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Background aims Multiple steps are required to produce chimeric antigen receptor (CAR)-T cells, involving subset enrichment or depletion, activation, gene transduction and expansion. Open processing steps that increase risk of contamination and production failure are required. This complex process requires skilled personnel and costly clean-room facilities and infrastructure. Simplified, reproducible CAR-T-cell manufacturing with reduced labor intensity within a closed-system is highly desirable for increased availability for patients. Methods The CliniMACS Prodigy with TCT process software and the TS520 tubing set that allows closed-system processing for cell enrichment, transduction, washing and expansion was used. We used MACS-CD4 and CD8-MicroBeads for enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 ζ-cfrag vectors expressing scFv for CD19 or CD20/CD19 antigens for transduction, TexMACS medium-3%-HS-IL2 for culture and phosphate-buffered saline/ethylenediaminetetraacetic acid buffer for washing. Processing time was 13 days. Results Enrichment (N = 7) resulted in CD4/CD8 purity of 98 ± 4.0%, 55 ± 6% recovery and CD3+ T-cell purity of 89 ± 10%. Vectors at multiplicity of infection 5–10 resulted in transduction averaging 37%. An average 30-fold expansion of 108 CD4/CD8-enriched cells resulted in sufficient transduced T cells for clinical use. CAR-T cells were 82–100% CD3+ with a mix of CD4+ and CD8+ cells that primarily expressed an effector-memory or central-memory phenotype. Functional testing demonstrated recognition of B-cells and for the CAR-20/19 T cells, CD19 and CD20 single transfectants were recognized in cytotoxic T lymphocyte and interferon-γ production assays. Discussion The CliniMACS Prodigy device, tubing set TS520 and TCT software allow CAR-T cells to be manufactured in a closed system at the treatment site without need for clean-room facilities and related infrastructure.

Background Clinical success with chimeric antigen receptor (CAR)- based immunotherapy for leukemia has been accompanied by the associated finding that antigen-escape variants of the disease are responsible for relapse. To target hematologic malignancies with a chimeric antigen receptor (CAR) that targets two antigens with a single vector, and thus potentially lessen the chance of leukemic escape mutations, a tandem-CAR approach was investigated. Methods Antigen binding domains from the FMC63 (anti-CD19) and Leu16 (anti-CD20) antibodies were linked in differing configurations to transmembrane and T cell signaling domains to create tandem-CARs. Expression on the surface of primary human T cells was induced by transduction with a single lentiviral vector (LV) encoding the tandem-CAR. Tandem-CARs were compared to single antigen targeting CARs in vitro and in vivo, and to an admixture of transduced cells expressing each CAR in vivo in immunodeficient (NSG) disease-bearing mice. Results Tandem constructs efficient killed the Raji leukemia cell line both in vitro and in vivo. Tandem CARs generated less cytokine than the CD20 CAR, but similar to CD19 CARs, on their own. In co-culture experiments at low effector to target ratios with both single- and tandem- CAR-T cells, a rapid down-modulation of full-length CD19 expression was seen on leukemia targets. There also was a partial down-modulation of CD22, and to a lesser degree, of CD20. Our data also highlight the extreme sensitivity of the NALM-6 cell line to general lymphocyte-mediated cytotoxicity. While single and tandem constructs were effective in vivo in a standard setting, in a high-disease burden setting, the tandem CAR proved both effective and less toxic than an admixture of transduced T cell populations expressing single CARs. Conclusion Tandem CARs are equally effective in standard disease models to single antigen specificity CARs, and may be both more effective and less toxic in a higher disease burden setting. This may be due to optimized cell killing with more moderate cytokine production. The rapid co-modulation of CD19, CD20, and CD22 may account for the ability to rapidly evolve escape mutants by selecting for leukemic clones that not require these target antigens for continued expansion. Electronic supplementary material The online version of this article (doi:10.1186/s40425-017-0246-1) contains supplementary material, which is available to authorized users.

Malignant melanoma incidence has been increasing for over 30 years, and despite promising new therapies, metastatic disease remains difficult to treat. We describe preliminary results from a Phase I clinical trial (NCT01586403) of adoptive cell therapy in which three patients received autologous CD4+ and CD8+ T cells transduced with a lentivirus carrying a tyrosinase-specific TCR and a marker protein, truncated CD34 (CD34t). This unusual MHC Class I-restricted TCR produces functional responses in both CD4+ and CD8+ T cells. Parameters monitored on transduced T cells included activation (CD25, CD69), inhibitory (PD-1, TIM-3, CTLA-4), costimulatory (OX40), and memory (CCR7) markers. For the clinical trial, T cells were activated, transduced, selected for CD34t+ cells, then re-activated, and expanded in IL-2 and IL-15. After lymphodepleting chemotherapy, patients were given transduced T cells and IL-2, and were followed for clinical and biological responses. Transduced T cells were detected in the circulation of three treated patients for the duration of observation (42, 523, and 255 days). Patient 1 tolerated the infusion well but died from progressive disease after 6 weeks. Patient 2 had a partial response by RECIST criteria then progressed. After progressing, Patient 2 was given high-dose IL-2 and subsequently achieved complete remission, coinciding with the development of vitiligo. Patient 3 had a mixed response that did not meet RECIST criteria for a clinical response and developed vitiligo. In two of these three patients, adoptive transfer of tyrosinase-reactive TCR-transduced T cells into metastatic melanoma patients had clinical and/or biological activity without serious adverse events.

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.

