Cell Matrix Receptor System and Use In Cancer Diagnosis and Management

Background Art

The basement membrane is a ubiquitous/ specialized' type of extracellular matrix separating organ parenchymal cells from interstitial collagenous stroma. Interaction of cells with this matrix is an important aspect of both normal and neoplastic cellular processes. Normal cells appear to require an extracellular matrix for survival/ proliferation/ and differentiation/ while migratory cells/ both normal and neoplastic/ must traverse the basement membrane in moving from one tissue to another. In particular/ metastatic cancer cells arising in squamous or glandular epithelium must traverse the basement membrane to enter the circulatory and lymphatic systems (intravasation) ; the circulating neoplastic cells are typically arrested in the capillary beds of an organ/ invade the blood vessel walls/ and penetrate the basement membrane to extravascular tissue (extravasation)/ where a secondary neoplasm is then established. The mechanisms of cellular interaction with the basement membrane are thus of great interest.

The interaction of cells with extracellular matrices is dependent upon the ability of the cells to attach themselves to the matrix; it is known that this attachment may be mediated by specific glycoproteins which typically bind cells to discrete collagen types

present in the matrix. Fibronec in-medi ted attachment of fibroblastS/ myoblasts, and smooth muscle cells to intersititial type I and type III collagen/ and chondronectin- ediated attachment of chondrocytes to type II cartilage collagen/ are exemplary.

It has been found that the attachment of both normal and neoplastic cells to the basement membranes is similarly mediated. The primary constituents of the basement membrane are type IV collagen/ glycoproteins and prot-eoglycans. The glycoprotein laminin mediates the attachment of both epithelial and neoplastic calls to the basement membrane/ binding the cells to type IV collagen by mechanisms to be described hereinafter. Since/ as previously noted/ metastasizmg tumor cells must traverse basement membranes at multiple stages in the metastatic process/ and since the first step in this process is tumor cell attachment to the basement membrane/ the elucidation of this mechanism and the corollary characterization of specific attachment factors which promote or inhibit tumor cell attachment to this membrane has important implications for cancer diagnosis and management.

Disclosure of the Invention

Methods for the diagnosis and management of cancer according to the invention have been predicated on the successful isolation and purification of a unique cell matrix receptor for laminin/ and the characterization of substrate-specific binding site domains on the laminin molecule. Isolation of these domains according to substrate on protease-deπved laminin fragments permits selection of fragments having specific binding capacities/ since each fragment contains at least one,

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but not a1.1/ of the domains present on the intact molecule*. Of particular interest are fragments which retain only binding sites for the cell matrix receptor. This receptor/ which has a high affinity for receptor binding domains in the laminin molecule/ is characteristic of human cancer cells and perhaps epithelial cells. The receptor is present on the surface of ^' these cells and can be isolated from plasma membrane extracts.

Bioassays based on this model within the scope of the invention include immunoassays/ especially prognostic radioimmunoassays ₇ applied to clinical specimens for the detection and quantitation of laminin cell receptors expressed by metastatasizing cells/ or for the isolation of highly metastatic tumor cells from a mixed population. Therapeutic procedures include the treatment of hosts with laminin fragments agonistic or antagonistic to the laminin molecule to block attachment of tumor cells to type IV collagen and reduce hematogenous formation of metastases. Suitable laminin fragments may also be used as ligands for known or experimental chemotherapeutic agent: the conjugation of toxins with these fragments permits direct introduction of the conjugate to the metastasizing tumor cell for in vivo treatment of cancer-/ or for drug evaluation in vivo or in vitro. The described model is also useful in the evaluation of synthetic binding site analogues for therapeutic use in cancer management. Other proposed therapeutic uses include the use of laminin or appropriate laminin fragments to promote cell adhesion to type IV collagen/ for example to promote adhesion and growth of epithelial cells in burn therapy. These fragments/ and laminin itself/ may broadly act _. as a growth factor on receptive cells

pro oting cell attachment and dispersion/ and stimulating cell division.

