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Patients with relapsed or refractory large B-cell lymphoma after first-line treatment who are not intended for haematopoietic stem-cell transplantation (HSCT) have poor outcomes and limited treatment options. We assessed the antitumour activity and safety of lisocabtagene maraleucel, an autologous, CD19-directed chimeric antigen receptor (CAR) T-cell product, as second-line treatment in adults with relapsed or refractory large B-cell lymphoma not intended for HSCT.PILOT, an open-label, phase 2 trial done at 18 clinical sites in the USA, included adults aged 18 years or older who had relapsed or refractory large B-cell lymphoma and PET-positive disease, had received first-line therapy containing an anthracycline and a CD20-targeted agent, were not intended for HSCT by their physician, and met at least one prespecified transplantation not intended criterion. Patients received lymphodepleting chemotherapy (intravenous fludarabine 30 mg/mBetween July 26, 2018, and Sept 24, 2021 (data cutoff for the primary analysis), 74 patients underwent leukapheresis and 61 received lisocabtagene maraleucel (efficacy and safety sets); median age was 74 years (IQR 70-78), 24 (39%) patients were women versus 37 (61%) men, and 54 (89%) patients were White. 16 (26%) of 61 patients had an Eastern Cooperative Oncology Group performance status of 2, 33 (54%) had refractory disease, 13 (21%) relapsed within 1 year of first-line therapy, and 15 (25%) relapsed after 12 months of first-line therapy. Median on-study follow-up was 12·3 months (IQR 6·1-18·0). 49 (80% [95% CI 68-89]; p0·0001) patients had an overall response. The most common grade 3 or worse treatment-emergent adverse events were neutropenia (29 [48%] patients), leukopenia (13 [21%]), and thrombocytopenia (12 [20%]). Lisocabtagene maraleucel-related serious treatment-emergent adverse events were reported in 13 (21%) patients. There were no treatment-related deaths. Cytokine release syndrome occurred in 23 (38%; grade 3 in one) patients and neurological events in 19 (31%; grade 3 in three) patients, with no grade 4 events or deaths.These results support lisocabtagene maraleucel as a potential second-line treatment in patients with large B-cell lymphoma for whom HSCT is not intended.Juno Therapeutics, a Bristol-Myers Squibb company.

Supplementary Methods; Supplementary Figures S1-S9; Supplementary Tables S1-S7

In the TRANSCEND NHL 001 study, 53% of patients with relapsed/refractory large B-cell lymphoma (LBCL) treated with lisocabtagene maraleucel (liso-cel) achieved a complete response (CR). To determine characteristics of patients who did and did not achieve a CR, we examined the tumor biology and microenvironment from lymph node tumor biopsies. LBCL biopsies from liso-cel–treated patients were taken pretreatment and ∼11 days posttreatment for RNA sequencing (RNA-seq) and multiplex immunofluorescence (mIF). We analyzed gene expression data from pretreatment biopsies (N = 78) to identify gene sets enriched in patients who achieved a CR to those with progressive disease. Pretreatment biopsies from month-3 CR patients displayed higher expression levels of T-cell and stroma-associated genes, and lower expression of cell-cycle genes. To interpret whether LBCL samples were “follicular lymphoma (FL)–like,” we constructed an independent gene expression signature and found that patients with a higher “FL-like” gene expression score had longer progression-free survival (PFS). Cell of origin was not associated with response or PFS, but double-hit gene expression was associated with shorter PFS. The day 11 posttreatment samples (RNA-seq, N = 73; mIF, N = 53) had higher levels of chimeric antigen receptor (CAR) T-cell densities and CAR gene expression, general immune infiltration, and immune activation in patients with CR. Further, the majority of T cells in the day 11 samples were endogenous. Gene expression signatures in liso-cel–treated patients with LBCL can inform the development of combination therapies and next-generation CAR T-cell therapies.

Bruton tyrosine kinase inhibitors (BTKi) and venetoclax are currently used to treat newly diagnosed and relapsed/refractory chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). However, most patients eventually develop resistance to these therapies, underscoring the need for effective new therapies. We report results of the phase 1 dose-escalation portion of the multicenter, open-label, phase 1/2 TRANSCEND CLL 004 (NCT03331198) study of lisocabtagene maraleucel (liso-cel), an autologous CD19-directed chimeric antigen receptor (CAR) T-cell therapy, in patients with relapsed/refractory CLL/SLL. Patients with standard- or high-risk features treated with ≥3 or ≥2 prior therapies, respectively, including a BTKi, received liso-cel at 1 of 2 dose levels (50 × 106 or 100 × 106 CAR+ T cells). Primary objectives included safety and determining recommended dose; antitumor activity by 2018 International Workshop on CLL guidelines was exploratory. Minimal residual disease (MRD) was assessed in blood and marrow. Twenty-three of 25 enrolled patients received liso-cel and were evaluable for safety. Patients had a median of 4 (range, 2-11) prior therapies (100% had ibrutinib; 65% had venetoclax) and 83% had high-risk features including mutated TP53 and del(17p). Seventy-four percent of patients had cytokine release syndrome (9% grade 3) and 39% had neurological events (22% grade 3/4). Of 22 efficacy-evaluable patients, 82% and 45% achieved overall and complete responses, respectively. Of 20 MRD-evaluable patients, 75% and 65% achieved undetectable MRD in blood and marrow, respectively. Safety and efficacy were similar between dose levels. The phase 2 portion of the study is ongoing at 100 × 106 CAR+ T cells. This trial was registered at clinicaltrials.gov as NCT03331198.

