Immunomic, genomic and transcriptomic characterization of CT26 colorectal carcinoma

Background Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. Here, we present an integrated map of the genome, transcriptome and immunome of an epithelial mouse tumor, the CT26 colon carcinoma cell line. Results We found that Kras is homozygously mutated at p.G12D, Apc and Tp53 are not mutated, and Cdkn2a is homozygously deleted. Proliferation and stem-cell markers, including Top2a, Birc5 (Survivin), Cldn6 and Mki67, are highly expressed while differentiation and top-crypt markers Muc2, Ms4a8a (MS4A8B) and Epcam are not. Myc, Trp53 (tp53), Mdm2, Hif1a, and Nras are highly expressed while Egfr and Flt1 are not. MHC class I but not MHC class…

Expression of serologically identified tumor antigens in acute leukemias.

Cancer/testis antigens (CTA) are an expanding family of immunogenic proteins selectively expressed in human neoplasms. As little is known about the expression of serologically identified CTA in leukemias so far, we investigated the expression of 5 CT genes (SSX-1, HOM-MEL-40/SSX-2, HOM-TES-14/SCP-1, SCP-3 and NY-ESO-1) in leukemic blood samples obtained from patients with either acute lymphatic leukemias (ALL) or myelocytic leukemia (AML). RT-PCR-analyses showed no expression of any of the CT-genes in the leukemia samples of 19 patients with AML, whereas frequent expression was found in ALL. In the 17 ALL cases studied, SCP3a, SSX-1, HOM-MEL-40/SXX-2 and HOM-TES-14/SCP-1 were expressed in 4…

Investigation of pH-Responsiveness inside Lipid Nanoparticles for Parenteral mRNA Application Using Small-Angle X-ray Scattering.

Messenger ribonucleic acid (mRNA)-based nanomedicines have shown to be a promising new lead in a broad field of potential applications such as tumor immunotherapy. Of these nanomedicines, lipid-based mRNA nanoparticles comprising ionizable lipids are gaining increasing attention as versatile technologies for fine-tuning toward a given application, with proven potential for successful development up to clinical practice. Still, several hurdles have to be overcome to obtain a drug product that shows adequate mRNA delivery and clinical efficacy. In this study, pH-induced changes in internal molecular organization and overall physicochemical characteristics of lipoplexes comprising ionizable li…

Targeting the tumor mutanome for personalized vaccination therapy

Next generation sequencing enables identification of immunogenic tumor mutations targetable by individualized vaccines. In the B16F10 melanoma system as pre-clinical proof-of-concept model, we found a total of 563 non-synonymous expressed somatic mutations. Of the mutations we tested, one third were immunogenic. Immunization conferred in vivo tumor control, qualifying mutated epitopes as source for effective vaccines.

mTOR Inhibition Improves Antitumor Effects of Vaccination with Antigen-Encoding RNA

Abstract Vaccination with in vitro transcribed RNA encoding tumor antigens is an emerging approach in cancer immunotherapy. Attempting to further improve RNA vaccine efficacy, we have explored combining RNA with immunomodulators such as rapamycin. Rapamycin, the inhibitor of mTOR, was used originally for immunosuppression. Recent reports in mouse systems, however, suggest that mTOR inhibition may enhance the formation and differentiation of the memory CD8+ T-cell pool. Because memory T-cell formation is critical to the outcome of vaccination aproaches, we studied the impact of rapamycin on the in vivo primed RNA vaccine-induced immune response using the chicken ovalbumin-expressing B16 mela…

Abstract A004: Systemic RNA vaccines: Connecting effective cancer immunotherapy with antiviral defense mechanisms

Abstract Mechanisms of antiviral host defense are important for survival and evolutionarily optimized for high sensitivity and potency. Intending to harvest the multitude of highly specialized and intertwined pathogen immune defense programs for cancer immunotherapy, we simulated a systemic pathogen intrusion into the blood stream by intravenous injection of lipid-formulated, tumor antigen-encoding mRNA nanoparticles. These RNA-lipoplexes (RNA-LPX) were directed to various lymphoid tissues, including the spleen, lymph nodes and bone marrow, which provide the ideal microenvironment for efficient priming and amplification of T cell responses. Solely the RNA-to-lipid ratio was discovered to de…

Aberrantly activated claudin 6 and 18.2 as potential therapy targets in non-small-cell lung cancer

Claudins (CLDNs) are central components of tight junctions that regulate epithelial-cell barrier function and polarity. Altered CLDN expression patterns have been demonstrated in numerous cancer types and lineage-specific CLDNs have been proposed as therapy targets. The objective of this study was to assess which fraction of patients with non-small-cell lung cancer (NSCLC) express CLDN6 and CLDN18 isoform 2 (CLDN18.2). Protein expression of CLDN6 and CLDN18.2 was examined by immunohistochemistry on a tissue microarray (n=355) and transcript levels were supportively determined based on gene expression microarray data from fresh-frozen NSCLC tissues (n=196). Both were analyzed with regard to …

Cascades of transcriptional induction during dendritic cell maturation revealed by genome-wide expression analysis.

Dendritic cells (DC) are central regulators of immunity. Signal-induced maturation of DCs is assumed to be the starting point for specific immune responses. To further understand this process, we analyzed the alteration of transcript profiles along the time course of CD40 ligand-induced maturation of human myeloid DCs by Affymetrix GeneChip microarrays covering >6800 genes. Besides rediscovery of genes already described as associated with DC maturation proving reliability of the methods used, we identified clusterin as novel maturation marker. Looking across the time course, we observed synchronized kinetics of distinct functional groups of molecules whose temporal coregulation underscores …

Abstract CT034: A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent melanoma immunotherapy

Abstract Therapeutic vaccination with tumor antigen-encoding RNAs by local administration is currently being successfully employed in various clinical trials. Advancing from local to more efficient systemic targeting of antigen-presenting cells (APCs), we have developed pioneering RNA-lipoplex (RNA(LIP)) immunotherapeutics for intravenous application based on the employment of well-known lipid carriers without the need for functionalization of particles with molecular ligands. The novel RNA(LIP) formulation has been engineered to preserve RNA integrity after intravenous injection and physicochemically optimized for efficient uptake and expression of the encoded antigen by APCs in various ly…

Abstract 1778: Preclinical characterization of the safety and antitumor activity of IMAB027-vcMMAE, an anticlaudin 6 antibody-drug conjugate

Abstract Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers. The anti-CLDN6 monoclonal antibody (mAb), IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. Conjugation of IMAB027 with monomethyl auristatin E (MMAE) may utilize the precision tumor-targeting of the mAb to deliver a highly effective cytotoxic drug to the tumor. In this report we present the preclinical characterization of this antibody–drug conjugate, IMAB027–vcMMAE. Methods Internalization of IMAB027 in various CLDN6+ human ovarian (OC) and…

Specific hepatic delivery of procollagen α1(I) small interfering RNA in lipid‐like nanoparticles resolves liver fibrosis

Fibrosis accompanies the wound-healing response to chronic liver injury and is characterized by excessive hepatic collagen accumulation dominated by collagen type I that often progresses to cirrhosis. Here we present ample in-vivo evidence of an up to 90% suppression of procollagen α1(I) expression, a reduction of septa formation and a 40–60% decrease of collagen deposition in mice with progressive and advanced liver fibrosis, that received cationic lipid nanoparticles loaded with small interfering RNA to the procollagen α1(I) gene (LNP-siCol1a1). After intravenous injection up to ninety percent of LNP-siCol1a1 were retained in the liver of fibrotic mice and accumulated in nonparenchymal > …

Zolbetuximab combined with EOX as first-line therapy in advanced CLDN18.2+ gastric (G) and gastroesophageal junction (GEJ) adenocarcinoma : Updated results from the FAST trial.

16 Background: Physiologically, the tight junction protein CLDN18.2 is present only in the gastric mucosa. Upon malignant transformation, CLDN18.2 epitopes are exposed on the cell surface and accessible to targeted therapy. Zolbetuximab (formerly IMAB362) is a chimeric mAb that mediates specific killing of CLDN18.2+ cancer cells through immune effector mechanisms; single-agent activity has been reported in G/GEJ cancer. Methods: Patients (pts) with advanced HER2-negative (HER–) G/GEJ cancer with CLDN18.2 expression of ≥ 2+ staining intensity with the anti-CLDN18 43-14A mAb in ≥ 40% tumor cells were eligible (NCT01630083). Patients were randomized 1:1 to receive first-line EOX ± zolbetuxima…

Expression profiling of autoimmune regulator AIRE mRNA in a comprehensive set of human normal and neoplastic tissues.

Defects in the autoimmune regulator (AIRE) gene cause the monogenic autoimmune disease autoimmune polyendocrinopathy syndrome type 1 (APS-1), which is characterized by a loss of self-tolerance to multiple organs. In concordance with its role in immune tolerance, AIRE is strongly expressed in medullary thymic epithelial cells (mTECs). Data on mechanisms controlling AIRE activation and the expression of this gene in other tissues are fragmentary and controversial. We report here AIRE mRNA expression profiling of a large set of normal human tissues and cells, tumor specimen and methylation deficient cell lines. On this broad data basis we found that AIRE mRNA expression is confined to mTECs in…

PO-516 E6/E7 RNA(LIP): a novel liposomal RNA vaccine for treatment of patients with HPV16-positive malignancies

Introduction Human papillomavirus (HPV) has emerged as a major risk factor for Head Neck squamous cell carcinoma (HNSCC) and the incidence of HPV-positive HNSCC continues to rise. Standard treatment of HNSCC given with curative intent causes substantial and long-term physical and functional impairments, but nonetheless, approx. 50% of patients die of their disease. Alternative treatments are urgently needed to improve survival but also to reduce treatment-associated morbidity. Both CD8+ and CD4+ T cells are important for viral clearance and regression of HPV-positive premalignant lesions; density of tumor-infiltrating lymphocytes (TILs) is a strong predictor of the outcome of HPV-positive o…

A noninflammatory mRNA vaccine for treatment of experimental autoimmune encephalomyelitis.

Precision therapy for immune tolerance Autoimmune diseases, such as multiple sclerosis (MS), result from a breach of immunological self-tolerance and tissue damage by autoreactive T lymphocytes. Current treatments can cause systemic immune suppression and side effects such as increased risk of infections. Krienke et al. designed a messenger RNA vaccine strategy that lacks adjuvant activity and delivers MS autoantigens into lymphoid dendritic cells. This approach expands a distinct type of antigen-specific effector regulatory T cell that suppresses autoreactivity against targeted autoantigens and promotes bystander suppression of autoreactive T cells against other myelin-specific autoantigen…

Phase 1 Study of IMAB362 with immunomodulation in patients with advanced gastric cancer

FAST: An international, multicenter, randomized, phase II trial of epirubicin, oxaliplatin, and capecitabine (EOX) with or without IMAB362, a first-in-class anti-CLDN18.2 antibody, as first-line therapy in patients with advanced CLDN18.2+ gastric and gastroesophageal junction (GEJ) adenocarcinoma.

