DESIGN OF HORMONE-LIKE ANTIBODIES WITH AGONISTIC AND ANTAGONISTIC FUNCTIONS

Abstract: The present invention relates to an agonistic anti-human TrkA mAb 5C3 which recognizes the NGF docking site. Such antibodies may be used for the treatment, diagnosis or prevention of neurological diseases, neuromas and neoplastic tumors which express TrkA receptors. Also these antibodies may be used to develop and screen for pharmaceutical agents which are agonistic or antagonistic to NGF binding to the TrkA receptors.

Description: DESIGN OF HORMONE-LIKE ANTIBODIES WITH AGONISTIC AND ANTAGONISTIC FUNCTIONS BACKGROUND OF THE INVENTION (a) Field of the Invention The invention relates to a method of designing agonistic and/or antagonistic antibodies for any hor¬ mone receptor more specifically antibodies which are capable of blocking nerve growth factor (NGF) binding and antibodies which can mimic NGF binding to its receptor. (b) Description of Prior Art The TrkA receptor is a 140 kDa transmembrane glycoprotein with tyrosine kinase activity that func- tions as the Nerve Growth Factor (NGF) receptor. NGF also binds with low affinity to a p75 receptor whose signaling function is unclear. Homo or heterodimers or oligomers of TrkA and p75 bind NGF with higher affin¬ ity (Jing, S. et al. (1992) Neuron , 9: 1067-1079) suggesting that specific receptor conformations may play specific functions. NGF promotes the differentiation of certain neuronal cells, is itogenic for TrkA-transfected fibroblasts, and allows survival in serum-deprived conditions for both cell types. Activation of the tyrosine kinase activity of TrkA via NGF binding leads to receptor trans- and auto-tyrosine phosphorylation (PY), and PY of second messengers including phos- phatidylinositol-3 kinase (PI-3 kinase). PI-3 kinase is involved in protein trafficking and endocytosis of ligand-receptor complexes (reviewed by Kaplan, D.R. et al. (1994) J. Neurobiol . , 25: 1404-1417). Since microinjection of NGF into cells does not result in NGF biological signals, cell surface receptor ligation and internalization of TrkA or NGF-TrkA complexes must mediate these effects. TrkA, like most kinase growth factor receptors, signals through receptor oligomerization. Thus, mono¬ valent TrkA-binding agents are antagonistic or have no biological effects (LeSauteur, L. et al.(1995) J. Biol . Chem. , 270:6564-6569), whereas bivalent receptor-binding agents such as NGF (a homodimer) or antibodies can be agonistic. The principle of using polyclonal antibodies to activate neural receptors has been demonstrated (Twyman, R.E. et al. (1995) Neuron, 14: 755-762). In contrast, only a limited number of anti-receptor monoclonal antibodies (mAb) mimic ligand functions (Taub, R. et al. (1992) Biochemistry, 31: 7431-7435), and none exist against neurotrophin receptors. It would be highly desirable to be provided with an agonistic or antagonistic anti-human TrkA mAb which recognizes the NGF docking site. Such antibod¬ ies may be used for the diagnosis, treatment or pre¬ vention of neurological diseases, neuromas and neo- plastic tumors which express TrkA receptors. Also these antibodies may be used to develop and screen for pharmaceutical agents which are agonistic or antago¬ nistic by binding to the TrkA receptors. SUMMARY OF THE INVENTION One aim of the present invention is to report the development and characterization of an agonistic anti-human TrkA mAb 5C3 which recognizes at least one NGF docking site. This MAb 5C3 was used to characterize the pattern of TrkA protein expression in normal human brain, and the NGF binding features of the receptor. MAb 5C3 behaves like NGF in bioassays, and monomeric 5C3 F a bs retained binding and functional agonistic activity. MAb 5C3 will be useful to identify the NGF docking site on TrkA and possibly as a pharmacological lead in the development of small mimetics. In accordance with the present invention there is provided an antibody or functional fragment thereof which binds to at least the TrkA receptor under physi¬ ological conditions, and wherein the binding to the receptor at least partially mimics or inhibits nerve growth factor biological activity. In accordance with the present invention there is also provided a method of screening pharmacological agents which are capable to mimic or inhibit nerve growth factor biological activity, which comprises using the antibody of the present invention to screen for pharmacological agents capable of binding to the complementary determining region of the antibody, wherein the screened pharmacological agents can mimic or inhibit nerve growth factor biological activity. In accordance with the present invention there is also provided the use of pharmacological agents obtained by the process of the present invention, for the in vi vo inhibition of nerve growth factor binding to TrkA receptor or the internalization or down odula- tion of the receptor. In accordance with the present invention there is also provided the use of the antibody of the pre¬ sent invention, for the in vi vo inhibition of nerve growth factor binding to TrkA receptor or the inter¬ nalization or downmodulation of the receptor, such as for inhibiting tumor growth in si tu, for the treatment or prevention of neurological diseases, neuromas and neoplastic tumors which express TrkA receptors, for mapping hormone-receptor interactive sites and recep¬ tor domain-function correlation such as mapping TrkA docking sites, for screening pharmacological agents which bind to the hormone-receptor interactive sites. In accordance with the present invention there is also provided an antibody having CDR-like domains of hormones, wherein the antibody or functional frag¬ ment thereof binds to at least TrkA receptor under physiological conditions, and wherein the binding to the receptor at least partially mimics or inhibits nerve growth factor biological activity. In accordance with the present invention there is also provided a method for the treatment of neuro- logical diseases, neuromas and neoplastic tumors which express TrkA receptors in a patient, which comprises administering an effective amount of an antibody of the present invention or a functional fragment thereof to a patient. In accordance with the present invention there is also provided a pharmaceutical composition for the treatment of neurological diseases, neuromas and neo¬ plastic tumors which express TrkA receptors, which comprises an effective amount of an antibody of the present invention or a functional fragment thereof in association with a pharmaceutically acceptable car¬ rier. In accordance with the present invention there is also provided a method for immunization of a mammal against an antibody of the present invention or a functional fragment thereof, which comprises adminis¬ tering by systemic injection an immunizing amount of at least one of the antibody or the fragment thereof in an immunogenic form in association with a phar a- ceutically acceptable carrier. In accordance with the present invention there is also provided a method for the prognosis or diagno¬ sis of human tumors which comprises: a) biopsy and immunocytochemistry of tumors using the antibody of the present invention and fragments thereof; or b) radiolabeling of the antibody of the present invention and fragments thereof and nuclear imaging analysis. In accordance with the present invention there is also provided a method for the treatment of human tumor of a patient which comprises the steps of: a) coupling cytotoxic agents to the antibody of the present invention and fragments thereof; b) administering the coupled antibody of step a) to the patient. In accordance with the present invention there is also provided a pharmaceutical composition for the targeting of pharmaceutical agents to tissues of the central and/or peripheral nervous system, which comprises an effective amount of an antibody of the present invention or a functional fragment thereof coupled to a pharmaceutical agent in association with a pharmaceutically acceptable carrier. The pharmaceutical agent may be selected from the group consisting of radioligands, nucleic acid molecules, toxins, growth factors and gangliosides. In accordance with the present invention there is also provided a TrkA docking site which binds to the antibody of the present invention. In accordance with the present invention there is also provided the use of the docking site of the present invention for screening pharmacological agents which are capable to mimic or inhibit nerve growth factor biological activity and said antibody biological antibody. