ALBERT

Description: Introduction: The autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, axicabtagene ciloleucel (Axi-cel) improved long-term survival of patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the pivotal ZUMA-1 trial indicates a 2-year PFS of ~40% (Locke, Lancet Oncology 2018). Early identification of patients with increased relapse risk may allow for early intervention and improved outcomes. In a pilot study of 6 ZUMA-1 patients, minimal residual disease (MRD) evaluation via a next-generation sequencing MRD assay (Adaptive Biotechnologies, Seattle, WA) to assess for circulating tumor (ct)DNA, mirrored clinical outcome as assessed by PET-CT (Hossain et. al. Leukemia & Lymphoma 2019). Based on these promising results, a multi-institutional prospective study utilizing cell-free MRD assessments to predict outcomes in r/r DLBCL patients after Axi-cel therapy was initiated. Methods: To identify tumor clonotype(s), tumor DNA extracted from archival paraffin-embedded tissue underwent PCR amplification of IgH-VDJ, IgH-DJ and IgKappa/Lambda regions using universal consensus primers. CtDNA levels were measured pre-LD, 0, 7, 14, 21, 28, 56, 90, 180, 270, and 365 days following Axi-cel infusion. PET-CT scans were obtained at baseline, Day 28, Month 3, 6, and 12 with response assessed per Lugano criteria. Deauville 1-3 was considered PET-negative. The protocol prespecified that patients with less than Day 28 follow-up be excluded from analysis. Any detectable ctDNA was considered MRD positive. Results: Here we report on the pre-planned analysis of the first 50 study patients with at least a Day 28 MRD assessment and 3 months of follow up. An additional 4 patients with at least 3 months of follow-up but who did not have a Day 28 MRD assessment were also included. Baseline characteristics and clinical outcomes of patients were similar to ZUMA-1 and a real-world analysis of 295 patient who received Axi-cel (Nastoupil et al ASH 2018). The median age was 61 years old (range 19-76) (53.7% male, 46.3% female) and 59% of patients received 3 or more prior lines of therapy (range 1-6). After a median follow-up of 7.5 months, the best overall response rate was 87% (47 of 54) and complete response rate was 57% (31 of 54). The median OS was not reached and median PFS was 4.6 months (panel A). At Day 28, 56% (28 of 50) of patients were MRD negative (MRD-neg) and 44% (22 of 50) were MRD positive (MRD-pos). As compared to MRD-pos, MRD-neg correlated with improved median PFS (not reached vs. 2.96 months, p

Description: Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.

Description: Introduction: Chronic lymphocytic leukemia (CLL) cells typically carry one or more clonally rearranged immunoglobulin (Ig) genes. The specific sequence of an Ig rearrangement, or clonotype, can serve as a marker of minimal residual disease (MRD) in blood or bone marrow samples. The clonoSEQ assay (Adaptive Biotechnologies Corp) employs a next-generation sequencing (NGS) based MRD quantification method that uses consensus primers to amplify and then sequence the entire repertoire of Ig genes in a clinical sample. Each Ig clonotype, including the one marking a patient's CLL, can then be individually quantified with high precision. We previously demonstrated the utility of this NGS approach for predicting relapse-free survival in a single-institution cohort of CLL patients undergoing reduced-intensity allogeneic hematopoietic cell transplantation (RIC alloHCT). In this landmark analysis, we now assess the prognostic utility of molecular MRD quantification one year following RIC alloHCT in a multi-institution cohort. Methods: We retrospectively evaluated the outcomes and MRD status of patients surviving without relapse to one year after RIC alloHCT for CLL. Patients were assessable for MRD based on availability of a tumor specimen from which to determine the CLL-associated Ig heavy chain (IgH) clonotypes and a cryopreserved peripheral blood mononuclear cell (PBMC) aliquot obtained one year post-HCT. Forty-six