Expression data from chimeric antigen receptor transduced (CAR) human CD4+ T cells during expansion
ABSTRACT: In this data set we include expression data from human CD4+ T cells isolated on day 0, 6, 11 and 24 follow anti-CD3/anti-CD28 magnetic bead stimulation and chimeric antigen receptor transduction. 30 samples were submitted. Samples represented three biological replicates of normal donors transduced with various CARs. CARs used were a cMet 28z specific CAR comprised of the IgG4 hinge, CD28 transmembrane and CD28 and CD3zeta intracellular domains. A CD19 CD28 CAR was specific to CD19, and was comprised of a CD8a hinge, CD28 transmembrane and CD28 and CD3zeta intracellular domain. A third CAR, the CD19 BBz, was used that was specific to CD19 was comprised of a CD8a hinge, CD8a transmembrane and 4-1BB and CD3zeta intracellular domains. Expression data was analyzied on day 0, 6, 11 and 24.
Project description:Anti-CD19 chimeric antigen receptor (CAR) T cells have caused remissions of B cell malignancies, but problems including cytokine-mediated toxicity and short persistence of CAR T cells in vivo might limit the effectiveness of anti-CD19 CAR T cells. Anti-CD19 CARs that have been tested clinically had single-chain variable fragments (scFvs) derived from murine antibodies. We have designed and constructed novel anti-CD19 CARs containing a scFv with fully human variable regions. T cells expressing these CARs specifically recognized CD19+ target cells and carried out functions including degranulation, cytokine release, and proliferation. We compared CARs with CD28 costimulatory moieties along with hinge and transmembrane domains from either the human CD28 molecule or the human CD8? molecule. Compared with T cells expressing CARs with CD28 hinge and transmembrane domains, T cells expressing CARs with CD8? hinge and transmembrane domains produced lower levels of cytokines and exhibited lower levels of activation-induced cell death (AICD). Importantly, CARs with hinge and transmembrane regions from either CD8? or CD28 had similar abilities to eliminate established tumors in mice. In anti-CD19 CARs with CD28 costimulatory moieties, lower levels of inflammatory cytokine production and AICD are potential clinical advantages of CD8? hinge and transmembrane domains over CD28 hinge and transmembrane domains.
Project description:B7-H3 is actively being explored as an immunotherapy target for pediatric patients with solid tumors using monoclonal antibodies or T cells expressing chimeric antigen receptors (CARs). B7-H3-CARs containing a 41BB costimulatory domain are currently favored by several groups based on preclinical studies. In this study, we initially performed a detailed analysis of T cells expressing B7-H3-CARs with different hinge/transmembrane (CD8? versus CD28) and CD28 or 41BB costimulatory domains (CD8?/CD28, CD8?/41BB, CD28/CD28, CD28/41BB). Only subtle differences in effector function were observed between CAR T cell populations in vitro. However, CD8?/CD28-CAR T cells consistently outperformed other CAR T cell populations in three animal models, resulting in a significant survival advantage. We next explored whether adding 41BB signaling to CD8?/CD28-CAR T cells would further enhance effector function. Surprisingly, incorporating 41BB signaling into the CAR endodomain had detrimental effects, while expressing 41BBL on the surface of CD8?/CD28-CAR T cells enhanced their ability to kill tumor cells in repeat stimulation assays. Furthermore, 41BBL expression enhanced CD8?/CD28-CAR T cell expansion in vivo and improved antitumor activity in one of four evaluated models. Thus, our study highlights the intricate interplay between CAR hinge/transmembrane and costimulatory domains. Based on our study, we selected CD8?/CD28-CAR T cells expressing 41BBL for early phase clinical testing.
Project description:BACKGROUND:Multiple iterations of chimeric antigen receptors (CARs) have been developed, mainly focusing on intracellular signaling modules. However, the effect of non-signaling extracellular modules on the expansion and therapeutic efficacy of CARs remains largely undefined. METHODS:We generated two versions of CAR vectors, with or without a hinge domain, targeting CD19, mesothelin, PSCA, MUC1, and HER2, respectively. Then, we systematically compared the effect of the hinge domains on the growth kinetics, cytokine production, and cytotoxicity of CAR T cells in vitro and in vivo. RESULTS:During in vitro culture period, the percentages and absolute numbers of T cells expressing the CARs containing a hinge domain continuously increased, mainly through the promotion of CD4+ CAR T cell expansion, regardless of the single-chain variable fragment (scFv). In vitro migration assay showed that the hinges enhanced CAR T cells migratory capacity. The T cells expressing anti-CD19 CARs with or without a hinge had similar antitumor capacities in vivo, whereas the T cells expressing anti-mesothelin CARs containing a hinge domain showed enhanced antitumor activities. CONCLUSIONS:Hence, our results demonstrate that a hinge contributes to CAR T cell expansion and is capable of increasing the antitumor efficacy of some specific CAR T cells. Our results suggest potential novel strategies in CAR vector design.
