WO2011050342A2 - Conception rationnelle d'un polymère de laminine-111 biocompatible et utilisations correspondantes - Google Patents

Conception rationnelle d'un polymère de laminine-111 biocompatible et utilisations correspondantes Download PDF

Info

Publication number
WO2011050342A2
WO2011050342A2 PCT/US2010/053877 US2010053877W WO2011050342A2 WO 2011050342 A2 WO2011050342 A2 WO 2011050342A2 US 2010053877 W US2010053877 W US 2010053877W WO 2011050342 A2 WO2011050342 A2 WO 2011050342A2
Authority
WO
WIPO (PCT)
Prior art keywords
poly
polymer
synthetic
basement membrane
peptides
Prior art date
Application number
PCT/US2010/053877
Other languages
English (en)
Other versions
WO2011050342A3 (fr
Inventor
Mark A. La Barge
Cyrus M. Ghajar
Kevin E. Healy
Mina J. Bissell
Original Assignee
The Regents Of The University Of California
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Publication of WO2011050342A2 publication Critical patent/WO2011050342A2/fr
Publication of WO2011050342A3 publication Critical patent/WO2011050342A3/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds

Definitions

  • the invention relates to synthetic polymers and uses in mimicking functional synthetic extracellular matrix components.
  • Laminins are important structural and signaling components of basement membrane, a specialized form of extracellular matrix (ECM). Laminins serve as mediators of organ architecture, morphogenesis, and tissue-specific gene expression. Mutations in genes encoding individual laminin chains or their receptors are either embryonic lethal or cause serious defects in adult tissues including muscular dystrophies and blistering disorders [1]. In mammary gland, the presence of laminin- 111 (Ln-1) in an appropriate context is required to confer structure and function to mammary epithelial cells (MECs) within the gland. Not only cellular messaging to promote tissue-specific expression of milk proteins (e.g., ⁇ -casein) [4- 11].
  • MECs mammary epithelial cells
  • Ln-1 facilitates these functions is crucial from developmental and homeostasis standpoints, and is of further interest because losses of polarity and of tissue- specificity are hallmarks of tumorigenesis [12].
  • a particular gap in our understanding is that the specific domains of Ln-1 that confer tissue-specific behaviors to MECs are unknown.
  • Ln-1 is a trimeric glycoprotein comprised of ⁇ -, ⁇ , and ⁇ - chains, and is host to a large number of putative receptor binding sites [13].
  • Direct antagonism of Ln-1 receptors integrin ⁇ 6 ⁇ 1, ⁇ 6 ⁇ 4, [6, 8], and the non-integrin cell surface receptor dystroglycan (DG) [3] has demonstrated that these receptors mediate Ln-1 -induced functions in MECs.
  • DG non-integrin cell surface receptor dystroglycan
  • MECs were provided with a fragment of Ln-1 derived from the E3 domain of the al chain, which was known to inhibit milk protein synthesis in the presence of Ln-1, it was not sufficient to confer this tissue-specific function to MECs on its own [10]. This result suggested that simple ligation of integrin and/or non-integrin receptors by laminin-derived moieties may be sufficient to promote cell adhesion, but more is required to induce differentiation.
  • the present invention provides for synthetic cellular basement membrane comprising a polymer composition having a peptide conjugated to the polymer, whereby the polymer composition is functional mimetic of Laminin-111.
  • the polymer composition having a Laminin-111 peptide conjugated to the polymer can be a peptide comprising at least 60% identity to any of SEQ ID NOS: 1-9.
  • the peptides AGIO SEQ ID NO:3
  • AG32 SEQ ID NO:4
  • AG73 SEQ ID NO:5
  • P3 SEQ ID NO:8
  • the synthetic cellular basement membrane polymer is a biopolymer hyaluronic acid (HyA) mixed with a copolymer of N-Isopropylacrylamide (NIPAAm) and poly(ethylene glycol).
  • HyA biopolymer hyaluronic acid
  • NIPAAm N-Isopropylacrylamide
  • poly(ethylene glycol) poly(ethylene glycol).
  • the invention also provides a synthetic basement membrane that can be used for tissue and/or organ regeneration, for support of alveogenesis and the generation of breast tissue stability in polarized epithelia, including breast tissue. Methods for such uses are also provided by the present invention.
  • the invention also provides for a functional mimetic of laminin-111 generated by conjugation of inhibitory peptides to a biocompatible linear polymer.
  • the invention further describes a method of making a functional mimetic of laminin- 111 comprising the steps of: (a) providing a biocompatible linear polymer, (b) providing a Laminin-111 peptide conjugated to a second polymer; (c) mixing said peptide -polymer with the biocompatible linear polymer.
  • the Laminin-111 peptide can be peptides AGIO (SEQ ID NO:3), AG32 (SEQ ID NO:4) , or AG73 (SEQ ID NO:5) and P3 (SEQ ID NO:8) present in the polymer in equal concentrations
  • the biocompatible linear polymer can be a polymer or copolymer derived from polydioxane, polyphosphazene, polysulphone resins, poly(acrylic acid), poly(acrylic acid) butyl ester, poly(ethylene glycol), poly(propylene), polyurethane resins, poly(methacrylic acid), poly(methacrylic acid)-methyl ester, poly(methacrylic acid)-n butyl ester, poly(methacrylic acid)-t butyl ester, polytetrafluoroethylene, polyperfluoropropylene, poly N- vinyl carbazole, poly(methyl isopropenyl ketone), poly alphamethyl styrene, polyvinylacetate, poly(oxymethylene), poly(ethylene-co-vinyl acetate), a polyurethane, a poly(vinyl alcohol), and polyethylene terephthalate; ethylene vinyl alcohol copolymer (commonly known by the generic name EVOH or by
  • the second polymer that the peptide is conjugated can comprise multiple subunits selected from hyaluronic acid, acrylic acid, ethylene glycol, vinyl, propylene, methyl methacrylate, methacrylic acid, acrylarnide, hydroxyethyl methacrylate, tetrafluoroethylene, oxymethylene, a sugar (e.g., glucose, mannitol, maltose, arabinose, etc.), taurine, betaine, modified celluloses, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, modified starches, hydrophobically modified starch, hydroxyethyl starch, hydroxypropyl starch, amylose, amylopectin, oxidized starch, an amino acid, or copolymers of any of the foregoing.
  • the second polymer is hyaluronic acid or
  • Figure 1 shows Determinants of Functional Mammary Epithelial Cell Differentiation as previously found by others.
  • Figure 2 shows images which previously show that myoepithelial cells use Ln-1 as a tool to organize luminal epithelium.
  • FIG. 3 Putative receptors are labeled showing peptides derived from Laminin-111 to block interaction with cells at specific sites and a table of the peptides.
  • Ln-1 laminin-111
  • FIG.3(B) A table shows the compositions of the 9 different combinations of P3-, AGIO-, AG32, and AG73-peptide conjugated HyA polymers that were tested for their ability to stimulate Casein promoter activity
  • Figure 4 shows a schematic of two main rationales for experimental design to study the effect of disrupting MEC/Ln-1 interactions on acinar morphogenesis and polarity : (1) Peptide-mediated inhibition of interaction with Ln-1 domains or (2) Promotion of tissue- specific behavior with Ln-1 derived peptide(s).
  • the prior art had previously conducted morphogenesis/structure, ⁇ -casein expression and branching morphogenesis. Our goal was to study the promotion of tissue-specific behavior with Ln-1 derived peptide(s) to create a designer microenvironment.
  • Figure 5 shows distinct regions of laminin-111 are critical for establishing apical polarity in mammary epithelial cells.
  • A Composite images of 30 acini are shown as heat maps to observe changes in distributions of the basal polarity marker a6 integrin subunit and the apical polarity marker ZO-1 when the acini were cultured in 3D IrECM in the presence of Ln-1 peptides. Heat maps of nuclear distributions are shown in hues of blue.
  • B shows confocal microscope methods for imaging cells to determine polarity.
  • Figure 6 Casein promoter activity requires signaling input from specific domains of Laminin-111.
  • A Representative images of CFP expression driven by the Casein promoter in engineered EpH4 MECs in response to IrECM drip and the indicated peptides. Hoechst 33342 labeled nuclei are false-colored red to facilitate visualization. Scale bar represents 100 ⁇ . Inset shows no peptide control.
  • B A bar graph shows mean CFP expression per cell, generated from nine lOx magnification microscope fields per condition. Error bars represent S.E.M.
  • Figure 7 shows salivary glands cultured in Matrigel demonstrate laminin dependence for normal morphogenesis as determined by Hosokawa et al, 1999.
  • Figure 8 Distinct domains of Laminin-111 are required for alveogenesis.
  • A Representative images are shown of primary mouse mammary organoids embedded within IrECM 5 days after stimulation by 9 nM TGFa. Scale Bars represent 100 ⁇ . Inset shows the no peptide control.
  • B A bar graph showing the branching factor for each condition. Numbers underneath the indicated treatment denote the number of organoids analyzed per condition.
  • Figure 9 shows images of cells that show that the linear peptides that function as inhibitors in solution do not serve as agonists in 2D cultures.
  • Figure 10 shows a schematic for making synthetic laminin- like matrices. inhibitory peptides to a biocompatible linear polymer.
  • a bar graph shows mean CFP fluorescence per cell in cas-CFP EpH4 reporter cells. Values were calculated from four lOx magnification microscope fields per condition. Error bars represent standard deviation..
  • B Graph of the factor level effect (m) shows the optimal concentration of each peptide- conjugated polymer and predicts synergistic relationship among the peptides.
  • C Representative high magnification images of cas-CFP EpH4 reporter cells treated with the indicated combinations of synthetic Ln-1 polymers or controls. Bars represent 50 ⁇ .
  • the present invention provides for three-dimensional (3D), laminin-rich extracellular matrix (lrECM)-based organotypic compositions which recapitulate many features of the mammary microenvironment.
  • Specific peptides derived laminin-111 and a6 integrin proteins which were then conjugated to a polymer and employed to functionally test the domains of Ln-1 required to induce a variety of mammary tissue-specific behaviors by murine MECs.
  • these compositions revealed specific regions within the E3 and E8 domains of Ln-1 crucial for maintaining cytostructure, inducing milk protein promoter activity, and facilitating morphogenic behaviors.
  • the present invention provides for methods and compositions for a synthetic basement membrane which promote tissue-specific behavior of epithelial cells.
  • the present invention further provides for methods of designing and making such compositions and compositions useful for tissue and/or organ regeneration or to enhance post-surgical tissue stability in polarized epithelia, including breast tissue, and other more specific uses involving mammary epithelial cells and function.
  • Laminin-111 (also referred to herein as Ln-1 and Laminin-1) is essential in the mammary gland for maintaining structure and function.
  • Laminins a family of extracellular They have been implicated in a wide variety of biological processes including cell adhesion, differentiation, migration, signaling, neurite outgrowth and metastasis.
  • Laminins are composed of 3 non identical chains: laminin alpha, beta and gamma (formerly A, Bl, and B2, respectively) and they form a cruciform structure consisting of 3 short arms, each formed by a different chain, and a long arm composed of all 3 chains.
  • Each laminin chain is a multidomain protein encoded by a distinct gene. Several isoforms of each chain have been described.
  • alpha 1 beta 1 gamma 1 heterotrimer is laminin 1.