2062 Background: GBM is the most common malignant brain tumor with a median survival of 15 months despite surgery and radio-chemotherapy. The most important mechanism of TMZ resistance is the O6-methylguanine-DNA methyltransferase (MGMT) gene which repairs temozolamide-induced DNA methylation. The MGMT inhibitor O6-benzylguanine (BG) demonstrated efficacy in depleting MGMT and maximizing tumor response in early phase clinical trials. However, MGMT expression is also low in hematopoietic cells, so this approach led to unacceptable bone marrow toxicity and thus has been abandoned. We hypothesized that chemoprotection of hematopoietic HPC with an MGMT mutant (MGMT-P140K) characterized by normal methyltransferase activity, coupled with low affinity for BG would maximize anti-tumor response while enabling patients to tolerate TMZ & BG dose escalation with minimal toxicity. A phase I trial was performed to test this hypothesis. Methods: 10 adults with newly diagnosed MGMT unmethylated, IDH-1 WT, GBM underwent standard surgery and radiation, followed by transplantation with autologous CD34+ HPC engineered to express MGMT-P140K using a lentiviral vector. We tested tolerance and efficacy of three different paradigms for conditioning bone marrow and re-infusion of HPC. To assess chemo-protection, patients’ blood counts and transgene marking were monitored during and after treatment, as was toxicity, response, and progression-free and overall survival. Results: Treatment was moderately toxic with 3/10 patients suffering grade 3-4 hematologic toxicity; no high grade non-hematologic toxicity was observed . Viral transduction rates ranged from 3-75% and were clearly improved in Arm III utilizing BCNU conditioning and intra-patient dose escalation of TMZ/O6GB. In patients tolerating 3 cycles or more, P140K-MGMT gene markings in peripheral blood and bone marrow cells increased 3-26-fold with only mild (Grade 2-3) mylosuppression consistent with chemo-protection as hypothesized. Median PFS and OS was 22 and 31 months respectfully, and three patients in Arm III are healthy and progression free at 36-39 months. OS exceeded RPA predicted survival by 3.3-fold suggesting clinical benefit. Viral insertion site analysis demonstrate lack of clonal dominance. Conclusions: P140K-MGMT transfected HPC enables TMZ/ BG dose escalation with acceptable toxicity and increased survival in a small cohort of selected patients. A phase II study is ongoing. Clinical trial information: NCT01269424.

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.

Adoptive cell therapy with autologous CAR-T cells has induced remarkable responses in patients with treatment-refractory hematologic malignancies, but limitations exist with off-site commercial CAR-T production: (1) manufacturing can take several weeks and requires shipping patient cells; (2) off-site manufacturing limits treatment options for patients with rapidly progressive disease; (3) high cost of the products may limit their availability. To address these challenges, we used the fully automated Miltenyi CliniMACS Prodigy GMP-compliant device to manufacture autologous CAR-T cells for a Phase I trial (NCT03019055) evaluating a first-in-human bi-specific CAR that targets CD19 and CD20 (CD20.19 CAR). Using reagents from Miltenyi Biotec, production was performed within the Medical College of Wisconsin (MCW) Cell Therapy Laboratory. Manufacturing time was 14 days, and production was as follows. First, peripheral blood mononuclear cells (MNC) collected from patients by apheresis were loaded onto the Prodigy, and CD4 and CD8 T cells enriched by positive immunomagnetic selection. The T cells were cultured in 200 U/mL IL-2, and TransACT reagent was added to stimulate the T cells in the Prodigy cell culture chamber. Next day, lentiviral vector expressing anti-CD19 and anti-CD20 (in tandem) with CD3z and 4-1BB stimulatory domains was added to the cells. Culture washes and feedings were done on days 5, 6, 8, 10 and 12 of manufacture, and final products harvested on day 14. Eligible patients either received fresh CAR-T cells, or cells that had been cryopreserved and administered on a later date. CAR-T cell products have been successfully generated for all 8 patients enrolled thus far on the Phase 1 clinical trial with no production failures, and all products passed release criteria for infusion. Three patients received cryopreserved product and 5 patients received fresh product. Average T cell recovery from the apheresis cell products was 66.0% (56.0-76.0%), and the enriched T cells were 94.3% CD3+ (87.8-97.4%) (Table 1). Protein L staining of the final cell products indicated 19.6% average CD20.19 CAR expression. Patient CAR-T cells were able to kill CD19+ and CD20+ target cells in vitro and produce IFNg upon antigen stimulation. An average yield of 5.27e+8 CAR T cells was obtained at harvest, exceeding the required cell dose for all patients. The CAR-T cells were comprised of both CD4 and CD8 T cells, with greater percentages of CD4 T cells. The majority of T cells (avg. 83.2%) had an effector-memory phenotype. In summary, we have successfully demonstrated feasibility for point-of-care CAR-T cell production for clinical use from patient apheresis products utilizing the CliniMACS Prodigy device. A major clinical advantage of CAR-T cells generated on-site is flexibility in treatment, with either immediate (i.e., fresh) or later (cryopreserved/thaw) infusion, dependent on patient need.

Reference standards are important tools for the calibration of medical products and procedures. They are particularly important when dosing and potency are critical parameters for patient safety and product efficacy.1 Reference standards benefit the field by fostering best practices for the manufacturing and testing of safe and efficacious medicinal products. As the first commercial gene therapy products are now reaching market approval in the United States,2,3 there is increased discussion of the need to generate reference standards for the industry.

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.

