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Supplementary figure S1-S6

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.

A model for ICAM1-CAR T and anti-PD1 antibody activities in advanced thyroid cancers.

Purpose:Advanced thyroid cancers, including poorly differentiated and anaplastic thyroid cancer (ATC), are lethal malignancies with limited treatment options. The majority of patients with ATC have responded poorly to programmed death 1 (PD1) blockade in early clinical trials. There is a need to explore new treatment options.Experimental Design:We examined the expression of PD-L1 (a ligand of PD1) and intercellular adhesion molecule 1 (ICAM1) in thyroid tumors and ATC cell lines, and investigated the PD1 expression level in peripheral T cells of patients with thyroid cancer. Next, we studied the tumor-targeting efficacy and T-cell dynamics of monotherapy and combination treatments of ICAM1-targeting chimeric antigen receptor (CAR) T cells and anti-PD1 antibody in a xenograft model of ATC.Results:Advanced thyroid cancers were associated with increased expression of both ICAM1 and PD-L1 in tumors, and elevated PD1 expression in CD8+ T cells of circulating blood. The expression of ICAM1 and PD-L1 in ATC lines was regulated by the IFNγ–JAK2 signaling pathway. ICAM1-targeted CAR T cells, produced from either healthy donor or patient T cells, in combination with PD1 blockade demonstrated an improved ability to eradicate ICAM1-expressing target tumor cells compared with CAR T treatment alone. PD1 blockade facilitated clearance of PD-L1 high tumor colonies and curtailed excessive CAR T expansion, resulting in rapid tumor clearance and prolonged survival in a mouse model.Conclusions:Targeting two IFNγ-inducible, tumor-associated antigens—ICAM1 and PD-L1—in a complementary manner might be an effective treatment strategy to control advanced thyroid cancers in vivo.

Expression of ICAM1 and PD-L1 in anaplastic thyroid tumor cell lines.

PD1 inhibition enhances survival benefit of CAR T treatment in KHM-5M xenografts.

The ability to image adoptively transferred T cells in the body and to eliminate them to avoid toxicity will be vital for chimeric antigen receptor (CAR) T cell therapy, particularly against solid tumors with higher risk of off-tumor toxicity. Previously, we have demonstrated the utility of somatostatin receptor 2 (SSTR2) for CAR T cell imaging, illustrating the expansion and contraction of CAR T cells in tumor as well as off-tumor expansion. Using intercellular adhesion molecule 1 (ICAM-1)-specific CAR T cells that secrete interleukin (IL)-12 as a model, herein we examined the potential of SSTR2 as a safety switch when combined with the SSTR2-specific maytansine-octreotate conjugate PEN-221. Constitutive secretion of IL-12 led to continuous expansion of CAR T cells after rapid elimination of tumors, causing systemic toxicity in mice with intact MHC expression. Treatment with PEN-221 rapidly reduced the abundance of CAR T cells, decreasing the severity of xenogeneic graft-versus-host disease (GvHD), and prolonged survival. Our study supports the development of SSTR2 as a single genetic marker for CAR T cells that is readily applicable to humans both for anatomical detection of T cell distribution and an image-guided safety switch for rapid elimination of CAR T cells.

Purpose: Advanced thyroid cancers, including poorly differentiated and anaplastic thyroid cancer (ATC), are lethal malignancies with limited treatment options. The majority of patients with ATC have responded poorly to programmed death 1 (PD1) blockade in early clinical trials. There is a need to explore new treatment options. Experimental Design: We examined the expression of PD-L1 (a ligand of PD1) and intercellular adhesion molecule 1 (ICAM1) in thyroid tumors and ATC cell lines, and investigated the PD1 expression level in peripheral T cells of patients with thyroid cancer. Next, we studied the tumor-targeting efficacy and T-cell dynamics of monotherapy and combination treatments of ICAM1-targeting chimeric antigen receptor (CAR) T cells and anti-PD1 antibody in a xenograft model of ATC. Results: Advanced thyroid cancers were associated with increased expression of both ICAM1 and PD-L1 in tumors, and elevated PD1 expression in CD8+ T cells of circulating blood. The expression of ICAM1 and PD-L1 in ATC lines was regulated by the IFNγ–JAK2 signaling pathway. ICAM1-targeted CAR T cells, produced from either healthy donor or patient T cells, in combination with PD1 blockade demonstrated an improved ability to eradicate ICAM1-expressing target tumor cells compared with CAR T treatment alone. PD1 blockade facilitated clearance of PD-L1 high tumor colonies and curtailed excessive CAR T expansion, resulting in rapid tumor clearance and prolonged survival in a mouse model. Conclusions: Targeting two IFNγ-inducible, tumor-associated antigens—ICAM1 and PD-L1—in a complementary manner might be an effective treatment strategy to control advanced thyroid cancers in vivo.

