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Chimeric Antigen Receptor (CAR) T cell therapy is an accepted standard of care for relapsed/refractory B cell malignancies. However, the high cost of existing industry-driven centralized production makes this therapy unaffordable in low and middle-income countries. Decentralized or point of care manufacturing has the potential to overcome some of these challenges. Here we demonstrate a decentralized manufacturing process for anti-CD19-CAR-T cells using a fully automated closed system (Miltenyi CliniMACS Prodigy®) is feasible in a developing country setting. Validation run data, as part of a pre-clinical trial safety evaluation, demonstrates the successful and robust manufacturing of anti-CD19 CAR-T cells with T cell expansion of 25 to 47-fold. The median transduction efficiency was 48.8%, with a median viability of 98% and fulfillment of all standard release criteria assays for clinical application. Evaluation of production costs in an academic, not for profit setting in India provide a benchmark for low and middle-income pricing which could greatly increase access to this therapy. Based on our analysis, the cost per product would be approximately $35,107 US dollars. Our data highlights the safety, efficacy, and reproducibility of the process for use in planned future clinical trials.

Chimeric antigen receptor T-cell (CAR-T cell) therapy directed at CD19 produces durable remissions in the treatment of relapsed/refractory non-Hodgkin lymphoma (NHL). Nonetheless, many patients receiving CD19 CAR-T cells fail to respond for unknown reasons. To reveal changes in 4-1BB–based CD19 CAR-T cells and identify biomarkers of response, we used single-cell RNA sequencing and protein surface marker profiling of patient CAR-T cells pre- and postinfusion into patients with NHL. At the transcriptional and protein levels, we note the evolution of CAR-T cells toward a nonproliferative, highly differentiated, and exhausted state, with an enriched exhaustion profile in CAR-T cells of patients with poor response marked by TIGIT expression. Utilizing in vitro and in vivo studies, we demonstrate that TIGIT blockade alone improves the antitumor function of CAR-T cells. Altogether, we provide evidence of CAR-T cell dysfunction marked by TIGIT expression driving a poor response in patients with NHL. Significance: This is the first study investigating the mechanisms linked to CAR-T patient responses based on the sequential analysis of manufactured and infused CAR-T cells using single-cell RNA and protein expression data. Furthermore, our findings are the first to demonstrate an improvement of CAR-T cell efficacy with TIGIT inhibition alone. This article is highlighted in the In This Issue feature, p. 1825

Supplementary Data from Sequential Single-Cell Transcriptional and Protein Marker Profiling Reveals TIGIT as a Marker of CD19 CAR-T Cell Dysfunction in Patients with Non-Hodgkin Lymphoma

Chimeric antigen receptor T-cell (CAR-T cell) therapy directed at CD19 produces durable remissions in the treatment of relapsed/refractory non-Hodgkin lymphoma (NHL). Nonetheless, many patients receiving CD19 CAR-T cells fail to respond for unknown reasons. To reveal changes in 4-1BB–based CD19 CAR-T cells and identify biomarkers of response, we used single-cell RNA sequencing and protein surface marker profiling of patient CAR-T cells pre- and postinfusion into patients with NHL. At the transcriptional and protein levels, we note the evolution of CAR-T cells toward a nonproliferative, highly differentiated, and exhausted state, with an enriched exhaustion profile in CAR-T cells of patients with poor response marked by TIGIT expression. Utilizing in vitro and in vivo studies, we demonstrate that TIGIT blockade alone improves the antitumor function of CAR-T cells. Altogether, we provide evidence of CAR-T cell dysfunction marked by TIGIT expression driving a poor response in patients with NHL.Significance:This is the first study investigating the mechanisms linked to CAR-T patient responses based on the sequential analysis of manufactured and infused CAR-T cells using single-cell RNA and protein expression data. Furthermore, our findings are the first to demonstrate an improvement of CAR-T cell efficacy with TIGIT inhibition alone.This article is highlighted in the In This Issue feature, p. 1825

Despite the success of antiretroviral therapy (ART) for people living with HIV, lifelong treatment is required and there is no cure. HIV can integrate in the host genome and persist for the life span of the infected cell. These latently infected cells are not recognized as foreign because they are largely transcriptionally silent, but contain replication-competent virus that drives resurgence of the infection once ART is stopped. With a combination of immune activators, neutralizing antibodies, and therapeutic vaccines, some nonhuman primate models have been cured, providing optimism for these approaches now being evaluated in human clinical trials. In vivo delivery of gene-editing tools to either target the virus, boost immunity or protect cells from infection, also holds promise for future HIV cure strategies. In this Review, we discuss advances related to HIV cure in the last 5 years, highlight remaining knowledge gaps and identify priority areas for research for the next 5 years. An effective and scalable cure strategy is a top priority for the HIV research field; this Review discusses recent advances, knowledge gaps, and priority research areas for the next 5 years.

The gene and cell therapy field saw its first approved treatments in Europe in 2012 and the United States in 2017 and is projected to be at least a $10B USD industry by 2025. Despite this success, a massive gap exists between the companies, clinics, and researchers developing these therapeutic approaches, and their availability to the patients who need them. The unacceptable reality is a geographic exclusion of low-and middle-income countries (LMIC) in gene therapy development and ultimately the provision of gene therapies to patients in LMIC. This is particularly relevant for gene therapies to treat human immunodeficiency virus infection and hemoglobinopathies, global health crises impacting tens of millions of people primarily located in LMIC. Bridging this divide will require research, clinical and regulatory infrastructural development, capacity-building, training, an approval pathway and community adoption for success and sustainable affordability. In 2020, the Global Gene Therapy Initiative was formed to tackle the barriers to LMIC inclusion in gene therapy development. This working group includes diverse stakeholders from all sectors and has set a goal of introducing two gene therapy Phase I clinical trials in two LMIC, Uganda and India, by 2024. Here we report on progress to date for this initiative.

