ANX005, an Inhibitory Antibody Against C1q, Blocks Complement Activation Triggered By Cold Agglutinins in Human Disease
Abstract Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia characterized by the presence of autoantibodies (cold agglutinins) that bind to red blood cells (RBC) at low temperatures. Cold agglutinin binding to RBCs results in antibody-mediated classical complement activation with deposition of complement C4 and C3 fragments onto the RBC surface. Complement-opsonized RBCs are removed from the circulation by macrophage-driven phagocytosis in the liver or spleen and via intravascular hemolysis following assembly of the terminal lytic complex (C5b - C9), together resulting in clinical anemia. C1q is the initiating molecule for cold agglutinin-mediated complement activation on the surface of human RBCs. We hypothesized that directly blocking C1q recruitment onto cold agglutinin-sensitized RBCs will prevent complement activation and opsonization and reduce hemolysis. To this end, we have developed a humanized monoclonal antibody (ANX005) that binds with high-affinity (~10 pM) to C1q and blocks classical complement activation & hemolysis in an in vitro sheep RBC assay. We evaluated the impact of ANX005 on hemolysis and complement deposition on human RBCs that were pre-sensitized with sera from CAD subjects. ANX005 showed a dose-dependent reduction in hemolysis using both individual and pooled CAD sera as the source of cold agglutinin. We further demonstrated that C1q blockade led to a robust reduction in C4 and C3 fragment deposition onto human RBCs. These results demonstrate that C1q inhibition is an effective way to impede C4 and C3 activation and downstream assembly of the lytic complex in sera from CAD patients, and support the clinical development of ANX005 in CAD and other antibody-mediated diseases. Disclosures Gertz: Prothena Therapeutics: Research Funding; Sandoz Inc: Honoraria; NCI Frederick: Honoraria; Celgene: Honoraria; Med Learning Group: Honoraria, Speakers Bureau; Research to Practice: Honoraria, Speakers Bureau; Alnylam Pharmaceuticals: Research Funding; Novartis: Research Funding; Ionis: Research Funding; Annexon Biosciences: Research Funding; GSK: Honoraria. Qiu:Annexon Biosciences: Employment, Equity Ownership. Kendall:Annexon Biosciences: Employment, Equity Ownership. Saltarelli:Mallinckrodt: Equity Ownership; Abbvie: Equity Ownership; Annexon Biosciences: Employment, Equity Ownership, Patents & Royalties. Yednock:Annexon, Inc: Employment, Equity Ownership. Sankaranarayanan:Annexon Biosciences: Employment, Equity Ownership.
Therapeutic Rationale and Clinical Development of TNT009, an Upstream Classical Pathway Inhibitor, for Cold Agglutinin Disease
Abstract Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia (AIHA) characterized by the presence of autoantibodies (cold agglutinins) that bind red blood cells (RBCs) and activate the classical complement pathway (CP). We have previously shown in vitro that in contrast to C5 inhibition, inhibition of the CP specific protease C1s prevents complement opsonin deposition on cold agglutinin-sensitized RBCs and protects them from phagocytosis, underscoring the necessity to block upstream CP activity (Shi et al., Blood, 2014). Based on the strong scientific rationale and nonclinical data, a Phase 1 clinical trial for TNT009, a monoclonal antibody (mAb) inhibitor of C1s, has commenced at the Medical University of Vienna, Austria. Phase 1a consists of healthy volunteer cohorts in single- and multiple-ascending dose protocols for which interim study results will be presented. In the integrated protocol design of Phase 1b, TNT009 will be dosed in patients with diseases in which pathological CP activity has been implicated, including CAD, warm AIHA and additional non-hematologic indications. In anticipation of the clinical trial, we initiated a screening campaign in Vienna to find prospective CAD patients with serological