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Recently our group and others have identified DDX41 mutations both as germ line and acquired somatic mutations in families with multiple cases of late onset myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML), suggesting that DDX41 acts as a tumor suppressor. To determine whether novel DDX41 mutations could be identified in families with additional types of hematologic malignancies, our group screened two cohorts of families with a diverse range of hematologic malignancy subtypes. Among 289 families, we identified nine (3%) with DDX41 mutations. As previously observed, MDS and AML were the most common malignancies, often of the erythroblastic subtype, and 1 family displayed early-onset follicular lymphoma. Five novel mutations were identified, including missense mutations within important functional domains and start-loss and splicing mutations predicted to result in truncated proteins. We also show that most asymptomatic mutation carriers have normal blood counts until malignancy develops. This study expands both the mutation and phenotypic spectra observed in families with germ line DDX41 mutations. With an increasing number of both inherited and acquired mutations in this gene being identified, further study of how DDX41 disruption leads to hematologic malignancies is critical.

Recently our group and others have identified DDX41mutations both as germ line and acquired somatic mutations in families with multiple cases of late onset myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML), suggesting that DDX41 acts as a tumor suppressor. To determine whether novel DDX41mutations could be identified in families with additional types of hematologic malignancies, our group screened two cohorts of families with a diverse range of hematologic malignancy subtypes. Among 289 families, we identified nine (3%) with DDX41mutations. As previously observed, MDS and AML were the most common malignancies, often of the erythroblastic subtype, and 1 family displayed early-onset follicular lymphoma. Five novel mutations were identified, including missense mutations within important functional domains and start-loss and splicing mutations predicted to result in truncated proteins. We also show that most asymptomatic mutation carriers have normal blood counts until malignancy develops. This study expands both the mutation and phenotypic spectra observed in families with germ line DDX41mutations. With an increasing number of both inherited and acquired mutations in this gene being identified, further study of how DDX41 disruption leads to hematologic malignancies is critical.

The interaction between the hormone hepcidin and the iron exporter ferroportin (Fpn) regulates plasma iron concentrations. Hepcidin binds to Fpn and induces its internalization and degradation, resulting in decreased iron efflux from cells into plasma. Fpn mutations in N144, Y64N, and C326 residue cause autosomal dominant disease with parenchymal iron overload, apparently due to the resistance of mutant Fpn to hepcidin-mediated internalization. To define the mechanism of resistance, we generated human Fpn constructs bearing the pathogenic mutations. The mutants localized to the cell surface and exported iron normally, but were partially or completely resistant to hepcidin-mediated internalization and continued to export iron despite the presence of hepcidin. The primary defect with exofacial C326 substitutions was the loss of hepcidin binding, which resulted in the most severe phenotype. The thiol form of C326 was essential for interaction with hepcidin, suggesting that C326-SH homology is located in or near the binding site of hepcidin. In contrast, N144 and Y64 residues were not required for hepcidin binding, but their mutations impaired the subsequent internalization of the ligand-receptor complex. Our observations explain why the mutations in C326 Fpn residue produce a severe form of hemochromatosis with iron overload at an early age.

The interaction between the hormone hepcidin and the iron exporter ferroportin (Fpn) regulates plasma iron concentrations. Hepcidin binds to Fpn and induces its internalization and degradation, resulting in decreased iron efflux from cells into plasma. Fpn mutations in N144, Y64N, and C326 residue cause autosomal dominant disease with parenchymal iron overload, apparently due to the resistance of mutant Fpn to hepcidin-mediated internalization. To define the mechanism of resistance, we generated human Fpn constructs bearing the pathogenic mutations. The mutants localized to the cell surface and exported iron normally, but were partially or completely resistant to hepcidin-mediated internalization and continued to export iron despite the presence of hepcidin. The primary defect with exofacial C326 substitutions was the loss of hepcidin binding, which resulted in the most severe phenotype. The thiol form of C326 was essential for interaction with hepcidin, suggesting that C326-SH homology is located in or near the binding site of hepcidin. In contrast, N144 and Y64 residues were not required for hepcidin binding, but their mutations impaired the subsequent internalization of the ligand-receptor complex. Our observations explain why the mutations in C326 Fpn residue produce a severe form of hemochromatosis with iron overload at an early age.