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.

Anti-CD19 chimeric antigen receptor T (CAR-T) cells have demonstrated activity against relapsed/refractory lymphomas. Cytokine release syndrome (CRS) and immune effector cell – associated neurotoxicity syndrome (ICANS) are well-known complications. Tocilizumab, a monoclonal antibody targeting the interleukin-6 (IL-6) receptor was administered 1 hour prior to infusion of anti-CD19 CAR-T cells with CD3ζ/4-1BB costimulatory signaling used to treat non-Hodgkin lymphoma patients. Relapsed/refractory lymphoma patients treated with anti-CD19 CAR-T cells were included in this analysis. Cytokine plasma levels were measured by electrochemiluminescence before lymphodepleting chemotherapy, prior to infusion and then on days 2, 4,6, and 14 days after treatment. Twenty patients were treated. Cell products included locally manufactured anti-CD19 CAR-T (n=18) and tisagenlecleucel (n=2). There were no adverse events attributed to tocilizumab. Ten patients had grade 1–2 CRS at a median of 4 (range 3-7) days. There were no cases of grade ≥3 CRS. Five patients had ICANS, grade 1 (n=4) and grade 4 (n=1). Laboratory studies obtained prior to lymphodepleting chemotherapy were comparable between patients with and without CRS, except for interleukin (IL)-15 plasma concentrations. patients with CRS had higher post-infusion ferritin and C reactive protein, with more marked increases in inflammatory cytokines, including IL-6, IL-15, IFN-γ, fractalkine and MCP-1. Fifteen patients (75%) achieved CR and 2 (10%), PR. One-year OS and PFS estimates were 83% and 73%. Prophylactic tocilizumab was associated with low CRS incidence and severity. There were no adverse events associated with tocilizumab, no increase in frequency or severity of ICANS and excellent disease control and overall survival.

Chimeric antigen receptor (CAR) T cells typically use a strong constitutive promoter to ensure maximal long-term CAR expression. However, recent evidence suggests that restricting the timing and magnitude of CAR expression is functionally beneficial, whereas constitutive CAR activation may lead to exhaustion and loss of function. We created a self-driving CD19-targeting CAR, which regulates its own function based on the presence of a CD19 antigen engaged by the CAR itself, by placing self-driving CAR19 constructs under transcriptional control of synthetic activator protein 1 (AP1)-nuclear factor κB (NF-κB) or signal transducer and activator of transcription (STAT)5 promoters. CD19 antigen-regulated expression was observed for self-driving AP1-NFκB-CAR19, with CAR19 upregulation within 18 h after exposure to target CD19, and corresponded to the level of tumor burden. Self-driving CAR-T cells showed enhanced tumor-dependent activation, expansion, and low exhaustion in vitro as compared to constitutively expressed EF1α and murine stem cell virus (MSCV) CARs and mediated tumor regression and survival in Raji-bearing NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) mice. Long-term CAR function correlated with upregulated CAR expression within 24 h of exposure to tumor antigen. The self-driving AP1-NFκB-CAR19 circuit was also used to inducibly express dominant-negative transforming growth factor β receptor II (TGFBRIIdn), which effectively countered the negative effects of TGF-β on CAR-T activation. Thus, a self-driving CAR approach may offer a new modality to express CAR and auxiliary proteins by enhancing CAR-T functional activity and limiting exhaustion.

Chimeric antigen receptor T cell (CAR-T cell) therapy is known to produce durable remissions in the treatment of CD19+relapsed/refractory B cell malignancies. Nonetheless, a significant portion of patients receiving the therapy experience poor outcomes in the acute response for unknown reasons. Given the decreased expansion and persistence of CD8 CAR-T cells in poor outcome groups, this failure may be attributed to CAR-T cell dysfunction. However, a comparison of the post-infusion transcriptional profiles and phenotypes between CAR-T cells of poor and favorable response groups has not been performed. Here, we employed single cell RNA sequencing and protein surface marker profiling of serial CAR-T cell blood samples from patients with CD19+relapsed/refractory non-Hodgkin’s lymphoma (NHL) to reveal CAR-T cell evolution, identify biomarkers of response, and test for evidence of exhaustion in CAR-T cells of poor responders. At the transcriptional and protein levels, we note the evolution of a majority of CAR-T cells toward a non-proliferative and highly-differentiated state. In poor outcome patients, we observed a more marked enrichment of an exhaustion profile as compared to favorable outcome patients. Lastly, we identified the checkpoint receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains) as a novel prognostic biomarker and potential driver of CAR-T cell exhaustion. Altogether, we provide evidence of CAR-T cell dysfunction marked by TIGIT expression driving poor response in NHL patients.

A substantial number of patients with leukemia and lymphoma treated with anti-CD19 or anti-CD22 monoCAR-T cell therapy relapse because of antigen loss or down-regulation. We hypothesized that B cell tumor antigen escape may be overcome by a chimeric antigen receptor (CAR) design that simultaneously targets three B cell leukemia antigens. We engineered trispecific duoCAR-T cells with lentiviral vectors encoding two CAR open reading frames that target CD19, CD20, and CD22. The duoCARs were composed of a CAR with a tandem CD19- and CD20-targeting binder, linked by the P2A self-cleaving peptide to a second CAR targeting CD22. Multiple combinations of intracellular T cell signaling motifs were evaluated. The most potent duoCAR architectures included those with ICOS, OX40, or CD27 signaling domains rather than those from CD28 or 4-1BB. We identified four optimal binder and signaling combinations that potently rejected xenografted leukemia and lymphoma tumors in vivo. Moreover, in mice bearing a mixture of B cell lymphoma lines composed of parental triple-positive cells, CD19-negative, CD20-negative, and CD22-negative variants, only the trispecific duoCAR-T cells rapidly and efficiently rejected the tumors. Each of the monoCAR-T cells failed to prevent tumor progression. Analysis of intracellular signaling profiles demonstrates that the distinct signaling of the intracellular domains used may contribute to these differential effects. Multispecific duoCAR-T cells are a promising strategy to prevent antigen loss-mediated relapse or the down-regulation of target antigen in patients with B cell malignancies.