markers of anemia and hemolysis. To date, plasma and serum samples have been collected from 15 CAD patients. Serum samples from 10 patients induce robust complement activation (C3b/iC3b deposition and/or hemolysis) on AET-treated human RBCs incubated in the patient's own complement-containing serum. In contrast to isotype control (IC), 100 mcg/mL of TNT003 (mouse parental mAb of TNT009), showed near complete inhibition of patient serum mediated C3b/iC3b deposition (90 ± 4%, n = 10; p< 1 x 10-5) and hemolysis (93 ± 5 %, n = 9; p< 1 x 10-5) (Fig. 1). To further support the rationale of C1s inhibition in CAD, we asked whether serological signs of anemia and hemolysis were associated with evidence of increased in vivo CP activity in patient samples. We first examined how well experimental laboratory results agreed with standard clinical readouts. We found good concordance between patient sample induced C3 deposition on RBCs (FACS) and clinical C3 DAT scores (p< .05). Furthermore, IgM staining on RBCs incubated in patient samples (FACS) correlated well with cold agglutinin titers determined in the clinic (p < .001). Next, we observed that the extent of in vitro hemolysis correlated with C3d DAT scores (p< .05), LDH levels (p< .05), and bilirubin levels (p= .05). The agreement between the results from our in vitro patient sample-induced hemolysis assay with serologicalmarkers of complement activity, hemolysis and anemia used in the clinic suggest that our in vitro paradigm serves as a good model for in vivo complement activity in CAD patients. We then measured plasma C4 levels and CP activity in CAD serum samples (Wieslab Classical Pathway ELISA). We found that plasma C4 positively correlated with hemoglobin levels (p = .05). Additionally, we found an inverse correlation between serum CP activity and reticulocyte count (p < .05) and bilirubin levels (p = .05). These data demonstrate that in vivo consumption of the CP and its components (low CP activity, low C4) is associated with markers of anemia and hemolysis (low hemoglobin, high reticulocyte counts, high bilirubin). Finally, an emerging literature calls attention to an increased thromboembolic risk in AIHA, similar to that seen in patients with other hemolytic anemias such as paroxysmal nocturnal hemoglobinuria. We therefore measured D-dimer levels and found it significantly elevated in CAD patient plasma compared to healthy controls (p < .0001). Preliminary analyses show an inverse correlation of C4 and D-dimer in patient plasma (p < .05) suggesting that in vivo CP activity may contribute to the elevated thromboembolic risk in patients (Fig. 2). On-going analyses for other markers of thrombosis, in addition to other experimental approaches to assess the hypercoagulable state in these patients will seek to corroborate this finding. The successful identification of CAD patients with altered complement and hematological profiles provides a unique opportunity to assess proof-of-concept early in the clinical development of TNT009. Figure 1. TNT009 Parental mAb (TNT003) Inhibits CAD Serum Mediated Complement Activation on AET-Treated Human RBCs Figure 1. TNT009 Parental mAb (TNT003) Inhibits CAD Serum Mediated Complement Activation on AET-Treated Human RBCs Figure 2. Elevated D-dimer Correlates with Lower C4 Levels in CAD Patient Plasma Figure 2. Elevated D-dimer Correlates with Lower C4 Levels in CAD Patient Plasma Disclosures Jaeger: Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; True North Therapeutics, Inc.: Research Funding; Hoffmann La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rose:True North Therapeutics, Inc.: Employment, Equity Ownership. Singh:True North Therapeutics, Inc.: Employment, Equity Ownership. Jilma:True North Therapeutics, Inc.: Consultancy, Research Funding. Gilbert:True North Therapeutics, Inc.: Employment, Equity Ownership. Panicker:True North Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties.
