Single Chimeric Antigen Receptor Research Articles

Simultaneous targeting of Tim3 and A2a receptors modulates MSLN-CAR T cell antitumor function in a human cervical tumor xenograft model.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of hematological malignancies. However, its efficacy in solid tumors is limited by the immunosuppressive tumor microenvironment that compromises CAR T cell antitumor function in clinical settings. To overcome this challenge, researchers have investigated the potential of inhibiting specific immune checkpoint receptors, including A2aR (Adenosine A2 Receptor) and Tim3 (T cell immunoglobulin and mucin domain-containing protein 3), to enhance CAR T cell function. In this study, we evaluated the impact of genetic targeting of Tim3 and A2a receptors on the antitumor function of human mesothelin-specific CAR T cells (MSLN-CAR) in vitro and in vivo. Second-generation anti-mesothelin CAR T cells were produced using standard cellular and molecular techniques. A2aR-knockdown and/or Tim3- knockdown anti-mesothelin-CAR T cells were generated using shRNA-mediated gene silencing. The antitumor function of CAR T cells was evaluated by measuring cytokine production, proliferation, and cytotoxicity in vitro through coculture with cervical cancer cells (HeLa cell line). To evaluate in vivo antitumor efficacy of manufactured CAR T cells, tumor growth and mouse survival were monitored in a human cervical cancer xenograft model. In vitro experiments demonstrated that knockdown of A2aR alone or in combination with Tim3 significantly improved CAR T cell proliferation, cytokine production, and cytotoxicity in presence of tumor cells in an antigen-specific manner. Furthermore, in the humanized xenograft model, both double knockdown CAR T cells and control CAR T cells could effectively control tumor growth. However, single knockdown CAR T cells were associated with reduced survival in mice. These findings highlight the potential of concomitant genetic targeting of Tim3 and A2a receptors to augment the efficacy of CAR T cell therapy in solid tumors. Nevertheless, caution should be exercised in light of our observation of decreased survival in mice treated with single knockdown MSLN-CAR T cells, emphasizing the need for careful efficacy considerations.

Joint single-cell transcriptomic and epigenomic analysis reveals key regulators of CAR T cell stemness and antitumor immunity

Abstract The differentiation of antitumor T cells is tightly controlled by transcriptional and epigenetic pathways. Understanding the programs governing the differentiation of therapeutic T cells such as chimeric antigen receptor (CAR) T cells is crucial for improving their efficacy. Using a single-cell multi-omics approach, we simultaneously profiled chromatin accessibility and transcription within the same single CAR T cell in mice with B-cell leukemia or melanoma. One of the CAR T-cell subsets displayed stem cell-like properties and greater proliferative potential upon tumor challenge. In addition, we uncovered primed chromatin states foreshadowing transcriptional changes and lineage-defining genes heavily regulated at the epigenetic level. By constructing gene regulatory networks in CAR T cells based on the single-cell multi-omics data, we identified hub transcription factors of stem-like CAR T cells including BACH2. By knocking out BACH2 in CAR T cells, we demonstrated an essential role of BACH2 in the development of the stem-like subset and antitumor immunity of CAR T cells. Thus, our study elucidated the transcriptional and epigenetic mechanisms underlying CAR T-cell differentiation and revealed key transcriptional regulators controlling the differentiation and antitumor immunity of CAR T cells.

Single Cell Multi-Omic Dissection of Response and Resistance to Chimeric Antigen Receptor T Cells Against BCMA in Relapsed Multiple Myeloma

Background: Chimeric antigen receptor (CAR) T cell therapy has revolutionized treatment of relapsed/refractory multiple myeloma (RRMM). Robust variables that predict long-term response are currently missing. Limited data are especially available on the impact of bridging therapies on manufacturing and outcome. We conducted a longitudinal single-cell multi-omics study to identify factors that predict response to BCMA-directed CAR T cells. Changes in the immune microenvironment associated with response were analyzed as well as the impact of prior bridging therapy with bispecific antibodies on subsequent CAR T cell manufacturing and outcome. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated from 29 consecutive MM patients treated with commercially available anti-BCMA CAR T cells on the day of leukapheresis as well as days 30 and 100 after CAR T cell infusion. PBMCs were subjected to single cell RNA, T-cell receptor (TCR) and B-cell receptor (BCR) sequencing. A custom panel of 57 oligonucleotide-coupled antibodies was used to study surface proteomics. Downstream analyses were performed with Seurat. Differences in cellular compositions at all three time points were analyzed with scCODA. To analyze CAR T cell functionality, CAR T cells from peripheral blood were isolated 7 days after infusion and subjected to an in vitro cytotoxicity assay after expansion and stimulation. Patients were grouped based on their best response following CAR T cell infusion (CR: n=12, non CR: n=17). Results: In total, 375,338 cells were sequenced (median 7246 cells/sample, range 1,569-10,972 cells) and 354,878 cells (94.5%) passed quality assessment. Quantitative and qualitative differences in the cellular composition of peripheral blood between CR and non CR patients were detected at the time of leukapheresis as well as on days 30 and 100 following infusion. CR patients harbored significantly more CD8+ effector memory T cells (TEM) at leukapheresis and less NK cells on day 30 after therapy compared to non CR patients. Regulatory T cells isolated at the time of leukapheresis from non CR patients exhibited significantly higher surface protein levels of CXCR3, CD40, CD95 and KLRG1 (p<0.015, respectively) that have been associated with T cell senescence and impaired tumor immunity. No significant differences in cell numbers between CR and non CR were detected on day 100 after CAR T cell infusion. However, single cell TCR analysis revealed an increasing diversity in the TCR repertoire over time in patients with CR, while Shannon diversity decreased from leukapheresis over day 30 to day 100 in non CR patients (p=0.004). The prior administration of the bispecific antibody teclistamab had no significant impact on the quantitative cellular composition at the time of leukapheresis. However, termination of manufacturing in the first attempt occurred in all patients with a close proximity of teclistimab administration and apheresis. Differential gene expression analysis showed that the application of teclistamab was associated with impaired T cell activation and exhaustion indicated by upregulation of e.g. CTLA4, TIGIT, LAG3 and GZMK. After discontinuation of teclistamab (median 4 weeks) and successful manufacturing of CAR T cells, we found no significant differences for in vitro cytotoxicity and in vivo expansion of CAR T cells. CAR T cells isolated at day 7 post-infusion from patients in CR, non CR or with prior teclistamab exposure, effectively eliminated MM cells (U-266). Tracking of single CAR T cells over time showed that the majority of CAR+ cells were CD8+ TEMs regardless of remission achievement or prior teclistamab exposure. Conclusion: We demonstrate that differences between MM patients achieving a CR and patients with suboptimal response upon anti-BCMA CAR T cell therapy can already be identified at the time of leukapheresis. Long-term changes associated with CR include a diversification of the TCR repertoire. Successful CAR T cell manufacturing is hampered by exposure to bispecific antibodies but can be successfully achieved by allowing for a wash-out phase of ca. 4 weeks.

