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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.

Supplementary figure legend

Supplementary Figure 1: Murine SAR expression and immunophenotyping of transduced T cells

Supplementary Figure 5: Effect of pre-activated SAR T cells on hepatocellular carcinoma and glioblastoma cells


Purpose:Genetically engineered T cells are powerful anticancer treatments but are limited by safety and specificity issues. We herein describe an MHC-unrestricted modular platform combining autologous T cells, transduced with a targetable synthetic agonistic receptor (SAR), with bispecific antibodies (BiAb) that specifically recruit and activate T cells for tumor killing.Experimental Design:BiAbs of different formats were generated by recombinant expression. T cells were retrovirally transduced with SARs. T-cell activation, proliferation, differentiation, and T-cell–induced lysis were characterized in three murine and human tumor models in vitro and in vivo.Results:Murine T cells transduced with SAR composed of an extracellular domain EGFRvIII fused to CD28 and CD3ζ signaling domains could be specifically recruited toward murine tumor cells expressing EpCAM by anti-EGFRvIII × anti-EpCAM BiAb. BiAb induced selective antigen-dependent activation, proliferation of SAR T cells, and redirected tumor cell lysis. Selectivity was dependent on the monovalency of the antibody for EGFRvIII. We identified FAS ligand as a major mediator of killing utilized by the T cells. Similarly, human SAR T cells could be specifically redirected toward mesothelin-expressing human pancreatic cancer cells. In vivo, treatment with SAR T cells and BiAb mediated antitumoral activity in three human pancreatic cancer cell xenograft models. Importantly, SAR activity, unlike CAR activity, was reversible in vitro and in vivo.Conclusions:We describe a novel ACT platform with antitumor activity in murine and human tumor models with a distinct mode of action that combines adoptive T-cell therapy with bispecific antibodies.

Supplementary Figure 4: Human SAR T cells are specifically stimulated by anti-human mesothelin x anti-EGFRv3 2 + 1 BiAb and redirect lysis to mesothelin+ target cells in vitro and in vivo

Supplementary Figure 3: In contrast to SAR T cells, CAR T cells rely on the release of perforin for efficient killing.

Supplementary Figure 2: Soluble and bound EGFR ligands do not trigger unwanted SAR T cell activation, while antibodies monovalent and bivalent for SAR can trigger T cell activation to the same extent.

BackgroundMelanoma 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.MethodsHuman 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.ResultsMCSP 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.ConclusionThe 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.

Adoptive T cell therapy (ACT) is a therapeutic approach which employs genetically manipulated autologous T cells to target and eliminate a patient's malignancy. This novel therapeutic approach, when employing a chimeric antigen receptor (CAR) targeting CD19-expressing B cells, has shown remarkable success in treating acute B-cell lymphocytic leukemia. However, blood born malignancies represent only a fraction of cancers which affect patients. Unfortunately, the utilization of ACT to target solid malignancies has only shown marginal success rates. There are many known obstacles which hinder CAR T cell therapy in patients suffering from solid cancer, one notable obstacle is the effective trafficking of CAR T cells to the tumor site. With the rapid advancement of novel approaches and targets which may enhance CAR T cell infiltration into solid tumors, a standardized approach to assess and measure CAR T cell infiltration becomes imperative in order to compare these different approaches across platforms. Here we describe a flow cytometry method which enables the rapid detection and quantification of CAR T cells which have reached and entered the tumor mass following intravenous injection. Competence with single cell preparation and flow cytometry is required for optimal results.

BackgroundNatural killer (NK) cells require a functional lytic granule machinery to mediate effective antitumor responses. Evading the lytic cargo deployed at the immune synapse (IS) could be a critical step for cancer progression through yet unidentified mechanisms.MethodsNK cell antibody-dependent cellular cytotoxicity (ADCC) is a major determinant of the clinical efficacy of some therapeutic antibodies including the anti-HER2 Trastuzumab. Thus, we screened sera of Trastuzumab-resistant HER2 +patients with breast cancer for molecules that could inhibit NK cell ADCC. We validated our findings in vitro using cytotoxicity assays and confocal imaging of the lytic granule machinery and in vivo using syngeneic and xenograft murine models.ResultsWe found that sera from Trastuzumab-refractory patients could inhibit healthy NK cell ADCC in vitro. These sera contained high levels of the inflammatory protein chitinase 3-like 1 (CHI3L1) compared with sera from responders and healthy controls. We demonstrate that recombinant CHI3L1 inhibits both ADCC and innate NK cell cytotoxicity. Mechanistically, CHI3L1 prevents the correct polarization of the microtubule-organizing center along with the lytic granules to the IS by hindering the receptor of advanced glycation end-products and its downstream JNK signaling. In vivo, CHI3L1 administration drastically impairs the control of NK cell-sensitive tumors, while CHI3L1 blockade synergizes with ADCC to cure mice with HER2 +xenografts.ConclusionOur work highlights a new paradigm of tumor immune escape mediated by CHI3L1 which acts on the cytotoxic machinery and prevents granule polarization. Targeting CHI3L1 could mitigate immune escape and potentiate antibody and cell-based immunotherapies.

