Based on analyses of patient tumor samples, ovarian carcinomas appear to be relatively immunogenic tumors. Specifically, researchers have found a direct correlation between prognosis following surgery and chemotherapy and the quantity of TILs in pretreatment tumor samples (25
). Furthermore, others have described an inverse correlation between prognosis following therapy and pre-treatment levels of Tregs within the tumor, which in turn presumably inhibit the anti-tumor function of tumor specific effector TILs (26
). Both of these findings imply a role for an endogenous effector T cell response to tumor in controlling disease progression both prior to and following initial therapy and strongly support the contention that ovarian carcinomas may be susceptible to killing by adoptive infusion of autologous T cells targeted to ovarian tumor cell antigens.
While endogenous effector TILs are one source for presumably tumor specific T cells, an alternative approach to adoptive T cell therapy is to isolate autologous peripheral blood T cells which in turn may be genetically modified ex vivo
to target tumor cell antigens. One such genetic approach is to retrovirally transduce patient T cells with CARs targeted to surface exposed antigens either unique to or over-expressed by the tumor. To this end, promising preclinical studies utilizing this approach in other malignancies have recently been translated to the clinical setting (6
Application of this approach to ovarian carcinomas requires the identification of suitable target antigens expressed on the tumor cell surface. To this end, other investigators have studied this approach in vivo
in the pre-clinical setting utilizing CAR+
T cells targeted to disparate antigens over-expressed on ovarian carcinomas including the α-folate receptor, the Lewis Y antigen, Her2/neu, NKG2D ligands (MICA, MICB, and UL-16 binding proteins), mesothelin, and MUC-1 (4
). Specifically, in an elegant series of studies utilizing a syngeneic orthotopic 1D8 tumor model of ovarian carcinoma in C57BL6 mice, Barber et al demonstrate efficient eradication of well established i.p. tumors when treated with i.p. injections of syngeneic murine T cells modified to express the chNKG2D CAR (54
). Furthermore, Hwu et al demonstrated significant delays in tumor progression in immune compromised nude mice bearing orthotopic human IGROV tumors following a single infusion of murine T cells modified to express an α-folate receptor-targeted CAR (4
). In the xenogeneic setting, several groups have demonstrated delayed tumor progression or complete anti-tumor responses of subcutaneous human ovarian carcinoma cell lines in immune compromised mice following intratumoral and/or intravenous infusion of human T cells expressing CARs specific to the Her2/neu and Lewis-Y antigens (59
). Similarly, Wilkie et al and Carpentino et al have recently published reports demonstrating efficient anti-tumor efficacy in xenotransplant models of human breast and mesothelioma tumors treated with human T cells modified respectively with CARs targeted to the MUC-1 and mesothelin antigens, antigens also over-expressed on ovarian carcinomas(57
In the clinical setting, Kershaw et al recently published the results of a phase I dose escalation trial treating patients with relapsed ovarian carcinomas with autologous T cells modified to express a first generation CAR specific to the α-folate receptor (6
). The authors of this study found that therapy with targeted T cells was well tolerated, but noted a lack of anti-tumor response in these studies related to poor persistence of modified T cells over time as well as a yet undefined T cell inhibitory factor in the serum of several treated patients.
