EP0716659A1 - Epiligrine, ligand epithelial pour les integrines - Google Patents

Epiligrine, ligand epithelial pour les integrines

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Publication number
EP0716659A1
EP0716659A1 EP94929787A EP94929787A EP0716659A1 EP 0716659 A1 EP0716659 A1 EP 0716659A1 EP 94929787 A EP94929787 A EP 94929787A EP 94929787 A EP94929787 A EP 94929787A EP 0716659 A1 EP0716659 A1 EP 0716659A1
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EP
European Patent Office
Prior art keywords
epiligrin
cells
leu
epithelial
ser
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EP94929787A
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German (de)
English (en)
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EP0716659A4 (fr
Inventor
William G. Carter
Susana G. Gil
Maureen C. Ryan
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Fred Hutchinson Cancer Center
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Fred Hutchinson Cancer Research Center
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Priority to EP07075129A priority Critical patent/EP1808439A3/fr
Publication of EP0716659A1 publication Critical patent/EP0716659A1/fr
Publication of EP0716659A4 publication Critical patent/EP0716659A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates generally to epithelial cell receptors and ligands which are useful for adhering epithelial cells to a substratum.
  • the invention is predicated upon a basic understanding of epithelial cells and tissues studied.
  • epithelia which cover free surfaces and line body cavities and ducts, have been studied microscopically for at least three centuries.
  • biochemistry and molecular biology of epithelial cells and tissues have been extensively investigated.
  • the seemingly simple question of how the cells in epithelial tissues are driven to become specialized has remained unanswered.
  • the present invention provides reagents that allow us for the first time to unravel the inter- and intracellular signals that direct epithelial cell differentiation.
  • the subject reagents permit one to finally decipher what has been a tangled web of suspected interactions involving a wide variety of cell types, some of them non- epithelial, in order to understand and modulate at a molecular level how the cells are driven to differentiate to fulfill specialized functions in the body.
  • Pertinent background information concerning these heretofore disparate systems follows. 2 ⁇ Abbreviations: By way of introduction, the following abbreviations are used in this disclosure:
  • BPA Bullous Pemphigoid Antigen
  • CD3, cellular determinant #3 a lymphocyte surface antigen marker
  • CP cicatrical pemphigoid
  • EBA epidermolysis bullosa acquisita, an autoimmune dermatological disease
  • ECM extracellular matrix
  • FAs focal adhesions
  • HD-BSA heat denatured bovine serum albumin
  • HFK(s) human foreskin keratinocyte(s)
  • HFK-ECM human foreskin keratinocyte-extracellular matrix
  • kd kilodaltons of molecular mass as determined by SDS-PAGE
  • MAbs monoclonal antibodies
  • Mr molecular radius by SDS-PAGE, approximating molecular mass
  • SACs stable anchoring contacts
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
  • E200, E170, E145, E135, E100, and E36 refer to the constituent and associated glycoproteins of the subject epithelial ligand complex epiligrin, having apparent molecular weights of 200 ⁇ 20 kd, 170 ⁇ 20 kd, 145 ⁇ 20 kd, 135 ⁇ 15 kd, lOO ⁇ lO kd, and 36 ⁇ 5 kd, respectively.
  • Ep-1, 1-1, and 8-6 refer to the disclosed cDNA clones deposited under ATCC accession numbers 75540, 75539, and 75538, respectively.
  • the invention is predicated upon a basic understanding of epithelial cells and tissues studied.
  • epithelia which cover free surfaces and line body cavities and ducts, have been studied microscopically for at least three centuries.
  • biochemistry and molecular biology of epithelial cells and tissues have been extensively investigated.
  • the seemingly simple question of how the cells in epithelial tissues are driven to become specialized has remained unanswered.
  • the present invention provides reagents that allow us for the first time to unravel the inter- and intracellular signals that direct epithelial cell differentiation.
  • the subject reagents permit one to finally decipher what has been a tangled web of suspected interactions involving a wide variety of cell types, some of them non- epithelial, in order to understand and modulate at a molecular level how these cells are driven to differentiate to fulfill their specialized functions in the body.
  • Pertinent background information concerning these heretofore disparate systems follows.
  • epithelial tissues as a protective barrier is readily apparent in the body as the lining of body cavities, blood vessels, digestive tract, mammary glands, urogenital, endocrine, reticuloendothelial systems, respiratory surfaces, placenta, and surrounding the nerves and brain.
  • the epithelia also forms the basis for the epidermis, cornea, and conjunctiva.
  • Epithelial tissues are rather unique in their ability for continuous regulated self-renewal, and in their ability to polarize and control cellular division and the subsequent differentiation of the daughter cells. In attempting to explain how epithelial cells may decide how and when to differentiate, it has been suggested that perhaps gradients of growth factors or interactions with extracellular matrix (ECM) may influence the cells. However, the biochemical mechanisms remain largely unknown.
  • ECM extracellular matrix
  • the epithelial basement membrane is a common histological feature of columnar, stratified, and squamous epithelia. Another prominent feature is a proliferative basal (stem) cell layer resting on a basement membrane.
  • an epithelial basement membrane may include lucent and dense regions termed, respectively, the Lamina lucida and Lamina densa, -which are sandwiched between an overlying cellular stroma (stroma), made up of basal stem cells and fibroblasts, and an underlying collagenous matrix.
  • stroma cellular stroma
  • Basement membranes are thin but continuous sheets that separate epithelium from stroma and surround nerves, muscle fibers, smooth muscle cells, and fat cells (1-4).
  • the molecular composition of the basement membrane varies with specialized cellular functions and with the developmental stages, shape, structure, and architecture of different epithelia (5).
  • basement membranes contain at least type IN collagen (1, 6-8), laminin (7-8), entactin (9), and heparin sulfate proteoglycans (10-11).
  • EHS tumor laminin was reported to have apparent molecular sizes of 400 kd and 200 kd, entactin was 158 kd, and nidogen was 100 kd (Kleinman et al., Biochemistry 25:312-318 (1986)).
  • the human skin for example, is an epithelial tissue composed of the epidermis and the dermis.
  • the dermis is relatively acellular and composed of secreted cell products, e.g., collagens and heparin-sulfate- and chondroitin-sulfate- proteoglycans.
  • the epidermis is essentially cellular, containing a layer of cells resting on a basement membrane, termed the basal (stem) cells that are covered by a layer of cornified cells, termed the stratum corneum.
  • the viable inner malpighian layers of the skin are composed of the basal cell layer, the stratum spinosum and the stratum granulosum.
  • the cell types in these areas include at least keratinocytes, melanocytes, Merkel cells, Langerhans cells, and migratory immune cells.
  • Cell division in the basal (stem) cell layer forms the basis for the continuous self-renewal of the skin, and it is thought that decisions on the fate of the daughter cells are made in this layer.
  • Two types of daughter cells appear to be created by cell division in the basal (stem) cell layers of the skin.
  • the first daughter cell which will continue to divide; and the second daughter cell, which will differentiate and ultimately become cornified.
  • Distinctive cellular features that may define stages in the differentiation of the second daughter cell include at least the acquisition first, of a flattened cell shape with cytoplasmic keratohyalin granules, ivolucrin, and cytokeratin filaments (characteristics of cells in the stratum spinosum); second, of greater amounts of cytoplasmic keratin and a submembranous envelope formed of proteins cross-linked by epidermal transglutaminase (characteristics of cells in the stratum granulosum); and third, the acquisition of distinguishing features associated with cornified anuclear cells such as extensively cross-linked dense submembranous envelopes (i.e., characteristics of cells in the stratum corneum).
  • first daughter and second daughter are largely unknown, but these mechanisms appear to be coordinated; i.e., cells enter and leave the malpighian layer at approximately the same rate; they appear to be polarized, i.e., from the basal (stem) cell layer to the apical cornified layers; and they appear to be self-regulating, i.e., processes by which the cornified layers are renewed can effectively compensate for variation in the rate of mechanical sloughing of cells from the surface in different parts of the body.
  • the molecular processes by which this remarkable coordination of cells is achieved in skin or other epithelial tissues are largely unknown, at present.
  • the ultrastructure of the attachment points where basal cells are in contact with the basement membrane exhibits characteristic features that are identifiable in appropriately fixed and stained tissues (and cultures).
  • the ultrastructural features have been termed hemidesmosomes (14-16), focal adhesions (17, 18), and hemidesmosome-like stable anchoring contacts (SACs) (19).
  • Focal adhesions and SAC/hemidesmosomes are structurally and functionally distinct adhesion structures (19, 20). Focal adhesions have been observed in motile cells in association with actin-containing stress fibers (20, 21), while SACs appear to be distinguished as a structural component of stationary cells which only form in vitro after cells stop migrating.
  • SACs and focal adhesions are currently not clear, either with respect to their possible role in motility or to other possible roles in the cell biology of the epidermis.
  • the lamina densa may be connected to stroma through anchoring fibrils (22), such as those observed in cells which appear to be linked to hemidesmosomes (23-25).
  • SAC/hemidesmosome structures have also been observed to be associated with cytoplasmic intermediate filaments (26, 27) which have a Bullous pemphigoid antigen (BPA) identifiable by indirect immunofluorescence.
  • BPA Bullous pemphigoid antigen
  • Human keratinocytes have been isolated and cultured from stratified or squamous epithelia in vitro under controlled conditions either using fibroblast feeder layers and conditioned medium (28-30); medium containing at least epidermal growth factor (31); keratinocyte growth medium (KGM) containing at least hydrocortisone, low-calcium, insulin, and insulin ⁇ like growth factor-1 (32, 33) serum-free (34, 35) or supplemented MCDB 153 basal nutrient medium (36).
  • fibroblast feeder layers and conditioned medium 28-30
  • KGM keratinocyte growth medium containing at least hydrocortisone, low-calcium, insulin, and insulin ⁇ like growth factor-1 (32, 33) serum-free (34, 35) or supplemented MCDB 153 basal nutrient medium (36).
  • keratinocyte clones derived from growing and cloning normal human skin keratinocytes, may be derived from the basal (stem) cell layer and 10-15% from the suprabasal layers of the epidermis (36).
  • the presumptive "suprabasal" keratinocytes expressed markers of terminal differentiation (i.e., ivolucrin) but still possessed the ability to synthesize DNA.
  • Ivolucrin is one marker for keratinocyte differentiation in vitro. It is a cytosolic protein of human keratinocytes with a reported apparent Mr of 140 kd on SDS-PAGE (38); the gene has recently been reportedly cloned (39) and its regulation studied in cells in vitro (40). Ivolucrin is useful as a marker for an early stage in the terminal differentiation of keratinocytes since it is synthesized shortly after keratinocytes leave the basal (stem) cell layer, at a time when cellular enlargement has begun, but before onset of envelope cross- linking (41, 42).
  • Cytokeratins are a second useful marker for keratinocyte differentiation in vitro.
  • cytokeratins which may be expressed by keratinocytes in vitro using Western immunoblot analysis and commercially available monoclonal antibodies AE1 and AE3: these include cytokeratins No. 5 (58 kd), No. 6 (56 kd); No. 14/15 (50 kd); No. 16 (48 kd); and No. 17 (46 kd) (45).
  • Keratinocyte differentiation can be induced in vitro, at least to the extent that the cells change morphology into cells resembling cornified epithelia. This process can be induced in tissue culture with calcium or with ionophores (46, 47). When such keratinocyte differentiation is induced in tissue culture, epidermal transglutaminase can become activated in the cells with coincident development of a cross-linked submembranous protein envelope. During cross-linking, cytosolic ivolucrin becomes associated with the submembranous protein envelope as do two other proteins which are reportedly found in keratinocytes but not in fibroblasts.
  • keratinocytes are useful as an in vitro model for some molecular processes involved in epithelial differentiation, they are not basal (stem) cells and are clearly distinguished from them with at least ivolucrin as a marker.
  • the past studies of keratinocytes has not approached at a molecular level the possible interactions which may occur between receptors in basal (stem) cells and ligands in the basement membrane.
  • the known basement membrane components in the lamina lucida layer of the epithelium include at least laminin, nidogen, and heparin sulfate proteoglycan, and in the lamina densa they include types IN and Nil collagen (5, 50, 51).
  • the possible cellular receptors which may bind to these ligands include at least the integrin adhesion receptors (for reviews see 52-55).
  • Integrins are a family of receptor glycoproteins with two noncovalently associated polypeptide chains of different molecular sizes (the larger termed the a chain and the smaller the b), forming a structure termed a heterodimer.
  • the respective chains have amino acid sequence homology, and the integrins serve a similar function at least as receptors for cellular adhesion to extracellular matrix glycoproteins.
  • Six a chains and at least four b chains have recently been identified, giving at least 24 different theoretical heterodimers which could act as receptors for cellular adhesion.
  • An alignment of the ctg chain amino acid sequence with the 0:3 chain reportedly showed approximately 37% identity (#56).
  • T-lymphocytes as opposed to epithelial cells, the activation of cells with interleukin-2 is correlated with induction of expression of the 0:3 ⁇ i integrin on the cell surface (#57).
  • integrins in tissues and cells include (1) the possible mediation of the attachment of T- and B-lymphocytes and platelets to basement membrane via integrins o ⁇ i, o-2 ⁇ and 0 ⁇ 4 and (2) a possible role in hemostasis and homeostasis for these integrins, the latter by contributing to the maintenance of the structure of the integument and epithelia (#s 1 -21; 57, 58).
  • Laminin is a collagen receptor in human fibrosarcoma cells (59, 60) with affinity for laminin in some cells (61, 62).
  • Laminin is a disulfide- bonded glycoprotein complex composed of three distinct polypeptide chains. Laminin was first isolated from mouse Engelbreth-Holm- Swarm (Elts) tumor (#7), and the subunits were originally designated as follows: A (400 KDa), Bl (220 KDa), and B2 (210 KDa).
  • laminin- 1 the original subunits of EHS laminin (laminin- 1) have now been designated as ⁇ l (400 KDa), ⁇ l (220 KDa), and ⁇ l (210 KDa) (#125).
  • a ⁇ 4 integrin has also been suggested as a laminin receptor in human colon carcinoma cells (63), but it reportedly does not bind to the E8 domain of laminin, a ligand domain of laminin that interacts with ⁇ 6 ⁇ j integrin (64).
  • oc3 ⁇ j is reportedly one of the most widely expressed integrins in tissues and in cultured epithelial and non-epithelial cells.
  • Laminin is reportedly a poor ligand for adhesion of cultured human foreskin keratinocytes (20, 21). In tissue culture, antibody reactive with ⁇ 3 ⁇ 1 reportedly substantially inhibited adhesion of human foreskin keratinocytes to HFK-extracellular matrix.
  • c ⁇ 4 , ⁇ 3 ⁇ l5 and ⁇ 2 ⁇ appear only in the proliferating basal cell layer; 0 ⁇ 4 appears to be restricted to regions of the stem cell basal plasma membrane (58, 20, 21); o ⁇ -. appears on basal lateral and apical regions of the stem cell plasma membrane; and 2 ⁇ appears primarily on the apical and lateral regions of the stem cell plasma membrane (59, 20, 21).
  • integrins o ⁇ and ⁇ g ⁇ 4 have been recognized as glycoproteins involved in cell-substrate contact in vitro, the available information has created a tangled web which does not permit a determination of which interactions may be physiologically meaningful in vivo.
  • Integrins are reported to play a possible role in lymphocyte activation. It has been reported recently that in T-lymphocytes, the interaction of cells containing o ⁇ ! integrin with collagen and a second signal such as initiated by binding of antibody to CD3 to the cell surface integrin may trigger cellular activation. Whether such effects may also be triggered by integrins in non-lymphoid cells is not known, at present.
  • the non-malignant phenotype in the fused cells was reportedly correlated with the continued expression of ivolucrin as a marker of keratinocyte terminal differentiation, i.e., cells which reportedly lost the ability to produce ivolucrin during in vitro culture also reacquired the ability to grow progressively in animals (80).
  • BPA BP antigens
  • Epiligrin is a recently elucidated epithelial basement membrane component that mediates cell adhesion via integrins ⁇ 3 ⁇
  • Epiligrin a complex of several glycoproteins, is located in the lamina lucida of the basement membrane where the complex comes in direct contact with the overlying epithelial cells.
  • a major constituent of this complex is a 170 kd protein (El 70) that is encoded by the LamA3 gene (#135).
  • E170 is the ⁇ 3 chain of epiligrin, not to be confused with the 0:3 chain of integrin (#135).
  • junctional epidermolysis bullosa is a blistering disorder of the skin that is characterized by a separation of basal cells from - l i ⁇
  • Epiligrin is the major adhesion ligand present in epidermal basement membranes and it has been shown to mediate basal cell adhesion via integrins o ⁇ i in focal adhesions and 01 ⁇ 4 in hemidesmosome adhesion structures (113, 133, 134).
  • the present invention is predicated upon the discovery that adhesion of epithelial and lymphoid cells is modified by epiligrin, an epithelial ligand complex composed of disulfide-linked glycoproteins of 200+20 kd, 170 ⁇ 20 kd, 145 ⁇ 20 kd, 135+15 kd, and lOO ⁇ lO kd, with an associated intracellular 36+15 kd glycoprotein.
  • epiligrin, as well as constituent epithelial ligand glycoproteins and peptides disclosed herein, are ligands for the ot 3 ⁇ and 0 ⁇ 4 integrins.
