HK1024250A - Antibodies that bind to the nidogen-binding domain of laminin, their production and use - Google Patents

Description

Antibodies that bind to the nidogen-binding domain of laminin and their preparation and use

The present invention relates to monoclonal and polyclonal antibodies and parts thereof that specifically bind to the nidogen-binding domain of laminin, to methods for the production of said antibodies and parts thereof, and to their use as medicaments, as diagnostic reagents for the detection of laminin isoforms, as model substances for the study and evaluation of substances that influence the nidogen/laminin interaction. The antibodies of the invention or portions thereof preferably bind to the γ 1III 4 region of laminin, particularly to the essential nestin binding site in the highly conserved regions of loop a or loop-a and c, and are capable of inhibiting the binding of laminin and nestin.

The binding of laminin (an 800kDa glycoprotein) and nidogen (a 160kDa glycoprotein) is considered to be a critical biomolecular mechanism in basement membrane synthesis and stabilization [ Mayer, U. & Timpl, R. (1994): extracellular matrix assembly and Structure (P.D. Yurchenco et al eds.) p389-416, Academic Press, Orlando, FL ]. Since nestin can associate with all major components of basement membranes, such as the gamma 1-containing laminin isoform [ for its nomenclature, see burgenson, r.e. et al (1994), matrix biology, 14: 209-211), collagen IV, perlecan and fibulin and their respective binding structures form a ternary complex, so that nestin may function to connect members, interconnect, sterically recognize and stabilize macrostructures different from each other (Fox, J.W et al (1991) EMBO J.10: 3137-3146; Aumailley, M. et al (1993) Kidney int.43: 7-12).

Experiments using polyclonal anti-laminin antibodies provide clear evidence that: laminin/nidogen interactions play a major role in the synthesis of functional basement membranes. These antibodies were obtained by immunizing rabbits with laminin P1 or recombinantly produced laminin fragment γ 1III 3-5, and concentrated by affinity chromatography on laminin P1 or laminin γ 1III 3-5 matrix. In inhibition assays, the antibody completely inhibited laminin/nidogen binding, however, this inhibition was antibody-based, with the binding region of the antibody being located only near the nidogen binding sequence of laminin, spatially blocking access of nidogen to laminin (Mayer, U et al (1993) EMBO J.12: 1879-. In embryonic organ culture, these antibodies inhibit tubular development and also inhibit alveolar formation. Both of these processes are newly synthesized ontogeny programs that rely on the basement membrane without being hindered (Ekbum, P et al (1994) development 120: 2003-.

The position and sequence of the nidogen-binding domain of laminin and its spatial structure (X-ray crystal structure and NMR structure) have been clearly identified and described (Mayer, U et al (1993) EMBO J.12: 1879-. The binding domain is located within the "LE module" (laminin-type, epidermal growth factor-like) in the γ 1III 4 region of the γ 1 chain short arm of laminin. An "LE module" is a structural motif consisting of 50-60 amino acids that exhibits a complex folding pattern with 4 disulfide bonds similar to epidermal growth factor (Bairoch, A. (1995) nomenclature of the extracellular domain, SWISS-PROT protein sequence database, Release 310; Engel, J. (1989) FEBS Letters 251: 1-7).

It has been demonstrated that for mouse EHS tumor laminin P1, human placental laminin 2 and laminin 4, and drosophila laminin, nestin binds with high affinity to complementary laminin regions. The reason for the occurrence of such interspecies overlapping binding specificities is: there are abnormally high levels of amino acid sequence identity in the various laminin γ 1III 4 regions studied. When the whole module is considered, the above sequence identity between human and mouse reaches 97%, and the above identity between human and Drosophila reaches 61%. If the comparison is limited to the region of the a to c loop containing the necessary nestin binding site, this is increased to 100% and 75%, respectively (Pikkarinen, T et al (1987) J. biochem., 263: 6751-6758; Chi, H-C. & Hui, C.F. (1989) J. biochem., 264: 1543-1550).

In addition to elucidating the dependence of nestin binding on the entire three-dimensional structure, it is also possible to identify well-defined sequence regions within the S-S stabilized a and c loops of the laminin gamma 1III 4 region. 5 essential amino acids were identified: 4 lie within a segment of 7 amino acids in the a-loop, one tyrosine in the c-loop side chain (Poschl, E et al (1994) EMBO J, 13: 3741-3747; Mayer, U et al (1993) EMBO J.12: 1879-1885; Poschl, E et al (1996) EMBO J, 15: 5154-5159).

In specific binding assays, synthetic peptides obtainable from the corresponding region of the gamma 1III 4 region of laminin were able to completely inhibit laminin/nidogen binding (US5,493,008). However, this synthetic peptide showed about 400-fold lower activity in inhibition assays than that of intact laminin P1 or laminin γ 1III 3-5 (Poschl, E et al (1994) EMBO J, 13: 3741-3747; U.S. Pat. No. 3,000). Laminin/entactin interactions are affected by a powerful conformational component (Mayer, U et al (1993) EMBO J.12: 1879) -1885). In aqueous solution, peptides can adopt countless different conformations, with the result that only a part of the peptides is present in a conformation with biological activity, which may explain the reason why the inhibitory effect of synthetic peptides is weak.

The use of these Peptides as pharmaceuticals is greatly limited due to their flexibility in configuration, instability in the presence of proteases and poor bioavailability and potency (Milner-White, E.J, (1989) Trends Pharmacol. Sci.10: 70-74; Hruby, V.J, (1994): Peptides, Proc. third American peptide Symposium; (Hodges, R.S. & Smith, J.A eds.) p 3-17; ESCOM: Leiden, Netherlands).

Antibodies that specifically bind to the nidogen-binding domain of laminin and competitively inhibit binding between laminin and nidogen at low concentrations are more suitable for use as therapeutic agents in the treatment of diseases because of their higher affinity, higher degree of stability and satisfactory pharmacokinetics. In addition, they can be used as diagnostic reagents or as aids in the study and evaluation of biological and pharmaceutical models of substances that influence the laminin/entactin interaction.

