Eosinophils have been identified as a prognostic marker in immunotherapy of melanoma and suggested to contribute to anti-tumor host defense. However, the influence of immune checkpoint inhibitors (ICI) on the eosinophil population is poorly studied. Here, we applied routine laboratory tests, multicolor flow cytometry, RNA microarray analysis, and bio-plex assay to analyze circulating eosinophils and related serum inflammatory factors in 32 patients treated with pembrolizumab or the combination of nivolumab and ipilimumab. We demonstrated that clinical responses to ICI treatment were associated with an eosinophil accumulation in the peripheral blood. Moreover, immunotherapy led to the alteration of the eosinophil genetic and activation profile. Elevated serum concentrations of IL-16 during ICI treatment were found to be associated with increased frequencies of eosinophils in the peripheral blood. Using immunohistochemistry, we observed an enhanced eosinophil degranulation and a positive correlation between eosinophil and CD8 + T cell infiltration of tumor tissues from melanoma patients treated with ICI. Our findings highlight additional mechanisms of ICI effects and suggest the level of eosinophils as a novel predictive marker for melanoma patients who may benefit from this immunotherapy.

Despite melanoma immunogenicity and remarkable therapeutic effects of negative immune checkpoint inhibitors, a significant fraction of patients does not respond to current treatments. This could be due to limitations in tumor immunogenicity and profound immunosuppression in the melanoma microenvironment. Moreover, insufficient tumor antigen processing and presentation by dendritic cells (DC) may hamper the development of tumor-specific T cells. Using two genetically engineered mouse melanoma models ( and transgenic mice), in which checkpoint inhibitor therapy alone is not efficacious, we performed proof-of-concept studies with an improved, multivalent DC vaccination strategy based on our recently developed genetic mRNA cancer vaccines. The expression of multiple chimeric MHC class I receptors allows a simultaneous presentation of several melanoma-associated shared antigens tyrosinase related protein (TRP)-1, tyrosinase, human glycoprotein 100 and TRP-2. The DC vaccine induced a significantly improved survival in both transgenic mouse models. Vaccinated melanoma-bearing mice displayed an increased CD8 T cell reactivity indicated by a higher IFN-γ production and an upregulation of activation marker expression along with an attenuated immunosuppressive pattern of myeloid-derived suppressor cells (MDSC) and regulatory T cells (Treg). The combination of DC vaccination with ultra-low doses of paclitaxel or anti-PD-1 antibodies resulted in further prolongation of mouse survival associated with a stronger reduction of MDSC and Treg immunosuppressive phenotype. Our data suggest that an improved multivalent DC vaccine based on shared tumor antigens induces potent anti-tumor effects and could be combined with checkpoint inhibitors or targeting immunosuppressive cells to further improve their therapeutic efficiency.

Malignant melanoma is characterized by a rapid progression, metastasis to distant organs and resistance to chemo and radiotherapy. Although melanoma is capable of eliciting an immune response, the disease progresses and the overall results of immunotherapeutic clinical studies are not satisfactory. Recently, we have developed a novel genetic platform for improving an induction of peptide-specific CD8 C T cells by dendritic cell (DC) based on membrane-anchored b2-microglobulin (b2m) linked to a selected antigenic peptide at the N-terminus and to the cytosolic domain of TLR4 at the C-terminus. In vitro transcribed mRNA transfection of antigen-presenting cells (APCs) resulted in an efficient coupling of peptide presentation and cell activation. In this research, we utilize the chimeric platform to induce an immune response in ret transgenic mice that spontaneously develop malignant skin melanoma and to examine its effect on the overall survival of tumor-bearing mice. Following immunization with chimeric construct system, we observe a significantly prolonged survival of tumor-bearing mice as compared to the control group. Moreover, we see elevations in the frequency of CD62L hi CD44 hi central and CD62L lo CD44 hi effector memory CD8C T-cell subsets. Importantly, we do not observe any changes in frequencies of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in the vaccinated groups. Our data suggest that this novel vaccination approach could be efficiently applied for the immunotherapy of malignant melanoma.

For many years, clinicians and scientists attempt to develop methods to stimulate the immune system to target malignant cells. Recent data suggest that effective cancer vaccination requires combination immunotherapies to overcome tumor immune evasion. Through presentation of both MHC-I and II molecules, DCs-based vaccine platforms are effective in generating detectable CD4 and CD8 T cell responses against tumor-associated antigens. Several platforms include DC transfection with mRNA of the desired tumor antigen. These DCs are then delivered to the host and elicit an immune response against the antigen of interest. We have recently established an mRNA genetic platform which induced specific CD8 cytotoxic T cell response by DC vaccination against melanoma. In our study, an MHC-II mRNA DCs vaccine platform was developed to activate CD4 T cells and to enhance the anti-tumor response. The invariant chain (Ii) was modified and the semi-peptide CLIP was replaced with an MHC-II binding peptide sequences of melanoma antigens. These chimeric MHC-II constructs are presented by DCs and induce proliferation of tumor specific CD4 T cells. When administered in combination with the MHC-I platform into tumor bearing mice, these constructs were able to inhibit tumor growth, and improve mouse survival. Deciphering the immunological mechanism of action, we observed an efficient CTLs killing in addition to higher levels of Th1 and Th2 subsets in the groups immunized with a combination of the MHC-I and MHC-II constructs. These universal constructs can be applied in multiple combinations and offer an attractive opportunity to improve cancer treatment.

