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

Neurotrophins signal via Trk tyrosine kinase receptors. Nerve growth factor (NGF) is the cognate ligand for TrkA, the brain-derived neurotrophic factor for TrkB, and NT-3 for TrkC. NT-3 also binds TrkA as a lower affinity heterologous ligand. Because neurotrophin-3 (NT-3) interactions with TrkA are biologically relevant, we aimed to define the TrkA "hot spot" functional docking sites of NT-3. The Trk extracellular domain consists of two cysteine-rich subdomains (D1 and D3), flanking a leucine-rich subdomain (D2), and two immunoglobulin-like subdomains IgC1(D4) and IgC2(D5). Previously, the D5 subdomain was defined as the primary ligand-binding site of neurotrophins for their cognate receptors (e.g. NGF binds and activates through TRKA-D5 hot spots). Here binding studies with truncated and chimeric extracellular subdomains show that TRKA-D5 also includes an NT-3 docking and activation hot spot (site 1), and competition studies show that the NGF and NT-3 hot spots on TRKA-D5 are distinct but partially overlapping. In addition, ligand binding studies provide evidence for an NT-3-binding/allosteric site on TRKA-D4 (site 2). NT-3 docking on sites 1 and/or 2 partially blocks NGF binding. Functional survival studies showed that sites 1 and 2 regulate TrkA activation. NT-3 docking on both sites 1 and 2 affords full agonism, which can be additive with NGF activation of Trk. However, NT-3 docking solely on site 1 is partially agonistic but noncompetitively antagonizes NGF binding and activation of Trk. This study demonstrates that Trk signaling is more complex than previously thought because it involves several receptor subdomains and hot spots.

Overexpression of nerve growth factor in peritoneal fluid from women with endometriosis may promote neurite outgrowth in endometriotic lesions

Simple SummaryChronic pain is a major health problem that affects both humans and animals, often reducing quality of life and limiting daily activities. A key player in the development of this type of pain is a substance called Nerve Growth Factor (NGF), which normally helps nerves grow and stay healthy. However, in disease conditions, NGF can become overactive and make pain signals stronger and long-lasting. New treatments called anti-NGF monoclonal antibodies have been developed to block this process. These drugs have already shown promising results in treating painful joint disease, such as osteoarthritis, in animals like dogs and cats. Importantly, early studies suggest that their benefits may go beyond arthritis, potentially helping with other painful conditions such as certain cancers, bladder disease, gut inflammation, and nerve-related disorders. The aim of this review is to highlight how these new drugs might be used more widely in the future, what scientific evidence supports their use, and what challenges still need to be overcome. By expanding treatment options for chronic pain, these therapies could improve wellbeing for both people and animals, reducing suffering and helping patients live more comfortable lives.Nerve Growth Factor (NGF) is a neurotrophin essential for the maintenance and growth of sensory and sympathetic nerve fibers. In pathological conditions, NGF is widely implicated in peripheral and central sensitization mechanisms, significantly contributing to the genesis and maintenance of chronic pain. Anti-NGF monoclonal antibodies, developed for the management of osteoarthritis pain, have demonstrated clinical efficacy and good tolerability in several animal species, particularly dogs and cats. Although their use is currently limited to the management of osteoarthritis pain, preclinical and clinical evidence indicates their potential therapeutic role in other pathological conditions characterized by chronic pain, such as oncological conditions like osteosarcoma, idiopathic cystitis, inflammatory bowel disease, and neuropathies. This review aims to provide an updated overview of the potential clinical applications of anti-NGF monoclonal antibodies beyond osteoarthritis, analyzing their pathophysiological rationale, available scientific evidence, possible therapeutic advantages, and limitations that remain to be addressed.

