Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
Add more filters










Publication year range
1.
Cancer Res ; 76(13): 3942-53, 2016 07 01.
Article in English | MEDLINE | ID: mdl-27216193

ABSTRACT

Non-small cell lung cancers (NSCLC) with activating EGFR mutations become resistant to tyrosine kinase inhibitors (TKI), often through second-site mutations in EGFR (T790M) and/or activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of single receptor-binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-61186372 treatment resulted in tumor regression through inhibition of signaling/receptor downmodulation and Fc-driven effector interactions. Complete and durable regression of human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third-generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372 resulted in no major toxicities, including absence of skin rash observed with other EGFR-directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit the spectrum of mutations driving EGFR TKI resistance in NSCLC. Cancer Res; 76(13); 3942-53. ©2016 AACR.


Subject(s)
Antibodies, Bispecific/pharmacology , Antineoplastic Agents/pharmacology , Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Resistance, Neoplasm/drug effects , ErbB Receptors/antagonists & inhibitors , Lung Neoplasms/drug therapy , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Animals , Apoptosis/drug effects , Blotting, Western , Carcinoma, Non-Small-Cell Lung/metabolism , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation/drug effects , ErbB Receptors/genetics , Female , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Macaca fascicularis , Mice , Mice, Inbred BALB C , Mice, Nude , Mutation/genetics , Phosphorylation/drug effects , Proto-Oncogene Proteins c-met/genetics , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
2.
Protein Expr Purif ; 121: 133-40, 2016 May.
Article in English | MEDLINE | ID: mdl-26826313

ABSTRACT

Bispecific antibody generation is actively pursued for therapeutic and research antibody development. Although there are multiple strategies for generating bispecific antibodies (bsAbs); the common challenge is to develop a scalable method to prepare bsAbs with high purity and yield. The controlled Fab-arm exchange (cFAE) method combines two parental monoclonal antibodies (mAbs), each with a matched point mutation, F405L and K409R in the respective CH3 domains. The conventional process employs two steps: the purification of two parental mAbs from culture supernatants followed by cFAE. Following a reduction/oxidation reaction, the bispecific mAb is formed with greater than 95% heterodimerization efficiency. In this study, cFAE was initiated in culture supernatants expressing the two parental mAbs, thereby eliminating the need to first purify the parental mAbs. The bsAbs formed in culture supernatant was then purified using a Protein A affinity chromatography. The BsAbs generated in this manner had efficiency comparable to the conventional method using purified parental mAbs. BsAbs prepared by two different routes showed indistinguishable characteristics by SDS capillary electrophoresis, analytical size exclusion, and cation exchange chromatography. This alternative method significantly shortened timelines and reduced resources required for bsAb generation, providing an improved process with potential benefits in large-scale bsAb preparation, as well as for HTP small-scale bsAb matrix selection.


Subject(s)
Antibodies, Bispecific/biosynthesis , Antibodies, Monoclonal/biosynthesis , Chromatography , Antibodies, Bispecific/immunology , Antibodies, Bispecific/isolation & purification , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Culture Media , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/immunology , Point Mutation
3.
Int J Biol Sci ; 8(3): 310-27, 2012.
Article in English | MEDLINE | ID: mdl-22355267