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates surface immunofluorescence studies with mAb 5C3; Fig. 2 illustrates the direct detection of pl40 TrkA by western blotting; Fig. 3 illustrates TrkA immunoreactivity in normal human brain; Fig. 4 illustrates the induction of TrkA-tyro- sine phosphorylation by 5C3; Fig. 5 illustrates 5C3 binding studies and Scatchard plot analysis; Fig. 6 illustrates the protection from apop¬ totic death by 5C3 and 5C3 Fabs; Fig. 7 illustrates nuclear imaging of tumors in vi vo with 5C3; Fig. 8 illustrates the survival of TrkA- expressing cells in serum-free media by 5C3 and derivatives; Fig. 9 illustrates the differentiation/neurito- genesis of human TrkA-expressing cells in serum media; Fig. 10 illustrates Mab5C3 prevents TrkA- expressing tumor growth in vi vo ; and Fig. 11 illustrates the topography of the CDRs of the present invention. DETAILED DESCRIPTION OF THE INVENTION Monoclonal antibody (mAb) 5C3 directed against human pl40 TrkA is a structural and functional mimic of NGF and an artificial receptor agonist. MAb 5C3 binds in the Nerve Growth Factor (NGF) docking site, and like NGF it promotes TrkA internalization; TrkA and phosphatidylinositol-3 kinase tyrosine phosphory¬ lation; and increased transformation of TrkA-expressing fibroblasts. More importantly, mAb 5C3 protects human TrkA-expressing cells from apoptotic death in serum-free media. Interestingly, agonistic activity is observed with monomeric F a b 5C3 fragments. The affinity of mAb 5C3 is ~2 nM and was used to study features of ligand binding by TrkA and the distribu- tion of TrkA protein in normal human brain. Antibodies: Female Balb/c mice were immunized with human TrkA, and splenocytes fused to SP2/0 myelomas. Hybri¬ domas were screened by differential binding between untransfected and TrkA-transfected cells using a Fluo¬ rescent Activated Cell Scanner™ (FACScan) (Becton Dickinson, CA) . MAb 5C3 was identified and subcloned 3 times. Rat anti-mouse IgG (αmlgG) (Sigma, Saint Louis, MO), anti-phosphotyrosine mAb 4G10 (UBI, Lake Placid, NY), and anti-PI-3 kinase polyclonal serum (UBI) were purchased, mouse anti-rat p75 mAb MC192 ascites were a gift from P. Barker, and anti-p65 mAb 87.92.6 was grown in the laboratory. Monomeric mAb 5C3 Fabs : MAb 5C3 was purified (1 mg/ml) with Protein G-Sepharose™ (Sigma) and digested with papain (10 μg/ml) (GIBCO, Toronto, Ontario). F a bs were repuri- fied on KappaLock-Sepharose™ (UBI) and Protein G- Sepharose™ and dialyzed against phosphate buffered saline (PBS). All products were characterized by SDS- PAGE under non-reducing or reducing conditions (100 M 2-mercaptoethanol) to >98% purity. Control F a bs from anti-rat p75 mAb MC192 were similarly prepared. Cell lines: Mouse SP2/0 myelomas, mouse Rl.l and EL4 thymo- mas, mouse NIH-3T3 fibroblasts, mouse 2B4 T cell hybridomas, NGF responsive-rat PC12 pheochromocytoma cells, human Jurkat T lymphomas, and human HeLa fibro¬ blasts were used. NIH-3T3 cells transfected either with human pl40 trkA cDNA (E25 cells), with p75 cDNA (Z91 cells), or p75 and pl40tr Λ cDNAs (R7 cells) (Jing, S. et al . (1992) Neuron , 9: 1067-1079). The trk negative rat B104 neuronal cell line (expressing endogenous rat p75), and B104 transfected with human trkA cDNA (4-3.6 cells, expressing human TrkA and rat p75) were kindly provided by Dr. E. Bogenmann (Bogenmann, E. et al. (1995) Oncogene, 10: 1915-1925). PC12 cells transfected with human trkA cDNA were obtained from Dr. Kaplan. All cells were cultured in RPMI media supplemented with 5-10% Fetal Bovine Serum (FBS) and antibiotics (GIBCO). Transfectants were added the appropriate drug selection. FACScan: 5x10-*- cells in 0.1 ml of binding buffer (Hanks' Balanced Salt Solution (HBSS), 0.1% BSA, 0.1% aN3 ) were incubated with the indicated concentration of Abs or F a bs f° r 30 min at 4°C, washed in binding buffer to remove excess primary antibody, and immu¬ nostained with fluorescinated (FITC) goat anti-mouse IgG (FITC-GαmlgG) , or anti-mouse Fab (FITC-GαmF a fc>) (Sigma) secondary antibody for 30 min at 4°C. Cells were acquired and analyzed on a FACScan (Becton Dick¬ inson, CA) using the LYSIS II™ program. As negative controls (background fluorescence) either mouse IgG (Sigma), mAb 192 or 192 F a fc> s were used as primary, followed by appropriate secondary. Characterization of mAb 5C3: FACScan analysis of non-permeabilized cells demonstrated that mAb 5C3 recognizes the extracellular domain of human TrkA receptors (Table 1, Fig. IA) . TABLE 1 Surface phenotyping with mAb 5C3 CELLS 5C3 BINDING E25 (hTrkA) +++++ R7 (hTrkA/p75) +++ Z91 (p75) 4-3.6 (hTrkA/p75) +++ B104 (p75) PC12 (rTrkA/p75) transient NIH-3T3 transfections h trkA cDNA ++ h trkB cDNA rtrkB cDNA rtrkA cDNA - The indicated cell lines expressing human TrkA (hTrkA), rat TrkA (rTrkA) and/or p75 were analyzed by surface immunofluorocyto etry with mAb 5C3 versus con¬ trol mlgG. Transient transfections (48 hrs) were done by eiectroporation of cDNAs. Relative intensities of staining are indicated as +++++ (high staining) or - (no staining) (see Fig. 1). Saturating doses of mAb were used, and differences represent receptor number. Other cells tested include wild type NIH-3T3, Jurkat, Rl.l, EL4, 2B4, and HeLa cells which are all negative. Human TrkA-transfectant lines 4-3.6, E25, and R7, bound mAb 5C3. In contrast, rat PC12 (expressing rat TrkA and rat p75), rat B104 (parental cells of 4- 3.6, expressing rat p75), Z91 (NIH-3T3 transfected with p75), wild type NIH-3T3, or NIH-3T3 cells tran- siently transfected with human trkB, rat trkA, or rat trkB cDNA did not bind mAb 5C3. Thus, mAb 5C3 is spe¬ cific for human TrkA, and co-expression of rat or human p75 does not interfere with binding. The concentration of mAb 5C3 required to satu¬ rate TrkA receptors was determined in E25 cells by testing increasing amounts of antibody in FACScan assays (Fig. IA) . Similar analysis with mAb 5C3 F a fc > s demonstrated that specificity and saturability were similar to that obtained with intact mAb but that 3-fold lower F a b protein concentrations were required (Fig. IB). Since the molecular weight of 5C3 F a is 3-fold lower than 5C3 IgG (~50 versus ~150 kDa), equi- molar concentrations were required for saturation. E25 cells expressing human TrkA were analyzed by indirect immunofluorescence in a FACScan with vari¬ ous doses of mAb 5C3 or 5C3 F arS to assess