RESUMO
Bispecific antibodies (BsAb) have been traditionally utilized to redirect cytotoxic effector cells and agents to kill tumor cells expressing the target antigens. Recently a new concept is emerging to develop BsAb that simultaneously block the functions of two tumor-associated targets, eg., growth factor receptors, for enhanced antitumor efficacies. Broad clinical applications of BsAb have been, and still are, significantly hampered by the difficulty in producing the materials in sufficient quantity and quality by traditional approaches. Here we describe a recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, using a single variable domain (sVD) antibody as a versatile building block. In this method, a sVD of a defined specificity is genetically fused to either the N-terminus of the light chain or the C-terminus of the heavy chain of a functional IgG antibody of a different specificity. A model BsAb was constructed using a sVD to mouse platelet derived growth factor receptor alpha and a conventional IgG antibody to mouse platelet derived growth factor receptor beta. The BsAb were expressed in mammalian cells and purified to homogeneity by a one-step Protein A affinity chromatography. Further, the BsAb retained the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. Importantly, the BsAb inhibited the activation of both its target receptors in tumor cells stimulated by both platelet derived growth factor AA and BB, whereas the parent monospecific antibody only inhibited the activation of a single receptor stimulated by its cognate ligand. This format of BsAb should be readily applicable to the production of other BsAb recognizing any pairs of antigens.
Assuntos
Anticorpos Biespecíficos/imunologia , Região Variável de Imunoglobulina/genética , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/farmacologia , Afinidade de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Becaplermina , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos/genética , Humanos , Imunoglobulina G/imunologia , Região Variável de Imunoglobulina/imunologia , Cadeias kappa de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/imunologia , Cinética , Camundongos , Modelos Moleculares , Fosforilação/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/química , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas c-sis , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/imunologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas/imunologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , TransfecçãoRESUMO
Platelet-derived growth factor receptor beta (PDGFRbeta) is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRbeta from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRbeta and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRbeta and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRbeta antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias/tratamento farmacológico , Receptor beta de Fator de Crescimento Derivado de Plaquetas/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Citometria de Fluxo , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Neoplasias/patologia , Biblioteca de Peptídeos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Both laboratory and early clinical studies to date have demonstrated that bispecific antibodies (BsAb) may have potentially significant application in cancer therapy. The clinical development of BsAb as therapeutics has been hampered, however, by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods has been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. Here we describe a novel recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a single variable domain antibody to the N terminus of the light chain of a functional IgG antibody of different specificity. A model BsAb was constructed using a single variable domain antibody to mouse platelet-derived growth factor receptor alpha and a conventional IgG antibody to mouse vascular endothelial growth factor receptor 2. The BsAb was expressed in mammalian cells and purified to homogeneity by one-step protein A affinity chromatography. Furthermore, the BsAb retains the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. This design and expression of Fc domain-containing, IgG-like BsAb should be applicable to the construction of similar BsAb from antibodies recognizing any pair of antigens.