CAR T cells: Building on the CD19 paradigm.

Spearheaded by the therapeutic use of chimeric antigen receptors (CARs) targeting CD19, synthetic immunology has entered the clinical arena. CARs are recombinant receptors for antigen that engage cell surface molecules through the variable region of an antibody and signal through arrayed T-cell activating and costimulatory domains. CARs allow redirection of T-cell cytotoxicity against any antigen of choice, independent of MHC expression. Patient T cells engineered to express CARs specific for CD19 have yielded remarkable outcomes in subjects with relapsed/refractory B- cell malignancies, setting off unprecedented interest in T-cell engineering and cell-based cancer immunotherapy. In this review, we present the challenges to extend the use of CAR T cells to solid tumors and other pathologies. We further highlight progress in CAR design, cell manufacturing, and genome editing, which in aggregate hold the promise of generating safer and more effective genetically instructed immunity. Novel engineered cell types, including innate T-cell types, natural killer (NK) cells, macrophages, and induced pluripotent stem cell-derived immune cells, are on the horizon, as are applications of CAR T cells to treat autoimmunity, severe infections, and senescence-associated pathologies.

T Cells Retain Pivotal Antitumoral Functions under Tumor-Treating Electric Fields.

Tumor-treating fields (TTFields) are a localized, antitumoral therapy using alternating electric fields, which impair cell proliferation. Combining TTFields with tumor immunotherapy constitutes a rational approach; however, it is currently unknown whether TTFields' locoregional effects are compatible with T cell functionality. Healthy donor PBMCs and viably dissociated human glioblastoma samples were cultured under either standard or TTFields conditions. Select pivotal T cell functions were measured by multiparametric flow cytometry. Cytotoxicity was evaluated using a chimeric Ag receptor (CAR)-T-based assay. Glioblastoma patient samples were acquired before and after standard chemoradiation or standard chemoradiation + TTFields treatment and examined by immunohistochemistry and by RNA sequencing. TTFields reduced the viability of proliferating T cells, but had little or no effect on the viability of nonproliferating T cells. The functionality of T cells cultured under TTFields was retained: they exhibited similar IFN-γ secretion, cytotoxic degranulation, and PD1 upregulation as controls with similar polyfunctional patterns. Glioblastoma Ag-specific T cells exhibited unaltered viability and functionality under TTFields. CAR-T cells cultured under TTFields exhibited similar cytotoxicity as controls toward their CAR target. Transcriptomic analysis of patients' glioblastoma samples revealed a significant shift in the TTFields-treated versus the standard-treated samples, from a protumoral to an antitumoral immune signature. Immunohistochemistry of samples before and after TTFields treatment showed no reduction in T cell infiltration. T cells were found to retain key antitumoral functions under TTFields settings. Our data provide a mechanistic insight and a rationale for ongoing and future clinical trials that combine TTFields with immunotherapy.

P32-specific CAR T cells with dual antitumor and antiangiogenic therapeutic potential in gliomas.

Glioblastoma is considered one of the most aggressive malignancies in adult and pediatric patients. Despite decades of research no curative treatment is available and it thus remains associated with a very dismal prognosis. Although recent pre-clinical and clinical studies have demonstrated the feasibility of chimeric antigen receptors (CAR) T cell immunotherapeutic approach in glioblastoma, tumor heterogeneity and antigen loss remain among one of the most important challenges to be addressed. In this study, we identify p32/gC1qR/HABP/C1qBP to be specifically expressed on the surface of glioma cells, making it a suitable tumor associated antigen for redirected CAR T cell therapy. We generate p32 CAR T cells and find them to recognize and specifically eliminate p32 expressing glioma cells and tumor derived endothelial cells in vitro and to control tumor growth in orthotopic syngeneic and xenograft mouse models. Thus, p32 CAR T cells may serve as a therapeutic option for glioblastoma patients.

Treatment of Multiple Myeloma Using Chimeric Antigen Receptor T Cells with Dual Specificity.

