Longitudinal Antibody Signatures Following FVIII Replacement Therapy in Previously Untreated Patients with Severe Hemophilia Α- New Insights from the Hemophilia Inhibitor PUP Study (HIPS)

Abstract Background and objectives The Hemophilia Inhibitor PUP Study (HIPS, clinicaltrials.gov NCT01652027) is a prospective multicenter observational study of PUPs with severe hemophilia A during their first 50 exposure days (EDs) to recombinant Factor VIII (FVIII, Advate TM). The objective is to elucidate immune system changes and identify potential early biomarkers of inhibitor development in PUPs. Previously we reported that the appearance of high-affinity anti-FVIII antibodies preceded clinical diagnosis of FVIII inhibitors in patients (Reipert 2016). Here we present longitudinal antibody data including titers of FVIII-specific IgM, IgG1-4, IgA, apparent affinities of antibodies and titers of FVIII inhibitors for the first 15 patients who completed the antibody analysis of the HIPS study. Moreover, we present longitudinal monitoring data for key circulating immune cells such as FoxP3+ T cells (Tregs), pro-inflammatory Th17 cells, total T cells and granulysin+ NK cells. Methods FVIII inhibitor testing (Nijmegen methodology, performed in the central laboratory Medical College of Vienna) and antibody analytics were done prior to first exposure, 7-9 days after ED 1 and 5-7 days after EDs 5, 10, 20, 30, 40, and 50. FVIII-specific antibody analytics, including specifications of isotypes, IgG subclasses and apparent affinities were done using methodology described by Whelan 2013 and Hofbauer 2015. Circulating immune cells including FoxP3+ T cells (Tregs), pro-inflammatory Th17 cells, total T cells and granulysin+ NK cells were monitored by epigenetic cell counting in whole blood using quantitative PCR-based methylation assays (www.epiontis.com) Results 25 subjects have been enrolled among 15 study sites. Data for 15 subjects who had completed 50 exposure days were available for analysis. Among these 15 subjects, 4 developed FVIII inhibitors by study criteria (> 0.6 B.U. x 2 measurements in central laboratory) at exposure days 6, 10 and 20. All inhibitors were associated with the development of high affinity FVIII-specific antibodies. High-affinity IgG1 antibodies were detected first and subsequently switched to IgG3 and IgG4, switching to IgG2 was observed only in one of the 4 patients. Two of the 4 inhibitor patients initially developed low affinity IgG1 antibodies which were subsequently accompanied by high affinity IgG1 before switching to high-affinity IgG3 and IgG4. One of the 4 inhibitor patients expressed non-neutralizing FVIII-specific IgG1 antibodies prior to first FVIII infusion already and developed a high-titer inhibitor as early as ED6 Six of the 15 patients who had completed 50 exposure days developed non-neutralizing antibodies (detected on at least 2 time points). Five of them expressed low affinity IgG1 which was never accompanied by high affinity IgG1 or by any other IgG subclass. One of the 6 patients initially expressed low affinity IgG1 which was later accompanied by non-neutralizing high affinity IgG1. Switching to other IgG subclasses was not observed. Three patients had no antibodies detected and 2 had non-neutralizing antibodies detected at a single time point only. Levels of circulating FoxP3+ Tregs in the infant patients were in the same range as in healthy adults with some variation between individuals. In contrast, circulating TH17 cells were barely detectable. The levels of granulysin+ NK cells showed substantial variations between patients as well as during longitudinal monitoring in the same patient. Conclusion Antibody data obtained from the first 15 patients who completed 50 exposure days in the HIPS study confirms that FVIII inhibitor development is preceded by the detection of high-affinity FVIII-specific antibodies. High affinity IgG1 antibodies appeared first and subsequently switched to IgG3 and IgG4 which indicates a T-cell dependent pathway in FVIII inhibitor development. Switching to IgG2 was only seen in 1 of the 4 inhibitor patients. In contrast, patients with non-neutralizing antibodies only expressed IgG1 antibodies and never switched to another IgG subclass. 5 of the 6 patients with non-neutralizing antibodies only expressed low affinity IgG1 which might reflect a T-cell independent pathway of antibody development. In summary, our data indicate that comprehensive analysis of FVIII-specific antibodies might provide early biomarkers which indicate evolving FVIII inhibitors. Disclosures Gangadharan: Shire: Employment. Reipert: Shire: Employment. Scheiflinger: Shire: Employment. Bowen: Shire: Research Funding. Fijn van Draat: ZonMW,CSL Behring: Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees; Pfizer, Novo Nordisk: Other: Lectures. Gruppo: Shire: Other: Honorarium for participation on physician advisory board. Male: Bayer, Baxalta, Biotest, CSL Behring, Novo Nordisk, Pfizer: Other: Speaker Honoraria and/Travel support. McGuinn: Shire/Baxalta: Consultancy; Shire / Baxalta, Spark, Biogen, Roche/Genetehc: Research Funding. Recht: Baxalta, Novo Nordisk, Biogen, Pfizer: Research Funding; Biogen , Kediron: Consultancy. Ragni: A Anylam,Biomarin,Tecere, Benitec: Honoraria; Alnylam, CSL Behring, Dimensions, Genetech/Roche,Pfizer, Shire, SPARK: Research Funding. Yaish: Baxalta, Bayer and Octapharma, CSL behring: Consultancy, Membership on an entity's Board of Directors or advisory committees; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees. Santagostino: : Bayer, Shire, Pfizer, Novo Nordisk, Kedrion, Roche, Sobi, Bioverativ, CSL Behring, Grifols, Octapharma: Speakers Bureau; Bayer, Shire, Pfizer, Novo Nordisk, Kedrion, Roche, Sobi, Bioverativ: Other: Advisory board. Brown: Shire: Research Funding.