Best Mode for Carrying Out the Invention

The glycoprotem laminin is uniquely localized in the basement membranes and is the major glycoprotem constituent thereof. While cells bearing the laminin receptor are capable of directly attaching to the basement membrane laminin component/ the preferred pathway- of invading cells is by laminin- ediated attachment to the type IV collagen constituent of the basement membrane. The secretion of laminin by etastasizing cells facilitates the invasion process.

Laminin (M _r 10 ) upon reduction migrates on polyacrylamide gels as two subunits with apparent molecular weights of 200/000 ( α3 subunit) and

400/000 (β subunit). Electron microscopy techniques have demonstrated that the configuration of the laminin molecule is that of a Latin cross having one long arm (77 nm) and three identical short arms (37 nm). The three short arms of the cross comprise the α3 subunit/ consisting of 3 similar (M _r 200/000) chains, while the long arm of the cross comprises the β subunit (M _r 400/000; all arms have globular end units.

Laminin binds with high affinity to the cell matrix receptor (κ _d =2nm), to type IV collagen/ and also to heparin. According to the invention/ substrate- specific binding domains residing on distinct regions of the molecule were characterized by cell attachment and collagen-binding assays using enzyme digestion products of laminin. Digestion of the molecule with -thrombin/ pepsin/ and cathepsm G yielded fragments

having more limited binding capacity than that exhibited by native laminin. Structural properties of native laminin and these fragments are shown in FIGURE 1. The α3 fragment (M _r 600,000) derived from α-thrombin digestion of laminin lacks the long arm (M _r 400/000) fragment of the molecule, and retains the short arms with their globular end regions. Digestion of laminin with pepsin or cathepsm G yields Pi (M _r 280/000) and Cl (M _r 350,000) fragments, respectively, wherein the long arm of the molecule, is removed and also the globular end regions of the short arms are altered. Functional properties of native laminin and the α3, Cl and Pi fragments are summarized in FIGURE 1 and Table 1. Cl and PI fragments having similar molecular weights and binding capacities are also obtained by digestion of laminin with plasmin or chymotrypsin.

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TABLE I

Ability of laminin and laminin fragments to bind to type IV collagen and to mediate human breast carcinoma cell adhesion to type IV collagen.

% a'ttachment

Laminin or % binding fragment to collagen MCF- ^■ 7 T47-D

Laminin 80 84 28 α3 85 81 29

PI 15 13 11

Cl 10 11 10

None - 48 22

- Binding of whole laminin or puri ied protease- derived fragments of laminin to .procollagen type IV immobilized on nitrocellulose was measured. Ten micrograms of the ligand was applied to 25 μg of type IV collagen. Percent attachment of MCF-7 and T47-D cell to type IV collagen substrate in the presence of various laminin fragments is indicated. Freshly trypsinized cells were incubated for 3 hours in serum- free Dulbecco's modified Eagle's medium with laminin added at 1 yg/ml, M _r 600/000 α3 laminin fragment at 1 yg/ml/ Pi laminin fragment at 100 μg/ml/ or Cl laminin fragment at 100 μg/ml. Unattached cells were removed and counted/ the dishes were washed with the attached cells were removed with 0.01% trypsin 0.1% EDTA and counted electronically. The data are the mean of quadruplicates with a range less than 10% of the mean; sensitivity was as low as 100 ng/ml.

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Digestion of laminin with α-thrombin does not affect the binding capacity of the α3 fragment for the cell matrix receptor or type IV collagen. The PI or Cl fragments are not capable of mediating attachment of human breast carcinoma cell lines to type IV collagen (Table 1), while the α3 fragment is comparable to native laminin in its mediating ability: both stimulate attachment. As seen in FIGURE 2, the Pi fragment blocks attachment of human breast carcinoma cells to. type IV collagen; the Cl fragment also blocks attachment of carcinoma cells to type IV collagen, completely inhibiting attachment of MCF-7 cells to type IV collagen when used in concentrations of 1 μg/ml in a similar protocol (data not given). For comparison/ a dose-response curve for laminin-mediated attachment of human carcinoma cells is shown in FIGURE 3; while fibronectin mediated attachment of the cells to type I collagen/ laminin in the same dose range failed to stimulate attachment of the cells to type I collagen (data not shown). Normal binding capacities of the cell lines employed for types I and IV collagen in the absence of specific attachment factors is summarized in Table 2.