ABSTRACT

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a promising therapeutic target for treating coronary heart disease. We report a novel antibody 1B20 that binds to PCSK9 with sub-nanomolar affinity and antagonizes PCSK9 function in-vitro. In CETP/LDLR-hemi mice two successive doses of 1B20, administered 14 days apart at 3 or 10 mpk, induced dose dependent reductions in LDL-cholesterol (≥ 25% for 7-14 days) that correlated well with the extent of PCSK9 occupancy by the antibody. In addition, 1B20 induces increases in total plasma antibody-bound PCSK9 levels and decreases in liver mRNA levels of SREBP-regulated genes PCSK9 and LDLR, with a time course that parallels decreases in plasma LDL-cholesterol (LDL-C). Consistent with this observation in mice, in statin-responsive human primary hepatocytes, 1B20 lowers PCSK9 and LDLR mRNA levels and raises serum steady-state levels of antibody-bound PCSK9. In addition, mRNA levels of several SREBP regulated genes involved in cholesterol and fatty-acid synthesis including ACSS2, FDPS, IDI1, MVD, HMGCR, and CYP51A1 were decreased significantly with antibody treatment of primary human hepatocytes. In rhesus monkeys, subcutaneous (SC) dosing of 1B20 dose-dependently induces robust LDL-C lowering (maximal ~70%), which is correlated with increases in target engagement and total antibody-bound PCSK9 levels. Importantly, a combination of 1B20 and Simvastatin in dyslipidemic rhesus monkeys reduced LDL-C more than either agent alone, consistent with a mechanism of action that predicts additive effects of anti-PCSK9 agents with statins. Our results suggest that antibodies targeting PCSK9 could provide patients powerful LDL lowering efficacy on top of statins, and lower cardiovascular risk.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Anticholesteremic Agents/therapeutic use , Cholesterol, LDL/blood , Gene Expression Regulation/drug effects , Hepatocytes/drug effects , Immunization, Passive , Metabolic Syndrome/therapy , Proprotein Convertases/antagonists & inhibitors , Proprotein Convertases/immunology , Serine Endopeptidases/immunology , Simvastatin/therapeutic use , Sterol Regulatory Element Binding Proteins/physiology , Animals , Antibodies, Monoclonal/pharmacology , Antibody Affinity , Anticholesteremic Agents/administration & dosage , Cells, Cultured/drug effects , Cells, Cultured/metabolism , Cholesterol Ester Transfer Proteins/genetics , Cholesterol Ester Transfer Proteins/metabolism , Gene Expression Profiling , Hep G2 Cells/drug effects , Hep G2 Cells/metabolism , Hepatocytes/metabolism , Humans , Lipid Metabolism/genetics , Liver/drug effects , Liver/metabolism , Macaca mulatta , Metabolic Syndrome/drug therapy , Metabolic Syndrome/genetics , Mice , Mice, Transgenic , Proprotein Convertase 9 , Proprotein Convertases/biosynthesis , Proprotein Convertases/genetics , RNA, Messenger/metabolism , Receptors, LDL/biosynthesis , Receptors, LDL/genetics , Recombinant Proteins/metabolism , Serine Endopeptidases/biosynthesis , Serine Endopeptidases/genetics , Simvastatin/administration & dosage
4.
J Pharmacol Exp Ther ; 338(2): 568-78, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21531794

ABSTRACT

Genetic studies have linked both osteoporotic and high bone mass phenotypes to low-density lipoprotein receptor-related proteins (LRP4, LRP5, and LRP6). LRPs are receptors for inhibitory Dickkopf-1 (DKK1) protein, and treatment modalities that modulate LRP/DKK1 binding therefore may act as stimulators of bone mass accrual. Here, we report that RH2-18, a fully human monoclonal anti-DKK1 antibody elicits systemic pharmacologic bone efficacy and new bone formation at endosteal bone surfaces in vivo in a mouse model of estrogen-deficiency-induced osteopenia. This was paralleled by partial-to-complete resolution of osteopenia (bone mineral density) at all of the skeletal sites investigated in femur and lumbar-vertebral bodies and the restoration of trabecular bone microarchitecture. More importantly, testing of RH2-18 in adult, osteopenic rhesus macaques demonstrated a rate-limiting role of DKK1 at multiple skeletal sites and responsiveness to treatment. In conclusion, this study provides pharmacologic evidence for the modulation of DKK1 bioactivity in the adult osteopenic skeleton as a viable approach to resolve osteopenia in animal models. Thus, data described here suggest that targeting DKK1 through means such as a fully human anti-DKK1-antibody provides a potential bone-anabolic treatment for postmenopausal osteoporosis.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Disease Models, Animal , Intercellular Signaling Peptides and Proteins/physiology , Osteogenesis/immunology , Osteoporosis, Postmenopausal/metabolism , Osteoporosis, Postmenopausal/therapy , Animals , Bone Density/immunology , CHO Cells , Cricetinae , Cricetulus , Female , HEK293 Cells , Humans , Macaca mulatta , Male , Mice , Mice, Inbred C57BL , Osteoporosis, Postmenopausal/pathology
5.
J Lipid Res ; 52(1): 78-86, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20959675

ABSTRACT

Proprotein convertase subtilisin-like/kexin type 9 (PCSK9) regulates LDL cholesterol levels by inhibiting LDL receptor (LDLr)-mediated cellular LDL uptake. We have identified a fragment antigen-binding (Fab) 1D05 which binds PCSK9 with nanomolar affinity. The fully human antibody 1D05-IgG2 completely blocks the inhibitory effects of wild-type PCSK9 and two gain-of-function human PCSK9 mutants, S127R and D374Y. The crystal structure of 1D05-Fab bound to PCSK9 reveals that 1D05-Fab binds to an epitope on the PCSK9 catalytic domain which includes the entire LDLr EGF(A) binding site. Notably, the 1D05-Fab CDR-H3 and CDR-H2 loops structurally mimic the EGF(A) domain of LDLr. In a transgenic mouse model (CETP/LDLr-hemi), in which plasma lipid and PCSK9 profiles are comparable to those of humans, 1D05-IgG2 reduces plasma LDL cholesterol to 40% and raises hepatic LDLr protein levels approximately fivefold. Similarly, in healthy rhesus monkeys, 1D05-IgG2 effectively reduced LDL cholesterol 20%-50% for over 2 weeks, despite its relatively short terminal half-life (t(1/2) = 3.2 days). Importantly, the decrease in circulating LDL cholesterol corresponds closely to the reduction in free PCSK9 levels. Together these results clearly demonstrate that the LDL-lowering effect of the neutralizing anti-PCSK9 1D05-IgG2 antibody is mediated by reducing the amount of PCSK9 that can bind to the LDLr.


