RESUMO
The antigen-binding fragment of functional heavy chain antibodies (HCAbs) in camelids comprises a single domain, named the variable domain of heavy chain of HCAbs (VHH). The VHH harbors remarkable amino acid substitutions in the framework region-2 to generate an antigen-binding domain that functions in the absence of a light chain partner. The substitutions provide a more hydrophilic, hence more soluble, character to the VHH but decrease the intrinsic stability of the domain. Here we investigate the functional role of an additional hallmark of dromedary VHHs, i.e. the extra disulfide bond between the first and third antigen-binding loops. After substituting the cysteines forming this interloop cystine by all 20 amino acids, we selected and characterized several VHHs that retain antigen binding capacity. Although VHH domains can function in the absence of an interloop disulfide bond, we demonstrate that its presence constitutes a net advantage. First, the disulfide bond stabilizes the domain and counteracts the destabilization by the framework region-2 hallmark amino acids. Second, the disulfide bond rigidifies the long third antigen-binding loop, leading to a stronger antigen interaction. This dual beneficial effect explains the in vivo antibody maturation process favoring VHH domains with an interloop disulfide bond.
Assuntos
Cisteína/química , Dissulfetos/química , Cadeias Pesadas de Imunoglobulinas/química , Anticorpos de Cadeia Única/química , Animais , Camelus , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/metabolismo , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismoRESUMO
UNLABELLED: Nanobodies are a novel type of immunoglobulinlike, antigen-binding protein with beneficial pharmacologic and pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. However, because of their camelid, nonhuman origin, the possible immunogenicity of Nanobodies when used in the clinic is a concern. Here we present a new strategy to quickly generate humanized Nanobodies for molecular imaging purposes. METHODS: We genetically grafted the antigen-binding loops of NbCEA5, a Nanobody with specificity for the colon carcinoma marker carcinoembryonic antigen (CEA), onto the framework of a humanized Nanobody scaffold. This scaffold has been previously characterized in our laboratory as a stable Nanobody that can serve as a universal loop acceptor for antigen-binding loops from donor Nanobodies and has been additionally mutated at about 10 crucial surface-exposed sites to resemble the sequence of human variable immunoglobulin domains. The 3 recombinant Nanobodies (NbCEA5, humanized scaffold, and humanized CEA5 graft) were produced in bacteria and purified. Unlabeled and (99m)Tc-labeled Nanobodies were biochemically characterized in vitro and tested as probes for SPECT/CT of xenografted tumors. RESULTS: The success of loop-grafting was confirmed by comparing these Nanobodies for their capacity to recognize soluble CEA protein in enzyme-linked immunosorbent assay and by surface plasmon resonance and to bind to CEA-positive LS174T colon carcinoma cells and CEA-transfected but not untransfected Chinese hamster ovary cells in flow cytometry. Specificity of binding was confirmed by competition studies. All Nanobodies were heat-stable, could be efficiently labeled with (99m)Tc, and recognized both soluble and membrane-bound CEA protein in binding studies. Finally, biodistribution experiments were performed with intravenously injected (99m)Tc-labeled Nanobodies in LS174T tumor-bearing mice using pinhole SPECT/micro-CT. These in vivo experiments revealed specificity of tumor targeting and rapid renal clearance for all Nanobodies, with low signals in all organs besides the kidneys. CONCLUSION: This study shows the potency of antigen-binding loop-grafting to efficiently generate humanized Nanobodies that retain their targeting capacities for noninvasive in vivo imaging of tumors.
Assuntos
Proteínas de Transporte , Compostos Radiofarmacêuticos , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Células CHO , Antígeno Carcinoembrionário/imunologia , Proteínas de Transporte/síntese química , Proteínas de Transporte/farmacocinética , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Sistemas de Liberação de Medicamentos , Ensaio de Imunoadsorção Enzimática , Humanos , Processamento de Imagem Assistida por Computador , Marcação por Isótopo , Camundongos , Dados de Sequência Molecular , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Compostos de Tecnécio/síntese química , Distribuição Tecidual , Tomografia Computadorizada de Emissão , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Several antibody fragment engineering techniques aim at intrinsic stability enhancement, but are not applied in a truly generic way. Here, a strategy is proposed whereby consistent gain in stability is accomplished by introducing a specific disulfide bond between two opposite beta-strands in the hydrophobic core of the immunoglobulin heavy-chain variable domain of heavy-chain antibodies (Nanobody). Besides the rational design of a disulfide bond between residues 39 and 87, a Nanobody harboring an extra naturally occurring cystine between residues 54 and 78 was compared to an equivalent Nanobody without that cystine. Both novel disulfide cross-links were introduced in several Nanobodies in various combinations. Interestingly, only the extra naturally occurring cystine consistently increased the conformational and thermal stabilities of wild-type Nanobodies without affecting antigen binding.