ALBERT

Description: Background After European Medicines Agency (EMA) approval of axicabtagene ciloleucel and tisagenlecleucel for the treatment of relapsed/refractory (r/r) high-grade lymphoma in 2018, England was one of the first European countries granting fully funded access to these CD19 CAR-T therapies. Both products are available through the National Health Service England (NHSE) Cancer Drug Fund until their cost-effectiveness has been determined. The NHSE CAR-T program has been set up in a structure aiming to implement robust and transparent criteria for patient selection and to ensure equity of treatment access: CAR-T slots are approved by a weekly National CAR-T Clinical Panel (NCCP), consisting of independent clinical experts, patient representatives, and delegates from each CAR-T centre; treatment is delivered in 7 geographically spread commissioned CAR-T centres (Birmingham, Bristol, King's College Hospital London, University Hospital London, The Christie Manchester, Manchester Royal Infirmary, Newcastle). Here, we report prospective data on the first 122 lymphoma patients approved by the NCCP. Methods Patients with r/r high-grade lymphoma referred to the NCCP between December 2018 and July 2019 and deemed eligible for treatment with CD19 CAR-T were analysed. Eligibility was assessed in the CAR-T centre's tumor board, based on organ function and fitness (performance status 0/1), absence of active CNS disease, and biopsy confirmation of r/r high-grade lymphoma. The final decision on patient eligibility was made by consensus through the NCCP independent clinical panel. CAR-T product selection for each patient was done by the CAR-T centre, mainly on the basis of manufacturing slot availability. Results 122 patients were approved for treatment with CD19 CAR-T therapy by the panel. CAR-T centres selected 76 patients for axicabtagene ciloleucel and 46 for tisagenlecleucel. Patients' median age was 56 years (range 18-75). 62% were male. 87 (71%) patients had de novo diffuse large B-cell lymphoma, 29 (24%) transformed lymphoma (23 from follicular- and 6 from marginal zone lymphoma), and 6 (5%) primary mediastinal B-cell lymphoma. 96 (79%) patients had biopsy confirmation of disease prior to submission. 71 (58%) patients had received 2 prior lines of therapy for high-grade lymphoma, 51 (42%) patients 3 or more treatment lines (maximum 6). 5 patients had previous allogeneic, 19 previous autologous transplant. 88% of patients (107/122) were refractory to the last line of treatment (stable- or progressive disease (PD) or relapse within 6 months). Among 122 patients, 112 completed leukapheresis, 3 are awaiting the procedure, and 7 patients did not proceed (6 due to PD, 1 opted for radical radiotherapy). 57 of 112 patients were infused at the time of abstract submission, 42 are awaiting CAR-T infusion. 10 patients did not proceed to infusion due to disease progression and clinical deterioration (3 with CNS relapse), 2 due to manufacturing failure. One patient achieved a complete response following bridging therapy and is currently monitored. 84% (88/105) patients received bridging therapy between the time of NCCP approval and CAR-T infusion (median 64 days), 62 had chemotherapy, 9 radiotherapy, and 17 steroids only. Details on bridging therapy, treatment-related toxicities and outcomes will be provided at the meeting, by which time approximately 62 patients will have completed their 3 months PET response assessment. Conclusion NHSE has successfully implemented a national structure for providing licenced CAR-T products in England, enabling equity of access and oversight on capacity and patient outcomes, which can serve as a model for newly licenced, cost-intense and complex cell- and gene therapies in the future. The prospective and centralised nature of this dataset offers a true reflection of the real-world patient population undergoing CAR-T therapy in England. Disclosures Kuhnl: Kite Gilead: Honoraria. Roddie:Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Novartis: Consultancy. Menne:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Kyowa Kirin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Daiichi Sankyo: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astra Zeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel grant, Research Funding, Speakers Bureau. Sanderson:Kite/Gilead: Honoraria. Osborne:Novartis: Other: Travel; Pfizer: Honoraria, Speakers Bureau; MSD: Consultancy; Takeda: Consultancy, Honoraria, Other: Travel, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Speakers Bureau; Servier: Consultancy; Gilead: Consultancy. Radford:AstraZeneca: Equity Ownership, Research Funding; Takeda: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; ADC Therapeutics: Consultancy, Research Funding; GSK: Equity Ownership; Seattle Genetics: Consultancy, Honoraria. Patten:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria; Roche: Honoraria, Research Funding. O'Reilly:Kite Gilead: Honoraria. Bloor:Abvie, Gilead, Novartis, Autolus, Celgene, etc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational grant. Rowntree:Novartis: Consultancy. Bowles:Abbvie: Research Funding; Janssen: Research Funding. Collins:Gilead: Consultancy, Honoraria. McMillan:BMS: Honoraria; Celgene: Honoraria, Speakers Bureau; F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Gilead: Honoraria; Novartis: Honoraria; Sandoz: Honoraria; Pfizer: Honoraria, Research Funding; MSD: Honoraria.