TABLE II

Attachment of human breast carcinoma cells

% attachment

Cells Type I Type IV

MCF-7 22 68

ZR-75-1 54 44

T47-D 62 33

Attachment of cells to types I and IV collagen- coated dishes was measured after 3-hrs incubation in serum-free Dulbeccos ¹ modified Eagle's medium.

Attachment assay is detailed in the legend to Figure 3. The data are the means of quadruplicates, which did not differ by more than 5%.

The binding domains present on the laminin molecule were accordingly characterized and mapped as illustrated in FIGURE 1. The cell matrix receptor present on the cell surface binds to a protease- resistant, disulfide bonded intersection region of the three short arms of the laminin molecule. The type IV collagen-binding domain resides on or near the globular regions of the short arms, while the long arm of the molecule binds to heparin sulfate proteoglycan. Since the α3 ₍ fragment retains both the cell-binding and collagen-binding domains/ this fragment exhibits a mediating capability similar to that of the intact molecule. The removal of the globular regions of the short arms by pepsin or cathepsin G eliminates the binding domains for type IV collagen, and the corresponding fragments thus lack the ability to mediate cell attachment to the basement membrane. The Pi and Cl fragments, however/ retain the cell receptor binding domains/ and are thus able to saturate the receptors in successful competition with native laminin.

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Isolation and purification of the cell matrix receptor was accomplished by detergent extraction from plasma membranes and purification using laminin affinity chromatography. The purified receptor has a molecular weight of about 50,000 to about 75,000 (SDS polyacrylamide gel electrophoresis) and retains a high binding affinity for laminin (K _c * _j =2nm) close to that of the plasma membranes or whole cells. The receptor has been identified on both human and murine tumor cells.

Diagnostic assays for cells bearing the receptor moiety such as human cancer cells, especially carcinoma cells. have been developed which are contemplated as useful in the diagnosis and prognosis of cancer, based on clinical specimens such as tissue or cytological biopsies. In general, known binding assays of the immunoassay type are useful, such as radioimmunoassays or enzyme immunoassays employing as labelled ligand laminin, appropriate laminin fragments retaining biologically active cell receptor sites, or antibodies raised in heterologous species animals such as rabbit or goat against purified cell receptor, or monoclonal antibodies having the desired affinity and specificity characteristics. Typical assays include conventional competitive binding assays on plasma membrane extracts of the cell receptors, employing immobilized ligand or receptor. Diagnostic kits for performing these binding assays are within the scope of the invention and include, for example, free or immobilized radiolabelled laminin or laminin fragment, or if antibody is employed, sandwich-type kits including, for example, bound labelled antibody and anti-antibody. Results are suitable evaluated by Scatchard analysis of the specific binding (Scatchard, Ann. N.Y. Acad. Sci. 51: 660-672, 1949) of the specimen, and comparison with

binding affinity of cultured carcinoma cells of the relevant type. Scatchard binding analysis of human breast carcinoma cells indicate an estimated K _d of 50- 2.2nm for metastasizing carcinoma (MCF-7) cells, with calculations suggesting about 10,000 to 100,000 receptors per cell for all types of carcinoma. In contrast, no specific binding was found by Scatchard analysis in samples of mammary fibroschlerosis tissue containing no neoplastic cells.

Laminin fragments containing cell receptor bindxng sites but devoid of type IV collagen binding domains (Pi and Cl), in addition to being useful in diagnostic binding assays, are contemplated as useful in the treatment of cancer to inhibit formation of metastases. In in vitro mouse studies/ fragments blocked attachment of BL6 murine melanoma cells to type IV collagen in the presence of exogenous laminin. The Cl fragment, when preincubated with BL6 melanoma cells/ markedly inhibited or abolished hematogenous metastases formation in vivo on a dose dependent basis. Further/ the fragments are non-toxic/ with no evidence of adverse im unogenic side effects in experimental animals. In addition/ such fragments are useful in targeting effective chemotherapeutic agents/ or as carriers for known effective anti-tumor drugs. By conjugation of the drug with the ligand/ toxic agents can be localized on the surface of invasive tumor cells; drugs such as ricin may be conjugated to the ligand by known methods/ such as these used to conjugate such drugs to antibodies for tumor-associated antigens.