Subject(s)
Cholesterol, LDL/blood , Immunoglobulin Fab Fragments/pharmacology , Receptors, LDL/chemistry , Serine Endopeptidases/immunology , Animals , Antibodies, Monoclonal/metabolism , Binding Sites , Cholesterol Ester Transfer Proteins/metabolism , Fluoroimmunoassay , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Macaca mulatta , Male , Mice , Mice, Transgenic , Proprotein Convertase 9 , Proprotein Convertases , Receptors, LDL/metabolism , Serine Endopeptidases/chemistry
6.
J Biol Chem ; 285(51): 40135-47, 2010 Dec 17.
Article in English | MEDLINE | ID: mdl-20929859

ABSTRACT

Wnt/LRP5 signaling is a central regulatory component of bone formative and resorptive activities, and the pathway inhibitor DKK1 is a suppressor of bone formation and bone mass accrual in mice. In addition, augmented DKK1 levels are associated with high bone turnover in diverse low bone mass states in rodent models and disease etiologies in human. However, examination of the precise role of DKK1 in the normal skeleton and in higher species requires the development of refined DKK1-specific pharmacological tools. Here, we report the strategy resulting in isolation of a panel of fully human anti-DKK1 antibodies applicable to studies interrogating the roles of mouse, rhesus, and human DKK1. Selected anti-DKK1 antibodies bind primate and human DKK-1 with picomolar affinities yet do not appreciably bind to DKK2 or DKK4. Epitopes mapped within the DKK1 C-terminal domain necessary for interaction with LRP5/6 and consequently effectively neutralized DKK1 function in vitro. When introduced into naïve normal growing female mice, IgGs significantly improved trabecular bone volume and structure and increased both trabecular and cortical bone mineral densities in a dose-related fashion. Furthermore, fully human DKK1-IgG displayed favorable pharmacokinetic parameters in non-human primates. In summary, we demonstrate here a rate-limiting function of physiologic DKK1 levels in the regulation of bone mass in intact female mice, amendable to specific pharmacologic neutralization by newly identified DKK1-IgGs. Importantly the fully human IgGs display a profile of attributes that recommends their testing in higher species and their use in evaluating DKK1 function in relevant disease models.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Neutralizing/pharmacology , Antibody Specificity , Bone Density/drug effects , Intercellular Signaling Peptides and Proteins/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Bone Density/immunology , Bone Diseases/drug therapy , Bone Diseases/immunology , Bone Diseases/metabolism , Dose-Response Relationship, Drug , Female , Humans , Intercellular Signaling Peptides and Proteins/immunology , LDL-Receptor Related Proteins/immunology , LDL-Receptor Related Proteins/metabolism , Low Density Lipoprotein Receptor-Related Protein-5 , Low Density Lipoprotein Receptor-Related Protein-6 , Macaca fascicularis , Macaca mulatta , Mice , Osteogenesis/drug effects , Osteogenesis/immunology
7.
PLoS One ; 5(2): e9094, 2010 Feb 08.
Article in English | MEDLINE | ID: mdl-20161710

ABSTRACT

BACKGROUND: Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target. PRINCIPAL FINDINGS: Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD), and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR). The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50) values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL). In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell-penetrating gamma-secretase inhibitors. CONCLUSIONS/SIGNIFICANCE: Antibodies that compete with Notch1 ligand binding or that bind to the negative regulatory region can act as potent inhibitors of Notch1 signaling. These antibodies may have clinical utility for conditions in which inhibition of signaling by wild-type Notch1 is desired, but are likely to be of limited value for treatment of T-ALLs associated with aberrant Notch1 activation.


