Evading pre-existing anti-hinge antibody binding by hinge engineering.
Antigen-binding fragments (Fab) and F(ab')2antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab')2C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab')2in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T225L variant and the natural C-terminal D221as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab')2for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.
a Department of Antibody Engineering , Genentech Research and Early Development , South San Francisco , CA , USA.
Hok Seon
HS
Kim
a Department of Antibody Engineering , Genentech Research and Early Development , South San Francisco , CA , USA.
Ingrid
I
Kim
b Department of Biochemical and Cellular Pharmacology , Genentech Research and Early Development , South San Francisco , CA , USA.
Linda
L
Zheng
b Department of Biochemical and Cellular Pharmacology , Genentech Research and Early Development , South San Francisco , CA , USA.
Jean-Michel
JM
Vernes
b Department of Biochemical and Cellular Pharmacology , Genentech Research and Early Development , South San Francisco , CA , USA.
Y Gloria
YG
Meng
a Department of Antibody Engineering , Genentech Research and Early Development , South San Francisco , CA , USA.
Christoph
C
Spiess
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology*; D018604:Epitope Mapping; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000276:immunology*; D007074:Immunoglobulin G / Q000276:immunology*; D015202:Protein Engineering
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D018985:Epitopes, B-Lymphocyte; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G
Antibody engineering; epitope mapping; immunogenicity; immunoglobulin G (IgG); monoclonal antibody; protease