Abstract: CD19 CAR T cells have revolutionized the treatment of relapsed and refractory (R/R) large B cell lymphomas (LBCL), mediating durable complete responses in approximately 40-50% of patients. Besides a loss or decrease in CD19 expression, no studies have identified tumor specific factors driving inherent or acquired resistance to CAR T cells in LBCL. Mutations in and loss of expression of LFA-3 (CD58) have been described in approximately 20% of cases of LBCL. As the ligand for CD2 on T cells, CD58 provides costimulation to T cells and CD58 loss or mutation has been linked to immune resistance in LBCL. We evaluated CD58 status in fifty-one R/R LBCL patients treated at Stanford with commercial axicabtagene ciloleucel (axi-cel) through immunohistochemistry (IHC) on tumor biopsy samples and/or deep sequencing of circulating tumor DNA by CAPP-Seq. We identified 12/51 (24%) patients with a CD58 aberration (lack of expression by IHC or mutation by CAPP-Seq). Progression-free survival (PFS) was significantly decreased in patients with a CD58 aberration (median PFS for CD58 aberration 3 months vs. not reached for CD58 intact, p & lt;0.0001). In fact, only 1/12 patients with a CD58 alteration achieved a durable, complete response to axi-cel, while the remaining 11 patients progressed, most commonly after a period of initial response. Partial responses were more common among patients with CD58 aberrations (58% for CD58 aberration vs 10% for CD58 intact, p & lt;0.001), and complete responses were less common (25% for CD58 aberration vs 82% for CD58 intact, p & lt;0.0001). To probe the biology of CAR T cell responses towards tumors lacking functional CD58, we generated a CD58 knockout Nalm6 model. CD19.CD28.ζ, CD19.4-1BB.ζ, and CD22.4-1BB.ζ CAR T cells demonstrated significantly reduced cytokine production and cytolytic activity in response to CD58 KO vs wildtype (WT) tumor cells. Additionally, while mice inoculated with WT Nalm6 and treated with any of the three CARs demonstrate complete responses and prolonged leukemia-free survival, mice inoculated with CD58KO Nalm6 demonstrated only partial responses, eventual tumor progression, and death from leukemia. CD2, the T cell ligand for CD58, plays both an adhesive role and a costimulatory role in T cells. CD2 knockout resulted in significantly reduced cytokine production after CAR stimulation. Re-expression of only the CD2 extracellular domain did not rescue CAR function, indicating that CD2 signaling is essential for full CAR activation. Additionally, when we stimulated CD19 CAR T cells with anti-idiotype antibody (CAR stimulation), soluble CD58 (CD2 stimulation), or both, we observed significantly enhanced phosphorylation of both CD3ζ and ERK by western blot in CAR T cells stimulated through both the CAR and CD2. Phosphorylation analysis by mass spectrometry revealed that CD2 stimulation enhances phosphorylation of proximal signaling molecules in the TCR pathway (LCK, LAT, CD3ε among others) and also mediators of actin-cytoskeletal rearrangement in CAR T cells, consistent with effects in natural T cell responses. To overcome CD58 loss in LBCL, we generated second- and third-generation CAR T cell constructs integrating CD2 costimulatory domains within the CAR molecule. While these cis constructs demonstrated increased potency against CD58KO cells in vitro, they were unable to ultimately overcome CD58 loss in vivo. However, when CARs were co-expressed with an additional CD2 receptor in trans, they mediated significant anti-tumor activity in vivo, overcoming CD58 knockout in tumor cells. In conclusion, we have identified that CD58 status is an important biomarker for durable response to CAR T cells in LBCL. We modeled the biologic basis for this finding and generated CAR T cells capable of overcoming CD58 loss in B cell malignancies. CD58 mutations have been reported in many cancers, including multiple myeloma and colon cancer, and are likely to play a role in immune evasion for CAR T cells as they are developed for additional histologies. These data provide rationale for investigating CD58 status for patients receiving CAR based therapeutics and devising next generation CARs capable of overcoming this newly discovered mechanism of resistance. Disclosures Majzner: Xyphos Biopharma: Consultancy; Zai Lab: Consultancy; Lyell Immunopharma: Consultancy; GammaDelta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aprotum Group: Consultancy; Illumina Radiopharmaceuticals: Consultancy. Kurtz:Roche: Consultancy; Genentech: Consultancy; Foresight Diagnostics: Other: Ownership. Sotillo:Lyell Immunopharma: Consultancy, Other: Consultancy. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding. Miklos:Miltenyi Biotec: Research Funding; Janssen: Consultancy, Other: Travel support; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Apricity Health: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy; Lyell Immunopharma: Consultancy, Current equity holder in private company.

