Atherosclerosis is characterized by the deposition of lipoproteins in the vessel wall leading to inflammatory response, neointimal thickening, and occlusive plaque. Emerging data suggests that the cell surface marker CD47 is upregulated in atherosclerotic lesions. CD47 produces a ‘don’t eat me’ signal that prevents efficient clearance of diseased vascular cells by phagocytes (a process referred to as ‘efferocytosis’), and it has been hypothesized that dysregulated CD47 may contribute to expansion of atherosclerotic lesions by allowing the accumulation of inflammatory debris in the necrotic core. Blockade of CD47 has been shown to reverse this defect in efferocytosis and attenuate atherosclerosis in mouse models. Here, we evaluated the efficacy of BRB-002, a novel hybrid protein biologic inhibitor of CD47, in a mouse model of atherosclerosis. Four-week-old apolipoprotein E-deficient (ApoE -/-) male mice were fed a high fat diet for 12 weeks during which they were treated with BRB-002 at 2.5 mg/kg, BRB-002 at 10 mg/kg, or IgG isotype control (10 mg/kg) three times a week. Animals were monitored by serial measurements of hematologic parameters, pharmacokinetics, and CD47 receptor occupancy. After 12 weeks, mice were injected with a cathepsin B fluorescent probe and then necropsied to examine their descending aortas. Probes of cathepsin B activity have been used to detect atherosclerotic lesions and shown to correlate with areas of active inflammation and plaque burden. BRB-002 was generally well tolerated in ApoE -/- mice with similar hemoglobin, hematocrit, and red blood cell count between groups. Evaluation of aortas by cathepsin B activity showed that animals treated with BRB-002 at both 2.5 mg/kg and 10 mg/kg dose groups showed significantly decreased cathepsin B activity compared to mice treated with isotype control. These results suggest that blocking CD47 with BRB-002 can attenuate inflammation and atherogenesis in mice.

The CD47/signal regulatory protein α (Cd47/SIRPα)interaction provides a macrophage immune checkpoint pathway that plays a critical role in cancer immune evasion across multiple cancers. Here, we report the engineering of a humanized anti-SIRPα monoclonal antibody (1H9) for antibody target cancer therapy. 1H9 has broad activity across a wide range of SIRPα variants. Binding of 1H9 to SIRPα blocks its interaction with CD47, thereby promoting macrophage-mediated phagocytosis of cancer cells. Preclinical studies in vitro and in vivo demonstrate that 1H9 synergizes with other therapeutic antibodies to promote phagocytosis of tumor cells and inhibit tumor growth in both syngeneic and xenograft tumor models, leading to survival benefit. Thus, 1H9 can potentially act as a universal agent to enhance therapeutic efficacy when used in combination with most tumor-targeting antibodies. We report a comparison of anti-SIRPα and anti-CD47 antibodies in CD47/SIRPα double-humanized mice and found that 1H9 exhibits a substantially reduced antigen sink effect due to the limited tissue distribution of SIRPα expression. Toxicokinetic studies in nonhuman primates show that 1H9 is well tolerated, with no treatment-related adverse effects noted. These data highlight the clinical potential of 1H9 as a pan-therapeutic with the desired properties when used in combination with tumor-targeting antibodies.

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.

Purpose: CXCR4 has been identified as a prognostic marker for acute myeloid leukemia (AML) and other malignancies. We describe the development and characterization of a fully human antibody to CXCR4 and its application for therapy of AML, non–Hodgkin lymphoma (NHL), chronic lymphoid leukemia (CLL), and multiple myeloma. Experimental Design: Human transgenic mice were immunized with CXCR4-expressing cells, and antibodies reactive with CXCR4 were analyzed for apoptosis induction and ability to interfere with CXCL12-induced migration and calcium flux. In vivo efficacy was determined in multiple AML, NHL, and multiple myeloma xenograft tumors in severe combined immunodeficient mice. Results: BMS-936564/MDX-1338 is a fully human IgG4 monoclonal antibody that specifically recognizes human CXCR4. In vitro studies show that MDX-1338 binds to CXCR4-expressing cells with low nanomolar affinity, blocks CXCL12 binding to CXCR4-expressing cells, and inhibits CXCL12-induced migration and calcium flux with low nanomolar EC50 values. When given as monotherapy, MDX-1338 exhibits antitumor activity in established tumors including AML, NHL, and multiple myeloma xenograft models. In addition, we show that MDX-1338 induced apoptosis on a panel of cell lines and propose that antibody-induced apoptosis is one of the mechanisms of tumor growth inhibition. Conclusions: BMS-936564/MDX-1338 is a potent CXCR4 antagonist which is efficacious as monotherapy in tumor-bearing mice and is currently in phase I for the treatment of relapsed/refractory AML, NHL, CLL, and multiple myeloma. Clin Cancer Res; 19(2); 357–66. ©2012 AACR.

