Search

Purpose: Current understanding of resistance to CAR-T cell therapy in solid tumors implicates inadequate CAR-T cell potency in the immunosuppressive tumor microenvironment (TME). We have previously developed a platform using somatostatin receptor 2 (SSTR2) as a positron emission tomography (PET) reporter to detect CAR-T cell expansion and trafficking. The current study aimed to leverage SSTR2 for low-dose targeted radionuclide therapy (177Lu-DOTATATE, Lutathera), which under dosimetry guidance might enhance antitumor immunity by reprograming the TME and promoting T-cell reinvigoration. Methods: Using intercellular adhesion molecule 1 (ICAM-1) as a model antigen, we evaluated the immunomodulatory effects of low-dose radiation in a gastric cancer animal model. NSG mice were inoculated subcutaneously with firefly luciferase-expressing Hs 746T cells (0.1 × 106 per mouse) and treated 5 days later with 10 × 106 SSTR2-expressing ICAM-1 CAR-T cells. CAR-T cell expansion was monitored weekly by PET scan using 18F-NOTA-Octreotide, a radiotracer targeting SSTR2. Three weeks post-T cell infusion, a cohort of mice were injected with 7.4 MBq of 177Lu-DOTATATE via the tail vein. SPECT imaging was performed for dosimetry analysis. Tumor growth was monitored by bioluminescence imaging and tumor size measurement. Serum cytokines were analyzed. Results: Single dose 177Lu-DOTATATE treatment (delivering 1-6 Gy to tumor) improved response of established gastric cancer tumor (>1,000 mm3) that was not responsive to CAR-T cell treatment alone and significantly prolonged survival. All mice receiving CAR-T cells plus 177Lu-DOTATATE displayed rapid tumor shrinkage, with 83% of mice achieving complete remission within 3 weeks of 177Lu-DOTATATE treatment. SPECT imaging confirmed specific delivery of 177Lu-DOTATATE by tumor-infiltrating CAR-T cells, with tumor uptake of 0.43 ± 0.24 and 0.19 ± 0.08 MBq/g at 24 and 144 hours post 177Lu-DOTATATE injection, respectively. Most radioactivity reduction over time (73%) is explained by physical decay of 177Lu, indicating persistent tumor retention of 177Lu-DOTATATE. In contrast, rapid clearance of 177Lu-DOTATATE was observed in liver and kidneys. Importantly, longitudinal CAR-T cell imaging using 18F-NOTA-Octreotide revealed increased CAR-T cell expansion induced by low-dose radiation. Furthermore, we detected high levels of IFN-γ and perforin in serum after 177Lu-DOTATATE treatment, which were >10 folds higher than that in control mice receiving CAR-T cell only, indicating activation of T cells by low-dose radiation. Conclusions: We developed a translatable radioimmunotherapy platform that incorporates a FDA-approved theranostic endoradiotherapy (Lutathera) to improve tumor response to CAR-T cell therapy. The next steps would be to explore the immunomodulatory effects of low-dose radiation systematically and elucidate the mechanism of action. Citation Format: Yanping Yang, Yago Alcaina, Yogindra Vedvyas, Maria Cristina Riascos, Edward K. Fung, Brett Vaughn, Sarah M. Cheal, Irene M. Min, Claire Vanpouille-Box, Moonsoo M. Jin. Targeted delivery of low-dose radiation alleviates tumor resistance to CAR-T cell therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5084.

