ABSTRACT:The success of targeted therapies for hematological malignancies has heralded their potential as both salvage treatment and early treatment lines, reducing the need for high-dose, intensive, and often toxic chemotherapeutic regimens. For young patients with classic Hodgkin lymphoma (cHL), immunotherapies provide the possibility to lessen long-term, treatment-related toxicities. However, suitable therapeutic targets are lacking. By integrating single-cell dissection of the tumor landscape and an in-depth, single-cell-based off-tumor antigen prediction, we identify CD86 as a promising therapeutic target in cHL. CD86 is highly expressed on Hodgkin and Reed-Sternberg cancer cells and cHL-specific tumor-associated macrophages. We reveal CD86-CTLA-4 as a key suppressive pathway in cHL, driving T-cell exhaustion. Cellular therapies targeting CD86 had extraordinary efficacy in vitro and in vivo and were safe in immunocompetent mouse models without compromising bacterial host defense in sepsis models. Our results prove the potential value of anti-CD86 immunotherapies for treating cHL.

Chimeric antigen receptor-T cells have spearheaded the field of adoptive cell therapy and have shown remarkable results in treating hematological neoplasia. Because of the different biology of solid tumors compared to hematological tumors, response rates of CAR-T cells could not be transferred to solid entities yet. CAR engineering has added co-stimulatory domains, transgenic cytokines and switch receptors to improve performance and persistence in a hostile tumor microenvironment, but because of the inherent cell type limitations of CAR-T cells, including HLA incompatibility, toxicities (cytokine release syndrome, neurotoxicity) and high costs due to the logistically challenging preparation process for autologous cells, the use of alternative immune cells is gaining traction. NK cells and γδ T cells that do not need HLA compatibility or macrophages and dendritic cells with additional properties such as phagocytosis or antigen presentation are increasingly seen as cellular vehicles with potential for application. As these cells possess distinct properties, clinicians and researchers need a thorough understanding of their peculiarities and commonalities. This review will compare these different cell types and their specific modes of action seen upon CAR activation.

Background In recent years, T cell-based immunotherapies have shown promising results in hematologic malignancies. However, these strategies seem to be limited in solid cancers, posing more complex challenges including a hostile TME with nutrient deprivation and tissue hypoxia [1]. Additionally, metabolic reprogramming has been identified as a crucial factor for proper cytotoxic T-cell functions upon their activation. Such energy demands are answered by the upregulation of glycolysis, oxidative phosphorylation, and upregulation of nutrient transporters represented by SLCs [2,3]. Within the TME, tumor and immune cells compete for nutrients and shape a distinct metabolic milieu, resulting in an ineffective effector function [4]. Herein, we aim to metabolically engineer T cells to improve their fitness in the glucose-deprived TME and optimize ACT. Materials and Methods We retrovirally overexpressed the glucose transporter Slc2a1/GLUT1 in murine CD8+ T cells (CD8+Slc2a1). To assess T-cell fitness we conducted experiments in physiologic (5mM) and hypoglycemic (0.5mM) media conditions. CellTraceTM-based proliferation experiments and killing assays in the OT1-OVA model are used to examine differences to MOCK in functionality and were analyzed via flow cytometry and microscopy, respectively. Furthermore, Seahorse analyses, bulk RNA-Seq, and metabolomic analyses were performed to examine the mechanical background. Murine in vivo studies are performed to approach the translatability of this system into living organisms. Results CD8+Slc2a1 cells possessed a higher proliferative capacity in all conditions tested but most prominently in hypoglycemic (0.5mM) media. This better functional activity of CD8+Slc2a1 was also translated to higher killing rates in coculture assays with tumor cells, especially in low-glucose environments. Metabolic flux analyses and multi-omics suggested greater metabolic activity of CD8+Slc2a1 and revealed higher ROS production and upregulation of correlating anti-oxidative pathways, especially the pentose-phosphate pathway. Preliminary in vivo studies support the in vitro killing in a syngeneic tumor model. Furthermore, signs of altered memory formation were visible, expressed in a higher proportion of effector memory cells. Conclusions Our data point to the role of GLUT1 overexpression in T cells for improved cytotoxic activity, proliferation, and long-term persistence. Therefore, combinatorial approaches with GLUT1 overexpression could serve as a potential approach to increase efficacy in ACT against solid cancer. We also identified GLUT1-dependent reprogramming in CD8+Slc2a1 cells which is further investigated in ongoing studies. Additionally, we are evaluating the potential risk of this approach to neoplastic formation. References Treating hematological malignancies with cell therapy: where are we now? Landoni E, Savoldo B.; Expert Opin Biol Ther. 2018. Anticancer targets in the glycolytic metabolism of tumors: a comprehensive review; Paolo E. Porporato et al. Frontiers in Pharmacology 2011. Glucose Metabolism on Tumor Plasticity, Diagnosis, and Treatment; Lin Xiaoping et al. Frontiers in Oncology 2020 Fighting in a wasteland: deleterious metabolites and antitumor immunity. Watson MJ, Delgoffe GM. J Clin Invest. 2022. M.E. Kirmaier: None. B.L. Cadilha: None. A. Hadzic: None. W. Schmitz: None. M.R. Benmebarek: None. D. Briukhovetska: None. S. Michaelides: None. V. Buschinger: None. B. Tast: None. C.H. Bönigk: None. S. Oganesian: None. L. Vona: None. A. Tischmacher: None. V. Heissmeyer: None. M. Vaeth: None. V.R. Buchholz: None. M. Eilers: None. M. von Bergwelt-Baildon: None. M. Subklewe: None. S. Kobold: None. S. Theurich: None.

