CART19 Cell Research Articles

Abstract PR01: EZH2 inhibitors improve CAR-T therapy by enhancing lymphoma B cell immunogenicity and CAR-T cell functions

Abstract Although CAR-T19 therapy is widely used for relapsed/refractory B cell lymphomas, approximately half of patients experience post CAR-T relapse. The mechanisms underlying this resistance remain largely unknown and efforts to overcome this limitation are hindered by the lack of suitable pre-clinical models for study. To address these unmet needs, we generated a novel genetically engineered mouse model with conditional expression of Ezh2Y641F and BCL2 in germinal center (GC) B cells. Ezh2/BCL2 mice developed follicular lymphoma (FL) which histologically and transcriptionally recapitulates human FL. Moreover, we established a cell line derived from an Ezh2/BCL2 mouse that developed transformed FL (tFL). Injection of the cell line named “tFL-P6” in immunocompetent C57BL6 recipients resulted in rapid disease progression that histologically and transcriptionally mimics human tFL. Treatment of tFL-P6 with EZH2 inhibitors (EZH2i) didn’t affect proliferation and viability, however, the number of pretreated cells was significantly reduced when co-cultured with T cells. EZH2i pretreated tFL-P6 displayed extended interaction with T cells, as measured by live imaging. EZH2i reprogrammed tFL-P6 to restore T cell engagement genes such as ICOSL, ICAM1, OX40L, and integrins, as well as cytokines and chemokines involved in T cell recruitment, therefore enhancing immunogenicity. Notably, pretreated tFL-P6 cells were rejected by C57BL6 whereas they developed lethal disease in immunodeficient recipients. Pre-treatment of tFL-P6 as well as human DLBCL and PDX-derived cell lines significantly enhanced CAR-T19 cell killing effects in vitro. Administering CAR-T cells in mice engrafted with EZH2i-pretreated tFL-P6 significantly reduced the tumor burden and prolonged their survival (100% vs 30%, p<0.01). To assess the direct interactions between lymphoma cells and CAR-T cells in vivo, we performed intravital 2-photon imaging of popliteal lymph nodes using dTomato labeled CAR-T cells and GFP+ tFL-P6 cells. Pretreatment of tFL-P6 cells with EZH2i doubled the recruitment of CAR-T cells in the microenvironment and enhanced the duration of contact and surface engagement with CAR-T cells. To explore the impact of EZH2 inhibition on CAR-T cells, we manufactured CAR-T cells from splenic T cells of mice treated with EZH2i or vehicle for 14 days. Prior exposure to EZH2i didn’t affect proliferation and transduction during CAR-T production. Ex vivo assays with those CAR-T cells and tFL-P6 cells showed a superior killing effect and expansion of EZH2i-exposed CAR-T (p<0.001). Mice bearing tFL-P6 lymphomas displayed a longer survival when infused with EZH2i-pretreated CAR-T cells (p=0.06). Pretreatment with EZH2i resulted in an increased memory/effector ratio (p<0.01) and reduction of PD1+CD38+ exhausted CD8CAR-T cells (p<0.001). Overall, EZH2i improved CAR-T therapy by enhancing lymphoma cell immunogenicity and CAR-T cell functions. These results prompted the initiation of a clinical trial to evaluate the safety and efficacy of this combination in R/R B cell lymphomas (NCT05934838). Citation Format: Yusuke Isshiki, Xi Chen, Matt Teater, Ioannis Karagiannidis, Henna Nam, Amy Chadburn, Ari Melnick, Wendy Béguelin. EZH2 inhibitors improve CAR-T therapy by enhancing lymphoma B cell immunogenicity and CAR-T cell functions [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PR01.

Abstract 3849: Frontline ibrutinib-based therapy enhances CART cell functions in patients with CLL: Analysis of the phase 3 randomized clinical trial ECOG-ACRIN E1912

Abstract Introduction: Despite the remarkable activity of CD19-directed chimeric antigen receptor T cell therapy (CART19), durable remissions remain low, especially in chronic lymphocytic leukemia (CLL). Ibrutinib is an oral irreversible inhibitor of Bruton’s tyrosine kinase and is a first line therapy for CLL. Preclinical and early clinical data indicate that ibrutinib plus CART19 may be synergistic through inhibition of IL2-inducible T cell kinase. To systematically study the effects of prior ibrutinib treatment on subsequent CART functions, we utilized blood samples from the pivotal clinical trial E1912 (NCT02048813). E1912 was a phase 3 randomized trial comparing treatment with ibrutinib/rituximab (IR) to fludarabine/cyclophosphamide/rituximab (FCR) in previously untreated patients with CLL, which led to the FDA approval of ibrutinib as a first-line therapy in CLL. Methods: We evaluated the ex vivo expansion and effector functions of T cells and CART19 generated from patients (n=12) with CLL progression following treatment with IR or FCR. We generated CART19 from longitudinal samples obtained at baseline (pretreatment), 6 months post-treatment, 12 months post-treatment, and disease progression. Isolated T cells were lentivirally transduced to express CD28- or 41BB-costimulated CAR19. To assess antigen specific killing and proliferation, untransduced T cells or CART19 were co-cultured at a 1:1 ratio with the CD19+ cell line, JeKo-1, as a surrogate for CLL cells. Results: Ex vivo T cell expansion increased from an average of 1.5-fold at baseline to 6.7-fold at 6 months, 11.6-fold at 12 months, and 16.9-fold at progression, following ibrutinib-based therapy (p = 0.02, 0.04, and 0.04, respectively). Improved T cell expansion was independent of disease burden (p = 0.89, 0.73, and 0.54 for expansion vs B cell count at 6 months, 12 months, and progression). T cell expansion significantly worsened following treatment with FCR-based therapy compared to ibrutinib-based therapy (0.6- vs 11.6-fold at 12 months and 0.9- vs 16.9-fold at progression; p = 0.031 and 0.039, respectively). Starting at 6 months post-IR, CART19 demonstrated improved antitumor activity and antigen-specific proliferation against JeKo-1. There was no significant difference in the expansion of 41BB- vs CD28-costimulated CART19 following ibrutinib treatment. However, 41BB-CART19 showed enhanced antigen-specific proliferation upon stimulation with JeKo-1 (p = 0.008, 0.02, and 0.02 at 6 months, 12 months, and progression). Conclusion: Our results indicate significant improvement in T cell expansion and CART19 cell functions in patients with CLL following frontline ibrutinib-based therapy, and worsening T cell functions after FCR treatment. Improvement in T cell functions is independent of malignant B cell eradication and is more significant in 41BB-costimulated CART19. Citation Format: Truc Ngoc Huynh, Victoria Wang, Tait Shanafelt, Elizabeth L. Siegler, Carli M. Stewart, Kun Yun, Long K. Mai, Olivia Sirpilla, James H. Girsch, Claudia Manriquez-Roman, Jennifer M. Feigin, Makena Rodriguez, Brooke L. Kimball, Hong Xia, Reona L. Sakemura, Ismail Can, Omar L. Gutierrez-Ruiz, Ekene J. Ogbodo, Erin E. Tapper, Lionel A. Fonkoua, Mehrdad T. Hefazi, Michael W. Ruff, Sameer Parikh, Neil E. Kay, Saad J. Kenderian. Frontline ibrutinib-based therapy enhances CART cell functions in patients with CLL: Analysis of the phase 3 randomized clinical trial ECOG-ACRIN E1912 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3849.

Treatment strategies for relapse after CAR T-cell therapy in B cell lymphoma.