Although some anti-laminin P1 or anti-laminin γ 1III 3-5 antibodies previously produced were able to inhibit nestin/laminin binding, they did not directly recognize the nestin binding site of the laminin γ 1 chain; rather, nestin/laminin binding is inhibited as a result of steric interaction (Mayer, U et al (1993) EMBO J.12: 1879) -1885). The nidogen-binding domain of laminin gamma 1 chain is unusually highly conserved in an interspecies overlapping manner. In addition, laminin is an extracellular protein which is continuously exposed to the immune system, either as an integral component of the basement membrane or as a circulating serum component (EP 0696597 a 2). Since the immune system can distinguish "i" from "non-i", it can be concluded that each immunized species recognizes a highly conserved immunizing antigen as its own component, and therefore does not produce any antibodies against this component, and therefore, cannot be expected to produce a specific antibody titer. The above generally accepted doctrine has been further confirmed by the fact that, to date, no antibodies against the laminin nidogen binding domain could be generated by immunizing rabbits with laminin P1 and laminin γ 1III 3-5 (Mayer, U et al (1993) EMBO J.12: 1879-. However, the above-mentioned polyclonal antibodies that bind to a non-specifically defined epitope outside the nidogen-binding domain of laminin are not well suited or entirely unsuitable for use as therapeutic agents, diagnostic agents, or as model substances for the study and evaluation of substances that influence laminin/nidogen interactions: since steric inhibition depends on the steric extension of the inhibitor, it is almost impossible to use portions of these antibodies as therapeutic agents (which are pharmaceutically preferred). In addition, possible cross-reactivity with analytes that are not intended to be detected limits the use of these antibodies in diagnostic assays.

The aim of the present invention is to produce antibodies which specifically bind to the nidogen-binding domain of laminin, i.e. which are able to directly recognize the nidogen-binding domain of the laminin gamma 1 chain, and which are suitable for use as medicaments, as diagnostic reagents for the detection of laminin isoforms and as model substances for the development and evaluation of substances which influence the laminin/nidogen interaction.

The objects of the invention are achieved by the antibodies described below, as well as methods of making and using these antibodies.

The antibodies of the invention, or portions thereof, characteristically bind to the nidogen-binding domain of laminin (i.e., the laminin γ 1III 4 region), preferably to a highly conserved region of the a-loop or a and c-loops of the laminin γ 1III 4 region. It is particularly preferred that the antibodies of the invention bind to the highly conserved regions of loop a or loops a and c either directly or in an overlapping manner in a conformation-dependent manner related to the epitope (i.e. recognition of the native conformation of the nidogen binding site of laminin; see example 6). In particular, the invention includes antibodies or portions thereof that bind to at least one of the peptides shown in table 1. The invention provides polyclonal and monoclonal antibodies. Preferably, the antibodies of the invention are chimeric, humanized, bispecific or oligospecific antibodies. It is particularly preferred that the antibodies of the invention competitively or partially competitively inhibit laminin/entactin binding (see examples)7). Table 1: amino acid sequence of a peptide for immunization (1): DNIDPNAVGNL

The antibody of the present invention can be obtained by immunizing an immunologically active vertebrate such as rabbit, mouse, sheep, goat, guinea pig, rat and chicken with laminin, laminin P1, laminin γ 1III 3-5 or laminin γ 1III 4, particularly with a peptide containing the essential nidogen-binding site of the laminin γ 1III 4 region but not the entire amino acid sequence thereof, particularly preferably with one or two peptides shown in table 1 as the immunizing antigen.

Antibodies were identified using laminin gamma 1 III-3-5 and/or laminin gamma 1 III-4 when immunized with laminin or laminin P1, and ultimately tested for their ability to competitively or partially competitively inhibit laminin/nidogen binding.

Antibodies were identified using laminin and/or laminin P1 when immunized with laminin γ 1III 3-5, and ultimately tested for their ability to competitively or partially competitively inhibit laminin/nidogen binding.

It is particularly preferred to use laminin gamma 1III 4 or one or more peptides shown in Table 1 as immunizing antigen. Antibodies obtainable by immunization with these immunizing antigens are preferably identified using laminin and/or laminin P1. It is advantageous to test the ability of the identified antibodies to competitively or partially competitively inhibit the laminin binding site.

In addition to polyclonal antibodies, in the examples below, monoclonal antibodies (mabs) are also obtained using hybridoma cells that are initially produced to produce monoclonal antibody mabs. The antibodies of the invention can also be purified from antibody-containing material (e.g., antisera from immunized animals, hybridoma cell culture supernatant, ascites fluid, or cells) using affinity chromatography, preferably using affinity chromatography with laminin and/or laminin P1 as the affinity matrix, to obtain the antibodies in purified form.

The antibodies or portions thereof of the invention inhibit laminin/nidogen interactions and, as a general term, they also include corresponding chimeric, humanized, bispecific or oligospecific antibodies, and antibody analogs, which are described in detail elsewhere.

The invention also includes cells and cell lines of animals, plants and prokaryotes, preferably the hybridoma DSMACC2327, which is deposited on Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of microorganisms and cell cultures) (DSMZ, Marsche der Weg 1b, D-38124 Braunweischig, Germany) on day 27, 10.1997, according to the Budapest treaty. The invention also relates to a monoclonal antibody produced by the hybridoma deposited with the accession number DSMACC 2327.

The present invention further includes a method for producing the above antibody, wherein the above antibody is produced using an immunologically active vertebrate such as rabbit, mouse, sheep, goat, guinea pig, rat and chicken, using laminin, laminin P1, laminin γ 1III 3-5 and laminin γ 1III 4, particularly preferably using a peptide not containing the complete amino acid sequence of the region of laminin γ 1III 4, and more particularly preferably using one or two peptides shown in table 1. It is understood that the term "peptide" includes oligopeptides, polypeptides, and also includes proteins and protein fragments. When used as an immunizing antigen, it is preferred to use a peptide coupled to a carrier, such as a protein (e.g., ovalbumin, albumin, or hemocyanin), or a polymer (e.g., polyethylene glycol, polyacrylamide, or poly-d-glutamine-d-lysine).

Antibodies were identified using laminin gamma 1III 3-5 and/or laminin gamma 1III 4 when immunized with laminin or laminin P1 and tested for their ability to competitively or partially competitively inhibit laminin/nidogen binding.

Antibodies were identified using laminin and/or laminin P1 when immunized with laminin γ 1III 3-5 and tested for their ability to competitively or partially competitively inhibit laminin/nidogen binding.

Preferably, laminin γ 1III 4 or, if desired, one or two peptides as shown in table 1, preferably coupled with a carrier, are used in the method of the invention.

