CD19-specific Chimeric Antigen Receptor Research Articles

Cellular Immunotherapy in Lymphoma: Beyond CART Cells.

Cellular immunotherapy has been rapidly evolving and increasingly utilized in the management of relapsed and refractory lymphoma. CD19-specific chimeric antigen receptor T cells (CARTs) have achieved impressive results in pivotal clinical trials. Although CART development continues, these products have fundamental limitations that may make them less desirable in particular settings. For example, CARTs can only target cell surface antigens and thus are incapable of targeting intracellular tumor-associated proteins. In contrast to CARTs, conventional T cell receptors (TCR) allow T cells to target any cellular antigen, including intracellular proteins, since they interact with peptides presented by MHC I and II molecules. T cells recognizing EBV antigens through native TCRs have been successfully employed for treatment and prophylaxis of EBV-associated lymphomas, including post-transplant lymphoproliferative disorder. Currently, transgenic TCR-transduced T cells targeting nonviral tumor antigens remain experimental but, if successful, could become an invaluable cellular therapy option. Because the manufacturing process of autologous T cell products, including CARTs and other tumor-specific T cells, takes several weeks, patients often need bridging therapy to maintain disease control, which may be challenging. Novel cellular platforms, such as genetically modified NK and NKT cells, may be amenable to allogeneic use and thus may allow production as a readily available, "off-the-shelf" product. As cellular therapies beyond CART continue to grow, available therapeutic options for relapsed and refractory lymphoma patients are expected to expand further.

High Cytotoxic Efficiency of Lentivirally and Alpharetrovirally Engineered CD19-Specific Chimeric Antigen Receptor Natural Killer Cells Against Acute Lymphoblastic Leukemia.

Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CAR-T cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs delivered by alpharetroviral/RD114-TR and lentiviral/RD114-TR vectors outperformed lentiviral/VSV-G vectors. The final generated CD19-CAR-NK cells displayed superior cytotoxic activity against CD19-expressing target cells when compared to non-transduced NK cells achieving up to 90% specific killing activity. In summary, our findings present the use of RD114-TR pseudotyped retroviral particles in combination with Vectofusin-1 as a successful strategy to genetically modify PB-derived NK cells to achieve highly cytotoxic CD19-CAR-NK cells at high yield.

Allogeneic Chimeric Antigen Receptor-Invariant Natural Killer T Cells Exert Both Direct and Indirect Antitumor Effects through Host CD8 T Cell Cross-Priming

Introduction Chimeric antigen receptor (CAR) T cells have shown impressive results in B-cell malignancies but, unfortunately, their use is still limited by the logistical and financial burdens related to the need of generating autologous cell products. The development of universal allogeneic CAR T cells to be used off-the-shelf has faced major limitations, namely the risk of Graft-versus-Host-Disease (GvHD) induction and the rejection of the cells by the host immune system. Invariant Natural Killer-T (iNKT) cells are innate lymphocytes deprived of GvHD induction potential and displaying antitumor effects, both directly and indirectly, through enhancement of CD8 T cell responses. Preclinical studies in xenogeneic mouse models demonstrated the feasibility of using iNKT cells as a platform for CAR-based therapies, and two clinical trials are currently ongoing. Objective To assess the immunoadjuvant effect of allogeneic CD19-specific iNKT CAR on the host immune system. Methods We transduced murine iNKT cells from FVB/N (H-2Kq) mice with a CD19-specific CD28/CD3ζ CAR and assessed their antitumor effect in vitro and in vivo. Results CD19-iNKT CAR had a direct cytotoxic effect in vitro against the CD19+ A20 lymphoma cells and significantly improved survival of mice after administration to major histocompatibility complex (MHC)–mismatched, immunodeficient BALB/c (H-2Kd) Rag2−/− gamma-chain−/− mice receiving A20 cells (2 × 10e4; Figure 1A) without inducing any signs of GvHD. To test the efficacy of iNKT CAR cells in the presence of host immune cells, we employed BALB/c mice receiving sublethal irradiation (4.4 Gy), resulting in only a partial and transient lymphopenia. The antitumor effect of allogeneic iNKT CAR cells was greatly enhanced (Figure 1B) suggesting the participation of host cells in the antitumor effect. Interestingly, the iNKT CAR effect was partially abrogated when we employed as recipients BALB/c BATF3−/− mice, in which CD8 T cell cross-priming is impaired as a result of the absence of BATF3-dependent CD103+ CD8a+ dendritic cells known to play a role in iNKT cell interactions with other immune effector cells (Figure 1C). Co-administration of allogeneic FVB/N iNKT CAR with autologous BALB/c CD8 T cells at the time of transfer of T-cell-depleted autologous bone marrow cells and A20 lymphoma cells into lethally irradiated BALB/c recipients resulted in a synergistic effect (Figure 1D). Due to their immuno-adjuvant effect on host CD8 T cells, low numbers of allogeneic iNKT CAR outperformed allogeneic conventional CAR T (Tcon CAR) when administered to sublethally irradiated BALB/c mice receiving A20 cells (Figure 1E-F). Conclusion Collectively, these results demonstrate the potent immunoadjuvant effect exerted by allogeneic iNKT CAR cells toward the host immune system suggesting that iNKT CAR cells are an attractive off the shelf product for adoptive immunotherapy.

