Single Cell-Level Signaling Profiling of Acute Myeloid Leukemia Following Treatment with Axl Kinase Inhibitor BGB324

Abstract Axl is a member of the Tyro3, Axl, Mer (TAM) receptor tyrosine kinase family that regulate a wide range of cellular functions, including cell survival, proliferation, migration/invasion and adhesion. Axl has been shown to play a key role in the survival and metastasis of many tumors, and has also been found to be upregulated and constitutively active in human AML. Indeed, Axl has been reported as an independent prognostic marker and a potential novel therapeutic target in AML. BGB324 is a first-in-class highly selective small molecule inhibitor of Axl. BGB324 has been shown to be safe and well tolerated in clinical safety studies in healthy volunteers at doses up to 1500 mg/day with a predictable PK profile and long plasma half-life, and is currently in phase I b clinical trials for AML and non-small cell lung cancer. In this study, we use phosphoflow cytometry to measure changes in signal transduction nodes in single AML cells treated with BGB324. We are applying this approach to monitor signaling profiles in primary AML cells harvested from patients undergoing BGB324 treatment. Results: The human AML cell line MOLM13 was treated in vitro with BGB324 (0.5 and 1µM for 1 hour) and analyzed for signal transduction changes by phosphoflow cytometry. We found a significant reduction in phosphorylation of Axl (pY779), Akt(pS473), Erk1/2(pT202/Y204) and PLCɣ1(pY783). Next we established a systemic MOLM13 preclinical AML model in NOD/SCID mice. The mice were treated with 25 or 50 mg/kg BGB324 until moribund (up to 16 days). We found a dose-dependent and significant increase in overall survival in BGB324-treated mice. We further investigated intracellular signaling in BGB324-treated cells in vivo. Mice carrying systemic AML disease (MOLM13) were treated with BGB324 at 50mg/kg for 4 days, and we monitored CD33/45-positive MOLM13 cells harvested from spleen and bone marrow by flow cytometry. BGB324-treated mice showed a significant reduction in pErk and pPLCɣ1 relative to mice in the control group. PBMCs from peripheral blood of AML patients treated with BGB324 400 mg x1 at day 1 and 2, and thereafter 100 mg daily were collected for single cell signal profiling of signal transduction changes by conventional flow cytometry (phospho-flow) and mass cytometry (CyTOF). Preliminary phopho-flow analyses show decrease of pAkt(T308) and pPLCgamma1(Y783) in one patient. Further analyses are ongoing and will be presented. Figure 1. In vitro response to 1 hour BGB324 treatment in human AML cell line MOLM13 at 0.5 and 1µM doses. Response was evaluated in pAxl, pErk1/2, pAkt and pPLCγ1. n=3, *p≤0.05, **p≤0.005. Figure 1. In vitro response to 1 hour BGB324 treatment in human AML cell line MOLM13 at 0.5 and 1µM doses. Response was evaluated in pAxl, pErk1/2, pAkt and pPLCγ1. n=3, *p≤0.05, **p≤0.005. Figure 2. Dose-dependent response in overall survival in a MOLM13 systemic xenograft model (n=10). Figure 2. Dose-dependent response in overall survival in a MOLM13 systemic xenograft model (n=10). Figure 3. Response to BGB324-treatment in pErk, pPLCγ1 and pAkt in CD33/CD45-positive cells harvested from spleens (left) and bone marrows (right) of mice with systemic MOLM13 xenografts. n=5, *p≤0.05, **p≤0.005. Figure 3. Response to BGB324-treatment in pErk, pPLCγ1 and pAkt in CD33/CD45-positive cells harvested from spleens (left) and bone marrows (right) of mice with systemic MOLM13 xenografts. n=5, *p≤0.05, **p≤0.005. Disclosures Hellesøy: BerGenBio AS: Other: Previous employee. Stock option holder. Wnuk-Lipinska:BerGenBio AS: Employment. Boniecka:BerGenBio AS: Employment. Nævdal:BerGenBio AS: Employment. Loges:BerGenBio: Honoraria, Other: travel support, Research Funding. Cortes:Teva: Research Funding; BMS: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; BerGenBio AS: Research Funding; Ariad: Consultancy, Research Funding; Astellas: Consultancy, Research Funding; Ambit: Consultancy, Research Funding; Arog: Research Funding; Celator: Research Funding; Jenssen: Consultancy. Lorens:BerGenBio AS: Employment, Equity Ownership. Micklem:BerGenBio AS: Employment, Equity Ownership. Gausdal:BerGenBio AS: Employment. Gjertsen:Haukeland University Hospital: Research Funding.

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FcRL5 as a Target of Antibody-Drug Conjugates for the Treatment of Multiple Myeloma.

Blood ◽

10.1182/blood.v114.22.3836.3836 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3836-3836 ◽

Cited By ~ 1

Author(s):

Andrew G. Polson ◽

Bing Zheng ◽

Kristi Elkins ◽

Jeffery Lau ◽

Mary Ann T. Go ◽

...

Keyword(s):

