ABSTRACT:T-cell-based immunotherapies have revolutionized treatment paradigms in B-cell malignancies, yet their translation to acute myeloid leukemia (AML) has been hindered by a scarcity of tumor-restricted antigens and the risk of on-target off-leukemia toxicity. FLT3 has emerged as a promising therapeutic target with limited expression in healthy hematopoietic tissues. Here, we performed a head-to-head preclinical comparison of an FMS-like tyrosine kinase 3 (FLT3)-directed bispecific T-cell engager (BiTE) molecule and second-generation FLT3-specific chimeric antigen receptor (CAR) T cells. Both approaches induced potent cytotoxicity against AML cell lines and primary patient-derived cells but spared healthy hematopoietic stem and progenitor cells in vitro. Despite similar short-term efficacy, prolonged antigen exposure demonstrated progressive functional decline and metabolic exhaustion; however, CAR T cells maintained cytotoxic capacity and proliferative potential over time. In AML xenograft models, CAR T cells achieved superior tumor control, prolonged survival, and greater T-cell infiltration than BiTE molecule-treated counterparts. Transcriptomic profiling of T cells recovered from the bone marrow further revealed a distinct exhaustion-associated gene signature in samples from mice that had been treated with the FLT3 BiTE molecule. Importantly, provision of CD86-mediated costimulation enhanced antitumor activity of BiTE-redirected T cells in vitro and in vivo. These findings establish FLT3 as a viable and selective immunotherapeutic target in AML and underscore the functional and transcriptional differences between BiTE molecule-redirected T cells and CAR T cells. Moreover, they reveal a critical role for costimulatory signaling in sustaining the efficacy of T-cell-based therapies in vivo, offering a rationale for improving T cell-redirection strategies in myeloid malignancies.

T cell-based immunotherapy has revolutionized the treatment of B-cell malignancies, yet applying it to acute myeloid leukemia (AML) is challenging due to difficulties in identifying suitable target antigens without on-target off-leukemia toxicity. Prior studies identified FLT3 as a promising target antigen with restricted expression in the healthy hematopoietic compartment1. Here, we evaluate a FLT3-directed BiTE® molecule and 2nd generation FLT3-specific CAR T cells in a preclinical AML model. We hypothesize that positive costimulatory molecules on AML cells enhance BiTE® molecule effectiveness, while CAR T cells may be less dependent due to their built-in costimulatory domain. Cytotoxicity was assessed in cocultures over time using MPFC. The impact of positive costimulation was tested using our established Ba/F3 model system lacking any human costimulatory molecules. On-target off-leukemia toxicity was evaluated in cytotoxicity and CFU assays of pAML cells and healthy bone marrow (hBM). T-cell exhaustion was examined in a longterm culture system with continuous antigen exposure. Lastly, xenograft AML models and RNA sequencing explored differences between the two platforms in vivo. BiTE® molecule and CAR effectively mediated cytotoxicity against various AML cell lines and pAML cells. Overexpression of the costimulatory CD86 on the target cells significantly increased BiTE® molecule-mediated cytotoxicity cells, while CAR-mediated lysis was unaltered. In both scenarios, low E:T-dependent toxicity was observed against hBM. Mixing experiments showed efficient pAML cell lysis with minimal impact on hBM. CFU assays indicated no effect on hematopoiesis of healthy CD34+ stem cells. 28-day cocultures led to T-cell exhaustion in both settings, marked by reduced proliferation, cytokine secretion, metabolic fitness, and cytotoxicity. CAR T cells demonstrated superior antileukemic activity, with enhanced proliferation and splenic homing in an AML xenograft model. RNA sequencing revealed more exhaustion-related markers in BiTE® molecule-redirected T cells, while CAR T cells showed upregulated glycolysis and fatty acid metabolism. Repeating our in vivo xenograft experiment with CD86-overexpressing AML cells supported our hypothesis that costimulation might be a key factor providing a head start to CAR T cells by increasing T-cell proliferation and persistence in vivo. Our in vitro data show similar cytotoxicity and specificity, while xenograft mouse models suggest CAR T cells offer a significant survival advantage over BiTE® molecules. Our data support the hypothesis that positive costimulation integrated within the CAR constructs boosts T-cell activation, homing, and efficacy, thereby delaying T-cell exhaustion in vivo. Future studies are needed to further dissect differences between BiTE® molecule vs CAR T cell-based immunotherapy and identify suitable patient groups. 1Brauchle et al 2020 Lisa Rohrbacher, Daniel Nixdorf, Helena Stadler, Bettina Brauchle, Florian Märkl, Adrian Gottschlich, Gordon Hoffmann, Nora Philipp, Gerulf Hänel, Martin Kirmaier, Anetta Marcinek, Maryam Kazerani, Alica-Joana Emhardt, Giulia Magno, Rebecca L. Goldstein, Sebastian Theurich, Tobias Straub, Sebastian Kobold, Tara Arvedson, Veit L. Bücklein, Marion Subklewe. BiTE® molecule vs CAR-T targeting FLT3 in AML: How positive co-stimulation tips the scale [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3174.