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Appearance of High-Affinity Antibodies Precedes Clinical Diagnosis of FVIII Inhibitors - Preliminary Analysis from the Hemophilia Inhibitor PUP Study (HIPS)

Blood ◽

10.1182/blood.v128.22.328.328 ◽

2016 ◽

Vol 128 (22) ◽

pp. 328-328 ◽

Cited By ~ 2

Author(s):

Birgit M Reipert ◽

Bagirath Gangadharan ◽

Christoph J Hofbauer ◽

Fritz Scheiflinger ◽

Joel Bowen ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Hemophilia A ◽

Advisory Committees ◽

Inhibitor Development ◽

Specific Antibodies ◽

High Affinity ◽

Early Biomarkers ◽

Fviii Inhibitor ◽

Bayer Healthcare

Abstract Background and Objectives The development of neutralizing antibodies (inhibitors) to factor VIII (FVIII) represents the major complication following replacement therapy with FVIII products in patients with hemophilia A. Why some patients develop FVIII inhibitors while others do not is poorly understood. Identification of early biomarkers which predict inhibitor development in previously untreated patients (PUPs) with hemophilia A will assist in risk identification and possible early intervention strategies. Previous analysis of FVIII-specific antibodies in different cohorts of patients with severe hemophilia A and in healthy individuals indicated distinct spectra of neutralizing and non-neutralizing antibodies (Whelan SFJ et al. Blood 2013). FVIII-specific antibodies found in hemophilia A patients with inhibitors have approximately 100-fold higher apparent affinities than that of antibodies found in patients without inhibitors or in healthy individuals (Hofbauer CJ et al. Blood 2015). In one individual, high affinity FVIII-specific antibodies were detectable prior to the diagnosis of FVIII inhibitors. The Hemophilia Inhibitor PUP Study (HIPS) is a prospective multicenter observational study of PUPs with severe hemophilia A during their first 50 exposure days (EDs) to recombinant FVIII (Advate TM). The objective is to elucidate immune system changes and identify potential early biomarkers of inhibitor development in PUPs. This is the first report of FVIII-specific antibody development in the first 10 subjects who have completed the study. Methods HIPS study procedures have been previously described (Hofbauer CJ et al. 2015; clinicaltrials.gov NCT01652027). FVIII inhibitor testing by Nijmegen methodology performed in a central laboratory (Medical College of Vienna) and FVIII-specific antibody analytics were done prior to first exposure, 7-9 days after exposure day (ED) 1 and 5-7 days after EDs 5, 10, 20, 30, 40, and 50. FVIII-specific antibody analytics, including specifications of isotypes, IgG subclasses and apparent affinities were done using methodology described in Whelan 2013 and Hofbauer 2015. Results: Currently, 24 subjects have been enrolled among 19 study sites. Data for 10 subjects were available for analysis. Among these 10 subjects, 2 developed FVIII inhibitors by study criteria (B.U. > 0.6 B.U. x 2 measurements in central laboratory) by exposure days 6 and 20 (see figure 1). A third subject is expected to meet study criteria for FVIII inhibitor due to multiple high titer measurements reported over time at a local laboratory. All three inhibitor patients revealed antibody class switching from IgG1 to IgG4 and affinity maturation resulting in the generation of high affinity FVIII-specific antibodies. High-affinity FVIII-specific IgG1 antibodies were detectable prior to inhibitor diagnosis in all 3 inhibitor subjects. One subject with a transient FVIII inhibitor showed medium affinity FVIII-specific IgG1 antibodies which persisted until ED50, but never developed high-affinity antibodies. Another subject without a detectable inhibitor developed low titer, low affinity IgG1 antibodies that persisted to ED 50 without the development of high-affinity antibodies. The remaining subjects had no inhibitor and no detectable FVIII-specific antibodies. Conclusions: Interim data from HIPS suggests that FVIII inhibitor development is preceded by the detection of high-affinity FVIII-specific antibodies which undergo class switch from IgG1 and IgG3 to IgG4. The findings confirm affinity maturation of FVIII-specific antibodies which require T-cell dependent antibody responses resulting from germinal center reactions. While low affinity IgG1 antibodies were sometimes seen in subjects without inhibitors, high-affinity antibodies were only seen in subjects who developed inhibitors. Our data indicate that high affinity FVIII-specific antibodies preceded the clinical diagnosis of an inhibitor and may serve as early biomarkers of FVIII inhibitor development facilitating early immune intervention. Figure 1. Kinetic of FVIII-binding antibodies and FVIII inhibitors in patient 0902. High-affinity FVIII-specific IgG1 antibodies were first detected after exposure day 6, FVIII inhibitors were first diagnosed after exposure day 20. Figure 1. Kinetic of FVIII-binding antibodies and FVIII inhibitors in patient 0902. High-affinity FVIII-specific IgG1 antibodies were first detected after exposure day 6, FVIII inhibitors were first diagnosed after exposure day 20. Disclosures Reipert: Shire: Employment, Research Funding. Gangadharan:Shire: Employment, Research Funding. Hofbauer:Shire: Employment. Scheiflinger:Shire: Employment, Research Funding. Bowen:Baxalta: Research Funding. Donnachie:Baxalta: Research Funding; CSL Behring: Research Funding; Bayer Healthcare: Research Funding. Fijvandraat:CSL Behring: Research Funding; Bayer Healthcare: Research Funding; Baxalta: Membership on an entity's Board of Directors or advisory committees. Gruppo:Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees. Male:Baxalta: Other: travel support; Novo Nordisk: Other: travel support; Biotest: Other: travel support; Bayer Healthcare: Other: travel support; CSL Behring: Other: travel support; Pfizer: Other: travel support. McGuinn:Baxalta: Research Funding; Novo Nordisk: Research Funding; Spark: Research Funding; Biogen: Research Funding. Meeks:Genentech: Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Membership on an entity's Board of Directors or advisory committees; Grifols: Membership on an entity's Board of Directors or advisory committees; Biogen: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees. Recht:Novo Nordisk: Consultancy, Research Funding; Baxalta: Research Funding; Biogen Idec: Research Funding; Kedrion: Consultancy. Ragni:Biogen: Research Funding. Yaish:Octapharma: Consultancy; Bayer Healthcare: Consultancy; Baxalta: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Santagostino:Sobi: Consultancy; Biogen Idec: Consultancy; Novo Nordisk: Consultancy; Pfizer: Consultancy; Octapharma: Consultancy; Baxalta: Consultancy; Kedrion: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Grifols: Consultancy; Roche: Consultancy. Brown:Baxalta/Shire: Research Funding.