LBA4001 Background: Claudin18.2 (CLDN18.2) is a tight junction protein expressed by several cancers including gastric and GEJ adenocarcinoma. IMAB362 is a chimeric monoclonal antibody that mediates specific killing of CLDN18.2-positive cancer cells by activation of immune effector mechanisms. IMAB362 has demonstrated single-agent activity and was safe and tolerable in patients (pts) with pretreated gastric cancer. Methods: Pts with advanced/recurrent gastric and GEJ cancer were centrally evaluated for CLDN18.2 expression by IHC (validated CLAUDETECT18.2 Kit). Eligible pts had a CLDN18.2 expression of ≥ 2+ in ≥ 40% tumor cells, an ECOG PS of 0–1 and were not eligible for trastuzumab. Pts we…

Abstract CT020: MERIT: introducing individualized cancer vaccines for the treatment of TNBC - a phase I trial

Abstract The majority of metastatic cancers remain incurable since the current methods of treatment often fail to target the heterogeneous nature of each individual patient's tumor. Personalized approaches targeting each individual patient's tumor may therefore bring significant improvements. The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically validate a pioneering RNA-based immunotherapy concept for the treatment of triple negative breast cancer (TNBC) by targeting shared tumor antigens and individual neo-antigens in TNBC patients. MERIT combines two personalized treatment concepts: (i) treatment with vaccines containing “off-the-shelf” mRNAs selected from a pre-s…

The European regulatory environment of rna-based vaccines

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 t…

Abstract A110: Mutant MHC class II epitopes drive therapeutic immune responses to cancer

Abstract Mutations are regarded as ideal targets for cancer immunotherapy. As neoepitopes with strict lack of expression in any healthy tissue, they are expected to be safe and could bypass the central tolerance mechanisms. Recent advances in nucleic acid sequencing technologies have revolutionized the field of genomics, allowing the readily targeting of mutated neoantigens for personalized cancer vaccination. We demonstrated in three independent murine tumor models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that unexpectedly the immunogenic mutanome is pre-dominantly recognized by CD4+ T cells. RNA vaccination with such MHC class II restricted immuno…

Abstract CT202: IVAC MUTANOME: Individualized vaccines for the treatment of cancer

Abstract Cancer arises from the accumulation of genomic alterations and epigenetic changes that constitute a hallmark of cancer. Owing to the molecular heterogeneity in cancer, only a minor fraction of patients profit from approved therapies. Available targeted therapies can only address alterations common to a particular type of cancer and induce transient effects due to the generation of resistant sub-clones. In contrast, the IVAC MUTANOME project aims to immunologically target multiple cancer mutations uniquely expressed in a given patient's tumor. The IVAC MUTANOME approach should be applicable to the majority of patients irrespective of the tumor entity and offers the potential to expl…

Galaxy LIMS for next-generation sequencing.

Abstract Summary: We have developed a laboratory information management system (LIMS) for a next-generation sequencing (NGS) laboratory within the existing Galaxy platform. The system provides lab technicians standard and customizable sample information forms, barcoded submission forms, tracking of input sample quality, multiplex-capable automatic flow cell design and automatically generated sample sheets to aid physical flow cell preparation. In addition, the platform provides the researcher with a user-friendly interface to create a request, submit accompanying samples, upload sample quality measurements and access to the sequencing results. As the LIMS is within the Galaxy platform, the …

Claudin 18.2 is a target for IMAB362 antibody in pancreatic neoplasms

The majority of pancreatic neoplasms are characterized by a generally lethal progress within a short period of time after primary diagnosis and the mortality of patients is expected to increase further. Due to lack of efficient screening programs and moderate response to treatments, novel compounds for treatment are needed. We investigated the CLDN18.2 expression in affected patients as in vitro feasibility study for a potential treatment with the novel antibody IMAB362. Therefore, we analyzed the expression of CLDN18.2 in normal pancreatic tissues (N = 24), primary lesions (N = 202), metastases (N = 84) and intra-individually matched samples (N = 48) of patients with pancreatic ductal aden…

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

Abstract 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 p…

Genome-based in silico identification of new Mycobacterium tuberculosis antigens activating polyfunctional CD8+ T cells in human tuberculosis.

Although CD8(+) T cells help control Mycobacterium tuberculosis infection, their M. tuberculosis Ag repertoire, in vivo frequency, and functionality in human tuberculosis (TB) remains largely undefined. We have performed genome-based bioinformatics searches to identify new M. tuberculosis epitopes presented by major HLA class I supertypes A2, A3, and B7 (covering 80% of the human population). A total of 432 M. tuberculosis peptides predicted to bind to HLA-A*0201, HLA-A*0301, and HLA-B*0702 (representing the above supertypes) were synthesized and HLA-binding affinities determined. Peptide-specific CD8(+) T cell proliferation assays (CFSE dilution) in 41 M. tuberculosis-responsive donors ide…

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

Abstract 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% a…

Synthetic mRNAs with Superior Translation and Stability Properties

The translational efficiency and stability of synthetic mRNA in both cultured cells and whole animals can be improved by incorporation of modified cap structures at the 5'-end. mRNAs are synthesized in vitro by a phage RNA polymerase transcribing a plasmid containing the mRNA sequence in the presence of all four NTPs plus a cap dinucleotide. Modifications in the cap dinucleotide at the 2'- or 3'-positions of m(7)Guo, or modifications in the polyphosphate chain, can improve both translational efficiency and stability of the mRNA, thereby increasing the amount and duration of protein expression. In the context of RNA-based immunotherapy, the latter is especially important for antigen producti…

BNT162b2 induces SARS-CoV-2-neutralising antibodies and T cells in humans

BNT162b2, a lipid nanoparticle (LNP) formulated nucleoside-modified messenger RNA (mRNA) encoding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S) stabilized in the prefusion conformation, has demonstrated 95% efficacy to prevent coronavirus disease 2019 (COVID-19). Recently, we reported preliminary BNT162b2 safety and antibody response data from an ongoing placebo-controlled, observer-blinded phase 1/2 vaccine trial1. We present here antibody and T cell responses from a second, non-randomized open-label phase 1/2 trial in healthy adults, 19-55 years of age, after BNT162b2 prime/boost vaccination at 1 to 30 µg dose levels. BNT162b2 elicited strong antibody …

Dexamethasone premedication suppresses vaccine-induced immune responses against cancer

ABSTRACT Glucocorticosteroids (GCS) have an established role in oncology and are administered to cancer patients in routine clinical care and in drug development trials as co-medication. Given their strong immune-suppressive activity, GCS may interfere with immune-oncology drugs. We are developing a therapeutic cancer vaccine, which is based on a liposomal formulation of tumor-antigen encoding RNA (RNA-LPX) and induces a strong T-cell response both in mice as well as in humans. In this study, we investigated in vivo in mice and in human PBMCs the effect of the commonly used long-acting GCS Dexamethasone (Dexa) on the efficacy of this vaccine format, with a particular focus on antigen-specif…

Intravenous delivery of the toll-like receptor 7 agonist SC1 confers tumor control by inducing a CD8+ T cell response

TLR7 agonists are considered promising drugs for cancer therapy. The currently available compounds are not well tolerated when administered intravenously and therefore are restricted to disease settings amenable for topical application. Here we present the preclinical characterization of SC1, a novel synthetic agonist with exquisite specificity for TLR7. We found that intravenously administered SC1 mediates systemic release of type I interferon, but not of proinflammatory cytokines such as TNFα and IL6, and results in activation of circulating immune cells. Tumors of SC1-treated mice have brisk immune cell infiltrates and are polarized towards a Th1 type signature. Intratumoral CD8(+) T cel…

Preclinical characterization of IMAB362-vcMMAE, an anti-CLDN18.2 antibody–drug conjugate

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

AbstractAdoptive 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 (R…

The human X chromosome is enriched for germline genes expressed in premeiotic germ cells of both sexes.

The role of X-chromosomal genes in spermatogenesis has been subject to a number of studies in different organisms. Recently, it was proposed that the X chromosome has a predominant role in premeiotic stages of mammalian spermatogenesis. We analyzed the expression of a representative set of 17 X-linked and 48 autosomal germline-restricted genes in different stages of human germ cell development. In accordance with data from other species, we show that the human X chromosome is indeed significantly enriched for genes activated in premeiotic stages of spermatogenesis. In contrast to recent studies, however, we found that expression of these genes is not restricted to spermatogenesis, but is ac…

Abstract IA06: Targeting the mutanome for individualized cancer immunotherapy

Abstract Mutations are regarded as ideal targets for cancer immunotherapy. As neo-epitopes with strict lack of expression in any healthy tissue, they are expected to be safe. The systematic use of mutations for vaccine approaches, however, is hampered by the uniqueness of the repertoire of mutations (the mutanome) in every patient's tumor. We have recently proposed a personalized immunotherapy approach targeting the spectrum of individual mutations. Preclinically we could show in three independent murine tumor models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that unexpectedly the immunogenic mutanome is pre-dominantly recognized by CD4+ T cells (the …

Targeting the Heterogeneity of Cancer with Individualized Neoepitope Vaccines

Abstract Somatic mutations binding to the patient's MHC and recognized by autologous T cells (neoepitopes) are ideal cancer vaccine targets. They combine a favorable safety profile due to a lack of expression in healthy tissues with a high likelihood of immunogenicity, as T cells recognizing neoepitopes are not shaped by central immune tolerance. Proteins mutated in cancer (neoantigens) shared by patients have been explored as vaccine targets for many years. Shared (“public”) mutations, however, are rare, as the vast majority of cancer mutations in a given tumor are unique for the individual patient. Recently, the novel concept of truly individualized cancer vaccination emerged, which explo…

Tailoring the stealth properties of biocompatible polysaccharide nanocontainers.

Fundamental development of a biocompatible and degradable nanocarrier platform based on hydroxyethyl starch (HES) is reported. HES is a derivative of starch and possesses both high biocompatibility and improved stability against enzymatic degradation; it is used to prepare nanocapsules via the polyaddition reaction at the interface of water nanodroplets dispersed in an organic miniemulsion. The synthesized hollow nanocapsules can be loaded with hydrophilic guests in its aqueous core, tuned in size, chemically functionalized in various pathways, and show high shelf life stability. The surface of the HES nanocapsules is further functionalized with poly(ethylene glycol) via different chemistri…

Characterization of zolbetuximab in pancreatic cancer models

ABSTRACT In healthy tissue, the tight junction protein Claudin 18.2 (CLDN18.2) is present only in the gastric mucosa. Upon malignant transformation of gastric epithelial tissue, perturbations in cell polarity lead to cell surface exposure of CLDN18.2 epitopes. Moreover, CLDN18.2 is aberrantly expressed in malignancies of several other organs, such as pancreatic cancer (PC). A monoclonal antibody, zolbetuximab (formerly known as IMAB362), has been generated against CLDN18.2. In a phase 2 clinical trial (FAST: NCT01630083), zolbetuximab in conjunction with chemotherapy prolonged overall and progression-free survival over chemotherapy alone and improved quality of life. In this study, the mech…

MS4A12 is a colon-selective store-operated calcium channel promoting malignant cell processes.

AbstractUsing a data mining approach for the discovery of new targets for antibody therapy of colon cancer, we identified MS4A12, a sequence homologue of CD20. We show that MS4A12 is a cell surface protein. Expression analysis and immunohistochemistry revealed MS4A12 to be a colonic epithelial cell lineage gene confined to the apical membrane of colonocytes with strict transcriptional repression in all other normal tissue types. Expression is maintained upon malignant transformation in 63% of colon cancers. Ca2+ flux analyses disclosed that MS4A12 is a novel component of store-operated Ca2+ entry in intestinal cells. Using RNAi-mediated gene silencing, we show that loss of MS4A12 in LoVo co…

Enhanced protection of C57 BL/6 vs Balb/c mice to melanoma liver metastasis is mediated by NK cells.