patients underwent conditioning with total lymphoid irradiation and anti-thymocyte globulin (TLI/ATG) and 56 were conditioned with fludarabine and busulfan (Flu/Bu) regimens. Clonal IgH rearrangements were identified by NGS-based IgH profiling in CLL-bearing blood or marrow samples obtained prior to HCT and these clonotypes were quantified in the IgH repertoire of PBMC samples from one year (+/- 90 days) post-HCT. Results: One hundred two patients were assessable for outcomes beyond the one year post-HCT landmark. Amongst 97 patients with a diagnostic sample available, the IgH clonotype calibration rate was 90%. Five archived diagnostic samples yielded insufficient DNA to proceed with clonotype identification. 87/102 (85%) patients had paired tumor and one year PBMC samples suitable for MRD quantification. Patients receiving TLI/ATG or Flu/Bu conditioning had similar clinical characteristics. Median relapse-free survival (RFS) from the one year landmark was 4.7 years with TLI/ATG and 6.8 years with Flu/Bu (p=0.08; Figure 1A), and overall survival was not significantly different (Figure 1B). Acute GVHD (grades 2-4) occurred in 15.6% with TLI/ATG and 37.5% with Flu/Bu. Chronic GVHD occurred in 53% and 77%, respectively, with less extensive grade chronic GVHD using TLI/ATG (39%) compared with Flu/Bu (73%). Amongst patients alive and in remission one year after RIC alloHCT, peripheral blood MRD

Description: Introduction: High dose chemotherapy followed by autologous stem cell transplantation (ASCT) cures a subset of patients with chemosensitive relapsed or refractory (rel/ref) diffuse large B-cell lymphoma (DLBCL). Several factors associated with post-ASCT outcome have been identified, including pre-ASCT PET status, but better biomarkers are needed in order to optimally select candidates for the procedure. In other lymphoma subtypes with defining chromosomal translocations, PCR detection of pre- and post-ASCT minimal residual disease (MRD) in peripheral blood and of tumor contamination in the stem cell product is associated with inferior outcome. Until recently, MRD detection in DLBCL was limited by the rarity of detectable circulating disease using conventional techniques. The immunosequencing platform (Adaptive Biotechnologies, Corp.) is a next-generation-sequencing (NGS)-based MRD assay that detects small amounts of circulating tumor DNA (CTD) in patients with lymphoid malignancies. The assay detects CTD at diagnosis in most DLBCL patients and CTD levels track with response to induction therapy (Armand, 2013). Persistence of CTD or recurrence of CTD after completion of therapy is highly associated with DLBCL relapse (Kurtz, 2015; Roschewski, 2015). We evaluated whether CTD in autologous stem cell grafts was predictive of outcome in patients with rel/ref DLBCL undergoing ASCT. Methods: We retrospectively studied patients with rel/ref DLBCL, including transformed indolent lymphoma (TIL), who had paired archival tumor and autologous stem cell specimens and underwent ASCT at Brigham and Women's Hospital/Dana-Farber Cancer Institute from 2003-2013. Genomic tumor DNA was extracted from archival formalin-fixed paraffin-embedded (FFPE) tissue and analyzed using the NGS-based MRD assay. PCR amplification of IGH-VDJ, IGH-DJ,and IGK regions using universal consensus primers was performed followed by NGS to determine the tumor clonotype(s), defined as having a frequency 〉 5% in the tumor specimen. DNA from all available autologous peripheral blood or bone marrow stem cell specimens from each patient was amplified using universal consensus primers and sequenced to determine the level of CTD, defined as the number of lymphoma molecules per diploid genome. Results: We identified 98 eligible patients with rel/ref DLBCL/TIL. The median age was 60 (range 22-77) years; 63% were male; 65% had DLBCL, 29% had TIL, and 5% had primary mediastinal DLBCL; the median number of prior lines of therapy was 2 (range 2-5); all had received prior rituximab; 38% had primary refractory disease; 60% were in complete