CAR T Cell Generation by piggyBac Transposition from Linear Doggybone DNA Vectors Requires Transposon DNA-Flanking Regions

CD19-specific chimeric antigen receptor (CAR19) T cells, generated using viral vectors, are an efficacious but costly treatment for B cell malignancies. The nonviral piggyBac transposon system provides a simple and inexpensive alternative for CAR19 T cell production. Until now, piggyBac has been plasmid based, facilitating economical vector amplification in bacteria. However, amplified plasmids have several undesirable qualities for clinical translation, including bacterial genetic elements, antibiotic-resistance genes, and the requirement for purification to remove endotoxin. Doggybones (dbDNA) are linear, covalently closed, minimal DNA vectors that can be inexpensively produced enzymatically in vitro at large scale. Importantly, they lack the undesirable features of plasmids. We used dbDNA incorporating piggyBac to generate CAR19 T cells. Initially, expression of functional transposase was evident, but stable CAR expression did not occur. After excluding other causes, additional random DNA flanking the transposon within the dbDNA was introduced, promoting stable CAR expression comparable to that of using plasmid components. Our findings demonstrate that dbDNA incorporating piggyBac can be used to generate CAR T cells and indicate that there is a requirement for DNA flanking the piggyBac transposon to enable effective transposition. dbDNA may further reduce the cost and improve the safety of CAR T cell production with transposon systems.

Myeloid cell–targeted miR-146a mimic inhibits NF-κB–driven inflammation and leukemia progression in vivo

AbstractNF-κB is a key regulator of inflammation and cancer progression, with an important role in leukemogenesis. Despite its therapeutic potential, targeting NF-κB using pharmacologic inhibitors has proven challenging. Here, we describe a myeloid cell–selective NF-κB inhibitor using an miR-146a mimic oligonucleotide conjugated to a scavenger receptor/Toll-like receptor 9 agonist (C-miR146a). Unlike an unconjugated miR146a, C-miR146a was rapidly internalized and delivered to the cytoplasm of target myeloid cells and leukemic cells. C-miR146a reduced expression of classic miR-146a targets (IRAK1 and TRAF6), thereby blocking activation of NF-κB in target cells. IV injections of C-miR146a mimic to miR-146a–deficient mice prevented excessive NF-κB activation in myeloid cells, and thus alleviated myeloproliferation and mice hypersensitivity to bacterial challenge. Importantly, C-miR146a showed efficacy in dampening severe inflammation in clinically relevant models of chimeric antigen receptor (CAR) T-cell–induced cytokine release syndrome. Systemic administration of C-miR146a oligonucleotide alleviated human monocyte-dependent release of IL-1 and IL-6 in a xenotransplanted B-cell lymphoma model without affecting CD19-specific CAR T-cell antitumor activity. Beyond anti-inflammatory functions, miR-146a is a known tumor suppressor commonly deleted or expressed at reduced levels in human myeloid leukemia. Using The Cancer Genome Atlas acute myeloid leukemia data set, we found an inverse correlation of miR-146a levels with NF-κB–related genes and with patient survival. Correspondingly, C-miR146a induced cytotoxic effects in human MDSL, HL-60, and MV4-11 leukemia cells in vitro. The repeated IV administration of C-miR146a inhibited expression of NF-κB target genes and thereby thwarted progression of disseminated HL-60 leukemia. Our results show the potential of using myeloid cell–targeted miR-146a mimics for the treatment of inflammatory and myeloproliferative disorders.

Lenalidomide Enhances CAR-T Cell Activity Against Solid Tumor Cells

Chimeric antigen receptor (CAR) T-cell immunotherapy still faces many challenges in the treatment of solid tumors, one of which is T-cell dysfunction or exhaustion. Immunomodulator lenalidomide may improve CAR T-cell function. In this study, the effects of lenalidomide on CAR T-cell functions (cytotoxicity, cytokine secretion, and cell proliferation) were investigated. Two different CAR T cells (CD133-specific CAR and HER2-specific CAR) were prepared, and the corresponding target cells including human glioma cell line U251 CD133-OE that overexpress CD133 and human breast cancer cell line MDA-MB-453 were used for functional assay. We found that lenalidomide promoted the killing of U251 CD133-OE by CD133-CAR T cells, the cytokine secretion, and the proliferation of CD133-CAR T cells. Lenalidomide also enhanced the cytotoxicity against MDA-MB-453 and the cytokine secretion of HER2-CAR T cells but did not affect their proliferation significantly. Furthermore, lenalidomide may regulate the function of CAR T cells by inducing the degradation of transcription factors Ikaros and Aiolos.

CAR T-cell immunotherapy of B-cell malignancy: the story so far.

Chimeric antigen receptor (CAR) T-cell immunotherapy has achieved unprecedented efficacy in the treatment of chemotherapy-resistant or refractory B-cell malignancies. Promising results from pivotal anti-CD19 CAR T-cell phase II trials have led to landmark approvals of two CD19-specific CAR T-cell products by the United States Food and Drug Administration and European Medicines Agency. However, several issues associated with CAR T-cell treatment remain unresolved, such as the management of severe toxicities and the frequent occurrence of both antigen-positive and antigen-negative relapse. Nonetheless, pre-clinical research is advancing at an unprecedented pace to develop innovative solutions to address these issues. Herein, we summarise recent clinical developments and outcomes of CD19-targeted CAR T-cell immunotherapy and discuss emerging strategies that may further improve the success, safety and broadened applicability of this approach.

