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1.
J Inflamm Res ; 14: 3823-3835, 2021.
Article in English | MEDLINE | ID: mdl-34408465

ABSTRACT

BACKGROUND: Interleukin-33 (IL-33) is an alarmin that is released following cellular damage, mechanical injury, or necrosis. It is a member of the IL-1 family and binds to a heterodimer receptor consisting of ST2 and IL-1RAP to induce the production of a wide range of cellular mediators, including the type 2 cytokines IL-4, IL-5 and IL-13. This relationship has led to the hypothesis that the IL-33/ST2 pathway is a driver of allergic disease and inhibition of the IL-33 and ST2 association could have therapeutic benefit. METHODS: In this paper, we describe the selection of a phage antibody through the ability to bind human IL-33 and block IL-33/ST2 interaction. This hit antibody was then affinity matured by site-directed mutagenesis of the antibody complementarity-determining regions (CDRs). Further characterization of a fully human monoclonal antibody (mAb), torudokimab (LY3375880) included demonstration of human IL-33 neutralization activity in vitro with an NFκB reporter assay and IL-33 induced mast cell cytokine secretion assay, followed by an in vivo IL-33-induced pharmacodynamic inhibition assay in mice that used IL-5 production as the endpoint. RESULTS: Torudokimab is highly specific to IL-33 and does not bind any of the other IL-1 family members. Furthermore, torudokimab binds human and cynomolgus monkey IL-33 with higher affinity than the binding affinity of IL-33 to ST2, but does not bind mouse, rat, or rabbit IL-33. Torudokimab's half-life in cynomolgous monkey projects monthly dosing in the clinic. CONCLUSION: Due to torudokimab's high affinity, its ability to completely neutralize IL-33 activity in vitro and in vivo, and the observed cynomolgus monkey pharmacokinetic properties, this molecule was selected for clinical development.

2.
MAbs ; 2(2): 190-8, 2010.
Article in English | MEDLINE | ID: mdl-20150758

ABSTRACT

The pathogenicity of Clostridium difficile (C. difficile) is mediated by the release of two toxins, A and B. Both toxins contain large clusters of repeats known as cell wall binding (CWB) domains responsible for binding epithelial cell surfaces. Several murine monoclonal antibodies were generated against the CWB domain of toxin A and screened for their ability to neutralize the toxin individually and in combination. Three antibodies capable of neutralizing toxin A all recognized multiple sites on toxin A, suggesting that the extent of surface coverage may contribute to neutralization. Combination of two noncompeting antibodies, denoted 3358 and 3359, enhanced toxin A neutralization over saturating levels of single antibodies. Antibody 3358 increased the level of detectable CWB domain on the surface of cells, while 3359 inhibited CWB domain cell surface association. These results suggest that antibody combinations that cover a broader epitope space on the CWB repeat domains of toxin A (and potentially toxin B) and utilize multiple mechanisms to reduce toxin internalization may provide enhanced protection against C. difficile-associated diarrhea.


Subject(s)
Antibodies, Monoclonal/metabolism , Antitoxins/metabolism , Clostridioides difficile/physiology , Enterocolitis, Pseudomembranous/drug therapy , Enterocolitis, Pseudomembranous/immunology , Animals , Antibodies, Monoclonal/immunology , Antitoxins/immunology , Bacterial Proteins/adverse effects , Bacterial Proteins/immunology , Bacterial Toxins/adverse effects , Bacterial Toxins/immunology , Binding Sites, Antibody/immunology , CHO Cells , Clostridioides difficile/pathogenicity , Cricetinae , Cricetulus , Diarrhea/etiology , Diarrhea/prevention & control , Drug Combinations , Enterocolitis, Pseudomembranous/microbiology , Enterocolitis, Pseudomembranous/physiopathology , Enterotoxins/adverse effects , Enterotoxins/immunology , Epitope Mapping , Humans , Mice , Protein Binding
3.
Protein Eng Des Sel ; 19(7): 325-36, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16672248

ABSTRACT

The recombinant expression of immunoglobulin domains, Fabs and scFvs in particular, in Escherichia coli can vary significantly from antibody to antibody. We hypothesized that poor Fab expression is often linked to poor intrinsic stability. To investigate this further, we applied a novel approach for stabilizing a poorly expressing anti-tetanus toxoid human Fab with a predisposition for being misfolded and non-functional. Forty-five residues within the Fab were chosen for saturation mutagenesis based on residue frequency analysis and positional entropy calculations. Using automated screening, we determined the approximate midpoint temperature of thermal denaturation (TM) for over 4000 library members with a maximum theoretical diversity of 855 unique mutations. This dataset led to the identification of 11 residue positions, primarily in the Fv region, which when mutated enhanced Fab stability. By combining these mutations, the TM of the Fab was increased to 92 degrees C. Increases in Fab stability correlated with higher expressed Fab yields and higher levels of properly folded and functional protein. The mutations were selected based on their ability to increase the apparent stability of the Fab and therefore the exact mechanism behind the enhanced expression in E.coli remains undefined. The wild-type and two optimized Fabs were converted to an IgG1 format and expressed in mammalian cells. The optimized IgG1 molecules demonstrated identical gains in thermostability compared to the Fabs; however, the expression levels were unaffected suggesting that the eukaryotic secretion system is capable of correcting potential folding issues prevalent in E.coli. Overall, the results have significant implications for the bacterial expression of functional antibody domains as well as for the production of stable, high affinity therapeutic antibodies in mammalian cells.