  • Epiligrin and its constituents are useful for modifying adhesion of epithelial and lymphoid cells. Certain embodiments of the invention thereby provide reagents and methods for restoring normal growth in epithelial cells, e.g., in autoimmune disease and carcinoma. Other embodiments provide reagents and methods for inhibiting the binding of activated lymphoid cells to epithelial cells through the integrins, e.g., for controlling inflammation in epithelial tissues.
  • the present claims are directed to nucleic acids that encode El 70 epithelial ligand glycoproteins, and particularly to such nucleic acids capable of hybridizing under stringent conditions to one or more of the disclosed "Ep-1” (ATCC No. 75540), "1-1” (ATCC No. 75539; deposited under the strain designation "NAS-3 1-1”) , and "8-6" (ATCC No. 75538; deposited under the strain designation "NAS-3 8-6") nucleotide sequences.
  • the gene encoding E170 is called LamA3, and is located at human chromosome 18ql l.2.
  • the subject nucleic acids are useful in expression systems for production of El 70 epithelial ligand glycoproteins, and they also find use as diagnostic and therapeutic agents in identifying and treating patients with the gravis form of junctional epidermolysis bullosa and other epithelial diseases.
  • the claims are directed also to antibodies directed against proteins, as well as to the epiligrin complex itself and the proteins encoded by the claimed nucleic acids.
  • FIGURE 1 shows glycoproteins extracted as the epithelial ligand glycoprotein complex (epiligrin) from extracellular matrix.
  • FIGURE 2 depicts glycoproteins in the epiligrin complex which are not related to known basement membrane components.
  • FIGURE 3 shows that the purified HFK-ECM does not contain fibronectin or laminin.
  • FIGURES 4A-4K illustrate the SACs "ring structures" in which epiligrin is deposited in ECM.
  • FIGURES 5A-5K depict the co-localization of ⁇ 6 ⁇ 4 (GoH3, FIGURE 5B), the subject epithelial ligand integrins (PlEl, FIGURES 5A 5C and 5E), BPA (FIGURE 5D), and ⁇ 3 ⁇ 1 (P1F2, FIGURE 5F) in the SACs contained in the purified HFK-ECM.
  • FIGURES 6A-6F demonstrate the attachment of human foreskin fibroblasts to HFK-ECM (FIGURES 6D-6F) but not other ECM components.
  • FIGURE 7 illustrates a specific test cell assay for epiligrin in which specific adherence of cells to epiligrin is modulated.
  • FIGURES 8A-8P illustrate the localization of epiligrin in epithelial basement membranes of skin, tonsil, and lung, and shows epiligrin distribution in sweat glands, lymphoid follicle germinal centers, and in submucosal glands.
  • FIGURES 9A-9F illustrate the ultrastructural localization of epiligrin in epithelium.
  • FIGURE 10A schematically depicts the strategy used to clone the El 70 component of epiligrin .
  • FIGURES 10B- 10C depict a comparison of the domains of ⁇ l laminin- 1 and 03 epiligrin, as well as the overlapping cDNA clones (FIGURE 10D) used in Example 15 to compile the nucleotide sequence encoding El 70 epithelial ligand glycoprotein, i.e., as shown in FIGURES 10F, 11 A-l 1C, AND 15A-15F.
  • FIGURE 10F shows the nucleotide sequence of El 70 epithelial ligand glycoprotein cDNA clone "1-1" from position 1 to position 664 (SEQ ID No. 1). (The sequence of clone 1-1 was edited to remove a common cloning artefact, the first 150bp of the Ep-1 cDNA consisting of a cloned fragment of a rRNA.) - 12/1 -
  • FIGURES 11 A-l lC show 1994 bp (SEQ ID No. 2) of the nucleotide sequence of El 70 epithelial ligand glycoprotein compiled from cloned cDNAs as depicted in FIGURE 10A.
  • PCR primers used in cDNA cloning were:
  • MR-12 corresponding to (nucleotides 183-198 in FIGURE 11 A), MR-11 (nucleotides 340-357 in FIGURE 11 A), MR-8 (nucleotides 640-657 in FIGURE 11 A), MR-6 (nucleotides 700-719 in FIGURE 11 A), MR-7 (nucleotides 992-1012 in FIGURE 1 IB), MR-5 complement to (nucleotides 1055-1073 in FIGURE 1 IB), MR-4 (nucleotides 1277-1296 in FIGURE 1 IB) and MR-3 (nucleotides 1709-1723 in FIGURE 11C)).
  • FIGURE 11A shows the sequence from position 1 to position 720.
  • FIGURE 1 IB shows the sequence from position 721 to position 1500.
  • FIGURE 11C shows the sequence from position 1501 to position 1994.
  • FIGURES 11D-1 IE show schematically the positions of restriction endonuclease sites within the El 70 nucleotide sequence shown in FIGURES 11A- 11C.
  • FIGURE 12A shows the relative positions of the primers within the EP-1 El 70 sequence.
  • FIGURE 12B shows the nucleotide sequences of 8 primers useful in PCR methods for isolating nucleic acids encoding El 70 epithelial ligand glycoprotein, as described in Example 15.
  • FIGURE 13 depicts schematically the steps in a representative PCR assay method for isolating nucleic acids encoding El 70 epithelial ligand glycoprotein.
  • FIGURE 14 depicts schematically the steps in a representative 5' RACE system for PCR cloning of cDNAs encoding El 70 epithelial ligand glycoprotein, as described in Example 15.
  • FIGURES 15A-15F depict the nucleotide sequence compiled from sequencing cDNA clones corresponding to the ⁇ 3 EpA transcript.
  • FIGURES 16A-16B show experiments demonstrating that the clone Ep-1 expresses a fusion protein that corresponds to at least a portion of the 170 kDa subunit of epiligrin.
  • FIGURES 17A-17B show a Northern blot analysis of ⁇ 3 Ep mRNA and illustrates that two distinct transcripts are detectable.
  • FIGURES 18A-18C illustrate the sequence variability in domain Ilia, near the amino-terminal portion of the protein encoded by ⁇ 3 Ep .
  • FIGURES 19A-19R show the amino acid sequence encoded by ⁇ 3 EpA
  • FIGURES 20A-20H illustrate the localization of epiligrin mRNA and protein in 48 hour human wounds, using in situ hybridization with probes derived from ⁇ 3 Ep - 13/1 -
  • FIGURES 20A,20C, AND 20E show the wound site, or compound to normal skin shown in FIGURES 20B, 20D, and 20F.
  • FIGURES 20A-20B are labeled with epiligrin anti-sense probe, FIGURES 20C-20D with epiligrin sense probe, and FIGURES 20E-20F with Keratin anti-sense probe.
  • FIGURES 20g and 20H show wound sites labeled with anti-epiligrin and anti- alpha3 antibodies, respectively.
  • FIGURE 21 shows the localization of the human LamA3 gene to chromosome 18ql 1.2.
  • FIGURES 22A-22B are graphical representations depicting that integrins ⁇ 6 ⁇ 4 and ⁇ 3 ⁇ 1 mediate anchorage and motility, respectively, on epiligrin via distinct signal pathways, as described in Example 18.
  • keratinocytes human fetal keratinocytes (HFKs) expressed both the o ⁇ i and ⁇ g ⁇ 4 integrins.
  • HTKs human fetal keratinocytes
  • keratinocytes were recognized by the literature to be a differentiated form of epithelial cell, both with respect to their microscopic appearance and their biosynthetic activities, the inventors reasoned that cultures of these cells might synthesize and secrete the novel extracellular matrix ligand, and might be suitable for in vitro study.
  • epiligrin is a covalently linked glycoprotein complex that mediates epithelial cell attachment to the basement membrane through 0:3 ⁇ j integrin acting as a cellular receptor.
  • epiligrin is a covalently linked glycoprotein complex that mediates epithelial cell attachment to the basement membrane through 0:3 ⁇ j integrin acting as a cellular receptor.
  • the terms "epiligrin” and “epithelial ligand glycoproteins” are used interchangeably to refer to the same glycoprotein complex.
  • epiligrin Individual protein components of epiligrin are sometimes referred to as "epithelial ligand glycoproteins.”
  • Epiligrin is present in the lamina lucida of basement membrane and is associated with those cell membrane ultrastructural features previously termed focal adhesions. These focal adhesions are located on the basal surface of the cells in areas of contact with the basement membrane substratum, and they are also involved in cell motility.
  • Epiligrin also interacts with the 0 ⁇ 4 integrin, a cellular receptor that is present in ultrastructural membrane features previously termed hemidesmosomes and stable adhesion complexes.
  • the invention provides an understanding, for the first time, of how these two different ultrastructural features (frozen in time by fixation for electron microscopy) can function in a living cell to mediate adhesion, control of cell growth, and determination of the fate of daughter cells derived from cell division in the basal layer of the epithelium.
  • Burgeson et al. (#125) have proposed nomenclature that categorizes epiligrin as a kind of "laminin.” Accordingly, the inventors have denoted the gene encoding El 70 as "LamA3.” However, following this nomenclature can convey the misleading impression that E170 is demonstrably similar to the ⁇ 3 chains of laminin 5, 6, and 7.
  • o ⁇ i, integrin which binds epiligrin is one of the most widely expressed of all integrins in tissue, but its physiological ligand has not been identified until now. Novel test cell assays, extracellular matrix compositions, and immunochemical reagents were created which allowed identification for the first time of epiligrin, in basement membranes as the physiologically significant ligand for plasma membrane- based o ⁇ i and o ⁇ 4 integrins.
  • Only a few epithelial cells in culture e.g., keratinocytes express significant quantities of epiligrin, and this glycoprotein complex is of a large molecular size and has poor solubility in aqueous solutions. Epiligrin's size and poor solubility have undoubtedly contributed to the lack of previous recognition of this ligand in binding to integrins.
  • 0 ⁇ 4 integrin As a receptor for the epithelia ligand(i.e., in stable adhesion complexes) is less impressive than the adhesion mediated by the -o ⁇ i integrin but is potentially more significant.
  • the findings described below indicate that 0 ⁇ 4 integrin is involved in cellular adhesion to basement membranes, and it may also localize the focal adhesions in a pattern which encircles the regions of the stable anchoring contracts. This process of encirclement, as well as the localization of 0 ⁇ 4 integrin in cell-cell adhesion sites, determines the fate of the daughter cells formed by division in the basal (stem) cell layer of the epithelium.
  • Migration of epithelial cells is an important aspect of at least wound healing, inflammation, and tumor metastasis.
  • Focal adhesions containing integrin are involved in cell movement, and the stable anchoring contacts containing 0 ⁇ 4 integrin are involved in stopping cell movement.
  • Epiligrin binds to both o ⁇ j and 01 ⁇ 4 integrins.
  • the invention also provides, for the first time, an understanding at the molecular level of how polarized self-regulated growth and differentiation are achieved in epithelial tissues through the binding of the transmembrane integrins in the plasma membrane to extracellular epiligrin, the epithelial ligand complex, and possibly through intracellular signaling accomplished by the 36+15 kd epiligrin glycoprotein. These events occur at discrete plasma membrane sites in the stable anchoring contacts, and a second cytoplasmic polypeptide was also discovered to be a recognized SACs protein termed Bullous pemphigoid antigen.
  • Epiligrin was the major component of the extracellular matrix synthesized by human foreskin keratinocytes. In cultures of stationary keratinocytes, epiligrin was deposited and co-distributed with the transmembrane ⁇ 6 ⁇ 4 integrin and with cytoplasmic bullous pemphigoid antigens which are recognized components of hemidesmosome-like stable adhesion complexes. All three of these components in the stable adhesion complexes were resistant to sequential extraction with detergent, 2 M Urea/l M NaCl, and 8 M Urea. In contrast, the ⁇ j -containing integrins in the focal adhesions were not stable to this extraction. The 0.
  • the subject epiligrin derived from HFK is an epithelial ligand glycoprotein complex that includes at least three major covalently linked disulfide-bonded glycoproteins having apparent molecular sizes of 170 kd, 145 kd, and 135 kd. A glycoprotein of 36 kd is also associated with the epiligrin complex. Other observed components of epiligrin include a 100 kd protein that is antigenically related to the 145 kd protein, and a 200 kd protein that is antigenically related to the 170 kd protein.
  • the individual epithelial ligand glycoproteins are visible following reduction and SDS-PAGE (under reducing conditions).
  • the constituent glycoproteins are at times referred to herein by reference to their apparent molecular weight on SDS-PAGE, i.e., E200, E170, E145, E135, ElOO, and E36, respectively.
  • the 145 kd protein in some instances is referred to as E145/100.
  • E36 may be found in the culture supernatant (CS) and extracellular matrix of HFK cells (HFK-ECM); ElOO accumulates in CS; El 70 is usually not found in CS but may be found in a Triton X-100 extract of HFK-ECM as well as in the insoluble HFK-ECM fraction after the Triton X-100 extraction; and E200, El 40, and EDO are usually not found in CS or the Triton X-100 soluble fraction of HFK-ECM, but only in the Triton X-100 insoluble fraction of HFK-ECM.
  • a variety of biochemical and immunochemical methods may be utilized to purify the subject epiligrin glycoproteins, e.g., affinity chromatography in buffers containing Triton X-100 and/or mixtures of ionic, nonionic, or zwitterionic detergents.
  • Treatment with proteases e.g., trypsin
  • proteases may be useful for preparation of soluble epithelial ligand glycopeptides, some of which, while failing to mediate cellular adhesion to a surface may still retain the ability to block cellular adherence to epithelial ligand coated surfaces.
  • E200 appears to be antigenically related to El 70
  • El 45 appears antigenically related to ElOO
  • El 35 does not appear to be related to other glycoproteins in the epithelial ligand complex.
  • El 70 may be derived from E200 by proteolytic degradation (and/or processing) and, in a similar manner, ElOO may be derived from El 45.
  • Epiligrin antigens are associated with the basal surfaces of basal (stem) cells in epithelia at limited points of cellular contact with basement membranes.
  • Embodiments of the invention also relate to the isolation of epiligrin glycoprotein complexes for modifying adhesion of cells to substrata and for achieving polarized and self-regulated growth and differentiation in cells of epithelial origin.
  • Other embodiments relate to antibodies to the epiligrin glycoprotein complex for modifying cellular adhesion to substrata and for identifying epiligrin-like antigens in biological fluids, as well as epiligrin antigens for identifying antibodies in patient samples.
  • Still other embodiments relate to nucleotide sequences of El 70 epithelial ligand glycoprotein useful as specific probes for measuring the presence of epithelial ligand mRNA in a tissue, as well as the level of expression in different cells in the tissue.
  • Embodiments of the invention provide compositions and test methods for identifying - 19 -
  • the subject test methods and compositions are useful for determining the level of expression of epiligrin in a tissue.
  • Expression of epiligrin is a hallmark of a regenerating epithelial tissue (see Example 15).
  • the level of expression of El 70 epiligrin glycoprotein was found to provide a tool useful for distinguishing between regenerating epithelial tissues (where expression was high) and non-regenerative epithelial tissues or malignant tissues (where expression was low). Diagnostic histopathology is frequently complicated because it is not easy to distinguish tissue repair (e.g., resulting from traumatic injury or infection) from an abnormality that might be a neoplastic or preneoplastic event.
  • the invention provides nucleic acids capable of hybridizing under stringent conditions to at least one nucleotide sequence selected from the group consisting of the nucleotide sequence shown in FIGURES 11 A-l 1C, the cDNA clone Ep-1 (ATCC No. 75540) shown in FIGURE 10F, the cDNA clone 1-1 (ATCC No. 75539), and the cDNA clone 8-6 (ATCC No. 75538), or the nucleotide sequences shown in FIGURES 15A-15F.
  • the subject nucleic acids are preferably capable of encoding an El 70 epithelial ligand glycoprotein.
  • FIGURES 11A-11C A partial nucleotide sequence of nucleic acid encoding El 70 epithelial ligand glycoprotein is provided in FIGURES 11A-11C compiled from the cDNAs shown in FIGURE 10D as schematically depicted in FIGURE 10 A.
  • the entire nucleotide region encoding El 70 is depicted in FIGURES 15A-15F, and corresponds to the sequence of 3 EpA , one of the two distinct ⁇ 3 Ep transcripts discovered by the Applicants.
  • FIGURES 15A-15F consist of a composite sequence derived from the several overlapping clones shown in FIGURE 10D.
  • FIGURES 10F, 11 A-l 1C, and 15A-15F Although only a single (+) strand of the cDNA is shown in FIGURES 10F, 11 A-l 1C, and 15A-15F, those skilled in the art will recognize that the complementary (-) strands are thereby disclosed as well. According to the convention used herein to describe PCR primers, the "(-) strand” is complementary to El 70 mRNA. - 20
  • nucleic acid molecule DNA R and/or synthetic nucleotide sequences such as oligonucleotides that are the same as, homologous with, or complementary to, at least one helical turn (about 10 to 15 nucleotides) of the illustrated El 70 epithelial ligand glycoprotein nucleotide sequence.
  • El 70 transcripts are disclosed herein in HFK cells, one mRNA of about 5 kb and another of about 6 kb. Both mRNA species are identifiable to those skilled in the art in RNA from HFK by standard Northern blotting methods (e.g., using radiolabeled Ep-1 as a probe as illustrated in Example 15).
  • the invention relates to at least four classes of El 70 encoding nucleotide sequences, 1) alternative splicing transcript sequences, 2) sequences resulting from genetic polymorphism of El 70, 3) sequences resulting from translocation of El 70 in tumorigenesis and genetic diseases, and 4) sequences of El 70 family members having greater than 75% homology with El 70 over a conserved region of at least 30 nucleotides.