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., Graphical Abstract, In this study, we demonstrated the therapeutic potential of CAR T cells targeting ICAM-1 in preclinical models of systemic and intraperitoneal metastases of gastric cancer. A combination with paclitaxel or armoring CAR T cells with inducible IL-12 was found to significantly enhance the treatment effect of CAR T cells.

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 × 109 cells. 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.

Zhang, Weiqi/0000-0003-4315-6809; Sayman, Haluk Burcak/0000-0002-0954-5707; Rodriguez, Erik/0000-0001-9143-2535 WOS:000476957100009 PubMed ID: 31120734 Clinical trials involving genome-edited cells are growing in popularity, where CAR-T immunotherapy and CRISPR/Cas9 editing are more recognized strategies. Genetic reporters are needed to localize the molecular events inside these cells in patients. Specifically, a nonimmunogenic genetic reporter is urgently needed as current reporters are immunogenic due to derivation from nonhuman sources. Prostate-specific membrane antigen (PSMA) is potentially nonimmunogenic due to its natural, low-level expression in select tissues (self-MHC display). PSMA overexpression on human prostate adenocarcinoma is also visible with excellent contrast. We exploit these properties in a transduced, two component, Human-Derived, Genetic, Positron-emitting, and Fluorescent (HD-GPF) reporter system. Mechanistically analogous to the luciferase and luciferin reporter, PSMA is genetically encoded into non-PSMA expressing 8505C cells and tracked with ACUPA-Cy3-BF3, a single, systemically injected small molecule that delivers positron emitting fluoride (F-18) and a fluorophore (Cy3) to report on cells expressing PSMA. PSMA-lentivirus transduced tissues become visible by Cy3 fluorescence,[F-18]-positron emission tomography (PET), and gamma-scintillated biodistribution. HD-GPF fluorescence is visible at subcellular resolution, while a reduced PET background is achieved in vivo, due to rapid ACUPA-Cy3-BF3 renal excretion. Co-transduction with luciferase and GFP show specific advantages over popular genetic reporters in advanced murine models including, a "mosaic" model of solid-tumor intratumoral heterogeneity and a survival model for observing postsurgical recurrence. We report an advanced genetic reporter that tracks genetically modified cells in entire animals and with subcellular resolution with PET and fluorescence, respectively. This reporter system is potentially nonimmunogenic and will therefore be useful in human studies. PSMA is a biomarker of prostate adenocarcinoma and ACUPA-Cy3-BF3 potential in radical prostatectomy is demonstrated. National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [EB013904, R01CA178007, P30CA008748]; NATIONAL CANCER INSTITUTEUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [R01CA178007, R01CA217059, R01CA217059, R01CA217059] Funding Source: NIH RePORTER The project was supported by National Institutes of Health Grants EB013904, R01CA178007, and P30CA008748.

