Stephen Gottschalk

Human Epidermal Growth Factor Receptor 2 (HER2) –Specific Chimeric Antigen Receptor–Modified T Cells for the Immunotherapy of HER2-Positive Sarcoma

PURPOSE The outcome for patients with metastatic or recurrent sarcoma remains poor. Adoptive therapy with tumor-directed T cells is an attractive therapeutic option but has never been evaluated in sarcoma. PATIENTS AND METHODS We conducted a phase I/II clinical study in which patients with recurrent/refractory human epidermal growth factor receptor 2 (HER2) -positive sarcoma received escalating doses (1 × 10(4)/m(2) to 1 × 10(8)/m(2)) of T cells expressing an HER2-specific chimeric antigen receptor with a CD28.ζ signaling domain (HER2-CAR T cells). RESULTS We enrolled 19 patients with HER2-positive tumors (16 osteosarcomas, one Ewing sarcoma, one primitive neuroectodermal tumor, and one desmoplastic small round cell tumor). HER2-CAR T-cell infusions were well tolerated with no dose-limiting toxicity. At dose level 3 (1 × 10(5)/m(2)) and above, we detected HER2-CAR T cells 3 hours after infusion by quantitative polymerase chain reaction in 14 of 16 patients. HER2-CAR T cells persisted for at least 6 weeks in seven of the nine evaluable patients who received greater than 1 × 10(6)/m(2) HER2-CAR T cells (P = .005). HER2-CAR T cells were detected at tumor sites of two of two patients examined. Of 17 evaluable patients, four had stable disease for 12 weeks to 14 months. Three of these patients had their tumor removed, with one showing ≥ 90% necrosis. The median overall survival of all 19 infused patients was 10.3 months (range, 5.1 to 29.1 months). CONCLUSION This first evaluation of the safety and efficacy of HER2-CAR T cells in patients with cancer shows the cells can persist for 6 weeks without evident toxicities, setting the stage for studies that combine HER2-CAR T cells with other immunomodulatory approaches to enhance their expansion and persistence.

Sustained Complete Responses in Patients With Lymphoma Receiving Autologous Cytotoxic T Lymphocytes Targeting Epstein-Barr Virus Latent Membrane Proteins

PURPOSE Tumor cells from approximately 40% of patients with Hodgkin or non-Hodgkin lymphoma express the type II latency Epstein-Barr virus (EBV) antigens latent membrane protein 1 (LMP1) and LMP2, which represent attractive targets for immunotherapy. Because T cells specific for these antigens are present with low frequency and may be rendered anergic by the tumors that express them, we expanded LMP-cytotoxic T lymphocytes (CTLs) from patients with lymphoma using autologous dendritic cells and EBV-transformed B-lymphoblastoid cell lines transduced with an adenoviral vector expressing either LMP2 alone (n = 17) or both LMP2 and ΔLMP1 (n = 33). PATIENTS AND METHODS These genetically modified antigen-presenting cells expanded CTLs that were enriched for specificity against type II latency LMP antigens. When infused into 50 patients with EBV-associated lymphoma, the expanded CTLs did not produce infusional toxicities. RESULTS Twenty-eight of 29 high-risk or multiple-relapse patients receiving LMP-CTLs as adjuvant therapy remained in remission at a median of 3.1 years after CTL infusion. None subsequently died as a result of lymphoma, but nine succumbed to complications associated with extensive prior chemoradiotherapy, including myocardial infarction and secondary malignancies. Of 21 patients with relapsed or resistant disease at the time of CTL infusion, 13 had clinical responses, including 11 complete responses. T cells specific for LMP as well as nonviral tumor-associated antigens (epitope spreading) could be detected in the peripheral blood within 2 months after CTL infusion, but this evidence for epitope spreading was seen only in patients achieving clinical responses. CONCLUSION Autologous T cells directed to the LMP2 or LMP1 and LMP2 antigens can induce durable complete responses without significant toxicity. Their earlier use in the disease course may reduce delayed treatment-related mortality.

TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy

Targeted T cells are emerging as effective non-toxic therapies for cancer. Multiple elements, however, contribute to the overall pathogenesis of cancer through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy. We created a functional chimeric antigen receptor—the TanCAR, a novel artificial molecule that mediates bispecific activation and targeting of T cells. We demonstrate the feasibility of cumulative integration of structure and docking simulation data using computational tools to interrogate the design and predict the functionality of such a complex bispecific molecule. Our prototype TanCAR induced distinct T cell reactivity against each of two tumor restricted antigens, and produced synergistic enhancement of effector functions when both antigens were simultaneously encountered. Furthermore, the TanCAR preserved the cytolytic ability of T cells upon loss of one of the target molecules and better controlled established experimental tumors by recognition of both targets in an animal disease model. This proof-of-concept approach can be used to increase the specificity of effector cells for malignant versus normal target cells, to offset antigen escape or to allow for targeting the tumor and its microenvironment.

Rapidly Generated Multivirus-specific Cytotoxic T Lymphocytes for the Prophylaxis and Treatment of Viral Infections

Severe and fatal viral infections remain common after hematopoietic stem cell transplantation. Adoptive transfer of cytotoxic T lymphocytes (CTLs) specific for Epstein–Barr virus (EBV), cytomegalovirus (CMV), and adenoviral antigens can treat infections that are impervious to conventional therapies, but broader implementation and extension to additional viruses is limited by competition between virus-derived antigens and time-consuming and laborious manufacturing procedures. We now describe a system that rapidly generates a single preparation of polyclonal (CD4+ and CD8+) CTLs that is consistently specific for 15 immunodominant and subdominant antigens derived from 7 viruses (EBV, CMV, Adenovirus (Adv), BK, human herpes virus (HHV)-6, respiratory syncytial virus (RSV), and Influenza) that commonly cause post-transplant morbidity and mortality. CTLs can be rapidly produced (10 days) by a single stimulation of donor peripheral blood mononuclear cells (PBMCs) with a peptide mixture spanning the target antigens in the presence of the potent prosurvival cytokines interleukin-4 (IL4) and IL7. This approach reduces the impact of antigenic competition with a consequent increase in the antigenic repertoire and frequency of virus-specific T cells. Our approach can be readily introduced into clinical practice and should be a cost-effective alternative to common antiviral prophylactic agents for allogeneic hematopoietic stem cell transplant (HSCT) recipients.

T Cells Redirected to EphA2 for the Immunotherapy of Glioblastoma

Outcomes for patients with glioblastoma (GBM) remain poor despite aggressive multimodal therapy. Immunotherapy with genetically modified T cells expressing chimeric antigen receptors (CARs) targeting interleukin (IL)-13Rα2, epidermal growth factor receptor variant III (EGFRvIII), or human epidermal growth factor receptor 2 (HER2) has shown promise for the treatment of gliomas in preclinical models and in a clinical study (IL-13Rα2). However, targeting IL-13Rα2 and EGFRvIII is associated with the development of antigen loss variants, and there are safety concerns with targeting HER2. Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) has emerged as an attractive target for the immunotherapy of GBM as it is overexpressed in glioma and promotes its malignant phenotype. To generate EphA2-specific T cells, we constructed an EphA2-specific CAR with a CD28-ζ endodomain. EphA2-specific T cells recognized EphA2-positive glioma cells as judged by interferon-γ (IFN-γ) and IL-2 production and tumor cell killing. In addition, EphA2-specific T cells had potent activity against human glioma-initiating cells preventing neurosphere formation and destroying intact neurospheres in coculture assays. Adoptive transfer of EphA2-specific T cells resulted in the regression of glioma xenografts in severe combined immunodeficiency (SCID) mice and a significant survival advantage in comparison to untreated mice and mice treated with nontransduced T cells. Thus, EphA2-specific T-cell immunotherapy may be a promising approach for the treatment of EphA2-positive GBM.

