Interfacial biomaterials (02-Oct-2003)

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on and claims priority to U.S. provisional patent application serial No. 60/331,843, filed Nov. 20, 2001, herein incorporated by reference in its entirety.

GRANT STATEMENT

FIELD OF THE INVENTION

[0003] The present invention generally relates to interfacial biomaterials that mediate interaction between a non-biological substrate and a biological substrate, and methods for preparing and using the same. More particularly, the present invention relates to binding agents that create a binding interface between substrates via specific binding of each substrate. The present invention also relates to binding agents that create a non-binding interface between substrates via specific binding to a non-biological substrate and substantially no binding to a biological substrate. 1

Table of Abbreviations

	AFM	atomic force microscope
	Ang1	Angiopoitin-1
	BAP	bacterial alkaline phosphatase
	BNHS	biotin N-hydroxysuccinimide ester
	BSA	bovine serum albumin
	DMSO	dimethyl sulfoxide
	DWI	diffusion-weighted imaging
	ELISA	enzyme-linked immunosorbent assay
	ExFms	purified extracellular domain of the Fms
		receptor
	ExTek	purified extracellular domain of the Tie2
		receptor
	FMOC	N-9-fluorenylmethyloxycarbonyl
	fMRI	functional MR imaging
	FTIR	Fourier Transform Infrared spectroscopy
	GFP	green fluorescent protein
	GST	glutathione-S-transferase
	HPLC	high performance liquid chromatography
	HRP	horseradish peroxidase
	IFBM	interfacial biomaterial
	IgG	immunoglobulin type G
	ITO	indium tin oxide
	I.U.B.	International Union of Biochemists
	Ka	association constant
	MRS	proton magnetic resonance spectroscopy
	MTI	magnetization transfer imaging
	NIH	National Institutes of Health
	pIII	M13 phage gene encoding coat protein
	PIII	M13 phage coat protein
	PBS	phosphate buffered saline
	PBS-T	PBS + 1% TRITON-X ® detergent
	PEG	polyethylene glycol
	PELL	pellethane
	PEPT	polyethylene terephthalate
	PET	positron emission tomography
	PFU	plaque-forming unit
	PGA	polyglycolic acid
	PHEMA	2-hydroxyethyl methacrylate
	PLA	polylactate
	PMMA	polymethylmethacrylate
	PPACK	D-phenylalanyl-L-prolyl-L-arginine
		chloromethylketone
	TNF	tumor necrosis factor
	scFv	single chain fragment variable antibody
	SPECT	single photon emission computed
		tomography
	SPR	surface plasmon resonance
	TG1	a strain of E. coli cells
	TSAR	totally synthetic affinity reagents
	VEGF	vascular endothelial growth factor

[0004] 2

Amino Acid Abbreviations and Corresponding mRNA Codons

Amino Acid	3-Letter	1-Letter	mRNA Codons
Alanine	Ala	A	GCA GCC GCG GCU
Arginine	Arg	R	AGA AGG CGA CGC CGG CGU
Asparagine	Asn	N	AAC AAU
Aspartic Acid	Asp	D	GAC GAU
Cysteine	Cys	C	UGC UGU
Glutamic Acid	Glu	E	GAA GAG
Glutamine	Gln	Q	CAA CAG
Glycine	Gly	G	GGA GGC GGG GGU
Histidine	His	H	CAC CAU
Isoleucine	Ile	I	AUA AUC AUU
Leucine	Leu	L	UUA UUG CUA CUC CUG CUU
Lysine	Lys	K	AAA AAG
Methionine	Met	M	AUG
Proline	Pro	P	CCA CCC CCG CCU
Phenylalanine	Phe	F	UUC UUU
Serine	Ser	S	ACG AGU UCA UCC UCG UCU
Threonine	Thr	T	ACA ACC ACG ACU
Tryptophan	Trp	W	UGG
Tyrosine	Tyr	Y	UAC UAU
Valine	Val	V	GUA GUC GUG GUU

BACKGROUND OF THE INVENTION

[0005] The remarkable specificity of binding and function displayed by organic molecules has motivated efforts to employ these binding and functional activities in new ways. Molecular display technologies have facilitated these efforts by permitting rapid identification of specific binding agents for almost any target molecule. In particular, phage display of peptides and proteins (including antibodies) have led to the discovery of natural and designer binding sites.

[0006] Phage display systems use highly diverse libraries constructed by fusing degenerate sequences of DNA to a gene encoding a phage coat protein, such that the encoded variable protein sequence is displayed on the phage coat. Individual phage with desired binding specificities are isolated by binding to an immobilized or selectable target molecule. The peptides or proteins that confer binding are identified by sequencing the DNA within selected phage.

[0007] Peptides and proteins having unique binding and functional properties can be used as therapeutic agents (Raum et al., 2001), as templates for molecular design, including drug design (Ballinger et al., 1999; Bolin et al., 2000; Wolfe et al., 2000; Mourez et al., 2001; Rudgers & Palzkill, 2001), as homing molecules for drug delivery (Arap et a!., 1998; Nilsson et al., 2000; Ruoslahti, 2000), and as compositions to promote cellular attachment in cases of tissue healing or repair (e.g., U.S. Pat. Nos. 5,856,308; 5,635,482; and 5,292,362).

[0008] Phage display has also been used to select peptides that bind to inorganic surfaces with high specificity. Semiconductor surface-binding peptides that also bind a second molecule are suggested for assembly of electronic structures. See Whaley et al., 2000.

[0009] Recent interest has developed in compositions that mimic recognition and functional capabilities of biological molecules to mediate interactions involving non-biological materials. For example, peptides can be used to coat prosthetic devices to thereby promote attachment of endothelial cells following implantation. See U.S. Pat. Nos. 6,280,760; 6,140,127; 4,960,423; and 4,378,224.

[0010] Prior to the disclosure of the present invention, preparation of peptide-coated surfaces and devices has been accomplished by non-specific adsorption, by coupling of the peptide to a derivatized surface, or by coupling of the peptide to a linker molecule covalently attached to the surface. These procedures are relatively tedious and time-consuming, and they generally require multiple steps for effective association of the peptide and the substrate. However, the potential benefits of non-biological surfaces and devices that include a biological coat are clear.

[0011] Thus, there exists a long-felt need in the art to develop an efficient and widely applicable method for promoting specific interactions between non-biological substrates and biological substrates. In addition, there exists a continuing need to develop methods for directing interactions among molecules and/or cells, particularly in the context of diagnostic and therapeutic treatments.

[0012] To meet this need, the present invention provides interfacial biomaterials that can mediate selective interactions between biological and non-biological substrates, novel binding agents that can specifically bind a target non-biological substrate and/or a target biological substrate, and methods for making and using the same.

SUMMARY OF INVENTION

[0013] The present invention provides an interfacial biomaterial comprising a plurality of binding agents wherein each binding agent comprises a first ligand that specifically binds a non-biological substrate and a second ligand that specifically binds a biological substrate, and wherein the plurality of binding agents comprise an interface between the non-biological substrate and the biological substrate.

[0014] The present invention also provides an interfacial biomaterial comprising a plurality of binding agents wherein each binding agent comprises first and second ligands that specifically bind a biological substrate, and wherein the plurality of binding agents comprise an interface between the biological substrates. In one embodiment, the first and second ligands bind the same biological substrate. In another embodiment, the first and second ligands bind different biological substrates.

[0015] The present invention also provides an interfacial biomaterial comprising a plurality of binding agents, wherein each binding agent comprises a ligand that specifically binds a target non-biological substrate and a non-binding domain that substantially lacks binding to a target biological substrate.

[0016] The interfacial biomaterial can comprise a plurality of identical or non-identical binding agents. When the interfacial biomaterial comprises a plurality of non-identical binding agents, each of the plurality of non-identical binding agents comprises in one embodiment an identical ligand that specifically binds a non-biological substrate.

[0017] The present invention further provides a patterned interfacial biomaterial, wherein the binding agents are spatially restricted within the interface.

[0018] Representative non-biological substrates include but are not limited to a non-biological substrate comprising a synthetic polymer, plastic, metal, a metal oxide, a non-metal oxide, silicone, a ceramic material, a drug, or a drug carrier. In one embodiment, a synthetic polymer comprises polyglycolic acid. In another embodiment, a synthetic polymer comprises a nylon suture. In one embodiment, a plastic comprises polycarbonate, in another embodiment polystyrene, and in yet another embodiment polyurethane. In one embodiment, a metal comprises titanium. In another embodiment, a metal comprises stainless steel.

[0019] Representative biological substrates include but are not limited to a tissue, a cell, or a macromolecule. In one embodiment, a target biological substrate comprises collagen. In another embodiment, a biological substrate comprises a Tie2 receptor.

[0020] Also provided are methods for preparing an interfacial biomaterial. Thus, in one embodiment of the invention, the method comprises: (a) applying to a non-biological substrate a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds to the non-biological substrate and a second ligand that specifically binds a target biological substrate, and wherein the applying is free of coupling; (b) contacting the non-biological substrate, wherein the plurality of binding agents are bound to the non-biological substrate, with a sample comprising the target biological substrate; and (c) allowing a time sufficient for binding of the target biological substrate to the plurality of binding agents, wherein an interfacial biomaterial is prepared. In accordance with the disclosed invention, the contacting can comprise contacting in vitro, ex vivo, or in vivo.

[0021] In another embodiment of the invention, an interfacial biomaterial comprises a biological array. In one embodiment, a method for preparing an interfacial biomaterial comprises: (a) providing a non-biological substrate having a plurality of positions; (b) applying to each of the plurality of positions a binding agent comprising a first ligand that specifically binds the non-biological substrate and a second ligand that specifically binds a target biological substrate, wherein the applying is free of coupling; (c) contacting the non-biological substrate, wherein a plurality of binding agents are bound to the non-biological substrate, with a sample comprising the target biological substrate; and (d) allowing a time sufficient for binding of the target biological substrate to the plurality of binding agents, whereby a biological array is prepared. In one embodiment, a method for applying the plurality of binding agents comprises dip-pen printing.

[0022] In still another embodiment of the invention, a method for preparing an interfacial biomaterial comprises: (a) applying to a non-biological substrate a plurality of binding agents, wherein each of the plurality of binding agents comprises a ligand that specifically binds to the non-biological substrate and a non-binding domain that shows substantially no binding to a target biological substrate, and wherein the applying is free of coupling; and (b) contacting the non-biological substrate, wherein the plurality of binding agents are bound to the non-biological substrate, with a sample comprising the target biological substrate, whereby an interfacial biomaterial is prepared.

[0023] The present invention further provides methods for preparing binding agents. In one embodiment of the invention, the method comprises: (a) panning a library of diverse molecules over a target non-biological substrate, whereby a first ligand that specifically binds a target non-biological substrate is identified; and (b) linking the first ligand to a second ligand, wherein the second ligand specifically binds a target biological substrate, whereby a binding agent is prepared. The method can further comprise panning a ligand over a target biological substrate, whereby a ligand that specifically binds a target biological substrate is identified.

[0024] In another embodiment of the invention, a method for preparing a binding agent comprises: (a) panning a library of diverse molecules over a target non-biological substrate, whereby a ligand that specifically binds a target non-biological substrate is identified; and (b) linking the ligand to a non-binding domain, wherein the non-binding domain shows substantially no binding to a target biological substrate, whereby a binding agent is prepared. The method can further comprise panning a ligand over a target biological substrate, whereby a non-binding domain that shows substantially no binding to a target biological substrate is identified.

[0025] Also provided are binding agents produced by the method. In one embodiment of the invention, a binding agent further comprises a linker that links the first ligand and the second ligand, or a linker that links the first ligand and non-binding domain.

[0026] In one embodiment of the invention, the first ligand comprises a peptide or single chain antibody that specifically binds a non-biological substrate. Representative plastic-binding ligands are set forth as SEQ ID NOs:1-23 and 66-71, and representative metal-binding ligands are set forth as SEQ ID NOs:24-36 and 51-65. In one embodiment, the second ligand or non-binding region comprises a peptide or single chain antibody.

