Systematic assessment of LCMV based vaccine vectors expressing melanocyte differentiation antigens in human in vitro assays and in mouse melanoma models.

e14299 Background: Antibodies blocking the immune checkpoint pathways represent an important milestone in the treatment of patients with metastatic melanoma. However, individuals presenting cold tumors do usually not show a clinical benefit. New cancer vaccine approaches are needed to cover this gap. Methods: A novel replication attenuated vaccine vector based on lymphocytic choriomeningitis virus expressing full length melanocyte differentiation antigens is evaluated in mouse melanoma models and in PBMCs and T cell cultures from melanoma patients. Results: Here, we demonstrate that intratumoral but not intravenous injection of a recombinant propagating LCMV vector expressing the melanoma-associated antigen TRP2 leads to T cell-dependent eradication of established s.c. melanoma. Importantly, intratumoral vaccination shows an abscopal effect on distant lung metastasis and protects from a rechallenge with melanoma. Confocal microscopy and flow cytometry reveal that intratumoral injection of rLCMV vectors reprograms the tumor microenvironment resulting in sustained T cell fitness. In addition, we demonstrate that rLCMV vectors can efficiently transduce human antigen presenting cells. Moreover, in vitro data confirm that rLCMV efficiently induces T cells against various melanoma-associated antigens in PBMCs from melanoma patients. Conclusions: Preclinical assessment of propagating rLCMV vectors shows unique features of this cancer vaccine resulting in a profound and multistep activation of the cancer immunity cycle resulting in eradication of established melanomas in the B16F10 mouse model after one single immunization. Positive proof of principle experiments using PBMCs from melanoma patients suggest a rapid evaluation in clinical trials.

Identification of CAR As a Novel Mediator of Erythroid Differentiation and Migration That Is Specifically Downregulated in Erythropoietic Progenitor Cells in Patients with MDS

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by peripheral cytopenia, an accumulation of dysplastic cells in the bone marrow (BM) and a high risk of transformation to acute myeloid leukemia. Transfusion-dependent anemia develops in most patients suffering from MDS. In patients with refractory anemias the accumulation of dysplastic erythroid BM progenitor cells (EryPC) which paradoxically accompanies peripheral anemia, is a typical finding. Although various hypotheses have been discussed, the biochemical basis of the maturation defect of erythroid cells and their abnormal accumulation in the BM in MDS remains unknown. In order to learn more about abnormally regulated and functionally relevant genes expressed in EryPC, we have recently established a screening program involving mRNA expression profiling studies of EryPC in patients with low-risk MDS (IPSS-R score<7) and control BM samples obtained from patients with other BM neoplasms as well as patients with unexplained cytopenia, normal BM (staging for lymphomas) or reactive/deficiency cytopenias. EryPC were defined as CD45low/CD105+ cells and purified from BM mononuclear cells (MNC) by multicolor flow cytometry (MFC) and cell sorting (purity>95%). mRNA expression profiles were analyzed by Affymetrix array technology (GeneChip U133 Plus 2.0 arrays) and confirmed for a panel of selected mRNA species by qPCR. In mRNA- and MFC-validation experiments, we found that the major Coxsackie-Adenovirus Receptor (CAR) is markedly and specifically down-regulated in CD45low/CD105+ EryPC in patients with MDS when compared to EryPC in normal BM or other control cohorts. In line with this observation, the immature erythroblastic cell lines HEL, K562 and KU812 all stained negative for CAR in flow cytometry analyses. Lentiviral transduction of the full-length CAR gene into these cells resulted in a significantly increased expression of various erythroid differentiation antigens, including CD36, CD71 and CD235a (Glycophorin-A) as determined by flow cytometry and qPCR. As assessed by flow cytometry, the levels of CD235a increased to 158±46% in HEL cells, to 211±23% in K562 cells and to 170±30% in KU812 cells after CAR transduction compared to the empty vector control (p<0.05). In addition, CAR transduction resulted in an increased migration of HEL cells against a serum protein-gradient in a transwell assay (empty vector control: 185±67% vs CAR-transfected cells: 356±74%, p<0.05). Transfection with truncated variants of CAR did not result in an increased expression of erythroid antigens or an increased directed migration. In conclusion, our data show that CAR is a functionally relevant antigen that promotes the expression of early erythroid differentiation antigens on myeloid progenitor cells and their migration against blood serum proteins. In patients with MDS, CAR is specifically downregulated on EryPC, which may have clinical implications both in term of the pathogenesis of the disease and the application of this novel marker in diagnostic MFC algorithms. With regard to functional consequences, we hypothesize that CAR-deficiency is pathogenetically relevant as it may not only contribute to the maturation-defect of erythroid progenitor cells in MDS EryPC but also to the related accumulation of erythroid cells in the BM that is accompanying the peripheral anemia in these patients. Disclosures No relevant conflicts of interest to declare.