Humanization of a phosphothreonine peptide-specific chicken antibody by combinatorial library optimization of the phosphoepitope-binding motif

Baek, Du-San; Kim, Yong‐Sung

doi:10.1016/j.bbrc.2015.05.086

Cited by 21 publications

(27 citation statements)

References 25 publications

(59 reference statements)

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“…The immunogenic potential of these structures in humans is unknown at present, and as such may pose a challenge for humanization, although examples of humanizing these types of antibodies have been recently published 26, 27 . Replacing the cysteines via mutagenesis may have an unpredictable effect on the antibodies' affinity, binding mode or may even disrupt antigen recognition altogether.…”

Section: Discussionmentioning

confidence: 99%

Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor

Könitzer

Pramanick²,

Pan

et al. 2017

mAbs

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Raising functional antibodies against G protein-coupled receptors (GPCRs) is challenging due to their low density expression, instability in the absence of the cell membrane's lipid bilayer and frequently short extracellular domains that can serve as antigens. In addition, a particular therapeutic concept may require an antibody to not just bind the receptor, but also act as a functional receptor agonist or antagonist. Antagonizing the glucose-dependent insulinotropic polypeptide (GIP) receptor may open up new therapeutic modalities in the treatment of diabetes and obesity. As such, a panel of monoclonal antagonistic antibodies would be a useful tool for in vitro and in vivo proof of concept studies. The receptor is highly conserved between rodents and humans, which has contributed to previous mouse and rat immunization campaigns generating very few usable antibodies. Switching the immunization host to chicken, which is phylogenetically distant from mammals, enabled the generation of a large and diverse panel of monoclonal antibodies containing 172 unique sequences. Three-quarters of all chicken-derived antibodies were functional antagonists, exhibited high-affinities to the receptor extracellular domain and sampled a broad epitope repertoire. For difficult targets, including GPCRs such as GIPR, chickens are emerging as valuable immunization hosts for therapeutic antibody discovery.

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Section: Discussionmentioning

confidence: 99%

Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor

Könitzer

Pramanick²,

Pan

et al. 2017

mAbs

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“…In only one case was a framework back mutation necessary to include during humanization (V L L46T, anti-pTau). Interestingly, a classical humanization study performed very recently (by another group) on the anti-pTau antibody also sampled the L46 position and found it to be a necessary back mutation to humanize this antibody (54). Importantly, however, four other back mutations in multiple FW regions were also found to be necessary.…”

Section: Discussionmentioning

confidence: 99%

Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies

Townsend

Fennell

Apgar

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.antibody | paratope | plasticity | humanization | immunogenicity

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“…Yeast strains and media composition have been previously described254849. The synthetic human HC library displayed in the format of VH library-CH1 on the surface of Saccharomyces cerevisiae JAR200 haploid cells with mating type a (MATa) was used2549.…”

Section: Methodsmentioning

confidence: 99%

“…The synthetic human HC library displayed in the format of VH library-CH1 on the surface of Saccharomyces cerevisiae JAR200 haploid cells with mating type a (MATa) was used2549. For HC haploid library screening against KRas G12D ·GppNHp, one round of MACS and then one round of FACS were sequentially performed using 1 and 0.5 μM of biotinylated KRas G12D ·GppNHp in the presence of 10-fold excess molar concentrations of non-biotinylated KRas G12D ·GDP as a competitor254748. After the first round of FACS, the enriched HC yeast haploid library cells were mated with the other mating type of S. cerevisiae YVH10 cells (MATα) that secreted a fixed LC with the VL-CL (LC constant domain) of TMab4 to generate an enriched Fab library on the diploid yeast cells, as illustrated in Fig.…”

Section: Methodsmentioning

confidence: 99%

Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

et al. 2017

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Oncogenic Ras mutants, frequently detected in human cancers, are high-priority anticancer drug targets. However, direct inhibition of oncogenic Ras mutants with small molecules has been extremely challenging. Here we report the development of a human IgG1 format antibody, RT11, which internalizes into the cytosol of living cells and selectively binds to the activated GTP-bound form of various oncogenic Ras mutants to block the interactions with effector proteins, thereby suppressing downstream signalling and exerting anti-proliferative effects in a variety of tumour cells harbouring oncogenic Ras mutants. When systemically administered, an RT11 variant with an additional tumour-associated integrin binding moiety for tumour tissue targeting significantly inhibits the in vivo growth of oncogenic Ras-mutated tumour xenografts in mice, but not wild-type Ras-harbouring tumours. Our results demonstrate the feasibility of developing therapeutic antibodies for direct targeting of cytosolic proteins that are inaccessible using current antibody technology.

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Humanization of a phosphothreonine peptide-specific chicken antibody by combinatorial library optimization of the phosphoepitope-binding motif

Cited by 21 publications

References 25 publications

Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor

Generation of a highly diverse panel of antagonistic chicken monoclonal antibodies against the GIP receptor

Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies

Antibody targeting intracellular oncogenic Ras mutants exerts anti-tumour effects after systemic administration

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