Multiple Myeloma ◽

B Cells ◽

Cell Line ◽

Research Funding ◽

Employment Equity ◽

Antibody Drug Conjugates ◽

Drug Conjugates ◽

Equity Ownership ◽

Antibody Drug

Abstract Abstract 3836 Poster Board III-772 Antibody-drug conjugates (ADCs), potent cytotoxic drugs linked to antibodies via specialized chemical linkers, provide a means to increase the effectiveness of chemotherapy by targeting the drug to neoplastic cells while reducing side effects. Target selection is a key component to the success of an ADC. In addition to expression on the tumor, the target should have limited expression on normal tissues. The most prevalent and best-studied surface antigens on multiple myeloma (MM) cells, CD138, CD38, CD72, and CD56, all have relatively broad expression patterns that may lead to target-dependent toxicities with ADCs. We have previously shown that FcRL5/FcRH5/IRTA2 is expressed only on B-cells and plasma cells. Here we show that FcRL5 is expressed on the surface of MM cells from 85% of patients and thus could be a target for antibody and ADC treatments of MM. As experience in humans with anti-CD20 antibodies suggests that depletion of B-cells does not present a major safety issue, FcRL5 appears to have an excellent expression pattern as an ADC target for MM. Internalization of target antigens is known to increase the efficacy of ADCs and we found that FcRL5 is internalized upon antibody binding, an ideal trait. We made two different ADCs consisting of anti-FcRL5 antibodies 1) conjugated through cysteines to monomethylauristatin E (MMAE) by a maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (MC-vcPAB) linker that is designed to be cleaved by cathepsins (Anti-FcRL5-MC-vcPAB-MMAE); and 2) conjugated via lysines to the maytansinoid DM4 through a disulified linker designed to be cleaved by reducing conditions (anti-FcRL5-SPDB-DM4). These ADCs were tested for cell killing in vitro and in xenograft studies using OPM2 cells stably expressing FcRL5. While unconjugated anti-FcRH5 and control ADCs were not effective, anti-FcRL5-MC-vcPAB-MMAE and anti-FcRL5-SPDB-DM4 were effective at killing this MM cell line both in vitro and in vivo. Furthermore, the anti-FcRL5-SPDB-DM4 conjugate was effective in a SCID-rabbit bone model of MM using the LD cell line. These data suggest that FcRL5 ADCs could be an effective treatment for MM. Disclosures: Polson: Genentech, Inc.: Employment, Equity Ownership. Zheng:Genentech, Inc.: Employment, Equity Ownership. Elkins:Genentech, Inc.: Employment, Equity Ownership. Lau:Genentech, Inc.: Employment, Equity Ownership. Go:Genentech, Inc.: Employment, Equity Ownership. Scales:Genentech, Inc.: Employment, Equity Ownership. Yu:Genentech, Inc.: Employment, Equity Ownership. Chesi:Genentech, Inc.: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Bergsagel:Genentech, Inc.: Consultancy; Amgen: Consultancy; Celgene: Consultancy; Merck: Research Funding. Ebens:Genentech, Inc.: Employment, Equity Ownership, Patents & Royalties.

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Optimized Fully-Synthetic Salicylamide Heparin Antagonists Have Greater Efficacy Versus LMWHs and Display Improved Hemodynamic Responses as Compared to Protamine

Blood ◽

10.1182/blood.v116.21.191.191 ◽

2010 ◽

Vol 116 (21) ◽

pp. 191-191

Author(s):

Richard W. Scott ◽

Michael J. Costanzo ◽

Katie B. Freeman ◽

Robert W. Kavash ◽

Trevor M. Young ◽

...

Keyword(s):

Blood Pressure ◽

Cell Line ◽

Factor Xa ◽

In Vivo Model ◽

Hemodynamic Effects ◽

Employment Equity ◽

Human Liver Cell ◽

Equity Ownership

Abstract Abstract 191 A series of salicylamides, fully synthetic cationic foldamers designed to disrupt the binding of the pentasaccharide unit of heparin to antithrombin III, were found to be potent neutralizers of the activity of unfractionated heparin (UFH) and low molecular weight heparins (LMWHs). A compound from this series, PMX-60056, is currently in human clinical trials for neutralization of UFH and LMWHs. PMX-60056 potently neutralizes UFH and LMWHs but is not as efficacious versus fondaparinux (FPX). The goal of the present research was to 1) identify back-up compounds to optimize activity against the LMWHs and FPX and 2) mitigate the hemodynamic effects commonly associated with protamine and observed clinically with PMX-60056 in the absence of heparin. Compounds were first tested for their ability to neutralize the anticoagulant activity of enoxaparin (ENX), tinzaparin or FPX in an in vitro amidolytic assay for factor Xa activity. While only minor improvements were observed in the neutralization of ENX and tinzaparin, compounds were identified which had 6 to 40 fold increase in activity against FPX (EC50s of 0.09 – 0.58 uM) in comparison to PMX-60056 (EC50 3.64 uM). Activated partial thromboplastin time (aPTT) assays demonstrated that these compounds maintained activity against heparin in a plasma based clotting assay. Rotation thromboelastometry (ROTEM) was used to show that these compounds are able to neutralize heparin and ENX in human whole blood, restoring normal coagulation profiles. As an initial test for safety, compounds were tested in hemolysis and cytotoxicity assays using isolated human erythrocytes, a transformed human liver cell line (HepG2 cells) and a mouse fibroblast cell line (NIH3T3). Lead back-up compounds were not cytotoxic (or hemolytic) at >100 fold concentrations over their EC50 concentrations in the anti-coagulation assays, indicating a high selectivity index between toxicity and efficacy. Five compounds were selected for further studies based on their in vitro profiles. The in vivo efficacy of these compounds was evaluated in a rat coagulation model for neutralization of ENX (2 mg/kg). Three minutes following IV dosing with ENX, either saline, protamine or one of the five salicylamide test compounds was administered. Blood was collected before dosing with ENX, and at 1, 3, 10, and 60 min after dosing, for aPTT and factor Xa analysis. Three of the five salicylamides (PMX640, PMX686 and PMX747) were more efficacious than protamine; with PMX640 and PMX686 neutralizing 91 – 100% and PMX747 neutralizing 78–100% of the ENX anti-factor Xa activity over the entire 60 minute time course. In a second in vivo model, PMX747 and PMX686 (2 mg/kg) completely neutralized the prolonged bleeding times in a rat tail bleeding model caused by treatment with 2 mg/kg ENX. Significantly, with protamine at a 5 mg/kg dosage, only partial restoration was obtained. Protamine routinely causes a transient decrease in blood pressure upon dosing, and hemodynamic effects have also been observed with PMX-60056 in human subjects in the absence of heparin. To address this issue, structural features that have successfully reduced hemodynamic liabilities in other cationic compounds were incorporated into the design of the back-up salicylamides. The effect of compounds on blood pressure and heart rate was measured via arterial catheters in rats following IV administration of protamine, PMX-60056, or test agents. As expected, in rats treated with a low dose of UFH (50 u/kg) and high dosages of antagonist, both protamine and PMX-60056 displayed transient or prolonged blood pressure reductions at 8 and 16 mg/kg, respectively. However, the lead back-up salicylamides, PMX640, PMX686 and PMX747 had little to no effect on blood pressure at these same dosages. In conclusion, we have discovered compounds in the salicylamide series that have greater efficacy versus LMWHs and that have significantly reduced hemodynamic liabilities in rats as compared to protamine. Furthermore, these compounds potently neutralize FPX activity in vitro; exceeding the activity of protamine and our clinical lead salicylamide, PMX-60056, by up to 40 fold. Thus we have been able to optimize the salicylamide series, identifying compounds that offer the potential to greatly improve upon the current clinical heparin antagonist, protamine, in respect to both activity against LMWHs and side effect profile. Disclosures: Scott: PolyMedix Inc.: Employment, Equity Ownership. Costanzo:PolyMedix Inc.: Employment, Equity Ownership. Freeman:PolyMedix Inc.: Employment, Equity Ownership. Kavash:PolyMedix Inc.: Employment, Equity Ownership. Young:PolyMedix, Inc.: Employment, Equity Ownership. DeGrado:PolyMedix, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Jeske:PolyMedix, Inc.: Research Funding.

