2020) GD2-targeted chimeric antigen receptor T cells prevent metastasis formation by elimination of breast cancer stem-like cells, OncoImmunology, 9:1, 1683345, ABSTRACT Expression of the disialoganglioside GD2 has been identified as a marker antigen associated with a breast cancer stem-like cell (BCSC) phenotype. Here, we report on the evaluation of GD2 as a BCSCspecific target antigen for immunotherapy. GD2 expression was confirmed at variable degree in a set of breast cancer cell lines, predominantly in triple-negative breast cancer (TNBC). To target GD2, we have generated novel anti-GD2 chimeric antigen receptors (GD2-CAR), based on single-chain variable fragments (scFv) derived from the monoclonal antibody (mAb) ch14.18, also known as dinutuximab beta. Expressed on T cells, GD2-CARs mediated specific GD2-dependent T-cell activation and target cell lysis. In contrast to previously described GD2-CARs, no signs of exhaustion by tonic signaling were found. Importantly, application of GD2-CAR expressing T cells (GD2-CAR-T) in an orthotopic xenograft model of TNBC (MDA-MB-231) halted local tumor progression and completely prevented lung metastasis formation. In line with the BCSC model, GD2 expression was only found in a subpopulation (4-6%) of MDA-MB-231 cells before injection. Significant expansion of GD2-CAR-T in tumor-bearing mice as well as T-cell infiltrates in the primary tumor and the lungs were found, indicating site-specific activation of GD2-CAR-T. Our data strongly support previous findings of GD2 as a BCSC-associated antigen. GD2-targeted immunotherapies have been extensively studied in human. In conclusion, GD2-CAR-T should be considered a promising novel approach for GD2-positive breast cancer, especially to eliminate disseminated tumor cells and prevent metastasis formation. ARTICLE HISTORY

Several COVID-19 vaccines are approved to prevent severe disease outcome following SARS-CoV-2 infection. Whereas the induction and functionality of anti-viral antibody response is largely studied, the induction of T cells upon vaccination with the different approved COVID-19 vaccines is less studied. Here, we reported on T-cell immunity four weeks and six months after different vaccination regimens and four weeks after an additional booster vaccination, in comparison to SARS-CoV-2 T-cell responses in convalescents and prepandemic donors using interferon-gamma ELISpot assays and flow cytometry. Increased T-cell responses and cross-recognition of B.1.1.529 Omicron variant-specific mutations were observed ex vivo in mRNA- and heterologous-vaccinated donors compared to vector-vaccinated donors. Nevertheless, potent expandability of T cells targeting the spike protein was observed for all vaccination regimens with frequency, diversity and the ability to produce several cytokines of vaccine-induced T-cell responses comparable to those in convalescent donors. T-cell responses for all vaccinated donors significantly exceeded preexisting cross-reactive T-cell responses in prepandemic donors. Booster vaccination led to a significant increase of anti-spike IgG responses, which showed a marked decline 6 month after complete vaccination. In contrast, T-cell responses remained stable over time following complete vaccination with no significant effect of booster vaccination on T-cell responses and cross-recognition of Omicron BA.1 and BA.2 mutations.This suggested that booster vaccination is of particular relevance for the amelioration of antibody response. Together, our work shows that different vaccination regimens induce broad and long-lasting spike-specific CD4 + and CD8 + T-cell immunity to SARS-CoV-2.

Chimeric antigen receptor (CAR)-T therapy holds great promise to sustainably improve cancer treatment. However, currently, a broad applicability of CAR-T cell therapies is hampered by limited CAR-T cell versatility and tractability and the lack of exclusive target antigens to discriminate cancerous from healthy tissues. To achieve temporal and qualitative control on CAR-T function, we engineered the Adapter CAR (AdCAR) system. AdCAR-T are redirected to surface antigens via biotin-labeled adapter molecules in the context of a specific linker structure, referred to as Linker-Label-Epitope. AdCAR-T execute highly specific and controllable effector function against a multiplicity of target antigens. In mice, AdCAR-T durably eliminate aggressive lymphoma. Importantly, AdCAR-T might prevent antigen evasion by combinatorial simultaneous or sequential targeting of multiple antigens and are capable to identify and differentially lyse cancer cells by integration of adapter molecule-mediated signals based on multiplex antigen expression profiles. In consequence the AdCAR technology enables controllable, flexible, combinatorial, and selective targeting.

The DNAJB1-PRKACA fusion transcript is the oncogenic driver in fibrolamellar hepatocellular carcinoma, a lethal disease lacking specific therapies. This study reports on the identification, characterization, and immunotherapeutic application of HLA-presented neoantigens specific for the DNAJB1-PRKACA fusion transcript in fibrolamellar hepatocellular carcinoma. DNAJB1-PRKACA-derived HLA class I and HLA class II ligands induce multifunctional cytotoxic CD8+ and T-helper 1 CD4+ T cells, and their cellular processing and presentation in DNAJB1-PRKACA expressing tumor cells is demonstrated by mass spectrometry-based immunopeptidome analysis. Single-cell RNA sequencing further identifies multiple T cell receptors from DNAJB1-PRKACA-specific T cells. Vaccination of a fibrolamellar hepatocellular carcinoma patient, suffering from recurrent short interval disease relapses, with DNAJB1-PRKACA-derived peptides under continued Poly (ADP-ribose) polymerase inhibitor therapy induces multifunctional CD4+ T cells, with an activated T-helper 1 phenotype and high T cell receptor clonality. Vaccine-induced DNAJB1-PRKACA-specific T cell responses persist over time and, in contrast to various previous treatments, are accompanied by durable relapse free survival of the patient for more than 21 months post vaccination. Our preclinical and clinical findings identify the DNAJB1-PRKACA protein as source for immunogenic neoepitopes and corresponding T cell receptors and provide efficacy in a single-patient study of T cell-based immunotherapy specifically targeting this oncogenic fusion.

