Bispecific minibodies targeting HER2/neu and CD16 exhibit improved tumor lysis when placed in a divalent tumor antigen binding format.
Unconjugated monoclonal antibodies have emerged as important therapeutic agents for selected malignancies. One mechanism by which antibodies can exert cytotoxic effects is antibody-dependent cellular cytotoxicity (ADCC). In an effort to increase the efficiency of ADCC at tumor sites, we have focused on the construction of bispecific antibodies specific for the tumor antigen HER2/neu and the Fc gamma RIII-activating receptor (CD16) found on NK cells, mononuclear phagocytes, and neutrophils. Here, we describe the production of bispecific minibodies in two distinct binding formats. The parent minibody was constructed such that the IgG1 C(H)3 constant domain serves as the oligomerization domain and is attached to an anti-CD16 and an anti-HER2/ neu single-chain Fv via 19- and 29-amino acid linkers, respectively. This molecule can be expressed in mammalian cells from a dicistronic vector and has been purified using sequential affinity purification techniques. Analysis by surface plasmon resonance shows that the bispecific minibody can bind to HER2/neu and CD16, both individually and simultaneously. Furthermore, cytotoxicity studies show that the minibody can induce significant tumor cell lysis at a concentration as low as 20 nm. A trimeric, bispecific minibody (TriBi) that binds dimerically to HER2/neu and monomerically to CD16 induces equivalent cytotoxicity at lower antibody concentrations than either the parent minibody or the corresponding single-chain dimer. Both minibody constructs are stable in mouse and human serum for up to 72 h at 37 degrees C. These minibodies have the potential to target solid tumors and promote tumor lysis by natural killer cells and mononuclear phagocytes.
Shahied
Lillian S
LS
Department of Medical Oncology, Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111, USA.
Tang
Yong
Y
Alpaugh
R Katherine
RK
Somer
Robert
R
Greenspon
Dana
D
Weiner
Louis M
LM
D000818:Animals; D018033:Antibodies, Bispecific / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D000918:Antibody Specificity; D000920:Antibody-Dependent Cell Cytotoxicity; D000951:Antigens, Neoplasm / Q000276:immunology*; D001666:Binding Sites, Antibody; D001769:Blood; D019556:COS Cells; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004355:Drug Stability; D004622:Embryo, Mammalian; D005260:Female; D015870:Gene Expression; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000276:immunology; D007668:Kidney; D051379:Mice; D009369:Neoplasms / Q000276:immunology*; D010051:Ovarian Neoplasms; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology*; D017452:Receptors, IgG / Q000276:immunology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D018033:Antibodies, Bispecific; D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D007074:Immunoglobulin G; D018719:Erb-b2 Receptor Tyrosine Kinases; D017452:Receptors, IgG