CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. application no. 61/783,779, filed

March 14, 2013, the disclosure of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under CA077739 and

HHSN261201000119C awarded by the National Institutes of Health, and W81XWH-04-1- 0264 awarded by the United States Army Medical Research and Materiel Command. The government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to targeted peptide delivery and more specifically to novel peptides and conjugates thereof for use as therapeutic and imaging agents.

BACKGROUND OF THE INVENTION

[0004] There is an ongoing and unmet need to develop compositions and methods for targeting tissue architecture that is involved in disease etiology, and especially for vascular structures that are involved in the progression and/or treatment of cancer. The present invention meets these needs.

SUMMARY OF THE INVENTION

[0005] The present disclosure provides compositions and method related to novel peptides, method of making the peptides, fusion proteins comprising the peptides, peptides conjugated to detectable labels, and chemotherapeutic agents. In embodiments, the chemotherapeutic agents comprise or consist of polypeptide sequences.

[0006] The disclosure includes peptides and recombinant fusion proteins comprising them, wherein the peptide sequences include every peptide sequence disclosed herein. In embodiments, the disclosure includes a peptide having a peptide sequence selected from G AMHLPWHM GTL (SEQ ID NO:5), KPPQNTSAPYLP (SEQ ID NO:6);

SPHWQPNAIFVN (SEQ ID NO:8); TLQRTHFPPAFS (SEQ ID NO:9), TMGFTAPRFPHY (SEQ ID NO: 10 ), SPHWQPNAI (SEQ ID NO: 19), and G AMHLPWHM GT (SEQ ID NO:100). In embodiments the peptide sequence does not comprise bacteriophage coat protein amino acid sequence. In embodiments, the peptide sequence is present in a fusion protein, wherein the fusion protein does not comprise bacteriophage coat protein. In embodiments, the fusion protein that comprises the peptide sequence comprises a detectable label, such as a radio label, or the fusion protein comprises a chemotherapeutic agent, including but not limited to chemotherapeutic agents that are enzymatically active toxins. It will be recognized by those skilled in the art that the peptides provided by the invention are artificial and have properties that make the markedly different from that which occurs in nature.

[0007] In one embodiment, the disclosure includes a peptide or fusion protein comprising the sequence of a peptide, wherein the peptide or fusion protein is bound to a human cell or tissue. Thus, complexes comprising the peptides/fusion proteins and human epithelial cells and tissues are included. In embodiments the complex is formed between a peptide or fusion protein comprising the peptide sequence and an endothelial cell and/or a component, such as a marker, of the vascular basement membrane. In embodiments, the peptide binding location is exposed during a period subsequent to initiation of androgen deprivation in a subject. Method of forming such complexes are included, and can be part of a prophylactic and/or therapeutic approach for prostate cancer and other prostate related disorders, such as benign prostatic hypertrophy (BPH). The disclosure is not necessarily limited to prostate disorders.

[0008] The disclosure also includes pharmaceutical compositions comprising a peptide having a peptide sequence disclosed herein. In embodiments, the peptides are conjugated to other agents. The pharmaceutical compositions comprise a pharmaceutically acceptable additive, including but not necessarily limited to a pharmaceutically acceptable carrier.

[0009] In one approach, the disclosure includes a method for prophylaxis and/or therapy of an individual in need thereof comprising administering to the individual a peptide having a peptide sequence disclosed herein, or fusion proteins comprising such peptide sequences, and compositions comprising such peptide sequences. In embmodiments the individual is in need of therapy for prostate cancer or benign prostatic hypertrophy. In embodiments, the individual is undergoing androgen deprivation therapy.

[0010] The disclosure also includes all methods of making the peptides and fusion proteins and peptide conjugates, including by chemical synthesis and recombinant methods.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The present disclosure provides compositions and methods that are useful for, among other purposes, specifically binding to certain markers that are exposed during a transient period the follows androgen deprivation therapy. In this regard, the disclosure provides peptides and methods of using the peptides for targeting tissue structures, and particularly for targeting epithelial cells, and endothelial cells, and the vascular basement membrane, particularly in prostate tissue. The invention accordingly provides compositions comprising the peptides and methods for using them in therapeutic interventions for treating diseases that include but are not necessarily limited to cancer, such as benign prostatic hypertrophy (hyperplasia).