Description: BACKGROUND: Multiple myeloma (MM) is the most common hematologic malignancy in the African American population, with an incidence more than 2 times higher than Caucasian population [Landgren O et al Blood 2006]. Historically, the African American MM patients have had better outcomes compared with other races [Ailawadhi S et al Br J Haematol 2012], but no biologic explanations exist for this observation. Greenberg et al [Blood Cancer J 2015] have recently reported on differences in commonly observed baseline cytogenetic abnormalities (CA) between African American and Caucasian MM patients seen at Mayo Clinic (Rochester, MN), Cook County Hospital (Chicago, IL) and University of Maryland (Baltimore, MD). We examined the MM cohort at our referral center to validate these observations. PATIENTS & METHODS: The Levine Cancer Institute MM database was interrogated for all patients presenting with MM between January 2012 and April 2015. Baseline clinical and pathology variables were compared between the African American and Caucasian cohorts. Continuous variables were compared using nonparametric rank tests, while incidences and proportions (e.g. CAs including t(11;14), t(4;14), monosomy13/del13q and del17p) were compared using Fisher's exact tests. RESULTS: A total of 662 patients were identified; excluding those with MGUS classification, 368 patients were included in the analysis (African Americans n = 130, Caucasian n = 238). The median age of African American MM patients was significantly younger than Caucasian MM patients (median age 60 years vs. 65 years, p=0.010), with similar gender distribution. There was a numerically larger proportion of African American patients with anemia (40.8% vs 30.8%, p =0.166), however, there was no significant difference in degree of BM plasmacytosis amongst the two groups. The overall distribution of MM patients by IMWG risk stratification (Chng et al, Leukemia 2013) was also similar between the two groups. The African American MM patients had a numerically higher incidence of a metaphase abnormality on conventional cytogenetics (21.7% vs. 13.9%, p =0.154). They had a significantly lower incidence of t(11;14) [7.7% vs. 16%, p=0.024], a numerically higher incidence of t(4;14) [6.2% vs. 3.8%, p=0.309], and similar incidence of deletion 13/del13q [22.3% vs. 18.9%, p=NS ] and del17p [7.7% vs. 7.6%, p=NS ]. CONCLUSIONS: The present dataset is the largest single institution report on CA racial differences in MM patients. We found that unlike previous reports of lower incidence of t(4;14) or del17 p in African American MM patients by Greenberg et al, we have observed a higher incidence of t(4;14) and similar incidence of del17p in our experience compared to Caucasian MM patients. The different pattern of CA distribution compared to published literature may represent geographic heterogeneity and potentially influence survival outcomes. Disclosures Cogdill: Celgene: Speakers Bureau; Onyx: Speakers Bureau; Millennium: Speakers Bureau; Novartis: Speakers Bureau. Usmani:Celgene: Honoraria, Speakers Bureau; Onyx: Honoraria, Research Funding, Speakers Bureau; Janssen Oncology: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Pharmacyclics: Research Funding; Millennium: Honoraria, Speakers Bureau; Array BioPharma: Honoraria, Research Funding.

