CD19 CAR Research Articles

Low incidence of invasive fungal disease following CD19 chimeric antigen receptor T-cell therapy for non-Hodgkin lymphoma

AbstractCAR T-cell therapy has revolutionized the treatment of hematologic malignancies, although its use may be complicated by toxicities, including cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and infections. Invasive fungal disease (IFD) has been reported after CAR T-cell therapy, but the incidence in the absence of antifungal prophylaxis is unknown. Optimal prophylaxis strategies are widely debated. We performed a single-center retrospective study of 280 adults receiving CD19 CAR T-cell therapy for non-Hodgkin lymphoma (NHL) from December 2017 through September 2021. Patients did not receive routine antiyeast or antimold prophylaxis. IFD was identified between day of cell infusion and last follow-up. Cumulative incidence functions were calculated at 100 days and 18 months based on time to IFD, using dates of IFD-free death, initiation of salvage treatment, and hematopoietic cell transplantation as competing risks. Eight patients (2.9%) developed IFD, including 3 Pneumocystis jirovecii pneumonia, 3 invasive mold infections (IMIs), and 2 invasive yeast infections (IYIs). The 100-day cumulative incidence of IFD accounting for competing risks was 1.8% (95% confidence interval [CI], 0.8% to 4.4%). Among the 280 patients, early toxicities including CRS (85%) and ICANS (55%) and late toxicities after day 30 including grades 3 and 4 neutropenia (41%) and low CD4 T-cell count (20%) were common. IFD was rare among patients who received CD19 CAR T-cell therapy for NHL in the absence of routine antifungal prophylaxis, despite frequent toxicities. These results suggest that, in settings with low institutional rates of IFD, routine antifungal prophylaxis may not be indicated.

P1447: INFERIOR OUTCOMES OF EU VS. US PATIENTS WITH RELAPSED/REFRACTORY LARGE B-CELL LYMPHOMA AFTER CD19 CAR T-CELL THERAPY ARE IMPACTED BY BASELINE RISK FACTORS AND CAR PRODUCT CHOICE

Background: CD19 CAR T cells have significantly improved the prognosis of patients with relapsed/refractory (r/r) large B-cell lymphoma (LBCL). Real-world evidence (RWE) in the US has confirmed outcome data from pivotal trials. In contrast, several European consortia reported inferior real-world response rates and survival. Aims: To understand these divergent outcomes, we sought to characterize differences in patients treated with CD19 CAR T cells in Europe vs. the US. Methods: In this retrospective observational study, we assessed CAR T cell logistics, patient/disease characteristics, toxicities, response, and survival of r/r LBCL patients treated with Axi-cel or Tisa-cel at five EU and one US CAR T center. We evaluated variables for their association with PFS by univariate and multivariate stepwise Cox regression. Results: A total of 386 patients were included (EU: 175, USA: 211). Indeed, the objective response rate was lower in the EU (65% vs. 82% in the US, p<0.001). Median PFS (Fig. 1, 3 vs. 8 months, p<0.001) was significantly shorter for EU compared to US patients. Tisa-cel was applied more frequently in the EU cohort (74% vs. 14%, p<0.001). While severe CRS rates did not significantly differ between cohorts, grade ≥3 ICANS was more common in US patients (25% vs. 9%, p<0.001), likely reflecting the more frequent use of of Axi-cel. In terms of baseline characteristics, median LDH (321 vs. 272 U/l, p=0.02) and ferritin levels (682 vs. 449 ng/ml, p=0.002) were significantly higher in the EU cohort. EU patients received fewer treatment lines (p=0.004), though the percentage of prior auto-SCT (35% vs. 18%, p<0.001) and active/history of CNS disease (17% vs. 7%, p=0.005) was higher. Analysis of CAR-T logistics revealed longer EU vein-to-vein intervals (43 vs. 28 days, p<0.001). The proportion of patients receiving “holding” therapy, defined as lymphoma treatment between indication-to-CAR and apheresis, was comparable between EU and US centers (22% vs. 27%, p=0.32). However, significantly more patients were treated with bridging therapy (between apheresis and transfusion) in the EU (89% vs. 69%, p<0.001). In univariate analyses, shorter PFS was associated (p<0.05) with lack of response to the latest therapy, longer indication-to-CAR intervals, higher ECOG, stage III/IV disease, IPI, extranodal disease (END), higher LDH, ferritin, CRP levels, and Tisa-cel use. On multivariate regression (n=319), ECOG (adjusted HR 1.4), ferritin (HR 1.5), refractory disease (HR 1.5), END (HR 1.4), and Tisa-cel use (HR 1.5) represented independent risk factors. Of note, the aggregate of these four independent baseline risk factors (ECOG, ferritin, refractory disease, END) did not significantly differ between EU vs. US patients (p=0.18), indicating that product choice likely influenced the observed differences in PFS. Interestingly, although Axi- vs. Tisa-cel did not confer different outcomes for patients with 0-1 risk factors (p=0.47), Axi-cel led to significantly improved PFS compared to Tisa-cel in patients with 2-4 risk factors (p<0.001). Image:Summary/Conclusion: This is the first analysis to comprehensively compare CAR T cell treatment characteristics in Europe vs. the US in a large real-world cohort. Response rates and PFS were significantly inferior in the EU, potentially due to the use of Tisa-cel in adverse-risk patients. Our data suggest that Tisa- and Axi-cel are equally effective in low-risk patients (≤1 risk factor - ECOG, ferritin, refractory disease, END), but that Axi-cel facilitates improved outcomes in patients with 2-4 risk factors, with a representative toxicity profile.

P1459: SAFETY AND EFFICACY FINDINGS OF AUTO1, A FAST-OFF RATE CD19 CAR, IN RELAPSED/REFRACTORY B-CELL NON-HODGKIN’S LYMPHOMA (B-NHL), AND CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) / SMALL LYMPHOCYTIC LYMPHOMA (SLL)

Background: We have previously described AUTO1, a CD19 CAR with a fast off-rate CD19 binding domain, designed to reduce CAR T-cell immune toxicity and improve engraftment. Its clinical activity has been tested in r/r paediatric and adult B-ALL (Ghorashian S et al., Nat Med 2019; Roddie C et al., JCO 2021). This data confirms the intended function of the receptor, with low levels of CRS/ICANS and long-term engraftment of CAR T-cells observed in both patient groups. Aims: We have initiated testing of AUTO1 in the setting of B-NHL and CLL/SLL (NCT02935257). Methods: Manufacturing: CAR T-cell products were generated using a semi-automated closed process from non-mobilised patient leukapheresate. Study design: Subjects ≥ 16y underwent lymphodepletion with fludarabine (30mg/m x3) and cyclophosphamide (60mg/kg x1) prior to AUTO1 infusion, with the exception of the DLBCL cohort who additionally received a single dose of pembrolizumab (200mg) on day -1 to potentiate CAR-T expansion. AUTO1 dose varies based on the indication. Split dosing of 230 x106 CD19 CAR T-cells at day 0 and day 9 is employed in the CLL cohort. A single dose of 200 x106 CD19 CAR T-cells is delivered to patients with B-NHL. Study endpoints include feasibility of manufacture, grade 3-5 toxicity and remission rates at 1 and 3 months. Results: As of 8th February 2022, we enrolled 23 patients: 11 low grade NHL (LG-NHL:7 with FL and 3 with MCL), 7 DLBCL and 5 CLL. Apheresis was successful in all 23 patients and product manufacture was successful in 22 (pending in the last). 19 patients were infused: 10 with LG-NHL, 6 with DLBCL and 3 with CLL. 1 CLL patient was pending infusion at time of data cut-off and 2 patients died pre-infusion: 1 MCL patient, from COVID-19 and 1 CLL patient, from intracerebral haemorrhage. Patients treated with AUTO1 had a median age of 60 years (range 39-79), had received a median of 3 prior lines of treatment (range 2-8). Grade 1 CRS was reported in 6/19 and Grade 2 CRS in 3/19. No ICANS was observed in the B-NHL and CLL cohorts. CAR engraftment was observed in 13/13 patients evaluated by qPCR with ongoing persistence in 12/13 patients at last follow-up. In the LG-NHL and DLBCL cohorts 10/10 and 4/5 evaluable patients respectively were in CMR by 18FDG PET-CT post-treatment. Responses were ongoing in 9/10 LG-NHL at 12 months and in 4/4 DLBCL at months 1, 3, 3 and 6. In the CLL cohort, 2/3 evaluable patients achieved MRD negative remission in the bone marrow with residual small volume lymph nodes by CT at 6 and 3 months of follow-up respectively. 1 CLL patient did not engraft and had SD at month 1. Summary/Conclusion: AUTO1 has a tolerable safety profile in patients with r/r B-NHL and CLL despite high disease burden. Early data shows excellent complete remission rates and excellent CAR engraftment/expansion. Additional patients, updated data and longer follow up will be presented.

