Patent Publication Number: US-2022211648-A1

Title: Compositions, delivery systems, and methods useful in tumor therapy

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation of International Application No. PCT/US2021/057880 filed Nov. 3, 2021, which claims the benefit of and priority to U.S. Application No. 63/109,186 filed Nov. 3, 2020, and U.S. Application No. 63/256,885 filed Oct. 18, 2021, the contents of which are incorporated by reference in their entirety. 
    
    
     REFERENCE TO SEQUENCE LISTING 
     The Sequence Listing submitted Jan. 17, 2022, as a text file named “INDI_111_CON_ST25.txt,” created on Jan. 11, 2022, and having a size of 14,065 bytes is hereby incorporated by reference. 
     FIELD OF THE INVENTION 
     The disclosed invention is generally in the field of composition delivery systems and in particular in the field of composition delivery systems for reducing toxicity or toxic effects of compounds and compositions administered to patients. 
     BACKGROUND OF THE INVENTION 
     Targeted delivery of compositions to a tumor or organ for a therapeutic effect may require modification of the drug or composition for delivery to the site of interest. We describe a modification to a PCC that may cleave to deliver a therapeutic to a specific target. The modification is organ protective. 
     Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application. 
     BRIEF SUMMARY OF THE INVENTION 
     Disclosed are compounds, compositions, systems, and methods useful for treating disease (such as cancer and tumors), or binding, detecting, and affecting compounds, compositions, cells, tissues, and organs, where the compounds, compositions, systems, and methods include or involve toxic compounds and where the toxic effect of the compounds, compositions, systems, and methods is reduced by providing a cleavage site to facilitate separation of the toxic component from other components of the compound, composition, or system. Preferably the system is a composition delivery system comprising or using a composition as disclosed herein. In some forms, the cleavage of the composition delivers a therapeutic agent and avoids organ damage by the composition. 
     Disclosed are compositions comprising a first component and a second component, where the first and second components are coupled via a linking component, where the linking component comprises a neprilysin (NEP) cleavage site, where the NEP cleavage site can be cleaved by NEP, where cleavage of NEP cleavage site separates the first component from the second component. 
     In some forms, the first component is toxic. In some forms, the first component is toxic to a cell, to an organ, or to both. In some forms, the first component is toxic to the kidney, lung or heart. In some forms, the first component is nephrotoxic. 
     In some forms, the separation of the first component from the second component reduces toxic effect of the first component to the cell, the organ, a subject containing the cell, the organ, or both, or a combination thereof, compared to the toxic effect of the uncleaved composition. In some forms, the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to a tumor compared to the delivery percentage of the uncleaved composition. In some forms, the reduction in the toxic effect is at least partially due to an increased delivery rate of the separated first component to a tumor compared to the delivery rate of the uncleaved composition. In some forms, the reduction in the toxic effect is at least partially due to an increased rate of clearance of the separated first component from the subject compared to the rate of clearance of the uncleaved composition. In some forms, the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to a second cell, to a second organ, or to both compared to the delivery percentage of the uncleaved composition. In some forms, the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to the cell, to the organ, or to both compared to the delivery percentage of the uncleaved composition. 
     Where the first component is toxic to the kidney, separation of the first component from the second component reduces toxic effect of the first component to the kidney by, for example, increasing clearance by the kidney of the toxic first component, thus reducing toxicity of the composition. 
     In some forms, the NEP cleavage site comprises Gly-Phe-Lys or Met-Val-Lys. In some forms, the first component comprises a therapeutic agent, a detection agent, or a combination thereof. In some forms, the first component comprises a radioisotope. In some forms, the radioisotope is  177 Lu,  225 Ac,  99m Tc,  51 Cr,  67 Ga,  68 Ga,  47 Sc,  51 Cr,  167 Tm,  141 Ce,  111 In,  168 Yb,  175 Yb,  140 La,  90 Y,  88 Y,  153 Sm,  166 Ho,  165 Dy,  166 Dy,  62 Cu,  64 Cu,  67 Cu,  97 Ru,  103 Ru,  186 Re,  188 Re,  203 Pb,  212 Bi,  212 Bi,  213 Bi,  214 Bi,  105 Rh,  109 Pd,  117 mSn,  149 Pm,  161 Tb  198 Au,  199 Au,  18 F,  89 Zr,  124 I,  86 Y,  94m Tc,  110m In,  11 C, or  76 Br. In some forms, the radioisotope is  177 Lu or  68 Ga. 
     In some forms, the second component comprises a ligand. In some forms, the ligand can bind to a target. In some forms, the second component comprises a biligand. In some forms, the second component comprises a heterobiligand. In some forms, the biligand and the heterobiligand each comprise two ligands, where both of the two ligands of the biligand and heterobiligand can bind either two separate parts of the same target or two different targets. 
     In some forms, each target is, independently, a detection target, a therapeutic target, both a detection target and a therapeutic target, or a combination thereof. 
     In some forms, one or more of the second component, the linking component, and the first component further comprise an albumin binding moiety. In some forms, the albumin binding moiety is 4-methylphenyl butyric acid (4-MPBA) or 4-iodophenyl butyric acid (IPBA). 
     In some forms, one or more of the second component, the linking component, and the first component further comprise a reporter moiety. 
     In some forms, the composition comprises the structure (I): 
     
       
         
         
             
             
         
       
     
     or a salt, tautomer, prodrug or stereoisomer thereof, where: 
     L 1  and L 2  are each individually a bond or an optionally substituted linker moiety, where each linker moiety optionally comprises a linkage to the NEP cleavage site and the first component, a linkage to the first component, a linkage to a ligand, a linkage to a reporter moiety, a linkage to an albumin binding moiety, a linkage to a peptide ligand, or combinations thereof; 
     G is a triazole, a carbon-carbon double bond or an amide; 
     M is methionine; 
     R is H or an optionally substituted linker moiety, wherein each linker moiety optionally comprises a linkage to the NEP cleavage site and the first component, a linkage to the first component, a linkage to a ligand, a linkage to a reporter moiety, a linkage to an albumin binding moiety, a linkage to a peptide ligand, or combinations thereof; 
     R 1  is H or C 1 -C 6  alkyl; 
     Y 1  and Y 2  are each individually 0 or 1; and 
     SEQ is an amino acid sequence comprising from 2 to 20 amino acids selected from natural and non-natural amino acids. 
     In some forms, L 1  is —C(HR 2 )— wherein R 2  is H, —R 5 -L 3 -A 1 , —R 5 —C(═O)-L 3 -A 1 , —R 5 -A 2 -L 3 -A 1 , —R 5 —C(═O)-A 2 -L 3 -A 1 , —R 5 -L 3 (-A 2 )-A 1 , or —R 5 —C(═O)-L 3 (-A 2 )-A 1 , where —R 5  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 3  is a linker moiety, and where A 1  and A 2  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     In some forms, L 2  is —C(HR 4 )—, wherein R 4  is H, —R 6 -L 5 -A 3 , —R 6 —C(═O)-L 5 -A 3 , —R 6 -A 4 -L 5 -A 3 , —R 6 —C(═O)-A 4 -L 5 -A 3 , —R 6 -L 5 (-A 4 )-A 3 , or —R 6 —C(═O)-L 5 (-A 4 )-A 3 , where —R 6  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 5  is a linker moiety, and where A 3  and A 4  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     In some forms, R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     In some forms, L 1  is —C(HR 2 )—, wherein R 2  is H, —R 5 -L 3 -A 1 , —R 5 —C(═O)-L 3 -A 1 , —R 5 -A 2 -L 3 -A 1 , —R 5 —C(═O)-A 2 -L 3 -A 1 , —R 5 -L 3 (-A 2 )-A 1 , or —R 5 —C(═O)-L 3 (-A 2 )-A 1 , where —R 5  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 3  is a linker moiety, and where A 1  and A 2  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     In some forms, L 2  is —C(HR 4 )—, wherein R 4  is H, —R 6 -L 5 -A 3 , —R 6 —C(═O)-L 5 -A 3 , —R 6 -A 4 -L 5 -A 3 , —R 6 —C(═O)-A 4 -L 5 -A 3 , —R 6 -L 5 (-A 4 )-A 3 , or —R 6 —C(═O)-L 5 (-A 4 )-A 3 , where —R 6  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 5  is a linker moiety, and where A 3  and A 4  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     In some forms, R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     In some forms, one or more of A 1 , A 2 , A 3 , A 4 , A 5 , and A 6  individually and independently comprise a combination of one or more of the following: the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, and a peptide ligand. 
     In some forms, the composition has one of the following structures (Ia) or (Ib): 
     
       
         
         
             
             
         
       
     
     where: 
     R 3  is H, -L 3 -A 1 , —C(═O)-L 3 -A 1 , -A 2 -L 3 -A 1 , —C(═O)-A 2 -L 3 -A 1 , -L 3 (-A 2 )-A 1 , or —C(═O)-L 3 (-A 2 )-A 1 , where L 3  is a linker moiety and A 1  and A 2  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof; and 
     x and y are each independently an integer from 1 to 8. 
     In some forms, R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     In some forms, R 3  is H, -L 3 -A 1 , —C(═O)-L 3 -A 1 , -A 2 -L 3 -A 1 , —C(═O)-A 2 -L 3 -A 1 , -L 3 (-A 2 )-A 1 , or —C(═O)-L 3 (-A 2 )-A 1 , where L 3  is a linker moiety and A 1  and A 2  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     In some forms, R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     In some forms, the composition has one of the following structures: 
     
       
         
         
             
             
         
       
     