Preferably, laminin and/or laminin P1 are used to identify antibodies raised by immunization with laminin γ 1III 4 or with one or two peptides shown in table 1, which antibodies advantageously detect the ability to competitively or partially competitively inhibit laminin/nidogen binding.

The method also optionally includes generating hybridoma cells that produce the MAb. It has proven advantageous to purify the antibodies of the invention or parts thereof from antibody-containing material, such as antisera from immunized animals, hybridoma cell culture supernatants, ascites fluids or cells, by means of, for example, affinity chromatography, preferably using affinity chromatography with laminin and/or laminin P1 as affinity matrix.

The antibodies of the invention or parts thereof can be used in a number of different ways, for example as pharmaceuticals, as diagnostic reagents, as aids in the development and evaluation of biological and pharmaceutical models of substances capable of influencing the nidogen/laminin interaction, for example as model substances, for the evaluation of the spatial structure of contact zones complementary to the nidogen-binding site of laminin and the potential binding potency of such contact zones, and for the study of the biosynthesis of basement membrane and of the influence of basement membrane in different physiological processes, such as organ development, angiogenesis or embryogenesis. The invention also includes pharmaceutical and diagnostic reagents comprising one or more antibodies or antibody portions of the invention.

The invention also includes the use of one or more antibodies or antibody portions of the invention for: for the preparation of medicaments for the treatment of diseases of the basement membrane, in particular of the excessive or unnecessary synthesis of the fibrous membrane, especially alcoholic liver fibrosis and pulmonary fibrosis, and of all forms of diabetic late complications accompanied by thickening of the basement membrane, especially in the kidney, eye and vascular system, and also of arteriosclerosis and all diseases with worsening clinical symptoms due to angiogenesis, such as cancer, diabetic retinopathy, and diseases with strong inflammatory factors, such as rheumatoid arthritis, osteoarthritis, vasculitis, hemangiomas and psoriasis; for the preparation of diagnostic reagents for the detection of gamma 1-containing laminin isoforms in biological samples, for example body fluids (such as blood, serum, plasma, urine, saliva or cerebrospinal fluid) and tissues. The term diagnostic agent includes, for example, different embodiments of reagents used in xenogeneic and allogeneic immunoassays, immunohistochemical detection systems, and in vivo detection methods such as immunoscintigraphy. Articles of manufacture comprising the antibodies or antibody portions of the invention may also be used in the form of diagnostic kits or in combination with other auxiliary reagents (e.g., buffers, wash solutions, emission signal detection solutions) and/or other aids (e.g., cuvettes).

Hereinafter, the invention will be described and illustrated in more detail by means of a number of examples:

surprisingly: antibodies to amino acid sequences highly conserved at the nidogen-binding domain of laminin can be generated using peptides listed in table 1 that do not form any folding pattern as seen in the LE module. For this purpose, the peptides shown in table 1 were conjugated to ovalbumin using carbodiimide, and then rabbits were immunized with these conjugates. The production of specific antibody titers against laminin P1 and laminin gamma 1III 3-5 was analyzed by means of an enzyme immunoassay.

The antibodies were concentrated by passing polyclonal antibodies of the desired specificity through a matrix that bound human placental laminin P1 for affinity chromatography, followed by molecular sieve chromatography. Methods for the purification and characterization of human placental laminin are described in EP 0696597 a2, as well as the use of human placental laminin for immunization of mice and the isolation of hybridomas that synthesize antibodies against laminin P1.

Antibodies concentrated after affinity chromatography of laminin P1 on antiserum showed binding specificity to human placental laminin P1/mouse laminin P1(EHS tumor) and rat laminin (yolk sac). In addition to recognizing this specific sequence, the antibody also recognizes a biologically active conformation of the nidogen-binding domain of laminin. They completely inhibit laminin/nidogen binding.

Preferably, however, the antibodies of the invention are also obtained by immunizing other vertebrates, such as mice, sheep, goats, guinea pigs, rats and chickens, which are immunologically active with laminin γ 1III 4, in particular with peptides which contain the important nidogen binding site of the region of laminin γ 1III 4 but which do not contain their complete amino acid sequence, and particularly preferably with the peptides shown in table 1. Polyclonal antibodies of the invention can be purified from antisera from the immunized animal. For the production of corresponding monoclonal antibodies, well known methods can be used (see e.g., Harlow, E.&Lane, d. (1988) antibodies: a laboratory manual, cold spring harbor laboratory, cold spring harbor), immune cells from an immunized animal (e.g., mouse) are fused with myeloma cells to produce hybridoma cells that produce mabs, and appropriate clones are isolated. The desired Mab producing clones are selected using specific screening methods. Preferably, enzymatic or radioimmunoassays, as well as Western blots, are used herein to examine the specificity of antibody binding, e.g., to immune antigens, immune antigen carriers, and native and recombinant laminins and/or fragments thereof, released into the culture supernatant. Another possible selection criterion is the ability of the antibody to prevent nestin/laminin binding. This ability can be assessed using, for example, the inhibition assay described in detail in the examples. Hybridomas that produce mabs that specifically bind the nestin-binding domain of laminin have been cloned and can then be used for long periods to produce mabs. According to the desired purpose, only a part of the antibody is used, e.g. F (ab)₂Fab', or Fab fragments may be more advantageous. Using e.g. the fieldThese fragments can be generated by enzymatic cleavage methods known to the skilled person (see e.g. Harlow, E.&Lane, d. (1988) antibodies: a laboratory manual, cold spring harbor laboratory, cold spring harbor).

The antigen binding site of an antibody is located in the so-called variable region, which is encoded by the corresponding V gene. If the sequence is not known as a result of amino acid sequencing, the corresponding nucleic acid sequence of an antibody that binds to the nidogen-binding domain of laminin can also be determined using known methods of genetic manipulation (see, for example, molecular cloning of Sambrook, J et al (1989): A laboratory Manual, Cold spring harbor laboratory, Cold spring harbor, 2 nd edition; McCafferty, J et al (1990) Nature 348: 552-554) to thereby presume the corresponding amino acid sequence. Hybridoma cells or immune cells producing antibodies from an immunized mammal are used as starting materials for the assay.