Abstract Abstract 1543 BMS-936564/MDX-1338 is a fully human monoclonal antibody that specifically recognizes human CXCR4 and is currently in phase 1 studies in patients with relapsed/refractory acute myeloid leukemia (AML) and multiple myeloma (MM). CXCR4 has been identified as a prognostic indicator for AML and other malignancies, in which greater expression of CXCR4 correlates with disease severity. CXCR4 is a seven-transmembrane, G-protein-coupled receptor in the CXC chemokine receptor family. In response to stimulation by its ligand, the chemokine CXCL12, CXCR4 activates calcium flux, chemotaxis and mediates directional migration of hematopoietic cells. In healthy adults, the receptor is predominantly expressed on B and T cells, monocytes, macrophages, NK and dendritic cells, as well as lymphoid and myeloid precursor cells. Expression of CXCR4 is elevated in a variety of cancers and the interaction of CXCR4 on tumor cells with CXCL12 in the bone marrow promotes tumor cell survival and growth. An antagonist of this pathway is predicted to be efficacious in a variety of hematologic malignancies. In vitro studies demonstrate that BMS-936564/MDX-1338 binds to CXCR4expressing cells with low nanomolar affinity. The antibody blocks CXCL12 binding to CXCR4 expressing cells and inhibits CXCL12 induced migration and calcium flux with low nanomolar EC50 values. When given as monotherapy on established tumors, the antibody exhibits anti-tumor activity in multiple AML, NHL and MM xenograft models. BMS-936564/MDX-1338 is an IgG4 and thus does not elicit complement dependent cytotoxicity (CDC) or antibody dependent cell mediated cytotoxicity (ADCC). In vitro and in vivo studies suggest that BMS-936564/MDX-1338 induces apoptosis as one mechanism of tumor growth inhibition. Here we describe the in vitro and in vivo characterization and activities of BMS-936564/MDX-1338. Disclosures: Kuhne: Bristol-Myers Squibb: Employment. Mulvey:Bristol-Myers Squibb: Employment. Chen:Bristol-Myers Squibb: Employment. Pan:Bristol-Myers Squibb: Employment. Chong:Bristol-Myers Squibb: Employment. Niekro:Bristol-Myers Squibb: Employment. Kempe:Bristol-Myers Squibb: Employment. Henning:Bristol-Myers Squibb: Employment. Cohen:Bristol-Myers Squibb: Employment. Korman:Bristol-Myers Squibb: Employment. Cardarelli:Bristol-Myers Squibb: Employment.

A human anti-CD19 antibody was expressed in fucosyltransferase-deficient CHO cells to generate nonfucosylated MDX-1342. Binding of MDX-1342 to human CD19-expressing cells was similar to its fucosylated parental antibody. However, MDX-1342 exhibited increased affinity for FcγRIIIa-Phe158 and FcγRIIIa-Val158 receptors as well as enhanced effector cell function, as demonstrated by increased potency and efficacy in antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis assays. MDX-1342 showed dose-dependent improvement in survival using a murine B-cell lymphoma model in which Ramos cells were administered systemically. In addition, low nanomolar binding to cynomolgus monkey CD19 and increased affinity for cynomolgus monkey FcγRIIIa was observed. In vivo administration of MDX-1342 in cynomolgus monkeys revealed potent B-cell depletion, suggesting its potential utility as a B-lymphocyte depletive therapy for malignancies and autoimmune indications.

CXCR4 has been identified as a prognostic indicator for acute myeloid leukemia (AML) in which greater expression of CXCR4 correlates with disease severity. In the normal setting CXCR4 is predominantly expressed on B and T cells, monocytes, macrophages, NK and dendritic cells, as well as lymphoid and myeloid precursor cells. CXCR4 is a seven-transmembrane, G-protein-coupled receptor in the CXC chemokine receptor family. In response to stimulation with the chemokine CXCL12, CXCR4 activates chemotaxis and mediates directional migration of hematopoietic cells. MDX-1338 is an anti-human CXCR4-specific, fully human monoclonal antibody. In vitro studies demonstrate that MDX-1338 binds to CXCR4-expressing cells with low nanomolar affinity. MDX-1338 blocks CXCL12 ligand binding to CXCR4 expressing cells and inhibits CXCL12 induced migration and calcium flux with low nanomolar EC 50 values. MDX-1338 is an IgG 4 , and thus lacks complement dependent cytotoxicity (CDC) activity and antibody dependent cell mediated cytotoxicity (ADCC) activity. MDX-1338 induces apoptosis in a range of CXCR4 expressing cell lines and also has anti-tumor activity in multiple AML and lymphoma tumor xenograft models. In a therapeutic human Burkitt\#8217;s lymphoma model, in which Ramos cells are implanted subcutaneously, MDX-1338 inhibited tumor growth approximately 58%-78% in a dose range of 1-30 mg/kg. In two Ara-C refractory AML models, Nomo-1 and HL60, MDX-1338 induced significant tumor growth inhibition at a dose of 10 mg/kg. MDX-1338 is being developed initially for the treatment of relapsed/refractory AML and ALL. Here we demonstrate the characterization and activities of MDX-1338 in vitro and in vivo. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr LB-150.