Description: BACKGROUND: Multiple Myeloma (MM) is a heterogenous disease with variable outcomes based on both disease biology and burden. The revised international staging system (R-ISS) can distinguish 5-year overall survival (OS) however, it does not incorporate novel imaging that provide more accurate disease burden assessment (e.g., PET-CT). Previous studies (Usmani SZ et al Blood 2013) showed that the presence of more than 3 focal lesions (FL)s detected on PET-CT scan at baseline in newly diagnosed MM patients, or the persistence of any FL post induction therapy or post- autologous stem cell transplantation (ASCT), were associated with shorter progression free survival (PFS) and overall survival (OS) in Total Therapy trials. There is limited information available on the prognostic significance of PET-CT in the context of current standard 3-drug induction followed by ASCT in clinical practice. Herein, the investigators examined the prognostic implications of post-therapy PET-CT positivity in routine clinical practice. PATIENTS AND METHODS: The LCI MM database was interrogated (3/2014-5/2018) for NDMM patients who had undergone upfront ASCT and had PET-CT data available. Continuous variables were summarized with descriptive statistics, while categorical variables were summarized with frequencies and proportions. Survival distributions were estimated with Kaplan Meier techniques and compared using a log rank test PET-CT scan results were reported as negative or positive (defined as one FL having higher 18F-FDG uptake relative to uptake in mediastinum or surrounding background, with no similar activity seen on the contralateral side, or increased activity at any location incompatible with normal physiological distribution). RESULTS: A total of 198 MM patients were identified, 62 had pre-ASCT PET-CT scans and 80 patients had post-ASCT PET-CT. Median age at transplant was 62 years (range 25 - 77), 52.0% were female. R-ISS stage I (17.2%), stage II (41.4%), stage III (1.7%). The 3 years overall survival (OS) in patients with negative PET-CT post-ASCT vs. positive PET-CT post-ASCT was 96.6% vs. 74.2% (p-value 0.002) and median progression free survival (PFS) was 35 months vs. 13 months (p-value

Description: Background: Targeting B-cell maturation antigen (BCMA) with antibody-drug conjugates (ADCs), bispecific antibodies, or chimeric antigen receptor t-cells (CAR-Ts) has proven safe and effective in recent clinical trials, but relapses remain common. As most patients treated with BCMA targeting therapies are refractory to conventional anti-myeloma therapies, management of these patients poses unique challenges once they progress, with no data available to guide subsequent therapies. Methods: We performed a retrospective chart review of all relapsed refractory multiple myeloma (RRMM) patients at our institution who progressed while on or after a BCMA targeting therapy and were treated with subsequent therapies. We evaluated the best response achieved and overall survival (OS) measured from progression on BCMA targeting therapies. Kaplan Meier methods were used to estimate OS curves and landmarks between classes of BCMA targeting therapy received (ADC, bispecific antibody or CAR-T), and by type of subsequent therapy. Results: At a median follow up of 6 months, a total of 47 patients were treated with a BMCA targeting therapy. Of those, a total of 21 (44.7%) patients have progressed, with 18 (38.3%) receiving another therapy. Twelve-month overall survival of the patients who received a subsequent treatment was 51.1% (figure 1a), but varied considerably based on the class of BCMA therapy they received (figure 1b). Patients who progressed after a BCMA CAR-T had the best OS (N =2, 6 mo OS: 100%, 12 mo OS: Of the 18 patients who progressed and were treated with subsequent therapies, 7 (38.9%) received 2 lines of therapy, 5 (27.8%) received 3 lines of therapy, and 1 patient (5.6%) received 5 lines of therapy. In the first relapse, 4 (22.2%) patients received infusional chemotherapy with CAR-D PACE or CAR-DCEP, 4 (22.2%) received the combination of elotuzumab, pomalidomide, and dexamethasone (Elo-Pd; one of which was first treated with CAR-DCEP), 3 (16.7%) received selinexor based regimens. The best response seen after first-line post BCMA