The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics.
A drawback of targeting soluble antigens such as cytokines or toxins with long-lived antibodies is that such antibodies can prolong the half-life of the target antigen by a "buffering" effect. This has motivated the design of antibodies that bind to target with higher affinity at near neutral pH relative to acidic endosomal pH (~pH 6.0). Such antibodies are expected to release antigen within endosomes following uptake into cells, whereas antibody will be recycled and exocytosed in FcRn-expressing cells. To understand how the pH dependence of antibody-antigen interactions affects intracellular trafficking, we generated three antibodies that bind IL-6 with different pH dependencies in the range pH 6.0-7.4. The behavior of antigen in the presence of these antibodies has been characterized using a combination of fixed and live cell fluorescence microscopy. As the affinity of the antibody:IL-6 interaction at pH 6.0 decreases, an increasing amount of antigen dissociates from FcRn-bound antibody in early and late endosomes, and then enters lysosomes. Segregation of antibody and FcRn from endosomes in tubulovesicular transport carriers (TCs) into the recycling pathway can also be observed in live cells, and the extent of IL-6 association with TCs correlates with increasing affinity of the antibody:IL-6 interaction at acidic pH. These analyses result in an understanding, in spatiotemporal terms, of the effect of pH dependence of antibody-antigen interactions on subcellular trafficking and inform the design of antibodies with optimized binding properties for antigen elimination.
Department of Immunology; University of Texas Southwestern Medical Center; Dallas, TX, USA.
Siva Charan
SC
Devanaboyina
Department of Immunology; University of Texas Southwestern Medical Center; Dallas, TX, USA.
Sandra M
SM
Lynch
Department of Immunology; University of Texas Southwestern Medical Center; Dallas, TX, USA; Department of Electrical Engineering; University of Texas at Dallas; Richardson, TX, USA.
Raimund J
RJ
Ober
Department of Immunology; University of Texas Southwestern Medical Center; Dallas, TX, USA; Department of Electrical Engineering; University of Texas at Dallas; Richardson, TX, USA.
Sripad
S
Ram
Department of Immunology; University of Texas Southwestern Medical Center; Dallas, TX, USA; Department of Electrical Engineering; University of Texas at Dallas; Richardson, TX, USA.
Dongyoung
D
Kim
Department of Immunology; University of Texas Southwestern Medical Center; Dallas, TX, USA.
Alberto
A
Puig-Canto
Department of Oncology; Medimmune; Gaithersburg, MD, USA.
Shannon
S
Breen
Department of Antibody Discovery & Protein Engineering; Medimmune; Gaithersburg, MD, USA.
Srinath
S
Kasturirangan
Department of Antibody Discovery & Protein Engineering; Medimmune; Granta Park, Cambridge, UK.
Susan
S
Fowler
Department of Antibody Discovery & Protein Engineering; Medimmune; Gaithersburg, MD, USA.
Li
L
Peng
Department of Oncology; Medimmune; Gaithersburg, MD, USA.
Haihong
H
Zhong
Department of Antibody Discovery & Protein Engineering; Medimmune; Granta Park, Cambridge, UK.
Lutz
L
Jermutus
Department of Antibody Discovery & Protein Engineering; Medimmune; Gaithersburg, MD, USA.
Herren
H
Wu
Department of Antibody Discovery & Protein Engineering; Medimmune; Granta Park, Cambridge, UK.
Carl
C
Webster
Department of Immunology; University of Texas Southwestern Medical Center; Dallas, TX, USA.
E Sally
ES
Ward
Department of Antibody Discovery & Protein Engineering; Medimmune; Gaithersburg, MD, USA.
Changshou
C
Gao
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000378:metabolism*; D000941:Antigens / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D006863:Hydrogen-Ion Concentration; D015850:Interleukin-6 / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D021381:Protein Transport
D000911:Antibodies, Monoclonal; D000941:Antigens; D015850:Interleukin-6
antigen buffering; antigen-antibody trafficking; pH-dependent