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Tumor cell–derived extracellular vesicles (EV) convert normal myeloid cells into myeloid-derived suppressor cells (MDSC), inhibiting antitumor immune responses. Here, we show that EV from Ret mouse melanoma cells upregulate the expression of programmed cell death ligand 1 (PD-L1) on mouse immature myeloid cells (IMC), leading to suppression of T-cell activation. PD-L1 expression and the immunosuppressive potential of EV-generated MDSC were dependent on the expression of Toll-like receptors (TLR). IMC from Tlr4−/− mice failed to increase T-cell PD-L1 expression and immunosuppression with Ret-EV treatment, and this effect was dependent on heat-shock protein 86 (HSP86) as HSP86-deficient Ret cells could not stimulate PD-L1 expression on normal IMC; IMC from Tlr2−/− and Tlr7−/− mice demonstrated similar results, although to a lesser extent. HSP86-deficient Ret cells slowed tumor progression in vivo associated with decreased frequency of tumor-infiltrating PD-L1+CD11b+Gr1+ MDSC. EV from human melanoma cells upregulated PD-L1 and immunosuppression of normal monocytes dependent on HSP86. These findings highlight a novel EV-mediated mechanism of MDSC generation from normal myeloid cells, suggesting the importance of EV targeting for tumor therapy.Significance:These findings validate the importance of TLR4 signaling in reprogramming normal myeloid cells into functional myeloid-derived suppressor cells.

Figure S1 shows PD-L1 upregulation induced by EV that is mediated by NF-kB activation; Figure S2 shows an induction of PD-L1 and immunosuppressive capacity of myeloid cells through TLR signaling; Figure S3 presents characterization of EV derived from human melanoma cells; Figure S4 shows induction of PD-L1 expression on normal monocytes by melanoma EV; Figure S5 shows HSP86 expression in HT-144 human melanoma cells and HT-144-EV; Table S1 presents the sequences of primers for the quantitative real-time PCR

Background Recent and soon-to-expect approvals have significantly increased the number of patients treated with CAR-T cells and clinical responses can be very impressive. However, side effects including cytokine storm, neurotoxicity or on-target/off-tumor toxicity can be challenging to manage. Further, malignant transformation of piggyBac modified CAR-T cells was recently reported in a phase I clinical trial as a potentially life threatening event. Reliable strategies to control or even deplete CAR-T cells are thus urgently needed. Methods We generated SLAMF7 CAR-T cells as previously described and exposed the cells to clinically relevant doses of the following drugs: dexamethasone, fludarabine, mafosfamide, dasatinib, cetuximab, and belantamab-mafodotin. After defined time periods, we evaluated absolute numbers of CAR-T cells and their effector functions against myeloma cell lines (cytotoxicity, cytokine secretion, proliferation). Statistical analysis was performed using one-way ANOVA, followed by Dunnett’s multiple comparisons test if appropriate. The study was approved by the local ethics committee. Results Dexamethasone, used in the setting of severe CRS or ICANS, minimally decreased T cell numbers and cytokine release, but did not interfere with lytic capacity of the CAR-T cells. Fludarabine and the pre-activated cyclophosphamide analog mafosfamide, both drugs with potent lymphodepleting properties, significantly depleted numbers of CAR-T cells, reduced antigen-specific proliferation and led to an overall decrease in target cell elimination, even if specific lysis and cytokine release of the few surviving cells was preserved. Of note, fludarabine and mafosfamide similarly depleted unmanipulated T cells. Dasatinib, previously identified as a potent inhibitor of intracellular CAR signaling, had no impact on absolute T cell numbers, however lysis of myeloma cells, cytokine secretion and antigen-specific proliferation were entirely blocked. Evaluation of selective elimination of CAR-T cells with cetuximab, targeting the EGFR transduction marker of our CAR, showed modest CAR-T cell depletion in an ADCC assay. This was potentially related to competing natural killer cell elimination by the SLAMF7 CAR-T cells. To circumvent the problem of impaired ADCC, T cells were genetically engineered to express BCMA to enable simultaneous depletion of T cells and myeloma cells. Exposure to the BCMA-directed antibody-drug conjugate belantamab-mafodotin resulted in potent elimination of BCMA-positive T cells while unmanipulated cells were largely spared. Conclusions In aggregate, our data show that the different drugs can have justification in different clinical settings of CAR-T cell toxicity. While dasatinib and dexamethasone mainly act on T cell function, fludarabine and mafosfamide effectively deplete T cells in an unselective manner. Targeted elimination of CAR-T cells with antibodies can be augmented further by the use of antibody-drug-conjugates.