ABSTRACT The B16F10 murine melanoma cell line displays a low expression of MHC class I molecules favoring immune evasion and metastases in immunocompetent C57 BL/6 wild-type mice. Here, we generated metastases to the liver, an organ that is skewed towards immune tolerance, by intrasplenic injection of B16F10 cells in syngeneic C57 BL/6 compared to allogeneic Balb/c mice. Surprisingly, Balb/c mice, which usually display a pronounced M2 macrophage and Th2 T cell polarization, were ∼3 times more susceptible to metastasis than C57 BL/6 mice, despite a much higher M1 and Th1 T cell immune response. The anti-metastatic advantage of C57 BL/6 mice could be attributed to a more potent NK-cell mediat…

Identification of a tumor-reactive T-cell repertoire in the immune infiltrate of patients with resectable pancreatic ductal adenocarcinoma

The devastating prognosis of patients with resectable pancreatic ductal adenocarcinoma (PDA) presents an urgent need for the development of therapeutic strategies targeting disseminated tumor cells. Until now, T-cell therapy has been scarcely pursued in PDA, due to the prevailing view that it represents a poorly immunogenic tumor.We systematically analyzed T-cell infiltrates in tumor biopsies from 127 patients with resectable PDA by means of immunohistochemistry, flow cytometry, T-cell receptor (TCR) deep-sequencing and functional analysis ofProminent T-cell infiltrates, as well as tertiary lymphoid structures harboring proliferating T-cells, were detected in the vast majority of biopsies f…

Frequent nonrandom activation of germ-line genes in human cancer.

Abstract The growing class of cancer/germ-line genes is characterized by a unique expression pattern with transcription restricted to germ cells and cancer cells. It is not known which fraction of germ-line genes is ectopically activated in tumor cells and whether this fraction displays common features as compared with strictly germ-line genes remaining silent in cancer. Using an unbiased genome-wide scanning approach, representative samples of both cancer/germ-line genes as well as strictly germ-line-specific genes were determined. Comparative analysis disclosed highly significant diametric characteristics for these two categories of genes with regard to sex specificity, developmental stag…

Autoimmunity seen through the SEREX-scope.

Autoantibodies can be detected in autoimmune diseases with a long prodromal phase and may serve as early indicators of disease activity. Autoantibody-based screening methods are therefore potent tools for the identification of target antigens. The SEREX method (serological identification of antigens by recombinant expression cloning) has been developed for the serological definition of immunogenic tumor antigens. Recent studies indicate that the SEREX approach may also be utilized for the analysis of complex immune responses involved in autoimmune diseases.

A catalog of HLA type, HLA expression, and neo-epitope candidates in human cancer cell lines

Cancer cell lines are a tremendous resource for cancer biology and therapy development. These multipurpose tools are commonly used to examine the genetic origin of cancers, to identify potential novel tumor targets, such as tumor antigens for vaccine devel-opment, and utilized to screen potential therapies in preclinical studies. Mutations, gene expression, and drug sensitivity have been determined for many cell lines using next-generation sequencing (NGS). However, the human leukocyte antigen (HLA) type and HLA expression of tumor cell lines, characterizations necessary for the development of cancer vaccines, have remained largely incomplete and, such information, when available, has been …

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model.

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was foll…

Tumor vaccination using messenger RNA: prospects of a future therapy.

While the endeavor to vaccinate against cancer has been pursued for over 20 years, only recently was the first tumor vaccine approved. Among the different antigen formats assessed for vaccination, coding messenger RNA (mRNA) is emerging as a particularly attractive option. It can code for all types of transcript based proteins, is easy and cost efficient to produce, has a favorable safety profile and enables induction of combined immune responses. Within the last few years major developments have been achieved in this field. Clinical approaches use mRNA either for direct administration or for engineering of adoptively transferred dendritic cells. However, there are still challenges to be ov…

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 alc…

Prognostic Significance of Interferon-γ and Its Signaling Pathway in Early Breast Cancer Depends on the Molecular Subtypes

Interferons are crucial for adaptive immunity and play an important role in the immune landscape of breast cancer. Using microarray-based gene expression analysis, we examined the subtype-specific prognostic significance of interferon-&gamma

Investigation of the immune infiltrate of melanoma metastases under immune checkpoint inhibition.

9570 Background: Tumor infiltrating lymphocytes (TIL) play a crucial role in the therapeutic impact of immune checkpoint blockers. Methods: We investigated metastases from 56 melanoma patients before and during treatment with immune checkpoint blockers (i) immunohistochemically, (ii) with TCR repertoire profiling and (iii) analysis of the transcriptome. The patients were treated with ipilimumab (n = 25) or pembrolizumab (n = 23) or ipilimumab/nivolumab (n = 7); half of them had a disease control, the other half progressed as best response to treatment. Results: In contrast to previous reports immunohistochemical analysis of the immune infiltrate revealed no significant difference in the nu…

Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3' UTRs Identified by Cellular Library Screening.

Synthetic mRNA has emerged as a powerful tool for the transfer of genetic information, and it is being explored for a variety of therapeutic applications. Many of these applications require prolonged intracellular persistence of mRNA to improve bioavailability of the encoded protein. mRNA molecules are intrinsically unstable and their intracellular kinetics depend on the UTRs embracing the coding sequence, in particular the 3′ UTR elements. We describe here a novel and generally applicable cell-based selection process for the identification of 3′ UTRs that augment the expression of proteins encoded by synthetic mRNA. Moreover, we show, for two applications of mRNA therapeutics, namely, (1) …

Abstract CT032: A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent cancer immunotherapy in patients with malignant melanoma

Abstract Immunotherapeutic approaches have evolved as promising and valid alternatives to available conventional cancer treatments. Amongst others, vaccination with tumor antigen-encoding RNAs by local administration is currently successfully employed in various clinical trials. To allow for a more efficient targeting of antigen-presenting cells (APCs) and to overcome potential technical challenges associated with local administration, we have developed a novel RNA immunotherapeutic for systemic application based on a fixed set of four liposome complexed RNA drug products (RNA(LIP)), each encoding one shared melanoma-associated antigen. The novel RNA(LIP) formulation was engineered (i) to p…

3D cryo-electron microscopy, molecular modelling and structural fitting with recombinant expressed virus like particles as part of drug design

Extended abstract of a paper presented at MC 2007, 33rd DGE Conference in Saarbrücken, Germany, September 2 – September 7, 2007

Here we present a comprehensive molecular mapping of virus-induced autoimmune B cell responses obtained by serological identification of antigens by recombinant expression cloning analysis. Immunoscreening of cDNA expression libraries of various organs (lung, liver, and spleen) using sera from mice infected with cytopathic (vaccinia virus [VV]) or noncytopathic (lymphocytic choriomeningitis virus [LCMV]) viruses revealed a broad specificity of the elicited autoantibody response. Interestingly, the majority of the identified autoantigens have been previously described as autoantigens in humans. We found that induction of virus-induced autoantibodies of the immunoglobulin G class largely depe…

A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles encoding shared tumor antigens for potent melanoma immunotherapy

Mutated tumor alleles are expressed according to their DNA frequency

AbstractThe transcription of tumor mutations from DNA into RNA has implications for biology, epigenetics and clinical practice. It is not clear if mutations are in general transcribed and, if so, at what proportion to the wild-type allele. Here, we examined the correlation between DNA mutation allele frequency and RNA mutation allele frequency. We sequenced the exome and transcriptome of tumor cell lines with large copy number variations, identified heterozygous single nucleotide mutations and absolute DNA copy number and determined the corresponding DNA and RNA mutation allele fraction. We found that 99% of the DNA mutations in expressed genes are expressed as RNA. Moreover, we found a hig…

NFATc1 supports imiquimod-induced skin inflammation by suppressing IL-10 synthesis in B cells

Epicutaneous application of Aldara cream containing the TLR7 agonist imiquimod (IMQ) to mice induces skin inflammation that exhibits many aspects of psoriasis, an inflammatory human skin disease. Here we show that mice depleted of B cells or bearing interleukin (IL)-10-deficient B cells show a fulminant inflammation upon IMQ exposure, whereas ablation of NFATc1 in B cells results in a suppression of Aldara-induced inflammation. In vitro, IMQ induces the proliferation and IL-10 expression by B cells that is blocked by BCR signals inducing NFATc1. By binding to HDAC1, a transcriptional repressor, and to an intronic site of the Il10 gene, NFATc1 suppresses IL-10 expression that dampens the pro…

Molecular Characterization of Virus-induced Autoantibody Responses

Here we present a comprehensive molecular mapping of virus-induced autoimmune B cell responses obtained by serological identification of antigens by recombinant expression cloning analysis. Immunoscreening of cDNA expression libraries of various organs (lung, liver, and spleen) using sera from mice infected with cytopathic (vaccinia virus [VV]) or noncytopathic (lymphocytic choriomeningitis virus [LCMV]) viruses revealed a broad specificity of the elicited autoantibody response. Interestingly, the majority of the identified autoantigens have been previously described as autoantigens in humans. We found that induction of virus-induced autoantibodies of the immunoglobulin G class largely depe…

Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion

mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent and time-consuming and mainly restricted to fibroblasts, thereby demonstrating the need for a simple but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Nonmodified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence, to overcome RNA…

mRNA: A Versatile Molecule for Cancer Vaccines

mRNA vaccines are finally ready to assume their rightful place at the forefront of nucleic acid- based vaccines. Major achievements within the last two decades have turned this highly versatile molecule into a safe and very attractive pharmaceutical platform that combines many positive attributes able to address a broad range of diseases, including cancer. The simplicity of mRNA vaccines greatly reduces complications generally associated with the production of biological vaccines. Intrinsic costimulatory and inflammatory triggers in addition to the provision of the antigenic information makes mRNA an all- in-one molecule that does not need additional adjuvants and that does not pose the ris…

Preclinical characterization of IMAB362 for the treatment of gastric carcinoma

ArtiFuse—computational validation of fusion gene detection tools without relying on simulated reads

Abstract Motivation Gene fusions are an important class of transcriptional variants that can influence cancer development and can be predicted from RNA sequencing (RNA-seq) data by multiple existing tools. However, the real-world performance of these tools is unclear due to the lack of known positive and negative events, especially with regard to fusion genes in individual samples. Often simulated reads are used, but these cannot account for all technical biases in RNA-seq data generated from real samples. Results Here, we present ArtiFuse, a novel approach that simulates fusion genes by sequence modification to the genomic reference, and therefore, can be applied to any RNA-seq dataset wit…

RNA-Seq Atlas—a reference database for gene expression profiling in normal tissue by next-generation sequencing

Abstract Motivation: Next-generation sequencing technology enables an entirely new perspective for clinical research and will speed up personalized medicine. In contrast to microarray-based approaches, RNA-Seq analysis provides a much more comprehensive and unbiased view of gene expression. Although the perspective is clear and the long-term success of this new technology obvious, bioinformatics resources making these data easily available especially to the biomedical research community are still evolving. Results: We have generated RNA-Seq Atlas, a web-based repository of RNA-Seq gene expression profiles and query tools. The website offers open and easy access to RNA-Seq gene expression pr…

Abstract LB-130: Combinatorial treatment with intratumoral cytokine mRNAs results in high frequency of tumor rejection and development of anti-tumor immunity across a range of preclinical cancer models

Abstract Cancer immunotherapy localized to the tumor microenvironment holds great potential to promote innate and adaptive immune responses against tumors, while avoiding toxicities related to systemic administration of immuno-modulatory therapeutics. Current strategies for tumor-targeted, gene-based delivery of immune therapies face limitations in the clinic due to suboptimal target expression, anti-vector immunity, potential for unwanted genomic rearrangements and other off target effects. We developed a highly potent synthetic mRNA-based platform for in vivo transfection and sustained intratumoral expression of immuno-modulatory molecules that is capable of inducing immunity to tumor spe…

24 Combined analysis of antigen presentation and T cell recognition reveals restricted immune responses in melanoma

Introduction Studies in the past few years have suggested a key role for neo-antigens in cancer immunotherapy. Since neo-antigens are specifically expressed on the tumour, targeting them is not likely to induce tolerance or normal tissue toxicity, making them candidates for immunotherapy. Despite encouraging results in clinical trials using neo-antigens, peptide or RNA vaccines and adoptive cell transfer (ACT), only a handful of neo-antigens and their corresponding T-cells have been identified in patients. Material and methods In this study we are using a combination of a novel neo-antigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to unbiasedly identify tumour associ…

Integrative analysis of structural variations using short-reads and linked-reads yields highly specific and sensitive predictions.