Inadequate hepcidin synthesis leads to iron overload in HFE-related hemochromatosis. We explored the regulation of hepcidin by iron in 88 hemochromatosis patients (61 C282Y/C282Y, 27 C282Y/H63D) and 23 healthy controls by analyzing urinary hepcidin before and 24 hours after a 65-mg oral iron dose. Thirty-four patients were studied at diagnosis and had iron overload, and 54 patients were iron depleted. At diagnosis, hepcidin values in C282Y homozygotes were similar to controls, whereas values in C282Y/H63D heterozygotes were higher (P = .02). However, the hepcidin/ferritin ratio was decreased in both homozygotes (P < .001) and heterozygotes (P = .017), confirming the inadequate hepcidin production for the iron load with both genotypes. In iron-depleted patients of both genotypes studied at a time remote from phlebotomy, basal hepcidin was still lower than in controls (P < .001 for C282Y/C282Y and P = .002 for heterozygotes). After an iron challenge, mean urinary hepcidin excretion increased in controls (P = .001) but not patients, irrespective of genotype and iron status. Significant hepcidin increase ( ≥ 10 ng/mg creatinine) was observed in 74% of controls, 15% of homozygotes, and 32% of heterozygotes. The hepcidin response to oral iron is blunted in HFE-related hemochromatosis and not improved after iron depletion. The findings support the involvement of HFE in iron sensing and subsequent regulation of hepcidin.

Inadequate hepcidin synthesis leads to iron overload in HFE-related hemochromatosis. We explored the regulation of hepcidin by iron in 88 hemochromatosis patients (61 C282Y/C282Y, 27 C282Y/H63D) and 23 healthy controls by analyzing urinary hepcidin before and 24 hours after a 65-mg oral iron dose. Thirty-four patients were studied at diagnosis and had iron overload, and 54 patients were iron depleted. At diagnosis, hepcidin values in C282Y homozygotes were similar to controls, whereas values in C282Y/H63D heterozygotes were higher (P= .02). However, the hepcidin/ferritin ratio was decreased in both homozygotes (P< .001) and heterozygotes (P= .017), confirming the inadequate hepcidin production for the iron load with both genotypes. In iron-depleted patients of both genotypes studied at a time remote from phlebotomy, basal hepcidin was still lower than in controls (P< .001 for C282Y/C282Y and P= .002 for heterozygotes). After an iron challenge, mean urinary hepcidin excretion increased in controls (P= .001) but not patients, irrespective of genotype and iron status. Significant hepcidin increase ( ≥ 10 ng/mg creatinine) was observed in 74% of controls, 15% of homozygotes, and 32% of heterozygotes. The hepcidin response to oral iron is blunted in HFE-related hemochromatosis and not improved after iron depletion. The findings support the involvement of HFE in iron sensing and subsequent regulation of hepcidin.

Glycoprotein (GP)IIb/IIIa, an integrin complex found on the surface of platelets, is a receptor for fibrinogen and other ligands, and is involved in platelet aggregation. Because GPIIb is specifically expressed in megakaryocytes, we have studied the 52-flanking region of the rat (r) GPIIb gene as a model of a megakaryocyte-specific gene. The studies presented here used a rat marrow expression system, which allows the study of primary cells undergoing terminal differentiation into megakaryocytes. The determination of megakaryocyte-specific expression of DNA constructs was possible by immunomagnetically separating megakaryocytes from total bone marrow cells. Transient expression constructs, containing varying lengths of the 52-flanking region from -39 to -912 bp, localized a regulatory element between -460 and -439 bp upstream of the transcriptional start site. This region contains a GATA consensus binding element between -457 and -454 (GATA454). Further constructs demonstrated that this GATA binding element was indeed essential for expression. A 25-bp substitution, covering the region -450 to -426 immediately downstream of the GATA454, demonstrated that this region was essential for full expression, which suggests that this region may interact with the GATA454 site in promoting high-level lineage-specific expression. To define regulatory elements between the GATA454 and the transcriptional start site further, we tested additional constructs derived from the original -912 construct; each of which contained the GATA454 but had different 50-bp deletions from -450 to the start site. Virtually all of these constructs continued to show high-level tissue-specific expression. The deleted -150 to -101 construct had twice the level of expression of the full-length wild-type construct; therefore, this region may contain a negative regulatory element. Comparison of our data with expression studies performed with the 52-region of the human GPIIb gene using HEL cells, a cell line with some megakaryocytic properties, demonstrates significant differences, which may reflect our use of primary rate bone marrow cells. In particular, our study points to the importance of the GATA454 for high levels of GPIIb expression in developing megakaryocytes.