  • the latter four classes of El 70 nucleotide sequences are identifiable as hybridizing under stringent conditions with an El 70 nucleotide sequence of FIGURES 11A-1 IC, e.g., cDNA clone 1-1, Ep-1, or 8-6.
  • FIGURE 10D While the several clones depicted in FIGURE 10D encompass the entire nucleotide sequence encoding an El 70 epithelial ligand glycoprotein, skilled artisans will recognize that additional cDNA clones may be obtained using nucleotide sequences contained within the subject cDNAs as probes and primers for obtaining additional cDNA clones.
  • An illustrative example of a PCR cloning method for obtaining additional cDNA clones through PCR cloning is provided in Example 15, below.
  • PCR primers are additionally provided in FIGURE 12B and Table 1, below, and the steps of an illustrative PCR method are outlined in FIGURE 13. TABLE 1
  • Primer 4 SEQUENCE ID NO: 9
  • Primer 5 SEQUENCE ID NO: 10: 5' AAGTCACCTGAAGGCACG 3'
  • Primer 6 SEQUENCE ID NO: 11: 5' TGGACGTGCGACTTGACCAG 3 *
  • Primer 13 (SEQUENCE ID NO 12): 5' AACTCGCTTGCAGTTGAC 3'
  • Primer 14 SEQUENCE ID NO 13: 5' GATGGCTGTGGATCTTTG 3'
  • Primer 15 (SEQUENCE ID NO 14): 5' TCCACAGCAAGTGCTATG 3'
  • Primer 16 SEQUENCE TD NO 15: 5' ATGACAGTGCTGTCTGGAC 3'
  • Primer 17 (SEQUENCE ID NO 16): 5' TCTCCGAGATGGTCTTCATG 3'
  • Primer 18 SEQUENCE ID NO 17: 5' TTATCTGCATCAGTCAGAGC 3'
  • Primer 20 (SEQUENCE ID NO 18): 5' TGACCAGTGAGCTGTACATC 3'
  • Primer 29 SEQUENCE ID NO 19: 5' AGAGACCATTCGATTCAGAT 3'
  • Primer 30 SEQUENCE ID NO 20: 5' AGCTTCTGAGAAATAGCAAA 3' - 21 -
  • the PCR method in FIGURE 13 was used successfully to isolate mRNA encoding El 70 from normal epidermal tissue as well as from cells of patients with Epidermolysis bullosa.
  • Primers MR-4 and MR-7 and primers MR-5 and MR-7 (FIGURES 12A-12B) and the primers shown in Table 1 have also been used for PCR amplification and isolation of genomic DNA from normal and patient samples.
  • the latter isolated gemonic DNA contained both intron and exon sequences.
  • the subject nucleic acid capable of hybridizing under stringent conditions to a nucleotide sequence in FIGURES 11A-11C and FIGURE 15, e.g., cDNA clones "Ep-1", 3-1-1, 5-4-2, 3-8-6, 5-4-1, 3-8-2, or 8-6-1
  • a nucleotide sequence in FIGURES 11A-11C and FIGURE 15 e.g., cDNA clones "Ep-1", 3-1-1, 5-4-2, 3-8-6, 5-4-1, 3-8-2, or 8-6-1
  • the nucleic acids are useful (as illustrated in Example 15) in expression systems that produce El 70 epithelial ligand glycoprotein.
  • the expressed epiligrin glycoproteins find a variety of uses: e.g., as adhesive agents for cells; as antigens for production of antibodies; and, as antigens useful in detection of patient autoantibodies such as those described in the serum of patients with acquired subepidermal blistering diseases (Domologe- Hultsch et al., citation #114, incorporated herein by reference).
  • the subject nucleotide sequences of the subject nucleic acids are useful for constructing antisense oligonucleotides (as illustrated in Example 15, below).
  • the antisense oligonucleotides have nucleotide sequences capable of hybridizing under stringent conditions with the subject nucleic acids and are complementary with a nucleotide sequence encoding an El 70 epithelial ligand glycoprotein.
  • the subject antisense nucleotides have been used successfully for in situ hybridization, as shown in FIGURE 21.
  • the subject antisense nucleotides may be further characterized by their ability to transiently inhibit expression of an epiligrin gene in a cell, e.g., by transiently binding and inhibiting translation of an mRNA encoding an epiligrin constituent.
  • Epithelial cells whose expression of epiligrin was transiently blocked by antisense oligonucleotides did not adhere as strongly to HFK-ECM in vitro, and they became more rounded in appearance and form multicellular aggregates in suspension. The cells in the aggregates were observed to be differentiating. Thus, it is considered most likely that one or more regulatory feedback mechanisms exist in epithelial cells through which the binding of epiligrin to its ⁇ 3 ⁇ j receptor transduces a signal through a second messenger pathway that stops cellular proliferation and induces differentiation. It is thought highly likely that - 22 -
  • abnormalities in the latter signal transduction pathway will exist in certain epithelial cells because of defects in expression levels of epiligrin, or abnormalities in one or more epiligrin glycoproteins or in the epithelial ⁇ 3 ⁇ j integrin.
  • the affected cells may exhibit a phenotype of either uncontrolled growth or premature differentiation.
  • Antisense nucleic acids e.g., oligonucleotides
  • Antisense nucleic acids may be useful therapeutically for inducing epithelial differentiation in ulcers, and for restoring normal differentiation to prevent keloid formation and scaring, conditions.
  • the subject antisense nucleic acids may be introduced into a host cell by transfection (e.g., of an oligonucleotide) or by transduction of a nucleic acid encoding an antisense nucleic acid (e.g., using retroviral vectors ).
  • the subject antisense nucleic acids are all characterized by their ability to hybridize under stringent conditions with a (+) or a (-) strand of a nucleic acid encoding an El 70 epithelial ligand glycoprotein, e.g., as represented in FIGURE 10F, FIGURES 11A-11C, and FIGURE 15.
  • TGF ⁇ or TGF ⁇ Methods are disclosed in Example 16, below, for up-regulating expression of epiligrin through the addition of TGF ⁇ or TGF ⁇ to epithelial cells. These methods may be useful for increasing expression of epiligrin in patients suffering diminished synthetic capacity, e.g., in patients with a variety of blistering disorders and idiopathic urticarias (hives). Skilled practitioners will note that an effective dosage of TGF ⁇ or TGF ⁇ may be determined in screening assays (i.e., in vitro and in vivo in animal models) where the dosage in contact with the epithelial cells is escalated in a stepwise manner until synthesis of an epiligrin glycoprotein is increased (i.e., as measured by mRNA or protein).
  • a variety of systemic and topical methods for application may be tested by examining the levels of expression of an epiligrin glycoprotein in the treated cells before and after the treatment.
  • the subject nucleic acids also find use in gene therapy for inducing over- expression of epiligrin in diseased cells, and for gene replacement therapy in genetic - 23 -
  • junctional epidermolysis bullosa gravis can be a lethal genetic disease of infants that is associated with failure to normally express epiligrin.
  • Gene transfer may be accomplished using vectors (e.g., a retroviral vector) containing a construct that has in serial array: a promoter, a subject nucleic acid that encodes one or more epithelial ligand glycoproteins, e.g., E170, and a polyA tail.
  • constitutive expression of epiligrin in vivo may result in establishment of epithelium- basement membrane integrity in diseased epidermal tissues as well as in the lung, urogenital tract, gastrointestinal tract, and other sites of epiligrin expression (representatively illustrated in the Examples, below).
  • Epithelial ligand glycoprotein means a constituent glycoprotein of the epithelial ligand complex epiligrin.
  • Substantially-pure means of a purity sufficient that more than 70% of the polypeptides in the preparation can be determined by SDS-PAGE and protein staining to be the composition so specified.
  • Covalently linked means polypeptides chemically bonded to one another, as through for example (but not limited to) disulfide-bonds, thiol-ester bonds, ester bonds, amide bonds, or the like.
  • Capable of binding means physical interaction between two materials, such as between a specific binding partner and a ligand, where the interaction is sufficiently strong to permit measurement of a chemical association (or dissociation) constant (i.e., Ka or Kd).
  • Capable of hybridizing under stringent conditions means annealing of a nucleic acid molecule to at least a region of the disclosed El 70 epithelial ligand glycoprotein nucleic acid sequence (whether as cDNA, cRNA mRNA, or genomic DNA), or to its complementary strand under standard conditions, e.g., high temperature and/or low salt content, which tend to disfavor hybridization of noncomplementary nucleotide sequences.
  • a suitable protocol is described in Maniatis, T., et al. (#118 which is hereby incorporated by reference), at pages 387-389), wherein following the hybridization step filters are washed in 0.1X SSC, 68°C for 2 hours.
  • Other protocols for achieving stringent hybridization are well-known to those skilled in the art, and can be selected from those presented in - 24 -
  • hybridizing molecules may be related to the disclosed sequence by deletion, point mutation, base substitute, frameshift, alternative ORFs, mRNA splicing or processing, or post-transcriptional modification (e.g., methylation and the like).
  • Substratum means an insoluble material upon which cells may be deposited by gravity.
  • Non-adhesive substratum means a substratum to which fewer than 20% of the cells will bind in 24 hours at 37°C and from which 80% of the cells can be removed by washing with medium, e.g., such a substratum is provided by microbiological grade polystyrene plastic petri dishes.
  • Epithelial cells means, in this disclosure, the cells originating through mitosis in epithelial tissues which cover the free surfaces of the body and line the body cavities and ducts, as well as cells of epithelial origin such as malignant carcinoma cells. Further examples of epithelial cells as they are commercially available are provided in Table 2, below, as listed in the "Catalogue of Cell Lines and Hybridomas", 6th Edition, 1988, the American Type Culture Collection, Rockville, Maryland.
  • Modulate means to effect an increase or decrease of a specified parameter to a measurable extent.
  • Adhesion assay means an assay conducted with test cells, such as HT1080 in Example 6 below, to measure adhesion of cells to a protein-coated "non-adhesive" substratum under defined test conditions of tissue culture.
  • “Differentiation” means a staged process, e.g., in development, through which a cell progressively acquires distinguishably new phenotypic attributes.
  • Constant cell culture means a culture in which more than 85% of the cells are observed microscopically to be in physical contact with their neighboring cell.
  • Resistant to digestion means that no substantial change in physical properties is observed following incubation of the polypeptide with an enzyme for a substantial period of time.
  • Co-migrate means substantially the same electrophoretic migration when two polypeptides are either run together in the same lane of an SDS-PAGE gel, or when they are run side-by-side in adjacent lanes.
  • Molecular size means the apparent molecular radius of the polypeptide as observed under denaturing conditions in SDS-PAGE, and as recorded in kilodaltons (kd ⁇ ) of mass as determined by comparison with other polypeptides of known molecular mass. - 25 -
  • HFK-ECM extracellular matrix synthesized and secreted by HFKs
  • HFF-ECM synthesized and secreted by HFFs
  • Endogenous HFK-ECM is that which is intracellular or plasma membrane associated.
  • HFK-ECM secreted into the conditioned culture medium during the time course of an assay, or that which can be purified from culture dishes or glass cover slips (after the removal and/or extraction of the HFKs, as by the three-step extraction procedure detailed below), is referred to as exogenous HFK-ECM.
  • exogenous HFK-ECM secreted into the conditioned culture medium during the time course of an assay, or that which can be purified from culture dishes or glass cover slips (after the removal and/or extraction of the HFKs, as by the three-step extraction procedure detailed below
  • HFK-ECM and HFK were prepared by incubating HFK in culture dishes for 15 hours in KGM containing 3 5 S-methionine, 3H-glucosamine, or 3 5 SO 4 - 2 , and 1 mg/ml HD-BSA (Sigma) as a carrier protein.
  • Radiolabeled HFKs were sequentially extracted in a sequential three-step extraction procedure, as described previously (Wayner and Carter, 1987): (1) with 1% (w/v) Triton X- 100 (Sigma; to solubilize membranes and cytoplasmic constituents) and 2 mM N-ethylmaleimide (Sigma, to prevent intramolecular cross-linking); (2) with a solution containing 2 M Urea and 1 M NaCl (to remove nuclear and cytoskeletal components); and (3) with 8 M Urea (to solubilize residual cellular components). All extraction buffers contained 1 mM phenylmethyl sulfonyl fluoride (PMSF; Sigma Chemical Co., St.
  • PMSF phenylmethyl sulfonyl fluoride
  • FIGURE 1 shows the glycoproteins extracted in the steps 1, 2, and 3 (above), respectively.
  • glycoproteins were developed essentially of at least five major glycoproteins visualized by protein staining with Coomassie brilliant blue (FIGURE 1; lane 8) or following biosynthetically radiolabeled with 3 5 S-methionine (FIGURE 1, lane 4), or 3H-glucosamine (FIGURE 1, lane 9); these glycoproteins having apparent Mr of 200 kDa, 170 kDa, 145 kDa, 135 kDa, and 36 kDa (FIGURE 1, lane 9).
  • the HFK-ECM glycoproteins detected with protein stain showed slightly decreased amounts of the 200 kd glycoprotein (FIGURE 1, lane 8).
  • the five major glycoproteins were designated E200, El 70, E145, E135, and E36, based on relative molecular mass under reducing conditions on 8% SDS-PAGE.
  • the E170 band was inconsistently resolved into two bands (FIGURE 1, lane 9). Under non-reducing conditions the five glycoproteins did not enter the polyacrylamide gel (FIGURE 1, lane 5), indicating that they were subunits of - 27 -
  • proteins in conditioned medium from HFK cells contained a multiplicity of proteins, some of which co-migrated with the protein standards (FIGURE 2, HFK CS).
  • FOGURE 2, HFK CS protein standards
  • fibronectin or laminin
  • three types of experiments were conducted. First, the glycoproteins in exogenous HFK-ECM were separated on SDS-PAGE, blotted onto nitrocellulose as described previously (98) and tested for their immunoblot reactivity with rabbit antibodies directed toward laminin (Anti-LN; FIGURE 2) or fibronectin (Anti-FN; FIGURE 2).
  • HFK-conditioned medium HFK CS
  • FN fibronectin
  • H LN human placental laminin
  • EHS LN sarcoma EHS laminin
  • HFK-ECM HFK-ECM
  • HFK-ECM monoclonal antibodies
  • Binding Partners as Exemplified by Monoclonal Antibody to HFK-ECM Binding partners as exemplified by MAbs to HFK-ECM were produced by the methods of Oi and Herzenberg (99) and Taggart and Samloff (100) as described (59). Spleen cells from RBF/DN mice immunized with cultured HFKs were fused with NS-1 FOX-NY myeloma cells. Niable heterokaryons were selected in RPMI 1640 medium supplemented with adenine/aminopterine/thymidine.
  • Hybridomas PlEl (ATCC ⁇ o. HB10681) and P1H8 (ATCC No. HB10682)producing antibody specifically directed to HFK-ECM were selected using immunofluorescence microscopy and HFK-ECM or HFF-ECM on glass cover slips.
  • MAbs PlEl and P1H8 that reacted with HFK-ECM but not HFF-ECM produced by the dermal fibroblasts.
  • PlEl and P1H8 were cloned by limiting dilution.
  • PlEl and P1H8 (with rabbit anti-mouse IgG) immunoprecipitated five relatively minor disulfide-bonded subunits from the conditioned culture medium of 3 5 S-methionine-labeled HFKs.
  • the results presented in FIGURE 2 show examples of three (1, 2, 3) such immunoprecipitation experiments conducted with PlEl in which HFK cells were metabolically radiolabeled with 3 5 S-methionine, as described above, and antigens in the conditioned medium (FIGURE 2; CS) and HFK-ECM (FIGURE 2; ECM) were immunoprecipitated.
  • the five subunits of the PlEl antigen(s) co-migrated with the five major glycoprotein subunits of the exogenous HFK-ECM (FIGURE 2, compare ECM in the far left lane of the figure with PlEl, experiment 1).
  • glycoproteins i.e., E200, E170, E145, E135, or E36
  • E200 in the complex is not laminin
  • glycoprotein complex may be associated with laminin so that it forms a complex that can be precipitated with the anti-laminin antibody. This type of interaction of laminin has not been reported previously, and the composition of the precipitate differs significantly from previously reported complexes of laminin interacting with other glycoproteins.
  • the PlEl and P1H8 antigens correspond in molecular sizes to the glycoproteins in exogenous HFK-ECM, which consists of at least five subunits: E200, E170, E145, E135, and E36, which are visualized on SDS-PAGE after reduction, and that are distinct from any previously identified adhesion ligand(s) present in basement membranes or extracellular matrix.
  • the complex recognized by PlEl and P ⁇ H8 is the major component of exogenous HFK-ECM and also a minor component in HFK- conditioned culture medium.
  • HFKs were grown for 24 hours on glass cover slips coated with either fibronectin (FIGURES 4A-4B) or BSA (FIGURES 4C-4K), as described above (see “Cellular Adhesion to Extracellular Matrix Adhesive Ligand-Coated Substrates”). Glass cover slips and cells were then incubated with mouse or rat MAbs or rabbit polyclonal primary antibodies diluted in 1% heat HD-BSA overnight as previously described (21).
  • the cover slips were washed with PBS; incubated with dilutions of affinity-purified, species-specific, FITC-conjugated goat anti-mouse/rat IgG or rhodamine-conjugated goat anti-rabbit IgG secondary antibodies (respectively) for 1 hour, washed with PBS, and fixed with 2% formaldehyde prior to immunofluorescence microscopy.
