Suksanpaisan, Lukkana

Abstract Although chimeric antigen receptor T (CART)–cell therapy has been successful in treating certain hematologic malignancies, wider adoption of CART-cell therapy is limited because of minimal activity in solid tumors and development of life-threatening toxicities, including cytokine release syndrome (CRS). There is a lack of a robust, clinically relevant imaging platform to monitor in vivo expansion and trafficking to tumor sites. To address this, we utilized the sodium iodide symporter (NIS) as a platform to image and track CART cells. We engineered CD19-directed and B-cell maturation antigen (BCMA)–directed CART cells to express NIS (NIS+CART19 and NIS+BCMA-CART, respectively) and tested the sensitivity of 18F-TFB-PET to detect trafficking and expansion in systemic and localized tumor models and in a CART-cell toxicity model. NIS+CART19 and NIS+BCMA-CART cells were generated through dual transduction with two vectors and demonstrated exclusive 125I uptake in vitro. 18F-TFB-PET detected NIS+CART cells in vivo to a sensitivity level of 40,000 cells. 18F-TFB-PET confirmed NIS+BCMA-CART-cell trafficking to the tumor sites in localized and systemic tumor models. In a xenograft model for CART-cell toxicity, 18F-TFB-PET revealed significant systemic uptake, correlating with CART-cell in vivo expansion, cytokine production, and development of CRS-associated clinical symptoms. NIS provides a sensitive, clinically applicable platform for CART-cell imaging with PET scan. 18F-TFB-PET detected CART-cell trafficking to tumor sites and in vivo expansion, correlating with the development of clinical and laboratory markers of CRS. These studies demonstrate a noninvasive, clinically relevant method to assess CART-cell functions in vivo.

Since its emergence at the end of 2019, SARS-CoV-2, the causative agent of COVID-19, has caused over 100 million infections and 2.4 million deaths worldwide. Recently, countries have begun administering approved COVID-19 vaccines, which elicit strong immune responses and prevent disease in most vaccinated individuals.