Immune checkpoint blockade using antibodies targeting the cell surface expressed proteins CTLA-4, PD-1 and PD-L1 has revolutionized cancer care and its clinical impact in several indications has prompted a search for complementary immunostimulatory approaches that can further increase the efficacy of these drugs. Mitogen-activated protein kinase kinase kinase kinase 1 (MAP4K1; HPK1), a serine/threonine kinase expressed exclusively in hematopoietic cell lineages, mediates a negative feedback signal downstream of T-cell receptor stimulation. Its activity is enhanced by PGE2 and TGFβ, factors commonly present in the tumor microenvironment. Mice deficient for MAP4K1 or expressing a kinase-dead variant of MAP4K1 exhibit enhanced T-cell function, including increased anti-tumor immunity. We developed the small molecule inhibitor BAY-405 that displays potent nanomolar MAP4K1 inhibition in biochemical and cellular assays, good kinase selectivity, and in vivo exposure after oral dosing. Pharmacological inhibition of MAP4K1 enhances T-cell immunity and overcomes the suppressive impact of PGE2, TGFβ and CD4+ T-regulatory cells. Single agent treatment of tumor-bearing mice results in suppression of tumor outgrowth in several syngeneic models. This is accompanied by an increase in the anti-tumor T-cell response, dependent on an intact T-cell compartment, while not involving direct anti-tumor cytotoxicity. Inhibition of MAP4K1 in conjunction with PD-L1 blockade results in further suppression of tumor outgrowth. Moreover, we found that MAP4K1 is expressed in both PD-L1-high and PD-L1-low human cancers. In summary, our data show that selective inhibition of MAP4K1 by means of small molecule drugs may be used to expand the patient population responding to immune checkpoint inhibition. Citation Format: Gabriele Leder, Rafael Carretero, Jeffrey Mowat, Sandra Berndt, Roland Neuhaus, Nuria Aiguabella Font, Ulf Boemer, Oliver von Ahsen, Uwe Eberspaecher, Judith Guenther, Mareike Grees, Corinna Link, Barbara Nicke, Daniel Baumann, Martina Schaefer, Mine Oezcan-Wahlbrink, Nicolas D. Werbeck, Ingo Hartung, Bertolt Kreft, Rienk Offringa. Enhancement of anti-tumor T-cell immunity by means of an oral small molecule targeting the intracellular immune checkpoint MAP4K1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1722.

The second messenger diacyl glycerol (DAG) plays a key role in T cell receptor downstream signaling and thereby in T-cell activation. Diacylglycerol kinase zeta (DGK-ζ) is a lipid kinase that can down-modulate T-cell activation by catalyzing the conversion of DAG to phosphatidic acid, thereby acting as a ligand independent, intracellular immune checkpoint. Inhibition of DGK- ζ offers the potential to enhance T cell priming against suboptimal tumor antigens and to overcome multiple immune-suppressive mechanisms in the tumor microenvironment. Bayer AG in collaboration with the German Cancer Research Center (DKFZ) have developed BAY 2965501, a highly selective, orally available DGK-ζ inhibitor which shows a unique binding mode and good DMPK profile. The compound enhances the reactivity of human and mouse T-cells, both in the priming and the effector phase, and overcomes the inhibitory impact of suppressive factors, such as adenosine and prostaglandin E2. Oral dosing of BAY 2965501 shows T cell dependent efficacy in syngeneic, murine tumor models. Furthermore, this results in reactivation of exhausted T cells in vivo. Preclinical safety studies showed only mild findings, so that good clinical tolerability is expected. Collectively, the preclinical data support first in human (FiH) phase I testing of therapeutic potential. A FiH trial with BAY 2965501 in patients with advanced solid tumors including NSCLC, gastric/GEJ AdCa, ccRCC, and melanoma patients, is currently enrolling patients (NCT05614102). This study will evaluate the safety, tolerability, maximum tolerated or administered dose, pharmacokinetics, pharmacodynamics, and tumor response profile of BAY 2965501. Citation Format: Rienk Offringa, Catherine Olesch, Frederik Cichon, Mareike Grees, Norbert Schmees, Ulrike Roehn, Florian Prinz, Judith Guenther, Detlef Stoeckigt, Michael Erkelenz, Thi Thanh Uyen Nguyen, Ulf Boemer, Nicolas Werbeck, Marc Kunze, Kirstin Petersen, Dennis Kirchhoff. BAY 2965501: A highly selective DGK- ζ inhibitor for cancer immuno-therapy with first-in-class potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr ND04.

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