Adoptive cell therapy with autologous CAR-T cells has induced remarkable responses in patients with treatment-refractory hematologic malignancies, which has led to FDA approvals for two CAR-T products. However, limitations exist with commercial CAR-T centralized production: (1) off-site manufacturing can take several weeks and requires shipping from and to the treating facility; (2) off-site manufacturing limits treatment options for progressing patients; (3) high cost of the commercial products may limit their availability. To address these challenges, we used the fully automated Miltenyi CliniMACS Prodigy device, a GMP-compliant closed system, to manufacture autologous CAR-T cells for a Phase I trial (NCT03019055) evaluating a first-in-human bi-specific CAR that targets CD19 and CD20 (CD20.19 CAR). CAR-T manufacturing was performed exclusively using the CliniMACS Prodigy device and reagents obtained from Miltenyi Biotec. Production was performed within the Medical College of Wisconsin (MCW) Cell Therapy Laboratory, an ISO7 air handling environment. Manufacturing was set at 14 days, and production was as follows. First, peripheral blood mononuclear cells (MNC) were collected by apheresis, with a collection goal of 4 blood volumes to eliminate risk of a low CD3 yield in heavily pre-treated patients. Next, MNC were loaded onto the Prodigy, and CD4 and CD8 T cells enriched by positive immunomagnetic selection. To start the culture process, enriched T cells were suspended in TexMACS medium supplemented with 3% human AB serum and 200 U/mL IL-2, and TransACT reagent was added to stimulate the T cells in the Prodigy cell culture chamber. The following day (day 1), lentiviral vector expressing anti-CD19 and anti-CD20 (in tandem) with CD3ζ and 4-1BB stimulatory domains was added to the stimulated cells. Culture washes and feedings were done on days 5, 6, 8, 10 and 12 of manufacture, and final products harvested on Day 14. Protein L staining was used to detect expression of CD20.19 CAR on the T cells. On Day 14, eligible patients received fresh CAR-T cells, while for others the product was cryopreserved and administered on a later date. To date, the MCW Cell Therapy Laboratory has successfully generated CAR-T cell products for all 6 patients enrolled thus far on the Phase 1 clinical trial with no production failures (Table 1). Three patients received cryopreserved product and 3 patients received fresh product. The enriched T cells were 94.3% CD3+ (87.8-97.4%), and average T cell recovery from the apheresis cell products was 65.2% (54.2-80.0%). Protein L staining indicated 20.8% average CD20.19 CAR expression. Patient CAR-T cells were able to kill CD19+ and CD20+ target cells in vitro and produce IFN-gamma in response to the same target cells. An average yield of 5.9e+8 (4.3-7.9e+8) CAR T cells was obtained at harvest, which exceeded the required cell dose for all patients. The CAR-T cells were comprised of both CD4 and CD8 T cells, with higher expression on CD4 T cells; average CAR-T CD4:CD8 ratio on the final products was 2.8. The majority of T cells (average of 81.5%) had an effector-memory phenotype. In-process testing performed on Day 8 of manufacturing demonstrated sufficient numbers of CAR-T cells needed for patient infusions were already present, and that the CAR-T cells only expanded an additional 1.9 to 3.5-fold between Days 8 and 14. In conclusion, we have successfully demonstrated feasibility for point-of-care CAR-T cell production for clinical use from patient apheresis products utilizing the CliniMACS Prodigy device. Time to production was efficient (14 days), and patient-derived CAR-T cell products were reproducibly generated in a standard cell processing laboratory within an academic medical center. A major clinical advantage of CAR-T cells generated on-site is the flexibility in treatment. Patients can receive cells either immediately (i.e., fresh) or the cells can be cryopreserved for later infusion if the patient is not able to receive fresh cells. Based on our results, we intend to decrease the cell processing time to 10 days. Disclosures Zhu: Lentigen Technology Inc., A Miltenyi Biotec Company: Research Funding. Shah:Juno Pharmaceuticals: Honoraria; Oncosec: Equity Ownership; Geron: Equity Ownership; Exelexis: Equity Ownership; Miltenyi: Other: Travel funding, Research Funding; Lentigen Technology: Research Funding. Schneider:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Keever-Taylor:Medical College of Wisconsin: Research Funding. Dropulic:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen Technology Inc., A Miltenyi Biotec Company: Employment. Hari:Bristol-Myers Squibb: Consultancy, Research Funding; Amgen Inc.: Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Janssen: Honoraria; Kite Pharma: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding; Spectrum: Consultancy, Research Funding; Sanofi: Honoraria, Research Funding. Johnson:Miltenyi: Research Funding.

Introduction CD19 CAR-T cell products were recently approved as therapy for advanced B-cell lineage malignancies. The predominant manufacturing model for this type of therapy is a centralized industrial-type production process. An attractive model of CAR-T cell production and delivery to the patient is via a point-of care-manufacturing process. We report on first cases of implementation of this approach in a setting of compassionate use program. Patients and methods Four patients with relapsed/refractory B-cell lineage malignancies were eligible for compassionate use of CD19 CAR-T cell therapy. Three patients, median age 15 years, had relapsed B-cell lineage acute lymphoblastic leukemia (B-ALL) after haploidentical hematopoietic stem cell transplantation (HSCT). Bone marrow disease burden at therapy start was 12%, 74% and 0,159. The patient with low minimal residual disease (MRD) in the bone marrow had skeletal involvement with multiple lymphomatous mass lesions. One patient had refractory primary mediastinal B-cell lymphoma (PMBCL). The CliniMACS Prodigy T cell transduction (TCT) process was used to produce CD19 CAR-T cells from patient-derived leukapheresis product. Automatic production included CD4/CD8 selection, CD3/CD28 stimulation with MACS GMP T Cell TransAct, transduction with lentiviral (second generation CD19.4-1BB zeta) vector (Lentigen, Miltenyi Biotec company) and expansion over 12 days in the presence of TexMACS GMP Medium supplemented with MACS GMP IL-7/IL-15 combination. Final product was administered without cryopreservation to the patients after fludarabine/cyclophosphamide preconditioning. All patients received prophylactic tocilizumab 1 hour before CAR-T cell infusion at 8mg/kg. Results All production cycles were successful. Median transduction efficacy achieved was 57%(52-63). Median expansion of T cells was x26(24-43). CD4/CD8 ratio in the final product was 0,750,22-6). All final products passed the in-process and quality controls. The cell products were administered at a dose of 1*106/kg of CAR-T cells. No immediate infusion reactions were reported. In 2 cases mild cytokine release syndrome (CRS) was diagnosed. In one case mild CAR-T cell related encephalopathy developed. In one case additional dose of tocilizumab and one dose of dexamethasone were administered to control CRS and encephalopathy. All patients survived to the point of response evaluation. In 3 cases CAR-T cell expansion was detected to a maximum 25%. MRD-negative responses detected by flow cytometry and PCR were achieved in 2 cases with bone marrow involvement. In two cases with prominent mass lesions an objective response was documented. In the patient with 74% blast in bone marrow at start of therapy, neither CAR-T cell expansion nor leukemia response were documented. Details of therapy and response are summarized in table 1. Conclusion Production of CAR-T cells with the CliniMACS Prodigy TCT process is a feasible and an attractive option that provides a point-of-care manufacturing approach to enable rapid delivery of CAR-T cells to patients in need. Robustness and consistency of this approach provides a solid basis for multi-center academic trials in the field of adoptive cell therapy. Table 1. Table 1. Disclosures Dropulic: Lentigen, A Miltenyi Biotec company: Employment. Shneider:Lentigen, A Miltenyi Biotec company: Employment. Orentas:Lentigen, A Miltenyi Biotec company: Employment. Alex:Miltenyi Biotec: Employment. Essl:Miltenyi Biotec: Employment.