Purpose: This study was undertaken to evaluate the effects of MDX-1401, a nonfucosylated fully human monoclonal antibody that binds to human CD30, and to determine whether it exhibits greater in vitro and in vivo activity than its parental antibody.Experimental Design: Assays measuring antibody binding to CD30-expressing cells and Fc gamma RIIIa (CD16) transfectants as well as antibody-dependent cellular cytotoxicity (ADCC) were conducted. Antitumor activity was determined using a Karpas-299 systemic model.Results: The binding of MDX-1401 to CD30 antigen was identical to fucose-containing parental anti-CD30 antibody (MDX-060). In contrast, MDX-1401 showed increased binding affinity to Fc gamma RIIIa-transfected cells resulting in increased effector function. MDX-1401 greatly improved ADCC activity as evidenced by a decrease in half-maximal effective concentration (EC50) and an increase in maximum cell lysis when compared with MDX-060. Increased ADCC activity was observed among a panel of cell lines, including one with very low CD30 antigen expression in which parental antibody failed to induce any detectable ADCC. MDX-1401 activity with all Fc gamma RIIIa polymorphic variants, including less active Phe/Phe158 and Phe/Val158 effector cells, was shown. Furthermore, MDX-1401 was efficacious in inhibiting tumor growth in CD30(+) lymphoma xenografts.Conclusions: The low doses of antibody required for ADCC activity irrespective of donor genotype, the ability to mediate ADCC in target cells expressing low levels of CD30, and increased in vivo efficacy support the development of MDX-1401 for treatment of malignant lymphoma.

Therapeutic monoclonal antibodies kill target cells by multiple mechanisms including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), complement-dependent cytotoxicity (CDC), and direct induction of apoptosis. Antibody binding to FcgRIIIA (CD16), which is expressed on NK cells, macrophages, and CD8+ cells, is essential for ADCC. MDX-1401 is a fully human monoclonal antibody of IgG1 (kappa) isotype that binds to human CD30. This second generation anti-CD30 antibody was derived from parental antibody MDX-060 but differs in that the oligosaccharides of the IgG1 lack fucose, a property that conveys increased antibody affinity for Fc receptor CD16 (FcgRIIIa) and thus stimulates both more potent and efficacious ADCC than a fucosylated IgG1. The KD of the parental antibody for CD30 expressed on L540 Hodgkin's lymphoma cells is approximately 1.4 nM while the KD of MDX-1401 is 1.9 nM, demonstrating that binding affinity for antigen are nearly identical. However, the EC50 for MDX-1401 binding to CD16-transfected cells is approximately 3 nM compared to barely detectable binding by parental antibody, confirming that the absence of fucose conveys increased affinity for FcgRIIIa. Data is presented that demonstrates MDX-1401 has significantly improved potency and efficacy in an ADCC assay as evidenced by a decrease in EC50 as well as an increase in maximum specific lysis. Importantly, the enhanced ADCC activity is observed among a large panel of cell lines with varying numbers of CD30 receptors per cell, including one cell line with low antigen expression in which parental antibody failed to induce ADCC. MDX-1401 enhances ADCC activity with all polymorphic variants including lower affinity FcgRIIIa Phe/Phe158 and FcgRIIIa Phe/Val158 effector cells. Furthermore, MDX-1401 is efficacious in inhibiting growth of CD30+ tumor xenografts in mice. The low doses of antibody required for ADCC activity irrespective of donor genotype, the capacity to mediate ADCC of target cells expressing low levels of CD30, and increased in vivo efficacy support the development of MDX-1401 for treatment of malignant lymphoma. Phase 1 studies in subjects with Hodgkin's lymphoma are currently underway.

The chemokine, CXCL10 or interferon γ inducible protein-10 (IP-10) is a chemotactic cytokine for activated T cells and monocytes and plays an important role in migration of cells into sites of inflammation. The receptor for CXCL10, CXCR3, is expressed by activated T cells, eosinophils, NK, and endothelial cells. CXCL10 levels are elevated in ulcerative colitis (UC) amongst other inflammatory diseases. In preclinical animal models of UC, antibodies against CXCL10 have been shown to modify disease progression. Medarex, Inc. has developed a fully human monoclonal antibody (MDX-1100) that binds selectively to CXCL10. This antibody binds to the ligand with high affinity and effectively competes for ligand binding to CXCR3 expressing cells. MDX-1100 blocks CXCL10 induced calcium flux and cell migration with an estimated IC50 in the low nM range. In order to identify potential pharmacodynamic markers for CXCL10 activity, gene chip analysis was performed with RNA purified from CXCL10-stimulated human PBMCs. We identified cell surface receptors, intracellular and soluble markers that are CXCL10 responsive and confirmed the induction of a subset of these genes using quantitative RT-PCR. In summary, MDX-1100, an antibody that binds and neutralizes the activity of CXCL10 is predicted to reduce disease severity in patients with UC and thus a Phase I clinical trial has been initiated.