Jens Fritsche

Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival

IMA901 is the first therapeutic vaccine for renal cell cancer (RCC) consisting of multiple tumor-associated peptides (TUMAPs) confirmed to be naturally presented in human cancer tissue. We treated a total of 96 human leukocyte antigen A (HLA-A)*02+ subjects with advanced RCC with IMA901 in two consecutive studies. In the phase 1 study, the T cell responses of the patients to multiple TUMAPs were associated with better disease control and lower numbers of prevaccine forkhead box P3 (FOXP3)+ regulatory T (Treg) cells. The randomized phase 2 trial showed that a single dose of cyclophosphamide reduced the number of Treg cells and confirmed that immune responses to multiple TUMAPs were associated with longer overall survival. Furthermore, among six predefined populations of myeloid-derived suppressor cells, two were prognostic for overall survival, and among over 300 serum biomarkers, we identified apolipoprotein A-I (APOA1) and chemokine (C-C motif) ligand 17 (CCL17) as being predictive for both immune response to IMA901 and overall survival. A randomized phase 3 study to determine the clinical benefit of treatment with IMA901 is ongoing.

Predicting immunogenic tumour mutations by combining mass spectrometry and exome sequencing

Human tumours typically harbour a remarkable number of somatic mutations. If presented on major histocompatibility complex class I molecules (MHCI), peptides containing these mutations could potentially be immunogenic as they should be recognized as ‘non-self’ neo-antigens by the adaptive immune system. Recent work has confirmed that mutant peptides can serve as T-cell epitopes. However, few mutant epitopes have been described because their discovery required the laborious screening of patient tumour-infiltrating lymphocytes for their ability to recognize antigen libraries constructed following tumour exome sequencing. We sought to simplify the discovery of immunogenic mutant peptides by characterizing their general properties. We developed an approach that combines whole-exome and transcriptome sequencing analysis with mass spectrometry to identify neo-epitopes in two widely used murine tumour models. Of the >1,300 amino acid changes identified, ∼13% were predicted to bind MHCI, a small fraction of which were confirmed by mass spectrometry. The peptides were then structurally modelled bound to MHCI. Mutations that were solvent-exposed and therefore accessible to T-cell antigen receptors were predicted to be immunogenic. Vaccination of mice confirmed the approach, with each predicted immunogenic peptide yielding therapeutically active T-cell responses. The predictions also enabled the generation of peptide–MHCI dextramers that could be used to monitor the kinetics and distribution of the anti-tumour T-cell response before and after vaccination. These findings indicate that a suitable prediction algorithm may provide an approach for the pharmacodynamic monitoring of T-cell responses as well as for the development of personalized vaccines in cancer patients.

IMA901, a multipeptide cancer vaccine, plus sunitinib versus sunitinib alone, as first-line therapy for advanced or metastatic renal cell carcinoma (IMPRINT): a multicentre, open-label, randomised, controlled, phase 3 trial

