SE465573B - NERVOUS GROWTH FACTOR Peptides, ANTIBODY CORRESPONDING MATERIALS AND PROCEDURES FOR DETERMINING NATIVE NERVOUS GROWTH FACTORS - Google Patents

Description

5 10 15 20 35 llåß 573 Another peptide encompassed by the invention has the following formula: Gly-Asp-Lys-'llir-Thr-Ala-'Iltr-Asp-lle-Lys-Gly-Lys-Glu, and this peptide corresponds to a conserved region in the NGF protein correspondingly amino acid residues 23 to 35 in mature NGF protein. Tests conducted in connection with research leading to this invention showing that antibodies formed against this peptide recognizes native NGF protein.

Relevant antibodies may be directed to either of the peptides described above or against both such peptides and native NGF.

The antibodies can be either polyclonal or monoclonal.

The invention also encompasses a method of determination of the presence of mature NGF or its precursor in biological testing material. Said procedure is based on interaction between antibodies obtained by immunization against a peptide according to fínníngen or NGF or its precursor. Such determination can , be qualitative or quantitative depending on the type of diagnosis performed and the purpose of the determination.

The sample material for such determination may be mammalian brain tissue including brain tissue of human origin.

In the following, when reference is made to living animals or living things Mammals are meant to include humans as well.

This invention will be further illustrated below with reference to the accompanying drawings, in which: Figure 1A shows homologous antibody binding to NGF peptides as one function of antiserum concentration; Fig. 1B shows binding of degenerate-purified peptide cells to theirs homologous peptides as a function of concentration of the immunoglobulins (Ig); Fig. 1C shows antibody binding to various NGF peptides as a function of antiserum concentration; Figure 2A shows binding of antibodies to peptides to the β-NGF protein as a function of concentration of the affinity purified immunoglobulins; Fig. 2B shows a comparison of how different antisera to ß-NGF bind to NGF peptide P3; and.

Fig. 2C shows how an antiserum to mouse ß-NGF binds to various synthetics NGF peptides. 10 15 20 35 465 573 EXAMPLE 1 Synthesis of NGF peptides Hydrophilic lots selected using hydrophilicity values (TP Jump and KR Woods (1981): Prediction of protein antigenic determinants from amino acid sequences. Proc. N atl. Acad. Sci. USA 78: 3824-3828) in proNGF and mature NGF were selected for peptide synthesis (Table I). Peptides P1 to P6 were synthesized by Cambridge Research Biochemicals Ltd., England, with an estimated purity better than 80% controlled by amino acid analysis, HPLC and mass spectrometry.

Peptides P7 and P8 were kindly synthesized by Doctors Tamas Bartfai and Ianis Abens at the Arrhenius Laboratory, Department of Biochemistry, Stockholm university. Similarities with protein sequences other than NGF for each of these Peptides were controlled using "GeneBank" without any extensive similarities could be detected except for NGF.

EXAMPLE 2 Conjugation and production of peptide antibodies All peptides were conjugated to "keyhole limpet hemocyanin CKLHY" before use for immunization. KLH (8-10 mg) was coupled for each conjugation with 3-10 mg peptide. The couplings were obtained with m-maleimidobenzoic acid N- hydroxy-succinintide or N-succinimidyl 3- (2-pyridyldithio) propionate to obtain N-terminally bound conjugates of P1-PS and P7-P8 and a C-terminal bound conjugate of P6. Following dialysis, the peptide-KLH conjugates were used to immunize rabbits (Table II). Each rabbit received an initial dose of approx. 400 pg conjugate in Freund's complete adjuvant intramuscularly as well as via multiples intradermal injections. After one month, the rabbits received four booster injections at one-week intervals, each consisting of approx. 200 pg conjugate in incomplete adjuvant before killing.