ligand con¬ centrations that achieve receptor saturation. (Fig. IA) mAb 5C3 doses: 0.02 μg/ml (thick line, c); 0.2 μg/ml (thin line, b); 2 μg/ml (dotted line, a). For background fluorescence mlgG at 2 μg/ml (crossed line, d) was used. (Fig. IB) 5C3 F aDS doses: 0.007 μg/ml (thick line, c); 0.07 μg/ml (thin line, b); 0.7 μg/ml (dotted line, a). For background fluorescence 192 F aD at 0.7 μg/ml (crossed line, d) was used. Increased fluorescence intensity (X axis of histograms) reflect increased staining by mAb 5C3 or 5C3 a b s . The areas under the curves represent the total number of cells acquired for each sample (constant 5000 cells). Histogram heterogeneity is due to individual cell receptor density. Western blot analysis with mAb 5C3 revealed heterogeneous material of M r 140,000 (pl40) for sam- pies from E25 and 4-3.6 cells, but not for control cells (Fig. 2A). In these cells a band of ~110 kDa (pllO) was also observed, previously thought to be intracellular TrkA precursors. The pl40 band was also immunoblotted in samples dissected from normal human cortex or nucleus basalis of Meynert (Fig. 2B). The pllO band was not seen perhaps due to different post- translational processing in neuronal tissues with respect to transfected cell lines. MAb 5C3 was effec¬ tive in western blot analysis only when samples were prepared under non-reducing or mildly reducing condi¬ tions, indicating that a disulfide bond-stabilized conformational epitope is recognized. Whole cell detergent lysates (2x10*^ cell equivalents/lane) were resolved by SDS-PAGE under non-reducing conditions and analyzed by western blot¬ ting with mAb 5C3. (Fig. 2A) Lane 1, Jurkat; lane 2, PC12; lane 3, NIH-3T3; lane 4, Rl.1; lane 5, Z91; lane 6, E25. Thick arrow, pl40 TrkA; thin arrow, pllO. (Fig. 2B) Dissected human brain tissues: nucleus basalis (NB; lanes 1, 2, 6, 7), and cortex (Fig. 2C lanes 3, 4, 8, 9) were compared to E25 cells (lane 5 2x10*-*' cell equivalents). Lanes 1 and 3, 300 μg/lane lanes 2 and 4, 150 μg/lane; lanes 6 and 8, 75 μg/lane lanes 7 and 9, 33 μg/lane. Thick arrow, pl40 TrkA thin arrow, pllO. Note that pllO is not seen in the human brain tissues. Biochemical analysis: Cell lysates 33x10*-* cells/ml were detergent solubilized (lysis buffer 2% NP-40™, 150 mM NaCl, 50 mM Tris-Glycine, 10 mM NaF, 50 μM Na3Vθ4, 30 mM Na Pyro¬ phosphate 10 mM benzamidine, 20 mM iodoacetamide, pH 7.8) supplemented with protease inhibitors (2 μg/ml soybean trypsin inhibitor, 10 μg/ml aprotinin, 5 mM PMSF, and 10 μg/ml leupeptin) for 30 min at 4°C, fol¬ lowed by a 15 minute centrifugation at 14,000 g. Cleared supernatants were analyzed by SDS-PAGE directly (whole cell lysates) or after immunoprecipi- tation. Gel analysis Cell lysates were prepared in Laemmli electro¬ phoresis sample buffer and analyzed by SDS-PAGE under reducing (100 mM 2-mercaptoethanol) or non-reducing conditions. Prestained protein markers (GIBCO) were used as reference. Protein concentrations were quanti¬ tated by the biuret assay (Bio-Rad, Melville, NY), and by parallel Coomassie blue staining of SDS-PAGE gels. For western blotting samples were electrotransfered to PVDF (Xy otech Biosystems, Mt. Royal, QC), blocked overnight in TBST (0.05 M Tris base, 0.2 M NaCl, 0.5% Tween™-20, pH 7.6) containing 1% BSA (Sigma), and immunoblotted with the indicated primary mA s. Secon¬ dary antibodies were either horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (HRP-GαR), or goat anti-mouse IgG (HRP-GαM) (Sigma). For detection the enhanced chemiluminescence (ECL) reagents (Amersham, Oakville, Ont. ) were used following the manufacturer's instructions. Densitometric analysis waε performed with a Masterscan Interpretive Densi¬ tometer CSPI™ and a Howtek Scanmaster™ (Scanalytics, Billerika, MA). Binding, competition, and internalization assays: MAb 5C3 was 12 -^I-labeled by the Iodogen (Pierce) method (Harlow, E., Lane, D. (1988) A Laboratory Manual . Cold Spring Harbour Laboratory Publishing. Chapter 9:332-333) to a specific activity of 1.8 Ci/mg. 125 I[5C3] was repurified from free 12 5i with Sephadex G25 columns (15x1 cm) to >96% trichloroacetic acid precipitable incorporation. Bind¬ ing studies were performed with serial dilutions of 125 I[5C3] on 0.5xl0 6 E25 or 4-3.6 cells (and their respective controls NIH-3T3 and B104 cells) for 1 hour at 4°C. Cell-associated 125 I[5C3] and free 125 I[5C3] were counted after washing unbound ligand. Parallel 125 I[NGF] (70 mCi/mg) (NEN-DuPont, Mississauga, Ont. ) binding assays were performed as control. Competition of 125 I[5C3] binding was done in binding assays in the presence of unlabeled mAb 5C3 (100-fold molar excess), or unlabeled NGF (500-fold molar excess). Competition of -*- 5l[NGF] binding was performed by first incubating cells with excess unlabeled mAb 5C3, NGF, mAb 87.92.6 or vehicle binding buffer for 30 min at 4°C. 125 I[NGF] was then added to a final satu- rating concentration of ~1 nM, the mixtures incubated for an additional 45 min at 4°C, cells were washed and cell-associated -*- 2 *-* 1 ! [NGF] were determined. Based on the competition assays above, it was desirable to identify the mAb 5C3 receptor docking site which is presumed to be a functional or energy- favorable site where the ligand-binding causes receptor-mediated signals. This view is supported by similar findings in the EPO receptor system (Wrighton et al. , 1996, Sci ence, 273:458) wherein a "docking hot spot" was found; and by our finding that mAb 5C3 and NGF block each other's binding. The docking site of mAb 5C3 was identified within the juxtamembrane/IgG2 domain of human TrkA receptors. The sequence is as follows: Gin Val Asn Val Ser Phe Pro Ala Ser Val Gin Leu His Thr Ala Val 1 5 10 15 Glu Met His His Trp Ser lie Pro Phe Ser Val Asp Gly Gin Pro Ala 20 25 30 Pro Ser Leu Arg Trp Leu Phe Asn Gly Ser Val Leu Asn Glu Thr Ser 35 40 45 Phe lie Phe Thr Glu Phe Leu Glu Pro Ala Ala Asn Glu Thr Val Arg 50 55 60 His Gly Cys Leu Arg Leu Asn Gin Pro Thr His Val Asn Asn Gly Asn 65 70 75 80 Tyr Thr Leu Leu Ala Ala Asn Pro Phe Gly Gin Ala Ser Ala Ser lie 85 90 95 Met Ala Ala Phe Met Asp Asn Pro Phe Glu Phe Asn Pro Glu Asp Pro 100 105 110 lie Pro Asp Thr Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp 115 120 125 Glu Thr Pro 130 (SEQ ID N0:9) For receptor internalization studies cells were incubated with TrkA-binding agents (0.01 μg mAb 5C3 ; 2 nM NGF) or controls (mlgG; HBSS) for 20 min either at 37°C (internalization permissive temperature) or at 4°C (internalization non-premissive temperature). After washing, cells were processed for surface TrkA immunofluorescence with mAb 5C3 primary and FITC-GαmIgG secondary as above, and analyzed by FACScan. Binding studies: Scatchard plot analysis of **- 2 -^I[5C3] binding assays demonstrated that in the E25 cell surface there are ~250,000 5C3 binding sites/cell with a Kd of 1.6 nM (Fig. 5), and in the 4-3.6 cell surface there are " 200,000 5C3 binding sites/cell with a Kd of 3.0 nM. Serial dilutions **- 2 -^I[5C3] without competition (open triangles) were used in binding studies with a constant number of E25 cells. Binding was competed with molar excess of unlabeled NGF (filled circles) or mAb 5C3 (open squares). In 3 independent experiments the average Kd of mAb 5C3 in E25 cells was 1.6 nM. Competition with NGF reduced the number of 5C3 binding sites in E25 cells but the affinity of mAb 5C3 was not affected. No 125 [5C3] binding was observed for parental NIH-3T3 or B104 cells. NGF competition reduced the number of 5C3 binding sites in E25 cells by 25%. How¬ ever, NGF caused no change in the affinity of mAb 5C3 for TrkA receptors . Similar data was obtained measur¬ ing mAb 5C3 binding sites by FACScan analysis, where a decrease was observed after NGF treatment (Table 2). TABLE 2 mAb 5C3-induced TrkA receptor internalization TREATMENT TEMP.