[0027] Thus, the present invention also provides synthetic peptides comprising polystyrene-binding, polyurethane-binding, polycarbonate-binding, polyglycolic acid-binding, titanium-binding, stainless steel-binding ligands. In one embodiment, the synthetic ligands comprise less than about 20 amino acid residues. Representative polystyrene-binding peptide ligands are set forth as SEQ ID NOs:1-22, a representative polyurethane-binding ligand is set forth as SEQ ID NO:23, representative polycarbonate-binding ligands are set for as SEQ ID NOs:66-71, representative titanium-binding peptide ligands are set forth as SEQ ID NOs:24-36, and representative stainless steel-binding ligands are set forth as SEQ ID NOs:51-65.

[0028] The present invention further provides representative methods for using an interfacial biomaterial, including, but not limited to a method for cell culture, a method for implanting a device in a subject, a method for modulating an activity of a biological substrate, a method for preparing a non-fouling coating, a method for drug delivery, and a method for screening for screening a test substance for interaction with a biological substrate.

[0029] A method for cell culture, in accordance with the present invention, can comprise: (a) applying to a non-biological substrate a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds the non-biological substrate and a second ligand that specifically binds cells, macromolecules or a combination thereof, wherein the applying is free of coupling; (b) contacting the non-biological substrate, wherein the plurality of binding agents are bound to the non-biological substrate, with cells; (c) allowing a time sufficient for binding of the cells to the plurality of binding agents; and (d) culturing the cells.

[0030] The present invention also provides methods for implanting a device in a subject. In one embodiment of the invention, the method comprises: (a) applying to an implant a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds the implant and a second ligand that specifically binds cells at an implant site, wherein the applying is free of coupling; and (b) placing the implant in a subject at the implant site. When implanted in a subject, a device so prepared can promote cell attachment to the device.

[0031] The present invention also provides a method for creating a lubricant interface comprising: applying to a first substrate a plurality of binding agents, wherein the applying is free of coupling, and wherein each of the plurality of binding agents comprises: (a) a ligand that specifically binds to the first substrate; and (b) a non-binding domain that shows substantially no binding to a second substrate. The first substrate can comprise a non-biological or a biological substrate.

[0032] Thus, in another embodiment of the invention, a method for implanting a device in a subject can comprise: (a) applying to the implant a plurality of binding agents, wherein each of the plurality of binding agents comprises a ligand that specifically binds the implant and a non-binding domain that shows substantially no binding to cells at an implant site, wherein the applying is free of coupling; and (b) placing the implant in a subject at the implant site. When implanted in a subject, a device so prepared can provide a lubricating activity at the implant site.

[0033] A method for preparing an interfacial biomaterial comprising a boundary lubricant can also comprise: (a) administering to a subject a plurality of binding agents, wherein each of the plurality of binding agents comprises a ligand that specifically binds a first biological substrate and a non-binding domain that shows substantially no binding to a second biological substrate; and (b) allowing a time sufficient for binding of the plurality of binding agents to the first biological substrate, whereby a lubricant interface is created.

[0034] Also provided is a method for modulating an activity of a biological substrate, the method comprising: (a) coating a biodegradable, non-biological substrate with a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds the biodegradable, non-biological substrate and a second ligand that specifically binds the biological substrate, wherein the coating is free of coupling; (b) placing the coated biodegradable, non-biological substrate at a target site, wherein the biological substrate is present at the target site; and (c) allowing a time sufficient for binding of the biological substrate at the target site to the binding agents, wherein the binding modulates the activity of the biological substrate. In one embodiment, a biological substrate is a vascular endothelial cell. In another embodiment, biological substrate is a tumor vascular endothelial cell. In yet another embodiment, a biological substrate is a Tie2 receptor. In one embodiment, a target site is a wound site, and the modulating promotes wound healing. In another embodiment, a target site is an angiogenic site and the modulating inhibits angiogenesis, including, but not limited to tumor angiogenesis.

[0035] The present invention further provides a method for preparing a non-biological substrate with a non-fouling coating. The coating comprises a plurality of binding agents, wherein each of the plurality of binding agents comprises: (a) a ligand that specifically binds the non-biological substrate; and (b) a non-binding domain that shows substantially no binding to a fouling agent.

[0036] The present invention also provides a method for drug delivery involving an interfacial biomaterial. The method comprises: (a) applying to a non-biological drug, or to a non-biological carrier of the drug, a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds the drug or the drug carrier and a second ligand that specifically binds a target cell; (b) administering the drug to a subject; and (c) allowing a sufficient time for binding of the plurality of binding agents to the target cell.

[0037] Also provided is a method for screening a test substance for interaction with a biological substrate. In one embodiment, the method comprises: (a) preparing a biological array comprising a plurality of biological substrates, wherein each of the plurality of biological substrates is specifically bound to one of a plurality of positions on a non-biological substrate; (b) contacting the biological array with a candidate substance; (c) allowing a time sufficient for binding of the candidate substance to the biological array; and (d) assaying an interaction between one or more of the biological substrates and the candidate substance, whereby an interacting molecule is identified.

[0038] Accordingly, it is an object of the present invention to provide interfacial biomaterials that can mediate direct binding and non-binding interactions between substrates. This object is achieved in whole or in part by the present invention.

[0039] An object of the invention having been stated above, other objects and advantages of the present invention will become apparent to those skilled in the art after a study of the following description of the invention and non-limiting Examples.

DETAILED DESCRIPTION OF THE INVENTION

[0040] I. Definitions

[0041] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the invention.

[0042] The term “ligand” as used herein refers to a molecule or other chemical entity having a capacity for binding to a substrate. A ligand can comprise a peptide, an oligomer, a nucleic acid (e.g., an aptamer), a small molecule (e.g., a chemical compound), an antibody or fragment thereof, a nucleic acid-protein fusion, a polymer, a polysaccharide, and/or any other affinity agent.

[0043] The term “non-binding domain” as used herein refers to a molecule, macromolecule, or other chemical entity that shows substantially no binding to a target substrate. A non-binding domain can comprise a peptide, an oligomer, a nucleic acid (e.g., an aptamer), a small molecule (e.g., a chemical compound), an antibody or fragment thereof, a nucleic acid-protein fusion, a polymer, a polysaccharide, and/or any other agent that shows substantially no binding to a target substrate.

[0044] The term “substrate” as used herein refers to a biological or non-biological composition used to prepare an interfacial biomaterial. Thus, the term “substrate” encompasses compositions having a capacity for binding to a ligand of the invention as well as compositions showing substantially no binding to a non-binding domain of the invention.

[0045] The term “target” is typically used to qualify a description of a substrate as one of multiple substrates having different binding specificities. Thus, the term “target” generally refers to a substrate that is specifically bound by a ligand of the present invention, or to a substrate that shows substantially no binding to a non-binding domain of the present invention.

[0046] The term “binding” refers to an affinity between two molecules, for example, between a peptide and a substrate. As used herein, “binding” refers to a preferential binding of a peptide for a substrate in a mixture of molecules. The binding of a peptide to a substrate can be considered specific if the binding affinity is about 1×10⁴ M⁻¹ to about 1×10⁶ M⁻¹ or greater.

[0047] The phrase “specifically (or selectively) binds”, when referring to the binding capacity of a ligand, refers to a binding reaction that is determinative of the presence of the substrate in a heterogeneous population of other substrates. Specific binding excludes non-specific adsorption, covalent linkage via a chemical reaction, and coupling via a linking moiety.

[0048] The term “time sufficient for binding” generally refers to a temporal duration sufficient for specific binding of a ligand and a substrate.

[0049] The phases “substantially lack binding” or “substantially no binding”, as used herein to describe binding of a ligand or non-binding domain to a substrate, refers to a level of binding that encompasses non-specific or background binding, but does not include specific binding.

[0050] The term “subject” as used herein refers to any invertebrate or vertebrate species. The methods of the present invention are particularly useful in the treatment and diagnosis of warm-blooded vertebrates. Thus, the invention concerns mammals and birds. More particularly, contemplated is the treatment and/or diagnosis of mammals such as humans, as well as those mammals of importance due to being endangered (such as Siberian tigers), of economical importance (animals raised on farms for consumption by humans) and/or social importance (animals kept as pets or in zoos) to humans, for instance, carnivores other than humans (such as cats and dogs), swine (pigs, hogs, and wild boars), ruminants (such as cattle, oxen, sheep, giraffes, deer, goats, bison, and camels), and horses. Also contemplated is the treatment of birds, including the treatment of those kinds of birds that are endangered, kept in zoos, as well as fowl, and more particularly domesticated fowl, e.g., poultry, such as turkeys, chickens, ducks, geese, guinea fowl, and the like, as they are also of economical importance to humans. Thus, contemplated is the treatment of livestock, including, but not limited to, domesticated swine (pigs and hogs), ruminants, horses, poultry, and the like.

[0051] The term “about”, as used herein when referring to a measurable value such as a number of amino acids, etc. is meant to encompass variations of in one embodiment ±20% or ±10%, in another embodiment ±5%, in another embodiment ±1%, and in yet another embodiment ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods.

[0052] II. Interfacial Biomaterials

[0053] The present invention provides an interfacial biomaterial comprising a plurality of binding agents. In one embodiment of the invention, each binding agent specifically binds a non-biological substrate and a biological substrate, to thereby create an interface between the non-biological substrate and the biological substrate. Also provided are binding agents and methods for making the same, as described further herein below.

[0054] The term “interfacial biomaterial” is used herein to broadly refer to a composition comprising a plurality of binding agents, wherein the plurality of binding agents creates a functional interface between two or more substrates. Each of the binding agents comprises two or more desired binding specificities, or a desired combination of binding specificities, including: (a) specific binding of at least one non-biological substrate; (b) and specific binding of at least one target biological substrate or substantially no binding of a target biological substrate.

[0055] Several prior studies have described ligands having two or more binding specificities. For example, U.S. Pat. No. 5,948,635 to Kay et al. discloses totally synthetic affinity reagents (TSARs) comprising bivalent fusion peptides. As defined therein, a bivalent peptide comprises two functional regions: a binding domain and an effector domain that is useful for enhancing expression and/or detection of the expressed TSAR. In contrast to the bivalent peptides described in U.S. Pat. No. 5,948,635 to Kay et al., an interfacial biomaterial of the present invention comprises two or more binding domains and does not require an element for enhancing expression and/or detection of the interfacial biomaterial. In addition, U.S. Pat. No. 5,948,635 to Kay et al. does not disclose creation of an interfacial biomaterial comprising a plurality of binding agents, wherein the plurality of binding agents creates a functional interface between two or more substrates.

[0056] The term “functional interface” refers to an interface, wherein the functionality of the interface requires a plurality of binding agents. More particularly, a functional interface is not created by a binding reaction between a single binding agent and a substrate. For example, a binding interaction between a solid support, such as a purification column, and a molecule of interest does not comprise a functional interface in that the functionality of the interaction (purification) can comprise a single reagent and a single molecule of interest.

[0057] Representative functional interfaces include coatings, wherein the plurality of binding agents comprises a binding interface, a non-binding interface, or a combination thereof. The term “binding interface” refers to an interface created using binding agents comprising a first ligand that specifically binds a first substrate (e.g., a non-biological substrate) and a second ligand that specifically binds a second substrate (e.g., a biological substrate. Thus, a binding interface mediates interaction between two or more substrates by providing an affinity for each of the two or more substrates. In one embodiment, the two or more substrates are all the same. In another embodiment, the two or more substrates are not all the same.

[0058] The term “non-binding interface” refers to an interface created using binding agents comprising a first ligand that specifically binds a first substrate (e.g., a non-biological substrate) and a second ligand that shows substantially no binding to a second substrate (e.g., a target biological substrate). Additionally, a non-binding interface can be created using binding agents comprising a first ligand that shows substantially no binding to a target non-biological substrate and a second ligand that specifically binds a biological substrate. A non-binding interface thus ensures a lack of interaction between two or more substrates.

