Conventional Chimeric Antigen Receptor Research Articles

Abstract 934: γδ CAR T-cell therapy significantly mitigates bone metastatic castrate-resistant prostate cancer

Abstract Bone metastasis is a frequent complication in advanced prostate cancer, with the resultant lesions significantly contributing to patient morbidity and mortality. While next generation hormone ablation therapies and bone protecting bisphosphonates alleviate these symptoms, the disease remains incurable, and new therapeutic approaches are of urgent need. In this regard, we have focused on tapping the tumor-seeking potential of chimeric antigen receptor (CAR) T cells. CAR T cells have shown remarkable anti-tumor responses in hematologic malignancies, but unique physical and biological challenges have hindered their activity in solid tumors. Interestingly, patients treated with bisphosphonates such as zoledronate exhibit enhanced recruitment and activation of the γδ subset of T cells in bone due intracellular accumulation of isopentenyl pyrophosphate (IPP) phosphoantigen. Whereas conventional CAR T cell therapies utilize αβ T cells, we posit that designing γδ CAR T cells could improve homing to bone metastases when administered with bisphosphonates. First, to test the impact of prostate specific stem cell antigen (PSCA)-specific γδ CAR T cells against bone metastatic prostate cancer in vivo, NSG mice (n=10) were intratibially injected with PSCA/luciferase-expressing C4-2B (2x105) castrate resistant prostate cancer cells. Tumors were allowed to establish for 10 days and then randomized into control or γδ CAR T (1.5x107 via tail vein) groups. Subsequent bioluminescent imaging indicated a rapid and significant (p=0.0006) regression of tumors in the γδ CAR T cell group, leading to increased overall survival (5/5 γδ CAR T vs. 0/5 control after 68 days, p=0.0002). Ex vivo bone morphometry analysis also demonstrated the significant protective effect of γδ CAR T associated bone disease. To determine whether bisphosphonates could further enhance the homing of γδ CAR T to bone, NSG mice (n=30) were intratibially injected with C4-2B (2x105), and randomized into control and zoledronate (30µg/kg) groups. After 10 days, mice received γδ T cells (3x106). Subsets were sacrificed at 1, 3, and 5 days post-T cell administration, and peripheral blood, tibia bone marrow, and spleens isolated. CD3-Vδ2 flow cytometry indicated increased γδ T cells in the tibia bone marrow from zoledronate groups (Day 1=61%, Day 3=32%, and Day 5=57%). Furthermore, decreased tumor growth rates were observed in the zoledronate group, suggesting that increased homing of γδ T cells induced anti-tumor effects. Our data to date demonstrate that γδ CAR T cells significantly mitigate bone metastatic prostate cancer and associated bone disease. Further, bisphosphonates (already used in the clinical setting) enhance the homing of γδ CAR T cells to the tumor-bone microenvironment. We posit that γδ CAR T will be an effective immunotherapy approach for the treatment of men with incurable bone metastatic prostate cancer. Citation Format: Jeremy Steven Frieling, Maria Cecilia Ramello, Ismahene Benzaid, Emiliano Roselli, Chen Hao Lo, Conor C. Lynch, Daniel Abate-Daga. γδ CAR T-cell therapy significantly mitigates bone metastatic castrate-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 934.

Abstract 1424: Preclinical development of TAC01-CD19, a CD19 specific T-cell Antigen Coupler (TAC) therapy for the treatment of CD19-positive B cell malignancies

Abstract Background: The T cell antigen coupler (TAC) is a chimeric receptor co-opting the natural TCR receptor signaling complex to target tumor antigens in an MHC-independent manner (Helsen et. al., Nat. Comm. 2018). TAC engineered T cells mediate biological effects that are distinct from conventional chimeric antigen receptors (CARs) and may offer safety advantages due to greater target selectivity and no detectable off-target toxicity. Here, we present preclinical data of TAC01-CD19, an autologous CD19-specific TAC-T cell product candidate for clinical testing in patients with CD19-positive B cell malignancies Materials and Methods: T cells from healthy donors and lymphoma patients were engineered with a CD19-specific TAC using lentivirus. Anti-tumor activity, T cell expansion and persistence were measured in the CD19-positive NALM-6 and Jeko-1 mouse tumor xenograft models. We used flow cytometry to determine surface expression of CD19-TAC, cytokine production and in vitro T cell proliferation. Cytotoxicity was evaluated in vitro using luciferase-based killing assay. Results: Cytotoxicity was measured against multiple CD19 positive tumors cells (NALM6/Jeko-1/Raji) showing strong cytotoxicity in all cases. Treatment of established NALM-6 (acute lymphoblastic leukemia) and Jeko-1 xenografts (mantle cell lymphoma) with CD19 TAC-T cells resulted in clearance of tumors within a few weeks of T cell infusion. In vivo efficacy was tested across multiple donors, establishing consistent and reproducible anti-tumor efficacy. Monitoring of TAC T cells post-infusion revealed robust expansion that peaked in the peripheral blood 1-2 weeks post-infusion. Mice that cleared tumors following TAC-T cell treatment were resistant to subsequent challenge with fresh tumor cells demonstrating persistence of TAC-T cells. In contrast, treatment with control TAC-T had no impact on tumor growth. A histological examination of normal mouse tissues failed to detect TAC-T cell induced toxicity. Conclusion: The pre-clinical evaluation of TAC01-CD19 has demonstrated specific anti-tumor responses without signs of toxicity. Results were reproducible across multiple donors demonstrating the robustness of the CD19 TAC system. TAC01-CD19 is expected to enter clinical testing in patients with CD19-positive hematological malignancies in Q2 2019. Citation Format: Christopher W. Helsen, Danielle Hayes, Joanne Hammill, Criag Aarts, Andreas Bader, Donna Rill, Jonathan Bramson. Preclinical development of TAC01-CD19, a CD19 specific T-cell Antigen Coupler (TAC) therapy for the treatment of CD19-positive B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1424.