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.

Rhabdomyosarcoma (RMS) is an aggressive soft tissue sarcoma originating from skeletal muscle in children and adolescent young adults. Despite multi-modal aggressive therapies, relapsed, refractory or metastatic rhabdomyosarcoma remains a lethal disease with no significant improvement in outcome over decades of clinical trials. Therefore novel therapies are needed. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase that is overexpressed in RMS when compared with normal tissues, and mutationally activated in about 7.5% of RMS. Recently we showed that PAX3-FOXO1 establishes a super-enhancer in the FGFR4 genomic locus driving its high expression in fusion positive RMS. CAR T-cell therapy is effective in treating refractory and relapsed B-cell leukemia and lymphoma, with three CARs targeting CD19 approved by the FDA. Multiple CART trials are currently underway for solid tumors. Since FGFR4 is a cell surface protein, we hypothesized that FGFR4 will provide a rational target for immunotherapy in RMS. We confirmed by immunohistochemistry staining, western analysis, and Meso Scale Discovery that FGFR4 protein is highly differentially expressed in RMS samples. We developed a murine anti-FGFR4 antibody, 3A11, by immunizing mouse with FGFR4-IG fusion protein. 3A11 showed high affinity and specificity of binding to FGFR4. We then developed a second-generation CAR using the VL and VH domain of 3A11 antibody and found that the scFvFc retained its specificity and high affinity at nanomolar range. Human T cells transduced with 3A11 CAR construct were found to be highly potent at inducing IFN-γ, TNF-α, IL-2 and cytotoxicity when the FGFR4-CART was co-cultured with RMS cells, but not with RMS cells with FGFR4 knocked out or FGFR4 negative cells. 3A11 CART incubated with human primary cells obtained from liver, kidney, heart, and pancreas, did not elicit a cytokine response, indicating a low potential for “on-target off-tumor” toxicity. In vivo testing also found that 3A11 CART eliminated RMS cells in both murine xenograft metastatic and localized subcutaneous models. Therefore we have developed a CART targeting FGFR4 that shows high potency for treating RMS. A phase 1 FGFR4-CART clinical trial is planned for children and adolescent young adults with relapsed/refractory rhabdomyosarcoma. Citation Format: Adam Tai Chi Cheuk, Meijie Tian, Nityashree Shivaprasad, Steven Highfill, David Milewski, G Tom Brown, Peter Azorsa, Dina Schneider, Berkley Gryder, Jun S Wei, Young Kwok Song, Hsien-Chao Chou, Jerry Wu, Joon-Yong Chung, Brian Belyea, Corinne Linardic, Stephen M Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Potent antitumor activity of a FGFR4 CAR-T in rhabdomyosarcoma [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 LB213.

Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.

INTRODUCTION GBM has median survival of 12 months despite current SOC therapy. The most important mechanism of TMZ resistance is the O6-methylguanine-DNA methyltransferase (MGMT) gene. The MGMT inhibitor O6-benzylguanine (BG) demonstrated efficacy in depleting MGMT but approach led to unacceptable bone marrow toxicity and has thus 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 transfected with MGMT-P140K using a lentiviral vector. We tested tolerance and efficacy of three different paradigms for conditioning bone marrow and re-infusion of HPC. 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) myelosuppression consistent with chemo-protection as hypothesized. Median PFS and OS was 24 and 33 months respectfully, and three patients in Arm III were progression free at 36 months with one progression free at 48 months. OS exceeded RPA & Nomogram predicted survival by 3.6-fold, suggesting clinical benefit. Viral insertion site analysis demonstrate no 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 U-01 funded phase II study is ongoing at UH and NIH.

Gene-based therapies represent a promising treatment for HIV-1 infection, as they offer the potential for sustained viral inhibition and reduced treatment interventions. One approach developed here involves using conditionally replicating vectors (CR-vectors). CR-vectors utilize HIV-expressed proteins to replicate and disseminate along with HIV into the budding viral particles, thereby co-infecting target cellular reservoirs. We generated and characterized several CR-vectors carrying various therapeutic payloads of non-coding RNAs targeted to HIV-1, both transcriptionally and post-transcriptionally. Both virus and vector expression was followed in cell culture systems and T cells 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 manner and that transcriptional targeting of and epigenetic silencing of HIV can be passaged to newly infected cells by the action of the CR-vector, ultimately establishing a sustained parasitism of HIV. Our findings suggest that CR-vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term sustained suppression of HIV-1, leading ultimately to a functional cure. Keywords: non-coding RNA, HIV-1, conditionally replicating lentiviral vectors, epigenetic silencing, transcription