P1444: IMPACT OF CO-MORBIDITIES ON NON-RELAPSE MORTALITY AND CYTOKINE-RELEASE SYNDROME AFTER CD19 CAR-T CELL THERAPY - A RETROSPECTIVE STUDY FROM THE TCWP OF THE EBMT AND GOCART COALITION

Background: CAR T-cell therapies have been associated with a profile of significant toxicities and previously unknown side effects. For that reason, the registration of clinical trials with commercial and non-commercial products included very strict inclusion and exclusion criteria, to minimize severe adverse events and maximize therapeutic effect. The real-life analyses revealed that a proportion of patients are treated outside of these specifications, with variable outcomes. Aims: The goal of this study was to determine the cumulative incidence of non-relapse mortality (NRM) and cytokine-release syndrome (CRS) after anti-CD19 CAR T-cell therapy, and impact of patients’ co-morbidities expressed as the hematopoietic cell transplantation-co-morbidity index (HCT-CI) scores, on the major outcome measures. Methods: This is a retrospective multicentre analysis from the Transplant Complications Working Party (TCWP) of the EBMT using the data of the GoCART coalition. Eligibility criteria for this analysis include diffuse large B-cell lymphoma (DLBCL) and acute lymphocytic leukemia (ALL) patients that underwent CD19 CAR T-cell therapy in an EBMT member centre before 31 January 2021. Exclusion criteria were lack of information on comorbidities, survival or relapse status, or age <18 years. Outcomes were measured from the date of CAR T-cell infusion for all endpoints. NRM was defined as death without relapse/progression. Cumulative incidence functions were used to estimate the incidence of complications. Multivariate analyses were performed using the Cox cause-specific model for all endpoints. The primary endpoint was NRM at 3 and at 12 months post CAR T-cell therapy, while secondary endpoints included cumulative incidence of CRS as well as NRM and CRS incidence in subgroups based on HCT-CI. Statistical analyses were performed with R 3.4.2 software packages. Results: The study identified 586 patients treated with CAR T-cells in the years 2018-2021 for DLBCL (96.4%) or ALL (3.6%). The median age was 60 (range, 18-84). 166/437 (38%) patients had a Karnofsky performance status <90%, while the HCT-CI was available for 483 (82%) patients and was intermediate (1-2) in 17.6% and high (≥3) in 13.9% of these patients. The most common co-morbidities were pulmonary (16.1%), cardiac (6.8%), obesity (6.8%), diabetes (6.4%), arrythmia (5.5%), history of solid tumor (5.3%), and active infection before treatment (4.3%). The median follow up was 12.5 months. The NRM was reported to be very low: 1.7% (95%CI 0.9-3) at 3 months and 2.9% (95%CI 1.7-4.5) at 12 months. The cumulative incidence of CRS (all grades) was 35.3% (95%CI 30.6-40.1) at 3 days and 82.7% (95%CI 78.5-86.1) at 12 days. Unexpectedly, no significant impact of HCT-CI on NRM, CRS incidence, or overall survival was observed (Fig. 1) both in univariate and multivariate analyses. Most deaths were caused by the original disease (75.4%) with concomitant infection (5.9%) or with multiorgan failure (4.3%). Image:Summary/Conclusion: Our real-life data from the GoCART registry revealed the profile and frequency of co-morbidities of patients undergoing CAR T-cell treatments at EBMT centers. While the HCT-CI score was high in a substantial proportion of patients, it did not influence any major outcome. The incidence of CRS was also high and not influenced by the HCT-CI. Our preliminary results might support application of more liberal criteria for patient qualification for CAR T-cell therapies based on co-morbidities, but this needs to be confirmed in further studies.

P1450: AUTOLOGOUS STEM CELL TRANSPLANTATION INDUCES HIGHER LEVEL OF EXHAUSTED T-CELLS IN DLBCL PATIENTS BEFORE LEUKAPHERESIS FOR CAR-T CELL THERAPY.

Background: Chimeric antigen receptor T-cells (CAR-T) are an highly effective therapy in relapse/refractory DLBCL, PMBCL and ALL, but almost half of treated patients (pts) still relapse. CAR-T cells generated from T-lymphocytes (T-Ly) enriched for early lineage T cells has shown durable persistence and replicative potential both in animal and in vivo models, while CAR-T cells from “exhausted” T-Ly have been related with poorer response. In the prospective BioCART BS study, we assessed the T-cell repertoire before the Ly-apheresis in DLBCL patients for evaluating the impact of previous treatments and efficacy of CAR-T therapy. Aims: To evaluate T-cells repertoire before the Ly-apheresis in high risk DLBCL pts. Methods: Between April and December 2021, a total of 11 DLBCL pts underwent Ly-apheresis for CAR-T manufacturing. Since cryopreservation of the apheresed Ly is allowed for Tisa-cel, DLBCL pts with poor prognostic risk factor (refractory to first line of treatment; PET positivity before Autologous Stem Cell Transplantation [ASCT]; first complete response less than 12 months) were enrolled in an early/“pre-emptive” Ly-apheresis program, scheduling leukapheresis as soon as possible, with the aim to collect more “fit” Ly. Combinations of monoclonal antibodies directed against CD45RA, CCR7, CD3, CD4, and CD8 were used by Flow Cytometry to evaluate the following CD4+/CD8+ T-ly subsets: T-naïve (CD45RA+CCR7+); central memory (CM, CD45RA-CCR7+); effector memory (EM, CD45RA-CCR7-); terminally differentiated (TD; CD45RA+CCR7-). For evaluating CAR-T cells expansion, pts’ cells were tested with a specific biotinilated CD19 CAR reagent followed by incubation with an anti-biotin PE conjugated MoAb. The time points were: at day +1, +3, +7, +10, +14, +21 after CAR-T cells infusion, and then monthly until 1 year or at any time for relapse onset. All stained samples were acquired on a Canto II (BD Bioscience) flow cytometer and analyzed using DIVA software version 8.0.2. Continuous and categorical variables were compared using an unpaired T-test, Mann-Whitney U test, or Chi-square test, as appropriate. Results: Table 1 shows the main patients’ characteristics. 5 out of the 11 (45%) pts performed Ly-apheresis before ASCT. At the time of Ly-apheresis, no significative differences were observed between pts who previously underwent ASCT and those who did not, except for age. ASCT induced a more “exhausted” T-cells repertoire. Indeed, pts who previously underwent ASCT had lower CD4+/CD8+ ratio (p 0.04) (Fig 1C) as well as lower levels of CD4+ Naïve T cells, both in terms of absolute count (p 0.05) and as percentage of CD4+ T cells (p 0, 01), and lower prevalence of CD8+ Naïve T cells (p 0.03) (Fig 1A). Moreover they also presented higher prevalence of CD4+ EM T cells (p 0.01) and of CD4+ TD T-cells (p 0.05), as well as higher level of both CD8+ CM (p 0.03) and TD (p 0.05) T cells (Fig 1B). 3 out of the 6 pts who underwent ASCT before Ly-apheresis and 2 of the 5 who did not, sent Ly for manufacturing. To date, no differences were apparently observed on CAR-T cells expansion comparing the two groups, but the number of pts analyzed is too small to make any consideration (Fig 1D for pts with longer follow up). - Ly-apheresis PRE ASCT Ly-apheresis POST ASCT p value Number 5 6 Age 51 (29-60) 67 (57-70) 0.02 Disease status @Ly-apheresis 1 2 0.08 Refractory 0 3 Relapse 4 1 Partial response Image:Summary/Conclusion: ASCT before Ly-apheresis may result in a higher prevalence of “exhausted” T-cells. In DLBCL pts primary refractory or at high risk of relapse after second line of treatment, timely pre-ASCT Ly-apheresis would be considered.

