Chimeric Antigen Receptor Design Research Articles

Abstract 3505: Preclinical testing of mRNA engineered chimeric antigen receptor T cells: Comparison of a multi-infusion protocol to a single injection of stably expressed CARs in ALL xenograft models

Abstract T cells engineered to express chimeric antigen receptors (CARs), consisting of an anti-tumor targeting domain fused to Signal 1 and 2 activation domains, are a promising approach to cell therapy. We compared T cells engineered to stably express CARs using integrating viral vectors with T cells transiently expressing CARS via direct transduction with non-integrating mRNA. In early clinical testing, this CAR design using lentiviral transduction has been tested in 3 patients with advanced CLL. In these refractory patients with very high disease burdens, dramatic clinical responses have been observed, with two patients achieving CRs and one a PR, all sustained at >12 months. Transient expression has several advantages: short-term expression may allow initial clinical testing while limiting toxicity, and expediting manufacturing and scaling to GMP of CAR modifications, allowing rapid iterative improvements of CAR design. To evaluate the in vivo efficacy of mRNA CAR+T cells, we used xenograft models of human ALL (Nalm-6 and a primary patient leukemia, both CD19+) and high-level human T cell engraftment. NSG mice were injected with ALL at a dose producing 100% BM engraftment by 5d with 100% mortality by 25d (Nalm-6) or 36d (Primary). T cells were expanded ex vivo using CD3/CD28 beads and underwent lentiviral gene transfer or mRNA electroporation. mRNA transfection produced high but transient CAR surface expression, peaking at 4 hours and then declining over 8d. Using a novel method of simultaneous bioluminescent tracking of CARs and leukemia, we observed rapid migration of CAR T cells to all sites of disease and 100-fold reduction in ALL burden after only 24h. Using low dose cyclophosphamide (CTX; 60 mg/kg IP) 24h before the next infusion, we were able to deplete non-CAR expressing T cells and observe continued effects on leukemia from subsequent mRNA CAR infusions resulting in significantly prolonged survival in mice treated with mRNA CARs. Short-term efficacy of a single dose of mRNA vs. lentiviral CAR+ T cells was similar. Neither CTX nor untargeted repeat CAR T cells infusions alone had an effect on leukemia burden or survival. While lentiviral CAR+ T cells persisted and were active longer, median overall survival was not different between repeat mRNA CARs (52d Nalm-6, 76d primary) and a single dose of lentiviral transduced CARs (54d Nalm-6, 87d primary). Only stably expressed CARs resulted in any long term cures in this model. Relapse patterns suggest that the efficacy of mRNA and lentivirus CAR T cells are similar, as peripheral disease can be repeatedly cleared with subsequent mRNA CAR infusions but immune privileged xenograft artifact sites such as the tooth root are more resistant. These pre-clinical results demonstrate an important advance in T cell engineering, and are leading directly to clinical trials testing mRNA CAR CTLs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3505. doi:1538-7445.AM2012-3505

A New Cellular Weapon to Kill Leukaemic B-Cells = سلاح خلوي جديد لقتل خلايا ( ب ) المسببة لإبيضاض الدم

A New Cellular Weapon to Kill Leukaemic B-Cells = سلاح خلوي جديد لقتل خلايا ( ب ) المسببة لإبيضاض الدم

Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR) expression by flow cytometry

BackgroundThere has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL) with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes.MethodsCurrently anti-fragment antigen binding (Fab) conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig)-binding protein that binds to the variable light chains (kappa chain) of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv) and Fab fragments.ResultsWe used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2), two murine antibody derived CARs (anti-CSPG4, and anti-CD19), and two humanized mouse antibody based CARs (anti-ERBB2, and anti-PSCA). Transduced cells were stained first with biotin labeled protein L followed by phycoerythrin (PE)-conjugated streptavidin (SA) and analyzed by flow cytometry. For comparison, cells were stained in parallel with biotin conjugated goat-anti-mouse Fab or CAR specific fusion proteins. Using protein L, all CAR transduced lymphocytes exhibited specific staining pattern ranging from 40 to 80% of positive cells (compared to untransduced cells) and staining was comparable to the pattern observed with anti-Fab antibodies.ConclusionOur data demonstrate the feasibility of employing Protein L as a general reagent for the detection of CAR expression on transduced lymphocytes by flow cytometry.