     In some forms, the linker moieties independently comprise ethylene glycol, triazole, lysine, ethylene diamine, or combinations thereof. 
     In some forms, SEQ comprises from 2 to 9 amino acids. In some forms, SEQ comprises from 5 to 7 amino acids. In some forms, SEQ comprise natural amino acids. In some forms, SEQ comprises non-natural amino acids. In some forms, SEQ comprises natural and non-natural amino acids. 
     Also disclosed are methods of using the disclosed compounds, compositions, and systems to treat subjects in need thereof. For example, disclosed are methods of providing a therapeutic agent to treat a tumor, wherein the agent cleaves to deliver the agent and reduce organ damage. Also disclosed are methods of treating a subject having a tumor, the method comprising administering to the subject a composition as disclosed herein, where the first component is toxic to a cell, to an organ, or to both, where the separation of the first component from the second component reduces toxic effect of the first component to the cell, the organ, a subject containing the cell, the organ, or both, or a combination thereof, compared to the toxic effect of the uncleaved composition. 
     In some forms, the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to a tumor compared to the delivery percentage of the uncleaved composition. In some forms, the reduction in the toxic effect is at least partially due to an increased delivery rate of the separated first component to a tumor compared to the delivery rate of the uncleaved composition. In some forms, the reduction in the toxic effect is at least partially due to an increased rate of clearance of the separated first component from the subject compared to the rate of clearance of the uncleaved composition. In some forms, the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to a second cell, to a second organ, or to both compared to the delivery percentage of the uncleaved composition. In some forms, the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to the cell, to the organ, or to both compared to the delivery percentage of the uncleaved composition. In some forms, the organ is the kidney, lung or heart. 
     Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions. 
         FIG. 1  is a diagram of an example of screening for PCC ligands of FOLR1 using synthetic epitopes (SynEps). 
         FIG. 2  is a diagram of an example of screening for PCC ligands of FOLR1 using synthetic epitopes (SynEps). 
         FIG. 3  is a diagram of an example of screening for PCC ligands of FOLR1 using FOLR1 and a folate-Az4-biotin. 
         FIG. 4  is a diagram of an example of screening for PCC ligands of FOLR1 using FOLR1 and a folate-Az4-biotin. 
         FIG. 5  is a diagram of ELISA assay of FOLR1 binding of heterobiligands. 
         FIG. 6  is a graph binding of ligands and heterobiligands to FOLR1+ and FOLR1− cells assessed by flow cytometry. 
         FIG. 7  is a graph binding of ligands and heterobiligands to FOLR1+ and FOLR1− cells assessed by flow cytometry. 
         FIG. 8  is a graph binding of heterobiligands with different linkers to FOLR1+ and FOLR1− cells assessed by flow cytometry. 
         FIG. 9  is a graph binding of different concentrations of heterobiligands to FOLR1+ and FOLR1− cells assessed by flow cytometry. 
         FIG. 10  is a diagram of an assay for assessing cell internalization FOLR1 heterobiligands. 
         FIG. 11  is PET imaging data for an  18 F-labeled heterobiligand detected in a tumor-bearing mouse over time. T, tumor; L, liver; K, kidney; B, bladder; Int, intestine. 
         FIG. 12  is the time-course biodistribution analysis to quantify the uptake of an  18 F heterobiligand in a tumor-bearing mouse. 
         FIG. 13  is a cross trial comparison of three mice showing radiotracer accumulation in the tumor. 
         FIG. 14  is a graph binding of heterobiligands with DOTA, MPBA, or both to FOLR1+ and FOLR1− cells assessed by flow cytometry. 
         FIG. 15  is PET imaging data for a  68 Ga-labeled heterobiligand detected in a mouse over time at early tumor size. 
         FIG. 16  is PET imaging data for a  68 Ga-labeled heterobiligand detected in a mouse over time at larger tumor size. 
         FIGS. 17A-17F  are graphs of  68 Ga-labeled heterobiligand over time in various tissues of two mice: mouse 4, with a tumor size of 150 mm 3 , and mouse 6, with a tumor size of 490 mm3.  FIG. 17A  is tumor in mouse 4.  FIG. 17B  is tumor in mouse 6.  FIG. 17C  is kidney in mouse 4.  FIG. 17D  is kidney in mouse 6.  FIG. 17E  is tumor/muscle in mouse 4.  FIG. 17F  is tumor/muscle in mouse 6.  FIG. 17G  is liver in mouse 4.  FIG. 17H  is liver in mouse 6.  FIG. 17I  is heart in mouse 4.  FIG. 17J  is heart in mouse 6.  FIG. 17K  is bladder in mouse 4.  FIG. 17L  is bladder in mouse 6. 
         FIG. 18  is a graph of the accumulation of heterobiligand in large tumors compared to smaller tumors. 
         FIG. 19  is a diagram of the combination of tumor imaging and tumor treatment using the same heterobiligand loaded with an imaging radionuclide or a therapeutic radionuclide, respectively. 
         FIG. 20  are graphs of tumors over time under treatment with different radionuclide doses delivered via heterobiligands. 
         FIG. 21  is a graph binding of ligands and heterobiligands to FOLR1+ and FOLR1− cells assessed by flow cytometry. 
         FIG. 22  is a diagram showing half-life extension of compositions by albumin binding. 
         FIG. 23  are dorsal images of label detected in a mouse over time. 
         FIG. 24  are transverse slice images of label detected in a mouse over time. 
         FIGS. 25A-25E  are graphs of labeled compositions in lung ( FIG. 25A ), kidney ( FIG. 25B ), heart ( FIG. 25C ), bladder ( FIG. 25D ), and liver ( FIG. 25E ) over time. 
         FIG. 26  is a graph of body weight change over days of treatment with Lu177-#6305. 
         FIG. 27  is a graph of tumor size over days of treatment with Lu177-#6305 treatment. 
         FIG. 28  is a graph of radioactivity (CPM/g) in various tissues 7 days after Lu177-#6305 injection. 
         FIG. 29  is a graph of radioactivity (CPM/g) in various tissues at study endpoint (Day 28 post-injection) in various tissues. 
         FIG. 30  is a graph of tumor size on day −2 for the  177 Lu-#2809 treatment. 
         FIG. 31  is a graph of tumor size on day 0 for the  177 Lu-#1307 treatment. 
         FIG. 32  is a graph of tumor size on day 0 for the  177 Lu-#6305 treatment. 
         FIGS. 33A-33C  are graphs of tumor size over days of treatment for OVCAR3 xenografts treated with Lu177-#2809 ( FIG. 33A ), for OVCAR3 xenografts treated with Lu177-#1307 ( FIG. 33B ), and for OVCAR3 xenografts treated with Lu177-#6305 ( FIG. 33C ). 
         FIG. 34  is a graph of percent body weight change versus time (in days) post-injection of different radioactivity doses of  177 Lu-7327. 
         FIG. 35  is a graph of percent body weight change versus time (in days) post-injection of different radioactivity doses of  177 Lu-7327. 
         FIG. 36A  is a graph of percent body weight change versus time (in days) post-injection of different radioactivity doses of  177 Lu-7327.  FIG. 36B  is a graph of tumor size versus time (in days) post-injection of different radioactivity doses of  177 Lu-7327. 
         FIGS. 37A-37D  are graphs of tumor size versus time (in days) post-injection of different radioactivity doses of  177 Lu-7327 in different mice.  FIG. 37A  used 0 MBq of  177 Lu-7327.  FIG. 37B  used 9.25 MBq of  177 Lu-7327.  FIG. 37C  used 14.8 MBq of  177 Lu-7327.  FIG. 37D  used 29.6 MBq of  177 Lu-7327. 
         FIG. 38A  is a graph of percent body weight change versus time (in days) post-injection of different radioactivity doses of  177 Lu-7327.  FIG. 38B  is a graph of percent tumor size change versus time (in days) post-injection of different radioactivity doses of  177 Lu-7327. 
         FIG. 39  is a graph of percent body weight change versus time (in days) post-injection of different radioactivity doses of  177 Lu-6305. 
         FIG. 40  is a graph of percent body weight change versus time (in days) post-injection of different radioactivity doses of  177 Lu-6305. 
         FIG. 41  is a graph of tumor size versus time (in days) post-injection of 0 MBq and 74 MBq of  177 Lu-7327. 
         FIG. 42  is a diagram of the compound species resulting from incubation of compound #7327 (folate-hshta-Gly-Phe-Lys(DOTA)) with different concentration of NEP and shows that compound #7327 is progressively cleaved by increasing NEP into a single predominant species corresponding to cleavage of the G-F-K peptide linker in compound #7327. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description. 
     Disclosed are compounds, compositions, systems, and methods useful for treating disease (such as cancer and tumors), or binding, detecting, and affecting compounds, compositions, cells, tissues, and organs, where the compounds, compositions, systems, and methods include or involve toxic compounds and where the toxic effect of the compounds, compositions, systems, and methods is reduced by providing a cleavage site to facilitate separation of the toxic component from other components of the compound, composition, or system. Preferably the system is a composition delivery system comprising or using a composition as disclosed herein. In some forms, the cleavage of the composition delivers a therapeutic agent and avoids organ damage by the composition. 
     In some forms, the first component is toxic. In some forms, the first component is toxic to a cell, to an organ, or to both. In some forms, the first component is toxic to the kidney, lung or heart. In some forms, the first component is nephrotoxic. 
     It has been discovered that that compositions and systems that include or involve a toxic moiety can be made less toxic by facilitating delivery of the composition or the toxic component to its target (such as a tumor). Cleavage of the toxic component form the composition or system results in separation and delivery of the toxic component to its target, thus reducing toxicity of the compositions. For example, disclosed are compositions comprising a first component and a second component, where the first component is toxic, where the first and second components are coupled via a linking component, where the linking component comprises a neprilysin (NEP) cleavage site, where the NEP cleavage site can be cleaved by NEP, where cleavage of NEP cleavage site separates the first component from the second component. 
     Kidneys are the major organ of elimination. Kidney retention of radiotracers and other nephrotoxins is a source of nephrotoxicity that can hinder development of theranostic agents via radiation-mediated DNA damage and other therapeutic agents that include nephrotoxins. It has also been discovered that compositions that include or involve a nephrotoxic moiety can be made less nephrotoxic by facilitating clearance of the nephrotoxic moiety by including a kidney-specific cleavage site that results in separation and clearance by the kidney of the nephrotoxic moiety, thus reducing nephrotoxicity of the compositions. For example, disclosed are compositions comprising a first component and a second component, where the first component is nephrotoxic, where the first and second components are coupled via a linking component, where the linking component comprises a neprilysin (NEP) cleavage site, where the NEP cleavage site can be cleaved by NEP, where cleavage of NEP cleavage site separates the first component from the second component. 
     A. Definitions 
     As used herein, the term “mammal” includes humans and both domestic animals such as laboratory animals and household pets (e.g., cats, dogs, swine, cattle, sheep, goats, horses, rabbits), and non-domestic animals such as wildlife and the like. 
     The term “capture agent” as used herein refers to a composition that comprises one or more target-binding moieties, or ligands, 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 some forms, 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 can 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. In some forms, capture agents comprising one or more peptide ligands that specifically bind a target are also referred to as epitope-targeted macrocyclic peptide ligands against the target. 
     Reference to “capture agents” further refers to pharmaceutically acceptable salts thereof. “Pharmaceutically acceptable salt” includes both acid and base addition salts. 
     “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like. 
     “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2 dimethylaminoethanol, 2 diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. 
     The capture agents described herein, or their pharmaceutically acceptable salts, may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that can be defined, in terms of absolute stereochemistry, as (R) or (S) or, as (D) or (L) for amino acids. The disclosed compositions and methods are meant to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (−), (R) and (S), or (D) and (L) isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, for example, chromatography and fractional crystallization. Conventional techniques for the preparation/isolation of individual enantiomers include chiral synthesis from a suitable optically pure precursor or resolution of the racemate (or the racemate of a salt or derivative) using, for example, chiral high pressure liquid chromatography (HPLC). When the compositions described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compositions include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. (D)-amino acids (also referred to as D-amino acids) are referred to herein in lower case letters (e.g. D-valine is referred to as “v”), while (L)-amino acids (also referred to herein as L-amino acids) are referred to in upper case letters (e.g. L-valine or valine is referred to as “V”). Glycine is non-chiral and is referred to as “G.” 
     A “stereoisomer” refers to a compound made up of the same atoms bonded by the same bonds but having different three-dimensional structures, which are not interchangeable. The disclosed compositions and methods contemplate various stereoisomers and mixtures thereof and include “enantiomers,” which refers to two stereoisomers whose molecules are non-superimposable mirror images of one another. 
     A “tautomer” refers to a proton shift from one atom of a molecule to another atom of the same molecule. The disclosed compositions and methods include tautomers of any said compounds. 
     The term “epitope” as used herein refers to a distinct molecular surface of a protein. Typically, the epitope is a polypeptide and it can act on its own as a finite sequence of 10-40 amino acids. 
     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. 
     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 can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. 
     The term “artificial amino acid” as used herein refers to an amino acid that is different from the twenty naturally occurring amino acids (alanine, arginine, glycine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, serine, threonine, histidine, lysine, methionine, proline, valine, isoleucine, leucine, tyrosine, tryptophan, phenylalanine) in its side chain functionality. The artificial amino acid can be a close analog of one of the twenty natural amino acids, or it can introduce a completely new functionality and chemistry, as long as the hydrophobicity of the artificial amino acid is either equivalent to or greater than that of the natural amino acid. The artificial amino acid can either replace an existing amino acid in a protein (substitution), or be an addition to the wild type sequence (insertion). The incorporation of artificial amino acids can be accomplished by known chemical methods including solid-phase peptide synthesis or native chemical ligation, or by biological methods. 
     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 10 −8  M, 10 −9  M or 10 −10  M or even lower. 
     The term “K D ” as used herein refers to the dissociation equilibrium constant of a particular capture agent-antigen interaction. 
     The term “k d ” (sec −1 ) as used herein refers to the dissociation rate constant of a particular capture agent-antigen interaction. Said value is also referred to as the k off  value. 
     The term “k a ” (M −1 ×sec −1 ) as used herein refers to the association rate constant of a particular capture agent-antigen interaction. 
     The term “K D ” (M) as used herein refers to the dissociation equilibrium constant of a particular capture agent-antigen interaction. 
     The term “K A ” (M −1 ) as used herein refers to the association equilibrium constant of a particular capture agent-antigen interaction and is obtained by dividing the k a  by the k d . 
     The term “imaging agent” refers to capture agents that have been labeled for detection. In some forms, imaging agents are isotopically-labelled by having one or more atoms replaced by an atom having a different atomic mass or mass number. Examples of isotopes that can be incorporated into the disclosed compositions include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorous, fluorine, chlorine, and iodine, such as  2 H,  3 H,  11 C,  13 C,  14 C,  13 N,  15 N,  15 O,  17 O,  18 O,  31 P,  32 P,  35 S,  18 F,  36 Cl,  123 I, and  125 I, respectively. These radiolabeled compositions could be useful to help determine or measure the effectiveness of the compositions, by characterizing, for example, the site or mode of action, or binding affinity to a pharmacologically important site of action. Certain isotopically-labelled disclosed imaging agents, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The radioactive isotopes tritium, i.e.  3 H, and carbon-14, i.e.  14 C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection. 
     Substitution with heavier isotopes such as deuterium, i.e.  2 H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances. 
     Substitution with positron emitting isotopes, such as  11 C,  18 F,  15 O and  13 N, can be useful in Positron Emission Tomography (PET) studies for examining substrate receptor occupancy. Isotopically-labeled imaging agents can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the Examples as set out below using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed. 
     The present disclosure is also meant to encompass the in vivo metabolic products of the disclosed imaging agents. Such products can result from, for example, the oxidation, reduction, hydrolysis, amidation, esterification, and the like of the administered composition, primarily due to enzymatic processes. Accordingly, disclosed are compositions produced by a process comprising administering a composition as disclosed to a mammal for a period of time sufficient to yield a metabolic product thereof. Such products are typically identified by administering a radiolabeled the disclosed compositions in a detectable dose to an animal, such as rat, mouse, guinea pig, monkey, or to human, allowing sufficient time for metabolism to occur, and isolating its conversion products from the urine, blood or other biological samples. 
     A “pharmaceutical composition” refers to a formulation of a composition as disclosed and a medium generally accepted in the art for the delivery of the biologically active composition to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefor. 
     The term “condition” as used herein refers generally to a disease, event, or a change in health status. A change in health status may be associated with a particular disease or event, in which case the change may occur simultaneously with or in advance of the disease or event. In those cases where the change in health status occurs in advance of a disease or event, the change in health status may serve as a predictor of the disease or event. For example, a change in health status may be an alteration in the expression level of a particular gene associated with a disease or event. Alternatively, a change in health status may not be associated with a particular disease or event. 
     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. 
     An “effective amount” or “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 a disclosed capture agent can 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. 
     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. 
     The term “synthetic” as used herein with regard to a protein catalyzed capture agent refers to the capture agent having been generated by chemical rather than biological means. 
     Nephrotoxicity is defined as rapid deterioration in the kidney function due to toxic effect of medications and chemicals. There are various forms, and some drugs may affect renal function in more than one way. Nephrotoxins are substances displaying nephrotoxicity. Different mechanisms lead to nephrotoxicity, including renal tubular toxicity, inflammation, glomerular damage, crystal nephropathy, and thrombotic microangiopathy. The traditional markers of nephrotoxicity and renal dysfunction are blood urea and serum creatinine. Kidney injury molecule-1, Cystatin C, and neutrophil gelatinase-associated lipocalin sera levels are more sensitive than blood urea and serum creatinine in the detection of acute kidney injury during nephrotoxicity (Al-Naimi et al., J. Adv. Pharm. Technol. Res. 10(3):95-99) (2019). As used herein, “nephrotoxic moiety” refers to a part of a compound or composition that displays nephrotoxicity. 
     Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al., (1997) Nucleic Acids Res. 25:3389-402). 
     As those of ordinary skill in the art will understand, BLAST searches assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of nonrandom sequences, which can be homopolymeric tracts, short-period repeats, or regions enriched in one or more amino acids. Such low-complexity regions can be aligned between unrelated proteins even though other regions of the protein are entirely dissimilar. A number of low-complexity filter programs can be employed to reduce such low-complexity alignments. For example, the SEG (Wooten and Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie and States, (1993) Comput. Chem. 17:191-201) low-complexity filters can be employed alone or in combination. 
     As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences, which are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences, which differ by such conservative substitutions, are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif., USA). 
     As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window can comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. 
     The term “substantial identity” or “substantially identical” of polynucleotide sequences means that a polynucleotide comprises a sequence that has between 50-100% sequence identity, preferably at least 50% sequence identity, preferably at least 60% sequence identity, preferably at least 70%, more preferably at least 80%, more preferably at least 90% and most preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of between 55-100%, preferably at least 55%, preferably at least 60%, more preferably at least 70%, 80%, 90% and most preferably at least 95%. 
     B. Development of Capture Agents 
     Antibodies are currently the default detection agent for use in diagnostic and therapeutic 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. 
     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). In humans, this loop ranges in size from 1-35 residues (15 on average), can adopt a wide range of structural conformations, 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. 
     In situ click chemistry 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 protein effectively plays the role of an extremely selective variant of the Cu(I) catalyst that is commonly used for such couplings. 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. 
     Sequential in situ click chemistry extends the in situ click chemistry concept to enable the discovery of multiligand capture agents (U.S. Publication No. 20100009896, incorporated herein by reference). This process was used previously to produce a triligand capture agent against the model protein carbonic anhydrase II (CAII). 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 can be identified by screening the protein against a large (&gt;10 6  element) one-bead-one-compound (OBOC) peptide library, where the peptides themselves can 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, 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. 
     C. Capture Agents 
     In one aspect, provided herein is a stable, synthetic capture agent that specifically binds a target, where the capture agent comprises one or more “anchor” ligands (also referred to as simply “ligands” herein), a linker, and one or more additional ligands, and wherein the ligands selectively bind the same target. These are referred to herein as capture agents. The disclosed compositions are or include capture agents. For example, the second component of the composition can be or comprise a capture agent. 
     Ligands are target-binding moieties (also referred to as binding molecules, specific binding molecules, target-binding molecules, binding moieties, and specific binding moieties). 
     In some forms, two separate ligands that bind to two different regions of the same protein (the target) are chemically linked together to form a biligand. By optimizing a linker of the two ligands, the biligand formed by the ligands and linker can exhibit a binding affinity that is far superior to either of the individual ligands. This enhanced binding effect is called binding cooperativity. For an ideal cooperative binder, the thermodynamic binding energies of the individual ligands to the target will sum to yield the binding energy of the linked biligand. This means that the binding affinity constant (K D ) of the linked biligand will be the product of the binding affinity of the individual ligands (i.e. K D =K D1 ×K D2 , where the subscripts 1 and 2 refer to the two ligands). In practice, full cooperative binding is rarely, if ever, achieved. Thus, a comparison of the properties of a linked biligand against those of a fully cooperative binder provides a measurement of how optimally the two ligands were linked. 
     A capture agent having two ligands can be referred to as a biligand capture agent, or just as a biligand. Where the two ligands have different character, such as a peptide ligand and a small molecule ligand, the biligand capture agent can be referred to as a heterobiligand, or just as a heterobiligand. 
     A capture agent having three ligands can be referred to as a triligand capture agent, or just as a triligand. Where two or three of the ligands have different character, such as two peptide ligands and one small molecule ligand, the triligand capture agent can be referred to as a heterotriligand, or just as a heterotriligand. 
     If the protein target has a known and well-defined tertiary (folded) structure, then ligands that bind to preferred regions of the protein can be used and linked together to optimize their binding to their respective regions. 
     In some forms, a ligand comprises one or more polypeptides or peptides. In some forms, 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 azidoalkyl, substituted and unsubstituted polyethyleneglycolyl, and substituted and unsubstituted 1,2,3-triazole. In some forms, the ligand comprises a peptide comprising D-amino acids and artificial amino acids. 
     In some forms, the ligands are linked to one another via a covalent linkage through a linker. In some forms, the ligand and linker 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 
     In those forms where the ligands and linker are linked to one another via a 1,4-disubstituted-1,2,3-triazole linkage, the 1,4-disubstituted-1,2,3-triazole linkage can be formed by Cu-Catalyzed Azide/Alkyne Cycloaddition (CuAAC). 
     In some forms, the ligands and linker are linked to one another by a Tz4 linkage having the following structure: 
     
