Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer.

T cells directed against mutant neo-epitopes drive cancer immunity. However, spontaneous immune recognition of mutations is inefficient. We recently introduced the concept of individualized mutanome vaccines and implemented an RNA-based poly-neo-epitope approach to mobilize immunity against a spectrum of cancer mutations. Here we report the first-in-human application of this concept in melanoma. We set up a process comprising comprehensive identification of individual mutations, computational prediction of neo-epitopes, and design and manufacturing of a vaccine unique for each patient. All patients developed T cell responses against multiple vaccine neo-epitopes at up to high single-digit percentages. Vaccine-induced T cell infiltration and neo-epitope-specific killing of autologous tumour cells were shown in post-vaccination resected metastases from two patients. The cumulative rate of metastatic events was highly significantly reduced after the start of vaccination, resulting in a sustained progression-free survival. Two of the five patients with metastatic disease experienced vaccine-related objective responses. One of these patients had a late relapse owing to outgrowth of ß2-microglobulin-deficient melanoma cells as an acquired resistance mechanism. A third patient developed a complete response to vaccination in combination with PD-1 blockade therapy. Our study demonstrates that individual mutations can be exploited, thereby opening a path to personalized immunotherapy for patients with cancer.

Generation of TCR-engineered T cells and their use to control the performance of T cell assays.

The systematic assessment of the human immune system bears huge potential to guide rational development of novel immunotherapies and clinical decision making. Multiple assays to monitor the quantity, phenotype, and function of Ag-specific T cells are commonly used to unravel patients' immune signatures in various disease settings and during therapeutic interventions. When compared with tests measuring soluble analytes, cellular immune assays have a higher variation, which is a major technical factor limiting their broad adoption in clinical immunology. The key solution may arise from continuous control of assay performance using TCR-engineered reference samples. We developed a simple, stable, robust, and scalable technology to generate reference samples that contain defined numbers of functional Ag-specific T cells. First, we show that RNA-engineered lymphocytes, equipped with selected TCRs, can repetitively deliver functional readouts of a controlled size across multiple assay platforms. We further describe a concept for the application of TCR-engineered reference samples to keep assay performance within or across institutions under tight control. Finally, we provide evidence that these novel control reagents can sensitively detect assay variation resulting from typical sources of error, such as low cell quality, loss of reagent stability, suboptimal hardware settings, or inaccurate gating.

Luciferase mRNA Transfection of Antigen Presenting Cells Permits Sensitive Nonradioactive Measurement of Cellular and Humoral Cytotoxicity.

Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.

Targeting Carcinoembryonic Antigen with DNA Vaccination: On-Target Adverse Events Link with Immunologic and Clinical Outcomes.

PURPOSE: We have clinically evaluated a DNA fusion vaccine to target the HLA-A*0201-binding peptide CAP-1 from carcinoembryonic antigen (CEA605-613) linked to an immunostimulatory domain (DOM) from fragment C of tetanus toxin. EXPERIMENTAL DESIGN: Twenty-seven patients with CEA-expressing carcinomas were recruited: 15 patients with measurable disease (arm-I) and 12 patients without radiological evidence of disease (arm-II). Six intramuscular vaccinations of naked DNA (1 mg/dose) were administered up to week 12. Clinical and immunologic follow-up was up to week 64 or clinical/radiological disease. RESULTS: DOM-specific immune responses demonstrated successful vaccine delivery. All patients without measurable disease compared with 60% with advanced disease responded immunologically, while 58% and 20% expanded anti-CAP-1 CD8+ T cells, respectively. CAP-1-specific T cells were only detectable in the blood postvaccination but could also be identified in previously resected cancer tissue. The gastrointestinal adverse event diarrhea was reported by 48% of patients and linked to more frequent decreases in CEA (P < 0.001) and improved global immunologic responses [anti-DOM responses of greater magnitude (P < 0.001), frequency (P = 0.004), and duration] compared with patients without diarrhea. In advanced disease patients, decreases in CEA were associated with better overall survival (HR = 0.14, P = 0.017). CAP-1 peptide was detectable on MHC class I of normal bowel mucosa and primary colorectal cancer tissue by mass spectrometry, offering a mechanistic explanation for diarrhea through CD8+ T-cell attack. CONCLUSIONS: Our data suggest that DNA vaccination is able to overcome peripheral tolerance in normal and tumor tissue and warrants testing in combination studies, for example, by vaccinating in parallel to treatment with an anti-PD1 antibody. Clin Cancer Res; 22(19); 4827-36. ©2016 AACR.

