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Chimeric antigen receptor (CAR) T cells targeting CD19 are a breakthrough treatment for relapsed, refractory B cell malignancies1–5. Despite impressive outcomes, relapse with CD19− disease remains a challenge. We address this limitation through a first-in-human trial of bispecific anti-CD20, anti-CD19 (LV20.19) CAR T cells for relapsed, refractory B cell malignancies. Adult patients with B cell non-Hodgkin lymphoma or chronic lymphocytic leukemia were treated on a phase 1 dose escalation and expansion trial (NCT03019055) to evaluate the safety of 4-1BB–CD3ζ LV20.19 CAR T cells and the feasibility of on-site manufacturing using the CliniMACS Prodigy system. CAR T cell doses ranged from 2.5 × 105–2.5 × 106 cells per kg. Cell manufacturing was set at 14 d with the goal of infusing non-cryopreserved LV20.19 CAR T cells. The target dose of LV20.19 CAR T cells was met in all CAR-naive patients, and 22 patients received LV20.19 CAR T cells on protocol. In the absence of dose-limiting toxicity, a dose of 2.5 × 106 cells per kg was chosen for expansion. Grade 3–4 cytokine release syndrome occurred in one (5%) patient, and grade 3–4 neurotoxicity occurred in three (14%) patients. Eighteen (82%) patients achieved an overall response at day 28, 14 (64%) had a complete response, and 4 (18%) had a partial response. The overall response rate to the dose of 2.5 × 106 cells per kg with non-cryopreserved infusion (n = 12) was 100% (complete response, 92%; partial response, 8%). Notably, loss of the CD19 antigen was not seen in patients who relapsed or experienced treatment failure. In conclusion, on-site manufacturing and infusion of non-cryopreserved LV20.19 CAR T cells were feasible and therapeutically safe, showing low toxicity and high efficacy. Bispecific CARs may improve clinical responses by mitigating target antigen downregulation as a mechanism of relapse. A new bispecific CAR T cell product targeting the CD20 and CD19 antigens demonstrates an excellent safety profile and high clinical efficacy in patients with B cell non-Hodgkin lymphoma and chronic lymphocytic leukemia.

Gene targeting (GT) for precise gene insertion or swap into pre-defined genomic location has been a bottleneck for expedited soybean precision breeding. We report a robust selectable marker-free GT system in soybean, one of the most economically important crops. An efficient Oh H1-8 (Ochrobactrum haywardense H1-8)-mediated embryonic axis transformation method was used for the delivery of CRISPR-Cas9 components and donor template to regenerate T0 plants 6–8 weeks after transformation. This approach generated up to 3.4% targeted insertion of the donor sequence into the target locus in T0 plants, with ∼ 90% mutation rate observed at the genomic target site. The GT was demonstrated in two genomic sites using two different donor DNA templates without the need for a selectable marker within the template. High-resolution Southern-by-Sequencing analysis identified T1 plants with precise targeted insertion and without unintended plasmid DNA. Unlike previous low-frequency GT reports in soybean that involved particle bombardment–mediated delivery and extensive selection, the method described here is fast, efficient, reproducible, does not require a selectable marker within the donor DNA, and generates nonchimeric plants with heritable GT.

Genetically engineered chimeric antigen receptor (CAR) T cells have exhibited distinct effectiveness against chemotherapy refractory CD19 expressing B cell malignancies in both adults and children. This phase I clinical trial tests a novel anti-CD19 CAR T cell product in adults with relapsed/refractory (R/R) B-cell Non-Hodgkin’s Lymphoma (B-NHL). The CAR construct is comprised of the short chain variable regions of the anti-CD19 monoclonal antibody FMC63, the TNFRSF19-derived transmembrane domain, the 4-1BB costimulatory domain, and the CD3-zeta signaling domain. CD19 CAR T cells were manufactured utilizing the CliniMACS Prodigy® T Cell Transduction Process (CD3/CD28 TransAct™ reagent) allowing for highly automated production, with IL-7 and IL-15 used for T cell expansion for 8-12 days. To date, 7 patients have been treated with an average dose of 1.2 ± 0.2 x 108 CAR T cells. The histology includes marginal zone lymphoma (n=1), follicular lymphoma Grade IIIA (n=1), transformed lymphoma (n=3), follicular lymphoma low grade (n=2), and diffuse large B-cell lymphoma (n=1). One patient required a second apheresis due to poor cell expansion. Despite heterogeneity in disease subtype and leukapheresis product quality, CAR T production and expansion have been consistent with final transduction efficiencies between 14-45%, cell viability between 88-91%, and an overall average yield of 3.2 ± 0.3 x 109 cells before harvest, allowing for product banking. No safety-related out of specifications (OOS) events have occurred, however, 2 patients had OOS product infused due to low transduction efficiency (both at 14% rather than the ≥ 20% release criteria). Two patients experienced Grade 2 CRS, 1 patient experienced Grade 2 neurotoxicity; otherwise, no new safety signals were detected. Disease response was assessed on Days 90, 180, 270, and 360 post-infusion. The assessments were based on 2014 Lugano criteria. Even with 2 OOS products, Day 90 scans showed a complete metabolic remission (CMR) in 6 evaluable patients to date. Of the 6 patients with CMR, 1 patient progressed at Day 180 and the others remain in remission (median f/u = 12 months). Flow cytometry was utilized to measure CAR T cell peak expansion and persistence in 5 patients. Peak CAR T cell expansion (2.9-44.4% of CD3 cells) ranged from Day 5 to 15. Cell persistence was detected for the 5 patients through at least Day 180. ddPCR is currently in development to perform persistence testing in parallel. Additionally, cytokine concentrations including INFγ, IL-10, IL-12p70, IL-13, IL1β, IL-2, IL-4, IL-6, IL-8, and TNFα were evaluated over the first 30 days. Overall, 7 patients diagnosed with 5 different B-NHL subtypes have been treated with the CD19 CAR T cell product. Manufacturing was successful for all patients with no safety related OOS, and no new post-infusion safety signals detected. To date, 6 out of 7 patients are alive, 5 with CMR and with CD19 CAR T cell persistence through at least 180 days. Citation Format: Manali Kamdar, Cheri Adams, Steven Bair, Boro Dropulic, Jonathon Gutman, Bradley Haverkos, Kimberly Jordan, Rebecca Mallo, Russell Marians, Felicia Mast, Lindsey Murphy, Andrew Roth, Matthew Seefeldt, Andrew Worden, Mike Kadan, Ying Xiong, Dina Schneider, Rimas Orentas, Terry Fry, Michael Verneris. Feasibility and safety of a novel CD19 CAR T cell therapy in adults with R/R B-NHL [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 CT522.