Anti-TACI single and dual-targeting CAR T cells overcome BCMA antigen loss in multiple myeloma

Chimeric Antigen Receptor (CAR) T cells directed to B cell maturation antigen (BCMA) mediate profound responses in patients with multiple myeloma, but most patients do not achieve long-term complete remissions. In addition, recent evidence suggests that high-affinity binding to BCMA can result in on-target, off-tumor activity in the basal ganglia and can lead to fatal Parkinsonian-like disease. Here we develop CAR T cells against multiple myeloma using a binder to targeting transmembrane activator and CAML interactor (TACI) in mono and dual-specific formats with anti-BCMA. These CARs have robust, antigen-specific activity in vitro and in vivo. We also show that TACI RNA expression is limited in the basal ganglia, which may circumvent some of the toxicities recently reported with BCMA CARs. Thus, single-targeting TACI CARs may have a safer toxicity profile, whereas dual-specific BCMA-TACI CAR T cells have potential to avoid the antigen escape that can occur with single-antigen targeting.

Data driven model discovery and interpretation for CAR T-cell killing using sparse identification and latent variables.

In the development of cell-based cancer therapies, quantitative mathematical models of cellular interactions are instrumental in understanding treatment efficacy. Efforts to validate and interpret mathematical models of cancer cell growth and death hinge first on proposing a precise mathematical model, then analyzing experimental data in the context of the chosen model. In this work, we present the first application of the sparse identification of non-linear dynamics (SINDy) algorithm to a real biological system in order discover cell-cell interaction dynamics in in vitro experimental data, using chimeric antigen receptor (CAR) T-cells and patient-derived glioblastoma cells. By combining the techniques of latent variable analysis and SINDy, we infer key aspects of the interaction dynamics of CAR T-cell populations and cancer. Importantly, we show how the model terms can be interpreted biologically in relation to different CAR T-cell functional responses, single or double CAR T-cell-cancer cell binding models, and density-dependent growth dynamics in either of the CAR T-cell or cancer cell populations. We show how this data-driven model-discovery based approach provides unique insight into CAR T-cell dynamics when compared to an established model-first approach. These results demonstrate the potential for SINDy to improve the implementation and efficacy of CAR T-cell therapy in the clinic through an improved understanding of CAR T-cell dynamics.

Associations of granulocyte colony-stimulating factor with toxicities and efficacy of chimeric antigen receptor T-cell therapy in relapsed or refractory multiple myeloma

Background aimsFew studies have reported the associations of granulocyte colony-stimulating factor (G-CSF) with cytokine release syndrome (CRS), neurotoxic events (NEs) and efficacy after chimeric antigen receptor (CAR) T-cell therapy for relapsed or refractory (R/R) multiple myeloma (MM). We present a retrospective study performed on 113 patients with R/R MM who received single anti-BCMA CAR T-cell, combined with anti-CD19 CAR T-cell or anti-CD138 CAR T-cell therapy. MethodsEight patients were given G-CSF after successful management of CRS, and no CRS re-occurred thereafter. Of the remaining 105 patients that were finally analyzed, 72 (68.6%) received G-CSF (G-CSF group), and 33 (31.4%) did not (non G-CSF group). We mainly analyzed the incidence and severity of CRS or NEs in two groups of patients, as well as the associations of G-CSF timing, cumulative dose and cumulative time with CRS, NEs and efficacy of CAR T-cell therapy. ResultsBoth groups of patients had similar duration of grade 3–4 neutropenia, and the incidence and severity of CRS or NEs.There were also no differences in the incidence and severity of CRS or NEs between patients with the timing of G-CSF administration ≤3 days and those >3 days after CAR T-cell infusion. The incidence of CRS was greater in patients receiving cumulative doses of G-CSF >1500 μg or cumulative time of G-CSF administration >5 days. Among patients with CRS, there was no difference in the severity of CRS between patients who used G-CSF and those who did not. The duration of CRS in anti-BCMA and anti-CD19 CAR T-cell–treated patients was prolonged after G-CSF administration. There were no significant differences in the overall response rate at 1 and 3 months between the G-CSF group and the non–G-CSF group. ConclusionsOur results showed that low-dose or short-time use of G-CSF was not associated with the incidence or severity of CRS or NEs, and G-CSF administration did not influence the antitumor activity of CAR T-cell therapy.

Human EGFRvIII chimeric antigen receptor T cells demonstrate favorable safety profile and curative responses in orthotopic glioblastoma.

Glioblastoma is a highly aggressive and fatal brain malignancy, and effective targeted therapies are required. The combination of standard treatments including surgery, chemotherapy and radiotherapy is not curative. Chimeric antigen receptor (CAR) T cells are known to cross the blood-brain barrier, mediating antitumor responses. A tumor-expressed deletion mutant of the epidermal growth factor receptor (EGFRvIII) is a robust CAR Tcell target in glioblastoma. Here, we show our de novo generated, high-affinity EGFRvIII-specific CAR; GCT02, demonstrating curative efficacy in human orthotopic glioblastoma models. The GCT02 binding epitope was predicted using Deep Mutational Scanning (DMS). GCT02 CAR T cell cytotoxicity was investigated in three glioblastoma models in vitro using the IncuCyte platform, and cytokine secretion with a cytometric bead array. GCT02 in vivo functionality was demonstrated in two NSG orthotopic glioblastoma models. The specificity profile was generated by measuring T cell degranulation in response to coculture with primary human healthy cells. The GCT02 binding location was predicted to be located at a shared region of EGFR and EGFRvIII; however, the in vitro functionality remained exquisitely EGFRvIII specific. A single CAR T cell infusion generated curative responses in two orthotopic models of human glioblastoma in NSG mice. The safety analysis further validated the specificity of GCT02 for mutant-expressing cells. This study demonstrates the preclinical functionality of a highly specific CAR targeting EGFRvIII on human cells. This CAR could be an effective treatment for glioblastoma and warrants future clinical investigation.

