Adoptive transfer of high-affinity chimeric antigen receptor (CAR) T cells targeting hematological cancers has yielded impressive clinical results. However, safety concerns regarding target expression on healthy tissue and poor efficacy have hampered application to solid tumors. Here, a panel of affinity-variant CARs were constructed targeting overexpressed ICAM-1, a broad tumor biomarker, using its physiological ligand, LFA-1. Anti-tumor T cell potencyin vitrowas directly proportional to CAR affinity and ICAM-1 density. In a solid tumor mouse model allowing simultaneous monitoring of anti-tumor potency and systemic off-tumor toxicity, micromolar affinity CAR T cells demonstrated superior anti-tumor efficacy and safety compared to their nanomolar counterparts. Longitudinal T cell tracking by PET/CT and concurrent cytokine measurement revealed superior expansion and contraction kinetics of micromolar affinity CAR T cells. Therefore, we developed an ICAM-1 specific CAR with broad anti-tumor applicability that utilized a reduced affinity targeting strategy to significantly boost efficacy and safety.

Purpose: Poorly differentiated thyroid cancer and anaplastic thyroid cancer (ATC) are rare yet lethal malignancies with limited treatment options. Many malignant tumors, including papillary thyroid cancer (PTC) and ATC, are associated with increased expression of ICAM-1, providing a rationale for utilizing ICAM-1–targeting agents for the treatment of aggressive cancer. We developed a third-generation chimeric antigen receptor (CAR) targeting ICAM-1 to leverage adoptive T-cell therapy as a new treatment modality. Experimental Design: ICAM-1 CAR T cells were applied to multiple malignant and nonmalignant target cells to investigate specific target cell death and “off-tumor” toxicity in vitro. In vivo therapeutic efficacy of ICAM-1 CAR T cells was examined in ATC mouse models established from a cell line and patient-derived tumors that rapidly develop systemic metastases. Results: ICAM-1 CAR T cells demonstrated robust and specific killing of PTC and ATC cell lines in vitro. Interestingly, although certain ATC cell lines showed heterogeneous levels of ICAM-1 expression, addition of cytotoxic CAR T cells induced increased ICAM-1 expression such that all cell lines became targetable. In mice with systemic ATC, a single administration of ICAM-1 CAR T cells mediated profound tumor killing that resulted in long-term remission and significantly improved survival. Patient-derived ATC cells overexpressed ICAM-1 and were largely eliminated by autologous ICAM-1 CAR T cells in vitro and in animal models. Conclusions: Our findings are the first demonstration of CAR T therapy against both a metastatic, thyroid cancer cell line and advanced ATC patient-derived tumors that exhibit dramatic therapeutic efficacy and survival benefit in animal studies. Clin Cancer Res; 23(24); 7569–83. ©2017 AACR.

Clinical monitoring of adoptive T cell transfer (ACT) utilizes serial blood analyses to discern T cell activity. While useful, these data are 1-dimensional and lack spatiotemporal information related to treatment efficacy or toxicity. We utilized a human genetic reporter, somatostatin receptor 2 (SSTR2), and PET, to quantitatively and longitudinally visualize whole-body T cell distribution and antitumor dynamics using a clinically approved radiotracer. Initial evaluations determined that SSTR2-expressing T cells were detectable at low densities with high sensitivity and specificity. SSTR2-based PET was applied to ACT of chimeric antigen receptor (CAR) T cells targeting intercellular adhesion molecule-1, which is overexpressed in anaplastic thyroid tumors. Timely CAR T cell infusions resulted in survival of tumor-bearing mice, while later infusions led to uniform death. Real-time PET imaging revealed biphasic T cell expansion and contraction at tumor sites among survivors, with peak tumor burden preceding peak T cell burden by several days. In contrast, nonsurvivors displayed unrelenting increases in tumor and T cell burden, indicating that tumor growth was outpacing T cell killing. Thus, longitudinal PET imaging of SSTR2-positive ACT dynamics enables prognostic, spatiotemporal monitoring with unprecedented clarity and detail to facilitate comprehensive therapy evaluation with potential for clinical translation.