Adoptive Transfer of EBV-specific T Cells Results in Sustained Clinical Responses in Patients With Locoregional Nasopharyngeal Carcinoma

Patients with recurrent or refractory Epstein Barr Virus (EBV)-positive nasopharyngeal carcinoma (NPC) continue to have poor outcomes. Our earlier Phase I dose escalation clinical study of 10 NPC patients showed that infusion of EBV-specific cytotoxic T cells (EBV-CTLs) was safe and had antitumor activity. To better define the overall response rate and discover whether disease status, EBV-antigen specificity, and/or in vivo expansion of infused EBV-CTLs predicted outcome, we treated 13 additional NPC patients with EBV-CTLs in a fixed-dose, Phase II component of the study. We assessed toxicity, efficacy, specificity, and expansion of infused CTLs for all 23 recurrent/refractory NPC patients treated on this Phase I/II clinical study. At the time of CTL infusion, 8 relapsed NPC patients were in remission and 15 had active disease. No significant toxicity was observed. Of the relapsed patients treated in their second or subsequent remission, 62% (5/8) remain disease free (at 17 to 75 mo), whereas 48.7% (7/15) of those with active disease had a CR/CRu (33.3%) or PR (15.4%). In contrast to locoregional disease, metastatic disease was associated with an increased risk of disease progression (HR: 3.91, P=0.015) and decreased overall survival (HR: 5.55, P=0.022). Neither the specificity of the infused CTLs for particular EBV antigens nor their measurable in vivo expansion discernibly influenced outcome. In conclusion, treatment of patients with relapsed/refractory EBV-positive NPC with EBV-CTLs is safe and can be associated with significant, long-term clinical benefit, particularly for patients with locoregional disease.

Characterization and treatment of chronic active Epstein-Barr virus disease: a 28-year experience in the United States

Chronic active EBV disease (CAEBV) is a lymphoproliferative disorder characterized by markedly elevated levels of antibody to EBV or EBV DNA in the blood and EBV RNA or protein in lymphocytes in tissues. We present our experience with CAEBV during the last 28 years, including the first 8 cases treated with hematopoietic stem cell transplantation in the United States. Most cases of CAEBV have been reported from Japan. Unlike CAEBV in Japan, where EBV is nearly always found in T or natural killer (NK) cells in tissues, EBV was usually detected in B cells in tissues from our patients. Most patients presented with lymphadenopathy and splenomegaly; fever, hepatitis, and pancytopenia were common. Most patients died of infection or progressive lymphoproliferation. Unlike cases reported from Japan, our patients often showed a progressive loss of B cells and hypogammaglobulinemia. Although patients with CAEBV from Japan have normal or increased numbers of NK cells, many of our patients had reduced NK-cell numbers. Although immunosuppressive agents, rituximab, autologous cytotoxic T cells, or cytotoxic chemotherapy often resulted in short-term remissions, they were not curative. Hematopoietic stem cell transplantation was often curative for CAEBV, even in patients with active lymphoproliferative disease that was unresponsive to chemotherapy. These studies are registered at http://www.clinicaltrials.gov as NCT00032513 for CAEBV, NCT00062868 and NCT00058812 for EBV-specific T-cell studies, and NCT00578539 for the hematopoietic stem cell transplantation protocol.

