WO2002046213A2 - Methods and reagents for isolating angiogenic modulators - Google Patents

Abstract

The present invention relates to a method for isolating peptide domains capable of modulating angiogenesis. The method utilizes a peptide domain display library which may be used in both a 'display mode', attached to the surface of a microorganism, and in a 'secretion mode', wherein the peptide domains are secreted in soluble form.

Description

Methods and Reagents for Isolating Angiogenic Modulators

Background of the Invention

Angiogenesis

Blood vessels are the means by which oxygen and nutrients are supplied to living tissues and waste products are removed from living tissue. Angiogenesis refers to the process by which new blood vessels are formed. See, for example, the review by Folkman and Shing, J. Biol. Chem. 267 (16), 10931-10934 (1992). Under normal physiological conditions, humans or animals undergo angiogenesis only in very specific restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonal development and formation of the corpus luteum, endometrium and placenta. In adults, the proliferation rate of endothelial cells is typically low compared to other cell types in the body. The turnover time of these cells can exceed one thousand days.

The rate of angiogenesis involves a change in the local equilibrium between positive and negative regulators of the growth of microvessels. The therapeutic implications of angiogenic growth factors were first described by Folkman and colleagues over two decades ago (Folkman, N. Engl. J. Med., 285:1182-1186 (1971)). Abnormal angiogenesis occurs when the body loses at least some control of angiogenesis, resulting in either excessive or insufficient blood vessel growth. For instance, conditions such as ulcers, strokes, and heart attacks may result from the absence of angiogenesis normally required for natural healing. In contrast, excessive blood vessel proliferation can result in tumor growth, tumor spread, blindness, psoriasis and rheumatoid arthritis.

There are instances where a greater degree of angiogenesis is desirable— increasing blood circulation, wound healing, and ulcer healing. For example, recent investigations have established the feasibility of using recombinant angiogenic growth factors, such as fibroblast growth factor (FGF) family (Yanagisawa-Miwa, et al., Science, 257:1401-1403 (1992) and Baffour, et al., J Vase Surg, 16:181-91 (1992)), endothelial cell growth factor (ECGF) (Pu, et al, J Surg Res, 54:575-83 (1993)), and more recently, vascular endothelial growth factor (NEGF) to expedite and/or augment collateral artery development in animal models of myocardial and hindlimb ischemia (Takeshita, et al, Circulation, 90:228-234 (1994) and Takeshita, et al., J Clin Invest, 93 :662-70 (1994)).

Conversely, there are instances, where inhibition of angiogenesis is desirable. For example, many diseases are driven by persistent unregulated angiogenesis, also sometimes referred to as "neovascularization." In arthritis, new capillary blood vessels invade the joint and destroy cartilage. In diabetes, new capillaries invade the vitreous, bleed, and cause blindness. Ocular neovascularization is the most common cause of blindness. Tumor growth and metastasis are angiogenesis-dependent. A tumor must continuously stimulate the growth of new capillary blood vessels for the tumor itself to grow.

Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the erosion of the basement membrane by enzymes released by endothelial cells and leukocytes. The endothelial cells, which line the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimulants induce the endothelial cells to migrate through the eroded basement membrane. The migrating cells form a "sprout" off the parent blood vessel, where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating the new blood vessel.

Although angiogenesis is a highly regulated process under normal conditions, many diseases (characterized as "angiogenic diseases") are driven by persistent unregulated angiogenesis. Otherwise stated, unregulated angiogenesis may either cause a particular disease directly or exacerbate an existing pathological condition. For example, ocular neovacularization has been implicated as the most common cause of blindness and dominates approximately 20 eye diseases. In certain existing conditions such as arthritis, newly formed capillary blood vessels invade the joints and destroy cartilage. In diabetes, new capillaries formed in the retina invade the vitreous, bleed, and cause blindness. Growth and metastasis of solid tumors are also angiogenesis-dependent (Folkman, J., Cancer Research, 46: 467-473 (1986), Folkman, J., Journal of the National Cancer Institute, 82: 4-6 (1989)). It has been shown for example that tumors which enlarge to greater than 2 mm, must obtain their own blood supply and do so by inducing the growth of new capillary blood vessels. Once these new blood vessels become embedded in the tumor, they provide a means for tumor cells to enter the circulation and metastasize to distant sites, such as liver, lung or bone (Weidner, N., et al., The New England Journal of Medicine, 324(1): 1-8 (1991)). Several lines of direct evidence now suggest that angiogenesis is essential for the growth and persistence of solid tumors and their metastases (Folkman, 1989; Hori et al., 1991; Kim et al., 1993; Millauer et al, 1994). To stimulate angiogenesis, tumors upregulate their production of a variety of angiogenic factors, including the fibroblast growth factors (FGF and BFGF) (Kandel et al, 1991) and vascular endothelial cell growth factor/vascular permeability factor (NEGF/NPF). However, many malignant tumors also generate inhibitors of angiogenesis, including angiostatin and thrombospondin (Chen et al., 1995; Good et al., 1990; O'Reilly et al., 1994). It is postulated that the angiogenic phenotype is the result of a net balance between these positive and negative regulators of neovascularization (Good et al., 1990; O'Reilly et al., 1994; Parangi et al., 1996; Rastinejad et al., 1989). Several other endogenous inhibitors of angiogenesis have been identified, although not all are associated with the presence of a tumor. These include, platelet factor 4 (Gupta et al., 1995; Maione et al, 1990), interferon-alpha, interferon-inducible protein 10 (Angiolillo et al., 1995; Strieter et al., 1995), which is induced by interleukin-12 and/or interferon-gamma (Noest et al, 1995), gro-beta (Cao et al., 1995), and the 16 kDa Ν-terminal fragment of prolactin (Clapp et al., 1993). The only known angiogenesis inhibitor which specifically inhibits endothelial cell proliferation is angiostatin (O'Reilly et al. 1994).

Angiogenic Inhibitors

There are a number of well known proteins with anti-angiogenic activities, including thrombospondin I (Kosfeld et al, J. Biol. Chem., 267:16230-16236 (1993); Tolsma et al, J. Cell Biol., 122:497-511 (1993); and Dameron et al, Science, 265:1582-1584 (1995)), IL-12 (Voest et al, J. Νatl. Cancer Inst.,87:581-586 (1995)), protamine (Ingber et al, Nature, 348:555-557 (1990)), angiostatin (O'Reilly et al, Cell, 79:315-328 (1994)), laminin (Salcamoto et al, Cancer Res., 5:903-906 (1991)), endostatin (O'Reilly et al., Cell, 88:277-285 (1997)), and a prolactin fragment (Clapp et al, Endocrinol., 133:1292-1299 (1993)). In addition, several anti- angiogenic peptides have been isolated from these proteins (Maione et al, Science, 247:77-79 (1990); Woltering et al, J. Surg. Res., 50:245-251 (1991); and Eijan et al, Mol. Biother., 3:38-40 (1991)).

Thrombospondin I (TSP1, MW 450,000) is a large trimeric glycoprotein composed of tliree identical 180 kDa subunits (Lahav et al, Semin. Tliromb. Hemostasis, 13:352-360 (1987)) linked by disulfide bonds (Lawer et al, J. Cell Biol, 103:1635-1648 (1986); and Lahav et al, Eur. J. Biochem., 145:151-156 (1984)). The majority of anti-angiogenic activity is found in the central 70 kDa stalk region of this protein (Tolsma et al, supra). There are at least two different structural domains within this central stalk region that inhibit neovascularization (Tolsma et al, supra).

Besides TSPI, there are six other proteins (fibronectin, laminin, platelet factor-4, angiostatin, endostatin and prolactin fragment) in which peptides have been isolated that inhibit angiogenesis. In addition, the dominant negative fragment of F1K1 and analogues of the peptide somatostatin are known to inhibit angiogenesis. Fibronectin (FN) is a major surface component of many normal cells, as well as a potent cell spreading factor. During transformation, the loss of cellular FN has been observed. Furthermore, the addition of fibronectin to transformed cells restores the normal phenotype. It has been found that either heparin-binding or cell-adhesion fragments from FN can inhibit experimental metastasis, suggesting that cell surface proteolyglycans are important in mediating the adhesion of metastatic tumor cells (McCarthy et al, J. Natl. Cancer Inst, 80:108-116 (1988)). It has also been found that FN and one of its peptides inhibits in vivo angiogenesis (Eijan et al, Mol. Biother., 3:38-40 (1991)).

Laminin is a major component of the basement membrane, and is known to have several biologically active sites that bind to endothelial and tumor cells. Laminin is a cruciform molecule that is composed of three chains, an A Chain and two B chains. Several sites in laminin have been identified as cell binding domains. These sites promote cellular activities in vitro, such as cell spreading, migration, and cell differentiation. Two peptides from two sites of the laminin Bl chain are known to inhibit angiogenesis (Grant et al, Path. Res. Pract, 190:854-863 (1994)). Platelet factor-4 (PF4) is a platelet α-granule protein originally characterized by its high affinity for heparin. The protein is released from platelets during aggregation as a high molecular weight complex of a tetramer of the PF4 polypeptide and chondroitin sulfate, which dissociates at high ionic strength. PF4 has several biological properties including immunosuppression, chemotactic activity for neutrophils and monocytes as well as for fibroblasts, inhibition of bone resorption, and inhibition of angiogenesis. The angiostatic properties of human PF4 are associated with the carboxyl-terminal, heparin binding region of the molecule. A 12 amino acid synthetic peptide derived therefrom has been discovered to have marked angiostatic affects (Maione et al, Science, 247:77-79 (1990)).

Endostatin is a 20 kDa protein fragment of collagen XNIII. It has recently been found to be a potent inhibitor of tumor angiogenesis and tumor growth (O'Reilly et al, Cell, 88, 277-285, 1997).

Although somatostatin is not a protein, it is a naturally-occurring cyclic 14 amino acid peptide whose most-recognized function is the inhibition of growth hormone (GH) secretion. Somatostatin is widely distributed in the brain, in which it fulfills a neuromodulatory role, and in several organs of the gastrointestinal tract, where it can act as a paracrine factor or as a true circulating factor. The role played by the neuropeptide somatostatin, also known as somatotropin release inhibitory factor (SRIF), in human cancer is not well understood. Recent investigations involving somatostatin receptors in normal and neoplastic human tissues suggest that the action is complex, and involves both direct and indirect mechanisms. One of the anti-tumor mechanisms of these synthetic somatostatin analogues may be an anti-angiogenic effect (Woltering et al, J. Surg. Res., 50:245-50 (1990)). In a recent study, the ability of native somatostatin and nine somatostatin analogues to inhibit angiogenesis were evaluated. The most potent somatostatin analogue was found to be approximately twice as potent as the naturally-occurring somatostatin (Barrie et al, J. Surg. Res., 55:446-50 (1993)).

Angiostatin is a 38 kDa polypeptide fragment of which is an internal fragment of plasminogen. Whereas plasminogen has no fibrinogenic activity, angiostatin has marked angiogenic activity (O'Rielly MS, et al Cell, 79:315-28 (1994)). Angiostatin has been shown to reduce tumor weight and to inhibit metastasis in certain tumor models.

The Flkl receptor is a receptor for vascular endothelial growth factor (NEGF). F1K-1 is exclusively expressed on the surface of the endothelial cells. Once NEGF. binds to the receptor, the Flk-1 receptor then homodimerizes to stimulate the endothelial cell to divide. If a mutant receptor of Flk-1 is transfected into the endothelial cells, the mutant receptor dimerizes with the wild-type Flk-1 receptor. In this endothelial transfected with the mutant Flk-1 receptor, VEGF is unable to stimulate the endothelial cells to divide. Co-administration of a retrovirus carrying the Flk-1 cDΝA (Millauer B. et a., Nature, 367, 1994) inhibits tumor growth. This emphasizes that the receptor plays a critical role in the angiogenesis of solid tumors.

Finally, a 16 kDa fragment of prolactin has been found to be antiangiogenic. Similar to plasminogen, prolactin is not anti-angiogenic but the prolactin fragment is a potent in vivo and in vitro inhibitor of angiogenesis (Clapp C. et al. Endocrinology. 133:1292-1299 (1993).

An emerging common theme among many of those identified angiogenesis modulators, such as the naturally occurring angiogenesis inhibitors angiostatin, endostatin or antiangiogenic antithrombin (aaAT), is that they are generated by a proteolytic cleavage of larger non-angiogenic precursor proteins. For example, as mentioned before, angiostatin is a fragment of plasminogen, endostatin is a domain of collagen XNIII and aaAT is an internal fragment of antithrombin III. Intriguingly, none of the large precursor molecules have antiangiogenic effect on their own, which makes the isolation of those inhibitors difficult through conventional molecular biology techniques such as expression cloning. Therefore, there is a need for an improved method to identify novel modulators of angiogenetic peptides.

Although several angiogenesis inhibitors are currently under development for use in treating angiogenic diseases (Gasparini, G. and Harris, A. L., J Clin Oncol 13(3): 765-782, (1995)), there are disadvantages associated with several of these compounds. For example, suramin is a potent angiogenesis inhibitor, but causes (at doses required to reach antitumor activity) severe systemic toxicity in humans. Other compounds, such as retinoids, interferons and antiestrogens are safe for human use but have only a weak anti-angiogenic effect. Still other compounds may be difficult or costly to make. Although preliminary results with the antiangiogenic proteins are promising, they are relatively large in size and are difficult to use and produce. Moreover, proteins are subject to enzymatic degradation. Thus, new agents that inhibit angiogenesis are needed. New antiangiogenic proteins or peptides that show improvement in size, ease of production, stability and/or potency would be desirable.

High Throughput Screening and Drug Discovery

High throughput screening has become a dominant tool in the pharmaceutical industry for the discovery of lead compounds that can be modified into candidates for drug development. For instance, it is abundantly used for identification of ligands with high affinity for receptors. In this regard, combinatorial techniques have provided approaches to generating and deconvoluting large libraries of test compounds in high throughput screens. It involves selection and amplification of a subset of molecules with desired biological properties from complex libraries.

One technique which has emerged for identification of peptide leads involves the use of peptide display methodologies such as phage display. Phage-displayed peptide libraries can comprise vast collections of short, randomized polypeptides that are displayed on the surface of a filamentous bacteriophage particle. Thus, each "peptide" is actually the N-terminal sequence of a phage-coat protein, that is encoded by a randomly-mutated region of the phage genome responsible for the production of the coat protein. In this manner, each unique peptide in the library is physically linked with the DNA molecule encoding it. Antibodies and other binding molecules can be used as "targets" to specifically select rare phage clones bearing ligand peptides, and sequencing of the corresponding viral DNA will reveal their amino acid sequences. Relatively high-affinity peptides for a variety of peptide- and non-peptide-binding targets have been affinity-isolated from epitope libraries. This technology has been used to map epitopes on proteins and to find peptide mimics for a variety of target molecules. Many powerful applications can be envisioned in the areas of drug design and the development of diagnostic markers, vaccines and toleragens. For the purposes of drug discovery, there are potential advantages in the use of genetically encoded libraries, such as phage display (Scott et al, Science 249. 386 (1990); Devlin et al., Science 249, 386 (1990)), "peptide on plasmid" (Cull et al. PNAS 89, 1865 (1992)), and in vitro translation-based systems (Mattheakis et al. PNAS 91, 9022 (1994)), compared to the use of synthetic small molecule libraries (Bunin et al. PNAS 91, 4708 (1994); Gordon et al. J. Med. Chem. 37, 1385 (1994); and Dooley et al., Science 266, 2019 (1994)). The genetic encoding of libraries allows the resynthesis and rescreening of molecules with a desired binding activity. The resulting amplification of interacting molecules in subsequent rounds of selection can lead to the isolation of extremely rare, specific binders from a large pool of molecules.

However, despite the success of these methods, they suffer from numerous sources of error and bias, such as very low initial concentrations of species, nonspecific binding, and, significantly, the sampling of only a fraction of the library at the end of an experiment.

Another problem with the traditional random peptide display technique is that many of these randomly generated peptides or mutations of natural peptides will potentially elicit adverse immune responses when administered to a subject as a therapeutic since the body may perceive these peptides as "foreign." As a consequence of using these peptides, not only the effective concentration of the therapeutics may be reduced but also the possibility of causing undesirable side effects is raised. Therefore, there is a need to identify peptides that have retained desirable biological effects while having minimum side effects.

Summary of the Invention

One aspect of the invention provides a method for isolating a peptide domain capable of modulating angiogenic activity, comprising the steps of:

(i) providing a peptide domain display library comprising a variegated population of test peptide domains expressed on the surface of a population of display packages; (ii) in a display mode, isolating, from the peptide domain display library, a sub-population of display packages enriched for test peptide domains which have a binding specificity for an endothelial cell or a component thereof;

(iii) in a secretion mode, simultaneously expressing the enriched test peptide domain sub-population under conditions wherein the test peptide domains are secreted and are free of the display packages;

(iv) assessing the ability of the secreted test peptide domains to regulate a biological process of an endothelial cell; and

(v) assessing the ability of the test peptide domains capable of regulating a biological process of an endothelial cell for the ability to regulate angiogenesis, thereby isolating a peptide domain capable of modulating angiogenic activity.

The method may be used to isolate peptide domains capable of stimulating angiogenesis or peptide domains capable of inhibiting angiogenesis.

In one embodiment, the peptide domain display library is encoded by a nucleic acid library constructed by random priming of mRNA.

In another embodiment, the peptide domain display library can be a phage display library, e.g., which utilizes phage particles such as M13, fl, fd, Ifl, Ike, Xf, Pfl, Pf3, λ, T4, T7, P2, P4, φX-174, MS2 or f2. In preferred embodiments, the phage display library is generated with a filamentous bacteriophage specific for Escherichia coli and the phage coat protein is coat protein III or coat protein VIII. For instance, the filamentous bacteriophage can be Ml 3, fd, or fl.

In other embodiments, the peptide domain display library is a bacterial cell- surface display library or a spore display library.

In certain embodiments, the test peptide domains are enriched from the peptide domain display library in the display mode by a differential binding step comprising affinity separation of test peptide domains which specifically bind the endothelial cell or component thereof from test peptide domains which do not. For example, the differential binding step can include panning the peptide domain display library on whole endothelial cells, affinity chromatographic step in which a component of an endothelial cell is provided as part of an insoluble matrix (e.g., a cell surface protein attached to a polymeric support), and/or immunoprecipitating the display packages.