Nerve growth factor (NGF), a member of the neurotrophic factor family, is involved in mediating the growth, development, differentiation, survival, and damage repair of nerve cells. It also plays an important regulation role in the functional expression of central and peripheral neurons. The existing studies have shown that the interaction of NGF with its receptors plays a critical role in the development and progression of pain. Anti-NGF monoclonal antibody therapy is a promising new therapy for nociceptive and neuropathic pain, and related antibodies have entered clinical trial phase. In this paper, the biological function of anti-NGF monoclonal antibody and progress of related clinical studies are reviewed. Key words: Nerve growth factor; Antibodies, monoclonal; Analgesics

The effects of treatment with L-thyroxine (T4), 2.5 S nerve growth factor (NGF), monoclonal anti-NGF and monoclonal anti-NGF receptor antibodies, separately or together, on the two main processes of cerebellar histogenesis, the disappearance of the external granular layer (egl) and Purkinje cell (PC) morphogenesis, were studied in 10-day-old (1 day after the last injection) and 15-day-old normal and hypothyroid rats. The results provide the following information. (1) Anti-NGF antibodies given to normal rats alter more markedly the growth of PC soma and dendrite than the developmental pattern of egl. In contrast, anti-NGF receptor antibodies mainly delay the disappearance of egl, with minor changes in PC morphogenesis. This is the first evidence for a physiological role of NGF in neuronal maturation in both pre- and postmigratory phases. (2) The delays in the disappearance of egl and hypotrophy of PC due to hypothyroidism are greater than those induced in normal rats by anti-NGF antibodies, and T4 therapy in hypothyroid rats is more effective than that with NGF. The effects of combined T4/NGF treatment on PC size (including soma and dendrite) were approximately the sum of individual effects, with no apparent positive cooperation. Moreover, the effects of NGF treatment, but not those of T4, disappear over the long term. (3) Thyroid deficiency strongly reduces NGF receptor immunoreactivity. Anti-NGF antibodies given to thyroid-deficient rats partly counteract T4 therapy on the cerebellar growth and cortex layering, whereas they potentiate the action of the T4 on the growth of PC nuclei. The PC somas of thyroid-deficient rats assume a normal shape only after T4/NGF treatment. The perisomatic processes of immature PC in thyroid-deficient rats disappear after T4 therapy whereas they grow after NGF treatment. These results strongly suggest that NGF is complementary to thyroid hormone, and that the T4 action is partly mediated and regulated by NGF. Finally, thyroid hormone appears to have a long-term permissive role, while NGF may be a local and short-term limiting neurotrophic factor. Such a balance is essential for ensuring a normal time course of cerebellar histogenesis.

Nerve growth factor (NGF) plays a central role in multiple chronic pain conditions. As such, anti-NGF monoclonal antibodies (mAbs) that function by antagonizing NGF downstream signaling are leading drug candidates for non-opioid pain relief. To evaluate anti-canine NGF (cNGF) mAbs we sought a yeast surface display platform of cNGF. Both mature cNGF and pro-cNGF displayed on the yeast surface but bound conformationally sensitive mAbs at most 2.5-fold in mean fluorescence intensity above background, suggesting that cNGF was mostly misfolded. To improve the amount of folded, displayed cNGF, we used comprehensive mutagenesis, FACS, and deep sequencing to identify point mutants in the pro-region of canine NGF that properly enhance the folded protein displayed on the yeast surface. Out of 1,737 tested single point mutants in the pro region, 49 increased the amount of NGF recognized by conformationally sensitive mAbs. These gain-of-function mutations cluster around residues A-61-P-26. Gain-of-function mutants were additive, and a construct containing three mutations increased amount of folded cNGF to 23-fold above background. Using this new cNGF construct, fine conformational epitopes for tanezumab and three anti-cNGF mAbs were evaluated. The epitope revealed by the yeast experiments largely overlapped with the tanezumab epitope previously determined by X-ray crystallography. The other mAbs showed site-specific differences with tanezumab. As the number of binding epitopes of functionally neutralizing anti-NGF mAbs on NGF are limited, subtle differences in the individual interacting residues on NGF that bind each mAb contribute to the understanding of each antibody and variations in its neutralizing activity. These results demonstrate the potential of deep sequencing-guided protein engineering to improve the production of folded surface-displayed protein, and the resulting cNGF construct provides a platform to map conformational epitopes for other anti-neurotrophin mAbs.

