Vascular Remodeling Underlies Re-Bleeding in Hemophilic Arthropathy

Abstract Hemophilic arthropathy is a debilitating condition that can develop as a consequence of frequent joint bleeding despite adequate clotting factor replacement. The mechanisms leading to repeated spontaneous bleeding are unknown. To better understand these mechanisms, we investigated the time course of synovial, vascular, stromal and cartilage changes in response to a single induced hemarthrosis in FVIII-deficient mice, compared the observed changes in vascular architecture to the changes in mouse models of rheumatoid arthritis (RA) and osteoarthritis (OA), and explored the extent to which these findings correlated with the development of hemophilic arthropathy in patients. Following a single induced hemarthrosis in the FVIII-deficient mouse, we found soft tissue hyperproliferation with marked induction of neoangiogenesis and evolving abnormal vascular architecture in the injured joint. While soft tissue changes were rapidly reversible, and cartilage changes were mild and mostly reversible, ongoing structural changes of abnormal vascularity persisted for months. These vascular changes involved pronounced remodeling with expression of α-Smooth Muscle Actin (SMA), Endoglin (CD105) and vascular endothelial growth factor (VEGF), as well as altered joint perfusion as determined by in vivo imaging. Surprisingly, these vascular changes were also seen in uninjured joints, suggesting systemic mediation of the observed effects. Changes in vascular architecture and pronounced expression of α-SMA were unique to hemophilia, and were not found in joint tissue obtained from RA or OA mice or from tissue from patients with OA or RA. Since FVIII-deficient mice do not suffer from spontaneous joint bleeding, human studies needed to be designed to reveal if such vascular changes were associated with joint bleeding and joint deterioration. Therefore, to determine if vascular perfusion changes were also present in hemophilia patients and to what extent they were related with joint bleeding and the degree of arthropathy, a cohort of 26 adult patients with hemophilia was studied prospectively. Radiographic Pettersson Scores and clinical Hemophilia Joint Health Scores (HJHS) were assessed and joint bleed status and vascularity changes were investigated by dynamic in vivo imaging using power Doppler (PD) during high resolution musculoskeletal ultrasound examinations of 156 joints (both elbows, knees and ankles of each patient) at baseline and, subsequently of 10 joints during painful episodes. Subclinical joint bleeding was present in 41% of joints at baseline. Positive PD signals were more frequently present in joints with subclinical bleeding than in joints without (83% vs. 55%; p< 0.01), while the mean PD signal score was significantly higher in bleeding joints (3.1 vs. 1.5; p < 0.01). Importantly, joint bleed status was independently associated with PD signal score (odds ratio [OR] = 1.45 [95% CI: 1.13, 1.86, p = 0.0035]) and with Pettersson score (OR = 1.21 [95%CI: 1.03, 1.43, p = 0.0185]) after adjusting for HJHS. Acute bleeding episodes, irrespective of occurring in previously non-bleeding or in previously subclinically bleeding joints only happened in joints where vascularity changes were present at baseline. Moreover, during these episodes, mean PD signal increased significantly from 3.5 at baseline to 6.0 (maximum 9 per joint; p≤0.05). Therefore, in vivo imaging demonstrated that those vascular changes, first observed in the FVIII-deficient mouse, were significantly associated with bleeding and joint deterioration in hemophilia patients. In aggregate, our observations in hemophilic mice and patients provide strong evidence that abnormal blood vessel formation and dynamic vascular remodeling facilitate and perpetuate joint bleeding in response to local and/or systemic stimuli. While changes in vascularity appear to be an important link to repeated bleeding and progression of arthropathy, the interrelations of bleeding and vascular changes are complicated and will require further study. These findings not only provide new insights into the pathobiology of progression of hemophilic arthroapthy, but also open new avenues to study molecular targets for angiogenesis inhibition to prevent aberrant vessel formation, potentially increasing the effectiveness of clotting factor replacement therapy. Disclosures Von Drygalski: CSL Behring: Honoraria, Speakers Bureau; Novo Nordisk: Honoraria, Research Funding; Biogen: Honoraria, Research Funding; Hematherix Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria, Research Funding; Pfizer: Honoraria; Baxalta: Honoraria, Research Funding. Mosnier:Hematherix Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

The Propagation of Hemophilic Arthropathy - the Role of Abnormal Angiogenesis and Vascular Remodeling in Recurrent Joint Bleeding in Adults with Hemophilia

Blood ◽

10.1182/blood.v126.23.3538.3538 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3538-3538

Author(s):

Kidder Wesley ◽

Eric Y Chang ◽

Colleen Moran ◽

Steven Rose ◽

Annette von Drygalski

Keyword(s):

Board Of Directors ◽

Vascular Remodeling ◽

Research Funding ◽

Clotting Factor ◽

Activity Levels ◽

Factor Activity ◽

Vascular Changes ◽

Advisory Committees ◽

Hemophilic Arthropathy ◽

Equity Ownership

Abstract Neoangiogenesis and vascular remodeling in the hypertrophic synovium of arthropathic hemophilic joints may disturb vascular integrity and contribute to repeated bleeding (A von Drygalski, et al. Vascular Remodeling Underlies Re-Bleeding in Hemophilic Arthropathy; ASH 2015; Abstract ID 78677). However, it is difficult to distinguish the importance of the contribution of abnormal vascular structures to hemophilic joint bleeding from the contribution of low clotting factor levels. This is because most prophylactic clotting factor replacement strategies are designed to avoid trough levels below 1-5%, but not to fully correct the factor deficiency. This report reveals the importance of aberrant angiogenesis for joint bleeding in two hemophilia patients whose clotting factor activity levels were constantly normal or near normal. The first patient had severe Hemophilia A with normalized Factor (f)VIII activity levels after liver transplantation 6 years previously. Abnormal vascularization of elbows, knees and ankles (absent bleeding) was detected by musculoskeletal ultrasound and Power Doppler (PD). Three months after the baseline exam the patient experienced severe elbow bleeding associated with new vascular changes on PD. These changes were characterized by pronounced confluent, patchy vascular signals and numerous enlarged and thickened vessels by angiography consistent with acute and chronic vascular remodeling. Bleeding could not be controlled by increasing fVIII activity to supra-normal levels, and ultimately required synovial embolization. The second patient has severe Hemophilia B and presented shortly after left knee joint replacement with fluctuating knee bleeding for many months associated with pronounced dynamic vascular changes on PD. Neither bleeding nor vascular changes were prevented by daily fIX treatment which maintained near normal plasma fIX activity levels. Within one year, additional chronic bleeding with vascular changes developed in the left elbow and left ankle and remained unresponsive to intense factor replacement. These cases provide several insights. First, vascular changes in hemophilic arthropathy persist and evolve in dynamic fashion even in the presence of normal clotting factor activities. Second, vascular disturbance and leakiness contributed to bleeding independent of clotting factor activity levels as exemplified by the first patient and as suggested by the second patient. Third, the sequence of bleeding events in the second patient suggests that at least some of the observed vascular abnormalities are mediated systemically. These observations provide clinical evidence in support of the hypothesis that neoangiogenesis and vascular remodeling contribute to hemophilic joint bleeding and should lead to new studies of the pathophysiology of vascular remodeling in such joints. Targeting angiogenesis may emerge as a new treatment strategy to enhance the effectiveness of clotting factor replacement hemophilic arthropathy. Disclosures Chang: Biogen: Research Funding. Rose:Sirtex Medical: Equity Ownership, Honoraria; XLSciTech: Consultancy; Surefire Medical: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Embolx: Consultancy. von Drygalski:Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Research Funding; Hematherix Inc: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text

