Evaluation of Targeting Efficiency of Joints with Anticollagen II Antibodies

Abstract

Diseases of the joints affect over 10% of the world's population, resulting in significant morbidity. There is an unmet need in strategies for specific delivery of therapeutics to the joints. Collagen type II is synthesized by chondrocytes and is mainly restricted to the cartilage and tendons. Arthrogen-CIA is a commercially available anti-collagen II antibody cocktail that reacts with 5 different epitopes on human, bovine, and mouse collagen II. Arthrogen has been used for induction of experimental rheumatoid arthritis (RA) in mice because of high complement activation on the cartilage surface. Native collagen II might serve as a useful target for potential delivery of therapeutics to the joint. To evaluate the efficiency and specificity of targeting collagen II, Arthrogen was labeled with near-infrared (NIR) dye IRDye 800 or IRDye 680. Using ex vivo

C2 IgM Natural Antibody Enhances Inflammation and Its Use in the Recombinant Single Chain Antibody-Fused Complement Inhibitor C2-Crry to Target Therapeutics to Joints Attenuates Arthritis in Mice

“…In separate studies, mice injected with 1 × PBS, appears identical to the non-injected mice (data not shown) and we have published it (61). No non-specific binding of IRD800 labeled anti-CII Ab was seen in other organs such as kidney, liver and lung which is consistent with our previous publication therefore no images were taken (61). These data show that IRDye 800 labeled antibodies or proteins can be detected in injured joints.…”

“…This binding of IRDye 800 anti-CII Abs also induced a low level of disease and it can compete with the unlabeled arthrogen ( 60 ). Our previous study show that IRDye 800 labeled anti-CII also does not bind non-specifically to spleen, kidney, lung, heart, liver and intestine but only to the arthritic joints ( 61 ). At day 15, bones of forelimbs and hind limbs were cleaned of muscle, sandwiched between two glass slides and scanned with a Li-COR Odyssey at 700 nm channel for autofluorescence and 800 nm channel for IRDye 800.…”

“…To show the binding of C2-Crry in vitro to apoptotic FLS, we labeled the protein with IRDye800-NHS ester (Li-COR Biosciences) as we have shown previously (60,61). C2-Crry fusion protein was incubated with a 10-fold molar excess of dye for 5 h at 4 • C in PBS.…”

“…Unbound dye was removed with a 7 kDa Zeba desalting column (Thermo Fisher). After labeling C2-Crry with IRDye800 and addition to a Zeba column, the protein was eluted from the column with PBS and stored at 4 • C before use according to our previously published study (60,61). Similarly, Arthrogen (anti-CII) mAbs were labeled with IRDye 800 (60,61).…”

“…After two weeks of antibody wash out period (15 days) mice were sacrificed to detect IRDye 800 labeled anti-CII Abs in the joints. IRDye 800 labeled anti-CII Abs clears very slowly from the circulation but firmly binds to the cartilage in joints even after a long wash period according to our published methods (60,61). This binding of IRDye 800 anti-CII Abs also induced a low level of disease and it can compete with the unlabeled arthrogen (60).…”