Retrieval of functional TCRs from single antigen-specific T cells: Toward individualized TCR-engineered therapies.

We have developed a highly versatile platform for the systematic retrieval of T-cell receptors (TCRs) from single-antigen-reactive T cells and for characterization of their function and specificity. This approach enables rapid extraction of multiple TCRs from repertoires in individuals and not only broadens the diversity of TCRs suitable for clinical use, but also sets the stage for actively personalized immunotherapeutic strategies.

Humoral immune responses of lung cancer patients against the Transmembrane Phosphatase with TEnsin homology (TPTE).

OBJECTIVE: The cancer/testis (C/T) antigen Transmembrane Phosphatase with TEnsin homology (TPTE) is aberrantly expressed in many tumors including lung cancer. In the present study, we analyzed TPTE-auto-antibodies in lung cancer patients. METHODS: Using a crude-lysate ELISA, we analyzed a large cohort of 307 sera from lung cancer patients and 47 healthy donors for TPTE-specific autoantibodies. Sero-reactivity was correlated with clinical parameters and patients' survival. RESULTS: TPTE-specific antibodies were detected in 41 of 307 (13.4%) sera from lung cancer patients. Based on an optimal cut-off value calculated by ROC curve analysis sensitivity for diagnosing lung cancer was 52% and specificity was 72%. TPTE sero-positivity was not associated with tumor stage, tumor histology, gender or age. Multivariate analysis indicated that TPTE sero-positivity is associated with prolonged survival in patients with lung cancer, but established prognostic factors for survival prediction such as stage and histology remain indispensable. CONCLUSION: Autoantibodies against TPTE occur spontaneously in lung cancer patients. TPTE sero-reactivity has moderate sensitivity and specificity for diagnosing lung cancer and is a positive prognostic marker.

CXorf61 is a target for T cell based immunotherapy of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a high medical need disease with limited treatment options. CD8+ T cell-mediated immunotherapy may represent an attractive approach to address TNBC. The objectives of this study were to assess the expression of CXorf61 in TNBCs and healthy tissues and to evaluate its capability to induce T cell responses. We show by transcriptional profiling of a broad comprehensive set of normal human tissue that CXorf61 expression is strictly restricted to testis. 53% of TNBC patients express this antigen in at least 30% of their tumor cells. In CXorf61-negative breast cancer cell lines CXorf61 expression is activated by treatment with the hypomethylating agent 5-aza-2'-deoxycytidine. By vaccination of HLA-A*02-transgenic mice with CXorf61 encoding RNA we obtained high frequencies of CXorf61-specific T cells. Cloning and characterization of T cell receptors (TCRs) from responding T cells resulted in the identification of the two HLA-A*0201-restricted T cell epitopes CXorf6166-74 and CXorf6179-87. Furthermore, by in vitro priming of human CD8+ T cells derived from a healthy donor recognizing CXorf6166-74 we were able to induce a strong antigen-specific immune response and clone a human TCR recognizing this epitope. In summary, our data confirms this antigen as promising target for T cell based therapies.

Functional TCR retrieval from single antigen-specific human T cells reveals multiple novel epitopes.

The determination of the epitope specificity of disease-associated T-cell responses is relevant for the development of biomarkers and targeted immunotherapies against cancer, autoimmune, and infectious diseases. The lack of known T-cell epitopes and corresponding T-cell receptors (TCR) for novel antigens hinders the efficient development and monitoring of new therapies. We developed an integrated approach for the systematic retrieval and functional characterization of TCRs from single antigen-reactive T cells that includes the identification of epitope specificity. This is accomplished through the rapid cloning of full-length TCR-α and TCR-ß chains directly from single antigen-specific CD8(+) or CD4(+) T lymphocytes. The functional validation of cloned TCRs is conducted using in vitro-transcribed RNA transfer for expression of TCRs in T cells and HLA molecules in antigen-presenting cells. This method avoids the work and bias associated with repetitive cycles of in vitro T-cell stimulation, and enables fast characterization of antigen-specific T-cell responses. We applied this strategy to viral and tumor-associated antigens (TAA), resulting in the retrieval of 56 unique functional antigen-specific TCRs from human CD8(+) and CD4(+) T cells (13 specific for CMV-pp65, 16 specific for the well-known TAA NY-ESO-1, and 27 for the novel TAA TPTE), which are directed against 39 different epitopes. The proof-of-concept studies with TAAs NY-ESO-1 and TPTE revealed multiple novel TCR specificities. Our approach enables the rational development of immunotherapy strategies by providing antigen-specific TCRs and immunogenic epitopes.