BACKGROUND In a phase 2 study in patients with metastatic renal cell carcinoma, overall survival was associated with T-cell responses against IMA901, a vaccine consisting of ten tumour-associated peptides. In this phase 3 trial, we aimed to determine the clinical effect of adding IMA901 to sunitinib, the standard first-line treatment in metastatic renal cell carcinoma with postulated favourable immunomodulatory effects. METHODS The IMPRINT study is an open-label, randomised, controlled, phase 3 trial done at 124 clinical sites in 11 countries. HLA-A*02-positive patients (aged ≥18 years) with treatment-naive, histologically confirmed metastatic or locally advanced (or both) clear-cell renal cell carcinoma were randomly assigned (3:2) to receive sunitinib plus up to ten intradermal vaccinations of IMA901 (4·13 mg) and granulocyte macrophage colony-stimulating factor (75 μg), with one dose of cyclophosphamide (300 mg/m2) 3 days before the first vaccination, or to receive sunitinib alone. Sunitinib (50 mg) was given orally once daily, with each cycle defined as 4 weeks on treatment followed by 2 weeks off treatment, until progression of disease as determined by the investigator, death, or withdrawal of consent. Block randomisation (block size five) was done centrally using an interactive web response system, stratified by prognostic risk, geographical region, and previous nephrectomy. Patients and investigators were not masked to treatment allocation. The primary endpoint was overall survival from randomisation until death of any cause as determined by the investigator, analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01265901. FINDINGS Between Dec 22, 2010, and Dec 15, 2012, we screened 1171 patients, of whom 339 were randomly assigned to receive sunitinib plus IMA901 (n=204) or sunitinib monotherapy (n=135). Patients had a median follow-up of 33·27 months (IQR 29·92-35·64). Median overall survival did not differ significantly between the groups (33·17 months [95% CI 27·81-41·36] in the sunitinib plus IMA901 group vs not reached [33·67-not reached] in the sunitinib monotherapy group; hazard ratio 1·34 [0·96-1·86]; p=0·087). 116 (57%) of 202 patients in the sunitinib plus IMA901 group and 62 (47%) of 132 in the sunitinib group had grade 3 or worse adverse events, the most common of which were hypertension, neutropenia, and anaemia in both groups, and mild-to-moderate transient injection-site reactions (eg, erythema, pruritus) were the most frequent IMA901-related side-effect in the sunitinib plus IMA901 group. Serious adverse events leading to death occurred in four (2%) patients (one respiratory failure and circulatory collapse [possibly related to sunitinib], one oesophageal varices haemorrhage [possibly related to sunitinib], one cardiac arrest [possibly related to sunitinib], and one myocardial infarction) and eight (6%) patients in the sunitinib group (one case each of renal failure, oesophageal varices haemorrhage, circulatory collapse, wound infection, ileus, cerebrovascular accident [possibly treatment related], and sepsis). INTERPRETATION IMA901 did not improve overall survival when added to sunitinib as first-line treatment in patients with metastatic renal cell carcinoma. The magnitude of immune responses needs to be improved before further development of IMA901 in this disease is indicated. FUNDING Immatics Biotechnologies.

Immunogenic stress and death of cancer cells: Contribution of antigenicity vs adjuvanticity to immunosurveillance

Cancer cells are subjected to constant selection by the immune system, meaning that tumors that become clinically manifest have managed to subvert or hide from immunosurveillance. Immune control can be facilitated by induction of autophagy, as well as by polyploidization of cancer cells. While autophagy causes the release of ATP, a chemotactic signal for myeloid cells, polyploidization can trigger endoplasmic reticulum stress with consequent exposure of the “eat‐me” signal calreticulin on the cell surface, thereby facilitating the transfer of tumor antigens into dendritic cells. Hence, both autophagy and polyploidization cause the emission of adjuvant signals that ultimately elicit immune control by CD8+ T lymphocytes. We investigated the possibility that autophagy and polyploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. Mass spectrometry led to the identification of peptides that were presented on major histocompatibility complex (MHC) class I molecules in an autophagy‐dependent fashion or that were specifically exposed on the surface of polyploid cells, yet lost upon passage of such cells through immunocompetent (but not immunodeficient) mice. However, the preferential recognition of autophagy‐competent and polyploid cells by the innate and cellular immune systems did not correlate with the preferential recognition of such peptides in vivo. Moreover, vaccination with such peptides was unable to elicit tumor growth‐inhibitory responses in vivo. We conclude that autophagy and polyploidy increase the immunogenicity of cancer cells mostly by affecting their adjuvanticity rather than their antigenicity.

Front Cover: Translating Immunopeptidomics to Immunotherapy-Decision-Making for Patient and Personalized Target Selection

Immunotherapy is revolutionizing cancer treatment and has shown success in particular for tumors with a high mutational load. These effects have been linked to neoantigens derived from patient‐specific mutations. To expand efficacious immunotherapy approaches to the vast majority of tumor types and patient populations carrying only a few mutations and maybe not a single presented neoepitope, it is necessary to expand the target space to non‐mutated cancer‐associated antigens. Mass spectrometry enables the direct and unbiased discovery and selection of tumor‐specific human leukocyte antigen (HLA) peptides that can be used to define targets for immunotherapy. Combining these targets into a warehouse allows for multi‐target therapy and accelerated clinical application. For precise personalization aimed at optimally ensuring treatment efficacy and safety, it is necessary to assess the presence of the target on each individual patients tumor. Here we show how LC‐MS paired with gene expression data was used to define mRNA biomarkers currently being used as diagnostic test IMADETECT™ for patient inclusion and personalized target selection within two clinical trials (NCT02876510, NCT03247309). Thus, we present a way how to translate HLA peptide presentation into gene expression thresholds for companion diagnostics in immunotherapy considering the peptide‐specific correlation to its encoding mRNA.