The applicability of immunotherapy would be dramatically broadened to a greater number of recipients if direct “off-the-shelf” products could be engineered to engender functionally potent immune responses against true “self”-tumor antigens. This would obviate the need for ex vivo culture of dendritic cells or T cells on a patient-by-patient basis, for example. The carcinoembryonic antigen (CEA) is a glycoprotein expressed in normal gut epithelium that is up-regulated in the majority of colon cancers, non-small cell lung cancers, and half of all breast cancers. Such properties make CEA an excellent and important target for cancer immunotherapy. In this study, we show stabilization of 14-day established s.c. mGC4CEA tumors in human CEA (huCEA) transgenic mice following two direct low-dose injections of 0.15 × 106 transducing units of a lentiviral vector (LV) that directs expression of huCEA (LV-huCEA). This stabilization result was reproducible and detailed analyses including antibody assays, multiplex cytokine analyses on unstimulated splenocytes, lymph node cell characterizations, tetramer staining, and immunofluorescence staining of tumor sections showed that this outcome correlated with both a cellular and humoral immune response. Similar tumor outcomes were not seen when mice were vaccinated with a control LV that engineered expression of enGFP only. The long-term potency of this vaccination strategy was also studied and revealed the requirement for maintenance of tumor antigen-specific immunity for efficient tumor control. These data support the use of direct injections of low doses of LV-huCEA for enhancement of tumor immunotherapy directed against CEA. [Mol Cancer Ther 2009;8(3):OF692–11]

We report findings from a clinical evaluation of lentiviral vectors in a phase I open-label nonrandomized clinical trial for HIV. This trial evaluated the safety of a conditionally replicating HIV-1-derived vector expressing an antisense gene against the HIV envelope. Five subjects with chronic HIV infection who had failed to respond to at least two antiviral regimens were enrolled. A single i.v. infusion of gene-modified autologous CD4 T cells was well tolerated in all patients. Viral loads were stable, and one subject exhibited a sustained decrease in viral load. CD4 counts remained steady or increased in four subjects, and sustained gene transfer was observed. Self-limiting mobilization of the vector was observed in four of five patients. There is no evidence for insertional mutagenesis after 21–36 months of observation. Immune function improved in four subjects. Lentiviral vectors appear promising for gene transfer to humans.

More than 40 million people are infected with human immunodeficiency virus (HIV), and a successful vaccine is at least a decade away. Although highly active antiretroviral therapy prolongs life, the maintenance of viral latency requires life-long treatment and results in cumulative toxicities and viral escape mutants. Gene therapy offers the promise to cure or prevent progressive HIV infection by interfering with HIV replication and CD4+ cell decline long term in the absence of chronic chemotherapy, and approximately 2 million HIV-infected individuals live in settings where there is sufficient infrastructure to support its application with current technology. Although the development of HIV/AIDS gene therapy has been slow, progress in a number of areas is evident, so that studies to date have significantly advanced the field of gene-based immunotherapy. Advances have helped to define a series of ongoing and planned trials that may shed light on potential mechanisms for the successful clinical gene therapy of HIV.

We examined the ability of a HIV-1-based vector (VRX494) encoding a 937-bp antisense HIV-1 envelope sequence to inhibit the replication of chimeric SIV/HIV-1 viruses encoding the HIV-1 envelope. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the potential efficacy of the VRX494 vector for stem cell gene therapy, rhesus CD34(+) bone marrow cells were transduced with VRX494 and then cultured on thymus stroma to induce T cell differentiation. Transduction conditions for CD34(+) cells were optimized to yield high transduction efficiency with minimal effective multiplicity of infection. Purified CD4(+) GFP(+) T cells derived from VRX494-transduced CD34(+) cells strongly inhibited SHIV HXBC2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of VRX494-transduced T cell clones revealed a subset of cells with multiple proviral copies per cell. Expression of GFP and the antisense inhibitor in VRX494-transduced cells was upregulated by Tat. Analysis of HIV-1 envelope sequences in VRX494-transduced cells revealed modifications consistent with those mediated by double-stranded RNA-dependent adenosine deaminase. These results indicate that the macaque/SHIV model should serve as a useful preclinical model to evaluate this lentiviral vector expressing an HIV-1 antisense inhibitor for stem cell gene therapy for AIDS.

Background An Erratum has been published for this article in Journal of Gene Medicine 7(6), 2005, 835. A stable packaging cell line facilitates large-scale lentivirus vector manufacture. However, it has been difficult to produce clinical-scale HIV-1-based lentiviral vectors using a packaging cell line, in part due to toxicity of packaging genes, and gene silencing that occurs during the long culture period necessary for sequential addition of packaging constructs. Methods To avoid these problems, we developed a three-level cascade gene regulation system designed to remove tetracycline transactivator (tTA) from cytomegalovirus immediate early promoter (CMV)-controlled expression to reduce cytotoxicity from constitutive expression of tTA and leaky expression of packaging genes. We also performed a one-step integration of the three packaging plasmids to shorten the culture time for clonal selection. Results Although leaky expression of p24 and vector production still occurred despite the three-level regulation system, little cytotoxicity was observed and producer cells could be expanded for large-scale production. Producer cells yielded remarkably stable vector production over a period greater than 11 days with the highest titer 3.5 × 107 transducing units (TU)/ml and p24 300 ng/ml, yielding 2.2 × 1011 TU and 1.8 milligram (mg) p24 from one cell factory. No replication-competent lentivirus (RCL) was detected. Long-term analysis demonstrated that, although the cells are genetically stable, partial gene silencing occurs after 2–3 months in culture; however, the one-step construct integration allowed prolonged vector production before significant gene silencing. Concentrated vector resulted in 90% transduction in CD4+ lymphocytes at 20 TU per cell. CD34+ progenitor cells were transduced at 41–46% efficiency, and long-term initiating culture (LTC-IC) was transduced at 45–51%. Conclusions These results demonstrate for the first time HIV-1-based lentiviral vector production on the large scale using a packaging cell line. Copyright © 2005 John Wiley & Sons, Ltd.

