Advances in nanobody multimerization and multispecificity: from in vivo assembly to in vitro production

Abstract

NANOBODIES® (Nbs) have emerged as valuable tools across therapeutic, diagnostic, and industrial applications owing to their small size and consequent ability to bind unique epitopes inaccessible to conventional antibodies. While Nbs retrieved from immune libraries normally possess sufficient affinity and specificity for their cognate antigens in the practical use case, their multimerization will often increase functional affinity via avidity effects. Therefore, to rescue binding affinity and broaden targeting specificities, recent efforts have focused on conjugating multiple Nb clones - of identical or unique antigen cognates - together. In vivo and in vitro approaches, including flexible linkers, antibody domains, self-assembling coiled coils, chemical conjugation, and self-clustering hydrophobic sequences, have been employed to produce multivalent and multispecific Nb constructs. Examples of successful Nb multimerization are diverse, ranging from immunoassaying reagents to virus-neutralizing moieties. This review aims to recapitulate the in vivo and in vitro modalities to produce multivalent and multispecific Nbs while highlighting the applications, advantages, and drawbacks tied to each method.

Keywords: NANOBODIES®; affinity; antibody domains; avidity; coiled coils; conjugation; diagnostics; hydrophobic effect; linkers; multimerization; multispecificity; peptidisc; self-assembly; sortase A; therapeutics.

Figure 1. Flexible linkers as a primarily in vivo tandem-linking method.

(A) The (Gly₄Ser)₃ linker is amended in sequence between Nb1 and Nb2, affording a bivalent polybody construct. (B) The (Gly₄Ser)₃ linker formalizes the linkage between two distinct Nb–antigen interactions (bispecific polybody), thereby forming a clamp that proximalizes the two cells along which said antigens occur. (C) ELP accommodates three unique Nb structures, though exceptionally by in vitro conjugation to afford a trispecific polybody. Nb1, nanobody 1; Nb2, nanobody2.

Figure 2. Amending antibody domains as an in vivo method.

(A) The hinge region is amended in sequence between Nb1 and Nb2, affording a bivalent polybody construct. (B) The crystallizable Fc region is amended in sequence between Nb1 and Nb2, similarly affording a bivalent polybody construct. Nb1, nanobody 1; Nb2, nanobody 2.

Figure 3. Self-assembling coiled coils as an in vivo, multimerization domain method.

(A) The sequence encoding for a self-assembling coiled coil (e.g., RHCC, COMP, or C4bp) is amended to a sequence encoding for a Nb. The Nb-coiled coil conjugate self assembles with other chimeras in stoichiometry prescribed by the identity of the appended coiled coil, here designated by N. (B) The SpyTag-SpyCatcher system is employed to conjugate a Nb-to-vasodilator-stimulated phosphoprotein (VASP) conjugate that affords a tetrameric polybody assembly. COMP, cartilage oligomeric matrix glycoprotein; C4bp-binding protein; RHCC, right-handed coiled coil; Nb, nanobody.

Figure 4. Self-assembling subunits as an in-vivo, multimerization domain method.

(A) The sequence encoding for a VT1B subunit is amended to a sequence encoding for an Nb, lending to spontaneous pentavalent assembly. (B) The sequence encoding for a ferritin subunit is amended to a sequence encoding for an Nb, lending to spontaneous 24-mer assembly. (C) A SpyCatcher-AaLS chimera is introduced to a SpyTag-Nb chimera, lending to a 60-mer assembly. Nb, nanobody; VT1B, verotoxin 1B subunit.

Figure 5. Chemical and enzymatic mediation as an in vitro method.

(A) Sortase A transpeptidation in theory can formalize Nb–Nb conjugation in the presence of an LPXTG tag and an attacking glycine residue through an acyl intermediate. (B) FGE in theory can catalyze Nb–Nb conjugation in the presence of an LCTPSR tag and a biorthogonal nucleophilic carrier. (C) Sulfo-SMCC in theory can cross-link a primary amine-carrying Nb and sulfhydryl-carrying Nb via thioether and amide bonds. FGE, formyl-glycine generating enzyme; Nb, nanobody.

Figure 6. Transmembrane segment appendage with membrane mimetics as an in vitro method.

TMS–Nb chimeras are expressed in the cell membrane and can be liberated using mild detergent. The amphipathic peptidisc mimetic stabilizes hydrophobic interactions at TMS junctions. Nb, nanobody; TMS, transmembrane segment.