Abstract Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia in which autoantibodies bind to red blood cells (RBC) at temperatures below 37°C, resulting in activation of the classical complement pathway (CCP). CCP activation leads to hemolysis either intravascularly, by formation of the membrane attack complex, or extravascularly, when C3/C4 fragment deposition onto the RBC surface results in sequestration by the reticuloendothelial system. Here we describe the in vitro and in vivo activity of TNT003 and TNT009, inhibitors of a serine protease specific to the CCP, in pre-clinical models of CAD. TNT003 is a mouse monoclonal IgG2a antibody with sub-nanomolar affinity. TNT009 is the humanized form (IgG4) of TNT003 and retains affinity and specificity to its target. In vitro assays using IgM-sensitized sheep RBC and human or non-human primate (NHP) serum showed that TNT003 and TNT009 potently inhibited antibody-mediated hemolysis in a concentration dependent manner. Additionally, TNT003 and TNT009 inhibited CCP-mediated production of the anaphylatoxins C4a, C3a, and C5a. Flow cytometry analysis showed that both antibodies also prevented C3 fragment deposition on the RBC surface. Activity was proportional to the amount of serum used, and at 80% human or NHP serum, TNT003 completely inhibited hemolysis with an IC50 of ∼13 µg/mL. Using an ELISA-based assay, TNT003 inhibited C5b-9 deposition driven by the CCP but not by the alternative (CAP) or lectin (CLP) pathways. These data suggest that TNT003 and TNT009 are specific and potent inhibitors of the CCP. To demonstrate the utility of a CCP inhibitor in disease, we tested the ability of TNT003 and TNT009 to inhibit the CCP in ex vivo hemolysis assays using CAD patient autoantibodies. Type O- RBC were incubated in the presence of CAD plasma to sensitize the cells with autoantibody. RBC were then washed and 25% normal human serum (NHS) added as a source of complement. Thirteen of the seventeen CAD samples tested (76%) mediated C3 fragment deposition on the RBC surface as determined by flow cytometry. TNT003 significantly inhibited C3 fragment deposition by all patient samples that deposited complement (88 ± 2.6% inhibition, n = 13) with an average IC50 of 4.7 ± 0.4 µg/mL. One patient sample induced complement-dependent hemolysis of ∼50% of the RBC upon addition of NHS. In a concentration dependent manner, TNT003 and TNT009, but not control IgG, completely inhibited CAD autoantibody-mediated hemolysis (Fig. 1), as well as C4a, C3a and C5a generation. We further characterized each patient sample to determine cold agglutinin titer. We found that cold agglutinin titer correlated with the percent RBC staining positive for cell surface C3 fragments (R2 = 0.3566; p < .01; n = 17 samples; Fig. 2).Figure 1TNT003 and TNT009 inhibit CAD autoantibody-mediated hemolysisFigure 1. TNT003 and TNT009 inhibit CAD autoantibody-mediated hemolysisFigure 2Cold agglutinin titers correlate with C3 fragment deposition on RBCFigure 2. Cold agglutinin titers correlate with C3 fragment deposition on RBC Extravascular hemolysis of C3 fragment-coated RBC by liver macrophages is believed to be the primary mechanism of RBC destruction in CAD. We therefore tested the hypothesis that CAD patient plasma-induced C3 fragment deposition on RBC would promote phagocytosis by the monocytic cell line THP-1. We found that RBC sensitized in CAD plasma and exposed to NHS were engulfed in an FcgR-independent mechanism by THP-1 cells. RBC phagocytosis was significantly inhibited if NHS exposure occurred in the presence of TNT003 (100 µg/mL), but not a control IgG. The selective CCP inhibitory activity of TNT003 was evaluated in vivo in cynomolgus monkeys. TNT003 administered as a single IV injection at 30 mg/kg resulted in a Cmax of ∼330 µg/mL and detectable serum TNT003 thru ≥72 hours. Using an ELISA-based assay, we observed specific inhibition (≥95%) of the CCP for ≥72 hours. In contrast, CAP activity was modestly and transiently inhibited for 4 - 8 hours. At Cmax, endogenous C4a levels were reduced by >90% and returned to baseline levels by ≥96 hours. Serum samples containing TNT003 showed complete (100%) inhibition of hemolysis and C3 fragment deposition in vitro. CCP activity was completely restored to baseline after TNT003 concentrations fell below a predictable, threshold level. Collectively, these data indicate that TNT003 and TNT009 are potent and specific inhibitors of CCP activity and C3 fragment deposition in vitro and in vivo. These findings support the preclinical development of TNT009 for the treatment of CCP-mediated diseases including CAD. Disclosures: Panicker: True North Therapeutics, Inc.: Employment, Equity Ownership. Shi:True North Therapeutics, Inc.: Employment, Equity Ownership. Rose:True North Therapeutics, Inc.: Employment, Equity Ownership. Hussain:True North Therapeutics, Inc.: Employment, Equity Ownership. Tom:True North Therapeutics, Inc.: Employment, Equity Ownership. Strober:True North Therapeutics, Inc.: Employment. Sloan:True North Therapeutics, Inc.: Consultancy. Parry:True North Therapeutics, Inc.: Employment, Equity Ownership. Stagliano:True North Therapeutics, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.