Introduction: The application of chimeric antigen receptor (CAR) T cells in solid tumors has met many challenges, arising from the paucity of effective yet safe targets and T cell dysfunction within the immunosuppressive milieu. Clinical trials over the years have reported severe on-target off-tumor toxicities associated with CAR T cell therapies targeting tumor-associated antigens (TAAs). Here, we propose a strategy to target TAAs effectively and safely: 1) improve tumor-targeting specificity by affinity-tuning; 2) armor T cells with antigen-dependent expression of interleukin-12 (IL-12) to compensate loss of CAR T cell activity caused by affinity-tuning. In this system, IL-12 expression is tightly regulated by the nuclear factor of the activated T cells promoter (NFAT) to limit IL-12 to the environ of activated affinity-tuned CAR T cells within the tumor. We demonstrate here the feasibility of this strategy using epithelial cell adhesion molecule (EpCAM) as a model TAA. Methods: Affinity variants were generated by alanine scanning mutagenesis in the complementary-determining region 3 (CDR3) of an EpCAM antibody (UBS54). The affinity of CAR variants was determined by flow cytometry-based saturation binding assay. On-target off-tumor cytotoxicity of CAR T cells was examined against human primary normal epithelial cells. In vivo anti-tumor efficacy was evaluated in mouse models of gastric cancer with cell line-derived (SNU-638 and MKN-45) and patient-derived xenografts. Results: Substitution of alanine into the UBS54 CDR3 led to identification of a tyrosine (6th residue in heavy chain CDR3) as a hot spot for affinity tuning. Among several more subtle amino acid substitutions for Tyr-6, we found that CAR molecule with valine substituent (Y6V) possesses 10 µM affinity toward EpCAM, rendering CAR T cells to be selective to EpCAM-high tumors while being not reactive to primary normal epithelial cells in vitro. In gastric cancer mouse models, compared to UBS54 CAR T cells that mediated rapid tumor remission, Y6V CAR T cells produced mainly partial responses. To revitalize affinity-tuned CAR T cells, we further engineered CAR T cells to release IL-12 under NFAT. We found that both the activity of the NFAT promoter and the level of IL-12 release were tightly regulated by the antigen density of targets when inducible IL-12 is combined with 10 µM affinity Y6V CAR; however, such dependence is lost when 1 µM affinity UBS54 was examined. In mouse models of gastric cancer, inducible lL-12 armored Y6V CAR T cells produced enhanced anti-tumor responses without elevating systemic exposure to IL-12, evidenced by low levels of IL-12 in mouse sera (below 100 pg/ml). Conclusions: Combination of affinity-tuned CAR with inducible expression of IL-12 is a promising strategy to develop CAR T cell therapy against TAAs with the potential for reduced on-target off-tumor toxicity and tumor resistance. Citation Format: Yanping Yang, Huan Yang, Yago Alcaina, Jaclyn E. McCloskey, Janusz Puc, Alyssa Birt, Yogindra Vedvyas, Juan Gonzalez-Valdivieso, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5568.

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.

Background: Adoptive cell transfer therapy with genetically engineered chimeric antigen receptor (CAR)-T cells is rapidly revolutionizing treatment against cancers. However, limited success of CAR T cells against large metastatic solid tumors has been made to date. It is largely due to safety concerns regarding on-target off-tumor toxicity to vital healthy tissues, and poor efficacy after adoptive transfer. Epithelial cell adhesion molecule (EpCAM) was found to be frequently over-expressed in a wide variety of carcinomas, including colon, gastric, pancreas, and breast cancers, making EpCAM an attractive molecule for targeted therapeutics. However, clinical trials with anti-EpCAM antibodies (ING-1, 3622W94) and bispecific T cell engager antibodies (Solitomab) have shown significant dose-limiting toxicities, resulting in very limited clinical responses. To selectively target EpCAM high tumors, we are developing affinity tuned EpCAM CAR T cells with varying affinities to EpCAM. Methods: The human glioblastoma cell line U251, breast cancer cell lines MDA-MB-231 and SK-BR-3, gastric cancer cell lines MKN28 and MKN45, pancreatic cancer cell lines SW1990 and Capan2, and colon cancer cell line HT29 were transduced with Luciferase-F2A-GFP (FLuc/GFP) lentivirus. EpCAM expression levels on tumor cell lines were measured by flow cytometry. Primary human T cells were transduced with EpCAM CAR lentivirus twice at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. NSG mice bearing intraperitoneal or subcutaneous MKN28 xenografts, systemic metastatic MKN45 xenografts, and orthotopic Capan2 xenografts were treated with EpCAM CAR T cells to test the in vivo efficacy. PET/CT imaging was adopted to monitor T cell dynamics using 18F-NOTA-octreotide which binds specifically to somatostatin receptor 2 (SSTR2) introduced in CAR T cells. Results: Jurkat cells transduced with C215 and UBS54 CARs displayed 3.5 nM and 264.3 nM binding affinities to soluble EpCAM, respectively. EpCAM CAR expression in primary human T cells was higher than 50%. CD8+ to CD4+ T cell ratios were 1:1. Compared to high-affinity C215 CAR T cells, low-affinity UBS54 CAR showed equivalent cytotoxicity against high-density EpCAM-expressing targets (MKN28, MKN45 and HT29), but slower killing of low-density EpCAM-expressing cell line MDA-MB-231. T cell activation as monitored by CD69 upregulation and cytokine secretion (IL2, IFN-γ, TNF-α, GM-CSF and MIP-1β) of C215 and UBS54 CAR T cells were similar, and correlate well with EpCAM density on tumor cell surfaces. In immunodeficient mouse models, low-affinity UBS54 CAR potently eliminated intraperitoneal and subcutaneous gastric tumors (MKN28), systemic metastatic gastric tumor (MKN45), and orthotopic pancreatic tumor (Capan2). Notably, the level of T cell expansion in the course of tumor killing, observed by PET/CT imaging, was significantly lower for UBS54 CAR than high-affinity C215 CAR. Conclusions: Low-affinity EpCAM targeting CAR T cells demonstrated durable antitumor activity against various tumor models in vivo. To enhance safety and therapeutic efficacy, micromolar affinity anti-EpCAM CAR T cells are currently being evaluated to limit cell killing to EpCAM-positive tumor cells while sparing normal cells with basal levels of EpCAM expression. Citation Format: Yanping Yang, Jaclyn E. McCloskey, Huan Yang, Janusz Puc, Angel A. Gomez Gallegos, Yogindra Vedvyas, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Eradication of EpCAM expressing solid tumors by low-affinity CAR T cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6598.

