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1.
Mol Cancer Ther ; 15(1): 114-24, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26516157

ABSTRACT

Death receptors of the TNF family are found on the surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors 4 and 5 (DR4 and DR5) is TNF-related apoptosis-inducing ligand, TRAIL (Apo2L). As most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing proapoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 monospecific antibodies. Taken together, Surrobody shows promising preclinical proapoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent.


Subject(s)
Antibodies, Monoclonal/pharmacology , Apoptosis/drug effects , Receptors, TNF-Related Apoptosis-Inducing Ligand/agonists , Animals , Caspases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Disease Models, Animal , Drug Resistance, Neoplasm , Gene Knockout Techniques , Humans , Male , Mice , Neoplasms/drug therapy , Neoplasms/metabolism , Neoplasms/pathology , Receptors, TNF-Related Apoptosis-Inducing Ligand/genetics , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Xenograft Model Antitumor Assays
2.
Nature ; 489(7417): 526-32, 2012 Sep 27.
Article in English | MEDLINE | ID: mdl-22982990

ABSTRACT

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibodies, Viral/chemistry , Antibodies, Viral/immunology , Antibody Specificity/immunology , Influenza A virus/classification , Influenza A virus/immunology , Animals , Antibodies, Neutralizing/genetics , Antibodies, Viral/genetics , Antibody Specificity/genetics , Antigens, Viral/chemistry , Antigens, Viral/immunology , Binding Sites , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , Conserved Sequence , Cross Reactions/genetics , Cross Reactions/immunology , Crystallography, X-Ray , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Epitopes/immunology , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Influenza A Virus, H1N1 Subtype/chemistry , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/chemistry , Influenza A Virus, H3N2 Subtype/immunology , Influenza A virus/chemistry , Influenza Vaccines/immunology , Mice , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Orthomyxoviridae Infections/immunology , Orthomyxoviridae Infections/prevention & control , Orthomyxoviridae Infections/virology , Protein Conformation
3.
Mol Cancer Ther ; 11(7): 1411-20, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22553357

ABSTRACT

ErbB3 is an important regulator of tumorigenesis and is implicated in development of resistance to several currently used oncology drugs. We have identified ErbB3 inhibitors based on a novel biologic scaffold termed a surrobody. Two of these inhibitors appear to work by a previously unrecognized mechanism of action. As a consequence, they not only inhibited cell proliferation and intracellular signaling driven by stimulation with the ErbB3 ligand neuregulin (NRG), but also inhibited signaling and proliferation that was driven by overexpression of ErbB2 in the absence of ligand stimulation. In addition, the surrobodies inhibited tumor growth in vivo in both ErbB2-overexpressing and nonoverexpressing cells. In ErbB2-overexpressing cells, both of the anti-ErbB3 surrobodies significantly augmented the activities of trastuzumab, lapatinib, and GDC-0941, agents that inhibit cell proliferation by different mechanisms. Moreover, although NRG diminished the efficacy of these agents, when they were combined with anti-ErbB3 surrobodies the affect of NRG was abrogated. In this capacity, the anti-ErbB3 surrobodies were more effective than the ErbB2/ErbB3 dimerization inhibitory antibody pertuzumab. Despite the fact that these surrobodies appear to engage ErbB3 differently than previously described anti-ErbB3 antibodies, they retain all of the beneficial characteristics of this class of agents, including the ability to augment drugs that inhibit EGF receptor. These anti-ErbB3 agents, therefore, show substantial promise for development as single agents or in combination with other ErbB-directed antibodies or small molecules and may provide for a broader range of therapeutic indications than previously described anti-ErbB3 antibodies.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Neoplasms/metabolism , Receptor, ErbB-3/antagonists & inhibitors , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , ErbB Receptors/antagonists & inhibitors , Gene Expression , Humans , Mice , Mice, Nude , Neoplasms/genetics , Neoplasms/pathology , Neuregulin-1/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Receptor, ErbB-3/genetics , Receptor, ErbB-3/metabolism , Signal Transduction/drug effects , Tumor Burden/drug effects
4.
PLoS Pathog ; 6(7): e1000990, 2010 Jul 08.
Article in English | MEDLINE | ID: mdl-20628565

ABSTRACT

Influenza viruses elude immune responses and antiviral chemotherapeutics through genetic drift and reassortment. As a result, the development of new strategies that attack a highly conserved viral function to prevent and/or treat influenza infection is being pursued. Such novel broadly acting antiviral therapies would be less susceptible to virus escape and provide a long lasting solution to the evolving virus challenge. Here we report the in vitro and in vivo activity of a human monoclonal antibody (A06) against two isolates of the 2009 H1N1 pandemic influenza virus. This antibody, which was obtained from a combinatorial library derived from a survivor of highly pathogenic H5N1 infection, neutralizes H5N1, seasonal H1N1 and 2009 "Swine" H1N1 pandemic influenza in vitro with similar potency and is capable of preventing and treating 2009 H1N1 influenza infection in murine models of disease. These results demonstrate broad activity of the A06 antibody and its utility as an anti-influenza treatment option, even against newly evolved influenza strains to which there is limited immunity in the general population.