Abstract Abstract 4591 Introduction: Clinicians and investigators appreciate the value of measuring HRQOL for monitoring CLL and the impact of treatments, and commonly use ECOG performance status (PS) and clinician-reported patient fatigue as surrogates for HRQOL in clinical practice. However, limited data exist on the relationships between PS, fatigue, and HRQOL in CLL patients (pts) undergoing treatment in clinical practices. We examined the associations between these measures and 3 psychometrically validated, patient-reported, HRQOL instruments: the Brief Fatigue Inventory (BFI), EQ-5D, and Functional Assessment of Cancer Therapy-Leukemia (FACT-Leu). Methods: Data were collected as part of Connect CLL®, a prospective observational registry initiated in March 2010 involving US practices. Data on pt demographics and clinical characteristics were provided by clinicians. HRQOL was self-reported by pts at enrollment using the BFI, EQ-5D, and FACT-Leu. Mean BFI, EQ-5D and FACT-Leu scores were analyzed by ECOG PS and clinician-reported fatigue. Differences in HRQOL scores between sub-cohorts were assessed by ANOVA. Results: HRQOL data were reported by 604 pts enrolled from 10 academic, 148 community, and 3 government centers. Pts were predominantly male (62%) and white (90%); mean age was 70 (standard deviation 11) years. BFI data (scale: 0 [no fatigue] - 10 [worst fatigue]) indicated that on average pts report that global fatigue, fatigue severity and fatigue-related interference worsen by ECOG severity (Table 1) and are statistically associated with clinician-reported fatigue (Table 2). Mean EQ-5D overall HRQOL as measured by a Visual Analogue Scale (VAS) from 0 (worst) to 100 (best) worsens by ECOG severity and is significantly worse in pts with fatigue. Mean EQ-5D domain scores (scale: 1 [no problem], 2 [some problems], 3 [incapacity]) indicated that pain/discomfort, mobility and usual activities increase in severity as ECOG worsens and in pts with fatigue. FACT-Leu domains except social/family were statistically worse with worse ECOG PS and in pts with fatigue. Conclusions: Initial results from Connect CLL® indicate that HRQOL worsens with worsening ECOG PS, especially in physical / functioning domains, pain/discomfort, and mobility, and worsens across multiple domains among pts whose physicians reported fatigue. Future analyses should be conducted on how HRQOL, PS and fatigue may change over time with changes in CLL, and how they are influenced by therapies. These results may serve as baseline reference. Disclosures: Pashos: Celgene: Membership on an entity's Board of Directors or advisory committees. Flowers:Genentech/Roche (unpaid): Consultancy; Celgene: Consultancy; Millennium/Takeda: Research Funding; Wyeth: Research Funding; Novartis: Research Funding. Weiss:Celgene: Membership on an entity's Board of Directors or advisory committees. Lamanna:Celgene: Membership on an entity's Board of Directors or advisory committees. Farber:Celgene: Membership on an entity's Board of Directors or advisory committees. Kipps:Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbot Industries: Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding. Lerner:Celgene: Membership on an entity's Board of Directors or advisory committees. Kay:Celgene: Membership on an entity's Board of Directors or advisory committees. Sharman:Celgene: Membership on an entity's Board of Directors or advisory committees. Grinblatt:Celgene: Membership on an entity's Board of Directors or advisory committees. Flinn:Celgene: Membership on an entity's Board of Directors or advisory committees. Kozloff:Celgene: Membership on an entity's Board of Directors or advisory committees. Swern:Celgene Corporation: Employment, Equity Ownership. Kahn:Celgene Corporation: Employment, Equity Ownership. Street:Celgene: Employment, Equity Ownership. Sullivan:Celgene: Employment, Equity Ownership. Keating:Celgene: Membership on an entity's Board of Directors or advisory committees.
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.
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.
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.
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.