  • the organization of epiligrin was dependent on the ligand to which the HFKs were attached. When HFKs attached to fibronectin (FIGURE 4A), collagen, or laminin, the cells migrated over the ligand surface leaving trails of epiligrin.
  • HFKs were incubated with: (1) mouse MAb anti- ⁇ g (GoH3); followed by (2) incubation with rhodamine-conjugated goat-antimouse IgG and IgM; after which the cells were fixed and reacted with (3) biotinylated with mouse PlEl -MAb; followed by (4) fluorescein Avidin.
  • ⁇ 6 ⁇ 4 was expressed on the apical surface of the cells and at the trailing edge. Small quantities of ⁇ co-distributed with the epiligrin antigen in the trails of these cells (FIGURE 4B).
  • FIGURES 4B and 4D), and BPA (FIGURE 4G) was most strikingly similar in the "ring structures.”
  • interference reflection microscopy IRM was performed basically as described (Izzard and Lochner, 1976) and was used to identify focal adhesions (FAs) in the same field as the two color immunofluorescence which identified the SACs. FAs were also localized by the antibody exclusion technique (101).
  • Epiligrin identified by PlEl
  • FIGURES 4E and 4H The co- localization and similar stabilities indicated that the deposits of epiligrin were at adhesion sites linked to ⁇ 6 ⁇ 4/BP A-S ACs.
  • Epiligrin co-distributed with the ⁇ g (GoH3; FIGURE 5B), and BPA (FIGURE 5D) in the extracted HSK-ECM Epiligrin co-distributed with the ⁇ g (GoH3; FIGURE 5B), and BPA (FIGURE 5D) in the extracted HSK-ECM.
  • the co- distribution of epiligrin with ⁇ 6 ⁇ 4 and BPA in both non-extracted and sequentially extracted HFKs Triton X-100, 2 M urea IM NaCl, above
  • the Epithelial Ligand Complex Epiligrin is a Ligand f or a ⁇ -FAs
  • ⁇ 3 ⁇ i in HFK-ECM is localized into FAs in proximity to, but excluded from, ⁇ 6 ⁇ 4 /BPA-SACs (20, 21).
  • HFKs that deposited epithelial ligand in SACS usually localized ⁇ ⁇ to FAs at the periphery of SACs as detected by interference reflection microscopy (FIGURES 4I-4K).
  • FIGURES 4I-4K interference reflection microscopy
  • Ovcar-4 an ovarian carcinoma cell line, and FEPE1L-8, FE-A, and FE-H18L, the human papilloma virus-transformed-HFKs (94, 95), expressed epithelial ligand but at low levels relative to HFKs.
  • Expression of ⁇ 6 ⁇ 4 was also investigated with immunofluorescence microscopy. Of the nine cell lines and primary HFKs cells, only the HFKs, T-47D, FEPE1L8, FE-A, and FE-H18L cells expressed ⁇ .
  • HFF and HT1080 fibrosarcoma cells expressed ⁇ 3 ⁇ j , but not epithelial ligand or ⁇ and provided us a model system to study the interactions of ⁇ 3 ⁇ with the epithelial ligand glycoprotein complex.
  • HFFs localized the a 5 ⁇ j and a 2 ⁇ integrins in focal adhesions when the cells were attached to fibronectin- and collagen-coated surfaces respectively (21), and this property was considered to be associated with ligand-induced receptor redistribution on the cell surface.
  • the ⁇ 3 ⁇ j integrin was not previously examined during the interaction of these cells with fibronectin or collagen.
  • HFFs were attached to surfaces coated with fibronectin (FIGURE 6A), type I collagen (FIGURE 6B), laminin (FIGURE 6C), or HFK-ECM (FIGURES 6D-6F) for 1 hour.
  • the fixed and permeabilized cells were - 33 -
  • FIGURE 6D-6F are all the same field.
  • the field in FIGURE 6E was stained for epiligrin (PlEl), and FAs were detected by interference reflection microscopy as shown in Panel F.
  • Arrows in FIGURES 6D-6F indicate localization of ⁇ 3 ⁇ 2 in FAs that contact the adhesion surface and exclude anti-epiligrinantibody.
  • HFFs attached to fibronectin or collagen they distributed ⁇ 3 ⁇ , over the entire cell surface with no localization of ⁇ 3 ⁇ i in FAs (FIGURES 6 A and 6B).
  • the laminin When attached to surfaces coated with laminin, the laminin induced thin FAs that were weakly positive for ⁇ 3 ⁇ ! (FIGURE 6C).
  • fibronectin, collagen, nor laminin appears to constitute a major ligand capable of redistributing the ⁇ 3 ⁇ integrin on the surface of human foreskin fibroblasts in vitro.
  • HFFs that attached to HFK-ECM for only 1 hour localized ⁇ 3 ⁇ ] into FAs as determined by interference reflection microscopy and exclusion of the PlEl antibody (FIGURES 6D-6F).
  • the ⁇ 3 ⁇ 1 co- distributed with epiligrin in both FAs and the ring structures characteristic of SACs.
  • Epiligrin was immunopurified from conditioned culture medium. This was accomplished in a stepwise fashion, first, by affinity-purification of MAb PlEl from hybridoma culture medium on Protein G-Sepharose (Pharmacia, Piscataway, NJ). Second, the purified monoclonal antibody was covalently coupled to Affigel-10 (Bio- Rad Laboratories, Richmond, CA; forming the PlEl -affinity-column). Third, conditioned culture medium from confluent cultures of HFKs was passed over a gelatin-Sepharose column (Pharmacia) to remove fibronectin. Fourth, the flow- through from the gelatin sepharose column was then passed over the PlEl -affinity- column.
  • the epiligrin purified on the PlEl affinity-column contained the complex of E200, E170, E145, and E135 covalently linked subunits as well as the E36 and ElOO, although lower levels of E200 were present than in HFK-ECM.
  • ⁇ 3 ⁇ j was evaluated by testing for inhibition of adhesion with anti ⁇ j , MAb (P1B5).
  • epiligrin EN; 1 ⁇ /ml
  • human plasma fibronectin FN
  • type I collagen CN; 10 ⁇ /ml
  • EHS laminin LN; 10 ⁇ /ml
  • BSA 5 mg/ml
  • the cells were labeled with Na 51 CrO 2 (New England Nuclear; 50 ⁇ Ci/ml for 2-4 hours) and were allowed to adhere (in the presence of the following inhibitory antibodies) to the protein-coated surfaces in the presence of the hybridoma supernatants for 1.5 hours.
  • the inhibitory antibodies indicated in FIGURE 7 include: SP2 as a control, non-inhibitory antibody.
  • P4C1O inhibits cell adhesion via all ⁇ j -containing integrins. GoH3, anti- ⁇ g j ) laminin receptor in HT1080.
  • P1H5 anti- ⁇ 2 ⁇ collagen receptor.
  • P1D6 anti- ⁇ s ⁇ j fibronectin receptor.
  • P1B5 anti- ⁇ 3 ⁇ j epiligrin receptor.
  • the bars in FIGURE 7 represent the mean values of three assays. Unattached cells were removed by washing and the adherent cells dissolved in SDS/NaOH and quantitated in a gamma counter.
  • the results presented in FIGURE 7 show that purified epiligrin mediated adhesion of HT 1080 cells to the previously non-adhesive plastic surface, and the cellular adhesion to epiligrin-coated plastic was blocked in a specific manner by the P1B5 MAb to the a i integrin. More specifically, the data presented in FIGURE 7 show that HT1080 cells attached to epiligrin-, fibronectin-, and collagen-coated and non-adhesive plastic surfaces and laminin (FIGURE 7).
  • FIGURES 8E-8H The magnification in FIGURES 8A-8R prior to photographic enlargement was 160x.
  • Cryostat sections were prepared from human tissues embedded in OCT medium after snap freezing in isopentant/liquid nitrogen. All sections were fixed with 2% formaldehyde in 0.1 M NaCacodylate pH 7.2 in 0.1 M sucrose for 20 minutes and then permeabilized with 1% Triton X- 100 for 15 minutes. The sections were incubated with primary antibodies and peroxidase-conjugated secondary antibodies.
  • epiligrin and laminin localized to the BM of ciliated epithelium (E) and submucosal glands (SMG) in the bronchus.
  • E ciliated epithelium
  • SMG submucosal glands
  • ⁇ 3 ⁇ ! was adjacent to epiligrin antigen in the basal plasma membranes of the ciliated epithelial cells.
  • ⁇ 4 was only sporadically expressed along the basal plasma membrane (E).
  • Both laminin and ⁇ 3 ⁇ j were strongly expressed in bundles of smooth muscle cells (M) and veins (N) while epiligrin was absent.
  • Epiligrin was absent from BM of heart muscle, mesothelium, brain, and glomerulus and tubules in kidney, while laminin was expressed. Epiligrin was present in the BM separating the intestinal epithelium from the lamina propria. These results identified epiligrin as a component of epithelial BMs particularly in organs of endodermal and ectodermal (but not neural) origin. Epiligrin was not present in muscle, neural, and endothelial BMs.
  • FIGURE 9A-9F The ultrastructural localization of epiligrin, a ⁇ 3 ⁇ , and ⁇ 4 was determined by immunoelectron microscopy of human skin. Electron micrographs are presented in FIGURE 9A-9F of immunoperoxidase-stained human neonatal foreskin which was stained with the following antibodies and achieving the following results: (FIGURE 9A) control staining with SP2 culture (supernatant is negative; hemidesmosomes and desmosomes are detectable); (FIGURE 9B) P1F2 ( ⁇ 3 ⁇ is detectable on the apical, lateral, and basal membranes; staining was also observed in desmosomes); (FIGURE 9C) and (FIGURE 9D) E31 (the ⁇ 4 subunit is localized to the basal surface and increased staining localized to hemidesmosomes at the origin of keratin filaments); and (FIGURE 9E) and (FIGURE 9F) PlEl (epiligrin
  • BM basement membrane
  • IC intercellular contacts
  • BS basal membrane surface
  • KF intermediate filaments
  • HD hemidesmosomes
  • D desmosomes
  • C collagen filaments .
  • HFKs Human foreskin keratinocytes
  • Keratinocytes have a distinctive composition of glycoproteins which is extracted with SDS sample buffer (12) and is visualized by SDS-PAGE (12): namely, (a) cytokeratins glycoproteins No. 5 (58 kd); No. 6 (56 kd); No. 14/15 (50 kd);
  • HFFs Human foreskin fibroblasts
  • HFK-ECM or HFF-ECM of HFK or HFF cells were seeded in KGM into 7.5 cm diameter tissue culture plastic dishes, and the dishes were incubated at 37°C in a humidified atmosphere consisting of 95% air/5% CO2 preferably for 1-3 days.
  • the HFK-ECM or HFF-ECM was prepared by a three-step extraction procedure.
  • the adherent HFK and HFF cells and their membrane and cytoplasmic constituents were removed by extraction with detergent in the continuous presence of protease inhibitors and 2 mM N-ethylmalaeimide (to inhibit intramolecular cross-linking).
  • Suitable detergents and concentrations for this first step include for example 1% (v/v) Triton X-100-tm anionic detergent (Sigma), Empigen BB-tm Zwitterionic detergent or 100 mM octyl glucoside.
  • Suitable protease inhibitors include diisopropyl fluorophosphate (DFP; Sigma), benzamidine, polybrene, kallikrein inhibitor, or phenyl methyl sulfonyl fluoride (PMSF; Sigma), which may be used individually or in combination as necessary to inhibit cellular protease activity (as evidenced by successful preparation by the complete three-step extraction procedure of HFK-ECM capable of adhering HFF or HT1080 cells in a manner inhibitable with antibodies to the ⁇ 3 ⁇ ! integrin; see below; Example 6, "Functional Properties of Epithelial Ligand").
  • DFP diisopropyl fluorophosphate
  • PMSF phenyl methyl sulfonyl fluoride
  • the second step in the three-step extraction procedure, involves solubilizing and removing nuclear and cytoskeletal components with a solution containing 2 M urea (ammonium-free), 1 M NaCl, and protease inhibitors; and the third step involves solubilization of any residual cellular components with a solution of 8 M urea and protease inhibitors.
  • the ECM remaining attached to the culture dish after the three-step extraction procedure of HFK culture dishes, or HFF culture dishes is referred to as HFK-ECM or HFF-ECM, respectively.
  • HFK-ECM can be solubilized into 0.5% w/v SDS by scraping the culture dishes with a rubber policeman; its constituent glycoproteins consist essentially of four - 39 -
  • the matrix produced by cultured fibroblasts consists essentially of a non-covalently linked dissociable complex of type I collagen, fibronectin, and heparin-containing and chondroitin-sulfate-containing proteoglycans (108), most of which is extracted by the three-step sequential extraction procedure. 7.
  • Example 2
  • Useful antigens for producing monoclonal and polyclonal antibodies include HFK cells, and also include HFK-ECM (prepared as described in Example 1), or individual glycoproteins present in HFK-ECM which are physically separable by techniques obvious to those skilled in the art including at least SDS-PAGE.
  • HFK-ECM Various procedures well known in the art are useful for the production of polyclonal antibodies to antigenic epitopes in HFK-ECM.
  • Various host animals can be immunized by injection with HFK-ECM proteins, fragments thereof, or synthetic peptides constructed to mimic the amino acid sequence in an HFK-ECM protein.
  • Adjuvants may be used to augment the immune response and immunogenicity of small proteins, and peptides may be enhanced by coupling them to larger "carrier" molecules.
  • Monoclonal antibodies for therapeutic use may be human monoclonal antibodies or chimeric human-mouse (or other species) of monoclonal antibodies.
  • HFK cells for use as an immunogen to produce monoclonal antibodies were prepared as follows. First, HFK cells were seeded in plastic tissue culture dishes. - 40 -
  • HFK-ECM was collected from the surface of culture dishes by scraping with a rubber policeman into 0.5% SDS and dialyzing against PBS to remove the SDS. This HFK-ECM aggregated protein suspension was mixed with an equal volume of Complete Freund's Adjuvant and lOO ⁇ l of the adjuvant solution was injected at each of 2sc and 2im sites in New Zealand white rabbits. The animals were boosted at 14 and 21 days.
  • Immune spleen cells from HFK-immunized mice were prepared and fused with NS-1/FOX-NY murine myeloma cells using polyethylene glycol as described (59, 99, 100). (These and the following methods (below) are also useful for producing monoclonal antibodies from mice immunized with HFK-ECM and individual glycoproteins present in HFK-ECM).
  • Niable heterokaryons were selected in RPMI 1640 supplemented with adenine/aminopterin thymidine (AAT; 100).
  • Heterokaryons producing antibodies (termed "hybridomas") specific for HFK-ECM and not binding to HFF-ECM were selected.
  • the clones designated PlEl and P1H8 were derived, as an example, by this method.
  • Other immunochemical methods for selecting positive hybridomas producing antibodies reacting with epithelial ligand glycoprotein complexes will be obvious to those skilled in the art, including at least selection by ELISA, RIA, and Western blotting using purified epithelial ligand antigens (Example 3, below) and/or HFK-ECM and HFF-ECM. It will also be understood that antibodies other than monoclonal antibodies may be produced (e.g., by immunizing rabbits, goats, or other animals), and will be equally useful.
  • Example 3 The specific immunochemical properties of monoclonal (or other) antibodies specific for epiligrin antigens are detailed below in Example 5. 8. Example 3
  • Epiligrin epiligand complex
  • HFK-ECM prepared in Example 1
  • SDS 0.5% SDS
  • a mixture containing predominantly a covalently-linked epithelial ligand glycoprotein complex was substantially purified from the conditioned medium of HFK cells as outlined in FIGURE 3. This was accomplished in a stepwise fashion involving, first, affinity purification of MAb PlEl from hybridoma culture medium on Protein G- sepharose using the methods recommended by the manufacturer (Pharmacia, Piscatawy, NJ). Second, the PlEl antibody was immobilized on a matrix (i.e., to form a PlEl -affinity-column) by covalently coupling purified monoclonal antibody to Affigel-10 according to the manufacturer's instructions (Bio-Rad Laboratories, Richmond, CA).
  • conditioned medium from confluent cultures of HFK cells was passed over a gelatin-sepharose column (Pharmacia) to remove fibronectin.
  • the flow-through from the gelatin-sepharose column was passed over the PlEl -affinity-column; unbound protein was removed by washing with PBS until the wash had an OD280 of less than 0.001 units; the bound epithelial ligand glycoprotein complex was eluted with 3 M KSCN, and the fractions containing the eluted protein were pooled and dialyzed overnight at 4°C against at least 10 volumes of PBS.
  • the substantially-purified covalently-linked epithelial ligand glycoprotein complex purified in this manner comprised predominantly E170, E145, El 35, and E36, although low levels of E200 and ElOO were also present by SDS-PAGE.
  • MAb PlEl is purified using affinity chromatography on other chromatographic resins containing compositions binding murine Ig (i.e., protein A-sepharose, or anti-IgG or protein M-sepharose); or alternatively, by specific binding of MAb PlEl to epithelial ligand complex or epithelial ligand glycoprotein covalently bound to a matrix (e.g., Affigel" 10 , Bio-Rad or CNBr-sepharose, Pharmacia).
  • a matrix e.g., Affigel" 10 , Bio-Rad or CNBr-sepharose, Pharmacia.
