John Maher

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.

Retargeting of Human T Cells to Tumor-Associated MUC1: The Evolution of a Chimeric Antigen Receptor

MUC1 is a highly attractive immunotherapeutic target owing to increased expression, altered glycosylation, and loss of polarity in >80% of human cancers. To exploit this, we have constructed a panel of chimeric Ag receptors (CAR) that bind selectively to tumor-associated MUC1. Two parameters proved crucial in optimizing the CAR ectodomain. First, we observed that the binding of CAR-grafted T cells to anchored MUC1 is subject to steric hindrance, independent of glycosylation status. This was overcome by insertion of the flexible and elongated hinge found in immunoglobulins of the IgD isotype. Second, CAR function was highly dependent upon strong binding capacity across a broad range of tumor-associated MUC1 glycoforms. This was realized by using an Ab-derived single-chain variable fragment (scFv) cloned from the HMFG2 hybridoma. To optimize CAR signaling, tripartite endodomains were constructed. Ultimately, this iterative design process yielded a potent receptor termed HOX that contains a fused CD28/OX40/CD3ζ endodomain. HOX-expressing T cells proliferate vigorously upon repeated encounter with soluble or membrane-associated MUC1, mediate production of proinflammatory cytokines (IFN-γ and IL-17), and elicit brisk killing of MUC1+ tumor cells. To test function in vivo, a tumor xenograft model was derived using MDA-MB-435 cells engineered to coexpress MUC1 and luciferase. Mice bearing an established tumor were treated i.p. with a single dose of engineered T cells. Compared with control mice, this treatment resulted in a significant delay in tumor growth as measured by serial bioluminescence imaging. Together, these data demonstrate for the first time that the near-ubiquitous MUC1 tumor Ag can be targeted using CAR-grafted T cells.

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.

Cytokine overproduction in healthy first degree relatives of patients with IDDM

Summary Healthy family members of patients with insulin-dependent diabetes mellitus (IDDM) are known to share a number of immunological abnormalities with their affected relatives. Since monocyte and type 1 T-cell-derived cytokines contribute to the pathogenesis of IDDM, we studied the production of these cytokines in the healthy first degree relatives of 29 children with IDDM. We report that circulating tumour necrosis factor-α (TNF-α) and soluble interleukin-2 (sIL-2) receptor were present in increased amounts in non-diabetic family members at levels similar to those found in the diabetic children (duration of disease 3 months–5 years). Furthermore, marked hypersecretion of IL-1α and TNF-α by mitogen-stimulated peripheral blood mononuclear cells was found in both diabetic and healthy family members. Abnormalities of cytokine production in healthy relatives did not correlate with the presence of islet cell antibodies or with HLA DR type. These data indicate that healthy family members of patients with IDDM exhibit overproduction of a number of cytokines that have been implicated in diabetogenesis. [Diabetologia (1998) 41: 343–349]

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.

Preclinical In Vivo Modeling of Cytokine Release Syndrome Induced by ErbB-Retargeted Human T Cells: Identifying a Window of Therapeutic Opportunity?

The ErbB network is dysregulated in many solid tumors. To exploit this, we have developed a chimeric Ag receptor (CAR) named T1E28z that targets several pathogenetically relevant ErbB dimers. T1E28z is coexpressed with a chimeric cytokine receptor named 4αβ (combination termed T4), enabling the selective expansion of engineered T cells using IL-4. Human T4+ T cells exhibit antitumor activity against several ErbB+ cancer types. However, ErbB receptors are also expressed in several healthy tissues, raising concerns about toxic potential. In this study, we have evaluated safety of T4 immunotherapy in vivo using a SCID beige mouse model. We show that the human T1E28z CAR efficiently recognizes mouse ErbB+ cells, rendering this species suitable to evaluate preclinical toxicity. Administration of T4+ T cells using the i.v. or intratumoral routes achieves partial tumor regression without clinical or histopathologic toxicity. In contrast, when delivered i.p., tumor reduction is accompanied by dose-dependent side effects. Toxicity mediated by T4+ T cells results from target recognition in both tumor and healthy tissues, leading to release of both human (IL-2/IFN-γ) and murine (IL-6) cytokines. In extreme cases, outcome is lethal. Both toxicity and IL-6 release can be ameliorated by prior macrophage depletion, consistent with clinical data that implicate IL-6 in this pathogenic event. These data demonstrate that CAR-induced cytokine release syndrome can be modeled in mice that express target Ag in an appropriate distribution. Furthermore, our findings argue that ErbB-retargeted T cells can achieve therapeutic benefit in the absence of unacceptable toxicity, providing that route of administration and dose are carefully optimized.