Genetic diseases are driven by aberrations of the human genome. Identification of such aberrations including structural variations (SVs) is key to our understanding. Conventional short-reads whole genome sequencing (cWGS) can identify SVs to base-pair resolution, but utilizes only short-range information and suffers from high false discovery rate (FDR). Linked-reads sequencing (10XWGS) utilizes long-range information by linkage of short-reads originating from the same large DNA molecule. This can mitigate alignment-based artefacts especially in repetitive regions and should enable better prediction of SVs. However, an unbiased evaluation of this technology is not available. In this study, w…

In Silico HLA Typing Using Standard RNA-Seq Sequence Reads

Next-generation sequencing (NGS) enables high-throughput transcriptome profi ling using the RNA-Seq assay, resulting in billions of short sequence reads. Worldwide adoption has been rapid: many laboratories worldwide generate transcriptome sequence reads daily. Here, we describe methods for obtaining a sample’s human leukocyte antigen (HLA) class I and II types and HLA expression using standard NGS RNA- Seq sequence reads. We demonstrate the application using our algorithm, seq2HLA, and a publicly available RNA-Seq dataset from the Burkitt lymphoma cell line Raji.

Comparison of Claudin 18.2 expression in primary tumors and lymph node metastases in Japanese patients with gastric adenocarcinoma.

CLDN18.2 expression is highly prevalent in Japanese patients with gastric cancer, making it a targetable alteration, and supporting development of zolbetuximab as a therapeutic agent for this patient population.

Hybrid Biopolymer and Lipid Nanoparticles with Improved Transfection Efficacy for mRNA

Cells 9(9), 2034 (1-19) (2020). doi:10.3390/cells9092034

A Trans-amplifying RNA Vaccine Strategy for Induction of Potent Protective Immunity

Here, we present a potent RNA vaccine approach based on a novel bipartite vector system using trans-amplifying RNA (taRNA). The vector cassette encoding the vaccine antigen originates from an alphaviral self-amplifying RNA (saRNA), from which the replicase was deleted to form a transreplicon. Replicase activity is provided in trans by a second molecule, either by a standard saRNA or an optimized non-replicating mRNA (nrRNA). The latter delivered 10- to 100-fold higher transreplicon expression than the former. Moreover, expression driven by the nrRNA-encoded replicase in the taRNA system was as efficient as in a conventional monopartite saRNA system. We show that the superiority of nrRNA- ov…

Humoral immune responses of lung cancer patients against tumor antigen NY-ESO-1

The cancer-associated antigen NY-ESO-1 is expressed in a number of malignancies of different histological type. Patients with NY-ESO-1 expressing tumors have been shown to bear circulating autoantibodies against this antigen. In this study, we have assessed the NY-ESO-I autoantibody response in patients with lung cancer by a serum ELISA. Using a serum dilution of 1:400 we detected seroreactivity in 35 of 175 (20%) of patients. Incidence of autoantibodies was significantly higher in patients suffering from non small cell lung cancer (NSCLC, 23%) as compared to those with small cell lung cancer (SCLC, 9%). In the NSCLC group, NY-ESO-I antibody was significantly more frequent in patients with …

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 r…

The regulatory landscape for actively personalized cancer immunotherapies

An RNA toolbox for cancer immunotherapy.

Cancer immunotherapy has revolutionized oncology practice. However, current protein and cell therapy tools used in cancer immunotherapy are far from perfect, and there is room for improvement regarding their efficacy and safety. RNA-based structures have diverse functions, ranging from gene expression and gene regulation to pro-inflammatory effects and the ability to specifically bind different molecules. These functions make them versatile tools that may advance cancer vaccines and immunomodulation, surpassing existing approaches. These technologies should not be considered as competitors of current immunotherapies but as partners in synergistic combinations and as a clear opportunity to r…

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.

Among nucleic acid–based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdow…

Harnessing Tumor Mutations for Truly Individualized Cancer Vaccines

T cells are key effectors of anticancer immunity. They are capable of distinguishing tumor cells from normal ones by recognizing major histocompatibility complex–bound cancer-specific peptides. Accumulating evidence suggests that peptides associated with T cell–mediated tumor rejection arise predominantly from somatically mutated proteins and are unique to every patient's tumor. Knowledge of an individual's cancer mutanome (the entirety of cancer mutations) allows harnessing this enormous tumor cell–specific repertoire of highly immunogenic antigens for individualized cancer vaccines. This review outlines the preclinical and clinical state of individualized cancer vaccine development and t…

Claudin-18 gene structure, regulation, and expression is evolutionary conserved in mammals

Claudin-18 isoform 2 (CLDN18.2) is one of the few members of the human claudin family of tight junction molecules with strict restriction to one cell lineage. The objective of the current study was to compare molecular structure and tissue distribution of this gastrocyte specific molecule in mammals. We show here that the CLDN18.2 protein sequence is highly conserved, in particular with regard to functionally relevant domains in mouse, rat, rabbit, dog, monkey and human and also in lizards. Moreover, promoter regions of orthologs are highly homologous, including the binding site of the transcription factor cyclic AMP-responsive element binding protein (CREB), which is known to regulate acti…

A placenta-specific gene ectopically activated in many human cancers is essentially involved in malignant cell processes.

Abstract The identification and functional characterization of tumor-specific genes is a prerequisite for the development of targeted cancer therapies. Using an integrated data mining and experimental validation approach for the discovery of new targets for antibody therapy of cancer, we identified PLAC1. PLAC1 is a placenta-specific gene with no detectable expression in any other normal human tissue. However, it is frequently aberrantly activated and highly expressed in a variety of tumor types, in particular breast cancer. RNAi-mediated silencing of PLAC1 in MCF-7 and BT-549 breast cancer cells profoundly impairs motility, migration, and invasion and induces a G1-S cell cycle block with n…

BNT162b vaccines are immunogenic and protect non-human primates against SARS-CoV-2

AbstractA safe and effective vaccine against COVID-19 is urgently needed in quantities sufficient to immunise large populations. We report the preclinical development of two BNT162b vaccine candidates, which contain lipid-nanoparticle (LNP) formulated nucleoside-modified mRNA encoding SARS-CoV-2 spike glycoprotein-derived immunogens. BNT162b1 encodes a soluble, secreted, trimerised receptor-binding domain (RBD-foldon). BNT162b2 encodes the full-length transmembrane spike glycoprotein, locked in its prefusion conformation (P2 S). The flexibly tethered RBDs of the RBD-foldon bind ACE2 with high avidity. Approximately 20% of the P 2S trimers are in the two-RBD ‘down,’ one-RBD ‘up’ state. In mi…

Discovery and Subtyping of Neo-Epitope Specific T-Cell Responses for Cancer Immunotherapy: Addressing the Mutanome

Cancer accumulates 10s to 1000s of genomic mutations of which a fraction is immunogenic and may serve as an Achilles' heel of tumor cells. Mutation-specific T cells can recognize these antigens and destroy malignant cells. Strategies to immunotherapeutically address individual tumor mutations employing peptide or mRNA based vaccines are now actively investigated in mice and humans. An important step of determining the therapeutic potential of a mutanome vaccine is the detection of mutation reactive T-cell responses. In this chapter we provide protocols to identify and subtype mutation specific T cells in mice based on IFN-γ ELISpot and flow cytometry.

A novel tumour associated leucine zipper protein targeting to sites of gene transcription and splicing

We describe here the definition and characterization of antigen CT-8/HOM-TES-85 encoded by a previously unknown gene and identified by serological expression screening using antibodies from a seminoma patient. Intriguingly, the leucine zipper region of CT-8/HOM-TES-85 shows an atypical amphipathy with clusters of hydrophobic residues that is exclusively shared by the N-myc proto-oncogene. CT-8/HOM-TES-85 gene is tightly silenced in normal tissues except for testis. However, it is frequently activated in human neoplasms of different types including lung cancer, ovarian cancer, melanoma and glioma. Endogenous as well as heterogeneously expressed CT-8/HOM-TES-85 targets predominantly to the nu…

SeroGRID: an improved method for the rapid selection of antigens with disease related immunogenicity

Screening of cDNA expression libraries derived from human tumors with autologous sera (SEREX) permits the definition of immunogenic antigens in individual cancer patients. However, only a minority of SEREX-derived cDNA clones show a clear cancer-relatedness in the sense that circulating autoantibodies to them occur exclusively in the sera of tumor patients but not in healthy individuals. Evaluation of multiple SEREX-defined clones in serological assays using panels of allogeneic sera from cancer patients as well as appropriate control groups is an important step towards focussing on the relevant antigens. This in turn is the basis for defining disease parameters of diagnostic and prognostic…

Local delivery of mRNA-encoded cytokines promotes antitumor immunity and tumor eradication across multiple preclinical tumor models

Local immunotherapy ideally stimulates immune responses against tumors while avoiding toxicities associated with systemic administration. Current strategies for tumor-targeted, gene-based delivery, however, are limited by adverse effects such as off-targeting or antivector immunity. We investigated the intratumoral administration of saline-formulated messenger (m)RNA encoding four cytokines that were identified as mediators of tumor regression across different tumor models: interleukin-12 (IL-12) single chain, interferon-α (IFN-α), granulocyte-macrophage colony-stimulating factor, and IL-15 sushi. Effective antitumor activity of these cytokines relied on multiple immune cell populations and…

The Wnt/beta-Catenin Pathway Attenuates Experimental Allergic Airway Disease

Abstract Signaling via the Wnt/β-catenin pathway plays crucial roles in embryogenesis and homeostasis of adult tissues. In the lung, the canonical Wnt/β-catenin pathway has been implicated in remodeling processes, development of emphysema, and fibrosis. However, its relevance for the modulation of allergic responses in the lung remains unclear. Using genetically modified mice with lung-specific inducible (doxycycline) Wnt-1 expression (CCSP-rtTA × tetO-Wnt1), the impact of Wnt on the development of allergic airway disease was analyzed. Overexpression of Wnt during the allergen challenge phase attenuated the development of airway inflammation in an acute model, as well as in a more therapeut…

Expression of Claudin 18.2 and HER2 in gastric, gastroesophageal junction, and esophageal cancers : Results from the FAST study

4038 Background: Claudin 18.2 (CLDN18.2), a gastric mucosa tight junction protein, is aberrantly expressed in various cancers. In the FAST Phase 2 trial (NCT01630083), IMAB362, an anti-CLDN18.2 monoclonal antibody, administered in combination with EOX chemotherapy, prolonged survival compared to EOX alone in patients with advanced/recurrent gastric, gastroesophageal junction (GEJ), and esophageal cancers ineligible for trastuzumab. The aim of the present analysis was to assess tumor CLDN18.2 expression and co-expression with HER2 in the FAST population. Methods: Tumor tissue samples from patients screened for inclusion into the FAST trial were analyzed for CLDN18.2 expression using a CE-ma…

A shared tumor-antigen RNA-lipoplex vaccine with/without anti-PD1 in patients with checkpoint-inhibition experienced melanoma.