CAR-Based Strategies beyond T Lymphocytes: Integrative Opportunities for Cancer Adoptive Immunotherapy.

Chimeric antigen receptor (CAR)-engineered T lymphocytes (CAR Ts) produced impressive clinical results against selected hematological malignancies, but the extension of CAR T cell therapy to the challenging field of solid tumors has not, so far, replicated similar clinical outcomes. Many efforts are currently dedicated to improve the efficacy and safety of CAR-based adoptive immunotherapies, including application against solid tumors. A promising approach is CAR engineering of immune effectors different from αβT lymphocytes. Herein we reviewed biological features, therapeutic potential, and safety of alternative effectors to conventional CAR T cells: γδT, natural killer (NK), NKT, or cytokine-induced killer (CIK) cells. The intrinsic CAR-independent antitumor activities, safety profile, and ex vivo expansibility of these alternative immune effectors may favorably contribute to the clinical development of CAR strategies. The proper biological features of innate immune response effectors may represent an added value in tumor settings with heterogeneous CAR target expression, limiting the risk of tumor clonal escape. All these properties bring out CAR engineering of alternative immune effectors as a promising integrative option to be explored in future clinical studies.

S911 A COMBINATORIAL RECEPTOR STRATEGY TO IMPROVE MULTIPLE MYELOMA THERAPY WITH CHIMERIC ANTIGEN RECEPTOR‐ENGINEERED T LYMPHOCYTES

Background:Multiple Myeloma (MM), a disease characterized by a clonal expansion of plasma cells, still remains incurable with current treatment options. The genetic engineering of T lymphocytes with chimeric antigen receptors (CAR) has recently emerged as a promising therapeutic approach and BCMA has shown to be a good MM‐specific antigenic target. Despite its specificity, the intensity of BCMA is variable in MM patients and may not be uniform across different subclonal populations which might explain why first clinical trials of BCMA‐CAR‐T cells report lower complete remission rates as well as relapses of BCMA‐low clonal variants. Another MM‐related antigen, CD38, is uniformly highly expressed in all malignant MM cells.Aims:We hypothesized that targeting 2 MM‐related antigens would increase cytotoxic ability of CAR‐T cells and the incorporation of a dual costimulation design would confer increased persistence. Thus, we propose to improve CAR‐T cell therapy for MM by dual targeting of BCMA and CD38 and incorporating full costimulation with 4‐1BB and CD28 signaling by using the combination of a CAR and a chimeric costimulatory receptor (CCR).Methods:To this end, we designed first and second generation CAR constructs targeting BCMA (BCMA‐z and BCMA‐28z), CCR constructs targeting CD38 (CD38‐28BB and CD38‐BB) as well as a CD38‐engager construct lacking intracellular domain (CD38del). We then evaluated in vitro the anti‐MM cytotoxic potential, cytokine production, proliferative capacity and exhaustion of the double‐targeting CAR‐T cells (BCMA‐z/38‐28BB, BCMA‐28z/CD38‐BB or BCMA‐z/CD38del) compared to single‐targeting T cells carrying a conventional CAR (BCMA‐28z, BCMA‐BBz, BCMA‐z), or CCR (38‐28z or 38‐28BB).Results:We found that double‐targeting BCMA‐z/38‐28BB CAR‐T cells showed significantly increased cytotoxic capacity against BCMA+CD38+ MM cell lines and primary MM cells, even in low effector to target ratios, as compared to the single‐targeting BCMA‐CAR‐T cells. This effect was diminished when a BCMA+CD38‐ cell line was used as target, while cells carrying only a CCR had no cytotoxic potential. Interestingly, BCMA‐z/CD38del T cells, where CD38‐engagement does not mediate any costimulation signal, had similar cytotoxic ability as BCMA‐z/38‐28BB cells, therefore the increased killing capacity of double‐targeting CAR‐T cells is due to CD38 engagement and the consecutive increase in avidity. Finally, double‐targeting BCMA‐z/38‐28BB CAR‐T cells showed significantly higher cytokine secretion (IL‐2, IFN‐γ, TNF‐α), proliferative capacity and induction of PD‐1 expression than single‐targeting BCMA‐CAR‐T cells, which was not reproduced by BCMAz/CD38del T cells indicating that this effect is a result of combining both CD28 and 4‐1BB costimulation.Summary/Conclusion:In conclusion, we here show that double targeting MM with a BCMA‐CAR and a CD38‐CCR provides higher effector‐MM avidity and full costimulation. Engagement of the CD38‐CCR enhances the anti‐MM cytotoxicity and persistence and reduces the exhaustion of double‐targeting compared to conventional single‐targeting CAR T cells. Therefore double‐targeting of BCMA and CD38 is a powerful strategy to improve clinical outcomes of BCMA‐CAR T cell therapy.

Development of a CD19-TAC therapy in preparation of human trials

Background & Aim The T cell antigen coupler (TAC) is a novel chimeric receptor designed to redirect T cells in an MHC-independent fashion and to activate T cells by co-opting the natural TCR receptor. In vitro and in vivo assessments of TAC T cells in solid tumor models (Helsen et. al. Nature Communications) have revealed that TACs mediate biological effects that are distinct from conventional chimeric antigen receptors (CARs), providing safety advantages, including greater target selectivity and reduced off-target toxicity. Here, we present the preclinical data of a CD19 targeted TAC T cell product in preparation of Phase I clinical trials. Methods, Results & Conclusion CD19-specific anti-cancer activity was established in vitro and in vivo using multiple liquid tumor models such as NALM6 (acute lymphoblastic leukemia), Jeko-1 (Mantle Cell Lymphoma) and Raji (Burkitt's lymphoma). In all cases, administration of TAC engineered T cells led to tumor regression and long-term tumor control. Mice that responded to treatment were resistant to tumor regrowth in tumor re-challenge experiments. This resistance was specific to CD19-positive tumors and absent in CD19-negative control tumor cells, suggesting the development of TAC T cell persistence. Mice treated with two dose levels were subject to detailed health monitoring and included an assessment of serum cytokines, T cell expansion, clinical chemistries and organ histopathology. These data confirm our previous results produced by other TAC constructs and provide further evidence highlighting its potential as an effective and well tolerated T cell therapy that may be accessible to a broad patient population. A CD19 TAC therapy is expected to enter clinic testing in H1 2019 in patients with DLBCL.

