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Review
. 2017 Nov 22;8:1603.
doi: 10.3389/fimmu.2017.01603. eCollection 2017.

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

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Free PMC article
Review

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics

Peter Bannas et al. Front Immunol. .
Free PMC article

Abstract

Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single-chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g., to generate a bispecific T-cell engager, or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or Nb). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy chain antibodies as antitumor therapeutics.

Keywords: antitumor therapeutics; heavy chain antibodies; nanobodies; nanobody fusion proteins; nanobody-conjugates; sortagging of nanobodies.

Figures

Figure 1
Figure 1
Advantageous features of camelid heavy chain antibodies. Heavy chain antibodies are composed of two heavy chains. The target-binding module is composed of a single VHH domain. A recombinant VHH domain, designated nanobody (Nb) is highly soluble and does not show any tendency to associate with other hydrophobic protein surfaces. Conventional antibodies are composed of two heavy and two light chains. The target-binding module is composed of two non-covalently associated variable domains VH and VL. In intact antibodies, the proper orientation of these domains is mediated by a hydrophobic interface (see Figure 1) and is further stabilized by the disulfide-linked CL and CH1 domains. A pair of VH and VL domains can be linked genetically into a single-chain variable fragment (scFv) in which the proper orientation of domains is mediated alone by the hydrophobic interface between the two V-domains.
Figure 2
Figure 2
Comparison of the VHH domain (nanobody) of a camelid heavy chain antibody with its VH-counterpart of a conventional antibody. The three complementarity determining regions (CDRs) of the antigen-binding paratope are indicated in red, the framework region is indicated in cyan (camelid VHH) and yellow (human VH and VL). The CDR3 loop contributes most to the diversity and specificity of the paratope since its coding region is newly generated during B-cell development, i.e., by genetic fusion of a D element with flanking V and J elements and deletion or insertion of nucleotides at the junctions. The CDR3 loop of a camelid VHH typically is much longer than that of a human VH. A key distinguishing feature of a camelid VHH is that it binds its target with a single domain, whereas a human VH binds its target together with a non-covalently associated VL. A second distinguishing feature of a VHH is its entirely hydrophilic framework, whereas a VH domain contains a hydrophobic side facing the VL domain (indicated in black). This hydrophobic interface helps to maintain the proper orientation of the six CDR loops of the target-binding paratope. This hydrophobic interface accounts for the inherent stickiness of isolated VH domains, and for the propensity of “mispairing” of VH and VL domains in bispecific antibody (bsAb) constructs. The corresponding region in a VHH is hydrophilic (indicated by dashed lines), accounting for the superior stability and solubility of a VHH over a VH. A third distinguishing feature of a VHH is a long CDR3 that can form finger-like extensions and reach cavities on target antigens inaccessible to conventional antibodies. The long CDR3 loop of a VHH often partially folds over the side of the framework corresponding to the side in the VH domain facing the VL. Such folded over loops sterically preclude binding to a VL.
Figure 3
Figure 3
Chimeric and humanized heavy chain antitumor antibodies. Second generation antitumor antibodies such as daratumumab are fully human antibodies, derived from human-Ig transgenic mice or synthetic libraries. Successful antitumor antibodies, such as rituximab and cetuximab, are chimeric antibodies composed of VH and VL domains from mouse monoclonal antibodies (mAbs, green) fused to the constant domains of human IgG1 and kappa, respectively. Chimeric antitumor heavy chain antibodies are easily generated by genetic fusion of a VHH domain (blue) to the hinge and Fc domains of human IgG1. Such chimeric heavy chain antibodies combine the advantageous features of a nanobody (Nb), i.e., high solubility and stability, with the effector functions of a human IgG. Fully human heavy chain antibodies often suffer from the poor solubility and stability of a partnerless VH domain with a vacant sticky hydrophobic side (indicated in black). By substituting divergent framework residues, camelid VHH domains can be “humanized” (yellow dots) and human VH domains can be “camelized” (blue dots) to reduce immunogenicity and to improve solubility, respectively.