Cancer-specific TCF1+ stem-like CD8+ T cells can drive protective anticancer immunity through expansion and effector cell differentiation1-4; however, this response is dysfunctional in tumours. Current cancer immunotherapies2,5-9 can promote anticancer responses through TCF1+ stem-like CD8+ T cells in some but not all patients. This variation points towards currently ill-defined mechanisms that limit TCF1+CD8+ T cell-mediated anticancer immunity. Here we demonstrate that tumour-derived prostaglandin E2 (PGE2) restricts the proliferative expansion and effector differentiation of TCF1+CD8+ T cells within tumours, which promotes cancer immune escape. PGE2 does not affect the priming of TCF1+CD8+ T cells in draining lymph nodes. PGE2 acts through EP2 and EP4 (EP2/EP4) receptor signalling in CD8+ T cells to limit the intratumoural generation of early and late effector T cell populations that originate from TCF1+ tumour-infiltrating CD8+ T lymphocytes (TILs). Ablation of EP2/EP4 signalling in cancer-specific CD8+ T cells rescues their expansion and effector differentiation within tumours and leads to tumour elimination in multiple mouse cancer models. Mechanistically, suppression of the interleukin-2 (IL-2) signalling pathway underlies the PGE2-mediated inhibition of TCF1+ TIL responses. Altogether, we uncover a key mechanism that restricts the IL-2 responsiveness of TCF1+ TILs and prevents anticancer T cell responses that originate from these cells. This study identifies the PGE2-EP2/EP4 axis as a molecular target to restore IL-2 responsiveness in anticancer TILs to achieve cancer immune control.

