Sjoukje J. C. van der Stegen

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.

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.

Synergistic Chemoimmunotherapy of Epithelial Ovarian Cancer Using ErbB-Retargeted T Cells Combined with Carboplatin

Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy, underscoring the need for better therapies. Adoptive immunotherapy using genetically targeted T cells represents a promising new treatment for hematologic malignancies. However, solid tumors impose additional obstacles, including the lack of suitable targets for safe systemic therapy and the need to achieve effective T cell homing to sites of disease. Because EOC undergoes transcœlomic metastasis, both of these challenges may be circumvented by T cell administration to the peritoneal cavity. In this study, we describe such an immunotherapeutic approach for EOC, in which human T cells were targeted against the extended ErbB family, using a chimeric Ag receptor named T1E28z. T1E28z was coexpressed with a chimeric cytokine receptor named 4αβ (combination termed T4), enabling the selective ex vivo expansion of engineered T cells using IL-4. Unlike control T cells, T4+ T cells from healthy donors and patients with EOC were activated by and destroyed ErbB+ EOC tumor cell lines and autologous tumor cultures. In vivo antitumor activity was demonstrated in mice bearing established luciferase-expressing SKOV-3 EOC xenografts. Tumor regression was accompanied by mild toxicity, manifested by weight loss. Although efficacy was transient, therapeutic response could be prolonged by repeated T cell administration. Furthermore, prior treatment with noncytotoxic doses of carboplatin sensitized SKOV-3 tumors to T4 immunotherapy, promoting enhanced disease regression using lower doses of T4+ T cells. By combining these approaches, we demonstrate that repeated administration of carboplatin followed by T4+ T cells achieved optimum therapeutic benefit in the absence of significant toxicity, even in mice with advanced tumor burdens.

Adoptive Immunotherapy of Epithelial Ovarian Cancer with Vγ9Vδ2 T Cells, Potentiated by Liposomal Alendronic Acid

Adoptive immunotherapy using γδ T cells harnesses their natural role in tumor immunosurveillance. The efficacy of this approach is enhanced by aminobisphosphonates such as zoledronic acid and alendronic acid, both of which promote the accumulation of stimulatory phosphoantigens in target cells. However, the inefficient and nonselective uptake of these agents by tumor cells compromises the effective clinical exploitation of this principle. To overcome this, we have encapsulated aminobisphosphonates within liposomes. Expanded Vγ9Vδ2 T cells from patients and healthy donors displayed similar phenotype and destroyed autologous and immortalized ovarian tumor cells, following earlier pulsing with either free or liposome-encapsulated aminobisphosphonates. However, liposomal zoledronic acid proved highly toxic to SCID Beige mice. By contrast, the maximum tolerated dose of liposomal alendronic acid was 150-fold higher, rendering it much more suited to in vivo use. When injected into the peritoneal cavity, free and liposomal alendronic acid were both highly effective as sensitizing agents, enabling infused γδ T cells to promote the regression of established ovarian tumors by over one order of magnitude. Importantly however, liposomal alendronic acid proved markedly superior compared with free drug following i.v. delivery, exploiting the “enhanced permeability and retention effect” to render advanced tumors susceptible to γδ T cell–mediated shrinkage. Although folate targeting of liposomes enhanced the sensitization of folate receptor–α+ ovarian tumor cells in vitro, this did not confer further therapeutic advantage in vivo. These findings support the development of an immunotherapeutic approach for ovarian and other tumors in which adoptively infused γδ T cells are targeted using liposomal alendronic acid.