Safety and Anti-Leukemic Activity of CD123-CAR T Cells in Pediatric Patients with AML: Preliminary Results from a Phase 1 Trial

Background The prognosis of pediatric patients with relapsed or refractory (r/r) Acute Myeloid Leukemia (AML) remains dismal. Evaluation of novel therapies such as chimeric antigen receptor (CAR) T cells is therefore urgently needed. CD123 is overexpressed on AML blasts and leukemic stem cells (LSC), making it an attractive target for CAR T-cell directed therapy. Methods and Results We designed a 'bridge to allogeneic hematopoietic cell transplant (HCT)’ Phase 1 study to evaluate the safety and feasibility of treating pediatric patients with r/r AML with CD123-CAR T-cell on four dose levels (DL): DL1: 3x105/kg, DL2: 1x106/kg, DL3: 3x106/kg, DL4: 1x107/kg (NCT04318678). CD123-CAR T-cells were generated from CD4/CD8-selected autologous leukapheresis products using a lentiviral vector, which encoded a CD123-CAR with CD28.z signaling domain and a CD20 safety switch. Patients received lymphodepleting chemotherapy with Fludarabine and Cyclophosphamide followed by a single CAR T cell infusion. To date we have enrolled 12 patients. The median age of treated patients was 17 years (range 12-21years). Except for the first patient who had primary refractory disease, all other patients had relapsed following HCT (n=1-4 previous HCTs). Despite patients being heavily pre-treated, we successfully manufactured products on all patients. The infused products had a predominantly CD4+ effector memory immunophenotype, with a median CD123-CAR+/CD20+ expression of 60.2% (range 46.1-79.4%, N=6). All infused CAR T cell products exhibited potent antitumor activity in vitro when cultured against CD123+ targets (including autologous blasts for 2 patients). We infused 2 patients on DL1 and 3 patients on DL2. Two patients were infused on single patient protocols. All infusions were well tolerated, without any adverse infusion events. Post infusion, we observed isolated fevers that resolved within 24 hours (possible Grade 1 Cytokine Release Syndrome (CRS)). No Grade 2 ≥ CRS or neurotoxicity was seen. Multiplex cytokine analysis showed statistically significant increase of IL15 after lymphodepletion without significant increase in Th1 cytokines or IL6 after T cell infusion. While transient cytopenias were observed, patients did not develop persistent marrow aplasia. No dose limiting toxicities were noted. Disease evaluation was performed at 4-6 weeks following CAR T cell infusion. The 2 patients on DL1 showed no response. In the 3 patients on DL2, we observed: reduction in blast percentage without complete remission (CR) in 1 patient, no response in 1 patient and CR in 1 patient. The patient with CR had isolated extramedullary disease, showing complete resolution of all lesions by week 4 by PET imaging (Figure 1). She recurred 2 months following infusion, coinciding with loss of CAR T-cell detection by qPCR. She received a second infusion off study and again achieved a short-lived CR. One additional patient was infused on dose level 2 off protocol. She achieved morphological CR at day 28 with low level minimal residual disease (0.19%). Correlative studies revealed CD123-CAR T cell expansion in patients on DL2 (Figure 2), but not on DL1. However, peak expansion on DL2 was low in comparison to published reports on CAR T cells for Acute Lymphoblastic Leukemia. Detailed phenotypic analysis revealed that CD123-CAR T cell products were predominantly effector memory (CD45RO+CCR7-) with <2% naïve-like (CD45RO-CCR7+ or CD45RO-CD62L+) T cells. In addition, PD1, TIM3 and CD39 were expressed at a median of 43, 81 and 16% in CD4+ and 3, 72 and 45% in CD8+ T cells. This phenotype could be completely reversed by inhibiting CAR signaling with dasatinib during the manufacturing process, resulting in a CAR T cell product dominated by naïve-like T cells that did not express TIM3, PD1 or CD39, leading to enhanced T-cell effector function. Mechanistic analysis revealed that the observed phenotype could be explained by the transient, increased cell surface expression of CD123 on T cells upon activation during manufacturing, and not by tonic CAR signaling. Conclusions Our initial clinical experience with CD123-CAR T cells for pediatric patients with r/r AML demonstrates feasibility, safety, and evidence of anti-leukemic activity. Upcoming CD123-CAR T cell products will be manufactured in the presence of dasatinib to limit T-cell differentiation and exhaustion as we continue to explore our CD123-CAR T cell therapy approach for pediatric r/r AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Parallel CD19/CD20 CAR-Activated T-Cells Are More Effective for Refractory B-Cell Lymphoma In Vitro and In Vivo.

Anti-CD19 chimeric antigen receptor (CAR) T-cells are an effective treatment for refractory B-cell lymphoma, but CD19 deletion is prone to relapse. We conducted this study to find more effective dual CAR19/20 T-cells to target B-cell lymphoma and prevent antigen loss leading to recurrence. In this study, we transduced CD19 and CD20 CARs into human T cells in parallel and compared parallel dual CAR19/20, single CAR, and tandem CAR19/20 in vitro and in vivo. After transduction with the corresponding vectors, CD19 and CD20 CARs were dually expressed in human T cells. It was observed that parallel CAR19/20 T-cells contained a substantial proportion of naive subpopulations and were able to proliferate in vitro. Treatment with parallel CAR19/20, single CAR, or tandem CAR19/20 T-cells sustainably induced complete lysis of leukemia cells in a 5 : 1 ratio. Compared with single or tandem CAR T-cell-transplanted mice, parallel CAR19/20 T-cell-transplanted mice exhibited smaller tumor volume, more stable body weight, and longer survival. This suggests that parallel CAR19/20 has superior antilymphoma activity in vivo. In addition, parallel CAR19/20 T-cells were also able to kill patients' lymphoma cells in vitro. Therefore, it can be considered that parallel CAR19/20 is equally effective against single CAR and tandem CAR19/20 in vitro but more effective against lymphoma cells in vivo. This is a promising treatment to prevent the recurrence of antigen loss following CD19-targeted therapy in B lymphoma.

