2015-4704

Immediate-Early Expression of a Recombinant Antigen by Modified Vaccinia Virus Ankara Breaks the Immunodominance of Strong Vector-Specific B8R Antigen in Acute and Memory CD8 T-Cell Responses

Efficient T-cell responses against recombinant antigens expressed by vaccinia virus vectors require expression of these antigens in the early phase of the virus replication cycle. The kinetics of recombinant gene expression in poxviruses are largely determined by the promoter chosen. We used the highly attenuated modified vaccinia virus Ankara (MVA) to determine the role of promoters in the induction of CD8 T-cell responses. We constructed MVA recombinants expressing either enhanced green fluorescent protein (EGFP) or chicken ovalbumin (OVA), each under the control of a hybrid early-late promoter (pHyb) containing five copies of a strong early element or the well-known early-late p7.5 or pS promoter for comparison. In primary or cultured cells, EGFP expression under the control of pHyb was detected within 30 min, as an immediate-early protein, and remained higher over the first 6 h of infection than p7.5- or pS-driven EGFP expression. Repeated immunizations of mice with recombinant MVA expressing OVA under the control of the pHyb promoter led to superior acute and memory CD8 T-cell responses compared to those to p7.5- and pS-driven OVA. Moreover, OVA expressed under the control of pHyb replaced the MVA-derived B8R protein as the immunodominant CD8 T-cell antigen after three or more immunizations. This is the first demonstration of an immediate-early neoantigen expressed by a poxviral vector resulting in superior induction of neoantigen-specific CD8 T-cell responses.