[0059] A functional interface can also comprise a biological array, wherein each of the plurality of binding agents is adhered to a substrate at a prescribed position, and the sum of each of the plurality of binding agents comprises a pattern. In one embodiment of a patterned interfacial biomaterial in accordance with the present invention, binding agents of the present invention are applied to a non-biological interface in a spatially restricted manner, as described further herein below.

[0060] An interfacial biomaterial of the present invention can comprise a homogeneous interfacial biomaterial, wherein each of the plurality of binding agents is identical. Alternatively, an interfacial biomaterial can be heterogeneous by constructing the interfacial biomaterial using a plurality of non-identical binding agents. In one embodiment, each of the plurality of non-identical binding agents comprises: (a) an identical ligand that specifically binds a first substrate (preferably a non-biological substrate); and (b) a variable domain. The variable domain can be selected from among any of a variety of ligands or non-binding domains for substrates (in one embodiment, a biological substrate), so that a plurality of substrates (in one embodiment, a biological substrate) can be bound and/or not bound.

[0061] For example, a heterogeneous interfacial biomaterial can comprise a plurality of non-identical binding agents, wherein each of the plurality of non-binding agents comprises: (a) a first ligand that specifically binds polystyrene; and (b) a second ligand that specifically binds one of a variety of cell types. The plurality of binding agents can be adhered to a polystyrene substrate. A sample comprising a mixed cell population, wherein each of a different type of cell in the mixed cell population specifically binds one of the plurality of second ligands, can be provided to the polystyrene substrate. Following a time sufficient for binding of the mixed cell population to the plurality of binding agents, a heterogeneous interfacial biomaterial is formed between the polystyrene substrate and the mixed cell populations.

[0062] In one embodiment of the invention, preparation of a heterogeneous interfacial biomaterial can comprise: (a) adhering at random positions on a non-biological substrate each of a plurality of non-identical binding agents; or (b) adhering at known positions on a non-biological substrate each of a plurality of non-identical binding agents. Thus, a heterogeneous interfacial biomaterial can comprise a randomly heterogeneous or a patterned heterogeneous interfacial biomaterial.

[0063] A patterned interfacial biomaterial can be prepared in one embodiment by delivering each of a plurality of binding agents to a discrete position on a non-biological substrate using any technique suitable for dispensing a binding agent, including but not limited to spraying, painting, ink-jetting, dip-pen writing (Example 15), microcontact printing (U.S. Pat. Nos. 6,180,239 and 6,048,623), stamping (U.S. Pat. Nos. 5,512,131 and 5,776,748), or lithography (Bhatia et al., 1993), PCT International Publication No. WO 00/56375.

[0064] The present invention further provides methods for preparing an interfacial biomaterial. In one embodiment of the invention, a method for preparing a binding interfacial biomaterial comprises: (a) applying to a non-biological substrate a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds to the non-biological substrate and a second ligand that specifically binds a target biological substrate, and wherein the applying is free of coupling; (b) contacting the non-biological substrate, wherein the plurality of binding agents are bound to the non-biological substrate, with a sample comprising the target biological substrate; and (c) allowing a time sufficient for binding of the target biological substrate to the plurality of binding agents, whereby an interfacial biomaterial is prepared.

[0065] Alternatively, binding of the plurality of binding agents to each of a non-biological substrate and a biological substrate can be performed simultaneously or in the reverse order, depending on a particular application. Thus, a method for preparing a binding interfacial biomaterial can also comprise: (a) contacting a plurality of binding agents, wherein each of the binding agents comprises a first ligand that specifically binds to the non-biological substrate and a second ligand that specifically binds a target biological substrate, and wherein the applying is free of coupling; (b) applying to a non-biological substrate a plurality of binding agents; and (c) allowing a time sufficient for binding of the non-biological substrate to the plurality of binding agents, whereby an interfacial biomaterial is prepared.

[0066] In another embodiment of the invention, a method for preparing a non-binding interfacial biomaterial comprises: (a) applying to a non-biological substrate a plurality of binding agents, wherein each of the plurality of binding agents comprises a ligand that specifically binds to the non-biological substrate and a non-binding domain that shows substantially no binding to a target biological substrate, and wherein the applying is free of coupling and free of covalent linkage; and (b) contacting the non-biological substrate, wherein the plurality of binding agents are bound to the non-biological substrate, with a sample comprising the target biological substrate, whereby an interfacial biomaterial is prepared.

[0067] II.A. Non-Biological Substrates

[0068] The term “non-biological substrate” is used herein to describe a substrate that is not a quality or component of a living system. Representative non-biological substrates include but are not limited to common plastics (e.g., polystyrene, polyurethane, polycarbonate), silicone, synthetic polymers, metals (including mixed metal alloys), metal oxides (e.g., glass), non-metal oxides, ceramics, drugs, drug carriers, and combinations thereof.

[0069] A non-biological substrate can comprise any form suitable to its intended use including but not limited to a planar surface (e.g., a culture plate), a non-planar surface (e.g., a dish, an implant, or a tube), or a substrate in solution. In one embodiment, a non-biological substrate comprises a minimum dimension of at least about 20 nm. For example, a non-biological substrate can comprise a minimum dimension of about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1 μm, about 50%m, about 100 μm, about 200 μm, about 500 μm, or about 1 mm.

[0070] Representative synthetic polymers include but are not limited to polytetrafluoroethylene, expanded polytetrafluoroethylene, GORE-TEX® (Gore & Associates, Inc. of Newark, Del.), polytetrafluoroethylene, fluorinated ethylene propylene, hexafluroropropylene, polymethylmethacrylate (PMMA), pellethane (a commercial polyurethane, PELL), 2-hydroxyethyl methacrylate (PHEMA), polyethylene terephthalate (PEPT), polyethylene, polypropylene, nylon, polyethyleneterephthalate, polyurethane, silicone rubber, polystyrene, polysulfone, polyester, polyhydroxyacids, polycarbonate, polyimide, polyamide, polyamino acids, and combinations thereof. In one embodiment, a synthetic polymer comprises an expanded or porous polymer. In another embodiment, a synthetic polymer comprises a nylon suture.

[0071] Representative metals that can be used in accordance with the methods of the present invention include but are not limited to titanium, stainless steel, gold, silver, rhodium, zinc, platinum, rubidium, and copper. Suitable ceramic materials include but are not limited to silicone oxides, aluminum oxides, alumina, silica, hydroxyapapitites, glasses, quartz, calcium oxides, calcium phosphates, indium tin oxide (ITO), polysilanols, phosphorous oxide, and combinations thereof.

[0072] Other non-biological substrates include carbon-based materials such as graphite, carbon nanotubes, carbon “buckyballs”, and metallo-carbon composites.

[0073] Preparation of an interfacial biomaterial for drug delivery can employ a non-biological substrate comprising a drug or drug carrier. The term “drug” as used herein refers to any substance having biological or detectable activity. Thus, the term “drug” includes a pharmaceutical agent, a detectable label, or a combination thereof. The term “drug” also includes any substance that is desirably delivered to a target cell.

[0074] The term “drug carrier”, as used herein to describe a non-biological substrate, refers to a composition that facilitates drug preparation and/or administration. Any suitable drug delivery vehicle or carrier can be used, including but not limited to a gene therapy vector (e.g., a viral vector or a plasmid), a microcapsule (for example, a microsphere or a nanosphere, Manome et al., 1994; Saltzman & Fung, 1997), a fatty emulsion (U.S. Pat. No. 5,651,991), a nanosuspension (U.S. Pat. No. 5,858,410), a polymeric micelle or conjugate (Goldman et al., 1997; U.S. Pat. Nos. 4,551,482, 5,714,166, 5,510,103, 5,490,840, and 5,855,900), a liposome (U.S. Pat. Nos. 6,214,375; 6,200,598; 6,197,333); and a polysome (U.S. Pat. No. 5,922,545).

[0075] The term “detectable label” refers to any substrate that can be detected, including, but not limited to an agent that can be detected using non-invasive methods such as scintigraphic methods, magnetic resonance imaging, ultrasound, spectroscopic, enzymatic, electrochemical, and/or fluorescence. Representative substrates useful for non-invasive imaging are described herein below.

[0076] A non-biological substrate is selected for a desired application based on a number of factors including but not limited to biocompatibility, degradability, surface area to volume ratio, and mechanical integrity. For clinical applications, a non-biological substrate can comprise a biocompatible non-biological substrate such as titanium, synthetic polymers (e.g., silicone), and any other biocompatible non-biological substrate. A non-biological substrate can also be rendered biocompatible by application of a plurality of binding agents as disclosed herein. Selection of a suitable non-biological substrate is within the skill of one in the art.

[0077] II.B. Biological Substrates

[0078] The term “biological substrate” as used herein refers to a quality or component pertaining to living systems. As such, a “biological substrate” can comprise an organ, a tissue, a cell, or components thereof. Thus, a biological substrate can comprise a macromolecule including, but not limited to a protein (e.g., an antibody, collagen, a receptor), a peptide, a nucleic acid (e.g., an aptamer), an oligomer, a small molecule (e.g., a chemical compound), a nucleic acid-protein fusion, and/or any other biological affinity agent. The term “biological substrate” also encompasses substrates that have been isolated from a living system and substrates that have been recombinantly or synthetically produced.

[0079] III. Binding Agents

[0080] The term “binding agent” refers to a composition that mediates a binding or non-binding interaction between two substrates. In one embodiment, a binding agent mediates an interaction between a non-biological substrate and a biological substrate. Thus, in one embodiment of the present invention, a binding agent comprises: (a) a ligand that specifically binds a non-biological substrate; and (b) a ligand that specifically binds a biological substrate. In another embodiment of the invention, a binding agent comprises: (a) a ligand that specifically binds a non-biological substrate; and (b) a non-binding domain that shows substantially no binding to a target biological substrate.

[0081] A ligand that specifically binds a non-biological substrate shows specific binding in the absence of covalent linkage or coupling via a linking moiety. For example, the binding between the ligand and the non-biological substrate is free of any of the forms of linking described herein below as they pertain to, for example, linking a first and second ligand of a binding agent.

[0082] A ligand that specifically binds a biological substrate can possess additional bioactivity as a result of specific binding. For example, a ligand can additionally show kinase activity, phosphatase activity, DNA repair activity, oncogene activity, tumor suppressor activity, angiogenesis stimulatory activity, angiogenesis inhibitory activity, mitogenic activity, signaling activity, transport activity, enzyme activity, anti-fouling activity, anti-bacterial activity, anti-viral activity, antigenic activity, immunogenic activity, apoptosis-inducing activity, anti-apoptotic-inducing activity, cytotoxic activity, lubricant activity, and combinations thereof.

[0083] A binding agent can be constructed by linking a first and second ligand, or a ligand and a non-binding domain, to form a single molecule or complex. Linking can comprise fusing two or more peptide ligands during synthesis, as described in Examples 12 and 13. Optionally, a peptide linker region between the two domains can also be incorporated during synthesis. Alternatively, a first and second ligand, or a ligand and a non-binding domain, can be combined via a linker by covalent bonding or chemical coupling, as described further herein below.

[0084] III.A. Peptides

[0085] In one embodiment of the invention, a ligand comprises a peptide ligand that specifically binds to a non-biological substrate and/or to a biological substrate. Similarly, in one embodiment a non-binding domain comprises a peptide that shows substantially no binding to a target biological substrate.

[0086] The term “peptide” broadly refers to an amino acid chain that includes naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non-genetically encoded amino acids, and combinations thereof. Peptides can include both L-form and D-form amino acids. A peptide of the present invention can be subject to various changes, substitutions, insertions, and deletions where such changes provide for certain advantages in its use. Thus, the term “peptide” encompasses any of a variety of forms of peptide derivatives including amides, conjugates with proteins, cyclone peptides, polymerized peptides, conservatively substituted variants, analogs, fragments, chemically modified peptides, and peptide mimetics.