Subject(s)
Antibodies, Monoclonal/pharmacology , Mutation , Receptor, Notch1/immunology , Signal Transduction/drug effects , 3T3 Cells , Animals , Antibody Specificity/immunology , Binding Sites/genetics , Binding Sites/immunology , Binding, Competitive , Cell Line , Cell Line, Tumor , Cell Proliferation/drug effects , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , Humans , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Jagged-2 Protein , Ligands , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Receptor, Notch1/genetics , Receptor, Notch1/metabolism , Reverse Transcriptase Polymerase Chain Reaction
8.
MAbs ; 1(6): 572-9, 2009.
Article in English | MEDLINE | ID: mdl-20073128

ABSTRACT

The Fc region of an antibody mediates effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and plays a key role in the in vivo half-life of an antibody. In designing antibody therapeutics, it is sometimes desirable that the antibody has altered Fc-mediated properties. In the case of a "benign blocker" antibody, it is often desirable to diminish or abolish the ADCC and CDC functions while retaining its PK profile. Here, we report a novel engineered IgG isotype, IgG2m4, with reduced Fc functionality. IgG2m4 is based on the IgG2 isotype with four key amino acid residue changes derived from IgG4 (H268Q, V309L, A330S and P331S). An IgG2m4 antibody has an overall reduction in complement and Fc gamma receptor binding in in vitro binding analyses while maintaining the normal in vivo serum half-life in rhesus.


Subject(s)
Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Complement C1q/metabolism , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Protein Engineering , Receptors, IgG/metabolism , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized , Antibody-Dependent Cell Cytotoxicity/genetics , Antibody-Dependent Cell Cytotoxicity/immunology , Complement C1q/genetics , Complement C1q/immunology , Cricetinae , Half-Life , Immunoglobulin Fc Fragments/genetics , Immunoglobulin Fc Fragments/immunology , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Mutagenesis, Site-Directed , Protein Binding/genetics , Protein Binding/immunology , Receptors, IgG/genetics , Receptors, IgG/immunology
9.
MAbs ; 1(5): 462-74, 2009.
Article in English | MEDLINE | ID: mdl-20065653

ABSTRACT

The human D5 monoclonal antibody binds to the highly conserved hydrophobic pocket on the N-terminal heptad repeat (NHR) trimer of HIV-1 gp41 and exhibits modest yet relatively broad neutralization activity. Both binding and neutralization depend on residues in the complementarity determining regions (CDRs) of the D5 IgG variable domains on heavy chain (VH) and light chain (VL). In an effort to increase neutralization activity to a wider range of HIV-1 strains, we have affinity matured the parental D5 scFv by randomizing selected residues in 5 of its 6 CDRs. The resulting scFv variants derived from four different CDR changes showed enhanced binding affinities to gp41 NHR mimetic (5-helix) which correlated to improved neutralization potencies by up to 8-fold. However, when converted to IgG1s, these D5 variants had up to a 12-fold reduction in neutralization potency over their corresponding scFvs despite their slightly enhanced in vitro binding affinities. Remarkably, D5 variant IgG1s bearing residue changes in CDRs that interact with epitope residues N-terminal to the hydrophobic pocket (such as VH CDR3 and VL CDR3) retained more neutralization potency than those containing residue changes in pocket-interacting CDRs (such as VH CDR2). These results provide compelling evidence for the existence of a steric block to an IgG that extends to the gp41 NHR hydrophobic pocket region, and can be a useful guide for developing therapeutic antibodies and vaccines circumventing this block.


Subject(s)
Antibodies, Monoclonal/immunology , Antibody Affinity/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp41/immunology , HIV-1/immunology , Immunoglobulin Fragments/immunology , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Complementarity Determining Regions/chemistry , HIV Antibodies/chemistry , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/immunology , Molecular Sequence Data , Neutralization Tests , Peptide Library
10.
Assay Drug Dev Technol ; 3(1): 7-15, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15798391

ABSTRACT

In this article we describe the use of division-arrested cells for cell-based assays designed for high-throughput screening. Cells are the most critical and variable reagent for cell-based high-throughput screening. The robustness of robotic screening depends on the quality and consistency of cell reagents. We demonstrate that for most cell types commonly used for high-throughput screening, cells can be irreversibly division-arrested by mitomycin C treatment at doses that cause no apparent toxicity or obvious change to the cell signaling properties we measured. Our data also suggest that division-arrested cells perform favorably compared to regular growing cells in reporter and calcium flux assays, two platforms most commonly used in robotic screening. Division arrest technology effectively uncouples the process of cell production from robotic screening and brings the convenience of having quality-approved cell reagent on demand for cell-based high-throughput screening.


Subject(s)
Biological Assay/methods , Cell Culture Techniques/methods , Cell Cycle/physiology , Drug Design , Kidney/cytology , Kidney/drug effects , Mitomycin/pharmacology , Robotics/methods , Animals , Cell Cycle/drug effects , Cell Line , Cryopreservation , Dose-Response Relationship, Drug , Humans , Mice , NIH 3T3 Cells , Reagent Kits, Diagnostic , Reproducibility of Results , Sensitivity and Specificity
SELECTION OF CITATIONS
SEARCH DETAIL
...