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.

In multiple myeloma (MM), B cell maturation antigen (BCMA)-directed CAR T cells have emerged as a novel therapy with potential for long-term disease control. Anti-BCMA CAR T cells with a CD8-based transmembrane (TM) and CD137 (41BB) as intracellular costimulatory domain are in routine clinical use. As the CAR construct architecture can differentially impact performance and efficacy, the optimal construction of a BCMA-targeting CAR remains to be elucidated. Here, we hypothesized that varying the constituents of the CAR structure known to impact performance could shed light on how to improve established anti-BCMA CAR constructs. CD8TM.41BBIC-based anti-BCMA CAR vectors with either a long linker or a short linker between the light and heavy scFv chain, CD28TM.41BBIC-based and CD28TM.CD28IC-based anti-BCMA CAR vector systems were used in primary human T cells. MM cell lines were used as target cells. The short linker anti-BCMA CAR demonstrated higher cytokine production, whereas in vitro cytotoxicity, T cell differentiation upon activation and proliferation were superior for the CD28TM.CD28IC-based CAR. While CD28TM.CD28IC-based CAR T cells killed MM cells faster, the persistence of 41BBIC-based constructs was superior in vivo. While CD28 and 41BB costimulation come with different in vitro and in vivo advantages, this did not translate into a superior outcome for either tested model. In conclusion, this study showcases the need to study the influence of different CAR architectures based on an identical scFv individually. It indicates that current scFv-based anti-BCMA CAR with clinical utility may already be at their functional optimum regarding the known structural variations of the scFv linker.

The concept of precision cell therapy targeting tumor-specific mutations is appealing but requires surface-exposed neoepitopes, which is a rarity in cancer. B cell receptors (BCR) of mature lymphoid malignancies are exceptional in that they harbor tumor-specific-stereotyped sequences in the form of point mutations that drive self-engagement of the BCR and autologous signaling. Here, we use a BCR light chain neoepitope defined by a characteristic point mutation (IGLV3-21 R110 ) for selective targeting of a poor-risk subset of chronic lymphocytic leukemia (CLL) with chimeric antigen receptor (CAR) T cells. We develop murine and humanized CAR constructs expressed in T cells from healthy donors and CLL patients that eradicate IGLV3-21 R110 expressing cell lines and primary CLL cells, but neither cells expressing the non-pathogenic IGLV3-21 G110 light chain nor polyclonal healthy B cells. In vivo experiments confirm epitope-selective cytolysis in xenograft models in female mice using engrafted IGLV3-21 R110 expressing cell lines or primary CLL cells. We further demonstrate in two humanized mouse models lack of cytotoxicity towards human B cells. These data provide the basis for advanced approaches of resistance-preventive and biomarker-guided cellular targeting of functionally relevant lymphoma driver mutations sparing normal B cells.

