Sophie Burbridge

Dual Targeting of ErbB2 and MUC1 in Breast Cancer Using Chimeric Antigen Receptors Engineered to Provide Complementary Signaling

PurposeChimeric antigen receptor (CAR) engineered T-cells occupy an increasing niche in cancer immunotherapy. In this context, CAR-mediated CD3ζ signaling is sufficient to elicit cytotoxicity and interferon-γ production while the additional provision of CD28-mediated signal 2 promotes T-cell proliferation and interleukin (IL)-2 production. This compartmentalisation of signaling opens the possibility that complementary CARs could be used to focus T-cell activation within the tumor microenvironment.MethodsHere, we have tested this principle by co-expressing an ErbB2- and MUC1-specific CAR that signal using CD3ζ and CD28 respectively. Stoichiometric co-expression of transgenes was achieved using the SFG retroviral vector containing an intervening Thosea asigna peptide.ResultsWe found that “dual-targeted” T-cells kill ErbB2+ tumor cells efficiently and proliferate in a manner that requires co-expression of MUC1 and ErbB2 by target cells. Notably, however, IL-2 production was modest when compared to control CAR-engineered T-cells in which signaling is delivered by a fused CD28 + CD3ζ endodomain.ConclusionsThese findings demonstrate the principle that dual targeting may be achieved using genetically targeted T-cells and pave the way for testing of this strategy in vivo.

Selective Expansion of Chimeric Antigen Receptor-targeted T-cells with Potent Effector Function using Interleukin-4

Polyclonal T-cells can be directed against cancer using transmembrane fusion molecules known as chimeric antigen receptors (CARs). Although preclinical studies have provided encouragement, pioneering clinical trials using CAR-based immunotherapy have been disappointing. Key obstacles are the need for robust expansion ex vivo followed by sustained survival of infused T-cells in patients. To address this, we have developed a system to achieve selective proliferation of CAR+ T-cells using IL-4, a cytokine with several pathophysiologic and therapeutic links to cancer. A chimeric cytokine receptor (4αβ) was engineered by fusion of the IL-4 receptor α (IL-4Rα) ectodomain to the βc subunit, used by IL-2 and IL-15. Addition of IL-4 to T-cells that express 4αβ resulted in STAT3/STAT5/ERK phosphorylation and exponential proliferation, mimicking the actions of IL-2. Using receptor-selective IL-4 muteins, partnering of 4αβ with γc was implicated in signal delivery. Next, human T-cells were engineered to co-express 4αβ with a CAR specific for tumor-associated MUC1. These T-cells exhibited an unprecedented capacity to elicit repeated destruction of MUC1-expressing tumor cultures and expanded through several logs in vitro. Despite prolonged culture in IL-4, T-cells retained specificity for target antigen, type 1 polarity, and cytokine dependence. Similar findings were observed using CARs directed against two additional tumor-associated targets, demonstrating generality of application. Furthermore, this system allows rapid ex vivo expansion and enrichment of engineered T-cells from small blood volumes, under GMP-compliant conditions. Together, these findings provide proof of principle for the development of IL-4-enhanced T-cell immunotherapy of cancer.

Trafficking of CAR-Engineered Human T Cells Following Regional or Systemic Adoptive Transfer in SCID Beige Mice

Adoptive immunotherapy using chimeric antigen receptor-engrafted T cells is a promising emerging therapy for cancer. Prior to clinical testing, it is mandatory to evaluate human therapeutic cell products in meaningful in vivo pre-clinical models. Here, we describe the use of fused single-photon emission CT–CT imaging to monitor real-time migration of chimeric antigen receptor-engineered T cells in immune compromised (SCID Beige) mice. Following intravenous administration, human T cells migrate in a highly similar manner to that reported in man, but penetrate poorly into established tumors. By contrast, when delivered via intraperitoneal or subcutaneous routes, T cells remain at the site of inoculation with minimal systemic absorption—irrespective of the presence or absence of tumor. Together, these data support the validity of pre-clinical testing of human T-cell immunotherapy in SCID Beige mice. In light of their established efficacy, regional administration of engineered human T cells represents an attractive therapeutic option to minimize toxicity in the treatment of selected malignancies.