Abstract 576: Shark VNAR single domain-based CAR T cells targeting PD-L1 for cancer therapy

Abstract Background: Chimeric antigen receptor (CAR)-T therapy shows great potency against hematological malignancies, whereas it is difficult to transfer CAR T into solid tumors due to lack of appropriate antigenic targets and immunosuppressive tumor microenvironment. Checkpoint molecule PD-L1 is widely overexpressed on multiple tumor types, and PD-1/PD-L1 interaction is a primary mediator of immunosuppression in the TME. Here, we generated anti-PD-L1-specific shark single domain VNAR-based CAR T cell strategy and explored its anti-tumor efficacy in xenograft mouse models. Methods: We isolated anti-PD-L1 single domain antibodies from semi-synthetic shark VNAR phage display libraries. The binding of isolated VNARs was validated by ELISA, flow cytometry, and Octet. A peptide library based on human PD-L1 was synthesized to predict the epitope of select VNARs. Anti-tumor efficacy of CAR T cells was determined via cell luciferase-based cell assay as well as xenograft mouse models. Two tumor models, MDA-MB-231 (triple-negative breast cancer) and Hep3B (hepatocellular carcinoma or HCC) were used in the present study. Glypican-3 (GPC3) is a tumor-specific target in the Hep3B model. Results: We identified three anti-PD-L1 phage binders, B2, A11, and F5 from our shark phage libraries. All three binders showed cross-activity to human, mouse, and canine antigens, whereas only B2 functionally blocked the interaction of human PD-1 to PD-L1. Moreover, CAR (B2) T cells specifically lysed both MDA-MB-231 and Hep3B tumor cells by targeting constitutive and inducible expression of PD-L1. Importantly, the combination of anti-GPC3 CAR (hYP7) T cells with CAR (B2) T cells regress Hep3B tumors synergistically in mice. Conclusions: PD-L1-targeted shark VNAR single domain-based CAR T cell therapy is a novel strategy to treat breast cancer and liver cancer. This work provides a rationale for a potential use of anti-PD-L1 CAR T cells as a monotherapy or combination with a tumor-specific therapy in clinical studies for solid tumors. Citation Format: Dan Li, Hejiao English, Jessica Hong, Tianyuzhou Liang, Glenn Merlino, Chi-Ping Day, Mitchell Ho. Shark VNAR single domain-based CAR T cells targeting PD-L1 for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 576.

Abstract 2826: Rewiring CAR T-cells for absolute solid tumor specificity and safety

Abstract Translation of the unprecedented efficacy of chimeric antigen receptor (CAR) T-cell technology into solid tumors requires a solution to the problem of on-target damage of vital organs. We have developed an inhibitory CAR platform (iCAR) that imposes self-regulation in CAR T-cells to restrict inappropriate activation against normal cells without compromising tumor efficacy. The system consists of an iCAR that is co-expressed with a conventional activating CAR (aCAR) designed to activate T-cells. The iCAR and aCAR scFvs bind distinct cell-surface antigens that are ubiquitously expressed across all solid tumor histologies and normal tissues. The iCAR is allele-specific whereas the aCAR is pan-allelic. The iCAR technology was validated in functional assays and NSG models with cancer cell lines that mimic loss-of-heterozygosity (LOH) which is common in most human cancers due to increased chromosomal instability. In this context, loss of iCAR target expression due to LOH generates irreversible target specificity. Tumor-specific overexpression of aCAR and iCAR targets, a common barrier to the clinical translation of potent agents targeting the cell-surface proteins is not required. We have shown using in vitro and in vivo models that CAR T-cell activation and target killing are fully inhibited by dual iCAR + aCAR antigen engagement. This outcome validates the concept of iCAR-mediated protection of normal cells in vital organs. In contrast, tumors that mimic iCAR target LOH through CRISPR editing were fully eradicated with aCAR engagement alone. T-cell activation, proliferation, and cytotoxic activity after exposure to target cells lacking iCAR target antigen was found to be quantitatively indistinguishable from the results obtained with single input aCAR CAR T-cells. These results suggest that iCAR technology can enable treatment of cancer patients with solid tumors using CAR T-cells that maximize potency and efficacy by mitigating on-target CRS, neurotoxicity, and inevitable organ damage. The restriction of CAR T activation to the local tumor environment by iCAR-mediated self-regulation directly addresses a fundamental technology gap that cannot be solved by CAR T dosing or aCAR modification alone. Citation Format: Michael R. Weist, Adi Sharbi-Yunger, Jason Yi, Caitlin Schnair, David Bassan, Tanya Kim, Sarit Tabak, Yael Lopesco, Leehee Weinberger, Nir Bujanover, Neta Chaim, Kristina Vucci, Orit Foord, Frank J. Calzone, Rick Kendall, Gregor B. Adams. Rewiring CAR T-cells for absolute solid tumor specificity and safety [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2826.

Combinatorial antigen targeting strategies for acute leukemia: application in myeloid malignancy

Background aimsEfforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia-associated antigen with chimeric antigen receptor (CAR) T cells have met with limited success, due in part to heterogeneous expression of myeloid antigens. The authors hypothesized that T cells expressing CARs directed toward two different AML-associated antigens would eradicate tumors and prevent relapse. MethodsFor co-transduction with the authors’ previously optimized CLL-1 CAR currently in clinical study (NCT04219163), the authors generated two CARs targeting either CD123 or CD33. The authors then tested the anti-tumor activity of T cells expressing each of the three CARs either alone or after co-transduction. The authors analyzed CAR T-cell phenotype, expansion and transduction efficacy and assessed function by in vitro and in vivo activity against AML cell lines expressing high (MOLM-13: CD123 high, CD33 high, CLL-1 intermediate), intermediate (HL-60: CD123 low, CD33 intermediate, CLL-1 intermediate/high) or low (KG-1a: CD123 low, CD33 low, CLL-1 low) levels of the target antigens. ResultsThe in vitro benefit of dual expression was most evident when the target cell line expressed low antigen levels (KG-1a). Mechanistically, dual expression was associated with higher pCD3z levels in T cells compared with single CAR T cells on exposure to KG-1a (P < 0.0001). In vivo, combinatorial targeting with CD123 or CD33 and CLL-1 CAR T cells improved tumor control and animal survival for all lines (KG-1a, MOLM-13 and HL-60); no antigen escape was detected in residual tumors. ConclusionsOverall, these findings demonstrate that combinatorial targeting of CD33 or CD123 and CLL-1 with CAR T cells can control growth of heterogeneous AML tumors.