       
         
         
             
             
         
       
     
     In some forms, the ligands and linker are linked to one another by a Tz5 linkage having the following structure: 
     
       
         
         
             
             
         
       
     
     In those forms wherein one or more of the ligands and linker are linked to one another via amide bonds, the amide bond can be formed by coupling a carboxylic acid group and an amine group in the presence of a coupling agent (e.g., O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HATU), N-hydroxy-7-aza-benzotriazole (HOAt), or diisopropylethylamine (DIEA) in DMF). 
     In some forms, 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 some forms, 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 some forms the capture agents provided herein are more resistant to proteolytic degradation than an antibody binding to the same target protein. 
     In some forms, 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 some forms, the capture agents have a shelf-life of one year or greater, two years or greater, or more than three years. In some forms, the capture agents are stored as a lyophilized powder. In some forms, the capture agents provided herein have a longer shelf-life than an antibody binding to the same target protein. 
     In some forms, the capture agents provided herein are stable at temperatures ranging from about −80° to about 120° C. In some forms, 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 some forms, 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. 
     In some forms, the capture agents provided herein are stable at a pH range from about 3.0 to about 8.0. In some forms, the range is about 4.0 to about 7.0. In some forms, the range is about 7.0 to about 8.0. 
     In some forms, the capture agents provided herein are stable in human serum for more than 12 hours. In some forms, the capture agents are stable in human serum for more than 18 hours, more than 24 hours, more than 36 hours, or more than 48 hours. In some forms, 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. In some forms, the capture agents are stable as a powder for two months at a temperature of about 60° C. 
     In some forms, the capture agents provided herein can comprise one or more detection labels, including for example biotin, copper-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (copper-DOTA),  64 Cu DOTA,  68 Ga DOTA,  18 F,  64 Cu,  68 Ga,  89 Zr,  124 I,  86 Y,  94m Tc,  110m In,  11 C,  76 Br,  123 I,  131 I,  67 Ga,  111 In and  99m Tc, or other radiolabeled products that can include gamma emitters, proton emitters, positron emitters, tritium, or covered tags detectable by other methods (i.e., gadolinium) among others. In some forms, the detection label is  18 F. The capture agents can be used as diagnostic agents. 
     In some forms, the capture agents provided herein can 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 can 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 can be synthesized to bind to non-canonical or non-biological epitopes on proteins, thereby increasing their versatility. In some forms, the capture agent can be modified by modifying the synthesis blocks of the target-binding moieties before the coupling reaction. 
     In some forms, the capture agents provided herein are stable across a wide range of temperatures, pH values, storage times, storage conditions, and reaction conditions, and in some forms the imaging agents are more stable than a comparable antibody or biologic. In some forms, the capture agents are stable in storage as a lyophilized powder. In some forms, the capture agents are stable in storage at a temperature of about −80° C. to about 60° C. In some forms, the capture agents are stable at room temperature. In some forms, the capture agents are stable in human serum for at least 24 hours. In some forms, the capture agents are stable at a pH in the range of about 3 to about 12. In some forms, the capture agents are stable as a powder for two months at a temperature of about 60° C. 
     As shown in the Example, tuning of heterobiligand capture agents can enhance, for example, binding to the target, in vivo half-life, and combinations of these. For example, incorporation of unnatural or modified amino acids can increase binding. Deletion of amino acids in a hit peptide ligand can increase, for example, binding to the target (deletions can also decrease these properties). Related to these, substitution of amino acids can increase, for example, binding to the target. Alanine scanning of hit peptides is useful for identify amino acids that can be modified without reducing binding or other properties. 
     The length and composition of linkers can be tuned to optimize, for example, binding to the target, in vivo half-life, and combinations of these. For example, in addition to PEG linkers, all carbon (e.g., alkyl) linkers, linkers with mixtures of PEG and alkyl, peptide linkers, linkers with mixtures of PEG and peptides (amino acids), linkers with mixtures of alkyl and peptides (amino acids), and mixtures of PEG, alkyl, and amino acids can be used. In particular, inclusion of an alkyl on the end of the linker that couples to a ligand (or inclusion of an alkyl tail on the ligand for coupling to the linker) is useful for tuning the heterobiligand. 
     Methylation of amines in heterobiligands, preferably in the peptide ligand, but also in the linker, can increase cell penetration. Addition of a cell penetrating peptide sequence in the heterobiligand can increase cell penetration. Lipidating groups can be added to the heterobiligand, such as in the peptide ligand or in the linker, to increase lipophilicity of the heterobiligand. The closure (cyclization) of the peptide ligand can be accomplished using different chemistries and different groups. For example, triazole linkages can be used. 
     Combinations of these modifications (tunings) can be used to increase or modulate these effects. 
     In some forms, the composition is a cyclic peptide having the following structure (I): 
     