  • the relatively large molecular size of the epithelial ligand glycoprotein complex (calculated to at least greater than 450 kd to 650 kd, assuming equimolar amounts of each epithelial ligand glycoprotein), and insolubility of HFK-ECM glycoproteins in aqueous solutions will be used to advantage in purification schemes designed to separate these complexes from numerous smaller soluble cellular components. Purification of epithelial ligand glycoprotein complex from HFK-ECM or HGK-conditioned medium by conventional column chromatography was not possible due to the relatively poor solubility of the - 42 -
  • the epiligrin (El 70) protein was substantially purified from either HFK-ECM or the affinity-purified epithelial ligand glycoprotein complex by SDS-PAGE. It is understood by those skilled in the art that epithelial ligand for integrins is also purified by other conventional means from conditioned media of other epithelial cells under other conditions of growth.
  • HFKs were seeded into 7.5 mm plastic tissue culture dishes, as described in Example 1, and incubated in KGM medium (supplemented as described in Example 1) and containing 3 5 S-methionine or alternatively 3H-glucosamine.
  • HFK-ECM was prepared according to Example 1, which includes sequential extraction with 1% Triton X-100, 2 M urea 1 M NaCl, and then 8 M urea. These conditions are known by those skilled in the art to frequently be sufficient to dissociate non-covalently associated proteins, and also, to be sufficient to denature other proteins.
  • HFK-ECM is also stable to extraction with 6 M guanidine hydrochloride and 4 M sodium trichloroacetate.
  • the epiligrincomplex is relatively stable to denaturing conditions, and relatively resistant to extraction from the plastic tissue culture substrata (although it has been observed that some low levels of epithelial ligand glycoprotein complex are extracted with 8 M urea).
  • Non-radiolabeled glycoproteins were visualized by staining with Coomassie Brilliant Blue (Bio-Rad) and the radiolabeled glycoproteins were visualized by fluorography although other methods known to those skilled in the art for detecting biosynthetically radiolabeled glycoproteins are equally useful.
  • the non-radiolabeled sample of HFK-ECM appeared to contain lesser amounts of the E200 precursor (relative to E170, E145, or E135) than that present in biosynthetically-radiolabeled samples. This may be because there are differences in the amounts of E200 in the epithelial ligand glycoprotein complexes at different stages in HFK cell growth (e.g., subconfluent vs. confluent).
  • the five glycoproteins in the epithelial ligand complex were not biosynthetically radiolabeled with 3 5 SO 4 " 2 (FIGURE 1, lane 10) indicating that they are not sulfated proteoglycans.
  • the HFK-ECM biosynthetically radiolabeled for 15 hours with 50 ⁇ Ci/ml of 3 5 SO4" 2 in KGM containing 1 mg/ml HD-BSA as a carrier protein
  • the first sulfated proteoglycan did not enter the 3.5% stacking gel and the second barely entered the stacking gel but did not enter the 8% running gel.
  • PlEl did not bind to any of the epithelial ligand glycoproteins when they had been reduced and subjected to SDS-PAGE under reducing conditions, suggesting that the PlEl antigenic epitope may be conformational and denatured in these treatments.
  • the glycoproteins immunoprecipitated by PlEl include relatively greater amounts of E170 than E135, E145, or E200.
  • Verification that monoclonal (or polyclonal) antibody is directed to epiligrin complex is obtained by 3 5 S-methionine or 3H-glucosamine biosynthetically- radiolabeling HFK cells, collecting the conditioned medium, and using the antibody in question to form an immunoprecipitate with the biosynthetically radiolabeled antigens in the conditioned medium.
  • Antigens in the epiligrin complex exhibit characteristic molecular weights of 170 ⁇ 20 kd, 145+20 kd, 135+20 kd, 36 ⁇ 15 kd (with variable amounts of E200 ⁇ 20 kd) under reducing conditions on 8% SDS-PAGE and when run according to Laemmli (12). Additional verification that monoclonal (or polyclonal) antibody is directed to epiligrin can be obtained by examining the pattern of staining of cells in epithelial - 45 -
  • tissues by immunoperoxidase staining with cryostat sections of tissues (prepared as described above, see “Epithelial Ligand Localization in Normal Epithelial BM") (see “Epithelial Ligand, ⁇ 3 ⁇ , and ⁇ localization at the cell-BM Junction”).
  • Antibodies to epiligrin stain basement membrane materials in normal skin, tonsil, and lung essentially as described see “Epiligrin Localization in Normal Epithelial BM”; “Epiligrin Localization in Lymphoid Tissues”; “Epiligrin Localization in Lung Tissues”; above).
  • Antibodies to epiligrin do not stain BM materials of heart muscle, mesothelium, brain, or glomerulus and tubules in kidney (see “Epiligrin Localization in Other Tissues and Organs”).
  • the epiligrin complex is contained within characteristic "ring structures" in the ECM that are visualized by immunofluorescent staining with PlEl (see, “Epiligrin Distribution in Motile and Non-motile HFKs”).
  • FIGURES 4A, 4C, and 4F An example of these characteristic "ring structures” formed by epithelial ligand glycoprotein complex in HFK-ECM is provided in FIGURES 4A, 4C, and 4F.
  • Test cells which express functional a.$ ⁇ integrin will bind to ligand in the epiligrin complex, and if epiligrin is coated onto the surface of a normally non- adhesive substratum (epithelial ligand-coated substratum) the interaction between the epiligrin and the ⁇ 3 ⁇ ! integrin is sufficient to modulate adhesion of the test cells, in this case, by increasing their adhesion to the epiligrin-coated substratum.
  • the adhesion of the test cells to the substratum will be optimal after 24 hours of - 46 -
  • test cells which do not express the ⁇ g ⁇ 4 receptor or epiligrin in amounts sufficient to be detected by immunofluorescence microscopy, and HFF and HT1080 fibrosarcoma cells are examples of such test cells.
  • Polystyrene plastic petri dishes i.e., bacteriological grade petri dishes as opposed to tissue culture plastic dishes
  • non-adhesive substratum which will be useful for coating with epiligrin.
  • the epiligrin coating on the non-adhesive substrate may be applied by any of a variety of means known to those skilled in the art (e.g., by soaking, spraying, dipping, etc.) using a concentration of protein sufficient to accomplish the desired result of test cell adhesion to the epiligrin-coated substratum.
  • Epiligrin useful for coating the non-adhesive substratum will be, as an example, the epithelial ligand glycoprotein complex purified in Example 3, above, from HFK-conditioned medium, although other cellular sources, preparative purification methods, and substantially equivalent epithelial ligand compositions will also be useful.
  • the epiligrin-coated substratum involves a specific binding interaction between ⁇ 3 ⁇ j integrin and the epiligrin, it will be obvious to one skilled in the art that controls will be required including at least non-coated non- adhesive substrata incubated for the same period of time with the test cells.
  • the receptor e.g., monoclonal antibodies, such as P1B5 to the ⁇ 3 ⁇ j integrin, or MAb to the ⁇ 3 or ⁇ j chains; or peptide portions of the epiligrin complex which will competitively or non-competitively inhibit specific binding of the receptor
  • the ligand e.g., monoclonal antibodies to epiligrin which will inhibit adhesion of test cells in a specific manner or peptide portions of L ⁇ ⁇ integrin polypeptide chains which will inhibit the adhesion of test cells in a specific manner.
  • LAK cells lymphokine-activated killer cells
  • receptors bind in a specific manner to epiligrin-coated non-adhesive substrata in the cell adhesion assay described in Example 6, above, or Example 11, below.
  • the binding of activated lymphoid cells to epiligrin-coated substrata is inhibited in a specific manner by reagents specifically binding to either the ⁇ 3 ⁇ ! , receptor or to epiligrin, such as that described in Example 6, above and Example 11, below.
  • reagents e.g., peptides mimicking the receptor binding domain in epithelial ligand, or alternatively, integrin peptides mimicking the receptor domain which specifically inhibit binding of activated lymphoid cells to epiligrin.
  • reagents are also useful for specifically inhibiting the binding of activated lymphoid cells at sites of chronic and acute inflammation, for example, but not limited to, autoimmune dermatological diseases, rheumatoid arthritis, graft-versus-host disease and transplant rejection sites.
  • Embodiments of the invention relate to activated lymphoid cells which include but are not limited to LAK cells; interleukin, cytokine, and specific antigen-activated T- and B-lymphocytes; and activated mononuclear phagocytes (e.g., but not limited to, treatment with LPS), and/or antigen-activated mast cells (e.g., but not limited to, treatment with allergens).
  • activated lymphoid cells which include but are not limited to LAK cells; interleukin, cytokine, and specific antigen-activated T- and B-lymphocytes; and activated mononuclear phagocytes (e.g., but not limited to, treatment with LPS), and/or antigen-activated mast cells (e.g., but not limited to, treatment with allergens).
  • Example 8 Wound Healing Compositions Wound strength depends on cellular and molecular factors which include granulation tissue deposition, deposition of extracellular matrix, and re- epithelialization. Re-epithelialization depends upon migration of epithelial cells from the periphery of the wound site in a migratory tongue into the wound site. This migratory process is encouraged and promoted by epiligrin complex, epiligrin component glycoproteins, and portions thereof; particularly receptor binding portions of epiligrin glycoproteins. Agents which stimulate increased - 48 -
  • ⁇ g ⁇ 4 and ⁇ 3 ⁇ integrins on cells also promote cellular migration, which is advantageous in wound healing.
  • the epiligrin compositions and receptor binding portions disclosed herein promote the formation of SACS, and the proliferation of basal (stem) cells in epithelial tissues by cytokines, since they act as "second signals" to potentiate the action of cytokines.
  • the binding of ⁇ 6 ⁇ 4 and ⁇ 3 ⁇ j receptors to the epiligrin complex serves as a nucleation site for the formation of SACs, and stimulates synthesis of epiligrin glycoproteins which ultimately results in a migratory cell becoming stationary.
  • migratory behavior also promoted by agents which down-regulate epiligrin glycoprotein synthesis, or interfere with formation of the epiligrin complex.
  • basal (stem) cells The premature terminal differentiation of basal (stem) cells in wound sites slows the process of wound healing and contributes to wounds having lesser tensile strength than wounds in which terminal differentiation of epithelial cells can be slowed or completely retarded to allow proliferation of basal (stem) cells.
  • epiligrin complex provides a natural basement membrane material for basal (stem) cells and epithelial tissue explants which favors terminal differentiation of the epithelial cells into complex structures such as sweat glands and hair follicles, this process is not currently possible with existing wound healing compositions.
  • Epiligrin is also useful for screening reagent compositions in vitro that promote wound healing and epithelial cell growth in vivo, for example, but not limited to, cytokines and growth factors, epithelial ligand peptides, and ⁇ 3 ⁇ j receptor binding partners, such as described in Example 6, above, and in Examples 10 and 11, below.
  • Asymmetric cell division of epithelial basal cells is characterized by retention of one proliferative daughter cell at the BM (the first daughter cell) and dissociation of the other differentiating daughter (second daughter cell) from the basal layer with movement into the upper layers (i.e., the Malpighian layer of the skin and spinous strata in nervous tissue) (109).
  • ⁇ 3 ⁇ j and epiligrin are associated with proliferating basal cells (57, 110, 111), and epiligrin synthesis ceases as cells leave the basal layer.
  • Cellular control of asymmetric cell division is manifest through cytoplasmic polarization created by asymmetrical localization of adhesion sites (e.g., - 49 -
  • SACs and FAs on the cell plasma membrane of the basal stem cells.
  • Such asymmetry involves cytoplasmic glycoproteins associated with SACs and FAs and their interactions with epiligrin and the ⁇ 3 ⁇ and ⁇ integrins in these structures, e.g., cytoplasmic actin and the 36+15 kd epithelial ligand associated glycoprotein.
  • cytoplasmic glycoprotein components of SACs and FAs bind to cytoplasmic cytoskeletal elements and create the cytoplasmic polarization sufficient to create a first daughter cell which is distinctly different in cytoplasmic organization from the second daughter cell.
  • ⁇ 6 ⁇ 4 and ⁇ 3 ⁇ j interact with epiligrin in the proliferative first daughter at the basal surface of the cell (i.e., in association with BM).
  • ⁇ 3 ⁇ relocates to proximal sites of intercellular cell-cell adhesion in the basal cell which creates an asymmetrical force upon the differentiating second daughter cell.
  • the lack of physical binding of the second daughter cell to the basement membrane, the down-regulation of epiligrin synthesis, and increased cell-cell adhesion in the upper Malpighian or spinous layers creates a physical force to draw the differentiating second daughter away from the proliferative first daughter.
  • Epiligrin functions to maintain the proliferative functions of the basal (stem) cells through dual roles in anchoring the cell to the substratum and promoting (as a second signal) the activities of cytokines. Lack of epiligrin functions to create the class of "second daughter cells" committed to differentiation.
  • the epiligrin complex, epithelial ligand glycoproteins, and portions thereof are useful for promoting basal (stem) cell growth in epithelial cells and modulation of epiligrin synthesis promotes differentiation of epithelial cells such as cancer cells, cells in autoimmune disease states, and cells in psoriasis.
  • epithelial cells such as cancer cells, cells in autoimmune disease states, and cells in psoriasis.
  • ⁇ 3 ⁇ receptor specific binding partners such as (for example) antibody to the receptor, promotes aggregation of cells lacking epiligrin, and this also is useful for mimicking the effects of epiligrin in inducing proliferation and differentiation of epithelial cells .
  • epiligrin complex binds to both ⁇ 3 ⁇ j and ⁇ 6 ⁇ provides for the first time a relationship between these two integrins and a functional basis for regions of conserved amino acid sequence(s). Knowing that there is a 37% sequence identity between ⁇ 3 and ⁇ 6 , and that both ⁇ 3 ⁇ ! and ⁇ 6 ⁇ bind to epiligrin, one can select and employ conserved peptide sequence(s) to modulate binding of epiligrin to the ⁇ 3 ⁇ 4 and ⁇ 6 ⁇ 4 integrins as follows.
  • “Limited sequence portions” of the ( ⁇ 3 and ⁇ 6 chains i.e., peptides which are at least 30% homologous or identical over at least 3 to 30 amino acids, are identified by direct comparison of the aligned amino acid sequences.
  • homologous peptides are constructed (" ⁇ 3 or ⁇ g test peptides") which are composed of amino acid sequences that are at least 30%) homologous and/or identical to the amino acid sequence in the "limited sequence portions.”
  • Such “limited sequence portions” and/or “ ⁇ 3 and ⁇ g test peptides” are then assayed pursuant to this disclosure to determine and select "reagent peptides" which will function as inhibitors (" ⁇ 3 and ⁇ g inhibitory test peptides”), antagonists, and agonists of the natural binding of ⁇ 3 ⁇ j and ⁇ g ⁇ integrins to the epiligrin.
  • cellular adhesion assays such as those described in Example 6, above, are used to determine and select which of the "limited sequence portions" and " ⁇ 3 and ⁇ g, inhibitory test peptides" inhibit cellular adhesion by at least 20% to epiligrin-coated substrata at a physiologically significant molar concentration (i.e., to determine the " ⁇ 3 or ⁇ g inhibitory peptides").
  • Test cells are allowed to adhere to epiligrin-coated substrata in tissue culture medium within 2 to 24 hours, and preferably 2 to 18 hours, and this adherence of the test cells is inhibited by at least 20% when the " ⁇ 3 or ⁇ 6 inhibitory test peptide" is added to the tissue culture medium at a physiologically significant molar concentration, i.e., of lO" 5 M to 10" 10 M and preferably 10" 6 M to 10" 10 M M.
  • a plurality of assays with a plurality of test cells and " ⁇ 3 or ⁇ g inhibitory test peptides" are run to identify the " ⁇ 3 or ⁇ g inhibitory peptide(s)" which inhibit test cell adherence in the assay by at least 20% at a physiologically significant molar concentration.
  • the selected inhibitors, antagonists, and agonists may be engineered to, e.g., improve their stability, half-life in blood or binding affinity for ligands. Such improvements are made either by substitution of one or more amino acid(s) in the it " ⁇ 3 or ⁇ g inhibitor peptide" for a "natural amino acid” (the "natural amino acid” is that amino acid that is present more than 40% of the time at that particular position when the ⁇ 3 and ⁇ g chains from at least 5 different animal species are properly aligned).
  • the “natural amino acid” is that amino acid that is present more than 40% of the time at that particular position when the ⁇ 3 and ⁇ g chains from at least 5 different animal species are properly aligned).
  • Improvements are also made by biochemical or chemical modification of the " ⁇ 3 or ⁇ g inhibitor peptide” to create at least one synthetic amino acid, or a plurality of synthetic amino acids, in the amino acid sequence of the " ⁇ 3 or ⁇ g inhibitor peptide” to produce “ ⁇ 3 and ⁇ g inhibitor analogues.”
  • compositions described in Example 10 are useful for inhibiting the binding of activated T lymphocytes through the integrins to basement membrane compositions having at least epiligrin.
  • test cell assay disclosed in Example 7 it is possible to assay a plurality of the inhibitor, antagonist, and agonist compositions disclosed in Example 10 to determine which compositions inhibit by at least 20% the adherence of lymphoid test cells at a physiologically meaningful concentration, e.g., between IO" 5 M to 10" 10 M (i.e., "lymphoid inhibitory compositions").
  • the "lymphoid inhibitory compositions” are useful for preventing and slowing the accumulation of activated lymphoid cells (as defined in Example 7) at sites of chronic or acute inflammation, for example (but not limited to) in graft vs. host disease, transplant rejection, autoimmune dermatological and rheumatic diseases, such as rheumatoid and nonrheumatoid arthritis, Bullous pemphigoid, CP, and EBA.
