WO2014074907A1

WO2014074907A1 - C-met-specific capture agents, compositions, and methods of using and making

Info

Publication number: WO2014074907A1
Application number: PCT/US2013/069282
Authority: WO
Inventors: Heather Dawn AGNEW; Suresh Mark Pitram; Bert Tsunyin LAI; Rosemary Dyane ROHDE; Paul Edward Kearney
Original assignee: Indi Molecular, Inc.
Priority date: 2012-11-09
Filing date: 2013-11-08
Publication date: 2014-05-15

Abstract

The present application provides stable peptide-based c-MET capture agents and the use thereof as detection, diagnosis, and treatment agents. The application further provides novel methods of developing stable peptide-based capture agents, including c-MET capture agents, using iterative on-bead in situ click chemistry.

Description

C-MET-SPECIFIC CAPTURE AGENTS, COMPOSITIONS, AND METHODS

OF USING AND MAKING

BACKGROUND

[0001] The early detection of diseases including cancer requires multiplex measurements of key protein biomarkers in biological samples. The availability of high-affinity, highly selective molecular moieties that recognize biomarkers from complex biological mixtures is a critical component for accurate detection of proteins that may indicate disease.

[0002] The c-Met gene is a proto-oncogene that encodes the protein hepatocyte growth factor receptor (HGFR, or c-MET). Activation of c-MET by HGF induces kinase activity in c-MET. Interrupting the activation of c-MET by HGF slows tumor progression in animal models. In addition to stimulating proliferation of certain cancer cells through activation of c-Met, HGF also protects against DNA-damaging agent-induced cytotoxicity in a variety of cell lines susceptible to hyperproliferative phenotypes. Therefore, preventing HGF from binding to c-Met could predispose certain cancer cells to the cytotoxicity of certain drugs. c-MET overexpression and/or hyperactivation is implicated in tumor growth, angiogenesis and metastasis.

Therefore, c-MET provides an attractive target as a biomarker for specific cancer types, as well as a potential therapeutic. Most current biomarker assays utilize antibodies. It is challenging to produce stable antibodies for complex targets. Thus, there is a need in the art for synthetic, stable capture agents that can be used reproducibly and effectively in bioassays and as a therapeutic treatment.

SUMMARY

[0003] Provided herein are stable, synthetic capture agent that specifically binds c-Met, wherein the capture agent comprises a designed anchor ligand, a designed secondary ligand, optionally, a designed tertiary ligand, and optionally a designed quarternary ligand and wherein the ligands selectively bind c- Met. In certain embodiments, the capture agents provided herein bind to a non-ATP binding site of c-MET. In one embodiment, the binding of said capture agent to c- Met inhibits c-Met activity. In yet another embodiment, the capture agents bind to a ligand binding site of c-MET. In still another embodiment, the capture agent inhibits binding of hepatocyte growth factor (HGF) to c-Met.

Anchor Ligand

[0004] In one embodiment of the capture agent, the anchor ligand comprises an amino acid sequence 80 or 1 00% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:1 -27. In a particular embodiment, the anchor ligand comprises an amino acid sequence 80 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:3, 4, 5, 9, 1 1 , 1 2, 13, 16, 19, 22, 24, 25 and 26.

[0005] In some embodiments of the capture agent, the anchor ligand comprises an amino acid sequence of trwX1 X2, wherein X1 and/or X2 are independently any D-amino acid or glycine or not present. In a particular embodiment, X1 is selected from the group consisting of D-alanine, D-valine, D- leucine, D-isoleucine, D-proline, glycine and not present. In a more particular embodiment, X1 is selected from the group consisting of D-valine, D-leucine, and D- isoleucine. In another particular embodiment, X2 is selected from the group consisting of D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, glycine, D- histidine, D-lysine, D-arginine and not present. In a more particular embodiment, X2 is selected from the group consisting of D-isoleucine, D-proline and D-arginine.

[0006] In another embodiment of the capture agent, the anchor ligand comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 , 2 and 3. In a particular embodiment, the anchor ligand comprises an amino acid sequence of SEQ ID NO 3.

[0007] In yet another embodiment of the capture agent, the anchor ligand comprises an amino acid sequence of tX3dll, wherein X3 is independently any D- amino acid or glycine or not present. In a particular embodiment, X3 is selected from the group consisting of D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, glycine, D-histidine, D-lysine, D-arginine, D-serine, D-threonine, D-asparagine, D- glutamine and not present. In a more particular embodiment, X3 is selected from the group consisting of D-leucine and D-asparagine. [0008] In certain embodiments of the capture agent, the anchor ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs: 4, 5 and 13.

[0009] In one embodiment of the capture agent, the anchor ligand comprises the peptide sequence trwlr.

[0010] In another embodiment of the capture agent, the anchor ligand comprises the peptide sequence trwlr- Az4-CONH₂ or irnwk-Az4-CONH₂.

Secondary Ligand

[0011] In one embodiment of the capture agent, the secondary ligand comprises an amino acid sequence 80 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 28-95. In another embodiment, the secondary ligand comprises an amino acid sequence 80 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 , 35, 37, 42, 46, 48, 49, 50, 52, 54, 55, 58, 59, 66, 69, 71 , 76, 85, 86 and 92.

[0012] In some embodiments of the capture agent, the secondary ligand comprises an amino acid sequence of X4krhG, wherein X4 is independently any D- amino acid or glycine or not present. In a particular embodiment, X4 is selected from the group consisting D-phenylalanine, D-tryptophan, D-tyrosine, of D-alanine, D- valine, D-leucine, D-isoleucine, D-proline, glycine and not present. In a more particular embodiment, X4 is selected from the group consisting of D-phenylalanine, D-isoleucine and D-proline.

[0013] In certain embodiments, the secondary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 29, 30 and 31 . In a particular embodiment, the secondary ligand comprises an amino acid sequence of SEQ ID NO 31 .

[0014] In some embodiments of the capture agent, the secondary ligand comprises an amino acid sequence of X5hGX6p, wherein X5 is independently any D-amino acid or glycine or not present. In a particular embodiment, X5 is selected from the group consisting of D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, glycine, D-serine, D-threonine, D-asparagine, D-glutamine and not present. In a more particular embodiment, X5 is selected from the group consisting of D-alanine, D-proline, D-leucine, D-isoleucine and D-asparagine. [0015] In some embodiments of the capture agent, X6 is selected from the group consisting of D-phenylalanine, D-tryptophan, D-tyrosine, D-aspartate, D- glutamate, D-histidine, D-lysine, D-arginine and not present. In a particular embodiment, X6 is selected from the group consisting of D-phenylalanine, D- glutamate and D-lysine.

[0016] In certain embodiments, the secondary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 43-47.

[0017] In some embodiments of the capture agent, the secondary ligand comprises an amino acid sequence of X7X8rhG, wherein X7 is independently any D- amino acid or glycine or not present. In a particular embodiment, X7 is selected from the group consisting of D-phenylalanine, D-tryptophan, D-tyrosine, D-alanine, D- valine, D-leucine, D-isoleucine, D-proline, glycine, D-serine, D-threonine, D- asparagine, D-glutamine, D-histidine, D-lysine, D-arginine and not present. In a more particular embodiment, X7 is selected from the group consisting of D- phenylalanine, D-isoleucine, D-proline, D-leucine, glycine, D-valine, D-threonine, D- asparagine and D-lysine.

[0018] In some embodiments of the capture agent, X8 is independently any

D-amino acid or glycine or not present. In a particular embodiment, X8 is selected from the group consisting of D-phenylalanine, D-tryptophan, D-tyrosine, D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, glycine, D-aspartate, D-glutamate, D- histidine, D-lysine, D-arginine and not present. In a more particular embodiment, X8 is selected from the group consisting of D-phenylalanine, D-lysine, D-glutamate, D- histidine, D-valine and D-tryptophan.

[0019] In certain embodiments, the secondary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 28-37. In a particular embodiment, the secondary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 31 , 35, 37 and 52.

[0020] In one embodiment of the capture agent, the secondary ligand comprises the peptide sequence kvrhG.

[0021] In another embodiment of the capture agent, the secondary ligand comprises a peptide sequence selected from the group consisting of (D-Pra)- kvrhG-CONH₂, (D-Pra)-pkrhG-CONH₂, (D-Pra)-efwhG-CONH₂, (D-Pra)-hvwhG- CONH₂ and (D-Pra)-skrhe-CONH₂.

Tertiary Liqand

[0022] In certain embodiments of the capture agent, the tertiary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 96-144, 170 and 171 . In a particular embodiment, the tertiary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 96, 97, 100, 1 02, 1 03, 104, 106, 107, 1 14, 1 22, 124, 128, 132, 1 39, 141 , 144, 170 and 171 . In a more particular embodiment, the tertiary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 96, 97, 100, 103, 104, 1 06, 1 14, 141 , 170 and 171 .

[0023] In some embodiments of the capture agent, the tertiary ligand comprises an amino acid sequence of X9swwr, wherein X9 is independently any D- amino acid or glycine or not present. In a particular embodiment, X9 is selected from the group consisting of D-phenylalanine, D-tryptophan, D-tyrosine, D-alanine, D- valine, D-leucine, D-isoleucine, D-proline, glycine and not present. In a more particular embodiment, X9 is selected from the group consisting of D-phenylalanine, D-proline and D-tyrosine. In certain embodiments, the tertiary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 96 and 97.

[0024] In some embodiments of the capture agent, the tertiary ligand comprises an amino acid sequence of fpfXI Or, wherein X10 is independently any D- amino acid or glycine or not present. In a particular embodiment, X1 0 is selected from the group consisting of D-alanine, D-valine, D-leucine, D-isoleucine, D-proline, glycine and not present. In a more particular embodiment, X10 is selected from the group consisting of D-leucine and D-isoleucine.

[0025] In some embodiments of the capture agent, the tertiary ligand comprises an amino acid sequence of X1 1 wrqw, wherein X1 1 is independently any D-amino acid or glycine or not present. In a particular embodiment, X1 1 is selected from the group consisting D-asparagine, D-glutamine, D-serine, D-threonine and not present. In a more particular embodiment, X1 1 is selected from the group consisting of consisting of D-asparagine and D-glutamine. [0026] In some embodiments of the capture agent, the tertiary ligand comprises an amino acid sequence of X12wwlr, wherein X12 is independently any D-amino acid or glycine or not present. In a particular embodiment, X12 is selected from the group consisting of D-threonine, D-serine, D-asparagine, D-glutamine, D- lysine, D-histidine, D-arginine, D-tryptophan, D-tyrosine, D-phenylalanine and not present. In a more particular embodiment, X12 is selected from the group consisting of D-threonine, D-serine, D-lysine and D-tryptophan.

Quarternary Ligand

[0027] In certain embodiments of the capture agent, the quarternary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 145-169, and 172. In a particular embodiment, the quarternary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 160, 1 61 , 167 and 169.

[0028] In some embodiments, of the capture agent, anchor ligand comprises the peptide sequence trwlr or irnwk. In other embodiments, the secondary ligand comprises a peptide sequence selected from the group consisting of kvrhG, pkrhG, efwhG, hvwhG and skrhe. In other embodiments, the tertiary ligand comprises a peptide sequence selected from the group consisting of pswwr, fswwr, fpflr, wqwlr, pwrqw, Iwrqw, wkkdr, and kwwlr. In other embodiments, the quarternary ligand comprises a peptide sequence selected from the group consisting of shirt, kGfkf, rkekw and rnpwk.

[0029] In another embodiment of the capture agent, the tertiary ligand comprises a peptide sequence selected from the group consisting of (D-Pra)-pswwr- CONH₂, (D-Pra)-fswwr-CONH₂, (D-Pra)-fpflr-CONH₂, (D-Pra)-wqwlr-CONH₂, (D- Pra)-pwrqw-CONH₂, (D-Pra)-lwrqw-CONH₂, (D-Pra)-wkkdr-CONH₂, and (D-Pra)- kwwlr-CONH₂.

Triazole Linkage

[0030] In one embodiments of the capture agent, the anchor ligand and secondary ligand are linked together via a 1 ,4-substituted-1 ,2,3-triazole residue (Tz4). In another embodiment, the secondary ligand and the tertiary ligand are linked together via a 1 ,4-substituted-1 ,2,3-triazole residue (Tz4). In yet another embodiment, the tertiary ligand and the quarternary ligand are linked together via a 1 ,4-substituted-1 ,2,3-triazole residue (Tz4). In yet another embodiment, the anchor ligand and secondary ligand are linked together via a 1 ,4-substituted-1 ,2,3-triazole residue, and the secondary ligand and the tertiary ligand are linked together via a 1 ,4-substituted-1 ,2,3-triazole residue. In yet another embodiment, the anchor ligand and secondary ligand are linked together via a 1 ,4-substituted-1 ,2,3-triazole residue, the secondary ligand and the tertiary ligand are linked together via a 1 ,4-substituted- 1 ,2,3-triazole residue and the tertiary ligand and the quarternary ligand are linked together via a 1 ,4-substituted-1 ,2,3-triazole residue.

Bilaqands, Triliqands and Tetraliqands

[0031] In certain embodiments, the c-MET capture agents provided herein have structures selected from the group consisting of:





10

11

wherein X is selected from the group consisting of CH₃, biotin-PEG3, biotin, aminooxyacetate, aminooxyacetate-PEG3, ¹⁹FB, ¹⁸FB, ¹⁹FB-PEG3, ¹⁸FB-PEG3 and FITC-PEG3.

Properties

[0032] In certain embodiments, the c-MET capture agents provided herein are stable across a wide range of temperatures, pH's, storage times, storage conditions, and reaction conditions, and in certain embodiments the capture agents are more stable than a comparable antibody or biologic. In certain embodiments, the capture agents are stable in storage as a lyophilized powder. In certain embodiment, the capture agents are stable in storage at a temperature of about -80⁵C to about 40⁵C. In certain embodiments, the capture agents are stable at room temperature. In certain embodiments, the capture agents are stable in human serum for at least 24 hours. In certain embodiments, the capture agents are stable at a pH in the range of about 3 to about 12.

Detectable Labels [0033] In some embodiments, the capture agent is labeled with a label selected from the group consisting of biotin, copper-DOTA, biotin-PEG3, aminooxyacetate, ¹⁹FB, ¹⁸FB and FITC-PEG3. In other embodiments, the capture agent is labeled with the detectable moiety consisting of ⁶⁴Cu DOTA, ⁶⁸Ga DOTA, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, ¹²⁴l, ⁸⁶Y, ^94mTc, ^110mln, ¹¹C and ⁷⁶Br. In other embodiments, the label is a fluorescent label. In a particular embodiment, the detectable label is ¹⁸F.

Methods and Uses

[0034] Provided herein is a method of inhibiting c-Met signaling in a subject comprising administering to the subject a capture agent as described herein. In certain embodiments, methods are provided for inhibiting c-MET activity in vivo or in vitro using a c-MET capture agent as provided herein. In certain embodiments, the c-MET capture agents inhibit c-MET activity by modulating or inhibiting the binding of an endogenous ligand to c-MET. In a particular embodiment, the endogenous ligand is HGF. In certain embodiments, inhibition of c-MET activity results in an effective decrease in c-MET levels and/or a change in c-MET

conformation.

[0035] Also provided herein is the use of a capture agent as described herein as a detection agent for detecting c-MET in a biological sample.

[0036] Also provided herein is a method of detecting c-Met in a biological sample using an immunoassay, wherein the immunoassay utilizes a capture agent as described herein, and wherein said capture agent replaces an antibody or its equivalent in the immunoassay. In certain embodiments, methods are provided for identifying, detecting, quantifying, or separating c-MET in a biological sample using the capture agents as described herein. In one embodiment of the method, the immunoassay is selected from the group of Western blot, pull-down assay, dot blot, and ELISA.

[0037] Also provided herein is a method of treating a condition associated with increased c-Met expression and/or activity in a subject in need thereof, comprising administering a therapeutically effective amount of a capture agent as described herein.

[0038] In certain embodiments of the method, said condition is selected from the group consisting of multiple myeloma, chronic myelogenous leukemia, pancreatic cancer, non-small cell lung cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, prostate cancer, malignant melanoma, non-melanoma skin cancers, gastrointestinal stromal tumors, hematologic tumors, hematologic

malignancies, childhood leukemia, childhood lymphomas, multiple myeloma,

Hodgkin's disease, lymphomas of lymphocytic origin, lymphomas of cutaneous origin, acute leukemia, chronic leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm and cancers associated with AIDS. In a particular embodiment, the cancer is selected from the group consisting of lung, breast and prostate cancer. In a particular embodiment, said condition is selected from the group consisting of lung cancer, breast cancer and prostate cancer. In a more particular embodiment, said condition is prostate cancer. In certain embodiments, the c-MET capture agents provided herein function as immunotherapeutics.

[0039] Also provided herein is a method of diagnosing a c-Met expressing cancer in a human or mouse subject, the method comprising the steps of: a) administering to the subject the c-Met capture agent, as described herein, linked to a detectable moiety; and b) detecting the moiety linked to the c-MET capture agent in the subject; wherein detection of the moiety diagnoses a c-Met -expressing cancer in the subject.

[0040] Also provided herein is a method of detecting c-MET in a sample comprising a) exposing the sample to the c-MET capture agent, as described herein, linked to a detectable moiety; and b) detecting the moiety linked to the c-MET capture agent in the subject; wherein detection of the moiety diagnoses a c-MET- expressing cancer in the subject.

[0041] Also provided herein is a method of monitoring treatment of a subject receiving c-Met-directed therapy comprising administering to the patient a small- molecule positron-emission-tomography ligand (PET ligand) that is bound to the c- Met capture agent, as described herein, on or near a c-Met-expressing cancer in the subject. In some embodiments, the cancer is selected from the group consisting of multiple myeloma, chronic myelogenous leukemia, pancreatic cancer, non-small cell lung cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, prostate cancer, malignant melanoma, non-melanoma skin cancers, gastrointestinal stromal tumors, hematologic tumors, hematologic malignancies, childhood leukemia, childhood lymphomas, multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic origin, lymphomas of cutaneous origin, acute leukemia, chronic leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm and cancers associated with AIDS. In a particular embodiment, the cancer is selected from the group consisting of lung, breast and prostate cancer.

[0042] Also provided herein is the use of one or more c-MET capture agents is provided for use in preparing a medicament for treating a condition associated with increased c-MET expression and/or activity in a subject in need thereof.

Kits

[0043] In certain embodiments, kits are provided that comprise one or more of the c-MET capture agents provided herein. In certain of these embodiments, the kits include instructions for use.

Synthesis of Capture Agents

[0044] In certain embodiments, methods are provided for synthesizing the c-MET capture agents disclosed herein.

[0045] In certain embodiments, methods are provided for generating a capture agent for a target protein. In certain embodiments, the target protein is a kinase, and in certain of these embodiments the kinase is c-MET. In certain embodiments, these methods comprise the following steps:

(a) identifying an anchor ligand by the following steps:

(i) preparing a synthetic target polypeptide corresponding to an epitope of the target protein;

(ii) preparing a first plurality of candidate peptides to screen against the target polypeptide;

(iii) contacting the target polypeptide with the first plurality of candidate peptides;

(iv) selecting a candidate peptide with affinity for the target polypeptide as the anchor ligand, wherein the candidate peptide binds to the target polypeptide; and (v) sequencing the anchor ligand;

(b) identifying a secondary ligand by the following steps:

(i) preparing an anchor ligand selection block comprising the anchor ligand and an azido group or an alkynyl group;

(ii) preparing a second plurality of candidate peptides to select a

secondary ligand for the target protein, the second plurality of peptides comprising an azido group or an alkynyl group if the anchor ligand selection block comprises an alkynyl group and azido group respectively;

(iii) contacting the anchor ligand selection block and the second plurality of peptides with the target protein;

(iv) providing a capture agent biligand by forming a disubstituted 1 ,2,3- triazole linkage between the anchor ligand selection block and the secondary ligand wherein the azido and alkynyl group of the anchor ligand selection block and the secondary ligand are brought in close proximity by binding to the target protein;

(v) selecting the capture agent biligand that has an affinity with the target protein; and

(vi) sequencing the secondary ligand;

(c) identifying a tertiary ligand by the following steps:

(i) preparing a biligand selection block comprising an azido group or an alkynyl group; and

(ii) repeating steps (b)(ii) to (b)(vi) using a third plurality, fourth plurality, etc., of candidate peptides until a capture agent having desired binding affinity to the target protein is obtained;

(d) identifying a quarternary ligand and, optionally, additional ligands by the following steps:

(i) preparing a triligand selection block comprising an azido group or an alkynyl group; and (ii) repeating steps (c)(ii) to (c)(vi) using a fourth plurality, fifth plurality, etc., of candidate peptides until a capture agent having desired binding affinity to the target protein is obtained.

[0046] In certain embodiments, the active site is a substrate peptide binding site.

[0047] The disclosure also provides a multiplex capture agent comprising a mixture of capture agents that binds specifically to two or more of c-Met, PSMA, and MUC1 . In certain embodiments, the capture agent binds to all three of c- Met, PSMA, and MUC1 . In other embodiments, the capture agent comprises a designed anchor ligand, a designed secondary ligand, optionally, a designed tertiary ligand and optionally, a designed quarternary ligand.

[0048] The disclosure also provides a method of treating a disease comprising administering an effective amount of the multiplex capture agent described above to a subject in need thereof.

[0049] The disclosure also provides a method of diagnosing a disease comprising a) administering to the subject the multiplex capture agent of described above linked to a detectable moiety; and b) detecting the moiety linked to the multiplex capture agent in the subject; wherein detection of the moiety diagnoses a disease in the subject.

[0050] In either of these methods, the disease can be cancer. In certain embodiments, the cancer is selected from the group consisting of multiple myeloma, chronic myelogenous leukemia, pancreatic cancer, non-small cell lung cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, prostate cancer, malignant melanoma, non-melanoma skin cancers, gastrointestinal stromal tumors, hematologic tumors, hematologic malignancies, childhood leukemia, childhood lymphomas, multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic origin, lymphomas of cutaneous origin, acute leukemia, chronic leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm and cancers associated with AIDS. In a particular embodiment, the cancer is selected from the group consisting of lung, breast and prostate cancer. [0051] The disclosure also provides an agent comprising a first and a second capture agent, wherein the first capture agent specifically binds one of c-Met, PSMA, fPSA and MUC1 and the second capture agent specifically binds one of c- Met, PSMA, fPSA and MUC1 , wherein the first and the second capture agents bind distinct proteins. In certain embodiments, the agent further comprises a third capture agent, wherein the third capture agent specifically binds one of c-Met, PSMA, fPSA and MUC1 , wherein the first, the second and the third capture agents bind distinct proteins. In other embodiments, the agent further comprises a fourth capture agent, wherein the fourth capture agent specifically binds one of c-Met, PSMA, fPSA and MUC1 , wherein the first, the second, the third and the fourth capture agents bind distinct proteins.

[0052] The disclosure also provides a method of treating a disease comprising administering an effective amount of the multiplex capture agent described above to a subject in need thereof.

[0053] The disclosure also provides a method of diagnosing a disease comprising a) administering to the subject the multiplex capture agent of described above linked to a detectable moiety; and b) detecting the moiety linked to the multiplex capture agent in the subject; wherein detection of the moiety diagnoses a disease in the subject.

[0054] In either of these methods, the disease can be cancer. In certain embodiments, the cancer is selected from the group consisting of multiple myeloma, chronic myelogenous leukemia, pancreatic cancer, non-small cell lung cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, prostate cancer, malignant melanoma, non-melanoma skin cancers, gastrointestinal stromal tumors, hematologic tumors, hematologic malignancies, childhood leukemia, childhood lymphomas, multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic origin, lymphomas of cutaneous origin, acute leukemia, chronic leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm and cancers associated with AIDS. In a particular embodiment, the cancer is selected from the group consisting of lung, breast and prostate cancer. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 : The crystal structure of human c-MET in complex with HGF (1 SHY) (Example 1 ).

[0056] Figure 2: The in vitro blocking potential of Polypeptides 1 and 2 (Example 2).

[0057] Figure 3: MALDI-MS and MS/MS analysis of anchor peptides cleaved from single beads (Example 5). Each peptide is labeled with its SEQ ID NO.

[0058] Figure 4: Bioinformatic clustering analysis of anchor candidates (Example 5).

[0059] Figure 5: Results of the pull-down assay for DAnchors 1 to 8.

[0060] Figure 6: Sequence homology-based selection of biligand candidates

(Example 8). Each peptide is labeled with its SEQ ID NO.

[0061] Figure 7: Bioinformatics-based selection of biligand candidates (Example 8).

[0062] Figure 8: Competitive binding assay for DAnchorl (trwlr) and Biligands 1 (kvrhG) and 3 (pkrhG) demonstrates that biligands attenuate the binding of c-MET to HGF (Example 1 0).

[0063] Figure 9: Affinity of DAnchorl (trwlr) and Biligands 1 and 3 (Example 1 1 ).

[0064] Figure 1 0: Results of the pull-down assay for Biligands 1 to 3 (Example 12).

[0065] Figure 1 1 : Results of the pull-down assay for Biligands 4 to 6 (Example 12).

[0066] Figure 1 2: Results of the pull-down assay for Biligands 7 to 9 (Example 12).

[0067] Figure 1 3: Results of the pull-down assay for Biligands 10 to 13 (Example

12) .

[0068] Figure 14: Sequence homology-based selection of triligand candidates (Example 13). Each peptide is labeled with its SEQ ID NO.

[0069] Figure 1 5: Bioinformatics-based selection of triligand candidates (Example

13) .

[0070] Figure 1 6: Results of the pull-down assay for Triligands 1 to 3 (Example 15).

[0071] Figure 1 7: Results of the pull-down assay for Triligands 4 to 7 (Example 15).

[0072] Figure 1 8: Results of the pull-down assay for Triligands 8 to 1 1 (Example

15). [0073] Figure 1 9: Radiosynthesis of 4-[¹⁸F] fluorobenzaldehyde and conjugation to aminooxy-modified PCC peptides (Example 16).

[0074] Figure 20: Fluorescence imaging with confocal microscope (Example 1 ^'

[0075] Figure 21 : Fluorescence intensities quantitated using ImageJ (Example

17.1 ).

[0076] Figure 22: RIMChip/betabox experiments (Example 17.2).

[0077] Figure 23: Structure of azido amino acid Az4.

[0078] Figure 24: Results of the pull-down assay for Anchor, Biligands and

Triligands

[0079] Figure 25: Biligand 1 : X-trwlr-Tz4-kvrhG.

[0080] Figure 26: Biligand 1 -Az4: X-trwlr-Tz4-kvrhG-Az4.

[0081] Figure 27: Triligand 1 : X-trwlr-Tz4-kvrhG-Tz4-pswwr.

[0082] Figure 28: Triligand 2: X-trwlr-Tz4-kvrhG-Tz4-fswwr.

[0083] Figure 29: Triligand 3: X-trwlr-Tz4-kvrhG-Tz4-fpflr.

[0084] Figure 30: Triligand 6: X-trwlr-Tz4-kvrhG-Tz4-wqwlr.

[0085] Figure 31 : Triligand 9 = X-trwlr-Tz4-kvrhG-Tz4-pwrqw.

[0086] Figure 32: Triligand 10 = X-trwlr-Tz4-kvrhG-Tz4-lwrqw.

[0087] Figure 33: Triligand 1 1 = X-trwlr-Tz4-kvrhG-Tz4-kwwlr.

[0088] Figure 34: Biligand 3 = X-trwlr-Tz4-pkrhG

[0089] Figure 35: Biligand 3-Az4 = X-trwlr-Tz4-pkrhG-Az4

[0090] Figure 36: Triligand 12 = X-trwlr-Tz4-pkrhG-Tz4-wkkdr

[0091] Figure 37: Triligand 12-Az4 = X-trwlr-Tz4-pkrhG-Tz4-wkkdr-Az4

[0092] Figure 38: Tetraligand 1 = X-trwlr-Tz4-pkrhG-Tz4-wkkdr-Tz4-rkekw

[0093] Figure 39: Tetraligand 2 = X-trwlr-Tz4-pkrhG-Tz4-wkkdr-Tz4-kGfkf

[0094] Figure 40: Tetraligand 3 = X-trwlr-Tz4-pkrhG-Tz4-wkkdr-Tz4-rnpwk

[0095] Figure 41 A: Schematic showing multiplex capture agent. [0096] Figure 41 B: Schematic showing an agent comprising multiple capture agents binding distinct proteins.

[0097] Figure 42: ¹⁸F labeled oxime biligand capture agent trwlr-Tz4-kvrhG

[0098] Figure 43: ¹⁸F labeled hydrazone biligand capture agent trwlr-Tz4-kvrhG

[0099] Figure 44: Binding between PCC ligands and HRP-conjugated c-MET.

[00100] Figure 45: Repeating Selection of PCC Triligand by In Situ Click Chemistry: A. Pre-clear; B: Product screen; C. Anti-screen.

[00101] Figure 46: Sequence homology and bioinformatics analysis used for triligand candidate selection. Each peptide is labeled with its SEQ ID NO.

[00102] Figure 47:Unsupervised clustering of sequenced ligands by amino acid similarity. The circled ligands are listed in Figure 46.

[00103] Figure 48: Affinity of the biligands and triligands measured by ELISA.

[00104] Figure 49: Binding between PCC ligands and HRP-conjugated c-MET.

[00105] Figure 50: Pull-down detection of c-MET using modified immunoprecipitation technique.

[00106] Figure 51 : Sequence homology and bioinformatics analysis for election of tetraligands. Each peptide is labeled with its SEQ ID NO.

[00107] Figure 52: Unsupervised clustering of sequenced ligands by amino acid similarity. The circled ligands are listed in Figure 51 .

[00108] Figure 53: Sequence homology and bioinformatics analysis for election of tetraligands. Each peptide is labeled with its SEQ ID NO.

[00109] Figure 54: Unsupervised clustering of sequenced ligands by amino acid similarity. The peptides (except for 5mers of glycine, arginine, glutamate and aspartate) are listed in Figure 53.

[00110] Figure 55: Affinity of the biligand, triligand, and tetraligand candidates measured by ELISA.

[00111] Figure 56: Tetraligand binding to c-MET in human serum.

[00112] Figure 57: Binding between PCC and c-MET-HRP in the presence of HGF. [00113] Figure 58: Pull-down assays of triligands and tetraligands using dilutions of human serum.

[00114] Figure 59: Pull-down assays of biligands, triligands and tetraligands using dilutions of human serum.