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Preclinical Characterization of E7438, a Potent, Selective Inhibitor of Protein Methyltransferase EZH2 with Robust Antitumor Activity Against EZH2 Mutated Non-Hodgkin Lymphoma Xenografts in Mice

Blood ◽

10.1182/blood.v120.21.3712.3712 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3712-3712 ◽

Cited By ~ 1

Author(s):

Heike Keilhack ◽

Akira Yokoi ◽

Sarah K Knutson ◽

Tim Wigle ◽

Natalie Warholic ◽

...

Keyword(s):

Repeated Measures ◽

Tumor Regression ◽

Xenograft Model ◽

Lymphoma Cells ◽

Employment Equity ◽

H3k27 Methylation ◽

Non Hodgkin Lymphoma ◽

Mouse Xenograft Model ◽

Equity Ownership ◽

Dose Dependent

Abstract Abstract 3712 The coupled enzymatic activity of wild-type and mutant EZH2 results in hyper-trimethylation of histone H3 lysine 27 (H3K27), which drives lymphomagenesis in heterozygous patients bearing the EZH2 mutations. Our group has previously reported that selective inhibition of EZH2 in cell culture results in selective killing of lymphoma cells bearing EZH2 mutations, with minimal effect on non-mutant lymphoma cells, suggesting that EZH2 enzymatic activity is a required driver of proliferation in the mutant-bearing cells [Knutson et al. (2012) Nature Chemical Biology, in press]. Through iterative medicinal chemistry we have developed a selective inhibitor of EZH2 with good pharmacological properties, E7438. E7438 binds to the enzyme in a manner competitive with S-adenosyl methionine (SAM) and a Ki for wild-type EZH2 of 2.5 ± 0.5 nM. The compound potently inhibits all known mutants of EZH2 that have been identified in non-Hodgkin lymphoma (NHL) patient samples. E7438 displays about 35-fold less activity against the closely related enzyme EZH1, and is >4500-fold selective with respect to all other protein methyltransferases tested. Lymphoma cells treated with E7438 display concentration- and time-dependent loss of H3K27 methylation with no effect on the methylation status of any other histone sites. The loss of H3K27 methylation results in selective killing of EZH2 mutant-bearing lymphoma cell lines. E7438 displays good oral bioavailability and pharmacokinetic properties. Various EZH2 mutant-bearing human lymphoma tumors were subcutaneously implanted in nude, SCID or NSG mice. Oral administration of E7438 to tumor bearing mice resulted in significant anti-tumor activity. The responses ranged from dose-dependent tumor growth inhibition to complete and sustained regressions. For example, KARPAS422 tumors in nude mice showed complete tumor elimination after 28 days of dosing, with mice remaining tumor free for up to 90 days after treatment cessation. Figure 1. E7438 causes complete and sustained tumor regression in a KARPAS422 nude mouse xenograft model of EZH2-mutated NHL. Dosing was on a BID schedule. * P< 0.05, Repeated measures ANOVA, Dunnett's post test vs. vehicle. Figure 1. E7438 causes complete and sustained tumor regression in a KARPAS422 nude mouse xenograft model of EZH2-mutated NHL. Dosing was on a BID schedule. * P< 0.05, Repeated measures ANOVA, Dunnett's post test vs. vehicle. Mice and rats tolerated E7438 administration well at doses representing high multiples of doses that show antitumor activity in mice. Activity against the EZH2 target in both species was demonstrated by dose-dependent diminution of H3K27me3 levels, assessed by ELISA, in samples of tumor, bone marrow, skin and peripheral blood mononuclear cells (PBMCs). Highly sensitive detection of existing H3K27me3 signal could also be observed with this ELISA assay in drug-naïve samples of human PBMCs. The ability to measure dose-dependent changes in H3K27me3 levels in skin and PBMCs portends the use of signal from these surrogate tissues as a non-invasive pharmacodynamics biomarker in human clinical trials. Disclosures: Keilhack: Epizyme Inc.: Employment, Equity Ownership. Yokoi:Eisai Co., Ltd.: Employment. Knutson:Epizyme Inc.: Employment, Equity Ownership. Wigle:Epizyme Inc.: Employment, Equity Ownership. Warholic:Epizyme Inc.: Employment, Equity Ownership. Kawano:Eisai Co., Ltd.: Employment. Minoshima:Eisai Co., Ltd.: Employment. Huang:Eisai Inc.: Employment. Kuznetsov:Eisai Inc.: Employment. Kumar:Eisai Inc.: Employment. Klaus:Epizyme, Inc.: Employment, Equity Ownership. Allain:Epizyme Inc.: Employment, Equity Ownership. Raimondi:Epizyme Inc.: Employment, Equity Ownership. Porter Scott:Epizyme: Employment, Equity Ownership. Chesworth:Epizyme: Employment, Equity Ownership. Moyer:Epizyme: Employment, Equity Ownership. Uenaka:Eisai Co., Ltd.: Employment. Copeland:Epizyme Inc.: Employment, Equity Ownership. Richon:Epizyme, Inc.: Employment, Equity Ownership. Pollock:Epizyme Inc.: Employment, Equity Ownership. Kuntz:Epizyme Inc.: Employment, Equity Ownership.

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Stroma-Free Ex Vivo Expanded, CD34+ Cord Blood-Derived NK Cells Retain Lytic Activity After Long-Term Engraftment in NSGS Mice

Blood ◽

10.1182/blood.v120.21.580.580 ◽

2012 ◽

Vol 120 (21) ◽

pp. 580-580

Author(s):

Mark Wunderlich ◽

Mahesh Shrestha ◽

Lin Kang ◽

Eric Law ◽

Vladimir Jankovic ◽

...