British Society for Gene and Cell Therapy Autumn Conference Friday 23 November 2018 Regent's University Conference Centre, London, UK www.bsgct.org

British Society for Gene and Cell Therapy Autumn Conference Friday 23 November 2018 Regent's University Conference Centre, London, UK www.bsgct.org

Abstract A031: Engineering antigen density sensors for T-cell immunotherapy

Abstract T-cells can be redirected to kill tumor cells via synthetic T-cell receptors known as chimeric antigen receptors (CARs); this approach is becoming a highly promising therapeutic strategy for cancer treatment. Current CAR T-cells, while effective at killing cells expressing the target antigen, fail to discriminate between cells expressing high and low levels of common antigens. Potential target antigens are often also found on normal cells at lower expression levels, and CAR T-cells get activated even by low amounts of target antigen, resulting in potentially deadly toxic effects. We are approaching this problem of antigen density discrimination by using common biochemical mechanisms for cooperative recognition to design new synthetic T-cell receptors. CAR T-cells are highly sensitive to even low densities of the target antigen. Current strategies for generating CAR T-cells consist of selecting antibodies with high affinity since previous studies have shown that the CAR T-cell activity is inversely correlated with the antibody’s affinity. However, these CARs are unable to discriminate between cancer and normal cells based on antigen density. Recent studies suggest that lowering the receptor affinity on a CAR T-cell could increase its selectivity against targeT-cells with different antigen densities. Nevertheless, lowering the affinity is unlikely to provide a sharp density threshold of activation. Instead for a sharp threshold transition from off to on, one would require cooperativity and nonlinear recognition. We have designed a two-step recognition-activation circuit that involves two receptors. In this circuit an initial recognition event alters the potency of a subsequent response. We combined a weak receptor that turns on the activity of a high-affinity receptor that fully activates the cell. For this circuits we have made used of a new class of modular receptors called synthetic notch receptors (SynNotch). SynNotch receptors use antibody-based domains to recognize a target antigen, but when activated, they control transcription via a cleaved transcriptional domain. We have designed and expressed several synNotch/CAR circuits and have tested in vitro their ability to achieve antigen density sensing. These circuits use a synNotch receptor to control the expression of a CAR—all recognizing the same Her2 ligand. The constructs are introduced into T-cells via standard lentiviral infection, and sorted for receptor expression using flow cytometry (anti-myc stain). We have screened candidate receptors for expression and activation response in the human Jurkat T-cell line and human primary CD4+ and CD8+ T-cells from several donors, using standard flow-based assays (e.g. CD69 Activation, T-cell proliferation) and cytokine assays (e.g., IL-2, INF-γ). We are now taking the best circuit candidate and proceeding in mouse models. We have first characterized the growth curves of tumors made by engineered targeT-cells to measure the baseline tumor growth and then I have tested either conventional CAR T-cells or my new engineered T-cells in bilateral tumors into NSG immunocompromised mice, in which one side contains a low-density Her2 K562 tumor, and the other side will contain a higher-density Her2 K562 tumor. The engineered T-cells are injected into the tail vein, and allowed to traffic freely to either tumor. We are monitoring tumor size by caliper over the course of 4 weeks, and tracking the differential clearance of the high- and low-density tumors. We have confirmed that when using a fixed affinity standard CAR T-cell, then cells kill either high- or low-expression-level Her2 tumors with equal efficiency. I have begun parallel mouse experiments with the two receptor circuits that show improved antigen density discrimination in vitro. While still in progress, preliminary results look promising. Citation Format: Rogelio A. Hernandez-Lopez, Wendell A. Lim, Wei Yu. Engineering antigen density sensors for T-cell immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A031.

T Cells Engineered with T Cell Antigen Coupler (TAC) Receptors for Haematological Malignancies

Background:We recently described the T cell antigen coupler (TAC) technology (Helsen et. al. Nature Communications) which is a chimeric receptor that targets antigens in an MHC-independent fashion and activates T cells by co-opting the natural TCR receptor. In vitro and in vivo assessments of TAC T cells in solid tumor models have revealed that TACs mediate biological effects that are distinct from conventional chimeric antigen receptors (CARs) and offer safety advantages, including greater target selectivity and reduced off-target toxicity. Here, we present in vitro and in vivo data showing that TAC-engineered T cells directed against CD19 and BCMA demonstrate robust anti-tumor efficacy in haematological malignancies with no detectable side effects.Materials and Methods:T cells from health donors were engineered with TAC receptors directed against CD19 or BCMA using lentivirus vectors. Flow cytometry was employed to measure surface expression of TAC receptors, cytokine production and proliferation of TAC T cells following stimulation with relevant target cells. Antigen-specific toxicity was measured using a luciferase-based killing assay. Anti-tumor activity was measured against acute lymphoblast leukemia for CD19 and multiple myeloma for BCMA xenografts in immunodeficient NRG mice.Results:Engineering T cells with TAC receptors targeted against either CD19 or BCMA revealed antigen-specific activation of cytokine production, cytotoxic function and proliferation. TAC T cells, but not CAR engineered T cells, show significant selectivity towards the context of antigen presentation. This is reflected by the differential proliferative response to a diverse framework of antigen surface arrangement, potentially indicating that TAC T cells are less susceptible to off target activation and the resulting toxicities.Treatment of established NALM-6 xenografts (acute lymphoblastic leukemia) and KMS-11 xenografts (multiple myeloma) with CD19 TAC T cells and BCMA TAC T cells, respectively, resulted in clearance of tumors within a few weeks of T cell infusion. Mice that cleared tumors following TAC T cell treatment were resistant to subsequent challenge with fresh tumor cells demonstrating persistence of TAC T cells. Treatment with control TAC T cells that carry no binding domain had no impact on tumor growth. Monitoring of TAC T cells post-infusion revealed robust expansion that peaked in the peripheral blood 1-2 weeks post-infusion. A clinical manufacturing protocol has been developed for the CD19 TAC T cells in anticipation of human trials.Conclusion:Our pre-clinical evaluation suggests that TAC therapy has the potential to becoming a safer and more effective alternative to conventional CAR therapy. A first in human Phase I/II trial with CD19 TAC T cells is expected to start in the first half of 2019. DisclosuresHelsen:Triumvira Immunologics: Employment, Patents & Royalties. Hammill:Triumvira Immunologics: Other: Holding shares, Patents & Royalties. Mwawasi:Triumvira Immunologics: Other: Holding shares, Patents & Royalties. Hayes:Triumvira Immunologics: Employment. Afsahi:Triumvira Immunologics: Patents & Royalties. Denisova:Triumvira Immunologics: Patents & Royalties. Bramson:Triumvira Immunologics: Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Calibrated CAR Activation Potential Directs Alternative T Cell Fates and Therapeutic Potency