[00115] Figure 60: A. Biotin-PEG3 tetraligand 1 buffer stability; B. Biotin-PEG3 tetraligand 2 buffer stability; C. Biotin-PEG3 tetraligand 3 buffer stability.

[00116] Figure 61 : Fluorescence imaging experimental data of Example 28.

[00117] Figure 62: Flow cytometry experimental data of Example 28.

[00118] Figure 63: A. Coronal (left) and sagittal (right) plane sections showing liver and kidney uptake of 18FB-triligand 1 1 ; B. Biodistribution data over time for the average percent of total injected dose for liver, kidneys, and bladder.

[00119] Figure 64: Radiation dosimetry data of Example 29.

[00120] Figure 65: A. and C. Coronal (left) and sagittal (right) plane sections showing liver and kidney uptake of 1 8FB-PEG3-triligand 1 1 ; B. and D. Biodistribution data over time for the average percent of total injected dose for liver, kidneys, and bladder.

[00121] Figure 66: A. and C. Coronal (left) and sagittal (right) plane sections showing uptake of 18FB-PEG3-Biligand 3; B. and D. Biodistribution data over time for the average percent of total injected dose for liver, kidneys, and bladder.

[00122] Figure 67: A. and B. Coronal (left) and sagittal (right) plane sections showing uptake of 18FB-PEG3-Triligand 1 2; C. Biodistribution data over time for the average percent of total injected dose for liver, kidneys, and bladder.

[00123] Figure 68: A. The chemical structure of 1 8FB; B. MicroPET-CT imaging for 18FB in normal mice, biodistribution data over time for the average percent of total injected dose for liver, kidneys, and bladder.

[00124] Figure 69: A. Coronal (left) and sagittal (right) plane sections showing uptake of 18FB-PEG3-labeled Triligand 12 (for study m33018); B. Representative coronal plane sections for kidney at 0 (left) and 60 min (center) post injection; C. Biodistribution data over time for the average percent of total injected dose for lung, heart, liver, kidneys, and bladder. [00125] Figure 70: A., B. and C. Binding of 19FB-PEG3-labeled PCCs in human plasma; D. Microsomal stability of 1 9FB-PEG3-labeled Biligand 3.

DETAILED DESCRIPTION OF THE INVENTION

[00126] The following description of the invention is merely intended to illustrate various embodiments of the invention. As such, the specific modifications discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and

modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein.

Definitions:

[00127] The term "capture agent" as used herein refers to a composition that comprises one or more target-binding moieties and which specifically binds to a target protein via those target-binding moieties. Each target-binding moiety exhibits binding affinity for the target protein, either individually or in combination with other target-binding moieties. In certain embodiments, each target-binding moiety binds to the target protein via one or more non-covalent interactions, including for example hydrogen bonds, hydrophobic interactions, and van der Waals interactions. A capture agent may comprise one or more organic molecules, including for example polypeptides, peptides, polynucleotides, and other non-polymeric molecules. In some aspects a capture agent is a protein catalyzed capture agent (PCC).

[00128] The term "epitope" as used herein refers to a distinct molecular surface of a target protein capable of catalyzing the assembly of a PCC from a library of molecular building blocks. Typically, the epitope is a polypeptide and it can act on its own as a finite sequence of 20-40 amino acids.

[00129] The term "epitope targeting" as used herein referes to a process by which an anchor ligand is selected by an epitope-catalyzed process where a synthetic polypeptide epitope presenting a first functional group interacts with a library of possible anchor ligands presenting a second functional group to result in the formation of a covalent linkage between the polypeptide and anchor ligand. The selected anchor ligand displays affinity toward both the polypeptide epitope and the full-length (native) target protein. The polypeptide epitope dictates the sequence and binding site of the anchor ligand, and ultimately the capture agent or PCC.

[00130] The same epitope, now existing as part of the larger protein, can be involved in catalyzing the assembly of a PCC biligand from the previously selected anchor ligand (modified with a second functional group) and a library of molecular building blocks (modified with a first functional group) in a protein- catalyzed process. This protein-catalyzed process can then be repeated to assemble a PCC triligand from the previously selected biligand (modified with a third functional group) and a library of molecular building blocks (modified with a fourth functional group).

[00131] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to an amino acid sequence comprising a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymers.

[00132] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids, and isomers thereof. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, carboxyglutamate, O- phosphoserine, and isomers thereof. The term "amino acid analogs" refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. The term "amino acid mimetics" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols

recommended by the lUPAC-IUB Biochemical Nomenclature Commission. [00133] The terms "specific binding," "selective binding," "selectively binds," or "specifically binds" as used herein refer to capture agent binding to an epitope on a predetermined antigen. Typically, the capture agent binds with an affinity (K_D) of approximately less than 10^"7 M, such as approximately less than 1 CT M, 10^"9 M or 10^"10 M or even lower.

[00134] The term "K_D" as used herein refers to the dissociation equilibrium constant of a particular capture agent-antigen interaction. Typically, the capture agents of the invention bind to c-MET with a dissociation equilibrium constant (K_D) of less than approximately 10^"6 M, 10^"7 M, such as less than approximately 10^"8 M, 1 0^"9 M or 1 0^"10 M or even lower, for example, as determined using surface plasmon resonance (SPR) technology in a Biacore instrument using the antigen as the ligand and the antibody as the analyte, and binds to the predetermined antigen with an affinity corresponding to a K_D that is at least ten-fold lower, such as at least 1 00 fold lower, for instance at least 1 000 fold lower, such as at least 1 0,000 fold lower, for instance at least 100,000 fold lower than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the predetermined antigen or a closely-related antigen. The amount with which the affinity is lower is dependent on the K_D of the antibody, so that when the K_D of the antibody is very low (that is, the antibody is highly specific), then the amount with which the affinity for the antigen is lower than the affinity for a non-specific antigen may be at least 10,000 fold.

[00135] The term "kd" (sec^-1) as used herein refers to the dissociation rate constant of a particular antibody-antigen interaction. Said value is also referred to as the koff value.

[00136] The term "k_a" (M^~1 xsec^~1) as used herein refers to the association rate constant of a particular antibody-antigen interaction.

[00137] The term "K_D" (M) as used herein refers to the dissociation equilibrium constant of a particular antibody-antigen interaction.

[00138] The term "K_A" (M^~1) as used herein refers to the association equilibrium constant of a particular antibody-antigen interaction and is obtained by dividing the k_a by the kd. [00139] The terms "treat," "treating," or "treatment" as used herein generally refer to preventing a condition or event, slowing the onset or rate of development of a condition or delaying the occurrence of an event, reducing the risk of developing a condition or experiencing an event, preventing or delaying the development of symptoms associated with a condition or event, reducing or ending symptoms associated with a condition or event, generating a complete or partial regression of a condition, lessening the severity of a condition or event, or some combination thereof.

[00140] A "therapeutically effective amount" as used herein refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of an antibody may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the capture agent to elicit a desired response in the individual.

[00141] The term "c-MET" as used herein refers to the human protein shown in Table 1 below.

[00142] Table 1 . Amino acid sequence of human c-Met.

1 mkapavlapg ilvllftlvq rsngeckeal aksemnvnmk yqlpnftaet

piqnvilheh

61 hiflgatnyi yvlneedlqk vaeyktgpvl ehpdcfpcqd csskanlsgg

vwkdninmal

121 vdtyyddql iscgsvnrgt cqrhvfphnh tadiqsevhc ifspqieeps

qcpdc vsal

181 gakvlssvkd rfinffvgnt inssyfpdhp lhsisvrrlk etkdgfmflt

dqsyidvlpe

241 frdsypikyv hafesnnfiy fltvqretld aqtfhtriir fcsinsglhs

ymemplecil

301 tekrkkrstk kevfnilqaa yvskpgaqla rqigaslndd ilfgvfaqsk

pdsaepmdrs

361 amcafpikyv ndffnkivnk nnvrclqhfy gpnhehcfnr tllrnssgce

arrdeyrtef

421 ttalqrvdlf mgqfsevllt sistfikgdl tianlgtseg rfmq vvsrs

gpstphvnf1 481 ldshpvspev ivehtlnqng ytlvitgkki tkiplnglgc rhfqscsqcl sappf qcgw

541 chdkcvrsee clsgtwtqqi clpaiykvfp nsapleggtr lticgwdfgf rrnnkfdlkk

601 trvllgnesc tltlsestmn tlkctvgpam nkhfnmsiii snghgttqys tfsyvdpvit

661 sispkygpma ggtlltltgn ylnsgnsrhi siggktctlk svsnsilecy tpaqtistef

721 avklkidlan retsifsyre dpivyeihpt ksfisggsti tgvgknlnsv svprmvinvh

781 eagrnftvac qhrsnseiic cttpslqqln lqlplktkaf fmldgilsky fdliyvhnpv

841 fkpfekpvmi smgnenvlei kgndidpeav kgevlkvgnk scenihlhse avlctvpndl

901 lklnselnie wkqaisstvl gkvivqpdqn ftgliagvvs istalllllg fflwlkkrkq

961 ikdlgselvr ydarvhtphl drlvsarsvs pttemvsnes vdyratfped qfpnssqngs

1021 crqvqypltd mspiltsgds disspllqnt vhidlsalnp elvqavqhvv igpsslivhf

1081 nevigrghfg cvyhgtlldn dgkkihcavk slnritdige vsqfltegii mkdfshpnvl

1141 sllgiclrse gsplvvlpym khgdlrnfir nethnptvkd ligfglqvak gmkylaskkf

1201 vhrdlaarnc mldekftvkv adfglardmy dkeyysvhnk tgaklpvkwm aleslqtqkf

1261 ttksdvwsfg vllwelmtrg appypdvntf ditvyllqgr rllqpeycpd plyevmlkcw

1321 hpkaemrpsf selvsrisai fstfigehyv hvnatyvnvk cvapypslls sednaddevd

1381 trpasfwets (SEQ ID NO:108) [00143] Human "c-Met" also refers to NCBI Reference Sequence:

NP_000236.2.

[00144] The term "c-MET" as used herein also refers to the

corresponding c-Met protein in other mammals including mice, rats, non-human primates, cats, dogs, hamsters, rabbits, sheep, goats, camels and llamas.

[00145] The term "kinase" as used herein refers to a polypeptide or enzyme whose natural activity is to transfer phosphate groups from high-energy donor molecules such as ATP to specific substrates.

[00146] The term "antibody" as used herein refers to a protein of the kind that is produced by activated B cells after stimulation by an antigen and can bind specifically to the antigen promoting an immune response in biological systems. Full antibodies typically consist of four subunits including two heavy chains and two light chains. The term antibody includes natural and synthetic antibodies, including but not limited to monoclonal antibodies, polyclonal antibodies or fragments thereof. Exemplary antibodies include IgA, IgD, IgGI, lgG2, lgG3, IgM and the like.

Exemplary fragments include Fab, Fv, Fab', F(ab')₂ and the like. A monoclonal antibody is an antibody that specifically binds to and is thereby defined as

complementary to a single particular spatial and polar organization of another biomolecule which is termed an "epitope." In some forms, monoclonal antibodies can also have the same structure. A polyclonal antibody refers to a mixture of different monoclonal antibodies. In some forms, polyclonal antibodies can be a mixture of monoclonal antibodies where at least two of the monoclonal antibodies binding to a different antigenic epitope. The different antigenic epitopes can be on the same target, different targets, or a combination. Antibodies can be prepared by techniques that are well known in the art, such as immunization of a host and collection of sera (polyclonal) or by preparing continuous hybridoma cell lines and collecting the secreted protein (monoclonal).

[00147] The term "stable" as used herein with regard to a capture agent protein catalyzed capture agent or pharmaceutical formulation thereof refers to the agent or formulation retaining structural and functional integrity for a sufficient period of time to be utilized in the methods described herein. [00148] The term "synthetic" as used herein with regard to a protein catalyzed capture agent or capture agent refers to the capture agent has been generated by chemical rather than biological means.

Development of c-MET capture agents:

[00149] Antibodies are currently the default detection agent for use in

diagnostic platforms. However, antibodies possess several disadvantages, including high cost, poor stability, and, in many cases, lack of proper characterization and high specificity. The ideal replacement for use in diagnostic assays should be synthetic, stable to a range of thermal and chemical conditions, and display high affinity and specificity for the target of interest.

[00150] A high quality monoclonal antibody possesses low-nanomolar affinity and high target specificity. Interestingly, structural and genetic analyses of the antigen recognition surface have shown that the majority of the molecular diversity of the variable loops is contained in a single highly variable loop (CDR-H3) (Xu 2000). In humans, this loop ranges in size from 1 -35 residues (1 5 on average) (Zemlin 2003), can adopt a wide range of structural conformations (Chothia 1 989), and is responsible for most of the interactions with the antigen. The other five loops are significantly less diverse and adopt only a handful of conformations. This suggests that a carefully selected "anchor" peptide can dominate the mode and strength of the interaction between a capture agent and its target protein. It also suggests that other peptide components, while providing only modest contributions to the total

interaction energy, can supply important scaffolding features and specificity elements.

[00151] In situ click chemistry (Manetsch 2004; Mocharla 2004; Whiting 2006) is a technique in which a small molecule enzymatic inhibitor is separated into two moieties, each of which is then expanded into a small library - one containing acetylene functionalities, and the other containing azide groups. The enzyme itself then assembles the 'best fit' inhibitor from these library components by selectively promoting 1 ,3-dipolar cycloaddition between the acetylene and azide groups to form a triazole linkage (the 'click' reaction). The enzyme promotes the click reaction only between those library components that bind to the protein in the right orientation. The resultant inhibitor can exhibit far superior affinity characteristics relative to the initial inhibitor that formed the basis of the two libraries (Jencks 1981 ; Murray 2002). [00152] Sequential in situ click chemistry extends the in situ click chemistry concept to enable the discovery of multiligand capture agents (see: USSN 20100009896, incorporated herein by reference). This process was used previously to produce a triligand capture agent against the model protein carbonic anhydrase II (CAN) (Agnew 2009). Sequential in situ click chemistry has several advantages. First, structural information about the protein target is replaced by the ability to sample a very large chemical space to identify the ligand components of the capture agent. For example, an initial ligand may be identified by screening the protein against a large (> 10⁶ element) one-bead-one-compound (OBOC) (Lam 1991 ) peptide library, where the peptides themselves may be comprised of natural, non- natural, and/or artificial amino acids. The resultant anchor ligand is then utilized in an in situ click screen, again using a large OBOC library, to identify a biligand binder. A second advantage is that the process can be repeated, so that the biligand is used as an anchor to identify a triligand, and so forth. The final capture agent can then be scaled up using relatively simple and largely automated chemistries, and it can be developed with a label, such as a biotin group, as an intrinsic part of its structure. This approach permits the exploration of branched, cyclic, and linear capture agent architectures. While many strategies for protein-directed multiligand assembly have been described (Shuker 1 996; Erlanson 2000), most require detailed structural information on the target to guide the screening strategy, and most (such as the original in situ click approach), are optimized for low-diversity small molecule libraries.

[00153] As disclosed herein, an iterative in situ click chemistry approach was utilized to synthesize an epitope-targeted triligand capture agent that specifically binds c-MET. This in situ click chemistry approach utilized two novel screening strategies. First, a synthetic polypeptide derived from c-MET was used as the initial screening target, providing a means for developing an epitope-targeted anchor ligand. Second, the selection process took advantage of the fact that an in situ click screen in which an anchor ligand and full-length protein target are screened against a large OBOC library will selectively generate multiligand products on the hit beads. This concept was expanded in the form of "product screens," in which the presence of on-bead clicked product is taken to be the signature of a hit bead. As shown herein, such a product screen can be utilized to increase both the affinity and/or selectivity of the final multiligand capture agent. [00154] The triligand c-MET capture agents generated by the methods disclosed herein were found to display nanomolar binding affinity, excellent specificity, and low μΜ level inhibitory potency for c-MET. The capture agents also exhibited inhibition kinetics consistent with binding to c-MET at a site that coincides with the binding site of HGF. The capture agents were shown to function as both capture and detection agents in ELISA assays, efficiently immunoprecipitate c-MET from dilute human serum, and label c-MET in live and fixed cancer line cells.

[00155] Based on the results disclosed herein, the present application provides c-MET capture agents comprising three c-MET binding moieties, as well as methods of using these capture agents to identify, detect, quantify, and separate c-MET and to diagnose, classify, and treat various conditions associated with increased c-MET expression and/or activity. The present application also provides novel in situ click chemistry methods for generating epitope-targeted capture agents with high affinity and specificity.

c-MET capture agents:

[00156] Provided herein in certain embodiments are triligand c-MET capture agents comprising three target-binding moieties. The first target-binding moiety is referred to as an anchor ligand, the second is referred to as a secondary ligand, the third is referred to as a tertiary ligand and the fourth is referred to as a quarternary ligand. In certain embodiments, triligand capture agents inhibit c-MET activity by modulating or inhibiting the binding of an endogenous ligand to c-MET. In a particular embodiment, the endogenous ligand is HGF. In other embodiments, tetraligand capture agents inhibit c-MET activity by modulating or inhibiting the binding of an endogenous ligand to c-MET. In a particular embodiment, the endogenous ligand is HGF.

[00157] In certain embodiments, a target-binding moiety comprises one or more polypeptides or peptides. In certain of these embodiments, a target-binding moiety comprises one or more peptides comprising D-amino acids, L-amino acids, and/or amino acids substituted with functional groups selected from the group consisting of substituted and unsubstituted alkyl, substituted and unsubstituted azido, substituted and unsubstituted alkynyl, substituted and unsubstituted biotinyl, substituted and unsubstituted azioalkyl, substituted and unsubstituted

polyethyleneglycolyl, and substituted and unsubstituted 1 ,2,3-triazole. [00158] In certain embodiments, the anchor ligand and secondary ligand are linked to one another via a covalent linkage to form a capture agent biligand. In certain of these embodiments, the anchor ligand and secondary ligand are linked to one another via an amide bond or a 1 ,4-disubstituted-1 ,2,3-triazole linkage as shown below:

1 ,4-disubstituted-1 ,2,3-triazole linkage.

[00159] In those embodiments where the anchor and secondary ligands are linked to one another via a 1 ,4-disubstituted-1 ,2,3-triazole linkage, the 1 ,4- disubstituted -1 ,2,3-triazole linkage may be formed by Cu-Catalyzed Azide/Alkyne Cycloaddition (CuAAC).

[00160] In certain embodiments, the anchor and secondary ligands are linked to one another by a Tz4 linkage having the following structure:

[00161] In certain embodiments, the tertiary and/or quarternary ligand is linked to the capture agent biligand by a covalent linkage, preferably via the secondary ligand in the biligand. In certain of these embodiments, the tertiary ligand and the biligand and/or the quarternary ligand and the tertiary ligand are linked to one another by a Tz4 linkage.

[00162] In those embodiments wherein one or more of the anchor, secondary, tertiary, and/or quarternary ligands are linked to one another via amide bonds, the amide bond may be formed by coupling a carboxylic acid group and an amine group in the presence of a coupling agent (e.g., 0-(7-azabenzotriazol-1 -yl)- Ν,Ν,Ν',Ν'-tetramethyluronium hexafluorophosphate (HATU), N-hydroxy-7-aza- benzotriazole (HOAt), or diisopropylethylamine (DIEA) in DMF).

[00163] In certain embodiments, the capture agents provided herein comprise the anchor ligands trwlr or irnwk.

[00164] In certain embodiments, the capture agents provided herein comprise the secondary ligands kvrhG, pkrhG, efwhG, hvwhG or skrhe.

[00165] In certain embodiments, the capture agents provided herein comprise the tertiary ligands pswwr, fswwr, fpflr, wqwlr, pwrqw, Iwrqw, wkkdr, or kwwlr.

[00166] In certain embodiments, the capture agents provided herein comprise quarternary ligands shirt, kGfkf, rkekw and rnpwk.

[00167] In certain embodiments, the capture agents provided herein have the structures set forth in Figures 25-40.

[00168] In certain embodiments, the c-MET capture agents provided herein bind to an HGF binding site of c-MET. In a particular embodiment, the capture agents inhibit the binding of HGF to c-MET.

[00169] In certain embodiments, the capture agents provided herein are stable across a range of reaction conditions and/or storage times. A capture agent that is "stable" as used herein maintains the ability to specifically bind to a target protein. In certain embodiments, the capture agents provided herein are more stable than an antibody binding to the same target protein under one or more reaction and/or storage conditions. For example, in certain embodiments the capture agents provided herein are more resistant to proteolytic degradation than an antibody binding to the same target protein.

[00170] In certain embodiments, the capture agents provided herein have a shelf-life of greater than six months, meaning that they are stable in storage for greater than six months. In certain of these embodiments, the capture agents have a shelf-life of one year or greater, two years or greater, or more than three years. In certain of these embodiments, the capture agents are stored as a lyophilized powder. In certain embodiments, the capture agents provided herein have a longer shelf-life than an antibody binding to the same target protein.

[00171] In certain embodiments, the capture agents provided herein are stable at temperatures ranging from about -80° to about 1 20 °C. In certain of these embodiments, the capture agents are stable within a temperature range of -80° to - 40 °C; -40° to -20 °C; -20° to 0°C; 0° to 20 °C; 20° to 40 °C; 40° to 60 °C; 60° to 80 °C; and/or 80° to 120°C. In certain embodiments, the capture agents provided herein are stable across a wider range of temperatures than an antibody binding to the same target protein, and/or remain stable at a specific temperature for a longer time period than an antibody binding to the same target protein.

[00172] In certain embodiments, the capture agents provided herein are stable at a pH range from about 3.0 to about 8.0. In certain embodiments, the range is about 4.0 to about 7.0. In certain embodiments, the range is about 7.0 to about 8.0.

[00173] In certain embodiments, the capture agents provided herein are stable in human serum for more than 12 hours. In certain of these embodiments, the capture agents are stable in human serum for more than 1 8 hours, more than 24 hours, more than 36 hours, or more than 48 hours. In certain embodiments, the capture agents provided herein are stable for a longer period of time in human serum than an antibody binding to the same target protein.

[00174] In certain embodiments, the capture agents provided herein may comprise one or more detection labels, including for example biotin, copper-1 ,4,7,10- tetraazacyclododecane-1 ,4,7, 10-tetraacetic acid (copper- DOTA), ⁶⁴Cu DOTA, ⁶⁸Ga DOTA, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, ¹²⁴l, ⁸⁶Y, ^94mTc, ^110mln, ¹¹C, ⁷⁶Br, ¹²³l, ¹³¹ l, ⁶⁷Ga, ^{1 11} ln and ^99mTc, or other radiolabeled products that may include gamma emitters, proton emitters, positron emitters, tritium, or covered tags detectable by other methods (i.e., gadolinium) among others. In a particular embodiment, the detection label is ¹⁸F. In certain embodiments, the capture agents may be modified to be used as imaging agents. The imaging agents may be used as diagnostic agents.

[00175] In certain embodiments, the capture agents provided herein may be modified to obtain a desired chemical or biological activity. Examples of desired chemical or biological activities include, without limitation, improved solubility, stability, bioavailability, detectability, or reactivity. Examples of specific modifications that may be introduced to a capture agent include, but are not limited to, cyclizing the capture agent through formation of a disulfide bond; modifying the capture agent with other functional groups or molecules. Similarly, a capture agent may be synthesized to bind to non-canonical or non-biological epitopes on proteins, thereby increasing their versatility. In certain embodiments, the capture agent may be modified by modifying the synthesis blocks of the target-binding moieties before the coupling reaction.

[00176] Provided herein in certain embodiments are pharmaceutical formulations comprising one or more of the capture agents provided herein. In certain embodiments, these pharmaceutical formulations comprise one or more pharmaceutically acceptable carriers, excipients, or diluents. These carriers, excipients, or diluents may be selected based on the intended use and/or route of administration of the formulation.

[00177] Provided herein in certain embodiments are kits comprising one or more of the capture agents disclosed herein. In certain embodiments, these kits may be used for identifying, detecting, quantifying, and/or separating c-MET, and in certain of these embodiments the kits may be used in the diagnosis and/or staging of a cancer associated with increased c-MET expression and/or activity. In certain embodiments, a kit as provided herein comprises: (a) a substrate comprising an adsorbent thereon, wherein the adsorbent is suitable for binding c-MET, and (b) a washing solution or instructions for making a washing solution, wherein the combination of the adsorbent and the washing solution allows detection of c-MET. In other embodiments, the kits provided herein may be used in the treatment of a condition associated with increased c-MET expression and/or activity.

[00178] In certain embodiments, the kits provided herein may further comprise instructions for suitable operational parameters in the form of a label or a separate insert. For example, the kit may have standard instructions informing a consumer/kit user how to wash the probe after a sample of plasma or other tissue sample is contacted on the probe.

[00179] In certain embodiments, a kit as provided herein comprises (a) one or more c-MET capture agents that specifically bind c-MET; and (b) a detection reagent. Such kits can be prepared from the materials described herein. [00180] The kits provided herein may optionally comprise a standard or control information, and/or a control amount of material, so that the test sample can be compared with the control information standard and/or control amount to determine if the test amount of c-MET detected in a sample is an amount consistent with a diagnosis of a particular condition.

Methods of making/screening capture agents:

[00181] Provided herein in certain embodiments are methods of screening target-binding moieties and/or making capture agents that comprise these target- binding moieties. In certain of these embodiments, the resultant capture agent is a kinase capture agent, and in certain of these embodiments the kinase capture agent is a c-MET capture agent.

[00182] The capture agent production methods disclosed herein begin with identification of a short-chain anchor peptide, then proceed by adding additional covalently coupled peptide ligands via a process that is promoted by the target protein. The specificity and inhibitory potency of the final multiligand capture agent are augmented by the peripheral peptide ligands.

[00183] In certain embodiments, the methods provided herein comprise the following steps:

(a) identifying an anchor ligand by the following steps:

(iv) selecting a candidate peptide with affinity for the target polypeptide as the anchor ligand, wherein the candidate peptide binds to the target polypeptide; and

(v) sequencing the anchor ligand;

(b) identifying a secondary ligand by the following steps: (i) preparing an anchor ligand selection block comprising the anchor ligand and an azido group or an alkynyl group;

(ii) preparing a second plurality of candidate peptides to select a

(vi) sequencing the secondary ligand;

(c) identifying a tertiary ligand by the following steps:

(ii) repeating steps (b)(ii) to (b)(vi) using a third plurality of candidate peptides until a capture agent having desired binding affinity to the target protein is obtained;

(i) preparing a triligand selection block comprising an azido group or an alkynyl group; and

(ii) repeating steps (c)(ii) to (c)(vi) using a fourth plurality, fifth plurality, etc., of candidate peptides until a capture agent having desired binding affinity to the target protein is obtained. [00184] In certain embodiments, one or more of the above steps may be omitted. For example, in certain embodiments a known anchor ligand is used. In these embodiments, step (a) is omitted, and the known anchor ligand is used to identify the secondary ligand in step (b). In those embodiments where the target protein is c-MET, the anchor ligand may comprise the peptide sequence trwlr. In certain embodiments, this anchor ligand may be modified with an N- or C-terminal biotin prior to step (b).

[00185] In certain embodiments, steps (b)(ii) to (b)(vi) are repeated one time, resulting in production of a capture agent triligand.

[00186] In certain embodiments, the first, second, and any additional pluralities of candidate peptides comprise a "one bead one compound" (OBOC) peptide library, wherein the peptides comprise 5 to 7 D-amino acid residues and coupled with a D-propargylglycine at the N-terminus. In certain embodiments, the pluralities of candidate peptides may be different. In other embodiments, one or more of the pluralities may contain the same peptide pool.

[00187] In certain embodiments, the methods provided herein utilize a known anchor ligand. In certain of these embodiments, the anchor ligand is trwlr-Az4- CONH₂ or irnwk-Az4-CONH₂.

[00188] In certain embodiments, the anchor ligand used for the screening process may be modified with a biotin. For example, the anchor ligand used for the screening process may be Biotin-(PEG)₃-trwlr-Az4-CONH₂ or Biotin-(PEG)₃-irnwk- CONH2 wherein "Biotin" is an N-terminal label. In these embodiments, the screening/preparation process comprises the following steps:

a) contacting c-METwith Biotin-(PEG)₃-trwlr-Az4 ("azide-modified c-

METcapture agent anchor ligand selection block (I)") to provide an c-MET- anchor complex;

b) contacting the c-MET-anchor complex with a first plurality of candidate peptides to select a secondary ligand, the peptides coupled with an D- propargylglycine at its N-terminus;

c) providing an c-METcapture agent biligand by forming a disubstituted-1 ,2,3- triazole linkage between the anchor ligand selection block and the secondary ligand, wherein the azido and alkynyl group of the anchor ligand selection block and the secondary ligand are brought in close proximity by binding to the target protein to provide a bead modified with the c-METcapture agent biligand;

d) selecting the beads modified with the c-METcapture agent biligand;

e) removing the c-MET capture agent biligands from the beads modified with the c-METcapture agent biligand;

f) sequencing the c-METcapture agent secondary ligand of the c-METcapture agent biligand;

g) preparing the c-METcapture agent biligand with an N-terminal Biotin- (PEG)₃ label and a C-terminal Az4 ("azide-modified capture agent biligand selection block (I)"); and

h) repeating the above steps until a c-METcapture agent having the desired properties is identified.

[00189] In certain embodiments, methods are provided for synthesizing a capture agent as provided herein. In certain embodiments, these methods comprise: a) preparing a synthesis block of a target-binding moiety, the synthesis block comprising the target-binding moiety and at least one reactive group that can form a desired linkage with another synthesis block, wherein: i) the linkage is selected from the group consisting of amide linkage, 1 ,4- disubstituted 1 ,2,3-triazole linkage, and 1 ,5- disubstituted 1 ,2,3-triazole linkage; and

ii) all other active functional groups of the target-binding moiety are protected to avoid undesired reactions; and

b) coupling the synthesis blocks of the target-binding moieties to provide the capture agent.