Abstract: The prognosis of patients with large B-cell lymphoma (LBCL) that progresses after treatment with chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CAR19) is poor. We report on the first 3 consecutive patients with autologous CAR19-refractory LBCL who were treated with a single infusion of autologous 1 × 106 CAR+ T cells per kilogram targeting CD22 (CAR22) as part of a phase 1 dose-escalation study. CAR22 therapy was relatively well tolerated, without any observed nonhematologic adverse events higher than grade 2. After infusion, all 3 patients achieved complete remission, with all responses continuing at the time of last follow-up (mean, 7.8 months; range, 6-9.3). Circulating CAR22 cells demonstrated robust expansion (peak range, 85.4-350 cells per microliter), and persisted beyond 3 months in all patients with continued radiographic responses and corresponding decreases in circulating tumor DNA beyond 6 months after infusion. Further accrual at a higher dose level in this phase 1 dose-escalation study is ongoing and will explore the role of this therapy in patients in whom prior CAR T-cell therapies have failed. This trial is registered on clinicaltrials.gov as #NCT04088890.

Abstract: Introduction: Autologous anti-CD19 chimeric antigen receptor T cells (CAR19) have shown dramatic clinical responses in relapsed-refractory large B cell lymphomas, but more than 50% of patients will have disease progression. Here we characterize the observed mechanisms of treatment failure following Axicabtagene Ciloleucel (Axi-cel) therapy. Methods: Sixty-nine patients with refractory large B cell lymphoma were referred for CAR19 therapy from October 2017 to June 2018. The WHO diagnosis and B cell antigen expression on lymphoma cells were assessed by immunohistochemistry and/or flow cytometry before and at the time of progression. We assessed peripheral blood CAR-T cell numbers at Days 7, 14, 21, and 28 by flow cytometry immunophenotyping and monitored disease response with PET-CT at Day 28, 3 months, and 6 months post-infusion. Results: Twenty-two patients who received CAR19 therapy, including patients with transformed large B cell lymphoma (N =5), diffuse large B cell lymphoma, not otherwise specified (N =11), high grade B cell lymphoma, not otherwise specified (N = 2), primary mediastinal large B cell lymphoma (N = 2), and high grade B cell lymphoma with rearrangement of MYC and BCL2 or BCL6 (N =2). The Day 28 ORR was 86%: 10 patients had complete response, 9 had a partial response, 1 had stable disease, and 2 had progressive disease. There was no statistically significant difference in age, gender, underlying disease, or number of prior treatment regimens between patients who achieved a clinical response versus those who failed therapy. Both patients (2 of 2) with progressive disease at Day 28 showed dim or partial CD19 expression prior to CAR-T infusion but nonetheless demonstrated robust Axi-cel expansion. In contrast, one patient with Day 28 stable disease showed no CAR-T cell expansion despite intact CD19 expression. Overall, there was no statistical difference in relative or absolute CAR+ T cells in patients who responded versus those who did not at Day 28 (Figure 1). Day 90 response was available for 12 patients with either CR or PR at Day 28. Five patients (26%) developed progressive disease, and 4 of 5 underwent repeat biopsy. Of these patients, 2 had complete loss of tumor CD19 (Figure 2) and another had downregulation of CD19 with variable expression of other B cell antigens. Conclusion: Eight of 22 (36%) of patients who underwent CAR19 infusion did not respond or relapsed after Day 28 response. Five patients (62%) who failed therapy had loss or downregulation of CD19, which emphasizes that single target antigen loss is a common mechanism of CAR-T failure. However, lack of CAR-T cell expansion was noted in multiple patients, suggesting that there may be T cell intrinsic causes of treatment failure. Further studies are necessary to help identify and predict which patients will benefit from targeted immunotherapy. Disclosures No relevant conflicts of interest to declare.

