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IMA101 is an actively personalized, multi-targeted adoptive cell therapy (ACT), whereby autologous T cells are directed against multiple novel defined peptide-HLA (pHLA) cancer targets. HLA-A*02:01-positive patients with relapsed/refractory solid tumors expressing ≥1 of 8 pre-defined targets underwent leukapheresis. Endogenous T cells specific for up to 4 targets were primed and expanded in vitro. Patients received lymphodepletion (fludarabine, cyclophosphamide), followed by T-cell infusion and low-dose interleukin-2 (Cohort 1). Patients in Cohort 2 received atezolizumab for up to 1 year (NCT02876510). Overall, 214 patients were screened, 15 received lymphodepletion (13 women, 2 men; median age, 44 years), and 14 were treated with T-cell products. IMA101 treatment was feasible and well tolerated. The most common adverse events were cytokine release syndrome (Grade 1, n=6; Grade 2, n=4) and expected cytopenias. No patient died during the first 100 days after T-cell therapy. No neurotoxicity was observed. No objective responses were noted. Prolonged disease stabilization was noted in three patients lasting for 13.7, 12.9, and 7.3 months. High frequencies of target-specific T cells (up to 78.7% of CD8+ cells) were detected in the blood of treated patients, persisted for >1 year, and were detectable in post-treatment tumor tissue. Individual TCRs contained in T-cell products exhibited broad variation in TCR avidity, with the majority being of low-avidity. High-avidity TCRs were identified in some patients’ products. This study demonstrates the feasibility and tolerability of an actively personalized ACT directed to multiple defined pHLA cancer targets. Results warrant further evaluation of multi-target ACT approaches using potent high-avidity TCRs.

Supplementary Table S1. TUMAPs contained in IMA950 and associated source antigens. Supplementary Table S2. Statistical analysis underlying the recruitment of 20 immune evaluable patients per cohort. Supplementary Table S3. Reasons for HLA-A*02 positive patients not entering the clinical trial. Supplementary Figure S1. Study schedule. Supplementary Figure S2. Exemplary gating strategy used for the primary multimer assay. Supplementary Figure S3. Exemplary immune responses in patients as determined by multimer assay after in vitro sensitization. Supplementary Figure S4. Apparent diffusion coefficient (ADC) in Cohort 1 at each of the four scan points.

Purpose:Immunotherapy for hepatocellular carcinoma (HCC) shows considerable promise in improving clinical outcomes. HepaVac-101 represents a single-arm, first-in-human phase I/II multicenter cancer vaccine trial for HCC (NCT03203005). It combines multipeptide antigens (IMA970A) with the TLR7/8/RIG I agonist CV8102. IMA970A includes 5 HLA-A*24 and 7 HLA-A*02 as well as 4 HLA-DR restricted peptides selected after mass spectrometric identification in human HCC tissues or cell lines. CV8102 is an RNA-based immunostimulator inducing a balanced Th1/Th2 immune response.Patients and Methods:A total of 82 patients with very early- to intermediate-stage HCCs were enrolled and screened for suitable HLA haplotypes and 22 put on study treatment. This consisted in a single infusion of low-dose cyclophosphamide followed by nine intradermal coadministrations of IMA970A and CV8102. Only patients with no disease relapse after standard-of-care treatments were vaccinated. The primary endpoints of the HepaVac-101 clinical trial were safety, tolerability, and antigen-specific T-cell responses. Secondary or exploratory endpoints included additional immunologic parameters and survival endpoints.Results:The vaccination showed a good safety profile. Transient mild-to-moderate injection-site reactions were the most frequent IMA970A/CV8102-related side effects. Immune responses against ≥1 vaccinated HLA class I tumor-associated peptide (TAA) and ≥1 vaccinated HLA class II TAA were respectively induced in 37% and 53% of the vaccinees.Conclusions:Immunotherapy may provide a great improvement in treatment options for HCC. HepaVac-101 is a first-in-human clinical vaccine trial with multiple novel HLA class I– and class II–restricted TAAs against HCC. The results are initial evidence for the safety and immunogenicity of the vaccine. Further clinical evaluations are warranted.

Supplementary Data from Phase I/II Multicenter Trial of a Novel Therapeutic Cancer Vaccine, HepaVac-101, for Hepatocellular Carcinoma

Purpose: To perform a two-cohort, phase I safety and immunogenicity study of IMA950 in addition to standard chemoradiotherapy and adjuvant temozolomide in patients with newly diagnosed glioblastoma. IMA950 is a novel glioblastoma-specific therapeutic vaccine containing 11 tumor-associated peptides (TUMAP), identified on human leukocyte antigen (HLA) surface receptors in primary human glioblastoma tissue.Experimental Design: Patients were HLA-A*02–positive and had undergone tumor resection. Vaccination comprised 11 intradermal injections with IMA950 plus granulocyte macrophage colony-stimulating factor (GM-CSF) over a 24-week period, beginning 7 to 14 days prior to initiation of chemoradiotherapy (Cohort 1) or 7 days after chemoradiotherapy (Cohort 2). Safety was assessed according to NCI CTCAE Version 4.0 and TUMAP-specific T-cell immune responses determined. Secondary observations included progression-free survival (PFS), pretreatment regulatory T cell (Treg) levels, and the effect of steroids on T-cell responses.Results: Forty-five patients were recruited. Related adverse events included minor injection site reactions, rash, pruritus, fatigue, neutropenia and single cases of allergic reaction, anemia and anaphylaxis. Two patients experienced grade 3 dose-limiting toxicity of fatigue and anaphylaxis. Of 40 evaluable patients, 36 were TUMAP responders and 20 were multi-TUMAP responders, with no important differences between cohorts. No effect of pretreatment Treg levels on IMA950 immunogenicity was observed, and steroids did not affect TUMAP responses. PFS rates were 74% at 6 months and 31% at 9 months.Conclusions: IMA950 plus GM-CSF was well-tolerated with the primary immunogenicity endpoint of observing multi-TUMAP responses in at least 30% of patients exceeded. Further development of IMA950 is encouraged. Clin Cancer Res; 22(19); 4776–85. ©2016 AACR.See related commentary by Lowenstein and Castro, p. 4760

Summary 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/m 2 ) 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.

