Exploiting sequence and stability information for directing nanobody stability engineering.

BACKGROUND: Variable domains of camelid heavy-chain antibodies, commonly named nanobodies, have high biotechnological potential. In view of their broad range of applications in research, diagnostics and therapy, engineering their stability is of particular interest. One important aspect is the improvement of thermostability, because it can have immediate effects on conformational stability, protease resistance and aggregation propensity of the protein. METHODS: We analyzed the sequences and thermostabilities of 78 purified nanobody binders. From this data, potentially stabilizing amino acid variations were identified and studied experimentally. RESULTS: Some mutations improved the stability of nanobodies by up to 6.1°C, with an average of 2.3°C across eight modified nanobodies. The stabilizing mechanism involves an improvement of both conformational stability and aggregation behavior, explaining the variable degree of stabilization in individual molecules. In some instances, variations predicted to be stabilizing actually led to thermal destabilization of the proteins. The reasons for this contradiction between prediction and experiment were investigated. CONCLUSIONS: The results reveal a mutational strategy to improve the biophysical behavior of nanobody binders and indicate a species-specificity of nanobody architecture. GENERAL SIGNIFICANCE: This study illustrates the potential and limitations of engineering nanobody thermostability by merging sequence information with stability data, an aspect that is becoming increasingly important with the recent development of high-throughput biophysical methods.

Protein Profiling Gastric Cancer and Neighboring Control Tissues Using High-Content Antibody Microarrays.

In this study, protein profiling was performed on gastric cancer tissue samples in order to identify proteins that could be utilized for an effective diagnosis of this highly heterogeneous disease and as targets for therapeutic approaches. To this end, 16 pairs of postoperative gastric adenocarcinomas and adjacent non-cancerous control tissues were analyzed on microarrays that contain 813 antibodies targeting 724 proteins. Only 17 proteins were found to be differentially regulated, with much fewer molecules than the numbers usually identified in studies comparing tumor to healthy control tissues. Insulin-like growth factor-binding protein 7 (IGFBP7), S100 calcium binding protein A9 (S100A9), interleukin-10 (IL-10) and mucin 6 (MUC6) exhibited the most profound variations. For an evaluation of the proteins' capacity for discriminating gastric cancer, a Receiver Operating Characteristic curve analysis was performed, yielding an accuracy (area under the curve) value of 89.2% for distinguishing tumor from non-tumorous tissue. For confirmation, immunohistological analyses were done on tissue slices prepared from another cohort of patients with gastric cancer. The utility of the 17 marker proteins, and particularly the four molecules with the highest specificity for gastric adenocarcinoma, is discussed for them to act as candidates for diagnosis, even in serum, and targets for therapeutic approaches.

Methods for analyzing and quantifying protein-protein interaction.

Genome sequencing has led to the identification of many proteins, which had not been recognized before. In consequence, the basic set of human proteins is generally known. Far less information, however, exists about protein-protein interactions, which are required and responsible for cellular activities and their control. Many protein isoforms that result from mutations, splice-variations and post-translational modifications also come into play. Until recently, interactions of only few protein partners could be analyzed in a single experiment. However, this does not meet the challenge of investigating the highly complex interaction patterns in cellular systems. It is made even more demanding by the need to determine the intensity of interactions quantitatively in order to properly understand protein interplay. Currently available techniques vary with respect to accuracy, reliability, reproducibility and throughput and their performances range from a mere qualitative demonstration of binding to a quantitative characterization of affinities. In this article, an overview is given of the methodologies available for analysis of protein-protein interactions.

The Global Sequence Signature algorithm unveils a structural network surrounding heavy chain CDR3 loop in Camelidae variable domains.

BACKGROUND: A large fraction of camelid (camels and llamas) antibodies is composed of heavy chain-only homodimers, able to recognise antigens with their variable domain. Events in somatic assembly and maturation of antibodies such as hypermutations and rearrangement of variable loops (CDRs - complementary determining regions) and selection among a wide range of framework variants are generally considered to be random processes. METHODS: An original algorithmic approach (Global Sequence Signature-GSS) was developed, able to take into account multiple functional and/or local sequence properties to detect scattered evolutionary constraints into sequences. RESULTS: Using the GSS approach, we show that the length of the main hypervariable loop (CDR3) is linked to the nature of 19 surrounding residues on the scaffold. Surprisingly, the relation between CDR3 size and scaffold residues strongly depends on the considered species, illustrating either significant differences in selection mechanisms or functional constraints during antibody maturation. CONCLUSIONS: Combined with the statistical coupling analysis (SCA) approach at the level of scaffold residues, this study has unravelled a robust interaction network on antibody structure surrounding the CDR3 loop. GENERAL SIGNIFICANCE: In addition to the general applicability of the GSS algorithm, which can bring together functional and sequence data to locate hot spots of constrained evolution, the relationship between CDR3 and scaffold discussed here should be taken into account in protein engineering when designing antibody libraries.