remission at ASCT; 96% received CBV conditioning. Median follow-up was 56 (range 19-123) months. The 4y progression-free survival (PFS) and overall survival (OS) in the entire cohort were 46% and 64%, respectively. Among 83 patients (85%) with sufficient DNA for clonotype determination, a clonotype was identified in 59 (71%). CTD data was complete in 53 patients (52 received peripheral blood stem cells (PBSC) and 1 received bone marrow). Eight patients (15%) had detectable CTD (CTD+) in the stem cell autografts (all PBSC) and 6/8 relapsed after ASCT. One CTD+ patient had early non-relapse mortality less than 1 month after ASCT and was never restaged. Seven of 8 CTD+ patients had TIL histology, 5 of whom relapsed (4 with aggressive lymphoma). The 4y PFS and OS in CTD+ v CTD- patients were 13% v 48% (p=0.01), and 38% v 67% (p=0.013), respectively [Figure 1]. In multivariable models including CTD status and pre-ASCT characteristics, CTD+ was the only factor associated with OS (HR 3.1, p=0.018), but was not significantly associated with PFS. Discussion: In patients with rel/ref DLBCL undergoing ASCT, the presence of CTD in the autologous stem cell graft is associated with inferior survival. CTD detection in the autograft may be more common in patients with TIL. In studies evaluating CTD detection in DLBCL, the plasma compartment has been more sensitive for detecting CTD than mononuclear cells. The use of concentrated cell specimens in this study may have decreased the sensitivity of the assay. Nevertheless, if the present findings are confirmed in a larger population, CTD detection may permit the identification of a subgroup of patients with a particularly poor outcome after ASCT, for whom alternative approaches could be considered. Figure 1. Overall (A) and Progression-Free (B) Survival in CTD+ vs CTD- Patients Figure 1. Overall (A) and Progression-Free (B) Survival in CTD+ vs CTD- Patients Figure 2. Figure 2. Disclosures Herrera: Sequenta, Inc.: Research Funding; Pharmacyclics: Research Funding; Genentech: Research Funding. Kong:Adaptive Biotechnologies, Corp.: Employment, Other: Stockholder. Davids:Genentech: Other: ad board; Pharmacyclics: Consultancy; Janssen: Consultancy. Rodig:Perkin Elmer: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding. Faham:Adaptive Biotechnologies Corp.: Employment, Other: Stockholder. Armand:BMS: Research Funding; Infinity: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Sequenta, Inc.: Research Funding.

Description: Background: Classical Hodgkin lymphoma (CHL) has been established as B-cell malignancy characterized by a clonal expansion of pathognomonic Reed Sternberg cells (Marafioti et al. Blood 2000). A previous report suggests that clonotypic B-cells may be present in the blood of patients with CHL; however, the relationship between these circulating clonotypic B-cells and CHL is unclear. We utilized the LymphoSIGHT™ method, a next-generation sequencing approach, to detect lymphoma-specific clonotypes in peripheral blood in patients with CHL at diagnosis or disease recurrence as well as in follow-up blood samples. This method has the sensitivity to detect one lymphoma cell per million leukocytes in peripheral blood, and has been applied to minimal residual disease (MRD) detection in multiple B-cell malignancies. We evaluated the extent of somatic hypermutation in the lymphoma clonotypes, and performed sequence and expression level analyses of the lymphoma clonotype repertoire. Methods: Frozen primary tumor biopsy samples were first analyzed for clonality at the immunoglobulin heavy chain (IGH) and kappa chain (IGK) loci using the LymphoSIGHT method. Rearranged immunoglobulin gene segments (IGH-VDJ, IGH-DJ and IGK) in the genomic DNA and/or RNA were amplified with locus-specific primer sets, sequenced, and analyzed using standardized algorithms for clonotype determination. Clonotypes with a frequency 〉5% in the B-cell repertoire of the tumor biopsy were considered to represent tumor clonotypes. IGH-VDJ clonotypes with a frequency 〉2% in DNA