Purpose: To perform a two-cohort, phase I safety and immunogenicity study of IMA950 in addition to standard chemoradiotherapy and adjuvant temozolomide in patients with newly diagnosed glioblastoma. IMA950 is a novel glioblastoma-specific therapeutic vaccine containing 11 tumor-associated peptides (TUMAP), identified on human leukocyte antigen (HLA) surface receptors in primary human glioblastoma tissue. Experimental Design: Patients were HLA-A*02–positive and had undergone tumor resection. Vaccination comprised 11 intradermal injections with IMA950 plus granulocyte macrophage colony-stimulating factor (GM-CSF) over a 24-week period, beginning 7 to 14 days prior to initiation of chemoradiotherapy (Cohort 1) or 7 days after chemoradiotherapy (Cohort 2). Safety was assessed according to NCI CTCAE Version 4.0 and TUMAP-specific T-cell immune responses determined. Secondary observations included progression-free survival (PFS), pretreatment regulatory T cell (Treg) levels, and the effect of steroids on T-cell responses. Results: Forty-five patients were recruited. Related adverse events included minor injection site reactions, rash, pruritus, fatigue, neutropenia and single cases of allergic reaction, anemia and anaphylaxis. Two patients experienced grade 3 dose-limiting toxicity of fatigue and anaphylaxis. Of 40 evaluable patients, 36 were TUMAP responders and 20 were multi-TUMAP responders, with no important differences between cohorts. No effect of pretreatment Treg levels on IMA950 immunogenicity was observed, and steroids did not affect TUMAP responses. PFS rates were 74% at 6 months and 31% at 9 months. Conclusions: IMA950 plus GM-CSF was well-tolerated with the primary immunogenicity endpoint of observing multi-TUMAP responses in at least 30% of patients exceeded. Further development of IMA950 is encouraged. Clin Cancer Res; 22(19); 4776–85. ©2016 AACR. See related commentary by Lowenstein and Castro, p. 4760

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.

A variety of different mRNA-based drugs are currently in development. This became possible, since major breakthroughs in RNA research during the last decades allowed impressive improvements of translation, stability and delivery of mRNA. This article focuses on antigen-encoding RNA-based vaccines that are either directed against tumors or pathogens. mRNA-encoded vaccines are developed both for preventive or therapeutic purposes. Most mRNA-based vaccines are directly administered to patients. Alternatively, primary autologous cells from cancer patients are modified ex vivo by the use of mRNA and then are adoptively transferred to patients. In the EU no regulatory guidelines presently exist that specifically address mRNA-based vaccines. The existing regulatory framework, however, clearly defines that mRNA-based vaccines in most cases have to be centrally approved. Interestingly, depending on whether RNA-based vaccines are directed against tumors or infectious disease, they are formally considered gene therapy products or not, respectively. Besides an overview on the current clinical use of mRNA vaccines in various therapeutic areas a detailed discussion of the current regulatory situation is provided and regulatory perspectives are discussed.

T cell receptors (TCRs) naturally recognize human leukocyte antigen (HLA)-bound peptides derived from foreign and endogenous proteins regardless of their extracellular or intracellular location. Preferentially expressed antigen in melanoma (PRAME) has been shown to be expressed at high levels in a variety of cancer cells while being absent or present only at very low levels in normal adult tissues except testis. In contrast to most other cancer/testis antigens, PRAME is expressed not only in solid tumors but also in leukemia and myeloma cells. Immunotherapy with bispecific T cell engagers has emerged as a novel and promising treatment modality for malignant diseases, however, antibody-based approaches (ie. blinatumomab) are restricted to few surface antigens such as CD19 or BCMA. Immatics has developed bispecific T cell-engaging receptors (TCER®) that are fusion proteins consisting of an affinity-maturated TCR and a humanized T cell-recruiting antibody with an effector function-silenced IgG1 Fc part. TCER® molecules confer extended half-life together with antibody-like stability and manufacturability characteristics. The molecular design allows for effective redirection of T cells towards target peptide-HLA selectively expressed in tumor tissues. Here we present proof-of-concept data from a TCER® program targeting a PRAME-derived peptide bound to HLA-A*02:01. We confirmed the abundant presence of the target peptide-HLA in several cancer indications and its absence in relevant human normal tissues by using the XPRESIDENT® target discovery engine, which combines quantitative mass spectrometry, transcriptomics and bioinformatics. Yeast surface display technology was used to maturate the stability and affinity of a parental human TCR recognizing PRAME with high functional avidity and specificity. During maturation we applied XPRESIDENT®-guided off-target toxicity screening, incorporating the world's largest normal tissue immunopeptidome database, to deselect cross-reactive candidate TCRs. The maturated TCRs were engineered into the TCER® scaffold and production in Chinese hamster ovary (CHO) cells generated highly stable molecules with low tendency for aggregation as confirmed during stress studies. Following TCR maturation, the TCER® molecules exhibited an up to 10,000-fold increased binding affinity towards PRAME when compared to the parental TCR. The high affinity correlated with potent in vitro anti-tumor activity requiring only low picomolar concentrations of TCER® molecules to induce half-maximal lysis of tumor cells expressing the target at physiological levels. Furthermore, using a tumor xenograft model in immunodeficient NOG mice, we could demonstrate significant growth inhibition of established tumors upon intravenous injection of TCER® molecules. Pharmacokinetic profiling in NOG mice determined a terminal half-life of more than 4 days, compatible with a once weekly dosing regimen in patients. For the safety assessment, we measured killing of more than 20 different human normal tissue cell types derived from high risk organs. Notably, we could confirm a favorable safety window for selected TCER® molecules, which induced killing of most normal tissue cells only at significantly higher concentrations than required for killing of tumor cells. To further support safety of TCER® molecules, we also performed a comprehensive characterization of potential off-target peptides selected from the XPRESIDENT® normal tissue database based on its high similarity to the sequence of the target peptide or based on data from alternative screening approaches. In summary, the efficacy, safety and manufacturability data to be presented provide preclinical proof-of-concept for a novel bispecific T cell-engaging receptor (TCER®) molecule targeting PRAME for treatment of various malignant diseases. Disclosures Bunk: Immatics: Employment. Hofmann:Immatics: Employment. Unverdorben:Immatics: Employment. Hutt:Immatics: Employment. Pszolla:Immatics: Employment. Schwöbel:Immatics: Employment. Wagner:Immatics: Employment. Yousef:Immatics: Employment. Schuster:Immatics: Employment. Missel:Immatics: Employment. Schoor:Immatics: Employment. Weinschenk:Immatics: Employment, Equity Ownership. Singh-Jasuja:Immatics: Employment, Equity Ownership. Maurer:Immatics: Employment. Reinhardt:Immatics: Employment, Equity Ownership.