Abstract: Background: Axicabtagene ciloleucel (axi-cel), an autologous anti-CD19 chimeric antigen receptor (CAR-T), showed significant clinical responses in patients with relapsed-refractory large-B cell lymphomas in the Zuma-1 trial (Neelapu et al, NEJM 2017). Zuma-1 analysis showed blood CAR-T cell expansion was associated with clinical response and toxicity. Herein, we report on 25 patients treated with commercial axi-cel and describe CAR-T expansion by immunophenotyping and its correlation with clinical outcomes. Methods: Twenty-five patients with aggressive lymphoma consecutively apheresed at Stanford University prior to June 30, 2018 were studied on an IRB approved biorepository-clinical outcome protocol. Cytokine release syndrome (CRS) was graded by Lee criteria (Blood 2014) and neurotoxicity according to Neelapu et. al (Nat. Rev. Clin. Onc. 2017). CAR-T cell immunophenotyping was assessed by peripheral blood flow cytometry on days 7, 14, 21 and 28 and then monthly. CAR-T cells were identified by gating on singlet+, live+, CD45+, CD14-, CD3+, anti-CD19-specific CAR mAb (clone 136.20.1; Jena et. al Plos 2013) and characterized as either CD4+ or CD8+. Results: Of 25 apheresed patients, 3 patients died prior to axi-cel infusion due to progressive lymphoma. Of 22 infused patients, 14 (64%) would have been eligible for the Zuma-1 trial. Reasons for ineligibility included symptomatic DVT (n=2), renal insufficiency (n=1), transaminitis (n=1), thrombocytopenia (n=1), MDS (n=1), pleural effusion (n=1) and 1 was ineligible by multiple criteria. Median time from initial clinic visit to infusion was 47 days (range 34-117); median time from apheresis to infusion was 22 days (range 19-38). Nine patients received bridging therapy prior to lymphodepletion chemotherapy (chemo = 4, radiation = 2, high dose dexamethasone = 3). Axi-cel infusion occurred in hospital and patients were followed expectantly for a minimum of 7 days or until adverse events resolved to 〈 Grade 2; median hospitalization was 13.5 days (range 7-44). Ninety-five percent of patients developed CRS (Grade 2 = 73%, none ≥Grade 3). Median number of tocilizumab doses was 1 (range 0-4). Neurotoxicity occurred in 64%, Grade 3 or 4 in 27%. Corticosteroid therapy was required in 82% (77% received both tocilizumab and steroids). Median duration of steroids was 8.5 days (range 1-30); 12 patients required at least 1 week and 4 patients ≥2 weeks. Of patients infused, complete response (CR) at day 28 was 45% (ORR 86%). Of 15 patients evaluable at 3 months, ORR was 53% (CR = 7, PR = 1) and 47% progressed, similar to Zuma-1. Ineligibility for Zuma-1 was not associated with inferior outcomes. Overall, median day 7 peak in vivo axi-cel expansion using anti-CAR19 flow cytometry was 38 CAR-T/ul (Fig. 1A), matching RT-PCR measured levels reported in Zuma-1. As shown in Fig. 1A, the majority of CAR-T cells were CD8+. Patients with Grade 2 CRS had significantly higher peak expansion of CAR-T cells (both CD4+ and CD8+) as compared to those with either Grade 0 or 1 CRS (Fig. 1B). Grades 2-4 neurotoxicity were significantly associated with peak total and CD8+ CAR-T but not CD4+ (Fig. 1B). Illustratively, 2 patients with the most robust CAR-T expansion (▪, ▼ Fig. 1A) experienced Grade 4 neurotoxicity including status epilepticus requiring multiple anti-epileptics and intubation. Peak CAR-T expansion in blood did not correlate with CR or ORR at day 28; expansion did not differ between patients who did or did not require steroids. Fine needle aspirates (FNA) on a subset of patients with FDG-avid lymph nodes 2-3 days post axi-cel showed significant CAR-T expansion within the node despite low detectable circulating CAR-T. Figure 1C depicts a 76-year-old male with double expressor DLBCL who attained a CR at day 28; day 14 blood CAR-T expansion was below average (6 CAR-T/ul), while his day 2 FNA showed 〉 35% of CD3+ T-cells expressed CAR19. As of submission, 34 patients were apheresed and updated blood and FNA results will be presented. Conclusion: Our analysis of 22 infused axi-cel patients showed an ORR of 86% and CR of 45%, despite 36% Zuma-1 ineligibilities and steroid use in 82%. Blood CAR-T expansion was associated with both CRS and neurotoxicity but not clinical response. Detection of high concentration of CAR-T cells in affected lymph nodes 2 days post infusion suggests quantification of CAR-T cells at disease sites could be predictive of clinical responses. J.Y.S and B.S are co-first authors Disclosures Latchford: Kite a Gilead Company: Speakers Bureau. Muffly:Adaptive Biotechnologies: Research Funding; Shire Pharmaceuticals: Research Funding. Miklos:Kite - Gilead: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Genentech: Research Funding; Novartis: Consultancy, Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding.