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Reducing on-Target Off-Tumor Effects of CD38-Chimeric Antigen Receptors By Affinity Optimization

Blood ◽

10.1182/blood.v128.22.2170.2170 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2170-2170 ◽

Cited By ~ 7

Author(s):

Esther Drent ◽

Maria Themeli ◽

Renée Poels ◽

Niels W.C.J. van de Donk ◽

Henk M. Lokhorst ◽

...

Keyword(s):

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Hematopoietic Cells ◽

Antigen Receptors ◽

Chimeric Antigen Receptors ◽

Advisory Committees ◽

Lower Affinity ◽

High Affinity ◽

Car T

Abstract Chimeric Antigen Receptors (CARs) are engineered transmembrane proteins consisting of an antibody (ab)-derived antigen recognition domain linked to intracellular T cell signaling domains. Cytotoxic T cells endowed with tumor-reactive CARs are highly promising tools for immunotherapy of cancer. There are however only a few truly tumor specific molecules that can be targeted by CARs, a drawback for the broad application of CAR T cell therapy. Indeed, when we recently aimed at targeting CD38high multiple myeloma (MM) with T cells transduced with high affinity CD38CARs, we observed that they not only lysed the CD38high MM cells but also CD38+ normal hematopoietic cells, pointing towards potential safety issues of such tumor-associated, but not entirely tumor-specific CARs. Therefore, using CD38 as a model for antigen we now tested whether it would be possible to reduce the on target, off-tumor effects of such CARs by optimizing their target cell affinity. To this end, we generated a new panel of CD38 abs through the "light chain exchange" method, in which heavy chains of two high affinity CD38 abs were combined with 176 different germ line light chains. This approach revealed around 100 new abs, which displayed 10- >1000 fold lower affinity to CD38 as compared to the parental abs. After categorizing them in three classes based on CD38 binding affinity, we used 8 abs from each class to generate 24 different CD38-CAR constructs. Testing the cytotoxic activity of T cells transduced with these CD38-CARs against CD38++ MM cell lines, primary MM cells and CD38+ normal hematopoietic cells in vitro and in vivo demonstrated that CD38-CAR T cells with ca. 1000 fold lower affinity to CD38 could still effectively lyse CD38++ MM cells while there was little or no cytotoxicity against CD38+ healthy hematopoietic cells. The results of this study reveal that it is possible to reduce the on-target off-tumor effects of CARs by optimizing their affinity. Thus, tailored affinity of the ab binding domain may open up new roads for CAR therapy. Disclosures van de Donk: Janssen: Research Funding; Celgene: Research Funding; BMS: Research Funding; Amgen: Research Funding. Lokhorst:Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Genmab: Research Funding. Mutis:Celgene: Research Funding; Genmab: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Extended Survival in CLL Patients Who Discontinued Idelalisib Because of Colitis Correlates with Higher Th17 Levels in Blood and Colon Tissue

Blood ◽

10.1182/blood-2018-99-116910 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1848-1848

Author(s):

Rukhsana Aslam ◽

Jacqueline C Barrientos ◽

Gerardo Ferrer ◽

Pui Yan Chiu ◽

Kanti R Rai ◽

...

Keyword(s):

T Cells ◽

B Cell ◽

Board Of Directors ◽

Th17 Cells ◽

Research Funding ◽

Treg Cells ◽

Drug Discontinuation ◽

Advisory Committees ◽

Extended Survival

Abstract Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries. Despite significant clinical and molecular advances, CLL remains an incurable disease. In CLL there is ongoing communication between leukemic B cells and non-leukemic cells in the tissue microenvironment. Idelalisib, a PI3Kδ inhibitor, is efficacious in CLL although side effects like inflammatory reactions in different tissues (e.g., colitis, transaminitis and pneumonitis) and progression of disease can lead to discontinuation of the drug. We previously presented an intriguing finding that a cohort of patients who discontinued idelalisib therapy due to colitis had extended survival compared to other patients who discontinued drug but did not have colitis. Of note, the colon tissues from patients with ulcerative colitis (UC) often contain elevated levels of Th17 cells and decreased levels of Treg (Foxp3+) cells. Moreover, Th17 cells from humans and mice have been shown to promote autoimmune B-cell maturation. In this regard, we have previously published that circulating Th17 levels are elevated in CLL patients and Th17 numbers correlate with extended survival. For these reasons, we have now quantified the numbers of Th17 and Treg cells in the blood and colon tissues of a cohort of CLL patients with drug-associated colitis and extended survival and have investigated the effects of in vitro exposure to idelalisib on Th17 generation. Circulating Th17A levels were quantified in a cohort of patients (n=11) before starting idelalisib-treatment and at the time the drug was discontinued due to colitis by flow cytometric analysis of intracellular IL-17A in CD4+ T cells. In addition, colon tissues from those CLL patients who developed colitis during idelalisib treatment taken at the time of drug discontinuation and patients with UC were examined to quantify the infiltration of T (CD3+) cells, Th17 (IL17A+) cells and Treg (Foxp3+) cells by immunohistochemistry (IHC). As negative control tissues (CT) for these studies we used colectomy samples from patients with inactive diverticulosis. FACS analysis of peripheral blood mononuclear cells from the CLL patients treated with idelalisib and having extended survival showed significantly higher circulating Th17A levels (P < 0.01) at the time of drug discontinuation compared to pre-treatment levels. In addition, IHC analyses on tissues from a subset of these same patients indicated that the ratio of % IL-17A+ to CD3+ cells was significantly higher in CLL than in CT (P = 0.0002). It was also significantly higher in CLL than UC (P = 0.001), even though the average number of CD3+ cells in UC was higher than in CLL (P = 0.0001). We also determined the ratio of % FoxP3+ to CD3+ cells in the colon tissues of 12 CT, 16 UC and 6 CLL patients from the cohort studied above. This revealed the average percentage of FoxP3+ cells within the total number of infiltrating T cells was significantly higher in CLL than CT (P = <0.0001), but not significantly different from UC patients (P = 0.90). When analyzing solely the CLL samples, there was a reciprocal relationship between Th17A+ cells and FoxP3+ cells, with the former being higher and the latter lower. To examine the effect of idelalisib on Th17-cell generation, naïve CD4+ CLL T cells from 6 patients (3 M-CLL + 3 U-CLL) were activated in vitro by anti-CD3/28 ligation plus IL-2 in the presence or absence of idelalisib. After 7 days, T helper subset profiling was performed by intracellular cytokine staining. Significantly higher percentages of Th17A cells and significantly lower percentages of Th1 cells (IFNg+) (P= 0.006) were found in cultures containing idelalisib. The percentages of Tregs were unchanged in the same cultures. These findings suggest that the higher numbers of Th17s in blood and tissue of patients after idelalisib treatment may reflect the action of idelalisib on Th17 generation. In conclusion, CLL patients who discontinued idelalisib due to colitis and yet had extended survival have increased circulating and tissue-resident Th17 cells and decreased levels of Treg cells. These findings suggest that an immune imbalance between Th17 and Treg cell numbers and function promotes colitis and at the same time favors improved clinical outcome. This is consistent with the known action of Th17 cells enhancing autoimmune B-cell responses. Disclosures Barrientos: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rai:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