We present preclinical studies that demonstrate in vitro the feasibility and efficacy of lentivirus-based vector antisense gene therapy for control of HIV replication in primary T lymphocytes isolated from HIV-infected patients discordant for clinical status. VRX496 is a VSV-G-pseudotyped HIV-based vector that encodes an antisense payload against the HIV envelope gene. The antisense payload is under the control of the native LTR promoter, which is highly transactivated by tat upon HIV infection in the cell. Transfer of autologous CD4(+) T lymphocytes genetically modified with VRX496 (VRX496T) into HIV-infected patients is intended to provide a reservoir of cells capable of controlling HIV, potentially delaying AIDS onset. To determine the patient population likely to respond to VRX496 for optimal efficacy, we examined the ability of our research vector, VRX494, to modify and suppress HIV in vitro in lymphocytes isolated from 20 study subjects discordant for CD4 count and viral load. VRX494 is analogous to the clinical vector VRX496, except that it contains GFP as a marker gene instead of the 186-tag marker in the clinical vector. To transfer VRX494 to target cells we developed a novel scalable two-step transduction procedure that has been translated to the clinic in an ongoing clinical trial. This procedure achieved unprecedented transduction efficiencies of 94 +/- 5% in HIV(+) study subject cells. In addition the vector inhibited HIV replication >/=93% in culture regardless of the viral load or CD4 count of the subject or tropism of the virus strain with which they were infected. These findings demonstrate that VRX496T therapy is expected to be beneficial to patients that differ in their status in term of CD4 count and viral load. The methods described represent significant technical advances facilitating execution of lentivirus vector-mediated gene therapy for treatment of HIV and are currently being employed in the first trial evaluating lentivirus vector safety in humans.

We have developed an HIV-based lentiviral vector, VRX496, which efficiently transduces human CD34+ progenitors and CD4+ T lymphocytes. VRX496 contains an antisense sequence against the HIV envelope and is currently being evaluated for safety in a clinical trial for treatment of HIV. Selective outgrowth of transduced hematopoietic cells in vivo is anticipated to increase the therapeutic efficacy of this treatment by maximizing the persistence of virus-resistant cells in the body. Although HIV resistance is selective, additional selection may aid in treatment efficacy due to the vast quantity of target cells. Therefore, we engineered VRX496 to express the P140K MGMT gene to drive potent drug-mediated in vivo selection for transduced hematopoietic long-term repopulating cells. Suboptimally transduced T cell cultures treated with O6-benzylguanine and BCNU were selected from 3 to 100%, and after selection cultures did not support HIV replication. Primary CD34+ progenitors derived from G-CSF-mobilized peripheral blood were transduced at 27 to 35% efficiency. Approximate sixfold selection was observed for transduced CD34+ progenitors, colony-forming units, and long-term culture-initiating cells. Multilineage in vivo selection was demonstrated for transduced murine hematopoietic cells in human CD34(+)-derived hematopoietic cells in NOD-SCID mice. These results establish efficient ex vivo and in vivo selection for hematopoietic cells transduced with lentiviral vectors and support the potential therapeutic benefit of this strategy in human gene therapy.

Chimeric antigen receptor T cells redirected to the B cell antigen CD19 (CAR19) have shown efficacy in the treatment of B-lineage acute lymphocytic leukemia, and are being evaluated in other hematologic malignancies as well. The prevalent configuration of CAR19 for clinical application is comprised of the extracellular single chain targeting domain (scFv) derived from the FMC63 antibody, a CD8 linker and transmembrane domain, a CD137 signaling domain, and a CD3-zeta signaling domain. Transmembrane domains from other immunologically-active proteins, including CD3, CD28, or CD4, and spacer domains from IgG have been employed as structural components of CARs as well. The relationship between the composition of the CAR linker and transmembrane domain and CAR T function is, however, poorly understood. We hypothesized that CAR transmembrane and linker domains derived from proteins of the tumor necrosis factor receptor superfamily (TNFRSF), whose expression is associated with normal T cell functions, may optimize the anti-tumor activity of CAR19. Therefore, primary human T cells were transduced with lentiviral expression vectors (LV) expressing new combinations of linker and transmembrane sequences encoded by various TNFRSF members. Specifically, the linker:transmembrane domain combinations were: CD8:CD4, CD8:TNFRSF19, TNFRSF19:TNFRSF19, truncated TNFRSF19:TNFRSF19, TNFRSF9:TNFRSF9 and TNFRSF16:TNFRSF16. We found that CAR T cells comprised of transmembrane domains derived from either TNFRSF19 or TNFRSF9 were superior to the CD8 transmembrane CAR 19 in tumor killing, and in the induction of IFN gamma, TNF alpha, IL-2, and GM-CSF cytokine secretion in vitro, upon co-culture with leukemia cell line targets. Moreover, TNFRSF19-expressing CAR19 T cells were superior to the CD8 transmembrane CAR19 in the elimination of engrafted Raji cells (a Burkitt’s lymphoma line) in an NSG leukemia mouse model. On the other hand, the transmembrane domain derived from TNFRSF16/LNGFR, a low affinity neurotrophin receptor, created a CAR19 construct that had no functional activity either in vitro or in vivo, despite high levels of surface CAR expression in human T cells. In conclusion, the magnitude of CAR T response can be enhanced by the use of novel linker and transmembrane domains derived from TNFRSF19 and TNFRSF9. Thus, the transmembrane domain can play a determining role in CAR T function beyond a direct topographical connection between the extracellular and the intracellular domains of the chimeric protein. Studies are underway to elucidate the mechanistic role of TNFRSF-derived linker and transmembrane domains in CAR T function. Citation Format: Dina Schneider, Ying Xiong, Darong Wu, Boro Dropulic, Rimas Orentas. Plasma membrane spanning and linker-domains from tumor necrosis factor receptor superfamily (TNFRSF) proteins provide novel functionality to chimeric antigen receptors (CARs) expressed in human T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3746. doi:10.1158/1538-7445.AM2017-3746

The recent successes of adoptive T-cell immunotherapy for the treatment of hematologic malignancies have highlighted the need for manufacturing processes that are robust and scalable for product commercialization. Here we review some of the more outstanding issues surrounding commercial scale manufacturing of personalized-adoptive T-cell medicinal products. These include closed system operations, improving process robustness and simplifying work flows, reducing labor intensity by implementing process automation, scalability and cost, as well as appropriate testing and tracking of products, all while maintaining strict adherence to Current Good Manufacturing Practices and regulatory guidelines. A decentralized manufacturing model is proposed, where in the future patients' cells could be processed at the point-of-care in the hospital.