[0012] The instant invention is based in part on our discovery of novel peptides that can target microvasculature in human prostate tissue exposed by androgen deprivation. In more detail, and as disclosed in U.S. Patent No. 8,048,640, (the '640 patent) the disclosure of which is incorporated herein by reference, the vasculature of the human prostate is unique among organs that are major sites of human disease in that the endothelial compartment appears significantly less stable than the vascular compartment of other organs. The epithelial compartment of the prostate demonstrates a proliferative index as much as two orders of magnitude greater than other organs and as much as 50-70% of the small vessels in benign and malignant prostate lack association with vascular smooth muscle cells that signify vascular maturation and stability. Studies using human prostate xenografts transplanted to immunocompromised mouse as described in the '640 patent demonstrate that the endothelial cells of the human vasculature express androgen receptor, have a peak of apoptotic death at two days after androgen deprivation therapy, and disruption of the endothelial cells causes the vessels to leak plasma components into the interstitial space. The death of the endothelial compartment is followed by apoptotic death of the glandular epithelial compartment at 6-7 days after androgen deprivation. However, in response to the hypoxia induced by the rapid vascular involution, proliferation of the endothelial compartment with revascularization of the prostate tissue begins between 4 and 7 days after androgen-deprivation. Thus, the period between 1 and 7 days, and optimally between 1 and 4 days after androgen deprivation, represents an important but transient therapeutic window during which areas of labilized vasculature permit improved access to prostate cancer/epithelial cells.

[0013] In the present disclosure we harnessed this model in novel approaches to develop new peptides that can target the prostatic microenvironment exposed by androgen deprivation. Specifically, the experimental model in which the pro state- specific targeting peptides were identified were primary xenografts of human prostate cancer tissue transplanted to immuno-compromised mouse hosts that were pre-castrated and implanted with silastic tubing packed with testosterone to maintain human levels of circulating testosterone, and to allow the selective induction of androgen deprivation therapy (ADT) by removal of the silastic tubing. An important enabling characteristic of this model is that the vasculature of the xenografts is of human origin, and that the vascular endothelial cells undergo rapid apoptotic death in response to short-term androgen deprivation, resulting in transient destruction of the endothelial cell permeability barrier and access of a systemically introduced peptide library to endothelial cells, damaged endothelial cells, denuded vascular basement membrane and the interstitial tissue space including prostate cancer cells, benign prostate epithelial cells in benign prostatic hypertrophy (hyperplasia) and prostate cancer stem cells/benign prostate stem cells. The loss of the vascular permeability barrier allows access of large molecules into the interstitial tissue space, such as fibrin, indicating that the peptides will have access to all cellular types within the prostate tissue space, such as endothelial cells and vascular basement membrane.

[0014] The peptides of the invention include but are not limited to peptides comprising or consisting of amino acid sequences disclosed herein, fragments of the amino acid sequences, and modifications of peptides. In certain embodiments, peptides encompassed within the scope of the invention will function as agents that can bind to certain target tissues and/or cells. In certain embodiments, peptides of the invention will function to bind to tissue which comprises a human prostatic microenvironment. In certain

embodiments, a peptide of the invention will selectively or specifically bind to human prostatic microenvironment exposed by androgen deprivation. In embodiments, a peptide of the invention, including in embodiments a fusion protein comprising a peptide sequence of the invention, will form a complex with a human endothelial cell or vascular basement membrane. The vascular basement membrane is understood to those skilled in the art. In general, it is a thin sheet of fibers, i.e., a matrix, that underlies the epithelium, which lines the cavities and surfaces of organs, or the endothelium which lines the interior surface of blood vessels. It is expected that the peptides disclosed herein can be modified and still retain these desirable properties. In this regard, the invention includes peptides of various lengths, and with various amino acid substitutions. For example, the invention includes peptides which have been modified by conservative amino acid substitutions that are based generally on relative similarity of R- group substituents. Non- limiting examples of such substitutions contemplated in the present invention include, but are not limited to: gly or ser for ala; lys for arg; gin or his for asn; glu for asp; ser for cys; asn for gin; asp for glu; ala for gly; asn or gin for his; leu or val for ile; ile or val for leu; arg for lys; leu or tyr for met; thr for ser; tyr for trp; phe for tyr; and ile or leu for val. Thus, peptides that comprise any single conservative amino acid substitution, or any combination of conservative amino acid substitutions, are included in the invention. Any peptide provided by the invention can comprise one, two, three, four, or five conservative amino acid substitutions.

[0015] Non-conservative substitutions that enhance desirable characteristics of the peptides, such as their capability to bind to human epithelial cells and/or human prostatic microenvironment, activity, circulation time, bioavailability, stability, targeting, imaging properties, etc., are also included in the invention.

