Single Domain Intracellular Antibodies from Diverse Libraries

Abstract

Interfering intracellular antibodies are valuable for biological studies as drug surrogates and as potential macromolecular drugs per se. Their application is still limited because of the difficulty of acquisition of functional intracellular antibodies. We describe the use of the new intracellular antibody capture procedure (IAC 3 ) to facilitate direct isolation of functional single domain antibody fragments using four independent target molecules (LMO2, TP53, CRAF1, and Hoxa9) from a set of diverse libraries. Initially, these have variability in only one of the three antigen-binding CDR regions of VH or VL and first round single domains are affinity matured by iterative randomization of the two other CDRs and reselection. We highlight the approach using a single domain binding to LMO2 protein. Our results show that interfering with LMO2 protein function demonstrates a role specifically in erythroid differentiation, confirm a necessary and sufficient function for LMO2 as a cancer therapy target in T-cell neoplasia and allowed for the first time production of soluble recombinant LMO2 protein by co-expression with intracellular domain antibodies. Cocrystallization of LMO2 and the anti-LMO2 VH protein was successful. These results demonstrate that this third generation IAC 3 offers a robust toolbox for various biomedical applications and consolidates functional features of the LMO2 protein complex, which includes the importance of Lmo2-Ldb1 protein interaction.Antibodies and derivative fragments are molecules that can capture a huge variety of antigens with high specificity and affinity and are indispensable tools and reagents in bioscience and in medicine (1). The utilities of intracellular application of antibody fragments is that, unlike gene knock-out and interfering RNAs or antisense, these molecules interfere with cellular functions directly by binding to target proteins and can therefore target specific properties of proteins such as particular signal transduction via blockading protein-protein interaction (2, 3). This not only provides valuable and robust tools in biological analysis such as functional genomics and proteomics but also serve as drug surrogates to establish the target validity for drug development by ascertaining the biological effects from targeting the protein. To make these options possible on a broad basis, robust screening methods with high quality libraries are needed to allow selection of target-specific intracellular antibodies.Antibody binding sites comprise heavy chain variable domain (VH) 4 and light chain variable domains (VL), each with three complementarity determining regions (CDRs), directly contacting antigen and concerned in binding affinity and specificity. Single domain antibody fragments (DAbs), comprising either VH or VL, encompass a minimal antigen recognition can also be used for intracellular applications, so-called intracellular domain antibodies (iDAbs) (4). Structural analysis of V regions based on a consensus framework sequence have shown that essentially no diff...

Conformational flexibility of the oncogenic protein LMO2 primes the formation of the multi-protein transcription complex

“…All our previous attempts to produce recombinant LMO2 protein have proved unsuccessful due to the intransigent insolubility of the protein in induction systems (unpublished). However, we found that co-expressing LMO2 with the anti-LMO2 single domain resulted in efficient production of soluble protein that was readily purified and crystallized in the dimeric state35. This has two main implications.…”

Engineered antibody therapies to counteract mutant huntingtin and related toxic intracellular proteins

“…Camelid VHH intrabodies have a higher probability of correct intracellular folding, although they do contain cysteines that can form disulfide bonds (Colby et al ., 2004b; Saerens et al ., 2005). The method that is designed to directly select for stable intrabodies in a high-throughput manner is intracellular antibody capture, which uses an in situ two-hybrid screen to select for antigen-scFv interactions within the cytoplasm of yeast (Auf der Maur et al ., 2001; Colby et al ., 2004c; Feldhaus et al ., 2003; Tanaka et al ., 2011; Visintin et al ., 1999). Alternatively, the antigen-binding complimentary determining regions of an unstable scFv can theoretically be “loop- grafted” into several well-characterized intrabody consensus scaffolds that are reported to exhibit improved intracellular folding and stability (Ewert et al ., 2004).…”

“…However, initial screening generally reveals valuable lead candidate intrabodies for therapeutic or mechanistic studies that can be further characterized in situ (Kvam et al ., 2010). Another method that is designed to directly select for intrabodies that recognize intracellular conformers of target proteins in context is intracellular antibody capture, which uses an in situ two-hybrid screen to select for antigen-scFv interactions within the cytoplasm of yeast or within mammalian cells (Auf der Maur et al ., 2001; Feldhaus et al ., 2003; Tanaka et al ., 2011; Visintin et al ., 1999). …”

Biotechnological applications of recombinant single-domain antibody fragments

“…The stability and functionality of independent VHs is maintained also when they are expressed as intrabodies [32,33]. …”