Preemptive mitigation of CD19 CAR T-cell cytokine release syndrome without attenuation of antileukemic efficacy

AbstractImmunotherapy with the adoptive transfer of T cells redirected with CD19-specific chimeric antigen receptors (CARs) for B-lineage acute lymphoblastic leukemia (ALL) can salvage >80% of patients having relapsed/refractory disease. The therapeutic index of this emerging modality is attenuated by the occurrence of immunologic toxicity syndromes that occur upon CAR T-cell engraftment. Here, we report on the low incidence of severe cytokine release syndrome (CRS) in a subject treated with a CAR T-cell product composed of a defined ratio CD4:CD8 T-cell composition with a 4-1BB:zeta CAR targeting CD19 who also recieved early intervention treatment. We report that early intervention with tocilizumab and/or corticosteroids may reduce the frequency at which subjects transition from mild CRS to severe CRS. Although early intervention doubled the numbers of subjects dosed with tocilizumab and/or corticosteroids, there was no apparent detrimental effect on minimal residual disease–negative complete remission rates or subsequent persistence of functional CAR T cells compared with subjects who did not receive intervention. Moreover, early intervention therapy did not increase the proportion of subjects who experience neurotoxicity or place subjects at risk for infectious sequelae. These data support the contention that early intervention with tocilizumab and/or corticosteroids in subjects with early signs of CRS is without negative impact on the antitumor potency of CD19 CAR T cells. This intervention serves to enhance the therapeutic index in relapsed/refractory patients and provides the rationale to apply CAR T-cell therapy more broadly in ALL therapy. This trial was registered at www.clinicaltrials.gov as #NCT020284.

Autologous cryopreserved leukapheresis cellular material for chimeric antigen receptor–T cell manufacture

Tisagenlecleucel, a CD19-specific autologous chimeric antigen receptor (CAR)–T cell therapy, is efficacious for the treatment of relapsed/refractory B-cell precursor acute lymphoblastic leukemia and diffuse large B-cell lymphoma. The tisagenlecleucel manufacturing process was initially developed in an academic setting and subsequently transferred to industry for qualification, validation and scaling up for global clinical trials and commercial distribution. Use of fresh leukapheresis material was recognized early on in the transfer process as a challenge with regard to establishing a global supply chain. To maximize manufacturing success rates and to overcome logistical challenges, cryopreservation was adapted into the Novartis manufacturing process from the beginning of clinical trials. Tisagenlecleucel manufactured in centralized facilities with cryopreserved leukapheresis material has been used successfully in global clinical trials at more than 50 clinical centers in 12 countries. Cryopreservation provides flexibility in scheduling leukapheresis when the patient's health is optimal to provide T cells; it also provides protection from external factors, such as shipping delays, and removes manufacturing time constraints. Several studies were performed to establish comparability of fresh versus cryopreserved leukapheresis material, to evaluate and optimize the cryopreservation process, to determine the optimal temperature and maximum hold time prior to cryopreservation and to determine the optimal temperature range for shipment and storage. Using the current validated industry manufacturing process, high success rates were achieved with regard to manufacturing tisagenlecleucel batches that met specifications and were released to patients. Consistent product quality and positive clinical outcomes support the use of cryopreserved non-mobilized peripheral mononuclear blood cells collected using leukapheresis for CAR-T cell manufacturing.

Identification and Validation of Predictive Biomarkers to CD19- and BCMA-Specific CAR T-Cell Responses in CAR T-Cell Precursors

Identification and Validation of Predictive Biomarkers to CD19- and BCMA-Specific CAR T-Cell Responses in CAR T-Cell Precursors

Low Achievement of End of Life Quality Measures in Large B-Cell Lymphoma Patients Who Progressed after CD19-Specific CAR-T Cell Therapy

Low Achievement of End of Life Quality Measures in Large B-Cell Lymphoma Patients Who Progressed after CD19-Specific CAR-T Cell Therapy

Cytogenetic Characteristics and Outcomes of Patients Receiving CTL019 CAR T Cell Therapy