Keyword(s):

Nk Cells ◽

Research Funding ◽

Nk Cell ◽

Ex Vivo ◽

Employment Equity ◽

Cell Product ◽

Equity Ownership ◽

Cellular Therapeutics

Abstract Abstract 580 Generating a large number of pure, functional immune cells that can be used in human patients has been a major challenge for NK cell-based immunotherapy. We have successfully established a cultivation method to generate human NK cells from CD34+ cells isolated from donor-matched cord blood and human placental derived stem cells, which were obtained from full-term human placenta. This cultivation method is feeder-free, based on progenitor expansion followed by NK differentiation supported by cytokines including thrombopoietin, stem cell factor, Flt3 ligand, IL-7, IL-15 and IL-2. A graded progression from CD34+ hematopoietic progenitor cells (HSC) to committed NK progenitor cells ultimately results in ∼90% CD3-CD56+ phenotype and is associated with an average 10,000-fold expansion achieved over 35 days. The resulting cells are CD16- and express low level of KIRs, indicating an immature NK cell phenotype, but show active in vitro cytotoxicity against a broad range of tumor cell line targets. The in vivo persistence, maturation and functional activity of HSC-derived NK cells was assessed in NSG mice engineered to express the human cytokines SCF, GM-CSF and IL-3 (NSGS mice). Human IL-2 or IL-15 was injected intraperitoneally three times per week to test the effect of cytokine supplementation on the in vivo transferred NK cells. The presence and detailed immunophenotype of NK cells was assessed in peripheral blood (PB), bone marrow (BM), spleen and liver samples at 7-day intervals up to 28 days post-transfer. Without cytokine supplementation, very few NK cells were detectable at any time-point. Administration of IL-2 resulted in a detectable but modest enhancement of human NK cell persistence. The effect of IL-15 supplementation was significantly greater, leading to the robust persistence of transferred NK cells in circulation, and likely specific homing and expansion in the liver of recipient mice. The discrete response to IL-15 versus IL-2, as well as the preferential accumulation in the liver have not been previously described following adoptive transfer of mature NK cells, and may be unique for the HSC-derived immature NK cell product. Following the in vivo transfer, a significant fraction of human CD56+ cells expressed CD16 and KIRs indicating full physiologic NK differentiation, which appears to be a unique potential of HSC-derived cells. Consistent with this, human CD56+ cells isolated ex vivo efficiently killed K562 targets in in vitro cytotoxicity assays. In contrast to PB, spleen and liver, BM contained a substantial portion of human cells that were CD56/CD16 double negative (DN) but positive for CD244 and CD117, indicating a residual progenitor function in the CD56- fraction of the CD34+ derived cell product. The BM engrafting population was higher in NK cultures at earlier stages of expansion, but was preserved in the day 35- cultured product. The frequency of these cells in the BM increased over time, and showed continued cycling based on in vivo BrdU labeling 28 days post-transfer, suggesting a significant progenitor potential in vivo. Interestingly, DN cells isolated from BM could be efficiently differentiated ex vivo to mature CD56+CD16+ NK cells with in vitro cytotoxic activity against K562. We speculate that under the optimal in vivo conditions these BM engrafting cells may provide a progenitor population to produce a mature NK cell pool in humans, and therefore could contribute to the therapeutic potential of the HSC-derived NK cell product. The in vivo activity of HSC-derived NK cells was further explored using a genetically engineered human AML xenograft model of minimal residual disease (MRD) and initial data indicates significant suppression of AML relapse in animals receiving NK cells following chemotherapy. Collectively, our data demonstrate the utility of humanized mice and in vivo xenograft models in characterizing the biodistribution, persistence, differentiation and functional assessment of human HSC-derived cell therapy products, and characterize the potential of HSC-derived NK cells to be developed as an effective off-the-shelf product for use in adoptive cell therapy approaches in AML. Disclosures: Wunderlich: Celgene Cellular Therapeutics: Research Funding. Shrestha:C: Research Funding. Kang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Law:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Jankovic:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Zhang:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Herzberg:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Abbot:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hariri:Celgene Cellular Therapeutics: Employment, Equity Ownership, Patents & Royalties. Mulloy:Celgene Cellular Therapeutics: Research Funding.

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A Rare Genetic Polymorphism In C5 Confers Poor Response To The Anti-C5 Monoclonal Antibody Eculizumab In 11 Japanese Patients With PNH

Blood ◽

10.1182/blood.v122.21.3709.3709 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3709-3709

Author(s):

Jun-ichi Nishimura ◽

Masaki Yamamoto ◽

Shin Hayashi ◽

Kazuma Ohyashiki ◽

Kiyoshi Ando ◽

...

Keyword(s):

Healthy Volunteers ◽

Hemolytic Activity ◽

Research Funding ◽

Poor Response ◽

Heterozygous Mutation ◽

Poor Responders ◽

Classical Pathway ◽

Employment Equity ◽

Equity Ownership

Abstract Eculizumab is a humanized monoclonal antibody targeting the terminal complement protein C5 and inhibiting terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). In the Japanese AEGIS PNH-eculizumab study, 2 poor-responders were identified out of 29 cases. Currently, more than 300 patients have been treated with eculizumab, and a total of 11 poor-responders were identified all of whom are Japanese. To clarify the mechanism of difference in the responsiveness of eculizumab, blood samples from poor and good responders were analyzed after obtaining informed consent. Approval for these studies was obtained from the institutional review boards at each study site taking care of patients as well as from Osaka University. The levels of lactate dehydrogenase in these two patients were markedly elevated before eculizumab treatment, and were not decreased during the 12 weeks AEGIS study. From the pharmacokinetic analysis, peak and trough levels of eculizumab during the study were well above the minimal level required to completely inhibit complement-mediated hemolysis in PNH patients. The pharmacodynamics of eculizumab were determined by measuring the capacity of the patients’ serum to lyse chicken erythrocytes in a standard hemolytic assay. Serum samples analysed from these two patients failed over the entire treatment period, to show a suppression of hemolysis, prompting further study of the effect of exogenous eculizumab on the hemolytic activity of patient pre-drug sera. Eculizumab up to 2000μg/mL did not block hemolytic activity in the sera of either poor-responder. However, hemolytic activity both in the two poor-responders and in control patient was blocked completely using a different anti-C5 antibody (N19/8) at 50μg/mL. Therefore, the DNA of C5 from Japanese PNH patients with a good or poor response to eculizumab was sequenced, and a single missense C5 heterozygous mutation at exon 21, c.2654G>A, which predicts p.Arg885His, was found in all of the 11 poor responders identified to date, but not in any of the responders. Among about 300 Japanese patients treated with eculizumab, 11 patients (about 3.7%) have been identified as poor responders. A similar prevalence (3.5%) was seen in healthy volunteers, since we determined that 10 out of 288 Japanese healthy volunteers have the same mutation. This polymorphism was also identified in 1 out of 120 China Han healthy volunteers, but not in 100 persons of British ancestry living in England and Scotland, and not in 90 persons of Mexican ancestry in Los Angels. To close the genotype-phenotype loop, electrophoretically pure recombinant C5 (rC5) and rC5 mutant (rC5m) containing c.2654G>A were generated and functionally compared in various in vitro experiments. As a preliminary experiment, we confirmed that natural C5, rC5, and rC5m restored classical pathway lysis equivalently when added to C5-depleted serum. Eculizumab did not block classical pathway lysis reconstituted with rC5m but did block rC5 and nC5-dependent lysis. By contrast, as observed with patient sera, N19/8 inhibited lysis reconstituted with nC5, rC5, and rC5m. Finally, while eculizumab bound nanomolar concentrations of rC5 using surface plasmon resonance, with clear association and dissociation phases, there was no detectable binding with rC5m in the same assay up to the highest concentration (1 µM) of eculizumab examined. A single missense C5 heterozygous mutation, c.2654G>A, which predicts p.Arg885His, was commonly identified in poor-responders, but not in responders. This polymorphism had at least spread to other East Asian countries. After determining that the poor responders likely express both wild-type C5 and a structural variant C5, we then showed that the hemolytic activity supported by this structural variant in vitro, like the effects on patient sera, was not blocked by eculizumab but was fully blocked by N19/8, and that the variant was incapable of binding eculizumab. Collectively, these data are consistent with the hypothesis that the functional capacity of the mutant C5 together with its inability to bind to and undergo blockade by eculizumab fully account for the poor response in patients carrying this mutation. (JN and MY contributed equally to this work) Disclosures: Nishimura: Alexion Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Yamamoto:Alexion Pharm: Research Funding. Ohyashiki:Alexion: Research Funding. Noji:Alexion Pharmaceuticals: Honoraria. Shichishima:Alexion Pharmaceuticals: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding. Hase:Alexion Phama: Employment, Equity Ownership. Lan:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Johnson:Alexion Pharmaceuticals, Inc.: Employment. Tamburini:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership, Patent inventor but do not receive royalties, Patent inventor but do not receive royalties Patents & Royalties. Kinoshita:Alexion: Honoraria. Kanakura:Alexion Pharmaceuticals: Research Funding, Speakers Bureau.