Integrin alpha4/beta7 on circulating lymphocytes identifies them as gut-tropic, and can be targeted by the humanized antibody vedolizumab to treat inflammatory bowel disease (IBD). We found lymphocytes expressing alpha4/beta7 were significantly more responsive to the pro-inflammatory cytokines IL-6, IL-7, and IL-21, and less responsive to the regulatory T cell (Treg)-supporting cytokine IL-2. Alpha4/beta7 was expressed by a smaller percent of FOXP3 + Helios+ thymically-derived Tregs (tTregs) than FOXP3 + Helios- peripherally-derived Tregs (pTregs) or FOXP3- effector T cells. Integrin alpha4/beta7+ CD4 T cells were also rare among cells expressing the Th2 marker CRTh2, but enriched in cells bearing the circulating T follicular helper cell marker CXCR5. Thus the effect of this anti-integrin therapy on the mucosal immune system may be more qualitative than quantitative, and selectively replace pro-inflammatory effector cells with Tregs and Th2 cells to facilitate immune tolerance in the mucosa without globally depleting lymphocytes from the intestinal mucosa.

Background: Liso-cel is an autologous, CD19-directed, defined composition, 4-1BB CAR T cell product administered at equal target doses of CD8 + and CD4 + CAR + T cells. In the pivotal TRANSCEND NHL 001 study (NCT02631044) in patients (pts) with R/R LBCL after ≥ 2 prior lines of therapy, liso-cel demonstrated significant clinical activity with an objective response rate of 73%, complete response (CR) rate of 53%, and median duration of response (DOR) not reached at 12 months' median DOR follow-up, and a low incidence of severe cytokine release syndrome (2%) and neurological events (10%; Abramson et al. Lancet 2020;396:839-852). While CAR T cells have shown remarkable efficacy in treating LBCL, with a well-understood mechanism of direct CAR T cell tumor killing, the contribution of the endogenous immune system in this response is currently unclear. Previously, histologic and gene expression analysis of pre- and posttreatment tumor biopsies from pts who received liso-cel in TRANSCEND NHL 001 showed a relationship between tumor-infiltrating CAR-negative (endogenous) T cells and early and durable response [Reiss et al. Blood 2019;134(suppl 1):202]. Here we examine, by gene expression and flow cytometry, post-infusion characteristics of liso-cel and peripheral endogenous T cells in pts from TRANSCEND NHL 001 with early (CR at 1 or 3 months) and long-term (CR at 9 or 12 months) response versus pts with progressive disease (PD) before or at the same time points. Methods: CAR and endogenous T cells were assayed using a novel cell-sorting and low-input RNA-seq method. CAR and endogenous T cells were isolated from peripheral blood mononuclear cell samples from 39 pts at time to maximum concentration of liso-cel (T max; Day 8-22), 1 month, and 2 months after liso-cel infusion. In addition, samples from 44 pts were assayed only at T max by RNA-seq. A subset of these samples was assessed by flow cytometry for expression of the T cell differentiation markers CD4, CD8, CD45RA, CCR7, CD27, and CD28. All RNA-seq analyses were performed in R version 4.0.2. Differential expression analysis was performed with the DESeq2 package with gene set enrichment analysis using clusterProfiler and GSVA packages with gene sets obtained from MSigDB. Comparisons of 2 groups were made with a Wilcoxon rank sum test. Association with the time-to-event outcome of progression-free survival (PFS) was assessed using Cox proportional hazards model or log rank test. Results: Characteristics consistent with less differentiated CAR T cells and endogenous T cells were significantly associated with early and long-term response. Elevated CAR T cell expression of CD27 at T max was associated with CR at 1 month and longer PFS. Expression of CCR7 by CAR T cells at 1 and 2 months was associated with CR at 9 months. Additionally, at T max, gene sets associated with T cell effector differentiation and metabolism were enriched in CAR T cells from pts with early relapse (pts with CR vs PD at 3 months). In endogenous T cells, elevated expression of CCR7 and CD28 at T max was associated with early response (CR at 1 and 3 months). Additionally, expression of CCR7 and CD28 in T cells isolated at 1 and 2 months was associated with long-term response (CR at 9 and 12 months). Furthermore, expression of the immune checkpoint receptor LAG3 by endogenous T cells was associated with PD from month 3 to month 12. Conclusions: In summary, the data from our study show the relationship between CAR T cell differentiation status and achieving response over time, and provide new evidence for the involvement of endogenous T cells in the therapeutic efficacy of CAR T cell therapy. Disclosures Thorpe: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Jiang: Bristol Myers Squibb: Ended employment in the past 24 months. Rytlewski: Adaptive Biotechnologies: Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months; Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Kostic: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Kim: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company. Peiser: Bristol Myers Squibb: Current Employment, Current equity holder in publicly-traded company.