Background: AntiCD19 CAR-T cells are effective against chemorefractory B cell lymphoma. Patients (pts) with rapidly progressive disease and urgent need for therapy have very poor prognosis and may not be able to receive CAR-T cells in time. Decreasing the apheresis to infusion time can make CAR-T cells rapidly available. We conducted a dual-center phase I trial using on-site manufacture of CAR-T cells for treatment of relapsed and refractory (r/r) B cell lymphoma. 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, TNFRS19 transmembrane region, and 4-lBB/CD3z intracellular signaling domains. GMP-compliant manufacture was done using CliniMACS Prodigy in a 12-day culture, subsequently shortened to 8 days. Dose escalation was done using 3+3 design. Lymphodepletion included cyclophosphamide (60mg/kg x 1) and fludarabine (25mg/m2/d x 3). Cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) were graded using the Lee and CARTOX criteria, respectively. CAR-T persistence was measured with qPCR and flow cytometry. Plasma cytokine concentrations were measured using electrochemiluminescence (MesoScale Diagnostics, Inc). Results: Thirty-one pts were enrolled and treated. Baseline patient and disease characteristics are listed in table 1. Twenty-nine (94%) pts were refractory to the prior line of therapy and 21 (68%) had symptomatic disease at the time of lymphocyte collection. CAR-T cell product manufacture was successful in all pts. Median transduction rate was 45% [range 15-66], median culture expansion was 36-fold [range 3-79]. CAR-T cell doses were 0.5 x 10 6/kg (n = 4), 1 x 10 6/kg (n = 16), and 2 x 10 6/kg (n = 11). Median time from apheresis to lymphodepletion was 7 days (range 2 - 15) and median time from apheresis to CAR-T cell infusion time was 13 days (range 9 - 20). Twenty-eight pts were infused fresh product. Seventeen pts (55%) experienced CRS. Grade 1-2 CRS was observed in 15 pts (48%), grade ≥ 3 was observed in 3 pts (10%). One patient had grade 4 CRS that was later complicated by hemophagocytic syndrome and died on day 21; a second patient had grade 5 CRS in the context of bulky disease and died on day 8. Ten pts (32%) had ICANS and 4 pts had grade 3-4 ICANS. Treatment for CRS / ICANS included tocilizumab (n = 12), siltuximab (n = 4), anakinra (n = 3) and corticosteroids (n = 10). The most common all grade non - hematologic toxicity was fatigue, observed in 19 pts, all grade 1. Hematologic toxicity was common, with grade ≥ 3 neutropenia observed in all subjects. Twenty-five (81%) presented disease response and twenty-two pts (71%) achieved complete response (CR). There were no statistically significant differences in the overall and complete response rates between dose levels. After a median follow up of 18 months (range 1 - 32), 5 pts relapsed, and 7 pts have died. Causes of death include progressive disease (n=5), CRS (n=1) and CRS/HLH (n=1). Two-year estimates of PFS and OS for the whole cohort were 67% (95%CI 52-88%) and 75% (95%CI 60-93%)(fig1), respectively. Two-year estimates for patients achieving disease response (CR or PR) were 82% (95%CI 67-99%) and 90% (95%CI 78-100%), respectively. The median duration of response has not been reached (95% CI 74-100). Among pts achieving CR, 94% (95% CI 61-100%) had sustained remission at 12 months. Median time to peak CAR-T expansion, measured by PCR, was 14 days (IQR 14-19), without differences between dose levels, culture duration or fresh vs. cryopreserved infusion. 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 2). Cytokine measurements have been conducted in 19 pts, with area under the curve (AUC) analyses showing pts with CRS had higher plasma concentrations of multiple cytokines (fig 3). Patients achieving CR had higher plasma concentrations of MIP3B. Conclusions: Second generation antiCD19 CAR-T cells with TNFRS19 transmembrane domain have potent clinical activity. On-site manufacture was successful in all pts. This strategy, in combination with fresh product infusion, can make CAR-T cell therapy rapidly available for pts with high-risk r/r B cell lymphoma. Figure 1 Figure 1. Disclosures Caimi: Amgen Therapeutics.: Consultancy; TG Therapeutics: Honoraria; XaTek: Patents & Royalties: Royalties from patents (wife); Kite Pharmaceuticals: Consultancy; Genentech: Research Funding; ADC Theraputics: Consultancy, Research Funding; Seattle Genetics: Consultancy; Verastem: Consultancy. Ghobadi: Wugen: Consultancy; Atara: Consultancy; Amgen: Consultancy, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Celgene: Consultancy. Schneider: Lentigen Technology: Current Employment. Boughan: Beigene: Speakers Bureau. Metheny: Incyte: Speakers Bureau; Pharmacosmos: Honoraria. Krueger: Lentigen: Current Employment. Kadan: Lentigen: Current Employment. Orentas: Lentigen: Patents & Royalties. Dropulic: Lentigen: Ended employment in the past 24 months, Patents & Royalties. de Lima: Miltenyi Biotec: Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: AntiCD19 CAR-T cells with TNFRSF19 transmembrane domain for treatment of relapsed and refractory B cell lymphomas.

Chimeric antigen receptor (CAR) T cells targeting CD19 mediate potent effects in relapsed and/or refractory pre-B cell acute lymphoblastic leukemia (B-ALL), but antigen loss is a frequent cause of resistance to CD19-targeted immunotherapy. CD22 is also expressed in most cases of B-ALL and is usually retained following CD19 loss. We report results from a phase 1 trial testing a new CD22-targeted CAR (CD22-CAR) in 21 children and adults, including 17 who were previously treated with CD19-directed immunotherapy. Dose-dependent antileukemic activity was observed, with complete remission obtained in 73% (11/15) of patients receiving ≥1 × 106 CD22-CAR T cells per kg body weight, including 5 of 5 patients with CD19dim or CD19- B-ALL. Median remission duration was 6 months. Relapses were associated with diminished CD22 site density that likely permitted CD22+ cell escape from killing by CD22-CAR T cells. These results are the first to establish the clinical activity of a CD22-CAR in B-ALL, including leukemia resistant to anti-CD19 immunotherapy, demonstrating potency against B-ALL comparable to that of CD19-CAR at biologically active doses. Our results also highlight the critical role played by antigen density in regulating CAR function.

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.