Abstract 560: Generation of tertiary lymphoid structures and CD3+ CD8+ CD56+ NKG2D+ CAR TaNK cells following subcutaneous injection of CD3-directed lentiviral-loaded PBMCs

Abstract Background: We have previously established the ability of CD3-directed lentiviral vectors encoding for CD19 or CD22 CARs to mediate robust anti-tumor immunity in humanized lymphoreplete mouse models. We now present additional mechanistic data for this novel subcutaneous (SC) CAR-T approach. Methods: Human PBMCs loaded with a self-inactivating lentiviral vector (LV) encoding CD19 CAR with a synthetic driver element were injected SC into autologous PBMC humanized NSG MHC I/II double knock out (DKO) mice. The LV was packaged with a modified envelope with the ability to target and activate CD3+ T cells. To first track the site of CAR formation in vivo following SC injection, PBMCs were loaded with an LV encoding a CD19 CAR and luciferase, and bioluminescence imaging (BLI) was performed. Additionally, histopathology of the site of injection and distal organs were examined. Evaluation for CAR+ cells was performed through immunohistochemistry and PCR detection. To examine the tropism of the CD3-directed CD19 CAR LV and characterize CAR+ cells, the phenotype of in vivo expanded CAR+ cells was evaluated. Further characterization of CAR+ cells was performed with in vitro studies. Results: Following SC injection of LV-loaded PBMCs, the first evidence of transgene expression utilizing BLI for luciferase was detected four to five days following SC injection. At the same time point, histologic examination of the SC site of injection revealed the formation of tertiary lymphoid structures (TLS) consisting of human CD8+ and CD4+ T cells, CD68+ macrophages, CD68+ dendritic cells, and a few CD20+ B cells. On day 13 post-SC injection, BLI detected the presence of CAR+ cells systemically beyond the site of injection and within subcutaneous Raji tumors implanted on the contralateral side. On day 14 post-SC injection, histologic examination showed sustained TLS within the SC tissue without signs of dermal acute inflammation or ulceration and evidence of CAR+ cells appearing in the spleen. CAR+ cells exhibited robust anti-tumor immunity with expansion into peripheral blood. CAR+ cells consisted of a distinct population of CD8+ T cells with NK-like features (TaNKs) and a CD3+ CD8+ CD56+ NKG2D+ cell phenotype. In vitro transduction of CD56 NK cell-depleted PBMC with the CD3-directed LV also led to CAR-TaNK formation. Conclusion: The subcutaneous injection of CD3-directed LV-loaded PBMCs leads to the formation of tertiary lymphoid structures at the site of injection and the development of distinct CD3+ CD8+ CD56+ NKG2D+ CAR-TaNK cells. These cells possess enhanced systemic proliferative capacity compared to traditional ex vivo manufactured 41BB CAR-T cells in a lymphoreplete mouse model and the ability to eliminate target cells in vivo with low numbers of starting cells (10,000 cells). Citation Format: Ani Kundu, Dongming Zhang, Frederic Vigant, Junyi Zhang, Greg Schreiber, Ewa Jaruga-Killeen, Alissa Kerner, Michelle Andraza, Wei Zhang, John Henkelman, Renata Soares, Gregory I. Frost, Sid P. Kerkar. Generation of tertiary lymphoid structures and CD3+ CD8+ CD56+ NKG2D+ CAR TaNK cells following subcutaneous injection of CD3-directed lentiviral-loaded PBMCs [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 560.

Abstract 3910: Cellular profiling of leukapheresis and infusion products of patients enrolled in CD19 CAR T cell therapy identifies biomarkers associated with clinical outcomes

Abstract CD19 CAR T cell therapy has emerged as a promising therapy in the treatment of B cell malignancies, such as B cell acute lymphoblastic leukemia (B-ALL). However, questions remain regarding the heterogeneity of the CAR T cell infusion products that lead to differential clinical outcomes and side effects, including cytokine release syndrome and neurotoxicity. The purpose of this study is to identify biomarkers and T cell subpopulations within 1) leukapheresis samples of patients receiving CD19 CAR T cell therapy and 2) pre-infusion manufactured CAR T cell products to identify correlates with clinical outcomes. We hypothesize that different manufacturing practices used to generate CAR T cells from the patient’s leukapheresis T cells drive heterogeneity within the infusion products that drive differential patient outcomes, and that cell intrinsic properties within the leukapheresis T cells drive phenotypic dispositions observed in the infusion products. Leukapheresis samples and pre-infusion CAR T cells were obtained from pediatric and adult B-ALL patients enrolled in CD19 CAR T cell clinical trials. Briefly, leukapheresis T cells from pediatric patients were CD4/8 sorted and then stimulated with CD3/CD28 beads followed by CAR transduction. Pediatric CD4 CAR T cells were cultured with IL-7 + IL-15 while CD8 CAR T cells were cultured with IL-2 + IL-15. CAR T cells from adults were stimulated with LCLs expressing CD19. To identify T cell subpopulations and biomarkers leukapheresis T cells and CAR T cells from each patient were stained with two mass cytometry panels: 1) a phenotype panel of antibodies to characterize surface markers, transcription factors and chemokine receptors and 2) a functional panel of antibodies specific for cytokines to broadly characterize the secretion profile of leukapheresis and CAR T cells upon stimulation with PMA and Ionomycin. Additional multi-omic profiling was performed with CITE-seq and 10X Chromium on leukapheresis samples and CAR T cells of select adult patients to provide in depth analysis on transcriptomic drivers of phenotypes observed within CAR T cell infusion products. We hierarchically clustered leukapheresis T cells and CAR T cells and correlated the abundance of T cell subclusters within each patient to their neurotoxicity grade (0-5). We determined that CD4 CAR T cells that secrete elevated levels of IL-3 upon stimulation correlate with an increased risk of severe neurotoxicity (designated as grades 3-5) within our pediatric cohort. Identification of IL-3 as a driver of neurotoxicity within the CD4 infusion products may inform strategies to improve manufacturing practices used to generate safer CAR T cell products. Citation Format: Tony Chour, Alexandre Hirayama, Ye Zheng, Alyssa Sheih, Summer Zhuang, Ashley Wilson, Vicky Wu, Raphael Gottardo, Cameron Turtle, Rebecca Gardner, Evan Newell. Cellular profiling of leukapheresis and infusion products of patients enrolled in CD19 CAR T cell therapy identifies biomarkers associated with clinical outcomes [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 3910.