[0087] In one embodiment of the invention, the peptide comprises an amino acid sequence comprising at least about 3 residues, in another embodiment about 3 to about 50 residues, and in yet another embodiment about 3 to about 25 residues. Any peptide ligand that shows specific binding features can be used in the practice of the present invention. In one embodiment, peptide fragments containing less than about 25 amino acid residues are employed. In another embodiment, peptide fragments less than about 20 amino acids are employed.

[0088] Representative non-genetically encoded amino acids include but are not limited to 2-aminoadipic acid; 3-aminoadipic acid; β-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid (piperidinic acid); 6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3-aminoisobutyric acid; 2-aminopimelic acid; 2,4-diaminobutyric acid; desmosine; 2,2′-diaminopimelic acid; 2,3-diaminopropionic acid; N-ethylglycine; N-ethylasparagine; hydroxylysine; allo-hydroxylysine; 3-hydroxyproline; 4-hydroxyproline; isodesmosine; allo-isoleucine; N-methylglycine (sarcosine); N-methylisoleucine; N-methylvaline; norvaline; norleucine; and ornithine.

[0089] Representative derivatized amino acids include for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine.

[0090] The term “conservatively substituted variant” refers to a peptide having an amino acid residue sequence substantially identical to a sequence of a reference peptide in which one or more residues have been conservatively substituted with a functionally similar residue. In one embodiment, a conservatively substituted variant displays a similar binding specificity or non-binding quality when compared to the reference peptide. The phrase “conservatively substituted variant” also includes peptides wherein a residue is replaced with a chemically derivatized residue, provided that the resulting peptide has a binding specificity or non-binding quality as disclosed herein.

[0091] Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another; the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine; the substitution of one basic residue such as lysine, arginine or histidine for another; or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.

[0092] Peptides of the present invention also include peptides having one or more additions and/or deletions or residues relative to the sequence of a peptide whose sequence is disclosed herein, so long as the requisite binding specificity or non-binding quality of the peptide is maintained. The term “fragment” refers to a peptide having an amino acid residue sequence shorter than that of a peptide disclosed herein.

[0093] A peptide can be modified by terminal-NH₂ acylation (e.g., acetylation, or thioglycolic acid amidation) or by terminal-carboxylamidation (e.g., with ammonia or methylamine). Terminal modifications are useful to reduce susceptibility by proteinase digestion, and to therefore prolong a half-life of peptides in solutions, particularly in biological fluids where proteases can be present.

[0094] Peptide cyclization is also a useful modification because of the stable structures formed by cyclization and in view of the biological activities observed for such cyclic peptides. Representative methods for cyclizing peptides are described by Schneider & Eberle (1993) Peptides, 1992: Proceedings of the Twenty-Second European Peptide Symposium, Sep. 13-19, 1992, Interlaken, Switzerland, Escom, Leiden, The Netherlands. Typically, tertbutoxycarbonyl protected peptide methyl ester is dissolved in methanol, sodium hydroxide solution is added, and the admixture is reacted at 20° C. to hydrolytically remove the methyl ester protecting group. After evaporating the solvent, the tertbutoxycarbonyl-protected peptide is extracted with ethyl acetate from acidified aqueous solvent. The tertbutoxycarbonyl protecting group is then removed under mildly acidic conditions in dioxane co-solvent. The unprotected linear peptide with free amino and carboxyl termini so obtained is converted to its corresponding cyclic peptide by reacting a dilute solution of the linear peptide, in a mixture of dichloromethane and dimethylformamide, with dicyclohexylcarbodiimide in the presence of 1-hydroxybenzotriazole and N-methylmorpholine. The resultant cyclic peptide is then purified by chromatography.

[0095] Optionally, a ligand or non-binding domain of the present invention can comprise one or more amino acids that have been modified to contain one or more halogens, such as fluorine, bromine, or iodine, to facilitate linking to a linker molecule as described further herein below.

[0096] The term “peptoid” as used herein refers to a peptide wherein one or more of the peptide bonds are replaced by pseudopeptide bonds including but not limited to a carba bond (CH₂—CH₂), a depsi bond (CO—O), a hydroxyethylene bond (CHOH—CH₂), a ketomethylene bond (CO—CH₂), a methylene-oxy bond (CH₂—O), a reduced bond (CH₂—NH), a thiomethylene bond (CH₂—S), an N-modified bond (—NRCO—), and a thiopeptide bond (CS—NH). See e.g., Garbay-Jaureguiberry et al., 1992; Tung et al., 1992; Urge et al., 1992; Corringer et al., 1993; Pavone et al., 1993.

[0097] Representative peptides that specifically bind to a non-biological substrate are set forth as SEQ ID NOs:1-71. See Examples 2-8.

[0098] Peptide ligands that specifically bind a biological substrate include peptides with known binding specificities, including but not limited to: (a) cell-binding peptides listed in Table 1 (SEQ ID NOs:74-98); (b) other peptides known to specifically bind a target substrate; or (c) peptides discovered by display technology as described herein below. 3

TABLE 1
Binding Specificity	Peptide Sequence
Cell-binding epitopes of	GGWSHW (SEQ ID NO:74)
fibronectin	RGD (SEQ ID NO:75)
	YIGSR (SEQ ID NO:76)
	GRGD (SEQ ID NO:77)
	GYIGSR (SEQ ID NO:78)
	PDSGR (SEQ ID NO:79)
	IKVAV (SEQ ID NO:80)
	GRGDY (SEQ ID NO:81)
	GYIGSRY (SEQ ID NO:82)
	RGDY (SEQ ID NO:83)
	YIGSRY (SEQ ID NO:84)
	REDV (SEQ ID NO:85)
	GREDV (SEQ ID NO:86)
	RGDF (SEQ ID NO:87)
	GRGDF (SEQ ID NO:88)
lung cells	peptides of the format CX3CX3CX3C where
	X = any amino acid
	(e.g., CGFECVRQCPERC (SEQ ID NO:89))
fibroblast	RGD (SEQ ID NO:75)
	KRSR (SEQ ID NO:90)
heparin	KRSR (SEQ ID NO:90)
	KRSRGGG (SEQ ID NO:91)
muscle (myoblasts)	ASSLNIA (SEQ ID NO:92)
smooth muscle cells	KQAGDV (SEQ ID NO:93)
endothelial cells	YIGSR (SEQ ID NO:94)
	CRRGDWLC (SEQ ID NO:95)
fibroblasts and	RGD (SEQ ID NO:75)
endothelial cells	RGDS (SEQ ID NO:96)
osteoblasts	RGD (SEQ ID NO:75)
	KRSK (SEQ ID NO:97)
	KRSRGGG (SEQ ID NO:98)

[0099] Peptides of the present invention, including peptoids, can be synthesized by any of the techniques that are known to those skilled in the art of peptide synthesis. Synthetic chemistry techniques, such as a solid-phase Merrifield-type synthesis, are employed for reasons of purity, antigenic specificity, freedom from undesired side products, ease of production, and the like. A summary of representative techniques can be found in Stewart & Young (1969) Solid Phase Peptide Synthesis, Freeman, San Francisco, Calif., United States of America; Merrifield (1969) Adv Enzymol Relat Areas Mol Biol 32:221-296; Fields & Noble (1990) Int J Pept Protein Res 35:161-214; and Bodanszky (1993) Principles of Peptide Synthesis, 2nd Rev. Ed. Springer-Verlag, Berlin, N.Y., among other places. Representative solid phase synthesis techniques can be found in Andersson et al., (2000) Biopolymers 55:227-250, references cited therein, and in U.S. Pat. Nos. 6,015,561; 6,015,881; 6,031,071; and 4,244,946. Peptide synthesis in solution is described in Schröder & Lübke (1965) The Peptides, Academic Press, New York, N.Y., United States of America. Appropriate protective groups useful for peptide synthesis are described in the above texts and in McOmie (1973) Protective Groups in Organic Chemistry, Plenum Press, London, N.Y. In one embodiment of the invention, a peptide is produced using an automated peptide synthesizer as described in Examples 11-13.

[0100] Peptides can also be synthesized by native chemical ligation as described in U.S. Pat. No. 6,184,344. Briefly, the ligation step employs a chemoselective reaction of two unprotected peptide segments to produce a transient thioester-linked intermediate. The intermediate spontaneously rearranges to generate the full length ligation product.

[0101] Peptides, including peptides comprising non-genetically encoded amino acids, can also be produced in a cell-free translation system, such as the system described by Shimizu et al. (2001) Nat Biotechnol 19:751-755. In addition, peptides having a specified amino acid sequence can be purchased from commercial sources (e.g., Biopeptide Co., LLC of San Diego, Calif., United States of America, and PeptidoGenics of Livermore, Calif., United States of America).

[0102] Peptides possessing one or more tyrosine residues at an internal position or at the carboxyl terminus of the peptide can be conveniently labeled, for example, by iodination or radio-iodination.

[0103] The term “peptide mimetic” as used herein refers to a ligand that mimics the biological activity of a reference peptide, by substantially duplicating the antigenicity of the reference peptide, but it is not a peptide or peptoid. In one embodiment, a peptide mimetic has a molecular weight of less than about 700 daltons. A peptide mimetic can be designed or selected using methods known to one of skill in the art. See e.g., U.S. Pat. Nos. 5,811,392; 5,811,512; 5,578,629; 5,817,879; 5,817,757; and 5,811,515.

[0104] Any peptide or peptide mimetic of the present invention can be used in the form of a pharmaceutically acceptable salt. Suitable acids which can be used with the peptides of the present invention include, but are not limited to inorganic acids such as trifluoroacetic acid (TFA), hydrochloric acid (HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid or the like. In one embodiment, a pharmaceutically acceptable salt is HCl. In another embodiment, a pharmaceutically acceptable salt is TFA.

[0105] Suitable bases capable of forming salts with the peptides of the present invention include, but are not limited to inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide and the like; and organic bases such as mono-, di-, and tri-alkyl and aryl amines (e.g., triethylamine, diisopropyl amine, methyl amine, dimethyl amine and the like), and optionally substituted ethanolamines (e.g., ethanolamine, diethanolamine and the like).

[0106] A peptide ligand of the invention can further comprise one or more crosslinking moieties, such as a photocrosslinkable moiety, an ionically crosslinkable moiety, or terminally crosslinkable moiety. The crosslinking moieties can be used to create a two-dimensional or three-dimensional interfacial biomaterial.

[0107] III.B. Antibodies

[0108] In another embodiment of the invention, a ligand or non-binding domain can comprise a single chain antibody. The term “single chain antibody” refers to an antibody comprising a variable heavy and a variable light chain that are joined together, either directly or via a peptide linker, to form a continuous polypeptide. Thus, the term “single chain antibody” encompasses an immunoglobulin protein or a functional portion thereof, including, but not limited to a monoclonal antibody, a chimeric antibody, a hybrid antibody, a mutagenized antibody, a humanized antibody, and antibody fragments that comprise an antigen binding site (e.g., Fab and Fv antibody fragments).

[0109] Antibody ligands can be identified by the panning methods described herein below. Alternatively, known single chain antibodies having a desired binding specificity or a desired non-binding quality can be used. For example, U.S. Pat. No. 5,874,542 to Rockwell et al. discloses single chain antibodies that specifically bind to vascular endothelial growth factor (VEGF) receptor. VEGF is expressed in macrophages and proliferating epidermal keratinocytes and thus can be used to promote wound healing (Brown et al., 1992). A number of single chain antibodies have been identified that specifically bind to cancer cells (e.g., U.S. Pat. Nos. 5,977,322 and 5,837,243), to human immunodeficiency virus (U.S. Pat. No. 5,840,300), and to secreted signaling molecules (e.g., tumor necrosis factor (TNF); U.S. Pat. No. 5,952,087). These antibody ligands can be useful, for example, drug delivery and detection methods described herein below.