treatment was a partial response (PR) in 5 (27.8% of patients), whereas 8 (61.5%) patients who received second-line treatment post-BCMA therapy had a PR or better, including 3 (23.1%) who had a very good partial response (VGPR). In the third line post-BCMA, 1 (16.7%) had a VGPR, while 1 (16.7%) had stable disease as their best response. The use of Elo or Dara after anti-BCMA progression seemed to correlate with improved OS (see figure 1c below). While all these patients were Elo naïve, the majority (94.4%) were previously Dara exposed. Conclusions: Our data demonstrate that many RRMM patients who progress on BCMA targeting therapies still derive benefit from subsequent treatment. Early evidence from our experience suggests a survival advantage with monoclonal antibody-based therapies even in patients who had previously been exposed to these agents-suggesting a possible resensitization with BCMA directed therapy. Although our dataset is a single-center experience, to our knowledge it represents the first report of post-BCMA exposed management of RRMM and provides valuable insight into the treatment of this challenging and ever-expanding population. Disclosures Paul: Bristol-Myers Squibb: Other: Stock Ownership (prior employee); Amgen: Consultancy, Speakers Bureau; Regeneron: Membership on an entity's Board of Directors or advisory committees. Bhutani:BMS: Other: Clinical trial funding to institute, Speakers Bureau; Takeda: Other: Clinical trial funding to institute, Speakers Bureau; Prothena: Other: Clinical Trial Funding to Institute; Amgen: Speakers Bureau; MedImmune: Other: Clinical Trial Funding to Institute; Sanofi Genzyme: Consultancy; Janssen: Other: Clinical Trial Funding to Institute. Voorhees:Adaptive Biotechnologies: Other: Personal fees; Bristol-Myers Squibb: Other: Personal fees; Celgene: Other: Personal fees; Janssen: Other: Personal fees; Novartis: Other: Personal fees; Oncopeptides: Other: Personal fees; TeneoBio: Other: Personal fees; Levine Cancer Institute, Atrium Health: Current Employment. Usmani:Celgene: Other; Janssen: Consultancy, Honoraria, Other: Speaking Fees, Research Funding; SkylineDX: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Incyte: Research Funding; Pharmacyclics: Research Funding; Array Biopharma: Research Funding; GSK: Consultancy, Research Funding; Takeda: Consultancy, Honoraria, Other: Speaking Fees, Research Funding; Sanofi: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy; BMS, Celgene: Consultancy, Honoraria, Other: Speaking Fees, Research Funding; Amgen: Consultancy, Honoraria, Other: Speaking Fees, Research Funding. Atrash:BMS, Jansen oncology, Sanofi: Speakers Bureau; Takeda, Amgen, Karyopharm, BMS, Sanofi, Cellactar, Janssen and Celgene: Honoraria; Amgen, GSK, Karyopharm.: Research Funding.

Description: Background: In multiple myeloma, induction therapy (IT) before hematopoietic progenitor cells (HPC) collection reduces the tumor burden, improves the quality of the collection, and diminishes end-organ damage. The data concerning the impact of the response to IT on progression-free survival (PFS) after autologous stem cell transplant (ASCT) remains limited. IFM 2005-01 reported better PFS in patients who achieved at least a very good partial response (VGPR) after IT (PFS post-ASCT 41 vs. 31 months, p= 0.01). Also, studies have consistently shown that minimal residual disease (MRD) negativity impacted PFS/ overall survival (OS). Currently, rates of CR and MRD-negativity post-ASCT are sub-optimal. Given the clinical activity and the safety profile of daratumumab (Dara), evaluation of this novel agent pre- and post-ASCT is warranted, as it may improve the post-ASCT ≥ CR and MRD-negativity rates. Study Design and Methods: This study is a single-arm, two-stage phase II trial to estimate the CR rate post-ASCT in newly diagnosed MM (NDMM). Transplant eligible (TE) NDMM subjects who did not achieve at least a VGPR post initial IT are eligible for the trial. Enrollment will be planned post-induction and before HPC mobilization. Subjects will receive four weekly doses of Dara prior to HPC mobilization, then another four weekly doses of Dara after HPC engraftment post-ASCT. The primary objective is to evaluate the ≥ CR rate post-ASCT in NDMM subjects who did not achieve at least a VGPR