Abstract 3294: In vivo delivery of a novel CD3-targeted lentiviral vector generates CD19 CAR-T cells in two different humanized mouse models and results in complete B cell depletion

Abstract Introduction: Chimeric antigen receptor therapies (CAR-T) are highly effective against hematologic malignancies but require a lymphodepleting chemotherapy regimen and are faced with many challenges including manufacturing time, scalability, and cost of production due to the need for ex vivo culturing of cells and complex chain of custody requirements. In vivo delivery of self-inactivating lentiviral vectors (LV) encoding for CAR-T transgenes represents a promising strategy to improve the time to treatment, scalability, and cost of current CAR-T therapies. Methods: Lentiviral vectors encoding a CD19 CAR with a synthetic driver element were manufactured in a chemically defined cell substrate that incorporates a modified envelope designed to target and activate CD3+ T cells. Two different humanized mouse models were utilized. In a PBMC humanized mouse model, NSG MHC Class I/II knock-out mice (DKO) were injected with 1E7 human PBMC and followed one day later with 2E7 TU of LV encoding for CD19 CAR. In parallel, CD34+ humanized NSG SGM3 mice were also administered 2E7 TU LV encoding CD19 CAR. Flow cytometry of peripheral blood samples was evaluated at various time points for the presence of CAR+ cells and CD20+ B cells. Additionally, tissue samples were examined by histopathology and PCR for vector copy numbers. Results: The NSG SGM3 humanized CD34+ mouse model exhibited efficient chimerism of human CD45+ hematopoietic cells (&amp;gt;50% of live cells in peripheral blood). Apart from CD15+ neutrophils, all major human immune cell components were well represented in peripheral blood including CD14+ monocytes, CD20+ B cells, and CD3+ lymphocytes. At study initiation, the T cell compartment in the SGM3 mice exhibited skewing towards CD4+ T cells. Injection of CD19 CAR-LV resulted in a significant reduction of CD20+ B cells as early as 5 days post-injection. CD3+ CAR+ cells were detected in peripheral blood by day 5 with evidence of CAR+ cell expansion at subsequent time points. Loss of CD20+ B cells was stable throughout the observation period with some mice exhibiting a complete elimination of B cells. Similarly, the PBMC humanized NSG DKO mice injected with CD19 CAR-LV also showed evidence of CAR+ cells in peripheral blood. Conclusions: CD3-directed self-inactivating lentiviral vectors can efficiently deliver an integrating CAR gene into T lymphocytes following in vivo administration. These in vivo generated CD19 CAR T cells expand systemically and effectively eliminate pre-existing B cells. These data show that the targeting of CD3 through in vivo delivery can produce functional CAR T cells and represents an innovative therapeutic opportunity to potentially overcome current manufacturing times, scalability, and cost challenges facing cell therapies. Citation Format: Frederic Vigant, Ani Kundu, Dongming Zhang, Wei Zhang, Ewa Jaruga-Killeen, Michelle Andraza, Gregory Schreiber, Alissa Kerner, Junyi Zhang, John Henkelman, Renata Soares, Ramya Yarlagadda, Gregory I. Frost, Sid P. Kerkar. In vivo delivery of a novel CD3-targeted lentiviral vector generates CD19 CAR-T cells in two different humanized mouse models and results in complete B cell depletion [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 3294.

Abstract 3602: Mechanisms of CAR T cell dysfunction and identification of transcription factors that drive the exhaustion phenotype

Abstract Chimeric antigen receptor (CAR) T cell immunotherapy is FDA approved for the treatment of a subset of B cell malignancies but has shown limited clinical success in solid tumor therapy. T cell exhaustion is an important factor involved in treatment failure, and can, in part, result from continuous stimulation of the CAR by tumor cells expressing the cognate antigen. To gain deeper understanding of CAR T cell exhaustion induced by chronic antigen exposure (CAE), we developed and validated a robust in vitro model, in which mesothelin-redirected CAR T cells (M5CAR) were continuously stimulated with mesothelin-expressing AsPC-1 pancreatic tumor cells such that tumor cells were never cleared, and we characterized these CAE CAR T cells by gene expression at population and single-cell levels, and by epigenetic analyses. CAE M5CAR T cells recapitulate the hallmarks of T cell exhaustion, including reduced proliferation, down-modulation of surface CAR, decreased cytokine production, and reduced cytotoxicity. In addition, CAR T cells undergoing CAE have a transcriptional signature and an epigenetic landscape consistent with exhaustion. Further, transcriptomic analysis revealed that CAE M5CAR T cells undergo a transition from T cells to a post-thymic NK-like T cell phenotype. This plasticity was confirmed by TCR lineage tracing and was also detected in CD19 CAR T cells analyzed from post-infusion blood from DLBCL patients and in M5CAR T cells infiltrating relapsed tumors derived from a xenograft NSG/AsPC-1 mouse model. The dysfunctional signature and the NK phenotype were further detected in vivo by transcriptomic analysis in Ly95 TCR-specific TILs infiltrating NY-ESO-1 tumors. Among the genes included in the dysfunctional signature, the transcription factors SOX4 and ID3 were identified as potential regulators of dysfunction by differential gene expression and pathway analyses. To determine their role in the establishment of a dysfunctional phenotype, we generated ID3KO.M5 and SOX4KO.M5 CAR T cells using CRISPR technology in primary human lymphocytes. The cytotoxic potential of the M5 CAR T cell product generated was not modified by the genetic disruption of the transcription factors. However, when CAR T cells were challenged with chronic antigen stimulation, KO CAR T cells showed a significant reduction of the dysfunctional signature and the NK-like T cell gene expression. Importantly, CAE KO CAR T cells exhibit improved cytotoxicity as compared to Mock.M5CAR T cells. In summary, we have developed a robust in vitro model that recapitulates the hallmarks of T cell exhaustion and that facilitated the identification of a gene signature defining CAR dysregulation, a T-to-NK-like-T cell transition as a novel feature of CAR T cell dysfunction and the transcription factors SOX4 and ID3 as key regulators of CAR T cell exhaustion. Citation Format: M. Angela Aznar, Charly R. Good, Shunichiro Kuramitsu, Parisa Samareh, Sangya Agarwal, Greg Donahue, Kenichi Ishiyama, Nils Wellhausen, Austin K. Rennels, Yujie Ma, Lifeng Tian, Sonia Guedan, Katherine A. Alexander, Zhen Zhang, Philipp C. Rommel, Nathan Singh, Karl M. Glastad, Max W. Richardson, Keisuke Watanabe, Janos L. Tanyi, Mark H. O’Hara, Marco Ruella, Simon F. Lacey, Edmund K. Moon, Stephen J. Schuster, Steven M. Albelda, Lewis L. Lanier, Regina M. Young, Shelley L. Berger, Carl H. June. Mechanisms of CAR T cell dysfunction and identification of transcription factors that drive the exhaustion phenotype [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 3602.

Abstract 2760: Hypophosphatemia due to increased effector cell metabolic activity is associated with neurotoxicity symptoms in anti-CD19 CAR T cell therapy