Recent advances in the genetic modification of primary cells for ex vivo gene therapy have brought the field close to the promise envisioned almost a quarter of a century ago. The integration of vector systems that can stably transduce cells without genotoxicity, combined with superior cell processing methods, has been largely responsible for these advances. The result has been a series of ongoing clinical trials that are showing promising outcome for patients. Among the successes are adoptive T cell immunotherapy and stem cell therapies for cancer and other diseases.

Defective-interfering viruses are known to modulate virus pathogenicity. We describe conditionally replicating HIV-1 (crHIV) vectors that interfere with wild-type HIV-1 (wt-HIV) replication and spread. crHIV vectors are defective-interfering HIV genomes that do not encode viral proteins and replicate only in the presence of wt-HIV helper virus. In cells that contain both wt-HIV and crHIV genomes, the latter are shown to have a selective advantage for packaging into progeny virions because they contain ribozymes that cleave wt-HIV RNA but not crHIV RNA. A crHIV vector containing a triple anti-U5 ribozyme significantly interferes with wt-HIV replication and spread. crHIV vectors are also shown to undergo the full viral replicative cycle after complementation with wt-HIV helper-virus. The application of defective interfering crHIV vectors may result in competition with wt-HIVs and decrease pathogenic viral loads in vivo.

Introduction Chimeric Antigen Receptor T (CAR-T) cells redirected against tumor antigens are an effective therapy for B cell malignancies refractory to standard treatments. The production of patient-derived CAR-T cells is complicated and thus far is limited to institutions with experienced researchers and expensive GMP facilities, or to those invited to participate in industry sponsored clinical trials. The outsourcing of CAR T-cell production to third party vendors where cells are collected locally, shipped to the manufacturing site, and then sent back to the institution for infusion can be both costly and timely. As a result, CAR-T cell therapies are not widely available and only patients with means to travel to participating sites and with disease that is stable enough to wait the 2-3 months needed to collect and produce CAR-T cells are eligible for these treatments. At our instution we have explored the use of the CliniMACS® Prodigy (Miltenyi Biotec, Inc) for the production of CAR-T cells. The CliniMACS® Prodigy is an automated device that can be used for cell processing within a closed GMP-compliant system. Using the CAR-T system that includes software, specialized tubing sets, and optimized reagents we demonstrate the processing of CAR-T cells, with similar characteristics to those produced in a more traditional manner, in a closed system that is suitable for clinical use without the need for a clean room manufacturing facility. Methods In collaboration with Miltenyi Biotec, we obtained pre-release and final versions of the CliniMACS® Prodigy TCT process software and the TS520 tubing set that allows for cell enrichment, transduction, wash steps, and expansion all within a single set. Starting material was MNC cells recovered from a leukoreduction system chamber (LRSC) used during platelet collections by apheresis. Materials and reagents included MACS CD4 & CD8 reagents for cell enrichment, TransAct CD3/CD28 reagent for activation, lentiviral CD8 TM-41BB-CD3 zeta-cfrag vectors with either CD19 or CD20/CD19 Ab chains (Lentigen Technology Inc., A Miltenyi Biotec Company), TexMACS culture medium-3% HS-IL2, and PBS/EDTA buffer for wash steps. For two experiments, cells after CD4/CD8 enrichment were activated and transduced in 6 well plates and expanded after day 5 in G-Rex gas permerable devices. Total time for line preparation was 14±1 days. Transduction was measured by Protein L expression using flow cytometry. Line function was measured in 51Cr Release assays and by intracellular cytokine production. Results Starting cells were washed free of platelets and enriched for CD4+ and CD8+ cells using the Prodigy device. We achieved consistent high levels purity (99±3%) and good recovery (51.0±6%) of CD4+ and CD8+ cells (N=5). The enriched cells were 90±12% CD3+. The approximately 10% non-T cells were CD8+ NK cells, that were largely eliminated after cell activation through CD3/CD28 and expansion. A controlled number of 1 x 10E8 cells enriched for CD4+ plus CD8+ cells were retained in the Prodigy and in 2 experiments a smaller fraction of cells was cultured in 6 well plates for activation and initial transduction. Three preparations were conducted in the Prodigy, one using the CD19 vector and two with the CD19+CD20 vector. Transduction efficiency ranged from 21%-46% of total T cells with a modest preference for CD4+ cells. Expansion ranged from 26-40 fold and all of the lines recognized CD19 and/or CD20 targets based on 51Cr release assays or IFN-gamma production. The paired lines generated on the Prodigy versus manual methods showed similar overall transduction, phenotype, and function as shown in the figure for one representative preparation. Conclusions CAR-T cells generated in the Prodigy were similar to those prepared using manual methods in both phenotype and function. This process is timely, requiring 14 days for generation of the target CAR-T cell dose, and does not require outsourcing to third party vendors. All of the Prodigy CAR-T cell preparations met criteria for clinical use in our upcoming Phase I clinical trial. The ability to produce CAR-T cells suitable for clinical use in an entirely closed system without the need for a clean room should allow more centers and patients access to this novel form of immunotherapy. Disclosures Shah: Oncosec: Equity Ownership; Exelixis: Equity Ownership; Geron: Equity Ownership. Orentas:Lentigen Technology, Inc.: Employment. Dropulic:Lentigen Technology Inc. A Miltenyi Biotec Company: Employment. Hari:Merck: Research Funding; BMS: Honoraria.

