biomolecules Review An Inside Job: Applications of Intracellular Single Domain Antibodies Eline Soetens 1,2, Marlies Ballegeer 1,2 and Xavier Saelens 1,2,* 1 VIB-UGent Center for Medical Biotechnology, VIB, B-9052 Ghent, Belgium; [email protected] (E.S.); [email protected] (M.B.) 2 Department of Biochemistry and Microbiology, Ghent University, B-9000 Ghent, Belgium * Correspondence: [email protected] Received: 20 November 2020; Accepted: 10 December 2020; Published: 12 December 2020 Abstract: Sera of camelid species contain a special kind of antibody that consists only of heavy chains. The variable antigen binding domain of these heavy chain antibodies can be expressed as a separate entity, called a single domain antibody that is characterized by its small size, high solubility and oftentimes exceptional stability. Because of this, most single domain antibodies fold correctly when expressed in the reducing environment of the cytoplasm, and thereby retain their antigen binding specificity. Single domain antibodies can thus be used to target a broad range of intracellular proteins. Such intracellular single domain antibodies are also known as intrabodies, and have proven to be highly useful tools for basic research by allowing visualization, disruption and even targeted degradation of intracellular proteins. Furthermore, intrabodies can be used to uncover prospective new therapeutic targets and have the potential to be applied in therapeutic settings in the future. In this review we provide a brief overview of recent advances in the field of intracellular single domain antibodies, focusing on their use as research tools and potential therapeutic applications. Special attention is given to the available methods that allow delivery of single domain antibodies into cells. Keywords: single domain antibody; intrabody; delivery methods; therapy; research tool 1. Introduction In 1993, a serendipitous discovery that led to the identification of a new type of antibody in the sera of camelid species was reported [1]. It was demonstrated that the serum of these mammals not only contains classical immunoglobulins (IgGs), but also a second type of antibody that consists only of heavy chains. These so called heavy chain antibodies (HCAbs) make up 50–80% of the total serum IgG amount in camels and 10–25% in South-American camelids [2], suggesting that they play an important role in the humoral immune response of these animals. Similar alternatives to classical antibodies, named new antigen receptor (IgNAR) antibodies, were also found in the sera of some cartilaginous fish, such as nurse sharks and wobbegong sharks [3,4]. Although the sequences of these ancestral IgNAR antibodies differ significantly from camelid HCAbs, their structures show a remarkable resemblance, which points towards convergent evolution [4,5]. In the wake of the discovery of HCAbs in camelid sera, it was soon realized that the HCAb variable domain (VHH) could be expressed as a separate protein fragment, while retaining full antigen binding capacity [6]. VHHs have a size of about 2.5 nm in diameter and 4 nm in length and are the smallest natural antigen-binding entities known to date [7] (Figure1). Biomolecules 2020, 10, 1663; doi:10.3390/biom10121663 www.mdpi.com/journal/biomolecules Biomolecules 2020, 10, 1663 2 of 30 Biomolecules 2020, 10, x 2 of 29 Figure 1. Schematic representation of a conventional IgG antibody ( left) and aa camelidcamelid heavyheavy chainchain antibody (middle),), withwith theirtheir respectiverespective smallestsmallest antigen-bindingantigen-binding formats:formats: a singlesingle chainchain variablevariable fragment (ScFvs) and a single domain antibody (VHH). To thethe right,right, a foldedfolded VHHVHH domaindomain is schematically shownshown withwith CDRCDR 1,1, 2 and 3 indicated in orange, yellow and red, respectively. CH: heavy chain constant domain;domain; CL: light chain constant domain;domain; VH: heavy chain variable domain;domain; VL: light chain variable domain;domain; VHH:VHH: variable domaindomain ofof heavyheavy chainchain antibody.antibody. Heavy chainchain antibodies antibodies lack lack light light chains, chains, as well as as well the firstas the constant first domainconstant (CH1). domain To compensate(CH1). To forcompensate these differences, for these the difference variables, domains the variable of HCAbs domains have of adopted HCAbs distinct have adopted structural distinct features. structural Firstly, thefeatures. hallmark Firstly, residues the hallmark in framework residues region in framework 2 (FR2) of the region VHH, 2 which(FR2) of account the VHH, for the which interaction account with for the lightinteraction chain inwith classical the light antibodies, chain in have classical been