Generation of a high-fidelity antibody against nerve growth factor using library scanning mutagenesis and validation with structures of the initial and optimized Fab-antigen complexes

Nerve growth factor (NGF) is indispensable during normal embryonic development and critical for the amplification of pain signals in adults. Intervention in NGF signaling holds promise for the alleviation of pain resulting from human diseases such as osteoarthritis, cancer and chronic lower back disorders. We developed a fast, high-fidelity method to convert a hybridoma-derived NGF-targeted mouse antibody into a clinical candidate. This method, termed Library Scanning Mutagenesis (LSM), resulted in the ultra-high affinity antibody tanezumab, a first-in-class anti-hyperalgesic specific for an NGF epitope. Functional and structural comparisons between tanezumab and the mouse 911 precursor antibody using neurotrophin-specific cell survival assays and X-ray crystal structures of both Fab-antigen complexes illustrated high fidelity retention of the NGF epitope. These results suggest the potential for wide applicability of the LSM method for optimization of well-characterized antibodies during humanization.     

Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors

We describe the use of four complementary biosensors (Biacore 3000, Octet QK, ProteOn XPR36, and KinExA 3000) in characterizing the kinetics of human nerve growth factor (NGF) binding to a humanized NGF-neutralizing monoclonal antibody (tanezumab, formerly known as RN624). Tanezumab is a clinical candidate as a therapy for chronic pain. Our measurements were consistent with the NGF/tanezumab binding affinity being tighter than 10 pM due to the formation of an extremely stable complex that had an estimated half-life exceeding 100 h, which was beyond the resolution of any of our methods. The system was particularly challenging to study because NGF is an obligate homodimer, and we describe various assay orientations and immobilization methods that were used to minimize avidity in our experiments while keeping NGF in as native a state as possible. We also explored the interactions of NGF with its natural receptors, TrkA and P75, and how tanezumab blocks them. The Biacore blocking assay that we designed was used to quantify the potency of tanezumab and is more precise and reproducible than the currently available cell-based functional assays.     

Dissecting NGF interactions with TrkA and p75 receptors by structural and functional studies of an anti-NGF neutralizing antibody

The anti-nerve growth factor (NGF) monoclonal antibody αD11 is a potent antagonist that neutralizes the biological functions of its antigen in vivo. NGF antagonism is expected to be a highly effective and safe therapeutic approach in many pain states. A comprehensive functional and structural analysis of αD11 monoclonal antibody was carried out, showing its ability to neutralize NGF binding to either tropomyosine receptor kinase A (TrkA) or p75 receptors. The 3-D structure of the αD11 Fab fragment was solved at 1.7 Å resolution. A computational docking model of the αD11 Fab-NGF complex, based on epitope mapping using a pool of 44 NGF mutants and experimentally validated by small-angle X-ray scattering, provided the structural basis for identifying the residues involved in αD11 Fab binding. The present study pinpoints loop II of NGF to be an important structural determinant for NGF biological activity mediated by TrkA receptor.     

Neutralization of NGF-TrkA receptor interaction by the novel antagonistic anti-TrkA monoclonal antibody MNAC13: a structural insight

MNAC13, a mouse monoclonal antibody, recognizes with high affinity and specificity the neurotrophin receptor TrkA and displays a neutralizing activity toward the NGF/TrkA interaction. Detailed knowledge of the molecular basis determining the specificity of this antibody is of importance because of its potential use as a modulator of the TrkA-mediated NGF activity. Here, we report a full biochemical and structural characterization of the MNAC13 antibody. Epitope mapping studies, by serial deletion mutants and by phage display, reveal a conformational epitope that is localized on the carboxy-terminal region of the first immunoglobulin-like domain (d4) of TrkA. The X-ray crystal structure of the MNAC13 Fab fragment has been determined and refined to 1.8 A resolution. The antigen-binding site is characterized by a crevice, surrounded by hydrophilic-charged residues on either side, dipping deep toward three mainly hydrophobic subsites. Remarkably an isopropanol molecule has been found to bind in one of the hydrophobic crevices. Overall, the surface topology (shape and electrostatic potential) of the combining site is consistent with the binding data on TrkA ECD serial deletions mutants. The structure of the MNAC13 Fab fragment may assist in the rational structure-based design of high affinity humanized forms of MNAC13, appropriate for therapeutic approaches in neuropathy and inflammatory pain states.     