The B-cell maturation antigen (BCMA) is an immunotherapy target selectively expressed on multiple myeloma cells (MM). Despite recent success of experimental BCMA CAR-T cell therapy, clinical remissions in MM are often short, in part due to low persistence of the BCMA CAR-T cells. Additionally, immunosuppressive factors, notably TGF-β, are known to be elevated in the peripheral blood and tumor microenvironment in the bone marrow of MM patients, potentially contributing to the lack of durability of CAR-T cell therapy. We aimed to develop a fully-human BCMA CAR with long-term persistence and functional resistance to the suppressive effects of TGF-β. Initially, two fully human single chain variable fragments (scFv) specific for BCMA, derived by biopanning of a yeast display human scFv library, were characterized in a CAR format. Each of the two scFvs was cloned into a lentiviral vector CAR backbone, comprised of the CD8 hinge and transmembrane domain, 4-1BB co-stimulatory domain and CD3ζ signaling domain, and termed BCMA1 and BCMA2, respectively. BCMA2 and BCMA1 T cells, generated by lentiviral vector transduction of primary human T cells, exhibited high CAR expression at multiplicities of infection 10 to 40 (BCMA1: 64-81%; BCMA2: 84-94%), and consistently demonstrated potent cytotoxicity in an overnight co-culture with BCMA+ MM cell lines RPMI-8226 and MM1.S, but not the BCMA- 293T cells. BCMA2 CAR showed robust proliferative capacity in response to repeated, long-term exposure to MM1.S target cells, concordant with the sustained CD4 T-cell subset and high IL-2 production during the course of repeated exposure to target cells. This is in contrast to BCMA1, that showed precipitous decrease in CD4+ T-cell subset upon co-culture with MM cells, resulting in a CAR T-cell population dominated by CD8+ T-cells. Furthermore, the BCMA2 demonstrated prolonged potency in clearing tumor cells compared to BCMA1, even after continuous 20-day exposure to target cells. This was further confirmed in a mouse intradermal RPMI-8226 xenograft tumor model, in which infusion of BCMA2 T-cells resulted in the rapid and complete eradication of tumors, while BCMA1 showed a slower decline in tumor burden. To further improve the efficacy of the BCMA2, specifically within the TGF-β-rich immunosuppressive tumor microenvironment, we developed an armored BCMA2 CAR variant, which co-expresses the dominant negative TGF-β RII bicistronically via a 2A sequence (BCMA2-TbnegCAR). In a 10 day-long co-culture assay with MM1.S targets in the presence of spiked 10 ng/ml TGF-β, BCMA2-TbnegCAR retained high proliferative capacity and potent cytotoxicity, while the unarmored BCMA2 had diminished proliferation and substantially reduced cytokine and granzyme B production. Consistently, in the in vivo intradermal tumor model that utilizes RPMI-8226, which is a MM cell line that endogenously produces TGF-β, we observed that BCMA2-TbnegCAR treatment resulted in higher T-cell counts in the tumors and an earlier decrease of tumor burden compared to infusion with BCMA2, further demonstrating the increased efficacy and potency of the BCMA2-TbnegCAR. In conclusion, we have designed and characterized a new fully-human BCMA2-TbnegCAR, with a novel scFv that exhibits robust proliferative capacity and persistent cytotoxicity, and remains functionally resistant to the immunosuppressive effects of TGF-β. This novel BCMA CAR can potentially improve the effectiveness and durability of the current BCMA CAR-T cell therapy. Disclosures Alabanza: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Vu:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Wu:Lentigen, a Miltenyi Biotec Company: Current Employment. Zhu:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Schneider:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties.

INTRODUCTION: Chimeric Antigen Receptor (CAR)-T cell therapy is emerging as a powerful treatment for relapsed or refractory B cell lymphomas. However, a variety of escape mechanisms prevent CAR-T cell therapy from being more uniformly effective. To better understand mechanisms of CAR-T failure among patients treated with dual-targeted CAR-T cells, we performed single-cell RNA sequencing of samples from a Phase 1 trial (NCT03019055). The clinical trial used anti-CD20, anti-CD19 CAR-T cells for the treatment of relapsed/refractory B-cell non-Hodgkin Lymphoma. Clinical responses from this study are reported independently (Shah et al. in press in Nat Med). While robust clinical responses occurred, not all patients had similar outcomes. In single-antigen specific CAR-T cells, mechanisms of resistance include antigen down-regulation, phenotype switch, or PD-1 inhibition (Song et al. Int J Mol Sci 2019). However, very little is understood about the mechanisms of failure that are specific to dual-targeted CAR-T cells. Interestingly, loss of CD19 antigen was not observed in treatment failures in the study. METHODS: De-identified patient samples were obtained as peripheral blood mononuclear cells on the day of harvest ("pre" samples), at the peak of in vivo CAR-T cell expansion which varied from day 10 to day 21 after infusion ("peak" samples), and on day 28 post-infusion ("d28" samples). The CAR-T cell infusion product was obtained on day 14 of on-site manufacturing ("product" samples). All samples were cryopreserved and single cell preparation was performed with batched samples using 10X Genomics kits. Subsequent analysis was performed in R studio using the Seurat package (Butler et al. Nat Biotech 2018) with SingleR being used to identify cell types in an unbiased manner (Aran et al. Nat Immunol 2019). RESULTS: We found that distinct T cell clusters were similarly represented in the responder and non-responder samples. The patients' clinical responses did not depend on the level of CAR expression or the percentage of CAR+ cells in the infusion product. At day 28, however, there was a considerable decrease in the percentage of CAR+ cells in the responder samples possibly due to contracture of the CAR+ T cell compartment after successful clearance of antigen-positive cells. In all samples, the CAR-T cell population shifted from a CD4+ to a CD8+ T cell predominant population after infusion. We performed differentially-expressed gene analyses (DEG) of the total and CAR-T cells. In the pre samples, genes associated with T-cell stimulation and cell-mediated cytotoxicity were highly expressed in the responder samples. Since the responders had an effective anti-tumor response, we expected these pathways to also be enriched for in the peak samples; however, this was not the case. We hypothesize that differential expression of the above genes was masked due to homeostatic expansion of the T cells following conditioning chemotherapy. Based on the DEG results, we next interrogated specific genes associated with cytotoxicity, T cell co-stimulation, and checkpoint protein inhibition. Cytotoxicity-associated genes were highly expressed among responder CD8+ T cells in the pre samples, but not in the other samples (Figure 1). Few differences were seen in specific co-stimulatory and checkpoint inhibitor genes at any timepoint in the T cell clusters. We performed gene set enrichment analyses (GSEA). Gene sets representing TCR, IFN-gamma, and PD-1 signaling were significantly increased in the pre samples of the responders but not at later time points or in the infusion products. DISCUSSION: We found a correlation between expression of genes associated with T cell stimulation and cytotoxicity in pre-treatment patient samples and subsequent response to CAR-T cell therapy. This demonstrates that the existing transcriptome of T cells prior to CAR transduction critically shapes anti-tumor responses. Further work will discover biomarkers that can be used to select patients expected to have better clinical outcomes. Figure 1 Disclosures Johnson: Miltenyi Biotec: Research Funding; Cell Vault: Research Funding. Schneider:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Hari:BMS: Consultancy; Amgen: Consultancy; GSK: Consultancy; Janssen: Consultancy; Incyte Corporation: Consultancy; Takeda: Consultancy. Shah:Incyte: Consultancy; Cell Vault: Research Funding; Lily: Consultancy, Honoraria; Kite Pharma: Consultancy, Honoraria; Verastim: Consultancy; TG Therapeutics: Consultancy; Celgene: Consultancy, Honoraria; Miltenyi Biotec: Honoraria, Research Funding.