Abstract Background: A significant complication of CAR T cell therapy is immune effector cell-associated neurotoxicity syndrome (ICANS), which presents as aphasia, confusion, weakness, somnolence, seizures, and coma. This is similar to the neurological manifestations of serum hypophosphatemia, which typically results from sudden increased metabolic demand for phosphorylated intermediates, as in the setting of refeeding syndrome. Given these similarities, we investigated whether CAR T cell effector metabolic activity is associated with increased extracellular phosphate consumption, and the association of serum hypophosphatemia with ICANS. Methods: Human CAR T cells expressing anti-CD19scFV linked to either 4-1BB-CD28-CD3z or CD28-CD3z signaling domains were co-cultured with CD19+/firefly luciferase+ Raji cells. Media phosphorus consumption and cell cytotoxicity were assessed throughout co-culture. Single cell cytokine polyfunctionality of CAR T cells was measured via IsoPlexis platform. Oxidative phosphorylation and glycolytic activity of CAR T cells were measured via Agilent Seahorse assay. In parallel, we retrospectively analyzed 77 patients treated with CD28 or 41BB anti-CD19 CAR T cell therapy. A linear mixed effects model was generated from this dataset to assess the temporal relationship between longitudinal serum phosphorus measurements and ICANS/CARTOX-10 scores (clinical measures of CAR T neurotoxicity). Results: When co-cultured with Raji cells, 4-1BBz consumed 75.9% more phosphate (p=0.0019) and CD28z consumed 69.9% more phosphate (p=0.0048) compared to mock CAR T cells. Phosphate consumption was associated with increased Raji cell killing (4-1BBz: 97.0%, p=0.0011; CD28z: 89.5%, p=0.0015), increased single cell cytokine polyfunctionality, and increased cellular metabolic demand (oxidative and glycolytic ATP production rate). Of 77 patients treated with CD19 CAR T cells, 23 developed ICANS. A linear mixed effects model predicted a 0.29mg/dL decrease in serum phosphorus for each grade increment in ICANS score (p&amp;lt;0.0001) and a 0.11mg/dL decrease in serum phosphorus for each point of decrease in CARTOX-10 (p=0.0025). Importantly, this model predicts neurotoxicity only when drops in serum phosphorus bring patients into significant hypophosphatemia (&amp;lt;2.7mg/dL). The strength of the relationship is also dependent on time, with serum phosphorus decreases occurring shortly after cell infusion having stronger correlation with ICANS/CARTOX-10 scores. Conclusions: Our data suggest the increased metabolic demand of adoptively transferred CAR T cells, as a consequence of their cytokinetic polyfunctionality and antigen directed cytotoxic activity, may drive a state of hypophosphatemia, which is associated with ICANS in CAR T cell therapy. Early phosphate repletion may help mitigate the neurologic toxicity of CAR T cell therapy. Citation Format: Jack Pengfei Tang, Cole W. Peters, Crystal Quiros, Xiaoyan Wang, Theodore S. Nowicki. Hypophosphatemia due to increased effector cell metabolic activity is associated with neurotoxicity symptoms in anti-CD19 CAR T cell therapy [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 2760.

Abstract 1362: Metabolic engineering of CAR-T cells overcomes suppressive adenosine signaling and enhances functionality

Abstract Chimeric Antigen Receptor (CAR) T cell therapy has resulted in remarkable clinical outcomes in the context of acute and chronic lymphoblastic leukemia, but remains unsuccessful in the treatment of solid tumors. One reason for this failure is thought to be T cell dysfunction or exhaustion promoted by suppressive soluble factors within the tumor microenvironment (TME). High extracellular levels of the immunosuppressive factor adenosine (Ado) are generated in the TME via breakdown of ATP by ecto-enzymes CD39 and CD73 expressed on tumor-infiltrating immune cells. Binding of extracellular Ado to its receptor A2a on T cells results in inhibition of proliferation and effector function. Interestingly, CD39 has recently been described as a surrogate marker of exhaustion on human CAR-T cells and non-engineered T cells. Therefore, we hypothesized that CD39 expression on exhausted CAR-T cells promotes dysfunction through generation of extracellular adenosine. Using an in vitro model of T cell exhaustion, whereby human T cells express a CAR that tonically signals in an antigen-independent manner (HA CAR), we demonstrate that exhausted HA CAR T cells actively hydrolyze extracellular ATP via their elevated expression of CD39 and CD73. Moreover, exhausted CD39+ CAR T cells upregulate several genes associated with a Treg phenotype at the mRNA and protein levels, suggesting that this cell population might be suppressive. To assess whether CD39+/CD73+ CAR T cells exhibit suppressive functions, we co-cultured them with non-exhausted CD19-CAR T cells. Indeed, proliferation and secretion of IL-2 by CD19 CAR T cells were diminished when they were co-cultured with exhausted CD39+ CAR T cells, and that this suppression is dependent on the A2a receptor. Using this knowledge, we used gene-editing and overexpression approaches to engineer CAR-T cells with resistance to suppressive adenosine signaling. In contrast to genetic deletion of CD39 or CD73, which did not alleviate CAR T cell dysfunction, genetic deletion of adenosine receptor A2aR in exhausted CAR T cells resulted in phenotypic changes and a modest improvement in tumor-specific killing. Further, ectopic overexpression of adenosine deaminase (ADA) in CAR T cells led to decreased exhaustion marker expression and significantly enhanced effector function. These data indicate that ADA overexpression is an innovative approach to increase the functionality of CAR T cells through avoidance of suppressive adenosine signaling, and provides proof-of-concept that metabolic engineering of CAR-T cells can pave the way for responses in patients with solid tumors. Citation Format: Dorota Klysz, Meena Malipatlolla, Katherine Freitas, Malek Bashti, Louai Labanieh, Peng Xu, Cecilia Ramello, Amaury Lerust, Hyatt Balke Want, Kaithlen Zen Pacheco, Evan W. Weber, Shabnum Patel, Steven Feldman, Elena Sotillo, Crystal L. Mackall. Metabolic engineering of CAR-T cells overcomes suppressive adenosine signaling and enhances functionality [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 1362.

Abstract 3955: Targeting transcription elongation via CDK9 in mantle cell lymphoma patients with dual resistance to BTK inhibition and CAR T therapy

Abstract Introduction Clinical relapse following CD19 CAR T therapy after failure to BTK inhibitors (BTKi) is a novel and fast growing medical challenge in treating patients with mantle cell lymphoma (MCL). Thus, developing a novel therapy to overcome this BTKi-CAR T dual resistance (Dual-R) is an urgent need. Our unpublished data revealed MYC targets and cyclin-dependent kinase 9 (CDK9) are highly upregulated in the Dual-R compared to BTKi-R samples. CDK9 is a critical component of the positive transcription elongation factor b (P-TEFb) complex. Its inhibition induces acute decline of short-lived mRNA and proteins, especially MYC and MCL-1, in acute myeloid leukemia and diffuse large B-cell lymphoma. Therefore, we hypothesize that targeting CDK9 may turn off MYC-driven tumor survival and drug resistance. BAY-1251152 is a novel selective CDK9 inhibitor with nanomolar potency. However, whether it has the potential to overcome BTKi-CAR T dual resistance has not been assessed. Methods To validate the correlation of MYC and CDK9 with drug resistance and clinical outcome, we performed whole transcriptomic profiling using primary patient samples. To assess the in vitro efficacy of BAY-1251152, we performed cell viability assay and cell apoptosis assay using MCL cell lines and primary patient samples and followed up with functional studies. furthermore, we assessed its in vivo efficacy using patient-derived xenograft (PDX) models derived from MCL patients including one with Dual-R. Results The expression of MYC oncogene associates with ibrutinib resistance and Dual-R, and poor clinical outcome. CDK9 expression does not correlates with ibrutinib resistance but it does associate with Dual-R and poor clinical outcome in CAR T-relapsed patients. CDK9 inhibition by BAY-1251152 is highly potent in anti-MCL activity in MCL cell lines with low nanomolar range of IC50 (59.6-172.3 nM) by inducing robust cell apoptosis. BAY-1251152 induces dose and time-dependent CDK9 inhibition. A rapid decline of phosphorylation of RNA polymerase II, MYC, MCL-1, and Cyclin D1 can be observed as early as 2 hours. Furthermore, BAY-1251152 (10mg/kg, QW) significantly inhibited tumor growth (p = 0.000003) in a PDX model derived from a Dual-R patient without causing apparent toxicity in NSG mice. In addition, BAY-1251152 also significantly suppressed tumor growth in the PDX models derived from a BTKi-R patient (p=0.00015) and a BTKi-venetoclax dual-resistant patient (p=0.009). Conclusion Our findings showed that targeting CDK9 by its specific inhibitor BAY-1251152 led to potent in vitro anti-MCL activity. BAY-1251152 induces fast CDK9 inhibition and rapid decline of MYC, MCL-1 and Cyclin D1 to induce robust cell death. BAY-1251152 is also potent in inhibiting tumor growth in PDX models. These data support that CDK9 is a promising target to overcome BTKi-CAR T dual resistance in MCL, which is in urgent need. Citation Format: Vivian Changying Jiang, Lingzhi Li, Alexa Jordan, Yu Xue, Fangfang Yan, Joseph McIntosh, Yang Liu, Yuxuan Che, Yijing Li, Qingsong Cai, Angela Leeming, Lukas Simon, Zhongming Zhao, Jia Zhou, Michael Wang. Targeting transcription elongation via CDK9 in mantle cell lymphoma patients with dual resistance to BTK inhibition and CAR T therapy [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 3955.

