Integrin binding human antibody constant domains—Probing the C-terminal structural loops for grafting the RGD motif

Protein Engineering, Design and Selection

Wozniak‐Knopp

et al. 2013

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Antigen-binding Fc fragments (Fcab) are generated by engineering the C-terminal loop regions in the CH3 domain of human immunoglobulin G class 1-crystallizable fragment (IgG1-Fc). For an optimum library design with high percentage of well-folded clones for efficient binder selection, information about the correlation between primary structure and stability is needed. Here, we present a rapid method that allows determination of the overall stability of whole libraries of IgG1-Fc on the surface of yeast by flow cytometry. Libraries of IgG1-Fc mutants with distinct regions in AB-, CD- and EF-loops of the CH3 domains randomized or carrying therein insertions of five additional residues were constructed, incubated at increasing temperatures and probed for residual binding of generic Fc ligands. Calculated temperatures of half-maximal irreversible denaturation of the libraries gave a clear hierarchy of tolerance to randomization of distinct loop positions. Experimental data were evaluated by a computational approach and are discussed with respect to the structure of IgG1-Fc and variation in sequence and length of these loops in homologous Fc proteins. Generally, the described method allows for quick assessment of the effects of randomization of distinct regions on the foldability and stability of a yeast-displayed protein library.

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Stability assessment on a library scale: a rapid method for the evaluation of the commutability and insertion of residues in C-terminal loops of the CH3 domains of IgG1-Fc

Hasenhindl

Protein Engineering, Design and Selection

Wozniak‐Knopp

et al. 2013

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“…For example, IgG1-Fc has been engineered for binding to the tumor antigen Her2/neu or to αvβ3 integrin by mutating the C-terminal structural loops of the CH3 domains 37,38 . However, changing these loop sequences resulted in decreased thermal stability of the CH3 domains 37 . One of the critical factors determining the fitness of a randomly mutated library is the part of the protein that is chosen for randomization.…”

Section: Resultsmentioning

confidence: 99%

Construction of a Stability Landscape of the CH3 Domain of Human IgG1 by Combining Directed Evolution with High Throughput Sequencing

Journal of Molecular Biology

Hasenhindl

Hackl

et al. 2012

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One of the most important but still poorly understood issues in protein chemistry is the relationship between sequence and stability of proteins. Here, we present a method for analyzing the influence of each individual residue on the foldability and stability of an entire protein. A randomly mutated library of the crystallizable fragment of human immunoglobulin G class 1 (IgG1-Fc) was expressed on the surface of yeast, followed by heat incubation at 79 °C and selection of stable variants that still bound to structurally specific ligands. High throughput sequencing allowed comparison of the mutation rate between the starting and selected library pools, enabling the generation of a stability landscape for the entire CH3 domain of human IgG1 at single residue resolution. Its quality was analyzed with respect to (i) the structure of IgG1-Fc, (ii) evolutionarily conserved positions and (iii) in silico calculations of the energy of unfolding of all variants in comparison with the wild-type protein. In addition, this new experimental approach allowed the assignment of functional epitopes of structurally specific ligands used for selection [Fc γ‐receptor I (CD64) and anti-human CH2 domain antibody] to distinct binding regions in the CH2 domain.

“…An affinity matured variant was shown to trigger ADCC in vitro and the in vivo half life in mice was comparable to wild-type Fc (Fc-wt), demonstrating the possibility to generate antigen binding Fc proteins (Fcab, antigen binding Fc) with all antibody properties at only one third of the size (~ 50 kDa) of a whole IgG1 molecule, which could be an advantage regarding tissue penetration. In another work the integrin-binding motive “GCRGDCL” was incorporated into the C-terminal structural loops of the CH3-domains of IgG1-Fc [6]. Integrin binding Fcabs could be expressed and purified and all variants showed wild-type like secondary and tertiary structures and still bound to CD16, FcRn and Protein A.…”

Section: Introductionmentioning

confidence: 99%

Directed evolution of stabilized IgG1-Fc scaffolds by application of strong heat shock to libraries displayed on yeast

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Faissner

Stadlmayr

et al. 2012

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We have constructed IgG1-Fc scaffolds with increased thermal stability by directed evolution and yeast surface display. As a basis a new selection strategy that allowed the application of yeast surface display for screening of stabilizing mutations in proteins of already high intrinsic thermal stability and Tm-values up to 85 °C was developed. Besides library construction by error prone PCR, strong heat stress at 79 °C for 10 min and screening for well-folded proteins by FACS, sorting rounds had to include an efficient plasmid DNA isolation step for amplification and further transfection. We describe the successful application of this experimental setup for selection of 17 single, double and triple IgG1-Fc variants of increased thermal stability after four selection rounds. The recombinantly produced homodimeric proteins showed a wild-type-like elution profile in size exclusion chromatography as well as content of secondary structures. Moreover, the kinetics of binding of FcRn, CD16a and Protein A to the engineered Fc-molecules was very similar to the wild-type protein. These data clearly demonstrate the importance and efficacy of the presented strategy for selection of stabilizing mutations in proteins of high intrinsic stability within reasonable time.