Abstract Translation of the success of bispecific T cell engagers (BiTE®) and CAR T cells from B-cell to myeloid malignancies has been challenging. Identifying suitable target antigens in myeloid malignancies has been hampered by expected on-target-off leukemia toxicity. FLT3 is a promising target antigen with high expression in most AML samples independent of FLT3 mutation status (Brauchle et al., 2020). Here, we compared two T-cell based immunotherapy approaches, BiTE® vs CART, in targeting FLT3 in AML. We tested BiTE® and CAR mediated cytotoxicity against several AML cell lines and primary AML (pAML) cells in vitro. The impact of positive co-stimulation was evaluated using our previously established Ba/F3 model system, which is devoid of human costimulatory molecules. Conjugate formation as a surrogate for synapse formation and consecutive T-cell degranulation were analyzed. Combination with tyrosine kinase inhibition (TKI) was tested in conjunction with BiTE® and CAR to further enhance cytotoxicity against FLT3mut AML cells. On-target off-leukemia toxicity was tested in mixing assays of AML cells and healthy bone marrow (hBM). Finally, we compared CAR and BiTE® in an in vivo xenograft AML model. No differences in BiTE® vs CAR mediated cytotoxicity were observed against cell lines with different genetic background and FLT3 expression levels or pAML samples. However, the percentage of T cell-AML conjugates formed utilizing CART was significantly higher compared to BiTE® molecules. Also, faster and stronger degranulation of CD107a was detected using CAR T cells vs BiTE®. Interestingly, we showed that expression of the costimulatory CD86 increased the cytotoxicity of BiTE®-redirected T cells while CAR mediated lysis was unaltered. In case of the FLT3-ITD mutated cell line MV411, the addition of FLT3 TKIs significantly increased CAR and BiTE® mediated cytotoxicity. Looking at on-target off-leukemia toxicity, in particular hBM cells, we only observed low cytotoxicity. Moving to an in vivo AML xenograft model, we observed that CAR T cell treated mice had an improved OS compared to BiTE®-treated mice, accompanied by enhanced proliferation and homing of the CAR T cells to the spleen. We conclude, that FLT3 is a promising target antigen for T cell-based immunotherapy with limited on-target-off leukemia toxicity. Our preclinical in vitro data demonstrate similar cytotoxicity of both platforms against AML cell lines and pAML cells. Utilizing a xenograft mouse model, CAR T cells provided a significant survival benefit over BiTE® molecules. Our data support the hypothesis that positive co-stimulation either integrated within the construct (e.g. 2nd generation CAR) or provided by the target cell (e.g. CD86 expressing leukemia cells) propagates Tcell activation and proliferation. Future studies will be needed to further dissect differences between BiTE® vs CAR T cell based immunotherapy platforms and identify suitable combination partners. Citation Format: Lisa Rohrbacher, Daniel Nixdorf, Helena Stadler, Bettina Brauchle, Florian Märkl, Nora Philipp, Gerulf Hänel, Anetta Marcinek, Maryam Kazerani, Rebecca L. Goldstein, Michael von Bergwelt, Sebastian Kobold, Veit L. Bücklein, Tara Arvedson, Marion Subklewe. The race is on: BiTE vs CART targeting FLT3 in AML [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 894.

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.