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Final Analysis of Overall Survival from the Phase 3 Panorama 1 Trial of Panobinostat Plus Bortezomib and Dexamethasone Versus Placebo Plus Bortezomib and Dexamethasone in Patients with Relapsed or Relapsed and Refractory Multiple Myeloma

Blood ◽

10.1182/blood.v126.23.3026.3026 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3026-3026 ◽

Cited By ~ 5

Author(s):

Jesús F. San-Miguel ◽

Vania T.M. Hungria ◽

Sung-Soo Yoon ◽

Meral Beksac ◽

Meletios A. Dimopoulos ◽

...

Keyword(s):

Multiple Myeloma ◽

Overall Survival ◽

Board Of Directors ◽

Research Funding ◽

Final Analysis ◽

Employment Equity ◽

Phase 3 ◽

Advisory Committees ◽

Equity Ownership ◽

Study Therapy

Abstract Introduction: Panobinostat is a potent pan-deacetylase inhibitor (pan-DACi) that targets key aberrations in multiple myeloma (MM) cell biology, including epigenetics and protein metabolism. In the phase 3 clinical trial PANORAMA 1, panobinostat in combination with bortezomib and dexamethasone (PAN-BTZ-Dex) led to a statistically significant and clinically relevant increase in progression-free survival of approximately 4 months compared with that with placebo plus bortezomib and dexamethasone (Pbo-BTZ-Dex). Further analyses of patient outcomes by prior treatment demonstrated that the magnitude of PFS benefit was greatest among patients who received at least 2 prior regimens, including bortezomib and an immunomodulatory drug (IMiD; PAN-BTZ-Dex [n = 73]: 12.5 months [95% CI, 7.3-14.0 months]; Pbo-BTZ-Dex [n = 74]: 4.7 months (95% CI, 3.7-6.1 mo; HR 0.47 [95% CI, 0.32-0.72]). These data supported the regulatory approvals of PAN-BTZ-Dex for the treatment of patients with multiple myeloma who received at least 2 prior regimens, including bortezomib and an IMiD. Here we present the final analysis of overall survival (OS) for the entire patient population and among patients who received at least 2 prior regimens, including bortezomib and an IMiD. Methods: The study design for the PANORAMA 1 trial was described previously (San-Miguel. Lancet Oncol. 2014;15:1195-206). The key secondary endpoint was OS. As of June 29, 2015, the 415 events required to conduct the final analysis of OS had been observed. Kaplan-Meier estimation was utilized for OS analyses for the entire population (N = 768), the pre-specified subgroup of patients who received prior bortezomib and IMiD (n = 193), and patients who received at least 2 prior regimens including bortezomib and an IMiD (n = 147). Results: The median OS of patients who received PAN-BTZ-Dex in the overall population was 40.3 months (95% CI, 35.0-44.8 months) vs 35.8 months (95% CI, 29.0-40.6 months) for the Pbo-BTZ-Dex arm with HR 0.94 [95% CI, 0.78-1.14], P = .5435 (Fig 1A). The percentage of patients in each arm who received post-study therapy was 37.7% in the PAN-BTZ-Dex arm and 48.8% in the Pbo-BTZ-Dex arm. The median OS of patients who received at least 2 prior lines, including bortezomib and an IMiD, was 25.5 months (95% CI, 19.6-34.3 months) in the PAN-BTZ-Dex arm vs 19.5 months (95% CI, 14.1-32.5 months) in the Pbo-BTZ-Dex arm (Fig. 1B). The proportion of patients in this subgroup who received post-study therapy was 35.6% in the PAN-BTZ-Dex arm and 66.2% in the Pbo-BTZ-Dex arm. Conclusion: For the overall PANORAMA 1 study population, patients in the PAN-BTZ-Dex arm demonstrated an increase in median OS of 4.5 months vs patients in the Pbo-BTZ-Dex arm, but this result was not statistically significant (P = .5435). Median OS was also slightly longer for the PAN-BTZ-Dex arm among the more heavily pretreated subgroup of patients who received at least 2 prior regimens, including bortezomib and an IMiD. A higher percentage of patients on the Pbo-BTZ-Dex arm received post-study therapy vs the PAN-BTZ-Dex arm, which may have confounded the OS results. In summary, PAN-BTZ-Dex demonstrates statistically significant increases in PFS vs Pbo-BTZ-Dex in patients with relapsed or relapsed and refractory MM; however, this did not translate to a statistically significant increase in OS. Future trials will plan to focus on further optimization of dose and schedule of panobinostat and bortezomib to improve outcome, as well as novel combinations with other agents, including IMiDs and next-generation proteasome inhibitors. Figure 2. Figure 2. Disclosures Beksac: Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Consultancy, Speakers Bureau. Dimopoulos:Janssen: Honoraria; Janssen-Cilag: Honoraria; Onyx: Honoraria; Amgen: Honoraria; Genesis: Honoraria; Celgene: Honoraria; Novartis: Honoraria. Jedrzejczak:Onconova: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Siritanaratkul:Pfizer: Research Funding; Roche: Research Funding; Novartis: Research Funding; Janssen-Cilag: Research Funding. Schlossman:Millennium: Consultancy. Hou:Novartis: Membership on an entity's Board of Directors or advisory committees. Moreau:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lonial:Bristol-Myers Squibb: Consultancy, Research Funding; Millennium: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Onyx: Consultancy, Research Funding; Celgene: Consultancy, Research Funding. Sopala:Novartis Pharma: Employment, Equity Ownership. Bengoudifa:Novartis: Employment. Corrado:Novartis: Employment, Equity Ownership. Richardson:Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millennium Takeda: Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Membership on an entity's Board of Directors or advisory committees.