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.
Background: Current treatment options for red blood cell (RBC) transfusion-dependent (TD) patients with lower risk (LR) myelodysplastic syndromes (MDS) relapsed after or refractory to erythropoiesis-stimulating agents (ESAs) have limited efficacy and durability; new approaches are needed. Imetelstat is a 13-mer lipid-conjugated oligonucleotide that targets the RNA template of human telomerase and is a competitive inhibitor of telomerase enzymatic activity (Asai et al, Cancer Res 2003; Herbert et al, Oncogene 2005). Preclinical, in vivo xenograft models (Dikmen et al, Cancer Res 2005; Hochreiter et al, Clin Cancer Res 2006) and preliminary clinical data from a pilot study conducted at Mayo Clinic (Tefferi et al, Blood Cancer Journal 2016) supported initiation of a study in TD LR MDS patients. A Phase 2 study of imetelstat, IMerge, demonstrated an 8-week RBC transfusion independence (RBC-TI) rate of 42%, 24-week RBC-TI rate of 29%, and 68% erythroid hematologic improvement (HI-E) rate in 38 heavily TD patients (median prior RBC transfusion burden 8 units / 8 weeks over the 16 weeks pre-study period) with LR MDS. Responses were durable with median duration of 8-week RBC-TI of 85.9 weeks by Kaplan Meier estimates (range 8.0-140.9) (Steensma ASH 2018, Fenaux EHA 2019). These Phase 2 results provided further evidence of potential clinical benefit of imetelstat treatment in TD LR MDS, and supported initiation of a Phase 3 trial. Methods: IMerge is two-part, Phase 2/3 study (ClinicalTrials.gov: NCT02598661). The Phase 2 portion of the study described above is closed for enrollment. The Phase 3 portion of the study is open for enrollment of adult patients with International Prognostic Scoring System (IPSS) low or intermediate-1 risk, non-del(5q) MDS, who are TD, are relapsed after or refractory to ESAs, and have not received treatment with lenalidomide or hypomethylating agents. The study is a randomized (2:1) double-blind, placebo-controlled trial to compare efficacy of imetelstat vs. placebo that will enroll approximately 170 patients and will be conducted at approximately 90 centers in North America, Europe, Asia and Middle East. Imetelstat will be administered as 2-hour IV infusion every 4 weeks at 7.5 mg/kg. The primary endpoint of the study is to assess the rate of RBC-TI lasting ≥8 weeks. Secondary endpoints include safety, rate of RBC-TI ≥24 weeks, time to RBC-TI start, RBC-TI duration, rate of HI-E, the amount and relative change in RBC transfusions, rate of CR or PR, overall survival, progression of MDS, pharmacokinetics and effect of treatment on quality of life. Biomarkers relevant to the mechanism of action of imetelstat will be assessed to demonstrate target inhibition and their association with clinical responses. Cytogenetics and mutation analyses will be performed to evaluate the impact of imetelstat on reduction/depletion of malignant clones leading to disease modification. Disclosures Platzbecker: Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria. Steensma:Astex: Consultancy; Arrowhead: Equity Ownership; Summer Road: Consultancy; Onconova: Consultancy; Aprea: Research Funding; Pfizer: Consultancy; Stemline: Consultancy; H3 Biosciences: Other: Research funding to institution, not investigator.. Santini:Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Johnson & Johnson: Honoraria; Acceleron: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees. Germing:Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Jazz Pharmaceuticals: Honoraria; Amgen: Honoraria. Font:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees. Díez-Campelo:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Patnaik:Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees. Sherman:Geron Corporation: Employment, Equity Ownership. Dougherty:Geron Corporation: Employment, Equity Ownership. Feller:Geron Corporation: Employment. Sun:Geron Corporation: Employment, Equity Ownership. Wan:Geron Corporation: Employment, Equity Ownership. Huang:Geron Corporation: Employment, Equity Ownership. Rizo:Geron Corporation: Employment, Equity Ownership. Fenaux:Celgene Corporation: Honoraria, Research Funding; Aprea: Research Funding; Astex: Honoraria, Research Funding; Jazz: Honoraria, Research Funding.