Background: CTL019 is a therapy derived from autologous T cells expressing a CD19-specific chimeric antigen receptor (CAR) that was approved by the FDA in August 2017 (tisagenlecleucel). Complete and durable remissions have been seen in the setting of pediatric and young adult patients with relapsed and refractory B cell acute lymphoblastic leukemia (ALL) (Maude NEJM 2018). Initial case reports suggested that there may be differential outcomes mediated by cytogenetic characteristics of the leukemia at CAR T cell infusion. Here, we report results from a single institution experience of 112 patients. Methods: Patients with relapsed/refractory ALL were identified as having received CTL019 either in the context of a clinical trial (NCT02906371) or commercial product (tisagenlecleucel) at Children's Hospital of Philadelphia from October 2016 to April 2019. Patients who received prior CAR T therapy were excluded. Demographic, cytogenetic, and outcome data were manually abstracted from the medical record or clinical trial datasets. High risk lesions were defined as MLL(KMT2A) rearrangements, Philadelphia-chromosome (Ph+), Ph-like, hypodiploidy, and TCF3/HLF fusion. Favorable cytogenetics were defined as the presence of hyperdiploidy or ETV6/RUNX1fusion and intermediate were defined as iAMP21, IKZF1deletion, or TCF3/PBX1. Patients were classified according to their highest risk cytogenetic characteristic and stratified by cytogenetic risk category present at CAR T cell infusion. Relapse-free survival (RFS) and overall survival (OS) was described for cohorts with more than 10 patients. Results: One hundred and twelve patients were included in the analysis, with a median age of 11 years (range 1-29) at infusion, of which 32% had had a previous allogeneic hematopoietic stem cell transplant (alloHSCT). Disease burden at the time of CTL019 infusion was heterogenous, with 61% having detectable disease in the bone marrow and 21% having more than 25% blasts by flow cytometry. Thirty-six patients (32%) had leukemias with high-risk genetic lesions at infusion, including 12 with MLL rearrangements and 18 with Ph+ or Ph-like lesions (Table 2). Thirty-one patients (28%) had hyperdiploidy or ETV6/RUNX1; 3 additional were in conjunction with high-risk cytogenetics (t(17;19) and 2 with Ph+), and 3 in the setting of intermediate-risk cytogenetics (iAmp21, TCF3/PBX1, IKZF1deletion). Figure 1 demonstrates RFS for those patients in remission at day 28 following infusion, stratified by cytogenetic risk category. Complete remission (CR) rate in the high-risk cytogenetics group was 94%. RFS at 12 months was 69% (0.50-0.82), 69% (0.40-0.86), and 67% (0.48-0.80) for non-informative, favorable, and high-risk cytogenetic groups, respectively. Figure 2 shows OS of patients infused with CTL019, again stratified by cytogenetic categories of interest, with a maximum follow-up time of 30 months. OS at 12 months was 84% (0.68-0.93) and 76% (0.56-0.88) for the non-informative and high-risk cytogenetic groups, respectively. There were no deaths in that time period for the favorable risk category. There was no statistically significant difference in RFS or OS for patients with high-risk cytogenetics. The intermediate-risk cytogenetics group (n<10) was excluded from these analyses. Conclusion: Durable remissions can be achieved with CTL019 across several high-risk cytogenetic subtypes of B-ALL. Stratifying outcomes by cytogenetic risk category in this unadjusted analysis does not show a statistically significant difference in either RFS nor OS. Further investigation is needed to parse out the contribution of individual cytogenetic lesions as well as the effects of other relapse and survival risk factors at play. Figure Disclosures Rheingold: Novartis: Consultancy; Pfizer: Research Funding. Callahan:Novartis: Consultancy. Hunger:Bristol Myers Squibb: Consultancy; Amgen: Consultancy, Equity Ownership; Jazz: Honoraria; Novartis: Consultancy. Grupp:Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; Cure Genetics: Consultancy; Humanigen: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards. Maude:Kite: Consultancy; Novartis: Consultancy.

Academic, Phase I/II Trial on T Cells Expressing a Second Generation, CD19-Specific Chimeric Antigen Receptor (CAR) and Inducible Caspase 9 Safety Switch for the Treatment of B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) and B-Cell Non-Hodgkin Lymphoma (B-NHL) in Children