Clinical trials of anti-CD19 chimeric antigen receptor T (CART19) cell therapy have shown high overall response rates in patients with relapsed/refractory B-cell malignancies. CART19 cell therapy has been approved by the US Food and Drug Administration for patients who relapsed less than 12 months after initial therapy or who are refractory to first-line therapy. However, durable remission of CART19 cell therapy is still lacking, and 30%-60% of patients will eventually relapse after CART19 infusion. In general, the prognosis of patients who relapse after CART19 cell therapy is poor, and various strategies to treat this patient population have been investigated extensively. CART19 failures can be broadly categorized by the emergence of either CD19-positive or CD19-negative lymphoma cells. If CD19 expression is preserved on the lymphoma cells, a second infusion of CART19 cells or reactivation of previously infused CART19 cells with immune checkpoint inhibitors can be considered. When patients develop CD19-negative relapse, targeting different antigens (e.g., CD20 or CD22) with CAR T cells, investigational chemotherapies, or hematopoietic stem cell transplantation are potential treatment options. However, salvage therapies for relapsed large B-cell lymphoma after CART19 cell therapy have not been fully explored and are conducted based on clinicians' case-by-case decisions. In this review, we will focus on salvage therapies reported to date and discuss the management of relapsed/refractory large B-cell lymphomas after CART19 cell therapy.

CD19 occupancy with tafasitamab increases therapeutic index of CART19 cell therapy and diminishes severity of CRS

AbstractIn the development of various strategies of anti-CD19 immunotherapy for the treatment of B-cell malignancies, it remains unclear whether CD19 monoclonal antibody therapy impairs subsequent CD19-targeted chimeric antigen receptor T-cell (CART19) therapy. We evaluated the potential interference between the CD19-targeting monoclonal antibody tafasitamab and CART19 treatment in preclinical models. Concomitant treatment with tafasitamab and CART19 showed major CD19 binding competition, which led to CART19 functional impairment. However, when CD19+ cell lines were pretreated with tafasitamab overnight and the unbound antibody was subsequently removed from the culture, CART19 function was not affected. In preclinical in vivo models, tafasitamab pretreatment demonstrated reduced incidence and severity of cytokine release syndrome and exhibited superior antitumor effects and overall survival compared with CART19 alone. This was associated with transient CD19 occupancy with tafasitamab, which in turn resulted in the inhibition of CART19 overactivation, leading to diminished CAR T apoptosis and pyroptosis of tumor cells.

13 - Transient CD19 Occupancy with Tafasitamab at the Time of Anti-CD19 Chimeric Antigen Receptor T (CART19) Cell Infusion Diminishes Severity and Incidence of CRS and Improves the Therapeutic Index of CART19 Cell Therapy in Preclinical Models

13 - Transient CD19 Occupancy with Tafasitamab at the Time of Anti-CD19 Chimeric Antigen Receptor T (CART19) Cell Infusion Diminishes Severity and Incidence of CRS and Improves the Therapeutic Index of CART19 Cell Therapy in Preclinical Models

CD19 Antigen Occupancy on Cancer Cells with the CD19 Monoclonal Antibody Tafasitamab Improves the Activation, Antitumor Efficacy, and Safety Profile of CART19 Cell Therapy

Background Major limitations to CD19-targeted chimeric antigen receptor T (CART19) cell therapy include suboptimal antitumor activity and development of life-threatening toxicities such as cytokine release syndrome (CRS) and neurotoxicity. CART-associated toxicities are directly related to CART activation and proliferation in vivo. Emerging data suggest that CART over-activation contributes to therapeutic failure. Objective We hypothesized that modulation of CART activation reduces the risk of CRS while enhancing CART efficacy. Specifically, we hypothesized that using the clinically-approved CD19 monoclonal antibody tafasitamab prior to CART19 therapy can reduce tumor pyroptosis (inflammatory lytic programmed cell death), modulate CART activation, and enhance CART therapeutic index. Results First, we tested the optimal sequencing of tafasitamab and CART19 in preclinical models. We used a 4-1BB costimulated CART19 construct, similar to FDA-approved tisagenlecleucel. Our data indicate that sequential use of tafasitamab followed by CART19 ameliorated overactivation, prevented apoptosis, and improved antitumor activity of CART19 in xenograft models (Sakemura et al, ASH 2021). Given the reduction of CART19 apoptosis after treatment with tafasitamab, we aimed to determine the effect of tafasitamab on tumor pyroptosis. Here, we measured the level of high mobility group box-1 (HMGB-1) in CD19+ tumor cells after co-culture with CART19 and tafasitamab. CD19 modulation with tafasitamab resulted in a significant reduction of HMGB-1, which indicates reduced tumor pyroptosis. Given these in vitro results, we aimed to study how CD19 occupancy with tafasitamab prior to CART19 infusion impacts tumor pyroptosis in vivo. We utilized a mouse model that mimics CART toxicities. Immunocompromised NSG mice were inoculated via tail vein with 3-5x106 leukemic cells derived from patients with relapsed/refractory acute lymphoblastic leukemia (Sterner et al. Blood 2019, Sakemura et al. Cancer Immunol Res. 2021). Three weeks later, tumor burden was assessed by peripheral blood sampling. Leukemic cells were defined as murine CD45- and human CD45+CD19+CD20+ via flow cytometry, and volumetric absolute number was calculated. Mice were then randomized according to tumor burden to receive IgG control antibody one week before CART19 infusion [Group 1 (G1)], 5 mg/kg/day of tafasitamab one week before CART19 infusion (G2) or no treatment (G3). CRS was determined by weight loss, development of motor weakness, poor body posture, and elevated serum cytokine levels (e.g., IL-6, MCP-1, sCD40L, and GM-CSF). Prior to CART19 infusion, absolute number of CD19 molecules on tumor cells was quantified with antigen-binding capacity beads. G2 showed significant reduction of unbound/detectable CD19 molecules on the leukemic cell surface compared to G1 and G3. On day 0, 3x106 luciferase+CART19 were injected into G1 and G2 via tail vein. Mice were monitored with weekly bioluminescent imaging to track and quantify CART19 expansion and daily weighing and physical assessment to detect toxicity. G1 demonstrated significant weight reduction, motor weakness, and rapid CART19 expansion compared to G2. G1 reached an endpoint within 5 days of CART19 administration due to CRS, while G2 had delayed weight reduction and CART19 expansion. However, all mice from G2 remained healthy and did not display signs of CRS, including motor weakness and hunched posture. On day 5, CRS-related serum cytokines/chemokines were significantly elevated in G1 compared to G2. Overall survival was significantly longer in G2 (Fig 1A). Spleens were harvested, weighed, and assessed with flow cytometry. Spleens from G1 and G3 were significantly enlarged and were mainly composed of CD20+ leukemic cells compared to G2 spleens, which were smaller and infiltrated with CD3+ CART cells but not leukemic cells. Satellite mice were euthanized on day 1 and spleens were harvested. Western blot analysis revealed that mice from G1 showed significantly lower expression of apoptosis-inducing protein Gasdermin E (GSDME) and higher expression of cleaved GSDME on leukemic cells compared with G2, indicating reduced tumor pyroptosis in G2 (Fig 1B). Conclusions In summary, CD19 binding with tafasitamab improved leukemic disease control and decreased the incidence and severity of CRS after CART19 administration in vivo, in part through the reduction of tumor pyroptosis. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