Immunotherapy with second-generation chimeric antigen receptor (CAR) T cells has achieved great clinical success against hematological malignancies. CD19-specific CARs incorporating CD28 and CD3z signaling domains have demonstrated remarkable potency resulting in the frequent induction of complete remissions and were recently approved by the US FDA for use in some refractory B cell malignancies. We previously demonstrated that CD28-based CARs program strong effector functions, but impart a relatively limited T cell lifespan. Increasing functional T cell persistence is therefore likely to further enhance the therapeutic success of 1928z CAR T cells.We hypothesized that excessive signal strength arising from redundancy of combined CD3z and CD28 signals might foster terminal T cell differentiation and exhaustion. We therefore proceeded to titrate the activation potential of CD28-based CARs and assess the impact thereof on the function, longevity and therapeutic potency of CAR T cells.We analyzed the contribution of individual immunoreceptor tyrosine-based activation motifs (ITAMs) to the phenotype and function of 1928z CAR T cells. ITAM-mutated CAR T cells demonstrated similar expression levels in retrovirally transduced primary T cells and directed comparable short-term cytotoxicity and proliferation capacity in vitro. However, remarkable differences in their therapeutic potency emerged when T cells expressing different mutant CARs were tested in the pre-B acute lymphoblastic leukemia NALM6 mouse model. The CAR “stress test” model revealed that a CAR containing a single functional ITAM - depending on its position - outperformed wild-type 1928z CARs, achieving rapid and durable tumor eradication even at low T cell doses, by delaying T cell differentiation and exhaustion. CAR T cells retrieved from the bone marrow of treated mice demonstrated that inactivation of two ITAM domains augmented CAR persistence at the tumor sites with a higher percentage of central memory cells and a decreased proportion of terminally differentiated effector cells. Deletion mutants further revealed the importance of ITAM location within second-generation CAR T cells.These findings were rigorously tested by directing the mutant CAR cDNAs to the T-cell receptor α constant (TRAC) locus using CRISPR/Cas9 technology, thereby ruling out potentially confounding effects arising from different CAR expression levels. TRAC-1928z mutants demonstrated superior antitumor efficacy compared to conventional TRAC-1928z CARs and prevented terminal T cell differentiation and exhaustion.Genome-wide transcriptional profiles of TRAC-edited naïve peripheral blood T cells further demonstrated that CARs encoding different ITAMs direct T cells to different fates. While TRAC-1928z CARs demonstrated similarity to transcriptional profiles of effector cells, reducing the number of ITAMs to one ITAM preserved a less-differentiated T cell state and promoted greater T cell persistence. We identified one 1928z mutant CAR, which improved therapeutic potency and induced a transcriptional profile similar to that of stem cell memory T cells (TSCM). Another 1928z mutant CAR with further reduction of the activation potential resulted in a naïve-like phenotype with great proliferation potential and persistence, but greatly diminished anti-tumor efficacy.In conclusion, we demonstrate that the number and position of ITAMs in 1928z CAR T cells influence functional, phenotypic and transcriptional programs resulting in profound effects on therapeutic potency. Balancing T cell differentiation and acquisition of effector functions is essential to optimize therapeutic potency of CAR T cells and can be intrinsically regulated by defined mutations in the CD3z chain of 1928z CAR T cells.Improved therapeutic potency of CAR T cells can thus be achieved by calibrating activation strength, thus retaining memory functions and preventing exhaustion, without compromising effector functions. Importantly, we were able to identify a novel CAR design which programs a favorable balance of effector and memory signatures, inducing increased persistence of highly functional CARs with the replicative capacity of long-lived memory cells and potent effector functions. Clinical studies evaluating the new CAR design are in preparation. DisclosuresSadelain:Juno Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Fate Therapeutics Inc.: Research Funding.