Large scale purification and immunological characterization of human placental nerve growth factor

Nerve growth factor (NGF) is a protein which plays a critical role in the development and survival of not only peripheral neurons, but possibly also cholinergic brain neurons. The present study describes a procedure for large scale isolation of human NGF of placental origin, and its immunological characterization. A protein species of approximately 26 kDa was obtained, which crossreared characterization. A protein species of approximately 26 kDa was obtained, which crossreacted with antibodies to mouse NGF. Polyclonal and monoclonal anti-mouse NGF antibodies appeared to recognize different bands within this human NGF preparation. Although these polyclonal antibodies recognized both the dimeric and monomeric forms of mouse NGF, the monoclonal antibody recognized only a band corresponding to the dimeric form of mouse NGF.     

The Distribution of Nerve Growth Factor in the Male Sex Organs of Mammals

Abstract: The Nerve Growth Factor (NGF) content of male sex organs of the mouse, rat, guinea pig, hamster, rabbit, human, and bull has been investigated using both a biological assay and a two-site radioimmunoassay. The prostate glands of the rabbit and bull have been found to contain moderate levels of NGF, these being lower than the concentrations found in the guinea pig prostate and mouse submaxillary glands. The sex organs investigated of the mouse, rat, hamster, and human contained no detectable NGF activity. Genital organs, other than the prostate glands, of the guinea pig and rabbit were also devoid of NGF. The NGFs from the rabbit and bull are immunologically related to those found in the submaxillary glands of the mouse and the prostate glands of the guinea pig, but immunodiffusion and radioimmunoassay experiments show that there are also clear differences between the NGFs. The use of a two-site radioimmunoassay, based on purified antibodies against mouse submaxillary gland NGF, for the determination of NGF levels in species other than the mouse, is described. It is essential during such applications to compensate for the fact that the NGFs from different species are sufficiently distinct that only part of the antibody population (raised against mouse NGF) is capable of recognizing NGF from species other than the mouse. The results of radioimmunoassay and biological assay determinations are in reasonable agreement, if corrections for this feature are made.     

A Highly Sensitive Enzyme Immunoassay for Mouse β Nerve Growth Factor

Abstract: A sensitive two-site enzyme immunoassay system for mouse β nerve growth factor (NGF) was developed, based on the sandwiching of the antigen between anti-mouse β NGF antibody IgG coated to a polystyrene tube and anti-mouse β NGF antibody Fab'-linked β- d -galactosidase (β- d -galactoside hydrolase, EC 3.2.1.23). This method has the following advantages: (a) the procedures are simple and rapid compared to bioassay or two-site radioimmunoassay; (b) antibody Fab'-β- d -galactosidase complex is more stable than ¹²⁵I-labeled antibody; (c) purified β NGF is detectable at a concentration as low as 10 pg/ml. Our enzyme immunoassay was used to examine the levels of NGF in some tissues of mice. The submaxillary gland contained a high concentration of NGF. However, other tissues, such as the heart, brain, and skeletal muscle, and serum did not contain detectable NGF. These results support recent findings by other investigators that NGF was not found in the organs/tissues other than the submaxillary gland of mice.     

