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Introduction: Chimeric antigen receptor therapies (CAR-T) are highly effective against hematologic malignancies but require a lymphodepleting chemotherapy regimen and are faced with many challenges including manufacturing time, scalability, and cost of production due to the need for ex vivo culturing of cells and complex chain of custody requirements. In vivo delivery of self-inactivating lentiviral vectors (LV) encoding for CAR-T transgenes represents a promising strategy to improve the time to treatment, scalability, and cost of current CAR-T therapies. Methods: Lentiviral vectors encoding a CD19 CAR with a synthetic driver element were manufactured in a chemically defined cell substrate that incorporates a modified envelope designed to target and activate CD3+ T cells. Two different humanized mouse models were utilized. In a PBMC humanized mouse model, NSG MHC Class I/II knock-out mice (DKO) were injected with 1E7 human PBMC and followed one day later with 2E7 TU of LV encoding for CD19 CAR. In parallel, CD34+ humanized NSG SGM3 mice were also administered 2E7 TU LV encoding CD19 CAR. Flow cytometry of peripheral blood samples was evaluated at various time points for the presence of CAR+ cells and CD20+ B cells. Additionally, tissue samples were examined by histopathology and PCR for vector copy numbers. Results: The NSG SGM3 humanized CD34+ mouse model exhibited efficient chimerism of human CD45+ hematopoietic cells (>50% of live cells in peripheral blood). Apart from CD15+ neutrophils, all major human immune cell components were well represented in peripheral blood including CD14+ monocytes, CD20+ B cells, and CD3+ lymphocytes. At study initiation, the T cell compartment in the SGM3 mice exhibited skewing towards CD4+ T cells. Injection of CD19 CAR-LV resulted in a significant reduction of CD20+ B cells as early as 5 days post-injection. CD3+ CAR+ cells were detected in peripheral blood by day 5 with evidence of CAR+ cell expansion at subsequent time points. Loss of CD20+ B cells was stable throughout the observation period with some mice exhibiting a complete elimination of B cells. Similarly, the PBMC humanized NSG DKO mice injected with CD19 CAR-LV also showed evidence of CAR+ cells in peripheral blood. Conclusions: CD3-directed self-inactivating lentiviral vectors can efficiently deliver an integrating CAR gene into T lymphocytes following in vivo administration. These in vivo generated CD19 CAR T cells expand systemically and effectively eliminate pre-existing B cells. These data show that the targeting of CD3 through in vivo delivery can produce functional CAR T cells and represents an innovative therapeutic opportunity to potentially overcome current manufacturing times, scalability, and cost challenges facing cell therapies. Citation Format: Frederic Vigant, Ani Kundu, Dongming Zhang, Wei Zhang, Ewa Jaruga-Killeen, Michelle Andraza, Gregory Schreiber, Alissa Kerner, Junyi Zhang, John Henkelman, Renata Soares, Ramya Yarlagadda, Gregory I. Frost, Sid P. Kerkar. In vivo delivery of a novel CD3-targeted lentiviral vector generates CD19 CAR-T cells in two different humanized mouse models and results in complete B cell depletion [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 3294.

Background: We have previously established the ability of CD3-directed lentiviral vectors encoding for CD19 or CD22 CARs to mediate robust anti-tumor immunity in humanized lymphoreplete mouse models. We now present additional mechanistic data for this novel subcutaneous (SC) CAR-T approach. Methods: Human PBMCs loaded with a self-inactivating lentiviral vector (LV) encoding CD19 CAR with a synthetic driver element were injected SC into autologous PBMC humanized NSG MHC I/II double knock out (DKO) mice. The LV was packaged with a modified envelope with the ability to target and activate CD3+ T cells. To first track the site of CAR formation in vivo following SC injection, PBMCs were loaded with an LV encoding a CD19 CAR and luciferase, and bioluminescence imaging (BLI) was performed. Additionally, histopathology of the site of injection and distal organs were examined. Evaluation for CAR+ cells was performed through immunohistochemistry and PCR detection. To examine the tropism of the CD3-directed CD19 CAR LV and characterize CAR+ cells, the phenotype of in vivo expanded CAR+ cells was evaluated. Further characterization of CAR+ cells was performed with in vitro studies. Results: Following SC injection of LV-loaded PBMCs, the first evidence of transgene expression utilizing BLI for luciferase was detected four to five days following SC injection. At the same time point, histologic examination of the SC site of injection revealed the formation of tertiary lymphoid structures (TLS) consisting of human CD8+ and CD4+ T cells, CD68+ macrophages, CD68+ dendritic cells, and a few CD20+ B cells. On day 13 post-SC injection, BLI detected the presence of CAR+ cells systemically beyond the site of injection and within subcutaneous Raji tumors implanted on the contralateral side. On day 14 post-SC injection, histologic examination showed sustained TLS within the SC tissue without signs of dermal acute inflammation or ulceration and evidence of CAR+ cells appearing in the spleen. CAR+ cells exhibited robust anti-tumor immunity with expansion into peripheral blood. CAR+ cells consisted of a distinct population of CD8+ T cells with NK-like features (TaNKs) and a CD3+ CD8+ CD56+ NKG2D+ cell phenotype. In vitro transduction of CD56 NK cell-depleted PBMC with the CD3-directed LV also led to CAR-TaNK formation. Conclusion: The subcutaneous injection of CD3-directed LV-loaded PBMCs leads to the formation of tertiary lymphoid structures at the site of injection and the development of distinct CD3+ CD8+ CD56+ NKG2D+ CAR-TaNK cells. These cells possess enhanced systemic proliferative capacity compared to traditional ex vivo manufactured 41BB CAR-T cells in a lymphoreplete mouse model and the ability to eliminate target cells in vivo with low numbers of starting cells (10,000 cells). Citation Format: Ani Kundu, Dongming Zhang, Frederic Vigant, Junyi Zhang, Greg Schreiber, Ewa Jaruga-Killeen, Alissa Kerner, Michelle Andraza, Wei Zhang, John Henkelman, Renata Soares, Gregory I. Frost, Sid P. Kerkar. Generation of tertiary lymphoid structures and CD3+ CD8+ CD56+ NKG2D+ CAR TaNK cells following subcutaneous injection of CD3-directed lentiviral-loaded PBMCs [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 560.

Background Adoptive 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. Methods Lentiviral 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. Results Four-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. Regression 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 mice Conclusions We 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 Approval All 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.

Adult respiratory distress syndrome is often refractory to treatment and develops after entering the health care system. This suggests an opportunity to prevent this syndrome before it develops. The objective of this study was to demonstrate that early application of airway pressure release ventilation in high-risk trauma patients reduces hospital mortality as compared with similarly injured patients on conventional ventilation.Systematic review of observational data in patients who received conventional ventilation in other trauma centers were compared with patients treated with early airway pressure release ventilation in our trauma center. Relevant studies were identified in a PubMed and MEDLINE search from 1995 to 2012 and included prospective and retrospective observational and cohort studies enrolling 100 or more adult trauma patients with reported adult respiratory distress syndrome incidence and mortality data.Early airway pressure release ventilation as compared with the other trauma centers represented lower mean adult respiratory distress syndrome incidence (14.0% vs. 1.3%) and in-hospital mortality (14.1% vs. 3.9%).These data suggest that early airway pressure release ventilation may prevent progression of acute lung injury in high-risk trauma patients, reducing trauma-related adult respiratory distress syndrome mortality.Systematic review, level IV.

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.