P1440: BTK INHIBITORS IMPROVE CART19 CELL THERAPY BY MODULATING IMMUNE SYSTEM

Background: CD19-targeted chimeric antigen receptor-T (CART19) cell therapy has achieved extraordinary success in B-cell lymphoma and leukemia. However, recurrence and adverse effects, including cytokine release syndrome (CRS) and immune cell-associated neurotoxicity syndrome (ICANS), remain its obstacles. Recurrence, even resistance to CART19 cell therapy, might be attributed to CAR-T cell dysfunction and suppressor cells in the microenvironment. Cytokines required for CRS and ICANS are predominantly derived from monocytes and macrophages. Given that Bruton’s tyrosine kinase (BTK) is expressed on myeloid cells, we considered that BTK inhibitors (BTKi) could improve CAR-T cell therapy by modulating immune system. Aims: To systematically evaluate the effects of three BTKi, ibrutinib (IB), zanubrutinib (ZB) and orelabrutinib (OB), on T cells, CART19 cells and tumor microenvironment. Methods: T and CART19 cells were cultured with the three BTKi for 4 days, and the immune and differentiated phenotypes (PD1, TIM3, CTLA4, CD25, CD69 and CD28, CD45RA and CD62L) were analyzed by flow cytometry (FCM). The effects of BTKi on CART19 cytotoxicity against NALM-6 were assessed for 24-hour coculture. CART19, NALM-6 and macrophages differentiated from THP-1 upon PMA stimulation were cocultured and IL-6 in the supernatants was detected by cytometry bead assays. Lymphoma mouse models were established with BALB/c mice by subcutaneous injection of 5x106 A20 cells. IB, ZB and OB were administered to the BALB/c mice at 25, 10 and 10mg/kg respectively by oral gavage once daily for 28 days. Vehicle treatment was performed using 0.5% carboxymethylcellulose sodium. Mice in healthy controls were fed with no additional treatment. Splenocytes, bone marrow (BM) samples and tumor specimens were harvested, and single-cell suspensions were prepared to analyze immune phenotypes by FCM. Results: Compared with control T cells, IB-supplemented T cells significantly reduced the expression of PD-1 (62.28% vs 14.95%, P = 0.0024), TIM-3 (71.38% vs 7.88%, P <0.0001) and CTLA-4 (92.65% vs 15.5%, P <0.0001) following CD3/CD28 stimulation (Figure 1A). The downregulation of the suppressive markers was also observed in T cells treated with ZB, but not with OB (Figure 1A). In parallel, IB and ZB decreased activation markers expression and increased effector memory cell subsets, while decreased naïve T cells (Figure 1A and B). BTKi decreased IL-6 level in the supernatants of CART19, NALM-6 cells and macrophages (Figure 1C), and decreased CTLA-4 expression on CART19 (Figure 1D). The effects of BTKi on CART19 differentiated phenotypes were not observed (Figure E). Considering strong CD3/CD28 stimulation and tonic signaling could drive terminal differentiation and even apoptosis of CAR-T cells, we supposed BTKi could protect CART19 from excessive activation in long-term cultivation and stimulation (not shown). The three BTKi did not show synergistic effects on cytotoxicity of CART19 against NALM-6 cells (not shown). In lymphoma mice models, the three BTKi all increased the ratio of type 1 macrophages (M1) to type 2 macrophages (M2) and Th1 to Th2 in tumor, and reduced tumor-infiltrating macrophages (Figure 1F-H). BTKi induced significant decreases in exhaustion markers expression on T cells in BM tissues, but not in tumor specimens (Figure I). Image:Summary/Conclusion: Differentiation and exhaustion of CAR-T cells highly activated upon CD3/CD28 stimulation and tonic signaling can be prevented by BTKi. Moreover, BTKi can alter the tumor microenvironment and downregulate IL-6 level to improve the efficacy and safety of CART19 cell therapy.

GM-CSF disruption in CART cells modulates T cell activation and enhances CART cell anti-tumor activity.

Inhibitory myeloid cells and their cytokines play critical roles in limiting chimeric antigen receptor T (CART) cell therapy by contributing to the development of toxicities and resistance following infusion. We have previously shown that neutralization of granulocyte-macrophage colony-stimulating factor (GM-CSF) prevents these toxicities and enhances CART cell functions by inhibiting myeloid cell activation. In this report, we study the direct impact of GM-CSF disruption during the production of CD19-directed CART cells on their effector functions, independent of GM-CSF modulation of myeloid cells. In this study, we show that antigen-specific activation of GM-CSFKO CART19 cells consistently displayed reduced early activation, enhanced proliferation, and improved anti-tumor activity in a xenograft model for relapsed B cell malignancies. Activated CART19 cells significantly upregulate GM-CSF receptors. However, the interaction between GM-CSF and its upregulated receptors on CART cells was not the predominant mechanism of this activation phenotype. GM-CSFKO CART19 cell had reduced BH3 interacting-domain death agonist (Bid), suggesting an interaction between GM-CSF and intrinsic apoptosis pathways. In conclusion, our study demonstrates that CRISPR/Cas9-mediated GM-CSF knockout in CART cells directly ameliorates CART cell early activation and enhances anti-tumor activity in preclinical models.

Optimized Inhibition of GM-CSF in Preclinical Models of Anti-CD19 Chimeric Antigen Receptor T Cell Therapy

Anti-CD19 chimeric antigen receptor T (CART19) cell therapy has resulted in unprecedented outcomes in patients with relapsed/refractory B-cell malignancies, which led to the FDA approval for several indications. However, CART19 cell therapy is limited by the development of severe life-threatening toxicities, as well as by the limited rates of durable response. It has become apparent that myeloid cells contribute to the development of both CART cell toxicity and also to the inhibitory tumor microenvironment. We and others have identified that granulocyte-monocyte-colony-stimulating factor (GM-CSF) depletion results in decreased myeloid activation, reduced toxicities, and enhancement of CART19 cell therapy efficacy in pre-clinical models. Furthermore, we observed that GM-CSF knockout (GM-CSF k/o) in CART19 cells resulted in the improvement of their functions (in vitro and in vivo). These findings suggest that there is also a direct effect of GM-CSF on CART19 cells, which is independent of the GM-CSF impact on myeloid cell activation.To further evaluate this, we first examined GM-CSF receptor alpha (GM-CSFRα) expression by flow cytometry on resting and activated CART19 cells (using FMC63-41BBζ). When CART19 cells were stimulated with either anti-CD3/CD28 beads or lethally irradiated (120 Gy) CD19 + Nalm6 cells (B cell acute lymphoblastic leukemia cancer cell line), GM-CSFRα expression was upregulated upon both T cell receptor (TCR) (data not shown) and CAR stimulation (Figure 1A).Having demonstrated that GM-CSFRα is significantly upregulated on stimulated CART19 cells, we aimed to determine the impact of GM-CSF neutralization (clinical-grade anti-GM-CSF antibody, lenzilumab, 10 µg/mL) versus GM-CSFRα blockade (research-grade antibody, 10 µg/mL) on CART19 cell function and CART cell-monocyte interactions. An IgG isotype antibody was used as a control antibody. Neither the GM-CSF neutralizing antibody, nor GM-CSFRα blocking antibody, had any impact on CART19 cell antigen-specific killing against the CD19 + JeKo-1 cells (mantle cell lymphoma cancer cell line), in the presence or absence of CD14 + monocytes (ratio 1:1:1) isolated by magnetic beads from healthy donors (Figures 1B-C).Next, we compared the effects of GM-CSF neutralization versus GM-CSFRα blockade on CART19 cell antigen-specific proliferation. Here, CART19 cells were co-cultured with lethally irradiated CD19 + cell line JeKo-1 at 1:1 ratio in the presence of 10 µg/mL of the GM-CSF neutralizing antibody, increasing doses of the GM-CSFRα blocking antibody (10-100 µg/mL), or an IgG control. The absolute number of CART cells was measured by flow cytometry on day 5. GM-CSF neutralization did not affect CART19 cell proliferation, but GM-CSFRα blocking antibody significantly inhibited CART19 cell proliferation in a dose-dependent manner. Then, we assessed the effects of GM-CSF neutralizing antibody (20 µg/mL) versus GM-CSFRα blocking antibody (20 µg/mL) on CART19 cell antigen-specific proliferation in the presence of healthy donor monocytes (ratio 1:1:0.5) on day 3. Flow cytometric analysis revealed that GM-CSF neutralization, but not GM-CSFRα blockade, mitigated monocyte-suppression of CART19 antigen-specific proliferation (Figure 1E).In summary, our findings indicate significant differences on CART cell functions and CART cell-monocyte interactions when a specific cytokine, GM-CSF, is neutralized compared to blocking its receptor. Further mechanistic studies are ongoing to assess the functions of GM-CSFRα k/o and GM-CSF k/o CART cells. [Display omitted] DisclosuresSakemura: Humanigen: Patents & Royalties. Parikh: Pharmacyclics, MorphoSys, Janssen, AstraZeneca, TG Therapeutics, Bristol Myers Squibb, Merck, AbbVie, and Ascentage Pharma: Research Funding; Pharmacyclics, AstraZeneca, Genentech, Gilead, GlaxoSmithKline, Verastem Oncology, and AbbVie: Membership on an entity's Board of Directors or advisory committees. Kay: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Research Funding; CytomX Therapeutics: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Research Funding; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; TG Therapeutics: Research Funding; Acerta Pharma: Research Funding; Genentech: Research Funding; Rigel: Membership on an entity's Board of Directors or advisory committees; Behring: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding; Targeted Oncology: Membership on an entity's Board of Directors or advisory committees; Juno Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Agios Pharm: Membership on an entity's Board of Directors or advisory committees. Durrant: Humanigen Inc.: Current Employment. Ahmed: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company. Chappell: Humanigen Inc.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months. Cox: Humanigen: Patents & Royalties. Kenderian: Humanigen, Inc.: Consultancy, Honoraria, Research Funding.