Introduction: In the rapidly growing field of chimeric antigen receptor (CAR) engineered T cells new approaches aim to make CAR-T cell therapy safer and more effective. Recent, designs aim towards universal or modular CARs that do not directly recognize the target antigen itself, but instead facilitate contact to CAR-adaptor molecules, which in turn bind to the target antigen. Current CAR-adaptor molecules include human antibodies of the IgG1 isotype binding to CD16 or antibodies modified by peptide or hapten tags. We developed a modular CAR-T approach that recognizes the P329G mutation clinically used to silence the Fc effector function of therapeutic antibodies. These modular anti-P329G-CAR-T cells are only functional in the presence of antibodies that possess the P329G mutation. Methods: The anti-P329G interaction with P329G-containing Fc fragment was analyzed using surface plasmon resonance and co-crystallography. Lentivirus transfected anti-P329G CAR-T cells were characterized in vitro for their selectivity and potential to mediate antigen specific tumor cell lysis, cytokine secretion and proliferation. Immunological synapse formation was investigated using confocal microscopy. Results: Anti-P329G-CAR-T cells allow the precise recognition of the P329G mutation present in therapeutic IgG1 based adaptor-molecules. Crystal structure- and SPR-analysis revealed a 1:1 binding stoichiometry with low nanomolar affinity of the P329G-Fab fragment applied in the CAR for P329G-containing IgG1 antibodies. Potent tumor cell lysis was demonstrated for multiple tumor antigens e.g. CD20, HER2, FOLR1, EpCAM, FAP and others. For all tested antigens, a huIgG1 dose-dependent activation of anti-P329G-CAR-T cells as well as dose-dependent tumor cell lysis was observed. For selected antigens P329G-CAR-T activity was found comparable to the activity mediated by T cell bispecific antibodies recognizing the respective tumor antigen. Finally, the immunological synapse formed by P329-CAR-T cells was compared to the one formed by T cell bispecific antibodies in a 2+1 format. Conclusions: P329G-CAR-T cells mediate potent and specific tumor cell killing using various tumor targeted antibodies as adaptor molecules. Based on these data in vivo studies to investigate efficacy and safety of the approach are foreseen. Notably, this approach allows control of CAR-T activity and potential side effects by titrating the adaptor molecule, as well as the simultaneous targeting of more than one antigen at the same time with the goal to prevent tumor escape mechanisms. Combining the P329G-CAR with allogenic T-cells may provide a truly off-the-shelf P329G-CAR-T cell therapy approach. Citation Format: Diana Darowski, Christian Jost, Zeno Riester, Mohamed Benmebarek, Kay Stubenrauch, Anne Freimoser-Grundschober, Uwe Wessels, Jörg Benz, Ekkehard Mössner, Renier Myburgh, Floriana Cremasco, Mario Perro, Pablo Umana, Sebastian Kobold, Christian Klein. Anti-P329G-CAR-T cells as a novel universal CAR-T cell platform [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4229.

BackgroundMyeloid-derived suppressor cells (MDSC) play a major role in the immunosuppressive melanoma microenvironment. They are generated under chronic inflammatory conditions characterized by the constant production of inflammatory cytokines, chemokines and growth factors, including IL-6. Recruitment of MDSC to the tumor is mediated by the interaction between chemokines and chemokine receptors, in particular C–C chemokine receptor (CCR)5. Here, we studied the mechanisms of CCR5 upregulation and increased immunosuppressive function of CCR5+ MDSC.MethodsThe immortalized myeloid suppressor cell line MSC-2, primary immature myeloid cells and in vitro differentiated MDSC were used to determine factors and molecular mechanisms regulating CCR5 expression and immunosuppressive markers at the mRNA and protein levels. The relevance of the identified pathways was validated on the RET transgenic mouse melanoma model, which was also used to target the identified pathways in vivo.ResultsIL-6 upregulated the expression of CCR5 and arginase 1 in MDSC by a STAT3-dependent mechanism. MDSC differentiated in the presence of IL-6 strongly inhibited CD8+ T cell functions compared with MDSC differentiated without IL-6. A correlation between IL-6 levels, phosphorylated STAT3 and CCR5 expression in tumor-infiltrating MDSC was demonstrated in the RET transgenic melanoma mouse model. Surprisingly, IL-6 overexpressing tumors grew significantly slower in mice accompanied by CD8+ T cell activation. Moreover, transgenic melanoma-bearing mice treated with IL-6 blocking antibodies showed significantly accelerated tumor development.ConclusionOur in vitro and ex vivo findings demonstrated that IL-6 induced CCR5 expression and a strong immunosuppressive activity of MDSC, highlighting this cytokine as a promising target for melanoma immunotherapy. However, IL-6 blocking therapy did not prove to be effective in RET transgenic melanoma-bearing mice but rather aggravated tumor progression. Further studies are needed to identify particular combination therapies, cancer entities or patient subsets to benefit from the anti-IL-6 treatment.

Purpose of review Immunotherapy with gene-engineered chimeric antigen receptor (CAR)-T cells has curative potential in advanced malignancies and undergoes a surging preclinical and clinical development. Here, we present a selection of new targets and technologies that illustrate the progress that is being made with the aspiration to make CAR-T cell therapy a universally applicable and effective treatment in cancer medicine. Recent findings There is a rich pipeline of new target antigens for CAR-T cells in hematology and oncology that are rated based on uniformity but also stability of expression on tumor cells under therapeutic pressure. New technologies in CAR-T cell engineering are directed at neutralizing inhibitory ligands and factors in the tumor microenvironment, preventing CAR-T cell exhaustion and enhancing selectivity for tumor cells with 'smart' CAR designs. The manufacture of CAR-T cells using virus-free protocols is anticipated to reduce supply-chain complexity and to improve patient access. Summary CD19 CAR-T cell therapy is an approved treatment for B-cell leukemia and -lymphoma and considering the current 'target and technology' pipeline, we anticipate that additional CAR-T cell products will accomplish their 'breakthrough' and clinical proof-of-concept in other indications in hematology and in oncology. Technologies to enhance therapeutic index and facilitate manufacturing will be key for assuring availability and accessibility of CAR-T cell products and their implementation into routine clinical practice.