Abstract: Autologous CD19 directed CAR T-cell therapy has response rates of 〉 70% in adult acute lymphoblastic leukemia (ALL) and 〉 40% in adult diffuse large B cell lymphoma (DLBCL). Large trials (ZUMA-1/JULIET/TRANSCEND) have highlighted that many patients fail to achieve durable responses. Several groups have reported on the phenomenon of CD19 immune escape as a cause (Grupp et al, NEJM 2013, Neelapu et al, NEJM 2017) and the NIH Pediatric Oncology Branch has shown CD22 as an alternative target (Fry et al, Nat Med. 2018). We developed a bi-specific CAR construct targeting CD19 & CD22 with intracellular signaling domains incorporating 4-1BB and CD3ζ (CD19/CD22.BB.z) to overcome CD19 immune escape. Here, we present our Phase I experience with this bi-specific CAR in adults. This is a single institution phase I dose escalation study enrolling patients Age ≥ 18 years with relapsed/refractory B-ALL or DLBCL after standard therapies. Primary aim is to determine feasibility of manufacturing the bi-specific CAR and safety at three dose levels (1 x 106 CAR T cells/kg, 3 x 106 CAR T cells/kg, 1 x 107 CAR T cells/kg). Clinical response was evaluated as a secondary endpoint utilizing standard response criteria for ALL and DLBCL. All patients underwent lymphodepletion with cyclophosphamide (500mg/m2 daily x3 doses) and fludarabine (30mg/m2 daily x 3 doses) followed by CAR infusion two days later. Patients were assessed at pre-defined time-points (Day 28, Month 3, 6, 9, 12 then every 6-12 months) with correlative assessments including immunophenotyping, single cell RNAseq, CAR qtPCR, serum and single cell cytokine analysis. Seven adult patients (5 DLBCL, 2 ALL), aged 35 - 75 years have been enrolled and 6 treated, all at dose Level 1 [Table 1]. The first 3 patients received freshly harvested cells and the remaining received cryopreserved cells (1 patient treated twice received initial fresh then cryopreserved product). None received systemic bridging therapy before CAR T infusion. Six patients developed reversible cytokine release syndrome (CRS,4 with Grade 1, 2 with Grade 2), onset between Day 1 to 13, and 2 patients received tocilizumab & systemic steroids. Three patients developed neurotoxicity (1 with grade 2, Day 8-11 and the others grade 1) with grade 2 neurotoxicity managed with steroids. Four patients required growth factor support beyond Day 28 and all treated patients show persistent B-cell aplasia. Two patients achieved CR: an ALL patient with disease in bone marrow/blood/CNS was MRD negative at day 28 & 60; a 75yo DLBCL patient achieved PR at day 28 and CR at month 3. Three others have ongoing PR and one died of progressive disease after initial PR at Day 28. A patient with PD at Day 28 subsequently treated with radiation and 2-months of revlimid/rituximab, now has no detectable disease 6 months post CAR-T. One patient with initial 6-month PR received a second infusion due to PD, did not develop CRS or CRES with 2nd infusion and has SD at Day 28 Notably, the patient experienced a lack of CAR-T expansion with the second infusion, raising the possibility of an immunogenic response to the CAR-T cell infusion. Flow analysis of all patients' peripheral blood showed CAR expansion peaked at median Day 13 (range Day 10-20) and CARs remained detectable [Figure 1]. Multi-parametric CyTOF phenotyping of the CAR19-22 products showed significant numbers of transduced CAR-T memory stem cells (phenotype: CD3+CD8+CD45RA+CD127+CD27+CCR7+). Single cell cytokine secretion analysis (Isoplexis,Rossi et al Blood 2018) revealed high polyfunctional strength index (PSI) in both CD4+ and CD8+ cell subsets in each patient's pre-infusion CAR product that reflected phenotypic expansion in patients. Additional correlative studies, including cytokine analysis, qtPCR based CAR quantification and CyTOF phenotypic analysis of t he CAR-T cells will be presented. This first adult phase I trial of bi-specific CAR targeting CD19 & CD22 shows low toxicity with promising efficacy including achievement of CR in adult DLBCL and ALL patients. We have escalated dose to 3x 106 CAR T cells/kg and an expansion study of 60 patients will follow. CAR-T cells expanded within