A5077529516- Monoclonal and Polyclonal Antibodies Research
- Platelet Disorders and Treatments
- HER2/EGFR in Cancer Research
- Blood groups and transfusion
- Glycosylation and Glycoproteins Research
- Chronic Lymphocytic Leukemia Research
- Heparin-Induced Thrombocytopenia and Thrombosis
- Radiopharmaceutical Chemistry and Applications
- Antiplatelet Therapy and Cardiovascular Diseases
- Viral Infectious Diseases and Gene Expression in Insects
- Biochemical Analysis and Sensing Techniques
- Regulation of Appetite and Obesity
- Cell Adhesion Molecules Research
- Advanced Biosensing Techniques and Applications
- Blood Coagulation and Thrombosis Mechanisms
- Protease and Inhibitor Mechanisms
- T-cell and B-cell Immunology
- Myeloproliferative Neoplasms: Diagnosis and Treatment
- Multiple Myeloma Research and Treatments
- Autoimmune Bullous Skin Diseases
- Tissue Engineering and Regenerative Medicine
- Coagulation, Bradykinin, Polyphosphates, and Angioedema
- Complement system in diseases
- Immunodeficiency and Autoimmune Disorders
- Galectins and Cancer Biology
Genmab (Netherlands)
2006-2017
Genmab (United States)
2013-2014
University Medical Center Utrecht
2000-2009
Sanquin
2007
Maastricht University
2007
Hasselt University
2007
Utrecht University
1997-2000
Leiden University
1994
Rotterdam University of Applied Sciences
1994
Institut National de la Transfusion Sanguine
1994
Abstract CD38, a type II transmembrane glycoprotein highly expressed in hematological malignancies including multiple myeloma (MM), represents promising target for mAb-based immunotherapy. In this study, we describe the cytotoxic mechanisms of action daratumumab, novel, high-affinity, therapeutic human mAb against unique CD38 epitope. Daratumumab induced potent Ab-dependent cellular cytotoxicity CD38-expressing lymphoma- and MM-derived cell lines as well patient MM cells, both with...
Antibodies play a central role in immunity by forming an interface with the innate immune system and, typically, mediate proinflammatory activity. We describe novel posttranslational modification that leads to anti-inflammatory activity of antibodies immunoglobulin G, isotype 4 (IgG4). IgG4 are dynamic molecules exchange Fab arms swapping heavy chain and attached light (half-molecule) heavy-light pair from another molecule, which results bispecific antibodies. Mutagenesis studies revealed...
Abstract We have previously defined a panel of fully human CD20 mAb. Most these were unexpectedly efficient in their ability to recruit C1q the surface CD20-positive cells and mediate tumor lysis via activation classical pathway complement. This complement-dependent cytotoxicity (CDC) potency appeared relate unusually slow off-rate Abs. However, we now present epitope-mapping data, which indicates that all mAb bind novel region may influence CDC potency. Epitope mapping, using both...
Development of human therapeutic Abs has led to reduced immunogenicity and optimal interactions with the immune system in patients. Humanization had as a consequence that efficacy studies performed mouse models, which represent crucial step preclinical development, are more difficult interpret because gaps our knowledge activation murine effector cells by IgG (hIgG) remain. We therefore developed full sets isotype variants targeting epidermal growth factor receptor CD20 explore crosstalk...
Glycosylation of the antibody Fc fragment is essential for receptor-mediated activity. Carbohydrate heterogeneity known to modulate activity effector cells in blood, which fucosylation particularly affects NK cell-mediated killing. Here, we investigated how glycosylation profile 2F8, a human IgG(1) monoclonal against epidermal growth factor receptor clinical development, impacted function. Various 2F8 batches differing fucosylation, galactosylation, and sialylation complex-type...
Transient expression systems in mammalian cells have become the method of choice for producing research quantities antibodies. Both speed and yield available transient natural posttranslational modifications favor these above lower eukaryotes, prokaryotes or stable cell lines. We describe an optimized system, capable up to 400mg/L native secreted antibodies less than a week. The system is composed commercially components based on fast growing suspension line, FreeStyle™ 293-F (HEK-293F)....
A distinctive feature of human IgG4 is its ability to recombine half molecules (H chain and attached L chain) through a dynamic process termed Fab-arm exchange, which results in bispecific Abs. It becoming evident that the exchange conserved several mammalian species, thereby represents mechanism impacts humoral immunity more generally than previously thought. In humans, has been attributed core-hinge sequence (226-CPSCP-230) combination with unknown determinants third constant H domain...