Subject(s)
Escherichia coli/metabolism , Gene Expression Regulation , Immunoglobulin Fab Fragments/biosynthesis , Protein Engineering , Escherichia coli/genetics , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/isolation & purification , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/isolation & purification , Mutation , Pattern Recognition, Automated/methods , Peptide Library , Protein Denaturation , Protein Folding , Thermodynamics , Time Factors
4.
J Mol Biol ; 346(5): 1197-206, 2005 Mar 11.
Article in English | MEDLINE | ID: mdl-15713474

ABSTRACT

Clostridium difficile (C.difficile) is a nosocomially acquired intestinal bacillus which can cause chronic diarrhea and life-threatening colitis. The pathogenic effects of the bacillus are mediated by the release of two toxins, A and B. The C-terminal portions of both toxins are composed of 20 and 30 residue repeats known as cell wall binding (CWB) domains. We have cloned and expressed the CWB-domains of toxins A and B and several truncated CWB-domain constructs to investigate their structure and function. The smallest CWB-domain that folded in a cooperative manner was an 11 repeat construct of toxin A. This differentiates the C-terminal domains of toxins A and B from the CWB-domain of Streptococcus pneumoniae LytA, which only requires six repeats to fold. The 11 repeat toxin A construct bound Ca2+ directly with millimolar affinity and interacted with mammalian cell surfaces in a concentration and Ca2+-dependent fashion. Millimolar Ca2+ levels also accelerated toxin mediated CHO cell killing in an in vitro cell assay. Together, the data suggest a role for extracellular Ca2+ in the sensitization of toxin A/cell-surface interactions.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Toxins/chemistry , Calcium/metabolism , Cell Wall/metabolism , Clostridioides difficile , Enterotoxins/chemistry , Amino Acid Sequence , Animals , Apoptosis , Bacterial Proteins/metabolism , Bacterial Proteins/toxicity , Bacterial Toxins/metabolism , Bacterial Toxins/toxicity , CHO Cells , Cloning, Molecular , Cricetinae , Enterotoxins/metabolism , Enterotoxins/toxicity , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Streptococcus pneumoniae
5.
Hybrid Hybridomics ; 22(2): 97-108, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12831535

ABSTRACT

A panel of novel recombinant single-chain variable fragment (scFv) antibody against human immunodeficiency virus type-1 (HIV-1) was isolated and characterized. We generated human scFvs using RNA harvested from cervical B lymphocytes of Kenyan prostitutes who are highly exposed to HIV-1, but remain persistently seronegative. The variable regions of the heavy (VH) and light (VL) chain antibody genes were selected as hybrids using guided-selection with the VL and VH, respectively, of a derivative of IgGb(12) using the phagemid vector pComb3X. IgGb(12) is a previously well-characterized HIV-1 neutralizing human monoclonal antibody (MAb). One of the hybrid scFv, IgA6/4L, neutralizes HIV-1 infectivity in in vitro cell culture assay. The cervical VH and VL chain antibody genes were connected by a DNA linker and subcloned in pComb3X. The cervical scFv clones were functional in recognizing HIV-1 gp120 by enzyme-linked immunosorbant assay (ELISA) and on cells in flow cytometry. Whole IgGb(12) does not inhibit binding of clones IgA6/5k nor IgA6/30lambda to gp120, which suggests that they bind different epitopes. Nucleotide sequence analysis of the cervical scFv show the clones are unique and reveal interesting characteristics of human cervical V gene pools. This work demonstrates, for the first time, cloning of a functional scFv MAb to a sexually transmitted disease pathogen from local cervical B-cell pools in exposed humans.


Subject(s)
Antibodies, Monoclonal/genetics , Cervix Uteri/immunology , HIV Antibodies/genetics , HIV-1/immunology , Immunoglobulin Variable Region/genetics , Amino Acid Sequence , Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Base Sequence , Cervix Uteri/cytology , Female , HIV Antibodies/immunology , Humans , Immunoglobulin Variable Region/immunology , Kenya , Leukocytes, Mononuclear/immunology , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/immunology
6.
Hybrid Hybridomics ; 22(1): 23-31, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12713687

ABSTRACT

Dendritic cells (DC) are the professional antigen-presenting cells of the immune system. Previous studies have demonstrated that targeting foreign antigens to DC leads to enhanced antigen (Ag)-specific responses in vivo. However, the utility of this strategy for the generation of MAbs has not been investigated. To address this question we immunized mice with IgG-peptide conjugates prepared with the hamster anti-murine CD11c MAb N418. Synthetic peptides corresponding to two different exposed regions of DC-specific ICAM-3 grabbing nonintegrin (DC-SIGN), a human C-type lectin, were conjugated to N418 using thiol-based chemistry. The N418 MAb served as the targeting molecule and synthetic peptides as the Ag (MAb-Ag). A rapid and peptide specific serum IgG response was produced by Day 7 when the synthetic peptides were linked to the N418 MAb, compared to peptide co-delivered with the N418 without linkage. Spleen cells from N418-peptide immunized mice were fused on Day 10, and three IgG1/k monoclonal antibodies (MAbs) were selected to one of the peptide epitopes (MID-peptide). One of the MAbs, Novik 2, bound to two forms of recombinant DC-SIGN protein in enzyme-linked immunosorbent assay (ELISA), and was specifically inhibited by the MID-peptide in solution. Two of these MAbs show specific binding to DC-SIGN expressed by cultured human primary DC. We conclude that in vivo DC targeting enhances the immunogenicity of synthetic peptides and is an effective method for the rapid generation of MAbs to predetermined epitopes.


Subject(s)
Antibodies, Monoclonal/immunology , Dendritic Cells/immunology , Animals , Antibodies, Monoclonal/metabolism , CD11c Antigen/immunology , Cell Adhesion Molecules/analysis , Cell Adhesion Molecules/immunology , Flow Cytometry , Humans , Lectins, C-Type/analysis , Lectins, C-Type/immunology , Mice , Peptides/metabolism , Receptors, Cell Surface/analysis , Receptors, Cell Surface/immunology
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