BackgroundCLL is an incurable hematologic malignancy characterized by progressive accumulation of clonally derived CD19+ B-cell lymphocytes. CAR T cell therapies are promising treatments but inherent T cell dysfunction has limited their development in CLL. Preclinical studies suggest ibrutinib may modulate CAR T cells directly to improve their function independently of antitumor effects. Liso-cel is an autologous, CD19-directed, defined composition, 4–1BB CAR T cell product administered at equal target doses of CD8+ and CD4+ CAR+ T cells. The ongoing TRANSCEND CLL 004 study evaluates the efficacy and safety of liso-cel alone (monotherapy) or with concurrent ibrutinib (combination) in relapsed/refractory (R/R) CLL.1 We investigated the effect of ibrutinib on post-infusion CAR+ and endogenous T cells relative to monotherapy using a novel cell-sorting, low-input RNA-seq method for analysis of patient cells beyond peak expansion, including 1 and 2 months after liso-cel infusion.MethodsCAR+ and endogenous T cells from patients in TRANSCEND CLL 004 (NCT03331198; monotherapy, n=16; combination, n=19) were isolated from peripheral blood mononuclear cells by fluorescence-activated cell sorting. RNA-seq was performed using cell lysate as input material. CAR+ T cell pharmacokinetics were assessed by flow cytometry. Serum interleukin-6 was measured by electrochemiluminescent multiplex immunoassay. Minimal residual disease (ResultsGene set enrichment analyses revealed positive enrichment of cell proliferation-associated gene sets and negative enrichment of inflammation-associated gene sets at peak expansion in CAR+ T cells from combination relative to monotherapy patients. Similar, but less marked, proliferation- and inflammation-associated gene expression changes were also observed in endogenous T cells. Accordingly, increased CAR+ T cell expansion and reduced serum interleukin-6 were observed in combination patients. In addition, an independently derived CLL ibrutinib gene expression score2 was increased and sustained in CAR+ and endogenous T cells from combination but not monotherapy patients over time. A higher ibrutinib score trended with a higher rate of undetectable minimal residual disease, longer progression-free survival (PFS), and longer duration of response. Lastly, a T cell exhaustion-related gene signature was significantly reduced in CAR+ but not endogenous T cells with combination treatment, and this reduction was associated with improved PFS.ConclusionsConcurrent ibrutinib treatment in patients with R/R CLL resulted in measurable effects in CAR+ and endogenous T cells, including changes in gene signatures related to proliferation, inflammation, and T cell exhaustion. These changes were associated with enhanced CAR T cell function and efficacy.AcknowledgementsWe would like to thank the patients, caregivers, investigators, and study personnel. This study was funded by Juno Therapeutics, a Bristol-Myers Squibb Company. All authors contributed to and approved the abstract; writing and editorial assistance were provided by Allison Green, PhD, of The Lockwood Group (Stamford, CT, USA), funded by Bristol Myers Squibb.Trial RegistrationNCT03331198ReferencesWierda WG, Dorritie KA, Munoz J, et al. TRANSCEND CLL 004: phase 1 cohort of lisocabtagene maraleucel (liso-cel) combined with ibrutinib (IBR) for patients (pts) with R/R CLL/SLL. Hematol Oncol 2021;39(S2):141–143.Rendeiro AF, Krausgruber T, Fortelny N, et al. Chromatin mapping and single-cell immune profiling define the temporal dynamics of ibrutinib response in CLL. Nat Commun 2020;11:577.Ethics ApprovalThis study used patient cells obtained from a clinical trial that was done in accordance with with the Declaration of Helsinki, International Conference on Harmonization Good Clinical Practice guidelines, institutional review boards at participating institutions approved the study protocol and amendments, and all patients provided written informed consent.

Background: In CLL/SLL, ibrutinib treatment before leukapheresis improved in vivo and ex vivo expansion of the CD19-directed chimeric antigen receptor (CAR) T cell therapy tisagenlecleucel, and concurrent ibrutinib therapy improved engraftment and therapeutic efficacy of anti-CD19 CAR T cells in human xenograft mouse models (Fraietta et al. Blood. 2016;127:1117-27). Recent studies in patients with R/R CLL suggest that CD19-directed CAR T cell therapy combined with ibrutinib improves response rates with CTL119 and JCAR014 (Gill et al. Blood. 2018;132:298; Gauthier et al. Blood. 2020;135:1650-60). Liso-cel is an investigational, CD19-directed, defined composition, 4-1BB CAR T cell product administered at equal doses of CD8+ and CD4+ CAR+ T cells. We report initial safety and preliminary efficacy from the phase 1 liso-cel and ibrutinib combination cohort of the ongoing phase 1/2 TRANSCEND CLL 004 study (NCT03331198) in patients with R/R CLL/SLL. Methods: Eligible patients with CLL/SLL met ≥1 of the following: 1) received ibrutinib and progressed at time of study enrollment; 2) had high-risk features and received ibrutinib for ≥6 months (mo) with less than a complete response (CR); 3) had a Bruton tyrosine kinase (BTK) or PLCγ2 gene mutation, with or without progression on ibrutinib; 4) had received prior ibrutinib with no contraindication to reinitiating ibrutinib. Baseline disease assessments included bone marrow (BM) biopsy, complete blood count, lymphocyte enumeration, and CT scan. At enrollment, patients started or continued ibrutinib. Patients continued ibrutinib through leukapheresis and for ≥90 days after liso-cel infusion. Patients received liso-cel infusion at 50 × 106 (dose level [DL]1) or 100 × 106 (DL2) CAR+ T cells after 3 days of lymphodepletion with fludarabine/cyclophosphamide. Primary endpoints were safety and to determine the recommended dose (RD) of liso-cel in combination with ibrutinib for R/R CLL/SLL; overall response (OR) rate (CR + CR with incomplete blood count recovery [CRi] + partial response) and pharmacokinetics (PK) were exploratory endpoints. The RD was selected based on the modified toxicity probability interval algorithm. Results: At data cutoff, 19 patients received liso-cel (DL1, n=4; DL2, n=15) with ibrutinib. Median age was 60 (range, 50‒77) years, and 18 patients (95%) had high-risk cytogenetics (del[17p], n=8; TP53 mutation, n=6; unmutated IGHV, n=16). Patients had a median of 4 (range, 2‒11) prior therapies. All patients were R/R to prior ibrutinib; 14 patients (74%) had BTK inhibitor as last prior therapy and 10 (53%) had prior venetoclax. No dose-limiting toxicities were observed at either DL. The most common grade ≥3 treatment-emergent adverse events (TEAEs) were neutropenia/neutrophil count decrease (n=17; 89%), anemia (n=9; 47%), and febrile neutropenia (n=5; 26%; Table). Six patients had infections at DL2: grade 3 and grade 2 lung infection (n=1 each) and grade 2 coccidioidomycosis, scabies, skin, and gum infections (n=1 each). Ibrutinib-related AEs included diarrhea (n=7), hypertension (n=4), atrial fibrillation (n=1), and rash (n=1). No grade 5 TEAEs occurred. Fourteen patients (74%) had cytokine release syndrome (CRS; 1 grade 3) and 6 (32%) had neurological events (NEs; 3 grade ≥3). Seven patients (37%) required tocilizumab and/or corticosteroids to manage CRS and/or NEs. Preliminary PK data showed a median time to peak liso-cel expansion of 11 days across DLs (DL1, 12 days; DL2, 11 days). Of 19 patients with ≥1-mo follow-up, 18 (95%) had an OR (DL2, 100%; DL1, 75%) and 9 (47%) had a CR/CRi. One patient (5%) had stable disease. All ORs were achieved by Day 30 postinfusion, and 15 (83%) of 18 patients maintained their response at 3-mo follow-up. Of 19 patients evaluable for minimal residual disease (MRD), 17 (89%) achieved undetectable MRD in blood via flow cytometry and 15 (79%) in BM by next-generation sequencing (both sensitivity of ≤10-4). Conclusions: Preliminary data show that liso-cel in combination with ibrutinib is associated with manageable safety, including a low incidence of grade 3 CRS and grade ≥3 NEs, and promising efficacy in heavily pretreated patients with R/R CLL/SLL. No clear difference in safety was observed across DLs, and DL2 was selected as the RD for liso-cel in combination with ibrutinib in patients with R/R CLL/SLL. Updated results from the full combination cohort and additional PK/pharmacodynamic data will be reported. Table Disclosures Dorritie: Juno Therapeutics: Research Funding; Kite-Gilead: Research Funding. Munoz:Portola: Research Funding; Incyte: Research Funding; Acrotech/Aurobindo: Speakers Bureau; Alexion: Consultancy; Seattle Genetics: Consultancy, Honoraria, Research Funding, Speakers Bureau; Fosunkite: Consultancy; Pharmacyclics: Consultancy, Research Funding, Speakers Bureau; Innovent: Consultancy; Genentech/Roche: Research Funding, Speakers Bureau; Pfizer: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding, Speakers Bureau; Juno/Celgene/BMS: Consultancy, Research Funding, Speakers Bureau; Janssen: Consultancy, Research Funding, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Beigene: Consultancy, Speakers Bureau; Millenium: Research Funding; Verastem: Speakers Bureau; Merck: Research Funding; AbbVie: Consultancy, Speakers Bureau; AstraZeneca: Speakers Bureau; Kyowa: Consultancy, Honoraria, Speakers Bureau. Stephens:Innate: Consultancy; Verastem: Research Funding; Beigene: Consultancy; Karyopharm: Consultancy, Research Funding; Acerta: Research Funding; Gilead: Research Funding; Juno: Research Funding; MingSight: Research Funding; Arqule: Research Funding; Janssen: Consultancy; Pharmacyclics: Consultancy. Gillenwater:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb: Current equity holder in publicly-traded company. Gong:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb: Current equity holder in publicly-traded company. Yang:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb: Current equity holder in publicly-traded company. Ogasawara:Bristol-Myers Squibb: Current Employment; Bristol-Myers Squibb: Current equity holder in publicly-traded company. Thorpe:Bristol-Myers Squibb: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Siddiqi:Astrazenca: Membership on an entity's Board of Directors or advisory committees; PCYC: Membership on an entity's Board of Directors or advisory committees; Juno: Membership on an entity's Board of Directors or advisory committees; Kite: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; BeiGene: Other: DMC member; Juno Therapeutics, Pharmacyclics LLC, an AbbVie Company, AstraZeneca, Celgene, Kite Pharma, and BeiGene: Consultancy; Pharmacyclics LLC, an AbbVie Company, Juno Therapeutics, KITE Pharma, AstraZeneca, TG Therapeutics, Celgene, Oncternal, and BeiGene: Research Funding; Pharmacyclics LLC, an AbbVie Company, Seattle Genetics, Janssen, and AstraZeneca: Speakers Bureau; AstraZeneca: Other: Travel/accommodations/expenses.

Risk of autoimmunity is associated with multiple genetic variants. Genome-wide association studies have linked single-nucleotide polymorphisms in the phosphatases PTPN22 (rs2476601) and PTPN2 (rs1893217) to increased risk for multiple autoimmune diseases. Previous mouse studies of loss of function or risk variants in these genes revealed hyperactive T cell responses, whereas studies of human lymphocytes revealed contrasting phenotypes. To better understand this dichotomy, we established a robust gene editing platform to rapidly address the consequences of loss of function of candidate genes in primary human CD4+ T cells. Using CRISPR/Cas9, we obtained efficient gene disruption (>80%) of target genes encoding proteins involved in Ag and cytokine receptor signaling pathways including PTPN22 and PTPN2. Loss-of-function data in all genes studied correlated with previous data from mouse models. Further analyses of PTPN2 gene–disrupted T cells demonstrated dynamic effects, by which hyperactive IL-2R signaling promoted compensatory transcriptional events, eventually resulting in T cells that were hyporesponsive to IL-2. These results imply that altered phosphatase activity promotes evolving phenotypes based on Ag experience and/or other programming signals. This approach enables the discovery of molecular mechanisms modulating risk of autoimmunity that have been difficult to parse in traditional mouse models or cross-sectional human studies.

8040 Background: Patients (pts) with aggressive large B-cell NHL who are R/R after first-line immunochemotherapy and not eligible for high-dose chemotherapy and HSCT have a poor prognosis and no established standard of care. The ongoing, open-label phase 2 PILOT study is the first to assess the safety and efficacy of liso-cel, an investigational, CD19-directed, defined composition, 4-1BB CAR T cell product infused at equal target doses of CD8+ and CD4+ CAR+ T cells, as 2L therapy in TNE pts (NCT03483103). Methods: Eligible pts had aggressive R/R diffuse large B-cell lymphoma NOS (de novo or transformed follicular lymphoma [FL]), high-grade B-cell lymphoma, or FL grade 3B with 1 line of prior therapy containing an anthracycline and anti-CD20 agent. Pts were deemed TNE by meeting ≥1 criteria: age ≥70 y, ECOG PS 2, or impaired organ function (DLCO ≤60% [but SaO2 ≥92% and CTCAE ≤1 dyspnea], LVEF ≥40% to < 50%, creatinine clearance > 30 to < 60 mL/min, or AST/ALT > 2 to ≤5 × ULN). Liso-cel (100 × 106 CAR+ T cells) was administered 2–7 days after lymphodepletion (LD) with fludarabine/cyclophosphamide. The primary endpoint is ORR; key secondary endpoints are AEs and CR rate. Results: At data cutoff, 25 pts had LD followed by liso-cel infusion. Pt characteristics are summarized in the Table. Overall, 48% (n = 12) had high tumor burden and 48% were primary refractory. 18/25 (72%) pts had grade ≥3 treatment-emergent AEs, 40% of which were cytopenias. No grade 5 AEs occurred within the first 30 days after liso-cel. Five pts (20%) had cytokine release syndrome (CRS) and 3 (12%) had neurological events (NEs). No grade 3/4 CRS was observed; 2 pts (8%) had grade 3/4 NEs. Five pts (20%) received tocilizumab and/or dexamethasone for CRS/NEs. At a median follow-up of 3.5 mo, the ORR was 80% (95% CI, 59–93; n = 20); 48% of pts (n = 12) achieved CR. Conclusions: These interim data suggest that elderly and/or comorbid pts with R/R aggressive large B-cell NHL, who are not eligible for high-dose chemotherapy and HSCT, can receive 2L liso-cel with similar safety and efficacy to 3L+ pts as previously reported (Abramson, ASH 2019 #241). Updated data with longer follow-up will be presented. Clinical trial information: NCT03483103 . [Table: see text]

The IL-2/IL-2R pathway is implicated in type 1 diabetes (T1D). While its role in regulatory T cell (Treg) biology is well characterized, mechanisms that influence IL-2 responses in effector T cells (Teff) are less well understood. We compared IL-2 responses in 95 healthy control and 98 T1D subjects. In T1D, low IL-2 responsiveness was most pronounced in memory Teff. Unlike Treg, CD25 expression did not influence the Teff responses. Reduced IL-2 responses in memory Teff were not rescued by resting, remained lower after activation and proliferation, and were absent in type 2 diabetes. Comparing basal IL-2 responses in resting versus activated cells, memory Teff displayed lower, but more sustained, responses to IL-2 overtime. These results suggest that T1D-associated defects in the Teff compartment are due to intrinsic factors related to activation. Evaluation of both Teff and Treg IL-2R signaling defects in T1D subjects may inform selection of therapies.

Background: Patients (pts) with R/R aggressive large B cell NHL who fail first-line therapy with immunochemotherapy and are ineligible for high-dose chemotherapy and hematopoietic stem cell transplantation (HSCT) have a poor prognosis. Available treatment options include platinum/gemcitabine-based or bendamustine-based regimens in combination with rituximab, with or without radiotherapy, or clinical trials. However, long-term outcomes remain poor due to lack of a curative option. Liso-cel is an investigational, anti-CD19, defined composition, 4-1BB CAR T cell product administered at target doses of CD4+ and CD8+ CAR T cells. In the ongoing TRANSCEND NHL 001 study of liso-cel as third- or later-line treatment for pts with R/R large B cell NHL, preliminary data showed high overall response rates with a low incidence of grade ≥3 cytokine release syndrome (CRS) and neurological events (NEs) (Abramson et al, ASCO 2018). The open-label, phase 2 PILOT study is assessing the safety and efficacy of liso-cel as second-line therapy in TNE pts (NCT03483103). PILOT is the first study evaluating CAR T cell therapy focusing on this pt population. Methods: Eligible pts had R/R large B cell NHL (diffuse large B cell lymphoma [DLBCL], not otherwise specified [NOS], de novo or transformed indolent NHL, high-grade lymphoma with MYC and BCL2 and/or BCL6 [double/triple-hit lymphoma], or follicular lymphoma (FL) grade 3B) and had received only 1 prior line of immunochemotherapy containing an anthracycline and a CD20-targeted agent (eg, R-CHOP). Pts had to be deemed ineligible for high-dose chemotherapy followed by HSCT by meeting at least 1 of the following TNE criteria while still fulfilling the criteria for CAR T cell therapy: age ≥70 years, ECOG PS of 2, and/or impaired pulmonary (DLCO ≤60% but SaO2 ≥92% on room air and CTCAE ≤1 dyspnea), cardiac (LVEF ≥40% and 30 and 2 and ≤5 ×ULN). Liso-cel was administered at a target dose of 100×106 CAR+ T cells after lymphodepletion (LD) with fludarabine/cyclophosphamide for 3 days. Pts could be treated as outpatients at the investigator's discretion. Results: At data cutoff, 10 pts had been leukapheresed, and 9 pts had LD followed by liso-cel infusion; 1 pt is awaiting liso-cel treatment. Liso-cel was manufactured successfully in all pts. Five pts were infused and monitored as outpatients. Median age was 71 (range, 64-79) years; 5 pts were male. Histology included DLBCL NOS (n=7) and transformed FL (n=2); 2 pts had triple-hit, one of whom had transformed from FL. Five pts had relapsed from, and 4 pts had disease refractory to, prior therapy. Median SPD and LDH were 26.6 cm2 and 201 U/L, respectively. Four pts had high tumor burden with SPD ≥50 cm2 (n=4) and/or LDH ≥500 U/L (n=1). The median Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) score was 3 (range, 0-3). Six pts had 1 or more treatment-emergent adverse events (TEAEs) grade ≥3, which were primarily cytopenias. Three pts had prolonged grade ≥3 cytopenias at Day 29. Two pts had infections of any grade; no pts had grade ≥3 infections. No pts had CRS or NEs, and no pts received tocilizumab, corticosteroids, or vasopressors. There were no cases of macrophage activation syndrome, tumor lysis syndrome, infusion reactions, or grade 5 TEAEs. Among the 5 pts treated and monitored as outpatients, none were admitted to hospital for adverse events within the first 29 days post liso-cel infusion. All 9 pts achieved an objective response. Four pts achieved complete response; all are ongoing. Five pts achieved partial response (PR), with 2 PRs ongoing. Results were similar in inpatient vs outpatient pts. Median follow-up was 3.5 months. Median (range) time to peak CAR T cell expansion was 10 (7-21) days. Conclusions: These preliminary safety and efficacy data from the ongoing phase 2 PILOT study suggest that liso-cel can be successfully administered, including in the outpatient setting, as second-line therapy in pts with R/R aggressive B cell NHL who were ineligible for high-dose chemotherapy and HSCT by prespecified criteria. Updated safety and efficacy data with longer follow-up will be presented. Disclosures Sehgal: Kite/Gilead: Research Funding; Merck: Research Funding; Juno/Celgene: Research Funding. Pribble:Celgene/Juno: Employment. Wang:Celgene Corporation: Employment. Thorpe:Juno Therapeutics, a Celgene Company: Employment, Equity Ownership. Hildebrandt:Axim Biotechnologies: Equity Ownership; Abbvie: Equity Ownership; GW Pharmaceuticals: Equity Ownership; Endocyte: Equity Ownership; Clovis Oncology: Equity Ownership; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; CVS Health: Equity Ownership; Celgene: Equity Ownership; Axim Biotechnologies: Equity Ownership; Pharmacyclics: Research Funding; Sangamo: Equity Ownership; Cellectis: Equity Ownership; Bluebird Bio: Equity Ownership; Bristol-Myers-Squibb: Equity Ownership; crispr therapeutics: Equity Ownership; IDEXX laboratories: Equity Ownership; Johnson & Johnson: Equity Ownership; Pfizer: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Procter & Gamble: Equity Ownership; Vertex: Equity Ownership; Scotts-Miracle: Equity Ownership; Takeda: Research Funding; Bayer: Equity Ownership; Astellas: Other: Travel; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Novartis: Equity Ownership; Aetna: Equity Ownership; Juno Therapeutics: Equity Ownership; Cardinal Health: Equity Ownership; Novartis: Equity Ownership; Insys Therapeutics: Equity Ownership; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Immunomedics: Equity Ownership.

7501 Background: Eradication of MRD in CLL patients may be necessary for deep and durable responses. We assessed safety, pharmacokinetics, and efficacy of liso-cel, an investigational, anti-CD19 CAR T cell product administered as a defined composition of CD4+/CD8+ CAR T cells, in the ongoing phase 1/2 TRANSCEND CLL 004 study. Methods: Eligible pts had CLL/SLL, received ≥2 prior lines of therapy (including Bruton’s tyrosine kinase inhibitors [BTKi] unless medically contraindicated), and had ECOG PS ≤1. Post lymphodepleting chemotherapy, pts received liso-cel infusion at either dose level (DL)1 = 50 × 106 or DL2 = 100 × 106 total CAR+ T cells. Patients were monitored for dose-limiting toxicities (DLTs). Response was assessed by iwCLL 2008 criteria. MRD was assessed by flow cytometry in blood (10−4) and by NGS in bone marrow (BM; 10−6). Results: At data cutoff, 16 pts received liso-cel: DL1, n = 6; DL2, n = 10. 75% of pts had high-risk features ( TP53 mutation, complex karyotype, or del17p); 100% had prior ibrutinib and 50% had prior venetoclax. Median (range) number of prior lines of therapy was 4.5 (2‒11). There was 1 DLT of grade (G) 4 hypertension (DL2). The most common G3/4 treatment-emergent adverse events were cytopenias (thrombocytopenia, 75%; anemia, 69%; neutropenia, 63%; leukopenia, 56%). 1 pt had G3 cytokine release syndrome (CRS); 3 pts had G3 neurological events (NE). Best overall response rate (ORR) in 15 evaluable pts was 87% (13/15). 7 pts (47%) achieved complete remission with/without complete blood count recovery (CR/CRi). ORR at 6 mo was 83% (5/6). 10/15 pts (67%) achieved undetectable MRD (uMRD) in blood by day 30 and in 7/8 pts (88%) in BM. MRD-negative CRs were seen in patients who had failed both BTKi and venetoclax. Median time to peak blood CAR+ T cell level was 16 days (4‒30). Conclusions: In this study of heavily pretreated pts with standard- and high-risk CLL/SLL and previous ibrutinib treatment, liso-cel-related toxicities (ie, CRS and NEs), were manageable. Pts rapidly achieved CR/CRi and uMRD. Additional follow-up will be presented. Clinical trial information: NCT03331198.

Previous results from two proficiency panels of intracellular cytokine staining (ICS) from the Cancer Immunotherapy Consortium and panels from the National Institute of Allergy and Infectious Disease and the Association for Cancer Immunotherapy highlight the variability across laboratories in reported % CD8+ or % CD4+ cytokine-positive cells. One of the main causes of interassay variability in flow cytometry-based assays is due to differences in gating strategies between laboratories, which may prohibit the generation of robust results within single centers and across institutions. To study how gating strategies affect the variation in reported results, a gating panel was organized where all participants analyzed the same set of Flow Cytometry Standard (FCS) files from a four-color ICS assay using their own gating protocol (Phase I) and a gating protocol drafted by consensus from the organizers of the panel (Phase II). Focusing on analysis removed donor, assay, and instrument variation, enabling us to quantify the variability caused by gating alone. One hundred ten participating laboratories applied 110 different gating approaches. This led to high variability in the reported percentage of cytokine-positive cells and consequently in response detection in Phase I. However, variability was dramatically reduced when all laboratories used the same gating strategy (Phase II). Proximity of the cytokine gate to the negative population most impacted true-positive and false-positive response detection. Recommendations are provided for the (1) placement of the cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and 6) proper adjustment of the biexponential scaling.

Biologic treatment of type 1 diabetes (T1D) typically results in transient stabilization of C-peptide levels (a surrogate for endogenous insulin secretion) in some patients, followed by progression at the same rate as in untreated control groups. We used integrated systems biology and flow cytometry approaches with clinical trial blood samples to elucidate pathways associated with C-peptide stabilization in T1D patients treated with the anti-CD3 monoclonal antibody teplizumab. We identified a population of CD8 T cells that accumulated in patients with the best response to treatment (responders) and showed that these cells phenotypically resembled exhausted T cells by expressing high levels of the transcription factor EOMES, effector molecules, and multiple inhibitory receptors (IRs), including TIGIT and KLRG1. These cells expanded after treatment, with levels peaking after 3 to 6 months. To functionally characterize these exhausted-like T cells, we isolated memory CD8 TIGIT + KLRG1 + T cells from responders and showed that they exhibited expanded T cell receptor clonotypes (indicative of previous in vivo expansion), recognized a broad-based spectrum of environmental antigens and autoantigens, and were hypoproliferative during polyclonal stimulation, increasing expression of IR genes and decreasing cell cycle genes. Triggering these cells with a recombinant ligand for TIGIT during polyclonal stimulation further down-regulated their activation, demonstrating that their exhausted phenotype was not terminal. These findings identify and functionally characterize a partially exhausted cell type associated with response to teplizumab therapy and suggest that pathways regulating T cell exhaustion may play a role in successful immune interventions for T1D.

BACKGROUND: Identification of T-cell reactivity to β-cell antigen epitopes is an important goal for studying pathogenesis and for designing and monitoring of immunotherapeutic interventions in type 1 diabetes (T1D). METHODS: We performed a multicentre validation of known human leukocyte antigen (HLA) class I CD8+ T-cell epitopes. To this end, peripheral blood T-cell responses were measured in 35 recently (

Introduction Treatment of CLL is rapidly evolving, now including oral targeted agents and novel combinations. However, complete response (CR) rates remain low and continuous therapy is required. Eradication of minimal residual disease (MRD) is an increasingly important endpoint and an independent predictor of improved survival. Lisocabtagene maraleucel (liso-cel; JCAR017) is an investigational CD19-directed 4-1BB CAR T cell product administered in a defined composition of CD8:CD4 CAR T cells. TRANSCEND CLL 004 is an open-label Phase 1/2 trial of liso-cel in patients (pts) with relapsed/refractory (R/R) CLL (NCT03331198). Preliminary safety, pharmacokinetic (PK), and efficacy results from the Phase 1 monotherapy dose-finding portion of this study are reported. Methods Pts with CLL/SLL were eligible if they had received 3 (standard risk) or 2 [high risk: del(17p), TP53 mutation, unmutated IGVH, or complex karyotype] prior lines of therapy, including a Bruton's tyrosine kinase inhibitor (BTKi) unless medically contraindicated. Pts with active untreated CNS disease, ECOG >1, or Richter's transformation were excluded. After 3 days of lymphodepletion with fludarabine and cyclophosphamide, pts received liso-cel infusion. Two dose levels (DL) have been tested (DL1=5 × 107 CAR T cells; DL2=1 × 108 CAR T cells). Dose escalation followed a modified toxicity probability-interval-2 (mTIPI-2) algorithm. Dose-limiting toxicities (DLTs) were evaluated for 28 days post liso-cel infusion. Responses were assessed by iwCLL 2008 criteria. MRD was assessed at 10-4 sensitivity by 6-color flow cytometry using peripheral blood and at 10-6 sensitivity by clonoSEQâ (Adaptive) deep sequencing of bone marrow (BM) aspirates. Blood PK of liso-cel was determined using flow cytometry. Serum soluble chemokine and cytokine profiles for 39 analytes were assessed using V-PLEX immunoassays (MSD). Results At the time of data cut, 10 pts received liso-cel: 6 pts treated with DL1 and 4 pts with DL2. The median age was 64.5 years (range 51-76); 7/10 pts had high-risk disease. Pts had received a median of 4 prior therapies (range 3-8), including 9/10 pts who had received prior ibrutinib and 6/10 who previously received venetoclax and ibrutinib. No DLTs were identified. The most common adverse events (AEs) were cytokine release syndrome (CRS) (8/10 pts; all grade [G] 1/2), anemia (7/10 pts), thrombocytopenia (6/10 pts), and leukopenia (5/10 pts). Neurologic events (NE) were reported in 3/10 pts: G1 impaired concentration and aphasia, G3 encephalopathy, and G3 aphasia. The median time to onset of CRS and NE was 4.5 (range 1-9) and 11 (range 11-21) days respectively, and the median duration of CRS and NE was 5.5 (range 3-30) and 6 (range 2-20) days respectively. Six pts received tocilizumab and/or steroids for the management of CRS and/or NE. Serious AEs, all of which were G3/4, were reported in 5/10 pts. At 30 days post-dose, 6 of 8 pts who were evaluable for response had an objective response (75%), including 4 CRs (50%). Six of 7 pts (85.7 %) evaluable for MRD had undetectable disease by flow at the day 30 assessment. Of the 5 pts evaluable for response at 3 months post-dose, 4 had ongoing response and 1 progressed with Richter's transformation. All 4 pts with ongoing response continued to have undetectable MRD by flow at 3 months post-dose. Responses, including CRs and undetectable MRD responses, occurred in pts with high-risk and with standard-risk disease. Available BM analyses from clonoSEQâ data corroborate these findings and will be reported. Median Cmax was 219 CAR T cells/µl (range 0.35-583.46). Median time to peak expansion was 15.5 days (range 13-19) and median AUC was 1528 cells*day/μL (range 590-2847). In the one pt with a best response of progressive disease, minimal CAR T cell expansion was observed. CAR T cells persisted in pts maintaining their response at 3 months post-dose. Serum analysis showed elevated levels of multiple biomarkers, including CRP, IL6, PLGF, IL16, and IL15, in conjunction with CRS and NE. Conclusion Liso-cel toxicities were manageable, including events of CRS and NE, in these heavily pretreated pts with CLL. CRs and undetectable MRD were rapidly achieved in pts with both high-risk and standard-risk CLL who previously received ibrutinib, with the majority also having had received venetoclax. These preliminary data support continued development of single-dose liso-cel treatment in CLL. Disclosures Siddiqi: Juno Therapeutics: Other: Steering committee. Wierda:Genentech: Research Funding; AbbVie, Inc: Research Funding. Dubovsky:Juno Therapeutics: Employment, Equity Ownership. Gillenwater:Juno Therapeutics: Employment, Equity Ownership. Gong:Juno Therapeutics: Employment, Equity Ownership. Mitchell:Juno Therapeutics: Employment, Equity Ownership. Thorpe:Juno Therapeutics: Employment, Equity Ownership. Yang:Juno Therapeutics: Employment, Equity Ownership.

Fate determination for autoreactive T cells relies on a series of avidity-dependent interactions during T cell selection, represented by two general types of signals, one based on antigen expression and density during T cell development, and one based on genes that interpret the avidity of TCR interaction to guide developmental outcome. We used proinsulin-specific HLA class II tetramers to purify and determine transcriptional signatures for autoreactive T cells under differential selection in type 1 diabetes (T1D), in which insulin (INS) genotypes consist of protective and susceptible alleles that regulate the level of proinsulin expression in the thymus. Upregulation of steroid nuclear receptor family 4A (NR4A) and early growth response family genes in proinsulin-specific T cells was observed in individuals with susceptible INS-VNTR genotypes, suggesting a mechanism for avidity-dependent fate determination of the T cell repertoire in T1D. The NR4A genes act as translators of TCR signal strength that guide central and peripheral T cell fate decisions through transcriptional modification. We propose that maintenance of an NR4A-guided program in low avidity autoreactive T cells in T1D reflects their prior developmental experience influenced by proinsulin expression, identifying a pathway permissive for autoimmunity.

Previous results from two proficiency panels of intracellular cytokine staining (ICS) from the Cancer Immunotherapy Consortium and panels from the National Institute of Allergy and Infectious Disease and the Association for Cancer Immunotherapy highlight the variability across laboratories in reported % CD8+ or % CD4+ cytokine-positive cells. One of the main causes of interassay variability in flow cytometry-based assays is due to differences in gating strategies between laboratories, which may prohibit the generation of robust results within single centers and across institutions. To study how gating strategies affect the variation in reported results, a gating panel was organized where all participants analyzed the same set of Flow Cytometry Standard (FCS) files from a four-color ICS assay using their own gating protocol (Phase I) and a gating protocol drafted by consensus from the organizers of the panel (Phase II). Focusing on analysis removed donor, assay, and instrument variation, enabling us to quantify the variability caused by gating alone. One hundred ten participating laboratories applied 110 different gating approaches. This led to high variability in the reported percentage of cytokine-positive cells and consequently in response detection in Phase I. However, variability was dramatically reduced when all laboratories used the same gating strategy (Phase II). Proximity of the cytokine gate to the negative population most impacted true-positive and false-positive response detection. Recommendations are provided for the (1) placement of the cytokine-positive gate, (2) identification of CD4+ CD8+ double-positive T cells, (3) placement of lymphocyte gate, (4) inclusion of dim cells, (5) gate uniformity, and 6) proper adjustment of the biexponential scaling.