Targeting Wnt/β-Catenin and BCL-2, Two Critical Survival Factors, Synergistically Induces Apoptosis in AML Via Rac1-Mediated MCL-1 Inhibition

Blood ◽

10.1182/blood-2018-99-118367 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1442-1442

Author(s):

Xiangmeng Wang ◽

Po Yee Mak ◽

Wencai Ma ◽

Xiaoping Su ◽

Hong Mu ◽

...

Keyword(s):

Progenitor Cells ◽

Board Of Directors ◽

Cell Lines ◽

Research Funding ◽

Single Agent ◽

Advisory Committees ◽

Equity Ownership ◽

Critical Survival

Abstract Wnt/β-catenin signaling regulates self-renewal and proliferation of AML cells and is critical in AML initiation and progression. Overexpression of β-catenin is associated with poor prognosis. We previously reported that inhibition of Wnt/β-catenin signaling by C-82, a selective inhibitor of β-catenin/CBP, exerts anti-leukemia activity and synergistically potentiates FLT3 inhibitors in FLT3-mutated AML cells and stem/progenitor cells in vitro and in vivo (Jiang X et al., Clin Cancer Res, 2018, 24:2417). BCL-2 is a critical survival factor for AML cells and stem/progenitor cells and ABT-199 (Venetoclax), a selective BCL-2 inhibitor, has shown clinical activity in various hematological malignancies. However, when used alone, its efficacy in AML is limited. We and others have reported that ABT-199 can induce drug resistance by upregulating MCL-1, another key survival protein for AML stem/progenitor cells (Pan R et al., Cancer Cell 2017, 32:748; Lin KH et al, Sci Rep. 2016, 6:27696). We performed RNA Microarrays in OCI-AML3 cells treated with C-82, ABT-199, or the combination and found that both C-82 and the combination downregulated multiple genes, including Rac1. It was recently reported that inhibition of Rac1 by the pharmacological Rac1 inhibitor ZINC69391 decreased MCL-1 expression in AML cell line HL-60 cells (Cabrera M et al, Oncotarget. 2017, 8:98509). We therefore hypothesized that inhibiting β-catenin by C-82 may potentiate BCL-2 inhibitor ABT-199 via downregulating Rac1/MCL-1. To investigate the effects of simultaneously targeting β-catenin and BCL-2, we treated AML cell lines and primary patient samples with C-82 and ABT-199 and found that inhibition of Wnt/β-catenin signaling significantly enhanced the potency of ABT-199 in AML cell lines, even when AML cells were co-cultured with mesenchymal stromal cells (MSCs). The combination of C-82 and ABT-199 also synergistically killed primary AML cells (P<0.001 vs control, C-82, and ABT-199) in 10 out of 11 samples (CI=0.394±0.063, n=10). This synergy was also shown when AML cells were co-cultured with MSCs (P<0.001 vs control, C-82, and ABT-199) in all 11 samples (CI=0.390±0.065, n=11). Importantly, the combination also synergistically killed CD34+ AML stem/progenitor cells cultured alone or co-cultured with MSCs. To examine the effect of C-82 and ABT-199 combination in vivo, we generated a patient-derived xenograft (PDX) model from an AML patient who had mutations in NPM1, FLT3 (FLT3-ITD), TET2, DNMT3A, and WT1 genes and a complex karyotype. The combination synergistically killed the PDX cells in vitro even under MSC co-culture conditions. After PDX cells had engrafted in NSG (NOD-SCID IL2Rgnull) mice, the mice were randomized into 4 groups (n=10/group) and treated with vehicle, C-82 (80 mg/kg, daily i.p injection), ABT-199 (100 mg/kg, daily oral gavage), or the combination for 30 days. Results showed that all treatments decreased circulating blasts (P=0.009 for C-82, P<0.0001 for ABT-199 and the combination) and that the combination was more effective than each single agent (P<0.001 vs C-82 or ABT-199) at 2 weeks of therapy. The combination also significantly decreased the leukemia burden in mouse spleens compared with controls (P=0.0046) and single agent treated groups (P=0.032 or P=0.020 vs C-82 or ABT-199, respectively) at the end of the treatment. However, the combination did not prolong survival time, likely in part due to toxicity. Dose modifications are ongoing. These results suggest that targeting Wnt/β-catenin and BCL-2, both essential for AML cell and stem cell survival, has synergistic activity via Rac1-mediated MCL-1 inhibition and could be developed into a novel combinatorial therapy for AML. Disclosures Andreeff: SentiBio: Equity Ownership; Oncolyze: Equity Ownership; Oncoceutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Consultancy; Amgen: Consultancy, Research Funding; Eutropics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding; Aptose: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Reata: Equity Ownership; Astra Zeneca: Research Funding; Celgene: Consultancy; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer . Carter:novartis: Research Funding; AstraZeneca: Research Funding.