post-induction. The secondary objectives are to estimate: ≥VGPR rate, time to first response (TTFR), time to best response (TTBR), duration of response (DoR), PFS, time to progression (TTP), time to next treatment (TTNT), and OS. Also, the study will evaluate toxicities related to Dara and ASCT outcomes (HPC collection and engraftment parameters). The exploratory objectives are to explore: MRD rates of bone marrow and HPC product using Euro-flow criteria and DNA-PCR.The correlation between systemic immune profiling and the clinical response using cytokine profiling by multiplex protein assay and Blood immunotyping [including NK, NK-T, and T cell subsets distribution and activation analyses by flow cytometry.The correlation between circulating T cell receptor (TCR) repertoire immuno-sequencing by next-generation sequencing (NGS) with clinical response parameters.The changes in BM and BM plasma cells' biology before and after treatment by Whole Exome Sequencing, andThe correlation between PET/CT responses and endpoint. The main eligibility criteria are age ≥ 18 years, ECOG PS 0-2, measurable disease at the time of diagnosis, 〈 VGPR per IMWG 2016 criteria following a 3-drug IT regimen for TE-NDMM. However, patients must have achieved at least a minimal response, and treatment plan includes ASCT post-induction. Statistical methods: Post-ASCT ≥CR response is the primary endpoint for this study. For NDMM, standard therapy strategies provide ≥CR rates of approximately 50%. For this population of subjects treated with Dara-based in vivo purging, the aim is to achieve a post-ASCT ≥ CR rate of 70%. A minimax 2-stage design will be used to test the hypothesis that the ≥ CR rate post-ASCT is less than or equal to 50%. Twenty-three subjects will be enrolled in the first stage, and if at least 12 of the 23 subjects have ≥CR after ASCT, an additional 16 subjects will be enrolled (total of 39 subjects). If at least 24 of 39 subjects have ≥CR after ASCT, the null hypothesis will be rejected. Based on a one-sided, α = 0.10 significance level, this sample size will provide 90% power to reject the null, assuming the true ≥CR rate post-ASCT is 70%. The treatment schedule: Pre-mobilization Dara on days 1,8,15,22. Four weeks after the Day 22 dose of Dara, all subjects will undergo HPC mobilization with the hematopoietic growth factor G-CSF with or without the chemokine receptor type 4 (CXCR4) antagonist. Post-ASCT and after engraftment is complete, Dara will be restarted days +49, +56, +63, +70. ClinicalTrials.gov Identifier: NCT04230031 Figure 1 Disclosures Atrash: Amgen, GSK, Karyopharm.: Research Funding; Takeda, Amgen, Karyopharm, BMS, Sanofi, Cellactar, Janssen and Celgene: Honoraria; BMS, Jansen oncology, Sanofi: Speakers Bureau. Paul:Regeneron: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy, Speakers Bureau; Bristol-Myers Squibb: Other: Stock Ownership (prior employee). Symanowski:Casgen: Consultancy; Eli Lilly: Consultancy; Immatics: Consultancy; Novartis: Consultancy. Bhutani:Janssen: Other: Clinical Trial Funding to Institute; BMS: Other: Clinical trial funding to institute, Speakers Bureau; Amgen: Speakers Bureau; Prothena: Other: Clinical Trial Funding to Institute; Sanofi Genzyme: Consultancy; Takeda: Other: Clinical trial funding to institute, Speakers Bureau; MedImmune: Other: Clinical Trial Funding to Institute. Voorhees:Bristol-Myers Squibb: Other: Personal fees; Celgene: Other: Personal fees; Janssen: Other: Personal fees; Novartis: Other: Personal fees; Oncopeptides: Other: Personal fees; TeneoBio: Other: Personal fees; Levine Cancer Institute, Atrium Health: Current Employment; Adaptive Biotechnologies: Other: Personal fees. Usmani:Merck: Consultancy, Research Funding; Incyte: Research Funding; Pharmacyclics: Research Funding; Array Biopharma: Research Funding; GSK: Consultancy, Research Funding; Celgene: Other; Amgen: Consultancy, Honoraria, Other: Speaking Fees, Research Funding; BMS, Celgene: Consultancy, Honoraria, Other: Speaking Fees, Research Funding; Abbvie: Consultancy; Sanofi: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Honoraria, Other: Speaking Fees, Research Funding; Janssen: Consultancy, Honoraria, Other: Speaking Fees, Research Funding; SkylineDX: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding.

