IMMUNOMODULATION OF CANCER CELL MIGRATION AND INVASION USING RECOMBINANT CAMELID NANOBODIES AGAINST CYTOSKELETAL PROTEINS

Invasion and metastasis of cancer cells relies on dynamic reorganization of the actin cytoskeleton which is the driving force for cellular motility. Actin-associated or actin-binding proteins aid in this process by virtue of their ability to interact reversibly with actin and actin filaments. The expression level of quite a few actin binding proteins is up-regulated in cancer cells, and many studies have demonstrated a correlation between the expression of selected actin-binding proteins and cancer cell motility and/or invasion in vitro and in vivo. Instead of modulating the expression level of actin-associated proteins, we developed small recombinant antibodies (so-called nanobodies) raised against cell motility factors and use these as intrabodies. Obviously these antibodies do not affect the expression of their target antigen but inhibit biological functions. Nanobodies ( similar to 15 kDa) against gelsolin and other actin-associated proteins were raised in llamas or alpacas which have the unique property of expressing heavy chain antibodies that are devoid of light chains. The antigen-binding region of these antibodies (VHH) can be cloned, thus raising the possibility of developing small protein inhibitors against structural intracellular polypeptides. In addition, nanobodies can be used for tracing their target in living cells, thereby circumventing protein overexpression. They are also suitable for immunoprecipitation and Western blotting. We show that selected nanobodies bind their target with nanomolar affinity. Moreover, epitope mapping demonstrates their selective interaction with conformational epitopes (i.e. calcium-induced conformational changes in proteins). Examples are discussed showing inhibition of biochemical activities of L-plastin, an actin-bundling protein, by nanobodies. Moreover, this was associated with inhibition of filopodia formation in PC-3 cells. Importantly, expression of nanobodies against L-plastin or gelsolin in cancer cells reduced motility and in vitro invasion of these cells to the same extent as siRNA. This approach allows one to study the role of (actin-associated) proteins in tumorigenesis at the level of the protein. Nanobodies could be further developed into bona fide therapeutic molecules.