Vaccination with designed neopeptides induces intratumoral, cross-reactive CD4+ T cell responses in glioblastoma

Purpose: The low mutational load of some cancers is considered one reason for the difficulties to develop effective tumor vaccines. To overcome this problem, we developed a strategy to design neopeptides through single amino acid mutation to enhance their immunogenicity. Experimental Design: Exome- and RNA sequencing as well as in silico HLA-binding predictions to autologous HLA molecules were used to identify candidate neopeptides. Subsequently, in silico HLA-anchor placements were used to deduce putative T cell receptor contacts of peptides. Single amino acids of TCR contacting residues were then mutated by amino acid replacements. Overall, 175 peptides were synthesized and sets of 25 each containing both peptides designed to bind to HLA class I and II molecules applied in the vaccination. Upon development of a tumor recurrence, the tumor-infiltrating lymphocytes (TILs) were characterized in detail both at the bulk and clonal level. Results: The immune response of peripheral blood T cells to vaccine peptides, including natural peptides and designed neopeptides, gradually increased with repetitive vaccination, but remained low. In contrast, at the time of tumor recurrence, CD8+ TILs and CD4+ TILs responded to 45% and 100% respectively of the vaccine peptides. Further, TIL-derived CD4+ T cell clones showed strong responses and tumor cell lysis not only against the designed neopeptide but also against the unmutated natural peptides of the tumor. Conclusions: Turning tumor self-peptides into foreign antigens by introduction of designed mutations is a promising strategy to induce strong intratumoral CD4+ T cell responses in a cold tumor like glioblastoma

Identifizierung T-zellerkannter mutierter Neoantigene in einem humanen Melanommodell mittels Hochdurchsatzsequenzierung von Exomen und Transkriptomen

Somatische Punktmutationen in Tumoren können zur Generierung von tumorspezifischen Neoantigenen führen, die von patienteneigenen T-Zellen erkannt werden. Nach solchen immunogenen Mutationen wurde im humanen Melanommodell Ma-Mel-86 durch Sequenzieren der Exome und Transkriptome von vier Tumorzelllinien und einer EBV-immortalisierten B-Zelllinie aus dem Blut der gleichen Patientin gesucht. Die Melanomzelllinien waren aus Lymphknotenmetastasen etabliert worden, die über einen Zeitraum von sechs Jahren aufgetreten waren, wobei bei den Zelllinien aus den zuletzt aufgetretenen Metastasen ein partieller bzw. vollständiger Verlust der Oberflächenexpression von HLA-Klasse I-Molekülen vorlag. Durch die Kombination der Daten beider Sequenzierverfahren und die Verwendung von parallel prozessierten Replikaten war es möglich, 181 exprimierte, nicht-synonyme, somatische Punktmutationen sicher zu identifizieren. Mithilfe der Algorithmen von NetMHC und der Immune Epitope Database (IEDB) wurden bei 80 ausgewählten Mutationen für die Bindung an HLA-Klasse I-Allele der Patientin insgesamt 174 Okta-, Nona- und Dekamere vorhergesagt und synthetisiert. Die Immunogenität der Peptidkandidaten wurde in IFNγ-ELISpot-Assays mit autologen MLTC (engl. mixed lymphocyte tumor cell culture)-Responderpopulationen aus Blutlymphozyten überprüft. Insgesamt erwiesen sich mit diesem Verfahren 5% (4/80) der getesteten Punktmutationen als immunogen. Die erkannten Peptide wurden von den punktmutierten Genen HERPUD1-G161S (engl. homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-likedomainmember1), INSIG1-S238F (engl.insulininducedgene1), MMS22L-S437F (engl. MMS22-like, DNA repair protein) und PRDM10-S1050F (engl. PR domain containing 10) kodiert. HERPUD1-G161S war bereits zuvor durch cDNA-Bank-Screening als ein durch HLA-B*15:01 restringiertes Neoantigen identifiziert worden. Die drei bisher unbekannten Neoantigene wurden mit mutationsspezifischen CD8+ T-Zellklonen weiter charakterisiert. Dabei wurden HLA-A*24:02 als gemeinsames Restriktionsmolekül bestimmt, die C-Termini der Peptide definiert und deren intrazelluläre Prozessierung bestätigt. Es zeigte sich, dass alle vier mutierten Neoantigene nur auf der Tumorzelllinie erkannt werden konnten, die aus der frühesten Metastase generiert worden war, da diese als einzige noch die restringierenden HLA-Klasse I-Allele exprimierte. Die dargestellten Ergebnisse erweitern das Spektrum bekannter mutierter Tumorantigene und zeigen eine Strategie auf zur raschen Identifizierung von immunologisch relevanten tumorspezifischen Mutationen mithilfe der Hochdurchsatzsequenzierung vorzugsweise im frühen Krankheitsverlauf.Somatic point mutations in tumors can lead to the generation of tumor-specific neoantigens which can be recognized by the patient’s own T cells. Such immunogenic mutations were searched for in the human melanoma model Ma-Mel-86 by whole exome and transcriptome sequencing of four tumor cell lines and an autologous EBV-transformed B cell line. The melanoma cell lines had been established from lymph node metastases occurring over six years. Tumor cell lines derived from the later occurring metastases completely or partially lacked cell surface expression of HLA class I molecules. Altogether 181 expressed non-synonymous somatic point mutations could be identified by combining the data from both sequencing procedures and by processing replicates in parallel. Using the NetMHC and the Immune Epitope Database (IEDB) algorithms, for 80 selected mutations 174 octa-, nona- and decamers were predicted to bind to the patient’s HLA class I alleles. The immunogenicity of these peptide candidates was tested in IFNγ ELISpot assays with MLTC (mixed lymphocyte tumor cell culture) responder populations derived from the patient’s blood lymphocytes. With this approach 5% (4/80) of the tested point mutations were found to be immunogenic. The recognized peptides were encoded by the point-mutated genes HERPUD1-G161S (homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1), INSIG1-S238F (insulin-induced gene 1), MMS22L-S437F (MMS22-like, DNA repair protein) and PRDM10-S1050F (PR domain containing 10). HERPUD1-G161S had been identified before as a neoantigen restricted by HLA-B*15:01 via cDNA expression screening. The three hitherto unknown neoantigens were further characterized with mutation-specific CD8+ T cell clones. HLA-A*24:02 was found to serve as a common restriction molecule. The C-termini of the peptides were defined and their intracellular processing was validated. It turned out that all four mutated neoantigens could only be recognized on the tumor cell line established first during the patient’s clinical course. It was the only tumor line in this model system that expressed all of the patient’s HLA class I alleles. These results broaden the spectrum of known mutated tumor antigens and involved a strategy based on high throughput sequencing which is suitable for the systematic identification of tumor-specific immunogenic mutations in due time preferably in an early disease phase