       
         
         
             
             
         
       
     
     or a salt, tautomer, prodrug or stereoisomer thereof, wherein: 
     L 1  and L 2  are each individually a bond or an optionally substituted linker moiety, wherein each linker moiety optionally comprises a linkage to the NEP cleavage site and the first component, a linkage to the first component, a linkage to a ligand, a linkage to a reporter moiety, a linkage to an albumin binding moiety, a linkage to a peptide ligand, or combinations thereof; 
     G is a triazole, a carbon-carbon double bond or an amide; 
     M is methionine; 
     R is H or an optionally substituted linker moiety, wherein each linker moiety optionally comprises a linkage to the NEP cleavage site and the first component, a linkage to the first component, a linkage to a ligand, a linkage to a reporter moiety, a linkage to an albumin binding moiety, a linkage to a peptide ligand, or combinations thereof; 
     R 1  is H or C 1 -C 6  alkyl; 
     Y 1  and Y 2  are each individually 0 or 1; and 
     SEQ is an amino acid sequence comprising from 2 to 20 amino acids selected from natural and non-natural amino acids. 
     In some forms, L 1  is —C(HR 2 )— wherein R 2  is H, —R 5 -L 3 -A 1 , —R 5 —C(═O)-L 3 -A 1 , —R 5 -A 2 -L 3 -A 1 , —R 5 —C(═O)-A 2 -L 3 -A 1 , —R 5 -L 3 (-A 2 )-A 1 , or —R 5 —C(═O)-L 3 (-A 2 )-A 1 , where —R 5  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 3  is a linker moiety, and where A 1  and A 2  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. In some forms, A 1  and A 2  independently further comprise -L 4 -, wherein -L 4 - is a linker moiety. 
     In some forms, L 2  is —C(HR 4 )—, wherein R 4  is H, —R 6 -L 5 -A 3 , —R 6 —C(═O)-L 5 -A 3 , —R 6 -A 4 -L 5 -A 3 , —R 6 —C(═O)-A 4 -L 5 -A 3 , —R 6 -L 5 (-A 4 )-A 3 , or —R 6 —C(═O)-L 5 (-A 4 )-A 3 , where —R 6  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 5  is a linker moiety, and where A 3  and A 4  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. In some forms, A 3  and A 4  independently further comprise -L 6 -, wherein -L 6 - is a linker moiety. 
     In some forms, R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. In some forms, A 5  and A 6  independently further comprise -L 8 -, wherein -L 8 - is a linker moiety. 
     In some forms, L 1  is —C(HR 2 )—, wherein R 2  is H, —R 5 -L 3 -A 1 , —R 5 —C(═O)-L 3 -A 1 , —R 5 -A 2 -L 3 -A 1 , —R 5 —C(═O)-A 2 -L 3 -A 1 , —R 5 -L 3 (-A 2 )-A 1 , or —R 5 —C(═O)-L 3 (-A 2 )-A 1 , where —R 5  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 3  is a linker moiety, and where A 1  and A 2  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. In some forms, A 1  and A 2  independently further comprise -L 4 -, wherein -L 4 - is a linker moiety. 
     In some forms, L 2  is —C(HR 4 )—, wherein R 4  is H, —R 6 -L 5 -A 3 , —R 6 —C(═O)-L 5 -A 3 , —R 6 -A 4 -L 5 -A 3 , —R 6 —C(═O)-A 4 -L 5 -A 3 , —R 6 -L 5 (-A 4 )-A 3 , or —R 6 —C(═O)-L 5 (-A 4 )-A 3 , where —R 6  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 5  is a linker moiety, and where A 3  and A 4  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. In some forms, A 3  and A 4  independently further comprise -L 6 -, wherein -L 6 - is a linker moiety. 
     In some forms, R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. In some forms, A 5  and A 6  independently further comprise -L 8 -, wherein -L 8 - is a linker moiety. 
     In some forms, one or more of A 1 , A 2 , A 3 , A 4 , A 5 , and A 6  individually and independently comprise a combination of one or more of the following: the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, and a peptide ligand. 
     A preferred set of amino acids from which the amino acids of SEQ can be selected (Set 1) contains Cyclopropyl Alanine (CyA) and Gly (hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), and 4-Phenyl Phenylalanine (PhF) (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar side chain—neutral); His, Lys, Arg, Glu (polar side chain—charged); and β-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation). 
     Another preferred set of amino acids from which the amino acids of SEQ can be selected (Set 2) contains Cyclopropyl Alanine (CyA) and Gly (hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT), Thiazolyl Alanine (Thz), 4-Phenyl Phenylalanine (PhF), and Phe (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar side chain—neutral); His, Lys, Arg, Glu (polar side chain—charged); and N-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation). 
     Another preferred set of amino acids from which the amino acids of SEQ can be selected (Set 3) contains Cyclopropyl Alanine (CyA) and Gly (hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), Thiazolyl Alanine (Thz), 4-Phenyl Phenylalanine (PhF), and Phe (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar side chain—neutral); His, Lys, Arg, Glu (polar side chain—charged); and β-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation). 
     In some forms, G is a triazole. Such triazoles may be derived by reaction of an alkyne and azide on a precursor acyclic peptide. 
     In some forms, G is a carbon-carbon double bond. In some forms, these peptides are obtained by reactions of two carbon-carbon double bonds (alkenes) present in an acyclic precursor. Such reactions can be carried out using Grubbs metathesis chemistry, which is well-known to those of skill in the art. 
     In some forms of the foregoing, the cyclic peptide has one of the following structures (Ia) or (Ib): 
     
       
         
         
             
             
         
       
     
     wherein: 
     R 3  is H, -L 3 -A 1 , —C(═O)-L 3 -A 1 , -A 2 -L 3 -A 1 , —C(═O)-A 2 -L 3 -A 1 , -L 3 (-A 2 )-A 1 , or —C(═O)-L 3 (-A 2 )-A 1 , where L 3  is a linker moiety and A 1  and A 2  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof; and 
     x and y are each independently an integer from 1 to 8. 
     In some forms, A 1  and A 2  independently further comprise -L 4 -, wherein -L 4 - is a linker moiety. 
     In some forms, R 3  is H, -L 3 -A 1 , —C(═O)-L 3 -A 1 , -A 2 -L 3 -A 1 , —C(═O)-A 2 -L 3 -A 1 , -L 3 (-A 2 )-A 1 , or —C(═O)-L 3 (-A 2 )-A 1 , where L 3  is a linker moiety and A 1  and A 2  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. In some forms, A 1  and A 2  independently further include -L 4 -, wherein -L 4 - is a linker moiety. 
     In some forms of the compositions of structure (Ia) and (Ib), x is 1. In some forms, x is 2. In some forms, x is 3. In some forms, x is 4. In some forms, x is 5. In some forms, x is 6. In some forms, x is 7. In some forms, x is 8. 
     In some forms of the compositions of structure (Ia) and (Ib), y is 1. In some forms, y is 2. In some forms, y is 3. In some forms, y is 4. In some forms, y is 5. In some forms, y is 6. In some forms, y is 7. In some forms, y is 8. 
     In some forms, R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. In some forms, A 5  and A 6  independently further comprise -L 8 -, wherein -L 8 - is a linker moiety. 
     In some forms, R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. In some forms, A 5  and A 6  independently further include -L 8 -, wherein -L 8 - is a linker moiety. 
     In some forms the composition is biligand capture agent, and A 1 , A 2 , A 3 , A 4 , A 5 , and/or A 6  is a bond to a peptide ligand, for example a linear peptide ligand or a cyclic peptide ligand. In some forms, the peptide ligand further comprises a second peptide ligand, and the composition is thus a triligand capture agent. 
     The structure of the “linker moieties” (e.g., L 3 , L 4 , L 5 , L 6 , L 7 , and L 8 ) are not particularly limited. For example, in some forms, linkers comprising ethylene glycol of various lengths (e.g., 1-10 glycol repeating units, e.g., about 5-7) can be used. Ethylene diamine linkers can also be employed alone or in combination with other moieties (e.g., ethylene glycol). Linker moieties comprising triazole (e.g., resulting from reaction of an alkyne and azide) are also useful. 
     In some forms, y 1  and y 2  are each 0. 
     In some forms, the cyclic peptide has one of the following structures: 
     
       
         
         
             
             
         
       
     
     For example, in some forms SEQ comprises from 2 to 9 amino acids. In some forms, SEQ comprises from 5 to 7 amino acids. 
     In some forms, SEQ comprise natural amino acids. In some forms, SEQ comprises non-natural amino acids. In some forms, SEQ comprises natural and non-natural amino acids. 
     In some forms, the amino acids are selected from D and L stereoisomers of Ala, Gly, Leu, Ile, Val, Phe, Trp, Arg, His, Lys, Asp, Glu, Asn, Gln, Ser, Thr, Tyr and Pro. In some forms, the amino acids are selected from D and L stereoisomers of Ala, Gly, Leu, Val, Phe, Trp, Arg, His, Lys, Asp, Glu, Asn, Ser, Thr, Tyr and Pro. In some forms, the amino acids are selected from CyA, Gly, FP, MT, MeOPyr, PhF, Asn, Ser, Thr, His, Lys, Arg, Glu, BPhA, N-Me-a, and Pro (Set 1). In some forms, the amino acids are selected from CyA, Gly, FP, MT, Thz, PhF, Phe, Asn, Ser, Thr, His, Lys, Arg, Glu, N-Me-a, and Pro (Set 2). In some forms, the amino acids are selected from CyA, Gly, FP, MT, MeOPyr, Thz, PhF, Phe, Asn, Ser, Thr, His, Lys, Arg, Glu, BPhA, N-Me-a, and Pro (Set 3). 
     The amino acids in SEQ are selected to have affinity for the desired target, including allosteric binding sites such as protein epitopes. 
     Compositions comprising any of the foregoing compositions and a pharmaceutically acceptable carrier are also provided. In some forms, a library comprising a plurality of the forgoing compositions is provided. 
     In some forms, the compositions (also referred to herein as 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 some forms, the capture agents have a shelf-life of one year or greater, two years or greater, or more than three years. In some forms, the capture agents are stored as a lyophilized powder. In some forms, the capture agents provided herein have a longer shelf-life than a biologic binding to the same target protein. 
     In some forms, the capture agents provided herein are stable at temperatures ranging from about −80° C. to about 120° C. In some forms, the capture agents are stable within a temperature range of −80° C. to −40° C.; −40° C. to −20° C.; −20° C. to 0° C.; 0° C. to 20° C.; 20° C. to 40° C.; 40° C. to 60° C.; 60° C. to 80° C.; and/or 80° C. to 120° C. In some forms, the capture agents provided herein are stable across a wider range of temperatures than a biologic binding to the same target protein, and/or remain stable at a specific temperature for a longer time period than a biologic binding to the same target protein. 
     In some forms, the pH of a capture agent provided herein is in the range of about 3.0 to about 12.0. In some forms, the pH of the capture agent is in the range of about 5.0 to about 9.0. The pH of a capture agent may be adjusted to a physiologically compatible range using methods known in the art. For example, in some forms the pH of the capture agent may be adjusted to the range of about 6.5 to about 8.5. 
     In some forms, the capture agents provided herein are stable in blood serum for more than 12 hours. In some forms, the capture agents are stable in blood serum for more than 18 hours, more than 24 hours, more than 36 hours, more than 48 hours, or more than 96 hours. In some forms, the capture agents provided herein are stable for a longer period of time in blood serum than a biologic binding to the same target protein. 
     In some forms, the capture agents provided herein may comprise one or more reporter moieties (detection labels), including for example biotin, copper-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraaceticacid (copper-DOTA), desferrioxamine B (DFO), a ligand for radiolabeling with  68 Ga, 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 some forms, the capture agents provided herein comprise one or more reporter moieties. In some forms, the reporter moiety is copper-DOTA. In some forms, the reporter moiety is selected from  64 Cu DOTA,  68 Ga DOTA,  18 F,  64 Cu,  68 Ga,  89 Zr,  124 I,  86 Y,  94m Tc,  110m In,  11 C and  76 Br. In some forms, the reporter moiety is selected from  123 I,  131 I,  67 Ga,  111 In and  99m Tc. In some forms, the reporter moiety is a fluorescent label. 
     In some forms, the composition comprises a linkage to a reporter moiety, the reporter moiety selected from polyethylene glycol (PEG), biotin, thiol and fluorophores. For example, in some forms the fluorophores are selected from FAM, FITC, Cy5, TRITC, TAMRA. 
     Table 8 provides reporter moieties useful in various different applications of the compositions. Other useful reporter moieties can be derived by one of skill in the art. 
     