INTRODUCTION: Anti-CD19 chimeric antigen receptor T (CAR-T) cells have demonstrated activity against relapsed/refractory lymphomas. Cytokine release syndrome (CRS) and CAR-T related encephalopathy syndrome (CRES/ICANS) are well-known complications of CAR-T cell therapy. Tocilizumab, a humanized monoclonal antibody targeting the interleukin 6 (IL-6) receptor, is approved for treatment of CRS. Our institutional standard was modified to administer prophylactic tocilizumab before infusion CAR-T cell products. We present the outcomes of subjects treated with locally manufactured antiCD19 CAR-T cells (TNFRSF19 transmembrane domain, CD3Zeta/4-1BB costimulatory signaling) with and without prophylactic tocilizumab. METHODS: Relapsed / refractory (r/r) lymphoma patients (pts) treated with anti-CD19 CAR-T cells at our institution were included. Baseline demographic and clinical characteristics, as well as laboratory results were obtained from our Hematologic Malignancies and Stem Cell Therapy Database. Prior to institution of prophylactic tocilizumab, pts received this agent only if they presented evidence of CRS grade 2 or higher. In May 2019, our institutional practice changed to provide tocilizumab 8mg/kg, 1 hour prior to infusion of CAR-T cell product. CRS was measured according to the ASTCT Consensus Grading, whereas CRES was measured using the CARTOX-10 criteria. Comparisons between groups were done with the Mann-Whitney U test for continuous variables and Fisher's exact test for categorical variables. RESULTS: Twenty-three relapsed / refractory lymphoma pts were treated with antiCD19 CAR-T cells; 15 pts received prophylactic tocilizumab. Median follow up was 312 days (range 64 - 679) days. Baseline characteristics are listed in table 1. Both groups were similar: There were no statistically differences in the rate of bulky, refractory disease, prior ASCT or number or prior lines of therapy. Baseline lymphocyte counts, C - reactive protein (CRP) and were also comparable between groups (Table 2). We did not observe immune adverse reactions to tocilizumab infusion. There were no differences in the incidence of cytopenias or infectious complications between groups. CRS of any grade was observed in 6/8 (75%) of pts without prophylactic tocilizumab vs. 6/15 (40%) in pts treated with prophylactic tocilizumab (p = 0.23), whereas CRS grade >1 was observed in 5 pts (62.5%) without prophylactic tocilizumab and in 3 pts (20%) treated with prophylactic tocilizumab (p = 0.02). There was no significant difference in the incidence of all grade CRES (no prophylaxis, 3/8 [38%] pts; prophylaxis 5/15 [30%] pts, p = 0.2969). There was a statistically significant difference in the peak CRP and peak ferritin without difference in the peak lymphocyte count after CAR-T infusion (Table 2, Figure 1). Patients given prophylactic tocilizumab had higher IL-6 plasma concentrations on day 2 after infusion (Figure 2). Complete response was observed in 4/8 (50%) pts without prophylactic tocilizumab vs. 12/15 (80%) pts with prophylactic tocilizumab (p = 0.18). All pts had detectable Anti-CD19 CAR-T cells on day 30, both groups had peak CAR-T expansion on day 14, with no statistically significant differences in expansion rates between groups. All evaluable subjects have had CAR-T persistence on days 60, 90, 180, and 365. CONCLUSIONS: Use of prophylactic tocilizumab prior to infusion of antiCD19 CAR-T cells is associated with reduced incidence of severe CRS and decreased levels of clinical laboratory markers of inflammation, despite increases in plasma concentration of IL-6. This decreased rate of grade ≥2 CRS is not associated with impaired disease control and did not result in increased rates of neurologic toxicity. Prophylactic tocilizumab does not appear to affect CAR-T cell expansion or persistence. Figure 1 Disclosures Caimi: ADC therapeutics: Other: Advisory Board, Research Funding; Celgene: Speakers Bureau; Amgen: Other: Advisory Board; Bayer: Other: Advisory Board; Verastem: Other: Advisory Board; Kite pharmaceuticals: Other: Advisory Board. Worden:Lentigen, a Miltenyi biotec company: Current Employment. Kadan:Lentigen, a Miltenyi biotec company: Current Employment. Orentas:Lentigen Technology, a Miltenyi Biotec Company: Research Funding. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. de Lima:Celgene: Research Funding; Pfizer: Other: Personal fees, advisory board, Research Funding; Kadmon: Other: Personal Fees, Advisory board; Incyte: Other: Personal Fees, advisory board; BMS: Other: Personal Fees, advisory board. OffLabel Disclosure: Use of tocilizumab as prophylaxis for CRS is not approved, whereas use for treatment is approved and on label.