[0110] III.C. Other Ligands and Non-Binding Domains

[0111] A binding agent of the present invention can also comprise a ligand that shows specific binding other than a peptide or antibody ligand. Similarly, any suitable non-binding domain that shows substantially no binding to a target substrate can be used to prepare a binding agent. Thus, a ligand or non-binding domain of the invention can also comprise a protein, a synthetic polymer, a natural polymer, a polysaccharide, a nucleic acid (e.g., an aptamer), a small molecule (e.g., a chemical compound), a nucleic acid-protein fusion, and/or any other affinity or non-binding agent.

[0112] For example, a non-binding domain can comprise an anionic polymer or an anionic carbohydrate. These molecules show substantially no cellular binding and thus are useful for inhibiting fibrosis, scar formation, and surgical adhesions. See e.g., U.S. Pat. No. 5,705,177. Representative anionic polymers include but are not limited to natural proteoglycans, glycosaminoglycan moieties of proteoglycans, dextran sulfate, pentosan polysulfate, dextran sulfate, or cellulose derivatives. Anionic polymers can be obtained from commercial sources (e.g., Sigma Chemical Company of St. Louis, Mo., United States of America), purified from a natural source, or prepared synthetically. Methods for polymer purification and synthesis can be found in Budavari (1996) The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, 12th ed. Merck, Whitehouse Station, New Jersey, United States of America, among other places.

[0113] A non-binding domain can also comprise a polysaccharide that shows substantially no binding to platelets can be used as a calcification inhibitor as described in U.S. Pat. No. 4,378,224. Suitable calcification inhibitors include natural protein polysaccharides (e.g., chondroitin sulfates and hyaluronate), sulfated polysaccharides, diphosphonates, phosphoproteins, and other polyanions.

[0114] A ligand or non-binding domain can also comprise a small molecule. The term “small molecule” as used herein refers to a compound, for example an organic compound, with a molecular weight in one embodiment of less than about 1,000 daltons, in another embodiment of less than about 750 daltons, in another embodiment of less than about 600 daltons, and in yet another embodiment of less than about 500 daltons. In one embodiment, a small molecule has a computed log octanol-water partition coefficient in the range of about −4 to about +14, and in another embodiment, in the range of about −2 to about +7.5.

[0115] III.D. Linkers

[0116] Binding agents useful for preparation of an interfacial biomaterial optionally further comprise a linker between a first and second ligand, or between a ligand and a non-binding region. The linker can facilitate combination of two or more ligands. In addition, the linker can comprise a spacer function to minimize potential steric hindrance between the two or more domains.

[0117] In one embodiment, the linker does not abrogate or alter ligand binding strength, ligand binding specificity, or a quality of substantially no binding of a non-binding domain. In one embodiment, the linker is substantially biologically inert except for its linking and/or spacer activities.

[0118] Suitable linkers comprise one or more straight or branched chain(s) of 2 carbon atoms to about 50 carbon atoms, wherein the chain is fully saturated, fully unsaturated, or a combination thereof. Typically, a linker comprises between 2 and about one hundred sites for ligand attachment. The methods employed for linking will vary according to the chemical nature of each of a selected ligand, non-binding domain, and linker.

[0119] Suitable reactive groups of a linker include, but are not limited to amines, carboxylic acids, alcohols, aldehydes, and thiols. An amine group in a linker can form a covalent bond with a carboxylic acid group of a ligand, such as a carboxyl terminus of a peptide ligand. A carboxylic acid group or an aldehyde in a linker can form a covalent bond with the amino terminus of a peptide ligand or other ligand amine group. An alcohol group in a linker can form a covalent bond with the carboxyl terminus of a peptide ligand or other ligand carboxylic acid group. A thiol group in a linker can form a disulfide bond with a cysteine in a peptide ligand or a ligand thiol group.

[0120] Additional reactive groups that can be used for linking reactions include, but are not limited to a phosphate, a sulphate, a hydroxide, —SeH, an ester, a silane, urea, urethane, a thiol-urethane, a carbonate, a thio-ether, a thio-ester, a sulfate, an ether, or a combination thereof.

[0121] In one embodiment of the invention, a linker comprises a peptide. In one embodiment, a peptide linker comprises one (1) to about 40 amino acids. Sites for ligand attachment to a peptide ligand include functional groups of the amino acid side chains and the amino and carboxyl terminal groups. Representative peptide linkers with multiple reactive sites include polylysines, polyornithines, polycysteines, polyglutamic acid and polyaspartic acid. Alternatively, substantially inert peptide linkers comprise polyglycine, polyserine, polyproline, polyalanine, and other oligopeptides comprising alanyl, serinyl, prolinyl, or glycinyl amino acid residues.

[0122] Peptide linkers can be pennant or cascading. The term “pennant polypeptide” refers to a linear peptide. As with polypeptides typically found in nature, the amide bonds of a pennant polypeptide are formed between the terminal amine of one amino acid residue and the terminal carboxylic acid of the next amino acid residue. The term “cascading polypeptide” refers to a branched peptide, wherein at least some of the amide bonds are formed between the side chain functional group of one amino acid residue and the amino terminal group or carboxyl terminal group of the next amino acid residue.

[0123] In another embodiment of the invention, a linker can comprise a polymer, including a synthetic polymer or a natural polymer. Representative synthetic polymers include, but are not limited to polyethers (e.g., polyethylene glycol; PEG), polyesters (e.g., polylactic acid (PLA) and polyglycolic acid (PGA)), polyamides (e.g., nylon), polyamines (e.g., polymethylmethacrylate; PMMA), polyacrylic acids, polyurethanes, polystyrenes, and other synthetic polymers having a molecular weight of about 200 daltons to about 1000 kilodaltons. Representative natural polymers include, but are not limited to hyaluronic acid, alginate, chondroitin sulfate, fibrinogen, fibronectin, albumin, collagen, and other natural polymers having a molecular weight of about 200 daltons to about 20,000 kilodaltons. Polymeric linkers can comprise a diblock polymer, a multi-block copolymer, a comb polymer, a star polymer, a dendritic polymer, a hybrid linear-dendritic polymer, or a random copolymer.

[0124] A linker can also comprise a mercapto(amido)carboxylic acid, an acrylamidocarboxylic acid, an acrlyamido-amidotriethylene glycolic acid, and derivatives thereof. See U.S. Pat. No. 6,280,760.

[0125] Methods for linking a linker molecule to a ligand or to a non-binding domain will vary according to the reactive groups present on each molecule. Protocols for linking using the above-mentioned reactive groups and molecules are known to one of skill in the art. See Goldman et al., 1997; Cheng 1996; Neri et al., 1997; Nabel 1997; Park et al., 1997; Pasqualini et al., 1997; Bauminger & Wilchek 1980; U.S. Pat. Nos. 6,280,760 and 6,071,890; and European Patent Nos. 0 439 095 and 0 712 621.

[0126] IV. Identification of Ligands Using Phage Display

[0127] Display technology is an effective approach for the identification of ligands that specifically bind a substrate, for example phage display methods. According to this approach, a library of diverse ligands is presented to a target substrate, and ligands that specifically bind the substrate are selected. Conversely, ligands that show substantially no binding to a target substrate can also be recovered. Ligands and non-binding domains can be selected following multiple serial rounds of selection called panning.

[0128] Any one of a variety of libraries and panning methods can be employed to identify a peptide that is useful in the methods of the invention, as described further herein below.

[0129] V.A. Libraries

[0130] As used herein, the term “library” means a collection of molecules. A library can contain a few or a large number of different molecules, varying from about ten molecules to several billion molecules or more. A molecule can comprise a naturally occurring molecule, or a synthetic molecule, which is not found in nature. Optionally, a plurality of different libraries can be employed simultaneously for in vivo panning.

[0131] Representative libraries include but are not limited to a peptide library (Example 1 and U.S. Pat. Nos. 6,156,511, 6,107,059, 5,922,545, and 5,223,409), an oligomer library (U.S. Pat. Nos. 5,650,489 and 5,858,670), an aptamer library (U.S. Pat. Nos. 6,180,348 and 5,756,291), a small molecule library (U.S. Pat. Nos. 6,168,912 and 5,738,996), a library of antibodies or antibody fragments (U.S. Pat. Nos. 6,174,708, 6,057,098, 5,922,254, 5,840,479, 5,780,225, 5,702,892, and 5,667,988), a library of nucleic acid-protein fusions (U.S. Pat. No. 6,214,553), and a library of any other affinity agent that can potentially bind to a target substrate (e.g., U.S. Pat. Nos. 5,948,635, 5,747,334, and 5,498,538).

[0132] The molecules of a library can be produced in vitro, or they can be synthesized in vivo, for example by expression of a molecule in vivo. Also, the molecules of a library can be displayed on any relevant support, for example, on bacterial pili (Lu et al., 1995) or on phage (Smith, 1985).

[0133] A library can comprise a random collection of molecules. Alternatively, a library can comprise a collection of molecules having a bias for a particular sequence, structure, or conformation. See e.g., U.S. Pat. Nos. 5,264,563 and 5,824,483. Methods for preparing libraries containing diverse populations of various types of molecules are known in the art, for example as described in U.S. Patents cited herein above. Numerous libraries are also commercially available.

[0134] In one embodiment, a library to be used for the disclosed panning methods has a complexity of at least about 1×10⁸ to about 1×10⁹ different molecules per library. A typical panning experiment with an input of 1×10¹¹ phage therefore samples on average 100 copies to 1000 copies of each molecule in the library.

[0135] In one embodiment of the invention, the method for panning is performed using a phage library. Phage are used as a scaffold to display recombinant libraries and to also provide for recovery and amplification of ligands having a desired binding specificity.

[0136] The T7 phage has an icosahedral capsid made of 415 proteins encoded by gene 10 during its lytic phase. The T7 phage display system has the capacity to display peptides up to 15 amino acids in size at a high copy number (415 per phage). Unlike filamentous phage display systems, peptides displayed on the surface of T7 phage are not capable of peptide secretion. T7 phage also replicate more rapidly and are extremely robust when compared to other phage.

[0137] A phage library to be used in accordance with the panning methods of the present invention can also be constructed in a filamentous phage, for example M13 or M13-derived phage. In one embodiment, the ligands are displayed at the exterior surface of the phage, for example by fusion to M13 vital protein 8. Methods for preparing M13 libraries can be found in Sambrook & Russell (2001) Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., United States of America, among other places. Representative peptide libraries prepared in M13 phage and that are useful in the methods of the present invention are described in Example 1.

[0138] Other suitable phage vectors include the mAEK and mACK vectors, which are derived from an M13mp18 backbone. These versatile vectors are compatible with a wide range of screening formats, including cell-based, solution phase, and solid-phase panning. The mAEK vector provides an independent peptide epitope that is useful in quantitation of peptide for binding and functional assays beyond panning. The mAEK vector also includes a thrombin cleavage site for highly efficient and selective elution of specifically bound phage. Thrombin cleavage also permits “off-phage” assays, in which the peptide module is clipped from the phage prior to conducting the assay. This panning method can be used for experiments that produce unacceptably high background binding when the complete phage particle is present.

[0139] Phage vectors typically include a single allele of the viral coat gene pIII, and thus three copies to five copies of identical ligand-PIII fusion proteins are produced on the surface of each recombinant phage. This multiple valency results in increased avidity of selected ligands for target substrates. Thus, phage vectors can be used for primary screens where the goal is typically to identify one or several target-specific binding motifs for further characterization and where high affinity ligands are not essential.

[0140] In another embodiment of the invention, a library used for panning comprises a phagemid vector. A phagemid is a plasmid that includes both a phage f1 origin of replication, also acting as a packaging signal, and a single copy of the gene encoding PIII containing the expression cassettes described above. Useful phagemid vectors include the pAEK and pACK plasmids, which are derived from the vector pGEM-3z-f(+) (Promega Corporation, Madison, Wis., United States of America).