The success of targeted immunotherapies for hematological malignancies has heralded their potential as salvage therapies as well as in earlier treatment lines (Cappell & Kochenderfer, 2023). While conventional chemotherapy-based treatments can achieve long-term survival in up to 90 % of treated patients with classic Hodgkin lymphoma (cHL), these therapies are associated with treatment-related comorbidities, calling for more tailored and specific approaches (Schaapveld et al., 2015; Shanbhag & Ambinder, 2018). While targeted treatments, especially immunotherapies are taking oncology by storm, the utility in cHL is so far limited to CD30 and PD-1-targeting strategies and there is a clear lack of drugable relevant target structures in this disease. This can be partly attributed to technical difficulties of analyzing the malignant Hodgkin-Reed-Sternberg (HRS) cells specifically. Capitalizing on our previous work using large scale data mining to inform target discovery, we hypothesized that combining different analytical methods with large single-cell RNA-Sequencing (scRNA-Seq) datasets would permit selective target definition with functional relevance to the disease and thereby allow the development of novel immunotherapeutic strategies. Leveraging microarray profiles of laser-dissected HRS cells and a scRNA-Seq cohort of cHL patients (total of n = 44 primary samples; n = 34 cHL samples; n = 10 RLN (reactive lymph node) control samples), we screened for novel target antigens highly expressed on HRS cells with functional relevance in the tumor microenvironement (TME) of cHL. Unbiased in silico analyses revealed CD80, CD86 and PD-L1 as most suitable candidate target antigens with CD86 showing the highest expression on HRS cells. ScRNA-Seq analyses unveiled a shift of the CD80-CD86-CTLA-4-CD28 towards the immunosuppressive CTLA-4 axis in the TME of cHL compared to RLN controls. In advanced cell culture models, including iPSC-derived organoid models, blockage of CD86 lead to the decreased expression of PD-1 and CTLA-4 and an overall reversal of the exhaustive phenotype of cHL-associated T cells. High protein expression of CD86 on HRS cells and in the TME (cHL-infiltrating tumor-associated macrophages (cHL-TAM), B cells) was confirmed in different validation cohorts including relapsed and refractory cHL (r/r cHL) patients by conventional immunohistochemistry and multiplexed immunofluorescence (n = 34 cHL patients). Following target identification, CAR T cells redirected against CD86 were developed and the functionality of these CAR T cells was investigated in preclinical models both in vitro and in vivo. Anti-CD86 CAR T cells effectively deplete cHL-TAM and are highly effective in various in vitro and in vivo models of cHL, including models of CD30-negative disease. Given the fundamental role of the CD80-CD86-CTLA-4-CD28 axis in the generation of the adaptive immune response, detailed toxicity assessments were carried out leveraging murine surrogate anti-CD86 CAR T cells, with similar binding and activation thresholds as their human counterpart. These anti-mCD86 CAR T cells did not cause toxicities in lymphodepleted, immunocompetent mice. In addition, the impact of anti-CD86-directed immunotherapies (e.g. anti-CD86-blocking antibodies, anti-mCD86 CAR T cells) on bacterial host defense and formation of antigen-specific adaptive immunity was investigated in syngeic mouse models. Anti-CD86 immunotherapy did not lead to enhanced bacteremia in a model of gram-negative sepsis, while preclinical vaccination models revealed a mildy reduced formation of antigen-specific T cell development in mice. In summary, we provide a framework for unbiased, multi-dimensional target screening and highlight the functional relevance of the immunosuppressive CD86-CTLA-4 axis in cHL. CD86-directed immunotherapy could reverse the exhaustive phenotype of cHL-associated T cells, while demonstrating strong treatment efficacy in xenograft mouse models. Importantly, elaborate toxicity assessments of anti-CD86-targeted immunotherapies utilizing syngenic mouse models did not reveal measureable toxicity in mice. Overall, our data emphasizes the vast translational potential of CD86-targeted immunotherapies in cHL and provide a strong rationale for further clinical investigations.

Although T cells can exert potent anti-tumor immunity, a subset of T helper (Th) cells producing interleukin-22 (IL-22) in breast and lung tumors is linked to dismal patient outcome. Here, we examined the mechanisms whereby these T cells contribute to disease. In murine models of lung and breast cancer, constitutional and T cell-specific deletion of Il22 reduced metastases without affecting primary tumor growth. Deletion of the IL-22 receptor on cancer cells decreases metastasis to a degree similar to that seen in IL-22-deficient mice. IL-22 induced high expression of CD155, which bound to the activating receptor CD226 on NK cells. Excessive activation led to decreased amounts of CD226 and functionally impaired NK cells, which elevated the metastatic burden. IL-22 signaling was also associated with CD155 expression in human datasets and with poor patient outcomes. Taken together, our findings reveal an immunosuppressive circuit activated by T cell-derived IL-22 that promotes lung metastasis.

Chimeric antigen receptor T cells (CAR-T cells) have emerged as a powerful treatment option for individuals with B cell malignancies but have yet to achieve success in treating acute myeloid leukemia (AML) due to a lack of safe targets. Here we leveraged an atlas of publicly available RNA-sequencing data of over 500,000 single cells from 15 individuals with AML and tissue from 9 healthy individuals for prediction of target antigens that are expressed on malignant cells but lacking on healthy cells, including T cells. Aided by this high-resolution, single-cell expression approach, we computationally identify colony-stimulating factor 1 receptor and cluster of differentiation 86 as targets for CAR-T cell therapy in AML. Functional validation of these established CAR-T cells shows robust in vitro and in vivo efficacy in cell line- and human-derived AML models with minimal off-target toxicity toward relevant healthy human tissues. This provides a strong rationale for further clinical development.