Description: Incubation of human macrophages with endotoxin resulted in significantly enhanced colony-stimulating activity (CSA) production by these cells. Preincubation of endotoxin with mature granulocytes abolished this stimulatory effect. The stimulatory effect of endotoxin on macrophage CSA production was not abolished, however, by preincubation with NaF-treated granulocytes, granulocyte membranes, or nonphagocytic cells (lymphocytes or erythrocytes). These data suggest that mature granulocytes may play a role in the modulation of CSA production and granulopoiesis by inactivation of stimulatory materials such as endotoxin.

Description: Monoclonal antibody (mAb) treatment is an effective therapeutic strategy for many cancer types, though there remains meaningful opportunity to improve mAb efficacy by optimizing the interaction with natural killer (NK) cells to enhance antibody-dependent cellular cytotoxicity (ADCC). NK cells are an ideal effector cell for combined use with tumor-targeting mAbs, as NK cells effect both innate tumoricidal capacity and ADCC. CD38-targeting mAbs, such as daratumumab, are effective in treating multiple myeloma (MM) and achieve their efficacy through multiple mechanisms, including ADCC. However, because activated NK cells express high levels of CD38, daratumumab induces NK cell depletion through fratricide, potentially reducing treatment effectiveness. Adoptive NK cell immunotherapy therefore has the potential to augment daratumumab's ADCC activity if fratricide can be reduced or prevented. FT538 is an off-the-shelf adoptive NK cell immunotherapy product candidate designed for enhanced cellular persistence and ADCC while avoiding anti-CD38 mAb induced fratricide. It is derived from induced pluripotent stem cells (iPSC) engineered to lack CD38 expression, which we have previously shown to eliminate daratumumab-induced fratricide among iPSC-derived NK cells, resulting in enhanced long-term daratumumab-mediated ADCC. FT538 is engineered to express an IL-15 receptor alpha fusion protein (IL-15RF; IL-15 tethered to IL-15 receptor α) to enhance persistence and a high-affinity non-cleavable CD16 (hnCD16, FcRγIII) to increase ADCC. To support the clinical translation of FT538, and to enable the repeatable and scalable cell production to support off-the-shelf availability of a uniform NK cell product, a clinical-grade master pluripotent stem cell line was developed. The FT538 master pluripotent stem cell line was created by reprogramming donor fibroblasts into iPSCs using our non-integrating cellular reprogramming platform, and cells were further genetically edited by targeting IL-15RF and hnCD16 to the CD38 locus. Clonal iPSC lines were generated and screened for precise knock-in and knock-out edits at the CD38 locus and a lack of off-target genome integration (15% total success rate for CD38-/-IL-15RF+CD16+). Selected engineered iPSC clones were confirmed to be free of reprogramming transgenes and to maintain genomic stability. Engineered iPSC clones were additionally tested for their NK cell differentiation potential and function, and a single clone was selected to serve as the renewable starting material for cGMP manufacturing and clinical development. Upon differentiation and expansion FT538 demonstrated a mature NK cell phenotype with expression of NK cell receptors including NKp30, NKp46, NKG2D, KIR, NKG2A, and DNAM-1. The functional impact of CD38 knockout on FT538 NK cells was confirmed in an in vitro fratricide assay, where peripheral blood (PB)-NK cells exhibited fratricide at a frequency of 33% after 3 hr culture with increasing daratumumab concentrations. In contrast, FT538 cells were entirely resistant (

Description: Background: Venetoclax (VEN) monotherapy in patients (pts) with relapsed or refractory (R/R) chronic lymphocytic leukemia (CLL) has been efficacious with a complete remission rate of 16% and also durable with median time to progression of 33.2 months (Roberts et al., 2019). Despite these responses, many pts eventually relapse. To investigate possible mechanisms of resistance to VEN monotherapy, we performed a genome-wide CRISPR screen to identify genes whose loss of expression leads to reduction in drug sensitivity. To determine whether similar genomic alterations occur in pts treated with VEN, we analyzed the DNA and RNA of pre- and post-therapy specimens from CLL pts enrolled in two VEN monotherapy trials (NCT01328626 