Antigen-independent selection of intracellular stable antibody frameworks

Maur, Adrian Auf der; Tissot, Kathrin; Barberis, Alcide

doi:10.1016/j.ymeth.2004.04.004

Cited by 35 publications

(28 citation statements)

References 22 publications

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“…Framework Selection-A pool of 88-well folding and stable scFv antibodies previously isolated from a library generated by amplification and random combination of human VH and VL domains from a naive human spleen cDNA (22) was analyzed to select a suitable acceptor framework for rabbit CDR grafting. Human scFv sequences were ranked as follows: (a) according to expression level of the respective clone in yeast, and (b) according to homology to rabbit consensus at core residues.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Generic Approach for the Generation of Stable Humanized Single-chain Fv Fragments from Rabbit Monoclonal Antibodies

Borras¹,

Gunde²,

Tietz³

et al. 2010

Journal of Biological Chemistry

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Despite their favorable pharmacokinetic properties, singlechain Fv antibody fragments (scFvs) are not commonly used as therapeutics, mainly due to generally low stabilities and poor production yields. In this work, we describe the identification and optimization of a human scFv scaffold, termed FW1.4, which is suitable for humanization and stabilization of a broad variety of rabbit antibody variable domains. A motif consisting of five structurally relevant framework residues that are highly conserved in rabbit variable domains was introduced into FW1.4 to generate a generically applicable scFv scaffold, termed FW1.4gen. Grafting of complementarity determining regions (CDRs) from 15 different rabbit monoclonal antibodies onto FW1.4 and their derivatives resulted in humanized scFvs with binding affinities in the range from 4.7 ؋ 10 ؊9 to 1.5 ؋ 10Interestingly, minimalistic grafting of CDRs onto FW1.4gen, without any substitutions in the framework regions, resulted in affinities ranging from 5.7 ؋ 10 ؊10 to <1.8 ؋ 10 ؊12 M. When compared with progenitor rabbit scFvs, affinities of most humanized scFvs were similar. Moreover, in contrast to progenitor scFvs, which were difficult to produce, biophysical properties of the humanized scFvs were significantly improved, as exemplified by generally good production yields in a generic refolding process and by apparent melting temperatures between 53 and 86°C. Thus, minimalistic grafting of rabbit CDRs on the FW1.4gen scaffold presents a simple and reproducible approach to humanize and stabilize rabbit variable domains.Because of their favorable pharmacokinetic properties, single-chain Fv (scFv) 3 antibody fragments represent an attractive format for therapeutic applications (1, 2). scFvs are often derived from monoclonal antibodies isolated from animal or human lymphocytes. As an alternative to hybridoma screening, in vitro display technologies, e.g. phage and ribosome display, enable the selection of high affinity-binding variable domains from natural or synthetic genetic libraries. Despite the successful use of in vitro randomization and selection systems, generation of antibodies by immunization and subsequent screening of full-size antibodies (e.g. hybridoma supernatants) includes conceptual advantages. For example, in contrast to in vitro display systems, in vivo methods are less prone to preferential selection of well expressed clones, which in many cases results in loss of potentially interesting antibodies. Moreover, in vivo methods are preferred in particular for addressing complex antigens, such as integral membrane proteins that are notoriously difficult to purify. However, reducing a full-length monoclonal antibody to the scFv format frequently is challenging particularly due to solubility and stability problems, which often impair expression and purification. Therefore, technologies to humanize and stabilize the scFv format following isolation of a monoclonal antibody remain critical for the generation of scFv therapeutics. Numerous approaches have been describ...

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Section: Methodsmentioning

confidence: 99%

“…This scaffold was previously identified from a human library using a whole genome screening approach (22). We further identified a motif consisting of five rationally altered framework positions, which, when introduced into the human acceptor scaffold, improved protein stability and supported functional presentation of rabbit CDRs.…”

mentioning

confidence: 99%

Generic Approach for the Generation of Stable Humanized Single-chain Fv Fragments from Rabbit Monoclonal Antibodies

Borras¹,

Gunde²,

Tietz³

et al. 2010

Journal of Biological Chemistry

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“…The absence of activity of intracellular aP6 (Fig. 2D), best explained by its intracellular instability (Auf der Maur et al, 2004;Strube and Chen, 2004), precludes that small amounts of SaP6 escaping the secretion pathway could be at the origin of the observed phenotype. The importance of proper antibody folding was further demonstrated by the absence of activity of the SaP6-CS in which a cystein was modified into a serine, thus preventing the formation of disulfide bounds.…”

Section: Extracellular Pax6 Neutralization Prevents Opc Dispersalmentioning

confidence: 99%

Paracrine Pax6 activity regulates oligodendrocyte precursor cell migration in the chick embryonic neural tube

et al. 2011

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Homeoprotein transcription factors play fundamental roles in development, ranging from embryonic polarity to cell differentiation and migration. Research in recent years has underscored the physiological importance of homeoprotein intercellular transfer in eye field development, axon guidance and retino-tectal patterning, and visual cortex plasticity. Here, we have used the embryonic chick neural tube to investigate a possible role for homeoprotein Pax6 transfer in oligodendrocyte precursor cell (OPC) migration. We report the extracellular expression of Pax6 and the effects of gain and loss of extracellular Pax6 activity on OPCs. Open book cultures with recombinant Pax6 protein or Pax6 blocking antibodies, as well as in ovo gene transfer experiments involving expression of secreted Pax6 protein or secreted Pax6 antibodies, provide converging evidences that OPC migration is promoted by extracellular Pax6. The paracrine effect of Pax6 on OPC migration is thus a new example of direct non-cell autonomous homeoprotein activity.

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“…23 The stability of scFvs is sufficient in less than 1% of the cases for use as a high quality intrabody. 24,25 Further, every functional cytoplasmic intrabody needs to be capable of actively inhibiting the target function upon binding, a requirement that makes its selection much more tedious. Despite that, quite a number of studies of cytoplasmic proteins that were successfully targeted and function was affected, [26][27][28][29] or were visualized through expression of a scFv-green fluorescent protein (GFP) fusion protein, have been reported.…”

Section: O N O T D I S T R I B U T Ementioning

confidence: 99%