Bispecific T-cell engager (BiTE®) molecules recruit T cells to cancer cells through CD3ε binding, independently of T-cell receptor (TCR) specificity. Whereas physiological T-cell activation is dependent on signal 1 (TCR engagement) and signal 2 (co-stimulation), BiTE molecule-mediated T-cell activation occurs without additional co-stimulation. As co-stimulatory and inhibitory molecules modulate the strength and nature of T-cell responses, we studied the impact of the expression profile of those molecules on target cells for BiTE molecule-mediated T-cell activation in the context of acute myeloid leukemia (AML). Accordingly, we created a novel in vitro model system using murine Ba/F3 cells transduced with human CD33 ± CD86 ± PD-L1. T-cell fitness was assessed by T-cell function assays in co-cultures and immune synapse formation by applying a CD33 BiTE molecule (AMG 330). Using our cell-based model platform, we found that the expression of positive co-stimulatory molecules on target cells markedly enhanced BiTE molecule-mediated T-cell activation. The initiation and stability of the immune synapse between T cells and target cells were significantly increased through the expression of CD86 on target cells. By contrast, the co-inhibitory molecule PD-L1 impaired the stability of BiTE molecule-induced immune synapses and subsequent T-cell responses. We validated our findings in primary T-cell-AML co-cultures, demonstrating a PD-L1-mediated reduction in redirected T-cell activation. The addition of the immunomodulatory drug (IMiD) lenalidomide to co-cultures led to stabilization of immune synapses and improved subsequent T-cell responses. We conclude that target cells modulate CD33 BiTE molecule-dependent T-cell activation and hence, combinatorial strategies might contribute to enhanced efficacy.

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.

Translation of the success of bispecific T cell engagers (BiTE®) and CAR T cells from B-cell to myeloid malignancies has been challenging. Identifying suitable target antigens in myeloid malignancies has been hampered by expected on-target-off leukemia toxicity. FLT3 presents with a rather favorable expression profile due to broad AML expression and limited expression within the normal hematopoietic compartment (Brauchle et al., 2020). Importantly, expression on AML bulk cells and leukemic stem cells is observed independent of FLT3 mutational status. Here, we compared two T-cell based immunotherapy approaches, BiTE® vs CART, in targeting FLT3 in AML. We tested BiTE® and CAR-mediated cytotoxicity against several AML cell lines and primary AML (pAML) cells in vitro. On-target off-leukemia toxicity was evaluated in mixing assays of pAML cells and healthy donor bone marrow (hBM). The impact of positive costimulation on both approaches was tested using our previously established Ba/F3 model system (Marcinek et al., 2023), which is devoid of any human costimulatory molecules. As a surrogate for synapse formation, conjugate formation as well as consecutive T-cell degranulation were studied. Further, we studied the cell death pathway mediating cytotoxicity against AML target cells, e.g. FAS/FASL vs perforin/granzyme B. The impact of continuous stimulation by BiTE®-and CART on evolving T-cell exhaustion was tested using our previously established 28-day long-term culture assay (Philipp et al., 2022). Finally, we compared BiTE® and CAR in an in vivo xenograft AML model. In a second in vivo experiment, all mice were harvested 18 days after T-cell injection, followed by T-cell isolation from the bone marrow and subsequent bulk RNA sequencing. BiTE®- and CAR-redirected T cells led to comparable effector-to-target (E:T) ratio-dependent specific lysis of various AML cell lines and pAML cells. Using our in vitro mixing experiments, we could show that BiTE®- and CAR-redirected T cells efficiently killed pAML cells while mostly sparing the hBM. Using the Ba/F3 model system, we observed that the expression of the costimulatory molecule CD86 significantly increased the cytotoxicity of BiTE®-redirected T cells while CAR-mediated lysis was unaltered. Furthermore, we were able to show that the percentage of T cell-AML conjugates formed as well as degranulation of CD107a was significantly higher when using CART compared to BiTE® molecules. Interestingly, when we added a FAS-blocking antibody to our cocultures, we could show that CART-mediated lysis of the target cells was significantly reduced. This was not observed when using the BiTE® construct. Using our long-term culture system, we found that T-cell proliferation and cytokine secretion decreased with both constructs over time. The cytotoxic capacity of BiTE®-redirected T cells seemed to decrease faster compared to the CART construct. Moving to an in vivo AML xenograft model, we observed that CART-treated mice had an improved OS compared to the BiTE®-treated mice (Fig.1). This was accompanied by enhanced T-cell proliferation and splenic homing of the CAR T cells. Bulk RNA sequencing of T cells isolated from the murine bone marrow 18 days post T-cell injection revealed signs of exhaustion in BiTE®-redirected T cells compared to CAR T cells. We found an upregulation of several inhibitory immune receptors and transcription factors (Fig.2) that have been well described to promote T-cell exhaustion. Furthermore, gene set enrichment analysis revealed that genes upregulated in effector vs exhausted were also upregulated in CAR T cells. This was accompanied by an upregulated glycolysis and fatty acid metabolism in CAR T cells. We conclude, that FLT3 is a promising target antigen with limited on-target off-leukemia toxicity. Our preclinical in vitro data demonstrate similar cytotoxicity of both platforms against AML cell lines and pAML cells. Utilizing a xenograft mouse model, CAR T cells provided a significant survival benefit over BiTE® molecules. Our data support the hypothesis that positive costimulation integrated within the CAR construct provided an advantage in T-cell activation and might lead to better T-cell homing, resulting in improved efficacy and delayed T-cell exhaustion in vivo. Future studies will be needed to further dissect differences between BiTE® vs CAR T cell-based immunotherapy platforms and identify suitable combination partners.