In recent years, adoptive cell therapy using T cells engineered with chimeric antigen receptors (CAR T) specific for CD19 has shown marked clinical efficacy. Nevertheless, cases of tumor escape due to the loss of CD19 antigen have been reported. We hypothesized that tumor escape in B cell malignancies could be mitigated by CAR T approaches that target the CD19 and CD20 tumor antigens simultaneously. Second generation CARs containing scFv specific for CD19 and CD20 were generated by lentiviral transduction of human primary T cells. The configurations were: a) single targeting domain, b) tandem targeting domains, or c) two full-length CAR receptors co-expressed bicistronically in the same T cell. CAR T expression for all constructs was confirmed by Western blotting and protein L flow cytometry. CAR T cytolytic activity and IFN gamma secretion were evaluated by killing assays and ELISA, respectively. Single, tandem, and bicistronic CAR T cells exhibited CAR surface expression of 50-70%. All anti-CD19 and anti-CD20 CAR T cells demonstrated target-specific cells lysis and induction of IFN gamma. Interestingly, the bicistronic CAR19 CAR20 construct yielded high CAR T surface expression and IFN gamma production, but inferior in vitro cytolytic activity. To confirm CAR T specificity, K562 CD19+ and K562 CD20+ cell lines were generated. In flow-based 1h killing assays, CAR19 and CAR20 lysed their respective target lines only, whereas tandem CAR constructs lysed both K562 CD19+ and K562 CD20+ cells, but not K562 control. In a subsequent series of co-culture experiments, Raji target cells were combined with very low ratios of CAR T cells, to allow for the evolution of escape variants. On day 5 of co-incubation in the presence of CAR19 T cells, the surviving Raji cells numbers were comparable to control (85,000 cells/well vs 77, 000 cells/well in sham control). In comparison, viable Raji numbers decreased drastically post co-incubation with CAR20-, CAR19_20- and CAR20_19-T cells (546, 1,355 and 1,543 cells/well, respectively). Among the surviving Raji population, CD19 surface expression was reduced to 1.6%, vs 93.29% for sham control, whereas the expression of CD20 and CD22 remained high (97% and 78%, respectively). By contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration. Thus, targeting two tumor antigens simultaneously using tandem CAR T cells is a promising new strategy for mitigation of tumor escape via antigen down-regulation. Moreover, expression of two scFv in a single CAR format is superior to expression of two independent CARs from a bicitsronic vector. It also appears that the order of the targeting domains within the tandem CAR impacts anti-tumor activity. These findings are currently being explored further in animal model systems. Citation Format: Dina Schneider, Ying Xiong, Darong Wu, Boro Dropulic, Rimas Orentas. Tandem anti-CD20 and -CD19 scFV-based chimeric antigen receptors (CARs) mitigate tumor escape in hematologic malignancies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2297.

Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV, as they have the potential for sustained viral inhibition via reduced treatment interventions. One innovative approach developed here involves using conditionally replicating vectors (CR-Vectors), as these vectors utilize HIV-expressed proteins to replicate. These vector payloads can spread along with HIV into the budding viral particles, and co-infect target cells, essentially disseminating to HIV infected cells. We have generated and characterized CR-Vectors carrying therapeutic payloads consisting of various non-coding RNA regulatory expression cassettes, which modulate HIV both transcriptionally and post-transcriptionally, as well as CRISPR directed excision machinery. Notably, we have followed both virus and vector expression in T-cells and in vivo in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-Vectors functionally suppress HIV expression in a long-term stable and potent manner in both the presence and absence of MPA; and that transcriptional targeting is more potent at modulating stable suppression of HIV than post-transcriptional targeting or CRISPR directed excision of HIV. This suppression may be physiologically relevant, as it appears to drive HIV to a sustained non-pathogenic set point. Our findings suggest that CR-Vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term stable suppression of HIV leading ultimately to a functional cure.

In recent years, adoptive cell therapy using T cells engineered with chimeric antigen receptors (CAR T) specific for CD19 has shown marked clinical efficacy. Nevertheless, cases of tumor escape due to tumor escape in B cell malignancies could be mitigated by CAR T the loss of CD19 antigen have been reported. We hypothesized that approaches that target the CD19 and CD20 tumor antigens simultaneously. Second generation CARs containing scFv specific for CD19 and CD20 were generated by lentiviral transduction of human primary T cells. The configurations were: a) single targeting domain, b) tandem targeting domains, or c) two full-length CAR receptors co-expressed bicistronically in the same T cell. CAR T expression for all constructs confirmed by Western blotting and protein L flow cytometry. CAR T cytolytic activity and IFN gamma secretion were evaluated by killing assays and ELISA, respectively. Single, tandem, and bicistronic CAR T cells exhibited CAR surface expression of 50-70%. All anti- CD19, anti-CD20 CAR T cells demonstrated target-specific cells lysis and induction of IFN-gamma. Interestingly, bicistronic CAR19, CAR20 construct yielded high CAR T surface expression and IFN-gamma production, but inferior in vitro cytolytic activity. To confirm CAR T specificity, K562 CD19+ and K562 CD20+ cell lines were generated. In flow-based 1h killing assays, CAR19 and CAR20 lysed their respective target lines only, whereas tandem CAR constructs lysed both K562 CD19+ and K562 CD20+ cells, but not K562 control. In a subsequent series of co-culture experiments, Raji target cells were combined with very low ratios of CAR T cells, to allow for the evolution of escape variants. On day 5 of co-incubation in the presence of CAR19 T cells, the surviving Raji cells numbers were comparable to control (85,000 cells/well vs 77, 000 cells/well in sham transduction). In comparison, viable Raji numbers decreased drastically post co-incubation with CAR20-, CAR19_20- and CAR20_19-T cells (546, 1,355 and 1,543 cells/well, respectively). Among the surviving Raji population, CD19 surface expression was reduced to 1.6%, vs 93.29% for sham control, whereas the expression of CD20 and CD22 remained high (97% and 78%, respectively). By contrast, the CD20 surface marker was less prone to down-regulation by CAR-T cells expressing anti-CD20 scFv. Similar results were seen when experiments were of longer, 7 days, or shorter, 1 day, duration. Thus, targeting two tumor antigens simultaneously using tandem CAR T cells is a promising new strategy for mitigation of tumor escape via antigen down-regulation. Moreover, expression of two scFv in a single CAR format is superior to expression of two independent CARs from a bicitsronic vector. It also appears that the order of the targeting domains within the tandem CAR impacts anti-tumor activity. These findings are currently being explored further in animal model systems.