In the treatment of B cell leukemia, relapse due to antigen to loss or downregulation remains a major challenge. Tumor antigen escape may be mitigated by multi-targeting CAR T cells redirected to CD19 and CD22, and possessing a superior sensitivity to low-density antigens. Using lentiviral transduction of primary T cells, flow cytometry, cell-based assays, and xenograft mouse models, we systematically optimized 22-19 CAR architecture and co-stimulatory domains for best functionality. Fully-human tandem 22-19 CARs with co-stimulatory domains derived from 4-1BB, CD28, ICOS, OX40 or CD27, and hinge and transmembrane domains derived from CD8, CD28, or OX40 were evaluated. The tandem targeting ScFv domain orientation 22-19 was selected based on greater expression and cytotoxicity vs 19-22. All CARs achieved high T cell expression (mean 50-90%), and efficient dose-dependent killing of RajiCD19+CD22+, 293TCD19+, 293TCD22+, but not 293TCD19-CD22- target cells, and elaborated IL-2, IFN-γ, and TNF-a in antigen-dependent manner. CARs' potency in vitro varied by co-stimulatory domain: 4-1BB< OX40, ICOS, CD27 In RajiCD19+CD22+ mouse xenografts, the potency of tumor rejection by the 2nd generation tandem 22-19 CARs was also dependent on co-stimulatory domain, ranking 4-1 BB Low antigen density Raji clones were generated by CRISPR-Cas9-mediated disruption of both CD19 and CD22 expression, followed by lentiviral transduction to express a limited number of antigen molecules on the cell surface. When challenged with Raji CD22 low clone in vitro, 2nd generation tandem CARs with CD28 and ICOS co-stimulation, and the 3rd generation tandem CAR combining CD28 and 4-1BB co-stimulatory domains were more effective than 4-1BB-, CD27-, or OX40-containing tandem CARs. Against RajiCD19 low clone, CARs with CD27 and OX40 domains were more effective than CARs with 4-1BB, and CARs with ICOS or CD28 co-stimulation were the most potent. In summary, the fully-human tandem 22-19 CARs incorporating ICOS and CD28 co-stimulatory domains mitigate tumor antigen escape, exhibit robust anti-tumor function in pre-clinical models, enable superior lysis of CD22low and CD19low tumor clones, and may help improve clinical outcomes. Disclosures Hu: Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Xiong:Lentigen, a Miltenyi Biotec Company: Current Employment. Wu:Lentigen, a Miltenyi Biotec Company: Current Employment. Zhu:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Schneider:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties.

Background & Aim Currently in the Center for Cellular Engineering at the National Institutes of Health, a clinical trial which involves purifying CD8+CD62L+CD45RA+ naive T cells, culturing and transducing with a retroviral CD19 CAR, is underway. Mononuclear cells are enriched from apheresis product by automated density gradient, followed by 3 separate Strep-tactin® magnetic microbead manual selections. The selected Naive T cells are then transduced and cultured for 7 days in gas permeable cell culture bags. The entire process is open, especially purification procedures which are extremely labor intensive, takes between 16-18 hours and is performed over two days. Therefore, a less labor intensive, more streamlined and closed system process is needed. Methods, Results & Conclusion We developed a more efficient and GMP compliant process for generating CD19 and CD22 CAR Tscm product. A large scale CD8+ selection was performed on the CliniMacs Prodigy® platform followed by sterile, closed system cell sorting on the MACSQuant Tyto® for the purification of naive T cells (targets). These purified cells were placed back on the the Prodigy for culture and transduction using an automated customized program. While these enrichments worked well for donors with relatively normal target percentages, several older donors with low targets did not reach the desired purity. We then adopted a CD8+ releasable microbead selection, performed on the CliniMacs Plus device, followed by a CD62L+ microbead selection (Miltenyi Biotec) on the CliniMacs Prodigy to further enrich for target cells. We have performed many small scale selections and sorts and can consistently achieve 80-90% purities utilizing two selections. New MACSQuant Tyto® HS cartridges are now available which could potentially cut the sorting time in half, and along with new recently developed software program to decrease user setting manipulations, resulting in decreased sorting times and increased purities. In addition, after optimizing culture conditions in the CliniMacs Prodigy, as few as 25 million naive T cells can be initiated for culture, and a naive phenotype can be retained after 7 days in culture with a single lentiviral transduction. In summary, we developed a robust GMP compliant process for Tscm purification, culture and transduction which can be used to generate CAR Tscm products.

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.