  • the biological functions of the different chains and trimer molecules are largely unknown, but some of the chains have been shown to differ with respect to their tissue distribution, presumably reflecting diverse functions in vivo.
  • This gene encodes the beta chain isoform laminin, beta 1.
  • the beta 1 chain has 7 structurally distinct domains which it shares with other beta chain isomers.
  • Laminin, beta 1 is expressed in most tissues that produce basement membranes, and is one of the 3 chains constituting laminin 1 , the first laminin isolated from Engelbreth-Holm-Swarm (EHS) tumor.
  • EHS Engelbreth-Holm-Swarm
  • a sequence in the beta 1 chain that is involved in cell attachment, chemotaxis, and binding to the laminin receptor was identified and shown to have the capacity to inhibit metastasis, (provided by RefSeq in the publicly available GenBank descriptions of Laminin-111).
  • SEQ ID NO: 10 is laminin subunit alpha-1 precursor [Homo sapiens] (GenBank Accession No.: NP 005550.2 GL38788416); SEQ ID NO: 11 is laminin subunit beta-1 precursor [Homo sapiens] (GenGank Accession No. NP 002282.2 GL167614504; and SEQ ID NO: 12 is laminin subunit gamma-1 precursor [Homo sapiens] (GenBank Accession No.: NP 002284.3 GI: 145309326).
  • the sequences of Laminin-111 are provided herein to allow one of skill in the art to use various known methods for determining peptide sequences which may used in the present invention.
  • polypeptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.
  • polypeptide includes fusion sequence, fusions with heterologous and homologous leader sequences, with or without N- terminal methionine residues; immunologically tagged proteins; and the like.
  • polypeptide includes polypeptides comprising one or more of a fatty acid moiety, a lipid moiety, a sugar moiety, and a carhohydrate moiety.
  • polypeptides includes post- translationally modified polypeptides.
  • copolymer describes a polymer which contains more than one type of subunit.
  • the term encompasses polymer which include two, three, four, five, or six types of subunits.
  • Subjects and patients are used interchangeably herein to a member or members of any mammalian or non-mammalian species.
  • Subjects and patients thus include, without limitation, humans, non-human primates, canines, felines, ungulates (e.g., equine, bovine, swine (e.g., pig)), avians, rodents (e.g., rats, mice), and other subjects.
  • Non-human animal models particularly mammals, e.g. a non-human primate, a murine (e.g., a mouse, a rat), lagomorpha, etc. may be used for experimental investigations.
  • Treating" or "treatment” of a condition or disease includes: (1) preventing at least one symptom of the condition, i.e., causing a clinical symptom to not significantly develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its symptoms, or (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.
  • a “therapeutically effective amount” or “efficacious amount” means the amount of a compound that, when administered to a mammal or other subject for treating a disease, is sufficient, in combination with another agent, or alone in one or more doses, to effect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • unit dosage form refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of compounds of the present invention calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • the specifications for the novel unit dosage forms of the present invention depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host. compatible with living cells, e.g., predominantly aqueous conditions of a temperature, pH, salinity, etc. that are compatible with living cells.
  • a “pharmaceutically acceptable excipient,” “pharmaceutically acceptable diluent,” “pharmaceutically acceptable carrier,” and “pharmaceutically acceptable adjuvant” means an excipient, diluent, carrier, and adjuvant that are useful in preparing a pharmaceutical composition that are generally safe, non-toxic and neither biologically nor otherwise undesirable, and include an excipient, diluent, carrier, and adjuvant that are acceptable for veterinary use as well as human pharmaceutical use.
  • “A pharmaceutically acceptable excipient, diluent, carrier and adjuvant” as used in the specification and claims includes one and more than one such excipient, diluent, carrier, and adjuvant.
  • the present invention provides synthetic cellular basement membrane comprising a polypeptide-polymer conjugate.
  • Such conjugates should have controlled attachment stoichiometry.
  • a subject synthetic cellular basement membrane is useful in a variety of applications, which are also provided.
  • the present invention provides for methods of making and compositions providing a synthetic cellular basement membrane comprising a polymer composition wherein Laminin-1 derived peptides are conjugated to the polymer, whereby the synthetic basement membrane can be used in vivo for uses such as supporting alveogenesis or providing post-surgical tissue stability.
  • the biological activity of a polypeptide conjugated to the polymer substrate is enhanced relative to the activity of the polypeptide in soluble form, e.g., compared to the activity of the polypeptide not conjugated to the polymer.
  • the biological activity of the polypeptide of a subject polypeptide-polymer conjugate is at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15 -fold, at least about 20-fold, at least about 25 -fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75 -fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about WOO- fold, or more than WOO-fold, greater than the biological activity of the polypeptide in soluble (unconjugated) form.
  • the biological activity of the polypeptide of a subject polypeptidepolymer conjugate is at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 5 -fold, at least about 10-fold, at least about 15 -fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about WOO-fold, or more than WOO-fold, greater than the biological activity of the polypeptide in when conjugated to the polymer at al : 1 molar ratio.
  • the biological activity of the polypeptide of a subject polypeptidepolymer conjugate is at least about 25%, at least about 50%, at least about 75%, at least about 2-fold, at least about 5 -fold, at least about 10-fold, at least about 15 -fold, at least about 20-fold, at least about 25 fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, or at least about WOO-fold, or more than 1000-fold, greater than the biological activity of the polypeptide when present in admixture with the polymer.
  • Suitable synthetic substrates include biopolymeric materials comprising from about 50 to about 100,000 subunits, e.g., from about 50 subunits to about 100 subunits, from about 100 subunits to about 500 subunits, from about 500 subunits to about 1,000 subunits, from about 1,000 subunits to about 5,000 subunits, from about 5,000 subunits to about 10,000 subunits, from about 10,000 subunits to about 25,000 subunits, from about 25,000 subunits to about 50,000 subunits, or from about 50,000 subunits to about 100,000 subunits.
  • the linear polymer comprises more than 100,000 subunits.
  • the subunits can all be identical, e.g., the polymer is a homopolymer. In other embodiments, more than one species of subunit is present, e.g., the polymer is a heteropolymer or co-polymer. In some embodiments, the polymer is a linear polymer. In other embodiments, the polymer may include one or more branches.
  • Suitable polymers include natural polymers, semisynthetic polymers, and synthetic polymers.
  • Suitable natural polymers include hyaluronic acid, collagen, glycosaminoglycans, cellulose, polysaccharides, and the like.
  • Suitable synthetic polymers include, but are not limited to, polymers or copolymers derived from polydioxane, polyphosphazene, polysulphone resins, poly(acrylic acid), poly(acrylic acid) butyl ester, poly(ethylene glycol), poly(propylene), polyurethane resins, poly(methacrylic acid), poly(methacrylic acid)-methyl ester, poly(methacrylic acid)-n butyl ester, poly(methacrylic acid)-t butyl ester, polytetrafluoroethylene, polyperfluoropropylene, poly N-vinyl carbazole, poly(methyl isopropenyl ketone), poly alphamethyl styrene, polyvinylacetate, poly(oxymethylene), poly(ethylene-co-vinyl acetate), a polyurethane, a poly(vinyl alcohol), and polyethylene terephthalate; ethylene vinyl alcohol copolymer polybutylmethacrylate;
  • the polymer to which the biologically active polypeptide is conjugated can comprise multiple subunits selected from hyaluronic acid, acrylic acid, ethylene glycol, vinyl, propylene, methyl methacrylate, methacrylic acid, acrylarnide, hydroxyethyl methacrylate, tetrafluoroethylene, oxymethylene, a sugar (e.g., glucose, mannitol, maltose, arabinose, etc.), taurine, betaine, modified celluloses, hydroxyethyl cellulose, ethyl cellulose, methyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, modified starches, hydrophobically modified starch, hydroxyethyl starch, hydroxypropyl starch, amylose, amylopectin, oxidized starch, an amino acid, and copolymers of any of the foregoing.
  • the polymer does not include amino acids.
  • the polymer is hyaluronic acid or a hyaluronic acid derivative.
  • Hyaluronic acid derivatives include, e.g., a hyaluronic acid ester where part or all of the carboxylic functions are esterified with an alcohol of the aliphatic, aromatic, arylaliphatic, cycloaliphatic or heterocyclic series; a hemiester of succinic acid or a heavy metal salt of the hemiester of succinic acid with hyaluronic acid or with a partial or total ester of hyaluronic acid; sulphated or N-sulphated hyaluronic acid. described in, e.g., WO09120893, U.S.
  • suitable substrates include interpenetrating polymer networks (IPNs); a synthetic hydrogel; a semi-interpenetrating polymer network (sIPN); a thermo-responsive polymer; and the like.
  • IPNs interpenetrating polymer networks
  • a synthetic substrate comprises poly(ethylene glycol) (PEG).
  • a synthetic substrate comprises a co-polymer of polyacrylamide and poly(ethylene glycol).
  • the synthetic substrate comprises a co-polymer of polyacrylamide and PEG, and further comprises acrylic acid.
  • the polymer comprising N- Isopropylacrylamide (NIPAAm)-based polymer gels
  • NIPAAm N- Isopropylacrylamide
  • suitable biopolymers may comprise hyaluronic acid (HyA) such as thermoresponsive HyA-NIPAAm-based polymer gels.
  • the polypeptide component of the polypeptide-polymer conjugate in the synthetic basement membrane should be biologically active, e.g., exhibits one or more biological activities in vivo and/or in vitro such that the synthetic basement membrane promotes or mimics the effect of laminin-111.
  • the polypeptide component of a subject polypeptide- polymer conjugate can be a naturally-occurring polypeptide, a recombinant polypeptide, or a synthetic polypeptide.
  • the polypeptide can comprise one or more non-amino acid moieties, e.g., a lipid moiety, a sugar moiety, a carbohydrate moiety, etc.
  • a single species of polypeptide is attached to a polymer, e.g., a plurality of polypeptides, all having the same amino acid sequence, is attached to a polymer.
  • two or more species of polypeptides are attached to a polymer, where a first polypeptide has a first amino acid sequence, and a second polypeptide has a second amino acid sequence that is different from the first amino acid sequence (e.g., where the second amino acid sequence has from about 95% to about 99%, from about 90% to about 95%), from about 85% t about 90%>, from about 80%> to about 85%, from about 75% to about 80%), from about 70% to about 75%, from about 65% to about 70%, or less than 65%, amino acid sequence identity with the first amino acid sequence).
  • the first and the second polypeptides could target different cell surface receptors, e.g., the first polypeptide could provide for cell adhesion through an integrin receptor, and the second polypeptide could provide for activation of a bound cell, e.g., via growth factor receptors, etc. inhibitory peptides to a biocompatible linear polymer.