The Impact of Prior Treatment with a CD19 Targeting Monoclonal Antibody on Subsequent Treatment with CD19 Targeting CART Cell Therapy in Preclinical Models

The Impact of Prior Treatment with a CD19 Targeting Monoclonal Antibody on Subsequent Treatment with CD19 Targeting CART Cell Therapy in Preclinical Models

CD20-Specific Chimeric Antigen Receptor-Expressing T Cells As Salvage Therapy in Rituximab-Refractory CD20(+) B-Cell Non-Hodgkin Lymphoma

Background:CD20 antibody-based chemotherapy has been verified as a valid strategy and prolonged the overall survival (OS) of CD20(+) B-cell Non-Hodgkin Lymphoma (B-NHL) patients. However, over 40% of these patients finally would be relapsed or refractory(R/R) to CD20 antibody rituximab. Treatment of these R/R B-NHL patients has not been standardized yet. Furthermore, it is still a question that whether CD20-specific CAR T-cells could provide an alternative therapy in rituximab-relapsed/refractory B-NHL patients.Methods:Here, we conducted a prospective single-center study on patients with relapsed/refractory to CD20 antibody(No.ChiCTR2000036350). The aim of this study aims was to evaluate the efficacy and in vivo persistence of CART-20 cells in subjects with rituximab R/R CD20(+) B-NHL patients. Between October 2017 to December 2020, 15 patients with R/R B-NHL patients (including 14 DLBCL patents and 1 MCL patient) received anti-CD20 specific CAR T cell therapy. The efficacy of CAR T-cells therapy was evaluated at different assessment points by CT scan or 18FDG-PET. CAR-T expansion in blood was monitored regularly by real-time quantitative PCR (qPCR).Results:In this study, the clinical characteristic of these enrolled patients was a median age at 48 years (range 30 to 66), male n=7(47%). 10 (67%) patients were relapsed and 5 patients (33%) were refractory to rituximab-based chemotherapy.(Table 1) All of these patients were at advanced stage III/IV and 7 (47%) of them were diagnosed with bulky disease. The median time from latest rituximab utilization is 70 days (rang 31-471 days) before CAR T-20 cells infusion. The median number of lines of previous treatments was 4 (2-8). 14 patients (93%) received CAR T-20 with a dose of 1*10^7/kg, the median follow-up time for patients was 219 days (32 to 1231). CRS (100%) was observed in all 15 infusion patients: 11 patients grade I-II and 4 patients' grade ≥III. ICANS was recorded only in 1 patient (grade I). At 30 days after the infusion, the clinical response of all 15 infusion patients could be assessed: 4 (27%) CR, 11 (73%) PR, and ORR 100%.(Figure 1) 5 patients who were in PR at 30 days converted to CR, with a median time of 116 (6-858), and continued follow-up without relapse; 6 patients (40%) had relapse, with a median relapse time of 136 days (48- 1050), this might be related to the short time of rituximab before the CAR-T infusion.Although the cases are limited, we can still draw a preliminary conclusion that the use of rituximab within three months may not affect the efficacy of CD20-CAR-T, but may lead to early recurrence. Furthermore, the persistence time of CART-20 cells were comparably longer than that CART-19 cell infusions.Conclusions:In conclusion，these findings suggest that the use of CAR T-20 can be served as a salvage therapy in Rituximab-refractory CD20(+) B-cell Non-Hodgkin Lymphoma and raises the possibility of using CAR T-20 in an early disease stage. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Anti-CD19 Chimeric Antigen Receptor T Cell Treatment of Relapsed /Refractory Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Resistant to Bruton's Tyrosine Kinase (BTK) Inhibitor