Chimeric antigen receptor (CAR) T-cell therapy has shown remarkable successes in fighting B-cell leukemias/lymphomas. Promising response rates are reported in patients treated with B-cell maturation antigen (BCMA) CAR T cells for multiple myeloma. However, responses appear to be nondurable, highlighting the need to expand the repertoire of multiple myeloma-specific targets for immunotherapy and to generate new CAR T cells. Here, we developed a "dual-CAR" targeting two multiple myeloma-associated antigens and explored its safety and efficacy. To reduce the "off-target" toxicity, we used the recognition of paired antigens that were coexpressed by the tumor to induce efficient CAR T-cell activation. The dual-CAR construct presented here was carefully designed to target the multiple myeloma-associated antigens, taking into consideration the distribution of both antigens on normal human tissues. Our results showed that the CD138/CD38-targeted dual CAR (dCAR138-38) elicited a potent anti-multiple myeloma response both in vitro and in vivo NSG mice transplanted with a multiple myeloma cell line and treated with dCAR138-38 showed median survival of 97 days compared with 31 days in the control group treated with mock-lymphocytes. The dCAR138-38 showed increased specificity toward cells expressing both targeted antigens compared with single-antigen-expressing cells and low activity toward primary cells from healthy tissues. Our findings indicated that the dCAR138-38 may provide a potent and safe alternative therapy for patients with multiple myeloma.

Trastuzumab derived HER2-specific CARs for the treatment of trastuzumab-resistant breast cancer: CAR T cells penetrate and eradicate tumors that are not accessible to antibodies.

HER2-targeted monoclonal antibodies improve the outcome for advanced breast cancer patients; however, resistance to therapy is still frequent. Epitope masking and steric hindrance to antibody binding through matrix components are thought to be the major mechanism. We asked whether tumors resistant to trastuzumab can still be eliminated by CAR T cells redirected by the same antibody domain. While saturating doses of trastuzumab in the presence of CD16.176V.NK-92 effector cells and trastuzumab derived CAR T cells equally well recognized and killed HER2-positive tumor cells in a monolayer, only CAR T cells penetrated into the core region of tumor spheroids and exhibited cytotoxic activity in vitro, whereas antibodies failed. In NSG mice treatment with trastuzumab and CD16.176V.NK-92 cells only transiently retarded tumor growth but did not induce regression of clinically trastuzumab-resistant breast cancer xenografts. In contrast, one dose of HER2-specific CAR T cells eradicated established tumors resulting in long-term survival. Data indicate that CAR T cells can successfully combat antibody resistant tumors by targeting the same epitope suggesting that CAR T cells can penetrate the tumor matrix which is a barrier for antibodies.

Less is more: reducing the number of administered chimeric antigen receptor T cells in a mouse model using a mathematically guided approach.

Chimeric antigen receptor T cell (CAR-T) therapy is a novel approved treatment for hematological malignancies, still under development for solid tumors. Here, we use a rate equation-based mathematical model to discover regimens and schedules that maintain efficacy while potentially reducing toxicity by decreasing the amount of CAR-T infused. Tested on an in vivo murine model of spontaneous breast cancer, we show that our mathematical model accurately recapitulates in vivo tumor growth results achieved in the previous experiments. Moreover, we use the mathematical model to predict results of new therapy schedules and successfully prospectively validated these predictions in the in vivo. We conclude that using one tenth and even one percent of a full CAR-T dose used in preclinical trials can achieve efficacious results similar to full dose treatment.

Nanoparticulate vaccine inhibits tumor growth via improved T cell recruitment into melanoma and huHER2 breast cancer.

Nanoparticulate vaccines are promising tools to overcome cancer immune evasion. However, a deeper understanding on nanoparticle-immune cell interactions and treatments regime is required for optimal efficacy. We provide a comprehensive study of treatment schedules and mode of antigen-association to nanovaccines on the modulation of T cell immunity in vivo, under steady-state and tumor-bearing mice. The coordinated delivery of antigen and two adjuvants (Monophosphoryl lipid A, oligodeoxynucleotide cytosine-phosphate-guanine motifs (CpG)) by nanoparticles was crucial for dendritic cell activation. A single vaccination dictated a 3-fold increase on cytotoxic memory-T cells and raised antigen-specific immune responses against B16.M05 melanoma. It generated at least a 5-fold increase on IFN-γ cytokine production, and presented over 50% higher lymphocyte count in the tumor microenvironment, compared to the control. The number of lymphocytes at the tumor site doubled with triple immunization. This lymphocyte infiltration pattern was confirmed in mammary huHER2 carcinoma, with significant tumor reduction.