When nucleic acid and amino acid sequences are known, humanized or chimeric, bispecific or oligospecific antibodies and antibody analogs that specifically bind to the nestin-binding domain of laminin, such as peptides derived from complementarity determining regions ("minimal recognition units"), single-chain fragments and functional fusion products, such as recombinantly produced antibodies/enzymes or antibody/complement constructs, can be prepared using conventional genetic and molecular biological methods (see also Johnson, K.S. & Chiswell, D.J. (1993) structural biology recent findings, 3: 564 571). These molecules derived from the antibodies or antibody genes of the invention are included in the general term "antibody or portion thereof" as used herein. These molecules can be used as follows: for example, they may be used for reduced immunogenicity and/or increased efficacy when administered as a medicament, and/or may also be of benefit when used as a diagnostic agent or as an aid in the study and evaluation of substances affecting laminin/nidogen interactions. Antibodies or portions thereof can be produced in plants (e.g., yeast), animals, and prokaryotic cells.

When used for in vivo diagnosis, the antibodies and portions thereof of the present invention may be modified, for example, by labeling with radioisotopes or paramagnetic compounds, or may be modified by binding a pharmaceutically active substance thereto to produce even more effective drugs.

The examples described below illustrate various aspects of the invention by way of example. Example (b):

SDS gel electrophoresis, Western blotting and BCA protein assays were performed according to standard methods or manufacturer's instructions. Unless otherwise stated, the compounds used were obtained from Merck (Darmstadt), Sigma (Munich) or Riedel de Haen (Seelze). Example 1: synthesis of peptides

Peptides were synthesized in solid phase (ABI433 peptide synthesizer) using 148mg (0.1mmol) of FMOC-amide Anchor-PAM resin. After completion of the synthesis, an increase in resin weight to 513mg was observed. The resin was treated with a solution consisting of 10ml trifluoroacetic acid and 365. mu.l triethylsilane for 2 hours at room temperature. After the resin was filtered off, the solution was concentrated by rotary evaporation in vacuo, the residue was dissolved in 50ml of 10% acetic acid (AcOH), and the solution was lyophilized to give 145mg (yield 54%) of crude peptide. The crude peptide was then dissolved in 3ml 80% AcOH and this solution was added dropwise to a rapidly stirred solution (0.006mmol iodine and 0.006mmol sodium acetate in 55ml 80% AcOH). After 5 minutes, 0.1N ascorbic acid solution was added to terminate the reaction. The solution was concentrated to a volume of 2ml and loaded on a Sephadex  G25 column and developed with 0.1 MAcOH. The isolated peptide is then obtained in highly purified form using reverse phase HPLC chromatography. The yield is as follows: 36mg DNIDPNAVGNLCIKENTAGFYCDR-NH₂(13.5% of theory)

The structure was confirmed by mass spectrometry (molecular weight: 2673Da) and amino acid analysis.

Peptide DNIDPNAVGNL-NH was prepared in a similar manner₂. The yield is as follows: 268mg (67% of theory); molecular weight: 1140Da example 2: coupling peptides to ovalbumin

30mg of ovalbumin (Sigma A2512) was dissolved in 1ml of sodium phosphate buffer, pH7.4, and 200. mu.l of an aqueous solution containing 7mg of N-hydroxysulfosuccinimide sodium salt (Fluka 56485) and 300. mu.l of a solution containing 100mg of 1-ethyl-3- (3-diam) were added to the ovalbumin solutioninamidopropyl) carbodiimide, HCl (Sigma E6383) in water. After 5 minutes, a peptide solution (30mg of the appropriate peptide in 1ml of 10mM sodium phosphate buffer, pH7.4) was added. The coupling reaction was carried out in the dark for 16 hours at room temperature. At the end of the reaction period, the solution is centrifuged to remove any turbidity that may have occurred. Then, unreacted compounds and salts were removed by NAP 25 column (Pharmacia) chromatography. This transferred the ovalbumin/peptide conjugate to PBS + 0.04% Tween 20 in 50-55 mg. Conjugate 1: ovalbumin-DNIDPNAVGNL conjugate 2:example 3: immune rabbit

Hybrid rabbits weighing approximately 3kg were immunized with the ovalbumin/peptide conjugate. For this purpose, 1mg of each appropriate conjugate is dissolved in 0.5ml of Phosphate Buffered Saline (PBS), this solution is then mixed with the same volume of complete Freund's adjuvant, and the entire mixture is carefully emulsified. 1ml of the resulting emulsion was injected intradermally into rabbits; during this procedure, 1/5 volumes were applied to 1 of 5 different sites in the vicinity of regional lymph nodes each time. After 21 and 53 days, booster injections were performed with the antigen emulsion in semi-concentrated form (for this purpose, the antigen was emulsified with incomplete Freund's adjuvant). Example 4: development of titers in 4 immunized rabbits

The development of specific antibody titers in 4 animals was studied in detail by performing an enzyme immunoassay using plastic trays coated with laminin P1 or with laminin γ 1III 3-5. The corresponding animal sera are indicated by R2, R3, R4 and R905. Immunisation with conjugate 1 gave sera R3 and R4, whereas immunisation with conjugate 2 gave sera R2 and R905. In all rabbits, an immune response was elicited very quickly, which remained constant after 21 days (first booster) or decreased slowly but continuously. No restimulation of the immune response by the second booster injection was observed. The antibodies formed were reactive with either laminin P1 or the laminin γ 1III 3-5 region. However, the binding of antibodies to laminin P1 was somewhat less pronounced than to laminin γ 1III 3-5, and appeared to be less stable during the course of the immune response. This may indicate that there are several populations of antibodies in polyclonal serum that bind with different avidity to the nestin-binding motif in laminin P1 and laminin γ 1III 3-5, or their conformation. Example 5: affinity purification of antibodies

To further identify the antibodies, specific antibodies were separated from remaining immunoglobulins, other serum components and antibodies to ovalbumin in the animal serum. For this purpose, affinity chromatography was performed on laminin P1 affinity matrix using Fractogel  EMDazlactone 650(S) (Merck, Darmstadt) as support (gel matrix). Before coupling, in 6ml PBS, 1M Na₂SO₃0.3g of material was incubated at pH7.4 for 15 minutes and the liquid supernatant was then decanted off. During the incubation, the volume of the material swells to 1ml and the matrix can be used directly for covalent coupling with the desired ligand.