Flexible targeting of ErbB dimers that drive tumorigenesis by using genetically engineered T cells.

Pharmacological targeting of individual ErbB receptors elicits antitumor activity, but is frequently compromised by resistance leading to therapeutic failure. Here, we describe an immunotherapeutic approach that exploits prevalent and fundamental mechanisms by which aberrant upregulation of the ErbB network drives tumorigenesis. A chimeric antigen receptor named T1E28z was engineered, in which the promiscuous ErbB ligand, T1E, is fused to a CD28 + CD3ζ endodomain. Using a panel of ErbB-engineered 32D hematopoietic cells, we found that human T1E28z+ T cells are selectively activated by all ErbB1-based homodimers and heterodimers and by the potently mitogenic ErbB2/3 heterodimer. Owing to this flexible targeting capability, recognition and destruction of several tumor cell lines was achieved by T1E28z+ T cells in vitro, comprising a wide diversity of ErbB receptor profiles and tumor origins. Furthermore, compelling antitumor activity was observed in mice bearing established xenografts, characterized either by ErbB1/2 or ErbB2/3 overexpression and representative of insidious or rapidly progressive tumor types. Together, these findings support the clinical development of a broadly applicable immunotherapeutic approach in which the propensity of solid tumors to dysregulate the extended ErbB network is targeted for therapeutic gain.

Chemotherapy for the treatment of hormone-refractory prostate cancer.

Aims:  This review aims at analysing the published literature on chemotherapy for hormone‐refractory prostate cancer (HRPC) to provide recommendations for the treatment of this important patient group.

Clinical investigation of the role of Interleukin-4 and Interleukin-13 in the evolution of Prostate Cancer

Prostate cancer is the most common cancer in men, both in the USA and Europe. Although incurable, metastatic disease can often be controlled for years with anti-androgen therapy. Once the disease becomes castrate resistant, the median survival is 18 months. There is growing evidence that the immune system, and in particular cytokines, play an important role in prostate cancer immunosurveillance and progression. Here, we have undertaken a clinical investigation of the role of two closely related cytokines, IL-4 and IL-13 in prostate cancer. In the largest series studied to date, we show that serum IL-4, but not IL-13 is significantly elevated in castrate resistant, compared to androgen sensitive disease. Notably however, serum IL-4 levels are also raised in patients with benign prostatic disease. Analysis of benign and malignant prostate tissue demonstrates that the source of IL-4 is epithelial cells rather than infiltrating leukocytes. Together, our data are consistent with a dual role for IL-4 in prostate cancer development. In benign disease, our data add to the evidence that IL-4 serves a protective role. By contrast, the data support a direct role for IL-4 in the progression of prostate cancer from androgen responsive, to advanced castrate-resistant disease.

Role of lymphotoxin-a and interleukin-17A in human prostate cancer.

212 Background: Infiltrating immune cells have been implicated in prostate carcinogenesis. Murine studies suggest that B-cell-derived lymphotoxin (LT)a may be a key effector in the evolution to castrate-resistant prostate cancer (CRPC) (Ammirante et al, Nature 2011). Lymphotoxin a has also been implicated in the progression of murine fibrosarcoma, where it complements interleukin (IL)-17A, to synergistically enhance NFkB signaling. This interaction may also apply to human prostate cancer since IL-17-producing Th17 cells are abundant in these tumors. However, the role of LTα in human as distinct from murine prostate cancer remains unclear. Clinical validation is vital since murine models poorly recapitulate the behavior of prostate cancer in man. METHODS Serum levels of LTα and IL-17A were measured by ELISA in patients with benign (n=22) and malignant prostate disease (n=87). Samples were from 29 early prostate cancer patients (amenable to radical therapy), 28 with androgen-sensitive (AS) disease and 30 with CRPC. Data were not normally distributed and thus were analyzed by Mann-Whitney U test. RESULTS Serum LTαand IL-17A were significantly elevated in patients with both CRPC and early stage prostate cancer, compared to those with benign disease. LTαwas significantly raised in CRPC compared to AS disease. IL-17A was significantly higher in the early stage disease compared to AS prostate cancer. CONCLUSIONS Based upon these data, we hypothesize that B-cell derived LTα synergises with Th17 derived IL-17A from the earliest stage of primary tumor development. In support of this, blockade of LTα in prostate cancer-prone (TRAMP) mice delays primary tumor onset and inhibits metastasis. Furthermore, NSAIDs protect more strongly against lifetime risk of prostate cancer in those individuals with an LTα over-producer polymorphism. Prostate cancer preferentially metastasizes to local lymph nodes and bone - sites at which mature B-cells are plentiful. Consequently, tumor-trophic effects of LTα would become increasingly efficient with disease progression. Our data are incompatible with and thus refute the clinical relevance of a role for androgen ablation in promoting LTα-driven CRPC.

