Production and characterization of rNGFSP: a recombinant fusion immunogen eliciting dual anti-NGF and anti-Substance P therapeutic antibodies for Degenerative Joint Disease

• A novel recombinant fusion immunogen (rNGFSP) was designed to induce antibodies against both NGF and SP, key mediators of inflammation and chronic pain in OA. • rNGFSP was expressed in E. coli , purified under denaturing conditions, and structurally characterized by mass spectrometry and in silico modeling. • Immunization with rNGFSP in mice, rabbits, horses, and dogs, was safe and elicited robust IgG responses that neutralized NGF and SP biological activities in cell cultures. • rNGFSP administered to mammals as a vaccine represents a cost-effective and scalable biotechnological platform for active immunotherapy for the treatment of degenerative joint disease. Anti-NGF monoclonal antibodies have recently been approved for treating degenerative joint disease, including osteoarthritis pain, in dogs and cats. However, their widespread use is limited by high cost and the requirement for repeated injections. Nerve Growth Factor and Substance P play central roles in the initiation and maintenance of inflammation and chronic pain in OA. There is a pressing need for new, safe, cost-effective therapies that target the underlying mechanisms of OA chronic pain. Here, we designed and produced a novel recombinant fusion protein, termed rNGFSP, which functions as an immunogen due to its unique molecular structure combining amino acid sequences from NGF and SP in a non-native conformation. When formulated and administered as a vaccine, rNGFSP elicits dual anti-NGF and anti-SP therapeutic antibodies in the host. rNGFSP was produced in E. coli and purified from inclusion bodies using metal affinity chromatography under denaturing conditions. Mass spectrometry confirmed the expected molecular weight (17.5 kDa) and preserved amino acid sequence. Structural prediction using Alphafold2 revealed rNGFSP presented a non-natural folding, but a preserved NGF core and a flexible SP tail, supporting antigenic presentation. Vaccination of mice, rabbits, horses, and dogs, showed that rNGFSP elicited cross-reactive IgG antibodies against the native conformations of NGF and SP. Furthermore, immunoglobulins elicited in vaccinated dogs neutralized the biological activity of NGF and SP in cell cultures, suggesting a therapeutic potential. These findings support rNGFSP as a promising vaccine candidate simultaneously targeting endogenous NGF and SP species, providing a cost-effective alternative to monoclonal antibodies.