In the display mode, the test peptide domains can be enriched for those which bind to an endothelial cell specific marker and/or an endothelial cell surface receptor protein. For example, the test peptide domain library can be enriched in the display mode for test peptide domains which bind to a receptor for an endothelial cell mitogen or a receptor for an angiogenic factor. In still other embodiments, the test peptide domain library can be enriched in the display mode for test peptide domains which bind to a receptor for acidic fibroblast growth factor (aFGF), angiogenin, angiotensin, angiopoietin, angiotropin, antiangiogenic antithrombin (aaAT), atrial natriuretic factor (ANF), basic fibroblast growth factor (bFGF), betacellulin, endostatin, endothelial cell-derived growth factor (ECDGF), endothelial cell growth factor (ECGF), endothelial cell growth inhibitor, endothelial monocyte activating polypeptide (EMAP), endothelial cell-viability maintaining factor, endothelin (ET), endothelioma derived mobility factor (EDMF), epidermal growth factors (EGF), eryfhropoietin (Epo), fibroblast growth factors (FGF), heart derived inhibitor of vascular cell proliferation, hematopoietic growth factors, hepatic growth factor, insulin like growth factors (IGF), interferon (IFN), interleukins (IL), oncostatin M, placental growth factor (P1GF), platelet derived growth factor (PDGF), platelet derived endothelial cell growth factor (PD-ECGF), somatostatin, transferrin, transforming growth factor-beta (TGF-beta), thrombin, thrombospondin, tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor beta (TNF-b), vasoactive intestinal peptide or vascular endothelial growth factor (NEGF).

In preferred embodiments, the peptide domain display library includes at least IO³ different test peptide domains.

In preferred embodiments, the test peptide domains are 4-20 amino acid residues in length. In other preferred embodiments, the test peptide domains are 50- 300 amino acid residues in length. In certain embodiments, each of the test peptide domains are encoded by a chimeric gene comprising (i) a coding sequence for the test peptide domain, (ii) a coding sequence for a surface protein of the display package for displaying the test peptide domains on the surface of a population of display packages, and (iii) RNA splice sites flanking the coding sequence for the surface protein, wherein, in the display mode, the chimeric gene is expressed as fusion protein including the test peptide domain and the surface protein, whereas in the secretion mode, the test peptide domain is expressed without the surface protein as a result of the coding sequence for the surface protein being removed by RNA splicing. In preferred embodiments, the test peptide domains are expressed by a eukaryotic cell, more preferably a mammalian cell, in the secretion mode.

In preferred embodiments, the endothelial cell of step (ii) or (iv) is a mammalian cell such as a human cell, more preferably a human capillary endothelial cell. In certain embodiments, the biological process scored for in the secretion mode includes a change in cell proliferation, cell migration, cell differentiation or cell death.

In certain embodiments, the endothelial cell includes a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity of the cell surface receptor protein, expression of the reporter gene providing the detectable signal. For instance, the reporter gene can encode a gene product that gives rise to a detectable signal selected from the group consisting of: color, fluorescence, luminescence, cell viability relief of a cell nutritional requirement, cell growth, and drug resistance. In preferred embodiments, the reporter gene encodes a gene product selected from the group consisting of chloramphenicol acetyl transferase, beta-galactosidase and secreted alkaline phosphatase. In other preferred embodiments, the reporter gene encodes a gene product which confers a growth signal. In certain embodiments, the secretion mode includes assessing the ability of the secreted test peptide domains to modulate the biological activity induced on the endothelial cells by an exogenously added compound. In preferred embodiments, the compound is an endothelial cell mitogen, an angiogenic factor, or a compound selected from a group consisting of acidic fibroblast growth factor (aFGF), angiogenin, angiotensin, angiopoietin, angiotropin, antiangiogenic antithrombin (aaAT), atrial natriuretic factor (ANF), basic fibroblast growth factor (bFGF), betacellulin, endostatin, endothelial cell-derived growth factor (ECDGF), endothelial cell growth factor (ECGF), endothelial cell growth inhibitor, endothelial monocyte activating polypeptide (EMAP), endothelial cell- viability maintaining factor, endothelin (ET), endothelioma derived mobility factor (EDMF), epidermal growth factors (EGF), erythropoietin (Epo), fibroblast growth factors (FGF), heart derived inhibitor of vascular cell proliferation, hematopoietic growth factors, hepatic growth factor, insulin like growth factors (IGF), interferon (IFN), interleukins (IL), oncostatin M, placental growth factor (PIGF), platelet derived growth factor (PDGF)< platelet derived endothelial cell growth factor (PD-ECGF), somatostatin, transferrin, transforming growth factor-beta (TGF-beta), thrombin, thiOmbospondin, tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor beta (TNF-b), vasoactive intestinal peptide and vascular endothelial growth factor (VEGF). In an exemplary embodiment: in step (iv) above the ability of the secreted test peptide domains to inhibit proliferation of endothelial cells in the presence of an angiogenic amount of an endogenous growth factor can be assessed.

The subject invention also specifically contemplates that peptide domains identified in the secretion mode can be converted into peptidomimietics. Moreover, in certain embodiments, the subject method includes the further step of formulating, with a pharmaceutically acceptable carrier, one or more test peptide domains which modulate angiogenesis or peptidomimetics thereof.

Another aspect of the present invention provides a peptide domain display library enriched for test peptide domains having a binding specificity and/or affinity for an endothelial cell or a component thereof and which inhibit endothelial cell proliferation and/or migration in a target endothelial cell.

Still another aspect of the present invention relates to a vector comprising a chimeric gene for a chimeric protein, which chimeric gene comprises (i) a coding sequence for a test peptide domain, (ii) a coding sequence for a surface protein of a display package, and (iii) RNA splice sites flanking the coding sequence for the surface protein, wherein, in a display mode, the chimeric gene is expressed as a fusion protein including the test peptide domain and the surface protein such that the test peptide domain can be displayed on the surface of a population of display packages, whereas in the secretion mode, the test peptide domain is expressed without the surface protein as a result of the coding sequence for the surface protein being removed by RNA splicing.

In certain embodiments, the chimeric gene can include a secretion signal sequence for secretion of the test peptide domain in the secretion mode, e.g., secretion of the test peptide domain from eukaryotic cells, preferably mammalian cells.

Yet another aspect of the present invention provides a vector library, each vector comprising a chimeric gene for a chimeric protein, which chimeric gene comprises (i) a coding sequence for a test peptide domain, (ii) a coding sequence for a surface protein of a display package, and (iii) RNA splice sites flanking the coding sequence for the surface protein, wherein, in a display mode, the chimeric gene is expressed as fusion protein including the test peptide domain and the surface protein such that the test peptide domain can be displayed on the surface of a population of display packages, whereas in the secretion mode, the test peptide domain is expressed without the surface protein as a result of the coding sequence for the surface protein being removed by RNA splicing, the vector library collectively encodes a variegated population of test peptide domains.

In preferred embodiments, the vector library collectively encodes at least 10³ different test peptide domains. In preferred embodiments, the test peptide domains are 4-20 amino acid residues in length. In other preferred embodiments, the test peptide domains are 50- 300 amino acid residues in length.

Another aspect of the present invention is a cell composition comprising a population of cells containing the vector library described above.

Still another aspect of the present invention provides a method for isolating a peptide domain capable of inhibiting angiogenic activity, comprising:

(i) providing a peptide domain display library comprising a variegated population of test peptide domains expressed on the surface of a population of display packages ;

(ii) in a display mode, isolating, from the display packages, a sub- population of display packages enriched for test peptide domains which have a binding specificity for an endothelial cell or a component thereof;

(iv) in a secretion mode, simultaneously expressing the enriched test peptide domain sub-population under conditions wherein the test peptide domains are secreted and are free of the display packages;

(v) assessing the ability of the secreted test peptide domains to inhibit endothelial cell proliferation and/or migration; and

(vi) assessing the ability of the test peptide domains capable of inhibiting endothelial cell migration and/or proliferation for the ability to inhibit angiogenesis, thereby isolating a peptide domain capable of inhibiting angiogenic activity.

Another aspect of the invention provides a method of conducting a pharmaceutical business, comprising: by any of the suitable methods described above, identifying one or more peptide domains which are capable of modulating angiogenic activity; conducting therapeutic profiling of said identified peptide domain(s), or other homologs or peptidomimetics thereof, for using the peptide domain(s) to modulate angiogenesis; and, formulating a pharmaceutical preparation including one or more identified peptide domain(s) as a product having an acceptable therapeutic profile.

In one embodiment, the business method further comprises establishing a distribution system for distributing said product for sale. In another embodiment, the business method further comprises establishing a sales group for marketing the product.

Another aspect of the invention provides a method of conducting a pharmaceutical business, comprising: by any of the suitable methods described above, identifying one or more peptide domains which are capable of modulating angiogenic activity; conducting therapeutic profiling of said identified peptide domain(s), or other homologs or peptidomimetics thereof, for using the peptide domain(s) to modulate angiogenesis; and, licensing, to a third party, the rights for further development of agents to modulate angiogenesis.

Brief Description of the Drawings

Figure 1: Schematic of pAM7 & pAM9 M13/COS peptide expression plasmid.

Figure 2: a) Transwell cell culture chamber; b) Indicator plate antimicrobial assay.

Figure 3: Flowchart depicting utilization of the Ml 3 display/COS secretion method for identification of anti-angiogenic peptides.

Figure 4: Nucleotide level depiction of pAM7 M13/COS peptide expression plasmid.

Figure 5: Titration of plaque and colony forming units generated upon phagemid rescue.

Figure 6: Secretion of M13/COS plasmid encoded proteins from COS-7 cells.

Figure 7: Anti-pIII western blot detection of peptides incorporated into M13 phagemid capsids as pill fusions. Figure 8: Specific binding of pAM9-Kl phagemids to bovine capillary endothelial cells.

Figure 9: Inhibition BCE cell proliferation in Transwells by peptides secreted from COS-7 cells.

Detailed Description of the Invention

/. Overview

The present invention makes available a powerful directed approach for isolating biologically active peptide domains. One aspect of the present invention is the synthesis of a binary method that combines variegated peptide. domain display libraries, e.g., in a "display mode", with soluble secreted peptide domain libraries, e.g., in a "secretion mode", to yield a method for the efficient isolation of peptide domains having a desired biological activity.

Utilizing peptide domain display techniques, a peptide domain library can first be reduced in complexity by panning or other affinity purification techniques. In particular, the subject method selects .peptide domains having a certain affinity profile, e.g., a specificity and/or binding affinity for a discrete cell or protein or other cellular component thereof by (i) displaying the peptide domains on the outer surface of a replicable genetic display package to create a peptide domain display library, and (ii) using affinity selection techniques to enrich the population of display packages for those containing peptide domains which have a desired binding specificity for the target cell or cellular component (herein collectively referred to as the "target").

After the affinity enrichment step, the resulting sub-library is then utilized in a secretion mode whereby the test peptide domains are secreted as soluble extracellular factors and their effect as paracrine or autocrine factors is scored. That is, the secretion mode measures biological activity of the test peptide domains in order to distinguish between agonist, antagonist, and inactive peptide domains with regard to regulating a particular biological response of a test cell or tissue. In preferred embodiments, the display mode and secretion mode can be carried out without the need to sub-clone the test peptide domain coding sequence into another vector. To illustrate, Figures 1 shows exemplary vectors for sequential use in both the display and secretion modes. In bacterial cells, the vectors produce a fusion protein consisting of a secretion signal sequence, the test peptide domain and the remaining C-terminal portion of the gene III or VIII protein. The resulting chimeric protein is capable of being incorporated into an Ml 3 phage particle. However, in mammalian cells (such as COS cells), the Ml 3 coding sequences are removed from the mature mRNA by virtue of splice sites which flank the phage sequence. Thus, the mature mRNA, in mammalian cells, encodes a secretion signal sequence and test peptide domain alone, which is secreted as a soluble peptide domain from the cell.

One advantage to such embodiments of the subject method is the ability to reduce loss of peptide domain sequences from the sub-library by eliminating sub- cloning steps.

In an exemplary embodiment, the subject method can be used to identify peptide domains capable of modulating angiogenesis. The method may be used to identify peptide domains with angiogenic stimulatory activity or angiogenic inhibitory activity. In this regard, the present invention makes available a method for identifying angiogenic modulators which can be used to treat diseases related to insufficient or inappropriate angiogenesis.

II. Definitions

Before further description of the invention, certain terms employed in the specification, examples and appended claims are, for convenience, collected here. As used herein, the term "angiogenesis" means the generation of new blood vessels into a tissue or organ.

The term "angiogenic inhibitor" or "inhibitor of angiogenesis" refers to an agent capable of preventing neovascularization and/or reducing the amount of existing blood vessels. The term "angiogenic stimulator" or "inducer of angiogenesis" refers to an agent capable inducing the formation of new blood vessels.

As used herein, "cell surface receptor" refers to molecules that occur on the surface of cells, interact with the extracellular environment, and (directly or indirectly) transmit or transduce the information regarding the environment intracellularly in a manner that may modulate intracellular second messenger activities or transcription of specific promoters, resulting in transcription of specific genes.

The term "compound" as used herein is meant to include both exogenously added test compounds and peptide domains expressed from a peptide domain library.

The language "differential binding" as well as "affinity selection" and "affinity enrichment" refer to the separation of members of the peptide domain display library based on the differing abilities of peptide domains on the surface of each of the display packages of the library to bind to the target. The differential binding of a target by test peptide domains of the display can be used in the affinity separation of those peptide domains which specifically bind the target from those which do not. For example, the affinity selection protocol can also include a pre- or post-enrichment step wherein display packages capable of binding "background targets", e.g., as a negative selection, are removed from the library. Examples of affinity selection, while not limiting, include affinity chromatography, immunoprecipitation, fluorescence activated cell sorting, agglutination, and plaque lifts. As described below, the affinity chromatography may include bio-panning techniques using either purified, immobilized target proteins or the like, as well as whole cells.

The term "domain" or "fragment" as used in "peptide domain," "test peptide domain," "peptide domain library" refers to a fragment or a portion of a naturally occurring polypeptide encoded by a source nucleotide (i.e., mRNA, cDNA, etc.). It mimics the release of a polypeptide domain or fragment following proteolytic cleavage. In its broadest sense, the domain may or may not correspond to a naturally folding domain / motif occurring in the tertiary structure of a natural polypeptide. Therefore, in some cases, the domain / fragment may or may not fold properly or correspond to its natural folding. Each domain, however, does represent a naturally occurring (as opposed to non-natural or mutated) amino acid sequences. The polynucleotides encoding these domains can be obtained by, for example, random priming of a source polynucleotide (mRNA, etc.). The size of the domain / fragment can be selected to be a pre-determined size. For example, if 50 - 300 residue domains are to be used, the average source polynucleotide will be 150 - 900 bp.

The term "effective amount" refers to an amount sufficient to induce a statistically significant result.

The term "endothelial cell" refers to a cell of the squamous epithelium which lines the interiors of cavities, spaces or blood vessels of an animal.

The term "endothelial cell mitogen" refers to an agent capable of stimulating mitosis and/or cellular transformation of an endothelial cell. The term "endothelium" means a thin layer of flat epithelial cells that lines serous cavities, lymph vessels, and blood vessels.

As used herein, "extracellular signals" include a molecule or other change in the extracellular environment that is transduced intracellularly via cell surface proteins that interact, directly or indirectly, with the signal. An extracellular signal or effector molecule includes any compound or substance that in some manner alters the activity of a cell surface protein. Examples of such signals include, but are not limited to, molecules such as acetylcholine, growth factors and hormones, lipids, sugars and nucleotides that bind to cell surface and/or intracellular receptors and ion channels and modulate the activity of such receptors and channels. As used herein, "extracellular signals" also include as yet unidentified substances that modulate the activity of a cellular receptor, and thereby influence intracellular functions. Such extracellular signals are potential pharmacological agents that may be used to treat specific diseases by modulating the activity of specific cell surface receptors. The language "fusion protein" and "chimeric protein" are art-recognized terms which are used interchangeably herein, and include contiguous polypeptides comprising a first polypeptide covalently linked via an amide bond to one or more amino acid sequences which define polypeptide domains that are foreign to and not substantially homologous with any domain of the first polypeptide. According to the instant invention, one portion of the fusion protein comprises a test peptide domain, e.g., which can be generated by random priming. A second polypeptide portion of the fusion protein is typically derived from an outer surface protein or display anchor protein which directs the "display package" (as hereafter defined) to associate the test peptide domain with its outer surface. As described below, where the display package is a phage, this anchor protein can be derived from a surface protein native to the genetic package, such as a viral coat protein. Where the fusion protein comprises a viral coat protein and a test peptide domain, it will be referred to as a "peptide domain fusion coat protein". The fusion protein may further comprise a signal sequence, which is a short length of amino acid sequence at the amino terminal end of the fusion protein, that directs at least the portion of the fusion protein including the test peptide domain to be secreted from the cytosol of a cell and localized on the extracellular side of the cell membrane.

Gene constructs encoding fusion proteins are likewise referred to a "fusion genes" or "chimeric genes."

The language "helper phage" describes a phage which is used to infect cells containing a defective phage genome or phage vector and which functions to complement the defect. The defect can be one which results from removal or inactivation of phage genomic sequence required for production of phage particles. Examples of helper phage are M 13K07.

The term "indicator gene" generically refers to an expressible (e.g., able to be transcribed and optionally translated) DNA sequence which is, for example, expressed in response to a signal transduction pathway modulated by a target receptor or ion channel. Exemplary indicator genes include unmodified endogenous genes of the host cell, modified endogenous genes, or a reporter gene of a heterologous construct, e.g., as part of a reporter gene construct. The phrases "individually selective manner" and "individually selective binding", with respect to binding of a test peptide domain with a target protein, refers to the binding of a peptide domain to a certain protein target which binding is specific for, and dependent on, the molecular identity of the protein target. The term "ligand" refers to a molecule that is recognized by a particular protein, e.g., a receptor. Any agent bound by or reacting with a protein is called a "ligand," so the term encompasses the substrate of an enzyme and the reactants of a catalyzed reaction. The term "ligand" does not imply any particular molecular size or other structural or compositional feature other than that the substance in question is capable of binding or otherwise interacting with a protein. A "ligand" may serve either as the natural ligand to which the protein binds or as a functional analogue that may act as an agonist or antagonist.

The term "modulation of a signal transduction activity of a receptor protein" in its various grammatical forms, as used herein, designates induction and/or potentiation, as well as inhibition of one or more signal transduction pathways downstream of a receptor.

"Orphan receptors" is a designation given to a receptors for which no specific natural ligand has been described and/or for which no function has been determined. The terms "phage vector" and "phagemid" are art-recognized and generally refer to a vector derived by modification of a phage genome, containing an origin of replication for a bacteriophage, and preferably, though optional, an origin (ori) for a bacterial plasmid. The use of phage vectors rather than the phage genome itself provides greater flexibility to vary the ratio of chimeric peptide/coat protein to wild- type coat protein, as well as supplement the phage genes with additional genes encoding other heterologous polypeptides, such as "auxiliary polypeptides" which may be useful in the "dual" peptide display constructs described below.

The term "proliferation" refers to an increase in cell number.