CASE PRESENTATION: A 68-year-old male was diagnosed with CLL/SLL in November 2007. Bone marrow asp/bx: 36.5% lymphocytes, 78% CD19, 65% ATM (11q22 deleted) positive cells, 13.5% D13S25 (13q14.3 deleted). On December 10, 2009, the patient took FCR scheme for five cycles, followed by FR scheme for one cycle, and then a month of Chlorambucil. On September 5, 2013, the patient took BR scheme for four cycles with no effect. From March 2015 to Feb 2016, 420 mg of Ibrutinib was administered daily. On January 15, 2016, the patient developed swollen lymph nodes in his right neck with intermittent lumps, fever and nausea. He was admitted into the hospital at Feb 2, 2016. Test results: multiple swollen superficial lymph nodes over the body, with the biggest measuring 60×30mm on the right neck, with no tenderness. Supplementary tests: peripheral white blood cells (WBC) 11.94×10E9/L, lymphocyte 7.5×10E9/L, CD19 cells 6.73×10E9/L, bone marrow lymphocyte 62%, peripheral blood lymphocyte 52%. Immunophenotype: CD5, CD19, CD20dim, CD23, CD11b dim, HLA-DR expression, visible CD5+CD19+ cell clusters, and visible immunoglobulin cKappa with restricted expression. On March 10, 2016, peripheral blood platelet 60 × 10E9/L, CD19 cells 1.94×10E9/L, lactate dehydrogenase 460U/L, FER 115.6ng/ml, hepatitis B virus carrier. Diagnosis: CLL/SLL IV stage, ATM (11q22) deletion, D13S25 (13q14. 3) positive, CD19 positive. Relapse of CLL/SLL occurred again after four months and at this stage the patient was considered for therapy in a clinical trial of CD19-specific chimeric antigen receptor (CAR-) T cell therapy. Ethical approval and informed consent were obtained for anti-CD19 CAR T Cell treatment of ibrutinib resistance in relapsed/refractory CLL/SLL. We infused autologous T cells transduced with a CAR T 19 retroviral vector with CLL/SLL at doses of 3.3 × 10E8 CART19 cells on Mar. 16 2016. Patients were monitored for responses, toxic effects, and the expansion and persistence of circulating CART19 cells. After CART19 cells were infused, the patient experienced chills, fever, headache, weak, anorexia, nausea, shortness of breath, chest tightness, heart palpitation, hypotension and shock for 9 days. The serum levels of IFN-Υ were at their highest at day 7 after CAR T cells infusion. Serum interleukin 6 (IL-6) was at 680pg/ml and CD3+ cells were 97.5%, CD8+ cells 72.8% (18.7-32.8%), FER was 1529.5ng/ml (Normal No. 22-322ng/ml) 14 days after CAR-T cell infusion. The serum levels of IL-6 were at their highest at day14. The patient was diagnosed as having cytokine release syndrome. After the patient took the anti-IL-6R antibody and anti-TNF antibody, he began to recover gradually. Enlarge lymph nodes shrunk after being infused with CART19 cells for 7 days. The peripheral blood CD19 B lymphocytes were 0 on day 14 after infused with CAR T19 cells. Q-PCR was used to detect the amount of the peripheral blood CART19 cells, which stood at 5485 copies/μl, 924 copies/μl, 191 copies/μl respectively 2 weeks, 6 weeks and 3 months after infusing with CART19 cells. The peripheral blood CART 19 cells were not detectable 4 months after infusing with CART19 cells until present. The lymphadenopathy was decreased gradually after 14 days of infusion. The MRI test showed that lymphadenopathy reduced markedly or disappeared after 6 months of infusion. ATM (11q22 deleted) negative, D13S25 (13q14.3 deleted) negative. After treatment with CAR T 19 cell therapy for 53 months, the patient remained disease-free, the patient's lymph nodes, lymphocytes and I mmunoglobulins were normal. CONCLUSIONS : Cancer immunotherapy as a method of cancer treatment is the most effective after conventional treatments such as radiotherapy, chemotherapy, and surgery. For BTK Inhibitor resistance in relapsed and refractory CD19+ CLL/SLL, CD19 is a favorable target, because the expression of CD19 is limited to B cells and not present in other tissues or cells. Currently, the efficacy of this treatment in treating CLL/SLL remains to be seen. The effects of chemotherapy on the patient's B cell lymphoma are negligible, due to the fact that his CLL/SLL have become relapsed and refractory. As a result we chose the CAR T19 cell therapy genetic engineering technique as a method of treatment, to which the patient has responded well. Therefor, CAR T cell technology overcome the limitations of existing cancer therapies and has great potential for development and application. Disclosures No relevant conflicts of interest to declare.

Efficient Gene Editing of CART Cells with CRISPR-Cas12a for Enhanced Antitumor Efficacy

Chimeric antigen receptor T (CART) cells are engineered with an artificial receptor which redirects T cells to recognize cancer cells expressing a particular surface antigen. CART cell therapy has been astonishingly successful at eradicating certain B cell malignancies, but relapse is common, and efficacy is lacking in many cancers. Gene editing of CART cells is being investigated to enhance efficacy and safety and to develop off-the-shelf products. Currently, genome engineering tools used to modify CART cells include zinc finger nucleases, transposons, TALENs, and CRISPR-Cas9. CRISPR-Cas9 uses a trans-activating (tracrRNA): CRISPR RNA (crRNA) duplex to trigger imprecise DNA repair through targeted double stranded breaks, causing indels and often resulting in loss of protein function. Gene-edited CART cells have entered the clinic to provide an allogeneic cell source (TALEN TCRα knockout), safer treatment (CRISPR-Cas9 GM-CSF knockout), and resistance to exhaustion (CRISPR-Cas9 PD-1 knockout). CRISPR-Cas9 PD-1 knockout (PD-1k/o) CART cells were well-tolerated in a first-in-human clinical trial. However, clinically tested CRISPR-Cas9-edited CART cells showed only modest loss of function (~25%) of PD-1 upon infusion. Additionally, off-target editing has been observed in the clinic and remains a concern. We hypothesized that using next-generation CRISPR-Cas12a systems will result in enhanced editing efficiency and precision. CRISPR-Cas12a has a smaller protein component than CRISPR-Cas9, uses a single crRNA without a tracrRNA for simplified delivery and leaves staggered 5' overhangs. These properties, along with lower intrinsic off-target activity than Cas9, render Cas12a a powerful gene editing tool. First, we compared the knockout efficiency of Cas9 and Cas12a in three therapeutically relevant genetic targets in T cells by delivering ribonucleoprotein complexes containing the crRNA and Cas protein of interest. We showed that Cas12a more effectively knocked out CD3, GM-CSF, and PD-1 expression compared to Cas9 (Figure 1A), demonstrating the potential of Cas12a in further genetically editing T cell therapies. We then used electroporation with Cas9 and Cas12a to generate PD-1k/o in lentivirally transduced CD19-targeted CART (CART19) cells with the aim of making exhaustion-resistant CART19 cells through CRISPR gene editing. CART19 and PD-1k/o CART19 cells were repeatedly stimulated with CD19+ NALM6 target cells for one week, and exhaustion marker expression was measured over time with flow cytometry. The expression of CTLA4, TIM3, and LAG3 were similar between CART19 groups, but PD-1 expression was lower in Cas9 PD-1k/o CART19 cells and almost completely eradicated in Cas12a PD-1k/o CART19 cells compared to wildtype or mock shocked CART19 cells (Figure 1B). We then compared the functionality of wildtype, mock shocked, and Cas9 or Cas12a PD-1k/o CART19 cells in vitro to ensure that neither the electroporation process nor PD-1 knockout impaired CART19 cell antitumor activity. Over a range of effector-to-target ratios and with repeated stimulation with target cells, cytotoxicity was comparable across all CART19 cell groups (Figure 1C). All CART19 cell groups demonstrated robust proliferation in response to both nonspecific and antigen-specific stimulation and over one week of repeated antigen stimulation with NALM6 target cells (Figure 1D). We also confirmed that all CART19 cell groups demonstrated strong degranulation and cytokine production in response to nonspecific and antigen-specific stimulation, regardless of electroporation or PD-1 knockout (Figure 1E). In summary, our data demonstrate that Cas12a can be used as a gene editing tool to efficiently knock out therapeutically relevant genes in CART19 cell therapy. Additionally, Cas12a demonstrated improved knockout efficiency over Cas9 in three different genomic targets. PD-1 knockout via Cas9 or Cas12a reduced PD-1 expression on the CART19 cell surface, and PD-1 expression was almost completely ablated with Cas12a gene editing. Electroporation and PD-1 knockout did not impact the effector functions of the CART19 cells, including cytotoxicity, degranulation, cytokine secretion, or proliferation. In vivo studies assessing the antitumor efficacy and CART19 cell persistence are ongoing. Overall, Cas12a is a promising, efficient method of gene knockout to enhance the safety and efficacy of CART cells. Disclosures Sakemura: Humanigen: Patents &amp; Royalties. Cox:Humanigen: Patents &amp; Royalties. Kenderian:MorphoSys: Research Funding; Sunesis: Research Funding; Tolero: Research Funding; BMS: Research Funding; Juno: Research Funding; Gilead: Research Funding; Kite: Research Funding; Novartis: Patents &amp; Royalties, Research Funding; Torque: Consultancy; Humanigen: Consultancy, Patents &amp; Royalties, Research Funding; Mettaforge: Patents &amp; Royalties; Lentigen: Research Funding.