Glycoprotein (GP)llb/llla, an integrin complex found on the surface of platelets, is a receptor for fibrinogen and other ligands, and is involved in platelet aggregation. Because GPIIb is specifically expressed in megakaryocytes, we have studied the 5′-flanking region of the rat (r) GPIIb gene as a model of a megakaryocyte-specific gene. The studies presented here used a rat marrow expression system, which allows the study of primary cells undergoing terminal differentiation into megakaryocytes. The determination of megakaryocyte-specific expression of DNA constructs was possible by immunomagnetically separating megakaryocytes from total bone marrow cells. Transient expression constructs, containing varying lengths of the 5′-flanking region from –39 to –912 bp, localized a regulatory element between –460 and –439 bp upstream of the transcriptional start site. This region contains a GATA consensus binding element between –457 and –454 (GATA454). Further constructs demonstrated that this GATA binding element was indeed essential for expression. A 25-bp substitution, covering the region –450 to –426 immediately downstream of the GATA454, demonstrated that this region was essential for full expression, which suggests that this region may interact with the GATA454site in promoting high-level lineage-specific expression. To define regulatory elements between the GATA454 and the transcriptional start site further, we tested additional constructs derived from the original –912 construct; each of which contained the GATA454but had different 50-bp deletions from –450 to the start site. Virtually all of these constructs continued to show high-level tissue-specific expression. The deleted –150 to –101 construct had twice the level of expression of the full-length wild-type construct; therefore, this region may contain a negative regulatory element. Comparison of our data with expression studies performed with the 5′-region of the human GPIIb gene using HEL cells, a cell line with some megakaryocytic properties, demonstrates significant differences, which may reflect our use of primary rat bone marrow cells. In particular, our study points to the importance of the GATA454for high levels of GPIIb expression in developing megakaryocytes.

The low-density lipoprotein receptor-related protein (LRP) is a 600-kD scavenger receptor that binds a number of protein ligands with high affinity. Although some ligands do not compete with each other, binding of all is uniformly blocked by the 39-kD receptor-associated protein (RAP). RAP is normally found in the endoplasmic reticulum and seems to function as a chaperone for LRP. To identify the binding sites for RAP, lactoferrin, and plasminogen activator inhibitor-1 (PAI-1), a bacterial expression system has been developed to produce soluble LRP fragments spanning residues 783-1399. These residues overlap most of the CNBr fragment containing the second cluster of complement-type repeats (C). Solid phase binding assays show that 125I-RAP binds to fragments containing three successive complement-type repeats: C5-C7. PAI-1 and lactoferrin bind to the same fragments. A fragment containing C5-C7 also blocks uptake and degradation of 125I-RAP by fibroblasts in a concentration-dependent manner. Binding competition experiments show that RAP, PAI-1, and lactoferrin each inhibit the binding of the others, suggesting that at this site in LRP, RAP acts as a competitive, rather than an allosteric, inhibitor of PAI-1 and lactoferrin binding. © 1998 by The American Society of Hematology.

The low-density lipoprotein receptor-related protein (LRP) is a 600-kD scavenger receptor that binds a number of protein ligands with high affinity. Although some ligands do not compete with each other, binding of all is uniformly blocked by the 39-kD receptor-associated protein (RAP). RAP is normally found in the endoplasmic reticulum and seems to function as a chaperone for LRP. To identify the binding sites for RAP, lactoferrin, and plasminogen activator inhibitor-1 (PAI-1), a bacterial expression system has been developed to produce soluble LRP fragments spanning residues 783-1399. These residues overlap most of the CNBr fragment containing the second cluster of complement-type repeats (C). Solid phase binding assays show that 125I-RAP binds to fragments containing three successive complement-type repeats: C5-C7. PAI-1 and lactoferrin bind to the same fragments. A fragment containing C5-C7 also blocks uptake and degradation of 125I-RAP by fibroblasts in a concentration-dependent manner. Binding competition experiments show that RAP, PAI-1, and lactoferrin each inhibit the binding of the others, suggesting that at this site in LRP, RAP acts as a competitive, rather than an allosteric, inhibitor of PAI-1 and lactoferrin binding.