3136 Background: Cancer vaccines are considered unsuitable for patients with advanced tumours and have not been clinically successful. Methods: Lipo-MERIT is an ongoing phase 1/2 trial (NCT02410733) with melanoma FixVac, a liposomal RNA vaccine targeting four non-mutant shared tumour-associated antigens (TAAs) (MAGE-A3, NY-ESO-1, tyrosinase, TPTE). Patients with stage IIIB-C and IV melanoma are eligible. The trial comprises 7 dose escalation and 3 dose expansion cohorts, the latter with FixVac alone or combined with anti-PD1. Eight doses of FixVac are administered i.v. weekly/bi-weekly followed by optional continued monthly treatment. This abstract summarizes the findings of an exploratory…

Claudin-18 splice variant 2 is a pan-cancer target suitable for therapeutic antibody development

Abstract Purpose: Antibody-based cancer therapies have emerged as the most promising therapeutics in oncology. The purpose of this study was to discover novel targets for therapeutic antibodies in solid cancer. Experimental Design: We combined data mining and wet-bench experiments to identify strictly gastrocyte lineage–specific cell surface molecules and to validate them as therapeutic antibody targets. Results: We identified isoform 2 of the tight junction molecule claudin-18 (CLDN18.2) as a highly selective cell lineage marker. Its expression in normal tissues is strictly confined to differentiated epithelial cells of the gastric mucosa, but it is absent from the gastric stem cell zone. …

Development of an RNA-based kit for easy generation of TCR-engineered lymphocytes to control T-cell assay performance.

Cell-based assays to monitor antigen-specific T-cell responses are characterized by their high complexity and should be conducted under controlled conditions to lower multiple possible sources of assay variation. However, the lack of standard reagents makes it difficult to directly compare results generated in one lab over time and across institutions. Therefore TCR-engineered reference samples (TERS) that contain a defined number of antigen-specific T cells and continuously deliver stable results are urgently needed. We successfully established a simple and robust TERS technology that constitutes a useful tool to overcome this issue for commonly used T-cell immuno-assays. To enable users t…

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

Autoantibodies in breast cancer: their use as an aid to early diagnosis

There is increasing evidence that the immune system produces a humoral response to cancer-derived antigens. This study assessed the diagnostic potential of autoantibodies to multiple known tumour-associated proteins.Sera from normal controls (n = 94), primary breast cancer patients (n = 97) and patients with ductal carcinoma in situ (DCIS) (n = 40) were investigated for the presence of autoantibodies to p53, c-myc, HER2, NY-ESO-1, BRCA1, BRCA2 and MUC1 antigens by enzyme-linked immunosorbent assay.Reproducibly elevated levels of autoantibodies were seen in at least one of the six antigens in 64% of primary breast cancer patient sera and 45% of patients with DCIS at a specificity of 85%. No …

Large-scale analysis of SARS-CoV-2 spike-glycoprotein mutants demonstrates the need for continuous screening of virus isolates

AbstractDue to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it.We analyzed 146,917 SARS-CoV-2 genome assemblies and 2,393 NGS datasets from GISAID, NCBI Virus and NCBI SRA archives focusing on non-synonymous mutations in the spike protein.Only around 13.8% of the samples contained the wild-type spike protein with no variation from the reference. Among…

FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines

Intranodal immunization with antigen-encoding naked mRNA has proven to be an efficacious and safe approach to induce antitumor immunity. Thanks to its unique characteristics, mRNA can act not only as a source for antigen but also as an adjuvant for activation of the immune system. The search for additional adjuvants that can be combined with mRNA to further improve the potency of the immunization revealed Fms-like tyrosine kinase 3 (FLT3) ligand as a potent candidate. Systemic administration of the dendritic cell-activating FLT3 ligand prior to or along with mRNA immunization-enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tu…

88MO T-cell responses induced by an individualized neoantigen specific immune therapy in post (neo)adjuvant patients with triple negative breast cancer

Peptide microarrays enable rapid mimotope optimization for pharmacokinetic analysis of the novel therapeutic antibody IMAB362.

As membrane proteins play an important role in a variety of life-threatening diseases, the development of therapeutic monoclonal antibodies against membrane proteins is of significant interest. Among many other requirements, the process of antibody drug development requires a set of tailor-made assays for the characterization of the antibodies and for monitoring their activity. Designing assays to characterize antibodies directed to membrane proteins is challenging, because the natural targets are often not available in a format that is compatible with a biochemical assay setup. Thus, alternatives that mimic the targeted membrane proteins are needed. In this study, we developed optimal pept…

Generation of TCR-Engineered T Cells and Their Use To Control the Performance of T Cell Assays

Abstract 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 simp…

BNT162b2 Vaccine Encoding the SARS-CoV-2 P2 S Protects Transgenic hACE2 Mice against COVID-19.

BNT162b2 is a highly efficacious mRNA vaccine approved to prevent COVID-19. This brief report describes the immunogenicity and anti-viral protective effect of BNT162b2 in hACE2 transgenic mice. Prime-boost immunization with BNT162b2 elicited high titers in neutralizing antibodies against SARS-CoV-2, which correlated with viral clearance and alleviated lung lesions in these mice after viral challenge.

PO-324 Detection of high-risk prostate cancer biomarkers by RNA sequencing and qPCR method

Introduction New prognostic biomarkers for prostate cancer have the potential to overcome the clinical challenge of therapy decision and overtreatment. Present diagnostic and prognostic tests are still limited in specificity resulting in a large number of false positives and unnecessary biopsies. Furthermore, they do not enable a proper stratification between men with a high risk for an aggressive disease course requiring comprehensive therapy scheme after surgery and men with a low risk of disease recurrence cured after prostatectomy or eligible for active surveillance. In particular, patients with Gleason score 6 and 7 tumours (low and mid stage) are difficult to stratify for the appropri…

Abstract CT156: A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles encoding shared tumor antigens for immunotherapy of malignant melanoma

Abstract Therapeutic vaccination with tumor antigen-encoding RNAs is being investigated in various clinical trials. Typically, the RNA vaccine is administered intradermally, subcutaneously or intranodally with the intention to get expression of the encoded antigens in local antigen-presenting cells (APCs). We have developed a novel class of RNA-lipoplex (RNA(LIP)) immunotherapeutics for intravenous application, which allow systemic targeting of APCs. RNA(LIP) is a novel nanoparticulate formulation of lipid-complexed mRNA which selectively delivers the functional mRNA to APCs in lymphoid compartments body-wide for efficient mRNA uptake and expression of the encoded antigen by APCs. Moreover,…

A phase I dose-escalation study of IMAB362 (Zolbetuximab) in patients with advanced gastric and gastro-oesophageal junction cancer

Introduction IMAB362 (Zolbetuximab) is a chimeric monoclonal antibody that binds to Claudin-18.2, a target antigen specific to cancer cells. In vitro, IMAB362 mediates cell death through antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity; thus, IMAB362 may serve as a potent, targeted immunotherapeutic agent. Methods This first-in-human phase I study enroled adult patients (N = 15) with advanced gastric or gastro-oesophageal junction cancer into five sequential single dose-escalation cohorts (33, 100, 300, 600, and 1000 mg/m2) following a 3 + 3 design. Safety/tolerability, including determination of maximum tolerated dose and recommended phase II dose, were the pr…

Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine–elicited human sera

Vaccine protects against B1.1.7 variant The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B1.1.7 (VOC 202012/01) variant that emerged in late 2020 in the United Kingdom has many changes in the spike protein gene. Three of these are associated with enhanced infectivity and transmissibility, and there are concerns that B.1.1.7 might compromise the effectiveness of the vaccine. Muik et al. compared the neutralization efficacy of sera from 40 subjects immunized with the BioNTech-Pfizer mRNA vaccine BNT162b2 against a pseudovirus bearing the Wuhan reference strain or the lineage B.1.1.7 spike protein (see the Perspective by Altmann et al.). Serum was derived from 40 subjects in tw…

Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals.

Abstract Genetic modification of vaccines by linking the Ag to lysosomal or endosomal targeting signals has been used to route Ags into MHC class II processing compartments for improvement of CD4+ T cell responses. We report in this study that combining an N-terminal leader peptide with an MHC class I trafficking signal (MITD) attached to the C terminus of the Ag strongly improves the presentation of MHC class I and class II epitopes in human and murine dendritic cells (DCs). Such chimeric fusion proteins display a maturation state-dependent subcellular distribution pattern in immature and mature DCs, mimicking the dynamic trafficking properties of MHC molecules. T cell response analysis in…

Abstract 1907: Claudin 6 is a carcinoembryonic antigen with cancer stem cell marker features

Abstract Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but is aberrantly expressed in various human cancers, such as ovarian cancer (OC) and testicular cancer (TC). A monoclonal antibody against CLDN6, IMAB027, has shown promising antitumor activity in preclinical human CLDN6-positive (CLDN6+) cancer models. In this series of nonclinical studies, we investigated CLDN6 expression in normal and cancer tissues, as well as the localization and possible function of CLDN6 in cancer cells. Methods Expression of CLDN6 was assessed in a wide range of human tissues (eg, lung, colon, skin, ovary) and cultured cells b…

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

Abstract One of the hallmarks of cancer is the inherent instability of the genome leading to multiple genomic alterations and epigenetic changes that ultimately drive carcinogenesis. These processes lead to a unique molecular profile of every given tumor and to substantial intratumoral heterogeneity of cancer tissues. Recently, a series of independent reports revealed that pre-formed neoantigen specific T-cell responses are of crucial relevance for the clinical efficacy of immune checkpoint inhibitors. However, spontaneous immune recognition of neoantigens seems to be a rare event with only less than 1% of mutations inducing a T-cell response in the tumor-bearing patient. Accordingly, only …

549 An RNA-lipoplex (RNA-LPX) vaccine demonstrates strong immunogenicity and promising clinical activity in a Phase I trial in cutaneous melanoma patients with no evidence of disease at trial inclusion

BackgroundLipo-MERIT is an ongoing, first-in-human, open-label, dose-escalation Phase I trial investigating safety, tolerability and immunogenicity of BNT111 in patients with advanced melanoma. BNT111 is an RNA-LPX vaccine targeting the melanoma tumor-associated antigens (TAAs) New York esophageal squamous cell carcinoma 1 (NY-ESO-1), tyrosinase, melanoma-associated antigen 3 (MAGE-A3), and transmembrane phosphatase with tensin homology (TPTE). A previous exploratory interim analysis showed that BNT111, alone or combined with immune checkpoint inhibition (CPI), has a favorable adverse event (AE) profile, gives rise to antigen-specific T-cell responses and induces durable objective responses…

Determinants of intracellular RNA pharmacokinetics: Implications for RNA-based immunotherapeutics

RNAs with optimized properties are increasingly investigated as a tool to deliver the genetic information of complete antigens into professional antigen-presenting dendritic cells for HLA haplotype-independent antigen-specific vaccination against cancer. As the dose of the antigen and duration of its presentation are critical factors for generating strong and sustained antigen-specific immune responses, improvement of the immunobioavailability of RNA-based vaccines has been a recurrent subject of research. Substantial increase of the amount of antigen produced from RNA can be achieved by optimizing RNA stability and translational efficiency. Both features are determined by cis-acting elemen…

Mutanome directed cancer immunotherapy

Somatic mutations are important drivers of cancer development. Accumulating evidence suggests that a significant subset of mutations result in neo-epitopes recognized by autologous T cells and thus may constitute the Achilles' heel of tumor cells. T cells directed against mutations have been shown to have a key role in clinical efficacy of potent cancer immunotherapy modalities, such as adoptive transfer of autologous tumor infiltrating lymphocytes and immune checkpoint inhibitors. Whereas these findings strengthen the idea of a prominent role of neo-epitopes in tumor rejection, the systematic therapeutic exploitation of mutations was hampered until recently by the uniqueness of the reperto…

Identification of Tumor-Associated Autoantigens With SEREX

Serological analysis of tumor antigens by recombinant cDNA expression cloning (SEREX) allows the systematic cloning of tumor antigens recognized by the spontaneous autoantibody repertoire of cancer patients. For SEREX, cDNA expression libraries are constructed from fresh tumor specimens, packaged into lambda-phage vectors, and expressed recombinantly in Escherichia coli. Recombinant proteins expressed during the lytic infection of bacteria are transferred onto nitrocellulose membranes to be probed with diluted autologous patient serum for identification of clones reactive with high-titered IgG antibodies. This chapter describes the SEREX technology in detail.

