Anticancer Activity and Mechanisms of Action of MAPK pathway inhibitors

Similarly, genetic fusion to an engineered Fc-domain can endow nanobodies with potent effector functions, e

Similarly, genetic fusion to an engineered Fc-domain can endow nanobodies with potent effector functions, e.g., enhanced complement-dependent and antibody dependent cellular cytotoxicity (CDC, ADCC) (Schutze et al.?2018). the antigen is recognized by the variable domain of the heavy chain (VHH) (A). Monomeric or dimeric (bivalent or biparatopic) nanobodies can be linked to the constant region (Fc) of any isotype to mediate different effector functions (e.g., complement dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC)) (Schriewer et al.?2020) (B). VHHs can also be used as binding domains of chimeric antigen receptors (Nb-CAR) (Hambach et al.?2020) (C). Due to their modular structure, nanobodies can function as building blocks in multimeric constructs binding the same (multivalent) or different (multiparatopic) epitopes. The in vivo half-life of mono or multivalent nanobodies can be tuned, e.g., by genetic fusion to an albumin-specific nanobody (Tijink et al.?2008) (D). Monovalent VHHs can be conjugated chemically to radioisotopes (Huang et al.?2008) or fluorochromes (Fumey et al.?2017) and genetically to toxins (Mutter et al.?2018) and fluorescent proteins (Rothbauer et al.?2006) (E). Examples for nanobodies currently undergoing clinical trials include BCMA nanobody CAR T cells, Vobarilizumab, M1095, and Ozoralizumab, while Caplacizumab has already been FDA/EMA approved (F). scFv single-chain variable fragment, Ab antibody, hcAb heavy-chain antibody, ALB albumin, Nb nanobody, BCMA B cell maturation antigen, MM multiple myeloma, RA rheumatoid arthritis, SLE Systemic Lupus Erythematosus, SCP severe chronic psoriasis, TTP thrombotic thrombocytopenic purpura The molecular basis for the generation of heavy chain antibodies in camelids can be explained by the missing CH1 domain of the heavy chain in two of the IgG-isotypes (Muyldermans et al.?1994) (Fig. ?(Fig.1A).1A). Since the CH1 domain plays a major role in linking heavy and light chains, camelids can produce IgG isotypes GLPG0259 GLPG0259 consisting of only two heavy chains. In this case, antigen recognition is only performed by the variable domain of the GLPG0259 heavy chain. Interestingly, camelids possess two subsets of variable domains that preferentially pair either with heavy chain or with conventional antibodies. The single variable domain of heavy chain antibodies (referred to as VHH or nanobody) is considered to be the smallest naturally occurring antigen-recognizing domain produced by the adaptive OPD1 immune system (Muyldermans?2013). Nanobodies have unique properties. With their long CDR3 regions, they can penetrate into functional cavities of proteins that cannot or only poorly be reached by the comparatively planar paratope of conventional antibodies (De Genst et al.?2006; Jahnichen et al.?2010; Maussang et al.?2013). Since functional clefts on proteins often correspond to the active site of an enzyme or the ligand binding pocket of a receptor, many nanobodies are excellent functional antagonists, an important property rarely attainable with conventional antibodies (Danquah et al.?2016). Nanobodies have a number of other advantages over the variable domains of conventional antibodies, such as higher stability and solubility as well as better tissue penetration, reaching cell surface molecules in almost all major organs within minutes upon i.v. injection (Bannas et al.?2015; Bannas et al.?2014; Cheloha et al.?2020; Ingram et al.?2017; Rashidian et al.?2015). The high stability of VHHs is due to their structure consisting of two ?-folded sheets with a total of 9 ?-strands. The two ?-sheets are connected by a conserved canonical disulfide bridge connecting cysteine residues in framework (FR) 1 and 3. Some VHHs also have a second disulfide bridge linking the CDR3 with the CDR1 (lamas) or the CDR2 (camels) (Muyldermans et al.?1994). Nanobodies are highly soluble and, in contrast to conventional VH domains, do not tend to aggregate due to hydrophilic amino acids in the GLPG0259 FR2. The polarity of these amino acids promotes water solubility and also reduces the stickiness of the VHH domain. The corresponding FR2 region of the VH domain of conventional antibodies contains hydrophobic amino acids that mediate the association with the VL domain of the light chain (Wesolowski et al.?2009). The modular structure of VHHs allows easy conversion into multivalent formats and easy linkage to functional groups or proteins (Fig. ?(Fig.1BCE).1BCE). Importantly, the in vivo half-life of mono or multivalent nanobodies can be tuned, e.g., by genetic fusion to an albumin-specific nanobody (Tijink et al.?2008) (Fig. ?(Fig.1D).1D). Similarly, genetic fusion to an engineered Fc-domain can endow nanobodies with potent effector functions, e.g., enhanced complement-dependent and antibody dependent cellular cytotoxicity (CDC, ADCC) (Schutze et al.?2018). These extraordinary features underline the high potential for the use of nanobodies in different therapeutic.