[0016] In addition to amino acid substitutions, peptides of the invention may include fragments of any of the peptides described herein. Any peptide encompassed within the scope of the invention can comprise or consist of from 4 - 20 amino acids, inclusive, and including all integers there between. In certain embodiments, the peptides are 12-mers.

[0017] To the extent that any of the peptides/fragment sequences per se have been previously described, the present disclosure includes their use in methods for tissue targeting, diagnostic imaging, therapeutic interventions, and as pharmaceutical preparations, and in fusion proteins, where the fusion protein is distinct from any protein wherein a sequence of a peptide disclosed herein was included.

[0018] Each peptide sequence disclosed in the Tables provided herein is included within the scope of the invention. Compositions comprising any one of such peptides, or any combinations or subcombinations of them, are included in the invention. Thus, the invention includes compositions which include all, or only include one or some of the peptides disclosed herein. In certain aspects, the invention includes at least one of the ten distinct peptide sequences having the consensus sequences, as depicted in Table 1. The peptides comprising the consensus sequences are: KPPQNTSAPYLP (SEQ ID NO:6);

TLQRTHFPPAFS (SEQ ID NO:9), TMGFTAPRFPHY (SEQ ID NO: 10 ),

SPHWQPNAIFVN (SEQ ID NO:8); and SPHWQPNAI (SEQ ID NO:19), which is a fragment of SEQ ID NO:8. In addition to these peptide sequences, the sequence

G AMHLPWHM GTL (SEQ ID NO:5) exhibited high recovery, as further described below and in Table 2. In one embodiment, the M in position 9 of SEQ ID NO:5 is replaced with I to give GAMHLPWHIGTL (SEQ ID NO: 101. In one embodiment, the peptide of SEQ ID NO:5 does not include the C-terminal L (GAMHLPWHMGT - SEQ ID NO:100). Each of these consensus sequences permits certain diversity in sequence but still retains function as indicated by their having been selected in vivo as described in further detail above.

[0019] It is also contemplated that the peptides of the present invention may include additional amino acids, and may include modified amino acids that can improve any desirable property of the peptides. Thus, the peptide sequences presented herein could be part of larger polypeptides or proteins, such as fusion proteins, or they could be connected to other moieties. Accordingly, the peptides could be covalently or non-covalently associated with any desirable moiety that would be expected to improve their functional capabilities in accordance with the method of the invention. In this regard, the peptides of the invention are expected to have utility in at least tissue targeting and/or diagnostic imaging applications and can therefore be modified for these purposes. In the case of fusion proteins, a peptide having a peptide sequence selected from any of the peptide sequences disclosed herein can be present in the fusion protein. Thus it will be apparent that a peptide described herein can include a peptide sequence within the context of a fusion protein. In embodiments, the peptides and the fusion protein do not comprise any bacteriophage coat protein amino acid sequence. Bacteriophage proteins are well known in the art. In embodiments, the peptides and fusion proteins do not comprise any pill bacteriophage. In embodiments, the peptides do not comprise any Ml , or fd filamentous phage, or T4 or T7, or λ phage protein. In embodiments In embodiments, the peptides and fusion proteins do not comprise any phagemid protein or protein that is encoded by a phagemid, other than sequence of the peptide.

[0020] In one embodiment, the peptides can be coupled with a chemotherapeutic agent, or any other agent that has cytotoxic activity. For example, chemotherapeutic agents useful in the generation of such peptide conjugates include enzymatically active toxins and fragments thereof. Suitable enzymatically active toxins include docetaxel, mitoxanthrone, taxanes, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes, or any anti-angiogenic agent(s). In embodiments, the chemotherapeutic agents comprise or consist of polypeptides having enzymatic activity. In embodiments, the peptide is present at the amino-terminal end of the fusion protein. In embodiments, the N-terminal amino acid of a peptide disclosed herein is the N-terminus of a fusion protein, although the peptide can be positioned elsewhere in the fusion protein if desired, such as at the C-terminus, or within the fusion protein.

[0021] Conjugates of the peptide and chemotherapeutic agents may be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyriyldithiol) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate, iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l ,5-difluoro-2,4- dinitrobenzene).

[0022] In another embodiment, the peptides can be conjugated to a radioactive agent.