[0141] Phagemid libraries are maintained as plasmids, and they are rescued by superinfection with a packaging-deficient helper phage. Progeny viruses preferentially package the phagemid DNA, which lacks phage genes other than the pIII fusion gene. The helper virus provides copies of wild type pIII, while the phagemid expresses a lesser amount of recombinant ligand-PIII fusion protein. Thus, most recombinant viruses that express ligand-PIII fusion proteins express only a single copy. These monovalent libraries tend to result in higher affinity ligands because low affinity binding cannot be compensated by increased avidity. Thus, phagemid vectors can be used for secondary screens to optimize binding motifs and to produce high affinity ligands.

[0142] Plasmid expression systems can be used to generate sufficient quantities of ligands and non-binding domains for further characterization in standard binding assays. Alternatively, ligands and non-binding domains selected by panning can be synthesized to appropriate amounts.

[0143] As a precursor to chemical synthesis, it is often useful to determine activities of peptide ligands expressed as fusion proteins in standard expression cassettes such as glutathione-S-transferase (GST), green fluorescent protein (GFP), and bacterial alkaline phosphatase (BAP) (Yamabhai & Kay, 2001). These expression modules facilitate expression, stabilization, and purification of peptide ligands and can also serve as indicators of peptide binding.

[0144] Peptide Libraries. In one embodiment of the invention, a peptide library can be used to perform the disclosed panning methods. A peptide library comprises in one embodiment peptides comprising three or more amino acids, in another embodiment at least five, six, seven, or eight amino acids, in another embodiment up to 50 amino acids, in another embodiment up to 100 amino acids, in another embodiment up to about 200 amino acids, and in yet another embodiment up to about 300 amino acids. In one embodiment, a peptide library comprises peptides having a molecular weight of about 500 daltons to about 3500 daltons.

[0145] The peptides can be linear, branched, or cyclic, and can include non-peptidyl moieties. The peptides can comprise naturally occurring amino acids, synthetic amino acids, genetically encoded amino acids, non-genetically encoded amino acids, and combinations thereof.

[0146] A biased peptide library can also be used, a biased library comprising peptides wherein one or more (but not all) residues of the peptides are constant. For example, an internal residue can be constant, so that the peptide sequence is represented as:

(Xaa₁)_m-(AA)₁-(Xaa₂)_n

[0147] where Xaa₁ and Xaa₂ are any amino acid, or any amino acid except cysteine, wherein Xaa₁ and Xaa₂ are the same or different amino acids, m and n indicate a number Xaa residues, wherein in one embodiment m and n are independently chosen from the range of 2 residues to 20 residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and/or 20 residues), in another embodiment m and n are chosen from the range of 4 residues to 9 residues (e.g., 4, 5, 6, 7, 8, and/or 9), and AA is the same amino acid for all peptides in the library. In one embodiment, AA is located at or near the center of the peptide. In one embodiment m and n are not different by more than 2 residues; in another embodiment m and n are equal.

[0148] In one embodiment, libraries are those in which AA is tryptophan, proline, or tyrosine. In another embodiment, libraries are those in which AA is phenylalanine, histidine, arginine, aspartate, leucine, or isoleucine. In another embodiment, libraries are those in which AA is asparagine, serine, alanine, or methionine. In yet another embodiment, libraries are those in which AA is cysteine or glycine.

[0149] A representative library can be prepared using degenerate codons encoded as NNK, where N=A, C, G, or T and K=G or T. Restriction of the wobble position of the codon reduces, but does not eliminate, the codon bias intrinsic to the genetic code (e.g., 6 codons each for serine, arginine, and leucine, but only one each for methionine and tryptophan) and also eliminates two of the three stop codons. Additional library formats include, but are not limited to those presented in Table 2. In one embodiment, an X₆PX₆ library is employed. In another embodiment, an SCX₁₆S library is employed. In yet another embodiment, an X₆YX₆ library is employed. Representative approaches for library synthesis are also disclosed in the Examples (see e.g., Example 1). 4

TABLE 2
Library Format	Representation
X7	n-X-X-X-X-X-X-X-
CX7C	1 [Image Omitted]
SSX16S	n-S-S-X-X-X-X-X-X-X-X-X-X-X-X-X-X-X-X-S-
SCX16S	n-S-C-X-X-X-X-X-X-X-X-X-X-X-X-X-X-X-X-S-
SCX16C	2 [Image Omitted]
X6CX4CX6	3 [Image Omitted]
X6PX6	n-X-X-X-X-X-X-P-X-X-X-X-X-X-
X6NX6	n-X-X-X-X-X-X-N-X-X-X-X-X-X-
X6GX6	n-X-X-X-X-X-X-G-X-X-X-X-X-X-
X6YX6	n-X-X-X-X-X-X-Y-X-X-X-X-X-X-
X6HX6	n-X-X-X-X-X-X-H-X-X-X-X-X-X-
NOTE: X is any amino acid.
Solid lines indicate peptide bonds, and dotted lines indicate cysteine—cysteine bonds.

[0150] Antibody Libraries. In another embodiment of the invention, the panning methods employ an antibody library. Vectors for the construction of antibody libraries include the pCANTAB-5E or pCANTAB-6 vectors (Amersham Biosciences, Piscataway, N.J., United States of America). These vectors contain a constant region single chain fragment variable antibody (scFv) scaffold, and variable sequences are cloned into the vector sequences encoding antibody heavy and light chains. Antibody ligands can be displayed using, for example, an M13 phage vector as described herein above. Methods for constructing an antibody library in M13 or M13-derived phage can be found in U.S. Pat. Nos. 6,225,447; 5,580,717; and 5,702,892; among other places.

[0151] An antibody library used for the disclosed panning methods can comprise a naïve library or an immunized library. Naïve antibody libraries can be constructed using IgG hypervariable regions derived from peripheral blood lymphocytes pooled from normal and/or immunologically deficient subjects. Naïve libraries are particularly useful in screening targets comprising a poorly immunogenic epitope. Alternatively, an immunized library can be prepared, wherein IgG hypervariable regions are derived from splenocytes of mice previously immunized with the target substrate.

[0152] IV.B. Panning Methods

[0153] The panning techniques employed in the methods of the present invention can comprise solid phase screening, solution phase screening, antibody-directed proximity screening, cell-based screening, tissue-based screening, or a combination thereof. Screening formats are described further herein below. See also Examples 2-8.

[0154] Methods for recovering of ligands that bind to a substrate are selected based on one or more characteristics common to the molecules present in the library. For example, mass spectrometry and/or gas chromatography can be used to resolve molecules sharing a common core structure. Thus, where a library comprises diverse molecules based generally on the structure of an organic molecule, determining the presence of a parent peak for the particular molecule can identify a ligand.

[0155] Alternatively, each of the diverse molecules of a library can comprise a tag that facilitates recovery and identification. For example, a representative tag is an oligonucleotide or a small molecule such as biotin. See e.g., Brenner & Lerner 1992; Norris et al., 1999; Paige et al., 1999; U.S. Pat. No. 6,068,829.

[0156] A tag can also be a support or surface to which a molecule can be attached. For example, a support can be a biological tag such as a virus or virus-like particle such as a bacteriophage (“phage”); a bacterium; or a eukaryotic cell such as yeast, an insect cell, or a mammalian cell (e.g., an endothelial progenitor cell or a leukocyte); or can be a physical tag such as a liposome or a microbead. Where a molecule is linked to a support, the part of the molecule suspected of being able to interact with a substrate can be positioned so as be able to participate in the interaction.

[0157] Solid Phase Screening. Solid phase screening methods are used when the binding substrate comprises a non-biological surface. See Examples 2-8. Solid phase screening also encompasses panning methods in which a biological target is coated on a solid support (e.g., in wells of a microtiter plate), as described in Example 9. This approach requires that a target biological substrate retains at least an approximation of native structure and function when immobilized on a support.

[0158] Solution Phase Screening. This approach can be used to identify a ligand that specifically binds to biological substrate in solution. In particular, the method is suited for identification of a ligand that specifically binds to a biological substrate, wherein the biological substrate is bound to other biological components as part of a complex. Solution phase screening is also appropriate in cases in which the binding capacity of a biological substrate is diminished by immobilization on a substrate. According to this approach, the biological substrate, a component complexed therewith, or the ligand is modified to include a tag that facilitates recovery of the substrate, as described herein above.

What is claimed is:

1. An interfacial biomaterial comprising a plurality of binding agents, wherein each binding agent comprises a first ligand that specifically binds a target non-biological substrate and a second ligand that specifically binds a target biological substrate, and wherein the plurality of binding agents define an interface between the target non-biological substrate and the target biological substrate.

2. The interfacial biomaterial of claim 1, wherein the plurality of binding agents comprises a plurality of identical binding agents.

3. The interfacial biomaterial of claim 1, wherein the plurality of binding agents comprises a plurality of non-identical binding agents.

4. The interfacial biomaterial of claim 3, wherein each of the plurality of non-identical binding agents comprises an identical first ligand that specifically binds a target non-biological substrate.

5. The interfacial biomaterial of claim 1, wherein the plurality of binding agents further comprise a spatial pattern.

6. The interfacial biomaterial of claim 1, further comprising a linker, wherein the linker links the first ligand and the second ligand.

7. The interfacial biomaterial of claim 1, wherein the first ligand comprises a peptide or a single chain antibody.

8. The interfacial biomaterial of claim 1, wherein the first ligand specifically binds a target non-biological substrate, the target non-biological substrate being selected from the group consisting of a synthetic polymer, a plastic, a metal, a metal oxide, a non-metal oxide, a silicone material, a ceramic material, a drug, a drug carrier, and combinations thereof.

9. The interfacial biomaterial of claim 8, wherein the synthetic polymer comprises polyglycolic acid.

10. The interfacial biomaterial of claim 9, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:37-50.

11. The interfacial biomaterial of claim 8, wherein the synthetic polymer comprises nylon.

12. The interfacial biomaterial of claim 11, wherein the nylon forms a nylon suture.

13. The interfacial biomaterial of claim 8, wherein the first ligand specifically binds a plastic selected from the group consisting of polystyrene, polycarbonate, polyurethane, and combinations thereof.

14. The interfacial biomaterial of claim 13, wherein the first ligand specifically binds a plastic comprising polystyrene.

15. The interfacial biomaterial of claim 14, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:X-X. (polystyrene)

16. The interfacial biomaterial of claim 13, wherein the first ligand specifically binds a plastic comprising polyurethane.

17. The interfacial biomaterial of claim 16, wherein the first ligand comprises a peptide comprising an amino acid sequence of SEQ ID NO:X-X. (polyurethane)

18. The interfacial biomaterial of claim 13, wherein the first ligand specifically binds a plastic comprising polycarbonate.

19. The interfacial biomaterial of claim 18, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:X-X. (polycarbonate)

20. The interfacial biomaterial of claim 8, wherein the first ligand specifically binds a metal comprising titanium.

21. The interfacial biomaterial of claim 20, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:X-X. (titanium)

22. The interfacial biomaterial of claim 8, wherein the first ligand specifically binds a metal comprising stainless steel.

23. The interfacial biomaterial of claim 22, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:X-X. (stainless steel)

24. The interfacial biomaterial of claim 1, wherein the second ligand comprises a peptide or a single chain antibody.

25. The interfacial biomaterial of claim 1, wherein the second ligand specifically binds a target biological substrate, the target biological substrate being selected from the group consisting of a tissue, a cell, a macromolecule, and combinations thereof.

26. The interfacial biomaterial of either of claims 24 or 25, wherein the target biological substrate comprises collagen or a Tie2 receptor.

27. The interfacial biomaterial of claim 1 comprising a plurality of binding agents, wherein one or more of the plurality of binding agents comprises an amino acid sequence of SEQ ID NO:27 or 28. (linkers)

28. An interfacial biomaterial comprising a plurality of binding agents, wherein each binding agent comprises a ligand that specifically binds a target non-biological substrate and a non-binding domain that substantially lacks binding to a target biological substrate.