The term "peptide" or "polypeptide" refers to an oligomer in which the monomers are amino acids (usually alpha-amino acids) joined together through amide bonds. Peptides / polypeptides are two or more amino acid monomers long, but more often are between 5 to 10 amino acid monomers long and can be even longer, i.e. up to 20 amino acids or more. The term "protein" is well known in the art and usually refers to a very large polypeptide, or set of associated homologous or heterologous polypeptides, that has some biological function. For purposes of the present invention the terms "peptide," "polypeptide," and "protein" are largely interchangeable as all tliree types can be used to generate the display library and so are collectively referred to as peptides.

Agonists and antagonists are "receptor effector" molecules that modulate signal transduction via a receptor. Receptor effector molecules are capable of binding to the receptor, though not necessarily at the binding site of the natural ligand. Receptor effectors can modulate signal transduction when used alone, i.e. can be surrogate ligands, or can alter signal transduction in the presence of the natural ligand, either to enhance or inhibit signaling by the natural ligand. For example, "antagonists" are molecules that block or decrease the signal transduction activity of receptor, e.g., they can competitively, noncompetitively, and/or allosterically inhibit signal transduction from the receptor, whereas "agonists" potentiate, induce or otherwise enhance the signal transduction activity of a receptor.

The terms "receptor activator" and "surrogate ligand" refer to an agonist which induces signal transduction from a receptor.

As used herein, a "reporter gene construct" is a nucleic acid that includes a "reporter gene" operatively linked to at least one transcriptional regulatory sequence. Transcription of the reporter gene is controlled by these sequences to which they are linked. The activity of at least one or more of these control sequences can be directly or indirectly regulated by the target receptor protein. Exemplary transcriptional control sequences are promoter sequences. A reporter gene is meant to include a promoter-reporter gene construct which is heterologously expressed in a cell.

The language "replicable genetic display package" or "display package" describes a biological particle which has genetic information providing the particle with the ability to replicate. The package can display a fusion protein including a peptide domain derived from the variegated peptide domain library. The test peptide domain portion of the fusion protein is presented by the display package in a context which permits the peptide domain to bind to a target that is contacted with the display package. The display package will generally be derived from a system that allows the sampling of very large variegated peptide domain libraries. The display package can be, for example, derived from vegetative bacterial cells, bacterial spores, and bacterial viruses.

"Signal transduction" is the processing of physical or chemical signals from the cellular environment through the cell membrane, and may occur through one or more of several mechanisms, such as activation / inactivation of enzymes (such as proteases, or other enzymes which may alter phosphorylation patterns or other post- translational modifications), activation of ion channels or intracellular ion stores, effector enzyme activation via guanine nucleotide binding protein intermediates, formation of inositol phosphate, activation or inactivation of adenylyl cyclase, direct activation (or inhibition) of a transcriptional factor and/or activation. The term "simultaneously expressing" refers to the expression of a representative population of a peptide domain library, e.g., at least 50 percent, more preferably 75, 80, 85, 90, 95 or 98 percent of all the different peptide domain sequences of a library.

The term "solid support" refers to a material having a rigid or semi-rigid surface. Such materials will preferably talce the form of small beads, pellets, disks, chips, dishes, multi-well plates, wafers or the like, although other forms may be used. In some embodiments, at least one surface of the substrate will be substantially flat. The term "surface" refers to any generally two-dimensional structure on a solid substrate and may have steps, ridges, kinks, terraces, and the like without ceasing to be a surface.

In an exemplary embodiment of the present invention, the display package is a phage particle which comprises a peptide domain fusion coat protein that includes the amino acid sequence of a test peptide domain. Thus, a library of replicable phage vectors, especially phagemids (as defined herein), encoding a library of peptide domain fusion coat proteins is generated and used to transform suitable host cells. Phage particles formed from the chimeric protein can be separated by affinity selection based on the ability of the peptide domain associated with a particular phage particle to specifically bind a target. In a preferred embodiment, each individual phage particle of the library includes a copy of the corresponding phagemid encoding the peptide domain fusion coat protein displayed on the surface of that package. Exemplary phage for generating the present variegated peptide domain libraries include M13, fl, fd, Ifl, Ike, Xf, Pfl, Pf3, λ, T4, T7, P2, P4, φX- 174, MS2 and 2.

The term "vector" refers to a DNA molecule, capable of replication in a host cell, into which a gene can be inserted to construct a recombinant DNA molecule.

III. Exemplary Embodiments

A. Display Mode

In its "display mode", a library of test peptide domains is expressed by a population of display packages to form a peptide domain display library. With respect to the display package on which the variegated peptide domain library is manifest, it will be appreciated from the discussion provided herein that the display package will preferably be able to be (i) genetically altered to encode heterologous peptide domain, (ii) maintained and amplified in culture, (iii) manipulated to display the peptide domain-containing gene product in a manner permitting the peptide domain to interact with a target during an affinity separation step, and (iv) affinity separated while retaining the nucleotide sequence encoding the test peptide domain (herein "peptide gene") such that the sequence of the peptide domain gene can be obtained. In preferred embodiments, the display remains viable after affinity separation. Ideally, the display package comprises a system that allows the sampling of very large variegated peptide domain display libraries, rapid sorting after each affinity separation round, and easy isolation of the peptide domain gene from purified display packages or further manipulation of that sequence in the secretion mode. The most attractive candidates for this type of screening, are prokaryotic organisms and viruses, as they can be amplified quickly, they are relatively easy to manipulate, and large number of clones can be created. Preferred display packages include, for example, vegetative bacterial cells, bacterial spores, and most preferably, bacterial viruses (especially DNA viruses). However, the present invention also contemplates the use of eukaryotic cells, including yeast and their spores, as potential display packages.

In addition to commercially available kits for generating phage display libraries (e.g. the Pharmacia Recombinant Phage Antibody System, catalog no. 27- 9400-01; and the Stratagene SurβAP™ phage display kit, catalog no. 240612), examples of methods and reagents particularly amenable for use in generating the variegated peptide domain display library of the present invention can be found in, for example, the Ladner et al. U.S. Patent No. 5,223,409; the Kang et al. International Publication No. WO 92/18619; the Dower et al. International Publication No. WO 91/17271; the Winter et al. International Publication WO 92/20791; the Marldand et al. International Publication No. WO 92/15679; the Breitling et al. International Publication WO 93/01288; the McCafferty et al. International Publication No. WO 92/01047; the Garrard et al. International Publication No. WO 92/09690; the Ladner et al. International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) RN4S 88:7978-7982. These systems can, with modifications described herein, be adapted for use in the subject method.

When the display is based on a bacterial cell, or a phage which is assembled periplasmically, the display means of the package will comprise at least two components. The first component is a secretion signal which directs the recombinant peptide domain to be localized on the extracellular side of the cell membrane (of the host cell when the display package is a phage). This secretion signal can be selected so as to be cleaved off by a signal peptidase to yield a processed, "mature" peptide domain. The second component is a display anchor protein which directs the display package to associate the test peptide domain with its outer surface. As described below, this anchor protein can be derived from a surface or coat protein native to the genetic package.

When the display package is a bacterial spore, or a phage whose protein coating is assembled intracellularly, a secretion signal directing the peptide domain to the inner membrane of the host cell is unnecessary. In these cases, the means for arraying the variegated peptide domain library comprises a derivative of a spore or phage coat protein amenable for use as a fusion protein.

In some instances it may be necessary to introduce an unstructured polypeptide linker region between portions of the chimeric protein, e.g., between the test peptide domain and display polypeptide. This linker can facilitate enhanced flexibility of the chimeric protein allowing the test peptide domain to freely interact with a target by reducing steric hindrance between the two fragments, as well as allowing appropriate folding of each portion to occur. The linker can be of natural origin, such as a sequence determined to exist in random coil between two domains of a protein. Alternatively, the linker can be of synthetic origin. For instance, the sequence (Gly₄Ser)₃ (SEQ ID No. 1) can be used as a synthetic unstructured linker. Linkers of this type are described in Huston et al. (1988) PNAS 85:4879; and U.S. Patent Nos. 5,091,513 and 5,258,498. Naturally occurring unstructured linkers of human origin are preferred as they reduce the risk of immunogenicity.

In the instance wherein the display package is a phage, the cloning site for the test peptide domain gene sequences in the phagemid should be placed so that it does not substantially interfere with normal phage function. One such locus is the intergenic region as described by Zinder and Boeke, (1982) Gene 19:1-10.

The number of possible individual members in a peptide domain library can be quite large. To screen as many member as possible depends, in part, on the ability to recover large numbers of transformants. For phage with plasmid-like forms (as filamentous phage), electrotransformation provides an efficiency comparable to that of phage-transfection with in vitro packaging, in addition to a very high capacity for DNA input. This allows large amounts of vector DNA to be used to obtain very large numbers of transformants. The method described by Dower et al. (1988) Nucleic Acids Res., 16:6127 '-6145, for example, may be used to transform fd-tet derived recombinants at the rate of about IO⁷ transformants/μg of ligated vector into E. coli (such as strain MCI 061), and libraries may be constructed in fd-tet Bl of up to about 3 x 10⁸ members or more. Increasing DNA input and making modifications to the cloning protocol within the ability of the skilled artisan may produce increases of greater than about 10-fold in the recovery of transformants, providing libraries of up to 10¹⁰ or more recombinants.

As will be apparent to those skilled in the art, in embodiments wherein high affinity peptide domains are sought, an important criteria for the present selection method can be that it is able to discriminate between peptide domains of different affinity for a particular target, and preferentially enrich for the peptide domains of highest affinity. Applying the well known principles of peptide domain affinity and valence (i.e. avidity), it is understood that manipulating the display package to be rendered effectively monovalent can allow affinity enrichment to be carried out for generally higher binding affinities (i.e. binding constants in the range of 10⁶ to 10¹⁰ M^"1) as compared to the broader range of affinities isolable using a multivalent display package. To generate the monovalent display, the natural (i.e. wild-type) form of the surface or coat protein used to anchor the peptide domain to the display can be added at a high enough level that it almost entirely eliminates inclusion of the peptide domain fusion protein in the display package. Thus, a vast majority of the display packages can be generated to include no more than one copy of the peptide domain fusion protein (see, for example, Garrad et al. (1991) Bio/Technology 9:1373-1377). In a preferred embodiment of a monovalent display library, the library of display packages will comprise no more than 5 to 10 % polyvalent displays, and more preferably no more than 2 % of the display will be polyvalent , and most preferably, no more than 1 % polyvalent display packages in the population. The source of the wild-type anchor protein can be, for example, provided by a copy of the wild-type gene present on the same construct as the peptide domain fusion protein, or provided by a separate construct altogether. However, it will be equally clear that by similar manipulation, polyvalent displays can be generated to isolate a broader range of binding affinities. Such peptide domains can be useful, for example, in purification protocols where avidity can be desirable.

i) Phages As Display Packages

Bacteriophage are attractive prokaryotic-related organisms for use in the subject method. Bacteriophage are excellent candidates for providing a display system of the variegated peptide domain library as there is little or no enzymatic activity associated with intact mature phage, and because their genes are inactive outside a bacterial host, rendering the mature phage particles metabolically inert. In general, the phage surface is a relatively simple structure. Phage can be grown easily in large numbers, they are amenable to the practical handling involved in many potential mass screening programs, and they carry genetic information for their own synthesis within a small, simple package. As the peptide domain gene is inserted into the phage genome, choosing the appropriate phage to be employed in the subject method will generally depend most on whether (i) the genome of the phage allows introduction of the peptide domain gene either by tolerating additional genetic material or by having replaceable genetic material; (ii) the virion is capable of packaging the genome after accepting the insertion or substitution of genetic material; and (iii) the display of the peptide domain on the phage surface does not disrupt virion structure sufficiently to interfere with phage propagation.

One concern presented with the use of phage is that the morphogenetic pathway of the phage determines the enviromnent in which the peptide domain will have opportunity to fold. Periplasmically assembled phage are preferred as the displayed peptide domains may contain essential disulfides, and such peptide domains may not fold correctly within a cell. However, in certain embodiments in which the display package forms intracellularly (e.g., where the Ml 3 phage are used), it has been demonstrated in other instances that disulfide-containing peptide domains can assume proper folding after the phage is released from the cell. Another concern related to the use of phage, but also pertinent to the use of bacterial cells and spores as well, is that multiple infections could generate hybrid displays that carry the gene for one particular test peptide domain yet have two or more different test peptide domains on their surfaces. Therefore, it can be preferable, though optional, to minimize this possibility by infecting cells with phage under conditions resulting in a low multiple-infection.

For a given bacteriophage, the preferred display means is a protein that is present on the phage surface (e.g. a coat protein). Filamentous phage can be described by a helical lattice; isometric phage, by an icosahedral lattice. Each monomer of each major coat protein sits on a lattice point and makes defined interactions with each of its neighbors. Proteins that fit into the lattice by making some, but not all, of the normal lattice contacts are likely to destabilize the virion by aborting formation of the virion as well as by leaving gaps in the virion so that the nucleic acid is not protected. Thus in bacteriophage, unlike the cases of bacteria and spores, it is generally important to retain in the peptide domain fusion proteins those residues of the coat protein that interact with other proteins in the virion. For example, when using the Ml 3 cpVIII protein, the entire mature protein will generally be retained with the peptide domain fragment being added to the N- terminus of cp VIII, while on the other hand it can suffice to retain only the last 100 carboxy terminal residues (or even fewer) of the Ml 3 cpIII coat protein in the peptide domain fusion protein.

Under the appropriate induction, the test peptide domain library is expressed and exported, as part of the fusion protein, to the bacterial cytoplasm, such as when the Ml 3 phage is employed. The induction of the fusion protein(s) may be delayed until some replication of the phage genome, synthesis of some of the phage structural-proteins, and assembly of some phage particles has occurred. The assembled protein chains then interact with the phage particles via the binding of the anchor protein on the outer surface of the phage particle. The cells are lysed and the phage bearing the library-encoded test peptide domain (that corresponds to the specific library sequences carried in the DNA of that phage) are released and isolated from the bacterial debris. To enrich for and isolate phage which encodes a selected test peptide domain, and thus to ultimately isolate the nucleic acid sequences (the peptide domain gene) themselves, phage harvested from the bacterial debris are affinity purified. As described below, when a test peptide domain which specifically binds a particular target is desired, the target can be used to retrieve phage displaying the desired test peptide domain. The phage so obtained may then be amplified by infecting into host cells. Additional rounds of affinity enrichment followed by amplification may be employed until the desired level of enrichment is reached.

The enriched peptide domain-phage can also be screened with additional detection-techniques such as expression plaque (or colony) lift (see, e.g., Young and Davis, Science (1983) 222:778-782) whereby a labeled target is used as a probe.

a) Filamentous Phage

Filamentous bacteriophages, which include Ml 3, fl, fd, Ifl, Ike, Xf, Pfl, and Pf3, are a group of related viruses that infect bacteria. They are termed filamentous because they are long, thin particles comprised of an elongated capsule that envelopes the deoxyribonucleic acid (DNA) that forms the bacteriophage genome. The F pili filamentous bacteriophage (Ff phage) infect only gram-negative bacteria by specifically adsorbing to the tip of F pili, and include fd, fl and Ml 3.

Compared to other bacteriophage, filamentous phage in general are attractive and Ml 3 in particular is especially attractive because: (i) the 3-D structure of the virion is known; (ii) the processing of the coat protein is well understood; (iii) the genome is expandable; (iv) the genome is small; (v) the sequence of the genome is known; (vi) the virion is physically resistant to shear, heat, cold, urea, guanidinium chloride, low pH, and high salt; (vii) the phage is a sequencing vector so that sequencing is especially easy; (viii) antibiotic-resistance genes have been cloned into the genome with predictable results (Hines et al. (1980) Gene 11:207-218); (ix) it is easily cultured and stored, with no unusual or expensive media requirements for the infected cells, (x) it has a high burst size, each infected cell yielding 100 to 1000

Ml 3 progeny after infection; and (xi) it is easily harvested and concentrated (Salivar et al. (1964) Virology 24: 359-371). The entire life cycle of the filamentous phage Ml 3, a common cloning and sequencing vector, is well understood. The genetic structure of Ml 3 is well known, including the complete sequence (Schaller et al. in The Single-Stranded DNA Phages eds. Denliardt et al. (NY: CSHL Press, 1978)), the identity and function of the ten genes, and the order of transcription and location of the promoters, as well as the physical structure of the virion (Smith et al. (1985) Science 228:1315-1317; Raschad et al. (1986) Microbiol Dev 50:401-427; Kuhn et al. (1987) Science 238:1413-1415; Zimmerman et al. (1982) J Biol Chem 257:6529- 6536; and Banner et al. (1981) Nature 289:814-816). Because the genome is small (6423 bp), cassette mutagenesis is practical on RF Ml 3 (Current Protocols in Molecular Biology, eds. Ausubel et al. (NY: John Wiley & Sons, 1991)), as is single-stranded oligonucleotide directed mutagenesis (Fritz et al. in DNA Cloning, ed by Glover (Oxford, UK: IRC Press, 1985)). Ml 3 is a plasmid and transformation system in itself, and an ideal sequencing vector. Ml 3 can be grown on Rec- strains of E. coli. The Ml 3 genome is expandable (Messing et al. in The Single-Stranded DNA Phages, eds Denhardt et al. (NY: CSHL Press, 1978) pages 449-453; and Fritz et al., supra) and Ml 3 does not lyse cells. Extra genes can be inserted into Ml 3 and will be maintained in the viral genome in a stable manner.

The mature capsule or Ff phage is comprised of a coat of five phage-encoded gene products: cpNIII, the major coat protein product of gene NIII that forms the bulk of the capsule; and four minor coat proteins, cpIII and cpIV at one end of the capsule and cpVII and cpIX at the other end of the capsule. The length of the capsule is formed by 2500 to 3000 copies of cpVIII in an ordered helix array that forms the characteristic filament structure. The gene Ill-encoded protein (cpIII) is typically present in 4 to 6 copies at one end of the capsule and serves as the receptor for binding of the phage to its bacterial host in the initial phase of infection. For detailed reviews of Ff phage structure, see Rasched et al., Microbiol. Rev., 50:401-427 (1986); and Model et al., in The Bacteriophages, Volume 2, R. Calendar, Ed., Plenum Press, pp. 375-456 (1988).

The phage particle assembly involves extrusion of the viral genome through the host cell's membrane. Prior to extrusion, the major coat protein cpVIII and the minor coat protein cpIII are synthesized and transported to the host cell's membrane. Both cpVIII and cpIII are anchored in the host cell membrane prior to their incorporation into the mature particle. In addition, the viral genome is produced and coated with cpV protein. During the extrusion process, cpV-coated genomic DNA is stripped of the cpV coat and simultaneously recoated with the mature coat proteins. Both cpIII and cpVIII proteins include two domains that provide signals for assembly of the mature phage particle. The first domain is a secretion signal that directs the newly synthesized protein to the host cell membrane. The secretion signal is located at the amino terminus of the polypeptide and targets the polypeptide at least to the cell membrane. The second domain is a membrane anchor domain that provides signals for association with the host cell membrane and for association with the phage particle during assembly. This second signal for both cpVIII and cpIII comprises at least a hydrophobic region for spanning the membrane.