       
         
           
               
             
               
                 TABLE 8 
               
             
            
               
                   
               
               
                 Reporter Moieties 
               
            
           
           
               
               
            
               
                 Application 
                 Reporter 
               
               
                   
               
               
                 ELISA: microtiter plate 
                 Biotin 
               
               
                 ELISA: lateral flow test 
                 Biotin 
               
               
                 Immunoprecipitation (and other 
                 Biotin, thiol 
               
               
                 bead-based assays) 
                   
               
               
                 Dot blot 
                 Biotin 
               
               
                 Cell-based assay 
                 Biotin, fluorophore 
               
               
                 IHC 
                 Biotin, fluorophore 
               
               
                 In vivo imaging: PET 
                 Radioisotopes including  18 F, 
               
               
                   
                   68 Ga,  64 Cu,  89 Zr,  124 I 
               
               
                 In vivo imaging: SPECT 
                 Radioisotopes including  111 In, 
               
               
                   
                   90 Y,  99m Tc,  177 Lu 
               
               
                 In vivo imaging: MR 
                 Gd 3+   
               
               
                   
               
            
           
         
       
     
     In some forms, 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 some forms, the capture agent may be modified by modifying the synthesis blocks of the target-binding moieties before the coupling reaction. 
     Provided herein are pharmaceutical formulations comprising one or more of the capture agents provided herein. In some forms, 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. 
     Provided herein are kits comprising one or more of the capture agents disclosed herein. In some forms, 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. 
     It is understood that any form or instance of the peptides, as set forth above, and any specific substituent set forth herein for a R, R 1 , L 1 , L 2 , G, M, Y 1 , Y 2  or SEQ group in the peptides, as set forth above, may be independently combined with other forms and/or substituents of the peptides to form embodiments not specifically set forth above. In addition, in the event that a list of substituents is listed for any particular variable in a particular embodiment and/or claim, it is understood that each individual substituent may be deleted from the particular embodiment and/or claim and that the remaining list of substituents will be considered to be within the scope of the disclosed subject matter. 
     For the purposes of administration, the disclosed peptides may be administered as a raw chemical or may be formulated as pharmaceutical compositions. Pharmaceutical compositions of the disclosed subject matter can comprise a peptide of structure (I) and a pharmaceutically acceptable carrier, diluent or excipient. The peptide of structure (I) is present in the composition in an amount which is effective to treat a particular disease or condition of interest—that is, and preferably with acceptable toxicity to the patient. Activity of compositions of the peptides can be determined by one skilled in the art, for example, as described in the Examples. Appropriate concentrations and dosages can be readily determined by one skilled in the art. 
     Administration of the disclosed compositions, or their pharmaceutically acceptable salts, in pure form or in an appropriate pharmaceutical composition, can be carried out via any of the accepted modes of administration of agents for serving similar utilities. The disclosed pharmaceutical compositions can be prepared by combining a composition as disclosed with an appropriate pharmaceutically acceptable carrier, diluent or excipient, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, intratumoral, or infusion techniques. The disclosed pharmaceutical compositions can be formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units. Actual methods of preparing dosage forms are known, or will be apparent, to those skilled in this art; for example, see  Remington: The Science and Practice of Pharmacy,  current edition (Philadelphia College of Pharmacy and Science). The composition to be administered will, in any event, contain a therapeutically effective amount of a disclosed composition, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the description herein. 
     A pharmaceutical composition as disclosed may be in the form of a solid or liquid. In one aspect, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. 
     The pharmaceutical composition may be in the form of a liquid, for example, a solution, emulsion or suspension. The liquid may be for delivery by injection. When intended for injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included. 
     The disclosed liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer&#39;s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile. 
     A disclosed liquid pharmaceutical composition intended for parenteral administration should contain an amount of a disclosed composition such that a suitable dosage will be obtained. 
     The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining a compound or composition with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the composition so as to facilitate dissolution or homogeneous suspension of the composition in the aqueous delivery system. 
     The disclosed compositions, or their pharmaceutically acceptable salts, are administered in a therapeutically effective amount, which will vary depending upon a variety of factors including the activity of the specific composition employed; the metabolic stability and length of action of the composition; the age, body weight, general health, sex, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. 
     The disclosed compositions, or pharmaceutically acceptable derivatives thereof, may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy includes administration of a single pharmaceutical dosage formulation which contains a composition and one or more additional active agents, as well as administration of the composition and each active agent in its own separate pharmaceutical dosage formulation. For example, a composition and the other active agent can be administered to the patient together in a single dosage composition or each agent administered in separate dosage formulations. Where separate dosage formulations are used, the compositions and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially; combination therapy is understood to include all these regimens. 
     It is understood that in the present description, combinations of substituents and/or variables of the depicted formulae are permissible only if such contributions result in stable compositions. 
     It will also be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include t-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include —C(O)—R″ (where R″ is alkyl, aryl or arylalkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T. W. and P. G. M. Wutz,  Protective Groups in Organic Synthesis  (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin. 
     It will also be appreciated by those skilled in the art, although such protected derivatives of compounds may not possess pharmacological activity as such, they may be administered to a mammal and thereafter metabolized in the body to form the intended compounds which are pharmacologically active. Such derivatives may therefore be described as “prodrugs.” All prodrugs of the disclosed compounds and compositions are specifically contemplated. 
     Furthermore, all of the disclosed compounds and compositions that exist in free base or acid form can be converted to their pharmaceutically acceptable salts by treatment with the appropriate inorganic or organic base or acid by methods known to one skilled in the art. Salts of the compounds and compositions can be converted to their free base or acid form by standard techniques. 
     The disclosed peptides can be prepared by procedures known to those of skill in the art. For example, the peptides can be prepared using standard solid-phase peptide synthesis techniques, and modifications thereof. Modified amino acids may be employed to incorporate amino acids comprising alkyne and/or azide moieties and/or alkene moieties useful for cyclization. Methods for cyclizing the peptides using azide/alkyne chemistry and Grubbs metathesis chemistry are well-known in the art. Such methods are described in more detail in the examples. 
     It is understood that one skilled in the art may be able to make these compounds and compositions by similar methods or by combining other methods known to one skilled in the art. It is also understood that one skilled in the art would be able to make, in a similar manner as described below, other peptides not specifically illustrated in the examples below by using the appropriate starting components and modifying the parameters of the synthesis as needed. In general, starting components may be obtained from sources such as Sigma Aldrich, Lancaster Synthesis, Inc., Maybridge, Matrix Scientific, TCI, and Fluorochem USA, etc. or synthesized according to sources known to those skilled in the art (see, for example, Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th edition (Wiley, December 2000)) or prepared as described herein. 
     D. Identification of Capture Agents 
     Disclosed are methods for identification of cyclic peptides that are useful as capture agents for various targets. In general, the methods employ cyclic peptides, such as any of the cyclic peptides described herein, in methods for identification of mono-, bi- and/or tri-ligand capture agents. Higher order capture agents (tetra, penta, and the like) are also specifically contemplated. 
     In general, any methods employing the compounds and compositions described herein are specifically contemplated. For example, disclosed is a method for identifying a target binding compound (e.g., a protein capture agent) is provided, the method comprising 
     (a) providing a peptide library comprising a plurality of cyclic peptides comprising:
         (i) a sequence region comprising amino and carboxy termini and a variable peptide sequence of two to twenty amino acids selected from natural and non-natural amino acids; and   (ii) a linker region comprising a α-amino carbonyl, α-amido carbonyl, a methionine amino acid, or combinations thereof, and optionally comprising an alkyne, an azide, a linkage to a solid support or a linkage to a reporter moiety or a combination thereof, the linker region covalently linking the amino and carboxy termini of the sequence region.       

     (b) contacting the peptide library with a target or a truncated analogue thereof, the target or truncated analogue thereof comprising a binding site and optionally an alkyne, azide or reporter moiety or combinations thereof; 
     (c) identifying a peptide library member with affinity for the binding site 
     In some forms, a method for identifying a target binding compound (e.g., a protein capture agent) is provided, the method comprising: 
     (a) providing a first peptide library comprising a plurality of first peptide library members, the first peptide library members optionally comprising an alkyne, azide or reporter moiety or combinations thereof; 
     (b) contacting the first peptide library with a target or a truncated analogue thereof, the target or truncated analogue thereof comprising a first binding site and optionally an alkyne, azide or reporter moiety or combinations thereof; 
     (c) identifying a first peptide library member with affinity for the first binding site and optionally modifying the first peptide library member to include an alkyne or azide moiety; 
     and optionally: 
     (d) providing a second peptide library comprising a plurality of second peptide library members, the second peptide library members comprising an azide or alkyne or both; 
     (e) contacting the second peptide library with a composition comprising the target or truncated analogue thereof and the first peptide library member of step C; 
     (f) forming a triazole-linked conjugate between the first peptide library member of step C and a second peptide library member, the second peptide library member having affinity for a second binding site on the target or truncated analogue thereof, 
     wherein the first peptide library, the second peptide library, or both, comprise cyclic peptides comprising:
         (i) a sequence region comprising amino and carboxy termini and a variable peptide sequence of two to twenty amino acids selected from natural and non-natural amino acids; and   (ii) a linker region comprising a α-amino carbonyl, α-amido carbonyl, a methionine amino acid, or combinations thereof, and optionally comprising an alkyne, an azide, a linkage to a solid support or a linkage to a reporter moiety or a combination thereof, the linker region covalently linking the amino and carboxy termini of the sequence region.       