Background The efficacy of chimeric antigen receptor (CAR) T cells against solid tumors remains unsatisfactory due to impaired trafficking of the CAR T cells into the tumor microenvironment (TME) and the presence of immunosuppressive factors and cells. 5’- triphosphate double-stranded RNA (3p-RNA) is recognized by the intracellular pattern recognition receptor retinoic acid-induced gene I (RIG-I). RIG-I activates a downstream signaling cascade, triggering the expression of type I interferons (IFN), proinflammatory cytokines and chemokines enhancing immune surveillance in the TME. We hypothesized that priming the TME with RIG-I ligands increases the efficacy of CAR T cell therapy. Materials and Methods T110299 pancreatic tumor cells (derived from a genetically-engineered Kras and p53 mutant murine PDAC model) were engineered to express murine epithelial cell adhesion molecule (EpCAM) and used to induce subcutaneous or orthotopic tumors in C57BL/6J female mice. Mice bearing T110299 EpCAM+ tumors were treated with intratumoral or i.v. injections of 3p-RNA followed by i.v. injection of syngeneic murine T cells that were retrovirally tranduced to express anti-EpCAM CARs. Three days after CAR T cell injection, immune cell composition and CAR T cell infiltration in the TME were assessed by flow cytometry. Additionally, tumor growth and survival were monitored. Results Intratumoral injections of 3p-RNA reshaped the myeloid immune compartment in the TME by significantly reducing suppressive polymorphonuclear-MDSC and macrophages while increasing Ly6Chigh inflammatory monocytes. Moreover, antigen-presenting cells, such as dendritic cells and macrophages, were activated as evidenced by increased MHC-I expression levels. This was paralleled by a significant increase in the infiltration of CAR T cells into the TME in the combination therapy group. Interestingly, anti-EpCAM CAR T cells alone failed to control the tumor growth of T110299 EpCAM+ tumors, while monotherapy with 3p-RNA slightly delayed tumor growth in the subcutaneous model. Combination of 3p-RNA with anti-EpCAM CAR T cells induced a significant clinical benefit with tumor regression in 50% of the treated mice in the subcutaneous tumor model and prolonged survival in an orthotopic model. Conclusions Remodeling the immunosuppressive TME using RIG-I ligands is a promising strategy for overcoming therapeutic resistance of CAR T cells in solid tumors, such as pancreatic cancer. Funding The project was supported by the international doctoral program ‘i-Target: Immunotargeting of cancer’ funded by the Elite Network of Bavaria and the Stiftungen zu Gunsten der Medizinischen Fakultat. Disclosure Information A.M. Senz: None. S.L. Formisano: None. B. Cadilha: None. T. Lorenzini: None. S. Endres: None. S. Kobold: None. M. Schnurr: None. L.M. Konig: None.

Background Chimeric antigen receptor therapy – although very efficacious in B cell malignancies – is facing many challenges which limit its success in solid tumors, e.g. on-target off-tumor toxicities, antigen heterogeneity, lack of T cell migration into tumors and an immunosuppressive tumor microenvironment. To better control on-target off-tumor effects and address antigen heterogeneity we developed a modular approach where we equipped T cells with a synthetic agonistic receptor (SAR). The SAR is only activated in the presence of a bispecific antibody (BiAb) cross-linking the receptor with a tumor-associated antigen. While we could show efficacy of the SAR platform in different models, limited infiltration and immune suppression still hamper its function. We could previously demonstrate that T cell infiltration can be enhanced by transduction with carefully chosen chemokine receptors like CXCR6, CCR4 and CCR8. At the same time, gene silencing of checkpoint molecules like PD-1 can make T cells more resistant to immunosuppression, thus we assumed that combining these approaches might generate a desired T cell product. Materials and Methods All constructs had been generated previously by overlap-extension cloning. The EGFRvIII (E3) SAR consists of extracellular EGFRvIII, transmembrane CD28 and intracellular CD28 and CD3ζ. Human CXCR6-GFP, CCR4-GFP and CCR8-GFP are composed of the chemokine receptors fused to GFP via a 2A sequence. Primary human T cells were retrovirally transduced to stably express the SAR and chemokine receptors. We analyzed migration, cytotoxicity and activation of the single and double (E3 SAR and chemokine receptor) transduced T cells. In addition, PD-1 was knocked out using CRISPR-Cas9 and killing kinetics of target cells and T cell activation were assessed. Results Co-transduction with chemokine receptors significantly increased migration of E3 SAR T cells to their respective ligand while lysis of target-expressing tumor cell and T cell activation in the presence of BiAb were not affected in vitro. Additionally knocking out PD-1 enhanced killing kinetics and activation of E3 SAR and E3 SAR + CXCR6-GFP transduced T cells compared to corresponding mock electroporated T cells. Conclusions Using the controllable and modular SAR – BiAb platform SAR T cell activation can be limited by stopping BiAb dosing if adverse events occur. In addition, SAR T cells can be redirected to an alternative tumor-associated antigen by exchanging the BiAb in the case of antigen escape. Here we present add-ons to this approach for increased tumor infiltration and resistance to immunosuppression. Since migration is enhanced upon co-transduction with chemokine receptors and target cell lysis is accelerated upon PD-1 knockout in vitro these two additional modifications seem very promising options to further improve tumor control in vivo. Disclosure Information M. Schwerdtfeger: None. M. Benmebarek: None. F. Markl: None. C.H. Karches: A. Employment (full or part-time); Significant; Daiichi Sankyo Deutschland GmbH. A. Oner: None. M. Geiger: A. Employment (full or part-time); Significant; Roche. B. Cadilha: None. S. Endres: None. V. Desiderio: None. C. Klein: A. Employment (full or part-time); Significant; Roche. S. Kobold: None.

