Chimeric Antigen Receptor Design Research Articles

Multispecific Targeting with Synthetic Ankyrin Repeat Motif Chimeric Antigen Receptors.

The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DARPins) rather than scFvs for tumor recognition. A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR, EpCAM, and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo. The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR+ targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells, each expressing a single antigen, and displayed synergistic activity when tumor cells expressed more than one target antigen. DARPins can serve as high-affinity recognition motifs for CAR design, and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape.

CD19 CAR T Cells for the Treatment of Pediatric Pre-B Cell Acute Lymphoblastic Leukemia.

The development of cluster of differentiation (CD)-19-targeted chimeric antigen receptor (CAR) T cells for the treatment of pre-B-cell acute lymphoblastic leukemia (B-ALL) is an exciting new advancement in the field of pediatric oncology. Tisagenlecleucel and axicabtagene ciloleucel are the first US FDA-approved CD19-targeted CAR T cells. While various different CD19 CAR T cells are in development, tisagenlecleucel is the only CAR T cell approved for pediatric patients. The multicenter phase II trial that led to the approval of tisagenlecleucel demonstrated excellent responses in individuals with highly refractory disease. Other high-risk groups of patients with B-ALL who experience poor outcomes with standard therapy may also benefit from treatment with tisagenlecleucel. After receiving CAR T cells, patients must be closely monitored for unique toxicities, including cytokine release syndrome, neurotoxicity, and B-cell aplasia. The management of patients with relapsed or refractory disease after administration of CD19 CAR T cells can be challenging, and treatment options vary according to the characteristics of the disease present at relapse. In the many patients who experience a complete response, CAR T cells can lead to a durable remission. This review describes the current design and manufacturing of CAR T cells. Data in the selection and management of pediatric patients are highlighted, as are areas where further studies are needed.

CXCR1- or CXCR2-modified CAR T cells co-opt IL-8 for maximal antitumor efficacy in solid tumors

Chimeric antigen receptor (CAR) T-cell therapy targeting solid tumors has stagnated as a result of tumor heterogeneity, immunosuppressive microenvironments, and inadequate intratumoral T cell trafficking and persistence. Early (≤3 days) intratumoral presentation of CAR T cells post-treatment is a superior predictor of survival than peripheral persistence. Therefore, we have co-opted IL-8 release from tumors to enhance intratumoral T-cell trafficking through a CAR design for maximal antitumor activity in solid tumors. Here, we demonstrate that IL-8 receptor, CXCR1 or CXCR2, modified CARs markedly enhance migration and persistence of T cells in the tumor, which induce complete tumor regression and long-lasting immunologic memory in pre-clinical models of aggressive tumors such as glioblastoma, ovarian and pancreatic cancer.

Abstract 3207: Preclinical development of first-of-kind dual-targeted off-the-shelf CAR-NK cell product with engineered persistence for an effective treatment of B cell malignancies

Abstract The unprecedented success of chimeric antigen receptor (CAR) and monoclonal antibody (mAb) -based immune-therapies has provided a clear indication that a system as complex as human immunity can be harnessed, even enhanced, toward a growing number of hematological cancers. Here we describe pre-clinical progress to develop a multi-functional induced pluripotent stem cell (iPSC)-derived natural killer (iNK) cell platform that combines engineered longevity with CAR and mAb-based modalities to leverage the intrinsic polyfunctionality of NK cells. As frontrunners of immune surveillance, NK cells employ a diverse array of germline encoded receptors in distinct combinations, which engage multiple signaling pathways to deliver potent effector responses that can be directed toward tumor cells, drive rapid proliferation, and pave the way for recruitment of adaptive immunity. Specific engagement of multiple signaling pathways was achieved in iNK cells through design of an NK cell-centric CAR combining the transmembrane domain of activating receptor NKG2D with intracellular signaling domains of 2B4 and CD3ζ. Recombining an anti-CD19 scFv onto this signaling platform, CAR modified iNK cells produced specific in vitro recognition of CD19+ B cell lymphoma cells in short term and long term cytotoxicity assays (84% vs 40% clearance of tumor cells at 60H, p<0.001). Further introduction of a fusion receptor consisting of Interleukin-15 (IL15) with IL15 receptor α, enabling autonomous IL15 stimulation, greatly improved iNK longevity and functional persistence in animal models. Moreover, iNK cells modified with IL15 fusion receptor showed enhanced functional maturation including KIR expression and effector molecules such as granzyme B ( ≥2 fold). While iNK cells with anti-CD19 CAR delayed tumor progression in vivo prior to relapse, iNK cells engineered with anti-CD19 CAR and IL15/IL15 receptor were curative against B cell lymphoma, (p<0.002). Expression of CAR and IL15 fusion receptor was then combined with a third modality, a high affinity CD16a receptor modified to prevent proteolytic cleavage (hnCD16). These multifunctional iNK cells demonstrated enhanced directed cytotoxicity in vitro in combination with rituximab against CD19+ targets (>99% vs 90% clearance of tumor cells) and CD19- targets (>99% vs 50% clearance of tumor cells by iNK with anti-CD19 CAR alone, p<0.0001), revealing a unique opportunity to combine CAR with a universal targeting modality to mitigate antigen escape and address heterogeneity in the tumor population by a multi-node targeting strategy. The resulting product, FT519, is designed to provide a flexible, potent and persistent engineered immune cell that utilizes the intrinsic versatility of NK cells to enable a highly effective combination therapy in a single, standardized, scalable, off-the-shelf platform. Citation Format: Jode Goodridge, Sajid Mahmood, Huang Zhu, Svetlana Gaidarova, Robert Blum, Ryan Bjordahl, Frank Cichocki, Hui-Yi Chu, Greg Bonello, Tom Lee, Brian Groff, Karl-Johan Malmberg, Bruce Walcheck, Jeffrey Miller, Dan Kaufman, Bahram Valamehr. Preclinical development of first-of-kind dual-targeted off-the-shelf CAR-NK cell product with engineered persistence for an effective treatment of 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 3207.