Therapeutic Potential of T Cell Chimeric Antigen Receptors (CARs) in Cancer Treatment: Counteracting Off-Tumor Toxicities for Safe CAR T Cell Therapy.

A chimeric antigen receptor (CAR) is a recombinant fusion protein combining an antibody-derived targeting fragment with signaling domains capable of activating T cells. Recent early-phase clinical trials have demonstrated the remarkable ability of CAR-modified T cells to eliminate B cell malignancies. This review describes the choice of target antigens and CAR manipulations to maximize antitumor specificity. Benefits and current limitations of CAR-modified T cells are discussed, with a special focus on the distribution of tumor antigens on normal tissues and the risk of on-target, off-tumor toxicities in the clinical setting. We present current methodologies for pre-evaluating these risks and review the strategies for counteracting potential off-tumor effects. Successful implementation of these approaches will improve the safety and efficacy of CAR T cell therapy and extend the range of cancer patients who may be treated.

Functional comparison of engineered T cells carrying a native TCR versus TCR-like antibody-based chimeric antigen receptors indicates affinity/avidity thresholds.

Adoptive transfer of Ag-specific T lymphocytes is an attractive form of immunotherapy for cancers. However, acquiring sufficient numbers of host-derived tumor-specific T lymphocytes by selection and expansion is challenging, as these cells may be rare or anergic. Using engineered T cells can overcome this difficulty. Such engineered cells can be generated using a chimeric Ag receptor based on common formats composed from Ag-recognition elements such as αß-TCR genes with the desired specificity, or Ab variable domain fragments fused with T cell-signaling moieties. Combining these recognition elements are Abs that recognize peptide-MHC. Such TCR-like Abs mimic the fine specificity of TCRs and exhibit both the binding properties and kinetics of high-affinity Abs. In this study, we compared the functional properties of engineered T cells expressing a native low affinity αß-TCR chains or high affinity TCR-like Ab-based CAR targeting the same specificity. We isolated high-affinity TCR-like Abs recognizing HLA-A2-WT1Db126 complexes and constructed CAR that was transduced into T cells. Comparative analysis revealed major differences in function and specificity of such CAR-T cells or native TCR toward the same antigenic complex. Whereas the native low-affinity αß-TCR maintained potent cytotoxic activity and specificity, the high-affinity TCR-like Ab CAR exhibited reduced activity and loss of specificity. These results suggest an upper affinity threshold for TCR-based recognition to mediate effective functional outcomes of engineered T cells. The rational design of TCRs and TCR-based constructs may need to be optimized up to a given affinity threshold to achieve optimal T cell function.

Suppression of murine colitis and its associated cancer by carcinoembryonic antigen-specific regulatory T cells.

The adoptive transfer of regulatory T cells (Tregs) offers a promising strategy to combat pathologies that are characterized by aberrant immune activation, including graft rejection and autoinflammatory diseases. Expression of a chimeric antigen receptor (CAR) gene in Tregs redirects them to the site of autoimmune activity, thereby increasing their suppressive efficiency while avoiding systemic immunosuppression. Since carcinoembryonic antigen (CEA) has been shown to be overexpressed in both human colitis and colorectal cancer, we treated CEA-transgenic mice that were induced to develop colitis with CEA-specific CAR Tregs. Two disease models were employed: T-cell-transfer colitis as well as the azoxymethane-dextran sodium sulfate model for colitis-associated colorectal cancer. Systemically administered CEA-specific (but not control) CAR Tregs accumulated in the colons of diseased mice. In both model systems, CEA-specific CAR Tregs suppressed the severity of colitis compared to control Tregs. Moreover, in the azoxymethane-dextran sodium sulfate model, CEA-specific CAR Tregs significantly decreased the subsequent colorectal tumor burden. Our data demonstrate that CEA-specific CAR Tregs exhibit a promising potential in ameliorating ulcerative colitis and in hindering colorectal cancer development. Collectively, this study provides a proof of concept for the therapeutic potential of CAR Tregs in colitis patients as well as in other autoimmune inflammatory disorders.

The emergence of T-bodies/CAR T cells.