Background Patients (pts) with rapidly progressive lymphoma and urgent need for therapy have worse prognosis and may not be able to receive CAR-T cells. Decreasing apheresis to infusion time can make CAR-T cells available to this patient population. We present the results of a phase I trial using rapid on-site CAR-T manufacture for relapsed/refractory B cell non Hodgkin lymphoma (NHL). Methods Adult pts with CD19+ B NHL who failed ≥ 2 lines of therapy were enrolled. Autologous T cells were transduced with a lentiviral vector (Lentigen Technology, Inc) encoding antiCD19 binding motif, CD8 linker and TNFRS19 transmembrane region, and 4-lBB/CD3z costimulatory domains. GMP compliant manufacture was done using CliniMACS Prodigy, in a 12 day culture. Dose escalation was done using 3+3 design, with 3 dose levels (0.5, 1 and 2 × 106 CAR-T cells/kg). Lymphodepletion was done with cyclophosphamide (60mg/kg × 1) and fludarabine (25mg/m2/d × 3). Results As of September 30, 2019, 14 pts were enrolled and treated. Table 1 lists baseline characteristics. 12 pts had refractory NHL, 6 had bulky disease and 9 had symptomatic disease at the time of lymphocyte collection. CAR-T cell product manufacture was successful in all pts. Median transduction rate was 48% [range 29-62] with median culture expansion of 41-fold [range 30-79]. All pts received their infusion of antiCD19 CAR-T cells. CAR-T cell doses were 0.5 × 106/kg (n = 4) and 1 × 106/kg (n = 10). Median apheresis to infusion time was 13 days [range 13–20]. CAR-T persistence, based on vector sequence, peaked in peripheral blood MNCs between days 14-21. All evaluable subjects had persistent CAR-Ts on PCR measurements done on days 30, 60 and 90. CAR-T cell dose did not have an impact in the time to peak in vivo CAR-T cell expansion or in the rate of CAR-T cell persistence (fig 1). Six pts experienced CRS. Five pts had grade 1 – 2 CRS and 1 pt died on day 8 due to CRS. Two pts presented grade 4 CRES, resolved after corticosteroids. No other grade ≥3 non-hematologic toxicity was observed. The most common non – hematologic toxicity was fatigue (n=7). Hematologic toxicity was common, with grade ≥ 3 neutropenia observed in all pts. Among 12 evaluable pts, 8 have achieved complete response (CR) and two had partial response (PR). Two pts did not respond. The CR rate was 67% and overall response rate was 83%. Three pts have died, causes of death include progressive disease (n=2) and CRS (n=1). After a median follow up 4.5 months (range 1 – 14) all responding pts are alive; 1 pt relapsed 6 months after treatment with CD19+ disease and entered CR after antiCD19 antibody drug immunoconjugate. Conclusions AntiCD19 CAR-T cells with TNFRS19 transmembrane domain have potent clinical activity. The short manufacture times achieved by local CAR-T cell manufacture enables treatment of a very high risk NHL population that would otherwise not be able to receive CAR-T products due to rapidly progressive disease.

Adoptive immunotherapy using chimeric antigen receptor-modified T cells (CAR-T) has made substantial contributions to the treatment of certain B cell malignancies. Such treatment modalities could potentially obviate the need for long-term antiretroviral drug therapy in HIV/AIDS. Here, we report the development of HIV-1-based lentiviral vectors that encode CARs targeting multiple highly conserved sites on the HIV-1 envelope glycoprotein using a two-molecule CAR architecture, termed duoCAR. We show that transduction with lentiviral vectors encoding multispecific anti-HIV duoCARs confer primary T cells with the capacity to potently reduce cellular HIV infection by up to 99% in vitro and >97% in vivo. T cells are the targets of HIV infection, but the transduced T cells are protected from genetically diverse HIV-1 strains. The CAR-T cells also potently eliminated PBMCs infected with broadly neutralizing antibody-resistant HIV strains, including VRC01/3BNC117-resistant HIV-1. Furthermore, multispecific anti-HIV duoCAR-T cells demonstrated long-term control of HIV infection in vivo and prevented the loss of CD4+ T cells during HIV infection using a humanized NSG mouse model of intrasplenic HIV infection. These data suggest that multispecific anti-HIV duoCAR-T cells could be an effective approach for the treatment of patients with HIV-1 infection.

Wound classification helps predict wound-related complications and is useful in stratifying surgical site infection reporting. We sought to evaluate misclassification among commonly performed surgeries that are at least clean-contaminated.The National Surgical Quality Improvement Program database was queried from 2005 to 2016 by Current Procedural Terminology codes identifying common surgeries that are, by definition, not clean: colectomy, cholecystectomy, hysterectomy, and appendectomy. Univariate analysis and multivariate logistic regression were performed.Of the 1,208,544 operative cases reviewed, 22,925 (1.90%) were misclassified as clean. Hysterectomy was the most commonly misclassified operation (3.11%), and colectomy the least (0.82%). Misclassification was higher in laparoscopic cases (1.92% versus 1.82%; P 0.01). Misclassification increased from 2005 to 2016 (0.22% versus 3.11%; P 0.01). Misclassified patients were younger (46.7 versus 47.7 y; P 0.01); had lower rates of hypertension, chronic obstructive pulmonary disease, smoking history, and steroid use (P 0.01); and had shorter length of stay (2.2 versus 3.2 d; P 0.01), lower 30-d readmission rates (3.7% versus 5.0%; P 0.01), and less surgical site infections (1.7% versus 3.4%; P 0.01). Open hysterectomy was the most significant positive predictor for misclassification (odds ratio 3.34; P 0.01). Open appendectomy was the most significant negative predictor (odds ratio 0.20; P 0.01).There is an increasing trend of misclassifying wounds as clean. Misclassified patients have better outcomes, and misclassification may be affected by patient characteristics, operative approach, and type of procedure rather than reflecting the true infectious burden. Further research is warranted.