The concepts of the present invention have implications for a variety of biological processes

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including cell differentiation, mitogenesis, morphogenesis, and neoplasia. The methods described herein are in part predicated on the hypothesis that in all benign neoplasms and in carcinoma in situ; the basement membrane remains a continuous structure, and that, in the case of benign cells, the laminin receptors are occupied by attachment to the basement membrane. A hallmark of invasive cancer, however, is an absence of a formed basement membrane adjacent invading tumor cells; thus, the invading tumor cells may contain a large number of laminin receptors which are expressed on the cell surface, but are not bound to ligand, in contrast to epithelial cells, or benign neoplasms. The unique availability of laminin receptor sites on metastasizmg carcinoma cells may thus prove to be an important concept in cancer management and diagnosis.

EXAMPLES

The concepts exemplified are elaborated in the following publications, incorporated herein* by reference: Terranova, et al, Proc. Natl. Acad. Sci., 80: 444-448 (1983); and Rao, et al , J. Biol. Chem. , 257: 9740-9744 (Aug. 1982).

Example 1. Preparation of Laminin Fragments A. Materials and Methods

1. Purified α thrombin was kindly supplied by John W. Fenton (N.Y. Dept. of Health, Albany). Digestion with α thrombin was performed at pH 7.6 and 25°C using an enzyme to substrate weight ratio of 1:100 as described in Thro b. Res. , 21: 663-673. Thrombin digestion was arrested by addition of a 2-fold excess of hirudin (Sigma). Laminin digestion. with

pepsin, α thrombin and cathepsm G was performed as described in Cancer Res: 41: 4624-46 Hoppe-Seyler's Z. Physiol. Chem. , 361: 1651-1660 (1980); J. Mol. Biol., 257: 9740-9744 (1982), and Arch. Biochem. Biophys., 219: 65-70 (1982). Laminin digestion with chymotrypsin and plasmin is similarly described in these publications/ which are incorporated herein by reference.

2. Protease fragments were isolated by HPLC (Beckman) and studied by SDS gel electrophoresis on 5% polyacrylamide slab gels/ as described in Terranova/ et al and Rao/ et al/ supra. The Pi fragments obtained by digestion of laminin with plasmm or pepsin and the Cl fragments obtained by digestion of laminin with chymotrypsin or cathepsin G are characterized in the references noted in Example |A| / supra. The α3 fragment is similarly characterized.

Example II. Attachment Assays A. Materials and Methods

-• • Cell lines

The MCF-7 human breast cancer cell line was provided by the Michigan* Cancer Foundation. Characterization of the cells as to human and mammary origin has been summarized. The cells were shown to be invasiv and to metastasize in nude mice. They were also invasive in vitro when human amnion connective tissue was used as a barrier.

The ZR-75-1 and T47-D human breast carcinoma lines were provided by L. Engel (Laboratory of Pathology/ National Cancer Institute). These cells were verified

to be carcinomas by electron microscopy criteria/ to be human in origin by karyotypic analysis/ and to contain mammary gland specific secretory milk proteins. Both of these lines grew poorly in nude mice with no gross metastases/ even when subcutaneous xenotransplantation was performed in newborn nude mice. Normal human skin fibroblasts (CRL 1507 and CRL 1477) were obtained from the American Type Culture Collection.

2- Preparation of substrate/ attachment factors and laminin fragments

Laminin fragments were prepared as described in

Example I. Type I collagen was prepared from lathy itic rate skin (biochemistry, 3460-3473

(1966)). Type IV collagen and laminin were prepared from the Engelbreth-Holm-Swarm tumor (FEBS Lett. 127:

257-262, (1981); and J. Biol. Che ., 254: 9933-9937,

(1979). Laminin was iodinated by the lactoperoxidase method (Biochem. Biophys. Acta, 251: 363-369, 1969).