In an open clinical trial for Stage IV melanoma patients, we are testing a promising new therapy in which the patient’s own T cells are genetically engineered with a T cell receptor (TIL 1383I TCR) targeting the melanoma antigen tyrosinase. Included in the TIL 1383I TCR vector is a nonfunctional CD34 marker that can be readily tracked by flow cytometry. These transgenic T cells are infused into the patient with the goals of killing tumor cells and promoting anti-tumor inflammatory responses. We find that the transgenic T cells persist at low levels for at least twenty months in vivo. Many of the transgenic T cells are activated in the patient, indicating that they are responding to antigen. A subset of activated cells express inhibitory receptors, including PD-1 and CTLA-4. Upon further analysis of patient samples, we found that the percent of transgene-expressing T cells can be increased up to three-fold with either cytokine stimulation and/or CD3/CD28-bead stimulation. When cultured in vitro, transduced T cells lose transgene expression over time, and culturing in the absence of cytokines dramatically increases the rate of transgene expression loss. As seen ex vivo, restimulation of in vitro cultured T cells restores transgene expression. Cells undergoing division express substantially more transgene than resting cells. These data suggest that mitogenic factors causing cell division may promote transgene expression. We next plan to test mitogenic (high dose IL-2, IL-15) and anti-inhibition (PD-1 and CTLA-4 blockade) therapies with T cell transfer utilizing murine mouse models and potentially amend our protocol in the future to reduce the effects of exhaustion and transgene loss on transgenic anti-tumor T cells.

The field of possible application of mesenchymal stem cells in medicine and research expanded tremendously with the advent of improved Lentiviral-vectors capable of inserting stable copies of genes of interest and expressing proteins or biologically active RNA species ad libitum, performing delicate gene editing or active gene silencing or serving as advanced drug delivery systems utilized in ex vivo cell therapy. The combination of these two fields has created a number of new areas of research in the landscape of modern medicine which are now extensively studied and discussed here. These areas include tissue engineering, tissue repair, wound healing and tissue implants, anticancer therapies, angiogenesis, myocardial infarction and repair as well as understanding and treating acute lung damage and injury. In addition, genetically modified, tagged MSCs are being intensively deployed in research and therapeutic attempts of the various ailments of the central nervous system including Parkinson’s disease, Alzheimer’s disease, various phases of acute ischemia and trauma. The emergence of new and important data for type II diabetes research is being followed with treatment suggestions and studies of senescence to find novel applications for genetically engineered MSCs. We find in ­general that genetically modified MSCs are at the cusp of breaking through from basic research into the next phase of clinical trials.

Transduction with Lentiviral vectors has been shown to be the most efficient method for the stable delivery of nucleic acid sequences into mammalian cells. Lentiviral vectors have been widely used in research and have recently shown success in clinical trials for human gene therapy. In this paper, we describe the use of lentiviral vectors to generate genetically modified cell substrates for the manufacture of proteins and other complex biologics. The use of lentiviral vectors for the generation of genetically modified cell substrates for the production of biologic material has several advantages over other systems: (1) highly productive mammalian cell lines can be rapidly generated without selection or gene amplification; (2) the high number of vector copies are distributed throughout the open chromatin of the genome, resulting in cell lines that are extremely stable for high levels of gene expression and, consequently, protein production; and (3) high levels of protein glycosylation are maintained despite very high levels of protein production. These advantages offer the potential to significantly improve the quality, time-to-market, and manufacturing cost of biologics for human use.

Lentiviral vectors have been successfully used in the clinic and they are increasingly being used for nonclinical applications. They are capable of stably transducing a broad range of mammalian cell types, including nondividing cells, with high efficiency. This review summarizes the evolving molecular design of lentiviral vectors, describing how they have improved since their first description. Lentiviral vector safety and issues surrounding genotoxicity are discussed. Examples of successful application of lentiviral vectors in laboratory and preclinical research are described. These include functional genomics, target validation, protein manufacturing, in vivo imaging, transgenic animals, and stem cell research.

An approach that assesses the intracellular efficiency of ribozyme cleavage of HIV RNA is described. A viral replicon that contains both a ribozyme and its target sequence was constructed and tested for viability inside T lymphocytes. The viral replicon is an intact HIV- 1 genome into which we have engineered an RNA sequence that cleaves an RNA site in the HIV-1 U5. When this molecule was introduced into HeLa CD4 + cells followed by co-cultivation with MT4 cells, virus replication was initially inhibited, while control HIV-1 cultures showed normal growth kinetics. However, after several days in culture, virus production was observed from this HIV–U5–ribozyme molecule. This finding suggests that in this context a U5 ribozyme cannot permanently suppress HIV-1 production. This HIV ribozyme-containing model is useful for determining the intracellular efficiency of ribozymes targeted to different sites in the HIV genome. It could be used also to examine the nature and mechanism of resistance by HIV to ribozymes.

Patients with hemophilia A present with spontaneous and sometimes life-threatening bleeding episodes that are treated using blood coagulation factor VIII (fVIII) replacement products. Although effective, these products have limited availability worldwide due to supply limitations and product costs, which stem largely from manufacturing complexity. Current mammalian cell culture manufacturing systems yield around 100 µg/l of recombinant fVIII, with a per cell production rate of 0.05 pg/cell/day, representing 10,000-fold lesser production than is achieved for other similar-sized recombinant proteins (e.g. monoclonal antibodies). Expression of human fVIII is rate limited by inefficient transport through the cellular secretory pathway. Recently, we discovered that the orthologous porcine fVIII possesses two distinct sequence elements that enhance secretory transport efficiency. Herein, we describe the development of a bioengineered fVIII product using a novel lentiviral-driven recombinant protein manufacturing platform. The combined implementation of these technologies yielded production cell lines that biosynthesize in excess of 2.5 mg/l of recombinant fVIII at the rate of 9 pg/cell/day, which is the highest level of recombinant fVIII production reported to date, thereby validating the utility of both technologies.

We have designed a ribozyme that cleaves human immunodeficiency virus type 1 (HIV-1) RNA in U5 (at nucleotide +115). This ribozyme was tested in vitro and was found to give efficient and specific digestion of RNA containing the HIV-1 U5 sequence. When the U5 ribozyme was placed into the HIV-1 genome, virus replication was suppressed in tissue culture. Introduction of this ribozyme into cells by using an amphotropic retrovirus vector significantly reduced expression of U5-containing RNA in cells chronically infected with HIV-1. Naive T cells were cocultivated with packaging cells that produce defective amphotropic retroviruses containing the U5 ribozyme. These lymphocytes were found to be partially protected from HIV-1 infection.