(°C) % 5C3 STAINING NGF (2 nM) 4°C 83 ± 2.0 37°C 75 ± 3.6 5C3 (0.01 μg/ml) 4°C 96 ± 9.0 37°C 77 ± 5.5 TrkA surface immunostaining was performed on 4- 3.6 cells with mAb 5C3 after the indicated treatments, and measured by FACScan analysis. Data is presented as % staining ± sem, with reference to control vehicle treatment (100%) as per the following formula: (Treated sample staining - mlgG background staining) x 100% (maximum staining - mlgG background staining) In the converse experiment, mAb 5C3 inhibited ~60% of 125 I[NGF] binding to E25 cells. In these experiments background binding was assessed by block¬ ing with 5 μM NGF (100% inhibition), and maximal bind¬ ing was assessed with binding buffer vehicle only (0% inhibition) or by using irrelevant binding mAb 87.92.6 (Table 3) . TABLE 3 MAb 5C3 blocks NGF binding to TrkA TREATMENT % NGF BINDING mAb 5C3 39.3 ± 7.4 mAb 87.92.6 100 NGF (5 μM) 0 E25 cells expressing TrkA (but not p75 recep¬ tors) were incubated with -* *2 5l[NGF] in the presence of the indicated agents. 125 I[NGF] binding after treat¬ ment with mAb 87.92.6 was identical to treatment with vehicle binding buffer. Assays were done 3 times in duplicate. Data is expressed as % binding ± standard deviation (sd), where mAb 87.92.6 is maximum and 5 μM NGF is background binding as per the formula: (test - background) * 100 % (maximum - background) Proliferation/survival assays: 5,000 cells/well in serum-free media (SFM) (GIBCO) supplemented with 0.1% BSA were added to 96 well plates (Falcon, Lincoln Park, NJ) containing serial dilutions of NGF, mAb 5C3, control mA s, mAb 5C3 F a fc> fragments, control mAb 192 F a fragments or serum (final 5% FBS, normal growth conditions). Where indicated, F a bs were externally cross-linked with goat anti-mouse F a b (GαmF a b, Sigma). Wells containing all culture conditions but no cells were used as blanks. The proliferative/survival profile of the cells was quantitated using the tetrazolium salt reagent (MTT, Sigma) 48-72 hours after plating as initially described by T. Mos ann (Hansen, M.B. et al.(1989) Jour. Immunol . Meth . , 119: 203-210). Optical density readings of MTT were done in an EIA Plate Reader Model 2550™ (Bio-Rad) at 600 nm with the blanks subtracted. Assays were repeated at least 5 times in quadruplicates. Foci formation assays: 15xl0 4 E25 cells were plated in a 25% serum-containing 0.35% soft agar mixture in the pres¬ ence of either mlgG control (0.5 μg/ml), mAb 5C3 (0.5 μg/ml), or NGF (2 nM) . Conditions were replenished every 3 days and foci were counted after two weeks. Immunocytochemistry of human brain tissues: Human brain tissue was obtained from six males (age 71.7 ± 4.6) without signs of neurologic or psy¬ chiatric disorders. Tissue blocks were prepared (mean time post-mortem 16.2 ± 3.5 hrs) and stored at -80°C. Twenty μm thick cryostat sections were fixed (4% paraformaldehyde, 0.1 M phosphate, pH 7.4; 1 hour at 4°C), and rinsed in PBS for 1 hour at 4°C. Immunocyto¬ chemistry was performed using avidin-biotin complex (Vectastain Elite™ kit. Vector Labs) as described (Hsu, S-M. et al. (1981) J. Histochem. Cytochem . , 29: 577-580). Primary mAb 5C3 was used either as a 1:1000- 1:4000 dilution of ascites or a 1:4 dilution of serum free-media culture supernatant. Where indicated, 0.5% nickel ammonium sulfate was used to amplify the signal in the DAB revelation step. Some sections were also stained with cresyl violet to facilitate the cytoarchitectural analysis. Negative controls were performed without primary antibody or with normal mouse IgG as primary and in all cases yielded no detectable immunolabeling. Immunostaining in normal human brain: MAb 5C3 was used to map TrkA protein expression by immunocytochemistry of normal adult human brains. The striatum, the basal forebrain and the brainstem exhibited the strongest immunostaining, whereas only weak staining could be detected in the cerebral cortex and hippocampal formation (Fig. 3). Figs. 3A and 3B illustrate low power photomi¬ crographs of the nucleus basalis of Meynert showing large neurons (arrows) immunoreactive with mAb 5C3 (Fig. 3A) but lacking immunoreactivity with normal mouse IgG (Fig. 3B) in a consecutive section. Note in A that the labeled neuronal processes can often be followed (small arrows). Scale bar = 50 μm. Figs. 3C, 3D, and 3E illustrate high power pho¬ tomicrographs of TrkA-containing neurons in the nucleus basalis (Fig. 3C), the putamen (Fig. 3D), and the CA4 sub-field of the hippocampus (Fig. 3E). The perinuclear area displayed particular strong concen¬ tration of DAB precipitate (small arrows) often in granules. Labeled proximal processes could also be observed (arrows), n, nucleus. Scale bars = 10 μm. Fig. 3F illustrates in the pontine nuclei many weakly to strongly staining neurons (arrows) within the fiber network (small arrows) and around the non- labeled fiber bundles (Fig. 3F). Scale bar = 20 μm. Fig. 3G illustrates in the reticular formation of the brainstem numerous fibers (small arrows) con¬ stitute a network where some scattered neurons (arrow) are observed. Scale bar = 20 μm. Fig. 3H illustrates a photomicrograph of TrkA immunoreactivity in the frontal cerebral cortex show¬ ing weak labeling. A few neurons are weakly positive (arrows) with the staining residing mostly in puncta possibly corresponding to fibers (small arrows). Scale bar = 10 μm. All sectors of the basal nucleus contained large TrkA-positive neurons (Figs. 3A and 3C), most of them in groups embedded in a dense network of overlap¬ ping stained processes (Fig. 3A) . The cells had heterogeneous shapes, ranging from complex multipolar to fusiform. In the basal ganglia TrkA was detected in dis¬ tinct cellular compartments. The caudate nucleus, the nucleus accumbens and the putamen contained several immunoreactive cell bodies without apparent distinc- tion in density, perikaryal staining and shape. Fig. 3D shows typical labeled multipolar neurons that displayed strong granular immunoreactivity around the nucleus and in proximal processes. Moreover, numerous puncta and varicose fiber fragments were observed in these areas. The globus pallidus and the claustrum were mostly negative except for varicose fibers. Simi¬ larly, the interstitial elements and fiber bundles did not contain reactive fibers whereas the internal cap¬ sule displayed some labeled punctas and fibers par- ticularly near the putamen and caudate nucleus. The hippocampal formation showed weak immu¬ nostaining located principally in scattered fibers and puncta in the stratum granulosum of the dentate gyrus, as well as in the strati oriens and pyramidal of the Ammon 's horn. In addition, some weakly stained perikarya could be observed in the stratum pyramidal of the CA2 and CA3 sub-fields of the Ammon' s Horn and in the hilus of the dentate gyrus (CA4 sub-field, Fig. 3E). The perikarya of these neurons was rela- tively large in size, of ovoid to pyramidal shape, and bearing one prominent apical and radial dendritic process. The immunoreactivity appeared like in other stained cell types of the brain as small granular patches of precipitate located principally near the nuclear envelope and in some