Project description:Chimeric antigen receptors (CARs) are engineered receptors that mediate T cell activation. CARs are comprised of activating and co-stimulatory intracellular signaling domains derived from endogenous T cells that initiate signaling required for T cell activation, including ERK activation through the MAPK pathway. Understanding the mechanisms by which co-stimulatory domains influence signaling can help guide the design of next-generation CARs. Therefore, we constructed an experimentally validated computational model of anti-CD19 CARs in T cells bearing the CD3? domain alone or in combination with CD28. We performed a systematic analysis to explore the different mechanisms of CD28 co-stimulation on the ERK response time. Comparing these model simulations with experimental data indicates that CD28 primarily influences ERK activation by enhancing the phosphorylation kinetics of CD3?. Overall, we present a mechanistic mathematical modeling framework that can be used to gain insights into the mechanism of CAR T cell activation and produce new testable hypotheses.
Project description:Clinical trials of CD19-specific chimeric antigen receptor (CAR19) T cells have demonstrated remarkable efficacy against relapsed and refractory B cell malignancies. The piggyBac transposon system offers a less complex and more economical means for generating CAR19 T cells compared to viral vectors. We have previously optimized a protocol for the generation of CAR19 T cells using the piggyBac system, but we found that CAR19 T cells had poor in vivo efficacy and persistence, probably due to deleterious FcγR interactions with the CAR's IgG1 Fc-containing spacer domain. We therefore designed three CD19-specifc CARs that lacked the IgG1 Fc region, and we incorporated combinations of CD28 or 4-1BB transmembrane and co-stimulatory domains. PiggyBac-generated CAR19 T cells expressing these re-designed constructs all demonstrated reactivity in vitro specifically against CD19+ cell lines. However, those combining CD28 transmembrane and co-stimulatory domains showed CD4 predominance and inferior cytotoxicity. At high doses, CAR19 T cells were effective against B-ALL in a xenograft mouse model, regardless of co-stimulatory domain. At diminishing doses, 4-1BB co-stimulation led to greater potency and persistence of CAR19 T cells, and it provided protection against B-ALL re-challenge. Production of potent CAR T cells using piggyBac is simple and cost-effective, and it may enable wider access to CAR T cell therapy.
Project description:Costimulatory signals are required to achieve robust chimeric antigen receptor (CAR) T cell expansion, function, persistence and antitumor activity. These can be provided by incorporating intracellular signalling domains from one or more T cell costimulatory molecules, such as CD28 or 4-1BB, into the CAR. The selection and positioning of costimulatory domains within a CAR construct influence CAR T cell function and fate, and clinical experience of autologous anti-CD19 CAR T cell therapies suggests that costimulatory domains have differential impacts on CAR T cell kinetics, cytotoxic function and potentially safety profile. The clinical impacts of combining costimulatory domains and of alternative costimulatory domains are not yet clearly established, and may be construct- and disease-specific. The aim of this review is to summarise the function and effect of established and emerging costimulatory domains and their combinations within CAR T cells.
Project description:Genetically modifying autologous T cells to express an anti-CD19 chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19+ B cell malignancies in several clinical trials (CTs). Making this treatment available to our patients prompted us to develop a novel CART19 based on our own anti-CD19 antibody (A3B1), followed by CD8 hinge and transmembrane region, 4-1BB- and CD3z-signaling domains. We show that A3B1 CAR T cells are highly cytotoxic and specific against CD19+ cells in vitro, inducing secretion of pro-inflammatory cytokines and CAR T cell proliferation. In vivo, A3B1 CAR T cells are able to fully control disease progression in an NOD.Cg-Prkdc scid Il2rd tm1Wjl /SzJ (NSG) xenograph B-ALL mouse model. Based on the pre-clinical data, we conclude that our CART19 is clearly functional against CD19+ cells, to a level similar to other CAR19s currently being used in the clinic. Concurrently, we describe the implementation of our CAR T cell production system, using lentiviral vector and CliniMACS Prodigy, within a medium-sized academic institution. The results of the validation phase show our system is robust and reproducible, while maintaining a low cost that is affordable for academic institutions. Our model can serve as a paradigm for similar institutions, and it may help to make CAR T cell treatment available to all patients.
