Formatted anti–tumor necrosis factor α VHH proteins derived from camelids show superior potency and targeting to inflamed joints in a murine model of collagen‐induced arthritis

Abstract

Objective. The advent of tumor necrosis factor (TNF)-blocking drugs has provided rheumatologists with an effective, but highly expensive, treatment for the management of established rheumatoid arthritis (RA). Our aim was to explore preclinically the application of camelid anti-TNF VHH proteins, which are singledomain antigen binding (VHH) proteins homologous to human immunoglobulin V H domains, as TNF antagonists in a mouse model of RA.Methods. Llamas were immunized with human and mouse TNF, and antagonistic anti-TNF VHH proteins were isolated and cloned for bacterial production. The resulting anti-TNF VHH proteins were recombinantly linked to yield bivalent mouse and human TNFspecific molecules. To increase the serum half-life and targeting properties, an anti-serum albumin anti-TNF VHH domain was incorporated into the bivalent molecules. The TNF-neutralizing potential was analyzed in vitro. Mouse TNF-specific molecules were tested in a therapeutic protocol in murine collagen-induced arthritis (CIA). Disease progression was evaluated by clinical scoring and histologic evaluation. Targeting properties were evaluated by 99m Tc labeling and gamma camera imaging.Results. The bivalent molecules were up to 500 times more potent than the monovalent molecules. The antagonistic potency of the anti-human TNF VHH proteins exceeded even that of the anti-TNF antibodies infliximab and adalimumab that are used clinically in RA. Incorporation of binding affinity for albumin into the anti-TNF VHH protein significantly prolonged its serum half-life and promoted its targeting to inflamed joints in the murine CIA model of RA. This might explain the excellent therapeutic efficacy observed in vivo.Conclusion. These data suggest that because of the flexibility of their format, camelid anti-TNF VHH proteins can be converted into potent therapeutic agents that can be produced and purified cost-effectively.Tumor necrosis factor (TNF)-blocking drugs are widely considered to be among the most efficient treatments available for rheumatoid arthritis (RA). TNF blockade is also highly therapeutic for several other chronic inflammatory diseases, such as spondylarthropathies, psoriasis, and inflammatory bowel disease (1-3).

Llama Antibody Fragments with Cross-Subtype Human Immunodeficiency Virus Type 1 (HIV-1)-Neutralizing Properties and High Affinity for HIV-1 gp120

“…Engineered multivalent VHH to other antigens have previously been shown to exhibit better binding and neutralizing properties than their monovalent counterparts (13). It is possible that introduction of multivalency will increase the potency of the identified antigp120 VHH.…”

“…The VHH domain can be easily cloned and expressed to high levels in bacteria and yeast (26,27). This notion, together with advantageous characteristics in terms of stability and solubility (20,64,81), has led to successful development of camelid VHH in a number of applications against a range of biological targets (2,13,14,19,21,57,58,69,83,84), including neutralization of rotavirus (28,60). We hypothesized that the small size of VHH in combination with their protruding CDR3 loops and their preference for cleft recognition may allow them to recognize conserved motifs on gp120 that are occluded from conventional antibodies.…”

Single Domain Antibodies and Bioengineering Drugs on Their Basis: New Opportunities for Diagnostics and Therapy

“…Путем слияния анти-TNF наноантител были получены двухва-лентные комплексы с повышенной авидностью и более эффективной TNF-нейтрализующей ак-тивностью. Данные комплексы более эффектив-но блокировали TNF по сравнению с коммер-ческими блокаторами на основе классических моноклональных антител (инфликсимаб и ада-лимумаб) [24].…”

New Applications of Nanotechnology for Neuroimaging

“…They consist of heavy chains only and have desirable features such as small size, decreased immunogenicity, and high binding affinities. 56 Nanobodies have been designed for use in cancer therapy, 57,58 antivenoms, 59 inflammatory conditions, 60 and immunoimaging. [60][61][62][63][64] Nanobodies can detect vascular and parenchymal amyloid beta (A␤) deposits in vivo to differentiate cerebral ␤-amyloid indicative of Alzheimer disease and vascular A␤ plaques associated with cerebral amyloid angiopathy.…”

“…56 Nanobodies have been designed for use in cancer therapy, 57,58 antivenoms, 59 inflammatory conditions, 60 and immunoimaging. [60][61][62][63][64] Nanobodies can detect vascular and parenchymal amyloid beta (A␤) deposits in vivo to differentiate cerebral ␤-amyloid indicative of Alzheimer disease and vascular A␤ plaques associated with cerebral amyloid angiopathy. 65 With the transgenic Alzheimer Disease Cerebral Amyloid Angiopathy (AD/CAA) mouse model, 2 distinct A␤ targeting nanobodies, ni3A and pa2H, were administered to evaluate their ability in vivo to differentially detect vascular or parenchymal amyloid-␤ deposits and their ability to cross the blood-brain barrier.…”