     A preferred set of amino acids from which the amino acids of SEQ can be selected contains Cyclopropyl Alanine (CyA) and Gly (hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), and 4-Phenyl Phenylalanine (PhF) (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar side chain—neutral); His, Lys, Arg, Glu (polar side chain—charged); and β-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation). 
     Another preferred set of amino acids from which the amino acids of SEQ can be selected (Set 2) contains Cyclopropyl Alanine (CyA) and Gly (hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT), Thiazolyl Alanine (Thz), 4-Phenyl Phenylalanine (PhF), and Phe (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar side chain—neutral); His, Lys, Arg, Glu (polar side chain—charged); and N-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation). 
     Another preferred set of amino acids from which the amino acids of SEQ can be selected (Set 3) contains Cyclopropyl Alanine (CyA) and Gly (hydrophobic side chain—aliphatic); 4-Fluorophenyl Alanine (FP), Methyl Tryptophan (MT), 2-Methoxy Pyridylalanine (MeOPyr), Thiazolyl Alanine (Thz), 4-Phenyl Phenylalanine (PhF), and Phe (hydrophobic side chain—aromatic); Asn, Ser, Thr (polar side chain—neutral); His, Lys, Arg, Glu (polar side chain—charged); and β-Phenylalanine (BPhA), N-Methyl d-alanine (N-Me-a), and Pro (conformational perturbation). 
     E. Methods of Making/Screening Capture Agents 
     Provided herein in some forms are methods of screening target-binding moieties and/or making imaging agents that comprise these target-binding moieties. Methods for screening target-binding moieties and/or making imaging agents that comprise these target-binding moieties can also be found in International Publication Nos. WO 2012/106671, WO 2013/033561, WO 2013/009869 and WO 2014/074907, each of which is incorporated by reference, herein, in their entireties. 
     For developing a set of PCC binders against a target protein, first one or more PCCs that bind an epitope on the target protein are identified. Optionally, one or more different PCCs binding to a second epitope are identified. Additional PCCs that bind to a third, fourth, etc., epitope can be useful as well. The epitope targeted PCC method teaches that this can be accomplished by screening peptide libraries against synthetic epitopes (SynEps). A SynEp is a polypeptide that has the sequence of the naturally occurring target epitope, except that one position contains an artificial amino acid that presents an azide or acetylene chemical group, called a click handle. The SynEp is further modified to contain an assay handle, such as a biotin group, at the N- or C-terminus. The screening procedure can be done using any procedure disclosed herein or known in the art. By screening, one identifies at least one unique peptide binder to each of at least two epitopes on the target. Those peptide binders are validated via carrying out binding assays against the full protein target as well as against the SynEps. For those binding assays, the SynEps are prepared with the naturally occurring residue in place of the click handle. 
     Ideally, the different regions of the target protein to which the different ligands bind will be relatively close together (a few nanometers or less) in the tertiary protein structure. For even a single SynEp, a screen can produce PCCs that bind to two different sites. During the SynEp screening steps, PCCs that bind to the N-terminal side of the epitope or the C-terminal side can both be identified. 
     Once the epitope targeted PCCs are identified, there are several methods for selecting a linker. 
     In a first method, if the folded structure of the protein is known, and if the PCCs bind to that folded structure, then one can use that information, plus knowledge of which PCCs bind to which epitopes, to estimate an optimal linker length. Analysis of the binding arrangement, together with the structure of the protein from, for example, the Protein Data Bank, permits an estimate of the length of an optimized linker. Such an estimate can narrow down the choice of candidate linkers to a very small number. One example might be to use such a length estimate to select one or two length-matched polyethylene glycol oligomers for testing. The best linker is the one that brings the biligand affinity closest to that a fully cooperative binder. 
     In a second method, if the folded structure of the protein is not known, or if the protein simply does not have a well-defined folded structure, then one uses as much information as is available to determine the composition of a library of candidate linker molecules. That library is then screened to identify a best linker. 
     In a third method, if the folded structure of the protein is not known or if the protein simply does not have a well-defined folded structure, then, using what knowledge about the protein does exist, simply select a linker to append the two PCCs. 
     Even if an optimized, fully cooperative binder is not identified in this way, the linked biligand will almost certainly outperform either of the two monoligands because of cooperativity effects. 
     In some forms, linkers can include polyethylene glycol (PEG), alkane, alkene, triazole, amide, or peptides. 
     F. In Vitro 
     For detection of target in solution, a binding or capture agent as described herein can be detectably labeled (with a reporter moiety) to form an imaging agent, then contacted with the solution, and thereafter formation of a complex between the imaging agent and the target can be detected. As an example, a fluorescently labeled imaging agent can be used for in vitro target detection assays, wherein the imaging agent is added to a solution to be tested for target under conditions allowing binding to occur. The complex between the fluorescently labeled imaging agent and the target can be detected and quantified by, for example, measuring the increased fluorescence polarization arising from the complex-bound peptide relative to that of the free peptide. 
     Alternatively, a sandwich-type “ELISA” assay can be used, wherein a imaging agent is immobilized on a solid support such as a plastic tube or well, then the solution suspected of containing target is contacted with the immobilized binding moiety, non-binding materials are washed away, and complexed polypeptide is detected using a suitable detection reagent for recognizing target. 
     For detection or purification of soluble target from a solution, imaging agents as disclosed 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 an imaging agent/target complex. The non-binding portion of the solution can be removed and the complex can be detected, for example, using an anti-target antibody, or an anti-binding polypeptide antibody, or the target can be released from the binding moiety at appropriate elution conditions. 
     G. In Vivo Diagnostic Imaging 
     A particularly preferred use for the disclosed imaging agents is for creating visually readable images of target or target-expressing cells in a biological fluid, such as, for example, in human serum. The target imaging agents disclosed herein can be conjugated to a label appropriate for diagnostic detection. Preferably, an imaging agent exhibiting much greater specificity for target than for other serum proteins is conjugated or linked to a label appropriate for the detection methodology to be employed. For example, the imaging 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. 
     In some forms, rather than directly labeling an imaging agent with a reporter moiety (e.g., a detectable label or radiotherapeutic construct), one or more of the disclosed peptides or constructs can be conjugated with for example, avidin, biotin, or an antibody or antibody fragment that will bind the reporter moiety. 
     1. Magnetic Resonance Imaging 
     The target imaging 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 (III), 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(III) chelates have been used for clinical and radiologic MR applications since 1988, and approximately 30% of MRI exams currently employ a gadolinium-based contrast agent. 
     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-1,4,7,10-tetraacetic acid (DOTA), 1-substituted 1,4,7,-tricarboxymethyl-1,4,7,10-teraazacyclododecane (DO3A), ethylenediaminetetraacetic acid (EDTA), and 1,4,8,11-tetra-azacyclotetradecane-1,4,8,11-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 (0 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 N,N′,N″-triacetic acid, benzo-TETA, benzo-DOTMA, where DOTMA is 1,4,7,10-tetraazacyclotetradecane-1,4,7,10-tetra(methyl tetraacetic acid), and benzo-TETMA, where TETMA is 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-(methyl tetraacetic acid); derivatives of 1,3-propylene-diaminetetraacetic acid (PDTA) and triethylenetetraaminehexaacetic acid (TTNA); derivatives of 1,5,10-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 is DTPA, and the use of DO3A is particularly preferred. Examples of representative chelators and chelating groups that can be used in the disclosed compositions and methods are described in WO 98/18496, WO 86/06605, WO 91/03200, WO 95/28179, WO 96/23526, WO 97/36619, PCT/US98/01473, PCT/US98/20182, and U.S. Pat. Nos. 4,899,755, 5,474,756, 5,846,519 and 6,143,274, all of which are hereby incorporated by reference. 
     In accordance with the present disclosure, the chelator of the MRI contrast agent is coupled to the target imaging agent. The positioning of the chelate should be selected so as not to interfere with the binding affinity or specificity of the target imaging agent. The chelate also can be attached anywhere on the imaging agent. 
     In general, the target imaging 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 target binding moiety); derivatized or underivatized polyethylene glycols (PEGs), polyoxyethylene, or polyvinylpyridine chains; substituted or unsubstituted polyamide chains; derivatized or underivatized polyamine, polyester, polyethylenimine, polyacrylate, poly(vinyl alcohol), polyglycerol, or oligosaccharide (e.g., dextran) chains; alternating block copolymers; malonic, succinic, glutaric, adipic and pimelic acids; caproic acid; simple diamines and diols; any of the other linkers disclosed herein; or any other simple polymeric linkers known in the art (see, for example, WO 98/18497 and WO 98/18496). 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 100. In addition, it can be desirable to utilize a linker that is biodegradable in vivo to provide efficient routes of excretion for the disclosed imaging reagents. Depending on their location within the linker, such biodegradable functionalities can include ester, double ester, amide, phosphoester, ether, acetal, and ketal functionalities. 
     In general, known methods can be used to couple the metal chelate and the target imaging agent using such linkers (WO 95/28967, WO 98/18496, WO 98/18497 and discussion therein). The target 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. 
     MRI contrast reagents prepared according to the disclosures herein can be used in the same manner as conventional MRI contrast reagents. 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, 177: 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 target-expressing tissue and background tissues. Finally, magnetization transfer preparations also can improve contrast with these agents (Goodrich, K. et al., 1996. Invest. Radia, 31: 323-32). 
     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, intrathecally, interstitially, or intracavitarilly. For imaging target-expressing tissues, such as tumors, 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 target expression by at least 10%. After injection with the target imaging agent containing MRI reagent, the patient is scanned in the MRI machine to determine the location of any sites of target expression. In therapeutic settings, upon identification of a site of target expression (e.g., fluid or tissue), an anti-cancer agent (e.g., target heterobiligands coupled to an anti-cancer agent) can be immediately administered, if necessary, and the patient can be subsequently scanned to visualize viral load. 
     2. Nuclear Imaging (Radionuclide Imaging) and Radiotherapy 
     The disclosed target imaging 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 target imaging agents are conjugated with both a chelator for a radionuclide useful for diagnostic imaging and a chelator useful for radiotherapy are specifically contemplated. 
     For use as a PET agent a disclosed imaging agent can be complexed with one of the various positron emitting metal ions, such as  51 Mn,  52 Fe,  60 Cu,  68 Ga,  72 As,  94m Tc, or  110 In. The disclosed binding moieties can also be labeled by halogenation using radionuclides such as  18 F,  124 I,  125 I,  131 I,  123 I,  77 Br, and  76 Br. Preferred metal radionuclides for scintigraphy or radiotherapy include  99m Tc,  51 Cr,  67 Ga,  68 Ga,  47 Sc,  51 Cr,  167 Tm,  141 Ce,  111 In,  168 Yb  175 Yb,  140 La,  90 Y,  88 Y,  153 Sm,  166 Ho,  165 Dy,  166 Dy,  62 Cu,  64 Cu,  67 Cu,  97 Ru,  103 Ru,  186 Re,  188 Re,  203 Pb,  211 Bi,  212 Bi,  213 Bi,  214 Bi,  105 Rh,  109 Pd,  117 mSn,  149 Pm,  161 Tb,  177 Lu,  225 Ac,  198 Au and  199 Au. The choice of metal will be determined based on the desired therapeutic or diagnostic application. For example, for diagnostic purposes the preferred radionuclides include  64 Cu,  67 Ga,  68 Ga,  99m Tc, and  111 In. For therapeutic purposes, the preferred radionuclides include  64 Cu,  90 Y,  105 Rh,  111 In,  117 mSn,  149 Pm,  153 Sm,  161 T,  166 Tb,  166 Dy,  166 Ho,  175 Yb,  177 Lu,  225 Ac,  186/188 Re, and  199 Au.  99m Tc is useful for diagnostic applications because of its low cost, availability, imaging properties, and high specific activity. The nuclear and radioactive properties of  99m Tc 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  99 Mo- 99m Tc generator.  18 F, 4-[ 18 F]fluorobenzaldehyde ( 18 FB), Al[ 18 F]-NOTA,  68 Ga-DOTA, and  68 Ga-NOTA are typical radionuclides for conjugation to target imaging agents for diagnostic imaging. 