Aims: mesenchymal stromal/stem cells (MSCs) is one of the most promising cell therapies to treat immune related diseases such as GvHD, Crohn's disease, osteoarthritis as well as to support function of heart, liver, and central nervous system. Many advanced clinical trials has been taking place to investigate the safety and efficacy of MSC therapy, however, it remains challenging to compare the results of these trials due to variations in culture conditions and tissue origins. These variations pose the urgent need of a standardized large-scale production and quality control of these cells become more urgent, as the use of MSCs are spreading around the globe and disease backgrounds. Numerous culture protocols for MSCs are available using FBS-containing, human platelet lysate/plasma supplementing, or serum-free xeno-free condition, either commercial or self-prepared media. In this study, we have established a standardized expansion protocol that can be used for MSCs derived from bone marrow (BM), adipose tissue (AD), and umbilical cord (UC).

No relevant conflicts of interest to declare.

Feng: Forty Seven Inc: Employment, Equity Ownership. Gip:Forty Seven Inc: Equity Ownership. McKenna:Forty Seven Inc.: Equity Ownership. Zhao:Forty Seven Inc: Consultancy. Mata:Forty Seven Inc: Employment, Equity Ownership. Choi:Forty Seven Inc: Employment, Equity Ownership. Duan:Forty Seven Inc: Employment, Equity Ownership. Sompalli:Forty Seven Inc: Employment, Equity Ownership. Majeti:BioMarin: Consultancy; Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Weissman:Forty Seven, Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Takimoto:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Chao:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Chen:Forty Seven Inc: Consultancy, Equity Ownership. Liu:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties. Volkmer:Forty Seven Inc: Employment, Equity Ownership, Patents & Royalties.

CD47 is an anti-phagocytic signal and macrophage checkpoint that acute myeloid leukemia (AML) and other cancer cells utilize to evade innate immunity and establish disease. 5F9 is a humanized IgG4 monoclonal antibody (mAb) that binds to human CD47 and blocks its interaction with its macrophage receptor SIRPα, thereby promoting phagocytosis of cancer cells. We have found in numerous preclinical studies that anti-CD47 Abs synergize with targeted Abs (such as rituximab and cetuximab) by promoting phagocytosis, and also enable antigen cross-presentation and activation of cytotoxic T cells. These preclinical findings are being translated into clinical results as we have established in several clinical trials promising preliminary evidence of 5F9's therapeutic potential.

Moore: Xencor, Inc.: Employment, Equity Ownership. Lee:Xencor, Inc.: Employment, Equity Ownership. Schubbert:Xencor, Inc.: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Ardila:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Chu:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

CD38 is highly expressed on plasma cells and is an attractive target for multiple myeloma (MM) therapies. Several anti-CD38 antibodies including daratumumab and SAR650984 show promising results in clinical development, though such antibodies are not able to stimulate T cell-mediated killing of myeloma cells. To exploit a T cell immunotherapy mechanism while retaining the favorable drug properties of therapeutic antibodies, we designed bispecific antibodies that recruit T cells to CD38+MM cells. Such bispecifics act via redirected T cell-cytotoxicity (RTCC) to stimulate T cell-mediated target cell killing regardless of T cell receptor antigen specificity. These anti-CD38 × anti-CD3 antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain lacks binding to Fcγ receptors to minimize nonselective T cell activation, yet retains binding to FcRn to maintain long serum half-life.

Burnworth: HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Nguyen:HematoLogics Inc.: Employment. Verkamp:HematoLogics Inc.: Employment. Angela:Hematologics: Employment. Wentzel:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Loken:Hematologics: Employment, Equity Ownership. Wells:HematoLogics Inc.: Employment, Equity Ownership. Zehentner:HematoLogics Inc.: Employment, Equity Ownership.