29. The interfacial biomaterial of claim 28, wherein the plurality of binding agents comprises a plurality of identical binding agents.

30. The interfacial biomaterial of claim 28, wherein the plurality of binding agents comprises a plurality of non-identical binding agents.

31. The interfacial biomaterial of claim 30, wherein each of the plurality of non-identical binding agents comprises an identical ligand that specifically binds a non-biological substrate.

32. The interfacial biomaterial of claim 28, wherein the plurality of binding agents further comprise a spatial pattern.

33. The interfacial biomaterial of claim 28, wherein one or more of the plurality of binding agents comprises a linker that links the ligand and the non-binding domain.

34. The interfacial biomaterial of claim 29, wherein the ligand comprises a peptide or a single chain antibody.

35. The interfacial biomaterial of claim 29, wherein the ligand specifically binds a target non-biological substrate, the target non-biological substrate being selected from the group consisting of a synthetic polymer, a plastic, a metal, a metal oxide, a non-metal oxide, a silicone material, a ceramic material, a drug, a drug carrier, and combinations thereof.

36. The interfacial biomaterial of claim 35, wherein the synthetic polymer comprises polyglycolic acid.

37. The interfacial biomaterial of claim 35, wherein the synthetic polymer comprises nylon.

38. The interfacial biomaterial of claim 37, wherein the nylon forms a nylon suture.

39. The interfacial biomaterial of claim 35, wherein the ligand specifically binds a plastic selected from the group consisting of polystyrene, polycarbonate, polyurethane, and combinations thereof.

40. The interfacial biomaterial of claim 39, wherein the ligand specifically binds a plastic comprising polystyrene.

41. The interfacial biomaterial of claim 40, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:1-22.

42. The interfacial biomaterial of claim 39, wherein the ligand specifically binds a plastic comprising polyurethane.

43. The interfacial biomaterial of claim 42, wherein the ligand comprises a peptide comprising an amino acid sequence of SEQ ID NO:23.

44. The interfacial biomaterial of claim 35, wherein the ligand specifically binds a metal comprising titanium.

45. The interfacial biomaterial of claim 44, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:24-36.

46. The interfacial biomaterial of claim 35, wherein the ligand specifically binds a metal comprising stainless steel.

47. The interfacial biomaterial of claim 46, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:51-65.

48. The interfacial biomaterial of claim 28, wherein the domain comprises a peptide or a single chain antibody.

49. The interfacial biomaterial of claim 28, wherein the non-binding domain shows substantially no binding to a target biological substrate, the target biological substrate selected from the group consisting of a tissue, a cell, a macromolecule, and combinations thereof.

50. The interfacial biomaterial of claim 49, wherein the non-binding domain comprises a cytophobic agent.

51. The interfacial biomaterial of claim 50, wherein the cytophobic agent is polyethylene glycol.

52. The interfacial biomaterial of claim 28, wherein the interfacial biomaterial inhibits fouling of the target non-biological substrate.

53. A synthetic peptide that specifically binds polystyrene comprising a peptide having less than 20 amino acid residues.

54. The synthetic peptide of claim 53 comprising an amino acid sequence of any one of SEQ ID NOs:1-22

55. A synthetic peptide that specifically binds polyurethane comprising a peptide having less than 20 amino acid residues.

56. The synthetic peptide of claim 55 comprising an amino acid sequence of SEQ ID NO:23.

57. A synthetic peptide that specifically binds polycarbonate comprising a peptide having less than 20 amino acid residues.

58. The synthetic peptide of claim 57 comprising an amino acid sequence of any one of SEQ ID NOs:66-71.

59. A synthetic peptide that specifically binds polyglycolic acid comprising a peptide having less than 20 amino acid residues.

60. The synthetic peptide of claim 59 comprising an amino acid sequence of any one of SEQ ID NOs:37-50.

61. A synthetic peptide that specifically binds nylon comprising a peptide having less than 20 amino acid residues.

62. A synthetic peptide that specifically binds titanium comprising a peptide having less than 20 amino acid residues.

63. The synthetic peptide of claim 62 comprising an amino acid sequence of any one of SEQ ID NOs:24-36.

64. A synthetic peptide that specifically binds stainless steel comprising a peptide having less than 20 amino acid residues.

65. The synthetic peptide of claim 64 comprising an amino acid sequence of any one of SEQ ID NOs:51-65.

66. A synthetic peptide that specifically binds collagen comprising a peptide having less than 20 amino acid residues.

67. A synthetic peptide that specifically binds a Tie2 receptor comprising a peptide having less than 20 amino acid residues.

68. A method for preparing a binding agent, the method comprising: (a) panning a library of diverse molecules over a target non-biological substrate, whereby a first ligand that specifically binds a target non-biological substrate is identified; and (b) linking the first ligand to a second ligand, wherein the second ligand specifically binds a target biological substrate, whereby a binding agent is prepared.

69. The method of claim 68, wherein the first ligand comprises a peptide or a single chain antibody.

70. The method of claim 68, wherein the first ligand specifically binds a target non-biological substrate, the target non-biological substrate being selected from the group consisting of a synthetic polymer, a plastic, a metal, a metal oxide, a non-metal oxide, a silicone material, a ceramic material, a drug, a drug carrier, and combinations thereof.

71. The method of claim 70, wherein the synthetic polymer comprises polyglycolic acid.

72. The method of claim 70, wherein the synthetic polymer comprises nylon.

73. The method of claim 72, wherein the nylon forms a nylon suture.

74. The method of claim 70, wherein the first ligand specifically binds a plastic selected from the group consisting of polystyrene, polycarbonate, polyurethane, and combinations thereof.

75. The method of claim 74, wherein the first ligand specifically binds a plastic comprising polystyrene.

76. The method of claim 75, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:1-22.

77. The method of claim 74, wherein the first ligand specifically binds a plastic comprising polyurethane.

78. The method of claim 77, wherein the first ligand comprises a peptide comprising an amino acid sequence of SEQ ID NOs:23.

79. The method of claim 74, wherein the first ligand specifically binds a plastic comprising polycarbonate.

80. The method of claim 79, wherein the first ligand comprises a peptide comprising an amino acid sequence of SEQ ID NOs:66-71.

81. The method of claim 70, wherein the first ligand specifically binds a metal comprising titanium.

82. The method of claim 81, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:24-36.

83. The method of claim 70, wherein the first ligand specifically binds a metal comprising stainless steel.

84. The method of claim 83, wherein the first ligand comprises a peptide comprising an amino acid sequence of SEQ ID NOs:51-65.

85. The method of claim 68, wherein the second ligand comprises a peptide or a single chain antibody.

86. The method of claim 68, wherein the second ligand specifically binds a target biological substrate selected from the group consisting of a tissue, a cell, a macromolecule, and combinations thereof.

87. The method of any one of claims 85 or 86, wherein the target biological substrate comprises collagen or a Tie2 receptor.

88. The method of claim 68, further comprising panning a ligand over a target biological substrate, whereby a ligand that specifically binds a target biological substrate is identified.

89. A binding agent produced by the method of claim 68.

90. The binding agent of claim 89, wherein the binding agent comprises an amino acid sequence of either of SEQ ID NO:72 or 73.

91. A method for preparing a binding agent, the method comprising: (a) panning a library of diverse molecules over a target non-biological substrate, whereby a ligand that specifically binds a target non-biological substrate is identified; and (b) linking the ligand to a non-binding domain, wherein the non-binding domain shows substantially no binding to a target biological substrate, whereby a binding agent is prepared.

92. The method of claim 91, wherein the ligand comprises a peptide or a single chain antibody.

93. The method of claim 91, wherein the ligand specifically binds a target non-biological substrate selected from the group consisting of a synthetic polymer, plastic, metal, a metal oxide, a non-metal oxide, silicone, a ceramic material, a drug, a drug carrier, and combinations thereof.

94. The method of claim 93, wherein the synthetic polymer comprises polyglycolic acid.

95. The method of claim 94, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:37-50.

96. The method of claim 93, wherein the synthetic polymer comprises nylon.

97. The method of claim 96, wherein the nylon forms a nylon suture.

98. The method of claim 93, wherein the ligand specifically binds a plastic selected from the group consisting of polystyrene, polycarbonate, polyurethane, and combinations thereof.

99. The method of claim 98, wherein the ligand specifically binds a plastic comprising polystyrene.

100. The method of claim 99, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:1-22.

101. The method of claim 98, wherein the ligand specifically binds a plastic comprising polyurethane.

102. The method of claim 101, wherein the ligand comprises a peptide comprising an amino acid sequence of SEQ ID NOs:23.

103. The method of claim 98, wherein the ligand specifically binds a plastic comprising polycarbonate.

104. The method of claim 103, wherein the ligand comprises a peptide comprising an amino acid sequence of SEQ ID NOs:66-71.

105. The method of claim 93, wherein the ligand specifically binds a metal comprising titanium.

106. The method of claim 105, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:24-36.

107. The method of claim 93, wherein the ligand specifically binds a metal comprising stainless steel.

108. The method of claim 107, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:51-65.

109. The method of claim 91, wherein the non-binding domain comprises a peptide or a single chain antibody.

110. The method of claim 91, wherein the non-binding domain shows substantially no binding to a target biological substrate selected from the group consisting of a tissue, a cell, a macromolecule, and combinations thereof.

111. The method of claim 91, wherein the non-binding domain comprises a cytophobic agent.

112. The method of claim 111, wherein the cytophobic agent is polyethylene glycol.

113. The method of claim 91, further comprising panning a ligand over a target biological substrate, whereby a non-binding domain that shows substantially no binding to a target biological substrate is identified.

114. A binding agent produced by the method of claim 91.

115. A method for preparing an interfacial biomaterial, the method comprising: (a) applying to a non-biological substrate a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds to the non-biological substrate and a second ligand that specifically binds a target biological substrate, and wherein the applying is free of coupling; (b) contacting the non-biological substrate, wherein the plurality of binding agents are bound to the non-biological substrate, with a sample comprising the target biological substrate; and (c) allowing a time sufficient for binding of the target biological substrate to the plurality of binding agents, wherein an interfacial biomaterial is prepared.

116. The method of claim 115, wherein the applying comprises applying the plurality of binding agents in a spatially restricted manner.

117. The method of claim 115, wherein the non-biological substrate is selected from the group consisting of a synthetic polymer, a plastic, a metal, a metal oxide, a non-metal oxide, a silicone material, a ceramic material, a drug, a drug carrier, and combinations thereof.

118. The method of claim 117, wherein the synthetic polymer comprises polyglycolic acid.

119. The method of claim 117, wherein the synthetic polymer comprises nylon.

120. The method of claim 119, wherein the nylon forms a nylon suture.

121. The method of claim 120, wherein the plastic is selected from the group consisting of polystyrene, polycarbonate, polyurethane, and combinations thereof.

122. The method of claim 121, wherein the plastic comprises polystyrene.

123. The method of claim 121, wherein the plastic comprises polyurethane.

124. The method of claim 121, wherein the plastic comprises polycarbonate.

125. The method of claim 117, wherein the metal comprises titanium.

126. The method of claim 117, wherein the metal comprises stainless steel.

127. The method of claim 115, wherein the plurality of binding agents comprises a plurality of identical binding agents.

128. The method of claim 115, wherein the plurality of binding agents comprises a plurality of non-identical binding agents.

129. The method of claim 128, wherein each of the plurality of non-identical binding agents comprises an identical ligand that specifically binds the non-biological substrate.

130. The method of claim 115, wherein one or more of the plurality of binding agents comprises an amino acid sequence of SEQ ID NO:72 or 73.

131. The method of claim 115, wherein one or more of the binding agents comprises a linker that links the first ligand and the second ligand.

132. The method of claim 115, wherein the first ligand comprises a peptide or a single chain antibody.

133. The method of claim 132, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:37-50.

134. The method of claim 132, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:1-22.

135. The method of claim 132, wherein the first ligand comprises a peptide comprising an amino acid sequence of SEQ ID NO:23.