Survival rates of children with relapsed/refractory (r/r) BCP-ALL remain unsatisfactory and little progress has been made in the past 2 decades. Similarly, relapse of childhood B-NHL is usually associated with an aggressive disease and poor outcomes. Targeted immunotherapy with T-cells genetically modified to express a CD19-directed CAR showed an unprecedented antitumor efficacy, leading to the recent FDA and EMA approval of two CD19-CAR products for treatment of BCP-ALL and B-NHL. Relevant toxicities have, however, been reported, mainly related to the development of severe Cytokine Release Syndrome (CRS) and/or of neurotoxicity. At Ospedale Pediatrico Bambino Gesù (OPBG) in Rome, we developed a clinical-grade, 2nd generation, CD19-specific CAR construct, including 4.1bb as costimulatory domain and the inducible caspase-9 safety switch (iC9-CD19-CAR), vehiculated by a retroviral vector, to conduct an academic, phase I/II clinical trial in patients (age 1-25 yrs) affected by BCP-ALL or B-NHL. We now report on the results of the phase I and of the first 8 patients treated in the phase II portion of the study, in terms of feasibility, toxicity, maximum tolerated/recommended dose (MTD/RD) and data on response rate and biological correlates. The phase I, dose-escalation portion of the study included 3 dose levels (DL), namely: DL1, 0.5×106; DL2, 1.5×106; DL3, 3.0×106 CAR+ T cells per kg of recipient body weight. In the phase II portion, patients were treated at the RD identified in the phase I, namely 3.0×106 CAR+cells/kg. All patients received a lymphodepleting regimen consisting of fludarabine and cyclophosphamide for 3 days and iC9-CD19-CAR T cells were subsequently administered as single infusion. Patients were monitored for toxicity, expansion and persistence of iC9-CD19-CAR T cells. Seventeen children were enrolled into the trial and received iC9-CD19-CAR T cells between January 2018 and June 2019. Data were analyzed as of July 20, 2019. The characteristics of the patients are detailed in table 1. The designed dose concentration was successfully produced for all the enrolled patients and we did not observe any production failure. The median transduction rate in the drug product was 54% (range 21-73), while the median vector copy number was 3.8 (range 2.8-6.2). During the phase I portion of the study, no dose limiting toxicities (DLTs) have been recorded, defining the MTD as 3.0×106 CAR+ T cells per kg of recipient body weight. The treatment was overall tolerated and all the toxicities were reversible, the most severe being grade 3-4 neutropenia, thrombocytopenia and/or anemia, occurring in 16/17 (94.1%) patients; in 13/16 patients (81.2%) the hematological toxicity developed before the infusion and persisted after the administration of CAR T cells. Cytokine release syndrome (CRS) occurred in 10/17 patients (58.8%) and was overall moderate, reaching grade 3 (Lee criteria) in one patient only. Notably, none of the patients developed neurotoxicity and no activation of the safety switch was required. All patients were assessed for response at 4 weeks from iC9-CD19-CAR T cell infusion and 13/15 (86.7%) patients with ALL achieved complete remission (CR) with negativity of minimal residual disease (MRD), including 2/3 patients receiving the DL1, 9 patients who had failed a previous allogeneic haematopoietic stem-cell transplantation (HSCT) and 6 patients that had previously received blinatumomab, as CD19-directed immunotherapy. The iC9-CD19-CAR T cells expanded in vivo and were detectable by both flow-cytometry and molecular biology in the blood (Fig.1), bone marrow and cerebrospinal fluid of the responders. One CD19-negative relapse 3 months after infusion was recorded, while 3 additional patients relapsed with CD19+ leukemia blasts. Four patients received HSCT while in CR with MRD negativity because of regrowth of normal CD19+ B cells. The 18-month probability of overall survival for the BCP-ALL cohort is 72.2% (Fig.2). One of the 2 B-NHL patients showed a partial response. Our data indicate that iC9-CD19-CAR T cell in an academic setting is feasible, safe and extremely effective in treating highly resistant/relapsed BCP-ALL. In our trial, no major or life-threatening toxicities were observed and, despite the moderate CRS recorded, high rates of CR were achieved, suggesting that the combination of a retroviral platform and 4.1bb as costimulation is able to mediate a potent antitumor effect Disclosures Merli: Amgen: Honoraria; Novartis: Honoraria; Sobi: Consultancy; Bellicum: Consultancy. Algeri:Bluebird bio: Consultancy, Honoraria; Atara Biotherapeutics: Consultancy, Honoraria; Miltenyi: Honoraria. Locatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Miltenyi: Honoraria; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Good Tolerance and Durable Remission for Anti-CD19 Chimeric Antigen Receptor T-Cell Therapy in Refractory/Relapsed Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is one subtype of the B cell non-Hodgkin lymphomas (NHL) and has a distinguash course. Despite all efforts, MCL is nearly incurable that refractory or relapse(r/r) course will occur in almost all patients. Due to the rapid development of chemotherapy resistance, it is difficult to treat r/r MCL. A number of treatment regimens have been evaluated with various degrees of success. Cellular immunotherapy has the potential to lead significant and sustained clinical remissions in lymphoma patients, in which modification of T cells with chimeric antigen receptors (CARs) is a powerful regimen. T cells containing CARs with costimulatory domains show better activity against tumor cells. We conducted a clinical trial testing a "second-generation" CD19-specific CAR with 4-1BB costimulatory domains in patients with r/r MCL. Six patients with r/r MCL were enrolled and all were received T-cell infusions. The age of patients ranged from 52 to 81 years old. Five patients were male and one patient was female. Five patients had stage IV MCL with bone marrow involvement and one patient was stage II. Four patients had received more than 3 lines regimens and two patients had received first-line chemotherapy regimen. Four patients received FC regimen before CAR-T infusion and two patients were not received fludarabine due to age ≥80 years old. Treatment was well tolerated, two patients developed transient infusion reaction. No patients have grade 3-4 Cytokine Release Syndrome (CRS). Three patients have grade 2 CRS and two patients have grade 1 CRS. Objective response rate is 66.67%. One patient achieves complete remission(CR) and three patients have partial remission(PR). The response of the other two patients is stable disease(SD). The patient who has CR response has remained progression-free for 36 months and is still being followed up. Two patients who have PR response have progressed in 3 months and the other one has durable response for 7 months. In the patients who has response, the peak of fever appears at approximately day 10 after CAR-T infusion, moreover interleukin(IL)6,IL2αand interferon(IFN)-γ almost reaches the peak on the day 14. In conclusion, adoptive immunotherapy with CD19-specific T cells was well tolerated and was associated with antitumor activity. Further large-sample and multicenter studies are needed to fully evaluate this strategy for such patients. Disclosures No relevant conflicts of interest to declare.

Impact of Lab Abnormalities at the Time of Progression in Patients Receiving CD19-Specific CAR T-Cell Therapy for Relapsed/Refractory Large B-Cell Lymphomas

Impact of Lab Abnormalities at the Time of Progression in Patients Receiving CD19-Specific CAR T-Cell Therapy for Relapsed/Refractory Large B-Cell Lymphomas

Dissecting the Immune Cell Progeny of CAR-Expressing Hematopoietic Stem Cells in a Humanized Mouse Model

Background: Chemotherapy-refractory or recurrent B-lineage leukemias and lymphomas yield less than 50% of chance of cure. Therapy with autologous T-cells expressing chimeric antigen receptors (CAR) have led to complete remissions, but the effector cells may not persist, limiting clinical efficacy. Our hypothesis is the modification of hematopoietic stem cells (HSC) with anti-CD19 CAR will lead to persistent generation of multilineage target-specific immune cells, enhancing graft-versus-cancer activity and leading to development of immunological memory. Design/Methods: We generated second-generation CD28- and 4-1BB-costimulated CD19-specific CAR constructs using third-generation lentiviral vectors for modification of human HSC for assessment in vivo in NSG mice engrafted neonatally with human CD34-positive cells. Cells were harvested from bone marrows, spleens, thymus and peripheral blood at different time points for evaluation by flow cytometry and ddPCR for vector copy numbers. Cohorts of mice received tumor challenge with subcutaneous injection of lymphoma cell lines. Results: Gene modification of HSC with CD19-specific CAR did not impair differentiation or proliferation in humanized mice, leading to CAR-expressing cell progeny in myeloid, NK and T-cells. Humanized NSG engrafted with CAR-modified HSC presented similar humanization rates to non-modified HSC, with multilineage CAR-expressing cells present in all tissues with stable levels up to 44 weeks post-transplant. No animals engrafted with CAR-modified HSC presented autoimmunity or inflammation. T-cell populations were identified at higher rates in humanized mice with CAR-modified HSC in comparison to mice engrafted with non-modified HSC. CAR-modified HSC led to development of T-cell effector memory and T-cell central memory phenotypes, confirming the development of long-lasting phenotypes due to directed antigen specificity. Mice engrafted with CAR-modified HSC successfully presented tumor growth inhibition and survival advantage at tumor challenge with lymphoma cell lines, with no difference between both constructs (62.5% survival for CD28-costimulated CAR and 66.6% for 41BB-costimulated CAR). In mice sacrificed due to tumor development, survival post-tumor injection was directly correlated with tumor infiltration by CAR T-cells. Conclusions: CAR modification of human HSC for cancer immunotherapy is feasible and continuously generates CAR-bearing cells in multiple lineages of immune cells. Targeting of different malignancies can be achieved by adjusting target specificity, and this approach can augment the anti-lymphoma activity in autologous HSC recipients. It bears decreased morbidity and mortality and offers alternative therapeutic approach for patients with no available sources for allogeneic transplantation, benefiting ethnic minorities. Disclosures De Oliveira: National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London: Research Funding; NIAID, NHI: Research Funding; Medical Research Council: Research Funding; CIRM: Research Funding; National Gene Vector Repository: Research Funding.