     The metal radionuclides can be chelated by, for example, linear, macrocyclic, terpyridine, and N 3 S, N 2 S 2 , or N 4  chelants (see also, U.S. Pat. Nos. 5,367,080, 5,364,613, 5,021,556, 5,075,099, 5,886,142), and other chelators known in the art including, but not limited to, HYNIC, DTPA, EDTA, DOTA, DO3A, 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. Nos. 6,143,274; 6,093,382; 5,608,110; 5,665,329; 5,656,254; and 5,688,487. Certain N.sub.35 chelators are described in PCT/CA94/00395, PCT/CA94/00479, PCT/CA95/00249 and in U.S. Pat. Nos. 5,662,885; 5,976,495; and 5,780,006. The chelator also can include derivatives of the chelating ligand mercapto-acetyl-acetyl-glycyl-glycine (MAG3), which contains an N 3 S, and N 2 S 2  systems such as MAMA (monoamidemonoaminedithiols), DADS (N 2 S diaminedithiols), CODADS and the like. These ligand systems and a variety of others are described in, for example, Liu, S, and Edwards, D., 1999. Chem. Rev., 99:2235-2268, and references therein. 
     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. Nos. 5,183,653; 5,387,409; and 5,118,797, the disclosures of which are incorporated by reference herein, in their entirety. 
     The chelators can be covalently linked directly to the target imaging agent via a linker, as described previously, and then directly labeled with the radioactive metal of choice (see, WO 98/52618, U.S. Pat. Nos. 5,879,658, and 5,849,261). 
     Target imaging agents comprising  18 F, 4-[ 18 F]fluorobenzaldehyde ( 18 FB), Al[ 18 F]-NOTA,  68 Ga-DOTA, and  68 Ga-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 disclosed reagents, the technetium complex, preferably a salt of  99m Tc 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 disclosed reagent to be labeled and a sufficient amount of reducing agent to label the reagent with  99m Tc. Alternatively, the complex can be formed by reacting a disclosed peptide 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  99m Tc pertechnetate salts useful with the disclosed compositions and methods are included the alkali metal salts such as the sodium salt, or ammonium salts or lower alkyl ammonium salts. 
     Preparation of the disclosed complexes 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 NH 4 ReO 4  or KReO 4 . Re(V) is available as, for example, [ReOCl 4 ](NBu 4 ), [ReOCl 4 ](AsPh 4 ), ReOCl 3 (PPh 3 ) 2  and as ReO 2 (pyridine) 4+ , where Ph is phenyl and Bu is n-butyl. Other rhenium reagents capable of forming a rhenium complex also can be used. 
     Also disclosed are radioactively labeled PET, SPECT, or scintigraphic imaging agents that have 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 10 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. 
     Typical doses of a radionuclide-labeled target imaging agent can provide 10-20 mCi. After injection of the radionuclide-labeled target imaging 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. 
     Proper dose schedules for the disclosed radiotherapeutic compounds and compositions are known to those skilled in the art. The compounds and compositions 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 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 target-expressing tissue. In general, doses can range from a single dose of about 30-50 mCi to a cumulative dose of up to about 3 Ci. 
     The disclosed radiotherapeutic compositions can include physiologically acceptable buffers, and can require radiation stabilizers to prevent radiolytic damage to the compound or compositions prior to injection. Radiation stabilizers are known to those skilled in the art, and can include, for example, para-aminobenzoic acid, ascorbic acid, gentisic acid and the like. 
     Also disclosed are single or multi-vial kits that contain all of the components needed to prepare the disclosed complexes, other than the radionuclide. 
     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 γcyclodextrin 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. 
     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 disclosed complexes 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. 
     Also provided herein is a method to incorporate an  18 F radiolabeled prosthetic group onto a target imaging agent. In some forms, 4-[ 18 F]fluorobenzaldehyde ( 18 FB) is conjugated onto an imaging agent bearing an aminooxy moiety, resulting in oxime formation. In some forms, [ 18 F]fluorobenzaldehyde is conjugated onto an imaging agent bearing an acyl hydrazide moiety, resulting in a hydrazone adduct. 4-Fluorobenzaldehyde, can be prepared in  18 F form by displacement of a leaving group, using  18 F ion, by known methods. 
       18 F-labeled imaging agents can also be prepared from imaging agents possessing thiosemicarbazide moieties under conditions that promote formation of a thiosemicarbozone, or by use of a  18 F-labeled aldehyde bisulfite addition complex. 
     The above methods are particularly amenable to the labeling of imaging agents, e.g., the imaging 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 imaging 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-[ 18 F]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-[ 18 F]fluorobenzaldehyde typically is generated in situ by the acid-catalyzed decomposition of the addition complex of 4-[ 18 F]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-[ 18 F]fluorobenzaldehyde is consumed as it is formed in situ, resulting in the corresponding  18 F radiolabeled peptide sequence. 
     In the instances when the oxime, thiosemicarbazone or hydrazone linkages present in vivo instability, an additional reduction step can be employed to reduce the double bond connecting the peptide to the  18 F 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), 1189-1199, 1990 can also be used to form a Schiffs base involving a peptide and 4-[ 18 F]fluorobenzaldehyde and directly reducing the Schiff s base using reducing agents such as sodium cyanoborohydride. 
     The 4-[ 18 F]fluorobenzaldehyde can be prepared as described in Wilson et al., Journal of Labeled Compounds and Radiopharmaceuticals, XXVIII (10), 1189-1199, 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 18 F in water can be added to a mixture of Kryptofix and K 2 CO 3 . Anhydrous acetonitrile can be added and the solution is evaporated in a heating block under a stream of argon. Additional portions of acetonitrile can be added and evaporated to completely dry the sample. The 4-trimethylammoniumbenzaldehyde triflate can be dissolved in DMSO and added to the dried F-18. The solution can then be heated in the heating block. The solution can be cooled briefly, diluted with water and filtered through a Waters®. Oasis HLB LP extraction cartridge. The cartridge can be washed with 9:1 water:acetonitrile and water to remove unbound  18 F and unreacted 4-trimethylammoniumbenzaldehyde triflate. The 4-[ 18 F]fluorobenzaldehyde can then be eluted from the cartridge with methanol in fractions. 
     H. Therapeutic Applications 
     Provided herein in some forms are methods of using the capture agents disclosed herein to identify, detect, quantify, and/or separate the target in a biological sample. In some forms, these methods utilize an immunoassay, with the capture agent replacing an antibody or its equivalent. In some forms, the immunoassay can be a Western blot, pull-down assay, dot blot, or ELISA. 
     A biological sample for use in the methods provided herein can be selected from the group consisting of organs, tissue, bodily fluids, and cells. Where the biological sample is a bodily fluid, the fluid can 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, e.g., the adrenal glands, bladder, bones, brain, breasts, cervix, esophagus, eyes, gall bladder, genitals, heart, kidneys, large intestine, liver, lungs, lymph nodes, ovaries, pancreas, pituitary gland, prostate, salivary glands, skeletal muscles, skin, small intestine, spinal cord, spleen, stomach, thymus gland, trachea, thyroid, testes, ureters, and urethra. Tissues include, e.g., epithelial, connective, nervous, and muscle tissues. 
     Provided herein in some forms are methods of using the target imaging agents disclosed herein to diagnose and/or classify (e.g., stage) a condition associated with the target expression. In some forms, the methods comprise (a) obtaining a biological sample from a subject; (b) measuring the presence or absence of target in the sample with the target imaging agent; (c) comparing the levels of target to a predetermined control range for the target; and (d) diagnosing a condition associated with target expression based on the difference between target levels in the biological sample and the predetermined control. 
     In some forms, the capture agents disclosed herein are used as a mutant specific targeted therapeutic. In some forms, the capture agent is administered alone without delivering DNA, a radiopharmaceutical or another active agent. 
     The capture agents described herein also can be used to target genetic material to target expressing cells. 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. 
     In some forms, the disclosed capture agents are utilized in gene therapy. In some forms, genetic material, or one or more delivery vehicles containing genetic material can be conjugated to one or more capture agents of this disclosure and administered to a patient. 
     Therapeutic agents and the 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 target binding polypeptide can be coexpressed from the same synthetic gene, created using recombinant DNA techniques, as described above. The coding sequence for the target 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 target binding polypeptide. A particular advantage of this general approach is that concatamerization of multiple, tandemly arranged capture agents is possible, thereby increasing the number and concentration of target binding sites associated with each therapeutic protein. In this manner, target binding avidity is increased, which would be expected to improve the efficacy of the recombinant therapeutic fusion protein. 
     A residue of a monomer unit or moiety refers to the portion of the monomer or moiety that is the resulting product of the monomer unit or moiety in a particular reaction scheme or subsequent formulation or chemical product, regardless of whether the portion of the monomer or moiety is actually obtained from the monomer unit or moiety. Thus, an ethylene glycol residue in a polyester refers to one or more —OCH 2 CH 2 O— units in the polyester, regardless of whether ethylene glycol was used to prepare the polyester. Similarly, an amino acid residue in a peptide refers to one or more —CO—CHR—NH-moieties in the polyester, regardless of whether the residue is obtained by reacting the amino acid to obtain the peptide. 
     As used herein, the term “non-natural amino acid” refers to an organic compound that has a structure similar to a natural amino acid so that it mimics the structure and reactivity of a natural amino acid. The non-natural amino acid as defined herein generally increases or enhances the properties of a peptide (e.g., selectivity, stability) when the non-natural amino acid is either substituted for a natural amino acid or incorporated into a peptide. 
     As used herein, the term “peptide” refers to a class of compounds composed of amino acids chemically bound together. In general, the amino acids are chemically bound together via amide linkages (CONH); however, the amino acids can be bound together by other chemical bonds known in the art. For example, the amino acids can be bound by amine linkages. Peptide as used herein includes oligomers of amino acids and small and large peptides, including polypeptides. 
     The term “hit” refers to a test compound that shows desired properties in an assay. The term “test compound” refers to a chemical to be tested by one or more screening method(s) as a putative modulator. A test compound can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a carbohydrate, a lipid, or a combination thereof. Usually, various predetermined concentrations of test compounds are used for screening, such as 0.01 micromolar, 1 micromolar and 10 micromolar. Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target. 
     The terms “high,” “higher,” “increases,” “elevates,” or “elevation” refer to increases above basal levels, e.g., as compared to a control. The terms “low,” “lower,” “reduces,” or “reduction” refer to decreases below basal levels, e.g., as compared to a control. 
     The term “modulate” as used herein refers to the ability of a compound or a composition to change an activity in some measurable way as compared to an appropriate control. As a result of the presence of compounds and compositions in the assays, activities can increase or decrease as compared to controls in the absence of these compounds and compositions. Preferably, an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound or composition. Similarly, a decrease in activity is preferably at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound or composition. A compound or composition that increases a known activity is an “agonist.” One that decreases, or prevents, a known activity is an “antagonist.” 
     The term “inhibit” means to reduce or decrease in activity or expression. This can be a complete inhibition of activity or expression, or a partial inhibition. Inhibition can be compared to a control or to a standard level. Inhibition can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%. 
     The term “monitoring” as used herein refers to any method in the art by which an activity can be measured. 
     The term “providing” as used herein refers to any means of adding a compound, composition, or molecule to something known in the art. Examples of providing can include the use of pipettes, pipettemen, syringes, needles, tubing, guns, etc. This can be manual or automated. It can include transfection by any mean or any other means of providing nucleic acids to dishes, cells, tissue, cell-free systems and can be in vitro or in vivo. 
     The term “preventing” as used herein refers to administering a compound or composition prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition. 
     I. Albumin Binding Moieties 