Methods of assessing in situ click efficiency:

[00190] Methods for Targeting Specific Epitopes

[00191] Large biomolecules, such as proteins, can be characterized by a diverse landscape of chemical properties that vary significantly across different parts of the molecule. Specific regions of a biomolecule surface are referred to as epitopes. It is often desirable to develop molecules that bind specifically to one epitope on a protein, but not to other epitopes on that protein, or to other proteins. Monoclonal antibodies, which are biological products, are developed to bind to specific epitopes on specific proteins. However, there is not a good way, using chemical synthesis approaches, to target a particular epitope on a protein, unless that epitope also happens to fit very special criteria - i.e. the epitope contains a small molecule binding pocket, and so provides a unique energy well for attracting small molecule binders, relative to the rest of the protein. The vast majority of protein epitopes do not fit these special criteria. This invention describes an approach that can guide the development of highly specific molecular binders to general classes of protein epitopes.

[00192] An approach for synthesizing molecules that bind to specific parts (epitopes) of large protein biomolecules is described and demonstrated. The invention includes first preparing a peptide or polypeptide fragment of a specific protein. That polypeptide can be site-specifically modified near the region of the epitope of interest, by either substituting one of the naturally occurring amino acids for an artificial amino acid, or the polypeptide fragment is modified after synthesis by chemically altering a specific amino acid. In both cases, the modification results in the presentation of either an acetylene or an azide chemical group near the site- specific modification. That azide (acetylene) containing fragment is then incubated with a very large molecular library. This library, while typically chemically diverse, is also characterized by the fact that each element contains an acetylene (or, instead, each element contains an azide) group. The incubation can be done under conditions that the modified polypeptide fragment can provide a catalytic scaffold for promoting the covalent coupling between select library elements and the polypeptide fragment. In this embodiment, it promotes this coupling by catalyzing the formation of a triazole linkage that is the reaction product of the acetylene and azide groups. According to several embodiments, the selectivity of this catalyzed process is very high. This means that only a very small fraction of the elements in the molecular library will be coupled. Those elements are identified through analytical techniques, and then tested for binding to the polypeptide fragment, or to the entire protein biomolecule from which the polypeptide fragment was extracted. This approach provides a route towards identifying molecules that selectively bind to the intended epitope of the protein target. Approaches known in the art may then be utilized to increase the selectivity and the affinity of the identified binders, without sacrificing their epitope selective binding characteristics.

[00193] The following steps are performed in one embodiment of the epitope targeting process. A protein target (1 ) is selected. The protein target (1 ) has a specific epitope (2) that is of interest for developing capture agent molecule that will bind to that location. That epitope may be a specific amino acid residue (2) associated with a particular peptide or polypeptide fragment (3) of the entire protein (1 ), or it may be a larger region of the protein (1 ) containing several amino acids. The epitope is located within a region of the protein that is characterized by a known sequence of amino acids (3). An amino acid near (or within) the epitope (4) is identified for either substitution with an artificial amino acid, or some other specific chemical modification to introduce an azide or acetylene group onto that site. A polypeptide fragment (5) of the protein that contains the targeted epitope is synthesized, but with two modifications. First, (4) is either substituted or chemically modified so as to provide an azide or acetylene group. Second, a site on the polypeptide is modified (7) with a label (a fluorophore or biotin group, for example) for use during the screening steps. There are many ways through which this label can be introduced.

[00194] If a molecular library of 1 million molecules, designed to span a broad chemical space, is incubated with a -50-100 nM concentration solution of the modified polypeptide fragment (5), under standard blocking conditions to prevent non-selective binding, then that screen will generate about 20-100 hit molecules. Of those hit molecules, a small number (1 -10) will be molecules that specifically bind to the epitope of interest. Approaches described in the two above-referenced inventions can then be utilized to increase the affinity and specificity of those epitope specific binders.

In Vitro

[00195] U.S. Patent Publication No. 2010/0260672, discloses in vitro and in vivo methods of using c-Met binding moieties. This disclosure of U.S. Patent Publication No. 2010/0260672 is incorporated by reference, herein. [00196] For detection of HGF or c-Met in solution, a capture agent as disclosed herein can be detectably labeled then contacted with the solution, and thereafter formation of a complex between the capture agent and the c-Met target can be detected. As an example, a fluorescently labeled c-Met capture agent can be used for in vitro c-Met or HGF/c-Met complex detection assays, wherein the capture agent is added to a solution to be tested for c-Met or HGF/c-Met complex under conditions allowing binding to occur. The complex between the fluorescently labeled c-Met capture agent and c-Met or HGF/c-Met complex target can be detected and quantified by, for example, measuring the increased fluorescence polarization arising from the c-Met or HGF/c-Met complex-bound peptide relative to that of the free peptide.

[00197] Alternatively, a sandwich-type "ELISA" assay can be used, wherein a c-Met capture agent is immobilized on a solid support such as a plastic tube or well, then the solution suspected of containing c-Met or HGF/c-Met complex target is contacted with the immobilized binding moiety, non-binding materials are washed away, and complexed polypeptide is detected using a suitable detection reagent recognizing the c-Met or HGF/c-Met complex.

[00198] For detection or purification of soluble c-Met or HGF/c-Met complex in or from a solution, capture agents described herein can be immobilized on a solid substrate such as a chromatographic support or other matrix material, then the immobilized binder can be loaded or contacted with the solution under conditions suitable for formation of a capture agent/c-Met complex. The non-binding portion of the solution can be removed and the complex can be detected, for example, using an anti-HGF or anti-HGF/c-Met complex antibody, or an anti-binding polypeptide antibody, or the c-Met or HGF/c-Met complex target can be released from the binding moiety at appropriate elution conditions.

[00199] The biology of cellular proliferation and the roles of HGF and c-Met in initiating and maintaining it have been investigated by many researchers and continues to be an active field for research and development. In furtherance of such research and development, a method of purifying bulk amounts of c-Met or HGF/c- Met complex in pure form is desirable, and the capture agents described herein are especially useful for that purpose, using the general purification methodology described above. In Vivo

Diagnostic Imaging

[00200] A particularly preferred use for the capture agents described herein is for creating visually readable images of c-Met expressing tissue, such as, for example, neoplastic tumors, which exhibit hyperproliferation. The c-Met capture agents disclosed herein can be converted to imaging reagents by conjugating the capture agents with a label appropriate for diagnostic detection. Preferably, a capture agent exhibiting much greater specificity for c-Met or HGF/c-Met than for other serum proteins is conjugated or linked to a label appropriate for the detection methodology to be employed. For example, the capture agent can be conjugated with or without a linker to a paramagnetic chelate suitable for Magnetic Resonance Imaging (MRI), with a radiolabel suitable for x-ray, Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT) or scintigraphic imaging (including a chelator for a radioactive metal), with an ultrasound contrast agent (e.g., a stabilized microbubble, a microballoon, a microsphere or what has been referred to as a gas filled "liposome") suitable for ultrasound detection, or with an optical imaging dye.

[00201] In general, the technique of using a detectably labeled c-Met capture agent is based on the premise that the label generates a signal that is detectable outside a patient's body. For example, when the detectably labeled c-Met capture agent is administered to the patient in which it is desirable to detect, e.g., hyperproliferation, the high affinity of the c-Met binding moiety for c-Met causes the binding moiety to bind to the site of hyperproliferation and accumulate label at the site. Sufficient time is allowed for the labeled binding moiety to localize at the site of proliferation. The signal generated by the labeled peptide is detected by a scanning device that will vary according to the type of label used, and the signal is then converted to an image of the site of proliferation.

[00202] In another embodiment, rather than directly labeling a c-Met capture agent with a detectable label or radiotherapeutic construct, one or more peptides or constructs of the invention can be conjugated with for example, avidin, biotin, or an antibody or antibody fragment that will bind the detectable label or radiotherapeutic.

A. Magnetic Resonance Imaging [00203] The c-Met capture agents described herein can advantageously be conjugated with a paramagnetic metal chelate in order to form a contrast agent for use in MRI. Preferred paramagnetic metal ions have atomic numbers 21 -29, 42, 44, or 57-83. This includes ions of the transition metal or lanthanide series which have one, and more preferably five or more, unpaired electrons and a magnetic moment of at least 1 .7 Bohr magneton. Preferred paramagnetic metals include, but are not limited to, chromium (III), manganese (II), manganese (III), iron (II), iron (III), cobalt (II), nickel (II), copper (II), praseodymium (III), neodymium (III), samarium (III), gadolinium (III), terbium (III), dysprosium (III), holmium (III), erbium (III), europium (III) and ytterbium (III), chromium (II I), iron (III), and gadolinium (III). The trivalent cation, Gd³⁺, is particularly preferred for MRI contrast agents, due to its high relaxivity and low toxicity, with the further advantage that it exists in only one biologically accessible oxidation state, which minimizes undesired metabolysis of the metal by a patient. Another useful metal is Cr³⁺, which is relatively inexpensive. Gd(lll) chelates have been used for clinical and radiologic MR applications since 1988, and approximately 30% of MR exams currently employ a gadolinium-based contrast agent.

[00204] The paramagnetic metal chelator is a molecule having one or more polar groups that act as a ligand for, and complex with, a paramagnetic metal. Suitable chelators are known in the art and include acids with methylene phosphonic acid groups, methylene carbohydroxamine acid groups, carboxyethylidene groups, or carboxymethylene groups. Examples of chelators include, but are not limited to, diethylenetriaminepentaacetic acid (DTPA), 1 ,4,7,10-tetraazacyclo-tetradecane- 1 ,4,7,1 0-tetraacetic acid (DOTA), 1 -substituted 1 ,4,7,-tricarboxymethyl-1 ,4,7, 10- teraazacyclododecane (D03A), ethylenediaminetetraacetic acid (EDTA), and 1 ,4,8,1 1 -tetra-azacyclotetradecane- 1 ,4, 8, 1 1 -tetraacetic acid (TETA). Additional chelating ligands are ethylene bis-(2-hydroxy-phenylglycine) (EHPG), and derivatives thereof, including 5-CI-EHPG, 5-Br-EHPG, 5-Me-EHPG, 5-t-Bu-EHPG, and 5-sec-Bu-EHPG; benzodiethylenetriamine pentaacetic acid (benzo-DTPA) and derivatives thereof, including dibenzo-DTPA, phenyl-DTPA, diphenyl-DTPA, benzyl- DTPA, and dibenzyl DTPA; bis-2 (hydroxybenzyl)-ethylene-diaminediacetic acid (HBED) and derivatives thereof; the class of macrocyclic compounds which contain at least 3 carbon atoms, more preferably at least 6, and at least two heteroatoms (O and/or N), which macrocyclic compounds can consist of one ring, or two or three rings joined together at the hetero ring elements, e.g., benzo-DOTA, dibenzo-DOTA, and benzo-NOTA, where NOTA is 1 ,4,7-triazacyclononane Ν,Ν',Ν''-triacetic acid, benzo-TETA, benzo-DOTMA, where DOTMA is 1 ,4,7,10-tetraazacyclotetradecane-

1 .4.7.1 0- tetra(methyl tetraacetic acid), and benzo-TETMA, where TETMA is

1 .4.8.1 1 - tetraazacyclotetradecane-1 ,4,8,1 1 -(methyl tetraacetic acid); derivatives of 1 ,3-propylene-diaminetetraacetic acid (PDTA) and triethylenetetraaminehexaacetic acid (TTHA); derivatives of 1 ,5,1 0?N,N',N"-tris(2,3-dihydroxybenzoyl)-tricatecholate (LICAM); and 1 ,3,5-N,N',N"-tris(2,3-dihydroxybenzoyl)aminomethylbenzene (MECAM). A preferred chelator for use in the present invention is DTPA, and the use of DO3A is particularly preferred. Examples of representative chelators and chelating groups contemplated by the present invention are described in WO 98/18496, WO 86/06605, WO 91 /03200, WO 95/28179, WO 96/23526, WO 97/3661 9, PCT/US98/01473, PCT/US98/20182, and U.S. Pat. No. 4,899,755, U.S. Pat. No. 5,474,756, U.S. Pat. No. 5,846,519 and U.S. Pat. No. 6, 143,274, all of which are hereby incorporated by reference.

[00205] In accordance with the present invention, the chelator of the MRI contrast agent is coupled to the c-Met capture agent. The positioning of the chelate should be selected so as not to interfere with the binding affinity or specificity of the c-Met capture agent. The chelate also can be attached anywhere on the capture agent.

[00206] In general, the c-Met capture agent can be bound directly or covalently to the metal chelator (or other detectable label), or it can be coupled or conjugated to the metal chelator using a linker, which can be, without limitation, amide, urea, acetal, ketal, double ester, carbonyl, carbamate, thiourea, sulfone, thioester, ester, ether, disulfide, lactone, imine, phosphoryl, or phosphodiester linkages; substituted or unsubstituted saturated or unsaturated alkyl chains; linear, branched, or cyclic amino acid chains of a single amino acid or different amino acids (e.g., extensions of the N- or C-terminus of the c-Met binding moiety); derivatized or underivatized polyethylene glycols (PEGs), polyoxyethylene, or polyvinylpyridine chains; substituted or unsubstituted polyamide chains; derivatized or underivatized polyamine, polyester, polyethylenimine, polyacrylate, polyvinyl alcohol), polyglycerol, or oligosaccharide (e.g., dextran) chains; alternating block copolymers; malonic, succinic, glutaric, adipic and pimelic acids; caproic acid; simple diamines and dialcohols; any of the other linkers disclosed herein; or any other simple polymeric linkers known in the art (see, for example, WO 98/1 8497 and WO 98/1 8496). Preferably the molecular weight of the linker can be tightly controlled. The molecular weights can range in size from less than 100 to greater than 1000. Preferably the molecular weight of the linker is less than 1 00. In addition, it can be desirable to utilize a linker that is biodegradable in vivo to provide efficient routes of excretion for the imaging reagents of the present invention. Depending on their location within the linker, such biodegradable functionalities can include ester, double ester, amide, phosphoester, ether, acetal, and ketal functionalities.

[00207] In general, known methods can be used to couple the metal chelate and the c-Met capture agent using such linkers (WO 95/28967, WO 98/18496, WO 98/18497 and discussion therein). The c-Met binding moiety can be linked through an N- or C-terminus via an amide bond, for example, to a metal coordinating backbone nitrogen of a metal chelate or to an acetate arm of the metal chelate itself. The present disclosure contemplates linking of the chelate on any position, provided the metal chelate retains the ability to bind the metal tightly in order to minimize toxicity.

[00208] MRI contrast reagents prepared according to the disclosures herein can be used in the same manner as conventional MRI contrast reagents. When imaging a site of hyperproliferation, for example, certain MR techniques and pulse sequences can be preferred to enhance the contrast of the site to the background blood and tissues. These techniques include (but are not limited to), for example, black blood angiography sequences that seek to make blood dark, such as fast spin echo sequences (Alexander, A. et al., 1998. Magn. Reson. Med., 40: 298-310) and flow-spoiled gradient echo sequences (Edelman, R. et al., 1990. Radiology, 1 77: 45- 50). These methods also include flow independent techniques that enhance the difference in contrast, such as inversion-recovery prepared or saturation-recovery prepared sequences that will increase the contrast between angiogenic tumor and background tissues. Finally, magnetization transfer preparations also can improve contrast with these agents (Goodrich, K. et al., 1996. Invest. Radia, 31 : 323-32).

[00209] The labeled reagent is administered to the patient in the form of an injectable composition. The method of administering the MRI contrast agent is preferably parenterally, meaning intravenously, intraarterially, intrathecal^, interstitially, or intracavitarilly. For imaging active angiogenesis, intravenous or intraarterial administration is preferred. For MRI, it is contemplated that the subject will receive a dosage of contrast agent sufficient to enhance the MR signal at the site of angiogenesis at least 10%. After injection with the c-Met capture agent containing MRI reagent, the patient is scanned in the MRI machine to determine the location of any sites of hyperproliferation. In therapeutic settings, upon identification of a site of hyperproliferation (e.g., tumor), a tumoricidal agent or anti-hyperproliferative agent (e.g., inhibitors of HGF) can be immediately administered, if necessary, and the patient can be subsequently scanned to visualize tumor regression or arrest of angiogenesis.

B. Ultrasound Imaging

[00210] When ultrasound is transmitted through a substance, the acoustic properties of the substance will depend upon the velocity of the transmissions and the density of the substance. Changes in the acoustic properties will be most prominent at the interface of different substances (solids, liquids, gases). Ultrasound contrast agents are intense sound wave reflectors because of the acoustic differences between the agent and the surrounding tissue. Gas containing or gas generating ultrasound contrast agents are particularly useful because of the acoustic difference between liquid (e.g., blood) and the gas-containing or gas generating ultrasound contrast agent. Because of their size, ultrasound contrast agents comprising microbubbles, microballoons, and the like can remain for a longer time in the blood stream after injection than other detectable moieties; a targeted c-Met- specific ultrasound agent therefore could demonstrate superior imaging of sites of hyperproliferation (e.g., cancer) and angiogenesis.

[00211] In this aspect of the disclosure, the c-Met capture agent can be linked to a material that is useful for ultrasound imaging. For example, one or more c-Met capture agents can be linked to materials employed to form vesicles (e.g., microbubbles, microballoons, microspheres, etc.), or emulsions containing a liquid or gas, which functions as the detectable label (e.g., an echogenic gas or material capable of generating an echogenic gas). Materials for the preparation of such vesicles include surfactants, lipids, sphingolipids, oligolipids, phospholipids, proteins, polypeptides, carbohydrates, and synthetic or natural polymeric materials (WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18497, WO 98/18496, and WO 98/18501 , incorporated herein by reference in their entirety).

[00212] For contrast agents comprising suspensions of stabilized microbubbles (a preferred embodiment), phospholipids, and particularly saturated phospholipids are preferred. Examples of suitable phospholipids include esters of glycerol with one or two (the same or different) fatty acids molecules and with phosphoric acid, wherein the phosphoric acid residue is in turn bonded to a hydrophilic group, such as choline, serine, inositol, glycerol, ethanolamine, and the like groups. Fatty acids present in the phospholipids are in general long chain aliphatic acids, typically containing from 12 to 24 carbon atoms, preferably from 14 to 22, that can be saturated or can contain one or more unsaturations. Examples of suitable fatty acids are lauric acid, myristic acid, palmitic acid, stearic acid, arachidonic acid, behenic acid, oleic acid, linoleic acid, and linolenic acid. Mono esters of phospholipid are also known in the art as the "lyso" forms of the phospholipids. Further examples of phospholipid are phosphatidic acids, i.e., the diesters of glycerol-phosphoric acid with fatty acids, sphingomyelins, i.e., those phosphatidylcholine analogs where the residue of glycerol diester with fatty acids is replaced by a ceramide chain, cardiolipins, i.e., the esters of 1 ,3-diphosphatidylglycerol with a fatty acid, gangliosides, cerebrosides, etc.

[00213] As used herein, the term "phospholipids" includes naturally occurring, semisynthetic or synthetically prepared products that can be employed either singularly or as mixtures.

[00214] Examples of naturally occurring phospholipids are natural lecithins (phosphatidylcholine (PC) derivatives) such as, typically, soya bean or egg yolk lecithins. Examples of semisynthetic phospholipids are the partially or fully hydrogenated derivatives of the naturally occurring lecithins.

[00215] Examples of synthetic phospholipids are, e.g., dilauryloyl- phosphatidylcholine ("DLPC"), dimyristoylphosphatidylcholine ("DMPC"), dipalmitoyl- phosphatidylcholine ("DPPC"), diarachidoylphosphatidylcholine ("DAPC"), distearoyl- phosphatidylcholine ("DSPC"), 1 -myristoyl-2-palmitoylphosphatidylcholine ("MPPC"), 1 -palmitoyl-2-myristoylphosphatidylcholine ("PMPC"), 1 -palmitoyl-2- stearoylphosphatid-ylcholine ("PSPC"), 1 -stearoyl-2-palmitoyl-phosphatidylcholine ("SPPC"), dioleoylphosphatidylycholine ("DOPC"), 1 ,2 Distearoyl-sn-glycero-3- Ethylphosphocholine (Ethyl-DSPC), dilauryloyl-phosphatidylglycerol ("DLPG") and its alkali metal salts, diarachidoylphosphatidylglycerol ("DAPG") and its alkali metal salts, dimyristoylphosphatidylglycerol ("DMPG") and its alkali metal salts, dipalmitoyl- phosphatidylglycerol ("DPPG") and its alkali metal salts, distearolyphosphatidylglycerol ("DSPG") and its alkali metal salts, dioleoylphosphatidylglycerol ("DOPG") and its alkali metal salts, dimyristoyl phosphatide acid ("DMPA") and its alkali metal salts, dipalmitoyl phosphatidic acid ("DPPA") and its alkali metal salts, distearoyi phosphatidic acid ("DSPA"), diarachidoyl phosphatidic acid ("DAPA") and its alkali metal salts, dimyristoyl phosphatidyl-ethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine ("DPPE"), distearoyi phosphatidyl-ethanolamine ("DSPE"), dimyristoyl phosphatidylserine ("DMPS"), diarachidoyl phosphatidylserine ("DAPS"), dipalmitoyl phosphatidylserine ("DPPS"), distearoylphosphatidylserine ("DSPS"), dioleoylphosphatidylserine ("DOPS"), dipalmitoyl sphingomyelin ("DPSP"), and distearoyi sphingomyelin ("DSSP"). In a preferred embodiment, at least one of the phospholipid moieties has the structure described in U.S. Pat. No. 5,686,060, which is herein incorporated by reference.

[00216] Other preferred phospholipids include dipalmitoylphosphatidylcholine, dipalmitoylphosphatidic acid and dipalmitoylphosphatidylserine. The compositions also can contain PEG-4000 and/or palmitic acid. Any of the gases disclosed herein or known to the skilled artisan can be employed; however, inert gases, such as SF6 or fluorocarbons like CF4, C3F8 and C4F10, are preferred.

[00217] The preferred gas-filled microbubbles of the invention can be prepared by means known in the art, such as, for example, by a method described in any one of the following patents: EP 554213, U.S. Pat. No. 5,413,774, U.S. Pat. No. 5,578,292, EP 744962, EP 682530, U.S. Pat. No. 5,556,610, U.S. Pat. No. 5,846,518, U.S. Pat. No. 6,1 83,725, EP 474833, U.S. Pat. No. 5,271 ,928, U.S. Pat. No. 5,380,519, U.S. Pat. No. 5,531 ,980, U.S. Pat. No. 5,567,414, U.S. Pat. No. 5,658,551 , U.S. Pat. No. 5,643,553, U.S. Pat. No. 5,91 1 ,972, U.S. Pat. No. 6, 1 10,443, U.S. Pat. No. 6,1 36,293, EP 619743, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,597,549, U.S. Pat. No. 5,686,060, U.S. Pat. No. 6,187,288, and U.S. Pat. No. 5,908,610, which are incorporated by reference herein in their entireties. [00218] The preferred microbubble suspensions of the present disclosure can be prepared from phospholipids using known processes such as a freeze-drying or spray-drying solutions of the crude phospholipids in a suitable solvent or using the processes set forth in EP 554213; U.S. Pat. No. 5,413,774; U.S. Pat. No. 5,578,292; EP 744962; EP 682530; U.S. Pat. No. 5,556,610; U.S. Pat. No. 5,846,51 8; U.S. Pat. No. 6,183,725; EP 474833; U.S. Pat. No. 5,271 ,928; U.S. Pat. No. 5,380,51 9; U.S. Pat. No. 5,531 ,980; U.S. Pat. No. 5,567,414; U.S. Pat. No. 5,658,551 ; U.S. Pat. No. 5,643,553; U.S. Pat. No. 5,91 1 ,972; U.S. Pat. No. 6,1 10,443; U.S. Pat. No. 6, 136,293; EP 619743; U.S. Pat. No. 5,445,813; U.S. Pat. No. 5,597,549; U.S. Pat. No. 5,686,060; U.S. Pat. No. 6,187,288; and U.S. Pat. No. 5,908,61 0, which are incorporated by reference herein in their entireties. Most preferably, the phospholipids are dissolved in an organic solvent and the solution is dried without going through a liposome formation stage. This can be done by dissolving the phospholipids in a suitable organic solvent together with a hydrophilic stabilizer substance or a compound soluble both in the organic solvent and water and freeze- drying or spray-drying the solution. In this embodiment the criteria used for selection of the hydrophilic stabilizer is its solubility in the organic solvent of choice. Examples of hydrophilic stabilizer compounds soluble in water and the organic solvent are, e.g., a polymer, like polyvinyl pyrrolidone (PVP), polyvinyl alcohol (PVA), polyethylene glycol (PEG), etc., malic acid, glycolic acid, maltol, and the like. Such hydrophilic compounds also aid in homogenizing the microbubbles size distribution and enhance stability under storage. Any suitable organic solvent can be used as long as its boiling point is sufficiently low and its melting point is sufficiently high to facilitate subsequent drying. Typical organic solvents include, for example, dioxane, cyclohexanol, tertiary butanol, tetrachlorodifluoro ethylene (C.sub.2Cl.sub.4F.sub.2) or 2-methyl-2-butanol. 2-methyl-2-butanol and C.sub.2Cl.sub.4F.sub.2 are preferred.

[00219] Prior to formation of the suspension of microbubbles by dispersion in an aqueous carrier, the freeze dried or spray dried phospholipid powders are contacted with air or another gas. When contacted with the aqueous carrier the powdered phospholipids whose structure has been disrupted will form lamellarized or laminarized segments that will stabilize the microbubbles of the gas dispersed therein. This method permits production of suspensions of microbubbles, which are stable even when stored for prolonged periods, and are obtained by simple dissolution of the dried laminarized phospholipids, which have been stored under a desired gas, without shaking or any violent agitation.

[00220] Unless it contains a hyperpolarized gas, known to require special storage conditions, the lyophilized or freeze-dried residue can be stored and transported without need of temperature control of its environment and in particular it can be supplied to hospitals and physicians for on site formulation into a ready-to- use administrable suspension without requiring such users to have special storage facilities.

[00221] Preferably in such a case it can be supplied in the form of a two component kit. The two component kit can include two separate containers or a dual- chamber container. In the former case preferably the container is a conventional septum-sealed vial, wherein the vial containing the lyophilized residue of step b) is sealed with a septum through which the carrier liquid can be injected using an optionally pre-filled syringe. In such a case the syringe used as the container of the second component is also used then for injecting the contrast agent. In the latter case, preferably the dual-chamber container is a dual-chamber syringe and once the lyophilizate/freeze-dried residue has been reconstituted and then suitably mixed or gently shaken, the container can be used directly for injecting the contrast agent. In both cases means for directing or permitting application of sufficient bubble forming energy into the contents of the container are provided. However, as noted above, in the stabilized contrast agents the size of the gas microbubbles is substantially independent of the amount of agitation energy applied to the reconstituted dried product. Accordingly no more than gentle hand shaking is generally required to give reproducible products with consistent microbubble size.

[00222] It can be appreciated by one ordinary skilled in the art that other two- chamber reconstitution systems capable of combining the dried powder with the aqueous solution in a sterile manner are also within the scope of the present invention. In such systems, it is particularly advantageous if the aqueous phase can be interposed between the water-insoluble gas and the environment, to increase shelf life of the product. Where a material necessary for forming the contrast agent is not already present in the container (e.g., a c-Met capture agent of the invention to be linked to the phospholipid during reconstitution), it can be packaged with the other components of the kit, preferably in a form or container adapted to facilitate ready combination with the other components of the kit.

[00223] No specific containers, vial or connection systems are required; the present disclosure can use conventional containers, vials and adapters. The only requirement is a good seal between the stopper and the container. The quality of the seal, therefore, becomes a matter of primary concern; any degradation of seal integrity could allow undesirables substances to enter the vial. In addition to assuring sterility, vacuum retention is essential for products stoppered at ambient or reduced pressures to assure safe and proper reconstitution. As to the stopper, it may be a compound or multicomponent formulation based on an elastomer, such as poly(isobutylene) or butyl rubber.

[00224] Alternatively, microbubbles can be prepared by suspending a gas in an aqueous solution at high agitation speed, as disclosed, e.g., in WO 97/29783. A further process for preparing microbubbles is disclosed in co-pending European patent application no. 03002373, herein incorporated by reference, which comprises preparing an emulsion of an organic solvent in an aqueous medium in the presence of a phospholipid and subsequently lyophilizing said emulsion, after optional washing and/or filtration steps.

[00225] Additives known to those of ordinary skill in the art can be included in the suspensions of stabilized microbubbles. For instance, non-film forming surfactants, including polyoxypropylene glycol and polyoxyethylene glycol and similar compounds, as well as various copolymers thereof; fatty acids such as myristic acid, palmitic acid, stearic acid, arachidonic acid or their derivatives, ergosterol, phytosterol, sitosterol, lanosterol, tocopherol, propyl gallate, ascorbyl palmitate and butylated hydroxytoluene may be added. The amount of these non-film forming surfactants is usually up to 50% by weight of the total amount of surfactants but preferably between 0 and 30%.

[00226] In ultrasound applications the contrast agents formed by phospholipid stabilized microbubbles can, for example, be administered in doses such that the amount of phospholipid injected is in the range 0.1 to 200:g/kg body weight, preferably from about 0.1 to 30:g/kg. [00227] Other gas containing suspensions include those disclosed in, for example, U.S. Pat. No. 5,798,091 , WO 97/29783, also EP 881 91 5, incorporated herein by reference in their entireties. These agents can be prepared as described in U.S. Pat. No. 5,798,091 or WO97/29783.

[00228] Another preferred ultrasound contrast agent comprises microballoons. The term "microballoon" refers to gas filled bodies with a material boundary or envelope. More on microballoon formulations and methods of preparation can be found in EP 324 938 (U.S. Pat. No. 4,844,882); U.S. Pat. No. 5,71 1 ,933; U.S. Pat. No. 5,840,275; U.S. Pat. No. 5,863,520; U.S. Pat. No. 6,1 23,922; U.S. Pat. No. 6,200,548; U.S. Pat. No. 4,900,540; U.S. Pat. No. 5,123,414; U.S. Pat. No. 5,230,882; U.S. Pat. No. 5,469,854; U.S. Pat. No. 5,585,1 1 2; U.S. Pat. No. 4,718,433; U.S. Pat. No. 4,774,958; WO 95/01 1 87; U.S. Pat. No. 5,529,766; U.S. Pat. No. 5,536,490; and U.S. Pat. No. 5,990,263, the contents of which are incorporated herein by reference.