Background CAR T cell therapy remains ineffective in solid tumors. Scarce T cell infiltration and T cell suppression at the tumor site are two notable therapy limitations. T regulatory (Treg) cells are capable of suppressing effective anti-tumor responses through inhibitory factors such as transforming growth factor β (TGF-β). Treg cells expressing the C-C chemokine receptor 8 (CCR8) have been found to accumulate and to correlate with poor prognosis in breast cancer. 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. Materials and Methods CCR8 and DNR can be expressed in murine and human T cells upon retroviral transduction. T cell receptor (TCR) and chimeric antigen receptor (CAR) antigen specific models in murine and human systems were utilized. qPCR, IF microscopy, ELISA and the cancer genome atlas (TCGA) database were used in the steps of ligand identification and hypothesis generation. We employed flow cytometry and multi-photon intra-vital microscopy to interrogate infiltration, proliferation and phenotype of T cell products. Mechanistically, CRISPR was used to dissect the role of the CCL1-CCR8 positive feedback loop in T cell therapy. Results We identified that in an in vivo pancreatic murine model of cancer, the CCR8 gene was upregulated in tumor infiltrated lymphocytes compared to T cells that accumulated in the spleen. In this same tumor model, CCL1 could be detected in tumor explants. We identified that this secreted CCL1 from activated effector T cells potentiates a feedback loop for CCR8+ T cell recruitment to the tumor site. The introduction of CCR8 and DNR receptors in primary T cells improved migration towards CCL1 and improved proliferation capacity in the presence of TGF-β. Besides these effects, these receptors did not further impact effector and memory phenotype or secretome of T cell products. The CCR8-driven sustained and improved infiltration synergized with TGF-β-shielding conferred by the DNR for improved therapeutic efficacy, allowing tumor rejection in models that are otherwise completely resistant to CAR T cell therapy. Conclusions We conclude that the combination of CCR8- and DNR-transduction into antigen-specific T cells can exploit two critical biological axes to render T cell therapy effective in solid tumors such as pancreatic cancer. Beyond TGF-β, relieving other immunosuppressive axes may further sustain a CCL1 feedback loop mechanism to improve anti-tumoral function of CCR8+ ACT. This therapeutic approach could be extended to other Treg-rich solid tumor entities where limited infiltration into the tumor and intra-tumoral T cell proliferation prevent therapeutic success. Furthermore, the CCL1-CCR8 axis heralds the potential to be used as a target to improve the efficacy of immunotherapies beyond ACT. Disclosure Information B.L. Cadilha: None. M.R. Benmebarek: None. K. Dorman: None. A. Oner: None. T. Lorenzini: None. H. Obeck: None. S. Stoiber: None. D. Huynh: None. F. Markl: None. M. Seifert: None. K. Manske: None. S. Endres: None. S. Kobold: None.

Cytotoxic T lymphocyte (CTL) responses against tumors are maintained by stem-like memory cells that self-renew but also give rise to effector-like cells. The latter gradually lose their anti-tumor activity and acquire an epigenetically fixed, hypofunctional state, leading to tumor tolerance. Here, we show that the conversion of stem-like into effector-like CTLs involves a major chemotactic reprogramming that includes the upregulation of chemokine receptor CXCR6. This receptor positions effector-like CTLs in a discrete perivascular niche of the tumor stroma that is densely occupied by CCR7+ dendritic cells (DCs) expressing the CXCR6 ligand CXCL16. CCR7+ DCs also express and trans-present the survival cytokine interleukin-15 (IL-15). CXCR6 expression and IL-15 trans-presentation are critical for the survival and local expansion of effector-like CTLs in the tumor microenvironment to maximize their anti-tumor activity before progressing to irreversible dysfunction. These observations reveal a cellular and molecular checkpoint that determines the magnitude and outcome of anti-tumor immune responses.

Targeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+ and CD123+ AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment., Keypoints Modular platform enabling controlled targeting of AML by SAR-transduced T cells in combination with tandem scFv constructs. Efficient lysis of primary AML blasts in vitro and strong antitumoral effects and T cell persistence in xenograft models.