3.4mg of human laminin P1 were dissolved in 2ml of 0.2M ammonium bicarbonate, pH8.0, and this solution was incubated overnight (> 16 hours) with 1ml of activated (see above) support material at 4 ℃. The gel material was then washed with 0.2M ammonium bicarbonate, pH 8.0. Residual reactive groups on the support material were blocked by incubation at 4 ℃ in 5ml of 0.2M glycine, pH8.0, for 24 hours. By mixing in PBS, 1M Na₂SO₃(pH7.4), 0.1M sodium acetate (pH4.0) and 0.2M glycine (pH8.0) were incubated alternately, and 3 washing cycles were carried out to obtain a gel matrix for affinity binding. The column was packed with pharmaciHR 5/5 with the matrix and then equilibrated with PBS/0.04% Tween 20.

To purify antibodies binding to laminin P1 from animal sera, the relevant sera were diluted 1: 2 with PBS/0.04% Tween 20 and the diluted sera were passed through the column at a flow rate of 2 ml/min. Subsequently, the column was washed with buffer until the UV signal (220nm) flowing through the monitor again reached baseline. The bound antibody was finally eluted by changing the column buffer to 0.1M glycine/HCl, pH 2.7.

An enzyme immunoassay was performed using immobilized laminin γ 1III 3-5, and the column flow-through and column eluate were analyzed.

The above affinity purification was successfully used to purify antibodies from 4 animal sera, with an average yield of 0.3mg per 50ml of serum (the yield of serum R2 was only 0.1 mg). However, most antibodies (> 80%) in all sera did not bind to laminin P1, presumably either due to too low binding kinetics or due to only affinity for the sequence of the peptide used for immunization.

Affinity chromatography on a laminin P1 column allows for the selective enrichment of rapidly and stably bound antibody variants from serum (antibody search). Example 6: binding specificity of affinity purified antibodies

Western blot results confirmed that the affinity-purified antibody retained the binding specificity for laminin P1, and that the different preparations reacted more strongly with unreduced laminin P1 than with the portion of linear laminin P1 that is separable by reducing disulfide bonds (Gerl, M. et al (1991) European journal of biochemistry, 202: 167-. This is indirect evidence of conformation-dependent components in the binding specificity of the antibody. It was also found that the binding bias of the 4 antibody preparations was different. Two antibody preparations R3 and R4, immunized with conjugate 1, recognized the same laminin P1 band, obtained after SDS (sodium dodecyl sulfate) gel electrophoresis of the unreduced sample. While both antibody preparations R2 and R905 (anti-conjugate 2) also showed the same reaction pattern as each other, they recognized less laminin P1 band than both antibody preparations against conjugate 1.

In immunochemical detection of laminin P1 bands separated by reduction and SDS gel electrophoresis, we surprisingly found that all 4 antibody preparations had different binding biases for the laminin γ 1III 4-containing fraction. Example 7: inhibition assay-inhibition of laminin/nidogen binding with affinity purified antibodies

The inhibitory activity of the affinity purified antibodies can be identified using a coated tube assay that measures the binding of radiolabeled nestin to a tube coated with (human placental) laminin P1 in the presence of antibodies. Radiolabeling nestin with 125I

Recombinantly produced human nestin (35. mu.g, described for clones and culture and purification conditions see Mayer, U et al (1995), J. Eur. Biochem., 227: 681-¹²⁵I) Solution (═ 1.55 μ l, Nordion Europe), 10 μ l of 0.5M sodium phosphate (ph7.4) and 40 μ g of chloramine T (N-chloro-4-toluenesulfonamide sodium salt, Merck) dissolved in 100 μ l of 0.05M sodium phosphate (ph 7.4). The reaction was allowed to proceed for 60 seconds at room temperature, and a solution of 40. mu.g of sodium metabisulfite (Riedel-de-Haen) in 100. mu.l of 0.05M sodium phosphate (pH7.4) was added to terminate the reaction. This addition was continued for up to 30 seconds, then 900. mu.l of 1% BSA (Sigma) in PBS was added to the mixture. Free radioactivity and excess salt were separated by molecular sieve chromatography using a PD 10 column (Pharmacia). The iodine-labeled nestin eluted into PBS was collected and diluted with 1% BSA/0.05M sodium phosphate/0.01% sodium azide, pH7.4 to a concentration of 50 ng/ml. Coating reaction tube

Dissolved in carbonate buffer (0.159g sodium carbonate; 0.293g sodium bicarbonate; 0.02g NaN) at 4 ℃ in 4. mu.g/ml₃In 1 liter of distilled water), 20. mu.g/ml BSA (bovine serum albumin, Serva), laminin P1 solution in pH9.2 the reaction tube (Greiner, 75X 12, No.115061) was coated overnight. Free binding sites were blocked by incubation for 2 hours with 0.5ml 0.5% BSA in PBS/0.04% Tween 20. Inhibition assays (sequential inhibition) Using the "laminin mimetic" construct

200. mu.l of iodine-labeled nidogen (about 10ng, about 40,000cpm) and 200. mu.l of an inhibitor (e.g., a peptide obtainable from the region of laminin. gamma.1 III 4) or a standard (laminin. gamma.1 III 3-5) were shaken in a reaction vessel at room temperature. Both inhibitors and standards were dissolved in PBS/0.04% Tween 20. After 3 hours of incubation, 150. mu.l of this mixture was transferred to a coated tube and incubated for a further 2 hours at room temperature. Finally, the solution was removed, the tubes were washed 2 times with 1ml PBS/0.04% Tween 20, and the bound radioactivity (nestin) was then determined on a gamma counter. The amount of nestin bound in the inhibitor-containing solution correlates with the amount of nestin in the absence of the inhibitor. Inhibition assay (sequential inhibition) using the "nestin mimetic" structure

In a reaction vessel coated with laminin P1, 150 μ l of inhibitor (such as an antibody that binds to the laminin γ 1III 4 region or a peptide that is obtainable from the nestin sequence) or standard (recombinant nestin) was shaken for 3 hours. Both inhibitors and standards were dissolved in PBS/0.04% Tween 20. After the sample was absorbed, 150 μ l of iodine-labeled nestin (about 10ng, about 40,000cpm) was added and maintained for 2 hours to displace the bound inhibitor. Finally, the solution was removed, the containers were washed 2 times with 1ml PBS/0.04% tween 20, and then the bound radioactivity (nestin) was determined on a gamma counter. The amount of bound radioactive nestin is related to the concentration of the inhibitor or the concentration of the standard. Inhibition assay (Simultaneous inhibition)

In this test variant, no prior incubation was performed, but 75 μ l of iodine-labeled nestin (10ng) was transferred directly into a coated tube with 75 μ l of inhibitor or standard and then incubated at room temperature for 2 hours; apart from this difference, the present protocol is similar to the sequential inhibition assay variant. Results

The IC of laminin gamma 1III 3-5 standard was obtained from 10 independent experiments_50％0.22nM with a standard deviation of +/-15%. IC (integrated circuit)_50％Is defined as the concentration of the substance required to inhibit binding of nestin to laminin P1 by 50%. For comparison, IC's were obtained using the (equilibrium) inhibition assay described in US5,493,008_50％0.05nM with a standard deviation of +/-52%.