Chimeric antigen receptor (CAR)-T cell therapy has shown robust anti-cancer responses in hematologic malignancies. However, application of this therapeutic approach to solid tumors has been hindered by multiple challenges, one of which is on-target/off-tumor cytotoxicity to normal tissues. Tumor-specific antigens exclusively present on tumor cells are rare. Most CAR-T cells are designed to target tumor associated antigens (TAAs) expressed in high levels on tumor cells. Yet, normal tissues express these antigens as well, albeit at much lower densities.To mitigate the on-target/off-tumor cytotoxicity, we developed a strategy for fine tuning the affinity of CARs to selectively target tumor cells. Epithelial cell adhesion molecule (EpCAM) is highly expressed in epithelial cells and overexpressed in tumor cells in a variety of epithelial carcinomas. High-affinity (nM range) EpCAM-targeting CAR-T cells kill both normal human epithelial cells and EpCAM-high tumor cells in vitro. To develop CAR-T cells specific to EpCAM-high cancers, we identified low-affinity scFv variants by rational design of amino acid substitutions. The affinities of these scFvs were measured by Surface Plasmon Resonance (SPR). CAR-T cells equipped with low-affinity scFvs showed antigen-dependent activation, proliferation and Th1-like cytokine secretion when co-cultured with target cells having varied levels of EpCAM. Importantly, low-affinity CAR-T cells still lysed EpCAM-high tumor cells but spared normal human epithelial cells in vitro. Treatment of MKN28 xenograft mice with low-affinity CAR-T cells resulted in tumor regression and prolonged survival. Moreover, transcriptomic profiling of CAR-T cells revealed significant differences in gene expression levels between high- and low- affinity CARs, particularly as they relate to the functional state of these CAR-T cells, and their resistance to exhaustion. Our results show that rational design of scFv can aid in the identification of CARs that are minimally reactive toward normal tissues while effectively eliminating tumors. Furthermore, affinity-tuned CARs demonstrate better overall fitness and antitumor activity in vivo. This affinity fine-tuning approach shows promise as a general strategy for identifying a therapeutic window for CAR-T cells targeting novel TAAs that may have been overlooked because of basal expression levels in normal tissues. Citation Format: Huan Yang, Janusz Puc, Yanping Yang, Jaclyn E. McCloskey, Yogindra Vedvyas, Hongtao Li, Irene M. Min, Moonsoo M. Jin, Eric v. Hofe. Mitigating on-target off-tumor cytotoxicity of EpCAM CAR-T by affinity tuning [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-381.