The nickname "T-body" is used to denote a T cell expressing an antigen-specific or antibody-based chimeric receptor that combines antibody specificity with T-cell effector or regulatory function. Initially, we designed and constructed chimeric antibody-based receptors and expressed them in T cells to study the role of major histocompatibility complex in triggering T-cell activation. To this end, we replaced both variable domains (Vα and Vß of the native T-cell receptor chains) with antibody-derived VH and VL sequences. After transfection into T cells, the 2 chimeric chains paired, associated with the CD3 complex, and endowed transfectants with non-major histocompatibility complex-restricted antibody type specificity. In subsequent studies, we developed next generation of chimeric antibody-based receptors by fusing an antibody single-chain variable fragment to T-cell activation motifs. This modular configuration simplified gene transfer, avoided mixed pairing with endogenous T-cell receptor chains, and enabled simultaneous insertion of various domains as costimulatory moieties to generate T-bodies with efficient antitumor reactivity.

Allogeneic chimeric antigen receptor-modified cells for adoptive cell therapy of cancer.

INTRODUCTION: Chimeric antigen (or antibody) receptors (CAR) are fusion proteins typically combining an antibody-derived targeting fragment with signaling domains capable of activating immune cells. Recent clinical trials have shown the tremendous potential of adoptive cell transfer (ACT) of autologous T cells engineered to express a CD19-specific CAR targeting B-cell malignancies. Building on this approach, ACT therapies employing allogeneic CAR-expressing cytotoxic cells are now being explored. AREAS COVERED: The basic principles of CAR-ACT are introduced. The potential benefits as well as problems of using allogeneic CAR-modified cells against tumor antigens are discussed. Various approaches to allogeneic CAR therapy are presented, including donor leukocyte infusion, CAR-redirected γδ T cells and natural killer cells, strategies to avoid graft-versus-host disease, modulation of lymphocyte migration, and exploitation of graft-versus-host reactivity. EXPERT OPINION: CAR-modified allogeneic cells have the potential to act as universal effector cells, which can be administered to any patient regardless of MHC type. Such universal effector cells could be used as an 'off-the-shelf' cell-mediated treatment for cancer.

Elimination of progressive mammary cancer by repeated administrations of chimeric antigen receptor-modified T cells.

Continuous oncogenic processes that generate cancer require an on-going treatment approach to eliminate the transformed cells, and prevent their further development. Here, we studied the ability of T cells expressing a chimeric antibody-based receptor (CAR) to offer a therapeutic benefit for breast cancer induced by erbB-2. We tested CAR-modified T cells (T-bodies) specific to erbB-2 for their antitumor potential in a mouse model overexpressing a human erbB-2 transgene that develops mammary tumors. Comparing the antitumor reactivity of CAR-modified T cells under various therapeutic settings, either prophylactic, prior to tumor development, or therapeutically. We found that repeated administration of CAR-modified T cells is required to eliminate spontaneously developing mammary cancer. Systemic, as well as intratumoral administered CAR-modified T cells accumulated at tumor sites and eventually eliminated the malignant cells. Interestingly, within a few weeks after a single CAR T cells' administration, and rejection of primary lesion, tumors usually relapsed both in treated mammary gland and at remote sites; however, repeated injections of CAR-modified T cells were able to control the secondary tumors. Since spontaneous tumors can arise repeatedly, especially in the case of syndromes characterized by specific susceptibility to cancer, multiple administrations of CAR-modified T cells can serve to control relapsing disease.

CAR's made it to the pancreas.

Despite intensive treatment, pancreatic adenocarcinoma still has the worst prognosis among all malignancies. Using clinically relevant models, we demonstrated the therapeutic efficacy of adoptively transferred T cells engineered with a carcinoembryonic antigen (CEA)- and ERBB2-specific chimeric antigen receptor against pancreatic carcinoma. Targeting CD24, a putative cancer stem cell antigen expressed by a minority of carcinoma cells, was likewise effective.

Mixed alkanethiol monolayers on submicrometric gold patterns: a controlled platform for studying cell-ligand interactions.