Bispecific CD19-CD20 and CD19-CD22 CAR-T Cells with Glycogen Synthase Kinase (GSK)-3β Inhibitor TWS119 Treatment Have Superior Therapeutic Effects on Mantle Cell Lymphoma

Introduction: Chimeric antigen receptor (CAR) - expressing T cells have shown breakthrough clinical successes to hematological malignancies such as diffuse large B cell lymphoma. Intensive development on CAR-T cell therapies are being developed in many common lymphomas. However, the development of CAR-T cell therapies in mantle cell lymphoma (MCL), a rare lymphoma has lagged behind. So far only CD19 CAR-T has been applied to MCL treatment, and MCL frequent relapse from prior CD19 CAR-T therapy with CD19 antigen loss. Bispecific CAR-T cells target two tumor antigens at once could reduce the risk of relapse. In addition, immune checkpoint blockade such as GSK3β and PD-1inhibition has been successfully used to enhance CAR-T cell therapies. Therefore, we attempt to develop a dual CAR for mantle cell lymphoma with checkpoint blockade.Methods: CD19-CD20 and CD19-CD22 bispecific CAR-T constructs were generated by subcloning the fragments in-frame into the pSFG retroviral vector. The BiCAR-T cells were cultured with/without a small molecule GSK3β inhibitor TWS119. Then, we performed a serial of experiments using CARs. 1, We assayed the CAR expression and cell expansion rates in vitro. 2, We assessed the tumor-killing capacity in vitro using MCL cell lines and primary MCL patient samples. 3, We performed phenotypic analyses by determination of proteins expression profiles of exhaustion markers and stem cell markers by flow cytometry. 4, We examined in vivo antitumor efficacy using preclinical models of MCL cell line-derived xenograft (CDX) .Results: Firstly, we found CD19-CD20 and CD19-CD22 bispecific CAR-T cells showed similar CAR expression levels (55%-72% CAR+ ) and similar expansion rate as single CD19 or CD20 CAR. Secondly, bispecific CAR- T cells potently and specifically lysed tumor cells in vitro, and showed the better killing of MCL tumor than CD19 or CD20 single CAR after challenge with MCL cell lines in vitro. Finally, we also confirmed these effects in CDX mice models derived from MCL cell line, Jeko-1 in vivo. CD19-CD20 and CD19-CD22 bispecific CAR successfully eliminated MCL tumor, showed better antitumor efficacy, and prolonged mouse survival than single CAR treated groups (P < 0.001). We also found that CD19-CD20 and CD19-CD22 bispecific-CAR T cells treated with TWS119 express significantly fewer exhaustion markers, such as PD-1 (P < 0.001), and LAG3 (P < 0.001), and showed better antitumor efficacy than the nontreated control group and single CAR groups in vivo. Furthermore, CAR-T cells expressing bispecific CD19-CD20 and CD19-CD22 CAR with TWS119 treatment had superior anti-MCL activity in vitro. In total, significantly better anti-MCL effects were observed for CD19-CD20 and CD19-CD22 bispecific CARs than single CAR. Their anti-MCL functions were further improved by TWS119 treatment.Conclusion: Altogether, these findings suggest that bispecific CD19-CD20 and CD19-CD22 CAR-T cells are potent for treating MCL relapse and drug resistant patients. In addition, bispecific CD19-CD20 and CD19-CD22 CAR-T cells with TWS119 treatment will further bring up the full power of CARs. These data support the therapeutic potential of bispecific CARs in patients with MCL. DisclosuresWang: Newbridge Pharmaceuticals: Honoraria; Moffit Cancer Center: Honoraria; BGICS: Honoraria; Bayer Healthcare: Consultancy; Hebei Cancer Prevention Federation: Honoraria; Clinical Care Options: Honoraria; Lilly: Research Funding; Miltenyi Biomedicine GmbH: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria, Research Funding; Mumbai Hematology Group: Honoraria; Epizyme: Consultancy, Honoraria; Dava Oncology: Honoraria; Chinese Medical Association: Honoraria; The First Afflicted Hospital of Zhejiang University: Honoraria; Molecular Templates: Research Funding; Imedex: Honoraria; Physicians Education Resources (PER): Honoraria; VelosBio: Consultancy, Research Funding; InnoCare: Consultancy, Research Funding; Genentech: Consultancy; DTRM Biopharma (Cayman) Limited: Consultancy; CStone: Consultancy; Scripps: Honoraria; OMI: Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Pharmacyclics: Consultancy, Research Funding; Celgene: Research Funding; Oncternal: Consultancy, Research Funding; Loxo Oncology: Consultancy, Research Funding; BioInvent: Research Funding; Juno: Consultancy, Research Funding; CAHON: Honoraria; BeiGene: Consultancy, Honoraria, Research Funding; AstraZeneca: Consultancy, Honoraria, Research Funding; Anticancer Association: Honoraria; Acerta Pharma: Consultancy, Honoraria, Research Funding.

FLT3 OR CD33 NOT EMCN Logic Gated CAR-NK Cell Therapy (SENTI-202) for Precise Targeting of AML