cases within the cyto¬ plasm (Fig. 3E) . Within the cerebral cortex, particularly in the frontal area, TrkA immunoreactivity appeared more dis¬ crete. At high magnification, immunoreactive puncta and fiber fragments without a particular pattern of distribution are observed in all layers, but the lami¬ nae III-VI appeared more stained than superficial ones (Fig. 3H). Occasional, weakly staining, medium-sized perikarya were observed in layer IV (Fig. 3H). In the brainstem TrkA staining is also detected. The pontine nuclei contained numerous immu¬ noreactive medium sized globular perikarya and fibers between the pontocerebellar fibers (Fig. 3F). The reticular formation also displayed strong immunoreac¬ tivity for TrkA principally located in fiber networks (Fig. 3G). Some large neurons of bipolar or multipolar shape are also stained. No TrkA immunostaining was observed in the cerebellum. Functional agonis of mAb 5C3 Several functional assays of NGF bioactivity were used to test the agonistic potential of mAb 5C3. Receptor internalization 4-3.6 cells were treated with TrkA ligands at internalization permissive temperatures (37°C) or at non-permissive temperatures (4°C) (Table 2). NGF treatment reduced the % staining of mAb 5C3 to surface TrkA at both temperatures. Loss of surface 5C3 binding sites suggest direct blocking by NGF (Fig. 5). In con- trast, mAb 5C3 treatment reduced the number of surface 5C3 binding sites only at 37°C. This is likely due to receptor internalization, which does not occur effi¬ ciently at 4°C. Treatment with mlgG or binding buffer control did not reduce the number of surface 5C3 bind- ing sites at either temperature. Similar data was obtained with E25 cells. Receptor phosphorylation Anti-phosphotyrosine western blots of E25 or 4-3.6 whole cell detergent extracts revealed that TrkA phosphotyrosinylation (PY) increased significantly over basal levels after short treatment with mAb 5C3 or with NGF (Fig. 4). E25 cells were untreated (lane 1) or treated with mAb 5C3 (lane 2), NGF (lane 3), or mlgG (lane 4) for 15 min. at 37°C. Whole cell lysates were resolved in a 8% SDS-PAGE under reducing conditions and immuno¬ blotted with anti-phosphotyrosine mAb 4G10. A parallel gel under non-reducing conditions immunoblotted with mAb 5C3 (not shown) controlled for M r and equal load¬ ing of TrkA on all samples. Densitometric analysis of several blots from E25 and 4-3.6 cells is presented in Table 4. TABLE 4 TrkA tyrosine phosphorylation by mAb 5C3 TREATMENT E25 cells 4-3.6 cells mAb 5C3 2.7 ± 0.6 3.4 ± 1.5 NGF 6.5 ± 1.3 3.8 ± 0.8 E25 or 4-3.6 cells were untreated or treated with saturating concentrations of mAb 5C3 or NGF for 15 min. at 37°C. Whole cell lysates, or anti-PY immu- noprecipitates were resolved by SDS-PAGE under reduc¬ ing conditions, western transferred, immunoblotted with anti-phosphotyrosine (αPY) mAb 4G10, and developed using ECL techniques. Optical density (O.D.) readings were taken from X-ray films with film backgrounds subtracted (see Materials and Methods). Data is presented as fold increase in PY of TrkA with respect to untreated cells ± sd. n=3. Other proteins evidence increased PY, including ~95 kDa and ~60 kDa proteins, and the p85 subunit of PI-3 kinase (~2.5-fold increase). We have estimated that <10% of all p85 material was tyrosine phosphory¬ lated upon ligation of TrkA. Increased cellular transformation NGF treatment causes the transformation and an increase in anchorage-independent growth of TrkA-expressing E25 cells. MAb 5C3 caused a ~2-fold increase in the number and the size of foci as com¬ pared with mlgG treated cells ( Table 5 ) . TABLE 5 MAb 5C3 -induced anchorage -independent growth TREATMENT AVERAGE NUMBER OF TYPICAL FOLD INCREASE FOCI 3 CELLS/FOCI 3 IN FOCI b mlgG 416 ± 45 -24 1 ± 0.11 mAb 5C3 806 ± 178 >48 1.9 ± 0.22 NGF 676 ± 51 ^24 1.6 ± 0.08 E25 cells were cultured in soft agar in the presence of the indicated agents for 2 weeks. a Average number and typical size of foci ± sd are shown. -^Fold increase in foci was calculated with respect to mlgG treated cells (no increase). n=2. No change in the number or size of foci was observed in wild type NIH-3T3 cells upon mAb 5C3 treatment. Protection from cell death: Agonistic ligands of TrkA protect recep¬ tor-expressing cells from death in serum-free media (SFM). Both NGF and mAb 5C3 and 5C3 fragments or derivatives increased the number of surviving/proliferating E25 fibroblastoid cells (Fig. 6) and 4-3.6 cells (Fig. 8). Cells were cultured in Serum Free Media supplemented with the indicated conditions for 2-3 days, followed by the MTT assay. Similar data was obtained with neuronal 4-3.6 cells (Fig. 8). The % proliferation/survival was determined by standardizing serum containing wells to 100% with the use of the following formula: (O.D. of test) x 100% (O.D. of serum) Equivalent protection was also afforded by TrkA ligands to neuronal 4-3.6 cells. In most experiments mAb 5C3 protection is dose-dependent, although high dose antibody inhibition is sometimes seen (e.g. 1 mg/ml of mAb 5C3) . To ascertain whether cell death is apoptotic, DNA was prepared from serum-free cultured cells which showed a typical apoptotic fragmentation ladder. The DNA ladder was not seen in preparations from cells cultured in the presence of mAb 5C3 or NGF. Controls demonstrated the functional specific¬ ity of mAb 5C3. First, neither NGF nor mAb 5C3 pro¬ tected wild type NIH-3T3 cells. Second, PC12 cells were not protected by mAb 5C3 but were protected by NGF. Third, irrelevant mlgG, GαmF a b, or mAb 192 did not protect E25 cells (Fig. 6), or NIH-3T3 cells, or 4-3.6 cells (Fig. 8). Functional agonism of monomeric 5C3 Fabs Monovalent agents that bind TrkA behave as com- petitive antagonists (Clary, D.O. et al. (1994) Mol . Biol . Cell . , 5: 549-563; LeSauteur, L. et al.(1995) J. Biol . Chem . , 270:6564-6569) likely because they can not induce receptor dimerization. Therefore, it would be expected that monomeric 5C3 F aD s would be monovalent and not be able to mediate agonistic function. MAb 5C3 F a ],s afforded protection from apoptotic death to E25 cells (Fig. 6) and 4-3.6 cells in serum- free media. Moreover, anti-phosphotyrosine western blots revealed that cells treated with 5C3 F a bs had increased TrkA tyrosine phosphorylation similar to increases obtained with whole mAb 5C3. Monomeric 5C3 F a protection was dose-dependent. However, equivalent or better protec- tive effects were achieved when F a bs were externally cross-linked with GαmF a b antibodies. Specificity con¬ trols included those described in the previous section for whole mAb 5C3, plus 192 F a bs which had no protec¬ tive activity in E25 cells. DISCUSSION The availability of antibodies against pl40 TrkA and p75 has allowed the study of these NGF recep¬ tors. The mAb 5C3 reported herein is specific for human TrkA, functions in FACScan immunofluorescence analysis, immunoprecipitation, western blot analysis, and immunocytochemistry. Moreover, mAb 5C3 is a struc¬ tural and functional mimic of NGF. Aberrant expression of trkA mRNA and NGF responsiveness have been correlated with neurodegen¬ erative disorders and neoplastic malignancy. Hence, TrkA-binding agents will be useful clinical tools in diagnosis, prognosis and perhaps treatment of these diseases. Indeed, mAb 5C3 binding is a positive prognostic marker for certain human neoplasias. Table 6 TrkA expression in neuroblastoma Neuroblastomas* Number Positive Mixed Negative Group 1 60 38 17 5 Group 2 53 13 