136. The method of claim 132, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:66-71.

137. The method of claim 132, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:24-36.

138. The method of claim 132, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:51-65.

139. The method of claim 115, wherein the second ligand comprises a peptide or a single chain antibody.

140. The method of claim 115, wherein the second ligand specifically binds a target biological substrate selected from the group consisting of a tissue, a cell, a macromolecule, and combinations thereof.

141. The method of either of claims 139 or 140, wherein the target biological substrate comprises collagen or a Tie2 receptor.

142. The method of claim 115, wherein the contacting comprises contacting in vitro, ex vivo, or in vivo.

143. An interfacial biomaterial prepared according to the method of claim 115.

144. A method for preparing a biological array, the method comprising: (a) providing a non-biological substrate having a plurality of positions; (b) applying to each of the plurality of positions a binding agent comprising a first ligand that specifically binds the non-biological substrate and a second ligand that specifically binds a target biological substrate, wherein the applying is free of coupling; (c) contacting the non-biological substrate, wherein a plurality of binding agents are bound to the non-biological substrate, with a sample comprising the target biological substrate; and (d) allowing a time sufficient for binding of the target biological substrate to the plurality of binding agents, whereby a biological array is prepared.

145. The method of claim 144, wherein the non-biological substrate is selected from the group consisting of a synthetic polymer, a plastic, a metal, a metal oxide, a non-metal oxide, a silicone material, a ceramic material, a drug, a drug carrier, and combinations thereof.

146. The method of claim 145, wherein the synthetic polymer comprises polyglycolic acid.

147. The method of claim 145, wherein the synthetic polymer comprises nylon.

148. The method of claim 147, wherein the nylon forms a nylon suture.

149. The method of claim 145, wherein the plastic is selected from the group consisting of polystyrene, polycarbonate, polyurethane, and combinations thereof.

150. The method of claim 149, wherein the plastic comprises polystyrene.

151. The method of claim 149, wherein the plastic comprises polyurethane.

152. The method of claim 149, wherein the plastic comprises polycarbonate.

153. The method of claim 145, wherein the metal comprises titanium.

154. The method of claim 145, wherein the metal comprises stainless steel.

155. The method of claim 144, wherein the applying comprises dip-pen printing.

156. The method of claim 144, wherein the plurality of binding agents comprises a plurality of identical binding agents.

157. The method of claim 144, wherein the plurality of binding agents comprises a plurality of non-identical binding agents.

158. The method of claim 157, wherein each of the plurality of non-identical binding agents comprises an identical ligand that specifically binds the non-biological substrate.

159. The method of claim 144, wherein one or more of the plurality of binding agents comprises an amino acid sequence of SEQ ID NO:72 or 73.

160. The method of claim 144, wherein one or more of the plurality of binding agents comprises a linker that links the first ligand and the second ligand.

161. The method of claim 144, wherein the first ligand comprises a peptide or a single chain antibody.

162. The method of claim 161, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:37-50.

163. The method of claim 161, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:1-22.

164. The method of claim 161, wherein the first ligand comprises a peptide comprising an amino acid sequence of SEQ ID NO:23.

165. The method of claim 161, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:66-71.

166. The method of claim 161, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:24-36.

167. The method of claim 161, wherein the first ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:51-65.

168. The method of claim 144, wherein the second ligand comprises a peptide or a single chain antibody.

169. The method of claim 144, wherein the second ligand specifically binds a target biological substrate selected from the group consisting of a tissue, a cell, a macromolecule, and combinations thereof.

170. The method of claim one of claims 168 or 169, wherein the target biological substrate comprises collagen or a Tie2 receptor.

171. A biological array prepared according to the method of claim 144.

172. A method for preparing an interfacial biomaterial, the method comprising: (a) applying to a non-biological substrate a plurality of binding agents, wherein each of the plurality of binding agents comprises a ligand that specifically binds to the non-biological substrate and a non-binding domain that shows substantially no binding to a target biological substrate, and wherein the applying is free of coupling; and (b) contacting the non-biological substrate, wherein the plurality of binding agents are bound to the non-biological substrate, with a sample comprising the target biological substrate, whereby an interfacial biomaterial is prepared.

173. The method of claim 172, wherein the applying comprises applying the plurality of binding agents in a spatially restricted manner.

174. The method of claim 172, wherein the non-biological substrate is selected from the group consisting of a synthetic polymer, a plastic, a metal, a metal oxide, a non-metal oxide, a silicone material, a ceramic material, a drug, a drug carrier, and combinations thereof.

175. The method of claim 174, wherein the synthetic polymer comprises polyglycolic acid.

176. The method of claim 174, wherein the synthetic polymer comprises nylon.

177. The method of claim 176, wherein the nylon forms a nylon suture.

178. The method of claim 174, wherein the plastic is selected from the group consisting of polystyrene, polycarbonate, polyurethane, and combinations thereof.

179. The method of claim 178, wherein the plastic comprises polystyrene.

180. The method of claim 178, wherein the plastic comprises polyurethane.

181. The method of claim 178, wherein the plastic comprises polycarbonate.

182. The method of claim 174, wherein the metal comprises titanium.

183. The method of claim 174, wherein the metal comprises stainless steel.

184. The method of claim 172, wherein the plurality of binding agents comprises a plurality of identical binding agents.

185. The method of claim 172, wherein the plurality of binding agents comprises a plurality of non-identical binding agents.

186. The method of claim 185, wherein each of the plurality of non-identical binding agents comprises an identical ligand that specifically binds the non-biological substrate.

187. The method of claim 172, wherein one or more of the binding agents comprises a linker that links the ligand and the non-binding domain.

188. The method of claim 172, wherein the ligand comprises a peptide or a single chain antibody.

189. The method of claim 188, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:37-50.

190. The method of claim 188, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:1-22.

191. The method of claim 188, wherein the ligand comprises a peptide comprising an amino acid sequence of SEQ ID NO:23.

192. The method of claim 188, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:66-71.

193. The method of claim 188, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:24-36.

194. The method of claim 188, wherein the ligand comprises a peptide comprising an amino acid sequence of any one of SEQ ID NOs:51-65.

195. The method of claim 172, wherein the non-binding domain comprises a peptide or a single chain antibody.

196. The method of claim 172, wherein the non-binding domain shows substantially no binding to a biological substrate selected from the group consisting of a tissue, a cell, a macromolecule, and combinations thereof.

197. The method of claim 196, wherein the non-binding domain comprises a cytophobic agent.

198. The method of claim 197, wherein the cytophobic agent is polyethylene glycol.

199. The method of claim 172, wherein the contacting comprises contacting in vitro, ex vivo, or in vivo.

200. An interfacial biomaterial prepared according to the method of claim 179.

201. A method for cell culture, the method comprising: (a) applying to a non-biological substrate a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds the non-biological substrate and a second ligand that specifically binds cells, wherein the applying is free of coupling; (b) contacting the non-biological substrate, wherein the plurality of binding agents are bound to the non-biological substrate, with cells; (c) allowing a time sufficient for binding of the cells to the plurality of binding agents; and (d) culturing the cells.

202. A method for implanting a device in a subject, the method comprising: (a) applying to an implant a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds the implant and a second ligand that specifically binds cells at an implant site, wherein the applying is free of coupling; and (b) placing the implant in a subject at the implant site.

203. A method for modulating an activity of a biological substrate, the method comprising: (a) coating a non-biological substrate with a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds the biodegradable, non-biological substrate and a second ligand that specifically binds the biological substrate, wherein the coating is free of coupling; (b) placing the coated biodegradable, non-biological substrate at a target site, wherein the biological substrate is present at the target site; and (c) allowing a time sufficient for binding of the biological substrate at the target site to the binding agents, wherein the binding modulates the activity of the biological substrate.

204. The method of claim 203, wherein the biological substrate is selected from the group consisting of a tissue, a cell, a macromolecule, and combinations thereof.

205. The method of claim 204, wherein the cell is a vascular endothelial cell.

206. The method of claim 205, wherein the vascular endothelial cell is a tumor vascular endothelial cell.

207. The method of claim 204, wherein the macromolecule is a Tie2 receptor.

208. The method of claim 203, wherein the target site is a wound site and the modulating enhances wound healing.

209. The method of claim 203, wherein the target site is an angiogenic site and the modulating inhibits angiogenesis.

210. The method of claim 209, wherein the angiogenesis is tumor angiogenesis.

211. The method of claim 203, wherein the second ligand specifically binds a Tie2 receptor.

212. A method for creating a lubricant interface comprising applying to a first substrate a plurality of binding agents, wherein the applying is free of coupling, and wherein each of the plurality of binding agents comprises: (a) a ligand that specifically binds to the first substrate; and (b) a non-binding domain that shows substantially no binding to a second substrate.

213. The method of claim 212, wherein the first substrate comprises a non-biological substrate.

214. The method of claim 212 further comprising: (a) applying to an implant a plurality of binding agents, wherein each of the plurality of binding agents comprises a ligand that specifically binds the implant and a non-binding domain that shows substantially no binding to cells at an implant site, wherein the applying is free of coupling; and (b) placing the implant in a subject at the implant site, whereby a lubricant interface is created.

215. The method of claim 212, wherein the first substrate comprises a biological substrate.

216. The method of claim 212 further comprising: (a) administering to a subject a plurality of binding agents, wherein each of the plurality of binding agents comprises a ligand that specifically binds a first biological substrate and a non-binding domain that shows substantially no binding to a second biological substrate; and (b) allowing a time sufficient for binding of the plurality of binding agents to the first biological substrate, whereby a lubricant interface is created.

217. A method for preparing a non-biological substrate with a non-fouling agent comprising coating a non-biological substrate with a plurality of binding agents, wherein each of the plurality of binding agents comprises: (a) a ligand that specifically binds the non-biological substrate; and (b) a non-binding domain that shows substantially no binding to a fouling agent.

218. A method for drug administration to a subject, the method comprising: (a) applying to a non-biological drug, or to a non-biological carrier of the drug, a plurality of binding agents, wherein each of the plurality of binding agents comprises a first ligand that specifically binds the drug or the drug carrier and a second ligand that specifically binds a target cell; (b) administering the drug to a subject; and (c) allowing a sufficient time for binding of the plurality of binding agents to the target cell.

219. The method of claim 218, wherein the target cell has on its surface a Tie2 receptor.

220. The method of claim 218, wherein the second ligand binds the Tie2 receptor on the surface of the cell.

221. A method for screening a candidate substance for interaction with a biological substrate, the method comprising: (a) preparing a biological array comprising a plurality of biological substrates, wherein each of the plurality of biological substrates is specifically bound to one of a plurality of positions on a non-biological substrate; (b) contacting the biological array with a candidate substance; (c) allowing a time sufficient for binding of the candidate substance to the biological array; and (d) assaying an interaction between one or more of the biological substrates and the candidate substance, whereby an interacting molecule is identified.

222. The method of claim 221, wherein the interacting molecule is identified by a technique selected from the group consisting of spectroscopic, enzymatic, and electrochemical via a detectable label on one of the biological substrate or the non-biological substrate.

223. A kit comprising a first container containing an interfacial biomaterial of claim 1.

224. A kit containing a first container containing an interfacial biomaterial of claim 29.

225. A kit for preparing an interfacial biomaterial, the kit comprising: (a) a first binding agent comprising a ligand that specifically binds a non-biological substrate; and (b) a second binding agent comprising a ligand that specifically binds a biological substrate.

226. A kit for preparing an interfacial biomaterial, the kit comprising: (a) a binding agent comprising a ligand that specifically binds a non-biological substrate; and (b) a non-binding domain, wherein the non-binding domain shows substantially no binding to a target biological substrate.

227. The kit of any one of claims 225 and 226, further comprising a reagent for linking the binding agent and the non-binding domain.

228. The kit of any one of claims 223-226, further comprising a non-biological substrate.

229. The method of claim 203, wherein the non-biological substrate is biodegradable or non-biodegradable.