Mutanome Engineered RNA Immunotherapy: Towards Patient-Centered Tumor Vaccination

Advances in nucleic acid sequencing technologies have revolutionized the field of genomics, allowing the efficient targeting of mutated neoantigens for personalized cancer vaccination. Due to their absence during negative selection of T cells and their lack of expression in healthy tissue, tumor mutations are considered as optimal targets for cancer immunotherapy. Preclinical and early clinical data suggest that synthetic mRNA can serve as potent drug format allowing the cost efficient production of highly efficient vaccines in a timely manner. In this review, we describe a process, which integrates next generation sequencing based cancer mutanome mapping, in silico target selection and prioritization approaches, and mRNA vaccine manufacturing and delivery into a process we refer to as MERIT (mutanome engineered RNA immunotherapy)

CoVigator—A Knowledge Base for Navigating SARS-CoV-2 Genomic Variants

Background: The outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) resulted in the global COVID-19 pandemic. The urgency for an effective SARS-CoV-2 vaccine has led to the development of the first series of vaccines at unprecedented speed. The discovery of SARS-CoV-2 spike-glycoprotein mutants, however, and consequentially the potential to escape vaccine-induced protection and increased infectivity, demonstrates the persisting importance of monitoring SARS-CoV-2 mutations to enable early detection and tracking of genomic variants of concern. Results: We developed the CoVigator tool with three components: (1) a knowledge base that collects new SARS-CoV-2 genomic data, processes it and stores its results; (2) a comprehensive variant calling pipeline; (3) an interactive dashboard highlighting the most relevant findings. The knowledge base routinely downloads and processes virus genome assemblies or raw sequencing data from the COVID-19 Data Portal (C19DP) and the European Nucleotide Archive (ENA), respectively. The results of variant calling are visualized through the dashboard in the form of tables and customizable graphs, making it a versatile tool for tracking SARS-CoV-2 variants. We put a special emphasis on the identification of intrahost mutations and make available to the community what is, to the best of our knowledge, the largest dataset on SARS-CoV-2 intrahost mutations. In the spirit of open data, all CoVigator results are available for download. The CoVigator dashboard is accessible via covigator.tron-mainz.de. Conclusions: With increasing demand worldwide in genome surveillance for tracking the spread of SARS-CoV-2, CoVigator will be a valuable resource of an up-to-date list of mutations, which can be incorporated into global efforts