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Phase 1 Study of CB-839, a First-in-Class, Glutaminase Inhibitor in Patients with Multiple Myeloma and Lymphoma

Blood ◽

10.1182/blood.v126.23.3059.3059 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3059-3059 ◽

Cited By ~ 6

Author(s):

Dan T. Vogl ◽

Anas Younes ◽

Keith Stewart ◽

Keith William Orford ◽

Mark Bennett ◽

...

Keyword(s):

Multiple Myeloma ◽

Research Funding ◽

Blood Platelets ◽

Safety Data ◽

Employment Equity ◽

Efficacy Data ◽

Trough Concentrations ◽

Equity Ownership

Abstract Background: Malignant cells alter metabolism in order to enable their highly anabolic state. In addition to a massive increase in glycolysis, malignant cells frequently become dependent on glutamine to feed the TCA cycle and provide key building blocks for cell growth and proliferation. CB-839 is a first-in-class potent and selective inhibitor of glutaminase (GLS), the first step in glutamine metabolism, that has broad in vitro and in vivo anti-tumor activity in solid and heme malignancies, including multiple myeloma. GLS inhibition with CB-839 induces apoptosis and/or growth arrest in multiple myeloma and lymphoma cell lines and is synergistic with pomalidomide and lenalidomide in vitro and as well as in multiple myeloma xenograft models in vivo. Methods: CX-839-002 is an ongoing Ph1 evaluation of escalating doses of CB-839 in patients with relapsed/refractory multiple myeloma (MM) or non-Hodgkins lymphoma (NHL) with the primary objective of assessing the safety profile and selecting a recommended Phase 2 dose (RP2D). Pharmacokinetics (PK) was monitored on Days 1 and 15. Initially, CB-839 was given three times daily (TID) without food, but based on PK and safety data generated across three Ph1 studies in patients with solid and heme malignancies, the drug is now being given twice daily (BID) with meals. Results: Safety data are available for a total of 14 patients (9 MM, 4 follicular lymphoma, 1 diffuse large B cell lymphoma) that have enrolled to date during the dose escalation (100-400 mg TID and 600 mg BID). The patients have received a median of 7 prior lines of systemic therapy. CB-839 has been well tolerated with only three subjects experiencing a Gr3/4 AEs considered possibly related to study drug and there have been no discontinuations due to AEs. A similar tolerability profile has been observed across three Ph1 studies for CB-839. With a total of 119 pts treated with CB-839 across the three studies, Gr3/4 drug-related AEs have occurred in 16 subjects (13%) and 4.3% of discontinuations were due to AEs. Reversible, asymptomatic elevations in transaminases have been the primary Gr3 AEs, occurring primarily on the TID schedule in 6/59 (10.2%) pts; only one occurred among 60 pts (1.7%) receiving the BID regimen. BID dosing with 600 mg was determined to be the RP2D and combination studies with pomalidomide and dexamethasone have been initiated. The half-life of CB-839 is ~4 hr, exposure increases with dose, and trough concentrations generally remain above the target threshold of 200 ng/mL for patients receiving the RP2D. Six of 8 MM pts that received ≥ 400 mg TID achieved steady state (D15) trough concentrations above the PK target threshold while 0 of 5 pts that received ≤ 250 mg TID achieved the PK threshold. Pharmacodynamic assessment of GLS activity in MM patients was consistent with a broader PK/PD assessment (across all 3 Ph1 studies), which established clear exposure-dependent inhibition of the target in peripheral blood platelets 4 hr after the first dose of CB-839, with >90% inhibition being maintained for most patients at the RP2D. Preliminary efficacy data include confirmed stable disease in 4 of 9 evaluable MM patients. Updated efficacy data and correlative studies on clinical samples will also be presented. The first pt treated with the combination of CB-839 and pomalidomide/dexamethasone (Pd) during dose escalation received 400 mg CB-839 BID, pomalidomide at 4 mg/day (D1-21) and dexamethasone at 40 mg on Days 1, 8, 15 and 22 of each 28-day cycle. This pt had a 71% decreased in urine M-protein and an 83% reduction in serum free light chain after the first 2 cycles of treatment. This pt had 11 prior lines of therapy but not pomalidomide and had two stem cell transplants and was progressing rapidly prior to study entry. The pt has tolerated the combination well and is continuing on study. Conclusions: CB-839 has been well tolerated at and above doses that produced robust inhibition of GLS in blood platelets and in tumors. Dosing BID with food has improved the PK profile and mitigated the frequency and severity of LFT elevations, which was the primary safety signal using TID dosing. Strong preclinical combination data, an excellent clinical safety profile, and initial data with CB-839 combined with Pd provide a strong rationale for continued development of CB-839 this combination in pts with relapsed/refractory multiple myeloma. Disclosures Vogl: Constellation Pharmaceuticals: Research Funding; Calithera Biosciences: Research Funding; Celgene Corporation: Consultancy; Acetylon Pharmaceuticals, Inc.: Research Funding; Millennium Pharmaceuticals: Research Funding; GSK: Research Funding. Younes:Celgene: Honoraria; Curis: Research Funding; Sanofi-Aventis: Honoraria; Seattle Genetics: Honoraria, Research Funding; Novartis: Research Funding; Janssen: Honoraria; Takeda Millenium: Honoraria; Bristol Meyer Squibb: Honoraria; Bayer: Honoraria; Incyte: Honoraria; Johnson and Johnson: Research Funding. Orford:Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Siegel:Celgene Corporation: Consultancy, Speakers Bureau; Amgen: Speakers Bureau; Takeda: Speakers Bureau; Novartis: Speakers Bureau; Merck: Speakers Bureau. Berdeja:Curis: Research Funding; Acetylon: Research Funding; Novartis: Research Funding; Janssen: Research Funding; Takeda: Research Funding; BMS: Research Funding; Array: Research Funding; MEI: Research Funding; Abbvie: Research Funding; Celgene: Research Funding; Onyx: Research Funding.