the first 20 days and continue to be detectable through 6 months. Disclosures Muffly: Shire Pharmaceuticals: Research Funding; Adaptive Biotechnologies: Research Funding. Miklos:Janssen: Consultancy, Research Funding; Genentech: Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Kite - Gilead: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Abstract: BACKGROUND: Patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL) after failure of CD19-directed CAR T-cell therapy (CAR19) have a dire prognosis, with an overall response rate (ORR) of 29% to conventional salvage therapies, and a median overall survival (OS) of 6 months. CD22 is expressed on the majority of B-cell malignancies. Autologous CAR T-cells targeting CD22 (CAR22) have yielded an ORR of 70-90% in pediatric patients with R/R B-cell acute lymphoblastic leukemia (B-ALL), including those who had previously failed CAR19 therapy. Based on these encouraging results, we evaluated CAR22 in adult patients with R/R LBCL, focusing on those with CAR19-refractory disease. METHODS: This ongoing single-institution phase I dose escalation clinical trial (NCT04088890) is evaluating a CAR construct incorporating the m971 CD22 single chain variable fragments and 41BB/CD3z endodomains integrated within autologous T-cells via lentiviral transduction. After lymphodepletion (LD) with fludarabine and cyclophosphamide, patients are infused with cryopreserved CAR T-cells after a 7- to 11-day closed manufacturing process utilizing the CliniMACS Prodigy device (Miltenyi). Primary objectives assess the ability to successfully manufacture CAR22 and safety. Secondary objectives include efficacy and durability of responses. RESULTS: Twenty-one patients with LBCL [n=12 at dose level 1 (DL1), 1x10 6 CAR+ cells/kg; n=9 at dose level 2 (DL2), 3x10 6 CAR+ cells/kg] have been enrolled with a median age of 64 years (range, 36-79) and a median of 4 (range, 3-8) prior lines of therapy. All patients had at least one high risk feature, including failure of prior CAR19 therapy (n=20); refractory disease to second-line or later therapy (n=17); elevated lactate dehydrogenase (LDH) pre-LD (n=17); high tumor burden (n=9); a history of primary refractory disease (n=7); failure of prior autologous hematopoietic stem cell transplantation (HSCT) (n=6); never achieving CR to any therapy (n=5); or LBCL with MYC gene rearrangements (n=5). Successful manufacturing of cells was achieved in all patients. All patients reached day 28 post-infusion and are included in the safety and efficacy analysis presented here; updated results will be presented at the meeting. Every patient experienced cytokine release syndrome (CRS); 20/21 (95%) were Grade 1-2, 1/21 (5%) were Grade 3. Four patients (19%) experienced immune effector cell-associated neurotoxicity syndrome (ICANS); all cases were Grade 1-2 and resolved within 2 days. Five patients (24%) experienced a hyperinflammatory macrophage activation syndrome (MAS), manifested in all cases by pancytopenia and consumptive coagulopathy (DIC) requiring transfusion and/or growth factor support. One patient who received DL2 had a Grade 5 infectious event in the setting of ongoing MAS and pancytopenia. Relative to DL1, higher prevalence of Grade ≥3 cytopenias beyond D28 (89% vs. 50%) and MAS (33% vs. 17%) were observed at DL2; thus, DL1 was selected as the maximally tolerated dose (MTD). ORR at D28 was 86% (CR, n=11; PR, n=7), and was similar between DL1 and DL2 (92% vs. 78%; p=ns). 3/7 (43%) initial PR improved to CR at a median of 3 months post-infusion. All 14 patients (67% of cohort) who achieved CR remain in remission, with a mean follow-up of 7.3 months (range, 1.2-21.3); median progression free survival (PFS) and OS have not yet been reached. Five patients died from disease progression, and one patient died from septic shock in CR. CD22 expression by flow was downregulated or absent in 1/3 (33%) patients evaluated after relapse. Peak CAR-T expansion as detected by peripheral blood flow cytometry occurred at a median of 14 days, with a trend towards earlier and higher peak levels in DL2 patients. Significantly higher mean CAR-T levels occurred at peak expansion in patients who