were deemed to be cancer-specific if the clonotype was not present in the RNA. Such clonotypes were then quantitated in serum and peripheral blood mononuclear cells (PBMC), and DNA sequence and/or RNA expression level analysis was performed. Results: A total of 17 CHL patients were analyzed. A high-frequency clonal rearrangement was identified for at least one receptor (IGH-VDJ, IGH-DJ and IGK) in 12 of 17 cases (71%). Lymphoma-specific clonotypes were detected in blood samples from 8 of 11 patients (73%). Notably, a lymphoma-specific clonotype was detected in the serum compartment in 8 of 9 cases (89%) tested (Figure 1A), while it was detected in PBMC only in 3 of 9 cases (33%) tested (Figure 1B). Follow-up samples obtained from three patients in remission were negative for the tumor-specific clonotype in both the serum and cellular compartments. We conducted sequence and expression level analysis of each IGH-VDJ clonal rearrangement. We calculated the number of somatic mutations in each lymphoma-specific clonotype compared to the germline sequence in the interrogated region. In the ten patients with IGH-VDJ clonal rearrangements, we observed a median of 14 somatic mutations (range 0 to 27). This confirms that HRS cells correspond in their developmental stage to germinal or post-germinal center B-cells. While IGH-VDJ clonotypes were observed frequently in DNA obtained at diagnosis, IGH-VDJ clonotypes were not detected in the RNA from the same sample. We evaluated the relationship between the presence of lymphoma-specific clonotypes in the cellular compartment at diagnosis and eventual progression. All three untreated patients that were positive at baseline in the cellular compartment experienced relapse or progression (at 3, 11 and 17 months). In contrast, zero of 5 patients without detectable lymphoma-specific clonotypes in their cellular compartment at baseline experienced relapse (follow up duration 23-45 months, log-rank test p=0.004). Conclusions: This is the first clinical assay that can be used to detect and monitor MRD in CHL. Lymphoma-specific sequences can be identified in serum in 80% of cases. Our preliminary analysis suggests that the presence of lymphoma-specific clonotypes in PBMCs may indicate high risk for recurrence. This study demonstrates proof-of-principle and underscores the promise of a new methodology to measure disease burden and provide prognostic information from a blood test in patients with CHL. Figure 1. Lymphoma clonotype levels in A) cell-free plasma and B) PBMC samples are shown for the different patients. Figure 1. Lymphoma clonotype levels in A) cell-free plasma and B) PBMC samples are shown for the different patients. Figure 2 Figure 2. Disclosures Klinger: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Kong:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Background: The majority of multiple myeloma (MM) patients receiving autografts ultimately relapse, and tumor cell contamination of autografts may be a contributing factor. A clinical trial to assess tandem chemo-mobilization with the goal of reducing the myeloma cell burden in the patient and in the apheresis product was conducted and outcomes were previously reported (Chen et al., Bone Marrow Transplant 2012). The effect of myeloma cell depletion using tandem mobilization has not been previously quantified; however, clinical outcomes are comparable with other autotransplant regimens. Here we utilize a sequencing-based minimal residual disease (MRD) technology, termed the LymphoSIGHT™ platform, to assess the effect of tandem chemo-mobilization on myeloma cell contamination in apheresis collections. This sequencing-based method has a sensitivity to detect one tumor-associated immune receptor gene rearrangement molecule per million leukocytes and can be used for MRD quantification in lymphoid neoplasms (Faham et al., Blood 2012; Martinez-Lopez et al., Blood 2014). We analyzed cryopreserved apheresis samples from 19 MM patients to quantify the clonotypicmyeloma cell burden in tandem mobilized