Background: Chimeric antigen receptor (CAR) T cells have demonstrated continued success in the treatment of hematological malignancies. By contrast, limited efficacy of CAR T cells has been seen in solid tumors due to multiple obstacles including impaired T cell infiltration and immune suppressive tumor environment, resulting in lack of proliferation and function. Previously, we have developed a micromolar affinity tuned CAR T cells targeting overexpressed ICAM-1 against a variety of tumors. To combat the immunosuppressive microenvironment in solid tumors, we developed a novel lentivirus vector that incorporates expression cassettes for CAR, somatostatin receptor 2 (SSTR2) for PET/CT imaging of T cells, and CAR-activation dependent release of IL-12. Inducible IL-12 (iIL-12) expression was enabled by a synthetic promoter containing both NFkB and NFAT promoter elements by T cells to leverage IL-12's ability to promote Th1 response and at the same time to minimize systemic toxicity of IL-12. The iIL-12 CAR T exhibited much more robust killing of subcutaneous and peritoneal tumors, which showed partial to limited response to conventional CAR T cells without inducible cytokines. Methods: CAR T cells were generated by double lentiviral transduction of primary human T cells at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. NSG mice inoculated with subcutaneous anaplastic thyroid cancer cells (8505c) and triple negative breast cancer cells (MDA-MB-468), and intraperitoneal gastric cancer cells (MKN28) were treated with with or without iIL-12 CAR T cells. Tumor growth was monitored regularly by bioluminescence imaging. PET/CT imaging was implemented to monitor T cell localization and biodistribution using 18F-NOTA-octreotide, a radiotracer targeting SSTR2. Results: Jurkat cells expressing iIL-12 ICAM-1 CAR produced IL-12 in an antigen density dependent manner when co-incubated with ICAM-1 positive tumor cells or HEK 293T cells transduced to express human ICAM-1. In in vitro cytotoxicity assay, iIL-12 CAR T cells exhibited significantly augmented tumor-lytic activity than conventional ICAM-1 CAR T cells. In striking contrast to conventional ICAM-1 CAR T cells which did not control tumor growth, iIL-12 CAR T cells mediated complete regression of various solid tumors and prolonged mouse survival significantly. Based on PET/CT imaging, both conventional and iIL-12 CAR T cells showed high levels of T cell infiltration and localization in tumor lesions. These results suggest that conventional CAR T cells quickly exhausted and lost function after penetration, while locally released IL-12 played an important role in improving anti-tumor responses. Furthermore, serum levels of IL-12 in mice treated with iIL-12 CAR T cells were not detectable, indicating that IL-12 secretion was tightly controlled and limited to tumor sites. Conclusions: These findings demonstrate that local release of inducible IL-12 can help overcome hostile tumor microenvironment and augment anti-tumor immune responses. Studies are underway to evaluate the feasibility of inducible IL-12 armored ICAM-1 CAR T cells as a potential therapy with favorable safety and efficacy profiles. Citation Format: Yanping Yang, Jaclyn E. McCloskey, Yogindra Vedvyas, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Highly localized, inducible interleukin-12 release augments ICAM-1 CAR T cell activity against solid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6597.

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

Background: Triple negative breast cancer (TNBC) is an aggressive disease with a poor prognosis. TNBC patients do not respond to hormone receptor or HER2-targeted therapies owing to low expression of these cell surface biomarkers. The lacking of targeted therapy leaves conventional combination of surgery, chemotherapy and radiation therapy as the standard-of-care treatment options for TNBC. But even this combination usually fails to prevent disease recurrence. Intercellular adhesion molecule-1 (ICAM-1) was recently discovered to be upregulated in TNBC and could serve as an attractive molecular target (Guo et al., PNAS 2014). Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success against hematological malignancies, however there has been little success in the treatment of solid tumors. Here, we developed an ICAM-1 targeting CAR T cell-based immunotherapeutic strategy to redirect T cell to kill solid TNBC. Methods: Patient-derived xenograft (PDX) models of TNBC were utilized to determine ICAM-1 expression by immunohistochemistry. ICAM-1 surface protein expression in TNBC cell line MDA-MB-231 was measured by flow cytometry. Primary T cells (n = 4 donors) were isolated and transduced with ICAM-1 CAR lentivirus twice at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. In vitro killing ability was determined by co-incubation of GFP-Firefly Luciferase (GFP/Fluc) transduced target cells (HeLa, MDA-MB-231, and 293T) with transduced and non-transduced T cells. NSG mice bearing MDA-MB-231 xenografts were treated with CAR T cells to test the in vivo efficacy. Bioluminescence was used to quantify cell lysis in vitro and to track tumor growth in vivo. Results: 4 out of 8 TNBC PDX models (JAX, tumor markers: TER−/PR−/HER2−) showed strong IHC ICAM-1 staining. We validated by flow cytometry that ICAM-1 is highly expressed on TNBC cell line MDA-MB-231. These results provide further evidence supporting ICAM-1 as a molecular target for TNBC by CAR T cell therapy. Primary T cells were transduced to express ICAM-1 targeting CAR at approximately 50%. Co-incubation of CAR T cells or non-transduced T cells with ICAM-1 positive cell lines (HeLa and MDA-MB-231) or negative control cell line (293T) showed that CAR T cell-mediated killing was ICAM-1 expression dependent. After 48 hours, 70% of HeLa and 85% of MDA-MB-231 cells were specifically lysed at effector to target ratio of 5:1, while no obvious killing of 293T cells was observed. Additionally, non-transduced T cells produced little killing with less than 20% of target cell lysis. Ongoing in vivo studies will determine the efficacy of this ICAM-1 targeting CAR T cell against TNBC. Conclusions: We developed an ICAM-1 targeting CAR T cell with the ability to induce potent and specific killing of TNBC cells. Preclinical studies are being conducted to evaluate the feasibility of ICAM-1 specific CAR T cells as a potential therapy for patients with ICAM-1 positive TNBC. Citation Format: Yanping Yang, Yogindra Vedvyas, Jaclyn E. McCloskey, Irene M. Min, Moonsoo M. Jin. ICAM-1 targeting CAR T cell therapy for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2322.