Background: While chimeric antigen receptor (CAR) cell therapies have provided extraordinary clinical responses in some hematological malignancies, developing effective CAR cell therapies for acute myeloid leukemia (AML) has been challenging due to: (a) the lack of a single target antigen robustly expressed across both AML leukemic stem cell (LSC) and immature leukemic blast cell subpopulations, and (b) the lack of truly AML-specific target antigens, since current targets are also expressed on healthy tissues and may result in off-tumor toxicity. Using logic gated gene circuits, we are engineering our SENTI-202 CAR-NK cell therapy to overcome these long-standing challenges to treating AML patients.Methods: To maximize clearance of AML tumor subpopulations and minimize off-tissue toxicities, we used a proprietary bioinformatics paired antigen discovery platform to identify the optimal combinations of AML tumor-associated and healthy tissue antigens to target using an OR and NOT logic gated CAR gene circuit approach. The SENTI-202 therapeutic candidate is a FLT3 OR CD33 NOT Endomucin (EMCN) gene circuit-enabled allogeneic CAR-NK cell, designed to broadly target FLT3 and/or CD33-expressing AML tumor cells (including both LSCs and blasts) but not healthy hematopoietic stem cells (HSCs).Results: First, for the OR GATE portion of the logic circuit we demonstrated that engineered primary human NK cells expressing activating CARs (aCARs) that recognize both FLT3 and CD33 outperformed more traditional single target CAR approaches with FLT3 (p<0.05) or CD33 (p<0.01), and exhibited >80% cytotoxicity and significant cytokine secretion (GrB, IFN-g, and TNF-a) against multiple leukemia cell lines in vitro, including MOLM13, THP1, and SEM. We successfully engineered FLT3 OR CD33 CAR-NK cells using both bicistronic and bivalent CAR configurations, where bicistronic CARs possess separate FLT3 and CD33 CARs linked via a 2A peptide, and bivalent CARs use a loop structure to connect FLT3 and CD33 scFvs within the same CAR. While both approaches demonstrated robust efficacy against AML cells, the bivalent approach enabled greater CAR expression and cytotoxicity (p<0.05). Importantly, our FLT3 OR CD33 CAR-NK cells demonstrated significant cytotoxicity against primary AML patient samples (p<0.01-0.001) and significantly reduced tumor burden and improved mouse survival in MOLM13 (p<0.05) and MV4-11 (p<0.01) xenograft AML models. We believe that our strategy of concurrently targeting FLT3 and CD33 will result in a more robust synergistic anti-tumor effect, leading to a more durable remission with decreased risk of relapse due to single antigen escape.Second, for the NOT GATE portion of the logic circuit to protect healthy HSCs, we developed NK and T cell inhibitory CARs (iCARs) consisting of an scFv against a healthy cell antigen, hinge and transmembrane domains, and functional intracellular domains derived from inhibitory co-receptors containing immunoreceptor tyrosine-based inhibitory motifs. In the case of SENTI-202, the iCAR scFv recognizes EMCN, a surface antigen expressed on up to 76% of healthy HSCs but not on AML cells. Using two different iCAR configurations, we demonstrated that FLT3 (CD28z) aCAR-NK cells engineered with an EMCN-specific iCAR protected up to 67% (iCAR#1, p<0.01) or 50% (iCAR#2, p<0.01) of FLT3+ EMCN+ cells from FLT3 aCAR-mediated cytotoxicity. Next, to replicate a clinical context more closely, we mixed FLT3+ EMCN- (AML-like) and FLT3+ EMCN+ (HSC-like) target cells and demonstrated that FLT3 NOT EMCN CAR-NK cells exhibit preferential killing of FLT3+ EMCN- target cells (p<0.0001), demonstrating that our NOT GATED gene circuit controls NK-mediated responses on a cell-by-cell basis.Conclusion: SENTI-202 is a novel NK cell product candidate to be engineered with both OR and NOT logic gated CAR gene circuits, wherein the OR gate is designed to increase AML LSC/blast tumor clearance (to prevent relapse), and the NOT gate is designed to protect healthy HSCs from off-tumor toxicity, enabling regeneration of a healthy hematopoietic system and mitigating the need for a bone marrow transplant. Beyond AML, OR and NOT logic gated CAR-NK cell therapy has applicability to other cancer-associated antigen targets that are potentially limited by antigen escape and/or off-tumor toxicity, increasing the potential for enhanced efficacy and reduced risk of undesirable side effects. DisclosuresNo relevant conflicts of interest to declare.

Clinical Evidence That Deficiency of the Methylcytosine Dioxygenase TET2 Gene Promotes the Clonal Expansion and Therapeutic Efficacy of CD19-Directed T Cells

Adoptive transfer of autologous chimeric antigen receptor (CAR) T cells that target tumor antigens can induce remissions in patients with hematologic malignancies. A major obstacle to successful clinical responses in some patient populations has been infusion with ineffective cell populations that are terminally differentiated, exhausted, poorly proliferative and short-lived. Understanding and manipulating the mechanistic pathways that sustain T cell potency will potentially unlock durable responses to tumors. Here we present an unusual case that helps clarify determinants of CAR T cell persistence. A 78-year-old individual with advanced chronic lymphocytic leukemia (CLL) was infused with CAR T cells that target CD19 (CTL019). CAR T cell expansion was delayed and not evident until 50 days following infusion and anti-tumor activity was observed in the peripheral blood, lymph nodes as well as bone marrow, accompanied by complete remission that has been sustained for over 4.5 years. Retrospective analyses revealed that at the peak of in vivo CAR T cell activity, over 94% percent of CD8+ CTL019 cells originated from a single clone in which lentiviral vector-mediated insertion of the CAR transgene led to monoallelic loss of TET2 . Accordingly, examination of polyadenylated TET2 RNA populations showed the appearance of new chimeric RNAs that spliced from TET2 exon 9 into the vector and terminated, truncating the encoded protein. Further analysis revealed that this patient had a germline polymorphism in the other allele of TET2 . This was a missense variant at amino acid 1879 (exon 11), converting the wildtype residue, glutamic acid, to glutamine (protein change: pE1879Q; cDNA change: c.5635G>C; allele frequency of approximately 50%). TET2 deficient CTL019 cells exhibited reduced cytosine hydroxymethylation and a chromatin accessibility profile measured by ATAC-Seq consistent with altered T cell differentiation. Furthermore, at the peak of in vivo engraftment, CAR T cells from this subject showed an early (central) memory phenotype which differs from CTL019 cells of other complete responding patients that are instead characterized by late (effector) memory differentiation at the height of their response. Experimental knock-down of TET2 in healthy donor CAR T cells altered their differentiation and augmented CTL019 effector function in a manner that was consistent with the effects of TET2 deficiency observed in this subject. Our findings suggest that the profound clonal expansion of a single TET2 deficient CAR T cell resulted in a complete and durable remission of CLL, highlighting the potential for epigenetic modulation of ineffective patient T cells to generate curative T cell products. DisclosuresFraietta:Novartis: Patents & Royalties: patents, Research Funding. Morrissette:Novartis: Consultancy. Liu:Novartis: Patents & Royalties. Young:Novartis: Research Funding. Levine:Novartis Pharmaceuticals Corporation: Patents & Royalties, Research Funding; Tmunity Therapeutics: Equity Ownership, Research Funding; GE Healthcare: Consultancy; Brammer Bio: Consultancy. Zhao:Novartis: Patents & Royalties. Kalos:Novartis: Research Funding. Porter:Incyte: Honoraria; Servier: Honoraria, Other: Travel reimbursement; Genentech/Roche: Employment, Other: Family member employment, stock ownship - family member; Immunovative Therapies: Other: Member DSMB; Novartis: Honoraria, Patents & Royalties, Research Funding. Lacey:Novartis: Research Funding; Genentech: Honoraria. June:Tmunity Therapeutics: Equity Ownership, Research Funding; WIRB/Copernicus Group: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celldex: Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Patents & Royalties, Research Funding. Melenhorst:Novartis: Research Funding.