apheresis products. Methods: In the two-step chemo-mobilization process, MM patients were first treated with cyclophosphamide (4g/m2) followed by G-CSF (10mcg/kg/day) and an apheresis product collected at hematopoietic recovery was stored ("Cycle 1" apheresis sample). Following subsequent treatment with etoposide (2g/m2) followed by G-CSF (10mcg/kg/day), a second apheresis product was collected ("Cycle 2" apheresis sample). Using universal primer sets, we assessed clonal rearrangements at the immunoglobulin (IGH-VDJ, IGH-DJ and IGK) loci in diagnostic bone marrow aspirate (BMA) slides. Myeloma clonotypes were identified in the diagnostic BMA sample of each patient based on high-frequency within the B-cell repertoire. The level of the myeloma clonotype was then quantified in banked aliquots from the Cycle 1 and Cycle 2 apheresis products. Results: Paired Cycle 1 and Cycle 2 apheresis samples were analyzed, and the total number of rearranged immunoglobulin (Ig) molecules, an approximation of total B-cells, was calculated. An average 5.3 fold reduction in rearranged Ig molecules as a fraction of total cells was observed from Cycle 1 to Cycle 2 (p = 0.004, 2-tailed Wilcoxon) (Figure 1A). Myeloma cell contamination was present at a median level of 62.7 myeloma molecules/million PBMCs (range 0-11,951) after Cycle 1 and 43.6 myeloma molecules/million PBMCs (range 0-4,489) after Cycle 2. Quantitative comparison of MRD levels revealed no significant difference in the absolute number of myeloma molecules/million PBMCs between Cycle 1 and Cycle 2 apheresis products (p = 0.276) (Figure 1B). Analysis of the myeloma clonotype frequency illustrated an average 3.0-fold increase in myeloma clonotype frequency relative to total Ig rearrangements from Cycle 1 to Cycle 2 (p = 0.011) (Figure 1C). MRD levels less than 10-5 in autograftapheresis samples were associated with significantly longer PFS (40 vs 14.3 months, p = 0.003) (Figure 1D). Conclusions: Tandem mobilization fails to decrease myeloma contamination in the second apheresis product, and this practice has been discontinued based on this analysis. Highly sensitive quantification of myeloma clonotype levels in autograft samples may provide improved clinical prognostic information, complement post-transplant MRD quantification, and potentially informtreatment decisions about the timing and intensity of post-transplant maintenance therapies. Figure 1. Comparison of Cycle 1 and Cycle 2 apheresis products and clinical outcome data in MM patients. In Panels A-C, paired Cycle 1 and Cycle 2 values for each patient are connected by a line. Open circles represent A) Total rearranged Ig molecules (an approximation of total B-cells) as a fraction of total cells, B) Number of myeloma molecules per million PBMCs, and C) Myeloma clonotype frequency relative to total Ig rearrangements for each patient in the Cycle 1 and Cycle 2 apheresis products. D) Kaplan-Meier analysis of progression-free survival for 7 MRD negative (10-5) patients. Figure 1. Comparison of Cycle 1 and Cycle 2 apheresis products and clinical outcome data in MM patients. In Panels A-C, paired Cycle 1 and Cycle 2 values for each patient are connected by a line. Open circles represent A) Total rearranged Ig molecules (an approximation of total B-cells) as a fraction of total cells, B) Number of myeloma molecules per million PBMCs, and C) Myeloma clonotype frequency relative to total Ig rearrangements for each patient in the Cycle 1 and Cycle 2 apheresis products. D) Kaplan-Meier analysis of progression-free survival for 7 MRD negative (10-5) patients. Disclosures Klinger: Sequenta, Inc.: Employment, Equity Ownership. Kong:Sequenta, Inc.: Employment, Equity Ownership. Faham:Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Description: Key Points DLBCL can be detected in the blood by immunoglobulin high-throughput sequencing (Ig-HTS) with high specificity. Although DLBCL can be detected in leukocytes or plasma by Ig-HTS, plasma has greater sensitivity and more accurately reflects disease.