[00229] The preferred microballoons have an envelope including a biodegradable physiologically compatible polymer or, a biodegradable solid lipid. The polymers useful for the preparation of the microballoons of the present invention can be selected from the biodegradable physiologically compatible polymers, such as any of those described in any of the following patents: EP 458745; U.S. Pat. No. 5,71 1 ,933; U.S. Pat. No. 5,840,275; EP 554213; U.S. Pat. No. 5,413,774; and U.S. Pat. No. 5,578,292, the entire contents of which are incorporated herein by reference. In particular, the polymer can be selected from biodegradable physiologically compatible polymers, such as polysaccharides of low water solubility, polylactides and polyglycolides and their copolymers, copolymers of lactides and lactones such as .epsilon.-caprolactone, .gamma.-valerolactone and polypeptides. Other suitable polymers include poly(ortho)esters (see for instance U.S. Pat. No. 4,093,709; U.S. Pat. No. 4,131 ,648; U.S. Pat. No. 4,1 38,344; U.S. Pat. No. 4,180,646); polylactic and polyglycolic acid and their copolymers, for instance DEXON (Heller, J., 1980. Biomaterials, 1 :51 -57); poly(DL-lactide-co-e-caprolactone), poly(DL-lactide-co- .gamma.-valerolactone), poly(DL-lactide-co-.gamma.-butyrolactone), polyalkylcyanoacrylates; polyamides, polyhydroxybutyrate; polydioxanone; poly- .beta.-aminoketones (Polymer, 23:1693 (1982)); polyphosphazenes (Allcock, H., 1976. Science, 193:1 214-121 9); and polyanhydrides. The microballoons of the present invention can also be prepared according to the methods of WO 96/1 581 5, incorporated herein by reference, where the microballoons are made from a biodegradable membrane comprising biodegradable lipids, preferably selected from mono- di-, tri-glycerides, fatty acids, sterols, waxes and mixtures thereof. Preferred lipids are di- or tri-glycerides, e.g. di- or tri-myristin, -palmityn or -stearin, in particular tripalmitin or tristearin.

[00230] The microballoons can employ any of the gases disclosed herein of known to the skilled artisan; however, inert gases such as fluorinated gases are preferred. The microballoons can be suspended in a pharmaceutically acceptable liquid carrier with optional additives known to those of ordinary skill in the art and stabilizers.

[00231] Microballoons-containing contrast agents are typically administered in doses such that the amount of wall-forming polymer or lipid is from about 1 0:g/kg to about 20 μg/kg of body weight.

[00232] Other gas-containing contrast agent formulations include microparticles (especially aggregates of microparticles) having gas contained therein or otherwise associated therewith (for example being adsorbed on the surface thereof and/or contained within voids, cavities or pores therein). Methods for the preparation of these agents are as described in EP 01 22624; EP 0123235; EP 0365467; U.S. Pat. No. 5,558,857; U.S. Pat. No. 5,607,661 ; U.S. Pat. No. 5,637,289; U.S. Pat. No. 5,558,856; U.S. Pat. No. 5,1 37,928; WO 95/21631 or WO 93/1 3809, incorporated herein by reference in their entirety.

[00233] Any of these ultrasound compositions also should be, as far as possible, isotonic with blood. Hence, before injection, small amounts of isotonic agents can be added to any of above ultrasound contrast agent suspensions. The isotonic agents are physiological solutions commonly used in medicine and they comprise aqueous saline solution (0.9% NaCI), 2.6% glycerol solution, 5% dextrose solution, etc. Additionally, the ultrasound compositions can include standard pharmaceutically acceptable additives, including, for example, emulsifying agents, viscosity modifiers, cryoprotectants, lyoprotectants, bulking agents etc.

[00234] Any biocompatible gas can be used in the ultrasound contrast agents useful in the invention. The term "gas" as used herein includes any substances (including mixtures) substantially in gaseous form at the normal human body temperature. The gas may thus include, for example, air, nitrogen, oxygen, C0₂, argon, xenon or krypton, fluorinated gases (including for example, perfluorocarbons, SF₆, SeF₆) a low molecular weight hydrocarbon (e.g., containing from 1 to 7 carbon atoms), for example, an alkane such as methane, ethane, a propane, a butane or a pentane, a cycloalkane such as cyclopropane, cyclobutane or cyclopentene, an alkene such as ethylene, propene, propadiene or a butene, or an alkyne such as acetylene or propyne and/or mixtures thereof. However, fluorinated gases are preferred. Fluorinated gases include materials which contain at least one fluorine atom such as SF₆, freons (organic compounds containing one or more carbon atoms and fluorine, i.e., CF₄, C₂F ₆, C₃F₈, C₄F₈, C₄F₁₀, CBrF₃, CCI₂F₂, C₂CIF₅, and CBrCIF₂) and perfluorocarbons. The term perfluorocarbon refers to compounds containing only carbon and fluorine atoms and includes, in particular, saturated, unsaturated, and cyclic perfluorocarbons. The saturated perfluorocarbons, which are usually preferred, have the formula C_nFn₊₂, where n is from 1 to 1 2, preferably from 2 to 1 0, most preferably from 3 to 8 and even more preferably from 3 to 6. Suitable perfluorocarbons include, for example, CF₄, C₂F₆, C₃F₈ C₄F₈, C₄F₁₀, C₅F₁₂, C₆F₁₂, C₇F₁₄, C₈F₁₈, and C₉F₂₀. Most preferably the gas or gas mixture comprises SF₆ or a perfluorocarbon selected from the group consisting of C₃F₈ C₄F₈, C₄Fi₀, C₅Fi₂, C₆Fi₂, C₇Fi₄, C₈Fis, with C₄Fio being particularly preferred. See also WO 97/29783, WO 98/53857, WO 98/18498, WO 98/18495, WO 98/18496, WO 98/18497, WO 98/18501 , WO 98/05364, WO 98/1 7324.

[00235] In certain circumstances it can be desirable to include a precursor to a gaseous substance (e.g., a material that is capable of being converted to a gas in vivo, often referred to as a "gas precursor"). Preferably the gas precursor and the gas it produces are physiologically acceptable. The gas precursor can be pH- activated, photo-activated, temperature activated, etc. For example, certain perfluorocarbons can be used as temperature activated gas precursors. These perfluorocarbons, such as perfluoropentane, have a liquid/gas phase transition temperature above room temperature (or the temperature at which the agents are produced and/or stored) but below body temperature; thus they undergo a phase shift and are converted to a gas within the human body. [00236] The gas can comprise a mixture of gases. The following combinations are particularly preferred gas mixtures: a mixture of gases (A) and (B) in which, at least one of the gases (B), present in an amount of between 0.5-41 % by vol., has a molecular weight greater than 80 daltons and is a fluorinated gas and (A) is selected from the group consisting of air, oxygen, nitrogen, carbon dioxide and mixtures thereof, the balance of the mixture being gas A.

[00237] Since ultrasound vesicles can be larger than the other detectable labels described herein, they can be linked or conjugated to a plurality of c-Met binding polypeptides or multimeric polypeptide constructs in order to increase the targeting efficiency of the agent. Attachment to the ultrasound contrast agents described above (or known to those skilled in the art) can be via direct covalent bond between the c-Met binding polypeptide and the material used to make the vesicle or via a linker, as described previously. For example, see WO 98/53857 generally for a description of the attachment of a peptide to a bifunctional PEG linker, which is then reacted with a liposome composition (Lanza, G. et al., 1997. Ultrasound Med. Biol., 23:863-870)). The structure of these compounds typically comprises:

a) A hydrophobic portion, compatible with the material forming the envelope of the microbubble or of the microballoon, in order to allow an effective incorporation of the compound in the envelope of the vesicle; said portion is typically a lipid moiety (e.g., dipalmitin, distearoil);

b) A spacer (typically PEGs of different molecular weights), which can be optional in some cases (microbubbles may, for instance, prove difficult to freeze dry if the spacer is too long) or preferred in some others (e.g., peptides can be less active when conjugated to a microballoon with a short spacer);

c) A reactive group capable of reacting with a corresponding reactive moiety on the peptide to be conjugated (e.g., maleimido with the -SH group of cysteine).

[00238] A number of methods can be used to prepare suspensions of microbubbles conjugated to c-Met binding polypeptides. For example, one can prepare maleimide-derivatized microbubbles by incorporating 5% (w/w) of N-MPB- PE (1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-4-(p-maleimido-phenyl butyramide), (Avanti Polar-Lipids, Inc., Alabaster, Ala.) in the phospholipid formulation. Then, solutions of mercaptoacetylated c-Met-binding peptides (10 mg/mL in DMF), which have been incubated in deacetylation solution (50 mM sodium phosphate, 25 mM EDTA, 0.5 M hydroxylamine.HCI, pH 7.5) are added to the maleimide-activated microbubble suspension. After incubation in the dark, under gentle agitation, the peptide conjugated microbubbles can be purified by centrifugation.

[00239] Alternatively, c-Met-binding polypeptide conjugated microbubbles can be prepared using biotin/avidin. For example, avidin-conjugated microbubbles can be prepared using a maleimide-activated phospholipid microbubble suspension, prepared as described above, which is added to mercaptoacetylated-avidin (which has been incubated with deacetylation solution). Biotinylated c-Met-binding peptides (prepared as described herein) are then added to the suspension of avidin- conjugated microbubbles, yielding a suspension of microbubbles conjugated to c- Met-binding peptides.

[00240] Ultrasound imaging techniques, which can be used in accordance with the present invention, include known techniques, such as color Doppler, power Doppler, Doppler amplitude, stimulated acoustic imaging, and two- or three- dimensional imaging techniques. Imaging may be done in harmonic (resonant frequency) or fundamental modes, with the second harmonic preferred.

C. Optical Imaging, Sonoluminescence or Photoacoustic Imaging

[00241] In accordance with the present disclosure, a number of optical parameters can be employed to determine the location of c-Met or HGF/c-Met complex with in vivo light imaging after injection of the subject with an optically- labeled c-Met capture agent. Optical parameters to be detected in the preparation of an image may include transmitted radiation, absorption, fluorescent or phosphorescent emission, light reflection, changes in absorbance amplitude or maxima, and elastically scattered radiation. For example, biological tissue is relatively translucent to light in the near infrared (NIR) wavelength range of 650-1000 nm. N IR radiation can penetrate tissue up to several centimeters, permitting the use of the c-Met binding polypeptides or multimeric polypeptide constructs of the present invention for optical imaging of c-Met or HGF/c-Met complex in vivo. [00242] The c-Met capture agents can be conjugated with photolabels, such as, for example, optical dyes, including organic chromophores or fluorophores, having extensive delocalized ring systems and having absorption or emission maxima in the range of 400-1500 nm. The c-Met binding polypeptide or multimeric polypeptide construct can alternatively be derivatized with a bioluminescent molecule. The preferred range of absorption maxima for photolabels is between 600 and 1000 nm to minimize interference with the signal from hemoglobin. Preferably, photoabsorption labels have large molar absorptivities, e.g., greater than 10⁵ cm^"1 M^"1 , while fluorescent optical dyes will have high quantum yields. Examples of optical dyes include, but are not limited to those described in WO 98/18497, WO 98/1 8496, WO 98/18495, WO 98/18498, WO 98/53857, WO 96/17628, WO 97/18841 , WO 96/23524, WO 98/47538, and references cited therein. The photolabels can be covalently linked directly to the c-Met binding peptide or linked to the c-Met binding peptide or multimeric polypeptide construct via a linker, as described previously.

[00243] After injection of the optically-labeled c-Met capture agent, the patient is scanned with one or more light sources (e.g., a laser) in the wavelength range appropriate for the photolabel employed in the agent. The light used can be monochromatic or polychromatic and continuous or pulsed. Transmitted, scattered, or reflected light is detected via a photodetector tuned to one or multiple wavelengths to determine the location of c-Met or HGF/c-Met complex in the subject. Changes in the optical parameter can be monitored over time to detect accumulation of the optically-labeled reagent at the site of hyperproliferation. Standard image processing and detecting devices can be used in conjunction with the optical imaging reagents of the present invention.

[00244] The optical imaging reagents described above also can be used for acousto-optical or sonoluminescent imaging performed with optically-labeled imaging agents (see, U.S. Pat. No. 5,1 71 ,298, WO 98/57666, and references cited therein). In acousto-optical imaging, ultrasound radiation is applied to the subject and affects the optical parameters of the transmitted, emitted, or reflected light. In sonoluminescent imaging, the applied ultrasound actually generates the light detected. Suitable imaging methods using such techniques are described in WO 98/57666.

D. Nuclear Imaging (Radionuclide Imaging) and Radiotherapy [00245] The c-Met capture agents can be conjugated with a radionuclide reporter appropriate for scintigraphy, SPECT, or PET imaging and/or with a radionuclide appropriate for radiotherapy. Constructs in which the c-Met capture agents are conjugated with both a chelator for a radionuclide useful for diagnostic imaging and a chelator useful for radiotherapy are within the scope of the invention.

[00246] For use as a PET agent a peptide or multimeric polypeptide construct is complexed with one of the various positron emitting metal ions, such as ⁵¹ Mn, ⁵²Fe, ⁶⁰Cu, ⁶⁸Ga, ⁷²As, ⁹⁴mTc, or ¹¹⁰ln. The binding moieties of the invention can also be labeled by halogenation using radionuclides such as ¹⁸F, ¹²⁴l, ¹²⁵l, ¹³¹1, ¹²³l, ⁷⁷Br, and ⁷⁶Br. Preferred metal radionuclides for scintigraphy or radiotherapy include ^99mTc, ⁵¹Cr, ⁶⁷Ga, ⁶⁸Ga, ⁴⁷Sc, ⁵¹Cr, ¹⁶⁷Tm, ¹⁴¹Ce, ¹¹¹ In, ¹⁶⁸Yb, ¹⁷⁵Yb, ¹⁴⁰La, ⁹⁰Y, ⁸⁸Y, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁶⁵Dy, ¹⁶⁶Dy, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁹⁷Ru, ¹⁰³Ru, ¹⁸⁶Re, ¹⁸⁸Re, ²⁰³Pb, ^{21 1} Bi, ²¹²Bi, ²¹³Bi, ²¹⁴Bi, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹⁷mSn, ¹⁴⁹Pm, ¹⁶¹Tb, ¹⁷⁷Lu, ¹⁹⁸Au and ¹⁹⁹Au. The choice of metal will be determined based on the desired therapeutic or diagnostic application. For example, for diagnostic purposes the preferred radionuclides include ⁶⁴Cu, ⁶⁷Ga, ⁶⁸Ga, ^99mTc, and ¹¹¹ In. For therapeutic purposes, the preferred radionuclides include ⁶⁴Cu, ⁹⁰Y, ¹⁰⁵Rh, ^{1 11} ln, ¹¹⁷mSn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁷⁵Yb, ¹⁷⁷Ln, ^{186 188}Re, and ¹⁹⁹ Au. ^99mTc is useful for diagnostic applications because of its low cost, availability, imaging properties, and high specific activity. The nuclear and radioactive properties of ^99mTc make this isotope an ideal scintigraphic imaging agent. This isotope has a single photon energy of 140 keV and a radioactive half-life of about 6 hours, and is readily available from a "Mo-^99mTc generator. ¹⁸F, 4- [¹⁸F]fluorobenzaldehyde (¹⁸FB), AI[¹⁸F]-NOTA, ⁶⁸Ga-DOTA, and ⁶⁸Ga-NOTA are typical radionuclides for conjugation to c-MET capture agents for diagnostic imaging.

[00247] The metal radionuclides may be chelated by, for example, linear, macrocyclic, terpyridine, and N₃S, N₂S₂, or N₄ chelants (see also, U.S. Pat. No. 5,367,080, U.S. Pat. No. 5,364,613, U.S. Pat. No. 5,021 ,556, U.S. Pat. No. 5,075,099, U.S. Pat. No. 5,886, 142), and other chelators known in the art including, but not limited to, HYN IC, DTPA, EDTA, DOTA, D03A, TETA, NOTA and bisamino bisthiol (BAT) chelators (see also U.S. Pat. No. 5,720,934). For example, N.sub.4 chelators are described in U.S. Pat. No. 6,143,274; U.S. Pat. No. 6,093,382; U.S. Pat. No. 5,608,1 10; U.S. Pat. No. 5,665,329; U.S. Pat. No. 5,656,254; and U.S. Pat. No. 5,688,487. Certain N₃S chelators are described in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. No. 5,662,885; U.S. Pat. No. 5,976,495; and U.S. Pat. No. 5,780,006. The chelator also can include derivatives of the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N₃S, and N₂S₂ systems such as MAMA (monoamidemonoaminedithiols), DADS (N₂S diaminedithiols), CODADS and the like. These ligand systems and a variety of others are described in, for example, Liu, S, and Edwards, D., 1 999. Chem. Rev., 99:2235- 2268, and references therein.

[00248] The chelator also can include complexes containing ligand atoms that are not donated to the metal in a tetradentate array. These include the boronic acid adducts of technetium and rhenium dioximes, such as are described in U.S. Pat. No. 5, 183,653; U.S. Pat. No. 5,387,409; and U.S. Pat. No. 5,1 18,797, the disclosures of which are incorporated by reference herein, in their entirety.

[00249] The chelators can be covalently linked directly to the c-Met capture agent via a linker, as described previously, and then directly labeled with the radioactive metal of choice (see, WO 98/52618, U.S. Pat. No. 5,879,658, and U.S. Pat. No. 5,849,261 ).

[00250] c-MET capture agents comprising ¹⁸F, 4-[¹⁸F]fluorobenzaldehyde (¹⁸FB), AI[¹⁸F]-NOTA, ⁶⁸Ga-DOTA, and ⁶⁸Ga-NOTA are of preferred interest for diagnostic imaging. Complexes of radioactive technetium are also useful for diagnostic imaging, and complexes of radioactive rhenium are particularly useful for radiotherapy. In forming a complex of radioactive technetium with the reagents of this invention, the technetium complex, preferably a salt of ^99mTc pertechnetate, is reacted with the reagent in the presence of a reducing agent. Preferred reducing agents are dithionite, stannous and ferrous ions; the most preferred reducing agent is stannous chloride. Means for preparing such complexes are conveniently provided in a kit form comprising a sealed vial containing a predetermined quantity of a reagent of the invention to be labeled and a sufficient amount of reducing agent to label the reagent with ^99mTc. Alternatively, the complex can be formed by reacting a peptide of this invention conjugated with an appropriate chelator with a pre-formed labile complex of technetium and another compound known as a transfer ligand. This process is known as ligand exchange and is well known to those skilled in the art. The labile complex can be formed using such transfer ligands as tartrate, citrate, gluconate or mannitol, for example. Among the ^99mTc pertechnetate salts useful with the present invention are included the alkali metal salts such as the sodium salt, or ammonium salts or lower alkyl ammonium salts.

[00251] Preparation of the complexes of the present invention where the metal is radioactive rhenium can be accomplished using rhenium starting materials in the +5 or +7 oxidation state. Examples of compounds in which rhenium is in the Re(VII) state are NH₄Re0₄ or KRe0₄. Re(V) is available as, for example, [ReOCI₄](NBu₄), [ReOCI₄](AsPh₄), ReOCI₃(PPh₃)2 and as Re0₂(pyridine)⁴⁺, where Ph is phenyl and Bu is n-butyl. Other rhenium reagents capable of forming a rhenium complex also can be used.

[00252] Radioactively labeled PET, SPECT, or scintigraphic imaging agents provided by the present invention are encompassed having a suitable amount of radioactivity. Generally, the unit dose to be administered has a radioactivity of about 0.01 mCi to about 100 mCi, preferably 1 mCi to 20 mCi. The solution to be injected at unit dosage is from about 0.01 ml_ to about 1 0 ml_. It is generally preferred to form radioactive complexes in solutions containing radioactivity at concentrations of from about 0.01 mCi to 100 mCi per ml_.

[00253] Typical doses of a radionuclide-labeled c-Met capture agent according to the invention provide 10-20 mCi. After injection of the radionuclide-labeled c-Met capture agents into the patient, a gamma camera calibrated for the gamma ray energy of the nuclide incorporated in the imaging agent is used to image areas of uptake of the agent and quantify the amount of radioactivity present in the site. Imaging of the site in vivo can take place in a matter of a few minutes. However, imaging can take place, if desired, in hours or even longer, after the radiolabeled peptide is injected into a patient. In most instances, a sufficient amount of the administered dose will accumulate in the area to be imaged within about 0.1 of an hour to permit the taking of scintiphotos.

[00254] Proper dose schedules for the radiotherapeutic compounds of the present invention are known to those skilled in the art. The compounds can be administered using many methods including, but not limited to, a single or multiple IV or IP injections, using a quantity of radioactivity that is sufficient to cause damage or ablation of the targeted c-Met-expressing tissue, but not so much that substantive damage is caused to non-target (normal tissue). The quantity and dose required is different for different constructs, depending on the energy and half-life of the isotope used, the degree of uptake and clearance of the agent from the body and the mass of the tumor. In general, doses can range from a single dose of about 30-50 mCi to a cumulative dose of up to about 3 Ci.

[00255] The radiotherapeutic compositions of the invention can include physiologically acceptable buffers, and can require radiation stabilizers to prevent radiolytic damage to the compound prior to injection. Radiation stabilizers are known to those skilled in the art, and can include, for example, para-aminobenzoic acid, ascorbic acid, gentistic acid and the like.

[00256] A single, or multi-vial kit that contains all of the components needed to prepare the complexes of this invention, other than the radionuclide, is an integral part of this invention.

[00257] A single-vial kit preferably contains a chelating ligand, a source of stannous salt, or other pharmaceutically acceptable reducing agent, and is appropriately buffered with pharmaceutically acceptable acid or base to adjust the pH to a value of about 3 to about 9. The quantity and type of reducing agent used would depend on the nature of the exchange complex to be formed. The proper conditions are well known to those that are skilled in the art. It is preferred that the kit contents be in lyophilized form. Such a single vial kit can optionally contain labile or exchange ligands such as glucoheptonate, gluconate, mannitol, malate, citric or tartaric acid and can also contain reaction modifiers such as diethylenetriamine- pentaacetic acid (DPTA), ethylenediamine tetraacetic acid (EDTA), or α, β, or γ cyclodextrin that serve to improve the radiochemical purity and stability of the final product. The kit also can contain stabilizers, bulking agents such as mannitol, that are designed to aid in the freeze-drying process, and other additives known to those skilled in the art.

[00258] A multi-vial kit preferably contains the same general components but employs more than one vial in reconstituting the radiopharmaceutical. For example, one vial can contain all of the ingredients that are required to form a labile Tc(V) complex on addition of pertechnetate (e.g., the stannous source or other reducing agent). Pertechnetate is added to this vial, and after waiting an appropriate period of time, the contents of this vial are added to a second vial that contains the ligand, as well as buffers appropriate to adjust the pH to its optimal value. After a reaction time of about 5 to 60 minutes, the complexes of the present invention are formed. It is advantageous that the contents of both vials of this multi-vial kit be lyophilized. As above, reaction modifiers, exchange ligands, stabilizers, bulking agents, etc. can be present in either or both vials.

[00259] Also provided herein is a method to incorporate an ¹⁸F radiolabeled prosthetic group onto a c-MET targeted capture agent. In one embodiment, 4- [¹⁸F]fluorobenzaldehyde (¹⁸FB) is conjugated onto a capture agent bearing an aminooxy moiety, resulting in oxime formation, as shown in Figure 42. In another embodiment, [¹⁸F]fluorobenzaldehyde is conjugated onto a capture agent bearing an acyl hydrazide moiety, resulting in a hydrazone adduct, as shown in Figure 43. 4- Fluorobenzaldehyde, can be prepared in ¹⁸F form by displacement of a leaving group, using ¹⁸F ion, by known methods.

[00260] ¹⁸F-labeled capture agents can also be prepared from capture agents possessing thiosemicarbazide moieties under conditions that promote formation of a thiosemicarbozone, or by use of a ¹⁸F-labeled aldehyde bisulfite addition complex.

[00261] The above methods are particularly amenable to the labeling of capture agents, e.g., the capture agents described herein, which can be modified during synthesis to contain a nucleophilic hydroxylamine, thiosemicarbazide or hydrazine (or acyl hydrazide) moiety that can be used to react with the labeled aldehyde. The methods can be used for any capture agent that can accommodate a suitable nucleophilic moiety. Typically the nucleophilic moiety is appended to the N-terminus of the peptide, but the skilled artisan will recognize that the nucleophile also can be linked to an amino acid side chain or to the peptide C-terminus. Methods of synthesizing a radiolabeled peptide sequence are provided in which 4- [¹⁸F]fluorobenzaldehyde is reacted with a peptide sequence comprising either a hydroxylamine, a thiosemicarbazide or a hydrazine (or acyl hydrazide) group, thereby forming the corresponding oximes, thiosemicarbazones or hydrazones, respectively. The 4-[¹⁸F]fluorobenzaldehyde typically is generated in situ by the acid- catalyzed decomposition of the addition complex of 4-[¹⁸F]fluorobenzaldehyde and sodium bisulfite. The use of the bisulfite addition complex enhances the speed of purification since, unlike the aldehyde, the complex can be concentrated to dryness. Formation of the complex is also reversible under acidic and basic conditions. In particular, when the complex is contacted with a peptide containing a hydroxylamine, a thiosemicarbazide or a hydrazine (or acyl hydrazide) group in acidic medium, the reactive free 4-[¹⁸F]fluorobenzaldehyde is consumed as it is formed in situ, resulting in the corresponding F-18 radiolabeled peptide sequence.

[00262] In the instances when the oxime, thiosemicarbazone or hydrazone linkages present in vivo instability, an additional reduction step may be employed to reduce the double bond connecting the peptide to the F-18 bearing substrate. The corresponding reduced peptide linkage would enhance the stability. One of skill in the art would appreciate the variety of methods available to carry out such a reduction step. Reductive amination steps as described in Wilson et al., Journal of Labeled Compounds and Radiopharmaceuticals, XXVIII (10), 1 189-1 1 99, 1990 may also be used to form a Schiff's base involving a peptide and 4- [¹⁸F]fluorobenzaldehyde and directly reducing the Schiff's base using reducing agents such as sodium cyanoborohydride.

[00263] The 4-[¹⁸F]fluorobenzaldehyde may be prepared as described in Wilson et al., Journal of Labeled Compounds and Radiopharmaceuticals, XXVIII (10), 1 1 89-1 199, 1990; Iwata et al., Applied radiation and isotopes, 52, 87-92, 2000; Poethko et al., The Journal of Nuclear Medicine, 45, 892-902, 2004; and Schottelius et al., Clinical Cancer Research, 10, 3593-3606, 2004. The Na.sup.1 8F in water may be added to a mixture of kryptofix and K₂C0₃. Anhydrous acetonitrile may be added and the solution is evaporated in a heating block under a stream of argon. Additional portions of acetonitrile may be added and evaporated to completely dry the sample. The 4-trimethylammoniumbenzaldehyde triflate may be dissolved in DMSO and added to the dried F-1 8. The solution may then be heated in the heating block. The solution may be cooled briefly, diluted with water and filtered through a Waters. RTM. Oasis HLB LP extraction cartridge. The cartridge may be washed with 9:1 water:acetonitrile and water to remove unbound F-18 and unreacted 4- trimethylammoniumbenzaldehyde triflate. The 4-[¹⁸F]fluorobenzaldehyde may then be eluted from the cartridge with methanol in fractions.

Therapeutic Applications

[00264] Provided herein in certain embodiments are methods of using the c-MET capture agents disclosed herein to identify, detect, quantify, and/or separate c-MET in a biological sample. In certain embodiments, these methods utilize an immunoassay, with the capture agent replacing an antibody or its equivalent. In certain embodiments, the immunoassay may be a Western blot, pulldown assay, dot blot, or ELISA.

[00265] A biological sample for use in the methods provided herein may be selected from the group consisting of organs, tissue, bodily fluids, and cells. Where the biological sample is a bodily fluid, the fluid may be selected from the group consisting of blood, serum, plasma, urine, sputum, saliva, stool, spinal fluid, cerebral spinal fluid, lymph fluid, skin secretions, respiratory secretions, intestinal secretions, genitourinary tract secretions, tears, and milk. The organs include the prostate and lung. Tissues include breast tissue.

[00266] Provided herein in certain embodiments are methods of using the c- MET capture agents disclosed herein to diagnose and/or classify (e.g., stage) a condition associated with increased c-MET expression and/or activity, including for example various cancers. In certain of these embodiments, the methods comprise (a) obtaining a biological sample from a subject; (b) measuring the presence or absence of c-MET in the sample with the c-MET capture agent; (c) comparing the levels of c-MET to a predetermined control range for c-MET; and (d) diagnosing a condition associated with increased c-MET expression based on the difference between c-MET levels in the biological sample and the predetermined control.

[00267] Provided herein in certain embodiments are methods of treating a condition associated with increased c-MET expression and/or activity in a subject in need thereof by administering a therapeutically effective amount of one or more of the capture agents or pharmaceutical formulations disclosed herein. In certain of these embodiments, the capture agent(s) may be linked to one or more additional therapeutic agents, including for example a chemotherapeutic agent. In preferred embodiments, the capture agent is administered as a pharmaceutical composition.

[00268] A capture agent or pharmaceutical formulation may be administered to a patient in need of treatment via any suitable route. Routes of administration may include, for example, parenteral administration (including subcutaneous, intramuscular, intravenous, by means of, for example a drip patch). Further suitable routes of administration include (but are not limited to) oral, rectal, nasal, topical (including buccal and sublingual), infusion, vaginal, intradermal, intraperitoneal^, intracranial^, intrathecal and epidural administration or administration via oral or nasal inhalation, by means of, for example a nebulizer or inhaler, or by an implant.

[00269] A capture agent or pharmaceutical formulation may also be administered via microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in certain tissues including blood. Suitable examples of sustained release carriers include semi-permeable polymer matrices in the form of shared articles, e.g., suppositories or microcapsules. Examples of the techniques and protocols mentioned above and other techniques and protocols which may be used in accordance with the invention can be found in Remington's Pharmaceutical Sciences, 1 8th edition, Gennaro, A. R., Lippincott Williams & Wilkins; 20th edition (Dec. 15, 2000) ISBN 0-912734-04-3 and Pharmaceutical Dosage Forms and Drug Delivery Systems; Ansel, N. C. et al. 7th Edition ISBN 0-683305-72- 7, the entire disclosures of which is herein incorporated by reference.

[00270] Provided herein in certain embodiments is the use of the capture agents disclosed herein in the preparation of a medicament for treating a condition associated with increased c-MET expression and/or activity.