Download Full-text

Identification of Small Molecule Kinase Inhibitors That Potently and Reversibly Block Chimeric Antigen Receptor T Cell Proliferation and Cytotoxicity

Blood ◽

10.1182/blood-2019-126165 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2068-2068

Author(s):

Parmeshwar Amatya ◽

Matthew L. Cooper ◽

Alun J Carter ◽

John F. DiPersio

Keyword(s):

T Cell ◽

Research Funding ◽

Kinase Inhibitors ◽

Kinase Inhibitor ◽

Advisory Committees ◽

Car T Cell ◽

Car T ◽

Life Threatening ◽

Equity Ownership

Introduction: Despite remarkable clinical efficacy, CAR-T therapy has been limited by life threatening toxicities in over 30% of patients.1, 2 Toxicities primarily manifest as cytokine release syndrome (CRS) characterized by an early phase with fever, hypotension and elevations of cytokines including IFNγ, GM-CSF, TNF, IL-10, and IL-6 and a later phase associated with life-threatening or life-ending neurologic events. We hypothesized that reversible inhibition of CAR mediated signaling will enable controllable regulation of CAR-T cell activity in vivo and mitigate CAR-T mediated toxicities. There are specific protein kinases such as the SRC kinases, LCK, and ZAP-70 that are known to be involved in various cellular signaling pathways, especially T cell receptor mediated signaling and may also be appropriate targets for modulating (both enhancing and inhibiting) CAR-T function in a rapid and reversible fashion in vivo. Our hypothesis is that small molecule inhibitors of TCR signaling and downstream pathways could be identified using specific high throughput screens. Methods: To identify novel inhibitors of CAR-T cell proliferation, we developed a high throughput kinase inhibitor screen to identify compounds that reversibly inhibit CAR-T function. T cells containing a third generation CAR targeting CD19 cells (CAR19) and CD19+ tumor cells (Ramos cells expressing both GFP and luciferase) were incubated at an effector to target ratio of 1:1 in 96 well plates in the presence of 1µM of each inhibitor. After 24 hours, tumor cell death induced by CAR-T was measured using bioluminescence (BLI) imaging. Small molecules that inhibited CAR-T proliferation and cytotoxicity were determined by assessing the BLI signal in each well. Results: A protein kinase inhibitor library (Selleckchem, Texas) containing 644 independent compounds was tested (Figure 1). Of the 644 kinase inhibitors tested, 32 were found to be potent inhibitors of CART19 cell activation and cytotoxic killing of CD19+ target tumor cells, reducing anti-tumor viability in 24 hours by >50% compared to vehicle control. Compounds such as Nintedanib (C3), Dasatinib (C9), and Saracatinib (C12), all SRC kinase inhibitors, were able to inhibit cell killing by 99%, 84%, and 76% respectively. Next we assessed the reversibility of CAR-T cell mediated killing upon removal of inhibitors from the cultures. Reinitiation of potent, anti-tumor activity was observed within 24 hours after inhibitor removal, confirming reversible nature of CAR-T cell inhibition by the three most potent compounds. Conclusions: Recent publications (Weber et al Blood Adv, 2018, Westermann et al. Sci Transl Med, 2019) have also shown that dasatinib can reversibly suppresses CAR-T cell cytotoxicity, cytokine secretion, and proliferation in vitro and in vivo. 3, 4 Here we confirm the reports of others regarding dasatinib and that show for the first time that reversible inhibition of CAR-T activity by kinase inhibitors is not limited solely to dasatanib, but is observed with other small molecules targeting many different kinases. This work further demonstrates the potential applications of tyrosine kinase inhibitors as a safety switch to modulate CAR-T cell toxicity. 1. Maude, NEJM 2014 2. Davila, SciTransMed 2014 3. Mestermann SciTransMed 2019 4. Weber. Blood Adv 2019 Disclosures Cooper: Wugen: Consultancy, Equity Ownership, Patents & Royalties. DiPersio:WUGEN: Equity Ownership, Patents & Royalties, Research Funding; Magenta Therapeutics: Equity Ownership; Celgene: Consultancy; Karyopharm Therapeutics: Consultancy; RiverVest Venture Partners Arch Oncology: Consultancy, Membership on an entity's Board of Directors or advisory committees; Cellworks Group, Inc.: Membership on an entity's Board of Directors or advisory committees; NeoImmune Tech: Research Funding; Bioline Rx: Research Funding, Speakers Bureau; Macrogenics: Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding; Amphivena Therapeutics: Consultancy, Research Funding.

Download Full-text

Enhanced In Vivo Persistence and Proliferation of NK Cells Expanded in Culture with the Small Molecule Nicotinamide: Development of a Clinical-Applicable Method for NK Expansion

Blood ◽

10.1182/blood.v130.suppl_1.657.657 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 657-657 ◽

Cited By ~ 1

Author(s):

Tony Peled ◽

Guy Brachya ◽

Nurit Persi ◽

Chana Lador ◽

Esti Olesinski ◽

...

Keyword(s):