  • a synthetic cellular basement membrane comprising a polymerized hydrogel composition or polypeptide- polymer conjugate having peptides conjugated to the hydrogel or polymer that contact or interact with proteins such as laminin-111, a6 integrin and/or ⁇ integrin.
  • a synthetic cellular basement membrane comprising a polymerized hydrogel composition or polypeptide -polymer conjugate having peptides AGIO (SEQ ID NO:3), AG32 (SEQ ID NO:4) , AG73 (SEQ ID NO:5), and P3 (SEQ ID NO:8) conjugated to the hydrogel.
  • the Laminin-1 AGIO, AG32, and AG73 peptides and the P3 peptide from a-integrin 6 function synergistically to induce optimal ⁇ -casein promoter activity.
  • the peptides P3, AGIO, AG32, and AG73 are present in the polymer in equal concentrations.
  • the present invention provides peptides, e.g., isolated peptides or synthetic peptides, identified by the methods as described in the Examples below.
  • the peptides are useful for generating functional synthetic cellular basement membrane, as described herein.
  • Exemplary peptides are show in Table 1, below.
  • the peptides used in the polymer composition having at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of SEQ ID least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of AGIO (SEQ ID NO:3), AG32 (SEQ ID NO:4) , AG73 (SEQ ID NO:5), and P3 (SEQ ID NO:8).
  • the peptides comprising AGIO (SEQ ID NO:3), AG32 (SEQ ID NO:4) , AG73 (SEQ ID NO:5), and P3 (SEQ ID NO:8).
  • the subject peptide can have a length of from about 5 amino acids to about 50 amino acids, e.g., from about 12 amino acids to about 30 amino acids, from about 12 amino acids to about 20 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, from about 35 amino acids to about 40 amino acids, from about 40 amino acids to about 45 amino acids, or from about 45 amino acids to about 50 amino acids.
  • Peptides can be synthesized using standard methods for chemical synthesis of a peptide. Peptides can also be synthesized recombinantly, using standard methods.
  • the polypeptide component is recombinant, e.g., the polypeptide includes one or more amino acids that are not normally in amide bond linkage with the polypeptide.
  • the polypeptide can be engineered to include an amino acid that facilitates linkage to the polymer component of the polypeptide -polymer conjugate.
  • the polypeptide can be engineered to include a cysteine residue that facilitates linkage to the polymer component of the polypeptide-polymer conjugate.
  • the size of the polypeptide can range from 2 kDa to about 2000 kDa, e.g., from about 2 kDa to about 5 kDa, from about 5 kDa to about 10 kDa, from about 10 kDa to about 25 kDa, from about 25 kDa to about 50 kDa, from about 50 kDa to about 100 kDa, from about 100 kDa to about 250 kDa, from about 250 kDa to about 500 kDa, from about 500 kDa to about 1000 kDa, from about 1000 kDa to about 2000 kDa.
  • the polypeptide component of a subject polypeptide-polymer conjugate comprises a detectable label.
  • Suitable labels include, e.g., radioisotopes (e.g., 1251; 35S, and the like); enzymes whose products generate a detectable signal (e.g., luciferase,— galactosidase, horse radish peroxidase, alkaline phosphatase, and the like); fluorescent labels (e.g., fluorescein isothiocyanate, rhodamine, phycoerythrin, and the like); fluorescence emitting metals, e.g., 152Eu, or others of the lanthanide series, attached to the antibody through metal chelating groups such as EDTA; chemiluminescent compounds, e.g., luminol, isoluminol, acridinium salts, and the like; bioluminescent compounds, e.g., lucifer
  • Polypeptides that are of interest for attachment to a polymer, to generate a subject polypeptide-polymer conjugate include, e.g., growth factors, receptors, polypeptide ligands for receptors, enzymes, antibodies, coagulation factors, anti-coagulation factors, angiogenic factors, anti-angiogenic factors, etc.
  • Suitable polypeptides include linear polypeptides and cyclic polypeptides.
  • Suitable polypeptides include naturally occurring polypeptides, synthetic polypeptides, and the like.
  • the peptides are conjugated to a synthetic substrate.
  • the peptides are conjugated to the linear chains of a biopolymer and mixed with a hydrogel copolymer to form an associative network gel.
  • a subject polypeptide-polymer conjugate comprises a linker group that links the polypeptide to the polymer.
  • Suitable linkers include peptide linkers, and nonpeptide linkers.
  • a linker peptide may have any of a variety of amino acid sequences.
  • Exemplary peptide linkers are between about 6 and about 40 amino acids in length, or between about 6 and about 25 amino acids in length.
  • Exemplary linkers include poly(glycine) linkers (e.g., (GlY)m where n is an integer from 2 to about 10); linkers comprising Gly and Ser; and the like.
  • the gels are preferably a viscous liquid at room temperature and soft hydrogels above ⁇ 33°C, with an elastic modulus on par with that of normal tissue.
  • the composition having an elastic modulus similar to that of normal breast tissue ( ⁇ 175Pa).
  • a variety of conjugation methods and chemistries can be used to conjugate a polypeptide to a polymer.
  • Various zero-length, homo-bifunctional, and hetero-bifunctional crosslinking reagents can be used.
  • Zero-length crosslinking reagents include direct conjugation of two intrinsic chemical groups with no introduction of extrinsic material. Agents that catalyze formation of a disulfide bond belong to this category.
  • reagents that induce condensation of a carboxyl and a primary amino group to form an amide bond such as carbodiimides, ethylchloroformate, Woodward's reagent K (2ethyl-5- phenylisoxazolium-3'-sulfonate), and carbonyldiimidazole.
  • Homo-and hetero-bifunctional reagents generally contain two identical or two non-identical sites, respectively, which may be reactive with amino, sulfhydryl, guanidino, indole, or nonspecific groups. with a primary amine group on the polypeptide, or on a linker.
  • Suitable amino-reactive groups include, but are not limited to, N-hydroxysuccinimide (NHS) esters, imidoesters, isocyanates, acylhalides, arylazides, pnitrophenyl esters, aldehydes, and sulfonyl chlorides.
  • NHS N-hydroxysuccinimide
  • the polymer comprises a sulfhydryl-reactive group, e.g., for reacting with a cysteine residue in the polypeptide.
  • Suitable sulfhydryl-reactive groups include, but are not limited to, maleimides, alkyl halides, pyridyl disulfides, and thiophthalimides .
  • carbodiimides soluble in both water and organic solvent are used as carboxyl-reactive reagents. These compounds react with free carboxyl groups forming a pseudourea that can then couple to available amines, yielding an amide linkage.
  • a polypeptide is conjugated to a polymer using a homobifunctional crosslinker.
  • the homobifunctional crosslinker is reactive with primary amines.
  • Homobifunctional crosslinkers that are reactive with primary amines include NHS esters, imidoesters, isothiocyanates, isocyanates, acylhalides, arylazides, p-nitrophenyl esters, aldehydes, and sulfonyl chlorides.
  • Non-limiting examples of homobifunctional NHS esters include disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl) suberate (BS), disuccinimidyl tartarate (DST), disulfosuccinimidyl tartarate (sulfo-DST), bis-2- (succinimidooxycarbonyloxy)ethylsulfone (BSOCOES), bis-2-
  • Nonlimiting examples of homobifunctional imidoesters include dimethyl malonimidate (DMM), dimethyl succinimidate (DMSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-oxydipropionimidate (DODP), dimethy 1-3,3 '- (methylenedioxy)dipropionimidate (DMDP), dimethyl-,3'-
  • DTDP tetramethylenedioxydipropionimidate
  • DTBP dimethyl-3,3'dithiobispropionimidate
  • Non- limiting examples of homobifunctional isothiocyanates include: p- phenylenediisothiocyanate (DITC), and 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene toluene-2,4diisocyanate, toluene-2-isocyanate-4-isothiocyanate, 3-methoxydiphenyImethane- 4,4'-diisocyanate, 2,2'-dicarboxy-4,4'-azophenyldiisocyanate, and hexamethylenediisocyanate.
  • DITC p- phenylenediisothiocyanate
  • Non-limiting examples of homobifunctional arylhalides include l,5-difluoro-2,4-dinitrobenzene (DFDNB), and 4,4'-difluoro-3,3'dinitrophenyl-sulfone.
  • Non- limiting examples of homobifunctional aliphatic aldehyde reagents include glyoxal, malondialdehyde, and glutaraldehyde.
  • Non- limiting examples of homobifunctional acylating reagents include nitrophenyl esters of dicarboxylic acids.
  • Non- limiting examples of homobifunctional aromatic sulfonyl chlorides include phenol-2,4-disulfonyl chloride, and a- naphthol-2,4-disulfonyl chloride.
  • Non-limiting examples of additional amino-reactive homobifunctional reagents include erythritolbiscarbonate, which reacts with amines to give biscarbamates.
  • the homobifunctional crosslinker is reactive with free sulfhydryl groups.
  • Homobifunctional crosslinkers reactive with free sulfhydryl groups include, e.g., maleimides, pyridyl disulfides, and alkyl halides.
  • Non-limiting examples of homobifunctional maleimides include bismaleimidohexane (BMH), N,N'-(l,3-phenylene) bismaleimide, N,N'-(l,2-phenylene)bismaleimide, azophenyldimaleimide, and bis(N-maleimidomethyl)ether.
  • Non-limiting examples of homobifunctional pyridyl disulfides include l,4-di-3'-(2'-pyridyldithio)propionamidobutane (DPDPB).
  • Non- limiting examples ofhomobifunctional alkyl halides include 2,2'-dicarboxy- 4,4'-diiodoacetamidoazobenzene, a, a'-diiodo-p-xylenesulfonic acid, a, a'-dibromo-p- xylenesulfonic acid, N,N'-bis(b-bromoethyl)benzylamine, ⁇ , ⁇ '- di(bromoacetyl)phenylhydrazine, and 1 ,2-di(bromoacetyl)amino-3-phenylpropane.
  • a polypeptide is conjugated to a polymer using a heterobifunctional reagent.
  • Suitable heterobifunctional reagents include amino- reactive reagents comprising a pyridyl disulfide moiety; amino-reactive reagents comprising a maleimide moiety; amino-reactive reagents comprising an alkyl halide moiety; and amino- reactive reagents comprising an alkyl dihalide moiety.
  • Non-limiting examples ofhetero-bifunctional reagents with a pyridyl disulfide moiety and an amino-reactive NHS ester include N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl 6-3-(2-pyridyldithio)propionamidohexanoate (LC-SPDP), sulfosuccinimidyI6-3-(2pyridyldithio)propionamidohexanoate (sulfo-LCSPDP), 4- succinimidyloxycarbonyl-a-methyl-a-(2pyridyldithio)toluene (SMPT), and sulfosuccinimidyI6-a-methyl-a-(2pyridyldithio)toluamidohexanoate (sulfo-LC-SMPT).
  • SPDP N-succ
  • an amino-reactive NHS ester examples include succinimidyl maleimidylacetate (AMAS), succinimidyl 3maleimidylpropionate (BMPS), N-. gamma. -maleimidobutyryloxysuccinimide ester (GMBS)N.gamma.-maleimidobutyryloxysulfosuccinimide ester (sulfo-GMBS) succinimidyl 6maleimidylhexanoate (EMCS), succinimidy -maleimidylbenzoate (SMB), m- maleimidobenzoyl-Nhydroxysuccinimide ester (MBS), m-maleimidobenzoyl-N- hydroxysulfosuccinimide ester (sulfo-MBS), succinimidyl 4-(N- maleimidomethyl)cyclohexane-I-carboxylate (SMCC), sulfosuccinimidyl 4-(N-
  • Non-limiting examples ofheterobifunctional reagents comprising an alkyl halide moiety and an amino-reactive NHS ester include N-succinimidyl-(4- iodoacetyl)aminobenzoate (S1AB), sulfosuccinimidyl-(4-iodoacetyl)aminobenzoate (sulfo- SIAB), succinimidyl-6(iodoacetyl)aminohexanoate (SIAX), succinimidyl-6-(6-((iodoacetyl)- amino)hexanoylamino)hexanoate (SIAXX), succinimidyl-6-(((4-(iodoacetyl)-amino)methyl)- cyclohexane-I-carbonyl)aminohexanoate (SIACX), and succinimidyl-4((iodoacetyl)
  • a non-limiting example of a hetero-bifunctional reagent comprising an amino- reactive NHS ester and an alkyl dihalide moiety is N-hydroxysuccinimidyI2,3- dibromopropionate (SDBP).
  • a nonlimiting example of a hetero-bifunctional reagent comprising an alkyl halide moiety and an aminoreactive p-nitrophenyl ester moiety include p- nitrophenyl iodoacetate (NPIA).