  • Epiligrin complex and its constituent antigens are useful for identifying autoantibody in patients with autoimmune disease, for example (but not limited to) greater than 50% of the patients with cicatrical pemphigoid and less than 20% of the patients with BP or EBA; they are useful for distinguishing CP from BP and EBA.
  • Immunochemical diagnostic assay formats for example, which are useful include at least enzyme-linked immunoadsorbent assays (ELISA), radioimmunoassays
  • RIA fluorescence immunoassays
  • FFA fluorescence immunoassays
  • Western immunoblot assays time-resolved - 52 -
  • TRF fluorescence assays
  • particle agglutination assays e.g., latex, red cell, etc.
  • the bound antibody (or antigen) is separated from free antibody (or antigen) by physical means involving, for example, the use of a solid-phase adsorption of antibody (or antigen) in tubes, microtiter plates, and on polymeric membranes, dipsticks, and beads (e.g., magnetic beads or polystyrene or nylon-66-tm beads); or, alternatively, bound antibody (or antigen) is separated from free antibody (or antigen) through the use of a washing step wherein the assays are run in steps involving at least binding of antibody (or antigen), separation of bound antibody (or antigen) from the free antibody (or antigen) with washing, and assaying for the amount (or presence) of bound antibody (or antigen).
  • Epiligrin-complex-coated substratum is optimal for promoting the growth of epithelial basal (stem) cells and for preventing the differentiation of this population of cells into other cell types, such as keratinocytes in the skin.
  • KGM medium e.g., keratinocyte growth medium, containing growth factors such as PDGF, EGF, FGF, and insulin, and serum, such as 1-10% fetal bovine serum
  • basal (stem) cells in the samples continue to exhibit mitotic activity.
  • basal (stem) cells To confirm the mitotic activity of basal (stem) cells in these cultures, and to distinguish from the mitotic activity of other contaminating cell types (e.g., fibroblasts in skin biopsy samples) it is possible to observe the basal (stem) cells microscopically after fixing, staining, and embedding the cell layer for autoradiography, e.g., using a photographic emulsion.
  • contaminating cell types e.g., fibroblasts in skin biopsy samples
  • Basal (stem) cells cultured on epiligrin-coated substrata retain their mitotic activity for a period of time which is longer than that of biopsy samples grown on control substrata, i.e., HD-BSA; 7 to 14 days is a convenient length of time for assessing this activity (although it is also possible to visually assess the cultures on a daily basis by inverted microscopy, and those skilled in the art are readily able to determine the optimal time for determining mitotic activity in an individual experiment).
  • the mitotic activity is microscopically visible in the autoradiograph as increased numbers of silver grains over the nucleus of the basal (stem) cells.
  • epiligrin-complex-coated substrata to support mitosis and growth of epithelial basal (stem) cells creates, for the first time, the opportunity to establish tissue cultures of continuously proliferating and differentiating epithelia that mimic normal biological processes and provides a superior source of epithelial cell sheets for transplantation.
  • Such cell sheets are obtained from any epithelial tissue; for example, in the case of skin they are useful for skin transplants; in the case of epithelial cells in the bone marrow and lymphoid tissues they are useful in bone marrow transplantation methods; and, in the case of gastrointestinal ulcers, the cell sheets are useful for repopulating denuded areas of ulcerated tissues through non- invasive transplantation procedures, such as through the use of a catheter.
  • compositions may be administered to a human patient or other mammalian host in need of treatment by a variety of conventional routes of administration, including orally, parenterally, intravenously, intraperitoneally, intradermally, subcutaneously or intramuscularly.
  • Compositions may also be administered transdermally (as in a lipid- soluble vehicle for a timed-release skin patch), or by nasal or oral instillation into the lungs (as with a nebulizer).
  • these compounds will be administered at dosages between 1 and 250 mg per kg body weight of the subject to be treated per day. However, some variation in dosage will necessarily occur depending on the condition of the patient being treated, and the physician will, in any event, determine the appropriate dose for the individual patient.
  • salts can be readily prepared from sorbini! and sorbinil analogs by conventional methods.
  • such salts may be prepared by treating the sorbinil or sorbinil analog with an aqueous solution of the desired pharmaceutically acceptable metallic hydroxide or other metallic base and evaporating the resulting solution to dryness, preferably under reduced pressure.
  • a lower alkanoic solution of the sorbinil or sorbinil analog may be mixed with an alkoxide of the desired metal, and the solution subsequently evaporated to dryness.
  • the pharmaceutically acceptable hydroxides, bases, and alkoxides include those with cations that form metal salts with the acidic compounds of sorbinil and its analogs and that are nontoxic at the dosages administered to a patient in need of treatment. Suitable cations for this purpose include, but are not limited to, potassium, sodium, ammonium, calcium and magnesium. - 54 -
  • the compounds may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses.
  • suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions and various nontoxic organic solvents.
  • the pharmaceutical compositions formed by combining the sorbinil or sorbinil analog with the pharmaceutically acceptable carrier are then readily administered in a variety of dosage forms, such as tablets, powders, lozenges, syrups, injectable solutions and the like.
  • These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients, and the like.
  • tablets containing various excipients such as sodium citrate, calcium carbonate, and calcium phosphate may be employed along with various disintegrants such as starch, and preferably potato or tapioca starch, alginic acid, and certain complex silicates, together with binding agents such as polyvinylpyrrolidine, sucrose, gelatin, and acacia.
  • disintegrants such as starch, and preferably potato or tapioca starch, alginic acid, and certain complex silicates
  • binding agents such as polyvinylpyrrolidine, sucrose, gelatin, and acacia.
  • lubricating agents such as magnesium stearate, sodium luaryl sulfate, and talc are often useful for tableting purposes.
  • Solid compositions of a similar type may also be employed as fillers in salt and hard-filled gelatin capsules; preferred materials for this purpose include lactose or milk sugar and high molecular weight polyethylene glycols.
  • the essential active ingredient therein may be combined with various sweetening or flavoring agents, colored matter or dyes, and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof.
  • diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof.
  • solutions of the sorbinil or sorbinil analog in sesame or peanut oil or in aqueous propylene glycol may be employed, as well as sterile aqueous solutions of the corresponding water soluble pharmaceutically acceptable metal salts previously described.
  • Such an aqueous solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.
  • the sterile aqueous media employed are all readily obtainable by standard techniques well known to those skilled in the art. Additionally, it is also possible to administer the aforesaid compounds topically via an appropriate solution suitable for the present purposes at hand.
  • the subject compounds when formulated as described above, will typically be packaged with printed instructions specifying their use as anti-cancer or anti- inflammatory compounds, e.g., for reestablishing normal growth control in carcinoma cells, or for inhibiting adhesion of activated lymphoid cells to epithelium, respectively.
  • anti-cancer or anti- inflammatory compounds e.g., for reestablishing normal growth control in carcinoma cells, or for inhibiting adhesion of activated lymphoid cells to epithelium, respectively.
  • HFKs normal newborn human foreskin keratinocytes
  • KGM serum-free Keratinocyte Growth Medium
  • the FE-A, FEPE1L-8 and FE-H18L cell lines are HFKs that have been transfected with transforming genes E6 and E7 from human papilloma virus 16 and 18 (94; 95).
  • HFFs human foreskin fibroblasts
  • Peroxidase-, fluorescein-, and rhodamine-conjugated (goat) anti-mouse and anti-rat IgG and IgM (H and L chains) or peroxidase and rhodamine-conjugated (goat) anti-rabbit IgG and IgM (H and L chains) were obtained from Tago, Inc. (Burlingame, CA). 20.2 Antibodies and Immunochemical Reagents
  • Peroxidase-, fluorescein-, and rhodamine-conjugated (goat) anti-mouse and anti-rat IgG and IgM (H and L chains) or peroxidase and rhodamine-conjugated (goat) anti-rabbit IgG and IgM (H and L chains) were obtained from Tago, Inc. (Burlingame, CA). Fluorescein-conjugated avidin was from Vector Labs (Burlingame, CA). N-hydroxysuccinimido-Biotin was from CalBiochem (La Jolla, CA).
  • MAbs to the integrins ⁇ 3 ⁇ ! (P1B5, P1F2), ⁇ 2 ⁇ (P1H5), ⁇ 5 ⁇ ! (P1D6), and ⁇ i (P4C1O) have been described (20, 21, 57, 59, 96).
  • P1H5 and P1D6 inhibit fibroblast, keratinocyte, and platelet adhesion to collagen-coated and fibronectin- coated substrates, respectively (21, 57, 59, 97).
  • MAb P4C10 reacts with all ⁇ r containing integrins and inhibits cell adhesion to laminin, collagen, and fibronectin (20, 21).
  • SP2 is a control-conditioned culture medium from the SP2 mouse melanoma.
  • Monoclonal anti-tenascin, F9A5 was prepared in this lab (Maxwell and Carter, unpublished results).
  • Monoclonal anti- ⁇ 6 (GoH3) was from Dr. Arnoud Sonnenberg (Amsterdam, Holland) and inhibits platelet adhesion to laminin via ⁇ 6 ⁇ j (Sonnenberg et al., 1988) and carcinoma adhesion to laminin via ⁇ 6 ⁇ 4 (63).
  • Mouse MAb 3E1 against integrin b4 was a gift from Dr. Eva Engvall (La Jolla Cancer Res. Ctr., La Jolla, CA).
  • Rabbit polyclonal antiserum against the carboxy terminus of the Bullous pemphigoid antigen (BPA; R1086) was a gift from Dr. John R. Stanley (Dermatology Branch of the National Institutes of Health, Bethesda, MD). Rabbit polyclonal anti-entactin was from Upstate Biotechnology, Inc. (Lake Placid, NY).
  • Mouse laminin (derived from EHS sarcoma, grown in mice) was purchased from Collaborative Research Inc. (Bedford, MA) or prepared in this lab. Plasma fibronectin and collagen type I were prepared as described (Wayner et al., 1988).
  • Entactin was from Upstate Biotechnology, Inc. (Lake Placid, NY). Tenascin and pepsinized human placental laminin were from Telios (San Diego, CA).
  • HFK-ECM was prepared by growing HFKs (or HFF) for three days in KGM on acid-washed glass cover slips (25 mm diameter). The adherent cells were removed by a three-step sequential extraction procedure: first, with 1% v/v Triton x-100 detergent (Sigma) in 10 mM sodium phosphate, buffered, pH 7.4, 0.14M saline (PBS); second, with 2 M urea/1 M NaCl; and, third, with 8 M urea. The HFK-ECM was digested with DNase I for 30 minutes in 1% w/v HD-BSA (Sigma)/PBS.
  • cover slips were washed with PBS, and blocked with HD-BSA (i.e., to avoid nonspecific binding of test antibody to the glass).
  • HD-BSA i.e., to avoid nonspecific binding of test antibody to the glass.
  • acid-washed glass cover slips 25 mm diameter were derivatized with dimethyldichlorosilane (Pierce, Rockford, IL); then coated with purified ligands (1 to 10 "N" symbol protein/ml); and finally blocked with 1% w/v HD-BSA in PBS as previously described (20, 21). Cells were adhered to the cover slips in KGM medium for periods of 1 hour to 3 days.
  • epiligrin is a major component of epithelial basement membranes that mediates basal cell adhesion via integrin ⁇ 3 ⁇ !.
  • a cDNA expression library was screened using a - 57 -
  • polyclonal rabbit antibody prepared against the extracellular matrix of human foreskin keratinocytes i.e., HFK-ECM.
  • a lambda gtl l expression library generated from human keratinocytes was screened with an affinity purified polyclonal antibody prepared against HFK-ECM using a method previously described (124).
  • Ep-1 deposited at the American Type Culture Collection, Rockville, MD and assigned ATCC No. 75540
  • Ep-1 was shown by two independent immunological methods to express a polypeptide immunologically indistinguishable from the 170 kDa component of epiligrin, herein referred to as E170.
  • Ep-1 cDNA was expressed as a fusion protein, and the fusion protein was shown to serve as a specific antigen in preparation of immunosorbents that affinity purified anti-epiligrin antibodies from a polyclonal anti-HFK-ECM serum.
  • the Ep-1 fusion protein was immobilized on nitrocellulose and then incubated with anti-HFK-ECM polyclonal antiserum. The nitrocellulose blot was washed and the bound antibody eluted using 0.1 M glycine-HCl, pH 3.0.
  • the eluate was neutralized using 1 M Tris-HCl, pH 7.4, and dialyzed against 0.1M phosphate buffer, pH 7.4 0.14M saline (PBS).
  • PBS 0.1M phosphate buffer
  • the Ep-1 -expressed fusion-protein-adsorbed antibodies were tested for immunoreactivity against epiligrin, in this case purified by affinity chromatography on monoclonal PlEl (as described in the Examples above).
  • the adsorbed EP-1 fusion protein-reactive antibodies bound specifically to El 70 in epiligrin after electrophoresis in SDS-PAGE (under reducing conditions) and following blotting of the polypeptide onto nitrocellulose (i.e., in a Western blot).
  • Ep-1 cDNA was cloned into pGEX-lN (Amrad Corporation, Australia), which expresses a portion of the glutathione S-transferase gene.
  • the fusion protein encoded by the chimeric construct was purified using glutathione agarose beads and then SDS-PAGE.
  • the left panel shows a Coomassie Blue stain of purified epiligrin (PlEl Ag) under reduding (+2-ME) and nonreducing conditions (-2-ME).
  • the subunits of epiligrin are designated El 70, E150, E135, and ElOO, based on molecular size in kilodaltons. "A” identifies high molecular mass disulfide-bonded aggregates of epiligrin.
  • the right panel shows that the fusion protein expressed by the clone (Ep-1) can immunopurify antibodies from anti-HFK ECM polyclonal antiserum that react spedifically with the 170-kDa subunit of epiligrin (PlEl Ag). A second band of slightly lower molecular weight is detectable and may corespond to a degradation product or a second related polypeptide.
  • the negative control antibody (CONTROL) showed no reactivity to epiligrin.
  • Ep-1 fusion protein was used to generate a rabbit polyclonal antiserum.
  • Ep-1 immune serum reacts specifically with the 170- kDa subunit of epiligrin on an immunoblot containing purified epiligrin (PlEl Ag) and HFK-conditioned culture media (Cond. Media).
  • PlEl Ag purified epiligrin
  • HFK-conditioned culture media Cond. Media
  • a nonspecific band was detected by the preimmune serum in the lane containing conditioned media. It should be noted that this band does not comigrate with the second band of slightly lower molecular weight tnat is detected by the Ep-1 immune serum.
  • the negative control Pre ⁇ immune Serum
  • Ep-1 transcripts were present as a doublet of bands at about 5 kb and about 6kb, indicating two mRNA species for El 70, and the possibility of alternatively spliced forms of El 70.
  • the cDNA probe Ep-1 was used to examine factors that affect expression of epiligrin mRNA in cultured cells (FIGURES 17A-17B).
  • Total cellular RNA was isolated by the guanidine isothiocyanate/ultracentrifugation method (Kingston et al., 1991), run on a 1% denaturing agarose gel, and transferred to nitrocellulose (Brown, 1993). DNA was radiolabeled with 3 2 P by random priming, hybridized overnight at 42°C in 50% formamide solution (# 118, Maniatis), and washed at 54°C in 0.1 x SSC, 0.1% SDS.
  • FIGURE 17 A human papilloma virus
  • HFF cells which do not express epiligrin, were included in lane 4 as a control (FIGURE 17A). Positive hybridization to tubulin mRNA indicates that comparable amounts of RNA were loaded in FIGURE 17A lanes 1-4.
  • FIGURES 18A-18B These observations were consistent with the results of multiple sequence alignments which had indicated that there are two distinct ⁇ 3 transcripts that display variability within domain Ilia (see FIGURES 18A-18B).
  • the cDNA clone 5-4-1 was independently isolated and shown to encode an amino-terminal domain which is different from that presented in FIGURE 19.
  • This second distinct transcript referred to as ⁇ 3 EpB; maintains homology to ⁇ l laminin throughout domain III1 and into domain IV (FIGURE 18B).
  • primer 11 which corresponds to a region of nondivergent sequence in cDNA clones 3-1-1, 5-4-1, and 5-4-1, was used to synthesize cDNA from mRNA using reverse transcriptase.
  • primers 11 and 14, as well as primers 11 and 16 were used to amplify distinct PCR products.
  • a control RNA sample was included to ensure that the PCR products were generated from reverse-transcribed cDNA and not genomic DNA.
  • FIGURE 17B shows that when proliferating HFK cells are induced to differentiate by adding calcuim (1.3 mM Ca++, lane 2) or by increased cell density (lane 3), ⁇ 3 Ep expression is concommitantly down regulated.
  • RNA isolated from subconfluent cultures expressed significantly higher levels of ⁇ 3 Ep mRNA (FIGURE 17B, lane 1). Under these conditions, a third transcript wa detectable that appeared to be migrating behind the 7.5 Kb molecular weight marker (lane 1). Therefore, while the 6 Kb transcript appears to be the most abundant, there are clearly additional ⁇ 3 Ep transcripts present in HFK RNA.
  • El 70 epiligrin glycoprotein is an early marker for commitment of cells to differentiate into a cells of the epithelial lineage.
  • El 70 is down-regulated so that mature basal epithelial stem cells (and their progeny) express only low levels of El 70 mRNA.