220O Claudin 18.2 - a novel treatment target in the multicenter, randomized, phase II FAST study, a trial of epirubicin, oxaliplatin, and capecitabine (EOX) with or without the anti-CLDN18.2 antibody IMAB362 as 1st line therapy in advanced gastric and gastroesophageal junction (GEJ) cancer

Expression of multiple epigenetically regulated cancer/germline genes in nonsmall cell lung cancer.

Cancer/germline (CG) antigens represent promising targets for widely applicable mono- and multiantigen cancer vaccines for nonsmall cell lung cancer (NSCLC). Since little is known about their composite expression in this tumor type, we analyzed 7 CG genes (MAGE-A3, NY-ESO-1, LAGE-1, BRDT, HOM-TES-85, TPX-1 and LDHC) in 102 human NSCLC specimens. About 81% of NSCLC express at least 1 and half of the specimen at least 2 CG genes. Activation of most of these genes occurs more frequently in squamous cell cancer than in adenocarcinomas. Even though we found all genes but one to be regulated by genomic methylation, not all of them are co-expressed. In particular, combining CG genes not localized …

Studying Tumor-ReacTive T Cells: A Personalized Organoid Model.

Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer – T cell interactions for individual patients, and understand determinants of responsiveness, are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific T cell responses for epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich for tumor-reactive T cells from peripheral blood of patients with mismatch repair deficient colorectal cancer and non-small cell lung cancer. Furthermore, we …

A multicentre, phase IIa study of zolbetuximab as a single agent in patients with recurrent or refractory advanced adenocarcinoma of the stomach or lower oesophagus: the MONO study

Abstract Background Claudin 18.2 (CLDN18.2) is physiologically confined to gastric mucosa tight junctions; however, upon malignant transformation, perturbations in cell polarity lead to CLDN18.2 epitopes being exposed on the cancer cell surface. The first-in-class monoclonal antibody, zolbetuximab (formerly known as IMAB362), binds to CLDN18.2 and can induce immune-mediated lysis of CLDN18.2-positive cells. Patients and methods Patients with advanced gastric, gastro-oesophageal junction (GEJ) or oesophageal adenocarcinomas with moderate-to-strong CLDN18.2 expression in ≥50% of tumour cells received zolbetuximab intravenously every 2 weeks for five planned infusions. At least three patients …

Antitumor Vaccination with Synthetic mRNA: Strategies for In Vitro and In Vivo Preclinical Studies

Synthetic antigen-encoding mRNA is increasingly exploited as a tool for delivery of genetic information of complete antigens into professional antigen presenting dendritic cells for HLA haplotype-independent antigen-specific vaccination against cancer. Two strategies for mRNA-based antitumor vaccination have emerged into the clinical setting. One is transfection of autologous dendritic cells with synthetic mRNA for adoptive transfer into the patient. The other is direct injection of naked synthetic mRNA. Both methods have proven to be feasible and safe and to elicit antigen-specific immune responses. The design of novel synthetic vaccines employing synthetic mRNA requires further in-depth i…

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 p…

Translation of genomics-guided RNA-based personalised cancer vaccines: towards the bedside

Cancer is a disease caused by DNA mutations. Cancer therapies targeting defined functional mutations have shown clinical benefit. However, as 95% of the mutations in a tumour are unique to that single patient and only a small number of mutations are shared between patients, the addressed medical need is modest. A rapidly determined patient-specific tumour mutation pattern combined with a flexible mutation-targeting drug platform could generate a mutation-targeting individualised therapy, which would benefit each single patient. Next-generation sequencing enables the rapid identification of somatic mutations in individual tumours (the mutanome). Immunoinformatics enables predictions of mutat…

Abstract B041: A novel nanoparticular formulated tetravalent RNA cancer vaccine for treatment of patients with malignant melanoma

Abstract Immunotherapeutic approaches have evolved as promising and valid alternatives to available conventional cancer treatments. Amongst others, vaccination with tumor antigen-encoding RNAs by local administration is currently successfully employed in various clinical trials. To allow for a more efficient targeting of antigen-presenting cells (APCs) we have developed a novel RNA immunotherapeutic for systemic application based on a fixed set of four liposome complexed RNA drug products (RNA(LIP)) each encoding one shared melanoma-associated antigen. Similar to other liposomal drugs, the four injectable RNA(LIP) products constituting the investigational medicinal product will be prepared …

Large-scale analysis of SARS-CoV-2 spike-glycoprotein mutants demonstrates the need for continuous screening of virus isolates

Due to the widespread of the COVID-19 pandemic, the SARS-CoV-2 genome is evolving in diverse human populations. Several studies already reported different strains and an increase in the mutation rate. Particularly, mutations in SARS-CoV-2 spike-glycoprotein are of great interest as it mediates infection in human and recently approved mRNA vaccines are designed to induce immune responses against it. We analyzed 1,036,030 SARS-CoV-2 genome assemblies and 30,806 NGS datasets from GISAID and European Nucleotide Archive (ENA) focusing on non-synonymous mutations in the spike protein. Only around 2.5% of the samples contained the wild-type spike protein with no variation from the reference. Among…

NeoFox: annotating neoantigen candidates with neoantigen features

Abstract Summary The detection and prediction of true neoantigens is of great importance for the field of cancer immunotherapy. Wesearched the literature for proposed neoantigen features and integrated them into a toolbox called NEOantigen Feature toolbOX (NeoFox). NeoFox is an easy-to-use Python package that enables the annotation of neoantigen candidates with 16 neoantigen features. Availability and implementation NeoFox is freely available as an open source Python package released under the GNU General Public License (GPL) v3 license at https://github.com/TRON-Bioinformatics/neofox. Supplementary information Supplementary data are available at Bioinformatics online.

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 struc…

FAST: a randomised phase II study of zolbetuximab (IMAB362) plus EOX versus EOX alone for first-line treatment of advanced CLDN18.2-positive gastric and gastro-oesophageal adenocarcinoma.

Claudin 18.2 (CLDN18.2) is contained within normal gastric mucosa epithelial tight junctions; upon malignant transformation, CLDN18.2 epitopes become exposed. Zolbetuximab, a chimeric monoclonal antibody, mediates specific killing of CLDN18.2-positive cells through immune effector mechanisms.The FAST study enrolled advanced gastric/gastro-oesophageal junction and oesophageal adenocarcinoma patients (aged ≥18 years) with moderate-to-strong CLDN18.2 expression in ≥40% tumour cells. Patients received first-line epirubicin + oxaliplatin + capecitabine (EOX, arm 1, n = 84) every 3 weeks (Q3W), or zolbetuximab + EOX (loading dose, 800 mg/mIn the overall population, both PFS [hazard ratio (HR) = 0…

Preclinical evaluation of the anti-CLDN18.2 antibody, IMAB362, in pancreatic carcinoma

T-cell receptor transfer into human T cells with ecotropic retroviral vectors

Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the re…

Investigation of charge ratio variation in mRNA – DEAE-dextran polyplex delivery systems

Biomaterials 192, 612 - 620 (2019). doi:10.1016/j.biomaterials.2018.10.020

Steatohepatitis Impairs T-cell-Directed Immunotherapies Against Liver Tumors in Mice.

Background & Aims Nonalcoholic steatohepatitis causes loss of hepatic CD4+ T cells and promotes tumor growth. The liver is the most common site of distant metastases from a variety of malignancies, many of which respond to immunotherapy. We investigated the effects of steatohepatitis on the efficacy of immunotherapeutic agents against liver tumors in mice. Methods Steatohepatitis was induced by feeding C57BL/6NCrl or BALB/c AnNCr mice a methionine and choline–deficient diet or a choline-deficient l-amino acid–defined diet. Mice were given intrahepatic or subcutaneous injections of B16 melanoma and CT26 colon cancer cells, followed by intravenous injections of M30-RNA vaccine (M30) or intrap…

Racing for a SARS‐CoV‐2 vaccine

Interview with EMBO Members Özlem Türeci and Uğur Şahin, BioNTech, conducted by science journalist Kai Kupferschmidt.

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.

Abstract 882: The anti-claudin 6 antibody, IMAB027, induces antibody-dependent cellular and complement-dependent cytotoxicity in claudin 6-expressing cancer cells

Abstract Background Claudin 6 (CLDN6) is a tight junction membrane protein whose expression in normal tissue is confined to embryonic cells, but aberrantly expressed in various human cancer types, including some with a high medical need (eg, ovarian and uterine cancers). This tumor-specific expression in adult organs makes CLDN6 an attractive drug target; as such, IMAB027, an anti-CLDN6 monoclonal antibody (mAb), was developed. This report describes the preclinical characteristics of IMAB027. Methods IMAB027 was generated by hybridoma technology; the discovery process was set up so that mAbs that were good binders as well as inducers of the immune effector mechanisms of antibody-dependent c…

A non-functional neoepitope specific CD8+ T-cell response induced by tumor derived antigen exposure in vivo

Cancer-associated mutations, mostly single nucleotide variations, can act as neoepitopes and prime targets for effective anti-cancer T-cell immunity. T cells recognizing cancer mutations are critical for the clinical activity of immune checkpoint blockade (ICB) and they are potent vaccine antigens. High frequencies of mutation-specific T cells are rarely spontaneously induced. Hence, therapies that broaden the tumor specific T-cell response are of interest. Here, we analyzed neoepitope-specific CD8+ T-cell responses mounted either spontaneously or after immunotherapy regimens, which induce local tumor inflammation and cell death, in mice bearing tumors of the widely used colon carcinoma cel…

Functional TCR Retrieval from Single Antigen-Specific Human T Cells Reveals Multiple Novel Epitopes

Abstract 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-β chains directly from single antigen-spec…

Rapid molecular dissection of viral and bacterial immunomes

The development of preventive or therapeutic recombinant vaccines and the generation of serodiagnostic assays for infectious diseases depend essentially on the availability of molecularly defined antigens. A major bottleneck for the identification of suitable target antigens for many pathogens is the isolation of sufficient amounts of material for subsequent genomic or proteomic screening. Applying a highly efficient expression cloning strategy to the human pathogens vaccinia virus (VV) and Chlamydia pneumoniae (CP), we demonstrate that sub-nanogram amounts of isolated nucleic acids can be utilized to determine comprehensive sets of immunodominant antigens. Remarkably, the approach not only…

CrELISA: a fast and robust enzyme-linked immunosorbent assay bypassing the need for purification of recombinant protein

A multitude of antigens has been recently identified by screening of cDNA expression libraries derived from human tumors with autologous sera. Using a phage autoantibody assay and small panels of sera derived from cancer patients or controls it has been shown that some of these antigens display cancer-associated autoantibody responses. The diagnostic and prognostic significance of these potentially cancer-related autoantibodies remains unclear until large-scale assays are developed and serological data are available for hundreds of cancer patients and controls. The major bottleneck for the development of large-scale assays are the cloning, expression and the purification of each of the resp…

Combined Analysis of Antigen Presentation and T-cell Recognition Reveals Restricted Immune Responses in Melanoma.