[00271] Cancers that can be treated, diagnosed, and/or classified (e.g., staged) with c-Met capture agents disclosed herein include multiple myeloma, chronic myelogenous leukemia, pancreatic cancer, non-small cell lung cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, prostate cancer, malignant melanoma, non-melanoma skin cancers, gastrointestinal stromal tumors, hematologic tumors, hematologic malignancies, childhood leukemia, childhood lymphomas, multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic origin, lymphomas of cutaneous origin, acute leukemia, chronic leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm and cancers associated with AIDS. In a particular embodiment, the cancer is selected from the group consisting of lung, breast and prostate cancer. In another particular embodiment, the cancer is lung cancer. In yet another particular embodiment, the cancer is breast cancer. In still another particular embodiment, the cancer is prostate cancer.

[00272] The c-Met capture agents of the present disclosure can also be used to present, treat or improve the activity of therapeutic agents such as anti-proliferative or tumoricidal agents against undesired cellular proliferation (such as occurs in neoplastic tumors, e.g., cancer, by providing or improving their affinity for c-Met and their residence time at a HGF/c-Met complex on proliferating cells, such as, for example, epithelial cells) for diseases associated with c-Met, including, but not limited to, diseases related to c-Met activity. In this aspect of the invention, hybrid agents are provided by conjugating a c-Met capture agent according to the disclosure with a therapeutic agent. The therapeutic agent can be a radiotherapeutic, discussed above, a drug, chemotherapeutic or tumoricidal agent, genetic material or a gene delivery vehicle, etc. The c-Met capture agent causes the therapeutic to "home" to the sites of c-Met or HGF/c-Met complex, and to improve the affinity of the conjugate for the c-Met or HGF/c-Met complex, so that the therapeutic activity of the conjugate is more localized and concentrated at the sites of cellular proliferation. In addition, these c-Met binding moieties can inhibit HGF-mediated signaling events by preventing HGF from binding to c-Met. Such conjugates will be useful in treating hyperproliferative disorders, especially neoplastic tumor growth and metastasis, in mammals, including humans. The method comprises administering to a mammal in need thereof an effective amount of a c-Met binding polypeptide or multimeric polypeptide construct according to the invention conjugated with a therapeutic agent. The invention also provides the use of such conjugates in the manufacture of a medicament for the treatment of angiogenesis associated diseases, including cancers in mammals, including humans.

[00273] Suitable therapeutic agents for use in this aspect of the invention include, but are not limited to: antineoplastic agents, such as platinum compounds (e.g., spiroplatin, cisplatin, and carboplatin), methotrexate, adriamycin, mitomycin, ansamitocin, bleomycin, cytosine, arabinoside, arabinosyl adenine, mercaptopolylysine, vincristine, busulfan, chlorambucil, melphalan (e.g., PAM, L- PAM, or phenylalanine mustard), mercaptopurine, mitotane, procarbazine hydrochloride, dactinomycin (actinomycin D), daunorubcin hydrochloride, doxorubicin hydrochloride, taxol, mitomycin, plicamycin (mithramycin), aminoglutethimide, estramustine phosphate sodium, flutamide, leuprolide acetate, megestrol acetate, tamoxifen citrate, testoiactone, trilostane, amsacrine (m-AMSA), aparaginase (L-aparaginase), Erwina aparaginase, etoposide (VP-16), interferon CX-2a, Interferon CX-2b, teniposide (VM-26, vinblastine sulfate (VLB), vincristine sulfate, bleomycin sulfate, adriamycin, and arabinosyl; anti-angiogenic agents such as tyrosine kinase inhibitors with activity toward signaling molecules important in angiogenesis and/or tumor growth such as SU541 6 and SU6668 (Sugen/Pharmacia and Upjohn), endostatin (EntreMed), angiostatin (EntreMed), Combrestatin (Oxigene), cyclosporine, 5-fluorouracil, vinblastine, doxorubicin, paclitaxel, daunorubcin, immunotoxins; coagulation factors; antivirals such as acyclovir, amantadine azidothymidine (AZT or Zidovudine), ribavirin and vidarabine monohydrate (adenine arahinoside, ara-A); antibiotics, antimalarials, antiprotozoans such as chloroquine, hydroxychloroquine, metroidazole, quinine and meglumine antimonate; anti-inflammatories such as diflunisal, ibuprofen, indomethacin, meclofenamate, mefenamic acid, naproxen, oxyphenbutazone, phenylbutazone, piroxicam, sulindac, tolmetin, aspirin and salicylates.

[00274] In one embodiment, the therapeutic agent can be associated with an ultrasound contrast agent composition in which c-Met capture agents of the disclosure are linked to the material employed to form the vesicles as described herein. After administration of the ultrasound contrast agent and the optional imaging of the contrast agent bound to the tissue expressing c-Met or HGF/c-Met complex, the tissue can be irradiated with an energy beam (preferably ultrasonic, e.g., with a frequency of from 0.3 to 3 MHz), to rupture or burst the microvesicles. The therapeutic effect of the therapeutic agent can thus be enhanced by the energy released by the rupture of the microvesicles, in particular causing an effective delivery of the therapeutic agent to the targeted tissue. For instance, the therapeutic agent can be associated with the targeted ultrasound contrast agent and delivered as described in U.S. Pat. No. 6,258,378, herein incorporated by reference.

[00275] The c-Met capture agents of the present disclosure also can be used to target genetic material to c-Met-expressing cells. Thus, they can be useful in gene therapy, particularly for treatment of hyperproliferative disorders. In this embodiment, genetic material or one or more delivery vehicles containing genetic material useful in treating a hyperproliferative disorder can be conjugated to one or more c-Met capture agents of the disclosure and administered to a patient. The genetic material can include nucleic acids, such as RNA or DNA, of either natural or synthetic origin, including recombinant RNA and DNA and antisense RNA and DNA. Types of genetic material that can be used include, for example, genes carried on expression vectors such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs) and defective or "helper" viruses, antigene nucleic acids, both single and double stranded RNA and DNA and analogs thereof, such as phosphorothioate and phosphorodithioate oligodeoxynucleotides. Additionally, the genetic material can be combined, for example, with lipids, proteins or other polymers. Delivery vehicles for genetic material can include, for example, a virus particle, a retroviral or other gene therapy vector, a liposome, a complex of lipids (especially cationic lipids) and genetic material, a complex of dextran derivatives and genetic material, etc.

[00276] In a preferred embodiment the constructs of the invention are utilized in gene therapy for treatment of hyperproliferative disorders. In this embodiment, genetic material, or one or more delivery vehicles containing genetic material, e.g., useful in treating a hyperproliferative disorder, can be conjugated to one or more c- Met capture agents of this disclosure and administered to a patient.

[00277] Constructs including genetic material and the c-Met-capture agents of this disclosure can be used, in particular, to selectively introduce genes into proliferating cancer cells (e.g., epithelial cells), which can be useful to treat cancer.

[00278] Therapeutic agents and the c-Met capture agents disclosed herein can be linked or fused in known ways, optionally using the same type of linkers discussed elsewhere in this application. Preferred linkers will be substituted or unsubstituted alkyl chains, amino acid chains, polyethylene glycol chains, and other simple polymeric linkers known in the art. More preferably, if the therapeutic agent is itself a protein, for which the encoding DNA sequence is known, the therapeutic protein and c-Met binding polypeptide can be coexpressed from the same synthetic gene, created using recombinant DNA techniques, as described above. The coding sequence for the c-Met binding polypeptide can be fused in frame with that of the therapeutic protein, such that the peptide is expressed at the amino- or carboxy- terminus of the therapeutic protein, or at a place between the termini, if it is determined that such placement would not destroy the required biological function of either the therapeutic protein or the c-Met binding polypeptide. A particular advantage of this general approach is that concatamerization of multiple, tandemly arranged c-Met capture agents is possible, thereby increasing the number and concentration of c-Met binding sites associated with each therapeutic protein. In this manner c-Met binding avidity is increased, which would be expected to improve the efficacy of the recombinant therapeutic fusion protein. [00279] Additionally, constructs including c-Met capture agents disclosed herein can themselves be used as therapeutics to treat a number of diseases associated with c-Met activity. For example, where binding of a protein or other molecule (e.g., a growth factor, hormone etc.) is necessary for or contributes to a disease process and a binding moiety inhibits such binding, constructs including such binding moieties could be useful as therapeutics. Similarly, where binding of a binding moiety itself inhibits a disease process, constructs containing such capture agents also could be useful as therapeutics.

[00280] The binding of HGF to c-Met results in the activation of numerous intracellular signal transduction pathways leading to hyperproliferation of various cells. As such, in one embodiment, constructs including c-Met capture agents that inhibit the binding of HGF to c-Met (or otherwise inhibit activation of c-Met) can be used as anti-neoplastic agents. In addition, as binding of HGF and activation of c- Met is implicated in angiogenic activity, in another embodiment, constructs including c-Met capture agents that inhibit the binding of HGF to c-Met, or otherwise inhibit activation of c-Met, can be used as anti-angiogenic agents.

[00281] The capture agents described herein are useful as therapeutic agents for treating conditions that involve endothelial and/or epithelial cells expressing c-Met. Because an important function of endothelium is angiogenesis, or the formation of blood vessels, the capture agents are particularly useful for treating conditions that involve angiogenesis and/or hyperproliferation. Conditions that involve angiogenesis include, for example, solid tumors, tumor metastases and benign tumors. Tumors caused by c-Met activation or through angiogenesis are well known in the art and include, for example, breast, lung and prostate. Additional tumors and related disorders are listed in Table I of U.S. Pat. No. 6,025,331 , issued Feb. 15, 2000 to Moses, et al., the teachings of which are incorporated herein by reference. Benign tumors include, for example, hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas. Other relevant diseases that involve angiogenesis and/or hyperproliferation include for example, rheumatoid arthritis, psoriasis, and ocular diseases, such as diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rebeosis, Osier-Webber Syndrome, myocardial angiogenesis, plaque neovascularization, telangiectasia, hemophiliac joints, angiofibroma and wound granulation. Other relevant diseases or conditions that involve blood vessel growth include intestinal adhesions, atherosclerosis, scleroderma, and hypertropic scars, and ulcers. Furthermore, the capture agents of the present invention can be used to reduce or prevent uterine neovascularization required for embryo implantation, for example, as a birth control agent.

[00282] The capture agents can be administered to an individual over a suitable time course depending on the nature of the condition and the desired outcome. The capture agents can be administered prophylactically, e.g., before the condition is diagnosed or to an individual predisposed to a condition. The capture agents can be administered while the individual exhibits symptoms of the condition or after the symptoms have passed or otherwise been relieved (such as after removal of a tumor). In addition, the capture agents disclosed herein can be administered a part of a maintenance regimen, for example to prevent or lessen the recurrence or the symptoms or condition. As described herein, the capture agents described herein can be administered systemically or locally.

[00283] The quantity of material administered will depend on the seriousness of the condition. For example, for treatment of a hyperproliferative disorder, e.g., in the case of neoplastic tumor growth, the position and size of the tumor will affect the quantity of material to be administered. The precise dose to be employed and mode of administration must per force, in view of the nature of the complaint, be decided according to the circumstances by the physician supervising treatment. In general, dosages of the capture agents disclosed herein will follow the dosages that are routine for the therapeutic agent alone, although the improved affinity of a binding polypeptide or multimeric polypeptide construct of the invention for its target can allow for a decrease in the standard dosage.

[00284] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. EXAMPLES

[00285] Azido amino acids: The structure of the azido amino acid Az4 is shown in Figure 23.

[00286] Az4 synthesis was carried out as described previously (Agnew

2009).

Example 1.

Selection of epitope for PCC development

[00287] The crystal structure of human c-MET in complex with HGF (1 SHY) was downloaded from the Protein Data Bank. Polar contacts between the two proteins were visualized using PyMOL. In Figure 1 , it is demonstrated that Asp190 of c-MET is interacting strongly with Arg533 of HGF, while Arg191 of c-MET has polar interactions with Asp578 and Glu656 of HGF. Therefore, we selected this portion of protein as the epitope of interest.

[00288] To provide a "clickable" handle close to the interface between c-MET and HGF, the neighboring Lys189 was mutated into L-azidolysine. The structural similarity between lysine and L-azidolysine should provide minimal perturbation to the peptide structure. To determine the peptide length, attention was focused on the surrounding structural motif of the epitope of interest. The c-MET crystal structure reveals that the epitope assumes a hairpin structure, formed by two anti-parallel β- strands with hydrogen bonds. Therefore, the epitope was synthesized to include these two anti-parallel β-strands to ensure correct peptide folding. A similar exercise was performed to obtain other possible epitopes of interest.

[00289] The c-MET-derived peptides generated are listed as follows:

1 . ¹⁸¹GAKVLSSV-Az4-DRFINFFVGN¹⁹⁹

where Lys1 89→ L-azidolysine

2. ²¹⁶VRRLKETKDGFMFLTD²³¹-Az4

3. ¹¹⁶INMALVVDTYYDD-Az4-LISSGS¹³⁵

where Gln1 29→ L-azidolysine and Cys133→Ser

Example 2.

Evaluation of epitope targeting peptides through HGF assays [00266] To measure the ability of a c-MET-derived polypeptide to inhibit human HGF binding to c-MET, ELISA plates were coated with 2 μg/mL recombinant human HGFR/c-MET Fc chimera (R&D Systems, #358-MT-100/CF) in PBS at 25 °C for 2 h. After washing each well with TBS (3x), wells were blocked overnight at 4 °C with 1 % BSA in TBS containing 0.1 % (v/v) Tween 20. Three-fold serial dilutions of epitope polypeptide were incubated with 1 0 nM biotinylated HGF in TBS containing 0.1 % (v/v) Tween 20 for 2 h in tubes. The biotinylated HGF was produced by using EZ- Link Sulfo-NHS-LC-Biotinylation Kit (Pierce, #21435; Rockford, IL). The solutions from the tubes were then transferred to the ELISA plates and incubated for 5 min. The plate was washed with TBS containing 0.1 % (v/v) Tween 20 (5 x). Bound biotinylated HGF was detected using 0.2 μg/mL horseradish peroxidase-labeled streptavidin (Abeam, #ab7403) prepared in TBS containing 0.1 % (v/v) Tween 20. Wells were washed with TBS containing 0.1 % (v/v) Tween 20 (5x) followed by TBS (5x) and then developed by adding QuantaRed™ Enhanced Chemifluorescent HRP Substrate. Using an excitation wavelength of 535 nm, fluorescent emission at 595 nm was recorded by Beckman Coulter DTX880 photometer (Brea, CA) as a function of polypeptide concentration. The titration curves were fit using a four-parameter regression curve fitting program (Origin 8.5, Northampton, MA). Concentrations of peptides corresponding to the midpoint absorbance of the titration curve were calculated and used as the IC₅o values.

[00267] The in vitro blocking potential of Polypeptides 1 and 2 is shown in Figure 2. Concentration-dependent competitive binding was observed for

Polypeptidel , suggesting that this sequence can act as a mimic for full-length c- MET. Also, these results suggest specific targeting of the interface between c-MET and HGF, as was designed, using a synthetic polypeptide.

Example 3.

Selecting Epitope-tarqeted Anchor Selection by In Situ Click Screen

[00268] From the HGF assay, we chose the best epitope targeting peptide (Biotin-(PEG)₃-GAKVLSSV-Az4-DRFINFFVGN) to select a de novo anchor against c-MET. For the screen, a 200-mg portion of the OBOC library, coupled with D- propargylglycine at the N-terminus, was transferred into an 8-mL capacity Alltech vessel and pre-incubated in a blocking solution consisting of 0.1 % Tween 20 and 1 % BSA in TBS buffer overnight on a 360°-rotator at 4 °C. After draining the blocking solution from the beads, 3 ml_ of 1 :10,000 AP-linked Streptavidin (Promega) was incubated for 1 h at 25 °C. The screen was washed with 5 ^χ 3 ml_ Blocking Solution, 5 x 3 ml_ Wash 1 Buffer (25 mM Tris-CI, 10 mM MgCI₂, 700 mM NaCI, pH 7.5), followed by 5 x 3 mL wash 2 Buffer (25 mM Tris-CI, pH 7.5), and drained by vacuum. BCIP:NBT (Promega #S3771 ), freshly prepared in Alkaline Phosphatase Buffer (100 mM Tris-HCI [pH 9.0], 150 mM NaCI, 1 mM MgCI₂), was used to develop the screen. The most intensely colored purple beads ("background hits") were selected manually. Since these beads represent sequences that would non-specifically bind to AP-linked Streptavidin, they would be discarded. The selection process typically lasts for an hour, with 1 N HCI being added to the screen to eliminate further background development. The clear beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound proteins, followed by NMP.

[00269] Next, selected beads were incubated in blocking solution to prepare for a second round of screening. After the blocking solution was drained, a 2 mL volume of 50 μΜ epitope targeting peptide (2% (v/v) DMSO) was added to the beads for 5 h on a 360°-rotator at 25 °C. The beads were rinsed with TBS. Subsequently, the beads were incubated with 7.5 M guanidine hydrochloride (pH 2.0) to remove any non-specific binding peptides for 1 h. After washing the beads thoroughly with TBS, they were incubated in blocking buffer for 2 h. The subsequent steps involving AP-linked Streptavidin and development of library were repeated. The most intensely colored purple beads ("hits") were selected. The hit beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound proteins, followed by NMP for 6 h. The hit beads were then incubated in water overnight. Hit sequencing was performed with MALDI-TOF/TOF and a semi-automated algorithm (S. S. Lee et al. Anal. Chem. 82: 672-679 (201 0)).

Example 4.

CNBr Cleavage of Peptides from Single Beads.

[00270] A single bead was transferred using pure water (1 0 μί) to a 96-well plate, with one bead per well. The plate was purged with argon. Then, CNBr (10 μί, 0.50 M in 0.2 N HCI solution) was added to each well. The plate was again purged with argon and placed under microwave for 90 s (S. S. Lee et al. J. Comb. Chem. 10: 807-809 (2008)). The resulting solution was concentrated under centrifugal vacuum for 2 h. Example 5.

MALDI-MS and MS/MS Analysis of Peptides Cleaved from Single Beads.

[00271] To each well, a-cyano-4-hydroxycinnamic acid (CHCA, 0.5 μΙ_, 0.5% matrix solution in acetonitrile/water (70:30)) were added. The mixture solution was spotted onto a 384-well MALDI plate. Mass spectra were acquired and analyzed to find the parent ion peak. Another round of mass spectra was obtained after the instrument was calibrated. The MS/MS spectra were obtained by entering the pre- calibrated parent ion mass and analyzed with the following parameters:

1 . Optional Modification: Oxidation (W)" and Oxidation 02 (W)"

2. Fixed N-terminal modification:" Propargylglycine (G)"

3. Fixed modifications: "Homoserinelacton3"

4. Fixed C-terminal modifications: "Free Acid"

5. Set Mass Tol. MS at 0.3 Da

6. Set MS/MS Tol. At 0.5 Da

7. Enter calibrated parent mass under "Parent mass (MH+)"

8. Click "Calculate".

[00272] The top sequences supplied were noted. The MS/MS spectra were reanalyzed after changing MS/MS Tol. to 1 . If different sequences were obtained, these sequences were noted as well. The sequences can be further confirmed if tryptophan oxidation (M+16), isoleucine (M-42) and glutamine (M-71 ) substitution peaks were available.

[00273] A spreadsheet with all the sequences obtained was compiled. The frequencies of amino acids appearing at various positions were counted. For the samples with multiple possible sequences, analyze them using the information from the frequency table. Choose the sequence that follows the pattern from this frequency analysis. Regions of sequence homology were highlighted. For example, the motif trwxx has been repeated thrice, which txdll has appeared twice. Individual amino acids were also color-coded according to their physicochemical properties (Figure 3):

1 . Red (positive): Histidine, Lysine, and Arginine 2. Green (Aromatic): Phenylalanine, Tyrosine, and Tryptophan

3. Orange (polar): Serine, Threonine, Asparagine, and Glutamine

4. Black (non-polar): Alanine, Valine, Leucine, Isoleucine, Proline, and Glycine

5. Blue (negative): Aspartic Acid and Glutamic Acid

[00274] Even though dtltv and dhvtv only match by three amino acids, we can consider leucine and valine at their respective x4 positions as similar because of the physicochemical properties. Similarly, although takhp and tlhrk only match by the threonine at the x2 position, we can consider these two sequences similar to each other because of the neutral amino acids found at x3 and positive amino acids at x4 and x5.

[00275] The finalized sequences were submitted to bioinformatic clustering analysis. The analysis was focused on the sequences clustering in the perimeter of the universe, and special attention was paid to the sequences with high homology. Approximately 1 0 peptide candidates were chosen that represent the clusters located along the perimeter of the universe and share similar sequence (Figure 4). Candidates were chosen to ensure that most clusters are well represented. These anchor candidates were synthesized and evaluated by in vitro performance assays. Using the pair of tlhrk and takhp as an example, tlhrk is located at the perimeter of the universe, while takhp is more buried inside. Therefore, tlhrk has been selected to be synthesized. On the other hand, tldll

tndll have both been chosen because they can be found at the different clusters at the perimeter of the universe.

Example 6.

De novo anchor synthesis

[00276] After candidates of the de novo anchors were identified, large-scale production of material was required for in vitro assays. Each anchor was prepared using a combination of conventional Fmoc-based solid phase peptide synthesis (SPPS). Specifically, the differing de novo anchors were synthesized in parallel onto Rink amide resin using an AAPPTEC Titan 357 peptide synthesizer. Each amino acid coupling reaction incorporated 4 equiv of Fmoc-amino acid, 4 equiv of HBTU (O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate), and 10 equiv of DIEA. Deprotection of the Fmoc group required 20% piperidine/NMP. Each anchor was coupled with a PEG linker and biotin as a reporter. These anchors would then be released from the Rink resin through an acid cleavage step and purified using high-performance liquid chromatography.

Example 7.

Pull downs on de novo anchor candidates.

[00277] A pull-down assay was used to assess capture agent specificity for c- MET by measuring the ability of the capture agents to purify c-MET from buffer or complex media such as human serum. Capture agents were immobilized on streptavidin-functionalized magnetic beads, and the resultant resins were panned with c-MET-spiked serum or buffer.

[00278] Pull-down detection of c-MET was performed using a modified immunoprecipitation technique that incorporated PCCs rather than antibodies. First, biotinylated PCC (400 nM; 0.1 % DMSO, v/v) was incubated with 0.5 μg/mL recombinant human HGFR/c-MET Fc Chimera (R&D Systems, #358-MT-100/CF; Minneapolis, MN) in 1 ml_ TBS containing 0.02% Triton X-100 at 4 °C overnight. Separately, biotinylated PCC (400 nM; 0.1 % DMSO, v/v) was incubated with 0.5 μg/mL c-MET in 1 ml_ of 1 % (v/v) human AB male serum (Omega Scientific, #HS-20; Tarzana, CA) under the same conditions (4 °C, overnight). A vehicle-only control (0.1 % DMSO, v/v) accompanied each sample. Proteins were captured by

Dynabeads® M-280 Streptavidin (Invitrogen, 1 1 2-05D; Oslo, Norway) under rotation at 4 °C for 4 h (100 μΙ_ of 50% slurry per pull-down condition). Beads were separated from the serum or buffer matrix by application of the DynaMag™-Spin magnet (Invitrogen, #123-20D), and captured proteins were eluted from the beads in 30 μΙ_ of reducing Laemmli buffer. Eluted samples were subjected to 7.5% SDS- PAGE separation at 200 V for 30 min in 1 x TGS (25 mM Tris, 192 mM Glycine, 0.1 % SDS (w/v), pH 8.3). Samples were subsequently electrophoretically transferred to a nitrocellulose membrane in 25 mM Tris, 192 mM Glycine, pH 8.3, containing 20% (v/v) methanol (Bio-Rad Laboratories, Hercules, CA) at 1 00 V for 30 min at 4 °C.

[00279] Following transfer, the nitrocellulose membrane was blocked at 4 °C for 2 h in 5% non-fat dry milk in TBS. The membrane was then washed with TBS (3 ^χ), and 0.2 μg/mL goat anti-human HGFR/c-MET biotinylated antibody (R&D Systems, #BAF358) in 0.5% non-fat dry milk in TBS was incubated at 4 °C overnight. After washing with TBS containing 0.02% Tween20 (v/v) (5 χ), 0.1 μg/mL HRP-conjugated streptavidin (Abeam, #ab7403; Cambridge, MA) in 0.5% non-fat filtered milk in TBS was added to the membrane (4 °C, 1 h incubation). After washing with TBS containing 0.02% Tween20 (v/v) (5 ^χ), followed by TBS (5 ^χ), the membrane was developed with SuperSignal West Pico Chemiluminescent Enhancer and Substrate Solutions (Pierce, IL) and then immediately exposed to HyBlot CL AR film.

[00280] Results of the pull-down assay for DAnchors 1 to 8 are set forth in Figure 5. Probing the elutions via Western blot with a c-MET antibody suggests that DAnchorl (trwlr) and DAnchor2 (irnwk) display the largest c-MET bands in buffer (B), and correspondingly the highest affinity towards the target. At this stage of development, de novo anchors are so weakly binding that c-MET detection in 1 % (v/v) human serum (S) is not observed. Nevertheless, the pull-down assay has informed the prioritization of DAnchors 1 and 2 for selecting a biligand against c- MET. A biotinylated polyclonal antibody BAF358 (R&D Systems) has been validated as a positive control charting the maximum possible capture of c-MET in buffer and serum matrices.

Example 8.

Selecting PCC Biligand by In Situ Click Screen

[00281] From the pull down assay, we have chosen the best de novo anchor (Biotin-(PEG)₃-trwlr-Az4) to select a biligand targeting against c-MET. For the screen, a 200-mg portion of the OBOC library, coupled with D-propargylglycine at the N-terminus, was transferred into an 8-mL capacity Alltech vessel and pre- incubated in a blocking solution consisting of 0.1 % Tween 20 and 1 % BSA in TBS buffer overnight on a 360 °-rotator at 4 °C. After the blocking solution was drained, a 2 ml_ volume of 50 μΜ azide-modified de novo anchor (2% (v/v) DMSO) was added to the beads for 4 h on a 360 °-rotator at 25 °C. The beads were rinsed with TBS. Subsequently, the beads were incubated with 7.5 M guanidine hydrochloride (pH 2.0) to remove any non-specific binding peptides for 1 h. After washing the beads thoroughly with TBS, they were incubated in blocking buffer for 2 h. The subsequent steps involving AP-linked Streptavidin and development of library were repeated. Clear beads were removed, while the most intensely colored purple beads

("background hits") were left behind. The clear beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound materials, followed by NMP. [00282] Next, selected beads were incubated in blocking solution for 2 h at 25 °C to prepare for a second round of screening. In the meantime, 100 μg of c-MET (R&D Systems, 358-MT/CF) was dissolved with 2 ml_ volume of blocking solution. In addition, 50 μΜ azide-modified de novo anchor (2% (v/v) DMSO) was added to the c-MET solution and shaken for 2 h at 25 °C. This solution was subsequently added to the selected beads and incubated for 4 h. The protein/anchor solution was then drained. The beads were then shaken with 7.5 M guanidine hydrochloride (pH 2.0) to remove any non-covalently bound materials for 1 h. After washing the beads thoroughly with TBS, they were incubated in blocking buffer for 2 h. The subsequent steps involving AP-linked Streptavidin and development of library were repeated. The most intensely colored purple beads ("hits") were selected. The hit beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound materials, followed by NMP for 6 h. The beads were then incubated in water overnight. Hit sequences were subsequently identified following the protocols previously outlined.

[00283] Biligand candidates were then selected through sequence homology (Figure 6). It is observed that the motif xkrhG and xhGxp have been frequently repeated. The physicochemical properties of the sequences have been analyzed. It can be illustrated by grouping fiwrv and flrrp. Even though there are only two matching amino acids, position x3 and x6 can be considered similar because they are all neutral amino acids.

[00284] Bioinformatics analysis (Figure 7) was also employed to assist sequence selection. Using the frequently repeated xhGxp as an example, ihGep was chosen because it can be found at the perimeter of the universe. Conversely, fhGhp and nhGkp were not selected to be synthesized because they were found closer to the origin of the universe. It is also important to ensure that most clusters were well represented. For example, kvrhG and pkrhG were both selected as potential secondary ligands because they belonged to different clusters on the universe.

Example 9.

Biligand candidate synthesis.

[00285] After candidates of the secondary ligands were identified, large-scale production of material was required for in vitro assays. The differing secondary ligands were synthesized in parallel onto Rink amide resin using an AAPPTEC Titan 357 peptide synthesizer. Each amino acid coupling reaction incorporated 4 equiv of Fmoc-amino acid, 4 equiv of HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), and 1 0 equiv of DIEA. Deprotection of the Fmoc group required 20% piperidine/NMP. Each secondary ligand was coupled with Boc-D- propargylglycine at the N-terminus.

[00286] The biligands candidates were synthesized in parallel as described. Rink amide resin (25 mg, 0.5 mmol/g loading, 0.013 μιηοΙ) containing protected secondary ligands capped with Boc-D-propargylycine at the N-terminus was washed with NMP (3x), MeOH (3x), and CH₂CI₂ (3x). Side chain protected biotin-(PEG)₃- trwlr-Az4 (5 eq.) dissolved NMP (0.5 ml_) was added to the resin followed by copper(l)iodide (2.5 mg) and sodium ascorbate (0.1 ml_ of 1 .0 M aqueous solution). The mixture was agitated for 1 6 h, filtered, and then washed with NMP (3x), MeOH (3x), and CH₂CI₂ (3x). Peptides were cleaved from the resin (TFA/TIS/DODT/H₂0, 92.5 : 2.5 : 2.5 : 2.5) and purified via reversed phase HPLC.

Example 10.