Engrafting human regulatory T cells with a flexible modular chimeric antigen receptor technology

As regulatory T cells (Tregs) play a fundamental role in immune homeostasis their adoptive transfer emerged as a promising treatment strategy for inflammation-related diseases. Preclinical animal models underline the superiority of antigen-specific Tregs compared to polyclonal cells. Here, we applied a modular chimeric antigen receptor (CAR) technology called UniCAR for generation of antigen-specific human Tregs. In contrast to conventional CARs, UniCAR-endowed Tregs are indirectly linked to their target cells via a separate targeting module (TM). Thus, transduced Tregs can be applied universally as their antigen-specificity is easily adjusted by TM exchange. Activation of UniCAR-engrafted Tregs occurred in strict dependence on the TM, facilitating a precise control over Treg activity. In order to augment efficacy and safety, different intracellular signaling domains were tested. Both 4-1BB (CD137) and CD28 costimulation induced strong suppressive function of genetically modified Tregs. However, in light of safety issues, UniCARs comprising a CD137-CD3ζ signaling domain emerged as constructs of choice for a clinical application of redirected Tregs. In that regard, Tregs isolated from patients suffering from autoimmune or inflammatory diseases were, for the first time, successfully engineered with UniCAR 137/ζ and efficiently suppressed patient-derived effector cells. Overall, the UniCAR platform represents a promising approach to improve Treg-based immunotherapies for tolerance induction.

NKG2D-Based CAR T Cells and Radiotherapy Exert Synergistic Efficacy in Glioblastoma.

Chimeric antigen receptor (CAR) T-cell therapy is an emerging immunotherapy against several malignancies including glioblastoma, the most common and most aggressive malignant primary brain tumor in adults. The challenges in solid tumor immunotherapy comprise heterogenously expressed tumor target antigens and restricted trafficking of CAR T cells to and impaired long-term persistence at the tumor site, as well as the unaddressed integration of CAR T-cell therapy into conventional anticancer treatments. We addressed these questions using a NKG2D-based chimeric antigen receptor construct (chNKG2D) in fully immunocompetent orthotopic glioblastoma mouse models. ChNKG2D T cells demonstrated high IFNγ production and cytolytic activity in vitro Upon systemic administration in vivo, chNKG2D T cells migrated to the tumor site in the brain, did not induce adverse events, prolonged survival, and cured a fraction of glioma-bearing mice. Surviving mice were protected long-term against tumor rechallenge. Mechanistically, this was not solely the result of a classical immune memory response, but rather involved local persistence of chNKG2D T cells. A subtherapeutic dose of local radiotherapy in combination with chNKG2D T-cell treatment resulted in synergistic activity in two independent syngeneic mouse glioma models by promoting migration of CAR T cells to the tumor site and increased effector functions. We thus provide preclinical proof-of-concept of NKG2D CAR T-cell activity in mouse glioma models and demonstrate efficacy, long-term persistence, and synergistic activity in combination with radiotherapy, providing a rationale to translate this immunotherapeutic strategy to human glioma patients.Significance: These findings provide evidence for synergy of conventional anticancer therapy and CAR T cells and heralds future studies for other treatment combinations. Cancer Res; 78(4); 1031-43. ©2017 AACR.

Development of novel target modules for retargeting of UniCAR T cells to GD2 positive tumor cells

As the expression of a tumor associated antigen (TAA) is commonly not restricted to tumor cells, adoptively transferred T cells modified to express a conventional chimeric antigen receptor (CAR) might not only destroy the tumor cells but also attack target-positive healthy tissues. Furthermore, CAR T cells in patients with large tumor bulks will unpredictably proliferate and put the patients at high risk of adverse side effects including cytokine storms and tumor lysis syndrome. To overcome these problems, we previously established a modular CAR technology termed UniCAR: UniCAR T cells can repeatedly be turned on and off via dosing of a target module (TM). TMs are bispecific molecules which cross-link UniCAR T cells with target cells. After elimination of the respective TM, UniCAR T cells automatically turn off. Here we describe novel TMs against the disialoganglioside GD2 which is overexpressed in neuroectodermal but also many other tumors. In the presence of GD2-specific TMs, we see a highly efficient target-specific and -dependent activation of UniCAR T cells, secretion of pro-inflammatory cytokines, and tumor cell lysis both in vitro and experimental mice. According to PET-imaging, anti-GD2 TM enrich at the tumor site and are rapidly eliminated thus fulfilling all prerequisites of a UniCAR TM.

Driving better and safer HER2-specific CARs for cancer therapy.

Given the clinical efficacy of chimeric antigen receptor (CAR)-based therapy in hematological malignancies, CAR T-cell therapy for a number of solid tumors has been actively investigated. Human epidermal growth factor receptor 2 (HER2) is a well-established therapeutic target in breast, as well as other types of cancer. However, HER2 CAR T cells pose a risk of lethal toxicity including cytokine release syndrome from “on-target, off-tumor” recognition of HER2. In this review, we summarize the development of conventional HER2 CAR technology, the alternative selection of CAR hosts, the novel HER2 CAR designs, clinical studies and toxicity. Furthermore, we also discuss the main strategies for improving the safety of HER2 CAR-based cancer therapies.

Targeting Alpha-Fetoprotein (AFP)-MHC Complex with CAR T-Cell Therapy for Liver Cancer.

The majority of tumor-specific antigens are intracellular and/or secreted and therefore inaccessible by conventional chimeric antigen receptor (CAR) T-cell therapy. Given that all intracellular/secreted proteins are processed into peptides and presented by class I MHC on the surface of tumor cells, we used alpha-fetoprotein (AFP), a specific liver cancer marker, as an example to determine whether peptide-MHC complexes can be targets for CAR T-cell therapy against solid tumors. We generated a fully human chimeric antigen receptor, ET1402L1-CAR (AFP-CAR), with exquisite selectivity and specificity for the AFP158-166 peptide complexed with human leukocyte antigen (HLA)-A*02:01. We report that T cells expressing AFP-CAR selectively degranulated, released cytokines, and lysed liver cancer cells that were HLA-A*02:01+/AFP+ while sparing cells from multiple tissue types that were negative for either expressed proteins. In vivo, intratumoral injection of AFP-CAR T cells significantly regressed both Hep G2 and AFP158-expressing SK-HEP-1 tumors in SCID-Beige mice (n = 8 for each). Moreover, intravenous administration of AFP-CAR T cells in Hep G2 tumor-bearing NSG mice lead to rapid and profound tumor growth inhibition (n = 6). Finally, in an established intraperitoneal liver cancer xenograft model, AFP-CAR T cells showed robust antitumor activity (n = 6). This study demonstrates that CAR T-cell immunotherapy targeting intracellular/secreted solid tumor antigens can elicit a potent antitumor response. Our approach expands the spectrum of antigens available for redirected T-cell therapy against solid malignancies and offers a promising new avenue for liver cancer immunotherapy. Clin Cancer Res; 23(2); 478-88. ©2016 AACR.