  • HyA was first activated with a maleimide group by carbodiimide coupling to EMCH.
  • HyA 200 mg was added to 32 mL of MES buffer (0.1 M, pH 6.5) and stirred under low shear until dissolved.
  • Conjugation reagents 440 mg EDC, 123 mg sulfoNHS, and 53 mg EMCH were dissolved in 8.8 mL MES buffer. Eight mL of the conjugation reaction mixture was added through sterile-filter to the HyA solution and stirred.
  • each reagent for EMCH activation of HyA was: 5 mg/mL HyA, 10 mg/mL EDC, 2.8 mg/mL sulfoNHS, and 1.2 mg/mL EMCH.
  • the reaction was allowed to proceed for 2 hrs with stirring at room temperature. The solution was then 6.5).
  • the activated HyA-EMCH solution is split into 4 for reaction with each peptide.
  • the peptides were then reduced and added to the activated HyA-EMCH solution for conjugation via reaction of the maleimide moieties with the thiol group of the peptides.
  • 3 mg of NaOH and 5.7 mg of TCEP were added to 1 mL UPW.
  • the peptides (4.4 mg) were added to alkaline reducing solution, allowed to react for 30 min at 4° C, and added through sterile-filter to the activated HyA-EMCH solution. The final solution was reacted overnight at 4° C in the dark.
  • the conjugate solution was then dialyzed through a 10 kDa MWCO membrane against DPBS (0.08 M, pH 7.2). After dialysis, the solution was transferred to a 50 mL Steriflip tube (BD Bioscience) and lyophilized through the sterile filter cap. The dry product was stored at -20° C until characterization or use.
  • the HyA-peptide conjugates were characterized by SEC-MALS with refractive index (RI) and ultra-violet spectroscopy (UV) in order to confirm peptide conjugation and conjugate molecular weight.
  • the conjugates were mixed into the P(NIPAAm-co-mPEGMA) gels at 0.5 wt% (50 mM peptide). The solutions were then vortexed and centrifuged to make them homogeneous.
  • each peptide once conjugated to the biopolymer should be present in the hydrogel such that their concentration on the polymer chains ]such that the peptides can function synergistically to drive tissue specific cell behaviors.
  • a synthetic form of basement membrane comprising the polymerized hydrogel composition featuring the four peptides AGIO (SEQ ID NO:3), AG32 (SEQ ID NO:4) , AG73 (SEQ ID NO:5), and P3 (SEQ ID NO:8) in roughly equal concentrations.
  • peptides which mimic the effect of laminin-111 on surrounding tissues are known in the art.
  • methods of identifying peptide ligands of mammalian cell surface molecule e.g., a cell surface macromolecule such as a protein, a glycoprotein, etc.
  • the methods generally involve contacting a mammalian cell in vitro with a population of bacteria comprising individual bacteria, each of which displays on its surface a different heterologous peptide, forming a mixed mammalian cell population comprising mammalian cells bound to one or more bacteria and unbound bacterial cells; and separating the bound from the unbound bacterial cells.
  • Binding of a mammalian cell to a bacterium indicates that the bacterium displays on its cell surface a peptide that binds to a mammalian cell surface molecule.
  • the heterologous peptide displayed by the bacterium is considered a generating a peptide display library include, e.g., a method as described in U.S. Patent Publication No. 2007/0099247, and 2010/0189760 both of which are incorporated herein by reference.
  • a peptide-displaying bacterium displays a peptide at a density of from about 10. sup.3 to about 10. sup.5 peptides per bacterial cell, e.g., at a density of from about 10. sup.3 to about 5.times.l0.sup.3, from about 5. times.10. sup.3 to about 10. sup.4, from about 10.sup.4 to about 5. times.10. sup.4, or from about 5. times.10. sup.4 to about 10. sup.5, or more, peptide molecules per cell.
  • Each bacterium in a peptide-displaying bacterial population (or "library") displays a different peptide.
  • a peptide-displaying bacterial library can display two, three, four, five, six, seven, eight, nine, ten, from 10 to 25, from 25 to 50, from 50 to 100, from 10.sup.2 to 10.sup.4, from 10.sup.4 to 10. sup.6, from 10. sup.6 to 10. sup.8, from 10. sup.8 to 10. sup.9, or from 10. sup.9 to about 10. sup.10, or more, different peptides, each present on the surface of different bacteria in the library population.
  • a peptide displayed by a peptide-displaying bacterium is "heterologous," e.g., the peptide is one that is not normally synthesized by the bacterium.
  • Each heterologous peptide can have a length of from about 5 amino acids to about 50 amino acids, e.g., from about 5 amino acids to about 10 amino acids, from about 10 amino acids to about 15 amino acids, from about 15 amino acids to about 20 amino acids, from about 20 amino acids to about 25 amino acids, from about 25 amino acids to about 30 amino acids, from about 30 amino acids to about 35 amino acids, from about 35 amino acids to about 40 amino acids, from about 40 amino acids to about 45 amino acids, or from about 45 amino acids to about 50 amino acids.
  • the heterologous peptide is displayed as a fusion protein with an outer membrane protein (OMP) of a bacterium.
  • OMP outer membrane protein
  • the heterologous peptide is a C-terminal or N-terminal fusion protein with a circularly permuted variant of outer membrane protein X (CPX).
  • CPX is described in, e.g., Rice et al. (2006) Protein Science 15:825-836. See also, U.S. Patent Publication No. 2007/0099247.
  • the peptide-displaying bacteria are genetically modified to produce a fluorescent protein
  • the peptide-displaying bacteria are genetically modified by introduction into the bacteria of a nucleic acid comprising a nucleotide sequence encoding a fluorescent protein, where the nucleic acid is, e.g., an expression vector that provides for expression of the nucleotide sequence in the bacteria.
  • Suitable fluorescent proteins include, but are not limited to, a green fluorescent protein (GFP; Chalfie, et al, Science 263(5148):802-805 (Feb. 11, 1994); an enhanced GFP (EGFP), e.g., Genbank Accession 471 (1998); Heim, R. and Tsien, R. Y.
  • bound mammalian cells e.g., mammalian cells to which are bound one or more peptide-displaying bacteria
  • FACS fluorescence-activated cell sorting
  • the mammalian cells are genetically modified to produce a detectable signal upon binding of a peptide-displaying bacterium that displays a peptide that activates a cell signaling pathway.
  • Signaling pathways include, but are not limited to, phosphoinositide-3 kinase (PI 3-kinase), mitogen-activated protein kinase (MAPK), phospholipase C (PLC), protein kinase C (PKC), protein kinase A (PICA), protein kinase G (PKG), Sonic hedgehog (Shh), Wnt, Notch, Jak/STAT, and calcium/calmodulin dependent kinase (Cam kinase).
  • the assay used to detect activation of a cell signaling pathway can depend, e.g., on the mode of transmission of the signal and/or the nature of the components of the signaling pathway. For example, in some embodiments, detecting activation of a signaling pathway involves detecting activity of a kinase involved in the signaling pathway. In other embodiments, detecting activation of a signaling pathway involves detecting a change in intracellular calcium concentration ([Ca.sup.2+].sub.i). In other embodiments, detecting activation of a signaling pathway involves detecting relocalization of one or more molecules in the cell interior (e.g., relocalization of a protein from the cytoplasm to the nucleus, and the like).
  • a fluorescence resonance energy transfer (FRET)-based assay is used.
  • peptides A “phage display library” refers to a “library” of bacteriophages on whose surface is expressed exogenous peptides or proteins.
  • the foreign peptides or polypeptides are displayed on the phage capsid outer surface.
  • the foreign peptide can be displayed as recombinant fusion proteins incorporated as part of a phage coat protein, as recombinant fusion proteins that are not normally phage coat proteins, but which are able to become incorporated into the capsid outer surface, or as proteins or peptides that become linked, covalently or not, to such proteins.
  • exogenous nucleic acid sequence into a nucleic acid that can be packaged into phage particles.
  • exogenous nucleic acid sequences may be inserted, for example, into the coding sequence of a phage coat protein gene. If the foreign sequence is "in phase" the protein it encodes will be expressed as part of the coat protein.
  • libraries of nucleic acid sequences such as a genomic library from a specific cell or chromosome, can be so inserted into phages to create “phage libraries.”
  • a "peptide-display library” is generated. While a variety of bacteriophages are used in such library constructions, typically, filamentous phage are used (Dunn, 1996 Curr. Opin. Biotechnol. 7:547-553).
  • phage display libraries exploits the bacteriophage's ability to display peptides and proteins on their surfaces, i.e., on their capsids. Often, filamentous phage such as Ml 3, fd, or fl are used. Filamentous phage contain single-stranded DNA surrounded by multiple copies of genes encoding major and minor coat proteins, e.g., pill. Coat proteins are displayed on the capsid's outer surface. DNA sequences inserted in-frame with capsid protein genes are co-transcribed to generate fusion proteins or protein fragments displayed on the phage surface. Phage libraries thus can display peptides representative of the diversity of the inserted sequences.
  • these peptides can be displayed in "natural” folded conformations.
  • the fluorescent binding ligands expressed on phage display libraries can then bind target molecules, i.e., they can specifically interact with binding partner molecules such as antigens, e.g., (Petersen, 1995, Mol. Gen. Genet., 249:425-31), cell surface receptors (Kay, 1993, Gene 128:59-65), and extracellular and intracellular proteins (Gram, 1993, J. Immunol. Methods, 161 : 169-76).
  • binding partner molecules such as antigens, e.g., (Petersen, 1995, Mol. Gen. Genet., 249:425-31), cell surface receptors (Kay, 1993, Gene 128:59-65), and extracellular and intracellular proteins (Gram, 1993, J. Immunol. Methods, 161 : 169-76).
  • filamentous phages such as Ml 3 or fd
  • peptides displayed on phage surfaces to identify many potential ligands (see, e.g., Cwirla, 1990, Proc. Natl. Acad. Sci. USA, 87:6378-6382).
  • Cwirla 1990, Proc. Natl. Acad. Sci. USA, 87:6378-6382.
  • exogenous nucleic acids encoding the protein sequences to be displayed are inserted into a coat protein gene, e.g. gene III or gene VIII of the phage.
  • the resultant fusion proteins are displayed on the surface of the capsid.
  • Protein VIII is present in approximately 2700 copies per phage, compared to 3 to 5 copies for protein III (Jacobsson (1996), supra).
  • Multivalent expression vectors such as phagemids, can be used for manipulation of the nucleic acid sequences encoding the fluorescent binding library and production of phage particles in bacteria (see, e.g., Felici, 1991, J. Mol. Biol., 222:301-310).
  • Phagemid vectors are often employed for constructing the phage library. These vectors include the origin of DNA replication from the genome of a single-stranded filamentous bacteriophage, e.g., M13 or fl and require the supply of the other phage proteins to create a phage. This is usually supplied by a helper phage which is less efficient at being packaged into phage particles.
  • a phagemid can be used in the same way as an orthodox plasmid vector, but can also be used to produce filamentous bacteriophage particle that contain single-stranded copies of cloned segments of DNA.
  • the displayed protein does not need to be a fusion protein.
  • a fluorescent binding ligand may attach to a coat protein by virtue of a non-covalent interaction, e.g., a coiled coil binding interaction, such as jun/fos binding, or a covalent interaction mediated by cysteines (see, e.g., Crameri et al., 1994, Eur. J. Biochem., 226:53-58) with or without additional non-covalent interactions.