Background The rapid emergence of the omicron variant and its large number of mutations led to its classification as a variant of concern (VOC) by the WHO. Subsequently, omicron evolved into distinct sublineages (e.g. BA1 and BA2), which currently represent the majority of global infections. Initial studies of the neutralizing response towards BA1 in convalescent and vaccinated individuals showed a substantial reduction. Methods We assessed antibody (IgG) binding, ACE2 (Angiotensin-Converting Enzyme 2) binding inhibition, and IgG binding dynamics for the omicron BA1 and BA2 variants compared to a panel of VOC/VOIs, in a large cohort (n = 352) of convalescent, vaccinated, and infected and subsequently vaccinated individuals. Results While omicron was capable efficiently binding to ACE2, antibodies elicited by infection or immunization showed reduced binding capacities and ACE2 binding inhibition compared to WT. Whereas BA1 exhibited less IgG binding compared to BA2, BA2 showed reduced inhibition of ACE2 binding. Among vaccinated samples, antibody binding to omicron only improved after administration of a third dose. Conclusion omicron BA1 and BA2 can still efficiently bind to ACE2, while vaccine/infection-derived antibodies can bind omicron. The extent of the mutations within both variants prevent a strong inhibitory binding response. As a result, both omicron variants are able to evade control by pre-existing antibodies.

The rapid emergence of the Omicron variant and its large number of mutations has led to its classification as a variant of concern (VOC) by the WHO. Initial studies on the neutralizing response towards this variant within convalescent and vaccinated individuals have identified substantial reductions. However many of these sample sets used in these studies were either small, uniform in nature, or were compared only to wild-type (WT) or, at most, a few other VOC. Here, we assessed IgG binding, (Angiotensin-Converting Enzyme 2) ACE2 binding inhibition, and antibody binding dynamics for the omicron variant compared to all other VOC and variants of interest (VOI), in a large cohort of infected, vaccinated, and infected and then vaccinated individuals. While omicron was capable of binding to ACE2 efficiently, antibodies elicited by infection or immunization showed reduced IgG binding and ACE2 binding inhibition compared to WT and all VOC. Among vaccinated samples, antibody binding responses towards omicron were only improved following administration of a third dose. Overall, our results identify that omicron can still bind ACE2 while pre-existing antibodies can bind omicron. The extent of the mutations appear to inhibit the development of a neutralizing response, and as a result, omicron remains capable of evading immune control.

In individuals with low-to-moderate amounts of coronary artery calcium, 16-detector CT coronary angiography has high sensitivity and specificity for the diagnosis of significant coronary artery stenosis.

Despite recent advances and the approval of novel molecular therapies in acute myeloid leukemia (AML), the disease is still characterized by high relapse rates and poor overall survival due to the persistence of therapy-resistant residual leukemic progenitor cells (LPCs). T cell-based immunotherapy has been suggested as a novel therapeutic option to eliminate minimal residual disease and achieve long-lasting remissions. One main prerequisite for immunotherapy development is the selection of immunogenic targets that show natural, high-frequent, and tumor-exclusive presentation on the cell surface of malignant cells. In a previous study we characterized the antigenic landscape of AML by mass spectrometry to identify AML-specific T cell epitopes (Berlin et al. Leukemia 2015). Here, we aimed to analyze the immunopeptidome of primary AML progenitor cells (n = 10, purity >80% CD34+CD38-) to identify LPC-associated antigens that enable the specific targeting of AML LPCs. Additionally, we analyzed an extended set of AML patient samples (n = 47) to screen for naturally presented neoepitopes and to identify broadly applicable AML-associated target antigens that are presented on both AML blasts and LPCs. We identified more than 16,000 HLA class I- and 17,000 HLA class II-presented peptides on LPCs and 72,000 HLA class I and 61,000 HLA class II peptides in the total AML cohort. Comparative profiling of LPCs, AML blasts, and a benign tissue database (n = 332) revealed HLA class I- and HLA class II-presented LPC-exclusive as well as frequently presented AML-associated antigens on both AML blasts and LPCs. Besides these tumor-exclusive self-peptides, we detected naturally presented neoepitopes derived from two frequent mutations (NPM1 and IDH2) in this low-mutational burden malignancy. For immunogenicity analyses we selected 16 HLA class I- and 15 HLA class II-restricted peptides comprising unmutated as well as mutation-derived antigens. In vitro priming experiments showed the induction of peptide-specific, multi-functional, and cytotoxic CD8+ effector cells in samples of healthy volunteers and AML patients. We were able to detect strong preexisting memory T cell responses targeting LPC-associated antigens in AML patients with detection frequencies of up to 20%. Retrospective analyses revealed that patients with preexisting peptide-specific T cell responses showed improved overall survival compared to patients without any memory responses against our targets. Taken together, we identified novel, naturally presented, LPC-exclusive, and AML-associated self-antigens and neoepitopes presented on both AML blasts and LPCs. We could demonstrate the immunogenicity of 14/16 (88%) HLA class I and 13/15 (87%) HLA class II antigens highlighting their potential as promising targets for T cell-based immunotherapy approaches to eliminate minimal residual disease in AML patients. Citation Format: Annika Nelde, Heiko Schuster, Tatjana Bilich, Jens Bauer, Malte Roerden, Sarah Schroeder, Jonas S. Heitmann, Elke Rücker-Braun, Hans-Georg Rammensee, Helmut R. Salih, Juliane S. Walz. Immunopeptidome-defined acute myeloid leukemia progenitor cell-associated antigens are targeted in vivo by AML patients’ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3168.