Abstract The quest for tumor-associated antigens (TAA) and neoantigens is a major focus of cancer immunotherapy. Here, we combine a neoantigen prediction pipeline and human leukocyte antigen (HLA) peptidomics to identify TAAs and neoantigens in 16 tumors derived from seven patients with melanoma and characterize their interactions with their tumor-infiltrating lymphocytes (TIL). Our investigation of the antigenic and T-cell landscapes encompassing the TAA and neoantigen signatures, their immune reactivity, and their corresponding T-cell identities provides the first comprehensive analysis of cancer cell T-cell cosignatures, allowing us to discover remarkable antigenic and TIL similarities b…

Personalized vaccines for cancer immunotherapy

Cancer is characterized by an accumulation of genetic alterations. Somatic mutations can generate cancer-specific neoepitopes that are recognized by autologous T cells as foreign and constitute ideal cancer vaccine targets. Every tumor has its own unique composition of mutations, with only a small fraction shared between patients. Technological advances in genomics, data science, and cancer immunotherapy now enable the rapid mapping of the mutations within a genome, rational selection of vaccine targets, and on-demand production of a therapy customized to a patient’s individual tumor. First-in-human clinical trials of personalized cancer vaccines have shown the feasibility, safety, and immu…

Selective Activation of Trophoblast-specific PLAC1 in Breast Cancer by CCAAT/Enhancer-binding Protein β (C/EBPβ) Isoform 2

The trophoblast-specific gene PLAC1 (placenta-specific 1) is ectopically expressed in a wide range of human malignancies, most frequently in breast cancer, and is essentially involved in cancer cell proliferation, migration, and invasion. Here we show that basal activity of the PLAC1 promoter is selectively controlled by ubiquitous transcription factor SP1 and isoform 2 of CCAAT/enhancer-binding protein beta that we found to be selectively expressed in placental tissue and cancer cells. Binding of both factors to their respective elements within the PLAC1 promoter was essential to attain full promoter activity. Estrogen receptor alpha (ERalpha) signaling further augmented transcription and …

In Silico Typing of Classical and Non-classical HLA Alleles from Standard RNA-Seq Reads

Next-Generation Sequencing (NGS) enables the rapid generation of billions of short nucleic acid sequence fragments (i.e., "sequencing reads"). Especially, the adoption of gene expression profiling using whole transcriptome sequencing (i.e., "RNA-Seq") has been rapid. Here, we describe an in silico method, seq2HLA, that takes standard RNA-Seq reads as input and determines a sample's (classical and non-classical) HLA class I and class II types as well as HLA expression. We demonstrate the application of seq2HLA using publicly available RNA-Seq data from the Burkitt's lymphoma cell line DAUDI and the choriocarcinoma cell line JEG-3.

Translating nanoparticulate-personalized cancer vaccines into clinical applications: case study with RNA-lipoplexes for the treatment of melanoma

The development of nucleic acid based vaccines against cancer has gained considerable momentum through the advancement of modern sequencing technologies and on novel RNA-based synthetic drug formats, which can be readily adapted following identification of every patient's tumor-specific mutations. Furthermore, affordable and individual ‘on demand’ production of molecularly optimized vaccines should allow their application in large groups of patients. This has resulted in the therapeutic concept of an active personalized cancer vaccine, which has been brought into clinical testing. Successful trials have been performed by intranodal administration of sterile isotonic solutions of synthetic …

Characterization of the first-in-class T-cell-engaging bispecific single-chain antibody for targeted immunotherapy of solid tumors expressing the oncofetal protein claudin 6.

abstract The fetal tight junction molecule claudin 6 (CLDN6) is virtually absent from any normal tissue, whereas it is aberrantly and frequently expressed in various cancers of high medical need. We engineered 6PHU3, a T-cell-engaging bispecific single chain molecule (bi-(scFv)2) with anti-CD3/anti-CLDN6 specificities, and characterized its pharmacodynamic properties. Our data show that upon engagement by 6PHU3, T cells strongly upregulate cytotoxicity and activation markers, proliferate and acquire an effector phenotype. 6PHU3 exerts potent killing of cancer cells in vitro with EC50 values in the pg/mL range. Subcutaneous xenograft tumors in NSG mice engrafted with human PBMCs are eradicat…

HLA typing from RNA-Seq sequence reads.

We present a method, seq2HLA, for obtaining an individual's human leukocyte antigen (HLA) class I and II type and expression using standard next generation sequencing RNA-Seq data. RNA-Seq reads are mapped against a reference database of HLA alleles, and HLA type, confidence score and locus-specific expression level are determined. We successfully applied seq2HLA to 50 individuals included in the HapMap project, yielding 100% specificity and 94% sensitivity at a P-value of 0.1 for two-digit HLA types. We determined HLA type and expression for previously un-typed Illumina Body Map tissues and a cohort of Korean patients with lung cancer. Because the algorithm uses standard RNA-Seq reads and …

Polysarcosine-functionalized lipid nanoparticles for therapeutic mRNA delivery

Polysarcosine (pSar) is a polypeptoid based on the endogenous amino acid sarcosine (N-methylated glycine), which has previously shown potent stealth properties. Here, lipid nanoparticles (LNPs) for therapeutic application of messenger RNA were assembled using pSarcosinylated lipids as a tool for particle engineering. Using pSar lipids with different polymeric chain lengths and molar fractions enabled the control of the physicochemical characteristics of the LNPs, such as particle size, morphology, and internal structure. In combination with a suited ionizable lipid, LNPs were assembled, which displayed high RNA transfection potency with an improved safety profile after intravenous injection…

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 mutations. Here we show in three independent murine tumour models that a considerable fraction of non-synonymous cancer mutations is immunogenic and that, unexpectedly, the …

HPV16 RNA-LPX vaccine mediates complete regression of aggressively growing HPV-positive mouse tumors and establishes protective T cell memory

ABSTRACT HPV16 infections are associated with a variety of cancers and there is compelling evidence that the transforming activity of HPV16 critically depends on the expression of the viral oncoproteins E6 and E7. Therapeutic cancer vaccines capable of generating durable and specific immunity against these HPV16 antigens hold great promise to achieve long-term disease control. Here we show in mice that HPV16 E7 RNA-LPX, an intravenously administered cancer vaccine based on immuno-pharmacologically optimized antigen-encoding mRNA, efficiently primes and expands antigen-specific effector and memory CD8+ T cells. HPV-positive TC-1 and C3 tumors of immunized mice are heavily infiltrated with ac…

High Prevalence of Claudin 18.2 Expression in Japanese Patients with Gastric Cancer.

e15584 Background: Expression of the gastric mucosal tight junction protein, Claudin-18.2 (CLDN18.2), is altered in gastric cancer (GC). In a phase 2 clinical trial (NCT01630083; FAST), a monoclonal antibody against CLDN18.2 (IMAB362) significantly increased overall survival in European patients with CLDN18.2-positive (CLDN18.2+) gastric and gastroesophageal junction adenocarcinomas when added to an EOX chemotherapy regimen. As the GC occurrence is high in Japan, this study evaluated the prevalence of CLDN18.2 expression in Japanese patients with GC. Methods: CLDN18.2 expression was assessed in primary GC tumors and corresponding lymph node metastases (LNM) by cell membrane staining intens…

mRNA-based therapeutics — developing a new class of drugs

In vitro transcribed (IVT) mRNA has recently come into focus as a potential new drug class to deliver genetic information. Such synthetic mRNA can be engineered to transiently express proteins by structurally resembling natural mRNA. Advances in addressing the inherent challenges of this drug class, particularly related to controlling the translational efficacy and immunogenicity of the IVTmRNA, provide the basis for a broad range of potential applications. mRNA-based cancer immunotherapies and infectious disease vaccines have entered clinical development. Meanwhile, emerging novel approaches include in vivo delivery of IVT mRNA to replace or supplement proteins, IVT mRNA-based generation o…

Complex Cellular Responses of Helicobacter pylori-Colonized Gastric Adenocarcinoma Cells ▿

ABSTRACT Helicobacter pylori is an important class I carcinogen that persistently infects the human gastric mucosa to induce gastritis, gastric ulceration, and gastric cancer. H. pylori pathogenesis strongly depends on pathogenic factors, such as VacA (vacuolating cytotoxin A) or a specialized type IV secretion system (T4SS), which injects the oncoprotein CagA (cytotoxin-associated gene A product) into the host cell. Since access to primary gastric epithelial cells is limited, many studies on the complex cellular and molecular mechanisms of H. pylori were performed in immortalized epithelial cells originating from individual human adenocarcinomas. The aim of our study was a comparative anal…

Elimination of large tumors in mice by mRNA-encoded bispecific antibodies.

The potential of bispecific T cell-engaging antibodies is hindered by manufacturing challenges and short serum half-life. We circumvented these limitations by treating mice with in vitro-transcribed pharmacologically optimized, nucleoside-modified mRNA encoding the antibody. We achieved sustained endogenous synthesis of the antibody, which eliminated advanced tumors as effectively as the corresponding purified bispecific antibody. Because manufacturing of pharmaceutical mRNA is fast, this approach could accelerate the clinical development of novel bispecific antibodies.

First-in-human study of IMAB362, an anti-claudin 18.2 monoclonal antibody, in patients with advanced gastroesophageal cancer

mRNA Vaccination and Personalized Cancer Therapy

Nucleic acid vaccines link two prerequisites for success, namely, the delivery of molecularly defined antigens as vaccine targets of interest and an inherent adjuvant activity. As compared to DNA-based approaches, in vitro-transcribed messenger RNA (mRNA) is a safer drug format due to the adjustable, transient expression and lack of genomic integration. In contrast to viral vector vaccines, mRNA vaccination is not limited by the emergence of immune responses against antigens produced by the viral vector backbones. Thus, mRNA vaccines are particularly attractive for cancer immunotherapy for which induction of clinically meaningful antigen-specific immune responses depends on repeated immuniz…

Highly specific auto-antibodies against claudin-18 isoform 2 induced by a chimeric HBcAg virus-like particle vaccine kill tumor cells and inhibit the growth of lung metastases.