Background RNA interference (RNAi) technology has been widely used to knockdown target genes via post-transcriptional silencing. In plants, RNAi is used as an effective tool with diverse applications being developed such as resistance against insects, fungi, viruses, and metabolism manipulation. To develop genetically modified (GM) RNAi traits for insect control, a transgene is created and composed of an inversely-repeated sequence of the target gene with a spacer region inserted between the repeats. The transgene design is subject to form a self-complementary hairpin RNA (hpRNA) and the active molecules are > 60 bp doubled-stranded RNA (dsRNA) derived from the hpRNA. However, in some cases, an undesirable intermediate such as single-stranded RNA (ssRNA) may be formed, which is not an active molecule. The aforementioned characteristics of RNAi traits lead to increase the challenges for RNAi-derived dsRNA quantitation. Results To quantify the dsRNA and distinguish it from the ssRNA in transgenic maize, an analytical tool is required to be able to effectively quantify dsRNA which contains a strong secondary structure. Herein, we develop a modified qRT-PCR method (abbreviated as RNase If -qPCR) coupled with a ssRNA preferred endonuclease (i.e., RNase If). This method enables the precise measurement of the active molecules (i.e., dsRNA) derived from RNAi traits of GM crops and separately quantifies the dsRNA from ssRNA. Notably, we also demonstrate that the RNase If -qPCR is comparable to a hybridization-based method (Quantigene Plex 2.0). Conclusions To our best knowledge, this is the first report of a method combining RNase If with modified qRT-PCR protocol. The method represents a reliable analytical tool to quantify dsRNA for GM RNAi crops. It provides a cost-effective and feasible analytical tool for general molecular laboratory without using additional equipment for other methods. The RNase If -qPCR method demonstrates high sensitivity (to 0.001 pg/ μL of dsRNA), precision and accuracy. In this report, we demonstrated the deployment of this method to characterize the RNAi events carrying v-ATPase C in maize during trait development process. The method can be utilized in any application which requires the dsRNA quantification such as double-stranded RNA virus or sprayable dsRNA as herbicide. Electronic supplementary material The online version of this article (10.1186/s12896-018-0413-6) contains supplementary material, which is available to authorized users.

Background: Salvage regimens for chemorefractory aggressive lymphoma achieve response rates of approximately 30%. Anti-CD19 CAR-T cells have demonstrated anti-lymphoma activity, but patients (pts) with rapidly progressive disease and urgent need for therapy have worse prognosis and many are not able to receive CAR-T cells in time. Decreasing the time from apheresis to infusion can make CAR-T cells available to pts with rapid progression of their disease. We present the results of a phase I clinical trial using on-site CAR-T manufacture for treatment of relapsed / refractory (r/r) B cell non Hodgkin lymphoma (NHL). Methods: Adult pts with r/r CD19+ B cell lymphomas who failed ≥ 2 lines of therapy were enrolled. Autologous T cells were transduced with a lentiviral vector (Lentigen Technology, Inc, LTG1563) encoding an anti-CD19 binding motif, CD8 linker and tumor necrosis receptor superfamily 19 (TNFRS19) transmembrane region, and 4-lBB/CD3z intracellular signaling domains. GMP-compliant manufacture was done using CliniMACS Prodigy, in a 12-day culture. Dose escalation was conducted according to a 3+3 design. Lymphodepletion was done with cyclophosphamide (60mg/kg x 1) and fludarabine (25mg/m2/d x 3). Cytokine release syndrome (CRS) and CAR-T related encephalopathy syndrome (CRES) were graded using the Lee and CARTOX criteria, respectively. Results: As of July 30, 2019 , 12 pts were enrolled and treated. Baseline characteristics are listed in table 1. 10/12 pts were refractory to the prior line of therapy, 5 had bulky disease and 9 had symptomatic disease at the time of lymphocyte collection. CAR-T cell product manufacture was successful in all pts. Median transduction rate was 48% [range 29-62] with and median culture expansion of 43-fold [range 30-79]. High dimensional flow cytometry showed the infused CD4 and CD8 CAR-T cells express a central memory and transition to memory - like profile, with enrichment for CD27 and high CCR7 expression. In addition, a subset of CD4 and CD8 CAR-T cells expressed effector transcription factors T-BET and GATA3 while CD4 CAR-T clusters express low levels of immune checkpoint blockers PD-1 and BTLA. All enrolled pts received their infusion of anti-CD19 CAR-T cells. CAR-T cell doses were 0.5 x 106/kg (n = 4) and 1 x 106/kg (n = 8). Median apheresis to infusion time was 13 days [range 13-20], 10 products were infused fresh. CAR-T persistence, based on vector sequence, peaked in peripheral blood MNCs between days 14-21. All responding subjects have had CAR-T persistence on follow up PCR measurements (range 1 - 12 months). CAR-T cell dose did not have an impact in the time to peak in vivo CAR-T cell expansion or in the rate of CAR-T cell persistence. Five pts experienced CRS. Grade 1 - 2 CRS was observed in 4 pts; whereas 1 pt died as a consequence of severe CRS in the context of bulky disease. Pharmacologic interventions for CRS included tocilizumab (n = 5), siltuximab (n = 2) and corticosteroids (n = 2). Two subjects presented grade 4 CRES with resolution after corticosteroids, no other grade ≥3 non-hematologic toxicity was observed. The most common all grade non - hematologic toxicity was fatigue, observed in 6 subjects. Hematologic toxicity was common, with grade ≥ 3 neutropenia observed in all subjects, with 4 subjects presenting grade 3 neutropenia without fever beyond day 30. Among 11 pts evaluable for response, 8 pts have achieved complete response (CR) and one had partial response (PR). Two pts did not respond. For the intention to treat population (n=12), the CR rate was 67% and overall response rate (ORR) was 75%. Overall response rates were equal between both dose levels (75%), but CR rates were higher in pts treated with 1 x 106 CAR-T cells (75% vs. 50%). Two pts have died, causes of death include progressive disease (n=1) and CRS (n=1). After a median follow up 3 months (range 1 - 12) all responding pts are alive; 1 subject relapsed 6 months after treatment with CD19+ disease and entered CR after anti-CD19 antibody drug immunoconjugate treatment. Conclusions: In this phase 1 study, second generation anti-CD19 CAR-T cells with TNFRS19 transmembrane domain have potent clinical activity. The short manufacture times achieved by local CAR-T cell manufacture with the CliniMACS Prodigy enables treatment of a very high risk NHL population that would otherwise not be able to receive CAR-T products due to rapidly progressive disease. Disclosures Caimi: ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Genentech: Research Funding. Schneider:Lentigen Technology, A Miltenyi Biotec Company: Employment. Bakalarz:Genentech: Speakers Bureau. Kruger:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Worden:Lentigen Technology, A Miltenyi Biotec Company: Employment. Kadan:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Malek:Adaptive: Consultancy; Janssen: Speakers Bureau; Amgen: Speakers Bureau; Celgene: Consultancy; Takeda: Consultancy; Sanofi: Consultancy; Medpacto: Research Funding. Metheny:Takeda: Speakers Bureau; Incyte: Speakers Bureau. Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. OffLabel Disclosure: Clinical Trial of on - site manufactured CAR-T cells. This manufacturing process is under research.