Ligation of the CD2 co-stimulatory receptor enhances IL-2 production from first-generation chimeric antigen receptor T cells

T cells bearing chimeric antigen receptors (CARs) are broadly categorised into first- and second-generation receptors. Second-generation CARs contain a co-stimulatory signalling molecule and have been shown to secrete IL-2, undergo greater proliferation and to have enhanced persistence in vivo. However, we have shown that T cells bearing a first-generation CAR containing a CD19-targeting scFv (single-chain variable fragment) and the CD3ζ-signalling domain are able to produce IL-2 upon co-culture with CD19(+) B-cell lymphomas independent of CD28 activity. Here, we report that signalling through endogenous CD2 following ligation with its ligands, CD48 in mouse and CD58 in humans, drives IL-2 production by first-generation CD19-specific CAR. Moreover, the high levels of IL-2 produced by human T cells engrafted with a second-generation CD28-containing CAR during target-cell recognition are dependent to a degree upon CD2 receptor activity. These observations highlight the fact that the functional activity induced by T-cell-expressed CARs is dependent upon endogenous 'natural' receptor interactions. A deeper understanding of the role of these activities will serve to further refine the design of future CARs to either exploit or avoid these interactions.

Adoptive T-cell therapy of B-cell malignancies: Conventional and physiological chimeric antigen receptors

Hematological malignancies remain incurable diseases because of the high risk of relapse, even after complete remission. Adoptive T-cell therapy (ACT) using modified T cells with chimeric antigen receptors (CARs) targeted to specific tumor-associated antigens expressed by B-cell malignancies represents an attractive approach for cancer immunotherapy. Investigators optimized the design of CARs to enhance receptor mediated T cell signaling and demonstrated that second and third generation CARs, including various costimulatory molecules, resulted in enhanced T-cell persistence and sustained antitumor activity in both in vitro and in vivo mouse models as well as clinical trials. We highlight advances in the use of CARs in the treatment of B-cell malignancies and future challenges in the use of adoptive therapy with CAR-engineered T cells.

Treatment of Advanced Leukemia in Mice with mRNA Engineered T Cells

Cytotoxic T lymphocytes (CTLs) modified with chimeric antigen receptors (CARs) for adoptive immunotherapy of hematologic malignancies are effective in preclinical models and are being tested in several clinical trials. Although CTLs bearing stably expressed CARs generated by integrating viral vectors are efficacious and have potential long-term persistence, this mechanism of CAR expression can potentially result in significant toxicity. T cells were electroporated with an optimized in vitro transcribed RNA encoding a CAR against CD19. These RNA CAR CTLs were then tested in vitro and in vivo for efficacy. We found that T cells expressing an anti-CD19 CAR introduced by electroporation with optimized mRNA were potent and specific killers of CD19 target cells. CD19 RNA CAR T cells given to immunodeficient mice bearing xenografted leukemia rapidly migrated to sites of disease and retained significant target-specific lytic activity. Unexpectedly, a single injection of CD19 RNA CAR T cells reduced disease burden within 1 day after administration, resulting in a significant prolongation of survival in an aggressive leukemia xenograft model. The surface expression of the RNA CARs may be titrated, giving T cells with potentially tunable levels of effector functions such as cytokine release and cytotoxicity. RNA CARs are a genetic engineering approach that should not be subject to genotoxicity, and they provide a platform for rapidly optimizing CAR design before proceeding to more costly and laborious stable expression systems.