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Impact of Cardiac Stage and Hematologic Response on AL Amyloidosis Patients with Renal Involvement

Blood ◽

10.1182/blood.v128.22.2136.2136 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2136-2136

Author(s):

Sandy W. Wong ◽

Denis Toskic ◽

Melissa Warner ◽

Alejandro Moreno-koehler ◽

Daniel Fein ◽

...

Keyword(s):

Overall Survival ◽

Research Funding ◽

Cardiac Involvement ◽

Renal Involvement ◽

Al Amyloidosis ◽

Employment Equity ◽

Intention To Treat ◽

Hematologic Response ◽

Equity Ownership ◽

Stage 1

Abstract Cardiac stage and depth of hematologic remission are major predictors of survival for AL amyloidosis patients (Wechalekar et al., Blood, 2013; Dispenzieri et al., JCO, 2004; Palladini et al., JCO, 2012). Renal staging in AL amyloidosis (AL) has been studied in the context of renal survival (Palladini et al., Blood 2014). Influences on survival for renal patients have yet to be fully defined. We performed a retrospective study of all AL patients with renal involvement diagnosed at our center between 7/1/08 and 6/30/15. In this cohort of consecutive patients (n=80) median age was 63 (IQR 55-70) and 56% were men. Eighty-eight percent had lambda plasma cell disease and median involved FLC was 140mg/L (69-485). Thirty-nine percent were renal stage 1, 44% stage 2, and 16% stage 3. Median 24-hour proteinuria and serum creatinine were 6.23 g (3.47-10.70) and 1.03 mg/dL (0.80-1.80) respectively, and median eGFR was 72 mL/min (41-90). Fifty-eight percent had cardiac involvement, of whom 11% were cardiac stage 1, 54% stage 2, and 34% stage 3, while 18% had GI and 9% peripheral nerve involvement. As first-line therapy, 70% received bortezomib-based regimens and 25% melphalan-based autologous stem cell transplant. By intention-to-treat, at 6 months after beginning therapy, 54% of patients had a hematologic response of PR or better, and renal and cardiac responses occurred in 13% and 14% of patients respectively, while renal progression occurred in 6%. Median overall survival (OS) for this cohort (n=80) was 67 months. Those with cardiac involvement (n=45) had a median OS of 41 months and, while median OS was not reached for cardiac stage ≤ 2, it was 31 months for those who were stage 3 (P<0.05) (Figure). Median OS was also not reached for patients achieving hematologic response ≥ VGPR with a median follow-up of 19 months. In conclusion, for AL patients with renal involvement, both cardiac stage and depth of hematologic response are important contributors to overall survival. Furthermore, as this real world intention-to-treat analysis demonstrates, there is a continuing need for better therapies for both the hematologic disease and the organ damage associated with AL. Figure. Figure. Disclosures Oliver: Prothena Biosciences, Inc.: Employment, Equity Ownership. Guthrie:Prothena: Employment, Equity Ownership, Other: Leadership. Comenzo:Takeda: Consultancy, Research Funding; Prothena: Consultancy, Research Funding; Karyopharm: Research Funding; Janssen: Consultancy, Research Funding.

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Development of a Human Therapeutic L-Cyst(e)Ine-Degrading Enzyme for the Treatment of Hematological Malignancies

Blood ◽

10.1182/blood.v128.22.1587.1587 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1587-1587

Author(s):

Giulia Agnello ◽

Susan Alters ◽

Joseph Tyler ◽

Jinyun Liu ◽

Peng Huang ◽

...

Keyword(s):

Research Funding ◽

Hematological Malignancies ◽

Ex Vivo ◽

Redox Balance ◽

Lymphocytic Leukemia ◽

Employment Equity ◽

Equity Ownership ◽

Antioxidant Mechanisms

Abstract Cancer cells experience higher intrinsic oxidative stress than their normal counterparts and acquire adaptive antioxidant mechanisms to maintain redox balance. This increased antioxidant capacity has been correlated to malignant transformation, metastasis and resistance to standard anticancer drugs. This enhanced antioxidant state also correlates with cancer cells being more vulnerable to additional oxidative insults, therefore disruption of adaptive antioxidant mechanisms may have significant therapeutic implications. Hematological malignancies including Chronic Lymphocytic Leukemia (CLL), Acute Lymphocytic Leukemia (ALL), Acute Myeloid Leukemia (AML) and Multiple Myeloma (MM) are critically dependent on the cellular antioxidant glutathione (GSH), consistent with the higher intrinsic oxidative stress. L-cysteine is the rate-limiting substrate for GSH biosynthesis and adequate levels of cysteine are critical to maintain the intracellular homeostasis of GSH. CLL and a subset of ALL cells have been reported to rely on the stromal supply of cysteine to increase the synthesis of GSH in order to maintain redox balance, which in turn promotes cell survival and fosters drug resistance. One approach to target this cancer specific dependency is by therapeutic depletion of amino acids via enzyme administration; a clinically validated strategy for the treatment of ALL. Aeglea BioTherapeutics Inc. has developed a bioengineered cysteine and cystine degrading enzyme (Cyst(e)inase, AEB3103) and evaluated its therapeutic efficacy against hematological malignancies in in vitro, ex vivo and in vivo pre-clinical studies. The TCL1-TG:p53 -/- mouse model exhibits a drug resistant phenotype resembling human CLL with unfavorable cytogenetic alterations and highly aggressive disease progression. AEB3103 greatly decreased the viability of TCL1-TG:p53 -/- cells cultured in vitro, whereas the CLL therapeutic, fludarabine, showed minimal cytotoxic effects. In vivo treatment of TCL1-TG:p53 -/- mice with AEB3103 resulted in an increase in median survival time (7 months, p<0.0001) compared to the untreated control group (3.5 months, p<0.001) and a fludarabine treated group (5.3 months, p<0.001). These results indicate a superior therapeutic effect of AEB3103 compared to fludarabine. Additionally, evaluation of AEB3103 in in vitro 2D cultures of patient-derived CLL and MM cells, and in ex vivo 3D cultures of cells derived from ALL and AML PDx models resulted in significant cell growth inhibition with therapeutically relevant IC50 values. Collectively these results demonstrate the sensitivity of hematological malignancies to modulation of GSH levels via AEB3103-mediated cyst(e)ine depletion. Disclosures Agnello: Aeglea BioTherapeutics: Employment. Alters:Aeglea BioTherapeutics: Employment, Equity Ownership. Tyler:Aeglea BioTherapeutics: Employment, Equity Ownership. Huang:Aeglea BioTherapeutics: Research Funding. Stone:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Research Funding; University of Texas at Austin: Employment, Patents & Royalties: I am an inventor of technology related to this abstract. Georgiou:Aeglea Biotherapeutics: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Lowe:Aeglea BioTherapeutics: Employment, Equity Ownership. Rowlinson:Aeglea BioTherapeutics: Employment, Equity Ownership.