In our studies, we have chosen to target the MUC-16 glycoprotein which is over-expressed on a majority of ovarian carcinomas (1
). The utility of MUC-16 as a target antigen for adoptive T cell therapy is compromised by the fact that most of the extracellular portion of this molecule is cleaved by the tumor cell, secreted, and may be detected in the serum as the CA-125 tumor marker. However, following cleavage of this secreted fraction of MUC-16, there remains a residual extracellular fraction of the glycoprotein, termed MUC-CD, which is retained on the tumor cell surface and is therefore an attractive target for immune-based therapies. To this end, we utilized a murine hybridoma, 4H11, generated to the MUC-CD antigen (38
) to construct a first generation (4H11z) as well as a second generation co-stimulatory CAR (4H11-28z) specific to MUC-CD. Significantly, the antigen to the 4H11 antibody is highly expressed on a majority of pre-treatment ovarian carcinoma tumor samples obtained from patients treated at our institution as assessed by immunohistochemistry (38
Consistent with previous studies, we found that T cells transduced to express the second generation 4H11-28z CAR, but not the first generation 4H11z CAR, efficiently expanded upon co-culture with 3T3(MUC-CD) fibroblasts in the absence of exogenous co-stimulation. This conclusion is further supported by the finding that 4H11-28z+ T cells secreted significantly higher levels of IL-2, a cytokine indicative of T cell co-stimulation, and IFNγ upon co-culture on 3T3(MUC-CD) fibroblasts when compared to T cells transduced to express the first generation 4H11z CAR.
Specificity of the 4H11-28z CAR to the MUC-CD antigen was subsequently verified in vitro by comparing 4H11-28z+ T cell cytotoxicity and proliferation on a series of MUC-CD− ovarian carcinoma cell lines, as well as HeLA cells and the T80 immortalized normal ovarian surface epitherlial cell line, to the same cell lines further genetically modified to express the MUC-CD antigen. In order to additionally validate the clinical relevance of our findings, we subsequently demonstrated specific in vitro lysis of primary ascites-derived tumor cells isolated from untreated ovarian carcinoma patients by both healthy donor allogeneic 4H11-28z+ T cells and, more significantly, by autologous 4H11-28z+ patient peripheral blood T cells. These data strongly support the contention that treatment with autologous 4H11-based CAR+ T cells have promise in future clinical applications.
In order to assess the in vivo relevance of our in vitro findings, we next generated several orthotopic human ovarian cancer tumor models in SCID-Beige mice. These studies demonstrated eradication of early as well as more established BLI evident peritoneal tumors following i.p. injection of healthy donor 4H11-28z+ T cells. In the setting of delayed therapy, tumor imaging by BLI could demonstrate initial marked eradication of disease. However, loss of bioluminescent signal in these studies did not preclude future relapse of disease since a majority of apparently tumor free mice, as assessed by BLI at earlier time points of follow-up, developed relapsed disease within the peritoneum over time, consistent with the notion that BLI lacks the sensitivity to measure in vivo minimal residual disease in demonstrated persistent expression of the targeted MUC-CD antigen consistent with the notion that loss of target antigen expression by the tumor, or immune selection of MUC-CD− tumors was not the cause of tumor relapse. While the source of relapsed disease in these mice remains speculative, studies of i.p. infused 4H11-28z+ T cell persistence demonstrate rapidly declining numbers of T cells over time. Although modified T cells were still detectable in peritoneal washes out to 28 days post T cell infusion, these numbers were declining, suggesting a loss of modified T cell persistence as a potential source of disease relapse which typically occurred at later time points.
We further demonstrate trafficking of i.v. injected MUC-CD targeted T cells to peritoneal tumors by FACS. Significantly, tumor bearing mice treated with i.v. infused 4H11-28z+ T cells exhibited similar anti-tumor efficacy when compared to i.p. treated mice as assessed by combined survival data from 2 separate experiments using stratified log-rank analysis.
While insightful, these xenotransplant murine tumor models have significant limitations. Specifically, the biology of human T cells in immune compromised mice may significantly differ from autologous modified patient T cells in the clinical setting wherein these T cells encounter an intact immune system which may elicit an immune response to the CAR, and enter into a hostile tumor microenvironment containing immune suppressive regulatory T cells, immune inhibitory cytokines, including IL-10 and TGFβ, and myeloid derived suppressor cells (MDSCs) (64
). To address these limitations, we are currently generating a more clinically relevant syngeneic immune competent murine tumor model of ovarian carcinoma to further study the in vivo
biology, immunogenicity, and anti-tumor efficacy of MUC-CD targeted T cells.