Purification of antibodies Affinity columns were prepared for each of the 8 peptides, 3 ml ECH-Sepharose 4B (Pharmacia) was coupled with 2 mg of peptide (coupling via a extension arm to antino groups) using N-ethyl-N- (3- dimethylanenopropyl-carbodiimide hydrochloride according to the manufacturer instructions. Each gel was packed in a small column and antiserum diluted 15-fold in 0.1 M Tris-HCl (pH 8) with 0.5 M NaCl was allowed to pass fl several times through the column.

In most cases, the antiserum had first passed through another column containing a ' unrelated peptide to remove any unspecified binding components. After rinsing, the column was eluted with 4.5 M MgCl 3 in 0.05 M 15 4 6 5 5 7 3 NaAc buffer, pH 5.0, and the antibody peak in the eluate were located by absorption at 280 nm. The peak was collected, extensively dialyzed and kept frozen.

EXAMPLE 3 Others tested sera As a comparison material, normal rabbit antibodies were purified on protein A-.

Sepharos (Rabbit No. 3). Antibodies to mouse ß-NGF from rabbit (Ig 17) or sheep (Ig 28) were affinity purified against N GF coupled to CN Br-activated Sepharos 4B as_m previously described (Ebendal T, Olson L, Seiger Å (1983): The level of nerve growth factor (NGF) as a function of innervation. Exp. Cell Res. 148: 311-317; Ebendal T, Olson L, Seiger Å, Belew M (1984): Nerve growth factors in chick and rat tissue. In Black IB (ed): "Cellular and Molecular Biology of Neuronal Development, "New York: Plenum pp. 231-242). In addition, serum from another normal rabbit (No. 19) and from two rabbits immunized with mouse ß-NGF (Nos. 10 and 30). Finally, a rabbit (No. 46) was included as the first had been immunized with ß-NGF (200 μg) and then obtained four booster injections with P3-KLH (200 ug each time).

EXAMPLE 4 Enzyme immunoassay (ELISA) To the surface of 96-well immunoplates were bound various peptides, KLH or ß- NGF (all at the concentration of 1 μg / ml) in 0.05 M carbonate buffer (pH 9.6).

The plates were blocked with 1% bovine serum albumin (BSA), and then added dilutions of the peptide antibodies or the affinity purified antibodies to the bowls. They were incubated at 4 ° C overnight. After extensive washing was detected bound antibodies with biotinylated anti-rabbit antibodies (Vector Labs., CA) accompanied by streptavidin-conjugated β-galactosidase (Bethesda Research Labs., MD). Enzyric acid activity was measured with a fl uorometric plate reader (Dynatech Micnofluor) after administration of methylumbelliferyl-β-galactoside.

"Bíaassaay" of NGP inhibitory activity NGF activity was detected in an assay with sympathetic ganglia from chicken embryo explanted into a collagen angel (Ebendal T, Olson L, Seiger Å, Belew M (1984): Nerve growth factors in chick 'and rat tissue. In Black IB (ed): “Cellular and Molecular Biology of Neuronal Development, "New York: Plenum pp. 231-242). 10 15 20 465 573 The effect of increasing antiserum concentrations on the NGF-induced fiber growth was determined using this assay. ma-: wELs' Immunohistochemistry The NGF peptide antibodies were also immunohistocernically characterized in salivary glands from adult male mice kept isolated at least 24 hours before killing to guarantee dormancy in the salivary glands. The salivary glands were fixed by perfusing the animals with 4% paraformaldehyde and the glands was fixed by immersion in the same fixative for another 2 hours (Olson L, Ayer-LeLievre C, Ebendal T, Seiger Å (1987): Nerve growth factor-like immunoreactivities in rodent salivary glands and testis. Cell Tissue Res 248: 275- 286). Cryostat sections (5-14 μm) were incubated (4 ° C overnight) with the affinity purified an fi kfoppanta from 1 fi iizo ug / m1 in føsfaibuffraa coke mass (Pas) in presence of 0.3% Triton X-100. After washing, the bound antibodies were isolated through incubation for one hour at room temperature with fluorescein isotheliocyanate labeled anti-rabbit or anti-sheep antibodies. After further washing was mounted sections with coverslips in a mixture of glycerol and PBS (9: 1) containing 0.1% p-phenylene diamine to counteract fading (fluorescence losses at microscopy) (Johnson GD, de C Nogueira-Araujo GM (1981): A simple method of reducing the fading of immunofluroescence during microscopy. IN.