Chu: Xencor: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Chu: Xencor: Employment, Equity Ownership. Lee:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

Wang: HematoLogics Inc.: Employment. Fritschle:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Burnworth:HematoLogics Inc.: Employment. Bennington:HematoLogics Inc.: Employment. Wentzel:HematoLogics Inc.: Employment. Verkamp:HematoLogics Inc.: Employment. Nguyen:HematoLogics Inc.: Employment. Ghirardelli:HematoLogics Inc.: Employment. Broderson:HematoLogics Inc.: Employment. Wells:HematoLogics Inc.: Employment, Equity Ownership. Loken:HematoLogics Inc.: Employment, Equity Ownership. Zehentner:HematoLogics Inc.: Employment, Equity Ownership.

Chu: Xencor: Employment, Equity Ownership. Miranda:Xencor, Inc.: Employment, Equity Ownership. Phung:Xencor, Inc.: Employment, Equity Ownership. Chen:Xencor, Inc.: Employment, Equity Ownership. Rashid:Xencor, Inc.: Employment, Equity Ownership. Endo:Xencor, Inc.: Employment, Equity Ownership. Chan:Xencor, Inc.: Employment, Equity Ownership. Pong:Xencor, Inc.: Employment, Equity Ownership. Bonzon:Xencor, Inc.: Employment, Equity Ownership. Muchhal:Xencor, Inc.: Employment, Equity Ownership. Leung:Xencor, Inc.: Employment, Equity Ownership. Bernett:Xencor, Inc.: Employment, Equity Ownership. Moore:Xencor, Inc.: Employment, Equity Ownership. Szymkowski:Xencor, Inc.: Employment, Equity Ownership. Desjarlais:Xencor, Inc.: Employment, Equity Ownership.

CD38, being highly expressed on malignant plasma cells, is an attractive target of new therapies for multiple myeloma (MM). Several anti-CD38 antibodies including daratumumab are in clinical development; however, a limitation of such monospecific antibodies is their inability to stimulate cytotoxic T cell killing of myeloma cells. To exploit the potent mechanism of T cell immunotherapy yet preserve the favorable drug and dosing properties of therapeutic antibodies, we designed bispecific antibodies that recruit T cells to CD38+ MM cells. Such bispecifics act via a "redirected T cell-cytotoxicity" (RTCC) mechanism because they stimulate targeted T cell-mediated killing regardless of T cell receptor antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life).

Introduction:

CD20 is highly expressed on normal and malignant B cells, and is a well-established target of antibody therapeutics for B cell leukemias and lymphomas. However, a limitation of approved anti-CD20 antibodies such as rituximab, ofatumumab, and obinutuzumab is that they are unable to stimulate T cell-mediated killing of CD20+ B cells. To exploit the potent activity intrinsic to T cell immunotherapy while maintaining the favorable dosing regimen of a therapeutic antibody, we have designed novel humanized bispecific antibodies that bind to both CD20+ B cells and CD3+ T cells. Such antibodies act via a mechanism known as "redirected T cell-cytotoxicity" (RTCC), because they stimulate targeted T cell-mediated killing regardless of T cell receptor antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life).

CD123 (IL-3 receptor alpha) is highly expressed on acute myeloid leukemia stem cells and blasts, and represents a promising target of antibody therapies for AML. Anti-CD123 antibodies such as CSL-362 and KHK2823 are currently in clinical development; however, a limitation of these molecules is that they are unable to stimulate T cell-mediated killing of CD123+ AML cells. To exploit the potent activity inherent to T cell immunotherapy while maintaining the favorable dosing regimen of a therapeutic antibody, we have designed a novel bispecific antibody that recruits T cells to attack CD123+ AML stem and blast cells. Such antibodies act via a mechanism known as “redirected T cell-cytotoxicity” (RTCC), because they stimulate targeted T cell-mediated killing regardless of T cell antigen specificity. Unlike other bispecific formats, these antibodies possess a full Fc domain and spontaneously form stable heterodimers that are readily manufactured. Their Fc domain was also engineered to abolish binding to Fcγ receptors (to reduce the potential for nonselective T cell activation), yet preserve binding to human FcRn (to maintain long serum half-life).

Background:

No relevant conflicts of interest to declare.

Abstract 1726