The 50 amino acid mature gene VIII coat protein (cpVIII) is synthesized as a 73 amino acid precoat (Ito et al. (1979) PNAS 76:1199-1203). cpVIII has been extensively studied as a model membrane protein because it can integrate into lipid bilayers such as the cell membrane in an asymmetric orientation with the acidic amino terminus toward the outside and the basic carboxy terminus toward the inside of the membrane. The first 23 amino acids constitute a typical signal-sequence which causes the nascent polypeptide to be inserted into the inner cell membrane. An E. coli signal peptidase (SP-I) recognizes amino acids 18, 21, and 23, and, to a lesser extent, residue 22, and cuts between residues 23 and 24 of the precoat (Kuhn et al. (1985) J Biol. Chem. 260:15914-15918; and Kuhn et al. (1985) J Biol. Chem. 260:15907-15913). After removal of the signal sequence, the amino terminus of the mature coat is located on the periplasmic side of the imier membrane; the carboxy terminus is on the cytoplasmic side. About 3000 copies of the mature coat protein associate side-by-side in the inner membrane.

The sequence of gene VIII is known, and the amino acid sequence can be encoded on a synthetic gene. Mature gene VIII protein makes up the sheath around the circular ssDNA. The gene VIII protein can be a suitable anchor protein because its location and orientation in the virion are Icnown (Banner et al. (1981) Nature

289:814-816). Preferably, the peptide domain is attached to the amino terminus of the mature Ml 3 coat protein to generate the phage display library. As set out above, manipulation of the concentration of both the wild-type cpVIII and Ab/cpVIII fusion in an infected cell can be utilized to decrease the avidity of the display and thereby enhance the detection of high affinity peptide domains directed to the target(s). Another vehicle for displaying the peptide domain is by expressing it as a domain of a chimeric gene containing part or all of gene III, e.g., encoding cpIII. When monovalent displays are required, expressing the peptide domain as a fusion protein with cpIII can be a preferred embodiment, as manipulation of the ratio of wild-type cpIII to chimeric cpIII during formation of the phage particles can be readily controlled. This gene encodes one of the minor coat proteins of Ml 3. Genes VI, VII, and IX also encode minor coat proteins. Each of these minor proteins is present in about 5 copies per virion and is related to morphogenesis or infection. In contrast, the major coat protein is present in more than 2500 copies per virion. The gene VI, VII, and IX proteins are present at the ends of the virion; these three proteins are not posttranslationally processed (Rasched et al. (1986) Ann Rev. Microbiol. 41 :507-541). In particular, the single-stranded circular phage DNA associates with about five copies of the gene III protein and is then extruded through the patch of membrane-associated coat protein in such a way that the DNA is encased in a helical sheath of protein (Webster et al. in The Single-Stranded DNA Phages, eds Dressier et al. (NY:CSHL Press, 1978).

Manipulation of the sequence of cpIII has demonstrated that the C-terminal 23 amino acid residue stretch of hydrophobic amino acids normally responsible for a membrane anchor function can be altered in a variety of ways and retain the capacity to associate with membranes. Ff phage-based expression vectors were first described in which the cpIII amino acid residue sequence was modified by insertion of polypeptide "targets" (Parmely et al., Gene (1988) 73:305-318; and Cwirla et al., PNAS (1990) 87:6378-6382) or an amino acid residue sequence defining a single chain peptide domain (McCafferty et al, Science (1990) 348:552-554). It has been demonstrated that insertions into gene III can result in the production of novel protein domains on the virion outer surface. (Smith (1985) Science 228:1315-1317; and de la Cruz et al. (1988) J. Biol. Chem. 263:4318-4322). The peptide domain gene may be fused to gene III at the site used by Smith and by de la Cruz et al., at a codon corresponding to another domain boundary or to a surface loop of the protein, or to the amino terminus of the mature protein.

Generally, the successful cloning strategy utilizing a phage coat protein, such as cpIII of filamentous phage fd, will provide expression of a peptide domain chain fused to the N-terminus of a coat protein (e.g., cpIII) and transport to the inner membrane of the host where the hydrophobic domain in the C-terminal region of the coat protein anchors the fusion protein in the membrane, with the N-terminus containing the peptide domain chain protruding into the periplasmic space. Similar constructions could be made with other filamentous phage. Pf3 is a well Icnown filamentous phage that infects Pseudomonos aerugenosa cells that harbor an IncP-I plasmid. The entire genome has been sequenced ((Luiten et al. (1985) J. Virol. 56:268-276) and the genetic signals involved in replication and assembly are Icnown (Luiten et al. (1987) DNA 6:129-137). The major coat protein of PF3 is unusual in having no signal peptide to direct its secretion. The sequence has charged residues ASP-7, ARG-37, LYS-40, and PHE44 which is consistent with the amino terminus being exposed. Thus, to cause a peptide domain to appear on the surface of Pf3, a tripartite gene can be constructed which comprises a signal sequence Icnown to cause secretion in P. aerugenosa, fused in-frame to a gene fragment encoding the peptide sequence, which is fused in-frame to DNA encoding the mature Pβ coat protein. Optionally, DNA encoding a flexible linker of one to 10 amino acids is introduced between the peptide gene fragment and the Pf3 coat-protein gene. This tripartite gene^" is introduced into Pf3 so that it does not interfere with expression of any Pf3 genes. Once the signal sequence is cleaved off, the peptide is in the periplasm and the mature coat protein acts as an anchor and phage-assembly signal.

b) Bacteriophage φXl 74

The bacteriophage 0X174 is a very small icosahedral virus which has been thoroughly studied by genetics, biochemistry, and electron microscopy (see The Single Stranded DNA Phages (eds. Denhardt et al. (NY:CSHL Press, 1978)). Three gene products of 0X174 are present on the outside of the mature virion: F (capsid), G (major spike protein, 60 copies per virion), and H (minor spike protein, 12 copies per virion). The G protein comprises 175 amino acids, while H comprises 328 amino acids. The F protein interacts with the single-stranded DNA of the virus. The proteins F, G, and H are translated from a single mRNA in the viral infected cells. As the virus is so tightly constrained because several of its genes overlap, 0X174 is not typically used as a cloning vector due to the fact that it can accept very little additional DNA. However, mutations in the viral G gene (encoding the G protein) can be rescued by a copy of the wild-type G gene carried on a plasmid that is expressed in the same host cell (Chambers et al. (1982) Nuc Acid Res 10:6465- 6473). In one embodiment, one or more stop codons are introduced into the G gene so that no G protein is produced from the viral genome. The variegated peptide gene library can then be fused with the nucleic acid sequence of the H gene. An amount of the viral G gene equal to the size of peptide gene fragment is eliminated from the 0X174 genome, such that the size of the genome is ultimately unchanged. Thus, in host cells also transformed with a second plasmid expressing the wild-type G protein, the production of viral particles from the mutant virus is rescued by the exogenous G protein source. Where it is desirable that only one test peptide be displayed per ΦX174 particle, the second plasmid can further include one or more copies of the wild-type H protein gene so that a mix of H and test peptide/H proteins will be predominated by the wild-type H upon incorporation into phage particles.

c) Large DNA Phage

Phage such as λ or T4 have much larger genomes than do Ml 3 or φX174, and have more complicated 3-D capsid structures than M13 or φPX174, with more coat proteins to choose from. In embodiments of the invention whereby the test peptide library is processed and assembled into a functional form and associates with the bacteriophage particles within the cytoplasm of the host cell, bacteriophage λ and derivatives thereof are examples of suitable vectors. The intracellular morphogenesis of phage λ can potentially prevent protein domains that ordinarily contain disulfide bonds from folding correctly. However, variegated libraries expressing a population of functional peptides, which include such bonds, have been generated in λ phage. (Huse et al. (1989) Science 246:1275-1281; Mullinax et al. (1990) PNAS 87:8095-8099; and Pearson et al. (1991) RN4S 88:2432-2436). Such strategies talce advantage of the rapid construction and efficient transformation abilities of λ phage.

When used for expression of peptide sequences (ixogenous nucleotide sequences), may be readily inserted into a λ vector. For instance, variegated peptide libraries can be constructed by modification of λ ZAP II through use of the multiple cloning site of a λ ZAP II vector (Huse et al. supra).

ii) Bacterial Cells as Display Packages

Recombinant peptides are able to cross bacterial membranes after the addition of appropriate secretion signal sequences to the N-terminus of the protein (Better et al (1988) Science 240:1041-1043; and Skerra et al. (1988) Science 240:1038-1041). In addition, recombinant peptides have been fused to outer membrane proteins for surface presentation. For example, one strategy for displaying peptides on bacterial cells comprises generating a fusion protein by inserting the peptide into cell surface exposed portions of an integral outer membrane protein (Fuchs et al. (1991) Bio/Technology 9:1370-1372). In selecting a bacterial cell to serve as the display package, any well-characterized bacterial strain will typically be suitable, provided the bacteria may be grown in culture, engineered to display the test peptide library on its surface, and is compatible with the particular affinity selection process practiced in the subject method. Among bacterial cells, the preferred display systems include Salmonella typhirnurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Moraxella bovis, and especially Escherichia coli. Many bacterial cell surface proteins useful in the present invention have been characterized, and works on the localization of these proteins and the methods of determining their structure include Benz et al. (1988) Ann Rev Microbiol 42: 359-393; Balduyck et al. (1985) Biol Chem Hoppe-Seyler 366:9-14; Ehrmann et al (1990) RN4S 87:7574-7578; Heijne et al. (1990) Protein Engineering 4:109-112; Ladner et al. U.S. Patent No. 5,223,409; Ladner et al. WO88/06630; Fuchs et al. (1991) Bio/technology 9:1370-1372; and Goward et al. (1992) TIBS 18:136-140.

To further illustrate, the LamB protein of E coli is a well understood surface protein that can be used to generate a variegated library of test peptides on the surface of a bacterial cell (see, for example, Ronco et al. (1990) Biochemie 72:183- 189; van der Weit et al. (1990) Vaccine 8:269-277; Charabit et al. (1988) Gene 70:181-189; and Ladner U.S. Patent No. 5,222,409). LamB of E. coli is a porin for maltose and maltodextrin transport, and serves as the receptor for adsorption of bacteriophages λ and K10. LamB is transported to the outer membrane if a functional N-terminal signal sequence is present (Benson et al. (1984) PNAS 81 :3830-3834). As with other cell surface proteins, LamB is synthesized with a typical signal-sequence which is subsequently removed. Thus, the variegated peptide gene library can be cloned into the LamB gene such that the resulting library of fusion proteins comprise a portion of LamB sufficient to anchor the protein to the cell membrane with the test peptide fragment oriented on the extracellular side of the membrane. Secretion of the extracellular portion of the fusion protein can be facilitated by inclusion of the LamB signal sequence, or other suitable signal sequence, as the N-terminus of the protein. The E. coli LamB has also been expressed in functional form in S.

^■ typhimurium (Harldci et al. (1987) Mol Gen Genet 209:607-611), V. cholerae

(Harlcki et al. (1986) Microb Pathol 1:283-288), and K. pneumonia (Wehmeier et al.

(1989) Mol Gen Genet 215:529-536), so that one could display a population of test peptides in any of these species as a fusion to E. coli LamB. Moreover, K. pneumonia expresses a maltoporin similar to LamB which could also be used. In E. aeruginosa, the Dl protein (a homologue of LamB) can be used (Trias et al. (1988) Biochem Biophys Acta 938:493-496). Similarly, other bacterial surface proteins, such as PAL, OmpA, OmpC, OmpF, PhoΕ, pilin, BtuB, FepA, FhuA, IutA, FecA and FhuΕ, may be used in place of LamB as a portion of the display means in a bacterial cell. In another exemplary embodiment, the fusion protein can be derived using the FliTrx™ Random Peptide Display Library (Invitrogen). That library is a diverse population of random dodecapeptides inserted within the thioredoxin active-site loop inside the dispensable region of the bacterial flagellin gene (fliC). The resultant recombinant fusion protein (FLITRX) is exported and assembled into partially functional flagella on the bacterial cell surface, displaying the random peptide library.

Peptides are fused in the middle of thioredoxin, therefore, both their N- and C-termini are anchored by thioredoxin' s tertiary structure. This results in the display of a constrained peptide. By contrast, phage display proteins are fused to the N- terminus of phage coat proteins in an unconstrained manner. The unconstrained molecules possess many degrees of conformational freedom which may result in the lack of proper interaction with the target molecule. Without proper interaction, many potential protein-protein interactions may be missed. Moreover, phage display is limited by the low expression levels of bacteriophage coat proteins. FliTrx™ and similar methods can overcome this limitation by using a strong promoter to drive expression of the test peptide fusions that are displayed as multiple copies.

According to the present invention, it is contemplated that the FliTrx vector can be modified to provide, similar to the illustrated vectors of the attached figures, a vector which is differentially spliced in mammalian cells to yield a secreted, soluble test peptide.

iii) Bacterial Spores as Display Packages

Bacterial spores also have desirable properties as display package candidates in the subject method. For example, spores are much more resistant than vegetative bacterial cells or phage to chemical and physical agents, and hence permit the use of a great variety of affinity selection conditions. Also, Bacillus spores neither actively metabolize nor alter the proteins on their surface. However, spores have the disadvantage that the molecular mechanisms that trigger sporulation are less well worked out than is the formation of Ml 3 or the export of protein to the outer membrane of E. coli, though such a limitation is not a serious detractant from their use in the present invention

Bacteria of the genus Bacillus form endospores that are extremely resistant to damage by heat, radiation, desiccation, and toxic chemicals (reviewed by Losiclc et al. (1986) Ann Rev Genet 20:625-669). This phenomenon is attributed to extensive intermolecular cross-linking of the coat proteins. In certain embodiments of the subject method, such as those which include relatively harsh affinity separation steps, Bacillus spores can be the preferred display package. Endospores from the genus Bacillus are more stable than are, for example, exospores from Streptomyces. Moreover, Bacillus subtilis forms spores in 4 to 6 hours, whereas Streptomyces species may require days or weeks to sporulate. In addition, genetic knowledge and manipulation is much more developed for R. subtilis than for other spore-forming bacteria.

Viable spores that differ only slightly from wild-type are produced in R. subtilis even if any one of four coat proteins is missing (Donovan et al. (1987) J Mol Biol 196:1-10). Moreover, plasmid DNA is commonly included in spores, and plasmid encoded proteins have been observed on the surface of Bacillus spores (Debro et al. (1986) J Bacteriol 165:258-268). Thus, it can be possible during sporulation to express a gene encoding a chimeric coat protein comprising a peptide of the variegated gene library, without interfering materially with spore formation.

To illustrate, several polypeptide components of R. subtilis spore coat (Donovan et al. (1987) J Mol Biol 196:1-10) have been characterized. The sequences of two complete coat proteins and amino-terminal fragments of two others have been determined. Fusion of the test peptide sequence to cotC or cotD fragments is likely to cause the peptide to appear on the spore surface. The genes of each of these spore coat proteins are preferred as neither cotC or cotD are post-translationally modified (see Ladner et al. U.S. Patent No. 5,223,409).

iv) Selecting Peptide Doaminsfrom the Display Mode

Upon expression, the variegated peptide domain display is subjected to affinity enrichment in order to select for test peptide domains which bind preselected targets. The term "affinity separation" or "affinity enrichment" includes, but is not limited to: (1) affinity chromatography utilizing immobilized targets, (2) immunoprecipitation using soluble targets, (3) fluorescence activated cell sorting, (4) agglutination, and (5) plaque lifts. In each embodiment, the library of display packages are ultimately separated based on the ability of the associated test peptide domain to bind the target of interest. See, for example, the Ladner et al. U.S. Patent No. 5,223,409; the Kang et al. International Publication No. WO 92/18619; the Dower et al. International Publication No. WO 91/17271; the Winter et al. International Publication WO 92/20791; the Markland et al. International Publication No. WO 92/15679; the Breitling et al. International Publication WO 93/01288; the McCafferty et al. International Publication No. WO 92/01047; the Garrard et al. International Publication No. WO 92/09690; and the Ladner et al. International Publication No. WO 90/02809. In most preferred embodiments, the domain display library will be pre-enriched for peptide domains specific for the target by first contacting the domain display library with any negative controls or other targets for which differential binding by the test peptide domain is desired. Subsequently, the non-binding fraction from that pre-treatment step is contacted with the target and peptide domains from the display which are able to specifically bind the target are isolated. With respect to affinity chromatography, it will be generally understood by those skilled in the art that a great number of chromatography techniques can be adapted for use in the present invention, ranging from column chiOmatography to batch elution, and including ELISA and biopanning techniques. Typically, where the target is a component of a cell, rather than a whole cell, the target is immobilized on an insoluble carrier, such as sepharose or polyacrylamide beads, or, alternatively, the wells of a microtitre plate. As described below, in instances where no purified source of the target is readily available, such as the case with many cell surface receptors, the cells on which the target is displayed may serve as the insoluble matrix carrier. The population of display packages is applied to the affinity matrix under conditions compatible with the binding of the test peptide domain to a target. The population is then fractionated by washing with a solute that does not greatly effect specific binding of peptide domains to the target, but which substantially disrupts any non-specific binding of the display package to the target or matrix. A certain degree of control can be exerted over the binding characteristics of the peptide domains recovered from the display library by adjusting the conditions of the binding incubation and subsequent washing. The temperature, pH, ionic strength, divalent cation concentration, and the volume and duration of the washing can select for peptide domains within a particular range of affinity and specificity. Selection based on slow dissociation rate, which is usually predictive of high affinity, is a very practical route. This may be done either by continued incubation in the presence of a saturating amount of free hapten (if available), or by increasing the volume, number, and length of the washes. In each case, the rebinding of dissociated peptide domain- display package is prevented, and with increasing time, peptide domain-display packages of higher and higher affinity are recovered. Moreover, additional modifications of the binding and washing procedures may be applied to find peptide domains with special characteristics. The affinities of some peptide domains are dependent on ionic strength or cation concentration. This is a useful characteristic for peptide domains to be used in affinity purification of various proteins when gentle conditions for removing the protein from the peptide domain are required. Specific examples are peptides which depend on Ca for binding activity and which lose or gain binding affinity in the presence of EGTA or other metal chelating agent. Such peptides may be identified in the recombinant peptide library by a double screening technique isolating first those that bind the target in the presence of Ca^"1"1", and by subsequently identifying those in this group that fail to bind in the presence of EGTA.