Abstract 3569: Combining oncolytic adenovirus Delta-24-RGDOX with adoptive T cell therapy in localized treatment induces sustainable regression of disseminated solid tumors through antigen spreading

Abstract CAR T therapy greatly improves the survival of patients with hematological malignancies. But its effect in solid tumors is suboptimal. Intratumoral injections of oncolytic viruses, including Delta-24-RGDOX (DNX-2440) from our group, directly lyse cancer cells and activate tumor microenvironment, leading to adaptive antitumor immunity. To take advantage of instant antitumor activity of T cells and virus-mediated in situ autovaccination against heterogeneous cancer cells, we hypothesize that intratumorally injections of tumor-targeting T cells followed by Delta-24-RGDOX overcome antigen escape of CAR T therapy, leading to more sustainable systemic anti-cancer immunity. Thus, we used subcutaneous (s.c.)/s.c. B16 melanoma mouse models to assess the effect of localized treatment in disseminated tumors. OVA (or gp100)-specific CD8+ T cells or CD19 CAR T cells were injected into the first tumor, followed by three injections of Delta-24-RGDOX into the same tumor. T cells and tumor cells from the mice were profiled for surface markers with flow cytometry and immune staining. Activity of splenocytes against tumor cells and specific tumor-associated-antigens (TAAs) was measured with ELISA. Tumor growth was monitored through measuring tumor size. The animal survival curves were plotted according to the Kaplan-Meier method. We found TAA-specific T cells injected into the first s.c. tumor showed tropism for disseminated s.c. and intracranial tumors, tumor draining lymph nodes, but not for spleen, peripheral blood and normal brain. Unlike untreated and Delta-24-RGDOX-treated tumor, T cell-treated tumor showed decreased expression of the target TAA which was depleted in the recurrent tumor. Moreover, Delta-24-RGDOX increased total T cell presence within the tumors, and the activity of the splenocytes against the tumor cells and other antigens than the one targeted by injected T cells. Consequentially, the combination of OVA-specific T cells and Delta-24-RGDOX was more potent to inhibit the injected and untreated disseminated tumor growth and caused improved survival rate than either of the agent alone (p &amp;lt; 0.05). Importantly, we observed relapse of the regressed tumors in the group treated by T cells alone, but not in the combination group. The survivors from the combination therapy were protected from rechallenging with B16-OVA cells but not lung carcinoma cells, suggesting the development of immune memory. In summary, our study indicates the virus induces antigen spread, resulting in expansion of antitumor T cell repertoire to prevent cancer relapse in adoptive T cell therapy (ACT). Our data demonstrate that Delta-24-RGDOX collaborates with ACT to induce more potent systemic immunity against the tumors, leading to sustainable tumor regression. Citation Format: Hong Jiang, Dong Ho Shin, Yanhua Yi, Xuejun Fan, Joy Gumin, Marta M. Alonso, Frederick F. Lang, Candelaria Gomez-Manzano, Juan Fueyo. Combining oncolytic adenovirus Delta-24-RGDOX with adoptive T cell therapy in localized treatment induces sustainable regression of disseminated solid tumors through antigen spreading [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 3569.

Abstract 3603: Reverse fate mapping of CD19-targeted CAR T cells in patients with large B-cell lymphoma

Abstract Autologous T cells genetically engineered to express a chimeric antigen receptor targeting CD19 (CD19-CAR) have achieved high complete response rates in patients with hematologic malignancies, but &amp;gt;50% of patients progress following therapy. Here, we sought to understand key T-cell intrinsic factors impacting efficacy, namely CAR T-cell expansion, persistence, and homing to the tumor. Using an approach called reverse fate mapping, we followed individual T-cell clones at the single-cell level from pre-manufacture apheresis to the infusion product, tumor-involved lymph node, and blood at peak and late expansion in 12 adult patients with relapsed or refractory large B-cell lymphoma treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T-cell immunotherapy. The resulting CAR T-cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 322,028 cells from 44 samples, with 119,397 unique T-cell receptor (TCR) clonotypes identified. This atlas enabled us to ask questions like: “What were the phenotypes of the most successful CAR T-cell clones at the time of infusion or pre-manufacture apheresis?” We found that T-cell clonotypes with juvenile features at apheresis, including IL7R expression, were the most successful at expansion to higher frequencies in the infusion product, while clones with effector gene expression programs, such as those encoding perforin and granzymes, contracted between apheresis and product. Conversely, it was GZMK-expressing T cells in pre-manufacture apheresis that were dominant in the tumor early following CAR T-cell infusion. Further, T-cell clonotypes with active effector programs at infusion dominated at peak expansion. Finally, we defined active expression modules and pathways in the infusion product for CAR T-cell clones that homed to the tumor or became dominant at late expansion. These analyses pinpoint the molecular mechanisms that could be modulated to rationally steer CAR T-cell differentiation trajectories at the genetic or pharmacological level. This work was supported in part by the Parker Institute for Cancer Immunotherapy, California Institute for Regenerative Medicine, Kite Pharma, and Stanford Cancer Institute. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Sreevidya Kurra, Moksha Desai, Snehit Prabhu, Eric Yang, Michael G. Ozawa, Paul J. Hanson, Fang Wu, Matthew J. Frank, John H. Baird, Lori Muffly, Gursharan K. Claire, Juliana Craig, Katherine A. Kong, Dhananjay Wagh, John Coller, Sylvia K. Plevritis, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Reverse fate mapping of CD19-targeted CAR T cells in patients with large B-cell lymphoma [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 3603.

Abstract 3581: Multi-omic analysis identifies mechanisms of resistance to CD19 CAR T-cell therapy in children with acute lymphoblastic leukemia

Abstract Background: Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Despite the survival rate of 90% for newly diagnosed children with ALL, the outcome for relapsed patients is historically poor with a less than 30% survival. CD19 CAR T-cell therapy (CART19) has shown remarkable response rates, between 80-90% in relapsed/refractory disease. Little is known about antigen-independent factors that predict initial resistance to CART19. We hypothesized that leukemias that are resistant to CART19 are distinct from sensitive leukemias and that these differences can be detected prior to therapy. Methods: To interrogate differences between resistant and sensitive leukemias, we obtained pre-treatment bone marrow aspirates (BMAs) from patients enrolled in a clinical trial at Seattle Children’s Hospital (PLAT-02). Samples were categorized based on patient response, with non-response defined as not achieving and maintaining minimal residual disease negativity at Day +63. Our study included 7 resistant and 7 sensitive leukemias as controls. We performed whole exome sequencing, bulk RNA-seq, PacBio-seq of the CD19 locus, array-based methylation, ATAC-seq, scRNA-seq, and CyTOF. Results: We found that non-response to CART19 is independent of leukemic subtype. Despite blasts being CD19+ in all patients by flow cytometry, we identified alternative splicing of CD19 in one non-responder, while the remaining non-responders expressed high levels of wildtype CD19. We discovered a distinctive DNA methylation pattern in the non-responders characterized by hypermethylation of PRC2 targets in embryonic and cancer stem cells (p = 8.15E-25) Furthermore, using gene set enrichment analysis of ATAC-seq data, we found increased accessibility of chromatin at regions associated with stem cell proliferation (NES = 2.31; p &amp;lt; 0.0001) and cell cycling (NES = 2.27; p &amp;lt; 0.0001). We found a greater similarity between accessibility patterns of non-responders to hematopoietic progenitors, including hematopoietic stem cells (p = 0.037) and common myeloid progenitors (p = 0.047). These findings were supported by an increased frequency of cell subpopulations expressing a multi-lineage phenotype (CD19, CD20, CD33, CD34; p = 0.009). Moreover, we find decreased expression of antigen presentation and processing pathways across all leukemic cells relative to responders (p = 0.0001). Conclusions: This study, one of the most comprehensive multi-omic analyses of samples from patients treated with CAR T-cells, identified resistance mechanisms that can be detected prior to treatment. We report the novel association of a stem cell phenotype, lineage plasticity, and decreased antigen presentation with resistance. These results support further refinement of eligibility for CART19 for children with leukemia and highlights the need for alternative of complimentary approaches for these patients. Citation Format: Katherine E. Masih, Rebecca Gardner, Hsien-Chao Chou, Abdalla Abdelmaksoud, Young K. Song, Luca Mariani, Vineela Gangalapudi, Berkley E. Gryder, Ashley Wilson, Serifat O. Adebola, Benjamin Z. Stanton, Chaoyu Wang, Xinyu Wen, Gregoire Altan-Bonnet, Michael C. Kelly, Jun S. Wei, Martha L. Bulyk, Michael C. Jensen, Rimas J. Orentas, Javed Khan. Multi-omic analysis identifies mechanisms of resistance to CD19 CAR T-cell therapy in children with acute lymphoblastic leukemia [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 3581.