5 35 * 15 samples repeated after chemotherapy , at the time of second surgery or recurrence : 5C3 staining patterns remained unchanged in 14 tumors ; 1 negative tumor sub¬ sequently positive post chemotherapy in regions of maturing elements . Table 7 TrkA expression in other malignant tumors Malignant tumor N=42 TrkA-Pos Central nervous system tumors 6 0 R abdomyosarcomas 5 0 Primitive neuroectodermal tumors 6 0 Ewing's sarcomas 2 0 Wilm's tumors 6 1 Osteosarcomas 4 0 Melanomas 5 0 Breast carcinomas 5 0 Lung carcinomas 3 0 Table 8 TrkA detection by immunocytochemistry, RT-PCR and western blot IMMUNOCYTO. N RT-PCR Pos N West. Pos Group 1 15 15 2 2 5C3 Pos 5C3 Neg 4 4 2 1 5C3 Mixed 1 1 10 10 Group 2 11 11 11 11 5C3 Pos 5C3 Neg 11 4 9 5 5C3 Mixed 2 2 0 0 MAb 5C3 recognizes a disulfide-stabilized domain of TrkA and an extracellular epitope with these characteristics appears to be the NGF docking site. Cross-blocking studies indicated that mAb 5C3 and NGF can reciprocally block each other's binding to TrkA, further suggesting that the docking site of 5C3 may be similar to NGF. In addition, sequence comparison of both ligands revealed interesting homology between CDRs of mAb 5C3 and the variable turn regions of NGF. Since most CDRs are β-turns (Sibanda, B.L. et al. (1989) J. Mol . Bi ol . , 206: 759-777) and coincidentally the NGF structures that bind TrkA may also be β-turns (LeSauteur, L. et al.(1995) J. Biol . Chem. , 270:6564- 6569) we hypothesized that both mAb 5C3 and NGF bind to the same site on human TrkA, and cross-blocking is likely to be the result of direct competition rather than steric hindrance. Interestingly, mAb 5C3 was more efficient at blocking NGF binding than vice versa. Only ~25% of the mAb 5C3 binding sites on E25 cells were blocked by saturating doses of NGF. This data suggest that not all TrkA receptors in these cell lines bind NGF. It is unlikely that affinity considerations can account for these observations, as both ligands have roughly com¬ parable Kd for TrkA (mAb 5C3 K ~ 2 nM versus NGF Kd 0.7 nM (Jing, S. et al. (1992) Neuron, 9: 1067-1079)) and the affinity of mAb 5C3 was unchanged in the presence of NGF. Three non-exclusive possibilities can account for these observations: (i) TrkA receptors exist at equilibrium where ~ 25% are in an NGF binding conforma¬ tion (e.g. dimers) and the rest are in a non-NGF bind¬ ing conformation; (ii) specific post-translational modifications of TrkA receptors allow for NGF binding (iii) expression of other membrane proteins (e.g. p75 or unknown proteins) induce or favor the NGF binding conformation of TrkA. These hypotheses can be addressed by biochemical analysis after differential affinity purification of TrkA with mAb 5C3 versus NGF and by further binding studies in neuronal and fibro- blastoid cells expressing different receptors. Absence of mAb 5C3 binding to rat TrkA is intriguing. Binding by mAb 5C3 to rat TrkA was expected because of the homology between mAb 5C3 CDRs and the variable loops of NGF, particularly since NGF from one species does bind to TrkAs from other spe¬ cies. MAb 5C3 is a binding and structural mimic of NGF, with enhanced human receptor specificity. Remod- eling and mutating of the CDRs of mAb 5C3 will yield a pan-TrkA binding mAb. Further, analysis of the epitope of mAb 5C3 on TrkA revealed differences in the docking site of human and rat TrkAs. This information will be useful in screening receptor-binding analogs. To test functional mimicry by mAb 5C3, NGF bio- assays were performed using trkA transfected fibro¬ blast and neuronal cells. Functional mimicry by mAb 5C3 included TrkA internalization, TrkA tyrosine phos¬ phorylation, PI-3 kinase phosphorylation, increased anchorage-independent growth and proliferation/sur¬ vival of cells in serum-free media. By these criteria mAb 5C3 is agonistic. Increased TrkA receptor turnover or internali¬ zation is induced by NGF binding. MAb 5C3 increased the internalization of TrkA, as measured by loss of cell surface receptors. These results are consistent with data which showed that E25 cells internalize -** 2 -^I[NGF] within seconds upon shifting from 4°C to 37°C (Jing, S. et al. (1992) Neuron, 9: 1067-1079) and that this process does not require p75 receptors. Thus, artificial ligands of TrkA can induce receptor internalization and could be useful in delivering toxic agents to the cytoplasma of TrkA-expressing tumors. NGF ligation of TrkA causes receptor activation and autophosphorylation. MAb 5C3 induced TrkA tyrosine phosphorylation to a similar degree. Agonism in the absence of NGF suggests that TrkA dimerization and/or internalization are the required signaling event, rather than the formation of NGF-TrkA complexes. How- ever, we can not rule out that mAb 5C3-TrkA is the functional signal transducing complex. Ligand-induced tyrosine phosphorylation of the intracellular domain of TrkA allows for the recruit- ment of substrates and the activation of cytosolic proteins and nuclear oncoproteins. MAb 5C3 induces the tyrosine phosphorylation of proteins of M r 60, 85 and 95 kDa. The 85 kDa protein was identified as PI-3 kinase, whose activation correlates with the actions of growth factors and oncogenes . NGF stimulates neuronal survival and differen¬ tiation, and the proliferation of non-neuronal cells. NGF-activated TrkA induces transformation and morpho¬ logical changes in fibroblast cells. MAb 5C3 caused similar increases in anchorage-independent growth and foci formation in soft agar. Thus, mAb 5C3 can posi¬ tively modulate the growth of TrkA-expressing cells. Interestingly, the size of the mAb 5C3-induced foci were larger on average than NGF-induced foci. TrkA-expressing neuronal 4-3.6 cells or fibro- blastoid E25 cells undergo apoptotic death in serum free media but can be rescued by NGF or mAb 5C3. Syn¬ ergy between the two ligands occurred when combined at suboptimal doses, as would be expected if mAb 5C3 bound and activated unoccupied TrkA receptors. Fur¬ thermore, morphological changes and increased attach¬ ment to plastic were observed in both the NGF and 5C3 treated cells. Monomeric 5C3 F s protected E25 and 4-3.6 cells from apoptotic death. When F a bs were externally cross-linked using anti-F a b antibodies, a heightened response occurred. Since growth factor receptor acti¬ vation requires bivalent binding, the monomeric 5C3 F abs must have retained the ability to induce TrkA oligomerization. This could be explained in the foi- lowing 3 ways: (i) F a bs are relatively large molecules capable of aggregation; (ii) 5C3 F a binding could cause conformational changes in TrkA which induce receptor-receptor interactions; and (iii) monomeric 5C3 F a bs bind to two receptor molecules in a bivalent manner. The last possibility could occur by two CDRs binding to two different TrkAs. Homology of mAb 5C3 CDRs to NGF turn regions, and experiments using small recombinant antibody analogs support the last explana- tion. MAb 5C3 is the first report of an agonistic anti-neurotrophin receptor mAb and will be useful in studies of TrkA biology and for drug development. Antineoplastic effects with mAb 5C3 may be achieved either through terminal differentiation, anti¬ body-dependent cell cytotoxicity, or by the delivery of toxins or radionuclides. Furthermore, the structure of this mAb may be useful in designing peptidic and non-peptidic TrkA binding agents (Saragovi, H.U. et al. (1991) Science, 253: 792-795). Small non-peptidic agonists of TrkA should be useful pharmacological