Immunol. Methods 43: 349-350; Platt IL, Michael AF (1983): Retardation of fading and enhancement of intensity of immunofluorescence by p-phenylenediamine.

I. Histochem. Cytochem. 31: 840-842). The sections were evaluated using Epi-uorescence microscopy (Nikon Microphot FX) and distribution and intensity at the fluorescence was estimated on blind-labeled lenses. Fluorescence intensities was estimated semi-quantitatively on a scale from 0-5. 10 15 20 4 6 5 5 7 3 RESULTS Antisera to the 8 synthetic N GF peptides were obtained from rabbits (Table I) after the peptides are conjugated to KLH. All peptide conjugates resulted in antisera bound in the ELISA test to the respective NGF peptide (Table H). Serum titers (ald Successful dilutions) measured by ELISA ranged from l: l0.000 to 1: 1,000,000.

Peptide P2 gave high background values when used to cover immune bowls, necessitating conjugation with bovine serum albumin (BSA) before binding to the dishes to achieve efficacy estimates of the titers of antisera against P2. Titers saturated with KLH-covered bowls exceeded the titers for peptide binding and was in the order of 1: 1,000,000 more 1: l0.000.000.

All peptide antisera affinity chromatographed on columns to which the peptides had been coupled. The immunoglobulin stock was off on the order of 50-200 ug / ml antiserum (Table II). The balance corresponded only to some extent the relative titers of the various antisera.

Binding of some of the different peptide antisera to the respective NGF peptide as a function of serum dilution is shown in Figure 1A. Antisera tape to layer off respective peptides much more effective than normal rabbit serum at the corresponding concentration Some peptide antisera were particularly good, although strict ironing are not possible, as different peptides may be adsorbed differently the microscopes.

Similar results were obtained with affinity purified peptide antibodies (Figure IB). The signal vis-à-vis KLH-covered dishes was simultaneously reduced by 50- to SOO-fold (no illustration). Thus, it was concluded that the procedures used for affinity purification are useful for amplifying the specific signal e.g. for immunohistochemical studies.

The specificity of the peptide antibodies was further tested by ELISA by test antiserum against a given peptide against dishes with a series of different synthetics NGF peptides. An example is shown in Figure IC, from which it follows that the homolog the combination leads to the obviously best binding. However, some were seen binding also to peptides other than that used for immunization (Fig.

IC). ' 10 15 20 35 465 573 These findings show that antibodies specific for the various synthetics The NGF peptides are formed as a result of immunization of rabbits and the antibodies can be purified efficiently by affinity chromatography. ELISA was then used to examine whether any of the peptide antibodies recognized the corresponding peptide sequence in native ß-NGF protein. For comparison rabbit antibodies (No. 17) to mouse ß-NGF were included in ELISA with NGF-coated immune bowls (Fig. 2A). Of the various peptide antibodies, only Ig37 and 38 bound both targeted to peptide P3, strong to mature ß-NGF protein. Signal strength obtained with these two antibodies were close to those seen with affinity purified anti-NGF antibody (Fig. 2A). The other peptide antibodies bound to NGF at least two ten powers less efficient, some not above background levels obtained with normal rabbit immunoglobulin. Low binding levels against native NGF was also obtained with the use of precursor-specific peptide antibodies. Found suggests that peptide P3 is unique in causing the formation of antibodies such as recognizes mature native NGF protein.

It was further investigated whether P3 is an epitope in ß-NGF found in polyclonal antisera obtained by immunization with ß-NGF. Three antisera (AS 10, 17 and 30) were examined in a PS ELISA, where everyone recognized the synthetic the peptide clearly above background levels even though they differed in their relative ability to bind to P3 (Fig. 2B). The three NGF antisera bound to P3 approximately one thiopotency less effective than the anti-P3 antiserum AS 38 itself. An antiserum (AS 46) obtained by immunization with ß-NGF, followed by booster injections with PB-KLH, gave the same high binding to P3 dishes as homologous P3 antiserum (Fig. 2B).