After "washing" to remove non-specifically bound display packages, when desired, specifically bound display packages can be eluted by either specific desorption (using excess target) or non-specific desorption (using pH, polarity reducing agents, or chaotropic agents). In preferred embodiments, the elution protocol does not kill the organism used as the display package such that the enriched population of display packages can be further amplified by reproduction. The list of potential eluants includes salts (such as those in which one of the counter ions is Na⁺, NH₄ ⁺, Rb⁺, SO₄ ^2", H₂PO₄ ^", citrate, K⁺, Li⁺, Cs⁺, HSO₄ ^", CO₃ ^2", Ca²⁺, Sr²⁺, CI^", PO₄ ^2", HCO₃ ^", Mg ⁺, Ba ⁺, Br^", HPO₄ ^2", or acetate), acid, heat, and, when available, soluble forms of the target (or analogs thereof). Because bacteria continue to metabolize during the affinity separation step and are generally more susceptible to damage by harsh conditions, the choice of buffer components (especially eluates) can be more restricted when the display package is a bacteria rather than for phage or spores. Neutral solutes, such as ethanol, acetone, ether, or urea, are examples of other agents useful for eluting the bound display packages. In preferred embodiments, affinity enriched display packages are iteratively amplified and subjected to further rounds of affinity separation until enrichment of the desired binding activity is detected. In certain embodiments, the specifically bound display packages, especially bacterial cells, need not be eluted per se, but rather, the matrix bound display packages can be used directly to inoculate a suitable growth media for amplification.

Where the display package is a phage particle, the fusion protein generated with the coat protein can interfere substantially with the subsequent amplification of eluted phage particles, particularly in embodiments wherein the cpIII protein is used as the display anchor. Even though present in only one of the 5-6 tail fibers, some peptide constructs because of their size and/or sequence, may cause severe defects in the infectivity of their carrier phage. This causes a loss of phage from the population during reinfection and amplification following each cycle of panning. In one embodiment, the peptide can be derived on the surface of the display package so as to be susceptible to proteolytic cleavage which severs the covalent linkage of at least the target binding sites of the displayed peptide from the remaining package. For instance, where the cpIII coat protein of Ml 3 is employed, such a strategy can be used to obtain infectious phage by treatment with an enzyme which cleaves between the test peptide portion and cpIII portion of a tail fiber fusion protein (e.g. such as the use of an enterolcinase cleavage recognition sequence). To further minimize problems associated with defective infectivity, DNA prepared from the eluted phage can be transformed into host cells by electroporation or well known chemical means. The cells are cultivated for a period of time sufficient for marker expression, and selection is applied as typically done for DNA transformation. The colonies are amplified, and phage harvested for a subsequent round(s) of panning. After isolation of display packages which encode peptides having a desired binding specificity for the target, the test peptides for each of the purified display packages can be tested for biological activity in the secretion mode of the subject method.

B. Secretion Mode

In the "secretion mode," the peptide domain library, which has been enriched in the display mode, is transfected into and expressed by eukaryotic cells. In this mode, the test peptide domains are secreted by the host cells and screened for biological activity.

In preferred embodiments, and illustrated in the drawings, the subject vectors are constructed to include eukaryotic splice sites such that, in the mature mRNA, elements required for the display mode in prokaryotic cells are spliced out - at least those elements which would interfere with the secretion mode. A variety of naturally and non-naturally occurring splice sites are available in the art and can be selected for, e.g., optimization in particular eukaryotic cells selected. In preferred embodiments, the vectors of the subject invention are used to transfect a cell that can be co-cultured with a target cell. A biologically active protein secreted by the cells expressing the combinatorial library will diffuse to neighboring target cells and induce a particular biological response, such as to illustrate, proliferation or differentiation, or activation of a signal transduction pathway which is directly detected by other phenotypic criteria. The pattern of detection of biological activity will resemble a gradient function, and will allow the isolation (generally after several repetitive rounds of selection) of cells producing peptide domains having certain activity in the assay. Likewise, antagonists of a given factor can be selected in similar fashion by the ability of the cell producing a functional antagonist to protect neighboring cells from the effect of exogenous factor added to the culture media.

To further illustrate, target cells are cultured in 24-well microtitre plates. Other cells are transfected with the peptide domain library, recovered after the display mode step, and cultured in cell culture inserts (e.g. Collaborative Biomedical Products, Catalog #40446) that are able to fit into the wells of the microtitre plate. The cell culture inserts are placed in the wells such that recombinant test peptide domains secreted by the cells in the insert can diffuse through the porous bottom of the insert and contact the target cells in the microtitre plate wells. After a period of time sufficient for a secreted test peptide domain to produce a measurable response in the target cells, the inserts are removed and the effect of the peptide domains on the target cells determined. For example, where the target cell is a neural crest cell and the activity desired from the test peptide domains is the induction of neuronal differentiation, then fluorescently-labeled antibodies specific for Islet- 1 or other neuronal markers can be used to score for induction in the target cells as indicative of a functional neurotrophic peptide domain in that well. Cells from the inserts corresponding to wells which score positive for activity can be split and re-cultured on several inserts, the process being repeated until the active peptide domain is identified. When screening for bioactivity of test peptide domains, intracellular second messenger generation can be measured directly. For instance, a variety of intracellular effectors have been identified as being receptor- or ion channel- regulated, including adenylyl cyclase, cyclic GMP, phosphodiesterases, phosphoinositidases, phosphoinositol kinases, and phospholipases, as well as a variety of ions.

In one embodiment, the GTPase enzymatic activity by G proteins can be measured in plasma membrane preparations by determining the breakdown of γ- n

[ P] GTP using techniques that are known in the art (For example, see Signal

Transduction: A Practical Approach. G. Milligan, Ed. Oxford University Press, Oxford England). When receptors that modulate cAMP are tested, it will be possible to use standard techniques for cAMP detection, such as competitive assays which quantitate [³H]-cAMP in the presence of unlabelled cAMP.

Certain receptors and ion channels stimulate the activity of phospholipase C which stimulates the breakdown of phosphatidylinositol 4, 5, - bisphosphate to 1, 4, 5 - IP3 (which mobilizes intracellular Ca⁴ ) and diacylglycerol (DAG) (which activates protein kinase C). Inositol lipids can be extracted and analyzed using standard lipid extraction techniques. DAG can also be measured using thin-layer chromatography. Water soluble derivatives of all three inositol lipids (IP_l3 IP₂, IP₃) can also be quantitated using radiolabelling techniques or HPLC. The other product of PIP₂ breakdown, DAG can also be produced from phosphatidyl choline. The breakdown of this phospholipid in response to receptor- mediated signaling can also be measured using a variety of radiolabelling techniques.

The activation of phospholipase A2 can easily be quantitated using known techniques, including, for example, the generation of arachadonate in the cell.

In various cells, e.g., mammalian cells, specific proteases are induced or activated in each of several arms of divergent signaling pathways. These may be independently monitored by following their unique activities with substrates specific for each protease. In the case of certain receptors and ion channels, it may be desirable to screen for changes in cellular phosphorylation. Such assay formats may be useful when, for example, the assay is designed to detect an agonist or antagonist of a receptor kinase or phosphatase. For example, immunoblotting (Lyons and Nelson (1984) Proc. Natl. Acad. Sci. USA 81:7426-7430) using anti-phosphotyrosine, anti- phosphoserine or anti-phosphothreonine antibodies. In addition, tests for phosphorylation could be also useful when the receptor itself may not be a kinase, but activates protein lcinases or phosphatase that function downstream in the signal transduction pathway.

One such cascade is the MAP kinase pathway that appears to mediate both mitogenic, differentiation and stress responses in different cell types. Stimulation of growth factor receptors results in Ras activation followed by the sequential activation of c-Raf, MEK, and p44 and p42 MAP lcinases (ERK1 and ERK2). Activated MAP kinase then phosphorylates many key regulatory proteins, including p90RSK and Elk-1 that are phosphorylated when MAP kinase translocates to the nucleus. Homologous pathways exist in mammalian and yeast cells. For instance, an essential part of the S. cerevisiae pheromone signaling pathway is comprised of a protein kinase cascade composed of the products of the STE11, STE7, and FUS3/KSS1 genes (the latter pair are distinct and functionally redundant). Accordingly, phosphorylation and/or activation of members of this kinase cascade can be detected and used to quantitate receptor engagement. Phosphotyrosine specific antibodies are available to measure increases in tyrosine phosphorylation and phospho-specific antibodies are commercially available (New England Biolabs, Beverly, MA).

In yet another embodiment, the signal transduction pathway of interest may upregulate expression or otherwise activate an enzyme which is capable of modifying a substrate which can be added to the cell. The signal can be detected by using a detectable substrate, in which case lose of the substrate signal is monitored, or alternatively, by using a substrate which produces a detectable product. In preferred embodiments, the conversion of the substrate to product by the activated enzyme produces a detectable change in optical characteristics of the test cell, e.g., the substrate and/or product is chromogenically or fluorogenically active. In an illustrative embodiment the signal transduction pathway causes a change in the activity of a proteolytic enzyme, altering the rate at which it cleaves a substrate peptide (or simply activates the enzyme towards the substrate). The peptide includes a fluorogenic donor radical, e.g., a fluorescence emitting radical, and an acceptor radical, e.g., an aromatic radical which absorbs the fluorescence energy of the fluorogenic donor radical when the acceptor radical and the fluorogenic donor radical are covalently held in close proximity. See, for example, USSN 5,527,681, 5,506,115, 5,429,766, 5,424,186, and 5,316,691; and Capobianco et al. (1992) Anal Biochem 204:96-102. For example, the substrate peptide has a fluorescence donor group such as 1 -aminobenzoic acid (anthranilic acid or ABZ) or aminomethylcoumarin (AMC) located at one position on the peptide and a fluorescence quencher group, such as lucifer yellow, methyl red or nitrobenzo-2- oxo-l,3-diazole (NBD), at a different position near the distal end of the peptide. A cleavage site for the activated enzyme will be disposed between each of the sites for the donor and acceptor groups. The intramolecular resonance energy transfer from the fluorescence donor molecule to the quencher will quench the fluorescence of the donor molecule when the two are sufficiently proximate in space, e.g., when the peptide is intact. Upon cleavage of the peptide, however, the quencher is separated from the donor group, leaving behind a fluorescent fragment. Thus, activation of the enzyme results in cleavage of the detection peptide, and dequenching of the fluorescent group.

In still other embodiments, the detectable signal can be produced by use of enzymes or chromogenic/fluorescent probes whose activities are dependent on the concentration of a second messenger, e.g., such as calcium, hydrolysis products of inositol phosphate, cAMP, etc. For example , the mobilization of intracellular calcium or the influx of calcium from outside the cell can be measured using standard techniques. The choice of the appropriate calcium indicator, fluorescent, bioluminescent, metallochromic, or Ca^-sensitive microelectrodes depends on the cell type and the magnitude and time constant of the event under study (Borle (1990) Environ Health Perspect 84:45-56). As an exemplary method of Ca⁺⁺ detection, cells could be loaded with the Ca⁺⁺ sensitive fluorescent dye fura-2 or indo-1, using standard methods, and any change in Ca^44" measured using a fluorometer.

As certain embodiments described above suggest, in addition to directly measuring second messenger production, the signal transduction activity of a receptor or ion channel pathway can be measured by detection of a transcription product, e.g., by detecting receptor/channel-mediated transcriptional activation (or repression) of a gene(s). Detection of the transcription product includes detecting the gene transcript, detecting the product directly (e.g., by immunoassay) or detecting an activity of the protein (e.g., such as an enzymatic activity or chromogenic/fluorogenic activity); each of which is generally referred to herein as a means for detecting expression of the indicator gene. The indicator gene may be an unmodified endogenous gene of the host cell, a modified endogenous gene, or a part of a completely heterologous construct, e.g., as part of a reporter gene construct.

In one embodiment, the indicator gene is an unmodified endogenous gene.

For example, the instant method can rely on detecting the transcriptional level of such endogenous genes as the c-fos gene (e.g., in mammalian cells) or the Barl or

Fusl genes (e.g., in yeast cells) in response to such signal transduction pathways as originating from G protein coupled receptors.

In certain instances, it may be desirable to increase the level of transcriptional activation of the endogenous indicator gene by the signal pathway in order to, for example, improve the signal-to-noise of the test system, or to adjust the level of response to a level suitable for a particular detection technique. In one embodiment, the transcriptional activation ability of the signal pathway can be amplified by the overexpression of one or more of the proteins involved in the intracellular signal cascade, particularly enzymes involved in the pathway. For example, increased expression of Jun kinases (JNKs) can potentiate the level of transcriptional activation by a signal in an MEKK/JNKK pathway. Likewise, overexpression of one or more signal transduction proteins in the yeast pheromone pathway can increase the level of Fusl and/or Barl expression. This approach can, of course, also be used to potentiate the level of transcription of a heterologous reporter gene as well.

In other embodiments, the sensitivity of an endogenous indicator gene can be enhanced by manipulating the promoter sequence at the natural locus for the indicator gene. Such manipulation may range from point mutations to the endogenous regulatory elements to gross replacement of all or substantial portions of the regulatory elements. In general, manipulation of the genomic sequence for the indicator gene can be carried out using techniques Icnown in the art, including homologous recombination.

In another exemplary embodiment, the promoter (or other transcriptional regulatory sequences) of the endogenous gene can be "switched out" with a heterologous promoter sequence, e.g., to form a chimeric gene at the indicator gene locus. Again, using such techniques as homologous recombination, the regulatory sequence can be so altered at the genomic locus of the indicator gene.

In still another embodiment, a heterologous reporter gene construct can be used to provide the function of an indicator gene. Reporter gene constructs are prepared by operatively linking a reporter gene with at least one transcriptional regulatory element. If only one transcriptional regulatory element is included it must be a regulatable promoter. At least one the selected transcriptional regulatory elements must be indirectly or directly regulated by the activity of the selected cell- surface receptor whereby activity of the receptor can be monitored via transcription of the reporter genes.

Many reporter genes and transcriptional regulatory elements are Icnown to those of skill in the art and others may be identified or synthesized by methods known to those of skill in the art.

Examples of reporter genes include, but are not limited to CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-

869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al. (1987), Mol. Cell. Biol. 7:725-737); bacterial luciferase (Engebrecht and Silverman (1984), PNAS 1: 4154-4158; Baldwin et al.

(1984), Biochemistry 23: 3663-3667); alkaline phosphatase (Toh et al. (1989) Eur. J. Biochem. 182: 231-238, Hall et al. (1983) J. Mol. Appl. Gen. 2: 101), human placental secreted alkaline phosphatase (Cullen and Malim (1992) Methods in

Enzymol. 216:362-368); β-lactamase or GST.

Transcriptional control elements for use in the reporter gene constructs, or for modifying the genomic locus of an indicator gene include, but are not limited to, promoters, enhancers, and repressor and activator binding sites. Suitable transcriptional regulatory elements may be derived from the transcriptional regulatory regions of genes whose expression is rapidly induced, generally within minutes, of contact between the cell surface protein and the effector protein that modulates the activity of the cell surface protein. Examples of such genes include, but are not limited to, the immediate early genes (see Sheng et al. (1990) Neuron 4: 477-485), such as c-fos. Immediate early genes are genes that are rapidly induced upon binding of a ligand to a cell surface protein. The transcriptional control elements that are preferred for use in the gene constructs include transcriptional control elements from immediate early genes, elements derived from other genes that exhibit some or all of the characteristics of the immediate early genes, or synthetic elements that are constructed such that genes in operative linkage therewith exhibit such characteristics. The characteristics of preferred genes from which the transcriptional control elements are derived include, but are not limited to, low or undetectable expression in quiescent cells, rapid induction at the transcriptional level within minutes of extracellular simulation, induction that is transient and independent of new protein synthesis, subsequent shut-off of transcription requires new protein synthesis, and mRNAs transcribed from these genes have a short half-life. It is not necessary for all of these properties to be present. Other promoters and transcriptional control elements, in addition to those described above, include the vasoactive intestinal peptide (NIP) gene promoter (cAMP responsive; Fink et al. (1988), Proc. Νatl. Acad. Sci. 85:6662-6666); the somatostatin gene promoter (cAMP responsive; Montminy et al. (1986), Proc. Νatl. Acad. Sci. 8.3:6682-6686); the proenkephalin promoter (responsive to cAMP, nicotinic agonists, and phorbol esters; Comb et al. (1986), Nature 323:353-356); the phosphoenolpyruvate carboxy-ldnase gene promoter (cAMP responsive; Short et al. (1986), J. Biol. Chem. 261:9721-9726); the NGFI-A gene promoter (responsive to NGF, cAMP, and serum; Changelian et al. (1989). Proc. Natl. Acad. Sci. 86:377- 381); and others that may be known to or prepared by those of skill in the art. In the case of receptors which modulate cyclic AMP, a transcriptional based readout can be constructed using the cyclic AMP response element binding protein, CREB, which is a transcription factor whose activity is regulated by phosphorylation at a particular serine (SI 33). When this serine residue is phosphorylated, CREB binds to a recognition sequence known as a CRE (cAMP Responsive Element) found to the 5' of promoters known to be responsive to elevated cAMP levels. Upon binding of phosphorylated CREB to a CRE, transcription from this promoter is increased.

Phosphorylation of CREB is seen in response to both increased cAMP levels and increased intracellular Ca levels. Increased cAMP levels result in activation of PKA, which in turn phosphorylates CREB and leads to binding to CRE and transcriptional activation. Increased intracellular calcium levels results in activation of calcium/calmodulin responsive kinase II (CaM kinase II). Phosphorylation of CREB by CaM kinase II is effectively the same as phosphorylation of CREB by PKA, and results in transcriptional activation of CRE containing promoters. Therefore, a transcriptionally-based readout can be constructed in cells containing a reporter gene whose expression is driven by a basal promoter containing one or more CRE. Changes in the intracellular concentration of Ca+÷ (a result of alterations in the activity of the receptor upon engagement with a ligand) will result in changes in the level of expression of the reporter gene if: a) CREB is also co-expressed in the cell, and b) either an endogenous or heterologous CaM kinase phosphorylates CREB in response to increases in calcium or if an exogenously expressed CaM kinase II is present in the same cell. In other words, stimulation of PLC activity may result in phosphorylation of CREB and increased transcription from the CRE-construct, while inhibition of PLC activity may result in decreased transcription from the CRE-responsive construct.

As described in Bonni et al. (1993) Science 262:1575-1579, the observation that CNTF treatment of SK-N-MC cells leads to the enhanced interaction of STAT/p91 and STAT related proteins with specific DNA sequences suggested that these proteins might be key regulators of changes in gene expression that are triggered by CNTF. Consistent with this possibility is the finding that DNA sequence elements similar to the consensus DNA sequence required for STAT/p91 binding are present upstream of a number of genes previously found to be induced by CNTF (e.g., Human c-fos, Mouse c-fos, Mouse tisll, Rat junB, Rat SOD-1, and CNTF). Those authors demonstrated the ability of STAT/p91 binding sites to confer CNTF responsiveness to a non-responsive reporter gene. Accordingly, a reporter construct for use in the present invention for detecting signal transduction through STAT proteins, such as from cytokine receptors, can be generated by using -71 to +109 of the mouse c-fos gene fused to the bacterial chloramphenicol acetyltransferase gene (-71fosCAT) or other detectable marker gene. Induction by a cytokine receptor induces the tyrosine phosphorylation of STAT and STAT-related proteins, with subsequent translocation and binding of these proteins to the STAT-RE. This then leads to activation of transcription of genes containing this DNA element within their promoters.