Antibody-drug conjugates (ADC) are designed to be stable in circulation and release potent cytotoxic drugs intracellularly following antigen-specific binding, uptake, degradation tumor cells. Efficient internalization routing lysosomes where proteolysis can take place is therefore essential. For many cell surface proteins carbohydrate structures on cells, however, the magnitude of these processes insufficient allow for an effective ADC approach. We hypothesized that we could overcome this...
von Willebrand factor (vWF) mediates platelet adhesion at sites of vascular damage. It acts as a bridge between receptors on platelets and collagens present in the connective tissue. Two collagen binding have been identified A1 A3 domain vWF subunit. To study functional importance these sites, we made two deletion mutants that lack (residues 478-716; delta A1-vWF; Sixma et al. Eur. J. Biochem, 196, 369, 1991 [1]) or 910-1113; A3-vWF). After transfection baby hamster kidney cells...
We studied the variations in N-linked glycosylation of human IgG molecules derived from 105 different stable cell lines each expressing one six antibodies. Antibody expression was based on glutamine synthetase selection technology suspension growing CHO-K1SV cells. The glycans detected Fc fragment were mainly core-fucosylated complex type containing zero or galactose and little to no sialic acid. highly consistent for same line when grown multiple times, indicating robustness production...
Antibody engineering is increasingly being used to influence the properties of monoclonal antibodies improve their biotherapeutic potential. One important aspect this modulation glycosylation as a strategy efficacy. Here, we describe mutations Y407 in CH3 domain IgG1 and IgG4 that significantly increase sialylation, galactosylation, branching N-linked glycans CH2 domain. These also promote formation monomeric assemblies (one heavy-light chain pair). Hydrogen-deuterium exchange mass...
Abstract —We studied the role of von Willebrand Factor (vWF) in platelet thrombus formation flowing blood by using a perfusion system and mutant forms vWF lacking either interaction with glycoprotein Ib (GpIb) or IIb/IIIa (αIIb-β3). These mutants were added to patients severe Willebrand’s disease (vWD) normal reconstituted human albumin solution instead plasma. This was then perfused over collagen type III spray-coated on glass surface preincubated for 2 hours 20 μg/mL plasma vWF. In this...
We have analyzed a type IIB and I von Willebrand disease family for the presence of mutations in region coding glycoprotein Ib binding domain factor. Since this sequence is also present highly homologous factor pseudogene, we studied genomic DNA as well cDNA, which was produced from RNA isolated endothelial cells or platelets. In both families, detected multiple consecutive nucleotide substitutions 5' end exon 28 that result identical to pseudogene. These were found proves they are active...
Ab-dependent cellular cytotoxicity (ADCC) is recognized as a prominent cytotoxic mechanism for therapeutic mAbs in vitro. However, the contribution of ADCC to vivo efficacy, particularly treatment solid tumors, still poorly understood. For zalutumumab, epidermal growth factor receptor (EGFR)-specific mAb currently clinical development, previous studies have indicated signaling inhibition and induction important mechanisms action. To investigate role ADCC, panel EGFR-specific lacking specific...
Abstract Human IgG1 type I CD20 Abs, such as rituximab and ofatumumab (OFA), efficiently induce complement-dependent cytotoxicity (CDC) of CD20+ B cells by binding C1 to hexamerized Fc domains. Unexpectedly, we found that Ab F(ab′)2 fragments, well C1q-binding–deficient IgG mutants, retained an ability CDC, albeit with lower efficiency than for whole or unmodified IgG. Experiments using human serum depleted specific complement components demonstrated the observed lytic activity, which termed...
Rapid production of recombinant human IgG with improved antibody dependent cell-mediated cytotoxicity (ADCC) effector function is presented. The technique employs transient expression in suspension growing HEK-293F cells the presence glycosidase inhibitor kifunensine. procedure takes approximately 7 days, provided that plasmids encoding interest are available. Kifunensine inhibits N-linked glycosylation pathway endoplasmatic reticulum, resulting oligomannose type glycans lacking core-fucose....
The human epidermal growth factor receptor (HER)2 provides an excellent target for selective delivery of cytotoxic drugs to tumor cells by antibody-drug conjugates (ADC) as has been clinically validated ado-trastuzumab emtansine (KadcylaTM). While selecting a suitable antibody ADC approach often takes specificity and efficient antibody-target complex internalization into account, the characteristics optimal candidate remain poorly understood. We studied large panel HER2 antibodies identify...
Summary Binding of von Willebrand Factor (vWF) to sites vascular injury is the first step hemostasis. Collagen types I and III are important binding for vWF. We have previously determined threedimensional structure collagen A3 domain vWF (Huizinga et al., Structure 1997; 5: 1147). hypothesized that top face this might be collagen-binding site. Based on hypothesis, we made seven mutants (D934A/S936A, V1040A/ V1042A, D1046A, D1066A, D1069A, D1069R, R1074A). these was investigated in ELISA with...