Humanization of a chicken anti-IL-12 monoclonal antibody

Tsurushita, Naoya; Park, Minha; Pakabunto, Kanokwan; Ong, Kelly; Avdalovic, Anamarija; Fu, Helen; Jia, Audrey; Vásquez, Maximiliano; Kumar, Shankar

doi:10.1016/j.jim.2004.08.018

Cited by 52 publications

(27 citation statements)

References 37 publications

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“…CDR2 supporting Vernier residues 76A, 78I, and 80R were all highly conserved, whereas positions 82N and 87V were substituted more frequently to D (14%) and L (12%), respectively. In the case of 87V, homologous substitution to L may still be of considerable significance because this residue has been found to make CDR contacts and to be a critical position that had to be maintained to retain target-binding affinity during the humanization of two previously described chicken Abs (3,4). In addition, FW 3 V H -V L interface residue 103Y was frequently substituted for F (24%).…”

Section: Analysis Of Naive Diversity In the Fw Regions Of The Chickenmentioning

confidence: 99%

“…A relatively untapped source of such Abs is the immune repertoire of animals whose v-gene germline diversity is restricted, but still highly homologous to human sequences. A number of biotherapeutic research groups have, therefore, developed an interest in an underused option in biotherapeutic drug discovery: high-affinity, potently neutralizing rAbs from chickens (3)(4)(5). This approach exploits the fact that avians are phylogenetically distant from mammals but have theoretically uniform, humanizable v-gene sequences (3,4).…”

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confidence: 99%

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Fundamental Characteristics of the Immunoglobulin VH Repertoire of Chickens in Comparison with Those of Humans, Mice, and Camelids

Oficjalska

Lambert

et al. 2012

The Journal of Immunology

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Examination of 1269 unique naive chicken VH sequences showed that the majority of positions in the framework (FW) regions were maintained as germline, with high mutation rates observed in the CDRs. Many FW mutations could be clearly related to the modulation of CDR structure or the VH–VL interface. CDRs 1 and 2 of the VH exhibited frequent mutation in solvent-exposed positions, but conservation of common structural residues also found in human CDRs at the same positions. In comparison with humans and mice, the chicken CDR3 repertoire was skewed toward longer sequences, was dominated by small amino acids (G/S/A/C/T), and had higher cysteine (chicken, 9.4%; human, 1.6%; and mouse, 0.25%) but lower tyrosine content (chicken, 9.2%; human, 16.8%; and mouse 26.4%). A strong correlation (R2 = 0.97) was observed between increasing CDR3 length and higher cysteine content. This suggests that noncanonical disulfides are strongly favored in chickens, potentially increasing CDR stability and complexity in the topology of the combining site. The probable formation of disulfide bonds between CDR3 and CDR1, FW2, or CDR2 was also observed, as described in camelids. All features of the naive repertoire were fully replicated in the target-selected, phage-displayed repertoire. The isolation of a chicken Fab with four noncanonical cysteines in the VH that exhibits 64 nM (KD) binding affinity for its target proved these constituents to be part of the humoral response, not artifacts. This study supports the hypothesis that disulfide bond-constrained CDR3s are a structural diversification strategy in the restricted germline v-gene repertoire of chickens.

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Section: Analysis Of Naive Diversity In the Fw Regions Of The Chickenmentioning

confidence: 99%

mentioning

confidence: 99%

Fundamental Characteristics of the Immunoglobulin VH Repertoire of Chickens in Comparison with Those of Humans, Mice, and Camelids

Oficjalska

Lambert

et al. 2012

The Journal of Immunology

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“…Chicken V H and V λ regions are most closely related to human V H subgroup III and V λ subgroup III, respectively. These are well-established frameworks for humanization, and have been used previously to humanize mAbs elicited by immunization of chickens [24], [25]. The structure of a CDR is determined not only by the primary sequence of the CDR itself but also by a small number of nearby “Vernier zone" residues that contribute to shaping CDR structure [26].…”

Section: Resultsmentioning

confidence: 99%

Antibody Discovery Ex Vivo Accelerated by the LacO/LacI Regulatory Network

Yabuki

Cummings

Leppard³

et al. 2012

PLoS ONE

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Monoclonal antibodies (mAbs) can be potent and highly specific therapeutics, diagnostics and research reagents. Nonetheless, mAb discovery using current in vivo or in vitro approaches can be costly and time-consuming, with no guarantee of success. We have established a platform for rapid discovery and optimization of mAbs ex vivo. This DTLacO platform derives from a chicken B cell line that has been engineered to enable rapid selection and seamless maturation of high affinity mAbs. We have validated the DTLacO platform by generation of high affinity and specific mAbs to five cell surface targets, the receptor tyrosine kinases VEGFR2 and TIE2, the glycoprotein TROP2, the small TNF receptor family member FN14, and the G protein-coupled receptor FZD10. mAb discovery is rapid and humanization is straightforward, establishing the utility of the DTLacO platform for identification of mAbs for therapeutic and other applications.

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“…26 in addition, the two V-gene germline sequences found in chickens are highly homologous to the human Vλ and V h 3 germline families, both of which are associated with creating V-domains that are highly stable, highly soluble, and display efficiently on phage. 21,27,28 this leads to a more complete sampling of the encoded V-gene repertoire. chicken scFvs can be readily produced in large quantities and are tagged for rapid isolation and detection using highly specific secondary antibodies.…”

Section: Discussionmentioning

confidence: 99%

Developing Cell-Specific Antibodies to Endothelial Progenitor Cells Using Avian Immune Phage Display Technology

et al. 2011

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This study aims at generating immune chicken phage display libraries and single-chain antibodies (scFvs) specifically directed against cell surface markers of cultured peripheral blood mononuclear cells (PBMCs) that contain endothelial progenitor cells (EPCs). In contrast to previous approaches that use well-defined recombinant antigens attached to plastic surfaces that may alter the structure of the proteins, the authors describe a method that maintains the cell surface markers on live cells while providing the opportunity to rapidly screen entire libraries for antibodies that bind to unknown cell surface markers of progenitor/stem cells. Chickens immunized with live EPCs, consisting of a heterogeneous population of lymphocytes and monocytes, demonstrated a robust immune response. After three rounds of biopanning, the authors purified and characterized three unique scFvs called UG1-3. Codon-optimized recombinant UG1 (gUG-1) shows binding by flow cytometry to circulating CD14-positive cells in peripheral blood consistent with predominant expression of a target protein on monocyte subsets. The authors describe the successful use of immunization of chickens for the generation of scFvs against a heterogenous population of EPCs displaying unknown cell surface markers and demonstrate the strong potential of phage display technology in the development of reagents for the isolation and characterization of stem/progenitor cells.

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Humanization of a chicken anti-IL-12 monoclonal antibody

Cited by 52 publications

References 37 publications

Fundamental Characteristics of the Immunoglobulin VH Repertoire of Chickens in Comparison with Those of Humans, Mice, and Camelids

Fundamental Characteristics of the Immunoglobulin VH Repertoire of Chickens in Comparison with Those of Humans, Mice, and Camelids

Antibody Discovery Ex Vivo Accelerated by the LacO/LacI Regulatory Network

Developing Cell-Specific Antibodies to Endothelial Progenitor Cells Using Avian Immune Phage Display Technology

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