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The adoptive transfer of genetically engineered T cells expressing chimeric antigen receptors (CARs) has emerged as a transformative cancer therapy with curative potential, precipitating a wave of preclinical and clinical studies in academic centers and the private sector. Indeed, significant effort has been devoted to improving clinical benefit by incorporating accessory genes/CAR endodomains designed to enhance cellular migration, promote in vivo expansion/persistence or enhance safety by genetic programming to enable the recognition of a tumor signature. However, our efforts centered on exploring whether CAR Tcell potency could be enhanced by modifying pre-existing CAR components. We now demonstrate how molecular refinements to the CAR spacer can impact multiple biological processes including tonic signaling, cell aging, tumor localization, and antigen recognition, culminating in superior in vivo antitumor activity.

The success of adoptively transferred tumor-directed T cells requires them to survive and expand in vivo. Most tumors, however, employ immune evasion mechanisms, including the production of inhibitory cytokines that limit in vivo T-cell persistence and effector function. To protect tumor-directed T cells from such negative influences, we generated a chimeric cytokine receptor in which the interleukin (IL) 4 receptor exodomain was fused to the IL7 receptor endodomain. We thereby inverted the effects of tumor-derived IL4 so that the proliferation and activation of tumor directed cytotoxic T cells was enhanced rather than inhibited in the tumor microenvironment, resulting in superior antitumor activity. These transgenic T cells were only activated in the tumor environment since triggering required exposure to both tumor antigen (signal 1) and tumor-derived IL4 (signal 2). This selectivity supports future clinical adaptation.

Intraflagellar Transport (IFT) refers to a highly conserved process occurring in eukaryotic ciliated structures. In vertebrate photoreceptors, IFT mediates protein trafficking to the outer segments. The IFT particle is a multi-subunit complex and mutations in many individual components causes photoreceptor defects. In zebrafish, mutations in the ift57, ift88 and ift172 genes result in retinal degeneration by 5 days post fertilization (dpf). Although the effects of these mutations on photoreceptor survival have been described, early developmental morphogenesis remains poorly understood. We used transmission electron microscopy and immunohistochemistry to examine these mutants at 60, 72, and 96 hours post fertilization (hpf) and describe early photoreceptor morphogenesis defects.

BackgroundThe adoptive transfer of T cells redirected to tumor via chimeric antigen receptors (CARs) has produced clinical benefits for the treatment of hematologic diseases. To extend this approach to breast cancer, we generated CAR T cells directed against mucin1 (MUC1), an aberrantly glycosylated neoantigen that is overexpressed by malignant cells and whose expression has been correlated with poor prognosis. Furthermore, to protect our tumor-targeted cells from the elevated levels of immune-inhibitory cytokines present in the tumor milieu, we co-expressed an inverted cytokine receptor linking the IL4 receptor exodomain with the IL7 receptor endodomain (4/7ICR) in order to transform the suppressive IL4 signal into one that would enhance the anti-tumor effects of our CAR T cells at the tumor site.MethodsFirst (1G - CD3ζ) and second generation (2G - 41BB.CD3ζ) MUC1-specific CARs were constructed using the HMFG2 scFv. Following retroviral transduction transgenic expression of the CAR±ICR was assessed by flow cytometry. In vitro CAR/ICR T cell function was measured by assessing cell proliferation and short- and long-term cytotoxic activity using MUC1+ MDA MB 468 cells as targets. In vivo anti-tumor activity was assessed using IL4-producing MDA MB 468 tumor-bearing mice using calipers to assess tumor volume and bioluminescence imaging to track T cells.ResultsIn the IL4-rich tumor milieu, 1G CAR.MUC1 T cells failed to expand or kill MUC1+ tumors and while co-expression of the 4/7ICR promoted T cell expansion, in the absence of co-stimulatory signals the outgrowing cells exhibited an exhausted phenotype characterized by PD-1 and TIM3 upregulation and failed to control tumor growth. However, by co-expressing 2G CAR.MUC1 (signal 1 - activation + signal 2 - co-stimulation) and 4/7ICR (signal 3 - cytokine), transgenic T cells selectively expanded at the tumor site and produced potent and durable tumor control in vitro and in vivo.ConclusionsOur findings demonstrate the feasibility of targeting breast cancer using transgenic T cells equipped to thrive in the suppressive tumor milieu and highlight the importance of providing transgenic T cells with signals that recapitulate physiologic TCR signaling – [activation (signal 1), co-stimulation (signal 2) and cytokine support (signal 3)] - to promote in vivo persistence and memory formation.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0347-5) contains supplementary material, which is available to authorized users.

The adoptive transfer of chimeric antigen receptor (CAR)-modified T cells has produced tumor responses even in patients with refractory diseases. However, the paucity of antigens that are tumor selective has resulted, on occasion, in "on-target, off-tumor" toxicities. To address this issue, we developed an approach to render T cells responsive to an expression pattern present exclusively at the tumor by using a trio of novel chimeric receptors. Using pancreatic cancer as a model, we demonstrate how T cells engineered with receptors that recognize prostate stem cell antigen, TGFβ, and IL4, and whose endodomains recapitulate physiologic T-cell signaling by providing signals for activation, costimulation, and cytokine support, produce potent antitumor effects selectively at the tumor site. In addition, this strategy has the benefit of rendering our cells resistant to otherwise immunosuppressive cytokines (TGFβ and IL4) and can be readily extended to other inhibitory molecules present at the tumor site (e.g., PD-L1, IL10, and IL13). This proof-of-concept study demonstrates how sophisticated engineering approaches can be utilized to both enhance the antitumor efficacy and increase the safety profile of transgenic T cells by incorporating a combination of receptors that ensure that cells are active exclusively at the tumor site. .