  • cysteines see, e.g., Crameri et al., 1994, Eur. J. Biochem., 226:53-58
  • Morphosys have described a display system in which one cysteine is put at the C terminus of the scFv or Fab, and another is put at the N terminus of g3p. The two assemble in the periplasm and display occurs without a fusion gene or protein.
  • the coat protein does not need to be endogenous.
  • DNA binding proteins can be incorporated into the phage/phagemid genome (see, e.g., McGregor & Robins, 2001, present in the genome, the DNA binding protein becomes incorporated into the phage/phagemid. This can serve as a display vector protein. In some cases it has been shown that incorporation of DNA binding proteins into the phage coat can occur independently of the presence of the recognized DNA signal.
  • phage can also be used.
  • T7 vectors, T4 vector, T2 vectors, or lambda vectors can be employed in which the displayed product on the mature phage particle is released by cell lysis.
  • a "selectively infective phage” consists of two independent components. For example, a recombinant filamentous phage particle is made non-infective by replacing its N-terminal domains of gene 3 protein (g3p) with a protein of interest, e.g., an antigen. The nucleic acid encoding the antigen can be inserted such that it will be expressed. The second component is an "adapter" molecule in which the fluorescent ligand is linked to those N-terminal domains of g3p that are missing from the phage particle.
  • g3p gene 3 protein
  • the second component is an "adapter" molecule in which the fluorescent ligand is linked to those N-terminal domains of g3p that are missing from the phage particle.
  • analogous epitope display libraries can also be used.
  • the methods of the invention can also use yeast surface displayed libraries (see, e.g., Boder, 1997, Nat. BiotechnoL, 15:553-557 and Feldhaus et al, 2003, Nat. BiotechnoL, 21, 163-170), which can be constructed using such vectors as the pYDl yeast expression vector.
  • yeast surface displayed libraries see, e.g., Boder, 1997, Nat. BiotechnoL, 15:553-557 and Feldhaus et al, 2003, Nat. BiotechnoL, 21, 163-170.
  • Other potential display systems include mammalian display vectors and E. coli libraries.
  • In vitro display library formats known to those of skill in the art can also be used, e.g., ribosome displays libraries and mRNA display libraries.
  • proteins are made using cell-free translation and physically linked to their encoding mRNA after in vitro translation.
  • DNA encoding the sequences to be selected are transcribed in vitro and translated in a cell-free system.
  • the subject synthetic cell basement membrane can be in any of a variety of forms, e.g., a 3-dimensional form (e.g., suitable for implanting into a tissue such as breast tissue; or suitable for coating onto the surface of an implantable device, such as a scaffold, or silicone implants, etc.); and the like.
  • a 3-dimensional form e.g., suitable for implanting into a tissue such as breast tissue; or suitable for coating onto the surface of an implantable device, such as a scaffold, or silicone implants, etc.
  • compositions including pharmaceutical compositions, comprising a subject polypeptide-polymer conjugate.
  • a subject composition comprises a subject polypeptide- polymer conjugate, wherein the subject polypeptide-polymer conjugate is homogeneous, e.g., all of the polypeptides of the polypeptide-polymer conjugate comprise the same amino acid sequence.
  • a subject composition comprises a plurality of (e.g., multiple copies of) a subject polypeptide-polymer conjugate, where each polypeptide- polymer conjugate molecule comprises polypeptides that all have the same amino acid sequence.
  • a synthetic cell basement membrane comprises two or more species of a subject polypeptide-polymer conjugate, e.g., a subject composition comprises a first polypeptide-polymer conjugate, where the first polypeptide-polymer conjugate comprises polypeptides of a first amino acid sequence; and at least a second polypeptide- polymer conjugate, wherein the second polypeptidepolymer conjugate comprises polypeptides of a second amino acid sequence that is different from the first amino acid sequence.
  • a subject composition comprises a third or additional polypeptide-polymer conjugates.
  • a first polypeptide-polymer conjugate comprises a first polypeptide that provides for binding to an integrin; and a second polypeptide-polymer conjugate that comprises a second polypeptide that activates a cell signaling pathway.
  • first, second, etc., polypeptides can he used. The ratio of the first polypeptide-polymer conjugate to the second polypeptide-polymer
  • 3 3 conjugate in a subject composition can be varied, e.g., from about 0:001 to 10 to about 10 to 0.001.
  • a subject composition comprises a first, a second, and a third polypeptide-polymer conjugate
  • the ratios of the first, second, and third polypeptide-polymer conjugates can be varied.
  • a subject composition can comprise, in addition to a subject polypeptide-polymer conjugate, one or more of: a salt, e.g., NaCI, MgCI, KCI, MgS04, etc.; a buffering agent, e.g., a Tris buffer, N-(2Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N- Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N-tris[Hydroxymethyl]methyl-3- detergent such as Tween-20, etc.; a protease inhibitor; and the like.
  • a salt e.g., NaCI, MgCI, KCI, MgS04, etc.
  • a buffering agent e.g
  • the present invention provides compositions comprising a subject polypeptide-polymer conjugate and a pharmaceutically acceptable excipient.
  • Suitable excipient vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents or pH buffering agents. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17th edition, 1985.
  • composition or formulation to be administered will, in any event, contain a quantity of the agent adequate to achieve the desired state in the subject being treated.
  • pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are readily available to the public.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are readily available to the public.
  • the terms "pharmaceutically acceptable carrier” and “pharmaceutically acceptable excipient” are used interchangeably, and include any material, which when combined with a subject polypeptide-polymer conjugate does not substantially affect the biological activity of the conjugate, does not induce an immune response in a host, and does not have any substantial adverse physiological effect on the host.
  • examples include, but are not limited to, any of the standard pharmaceutical carriers such as a phosphate buffered saline solution, water, emulsions such as oil/water emulsion, and various types of wetting agents.
  • Other carriers may also include sterile solutions, tabletsincluding coated tablets and capsules.
  • Such carriers typically contain excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
  • excipients such as starch, milk, sugar, certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients.
  • Such carriers may also include flavor and color additives or other ingredients.
  • Compositions comprising such carriers are formulated by well-known conventional methods.
  • the pharmaceutical compositions may be formulated for a selected manner of administration, including for example, topical, oral, nasal, intravenous, intracranial, intraperitoneal, intratumoral, peritumoral, suhcutaneous, or intramuscular administration.
  • the carrier can comprise water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, Biodegradable microspheres (e.g., polylactate polyglycolate) may also be employed as carriers for a subject pharmaceutical composition. Suitable biodegradable microspheres are disclosed, for example, in U.S. Pat. Nos. 4,897,268 and 5,075,109.
  • compositions for parenteral administration which comprise a subject conjugate dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, phosphate-buffered saline, and the like.
  • an acceptable carrier e.g., water, buffered water, saline, phosphate-buffered saline, and the like.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, detergents and the like.
  • a subject composition can be sterilized by conventional sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration.
  • the pH of the preparations can range from 3 and 11, e.g., from about pH 5 to about pH 9, or from about pH 7 to about pH 8. Implantable tissues and devices.
  • a synthetic cell basement membrane is coated onto, layered onto, incorporated into, or forms, an implantable tissue or device, e.g., an artificial tissue; an implant into a tissue; a coating for an implantable device (such as an implant, a scaffold, a graft, etc.); an implantable drug delivery system; and the like.
  • Artificial tissues include, e.g., synthetic bulk breast tissue for providing tissue stability following a lumpectomy or biopsy.
  • Biomaterials include, e.g., films, gels, sponges, gauzes, nonwoven fabrics, membranes, microspheres, microcapsules, threads, guide channels, and the like.
  • a synthetic cell basement membrane is layered or coated onto or otherwise attached to a matrix, to form a synthetic implantable device.
  • a matrix also referred to as a "biocompatible substrate”
  • a biocompatible substrate is a material that is suitable for implantation into a subject and onto which a subject polypeptide-polymer conjugate is layered, coated, or otherwise attached.
  • a biocompatible substrate does not cause toxic or injurious effects once implanted in the subject.
  • the biocompatible substrate is a polymer with a surface that can be shaped into the desired structure that requires repairing or replacing.
  • the biocompatible substrate can also be shaped into a part of a structure that requires repairing or replacing.
  • the biocompatible substrate provides the supportive framework onto which a synthetic cell basement membrane can be layered, coated, or otherwise attached.
  • cell basement membrane comprising a subject polypeptide -polymer conjugate which further comprises one or more cells and/or one or more cell types bound to the matrix or scaffold comprising the polypeptide-polymer conjugate.
  • Such matrices or scaffolds are useful in the context of tissue engineering, cell culturing, cell transplantation, etc.
  • a drug delivery device comprises a synthetic cell basement membrane.
  • the drug release device can be based upon a diffusive system, a convective system, or an erodible system (e.g., an erosion-based system).
  • the drug release device can be an electrochemical pump, osmotic pump, an electroosmotic pump, a vapor pressure pump, or osmotic bursting matrix, e.g., where the drug is incorporated into a polymer and the polymer provides for release of drug formulation concomitant with degradation of a drug-impregnated polymeric material (e.g., a biodegradable, drug- impregnated polymeric material).
  • the drug release device is based upon an electrodiffusion system, an electrolytic pump, an effervescent pump, a piezoelectric pump, a hydrolytic system, etc.
  • a subject synthetic cell basement membrane further comprises one or more mammalian cells bound thereto; and is useful for, e.g., introducing the cells into a recipient individual (e.g., a mammalian subject).
  • a subject synthetic cell basement membrane is contacted in vitro with one or more mammalian cells; and the cells are cultured with the synthetic cell basement membrane in vitro.
  • mammalian cells are cultured with a subject synthetic cell basement membrane in vitro, then the cultured cells, which remain associated with the basement membrane, and introduced into a recipient individual.
  • a subject synthetic cell basement membrane without any bound mammalian cells is implanted into a recipient individual, where the synthetic cell basement membrane comprises one or more species of Laminin-1 peptides that recruit one or more cell types to the site of implantation.