BACKGROUND:In many situations, the therapeutic efficacy of CAR T cells is limited due to immune suppression and poor persistence. Immunostimulatory fusion protein (IFP) constructs have been advanced as a tool to convert suppressive signals into stimulation and thus promote the persistence of T cells, but no universal IFP design has been established so far. We now took advantage of a PD-1-CD28 IFP as a clinically relevant structure to define key determinants of IFP activity.METHODS:We compared different PD-1-CD28 IFP variants in a human leukemia model to assess the impact of distinctive design choices on CAR T cell performance in vitro and a xenograft mouse model.RESULTS:We observed that IFP constructs that putatively exceed the extracellular length of PD-1 induce T-cell response without CAR target recognition, rendering them unsuitable for tumour-specific therapy. IFP variants with physiological PD-1 length ameliorated CAR T cell effector function and proliferation in response to PD-L1+ tumour cells in vitro and prolonged survival in vivo. Transmembrane or extracellular CD28 domains were found to be replaceable by corresponding PD-1 domains for in vivo efficacy.CONCLUSION:PD-1-CD28 IFP constructs must mimic the physiological interaction of PD-1 with PD-L1 to retain selectivity and mediate CAR-conditional therapeutic activity.

Background Melanoma is an immune sensitive disease, as demonstrated by the activity of immune check point blockade (ICB), but many patients will either not respond or relapse. More recently, tumor infiltrating lymphocyte (TIL) therapy has shown promising efficacy in melanoma treatment after ICB failure, indicating the potential of cellular therapies. However, TIL treatment comes with manufacturing limitations, product heterogeneity, as well as toxicity problems, due to the transfer of a large number of phenotypically diverse T cells. To overcome said limitations, we propose a controlled adoptive cell therapy approach, where T cells are armed with synthetic agonistic receptors (SAR) that are selectively activated by bispecific antibodies (BiAb) targeting SAR and melanoma-associated antigens.Methods Human as well as murine SAR constructs were generated and transduced into primary T cells. The approach was validated in murine, human and patient-derived cancer models expressing the melanoma-associated target antigens tyrosinase-related protein 1 (TYRP1) and melanoma-associated chondroitin sulfate proteoglycan (MCSP) (CSPG4). SAR T cells were functionally characterized by assessing their specific stimulation and proliferation, as well as their tumor-directed cytotoxicity, in vitro and in vivo.Results MCSP and TYRP1 expression was conserved in samples of patients with treated as well as untreated melanoma, supporting their use as melanoma-target antigens. The presence of target cells and anti-TYRP1 × anti-SAR or anti-MCSP × anti-SAR BiAb induced conditional antigen-dependent activation, proliferation of SAR T cells and targeted tumor cell lysis in all tested models. In vivo, antitumoral activity and long-term survival was mediated by the co-administration of SAR T cells and BiAb in a syngeneic tumor model and was further validated in several xenograft models, including a patient-derived xenograft model.Conclusion The SAR T cell-BiAb approach delivers specific and conditional T cell activation as well as targeted tumor cell lysis in melanoma models. Modularity is a key feature for targeting melanoma and is fundamental towards personalized immunotherapies encompassing cancer heterogeneity. Because antigen expression may vary in primary melanoma tissues, we propose that a dual approach targeting two tumor-associated antigens, either simultaneously or sequentially, could avoid issues of antigen heterogeneity and deliver therapeutic benefit to patients.

During metastasis, cancer cells invade, intravasate, enter the circulation, extravasate, and colonize target organs. Here, we examined the role of interleukin (IL)-22 in metastasis. Immune cell-derived IL-22 acts on epithelial tissues, promoting regeneration and healing upon tissue damage, but it is also associated with malignancy. Il22-deficient mice and mice treated with an IL-22 antibody were protected from colon-cancer-derived liver and lung metastasis formation, while overexpression of IL-22 promoted metastasis. Mechanistically, IL-22 acted on endothelial cells, promoting endothelial permeability and cancer cell transmigration via induction of endothelial aminopeptidase N. Multi-parameter flow cytometry and single-cell sequencing of immune cells isolated during cancer cell extravasation into the liver revealed iNKT17 cells as source of IL-22. iNKT-cell-deficient mice exhibited reduced metastases, which was reversed by injection of wild type, but not Il22-deficient, invariant natural killer T (iNKT) cells. IL-22-producing iNKT cells promoting metastasis were tissue resident, as demonstrated by parabiosis. Thus, IL-22 may present a therapeutic target for prevention of metastasis.

