Date Published: January 19, 2017
Publisher: Public Library of Science
Author(s): Anna V. Lomonosova, Andrei B. Ulitin, Alexei S. Kazakov, Tajib A. Mirzabekov, Eugene A. Permyakov, Sergei E. Permyakov, Zhaozhong Han.
Small antibody mimetics, or alternative binding proteins (ABPs), extend and complement antibody functionality with numerous applications in research, diagnostics and therapeutics. Given the superiority of ABPs, the last two decades have witnessed development of dozens of alternative protein scaffolds (APSs) for the design of ABPs. Proteins from extremophiles with their high structural stability are especially favorable for APS design. Here, a 10X mutant of the 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii has been probed as an APS. A phage display library of L35Ae 10X was generated by randomization of its three CDR-like loop regions (repertoire size of 2×108). Two L35Ae 10X variants specific to a model target, the hen egg-white lysozyme (HEL), were isolated from the resulting library using phage display. The affinity of these variants (L4 and L7) to HEL ranges from 0.10 μM to 1.6 μM, according to surface plasmon resonance data. While L4 has 1–2 orders of magnitude lower affinity to HEL homologue, bovine α-lactalbumin (BLA), L7 is equally specific to HEL and BLA. The reference L35Ae 10X is non-specific to both HEL and BLA. L4 and L7 are more resistant to denaturation by guanidine hydrochloride compared to the reference L35Ae 10X (mid-transition concentration is higher by 0.1–0.5 M). Chemical crosslinking experiments reveal an increased propensity of L4 and L7 to multimerization. Overall, the CDR-like loop regions of L35Ae 10X represent a proper interface for generation of functional ABPs. Hence, L35Ae is shown to extend the growing family of protein scaffolds dedicated to the design of novel binding proteins.
Development of proteins capable of specific recognition of biological targets has numerous applications in biotechnology, diagnostics, therapy and research [1–13]. Though antibodies are traditionally used for these purposes [10–12], they suffer from several fundamental disadvantages related to their complex architecture (multi-subunit structure and abundance of post-translational modifications), including limited tissue penetration and access to antigen grooves, need for use of expensive eukaryotic expression systems, and the complicated process of their structural characterization. Antibody alternatives, such as small antibody mimetics, alternative binding proteins (ABPs), based on immunoglobulin-like or non-immunoglobulin folds (‘alternative protein scaffolds’, APSs) have the potential to address these shortcomings [1–9, 13]. An APS possesses a compact stable backbone supporting the target-binding regions, which are genetically randomized to provide a wide repertoire (105−1013) of variants with retained structural stability. The resulting combinatorial library serves as a source of proteins specific to a target of choice for in vitro display technologies, which give rise to ABPs possessing antibody-like specificity and selectivity to the target [1–9, 13]. The lower structural complexity of ABPs (single subunit structure and minimal post-translational modifications) enables the use of bacterial expression systems, providing higher protein yields and lower production costs, and facilitates their structural characterization. Furthermore, the smaller sizes of ABPs provide efficient tissue penetration, facilitate access to antigen grooves and clefts [13, 14], and promote more selective site blocking in extended targets. The greatly limited serum half-life of ABPs is favorable for tumor imaging and can be extended for therapeutic use by fusion of ABPs with high molecular weight compounds or other half-life increasing entities [7, 8]. ABPs fused with Fc domain attain natural effector functions of antibodies . Finally, ABPs are advantageous for design of multivalent or multispecific molecules [7, 8]. The properties of ABPs, which bridge those of antibodies and low molecular weight drugs/substances, and the ease of modifying ABPs to various applications, guarantee their growing use in resolution of critical problems in biotechnology, medicine and research.
