Brenda Aguilar

T cells expressing CD123-specific chimeric antigen receptors exhibit specific cytolytic effector functions and antitumor effects against human acute myeloid leukemia.

Induction treatments for acute myeloid leukemia (AML) have remained largely unchanged for nearly 50 years, and AML remains a disease of poor prognosis. Allogeneic hematopoietic cell transplantation can achieve cures in select patients and highlights the susceptibility of AML to donor-derived immunotherapy. The interleukin-3 receptor α chain (CD123) has been identified as a potential immunotherapeutic target because it is overexpressed in AML compared with normal hematopoietic stem cells. Therefore, we developed 2 chimeric antigen receptors (CARs) containing a CD123-specific single-chain variable fragment, in combination with a CD28 costimulatory domain and CD3-ζ signaling domain, targeting different epitopes on CD123. CD123-CAR-redirected T cells mediated potent effector activity against CD123+ cell lines as well as primary AML patient samples. CD123 CAR T cells did not eliminate granulocyte/macrophage and erythroid colony formation in vitro. Additionally, T cells obtained from patients with active AML can be modified to express CD123 CARs and are able to lyse autologous AML blasts in vitro. Finally, CD123 CAR T cells exhibited antileukemic activity in vivo against a xenogeneic model of disseminated AML. These results suggest that CD123 CAR T cells are a promising immunotherapy for the treatment of high-risk AML.

Recognition and Killing of Brain Tumor Stem-Like Initiating Cells by CD8+ Cytolytic T Cells

Solid tumors contain a subset of stem-like cells that are resistant to the cytotoxic effects of chemotherapy/radiotherapy, but their susceptibility to cytolytic T lymphocyte (CTL) effector mechanisms has not been well characterized. Using a panel of early-passage human brain tumor stem/initiating cell (BTSC) lines derived from high-grade gliomas, we show that BTSCs are subject to immunologic recognition and elimination by CD8(+) CTLs. Compared with serum-differentiated CD133(low) tumor cells and established glioma cell lines, BTSCs are equivalent with respect to expression levels of HLA class I and ICAM-1, similar in their ability to trigger degranulation and cytokine synthesis by antigen-specific CTLs, and equally susceptible to perforin-dependent CTL-mediated cytolysis. BTSCs are also competent in the processing and presentation of antigens as evidenced by the killing of these cells by CTL when antigen is endogenously expressed. Moreover, we show that CTLs can eliminate all BTSCs with tumor-initiating activity in an antigen-specific manner in vivo. Current models predict that curative therapies for many cancers will require the elimination of the stem/initiating population, and these studies lay the foundation for developing immunotherapeutic approaches to eradicate this tumor population.

Stem-like tumor-initiating cells isolated from IL13Rα2 expressing gliomas are targeted and killed by IL13-zetakine-redirected T Cells.

Purpose: To evaluate IL13Rα2 as an immunotherapeutic target for eliminating glioma stem–like cancer initiating cells (GSC) of high-grade gliomas, with particular focus on the potential of genetically engineered IL13Rα2-specific primary human CD8+ CTLs (IL13-zetakine+ CTL) to target this therapeutically resistant glioma subpopulation. Experimental Design: A panel of low-passage GSC tumor sphere (TS) and serum-differentiated glioma lines were expanded from patient glioblastoma specimens. These glioblastoma lines were evaluated for expression of IL13Rα2 and for susceptibility to IL13-zetakine+ CTL-mediated killing in vitro and in vivo. Results: We observed that although glioma IL13Rα2 expression varies between patients, for IL13Rα2pos cases this antigen was detected on both GSCs and more differentiated tumor cell populations. IL13-zetakine+ CTL were capable of efficient recognition and killing of both IL13Rα2pos GSCs and IL13Rα2pos differentiated cells in vitro, as well as eliminating glioma-initiating activity in an orthotopic mouse tumor model. Furthermore, intracranial administration of IL13-zetakine+ CTL displayed robust antitumor activity against established IL13Rα2pos GSC TS-initiated orthotopic tumors in mice. Conclusions: Within IL13Rα2 expressing high-grade gliomas, this receptor is expressed by GSCs and differentiated tumor populations, rendering both targetable by IL13-zetakine+ CTLs. Thus, our results support the potential usefullness of IL13Rα2-directed immunotherapeutic approaches for eradicating therapeutically resistant GSC populations. Clin Cancer Res; 18(8); 2199–209. ©2012 AACR.