In preferred embodiments, the reporter gene is a gene whose expression causes a phenotypic change which is screenable or selectable. If the change is selectable, the phenotypic change creates a difference in the growth or survival rate between cells which express the reporter gene and those which do not. If the change is screenable, the phenotype change creates a difference in some detectable characteristic of the cells, by which the cells which express the marker may be distinguished from those which do not. Selection is preferable to screening in that it can provide a means for amplifying from the cell culture those cells which express a test polypeptide which is a receptor effector.

The marker gene is coupled to the receptor signaling pathway so that expression of the marker gene is dependent on activation of the receptor. This coupling may be achieved by operably linldng the marker gene to a receptor-responsive promoter. The term "receptor-responsive promoter" indicates a promoter which is regulated by some product of the target receptor's signal transduction pathway.

Alternatively, the promoter may be one which is repressed by the receptor pathway, thereby preventing expression of a product which is deleterious to the cell. With a receptor repressed promoter, one screens for agonists by linking the promoter to a deleterious gene, and for antagonists, by linking it to a beneficial gene. Repression may be achieved by operably linking a receptor - induced promoter to a gene encoding mRNA which is antisense to at least a portion of the mRNA encoded by the marker gene (whether in the coding or flanking regions), so as to inhibit translation of that mRNA. Repression may also be obtained by linldng a receptor-induced promoter to a gene encoding a DNA binding repressor protein, and incorporating a suitable operator site into the promoter or other suitable region of the marker gene.

The marker gene may also be a screenable gene. The screened characteristic may be a change in cell morphology, metabolism or other screenable features. Suitable markers include β-galactosidase (Xgal, C₁₂FDG, Salmon-gal, Magenta-Gal (latter two from Biosynth Ag)), alkaline phosphatase, horseradish peroxidase, exo-glucanase (product of yeast exbl gene; nonessential, secreted); luciferase; bacterial green fluorescent protein; (human placental) secreted alkaline phosphatase (SEAP); and chloramphenicol transferase (CAT). Some of the above can be engineered so that they are secreted (although not β-galactosidase). A preferred screenable marker gene is beta-galactosidase; yeast cells expressing the enzyme convert the colorless substrate Xgal into a blue pigment. Again, the promoter may be receptor-induced or receptor-inhibited.

In certain assays it may be desirable to use changes in growth in the screening procedure. For example, one of the consequences of activation of the pheromone signal pathway in wild-type yeast is growth arrest. If one is testing for an antagonist of a G protein-coupled receptor, such as a human receptor engineered into a yeast cell, this normal response of growth arrest can be used to select cells in which the pheromone response pathway is inhibited. That is, cells exposed to a test compound will be growth arrested if the compound is an agonist, but will grow normally if the compound is neutral or an antagonist. Thus, the growth arrest response can be used to advantage to discover compounds that function as agonists or antagonists. Moreover, the effect of growth arrest can provide a selective advantage in the presence of an agent which is cytotoxic to mitotic cells. For example, during the growth arrest window, the cytotoxic agent is added to the culture. Cells which proceed through the cell-cycle, e.g., which are not growth arrested, will be killed. At some time after the addition of the cytotoxic agent, it can be washed from the culture, and surviving cells permitted to proceed with proliferation. Cells which were arrested by the test compound will be enriched in the surviving population. However, in certain embodiments the growth arrest consequent to activation of the pheromone response pathway is an undesirable effect since cells that bind agonists stop growing while surrounding cells that fail to bind peptide domains will continue to grow. The cells of interest, then, will be overgrown or their detection obscured by the background cells, confounding identification of agonistic peptide domains. To overcome this problem the present invention teaches engineering the cell such that: 1) growth arrest does not occur as a result of exogenous signal pathway activation (e.g., by inactivating the FAR1 gene); and/or 2) a selective growth advantage is conferred by activating the pathway (e.g., by transforming an auxotrophic mutant with a HIS3 gene under the control of a pheromone-responsive promoter, and applying selective conditions).

It is, of course, desirable that the exogenous receptor be exposed on a continuing basis to the peptide domains. Unfortunately, this is likely to result in desensitization of the pheromone pathway to the stimulus. For example, the mating signal transduction pathway is known to become desensitized by several mechanisms including pheromone degradation and modification of the function of the receptor, G proteins and/or downstream elements of the pheromone signal transduction by the products of the SST2, STE50, AFR1 (Konopka, J.B. (1993) Mol. Cell. Biol. 13:6876-6888) and SGN1, MSG5, and SIG1 genes. Selected mutations in these genes can lead to hypersensitivity to pheromone and an inability to adapt to the presence of pheromone. For example, introduction of mutations that interfere with function into strains expressing heterologous G protein-coupled receptors constitutes a significant improvement on wild type strains and enables the development of extremely sensitive bioassays for compounds that interact with the receptors. Other mutations e.g. STE50, sgvl, barl, ste2, ste3, pilcl, msg5, sigl, and aftl, have the similar effect of increasing the sensitivity of the bioassay. Thus desensitization may be avoided by mutating (which may include deleting) the SST2 gene so that it no longer produces a functional protein, or by mutating one of the other genes listed above. If the endogenous homolog of the receptor is produced by the yeast cell, the assay will not be able to distinguish between peptide domains which interact with the endogenous receptor and those which interact with the exogenous receptor. It is therefore desirable that the endogenous gene be deleted or otherwise rendered nonfunctional.

Suitable host cells for generating the target cells of subject assay include prokaryotes, yeast, or higher eukaryotic cells, including plant and animal cells, especially mammalian cells. Prokaryotes include gram negative or gram positive organisms. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman (1981) Cell 23:175) CN-1 cells (ATCC CCL 70), L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, HEK-293, SWISS 3T3, and BHK cell lines.

If yeast cells are used, the yeast may be of any species which are cultivable and in which an exogenous receptor can be made to engage the appropriate signal transduction machinery of the host cell. Suitable species include Kluyverei lactis, Schizosaccharomyces pombe, and Ustilaqo maydis; Saccharomyces cerevisiae is preferred. Other yeast which can be used in practicing the present invention are Neurospora crassa, Aspergillus niger, Aspergillus nidulans, Pichia pastoris, Candida tropicalis, and Hansenula polymorpha. The term "yeast", as used herein, includes not only yeast in a strictly taxonomic sense, i.e., unicellular organisms, but also yeast-like multicellular fungi or filamentous fungi. The choice of appropriate host cell will also be influenced by the choice of detection signal. For instance, reporter constructs, as described below, can provide a selectable or screenable trait upon transcriptional activation (or inactivation) in response to a signal transduction pathway coupled to the target receptor. The reporter gene may be an unmodified gene already in the host cell pathway. It may be a host cell gene that has been operably linlced to a "receptor-responsive" promoter. Alternatively, it may be a heterologous gene (e.g., a "reporter gene construct") that has been so linlced. Suitable genes and promoters are discussed below. In other embodiments, second messenger generation can be measured directly in the detection step, such as mobilization of intracellular calcium or phospholipid metabolism are quantitated. In yet other embodiments indicator genes can be used to detect receptor-mediated signaling. Accordingly, it will be understood that to achieve selection or screening, the host cell must have an appropriate phenotype. For example, generating a pheromone-responsive chimeric HIS3 gene in a yeast that has a wild-type HIS3 gene would frustrate genetic selection. Thus, to achieve nutritional selection, an auxotrophic strain is wanted.

A variety of complementations for use in the subject assay can be constructed. Indeed, many yeast genetic complementation with mammalian signal transduction proteins have been described in the art. For example, Mosteller et al. (1994) Mol Cell Biol 14:1104-12 demonstrates that human Ras proteins can complement loss of ras mutations in S. cerevisiae. Moreover, Toda et al. (1986) Princess Takamatsu Symp 17: 253-60 have shown that human ras proteins can complement the loss of RASl and RAS2 proteins in yeast, and hence are functionally homologous. Both human and yeast RAS proteins can stimulate the magnesium and guanine nucleotide-dependent adenylate cyclase activity present in yeast membranes. Ballester et al. (1989) Cell 59: 681-6 describe a vector to express the mammalian GAP protein in the yeast S. cerevisiae. When expressed in yeast, GAP inhibits the function of the human ras protein, and complements the loss of IRAl. IRAl is a yeast gene that encodes a protein with homology to GAP and acts upstream of RAS. Mammalian GAP can therefore function in yeast and interact with yeast RAS. Wei et al. (1994) Gene 151 : 279-84 describes that a human Ras-specific guanine nucleotide-exchange factor, Cdc25GEF, can complement the loss of CDC25 function in S. cerevisiae. Martegani et al. (1992) EMBO J 11: 2151-7 describe the cloning by functional complementation of a mouse cDNA encoding a homolog of CDC25, a Saccharomyces cerevisiae RAS activator. Vojtelc et al. (1993) J Cell Sci 105: 777-85 and Matviw et al. (1992) Mol Cell, Biol 12: 5033-40 describe how a mouse CAP protein, e.g., an adenylyl cyclase associated protein associated with ras- mediated signal transduction, can complements defects in S. cerevisiae. Papasawas et al. (1992) Biochem Biophys Res Commun 184:1378-85 also suggest that inactivated yeast adenyl cyclase can be complemented by a mammalian adenyl cyclase gene. Hughes et al. (1993) Nature 364: 349-52 describe the complementation of byrl in fission yeast by mammalian MAP kinase (MEK). Parissenti et al. (1993) Mol Cell Endocrinol 98: 9-16 describes the reconstitution of bovine protein kinase C (PKC) in yeast. The Ca(2+)~ and phospholipid-dependent Ser/Thr kinase PKC plays important roles in the transduction of cellular signals in mammalian cells. Marcus et al. (1995) PNAS 92: 6180-4 suggests the complementation of shlcl null mutations in S. pombe by the either the structurally related S. cerevisiae Ste20 or mammalian p65PAK protein kinases.

"Inactivation", with respect to genes of the host cell, means that production of a functional gene product is prevented or inhibited. Inactivation may be achieved by deletion of the gene, mutation of the promoter so that expression does not occur, or mutation of the coding sequence so that the gene product is inactive. Inactivation may be partial or total.

"Complementation", with respect to genes of the host cell, means that at least partial function of inactivated gene of the host cell is supplied by an exogenous nucleic acid. For instance, yeast cells can be "mammalianized", and even "humanized", by complementation of receptor and signal transduction proteins with mammalian homologs. To illustrate, inactivation of a yeast Byr2/Stel l gene can be complemented by expression of a human MEKK gene.

C. Generations of Peptide Domain Libraries

The variegated peptide domain libraries of the subject method can be generated by any of a number of methods.

One approach to generate a variegated peptide domain library is through random priming of isolated mRNA. Isolation of mRNA is well-known in the art. Commercial kits are available for isolation of total RNA from a variety of starting materials including tissue culture cells, dissected tumor or normal tissues, etc. (for example, see Qiagen RNeasy Total RNA System). Poly(A)⁺ mRNA purification following total RNA isolation is also well-known and commercial kits are available for such purposes (see, for example, Qiagen OligoTex mRNA Purification System). Random priming can be achieved by using random oligonucleotides from 6-10 bases, preferably 6-mers and a number of commercially available reverse transcriptases. The resulting cDNA can be size selected to enrich certain fragment sizes, for example, 50 — 300 amino acid residues. The (optionally) size-selected cDNA can then be ligated into a suitable vector, for example, the ones disclosed in the instant application, to construct a library encoding a peptide domain library. The advantage of the current peptide domain library is that it can mimic proteolytic cleavage products of naturally occurring polypeptide domains or fragments so that those domains or fragments of angiogenetic modulator activities can be effectively screened. An additional advantage of the system is that these naturally occurring polypeptide fragments poses a lesser possibility of eliciting undesirable immunological side effects, which may be a problem if random peptides are used. Alternatively, the peptide domain library mimicking natural sequences may be synthesized partially or in whole by conventional chemical synthesis using an automatic DNA synthesizer, and the synthetic nucleotides can then be ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is well known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) Recombinant DNA, Proc 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam: Elsevier pp273-289; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477. Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al. (1990) Science 249:386-390; Roberts et al. (1992) PNAS 89:2429-2433; Devlin et al. (1990) Science 249: 404-406; Cwirla et al. (1990) PNAS 87: 6378-6382; as well as U.S. Patents Nos. 5,223,409, 5,198,346, and 5,096,815).

As used herein, "variegated" refers to the fact that a population of peptides is characterized by having a peptide sequence which differ from one member of the library to the next. In a preferred embodiment of the present invention, the peptide display collectively produces a peptide domain library including at least 96 to IO⁷ different peptide domains, so that diverse peptide domains may be simultaneously assayed for the ability to interact with the target protein. In a preferred embodiment, the peptide domain library includes at least IO³ different test peptide domains. The mRNA sample used to generate the nucleic acid library may be from any source and can be chosen so as to enrich for a desired type of domain, for example proteins from a particular species, tissue type or cell type. For example, for treating a mammalian patient, mRNA from that particular mammalian (i.e., human) can be isolated from all sorts of adult or fetal tissues or cultured cells. Particularly, human endothelial cells, especially capillary endothelial cells can be used to generate the domain library. This type of library preferably represents all, or substantially all, naturally occurring protein and peptide sequences or fragments thereof, naturally occurring in the animal, tissue or cell from which the mRNA sample was derived. The diversity of this type of library may be further increased by using degenerate or mutational methods for generating the cDNA library from the mRNA sample. Such techniques are well Icnown in the art and are described in more detail above.

In certain preferred embodiments, the test peptide domain is flanked by cysteine residues in order to provide a constrained environment. For example, the test peptide domain may be represented in the general formula Cys-(Xaa)₃- ₃-Cys.

In certain embodiments, at least a portion of the peptide domains chosen for inclusion in the library are of known structure or function. Merely to illustrate, the domain library can be constructed from various domains of serum proteins, e.g., possessing a certain biological activity such as affecting angiogenesis. For example, the kringle family of polypeptides share a common domain named ringle domain, which is found in proteins involved in a variety of biological activities, such as blood coagulation (tlirombin, factor XII), fibrinolysis (tissue plasminogen activator, urokinase), liver regeneration (hepatocyte growth factor), and lipid metabolism (apolipoprotein A). These proteins may have a single kringle domain, as in urokinase, or multiple copies, as in apolipoprotein A, which has 38 repeated kringle domains. Kringle motif-containing proteins often have other separate motifs which perform different functions, such as growth factor and serine protease domains (Campbell I. D. et al, Philos. Trans. R. Soc. Lon. B. Biol. Sci., 332(1263), p. 165- 170, 1991). Kringle domains are thought to aid in binding to fibrin clots or membrane components, as well as to modulate protease activity in proteins having tins activity associated with them (Lijnen, H. R. et al, Ballieres Clin. Hematol., 8(2), p. 277-290, 1995). Other domains from serum proteins which can be included in the library are "finger" domains and an epidermal growth factor domains. It is also contemplated that the peptide domain library of the instant invention may also include combinations and permutations of multiple domains in the same construct. For instance, the library can be constructed so that each test domain construct includes from 1 to 10, though preferably 1 to 5 different domains. The synthesis of such library can employ a number of standard molecular biology techniques. For example, a certain peptide domain size can be pre-determined (i.e., 100 amino acids encoded by 300 bp). Linker sequences can be added to both ends of the size-selected DNA fragments to favor cloning of more than one DNA fragments into the same vector. The vector capacity can also be chosen in such a way that only certain insert size are favored. For example, if a minimum insert size of 1 kb is required, then at least 3-4 DNA fragments encoding 3-4 peptide domains may be present in one vector. Such combination of peptide domains may be advantageous in selecting certain non-naturally occurring peptide domain combinations while maintaining the benefit of having less likelihood of eliciting non-desirable immunological responses.

While random peptides sometimes elicits non-desirable side effects, it is still possible that certain modification of a natural peptide can dramatically improve efficacy without introducing a big undesirable side effect. Therefore, it is also contemplated that limited mutagenesis of certain initially identified peptide domains can be employed to improve the effect of these peptide domains as angiogenesis modulators. These mutations can be provided as a sub-library of synthesized peptides using automatic peptide synthesizer, which is commercially available. Alternatively, such mutations can be provided as a sub-library of synthesized DNA fragments using automatic DNA synthesizer, which is also commercially available. The sub-library may include replacing each of the amino acids of the initially identified peptide domains, or adding a few amino acids at either or both ends of the initially identified peptide domains.

In preferred embodiments, the size of any of the peptide domains described above are in the range of about 3-1000 amino acids in length, more preferably about 50-500, even more preferably about 75-400 amino acid residues in length, and most preferably about 100-300 amino acid residues in length. In other preferred embodiments, the size can be about 3-100 residues, more preferably 4-50 residues, and most preferably 4-20 residues. Preferably, the peptide domains of the library are of relatively uniform length. It will be understood that the length of the peptide domains does not reflect any extraneous sequences which may be present in order to facilitate expression, e.g., such as signal sequences or invariant portions of a fusion protein.

In other preferred embodiments, the peptide domain library can be synthesized as fusion proteins to known protein domains or motifs or fragments thereof. For example, the peptide domain library can be fused to a serum protein or an extracellular secreted protein (e.g., serum albumin, growth factors, immunoglobulin, Ig repeats, fibronectin or fibronectin repeats, Cadherins, etc). These proteins may confer overall improved stability of the fusion protein, and thus may be beneficial for using these fusion proteins in in vivo therapies. The peptide domain library may be fused to N- or C-terminus of the above mentioned proteins, or inserted in-frame into and/or replacing a stretch of the native sequence of those proteins.

In other preferred embodiments, the peptide domain library can be synthesized as fusion proteins to other Icnown protein domains or motifs or fragments thereof. This can be beneficial if a peptide domain is to be identified which binds to a known receptor on the cell surface. In this situation, the domain library can be fused to a know motif (e.g., ankyrin repeats, EGF-like repeats, Ig repeats, SH2, SH3, PDZ, etc.). Theoretically, any protein domains are within the scope of the invention. For example, Washington University at St. Louis, MO, maintains a large database named "PFAM," which is a large collection of multiple sequence alignments and hidden Markov models covering many common protein domains. Version 6.6 of Pfam (August 2001) contains alignments and models for 3071 protein families, based on the Swissprot 39 and SP-TrEMBL 14 protein sequence databases. The contents of this database is incorporated herein by reference. A skilled artisan will readily be able to find information regarding detailed knoweldge about individual protein domains with regard to the instant invention. The harnessing of biological systems for the generation of peptide diversity is now a well established technique which can be exploited to generate the peptide domain libraries of the subject method. Oligonucleotide synthesis is a well- characterized chemistry that allows tight control of the composition of the mixtures created.