Abstract 2145: Ex vivo phenotyping and potency monitoring of CD19 CAR T cells with a combined flow cytometry and impedance-based real time cell analysis workflow

Abstract Immunotherapy harnesses the immune system to attack cancer cells. While immunotherapy holds tremendous promise for cancer treatment, significant challenges remain in clinical translation. Further development is still required to expand treatment to more cancers and minimize adverse effects. T-cell differentiation and exhaustion influence the establishment and persistence of CAR-expressing T cells. Evaluation of the potency of CAR T cells is essential for effective immunotherapies. Here, we study the potency of CAR-T cells using a combined impendence-based Real-Time Cell Analysis (RTCA) and flow cytometry cell analysis workflow for ex vivo cytolytic potency monitoring of CD19 CAR T cells (CART19). CART19 cell killing of CD19 expressing HEK-293 cells with different E:T (Effector to Target) ratio was monitored by the xCELLigience RTCA system. Concurrently, T cell cytokine production was measured on the NovoCyte Quanteon. This workflow provides a thorough examination of CART cell function, RTCA data demonstrates that CART19 killing is highly potent as well as being antigen-specific and dose-dependent. In conjunction, more cytokine was produced with higher numbers of effector CART19 cells. It was also observed that CART19 cells express stem memory cell (TSCM) markers, expressing activation and exhaustion markers after expansion. Co-culture with HEK-293-CD19 also results in increased expression of CD25, decreased expression of CD127 and upregulation of exhaustion markers, PD-1, TIM-3, and LAG-3. In summary, this study demonstrates the combination of RTCA and flow cytometry cell analysis is a powerful workflow for immunotherapy research and can be employed for QC in CAR T cell manufacturing. Citation Format: Li Zhao, Ming Lei, Peifang Ye, Yan Lu, Xiaoping Ji, Yu Yan, Lauren Jachimowicz, Garret Guenther, Nancy Li. Ex vivo phenotyping and potency monitoring of CD19 CAR T cells with a combined flow cytometry and impedance-based real time cell analysis workflow [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2145.

Circulating Extracellular Vesicles Induce Chimeric Antigen Receptor T Cell Dysfunction in Chronic Lymphocytic Leukemia (CLL)

<h3>Introduction</h3> CD19 directed chimeric antigen receptor T cell (CART19) therapy has resulted in unprecedented clinical outcomes. However, its success in chronic lymphocytic leukemia (CLL) has been modest to date. An increasing body of evidence indicates that impaired CART cell fitness is the predominant mechanism of the relative dysfunction in CLL. The immunosuppressive microenvironment in CLL is well known and may be related to the abundance of circulating extracellular vesicles (EVs) bearing immunomodulatory properties. We hypothesized that CLL derived EVs contribute to CART cell dysfunction. <h3>Objectives</h3> We aimed to investigate the interaction between EVs from CLL patients and CART19 cells. <h3>Methods</h3> We characterized circulating EVs from platelet free plasma in untreated patients with CLL and normal controls using nanoscale flow cytometry. CLL derived EVs, CART19, and CLL B cells were imaged with super resolution microscopy. We stimulated CART19 cells with CD19⁺ JeKo1 cells at a 1:1 ratio in the presence of increasing concentrations of CLL derived EVs and measured inhibitory receptors by flow cytometry. To interrogate the transcriptome, we stimulated CART19 cells with irradiated JeKo1 cells in the presence of CLL derived EVs at ratios of 10:1 and 1:1 EV:CART19. Finally, immunocompromised mice were engrafted with the JeKo1 and randomized to treatment with untreated, CART19 cells, or CART19 cells co-cultured <i>ex vivo</i> with CLL derived EVs for six hours. <h3>Results</h3> Our findings indicate that CLL derived EVs impair normal donor CART19 antigen-specific proliferation (Fig 1A). Although there wasn't a difference in total EV particle count (Fig 1B), CLL derived EVs expressed significantly more PD-L1 than normal controls (Fig 1C). On super resolution microscopy, EVs were localized at the T cell-tumor junction (Fig 1D). Furthermore, CLL derived EVs were captured by T cells (Fig 1E). Having demonstrated this interaction, we sought to establish their functional impact on CART19 cells. There was a significant upregulation of inhibitory receptors on the T cells (Fig 1F), associated with a reduction in CART effector cytokines at higher concentrations of EVs (Fig 1G), suggesting a state of exhaustion. This was further supported by RNA sequencing, which indicated a significant upregulation of AP-1 and YY1 (Figs 1H), known T cell exhaustion pathways. Finally, to confirm the impact of CLL derived EVs on CART19 functions <i>in vivo</i>, treatment with CART19 cultured with EVs resulted in reduced anti-tumor activity compared to treatment with CART19 alone (Fig 1I). <h3>Conclusion</h3> Our results indicate that CLL derived EVs induce significant CART19 cell dysfunction <i>in vitro</i> and <i>in vivo</i>, by direct interaction with CART cells resulting in a downstream alteration of their exhaustion pathways. These studies illuminate a novel way that circulating and potentially systemic EVs can lead to CART cell dysfunction in CLL patients.

Targeting of CD19 By Tafasitamab Does Not Impair CD19 Directed Chimeric Antigen Receptor T Cell Activity in Vitro

Introduction CD19 directed chimeric antigen receptor T cell (CART19) therapy has shown remarkable activity in B cell lymphoma and acute lymphoblastic leukemia (ALL). With the emergence of therapeutic anti CD19 antibodies for the treatment of B cell malignancies, it remains to be elucidated whether such antibodies would interfere with the ability of CART19 to exert their antitumor effect in a subsequent therapy. Tafasitamab is an Fc enhanced humanized anti CD19 monoclonal antibody which mediates antibody dependent cellular toxicity (ADCC), antibody dependent cellular phagocytosis and direct cytotoxicity. It is currently being studied in phase 2/3 clinical trials in diffuse large B cell lymphoma (DLBCL) in combination with lenalidomide and bendamustine. Objectives To investigate the potential for functional interference between tafasitamab and CART19. Methods CART19 cells were generated through lentiviral transduction of healthy donor T cells with a second generation CD19 CAR construct (FMC63-CD8h-CD8TM-41BBζ). In vitro functional activity of tafasitamab was assessed through killing assays against CD19+ cell lines, JEKO (mantel cell lymphoma), Ly7 (DLBCL) and NALM6 (ALL). To study whether the observed CART19 activity may be influenced by tafasitamab in case of a direct CD19 binding competition we incubated NALM6 or JEKO with up to 100 μg/ml tafasitamab, to saturate the receptors, and then performed flow cytometry using FMC63 anti CD19 antibody (it carries the same CD19 binding domain as CAR19). To investigate the potential impact of such binding competition on CART19 cell effector functions, we cocultured tafasitamab with JEKO at increasing concentrations of up to 100 μg/ml, and then added CART19 at different effector to target (E:T) ratios to the cell culture. Results In a 24 hours ADCC (Fig 1A) and T cell cytotoxicity assays (CART19, E:T titrations) distinct activity was observed for both therapies on all tested cell lines. Secondly, after testing the CD19 binding competition, FMC63 antibody failed to detect CD19 expression, indicating a direct binding competition between FMC63 and tafasitamab (Fig 1B). Next, after coculturing tafasitamab and CART19 in JEKO, it was evident that the presence of tafasitamab, binding to the CD19 antigen, did not affect important CART cell effector functions such as antigen specific killing (Fig 1C), degranulation (Fig 1D), cytokine production or proliferation of CART19 (Fig 1E). Conclusions Our studies indicate that CART19 cells continue to exhibit potent antigen specific effector functions despite presence of tafasitamab and the related competition for CD19 binding. The question of therapeutic sequencing of tafasitamab and CART19 is being studied in xenograft models.