developed MAS (1070±915 vs. 196±209 CAR+ cells/μL; p=0.001). CONCLUSIONS: Infusion of CAR22 in R/R LBCL is safe and well tolerated at DL1. Manufacturing of CAR22 was uniformly successful. With a mean follow-up of 7.3 months, the ORR and CR rates are 18/21 (86%) and 14/21 (67%), respectively. These data demonstrate CAR22 to be an effective salvage therapy for CAR19-refractory or CD19-negative LBCL. Figure 1 Figure 1. Disclosures Frank: Allogene Therapeutics: Research Funding; Adaptive Biotechnologies: Research Funding; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees. Oak: Kite Pharma-Gilead: Research Funding. Arai: Magenta Therapeutics: Research Funding. Rezvani: Kaleido: Other: One-time scientific advisory board; Nohla Therapeutics: Other: One-time scientific advisory board; Pharmacyclics-Abbvie: Research Funding; US Department of Justice: Consultancy. Shiraz: Kite Pharma-Gilead: Research Funding. Sidana: Janssen: Consultancy, Research Funding; Allogene: Research Funding; Magenta Therapeutics: Consultancy, Research Funding; BMS: Consultancy. Weng: Kite Pharma: Research Funding. Davis: Novartis Pharmaceuticals: Honoraria; Jazz Pharmaceuticals: Research Funding. Feldman: Samsara Biocapital: Consultancy; Obsidian: Consultancy; Lonza PerMed: Consultancy; Gradalis: Consultancy. Mackall: Lyell: Consultancy, Current equity holder in publicly-traded company, Other: Founder; Syncopation Life Sciences: Consultancy, Current holder of individual stocks in a privately-held company, Other: Founder; Apricity: Consultancy, Current equity holder in publicly-traded company; Neoimmune Tech: Consultancy; Nektar: Consultancy, Research Funding. Miklos: Kite, a Gilead Company, Amgen, Atara, Wugen, Celgene, Novartis, Juno-Celgene-Bristol Myers Squibb, Allogene, Precision Bioscience, Adicet, Pharmacyclics, Janssen, Takeda, Adaptive Biotechnologies and Miltenyi Biotechnologies: Consultancy; Pharmacyclics: Patents & Royalties; Pharmacyclics, Amgen, Kite, a Gilead Company, Novartis, Roche, Genentech, Becton Dickinson, Isoplexis, Miltenyi, Juno-Celgene-Bristol Myers Squibb, Allogene, Precision Biosciences, Adicet, Adaptive Biotechnologies: Research Funding; Adaptive Biotechnologies, Novartis, Juno/Celgene-BMS, Kite, a Gilead Company, Pharmacyclics-AbbVie, Janssen, Pharmacyclics, AlloGene, Precision Bioscience, Miltenyi Biotech, Adicet, Takeda: Membership on an entity's Board of Directors or advisory committees. Muffly: Astellas, Jasper, Adaptive, Baxalta: Research Funding; Adaptive: Honoraria, Other: fees for non-CME/CE services: , Research Funding; Pfizer, Amgen, Jazz, Medexus, Pfizer: Consultancy. OffLabel Disclosure: CD22-directed CAR-T therapy for the treatment of adults with relapsed/refractory large B-cell lymphoma

Abstract: BACKGROUND: CD22 is expressed on the majority of B-cell malignancies. Autologous CAR T-cells targeting CD22 (CAR22) have yielded objective response rates (ORR) of 70-90% in pediatric patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (ALL), including those who had previously failed CD19-directed CAR T-cell (CAR19) therapy. Based on these encouraging results, we evaluated CAR22 in adult patients with R/R ALL and for the first time in patients with R/R large B-cell lymphoma (LBCL), including those who had failed prior autologous CAR19 therapy. METHODS: This single-institution phase I dose escalation clinical trial (NCT04088890) is evaluating a CAR construct incorporating the m971 CD22 single chain variable fragments and 41BB/CD3z endodomains integrated within autologous T-cells via lentiviral transduction. After lymphodepletion with fludarabine and cyclophosphamide, patients were infused with fresh or cryopreserved CAR T-cells after a 7- to 11-day closed manufacturing process utilizing the CliniMACS Prodigy device (Miltenyi). The current cohort includes patients treated at dose level 1 (DL1), which was 1x106 CAR+ cells/kg. Primary objectives assessed the ability to successfully manufacture CAR22 and safety. Overall response rate (ORR) at 28 days post-infusion (D28) was a secondary objective. RESULTS: Three patients with LBCL have been enrolled with a median age of 53 years (range, 51-57) and a median of 6 (range, 