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Automated Manufacture of Matched Donor-Derived Allogeneic CD19 CAR T-Cells for Relapsed/Refractory B-ALL Following Allogeneic Stem Cell Transplantation: Toxicity, Efficacy and the Important Role of Lymphodepletion

Blood ◽

10.1182/blood-2019-129640 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 776-776

Author(s):

Claire Roddie ◽

Maeve A O'Reilly ◽

Maria A V Marzolini ◽

Leigh Wood ◽

Juliana Dias Alves Pinto ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Peak Car ◽

Matched Donor

Introduction: 2nd generation CD19 CAR T cells show unprecedented efficacy in B-ALL, but several challenges remain: (1) scaling manufacture to meet patient need and (2) feasibility of generating products from lymphopenic patients post allogeneic stem cell transplant (allo-SCT). To overcome these issues we propose: (1) use of the CliniMACS Prodigy (Miltenyi Biotec), a semi-automated cGMP platform that simplifies CAR T cell manufacture and (2) the use of matched donor T cells to overcome the challenge posed by patient lymphopenia, albeit this may come with a heightened risk of graft versus host disease (GvHD). CARD (NCT02893189) is a Phase I study of matched donor derived CD19 CAR T cells generated on the CliniMACS Prodigy in 14 adult patients with relapsed/refractory (r/r) B ALL following allo-SCT. We additionally explore the requirement for lymphodepletion (LD) in the allogeneic CAR T cell setting and report on the incidence of GvHD with this therapy. Methods: Manufacturing: CARD utilises non-mobilised matched donor leucapheresate to manufacture 2nd generation CD19CAR T cells using a closed CliniMACS® Prodigy/ TransACTTM process. Study design: Eligible subjects are aged 16-70y with r/r B ALL following allo SCT. Study endpoints include feasibility of CD19CAR T cell manufacture from allo-SCT donors on the CliniMACS Prodigy and assessments of engraftment and safety including GvHD. To assess the requirement for LD prior to CD19CAR T cells in lymphopenic post-allo-SCT patients, the study is split into Cohort 1 (no LD) and Cohort 2 (fludarabine (30 mg/m2 x3) and cyclophosphamide (300mg/m2 x3)). To mitigate for the potential GvHD risk, cell dosing on study mirrors conventional donor lymphocyte infusion (DLI) schedules and is based on total CD3+ (not CAR T) cell numbers: Dose 1=1x106/kg CD3+ T cells; Dose 2= 3x106/kg CD3+ T cells; Dose 3= 1x107/kg CD3+ T cells. Results: As of 26 July 2019, 17 matched allo SCT donors were leukapheresed and 16 products were successfully manufactured and QP released. Patient demographics are as follows: (1) median patient age was 43y (range 19-64y); (2) 4/17 had prior blinatumomab and 5/17 prior inotuzumab ozogamicin; (3) 7/17 had myeloablative allo SCT and 10/17 reduced intensity allo SCT of which 6/17 were sibling donors and 12/17 were matched unrelated donors. No patients with haploidentical transplant were enrolled. To date, 12/16 patients have received at least 1 dose of CD19CAR T cells: 7/16 on Cohort 1 and 5/16 on Cohort 2 (2/16 are pending infusion on Cohort 2 and 2/16 died of fungal infection prior to infusion). Median follow-up for all 12 patients is 22.9 months (IQR 2.9-25.9; range 0.7 - 25.9). At the time of CAR T cell infusion, 7/12 patients were in morphological relapse with >5% leukemic blasts. Despite this, CD19CAR T cells were administered safely: only 2/12 patients experienced Grade 3 CRS (UPenn criteria), both in Cohort 1, which fully resolved with Tocilizumab and corticosteroids. No patients experienced ≥Grade 3 neurotoxicity and importantly, no patients experienced clinically significant GvHD. In Cohort 1 (7 patients), median peak CAR expansion by flow was 87 CD19CAR/uL blood whereas in Cohort 2 (5 patients to date), median peak CAR expansion was 1309 CD19CAR/uL blood. This difference is likely to reflect the use of LD in Cohort 2. CAR T cell persistence by qPCR in Cohort 1 is short, with demonstrable CAR in only 2/7 treated patients at Month 2. Data for Cohort 2 is immature, but this will also be reported at the meeting in addition to potential mechanisms underlying the short persistence observed in Cohort 1. Of the 10 response evaluable patients (2/12 pending marrow assessment), 9/10 (90%) achieved flow/molecular MRD negative CR at 6 weeks. 2/9 responders experienced CD19 negative relapse (one at M3, one at M5) and 3/9 responders experienced CD19+ relapse (one at M3, one at M9, one at M12). 4/10 (40%) response evaluable patients remain on study and continue in flow/molecular MRD negative remission at a median follow up of 11.9 months (range 2.9-25.9). Conclusions: Donor-derived matched allogeneic CD19 CAR T cells are straightforward to manufacture using the CliniMACS Prodigy and deliver excellent early remission rates, with 90% MRD negative CR observed at Week 6 in the absence of severe CAR associated toxicity or GvHD. Peak CAR expansion appears to be compromised by the absence of LD and this may lead to a higher relapse rate. Updated results from Cohorts 1 and 2 will be presented. Disclosures Roddie: Novartis: Consultancy; Gilead: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau. O'Reilly:Kite Gilead: Honoraria. Farzaneh:Autolus Ltd: Equity Ownership, Research Funding. Qasim:Autolus: Equity Ownership; Orchard Therapeutics: Equity Ownership; UCLB: Other: revenue share eligibility; Servier: Research Funding; Bellicum: Research Funding; CellMedica: Research Funding. Linch:Autolus: Membership on an entity's Board of Directors or advisory committees. Pule:Autolus: Membership on an entity's Board of Directors or advisory committees. Peggs:Gilead: Consultancy, Speakers Bureau; Autolus: Membership on an entity's Board of Directors or advisory committees.