Description: Chimeric Antigen Receptor (CAR) therapy targeting CD19 achieves complete remission (CR) rates of 70%-90% in relapsed/refractory B-ALL. Relapse due to loss of the CD19 targeted epitope presents a therapeutic challenge as evidenced by the largest global pediatric CD19-CAR experience which showed 15 of 16 relapses to be explained by CD19 downregulation (Maude et al, NEJM 2018). Alternatively targeting CD22 using CD22-CAR therapy has demonstrated a CR rate of approximately 70% in both CD19+ and CD19- B-ALL, however relapse due to CD22 downregulation limits the curative potential of singularly-targeting CD22 (Fry et al, Nat Med. 2018). We hypothesized that simultaneous targeting of CD19 and CD22 via a bispecific CAR-T cell would be a safe and tolerable treatment strategy in relapsed/refractory B-cell ALL and address immune evasion. Here, we report the first clinical experience in pediatric patients using bispecific CD19-CD22 CAR T cells. We describe a single institution phase I dose escalation study in pediatric patients with relapsed or refractory B cell ALL. We utilized lentiviral transduction of a bivalent CAR construct incorporating the fmc63 CD19 and m971 CD22 single chain variable fragments (scFvs) used in clinically tested CAR constructs and a 41BB costimulatory endodomain (Fry et al, Nat Med. 2018). Our primary objectives are feasibility of production of this bivalent CAR and safety at 3 dose escalation levels (1x106, 3x106 and 1x107 CAR T cells/kg). Clinical response assessment is evaluated as a secondary aim. All patients described received lymphodepletion with fludarabine (25mg/m2 x 3 days) and cyclophosphamide (900mg/m2 x 1) followed by fresh or cryopreserved CAR T cell infusion after a 7-9 day production time. Patients were prospectively monitored at predefined intervals for disease response and correlative assessments. Four pediatric patients with precursor-B ALL, age 2-17, have been enrolled and treated with CD19/CD22 bispecific CAR T cells at dose level 1 (1x106) [Table 1]. Three patients entered CAR therapy with low disease burden detected by minimal residual disease (MRD) alone and 1 patient initiated therapy with 12% bone marrow blasts. All patients were CNS1 at time of treatment. The toxicity profile mirrored that of the singular CD19 and CD22 CAR experience with 3 patients experiencing reversible CRS (2 Grade I, 1 Grade II), onset day 3-8, and 2 patients experiencing grade I neurotoxicity, onset day 3-9. In our cohort, we experienced lower grade toxicities than previously reported, likely due to a mean lower disease burden. Only 1 patient with CRS met criteria for tocilizumab and this patient was the singular study patient treated with higher burden disease. Neurotoxicity was managed with supportive care and fully reversible. Peripheral blood flow cytometry analysis detects circulating CAR by day 6 in all patients and demonstrates peak CAR expansion between day 6-10. Peak CAR T expansion reached levels of 10-25% of total T cells with inter-patient variability in CD4 and CD8 predominance, favoring CD8 expansion in 3 of 4 patients. Clinical symptoms and inflammatory markers expectedly correlate with peak CAR expansion. Four of 4 patients achieved complete remission (CR) at day 28 post-CD19/CD22 bispecific CAR therapy. Three of 4 patients demonstrated MRD- remissions by flow cytometry and of these, next generation sequencing (NGS) was negative where available (N=2). Multi-parametric CyTOF studies permitting CAR T cell phenotyping in conjunction with single cell TCR tracking, proteomics, epigenomics and cytokine profiling are ongoing and will be used to further characterize persisting CAR T cells and define inter-product and inter-patient variability. In this phase I study, we demonstrate safety and tolerability of this bispecific CD19/CD22 CAR at a dose of 1x106 CAR T cells/kg in pediatric patients with relapsed/refractory B cell ALL. The CD19/22-bispecific CAR mediated antileukemic activity in 100% of patients studied thus far. Long-term follow up and further accrual will be required to inform the effect of bispecific CAR targeting on surface antigen remodeling. Disclosures Muffly: Adaptive Biotechnologies: Research Funding; Shire Pharmaceuticals: Research Funding. Miklos:Genentech: Research Funding; Kite - Gilead: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics - Abbot: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

Description: 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 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.