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An All Antibody Approach for Conditioning Bone Marrow for Hematopoietic Stem Cell Transplantation with Anti-cKIT and Anti-CD47 in Non-Human Primates

Blood ◽

10.1182/blood-2019-131490 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4428-4428

Author(s):

Kristopher D Marjon ◽

James Y Chen ◽

Jiaqi Duan ◽

Timothy S Choi ◽

Kavitha Sompalli ◽

...

Keyword(s):

Bone Marrow ◽

Mast Cell ◽

Stem Cell ◽

Peripheral Blood ◽

Research Funding ◽

Mast Cell Degranulation ◽

Employment Equity ◽

Cell Counts ◽

Equity Ownership

Background Hematopoietic stem cell (HSC) transplantation (HSCT) is a well-established procedure that, with or without gene therapy, is curative for numerous severe life-threatening diseases including genetic blood disorders and blood cancers. While advances have been made, there are still substantial concerns since these chemo- and radiation therapy based procedures cause long-term toxicities such as infertility and secondary malignancies or even result in high mortality. We have previously established in a series of preclinical studies a novel chemo- and radiation-free non-toxic monoclonal antibody (Ab) -based conditioning regimen for autologous and allogeneic HSCT (Czechowicz et al., Akanksha et al. and George et al.). This cKIT-CD47 Ab-based regimen selectively depletes host HSCs for HSCT while sparing off-target toxicities caused by chemotherapy/radiation. By significantly decreasing morbidity/mortality associated with traditional conditioning regimens, antibody-mediated conditioning could expand the patient population eligible to receive HSCT for a variety of disorders. We developed a novel cKIT Ab (FSI-174), with an active Fc, and in combination with our CD47 magrolimab (previously 5F9, blocks the don't eat me pathway) could be utilized to translate the promising preclinical findings into clinical studies for safe and less toxic bone marrow conditioning for HSCT. Here we present the functional characterization of FSI-174 as single Ab and in combination with magrolimab in vitro and in non-human primate (NHP) studies. Methods We tested if FSI-174 could block stem cell factor signaling and we explored if FSI-174 alone or in combination with magrolimab could promote phagocytosis of cKIT positive cells (Kasumi-1). In addition, we determined if FSI-174 could cause mast cell degranulation. Subsequently, we explored the potential of FSI-174 alone (Phase A) or in combination with magrolimab (Phase B) to deplete HSCs in NHPs (rhesus macaques)in vivo. In Phase A, single doses of FSI-174 (0.3, 1, or 3 mg/kg) were administered alone. In Phase B, FSI-174 (0.3 or 3 mg/kg) was administered in combination with magrolimab (5mg/kg priming and 20 mg/kg maintenance dose). Bone marrow aspirates and core biopsies and peripheral blood were sampled before the study start and throughout the study. Frequency of bone marrow HSCs and cKIT receptor occupancy (RO) was determined by flow cytometry. In addition, the PK profile of FSI-174 was determined. Results In-vitro analysis demonstrated that FSI-174 decreases proliferation of HSPCs and enhances phagocytosis of cKIT positive cells, and the addition of magrolimab synergistically enhances the phagocytosis. Strikingly, FSI-174 did not cause mast cell degranulation in vitro. In the NHPs, complete (100%) cKIT receptor occupancy was achieved at all FSI-174 dose levels and was maintained for 1 to 9 days correlating with increasing doses and pharmacokinetics. The FSI-174 Cmax was found to be proportional to dose and mean Cmax increased from 6.25 ug/mL to 49.2 ug/mL. In Phase A, FSI-174 alone did not decrease the frequency of bone marrow HSCs compared to PBS control and had no effect on the peripheral blood cell counts. However, in Phase B, when FSI-174 was combined with magrolimab it significantly decreased the frequency of bone marrow HSCs with the nadir at day 9 and no recovery over 85 days compared to PBS control. Notably, there were no changes in peripheral blood cell counts over the course of the studies with no cytopenias in combination treatment. Conclusions We have developed a novel cKIT Ab (FSI-174) that meets the desired profile of stem cell factor block, promotion of phagocytosis, but without promoting mast cell degranulation. Furthermore, in the NHPs studies we have confirmed our chemo- and radiation-free cKIT-CD47 Ab -based conditioning approach with FSI-174 and magrolimab. As anticipated by our previous preclinical studies, monotherapy with FSI-174 does not deplete bone marrow HSCs in NHPs. Notably, no cytopenias are observed with either monotherapy or combination therapy. These data demonstrate the specificity, efficacy and safety of FSI-174/ magrolimab combination have great potential for conditioning regimen for HSCT in a chemotherapy and radiation free manner. Given the favorable safety profile of magrolimab across several clinical studies, these results are paving the way to the first-in-human trials for this novel conditioning for HSCT. Disclosures Marjon: Forty Seven Inc: Employment, Equity Ownership. Chen:Forty Seven Inc.: Consultancy, Equity Ownership. Duan:Forty Seven Inc.: Employment, Equity Ownership. Choi:Forty Seven inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Feng:Forty Seven Inc: Employment, Equity Ownership. Mata:Forty Seven inc: Employment, Equity Ownership. Chen:Forty Seven Inc: Employment, Equity Ownership. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding; Kymab: Consultancy; Jazz: Research Funding. Chao:Forty Seven Inc: Employment, Equity Ownership. Chao:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Takimoto:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties. Agoram:Forty Seven Inc.: Employment, Equity Ownership. Majeti:FortySeven: Consultancy, Equity Ownership, Other: Board of Director; BioMarin: Consultancy. Weissman:Forty Seven Inc.: Consultancy, Equity Ownership, Patents & Royalties. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven, Inc.: Employment, Equity Ownership, Patents & Royalties.

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