Highly sensitive enzyme immunoassay for beta-nerve growth factor (NGF): a tool for measurement of NGF level in rat serum

A sensitive two-site enzyme immunoassay (EIA) system was established for mouse beta nerve growth factor (NGF) isolated from mouse submaxillary gland. Our EIA system is based on the sandwiching of antigen between anti-mouse beta NGF antibody IgG coated on a polystyrene plate and biotinylated anti-mouse beta NGF antibody IgG. The bound antibody complex was quantified with streptavidin linked-beta-D-galactosidase (beta-D-galactosidase, EC 3.2.1.23). With this system NGF concentrations as low as 0.02 pg/well (corresponding to 8 x 10(-19) mol) could be measured reproducibly. The sensitivity of this EIA system permitted the quantification of endogenous immunoreactive beta NGF in rat serum. The mean level in serum of male rats (153.2 pg/ml) was found to be almost the same as that of female rats (127.6 pg/ml).     

Expression of recombinant human nerve growth factor in Escherichia coli.

Nerve growth factor (NGF) is a neurotrophic factor for basal forebrain cholinergic neurons and may be of benefit in neurodegenerative diseases of humans. A method is described to obtain significant amounts of biologically active recombinant human NGF (rhNGF) in one step. RhNGF was expressed in E. coli and the majority of the protein accumulated in inclusion bodies. It was immunoprecipitated by a serum against mouse NGF. Solubilization of the inclusion bodies was done in 3M guanidine HCl and renaturation was effected by dilution and air oxidation in the presence of 6 microM CuSO4. Recoveries were 10-12 micrograms of rhNGF per ml of bacterial suspension. Its biological activity was tested in a bioassay system employing sympathetic chick embryo ganglia and was inhibited by the monoclonal antibody 27/21 against mouse NGF.     

Association of antibody to nerve growth factor with ganglionic non-neurons (glia) and consequent interference with their neuron-supportive action

Most neurons dissociated from neonatal mouse dorsal root ganglia require for their attachment, neurite production, and survival in monolayer cultures, either the administration of exogenous Nerve Growth Factor (NGF) or the availability of ganglionic non-neuronal (glial) cells in adequate numbers. In the glia-supported cultures, the neuronal performance is drastically impaired by the presence of antibody against mouse submaxillary NGF, despite the assumed absence of such factor, implying the occurrence and functional importance in the test system of an NGFlike antigen. The present studies demonstrate that the same impairment of neuronal performance can be induced by pretreating with antibody the non-neuronal supplement prior to its presentation to the test neurons. The immune pretreatment achieved maximal effectiveness with antibody concentrations comparable to those needed for maximal interference during continuous presence of the antibody, and equivalent to those capable of blocking one unit of NGF in the standard NGF bioassay. Effective pretreatment required a minimum of 45 min of immune exposure, was unaffected by the presence or absence of ten-fold excess of fetal calf serum protein and appeared to resist brief exposure to trypsin. When antibody-pretreated non-neurons were reincubated in antibody-free medium before being used in the neuronal test system, they retained their full ability to impair neuronal performance for at least 4 h and lost it gradually thereafter over a 20-h period. The loss of interfering ability was accompanied by the appearance of ‘interfering ’ activity in the reincubation medium to a maximal level that matched the apparent depletion of active material noticeable in the medium initially used to pretreat the ganglionic nonneurons.     

Antibodies against mouse nerve growth factor interfere in vivo with the development of avian sensory and sympathetic neurones

The monoclonal antibody 27/21 directed against mouse nerve growth factor (NGF) interferes in vivo with the survival of sensory dorsal root ganglion (DRG) neurones during the development of the quail embryo: the number of DRG neurones at embryonic day 11 (E11) was reduced by about 30% in embryos treated with the antibody between E3 and E11. Neurone numbers in the nodose ganglion were not affected. The effect of NGF antibodies on sympathetic neurones was assessed by determining the levels of the adrenergic marker enzyme tyrosine hydroxylase. Both total tyrosine hydroxylase activity and protein levels in sympathetic chains were reduced by about 30% in embryos treated with 27/21 antibody but not in embryos treated with a control antibody. The 27/21 antibody cross-reacts with chick NGF-like activity as shown in vitro by the ability of the antibody to partially block the survival activity of chick-embryo-fibroblast-conditioned medium for E9 chick DRG neurones.     