In Vitro Inhibition of HGF binding to c-MET bv PCC Agents

[00287] To measure the ability of PCC Agents to inhibit human c-MET binding to HGF, ELISA plates were coated with 2 μg/mL HGF (R&D Systems, #294-HGN- 025/CF) in PBS at 25 °C for 2 h. After washing each well with TBS (3x), wells were blocked overnight at 4 °C with 1 % BSA in TBS containing 0.1 % (v/v) Tween 20. Three-fold serial dilutions of PCC Agent were incubated with 10 nM biotinylated c- MET in TBS containing 0.1 % (v/v) Tween 20 for 2 h in tubes. The biotinylated c- MET was produced by using EZ-Link Sulfo-NHS-LC-Biotinylation Kit (Pierce, #21435; Rockford, IL). The solutions from the tubes were then transferred to the ELISA plates and incubated for 5 min. The plate was washed with TBS containing 0.1 % (v/v) Tween 20 (5 x). Bound biotinylated c-MET was detected using 0.2 μg/mL horseradish peroxidase-labeled streptavidin (Abeam, #ab7403) prepared in TBS containing 0.1 % (v/v) Tween 20. Wells were washed with TBS containing 0.1 % (v/v) Tween 20 (5x) followed by TBS (5x) and then developed by adding QuantaRed™ Enhanced Chemifluorescent HRP Substrate. Using an excitation wavelength of 535 nm, fluorescent emission at 595 nm was recorded by Beckman Coulter DTX880 photometer (Brea, CA) as a function of PCC Agent concentration. The titration curves were fit using a four-parameter regression curve fitting program (Origin 8.5, Northampton, MA). Concentrations of peptides corresponding to the midpoint absorbance of the titration curve were calculated and used as the IC₅o values.

[00288] Results of the competitive binding assay for DAnchorl (trwlr) and Biligands 1 and 3 are shown in Figure 8. Concentration-dependent competitive binding by DBiligandl (kvrhG) and DBiligand3 (pkrhG) demonstrates that biligands attenuate the binding of c-MET to HGF. Results suggest that the targeted epitope lies in the growth factor binding region of c-MET, as was originally dictated by building the de novo anchor against a synthetic polypeptide. Receptor blocking by the biligands, as a class, can be described quantitatively as IC₅₀ ~ 10-20 μΜ.

Example 11.

Direct ELISA

[00289] A direct, solid-phase microplate enzyme-linked immunosorbent assay (ELISA) was used to measure in vitro binding of capture agents to c-MET Fc chimera (R&D Systems, #358-MT/CF). The equilibrium dissociation constant (KD) for the capture agents may be estimated as the concentration corresponding to half- maximal fluorescent emission. Assaying multiple capture agents in parallel permits relative comparison of in vitro binding.

[00290] A streptavidin-coated plate (Greiner Bio-One, #655997) was incubated with serially diluted biotinylated PCC Agent in TBS and incubated for 2 h at 25 °C. After washing each well with TBS (2x), followed by PBS (3 χ) , the plate was filled with 1 % BSA, 5% sucrose, 0.05% NaN₃ in PBS and blocked overnight at 4 °C. The plate was washed with PBS (2x), then PBS containing 0.05% (v/v) Tween 20 (3 χ) . HRP conjugation to recombinant human c-MET Fc chimera (R&D Systems, #358- MT/CF) was carried out following the protocol provided by the EZ-link Plus Activated Peroxidase Kit (Thermo Scientific, #31489). 15 nM HRP-conjugated recombinant human c-MET (Fc chimera) was incubated on the PCC-coated plate for 1 h at 25 °C. The plate was washed with PBS containing 0.05% (v/v) Tween 20 (5 χ) , followed by PBS (5 x) , and then developed by adding QuantaRed™ Enhanced Chemifluorescent HRP Substrate. Using an excitation wavelength of 535 nm, fluorescent emission at 595 nm was recorded by Beckman Coulter DTX880 photometer (Brea, CA) as a function of PCC Agent concentration. The titration curves were fit using a four- parameter regression curve fitting program (Origin 8.5, Northampton, MA). A control experiment was run using a serial dilution of human c-MET Biotinylated Affinity Purified Ab, Goat IgG (R&D, #BAF358) following the same protocol described above.

[00291] Results for the affinity of DAnchorl (trwlr) and Biligands 1 and 3 are shown in Figure 9, and are viewed as independent validation of the IC₅o results. As a detection agent, both biligands surpass the affinity of the de novo anchor. Affinity enhancements are a consequence of building length and morphology into the PCC per ligand addition. DBiligandl (kvrhG) shows ~100x to 1000x improvement in affinity over the de novo anchor. This affinity can be described quantitatively as KD * 250 nM.

Example 12.

Pull downs on biliqand candidates.

[00292] Again, pull-down detection of c-MET was performed using a modified immunoprecipitation technique that incorporated PCCs rather than antibodies. The biotinylated polyclonal antibody BAF358 (R&D Systems) has been implemented as a positive control in buffer and serum matrices. Results of the pull-down assay for Biligands 1 to 3 are set forth in Figure 10. Silver staining and Western blot results show superior pull-down of c-MET in buffer (B) by Biligands 1 and 3. These peptides also display effective pull-down of c-MET in 1 % (v/v) human serum (S), surpassing the de novo anchor. Thus, improved specificity is demonstrated by biligands.

[00293] Results of the pull-down assay for Biligands 4 to 6 are shown in Figure

1 1 . Specificity is not improved for these biligand candidates. Biligands 4 and 5 are weakly binding to c-MET, even in buffer (B). While Biligand 6 binds to c-MET effectively in buffer (B), this hydrophobic peptide shows significant off-target, high- MW interactions in 1 % (v/v) human serum (S).

[00294] Results of the pull-down assay for Biligands 7 to 9 are shown in Figure

12. Affinity is improved for these biligand candidates, particularly Biligand 9. Affinity improvements are marked by increased c-MET pull-down in buffer (B). However, specificity of Biligands 7 to 9 appears to be unimproved since no c-MET was detected from 1 % (v/v) human serum (S).

[00295] Results of the pull-down assay for Biligands 10 to 13 are shown in Figure 13. Silver staining and Western blot results show increased pull-down of c- MET in buffer (B) by Biligands 1 0 and 1 1 . These peptides also display effective pulldown of c-MET in 1 % (v/v) human serum (S), suggestive of improved specificity over de novo anchor. In contrast, Biligand 12 is weakly binding to c-MET, even in buffer. While Biligand 13 also binds to c-MET effectively in buffer (B), this hydrophobic peptide shows significant off-target, high-MW interactions in 1 % (v/v) human serum (S).

[00296] The pull-down assay has informed the prioritization of Biligands 1 and 3 for selecting a triligand against c-MET. It is noted that Biligands 1 , 3, and 10 share a common C-terminal motif "rhG" which may contribute to an overall similar specificity profile. Biligands 7 and 8 share a common C-terminal motif "whG" which may contribute to another discrete specificity profile. In addition, Biligands 10 and 1 1 share the same amino acid composition but differ in sequence and specificity, providing example of the precise targeting of c-MET that can be achieved with PCCs.

Example 13.

Selecting PCC Triligand by In Situ Click Screen

[00297] From the pull down assay, we have chosen the best biligand (Biotin- (PEG)₃-trwlr-Tz4-kvrhG-Az4) to select a triligand targeting against c-MET. For the screen, a 200-mg portion of the OBOC library, coupled with D-propargylglycine at the N-terminus, was transferred into an 8-mL capacity Alltech vessel and pre- incubated in a blocking solution consisting of 0.1 % Tween 20 and 1 % BSA in TBS buffer overnight on a 360 °-rotator at 4 °C. After the blocking solution was drained, a 2 ml_ volume of 25 μΜ azide-modified biligand (2% (v/v) DMSO) was added to the beads for 4 h on a 360°-rotator at 25 °C. The beads were rinsed with TBS.

Subsequently, the beads were incubated with 7.5 M guanidine hydrochloride (pH 2.0) to remove any non-specific binding peptides for 1 h. After washing the beads thoroughly with TBS, they were incubated in blocking buffer for 2 h. The subsequent steps involving AP-linked Streptavidin and development of library were repeated. Clear beads were removed, while the most intensely colored purple beads

("background hits") were left behind. The clear beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound materials, followed by NMP. [00298] Next, selected beads were incubated in blocking solution for 2 h at 25 °C to prepare for a second round of screening. In the meantime, 100 μg of c-MET (R&D Systems, 358-MT/CF) was dissolved with 2 ml_ volume of blocking solution. In addition, 25 μΜ azide-modified biligand (2% (v/v) DMSO) was added to the c-MET solution and shaken for 2 h at 25 °C. This solution was subsequently added to the selected beads and incubated for 4 h. The protein/biligand solution was then drained. The beads were then shaken with 7.5 M guanidine hydrochloride (pH 2.0) to remove any non-covalently bound materials for 1 h. After washing the beads thoroughly with TBS, they were incubated in blocking buffer for 2 h. The subsequent steps involving AP-linked Streptavidin and development of library were repeated. The most intensely colored purple beads ("hits") were selected. The hits beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound materials, followed by NMP for 6 h. The beads were then incubated in water overnight. Hit sequences were subsequently identified following the protocols previously outlined.

[00299] Triligand candidates were then selected through sequence homology (Figure 14). Many recurring motifs were observed, such as xswwr, fpfxr, xwwwt, xqrqw, wxqlr, xwwlr, fwrnx, and fwrix. Bioinformatics analysis (Figure 1 5) was also employed. Sequences to be synthesized as tertiary ligand candidates can be found at different clusters located at the perimeter of the universe.

Example 14.

Triligand candidate synthesis.

[00300] After candidates of the tertiary ligands were identified, large-scale production of material was required for in vitro assays. The differing tertiary ligands were synthesized in parallel onto Rink amide resin using an AAPPTEC Titan 357 peptide synthesizer. Each amino acid coupling reaction incorporated 4 equiv of Fmoc-amino acid, 4 equiv of HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium- hexafluoro-phosphate), and 1 0 equiv of DIEA. Deprotection of the Fmoc group required 20% piperidine/NMP. Each tertiary ligand was coupled with Boc-D- propargylglycine at the N-terminus.

[00301] The triligand candidates were synthesized in parallel as described. Rink amide resin (25 mg, 0.5 mmol/g loading, 0.013 μιηοΙ) containing protected tertiary ligands capped with Boc-D-propargylycine at the N-terminus was washed with NMP (3x), MeOH (3x), and CH₂CI₂ (3x). Side chain protected biotin-(PEG)₃- trwlr-Tz4-kvrhG-Az4 (5 eq.) dissolved NMP (0.5 mL) was added to the resin followed by copper(l)acetate (2.5 mg) and sodium ascorbate (0.1 mL of 1 .0 M aqueous solution). The mixture was agitated for 1 6 h, filtered, and then washed with NMP (3x), MeOH (3x), and CH₂CI₂ (3x). Peptides were cleaved from the resin

(TFA/TIS/DODT/H₂O, 92.5 : 2.5 : 2.5 : 2.5) and purified via reversed phase HPLC.

Example 15.

Pull downs on triliqand candidates.

[00302] Again, pull-down detection of c-MET was performed using a modified immunoprecipitation technique that incorporated PCCs rather than antibodies. The biotinylated polyclonal antibody BAF358 (R&D Systems) has been implemented as a positive control in buffer and serum matrices. Results of the pull-down assay for Triligands 1 to 3 are set forth in Figure 1 6. Probing the elutions via Western blot with a c-MET antibody confirms the increase in capture efficiency in buffer (B) and 1 % (v/v) human serum (S) as the combined affinity/specificity metrics of the PCC are improved from anchor to biligand to triligand. Analysis of the total

immunoprecipitated protein by SDS-PAGE shows low to tolerable non-selective binding for all PCCs, and correlates well with the capture efficiency for c-MET.

[00303] Results of the pull-down assay for Triligands 4 to 7 are shown in Figure 17. Affinity is the same or improved for Triligands 4, 5, and 7, as shown by c-MET pull-down in buffer (B). However, specificity of Triligands 4, 5, and 7 appears to be unimproved since no c-MET was detected from 1 % (v/v) human serum (S). Silver staining and Western blot results for Triligand 6 show increased capture of c-MET both in buffer (B) and 1 % (v/v) human serum (S), suggestive of improved specificity over the biligand.

[00304] Results of the pull-down assay for Triligands 8 to 1 1 are shown in Figure 18. Affinity is improved for Triligand 8, as shown by c-MET pull-down in buffer (B). However, specificity of Triligand 8 appears to be unimproved since no c- MET was detected from 1 % (v/v) human serum (S). Silver staining and Western blot results for Triligands 9, 10, and 1 1 show increased capture of c-MET both in buffer (B) and 1 % (v/v) human serum (S), suggestive of improved specificity over the biligand.

[00305] The pull-down assay has informed the prioritization of Triligands 1 , 2, 3, 6, 9, 10, and 1 1 for in vitro cellular binding characterization and in vivo imaging of c-MET in preclinical animal models.

[00306] As shown in Figure 24, another pull-down detection of c-MET was performed. Biotinylated capture agent (400 nM; 0.1 % DMSO, v/v) was incubated with a serial dilution of recombinant human HGFR/c-MET Fc Chimera (R&D Systems, #358-MT-1 00/CF; 1 μg/mL to 12 ng/mL) in 2 ml_ TBS containing 0.02% Triton X-1 00 at 4 °C overnight. The biotinylated polyclonal antibody BAF358 (R&D Systems; 2 nM) was implemented as a positive control for the same c-MET titration. Proteins were captured by Dynabeads® M-280 Streptavidin (Invitrogen, 1 12-05D) under rotation at 4 °C for 4 h (1 00 μΙ_ of 50% slurry per pull-down condition). Beads were separated from the buffer matrix by application of the DynaMag™-Spin magnet (Invitrogen, 123-20D), and captured proteins were eluted from the beads in 30 μΙ_ of reducing Laemmli buffer. Eluted samples were subjected to 7.5% SDS-PAGE separation at 200 V for 30 min in 1 x TGS (25 mM Tris, 192 mM Glycine, 0.1 % SDS (w/v), pH 8.3). Samples were subsequently electrophoretically transferred to a nitrocellulose membrane in 25 mM Tris, 192 mM Glycine, pH 8.3, containing 20% (v/v) methanol (Bio-Rad Laboratories) at 100 V for 40 min.

[00307] Following transfer, the nitrocellulose membrane was blocked at 4 °C for 2 h in 5% (w/v) non-fat dry milk in TBS. The membrane was then washed with TBS (3 x) , and incubated with 0.2 μg/mL goat anti-human HGFR/c-MET biotinylated antibody (R&D Systems, #BAF358) in 0.5% (w/v) non-fat dry milk in TBS at 4 °C overnight. After washing with TBS containing 0.02% Tween20 (v/v) (5 χ) , 0.1 μg/mL HRP-conjugated streptavidin (Abeam, #ab7403) in 0.5% (w/v) non-fat dry milk in TBS was added (4 °C, 1 h incubation). After washing with TBS containing 0.02% Tween20 (v/v) (5 χ) , followed by TBS (5 χ) , the membrane was developed with SuperSignal® West Pico Chemiluminescent Substrate (Pierce, 34087) and then immediately exposed to HyBlot CL AR film.

[00308] Results (Figure 24) show improvements in capture agent affinity by adding successive ligands. Biligand has ~3 times greater limit of detection than anchor, and triligand has >3 times greater limit of detection than biligand. The limits of detection for the DAnchor 1 , Biligand 1 , and Triligand 2 were, respectively, 0.1 μg/mL, -0.03 μg/mL, and <0.01 μg/mL c-MET.

Example 16.

Radiolabelinq of biligand and triligand candidates with an ¹⁸F-labeled prosthetic group.

[00309] The preparation of 4-[¹⁸F]fluorobenzaldehyde and site-specific conjugation of this aldehyde to aminooxy-functionalized biligand and triligand candidates were conducted based on modified procedures of T. Poethko et al. (J. Nucl. Med. 2004, 45, 892-902) (Figure 19).

Example 17.

Assays to confirm binding to target on c-MET-expressinq tumor cell lines in culture

[00310] Prostate cancer cell lines PC-3 (#CRL-1435), DU 145 (#HTB-81 ), 22Rv1 (#CRL-2505), and LNCaP (#CRL-1 740) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and grown in RPMI-1640 media supplemented with 1 0% (v/v) fetal bovine serum under standard cell culture conditions. PC-3 and DU 145 human prostate adenocarcinoma cells over- expressing c-MET, and 22Rv1 and LNCaP cells, which express low levels of c-MET, were used for these studies.

[00311] 17.1 . Fluorescence imaging experiments:

[00312] To analyze PCC binding to c-MET expressed on the surface of cultured tumor cells, fluorescently labeled biligand and triligand were synthesized by coupling fluorescein isothiocyanate (FITC) to the N-terminus. FITC-labeled Biligand 1 , FITC- labeled Triligand 2, or free fluorescein dye (as a control) were incubated with the prostate cancer cell lines at 50 μΜ in growth medium containing 1 % fetal bovine serum (FBS) as a blocking agent. Cells were treated for 1 h at 37 °C, washed twice with 0.1 % bovine serum albumin in phosphate buffer, and then imaged immediately using a Zeiss LSM 510 Meta confocal microscope with excitation and emission wavelengths set for FITC (Figure 20). Brightfield and fluorescence images were overlaid, and images represent 225 μιπ χ 225 μιπ in area. Fluorescence intensities were quantitated using ImageJ and are shown in Figure 21 for triligand (A) and biligand (B). [00313] The most intense fluorescence was visualized in the high c-MET- expressing cells treated with FITC-labeled Triligand 2 (Figures 20A, 21 A). Weak fluorescence was visualized in the high c-MET-expressing cells treated with FITC- labeled Biligand 1 (Figures 20B, 21 B). In part, this may be explained by the difference in affinity between biligand and triligand. Background autofluorescence for each cell line was visualized in the control assay (Figure 20C). These results suggest target-dependent binding of labeled PCCs to prostate cancer cells in vitro.

[00314] 17.2. RIMChip/betabox experiments:

[00315] 50-100 cells of PC-3, in quadruplicate, were exposed to 0 and 22 μΏ/ΓηΙ_ of ¹⁸F-labeled Triligand 2 for 30 min at 37 ⁵C on Radiopharmaceutical imaging chip (RIMChip) and then washed twice with fresh growth medium containing 1 % FBS. Emitted cell surface-associated radioactivity in the microfluidic platform was measured by β-camera (see N. T. Vu et al., J. Nucl. Med. 201 1 , 52, 815-821 ). To confirm binding of the ¹⁸F-labeled Triligand 2 to the designed epitope on c-MET, a third radioassay was performed in which PC-3 cells were pre-treated with HGF (25 ng/mL, 15 min) as a competitor.

[00316] Results from two experiments are shown in Figure 22. Activities (cpm) per cell and per chamber were quantitated from the images generated from the β- camera. Cells in assay rows treated with ¹⁸F-labeled Triligand 2 showed the highest activity, while cells receiving no ¹⁸F-labeled Triligand 2 showed negligible activity. Blocking the c-MET receptor with HGF caused a reduction in cell surface-associated activity and suggests that ¹⁸F-labeled Triligand 2 and HGF compete for a common epitope on c-MET.

Experimental details

[00314] Materials. Fmoc-protected amino acids were purchased from Anaspec (San Jose, CA) and AAPPTec (Louisville, KY) and used as received. TentaGel S- NH₂ resin (90 μιη, 0.31 mmol/g) was obtained from Anaspec (San Jose, CA) and utilized for OBOC library construction. Biotinylated peptides were synthesized by coupling biotin to the N-terminus. Biotin-PEG₃ modified peptides were prepared by coupling PEG₃ to the N-terminus via N-Fmoc-N"-succinyl-4,7,10-trioxa-1 ,13- tridecanediamine (Sigma-Aldrich, 671517-5G), followed by capping with biotin. Peptides and OBOC peptide libraries were synthesized on the CEM Liberty 1 (Matthews, NC) or Titan 357 (AAPPTec, Louisville, KY) peptide synthesizers. Amino acid coupling reactions were performed in 1 -methyl-2-pyrrolidinone (NMP, 99%) with HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate, AAPPTec Louisville, KY) and Ν,Ν-diisopropylethylamine (DIEA) (99%, Sigma-Aldrich, St. Louis, MO). For removal of N^a-Fmoc protecting groups, a solution of 20% piperidine in NMP was used (Sigma-Aldrich, St. Louis, MO). For final deprotection of peptides and resin cleavage, a solution of trifluoroacetic acid (TFA)/TIS/DODT/H₂O (92.5 : 2.5 : 2.5 : 2.5) was used (Sigma-Aldrich, St. Louis, MO).

[00315] Future studies: 50-1 00 cells of PC-3, in quadruplicate, will be exposed to varying concentrations of ¹⁸F-labeled PCC for 30 min at 37 ⁵C on RIMChip. The washed cells will be measured by β-camera for the dose-dependent cell surface-associated activity.

[00316] The radioassay will be expanded to evaluate binding of ¹⁸F- labeled PcC to low c-MET-expressing cells (22Rv1 , LNCaP) and another high c- MET-expressing cell line (DU 145). 50-100 cells of PC-3, DU 145, 22Rv1 , and LNCaP, each in quadruplicate, will be exposed to ¹⁸F-labeled PCC in the presence and absence of varying amounts of "cold" ¹⁹F-labeled PCC to test for binding specificity.

[00317] Peptide library Synthesis: Randomized OBOC libraries of hexapeptides were synthesized using the Titan 357 peptide synthesizer on 90 μιη polyethylene glycol-grafted polystyrene beads (TentaGel S-NH₂, 0.31 mrnol/g' 2.86 x 10⁶ beads/g). D amino acids were used for the synthesis of the library. The libraries used in this study are listed in the table 2.

Table 2:

Example 18.

[00318] Proposed in vivo imaging studies in mice Aim A. Demonstrate in vivo uptake of a c-MET-targeted Triligand in normal mice.

Total number of normal mice required = 12

[00319] 18.A.1 . MicroPET-CT imaging of ¹⁸F-labeled Triligand in normal mice. Female normal mice (n = 3) will be used for the study. The uptake of ¹⁸F-labeled Triligand will be evaluated by microPET-CT imaging. The PET-CT will be done after the intravenous (i.v.) injection of 100 μθί (-50 μg) of ¹⁸F-labeled Triligand. The imaging signal will be acquired by dynamic scan (0-90 min), followed by a static scan at 4 h post injection. The PET signal will be co-registered with CT signal. PET imaging data will be reconstructed, and 3D dynamic images will be generated and saved as mpg files. The coronal, sagittal, and transverse 2D images at each time point will be generated and saved as TIF files. The PET signal will be quantitated by ROI analysis and represented as percent injected dose/g tissue (%ID/g tissue).

[00320] 18.A.2. Ex vivo biodistribution of ¹⁸F-labeled Triligand in normal mice. The biodistribution of ¹⁸F-labeled Triligand will be evaluated in different organs of the animal to identify the level of activity associated with each tissue. Studies will be carried out on female normal mice (n = 9). Animals will receive 100 μθί (-50 μg) of ¹⁸F-labeled Triligand in 100 μΙ_ PBS via lateral tail vein injection. The animals will be euthanized at specified time points post injection (30 min, 90 min, 4 h, n = 3 per group). After the mice are sacrificed, tissues from different organs (blood, liver, heart, lung, bladder, kidney, spleen, gall bladder, brain, muscle, and femur) will be collected and the activity will be measured by gamma counter and normalized by measuring the tissue weight. The percentage of the injected dose per organ weight (%ID/g) then will be calculated and presented as mean ± SD.

Aim B. Demonstrate specific uptake/binding of Triligand in tumors expressing c-MET and compare against control imaging agents.

o Number of immunocompromised mice required = 21 + 6^*

o Note: Additional 6 animals are needed to allow for selection of animals with similar tumor volumes and because the anticipated tumors take rate is less than 100%.

[00321] 1 8.B.1 . MicroPET-CT imaging of ¹⁸F-labeled Triligand in immunocompromised mice bearing subcutaneous PC-3 xenograft tumors in their lower flanks. PC-3 human prostate adenocarcinoma cells over-expressing c-MET will be used for the study. Six-week-old female NU/J (nude) mice will be purchased from Jackson Laboratories and hosted in the pathogen-free animal facility in the Crump Institute at UCLA. Xenografts of the PC-3 prostate cancer cells will be established by intradermal^ injecting 3 x 1 0⁶ viable cells in RPMI 1 640 media into the flank/leg region of nude mice to produce subcutaneous tumors. Tumors will be allowed to grow until reaching a palpable volume (-200-400 mm³), at which time a group of 3 animals will receive 100 \iC\ (-50 μg) of ¹⁸F-labeled Triligand in 100 μί PBS via lateral tail vein injection. The tumor-specific uptake of ¹⁸F-labeled Triligand will be evaluated by microPET-CT imaging. The imaging signal will be acquired by dynamic scan (0-90 min), followed by a static scan at 4 h post injection. The PET signal will be co-registered with CT signal. PET imaging data will be reconstructed, and 3D dynamic images will be generated and saved as mpg files. The coronal, sagittal, and transverse 2D images at each time point will be generated and saved as TIF files. The PET signal will be quantitated by ROI analysis and represented as percent injected dose/g tissue (%ID/g tissue).

[00322] 1 8.B.2. Ex vivo biodistribution of ¹⁸F-labeled Triligand in PC-3 xenograft tumor-bearing nude mice. The biodistribution of ¹⁸F-labeled Triligand will be evaluated in different organs of the animal, including the PC-3 tumors, to identify the level of activity associated with each tissue. Studies will be carried out on female nude mice bearing PC-3 xenograft tumors (see 1 8.B.1 ). After reaching a palpable tumor volume (-200-400 mm³), 9 animals will receive 100 μ&\ (-50 μ9) of ¹⁸F- labeled Triligand in 100 μί PBS via lateral tail vein injection. The animals will be euthanized at specified time points post injection (30 min, 90 min, 4 h, n = 3 per group). After the mice are sacrificed, tissues from different organs (tumor, blood, liver, heart, lung, bladder, kidney, spleen, gall bladder, brain, muscle, and femur) will be collected and the activity will be measured by gamma counter and normalized by measuring the tissue weight. The percentage of the injected dose per organ weight (%ID/g) then will be calculated and presented as mean ± SD.

[00323] 18.B.3. Evaluation of in vivo binding specificity of ¹⁸F-labeled Triligand via co-treatment with "cold" ¹⁹F-labeled Triligand in PC-3 xenograft tumor-bearing nude mice. In this section of the study, we will evaluate the tumor-specific uptake of ¹⁸F-labeled Triligand. Studies will be carried out on female nude mice bearing PC-3 xenograft tumors (see 1 8.B.1 ). ¹⁸F-labeled Triligand (1 00 μθί, -50 μg) and unlabeled Triligand (-250 μg) will be co-injected into the PC-3 tumor model via a lateral tail vein (n = 3 animals). Excess "cold" ¹⁹F-labeled Triligand is expected to block the tumor c-MET that would lead to the decreased accumulation of ¹⁸F-labeled Triligand in the tumor. The imaging signal will be acquired by dynamic scan (0-90 min), followed by a static scan at 4 h post injection. The PET signal will be co- registered with CT signal. PET imaging data will be reconstructed, and 3D dynamic images will be generated and saved as mpg files. The coronal, sagittal, and transverse 2D images at each time point will be generated and saved as TIF files. The PET signal will be quantitated by ROI analysis and represented as percent injected dose/g tissue (%ID/g tissue). Ex vivo biodistribution will be studied following sacrifice of the animals after the final PET scan. For the ex vivo analysis, tissues from different organs (tumor, blood, liver, heart, lung, bladder, kidney, spleen, gall bladder, brain, muscle, and femur) will be collected and the activity will be measured by gamma counter and normalized by measuring the tissue weight. The percentage of the injected dose per organ weight (%ID/g) then will be calculated and presented as mean ± SD.

[00324] 18.B.4. MicroPET-CT imaging experiments with a non-avid PCC peptide (negative control) and another ¹⁸F-labeled c-MET binding PCC peptide ("TriligandX"). In this section of the study, the uptake of an ¹⁸F-labeled non-avid negative control PCC peptide in mice with PC-3 xenografts will be evaluated by microPET-CT imaging. Similarly, we will evaluate the in vivo distribution/kinetics of another ¹⁸F-labeled c-MET binding PCC peptide ("TriligandX") for comparison. For each PCC peptide, the PET-CT will be done after the intravenous (i.v.) injection of 100 μθϊ of the radiolabeled control article (2 groups, n = 3 animals per group). The imaging signal will be acquired by dynamic scan (0-90 min), followed by a static scan at 4 h post injection. The PET signal will be co-registered with CT signal. PET imaging data will be reconstructed, and 3D dynamic images will be generated and saved as mpg files. The coronal, sagittal, and transverse 2D images at each time point will be generated and saved as TIF files. The PET signal will be quantitated by ROI analysis and represented as percent injected dose/g tissue (%ID/g tissue). Ex vivo biodistribution will be studied following sacrifice of the animals after the final PET scan. For the ex vivo analysis, tissues from different organs (tumor, blood, liver, heart, lung, bladder, kidney, spleen, gall bladder, brain, muscle, and femur) will be collected and the activity will be measured by gamma counter and normalized by measuring the tissue weight. The percentage of the injected dose per organ weight (%ID/g) then will be calculated and presented as mean ± SD.

Example 19.

[00325] Testing Additional PCC Biligand Candidates

For in vitro diagnostic and in vivo imaging applications, PCC candidates must be able to efficiently recognize c-MET in a background of human serum. Serum protein binding and recovery assays were designed to permit rapid comparison of PCC candidates. To determine the serum sensitivity of PCC candidates, a 1 -point ELISA experiment was performed using a fixed concentration of PCC ligand and HRP-conjugated c-MET. Recombinant human HGF (cat. no. 294-HGN/CF; R&D Systems) was biotinylated and used as a positive control. The HRP-conjugated c- MET was produced by using EZ-Link Plus Activated Peroxidase Kit (Pierce, #31489). The binding between PCC ligand and HRP-conjugated c-MET was measured in buffer (= 1 % (w/v) BSA in PBS), 1 % (v/v) human serum (Omega Scientific, #HS-20), and 10% (v/v) human serum. The results from this experiment show that Biligand 3 (-pkrhG) was the optimal biligand to detect c-MET in buffer and 1 % serum (Figure 44). Also, Biligand 3 was found to recognize c-MET more efficiently in 1 % serum than Triligand 1 1 (-kwwlr). The rest of the tested PCC ligands displayed low binding signals in 1 % serum. No c-MET was detected by the PCC ligands in 10% serum. Based on this result, Biligand 3 was prioritized for selecting a new triligand against c- MET.