Introduction Chimeric Antigen Receptor modified T (CAR-T) cell therapies have revolutionized the relapsed, refractory B cell malignancy landscape. Due to the complex steps involved with cell production, some third-party companies require T cells to be cryopreserved prior to shipping, while most manufacturers deliver modified CAR-T cells to the treating center in a cryopreserved state. This is vastly different to the approach taken with traditional cell based therapies, specifically allogeneic transplant (allo-HCT), an immunological treatment that relies on a graft-versus-tumor (GVT) effect to prevent disease relapse. Historically, "fresh" stem cells were felt to be superior to cryopreserved products due to concerns that cryopreservation may damage T cells and other mononuclear cells delaying engraftment and limiting GVT reactivity. As a result, in clinical practice most allo-HCT products are still given as fresh infusions without cryopreservation. In a Phase 1 clinical trial evaluating the safety of a bispecific anti-CD19, anti-CD20 CAR (LV20.19CAR), CAR-T cells were produced in a point-of-care fashion utilizing the CliniMACS Prodigy device. Local manufacturing allowed flexibility to administer either fresh LV20.19CAR-T cells without cryopreservation, or if indicated, thawed CAR-T cells post-cryopreservation. Methods Patients (pts) were treated on a Phase 1 dose escalation + expansion trial (NCT03019055) to demonstrate safety of 41BB/CD3z LV20.19CAR-modified T cells for adults with relapsed, refractory B cell NHL including DLBCL, MCL, FL, and CLL. The starting dose was 2.5x10^5 cells/kg with a target dose of 2.5x10^6 cells/kg. All pts received low dose fludarabine (30 mg/m2) x 3 days +cyclophosphamide (500 mg/m2) x 1 day for lymphodepletion. In the Phase 1 dose-escalation cohorts, pts received fractionated CAR-T cells over two days (30% on Day 0 and 70% on Day+1), while expansion cohort pts received CAR-T cells as a single infusion. The goal for all pts was to infuse fresh CAR-T cell prior to cryopreservation, however, CAR-T cell could be cryopreserved and infused at a later date for clinical / logistical reasons. Results A total of 20 pts received LV20.19CAR T cell therapy (Table 1). Fourteen pts received fresh CAR-T cells immediately post-harvest, 5 pts received post-thaw CAR-T cells, and 1 patient received a mixed fresh/cryopreserved product and was not included in this analysis. Reasons for cryopreserved administration was delay due to active infection (N=3), patient preference (N=1), and unexplained neutropenia (N=1). Among 19 evaluable pts, the CR rate (79% vs 40%), mean ferritin, mean CRP, and incidence of CRS and neurotoxicity were all higher in the fresh infusion group (Table 1), but not statistically significant. In terms of LV20.19 CAR-T product characteristics, mean cell viability at infusion was 93% for the fresh infusion group versus 63% for cryopreserved pts (p Conclusions Cryopreservation is known to diminish cell viability and increase clinical costs associated with freezing and storage. To date, there is limited clinical data evaluating outcomes of pts receiving fresh CAR-T cells compared to thawed CAR-T cells post-cryopreservation. Although it is presumed that in-vivo CAR-T cell activity is comparable in both scenarios, among our pts, both cell viability and in-vivo expansion favored pts who received a fresh infusion. Unlike third-party CAR-T cell products where viability is unknown at the time of infusion, we adjusted the final dose to accommodate decreased cell viability. CR rates and incidence of CRS and NTX were higher among fresh infused pts suggesting greater in-vivo activity, although findings were not statistically significant, partially a result of the small sample size. While our findings are limited by small numbers in each cohort and variability in cell dose and diagnosis, these data suggest that cryopreservation of CAR-T cells may impact clinical responses and is a logistical step that needs further investigation. Disclosures Shah: Cell Vault: Consultancy, Equity Ownership; Oncosec: Equity Ownership; Lentigen: Honoraria, Research Funding; Exelexis: Equity Ownership; Geron: Equity Ownership; Celgene: Other: Advisory Board; Incyte: Consultancy; Oncosec: Equity Ownership; Kite Pharma: Other: Advisory Board. Zhu:Miltenyi Biotec: Research Funding. Schneider:Lentigen Technology, A Miltenyi Biotec Company: Employment. Krueger:Lentigen Technology, A Miltenyi Biotec Company: Employment. Worden:Lentigen Technology, A Miltenyi Biotec Company: Employment. Hamadani:Sanofi Genzyme: Research Funding, Speakers Bureau; Otsuka: Research Funding; ADC Therapeutics: Consultancy, Research Funding; Takeda: Research Funding; Celgene: Consultancy; Janssen: Consultancy; Pharmacyclics: Consultancy; Merck: Research Funding; Medimmune: Consultancy, Research Funding. Dropulic:Lentigen Technology, A Miltenyi Biotec Company: Employment. Hari:Celgene: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Kite: Consultancy, Honoraria; Amgen: Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding; Cell Vault: Equity Ownership; AbbVie: Consultancy, Honoraria. Johnson:Miltenyi Biotec: Research Funding; Cell Vault: Equity Ownership.