Abstract Strategies for antibody-mediated cancer immunotherapy, such as active immunization with virus-like particle (VLP)-based vaccines, are gaining increasing attention. We developed chimeric hepatitis B virus core antigen (HBcAg)-VLPs that display a surface epitope of the highly selective tumor-associated cell lineage marker claudin-18 isoform 2 (CLDN18.2) flanked by a mobility-increasing linker. Auto-antibodies elicited by immunization with these chimeric HBcAg-VLPs in 2 relevant species (mouse and rabbit) bind with high precision to native CLDN18.2 at physiologic densities on the surface of living cells but not to the corresponding epitope of the CLDN18.1 splice variant that differs b…

Final results of the FAST study, an international, multicenter, randomized, phase II trial of epirubicin, oxaliplatin, and capecitabine (EOX) with or without the anti-CLDN18.2 antibody IMAB362 as first-line therapy in patients with advanced CLDN18.2+ gastric and gastroesophageal junction (GEJ) adenocarcinoma

Background: IMAB362, a chimeric monoclonal antibody that mediates specific killing of cancer cells expressing the tight junction protein Claudin18.2 (CLDN18.2) by activation of immune effector mechanisms, has demonstrated single-agent activity and tolerability in patients ( pts) with heavily pretreated gastric cancer. Methods: Pts with advanced/recurrent gastric and GEJ cancer were centrally evaluated for CLDN18.2 expression by immunohistochemistry (CLAUDETECT® 18.2 Histology Kit). Eligible pts had a CLDN18.2 expression of ≥2+ in ≥40% tumor cells, an ECOG PS of 0–1 and were not eligible for trastuzumab. Pts were randomized 1:1 to first-line EOX (epirubicin 50 mg/m2 and oxaliplatin 130 mg/m2…

Identification and analysis of tumor-associated antigens in triple-negative breast cancer (TNBC)

Incorporation of mRNA in Lamellar Lipid Matrices for Parenteral Administration

Molecular pharmaceutics 15(2), 642 - 651 (2018). doi:10.1021/acs.molpharmaceut.7b01022

In silico strategy for detection of target candidates for antibody therapy of solid tumors

In contrast to earlier attempts for the identification of target candidates suitable for monoclonal antibody (mAb) based cancer therapies we concentrated on highly selective lineage-specific genes additionally preserved or even overexpressed in orthotopic cancers. In a script aided workflow we reduced all human entries of the RefSeq mRNA database to those encoding transmembrane domain bearing gene products and subjected them to BLAST analysis against the human EST database. All BLAST results were validated in a gene centric way allowing two types of data curation prior to expression profiling of matching ESTs in selected healthy tissues: (i) exclusion of questionable ESTs arising e.g. from …

Efficacy and Safety of Multiple Doses of Imab362 in Patients with Advanced Gastro-Esophageal Cancer: Results of a Phase Ii Study

ABSTRACT Aim: IMAB362 is a monoclonal antibody specifically targeting claudin 18 isoform 2 (CLDN18.2), which is expressed on gastric cancer cells, whereas it is only present on a fraction of healthy stomach cells. This may reduce the risk of target-related side effects. Single-agent IMAB362 appears safe in patients with advanced gastro-esophageal cancer (GEC) based on data from a phase I trial. Methods: This international, multicenter, non-randomized phase IIa study (NCT01197885) investigated the efficacy and safety of repeated doses of IMAB362 (300 and 600 mg/m2) in patients with metastatic, refractory/recurrent, CLDN18.2-positive GEC (i.e. cancer of the stomach, the lower esophagus and th…

Abstract CT201: The Mutanome Engineered RNA Immuno-Therapy (MERIT) project

Abstract The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically and industrially validate a pioneering RNA-based immunotherapy concept that targets individual tumor antigens and tumor-specific mutations in triple negative breast cancer (TNBC) patients. This biomarker-guided, personalized therapy is a collaborative effort of five partners from academia and industry and is funded by the European Commission's FP7 and led by BioNTech AG. TNBC is an aggressive, molecularly heterogeneous cancer that accounts for 20% of all breast cancer patients. The 5-year survival rate is less than 80%. The molecular heterogeneity across TNBCs results in a lack of common targetable molecu…

Abstract 2903: IMAB362, a novel first-in-class monoclonal antibody for treatment of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC), the most frequent subtype (>80%) of pancreatic cancer (PC) is characterized by a generally lethal progress within a short period of time after primary diagnosis. Despite high efforts, the treatment options are very limited and mainly of palliative nature. Therefore, we investigated whether IMAB362 might represent a potential treatment option in this patient population. IMAB362 is a highly potent and tumor-cell selective therapeutic antibody which is currently in clinical development in gastro-esophageal cancer (in phase II and IIb trials). IMAB362 is directed against the tight junction molecule CLDN18.2, a proliferation-promoting tran…

Correction for Schneider et al., Complex Cellular Responses of Helicobacter pylori-Colonized Gastric Adenocarcinoma Cells

Volume 79, no. 6, p. [2362–2371][1], 2011. Page 2368: Figure 5 should appear as shown below. ![Figure][2] [1]: /lookup/doi/10.1128/IAI.01350-10 [2]: pending:yes

Myeloid Cell-Derived Reactive Oxygen Species Induce Epithelial Mutagenesis

Increased oxidative stress has been suggested to initiate and promote tumorigenesis by inducing DNA damage and to suppress tumor development by triggering apoptosis and senescence. The contribution of individual cell types in the tumor microenvironment to these contrasting effects remains poorly understood. We provide evidence that during intestinal tumorigenesis, myeloid cell-derived H2O2 triggers genome-wide DNA mutations in intestinal epithelial cells to stimulate invasive growth. Moreover, increased reactive oxygen species (ROS) production in myeloid cells initiates tumor growth in various organs also in the absence of a carcinogen challenge in a paracrine manner. Our data identify an i…

Identification of new claudin family members by a novel PSI-BLAST based approach with enhanced specificity.

In an attempt to develop a novel strategy for the identification of new members of protein families by in silico approaches, we have developed a semi-automated procedure of consecutive PSI-BLAST (Position-Specific-Iterated Basic Local Alignment Search Tool) searches incorporating identificiation as well as subsequent validation of putative candidates. For a proof of concept study we chose the search for novel members of the claudin family. The initial step was an iterated PSI-BLAST search starting with the PMP22_Claudin domain of each known member of the claudin family against the human part of the RefSeq Database. Putative new claudin domains derived from the converged list were evaluated …

An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors.

A one-two, CAR-T cell punch Chimeric antigen receptor (CAR)–T cells have been clinically effective in killing certain hematological malignancies, but achieving long-term patient responses for solid tumors remains a challenge. Reinhard et al. describe a two-part “CARVac” strategy to overcome poor CAR-T cell stimulation and responses in vivo. They introduce the tight junction protein claudin 6 (CLDN6) as a new CAR-T cell target and designed a nanoparticulate RNA vaccine encoding a chimeric receptor directed toward CLDN6. This lipoplex RNA vaccine promotes CLDN6 expression on the surface of dendritic cells, which in turn stimulates and enhances the efficacy of CLDN6-CAR-T cells for improved tu…

Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy

Lymphoid organs, in which antigen presenting cells (APCs) are in close proximity to T cells, are the ideal microenvironment for efficient priming and amplification of T-cell responses. However, the systemic delivery of vaccine antigens into dendritic cells (DCs) is hampered by various technical challenges. Here we show that DCs can be targeted precisely and effectively in vivo using intravenously administered RNA-lipoplexes (RNA-LPX) based on well-known lipid carriers by optimally adjusting net charge, without the need for functionalization of particles with molecular ligands. The LPX protects RNA from extracellular ribonucleases and mediates its efficient uptake and expression of the encod…

Chromatin Immunoprecipitation Assay to Identify Genomic Binding Sites of Regulatory Factors

DNA-protein interactions are vital to fundamental cellular events including transcription, replication, DNA repair, and recombination. Thus, their study holds the key to our understanding of mechanisms underlying normal development and homeostasis as well as disease. Transcriptional regulation is a highly complex process that involves recruitment of numerous factors resulting in formation of multi-protein complexes at gene promoters to regulate gene expression. The studied proteins can be, for example, transcription factors, epigenetic regulators, co-activators, co-repressors, or ligand-activated nuclear receptors as estrogen receptor-α (ERα) bound either directly to the DNA or indirectly b…

Selective uptake of naked vaccine RNA by dendritic cells is driven by macropinocytosis and abrogated upon DC maturation.

Even though it is known for more than one decade that antigen-encoding RNA can deliver antigenic information to induce antigen-specific immunity against cancer, the nature and mechanism of RNA uptake have remained enigmatic. In this study, we investigated the pharmacokinetics of naked RNA administered into the lymph node. We observed that RNA is rapidly and selectively uptaken by lymph node dendritic cells (DCs). Furthermore, in vitro and in vivo studies revealed that the efficient internalization of RNA by human and murine DCs is primarily driven by macropinocytosis. Selective inhibition of macropinocytosis by compounds or as a consequence of DC maturation abrogated RNA internalization and…

Abstract CT301: A phase Ib study to evaluate RO7198457, an individualized Neoantigen Specific immunoTherapy (iNeST), in combination with atezolizumab in patients with locally advanced or metastatic solid tumors

Abstract Background: Neoantigens arising from somatic mutations are attractive targets for cancer immunotherapy as they may be recognized as foreign by the immune system. RO7198457, a systemically administered RNA-Lipoplex iNeST was designed to stimulate T cell responses against neoantigens. A first-in-human Phase Ib study of RO7198457, in combination with the aPD-L1 antibody atezolizumab is being conducted in patients with locally advanced or metastatic solid tumors. Methods: RO7198457 is manufactured on a per-patient basis and contains up to 20 tumor-specific neoepitopes. Nine doses of RO7198457 were administered i.v. in weekly and bi-weekly intervals during the 12-week induction stage an…

NF-κB factors control the induction of NFATc1 in B lymphocytes

In peripheral lymphocytes, the transcription factors (TFs) NF-κB, NFAT, and AP-1 are the prime targets of signals that emerge from immune receptors. Upon activation, these TFs induce gene networks that orchestrate the growth, expansion, and effector function of peripheral lymphocytes. NFAT and NF-κB factors share several properties, such as a similar mode of induction and architecture in their DNA-binding domain, and there is a subgroup of κB-like DNA promoter motifs that are bound by both types of TFs. However, unlike NFAT and AP-1 factors that interact and collaborate in binding to DNA, NFAT, and NF-κB seem neither to interact nor to collaborate. We show here that NF-κB1/p50 and c-Rel, th…

Confidence-based Somatic Mutation Evaluation and Prioritization

Next generation sequencing (NGS) has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR), to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the exist…

Past, present and future of immunology in Mainz.

Multi-Omics Characterization of the 4T1 Murine Mammary Gland Tumor Model

Background: Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. The 4T1 murine mammary cancer cell line is one of the most widely used breast cancer models. Here, we present an integrated map of the genome, transcriptome, and immunome of 4T1. Results: We found Trp53 (Tp53) and Pik3g to be mutated. Other frequently mutated genes in breast cancer, including Brca1 and Brca2, are not mutated. For cancer related genes, Nav3, Cenpf, Muc5Ac, Mpp7, Gas1, MageD2, Dusp1, Ros, Polr2a, Rragd, Ros1, and Hoxa9 are mutated. Markers for cell proliferation like Top2a, Birc5, and Mki67 are highly expressed, so are markers for metastasis like Msln, Ect2, and P…

Phosphorothioate cap analogs increase stability and translational efficiency of RNA vaccines in immature dendritic cells and induce superior immune responses in vivo

Vaccination with in vitro transcribed RNA coding for tumor antigens is considered a promising approach for cancer immunotherapy and has already entered human clinical testing. One of the basic objectives for development of RNA as a drug is the optimization of immunobioavailability of the encoded antigen in vivo. By analyzing the effect of different synthetic 5' mRNA cap analogs on the kinetics of the encoded protein, we found that m(2)(7,2'-O)Gpp(S)pG (beta-S-ARCA) phosphorothioate caps, in particular the D1 diastereoisomer, profoundly enhance RNA stability and translational efficiency in immature but not mature dendritic cells. Moreover, in vivo delivery of the antigen as beta-S-ARCA(D1)-c…