Introduction: Despite advances in treatment, gastric cancer (GC) remains among the most fatal malignancies. Intercellular adhesion molecule-1 (ICAM-1) is overexpressed and associated with various cancers, including GC. We developed a third-generation chimeric antigen receptor (CAR) targeting ICAM-1 (ICAM-1-CAR) and investigated ICAM-1 as a immunotherapeutic target for CAR T therapy in GC. Methods: We investigated ICAM-1 expression in tissues of curatively resected GC patients (n=134) by immunohistochemical staining to determine its prognostic value. Effector to target assays were performed with ICAM-1-CAR T cells co-cultured with 8 GC cell lines with varying levels of ICAM-1 expression to investigate specific target cell death. We created a firefly luciferase-expressing human GC model in mice to measure tumor growth and killing by whole body bioluminescence imaging. To find the best treatment route and optimal dose of T cells in GC mice model, ICAM-1-CAR was injected via tail vein and intraperitoneal administration (IP) at two different doses, i.e., low dose (1 million CAR T) and high dose (10 million live T cells). Results: The ICAM-1 expression was higher in advanced stages (22.2% in stage II vs. 48.8% in stage III, p=0.002) and the patients with high ICAM-1 expression showed significantly poor survival (disease free survival, hazard ration [HR], 4.55, p Conclusion: ICAM-1 specific CAR T cells demonstrated significant therapeutic efficacy in vitro and in vivo against preclinical GC models. ICAM-1-CAR T cells may be developed into a promising treatment strategy for patients with ICAM-1 positive GC cancers. Citation Format: Minkyu Jung, Marjan Zaman, Ygindra Vedvyas, Xianglan Zhang, Jaclyn E. McCloskey, Yanping Yang, Irene M. Min, Raza Zamegar, Yoon Young Choi, Jae-Ho Cheong, Sung Hoon Noh, Sun Young Rha, Hyun Cheol Chung, Moonsoo M. Jin. Chimeric antigen receptor T cell therapy targeting ICAM-1 in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1442.