Abstract 581: bbT369, a dual-targeted and CBLB gene-edited autologous CART product, demonstrates anti-lymphoma activity in preclinical mouse models

Abstract Anti-CD19 CAR T cell therapies have improved outcomes for non-Hodgkin lymphoma (NHL) patients. However, only 30-40% of patients treated with commercially available CART cell therapies obtain long term remission, highlighting the need for more efficacious and durable therapies. Emerging clinical data suggest several failure modes for CD19 CAR T cell therapies: including loss or downregulation of CD19 antigen, loss of co-stimulation pathways on tumor cells, exhaustion of CAR-T cells, and immunosuppressive microenvironments. To overcome these hurdles, we devised the next-generation autologous CAR-T cell therapy bbT369. bbT369 is dual targeted (CD79a/CD20) CAR T cell therapy that uses an OR gate design to limit antigen escape, has split 41BB and CD28 co-stimulatory domain architecture to augment T cell activation, and contains a knock-out of the CBLB gene to enhance potency and reduce T cell exhaustion. Here we report the first results with bbT369, demonstrating anti-lymphoma activity in in vitro assays and in vivo using xenograft mouse models. We demonstrate that CD79a and CD20 expression is B cell lineage restricted in normal human tissue and confirm that these proteins are co-expressed in diffuse large B cell samples. To target these antigens, we show a split dual-targeting CAR configuration is optimal for bbT369-directed tumor cell killing. Using an engineered megaTAL, we demonstrate high on-target activity of greater than 75% insertions and deletions (Indels) at the CBLB target site using clinical-scale manufacturing processes and low off-target activity (all off-targets less than 0.2%). In in vitro tumor co-culture assays, we show that inclusion of the CBLB gene edit in bbT369 increases Interleukin (IL)-2 production relative to an unedited anti-CD79a/CD20 CAR T cell control. Using various xenograft mouse models, we showed that bbT369 has similar or improved efficacy compared to anti-CD19 CAR drug product, including in low tumor-antigen models. In the Toledo subcutaneous xenograft model, bbT369 showed a 3-fold increase in T cell expansion compared with an unedited anti-CD79a/CD20 dual-targeting CAR T cell control. Furthermore, while a fraction of mice (3/5) receiving the unedited anti-CD79a/CD20 dual-targeting CAR T cells experienced late relapses (between 60-80 days following initial tumor clearance), all mice (n=5) receiving bbT369 were fully protected from late relapses (up to day 104 of follow-up). Collectively, the data support a first-in-human trial for bbT369 to evaluate initial safety and efficacy in NHL patients. Citation Format: Michael Certo, Christopher Baldeviano, Sharlene Adams, Martin Asimis, Alexander Astrakhan, Andy Chavkin, Maria L. Cabral, Jimmy Chu, Marie Debrue, Devina Desai, John Evans, Pinky Htun, Amanda Iniguez, Jordan Jarjour, Carl Johnson, Harini Kantamneni, Sema Kurtulus, Michael Magee, Unja Martin, Seamus McKenney, Sara Miller, Prashant Nambiar, Vinh Khang Nguyen, Mauris Nnamani, Jen Obrigewitch, Lisa Pechilis, Molly Perkins, Christopher Petersen, Jason Pinger, Cindy Rogers, Nick Rouillard, Kendal Sanson, Emily Thompson, Collin Walter, Roslyn Yi, Sarah Voytek, Philip Gregory. bbT369, a dual-targeted and CBLB gene-edited autologous CART product, demonstrates anti-lymphoma activity in preclinical mouse models [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 581.

Abstract 2824: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance anti-GPC3 CAR T cells to drive complete responses in solid epithelial tumor models

Abstract CAR T cell therapy has demonstrated clinical efficacy against hematological malignancies. However, prominent barriers including poor T cell effector function, lack of proliferation, and limited CAR T cell persistence have prevented CAR T cell therapies from reaching their full curative potential, especially in solid tumors. Interleukin-2 (IL-2) is a potent stimulator of T cell proliferation, survival, and cytotoxic function, thereby making it an attractive cytokine to support CAR T cell therapy. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations, including non-tumor reactive T cells and NK cells. To facilitate selective ex vivo and in vivo expansion of engineered T cells we have developed a human orthogonal (ortho) ligand/receptor system consisting of a IL-2 mutein (STK-009) that does not significantly stimulate cells expressing wild type IL-2 receptor and a mutated IL-2 Receptor Beta (orthoIL-2Rβ) that responds to STK-009 but not wild type IL-2. This system enables in vivo IL-2 signaling in engineered cells that express the orthoIL-2Rβ while avoiding stimulation of native T cells and NK cells. Previously, we demonstrated the ability of the STK-009/orthoIL-2Rβ receptor pair to selectively enhance the anti-tumor efficacy of orthoIL-2Rβ (hoRb) expressing CD19 CAR T cells (SYNCAR-001) in preclinical lymphoma mouse models. We also demonstrated that STK-009 is selective for the orthoIL-2Rβ expressing cells and therefore in a non-human primate model does not stimulate native T or NK cells. Here, we demonstrate the ability of the STK-009/hoRb system to enhance the anti-tumor activity and persistence of anti-glypican 3 (GPC3) CAR T cells. GPC3 overexpression is associated with various malignancies such as hepatocellular carcinoma (HCC), pediatric sarcomas, and non small cell lung carcinoma (NSCLC). Clinical trials of GPC3 CAR T therapy are ongoing, but early data suggests a need to boost CAR T cell function and persistence to achieve significant clinical responses. We incorporated the hoRb downstream of an anti-GPC3_28z CAR via a T2A cleavage peptide (SYNCAR-002). In vivo, STK-009 administration enhanced the anti-tumor efficacy of SYNCAR-002 in highly aggressive subcutaneous and intraperitoneal HCC models. STK-009 treatment resulted in significant expansion of SYNCAR-002 and drove infiltration of SYNCAR-002 into tumors. STK-009 treatment also induced intratumoral granzyme B+ and IFN-γ+ production by SYNCAR-002 indicating activation of effector T cell function. These findings validate that the orthogonal IL-2 platform has the potential to improve the efficacy and durability of CAR T therapy for solid tumor targets such as GPC3 by selectively expanding CAR-T cells in vivo, driving CAR-T cells into the tumor, and activating CAR-T cells in the tumor microenvironment. Citation Format: Paul-Joseph Aspuria, Marie Semana, Sandro Vivona, Mahalaksmi Ramadass, Navneet Ratti, Romina Riener, Michele Bauer, Mohammed Ali, Deepti Rokkam, Rob A. Kastelein, Patrick J. Lupardus, Martin Oft. Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance anti-GPC3 CAR T cells to drive complete responses in solid epithelial tumor models [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 2824.