Lung emphysema and chronic bronchitis are the two most common causes of chronic obstructive pulmonary disease. Excess macrophage elastase MMP-12, which is predominantly secreted from alveolar macrophages, is known to mediate the development of lung injury and emphysema. Here, we discovered the endolysosomal cation channel mucolipin 3 (TRPML3) as a regulator of MMP-12 reuptake from broncho-alveolar fluid, driving in two independently generated Trpml3-/- mouse models enlarged lung injury, which is further exacerbated after elastase or tobacco smoke treatment. Mechanistically, using a Trpml3IRES-Cre/eR26-τGFP reporter mouse model, transcriptomics, and endolysosomal patch-clamp experiments, we show that in the lung TRPML3 is almost exclusively expressed in alveolar macrophages, where its loss leads to defects in early endosomal trafficking and endocytosis of MMP-12. Our findings suggest that TRPML3 represents a key regulator of MMP-12 clearance by alveolar macrophages and may serve as therapeutic target for emphysema and chronic obstructive pulmonary disease.

BackgroundCD155 (poliovirus receptor, PVR) is an immunosuppressive molecule overexpressed in lung adenocarcinoma (LUAD) and breast cancers (BRCA). However, no mutation has been identified that could be linked to such overexpression, and therefore it is likely regulated on the transcriptional level. Previously we identified interleukin-22 (IL-22) signaling as one of the pathways that upregulate CD155 expression in mouse models of lung and breast cancer. However, it is difficult to assess the activity of the IL-22 axis in the publicly available datasets since IL-22 signaling involves several components that must be considered: IL22, IL22RA1 and IL10RB, which encode heterodimeric IL-22 receptors found on tumor cells, and IL22RA2, which encodes soluble IL-22 binding protein (IL-22BP), an antagonist of IL-22 secreted by myeloid cells. The expression of IL22 itself is often missing in the available data due to the insufficient depth of sequencing, which prompts scientists to utilize one of the available components of the axis as a surrogate.Materials and MethodsHere we used agglomerative clustering, a bottom-up method of hierarchical clustering, to stratify the dataset by gene expression patterns in an unsupervised way. For this, we used LUAD and BRCA sequencing datasets from The Cancer Genome Atlas (TCGA). In the current analysis, we focused on HER2+ samples of the BRCA dataset. For clustering, we utilized PVR, IL22RA1, IL22RA2, and IL10RB gene expression. In identified clusters, we compared overall (OS) and restricted mean survival time (RMST) for the first five-year follow-up.ResultsIn both cohorts, we identified three clusters that are characterized by the following patterns of gene expression: cluster 0 (IL22RA1high, IL22RA2low, IL10RBmed, PVRhigh), cluster 1 (IL22RA1low, IL22RA2high, IL10RBhigh, PVRlow), and cluster 2 (IL22RA1low, IL22RA2low, IL10RBlow, PVRmedium). Here, cluster 0, identified by a high expression of IL-22 receptor and CD155, and low expression of IL-22BP, was characterized by the poor OS in both cohorts. Moreover, the average difference in RMST between clusters 1 and 0 constituted 361 days in lung and 93 days in HER2+ breast cancer. This difference could be explained by the prevalence of advanced-stage patients in the lung cancer but not in the breast cancer cohort. Moreover, we identified that this difference in survival between clusters stems from differences in early (I and II), but not late-stage (III and IV) patient entries.ConclusionsHere we identified that early-stage lung and HER2+ breast cancer patients could be stratified according to their IL22RA1, IL22RA2, IL10RB, and PVR expression with cluster 0 predicting lower OS and shorter RMST. Mechanistically, the activity of such a pathway defines the immunosuppressive axis we identified previously.Disclosure InformationD. Briukhovetska: A. Employment (full or part-time); Significant; ♣ University Hospital, LMU Munich. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Novartis. J. Jobst: None. S. Endres: A. Employment (full or part-time); Significant; University Hospital, LMU Munich. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Deutsche Forschungsgemeinschaft, Elitenetzwerk Bayern, Bio-M, Munich, Germany, TCR2, Cambridge, MA, USA. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; Paul-Martini-Stiftung, Else Kröner-Fresenius Stiftung. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Carina Biotech Ltd, Mawson Lakes, Australia. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TCR2, Cambridge, MA, USA. F. Consultant/Advisory Board; Significant; Gilde Healthcare, Utrecht, Netherlands. S. Kobold: A. Employment (full or part-time); Significant; University Hospital, LMU Munich. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; European Union, Hector Foundation, Elite Network of Bavaria, Melanoma Research Alliance, EKFS, German Cancer Aid, Ernst-Jung-Stiftung, German Excellence Initiative, BMBF, Research Council, DFG, SFB-TRR 338/1, Fritz-Bender Foundation, José-Carreras Foundation, Bio-M, TCR2 Inc. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS, GSK. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; Novartis. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Carina Biotech Ltd, Mawson Lakes, Australia. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TCR2, Cambridge, MA, USA. F. Consultant/Advisory Board; Modest; Tabby therapeutic ltd. F. Consultant/Advisory Board; Significant; TCR2 Inc, Novartis.