Background CAR T cell therapy is remarkably successful in patients with hematological malignancies, in some cases inducing durable remissions. However, it remains ineffective in solid tumors, in part due to poor T cell infiltration into the tumor mass. Determinants of successful T cell infiltration to the tumor site remain to be defined. In contrast, tumors actively attract T regulatory (Treg) cells for immune suppression through the C-C chemokine receptor 8 (CCR8) - CCL1 axis. As this axis is functional across cancer entities, we postulated that CCR8 could also be used to target tumor-ablating T cells to the tumor site. Material and methods Murine and human CCR8 have been cloned in a retroviral expression vector. CCR8 can be expressed in murine and human T cells upon transduction. A chimeric antigen receptor (CAR) targeting the murine epithelial cell adhesion molecule (EpCAM) was used for syngeneic pancreatic tumor models and a CAR targeting human mesothelin was used for a xenograft pancreatic tumor model.Mechanistically, we use flow cytometry and multi-photon intra-vital microscopy to interrogate infiltration of CCR8-transduced CAR T cells. Results Here we show that genetically engineering CAR T cells to express CCR8 improves their migration into solid tumors and allows rejection of tumors that are otherwise resistant to CAR T cell therapy. We demonstrate the capacity of these enhanced CAR T cells to stunt solid tumor growth and improve survival in both murine syngeneic and human xenograft tumor models. Conclusion Our results demonstrate the viability of using CCR8 to confer Tregcell trafficking-properties in CAR T cells to enable their effectiveness in solid tumors. This receptor may be combined with other promising strategies to improve the efficacy of cellular approaches. Disclosure Information B. L. Cadilha: None. K. Dorman: None. D. Huynh: None. T. Lorenzini: None. M. Vanttinen: None. M. -R. Benmebarek: None. S. Stoiber: None. J. Suarez-Gosalvez: None. S. Endres: None. S. Kobold: None.

Background Immunotherapeutic approaches, including immune checkpoint blockade and adoptive T cell therapy (ACT) in the form of tumor-infiltrating lymphocytes (TILs), have had marked success in the treatment of melanoma. Despite these successes, many patients are refractory to treatment or relapse with therapy-resistant disease. To overcome these limitations, we propose a controlled ACT approach, where T cells are armed with synthetic agonistic receptors (SARs) that are conditionally activated only in the presence of a target melanoma-associated antigen, and a cross-linking bispecific antibody (BiAb) specific for both (SAR) T cell and tumour cell. Materials and Methods A SAR composed of an extracellular EGFRvIII, trans- membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap- extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. We validated our approach in two murine as well as two human cancer models expressing our melanoma-associated target antigens TYRP (murine) and MCSP (human). We confirmed conditional and specific stimulation and proliferation of our T cells, as well as their tumour-antigen-directed cytotoxicity, in vitro and in vivo. Results Crosslinking TYRP-EGFRvIII (murine) and MCSP-EGFRvIII (human) BiAb, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation of SAR-T cells and directed tumour cell lysis with specificity towards two TYRP-expressing murine melanoma and two MCSP-expressing human melanoma cancer models. In vivo, anti-tumoural activity was mediated by the co-administration of SAR-T cells and BiAb, in an A375 melanoma xenograft model. Further, overexpression of IDO (a key immunosuppressive enzyme implicated in the suppression of T cell function in the tumor microenvironment) in a melanoma model did not influence the killing kinetics of SAR T cells. Conclusions Here we apply the SAR x BiAb approach in efforts to deliver specific and conditional activation of synthetic agonistic receptor transduced T cells, and targeted tumour cell lysis. The modularity of our platform is key for a targeting approach in a tumor entity with a high mutational load such as melanoma and is fundamental in our drive towards personalised immunotherapies. Further, the SAR approach has demonstrated resistance to IDO-mediated inhibition in the context of melanoma, an interesting axis that requires further investigation. Disclosure Information M. Benmebarek: None. J. Keyl: None. F. Markl: None. M. Geiger: A. Employment (full or part-time); Significant; Roche. C. Karches: None. S. Rausch: None. A. Gottschlich: None. A. Oner: None. M. Feinendegen: None. J. Dorr: None. B. Cadilha: None. S. Endres: None. C. Klein: A. Employment (full or part-time); Significant; Roche. S. Kobold: None.

Background Chimeric antigen receptor (CAR) T cell therapy is currently approved for the treatment of some hematological malignancies. However, CAR T cells have so far lacked efficacy in the treatment of solid tumors. A major hurdle of CAR T cell therapy is the limited infiltration of CAR T cells into tumor tissue. Chemokine receptors enable immune cells to migrate along a chemokine gradient. Here, we show that overexpression of the C-X-C chemokine receptor 6 (CXCR6) enhances CAR T cell accumulation in C-X-C motif ligand 16 (CXCL16)-positive xenograft pancreatic cancer models, resulting in increased anti-tumor potency of anti-mesothelin CAR T cells. Materials and Methods Human T cells were retrovirally transduced with an anti-mesothelin CAR and CXCR6. NSG mice were injected subcutaneously with mesothelin-CXCL16-overexpressing tumor cells. Mice were treated once with CAR-, CAR-CXCR6- or mock-transduced T cells when tumors were palpable and tumor size was monitored with a caliper. In a separate tracking experiment, subcutaneous tumors were established as described above and the presence of T cells at the tumor site was determined by FACS analysis within one week after adoptive T cell transfer. For orthotopic xenograft experiments mesothelin-CXCL16-overexpressing tumor cells were directly injected into the pancreas of NSG mice and one-time treatment with CAR-, CAR-CXCR6- or mock T cells was performed 5 days post tumor injection. Results In a subcutaneous xenograft model of pancreatic cancer CXCR6-expressing CAR T cells displayed improved anti-tumoral potency compared to CAR T cells without CXCR6, resulting in prolonged survival of mice and tumor clearance in 9 out of 10 CAR-CXCR6-treated mice. A tracking experiment confirmed the increased accumulation of CAR-CXCR6 T cells compared to CAR T cells at the subcutaneous tumor site, suggesting increased migratory capacity of CAR-CXCR6-transduced T cells towards CXCL16-expressing tumors as the mode of action. Treatment of orthotopic pancreatic cancer xenografts similarly revealed prolonged survival of CAR-CXCR6-treated animals in comparison to CAR-treated animals, suggesting improved anti-tumor efficacy of CAR-CXCR6-transduced T cells. Conclusions Forced expression of CXCR6 in anti-mesothelin CAR T cells increased the accumulation of CAR T cells at the CXCL16-positive tumor site, resulting in improved survival of treated mice and in complete tumor rejection in the majority of cases. This data reveals the potential of CXCR6 to direct CAR T cells to the tumor site and this approach may therefore be an attractive strategy to target a major pitfall in the translation of CAR T cell therapy to solid tumors. Disclosure Information S. Stoiber: None. S. Lesch: None. J. Ogonek: None. B. Cadilha: None. M. Benmebarek: None. A. Gottschlich: None. P. Metzger: None. C. Horth: None. A. Nottebrock: None. S. Endres: None. S. Kobold: None.