Purpose: While there have been extensive studies about defining T cell states associated with response to checkpoint immunotherapy, relatively little is known about the crosstalk between tumor and tumor-infiltrating chimeric antigen receptor (CAR) T cells and how it leads to tumor resistance and CAR T cell dysfunction. Here we employ a two-pronged approach using CAR T cell imaging and single cell multi-omics techniques to elucidate the complex tumor-CAR T cell interactions in a gastric cancer xenograft model. Methods: Human CAR-T cells targeting epithelial cell adhesion molecule were engineered to co-express somatostatin receptor 2 (SSTR2) for positron emission tomography (PET) imaging using 18F-NOTA-Octreotide radiotracer. NSG mice were inoculated subcutaneously with firefly luciferase-expressing MKN-45 cells (1 × 106 per mouse) and treated 5 days later with 10 × 106 SSTR2-expressing CAR-T or non-transduced T cells. Tumor growth and CAR T cell expansion were monitored near synchronously by bioluminescence and PET-CT imaging, respectively. Resistant or relapsing tumors were harvested and MACS sorted into human CD45+ tumor-infiltrating T cells (TILs) and CD45-depleted tumor cells. CAR T cell products and MKN-45 tumor cell line prior to injection were included in single cell analysis for comparison. Results: TCR clonal analysis supports that CAR T cells in resistant tumors are proliferative and clonally expanded, which finding is corroborated by whole-body CAR T cell imaging. Highly expanded clones were observed within the CD4+ CAR T cell compartment, especially within the regulatory T (Treg) cell cluster. CD8+ CAR T cells underwent less clonal expansion with most clones found within the exhausted cells (LAG3, PDCD1, and TIGIT, LAYN and SOX4), whereas non-transduced control CD8+ T cells did not express high levels of exhaustion associated genes. Differential gene expression (DGE) analysis between TILs and pre-infusion CAR T cells showed increased expression of LAYN, CXCL13, LMNA, and DUSP4 in TILs. The most upregulated DUSP4 was previously found to be overexpressed in Treg and aged T cells, and the DUSP4 induction is linked to a defective adaptive immune response. In addition, we observed the loss of cell-surface CAR in TILs, likely from chronic exposure to antigens in resistant tumors. Furthermore, DGE analysis reveals that under CAR T cell treatment MKN-45 tumors upregulated interferon alpha inducible protein 27 (IFI27), TNFRSF14 and a group of pathways related to metabolic activity and antigen processing and presentation, implying the evolution of tumor cells to resist T cell killing. Conclusions: Our findings highlight the complex tumor-T cell interplay as tumor evades CAR T cell killing. Tumor resistance is associated with CAR T cell transition from cytotoxic to dysfunctional phenotypes, the suppression of expanded Treg cells, and the acquisition of resistant features by tumors. Citation Format: Yanping Yang, Raymond Louie, Janusz Puc, Yogindra Vedvyas, Yago Alcaina, Irene M. Min, Matt Britz, Fabio Luciani, Moonsoo M. Jin. Single cell analysis reveals tumor evolution and CAR T cell dysfunction associated with tumor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 912.

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.

Introduction: Prostate cancer is the third most frequent cause of cancer-related death in men worldwide. Since prostate-specific membrane antigen (PSMA) shows a significant overexpression on prostatic cancer cells, PSMA can be considered a promising target for prostate cancer imaging and treatment. Although chimeric antigen receptor (CAR) T cells have shown great clinical outcomes in hematological malignancies, there is still limited efficacy in solid tumors due to the lack of tumor-specific antigens, low T cell infiltration in the tumors, immune suppressive tumor microenvironment, and on-target off-tumor toxicity in healthy tissues. The antigen recognition module of CAR T cells is usually a single-chain variable fragment (scFv). In contrast, the variable domains of heavy-chain antibodies (VHH) are small, stable, single-domain antibody fragments with high affinity and do not require the supramolecular assembly. Severe fatalities recently halted clinical trials of PSMA CAR T cells, so affinity tuning may be required to avoid CAR T targeting normal tissues. Here, we developed VHH-based CAR T cells targeting PSMA, able to co-express human somatostatin receptor 2 (SSTR2) actioning as a PET reporter for tracking CAR T cell distribution, as an immunotherapeutic strategy for prostate cancer. Methods: Primary human T cells were isolated and transduced with PSMA CAR lentivirus at 24 and 48 hours after activation with anti-CD3/CD28 Dynabeads. Mouse models of 8505C cell line transduced to overexpress PSMA were utilized to test the efficacy of CAR T cells. Tumor growth was monitored regularly by bioluminescence imaging. PET/CT imaging was performed to monitor T cell localization and biodistribution using ¹⁸F-NOTA-octreotide radiotracer targeting SSTR2 introduced in CAR T cells. Results: By alanine substitution of CDR3 in the VHH, we identified several low affinity candidates. The affinity of the VHH-based PSMA CAR was determined to be 22 nM by a saturation binding assay using monomeric human PSMA protein. PSMA CAR expression in primary human T cells was higher than 90%. Compared to non-treated mice, PSMA targeting CAR T cells led to tumor reduction and improved survival in animal models. Moreover, high levels of T cell infiltration and localization in tumor tissues were found, based on PET/CT imaging. Considering possible treatment-associated toxicities, which led to 2 patient deaths and the halt of one phase 1 trial of PSMA CAR T cells, we will affinity tune VHH-based CAR to improve the safety profile. Thus, additional studies will be performed to test both activity and safety of affinity variants of PSMA CAR T cells. Conclusions: We developed PSMA targeting CAR T cells able to induce potent and specific killing of prostate cancer cells. This strategy demonstrated significant therapeutic efficacy in vivo against animal models. Therefore, CAR T cells targeting PSMA may be a promising treatment strategy for patients with prostate cancer. Citation Format: Juan Gonzalez-Valdivieso, Yanping Yang, Yogindra Vedvyas, Yago Alcaina, Moonsoo M. Jin. PSMA targeting CAR T cell immunotherapeutic strategy for prostate cancer [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 5577.