Nk Cells ◽

Board Of Directors ◽

Research Funding ◽

Nk Cell ◽

Culture Method ◽

Employment Equity ◽

Advisory Committees ◽

Nsg Mice ◽

Equity Ownership

Abstract Adoptive transfer of cytolitic Natural Killer (NK) cells is a promising immunotherapeutic modality for hematologic and other malignancies. However, limited NK cell in vivo persistence and proliferation have been challenging clinical success of this therapeutic modality. Here we present a reliable, scalable and GMP-compliant culture method for the expansion of highly functional donor NK cells for clinical use. Nicotinamide (NAM), a form of vitamin B-3, serves as a precursor of nicotinamide adenine dinucleotide (NAD) and is a potent inhibitor of enzymes that require NAD including ADP ribosyltransferases and cyclic ADP ribose/NADase. As such, NAM is implicated in the regulation of cell adhesion, polarity, migration, proliferation, and differentiation. We have previously reported that NAM augments tumor cytotoxicity and cytokine (TNFα and IFN-γ) secretion of NK cells expanded in feeder-free culture conditions stimulated with IL-2 or IL-15. Immunophenotype studies demonstrated NK cells expanded with NAM underwent typical changes observed with cytokine only-induced NK cell activation with no significant differences in the expression of activating and inhibitory receptors. CD200R and PD-1 receptors were expressed at low levels in resting NK cells, but their expression was up-regulated following activation in typical cytokine expansion cultures. Interestingly, the increase in CD200R and PD-1 was reduced by NAM, suggesting these NK cells to be less susceptible to cancer immunoevasion mechanisms (Fig 1). In vivo retention and proliferation is a pre-requisite for the success of NK therapy. We have reported that NK expanded with NAM displayed substantially better retention in the bone marrow, spleen and peripheral blood of irradiated NSG mice. Using a carboxyfluorescein succinimidyl ester (CFSE) dilution assay, we demonstrated increased in vivo proliferation of NAM-cultured NK cells compared with cells cultured without NAM. These results were recently confirmed using a BrdU incorporation assay in irradiated NSG mice (Fig.2). These findings were mechanistically supported by a substantial increase in CD62L (L-selectin) expression in cultures treated with NAM. CD62L is pivotal for NK cell trafficking and homeostatic proliferation and its expression is down regulated in IL-2 or IL-15 stimulated cultures (Fig. 3). These data provided the foundation for the development of a feeder cell-free scalable culture method for clinical therapy using apheresis units obtained from healthy volunteers. CD3+ cells were depleted using a CliniMACS T cell depletion set. Following depletion, the CD3- fraction was analyzed for phenotypic markers and cultured in closed-system flasks (G-Rex100 MCS, Wilson Wolf) supplemented with 20ng/ml IL-15 or 50ng/ml IL-2 GMP, 10% human serum, minimum essential medium-α and NAM USP for two weeks. While at seeding, NK cells comprised 5-20% of total culture seeded cells, at harvest, NK cells comprised more than 97% of the culture. Although overall contamination of the NK cultures was low with either IL-15 or IL-2, a lower fraction of CD3+ and CD19+ cells was observed with IL-15 vs IL-2 (0.2±0.1% vs. 0.4±0.2% and 1.3±0.4% vs. 2.4±0.6%, respectively). Consequently, we decided to use IL-15 for clinical manufacturing. Optimization of NAM concentration studies showed similar expansion with 2.5 and 5 mM and a decrease in expansion with 7.5 mM NAM. Since NAM at 5 mM had a stronger impact on CD62L expression and on the release of IFNγ and TNFα than NAM at 2.5 mM, we selected 5mM NAM for clinical manufacturing. Overall median NK expansion after two weeks in closed G-Rex flasks supplemented with IL-15 and 5mM NAM was 50-fold (range 37-87). An additional and significant increase in expansion was obtained after doubling the culture medium one week post seeding. While there was a marked advantage for single culture feeding, more feedings had less impact on NK expansion and had a negative effect on the in vivo retention potential. Our optimized expansion protocol therefore involved one feeding during the two weeks expansion duration resulting in 162±30.7-fold expansion of NK cells relative to their input number in culture. Based on these data, we have initiated a clinical trial at University of Minnesota, to test the safety and efficacy of escalating doses (2 x 107/kg - 2 x 108/kg) of our novel NAM NK cell product in patients with refractory non-Hodgkins lymphoma and multiple myeloma (NCT03019666). Disclosures Peled: Gamida Cell: Employment, Equity Ownership. Brachya: Gamida Cell: Employment. Persi: Gamida Cell: Employment. Lador: gamida Cell: Employment, Equity Ownership. Olesinski: gamida cell: Employment. Landau: gamida cell: Employment, Equity Ownership. Galamidi: gamida cell: Employment. Peled: Biokine: Consultancy; Biosight: Consultancy. Miller: Celegene: Consultancy; Oxis Biotech: Consultancy; Fate Therapeutics: Consultancy, Research Funding. Bachanova: Oxis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Zymogen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Seattle-Genetics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis Pharmaceuticals Corporation: 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.

Download Full-text

Significant In Vivo Sensitivity to Aurora Kinase Inhibition in TCF3-Hlf rearranged Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood-2018-99-115706 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 4026-4026

Author(s):

Bill H. Chang ◽

Jessica Leonard ◽

Joelle Wolf ◽

Michelle Degnin ◽

Kyle Lenz ◽

...

Keyword(s):

Board Of Directors ◽

Research Funding ◽

Lymphoblastic Leukemia ◽

Variable Region ◽

Drug Screen ◽

Significant Heterogeneity ◽

Type I ◽

Advisory Committees ◽

Equity Ownership

Abstract Acute Lymphoblastic Leukemia (ALL) harboring the t(17;19)(q22;p13) is a rare subtype of leukemia with a dismal prognosis. This recurring translocation produces an aberrant TCF3-HLF fusion with distinct gene expression profiles and drug sensitivity. Recent studies have shown that this subtype of ALL might be targeted using therapies inhibiting BCL-2 and the pre-B cell receptor through inhibition of SRC family kinases. However, preliminary validation of these studies have revealed significant heterogeneity of response to BCL-2 and SRC inhibitors. As such, we sought to identify other possible targets that could overcome this heterogeneity and improve response to therapy. Methods: One local as well as four other samples from the Children's Oncology Group's ALL Biorepository with TCF3-HLF ALL were expanded in immunodeficient NSG mice. All samples were verified by RT-PCR and Sanger sequencing for the fusion transcript. Samples were then interrogated with our functional drug screen that is comprised of compounds with activity against two-thirds of the tyrosine kinome as well as other non-tyrosine kinase pathways, including RAF/MEK/MAPKs, PI3K/AKT/mTOR, AMPK, ATM, Aurora kinases, CAMKs, CDKs, GSK3a/b, IKK, PKA, PKC, PLK1, and RAF as well as BCL2 family, BRD4, IDH1/2, Hedgehog, HSP90, NOTCH/g-secretase, proteasome, survivin, STAT3, and WNT/b-catenin. The samples were sequenced using the Agilent SureSelect Strand-Specific RNA Library Preparation Kit on the Bravo robot (Agilent). All five patient samples successfully engrafted into NSG mice and were tested for in vivo sensitivity as assessed for disease burden or survival. Results: Three patient samples were identified to carry Type I translocations fusing exon 13 of TCF3 with variable intronic insertions followed by exon 4 of HLF. All three type I translocations produced different fusions due to different lengths within the variable region. One sample predicted a truncation product of TCF3 ending in exon 13 with an early stop codon within the variable region. Two patient samples carried the identical type II translocation fusing Exon 12 of TCF3 with exon 4 of HLF. RNA-seq results of the five samples identified other individual translocations, but none involved other specific disease related lesions. Results from our drug screen showed significant heterogeneity in response to the majority of drugs assayed including the ABL/SRC inhibitor dasatinib and the BCL-2 inhibitor venetoclax. Further, in vivo studies exposing cohorts of animals to vehicle (n=5), dasatinib (40mg/kg/day; n=5), venetoclax (25-100mg/kg/day; n=5) or combination of dasatinib and venetoclax (n=5) identified only two samples with treatment benefit. Interestingly, review of the results of the drug screen suggested hypersensitivity to aurora kinase inhibitors. Each sample was tested in vivo in cohorts of vehicle (n=5) and alisertib (30mg/kg/day; n=5). All five ALL samples showed significant response (p<0.01 for all five samples compared to their respective vehicle controls by Chi Square analysis). All animals tolerated treatment and no animal showed significant hematologic toxicity from treatment with drugs. Conclusion: Our results suggest that TCF3-HLF ALL is a heterogeneous subset of ALL with both different gene expression patterns from TCF3-HLF to other fusions as well as functional drug response. In vivo validation in the murine model with these five samples suggests significant heterogeneity to current pursued targets such as BCL-2 and SRC compared to previously published reports. Most intriguing, all samples tested with alisertib identified significant in vivo response suggesting unique preclinical support to pursue further clinical testing within this rare and lethal subtype of ALL. Disclosures Leonard: Amgen: Research Funding. Mullighan:Loxo Oncology: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding; Cancer Prevention and Research Institute of Texas: Consultancy. Tyner:Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Array: Research Funding; Genentech: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Aptose: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding. Druker:GRAIL: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Research Funding; Leukemia & Lymphoma Society: Membership on an entity's Board of Directors or advisory committees, Research Funding; Patient True Talk: Consultancy; Fred Hutchinson Cancer Research Center: Research Funding; ARIAD: Research Funding; Beta Cat: Membership on an entity's Board of Directors or advisory committees; Oregon Health & Science University: Patents & Royalties; McGraw Hill: Patents & Royalties; Novartis Pharmaceuticals: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Consultancy, Membership on an entity's Board of Directors or advisory committees; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; ALLCRON: Consultancy, Membership on an entity's Board of Directors or advisory committees; MolecularMD: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Henry Stewart Talks: Patents & Royalties; Blueprint Medicines: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Monojul: Consultancy; Celgene: Consultancy; Third Coast Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aileron Therapeutics: Consultancy; Aptose Therapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Cepheid: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millipore: Patents & Royalties.