Background: Patients with anaplastic or poorly differentiated recurrent thyroid carcinomas have a poor prognosis, with a median survival of < 1 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, we designed CAR T cells possessing micromolar affinity to ICAM-1 to avoid cytotoxicity in normal cells with basal levels of ICAM-1 expression. Here, we report the automated process of CAR T cell manufacturing with CliniMACS Prodigy (Miltenyi Biotec) using cryopreserved peripheral blood leukopak cells as starting material. Methods: We have chosen to use CliniMACS Prodigy system for a closed, automated, and robust CAR T cell manufacturing process. Using a cryopreserved leukopak, TCT v2.0 protocol began with rapidly thawing the leukopak while directly welded to the Prodigy. T cell lentivirus transduction was performed 2x, 24 and 48 hr post activation. CAR T cells were released after ~10 days of culture and cryopreserved in ready-to-infuse formulation (AIC100). Products were then subject to in vitro cytotoxicity, in vivo efficacy and safety in xenografted mice with ATC cells (8505C), and PET/CT imaging using 18F-NOTA-octreotide to detect T cell dynamics. Results: Prodigy manufactured CAR T cells were subjected to qualification and functional assays (n=7): cell viability, cell number, CAR expression, virus copy number (VCN), T cell subset and phenotype, and E to T assays. Consistent with reported values for Prodigy, our manufacturing protocol produced 55±9% transduction rate, 96±1.8% viability, and 2.9±0.7x10^9 final cell number. The VCN was in the range of 1.0-2.1, below the criteria of < 5 copies per cell. In NSG mice xenografted with 8505C ATC cells and treated with CAR T cells (1X= 1 million live CAR T maximum tolerable dose (MTD)= 10 million live CAR T) or non-transduced (NT) T cells, tumor burden was quantitatively evaluated by whole body luminescence imaging. Compared to cohorts with no treatment (NoT) or NT, the cohorts of AIC100 exhibited complete or near-complete tumor elimination lasting variable times before tumor relapse was seen in some subjects. Median survival time for NoT and NT groups were similar (38.5 vs. 42 days), while it was significantly longer for AIC100 1X and MTD groups (72 vs. >100 days). Conclusions: 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 examine the safety and potential efficacy of micromolar affinity tuned CAR T cells against newly diagnosed anaplastic and refractory or recurrent thyroid cancers. Citation Format: Yogindra Vedvyas, Jaclyn E. McCloskey, Yanping Yang, Irene M. Min, Thomas J. Fahey, Yen-Michael S. Hsu, Jing-Mei Hsu, Koen V. Besien, Ian Gaudet, Ping Law, Joon Kim, Eric V. Hofe, Moonsoo M. Jin. Clinical manufacturing of CAR T cells targeting ICAM-1 for a phase I study against advanced thyroid cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2329.

Gene-engineered adoptive T cell therapies have recently been approved by the FDA. We have previously published data utilizing this technology to target intercellular adhesion molecule-1 (ICAM-1), a broad tumor biomarker. Using an affinity modified version of the physiological ligand of ICAM-1,leukocyte function-associated antigen-1 (LFA-1) I-domain, we have shown efficient clearance of a human metastatic anaplastic thyroid cancer (ATC). We were able to visualize and quantify the bio-distribution of CAR-T cells with a PET gene reporter, somatostatin receptor 2 (SSTR2) (Park et. al, Scientific Reports 7, Article number: 14366, 2017). To expand on this platform, we now want to control the activity of our CAR-T cells by exploiting these features using two FDA approved drugs. First, a somatostain analogue, Lanreotide, has been approved for treating Acromegaly and we have recently observed that Lanreotide can activate SSTR2-transduced T cells via calcium release. Second, Lovastatin, a drug clinically used for lowering cholesterol levels, was found to bind the LFA-1 I-domain and inhibit LFA-1 binding to ICAM-1. We hypothesized that in addition to our I-domain CAR-T cell, systemic use of Lanreotide will stimulate CAR-mediated tumor killing while systemic use of Lovastatin will inhibit CAR-mediated tumor killing. In-vitro testing involved effector to target (E:T) assays performed in the presence and absence of Lovastatin or Lanreotide. ICAM-1(+) ATC target cells or ICAM-1(-) 293T off-target cells were utilized, both of which were transduced with GFP-Firefly Luciferase (GFP/Fluc) for quantification. Effector T-cells derived from healthy donor PBMCs were either transduced with our I-domain CAR-T cell construct that contains SSTR2, or were left nontransduced. Similar assays were translated in-vivo using a previously established solid tumor model with GFP/Fluc ATC ICAM-1(+) cells. Luminescence was used to track tumor progression and regression throughout both in-vivo and in-vitro experiments. [Ga68] DOTA-TOC was administered via IV injections to visualize the bio-distribution of the CAR-T cells using SSTR2 via PET/CT. Both in-vitro and in-vivo experiments showed Lovastatins ability to compromise I-domain CAR-mediated tumor killing while Lanreotide expedited the rate of I-domain CAR-mediated killing. We report here that Lovastatin inhibits the interaction of our I-domain CAR with ICAM-1 that is present on our target tumor cells and compromises tumor killing. In addition, we have shown a unique phenomena where we can activate SSTR2-transduced CAR-T cells using Lanreotide. The ability to control CAR-T cell activity is an essential tool when addressing adverse effects and/or less than robust treatment outcomes. This will be crucial when translating our adoptive T cell therapy to the clinic. Citation Format: Yogindra Vedvyas, Jaclyn McCloskey, Yanping Yang, Irene M. Min, Moonsoo M. Jin. Pharmacological intervention to temporally stimulate or inhibit ICAM-1 targeting CAR-T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3592.