  • a subject synthetic cell basement membrane without any bound mammalian cells is coated onto the surface of an implantable device, forming a coated device.
  • the peptide(s) present in the cell platform coated onto the device recruits one or more cell types (e.g., endogenous cells present in the individual) to the site of implantation.
  • a subject synthetic cell basement membrane can be coated onto a device comprising any of a variety of materials, including, but not limited to, plastics, including any biocompatible plastic; glass, e.g., silicon dioxide, material that can be implanted into a recipient subject (e.g., a human) without causing substantial adverse effects.
  • a subject synthetic cell basement membrane is coated onto a silicone scaffold or implant, where the Laminin-1 peptides in the synthetic cell basement membrane recruit mammary epithelial cells.
  • the present invention thus provides an implantable device comprising a surface and a subject synthetic cell basement membrane coated onto the surface, forming a coated implantable device
  • the synthetic basement membrane can be formed as a bulk material and/or shaped for implantation in a subject for tissue and/or organ regeneration or to enhance post-surgical tissue stability in polarized epithelia, including breast tissue.
  • the synthetic basement membrane for support of alveogenesis and the generation of breast tissue in experimental in vitro systems or following surgical implantation of isogenic mammary epithelial cells intermixed with synthetic basement membrane.
  • the synthetic basement membrane for alveogenesis and synthetic breast milk production used in combination with in vitro and bioreactor systems.
  • the synthetic basement membrane can be used for support of alveogenesis and the generation of breast tissue. In another embodiment, the synthetic basement membrane to support alveogenesis and milk production.
  • EXAMPLE 1 The role of Ln-1 peptides in generating a synthetic basement membrane
  • Ln-1 The roles of Ln-1 in three important biological functions have been investigated individually in mammary gland: polarity, milk production, and morphogenesis. Here we have examined all three processes to determine whether or not they rely on the same or different regions of Ln-1, and we then used the results of our experiments to design a synthetic form of basement membrane. Because polarity and tissue-specificity are lost in carcinomas, understanding the mechanisms by which tissue-specificity is maintained is of value for understanding development and homeostasis.
  • MECs form acini in both IrECM and type 1 collagen gels, they exhibit inverted polarity in type 1 collagen gels [2, 3].
  • IrECM is composed of a variety of ECM molecules other than Ln-1 (e.g., collagens, nidogen, tenascins), basal polarity may be regulated by other ECM molecules or by other laminin domains that were not covered by our experiments.
  • Ln-1 e.g., collagens, nidogen, tenascins
  • Ln-1 receptor dystroglycan DG
  • polarity in collagen gels and ⁇ - casein production were disrupted [3].
  • DG organized endogenous Ln-1 on the surface of cells via its extracellular mucin domain, and its intracellular domain was not required for Ln-1 study, AG73, which is predicted to lie near the DG-receptor binding region [14], was a relatively potent inhibitor of mammary-specific functions.
  • thermoresponsive HyA-NIPAAm-based polymer gels seemed a natural fit, since they are biocompatible, biologically inert, and allow the simultaneous control of ligand concentration and mechanical elasticity [24]. [Whereas some of the present inventors had previously synthesis, we were unable to demonstrate that the intact E3 domain on its own was sufficient to induce ⁇ -casein production [10]. Thus, simply providing a crucial Ln-1 domain in an inappropriate context will not support this functional response.
  • Ln-1 Specific domains of Ln-1 are critical for establishing apical polarity in acinar structures.
  • the murine MEC line Eph4 was cultured in 3D IrECM in lactogenic differentiation medium with and without soluble linear Ln-1 -related peptides (Figure 1A).
  • the interaction between MECs and Ln-1 was disrupted at a number of previously identified sites [22, 27, 28], including regions within the ⁇ - and ⁇ -arms of the molecule as well as the globular domains of the a-chain.
  • Controls included a scrambled peptide constructed from the amino acids comprising AG73 (designated “scramble”) and a non-Ln-1 related peptide derived from a RGD-containing region within the laminin ⁇ -3 chain (designated “lamRGD”).
  • Peptide P3 is a synthetic peptide that was selected for its ability to block binding of the integrin a6 subunit to Ln-1 [29].
  • the estimated concentration of Ln-1 in the IrECM used in our 3D culture conditions was 5 to 6 ⁇ , thus the peptides were added at a 10-fold excess, 50 ⁇ , in order to impair the interaction of MECs with those specific sites on Ln-1.
  • EpH4 cells were engineered to express cyan fluorescent protein (CFP) driven by tandem ⁇ -casein promoters ⁇ CAS-CFP EpH4) [4].
  • Reporter cells were plated on tissue culture plastic and after 24 hours lactogenic differentiation medium containing a 2% (v/v) IrECM drip and the listed laminin-derived peptides was added to the cultures. After 72 hours, nuclei were labeled with cell permeable Hoechst 33342, and CFP and Hoechst fluorescence were imaged for all conditions tested ( Figure 2A).
  • alveogenesis the process of forming the alveoli in preparation for milk production, we tested their ability to inhibit alveoli formation in 3D organotypic cultures of primary mouse mammary epithelium.
  • Mammary gland organoids were isolated from 8-10 week old mice and cultured within 3D IrECM impregnated with 50 ⁇ of the indicated laminin-related peptides ( Figure 3 A). This same concentration of peptide was also present in the culture medium. Branching was analyzed for nearly 100 organoids per condition and normalized by the branching percentage obtained for the scrambled peptide control ( Figure 3B).
  • ⁇ -casein promoter activity was measured in CAS-CFP EpH4 after exposure to biocompatible polymers that were decorated with combinations of these peptides. Coating tissue culture plastic with a dried-down (dehydrated) layer of the different peptides alone or in combination was not sufficient to induce ⁇ -casein promoter activity (data not shown).
  • peptides P3, AGIO, AG32, and AG73 were conjugated to linear chains of the biopolymer hyaluronic acid (HyA) and mixed with a copolymer of N-Isopropylacrylamide (NIPAAm) and poly(ethylene glycol) to form an associative network gel with thermoresponsive properties [24]. Under these conditions the gels were a viscous liquid at room temperature and soft hydrogels above ⁇ 33°C, with an elastic modulus on par with that of normal breast tissue ( ⁇ 175Pa) [4, 30].
  • HyA biopolymer hyaluronic acid
  • NIPAAm N-Isopropylacrylamide
  • poly(ethylene glycol) poly(ethylene glycol)
  • Each peptide was conjugated to HyA such that their concentration on the polymer chains was 50 ⁇ , resulting in the polymers designated P3-HyA, AGlO-HyA, AG32-HyA, and AG73-HyA.
  • AG73 peptides and they have equal molar representations, however within a basement membrane that has many Ln-1 molecules in unknown orientations the numbers of each of the domains MECs interact with may differ. This presented a combinatorial optimization problem in which composition and concentration were simultaneously varied. Orthogonal array optimization was employed as an efficient means of sampling a large combinatorial space with relatively few experiments [25, 26], with a goal of predicting combinations of peptide-conjugated polymers that would generate maximal ⁇ -casein promoter activity.
  • Ln-1 peptides which otherwise served as inhibitors when used as linear peptides in solution, became agonists that mimicked some activities of Ln-1 when they were grafted to gel- forming biopolymers. Therefore, we have successfully imparted the functional ability of Ln- 1 onto a synthetic polymer and have unambiguously identified the functionally active core of Ln-1 as it may exist in mammary basement membrane. parts can be synthetically presented to reconstitute native laminin-induced functions may lead to new therapeutic and prophylactic strategies. This study presents a logic by which complex ECM molecules can be reduced into minimal units of function that confer tissue-specific functionality onto synthetic scaffolds
  • EpH4 cells were derived from IM-2 cells, originally isolated from the mammary tissue of a mid-pregnant mouse [31, 32].
  • the CFP- cas EpH4 reporter cell line carries a stable selected construct of 16 tandem repeats of the minimal ⁇ -casein reporter that drives eCFP expression [4].
  • EpH4 and cas-CFP EpH4 cells were maintained in growth medium consisting of Dulbecco's modified Eagle's medium/F-12 (UCSF cell culture facility) supplemented with 2% fetal bovine serum (Invitrogen), 50 ⁇ g/ml gentamycin (UCSF cell culture facility), and 5 ⁇ g/ml insulin (Sigma).
  • the cells were plated at a density of 10,000/cm 2 in growth medium and allowed to attach for 16-24 h and then for activity assays they were cultured in serum-free lactogenic differentiation medium composed of Dulbecco's modified Eagle's medium/F-12 medium supplemented with 50 ⁇ g/ml gentamycin, 5 ⁇ g/ml insulin, 1 ⁇ g/ml hydrocortisone (Sigma), and 3 ⁇ g/ml sheep prolactin (Sigma).
  • serum-free lactogenic differentiation medium composed of Dulbecco's modified Eagle's medium/F-12 medium supplemented with 50 ⁇ g/ml gentamycin, 5 ⁇ g/ml insulin, 1 ⁇ g/ml hydrocortisone (Sigma), and 3 ⁇ g/ml sheep prolactin (Sigma).
  • EpH4 Acinus polarity assay - EpH4 or CFP eas EpH4 were plated ontop of a thin layer of Ln-1- peptide infused growth factor reduced Matrigel (Beckton-Dikenson), 40 ⁇ 11 in a 24-well plate, at 50,000 cells/cm 2 and were given 10 minutes to adhere. Lactogenic differentiation medium with 2% growth factor reduced Matrigel that was also infused with the corresponding Ln-1 -peptide was then dripped on top of the cells.
  • Image analyses were performed with NIH ImageJ.
  • the hemispherical optical section from each acinus was identified by the longest circumference, then the smallest possible square-shaped region of interest was drawn to fit each hemisphere and the image of the optical slice was then extracted into an individual file.
  • the sections were elliptical with circularity ratios ranging from 0.95-0.86.
  • the sizes of all square-shaped images for a given treatment (at least 30 images/treatment) were adjusted to the same dimensions and the resulting images were compiled into a single stack.
  • the stacks were separated by channel, and the stacks for each channel were converted into binary images, which were then averaged to make a composite image of each channel using the 'Z-project' command.
  • ⁇ -Casein promoter activity assay - CFP eas EpH4 were seeded in 96-well plates at 3,000 cells/well in growth medium and incubated overnight. Growth medium was aspirated and exchanged with lactogenic differentiation medium alone, which serves as a negative control, or lactogenic differentiation medium with 2% growth factor reduced Matrigel, a positive control. Ln-1 peptides were added to 2% Matrigel medium to determine their impact on ⁇ - casein promoter activity. For the synthetic Ln-1 matrix experiments, the viscous gels were added to cells at room temperature and were then allowed to gel at 37°C for 20 minutes prior to addition of lactation differentiation medium.
  • Hoechst 3342 (Sigma, 1 : 1000) was added to the cells which were subsequently imaged with a Zeiss 710 LSM for CFP expression and nuclear staining. Acquisition parameters were held constant between conditions, and images were batch processed in a blinded and automated fashion using NIH ImageJ. For each well the nuclear stain image was used to create a binary mask which was used to create a selection map. The selection map was used to measure mean CFP intensity per cell in the corresponding CFP image.