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Identification of Two CAR T-Cell Populations Associated with Complete Response or Progressive Disease in Adult Lymphoma Patients Treated with Axi-Cel

Blood ◽

10.1182/blood-2019-130815 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 779-779 ◽

Cited By ~ 2

Author(s):

Zinaida Good ◽

Jay Y. Spiegel ◽

Bita Sahaf ◽

Meena B. Malipatlolla ◽

Matthew J. Frank ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Board ◽

Advisory Committees ◽

Car T Cells ◽

Scientific Advisory Board ◽

Car T ◽

Scientific Advisory

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

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Human CD83 Targeted Chimeric Antigen Receptor T Cell for the Prevention of Graft Versus Host Disease and Treatment of Myeloid Leukemia

Blood ◽

10.1182/blood-2019-124031 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 196-196

Author(s):

Bishwas Shrestha ◽

Kelly Walton ◽

Jordan Reff ◽

Elizabeth M. Sagatys ◽

Nhan Tu ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Research Funding ◽

Advisory Committees ◽

Graft Versus Host ◽

Car T Cells ◽

Car T Cell ◽

Car T ◽

Fund Research

Distinct from pharmacologic immunosuppression, we designed a programmed cytolytic effector T cell that prevents graft versus host disease (GVHD). CD83 is expressed on allo-activated conventional T cells (Tconv) and pro-inflammatory dendritic cells (DCs), which are implicated in GVHD pathogenesis. Therefore we developed a novel human CD83 targeted chimeric antigen receptor (CAR) T cell for GVHD prophylaxis. Here we demonstrate that human CD83 CAR T cells eradicate cell mediators of GVHD, significantly increase the ratio of regulatory T cells (Treg) to allo-activated Tconv, and provide lasting protection from xenogeneic GVHD. Further, we show human, acute myeloid leukemia (AML) expresses CD83 and can be targeted by CD83 CAR T cells. A 2nd generation CD83 CAR was generated with CD3ζ and 41BB costimulatory domain that was retrovirally transduced in human T cells to generate CD83 CAR T cells. The CD83 CAR construct exhibited a high degree of transduction efficiency of about 60%. The CD83 CAR T cells demonstrated robust IFN-γ and IL-2 production, killing, and proliferation when cultured with CD83+ target cells. To test whether human CD83 CAR T cells reduce alloreactivity in vitro, we investigated their suppressive function in allogeneic mixed leukocyte reactions (alloMLR). CD83 CAR T cells were added to 5-day alloMLRs consisting of autologous T cells and allogeneic monocyte-derived DCs at ratios ranging from 3:1 to 1:10. The CD83 CAR T cells potently reduced alloreactive T cell proliferation compared to mock transduced and CD19 CAR T cells. We identified that CD83 is differentially expressed on alloreactive Tconv, compared to Tregs. Moreover, the CD83 CAR T cell efficiently depletes CD83+ Tconv and proinflammatory DCs with 48 hours of engagement. To test the efficacy of human CD83 CAR T cells in vivo, we used an established xenogeneic GVHD model, where mice were inoculated with human PBMCs (25x106) and autologous CD83 CAR (1-10x106) or mock transduced T cells. The CD83 CAR T cells were well tolerated by the mice, and significantly improved survival compared to mock transduced T cells (Figure 1A). Mice treated with CD83 CAR T cells exhibited negligible GVHD target organ damage at day +21 (Figure 1B). Mice inoculated with CD83 CAR T cells demonstrated significantly fewer CD1c+, CD83+ DCs (1.7x106 v 6.2x105, P=0.002), CD4+, CD83+ T cells (4.8x103 v 5.8x102, P=0.005), and pathogenic Th1 cells (3.1x105 v 1.1x102, P=0.005) at day +21, compared to mice treated with mock transduced T cells. Moreover, the ratio of Treg to alloreactive Tconv (CD25+ non-Treg) was significantly increased among mice treated with CD83 CAR T cells (78 v 346, P=0.02), compared to mice injected with mock transduced T cells. Further, CD83 appears to be a promising candidate to target myeloid malignancies. We observed CD83 expression on malignant myeloid K562, Thp-1, U937, and MOLM-13 cells. Moreover, the CD83 CAR T cells effectively killed AML cell lines. Many AML antigens are expressed on progenitor stem cells. Thus, we evaluated for stem cell killing in human colony forming unit (CFU) assays, which demonstrated negligible on-target, off-tumor toxicity. Therefore, the human CD83 CAR T cell is an innovative cell-based approach to prevent GVHD, while providing direct anti-tumor activity against myeloid malignancies. Figure Disclosures Blazar: Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Fate Therapeutics, Inc.: Research Funding; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Davila:Atara: Research Funding; Celgene: Research Funding; Precision Biosciences: Consultancy; Bellicum: Consultancy; GlaxoSmithKline: Consultancy; Adaptive: Consultancy; Anixa: Consultancy; Novartis: Research Funding.