Carving the CAR

Chimeric antigen receptors (CARs) are major histocompatibility complex (MHC)-independent one-polypeptide chain receptor molecules that, when expressed in T cells, can redirect specificity towards tumor or viral antigens. The antigen-binding domain of the CAR is derived from a single-chain antibody fragment and the transmembrane and signalling domains from the T-cell receptor CD3z chain, and optionally includes a costimulatory molecule such as CD28. CARs therefore combine the antigen specificity of an antibody and the property to activate a cytotoxic T cell in a single fusion molecule. A spacer is required between the extracellular antigen-binding domain and the transmembrane moiety. Most commonly, the constant immunoglobulin G (IgG)1 hingeCH2-CH3 Fc domain is used as a spacer domain, resulting in dimerization and thereby increasing CAR expression on the cell surface. In this issue, Hombach et al. reveal that the IgG1 spacer used in most CARs may also bind to surface IgG Fc gamma receptors expressed on macrophages and natural killer cells. This binding activates both the engineered T cells and the innate immune cells independent of the specificity of the CAR binding domain. Activation of CAR-positive T cells and Fc receptor-bearing cells through FcgR binding led to an unwanted, ‘off-target’ pro-inflammatory immune response. Hombach et al. provide a solution to this problem by modifying the sequences required for Fc binding in the constant immunoreceptor Fc domain, which eliminated interaction with Fc-positive cells but preserved stable expression of CARs on the surface of T cells and specific antigen recognition. In recent years, a variety of CARs with different scFv-binding domains have been designed. These CARs possess binding specificities for tumor-associated antigens, such as carcinoembryonic antigen and melanoma-associated antigens, CD30 and CD19, or for virus-infected cells, such as HIV or hepatitis B virus. Clinical trials that use CAR-modified T cells for adoptive immunotherapy of malignant diseases have been implemented and additional trials are planned. Owing to its one-polypeptide chain design and modular composition, the use of CARs substantially simplifies redirecting T cells with defined specificity. Upon antigen engagement, the intracellular signalling domain initiates T-cell activation, resulting in T-cell proliferation, secretion of pro-inflammatory cytokines and cytolysis of the antigen-positive target cell. There are major differences between the natural T cell receptor and a CAR, which confer potential advantages and disadvantages. CAR recognition is independent from antigen presentation on MHC, and allows broad clinical application for patients of any HLA haplotype. The lack of MHC restriction may also allow effective therapy of tumors that escape MHC-restricted T cells by downregulating MHC expression or through defects in antigen processing. However, early versions of CARs often provided weak activation of engineered T cells, mainly because CAR expression was unstable and costimulatory molecules were not, or only inadequately, engaged. The modular nature of CARs has enabled researchers to modify the CAR structure to achieve better activity. In the early research on CARs, it became obvious that inserting a ‘spacer’ domain, the IgG1 Fc being most commonly used, between the antigen-binding and transmembrane moieties improved expression and binding. Later on, research focussed on improving the signalling capacity by fusing costimulatory domains, such as CD28, OX40, 4-1BB or the src family kinase lck, to the CD3z signalling domain. T cells carrying CARs with CD3z and CD28 signalling domains proved to be superior in vivo and were able to reject established tumors in mice. Using T cells with natural specificity for Epstein–Barr virus is another strategy to provide appropriate costimulation through intermittent T-cell receptor ligation, and improved the persistence and probably also the activity of CARgrafted T cells in tumor patients. Severe side effects reported from recent clinical trials of adoptive immunotherapy with CAR-redirected T cells are a poignant reminder that much remains to be learned to realize the therapeutic promise of this approach. In one case, reactivity of the CAR antibody-binding domain with normal antigen-expressing cells was implicated as the cause of a cytokine storm and organ toxicity, and an unexplained sepsis-like syndrome occurred in a second case. The work of Hombach et al. illustrates the potential for ‘offtarget’ activation of engineered T cells via FcR binding to induce unwanted cytokine release and tissue damage—and, importantly, provides an improvement in CAR design that may help to avoid these side effects. It is anticipated that further improvements in CAR design will focus on increasing the specificity of the CARredirected T-cell response and minimizing the inflammatory response that is elicited. This should improve the safety and efficacy of adoptive immunotherapy with CAR-engineered T cells.

Genetically Targeted T Cells Eradicate Systemic Acute Lymphoblastic Leukemia Xenografts

Human T cells targeted to the B cell-specific CD19 antigen through retroviral-mediated transfer of a chimeric antigen receptor (CAR), termed 19z1, have shown significant but partial in vivo antitumor efficacy in a severe combined immunodeficient (SCID)-Beige systemic human acute lymphoblastic leukemia (NALM-6) tumor model. Here, we investigate the etiologies of treatment failure in this model and design approaches to enhance the efficacy of this adoptive strategy. A panel of modified CD19-targeted CARs designed to deliver combined activating and costimulatory signals to the T cell was generated and tested in vitro to identify an optimal second-generation CAR. Antitumor efficacy of T cells expressing this optimal costimulatory CAR, 19-28z, was analyzed in mice bearing systemic costimulatory ligand-deficient NALM-6 tumors. Expression of the 19-28z CAR, containing the signaling domain of the CD28 receptor, enhanced systemic T-cell antitumor activity when compared with 19z1 in treated mice. A treatment schedule of 4 weekly T-cell injections, designed to prolong in vivo T-cell function, further improved long-term survival. Bioluminescent imaging of tumor in treated mice failed to identify a conserved site of tumor relapse, consistent with successful homing by tumor-specific T cells to systemic sites of tumor involvement. Both in vivo costimulation and repeated administration enhance eradication of systemic tumor by genetically targeted T cells. The finding that modifications in CAR design as well as T-cell dosing allowed for the complete eradication of systemic disease affects the design of clinical trials using this treatment strategy.

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<h3>SUMMARY</h3> Chimeric antigen receptors (CARs) are modular proteins capable of redirecting immune cells toward a wide variety of disease-associated antigens. Here, we explore the effects of CAR protein sequence and structure on CAR-T cell function. Based on the empirical observation that CD20 CARs with similar sequences exhibit divergent tonic-signaling and anti-tumor activities, we devised engineering strategies that aimed to improve CAR-T cell function by tuning the intensity of tonic signaling. We found that CARs designed to exhibit low but non-zero levels of tonic signaling show robust effector function upon antigen stimulation while avoiding premature functional exhaustion by CAR-T cells. Through alterations of the CAR’s ligand-binding domain and overall protein conformation, we generated CD20 CAR variants that outperform the CD19 CAR in mouse models of human lymphoma. We further demonstrate that rational modification of protein confirmation can be generalized to improve GD2 CAR-T cell efficacy against neuroblastoma. These findings point to tonic signaling and basal T-cell activation as informative parameters to guide the rational design of next-generation CARs for cancer therapy.