Circulating Extracellular Vesicles Induce Chimeric Antigen Receptor T Cell Dysfunction in Chronic Lymphocytic Leukemia (CLL)

Treatment with CD19-directed chimeric antigen receptor T cell (CART19) therapy has resulted in unprecedented clinical outcomes and was FDA-approved in acute lymphoblastic leukemia and non-Hodgkin B-cell lymphoma. However, its success in chronic lymphocytic leukemia (CLL) has been modest to date. An increasing body of evidence indicates that impaired CART cell fitness is the predominant mechanism of the relative dysfunction in CLL. The immunosuppressive microenvironment in CLL is well known and in part may be related to the abundance of circulating extracellular vesicles (EVs) bearing immunomodulatory properties. We hypothesized that CLL-derived EVs contribute to CART cell dysfunction. In this study, we aimed to investigate the interaction between circulating EVs isolated from CLL patient plasma (designated as CLL-derived EVs) and CART19 cells. We enumerated and immunophenotyped circulating EVs from platelet free plasma in untreated patients with CLL. We determined their interaction with CART19 cells using second generation, 41BB co-stimulated, lentiviral transduced CART19 cells generated in the laboratory from normal donors (FMC63-41BBζ CART cells). Our findings indicate that CLL-derived EVs impair normal donor CART19 antigen-specific proliferation against the CD19+ mantle cell lymphoma cell line Jeko-1 (Figure 1A). Next, we characterized CLL-derived EVs using nanoscale flow cytometric analysis of surface proteins and compared to healthy controls. Although the total EV particle count was not different between CLL and healthy controls (Figure 1B), there were significantly higher PD-L1+ EVs in patients with CLL (Figure 1C). Based on these results, we sought to assess the physical interaction between CLL-derived EVs and CART cells from normal individuals. When CLL-derived EVs were co-cultured with CART19 and CLL B cells and imaged with super-resolution microscopy, EVs were localized at the T cell-tumor junction (Figure 1D). Furthermore, CLL-derived EVs are captured by T cells as indicated by a significant reduction in the absolute count of EVs when co-cultured with resting T cells (Figure 1E). Having demonstrated that 1) there is an excess of PD-L1+ EVs in patients with CLL (Figure 1C) and 2) CLL-derived EVs physically interact with CART cells (Figures 1D-E), we sought to establish their functional impact on CART19 cells. Here, CART19 cells were stimulated with irradiated CD19+ JeKo-1 cells at a 1:1 ratio in the presence of increasing concentrations of CLL-derived EVs. There was a significant upregulation of inhibitory receptors such as PD-1 and CTLA-4 on the T cells (Figure 1F). This is associated with a reduction in CART effector cytokines (i.e., TNFβ) at higher concentrations of EVs (Figure 1G), suggesting a state of exhaustion in activated CART19 cells in the presence of CLL-derived EVs. This was further supported by transcriptome interrogation of CART19 cells. Here, CART19 cells were stimulated via 24-hour co-culture with the irradiated CD19+ cell line JeKo-1, in the presence of CLL-derived EVs at ratios of 10:1 and 1:1 EV:CART19 and then isolated by magnetic sorting. RNA sequencing of these activated CART19 cells indicated a significant upregulation of AP-1 (FOS-JUN) and YY1 (Figures 1H), known critical pathways in inducing T cell exhaustion. Finally, to confirm the impact of CLL-derived EVs on CART19 functions in vivo, we used our xenograft model for relapsed mantle cell lymphoma. Here, immunocompromised NOD-SCID-ɣ-/- mice were engrafted with the CD19+ luciferase+ cell line JeKo-1 (1x106 cells I.V. via tail vein injection). Engraftment was confirmed through bioluminescent imaging and mice were randomized to treatment with 1) untreated, 2) CART19 cells, or 3) CART19 cells co-cultured ex vivo with CLL-derived EVs for six hours prior to injection. A single low dose of CAR19 (2.5x105) was injected, to induce relapse. Treatment with CART19 cells that were co-cultured ex vivo with CLL-derived EVs resulted in reduced anti-tumor activity compared to treatment with CART19 alone (Figure 1I). Our results indicate that CLL-derived EVs induce significant CART19 cell dysfunction in vitro and in vivo, through a direct interaction with CART cells resulting in a downstream alteration of their exhaustion pathways. These studies illuminate a novel way through which circulating and potentially systemic EVs can lead to CART cell dysfunction in CLL patients. Disclosures Cox: Humanigen: Patents &amp; Royalties. Sakemura:Humanigen: Patents &amp; Royalties. Parikh:Ascentage Pharma: Research Funding; Janssen: Research Funding; AstraZeneca: Honoraria, Research Funding; Genentech: Honoraria; Pharmacyclics: Honoraria, Research Funding; MorphoSys: Research Funding; AbbVie: Honoraria, Research Funding; Acerta Pharma: Research Funding. Kay:Agios: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; MorphoSys: Other: Data Safety Monitoring Board. Kenderian:Humanigen: Other: Scientific advisory board , Patents &amp; Royalties, Research Funding; Lentigen: Research Funding; Novartis: Patents &amp; Royalties, Research Funding; Tolero: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.

Targeting of CD19 By Tafasitamab Does Not Impair CD19 Directed Chimeric Antigen Receptor T Cell Activity in Vitro

CD19 directed chimeric antigen receptor T cell (CART) therapy has shown remarkable activity in B cell lymphoma and acute lymphoblastic leukemia leading to the approval of two CART therapies. With the emergence of therapeutic anti-CD19 antibodies for the treatment of B cell malignancies, it remains to be elucidated whether such antibodies would interfere with the ability of CD19 targeting CARTs to exert their anti-tumor effect in a subsequent therapy. To address a part of this question, we investigated the potential for functional interference between the monoclonal anti-CD19 antibody tafasitamab (MOR208) and CD19 directed CART cells (CART19). CART19 cells were generated through lentiviral transduction of healthy donor T cells with a second generation CD19 CAR construct (FMC63-CD8h-CD8TM-41BBζ) which is similar to the construct used for the FDA-approved CART tisagenlecleucel. Tafasitamab, is an Fc-enhanced humanized monoclonal antibody which mediates antibody-dependent cellular toxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and direct cytotoxicity. It is currently being studied in phase 2 and 3 clinical trials in diffuse large B-cell lymphoma (DLBCL) in combination with the immunomodulatory agent lenalidomide (L-MIND) and the chemotherapeutic drug bendamustine (B-MIND). As a first step we confirmed the relevance of the tested CD19-positive target cell lines, JEKO (mantel cell lymphoma), Ly7 (DLBCL) and NALM-6 (ALL) based on functional activity of tafasitamab and CART19. In a 24 hours ADCC (tafasitamab titration plus natural killer (NK) cells; Figure 1A) and T cell cytotoxicity assays (CART19, E:T titrations; data not shown) distinct activity was observed for both therapies on all tested cell lines. Secondly, we studied whether the observed CART19 activity may be influenced by tafasitamab in case of a direct CD19 binding competition between tafasitamab and the CAR. To test for such binding competition we incubated the CD19+ cell lines NALM6 or JEKO with up to 100 µg/ml tafasitamab, to saturate the receptors. Subsequent flow cytometry analysis using the FMC63 antibody (carrying the same CD19 binding domain as contained in CART19) failed to detect CD19 expression, indicating a direct binding competition between FMC63 and tafasitamab (Figure 1B). Next, to investigate the potential impact of such binding competition on CART19 cell effector functions, we co-cultured tafasitamab CD19+ JEKO cell line at increasing concentrations of up to 100µg/ml, and then added CART19 cells at different effector to target ratios to the cell culture. The presence of tafasitamab, binding to the CD19 antigen, did not affect important CART cell effector functions such as antigen specific killing (Figure 1C), degranulation (Figure 1D), cytokine production or proliferation of CART19 (Figure 1E). In summary, our studies indicate that CART19 continue to exhibit potent antigen specific effector functions despite presence of tafasitamab and the related competition for CD19 binding. Besides the presented in vitro work the questions of therapeutic sequencing of tafasitamab and CART19 is being studied in xenograft models and will be presented at the meeting. Disclosures Sakemura: Humanigen: Patents &amp; Royalties. Cox:Humanigen: Patents &amp; Royalties. Schanzer:MorphoSys AG: Employment. Endell:MorphoSys AG: Employment, Patents &amp; Royalties. Nowakowski:Selvita: Membership on an entity's Board of Directors or advisory committees; NanoString: Research Funding; MorphoSys: Consultancy, Research Funding; Genentech, Inc.: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Curis: Research Funding; Bayer: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Kay:MorphoSys: Other: Data Safety Monitoring Board; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; Agios: Other: DSMB. Kenderian:Novartis: Patents &amp; Royalties, Research Funding; Tolero: Research Funding; Humanigen: Other: Scientific advisory board , Patents &amp; Royalties, Research Funding; Lentigen: Research Funding; Morphosys: Research Funding; Kite/Gilead: Research Funding.