A further limitation of xenotransplant models is the inability of these studies to address potential unforeseen off-target toxicities which may occur in the clinical setting wherein infused CAR-modified T cells recognize antigen not only on tumor cells but also on normal tissues. Based on previously published adverse events in recent clinical trials utilizing CAR-modified T cells, this is a very real concern with respect to the clinical feasibility of this adoptive T cell approach to cancer therapy. Specifically, three patients with metastatic renal carcinoma treated with autologous T cells transduced to express the G250 CAR specific to the TAA carboxyanhydrase IX (CAIX) developed significant liver toxicity (CTC grade 4 in patient 1, grade 2 in patient 2 and grade 3 in patient 3) due to an off target modified T cell response to CAIX expressed by bile duct epithelial cells (20
). More recently, Morgan et al reported the death of a patient with metastatic colon cancer treated with ERBB2 targeted autologous 4D5-CD8-28BBz+
T cells. Following an extensive post-mortem analysis, the investigators of this study postulate that the patient’s death resulted from off target recognition of the targeted ERBB2 antigen expressed by normal lung tissue resulting in marked modified T cell release of inflammatory cytokines, including TNF-α and IFN-γ, leading to pulmonary toxicity, edema, and a subsequent cascading cytokine storm resulting in multi-organ failure and death (67
While similar concerns may be raised in the setting of treating patients with autologous T cells targeted to the MUC-CD antigen, we have conducted extensive immunohistochemistry studies using the 4H11 antibody to assess for off target binding to normal human tissues. Significantly, these studies demonstrated no binding of the 4H11 MAb in normal adult colon, rectum, small intestine, ectocervix, ovary, liver, pancreatic ducts, spleen, kidney, brain and skin tissues. However, 4H11 did weakly bind cytoplasm of endocervical gland cells and thymic corpuscles, the luminal surface of esophageal glands, as well as intracytoplasmic granules of bronchial epithelium and gastric glands (38
). Significantly, these studies failed to demonstrate membrane bound antigen on the vascular surfaces of any normal human tissues. Nevertheless, the potential for off target toxicity in the clinical setting are not precluded by these studies. To this end, we acknowledge the likely requirement that further safety measures be added in the form of additional T cell modification with a suicide gene vector in order to enhance safety in the clinical setting.
In conclusion, herein we present the first published data demonstrating the feasibility of targeting MUC-16, an antigen over-expressed on a majority of ovarian carcinomas, through adoptive therapy with genetically modified T cells targeted to the retained MUC-CD portion of the MUC-16 antigen. Further, this report is the first to demonstrate efficient targeting of T cells in an orthotopic murine model of ovarian cancer, demonstrating efficacy of a single T cell infusion of modified T cells in the absence of exogenous IL-2 cytokine support. Collectively, these data support the further planned translation of this approach to the clinical setting in the form of a phase I clinical trial in patients with persistent or relapsed ovarian carcinomas following initial therapy with surgery and chemotherapy.
Statement of Translational Relevance
Ovarian carcinomas appear to be immunogenic tumors based on the fact that increased numbers of tumor infiltrating T cells (TILs) present in the pre-treatment tumor is associated with an enhanced survival following surgery and chemotherapy. Patient T cells may be genetically modified to recognize tumor associated antigens (TAAs) through the introduction of chimeric antigen receptors (CARs) specifically targeted to these antigens. We have generated a CAR, 4H11-28z, specific to the retained extracellular portion (MUC-CD) of MUC-16, a glycoprotein over expressed on most ovarian carcinomas. We demonstrate that T cells, when modified to express the 4H11-28z CAR, specifically lyse human ovarian cancer cells in vitro and further demonstrate efficient anti-tumor efficacy in orthotopic xenotransplant tumor models. Collectively, these data support the translation of these preclinical findings to the clinical setting in a planned phase 1 trial treating patients with relapsed or refractory ovarian cancer.