Despite the success of chimeric antigen receptor T (CART) cell therapy, it is limited by 1) lower rates of durable responses related to inadequate CART cell expansion and trafficking to tumor sites and 2) development of life-threatening complication such as cytokine release syndrome (CRS). Development of a strategy to efficiently and robustly image and track CART cells in the clinic would allow the in vivo characterization of T cell expansion and trafficking to tumor sites as well as the development of strategies to potentially overcome these limitations. The sodium iodide symporter (NIS) is a characterized and sensitive reporter system that has been used for cell imaging in the clinic. We hypothesized that the incorporation of NIS into CART cells would be a sensitive and efficient way to assess CART cell expansion, trafficking, and toxicity. To test our hypothesis, we used two CART cell constructs that are characterized in preclinical models and studied extensively in the clinic: CART19 (41BB costimulated) and BCMA-CART cells (41BB costimulated). First, we generated NIS+CART19 and NIS+BCMA-CART cells through dual transduction of lentiviral vectors (Fig A) and revealed the exclusive 125I uptake by these NIS+CARTs and its inhibition by the NIS inhibitor KClO4in vitro (Fig B). We then analyzed T cell functions of NIS+CART cells. Here, NIS+CART19 or CART19 cells were cultured with the CD19+ acute lymphoblastic leukemia (ALL) cell line NALM6. There was no difference in CART cell cytotoxicity (Fig C), proliferation, or cytokine production (not shown) between NIS+CART19 and CART19. This indicates that the incorporation of NIS into CART cells does not impair their antitumor activity. Next, we evaluated the sensitivity of NIS+CART19 cell detection by TFB-PET in vivo; imaging was performed using an Inveon TFB-PET/CT scanner. Mice received 250 μCi 18F-TFB 45 minutes prior to image acquisition. NIS+CART cells were detectable with TFB-PET when cells were subcutaneously injected at a dose of 1.25x106 cells (Fig D). Having demonstrated that the incorporation of NIS in CART cells provides a sensitive way of their detection by TFB-PET and does not interfere with their effector functions, we tested its efficiency to assess CART cell trafficking in vivo, using multiple myeloma (MM) xenografts. Here, immunocompromised NOD-SCID-ɣ-/- (NSG) mice were engrafted with the BCMA+OPM2 MM cell line (1x106 IV). After engraftment, the tumor burden was assessed by bioluminescence imaging (BLI) and mice were randomized to receive 1) BCMA-CART or 2) NIS+BCMA-CART cells (5x106 IV). Mice were then serially imaged for 1) bioluminescence as a measure of disease burden, and 2) with TFB-PET to assess CART cell expansion and trafficking. As expected, BLI demonstrated that MM predominantly engrafts in bones (Fig E). TFB-PET confirmed trafficking of the NIS+BCMA-CART cells to the bones, corresponding to the areas involved by MM based on BLI (Fig E, right). Both BCMA-CART and NIS+BCMA-CART cells exhibited similarly potent antitumor activity in this model (not shown). Finally, we aimed to explore whether TFB-PET can detect CART massive expansion in vivo and predict the development of CRS. Here, we used an established CRS model in our laboratory. NSG mice were engrafted with patient derived relapsed ALL blasts (5x106 IV). Engraftment was confirmed by peripheral blood sampling. When the leukemic burden is >10 CD19+ cell/µl, mice were treated with either high dose NIS+CART19 cells (5x106 IV) or PBS control. One week after NIS+CART19 cell treatment, mice developed muscle weakness, hunched bodies, and weight loss (Fig F), which correlate with an extreme elevation of cytokines (Sterner et al. Blood 2018). TFB-PET revealed a significant uptake in the bone marrow, spleen, liver, and lungs (Fig G) of the diseased mice but not control mice. Mice were then euthanized, and tissues were harvested. Flow cytometric analysis confirmed an extensive infiltration of CART cells in the liver and spleen. This demonstrates the ability of TFB-PET to detect NIS+CART cell expansion in vivo, correlating with the development of CRS. In summary, our results robustly show that the incorporation of NIS into CART cells provides a sensitive, clinically applicable platform to image CART cells using TFB-PET and to assess their expansion, trafficking to tumor sites, and the development of CRS. These studies illuminate a novel way to noninvasively assess CART cell functions in vivo. Figure Disclosures Sakemura: Humanigen: Patents & Royalties. Suksanpaisan:Imanis: Employment. Cox:Humanigen: Patents & Royalties. Parikh:Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding; Ascentage Pharma: Research Funding; Genentech: Honoraria. Kay:Agios: Other: DSMB; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; MorphoSys: Other: Data Safety Monitoring Board. Peng:Imanis: Equity Ownership. Russell:Imanis: Equity Ownership. Kenderian:Kite/Gilead: Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents & Royalties, Research Funding; Novartis: Patents & Royalties, Research Funding.

Abstract Introduction. VSV-IFNβ-NIS is an oncolytic vesicular stomatitis virus (VSV; Rhabdovirus family) with rapid replication kinetics and potent antitumor activity. VSV-IFNβ-NIS encodes the human interferon beta (IFNβ) gene as a STING agonist and the human sodium iodide symporter (NIS) as a reporter gene for tracking the pharmacokinetics (PK) of virus replication in infected tumors. VSV replicates selectively in cancer cells and has promising preclinical antitumor activity across a broad spectrum of cancer types. We report here the safety and correlative data from an FIH trial of intravenous (IV) administration of VSV-IFNβ-NIS in patients with stage IV or recurrent endometrial cancer (EC). Methods. There are two ongoing IV FIH trials using VSV-IFN-NIS, in patients with EC (NCT03120624) and one in patients with hematological malignancies (NCT03017820). In EC, it is a classical 3+3 phase I trial, starting at 5x109 TCID50 through 5x1011 TCID50, given as a single IV dose. The primary objective is safety and tolerability; secondary objectives include monitoring the PK of viral replication through SPECT/CT imaging with NIS gene, viremia, virus shedding, preliminary efficacy, changes in the immune profile of peripheral blood leukocytes, and immunohistochemistry for immune cell infiltrates in tumors. Results. Nine patients have received IV VSV-IFNβ-NIS to date; three with EC and six with hematologic malignancies. The highest dose administered to date is 1.7x1010 TCID50 and dose escalation is ongoing. No DLTs have been observed. Patients experienced the expected infusion related AEs including rigors, chills, nausea, fever, hypotension, and hot flashes. Multiple cytokines increased at 4h post infusion of virus, but most returned to baseline levels by 24h. Viremia was detectable in all patients at the end of infusion, and to varying levels at 30 mins, 1, 2, 4, 24, 48h or 72 hours post virus infusion. No persistent viremia was observed. No infectious virus was recovered in buccal swabs or urine and neutralizing anti-VSV antibodies were present by day 29. Extensive immune phenotyping for T cells, NK, MSDC, myeloid cells performed on peripheral blood cells collected at baseline and at day 3, 8, 15 and 29 post virus infusion showed a trend towards increased PD-1 expression on CD8+ cells. Early IHC data suggests an increase in CD3+ and CD8+ cells in tumor biopsies at day 29 and 3 months in patients with EC treated at the first dose level. Elispot assays for shared EC antigens are pending. Conclusions. IV administration of VSV-IFNβ-NIS up to doses of 1.7x1010 is safe and well tolerated. There is evidence of T cell activation with increased PD-1 expression in CD8+ T cells in the peripheral blood and increased in CD3+ and CD8+ cells in tumor biopsies. Updated results for the EC study will be reported. Citation Format: Jamie Bakkum-Gamez, Matthew S. Block, Nanda Packiriswamy, Bethany A. Brunton, Upreti Deepak, Jonathan M. Mitchell, Lukkana Suksanpaisan, Pamela Atherton, Amylou Dueck, Stephen J. Russell, Martha Q. Lacy, Kah-Whye Peng. First in human (FIH) dose escalation studies of intravenous administration of VSV-IFNβ-NIS (Voyager-V1™) in Stage IV or recurrent endometrial cancer [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 CT072.