agents for the treatment of neurodegenerative diseases. The present invention will be more readily un- derstood by referring to the following examples which are given to illustrate the invention rather than to limit its scope. EXAMPLE I PRELIMINARY STUDY OF THE EFFECT OF mAb 5C3 IN TUMOR GROWTH Nude mice were injected subcutaneously (right abdominal side) with 2X10^ human TrkA expressing tumor cells. Two days post-injections tumors in all mice had begun to form. Mice were randomized prior to treat- ment. A total of four intraperitoneal injections of 100 micrograms each on the left side were then admin- istered over a 12 day period with control mouse IgG or mAb 5C3 (Fig. 10). The mAb 5C3 dramatically reduced the primary tumor weight with no observable metastatic invasion. A small fibrotic mass was localized at the site of injection in mAb 5C3 treated mice. In contrast, IgG treated mice had large, vascularized tumor masses, which metastasized to the liver, peritoneum gut and spleen. All animals had similar body weights (~30 grams ) . Table 9 EXAMPLE II The use of Mab 5C3 and its derivatives for the diagnosis, prognosis and localization of tumors The in vivo targeting efficacy of agents that bind the NGF receptor pl40 TrkA was evaluated. Nuclear imaging studies were done after the injection of 9 m Tc-labeled compounds in nude mice bearing tumors. Kinetics of tumor targeting, blood clearance, and bioavailability were studied. Tumors that do not express TrkA were not targeted, demonstrating the specificity in vivo. This biodistribution study demonstrates that receptor-specific molecule analogs may be useful and may be effective agents for the detection, diagnosis, and possible treatment of neoplasias involving overexpressed oncogenic receptors such as TrkA (Fig. 7). Table 10 Biodistribution of ^*^ m Tc-[TrkA ligands] in mice. 99m Tc . ■ [5C3] Ligand % id/g T/nT 1 tumor 1.25 1 2 blood 0.1 13 3 muscle 0.06 20 4 heart 0.10 13 5 lung 0.17 7.30 6 liver 0.61 2.10 7 spleen 0.13 9.42 8 kidney 1.48 0.9 9 large bowel 2.2 0.7 EXAMPLE III 5C3 PROTEIN SEQUENCE Kappa light chain DILQTQSPAILSASPGEKVTMTCRASSSVSYMHWYQQKPGSSPKPWIYATSNLA SGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLEI (SEQ ID N0:1) Heavy chain (IgG2a) VQLQESGTVLARPGASVKMSCKASGYTFTSYWMHWVKQRPGQGLEWIGAIYPGDS DTSYNQKFKGEAKLTAVTSTSTAYMELSSLTNEDSAVYYCTLYGNYESYYAMDYW GQGILSHRLL (SEQ ID NO: 2) Complementarity Determining regions (according to Kabat, Sequences of Proteins of Immunological Interest , 4 th ed. Bethesda , MD, U. S. Dept . of Heal th and Human Servi ces . Public Health, NIH 1987) Kappa variable light chain VL CDR1: RASSSVΞYMH (SEQ ID NO:3) VL CDR2: ATSNLAS (SEQ ID NO:4) VL CDR3: QQWSSNPLT (SEQ ID NO: 5) Heavy chain variable (IgG2a) VH CDR1: SYWMH (SEQ ID NO: 6 ) VH CDR2: AIYPGDSDTSYNQKFKG (SEQ ID NO: 7 ) VH CDR3: YGNYESYYAMDY (SEQ ID NO: 8 ) Antibodies have 6 complementarity determining regions (CDRs) that combine in specific ways to create direct contacts with their antigen. Molecular cloning, recombination, mutagenesis and modeling studies of mAb 5C3 variable region indicated that 3 or less of its CDRs are relevant for binding TrkA. These were named CDR1, CDR2, and CDR3. Region CDR1 is connected to CDR2 by a 15 amino acid linker; CDR2 is connected to CDR3 by a 30 amino acid linker. Their secondary structures have been analyzed. The variable domains of mAb 5C3 heavy (VH) and light (LH) chains were cloned and sequenced. The predicted variable domain amino acid sequences are shown above . The hypervariable complementarity determining regions (CDRs) were resolved according to Kabat ( Sequences of Proteins of Immunological Interest , 4 th ed. Bethesda , MD, U. S . Dept . of Heal th and Human Servi ces . Public Health, NIH 1987). An example of the model of the structure of the CDRs (Insight II™, Biosy , CA) within the backbone of the variable domains is shown in Fig. 11. EXAMPLE IV MAb 5C3 AND DERIVATIVES: STRUCTURAL AND FUNCTIONAL MIMICS OF NGF Several criteria indicate that mAb 5C3 is a TrkA agonist. Previous functional studies in vi tro demonstrated that mAb 5C3 can: (i) induce tyrosine phosphorylation of TrkA, and Pl3-kinase; (ii) increase transformation of TrkA-expressing fibroblasts. Shown above are also experiments where the 5C3 and its derivatives protect cells from apoptotic death in serum-free media to the same degree as NGF does . Monovalent Fabs of 5C3 obtained after papain digestion are also agonistic, especially when externally cross¬ linked by anti-Fabs. A smaller fragment of mAb 5C3 called CDR(R) also protects cells from apoptosis (Fig. 8). Assays with recombinant CDRs and CDR-like synthetic polypeptides demonstrated they had agonistic bioactivies similar to intact mAb 5C3. Consequently a 6 kDa recombinant molecule "CDR(R)" (for recombinant CDR) was developed as a replacement for mAb 5C3 ; it is approximately five times smaller than the 25 kDa NGF molecule and is still agonistic. CDR(R) is composed of 3 selected CDRs (out of 6 possible ones) linked by long spacer regions. Preliminary studies have suggested that actually only 2 of the 3 CDRs are relevant for binding to TrkA. Further, it is expected that even smaller fragments can be designed, e.g. upon removal of the linker regions. 4-3.6 cells transfected with human TrkA were cultured in serum-free-media (SFM) to induce apoptosis (Fig. 8). The indicated agents were added to the cells and cell viability was measured after 48-72 h by the MTT assay. Proliferation was standardized to normal growth conditions (5% serum). NGF, mAb 5C3, 5C3 Fabs, and CDR(R) (recombinant 5C3 CDR analog) protect from apoptosis, but mouse IgG (control) does not. EXAMPLE V Use of Mab 5C3 The mAb 5C3 of the present invention and its derivatives induce differentiation/neuritogenesis of human TrkA-expressing cells. PC12 cells (expressing rat TrkA) and PC12 cells transfected with and expressing human trkA cDNA were cultured with various TrkA binding agents or controls (Fig. 9) as indicated NGF caused the differentiation of wild type PC12 and PC12 transfectants, whereas SC3 and CDR(R) only induced differentiation of PC12 transfectants. Ligand doses are as shown in Fig. 8. Further modifications of CDR(R) resulted in agents that support cell survival but not neuritogenesis . While the invention has been described in con¬ nection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any varia¬ tions, uses, or adaptations of the invention follow¬ ing, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims . SEQUENCE LISTING (1) GENERAL INFORMATION: (l) APPLICANT: (A NAME: McGILL UNIVERSITY (B STREET: 845 Sherbrooke Street West (C CITY: Montreal (D STATE: Quebec (E COUNTRY: Canada (F POSTAL CODE (ZIP) : H3A 1B1 (G TELEPHONE: 514-398-4200 (H TELEFAX: 514-398-8479 (A NAME: SARAGOVI, H. Un (B STREET: 1100 Dr. Penfield, Apt. 718 (C CITY: Montreal (D STATE: Quebec (E COUNTRY: Canada (F POSTAL CODE (ZIP) : H3A 1A8 (A NAME: LESAUTEUR, Lynne (B STREET: 210 Coronation (C CITY: Rosemere (D STATE: Quebec (E COUNTRY: Canada (F POSTAL CODE (ZIP) : J7A 1P3 (li) TITLE OF INVENTION: DESIGN OF HORMONE-LIKE ANTIBODIES WITH AGONISTIC AND ANTAGONISTIC FUNCTIONS (ill) NUMBER OF SEQUENCES: 9 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.30 (EPO) (v ) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: GB 9525180.7 (B) FILING DATE: 08-DEC-1995 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 105 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Asp lie Leu Gin Thr Gin Ser Pro Ala