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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.

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.