A variety of radioactive isotopes are available for conjugating to peptides such that cells or tissues to which the peptides bind can be imaged or selectively destroyed. For selective destruction of cells the peptides can be conjugated to a highly radioactive atom, such as At ²¹¹ , I ¹³¹ , 1 ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² and radioactive isotopes of Lu. For identifying cells, such as prostate cancer cells, or cells in metastatic foci or in tumors, the peptide conjugates can include a radioactive atom for scintigraphic studies, for example Tc ^99m (metastable technetium- 99), I ¹²³ , or a spin label for nuclear magnetic resonance and/or

123 131 111 19 13 15 17

magnetic resonance imaging, such as I , 1 , In , F , C , N , O or Gadlinium (III) or Manganese (II). The radio-labels may be incorporated in the peptides in known ways.

[0023] The use of peptides for identification of targeted tissues/cells can be performed using any suitable technique, given the benefit of the present disclosure. In certain embodiments, labeled peptides can be injected into patients diagnosed with or suspected of having a disease, such as a cancer. Information from such imaging can be used for diagnosing or staging of the disease status of the patient. The label used can be selected in accordance with the imaging system to be used. For example, Indium ¹¹¹ , Technetium ⁹⁹ or Iodine ¹³¹ can be used for planar scans or single photon emission computed tomography (SPECT). Positron emitting labels such as Fluorine ¹⁹ can be used in positron emission tomography. Paramagnetic ions such as Gadlinium (III) or Manganese (II) can used in magnetic resonance imaging (MRI). Localization of the label within a particular tissue of the individual permits identification of locations which the peptides have targeted.

[0024] Therapeutic formulations comprising conjugated or unconjugated peptides of the invention may be prepared by mixing with pharmaceutically acceptable carriers, excipients or stabilizers in the form of lyophilized formulations or aqueous solutions.

Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter- ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non- ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). In certain embodiments, compositions of the invention can include distinct peptides, and can include mixtures of peptides, and can include linear (non-cyclic) and cyclic peptides.

[0025] Compositions comprising the peptides can include other therapeutic agents, such as conventional chemotherapeutic agents. Further, any suitable pharmaceutical delivery vehicle can be used to enhance delivery and/or efficacy of the peptides.

[0026] The peptides may be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In certain embodiments, the peptides are administered to an individual diagnosed with or suspected of having prostate cancer to inhibit metastasis or to inhibit the growth of the prostate cancer, or to patients with benign prostatic hypertrophy (hyperplasia) to inhibit growth of a benign lesion.

[0027] The peptides of the invention can be prepared by any technique known to those skilled in the art or by techniques hereafter developed. For example, the peptides can be prepared using the so lid -phase synthetic technique (Merrifield, J. Am. Chem. Soc, 15:2149-2154 (1963); M. Bodanszky et al., (1976) Peptide Synthesis, John Wiley & Sons, 2d Ed.; Kent and Clark- Lewis in Synthetic Peptides in Biology and Medicine, p. 295-358, eds. Alitalo, K., et al. Science Publishers, (Amsterdam, 1985). The synthesis of peptides by solution methods may also be used, as described in The Proteins, Vol. II, 3d Ed., p. 105-237, Neurath, H., et al., Eds., Academic Press, New York, N.Y. (1976). The synthesized peptides may be substantially purified by preparative high performance liquid chromatography or other comparable techniques available in the art. The composition of the synthetic peptides can be confirmed by an technique for amino acid composition analysis. The peptides can also be made using any suitable recombination system. Thus, in embodiments, the disclosure includes expression vectors encoding the peptide sequences, cells comprising the expression vectors, and methods of making the peptides comprising separating the peptides and/or fusion proteins comprising the peptide sequences from a recombinant expression system. In embodiments, the expression system is not a bacteriophage expression system. Every polynucleotide encoding the peptide sequences of this disclosure are encompassed herein, and in particular embodiments such polynucleotide sequences are provided in expression vectors.

[0028] In one embodiment, the invention provides a method of inhibiting angiogenesis and/or tumor growth and/or metastasis. The method comprises administering a composition comprising one or more of the peptides of the invention, wherein the peptides may or may not comprise a cytotoxic agent, to an individual in an amount effective to inhibit to inhibit tumor growth and/or inhibit metastasis. In another embodiment, the invention provides a method for imaging a tissue and/or one or more cells. The method comprises administering a composition comprising one or more labeled peptides of the invention and detecting localized labeled peptide via any suitable imaging apparatus or method

[0029] The following techniques for phage selection, identification and production of peptides were used.