Abstract 2654: GAPVAC-101 phase I trial: First data of an innovative actively personalized peptide vaccination trial in patients with newly diagnosed glioblastoma

The Glioma Actively Personalized Vaccine Consortium (GAPVAC; funded by the European Union Framework 7 Program) aims at treating newly diagnosed glioblastoma (GB) patients with two distinct actively personalized vaccines (APVACs). Resected tumor material is analyzed for multiple biomarkers to characterize the tumor in depth and to enable the design of APVACs tailored to each individual patient: Tumor-specific mutations, the HLA peptidome and gene expression profile are assessed by next-generation sequencing, mass spectrometry and RNA microarray analysis, respectively. Further, the patient-individual immune status is investigated by assessment of leukapheresis samples utilizing an in vitro immunogenicity platform. Data are integrated to define two distinct APVACs for each patient: APVAC1 is composed of up to ten peptides selected from a pre-manufactured “warehouse”. The warehouse contains 59 HLA class I-binding and three class II-binding tumor-associated peptides frequently over-presented in GB. APVAC2 is composed of one or two peptides that are de novo synthesized for a given patient and preferentially represent mutation-bearing neo-epitopes. After a preparation phase in which the warehouse was generated and setup of APVAC selection and manufacturing processes took place, the GAPVAC-101 phase I clinical trial was initiated. Primary endpoints of the study are assessment of safety, feasibility of APVAC manufacturing and biological activity. The trial is conducted at six European centers and recruits HLA-A*02:01 or A*24:02-positive patients with newly diagnosed GB after gross total resection. Patients receive APVAC1 and APVAC2 vaccinations plus immunomodulators (poly-ICLC and GM-CSF) three and six months post study enrolment, respectively, and concurrent to maintenance temozolomide (TMZ). As of November 2015, 11 patients have been enrolled, of whom six already received APVAC vaccines. Composition and manufacturing are ongoing for four patients. All APVACs were generated in time without ultimate failures. APVAC1 vaccines differ substantially with 31 out of 59 warehouse peptides have been selected at least once, indicating the need for personalization due to tumor heterogeneity even for non-mutated epitopes. In patients’ tumor samples an average of 40 non-synonymous mutations (including known driver mutations) were identified. Injection site reactions were the most frequent toxicities so far. One brain edema (Grade 3) and one allergic reaction (Grade 4)were observed, both potentially related to the vaccinations. First data on biological activity of APVACs and updated clinical data will be presented at the Annual Meeting. In conclusion, the GAPVAC concept has been successfully translated into the clinics and so far demonstrated to be safe and feasible with its level of personalization matching the observed tumor heterogeneity. Citation Format: Norbert Hilf, Katrin Frenzel, Sabrina Kuttruff-Coqui, Sandra Heesch, Sebastian Kreiter, Arie Admon, Valesca Bukur, Sjoerd van der Burg, Cecile Gouttefangeas, Judith R. Kroep, Marij Schoenmaekers-Welters, Jordi Piro, Berta Ponsati, Hans Skovgaard Poulsen, Ulrik Lassen, Francisco Martinez-Ricarte, Jordi Rodon, Juan Sahuquillo, Monika Stieglbauer, Stefan Stevanovic, Per thor Straten, Marco Skardelly, Ghazaleh Tabatabai, Michael Platten, David Capper, Andreas von Deimling, Valerie Dutoit, Hideho Okada, Christian Ottensmeier, Randi Kristina Feist, Jens Fritsche, Karoline Laske, Peter Lewandrowski, Martin Lower, Regina Mendryzk, Miriam Meyer, Carsten Reinhardt, Bernhard Rossler, Anna Paruzynski, Nina Pawlowski, Colette Song, Stevermann Lea, Toni Weinschenk, Christoph Huber, Hans-Georg Rammensee, Pierre-Yves Dietrich, Wick Wolfgang, Ugur Sahin, Harpreet Singh-Jasuja. GAPVAC-101 phase I trial: First data of an innovative actively personalized peptide vaccination trial in patients with newly diagnosed glioblastoma. [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 2654.