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Pre-Existing T Cell Subsets Determine Anti-PD1 Blockade Response in Richter's Transformation

Blood ◽

10.1182/blood-2020-138601 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 42-43

Author(s):

Prajish Iyer ◽

Lu Yang ◽

Zhi-Zhang Yang ◽

Charla R. Secreto ◽

Sutapa Sinha ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Single Cell ◽

Board Of Directors ◽

Peripheral Blood ◽

Research Funding ◽

Gene Signature ◽

T Cell Subsets ◽

Rna Seq ◽

Advisory Committees

Despite recent developments in the therapy of chronic lymphocytic leukemia (CLL), Richter's transformation (RT), an aggressive lymphoma, remains a clinical challenge. Immune checkpoint inhibitor (ICI) therapy has shown promise in selective lymphoma types, however, only 30-40% RT patients respond to anti-PD1 pembrolizumab; while the underlying CLL failed to respond and 10% CLL patients progress rapidly within 2 months of treatment. Studies indicate pre-existing T cells in tumor biopsies are associated with a greater anti-PD1 response, hence we hypothesized that pre-existing T cell subset characteristics and regulation in anti-PD1 responders differed from those who progressed in CLL. We used mass cytometry (CyTOF) to analyze T cell subsets isolated from peripheral blood mononuclear cells (PBMCs) from 19 patients with who received pembrolizumab as a single agent. PBMCs were obtained baseline(pre-therapy) and within 3 months of therapy initiation. Among this cohort, 3 patients had complete or partial response (responders), 2 patients had rapid disease progression (progressors) (Fig. A), and 14 had stable disease (non-responders) within the first 3 months of therapy. CyTOF analysis revealed that Treg subsets in responders as compared with progressors or non-responders (MFI -55 vs.30, p=0.001) at both baseline and post-therapy were increased (Fig. B). This quantitative analysis indicated an existing difference in Tregs and distinct molecular dynamic changes in response to pembrolizumab between responders and progressors. To delineate the T cell characteristics in progressors and responders, we performed single-cell RNA-seq (SC-RNA-seq; 10X Genomics platform) using T (CD3+) cells enriched from PBMCs derived from three patients (1 responder: RS2; 2 progressors: CLL14, CLL17) before and after treatment. A total of ~10000 cells were captured and an average of 1215 genes was detected per cell. Using a clustering approach (Seurat V3.1.5), we identified 7 T cell clusters based on transcriptional signature (Fig.C). Responders had a larger fraction of Tregs (Cluster 5) as compared with progressors (p=0.03, Fig. D), and these Tregs showed an IFN-related gene signature (Fig. E). To determine any changes in the cellular circuitry in Tregs between responders and progressors, we used FOXP3, CD25, and CD127 as markers for Tregs in our SC-RNA-seq data. We saw a greater expression of FOXP3, CD25, CD127, in RS2 in comparison to CLL17 and CLL14. Gene set enrichment analysis (GSEA) revealed the upregulation of genes involved in lymphocyte activation and FOXP3-regulated Treg development-related pathways in the responder's Tregs (Fig.F). Together, the greater expression of genes involved in Treg activation may reduce the suppressive functions of Tregs, which led to the response to anti-PD1 treatment seen in RS2 consistent with Tregs in melanoma. To delineate any state changes in T cells between progressors and responder, we performed trajectory analysis using Monocle (R package tool) and identified enrichment of MYC/TNF/IFNG gene signature in state 1 and an effector T signature in state 3 For RS2 after treatment (p=0.003), indicating pembrolizumab induced proliferative and functional T cell signatures in the responder only. Further, our single-cell results were supported by the T cell receptor (TCR beta) repertoire analysis (Adaptive Biotechnology). As an inverse measure of TCR diversity, productive TCR clonality in CLL14 and CLL17 samples was 0.638 and 0.408 at baseline, respectively. Fifty percent of all peripheral blood T cells were represented by one large TCR clone in CLL14(progressor) suggesting tumor related T-cell clone expansion. In contrast, RS2(responder) contained a profile of diverse T cell clones with a clonality of 0.027 (Fig. H). Pembrolizumab therapy did not change the clonality of the three patients during the treatment course (data not shown). In summary, we identified enriched Treg signatures delineating responders from progressors on pembrolizumab treatment, paradoxical to the current understanding of T cell subsets in solid tumors. However, these data are consistent with the recent observation that the presence of Tregs suggests a better prognosis in Hodgkin lymphoma, Follicular lymphoma, and other hematological malignancies. Figure 1 Disclosures Kay: Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Oncotracker: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees; Juno Theraputics: Membership on an entity's Board of Directors or advisory committees; Agios Pharma: Membership on an entity's Board of Directors or advisory committees; Cytomx: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Membership on an entity's Board of Directors or advisory committees; Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol Meyer Squib: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acerta Pharma: Research Funding; Sunesis: Research Funding; Dava Oncology: Membership on an entity's Board of Directors or advisory committees; Abbvie: Research Funding; MEI Pharma: Research Funding. Ansell:AI Therapeutics: Research Funding; Takeda: Research Funding; Trillium: Research Funding; Affimed: Research Funding; Bristol Myers Squibb: Research Funding; Regeneron: Research Funding; Seattle Genetics: Research Funding; ADC Therapeutics: Research Funding. Ding:Astra Zeneca: Research Funding; Abbvie: Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees; MEI Pharma: Membership on an entity's Board of Directors or advisory committees; alexion: Membership on an entity's Board of Directors or advisory committees; Beigene: Membership on an entity's Board of Directors or advisory committees; DTRM: Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: pembrolizumab

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Emicizumab-Kxwh for Previously Untreated Patients with Haemophilia: The Conversation Begins

Blood ◽

10.1182/blood-2019-127270 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2409-2409

Author(s):

Tiffany Lin Lucas ◽

Shveta Gupta ◽

Joanna A. Davis ◽

Fernando F. Corrales-Medina

Keyword(s):