Tinkering in the garage - tuning CARs for safety.

The adoptive transfer of T cells expressing a chimeric antigen receptor (CAR) is an effective therapy for patients with relapsed or refractory CD19+ B cell malignancies, but can cause life-threatening toxicities. Herein we discuss a recent study suggesting that alterations to the design of anti-CD19 CARs can reduce cytokine release and the incidence of treatment-related complications.

Sensitive and adaptable pharmacological control of CAR T cells through extracellular receptor dimerization.

Chimeric antigen receptor (CAR) T cell therapies have achieved promising outcomes in several cancers, however more challenging oncology indications may necessitate advanced antigen receptor designs and functions. Here we describe a bipartite receptor system comprised of separate antigen targeting and signal transduction polypeptides, each containing an extracellular dimerization domain. We demonstrate that T cell activation remains antigen dependent but can only be achieved in the presence of a dimerizing drug, rapamycin. Studies performed in vitro and in xenograft mouse models illustrate equivalent to superior anti-tumor potency compared to currently used CAR designs, and at rapamycin concentrations well below immunosuppressive levels. We further show that the extracellular positioning of the dimerization domains enables the administration of recombinant re-targeting modules, potentially extending antigen targeting. Overall, this novel regulatable CAR design has exquisite drug sensitivity, provides robust anti-tumor responses, and is uniquely flexible for multiplex antigen targeting or retargeting, which may further assist the development of safe, potent and durable T cell therapeutics.

CAR T-cell therapy: Full speed ahead.

Chimeric antigen receptor (CAR) T-cell therapy has dramatically shifted the landscape of treatment for lymphoid malignancies, especially diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). However, there continue to be significant limitations of this therapy, such as incomplete or nonsustained responses and severe toxicities in a subset of patients. Furthermore, expanding the role of CAR T-cell therapy to new disease types is an important next step. In this review, we will highlight landmark trials for anti-CD19 CAR T cells and first-in-human trials of novel CARs, as well as discuss promising innovative CAR designs that are still undergoing preclinical development. Lastly, we will discuss toxicity and mechanisms of CAR T-cell resistance and failure, as well as potential future treatment approaches to these common issues.

Limitations in the Design of Chimeric Antigen Receptors for Cancer Therapy.

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

IL15 Enhances CAR-T Cell Antitumor Activity by Reducing mTORC1 Activity and Preserving Their Stem Cell Memory Phenotype.