Background Immunotherapies, like immune checkpoint inhibition and tumor infiltrating lymphocytes, have had remarkable success in treating melanoma. However, many patients do still not respond or relapse with therapy-resistant disease. To overcome said limitations, we propose a controlled adoptive cell therapy approach, where T cells are armed with EGFRvIII synthetic agonistic receptors (E3 SAR) that are selectively activated by a cross-linking bispecific antibody (BiAb) specific for both SAR T cell and melanoma-associated antigens. Materials and Methods Murine as well as human SAR constructs were generated and T cells were retrovirally transduced to stably express the SAR constructs. We validated our approach in murine, human and patient-derived cancer models expressing the melanoma-associated target antigens TYRP1 and MCSP. SAR T cells were functionally characterised by proving specific activation and proliferation of SAR T cells, as well as their tumor-directed cytotoxicity, in vitro and in vivo. Results Both on a mRNA and protein level, MCSP and TYRP1 were shown to be differentially expressed in treatment-naive as well as treatment-resistant melanoma patients compared to samples from healthy donors. Crosslinking anti-TYRP1 x anti-E3 and anti-MCSP x anti-E3 BiAb mediated conditional antigen-dependent activation, proliferation of SAR-T cells and lead to tumor cell lysis in all models tested. In vivo, anti-tumoral activity and tumor-free survival was mediated by the co-administration of SAR T cells and BiAb in a syngeneic tumor model and was further confirmed in several xenograft models. Conclusions Here, we apply the SAR x BiAb approach in an effortto deliver specific and conditional activation of SAR transduced T cells, and targeted tumor cell lysis in melanoma models. The modularity of our approach is key for targeting melanoma and is essential towards personalised immunotherapies addressing cancer heterogeneity. Due to variations of antigen expression in primary melanoma tissues, we propose that a dual-targeting approach, either simultaneous or sequential, could mitigate issues of heterogeneity and deliver therapeutic benefit to patients. Disclosure Information M. Benmebarek: A. Employment (full or part-time); Significant; Klinikum der Universität München. F. Märkl: A. Employment (full or part-time); Significant; Klinikum der Universität München. J. Keyl: None. B. Cadilha: A. Employment (full or part-time); Significant; Klinikum der Universität München. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Pantent in the immuno-oncology field. M. Geiger: A. Employment (full or part-time); Significant; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Stocks and patents with Roche. C. Karches: A. Employment (full or part-time); Significant; Klinikum der Universität München. H. Obeck: None. M. Schwerdtfeger: A. Employment (full or part-time); Significant; Klinikum der Universität München. D. Briukhovetska: A. Employment (full or part-time); Significant; Klinikum der Universität München. J. Jobst: None. P.J. Müller: None. M. Seifert: None. R. Grünmeier: None. M. Thomas: A. Employment (full or part-time); Significant; Helmholtz München. C. Marr: A. Employment (full or part-time); Significant; Helmholtz München. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; European Research Council. M. Levesque: A. Employment (full or part-time); Significant; University Hospital Zurich. M. Heppt: A. Employment (full or part-time); Significant; Universitätsklinikum Erlangen. S. Endres: A. Employment (full or part-time); Significant; Klinikum der Universität München. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Elite Network of Bavaria, LMU Munich's Institutional Strategy LMUexcellent. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Patents in the field of immuno-oncology. C. Klein: A. Employment (full or part-time); Significant; Roche. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Stocks and patents with Roche. S. Kobold: A. Employment (full or part-time); Significant; Klinikum der Universität München. B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Volkswagen Foundation, European Research Council, Hector Foundation, Elite Network of Bavaria, Melanoma Research Alliance, Else Kröner-Fresenius-Stiftung, German Cancer Aid, Ernst-Jung-Sfiftung, LMU Munich's Institutional Strategy LMUexcellent, Bundesministerium für Bildung und Forschung, European Research Council Grant, Fritz-Bender Foundation, José-Carreras Foundation. C. Other Research Support (supplies, equipment, receipt of drugs or other in-kind support); Significant; German Research Foundation. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Significant; TTCR2 Inc, Novartis, BMS, GSK. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; Patents in the field of immuno-oncology.