Nerve growth factor: biosynthetic products of the mouse salivary glands. Characterization of stable high molecular weight and 32,000-dalton nerve growth factors

Nerve growth factor (NGF) is a protein required for the growth and development of sensory and sympathetic neurons. The NGF is present in high concentrations in male mouse salivary glands, bovine seminal plasma, and snake venom. The physiological significance of NGF in these sources is not known: it might be a part of a high molecular weight (HMW) protein with possibly different biological function and be cleaved to the functional size by proteases. In an attempt to isolate a HMW protein containing as part of its structure the low molecular weight (LMW) NGF (2.5S), mouse salivary glands were homogenized in the presence of either 8 M urea or 6 M guanidine hydrochloride (Gdn X HCl) in order to denature proteases. This procedure revealed that the LMW NGF is a part of two HMW proteins that are biologically and immunologically homologous to the mouse 2.5S NGF. One of these HMW proteins (Mr 32,000 NGF) was purified and shown to be biologically active in the NGF bioassay. Furthermore, this Mr 32,000 NGF was cleaved by the gamma subunit of mouse HMW NGF to the 2.5S NGF. Evidence is also presented that there may be a HMW protein(s) with apparent molecular weights ranging from 94,000 to 200,000 and immunologically homologous to the three subunits (alpha, beta, gamma) of 7S NGF. This HMW NGF is biologically active in the NGF bioassay, and its activity is inhibited by antibody to the beta subunit. Furthermore, in contrast to mouse 7S NGF, this HMW NGF does not dissociate in either 8 M urea or 6 M Gdn X HCl.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single cycle structure-based humanization of an anti-nerve growth factor therapeutic antibody

Most forms of chronic pain are inadequately treated by present therapeutic options. Compelling evidence has accumulated, demonstrating that Nerve Growth Factor (NGF) is a key modulator of inflammatory and nociceptive responses, and is a promising target for the treatment of human pathologies linked to chronic and inflammatory pain. There is therefore a growing interest in the development of therapeutic molecules antagonising the NGF pathway and its nociceptor sensitization actions, among which function-blocking anti-NGF antibodies are particularly relevant candidates.In this respect, the rat anti-NGF αD11 monoclonal antibody (mAb) is a potent antagonist, able to effectively antagonize rodent and human NGF in a variety of in vitro and in vivo systems. Here we show that mAb αD11 displays a significant analgesic effect in two different models of persistent pain in mice, with a remarkable long-lasting activity. In order to advance αD11 mAb towards its clinical application in man, anti-NGF αD11 mAb was humanized by applying a novel single cycle strategy based on the a priori experimental determination of the crystal and molecular structure of the parental Fragment antigen-binding (Fab). The humanized antibody (hum-αD11) was tested in vitro and in vivo, showing that the binding mode and the NGF neutralizing biological activities of the parental antibody are fully preserved, with even a significant affinity improvement. The results firmly establish hum-αD11 as a lead candidate for clinical applications in a therapeutic area with a severe unmet medical need. More generally, the single-cycle structure-based humanization method represents a considerable improvement over the standard humanization methods, which are intrinsically empirical and require several refinement cycles.     