Background Indoleamine-2,3-dioxygenase 1 (IDO1) is a cytosolic enzyme that catalyzes the rate limiting reaction in the kynurenine pathway. Dendritic cells, macrophages and several tumor entities have been described to express IDO1. In the tumor microenvironment IDO1 promotes tryptophan starvation and accumulation of kynurenines which result in T effector cell proliferation arrest and T regulatory cell induction. Additionally, IDO1 possesses two immunoreceptor tyrosine-based inhibitory motifs (ITIM) that upon phosphorylation can act as docking sites for the recruitment and activation of the tyrosine phosphatases SHP–1 and SHP–2 and ultimately to an activation of the non-canonical NF-ΚB pathway. Whether IDO1 is expressed in T cells and its potential function is unknown. Materials and Methods Using IDO1-deleted splenocytes from CD4-Cre Ido1fl/fl mice and WT controls, we evaluated the induction of IDO1 in T cells, as well as the effect of IDO1 in T cell proliferation, differentiation and metabolism. Additionally, we compared in vitro and in vivo the cytotoxic activity of anti-epithelial cell adhesion molecule (EpCAM) chimeric antigen receptor (CAR) T cells using pancreatic tumor cell lines. Results IDO1 is inducible in primary mouse T cells upon T cell activation and type I and type II interferon signaling. Interestingly, the use of IDO1 knockout CAR T cells prolongs survival and improves tumor control compared to WT CAR T cell treatment in subcutaneous and orthotopic pancreatic cancer models. In vitro, T cell proliferation, differentiation and cytotoxic function is comparable in WT and IDO1-deleted T cells. RNA sequencing, metabolic and in vivo tracking studies are currently being performed to pin down IDO1-intrinsic effects on CAR T cells. Conclusions IDO1 is expressed in T cells upon T cell receptor and IFN stimulation and appears to negatively affect tumor control mediated by CAR T cells. Specific IDO1 deletion may improve therapeutic efficacy of CAR T cells in solid tumors, such as pancreatic cancer. Disclosure Information A.M. Senz: None. P. Metzger: None. R.K. Rubens: None. B. Cadilha: None. M. Kirmaier: None. S. Lesch: None. M.R. Benmebarek: None. S. Theurich: None. P. Murray: None. S. Endres: None. S. Kobold: None. L.M. Konig: None. P. Duewell: None. M. Schnurr: None.

Background Targeted immunotherapies have shown limited success in the context of acute myeloid leukemia (AML). Due to the mutational landscape and heterogeneity attributed to this malignancy and toxicities associated with the targeting of myeloid lineage antigens, it has become apparent that a modular and controllable cell therapy approach with the potential to target multiple antigens is required. We propose a controlled ACT approach, where T cells are armed with synthetic agonistic receptors (SARs) that are conditionally activated only in the presence of a target AML-associated antigen, and a cross-linking bispecific T cell engager (BiTE) specific for both (SAR) T cell and tumour cell. Materials and Methods A SAR composed of an extracellular EGFRvIII, trans-membrane CD28, and intracellular CD28 and CD3z domains was fused via overlap-extension PCR cloning. T cells were retrovirally transduced to stably express our SAR construct. SAR-specific bispecific T cell engagers (BiTE) that target AML-associated antigens were designed and expressed in Expi293FTMcells and purified by nickel affinity and size exclusion chromatography (SEC). We validated our approach in three human cancer models and patient-derived AML blasts expressing our AML-associated target antigen CD33. Results CD33-EGFRvIII BiTE, monovalently selective for our SAR, induced conditional antigen-dependent activation, proliferation and differentiation of SAR-T cells. Further, SAR T cells bridged to their target cells by BiTE could form functional immunological synapses, resulting in efficient tumor cell lysis with specificity towards CD33-expressing AML cells. SAR.BiTE combination could also mediate specific cytotoxicity against patient-derived AML blasts whilst driving SAR T cell activation. In vivo, treatment with SAR.BiTE combination could efficiently eradicate leukemia and enhance survival in an AML xenograft model. Furthermore, we could show selective activation of SAR T cells, as well as a controllable reversibility of said activation upon depletion of the T cell engaging molecule. Conclusions Here we apply the SAR x BiAb approach in efforts to deliver specific and conditional activation of agonistic receptor-transduced T cells, and targeted tumour cell lysis. The modularity of our platform will allow for a multi-targeting ACT approach with the potential to translate the ACT successes of B cell malignancies to AML. With a lack of truly specific AML antigens, it is invaluable that this approach possesses an intrinsic safety switch via its BiTE facet. Moreover, we are able to circumvent pan-T cell activation due to the specific targeting and activation of SAR T cells. Disclosure Information M. Benmebarek: None. B.L. Cadilha: None. M. Hermann: None. S. Lesch: None. C. Augsburger: None. B. Brauchle: None. S. Stoiber: None. A. Darwich: None. F. Rataj: None. C. Klein: A. Employment (full or part-time); Significant; Roche. K. Hopfner: None. M. Subklewe: None. S. Endres: None. S. Kobold: None.