IV. Exemplary Uses

Because of the flexibility of the system, the subject method can be used in a broad range of applications, including for the selection of peptide domains having effects on proliferation, differentiation, cell death, cell migration, etc. In preferred embodiments, the target used in the display mode is an extracellular component of a cell. However, it will be appreciated that the target for subject method can be an intracellular component and, during the secretion mode, the system can be augmented with agents which promote the cellular uptake of the test peptide domains.

In an illustrative embodiment, the subject method is utilized to identify peptide domains which are capable of modulating angiogenic activity. The method may be used to identify peptide domains capable of stimulating angiogenesis and peptide domains capable of inhibiting angiogenesis. The method as directed to isolation of angiogenic inhibitory peptide domains is outlined in Figure 3.

A peptide domain display library generated by any of the methods detailed above may be used as the starting material. In a preferred embodiment, the peptide domain library is derived from random priming of an mRNA sample in order to produce a cDNA library which will produce peptide fragments representing a protein fragment / domain library which mimics protein domains released by proteolytic cleavage. Use of this type of library will result in the isolation of proteins, peptides, or fragments thereof, naturally occurring in the animal from which the sample was obtained. Naturally occurring peptides will be less likely to produce an adverse immune response when administered to a subject as a therapeutic.

In the display mode, this peptide domain library can be panned against an endothelial cell or a component thereof to enrich the library for those peptide domains which have endothelial cell specific binding. For example, the peptide domains may be isolated based on their ability to bind to whole endothelial cells, an endothelial cell specific marker, an endothelial cell surface receptor, a receptor for an angiogenic factor, or a receptor for acidic fibroblast growth factor (aFGF), angiogenin, angiotensin, angiopoietin, angiotropin, antiangiogenic antithrombin (aaAT), atrial natriuretic factor (ANF), basic fibroblast growth factor (bFGF), betacellulin, endostatin, endothelial cell-derived growth factor (ECDGF), endothelial cell growth factor (ECGF), endothelial cell growth inhibitor, endothelial monocyte activating polypeptide (EMAP), endothelial cell-viability maintaining factor, endothelin (ET), endothelioma derived mobility factor (EDMF), epidermal growth factors (EGF), erythropoietin (Epo), fibroblast growth factors (FGF), heart derived inhibitor of vascular cell proliferation, hematopoietic growth factors, hepatic growth factor, insulin like growth factors (IGF), interferon (IFN), interleulcins (IL), oncostatin M, placental growth factor (PIGF), platelet derived growth factor (PDGF), platelet derived endothelial cell growth factor (PD-ECGF), somatostatin, transferrin, transforming growth factor-beta (TGF-beta), thrombin, thrombospondin, tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor beta (TNF-b), vasoactive intestinal peptide or vascular endothelial growth factor (NEGF).

The display packages may be put through several iterative rounds of enrichment and amplification (in E. coli) in order to produce a highly enriched domain library with specificity for binding to an endothelial cell or a component thereof.

In the secretion mode, the peptide domains are tested for the ability to regulate a biological process of an endothelial cell. For example, a change in the level of cell proliferation, cell migration, cell differentiation or cell death. For isolation of peptide domains which stimulate angiogenesis, peptides which stimulate endothelial cell proliferation and/or migration are preferably selected. For isolation of peptide domains which inhibit angiogenesis, peptides which inhibit endothelial cell proliferation and/or migration are preferably selected.

Peptide domains having activity for modulating endothelial cell proliferation and/or migration can then be produced recombinantly and tested for their ability to modulate angiogenesis. Peptide domains which show angiogenic stimulatory activity may be developed as biotherapeutics for patients in need of increased angiogenesis or neovascularization, for example, patients suffering from ischemia, wounds, ulcers, etc. Peptide domains which show angiogenic inhibitory activity may be developed as biotherapeutics for patients in need of preventing new blood vessel formation or reducing the number of existing blood vessels, for example patients suffering from arthritis, diabetes, cancer, etc..

Peptide domains capable of inhibiting angiogenesis may be tested for the ability to overcome the angiogenic activity of a growth factor such as, acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), endothelial cell-derived growth factor (ECDGF), endothelial cell growth factor (ECGF), epidermal growth factors (EGF), fibroblast growth factors (FGF), hematopoietic growth factors, hepatic growth factor, insulin like growth factors (IGF), placental growth factor (PIGF), platelet derived growth factor (PDGF), platelet derived endothelial cell growth factor (PD-ECGF), transforming growth factor-beta (TGF- beta), vascular endothelial growth factor (NEGF), etc.

Peptide domains capable of stimulating angiogenesis may be tested for the ability to overcome the anti-angiogenic effects of an angiogenic inhibitor such as thrombospondin I, IL-12, protamine, angiostatin, laminin, endostatin, somatostatin, antiangiogenic antithrombin (aaAT), prolactin fragments etc.

V. Pharmaceutical Preparations of Identified Agents

After identifying certain test peptide domains in the subject assay, e.g. as potential surrogate ligands, or receptor antagonists, the practitioner of the subject assay will continue to test the efficacy and specificity of the selected peptide domains both in vitro and in vivo. Whether for subsequent in vivo testing, or for administration to an animal as an approved drug, peptide domains identified in the subject assay, or peptidomimetics thereof, can be formulated in pharmaceutical preparations for in vivo administration to an animal, preferably a human.

In addition to the peptidyl compounds described herein, the inventor also contemplates that other sterically similar compounds may be formulated to mimic the key portions of the peptide structure. Such compounds, which include chemically modified peptides, peptide-like molecules containing non-naturally occurring amino acids, peptoids and the like, may be termed peptidomimetics. Peptidomimetics may be used in the same manner as the peptides of the invention and hence are also functional equivalents. Peptidomimetics provide various advantages over peptides, and are particularly useful for oral administration since they can be stable when administered to a subject during passage throughout the digestive tract. The generation of a structural functional equivalent may be achieved by the techniques of modeling and chemical design Icnown to those of skill in the art (see, for example, "Burger's Medicinal Chemistry and Drug Discovery" 5th ed., vols. 1 to 3 (ed. M. E. Wolff; Wiley Interscience 1995)). it will be understood that all such sterically similar constructs fall within the scope of the present invention.

The peptide domains selected in the subject assay, or a pharmaceutically acceptable salt thereof, may accordingly be formulated for administration with a biologically acceptable medium, such as water, buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like) or suitable mixtures thereof. The optimum concentration of the active ingredient(s) in the chosen medium can be determined empirically, according to procedures well known to medicinal chemists. As used herein, "biologically acceptable medium" includes any and all solvents, dispersion media, and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation. The use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the activity of the compound, its use in the pharmaceutical preparation of the invention is contemplated. Suitable vehicles and their formulation inclusive of other proteins are described, for example, in the book Remington 's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., USA 1985). These vehicles include injectable "deposit formulations". Based on the above, such pharmaceutical formulations include, although not exclusively, solutions or freeze-dried powders of the compound in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered media at a suitable pH and is osmotic with physiological fluids. In preferred embodiment, the peptide domains can be disposed in a sterile preparation for topical and/or systemic administration. In the case of freeze-dried preparations, supporting excipients such as, but not exclusively, mannitol or glycine may be used and appropriate buffered solutions of the desired volume will be provided so as to obtain adequate isotonic buffered solutions of the desired pH. Similar solutions may also be used for the pharmaceutical compositions of compounds in isotonic solutions of the desired volume and include, but not exclusively, the use of buffered saline solutions with phosphate or citrate at suitable concentrations so as to obtain at all times isotonic pharmaceutical preparations of the desired pH, (for example, neutral pH).

Exemplification

The invention now being generally described, it will be more readily understood by reference to the following examples which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention. Exemplary plasmids, pAM7 and pAM9 M13/COS peptide expression plasmids, are shown in Figure 1. The difference between pAM7 and pAM9 is the Ml 3 coat protein to which the peptide is fused in the display mode: pAM7 utilizes a pNIII fusion and pAM9 utilizes a pill fusion, as indicated by Ml 3 pNIII or pill. These vectors allow the display of protein domains as pill or pNIII fusions of the M13 phage. Protein domains displayed as pill fusions are presented in single copy while protein domains presented as pNIII fusion are presented in multiple copies on the surface of the phage.

As shown in Figure 1, pAM7 and pAM9 M13/COS peptide expression plasmid is designed to have distinct functions in prokaryotic and eukaryotic cells. In prokaryotic cells the plasmid functions in the display mode, which directs the display of encoded peptides on the surface of the Ml 3 phage within which it is packaged. The peptides are displayed as fusions with the major Ml 3 capsid protein pill or pVIII. The depicted elements which enable this mode are as follows: lac, the prokaryotic promoter; peptide domain, the coding sequence of the peptide domains (from a library) to be fused to an M13 coat protein; M13 pill or pVIII, the coding sequence of the pill or pVIII coat protein of Ml 3 (the STOP above it represents the stop codon and prokaryotic transcriptional terminator); ApR, the β-lactamase gene which allows ampicillin selection for cells carrying the plasmid; pUC ori, replication origin which directs high copy number replication of the plasmid within E. coli; M13(-) ori, replication origin and packaging signal which, in the presence of M13 helper phage, allows generation of single stranded DNA, and directs its packaging into phage particles.

In eukaryotic cells the plasmid functions in the secretion mode, which results in the secretion of the encoded peptide, apart from other sequences, into the extracellular media. The depicted elements which enable this mode are as follows: CMV enh/prom, the eukaryotic promoter; IgH secr.s., the immunoglobulin heavy chain signal sequence which directs extracellular secretion of proteins; peptide domain, the coding sequence of the peptide domain (from a library) to be secreted; SV40 poly A, RNA polyadenylation signal; globin splice donor/IgH splice acceptor pairs, direct processing of the RNA to a mature mRNA that is devoid of the unwanted intervening prokaryotic sequences; SV40 ori, origin of replication which results in high copy number replication of the plasmid in COS cells.

In these plasmids, as shown in Figure 1, the CMV enh/prom and lac promoters have been separated and placed adjacent to their respective eukaryotic IgH signal s and prokaryotic E. coli signal s signal sequences. The first pair of splice signals functions to remove the lac promoter and E. coli signal sequence from the mRNA. In addition, the E. coli signal s sequence was created from consensus signal sequences, and into its coding sequence the IgH splice acceptor sequence was silently engineered, which eliminates the addition of extra residues onto the amino terminus of the peptide domains. An illustrative transwell cell culture chamber is shown in Figure 2A. This illustration depicts the transwell system that is utilized in the secretion mode of the subject method. The lower compartment containing the target cells is a well in an ordinary tissue culture dish. The transwell, which fits into this well, is a chamber with solid sides and a permeable, microporous membrane bottom. The secreting COS cells are grown on this membrane through which their secreted peptides can diffuse and come into contact with the target cells.

In other embodiments, the subject method is used as part of an indicator plate antimicrobial assay. Figure 2B illustrates a method for identifying peptides with antimicrobial activity. Bacterial cells carrying plasmids that direct the secretion of test peptide domains are plated on top of an agar embedded culture of target microorganisms. Production of an inhibitory peptide domain by any of the bacterial colonies will result in an inhibition zone in the agar embedded target cell culture. This zone will be visualized as a clear area in the agar, due to the inability of the target cells to form a dense culture in the presence of the secreted peptide domain.

The method of the present invention has been utilized to identify antiangiogenic peptides. Figure 3 is a flowchart depicting utilization of the Ml 3 display/COS secretion method for identification of anti-angiogenic peptide domains.

Methods for assaying cell proliferation are well Icnown in the art. For example, in vitro proliferation of endothelial cells may be determined as described by Lingen, et al. in Laboratory Investigation, 74: 476-483 (1996), which is hereby incorporated herein by reference, using the Cell Titer 96 Aqueous Non-Radioactive

Cell Proliferation Assay kit (Promega Corporation, Madison, Wis.). Briefly, endothelial cells are plated at a density in a 96-well plate in Dulbecco's Modified Eagle Medium (DMEM) containing 10 % donor calf serum and 1 % BSA (bovine serum albumin, GIBCO BRL, Gaithersburg, Md.). After 8 hours, the cells are starved overnight in DMEM containing 0.1 % BSA then re-fed with media containing a test peptide domain and 5 ng/mL bFGF (basic fibroblast growth factor).

The results of the assay are corrected both for unstimulated cells (i.e. no bFGF added) as the baseline and for cells stimulated with bFGF alone (i.e. no test peptide domain added). The results may be represented as the percent change in cell number as compared to cells treated with bFGF alone.

Methods for assaying cell migration are well known in the art. For example, endothelial cell migration may be assayed essentially as described by Polverini, P. J. et al., Methods Enzymol, 198: 440-450 (1991), which is hereby incorporated herein by reference. Briefly, endothelial cells are starved overnight in DMEM containing 0.1 % bovine serum albumin (BSA). Cells are then harvested and resuspended in DMEM with 0.1 % BSA and added to the bottom of a 48-well modified Boyden chamber (Nucleopore Corporation, Cabin John, Md.). The chamber is assembled and inverted, and cells are allowed to attach for approximately 2 hours at 37°C to polycarbonate chemotaxis membranes (5 μm pore size) that had been soaked in 0.1 % gelatin overnight and dried. The chamber is then reinverted and test peptide domains are added to the wells of the upper chamber (to a total volume of 50 μL); the apparatus is then incubated for 4 hours at 37°C. Membranes are recovered, fixed and stained (DiffQuick, Fisher Scientific, Pittsburgh, Pa.) and the number of cells that migrate to the upper chamber are counted. Background migration to DMEM + 0.1 % BSA is subtracted and the percent change of migration compared to a positive control is determined.

The source of the test peptide domains for the endothelial cell proliferation and migration assays may be from conditioned media isolated from the transwell system as illustrated in Figures 2 A and 3.

Methods for testing the angiogenic' activity of a compound are well Icnown in the art. As many angiogenic factors are mitogenic and chemotactic for endothelial cells their biological activities can be determined in vitro by measuring the induced migration of endothelial cells or the effect of these factors on endothelial cell proliferation.

However, there are also a number of bioassays which allow direct determination of angiogenic activities and permit repeated, long-term quantitation of angiogenesis as well as physiological characterization of angiogenic vessels. The most frequently used in vivo models for angiogenesis are the rabbit corneal pocket, the hamster cheek pouch and the chicken chorioallantoic membrane (CAM) assays (Folkman, J. and Brem, H. (1992) Angiogenesis and inflammation. In: "Inflammation. Basic Principles and Clinical Correlates". Eds Gallin, J. I., Goldstein, I. M. and Snyderman, R. S., Raven Press, New York; Folkman, J. (1985) Adv. Cancer Res. 43: 175; Folkman, J. and Klagsbrun, M. (1987) Science 235: 442; Folkman, J. (1985) Perspect. Biol. Med. 29: 10), the rat aortic ring model, the placental fragment assay (Brown, K.J. et al. (1996) Laboratory Investigation 75(4): 539-555), the subcutaneous air sac model (Lichtenberg J et al. (1997) Pharmacol. Toxicology 81 (6): 280-284).

Several in vitro angiogenesis model which have also been described (O'Reilly, M.S. et al. (1994) Cell 79(2): 315-328; O'Reilly, M.S. et al. (1997) Cell 88: 277-285; Parish et al. U.S. Patent No. 5,976,782).

Any of the methods described above may be used in accord with the methods of the present invention to identify peptide domains capable of modulating angiogenic activity.

Figure 4 provides a more detailed look at the functional elements of the pAM7 and pAM9 M13/COS plasmid shown in Figure 1. In each of these examples, the peptide domain, flanked by distinct BstXl sites, is actually a Myc epitope-6xHis control peptide.

To test the plasmids, E. coli were transformed with the negative control pLITMUS plasmid and the Myc epitope-6xHis encoding plasmid pAM7. The cells were grown at 37°C to log phase and induced with 0.1 mM IPTG for 3 hours (+) or grown for 3 hours in the absence of IPTG (-). Whole cell lysates were separated by electrophoresis on a 16 % tricine SDS-PAGE, and immunoblotted with anti-myc antibody.

The results demonstrate that the pAM7 M13/COS vector express the Myc- 6xHis-pVIII fusion protein, and that the products of pAM7 appear to be properly processed. The incorporation of myc-6xHis-pVIII fusion protein into phagemid capsids was tested by anti-myc western blotting. E. coli were transformed with the negative control pLITMUS plasmid and the Myc epitope-6xHis encoding plasmid pAM7. The cells were grown at 37°C to log phase, induced with 0.1 mM IPTG, infected with Ml 3 helper phage and grown overnight. Phagemids contained in the culture media supernatant were separated by electrophoresis on a 16 % tricine SDS-PAG, and immunoblotted with anti-myc antibody.

The result demonstrates that the pAM7 vector results in properly processed

Myc-6xHis-pVIII fusion protein being incorporated into the capsid, Very little fusion protein is incorporated into the capsid of phagemids produced by pAM6 transformants, while nothing is detected in the negative control using the pLITMUS plasmid.

The ratio of native pVIII versus myc-6xHis-pVIII proteins in phagemid capsids was also determined. As above, E. coli were transformed with the negative control pLITMUS plasmid and the Myc epitope-6xHis encoding plasmid pAM7. The cells were grown at 37°C to log phase, induced with 0.1 mM IPTG, infected with Ml 3 helper phage and grown overnight. Phagemids contained in the culture media supernatant were separated by electrophoresis on a 16 % tricine SDS-PAG, and stained with coomassie blue. We observed that pAM7 vector results in properly processed Myc-6xHis- pVIII fusion protein being incorporated into the capsid at a ratio of 1-10 % of native pVIII. No fusion protein is detected in the pLITMUS lane.

Phagemids produced as described above were also tested for titration of plaque and colony forming units generated upon phagemid rescue by serially dilution and infection into log phase E. coli. (see Figure 5). Infected cells were either plated on soft agar to detect plaque forming units (p.f.u.), or on ampicillin to determine colony forming units (c.f.u.). c.f.u. represent those phage which have packaged a plasmid DNA, whereas p.f.u. represent phage which have packaged helper a phage genome DNA. The secretion of M13/COS plasmid encoded proteins from COS-7 cells, e.g., in the secretion mode, is examined. COS-7 cells were transfected with pIC400 negative control plasmid and with the pAM7 plasmid, which encodes p27 in the peptide domain insertion site. The normally intracellular p27 was chosen for this experiment to enable efficient western blot detection of the secreted protein in the cell media. 20 μl aliquots of media were collected on days 1, 2, 3 and 5 following transfection, separated by SDS-PAGE and immunoblotted with anti-p27 antibody. The results show that the level of secreted protein in the media increased over the time of the experiment for pAM7 (Figure 6). No p27 is detected with pIC400 negative control. Purified p27 is included as a size marker.