[0030] Biological System: Male SCID mice were castrated and pre-implanted with silastic tubing to provide sustained-release of testosterone to maintain human serum levels testosterone in the host mouse and to facilitate castration/androgen deprivation (ADT). Human prostate tumor tissue was prepared for transplantation by cutting the radical prostatectomy surgical remnant in ViaSpan into approximately 1.0mm ³ to 2.0mm ³ wedges. Tissue was transplanted, into individual incisions in the skin; four pieces of tissue were transplanted on each flank of mice anesthetized with isoflurane. Each piece of tumor tissue was dipped into Matrigel, inserted into an incision, and the incisions closed with tissue glue.

[0031] Anastamosis of the human vasculature of the prostate cancer xenografts to the host mouse vascular system occurred between Days 5 and 6 after tissue transplantation, and the vasculature had stabilized by Day 21 after transplantation. Initiation of ADT by removal of the silastic tubing containing testosterone at a time point between Day 30 and Day 45 after transplantation with tissue resulted in acute apoptotic death of human endothelial cells in the microvasculature of the prostate cancer xenografts. The ADT-induced involution of human prostate endothelial cells produced a "therapeutic window" during which vascular leakage of serum components into the interstitial tissue space was demonstrated using systemically administered fluorescent lectin, leakage of MRI contrast dye (Gd-DTPA or Magna vist), or deposition of fibrin/fibrinogen.

[0032] Seventy-two hours after initiation of ADT, the host mouse was anesthetized with acepromazine/ketamine/xylazine and the peptide-phage display library introduced systemically by intra-cardiac injection into the right ventricle. The Ph.D. -12 phage library (NEB, Ipswich, MA) was injected at a concentration of 1.2xlO ^u pfu in 100 μί. The phage library was allowed to circulate through the mouse host for 15 minutes. At the conclusion of the incubation the vascular volume was replaced by perfusion (40 ml) with PBS to remove unbound phage. During perfusion viable xenografts that anastomosed to the host vasculature were observed to blanche. The xenografts were excised and placed into an eppendorf tube containing 0.5 mL phage elution buffer (0.2 M Glycine-HCl pH 2.2, 0.5% BSA) and the xenografts homogenized with a rotor/stator homogenizer on ice. The phage containing homogenate was cleared by centrifugation at 20,800 x G for 5 minutes, and neutralization buffer (Tris-HCl pH 9.1) added to the supernatant. The concentration of phage in the eluate was established by titration: serial 10-fold dilutions of the neutralized phage eluate (10 μΐ) were added to 200 μL· of a mid-log culture of host bacteria (ER2738), the cells/phage mixed with 3.5 mL of molten top agar (48 °C), and plated onto LB. Plates were incubated at 37 °C overnight and blue plaques were counted for determination of pfu. Based on the pfu, an aliquot of the neutralized phage eluate that contained a standardized number of phage was amplified by addition to a 20 mL flask of LB/Tet inoculated with 200 μL· of an overnight culture of ER2738, and the culture incubated for 4 hours. The culture was cleared of bacteria by centrifugation, the supernatant treated with 1/6 volume of PEG/NaCl (20% (w/v) PEG- 8000, 2.5M NaCl), refrigerated overnight for phage precipitation, and phage recovered by centrifugation. The final phage pellet was resuspended in 200 mL of TBS and titered (as described above). For all in vivo selections the input phage held constant at 1.2xlO ^u phage per mouse.

[0033] Identification of Candidate Peptides: Following the third sequential round of in vivo selection against tissue from a single patient, the recovered phage population was titered, diluted and plated at a concentration to yield approximately 100 plaques per plate. Individual plaques were picked and amplified for conventional DNA sequencing to verify that a diversity of peptides was selected. Subsequently, the region of the phage genome that contained the random peptides embedded in the pill protein were amplified by PCR from all selected phage libraries in preparation for Next Gen Sequencing. PCR primers were designed that flanked the hyper- variable region of the phage genome, the region containing the peptide inserted into the pill protein: (FWD: -3gIII: 5 '-CCT TTC TAT TCT CAC TCT GCT-3' (SEQ ID NO:98); REV: -155gIII: 5 '-CCG TAA CAC TGA GTT TCG TCA-3' (SEQ ID NO:99)). The amplified segments were the templates for the NextGen DNA sequencing process. Templates were quantified and concentrations normalized and underwent 454 Next Gen sequencing using 500 ng of amplified template DNA at a concentration of 50 ng/mL. Raw data from the NextGen sequencing were analyzed to identify the DNA base sequence of the hyper- variable region in both the forward and reverse compliment strands to assure that all selected peptides were identified. The DNA base sequence was translated, the amino termini of peptides aligned, and the final data set evaluated to quantitate the number of times each peptide was recovered in each library. The data sets for the individual patients were compared to identify peptides recovered in all, or the majority, of the individual prostate cancer tissue specimens.