  • AWRC Animal Welfare and Research Committee
  • Mammary organoids were suspended within IrECM in 48 well plates (100-200 organoids/gel) overnight in basal media (DMEM/F12 with 1% insulin, transferrin, selenium, and penicillin/streptomycin), also containing 50 ⁇ of the indicated peptide.
  • basal media DMEM/F12 with 1% insulin, transferrin, selenium, and penicillin/streptomycin
  • basal media +/- peptides was supplemented with 9 nM TGFa (Sigma) to stimulate alveolar branching [20]. Every other day, samples were replenished with basal media +/- peptides.
  • branching organoids (defined as having 3 or more buds [20]) were tabulated. Branching factor was calculated by dividing the percentage of branching organoids for a given condition by that obtained for the scrambled peptide condition.
  • HyA-NIPAMM Ln-1 peptide synthesis - N-Isopropylacrylamide (NIPAAm) and methoxy poly(ethylene glycol) methacrylate (mPEGMA) (PEG MW 1 kDa) monomers (Polysciences, Inc.) were dissolved at 96:4 molar ratio in incomplete (without Ca++ or Mg++) phosphate buffered saline (PBS) (Invitrogen Corp.) at 10 wt% total monomer concentration. The solutions were purged of oxygen by bubbling with nitrogen for 30 minutes in a septum-capped conical flask. All reaction components were then cooled to 4°C.
  • ⁇ , ⁇ , ⁇ ', ⁇ '-tetramethylethylenediamine (TEMED) (Polysciences, Inc.) was added as catalyst to the solution at 15 mM through the septum via syringe and needle and gently swirled.
  • Ammonium persulfate (APS) (Sigma-Aldrich, Inc.) was added as initiator by dissolving granular APS in deionized water at 100 mg/mL and quickly adding it to monomer solution via syringe and needle for a final concentration of 1.5 mM.
  • the solution was sterile-filtered at 0.2 ⁇ and allowed to react under nitrogen and agitation at 4oC for 18 hours in the dark.
  • Conjugation reagents including l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (sulfoNHS), [ ⁇ - ⁇ -maleimidocaproic acid] hydrazide trifluoroacetic acid (EMCH), tris(2-carboxyethyl) phosphine (TCEP), and 2-(N- morpholino)ethanesulfonic acid (MES) were obtained from Pierce. All peptides were amino-terminus for coupling via thiol reaction with maleimide groups of an activated HyA backbone. All reactions were performed under aseptic conditions using sterile-filtered solutions and autoclaved containers and instruments.
  • HyA was first activated with a maleimide group by carbodiimide coupling to EMCH.
  • HyA 200 mg was added to 32 mL of MES buffer (0.1 M, pH 6.5) and stirred under low shear until dissolved.
  • Conjugation reagents 440 mg EDC, 123 mg sulfoNHS, and 53 mg EMCH were dissolved in 8.8 mL MES buffer. Eight mL of the conjugation reaction mixture was added through sterile-filter to the HyA solution and stirred.
  • each reagent for EMCH activation of HyA was 5 mg/mL HyA, 10 mg/mL EDC, 2.8 mg/mL sulfoNHS, and 1.2 mg/mL EMCH.
  • the reaction was allowed to proceed for 2 hrs with stirring at room temperature.
  • the solution was then thoroughly dialyzed through a 10 kDa MWCO membrane against MES buffer (0.1 M, pH 6.5).
  • the activated HyA-EMCH solution was split into 4 for reaction with each peptide.
  • the peptides were then reduced and added to the activated HyA-EMCH solution for conjugation via reaction of the maleimide moieties with the thiol group of the peptides.
  • 3 mg of NaOH and 5.7 mg of TCEP were added to 1 mL UPW.
  • the peptides (4.4 mg) were added to alkaline reducing solution, allowed to react for 30 min at 4° C, and added through sterile-filter to the activated HyA-EMCH solution. The final solution was reacted overnight at 4° C in the dark.
  • the conjugate solution was then dialyzed through a 10 kDa MWCO membrane against DPBS (0.08 M, pH 7.2). After dialysis, the solution was transferred to a 50 mL Steriflip tube (BD Bioscience) and lyophilized through the sterile-filter cap. The dry product was stored at -20 oC until characterization or use.
  • the HyA-peptide conjugates were characterized by SEC- MALS with refractive index (RI) and ultra-violet spectroscopy (UV) in order to confirm peptide conjugation and conjugate molecular weight.
  • the conjugates were mixed into the P(NIPAAm-co-mPEGMA) gels at 0.5 wt% (50 mM peptide). The solutions were then vortexed and centrifuged to make them homogeneous. Final copolymer mixtures were characterized for endotoxin levels by limulus amebocyte lysate (LAL) kinetic assay, contamination by agar microbial culture, peptide content by bicinchoninic acid (BCA) protein analysis, and final rheological and mechanical properties by DMA. assays were analyzed using one-way ANOVA followed by the Kruskal-Wallis post-test. * indicates /? ⁇ 0.05, ** indicates /? ⁇ 0.01, *** indicates /? ⁇ 0.001. One representative experiment out of three performed is shown.
  • LAL limulus amebocyte lysate
  • BCA bicinchoninic acid
  • ⁇ the mean CFP intensity/cell measured for reporter cells cultured in each synthetic matrix combination and normalized to the no gel control.
  • the effect of a given level of a given peptide (i.e. the factor effect) (m) was calculated as: m matr ix T
  • Senescent fibroblasts which accumulate in breast tissue with age, produce excess MMP-3 and reduce milk protein production in co-cultured EpH4 cells by nearly two-fold and also disrupt branching morphogenesis [41]. Combined with our observation that apical polarity can be disrupted by impaired binding of even single domains of Ln-1, we speculate that changes in the nature of MEC interactions with Ln-1 that occur as a function of age could predispose older women to cancer by abrogating normal polarity and destabilizing the tissue.
  • EXAMPLE 2 Alternative method for synthesis of the synthetic basement membrane.
  • Peptides that replicate laminin-11 lin vivo such as the AGIO (SEQ ID NO:3), AG32 (SEQ ID NO:4) , AG73 (SEQ ID NO:5), and P3 (SEQ ID NO:8) peptides are conjugated to linear polymers through a 2-step reaction using carbodiimide chemistry at the carboxylate group of the polymer and a maleimide reaction at the protein C-terminal cysteine.
  • the first step is the addition of [N-£-maleimidocaproic acid] hydrazide (EMCH, Pierce Biotechnology, Rockford, IL) to the linear polymer to allow for the subsequent attachment of the peptides.
  • This non-immunogenic hydrazide -maleimide hetereobifunctional crosslinker is added to the two linear polymers using the same general procedure, but with slightly different reaction conditions.
  • 450,000 Da pAAc Polysciences, Warrington, PA
  • EDC l-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
  • Sulfo-NHS Nhydroxysulfosuccinimide
  • EMCH 0.5 mg/ml EMCH at room temperature for 2 hours in pH 6.5 MES buffer as described for the attachment of small peptide sequences.
  • HyA For the activation of HyA, a method similar to that previously described for the attachment of hydrazides in Pouyani, T., and Prestwich, G. D. (1994) Functionalized Derivatives of Hyaluronic- Acid Oligosaccharides -Drug Carriers and Novel
  • Biomaterials. Bioconjugate Chemistry 5, 339-347, can be used with 10 Da MW HyA.
  • Activated polymers can be reacted with the peptides to produce conjugates of varying molecular substitution.
  • This reaction can be performed at 4°C overnight in 0.1 M MES buffer (pH 6.5) containing 50 I1M Tris (2-carboxyethyl) phosphine hydrochloride (TCEP, Pierce Biotechnology, Rockford, IL) to keep the C-terminus cysteine on the peptides reduced for duration of the reaction.
  • TCEP I1M Tris (2-carboxyethyl) phosphine hydrochloride
  • any remaining maleimide groups on the linear EXAMPLE 3 Using the synthetic basement membrane for post-surgical tissue stability after a mastectomy, lumpectomy, or biopsy.
  • Wound healing microevironments have been shown to be capable of pushing phenotypically normal pre-malignant cells into states of full malignancy [45, 46]. Disruption of normal tissue architecture is thought responsible for the deleterious change [47, 48].
  • laminin-111 was shown to be sufficient to communicate normal apical-basal polarity to mammary epithelial cells [2].
  • Current surgical practices are to perform mastectomies, lumpectomies, or biopsies and simply sew up the surgical incision and allow the normal wound healing process to take place.
  • any person undergoing these three procedures are in a higher risk category for harboring pre-malignant mammary epithelial cells, therefore, generation of a surgical wound healing microenvironment could be deleterious and result in recurrence.
  • Addition of synthetic basement membrane after tissue excision and prior to closing would bolster the cues that impart normal polarity in the remaining epithelium.
  • the synthetic basement membrane would therefore serve to minimize adverse wound healing effects and reduce the likelihood of future recurrence of malignancy.
  • EXAMPLE 4 Using the synthetic basement membrane for tissue regeneration or implantation in a patient.
  • synthetic basement membrane may be a superior material for breast augmentation. In principle it will be identified by the patient's body as less of a foreign object and it may have beneficial tissue stabilization effects as described above.
  • the synthetic basement membrane When made using the NIPAMM-PEG formulation of the polymer, the synthetic basement membrane would be thermo reversible, therefore a viscous liquid at room temp that would form a gel at body temp. The gel will not only stabilize the tissue, but it also has a tunable elastic modulus that will make the implants have the same elasticity as the surrounding tissue.
  • EXAMPLE 5 Coating a breast implant with a layer of synthetic basement membrane upon implantation to ensure long-term stability of surrounding tissue.
  • Coating existing silicone implants with synthetic basement membranes may improve stability of mammary epithelium that is damaged in the process of breast augmentation, specifically damaged regions of the epithelium that come in direct contact with the implant.
  • basement membrane induces tissue-specific gene expression in the absence of cell-cell interaction and morphological polarity. J Cell Biol, 1991. 115(5): p. 1383-95.
  • Hepatocyte growth factor /scatter factor induces a variety of tissue-specific morphogenic programs in epithelial cells. J Cell Biol, 1995. 131(6 Pt 1): p. 1573-86. Reichmann, E., R. Ball, B. Groner, and R.R. Friis, New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally. J Cell Biol, 1989. 108(3): p. 1 127-38.
  • stromelysin-1 acts as a natural mammary tumor promoter. Oncogene, 2000. 19(8): p. 1102-13.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dermatology (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Materials For Medical Uses (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention identifie et concerne l'unité minimale de fonction pour la laminine-111 (Ln-1) visant à aider à la conception des membranes de base synthétiques qui pourraient fonctionner à la place de la Ln-1. De petits peptides linéaires qui correspondent à différents domaines de liaison de récepteur de Ln-1 ont été identifiés comme médiateurs cruciaux de l'activité de promoteur de la protéine du lait, d'une polarité apicale, et de l'alvéogénèse. Les peptides ont été combinés à de l'acide hyaluronique biopolymère et combinés à un hydrogel pour créer un analogue synthétique de la molécule Ln-I. Des combinaisons spécifiques des conjugués peptidiques ont donné lieu de manière synergique à une activité de promoteur de protéine du lait. Ainsi, nous avons réussi à identifier l'unité minimale de fonction dans la Ln-1 responsable de la médiation d'un certain nombre de fonctions mammaires essentielles.
PCT/US2010/053877 2009-10-22 2010-10-22 Conception rationnelle d'un polymère de laminine-111 biocompatible et utilisations correspondantes WO2011050342A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US25417709P 2009-10-22 2009-10-22
US61/254,177 2009-10-22