Background and Aims Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties. Methods We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgcnull (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgcnull (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice. Results Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active β-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice. Conclusions In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.

Despite decades of multi-module therapies, RMS remains incurable once it has metastasized, thus new therapeutic strategies are warranted. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase, overexpressed in virtually all, mutationally activated in about 10% of RMS, and directly activated by PAX3-FOXO1 fusion protein which makes it a tractable target for immunotherapy. We generated fifteen binders against FGFR4 and characterized them further as candidate molecules for immune therapy. We found that 3A11, a mouse IgG antibody, bound to FGFR4 positive cell lines. The VL and VH domain of 3A11 was cloned and made into scFvFc format (mouse Fv and Human IgG1 Fc). The chimeric form of 3A11 antibody was successfully produced in vitro and retained its FGFR4 specificity with an observed binding affinity at nanomolar range. By ELISA using the extra cellular domain of human FGFR1, FGFR2, FGFR3 or FGFR4, 3A11 scFvFc showed dose dependent binding to FGFR4 only. We then made 3A11 into a second generation CAR. Human T cells transduced with 3A11 CAR construct were found to be highly potent at inducing IFN-γ, TNF-α, IL-2 and cytotoxicity when the FGFR4-CART was co-cultured with RMS cells, but not with RMS cells with FGFR4 knocked out and FGFR4 negative cells. Our in vivo testing also found that 3A11 CART was able to eliminate RMS cells in murine xenograft metastatic models. Here we report the successful generation of binders specific to human FGFR4. FGFR4 CAR developed from 3A11 was able to kill FGFR4 positive target cells both in-vivo and in-vitro. Thus, 3A11 CAR T cells targeting FGFR4 may provide effective immune therapies for rhabdomyosarcoma and other FGFR4 expressing cancers and clinical trials are planned. Citation Format: Adam Cheuk, Meijie Tian, Jeetendra Kumar, Peter Azorsa, Nityashree Shivaprasad, Dina Schneider, Berkley Gryder, Jun Wei, Young Song, Xinyu Wen, Sivasish Sindiri, Joon-Yong Chung, Zhongyu Zhu, Dimiter Dimitrov, Stephen Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Potent tumoricidal activity of a FGFR4 CART in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1885.

Introduction Anti CD19 CAR T-cell therapy is a breakthrough immunotherapeutic approach for relapsed, refractory B-cell malignancies. Despite initial excitement, long term progression free survival with single antigen targeted CD19 CAR T-cells range from 30-40% in aggressive B-cell NHL. Loss of target due to selective pressure against CD19 can lead to antigen downregulation and development of a CD19-negative clonal population. To overcome this resistance mechanism, we recently completed a Phase 1 anti-CD19, anti-CD20 (LV20.19) CAR T-cell trial demonstrating the safety of 2.5x10e6 cells/kg dose in patients with relapsed, refractory B-cell lymphomas (in press in Nature Medicine). We report below single cell cytokine studies from final LV20.19 CAR T-cell products using the Isoplexis single-cell proteomics device. Methods LV20.19 CAR T-cells were manufactured onsite using the CliniMACS Prodigy device on a fixed 14-day manufacturing platform with IL-2 for cell expansion (NCT03019055). Isoplexis cytokine analysis was limited to the cohort of patients treated at goal dose of 2.5x10e6 cells/kg (n=16). Leftover LV20.19 CAR T-cells from the original product were thawed, CD4 and CD8 T cells were enriched by immunomagnetic sorting, and the T-cell subsets separately stimulated with CD19-engineered K562 stimulators overnight. The stimulated cells were loaded onto single-cell, Adaptive Immune Isocode chips, and the chips read for 18 hours in an Isolight instrument to assess single-cell production of 32 individual cytokines. Isospeak software was used to analyze the single-cell data. Results 15 patients had adequate samples for analysis. Results were stratified by response (complete responders (CR) versus partial responders (PR) or progressive disease (PD)). The Isoplexis device was utilized to generate a polyfunctional (i.e., ability to produce more that 1 cytokine) index score (PSI) that has been previously reported (Rossi et al. Blood 2018) to correlate with CAR T-cell treatment outcomes. At Day 28 after LV20.19 CAR T-cell therapy, there were 11 CR patients and 4 PR/PD patients. CD4+ LV20.19 CAR T-cells from complete responders had a non-statistically significant decreased PSI score versus CD4+ cells from PR/PD patients (Figure 1A, p=0.38). The PSI score among CD8+ T-cells was similar (p=0.71) (Figure 1A). The increased PSI score among CR patients' CD4+ LV20.19 CAR T-cells was driven primarily by cells producing effector (IFNg and TNFb) cytokines. On a singular cytokine level, compared to PR/PD patient cells, CR patient CAR T-cells produced significantly higher levels of CCL-11, GM-CSF and IL-17A (Figure 1B; p Conclusion CD4+ LV20.19 CAR T-cells in responding patients trended towards a higher PSI than non-responding patients. At the single cell level, differences in the PSI among CD4+ LV20.19 CAR T-cells from responding patients were driven primarily by a few cytokines including IL-17A, CCL11, GM-CSF, IFNg, and TNFb, which may be key to clinical response. While our results are limited by sample size and only 4 PR/PD patients in this cohort, identification of cytokines correlating with response will allow selective modulation to improve clinical outcomes while limiting CRS. Ongoing studies with LV20.19 CAR T-cells will further delineate the role of IL-17A, GM-CSF and CCL-11 production as potential biomarkers of clinical response with bispecific CAR T-cell products. Disclosures Johnson: Cell Vault: Research Funding; Miltenyi Biotec: Research Funding. Fenske:Medical College of Wisconsin: Current Employment. Hamadani:Sanofi Genzyme, AstraZeneca: Speakers Bureau; Janssen R&D; Incyte Corporation; ADC Therapeutics; Celgene Corporation; Pharmacyclics, Omeros, AbGenomics, Verastem, TeneoBio: Consultancy; ADC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Takeda Pharmaceutical Company; Spectrum Pharmaceuticals; Astellas Pharma: Research Funding. Dropulic:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties: CAR-T immunotherapy. Schneider:Lentigen, a Miltenyi Biotec Company: Current Employment, Patents & Royalties. Hari:GSK: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Incyte Corporation: Consultancy; BMS: Consultancy; Amgen: Consultancy. Shah:Miltenyi Biotec: Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Incyte: Consultancy; TG Therapeutics: Consultancy; Verastim: Consultancy; Lily: Consultancy, Honoraria; Cell Vault: Research Funding; Kite Pharma: Consultancy, Honoraria.