CAR T-cell immunotherapy of MET-expressing malignant mesothelioma

ABSTRACT Mesothelioma is an incurable cancer for which effective therapies are required. Aberrant MET expression is prevalent in mesothelioma, although targeting using small molecule-based therapeutics has proven disappointing. Chimeric antigen receptors (CARs) couple the HLA-independent binding of a cell surface target to the delivery of a tailored T-cell activating signal. Here, we evaluated the anti-tumor activity of MET re-targeted CAR T-cells against mesothelioma. Using immunohistochemistry, MET was detected in 67% of malignant pleural mesotheliomas, most frequently of epithelioid or biphasic subtype. The presence of MET did not influence patient survival. Candidate MET-specific CARs were engineered in which a CD28+CD3ζ endodomain was fused to one of 3 peptides derived from the N and K1 domains of hepatocyte growth factor (HGF), which represents the minimum MET binding element present in this growth factor. Using an NIH3T3-based artificial antigen-presenting cell system, we found that all 3 candidate CARs demonstrated high specificity for MET. By contrast, these CARs did not mediate T-cell activation upon engagement of other HGF binding partners, namely CD44v6 or heparan sulfate proteoglycans, including Syndecan-1. NK1-targeted CARs demonstrated broadly similar in vitro potency, indicated by destruction of MET-expressing mesothelioma cell lines, accompanied by cytokine release. In vivo anti-tumor activity was demonstrated following intraperitoneal delivery to mice with an established mesothelioma xenograft. Progressive tumor regression occurred without weight loss or other clinical indicators of toxicity. These data confirm the frequent expression of MET in malignant pleural mesothelioma and demonstrate that this can be targeted effectively and safely using a CAR T-cell immunotherapeutic strategy.

Abstract CT118: T4 immunotherapy of head and neck squamous cell carcinoma using pan-ErbB targeted CAR T-cells

Striking progress has been achieved in CD19+ hematologic malignancies using chimeric antigen receptor (CAR) T-cells following lymphodepletion. Nonetheless, toxicity remains significant, due to cytokine release syndrome (CRS) and neurologic dysfunction. The frequent emergence of resistance due to antigen loss provides a strong rationale for engagement of multiple targets. Solid tumors impose additional challenges. Foremost, a paucity of targets that are tumor-specific, or restricted to dispensable tissues. Moreover, active CAR T-cells need to home to, penetrate and persist within profoundly immunosuppressive tumors. Cognizant of these obstacles, we designed T4 immunotherapy. T4+ T-cells are retroviral transduced to co-express (i) T1E28ζ, a CAR coupling a promiscuous ErbB ligand derived from EGF and TGFα to a fused CD28+CD3ζ endodomain; and (ii) 4αβ, a chimeric cytokine receptor containing the IL-4Rα ectodomain coupled to the IL-2Rβ endodomain. T1E28ζ engages 8/9 possible ErbB dimers, providing broad anti-tumor activity while minimizing risk of antigen escape. 4αβ enables IL-4-driven selective enrichment and expansion of CAR T-cells during manufacture. Pre-clinical data demonstrate potent anti-tumor activity in head and neck squamous cell carcinoma (HNSCC), mesothelioma, ovarian and breast cancer. However, risk of on-target off-tumor toxicity is significant, due to low-level ErbB expression in normal tissues. Indeed, CRS can be modeled when human T4+ T-cells are administered to the peritoneal cavity of SCID Beige mice. To de-risk T4 immunotherapy in man, a dose-escalation intra-tumoral Phase I trial was commenced, without lymphodepletion. HNSCC was selected due to the unmet need presented by locally advanced or recurrent disease. Ninety percent of patients were lymphopenic yet T4 immunotherapy was successfully generated from a 130mL blood draw, in a closed manufacturing process. Batches contained up to 7.5 x 109 cells, of which 63.8+ 12.1% were T4+, comprising a variable mixture of central and effector memory CD4+ and CD8+ T-cells. Cohorts of 1, 3 and 10 x 107 T4+ T-cells were treated. Patient 5 died of advanced HNSCC, prior to treatment. Intra-tumoral 1-2mL injections of T4 immunotherapy were administered as a single dose. Treatment-related AEs were Citation Format: Sophie Papa, Antonella Adami, Michael Metoudi, Daniela Achkova, May van Schalkwyk, Ana Parente Pereira, Leticia Bosshard-Carter, Lynsey Whilding, Sjoukje van der Stegen, David M. Davies, Teresa Guerrero-Urbano, Jean Pierre Jeannon, James Spicer, John Maher. T4 immunotherapy of head and neck squamous cell carcinoma using pan-ErbB targeted CAR T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT118. doi:10.1158/1538-7445.AM2017-CT118

Abstract A075: Immunotherapy of malignant pleural mesothelioma using cMET specific T-cells