  • Basal stem cells retain the capacity to express El 70 following wounding (as described in Example 16, below), and El 70 expression is increased in the epithelial "tongue" of cells that are migrating into wound sites.
  • El 70 is a useful marker for assessing damage to, and regeneration of, epithelial tissues.
  • FIGURE 18B compare the partial amino-terminal sequences of proteins encoded by the ⁇ 3 EpA and ⁇ 3 EpB cDNAs.
  • FIGURE 18C depicts the common protein domain of ⁇ 3 Ep and ⁇ 3 EpB , which is encoded by cDNA clones 3-1-1, 5-4-1, and 5-4-2 (FIGURE 10F). Nucleic acid sequencing of PCR products showed that HFK cells contain two distinct ⁇ 3 transcripts in which either sequences A and C are continuous or sequences B and C are continuous. Sequence Encoding El 70:
  • FIGURES 12A-12B were used to synthesize a 650bp fragment that overlaps with the Ep-1 cDNA. This 650bp fragment was then labeled with 32 P and used to rescreen the cDNA library (FIGURE 10A). Over 100 potential positive clones hybridized on a primary screening. Since this number of positive clones seemed too high, it was assumed that the amplified sequence was also hybridizing with a common sequence in several gene products. To impart specificity to the screening method the same filters were rescreened using 32 P-radiolabeled Ep-1 as a probe. Four positive clones were identified in this screening and were isolated for further testing: cDNA clones "l-l"(about 2kb in size), "2-3"(about l.
  • Clones 1-1 and 2-3 contain cDNA overlapping with each other and with cDNA in clone Ep-1, as well as with the 650bp PCR product (above).
  • Ep-1 was shown to contain 600bp encoding a helix region of the El 70 polypeptide.
  • a cloning artifact was also identified in the first 150bp of the clone sequence, namely, ribosomal RNA nucleotide sequence unrelated to E170. (The compiled nucleotide sequence in FIGURES 11A-11C and 15A-15F were edited to remove the unrelated rRNA sequence.) - 62 -
  • FIGURE 10D The overlapping cDNAs used to compile the El 70 nucleotide sequence (FIGURES 15A-15F) are shown in FIGURE 10D. Sequence analysis revealed that clone Ep-1 encodes a helix region of El 70 glycoprotein; clone "1-1 " an EGF-like region of El 70 (FIGURE 10F); and, clone "8-6” encodes a potentially globular region of El 70.
  • the complete El 70 nucleotide and protein sequences are shown in FIGURES 15A-15F and the composite nucleotide sequence of El 70 as contained in 1-1, the Race-product (above), and EP-1 is shown in FIGURES 11 A-l IC.
  • FIGURES 15A-15F depicts the entire ⁇ 3 EpA transcript.
  • the adenine residue presented as nucleotide 1 (FIGURE 15 A) is based on sequence analysis of a 5' RACE product that showed this residue to correspond to the 5' end of the ⁇ 3 EpA transcript.
  • FIGURES 15A-15F corresponds to the ⁇ 3 EpA transcript and encodes a probable signal peptide followed by domains Ilia, II I, and G.
  • FIGURES 15 A-l 5F depict the nucleotide sequence and FIGURES 19 A-l 9R the polypeptide sequence.
  • the initiator methionine (amino acid 1) was designated as such because it is followed by a sequence that likely corresponds to the signal peptide (FIGURE 19A), and it is flanked by a nucleotide sequence that is consistent with the consensus sequence reported for translation initiation of vertebrate mRNAs (#137).
  • the proposed signal peptide is followed by a short protein domain and then by a cysteine-rich domain comprised of multiple EGF repeats with similarity to those found in domain Ilia of the laminin ⁇ l chain (ref. Sasaki et al., 1988).
  • Domain Ilia FIGURES 19B-19C, residues 46-201 of the ⁇ 3 pA translation product contains two complete EGF repeats that show conserved spacing between cysteine residues 1, 2, and 8 and between cysteine residues 5, 6, and 7. This conserved spacing is strictly maintained in the EGF repeats that have been identified in all three laminin chains (Sasaki et al., 1987, 1988; Sasaki and Yamada, 1987).
  • the last EGF repeat of the ⁇ 3 EpA chain diverges from the ⁇ l chain of laminin and is a partial repeat containing 4 cysteine residues that conform with the arrangement described by Sasaki et al., (1988).
  • the sequence identity in domain Ilia between the human ⁇ 3 and ⁇ l (Nissinen et al., 1991) laminin chains was found to be 46%, whereas a comparison of the human ⁇ and ⁇ 2 merosin chains (Ehrig et al., 1990; Vuolteenaho et al., 1994) revealed a slightly higher sequence identity (48%).
  • FIGURES 19A-19R diagram several features of the translated amino acid sequence of ⁇ 3 EpA .
  • the proposed signal peptide (S.P.) is identified in FIGURE 19A.
  • Structural protein domains and the 3 '-untranslated sequence (3' UTR) are shown in FIGURES 19B-C, 19D-H, 19I-J, 19K-L, 19M-N, 19O-P, and 19Q-R. Cysteine residues are shown as "Cys".
  • the first cysteine residue of the EGF-repeats are marked with a black triangle and the first two cysteine residues in domains I II are marked with a black diamond.
  • Potential glycosylation sites are boxed, and consensus sequences for adhesion recognition sites are shaded.
  • the ⁇ 3 chain of epiligrin contains a series of heptad repeats (FIGURES 19D-H, residues 202-793) which are characteristic of proteins that form an ⁇ -helical coiled coil structure (ref, Cohen and Parry, 1986; Beck et al., 1990).
  • the presence of an ⁇ -helical domain in the ⁇ 3 chain of epiligrin suggests that it is capable of interacting with laminin chains to form a heterodimeric coiled structure (Hunter, 1990).
  • cysteine residues 202 and 205 could stabilize epiligrin heterotrimers by forming intermolecular disulfide bonds as was proposed previously for EGS laminin (Sasaki et al., 1988). These cysteine residues are also present in the ⁇ 3 chain of laminin-5 (Blk; Gerecke et al., 1994) and ⁇ 2 chain (B2t; Kallunki et al., 1992) suggesting that they may be important for stabilizing the isoforms of laminin that are found in skin.
  • the ⁇ 3 chain of epiligrin contains two potential cell adhesion recognition sequences, RGD (FIGURE 19G, residues 658-660) and LDV (FIGURE 19D, residues 313-314), within the ⁇ -helical domain. These sequences were shown to mediate cell adhesion to fibronectin through integrin ⁇ 5 ⁇ l (Wayner et al., 1989; Guan and Hunes, 1989; Komoriya et al., 1991), respectively.
  • a sequence SKVAV (FIGURE 19H, residues 765-769) was also identified at the carboxyl-terminal end of the ⁇ -helical domain which is homologous in sequence and position to a regulatory sequence in the laminin ⁇ l chain (SIKVAV) that mediated cell growth (Kubota et al., 1992) and metastases (Kanemoto et al., 1990).
  • Amino acids 794 through 1713 (FIGURE 191), comprise the G domain of the ⁇ 3 EpA transcript.
  • the G domain has shown to have an internal repeating structure composed of five tandem repeats with approximately 180 amino acids per repeat (Sasaki et al., 1988). Data compiled from a multiple - 64 -
  • a BLAST protein search (Altschul et al., 1990) revealed that the G4 and G5 subdomains of epiligrin are also related to the heparin sulfate proteoglycan core protein (Noonan et al., 1991) and the neurexin family of proteins (Ushkaryov et al., 1992).
  • El 70 glycoprotein deduced from the nucleotide sequence of FIGURE 15 has overall similarity to the structure found in laminin A chain, but (as shown in the Examples, above) antibodies to laminin fail to bind to epiligrin, PlEl fails to bind to laminin, polyclonal antibodies raised to epiligrin fail to bind to laminin, and 32 P-radiolabeled full length Ep-1 used as probe in Northern blots fails to detect any species of mRNA other than 5kb and 6kb transcripts that are appropriate in size for El 70 glycoprotein.
  • El 70 polypeptide, antibodies to El 70, and El 70 mRNA are markers useful in identifying abnormal patterns of epiligrin expression and thus monitoring basal cell differentiation in the epithelium.
  • epiligrin is not expressed in basal cell carcinoma and skin diseases such as junctional epidermolysis bullosa gravis (as disclosed in the Examples, above; and also independently confirmed, 114).
  • the amino acid sequences shown in FIGURES 19A-R provide one skilled in the art with the information needed to create synthetic polypeptides whose - 65 -
  • Synthetic polypeptides of the invention comprise at least 5 amino acids, corresponding to at least 15 consecutive nucleic acids from the squence of FIGURE 15. Methods are well known to those skilled in the art for synthesizing polypeptides having a predetermined amino acid sequence.
  • Ep-1 600 bp was cloned in both orientations into a Bluescript expression vector (Stratagene, La Jolla, CA) so that sense and anti-sense cRNA probes could be synthesized using the T7 promoter.
  • cRNA probes were labeled with digoxigenin-11-UTP (Boehringer Mannheim Biochemicals, St. Louis, MO) and then cleaved by alkaline hydrolysis to an average size of 100-150 nucleotides. Probes were incubated with tissue slides containing formaldehyde-fixed cryostat sections of punch biopsies from wounded epidermis (including adjacent non- wounded epidermis), and processed for immunohistochemical staining.
  • Bound probes were visualized using affinity purified anti-digoxigenin antibody coupled to alkaline phosphatase and a colorimetric assay was used to localize bound probe within the tissue. (Ep-1 sense probes were used as a control and they showed no signal above background on the indicated tissues.)
  • Ep-1 sense probes were used as a control and they showed no signal above background on the indicated tissues.
  • the highest observed steady state of ⁇ 3 Ep mRNA observed by the inventors has been in subconfluent keratinocytes that are still actively migrating. Taking this observation one step further, the inventors used the above-described methods to examine the expression of this mRNA in human skin harvested 55 h after the introduction of a wound. The wounding of skin results in the transition of keratinocytes from a sedentary to a migratory state (Grinnell, 1992).
  • FIGURE 20A the antisense cRNA probe localized ⁇ 3 Ep mRNA to keratinocytes in the wound site, but not to keratinocytes flanking the wound site (FIGURE 20B).
  • the sense probe (FIGURES 20C-20D) was negative as expected.
  • An antisense probe for keratin 14 (FIGURES 20E-20F) showed that keratin 14 mRNA is detectable in the basal cells of both wounded and normal epidermis.
  • FIGURES 20E and 20H illustrate the localization of epiligrin and integrin ⁇ 3 ⁇ ! in the wound site.
  • FIGURE 20H Immunoperoxidase staining using anti-epiligrin (mAb PlEl, FIGURE 20G) and anti- ⁇ 3 antibodies (mAb P1F2, FIGURE 20H) shows that epiligrin protein is present in the newly synthesized basement membrane (BM) (FIGURE 20H, arrows), and integrin relocalizes from the apical and lateral surfaces to the basal surface (FIGURE 20H, arrows).
  • the epidermis and dermis are marked as E and D, respectively.
  • the localization of the ⁇ 3 Ep transcript in the wound site is correlated with the reorganization of integrin ⁇ 3 ⁇ j from the apical and lateral to the basal surface of the keratinocytes (FIGURE 20H) in contact with the newly synthesized epiligrin present in the basement membrane (FIGURE 20G).
  • the reorganization of integrin ⁇ 3 ⁇ j suggests that keratinocytes may utilize integrin ⁇ 3 ⁇ j in cell/substrate adhesion mediated by epiligrin for motility during wound repair.
  • El 70 epithelial ligand glycoprotein in epidermal tissues distinguishes between regenerating epithelial tissues (where expression is high) and non-regenerative epithelial tissues or malignant tissues (where expression is low).
  • Many situations exist in histopathological examination where it would be most helpful to be able to distinguish between a normal regenerative event in an epithelial tissue (e.g., resulting from traumatic injury or infection) and an abnormality that might be a neoplastic or preneoplastic event.
  • Examining E170 epiligrin glycoprotein expression e.g., using immunochemical techniques or in situ hybridization provides a way in which normal regenerative events can be distinguished from premalignant and malignant events.
  • cDNA inserts were amplified using Taq Polymerase (Perkin-Elmer Cetus), purified on an agarose gel, and directly sequenced using either the Taq DyeDeoxyTM Terminator Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, CA) or a modified version of Sanger et al. (120) with Sequenase (USB, Cleveland, OH). Deduced amino acid sequence was compared to the available data bases through searches with both Blast/ncbi.nlm.nih.gov. and Blitz/EMBL- heidelberg.de using the homology matrix described by Henikoff and Henikoff (115).
  • RNA from each of these cell types was isolated by the guanidine isothyocyanate method, run on a 1% denaturing agarose gel, and transferred to nitrocellulose (118).
  • Ep-1 cDNA (0.6kb) was radiolabeled with 32 P by random priming, hybridized overnight at 42°C in 50% formamide solution (118), and washed at 54°C in 0. IX SSC/0.1% SDS.
  • Ep-1 600 bp was cloned in both orientations into a Bluescript expression vector (Stratagene, La Jolla, CA) so that sense and anti ⁇ sense cRNA probes could be synthesized using the T7 promoter.
  • cRNA probes were synthesized using T7 RNA polymerase and labeled with digoxigenin-11-UTP (Boehringer Mannheim Biochemicals, St. Louis, MO). Full length probes were then - 67 -
  • Probes were incubated with slides containing formaldehyde-fixed cryostat sections overnight at 50°C in hybridization buffer (50% formamide/5X SSC/lmg/ml yeast tRNA/lOO ⁇ g/ml heparin/lX Denhardts 0.1% Tween 20/0.1% CHAPS). Slides were treated for 30 minutes at 37°C with 20 ug/ml of RNase A, washed twice at room temperature with 2X SSC for 30 minutes, and washed twice at 50°C in 0.1X SSC for 30 minutes. Next, slides were equilibrated in PBS and processed for immunohistochemical staining. An affinity purified anti-digoxigenin antibody coupled to alkaline phosphatase (Boehringer Mannheim Biochemicals) followed by a colorimetric assay was used to localize bound probe within the tissue.
  • hybridization buffer 50% formamide/5X SSC/lmg/ml yeast tRNA/lOO ⁇ g/ml heparin/lX Denhard
  • Epidermal wound healing is characterized by migration of the regenerating epithelium in a migratory tongue across a bed of newly formed granulation tissue.
  • epiligrin is deposited in the basement membrane at the leading edge of the migratory tongue of epithelium 24, 48, and 72 hours after wounding in normal human volunteers.
  • epiligrin and ⁇ 3 ⁇ were localized to the basal surface of the cells in the migrating epithelial tongue.
  • In situ hybridization with antisense cRNA probes specific for El 70 identified elevated E170 mRNA levels in the epithelial cells in the wound site (see, e.g., FIGURE 20 A).
  • decreasing cell density and restoring epithelial cell contact with a material containing epiligrin may prove useful for therapeutic intervention in epithelial dysjunction diseases such as acquired and genetic epidermal disorders, ulcerative colitis, Crohns disease, and cholera.
  • epithelial dysjunction diseases such as acquired and genetic epidermal disorders, ulcerative colitis, Crohns disease, and cholera.
  • decreased expression of epiligrin in transformed cells contrasts markedly with the observed expression in wound sites, and that pathological metastasis of malignant cells into tissue sites may be distinguished from normal regeneration by examining the level of epiligrin expression in the cells (e.g., using in situ hybridization or immunocytochemical methods). In the latter case normal regenerating cells have high levels of epiligrin expression and tumor cells have low levels.
  • fibronectin is expressed in wounded epidermis, and no in normal regenerating epidermis, it is most likely that ⁇ 5 ⁇ j -fibronectin interactions inhibit differentiation during wound repair, while the ⁇ 3 ⁇ j -epiligrin interaction plays a role in normal epithelial regeneration by preventing basal stem cells from differentiating (i.e., while in contact with epithelial ligand) and by stimulating terminal differentiation in progeny cells (i.e., when released from contact with epiligrin).
  • TGF ⁇ and TGF ⁇ dermal growth factors
  • HFK cells were established in cell culture and grown to approximately 75% confluency so that expression was down-regulated and the cells assumed a resting state.
  • dermal growth factors 30ng/ml of TGF ⁇ , or 20ng/ml of TGF ⁇ , were added to separate cultures and the cells were cultured for an additional 24 hrs.
  • a control culture was included in which the medium was changed but no growth factors were added.
  • epithelial ligand expression was measured by Northern blotting for El 70 mRNA. The results obtained showed about a 2-fold increase in epiligrin expression with either TGF ⁇ or TGF ⁇ over the level of expression seen in the control culture.
  • Fluorescent in situ hybridization was performed using the PI genomic clone
  • DMPC-HFF#1-1034F10 which contained coding sequences for the human LamA3 gene.
  • the human laminin ⁇ chain (LamAl) was also found on chromosome 18 but mapped to 18pl 1.3 (Nagayoshi et al., 1989), a locus distinct from that of LamA3.
  • the probe for the chromosomal localization was prepared by nick-translation with biotin label and was hybridized to normal human metaphase cells as described previously (NanDevanter and Yirdaw, 1993). Hybridized probe was detected with Texas red avidin and chromosomes were identified by fluorescence R-banding with - 70 -
  • chromomycin A3/distamycin A (VanDevanter et al., 1994). Texas red and chromoymcin A3 signals were exited independently using a Nikon Optiphot-2 epifluorescence microscope and captured as 8-bit black and white digital TIFF images using an intensified CCD camera and a SAMBA 4000 image analysis system (Imaging Products International, Chantilly, VA). Chromomycin A3 intensities were linearly reduced to generate gray countersignals, digitally added to Texas red signals collected from the same fields, and printed as black and white images on a Phaser IISDX dye sublimation printer (Techtronix, Beaverton, OR).