Board Of Directors ◽

Real World ◽

Research Funding ◽

Hemophilia A ◽

The United States ◽

Haemophilia A ◽

Advisory Committees ◽

Inhibitor Development ◽

Increased Risk ◽

Prophylaxis Therapy

Introduction: With the Federal Drug and Administration approval of the use of emicizumab from birth to adulthood, clinicians will now grapple with when to choose and offer emicizumab for routine prophylaxis, especially in previously untreated patients (PUPs). Given the overall limited real-world reported data and experience using emicizumab in PUPs, we created and administered a survey to medical providers in the United States who care for paediatric patients with haemophilia to investigate real-world practice strategies and treatment selection for PUPs. Methods: After review and endorsement by the Haemostasis and Thrombosis Research Society (HTRS), the survey was electronically distributed by e-mail to all providers included in the HTRS core member list. The survey was also sent to those providers included in a list of Haemophilia Treatment Centre (HTC) physicians (with duplicate emails reconciled). Providers needed to self-identify as ones that treat pediatric patients to be included. The survey was developed as a tiered survey with questions presented to each recipient based on their prior responses. Results: Seventy-seven completed surveys were included and analysed. All participants were active providers at a comprehensive HTC and the majority (93.4%) were practicing at an academically affiliated site. In terms of characteristics of those that answered the survey, forty-eight percent of responders reported that 1-20% of their patients had expressed interest in emicizumab. 46% of participants (34/74) reported that they would personally consider emicizumab as their prophylaxis recommendation for the majority (>50%) of their hemophilia A patients without inhibitors. 57% (44/76) reported that 1-10% of their non-inhibitor hemophilia A patients were already prescribed emicizumab prophylaxis. Each participant was then asked about his or her consideration of emicizumab as prophylaxis therapy for a 2 month old PUP. Just over the majority were unsure or said no to this consideration (51.3%) and their concerns were lack of information on safety and efficacy in this young age group and increased risk for inhibitor development. If the 2 month old PUP had a high risk of inhibitor, the majority of providers who initially were hesitant to start emicizumab prophylaxis would remain so. Of note, those providers went on to be asked if the patient had gone on to complete 50 exposure days without inhibitor development, they would then become more likely to initiate emicizumab prophylaxis therapy. Use of concurrent factor replacement was posed to all participants and there were varied responses. Discussion: Overall, our results reflect a widespread practice variation and a not yet well-standardized or defined approach for the use of emicizumab in PUPs with haemophilia A. In this survey, patient preference and individual bleeding risk were the top reasons for which a provider would consider using switching to emicizumab prophylaxis in both severe and mild/moderate haemophilia A patients. This pattern of practice reflects the current era of individualized medicine. Overall, our findings reinforce the need for more studies to investigate the outcomes of a combined treatment approach with FVIII concentrates and emicizumab focusing in the potential benefit of this approach in decreasing the risk for inhibitor development PUPs. Clinicians also feel the need for further data to help clarifying the safety of emicizumab in this population. Figure Disclosures Gupta: Novartis: Honoraria, Speakers Bureau; CSL Behring: Research Funding; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Octapharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda-Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Davis:Sanofi: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda Shire: Consultancy; Spark Therapeutics: Consultancy. Corrales-Medina:Kedrion: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Membership on an entity's Board of Directors or advisory committees.

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Easix As Prediction Score before CAR-T Cells Vs Allogeneic Hematopoietic Cell Transplantation (alloHCT) for Relapsed/Refractory (r/r) Large B Cell Lymphoma (LBCL)

Blood ◽

10.1182/blood-2020-139615 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 11-12

Author(s):

Felix Korell ◽

Thomas Luft ◽

Michael Schmitt ◽

Sascha Dietrich ◽

Anita Schmitt ◽

...

Keyword(s):

T Cells ◽

Board Of Directors ◽

Research Funding ◽

B Cell Lymphoma ◽

Tumor Board ◽

Advisory Committees ◽

Educational Activities ◽

Bulky Disease ◽

Car T Cells ◽

Car T

BACKGROUND: In a previous study we have shown that CD19-directed chimeric antigen receptor (CAR)-T cells do not appear to be inferior to alloHCT when used as standard cellular immunotherapy (CI) for patients with multiply r/r LBCL (EBMT 2020). The purpose of the present follow-up analysis was to further compare the risk profile of the 2 cohorts by applying the EASIX score (lactate dehydrogenase (U/L) × creatinine (mg/dL)/thrombocytes (109 cells per L)), and to assess if EASIX could be used as outcome predictor in patients with r/r LBCL undergoing CAR-T and alloHCT, respectively. METHODS: Eligible were all patients referred to our institution with relapsed/refractory (R/R) DLBCL and a tumor board decision recommending treatment with CAR-T cells between 07/2018 and 02/2020 and those recommending allogeneic donor search between 2004 and 2019. Patients with DLBCL transformed from CLL were excluded. EASIX was evaluated retrospectively using uni- and multivariable analyses (with regards to age, gender and number of failed therapy lines) and mortality using Cox regression analyses. RESULTS: 41 patients intended for CAR-T cells and 60 patients intended for alloHCT were included. In both cohorts nearly all patients had active disease at indication. Cohorts were comparable for sex, time from diagnosis, ZUMA1 eligibility, and PS, but CAR-T patients tended to be older (median 56 vs 51 years, p=0.093), and had more often primary refractory and bulky disease (p=0.004 and p=0.04, respectively). Median EASIX score across both cohorts was 1.50 (0.27-70.5), with significantly higher scores in the CART group both at indication (EASIX-ind; median 1.79 and 1.22 for CAR-T and alloHCT, respectively, p=0.031) and at conditioning for CI (EASIX-pre, median 2.24 vs 1.26, p=0.005). Median OS from indication was 475d for the CAR-T cohort vs 285d for the alloHCT cohort (p=0.88). On multivariate analysis, EASIX-ind was significantly associated with adverse OS if alloHCT was intended (HR per 2fold increase 1.43, 95%CI 1.08-1.90, p=0.013), but not if CAR-T was intended (HR per 2fold increase 1.16, 95%CI 0.88-1.53, p=0.3). After CI, 12-month estimates for NRM, relapse incidence, PFS, and OS for CAR-T vs alloHCT were 3% vs 21% (p=0.04), 59% vs 44% (p=0.12), 39% vs 33% (p=0.97), and 68% vs 54% (p=0.32). EASIX-pre predicted overall survival (OS) in both CAR-T (HR per 2fold increase 2.11, 95%CI 1.21-3.7, p=0.009) and alloHCT (HR per 2fold increase 3.69, 95%CI 1.54-8.31, p=0.003) cohorts. In the alloHCT group, the EASIX effect was largely driven by higher NRM risk with increasing EASIX-pre, while in the CAR-T group poorer OS with increasing EASIX-pre was largely relapse-related. CONCLUSIONS: In patients undergoing CI for r/r LBCL, EASIX measured prior to conditioning can predict mortality after both CAR-T and alloHCT. If applied already at indication for CI, the predictive capacity of EASIX is weaker and no longer significant if CAR-T is intended. Further studies for validation of this data appear to be warrantable. Disclosures Schmitt: MSD: Membership on an entity's Board of Directors or advisory committees, Other: PI of clinical trials on letermovir; TolerogenixX Ltd: Other: Co-Founder and shareholder; Hexal: Other: Travel grants , Research Funding; Apogenix: Research Funding; Kite: Other: Travel grants, educational activities and conferences; Novartis: Other: educational activities and conferences, Research Funding. Dietrich:Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; KITE: Membership on an entity's Board of Directors or advisory committees. Schmitt:Hexal: Other: Travel grants ; TolerogenixX LtD: Other: Co-founder, Part-time employee ; Therakos/Mallinckrodt: Research Funding; Jazz Pharmaceuticals: Other: Travel grants . Dreger:Neovii: Research Funding; Roche: Consultancy, Speakers Bureau; Riemser: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy; Gilead: Consultancy, Speakers Bureau; AstraZeneca: Consultancy; AbbVie: Consultancy, Speakers Bureau.