Targeting Primary Pre-B Cell Acute Lymphoblastic Leukemia and CD19-Negative Relapses Using Trivalent CAR T Cells

B-Acute Lymphoblastic Leukemia (B-ALL) is the most common malignancy in children, and limited treatment options exist for patients with relapsed or refractory disease. Cellular immunotherapy, specifically chimeric antigen receptor (CAR) T cells targeting CD19, have demonstrated remarkable efficacy in treating B-ALL. However, recent reports show that up to 40% of patients who relapse after CD19 CAR T cell therapy have CD19-negative disease, justifying a need to expand CAR T cell therapy for B-ALL to include additional tumor-associated antigens.Here, we hypothesize that targeting three distinct leukemia antigens including CD19, CD20, and CD22 will improve B-ALL therapy outcomes and control disease progression during CD19-negative relapse.We designed two trivalent CAR T cell products with exodomains derived from single chain variable fragments (ScFv) targeting CD19 (FMC63 ScFv), CD20 (Rituximab ScFv), and CD22 (m971 ScFv). Using viral 2A intervening sequences for near equal expression, the first T cell product expresses the three CARs individually on the surface of a single T cell (TriCAR), and the second T cell product expresses a traditional single CAR targeting CD19 and a second bi-specific CAR targeting CD20 and CD22 through a tandem arrangement (SideCAR). All CARs are fused to the intracellular signaling domains of the co-stimulatory molecule 4-1BB and the T-cell receptor zeta (ζ) chain (2nd generation). Donor T cells were successfully engineered to express the CARs using a retroviral system and the surface expression of these CAR molecules was confirmed by flow cytometry. Using a target expression validated panel of patient derived B-ALL cells (US7 CD19/CD20/CD22 +++/++/++, LAX-56 +++/+/+, TXL-2 +++/++/+++), we observed that TriCAR and SideCAR T cells killed ALL cells more robustly than CD19 CAR T cells at low effector to target (E:T) ratios. TriCAR and SideCAR T cells secreted similar levels of IFN-γ/TNF-α when compared to CD19 CAR T cells suggesting a safety profile similar to the CD19 CAR T cells, but with enhanced killing. Further, we tested the efficacy of TriCAR and SideCAR T cells against primary CD19-negative relapsed bone marrow samples and CRISPR CD19 knockouts of the three primary ALL samples. Using these models of CD19 escape we demonstrated that trivalent CAR T cells effectively mitigated CD19 negative relapse, producing IFN-γ and TNF-α and killing CD19-negative primary ALL, while CD19 CAR T cells remained ineffective. Finally, we interrogated immune synapse (IS) microcluster formation and actin dynamics during interactions between CAR T cells and primary B-ALL cells by quantitative imaging flow cytometry. All CAR T cells exhibited higher actin polymerization compared to non-transduced T cell controls. TriCAR T cells formed significantly higher number of IS microclusters with different morphologies of actin polymerization compared to CD19 CAR T cells, suggesting distinct remodeling and dynamics of T cell polarization and immunoactivity, when interacting with TXL-2 primary B-ALL cells. TriCAR T cells formed IS microclusters, while CD19 CAR T cells failed to form IS with CD19 knockout TXL-2 cells.In conclusion, trivalent CAR T cells are effective at targeting primary ALL cells with varying antigen profiles and at mitigating CD19-negative relapse. This strategy has the potential for use as a front-line therapy for primary ALL as well as a salvage therapy for patients with CD19-negative disease relapse. DisclosuresNo relevant conflicts of interest to declare.

Enhanced Expression of CXCR4 Facilitates the Trafficking of Anti-c-Kit Chimeric Antigen Receptor (CAR) T Cells to Bone Marrow and Achieves Effective Donor Stem Cell Engraftment

Allogeneic hematopoietic stem cell (HSC) transplantation is one of the curative therapeutic options for patients with a variety of inherited disorders of blood cells, including immune deficiencies and hemoglobinopathies. In addition, considerable progress has been made toward achieving clinical benefit from autologous HSC ex vivo gene therapy for some of these disorders. Generally, some level of conditioning using chemotherapy and/or radiation is needed to achieve the required engraftment of allogeneic HSC or gene corrected autologous HSC. There is considerable interest in finding less toxic and more focused approaches to achieve the conditioning. Promising results were observed using antibody-based approaches including anti-c-kit (CD117) or anti-CD45 which directly target HSCs, and these results can be enhanced by linking the antibody to a toxin such as saponin. Here, we explored a related approach in mouse models in which chimeric antigen receptor (CAR)-T cells targeting c-kit (c-kit CAR-T cells) were employed as conditioning to achieve significant engraftment of congenic HSCs. Our initial attempts at efficient conditioning with c-kit CAR-T cells failed because there was insufficient trafficking of CAR-T cells to bone marrow (BM) and insufficient CAR-T cell expansion in vivo and because we did not eliminate the CAR-T cells before transplanting congenic HSCs.To address these barriers, before injecting CAR-T cells (derived from C57BL/6 Thy1.1/CD45.2 mouse spleen on Day1 and transduced with CAR retrovirus on Day2) we pre-treated the recipient mice (Thy1.2/CD45.2) with low-dose cyclophosphamide (CY, 125 mg/kg; Day2) and co-transduced the c-kit CAR-T cells with a CXCR4 (CD184) expression vector (Day2), followed by injection of 5×106 CAR-T cells on Day3. We later removed these CAR-T cells by treating the mice with anti-Thy1.1 antibody (Ab) on Day11 prior to HSC transplantation from congenic Thy1.2/CD45.1 mice (Day12; 3×106 cells/mouse).After single CAR retrovirus transduction, >90% of T cells expressed CAR. When used in vitro, CAR-T cells efficiently depleted BM c-kit+ population (10.5% reduced to 1.6%) and completely suppressed colony formation in nitrocellulose medium. In initial in vivo studies, we used a luciferase marker in the CAR-T cells to allow whole body imaging in addition to flow cytometry (FACS) of BM to assess trafficking. With no pre-treatment of mice, both in vivo imaging and FACS demonstrated poor trafficking of CAR-T cells to BM (< 1% of cells), and there was no decrease of the BM c-kit+ population. In contrast, pre-treatment with low-dose CY and overexpression of CXCR4 on the CAR-T cell surface improved the CAR-T cell expansion in vivo and the migration into BM (mean Thy1.1+ cells, 11.9%; n= 5), and this was associated with significant in vivo depletion of BM c-kit+ population (9.0% reduced to 0.0%). Severe pancytopenia was confirmed in peripheral blood (Hgb 2.2 g/dL compared to the normal, 13.0 g/dL) along with aplastic BM (Figure A), and these mice died within 4 weeks without BM transplant. Subsequent injection of anti-Thy1.1 Ab completely depleted CAR-T cells in viv o. When congenic BM cells were transplanted into mice conditioned in this manner (CY+CAR-T+Thy1.1 Ab), these mice achieved complete recovery of hematopoiesis with significant engraftment of donor BM in all lineages in peripheral blood (Figure B) and long-term HSCs in BM (> 20 % donor chimerism).Our findings provide proof-of-concept that c-kit CAR-T cells can be used to achieve sufficient conditioning to allow donor chimerism after congenic transplantation without use of high-dose chemotherapy or radiation. Low-dose CY was required to achieve the reduction of host T cells and stimulate in vivo expansion of CAR-T cells. A key element to achieve BM HSC depletion was the co-transduction of CXCR4 that greatly increased trafficking of the CAR-T cells to BM. This finding may be one of the most important aspects of this study, with broader implications for the CAR-T cell field in suggesting that use of trafficking receptors or ligands might be generalizable as a method to enhance targeting to designated organs or solid tumors. In summary, our studies add to the list of tools that should be further explored in the effort to seek less toxic conditioning regimens for BM transplantation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The antigen-binding moiety in the driver's seat of CARs.