HLA class I loss in metachronous metastases prevents continuous T cell recognition of mutated neoantigens in a human melanoma model

T lymphocytes against tumor-specific mutated neoantigens can induce tumor regression. Also, the size of the immunogenic cancer mutanome is supposed to correlate with the clinical efficacy of checkpoint inhibition. Herein, we studied the susceptibility of tumor cell lines from lymph node metastases occurring in a melanoma patient over several years towards blood-derived, neoantigen-specific CD8+ T cells. In contrast to a cell line established during early stage III disease, all cell lines generated at later time points from stage IV metastases exhibited partial or complete loss of HLA class I expression. Whole exome and transcriptome sequencing of the four tumor lines and a germline control were applied to identify expressed somatic single nucleotide substitutions (SNS), insertions and deletions (indels). Candidate peptides encoded by these variants and predicted to bind to the patient’s HLA class I alleles were synthesized and tested for recognition by autologous mixed lymphocyte-tumor cell cultures (MLTCs). Peptides from four mutated proteins, HERPUD1G161S, INSIG1S238F, MMS22LS437F and PRDM10S1050F, were recognized by MLTC responders and MLTC-derived T cell clones restricted by HLA-A*24:02 or HLA-B*15:01. Intracellular peptide processing was verified with transfectants. All four neoantigens could only be targeted on the cell line generated during early stage III disease. HLA loss variants of any kind were uniformly resistant. These findings corroborate that, although neoantigens represent attractive therapeutic targets, they also contribute to the process of cancer immunoediting as a serious limitation to specific T cell immunotherapy

A liposomal RNA vaccine inducing neoantigen-specific CD4+ T cells augments the antitumor activity of local radiotherapy in mice

Antigen-encoding, lipoplex-formulated RNA (RNA-LPX) enables systemic delivery to lymphoid compartments and selective expression in resident antigen-presenting cells. We report here that the rejection of CT26 tumors, mediated by local radiotherapy (LRT), is further augmented in a CD8+ T cell-dependent manner by an RNA-LPX vaccine that encodes CD4+ T cell-recognized neoantigens (CD4 neoantigen vaccine). Whereas CD8+ T cells induced by LRT alone were primarily directed against the immunodominant gp70 antigen, mice treated with LRT plus the CD4 neoantigen vaccine rejected gp70-negative tumors and were protected from rechallenge with these tumors, indicating a potent poly-antigenic CD8+ T cell response and T cell memory. In the spleens of CD4 neoantigen-vaccinated mice, we found a high number of activated, poly-functional, Th1-like CD4+ T cells against ME1, the immunodominant CD4 neoantigen within the poly-neoantigen vaccine. LRT itself strongly increased CD8+ T cell numbers and clonal expansion. However, tumor infiltrates of mice treated with CD4 neoantigen vaccine/LRT, as compared to LRT alone, displayed a higher fraction of activated gp70-specific CD8+ T cells, lower PD-1/LAG-3 expression and contained ME1-specific IFNγ+ CD4+ T cells capable of providing cognate help. CD4 neoantigen vaccine/LRT treatment followed by anti-CTLA-4 antibody therapy further enhanced the efficacy with complete remission of gp70-negative CT26 tumors and survival of all mice. Our data highlight the power of combining synergistic modes of action and warrants further exploration of the presented treatment schema

Comprehensive genomic and transcriptomic analysis of three synchronous primary tumours and a recurrence from a head and neck cancer patient

Synchronous primary malignancies occur in a small proportion of head and neck squamous cell carcinoma (HNSCC) patients. Here, we analysed three synchronous primaries and a recurrence from one patient by comparing the genomic and transcriptomic profiles among the tumour samples and determining the recurrence origin. We found remarkable levels of heterogeneity among the primary tumours, and through the patterns of shared mutations, we traced the origin of the recurrence. Interestingly, the patient carried germline variants that might have predisposed him to carcinogenesis, together with a history of alcohol and tobacco consumption. The mutational signature analysis confirmed the impact of alcohol exposure, with Signature 16 present in all tumour samples. Characterisation of immune cell infiltration highlighted an immunosuppressive environment in all samples, which exceeded the potential activity of T cells. Studies such as the one described here have important clinical value and contribute to personalised treatment decisions for patients with synchronous primaries and matched recurrences

Evolution of melanoma cross-resistance to CD8+ T cells and MAPK inhibition in the course of BRAFi treatment