[00326] Protocol. For the 1 -point ELISA, 0.7 μΜ biotinylated PCC candidate was prepared in 1 % (w/v) BSA in PBS (= 1 % Assay Buffer), 1 % serum, or 1 0% serum. Separately, 40 nM of HRP-conjugated c-MET protein was prepared in 1 % Assay Buffer, 1 % serum, or 10% serum. Note: c-MET was purchased as the recombinant human Fc chimera from R&D Systems (cat. no. 358-MT/CF). The solutions of 65 μΙ_ of biotinylated PCC candidate and 65 μΙ_ of HRP-conjugated c- MET were co-incubated in a polypropylene plate for 2 h at room temperature (RT). Next, a Pierce Streptavidin Coated High Binding Capacity Black 96 well plate (cat. no. 15503; Thermo Scientific) was equilibrated to RT. This Sa-plate was blocked with 5% (w/v) BSA in PBS (= 5% Blocking Buffer) for 2 h at RT. This was followed by washing the Sa-plate with 1 % Assay Buffer (3x). Next, 100 μΙ_ of the co- incubated solutions were transferred from the polypropylene plate to the corresponding wells of the Sa-plate and incubated for 5 min at RT. Wells were washed with PBS containing 0.05% (v/v) Tween-20 (6x) followed by PBS (7x) and then developed by adding QuantaRed™ Enhanced Chemifluorescent HRP Substrate (cat. no. 151 59; Thermo Scientific). Using an excitation wavelength of 535 nm, fluorescent emission at 595 nm was recorded by Beckman Coulter DTX880 photometer (Brea, CA).

Example 20.

[00327] Repeating Selection of PCC Triligand by In Situ Click Chemistry

From the assays described in Example 1 9, we have chosen the optimized biligand (= Biotin-PEG3-trwlr-Tz4-pkrhG-Az4) to select a triligand against c-MET. For the screen, a 200-mg portion of the OBOC library, coupled with D-propargylglycine at the N-terminus, was transferred into an 8-mL capacity Alltech vessel and pre- incubated in a blocking solution consisting of 0.1 % Tween-20 and 1 % BSA in TBS buffer overnight on a 360 °-rotator at 4 °C. After the blocking solution was drained, 50 μΜ of Biligand 3-Az4 (2% (v/v) DMSO: final) was added in a 2-mL volume to the beads for 4 h on a 360 °-rotator at 25 °C. The beads were washed with TBS and subsequently incubated with 7.5 M guanidine hydrochloride (pH 2.0) for 1 h. After washing thoroughly with TBS, the beads were placed back into blocking buffer for 2 h. The blocking solution was drained, and 3 ml_ of 1 :10,000 AP-linked Streptavidin (Promega) was added for 45 min at 25 °C. The screen was washed with 5 ^χ 3 ml_ Blocking Solution, 5 x 3 ml_ Wash 1 Buffer (25 mM Tris-CI, 10 mM MgCI₂, 700 mM NaCI, pH 7.5), followed by 5 x 3 ml_ Wash 2 Buffer (25 mM Tris-CI, pH 7.5), and drained by vacuum. BCIP:NBT (Promega #S3771 ), freshly prepared in Alkaline Phosphatase Buffer (100 mM Tris-HCI [pH 9.0], 150 mM NaCI, 1 mM MgCI₂), was used to develop the screen. Clear beads were selected manually, while the most intensely colored purple beads ("background hits") were left behind. Purple beads represented sequences that non-specifically bind to AP-linked Streptavidin and were discarded. The clear beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound materials, followed by NMP for 6 h (Figure 45A).

[00328] The selected beads were incubated in blocking solution for 2 h at 25 °C to prepare for a second round of screening. Separately, 100 μg of c-MET (R&D Systems, 358-MT/CF) was dissolved in 2 ml_ volume of blocking solution containing 50 μΜ of Biligand 3-Az4 (2% (v/v) DMSO: final) and incubated for 2 h at 25 °C. This solution was added to the selected beads after blocking and incubated for 4 h. The protein/biligand solution was then drained. The beads were then treated with 7.5 M guanidine hydrochloride (pH 2.0) to remove any non-covalently bound materials for 1 h. After washing the beads thoroughly with TBS, they were incubated in blocking buffer for 2 h. The subsequent steps involving AP-linked Streptavidin and development of library were repeated. The most intensely colored purple beads ("hits") were selected and represent those tertiary ligand sequences which conjugated with the biligand in a reaction templated by c-MET (Figure 45B). The hit beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound materials, followed by NMP for 6 h.

[00329] The selected beads were incubated in blocking solution for 2 h at 25 °C to prepare for a third round of screening. Those hits were then treated with 1 % (v/v) human serum (Omega Scientific, #HS-20) in blocking conditions for 1 h. Beads exhibiting off-target binding were detected using rabbit polyclonal pan anti-human serum antibodies (Abeam) and separated (Figure 45C). The remaining beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0), followed by NMP, and then water overnight. Hits were subsequently identified following the MALDI-TOF/TOF sequencing protocols previously outlined in Examples 4 and 5.

[00330] Triligand candidates were then selected through sequence homology and bioinformatics analysis (Figures 46 and 47). Sequences to be synthesized as tertiary ligand candidates can be found at different clusters located at the perimeter of the universe.

Example 21.

[00331] ELISA Testing of Triligand Candidates

After candidates of the tertiary ligands were identified, large-scale production of material was required for in vitro assays. The triligand candidates were synthesized in parallel as described in Example 14, cleaved from the resin, and purified via reversed phase HPLC. We tested the affinity of the biligand and triligand candidates by ELISA (Figure 48). We co-incubated HRP-conjugated c-MET with varying concentrations of biotinylated PCC ligands, and measured their binding interaction in buffer (= 1 % (w/v) BSA in PBS). As HGF is the natural ligand of c-MET, we generated a biotin-labeled recombinant human HGF (cat. no. 294-HGN/CF; R&D Systems) and used it as a positive control. The results from this experiment show that Biligand 3 (-pkrhG) binds to c-MET with KD ^ 375 nM. Triligand 1 2 (-wkkdr) (KD «s 60 nM) was found to be the optimal PCC candidate to detect c-MET in buffer, showing a 6-fold improvement in affinity for c-MET relative to the Biligand 3. Thus, the binding affinity is improved when successive ligands are added to the PCC agent. Additional candidates tested include Triligand 13 (-vnkrn) (SEQ ID NO:170) (KD ^ 125 nM) and Triligand 14 (-pwvhk) (SEQ ID NO:1 71 ) (KD ^ 220 nM). Compared to human HGF (KD ^ 3 nM), PCC triligands can bind to c-MET in vitro with promising performance but are much smaller in size. To achieve the KD of human HGF, we will perform screens to develop Triligand 1 2 into a tetraligand.

[00332] To determine the sensitivity of the selected triligands ligands in serum, a 1 -point ELISA experiment was performed as described in Example 19 using a fixed concentration of PCC ligand and HRP-conjugated c-MET. The binding between PCC ligand and HRP-conjugated c-MET was measured in buffer (= 1 % (w/v) BSA in PBS), 1 % (v/v) human serum, and 1 0% (v/v) human serum. Triligand 12 (-wkkdr) proved to be the optimal triligand to detect c-MET in buffer and 1 % serum, and it also showed improved performance over the biligand (Figure 49). The other triligands were more affected by the presence 1 % serum. No c-MET was detected by the PCC ligands in 10% serum. Based on this result, Triligand 1 2 was prioritized for selecting a tetraligand against c-MET.

[00333] Protocol. For the ELISA, titrations of biotinylated PCC candidate were prepared in 1 % (w/v) BSA in PBS (= 1 % Assay Buffer). Separately, 40 nM of HRP- conjugated c-MET protein (cat. no. 358-MT/CF; R&D Systems) was prepared in 1 % Assay Buffer. The HRP-conjugated c-MET was produced by using EZ-Link Plus Activated Peroxidase Kit (Pierce, #31489). The solutions of 65 μΙ_ of biotinylated PCC candidate and 65 μΙ_ of HRP-conjugated c-MET were co-incubated in a polypropylene plate for 2 h at RT. Next, a Pierce Streptavidin Coated High Binding Capacity Black 96 well plate (cat. no. 1 5503; Thermo Scientific) was equilibrated to RT. This Sa-plate was blocked with 5% (w/v) BSA in PBS (= 5% Blocking Buffer) for 2 h at RT. This was followed by washing the Sa-plate with 1 % Assay Buffer (3x). Next, 100 μΙ_ of the co-incubated solutions were transferred from the polypropylene plate to the corresponding wells of the Sa-plate and incubated for 5 min at RT. Wells were washed with PBS containing 0.05% (v/v) Tween-20 (6x) followed by PBS (7x) and then developed by adding QuantaRed™ Enhanced Chemifluorescent HRP Substrate. Using an excitation wavelength of 535 nm, fluorescent emission at 595 nm was recorded by Beckman Coulter DTX880 photometer (Brea, CA).

Example 22.

[00334] Pull-down Assays for Triligand Candidates.

Pull-down detection of c-MET was performed using a modified immunoprecipitation technique that incorporated PCCs rather than antibodies. Results of the pull-down assay for Biligand 3, Triligand 12, Triligand 15 (-yryee), and Triligand 16 (-Iqiqp) are set forth in Figure 50. Probing the elutions via Western blot with a c-MET antibody shows that Biligand 3, Triligand 12, and Triligand 1 5 display the largest c-MET bands in buffer (B), suggesting high affinity towards the target. The anti-c-MET Western analysis also shows that Triligand 12 most efficiently captures c-MET in 1 % (v/v) human serum (L). This suggests an increase in capture efficiency as the PCC agent is improved from biligand to triligand. Analysis of the total immunoprecipitated protein by SDS-PAGE shows tolerable non-selective binding for all PCCs, and correlates well with the capture efficiency for c-MET.

[00335] At this stage of development, c-MET detection in 25% (v/v) human serum (H) is not observed. Nevertheless, combined with the ELISA results, the pulldown assay has informed the prioritization of Triligand 12 for selecting a tetraligand against c-MET. Triligand 12 can be further characterized by in vitro cellular binding assays and in vivo imaging in a preclinical animal model.

[00336] Protocol. For the pull-down assay, 800 nM biotinylated PCC ligand (final: 0.2% (v/v) DMSO) first was incubated with 0.5 μg/mL recombinant human c- MET protein (cat. no. 358-MT/CF; R&D Systems) in 1 mL TBS + 0.02% Triton X-100 at 37 oC for 1 h. Separately, 800 nM biotinylated PCC ligand (final: 0.2% (v/v) DMSO) was incubated with 0.5 μg/mL recombinant human c-MET in 1 mL of 1 % (v/v) human AB male serum (cat. no. HS-20; Omega Scientific) in TBS + 0.02% Triton X- 100 under the same conditions (37 oC, 1 h). Finally, for the third sample, 800 nM biotinylated PCC ligand (final: 0.2% (v/v) DMSO) was incubated with 0.5 μg/mL recombinant human c-MET in 1 mL of 25% (v/v) human AB male serum in TBS + 0.02% Triton X-100 at 37 oC for 1 h. Proteins were captured by Dynabeads® M-280 Streptavidin (Invitrogen, #1 12-05D) under rotation at 37 oC for 1 h (1 00 μΙ_ of 50% slurry per pull-down condition). Beads were separated and washed from the serum or buffer matrix by application of the DynaMag™-Spin magnet (Invitrogen, #1 23- 20D), and captured proteins were eluted from the beads in 30 μΙ_ of reducing Laemmli buffer. Eluted samples were subjected to 7.5% SDS-PAGE separation at 200 V for 30 min in 1 x TGS (25 mM Tris, 192 mM Glycine, 0.1 % SDS (w/v), pH 8.3). Samples were subsequently electrophoretically transferred to a nitrocellulose membrane in 25 mM Tris, 192 mM Glycine, pH 8.3, containing 20% (v/v) methanol (Bio-Rad Laboratories, Hercules, CA) at 100 V for 30 min at 4 oC. A second gel was run and stained using Silver Stain Plus Kit (cat. no. 161 -0449; Bio-Rad) to visualize total captured proteins.

[00337] Following transfer of the first gel, the nitrocellulose membrane was blocked at 4 oC for 2 h in 5% non-fat dry milk in TBS. The membrane was then washed with TBS (3 ^χ), and 0.2 μg/mL goat anti-human HGFR/c-MET biotinylated antibody (R&D Systems, #BAF358) in 0.5% non-fat dry milk in TBS was incubated at 4 oC overnight. After washing with TBS containing 0.02% Tween-20 (v/v) (5 χ), 0.1 μg/mL HRP-conjugated streptavidin (Abeam, #ab7403; Cambridge, MA) in 0.5% non-fat filtered milk in TBS was added to the membrane (4 °C, 1 h incubation). After washing with TBS containing 0.02% Tween-20 (v/v) (5 ^χ), followed by TBS (5 ^χ), the membrane was developed with SuperSignal West Pico Chemiluminescent Enhancer and Substrate Solutions (Pierce, IL) and then immediately exposed to HyBlot CL AR film.

Example 23.

[00338] Selecting PCC Tetraligand by In Situ Click Screen

Protocol 1 . From the assays described in Examples 21 and 22, we have chosen the optimized triligand (= Biotin-PEG3-trwlr-Tz4-pkrhG-Tz4-wkkdr-Az4) to select a tetraligand against c-MET. For the screen, a 200-mg portion of the OBOC library, coupled with D-propargylglycine at the N-terminus, was transferred into an 8-mL capacity Alltech vessel and pre-incubated in a blocking solution consisting of 0.1 % Tween-20 and 1 % BSA in TBS buffer overnight on a 360°-rotator at 4 °C. After the blocking solution was drained, 50 μΜ of Triligand 12-Az4 (2% (v/v) DMSO: final) was added in a 2-mL volume to the beads for 4 h on a 360°-rotator at 25 °C. The beads were washed with TBS and subsequently incubated with 7.5 M guanidine hydrochloride (pH 2.0) for 1 h. After washing thoroughly with TBS, the beads were placed back into blocking buffer for 2 h. The blocking solution was drained, and 3 ml_ of 1 :1 0,000 AP-linked Streptavidin (Promega) was added for 45 min at 25 °C. The screen was washed with 5 χ 3 ml_ Blocking Solution, 5 χ 3 ml_ Wash 1 Buffer, followed by 5 x 3 mL Wash 2 Buffer, and drained by vacuum. BCIP:NBT, freshly prepared in Alkaline Phosphatase Buffer, was used to develop the screen. Clear beads were selected manually, while the most intensely colored purple beads ("background hits") were left behind. The clear beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound materials, followed by NMP for 6 h.

[00339] The selected beads were incubated in blocking solution for 2 h at 25 °C to prepare for a second round of screening. Separately, 100 μg of c-MET (R&D Systems, 358-MT/CF) was dissolved in 2 mL volume of blocking solution containing 50 μΜ of Triligand 12-Az4 (2% (v/v) DMSO: final) and incubated for 2 h at 25 °C. This solution was added to the selected beads after blocking and incubated for 4 h. The protein/triligand solution was then drained. The beads were then treated with 7.5 M guanidine hydrochloride (pH 2.0) to remove any non-covalently bound materials for 1 h. After washing the beads thoroughly with TBS, they were incubated in blocking buffer for 2 h. The subsequent steps involving AP-linked Streptavidin and development of library were repeated. The most intensely colored purple beads ("hits") were selected and represent those quaternary ligand sequences which conjugated with the triligand in a reaction templated by c-MET. The hit beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound materials, followed by NMP for 6 h.

[00340] The selected beads were incubated in blocking solution for 2 h at 25 °C to prepare for a third round of screening. Those hits were then treated with 1 % (v/v) human serum (Omega Scientific, #HS-20) in blocking conditions for 1 h. Beads exhibiting off-target binding were detected using rabbit polyclonal pan anti-human serum antibodies (Abeam) and separated. The remaining beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0), followed by NMP, and then water overnight. Hits were subsequently identified following the MALDI-TOF/TOF sequencing protocols previously outlined in Examples 4 and 5. [00341] Tetraligand candidates were then selected through sequence homology and bioinformatics analysis (Figures 51 and 52). Sequences to be synthesized as quaternary ligand candidates can be found at different clusters located at the perimeter of the universe.

[00342] Protocol 2. The above assay was repeated with the same optimized triligand (= Biotin-PEG3-trwlr-Tz4-pkrhG-Tz4-wkkdr-Az4) to select a second batch of tetraligands against c-MET. For the screen, the aforementioned protocol was repeated with 1 0 μΜ of Triligand 1 2-Az4 (2% (v/v) DMSO: final) was added in a 2-mL volume to the beads for 30 min on a 360°-rotator at 37 °C. The beads were then processed as previously described. Clear beads were selected manually, while the most intensely colored purple beads ("background hits") were left behind. The clear beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound materials, followed by NMP for 6 h.

[00343] The selected beads were incubated in blocking solution for 2 h at 25 °C to prepare for a second round of screening. Separately, 50 μg of c-MET (R&D Systems, 358-MT/CF) was dissolved in 2 ml_ volume of blocking solution containing 10 μΜ of Triligand 12-Az4 (2% (v/v) DMSO: final) and incubated for 30 min at 37 °C. This solution was added to the selected beads after blocking and incubated for 30 min. The protein/triligand solution was then drained. The beads were then treated with the procedure outlined above. The most intensely colored purple beads ("hits") were selected and represent those quaternary ligand sequences which conjugated with the triligand in a reaction templated by c-MET. The hit beads were decolorized with 7.5 M guanidine hydrochloride (pH 2.0) to remove bound materials, followed by NMP for 6 h.

[00344] The selected beads were subjected for a third round of screening outlined in Protocol 1 . Those hits were then treated with 2% (v/v) human serum (Omega Scientific, #HS-20) in blocking conditions for 1 h. Hits were subsequently identified following the MALDI-TOF/TOF sequencing protocols previously outlined in Examples 4 and 5. Tetraligand candidates were then selected through sequence homology and bioinformatics analysis (Figures 53 and 54). Sequences to be synthesized as quaternary ligand candidates can be found at different clusters located at the perimeter of the universe.

Example 24. [00345] Tetraligand Candidate Synthesis.

After candidates of the quaternary ligands were identified, large-scale production of material was required for in vitro assays. The differing quaternary ligands were synthesized in parallel onto Rink amide resin using an AAPPTEC Titan 357 peptide synthesizer. Each amino acid coupling reaction incorporated 4 equiv of Fmoc-amino acid, 4 equiv of HBTU (0-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro- phosphate), and 10 equiv of DIEA. Deprotection of the Fmoc group required 20% piperidine/NMP. Each quaternary ligand was coupled with Biotin and AEEEA (Chempep, #280103) at the N-terminus. Peptides were cleaved from the resin (TFA/TIS/DODT/H20, 92.5 : 2.5 : 2.5 : 2.5) and purified via reversed phase HPLC.

Example 25.

[00346] ELISA Testing of Tetraligand Candidates

We tested the affinity of the biligand, triligand, and tetraligand candidates by ELISA (Figure 55). We co-incubated HRP-conjugated c-MET with varying concentrations of biotinylated PCC ligands, and measured their binding interaction in buffer (= 1 % (w/v) BSA in PBS). We again generated a biotin-labeled recombinant human HGF and used it as a positive control. The results from this experiment show that Tetraligand 3 (-rnpwk) binds to c-MET with KD ^ 25 nM. This represents a 2-fold improvement in affinity for c-MET relative to the Triligand 12 (KD ^ 60 nM), and a 1 5-fold improvement relative to the affinity of the Biligand 3 (KD ^ 375 nM). Thus, the binding affinity is improved when successive ligands are added to the PCC agent. The affinity of the selected Tetraligand 3 is within a factor of 10 of the affinity of human HGF (KD ^ 3 nM), the natural ligand against c-MET.

[00347] To determine the sensitivity of the selected tetraligands ligands in serum, a 1 -point ELISA experiment was performed as described in Example 19 using a fixed concentration of PCC ligand and HRP-conjugated c-MET. The binding between PCC ligand and HRP-conjugated c-MET was measured in 5% (v/v) human serum (Omega Scientific, #HS-20), 7.5% (v/v) human serum, and 1 0% (v/v) human serum in PBS pH 7.4. Tetraligand 1 (-rkekw), Tetraligand 2 (-kGfkf), and Tetraligand 3 (-rnpwk) showed binding to c-MET under all conditions tested, even when c-MET was presented in a background of 10% serum (Figure 56). These tetraligands also showed improved performance over Triligand 1 2. Thus, the binding specificity is improved when successive ligands are added to the PCC agent.

[00348] To determine whether the PCC ligands have the ability to inhibit the binding between c-MET and human HGF, we carried out a competitive ELISA. For this experiment, biotinylated PCC ligand was pre-incubated with a fixed concentration of c-MET-HRP and recombinant human HGF. The results show that -1 00% of the binding between Biligand 3 and c-MET-HRP is lost if HGF is present. For Triligand 12, -80% of the binding to c-MET-HRP is lost in the presence of exogenous HGF (Figure 57). And, for Tetraligand 3 (-rnpwk), -40% of the binding to c-MET is lost in the presence of exogenous HGF. This modulation of the interaction between PCC ligand and c-MET-HRP by HGF suggests that PCC ligand and HGF share similar binding epitopes on the c-MET protein.

[00349] Protocol. For the competitive ELISA, 0.7 μΜ biotinylated PCC candidate was prepared in 1 % (w/v) BSA in PBS (= 1 % Assay Buffer). Separately, 40 nM of HRP-conjugated c-MET protein (cat. no. 358-MT/CF; R&D Systems) was prepared in 1 % Assay Buffer. The HRP-conjugated c-MET was produced by using EZ-Link Plus Activated Peroxidase Kit (Pierce, #31489). Solutions of 65 μΙ_ of biotinylated PCC candidate and 65 μΙ_ of HRP-conjugated c-MET were combined in a polypropylene plate. Recombinant human HGF (cat. no. 294-HGN/CF; R&D Systems) was treated to half of the wells at a final concentration of 50 nM for 2 h at RT. Next, a Pierce Streptavidin Coated High Binding Capacity Black 96 well plate (cat. no. 15503; Thermo Scientific) was equilibrated to RT. This Sa-plate was blocked with 5% (w/v) BSA in PBS (= 5% Blocking Buffer) for 2 h at RT. The Sa- plate was then washed with 1 % Assay Buffer (3x). Next, 1 00 μΙ_ of the co-incubated solutions were transferred from the polypropylene plate to the corresponding wells of the Sa-plate and incubated for 5 min at RT. Wells were washed with PBS containing 0.05% (v/v) Tween-20 (6x) followed by PBS (7x) and then developed by adding QuantaRed™ Enhanced Chemifluorescent HRP Substrate. Using an excitation wavelength of 535 nm, fluorescent emission at 595 nm was recorded by Beckman Coulter DTX880 photometer (Brea, CA).

Example 26.

[00350] Pull-down Assays for Tetraligand Candidates. To determine the specificity of the selected tetraligands, pull-down assays were performed as described in Example 22 using dilutions of human serum. Results of the pull-down assay for Triligand 1 2, Tetraligand 1 (-rkekw), Tetraligand 2 (-kGfkf), Tetraligand 3 (-rnpwk), and Tetraligand 4 (-shirt) are set forth in Figure 58. Probing the elutions via Western blot with a c-MET antibody shows that all PCC ligands are capable of binding to c-MET in 5% serum. The anti-c-MET Western analysis also shows that Tetraligand 1 captures c-MET in 7.5% serum and exceeds that captured by the triligand and other tetraligands in 7.5% serum. Analysis of the total immunoprecipitated protein by SDS-PAGE shows tolerable non-selective binding for all PCCs, and correlates well with the capture efficiency for c-MET.

[00351] An additional pull-down assay was performed to identify how much human serum can be added to the experiment until the binding between c-MET and Tetraligand 1 (-rkekw) is perturbed. Results of the pull-down assay for DAnchor 1 (- trwlr), Biligand 3, Triligand 12, and Tetraligand 1 (-rkekw) are set forth in Figure 59. Probing the elutions via Western blot with a c-MET antibody shows that neither DAnchor 1 nor Biligand 3 bind to c-MET in 5% serum. Triligand 12 shows increased specificity and recognition of the c-MET target in 7.5% serum, while Tetraligand 1 shows binding to c-MET in 12.5% serum. The increased capture of c-MET may be attributed to improvements in binding affinity and specificity as the PCC agent evolves from biligand to triligand, and from triligand to tetraligand.

Example 27.

[00352] Solution Stability Assays

Solution stability studies were performed on selected c-MET PCC tetraligands as described below. Tetraligands were found to be stable under the conditions tested.

[00353] For Biotin-PEG3 Tetraligand 1 , 0.5 μΙ_ of a 3.9 mM DMSO stock was diluted to 150 μΙ_ with pH 4.0 buffer (100 mM ammonium formate) giving a final concentration of 1 3.4 μΜ and incubated at ambient temperature. At every hour, 10 μΙ_ was removed and injected into an analytical HPLC using an autosampler. Injections started at time zero and continued to the 12th hour. Data is summarized on Figure 60A.

[00354] For Biotin-PEG3 Tetraligand 1 , 0.5 μΙ_ of a 3.9 mM DMSO stock was diluted to 150 μΙ_ with pH 7.4 phosphate saline buffer (PBS) giving a final concentration of 1 3.4 μΜ and incubated at ambient temperature. At every hour, 10 μΙ_ was removed and injected into an analytical HPLC using an autosampler. Injections started at time zero and continued to the 12th hour. Data is summarized on Figure 60A.

[00355] For Biotin-PEG3 Tetraligand 1 , 0.5 μΙ_ of a 3.9 mM DMSO stock was diluted to 1 50 μΙ_ with pH 9.5 borate buffer giving a final concentration of 13.4 μΜ and incubated at ambient temperature. At every hour, 10 μΙ_ was removed and injected into an analytical HPLC using an autosampler. Injections started at time zero and continued to the 1 2th hour. Data is summarized on Figure 60A.

[00356] For Biotin-PEG3 Tetraligand 2, 1 μΙ_ of a 7.8 mM DMSO stock was diluted to 200 μΙ_ with pH 4.0, 100 mM ammonium formate buffer giving a final concentration of 39 μΜ and incubated at ambient temperature. At every hour, 10 μΙ_ was removed and injected into an analytical HPLC using an autosampler. Injections started at time zero and continued to the 12th hour. Data is summarized on Figure 60B.

[00357] For Biotin-PEG3 Tetraligand 2, 1 μΐ of a 7.8 mM DMSO stock was diluted to 200 μί with pH 7.4 phosphate saline buffer (PBS) giving a final concentration of 39 μΜ and incubated at ambient temperature. At every hour, 10 μί was removed and injected into an analytical HPLC using an autosampler. Injections started at time zero and continued to the 12th hour. Data is summarized on Figure 60B.

[00358] For Biotin-PEG3 Tetraligand 2, 1 μΐ of a 7.8 mM DMSO stock was diluted to 200 μί with pH 9.5 borate buffer giving a final concentration of 39 μΜ and incubated at ambient temperature. At every hour, 1 0 μί was removed and injected into an analytical HPLC using an autosampler. Injections started at time zero and continued to the 12th hour. Data is summarized on Figure 60B.

[00359] For Biotin-PEG3 Tetraligand 3, 0.5 μΐ of a 3.9 mM DMSO stock was diluted to 150 μί with pH 4.0 buffer (100 mM ammonium formate) giving a final concentration of 1 3.4 μΜ and incubated at ambient temperature. At every hour, 10 μί was removed and injected into an analytical HPLC using an autosampler. Injections started at time zero and continued to the 12th hour. Data is summarized on Figure 60C. [00360] For Biotin-PEG3 Tetraligand 3, 0.5 μΙ_ of a 3.9 mM DMSO stock was diluted to 150 μΙ_ with pH 7.4 phosphate saline buffer (PBS) giving a final concentration of 1 3.4 μΜ and incubated at ambient temperature. At every hour, 10 μΙ_ was removed and injected into an analytical HPLC using an autosampler. Injections started at time zero and continued to the 12th hour. Data is summarized on Figure 60C.

[00361] For Biotin-PEG3 Tetraligand 3, 0.5 μΙ_ of a 3.9 mM DMSO stock was diluted to 1 50 μΙ_ with pH 9.5 borate buffer giving a final concentration of 13.4 μΜ and incubated at ambient temperature. At every hour, 10 μΙ_ was removed and injected into an analytical HPLC using an autosampler. Injections started at time zero and continued to the 1 2th hour. Data is summarized on Figure 60C.

Example 28.

[00362] Assays to Confirm Binding to Target on c-MET-expressing Tumor Cell Lines in Culture

[00363] Prostate cancer cell lines PC-3 (#CRL-1435), 22Rv1 (#CRL-2505), and LNCaP (#CRL-1740) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and grown in RPMI-1640 media supplemented with 10% (v/v) fetal bovine serum under standard cell culture conditions. PC-3 human prostate adenocarcinoma cells over-expressing c-MET, and 22Rv1 and LNCaP cells, which express low levels of c-MET, were used for these studies.

[00364] 28.1 . Fluorescence imaging experiments. To analyze PCC binding to c-MET expressed on the surface of cultured tumor cells, fluorescently labeled Triligand 1 1 (= FITC-PEG3-trwlr-Tz4-kvrhG-Tz4-kwwlr) was used. This triligand was incubated with the prostate cancer cell lines at 32 μΜ in growth medium containing 1 % fetal bovine serum (FBS) as a blocking agent. Cells were treated for 1 h at 37 °C, washed twice with 0.1 % bovine serum albumin in phosphate buffer, and then imaged using a Zeiss LSM 510 Meta confocal microscope with excitation and emission wavelengths set for FITC. Images 225 μιη in width were captured with brightfield and fluorescence shown in overlay (Figure 61 ). The most intense fluorescence was visualized in the high c-MET-expressing PC-3 cells treated with FITC-PEG3-labeled Triligand 1 1 . Blocking the c-MET receptor with HGF (the natural ligand, 25 ng/ml) caused a reduction in cell surface-associated fluorescence and suggests that Triligand and HGF compete for a common epitope on c-MET. The low c-MET- expressing 22Rv1 cells (control experiment) displayed only weak fluorescence signals and were less sensitive to co-incubation with HGF.

[00365] 28.2. Flow cytometry experiments. FITC-PEG3-labeled Triligand 1 1 was incubated with prostate cancer cells (1 0 million/ml) in phosphate-buffered saline, pH 7.4, containing 0.5% bovine serum albumin (BSA). FITC-PEG3-labeled Triligand 1 1 was tested at various concentrations ranging from 20 μΜ to 6.67 nM. Anti- Human c-Met (HGF Receptor) FITC (eBioscience, #1 1 -8858-41 ) was used as a positive control. Cells were treated for 1 h at 0-4 oC, and then analyzed on BD Biosciences FACSCalibur flow cytometer. Results show that FITC-PEG3-labeled Triligand binds differentially to the PC-3 and 22Rv1 cells, and with binding affinities that correlate with expression levels of c-MET on the cell surface (Figure 62).