3. Attachment assay

The assay was adapted from Klebe as described by Terranova, et al (Cell/ 22: 719-728, 1980).

4. Cell binding

Binding of laminin to MCF-7 and T47-D breast carcinoma cells and adult human fibroblasts CFL 1477 and CRL 1507 was preformed with monolayer cell cultures. All cell lines were replica plated in multiwell culture dishes (FB-6-TC, Limbro) in complete growth media. When the cells were 50-70%, the medium was changed to Dulbecco's modified Eagles' medium with 0.1% bovine serum albumin for a 2-hr. wash. The

binding medium consisted of Dulbecco's modified Eagle's medium/ 0.1% bovine serum albumin/ and 20raM Hepesbuffer (pH 7.4). ¹²⁵ l-Labeled laminin ( ¹²⁵ l-laminin) with either excess unlabeled laminin or excess unlabeled laminin fragments was added in 250 μl of phosphate- buffered saline (P ₁ /NaCl) to initiate the binding assay. After various times of incubation at 20°C and 37°C the binding medium was rapidly aspirated/ and monolayeres were washed three times with ice-cold P _j _/NaCl containing 0.2% bovine serum albumin to remove unbound material. The cells were then removed by using 0.02% EDTA in P _j _/NaCl and cell-bound radioactivity was determined in a Searle (Skokie/ IL) Autogamma counter. Specific binding was defined as the total radioactivity bound minus the amount bound in the prsence of a 100-fold excess of unlabeled material.

5. Collagen binding

Procollagen type IV was dissolved in 0.5 M acetic acid and neutralized with 0.05 M Tris HC1/0.9 M NaCl, pH 7.4. Five microliters of the solution (25 μg) was placed on a 13-mm SCWP nitrocellulose filter with 8- sa pore diameter (Millipore)'. The filters were then immersed in P--_/NaCl with 3% bovine serum albumin

Λ *~ at 4°C overnight. After washing/ ^iiJ I-*-labeled laminin (1 mg/ml) or purified ^^l-la ele laminin fragments were applied to the filters (10 μl) and incubated in a 100% relative humidity chamber for 20 min. After intensive washing in P^/NaCl the bound radioactivity was quantitated with a gamma counter.

6. Rotary shadowing and electron microscopy

These techniques were performed according to the

method of Engel et al modified as described in Rao/ supra .

B. Results

1- Cell attachment to collagen

When freshly trypsimzed MCF-7/ ZR-75-1/ and T47-D cells were added to various substrates the MCF-7 cells preferred type IV collagen over type I collagen or plastic (data for types I and _ IV collagen shown in Table 2) . In all experiments the MCF-7 cells attached more rapidly and to a greater extent to type IV collagen substrate compared to the ZR-75-1 and T47-D cell types. To determine whether the adherence of the cells to specific substrates was mediated by the presence of specific attachment factors/ the effect of exogenous laminin and fibronectin on cell attachment to type I and type IV collagen/ respectively was tested. To eliminate the effect the endogenous synthesis of these factors might have on adherence/ cells were treated with cycloheximide in the incubation media to inhibit protein synthesis. In the presence of cycloheximide/ laminin stimulated the attachment of all three cell lines to type IV collagen. The MCF-7 cells showed an 8-fold stimulation by laminin compared to a 4-fold stimulation for the ZR-75-1 cells and 2-fold stimulation for the T47-D cells (FIGURE 3).

Fibronectin/ when added to type I collagen in the presence of cycloheximide/ caused a 7-fold increase in the attachment of the T47-D cells/ a 5-fold increase in attachment of the ZR-75-1 cells/ and only a 2-fold increase in the attachment of the MCF-7 cells (FIGURE 3) . These data indicate that the MCF-7 cells prefer type IV collagen as a substrate and use laminin as an

attachment factor. Laminin in the same dose range failed to stimulate attachment of these cells to type I collagen (data not shown). In contrast/ the T47-D cells utilized fibronectin to bind preferentially to type I collagen/ whereas ZR-75-1 cells exhibited no preference for either laminin or fibronectin for attachment to type IV or type I collagen substrate/ respectively.