Tumor-Derived Chemokine MCP-1/CCL2 Is Sufficient for Mediating Tumor Tropism of Adoptively Transferred T Cells

To exert a therapeutic effect, adoptively transferred tumor-specific CTLs must traffic to sites of tumor burden, exit the circulation, and infiltrate the tumor microenvironment. In this study, we examine the ability of adoptively transferred human CTL to traffic to tumors with disparate chemokine secretion profiles independent of tumor Ag recognition. Using a combination of in vivo tumor tropism studies and in vitro biophotonic chemotaxis assays, we observed that cell lines derived from glioma, medulloblastoma, and renal cell carcinoma efficiently chemoattracted ex vivo-expanded primary human T cells. We compared the chemokines secreted by tumor cell lines with high chemotactic activity with those that failed to elicit T cell chemotaxis (Daudi lymphoma, 10HTB neuroblastoma, and A2058 melanoma cells) and found a correlation between tumor-derived production of MCP-1/CCL2 (≥10 ng/ml) and T cell chemotaxis. Chemokine immunodepletion studies confirmed that tumor-derived MCP-1 elicits effector T cell chemotaxis. Moreover, MCP-1 is sufficient for in vivo T cell tumor tropism as evidenced by the selective accumulation of i.v. administered firefly luciferase-expressing T cells in intracerebral xenografts of tumor transfectants secreting MCP-1. These studies suggest that the capacity of adoptively transferred T cells to home to tumors may be, in part, dictated by the species and amounts of tumor-derived chemokines, in particular MCP-1.

Phenotypic and functional attributes of lentivirus-modified CD19-specific human CD8+ central memory T cells manufactured at clinical scale.

A key determinant of the therapeutic potency of adoptive T-cell transfer is the extent to which infused cells can persist and expand in vivo. Ex vivo propagated virus-specific and chimeric antigen receptor (CAR)-redirected antitumor CD8+ effector T cells derived from CD45RA− CD62L+ central memory (TCM) precursors engraft long-term and reconstitute functional memory after adoptive transfer. Here, we describe a clinical scale, closed system, immunomagnetic selection method to isolate CD8+ TCM from peripheral blood mononuclear cells (PBMC). This method uses the CliniMACS device to first deplete CD14+, CD45RA+, and CD4+ cells from PBMC, and then to positively select CD62L+ cells. The average purity and yield of CD8+CD45RA− CD62L+ TCM obtained in full-scale qualification runs were 70% and 0.4% (of input PBMC), respectively. These CD8+ TCM are responsive to anti-CD3/CD28 bead stimulation, and can be efficiently transduced with CAR encoding lentiviral vectors, and undergo sustained expansion in interleukin (IL)-2/IL-15 over 3–6 weeks. The resulting CD8+ TCM-derived effectors are polyclonal, retain expression of CD62L and CD28, exhibit CAR-redirected antitumor effector function, and are capable of huIL-15-dependent in vivo homeostatic engraftment after transfer to immunodeficient NOD/Scid IL-2RgCnull mice. Adoptive therapy using purified TCM cells is now the subject of a Food and Drug Administration-authorized clinical trial for the treatment of CD19+ B-cell malignancies, and 3 clinical cell products expressing a CD19-specific CAR for IND #14645 have already been successfully generated from lymphoma patients using this manufacturing platform.