Figure 7 shows an anti-pill western blot detection of peptide domains incorporated into Ml 3 phagemid capsids as pill fusions. Briefly, oligonucleotides encoding the Myc epitope - 6 x His peptide domain, a thrombospondin derived peptide (tsp: SPWSSASVTCGDGVITRIR, SEQ ID No. 2), an α_vβ₃ integrin binding peptide containing the RGD motif (CDCRGDCFC, SEQ ID No. 3) and the first kringle domain of angiostatin (Kl : 80 amino acids) were inserted between the BstXI sites of pAM9. In E. coli, the plasmids direct the expression of the peptide- pIII fusion proteins. For phagemid production the cells were grown at 37°C to log phase, induced with 0.1 mM IPTG, infected with Ml 3 helper phage or an Ml 3 helper phage that carries an amber mutation in the pill gene and grown overnight. Phagemids contained in the culture media supernatant were separated by electrophoresis on a 16 % tricine SDS-PAG, and immunoblotted with anti-pill antibody. As a control M13K07 phage particle were used. The lower bands correspond to wild type pill proteins while the upper, higher molecular weight bands correspond to the peptide-pIII fusion proteins.

The data demonstrate that the pAM9 derived vectors express the predicted peptide domains as pill fusions and the fusion proteins being incorporated into the capsid of Ml 3 phagemids.

We observed specific binding of pAM9-Kl phagemids to bovine capillary endothelial cells (see Figure 8). Phagemid particles that display either the Myc epitope-6xHis peptide domain (pAM9-myc; negative control) or the first kringle domain of angiostatin (pAM9-Kl, positive control) as pill fusions were used to test the specificity of phagemid binding to bovine capillary endothelial (BCE) cells. Greater than 90 % confluent BCE cells in a well of a 6 well plate were incubated with ~5xl 0¹² Ml 3K07 p.f.u./well in 2.5 ml of peptide binding buffer (lxPBS, ImM CaCl₂, 10 mM MgCl₂, 0.1 % BSA) for 30 minutes at 37C. 10⁸ c.f.u. pAM9-myc or ρAM9-Kl phagemids were added to the mix and the incubation continued for 45 minutes at 37C. Excess phagemids were removed by washing the cells 5x5 ml Washing buffer (2 x PBS, ImM CaCl₂, 10 mM MgCl₂, 0.1% BSA) and phagemids bound to BCE cells were eluted by 2x1 ml 0.1 N HC1, pH 2.2 that were neutralized by the addition of 1 ml 1M Tris.HCl pH 8.0. The number of phagemids in the elution buffer was determined by infecting TGI cells with aliquots of the eluates and selecting pAM9-myc or pAM9-Kl transformants on LB+Amp (M13K07 phage do not form colonies on LB+Amp). The data demonstrate the feasibility to enrich peptide domain libraries in the display mode for peptide domains that specifically bind to endothelial cells.

Figure 9 shows inhibition of BCE cell proliferation in transwells by peptide domains secreted from COS-7 cells. COS-7 cells were transfected with pAM9-myc, pAM9-RGD and pAM9-Kl plasmids, respectively, that direct the expression and secretion of the Myc epitope-6xHis, the RGD and the angiostatin first kringle domain peptide domains. The transfected COS-7 cells were co-incubated in Transwells with BCE cells whose proliferation was stimulated by 1 ng/ml bFGF. As controls, untransfected COS-7 cells and bFGF stimulated BCE cells were similarly co-incubated and synthetic Myc-6xHis and RGD peptide domains as well as purified Kl were added to the media at the indicated concentrations. The proliferation of the bFGF stimulated BCE cells were measured 72 hrs later using the fluorescent CyQUANT proliferation kit (Molecular Probes).

The synthetic RGD peptide domain and the purified Kl as well as the COS-7 secreted RGD and Kl peptide domains inhibited bFGF stimulated BCE cell proliferation (positive controls). The negative control Myc epitope-6xHis peptide did not have inhibitory effect on BCE proliferation. The data demonstrate the feasibility of screening peptide domain libraries in the secretion mode for peptide domains that inhibit the proliferation of endothelial cells.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Patent No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., NN.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Nols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-TV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, Ν.Y., 1986).

All of the above-cited references and publications are hereby incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific method and reagents described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

We Claim:

1. A method for isolating a peptide domain capable of modulating angiogenic activity, comprising:

(i) providing a peptide domain display library comprising a variegated population of test peptide domains expressed on the surface of a population of display packages;

(ii) in a display mode, isolating, from the peptide domain display library, a sub-population of display packages enriched for test peptide domains which have a binding specificity for an endothelial cell or a component thereof;

(iii) in a secretion mode, simultaneously expressing the enriched test peptide domain sub-population under conditions wherein the test peptide domains are secreted and are free of the display packages;

(iv) assessing the ability of the secreted test peptide domains to regulate a biological process of an endothelial cell; and

(v) assessing the ability of the test peptide domains capable of regulating a biological process of an endothelial cell for the ability to regulate angiogenesis, thereby identifying a peptide domain capable of modulating angiogenetic activity.

2. The method of claim 1, wherein the isolated peptide domain is capable of stimulating angiogenesis.

3. The method of claim 1 , wherein the isolated peptide domain is capable of inhibiting angiogenesis.

4. The method of claim 1, wherein the peptide domain display library is encoded by a nucleic acid library constructed by random priming of mRNA.

5. The method of claim 1, wherein the peptide domain display library uses a phage display library as the display package.

6. The method of claim 5, wherein the display packages of the phage display library are phage particles selected from a group consisting of Ml 3, fl, fd, Ifl, Ike, Xf, Pfl, Pf3, λ, T4, T7, P2, P4, φX-174, MS2 and f2.

7. The method of claim 5, wherein the phage display library is generated with a filamentous bacteriophage specific for Escherichia coli and the phage coat protein is coat protein III or coat protein VIII.

8. The method of claim 7, wherein the filamentous bacteriophage is Ml 3, fd, or fl.

9. The method of claim 1, wherein the peptide domain display library is a bacterial cell-surface display library or a spore display library.

10. The method of claim 5, wherein the test peptide domains with binding specificity for an endothelial cell are enriched from the peptide domain display library in the display mode by a differential binding step comprising affinity separation of test peptide domains which specifically bind the endothelial cell or component thereof from test peptide domains which do not.

11. The method of claim 10, wherein the endothelial cell is a human cell.

12. The method of claim 11 , wherein the human cell is a capillary endothelial cell.

13. The method of claim 10, wherein the differential binding step comprises panning the peptide domain display library on whole endothelial cells.

14. The method of claim 10, wherein the differential binding step comprises an affinity chromatographic step in which a component of an endothelial cell is provided as part of an insoluble matrix.

15. The method of claim 14, wherein the insoluble matrix comprises an endothelial cell surface protein attached to a polymeric support.

16. The method of claim 10, wherein the differential binding step comprises immunoprecipitating the display packages.

17. The method of claim 1 , wherein the display mode enriches for test peptide domains which bind to an endothelial cell specific marker.

18. The method of claim 1 , wherein the display mode enriches for test peptide domains which bind to an endothelial cell surface receptor protein.

19. The method of claim 18, wherein the receptor protein is a receptor for an endothelial cell mitogen.

20. The method of claim 18, wherein the receptor protein is a receptor for an angiogenic factor.

21. The method of claim 18, wherein the receptor protein is a receptor for acidic fibroblast growth factor (aFGF), angiogenin, angiotensin, angiopoietin, angiotropin, antiangiogenic antithrombin (aaAT), atrial natriuretic factor (ANF), basic fibroblast growth factor (bFGF), betacellulin, endostatin, endothelial cell-derived growth factor (ECDGF), endothelial cell growth factor (ECGF), endothelial cell growth inhibitor, endothelial monocyte activating polypeptide (EMAP), endothelial cell- viability maintaining factor, endothelin (ET), endothelioma derived mobility factor (EDMF), epidermal growth factors (EGF), erythropoietin (Epo), fibroblast growth factors (FGF), heart derived inhibitor of vascular cell proliferation, hematopoietic growth factors, hepatic growth factor, insulin like growth factors (IGF), interferon (IFN), interleulcins (IL), oncostatin M, placental growth factor (PIGF), platelet derived growth factor (PDGF), platelet derived endothelial cell growth factor (PD-ECGF), somatostatin, transferrin, transforming growth factor-beta (TGF-beta), thrombin, thrombospondin, tumor necrosis factor- alpha (TNF-alpha), tumor necrosis factor beta (TNF-b), vasoactive intestinal peptide or vascular endothelial growth factor (NEGF).

22. The method of claim 1 , wherein the peptide domain display library includes at least IO³ different test peptide domains.

23. The method of claim 1, wherein the test peptide domains are 50-300 amino acid residues in length.

24. The method of claim 1, wherein each of the test peptide domains are encoded by a chimeric gene comprising (i) a coding sequence for the test peptide domain, (ii) a coding sequence for a surface protein of the display package for displaying the test peptide domains on the surface of a population of display packages, and (iii) RNA splice sites flanking the coding sequence for the surface protein, wherein, in the display mode, the chimeric gene is expressed as fusion protein including the test peptide domain and the surface protein, whereas in the secretion mode, the test peptide domain is expressed without the surface protein as a result of the coding sequence for the surface protein being removed by RNA splicing.

25. The method of claim 1 , wherein the test peptide domains are expressed by a eukaryotic cell in the secretion mode.

26. The method of claim 25, wherein the eukaryotic cell is a mammalian cell.

27. The method of claim 1, wherein the endothelial cell in step (iv) is a mammalian cell.

28. The method of claim 27, wherein the mammalian cell is a human cell.

29. The method of claim 28, wherein the human cell is a capillary endothelial cell.

30. The method of claim 1, wherein the biological process of an endothelial cell includes a change in cell proliferation, cell migration, cell differentiation or cell death.

31. The method of claim 1 , wherein the regulation of a biological process of an endothelial cell includes inhibition of cell proliferation and/or migration.

32. The method of claim 18, wherein the target cell further comprises a reporter gene construct containing a reporter gene in operative linkage with one or more transcriptional regulatory elements responsive to the signal transduction activity of the cell surface receptor protein, expression of the reporter gene providing the detectable signal.

33. The method of claim 32, wherein the reporter gene encodes a gene product that gives rise to a detectable signal selected from the group consisting of: color, fluorescence, luminescence, cell viability relief of a cell nutritional requirement, cell growth, and drug resistance.

34. The method of claim 33, wherein the reporter gene encodes a gene product selected from the group consisting of chloramphenicol acetyl transferase, beta-galactosidase and secreted alkaline phosphatase.

35. The method of claim 1 , wherein the secretion mode includes expression of the test peptide domains by a host cell co-cultured with the endothelial cell of step (iv).

36. The method of claim 35, wherein the co-cultured host and endothelial cells are separated by a membrane which is permeable to the test peptide domain.

37. The method of claim 1, wherein the secretion mode comprises assessing the ability of the secreted test peptide domains to inhibit the biological activity of an exogenously added compound on the endothelial cells.

38. The method of claim 37, wherein the exogenously added compound is an endothelial cell mitogen.

39. The method of claim 37, wherein the exogenously added compound is an angiogenic factor.

40. The method of claim 37, wherein the exogenously added compound is selected from the group consisting of acidic fibroblast growth factor (aFGF), angiogenin, angiotensin, angiopoietin, angiotropin, antiangiogenic antithrombin (aaAT), atrial natriuretic factor (ANF), basic fibroblast growth factor (bFGF), betacellulin, endostatin, endothelial cell-derived growth factor

(ECDGF), endothelial cell growth factor (ECGF), endothelial cell growth inhibitor, endothelial monocyte activating polypeptide (EMAP), endothelial cell-viability maintaining factor, endothelin (ET), endothelioma derived mobility factor (EDMF), epidermal growth factors (EGF), erythiOpoietin (Epo), fibroblast growth factors (FGF), heart derived inhibitor of vascular cell proliferation, hematopoietic growth factors, hepatic growth factor, insulin like growth factors (IGF), interferon (IFN), interleukins (IL), oncostatin M, placental growth factor (PIGF), platelet derived growth factor (PDGF), platelet derived endothelial cell growth factor (PD-ECGF), somatostatin, transferrin, transforming growth factor-beta (TGF-beta), thrombin, thrombospondin, tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor beta (TNF-b), vasoactive intestinal pepti,de and vascular endothelial growth factor (VEGF).

41. The method of claim 1 , wherein after step (ii) the isolated subset of test peptide domains which have a binding specificity for an endothelial cell or a component thereof, are amplified in E. coli.

42. The method of claim 41 , wherein the amplified test peptide domains are subjected to a second round of enrichment based on their binding specificity for an endothelial cell or a component thereof.

43. The method of claim 1, wherein: in step (iv), the ability of the secreted test peptide domains to inhibit proliferation of endothelial cell is assessed.

44. ^' The method of claim 43, wherein: in step (iv), the ability of the secreted test peptide domains to inhibit proliferation of endothelial cells in the presence of an angiogenic amount of an endogenous growth factor is assessed.

45. The method of claim 1 , comprising the further step of converting into peptidomimetics, one or more test peptide domains which regulate the biological process in the target cell.

46. The method of claim 1, comprising the further step of formulating, with a pharmaceutically acceptable carrier, one or more test peptide domains which regulate the biological process in the target cell or peptidomimetics thereof.

47. A peptide domain display library enriched for test peptide domains having a binding specificity and/or affinity for an endothelial cell or a component thereof and which inhibit endothelial cell proliferation and/or migration in a target endothelial cell.

48. A vector comprising a chimeric gene for a chimeric protein, which chimeric gene comprises (i) a coding sequence for a test peptide domain, (ii) a coding sequence for a surface protein of a display package, and (iii) RNA splice sites flanking the coding sequence for the surface protein, wherein,

in a display mode, the chimeric gene is expressed as a fusion protein including the test peptide domain and the surface protein such that the test peptide domain can be displayed on the surface of a population of display packages, whereas in the secretion mode, the test peptide domain is expressed without the surface protein as a result of the coding sequence for the surface protein being removed by RNA splicing.

49. The vector of claim 48, wherein the chimeric gene further comprises a secretion signal sequence for secretion of the test peptide domain in the secretion mode.

50. The vector of claim 49, wherein the secretion signal sequence causes secretion of the test peptide domain from eukaryotic cells.

51. The vector of claim 50, wherein the eukaryotic cells are mammalian cells.

52. The vector of claim 48, wherein the display package is a phage.

53. The vector of claim 52, wherein the phage is selected from a group consisting of M13, fl, fd, Ifl, Ike, Xf, Pfl, Pf3, λ, T4, T7, P2, P4, φX-174, MS2 and f2.

54. The vector of claim 52, wherein the phage is a filamentous bacteriophage specific for Escherichia coli and the surface protein is coat protein III or coat protein VIII.

55. The vector of claim 54, wherein the filamentous bacteriophage is Ml 3, fd, or fl.

56. A vector library, each vector comprising a chimeric gene for a chimeric protein, which chimeric gene comprises (i) a coding sequence for a test peptide domain, (ii) a coding sequence for a surface protein of a display package, and (iii) RNA splice sites flanking the coding sequence for the surface protein, wherein,

in a display mode, the chimeric gene is expressed as fusion protein including the test peptide domain and the surface protein such that the test peptide domain can be displayed on the surface of a population of display packages, whereas in the secretion mode, the test peptide domain is expressed without the surface protein as a result of the coding sequence for the surface protein being removed by RNA splicing, the vector library collectively encodes a variegated population of test peptide domains.

57. The vector library of claim 56, wherein the chimeric gene further comprises a secretion signal sequence for secretion of the test peptide domain in the secretion mode.

58. The vector library of claim 57, wherein the secretion signal sequence causes secretion of the test peptide domain from eukaryotic cells.

59. The vector library of claim 58, wherein the eukaryotic cells are mammalian cells.

60. The vector library of claim 56, wherein the display package is a phage.

61. The vector library of claim 60, wherein the phage is selected from a group consisting of M13, fl, fd, Ifl, Ike, Xf, Pfl, Pf3, λ, T4, T7, P2, P4, φX-174, MS2 and f2.

62. The vector library of claim 56, wherein the phage is a filamentous bacteriophage specific for Escherichia coli and the surface protein is coat protein III or coat protein VIII.

63. The vector library of claim 62, wherein the filamentous bacteriophage is M13, fd, or fl.

64. The vector library of claim 56, wherein the vector library collectively encodes at least IO³ different test peptide domains.

65. The vector library of claim 56, wherein the test peptide domains are 50-300 amino acid residues in length.

66. A cell composition comprising a population of cells containing the vector library of claim 56.

67. A method for modulating an angiogenic process in an animal, comprising administering to the animal a pharmaceutical preparation of claim 46.

68. A construct as shown in Figure 1.

69. A method for isolating a peptide domain capable of inhibiting angiogenic activity, comprising:

(i) providing a peptide domain display library comprising a variegated population of test peptide domains expressed on the surface of a population of display packages;

(ii) in a display mode, isolating, from the display packages, a sub- population of display packages enriched for test peptide domains which have a binding specificity for an endothelial cell or a component thereof;

(iv) in a secretion mode, simultaneously expressing the enriched test peptide domain sub-population under conditions wherein the test peptide domains are secreted and are free of the display packages;

(v) assessing the ability of the secreted test peptide domains to inhibit endothelial cell proliferation and/or migration; and

(vi) assessing the ability of the test peptide domains capable of inhibiting endothelial cell migration and/or proliferation for the ability to inhibit angiogenesis, thereby isolating a peptide domain capable of inhibiting angiogenic activity.

70. A method of conducting a pharmaceutical business, comprising:

(i) by the method of claim 1, identifying one or more peptide domains which are capable of modulating angiogenic activity; and, (ii) conducting therapeutic profiling of said identified peptide domain(s), or other homologs or peptidomimetics thereof, for using the peptide domain(s) to modulate angiogenesis; and,

(iii) formulating a pharmaceutical preparation including one or more identified peptide domain(s) as a product having an acceptable therapeutic profile.

71. The business method of claim 70, further comprising establishing a distribution system for distributing said product for sale.

72. The business method of claim 70, further including establishing a sales group for marketing the product.

73. A method of conducting a pharmaceutical business, comprising:

(i) by the method of claim 1, identifying one or more peptide domains which are capable of modulating angiogenic activity; (ii) conducting therapeutic profiling of said identified peptide domain(s), or other homologs or peptidomimetics thereof, for using the peptide domain(s) to modulate angiogenesis; and,

(iii) licensing, to a third party, the rights for further development of agents to modulate angiogenesis.