Abstract Introduction: Acute myeloid leukemia (AML) is characterized by heterogeneous cytogenetic and molecular aberrations. Deletions on the long arm of chromosome 9 (del(9q)) are observed in 2% of AML patients. In about 24% of the cases, del(9q) is observed as sole karyotypic abnormality, while in the remaining 76%, it is associated with a t(8;21) translocation or other aberrations. Among all del(9q) AML cases, 36%-50% exhibit an additional t(8;21), whereas 7%-14% of AML cases with t(8;21) show del(9q) as an additional aberration. A commonly deleted region (CDR) of del(9q) was defined and further analysis specified a minimally deleted region (MDR) composed of seven annotated genes (GKAP1, KIF27, C9ORF64, HNRNPK, RMI1, SLC28A3 and NTRK2) (Kronke J et al. Blood. 2013). However, the function of these genes and their impact on the pathogenesis of AML remain elusive. A recent study demonstrated that reduced expression of the HNRNPK gene product can contribute to leukemogenesis in AML (Gallardo M, Cancer Cell. 2015). The multifunctional protein hnRNP K interacts with other proteins, DNA and RNA, to modulate gene activity and gene expression on different levels. For example, hnRNP K not only regulates SRC gene transcription, but as well SRC mRNA translation and the activity of c-Src kinase. In the context of AML, hnRNP K was shown to interact with the mRNAs encoding C/EBPa (CEBPA) and p21 (CDKN1A). We analyzed a cohort of 31 del(9q) AML patients in order to further analyze the deleted region and to analyze the impact of HNRNPK deletion on leukemogenesis. Methods: 31 del(9q) patients were used for the characterization of the deleted region. mRNA level (determined by RT-qPCR analysis) and clinical parameters were compared with a cohort of 24 normal karyotype (NK) AML patients. HnRNP K immunoprecipitation was combined with RNA-Seq, a whole transcriptome shotgun sequencing application based on next generation sequencing and validated by RT-qPCR analysis. CRISPR-Cas9 genome editing has been applied to functionally characterize the impact of post-transcriptional control by hnRNP K in pathogenesis of AML. Results: Our analysis confirmed the MDR in a cohort of 31 AML del(9q) patients. Survival of patients and clinical parameters were not correlated with deletion size, further supporting the importance of the MDR, while other deleted genes seem to be less important for leukemogenesis. As demonstrated by qPCR analysis, the mRNA level of HNRNPK and other genes located in the MDR was reduced in patients carrying a del(9q) compared to NK patients. To further dissect a potential function of hnRNP K in AML del(9q), we characterized hnRNP K interacting mRNAs in the AML cell line KG-1a. Therefore, hnRNP K was immunoprecipitated from cytoplasmic extracts of KG-1a cells and interacting RNAs were identified by RNA-Seq analysis. This analysis revealed that 1076 RNAs are potentially associated with hnRNP K, among them the C/EBPa mRNA. Panther Protein Class analysis identified a high number of transcripts encoding nucleic acid binding proteins, mainly transcription factors. KG-1a cell lines harboring either a complete knock out of hnRNP K or a deletion of the RNA-binding KH-domain are currently generated by CRISPR-Cas9 genome editing to functionally analyze the impact of hnRNP K-mediated post-transcriptional control in AML. Conclusion and Outlook: The deletion of seven genes (GKAP1, KIF27, C9ORF64, HNRNPK, RMI1, SLC28A3 and NTRK2) in the MDR is indispensable, indicating a crucial function for the development of AML del(9q). Among them HNRNPK seems to be a particularly important factor in this process. The identification of hnRNP K interacting RNAs provides the basis to further improve our insight in molecular mechanisms, which drive the pathogenesis of AML del(9q). HNRNPK knock out cell lines will be used to analyze the effect of HNRNPK deletion on post-transcriptional control of identified target genes. Disclosures Ehninger: Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Brümmendorf:Merck: Consultancy; Janssen: Consultancy; Novartis: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Takeda: Consultancy. Rollig:Janssen: Research Funding; Bayer: Research Funding. Thiede:Novartis: Honoraria, Research Funding; AgenDix: Other: Ownership.