Non-specific immunotherapy has been for a long time a standard treatment option for patients with metastatic renal cell carcinoma but was redeemed by specific targeted molecular therapies, namely the VEGF and mTOR inhibitors. After moving treatment for mRCC to specific molecular agents with a well-defined mode of action, immunotherapy still needs this further development to increase its accuracy. Nowadays, an evolution from a rather non-specific cytokine treatment to sophisticated targeted approaches in specific immunotherapy led to a re-launch of immunotherapy in clinical studies. Recent steps in the development of immunotherapy strategies are discussed in this review with a special focus on peptide vaccination which aims at a tumor targeting by specific T lymphocytes. In addition, different combinatory strategies with immunomodulating agents like cyclophosphamide or sunitinib are outlined, and the effects of immune checkpoint modulators as anti-CTLA-4 or PD-1 antibodies are discussed. peerReviewed

Peptides presented at the cell surface reflect the protein content of the cell; those on HLA class I molecules comprise the critical peptidome elements interacting with CD8 T lymphocytes. We hypothesize that peptidomes from ex vivo tumour samples encompass immunogenic tumour antigens. Here, we uncover >6000 HLA-bound peptides from HLA-A*02(+) glioblastoma, of which over 3000 were restricted by HLA-A*02. We prioritized in-depth investigation of 10 glioblastoma-associated antigens based on high expression in tumours, very low or absent expression in healthy tissues, implication in gliomagenesis and immunogenicity. Patients with glioblastoma showed no T cell tolerance to these peptides. Moreover, we demonstrated specific lysis of tumour cells by patients' CD8(+) T cells in vitro. In vivo, glioblastoma-specific CD8(+) T cells were present at the tumour site. Overall, our data show the physiological relevance of the peptidome approach and provide a critical advance for designing a rational glioblastoma immunotherapy. The peptides identified in our study are currently being tested as a multipeptide vaccine (IMA950) in patients with glioblastoma.

BACKGROUND: There is a need for treatment personalization as every cancer is molecularly unique. In addition glioblastoma (GB) are immunologically regarded as resistant, cold tumor with few targetable antigens available from mutations, thus demanding new personalized immunotherapies. So far outside Neuro-Oncology, T cells orchestrate impressive anti-tumor effects with checkpoint inhibitors, but also vaccines. METHODS: The GAPVAC consortium established an immunotherapy, for which personalized selection of 2 peptide-based actively personalized vaccines (APVAC) per patient for treatment of newly diagnosed GB was based not only on whole-exome sequencing but also on human leukocyte antigen (HLA)-ligandome analyses providing insight into the actual presentation of relevant epitopes in the tumor. GAPVAC-101 (NCT02149225) enrolled 16 patients in a European phase I feasibility, safety and immunogenicity trial integrated into standard of care. For APVAC1, up to 7 peptides were selected from a trial specific warehouse based on individual biomarker data. Vaccination (i.d.) with GM-CSF and poly-ICLC in 15 patients started with the 1st adjuvant cycle of temozolomide (TMZ). For APVAC2, analyses revealed a median of 36 somatic, non-synonymous mutations in the patients tumors. From the 4th TMZ cycle, 11 patients received APVAC2 with usually 2 de novo antigens per patient selected according to mutation, actual or putative HLA presentation and immunogenicity. Overall 20 APVAC2 antigens incl. 14 mutated were vaccinated. RESULTS: Adverse events were largely reversible injection site reactions and two anaphylactic reactions and one increase in cerebral edema. Short, non-mutated APVAC1 antigens induced sustained CD8 responses with memory phenotype. Mutated APVAC2 antigens induced predominantly CD4 responses of favorable TH1 type. Median PFS and OS were 14.2 and 29 months from diagnosis, respectively, in patients that received 1 APVAC vaccination (N=15). CONCLUSION: Overall, GAPVAC displayed expected safety profiles and high biological activity indicating further development.

BACKGROUND: There is a need for treatment personalization as every cancer is molecularly unique. In addition glioblastoma (GB) are immunologically regarded as resistant, “cold” tumor with few targetable antigens available from mutations, thus demanding new personalized immunotherapies. So far outside Neuro-Oncology, T cells orchestrate impressive anti-tumor effects with checkpoint inhibitors, but also vaccines. MATERIAL AND METHODS: The GAPVAC consortium established an immunotherapy, for which personalized selection of 2 peptide-based actively personalized vaccines (APVAC) per patient for treatment of newly diagnosed GB was based not only on whole-exome sequencing but also on human leukocyte antigen (HLA)-ligandome analyses providing insight into the actual presentation of relevant epitopes in the tumor. GAPVAC-101 (NCT02149225) enrolled 16 patients in a European phase I feasibility, safety and immunogenicity trial integrated into standard of care. For APVAC1, up to 7 peptides were selected from a trial specific warehouse based on individual biomarker data. Vaccination (i.d.) with GM-CSF and poly-ICLC in 15 patients started with the 1(st) adjuvant cycle of temozolomide (TMZ). For APVAC2, analyses revealed a median of 36 somatic, non-synonymous mutations in the patients’ tumors. From the 4(th) TMZ cycle, 11 patients received APVAC2 with usually 2 de novo antigens per patient selected according to mutation, actual or putative HLA presentation and immunogenicity. Overall 20 APVAC2 antigens incl. 14 mutated were vaccinated. RESULTS: Adverse events were largely reversible injection site reactions and two anaphylactic reactions and one increase in cerebral edema. Short, non-mutated APVAC1 antigens induced sustained CD8 responses with memory phenotype. Mutated APVAC2 antigens induced predominantly CD4 responses of favorable TH1 type. Median PFS and OS were 14.2 and 29 months from diagnosis, respectively, in patients that received ≥1 APVAC vaccination (N=15). CONCLUSION: Overall, GAPVAC displayed expected safety profiles and high biological activity indicating further development.