Introduction Redirection of T cells has evolved as one of the most promising strategies for the treatment of hematologic diseases. The two main immunotherapeutic approaches consist of T-cell redirection via chimeric antigen receptor (CAR) T cells and T-cell engager (TCE) molecules. A growing arsenal of anti-CD19 and -CD20 directed CAR T, as well as TCE constructs, have been FDA and EMA approved in different precursor and mature B-cell malignancies. Translation into myeloid malignancies is challenged by the choice of target antigen and T-cell fitness. Based on previous work, we identified FLT3 as a promising target antigen for the treatment of AML with limited on-target-off-leukemia toxicity (Brauchle et al. 2020). Hence, we compared FLT3-directed CAR T cells with a bispecific T cell engager (BiTE®) molecule in a preclinical AML model with a focus on T-cell function and evolving T-cell exhaustion. Methods First, we implemented an AML cell line (MV-411, MOLM-13, and OCI-AML3) and primary AML in vitro model system to study CAR T- and BiTE®-mediated cytotoxicity using flow cytometry-based read-out assays. Impact of positive costimulation on T-cell function, in particular for the comparison of a CAR T containing a CD28 costimulatory domain vs a BiTE® molecule, were evaluated using our previously established Ba/F3 model system, which is devoid of any costimulatory molecules. The impact on conjugate formation as surrogate for immunological synapse formation and consecutive T-cell degranulation were studied. To further enhance cytotoxicity, CAR-T cells and BiTE® molecules were combined with PD-1 blockade or tyrosine kinase inhibition. To test if CAR-T cells and BiTE® molecules are able to induce bystander killing of tumor cells not expressing the target antigen, we set up a mixing model with FLT3+ and FLT3- cells including healthy bone marrow cells. Finally, we compared CAR-T cells and BiTE® molecules in an in vivo xenograft model using the OCI-AML3-LUC-GFP cell line. Results We observed that FLT3-targeted CAR-T cells and BiTE® molecules led to effector-to-target (E:T) ratio-dependent lysis of the FLT3+ cell lines and showed a similar cytotoxicity against all tested cell lines. Specific lysis of primary AML blasts was also similar in both conditions. However, using the Ba/F3 model system, we were able to show that the presence of the costimulatory ligand CD86 significantly increased the cytotoxic capacity of BiTE®-redirected T cells (p= 0.0312) while CAR-T cell-mediated lysis was unaltered. The percentage of conjugates formed after 10 and 30 minutes of coculture was significantly higher in the CAR-T cell condition (p = 0.0298 and p = 0.0225). This was accompanied by a faster and stronger degranulation of the CAR-T cells. Combinatorial treatment with PD-1 blockade slightly enhanced the specific lysis of AML cell lines in both cocultures. The addition of the tyrosine kinase inhibitor Quizartinib significantly increased the CAR-T and BiTE®-mediated lysis of the MV-411(FLT3-ITD/FLT3-ITD) cell line. Bystander killing of the target-antigen negative tumor cell line HEL92.1.7 was seen in both conditions, however low cytotoxicity against healthy primary bone marrow cells was observed. In contrast to our in vitro data, using an in vivoAML mouse model, the overall survival of mice treated with CAR-T cells was significantly prolonged when compared to the BiTE®-treated group (28 days vs 40 days, p = 0.0031). This was accompanied by enhanced proliferation and homing of the CAR-T cells as seen by significantly higher frequencies of T cells in the blood and spleen of these mice (p = 0.0019 and p = 0.0059). Conclusion Together, our data underline the power of T-cell-based immunotherapy platforms to combat myeloid malignancies. FLT3-directed CAR-T cells and BiTE® molecules mediated cytotoxicity in the same range, and similar results were observed in various in vitro model systems. However, using an in vivo AML xenograft model, CAR-T cells performed better and led to significantly prolonged overall survival of the mice in this group. This might be related to an integrated positive costimulatory domain within the CAR-T construct, enabling prolonged T-cell fitness and enhanced proliferation. Future studies will further dissect the impact of CAR-T cells versus BiTE® molecule treatment on T-cell fitness and how this translates into response and outcome.