To test if any of the other peptides would be recognized by antiserum to NGF, AS 10, 17 and 30 were added to the whole series of NGF peptides in one ELISA. Peptide P3 gave reproducibly high binding to the antibodies (Fig. 2C).

AS 17 was also tied to P4, which was less clear with AS 10 and 30.

The antiserum AS 46 (the combined NGF / P3 antiserum) also felt it only again P3 and not any of the other peptides.

The possibility that the peptide antibodies would interfere with NGF activity was examined in a bioassay using explanted sympathetic ganglia from chicken (Ebendal T, Olson L, Seiger Å, Belew M (1984): Nerve growth factors in chick and rat tissue. In Black IB (ed): “Cellular and Molecular Biology of Neuronal Development. "New York: Plenum pp. 231-242). NGF-induced fi beet growth (stimulated with mouse ß-NGF) was not blocked by any of 10 15 20 465 573 peptide antisera, not even when present in high concentrations (3%) in the bowls (no illustration). Affinity purified antibodies to peptide P3 could nor does it block the growth response (tested at concentrations up to 8 pg Ig 38 per ml). This property of peptide antibodies is reminiscent of the inability of monoclonal antibodies to NGF to block ß-NGF-induced fiber growth in ganglia tested. Peptide P3 (in the concentration 100 ng / ml) could not replace either NGF in terms of nerve fiber growth stimulation, and furthermore peptide P3, added in strongly molar excess interfere with biological activity of N GF. In a thousand-fold dilution, however, NGF antiserum AS 46 blocked the ß-NGF induced the fibrous growth response in sympathetic ganglia.

Immunohistochemistry Properties of peptide antisera and affinity purified antibodies was also evaluated using immunohistochemical techniques in male mice submandibular gland. Different immunoglobulin preparations from the 19 rabbits immunized with the eight different peptides (Table II) were tested immunohistochemically. In many cases, pre-absorption tests were performed with respective peptides. In addition, in the case of peptides 4 and 5, they were compared affinity purified preparations with the excluded fractions from the affinity columns. Finally, comparisons were made with the distribution of immunofluorescence in salivary gland induced with affinity purified antibodies.

EXAMPLE 6 The following peptide: Glu-Pro-His-Ser-Glu-Ser-Asn-Val-Pro-Ala-Gly-I-Iis-'Ilxr-Ile. was synthesized by Cambridge Resesarch Biochemicals Ltd., England, with a calculated purity of> 80% and checked by amino acid analysis, HPLC and mass spectrometry. The peptide corresponds to position -103 to -90 in the human NGF- the precursor.

With this peptide, the same treatments and procedures were performed as described in Examples 2-5 above and with the same useful results. The It was also noted that although the peptide corresponds to a sequence in the human NGF precursor, purified antibodies to the peptide could also bind to NGF precursor from rat, despite differences in amino acid composition.