Nerve growth factor is involved in the supportive effect by bone marrow- -derived stromal cells of the factor-dependent human cell line UT-7 [published erratum appears in Blood 1996 Oct 1;88(7):2818

Nerve Growth Factor Is Involved in the Supportive Effect by Bone Marrow-Derived Stromal Cells of the Factor-Dependent Human Cell Line UT-7

Forebrain cholinergic neurons are highly dependent on nerve growth factor (NGF) for phenotype maintenance. We have established that in addition to "target-derived" NGF neurotrophic stimulation, cholinergic neurons also respond dose-dependently, to intra-parenchymal NGF administration in the somato-dendritic region of the nucleus Basalis, thus illustrating the potential of alternative reparative therapies which would by-pass the undesirable effects of diffuse neurotrophin application. Moreover, our lab has also observed that the steady-state number of cortical cholinergic synapses is dependent on continuous NGF supply, as anti-NGF monoclonal antibodies and TrkA receptor antagonists deplete pre-existing cholinergic bouton numbers. Furthermore, the application of either NGF or TrkA NGF-mimetic agonists successfully rescues the age-dependent loss of cortical cholinergic boutons in aged-impaired rats. The vulnerability of the cortical cholinergic system has also been demonstrated in transgenic animal models of the Alzheimer's disease (AD) amyloid pathology. It is of interest to note however, that an up-regulation of cholinergic presynaptic boutons has been observed in certain transgenic mouse models prior to plaque formation. This observation is similar to the visibly increased immunoreactivity of cortical and hippocampal choline acetyltransferase (ChAT) fibers in patients with Mild Cognitive Impairment (MCI). A series of ex-vivo experiments conducted by our group have demonstrated that contrary to popular belief, proNGF, as opposed to mature NGF, is released from the cerebral cortex in an activity-dependent manner. In addition, proNGF appears to be released with a series of pro-enzymes and enzymes, which are involved in its subsequent maturation to NGF and degradation in the extracellular space. Given that proNGF is known to be upregulated in AD patients a dysregulation in the maturation or degradation of mature NGF might explain the preferential vulnerability of the cholinergic system in the AD pathology.

Nerve growth factor (NGF) is a neurotrophin that induces neuritogenic and trophic signals by binding to TrkA and/or p75 receptors. We report a comparative study of the binding, internalization, and biological activity of NGF versus that of NGF in association with an anti-NGF monoclonal antibody (mAb NGF30), directed against the C termini of NGF. NGF.mAb complexes do not bind p75 effectively but bind TrkA with high affinity. After binding, NGF. mAb complexes stimulate internalization faster and to a larger degree than NGF. NGF.mAb-induced activation of TrkA, Shc, and MAPK is transient compared with NGF-induced activation; yet NGF and NGF. mAb afford identical trophic responses. In contrast, NGF induces Suc-1-associated neurotrophic activating protein phosphorylation and neuritogenic differentiation, but NGF.mAb does not. Thus, an absolute separation of trophic and neuritogenic function is seen for NGF.mAb, suggesting that biological response modifiers of neurotrophins can afford ligands with selected activities.