Background: The use of chimeric antigen receptor (CAR) T cells for solid tumors has a number of challenges, such as lack of tumor-specific targets, CAR T cell exhaustion, and the immunosuppressive tumor microenvironment. To address these challenges, AffyImmune has developed technologies to affinity tune and track CAR T cells in patients. The targeting moiety is affinity tuned to preferentially bind to tumor cells overexpressing the target while leaving normal cells with low basal levels untouched, thereby increasing the therapeutic window and allowing for more physiological T cell killing. The CAR T cells are engineered to co-express somatostatin receptor 2 (SSTR2), which allows for the tracking of CAR T cells in vivo via PET/CT scan using FDA-approved DOTATATE. Methods: AIC100 was generated by affinity tuning the I-domain of LFA-1, the physiological ligand to ICAM-1. Various mutants with 106-fold difference in affinity were evaluated for structure activity relationships using targets with varying antigen densities. The AIC100 with micromolar affinity was clearly the most effective in non-clinical animal models. AIC100 is currently being evaluated to assess safety, CAR T expansion, tumor localization, and preliminary activity in patients with advanced thyroid cancer (ATC) in a phase I study (NCT04420754). Our study uses a modified toxicity probability interval design with three dosage groups of 10 × 106, 100 × 106, and 500 × 106 cells. Results: Preclinical studies demonstrated greater in vivo anti-tumor activity and safety with micromolar affinity CAR T cells. A single dose of AIC100 resulted in tumor elimination and significantly improved survival of animals bearing ATC xenografts. AIC100 activity was confirmed in other high ICAM-1 tumor models including breast cancer, gastric cancer, and multiple myeloma. In a Phase I patient given 10 × 106 CAR T cells, near synchronous imaging of FDG and DOTATATE revealed preliminary evidence of transient CAR T expansion and tumor reduction at multiple tumor lesions, with the peak of CAR T cell density coinciding with the spike in CAR T cell numbers in blood. Conclusion: We have developed affinity tuned CAR T cells designed to selectively target ICAM-1 overexpressing tumor cells and to spatiotemporally image CAR T cells. Near-synchronous FDG and DOTATATE scans will enhance patient safety by early detection of off-tumor CAR T activity and validation of tumor response. We anticipate that our “tune and track” technology will be widely applicable to developing potent yet safe CAR T cells against hard-to-treat solid cancers. Citation Format: Jingmei Hsu, Yen-Michael Hsu, Koen van Besien, Thomas J. Fahey, Jana Ivanidze, Janusz Puc, Karrie Du, Yanping Yang, Yogindra Vedvyas, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. First-in-human study of ICAM-1-specific affinity tuned CAR T cells against advanced thyroid cancer [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 5579.