2. Binding properties of laminin fragments

- In order to investigate which regions of the laminin molecule mediate the attachment of cells to type IV collagen/ protease-derived fragments of lam in were tested for their attachment properties, α thrombm-derived α3 fragment of laminin stimulated the attachment of MCF-7 cells to type IV collagen to the same extent as native laminin did (Table 1). The o3 fragment is as active as a microgram basis/ but it is less active than native laminin on a molar basis. The β component of laminin showed no attachment activity. The data supports the conclusion that the α3 component of laminin contains the biologically active sites for both cell and collagen binding. The effect of the α3 laminin component and a pepsin-derived "PI" M _r 280,000 laminin fragment on the abili y of the MCF-7 and T47-D cells to attach to type I and type IV collagen was measured in the presence of the native laminin molecule. The α3 component stimulated adherence to type IV collagen/ whereas the Pi fragment markedly inhibited attachment (Table 1). Neither fragment had an effect on attachment to type I collagen (data not shown). However/ the T47-D cells attached to type IV collagen by utilizing laminin or laminin fragments to a much lesser degree (Table 1). A dose-

response experiment using the Pi fragment is shown in FIGURe 4. Attachment of both the MCF-7 cells and the T47-D cells to type IV collagen was completely inhibited by the PI fragment at 1.0 μg/ml. A further laminin fragment (M _r 350,000) produced by cathepsin G digestion (named "Cl") completely inhibited the attachment of the MCF-7 cells to type IV collagen when used at concentrations of 1 μg/ml.

3. Laminin binding to cells

If laminin mediates attachment of epithelial cells to type IV collagen/ then these cells may possess specific surface receptors that are involved in recognizing laminin. Moreover/ cells such as fibroblasts, which utilize fibronectin rather than laminin as an attachment factor/ should lack these laminin-bmding sites. Experiments were conducted to determine whether ¹²⁵ l-laminin binds to cells with high affinity and specificity. Binding of laminin to the MCF-7/ T47-D/ and the CFL 1477 and 1507 fibroblast cell lines was time dependent. Equilibrium binding was reached after 90 min. for all cell lines tested (FIGURES 4 and 5). The human fibroblast cell lines/ CRL 1477 and 1507 showed no evidence of a specific laminin receptor (FIGURE 5) whereas the epithelial T47- D cells exhibited a low level of laminin binding when compared to the MCF-7 cells. Scatchard binding analysis (using MCF-7 cells) gave a roughly linear curve/ with an estimated K _d of 50-2.2 nM. Calculations suggest 10,000-100/000 binding sites per cell. The receptor for lam in could be extracted from the cell membrane by 0.1% Triton X-100 and had a molecular weight of 60/000-75/000 after isolation by lam in affinity chromatography. Laminin fragments 3 and Pi

^■ sy

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both competed for ¹²⁵ l-laminm, binding at a level similar to whole laminin (FIGURE 4) . Laminin bound to both attached and suspended MCF-7 cells. For the latter, binding was identified 2 hr. after trypsinization followed by incubation in Dulbecco's modified Eagle's medium containing 0.5% bovine serum albumin. Heat-denatured laminin and fibronectin were l/50th to l/500th as effective as native laminin in competing for ¹²⁵ l-laminin binding. Both ¹² ^l-laminm and ¹²⁵ l-labeled α3 laminin fragment maintained biological activity when used in an attachment assay. Therefore/ a major domain of the laminin molecule that binds to the MCF—7 and T47-D cells is retained on both the α3 laminin component and the Cl or Pi laminin fragment (FIGURE 4, Table 1).