2510 Background: Anti-CD19 CAR-T cell therapy is a breakthrough treatment (tx) for patients (pts) with relapsed/refractory (R/R) B-cell non-Hodgkin lymphoma (NHL). Despite impressive outcomes, non-response and relapse with CD19 negative disease remain challenges. Through dual B-cell antigen targeting of CD20 and CD19, with a first-in-human bispecific lentiviral CAR-T cell (LV20.19CAR), we aim to improve response rates while limiting CD19 negative relapse. Methods: Pts were treated on a Phase 1 dose escalation + expansion trial (NCT03019055) to demonstrate safety of a 41BB/CD3z LV20.19CAR T cell for adults with R/R B-cell NHL. Safety was assessed by incidence of dose limiting toxicities (DLTs) within 28 days post-infusion. Starting dose was 2.5 x 10^5 cells/kg with a target dose of 2.5 x 10^6 cells/kg. All pts received fludarabine+cyclophosphamide for lymphodepletion. Results: 11 pts have completed tx to date. 9 pts in dose escalation and 2 pts in expansion phase. Median age was 54 years (46-67) and histology included DLBCL = 5 pts, MCL = 4 pts, and CLL = 2 pts. In dose escalation, 3 pts were treated at 2.5 x 10^5 cells/kg, 3 pts at 7.5 x 10^5 cells/kg, and 3 pts at 2.5 x 10^6 cells/kg with no DLTs. As a result, 2.5 x 10^6 cells/kg was selected for expansion. In terms of safety, 6 pts developed Grade 1-2 cytokine release syndrome (CRS) and 3 pts had Grade 1-2 neurotoxicity (NTX). No patient had grade 3-4 CRS or NTX and none required ICU level care. 4 pts required 1-2 doses of tocilizumab for CRS. The day 28 overall response rate (ORR) for all pts was 82% (6/11 = complete response (CR) and 3/11 = partial response). All CR pts remain in remission, the longest > 1 year. All progressing pts underwent repeat biopsy, and all retained either CD19 or CD20 positivity. Additional pts are being enrolled in the expansion phase and updated data will be presented. Conclusions: Phase 1 results from the LV20.19 CAR T clinical trial demonstrate that infusion of 2.5 x 10^6 cells/kg is safe for further investigation with no DLTs among treated pts. Down-regulation of target antigens was not identified as a mechanism of resistance in progressing pts. With limited toxicity and encouraging ORR, dual targeted LV20.19CAR T cells merits further investigation. Clinical trial information: NCT03019055.

2539 Background: AntiCD19 CAR-T cells have shown encouraging anti-lymphoma activity. Decreasing the time from apheresis to CAR-T infusion can make this therapy available to pts with rapid progression. We present the interim results of a phase I clinical trial using on-site CAR-T manufacture. Methods: Adult pts with r/r CD19+ B cell lymphomas who failed ≥ 2 lines of therapy were enrolled. Autologous T cells were transduced with a lentiviral vector (Lentigen Technology, Inc,LTG1563) encoding an antiCD19 binding motif, CD8 linker and TNFRSF19 transmembrane region, and 4-lBB/CD3z domains. GMP-compliant manufacture was done using CliniMACS Prodigy, in a 12-day culture. Dose levels were 0.5, 1 and 2 x 106 CAR-T cells/kg. Lymphodepletion was done with cyclophosphamide (60mg/kg x 1) and fludarabine (25mg/m2/d x 3). Results: 7 pts (4 women, 3 men) were enrolled. Median age was 60y [range 43-69]. Diagnoses were DLBCL (n = 3) PMBCL, follicular lymphoma (FL), transformed FL, and transformed lymphoplasmacytic lymphoma; with a median of 4 previous treatments. Six pts had symptomatic refractory disease. CAR-T cell product manufacture was successful in all pts. Mean transduction rate was 44% [range 29-57]. CAR-T cell doses were 0.5 x 106/kg (n = 3) and 1 x 106/kg (n = 4). Median apheresis to infusion time was 13 days [range 13–20], 5 products were infused fresh. CAR-T persistence based on vector sequence, peaked in peripheral blood MNCs between days 14-21. Five pts are evaluable for safety. CRS grade 1 - 2 (Lee) occurred in 4 pts; with 3 requiring treatment. Grade 4 CRES (CARTOX-10) occurred in 1 pt, with resolution after corticosteroids; considered a DLT as it lasted more than 72 hours. No treatment-related mortality has occurred. 4/5 evaluable pts have achieved complete response. One pt did not respond and died. After a median follow up 3 months, all responding pts are alive and 1 relapsed 6 mo after treatment. Conclusions: Second generation antiCD19 CAR-T cells with TNFRS19 transmembrane domain have clinical activity against refractory NHL. Short manufacture time achieved by local CAR-T cell manufacture with the CliniMACS Prodigy enables treatment of a very high risk NHL population. Clinical trial information: NCT03434769.