Background While chimeric-antigen receptor (CAR) T cells have revolutionized the treatment of refractory B cell malignancies, they have yet to achieve success in the treatment of acute myeloid leukemia (AML).1 In AML, development of CAR therapy is hindered by expression of AML-associated antigens also on pivotal healthy tissues (e.g. hematopoietic stem and progenitor cells, HSPC). The revolution in single-cell technologies has generated massive expression data, providing precise information on the transcriptomic anatomy of healthy and malignant cells.2 However, these resources have rarely been used for de novo antigen predictions. We hypothesized that we could use these technologies to establish high resolution antigen projections, enabling the identification of novel target structures. Hence, we leveraged an atlas of RNA sequencing data of over 500,000 single cells from AML patients and healthy human tissues for target identification and subsequent testing of novel target structures for CAR T cell therapy. Materials and Methods 12 single cell data sets were harmonized and used for target prediction. Anti-murine and anti-human CAR constructs targeting the lead candidate - colony-stimulating factor 1 receptor (CSF1R) - were generated and transduced into primary murine and human T cells. AML cell lines and primary AML samples were used to verify expression of CSF1R and as target cell lines in vitro and in vivo. Off-target toxicities of CAR were analyzed in vitro and in vivo using a variety of different models. Results Using a newly developed single-cell RNA sequencing-based screening algorithm, CSF1R was identified as a promising target antigen for CAR T cell therapy in AML. Expression of CSF1R was verified on a large panel of AML cell lines and in primary AML samples. Newly developed anti-CSF1R-CAR T cells efficiently lysed AML target cells in vitro. In vivo, anti-CSF1R-CAR T cells induced strong and sustained remissions in cell line- and patient-derived xenograft models. Compared to anti-CD33-CAR T cells, anti-CSF1R-CAR T cells did not lyse healthy HSPC and proved to be safe when used in fully syngeneic mice models. Conclusions Aided by our screening algorithm, we identified CSF1R as a new promising target for CAR therapy in AML and proved the efficacy of newly developed CAR T cells. Our results highlight the remarkable translational potential of unbiased, high-resolution target screenings for cancer entities and warrant further clinical investigations of newly developed anti-CSF1R-CAR T cells. References Cummins, K.D. and S. Gill, Chimeric antigen receptor T-cell therapy for acute myeloid leukemia: how close to reality? Haematologica 2019; 104(7): p. 1302–1308. Papatheodorou, I., et al., Expression Atlas update: from tissues to single cells. Nucleic Acids Res, 2020. 48(D1): p. D77-D83. Disclosure Information A. Gottschlich: None. M. Thomas: None. R. Grünmeier: None. S. Lesch: None. L. Rohrbacher: None. V. Igl: None. D. Briukhovetska: None. M. Benmebarek: None. S. Dede: None. K. Müller: None. T. Xu: None. D. Dhoqina: None. Ö. Umut: None. F. Märkl: None. S. Robinson: None. A. Sendelhofert: None. H. Schulz: None. B. Vick: None. B.L. Cadilha: None. R. Brabenec: None. N. Röder: None. F. Rataj: None. M. Nüesch: None. J. Wellbrock: None. F. Modemann: None. W. Fiedler: D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Amgen, Gilead, Jazz Pharmaceuticals, Servier, Daiichi Sankyo, Novartis, Abbvie, Pfizer, Amgen. F. Consultant/Advisory Board; Modest; Amgen, ARIAD/Incyte, Pfizer, Novartis, Jazz Pharmaceuticals, Morphosys, Abbvie, Celgene, Stemline, Clinigen. C. Kellner: None. T. Herold: None. D. Paquet: None. I. Jeremias: None. L. von Baumgarten: None. S. Endres: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; TCR2. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Carina Biotech Ltd. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TCR2. F. Consultant/Advisory Board; Modest; Gilde Healthcare. M. Subklewe: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; Amgen, BMS, Gilead, Miltenyi, MorphoSys, Novartis, Roche, Seattle Genetics. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; Amgen, BMS, Gilead, Novartis, Pfizer, Takeda. F. Consultant/Advisory Board; Modest; Amgen, BMS, Gilead, Janssen, Novartis, Pfizer, Seattle Genetics, Takeda. C. Marr: None. S. Kobold: B. Research Grant (principal investigator, collaborator or consultant and pending grants as well as grants already received); Significant; TCR2 Inc. D. Speakers Bureau/Honoraria (speakers bureau, symposia, and expert witness); Modest; BMS, GSK, Novartis. E. Ownership Interest (stock, stock options, patent or other intellectual property); Modest; Carina Biotech Ltd. E. Ownership Interest (stock, stock options, patent or other intellectual property); Significant; TCR2. F. Consultant/Advisory Board; Modest; Tabby therapeutic ltd, TCR2 Inc. F. Consultant/Advisory Board; Significant; Novartis.