Example 29.

[00366] In vivo PET Imaging Studies in Mice

29.1 . Radiolabeling of Biligand and Triligand candidates with an 18F-labeled prosthetic group. The preparation of 4-[18F]fluorobenzaldehyde (18FB) and site- specific conjugation of this aldehyde to aminooxy-functionalized biligand and triligand candidates followed the modified procedures of T. Poethko et al. (J. Nucl. Med. 2004, 45, 892-902) (Example 1 6). Radiolabeling was performed at either the UCLA Crump Preclinical Imaging Technology Center or the biomedical cyclotron facility at the UCLA Ahmanson Biological Imaging Center. TLC and analytical HPLC analysis at 2 h post-formulation showed no evidence of free 1 8FB label, and suggesting in vitro stability of the probe. The pH (-5.5) of the probe was also found to be in the acceptable range.

[00367] 29.2. MicroPET-CT imaging and radiation dosimetry for 18FB-labeled Triligand 1 1 in normal mice. Three one - hour dynamic PET-CT imaging studies were performed, in the UCLA Crump Preclinical Imaging Technology Center using normal female SCID mice without tumors. The mice were injected with 26 - 127 μ&\ and imaged using either a Siemens Inveon DPET or Focus 220, followed by a CT scan. There was substantial residual activity in the syringe and catheter lines, which was decay corrected and subtracted from the drawn activity to provide the injected activity listed in the table below. Extensive testing has demonstrated that the exact same biodistribution results are obtained with mice imaged in any of the 3 PET scanners.

Session Injected Residua!

Date ID Scanner Activity Activity

1/17/2013 m31334 Inveon 32LS u i 79 TyCi 1/17/2013 ΠΊ31835 Focus 127.5 uCi 50.5 uCi 1/17/2013 m31836 inveon 28,3 uCi 26.3 uCi

[00368]

[00369] Image and Biodistribution Summary:

The 18FB-labeled Triligand 1 1 was very quickly bound to liver and kidney cortex. It appears that the probe was almost completely first pass extracted, with little change seen over the 1 hour scan. Some amount of activity accumulated in the bladder over the hour. There was no sign of defluorination, and all three scans were very consistent. The images in Figure 63A are from all 3 mice at 30 min post injection. There was no significant change over the 1 hour scan. Figure 63A shows representative coronal (left) and sagittal (right) plane sections for studies m31834,5,6 showing liver and kidney uptake. Images are from a 5 min acquisition frame acquired at 30 min post injection. Each image is individually scaled to its own maximum due to the range of injected doses.

[00370] Figure 63B shows the biodistribution data over time for the average percent of total injected dose for liver, kidneys, and bladder in the 3 mice, with standard deviation error bars. These three organs had the only visible uptake of 18FB-labeled Triligand 1 1 in vivo. The liver+kidney and bladder regions of interest (ROI) were drawn slightly larger than the actual organ sizes to include activity spillover from the limited scanner resolution. The kidney ROI was kept closer to the kidney size as determined using the CT scan to avoid excessive spillover signal from the liver. Liver activity was defined as the liver+kidney region minus the left and right kidney activity. The total activity in each organ was determined as the mean ROI ^* ROI size and was divided by the whole body ROI total (mean^*size) to obtain the percent injected dose for each organ. Note that biodistribution data obtained in this manner is slightly different than the method used for radiation dosimetry (described below). [00371] The bladder signal compared to other 18F-labeled probes is very low. Kidney values were fairly stable, rising slightly over time as additional activity was cleared from the bloodstream. Liver values were constant after the first 5 min. Each animal had very similar uptake patterns, with the greatest variability in the liver signal. The standard deviation measurements for kidney and bladder are extremely small compared to other PET imaging probes, indicating a high reproducibility of measurements with 1 8FB-labeled Triligand 1 1 .

[00372] Radiation Dosimetry:

Liver values were based on a representative region drawn in the center of the organ. Total liver activity was estimated using the ROI mean and assuming the liver represents 5.4% of the total body weight (data from investigation at UCLA in collaboration with Charles River and submitted for publication since no published organ weights for SCID mice exist). A representative kidney region was drawn for both left and right kidneys and the total activity was determined using the mean ROI value multiplied by the kidney percent body mass of 0.82%. For the bladder, a region was drawn inside of the bladder as determined by CT to provide a mean ROI value, which was multiplied by the bladder size as determined by the CT scan. A whole body ROI from the CT scan was used to determine the total body weight. The activity not included in the liver, kidneys and bladder was determined as the whole body activity minus these organs and listed as the remainder activity.

[00373] The total integrated activity was determined as the mean ROI value times the frame duration (5 min) times the mass of each organ (percent body weight times body weight) times a factor to undo the isotope decay correction in the image data. After the one hour imaging time, biological redistribution was assumed to be complete. Assuming no further change in activity location, the activity for each region was calculated for an additional 6 hours (~3 half lives) to bring the total integration count to greater than 99% of the total number of disintegrations. The sum of disintegrations per organ as expressed as a percentage of the total in the body was determined and divided by the isotope half life factor to obtain residency time equivalent values. These values were entered into OLINDA software licensed from Vanderbilt University (version 1 .0, 2003). The adult male model was used, with 18F as the isotope and tissue values entered for only liver, kidney, bladder and remainder activity. The urinary bladder voiding model was not used. (Note: OLINDA is not suitable for using microPET data from mice to accurately predict human dosimetry, since the organ size and inter - organ distances are not accurate when used with mouse data. This data only provides a preliminary guide for estimating limiting dosage for potential human use and should be followed with human dosimetry to determine the proper dose limitations.)

[00374] Based on the OLINDA values in Figure 64, the majority of radiation dose occurs in the gallbladder, kidneys, liver and urinary bladder (bold) and the dose limiting organ was the liver for m31834 and m31835 and urinary bladder for m31836 (bold italic). Urinary bladder activity could be lowered using a voiding model, however it is not likely to change the results since the liver is the dose limiting organ. Based on this estimation, a safe activity level to inject to keep below 5 REM would be 10.37 mCi.

[00375] Methods information:

The mice appeared to have normal respiration during the course of the study and showed no signs of distress. The mice recovered without any complications and appeared to suffer no ill effects from the injection or imaging process. Mice were kept warm by heating plates, heated induction boxes and imaging chamber. Gas isoflurane anesthesia at 1 - 2% was used. Mice were positioned using an imaging chamber that provided reproducible positioning, heating, anesthesia delivery and pathogen control. Data were acquired using Siemens Preclinical Solutions (Knoxville, TN) microPET Focus 220, Inveon DPET and MicroCAT II CT systems. PET data were acquired for 1 hour and reconstructed using filtered back projection into multiple 5 min frames. PET images are ~1 .8 mm resolution, 0.4 mm voxel size. CT images were acquired using a low dose 400 micron resolution acquisition with 200 micron voxel size. Images were co-registered and regions drawn using AMIDE software (Andreas Loening, amide.sourceforge.net, v1 .0).

[00376] 29.3. MicroPET-CT imaging for 1 8FB-PEG3-labeled Triligand 1 1 in normal mice. Three one - hour dynamic PET-CT imaging studies were performed in the UCLA Crump Preclinical Imaging Technology Center using normal female SCID mice without tumors (m32055,6,7). The PET-CT was done after the intravenous (i.v.) injection of ≤S 1 00 \iC\ of 18FB-PEG3-labeled Triligand 1 1 . The imaging signal was acquired by dynamic scan (0-60 min post injection). The PET signal was co- registered with CT signal. PET imaging data were reconstructed, and the coronal, sagittal, and transverse 2D images at each time point were generated and saved. The PET signal was quantitated by ROI analysis and represented as percent injected dose/g tissue (%ID/g tissue) for liver, kidneys, and bladder vs. time (min). There was no sign of defluorination, and all three scans were very consistent.

[00377] Figure 65A shows representative transverse (left), coronal (center), and sagittal (right) plane sections for study m32055 showing liver, kidney, and bladder uptake. Images are from acquisition frames acquired at 7.5 and 57.5 min post injection. Figure 65B shows the biodistribution data over time for the average percent of total injected dose for liver, kidneys, and bladder in the 3 mice, with standard deviation error bars. These three organs had the only visible uptake of 18FB-PEG3-labeled Triligand 1 1 in vivo.

[00378] The incorporation of a PEG3 linker between the 1 8FB label and PCC agent appears to have enhanced the in vivo biodistribution of the probe. The liver uptake is still substantial; however, it releases over time and causes the expected slightly higher kidney signals and increasing bladder signals.

[00379] 29.4. MicroPET-CT imaging for 18FB-PEG3-labeled Biligand 3 in normal mice. Two one - hour dynamic PET-CT imaging studies were performed in the UCLA Crump Preclinical Imaging Technology Center using normal female SCID mice without tumors (m32623,4). The PET-CT was done after the intravenous (i.v.) injection of =¾ 100 \iC\ of 18FB-PEG3-labeled Biligand 3. The imaging signal was acquired by dynamic scan (0-60 min post injection). The PET signal was co- registered with CT signal. PET imaging data were reconstructed, and the coronal, sagittal, and transverse 2D images at each time point were generated and saved. The PET signal was quantitated by ROI analysis and represented as percent injected dose/g tissue (%ID/g tissue) for lung, heart, liver, kidneys, and bladder vs. time (min). There was no sign of defluorination, and the two scans were very consistent.

[00380] Figure 66A,C shows representative coronal (left) and sagittal (right) plane sections for studies m32623,4. Images are from acquisition frames acquired at 0 and 55 min post injection. Figure 66B,D shows the biodistribution data over time for the average percent of total injected dose for lung, heart, liver, kidneys, and bladder in the 2 mice, with standard deviation error bars. There is very little probe in the liver and more in the lungs. The activity appears to persist in blood for some time, as evidenced by the lung, heart, kidney, and bladder profiles. Clearance is by a renal mechanism as would be expected for peptides. About 30 min elapsed before the kidneys peaked in signal, suggesting favorable plasma residency times. This suggests that there could be sufficient residency time for targeting of 1 8FB-PEG3- labeled Biligand 3 to tumors in vivo.

[00381] 29.5. MicroPET-CT imaging for 1 8FB-PEG3-labeled Triligand 12 in normal mice. Two 1 .5 - hour dynamic PET-CT imaging studies were performed in the UCLA Crump Preclinical Imaging Technology Center using normal female SCID mice without tumors (m32890,1 ). The PET-CT was done after the intravenous (i.v.) injection of 60 \iC\ of 18FB-PEG3-labeled Triligand 1 2. The imaging signal was acquired by dynamic scan (0-90 min post injection). The PET signal was co- registered with CT signal. PET imaging data were reconstructed, and the coronal, sagittal, and transverse 2D images at each time point were generated and saved. The PET signal was quantitated by ROI analysis and represented as percent injected dose/g tissue (%ID/g tissue) for lung, heart, liver, kidneys, and bladder vs. time (min). There was no sign of defluorination, and the two scans were very consistent.

[00382] Figure 67A,B shows representative coronal (left) and sagittal (right) plane sections for studies m32890, 1 . Images are from acquisition frames acquired at 0 and 90 min post injection. Figure 67C shows the biodistribution data over time for the average percent of total injected dose for lung, heart, liver, kidneys, and bladder in the 2 mice, with standard deviation error bars. The uptake pattern looks similar to the biodistribution of the biligand probe (see Example 29.4). Very little amounts of 1 8FB-PEG3-labeled Triligand 1 2 are found in the liver and lungs, and the probe clears by a renal mechanism. About 30 min elapsed before the kidneys peaked in signal, suggesting favorable plasma residency times. This suggests that there could be sufficient residency time for targeting of 1 8FB-PEG3-labeled Triligand 12 to tumors in vivo.

[00383] 29.6. MicroPET-CT imaging for 18FB in normal mice. A one - hour dynamic PET-CT imaging study was performed in the UCLA Crump Preclinical Imaging Technology Center using a normal female SCID mouse without tumors (m31827). As a control experiment, the PET-CT was done after the intravenous (i.v.) injection of 25 \iC\ of 18FB. The imaging signal was acquired by dynamic scan (0-60 min post injection). The PET signal was co-registered with CT signal. PET imaging data were reconstructed, and the coronal, sagittal, and transverse 2D images at each time point were generated and saved. The PET signal was quantitated by ROI analysis and represented as percent injected dose/g tissue (%ID/g tissue) for liver, kidneys, and bladder vs. time (min).

[00384] Figure 68A shows the chemical structure of 18FB. Figure 68B shows the biodistribution data over time for the average percent of total injected dose for liver, kidneys, and bladder in the mouse. This probe quickly cleared through the kidneys to the bladder with no liver uptake other than a tiny amount in the gall bladder. This is a very different uptake pattern than 18FB- or 18FB-PEG3-labeled PCC agents.

[00385] 29.7. MicroPET-CT imaging of 18FB-PEG3-labeled Triligand 12 in immunocompromised mice bearing subcutaneous HT-29 xenograft tumors. HT-29 (ATCC #HTB-38) human colorectal adenocarcinoma cells over-expressing c-MET were used for the study. Six-week-old female SCID mice were purchased from Charles River and hosted in the pathogen-free animal facility at UCLA Crump Preclinical Imaging Technology Center. Xenografts of the HT-29 colon cancer cells were established by intradermal^ injecting ~3 x 106 viable cells in 1 :1 (v/v) RPMI- 1640 and reconstituted basement membrane (MatrigelTM) into the shoulders of SCID mice. Subcutaneous tumors were allowed to grow for -10 days.

[00386] The tumor-specific uptake of 18FB-PEG3-labeled Triligand 12 was evaluated by microPET-CT imaging. Three 1 .5 - hour dynamic PET-CT imaging studies were performed in the UCLA Crump Preclinical Imaging Technology Center using the HT-29 xenograft mice (m33017,18,20). The PET-CT was done after the intravenous (i.v.) injection of ≤¾ 100 μθϊ of 18FB-PEG3-labeled Triligand 1 2. The imaging signal was acquired by dynamic scan (0-90 min post injection). The PET signal was co-registered with CT signal. PET imaging data were reconstructed, and the coronal, sagittal, and transverse 2D images at each time point were generated and saved. The PET signal was quantitated by ROI analysis and represented as percent injected dose/g tissue (%ID/g tissue) for lung, heart, liver, kidneys, and bladder vs. time (min). There was no sign of defluorination, and the three scans were very consistent.

[00387] Figure 69A shows representative coronal (left) and sagittal (right) plane sections for study m33018. Plane sections were drawn through the tumor. PET images are from acquisition frames acquired at 0 and 60 min post injection. Figure 69B shows representative coronal plane sections for kidney at 0 (left) and 60 min (center) post injection for study m3301 8. Also shown is the sagittal plane section for bladder at 60 min (right). Figure 69C shows the biodistribution data over time for the average percent of total injected dose for lung, heart, liver, kidneys, and bladder in the 3 mice, with standard deviation error bars. The uptake pattern looks similar to the biodistribution of the triligand probe in normal mice without tumors (see Example 29.5). Very little amounts of 1 8FB-PEG3-labeled Triligand 12 are found in the liver and lungs, and the probe clears renally to the bladder. The images and numbers both show little uptake of the probe into the tumors (% ID = 0.13). The activity mainly sticks to the kidney cortex and clears to bladder. The compound was very sticky and substantial activity remained in the syringe, despite taking care to only let the activity be in the syringe for a very short time between draw and injection.

Example 30.

[00388] In vitro Pharmacology Assays

30.1 . Plasma protein binding. Summary. The equilibrium dialysis technique was used to separate the fraction of a test compound that is unbound from the fraction that is bound to proteins. The assay was performed in a 96-well format in a dialysis block constructed from Teflon.

[00389] Materials and Methods. The protein matrix was spiked with the test compound at 10 μΜ (n=2) with a final DMSO concentration of 1 % (v/v). The dialysate compartment was loaded with 150 μΙ_ phosphate buffer (pH 7.5) and the sample side was loaded with equal volume of the spiked protein matrix. The dialysis plate was then sealed and incubated at 37 °C overnight (18 ± 2 h). After the incubation, samples were taken from each compartment, diluted with the phosphate buffer followed by addition of acetonitrile and centrifugation. The supernatants were then used for HPLC-MS/MS analysis. A control sample (n=2) was prepared from the spiked protein matrix in the same manner as the assay samples (without dialysis). This control sample served as the basis for the recovery determination. Samples were analyzed by HPLC-MS/MS using selected reaction monitoring. The HPLC conditions consisted of a binary LC pump with autosampler, a C1 8 column (2 x 20 mm), and gradient elution. The percent bound to proteins and the recovery were calculated as follows:

(Area_pe - Area_be) x ^

Protein binding (%) = - x 100

(Area_pe - Area_be) x + Area_be , (Vn_e x Area_De + V_be x Area_be)

Recovery (%) = ^ „ ^pe _Λ ^be ≥ _{x 0}0

V_pi x Area_cs

[00390]

[00391] Area_pe = Peak area of analyte in the protein matrix at equilibrium

[00392] Area_be = Peak area of analyte in the Assay Buffer at equilibrium

[00393] V_Pe = Volume of the protein matrix at equilibrium

[00394] Vpi = Initial volume of the protein matrix

[00395] Area_cs = Peak area of analyte in control sample

[00396] The recovery determination served as an indicator of reliability of the calculated protein binding value.

[00397] Results and Discussion. Increasing PPB (% Protein Bound) was observed as the PCC evolves from biligand to triligand, and from triligand to tetraligand (Figure 70A,B). The 19FB-PEG3 label contributes to the overall hydrophobicity of the compound and may contribute to increased PPB and reduced % Recovery for Triligand 12. 19FB-PEG3-Tetraligand 3 and 1 9FB-PEG3-Triligand 12 show a low % Recovery and may be suggestive of adhesion to the dialysis membrane.

[00398] 30.2. Stability in Human Plasma and Mouse Liver Microsomes. Summary. The stability of a test compound in human plasma, quantified at multiple time points by HPLC-MS/MS analysis. The stability of a test compound in pooled liver microsomes from mouse, quantified at multiple time points by HPLC-MS/MS analysis. [00399] Materials and Methods. Human plasma was pre-warmed at 37 ⁵C water bath for 5 min, followed by addition of 1 μΜ test compound with a final DMSO concentration of 0.5 % (v/v). The incubation was performed in a 37 ⁵C water bath for 0, 0.5, 1 , 1 .5, and 2 h. At each time point, an aliquot of the incubation mixture was transferred to acetonitrile. Samples were then mixed and centrifuged. Supernatants were used for HPLC-MS/MS analysis. Samples were analyzed by HPLC-MS/MS using selected reaction monitoring. The HPLC system consisted of a binary LC pump with autosampler, a C-18 column, and a gradient. Peak areas corresponding to the test compound were recorded. The compound remaining (%) was calculated by comparing the peak area at each time point to time zero.

[00400] The test compound was pre-incubated with pooled human liver microsomes (mixed gender, 0.3 mg/mL) in phosphate buffer (pH 7.4) for 5 min in a 37 ⁵C shaking waterbath. Concentration of the test compound was 1 μΜ with 0.01 % DMSO, 0.25% acetonitrile and 0.25% methanol. The reaction was initiated by adding NADPH-generating system (1 .3 mM NADP, 3.3 mM G6P, and 0.4 U/mL G6PDHase) and incubated for 0, 15, 30, 45, and 60 min. The reaction was stopped by transferring the incubation mixture to equal volume of acetonitrile/methanol (1 /1 , v/v). Samples were then mixed and centrifuged. Supernatants were used for HPLC- MS/MS analysis. Samples were analyzed by HPLC-MS/MS using selected reaction monitoring. The HPLC system consisted of a binary LC pump with autosampler, a C-18 column, and a gradient. Peak areas corresponding to the test compound were recorded. The compound remaining was calculated by comparing the peak area at each time point to time zero. The half-life was calculated from the slope of the initial linear range of the logarithmic curve of compound remaining (%) vs. time, assuming first order kinetics. In addition, the intrinsic clearance was calculated from the half- life.

[00401] Results and Discussion. 1 9FB-PEG3-labeled Biligand 3 was found to be stable in human plasma at 37 °C over the assay period of 2 h (Figure 70B,C). 19FB-PEG3-labeled Biligand 3 was also stable in human liver microsomes over the assay period of 60 min (Figure 70B,D).

Example 31 . Multiplex Probe Development [00402] Multiple capture agents can be used to diagnose, stage and treat cancers. As shown in Figure 41 A a combination probe can be used. In this example, a mixture of labeled capture agents are used that specifically bind to PSMA, fPSA, c- Met, and MUC1 . However, fewer, more or different probes could be used.

[00403] The advantages to these embodiments are the simple product format that allows for phenotypic loss of one of the target markers. The design also provides increased sensitivity over a single probe. Different capture agents sets could be set up for various disease states. Further, each of the capture agents can have the same label, allowing a one step label process.

[00404] As shown in Figure 41 B, multiple capture agents can be covalently bound together. Each of the capture agents can specifically bind to a distinct protein present in a single location and involved in a disease state. In this example, MUC1 , PSMA and c-Met are shown, but other targets can be used.

[00405] Similarly to the embodiment shown in Figure 41 A, this embodiment allows for phenotypic loss of one or more target markers. In certain embodiments, when each of the targets is expressed on a single cell, this multi-protein capture agent can provide increased target cell avidity as well as increased sensitivity. Further, this embodiment allows for detection of different disease stages as well as use of the same label for all of the linked capture agents.

Claims

What is claimed is:

1 . A stable, synthetic capture agent that specifically binds c-Met, wherein the capture agent comprises a designed anchor ligand, a designed secondary ligand, optionally, a designed tertiary ligand, and optionally a designed quarternary ligand and wherein the ligands selectively bind c-Met.

2. The capture agent of claim 1 , wherein the anchor ligand comprises an amino acid sequence 80 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:1 -27.

3. The capture agent of claim 2, wherein the anchor ligand comprises an amino acid sequence 80 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:3, 4, 5, 9, 1 1 , 12, 13, 16, 19, 22, 24, 25 and 26.

4. The capture agent of claim 1 , wherein the anchor ligand comprises an amino acid sequence of trwX1 X2, wherein X1 and/or X2 are independently any D-amino acid or glycine or not present.

5. The capture agent of claim 1 , wherein the anchor ligand comprises an amino acid sequence selected from the group consisting of SEQ ID NOs 1 , 2 and 3.

6. The capture agent of claim 5, wherein the anchor ligand comprises an amino acid sequence of SEQ ID NO 3.

7. The capture agent of claim 1 , wherein the anchor ligand comprises an amino acid sequence of tX3dll, wherein X3 is independently any D-amino acid or glycine or not present.

8. The capture agent of claim 1 , wherein the anchor ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 4, 5 and 13.

9. The capture agent of any of the preceding claims, wherein the secondary ligand comprises an amino acid sequence 80 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:28-95.

10. The capture agent of claim 9, wherein the secondary ligand comprises an amino acid sequence 80 or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs:31 , 35, 37, 42, 46, 48, 49, 50, 52, 54, 55, 58, 59, 66, 69, 71 , 76, 85, 86 and 92.

1 1 . The capture agent of any one of claims 1 -8, wherein the secondary ligand comprises an amino acid sequence of X4krhG, wherein X4 is independently any D- amino acid or glycine or not present.

12. The capture agent of any one of claims 1 -8, wherein the secondary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 29, 30 and 31 .

13. The capture agent of claim 12, wherein the secondary ligand comprises an amino acid sequence of SEQ ID NO 31 .

14. The capture agent of any one of claims 1 -8, wherein the secondary ligand comprises an amino acid sequence of X5hGX6p and wherein X5 is independently any D-amino acid or glycine or not present.

15. The capture agent of any one of claims 1 -8, wherein the secondary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 43-47.

16. The capture agent of any one of claims 1 -8, wherein the secondary ligand comprises an amino acid sequence of X7X8rhG and wherein X7 is independently any D-amino acid or glycine or not present.

17. The capture agent of any one of claims 1 -8, wherein the secondary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 28-37.

18. The capture agent of any one of claims 1 -8, wherein the secondary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 31 , 35, 37 and 52.

19. The capture agent of any one of claims 1 -18, wherein the tertiary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 96-144, 170 and 1 71 .

20. The capture agent of claim 19, wherein the tertiary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 96, 97, 100,

102, 1 03, 104, 106, 107, 1 14, 122, 1 24, 128, 132, 1 39, 141 , 144, 170 and 171 .

21 . The capture agent of claim 20, wherein the tertiary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 96, 97, 100,

103, 1 04, 106, 1 14, 141 , 170 and 171 .

22. The capture agent of any one of claims 1 -18, wherein the tertiary ligand comprises an amino acid sequence of X9swwr, wherein X9 is independently any D- amino acid or glycine or not present.

23. The capture agent of claim 22, wherein the tertiary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 96 and 97.

24. The capture agent of any one of claims 1 -18, wherein the tertiary ligand comprises an amino acid sequence of fpfX1 Or, wherein X1 0 is independently any D- amino acid or glycine or not present.

25. The capture agent of any one of claims 1 -24, wherein the quarternary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 145-169, and 172.

26. The capture agent of any one of claims 1 -24, wherein the quarternary ligand comprises an amino acid sequence of selected from the group consisting of SEQ ID NOs 160, 1 61 , 167 and 169.

27. The capture agent of claim 1 , wherein the anchor ligand comprises the peptide sequence trwlr or irnwk.

28. The capture agent of any one of claims 1 or 27, wherein the secondary ligand comprises a peptide sequence selected from the group consisting of kvrhG, pkrhG, efwhG, hvwhG and skrhe.

29. The capture agent of any one of claims 1 , 27 or 28, wherein the tertiary ligand comprises a peptide sequence selected from the group consisting of pswwr, fswwr, fpflr, wqwlr, pwrqw, Iwrqw, wkkdr, and kwwlr.

30. The capture agent of any one of claims 1 , 27, 28 or 29, wherein the quarternary ligand comprises a peptide sequence selected from the group consisting of shirt, kGfkf, rkekw and rnpwk.

31 . The capture agent of any of the preceding claims, wherein the anchor ligand and secondary ligand are linked together via a 1 ,4-substituted-1 ,2,3-triazole residue (Tz4).

32. The capture agent of any of the preceding claims, wherein the capture agent is labeled with a label selected from the group consisting of biotin, copper-DOTA, biotin-PEG3, aminooxyacetate, ¹⁹FB, ¹⁸FB and FITC-PEG3.

33. The capture agent of any of the preceding claims, wherein the capture agent is labeled with the detectable moiety consisting of ⁶⁴Cu DOTA, ⁶⁸Ga DOTA, ⁶⁸Ga NOTA, ¹⁸F, AI¹⁸F NOTA, ⁶⁴Cu, ⁶⁸Ga, ⁸⁹Zr, ¹²⁴l, ⁸⁶Y, ^94mTc, ^110mln, ¹¹C and ⁷⁶Br.

34. The use of any of the preceding claims as a detection agent for detecting c- Met in a biological sample.

35. A method of detecting c-Met in a biological sample using an immunoassay, wherein the immunoassay utilizes a capture agent of any one of claims 1 -33, and wherein said capture agent replaces an antibody or its equivalent in the

immunoassay.

36. A method of treating a condition associated with increased c-Met expression and/or activity in a subject in need thereof, comprising administering a

therapeutically effective amount of a capture agent of any one of claims 1 -33.

37. The method of claim 36, wherein said condition is prostate cancer.

38. A method of inhibiting c-Met signaling in a subject comprising administering to the subject a capture agent of any one of claims 1 -33.

39. A method of diagnosing a c-Met expressing cancer in a human or mouse subject, the method comprising the steps of: a) administering to the subject the c- Met capture agent of any one of claims 1 -33 linked to a detectable moiety; and b) detecting the moiety linked to the c-Met capture agent in the subject; wherein detection of the moiety diagnoses a c-Met -expressing cancer in the subject.

40. A method of monitoring treatment of a subject receiving c-Met-directed therapy comprising administering to the patient a small-molecule positron-emission- tomography ligand (PET ligand) that is bound to the c-Met capture agent of of any one of claims 1 -33 on or near a c-Met-expressing cancer in the subject.

41 . A method of detecting c-Met in a sample comprising a) exposing the sample to the c-Met capture agent of any one of claims 1 -33, linked to a detectable moiety; and b) detecting the moiety linked to the c-Met capture agent in the subject; wherein detection of the moiety diagnoses a c-Met-expressing cancer in the subject.

42. A multiplex capture agent comprising a mixture of capture agents that binds specifically to two or more of c-Met, PSMA, and MUC1 .

43. An agent comprising a first and a second capture agent, wherein the first cancer agent specifically binds one of c-Met, PSMA, fPSA and MUC1 and the second capture agent specifically binds one of c-Met, PSMA, fPSA and MUC1 , wherein the first and the second capture agents bind distinct proteins.

44. The agent of claim 43, wherein the agent further comprises a third capture agent, wherein the third capture agent specifically binds one of c-Met, PSMA, fPSA and MUC1 , wherein the first, the second and the third capture agents bind distinct proteins.

45. The agent of claim 44, wherein the agent further comprises a fourth capture agent, wherein the fourth capture agent specifically binds one of c-Met, PSMA, fPSA and MUC1 , wherein the first, the second, the third and the fourth capture agents bind distinct proteins.

46. A method of treating a disease comprising administering an effective amount of the agent of claim 42 to a subject in need thereof.

47. A method of diagnosing a disease comprising a) administering to the subject the agent of claim 42, wherein each of the capture agents is linked to a detectable moiety; and b) detecting the moiety linked to the capture agents in the subject;

wherein detection of the moiety diagnoses a disease in the subject.

48. The method of any one of claims 46 or 47, wherein the disease is cancer.

49. The method of claim 48, wherein the cancer is selected from the group consisting of multiple myeloma, chronic myelogenous leukemia, pancreatic cancer, non-small cell lung cancer, lung cancer, breast cancer, colon cancer, ovarian cancer, prostate cancer, malignant melanoma, non-melanoma skin cancers, gastrointestinal stromal tumors, hematologic tumors, hematologic malignancies, childhood leukemia, childhood lymphomas, multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic origin, lymphomas of cutaneous origin, acute leukemia, chronic leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm and cancers associated with AIDS. In a particular embodiment, the cancer is selected from the group consisting of lung, breast and prostate cancer.