Nanoscale organization of surface ligands often has a critical effect on cell-surface interactions. We have developed an experimental system that allows a high degree of control over the 2-D spatial distribution of ligands. As a proof of concept, we used the developed system to study how T-cell activation is independently affected by antigen density and antigen amount per cell. Arrays of submicrometer gold islands at varying surface coverage were defined on silicon by electron beam lithography (EBL). The gold islands were functionalized with alkanethiol self-assembled monolayers (SAMs) containing a small antigen, 2,4,6-trinotrophenyl (TNP), at various densities. Genetically engineered T-cell hybridomas expressing TNP-specific chimeric T-cell antigen receptor (CAR) were cultured on the SAMs, and their activation was assessed by IL-2 secretion and CD69 expression. It was found that, at constant antigen density, activation increased monotonically with the amount of antigen, while at constant antigen amount activation was maximal at an intermediate antigen density, whose value was independent of the amount of antigen.

Redirected T cells that target pancreatic adenocarcinoma antigens eliminate tumors and metastases in mice.

BACKGROUND & AIMS: Pancreatic adenocarcinoma (PAC) is often diagnosed at an advanced and inoperable stage, and standard systemic treatments are generally ineffective. We investigated the effects of adoptive transfer of tumor-specific T cells that express chimeric antibody-based receptors (CAR) to mice with primary and metastatic PAC xenografts. METHODS: Human effector T cells were genetically modified to express CAR against Her2/neu or CD24, a putative PAC stem cell antigen. The antitumor reactivity of the engineered T cells (T-bodies) was evaluated in SCID mice with different PAC xenografts. A total of 1 × 10(7) T-bodies were injected via the tail vein or directly administered to the subcutaneous tumor on 3 or 4 alternating days. Mice were then given twice-daily intraperitoneal injections of interleukin-2 for 10 days. RESULTS: Intratumor injection of human CD24 and Her2/neu-specific T-bodies completely eliminated the tumors from most animals. Intravenous injection of T-bodies reduced tumor size and prolonged survival of mice with orthotopically transplanted tumors; more than 50% of animals appeared to be disease-free more than 2 months later. Additional systemic administration of T-bodies 8 weeks after the initial injection eliminated primary tumors, along with liver and draining lymph node metastases. A single administration of the Her2/neu-specific T-bodies prolonged the survival of mice with tumors in which most of the cells expressed the target antigen. In contrast, the CD24-specific T-bodies prolonged survival of mice in which only a subpopulation of the tumor cells expressed the antigen. CONCLUSIONS: CAR-redirected T cells stop growth and metastasis of PAC xenografts in mice. T-bodies specific to CD24, a putative cancer stem cell antigen, were effective against PAC xenografts that had only a subset of antigen-expressing cells.

Electrically controlled molecular recognition harnessed to activate a cellular response.

Seamless embedment of electronic devices in biological systems is expected to add the outstanding computing power, memory, and speed of electronics to the biochemical toolbox of nature. Such amalgamation requires transduction of electronic signals into biochemical cues that affect cells. Inspired by biology, where pathways are directed by molecular recognition, we propose and demonstrate a generic electrical-to-biological transducer comprising a two-state electronic antigen and a chimeric cell receptor engineered to bind the antigen exclusively in its "on" state. T-cells expressing these receptors remain inactivated with the antigen in its "off" state. Switching the antigen to its "on" state by an electrical signal leads to its recognition by the T-cells and correspondingly to cell activation.

Tumor-specific allogeneic cells for cancer therapy.

Adoptive cell transfer (ACT) therapy involves transfer of therapeutic lymphocytes to patients mostly for the treatment of cancer and viral infections. One modality to generate therapeutic lymphocytes is to genetically engineer them to express a chimeric antigen receptor (CAR) capable of recognizing the desired target. Current ACT approaches employ the patient's own (syngeneic) lymphocytes, which is both economically and technically challenging. Using foreign (allogeneic) lymphocytes in ACT is problematic because of the severe immunological reaction that occurs between genetically mismatched individuals. However, recently our group has developed a protocol, which allows for safe and effective ACT therapy in a murine model of metastatic disease using allogeneic T cells redirected with a human EGFR2/neuregulin (Her2/neu)-specific CAR. Mild preconditioning of the recipient delayed the rejection of the allogeneic donor T cells such that they had enough time to destroy the tumor, but not enough to cause significant damage to the host. By modulating lymphocyte migration using FTY720, we were actually able to exploit the allogeneic anti-host reaction in order to augment therapeutic benefit while concurrently improving the safety of the treatment. Therefore, we suggest that CAR-based allogeneic ACT therapy could be universally used as a safe and potent 'off-the-shelf' treatment for cancer.