Improvements in the quality and fitness of chimeric antigen receptor (CAR)-engineered T cells, through CAR design or manufacturing optimizations, could enhance the therapeutic potential of CAR-T cells. One parameter influencing the effectiveness of CAR-T cell therapy is the differentiation status of the final product: CAR-T cells that are less-differentiated and less exhausted are more therapeutically effective. In the current study, we demonstrate that CAR-T cells expanded in IL15 (CAR-T/IL15) preserve a less-differentiated stem cell memory (Tscm) phenotype, defined by expression of CD62L+CD45RA+ CCR7+, as compared with cells cultured in IL2 (CAR-T/IL2). CAR-T/IL15 cells exhibited reduced expression of exhaustion markers, higher antiapoptotic properties, and increased proliferative capacity upon antigen challenge. Furthermore, CAR-T/IL15 cells exhibited decreased mTORC1 activity, reduced expression of glycolytic enzymes and improved mitochondrial fitness. CAR-T/IL2 cells cultured in rapamycin (mTORC1 inhibitor) shared phenotypic features with CAR-T/IL15 cells, suggesting that IL15-mediated reduction of mTORC1 activity is responsible for preserving the Tscm phenotype. CAR-T/IL15 cells promoted superior antitumor responses in vivo in comparison with CAR-T/IL2 cells. Inclusion of cytokines IL7 and/or IL21 in addition to IL15 reduced the beneficial effects of IL15 on CAR-T phenotype and antitumor potency. Our findings show that IL15 preserves the CAR-T cell Tscm phenotype and improves their metabolic fitness, which results in superior in vivo antitumor activity, thus opening an avenue that may improve future adoptive T-cell therapies.

Chimeric Antigen Receptors Incorporating D Domains Targeting CD123 Direct Potent Mono- and Bi-specific Antitumor Activity of T Cells.

Chimeric antigen receptor (CAR) Tcell therapies have demonstrated impressive initial response rates in hematologic malignancies.However, relapse rates are significant, and robust efficacies in other indications, such as solid tumors, will likely require novel therapeutic strategies and CAR designs. To that end, we sought to develop simple, highly selective targeting domains (D domains) that could be incorporated into complex, multifunctional therapeutics. Herein, we describe the identification and characterization of D domains specific for CD123, a therapeutic target for hematologic malignancies, including acute myelogenous leukemia (AML). CARs comprised of these D domains mediate potent Tcell activation and cytolysis of CD123-expressing target cells and induce complete durable remission in two AML xenograft models. We describe a strategy of engineering less immunogenic D domains through the identification and removal of putative Tcell epitopes and investigate the binding kinetics and affinity requirements of the resultant D domain CARs. Finally, we extended the utility of D domains by generating functional, bi-specific CARs comprised of a CD123-specific D domain and a CD19-specific scFv. The properties of D domains suggest that this class of targeting domain may facilitate the development of multi-functional CARs where conventional, scFv-based designs may be suboptimal.

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function.

The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and manufacturing optimizations. One challenge for these development efforts, however, has been the establishment of in vitro assays that can robustly inform selection of the optimal CAR T cell products for in vivo therapeutic success. Standard in vitro tumor-lysis assays often fail to reflect the true antitumor potential of the CAR T cells due to the relatively short co-culture time and high T cell to tumor ratio. Here, we describe an in vitro co-culture method to evaluate CAR T cell recursive killing potential at high tumor cell loads. In this assay, long-term cytotoxic function and proliferative capacity of CAR T cells is examined in vitro over 7 days with additional tumor targets administered to the co-culture every other day. This assay can be coupled with profiling T cell activation, exhaustion and memory phenotypes. Using this assay, we have successfully distinguished the functional and phenotypic differences between CD4+ and CD8+ CAR T cells against glioblastoma (GBM) cells, reflecting their differential in vivo antitumor activity in orthotopic xenograft models. This method provides a facile approach to assess CAR T cell potency and to elucidate the functional variations across different CAR T cell products.

CAR T Cells for Solid Tumors: New Strategies for Finding, Infiltrating, and Surviving in the Tumor Microenvironment.

Chimeric antigen receptor (CAR) T cells, T cells that have been genetically engineered to express a receptor that recognizes a specific antigen, have given rise to breakthroughs in treating hematological malignancies. However, their success in treating solid tumors has been limited. The unique challenges posed to CAR T cell therapy by solid tumors can be described in three steps: finding, entering, and surviving in the tumor. The use of dual CAR designs that recognize multiple antigens at once and local administration of CAR T cells are both strategies that have been used to overcome the hurdle of localization to the tumor. Additionally, the immunosuppressive tumor microenvironment has implications for T cell function in terms of differentiation and exhaustion, and combining CARs with checkpoint blockade or depletion of other suppressive factors in the microenvironment has shown very promising results to mitigate the phenomenon of T cell exhaustion. Finally, identifying and overcoming mechanisms associated with dysfunction in CAR T cells is of vital importance to generating CAR T cells that can proliferate and successfully eliminate tumor cells. The structure and costimulatory domains chosen for the CAR may play an important role in the overall function of CAR T cells in the TME, and “armored” CARs that secrete cytokines and third- and fourth-generation CARs with multiple costimulatory domains offer ways to enhance CAR T cell function.