Background: Malignant pleural mesothelioma (MPM) is an incurable cancer predominantly induced by asbestos exposure. This aggressive tumour commonly presents at an advanced stage of disease. Although surgery, combination chemotherapy and radiotherapy may be used in disease management, median survival from diagnosis is less than 12 months. New therapeutic approaches are required to improve outcome in this disease. Chimeric antigen receptors (CAR) are fusion molecules that couple the HLA- independent binding of a selected cell surface target antigen to the delivery of a tailored T-cell activating signal. Using CAR technology, we aim to develop a novel immunotherapy for MPM that is both safe and effective. These molecules are delivered to patient T-cells using retroviral gene-transfer, thereby stably re-directing their specificity for antigen. The receptor tyrosine kinase MET is overexpressed in >80% of MPM patients making it an attractive candidate for CAR directed immunotherapy. Methods: Three second-generation cMet-targeted CARs (containing a fused CD28/CD3ζ endodomain) have been developed, distinguished by peptide-binding motifs. Functionality of MET-re targeted T-cells has been assessed by co-cultivation of genetically enhanced test and control T-cells with MPM cell lines that express MET at various levels. Results: Studies to date have shown that human T-cells engineered to express cMet CARs can destroy mesothelioma tumour cell monolayers, accompanied by T-cell proliferation and cytokine production. To further potentiate the activity of the cMet targeted CARs, a dual targeting approach to both cMet and the ErbB family is currently under evaluation. The second advantage of this approach is in diminishing the potential toxicity risks associate with “on target, off tumour” toxicity. An MPM xenograft model has been established utilising bioluminescence to further evaluate functionality and toxicity in vivo. Conclusions: These findings demonstrate proof of concept for the utility of CAR engineered T-cells to recognise and destroy cMet-expressing MPM tumour cells. We envisage that regional i.e. intrapleural delivery of human CAR positive T-cells could be used to maximise therapeutic index of this approach. Citation Format: Thivyan Thayaparan, Sjoukje J.C. van der Stegen, Ana C. Parente Pereira Puri, Roseanna Maria Petrovic, James Spicer, John Maher. Immunotherapy of malignant pleural mesothelioma using cMET specific T-cells. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A075.

Abstract 2309: T-cell development from T cell-derived induced pluripotent stem cell

The ability to differentiate T cells of defined specificity and function from T-cell derived induced pluripotent stem cells (TiPSC) may be useful for the treatment of a range of pathologies, including cancer, infection and immune deficits. We recently reported that genetic engineering of TiPSC to express a Chimeric Antigen Receptor (CAR) is an effective strategy to combine the unlimited availability of TiPSC and facilitate reprogramming of the T cell specificity and functional potential. In vivo, CD19-retargeted human TiPS-derived CAR T cells (CARTiPSC-T) display therapeutic potency in a lymphoma model. Surprisingly, despite expression of the endogenous αβ TCR, the CARTiPSC-T cells possessed innate-like phenotype and function, most similar to γδ T cells. Although innate T cells have anti-tumor activity, they lack some vital features for therapeutic efficacy including long-term memory and in vivo persistence, which characterize mature CD8+ and CD4+ αβ TCR T cells. Additional research into the mechanisms underlying in vitro T cell differentiation of TiPSC is required to improve the development of mature TCRαβ T cells and facilitate the employment of their therapeutic potential. T cell lineage determination depends in part on the balance between Notch and TCR signaling, we are investigating their respective roles, as well as that of the CAR, in determining lineage commitment. We hypothesize that the combined CAR and early CD3/TCRαβ expression disrupts the TCR/Notch signaling balance, prohibiting mature TCRαβ T cell development. To study the effects of the different Notch ligands, TiPSC were differentiated on OP9 cells expressing one of four Notch ligands (DLL1, DLL4, Jagged-1 or Jagged-2). Preliminary data suggests that DLL4 is able to facilitate T cell development to CD4/CD8 double positive (DP) stage, however, this is hindered by CAR expression. To determine the role of early TCRαβ expression, elimination of TCRαβ expression was facilitated by targeted disruption of the TCRα constant region using the CRISPR/Cas9 system. Elimination of early TCRαβ expression showed improved development towards DP stage of TiPSC on OP9-DLL1. TiPSC are a valuable system for the study of human T cell differentiation. In addition, and most importantly they are further amenable to genetic engineering with TCRs or CARs, which may be useful for the generation of therapeutic “off-the-shelf”, antigen-specific T cells. Citation Format: Sjoukje J.C. van der Stegen, Maria Themeli, Justin Eyquem, Jorge Mansilla-Soto, Michel Sadelain. T-cell development from T cell-derived induced pluripotent stem cell. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2309.