Immuno‐oncology has been at the forefront of cancer treatment in recent decades. In particular immune checkpoint and chimeric antigen receptor (CAR)‐T cell therapy have achieved spectacular results. Over the years, CAR‐T cell development has followed a steady evolutionary path, focusing on increasing T cell potency and sustainability, which has given rise to different CAR generations. However, there was less focus on the mode of interaction between the CAR‐T cell and the cancer cell; more specifically on the targeting moiety used in the CAR and its specific properties. Recently, the importance of optimizing this domain has been recognized and the possibilities have been exploited. Over the last 10 years—in addition to the classical scFv‐based CARs—single domain CARs, natural receptor‐ligand CARs, universal CARs and CARs targeting more than one antigen have emerged. In addition, the specific parameters of the targeting domain and their influence on T cell activation are being examined. In this review, we concisely present the history of CAR‐T cell therapy, and then expand on various developments in the CAR ectodomain. We discuss different formats, each with their own advantages and disadvantages, as well as the developments in affinity tuning, avidity effects, epitope location, and influence of the extracellular spacer.

Evaluation of chimeric antigen receptor T cell therapy in non-human primates infected with SHIV or SIV.

Achieving a functional cure is an important goal in the development of HIV therapy. Eliciting HIV-specific cellular immune responses has not been sufficient to achieve durable removal of HIV-infected cells due to the restriction on effective immune responses by mutation and establishment of latent reservoirs. Chimeric antigen receptor (CAR) T cells are an avenue to potentially develop more potent redirected cellular responses against infected T cells. We developed and tested a range of HIV- and SIV-specific chimeric antigen receptor (CAR) T cell reagents based on Env-binding proteins. In general, SHIV/SIV CAR T cells showed potent viral suppression in vitro, and adding additional CAR molecules in the same transduction resulted in more potent viral suppression than single CAR transduction. Importantly, the primary determinant of virus suppression potency by CAR was the accessibility to the Env epitope, and not the neutralization potency of the binding moiety. However, upon transduction of autologous T cells followed by infusion in vivo, none of these CAR T cells impacted either acquisition as a test of prevention, or viremia as a test of treatment. Our study illustrates limitations of the CAR T cells as possible antiviral therapeutics.

Nanobody Based Tri-Specific Chimeric Antigen Receptor to Treat Acute Myeloid Leukaemia

Background: Chimeric Antigen Receptor (CAR) T-cell therapy represents a paradigm shift in therapy of advanced haematological malignancies in particular Acute Lymphoblastic Leukaemia. We have engineered multi-antigen specific CAR T-cells targeting three acute myeloid leukaemia (AML) associated antigens through incorporation of nanobody binding domains. Target antigens were selected for their broad expression on both on AML blasts as well as a high expression on Leukaemic Stem Cells (LSC). LSCs are a primitive self-renewing population that can contribute to relapse and may be phenotypically distinct from bulk AML blasts within the same patient. Aims: Our multi-antigenic approach thus aims to overcome antigen negative escape, overcome intra-tumour heterogeneity, and clear both LSCs and bulk disease by designing a tandem CAR capable of sensing CD33, CD123 and CLL1 on target cells. Methods: We initially screened libraries of nanobody binders for each antigen in a second generation CAR format. Once lead binders for each antigen were identified, Tandem CAR (TanCAR) structures were designed taking into consideration data on antigen structure from structural prediction servers and published crystal structures, nanobody domain mapping and antigen expression profiles. A TanCAR was then rationally designed comprising of three nanobodies joined by G4S linkers, fused to a hinge spacer, CD8 transmembrane domains, and 41BBz signalling domains. TanCAR T-cell function was compared to that of CAR T-cells targeting single antigens through in vitro co-culture assays with engineered target cells expressing single antigens (n=3). Antigen-specific cytotoxicity and proliferation were measured by flow cytometry assays; IL-2 and IFNy production was assayed by ELISA. Results: The ability of nanobody binding domains to bind individual antigens within the TanCAR format were confirmed using soluble antigen formats with a murine Fc tags via flow cytometry (Fig 1). In co-culture assays against engineered cell lines expressing single antigens (E:T=1:8, 24hrs) the TanCAR demonstrated antigen specific cytolytic activity (median cytotoxicity = 63%, 59%, and 47% against SupT1-CD33, SupT1-CD123, and SupT1-CLL1 respectively) approximately equal to or greater than single antigen targeting nanobody CAR T-cells (Fig 2a). In further co-culture experiments TanCAR T-cells produced high levels of IL-2 and IFNy when co cultured with cell lines expressing CD33 or CD123 only (n=3, median IL-2 = 9237pg/ml and 8190pg/ml, median IFNy 3924pg/ml and 4842pg/ml respectively) compared to SupT1 NT target cells, whereas low level cytokine production was observed with cell lines that express CLL-1 only (Fig 2b). Proliferation data matched this trend with greater levels of antigen specific proliferation observed when TanCAR T-cells were co-cultured with cells expressing CD33 and CD123 (7day co-culture, E:T=1:10, median fold expansion = 22, 8, and 5 fold for TanCAR co-cultured with cell lines expressing CD33, CD123, and CLL1 respectively), compared to antigen negative SupT1 target cells (Fig 2c). Summary/Conclusion: Collectively, our data demonstrate that TanCAR T-cells operate as an OR gate mediating potent reactivity against target cell lines expressing single AML antigens in vitro. We now aim to test for optimal TanCAR formats and potential additive or synergistic responses where TanCAR T cells are co-cultured with AML cell lines in-vivo (Molm14, PDX). Disclosures Ghorashian: Novartis: Honoraria; UCLB: Patents &amp; Royalties; Amgen: Honoraria. Pule:Mana Therapeutics: Other: entitled to share of revenue from patents filed by UCL; UCLB: Patents &amp; Royalties; Autolus: Current Employment, Other: owns stock in and receives royalties, Patents &amp; Royalties.