HuCAL PLATINUM, a Synthetic Fab Library Optimized for Sequence Diversity and Superior Performance in Mammalian Expression Systems

Prassler, Josef; Thiel, Stefanie; Pracht, Catrin; Polzer, Andrea; Peters, Solveig; Bauer, Marion; Nörenberg, Stephanie; Stark, Yvonne; Kölln, Johanna; Popp, Andreas; Urlinger, Stefanie; Enzelberger, Markus

doi:10.1016/j.jmb.2011.08.012

Cited by 144 publications

(145 citation statements)

References 51 publications

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“…Analysis of the amino acid distribution of antibodies from the human repertoire have revealed that tyrosine, glycine and serine are overrepresented in complementarity-determining regions (CDRs), 18 with tyrosine alone composing 10% of, and contributing up to 25% of, the antigen contact surface. [19][20][21] Based on this statistical consideration, designed synthetic antibody libraries composed of a single framework 19 or multiple frameworks 22 often have a high frequency of tyrosines in the CDRs. While these tyrosines can facilitate antigen recognition, we have observed that they can also increase the instability of scFvs by providing a pleiotropic interaction surface for aggregate nucleation.…”

Section: Introductionmentioning

confidence: 99%

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Zhang

Geddie

Kohli

et al. 2015

mAbs

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Antibody-targeted nanoparticles have the potential to significantly increase the therapeutic index of cytotoxic anticancer therapies by directing them to tumor cells. Using antibodies or their fragments requires careful engineering because multiple parameters, including affinity, internalization rate and stability, all need to be optimized. Here, we present a case study of the iterative engineering of a single chain variable fragment (scFv) for use as a targeting arm of a liposomal cytotoxic nanoparticle. We describe the effect of the orientation of variable domains, the length and composition of the interdomain protein linker that connects VH and VL, and stabilizing mutations in both the framework and complementarity-determining regions (CDRs) on the molecular properties of the scFv. We show that variable domain orientation can alter cross-reactivity to murine antigen while maintaining affinity to the human antigen. We demonstrate that tyrosine residues in the CDRs make diverse contributions to the binding affinity and biophysical properties, and that replacement of non-essential tyrosines can improve the stability and bioactivity of the scFv. Our studies demonstrate that a comprehensive engineering strategy may be required to identify a scFv with optimal characteristics for nanoparticle targeting.

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Section: Introductionmentioning

confidence: 99%

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Zhang

Geddie

Kohli

et al. 2015

mAbs

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show abstract

“…10,11 Phage display selection of large antibody repertoires routinely results in a high number of hits recognizing the target of interest. 12,13 Thus, the identification of a lead candidate with the greatest possibility of success in clinical development requires extensive screening, as well as functional 14 and physico-chemical characterization. 15 Testing for binding specificity is typically done at multiple stages of the discovery process.…”

Section: Resultsmentioning

confidence: 99%

“…mAb, IgG1 format) was selected from the HuCAL GOLD ® library by using lysozyme as a target. Other applied antibodies were obtained from the HuCAL PLATINUM ® library by phage display technology 13 via selection on the respective targets.…”

Section: Methodsmentioning

confidence: 99%

An automated immunoassay for early specificity profiling of antibodies

Frese

Eisenmann²,

Ostendorp³

et al. 2013

mAbs

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“…The diversity and quality of such an antibody gene library is of critical importance for success and the correlation between the number and affinity of antibodies that can be isolated and the library size has been demonstrated [5]. Thus, there has been significant effort to assemble very large antibody libraries, i.e., comprising between 10 8 and >10 11 members, so that, in principle, antibodies against any antigen can be identified [6][7][8].…”

Section: Introductionmentioning

confidence: 99%

Purpose-Oriented Antibody Libraries Incorporating Tailored CDR3 Sequences

et al. 2015

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Abstract:The development of in vitro antibody selection technologies has allowed overcoming some limitations inherent to the hybridoma technology. In most cases, large repertoires of antibody genes have been assembled to create highly diversified libraries allowing the isolation of antibodies recognizing virtually any antigen. However, these universal libraries might not allow the isolation of antibodies with specific structural properties or particular amino acid contents that are rarely found in natural repertoires. Purpose-oriented libraries specially designed to incorporate desired characteristics have been successfully used. However, the workload required for library construction has limited the attractiveness of this approach compared to the use of large universal libraries. We have developed an approach to capture synthetic or natural diversity into the complementarity determining regions 3 (CDR3) of human antibody repertoires using Type IIS restriction enzymes. In this way, we generated several libraries either biased in amino acid content or towards long CDRH3 loops. The latter were successfully used to identify antibodies inhibiting the enzymatic activity of horseradish peroxidase, whereas libraries enriched in histidines allowed for the isolation of antibodies binding to human Fc in a pH-dependent manner. These libraries indicate that tailored diversification of CDR3 is sufficient to generate purpose-oriented libraries and isolate antibodies with uncommon properties. OPEN ACCESSAntibodies 2015, 4 104

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HuCAL PLATINUM, a Synthetic Fab Library Optimized for Sequence Diversity and Superior Performance in Mammalian Expression Systems

Cited by 144 publications

References 51 publications

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

Comprehensive optimization of a single-chain variable domain antibody fragment as a targeting ligand for a cytotoxic nanoparticle

An automated immunoassay for early specificity profiling of antibodies

Purpose-Oriented Antibody Libraries Incorporating Tailored CDR3 Sequences

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