Optimised 'on demand' protein arraying from DNA by cell free expression with the 'DNA to Protein Array' (DAPA) technology.

We have previously described a protein arraying process based on cell free expression from DNA template arrays (DNA Array to Protein Array, DAPA). Here, we have investigated the influence of different array support coatings (Ni-NTA, Epoxy, 3D-Epoxy and Polyethylene glycol methacrylate (PEGMA)). Their optimal combination yields an increased amount of detected protein and an optimised spot morphology on the resulting protein array compared to the previously published protocol. The specificity of protein capture was improved using a tag-specific capture antibody on a protein repellent surface coating. The conditions for protein expression were optimised to yield the maximum amount of protein or the best detection results using specific monoclonal antibodies or a scaffold binder against the expressed targets. The optimised DAPA system was able to increase by threefold the expression of a representative model protein while conserving recognition by a specific antibody. The amount of expressed protein in DAPA was comparable to those of classically spotted protein arrays. Reaction conditions can be tailored to suit the application of interest. BIOLOGICAL SIGNIFICANCE: DAPA represents a cost effective, easy and convenient way of producing protein arrays on demand. The reported work is expected to facilitate the application of DAPA for personalized medicine and screening purposes.

Ribosome display and screening for protein therapeutics.

Ribosome display is a cell-free display technology which enables in vitro selection of antibodies from large recombinant DNA libraries. It also allows continuous introduction of mutations into the selected DNA pool by PCR-based mutagenesis in each cycle, enabling selection of antibody variants with improved affinity, specificity, and stability, thus providing a powerful "protein evolution" tool for optimizing antibody therapeutics. Ribosome display selects required molecules by linking individual proteins (phenotype) with their corresponding mRNAs (genotype) through the formation of stable Protein-Ribosome-mRNA (PRM) complexes. By affinity interaction with an immobilized ligand, the captured PRM complexes are recovered as cDNA using RT-PCR from the ribosome-attached mRNA. The DNA is then subjected to subsequent ribosome display cycles for further enrichment of rare species or cloning, expression, and sequencing to identify wanted candidates. Both prokaryotic and eukaryotic cell-free systems have been developed for ribosome display of different proteins. In this chapter, we describe ribosome display of antibodies using the eukaryotic rabbit reticulocyte system with an in situ single-primer DNA recovery method. A high-throughput Escherichia coli expression format is also described for screening of individual antibody binders from the ribosome-selected population.

Eukaryotic ribosome display with in situ DNA recovery.

Ribosome display is a cell-free display technology for in vitro selection and optimisation of proteins from large diversified libraries. It operates through the formation of stable protein-ribosome-mRNA (PRM) complexes and selection of ligand-binding proteins, followed by DNA recovery from the selected genetic information. Both prokaryotic and eukaryotic ribosome display systems have been developed. In this chapter, we describe the eukaryotic rabbit reticulocyte method in which a distinct in situ single-primer RT-PCR procedure is used to recover DNA from the selected PRM complexes without the need for prior disruption of the ribosome.

Cell free expression put on the spot: advances in repeatable protein arraying from DNA (DAPA).

We have previously described the 'DNA array to protein array' (DAPA) method for microarraying of proteins expressed by cell-free systems in situ on the array surface. In this technique, a DNA array on one slide acts as the template for generating a protein array on a second slide, mediated by a cell free lysate between the two juxtaposed slides. Here we explore the feature of the repeatability of the technology, in which the same DNA array is reused several times, and use the method to generate a microarray of 116 diverse proteins. The capabilities of DAPA technology in comparison with other protein array methods are discussed.

A single-step procedure of recombinant library construction for the selection of efficiently produced llama VH binders directed against cancer markers.

Heavy chain antibodies are naturally occurring in camelidae (camels and llamas). Their variable domain (VHH) can be efficiently produced as a recombinant protein in E. coli with a large range of applications in the fields of diagnostics and immunotherapy. Standard cloning approach involves resolution of VHH from the heavy chain variable domain of conventional antibodies (VH) by a nested PCR amplification followed by a phage display based selection. Present work illustrates that in contrast to usual finding, specific, good affinity and efficiently expressed VH domain of conventional antibodies can be selected from the co-amplification products of VH and VHH cDNAs. Sequence analysis illustrated that following the two first rounds of selection against cancer markers, similar number of VH and VHH binders were observed. However, after a third round, the more specific binders directed against p53, VEGF, BCL-2 proteins surprisingly contain only VH specific hallmarks. Characterisation of the specificity, affinity and productivity of selected VH binders is described. Because llama VHs show higher sequence and structural homology with the human VH III group than llama VHHs (Vu et al., 1997), they constitute very interesting agents in therapeutic applications, especially in human immunotherapy and cancer treatment.