The 10X mutant of 50S ribosomal protein L35Ae of the hyperthermophilic archaea, P. horikoshii, has previously been shown to possess favorable features for protein scaffolding use . Its unique six-stranded β-barrel motif is not found in existing scaffold proteins, and contains a nearly flat surface with CDR-like loops 1–3, which have target recognition potential (Fig 2). L35Ae 10X’s ability to serve as a framework for alternative binding proteins development was evaluated here via construction of a M13 phage display library of its variants, followed by isolation and characterization of the variants specific to HEL, a conventional model antigen. The residues of L35Ae 10X subjected to randomization form a double ‘paratopic’ region including two clusters of the residues provided by the CDR-like loop 1 and residues K74-G75 of loop 3, and loop 2 and residues G78-A80/D83 of loop 3, which are separated by residues L76-P77 (the model is based on tertiary structure of L35Ae from P. furiosus–see Fig 2). The analysis of residue conservation for archaeal L35Ae proteins (S1 Fig) shows low conservation of the loops 1 and 2 along with high conservation of the loop 3, which constrains its randomization. On the contrary, loop 2 resists elongation for up to three residues (S1 Fig), which enabled us to design a 10X(b) variant of L35Ae 10X with loop 2 elongated by three residues (Fig 1A). Overall, 20 or 24 residues of the loop regions 1–3 were subjected to randomization (Fig 1, S1 and S2 Figs) using mostly a degenerate NNK codon (see Table 1), covering all 20 amino acids . Apparently, theoretical limit of diversity of such library (up to 1031) greatly exceeds the limitations imposed by the use of phage display technology (library size up to 1012 ). Diversity of the constructed phage display library of L35Ae 10X reaches 2×108, which corresponds to only 10−23 of the theoretical limit. The L35Ae 10X library’s ability to provide binders for a model antigen with Kd values down to 0.1 μM (Table 3) despite sparse sampling of the sequence space is promising. The low coverage of the sequence space could explain low selectivity of L4 and L7 variants to HEL, manifested as their marked affinity to a homologous milk protein, BLA (Fig 6 and Table 3). The observed specificity of L4 and L7 to BLA is unexpected, given that the library was pre-depleted against BLA-containing MPBS and that incubations of HEL with the library during selections were performed in presence of MPBST. This contradiction could be resolved assuming that binding of skimmed milk components to BLA suppresses its affinity to the L35Ae 10X variants. L4 and L7 ‘s cross-reactivity with BLA could explain the high-background ELISA signals observed during examinations of specificity to HEL of the polyclonal phage particles after the selection rounds (Fig 4). Meanwhile, L4 or L7 variants expectedly do not recognize another major component of skimmed milk, BSA. The lack of affinity of reference protein (L35Ae 10X-myc) for HEL, BLA and BSA is an evidence that L35Ae 10X variants specificity and selectivity for HEL/BLA arose due to biopanning.
The first trial of extremophilic L35Ae 10X protein and its unique six-stranded β-barrel motif not found in the established scaffold proteins as a source of novel binding proteins is performed here using HEL as a model target. The random mutagenesis of three nearby CDR-like loop regions of the protein, followed by phage display isolation of HEL-specific L35Ae 10X variants, have shown that L35Ae’s structure successfully resists randomization in these protein regions and provides the variants with submicromolar to micromolar affinity to HEL, and marked cross-reactivity with its close homologue, BLA. These results are superior to those reported in a pioneer trial of 10Fn3, which is currently among the most advanced scaffold proteins (Adnectins™) [37, 38]. Meanwhile, properties of the HEL-specific L35Ae 10X binders are overall inferior to properties of target-specific binders isolated from combinatorial libraries of Sso7d and Sac7d proteins from extremophilic archaea [25–28] (Table 4). Negative features of the HEL-specific L35Ae binders, such as relatively low affinity and selectivity to HEL, relatively low resistance to GuHCl, and increased propensity to multimerization, could be suppressed by further optimization, including use of L35Ae proteins with initially superior properties, combining loop randomization with ‘flat surface’ randomization, use of more focused and more diverse protein libraries. Therapeutic use of L35Ae-based proteins will require addressing the issue of their potential immunogenicity; usage of L35Ae-based binding proteins for research and diagnostics represents a more straightforward path for potential practical application. In summary, though further refinement would be needed for application to practical use, this work shows that L35Ae 10X is a viable scaffold protein ripe for further development.