Abstract 1932: Targeting the extended erbb receptor family using chimeric antigen receptor (CAR)-grafted t-cells as a treatment for head and neck cancer

Over-expression of the epidermal growth factor receptor (ErbB1) is found in the majority of squamous cell carcinomas of the head and neck (SCCHN), making it an attractive therapeutic target. However, tumors often become refractory to ErbB1-specific therapies by upregulating other ErbB receptors. In keeping with this, increased expression of additional ErbB kinases is associated with poorer prognosis in SCCHN. Consequently, we hypothesized that simultaneous targeting of multiple ErbB receptors would represent a more robust therapeutic strategy. This hypothesis has been tested using adoptive T-cell therapy, in which primary human T-cells have been genetically manipulated to express a chimeric antigen receptor (CAR) named T28z. The broadened ErbB-targeting capacity of T28z is mediated by a chimeric peptide derived from transforming growth factor-α and EGF, known as T1E. Signaling is provided by a fused CD28+CD3ζ endodomain, ensuring full T-cell activation. To characterize the ErbB dimers recognized by T28z+ T-cells, co-cultivation experiments were performed with a panel of transfected 32D hematopoietic cells that express all ErbB receptors, alone or in pairs. We observed that a wide range of ErbB dimers are targeted, including those containing ErbB1 and the profoundly mitogenic ErbB2/ErbB3 heterodimer. T28z-grafted T-cells underwent activation on all of 13 SCCHN monolayers (displaying a range of ErbB expression profiles), when compared to T-cells expressing a control CAR with a truncated endodomain (T1NA). Importantly, T-cell activation was accompanied by tumor cell destruction, cytokine release and proliferation. Minimal T-cell activation was seen on control monolayers that express low ErbB levels. To study the in-vivo efficacy and toxicity of T28z-expressing T-cells, SCID/Beige mice were inoculated intraperitoneally (ip) with the SCCHN cell line, HN3, engineered to express firefly luciferase. Mice with established tumor were treated ip after 13 days with PBS or with 2×107 human T-cells that express T28z or T1NA. Tumor status was evaluated weekly using bioluminescent imaging (BLI). Compared to control groups, mice treated with T28z+ T-cells showed markedly delayed tumor growth and remain alive with minimal BLI signal >100 days post treatment. No toxicity was observed despite the capacity of human ErbB ligands (contained within T1E) to engage murine ErbB receptors. These data show that simultaneous targeting of the extended ErbB family may be achieved using a single T-cell population and that these cells are capable of inducing tumor regression in-vivo without untoward toxicity. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1932.

Genetic engineering of pharmacologically regulated T cells, specific for breast cancer target antigens

