The design and characterization of oligospecific antibodies for simultaneous targeting of multiple disease mediators.
Monoclonal antibodies are traditionally used to block the function of a specific target in a given disease. However, some diseases are the consequence of multiple components or pathways and not the result of a single mediator; thus, blocking at a single point may not optimally control disease. Antibodies that simultaneously block the functions of two or more disease-associated targets are now being developed. Herein, we describe the design, expression, and characterization of several oligospecific antibody formats that are capable of binding simultaneously to two or three different antigens. These constructs were generated by genetically linking single-chain Fv fragments to the N-terminus of the antibody heavy and light chains and to the C-terminus of the antibody C(H)3 domain. The oligospecific antibodies were expressed in mammalian cells, purified to homogeneity, and characterized for binding to antigens, Fcgamma receptors, FcRn, and C1q. In addition, the oligospecific antibodies were assayed for effector function, protease susceptibility, thermal stability, and size distribution. We demonstrate that these oligospecific antibody formats maintain high expression level, thermostability, and protease resistance. The in vivo half-life, antibody-dependent cellular cytotoxicity function, and binding ability to Fcgamma receptors and C1q of the test oligospecific antibodies remain similar to the corresponding properties of their parental IgG antibodies. The excellent expression, biophysical stability, and potential manufacturing feasibility of these multispecific antibody formats suggest that they will provide a scaffold template for the construction of similar molecules to target multiple antigens in complex diseases.
Dimasi
Nazzareno
N
Department of Antibody Discovery and Protein Engineering, MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
Gao
Changshou
C
Fleming
Ryan
R
Woods
Robert M
RM
Yao
Xiao-Tao
XT
Shirinian
Lena
L
Kiener
Peter A
PA
Wu
Herren
H
D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity / Q000276:immunology* / Q000528:radiation effects; D000920:Antibody-Dependent Cell Cytotoxicity / Q000276:immunology / Q000528:radiation effects; D000941:Antigens / Q000276:immunology; D015153:Blotting, Western; D002152:Calorimetry, Differential Scanning; D002850:Chromatography, Gel; D015922:Complement C1q / Q000276:immunology; D004194:Disease; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008027:Light; D051379:Mice; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000378:metabolism; D055550:Protein Stability / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D017452:Receptors, IgG / Q000276:immunology; D012031:Refractometry; D012542:Scattering, Radiation; D044967:Serum; D044366:Transition Temperature / Q000528:radiation effects
D000906:Antibodies; D000941:Antigens; D017452:Receptors, IgG; D015922:Complement C1q; D010447:Peptide Hydrolases