The clinical use of cellular immunotherapies is gaining momentum and the number of approved indications is steadily increasing. One class of cellular therapies—chimeric antigen receptor (CAR)-modified T cells—has achieved impressive results in distinct blood cancer indications. These existing cellular therapies treating blood cancers face significant relapse rates, and their application beyond hematology has been underwhelming, especially in solid oncology. Major reasons for resistance source largely in the tumor microenvironment (TME). The TME in fact functionally suppresses, restricts, and excludes adoptive immune cells, which limits the efficacy of cellular immunotherapies from the onset. Many promising efforts are ongoing to adapt cellular immunotherapies to address these obstacles, with the aim of reshaping the tumor microenvironment to ameliorate function and to achieve superior efficacy against both hematological and solid malignancies.

Immune checkpoint inhibition and chimeric antigen receptor (CAR) T cell therapy have demonstrated stunning clinical efficacy in many cancer types. However, most patients do not respond to immunotherapies or relapse after an initial response, stressing the need for improved strategies. Chemokines, as mediators of immune cell trafficking, play an important role in the composition of the tumor microenvironment and exert both pro- and antitumorigenic functions. Here, chemokines may represent valuable prognostic biomarkers of response to immunotherapy and a strategy to improve immunotherapies. In this review, the pleiotropic functions of chemokines in the tumor microenvironment (TME) and strategies of utilizing chemokines or chemokine antagonism in immunotherapy are discussed. The review highlights preclinical and clinical studies that apply or target chemokines in monotherapy or in combination therapies.

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.

CAR T cell therapy remains ineffective in solid tumors, due largely to poor infiltration and T cell suppression at the tumor site. T regulatory (Treg) cells suppress the immune response via inhibitory factors such as transforming growth factor-β (TGF-β). Treg cells expressing the C-C chemokine receptor 8 (CCR8) have been associated with poor prognosis in solid tumors. We postulated that CCR8 could be exploited to redirect effector T cells to the tumor site while a dominant-negative TGF-β receptor 2 (DNR) can simultaneously shield them from TGF-β. We identified that CCL1 from activated T cells potentiates a feedback loop for CCR8+ T cell recruitment to the tumor site. This sustained and improved infiltration of engineered T cells synergized with TGF-β shielding for improved therapeutic efficacy. Our results demonstrate that addition of CCR8 and DNR into CAR T cells can render them effective in solid tumors.