Table 2 shows the IC of the antibody preparations R3, R905, R1.2, R2.2 and R3.2 and the different free peptides_50％The value is obtained. Antibody preparations R1.2, R2.2 and R3.2 were obtained by a second round of rabbit immunizations with the novel conjugate II and by similar purification steps. These resultsIndicating reproducibility of the method. Table 2: inhibition of laminin/nidogen binding by antibodies of the invention

Inhibitors	R3	R905	R1.2	R2.2	R3.2	Peptide (1).)	Peptide (2).)
									IC50％nM	IC50％nM	IC50％nM	IC50％nM	IC50％NM	IC50％nM	IC50％nM
Sequential inhibition	72	150	500	80	350	60000	20000
								Simultaneous inhibition	110	-	600	80	500	60000	20000

*: the amino acid sequence is shown in table 1: type of test

The IC of an inhibitory peptide obtainable from the nestin binding domain is indicated in US5,493,008_50％Values were 22nM to 1000 nM. These values are not possible with the chosen tests, due to the significant reduction in incubation times; for example, in the assays described herein, the IC of peptide DNIDPNAVGNL_50％The value was only 60000 nM. Example 8: identification of binding kinetics using the BIAcore  System

The BIAcore  system from Pharmacia Biosensor can be used to monitor biospecific interactions on-line. The detection principle is based on an optical phenomenon (surface plasmon resonance) which is influenced by substances bound to the gold film. Briefly, the system is a miniaturized affinity chromatography of gold sensor surfaces. The amount of specifically bound ligand can be visualized as a resonance signal (Chaiken, I et al (1992) analysis of biochemistry, 201: 197-201; Karlsson, R et al (1992) antigen structure; (van Regenmortel eds.). p 127-148; CRC Press, Boca Raton, FI.).

Laminin P1 dissolved in 10mM sodium acetate (ph4.0) at a concentration of 200 μ g/ml was immobilized on the sensor sheet according to the instructions in the manual to give a matrix containing 4000RU bound laminin P1. Double pulsing was performed with 4. mu.l of 100mM HCl each time to regenerate the affinity matrix.

Nestin (20 μ g/ml) binds to laminin P1 at parabolic saturation kinetics and affinity purified antibody preparations R905 and R3 bind to laminin P1 in a linear antigen dependent manner in HBS buffer (10mM HEPES, 3.4mM EDTA, 150mM NaCl, 0.005% biakurvatant P20; pH7.4) at a flow rate of 2 μ l/min. The fact that no sign of a transition to the equilibrium state could be observed after 1400 seconds could be a reflection that the laminin P1-specific peptide sequence responsible for binding nestin was readily available for use by antibodies. Antibodies bind to this sequence whether it is in a biologically active conformation or a different conformation. Evidence of this view is the observation that nestin switches significantly to the saturated phase upon binding of only 600 RU. Thus, it is clear that not all of the immobilized laminin P1 molecules exist in a nestin-recognizable structure, as the theoretical maximum saturation value for the layer of 2500RU has not been reached. However, after a long time of exposure, both antibodies can reach this saturation. Surprisingly, the R905 sample had to be used at a 10-fold higher concentration (320. mu.g/ml) to achieve an association rate comparable to that of R3 (33. mu.g/ml). On the other hand, the binding of R905 to laminin P1 was more stable than that of R3, since the rate of R905 dissociation (as seen by the time to change to HBS buffer: 1500 seconds) was significantly slower than that of R3 preparation.

These findings explain the difference between R3 and R905 observed in the inhibition assay: ● antibody preparation R3 inhibited laminin/entactin binding better than R905 because R3 binding is characterized by more rapid binding kinetics. ● when incubated with nestin in a tube coated with laminin P1, the antibody preparation R3 also inhibited laminin/nestin binding because R3 has good binding kinetics at concentrations comparable to nestin. ● antibody preparation R905 was only able to inhibit laminin/entactin binding in the "sequential inhibition" assay variant, since it is characterised by a slow dissociation rate, so that subsequently added entactin can no longer readily substitute R905 from the antigen. When R905 and nestin compete for the binding site simultaneously, R905 is inferior to nestin due to its slow binding kinetics. Example 9: detection of the binding specificity of affinity-purified antibody preparations R3 and R905 by Western blotting

The interaction of antibody preparations R3 and R905 with conjugate 2 and ovalbumin was studied in Western blot analysis. For this purpose, according to NOVEX^TMInstructions, using 4-12% NuPAGE^TMGel (NOVEX)^TMSan Diego, CA) and MOPS buffers and then NuPAGE^TMTransfer buffer (NOVEX)^TM) The antigen was transferred to nitrocellulose membrane. After blocking the free binding sites on the membrane with 1. mu.g/ml polyvinyl alcohol (1min), the antigen was incubated with the detection antibody. Bound antibody was then detected with anti-rabbit IgG antibody covalently bound to alkaline phosphatase.

We found that affinity purified antibodies only bound to the peptide, as no interaction could be detected with the carrier protein ovalbumin. Nor was it observed with the blue "SeeBlue" standard marker (NOVEX)^TM) There is any reaction.

It has also been demonstrated that both R3 and R905 react with laminin and laminin derivatives derived from different species (human placental laminin and rat yolk sac laminin (Calbiochem), human placental laminin P1 and mouse EHS tumor laminin P1, as well as recombinantly produced mouse laminin γ 1III 3-5). This provides supporting evidence for binding of the antibody to conserved sequences within the nestin binding domain. Neither antibody preparation showed any cross-reactivity with human nestin or human type IV collagen. This is a prerequisite for the clear use of the above-mentioned antibodies as nestin antagonists. Example 10: preparation of monoclonal antibodies

To obtain monoclonal antibodies, suitable vertebrates, preferably mice or rats, are immunized using standard methods with, for example, laminin P1, laminin γ 1III 3-5 or laminin γ 1III 4, or with the conjugates mentioned in example 2. When antisera exhibit a specific immune response, the Mab-producing hybridomas are isolated using standard methods.