[0034] Construction of Peptide Display Vectors (pMAL-pIII) : The NEB PhD phage phage display libraries were constructed by insertion of a random peptide library into a KpnI/EagI site directly adjacent to the 5' leader signal sequence of the pill gene in the Ml 3 phage. Therefore, the peptide is displayed as the amino-terminal end of the peptide-pIII fusion protein. NEB sells an expression vector (pMAL-PIII) that utilizes the same KpnI/EagI restriction sites the flank the peptide sequence in the phage to allow transfer of a peptide of interest from the phage genome into the expression vector. The peptide is expressied as the amino-terminus portion of a peptide/Maltose Binding Protein (MBP) fusion protein. The expression construct can produce milligrams of purified fusion protein from a 500 ml culture.

[0035] Oligonucleotide duplexes that contained candidate peptides of interest were designed to contain a 3 ' triple glycine spacer, KpnI/EagI restriction sites and additional random nucleotides capping each end of the sequence. The oligonucleotide duplexes were used to construct a peptide/Maltose Binding Protein fusion by insertion into the pMAL-PIII vector. The pMAL-PIII vector was linearized by digestion with Acc65I and Eagl-HF in NEBuffer3/BSA for 60 minutes. The linearized vector is gel purified, the duplex

oligonucleotides containing the candidate peptides were ligated into the pMAL-PIII vector, and the vector containing the fusion protein construct transformed into NEB Turbo

Competent E.coli . Plasmid DNA was obtained from colonies with a Qiagen plasmid mini- prep kit and the presence of the correct insert confirmed by conventional DNA sequencing.

[0036] pMAL Fusion-Peptide Expression and Affinity Purification: The fusion protein is expressed in the cytoplasm, where the pill leader sequence aids in the translocation of the protein into the periplasmic space for post-translational removal of the leader sequence. Osmotic shock of the bacteria was used to release selective the processed fusion-protein from the periplasmic space. The cell lysate containing the fusion protein was applied to amylose- agarose beads (resin) that reversibly binds Maltose binding protein, and after washing the fusion protein was competitively eluted with lOmM maltose.

[0037] The construct containing the peptide of interest was transformed into NEB

Express cells, a protease-deficient bacterial strain. Transformed cells were propagated in f LB/AMP (100 μg/mL) + glucose (2.0 g/L to suppress the maltose genes on the bacterial chromosome, one of which will degrade the amylase affinity resin. At the conclusion of the culture the cell pellet was resuspended in ice-cold 5mM MgS0 ₄ , the slurry shaken in an ice bath, and the cell debris removed by centrifugation. The supernatant, the cold osmotic shock fluid that contains the fusion protein, was stabilized by addition of protease inhibitors and EDTA, and refrigerated until purification of the peptide-maltose binding protein fusion by affinity chromatography using amylose resin. After loading and washing, the fusion protein was competed off the amylose affinity matrix with a 10 mM maltose buffer. Fractions containing protein were concentrated to approximately 2.0 mg/ml, and glycerol added to a final concentration of 50% (1.0 mg/mL final protein concentration).

[0038] Identification of Candidate Peptides Specific for Binding to ADT-

Damaged Prostate Microvascular Targets. An aliquot of each the phage libraries recovered after one, two and three rounds of selection in vivo for binding to ADT-perturbed human prostate cancer vasculature underwent NextGen Sequence analysis. The average number of individual DNA sequences that contained peptide inserts reported for each of the 15 selected phage populations was 13,568. The DNA base sequences were converted to amino acid sequences, and the individual populations evaluated for the number of times each peptide was recovered to evaluate for enrichment across the three rounds of in vivo selection, and to identify peptides recovered in common across multiple patient specimens. All peptides selected as candidates for targeting ADT-perturbed prostate cancer

microvasculature/tissue demonstrated progressive enrichment through the three sequential rounds of selection.

[0039] The peptides are expressed in the pill coat protein of the phage (5 copies per phage) as a random 12 mer (12 amino acids). The 5 '-end of the coding sequence for the random peptides starts at the conclusion of a leader sequence, which is clipped during expression on the phage coat. Therefore, the 5 '-end of the peptide is free in solution.