  • Trvpsin-treated HFK-ECM "Epiligrin-ECM*"
  • differential extraction with Triton X-100 and urea may be used to prepare a tissue culture surface coated with substantially purified epiligrin complex.
  • the yield of epiligrin coated matrices is relatively low, and so studies were conducted to determine whether detaching HFK from ECM with trypsin (i.e., as cells were passaged) left an epiligrin that might be suitable for use in adhesion assays.
  • trypsin i.e., as cells were passaged
  • Epiligrin-ECM 1 was an adhesive substrate in adhesion assay, and surprisingly, the substrate adhered cells more rapidly (i.e., more than >90% of HFK were attached in less than 5 minutes) and tightly (i.e., not easily removed with strong shaking) than epiligrin substrates.
  • 4°C inhibits energy metabolism required for actin rearrangement in the cytoskeleton and test cells that express ⁇ 3 ⁇ , but not ⁇ 6 ⁇ 4 (e.g., HFF, HFM, and HT1080), adhere to epiligrin-ECM 1 and collagen at 37°C but not to any ligand at 4°C.
  • FAK focal adhesion kinase
  • ⁇ 6 ⁇ 4 mediated adhesion, the attachment of HFK occurred without spreading at 4°C, did not require energy metabolism and actin cytoskeletal rearrangement, and was inhibited by anti- ⁇ 6 alone. Thus, ⁇ 6 ⁇ 4 was capable of mediating attachment of HFK independent of ⁇ 3 ⁇ ! .
  • Hemidesmosome adhesion structures assembled on epiligrin at 4°C contained ⁇ 6 , ⁇ 4 and Bullous pemphigoid antigen I (BPAI) as a Triton X-100-insoluble complex.
  • BPAI Bullous pemphigoid antigen I
  • ⁇ 6 ⁇ 4 mediated adhesion of HFK to epiligrin was distinguished from that mediated by ⁇ j integrins in the following manner: (1) it was more rapid; (2) it occurred at 4°C; (3) it did not result in cell spreading; (4) it did not induce phosphorylation of FAK; (5) it did not induce - 72 -
  • Triton X-100 detergent-insoluble adhesion structures i.e., the type involved when both ⁇ 3 ⁇ ] and ⁇ ⁇ 4 mediate adhesion, below.
  • HFK failed to adhere to fibronectin and EHS laminin, indicating that under these conditions the assay is specific for ⁇ ⁇ 4 receptors, and that the ⁇ ⁇ 4 receptors form stable adhesion contact only with epiligrin at 4°C.
  • HFK attachment and motility at 37°C required both ⁇ 3 ⁇ j and ⁇ 6 ⁇ 4 ; monoclonal antibodies to ⁇ 3 blocked spreading but not attachment; and, anti- ⁇ 6 blocked neither spreading nor attachment, but the combination of anti- ⁇ 3 and anti- ⁇ g successfully blocked both spreading and attachment.
  • both ⁇ 3 ⁇ ! and ⁇ g ⁇ 4 appeared to mediate their effects through epiligrin, since anti-epiligrin monoclonal antibodies blocked adhesion through both receptors.
  • epithelial cells in contrast to many other cells that express only ⁇ 3 ⁇ receptors, control motility on epiligrin (i.e., through ⁇ 3 ⁇ and transmembrane signaling) and stable anchorage on epiligrin (i.e., through ⁇ g ⁇ ) by differential expression of these two integrin receptors in hemidesmosomes.
  • suspended cells (1) that express ⁇ 3 ⁇ l attach and spread on epiligrin via 03 ⁇ t at 37°C (3), but not at 4°C (2) or in the presence of anti- 3 or anti-epiligrin. Attachment and spreading involves formation of FAs and phosphorylation of FAK (FAK ⁇ P-FAK).
  • suspended cells (1) that express ⁇ 3 ⁇ j and ⁇ ⁇ 4 utilize ⁇ ⁇ for motility (attachment, spreading and migration) at 37°C (3) and ⁇ 6 ⁇ for stable anchorage without spreading at 4°C (2) and 37°C (3).
  • Adhesion at either temperature is blocked with anti-epiligrin monclonal antibodies.
  • HFKs anchored at 4°C via ⁇ ⁇ (2) will spread via ⁇ 3 ⁇ i when warmed to 37°C (3). This spreading is blocked with anti- ⁇ 3 ⁇ Anchorage via ⁇ g ⁇ 4 at 4°C does not require phosphorylation of FAK while spreading via ⁇ 3 ⁇ induces tyrosine phosphorylation of FAK (3).
  • epiligrin ligand protein amino acid sequence domain involved in integrin ⁇ 3 ⁇ j receptor binding to epiligrin-ECM 1 were different than those involved in ⁇ 3 ⁇ j or ⁇ g ⁇ 4 binding to non-trypsin-treated epiligrin.
  • monoclonal antibodies were selected that inhibited binding of cells to epiligrin-ECM 1 .
  • HFK were grown on cellophane sheets, and the HFK-ECM deposited on the sheets by the cells was used as an antigen for immunization of BALB/c mice.
  • Hybridoma clones were selected based on immunoreactivity in a solid phase ELISA with epiligrin (i.e., solid phase antigen). Three monoclonal antibodies from the resulting library of clones were selected for additional testing to for their ability to immunoprecipitate 35 S-methionine biosynthetically-radiolabeled HFK-ECM proteins (i.e., in the presence of Staphlococcal Protein A). Anti-epiligrin monoclonal antibody PlEl was used as a control. Immunoprecipitated proteins were evaluated on SDS-PAGE under reducing (i.e., using 2-mercaptoethanol) or non-reducing conditions.
  • monoclonal antibodies PlEl, C2-9, G3-3, and B4-6 specifically immunoprecipitate a disulfide-bonded high molecular mass complex from HFK-ECM (non-reduced) that contained only E170, E135, and E100/145 (i.e., following reduction).
  • Monoclonal antibodies C2-9, G3-3, and B4-6 were not reactive with human or mouse laminin, tenascin, thrombospondin, collagen types I, IV or VII, fibronectin, human placental basement membrane proteins, or BSA.
  • monoclonal antibody C2-9 identifies an amino acid sequence domain present in epiligrin-ECM 1 , and epiligrin, that is a functional requisite for binding by at least ⁇ 3 ⁇ ⁇ integrin receptors. - 74 -
  • HFK ⁇ g ⁇ 4 _receptor bearing test cells
  • the 4°C assay provides a simple and definitive test for identifying functional epiligrin ligand in normal and diseased epithelial tissues including e.g. epithelia in skin, gastric mucosa, lung, organs, endocrine glands, and the like.
  • Suitable ⁇ g ⁇ 4 _receptor bearing test cells that have been used in the assay include HFK and human papilloma virus transformed immortalized HFKs (i.e., E1L-8; Kaur et al., 1992).
  • ⁇ g ⁇ 4 _receptor-negative control cell As a negative control, (e.g., for maintaining a constant 4°C temperature during the assay), it may be desirable to use an ⁇ g ⁇ 4 _receptor-negative control cell on a parallel cryostat section of tissue. Suitable negative control cells that have been used include HFFs, melanocytes, HT1080 cells, and ⁇ 3 -K562 cells.
  • ⁇ g ⁇ 4 _receptor bearing test cells were incubated with the cryostat tissue section at 37°C (i.e., to allow both the ⁇ g ⁇ and ⁇ 3 ⁇ ! .receptor).
  • Epidermal basal stem cells in patients with Junctional Epidermolysis Bullosus (JEB) gravis are defective in epiligrin synthesis (Domloge-Hultsch et al.) and the patients exhibit a lethal blistering disease. It is thought highly likely that patients who synthesize functionally defective epiligrin (e.g., mutant epiligrin) will present clinically in a wide variety of seemingly unrelated skin diseases, which upon testing according to the assay of the invention will share a common underlying epiligrin-related etiology.
  • Skin biopsy samples collected from patients exhibiting less severe forms of blistering and skin diseases, e.g., non-lethal JEB, psoriasis, and the like, may be processed as follows.
  • the 4°C epiligrin-rosetting assay (developed above with FITC-labeled HFK cells), is used to identify locations in the patient tissue where functional epiligrin, (i.e., capable of binding an ⁇ g ⁇ .receptor positive test cell), is resident. It may be convenient to use a tissue sample of similar type from a normal individual as a positive control in the assay. Patients lacking a normal functional epiligrin are identified by - 76 -
  • the biosynthesis of a functionally defective epiligrin in a patient may be identified by fewer test cells binding, or a decreased rate of binding, at histological locations that would normally bind large numbers of test cells (i.e., in a normal tissue). Decreased numbers of test cells may be observed binding in the basement membrane zone in a skin biopsy sample within a 5 minute time interval.
  • patients having autoimmune anti-epiligrin antibodies may show decreased binding of the test cells in tissues as a result of auto-antibodies blocking epiligrin ligand binding sites for the ⁇ g ⁇ 4 _receptors on the test cells.
  • qualitative alterations in the distribution of epiligrin can be identified according to the assay, and these may result from defects in FA HD, or SAC formation in the tissues of a patient, e.g., an actin cytoskeletal defect (or related energy metabolism defect) that prevents epiligrin from co-localizing with ⁇ g, ⁇ , and BPAI in HDs.
  • ORGANISM Homo sapiens
  • xi SEQUENCE DESCRIPTION: SEQ ID N ⁇ :l: TGGCATTGAG GATGTTCTCG TAGG 24
  • ORGANISM Homo sapiens -87-
  • ORGANISM Homo sapiens
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATATGTCGAC AAGTCACCTG AAGGCACG 28
  • ORGANISM Homo sapiens
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:4: TGGACGTGAG ACTTGACCAG 20
  • ORGANISM Homo sapiens
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 5: ATGTCATCCG GAATGTGCAC 20
  • ORGANISM Homo sapiens -88-
  • MOLECULE TYPE cDNA to mRNA
  • MOLECULE TYPE cDNA to mRNA
  • A PCR primer; see TABLE 1
  • HYPOTHETICAL NO
  • ORGANISM Homo sapiens -89-
  • MOLECULE TYPE cDNA to mRNA
  • A PCR primer; see TABLE 1
  • HYPOTHETICAL NO
  • ORIGINAL SOURCE
  • ORGANISM Homo sapiens
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 11: TGGACGTGCG ACTTGACCAG 20
  • MOLECULE TYPE cDNA to mRNA
  • A PCR primer; see TABLE 1
  • HYPOTHETICAL NO
  • ORIGINAL SOURCE
  • MOLECULE TYPE cDNA to mRNA
  • A PCR primer; see TABLE 1
  • HYPOTHETICAL NO
  • ORIGINAL SOURCE
  • ORGANISM Homo sapiens -90-
  • MOLECULE TYPE cDNA to mRNA
  • A PCR primer; see TABLE 1
  • HYPOTHETICAL NO
  • ORIGINAL SOURCE
  • MOLECULE TYPE cDNA to mRNA
  • A PCR primer; see TABLE 1
  • HYPOTHETICAL NO
  • ORIGINAL SOURCE
  • ORGANISM Homo sapiens
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 16: TCTCCGAGAT GGTCTTCATG 20
  • MOLECULE TYPE cDNA to mRNA
  • A PCR primer; see TABLE 1
  • HYPOTHETICAL NO
  • ORIGINAL SOURCE
  • ORGANISM Homo sapiens -91-
  • MOLECULE TYPE cDNA to mRNA
  • A PCR primer; see TABLE 1
  • HYPOTHETICAL NO
  • ORIGINAL SOURCE
  • ORGANISM Homo sapiens
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 19: AGAGACCATT CGATTCAGAT 20
  • MOLECULE TYPE cDNA to mRNA
  • A PCR primer; see TABLE 1
  • HYPOTHETICAL NO
  • ORIGINAL SOURCE
  • ORGANISM Homo sapiens
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:20: AGCTTCTGAG AAATAGCAAA 20
  • ORGANISM Homo sapiens SEQUENCE DESCRIPTION: SEQ ID N0:21: CGGGGATGCC TCCAGCAGTG ACCCGGTCAG CCTGCAGCAT GGGATGGCTG TGGATCTTTG 60
  • ORGANISM Homo sapiens
  • SEQUENCE DESCRIPTION SEQ ID NO:22: CGGGGATGCC TCCAGCAGTG ACCCGGTCAG CCTGCAGCAT GGGATGGCTG TGGATCTTTG 60
  • CTTTCCAGAT CTGCTGGCAA AACATCCCTT GTGGAGGAGG CAGAAAAGCA CGCGCGGTCC 1620
  • GCT GGC AAA ACA TCC CTT GTG GAG GAG GCA GAA AAG CAC GCG CGG TCC 1642 Ala Gly Lys Thr Ser Leu Val Glu Glu Ala Glu Lys His Ala Arg Ser 515 520 525
  • ATC CAG ACA GAT GTG GAA AGA ATT AAG GAC ACC TAT GGG AGG ACA CAG 2170 lie Gin Thr Asp Val Glu Arg lie Lys Asp Thr Tyr Gly Arg Thr Gin 690 695 700

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Abstract

Séquences d'acide nucléique codant un ligand épithelial E170 et capable de s'hybrider dans des conditions strictes aux séquences nucléotidiques dérivées de clones d'ADNc illustrés dans la figure. L'invention se rapporte également à des vecteurs contenant les séquences d'acide nucléique, et à des cellules transformées au moyen de ces vecteurs. Des procédés de purification et d'utilisation d'épiligrine, un complexe de glycoprotéines épithéliales, et de ses glycoprotéines constitutives sont décrits, ainsi que des procédés de développement d'anticorps dirigés contre les constituants de ce complexe. Des méthodes de dosage destinées à l'identification d'épiligrine fonctionnelle dans des tissus sont également décrites.
EP94929787A 1993-09-02 1994-09-02 Epiligrine, ligand epithelial pour les integrines Withdrawn EP0716659A4 (fr)

Priority Applications (1)

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EP07075129A EP1808439A3 (fr) 1993-09-02 1994-09-02 Épiligrine, ligand épithélial pour les intégrines

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US115918 1987-11-02
US11591893A 1993-09-02 1993-09-02
PCT/US1994/010261 WO1995006660A1 (fr) 1993-09-02 1994-09-02 Epiligrine, ligand epithelial pour les integrines

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US5422264A (en) * 1993-11-12 1995-06-06 Desmos, Inc. Soluble factor stimulation of attachment and hemidesmosome assembly in epithelial cells
US5681587A (en) * 1995-10-06 1997-10-28 Desmos, Inc. Growth of adult pancreatic islet cells
US5672361A (en) * 1996-03-29 1997-09-30 Desmos, Inc. Laminin 5 for growth of pancreatic islet cells
EP0889743A1 (fr) * 1996-03-29 1999-01-13 Desmos, Inc. Fixation de cellules sur des dispositifs trans-epitheliaux recouverts de laminine 5
US5770448A (en) * 1996-03-29 1998-06-23 Northwestern University Human epithelial cell matrix and uses therefor
US5760179A (en) * 1996-06-10 1998-06-02 Desmos, Inc. Purification of soluble laminin 5
AU1207999A (en) * 1997-10-30 1999-05-24 Brigham And Women's Hospital Control of lymphocyte localization by leep-cam activity
US6294356B1 (en) 1998-01-16 2001-09-25 Northwestern University Methods and materials for making and using laminin-5
CA2348743A1 (fr) * 1998-09-01 2000-03-09 Desmos, Inc. Utilisation de laminine 5 dans le traitement de malformations osseuses
JP4798560B2 (ja) * 2003-06-04 2011-10-19 よこはまティーエルオー株式会社 ラミニン５ｂ

Non-Patent Citations (7)

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Title
BAUDOIN C. ET AL.: "The 150 kDa sub-unit of the basement membrane component nicein is a truncated isoform of laminin chain A." JOURNAL OF INVESTIGATIVE DERMATOLOGY, vol. 102, no. 4, April 1994, page 549 XP002068693 *
BOLOTIN,D. AND FUCHS,E.: "More than skin deep" NATURE, vol. 421, 6 February 2003 (2003-02-06), pages 594-595, *
DAJEE,M. ET AL.: "NF-kB blockade and oncogenic Ras trigger invasive human epidermal neoplasia" NATURE, vol. 421, 6 February 2003 (2003-02-06), pages 639-642, *
DATABASE EMBL Accession L20478, 5 May 1994 ABERDAM D.: "Mus musculus nicein chain A (NicA) mRNA sequence." XP002068571 *
ORTIZ-URDA ET AL.: "Type VII Collagen is required for Ras-driven human epidermal tumorigenesis" SCIENCE, vol. 307, 18 March 2005 (2005-03-18), pages 1773-1776, *
RYAN M.C. ET AL.: "Cloning of the LamA3 gene encoding the alpha-3 chain of the adhesive ligand epiligrin." THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 269, no. 36, 9 September 1994, pages 22779-22787, XP002068570 *
See also references of WO9506660A1 *

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EP0716659A4 (fr) 1998-09-02
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AU7872894A (en) 1995-03-22
CA2170777A1 (fr) 1995-03-09
EP1808439A3 (fr) 2008-03-12

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