Abstract Introduction Voyager-V1TM is derived from VSV, a bullet-shaped negative sense RNA virus with very low human seroprevalence; it is engineered to selectively replicate in and kill human cancer cells. Voyager-V1 encodes the human IFNβ gene to boost antitumoral immune responses and the thyroidal sodium iodide symporter NIS gene to permit noninvasive imaging of virus spread. FIH studies are underway via both IV and IT routes. Here we report safety and preliminary correlative data from the FIH IT study. Methods and objectives This is a classical 3+3 phase 1 design, using escalating single IT viral doses from 3 x 106 to 3 x 109 TCID50 into one target lesion. The primary objective is safety and tolerability, monitored by committee. Other objectives include PK by RT-PCR for viral genomes, serum IFNβ levels, Tc-99m SPECT/CT imaging to monitor virus infection in injected lesions, peripheral blood immunophenotyping with 11-color flow cytometry for activation markers on T cells, T-regs, NK cells, and MDSCs, and serial biopsies to assess the tumor microenvironment (TME). IHC was performed on tumor biopsies for CD3, CD8, CD4, FoxP3, CD68, PD-1 and PDL-1 pre and post treatment in non-injected and injected lesions. CD45 staining is ongoing. Results Dose level 3 is ongoing (n=8+). No DLTs have been observed to date. Most patients were male (75%), white (100%), with ECOG PS1 (75%) and a median of >6 lines of prior systemic therapy. AEs (in 63% patients) reported as related to study drug were mild-moderate, short-lived, and consisted of G1 fever, chills, hot flashes, nausea, vomiting, hyperhidrosis and G2 fatigue. Most patients had some mild AEs (pain, bruising, subclinical pneumothorax) related to biopsy and IT injection procedures and one had an SAE related to biopsy (G2 pneumothorax). There was no difference in incidence, intensity or duration of related AEs between dose levels so far. In patients analyzed to date, at the first three dose levels of 3 x 106, 1 x 107 and 3 x 107 TCID50 (n=7), viremia and serum IFNβ were below levels of detection after IT injection. There is evidence in all patients of an increase in PD-1 expression on CD4 and CD8 T cells, suggesting T cell activation post- single virus injection. One patient at dose level 2 had increased CD4 and CD8 T cells in the peripheral blood. TME analysis revealed increased infiltration of CD3 and CD8 cells in tumors of some patients. In addition, there are changes in numbers of FoxP3 and CD68 positive cells in the injected lesions compared to the pre-treatment and non-injected lesion. Two patients at dose level 3, one with an injected adrenal metastasis of colorectal cancer and another with a chest wall lesion from a head of pancreas primary, have positive SPECT/CTs on days 3-15 showing viral replication in tumor plus concomitant lymphocyte/neutrophil trafficking in the periphery. Conclusions IT injection of a single dose of the novel oncolytic virotherapy Voyager-V1 has proven safe at doses up to 3 x 107 TCID50. There was evidence of T cell activation with increased PD-1 expression in peripheral blood lymphocytes after a single IT injection. Tumor biopsies indicate increased lymphocyte infiltration. Two patients at the highest dose tested show signs of viral replication in the injected lesion plus evidence of systemic impact on leukocyte populations. Citation Format: Steven Powell, Manish R. Patel, Jaime R. Merchan, Timothy P. Cripe, James Strauss, Rosa M. Diaz, Nandakumar Packiriswamy, Bethany A. Brunton, Deepak Upreti, Rehan Khan, Lukkana Suksanpaisan, Rianna Vandergaast, Stephen J. Russell, Alice S. Bexon, Kah Whye Peng. Preliminary correlative and clinical data from a first-in-human (FIH) study of the intratumoral (IT) oncolytic virotherapy, Voyager-V1, in patients with solid tumors [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 CT051.