Identification of Small Molecule Kinase Inhibitors That Potently and Reversibly Block Chimeric Antigen Receptor T Cell Proliferation and Cytotoxicity

Introduction: Despite remarkable clinical efficacy, CAR-T therapy has been limited by life threatening toxicities in over 30% of patients.1, 2 Toxicities primarily manifest as cytokine release syndrome (CRS) characterized by an early phase with fever, hypotension and elevations of cytokines including IFNγ, GM-CSF, TNF, IL-10, and IL-6 and a later phase associated with life-threatening or life-ending neurologic events. We hypothesized that reversible inhibition of CAR mediated signaling will enable controllable regulation of CAR-T cell activity in vivo and mitigate CAR-T mediated toxicities. There are specific protein kinases such as the SRC kinases, LCK, and ZAP-70 that are known to be involved in various cellular signaling pathways, especially T cell receptor mediated signaling and may also be appropriate targets for modulating (both enhancing and inhibiting) CAR-T function in a rapid and reversible fashion in vivo. Our hypothesis is that small molecule inhibitors of TCR signaling and downstream pathways could be identified using specific high throughput screens. Methods: To identify novel inhibitors of CAR-T cell proliferation, we developed a high throughput kinase inhibitor screen to identify compounds that reversibly inhibit CAR-T function. T cells containing a third generation CAR targeting CD19 cells (CAR19) and CD19+ tumor cells (Ramos cells expressing both GFP and luciferase) were incubated at an effector to target ratio of 1:1 in 96 well plates in the presence of 1µM of each inhibitor. After 24 hours, tumor cell death induced by CAR-T was measured using bioluminescence (BLI) imaging. Small molecules that inhibited CAR-T proliferation and cytotoxicity were determined by assessing the BLI signal in each well. Results: A protein kinase inhibitor library (Selleckchem, Texas) containing 644 independent compounds was tested (Figure 1). Of the 644 kinase inhibitors tested, 32 were found to be potent inhibitors of CART19 cell activation and cytotoxic killing of CD19+ target tumor cells, reducing anti-tumor viability in 24 hours by &gt;50% compared to vehicle control. Compounds such as Nintedanib (C3), Dasatinib (C9), and Saracatinib (C12), all SRC kinase inhibitors, were able to inhibit cell killing by 99%, 84%, and 76% respectively. Next we assessed the reversibility of CAR-T cell mediated killing upon removal of inhibitors from the cultures. Reinitiation of potent, anti-tumor activity was observed within 24 hours after inhibitor removal, confirming reversible nature of CAR-T cell inhibition by the three most potent compounds. Conclusions: Recent publications (Weber et al Blood Adv, 2018, Westermann et al. Sci Transl Med, 2019) have also shown that dasatinib can reversibly suppresses CAR-T cell cytotoxicity, cytokine secretion, and proliferation in vitro and in vivo. 3, 4 Here we confirm the reports of others regarding dasatinib and that show for the first time that reversible inhibition of CAR-T activity by kinase inhibitors is not limited solely to dasatanib, but is observed with other small molecules targeting many different kinases. This work further demonstrates the potential applications of tyrosine kinase inhibitors as a safety switch to modulate CAR-T cell toxicity. 1. Maude, NEJM 2014 2. Davila, SciTransMed 2014 3. Mestermann SciTransMed 2019 4. Weber. Blood Adv 2019 Disclosures Cooper: Wugen: Consultancy, Equity Ownership, Patents &amp; Royalties. DiPersio:WUGEN: Equity Ownership, Patents &amp; Royalties, Research Funding; Magenta Therapeutics: Equity Ownership; Celgene: Consultancy; Karyopharm Therapeutics: Consultancy; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding; Amphivena Therapeutics: Consultancy, Research Funding.

YIA19-003: Size Matters: Larger Size CD19-Positive Extracellular Vesicles in Chronic Lymphocytic Leukemia (CLL) Inhibit Chimeric Antigen Receptor T-Cell Function

Background: Unprecedented clinical outcomes were reported after CD19 chimeric antigen receptor T-cell (CART19) therapy. However, the complete response rate in chronic lymphocytic leukemia (CLL) is much lower, at approximately 20%–30%. Several immune defects have been identified in CLL that result from the complex interaction between CLL cells and the microenvironment. This leukemic microenvironment is rich with extracellular vesicles (EVs) secreted by B-CLL cells. Here, we aimed to investigate the role of EVs play in the diminished CART response seen in some CLL patients. Methods: EVs were isolated from peripheral blood of 16 patients with untreated CLL at different Rai stages and risk profile by FISH. Cytometry was used to determine size, number of particles per μl, and CD19 expression. To investigate the impact of EVs on CAR T-cell functions, CART19 cells were stimulated with either CLL B cells or the CD19-positive cell line JEKO and different effector functions were analyzed. Results: Two patterns of EVs in CLL patients were identified; a single versus 2 distinct EV populations, characterized by size (small [EVssmall] and large [EVslarge], Fig 1A). In 25% of patients, EVs were CD19 positive (EVCD19+). CD19 positivity was detected only in patients with the EVslarge (Fig 1B). The EVs concentration, CD19 expression (EVsCD19+ vs EVsCD19-), or the size (EVssmall vs EVslarge) did not correlate with disease stage (early vs advanced Rai stage) or risk profile of CLL (low vs high risk). To investigate our hypothesis that EVs could modulate CART19 function, CART19 cell effector functions were examined in the presence of EVsCD19+, EVsCD19-, EVssmall, or EVslarge. EVs alone were insufficient to stimulate CART19 cells. However when CART19 cells were stimulated with the CD19-positive cell line JEKO, their effector functions were reduced only in the presence of EVsCD19+ but not EVsCD19-. This included a significant reduction in CART-specific killing (Fig 1C) and a reduction in cytokine production. The impairment of CART cell functions was independent of the size of EVs, ie, there was no impairment of CART functions with large or small size EVCD19- in co-culture. Conclusion: We identify CD19-positive large size EVs from patients with CLL and demonstrate that these EVs play a role in the leukemic microenvironment by reducing CAR T-cell activity. Studies are ongoing to define the mechanism(s).