T-cell receptor (TCR)-based immunotherapy has emerged as a promising treatment modality for malignant diseases. TCRs naturally recognize human leucocyte antigen (HLA)-bound peptides from foreign and endogenous antigens regardless of their source proteins' extracellular or intracellular location. Using its proprietary discovery engine XPRESIDENT®, Immatics can identify, quantify, and validate Tumor-Associated Peptides (TUMAPs) exclusively presented in cancer tissues. Immatics has established state-of-the-art technology to discover and affinity maturate TUMAP-specific TCRs originating from human T-cell repertoire. The maturated single-chain TCRs (scTv) are used to build a pipeline of highly potent T-cell engaging bispecific TCR molecules directed against TUMAPs. In brief, we use artificial antigen-presenting cells to selectively expand TUMAP-specific T-cells from which the coding TCR sequence is retrieved by 5'RACE after highly sensitive flow cytometry-based single cell sorting. About 50-150 TCRs per TUMAP are transiently re-expressed on human T-cells and extensively characterized for their functional properties. TCRs exhibiting highly active and specific TUMAP recognition are selected for yeast surface display to generate stabilized and affinity maturated scTv. The maturated scTv are engineered into Immatics' proprietary bispecific TCR scaffold comprising a humanized T-cell recruiting antibody domain for potent redirection and activation of T-cells against TUMAPs and an effector function-silenced IgG1 Fc domain. Here we present data of our bispecific TCR program targeting the TUMAP Ag008-01 bound to HLA-A*02. We confirmed the abundant presence of Ag008-01 in several cancer indications and its absence in human normal tissues by using XPRESIDENT® technology combining quantitative mass spectrometry, transcriptomics and bioinformatics. Based on the parental TCR showing highly active and specific recognition of Ag008-01, we generated stabilized and affinity maturated scTv variants with significant improvement in binding affinity towards Ag008-01 in the range of 1000-fold and higher. The maturated scTv variants showed no or minimal binding to off-target peptides selected from the XPRESIDENT® normal tissue database based on the criteria of highest sequence similarity to Ag008-01. By incorporating the maturated scTv into our proprietary bispecific TCR format, which outperformed five alternative TCR bispecific format designs, we obtained highly potent bispecific TCR molecules with picomolar activity. We observed half-maximal lysis of Ag008-01 expressing tumor cell lines at TCR bispecific concentrations below 100 picomolar while no reactivity was observed towards a panel of cell lines lacking Ag008-01 expression. Our data support proof-of-concept for the design of our novel class of T-cell engaging bispecific TCR-antibody fusion proteins. Citation Format: Sebastian Bunk, Martin Hofmann, Felix Unverdorben, Leonie Alten, Meike Hutt, Claudia Wagner, Oliver Schoor, Mathias Ferber, Jens Fritsche, Toni Weinschenk, Harpreet Singh-Jasuja, Dominik Maurer, Carsten Reinhardt. Development of highly potent T-cell receptor bispecifics with picomolar activity against tumor-specific HLA ligands [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 2789.

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 Immatics' 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 patient's 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 Hörzer, 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.

The tyrosine kinase inhibitors sorafenib and sunitinib are approved for the treatment of patients with malignant diseases. To analyze the possible use of these compounds in combination with immunotherapeutic approaches, we analyzed the effects of both inhibitors on the immunostimulatory capacity of human dendritic cells (DCs) and the induction of primary immune responses in vivo. Sorafenib, but not sunitinib, inhibits function of DCs, characterized by reduced secretion of cytokines and expression of CD1a, major histocompatibility complex, and costimulatory molecules in response to TLR ligands as well as by their impaired ability to migrate and stimulate T-cell responses. These inhibitory effects are mediated by inhibition of PI3 and MAP kinases and NFκB signaling. In contrast, sorafenib had no influence on the phenotype and proliferation of T cells. To analyze the effects of both TKIs on cytotoxic T-cell induction in vivo, C57BL/6 mice were pretreated with sorafenib or sunitinib and immunized with OVA257-264 peptide. Sorafenib, but not sunitinib, application significantly reduced the induction of antigen-specific T cells. Numbers of regulatory T cells were reduced in peripheral blood mononuclear cells from mice treated with sunitinib. These results indicate that sunitinib, but not sorafenib, is suitable for combination with immunotherapeutic approaches for treatment of cancer patients.

Human cytomegalovirus (HCMV) has evolved a multitude of molecular mechanisms to evade the antiviral immune defense of the host. Recently, using soluble recombinant molecules, the HCMV UL16 glycoprotein was shown to interact with some ligands of the activating immunoreceptor NKG2D and, therefore, may also function as a viral immunomodulator. However, the role of UL16 during the course of HCMV infection remained unclear. Here, we demonstrate that HCMV infection of fibroblasts induces expression of all known NKG2D ligands (NKG2DL). However, solely MICA and ULBP3 reach the cellular surface to engage NKG2D, whereas MICB, ULBP1 and ULBP2 are selectively retained in the endoplasmic reticulum by UL16. UL16-mediated reduction of NKG2DL cell surface density diminished NK cytotoxicity. Thus, UL16 functions by capturing activating ligands for cytotoxic lymphocytes that are synthesized in response to HCMV infection.

Among other heat shock proteins (HSPs), the ER-resident chaperone Gp96 has been described as a potent tumour vaccine in animal models. A growing list of data underlines that Gp96 triggers both arms of pathogen defence-innate and specific immunity-in a synergistic and most efficient way: It enables specific immune responses by transferring immunogenic peptides that have been acquired in the ER to the MHC class I pathway of antigen presenting cells (APCs). For this, two important features of Gp96 are required. First, its ability to bind immunogenic peptides. Secondly, its acquisition by specialized antigen presenting cells capable of inducing cellular immune responses. Due to specific receptors on the surface of APCs, this uptake from the extracellular space occurs very efficiently and rapidly. Serving the innate branch of immunity, Gp96 unspecifically activates APCs, which then provide a pro-inflammatory cytokine milieu and co-stimulation to cytotoxic T cells. Thus, Gp96 uses all resources of the immune system to trigger cytotoxic T cell responses against associated peptides.

The term cancer immunotherapy covers a huge range of different therapeutic interventions based on highly diverse medicinal products. Some immunotherapy products such as monoclonal antibodies have successfully been developed during the last years and contributed to major advances in cancer therapy. Development of cancer vaccines seemed to be more challenging. Nevertheless, some promising candidates are currently in late-stage clinical development, and one product (sipuleucel-T) was recently approved in the United States (USA) and in the European Union (EU). Additionally, cell therapy approaches based on adoptive lymphocyte transfer have recently come into focus due to surprisingly efficient antitumor effects on one side and a comparably good safety profile on the other. Here, we briefly summarize the regulatory classification of immunotherapy approaches. The second part focuses on the evolving regulatory landscape for nonpersonalized and personalized cancer vaccines that have been in the focus of the CIMT regulatory research group during the last years.

Renal cell carcinoma (RCC) is the adenocarcinoma of the kidney and the most common form of kidney cancer. More than 300,000 people are newly affected by RCC every year globally (Ferlay et al. 2012). The most common subtype of RCC is clear-cell RCC which comprises about 75 % of RCCs. For two decades, cytokine-based immunotherapy was the standard of care for the management of RCC. In the last 10 years, a better understanding of the tumor biology of RCC has led to the approval of a number of novel agents targeting angiogenesis and has led to an improvement of patient outcomes. However, almost all patients eventually develop a resistance to antiangiogenic agents, and the extension of overall survival (by each individual agent on its own) has remained relatively modest. These observations as well as recent remarkable developments in the field of cancer immunotherapy have strongly resurged the interest in immunotherapeutic approaches for treatment of RCC. This chapter summarizes the recent developments in the treatment of RCC through immunotherapies approved or in clinical development as well as the immunological role of targeted therapies.