5-8) prior lines of therapy. All three patients received prior CAR19 and had refractory disease to second-line or later therapy (n=3); had not undergone autologous hematopoietic stem cell transplantation (HSCT) (n=3); had MYC and BCL2 gene rearrangements (double-hit lymphoma; n=2); had high tumor burden (SPD & gt;50 cm2; n=2); had a history of primary refractory disease (n=1); or had never achieved CR to any therapy (n=1). Six patients with ALL have been enrolled with a median age of 43.5 years (range, 23-62) and a median of 6 (range, 4-8) prior lines of therapy. All six patients received prior allogeneic HSCT and had Ph-positive disease (n=3); had central nervous system (CNS) involvement (n=3); had extramedullary disease (n=2); had high disease burden (BM blasts & gt;5%; n=2); had received prior CD19-directed therapy (n=5); or had received prior CD22-directed therapy (n=3). Successful manufacturing of cells at DL1 was achieved in all patients. All patients (LBCL n=3, ALL n=6) reached day 28 and are included in the safety and response analysis presented here; updated results will be presented at the meeting. Eight patients (88.9%) experienced cytokine release syndrome (CRS); all were Grade 1-2. There were no cases of immune effector cell-associated neurotoxicity syndrome (ICANS). No differences in toxicities were seen across the patient age spectrum and no Grade 5 toxicities occurred following CAR22 infusion. In LBCL, all patients achieved a response at D28 (ORR=100%; CR, n=1, PR, n=2). Both patients with a D28 PR improved to CR by day 90 and 180. All patients remain in CR, with a median follow-up of 8.4 months (range, 6-9.3). In ALL, all patients achieved a CR at D28 (ORR=100%; MRD-, n=5, MRD+, n=1). After a median follow up of 5.1 months (range, 1-8.2), three patients relapsed at 2.5, 4, and 5.5 months after infusion; one patient died while undergoing subsequent therapy 7.3 months post-infusion. CD22 expression by flow cytometry was downregulated or absent in two patients after relapse. Peak CAR expansion as detected by peripheral blood flow cytometry reached a median level of 90.1 (LBCL; range, 85.4-350) and 43.4 (ALL; range, 0.9-399.6) CAR+ cells/µL between D14 and D21. In two LBCL patients with progression following CAR19, CAR22 levels were 11.7 and 55.9 fold higher than prior CAR19 levels at peak expansion. CONCLUSIONS: Infusion of CD22-targeting CAR T-cells in R/R LBCL and ALL is safe and well tolerated. Manufacturing of CAR22 was uniformly successful. To date, 3 of 3 heavily treated adult patients with LBCL whose disease relapsed after prior CAR19 have each achieved CR durable to at least 6 months. All adult ALL patients have achieved CR following CAR22, with some early relapses observed. Accrual is ongoing. Disclosures Negrin: Amgen: Consultancy; Biosource: Current equity holder in private company; UpToDate: Honoraria; KUUR Therapeutics: Consultancy; Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company; BioEclipse Therapeutics: Current equity holder in private company. Rezvani:Pharmacyclics: Research Funding. Shiraz:ORCA BioSystems: Research Funding; Kite, a Gilead Company: Research Funding. Sidana:Janssen: Consultancy. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Apricity Health: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy; Lyell Immunopharma: Consultancy, Current equity holder in private company. Miklos:Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Janssen: Consultancy, Other: Travel support; Miltenyi Biotec: Research Funding. Muffly:Amgen: Consultancy; Adaptive: Research Funding; Servier: Research Funding. OffLabel Disclosure: CD22-directed CAR T-cell Therapy for the treatment of adults with relapsed/refractory LBCL and B-ALL

Abstract: Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19 − or CD19 lo disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial ( NCT03233854 ) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL ( n  = 17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL ( n  = 21), 62% of patients responded with 29% CR. Relapses were CD19 −/lo in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22 −/lo disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency.