Juno Therapeutics (Germany)

Abstract Study question Can observation through Time Lapse Microscopy (TLM) be leveraged to standardize trophectoderm biopsy outcomes across different operators? Summary answer TLM-based blastocysts’ observation to pinpoint the ideal time-of-biopsy (t-biopsy) indicated comparable inconclusive PGT-A results rates among seven embryologists operating across eight years. What is known already Optimal timing for trophectoderm biopsy in blastocysts lacks clear guidelines, with current literature presenting mixed outcomes regarding its effect on molecular analysis reliability. Inappropriate biopsy timing can potentially yield inconclusive PGT diagnoses due to insufficient blastocyst expansion. Minimizing the need for a second biopsy is essential, as multiple manipulations can affect embryo reproductive competence. Thus, TLM and artificial intelligence (AI) offer valuable tools to objectively define biopsy timing based on blastocyst expansion. Study design, size, duration Observational analysis of 1,943 blastocysts cultured in TLM between 2013-2020 and biopsied with a sequential zona-pellucida opening and trophectoderm biopsy approach. Seven operators were trained to establish the stage of biopsy based on blastocysts’ full-expansion achieved between 110-169 hours-post-insemination (hpi; mean:134±15). AI (CHLOE, Fairtility) was retrospectively applied to annotate all timings and expansion as area of the embryo-proper at the median focal plane in µm2 at the time-of-expanding-blastocyst (tEB) and at the t-biopsy. Participants/materials, setting, methods We assessed all features possibly associated with the inconclusive result rate after trophectoderm biopsy: biopsy-operator, t-biopsy and embryo-proper area, blastocyst quality (based on Gardner and CHLOE embryo-quality score), day of the week (Monday-Sunday), hour of the working day (retrieved through RI electronic-witnessing-system; 8am-8pm), year (2013-2020), comprehensive-chromosome-testing technology (qPCR/NGS). Associations with euploidy and live-birth (LB) per euploid transfer were also tested. Logistic regression and Generalized Estimating Equations analyses were adopted to this end. Main results and the role of chance The overall inconclusive results rate was 3.3% (N = 64/1,943) varying 1%-5% across operators and years. The largest fluctuation occurred when transitioning from qPCR (2013-Oct2017; N = 35/1,222, 2.9%) to NGS (Oct2017-2020; 4%), but decreased to 2.3% (N = 2/88) in 2020. These differences were not-significant, suggesting that fluctuations can be minimized by monitoring operators’ performance systematically. Among all parameters investigated, none associated with inconclusive results. The embryo-proper area at t-biopsy was larger for day6-7 blastocysts (unstandardized coefficient-B day5 to day7:+730µm2, 95%CI:+301 to + 1,158,p<0.001), and smaller for poorer-quality blastocysts (unstandardized coefficient-B AA-grade to CC/BC/CB-grade:-1,115µm2, 95%CI:-1,332 to -897,p<0.001). The year was associated with embryo-proper area at t-biopsy (unstandardized coefficient-B 2013 to 2020:+309µm2, 95%CI:+168 to + 450,p<0.001) becoming significantly larger since 2017 (22,000-23,500µm2 to 24,000-25,000µm2), when NGS was implemented. No difference was reported among biopsy-operators, days of the week, nor hours of the working-day, except for sporadic cases of blastocysts biopsied between 8-9am showing lower expansion. Dispersion plots indicated more pronounced expansion from tEB to t-biopsy among day5 blastocysts, while this appeared less evident among day6-7 blastocysts. When adjusted for maternal-age and blastocyst-quality, embryo-proper area was associated with higher euploidy-rates (each 500µm2-increase OR:1.17,95%CI:1.07-1.28). Only blastocyst-quality and t-biopsy, instead, associated with LB per euploid-transfer. Limitations, reasons for caution Retrospective study to be confirmed prospectively with a larger sample size. Only the protocol entailing simultaneous laser-assisted zona pellucida opening and trophectoderm biopsy plus pulling was adopted. Wider implications of the findings The adoption of standard operating procedures and the continuous monitoring of operators’ performance minimized fluctuations in inconclusive PGT-A results, even when changing CCT technology or introducing new biopsy practitioners. TLM and AI can help monitoring and defining the optimal t-biopsy based on embryo-proper area, enhancing standardization, efficiency and safety. Trial registration number No

Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.

Introduction: Chimeric antigen receptor (CAR) T cell therapy has generally been limited to inpatient administration at university medical centers. Among US patients (pts) treated for relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL), about 80% receive treatment at nonuniversity medical centers where outpatient therapy is often at a distinct site from hospitalization for adverse event management. In the ongoing TRANSCEND NHL 001 study in R/R DLBCL, lisocabtagene maraleucel (liso-cel, JCAR017), an investigational, anti-CD19 CAR T cell product administered at a precise dose of CD4+/CD8+ CAR T cells, showed high overall response rates with a favorable safety profile (Abramson et al, ASCO 2018). Late onset and low rates of all grade and severe cytokine release syndrome (CRS) and neurological events (NE) support outpatient treatment with hospital admission at the first sign of fever. We report on 2 newly initiated open-label, phase 2 trials that allow outpatient treatment, assessing the safety and efficacy of liso-cel either as third-line therapy (OUTREACH; NCT03744676) or in second-line, transplant noneligible pts (PILOT; NCT03483103). Methods: In both trials, eligible pts had R/R aggressive B-cell NHL, adequate organ function, and prior systemic chemoimmunotherapy (OUTREACH: ≥2 prior lines of therapy and ECOG PS ≤1; PILOT: 1 prior line of therapy and deemed ineligible for autologous hematopoietic stem cell transplantation based on ECOG PS, organ function, and/or age). Liso-cel was administered at 100 × 106 total CAR+ T cells after lymphodepletion (LD) with fludarabine/cyclophosphamide. Both trials allowed outpatient treatment at nonuniversity (OUTREACH) or at both university and nonuniversity medical centers (PILOT). Outpatient treatment required pts to have a caregiver, safety monitoring education, and to stay close to the site of care. All sites were required to have a multidisciplinary CAR T cell team identified and standard operating procedures for outpatient administration and monitoring in place. Results: At data cutoff, 15 pts were leukapheresed (OUTREACH n=7; PILOT n=8). Age ranged from 60–79 years; 11 pts were female. 13 pts had DLBCL NOS, 2 had DLBCL transformed from indolent lymphoma. 7 pts had LD followed by liso-cel infusion, including 3 outpatients. The remaining 8 pts are awaiting liso-cel treatment. Among the 4 pts with 30 days of follow-up, no CRS, NE, or febrile neutropenia were reported. There were no grade ≥3 nonhematologic treatment-emergent adverse events (TEAEs), grade 5 TEAEs, or ICU admissions. Objective responses were observed in 4/4 pts (1 complete remission; 3 partial remissions). Updated enrollment, incidence of toxicity, intervention for CRS/NE and hospitalizations with longer follow-up data will be presented. Conclusions: In these ongoing phase 2 studies in R/R NHL, pts were successfully treated with liso-cel in the outpatient setting. Keywords: B-cell lymphoma; CD19; non-Hodgkin lymphoma (NHL). Disclosures: Sehgal, A: Research Funding: Juno/Celgene, Kite/Gilead, Merck. Hildebrandt, G: Consultant Advisory Role: Pfizer, Kite Pharma, Incyte, JAZZ Pharmaceuticals; Stock Ownership: Sangamo Bioscience, Juno Therapeutics, Kite Pharma, Novartis, Insys Therapeutics, Abbvie, GW Pharmaceuticals, Cardinal Health, Immunomedics, Endocyte, Clovis Oncology, Cellectis, Aetna, CVS Health, Celgene, Bluebird Bio, Bristol-Myers Squibb / Medarex, crispr therapeutics, IDEXX laboratories, Johnson & Johnson, Pfizer, Procter & Gamble, Vertex, JAZZ Pharmaceuticals; Research Funding: Takeda, JAZZ Pharmaceuticals, Pharmacyclics; Other Remuneration: Travel, expenses, accommodation: Kite Pharma, Incyte, Pfizer, Falk Foundation, JAZZ Pharmaceuticals, Astellas Pharma. Trede, N: Employment Leadership Position: Celgene; Stock Ownership: Celgene. Kostic, A: Employment Leadership Position: Celgene; Stock Ownership: Celgene. Wang, L: Employment Leadership Position: Celgene Corporation; Stock Ownership: Celgene Corporation. Lymp, J: Employment Leadership Position: Celgene; Stock Ownership: Celgene. Bachier, C: Honoraria: Sanofi.

Abstract Background: Adoptive T-cell therapy with engineered T-cell receptors (eTCR) has demonstrated promising, yet modest clinical benefit to date. Key obstacles for this technology are 1) competition with the endogenous TCR for CD3 components that are required for surface expression of a functional TCR complex and 2) mispairing of endogenous and exogenous TCRα and TCRβ chains. Substituting murine TCR constant domains has been shown to enhance eTCR expression, but increases the risk of immunologic rejection. Knockout or knockdown of the endogenous TCR has also been shown to improve eTCR expression. Current eTCR delivery approaches use semi-randomly integrating lentivirus or retrovirus to generate an eTCR expressing T-cell product. An alternative approach is to combine TCR knockout with targeted integration of eTCRs into the T-cell receptor alpha constant (TRAC) locus. In this study, we evaluate newly discovered eTCRs specific for human papillomavirus (HPV) type 16 oncogenic protein E7, expressed via these various gene engineering approaches, to optimize engineered T-cell functionality. Methods: HPV E7-specific eTCRs were introduced into primary human T-cells by lentiviral transduction or a dual CRISPR-Cas9/AAV eTCR delivery platform for targeted insertion into the TRAC locus using homology directed repair (HDR). Comparisons were made among TCR sufficient and TCR knockouT-cells with gene delivery by lentivirus or HDR. Engineered T-cell function was assessed both in vitro and in vivo against HPV-16+ head and neck squamous cell carcinoma cell lines. Results: CRISPR/Cas9-mediated TRAC editing eliminated endogenous TCR expression in >85% of T-cells. The impact of TRAC editing on eTCR expression and engineered T-cell function was variable across multiple eTCRαβ sequences. With many eTCRs, TRAC editing in lentivirus-derived populations resulted in increased expression and improved cytokine and killing responses. Targeted insertion of these eTCRs into the TRAC locus by HDR increased engineered cell product homogeneity and enhanced function compared to lentiviral integration. However, there were also examples of eTCRs that were minimally impacted by TRAC locus engineering. Conclusions: Our results demonstrate that elimination of the endogenous TCR, alone or combined with targeted insertion at the TRAC locus, improves eTCR expression and engineered T-cell function for the majority of eTCRs tested. While infrequent, some eTCRs were less impacted by TRAC locus engineering, suggesting a sequence-specific property that enables these eTCRs to out-compete the endogenous TCR for CD3 componentry. The cause for this variable response to TRAC locus engineering is still being explored. Citation Format: Alexandra Croft, Cameron Brandt, Stephen Burleigh, Eric Chadwick, Melissa Chin, Dean Toy, Bailey Donahue, Clay Patton, Stephen Goldfless, Brian Belmont, Ruth Salmon, Grant Welstead, Blythe D. Sather, David J. Huss. Targeted insertion of an HPV-16 E7-specific engineered T-cell receptor into the TRAC locus [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A027.

We show that defined lymphocytes can be rapidly purified by immunoaffinity chromatography starting directly from whole blood. The method relies on low-affinity Fab-fragments attached to a column-matrix combined with the reversible Strep-tag technology. Compared to established cell enrichment protocols, the Strep-tag affinity chromatography of cells is independent of erythrocyte lysis or centrifugation steps, allowing for simple cell-enrichment with good yields, high purities, and excellent functionality of purified cells.

120 Background: JCAR017 is a defined composition, CD19-directed 4-1BB CAR T cell product administered at a precise dose of CD8 and CD4 CAR T cells in a seamless design Phase 1 pivotal trial of relapsed/refractory (R/R) B-cell NHL (TRANSCEND NHL 001; NCT02631044). Methods: Patients (pts) with R/R DLBCL NOS, PMBCL, FL grade 3B, or MCL and adequate organ function are eligible. The FULL dataset includes all pts in the DLBCL cohort (i.e. excludes MCL). The CORE dataset includes pts meeting inclusion for a planned pivotal DLBCL cohort (DLBCL NOS [ de novo or transformed from follicular lymphoma], ECOG 0-1, no prior allo-SCT). Results: As of July 7, 2017, 69 pts were treated in the DLBCL cohort and evaluable for safety. In the FULL dataset, 21 pts (30%) had cytokine release syndrome (CRS), with 1 serious CRS event (1%; Gr 4). Neurotoxicity (NT) at any grade developed in 14 (20%), including 10 (14%) with Gr 3-4 events. There were no Gr 5 CRS or NT events. Median time to onset of first CRS and NT was 5 days and 10 days, respectively, 13 (19%) received anti-cytokine therapy and only one required vasopressor support. In the CORE dataset (n = 49), similar rates of CRS and NT were shown. The majority of pts, 64% (44/69), had no CRS or NT, and median onset of CRS and NT were 5 and 11 days, respectively, suggesting outpatient delivery of JCAR017 may be feasible. In the DLBCL cohort, 68 pts were evaluable for efficacy; best overall response, 3-month, and 6-month response rates in the FULL dataset were 75% (51/68), 49% (27/55), and 40% (14/35), respectively; and, in the CORE dataset, were 84% (41/49), 65% (26/40), and 57% (13/23). The best overall, 3-month, and 6-month CR rates were 61% (30/49), 53% (21/40), and 52% (12/23), respectively, in the CORE dataset. Among 16 double/triple hit pts, best ORR was 81%, and 3-month CR rate was 60%. A trend toward improved response rate at 3 months was observed in pts treated at DL2 compared to DL1. Conclusions: At the symposium, we will report updated efficacy and the preliminary safety profile for JCAR017, which supports potential outpatient administration in R/R Aggressive B-NHL patients. Clinical trial information: NCT02631044.