The desired antibody or corresponding hybridoma clone is selected using binding assays (e.g., dot blot or Western blot employing laminin γ 1III 3-5 and/or laminin γ 1III 4), particularly the inhibition assay described in example 7, among other methods. The selected clones constitute the source for the synthesis of large amounts of the antibodies of the invention.

Conventional methods, such as binding to protein G or protein a, can be used to purify the desired antibody. However, affinity chromatography on a laminin P1 column is preferably used. As for the polyclonal antibodies described above, this purification step makes it possible to sort out and selectively concentrate monoclonal antibodies having the best binding constants.

An example of an antibody according to the invention is the monoclonal antibody (MAb) produced by the monoclonal cell clone A6/2/4, which has been deposited under the provisions of the Budapest treaty at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of microorganisms and cell cultures) (DSMZ, Marscherer Weg 1b, D-38124Braunschweig, Germany) on 27 th 10.1997 with the accession number DSMACC 2327.

Hybridomas were obtained by immunizing mice with laminin P1 isolated from human placenta. Methods for purification of antigens and generation of hybridomas are described in EP 0696597 a 2. Based on its binding characteristics to laminin gamma 1III 3-5 and laminin gamma 1III 4, antibody A6/2/4 was identified. The antibody is monoclonal antibody of IgM subtype, which can be purified by molecular sieve chromatography. In part because the antibody is multivalent, it binds particularly strongly to laminin P1. For this reason, and due to the size of the IgM antibody, the antibody cannot be eluted from the laminin P1 affinity column (see above). The inhibition assay ("simultaneous inhibition assay variant", see above) has reflected strong binding of this antibody to laminin P1. MabA6/2/4 can inhibit laminin/nidogen binding，IC₅₀Was 30 nM.

Since its binding is clearly conformation dependent, the binding epitope within the region of laminin γ 1III 4 (the nidogen binding domain of laminin) cannot be well defined. However, the fact that the peptidic part (75-80%) of the nidogen binding sequence obtainable from laminin inhibits the interaction of the antibody with laminin P1 indicates that the binding epitope of Mab a6/2/4 overlaps with the binding epitope of nidogen.

The preparation of inhibitory monoclonal antibodies similar to the polyclonal antibodies of the present invention, which can be produced using peptide/ovalbumin conjugates, will be described below.

SJL/J strain mice were immunized subcutaneously with 50 μ g of peptide 2/ovalbumin conjugate (conjugate 2, supra) in the presence of complete Freund's adjuvant. After 4 and 8 weeks, 25 μ g each of conjugate 2 was injected subcutaneously in the presence of incomplete freund's adjuvant to boost the immune response for an additional 7 weeks. 3 days prior to fusion, the immune response was boosted by intraperitoneal injection of 25 μ g of conjugate 2.

For fusion, animals were sacrificed and splenocytes isolated. Spleen cells were fused with myeloma cell line P3X63AG8.653 in the presence of polyethylene glycol. Splenocytes x P3X63AG8.653 hybrids were selected by culturing the fusion mixture in hypoxanthine/aminopterin/thymidine medium for 3 weeks. To obtain stable cell lines, the resulting cell clones were subcloned several times. The resulting cell colonies were tested for antibody production in a variety of immunological binding assays. Cell lines E79/1/6 and E82/1/10 were selected according to the screening strategy described below. Experiments to characterize and identify specific monoclonal antibodies

Immunization of SJL/J mice with conjugate 2 resulted in a number of hybridoma clones producing antibodies. Antibodies in culture supernatants were shown to have strong immune responses to laminin gamma 1III 3-5, laminin P1, and ovalbumin.

Now, in order to find clones that produce monoclonal antibodies against the nidogen-binding domain of laminin with native structure, it is necessary to apply an appropriate screening method.

In this screening method, attention was focused mainly on the binding of antibodies to laminin P1 and laminin γ 1III 3-5. Also, in the subcloning process, it is noted that the antibody reacts negligibly with the carrier protein ovalbumin, and that an antibody specifically recognizing ovalbumin is selected.

The results obtained for the two clones identified in this way (E79 and E82) are given in tables 3 and 4. Table 3: determination of E79 binding in ELISA

	Laminin gamma 1III 3-5 coating: 2.5. mu.g/ml	Laminin P1 coating: 2.0. mu.g/ml	Coating with ovalbumin: 20 ug/ml
				Culture supernatant undiluted for hybridoma E79	1.33	0.68	1.63
E79/1/6, first clone, undiluted culture supernatant	2.03	0.16	0.27
				E79/1/6, purified antibody 2.5. mu.g/ml	0.56	0.33	0.05

Table 4: determination of E82 binding in ELISA

	Laminin gamma 1III 3-5 coating: 2.5. mu.g/ml	Laminin P1 coating: 2.0. mu.g/ml	Coating with ovalbumin: 20 ug/ml
				Culture supernatant undiluted for hybridoma E82	1.77	1.48	2.33
E82/1/10, first clone, undiluted culture supernatant	1.32	0.26	0.05
				E82/1/10, purified antibody 6.4. mu.g/ml	1.55	0.5	0.18

It is evident that cells immunoreactive with ovalbumin can be isolated by cloning. At the same time, in each case a cell clone producing antibodies capable of binding to laminin gamma 1III 3-5 and laminin P1 could be isolated. This fact and the fact that immunization was elicited with a peptide derived from the nidogen-binding domain of laminin indicate that antibodies are capable of binding to the nidogen-binding motif of laminin, which has a natural structure.

The "simultaneous binding" variant of the inhibition assay (see above) provides direct evidence of binding specificity, where the antibody competes directly with iodine-labeled nidogen for binding to the nidogen binding motif of complete laminin (laminin from mouse EHS tumor, Chemicon, No. cc095).

Antibody (IgG2a subtype) produced by cell clone E79/1/6 as IC₅₀Antibody (IgG1 subtype) generated by cell clone E82/1/10 at a level of 19nM to inhibit laminin-entactin binding as IC₅₀Laminin-nidogen binding was inhibited at a level of 190 nM.

Claims (42)

1. An antibody or portion thereof that binds to a laminin gamma 1 III-4 region that binds nestin.

2. An antibody or part thereof as claimed in claim 1 which binds to the a loop or the a and c loops of the laminin gamma 1III 4 region, or highly conserved regions of the immediate vicinity of these loops, which binds nestin.