Predicting N losses in flooded agricultural systems is complex because of the large number of potential N transformation pathways. Although coupled nitrification/denitrification at the flooded sediment near-surface oxic/anoxic interface is a recognized pathway of fertilizer N loss, the role of the newly discovered anaerobic ammonium oxidation (anammox) pathway in agricultural systems is poorly understood. A survey study was implemented to characterize sediment denitrification rates, anammox activity, and fluxes of ammonium and nitrate, and the vertical distribution of N functional genes were measured in Hawaiian taro (Colocasia esculenta) fields under three fertilizer management regimens (conventional, organic, and hybrid) as a model for flooded agroecosystems. Potential denitrification rates and anammox activity were measured using a slurry-based isotope pairing technique, and fluxes were determined by porewater modeling. Although significant numbers of anammox 16S rRNA genes were detected, only negligible anammox activity was found, illustrating the need to quantify not only the presence but also the activity of these bacteria. Quantitative polymerase chain reaction was performed for bacterial amoA, nirS, nosZ, and the 16S rRNA gene at 1-cm soil depth increments. Slurry isotope pairing technique-based potential denitrification rates (4.3-12.3 mmol N 2 m -2 day -1 ) were on the high end of previous anaerobic incubation studies. Flux-derived denitrification rates were up to three orders of magnitude lower than slurry isotope pairing technique rates, suggesting strong nitrification regulation. Organically managed fields exhibited significantly lower slurry and flux-based denitrification rates than conventional and hybrid systems. This study supports the use of alternative fertilizer management techniques in flooded agroecosystems by the mitigation of N losses through denitrification and decreased overall N flux rates.

Background: CAR-T cell therapy directed against the CD19 antigen is a breakthrough treatment for patients (pts) with relapsed/refractory (R/R) B-cell NHL. Despite impressive outcomes, not all pts respond and many that respond still relapse. Affordability and accessibility are further considerations that limit current commercial models of CAR-T products. Commercial CAR-T manufacturing is complex, time consuming, and expensive with a supply chain starting at the treating center with apheresis of mononuclear cells, cryopreservation, and shipping to and from a centralized third-party manufacturing site. We addressed these limitations in a Phase 1 clinical trial evaluating a first-in-human bispecific tandem CAR-T cell directed against both CD19 and CD20 (CAR-20.19-T) antigens for pts with R/R B-cell NHL. Through dual targeting we hope to improve response rates and durability of response while limiting antigen escape. We eliminated third party shipping logistics utilizing the CliniMACS Prodigy, a compact tabletop device that allows for automated manufacturing of CAR-T cells within a GMP compliant environment within the hospital. Most materials and reagents used to produce the CAR-T cell product were single-sourced from the device manufacturer. Methods: Phase 1 (NCT03019055), single center, dose escalation + expansion study to demonstrate feasibility and safety of locally manufactured second generation 41BB + CD3z CAR-20.19-T cells via the CliniMACS Prodigy. Feasibility was measured by ability to generate a target CAR-20.19-T cell dose for a minimum of 75% of subjects. Safety was assessed by the presence of dose limiting toxicities (DLTs) through 28 days post-infusion. Dose was escalated in a 3+3 fashion with a starting dose of 2.5 x 10^5 cells/kg, a target DLT rate CAR-T production was set for a 14-day manufacturing process. Day 8 in-process testing was performed to ensure quality and suitability of CAR-T cells for a potential fresh infusion. On Day 10, pts eligible for a fresh CAR-T infusion initiated lymphodepletion (LDP) chemotherapy with fludarabine 30 mg/m2 x 3 days and cyclophosphamide 500 mg/m2 x 1 day, and cells were administered after harvest on Day 14. Pts ineligible for fresh infusion received cryopreserved product and LDP was delayed accordingly. Results: 6 pts have been enrolled and treated with CAR-20.19-T cells: 3 pts at 2.5 x 10^5 cells/kg and 3 pts at 7.5 x 10^5 cells/kg. Median age was 53 years (48-62). Underlying disease was MCL in 3 pts, DLBCL in 2 pts, and CLL in 1 patient. Baseline data and prior treatments are listed in Table 1. CAR-T production was successful in all runs and all pts received their target dose. Three pts received fresh CAR-T cells and 3 pts received CAR-T cells after cryopreservation. To date there are no DLTs to report. No cases of Grade 3/4 cytokine release syndrome (CRS) or neurotoxicity (NTX) were observed. One patient had Grade 2 CRS and Grade 2 NTX requiring intervention. The other had self-limited Grade 1 CRS and Grade 1 NTX. Median time to development of CRS was Day +11 post-infusion. All pts had neutrophil recovery (ANC>0.5 K/µL) by Day 28. Response at Day 28 (Table 2) is as follows: 2/6 pts achieved a complete response (CR), 2/6 achieved a partial response (PR), and 2/6 had progressive disease (PD). One subject with a PR subsequently progressed at Day 90. The 3 pts who did progress all underwent a repeat biopsy, and all retained either CD19 or CD20 positivity. Pts are currently being enrolled at the target dose (2.5 x 10^6 cells/kg) and updated results will be provided at ASH. Conclusions: Dual targeted anti-CD19 and anti-CD20 CAR-T cells were successfully produced for all pts demonstrating the feasibility of a point-of-care manufacturing process via the CliniMACS Prodigy device. With no DLTs or Grade 3-4 CRS or NTX to report, and 2/6 heavily pre-treated pts remaining in CR at 3 and 9 months respectively our approach represents a feasible and promising alternative to existing CAR-T models and costs. Down-regulation of both target antigens was not identified in any patient following CAR-T infusion, and in-process studies suggest that a shorter manufacturing timeline is appropriate for future trials (10 days). Disclosures Shah: Juno Pharmaceuticals: Honoraria; Lentigen Technology: Research Funding; Oncosec: Equity Ownership; Miltenyi: Other: Travel funding, Research Funding; Geron: Equity Ownership; Exelexis: Equity Ownership. Zhu:Lentigen Technology Inc., A Miltenyi Biotec Company: Research Funding. Schneider:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Krueger:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Worden:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Hamadani:Sanofi Genzyme: Research Funding, Speakers Bureau; Merck: Research Funding; Janssen: Consultancy; MedImmune: Consultancy, Research Funding; Cellerant: Consultancy; Celgene Corporation: Consultancy; Takeda: Research Funding; Ostuka: Research Funding; ADC Therapeutics: Research Funding. Johnson:Miltenyi: Research Funding. Dropulic:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen Technology Inc., A Miltenyi Biotec Company: Other: Prior Employment. Hari:Takeda: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Kite Pharma: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Amgen Inc.: Research Funding; Sanofi: Honoraria, Research Funding.