Peripheral blood T cells can be genetically targeted against cancer using fusion receptors known as chimeric antigen receptors (CARs). Many preclinical studies have provided great encouragement for this approach. However, pioneering clinical trials have been less successful and identified poor T-cell survival in patients as a crucial limiting factor. To address this, what is needed is a system to achieve selective expansion of tumour-specific effector T cells, both in vitro and in vivo. Here, we describe such an approach using IL-4, a pharmaceutical that has been tested in cancer patients and which is normally a poor mitogen for T cells. A chimeric cytokine receptor named 4αβ was engineered in which the IL-4 receptor α (IL-4Rα) ectodomain was fused to the shared βc subunit, used by IL-2/IL-15. Addition of IL-4 to 4αβ-expressing T cells resulted in selective phosphorylation of STAT3/STAT5/ERK, mimicking the actions of IL-2 or IL-15. Using receptor-selective IL-4 muteins, partnering of 4αβ with γc was implicated in these findings. Next, human T cells were engineered to co-express 4αβ with CARs specific for two breast cancer targets: MUC1 or the extended ErbB family. These T cells exhibited an unprecedented capacity to undergo IL-4-dependent expansion in vitro and repeatedly destroyed breast cancer cultures, greatly exceeding the performance of IL-2-stimulated cells. Importantly, 4αβ-expressing T cells retained cytolytic specificity for target antigen and dependence upon IL-4 (or IL-2) for survival. We have also used this system to achieve rapid IL-4-driven ex vivo expansion and enrichment of CAR+ human T cells in bags (T-bags). Experiments were performed under closed and pseudo-good manufacturing practice conditions, scaling up for phase 1 clinical trials. T cells expanded in this manner demonstrate Th1 polarisation and potent tumour destructive activity, both in vitro and in vivo, in tumour-bearing SCID Beige mice. Together, these findings provide proof of principle for the development of pharmacologically regulated T-cell immunotherapy for breast and other cancers.

Abstract C242: Development of adoptive‐T‐cell therapy for prostate cancer incorporating a novel chimeric cytokine receptor

Introduction: Prostate cancer is the most common cancer in men. Standard therapy incorporates surgery, radiotherapy, anti‐androgen therapy and chemotherapy. Once anti‐androgen resistance occurs, median survival is only 18 months. Consequently, there is a pressing need for novel therapies. One promising strategy involves genetic re‐targeting of T‐cells using chimeric antigen receptor (CAR) technology. These fusion receptors are directed against native tumor antigens, thereby bypassing the need for either HLA expression or antigen processing. In prostate cancer, prostate specific membrane antigen (PSMA) represents a highly attractive target for this approach. However, one key limitation of this strategy is poor longevity of reinfused T‐cells. While parenteral interleukin‐2 (IL‐2) may help to circumvent this, it is not selective for engineered T‐cells and is frequently highly toxic. To address this need, we have developed a novel chimeric receptor, which enables T‐cells to receive an IL‐2 intracellular signal in response to IL‐4. Methods: The PSMA specific CAR, P28z was expressed in human T‐cells and the murine IL‐2 dependent cell line CTLL2 using the oncoretroviral vector SFG. A fusion receptor of IL4‐R and the intracellular domain of IL‐2R (MOHAIR) was co‐expressed with P28z using the T2A system. CAR grafted T‐cells were co‐cultured with antigen positive and negative tumor cell lines. Proliferation of CTLL2 cells or human T‐cells was determined by trypan exclusion. Production of IFN was measured by ELISA. Results: To test the function of MOHAIR, initial studies were performed using the IL‐2‐dependent CTLL2 cell line. We observed that all MOHAIR‐expressing CTLL2 cell lines proliferated equally well in human IL‐4 or IL‐2, unlike control cells which only proliferated in IL‐2. CTLL2s expressing MOHAIR and secreting IL4 became cytokine independent Next we tested function using primary human T‐cells. P28z and P28z‐MOHAIR (MOP) transduced T‐cells destroyed PSMA expressing prostate cancer cell lines and release IFN in a strictly antigen‐specific manner. Both T‐cell populations proliferated following multiple stimulations on PSMA positive cell lines in the presence of IL‐2. By contrast, these T‐cells died upon re‐stimulated in the absence of antigen or IL‐2. Uniquely, MOP transduced T‐cells proliferated over multiple antigen stimulations in the presence of IL‐4, whereas P28z transduced T‐cells died rapidly when cultured in IL‐4. When grown in the absence of antigen, MOP T‐cells expand preferentially in IL‐4, compared to MOP or P28z T‐cells grown in IL‐2. Conclusions: P28z confers PSMA antigen specificity on transduced human T‐cells. Mohair enables delivery of a potent IL‐2 intracellular signal in response to the weakly mitogenic cytokine, IL‐4. This provides a convenient system to achieve selective expansion of PSMA retargeted human T‐cells in response to exogenous IL‐4. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C242.