PurposeConsidering the adverse effects of nonsteroidal anti-inflammatory drugs (NSAIDs) and opioids for treating osteoarthritis (OA), development of drugs that are more effective and better tolerated than existing treatments is urgently needed. This systematic review aimed to evaluate the efficacy and safety of anti-nerve growth factor (NGF) monoclonal antibodies vs active comparator therapy, such as NSAIDs and oxycodone, in treating hip or knee OA.MethodsDatabases were comprehensively searched for randomized controlled trials (RCTs) published before January 2022. Efficacy and safety outcomes were assessed.ResultsSix RCTs that included 4325 patients were identified. Almost all the RCTs indicated that moderate doses of anti-NGF monoclonal antibody treatment significantly improved efficacy outcomes based on the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain score, the WOMAC physical function score and the Patient’s Global Assessment compared with those of the active comparator. At least half of the RCTs indicated that the incidence of severe adverse events, withdrawals due to adverse events (AEs) and total joint replacement were not significantly different between anti-NGF monoclonal antibody treatment and active comparator therapy, but the outcomes of some studies may have been limited by a short duration of follow-up. Most RCTs suggested that anti-NGF monoclonal antibody treatment had a lower incidence of gastrointestinal and cardiovascular AEs. However, the majority of RCTs reported a higher incidence of abnormal peripheral sensation with anti-NGF monoclonal antibody treatment. Furthermore, the higher incidence of rapidly progressive osteoarthritis (RPOA) with anti-NGF monoclonal antibody treatment should also not be overlooked, and the identification of patient characteristics that increase the risk of RPOA is critical in further studies.ConclusionBased on the current research evidence, anti-NGF monoclonal antibodies are not yet a replacement for analgesic drugs such as NSAIDs but might be a new treatment option for hip or knee OA patients who are intolerant or unresponsive to nonopioid or opioid treatment. Notably, however, considering the inconsistency and inconclusive evidence on the safety outcomes of recent studies, more research is needed, and long-term follow-up is required.

Background/Aims Osteoarthritis is a major cause of morbidity and disability. Much of this comes from joint pain, which is exacerbated by movement and exercise. Pharmacological analgesia therefore not only has the obvious benefit of alleviating pain, but in doing so, it also facilitates exercise (a pillar of conservative management). Non-steroidal anti-inflammatory drugs (NSAIDs) and opioids are currently the main analgesics used in this context. However, these agents can cause unwanted side effects and are contraindicated in some patients. We thus conducted a systematic review and meta-analysis using the Cochrane collaboration criteria to evaluate the efficacy of anti-nerve growth factor (anti-NGF) antibodies as potential alternative analgesics in osteoarthritis of the hip and/or knee. Whilst tanezumab has been studied extensively and monoclonal anti-NGF antibodies have been reviewed in other pain states, this is the first systematic review of three key anti-NGF antibodies: tanezumab, fulranumab and fasinumab in symptomatic hip and/or knee osteoarthritis. Methods An interdisciplinary work group conducted a literature search across seven electronic databases for the use of anti-NGF antibodies in osteoarthritis. All hip/knee osteoarthritis studies investigating anti-NGF antibodies regardless of dose regimen or phase of trial were included. Studies in which participants received NSAIDs or analgesics other than anti-NGF antibodies, or studies in which the only intervention was the administration of anti-NGF antibodies in combination with NSAIDs or other analgesics were excluded. The Jadad Scale score was used to assess the quality of each study. Results Thirteen studies involving 8,145 patients with a diagnosis of hip and/or knee osteoarthritis were analysed. Demographic information including duration of disease and Kellgren-Lawrence grades were also extracted. Anti-NGF antibodies showed significant improvements compared to placebo as rated on the Western Ontario and McMaster Universities Arthritis Index (WOMAC) scales for pain (SMD= -0.50, 95% CI -0.71 to -0.28, P < 0.00001; I2 = 88%), physical function (SMD= -0.82, 95% CI -1.09 to -0.55, P < 0.00001; I2 = 94%) and stiffness (SMD= -0.88, 95% CI -1.22 to -0.54, P < 0.00001; I2 = 95%). These agents were not associated with a significant increase in serious adverse events but were associated with a significant increase in discontinuation due to adverse events, abnormal peripheral sensations and peripheral neuropathy. Conclusion Anti-NGF antibodies appear very promising with regard to alleviating osteoarthritic hip/knee pain but more studies are needed to determine the optimal dosage and the overall risk-to benefit ratio, particularly with long-term use. Disclosure J. Rammanohar: None. J. Sutton: None. K. Seah: None. W.S. Khan: None. K. To: None.

Role of Low-Affinity Nerve Growth Factor Receptor Inhibitory Antibody in Reducing Pain Behavior and Calcitonin Gene-Related Peptide Expression in a Rat Model of Wrist Joint Inflammatory Pain

Knee Osteoarthritis Pain May Be Neuropathic

Characteristics and Potential Biomarkers of Adult Sickle Cell Patients with Chronic Pain