Peptides associated with the heat shock protein gp96 induce a specific T cell response against cells from which gp96 is isolated. Recently, we have shown that gp96 binds to a yet unknown receptor present on dendritic cells (DC) and that receptor-mediated uptake is required for cross-presentation of gp96-associated peptides by DC. We now describe that gp96 mediates maturation of DC as determined by up-regulation of MHC class II and CD86 molecules, secretion of the cytokines IL-12 and TNF-alpha and enhanced T cell stimulatory capacity. Heat-denatured gp96 is not able to induce DC maturation and cytokine secretion. Furthermore, we show that mature DC are no longer able to bind gp96 molecules. Hence, the gp96 receptor is down-regulated on mature DC, suggesting that this receptor behaves similar to other receptors involved in antigen uptake like the scavenger receptor CD36, the mannose receptor or the integrins alpha(v)beta(3) and alpha(v)beta(5). Together, our findings provide an additional explanation for the remarkable immunogenicity of gp96 as a cross-priming antigen carrier and direct activator of DC.

Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated protein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic cells (DCs), macrophages, and B cells, but not T cells, are able to bind gp96. The binding is saturable and can be inhibited using unlabeled gp96 molecules. Receptor binding by APCs leads to a rapid internalization of gp96, which colocalizes with endocytosed major histocompatibility complex (MHC) class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an adenovirus type 5 E1B epitope with the DC line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I–restricted re-presentation of gp96-associated peptides and CTL activation; non–receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the cross-presentation of antigens from cellular origin.

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.

The HEPAVAC Consortium aims to develop a highly innovative, novel cancer vaccine approach for hepatocellular carcinoma (HCC). The international project consortium consists of 9 European Partners from academia and the biotech industry with complementary and substantial expertise in developing immunotherapeutic strategies to treat cancer. The project has started in September 2013 and is supported by the European Commission's 7th Framework Program (www.hepavac.eu). HCC/normal adjacent tissue matched samples have been collected for HLA immunopeptidome analysis. 17 HCC samples from HLA-A*02+ patients and 15 samples from HLA-A*24+ patients have been analysed by mass spectrometry (LC-MS/MS). RNA-expression profiles have been established for 12 HCC samples. HLA-presentation/expression of peptides and mRNA on primary HCC samples are compared to >140 normal tissue samples from relevant organs (including heart, brain, lung, kidney, liver, nerve, skin etc.) from Immatics’ database. A total of 9051 HLA-A*02-restricted different tumor-associated peptides (TUMAPs) have been identified from HLA-A*02+ samples, while a total of 3286 different HLA-A*24-restricted TUMAPs have been identified from HLA-A*24+ samples. Of these, 33 HLA-A*02+ TUMAPS and 33 HLA-A*24+ TUMAPs have been validated, including peptide synthesis, immunogenicity testing and pharmaceutical evaluation. Most promising TUMAP candidates show selective expression only in HCC samples and no expression in normal tissues. In parallel, more than 6600 HLA-DR TUMAPs have been identified in Hep3B cells transfected with CIITA as well as an average of 1500 HLA-DR TUMAPs have been identified in HCC samples. A total of 16 newly identified HCC-specific epitopes have been selected for the HEPAVAC vaccine cocktail and are currently synthesized according to GMP standard. Of these, 7 are restricted to HLA- class I A*02; 5 HLA-A*24 and 4 HLA class II. In parallel, preclinical studies assessing the formulation and combination of the immunological RNA-based adjuvant (RNAdjuvant®) with peptide cocktails are underway. A multi Center phase I/II clinical trial in early HCC patients is predicted to start in July 2016. Citation Format: Sarah Kutscher, Roberto Accolla, Yuk T. Ma, Regina Heidenreich, Francesco Izzo, Alfred Koenigsrainer, Markus Loeffler, Phillip Mueller, Andrea Mayer, Hans-Georg Rammensee, Bruno Sangro, Sven Francque, Danila Valmori, Toni Weinschenk, Harpreet Singh-Jasuja, Luigi Buonaguro. Cancer vaccine development for hepatocellular carcinoma - HEPAVAC. [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 2354.

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, Valérie Dutoit, Hideho Okada, Christian Ottensmeier, Randi Kristina Feist, Jens Fritsche, Karoline Laske, Peter Lewandrowski, Martin Löwer, Regina Mendryzk, Miriam Meyer, Carsten Reinhardt, Bernhard Rössler, 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.

The HEPAVAC Consortium aims to develop a highly innovative, novel cancer vaccine approach for hepatocellular carcinoma (HCC). The international project consortium consists of 9 European Partners from academia and the biotech industry with complementary and substantial expertise in developing immunotherapeutic strategies to treat cancer. The project has started in September 2013 and is supported by the European Commission's 7th Framework Program (www.hepavac.eu). HCC/normal adjacent tissue matched samples have been collected for HLA immunopeptidome analysis. 17 HCC samples from HLA-A*02+ patients and 15 samples from HLA-A*24+ patients have been analysed by mass spectrometry (LC-MS/MS). RNA-expression profiles have been established for 12 HCC samples. HLA-presentation/expression of peptides and mRNA on primary HCC samples are compared to n>140 normal tissue samples from relevant organs (including heart, brain, lung, kidney, liver, nerve, skin etc.) from Immatics' database. A total of 9051 HLA-A*02-restricted different tumor-associated peptides (TUMAPs) have been identified from HLA-A*02+ samples, while a total of 3286 different HLA-A*24-restricted TUMAPs have been identified from HLA-A*24+ samples. Of these, 33 HLA-A*02+ TUMAPS and 33 HLA-A*24+ TUMAPs are currently in the process of validation, including peptide synthesis, immunogenicity testing and pharmaceutical evaluation. Most promising TUMAP candidates show selective expression only in HCC samples and no expression in normal tissues. In parallel, more than 6600 HLA-DR TUMAPs have been identified in Hep3B cells transfected with CIITA as well as an average of 1500 HLA-DR TUMAPs have been identified in HCC samples. Additional HLA-DR TUMAPs derived by distinct CIITA-transfected cell lines with different HLA-DR haplotypes are under scrutiny. The discovery phase of the HEPAVAC project is proceeding according to the proposed timelines. HCC-specific epitopes to be included in the vaccine cocktail will be selected in the coming weeks for GMP production. In parallel, preclinical studies assessing the formulation and combination of the immunological RNA-based adjuvant (RNAdjuvant®) with peptide cocktails are underway. Citation Format: Luigi Buonaguro, Sarah Kutscher, Roberto Accolla, Yuk T. Ma, Regina Heidenreich, Francesco Izzo, Alfred Koenigsrainer, Markus Loeffler, Phillip Mueller, Andrea Mayer, Hans-Georg Rammensee, Bruno Sangro, Sven Francque, Danila Valmori, Toni Weinschenk, Harpreet Singh-Jasuja. Cancer vaccine development for hepatocellular carcinoma – HEPAVAC. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A115.