BACKGROUND:Chimeric antigen receptor (CAR) T cell therapy has been successfully translated to clinical practice for the treatment of B cell malignancies. The suppressive microenvironment of many malignancies is a bottleneck preventing treatment success of CAR T cells in a broader range of tumours. Among others, the immunosuppressive metabolite adenosine is present in high concentrations within many tumours and dampens anti-tumour function of immune cells and consequently therapeutic response.METHODS:Here, we present the impact of the selective adenosine A2A and A2B receptor antagonist AB928/etrumadenant on CAR T cell cytokine secretion, proliferation, and cytotoxicity. Using phosphorylation-specific flow cytometry, we evaluated the capability of AB928 to shield CAR T cells from adenosine-mediated signalling. The effect of orally administered AB928 on CAR T cells was assessed in a syngeneic mouse model of colon carcinoma.RESULTS:We found that immunosuppressive signalling in CAR T cells in response to adenosine was fully blocked by the small molecule inhibitor. AB928 treatment enhanced CAR T cell cytokine secretion and proliferation, granted efficient cytolysis of tumour cells in vitro and augmented CAR T cell activation in vivo.CONCLUSIONS:Together our results suggest that combination therapy with AB928 represents a promising approach to improve adoptive cell therapy.

the homozygous cohort. Our findings demonstrate a reduced amount of potentially immunogenic peptides in EGA patients with HLA-homozygosity for at least one locus, which may result in impaired cancer immunosurveillance. In line with this observation, we also found increased levels of CTA expression in homozygous compared to heterozygous patients. After artificial modification of the genotype of homozygous patients to a heterozygous genotype, the set of predicted good-binding peptides was comparable to the heterozygous cohort. Conclusion Our results highlight the effect of HLA-I homozygosity on the immunopeptidome as important prerequisite of anti-tumor immunity. The high frequency of genomic HLA-I homozygosity observed in the EGA cohort may reflect an increased cancer risk for these patients. Together with previous reports demonstrating reduced survival after checkpoint therapy, our study suggests consideration of germ-line HLA-homozygosity for the design and interpretation of immunotherapeutic trials.

Interleukins and associated cytokines serve as the means of communication for innate and adaptive immune cells as well as non-immune cells and tissues. Thus, interleukins have a critical role in cancer development, progression and control. Interleukins can nurture an environment enabling and favouring cancer growth while simultaneously being essential for a productive tumour-directed immune response. These properties of interleukins can be exploited to improve immunotherapies to promote effectiveness as well as to limit side effects. This Review aims to unravel some of these complex interactions.