Consequently, the regions of conserved homology usually were located at the NITj-terminus of the peptide, with various amounts of sequence diversity (degeneracy) present in the 3 '-end of select peptides that share homology at the 5 ' -end. Table 1. Candidate Targeting Peptides

High-Priority Candidates Second Tier of Candidates

AETVESCLAKSH (SEQ ID NO:1 ) AELLKLFSKSHT (SEQ ID NO:11 )

APGTLPWASSNR (SEQ ID NO:2) VDAPLKSPLTAA (SEQ ID NO:12)

DAQSIYHFALAP (SEQ ID NO:3) AGNGTATATERT (SEQ ID NO:13)

DPKFPQNSHLIT (SEQ ID NO:4) HGLPVTTRGAFG (SEQ ID NO:14)

GAMHLPWHMGTL (SEQ ID NO:5) RLVNSSYLQLAS (SEQ ID NO:15)

KPPQNTSAPYLP (SEQ ID NO:6) SFRTLTDWNVTL (SEQ ID NO:16)

SHAHNSTTFLLA (SEQ ID NO:7) SPPPSHPSSGYL (SEQ ID NO:17)

SPHWQPNAIFVN (SEQ ID NO:8) THHHVTYWRSEA (SEQ ID NO:18)

TLQRTHFPPAFS (SEQ ID NO:9)

TMGFTAPRFPHY (SEQ ID NO:10)

[0040] Ten consensus peptides were identified as "high priority" candidates, and eight peptides were selected as "second tier" candidates. The peptides are listed in Table 1. The use of the terms "high priority" and "second tier" are used herein to reflect rank of interest and proposed order of study and are not meant to diminish the importance of second tier peptides which are encompassed within the scope of this disclosure.

[0041] The parental library also was subjected to NextGen sequence analysis. The ten "high priority" candidate peptides were distributed randomly among the 10 ⁹ peptides present in the parental phage library: none of the selected peptides were enriched relative to all of the random peptides displayed in the library. However, the ten "high-priority" candidate peptides accounted for -66% of the peptides recovered in the DNA sequence data sets after three rounds of selection in vivo for the two individual prostate cancer tissue specimens (Table 2), suggesting high levels of enrichment for these peptides as candidates capable of targeting specific epitopes unmasked or created by ADT.

[0042] For the high-priority candidates that were represented most frequently in the recovered peptides across all rounds of selection, not only was the consensus peptide recovered (the specific peptide recovered with the highest frequency), but multiple derivatives with small changes in the consensus sequence also were recovered. Table 3 presents a numerical tabulation of the recovery patterns from the individual in vivo selections after combining the data from the third round of selection for the two tumor specimens. The recovery of multiple degenerative derivatives of the consensus peptide provides evidence that the core sequence was selected specifically, and helps define the minimal core sequence necessary for maintenance of high- affinity binding to "epitopes" in the prostate cancer microvasculature and interstitial tissue. Table 2. Recovery of Candidate Peptides

Consensus Sequence Percent of Total Phage After 3 Cycles of Selection

CaP Tissue Specimen 1 CaP Tissue Specimen 2

TLQRTHFPPAFS (SEQ 9.58% 7.57%

ID NO:9)

TMGFTAPRFPHY (SEQ 26.5% 4.78%

ID NO:10

SPHWQPNAI (SEQ ID 3.29% 1 .53%

NO:19)

AETVESCLAKSH (SEQ 0.99% 0.5%

ID NO:1 )

KPPQTNTSAPYLP (SEQ 27.3% 5.48%

ID NO:20)

DQHGFMHIFDL (SEQ ID 0.4% 0.1 %

NO:21 )

GAMHLPWHMGT (SEQ 0.21 % 46.3%

ID NO:100)

DPKFPQNSHLIT (SEQ ID 0.63% 0.35%

NO:4)

HSAQPVKSKAWL (SEQ 0.3% 0.2%

ID NO:22)

LLADTTHHRWT (SEQ ID 0.1 % 0.1 %

NO:23)

onsensus equence = sec uence n

[0043] The "second-tier" candidates were selected from a group of peptides that were selected in vivo from all the CaP specimens. However, for these peptides, related or degenerate peptides with a common/shared consensus region, but with areas of divergence, were not observed (Table 4). Furthermore, none of these peptides represented more 1.2% of the total peptides recovered after the third round of in vivo selection. The eight candidate peptides were selected arbitrarily from this group because they represented the most frequently observed of this group of candidates (highlighted by shading).