BackgroundMajor limiting factors for cell therapy in solid tumors include clonal heterogeneity and the related lack of universally expressed tumor-specific antigens. As the only truly polyclonal cell product in advanced development, tumor infiltrating lymphocytes (TILs) offer the broadest diversity of tumor reactivity but can be limited by suboptimal effector function in situ. Here we present a novel platform designed to leverage the diverse TCR repertoire of TILs while amplifying their anti-tumor activity via a synthetic costimulatory antigen receptor (CoStAR).MethodsA CoStAR molecule encoding an extracellular folate receptor alpha (FOLR1)-targeting single-chain fragment variable (scFv) and intracellular CD28 and CD40 signaling sequences was transduced into peripherally harvested T cells (healthy donor) and primary ovarian cancer TILs. Coculture experiments were performed with engineered cell lines and autologous tumor digests expressing varying levels TCR-stimulus and/or FOLR1. Cytolytic activity, activation markers, proliferation and cytokine secretion were measured.ResultsAnti-FOLR1 CoStAR T cells activated with TCR stimulation and FOLR1 displayed increased activation markers (eg, CD137, > 50% increase), improved cytolytic activity, cytokine production (eg, IL-2, >15-fold) and ~7-fold increased proliferation when compared to either unmodified cells or when stimulated via TCR alone. Importantly, no T cell effector function, as measured by cytolytic activity, cytokine secretion, upregulation of activation markers or proliferation, was observed when CoStAR T cells were cocultured with targets expressing only FOLR1, underscoring the costimulation-only mechanism of action and reliance on native TCR repertoire for tumor recognition. Furthermore, increased activity was also observed when primary ovarian cancer anti-FOLR1 CoStAR TILs were cocultured with autologous tumor.ConclusionsCoStAR is a novel platform that leverages synthetic biology and T-cell-intrinsic circuits to create a product with markedly increased functional activity including cytotoxicity, proliferation, and cytokine expression while retaining broad, patient-specific neoantigen recognition to limit both antigen escape and off-tumor toxicity. Instil plans to initiate its first-in-human clinical trial with ITIL-306, an investigational anti-FOLR1 CoStAR TIL product in 1H 2022. Additional scFv targets are being evaluated for clinical application across a broad range of solid tumor histologies.

Introduction: Referred to as a “liquid tumor biopsy”, the ability to enrich Circulating Tumor Cells (CTCs) from blood samples allows for the analysis of cancer cells undergoing metastatic dissemination and has been a hallmark of biomarker discovery. However, the use of EpCAM-based enrichment platforms limits the type of tumor cells that can be recovered, which subsequently limits the use of CTCs as surrogates for the classical tumor biopsy. Therefore an EpCAM-independent enrichment platform is critically needed. Specific Aims: The performance of a novel antibody-independent dielectrophoretic field-flow fractionation based CTC isolation technology ApoStream was demonstrated in spiked cell model and cancer patient blood. Experimental Procedures: We used ApoStream, a novel antibody-independent technology, to enrich CTCs from prostate and breast cancer patient blood (high EpCAM expression), non-small cell lung cancer patient blood (NSCLC, low EpCAM expression) and melanoma (no EpCAM expression). Cells isolated from ApoStream were stained for cytokeratin (CK), CD45, and DAPI, and melanoma CTCs with tumor marker S100, CD45 and DAPI. Imaging and CTC enumeration were done using laser scanning cytometry (LSC). CTC morphology in lung cancer specimens was confirmed with H&E staining. To further demonstrate performance of ApoStream platform, EpCAM-negative ovarian cancer cells SKOV3 were spiked into peripheral blood mononuclear cells (PBMCs) from normal donor blood and isolated using the ApoStream device. Results: High CTC recovery from cancer patient blood samples was achieved with counts ranging from 0 − 2104 (lung, n=33), 0 − 3490 (prostate, n=15), 176 − 968 (breast, n=3), and 4 − 3120 (melanoma, n=11) CTCs per 7.5 ml blood. Positive CTC counts were obtained in ninety percent of NSCLC samples, 93% of prostate cancer samples, 100% breast cancer and melanoma specimens. There were no false-positive CTCs from normal donor blood controls demonstrating ApoStream's specificity. In cell spiking experiments, 80 ± 3% cells were recovered by ApoStream after spiking of EpCAM-negative ovarian cancer cells (SKOV3) into human peripheral blood mononuclear cells (PBMCs). EpCAM-negative CTCs were also recovered in all tested breast cancer patient specimens, confirming that ApoStream isolates cancer cells independent on their EpCAM status. Conclusions: Our data demonstrate that the ApoStream platform recovers large numbers of CTCs from the blood of patients with metastatic NSCLC, prostate, breast cancer and melanoma. Thus, the ApoStream CTCisolation platform provides a new effective tool with broad applications in cancer biomarker discovery and personalization of cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B20.