CAR T Cells Targeting the Tumor MUC1 Glycoprotein Reduce Triple-Negative Breast Cancer Growth

Antibody-derived chimeric antigen receptor (CAR) T cell therapy has achieved gratifying breakthrough in hematologic malignancies but has shown limited success in solid tumor immunotherapy. Monoclonal antibody, TAB004, specifically recognizes the aberrantly glycosylated tumor form of MUC1 (tMUC1) in all subtypes of breast cancer including 95% of triple-negative breast cancer (TNBC) while sparing recognition of normal tissue MUC1. We transduced human T cells with MUC28z, a chimeric antigen receptor comprising of the scFv of TAB004 coupled to CD28 and CD3ζ. MUC28z was well-expressed on the surface of engineered activated human T cells. MUC28z CAR T cells demonstrated significant target-specific cytotoxicity against a panel of human TNBC cells. Upon recognition of tMUC1 on TNBC cells, MUC28z CAR T cells increased production of Granzyme B, IFN-γ and other Th1 type cytokines and chemokines. A single dose of MUC28z CAR T cells significantly reduced TNBC tumor growth in a xenograft model. Thus, MUC28z CAR T cells have high therapeutic potential against tMUC1-positive TNBC tumors with minimal damage to normal breast epithelial cells

Targeting of Aberrant αvβ6 Integrin Expression in Solid Tumors Using Chimeric Antigen Receptor-Engineered T Cells.

Expression of the αvβ6 integrin is upregulated in several solid tumors. In contrast, physiologic expression of this epithelial-specific integrin is restricted to development and epithelial re-modeling. Here, we describe, for the first time, the development of a chimeric antigen receptor (CAR) that couples the recognition of this integrin to the delivery of potent therapeutic activity in a diverse repertoire of solid tumor models. Highly selective targeting αvβ6 was achieved using a foot and mouth disease virus-derived A20 peptide, coupled to a fused CD28+CD3 endodomain. To achieve selective expansion of CAR T cells ex vivo, an IL-4-responsive fusion gene (4αβ) was co-expressed, which delivers a selective mitogenic signal to engineered T cells only. In vivo efficacy was demonstrated in mice with established ovarian, breast, and pancreatic tumor xenografts, all of which express αvβ6 at intermediate to high levels. SCID beige mice were used for these studies because they are susceptible to cytokine release syndrome, unlike more immune-compromised strains. Nonetheless, although the CAR also engages mouse αvβ6, mild and reversible toxicity was only observed when supra-therapeutic doses of CAR T cells were administered parenterally. These data support the clinical evaluation of αvβ6 re-targeted CAR T cell immunotherapy in solid tumors that express this integrin

CAR T-Cells Targeting the Integrin αvβ6 and Co-Expressing the Chemokine Receptor CXCR2 Demonstrate Enhanced Homing and Efficacy against Several Solid Malignancies

Despite the unprecedented clinical success of chimeric antigen receptors (CAR) T-cells against haematological malignancy, solid tumors impose a far greater challenge to success. Largely, this stems from an inadequate capacity of CAR T-cells that can traffic and maintain function within a hostile microenvironment. To enhance tumor-directed T-cell trafficking, we have engineered CAR T-cells to acquire heightened responsiveness to interleukin (IL)-8. Circulating IL-8 levels correlate with disease burden and prognosis in multiple solid tumors in which it exerts diverse pathological functions including angiogenesis, support of cancer stem cell survival, and recruitment of immunosuppressive myeloid cells. To harness tumor-derived IL-8 for therapeutic benefit, we have co-expressed either of its cognate receptors (CXCR1 or CXCR2) in CAR T-cells that target the tumor-associated αvβ6 integrin. We demonstrate here that CXCR2-expressing CAR T-cells migrate more efficiently towards IL-8 and towards tumor conditioned media that contains this cytokine. As a result, these CAR T-cells elicit superior anti-tumor activity against established αvβ6-expressing ovarian or pancreatic tumor xenografts, with a more favorable toxicity profile. These data support the further engineering of CAR T-cells to acquire responsiveness to cancer-derived chemokines in order to improve their therapeutic activity against solid tumors

Generation and application of TGFβ-educated human Vγ9Vδ2 T cells

Clinical trials that tested the antitumor activity of γδ T cells have been mostly unsuccessful. To address this, we expanded human Vγ9Vδ2 T cells in TGFβ1, a cytokine which enhances their viability, trafficking properties, and intrinsic antitumor activity. This protocol summarizes the production and in vitro functional characterization of TGFβ1 educated human Vγ9Vδ2 cells and highlights their compatibility with chimeric antigen receptor (CAR) engineering. We also describe in vivo testing of the antitumor activity of these CAR T cells in mice. For complete details on the use and execution of this protocol, please refer to Beatson et al. (2021)

Synergistic chemo-immunotherapy of epithelial ovarian cancer using ErbB re-targeted T-cells combined with carboplatin.

Abstract 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.</jats:p

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

Abstract 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.</jats:p

Clinical use of programmed cell death-1 (PD-1) and its ligand (PD-L1) expression as discriminatory and predictive markers in ovarian cancer

Funding was received from Mann-Hodgson charitable trust and Rosie's charity