RESUMEN
Immunoglobulins and T cell receptors (TCRs) share common sequences and structures. With the goal of creating novel bispecific antibodies (BsAbs), we generated chimeric molecules, denoted IgG_TCRs, where the Fv regions of several antibodies were fused to the constant domains of the α/ß TCR. Replacing CH1 with Cα and CL with Cß, respectively, was essential for achieving at least partial heavy chain/light chain assembly. Further optimization of the linker regions between the variable and constant domains, as well as replacement of the large FG loop of Cß with a canonical ß-turn, was necessary to consistently obtain full heavy chain/light chain assembly. The optimized IgG_TCR molecules were evaluated biophysically and shown to maintain the binding properties of their parental antibodies. A few BsAbs were generated by co-expressing native Fabs and IgG_TCR Fabs within the same molecular construct. We demonstrate that the IgG_TCR designs steered each of the light chains within the constructs to specifically pair with their cognate heavy chain counterparts. We did find that even with complete constant domain specificity between the CH1/CL and Cα/Cß domains of the Fabs, strong variable domain interactions can dominate the pairing specificity and induce some mispairing. Overall, the IgG_TCR designs described here are a first step toward the generation of novel BsAbs that may be directed toward the treatment of multi-faceted and complex diseases.
Asunto(s)
Anticuerpos Biespecíficos , Fragmentos Fab de Inmunoglobulinas , Inmunoglobulina G , Ingeniería de Proteínas , Receptores de Antígenos de Linfocitos T alfa-beta , Proteínas Recombinantes de Fusión , Anticuerpos Biespecíficos/biosíntesis , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas/biosíntesis , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Inmunoglobulina G/genética , Receptores de Antígenos de Linfocitos T alfa-beta/biosíntesis , Receptores de Antígenos de Linfocitos T alfa-beta/química , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Robust generation of IgG bispecific antibodies has been a long-standing challenge. Existing methods require extensive engineering of each individual antibody, discovery of common light chains, or complex and laborious biochemical processing. Here we combine computational and rational design approaches with experimental structural validation to generate antibody heavy and light chains with orthogonal Fab interfaces. Parental monoclonal antibodies incorporating these interfaces, when simultaneously co-expressed, assemble into bispecific IgG with improved heavy chain-light chain pairing. Bispecific IgGs generated with this approach exhibit pharmacokinetic and other desirable properties of native IgG, but bind target antigens monovalently. As such, these bispecific reagents may be useful in many biotechnological applications.
Asunto(s)
Anticuerpos Biespecíficos/química , Fragmentos Fab de Inmunoglobulinas/química , Inmunoglobulina G/química , Ingeniería de Proteínas/métodos , Animales , Anticuerpos Biespecíficos/metabolismo , Biotecnología , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Inmunoglobulina G/metabolismo , Masculino , Ratones , Modelos Biológicos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The family of secretory phospholipase A2 (sPLA2) enzymes has been associated with inflammatory diseases and tissue injury including atherosclerosis. A-001 is a novel inhibitor of sPLA2 enzymes discovered by structure-based drug design, and A-002 is the orally bioavailable prodrug currently in clinical development. A-001 inhibited human and mouse sPLA2 group IIA, V, and X enzymes with IC50 values in the low nM range. A-002 (1 mg/kg) led to high serum levels of A-001 and inhibited PLA2 activity in transgenic mice overexpressing human sPLA2 group IIA in C57BL/6J background. In addition, the effects of A-002 on atherosclerosis in 2 ApoE mouse models were evaluated using en face analysis. (1) In a high-fat diet model, A-002 (30 and 90 mg/kg twice a day for 16 weeks) reduced aortic atherosclerosis by 50% (P < 0.05). Plasma total cholesterol was decreased (P < 0.05) by 1 month and remained lowered throughout the study. (2) In an accelerated atherosclerosis model, with angiotensin II-induced aortic lesions and aneurysms, A-002 (30 mg/kg twice a day) reduced aortic atherosclerosis by approximately 40% (P < 0.05) and attenuated aneurysm formation (P = 0.0096). Thus, A-002 was effective at significantly decreasing total cholesterol, atherogenesis, and aneurysm formation in these 2 ApoE mouse models.