Background: Despite decades of multimodule therapies, RMS remains incurable once it has metastasized; thus, new therapeutic strategies are warranted. FGFR4 is a developmentally regulated cell surface receptor tyrosine kinase, overexpressed in virtually all, mutationally activated in about 7.5% of RMS, and directly activated by PAX3-FOXO1 fusion protein, which makes it a tractable target for immunotherapy. Material and Methods: Using monoclonal antibody technologies and a yeast display B-cell library, we generated 15 human or mouse binders specific to human FGFR4 and engineered into human scFvFc. All binders were successfully produced in vitro, and we further characterized them using FACS and ELISA for their specificity. Octet was used to measure the binding affinity against human FGFR4. For those lead hits, they were made into different formats of therapeutic including CAR and BiTE. We then performed in vitro killing assays and/or in vivo xenograft model to determine the efficacy of those therapeutics in killing RMS cells. Results: m410 and m412 were two lead hits and scFvFcs of these two binders were successfully produced in vitro and showed FGFR4 specificity with a binding affinity at nanomolar concentration. By ELISA, these binders showed dose-dependent binding to FGFR4 protein but not to other FGFR family members. We then made m410 and m412 into CAR and BiTE format, respectively. T cells transduced with m410 CAR construct were found highly potent in inducing gamma interferon, TNF alpha, and cytotoxicity when the FGFR4-CART are cocultured with RMS cells. Our in vivo testing found them to be effective in eliminating RMS cells in murine xenograft models. When T cells were cocultured with RMS cells in the presence of m412 BiTE in vitro, potent selective antitumor effect was observed, suggesting this can be another promising strategy for RMS immunotherapy. Conclusions: Here our data demonstrated that we had successfully generated binders specific to human FGFR4. The CAR and BiTE developed from these binders were able to kill FGFR4-positive target cells. Our data suggest that these FGFR4 CARs and FGFR4 BiTEs could provide effective immune therapies for rhabdomyosarcoma and other FGFR4-expressing cancers. Citation Format: Adam Cheuk, Nityashree Shivaprasad, Dina Schneider, Marielle Yohe, Meijie Tan, Peter Azorsa, Ronald Sams, Silvia Pomella, Berkley Gryder, Rossella Rota, Ben Stanton, Jun Wei, Young Song, Xinyu Wen, Sivasish Sindiri, Jeetendra Kumar, Robert Hawley, Joon-Yong Chung, Doncho Zhelev, Zhongyu Zhu, Dimiter Dimitrov, Stephen Hewitt, Boro Dropulic, Rimas Orentas, Javed Khan. Development of FGFR4-specific chimeric antibody receptor (CAR) T cell and bispecific T cell engager (BiTE) for rhabdomyosarcoma (RMS) immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A08.

Background & Aim Current approved CAR-T cell manufacturing employs off-site production, a labor-intensive process that requires several weeks and involves shipping back and forth to the production site. This production model may limit availability and accessibility to CAR-T cells and delay treatment for patient (pt) s with rapidly progressive disease. To address these limitations, we utilized the Miltenyi CliniMACS Prodigy, a GMP-compliant device, to produce CAR-T cells on-site for a first-in-human bi-specific CD19/20 (LV20.19) CAR-T Phase I trial. Methods, Results & Conclusion CAR-T cells were manufactured within our Cell Therapy Laboratory, an ISO7 facility. Pt's mononuclear cell apheresis was collected and loaded onto the Prodigy to isolate CD4/CD8 T cells. The T cells were suspended in TexMACS supplemented with human AB serum and IL-2, activated with TransACT, and then transduced by LV20.19 the next day. Culture wash and feedings were done during the production process, and the final cell product was harvested at day 14. The process was automated with limited machine/operator interactions. CAR-T expression was assessed by protein L staining. CAR-T cells were either administered fresh or cryopreserved for later infusion. Dose was escalated in a 3+3 manner from 2.5e+5 cells/kg to a target dose of 2.5e+6 cells/kg. CAR-T cells have been successfully generated for 11/11 enrolled pts thus far with no manufacture failures, and all products met release criteria. Median T cell recovery from the apheresis products was 59.5% with a median CD3+ purity of 97.2% (Figure 1). Final product CAR expression was at a median of 17.6%. A median of 4.7e+8 CAR T cells was obtained at harvest, exceeding the required target dose. CAR-T cells contained both CD4 and CD8 T cells with higher CD4 content. The majority of CAR-T cells showed an effector-memory phenotype. CAR-T cells were able to kill CD19+/20+ target cells and produced IFN-gin response to antigens. Among the 11 treated pts 8 received fresh CAR-T cells and 3 received CAR-T cells after cryopreservation. As no DLTs were observed at the 3 dose levels, a dose of 2.5e+6 cells/kg has been selected for further expansion. Clinical response shows 6 pts have achieved a CR at Day +28 with a duration of response between 2 to 14 months. In summary, initial results demonstrated feasibility for point-of-care manufacturing of bispecific LV20.19CAR-T cells with safe and promising efficacy in treating heavily pretreated pts with R/R B-cell NHL, which warrants further investigations.