The profound but frequently transient clinical responses to BRAF(V600) inhibitor (BRAFi) treatment in melanoma emphasize the need for combinatorial therapies. Multiple clinical trials combining BRAFi and immunotherapy are under way to further enhance therapeutic responses. However, to which extent BRAF(V600) inhibition may affect melanoma immunogenicity over time remains largely unknown. To support the development of an optimal treatment protocol, we studied the impact of prolonged BRAFi exposure on the recognition of melanoma cells by T cells in different patient models. We demonstrate that autologous CD8(+) tumor-infiltrating lymphocytes (TILs) efficiently recognized short-term (3, 7days) BRAFi-treated melanoma cells but were less responsive towards long-term (14, 21days) exposed tumor cells. Those long-term BRAFi-treated melanoma cells showed a non-proliferative dedifferentiated phenotype and were less sensitive to four out of five CD8(+) T cell clones, present in the preexisting TIL repertoire, of which three recognized shared antigens (Tyrosinase, Melan-A and CSPG4) and one being neoantigen-specific. Only a second neoantigen was steadily recognized independent of treatment duration. Notably, in all cases the impaired T cell activation was due to a time-dependent downregulation of their respective target antigens. Moreover, combinatorial treatment of melanoma cells with BRAFi and an inhibitor of its downstream kinase MEK had similar effects on T cell recognition. In summary, MAP kinase inhibitors (MAPKi) strongly alter the tumor antigen expression profile over time, favoring evolution of melanoma variants cross-resistant to both T cells and MAPKi. Our data suggest that simultaneous treatment with MAPKi and immunotherapy could be most effective for tumor elimination

Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Tumour-specific mutations are ideal targets for cancer immunotherapy as they lack expression in healthy tissues and can potentially be recognized as neo-antigens by the mature T-cell repertoire. Their systematic targeting by vaccine approaches, however, has been hampered by the fact that every patient’s tumour possesses a unique set of mutations (‘the mutanome’) that must first be identified. Recently, we proposed a personalized immunotherapy approach to target the full spectrum of a patient’s individual tumour-specific mutations1. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the majority of the immunogenic mutanome is recognized by CD4+ T cells. Vaccination with such CD4+ immunogenic mutations confers strong antitumour activity. Encouraged by these findings, we established a process by which mutations identified by exome sequencing could be selected as vaccine targets solely through bioinformatic prioritization on the basis of their expression levels and major histocompatibility complex (MHC) class II-binding capacity for rapid production as synthetic poly-neo-epitope messenger RNA vaccines. We show that vaccination with such polytope mRNA vaccines induces potent tumour control and complete rejection of established aggressively growing tumours in mice. Moreover, we demonstrate that CD4+ T cell neo-epitope vaccination reshapes the tumour microenvironment and induces cytotoxic T lymphocyte responses against an independent immunodominant antigen in mice, indicating orchestration of antigen spread. Finally, we demonstrate an abundance of mutations predicted to bind to MHC class II in human cancers as well by employing the same predictive algorithm on corresponding human cancer types. Thus, the tailored immunotherapy approach introduced here may be regarded as a universally applicable blueprint for comprehensive exploitation of the substantial neo-epitope target repertoire of cancers, enabling the effective targeting of every patient’s tumour with vaccines produced ‘just in time’

Vaccination with Designed Neopeptides Induces Intratumoral, Cross-reactive CD4+ T-cell Responses in Glioblastoma

Purpose: The low mutational load of some cancers is considered one reason for the difficulty to develop effective tumor vaccines. To overcome this problem, we developed a strategy to design neopeptides through single amino acid mutations to enhance their immunogenicity. Experimental Design: Exome and RNA sequencing as well as in silico HLA-binding predictions to autologous HLA molecules were used to identify candidate neopeptides. Subsequently, in silico HLA-anchor placements were used to deduce putative T-cell receptor (TCR) contacts of peptides. Single amino acids of TCR contacting residues were then mutated by amino acid replacements. Overall, 175 peptides were synthesized and sets of 25 each containing both peptides designed to bind to HLA class I and II molecules applied in the vaccination. Upon development of a tumor recurrence, the tumor-infiltrating lymphocytes (TIL) were characterized in detail both at the bulk and clonal level. Results: The immune response of peripheral blood T cells to vaccine peptides, including natural peptides and designed neopeptides, gradually increased with repetitive vaccination, but remained low. In contrast, at the time of tumor recurrence, CD8+ TILs and CD4+ TILs responded to 45% and 100%, respectively, of the vaccine peptides. Furthermore, TIL-derived CD4+ T-cell clones showed strong responses and tumor cell lysis not only against the designed neopeptide but also against the unmutated natural peptides of the tumor. Conclusions: Turning tumor self-peptides into foreign antigens by introduction of designed mutations is a promising strategy to induce strong intratumoral CD4+ T-cell responses in a cold tumor like glioblastoma.ISSN:1078-0432ISSN:1557-326