CAR T Cells: A Snapshot on the Growing Options to Design a CAR.

Adoptive cell therapy of malignant diseases with chimeric antigen receptor (CAR) modified T cells rapidly advanced from pre-clinical models to commercial approvals within 2 decades. CARs redirect patient's T cells towards cancer cells and activate the engineered cells for a cytolytic attack resulting in the destruction of the cognate target cell. CAR T cells have demonstrated their powerful capacities in inducing complete and lasting remissions of leukemia/lymphoma in an increasing number of trials worldwide. Since the early 90's, the design of CARs went through various steps of optimization until the very recent developments which include CARs with logic gating in the recognition of antigen patterns on target cells and TRUCKs with a target recognition induced delivery of immune modulating agents. Here we review the generations in CAR design, the impact of specific modifications, the strategies to improve the safety of CAR T cell therapy, and the challenges to adapt the CAR design for broader applications.

CAR T-cell bioengineering: Single variable domain of heavy chain antibody targeted CARs.

Redirecting the recognition specificity of T lymphocytes to designated tumour cell surface antigens by transferring chimeric antigen receptor (CAR) genes is becoming an effective strategy to combat cancer. Today, CAR T-cell therapy has proven successful in the treatment of haematological malignancies and the first CD19 CAR T-cell products has already entered the market. This success is expanding CAR design for broader malignancies including solid tumours. Nevertheless, CARs such as those built on antigen-specific single chain antibody variable fragment (scFv) may induce some adverse effects. Here, we briefly review CAR T-cell bioengineering and discuss selected important initiatives for improved T-cell reprogramming, function and safety. In this respect, we further elaborate on unconventional CARs structured on single variable domain of heavy chain (VHH) antibodies (single-domain antibodies) as an alternative to scFv, because of their interesting immunological and physicochemical characteristics and unique structure, which shows a high degree of homology with human VH3 gene family.

Sustained Therapeutic Efficacy of Humanized Anti-CD19 Chimeric Antigen Receptors (CAR)-T in Relapsed/Refractory Acute Lymphoblastic Leukemia

Background: Immunogenicity derived from the murine scFv, a major molecular design of CAR, may limit the persistence of CAR-T cells, resulting in high relapase rate in cured relapsed/refractory acute lymphoblastic leukemia (r/r ALL) patients after successful treatment. In this study, we aimed to develop a humanized anti-CD19 scFv CAR-T (hCAR-T) and treated patients with r/r ALL. Methods: In this one-arm, open-labelled phaseⅠ/Ⅱ study, we infused the T cells modified with hCAR-T to patients with r/r ALL. Long term followup for response and safety to treatment were evaluated. Findings: Eleven patients with r/r ALL were recruited in this study. Nine patients were response-evaluable and all achieved CR at three months; Seven patients remain CR till now and five have been in CR for over 12 months without further treatment. Long persistence of hCAR-T cells was observed in the most of patients. Among these patients, three of them with high and rapidly progressive tumour burden (median 90%) experienced grade 3-4 of CRS. These severe CRS were successfully controlled by earlier use of tocilizumab, glucocorticoid and plasma exchange (PE). Interpretation: T cells expressing the humanized anti-CD19 scFv CAR exhibited the sustained therapeutic efficacy in treatment of r/r ALL. Low replase rate was assocated with long persistence of CAR-T cells. The severe CRS could be controlled by tocilizumab, glucocorticoid and plasma exchange (PE). Clinical Trial Number: The study was registered at Clinicaltrials.gov (NCT02349698). Funding Statement: This work was supported by National Key Research and Development Program (2016YFC1303405), National Natural Science Foundation of China 81520108025), Clinical Research Project of the Southwest Hospital (SWH2016ZDCX1005, SWH2016LCZD-02), Chongqing Precision Biotech Co., Ltd. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: This study was approved by the Ethics Committee of the Southwest Hospital of Third Military Medical University (Chongqing, China). It was performed according to the principles of the Declaration of Helsinki. All patients or their guardians provided written informed consent before they were recruited in this study.

Modeling anti-CD19 CAR T cell therapy in humanized mice with human immunity and autologous leukemia.