Forced expression of C-X-C chemokine receptor type 6 in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer.The efficacy of adoptive cell therapy for solid tumours is hampered by the poor accumulation of the transferred T cells in tumour tissue. Here, we show that forced expression of C-X-C chemokine receptor type 6 (whose ligand is highly expressed by human and murine pancreatic cancer cells and tumour-infiltrating immune cells) in antigen-specific T cells enhanced the recognition and lysis of pancreatic cancer cells and the efficacy of adoptive cell therapy for pancreatic cancer. In mice with subcutaneous pancreatic tumours treated with T cells with either a transgenic T-cell receptor or a murine chimeric antigen receptor targeting the tumour-associated antigen epithelial cell adhesion molecule, and in mice with orthotopic pancreatic tumours or patient-derived xenografts treated with T cells expressing a chimeric antigen receptor targeting mesothelin, the T cells exhibited enhanced intratumoral accumulation, exerted sustained anti-tumoral activity and prolonged animal survival only when co-expressing C-X-C chemokine receptor type 6. Arming tumour-specific T cells with tumour-specific chemokine receptors may represent a promising strategy for the realization of adoptive cell therapy for solid tumours.

Purpose: Genetically engineered T cells are powerful anticancer treatments but are limited by safety and specificity issues. We herein describe an MHC-unrestricted modular platform combining autologous T cells, transduced with a targetable synthetic agonistic receptor (SAR), with bispecific antibodies (BiAb) that specifically recruit and activate T cells for tumor killing. Experimental Design: BiAbs of different formats were generated by recombinant expression. T cells were retrovirally transduced with SARs. T-cell activation, proliferation, differentiation, and T-cell–induced lysis were characterized in three murine and human tumor models in vitro and in vivo. Results: Murine T cells transduced with SAR composed of an extracellular domain EGFRvIII fused to CD28 and CD3ζ signaling domains could be specifically recruited toward murine tumor cells expressing EpCAM by anti-EGFRvIII × anti-EpCAM BiAb. BiAb induced selective antigen-dependent activation, proliferation of SAR T cells, and redirected tumor cell lysis. Selectivity was dependent on the monovalency of the antibody for EGFRvIII. We identified FAS ligand as a major mediator of killing utilized by the T cells. Similarly, human SAR T cells could be specifically redirected toward mesothelin-expressing human pancreatic cancer cells. In vivo, treatment with SAR T cells and BiAb mediated antitumoral activity in three human pancreatic cancer cell xenograft models. Importantly, SAR activity, unlike CAR activity, was reversible in vitro and in vivo. Conclusions: We describe a novel ACT platform with antitumor activity in murine and human tumor models with a distinct mode of action that combines adoptive T-cell therapy with bispecific antibodies.

The remarkable success of chimeric antigen receptor (CAR)-engineered T cells in pre-B cell acute lymphoblastic leukemia (ALL) and B cell lymphoma led to the approval of anti-CD19 CAR T cells as the first ever CAR T cell therapy in 2017. However, with the number of CAR T cell-treated patients increasing, observations of tumor escape and resistance to CAR T cell therapy with disease relapse are demonstrating the current limitations of this therapeutic modality. Mechanisms hampering CAR T cell efficiency include limited T cell persistence, caused for example by T cell exhaustion and activation-induced cell death (AICD), as well as therapy-related toxicity. Furthermore, the physical properties, antigen heterogeneity and immunosuppressive capacities of solid tumors have prevented the success of CAR T cells in these entities. Herein we review current obstacles of CAR T cell therapy and propose strategies in order to overcome these hurdles and expand CAR T cell therapy to a broader range of cancer patients.

Immunotherapy has become an established pillar of cancer treatment improving the prognosis of many patients with a broad variety of hematological and solid malignancies. The two main drivers behind this success are checkpoint inhibitors (CPIs) and chimeric antigen receptor (CAR) T cells. This review summarizes seminal findings from clinical and translational studies recently presented or published at important meetings or in top-tier journals, respectively. For checkpoint blockade, current studies focus on combinational approaches, perioperative use, new tumor entities, response prediction, toxicity management and use in special patient populations. Regarding cellular immunotherapy, recent studies confirmed safety and efficacy of CAR T cells in larger cohorts of patients with acute lymphoblastic leukemia or diffuse large B cell lymphoma. Different strategies to translate the striking success of CAR T cells in B cell malignancies to other hematological and solid cancer types are currently under clinical investigation. Regarding the regional distribution of registered clinical immunotherapy trials a shift from PD-1 / PD-L1 trials (mainly performed in the US and Europe) to CAR T cell trials (majority of trials performed in the US and China) can be noted.