Intercellular adhesion molecule-1 (ICAM-1) participates in many important processes, including leukocyte endothelial transmigration, cell signaling, cell-cell interaction, cell polarity and tissue stability. ICAM-1 is highly expressed in inflammatory conditions, chronic diseases and several cancers, like anaplastic thyroid cancer (ATC) and triple negative breast cancer (TNBC). This rationale has motivated the pursuit of ICAM-1 as an emerging Immuno-Onco target, with many groups developing adoptive cell therapies against ICAM-1. Visualizing the ICAM-1 expression in the patients noninvasively is critical for a successful treatment. Identifying the ideal candidate patient, track the therapeutic effect of these treatments, the changes implicated in these diseases, or even after therapy for efficacy. PET imaging would be ideal due to its quantifiability, but unfortunately so far there is no such PET tracer to image ICAM-1 in patients. Here, we developed a nanobody based PET platform to image ICAM-1. Nanobodies are the smallest known functional antibody fragment. They are derived from heavy-chain only antibodies (HcAbs) in camelids. They retain high affinity and specificity for their target antigens, with low off-target accumulation due to their hydrophilic properties. Their nanoscale dimensions enable deep penetration of tumors, tolerance of high temperatures, elevated pressures, non-physiological pH, and even strong chemical denaturants. We generated these nanobodies by immunizing alpaca with Fc-fused human ICAM-1, produced and purified from mammalian cell transfection media. The harvested B cells from these immunized alpaca were used to generate a cDNA library, containing transcripts of the VHH domain of the HcAbs. This cDNA library was subsequently cloned into a yeast-surface display platform. After three rounds of panning we screened 160+ ICAM-1 binding clones from which we identified 8 unique clones with high to moderate binding to ICAM-1. Among which, clone A7 was utilized for nanobody-PET due to its high affinity (~115nM) and specificity to ICAM-1. With the goal of having a nanobody-PET that would be easy to execute at a moment’s notice and translated into clinic, we decided to utilize NOTA/aluminum-fluoride chemistry to radio label our PET tracer. For consistency in labeling, we utilized sortase to label the nanobody with NOTA at a 1:1 ratio. We demonstrated the utility of this ICAM-1 nanobody-PET tracer in subcutaneous tumor mouse xenograft model. PET/CT imaging shows clear localization to ICAM-1-positive xenograft, with low background uptake apart from minor gallbladder and kidney clearance. The success of this tracer in conjunction with the ease of NOTA/aluminum-fluoride chemistry opens up the possibility to carry over nanobody-PET to other targets (e.g., MSLN, EpCAM). Citation Format: Yogindra Vedvyas, Juan Gonzalez-Valdivieso, Yago Alcaina, Yanping Yang, Moonsoo M. Jin. Engineering easy-to-implement [18F] nanobody-PET for emerging immuno-onco target, ICAM-1 [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 5976.

Engineering CAR T cells to target solid tumors presents numerous challenges. Lack of tumor-specific antigens, high probability of CAR T cell exhaustion, and an often-immunosuppressive tumor microenvironment limit CAR T cell efficacy and outcomes in the clinic. To address these challenges, AffyImmune has developed “Tune & Track” technologies to reduce CAR affinity for antigens with disproportionally high density on tumor cells. Our CAR T cells are selective for tumor cells while leaving normal cells untouched, thereby increasing the therapeutic window and allowing for more physiological T cell killing. CAR T cells are also designed to express Somatostatin Receptor 2 (SSTR2), which allows for tracking in vivo via PET/CT scan using FDA-approved DOTATATE. Our primary CAR design called AIC100 expresses an affinity-tuned variant of LFA-1, which is the natural ligand for Intracellular Adhesion Molecule (ICAM)-1, a molecule that is highly expressed on some cancers. A single dose of AIC100 CAR T cells results in elimination of tumors in mice and significantly improves survival. We have tested the safety and anti-tumor activity of AIC100 in high ICAM-1-expressing animal tumor models including triple-negative breast cancer and gastric cancer. AffyImmune is currently conducting a Phase I study (NCT04420754) to evaluate the clinical efficacy of AIC100 in treating advanced thyroid cancer. After receiving 10 million AIC100 CAR T cells, FDG and DOTATATE imaging revealed preliminary evidence of CAR T expansion in the patient and tumor reduction at multiple lesions. To further optimize affinity-tuned CAR T cell activity within tumors, we are currently developing CAR designs that have the capability to deliver effector cytokines to the tumor microenvironment via a nuclear factor of activated T cells (NFAT)-inducible promoter. Because our CAR T cells are affinity-tuned, off-tumor cytokine secretion is minimized. Our ICAM-1-specific CAR design called AIC1010 releases interleukin (IL)-12 cytokine upon engagement with target antigen within tumors, which induces production of interferon (IFN)-γ from immune cells and surrounding tissues, enhancing not only CAR T cell effector function but also the immune response focused within the tumor microenvironment. AIC1010 CAR T cells demonstrate superior cancer elimination compared to AIC100 CAR T cells in subcutaneous and IP tumor models in mice. We anticipate that cytokine delivery, paired with our “Tune & Track” technology will be widely adaptable to develop potent and safe affinity-tuned CAR T cells against hard-to-treat solid cancers. Citation Format: Jingmei Hsu, Michael P. Gallagher, Yanping Yang, Eric von Hofe, Yen-Michael Hsu, Koen Van Besien, Thomas Fahey, Jana Ivanidze, Janusz Puc, Karrie Du, Moonsoo Jin. Focused IL-12 cytokine delivery enhances function of affinity-tuned and real-time tracked ICAM-1-specific CAR T cells in solid tumors [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 558.

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.

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.