BackgroundAdoptive immunotherapy using T cells expressing chimeric antigen receptors (CARs) targeting CD19 has produced remarkable clinical outcomes. However, much of the mechanisms of action, such as the development of memory responses and sources of immune cytokines, remain elusive largely due to the challenge of characterizing human CAR T cell function in vivo. The lack of a suitable in vivo model also hinders the development of new CAR T cell therapies.MethodsWe established a humanized mouse (hu-mouse) model with a functional human immune system and genetically-matched (autologous) primary acute B-lymphoblastic leukemia (B-ALL) that permits modeling of CD19-targeted CAR T cell therapy in immunocompetent hosts without allogeneic or xenogeneic immune responses.FindingsAnti-CD19 CAR T cells were detected in blood of leukemic hu-mice with kinetics and levels similar to those seen in patients receiving CAR T cell therapy. The levels of CAR T cells were correlated inversely with the burden of leukemia cells and positively with the survival times in anti-CD19 CAR T cell-treated leukemic hu-mice. Infusion of anti-CD19 CAR T cells also resulted in rapid production of T cell- and monocyte/macrophage-derived cytokines and an increase in frequency of regulatory T cells as reported in clinical studies.InterpretationThese results provide a proof-of-principle that this novel preclinical model has the potential to be used to model human CAR T cell therapy and facilitate the design of new CARs with improved antitumor activity.

Calibration of CAR activation potential directs alternative T cell fates and therapeutic potency.

Chimeric antigen receptors (CARs) are synthetic receptors that target and reprogram T cells to acquire augmented antitumor properties1. CD19-specific CARs that comprise CD28 and CD3ζ signaling motifs2 have induced remarkable responses in patients with refractory leukemia3-5 and lymphoma6 and were recently approved by the US Food and Drug Administration7. These CARs program highly performing effector functions that mediate potent tumor elimination4,8 despite the limited persistence they confer on T cells3-6,8. Extending their functional persistence without compromising their potency should improve current CAR therapies. Strong T cell activation drives exhaustion9,10, which may be accentuated by the redundancy of CD28 and CD3ζ signaling11,12 as well as the spatiotemporal constraints imparted by the structure of second-generation CARs2. Thus, we hypothesized that calibrating the activation potential of CD28-based CARs would differentially reprogram T cell function and differentiation. Here, we show that CARs encoding a single immunoreceptor tyrosine-based activation motif direct T cells to different fates by balancing effector and memory programs, thereby yielding CAR designs with enhanced therapeutic profiles.

37P - Pooling signaling and costimulatory domains in a flexible CARpool design

The present application relates to the field of immunotherapy, more particularly to the field of chimeric antigen receptors (CARs). Currently, second and third generation CAR designs are quite rigid in that they combine fixed costimulatory domains in cis on the same intracellular protein domain. Trans signaling is not equivalent as costimulatory receptors have different expression levels or stoichiometry. Here, a ‘mix and match’ approach is proposed where different signaling and costimulatory domains are present on separate chains within the same CAR complex, allowing increased flexibility and control of the nature and strength of the CAR-generated signal. Also proposed are polynucleotides, vectors encoding the transmembrane polypeptide chains and cells expressing such CARs. These cells are particularly suitable for use in immunotherapy, and strategies to treat diseases such as cancer using these cells are also provided.

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.

Chimeric Antigen Receptor Library Screening Using a Novel NF-κB/NFAT Reporter Cell Platform.

Chimeric antigen receptor (CAR)-T cell immunotherapy is under intense preclinical and clinical investigation, and it involves a rapidly increasing portfolio of novel target antigens and CAR designs. We established a platform that enables rapid and high-throughput CAR-screening campaigns with reporter cells derived from the Tcell lymphoma line Jurkat. Reporter cells were equipped with nuclear factor κB (NF-κB) and nuclear factor of activated Tcells (NFAT) reporter genes that generate a duplex output of enhanced CFP (ECFP) and EGFP, respectively. As a proof of concept, we modified reporter cells with CD19-specific and ROR1-specific CARs, and we detected high-level reporter signals that allowed distinguishing functional from non-functional CAR constructs. The reporter data were highly reproducible, and the time required for completing each testing campaign was substantially shorter with reporter cells (6days) compared to primary CAR-T cells (21days). We challenged the reporter platform to a large-scale screening campaign on a ROR1-CAR library, and we showed that reporter cells retrieved a functional CAR variant that was present with a frequency of only 6 in 1.05× 106. The data illustrate the potential to implement this reporter platform into the preclinical development path of novel CAR-T cell products and to inform and accelerate the selection of lead CAR candidates for clinical translation.