Identification of Dual Positive CD19+/CD3+ T Cells in an Apheresis Product Undergoing Chimeric Antigen Receptor (CAR) Transduction

Following CAR T cell therapy, many patients receive consolidative hematopoietic stem cell transplantation (HSCT), with the available donor pool recently expanding to include genetically engineered cell products. One such example with striking early promise is the α/Β T-cell depleted haploidentical HSCT (α/Β haplo-HSCT) given with bystander α/Β genetically modified T cells, termed BPX-501 cells. These cells are engineered to express an inducible caspase 9 (iCas9) suicide vector that can be activated by the AP1903 (Rimiducid) dimerizing agent in the event of graft-versus-host disease (GVHD) (US NCT03301168). Here, we report the identification of an unexpected dual expressing CD19+/CD3+ T cell subset in a 10 year old patient who underwent apheresis collection for CAR T cell manufacturing upon relapse six months after α/Β haplo-HSCT and BPX-501 cell addback. A leukapheresis product was collected and CD4 and CD8 T cells were selected from this product for CAR transduction using the Miltenyi CliniMACS Prodigy. T cell purity was assessed using flow cytometry. Characterization of his T cell product at this stage revealed an aberrant cell population expressing both surface CD3 and CD19 (Fig 1a) and lacking additional B cell surface markers, excluding leukemic origin of these cells. This patient was the recipient of the described BPX-501 cell product. BPX-501 cells are also engineered to express a truncated version of CD19 to permit tracking of these modified T cells in patients, post-infusion. We hypothesized that these dual positive CD19+/CD3+ cells were the BPX-501 cells derived from his paternal haploidentical donor and still circulating despite leukemia relapse. We utilized the patient's leukapheresis sample to better characterize these cells. Flow cytometry confirmed the presence of this dual positive CD19+/CD3+ population in the apheresis product. We subsequently treated the apheresis product with Rimiducid in vitro and observed elimination of the CD19+/CD3+ cell subset in a dose dependent manner, thus confirming that these cells were BPX-501 cells (Fig 1b). We additionally investigated the fate of the BPX-501 cells following CAR transduction and observed an absence of this subset post-CAR transduction (Fig 1a). Cells co-expressing CD19-targeting CARs and surface CD19 were not observed in the final manufactured product. The most likely explanation for this phenomenon is in vitro fratricide of the CD19+ T cell population by the CD19-specific CAR+ T cells in culture. We aim to bring attention to this cell phenotype that may be recognized with greater frequency as CAR T therapy and engineered α/Β haplo-HSCT are increasingly coupled. We additionally suggest consideration towards using alternative markers to CD19 as a synthetic identifier for post-transplant add-back products, as CD19-expression on effector T cells may complicate subsequent treatment using CD19-directed therapy. Disclosures Majzner: Xyphos Inc.: Consultancy; Lyell Immunopharma: Consultancy; Xyphos Inc.: Consultancy. Mackall:Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Feldman:Octane Biotech, Inc.: Employment; Personalized Medicine Initiative Science: Membership on an entity's Board of Directors or advisory committees.

Allogeneic Chimeric Antigen Receptor-Invariant Natural Killer T Cells Exert Both Direct and Indirect Antitumor Effects through Host CD8 T Cell Cross-Priming