Download Full-text

T Cells Educated By DC/AML Fusions in the Context of 4-1BB Costimulation As a Potent Strategy for Adoptive Cellular Therapy

Blood ◽

10.1182/blood-2019-131491 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2673-2673

Author(s):

Jessica Liegel ◽

Dina Stroopinsky ◽

Haider Ghiasuddin ◽

Adam Morin ◽

Marzia Capelletti ◽

...

Keyword(s):

T Cells ◽

Board Of Directors ◽

Research Funding ◽

Cellular Therapy ◽

Ex Vivo ◽

Advisory Board ◽

Adoptive Cellular Therapy ◽

Advisory Committees ◽

Equity Ownership

Introduction: Our group has developed a novel vaccine using patient-derived acute myeloid leukemia (AML) cells and autologous dendritic cells (DCs), capable of presenting a broad array of leukemia antigens. In a phase I/II clinical trial DC/AML vaccination led to an expansion of leukemia-specific T cells. We hypothesized that the fusion vaccine offered a unique platform for ex vivo expansion of functionally potent leukemia specific T cells with broad specificity targeting shared and tumor specific neoantigens. We postulated that incorporating 4-1BB (CD137) mediated co-stimulation would further enhance activation of antigen specific T cells and the development of a crucial memory response as well as promote survival and persistence. Here we describe therapeutic exploration of the use of 4-1BB to augment vaccine-educated T cells for adoptive cellular therapy in an immunocompetent murine model. Methods: DC/AML fusion vaccine was generated using DCs obtained from C57BL/6J mice and syngeneic C1498 AML cells as previously described. T cells were obtained from splenocytes after magnetic bead isolation and cultured with irradiated DC/AML fusion vaccine in the presence of IL-15 and IL-7. Following co-culture, 4-1BB positive T cells were ligated using agonistic 4-1BB antibody (3H3 clone, BioXCell) and further selected with RatIgG2a magnetic beads (Easy Sep). Subsequently T cells were expanded with anti-CD3/CD28 activation beads (Dynabeads). In vivo, mice underwent retro-orbital inoculation with C1498 and vaccination with irradiated fusion cells the following day. Agonistic mouse anti-4-1BB antibody was injected intraperitoneally on day 4 and day 7. In addition, C1498 cells were transduced with Mcherry/luciferase and a reproducible model of disease progression was established. Results: DC/fusion stimulated T cells showed increased immune activation as measured by multichannel flow cytometric analysis. Compared to unstimulated T cells, there was 5-fold increase in CD4+CD25+CD69+, and a 10-fold and 7-fold increase in 4-1BB and intracellular IFNƔ expression on CD8+ cells respectively. Following agonistic 4-1BB ligation and bead isolation, the proliferation rate was increased in the 4-1BB positive fraction as compared to both 4-1BB negative cells and unstimulated T cells. In addition, the 4-1BB positive fraction demonstrated increased cytotoxicity, as measured by a CTL assay detecting granzyme B with 1:10 tumor to effector cells. A shift from naïve to memory T cell phenotype was also observed. Following DC/fusion stimulation, CD44+CD62L- cells comprised 67% of CD8+ cells versus 20% without stimulation, the latter reflecting the effect of cytokines alone. Following 4-1BB ligation and anti-CD3/CD28 bead expansion, this phenotype was retained with the CD4+ and CD8+ effector memory and central memory compartments comprising the majority of T cells. Such findings are significant as presence of memory T cell populations are a critical component for successful adoptive cell transfer. The effect of agonistic 4-1BB antibody following vaccination was evaluated in vivo in an aggressive immunocompetent murine AML model. The combination of DC/AML fusion vaccine with 4-1BB antibody was associated with increased long-term survival (>120 days) of 40% versus 20% of mice treated with vaccine alone while all controls required euthanasia by 40 days. Conclusion: In the current study we have demonstrated the ability of DC/AML fusion vaccine to stimulate T cells ex-vivo as demonstrated by both early-activation (CD25,CD69), upregulation of antigen-specific markers (CD137) and cytokine secretion. Further enhancement of the cellular product using agonistic 4-1BB ligation and isolation simultaneously enriches for antigen-activated cells, as demonstrated by more potent cytotoxicity, as well as promoting memory phenotype and survival. Use of 4-1BB ligation for antigen-specific selection while providing an agonistic co-stimulatory signal is a potentially novel approach for development of non-engineered T cells. Ongoing experiments evaluating the efficacy of 4-1BB selected vaccine educated T cells using bioluminescence monitoring will be reported as well as in vitro use of patient-derived T cells. Disclosures Kufe: Canbas: Consultancy, Honoraria; Victa BioTherapeutics: Consultancy, Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees; Genus Oncology: Equity Ownership; Hillstream BioPharma: Equity Ownership; Reata Pharmaceuticals: Consultancy, Equity Ownership, Honoraria; Nanogen Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Rosenblatt:Dava Oncology: Other: Education; Partner Tx: Other: Advisory Board; Parexel: Consultancy; Celgene: Research Funding; BMS: Research Funding; Amgen: Other: Advisory Board; Merck: Other: Advisory Board; BMS: Other: Advisory Board ; Imaging Endpoint: Consultancy. Avigan:Takeda: Consultancy; Parexel: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees; Partners Tx: Membership on an entity's Board of Directors or advisory committees; Partner Tx: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy.