Dendritic cells (DC) play a key role in regulating immune responses and are the best professional antigen-presenting cells. Two major DC populations are defined in part according to cell surface CD11c expression levels. Unexpectedly, we observed that mouse DC strongly down-regulate the typical DC marker CD11c upon activation. To better characterize DC responses, we have analyzed CD11c expression on mouse and human myeloid DC after Toll-like receptor (TLR) triggering. Here we show that mouse bone marrow-derived DC (BMDC) as well as spleen DC down-regulate cell surface CD11c upon activation by TLR3/4/9 agonists. In all cases, full DC activation was reached, as determined by cytokine secretion, cell stimulation in mixed leukocyte reactions (MLR), and CD40/CD86/major histocompatibility complex (MHC) up-regulation. Interestingly, membrane CD11c down-regulation correlated with increased cytoplasmic pools of CD11c. In contrast to the up-regulation of CD40 and MHC class II molecules, lipopolysaccharide (LPS)-induced CD11c down-regulation was MyD88-dependent. Polyinosinic-polycytidylic acid (poly I:C), which does not signal through MyD88, also induced cell surface CD11c down-regulation. Notably, CD11c down-regulation was not observed upon activation of human DC, either through TLR-dependent or -independent cell activation. Thus, activated mouse DC may be transiently CD11c-negative in vivo, hampering the identification of those cells. On the other hand, cell surface CD11c down-regulation may serve as a new activation marker for mouse DC.

Heat shock proteins (Hsps) are able to induce protective immune responses against pathogens and tumors after injection into immunocompetent hosts. The activation of components of the adaptive immune system, including cytotoxic T lymphocytes specific for pathogen- or tumor-derived peptides, is crucial for the establishment of immuno- protection. Hsps acquire these peptides during intracellular protein degradation and when released during necrotic cell death, facilitate their uptake and Minor Histocompatibility Complex (MHC)-restricted representation by professional antigen-presenting cells (APCs). In addition, the interaction of Hsps with APCs, including the Endoplasmatic Reticulum (ER)-resident chaperone glycoprotein 96 (Gp96), induces the maturation of these cells by Toll-like receptor (TLR)- mediated signaling events. We now provide evidence that in contrast to lipopolysaccharides (LPS)-mediated dendritic cell (DC) maturation, the interaction of Gp96 with DCs leads to the preferential expansion of antigen-specific CD8- positive T cells in vitro and in vivo. This CD8 preference induced by mouse and human DCs did not correlate with enhanced levels of interleukin-12 secretion. Thus, despite the fact that both LPS and Gp96 activate DCs in a TLR4- dependent manner, the experiments of this study clearly demonstrate qualitative differences in the outcome of this maturation process, which preferentially favors the expansion of CD8-positive T cells.

Nucleosome is the major autoantigen in systemic lupus erythematosus. It is found as a circulating complex in the sera of patients and seems to play a key role in disease development. In this study, we show for the first time that physiologic concentrations of purified nucleosomes directly induce in vitro dendritic cell (DC) maturation of mouse bone marrow-derived DC, human monocyte-derived DC (MDDC), and purified human myeloid DC as observed by stimulation of allogenic cells in MLR, cytokine secretion, and CD86 up-regulation. Importantly, nucleosomes act as free complexes without the need for immune complex formation or for the presence of unmethylated CpG DNA motifs, and we thus identified a new mechanism of DC activation by nucleosomes. We have clearly demonstrated that this activation is nucleosome-specific and endotoxin-independent. Particularly, nucleosomes induce MDDC to secrete cytokines known to be detected in high concentrations in the sera of patients. Moreover, activated MDDC secrete IL-8, a neutrophil chemoattractant also detected in patient sera, and thus might favor the inflammation observed in patients. Both normal and lupus MDDC are sensitive to nucleosome-induced activation. Finally, injection of purified nucleosomes to normal mice induces in vivo DC maturation. Altogether, these results strengthen the key role of nucleosomes in systemic lupus erythematosus and might explain how peripheral tolerance is broken in patients.

There is substantial need for molecularly defined tumor antigens to prime cytotoxic T cells in vivo for cancer immunotherapy, especially in the case of tumor entities for which only a few tumor antigens have been defined so far. In this review, we present the “Tubingen approach” to identify, select, and validate large numbers of MHC/HLA class I–associated peptides derived from tumor-associated antigens. Step 1 is the identification of naturally presented HLA-associated peptides directly from primary tumor cells. Step 2 is selection of tumor-associated peptides from step 1 by differential gene expression analysis and data mining. Step 3 is validation of selected candidates by monitoring in vivo T-cell responses in the context of patient-individualized immunizations. Our approach combines methods from genomics, proteomics, bioinformatics, and T-cell immunology. The aim is to develop effective immunotherapeutics consisting of multiple tumor-associated epitopes in order to induce a broad and specific immune response against cancer cells.

The endoplasmic reticulum (ER)–resident heat shock protein Gp96 is involved in protein folding and is released into the extracellular space after necrotic cell death. In this context, Gp96 has immunostimulatory properties: it activates dendritic cells or macrophages and delivers associated peptides into the antigen presentation pathway, resulting in the induction of specific T-cell responses. The inflammatory response after necrotic tissue damage leads to the recruitment of polymorphonuclear neutrophils (PMNs) and monocytes, allowing them to make their first encounter with Gp96. We therefore investigated whether PMNs and monocytes interact with Gp96. We were able to show that PMNs and monocytes specifically bind fluorescein isothiocyanate (FITC)–conjugated Gp96. The binding of Gp96-FITC was competed by lipopolysaccharide (LPS) or fucoidan, a known inhibitor of scavenger receptors. Interestingly, the binding of LPS-FITC was also competed not only by fucoidan, but by Gp96, suggesting that LPS and Gp96 share a common receptor on PMNs. One important effector function of PMNs is the clearance of an inflammatory site by phagocytosis. We therefore assessed the influence of Gp96 on phagocytic activity using fluorochrome-labeled polystyrene beads. We found a marked enhancement of phagocytosis in the presence of Gp96 and concluded that PMNs not only bind Gp96, but are also activated by it. Additionally, Gp96-stimulated PMNs and especially monocytes release large amounts of interleukin-8, a potent neutrophil-attracting chemokine. In conclusion, we demonstrate that Gp96 specifically binds to and activates PMNs and monocytes, extending the function of Gp96 as a danger signal to additional members of the innate immune system.