lie Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 His Trp Tyr Gin Gin Lys Pro Gly Ser Ser Pro Lys Pro Trp lie Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu lie 100 105 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 120 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: Val Gin Leu Gin Glu Ser Gly Thr Val Leu Ala Arg Pro Gly Ala Ser 1 5 10 15 Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp 20 25 30 Met His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp lie Gly 35 40 45 Ala lie Tyr Pro Gly Asp Ser Asp Thr Ser Tyr Asn Gin Lys Phe Lys 50 55 60 Gly Glu Ala Lys Leu Thr Ala Val Thr Ser Thr Ser Thr Ala Tyr Met 65 70 75 80 Glu Leu Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys Thr 85 90 95 Leu Tyr Gly Asn Tyr Glu Ser Tyr Tyr Ala Met Asp Tyr Trp Gly Gin 100 105 110 Gly lie Leu Ser His Arg Leu Leu 115 120 (2) INFORMATION FOR SEQ ID NO: 3: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Arg Ala Ser Ser Ser Val Ser Tyr Met His 1 5 10 (2) INFORMATION FOR SEQ ID NO: 4: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (n) MOLECULE TYPE: protein ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Ala Thr Ser Asn Leu Ala Ser 1 5 (2) INFORMATION FOR SEQ ID NO: 5: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (n) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Gin Gin Trp Ser Ser Asn Pro Leu Thr 1 5 (2) INFORMATION FOR SEQ ID NO: 6: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: Ser Tyr Trp Met His 1 5 (2) INFORMATION FOR SEQ ID NO: 7: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Ala He Tyr Pro Gly Asp Ser Asp Thr Ser Tyr Asn Gin Lys Phe Lys 1 5 10 15 Gly (2) INFORMATION FOR SEQ ID NO: 8: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear ( i) MOLECULE TYPE: protein ( l) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Tyr Gly Asn Tyr Glu Ser Tyr Tyr Ala Met Asp Tyr 1 5 10 (2) INFORMATION FOR SEQ ID NO: 9: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 131 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (n) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Gin Val Asn Val Ser Phe Pro Ala Ser Val Gin Leu His Thr Ala Val 1 5 10 15 Glu Met H s His Trp Ser He Pro Phe Ser Val Asp Gly Gin Pro Ala 20 25 30 Pro Ser Leu Arg Trp Leu Phe Asn Gly Ser Val Leu Asn Glu Thr Ser 35 40 45 Phe He Phe Thr Glu Phe Leu Glu Pro Ala Ala Asn Glu Thr Val Arg 50 55 60 His Gly Cys Leu Arg Leu Asn Gin Pro Thr His Val Asn Asn Gly Asn 65 70 75 80 Tyr Thr Leu Leu Ala Ala Asn Pro Phe Gly Gin Ala Ser Ala Ser He 85 90 95 Met Ala Ala Phe Met Asp Asn Pro Phe Glu Phe Asn Pro Glu Asp Pro 100 105 110 He Pro Asp Thr Asn Ser Thr Ser Gly Asp Pro Val Glu Lys Lys Asp 115 120 125 Glu Thr Pro 130

Claims: WE CLAIM : 1. An antibody or functional fragment thereof which binds to at least the trkA receptor under physi¬ ological conditions, and wherein said binding to said receptor at least partially mimics or inhibits nerve growth factor biological activity. 2. A method of screening pharmacological agents which are capable to mimic or inhibit nerve growth fac¬ tor biological activity, which comprises using the antibody of claim 1 to screen for pharmacological agents capable of binding to the complementary deter¬ mining region of said antibody, wherein said screened pharmacological agents can mimic or inhibit nerve growth factor biological activity. 3. Use pharmacological agents obtained by the process of claim 2, for the in vivo inhibition of nerve growth factor binding to TrkA receptor or the inter¬ nalization or downmodulation of said receptor. 4. Use of the antibody of claim 1, for the in vivo inhibition of nerve growth factor binding to TrkA receptor or the internalization or downmodulation of said receptor. 5. The use of claim 3 or 4, for inhibiting tumor growth in si tu . 6. The use of claim 3 or 4, for the treatment or prevention of neurological diseases, neuromas and neo¬ plastic tumors which express TrkA receptors. 7. The use of the antibody of claim 1, for mapping hormone-receptor interactive sites and receptor domain- function correlation. 8. The use of the antibody of claim 1, for mapping trkA docking site. 9. The use of claims 7 and 8, for screening phar¬ macological agents which binds to said hormone-receptor interactive sites. 10. The use of the antibody of claim 1, for inducing partial nerve growth factor-like responses by cells expressing TrkA. 11. The use of claim 10, wherein said responses are selected from di ferentiation/neuritogenesis function and trophic function. 12. An antibody having CDR-like domains of hor¬ mones, wherein said antibody or functional fragment thereof binds to at least trkA receptor under physi¬ ological conditions, and wherein said binding to said receptor at least partially mimics or inhibits nerve growth factor biological activity. 13. An antibody having at least partial activity or binding of hormones, wherein said antibody or functional fragment thereof binds to at least trkA domain IgG2 under physiological conditions, and wherein said binding to said domain at least partially mimics or inhibits nerve growth factor biological activity. 14. A method for the treatment of neurological diseases, neuromas and neoplastic tumors which express TrkA receptors in a patient, which comprises adminis¬ tering an effective amount of an antibody of claim 1 or a functional fragment thereof to a patient. 15. A pharmaceutical composition for the treatment of neurological diseases, neuromas and neoplastic tumors which express TrkA receptors, which comprises an effective amount of an antibody of claim 1 or a func¬ tional fragment thereof in association with a pharma¬ ceutically acceptable carrier. 16. A method for immunization of a mammal against an antibody of claim 1 or a functional fragment thereof, which comprises administering by systemic injection an immunizing amount of at least one of the antibody or the fragment thereof in an immunogenic form in association with a pharmaceutically acceptable car¬ rier. 17. A method for the prognosis or diagnosis of human tumor which comprises: a) biopsy and immunocytochemistry of tumors using the antibody of claim 12 and fragments thereof; or b) radiolabeling of the antibody of claim 12 and fragments thereof and nuclear imaging analysis. 18. A method for the treatment of human tumor of a patient which comprises the steps of: a) coupling cytotoxic agents to the antibody of claim 12 and fragments thereof; b) administering the coupled antibody of step a) to said patient. 19. A pharmaceutical composition for the targeting of pharmaceutical agents to tissues of the central and/or peripheral nervous system, which comprises an effective amount of an antibody of claim 1 or a func¬ tional fragment thereof coupled to a pharmaceutical agent in association with a pharmaceutically acceptable carrier. 20. The pharmaceutical composition of claim 19, wherein the pharmaceutical agent is selected from the group consisting of radioligands, nucleic acid molecules, toxins, growth factors and gangliosides . 21. A TrkA docking site which binds to the antibody of claim 1. 22. Use of the docking site of claim 21 for screening pharmacological agents which are capable to mimic or inhibit nerve growth factor biological activity and said antibody biological antibody.

Inventors: LESAUTEUR LYNNE, SARAGOVI H URI, LESAUTER, LYNNE, SARAGOVI, H., URI
Applicants: LESAUTEUR LYNNE, SARAGOVI H URI, UNIV MCGILL, LESAUTER, LYNNE, MCGILL UNIVERSITY, SARAGOVI, H., URI
Application year: 1996
Main classification: C07K16
IPC codes: A61K38, A61K51, C07K14, C07K16