神经生长因子的发光免疫分析法

介绍了一种灵敏的神经生长因子化学发光免疫测定方法。小鼠神经生长因子（ｍＮＧＦ）的抗体ＩｇＧ用亲和层析纯化并用吖啶酯标记。该法可用于大鼠组织中ＮＧＦ含量的测定。方法的灵敏度为１０ｐｇ／ｍＬＮＧＦ;批内批间变异系数分别为８．７％及１３．２％;回收率平均为１０３％。ｍＮＧＦ抗体对人的ＮＧＦ也有极强的交叉反应,故本方法也可能用于病人血清或脑脊液中ＮＧＦ含量的测定。     

Production of a monoclonal antibody directed against the nerve growth factor receptor from sympathetic membranes

Spleen cells from BALB/c mice immunized with a plasma membrane-enriched fraction from rabbit sympathetic ganglia were fused with the mouse myeloma NS1. A hybrid clone was obtained that produced monoclonal antibody directed against the receptor for nerve growth factor (NGF). The antibody, identified as IgG, was able to immunoprecipitate solubilized NGF receptor in the presence or absence of bound NGF. The antibody bound specifically to sympathetic membranes with high affinity but did not affect the binding of 125I-NGF to its receptor in sympathetic or sensory neurons or PC12 cells.     

Development of sensitive enzyme immunoassay for human nerve growth factor.

We prepared anti-recombinant human nerve growth factor (hNGF) antibody IgG and characterized its property immunologically. This antibody IgG reacted with some animal NGFs, especially with bovine NGF, on immunodiffusion analysis. Using this antibody IgG, we developed a sensitive two-site enzyme immunoassay (EIA) system for human NGF, based on the biotin-streptoavidin system. NGF at a concentration as low as 0.02 pg/well (corresponding to 8 x 10(-19) mol) could be measured with high reproducibility. The sensitivity of this EIA was equal to that of our EIA for mouse NGF. With this EIA, the detection limit of other mammalian NGFs was reduced in parallel with the degree of decrease in amino acid sequence homology between them and hNGF.     

Nerve Growth Factor of Red Nucleus Involvement in Pain Induced by Spared Nerve Injury of the Rat Sciatic Nerve

Nerve growth factor (NGF), a member of the neurotrophin family, is essential for the development and maintenance of sensory neurons and for the formation of central pain circuitry. The current study was designed to evaluate the expression of NGF in the brain of rats with spared nerve injury (SNI), using immunohistochemical technique. The results showed that the level of NGF in the Red nucleus (RN) of SNI rats was apparently higher than that of sham-operated rats. To further study the effect of NGF in the development of neuropathic pain, different doses of anti-NGF antibody (20, 2.0 and 0.2 μg/ml) were microinjected into the RN contralateral to the nerve injury side of SNI rats. The data suggested that the higher doses of anti-NGF antibody (20 and 2.0 μg/ml) significantly attenuated the mechanical allodynia of neuropathic rats, while the 0.2 μg/ml antibody showed no analgesic effect. These results suggest that the NGF of RN is involved in the development of neuropathic allodynia in SNI rats.     

Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase

Summary The production of a bi-specific monoclonal antibody that simultaneously recognizes mouse kappa light chains and horseradish peroxidase (HRP) for use as a general developing reagent in a wide variety of immunobased techniques is described. This antibody, named McC10, was produced by the fusion of an aminopterin-sensitive interspecies hybridoma which secretes rat monoclonal antibodies against HRP (RAP₂·Ag) and splenocytes from a rat immunized with whole mouse immunoglobulin (Ig)G. The hybrid-hybridoma generated from this fusion expresses and secretes rat Igs of the IgG₁ and IgG_2a subclasses, as determined by radial immunodiffusion. In competitive binding solid-phase enzymatic assays, McC10 was found to cross-react with all four mouse IgG subclasses as well as mouse kappa light chains. In contrast, in this type of assay, McC10 did not appear to recognize mouse IgA, IgM or lambda light chains. However, IgM-bearing kappa light chains were recognized by immunocytochemistry. Epitope specificity of this bi-specific antibody was more clearly determined on immunoblots where McC10 was found to exclusively recognize mouse kappa light chains and display no cross-reactivity with mouse Ig heavy chains nor with kappa light chains from rat or rabbit. In addition, McC10 was used successfully in two-step immunocytochemistry (ICC) for the localization of enkephalin, nerve growth factor (NGF) receptor and paired helical filament-immunoreactive sites in rat brain, rat skin and human brain, respectively, using mouse IgG's and IgM's as primary antibodies. McC10 compared favourably with peroxidase-anti-peroxidase (PAP) ICC with respect to sensitivity but was markedly superior with respect to specificity when used in fixed human brain or rat skin. This study demonstrates some of the potential advantages of using an epitope specific monoclonal bi-specific developing reagent like McC10 in an immunobased technique like ICC. Its potential use in a variety of other immunobased procedures is discussed.     