and NCT01889186). Methods: CRISPR screen was performed in lymphoma SUDHL4 cell line using Brunello library. Peripheral blood or bone marrow specimens were collected pre-dose and after VEN discontinuation; CLL cells were enriched via CD19-positive (CD19+) selection using magnetic beads. DNA from 43 pts was analyzed by whole exome and two targeted sequencing panels. RNA-seq analysis was performed on paired pre- and post-therapy CD19+ samples from 24 pts. Results: The CRISPR screen identified genes whose loss of expression led to resistance to VEN. Several members of the pro-apoptotic machinery were among the strongest hits, including BAX, BCL2L11 (BIM) and PMAIP1 (NOXA). Outside of the apoptotic pathway, loss of ID3 or NFKBIA also led to decreased VEN sensitivity, suggesting multiple possible mechanisms of resistance. To compare these results to the potential mechanisms of resistance in pts treated with VEN, we sequenced the DNA from CD19+ B cells extracted before treatment initiation or upon treatment discontinuation. Of the 37 pts with disease progression, 15 acquired mutations in BCL2 at progression (N=14 CLL, N=1 Richters); 6 pts had a single mutation and 9 pts acquired 2-4 distinct BCL2 mutations. The prevalence of mutations increased with time on VEN therapy; mutations were observed in 12 pts progressing between 22 and 59 months and in only 3 pts progressing between 6 and 16 months. BCL2 mutations at G101V and D103 (E/Y/V) as previously described (Blombery et al., 2019; Tausch et al., 2019) were identified in 13/15 pts. Additionally, our analysis identified 1 patient with a mutation at F104L and 3 pts with mutations at A113G. These F104L and A113G mutations were previously identified in a preclinical NHL model of VEN resistance (Tahir et al., 2017) and in DLBLCL pts, respectively, but have not been previously reported in CLL pts. Two novel mutations in BCL2: a 4 amino acid insertion at position 110 and a substitution at V156(D) were acquired at CLL progression in 3 and 4 pts, respectively. Most BCL-2 mutations were subclonal (VAF 10%. In accordance with the CRISPR screen, we identified mutations in other pro-apoptotic genes including PMAIP1 (N=4), BAX (N=2) and BAD (N=1). In contrast to the acquired BCL2 mutations, mutations in these BCL-2 family members occurred with high allelic frequency in 4 of the 7 pts (27-35%). Interestingly, 4 pts had a mutation in BCL2 in addition to another member of the apoptotic machinery. In each case, allele frequency suggested that the mutations were found in different clones. Comparison of mRNA expression pre- and post-VEN treatment demonstrated increased expression at the time of progression of key apoptotic genes BCL2L1 (BCL-XL), MCL1 and BCL2A1, all of which are known resistance factors to VEN. Concomitant decreases in BCL2 and HRK expression, and to a lesser extent PMAIP1, were also observed. The changes in gene expression were found irrespective of the presence of BCL2 mutations. Conclusions: Preclinical investigation into potential resistance mechanisms indicated that reduction in several genes confer resistance to VEN. Some of these alterations were observed in clinical samples, including several distinct mutations in BCL2 and other BCL-2 family members.Our data suggest that BCL2 mutations are subclonal and further data are needed to evaluate their role in resistance to VEN monotherapy. Additional ongoing analyses of genomic data may identify genomic alterations in other biological pathways that potentially convey VEN resistance. Emergence of resistance is likely multifactorial with modulation of the apoptotic family members via both acquired mutations and changes in gene expression pattern. Disclosures Chyla: Abbvie, Inc: Employment, Other: Stock or options. Popovic:AbbVie: Employment, Other: Stock or options. Lu:AbbVie: Employment, Other: Stock or options. Dunbar:AbbVie: Employment, Other: Stock or options. Quarless:AbbVie: Employment, Other: Stock or options. Robinson:AbbVie: Employment, Other: Stock or options. Warder:AbbVie: Employment, Other: Stock or options. Jacobson:AbbVie: Employment, Other: Stock or options. Zhou:AbbVie: Employment, Other: Stock or options. Souers:AbbVie: Employment, Other: Stock or options. Waring:AbbVie: Employment, Other: Stock or options. Bhathena:AbbVie: Employment, Other: Stock or options. Leverson:AbbVie Inc: Employment, Other: Stock or options. Kim:AbbVie: Employment, Other: Stock or options.