Cancer therapy has entered a new era, transitioning from unspecific chemotherapeutic agents to increasingly specific immune-based therapeutic strategies. Among these, chimeric antigen receptor (CAR) T cells have shown unparalleled therapeutic potential in treating refractory hematological malignancies. In contrast, solid tumors pose a much greater challenge to CAR T cell therapy, which has yet to be overcome. As this novel therapeutic modality matures, increasing effort is being invested to determine the optimal structure and properties of CARs to facilitate the transition from empirical testing to the rational design of CAR T cells. In this review, we highlight how individual CAR domains contribute to the success and failure of this promising treatment modality and provide an insight into the most notable advances in the field of CAR T cell engineering.

International audience; Microphthalmia-associated transcription factor (MITF) is a key transcription factor in melanoma development and progression. MITF amplification and downregulation have been observed in a significant proportion of melanoma patients and correlate with clinical outcomes. Here, we have investigated the effect of MITF on melanoma chemokine expression and immune cell attraction. In B16F10 melanoma cells, MITF knockdown reduced expression of CXCL10, with concomitantly decreased attraction of immune cells and accelerated tumor outgrowth. Conversely, overexpression of MITF in YUMM1.1 melanoma cells also led to an increased immune cell attraction in vitro. Subcutaneous YUMM1.1 melanomas overexpressing MITF however showed a reduced immune infiltration of lymphocytes and an increased tumor growth. In human melanoma cell lines, silencing of MITF enhanced chemokine production and immune cell attraction, while overexpression of MITF led to lower immune cell attraction. In summary, our results show that MITF regulates chemokine expression in murine and in human melanoma cells, and affects in vivo immune cell attraction and tumor growth. These results reveal a functional relationship between MITF and immune cell infiltration, which may be exploited for cancer therapy.

Association of epithelial-mesenchymal transition with the metastatic potential of disseminated cells in breast cancer was studied., Carcinoma cells undergo epithelial-mesenchymal transition (EMT); however, contributions of EMT heterogeneity to disease progression remain a matter of debate. Here, we addressed the EMT status of ex vivo cultured circulating and disseminated tumor cells (CTCs/DTCs) in a syngeneic mouse model of metastatic breast cancer (MBC). Epithelial-type CTCs with a restricted mesenchymal transition had the strongest lung metastases formation ability, whereas mesenchymal-type CTCs showed limited metastatic ability. EpCAM expression served as a surrogate marker to evaluate the EMT heterogeneity of clinical samples from MBC, including metastases, CTCs, and DTCs. The proportion of epithelial-type CTCs, and especially DTCs, correlated with distant metastases and poorer outcome of patients with MBC. This study fosters our understanding of EMT in metastasis and underpins heterogeneous EMT phenotypes as important parameters for tumor prognosis and treatment. We further suggest that EpCAM-dependent CTC isolation systems will underestimate CTC numbers but will quantify clinically relevant metastatic cells.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (Treg) cells that restrict the function of effector T cells and thereby promote tumour growth1. The anti-tumour activity of effector T cells can be therapeutically unleashed, and is now being exploited for the treatment of some forms of human cancer. However, weak tumour-associated inflammatory responses and the immune-suppressive function of Treg cells remain major hurdles to broader effectiveness of tumour immunotherapy2. Here we show that, after disruption of the CARMA1-BCL10-MALT1 (CBM) signalosome complex, most tumour-infiltrating Treg cells produce IFNγ, resulting in stunted tumour growth. Notably, genetic deletion of both or even just one allele of CARMA1 (also known as Card11) in only a fraction of Treg cells-which avoided systemic autoimmunity-was sufficient to produce this anti-tumour effect, showing that it is not the mere loss of suppressive function but the gain of effector activity by Treg cells that initiates tumour control. The production of IFNγ by Treg cells was accompanied by activation of macrophages and upregulation of class I molecules of the major histocompatibility complex on tumour cells. However, tumour cells also upregulated the expression of PD-L1, which indicates activation of adaptive immune resistance3. Consequently, blockade of PD-1 together with CARMA1 deletion caused rejection of tumours that otherwise do not respond to anti-PD-1 monotherapy. This effect was reproduced by pharmacological inhibition of the CBM protein MALT1. Our results demonstrate that partial disruption of the CBM complex and induction of IFNγ secretion in the preferentially self-reactive Treg cell pool does not cause systemic autoimmunity but is sufficient to prime the tumour environment for successful immune checkpoint therapy.