4. Laminin binding to collagen

In contrast/ the same laminin fragments ( 3 and Pi) showed a marked difference in their ability to bind to type IV collagen immobilized on nitrocellulose. Native laminin and the α3 fragment bound equally well to type IV collagen (Table I). The Pi or Cl fragments exhibited no capacity to bind to type IV collagen (Table I) . The structual and binding properties of the various laminin fragments are summarized in FIGURE 1.

Example III. Isolation of Tumor Cell Laminin Receptor A. Materials and Methods

Metastatic BL6 melanoma cells were supplied by Dr. Ian Hart/ Frederick Maryland. The growth and attachment properties of these tumor cells have been described. BL6 cells were cultivated in RPMI 1640 media supplemented with 10% FCS. Plasma membranes were

isolated from cells in log phase of growth (J. Biol. Che . 255 1722-1731/ 1980). The plasma membrane homogenate was solubilized in 0.1% Triton X 100/ 1.0- 2.0 mg protein/ml. After centrifugation at 30/000 g for 45 in./ the supernate was collected and incubated with SM2 Bioheads (Bio-Rad) to remove the Triton. Iodination of the laminin ligand and the plasma membrane extract was performed using the lactoperoxidase method/ supra. Laminin receptors were measured on living cells in suspension. After trypsinization the BL6 cells were incubated in complete media under constant agitation at 37°C for 2 hr. The labeled ligand plus 250-fold excess unlabeled ligand was added and the incubation was continued for 2 hours at 25°C. The cell-bound and free ligand was separated by centrifugation. Binding assays on plasma membrane extracts were performed using one member of the ligand or receptor pair bound to solid phase nitrocellulose Millipore SCWP circles (Terranova/ et al, supra) or cyanogen bromide activated Sepharose 4B. In the latter case, 25 μg of laminin or plasma membrane extract protein bound to cyanogen bromide activated Sepharose 4B (100 μl) was mixed with an equal amount of 25 M Tris, 5 mM MgCl ₂ and CaCl ₂ / pH 7.4 and 100 μl of this buffer containing 0.1% BSA. ¹²⁵ l-labeled plasma membrane extract (10° cpm/mg) or ^-"l-labeled laminin (10^ cpm/mg) was added in a total volume of 100 μl, diluted with the buffer. Competition was performed with various concentrations of unlabeled laminin in solution or unlabeled plasma membrane extracts (2 to 20 μg). The binding assay mixture was incubated at 4°C overnight. The laminm-Sepharose beads were collected by centrifugation at 5,000 rpm for 30 min. and the pellet was washed twice with 2.0 ml of the buffer containing .0.1% BSA. The proteins in the

first spin supernate and the pellet were identified by electrophoresis on 7% slab gels by the method of Laemmli, followed by autoradiography, Laminin affinity chromatography was performed using purified laminin cross-linked to Sepharose 4B. ¹² ^l-labeled plasma membrane extract was incubated 15 hrs. in the laminm- Sepharose affinity column (1 x 15 cc) at 4°C. The unbound radioactivity was washed with 40 ml of 25 mM Tris, 5 mM CaCl ₂ / 5 mM MgCl ₂ / 0-9% NaCl, pH 7.4. The neutralized with 1.0 M Tris saline, and lyophilized. The proteins were identified by slab gel electrophoresis and autoradiography. The number of laminin receptor sites and the kd were calculated by Scatchard analysis.

B. Results

BL6 melanoma cells exhibited saturatable binding for laminin. Scatchard analysis demonstrated approximately 110,000 binding sites per cell with a high affinity: kd=2.2 nm (FIGURE 6A). Laminin binding to the tumor cells was abolished by trypsinization. The receptor regenerated after 2 hrs. of cell incubation in serum free or serum containing media. Collagen, denatured laminin, fibronectin or serum d d not compete for binding. Binding of ¹²⁵ l-lamιnιn to isolated cell plasma membranes also showed a high affinity: kd=1.5 nm (FIGURE 6B). Excess unbound laminin competed for binding of the solubilized membrane receptor to lam in immobilized to a solid phase (FIGURE 7). Gel electrophoresis of the solubilized membrane proteins bound to laminin before and after competition demonstrated a single molecule weight class for the receptor (FIGURE 7) . Laminin affinity chromatography was therefore used to isolate

the receptor (FIGURE 8 and FIGURE 7a) with a 900-fold purification relative to the crude membrane extract. The receptor molecular weight was 67/000 after reduction by polyacrylamide gel electrophoresis. The isolated receptor retained a high binding affinity for lammin: kd=2 nm (FIGURE 6C) .