Design of a phase I clinical trial to evaluate intratumoral delivery of ErbB-targeted chimeric antigen receptor T-cells in locally advanced or recurrent head and neck cancer.

Despite several advances, 5-year survival in patients with head and neck squamous cell carcinoma (HNSCC) remains unchanged at only 50%. The commonest cause of death is locally advanced/recurrent disease. Consequently, there is an unmet need for new approaches to improve local control in HNSCC. T4 immunotherapy is an autologous cell therapy in which peripheral blood T-cells are genetically engineered using a retroviral vector to coexpress two chimeric receptors: (i) T1E28z is a chimeric antigen receptor that engages multiple ErbB dimers that are commonly upregulated in HNSCC; (ii) 4αβ is a chimeric cytokine receptor that converts the weak mitogenic stimulus provided by interleukin (IL)-4 into a strong and selective growth signal, allowing preferential expansion and enrichment of T4(+) T-cells ex vivo. T4 immunotherapy exerts antitumor activity against HNSCC cell lines and tumors in vivo, without significant toxicity. Human T4(+) T-cells also engage mouse ErbB receptors, permitting safety testing in SCID Beige mice. Severe toxicity caused by cytokine release syndrome ensues when human T4(+) T-cells are administered at high doses to mice, particularly with advanced tumor burdens. However, such toxicity is not required for efficacy and is never seen if T-cells are administered by the intratumoral route. To exploit this, we have designed a first-in-man clinical trial in which T4(+) T-cells are administered to patients with locally advanced/recurrent HNSCC. Cells will be administered at a single sitting to multiple sites around the viable tumor circumference. A 3+3 dose escalation design will be used, starting at 10(7) cells (cohort 1), escalating to 10(9) cells (cohort 5). If maximum tolerated dose remains undefined, cohorts 6/7 will receive either low- or high-dose cyclophosphamide before 10(9) T4(+) T-cells. A panel of routine/in-house assays and imaging techniques will be used to monitor safety, efficacy, perturbation of endogenous antitumor immunity, immunogenicity, and T-cell trafficking.

Immunotherapy of Malignant Disease Using Chimeric Antigen Receptor Engrafted T Cells

Chimeric antigen receptor- (CAR-) based immunotherapy has been under development for almost 25 years, over which period it has progressed from a new but cumbersome technology to an emerging therapeutic modality for malignant disease. The approach involves the genetic engineering of fusion receptors (CARs) that couple the HLA-independent binding of cell surface target molecules to the delivery of a tailored activating signal to host immune cells. Engineered CARs are delivered most commonly to peripheral blood T cells using a range of vector systems, most commonly integrating viral vectors. Preclinical refinement of this approach has proceeded over several years to the point that clinical testing is now being undertaken at several centres, using increasingly sophisticated and therapeutically successful genetic payloads. This paper considers several aspects of the pre-clinical and clinical development of CAR-based immunotherapy and how this technology is acquiring an increasing niche in the treatment of both solid and haematological 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.

Targeting cytotoxic T lymphocytes for cancer immunotherapy

In light of their preeminent role in cellular immunity, there is considerable interest in targeting of cytotoxic T-lymphocytes to cancer. This review summarises the active and passive immunotherapeutic approaches under development to achieve this goal, emphasising how recent advances in tumour immunology and gene transfer have impacted upon this field.