Adoptive transfer of chimeric antigen receptor (CAR) T cells has demonstrated unparalleled responses in hematologic cancers, yet antigen escape and tumor relapse occur frequently. CAR T-cell therapy for patients with solid tumors faces even greater challenges due to the immunosuppressive tumor environment and antigen heterogeneity. Here, we developed a bispecific CAR to simultaneously target epithelial cell adhesion molecule (EpCAM) and intercellular adhesion molecule 1 (ICAM-1) to overcome antigen escape and to improve the durability of tumor responses. ICAM-1 is an adhesion molecule inducible by inflammatory cytokines and elevated in many types of tumors. Our study demonstrates superior efficacy of bispecific CAR T cells compared with CAR T cells targeting a single primary antigen. Bispecific CAR T achieved more durable antitumor responses in tumor models with either homogenous or heterogenous expression of EpCAM. We also showed that the activation of CAR T cells against EpCAM in tumors led to upregulation of ICAM-1, which rendered tumors more susceptible to ICAM-1 targeting by bispecific CAR T cells. Our strategy of additional targeting of ICAM-1 may have broad applications in augmenting the activity of CAR T cells against primary tumor antigens that are prone to antigen loss or downregulation.

While the majority of thyroid cancer patients are easily treatable, those with anaplastic or poorly differentiated recurrent thyroid carcinomas have a very poor prognosis with a median survival of less than a year. Previously, we have shown a significant correlation between ICAM-1 overexpression and malignancy in thyroid cancer, and have pioneered the use of ICAM-1 targeted CAR T cells as a novel treatment modality. For clinical translation of this novel modality, we designed CAR T cells possessing micromolar rather than nanomolar affinity to ICAM-1 to avoid cytotoxicity in normal cells with basal levels of ICAM-1 expression. Herein, we report the automated process of CAR T cell manufacturing with CliniMACS Prodigy (Miltenyi Biotec) using cryopreserved peripheral blood leukocytes from apheresis collections. Using Prodigy, thawed leukopak cells were enriched for CD4⁺and CD8⁺T cells, subjected to double transduction using lentiviral vector, and expanded in culture for a total of 10 days with a final yield of 2–4 × 10⁹cells. The resulting CAR T cells were formulated for cryopreservation to be used directly for infusion into patients after thawing with no further processing. We examined cross-reactivity of CAR T cells toward both human and murine ICAM-1 and ICAM-1 expression in human and mouse tissues to demonstrate that both efficacy and on-target, off-tumor toxicity can be studied in our preclinical model. Selective anti-tumor activity in the absence of toxicity provides proof-of-concept that micromolar affinity tuned CAR T cells can be used to target tumors expressing high levels of antigen while avoiding normal tissues expressing basal levels of the same antigen. These studies support the initiation of a phase I study to evaluate the safety and potential efficacy of micromolar affinity tuned CAR T cells against newly diagnosed anaplastic and refractory or recurrent thyroid cancers.

Cancer therapy utilizing adoptive transfer of chimeric antigen receptor (CAR) T cells has demonstrated remarkable clinical outcomes in hematologic malignancies. However, CAR T cell application to solid tumors has had limited success, partly due to the lack of tumor-specific antigens and an immune-suppressive tumor microenvironment. From the tumor tissues of gastric cancer patients, we found that intercellular adhesion molecule 1 (ICAM-1) expression is significantly associated with advanced stage and shorter survival. In this study, we report a proof-of-concept study using ICAM-1-targeting CAR T cells against gastric cancer. The efficacy of ICAM-1 CAR T cells showed a significant correlation with the level of ICAM-1 expression in target cells in vitro. In animal models of human gastric cancer, ICAM-1-targeting CAR T cells potently eliminated tumors that developed in the lungs, while their efficacy was more limited against the tumors in the peritoneum. To augment CAR T cell activity against intraperitoneal tumors, combinations with paclitaxel or CAR activation-dependent interleukin (IL)-12 release were explored and found to significantly increase anti-tumor activity and survival benefit. Collectively, ICAM-1-targeting CAR T cells alone or in combination with chemotherapy represent a promising strategy to treat patients with ICAM-1+ advanced gastric cancer.