Loss of p27Kip1, a cyclin-dependent kinase inhibitor, is observed in aggressive prostate cancers. We demonstrated that intratumoral injections of recombinant adenovirus overexpressing p27Kip1 (Adp27) reduced the growth of prostate cancer xenografts in nude mice. Presently, we studied the mechanism(s) of cell death induced by Adp27 in prostate cancer cell line PC-3. Cells were infected with Adp27 and compared with those infected by empty virus or were non-infected. Cell cycle and typical markers of apoptosis were analyzed by flow cytometry in the presence of the following reagents: cycloheximide, pan-caspase inhibitor ZVAD-fmk, neutralizing anti-TNFR1, and anti-TNFR2. Overexpression of p27Kip1 protein and cell cycle arrest were noted within 24 h after Adp27-infection. Sub-G1 fraction, chromatin margination, and phosphatidylserine exposure were evident by the third day of treatment. Cycloheximide elevated sub-G1 fraction in Adp27-infected cells by threefold, while ZVAD-fmk reduced sub-G1 to control levels. Caspase-dependent apoptosis occurred in a third of the population, while two-thirds were ZVAD-fmk insensitive but TUNEL-positive. Flow cytometry showed increased expression of TNFR1 and TNFR2 in Adp27-infected cells. Neutralizing anti-TNFR1 decreased TUNEL-positive score, while anti-TNFR2 did not affect p27Kip1-induced apoptosis. This is the first report showing that p27Kip1 induces caspase-dependent and -independent stages of cell death that may involve TNF-signaling through TNFR1.

Introduction: Direct in vivo delivery of lentiviral vectors (LV) to generate CD19 CAR+ cells without the need for ex vivo preparation represents a promising approach to transform autologous CAR therapy into an off-the-shelf treatment. In these studies, direct administration of a new LV encoding a CD19 CAR into humanized NOD SCID gamma (NSG) mice expressing human IL-3, GM-CSF, and SCF (NSG-SGM3) resulted in a dose-dependent elimination of B cells in the peripheral blood, peritoneal fluid, bone marrow, and tissue of treated mice. Methods: CD3-directed LV encoding a CD19 CAR with a novel synthetic driver element was manufactured utilizing a 25L clinical scale suspension-based process. NSG-SGM3 mice transplanted with human CD34+ cells from cord blood were injected with LV doses (1E6 IU, 1E7 IU, or 5E7 IU) intraperitoneally (IP) or 1E7 IU intravenously (IV). Quantification of CD19 CAR+ cells and CD20+ B cells in peripheral blood, peritoneal fluid, and bone marrow was assessed by flow cytometry. Additionally, immunohistochemical analysis was performed to evaluate the tissue-resident human B cells and for any other histopathological observations following test article administration. Results: All CD34+ humanized NSG-SGM3 mice were confirmed to exhibit efficient human hematopoietic engraftment by flow cytometry prior to test article administration (peripheral blood hCD45: 68.9% ± 4.93%; hCD19+ B cells: 53.7% ± 5.11%). Direct LV administration at the 1E7 IU and 5E7 IU doses demonstrated a dose-dependent reduction in circulating human B cells compared to the control and 1E6 IU dose (p < 0.05). The synthetic driver elements co-expressed with the CAR led to the formation of unique CD3+ CD8+ CD56+ T and NK-like (TaNK) CD19 CAR+ cells in circulation. The 1E7 IU IP dose demonstrated ablation of B cells (total cells/uL) in peripheral blood (control: 9.67 ± 3.72 vs. LV treated: 0.157 ± 0.117), intraperitoneal fluid (control: 0.322 ± 0.244 vs. LV treated: 0.038 ± 0.029), bone marrow (control: 9.16± 1.83 vs. LV treated: 0.734 ± 0.864), and splenic tissue. Both IP and IV routes of administration showed significant B cell depletion at the 1E7 IU dose. However, complete B cell elimination in splenic tissue was only observed at the 5E7 IU dose. Non-treated CD34+ humanized NSG-SGM3 mice exhibited hepatic portal inflammation and moderate graft-versus-host disease (GVHD) in the colon. Interestingly, mice treated with LV encoding CD19 CAR exhibited decreased inflammatory pathology, suggesting potential B cell involvement in the inflammatory response in this model. Conclusion: In this study, direct in vivo delivery of LV encoding CD19 CAR resulted in the generation of functionally active CD19 CAR TaNK cells capable of eliminating target B cells in peripheral blood, peritoneal fluid, bone marrow, and tissue. Citation Format: Frederic Vigant, Ani Kundu, Ramya Yarlagadda, Cody Gowan, Michael Betts, Jonathan Kato, Renata Soares, Alan Ponce, Lintao Liu, Junyi Zhang, Ewa Jaruga-Killeen, Michelle Andraza, Suraj Kachgal, Gregory Schreiber, Wei Zhang, Gregory Wade, Gregory I. Frost, Sid P. Kerkar. In vivo delivery of CD3-directed CD19-CAR lentivectors leads to the generation of CAR T and NK-like (CAR-TaNK) cells capable of complete ablation of B cells in the blood, bone marrow, and tissue of NSG-SGM3 CD34+ humanized mice [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 2918.