replacedantibodies, by have more been hydrophilic replaced residues by more [8– hy11].drophilic Indeed, itresidues has been [8 demonstrated–11]. Indeed, it that has swapping been demonstrated the corresponding that swapping hydrophobic the corresponding residues in a humanhydrophobic heavy chainresidues variable in a human domain heavy (VH) forchain smaller variable and domain more hydrophilic (VH) for ones,smaller promotes and more solubility hydrophilic and avoids ones, aggregationpromotes solubility [12]. The paratopeand avoids of VHHs aggregation consists of[12] three. The complementarity paratope of determiningVHHs consists regions of (CDRs) three comparedcomplementarity to six in determining classical antibodies. regions (CDRs) The adjacent compared CDR loopsto six ofin theclassical heavy antibodies. and light chain The adjacent variable 2 domainsCDR loops in classicalof the heavy antibodies and light form chain a paratope variable surface domains of 600–900 in classical Å . VHHs antibodies make up form for thea paratope reduced numbersurface of of 60 CDR0–900 loops Å 2. byVHHs increasing make theirup for length, the reduced in particular number CDR3 of CDR (on average loops by two increasing to four amino their 2 acidslength, longer in particular compared CDR3 to human (on average IgG [9 ]),two resulting to four inamino an average acids longer paratope compared surface to of human 600–800 IgG Å [[9]13),]. Asresulting a longer in CDR3 an average loop may paratope be entropically surface unfavorable of 600–800 for Å epitope2 [13]. binding,As a longer camel-derived CDR3 loop VHHs may often be containentropicall an additionaly unfavorable interloop for epitope cysteine binding, bridge between camel-derived CDR1 and VHHs CDR3, often effectively contain constraining an additional the movementinterloop cysteine of the CDR3 bridge loop between [14]. This CDR1 disulfide and CDR3, bond iseffectively notably less constraining common inthe llama movement VHHs, whichof the canCDR3 be loop explained [14]. This by the disulfide shorter bond CDRs is presentnotably inless these common VHHs in [ 9llama–11]. VHHs, Structural which differences can be explained between theby the variable shorter domains CDRs of present classical in antibodies these VHHs and HCAbs[9–11]. Structural can explain differences why these twobetween antibody the variantsvariable differdomains in their of classical target antigen antibodies preference. and HCAbs Classical can antibodies explain why typically these adopt two antibody a concave variants paratope differ surface, in makingtheir target them antigen more prone preference. to bind Classical to protruding antibo ordies flat surfaces.typically Onadopt the othera concave hand, paratope the prolate surface, shape ofmaking VHHs them combined more prone with theirto bind small to protruding size makes or them flat verysurfaces. suitable On the for other binding hand, clefts the on prolate the protein shape surface,of VHHs such combined as enzymatic with their sites small [13]. size makes them very suitable for binding clefts on the protein surface,VHH such coding as enzymatic sequences sites are usually[13]. isolated from immunized animals, after which specific binders are enrichedVHH coding by robust sequences selection are methods usually such isolated as phage from display, immunized although animals, other types after of which VHH librariesspecific (BOXbinders 1) are and enriched selection by methods robust selection (BOX 2) havemethods also such been as successfully phage display, used although to identify other high types affinity of binders.VHH libraries Several (BOX properties 1) and ofselection VHHs havemethods made (BOX them 2) attractive have also research been successfully tools and explain used to why identify they havehigh beenaffinity extensively binders. usedSeveral in manyproperties labs in of the VHHs almost have thirty made years them since attractive their discovery. research The tools classical and antibodyexplain why counterpart they have of been VHHs extensively are called used single-chain in many variable labs in fragmentsthe almost (scFvs) thirty andyears are since made their up ofdiscovery. the heavy The and classical light chain antibody variable counterpart domains ofof classicalVHHs are immunoglobulins, called single-chain connected variable by fragments a flexible peptide(scFvs) and linker. are Overmade the up years,of the heavy these scFvs and light have chain also provenvariable their domains value of as classical research immunoglobulins, tools [15], yet the mainconnected advantage by a VHHsflexible hold peptide over scFvslinker. is Over that theythe years, only consist these ofscFvs one domain,have also which proven makes their them value more as convenientresearch