Imaging Immune Response In vivo : Cytolytic Action of Genetically Altered T Cells Directed to Glioblastoma Multiforme

Purpose: Clinical trials have commenced to evaluate the feasibility of targeting malignant gliomas with genetically engineered CTLs delivered directly to the tumor bed in the central nervous system. The objective of this study is to determine a suite of magnetic resonance imaging (MRI) measurements using an orthotopic xenograft murine model that can noninvasively monitor immunologically mediated tumor regression and reactive changes in the surrounding brain parenchyma. Experimental Design: Our preclinical therapeutic platform is based on CTL genetic modification to express a membrane tethered interleukin-13 (IL-13) cytokine chimeric T-cell antigen receptor. This enables selective binding and signal transduction on encountering the glioma-restricted IL-13 α2 receptor (IL-13Rα2). We used MRI to visualize immune responses following adoptive transfer of IL-13Rα2-specific CD8+ CTL clones. Results: Based on MRI measurements, several phases following IL-13Rα2-specific T-cell adoptive transfer could be distinguished, all of which correlated well with glioblastoma regression confirmed on histology. The first detectable changes, 24 hours post-treatment, were significantly increased T2 relaxation times and strongly enhanced signal on T1-weighted postcontrast images. In the next phase, the apparent diffusion coefficient was significantly increased at 2 and 3 days post-treatment. In the last phase, at day 3 after IL-13Rα2-specific T-cell injection, the volume of hyperintense signal on T1-weighted postcontrast image was significantly decreased, whereas apparent diffusion coefficient remained elevated. Conclusions: The present study indicates the feasibility of MRI to visualize different phases of immune response when IL-13Rα2-specific CTLs are administered directly to the glioma tumor bed. This will further the aim of better predicting clinical outcome following immunotherapy.

Transgene-enforced co-stimulation of CD4+ T cells leads to enhanced and sustained anti-tumor effector functioning

Background The role of co-stimulation in CD4+ T cell activation by professional APC is well established, while less is known of the role co-stimulation plays when CD4+ T cells interact directly with tumor cells. Methods Through genetic engineering of human CD4+ T cells, we tested the hypothesis that integration of co-stimulatory signaling domains within a tumor-targeting chimeric Ag receptor (CAR), the IL-13Ralpha2-specific IL-13-zetakine (IL13zeta), would enhance CD4+ T cell mediated responses against tumors that fail to express ligands for co-stimulatory receptors. Results Compared with CD3zeta-mediated activation alone, CD4+ effector T cells expressing the IL13-CD28-41BBzeta CAR exhibited augmented/sustained MAPK and AKT activity, up-regulated Th1 cytokine production, and enhanced cytolytic potency against tumor targets. Moreover, upon recursive stimulation with tumor, the IL13-CD28-41BBzeta+ cells retained/recycled their lytic function, whereas IL-13zeta+ CD4+ cells became anergic/exhausted. These in vitro observations correlated with enhanced in vivo control of established orthotopic CNS glioma xenografts in immunodeficient mice mediated by adoptively transferred ex vivo-expanded CD4+ T cells expressing the co-stimulatory CAR. Discussion Together these studies demonstrate the importance of integrating co-stimulation with CD3zeta signaling events to activate fully CD4+ anti-tumor effector cells for sustained function in the tumor microenvironment.

Comparison of naïve and central memory derived CD8+ effector cell engraftment fitness and function following adoptive transfer

abstract Human CD8+ effector T cells derived from CD45RO+CD62L+ precursors enriched for central memory (TCM) precursors retain the capacity to engraft and reconstitute functional memory upon adoptive transfer, whereas effectors derived from CD45RO+CD62L− precursors enriched for effector memory precursors do not. Here we sought to compare the engraftment fitness and function of CD8+ effector T cells derived from CD45RA+CD62L+ precursors enriched for naïve and stem cell memory precursors (TN/SCM) with that of TCM. We found that cytotoxic T cells (CTLs) derived from TCM transcribed higher levels of CD28, FOS, INFγ, Eomesodermin (Eomes), and lower levels of BCL2L11, maintained higher levels of phosphorylated AKT, and displayed enhanced sensitivity to the proliferative and anti-apoptotic effects of γ-chain cytokines compared to CTLs derived from TN/SCM. Higher frequencies of CTLs derived from TCM retained CD28 expression and upon activation secreted higher levels of IL-2. In NOD/Scid IL-2RγCnull mice, CD8+ TCM derived CTLs engrafted to higher frequencies in response to human IL-15 and mounted robust proliferative responses to an immunostimulatory vaccine. Similarly, CD8+ TCM derived CD19CAR+ CTLs exhibited superior antitumor potency following adoptive transfer compared to their CD8+ TN/SCM derived counterparts. These studies support the use of TCM enriched cell products for adoptive therapy of cancer.