κ P H F L N Q s G S Y I SEQIDNO:87

L P Y R T T H I G P L P SEQIDNO:88

L Q S T S P A Y T H R M SEQIDNO:89

N H A V N K W T L S H A SEQIDNO:90

Q P R P T Q Y Q E H S A SEQIDNO:91

Q L T G P T G M K T P L SEQIDNO:92 u 1. V N S s Y 1. Q 1. Λ S SIX) II) NO:l ^r , s 1' 1' 1' S H 1' s S t; Y 1. SIX) II) N0:|- s F R T L T D w N V T L SEQIDN0:16 s H A H N s T T F L/C L/W A/R SEQIDNO:93 s T A G P M F P Q Q S R SEQIDN0:94

T II n II V T Y w K s i: Λ SKQ II) NO:IH

T H P G s s M R H T H H SEQIDNO:95

T H s H G L K P S E Y H SEQIDN0:96

V 1) Λ 1' 1. K S 1' 1. T Λ Λ S|-:Q II) N():I2

V M R T D N Y A P N T L SEQIDNO:97

[0044] Production of Fusion Protein and Peptide-Dis laying Phage Reagents to

Validate in vivo Targeting of ADT-Damaged Prostate Microvasculature. The format in which the peptide is displayed, as a free peptide chain anchored at the 3 '-terminus on the amino-terminus of the pill protein on the Ml 3 phage coat, suggests that peptides identified in this protocol are immediately applicable to targeting imaging/therapeutic agents to ADT- damaged prostate microvasculature after covalent attachment to platelets, or more importantly, conjugation to gold-nanoparticles that have been chemically derivatized to provide receptor sites to maximize covalent binding of targeting moieties. Table 5 summarizes t production of stocks of Ml 3 phage displaying the individual peptides, and the production and isolation of stocks of fusion protein (peptide fused to maltose binding protein). All of the peptides have been cloned into a shuttle vector, and subsequently recloned into the expression vector with the maltose binding fusion protein (pMAL-PIII) and into the phage genome (M13KE). The success of cloning into both vectors was verified by DNA sequencing. Some of the phage stocks expressing individual peptides have been expanded, clones picked and sequence verified, and the clonal phage population expanded to a sufficient level that phage were frozen. Eight of the expression constructs for the fusion protein have been expanded in bacterial cultures and the fusion protein isolated using the specific affinity column (yields listed in table). The protein yields are greater than expected. Several of the remaining expression vectors have been propagated large scale cultures and can be readily produced. Table 5: PEPTIDES SPECIFIC FOR IN VIVO TARGETS DURING THE "THERAPEUTIC WINDOW"

Sequence Sequence Phage Protein

Duplex Duplex Verified Verified Amplification Expressed

Formed Digested in pMAL Vector in M13KE Vector (ready for staining)

AELLKLFSKSHT

(SEQ ID NO:ll) X X X X X 3 mg

AGNGTATATERT

(SEQ ID NO:13) X X X X X 5.0 MG

APGTLPWASSNR

(SEQ ID NO:2) X X X X X 8.5 mg

DAQSIYHFALAP

(SEQ ID NO:3) X X X X X 6.9 mg

DPKFPQNSHLIT

(SEQ ID NO:4) X X X X X 3.7 MG

GAMHLPWHMG TL (SEQ ID NO:5) X X X X 6.7MG

HGLPVTTRGAFG

(SEQ ID NO:14) X X X 3.9 MG

KPPQNTSAPYLP

(SEQ ID NO:6) X X X X XX 3.6 mg

RLVNSSYLQLAS

(SEQ ID NO:15) X X X X X 2.6mg

SFRTLTDWNVTL

(SEQ ID NO:16) X X X X X 4.1 MG

SHAHNSTTFLLA

(SEQ ID NO:7) X X X X X 6.2 MG

SPHWQPNAIFVN

(SEQ ID NO:8) X X X X X 5.8 MG

SPPPSHPSSGYL

(SEQ ID N0:17) X X X X X 11.7 mg

THHHVTYWRSEA

(SEQ ID N0:18) X X X X X 3.1 MG

TLQRTHFPPAFS

(SEQ ID N0:9) X X X X X 10.5 mg

TMGFTAPRFPHY

(SEQ ID N0:10) X X X X X 6.4 MG

VDAPLKSPLTAA

(SEQ ID N0:12) X X X X X 7 MG

X - Finished

[0045] Those skilled in the art will recognize from the foregoing that the present disclosure includes a variety of peptides which will have specific utility for targeting specific binding partners that are present during the transient therapeutic window following androgen deprivation. It will also be recognized that the foregoing description of specific embodiments is for the purpose of illustration and is not to be construed as restrictive. From the teachings of the present invention, those skilled in the art will recognize that various modifications and changes may be made without departing from the spirit of the invention.