Inhibition of bovine sperm‐oocyte fusion by a monoclonal antibody recognising the TEC‐2 epitope on bovine oocytes

The TEC‐2 antigenic determinant is a carbohydrate epitope located on a glycoprotein carrier molecule. In the mouse, this epitope is expressed on the zona pellucida and plasma membrane of the oocyte and is associated with the ZP2 glycoprotein and involved in the secondary sperm receptor mechanism. On the bovine oocyte expression is confined to the plasma membrane. The aim of this study was to determine the role the TEC‐2 epitope plays during fertilization in the bovine species using the monoclonal antibody TEC‐02. Incubating oocytes with the TEC‐02 antibody prior to fertilization inhibited cleavage in a dose‐dependent manner—the cleavage rate decreased as the concentration of the antibody increased. Significantly more sperm were bound to oocytes exposed to TEC‐02 (12 sperm/oocyte) compared to oocytes that were not incubated with the antibody (4 sperm/oocyte). Oocytes treated with the TEC‐02 antibody had a 7.5 ± 3.2% fusion rate and no cortical granule exocytosis compared with oocytes not exposed to the antibody, with 86.5 ± 5.8% of sperm‐oocyte fusions and release of cortical granules. The block to sperm‐oocyte fertilization observed in the pretreated group was overcome using intracytoplasmic sperm injection as the method of fertilization that bypassed the fusion process. Although sperm were binding to the oolemma these results suggest that fusion was not occurring and this may be due to the antibody occupying TEC‐2 epitope sites involved in the fusion process. In conclusion, the TEC‐2 epitope seems to be involved in sperm‐oocyte interaction in the bovine species and appears to be involved specifically during the fusion events of fertilization. Mol. Reprod. Dev. 54:173–178, 1999. © 1999 Wiley‐Liss, Inc.     

Nerve Growth Factor Regulates Neurolymphatic Remodeling during Corneal Inflammation and Resolution

The cellular and physiologic mechanisms that regulate the resolution of inflammation remain poorly defined despite their widespread importance in improving inflammatory disease outcomes. We studied the resolution of two cardinal signs of inflammation–pain and swelling–by investigating molecular mechanisms that regulate neural and lymphatic vessel remodeling during the resolution of corneal inflammation. A mouse model of corneal inflammation and wound recovery was developed to study this process in vivo. Administration of nerve growth factor (NGF) increased pain sensation and inhibited neural remodeling and lymphatic vessel regression processes during wound recovery. A complementary in vivo approach, the corneal micropocket assay, revealed that NGF-laden pellets stimulated lymphangiogenesis and increased protein levels of VEGF-C. Adult human dermal lymphatic endothelial cells did not express canonical NGF receptors TrkA and p75^NTR or activate downstream MAPK- or Akt-pathway effectors in the presence of NGF, although NGF treatment increased their migratory and tubulogenesis capacities in vitro. Blockade of the VEGF-R2/R3 signaling pathway ablated NGF-mediated lymphangiogenesis in vivo. These findings suggest a hierarchical relationship with NGF functioning upstream of the VEGF family members, particularly VEGF-C, to stimulate lymphangiogenesis. Taken together, these studies show that NGF stimulates lymphangiogenesis and that NGF may act as a pathogenic factor that negatively regulates the normal neural and lymphatic vascular remodeling events that accompany wound recovery.