RNA editing derived epitopes function as cancer antigens to elicit immune responses

In addition to genomic mutations, RNA editing is another major mechanism creating sequence variations in proteins by introducing nucleotide changes in mRNA sequences. Deregulated RNA editing contributes to different types of human diseases, including cancers. Here we report that peptides generated as a consequence of RNA editing are indeed naturally presented by human leukocyte antigen (HLA) molecules. We provide evidence that effector CD8+ T cells specific for edited peptides derived from cyclin I are present in human tumours and attack tumour cells that are presenting these epitopes. We show that subpopulations of cancer patients have increased peptide levels and that levels of edited RNA correlate with peptide copy numbers. These findings demonstrate that RNA editing extends the classes of HLA presented self-antigens and that these antigens can be recognised by the immune system.RNA editing is a biological process that creates sequence variation. Here the authors show that peptides generated as a consequence of RNA editing are naturally presented by human leukocyte antigen (HLA) and serve as antigens to elicit anti-tumour immune responses.

Abstract 662: IMA_Detect: Mass spectrometry guided development and clinical application of a companion diagnostic for adoptive cellular therapy against tumor associated HLA peptides

Adoptive cellular therapy (ACT) has dramatically changed the landscape of cancer immunotherapy. ACTolog® and ACTengine® are actively personalized ACT approaches employing T-cell receptor products based on a warehouse of human leucocyte antigen (HLA)-bound peptide targets. Selecting the relevant target candidates requires the establishment of biomarkers predictive for HLA peptide presentation and their development into companion diagnostic devices. Here we describe the development of IMA_Detect, a diagnostic test based on gene expression analysis of primary patient tumor material by qPCR which is predictive for presentation of a target peptide by HLA. To establish mRNA expression levels which indicate actual peptide presentation, data of Immatics9 XPRESIDENT® target discovery platform was used by integrating quantitative immunopeptidomics data (label-free LC-MS) with paired transcriptomics data (RNA-Seq). The peptide-specific correlation between peptide presentation and expression of the coding exons was verified for each target individually. The resulting RNA-Seq thresholds were mapped to qPCR thresholds using calibration curves followed by validation of the qPCR assay. The IMA_Detect test is performed in a CLIA/CAP approved setting and is currently being applied in the phase I ACT trials IMA101-101 and IMA201-101 to determine whether a patient9s tumor expresses any of the tested targets at levels considered sufficient for potential benefit from the administered T-cell therapy. IMA101-101 uses autologous T-cell products (ACTolog®) for patients with solid cancers while IMA201-101 is based on TCR-engineered T Cells (ACTengine®) in NSCLC and HNSCC patients. We will present first results of the patient screening and the personalized target selection. Citation Format: Jens Fritsche, Arun Satelli, Helen Horzer, Barbara Rakitsch, Franziska Hoffgaard, Norbert Hilf, Oliver Schoor, Harpreet Singh-Jasuja, Weinschenk Toni. IMA_Detect: Mass spectrometry guided development and clinical application of a companion diagnostic for adoptive cellular therapy against tumor associated HLA peptides [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 662.

Abstract 2291: On- and off target toxicity profiling for adoptive cell therapy by mass spectrometry-based immunopeptidome analysis of primary human normal tissues