CIMT 2013: advancing targeted therapies--report on the 11th Annual Meeting of the Association for Cancer Immunotherapy, May 14-16 2013, Mainz, Germany.

The 11th Annual Meeting of Association for Cancer Immunotherapy (CIMT) welcomed more than 700 scientists around the world to Mainz, Germany and continued to be the largest immunotherapy meeting in Europe. Renowned speakers from various fields of cancer immunotherapy gave lectures under CIMT2013's tag: "Advancing targeted therapies" the highlights of which are summarized in this meeting report.

mRNA as a versatile tool for exogenous protein expression.

Several viral and non-viral vectors have been developed for exogenous protein expression in specific cells. Conventionally, this purpose is achieved through the use of recombinant DNA. But mainly due to the risks associated with permanent genetic alteration of cells, safety and ethical concerns have been raised for the use of DNA-based vectors in human clinical therapy. In the last years, synthetic messenger RNA has emerged as powerful tool to deliver genetic information. RNA vectors exhibit several advantages compared to DNA and are particularly interesting for applications that require transient gene expression. RNA stability and translation efficiency can be increased by cis-acting structural elements in the RNA such as the 5'-cap, the poly(A)-tail, untranslated regions and the sequence of the coding region. Here we review recent developments in the optimization of messenger RNA as vector for modulation of protein expression emphasizing on stability, transfection and immunogenicity. In addition, we summarize current pre-clinical and clinical studies using RNA-based vectors for immunotherapy, T cell, stem cell as well as gene therapy.

Characterization of a hepatitis B and hepatitis delta virus receptor binding site.

Insights into the early infection events of the human hepatitis B (HBV) and hepatitis delta virus (HDV) have been limited because of the lack of a cell culture system supporting the full replication cycle for these important pathogens. The human hepatoma cell line HepaRG allows the experimental induction of a differentiated state, thereby gaining susceptibility toward HBV and HDV infection. We recently identified HBV envelope protein-derived lipopeptides comprising amino acids 2 though 48 of the preS-domain of the L-surface protein, which block infection already at picomolar concentrations. To map the responsible sequence for the peptides' activity we describe an Escherichia coli expression system that permits myristoylation and investigated recombinant HBVpreS-GST fusion proteins with deletion- and point-mutations for their ability to prevent HBV and HDV infection. We found that (1) a myristoylated HBVpreS/2-48-GST fusion protein efficiently interferes with HBV infection of HepaRG cells; (2) deletions and point mutations in the highly conserved preS1 sequence between amino acids 11 through 21 result in the loss of infection inhibition activity; (3) hepatitis B viruses carrying single amino acid exchanges within this region lose infectivity; and (4) HDV infection of HepaRG cells can be inhibited by myristoylated HBVpreS peptides with the same specificity. In conclusion, HBV and HDV use at least one common step to enter hepatocytes and require a highly conserved preS1-sequence within the L-protein. This step is exceptionally sensitive toward inactivation by acylated HBVpreS1 peptides, which therefore represent a novel group of entry inhibitors that could be used for the treatment of hepatitis B and D.

Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells.

Adoptive transfer of dendritic cells (DCs) transfected with in vitro-transcribed, RNA-encoding, tumor-associated antigens has recently entered clinical testing as a promising approach for cancer immunotherapy. However, pharmacokinetic exploration of RNA as a potential drug compound and a key aspect of clinical development is still pending. While investigating the impact of different structural modifications of RNA molecules on the kinetics of the encoded protein in DCs, we identified components located 3' of the coding region that contributed to a higher transcript stability and translational efficiency. With the use of quantitative reverse transcription-polymerase chain reaction (RT-PCR) and eGFP variants to measure transcript amounts and protein yield, we showed that a poly(A) tail measuring 120 nucleotides compared with a shorter one, an unmasked poly(A) tail with a free 3' end rather than one extended with unrelated nucleotides, and 2 sequential beta-globin 3' untranslated regions cloned head to tail between the coding region and the poly(A) tail each independently enhanced RNA stability and translational efficiency. Consecutively, the density of antigen-specific peptide/MHC complexes on the transfected cells and their potency to stimulate and expand antigen-specific CD4+ and CD8+ T cells were also increased. In summary, our data provide a strategy for optimizing RNA-transfected DC vaccines and a basis for defining release criteria for such vaccine preparations.