Regional Delivery of Chimeric Antigen Receptor–Engineered T Cells Effectively Targets HER2+ Breast Cancer Metastasis to the Brain

Purpose: Metastasis to the brain from breast cancer remains a significant clinical challenge, and may be targeted with CAR-based immunotherapy. CAR design optimization for solid tumors is crucial due to the absence of truly restricted antigen expression and potential safety concerns with “on-target off-tumor” activity. Here, we have optimized HER2-CAR T cells for the treatment of breast to brain metastases, and determined optimal second-generation CAR design and route of administration for xenograft mouse models of breast metastatic brain tumors, including multifocal and leptomeningeal disease. Experimental Design: HER2-CAR constructs containing either CD28 or 4-1BB intracellular costimulatory signaling domains were compared for functional activity in vitro by measuring cytokine production, T-cell proliferation, and tumor killing capacity. We also evaluated HER2-CAR T cells delivered by intravenous, local intratumoral, or regional intraventricular routes of administration using in vivo human xenograft models of breast cancer that have metastasized to the brain. Results: Here, we have shown that HER2-CARs containing the 4-1BB costimulatory domain confer improved tumor targeting with reduced T-cell exhaustion phenotype and enhanced proliferative capacity compared with HER2-CARs containing the CD28 costimulatory domain. Local intracranial delivery of HER2-CARs showed potent in vivo antitumor activity in orthotopic xenograft models. Importantly, we demonstrated robust antitumor efficacy following regional intraventricular delivery of HER2-CAR T cells for the treatment of multifocal brain metastases and leptomeningeal disease. Conclusions: Our study shows the importance of CAR design in defining an optimized CAR T cell, and highlights intraventricular delivery of HER2-CAR T cells for treating multifocal brain metastases. Clin Cancer Res; 24(1); 95–105. ©2017 AACR.

Chimeric γc cytokine receptors confer cytokine independent engraftment of human T lymphocytes

Therapeutic responses following adoptive transfer of T cells correlate to levels of long-term T cell persistence. Lymphodepletion and exogenous γc cytokine administration can improve T cell persistence following adoptive transfer, but their effects are not uniform and toxicities are significant. To overcome these limitations, we designed a chimeric γc cytokine receptor (CγCR) composed of Interleukin-7 (IL-7) tethered to IL-7Rα/CD127 that confers exogenous cytokine independent, cell intrinsic, STAT5 cytokine signals. We additionally show that this design is modular in that the IL-2Rβ/CD122 cytoplasmic chain can be exchanged for that of IL-7Rα/CD127, enhancing Shc activity. When expressed in central memory-derived primary human CD8(+) CTL (T(E/CM)), these CγCRs signal according to their corresponding wild-type counterparts to support exogenous cytokine independent viability and homeostatic proliferation, while retaining full effector function. In vivo studies demonstrate that both CγCR-CD127(+) and CγCR-CD122(+) CD8(+) T((E/CM)) engraft in mice and persist in an absence of exogenous cytokine administration. Engrafted CγCR-CD127(+) CD8(+) T(E/CM) preferentially retain central memory marker expression in vivo demonstrating a dichotomy between CD127 versus CD122 signaling. Together, these results suggest that expression of CγCR in therapeutic T cells may aid in the in vivo persistence of these cells, particularly under conditions of limiting homeostatic cytokines.