Masked Chimeric Antigen Receptor for Tumor-Specific Activation

Adoptive cellular therapy based on chimeric antigen receptor (CAR)-engineered T (CAR-T) cells is a powerful form of cancer immunotherapy. CAR-T cells can be redirected to specifically recognize tumor-associated antigens (TAAs) and induce high levels of antitumor activity. However, they may also display "on-target off-tumor" toxicities, resulting from low-level expression of TAAs in healthy tissues. These adverse effects have raised considerable safety concerns and limited the clinical application of this otherwise promising therapeutic modality. To minimize such side effects, we have designed an epidermal growth factor receptor (EGFR)-specific masked CAR (mCAR), which consists of a masking peptide that blocks the antigen-binding site and a protease-sensitive linker. Proteases commonly active in the tumor microenvironment can cleave the linker and disengage the masking peptide, thereby enabling CAR-T cells to recognize target antigens only at the tumor site. Invitro mCAR showed dramatically reduced antigen binding and antigen-specific activation in the absence of proteases, but normal levels of binding and activity upon treatment with certain proteases. Masked CAR-T cells also showed antitumor efficacy invivo comparable to that of unmasked CAR. Our study demonstrates the feasibility of improving the safety profile of conventional CARs and may also inspire future design of CAR molecules targeting broadly expressed TAAs.

Abstract B099: The UniCAR system: Inducible CAR T cells for precise reactivity and high efficacy against hematopoietic malignancies

Abstract During the past years promising clinical outcomes have been reported using immunotherapy for treatment of solid tumors and malignancies of the hematopoietic system. Since T cells act as key players in immune surveillance, an encouraging approach is to genetically engineer T cells with lenti- or retroviral vectors, in order to express artificial chimeric antigen receptors (CARs), directing them against cancer cells and leading to a distinct cytotoxic response. CARs are synthetic fusion proteins consisting of an antigen-binding moiety, commonly a tumor-associated antigen (TAA)-specific single-chain fragment variable (scFv), combined with activating signaling domains like the CD3-zeta chain. Signaling can be enhanced by adding co-stimulatory domains, such as the intracellular regions of CD28 or CD137. Although, CAR T cells showed promising results in patients with B cell malignancies, a major drawback of conventional CARs, bearing a tumor-antigen-binding moiety, is the uncontrollable continuous T cell activity. Hence, CAR T cells can achieve a sustained anti-tumoral response. However, reactivity against any healthy tissues expressing the target antigen might cause severe adverse effects. A chance to gain safety on CAR T cell approaches by retaining efficacy is offered through a novel universal binary CAR system (UniCAR). The system is based on a second generation CAR with an extracellular scFv binding domain, which is directed against a short non-immunogenic peptide motif instead of a cell surface antigen. Thus, engineered T cells are inert per se, remaining in a “sleeping mode” after re-infusion. Small protein links (targeting modules, TMs), consisting e.g. of TAA-specific scFvs harboring the small peptide epitope recognized by the UniCAR are used to redirect UniCAR expressing T cells in an antigen-specific manner. Primary results demonstrate that UniCAR driven T cell activation and cytotoxic effects against tumor cells are strictly dependent on the presence of the antigen-specific TMs. UniCAR T cells show high anti-tumor efficacy even at picomolar TM concentrations and low effector to target cell (e:t) ratios in vitro. After those proof-of-concept studies we disclose positive evidence of high efficacy UniCAR T cells with reactivity against CD123-positive acute myeloid leukemia (AML) and CD19-bearing acute lymphoblastic leukemia (ALL) cells. UniCAR T cell anti-tumor efficacy proofed to be superior when compared to conventional CAR T cells at low e:t ratios adjusted to the situation usually found in patients. Furthermore, UniCAR T cells pre-decorated with CD123-specific TMs in vitro are capable of prohibiting tumor engraftment of CD123-positive AML in vivo and persist in immunodeficient NSG mice. Ex vivo isolated UniCAR T cells retained cytotoxic potential against MOLM-13 cells for at least four month post transplantation fortifying the therapeutic potential in vitro and in vivo. The tremendous benefit of the novel UniCAR platform is the precise control of CAR T cell activity by regulating TM administration and dosage, preventing off side-toxicities arising from conventional CAR approaches. Moreover, the modular UniCAR system also enables combinatorial targeting strategies against several TAAs to increase therapy performance and circumvent the occurrence of tumor resistance mechanisms due to selectable compositions of target structures. Leukemia-related target antigens like CD19, CD20, CD22, CD33 or CD123 are conceivable therapy settings, offering advantages compared to monotherapeutic CAR-based therapies. Taken together, the modular composition of the UniCAR platform maintains the high anti-tumor potential of CAR-engrafted T cells, while introducing tight control mechanisms and unparalleled target flexibility. These features will allow a more sophisticated application of CAR technology and a reduction of adverse events in the clinical setting. Citation Format: Simon Loff, Malte v. Bonin, Josephine Dietrich, Jan-Erik Meyer, Anja Feldmann, Claudia Arndt, Johannes Spehr, Cordula Gründer, Gerhard Ehninger, Michael P. Bachmann, Armin Ehninger, Marc Cartellieri. The UniCAR system: Inducible CAR T cells for precise reactivity and high efficacy against hematopoietic malignancies [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B099.