Description: Primary autologous as well as allogeneic and xenogeneic stroma will support human stem cell proliferation and differentiation for several months. In the present study, we investigated the capacity of porcine microvascular endothelial cells (PMVECs) together with combinations of cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF] + stem factor [SCF], interleukin-3 [IL-3] + SCF + IL-6, and GM-CSF + IL-3 + SCF + IL-6) to support the expansion and development of purified human CD34+ bone marrow cells. In short-term cultures (7 days), the greatest expansion of nonadherent hematopoietic cells and clonogenic progenitors was seen with CD34+ cells in direct contact with PMVEC monolayers (PMVEC contact), followed by PMVEC noncontact and liquid suspension cultures, respectively. Maximal expansion of nonadherent cells (42-fold) and total CD34+ cells (12.6-fold) occurred in PMVEC contact cultures treated with GM-CSF + IL-3 + SCF + IL-6, with similar increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), CFU-mix, erythroid burst-forming units (BFU-E), CFU-blast and CFU-megakaryocyte (CFU-Mk) progenitor cells. Moreover, the number of CD34+ CD38- and CD34+ CD38+ cells increased 148.1-fold and 8.0-fold, respectively. Replating studies show that cells from day 7 dispersed blast cell colonies generated on cytokine-treated PMVEC monolayers have a high replating potential for multilineage progenitor cells. In long- term PMVEC contact cultures, CD34+ cells seeded onto PMVEC monolayers with GM-CSF + IL-3 + SCF + IL-6 showed a total calculated expansion of over 5,000,000-fold of nonadherent cells over 35 days in culture. Maximal clonogenic cell production was observed at day 28, with 6,353-fold for total CFC and comparable increases for CFU-GM, CFU-mix, CFU-blast, BFU-E, and CFU-Mk. The total number of CD34+ cells increased 2,584-fold at day 28. Furthermore, the extended growth kinetics of these cultures indicates that these phenotypically primitive progenitor cells are also functionally expanded on PMVEC monolayers. These results support the hypothesis that direct contact with a PMVEC monolayer supports the initial expansion of hematopoietic progenitor cells with a high replating potential and, possibly, a more primitive phenotype (CD34+, CD34+/CD38-).

Description: 32P in membrane phosphatidylethanolamine (PE) and red cell 14C, reflecting cytoplasmic hemoglobin, were measured sequentially in rats given transfusions of doubly-labeled reticulocytes. With reticulocytes from normal rats there was a small decline in the levels of both the membrane and the cytoplasmic labels; the changes were almost parellel, although loss of membrane PE-32P exceeded that of 14C to a small extent. By contrast, with "stress reticulocytes" from bled donors, there was a markedly disproportionate loss of the membrane label; this asymmetrical loss of membrane material was diminished when recipients had been splenectomized. With transfusions of doubly-labeled reticulocytes from rats with severe iron deficiency anemia, there was a marked loss of both membrane PE-32P and red cell 14C which was only moderately asymmetrical. The asymmetrical loss of the membrane label found with stress reticulocytes supports the conclusion that these cells undergo a process of surface remodeling during their maturation in the peripheral blood. The spleen is partly responsible for this process. Normal reticulocytes also appear to undergo a minor degree of remodeling. On the other hand, the almost symmetrical loss of membrane and cytoplasmic label observed with reticulocytes from iron deficient rats indicates that many of the cells in this model of ineffective erythropoiesis are hemolyzed in their entirety. These experiments demonstrate that stress reticulocytes differ under different conditions and may lose cellular material by two, possibly interrelated, mechanisms: surface remodeling or whole-cell hemolysis.