Download Full-text

Antibody-Interferon-Alpha Fusion Protein Therapy for the Treatment of B-Cell Non-Hodgkin Lymphoma: Enhanced ADCC, Inhibition of Proliferation, and In Vivo Eradication of CD20+ Human Lymphomas

Blood ◽

10.1182/blood.v126.23.2762.2762 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2762-2762 ◽

Cited By ~ 1

Author(s):

John M. Timmerman ◽

Kristopher K Steward ◽

Reiko E Yamada ◽

Patricia A Young ◽

Dena M. Minning ◽

...

Keyword(s):

Fusion Protein ◽

B Cell ◽

Board Of Directors ◽

Research Funding ◽

Employment Equity ◽

Advisory Committees ◽

Term Survival ◽

Equity Ownership

Abstract Background: Interferon-alpha (IFNα) is a pleiotrophic cytokine with direct anti-tumor and immunostimulatory effects. Currently IFNα is approved for the treatment of multiple hematologic malignancies, including non-Hodgkin lymphoma (NHL). However, its clinical utility has been hindered by dose-limiting toxicitiy due to systemic activation of the interferon receptor. To overcome this limitation, we engineered anti-tumor antibody-IFNα fusion proteins to selectively increase delivery of IFN to the tumor site and reduce systemic toxicity. We previously reported that IGN002, an anti-CD20-IFNα fusion protein, exhibits enhanced complement-dependent cytotoxicity (CDC) compared to rituximab, and inhibits proliferation and induces apoptosis of human B-cell NHL (Yamada et al, ASCO 2013). We now extend these previous findings and show that IGN002 possesses enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) effector function and superior in vivo anti-tumor activity against B-cell NHL, compared to rituximab. Methods: IGN002 was evaluated against a panel of human Burkitt and diffuse large B-cell lymphoma (DLBCL) cell lines. Proliferation was measured by [3 H]-thymidine incorporation, STAT1 activation by flow cytometry, ADCC by lactate dehydrogenase release using human PBMC effectors, and IFN bioactivity by encephalomyocarditis (EMC) viral protection assay. NHL xenografts were grown in SCID mice. Results: IGN002 more potently inhibited the growth of NHL cell lines expressing CD20 than rituximab or unfused IFNα. Intrinsic IFNα activity of IGN002 was reduced in viral protection and anti-proliferation assays using cells lacking CD20 expression. STAT1 activation by IGN002 was enhanced on cells expressing the target antigen, whereas a control antibody-IFNα fusion protein showed reduced STAT activation activity compared to unfused IFNα. Together, these results indicate that fusion of IFNα to the antibody results in reduced IFN effects on cells not bearing the tumor antigen target. IGN002 exhibited enhanced ADCC activity compared to rituximab against Daudi, Ramos, and Raji NHL cells in long-term (overnight incubation) assays, demonstrating both higher potency and higher maximal cytotoxicity. This result is possibly due to activation of the effector cell populations by the fused IFNα moiety, as IFN is known to activate both NK cells and monocytes. The in vivo anti-tumor efficacy of IGN002 was compared to rituximab and a control antibody-IFNα fusion protein against 10-day established Raji NHL xenografts. IGN002 was superior to both rituximab and the control fusion protein, achieving a longer median survival and higher long-term survival rate (p = 0.0015 and < 0.0001 vs. rituximab and control fusion protein, respectively). The in vivo anti-tumor efficacy of IGN002 was also compared to rituximab at three equimolar dose levels (5 mg/kg, 1 mg/kg, and 0.2 mg/kg antibody) against 10-day established Daudi NHL xenografts. IGN002 showed superior efficacy compared to rituximab at all doses (p < 0.001), achieving tumor eradication (100% long-term survival) in all mice treated at all three dose levels, whereas rituximab only delayed tumor progression. Conclusions: IGN002 demonstrated more robust direct anti-proliferative and antibody effector functions than rituximab against human NHL cells in vitro, and also showed the ability to eradicate established NHL xenografts in vivo. Against cells expressing the CD20 target antigen, IGN002 exhibited greater anti-proliferative potency than unfused IFNα. In contrast, the anti-proliferative and anti-viral potency of IGN002 was reduced against cells lacking CD20, compared to unfused IFNα. These findings support the hypothesis that tumor antigen-targeted IFN therapeutics may possess a broader therapeutic index than unfused IFNα, inhibiting tumor growth by multiple mechanisms while reducing systemic toxicity. These results support the further development of IGN002 for the treatment of B-cell NHL, and a first-in-human phase I clinical study will begin later this year in the United States. Disclosures Timmerman: Janssen: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Valor Biotherapeutics: Research Funding. Steward:ImmunGene, Inc.: Employment. Minning:Valor Biotherapeutics, LLC: Consultancy. Sachdev:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Gresser:ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Khare:Valor Biotherapeutics: Membership on an entity's Board of Directors or advisory committees; ImmunGene, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Morrison:ImmunGene, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Download Full-text

Targeting of JAK/STAT Signaling to Reverse Stroma-Induced Cytoprotection Against BCL2 Antagonist Venetoclax in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.32.32 ◽

2016 ◽

Vol 128 (22) ◽

pp. 32-32

Author(s):

Riikka Karjalainen ◽

Mihaela Popa ◽

Minxia Liu ◽

Mika Kontro ◽

Mireia Mayoral Safont ◽

...

Keyword(s):