Abstract Clinical translation of intravenous therapies to treat disseminated or metastatic cancer is imperative. Comparative oncology, the evaluation of novel cancer therapies in animals with spontaneous cancer, can be utilized to inform and accelerate clinical translation. Preclinical murine studies demonstrate that single-shot systemic therapy with a vesicular stomatitis virus (VSV)-IFNβ-NIS, a novel recombinant oncolytic VSV, can induce curative remission in tumor-bearing mice. Clinical translation of VSV-IFNβ-NIS therapy is dependent on comprehensive assessment of clinical toxicities, virus shedding, pharmacokinetics, and efficacy in clinically relevant models. Dogs spontaneously develop cancer with comparable etiology, clinical progression, and response to therapy as human malignancies. A comparative oncology study was carried out to investigate feasibility and tolerability of intravenous oncolytic VSV-IFNβ-NIS therapy in pet dogs with spontaneous cancer. Nine dogs with various malignancies were treated with a single intravenous dose of VSV-IFNβ-NIS. Two dogs with high-grade peripheral T-cell lymphoma had rapid but transient remission of disseminated disease and transient hepatotoxicity that resolved spontaneously. There was no shedding of infectious virus. Correlative pharmacokinetic studies revealed elevated levels of VSV RNA in blood in dogs with measurable disease remission. This is the first evaluation of intravenous oncolytic virus therapy for spontaneous canine cancer, demonstrating that VSV-IFNβ-NIS is well-tolerated and safe in dogs with advanced or metastatic disease. This approach has informed clinical translation, including dose and target indication selection, leading to a clinical investigation of intravenous VSV-IFNβ-NIS therapy, and provided preliminary evidence of clinical efficacy and potential biomarkers that correlate with therapeutic response. Mol Cancer Ther; 17(1); 316–26. ©2017 AACR.

Transplantation of gene-corrected autologous hepatocytes can cure metabolic disease in a preclinical pig model of hereditary tyrosinemia type 1.

Cell transplantation is a potential treatment for the many liver disorders that are currently only curable by organ transplantation. However, one of the major limitations of hepatocyte (HC) transplantation is an inability to monitor cells longitudinally after injection. We hypothesized that the thyroidal sodium iodide symporter (NIS) gene could be used to visualize transplanted HCs in a rodent model of inherited liver disease: hereditary tyrosinemia type 1. Wild‐type C57Bl/6J mouse HCs were transduced ex vivo with a lentiviral vector containing the mouse Slc5a5 (NIS) gene controlled by the thyroxine‐binding globulin promoter. NIS‐transduced cells could robustly concentrate radiolabeled iodine in vitro, with lentiviral transduction efficiencies greater than 80% achieved in the presence of dexamethasone. Next, NIS‐transduced HCs were transplanted into congenic fumarylacetoacetate hydrolase knockout mice, and this resulted in the prevention of liver failure. NIS‐transduced HCs were readily imaged in vivo by single‐photon emission computed tomography, and this demonstrated for the first time noninvasive 3‐dimensional imaging of regenerating tissue in individual animals over time. We also tested the efficacy of primary HC spheroids engrafted in the liver. With the NIS reporter, robust spheroid engraftment and survival could be detected longitudinally after direct parenchymal injection, and this thereby demonstrated a novel strategy for HC transplantation. This work is the first to demonstrate the efficacy of NIS imaging in the field of HC transplantation. We anticipate that NIS labeling will allow noninvasive and longitudinal identification of HCs and stem cells in future studies related to liver regeneration in small and large preclinical animal models. Liver Transpl 21:442–453, 2015. © 2015 AASLD.