Project description:Chimeric antigen receptors (CARs) have an antigen-binding domain fused to transmembrane, costimulatory, and CD3? domains. Two CARs with regulatory approval include a CD28 or 4-1BB costimulatory domain. While both CARs achieve similar clinical outcomes, biologic differences have become apparent but not completely understood. Therefore, in this study we aimed to identify mechanistic differences between 4-1BB and CD28 costimulation that contribute to the biologic differences between the 2 CARs and could be exploited to enhance CAR T cell function. Using CD19-targeted CAR T cells with 4-1BB we determined that enhancement of T cell function is driven by NF-?B. Comparison to CAR T cells with CD28 also revealed that 4-1BB is associated with more antiapoptotic proteins and dependence on persistence for B cell killing. While TNF receptor-associated factor 2 (TRAF2) has been presupposed to be required for 4-1BB costimulation in CAR T cells, we determined that TRAF1 and TRAF3 are also critical. We observed that TRAFs impacted CAR T viability and proliferation, as well as cytotoxicity and/or cytokines, in part by regulating NF-?B. Our study demonstrates how 4-1BB costimulation in CAR T cells impacts antitumor eradication and clinical outcomes and has implications for enhanced CAR design.
Project description:The adoptive transfer of T cells modified to express a chimeric antigen receptor (CAR) comprised of an extracellular single-chain antibody (scFV) fragment specific for a tumor cell surface molecule, and linked to an intracellular signaling module, has activity in advanced malignancies. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a tumor-associated molecule expressed in prevalent B-lymphoid and epithelial cancers and is absent on normal mature B cells and vital tissues, making it a candidate for CAR T-cell therapy.We constructed ROR1-CARs from scFVs with different affinities and containing extracellular IgG4-Fc spacer domains of different lengths, and evaluated the ability of T cells expressing each CAR to recognize ROR1(+) hematopoietic and epithelial tumors in vitro, and to eliminate human mantle cell lymphoma (MCL) engrafted into immunodeficient mice.ROR1-CARs containing a short "Hinge-only" extracellular spacer conferred superior lysis of ROR1(+) tumor cells and induction of T-cell effector functions compared with CARs with long "Hinge-CH2-CH3" spacers. CARs derived from a higher affinity scFV conferred maximum T-cell effector function against primary CLL and ROR1(+) epithelial cancer lines in vitro without inducing activation-induced T-cell death. T cells modified with an optimal ROR1-CAR were equivalently effective as CD19-CAR-modified T cells in mediating regression of JeKo-1 MCL in immunodeficient mice.Our results show that customizing spacer design and increasing affinity of ROR1-CARs enhances T-cell effector function and recognition of ROR1(+) tumors. T cells modified with an optimized ROR1-CAR have significant antitumor efficacy in a preclinical model in vivo, suggesting they may be useful to treat ROR1(+) tumors in clinical applications.
Project description:The success of adoptive therapy using chimeric antigen receptor (CAR)-expressing T cells partly depends on optimal CAR design. CARs frequently incorporate a spacer/linker region based on the constant region of either IgG1 or IgG4 to connect extracellular ligand-binding with intracellular signaling domains. Here, we evaluated the potential for the IgG4-Fc linker to result in off-target interactions with Fc gamma receptors (Fc?Rs). As proof-of-principle, we focused on a CD19-specific scFv-IgG4-CD28-zeta CAR and found that, in contrast to CAR-negative cells, CAR+ T cells bound soluble Fc?Rs in vitro and did not engraft in NSG mice. We hypothesized that mutations to avoid Fc?R binding would improve CAR+ T cell engraftment and antitumor efficacy. Thus, we generated CD19-specific CARs with IgG4-Fc spacers that had either been mutated at two sites (L235E; N297Q) within the CH2 region (CD19R(EQ)) or incorporated a CH2 deletion (CD19Rch2?). These mutations reduced binding to soluble Fc?Rs without altering the ability of the CAR to mediate antigen-specific lysis. Importantly, CD19R(EQ) and CD19Rch2? T cells exhibited improved persistence and more potent CD19-specific antilymphoma efficacy in NSG mice. Together, these studies suggest that optimal CAR function may require the elimination of cellular Fc?R interactions to improve T cell persistence and antitumor responses.