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IGHV Unmutated Chronic Lymphocytic Leukemia (CLL) B Cells Are More Susceptible to Spontaneous Apoptosis Than Mutated CLL B Cells and Are Subject to the Anti-Apoptotic Effect of Environmental Signals

Blood ◽

10.1182/blood.v116.21.2431.2431 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2431-2431

Author(s):

Marta Coscia ◽

Francesca Pantaleoni ◽

Chiara Riganti ◽

Candida Vitale ◽

Micol Rigoni ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

Board Of Directors ◽

Stromal Cells ◽

Peripheral Blood ◽

Research Funding ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Spontaneous Apoptosis ◽

Advisory Committees

Abstract Abstract 2431 Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease. A very reliable prognosticator is the mutational status of the tumor immunoglobulin heavy chain variable region (IGHV): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. Soluble factors (i.e. IL-4 and CD40L) and cellular components of the local microenvironment [i.e. bone marrow stromal cells (BMSC) and nurse-like cells (NLCs)] are important survival factors for CLL B cells. It is currently unknown to what extent UM and M CLL cells depend on the local microenvironment for their survival. We have evaluated the spontaneous apoptotic rate of tumor cells isolated by immunomagnetic selection from the peripheral blood (PB) of M and UM CLL patients. Both M and UM CLL B cells underwent spontaneous apoptosis throughout the culture period. However, the UM CLL B cells showed a significantly higher degree of apoptosis in 7-day cultures as compared to M CLL B cells. In both M and UM CLL B cells, high basal levels of Bcl-2 expression and NF-kB activity were detected. On day 7, the percentage of Bcl-2+ leukemic cells was significantly lower in UM than in M CLL B cells. EMSA test showed that NF-kB was totally inactivated in UM CLL B cells and only partially reduced in M CLL B cells. Quantitative analysis of RelA and RelB subunits showed that NF-kB inactivation in UM CLL B cells consisted in a strong reduction of both RelA and RelB nuclear expression. CD40L, IL-4 and stromal cells significantly improved UM CLL B cells viability and significantly recovered Bcl-2 expression. The protective effect exerted by these stimuli was totally independent from the recovery of NF-kB expression. Indeed, after 7 days of culture, the UM CLL B cells had completely lost the nuclear form of NF-kB, and none of the stimuli was capable of restoring it. We observed that UM CLL cells were less susceptible to spontaneous apoptosis when cultured as unfractionated peripheral blood mononuclear cells (M or UM PBMC) as compared to purified leukemic cells (M and UM CLL B cells). The reduced apoptosis detected in UM PBMC was accompanied by a retained expression of Bcl-2 and by a restored activity of NF-kB and suggested the presence of a pro-survival element in the peripheral blood of these patients. To investigate the role of NLC in rescuing UM CLL B cells from apoptosis we first evaluated whether M and UM PBMC generated NLC with the same efficiency. Unexpectedly, the former generated significantly higher numbers of NLC than UM PBMC. Despite the lack of generation of NLC, CLL B cells viability was very similar in the non-adherent fraction of M and UM PBMC on day 7 and 14 of culture. This observation ruled out a role for NLC in supporting UM CLL B cells survival. Conversely, a pro-survival effect on UM CLL B cells was exerted by autologous T cells. Indeed, a significant reduction in the apoptotic rate of leukemic cells was observed when purified UM CLL B cells were cultured in the presence of autologous peripheral blood T cells (UM CLL B cell/T cell co-cultures). NF-kB activity was completely lost in UM CLL B cells cultured for 7 days in medium alone whereas it was restored in UM CLL B cells / T cells co-cultures. The prosurvival effect of circulating T cells was exerted both in cell-to-cell contact and in trans-well condition and was associated to increased secretions of tumor necrosis factor-alpha (TNF-α), platelet-derived growth factor (PDGF)-BB and interleukin-8 (IL-8) as detected by analyses of supernatants through a Multiplex system. These data indicate that despite their more aggressive features, UM CLL B cells are more susceptible to spontaneous apoptosis and depend from environmental prosurvival signals. This vulnerability of UM CLL B cells can be exploited as a selective target of therapeutic interventions. Disclosures: Boccadoro: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Massaia: Novartis: Honoraria, Research Funding.

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