Chimeric antigen receptor (CAR) T cells have shown impressive results in refractory B-cell malignancies. Unfortunately, to date, commercially available cells, as well as most products tested in clinical trials, are autologous CAR T cells whose widespread use is limited by the logistical and financial burdens related to their ad hoc generation. The development of universal allogeneic CAR T cell products to be used off-the-shelf across MHC-barriers has faced major limitations, namely the risk of Graft-versus-Host-Disease (GvHD) induction and the rejection of the administered cells by the host immune system. Invariant Natural Killer-T (iNKT) cells are innate lymphocytes that are deprived of any GvHD induction potential but that display antitumor effects both directly, through the production of cytotoxic effector molecules, and indirectly, through the enhancement of NK and CD8 T cell-mediated immune responses. Preclinical studies using xenogeneic mouse models have demonstrated the feasibility of using iNKT cells as a platform for CAR-based therapies, and two clinical trials are currently ongoing. In order to study the interaction of CD19-specific iNKT CAR cells with the host immune system, we transduced iNKT cells ex vivo expanded from FVB/N mice with a CAR composed of the variable region cloned from the 1D3 hybridoma recognizing murine CD19 linked to a portion of the murine CD28 molecule and to the cytoplasmic region of the murine CD3-ζ molecule. The cytotoxic potential of CD19-iNKT CAR was confirmed in an in vitro cytotoxic assay against the CD19-expressing A20 lymphoma cell line, revealing a strong, dose dependent cytotoxic effect of CD19-CAR iNKT cells. Accordingly, and similarly to what was previously reported in xenogeneic studies, FVB/N (H-2Kq) derived iNKT CAR (2x10e6 cells iv) significantly improved survival of mice after administration to major histocompatibility complex (MHC)-mismatched, immunodeficient BALB/c (H-2Kd) Rag2-/- gamma-chain-/- mice receiving A20 cells (2x10e4 cells iv; Figure 1A) without inducing any signs of GvHD. To test the efficacy of iNKT CAR cells in the presence of host immune cells, we tested the antitumor activity mediated by iNKT CAR against A20 cells in BALB/c mice receiving sublethal irradiation (4.4 Gy), resulting in only a partial and transient lymphopenia. In this model, the antitumor effect of iNKT CAR cells was greatly enhanced, leading to long-term survival of the great majority of treated mice (Figure 1B). Such a difference in iNKT CAR effect between mice that genetically lacked lymphocytes and mice with only partial lymphopenia suggested the participation of host derived lymphocytes in the antitumor effect. To test the hypothesis that host CD8 T cell activation via cross-priming could at least partially mediate the indirect antitumor effect of iNKT CAR cells, we repeated the experiment, employing as recipients BALB/c BATF3-/- mice, in which CD8 T cell cross-priming is impaired as a result of the absence of BATF3-dependent CD103+ CD8a+ dendritic cells that are thought to present antigens through CD1d to the invariant T cell receptor expressed by iNKT cells. Interestingly, the iNKT CAR effect was partially abrogated in A20-receiving BATF3-/- mice as compared to WT mice (Figure 1C), supporting the hypothesis that the impact of iNKT CAR cells is mediated at least partially by the activation of host CD8 T cells via their cross-priming. To formally demonstrate the synergistic effect between allogeneic iNKT CAR and autologous CD8 T cells, we employed an autologous bone marrow transplantation model, co-administering allogeneic iNKT CAR with autologous CD8 T cells at the time of transfer of T-cell-depleted autologous bone marrow cells and A20 lymphoma cells into lethally irradiated (8.8 Gy) BALB/c recipients. Co-administration of allogeneic iNKT CAR and autologous CD8 T cells resulted in a synergistic effect, significantly extending animal survival (Figure 1D) compared to mice receiving no treatment, as well as to mice receiving either allogeneic iNKT or autologous CD8 T cells alone. Collectively, these results represent the first demonstration of an immune adjuvant effect exerted by an allogeneic CAR cell product toward the autologous immune system of the host, suggesting that the effect of the administered cells would last longer than the physical persistence of the allogeneic cells after they will be rejected by the host immune system. Figure 1 Disclosures Mackall: Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board; Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Allogeneic CD34-Selected HSCT Following CAR T-Cells Is Associated with Low TRM and Favorable OS in Pediatric/Young Adult Patients with Relapsed/Refractory B-ALL

Background: Chimeric antigen receptor (CAR) T-cells have transformed the therapeutic options for patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). The tolerability and outcome of allo-HSCT following CAR T-cell therapy is yet to be defined. Methods: We retrospectively reviewed pediatric and young adult patients with R/R B-ALL who received allo-HSCT following CAR T-cells. Outcomes of interest were overall survival (OS), cumulative incidence of relapse, and transplant-related mortality (TRM). These were estimated using log-rank analysis. Variables of interest were disease status at time of allo-HSCT, time from CAR T-cell therapy to start of allo-HSCT, conditioning regimen, graft source and manipulation, GVHD prophylaxis, donor HLA matching, neutrophil/platelet engraftment, relapse and cause of death. Results: Sixteen patients, with a median age of 13 (range 1-20), at time of allo-HSCT were included. All patients achieved MRD-negative complete remission (CR) following CAR T-cells and proceeded to allo-HSCT as definitive therapy. CR status included CR1 (n=6), CR2 (n=5), CR3 (n=4), and CR4 (n=1). The median time from CAR T-cells to the start of allo-HSCT conditioning was 46.5 days (range 20 -126). Conditioning was TBI-based (n=13) or chemotherapy only (n=3). Graft source included PBSC (n=8) or BM (n=8). Graft manipulation included CD34-selected T-cell depleted (TCD) (Miltenyi) (n=9) or an un-modified HSCT (n=7). Donors were HLA-matched related (n=8), HLA-matched unrelated (n=3), or HLA-mismatched unrelated (n=5). GVHD prophylaxis in the non-manipulated grafts included a calcineurin inhibitor (CNI) plus MTX (n=5), CNI plus MMF (n=1) or post-transplant cyclophosphamide (n=1). Available data included: 15/15 patients achieved neutrophil engraftment (median 12 days; range 9-29) and 13/15 patients achieved platelet engraftment (median 30 days; range 16-265). The 12-month cumulative incidence of relapse was 0% in patients receiving CD34-selected transplant, versus 25% [95% CI: 0 to 67%] in patients receiving un-modified transplant (log-rank p=0.80) and the 24-month cumulative incidence of relapse for the whole cohort was 25% [0-47%]. Late relapse (>12 months from allo-HSCT) occurred in three (n=3) patients in the CD34-selected TCD cohort with a median time to relapse from allo-HSCT of 24.2 months (range 16.3 - 24.7 months). Two (n=2) patients achieved subsequent MRD-neg/CR following additional CD19-specific CAR T-cell infusions (CRs ongoing at 12.4 and 1.4 months). The other patient (n=1) experienced a CD19-negative relapse and is awaiting infusion of CD22-specific CAR T-cells. Early relapse occurred in one (n=1) patient (4.7 months from allo-HSCT) following un-modified allo-HSCT with subsequent MRD-neg/CR following treatment with CD19-specific CAR T-cells (CR ongoing at 5.2 month). Three (n=3) patients in the un-modified cohort died due to TRM (24-month TRM = 46% [7-86%]), including veno-occlusive disease (multi-organ failure/infection) (n=2) and GVHD (n=1), while no TRM was noted in the CD34-selected TCD cohort (p=0.03). The TRM for the entire cohort was 19% at 24 months [0-39%]. The 24-month OS for the entire cohort was 81% [61 -100%], with 100% OS in CD34-selected TCD recipients and 54% in the un-modified recipients (p=0.03). Conclusion: CAR T-cells when followed by a CD34-selected TCD allo-HSCT resulted in less TRM and favorable OS when compared to un-modified allo-HSCT. Disease control is not impacted by the type of allo-HSCT and late relapse was seen following CD34-selected TCD. Successful re-treatment with CAR T-cells to induce remission is possible and analysis as to the durability of this response is ongoing. Disclosures Margossian: Novartis: Membership on an entity's Board of Directors or advisory committees. Prockop:Atara Biotherapeutics: Other: Support for industry sponsored trails ; Mesoblast: Other: Support for industry sponsored trails . Shah:Jazz pharmaceuticals: Speakers Bureau. Boelens:Takeda: Consultancy; Magenta: Consultancy; Bluerock: Consultancy; Avrobio: Consultancy. Curran:Juno Therapeutics: Consultancy, Research Funding; Novartis: Consultancy.