Publications (2)

Publication Number Publication Date
WO2011050342A2 true WO2011050342A2 (fr) 2011-04-28
WO2011050342A3 WO2011050342A3 (fr) 2011-07-28

Family

ID=43901000

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2010/053877 WO2011050342A2 (fr) 2009-10-22 2010-10-22 Conception rationnelle d'un polymère de laminine-111 biocompatible et utilisations correspondantes

Country Status (1)

Country Link
WO (1) WO2011050342A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016183277A1 (fr) * 2015-05-12 2016-11-17 Northwestern University Matériaux pour la régénération tissulaire

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030013648A1 (en) * 1996-10-08 2003-01-16 Gerardo Castillo Peptides for the treatment of Alzheimer's disease and other beta-amyloid protein fibrillogenesis disorders
US20060127352A1 (en) * 1999-02-01 2006-06-15 Eidgenossische Technische Hochschule Zurich Conjugate addition reactions for the controlled delivery of pharmaceutically active compounds
US20060263878A1 (en) * 2003-03-24 2006-11-23 National Institute For Environmental Studies Cell culture medium and solidified preparation of cell adhesion protein or peptide
US20080281419A1 (en) * 2007-05-10 2008-11-13 Matheny Robert G Breast implants and compositions of extracellular matrix
US20090117166A1 (en) * 2007-08-15 2009-05-07 David Myung Sequential coupling of biomolecule layers to polymers

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030013648A1 (en) * 1996-10-08 2003-01-16 Gerardo Castillo Peptides for the treatment of Alzheimer's disease and other beta-amyloid protein fibrillogenesis disorders
US20060127352A1 (en) * 1999-02-01 2006-06-15 Eidgenossische Technische Hochschule Zurich Conjugate addition reactions for the controlled delivery of pharmaceutically active compounds
US20060263878A1 (en) * 2003-03-24 2006-11-23 National Institute For Environmental Studies Cell culture medium and solidified preparation of cell adhesion protein or peptide
US20080281419A1 (en) * 2007-05-10 2008-11-13 Matheny Robert G Breast implants and compositions of extracellular matrix
US20090117166A1 (en) * 2007-08-15 2009-05-07 David Myung Sequential coupling of biomolecule layers to polymers

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BARCELLOS-HOFF ET AL.: 'Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane.' DEVELOPMENT. vol. 105, no. 2, 1989, pages 223 - 35 *
BERKELEY_LAB. SYNTHETIC LAMININ FOR STABILIZING BREAST EPITHELIAL CELLS., [Online] 2010, Retrieved from the Internet: <URL:http://www.lbl.gov/tt/techs/lbnl2801.html> [retrieved on 2011-04-18] *
WANG ET AL.: 'Preadipocytes Stimulate Ductal Morphogenesis and Functional Differentiation of Human Mammary Epithelial Cells on 3D Silk Scaffolds.' TISSUE ENG PART A. vol. 15, no. 10, 16 July 2009, pages 3087 - 98 *
WITTY ET AL.: 'Matrix Metalloproteinases Are Expressed during Ductal and Alveolar Mammary Morphogenesis, and Misregulation of Stromelysin-1 in Transgenic Mice Induces Unscheduled Alveolar Development.' MOL BIOL CELL. vol. 6, no. 10, 1995, pages 1287 - 1303 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016183277A1 (fr) * 2015-05-12 2016-11-17 Northwestern University Matériaux pour la régénération tissulaire
EP3294312A4 (fr) * 2015-05-12 2019-01-09 Northwestern University Matériaux pour la régénération tissulaire
US10463745B2 (en) 2015-05-12 2019-11-05 Northwestern University Materials for tissue regeneration

Also Published As

Publication number Publication date
WO2011050342A3 (fr) 2011-07-28

Similar Documents

Publication Publication Date Title
Yan et al. Enhanced osteogenesis of bone marrow‐derived mesenchymal stem cells by a functionalized silk fibroin hydrogel for bone defect repair
JP6430595B2 (ja) 修飾自己組織化ペプチド
US10689425B2 (en) Collagen-binding synthetic peptidoglycans, preparation, and methods of use
Jue et al. The mouse Wnt-1 gene can act via a paracrine mechanism in transformation of mammary epithelial cells
Curvello et al. Engineered plant‐based nanocellulose hydrogel for small intestinal organoid growth
JP5057781B2 (ja) 修飾を組み込んだ自己集合ペプチドおよびそれを使用する方法
US8901084B2 (en) Self-assembling peptide incorporating modifications and methods of use thereof
JP6821908B2 (ja) 哺乳動物種における誘導組織再生のための組成物および方法
US20110200560A1 (en) Non-ionic self-assembling peptides and uses thereof
Pecci et al. Mutations responsible for MYH9-related thrombocytopenia impair SDF-1-driven migration of megakaryoblastic cells
CN110446500A (zh) 用于基质制备的方法和组合物
US20240041922A1 (en) Methods and compositions
WO2011050342A2 (fr) Conception rationnelle d&#39;un polymère de laminine-111 biocompatible et utilisations correspondantes
US11230586B2 (en) Exosome production method
Hosseinzadeh et al. A novel poly-ε-lysine based implant, Proliferate®, for promotion of CNS repair following spinal cord injury
Hazra et al. In vitro profiling of the extracellular matrix and integrins expressed by human corneal endothelial cells cultured on silk fibroin-based matrices
Bogorodskaya et al. Septins Coordinate with Microtubules and Actin to Initiate Cell Morphogenesis
Chan The Role of Adseverin in Regulating the Articular Chondrocyte Phenotype and Cartilage Homeostasis
Zhang Polyisocyanide hydrogels for modulating epithelial morphogenesis
JP2023548508A (ja) ヒドロゲル組成物及びその使用方法
CA2530482A1 (fr) Peptides autoassembles presentant des modifications et methodes d&#39;utilisation des peptides

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 10825795

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase in:

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 10825795

Country of ref document: EP

Kind code of ref document: A2