Specificin vivoknockdown of protein function by intrabodies

Abstract

Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed.

Intrabodies against the Polysialyltransferases ST8SiaII and ST8SiaIV inhibit Polysialylation of NCAM in rhabdomyosarcoma tumor cells

“…For inhibition of the function of transitory molecules IBs are targeted to the ER where they bind to their antigens very specifically [39]. Consequently, the translocation of the antigen to the final cell compartment is blocked.…”

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

“…This method is particularly suited for efficient engineering of antibodies designed to function in the intracellular environment. These intracellular antibodies are already being explored as potential therapeutics in a number of fields, including neurodegenerative diseases, cancer, and viral infections 31 . This technique will enable more widespread use of intracellular antibodies as tools for research and medicine in these fields and any other field where studying a protein target in situ is desired.…”

Single domain antibodies for the knockdown of cytosolic and nuclear proteins

“…In addition, stable human VH and VL domains can be constructed from human antibodies after framework mutations and randomization of CDRs . Camelid nanobodies can be stably expressed inside the cytoplasm even if the disulfide bridge is not formed . Folded nanobodies can find their antigen and are then stabilized by binding to the antigen.…”

“…ER‐intrabody knockdown function is based on retention of molecules passing the secretory pathway via the KDEL sequence fused to the C‐terminal end of the intrabody . In the past ER‐intrabodies have been the most promising molecules because they are correctly folded in the ER environment .…”