The invention is an important contribution to determination techniques in such a way that the levels of both NGF precursor and mature NGF per se can be determined by 4 6 5 5 7 3 using peptides in this invention. It is important when it comes to obtaining correct diagnosis of diseases of the nervous system, because the relationship between levels of precursor and level of mature NGF can be crucial for assessment of which treatment to resort to. Thus, abnormal levels of mature human NGF is caused either by overproduction thereof or by reduced consumption thereof. Thus, the precursor level can be decisive when it is important to assess the cause of the disease and thus enable it to be correct treatment. 465 573 TABLE 1 Synthetic NGF and Pro-NGP Peptides' Pep fi d Position No. Species Sequence Start End P1 Hen (Thy fl-Glu-'Iïïr-Lys-Cys-Arg-Asp-Pro- 54 69 Arg-Prc-Val-Ser-Ser-Gly-Cys-Arg-Gly P2 Hen Cys-Val-Leu-Ser-Arg-Lys-Ser-Gly-Arg-Pro 109 118 P3 Rat (mouse) (Cysßly-Asp-Lys-Thr-'Iïu-Ala-'Ilxr-Asp-23 35 hen, lle-Lys-Gly-Lys-Glu person) P4 Rat Cys-Arg-Ala-Pro-Asn-Pro-Val-Glu-Ser-Gly 58 67 P5 Rat (mouse) (Cys) -Glu-Pro-Tyr-'Iïxr-Asp-Ser-Asn-Val- -103 -90 Pro-Glu-Gly-Asp-Ser-Val P6 Rat (mouse, Ser-Prø-Arg-Val-Leu-Phe-Ser-'ITu-Gln- -38 -28 human) Pro- (Cys) P7 Rat (Lys) -Va1-Leu-Ser-Arg-Lys-A1a-Ala- 111 120 Arg-Arg-Gly P8 Rat (Cys) -Val-Lys-Ala-Leu-'Iïmr-'Ilxr-Asp- 87 97 Asp-Lys-Gln-Ala * Amino acid residues given in parentheses have 1 to enable conjugation. considered to the respective NGF sequences 4 6 5 5 7 3 TABLE H Studied NGF peptide antisera 5 Peptide no. Rabbit AS no. ELISA titer Retention (equal to IgG / 1111) * 10 P1 32 1: 200,000 60 33 1: 100,000 90 35 1: 10,000 15 44 1: 1,000,000 70 48 1: 200,000 120 15 P2 34 1: 10,000 30 36 1: 100,000 200 P3 37 1; 4oo.ooo 40 20 38 1: 1,000,000 80 P4 39 1: 1,000,000 100 P5 41 1: 100,000 60 25 42 1: 10,000 30 P6 43 1: 100,000 80 49 1: 100,000 100 30 P7 45 1: 10,000 150 50 1: 20,000 90 P8 51 1: 200,000 220 52 1: 100,000 40 35 * Values refer to the retention of specific peptide antibody per ml of antiserum after affinity chromatography

Claims (13)

465 575 'z PATENTKRAV A peptide of the formula: Glu-Pro-His-Ser-Glu-Ser-Asn-Val-Pro-Ala-Gly-His-Thr-Ile; Gly-Asp-Lys-Thr-Thr-A1a-Thr-Asp-Ile-Lys-Gly-Lys-Glu, or immunogenically corresponding extended or truncated forms thereof excluding native NGF and precursors thereof. A peptide according to claim 1 having the formula: Glu-Pro-Hís-Ser-Glu-Ser-Asn-Val-Pro-Ala-Gly-His-Thr-Ile. A peptide according to claim 2 having the formula: Gly-Asp-Lys-Thr-Thr-Ala-Thr-Asp-Ile-Lys-Gly-Lys-Glu. A peptide according to any one of the preceding claims for diagnostic use in the quantitative determination of NGP and its precursors in living animals or humans. Antibodies directed against peptide according to any one of the preceding claims. Antibodies according to claim 5 directed against the peptide according to claim 2. Antibodies according to claim 5 directed against the peptide according to claim 3. Antibodies according to any one of claims 5 to 7 which are polyclonal. Antibodies according to any one of claims 5 to 7 which are monoclonal. A method for determining the presence of mature NGF and / or a precursor thereof in a biological sample using the interaction between the antibodies according to any one of claims 5 to 9 and said NGF or said precursor. ll. A method according to claim 10 for the quantitative determination of a precursor to mature NGF in a biological sample of the interaction between the antibodies according to any one of claims 6, 8 and 9 and said precursor. A method according to claim 10 for the quantitative determination of a precursor to mature NGF in biological sample material by interaction between the antibodies according to any one of claims 7, 8 and 9 and said NGF. A method according to any one of claims 10 to 12, wherein said sample material consists of mammalian brain tissue.