BACKGROUND CD16-chimeric antigen receptors (CAR) T cells recognise the Fc-portion of therapeutic antibodies, which can enable the selective targeting of different antigens. Limited evidence exists as to which CD16-CAR design and antibody partner might be most effective. We have hypothesised that the use of high-affinity CD16 variants, with increased Fc-terminus antibody affinity, combined with Fc-engineered antibodies, would provide superior CD16-CAR T cell efficacy. METHODS CD16-CAR T (wild-type or variants) cells were co-cultured with Panc-1 pancreatic cancer, Raji lymphoma or A375 melanoma cells in the presence or absence of anti-CD20, anti-MCSP, wild-type or the glycoengineered antibody variants. The endpoints were proliferation, activation, and cytotoxicity in vitro. RESULTS The CD16 158 V variant of CD16-CAR T cells showed increased cytotoxic activity against all the tested cancer cells in the presence of the wild-type antibody directed against MCSP or CD20. Glycoengineered antibodies enhanced CD16-CAR T cell activity irrespective of CD16 polymorphisms as compared with the wild-type antibody. The combination of the glycoengineered antibodies with the CD16-CAR 158 V variant synergised as seen by the increase in all endpoints. CONCLUSION These results indicate that CD16-CAR with the high-affinity CD16 variant 158 V, combined with Fc-engineered antibodies, have high anti-tumour efficacy.

Solid tumours are infiltrated by effector T cells with the potential to control or reject them, as well as by regulatory T (T

Adoptive T cell therapy, namely chimeric antigen receptor (CAR) T cell therapy has been a groundbreaking and effective treatment of relapsed or refractory haematological malignancies. Still, many patients do not respond or relapse with treatment-resistant disease. Additionally, toxicities such as cytokine release syndrome remain problematic. Cancer heterogeneity, beyond cancer types and inter-patient differences, is present within every individual patient. This heterogeneity, especially in the context of solid tumors, has meant that targeted immunotherapies have fared relatively poorly, creating a need for a modular platform with a capacity to target multiple antigens simultaneously and/or sequentially. Likewise, treatment-related toxicities have limited the therapeutic efficacy and breadth of patient selection. To tackle these caveats through a modular and controllable approach, we equipped T cells with synthetic agonistic receptors (SARs) that are only activated when a tumor-associated antigen and a cross-linking bispecific antibody (BiAb) specific for both SAR T cell and tumor cell are also present. The SAR itself is constituted of an inert extracellular domain in the form of EGFRvIII, that is fused to the T cell activating domains CD28 and CD3ζ. The BiAb employed is a trivalent CrossMab, with two binding arms (2 x Fab) for the tumor-associated antigen (mesothelin), and one binding arm for the SAR receptor (EGFRvIII). We showed that BiAb triggering of the SAR is conditional upon the binding of the second BiAb specificity. What is particularly advantageous with this approach is that T cell activation may only occur when the BiAb is present and in proximity to the antibody-targeted tumor cell. This conditional T cell activation is an inherent safety feature of the platform, whereby if unwanted levels of T cell activation are observed, depletion of the BiAb from the system could result in the reversal of said activation, thus managing the potential toxicity. This work was able to determine the validity and efficacy of the approach. Through the generation of several human pancreatic cancer models, and extensive in vitro and in vivo testing, the platform could be characterized. Its translational relevance and significance as a next-generation adoptive T cell therapy with the potential to plug some gaping pitfalls of current ACT approaches were also shown.

Immunotherapy has successfully been implemented as the standard of care in a number of oncologic indications. A hallmark of cancer immunotherapy is the successful activation of T cells against cancer cells, leading to unparalleled efficacy for some tumour entities. However, current approved approaches are not specific, limiting both their activity and their safety. A more tailored way of using the therapeutic potential of T cells is adoptive T cell therapy, which encompasses ex vivo T cell manipulation and reinfusion to patients suffering from cancer. In haematologic malignancies such as acute lymphatic leukaemia of the B cell lineage, T cells modified with a chimeric antigen receptor against the B cell lineage antigen CD19 induce remissions in a high proportion of patients. In contrast, patients suffering from advanced solid tumours have shown little benefit from cell-based approaches. This is partly due to limited access of T cells to the tumour tissue, consequently restricting T cell activity. In this review, we focus on the limitations of T cell trafficking towards solid tumours. We summarise the existing knowledge on lymphocyte migration to understand how this pathway may be used to open therapeutic approaches for a broader range of indications. We also review new strategies targeting the tumour site that aid naturally occurring or gene-engineered T cells to migrate to solid tumours. Finally, we discuss how guiding T cells towards the tumour might contribute in harnessing their full cytolytic potential.

The combination of directed migration and immunosuppressive shielding enables effective T cell therapy in solid tumors., 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.

Effective adoptive T cell therapy (ACT) comprises the killing of cancer cells through the therapeutic use of transferred T cells. One of the main ACT approaches is chimeric antigen receptor (CAR) T cell therapy. CAR T cells mediate MHC-unrestricted tumor cell killing by enabling T cells to bind target cell surface antigens through a single-chain variable fragment (scFv) recognition domain. Upon engagement, CAR T cells form a non-classical immune synapse (IS), required for their effector function. These cells then mediate their anti-tumoral effects through the perforin and granzyme axis, the Fas and Fas ligand axis, as well as the release of cytokines to sensitize the tumor stroma. Their persistence in the host and functional outputs are tightly dependent on the receptor’s individual components—scFv, spacer domain, and costimulatory domains—and how said component functions converge to augment CAR T cell performance. In this review, we bring forth the successes and limitations of CAR T cell therapy. We delve further into the current understanding of how CAR T cells are designed to function, survive, and ultimately mediate their anti-tumoral effects.