Board Of Directors ◽

Stromal Cells ◽

Research Funding ◽

Stromal Cell ◽

Ex Vivo ◽

Intrinsic Resistance ◽

Advisory Committees ◽

Equity Ownership ◽

Refractory Aml

Abstract Several promising new, targeted agents are being developed for the treatment of AML. The BH3 mimetic venetoclax (ABT-199) is a specific inhibitor of BCL2, with results from a phase 2 study showing transient activity of venetoclax in relapsed/refractory AML (Konopleva et al, 2014). The bone marrow (BM) microenvironment is known to protect AML cells from drug therapy and we showed earlier that conditioned medium (CM) from BM stromal cells applied to AML patient cells conferred resistance to venetoclax, which could be reversed by the addition of the JAK1/2 inhibitor ruxolitinib (Karjalainen et al, 2015). Here, we investigated the mechanisms mediating the BM stromal cell induced resistance to venetoclax and its reversal by ruxolitinib. To identify the soluble factor(s) contributing to stroma-induced protection of BCL2 inhibition, we analyzed the cytokine content of 1) CM from the human BM stromal cell line HS-5, 2) CM from BM mesenchymal stromal cells (MSCs) isolated from AML patients, 3) supernatants from BM aspirates collected from AML patients, and 4) supernatants from BM aspirates collected from healthy donors. Although expression levels varied, the cytokines detected were similar among the different samples. In HS-5 CM, IL-6, IL-8 and MIP-3α were among the most abundant cytokines. In addition, gene expression analysis showed the receptors for these cytokines were expressed in AML patient samples. IL-6, IL-8 and MIP-3α were added individually to mononuclear cells collected from AML patients, which were then treated with venetoclax. However, none of the cytokines alone could mimic the reduced sensitivity to venetoclax conferred by the HS-5 CM suggesting that stromal cell induced cytoprotection is likely multi-factorial. Next we tested the effect of AML-derived BM MSCs on the ex vivo response of AML patient samples (n=8) to ruxolitinib or venetoclax alone or in combination in a co-culture setting. Apoptosis assays showed negligible effects of ruxolitinib at a concentration of 300 nM, while venetoclax at a dose of 100 nM induced reduction in the percentage of CD34+ AML cells. Co-treatment with venetoclax and ruxolitinib demonstrated synergistic effects in 6 out of 8 samples and significantly reduced the number of CD34+ AML cells. Mechanistic studies showed that ruxolitinib treatment inhibited the BM stromal medium-induced expression of BCL-XL mRNA on AML cells and the drugs in combination down-regulated BCL2, MCL1 and BCL-XL protein expression, which was in correlation with sensitivity to the drugs. To further evaluate the ability of the venetoclax and ruxolitinib combination to eradicate leukemic cells in vivo we used an orthotopic xenograft model of AML. NSG mice were injected with genetically engineered MOLM-13luc cells and after engraftment treated with venetoclax (25 mg/kg, i.p.), ruxolitinib (50 mg/kg BID, p.o) or both and imaged once per week for 4 weeks. At the end of the treatment period bioluminescent imaging showed significantly reduced leukemia burden in the ruxolitinib and venetoclax co-treated mice compared to controls demonstrating superior anti-tumor efficacy than either agent alone (Figure 1). In summary, our data demonstrate that the combined blockade of JAK/STAT and BCL2 pathways with ruxolitinib and ventoclax is synergistic in ex vivo co-culture models and in vivo in an AML mouse model. The addition of ruxolitinib was able to overcome intrinsic resistance to venetoclax by reducing expression of MCL1, a known escape mechanism of BCL2 inhibition. These results support further clinical investigation of this combination, particularly for relapsed/refractory AML. Disclosures Porkka: Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Wennerberg:Pfizer: Research Funding. Gjertsen:BerGenBio AS: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees; Kinn Therapeutics AS: Equity Ownership. Heckman:Celgene: Research Funding; Pfizer: Research Funding.

Download Full-text

Crispr/Cas9-Mediated In Vivo Gene Targeting Corrects Haemostasis in Newborn and Adult FIX-KO Mice

Blood ◽

10.1182/blood.v128.22.1174.1174 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1174-1174 ◽

Cited By ~ 2

Author(s):

Lili Wang ◽

Yang Yang ◽

John White ◽

McMenamin Deirdre ◽

Peter Bell ◽

...

Keyword(s):

Mouse Model ◽

Board Of Directors ◽

Partial Hepatectomy ◽

Research Funding ◽

Advisory Committees ◽

Exon 2 ◽

Equity Ownership ◽

Donor Template ◽

Vector Approach

Abstract CRISPR/Cas9, a powerful genome editing tool, is promising for efficient correction of disease-causing mutations. We recently developed a dual AAV vector approach for in vivo delivery of three key components of the homology directed repair (HDR) mediated by CRISPR/Cas9: Cas9 enzyme from Staphylococcus aureus (SaCas9), a single-guide RNA (sgRNA), and a donor template. In a mouse model of ornithine transcarbamylase (OTC) deficiency caused by a single nucleotide change, we demonstrated HDR-based correction of the G-to-A mutation in 10% of OTC alleles in the liver of newborn OTC mice and clinical benefits following in vivogenome editing. However, most monogenic genetic diseases including hemophilia are caused by different mutations scattered in a specific gene rather than a single predominant mutation. The vector developed for one mutation would not be applicable for a patient with a different mutation. In this study, we aim to develop a more universal CRISPR/Cas9 gene targeting approach in which the vector system could be applied to majority of the patients with a specific disease, for example, hemophilia B. To validate this new approach, we performed the experiment in a factor IX knockout (KO) mouse model. In this two-vector approach, vector 1 expresses the SaCas9 gene from a liver-specific TBG promoter, same as it was in our previous approach. The difference lies in vector 2. Besides the sgRNA sequence expressed from a U6 promoter that specifically targets to a region in the 5' end of exon 2 of murine FIX and a 1.8-kb donor fragments, it also contains a partial human FIX cDNA sequence spanning the remaining exon 2 to exon 8 followed by the bovine growth hormone polyA inserted in between the left and right arms of the donor template. In addition, the partial human FIX cDNA is codon optimized and carrying the hyperactive FIX Padua mutation. Following double strand breaks generated by Cas9 and HDR, the partial human FIX cDNA would be fused to 5' end of murine FIX and a hybrid of murine-human FIX transcript would be expressed from the native murine FIX promoter. In the control vector 2, it contains everything except for the 20-nt target sequence. Co-injection of the two vectors with varying doses in newborn and adult FIX-KO mice resulted in stable FIX activity at or above the normal levels for over 4 months. Eight weeks after the vector treatment, a subgroup of the newborn and adult treated FIX-KO mice were subjected to a two third partial hepatectomy, and all of them survived the procedure without any complications or interventions. FIX levels persisted at similar levels after partial hepatectomy indicate stable genomic targeting. FIX-expressing hepatocytes were detected in liver samples collected at the partial hepatectomy. This study provides convincing evidence for efficacy in a hemophilia B mouse model following in vivo genome editing by CRISPR/Cas9. Disclosures Wilson: Dimension Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; REGENXBIO: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Solid Gene Therapy: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Download Full-text

Predictors of T Cell Expansion and Clinical Responses Following B-Cell Maturation Antigen-Specific Chimeric Antigen Receptor T Cell Therapy (CART-BCMA) for Relapsed/Refractory Multiple Myeloma (MM)

Blood ◽

10.1182/blood-2018-99-119665 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1974-1974 ◽

Cited By ~ 2

Author(s):