Example IV. Measurement of Laminin Receptors in Human Breast Carcinoma Cells

A. Materials and Methods

1- Preparation of Membranes

Human breast cancer tissue samples were obtained at the time of mastectomy for biopsy proven infiltrating duct carcinoma. The tissues were frozen in liquid nitrogen and pulverized. The pulverized tissue was diluted in a volume ratio of 1 to 4 in 25 mM Tris 0.3 M sucrose pH 7.4/ and homogenized ^' using a polytron at 0°C. The homogenate was centrifuged 15/000 g for 20 minutes. The fat layer was discarded and the supernatant was removed and centrifuged at 100/000 g 4°C for 60 minutes. The pellet was suspended in a 25 mM Tris buffer containing 1.5 mM MgS0 and 0.15 mM CaCl pH 7.4. This membrane preparation was diluted to 100 mg/100 '1.

2- Binding Assay

Laminin or lammin fragments were purified and iodinated as described previously. The binding assay was conducted at room temperature for 90 rain, using 100

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'1 of the membrane fraction and ^iiJ l-laminin with a specific activity of 70/000 cpm per 2.3 x. 10 ^J M. Specific binding was determined by using cold

competitor ligand at a 10,000-fold excess concentration. The bound and free ligand was separated by centrifugation at 5,000 g for 30 minutes.

B. Results

Plasma membrane fractions from human breast cancer tissue exhibited saturatable binding of laminin in time course studies (FIGURE 9).' ^" The plateau of binding was reached at one hour (25°C). ^" After the plateau phase of binding was established, the addition of 1,000-fold excess cold ligand rapidly displaced the labeled ligand. Scatchard analysis of the specific binding was linear consistent with a single class of binding sites (r = 0.85) (FIGURE 10). No specific binding was noted in samples from mammary fibrosclerosis tissue containing no neoplastic cells. Heat denaturation of the membrane fraction abolished binding activity. Fibronectin, epidermal growth factor, or serum did not compete for binding. The use of purified fragments of the laminin molecule (FIGURE 11) enabled us to identify the domains of laminin participating in its various binding functions. Whole laminin appears as a four- armed cross-by-rotary shadowing electron microscopy (FIGURE 11). The fragment which lacks the long arm contains full binding activity for the laminin receptor, mediates cell attachment, and binds to type IV collagen, while the fragment which lacks the long arm and the globular end regions of the short arms competes for specific binding to the receptor with an affinity equal to whole laminin.

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Example V.. Preparation of Antibodies to the Laminin Cell Receptor

Antibodies to the receptor purified according to Example II were raised in New Zealand white rabbits by three injections of isolated receptor (250 μg per injection) emulsified in Freud's complete adjuvant. The specificity of the antibody was verified by standard solid phase immunoassay. The antibody is radiolabelled/ and employe in a conventional radioimmunoassy for laminin receptor.

Example ^" VI. In Vivo Therapy with Cl Laminin Fragment

BL6 muπne melanoma cells (Example III) were preincubated for three hours with laminin Cl fragment (obtained as in Example I) at concentrations of 1 mg Cl per ml cells/ and 10 mg Cl per ml cells. The incubated cells were harvested/ and administered in vivo to nude mice according to standard mouse protocols. The three control mice and six treated mice were sacrificed after 3 weeks _/ and the lungs examined. The control lungs showed a large number of metastases; the lungs of the three mice treated with Cl at 1 mg/ml showed a reduced number of metastases/ and the lungs of the three mice treated with Cl at 10 mg/ml were substantially free from metastases. The experiment was repeated seven times with comparable results. A representative photograph of the lungs from one experiment is set forth in FIGURE 12.