Safety and clinical efficacy of rapidly-generated trivirus-directed T cells as treatment for adenovirus, EBV, and CMV infections after allogeneic hematopoietic stem cell transplant

Adoptive transfer of virus-specific T cells can prevent and treat serious infections with Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenovirus (Adv) after allogeneic hematopoietic stem cell transplant. It has, however, proved difficult to make this approach widely available since infectious virus and viral vectors are required for T cell activation, followed by an intensive and prolonged culture period extending over several months. We now show that T cells targeting a range of viral antigens derived from EBV, CMV, and Adv can be reproducibly generated in a single culture over a 2-3-week period, using methods that exclude all viral components and employ a much-simplified culture technology. When administered to recipients of haploidentical (n = 5), matched unrelated (n = 3), mismatched unrelated (n = 1) or matched related (n = 1) transplants with active CMV (n = 3), Adv (n = 1), EBV (n = 2), EBV+Adv (n = 2) or CMV+Adv (n = 2) infections, the cells produced complete virological responses in 80%, including all patients with dual infections. In each case, a decrease in viral load correlated with an increase in the frequency of T cells directed against the infecting virus(es); both immediate and delayed toxicities were absent. This approach should increase both the feasibility and applicability of T cell therapy. The trial was registered at www.clinicaltrials.gov as NCT01070797.

Regression of Experimental Medulloblastoma following Transfer of HER2-Specific T Cells

Medulloblastoma is a common malignant brain tumor of childhood. Human epidermal growth factor receptor 2 (HER2) is expressed by 40% of medulloblastomas and is a risk factor for poor outcome with current aggressive multimodal therapy. In contrast to breast cancer, HER2 is expressed only at low levels in medulloblastomas, rendering monoclonal antibodies ineffective. We determined if T cells grafted with a HER2-specific chimeric antigen receptor (CAR; HER2-specific T cells) recognized and killed HER2-positive medulloblastomas. Ex vivo, stimulation of HER2-specific T cells with HER2-positive medulloblastomas resulted in T-cell proliferation and secretion of IFN-gamma and interleukin 2 (IL-2) in a HER2-dependent manner. HER2-specific T cells killed autologous HER2-positive primary medulloblastoma cells and medulloblastoma cell lines in cytotoxicity assays, whereas HER2-negative tumor cells were not killed. No functional difference was observed between HER2-specific T cells generated from medulloblastoma patients and healthy donors. In vivo, the adoptive transfer of HER2-specific T cells resulted in sustained regression of established medulloblastomas in an orthotopic, xenogenic severe combined immunodeficiency model. In contrast, delivery of nontransduced T cells did not change the tumor growth pattern. Adoptive transfer of HER2-specific T cells may represent a promising immunotherapeutic approach for medulloblastoma.

Tandem CAR T cells targeting HER2 and IL13Rα2 mitigate tumor antigen escape

In preclinical models of glioblastoma, antigen escape variants can lead to tumor recurrence after treatment with CAR T cells that are redirected to single tumor antigens. Given the heterogeneous expression of antigens on glioblastomas, we hypothesized that a bispecific CAR molecule would mitigate antigen escape and improve the antitumor activity of T cells. Here, we created a CAR that joins a HER2-binding scFv and an IL13Rα2-binding IL-13 mutein to make a tandem CAR exodomain (TanCAR) and a CD28.ζ endodomain. We determined that patient TanCAR T cells showed distinct binding to HER2 or IL13Rα2 and had the capability to lyse autologous glioblastoma. TanCAR T cells exhibited activation dynamics that were comparable to those of single CAR T cells upon encounter of HER2 or IL13Rα2. We observed that TanCARs engaged HER2 and IL13Rα2 simultaneously by inducing HER2-IL13Rα2 heterodimers, which promoted superadditive T cell activation when both antigens were encountered concurrently. TanCAR T cell activity was more sustained but not more exhaustible than that of T cells that coexpressed a HER2 CAR and an IL13Rα2 CAR, T cells with a unispecific CAR, or a pooled product. In a murine glioblastoma model, TanCAR T cells mitigated antigen escape, displayed enhanced antitumor efficacy, and improved animal survival. Thus, TanCAR T cells show therapeutic potential to improve glioblastoma control by coengaging HER2 and IL13Rα2 in an augmented, bivalent immune synapse that enhances T cell functionality and reduces antigen escape.