Adam D. Cohen ◽

J. Joseph Melenhorst ◽

Alfred L. Garfall ◽

Simon F Lacey ◽

Megan Davis ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Board Of Directors ◽

Cd8 T Cells ◽

Research Funding ◽

Advisory Committees ◽

Car T ◽

Equity Ownership

Abstract Background: Relapsed/refractory (rel/ref) MM is associated with progressive immune dysfunction, including reversal of CD4:CD8 T cell ratio and acquisition of terminally-differentiated T cell phenotypes. BCMA-directed CAR T cells have promising activity in MM, but the factors that predict for robust in vivo expansion and responses are not known. In a phase 1 study of CART-BCMA (autologous T cells expressing a human BCMA-specific CAR with CD3ζ/4-1BB signaling domains) in refractory MM patients (median 7 priors, 96% high-risk cytogenetics), we observed partial response (PR) or better in 12/25 (47%) (Cohen et al, ASH 2017, #505). Recently, we demonstrated in CLL pts receiving CD19-directed CAR T cells that certain T cell phenotypes prior to generation of the CAR T product were associated with improved in vivo expansion and clinical outcomes (Fraietta et al, Nat Med 2018). We thus sought to identify pre-treatment clinical or immunological features associated with CART-BCMA expansion and/or response. Methods: Three cohorts were enrolled: 1) 1-5 x 108 CART cells alone; 2) cyclophosphamide (Cy) 1.5 g/m2 + 1-5 x 107 CART cells; and 3) Cy 1.5 g/m2 + 1-5 x 108 CART cells. Phenotypic analysis of peripheral blood (PB) and bone marrow (BM) mononuclear cells, frozen leukapheresis aliquots, and phenotype and in vitro kinetics of CART-BCMA growth during manufacturing were performed by flow cytometry. CART-BCMA in vivo expansion was assessed by flow cytometry and qPCR. Responses were assessed by IMWG criteria. Results: Responses (≥PR) were seen in 4/9 pts (44%, 1 sCR, 2 VPGR, 1 PR) in cohort 1; 1/5 (20%, 1 PR) in cohort 2; and 7/11 (64%, 1 CR, 3 VGPR, 3 PR) in cohort 3. As of 7/9/18, 3/25 (12%) remain progression-free at 11, 14, and 32 months post-infusions. As previously described, responses were associated with both peak in vivo CART-BCMA expansion (p=0.002) as well as expansion over first month post-infusion (AUC-28, p=0.002). No baseline clinical or MM-related characteristic was significantly associated with expansion or response, including age, isotype, time from diagnosis, # prior therapies, being quad- or penta-refractory, presence of del 17p or TP53 mutation, serum hemoglobin, BM MM cell percentage, MM cell BCMA intensity, or soluble BCMA concentration. Treatment regimen given before leukapheresis or CART-BCMA infusions also had no predictive value. We did find, however, that higher CD4:CD8 T cell ratios within the leukapheresis product were associated with greater in vivo CART-BCMA expansion (Spearman's r=0.56, p=0.005) and clinical response (PR or better; p=0.014, Mann-Whitney). In addition, and similar to our CLL data, we found that a higher frequency of CD8 T cells within the leukapheresis product with an "early-memory" phenotype of CD45RO-CD27+ was also associated with improved expansion (Spearman's r=0.48, p=0.018) and response (p=0.047); Analysis of manufacturing data confirmed that higher CD4:CD8 ratio at culture start was associated with greater expansion (r=0.41, p=0.044) and, to a lesser degree, responses (p=0.074), whereas absolute T cell numbers or CD4:CD8 ratio in final CART-BCMA product was not (p=NS). In vitro expansion during manufacturing did associate with in vivo expansion (r=0.48, p=0.017), but was not directly predictive of response. At the time of CART-BCMA infusion, the frequency of total T cells, CD8+ T cells, NK cells, B cells, and CD3+CD56+ cells within the PB or BM was not associated with subsequent CART-BCMA expansion or clinical response; higher PB and BM CD4:CD8 ratio pre-infusion correlated with expansion (r=0.58, p=0.004 and r=0.64, p=0.003, respectively), but not with response. Conclusions: In this study, we found that CART-BCMA expansion and responses in heavily-pretreated MM patients were not associated with tumor burden or other clinical characteristics, but did correlate with certain immunological features prior to T cell collection and manufacturing, namely preservation of normal CD4:CD8 ratio and increased frequency of CD8 T cells with a CD45RO-CD27+ phenotype. This suggests that patients with less dysregulated immune systems may generate more effective CAR T cell products in MM, and has implications for optimizing patient selection, timing of T cell collection, and manufacturing techniques to try to overcome these limitations in MM patients. Disclosures Cohen: Celgene: Consultancy; Novartis: Research Funding; Oncopeptides: Consultancy; Janssen: Consultancy; Poseida Therapeutics, Inc.: Research Funding; Bristol Meyers Squibb: Consultancy, Research Funding; Kite Pharma: Consultancy; GlaxoSmithKline: Consultancy, Research Funding; Seattle Genetics: Consultancy. Melenhorst:Parker Institute for Cancer Immunotherapy: Research Funding; novartis: Patents & Royalties, Research Funding; Casi Pharmaceuticals: Consultancy; Incyte: Research Funding; Shanghai UNICAR Therapy, Inc: Consultancy. Garfall:Amgen: Research Funding; Kite Pharma: Consultancy; Bioinvent: Research Funding; Novartis: Research Funding. Lacey:Novartis Pharmaceuticals Corporation: Patents & Royalties; Parker Foundation: Research Funding; Tmunity: Research Funding; Novartis Pharmaceuticals Corporation: Research Funding. Davis:Novartis Institutes for Biomedical Research, Inc.: Patents & Royalties. Vogl:Karyopharm Therapeutics: Consultancy. Pruteanu:Novartis: Employment. Plesa:Novartis: Research Funding. Young:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Consultancy, Patents & Royalties, Research Funding; CRC Oncology: Consultancy; Incysus: Consultancy; Tmunity Therapeutics: Equity Ownership, Research Funding; Brammer Bio: Consultancy; Cure Genetics: Consultancy. June:Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Novartis Pharmaceutical Corporation: Patents & Royalties, Research Funding; Immune Design: Membership on an entity's Board of Directors or advisory committees; Celldex: Consultancy, Membership on an entity's Board of Directors or advisory committees; Tmunity Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Stadtmauer:Takeda: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AbbVie, Inc: Research Funding; Janssen: Consultancy. Milone:Novartis: Patents & Royalties.

Download Full-text