ShRNA-Interleukin-6 Modified CD19-Specific Chimeric Antigen Receptor T Cell Significantly Improves the Safety in Acute Lymphoblastic Leukemia

[Background] An urgent need exists to enhance the safety in treating hematologic malignancies with CAR-T therapy by reducing the CAR-T-related cytokine release syndrome (CRS) . Interleukin-6 (IL-6) is a central driver of CRS and neurotoxicity; hence, inhibition of the IL-6 of T cells via gene engineering may improve the safety of CAR-T therapy. [Objective] Investigation of the efficacy and safety of IL-6-targeting short hairpin (sh) RNA in the CART-19 (referred to ssCART-19) to determine whether the IL-6 shRNA in T cells can reduce the severe CRS incidence of ssCART-19 treatment. [Methods]We designed a short hairpin RNA sequence which targets the 3'UTR region of the human IL-6 transcript, and the sequence was added to a CAR construct containing the CD19 target single chain variable fragment (scFv), the EF1a promoter, the co-stimulated domain of 4-1BB and the CD3zeta domain. In vitro study, While there is no significant difference in the transduction efficiency, proliferation ability and cytotoxicity efficacy of ssCART-19 comparing to regular CART-19, there was clear inhibition of the IL-6 expression. IL-6 shRNA mediated gene silence of ssCART-19 significantly inhibited IL-6 gene expression at both the mRNA level (P<0.001) and the soluble cytokines level (P≤0.0001). IL-6 expression profile from ssCART-19 showed consistently maintained the lower level over the entire 150 hours of experiment period compared to regular CART-19 cells (P<0.001 ). And add the supernatants from regular CART-19/Raji co-culture and ssCART-19/Raji co-culture system to the primary induced monocytes, respectively, ssCART-19 could significantly reduce the monocytes derived IL-6 expression levels compared to regular CART-19. In vivo study, the preclinical study showed the consistent results that ssCART-19 significantly reduced the mouse serum IL-6 levels compared to regular CART-19, but with similar anti-tumor efficacy. In the clinical trail, 13 patients with the similar tumor burden baseline administrated with ssCART-19 (n=7) or regular CART-19 (n=6) cells with a dose of 5-10x106 CAR-T cells per kilogram over three consecutive days (10%, 30%, 60% split dose). While all patients from both groups achieved complete response and the CAR-T cells exhibit similar expansion ability, patients treated with ssCART-19 had lower CRS grade and significantly lower IL-6 level in the human serum compared to patients treated with regular CART-19 (the peak value of IL-6, P=0.0285, the IL-6 AUC(0-Tmax), P=0.0217). CRS emerged in 6/6 patients in regular CART-19 cohort and 6/7 patients in ssCART-19 cohort, severe CRS with grade 3 or higher was observed in 83.3% of the patients (5/6) treated with regular CART-19 cohort versus only 42.8% of the patients (3/7) treated with ssCART-19 cohorts. Tocilizumab was given to 66.7 % (4/6) of the patients in the regular CART-19 cohort and two patients needed more than one treatment with tocilizumab. In the regular CART-19 group one patient occurred CRES. There was no CAR T-related death. [Conclusion]Our study demonstrated that inhibition of CAR-T derived IL-6 expression by shRNA interfering technology could significantly reduce the severe CRS incidence without affecting their immune-oncotherapy efficacy in treating r/r B-ALL patients, which may provide a potential technology to improve the safety profile and promote the extended use of the CAR-T therapy without sacrificing efficacy. Disclosures No relevant conflicts of interest to declare.