274. One-Step Generation of Universal CAR T Cells

Adoptive immunotherapy using chimeric antigen receptors (CARs) has shown remarkable clinical results in the treatment of leukemia and is one of the most promising new strategies to treat cancer. Current clinical protocols utilize autologous T cells that are collected by apheresis and engineered with retroviral vectors to stably express the CAR. This approach therefore requires patient-specific cell manufacturing, which unavoidably results in patient-to-patient variability in the final cell product. Widespread implementation of this approach will further require progress in automation and miniaturization of cell manufacturing to meet the demand for CAR T cells. Furthermore, current approaches utilize randomly integrating vectors, including gamma-retroviral, lentiviral and transposons, which all result in semi-random integration and variable expression of the CAR owing to transgene variegation. Position effects may result in heterogeneous T cell function, transgene silencing and, potentially, insertional oncogenesis. Thus, the conjunction of autologous cell sourcing and random vector integration is prone to generating cell products with variable potency. Here we utilize gene editing to generate histocompatible T cell products with consistent and homogeneous CAR expression. Different tailored nucleases, including CRISPR/Cas9 system, Zinc Finger Nucleases or TAL effector nucleases (TALENs), have been previously used for gene disruption in a wide range of human cells including primary T cells. In some instances, these nucleases have been used to generate so-called “universal T cells” for allogeneic administration, by disrupting T cell receptor (TCR) or HLA class I expression, but viral vectors or the sleeping beauty transposon were used to deliver the CAR cDNA, all of which result in semi-random transgene integration and its downstream consequences. We present here a novel strategy for one-step generation of universal CAR T cells. We first compared the efficiency of TALEN and CRISPR/Cas9 to promote homologous recombination using AAV6 donor template in T cells and established conditions yielding more than 50% of universal CAR T cells combining target gene disruption and CAR insertion in a single single step. We molecularly confirmed the targeted integration of the CAR transgene, which results in highly homogeneous and stable CAR expression in human peripheral blood T cells. These T cells exhibited the same in vitro tumor lysis activity and proliferation than retrovirally transduced CAR T cells, which augur favorably for their in vivo anti-tumor activity. Deep sequencing analyses to evaluate off-target effects of the nucleases and random AAV integration are in progress, as are in vivo experiments comparing the anti-tumor activity and graft-versus-host disease potential of edited T cells vs conventional CAR T cells. The process we describe here, which combines the scalability of universal T cell manufacturing with the uniformity and safety of targeted CAR gene integration, should be useful for the development of off-the-shelf CAR therapy.

Abstract B021: Treatment with a novel targeting module, redirecting UniCAR T cells against PSCA, delays subcutaneous tumor growth and prolongs survival of tumor-bearing NSG mice

Abstract Background: The adoptive transfer of T cell engineered with chimeric antigen receptors (CARs) is currently considered a highly promising therapeutic option for treating so far incurable malignant diseases. In order to reduce adverse reactions associated with CAR engineered T cells, a flexible modular universal chimeric antigen receptor (UniCAR) platform is developed, which allows a precise control of CAR T cell reactivity to manage side effects while preserving efficacy. The UniCAR technology splits the signaling and antigen-binding aspects of conventional CARs into two individual components. The UniCAR is structurally similar to second-generation CARs, however the single chain variable fragment (scFv) does not recognize a cell surface antigen but a short peptide motif derived from a human nuclear autoantigen. Antigen-specificity and crosslinking of UniCAR expressing T cells to target cells is provided by a separate targeting module (TM) compromising a scFv, binding to specific cell surface antigens, fused to the aforementioned peptide motif. Thus, T cells engineered to express UniCARs remain inactivated after infusion, since the UniCAR target is not available for binding under physiological conditions. Only in the presence of the TM, antigen specific activation of UniCAR T cells occurs, which allows for a more sophisticated regulation. Methods: We developed a novel radio-immune target module (TM), which consists of an anti-PSCA (prostate stem cell antigen) scFv for redirecting of UniCARs to PSCA expressing tumor cells. Three different tags are fused in a row to the C-Terminus of the scFv to functionalize the molecule for combined immune- and radiotherapy. First, a ten amino acid (aa) long tag mediates the crosslink to T cells genetically modified with the UniCAR. Further downstream, a peptide tag (Rx) for enhanced conjugation of radionuclides and finally, a 6xhistidine (His) tag are located. TM-Rx efficacy was evaluated for target cell killing in-vitro and compared to a TM without Rx-tag. In-vivo efficacy of the TM-Rx was also investigated in a high and low-tumor burden mouse model. Results: Rx-TM and TM equivalently kill PSCA-expressing tumor cells in-vitro in presence of UniCARs in an E:T (Effector:Target) ratio of 1:1 after 24 hours. UniCAR T cell engrafted NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice show no clinical signs of autoimmunity neither in presence nor absence of the TM. One week treatment with TM delays subcutaneous tumor growth and prolongs survival of tumor bearing, UniCAR T cell engrafted NSG mice. Longterm infiltration of tumor tissue by UniCARs could be observed over 16 weeks in the high and low-tumor burden ectopic carcinoma model. Conclusion: The UniCAR technology shows efficacy both in-vitro and in-vivo. Extension of the TM with an additional tag does not impair functionality of the UniCAR platform. Short-treatment prolongs survival of tumor bearing mice but does not lead to cure, therefore combination with other treatment modalities e.g. radiation oncology seems reasonable. Citation Format: Elham Pishali Bejestani, Malte von Bonin, Marc Cartellieri, Armin Ehninger, Mechthild Krause, Michael Baumann, Gerhard Ehninger, Michael Bachmann. Treatment with a novel targeting module, redirecting UniCAR T cells against PSCA, delays subcutaneous tumor growth and prolongs survival of tumor-bearing NSG mice. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B021.