Figure 4
Figure 4
Bispecific heavy chain antibodies. A bispecific heavy chain antibody can be generated simply by linking two tandem VHH domains (blue) to the hinge and Fc domains of human IgG. Both specificities are contained in a single heavy chain. Therefore, there is no need to engineer the Fc-domain as in conventional biclonics. Two nanobodies with different specificities can be linked to one another without any mispairing issues to generate a bispecific construct (Nb-BiTE). Two different single-chain variable fragment (scFv) domains can be linked genetically to one another, e.g., to generate a bispecific T-cell engager (BiTE). Special precautions are required to prevent mispairing of VH and VL domains. Moreover, the hydrophobic interface of VH and VL domains can dissociate from one another and associate with other hydrophobic surfaces. This can severely limit the stability and solubility of scFv modules. Bispecific antibodies can be generated from two distinct heavy chains. In order to circumvent mispairing of VH–VL domains, a common “fixed” light chain is used that pairs with both heavy chains. In this case, target specificity is mediated largely if not entirely by the two VH domains. Fc-engineering is commonly used to favor formation of heteromeric over homomeric antibodies, e.g., by electrostatic steering to introduce negatively charged amino acid residues (DE) in one CH3 domain and matching basic residues (KK) in the other CH3 domain. Different line patterns are used to indicate different specificities of VHHs and VHs.
Figure 5
Figure 5
Schematic representation of di- and multimeric antitumor nanobodies. Owing to their high solubility and stability nanobodies can readily be fused genetically to other nanobodies without the mispairing and solubility issues inherent to single-chain variable fragment-based dimers and multimers. Flexible glycine–serine linkers are commonly used to fuse nanobodies, e.g., one or more tandem modules of G4S composed of four glycine residues to provide maximal flexibility and a hydrophilic serine residue to improve solubility. Tandem fusion of two identical nanobodies yields a bivalent dimer, often with improved avidity over the respective monomer. Tandem cloning of two distinct nanobodies that recognize non-overlapping epitopes of the same antigen yields a biparatopic binder. Fusion of two nanobodies recognizing distinct cell surface proteins yields a bispecific binder. The in vivo half-life can be extended by fusing one or more antitumor nanobodies to an albumin-specific nanobody. Piggy-backing on albumin reduces the loss of antitumor nanobodies by renal filtration.
Figure 6
Figure 6
Schematic representation of nanobodies as targeting moieties of effector domains for antitumor therapy. The schematics illustrate genetic fusion (A) and chemical conjugation (B,C) of nanobodies to effectors. For simplicity only a single nanobody is shown in each schematic. This nanobody can readily be replaced by any of the dimeric and multimeric constructs shown in Figure 5, usually without compromising solubility. Moreover, the unitag and sortag technologies can be used also with the Nb-hcAbs illustrated in Figures 2–4. (A) Nanobodies can be fused genetically to other proteins and/or to smaller peptides. Both, the N- and C-terminus are available for fusion. The diagrams illustrate the more commonly used C-terminus. Fusion to a toxin such as Pseudomonas exotoxin A (PE38) generates an immunotoxin. Fusion to a peptide tag provides a means to deliver a universal marker onto tumor cells, e.g., as a docking site for a universal tag-specific antibody or T cell transduced with a tag-specific chimeric antigen receptor. Fusion to a sortag provides a substrate for site-specific, sortase-catalyzed linkage to a synthetic peptide, e.g., GGGX. When X = lysine or cystein, almost any chemical moiety can be conjugated by amide or maleimide chemistry, including a chelator for a radionuclide. (B) Nanobodies can also be conjugated chemically to the side chains of lysine or cysteine residue. Amide conjugation usually introduces multiple modifications, since nanobodies typically contain several surface-exposed lysine residues and an N-terminal amine. Since most nanobodies contain only two deeply buried cysteine residues engaged in an intrachain disulfide bond, malemeide conjugation requires the introduction of one or more surface exposed cysteine residues by site directed mutagenesis. These techniques allow conjugation of radionuclides or near-infrared fluorochromes (NIRFs). (C) Nanobodies can be easily linked, e.g., via a C-terminal polyethyleneglycol moiety, to nanoparticles, such as liposomes containing anti-neoplastic drugs.

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