Abstract 2816: A novel in vivo model to simultaneously assess efficacy and toxicity of chimeric antigen receptor T-cell immunotherapy

Abstract Over the past few years, chimeric antigen receptor T (CAR T)-cell therapy has emerged as a novel treatment option for certain hematologic malignancies. Several CAR T-cell therapies produced highly efficacious responses from patients with hematologic malignancies. However, wider adoption of CAR T-cell therapy is challenged due to the potential development of life-threatening toxicities including cytokine release syndrome (CRS). CRS symptoms can range from mild fever to life-threatening events, including death. Preclinical animal models to assess the efficacy and toxicity of CAR T-cell therapies have been lacking. Here, we developed a novel mouse model to assess the efficacy and toxicity of CAR T-cell therapy simultaneously. Two different PBMC humanized NSG™ variants were used. NSG-MHC Class I/II double knock-out strain is known to have a delayed onset of GvHD, and NSG-SGM3xIL15 strain shows a higher engraftment level of human NK and myeloid cells, in addition to human T cells. Mice were humanized using PBMCs and treated with autologous or allogeneic CAR T cells, and efficacy and toxicity were assessed. Compared to the control treatment, CD19 CAR T-cells caused a decrease in the human CD19+ cell population in the blood and spleen, and induced cytokine release. Both T-cell and myeloid cytokine releases, including IFNgamma, IL-10, RANTES, and MIP-1alpha, were induced in our animal models. The in vivo platform can also be used to determine individual PBMC/CAR T donor differences and show the extent of efficacy and toxicity of each PBMC donor treated with autologous and allogeneic CAR T-cell therapy. We further confirmed anticancer efficacy using luciferase-tagged human B-cell lymphoma Raji tumor cells, which express a high level of CD19. In summary, we have developed a novel in vivo model to test the efficacy and toxicity of both autologous and allogeneic CAR T-cell therapy simultaneously. Citation Format: Jiwon Yang, Won Lee, Jing Jiao, Danying Cai, Heather Gustafson, Rebecca Gardner, Mingshan Cheng, James G. Keck. A novel in vivo model to simultaneously assess efficacy and toxicity of chimeric antigen receptor T-cell immunotherapy [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 2816.

Abstract 6225: Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models

Abstract Chimeric antigen receptor (CAR) - engineered natural killer (NK) cell therapy has emerged as a promising platform for adoptive immunotherapy for cancer. However, CAR-NK or CAR-T therapy so far has achieved limited efficacy in solid tumors compared with hematologic malignancies. One of the challenges is the lack of prevalent tumor-specific surface antigen target in solid tumors. Hence, we are developing a novel oncolytic vaccinia virus (VV) to deliver a dual functional glycol-immune checkpoint inhibitor and universal tumor cell marker combined with CAR-NK cell therapy to increase anti-tumor efficacy. Using the vaccinia virus deleted in both viral TK and VGF genes (vvDD) to drive the expression of a membrane-bound sialidase derived from actinomyces viscosus (avSial) under the viral late promoter, we observed efficient desialylation of both VV-infected and non-infected tumor cells, which would alleviate the suppression of sialic acid on NK and other immune cells within TME. Further, the surface bound avSial on VV-infected tumor cells can also serve as a universal target for avSial-CAR NK cells, with less concern for cross-reactivity to normal human tissues and antigen loss. We developed anti-avSial antibodies through mice immunization study. The selected single chain variable fragments (scFV) were constructed in CAR format containing CD28 CD3 ζ for co-stimulation and human IL-15 gene for better NK persistence and function. The binding affinity of anti-avSial scFv to avSialidase were further screened in CAR constructs-transfected 293T cells. The selected anti-avSial scFv CAR were packaged into gamma retroviral vectors to transduce activated and expanded NK cells derived from healthy donor peripheral blood. For proof-of-concept, target tumor cell lines A375, A549 and HT-29 expressing transmembrane sialidase and GFP were also generated. Compared with CD19 CAR NK and none transduced (NT) NK cells, avSial CAR NK had markedly increased cytotoxicity against avSial-expressing tumor cells in co-culture assays at low E:T ratios &amp;lt;= 1:2. Upon tumor cell rechallenge, avSial CAR NK also controlled the tumor growth significantly better than CD19 CAR NK or NT NK cells. Furthermore, avSial CAR NK completely eliminated A375 tumor spheroids expressing transmembrane sialidase whereas CD19 CAR NK or NT NK cells only transiently controlled the tumor spheroids growth. Taken together, we have developed an effective avSial targeting CAR IL15 NK that will be used to demonstrate enhanced efficacy and versatility in combination with avSial-armed VV for treatment of solid tumors. Citation Format: Xiaomei Wang, Guowei Wei, Keshav B. Karki, Winnie Chan, Mariya Viskovska, Andrew Williams, Nancy Chang, Haiyan Jiang. Developing a novel combination therapy using engineered chimeric antigen receptor natural killer cells targeting avsialidase with avsialidase-armed oncolytic vaccinia virus in solid tumor models [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 6225.

Abstract 2855: Antitumor activity of BAFF-R targeting CAR T-cells on chronic lymphocytic leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is the most common leukemia in adults with CD19-targeting CAR T-cell therapy showing an excellent initial curative response. However, 10-20% of patients who receive anti-C19 CAR T-cell treatment experience relapse due to CD19 antigen loss. B-cell activating factor receptor (BAFF-R) that is expressed on the surface of normal and neoplastic B cells mediates the survival of mature peripheral B cells along with the B-cell receptor. Since B-CLL is characterized by apoptotic disruption and subsequent accumulation of mature B cells, BAFF-R is a logical, novel target for CAR T-cell therapy directed against B-CLL, especially if CD19 antigen loss occurs. In this study, we first manufactured BAFF-R CAR T-cells by infecting healthy donor Tnaïve/memory (Tn/mem) cells with BAFF-R CAR lentivirus before evaluating the antitumor activity of BAFF-R CAR T-cells on wild type MEC-1 (a B-CLL cell line), modified MEC-1, and primary cell lines. Antigen-specific cytotoxicity and activation of the BAFF-R CAR T-cells or control CD19 CAR T-cells were determined upon incubation with B-CLL cell lines and patient-derived primary B-CLL by measuring degranulation and IFN-γ release, respectively. BAFF-R CAR T-cells demonstrated substantial cytotoxicity against MEC-1, CD19-deficient MEC-1, and primary B-CLL cell lines; CD19 CAR T-cells showed no effect on CD19-deficient MEC-1 cells but did show effects with primary B-CLL and MEC-1 cells. Similarly, the release of IFN-γ was greater when BAFF-R CAR T-cells were co-cultured with CD19-deficient MEC-1 or primary B-CLL compared to CD19 CAR T-cells co-cultured with CD19-deficient MEC-1. In summary, BAFF-R CAR T-cells exert significant antitumor properties not only on patient-derived primary B-CLL cells but also on a CD19-deficient B-CLL cell line. We, therefore, assert BAFF-R-targeting CAR T-cells to be a novel therapeutic avenue for patients newly diagnosed with B-CLL and patients experiencing relapsed B-CLL after anti-CD19 therapy. Citation Format: Yan Luo, Kaihin Lui, Tommy T. To, Andrew liu, Farah Yassine, Mohamed A. Kharfan Dabaja, Martha E. Gadd, Hong Qin. Antitumor activity of BAFF-R targeting CAR T-cells on chronic lymphocytic leukemia [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 2855.