BackgroundAdoptive cellular therapy with chimeric antigen receptor (CAR)-T cells has demonstrated remarkable clinical activity in a number of hematologic malignancies, but product chain of custody, individualized manufacturing, preparative chemotherapy, and patient management present technical and logistical hurdles to broader implementation.MethodsLentiviral constructs for CARs (either CD19- or CD22-directed) co-expressed with a synthetic driver domain were identified from a >6 × 10 diversity combinatorial library of proliferative elements, transmembrane domains, leucine zippers, and an EGFR epitope screened for cellular expansion in a lymphoreplete model. Modified serum-free-lentiviral manufacturing process was developed to reduce complexity of CAR-T and to introduce CD3-activating elements into the viral envelope allowing activation and transduction of resting lymphocytes from peripheral blood.ResultsFour-hour exposure of as little as 1 ml of blood to the CD3-directed CD19-targeted CAR encoding lentivirus followed by subcutaneous injection in NSG mice bearing CD19+/CD22+ Raji cells resulted in tumor regression (figure 1) and robust CAR-T cell expansion as determined by flow cytometry (figure 2) and qPCR (table 1), with peak levels >10,000 CAR-T cells/µl and less than three CAR copies per genome. In contrast, administration of the same products intravenously failed to support significant CAR-T expansion or control tumor growth (figure 3). Regression of established Raji tumors was also observed in NSG-(KbDb) (IA) animals following SC administration of CD19 or CD22 CARs with driver domains. CAR-T cells contracted in peripheral blood following tumor regression.Abstract 117 Figure 1Tumor RegressionRegression of Raji tumor from the initial median volume of 151 mm3 throughout 40 days post subcutaneous administration of the LV transduced (at MOI 1 or 5) CD19-directed CAR T product (1M or 5M cells) in the NSG miceAbstract 117 Figure 2CAR-T Cells expansion in Vivo Post SC injectionExpansion of CAR-T cells throughout 35 days post subcutaneous administration of the LV transduced (at MOI 1 or 5) CD19-directed CAR T product (1M or 5M cells) in the NSG mice bearing Raji tumor (groups G1-G4) or w/o tumor (G5-7G)Abstract 117 Figure 3CAR-T Cells expansion in vivo post IV injectionExpansion of CAR-T cells throughout 35 days post intravenous administration of the LV transduced (at MOI 1 or 5) CD19-directed CAR T product (1M or 5M cells) in the NSG mice bearing Raji tumor (groups G1-G7).Abstract 117 Table 1qPCR Analysis of LV IntegrationNumber of LV integrations (LV copies) in the PBMC genome (RNase P copies) post transduction at MOI 1 or 5.ConclusionsWe conclude that through a synthetic subcutaneous lymph node approach with modified lentiviruses and driver domains, rPOC SC may enable CAR-T generation with reduced complexity, while maintaining the ability of CAR-T cells to expand, persist and exert anti-tumor activity.Ethics ApprovalAll animal studies were IACUC approved.

Background: Adoptive cell therapies using Chimeric Antigen Receptors (CARs) show durable clinical benefit for patients with hematologic malignancies, however, challenges remain for enabling this personalized treatment to be delivered in a timely, cost effective, and logistically friendly manner. Methods: Lentiviral vectors (LV) encoding CD19 and CD22 CARs with a synthetic driver element were packaged with a VSV-G envelope designed with the capability to target and activate CD3pos T cells in whole blood. LV were directly reconstituted in peripheral whole blood for four hours and total nucleated cells (TNC) or peripheral blood mononuclear cells (PBMC) were rapidly isolated utilizing two different closed systems. Immediately following the four-hour exposure event, isolated TNC or PBMC were injected subcutaneously into mice with disseminated Raji luciferase tumor cells. For further characterization, LV-exposed TNC and PBMC were cultured in vitro for six days and functionally examined. Results: Following four-hour exposure to CD3-directed LVs, more than 90% of T cells, including naïve/naïve derived (CCR7pos CD45ROneg) and central memory (CCR7pos CD45ROpos) T cells present within isolated TNC or PBMC exhibited a significant decrease in CD3 surface expression. Subcutaneous injection of gene modified TNC or PBMC resulted in the in vivo generation and expansion of large numbers of circulating CAR-T positive cells with complete eradication of disseminated Raji tumors. In parallel cell culture experiments, TNC or PBMC isolated following four-hour LV whole blood exposure exhibited robust expansion without additional T-cell receptor (TCR) or CD3 stimulation, while TNC or PBMC not exposed to virus did not show any expansion. Following six days in culture, immunophenotyping by flow cytometry demonstrated that more than 90% of the cells were CD8pos and CD4pos T cells with CAR-T expression present on central memory (CCR7pos CD45ROpos) and effector memory (CCR7neg CD45ROpos) T cells. CAR-T antigen specificity to CD19 and CD22 was measured by IFN-gamma release co-culture assays. Conclusion: We conclude that large numbers of functionally active CAR-T positive cells can be generated both in vitro and in vivo following a four-hour peripheral whole blood exposure to CD3-directed LVs encoding for CARs with a synthetic driver element. These results provide the basis for an autologous same-day peripheral blood draw to subcutaneous injection rapid point-of-care (rPOC) approach. Citation Format: Dongming Zhang, Frederic Vigant, Qun He, Anirban Kundu, Wei Zhang, Hongliang Zong, Ewa Jaruga-Killeen, Gregory Schreiber, Michelle Andraza, Alissa R. Kerner, Gregory I. Frost, Sid P. Kerkar. Subcutaneous injection of total nucleated cells rapidly isolated following four-hour peripheral whole blood exposure to CD3-directed CAR-T lentiviruses with a synthetic driver results in robust CAR-T proliferation and anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1511.