Lentiviruses are well known for their species specificity, and consequently lentivirus-derived vectors exhibit the highest gene transfer efficiency in cells of the same species from which the vectors were derived. Therefore, to accurately model a gene therapy approach for HIV in a monkey model, a parallel simian immunodeficiency virus (SIV) vector should be developed to accurately model the vector-cell-virus interactions. Our first therapeutic HIV-derived vector VRX496 contains an antisense targeting HIV env region. We established the safety of this novel vector in a Phase I open label clinical trial, which was allowed to proceed without preliminary studies in monkeys due to 1) the engineered safety features of the vector and 2) the observation that a monkey study would not be informative regarding the dynamics of an HIV-based vector. After demonstrating safety of this vector with early indications of efficacy in humans, we hypothesized that subsequent generations of this vector that have an improved ability to mobilize may increase the therapeutic efficacy of our anti-HIV gene therapy. However, such "mobilizing" vectors are considered too risky to test directly in patients, due to concerns regarding insertional oncogenesis. Therefore, we chose to test this hypothesis in monkeys in order to safely model the dynamics of these new vectors in vivo. Accordingly, we developed three SIVmac239 based lentiviral vectors, one that parallels our clinical vector VRX496 that contains no viral proteins (pN1), a second generation (pN2) containing the gag-pol protein that may have an improved ability to mobilize as a result of reduced dependency on wtSIV-proteins, and a third (empty pN2) containing gag-pol and no antisense payload to serve as a control. We demonstrated that the SIV based vectors were able to transduce the primary simian CD4 cells significantly better than the counterpart of HIV based vector by an approximately 6-fold improvement in gene transfer. We also showed that the vector potently inhibited the SIVmac239 replication in both transduced CEMx174 cells and primary simian CD4 cells to below the limit of detection, as previously observed with VRX496 and HIV in primary human CD4 cells. Detailed analysis of these results and modelling of these vectors in a monkey SIV model will be discussed.

We established the first clinical ex vivo HIV-based vector gene therapy trial in humans with HIV+ CD4+ T-cells. Briefly, this therapy involves modifying patient CD4+ T-cells with our modified lentiviral vector carrying an anti-HIV payload. These cells are then activated and expanded, and re-infused back into the patient. However, cGMP regulations require the use of costly clinical grade reagents (i.e. Retronectin™, CD3/CD28 stimulating paramagnetic beads). In an attempt to reduce ex-vivo processing costs, but not at the expense of transduction levels, we sought to determine a way to directly activate CD4+ T-cells with modified lentiviral vectors. 293FT HEK cell lines, used for producing our lentiviral vectors, were modified to co-express the natural CD28 stimulatory ligand B7.2 (CD86) and ICAM-1 (CD54) proteins on their membrane for co-stimulation and anchoring purposes. When CliniMACS purified normal donor CD4+ T cells were co-cultured with CD54/CD86-expressing cells, in the presence of soluble OKT3 CD3 antibody, CD25 and CD69 activation markers were upregulated, indicating that functional proteins were being expressed at the cell membrane. These CD54 and/or CD86 expressing cells could subsequently be transfected with lentiviral vector plasmid constructs in order to produce host-derived CD54 and/or CD86 bearing HIV-based vectors. EGFP-expressing lentiviral vectors, VRX494, with CD54/CD86-modified envelopes were produced both in these cell lines and by transient transfection of all relevant plasmids, and titers were assayed on Hela-Tat cells by FACS. CD54 modified lentiviral vectors showed increased binding to CD4+ T-cells, as evidenced by significant cell clumping. CD86 (as well as CD54 plus CD86) modified lentiviral vector, with soluble OKT3 CD3 antibody, was shown to activate T-cells, above the levels seen with unmodified lentiviral vectors, as evidenced by the increase in cell surface CD25 and CD69 expression and also the increase in cell size. Cellular expansion of modified lentiviral vector transduced CD4+ T cells reached levels close to CD3:CD28 bead stimulated CD4+ T cell controls over a period of 2 to 3 weeks. The CD3/TCR repertoire was assessed by flow cytometry and, compared to the well-established CD3/CD28 coated M450 Dynabeads stimulatory system as a control, no skewing of the repertoire was observed. CD86 was shown to improve levels of transduction in pre-activated lymphocytes with CD3/CD28 coated M450 Dynabeads. However, CD86 co-expression was crucial for transducing minimally activated CD4+ T cells with only soluble OKT3 CD3 antibody. Levels of transduction and activation were on average 2 to 3 times higher with the modified lentiviral vectors. To our knowledge, we are reporting the first generation of lentiviral particles exhibiting an adhesion property with stimulatory abilities. The development of such a lentiviral vector has valuable implications for clinical application by reducing the number of exogenous reagents in large scale cell processing.