Subject(s)
Antibodies, Viral/therapeutic use , Immunotherapy/methods , Influenza A Virus, H1N1 Subtype/immunology , Influenza, Human/drug therapy , Orthomyxoviridae Infections/drug therapy , Animals , Antibodies, Monoclonal/therapeutic use , Cross Reactions/immunology , Disease Models, Animal , Disease Outbreaks , Humans , Influenza A Virus, H5N1 Subtype/immunology , Influenza, Human/prevention & control , Mice , Orthomyxoviridae Infections/prevention & control , Survivors
5.
J Mol Biol ; 397(1): 352-60, 2010 Mar 19.
Article in English | MEDLINE | ID: mdl-20100493

ABSTRACT

Surrobodies(2) are a unique type of binding protein based on the pre-B-cell receptor (pre-BCR). The pre-BCR is transiently expressed during development of the antibody repertoire. Unlike heterotetrameric canonical antibodies that are composed of identical pairs of heavy and light chains, the pre-BCR is a heterohexameric complex composed of identical pairs of heavy chains that are each paired with a two-subunit surrogate light chain (SLC). The SLC contains nonimmunoglobulin-like peptide extensions on each of the two SLC components. This arrangement provides unique opportunities for protein engineering by functional derivatization of these nonimmunoglobulin-like tails. Here we report recombinant fusions to these tails with either a fully active cytokine or with single-chain variable fragment (scFv) domains to generate Surrobodies with unique functions or Surrobodies that are bispecific with respect to targeted binding.


Subject(s)
Immunoglobulin Light Chains, Surrogate/chemistry , Protein Engineering , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Animals , Antibodies, Bispecific/biosynthesis , CHO Cells , Capsid Proteins/metabolism , Cricetinae , Cricetulus , Humans , Immunoglobulin Light Chains, Surrogate/genetics , Interleukin-2/metabolism , Protein Subunits/metabolism , Receptors, Cytokine/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification
6.
Proc Natl Acad Sci U S A ; 105(31): 10756-61, 2008 Aug 05.
Article in English | MEDLINE | ID: mdl-18664586

ABSTRACT

A unique type of combinatorial protein libraries has been constructed. These libraries are based on the pre-B cell receptor (pre-BCR). The pre-BCR is a protein that is produced during normal development of the antibody repertoire. Unlike that of canonical antibodies, the pre-BCR subunit is a trimer that is composed of an antibody heavy chain paired with two surrogate light chain (SLC) components. Combinatorial libraries based on these pre-BCR proteins in which diverse heavy chains are paired with a fixed SLC were expressed in mammalian, Escherichia coli, and phagemid systems. These libraries contain members that have nanomolar affinity for antigen. We term this type of antigen-binding protein a "surrobody" to distinguish it from the canonical antibody molecule.


Subject(s)
Antibodies/genetics , Models, Molecular , Peptide Library , Pre-B Cell Receptors/genetics
7.
Proc Natl Acad Sci U S A ; 105(16): 5986-91, 2008 Apr 22.
Article in English | MEDLINE | ID: mdl-18413603

ABSTRACT

The widespread incidence of H5N1 influenza viruses in bird populations poses risks to human health. Although the virus has not yet adapted for facile transmission between humans, it can cause severe disease and often death. Here we report the generation of combinatorial antibody libraries from the bone marrow of five survivors of the recent H5N1 avian influenza outbreak in Turkey. To date, these libraries have yielded >300 unique antibodies against H5N1 viral antigens. Among these antibodies, we have identified several broadly reactive neutralizing antibodies that could be used for passive immunization against H5N1 virus or as guides for vaccine design. The large number of antibodies obtained from these survivors provide a detailed immunochemical analysis of individual human solutions to virus neutralization in the setting of an actual virulent influenza outbreak. Remarkably, three of these antibodies neutralized both H1 and H5 subtype influenza viruses.


Subject(s)
Antibodies, Monoclonal/isolation & purification , Antibodies, Viral/isolation & purification , Disease Outbreaks/prevention & control , Immunization, Passive/methods , Influenza A Virus, H5N1 Subtype/immunology , Influenza in Birds/immunology , Influenza, Human/prevention & control , Peptide Library , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Viral/genetics , Antibodies, Viral/immunology , Birds/immunology , Birds/virology , Cell Culture Techniques , Cross Reactions , Dogs , Hemagglutinin Glycoproteins, Influenza Virus/immunology , Humans , Influenza A Virus, H1N1 Subtype/immunology , Influenza, Human/immunology , Mutation , Neutralization Tests , Protein Conformation , Turkey/epidemiology
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