3. An antibody or part thereof as claimed in claim 2 which binds to the highly conserved regions of loop a or loops a and c either directly in a conformation dependent manner associated with the epitope or in an overlapping manner.

4. An antibody or part thereof as claimed in one or more of claims 1 to 3, which binds to at least one peptide as shown in Table 1.

5. An antibody as claimed in one or more of claims 1 to 4 which is a polyclonal antibody.

6. An antibody as claimed in one or more of claims 1 to 4 which is a monoclonal antibody.

7. An antibody as claimed in claim 6 which is a chimeric, humanised, bispecific or oligospecific antibody.

8. An antibody as claimed in one or more of claims 1 to 7 which competitively or partially competitively inhibits laminin/nidogen binding.

9. An antibody obtainable by immunizing an immunocompetent vertebrate with laminin or laminin P1 as an immunizing antigen, followed by identifying the antibody using laminin γ 1III 3-5 and/or laminin γ 1III 4, and testing the resulting antibody for the ability to competitively or partially competitively inhibit laminin/nidogen binding.

10. An antibody obtainable by immunizing an immunocompetent vertebrate with laminin gamma 1III 3-5 as the immunizing antigen, followed by identifying the antibody using laminin and/or laminin P1, and testing the resulting antibody for the ability to competitively or partially competitively inhibit laminin/nidogen binding.

11. An antibody obtainable by immunizing an immunologically active vertebrate with laminin gamma 1III 4 and/or with a peptide that does not contain the complete amino acid sequence of the laminin gamma 1III 4 region but contains an important component of its nidogen binding site as an immunizing antigen.

12. An antibody as claimed in claim 11, wherein laminin γ 1III 4 is used as an immunizing antigen.

13. An antibody as claimed in claim 11 wherein one or two peptides shown in table 1 are used as immunizing antigens.

14. An antibody as claimed in one or more of claims 11 to 13 which is identified using laminin and/or laminin P1.

15. An antibody as claimed in one or more of claims 11 to 14, wherein the ability of the antibody to competitively or partially competitively inhibit laminin/nidogen binding is tested.

16. An antibody as claimed in any one of claims 9 to 15 wherein hybridoma cells are obtained which produce monoclonal antibodies.

17. An antibody as claimed in any one of claims 9 to 16 which has been purified from antibody-containing material by affinity chromatography.

18. An antibody as claimed in claim 18 wherein the affinity chromatography is carried out with laminin and/or laminin P1 as affinity matrix.

19. A cell or cell line producing an antibody or part thereof as claimed in one or more of claims 1 to 8.

20. Hybridoma DSMACC 2327.

21. An antibody produced by the hybridoma DSMACC 2327.

22. A method of preparing an antibody as claimed in any one of claims 1 to 8, which method comprises immunizing an immunocompetent vertebrate with laminin or laminin P1, identifying the antibody using laminin γ 1III 3-5 and/or laminin γ 1III 4, and testing the ability of the antibody to competitively or partially competitively inhibit laminin/nidogen binding.

23. A method of preparing an antibody as claimed in any one of claims 1 to 8, which method comprises immunizing an immunocompetent vertebrate with laminin γ 1III 3-5, identifying the antibody using laminin and/or laminin P1, and testing the ability of the antibody to competitively or partially competitively inhibit laminin/nidogen binding.

24. A method for the preparation of an antibody as claimed in any one of claims 1 to 8, which comprises immunizing an immunologically active vertebrate with laminin gamma 1III 4 and/or with a peptide which does not contain the complete amino acid sequence of the region of laminin gamma 1III 4 but contains an essential component of its nidogen binding site.

25. A method as claimed in claim 24, wherein laminin γ 1III 4 is used as an immunizing antigen.

26. A method as claimed in claim 24 wherein one or two peptides as shown in table 1 are used as immunizing antigens.

27. A method as claimed in claim 26 wherein the immunizing antigen is coupled to a carrier.

28. A method as claimed in one or more of claims 24 to 27, wherein antibodies are identified using laminin and/or laminin P1.

29. A method as claimed in one or more of claims 24 to 28, wherein the ability of the antibody to competitively or partially competitively inhibit laminin/nidogen binding is tested.

30. A method as claimed in one or more of claims 22 to 29, wherein hybridoma cells are produced which produce monoclonal antibodies.

31. A method as claimed in one or more of claims 22 to 30, wherein the antibody is purified from the antibody-containing material by affinity chromatography.

32. A process as claimed in claim 31, wherein the affinity chromatography is carried out with laminin and/or laminin P1 as affinity matrix.

33. An antibody or part thereof as claimed in any one of claims 1 to 18 or 21 for use as a medicament.

34. A medicament containing one or more antibodies or antibody portions as claimed in at least one of claims 1 to 18 and 21.

35. Use of one or more antibodies or antibody portions as claimed in at least one of claims 1 to 18 and 21 for the preparation of a medicament for the treatment of a disease characterized by an excess or unnecessary synthesis of basement membrane.

36. The use as claimed in claim 35, wherein the disease is in the form of late complications of diabetes accompanied by thickening of the basement membrane, in the form of arteriosclerosis, fibrosis, or a disease in which clinical symptoms worsen due to angiogenesis.

37. The use as claimed in claim 35 or 36 for the manufacture of a medicament for the treatment of diabetic retinopathy, alcoholic liver fibrosis, lung fibrosis, cancer, diabetic nephropathy or diseases with strong inflammatory factors, such as rheumatoid arthritis, osteoarthritis, vasculitis, hemangiomas and psoriasis.

38. An antibody or portion thereof as claimed in any one of claims 1 to 18 or 21 for use as a diagnostic reagent.

39. A diagnostic reagent comprising one or more antibodies or antibody portions as claimed in at least one of claims 1 to 18 and 21.

40. Use of one or more antibodies or antibody portions as claimed in at least one of claims 1 to 18 and 21 for the preparation of a diagnostic reagent for the detection of a gamma 1-containing laminin isoform in a biological sample, a body fluid or a tissue.

41. Use of an antibody or part thereof as claimed in any one of claims 1 to 18 or 21 as an aid in the study and evaluation of biological and pharmaceutical models of substances affecting laminin/entactin interactions.

42. The use as claimed in claim 41 in the study and evaluation of biological and pharmaceutical models of substances affecting laminin/entactin interactions.