ANTI-TNF BISPECIFIC ANTIBODIES: FUNCTIONAL PROPERTIES
Systemic TNF blockade is associated with the risk of undesirable side effects due to TNF role in host defense and in immune homeostasis [1, 2]. Previously it was reported that cellular sources of TNF specify its distinct functions in protective and autoreactive immune responses [3, 4]. It was shown that TNF derived from myeloid cells plays a non-redundant deleterious role in several disease models such as rheumatoid arthritis , severe sepsis, concanavalin A induced hepatitis  and EAE , making TNF from this cellular source a promising target for therapy.
For this purpose three anti-hTNF bispecific reagents were produced on the basis of unique single-domain antibodies (VHH) from camelids . To ensure myeloid cell specificity, BV-1 combined anti-F4/80- and anti-hTNF-binding domains allowing a selective capture of hTNF secreted by monocytes/macrophages. Due to insertion of several mutations into CDR- regions in F4/80-domain BV-1mut was unable to bind murine macrophages and therefore represented a reagent with systemic TNF-neutralizing activity. The third reagent was bispecific antibody BV-1c binding human TNF and lactoferrin. All bispecific proteins were expressed in Rosetta 2 (DE3) pLysS E. coli strain and purified by Ni-NTA liquid affinity chromatography.
The ability of these bispecific reagents to block TNF was tested by cytotoxic assay with WEHI 164 13 Cl cell line, sensitive to human TNF. According to the results, the following values of LD50 were obtained: 10 nM for BV-1, 15,5 nM for BV-1c and 57 nM for BV-1mut.
We also used cytotoxic assay to confirm the ability of BV-1 to specifically block TNF derived from myeloid cells. For this purpose we incubated macrophages derived from hTNFKI "humanized" mice with studied proteins and then incubated with LPS after washing step. After this we transferred the supernatant to WEHI cells. Survival curves showed dose-dependent neutralizing activity of BV-1 which provide complete WEHI cell survival in contrast to BV-1c and BV-1mut.
As myeloid cells are the principal source of deleterious TNF in mouse model of lethal LPS/D-Galactosamine (DGal) hepatotoxicity this model was used to evaluate the activity ability to block TNF in vivo. 30 min prior to injection of hepatotoxic agents the following substances were administered to mice: phosphate-buffered saline (PBS) as negative control for survival (all animals in this group died) or murine monoclonal antibody F10 as positive control (all mice survived) or different doses of the experimental neutralizing agents. Kaplan-Meier survival curves were plotted and different concentrations of the bispecific antibodies were compared. For BV-1 the dosage provided 100% survival of the experimental animals was 1,5 μg/g while for BV-1c it was 3 μg/g.
These results demonstrate that selective inhibition of macrophage-derived TNF is more effective than systemic TNF blockade in the mouse model of lethal LPS/DGal hepatotoxicity.
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