Immunizations using the endoplasmic reticulum–resident heat shock protein Gp96 induce specific immune responses. Specificity is based on the major histocompatibility complex class I–restricted cross-presentation of Gp96-associated peptides derived from endogenous proteins. Initiation of the immune response depends on the ability of Gp96 to induce the production of proinflammatory cytokines by macrophages and dendritic cells (DCs) and of their maturation in a fashion presumably independent of associated peptide. Both events are mediated by Gp96 receptors on antigen-presenting cells. It is known that Gp96 is released from cells at necrosis induced, for example, by virus infection. Although this event supports the efficient induction of immune responses, it might also interfere with processes that are susceptible to chronic inflammation, such as wound healing after tissue damage. Therefore, Gp96-mediated stimulation of the immune system requires tight regulation. Here we show that human thrombocytes specifically interact with Gp96 and that binding of Gp96 to platelets is enhanced more than 10-fold on activation by thrombin. Gp96 interferes with neither thrombin-induced platelet activation nor platelet aggregation. However, the presence of platelets during Gp96-mediated DC activation reduces the secretion of proinflammatory cytokines and the activation of DCs. This effect is independent of soluble platelet factors and cell-to-cell contact between DCs and thrombocytes. Thus, we provide evidence for a regulatory mechanism that neutralizes Gp96 molecules systemically, especially in the blood. This effect might be of significance in wounds in which chronic inflammation and immune responses against autoantigens have to be prevented.

The heat shock protein Gp96 has been shown to induce specific immune responses. On one hand, this phenomenon is based on the specific interaction with CD91 that mediates endocytosis and results in major histocompatibility complex class I-restricted representation of the Gp96-associated peptides. On the other hand, Gp96 induces activation of professional antigen-presenting cells, resulting in the production of pro-inflammatory cytokines and up-regulation of costimulatory molecules by unknown mechanisms. In this study, we have analyzed the consequences of Gp96 interaction with cells expressing different Toll-like receptors (TLRs) and with bone marrow-derived dendritic cells from mice lacking functional TLR2 and/or TLR4 molecules. We find that the Gp96-TLR2/4 interaction results in activation of nuclear factor kappaB-driven reporter genes and mitogen- and stress-activated protein kinases and induces IkappaBalpha degradation. Bone marrow-derived dendritic cells of C3H/HeJ and more pronounced C3H/HeJ/TLR2(-/-) mice fail to respond to Gp96. Interestingly, activation of bone marrow-derived dendritic cells depends on endocytosis of Gp96 molecules. Our results provide, for the first time, the molecular basis for understanding the Gp96-mediated activation of antigen-presenting cells by describing the simultaneous stimulation of the innate and adaptive immune system. This feature explains the remarkable ability of Gp96 to induce specific immune responses against tumors and pathogens.

Heat shock proteins (HSPs) have been described as potent tumor vaccines in animal models and are currently studied in clinical trials. The underlying immune response relies on immunogenic peptides that the HSPs have acquired intracellularly by interfering with the classical antigen processing pathways. There have been numerous reports shedding light on how HSPs are able to gain this function and a number of important requirements for HSP-mediated specific immunity have been described: first, the ability of HSPs to bind immunogenic peptides. Second, the acquisition of HSPs by specialized antigen presenting cells with efficient antigen processing pathways capable of inducing cellular immune responses. Third, the existence of specific receptors on the surfaces of antigen presenting cells, allowing efficient and rapid uptake of HSP-peptide complexes from the extracellular fluid. And fourth, the ability of heat shock proteins to activate antigen presenting cells, enabling the latter to prime cytotoxic T cell responses against the peptides associated to HSPs.

Combining immunotherapeutic approaches with targeted therapies like tyrosine-kinase inhibitors (TKIs) may give additional benefits to patients with VEGF-driven tumors (e.g. RCC). Previously, we have investigated the impact of first-generation TKIs sorafenib and sunitinib demonstrating distinct, in part opposing effects on immune cells (Hipp et al, Blood 2008, 111(12): 5610ff). Meanwhile, second-generation TKIs (pazopanib, axitinib, tivozanib) were approved or are expected to receive approval soon. In contrast to the multi-targeting first-generation TKIs, these TKIs act more selectively, therefore potentially inducing less off-target toxicities. However, so far little is known about the immunomodulatory properties of these new TKIs. Thus, we compared the influence of two first- and three second-generation TKIs on frequency and function of immune cells and on immune responses in mice. C57BL/6 mice were orally treated with vehicle, 25 mg/kg axitinib, 20 mg/kg tivozanib, 30 mg/kg pazopanib, 30 mg/kg sorafenib or 40 mg/kg sunitinib (daily for one week). Twelve hours after the last application, splenocytes were analyzed for CD4+ CD25+ FoxP3+ regulatory T cells (Tregs). Tivozanib and (as published before) sunitinib significantly reduced the frequency of Tregs among CD4+ cells, while no effect was observed for axitinib, pazopanib and sorafenib. Moreover, percentages of CD4+ as well as CD8+ T cells were increased in tivozanib- and sunitinib-treated mice. The functional status of isolated immune cells from TKI pre-treated mice was assessed in a mixed lymphocyte reaction (MLR). CD4+ T-cell proliferation of cells isolated from sorafenib-treated mice was diminished, while CD8+ T-cell proliferation of cells from sunitinib-treated mice was increased. In vitro activation assays revealed that - irrespective of the used TKI - the treatment led to reduced responsiveness of immune cells in terms of cytokine secretion and expression of surface activation markers. Finally, C57BL/6 mice were immunized subcutaneously twice (days 1 and 8) with ovalbumin peptide vaccine alone (negative control) or combined with poly-IC (positive control). To analyze the effect of TKIs in vivo, mice were pretreated (day -7 to -3) or concurrently treated (day 1 - 13) with axitinib, tivozanib or pazopanib in addition to vaccinations with peptide plus poly-IC. On day 15, induction of peptide-specific T cells was analyzed by MHC multimer staining as well as by IFN-γ ELISPOT. Pretreatment or concurrent treatment with all three second-generation TKIs did not negatively impact the induction of immune responses - in contrast to concurrent treatment with sorafenib reported before. In conclusion, these results suggest that immunotherapy and second-generation TKIs can be combined in the treatment of cancer, but the influence of these TKIs on the immune system requires further assessment in clinical trials. Citation Format: Nina Pawlowski, Helen Hoerzer, Harpreet Singh-Jasuja, Norbert Hilf. Impact of various first- and second-generation tyrosine-kinase inhibitors on frequency and functionality of immune cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3971. doi:10.1158/1538-7445.AM2013-3971