Improved Killing of AML Blasts By Dual-Targeting of CD123 and CD33 Via Unitarg a Novel Antibody-Based Modular T Cell Retargeting System

Acute myeloid leukemia (AML) is a hematologic malignancy of the myeloid line with high prevalence in older patients. As complete eradication of metastatic cancer cells is often not achieved by standard therapies, alternative treatment modalities are urgently needed.In recent years, bispecific antibodies (bsAbs) and chimeric antigen receptors (CARs) emerged as promising candidates for an antigen-specific cancer immunotherapy. Both bsAbs and CARs are able to redirect T cells for efficient tumor cell lysis. Nevertheless, the development of a novel TAA specific bsAb or a CAR is a long lasting process. Therefore, we recently introduced a novel antibody-based modular platform (UniTARG) that can be rapidly and easily adapted for redirection of T cells to any TAA in both a bsAb or CAR related manner. The modular UniTARG system distributes the effector arm (the anti-CD3 domain or CAR) and the anti-TAA binding domain to two separate molecules: (I) an exchangeable target module (TM) comprising an anti-TAA binding moiety and a short peptide epitope (E5B9), and (II) a universal effector unit. The effector systems represent either a bsAb with specificity for CD3 and a peptide epitope (E5B9) termed UniMAB or a CAR directed to the E5B9 epitope (UniCAR). Thus, TMs can form a complex with the respective effector system that facilitates the cross-linkage of tumor and T cells similar to conventional bsAbs or CARs. For redirection of T cells to any kind of TAA only the binding moiety of the TM has to be adapted what saves costs and time. To increase tumor specificity and to reduce the risk of tumor escape variants, the modular UniTARG system further offers the possibility to apply simultaneously different monospecific or even bispecific TMs recognizing two TAAs.For proof of concept of a dual targeting using the UniTARG system we selected as TAA on AML blasts the molecules CD33 and CD123. They represent promising target antigens as they are overexpressed on both rapidly proliferating terminal AML blasts and leukemic stem cells which might be responsible for disease relapse after initial chemotherapy. Thus, we generated an anti-CD123 TM and anti-CD33 TM that can be applied within the modular system for single-targeting or that can be combined for dual-targeting of AML blasts. By fusion of the anti-CD123 and anti-CD33 domains via the E5B9 epitope a bispecific TM was further constructed. As revealed by cytotoxicity assays with CD33+ CD123+ AML cell lines, the novel mono- and bispecific TMs can be easily applied to the modular systems to trigger highly potent tumor cell lysis at low E:T ratios and picomolar Ab concentrations. By using the dual-targeting approach we can show that lysis of CD123+ CD33+ AML blasts can be considerably improved in comparison to the mono-specific strategy.Overall, due to the ease and cost-effectiveness of development the UniTARG platform technology represents a promising tool in the field of both bsAbs and CARs with the advantage of simultaneous or consecutive dual or even multispecific targeting. This approach might additionally improve anti-tumor activity by increasing tumor specificity and diminishing off-target effects. DisclosuresCartellieri:Cellex Patient Treatment GmbH: Employment. Ehninger:GEMoaB Monoclonals GmbH: Employment, Patents & Royalties: related to the UniTARG system. Ehninger:GEMoaB Monoclonals GmbH: Equity Ownership, Patents & Royalties: related to the UniTARG system. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership, Patents & Royalties: related to the UniTARG system.

Unicar: A Novel Modular Retargeting Platform Technology for CAR T Cells

The adoptive transfer of T cells engineered with chimeric antigen receptors (CARs) is currently considered as a highly promising therapeutic option for treatment of otherwise incurable malignant diseases. CARs combine the cellular and humoral arm of the immune response by assembling a single-chain fragment variable (scFv) as binding moiety which provides the antigen-specificity and an activating immune receptor. It has been demonstrated both in vitro and in vivo, that CAR engrafted effector T cells mediate long-lasting anti-tumor responses. Despite encouraging clinical efficacy targeting CD19 in recent clinical trials, the appearance of potentially life-threatening adverse reactions and the lack of control mechanisms once initiated, prevent more widespread application of the CAR technology.To overcome limitations of conventional CAR T cells, a unique chimeric antigen receptor (UniCAR) technology was developed (Fig. 1) which allows precise control of CAR T cell reactivity, thus lowering the risk of side effects while preserving efficacy. Moreover, the UniCAR technology enables the retargeting of engrafted T cells against more than one antigen simultaneously or subsequently, thus reducing the risk for development of antigen-loss tumor variants under treatment. The UniCAR technology splits the signaling and antigen-binding aspects of conventional CAR into two individual components. T cells are engineered to express a universal CAR (UniCAR), which has specificity for a short peptide motif of 10 amino acids derived from a human nuclear protein. Thus, T cells engineered to express UniCAR remain inactivated after re-infusion, since the UniCAR target is not available for binding under physiological conditions. The ultimate antigen-specificity of the system is provided separately by targeting modules (TMs) comprising a binding domain e.g., a tumor-antigen specific scFv, fused to the nuclear antigen motif recognized by the UniCAR binding domain. Here we provide first in vitro and in vivo prove of concept for this new approach. Antigen-specific redirection of T cells armed with the universal CAR in the presence of different targeting modules against various antigens (CD33, CD123, CD19, CD20, PSCA, PSMA,) was effective at femtomolar concentrations of the targeting module both. Taken together, the modular nature of UniCAR technology will allow retargeting of autologous, patient-derived T cells to several antigens under controlled pharmacological conditions and has the potential to become a highly effective treatment option for late stage cancer patients with reduced risks for side effects. [Display omitted] DisclosuresCartellieri:Cellex Patient Treatment GmbH: Employment. Loff:GEMoaB Monoclonals GmbH: Employment. Ehninger:GEMoaB Monoclonals GmbH: Employment, Patents & Royalties: related to the UniTARG system. Ehninger:GEMoaB Monoclonals GmbH: Equity Ownership; Cellex Patient Treatment GmbH: Equity Ownership. Bachmann:GEMoaB Monoclonals GmbH: Equity Ownership, Patents & Royalties: related to the UniTARG system.