A major constraint for the broad and safe application of Adoptive Cellular Therapy (ACT) is the limited number of validated tumor targets, especially for solid tumors. For T-cell receptor (TCR)-based approaches, presentation of targeted HLA-peptides on normal tissues can lead to on-target toxicity, such as severe inflammatory colitis reported upon re-directing T cells to an HLA-A*02 restricted carcinoembryonic antigen (CEA) epitope. Independently, off-target cross-reactivity of TCRs occurred in previous ACT trials, e.g. when a MAGEA3-directed TCR cross-recognized an HLA-A*01 restricted epitope from titin expressed on heart, which led to fatal cardiac toxicities. Here we present a novel approach allowing the prediction of severe on- and off-target side effects before entering into clinical trials. We used a target discovery engine (XPRESIDENT) combining highly sensitive, quantitative mass spectrometry (LC-MS/MS), RNA-Seq-based differential transcriptomics, immunology and bioinformatics to characterize the human immunopeptidome directly on shock frozen primary human tissues. Over the last years we have built an according database for > 600 tumor samples from > 20 different tumor types and, importantly, > 300 samples from > 40 different normal tissue types, resulting in hundreds of thousands of unique HLA-peptide sequences. These data allow conclusions on which HLA peptides are actually presented on primary normal tissues in a quantitative manner, taking into account relative differences between normal tissues and tumors as well as absolute peptide copy numbers per cell. In order to assess the off-target risk for a TCR, we predict all theoretical HLA- and TCR-binding peptides in the proteome, ideally based on the binding motif of the TCR, and specifically search for actual peptide presentation by normal tissues. When analyzing the above described CEA case, we were able to detect the CEA-derived peptide IMIGVLVGV on HLA-A*02 positive colorectal cancer samples, but importantly also on normal colorectal samples. In the original study describing the titin case tremendous experimental efforts and sophisticated cell culture models were required to retrospectively identify cross-recognition of the peptide on cardiomyocytes as the cause of toxicity. In contrast, with our approach we easily and directly identified the critical peptide ESDPIVAQY as one of the most abundantly presented peptides on an HLA-A*01 positive primary human heart sample. We show that this approach can lead to noteworthy results also for other pre-clinical and clinical stage TCR candidates. In conclusion our data demonstrate that ultrasensitive LC-MS/MS of primary tissue may represent a fast, straightforward and meaningful complementary method to common in vitro or animal models for the prediction of on- and off-target toxicities in TCR-based immunotherapy approaches. Citation Format: Oliver Schoor, Jens Fritsche, Sarah Kutscher, Andrea Mahr, Lea Stevermann, Annika Sonntag, Franziska Hoffgaard, Dominik Vahrenhorst, Julia Leibold, Valentina Goldfinger, Leonie Alten, Sebastian Bunk, Dominik Maurer, Steffen Walter, Hans-Georg Rammensee, Harpreet Singh-Jasuja, Toni Weinschenk. On- and off target toxicity profiling for adoptive cell therapy by mass spectrometry-based immunopeptidome analysis of primary human normal tissues. [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 2291.

Abstract 5365: Prolonged survival of patients with advanced renal cancer responding to multi-peptide vaccine IMA901 after single-dose cyclophosphamide

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Translational research and the clinical development of therapeutic cancer vaccines requires stronger scientific rationalization. Here we demonstrate how immune response markers as well as biomarkers defining the immune regulatory environment were utilized as guiding tools from discovery to advanced clinical trials of IMA901, a novel therapeutic vaccine for the treatment of renal cell carcinoma (RCC). IMA901 consists of multiple tumor-associated peptides (TUMAPs) confirmed to be naturally presented in human RCC tissue by mass spectrometry, selected using differential transcriptomics and preclinically validated by systematic analysis of immunogenicity with artificial antigen-presenting cells. Two consecutive independent clinical studies in a total of 96 HLA-A*02+ advanced/metastatic RCC patients were conducted. The phase I study revealed that T-cell responses to multiple IMA901 antigens were significantly associated with disease control and negatively associated with the presence of FoxP3+ regulatory T cells (Tregs). The subsequent randomized phase II study demonstrated that pre-treatment with a single low dose of cyclophosphamide (Cy) reduced Treg frequencies and prolonged overall survival (OS) in patients who mounted an immune response to the IMA901 vaccine. Additionally, T-cell responses to multiple IMA901 antigens were again associated with clinical benefit. Furthermore, a comprehensive prognostic and predictive biomarker program was conducted. Among cellular biomarkers, highly significantly elevated levels of myeloid-derived suppressor cells (MDSC), IL-17-/IL-10-secreting T cells and dysfunctional T cells in RCC patients vs. healthy individuals were found. Two MDSC populations (CD14+ HLA-DR- and CD14- CD11b+ CD15+) were significantly negatively associated with survival in vaccinated RCC patients. Interestingly, both MDSC populations were also found to be negatively associated with OS in an independent trial in colorectal cancer patients (N=79) implying a broader role for these MDSC species. Additionally, among over 300 serum biomarkers tested, apolipoprotein A-I (ApoA1) and the chemokine CCL17 were found to be predictive for both immune responses to IMA901 and survival of the RCC patients. The knowledge acquired in these trials was used to design a randomized phase III study. In this ongoing study, IMA901 is combined with the tyrosine kinase inhibitor sunitinib based on the findings that sunitinib downmodulates the two MDSC populations described above. Furthermore, in this phase III study, the relevance of ApoA1/CCL17 will be explored by prospectively defined subgroup analyses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5365. doi:1538-7445.AM2012-5365