Translational research and the clinical development of therapeutic cancer vaccines require strong scientific rationale in order to better understand the mode-of-action and to incorporate the lessons learned in the optimization of cancer vaccines by (i) selecting appropriate immunomodulators to generate more effective vaccine regimens, (ii) combining vaccine regimens and standard-of-care therapeutics with synergistic potential and (iii) identifying patient populations that may have a better chance of responding to the vaccine. Here we demonstrate how preclinical and clinical immune response markers combined with cellular and serum biomarkers that define the immune regulatory environment were utilized as guiding tools in the development of three novel multi-peptide vaccines in renal cell cancer (IMA901), colorectal cancer (IMA910) and glioblastoma (IMA950). All three vaccine products comprise multiple tumor-associated peptides (TUMAPs) confirmed, by mass spectrometry, to be naturally presented in human cancer tissue. Each vaccine was selected using differential transcriptomics approaches and validated by in vitro analysis of immunogenicity with artificial antigen-presenting cells followed by in vivo analysis of immune responses in clinical trials. The vaccines contain HLA class I and II-restricted peptides designed to elicit CD8+ cytotoxic and CD4+ helper T-cell responses, respectively. IMA901 is a novel renal cell cancer (RCC) vaccine consisting of 9 HLA class I and 1 HLA class II TUMAPs with the majority of peptides confirmed to be naturally presented on and shared between RCC tissues. Consecutive independent Phase I and II clinical studies were conducted in HLA-A*02+ advanced/metastatic RCC patients (total 96) receiving repeated intradermal vaccinations with IMA901 plus human granulocyte-macrophage colony-stimulating factor (GM-CSF). The two studies demonstrated an association between overall survival and multiple vaccine-induced T-cell responses and an impact of single-dose cyclophosphamide (300 mg/m2) on regulatory T cells (Tregs) and overall survival. Furthermore, two myeloid-derived suppressor cell (MDSC) populations (CD14+ HLA-DR−/low and CD14− CD11b+ CD15+) were significantly negatively associated with survival in vaccinated RCC patients. The knowledge acquired in these trials was used to design a randomized, controlled Phase III study. Recruitment of 339 advanced/metastatic first-line RCC patients was recently completed. IMA901 is combined with the tyrosine kinase inhibitor sunitinib based on the findings that sunitinib down-modulates the two MDSC populations described above. Furthermore, in this Phase III study, the relevance of two serum biomarkers (Apoliprotein A1 and CCL17) found in the Phase II study to be associated with immune response and OS will be prospectively explored (Walter, Weinschenk et al., Nat Med 2012). IMA910 is a novel colorectal cancer (CRC) vaccine consisting of 10 HLA class I and 3 HLA class II TUMAPs with the majority of peptides confirmed to be naturally presented on and shared between CRC tissues. A Phase I/II trial was conducted in 92 HLA-A*02+ advanced colorectal cancer patients with stable (SD) or responding (PR/CR) disease after 12 weeks of first-line oxaliplatin-based therapy. Thereafter, first-line oxaliplatin-based chemotherapy was stopped and patients received a single dose of cyclophosphamide to reduce Tregs followed by repeated intradermal vaccinations with IMA910 plus GM-CSF (first cohort; n=66) with or without topically applied imiquimod (second cohort; n=26). IMA910 elicited immune responses towards multiple class I (in 43% of the subjects) and class II TUMAPs (65%). Similarly to the IMA901 vaccine trial in RCC, such vaccine-induced CD8+ as well as CD4+ T-cell responses to multiple TUMAPs were associated with increased overall survival. Patients who additionally received imiquimod were more often multi-peptide class I responders (p=0.016) as determined by intracellular cytokine staining (ICS) assay and showed a modestly (two-fold) increase of T-cell freqencies (p=0.12) as determined by HLA multimer assay. Interestingly, the same two populations of MDSC as described above in renal cell cancer patients were negatively associated with vaccine-induced immune response and overall survival implying a potential generalized role of these two MDSC populations. Further development of this vaccine is planned in a setting where an MDSC-modulating standard-of-care (e.g. chemotherapy) is applied. IMA950 is a novel glioblastoma vaccine consisting of 9 HLA class I and 2 HLA class II TUMAPs with the majority of peptides confirmed to be naturally presented on and shared between glioblastoma tissues. A Phase I study sponsored by Cancer Research UK is currently ongoing in the United Kingdom. A total of 45 newly diagnosed glioblastoma patients will be treated. The study is expected to complete recruitment in the first quarter of 2013. The primary objectives of this first-in-man study are to assess the safety, tolerability and immunogenicity of IMA950 plus GM-CSF when given alongside standard chemoradiotherapy followed by adjuvant chemotherapy with temozolomide. Patients enter the trial into one of two cohorts (start of vaccination either prior to or after initial chemoradiotherapy) in order to determine potential differences in the immune response in these two cohorts. First immunomonitoring data indicate a high immune as well as multi-TUMAP response rate in the study population. Updated and unpublished immune data will be presented at the meeting. Analysis of the in vitro immunogenicity data obtained from healthy donors during the discovery phases of these programs and comparison of these data with the in vivo immunogenicity data obtained in the clinical trials demonstrated that in all four clinical trials in a total of approx. 200 patients the in vitro data correlated with the in vivo data. This implies that such a standardized in vitro assay system may be utilized in future to further optimize vaccine regimens by selecting the most immunogenic novel peptides before entering clinical trials. In conclusion, we suggest that use of preclinically well characterized naturally presented tumor antigens combined with systematic clinical immunomonitoring and cellular/serum biomarker screening will be helpful to guide rational development of novel cancer vaccines. Citation Format: Harpreet Singh-Jasuja, Steffen Walter, Toni Weinschenk, Alexandra Kirner, Andrea Mayer-Mokler, Norbert Hilf, Oliver Schoor, Pierre-Yves Dietrich, James Richtie, Roy Rampling, Frank Mayer, Arnulf Stenzl, Brian Rini, Carsten Reinhardt, Hans-Georg Rammensee. Biomarker-guided development of novel multi-peptide cancer vaccines - from discovery to phase lll trials. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY27-03. doi:10.1158/1538-7445.AM2013-SY27-03