     In some forms, the disclosed molecules, compounds and compositions (e.g., heterobiligand, target binding compositions) can include one or more albumin-binding molecules or moieties. Such albumin-binding molecules or moieties can provide for altered pharmacodynamics of a molecule of interest (e.g., the disclosed heterobiligand), such as alteration of tissue uptake, penetration, or diffusion; enhanced efficacy; and increased half-life. For example, the serum half-life of a therapeutic, prophylactic or diagnostic molecule of interest can be increased by linking the molecule of interest to a serum albumin-binding moiety and administering the molecule/serum albumin-binding moiety to a subject. The resulting conjugate will associate with circulating serum albumin and will remain in the serum longer than if the molecule of interest were administered in the absence of a serum albumin-binding moiety. 
     Thus, in particular forms, albumin-binding molecules or moieties are used to increase the half-life and overall stability of a therapeutic, prophylactic or diagnostic compound or composition that is administered to or enters the circulatory system of a subject. In such methods, an albumin-binding moiety is used to link a therapeutic, prophylactic or diagnostic compound or composition to a serum albumin found in the blood of an individual who will receive the compound or composition. The albumin-binding moiety can be covalently or non-covalently linked, coupled or associated to a selected compound or composition at a site that keeps the albumin-binding site of the moiety intact and still capable of binding to a serum albumin, without compromising the desired diagnostic, prophylactic or therapeutic activity of the compound or composition. In this way, the albumin-binding moiety serves as a linker molecule to link the therapeutic, prophylactic or diagnostic compound or composition of interest to a serum albumin circulating in the blood. This is expected to be particularly useful in increasing the circulating half-life and/or overall stability of compounds and compositions that are normally subject to an undesirably rapid rate of degradation or clearance from circulation. Increasing the half-life or overall stability of a compound or composition in the circulatory system is likely to reduce the number and/or size of doses that must be administered to an individual to obtain a desired effect. 
     Exemplary albumin-binding molecules or moieties that can be used include, without limitation, fatty acids and derivatives thereof, small molecules, peptides, and proteins. See Zorzi A., et al., MedChemComm., 10(7):1068-1081 (2019), which is hereby incorporated by reference in its entirety, and which provides a review of albumin-binding ligands and their use in extending the circulating half-life of therapeutics. 
     Albumin acts as the key lipid delivery vehicle for tissues, binding up to seven molecules of long fatty acids simultaneously. Short- to medium-length fatty acids (6 to 12 carbons) bind albumin with affinities between 0.5 and 60 μM, while the longest ones (14 to 18 carbons) have 10-fold higher affinities (below 50 nM). This ability of serum albumin to bind fatty acids with a high affinity has inspired the use of post-translational acylation as a safe and effective platform for prolonging the half-life and the mode of action of peptides and small proteins. For example, acylation of insulin with saturated fatty acids containing 10-16 carbon atoms produces insulin with affinity for albumin (Kurtzhals P., et al., Biochem. J., 312:725-731 (1995); Markussen, J., et al., Diabetologia, 39:281-288 (1996)). Approved drugs relying on derivatization with albumin-binding fatty acids to prolong half-life include, LEVEMIR® (insulin detemir), TRESIBA® (insulin degludec), VICTOZA® (liraglutide), and OZEMPIC® (semaglutide). Thus, in some forms, fatty acids (e.g., myristic acid, lauric acid, or palmitic acid) and derivatives thereof, including those used in the aforementioned approved drugs can be used as albumin-binding molecules or moieties. 
     In addition to fatty acids, serum albumin can bind numerous small organic molecules by exploiting two major structurally dissimilar binding sites, known as Sudlow sites I and II. Site I, also known as the warfarin-azapropazone binding site, usually accommodates dicarboxylic acids and/or bulky heterocycles carrying a central negative charge, whereas site II, also known as the benzodiazepine binding site, can discriminate ligands based on their size and stereoselectivity. Because of their ability to non-covalently bind serum albumin, several organic moieties that are structurally similar to exogenous drugs (e.g., warfarin, ibuprofen and diazepam) and dye molecules (e.g., Evans blue) have also been used. Non-limiting examples of suitable albumin-binding small organic molecules include, 4-methylphenyl butyric acid (4-MPBA), 4-iodophenyl butyric acid (IPBA), naphthalene acyl sulfonamide moieties; diphenylcyclohexanol phosphate ester moieties; 9-fluorenylmethoxycarbonyl (Fmoc) moieties and derivatives (e.g., Fmoc linked to a 16-sulfanylhexadecanoic acid through a maleimide group); dicoumarol derivatives with a maleimide group; Evans blue derivatives with a maleimide group; divalent diflunisal-indomethacin moiety linked through a γGlu-Lys dipeptide coupled to a unit of 8-amino-3,6-dioxaoctanoic acid (020c) (also referred to as Diflunisal-γGlu-Lys(±020c)-indomethacin); lithocholic acid coupled to a γGlu linker; 6-(4-(4-iodophenyl)butanamido)hexanoate otherwise named AlbuTag; A083/B134; A083/B321; A077/B286; and A099/B344. See Zorzi A., et al., 2019; Table 1 and  FIG. 2 . 
     Additionally, an increasing number of peptides have been used as albumin-binding molecules. In contrast to small chemical moieties, albumin-binding peptides or proteins can easily be fused to any protein or peptide of interest, either recombinantly or chemically during solid-phase synthesis. Thus, in some forms, the albumin-binding molecule/moiety can be a peptide or protein. 
     Suitable albumin-binding peptides or proteins and methods of use thereof are known in the art, including those described in Patent Application Publication Nos. U.S. 2003/0069395, U.S. 2007/0269422, U.S. 2007/0202045, U.S. 2015/0037334, WO 1991/001743, WO 2001/045746, WO 2011/095545, WO 2012/069654, and U.S. Pat. Nos. 9,775,912 and 9,920,115, which are hereby incorporated by reference in the entirety, and in particular, for their description of compounds, peptides, epitopes, targets, and methods. Peptides that specifically bind to serum albumin, and that thereby can extend the in vivo half-life of other molecules linked/coupled to them, include variants of bacterial albumin-binding domains (see e.g., WO 2005/097202 and WO 2009/016043), small peptides (e.g., Dennis, M. S., et al., J. Biol. Chem., 277(3):35035-43 (2002) and WO 2001/045746), and fragments of immunoglobulins (see e.g., WO 2008/043822, WO 2004/003019, WO 2008/043821, WO 2006/040153, WO 2006/122787, and WO 2004/041865). 
     Suitable exemplary albumin-binding peptides/proteins include, without limitation, peptides or proteins containing one or more of the following amino acid sequences: WWEQDRDWDFDVFGGGTP (referred to as 89D03; SEQ ID NO:21), WWELDRDWDFDVFGGGTP (SEQ ID NO:22), YWWERRDWDFDVFGGGTP (SEQ ID NO:23), EWWWRRDWDFDVFGGGTP (SEQ ID NO:24), LFWWERDWDFDVFGGGTP (SEQ ID NO:25), and KWWEIRDWDFDVFGGGTPAKSDE (SEQ ID NO:26), all of which are known to bind tightly (K D ≤20 nM) to human serum albumin (see WO 2011/095545). Additional examples include peptides having the core amino acid sequence DICLPRWGCLW (SEQ ID NO:27), proteins or peptides including this core sequence (e.g., RLIEDICLPRWGCLWEDD (SEQ ID NO:28), MEDICLPRWGCLWGD (SEQ ID NO:29), QRLMEDICLPRWGCLWEDDE (SEQ ID NO:30), and QGLIGDICLPRWGCLWGRSV (SEQ ID NO:31)), and an acylated heptapeptide, named F-tag, which contains a fatty acid (palmitic acid) combined with a short linear peptide, EYEKEYE (SEQ ID NO:32) (Zorzi A., et al., Nat Commun., 8:16092 (2017)). 
     Additionally, a number of naturally occurring protein domains from bacteria are known to bind albumin. Called albumin-binding domains (ABDs), these domains have a molecular weight of ˜6 kDa, fold into a three-helix bundle domain, and interact with serum albumin primarily along one face of the bundle (Makrides S C., et al., J. Pharmacol. Exp. Ther., 277:534-542 (1996); Lejon et al., 2004; Cramer J F., et al., FEBS Lett., 581:3178-3182 (2007)). One such domain, a fragment of protein G from  Streptococcus  strain GI48, which binds to human serum albumin with an affinity of 1 nM (G148-GA), has been widely used to extend the serum half-life of proteins. Fusion to this domain has been shown to extend the half-life of soluble complement receptor type 1 (Makrides et al., 1996), a bispecific antibody (Stork R., et al., Protein Eng. Des. Sel., 20:569-576 (2007)), and Affibody scaffold molecules (Orlova A., et al., J. Nucl. Med., 54; 961-968 (2013); Malm M., et al., Biotechnol. J., 9:1215-1222 (2014)). Thus in some forms, suitable albumin-binding molecules or moieties include ABDs and derivatives or variants thereof, non-limiting examples of which include, G148-GA (LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAA; SEQ ID NO:33), ALB8-GA (LKNAKEDAIAELKKAGITSDFYFNAINKAKTVEEVNALKNEILKA; SEQ ID NO:34), ABD035 (Jonsson A., et al., Protein Eng., Des. Sel., 21(8):515-527 (2008)), ABD094 (Frejd F Y. and Kim K T., Exp. Mol. Med., 49(3):e306 (2017)), ABDCon (LKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA; SEQ ID NO:35), and ABDCon12 (TIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA; SEQ ID NO:36). Other suitable ABDs and derivatives or variants thereof, including albumin-binding designed ankyrin repeat proteins (DARPins®),  S. solfataricus  Sso7d derived ABDs, single domain antibodies (dAbs) also known as AlbudAbs, and stable variable domain of the heavy-chain-only (VHH) antibodies (Nanobodies®), are described in Jacobs S A., et al., Protein Eng. Des. Sel., 28(10):385-393 (2015) and Zorzi A., et al., MedChemComm., 10(7):1068-1081 (2019). 
     Other albumin-binding proteins suitable for used in accordance with the disclosed methods and compositions include anti-albumin antibodies and fragments thereof, such as the humanized anti-human serum albumin antibody, CA645, which binds to albumin across multiple species with similar affinity (Protein Data Bank (PDB) accession code: 5FUZ; see also Adams R., et al, MAbs, 8(7):1336-1346 (2016)). 
     The one or more albumin-binding molecules or moieties may be linked, coupled, conjugated, or otherwise associated to a molecule of interest such as a therapeutic, prophylactic or diagnostic compound or composition covalently or non-covalently. In some forms, more than one albumin-binding molecules or moieties can be used (e.g., two or more of the same or different albumin-binding moieties, optionally arranged in tandem and/or separated by linkers). A variety of methods for linking molecules together are known in the art. Such linkages include hydrophobic interactions, van der Waals forces, and ionic linkages. Useful covalent linkages include, but are not limited, to peptide linkages, disulfide linkages, maleimide linkages, ester linkages, and ether linkages. For example, an amino group of the side chain of a lysine residue present in an albumin-binding moiety may be used to covalently link the albumin-binding moiety to another peptide/protein via condensation to form a peptide bond. 
     In some forms, a fusion polypeptide containing the albumin-binding moiety and peptide/protein of interest may be synthesized directly using an automated peptide synthesizer or using any of the various standard recombinant DNA methods known in the art for producing fusion proteins. For example, a nucleic acid encoding peptide-based albumin-binding moiety can be operably linked to a nucleic acid encoding a peptide or protein of interest, optionally via a linker domain. The linker domain encompasses any group of molecules that provides a spatial bridge between the albumin-binding moiety and the compound or composition of interest. The linker domain can be of variable length and makeup. In some forms, the linker domain preferably allows for the albumin-binding moiety and/or the peptide or protein of interest to bind, substantially free of steric and/or conformational restrictions to the respective target molecule. 
     Depending on the type of linkage and its method of production, a peptide/protein based albumin-binding moiety may be joined via its N- or C-terminus to the N- or C-terminus of a peptide or protein of interest. In some forms, when the albumin-binding molecules or moieties are peptides or proteins, the peptides or proteins can be linear or cyclized. Cyclization can be achieved by the formation, for example, of a disulfide bond or a lactam bond between a first and a second residue capable of forming a disulfide bond, such as cysteine. 
     Whatever means is used to link the albumin-binding moiety to another molecule of interest, the desired final product is preferably a compound or composition in which there has been no significant loss of the desired characteristics of each of the component molecules. Particularly, in the case of the albumin-binding moiety component, there is preferably no significant reduction in the ability to bind serum albumin. In some preferred forms, linkage of an albumin-binding moiety with another molecule of interest results in enhanced properties, such as enhanced detectability, increased serum half-life, enhanced solubility, or enhanced therapeutic, prophylactic, or diagnostic efficacy. 
     The disclosed compositions and methods can be further understood through the following numbered paragraphs. 
     1. A composition comprising a first component and a second component, wherein the first and second components are coupled via a linking component, wherein the linking component comprises a neprilysin (NEP) cleavage site, wherein the NEP cleavage site can be cleaved by NEP, wherein cleavage of NEP cleavage site separates the first component from the second component. 
     2. The composition of paragraph 1, wherein the NEP cleavage site comprises Gly-Phe-Lys or Met-Val-Lys. 
     3. The composition of paragraph 1 or 2, wherein the first component comprises a therapeutic agent, a detection agent, or a combination thereof. 
     4. The composition of any one of paragraphs 1-3, wherein the first component comprises a radioisotope. 
     5. The composition of paragraph 4, wherein the radioisotope is  177 Lu,  225 Ac,  99m Tc,  51 Cr,  67 Ga,  68 Ga,  47 Sc,  51 Cr,  167 Tm,  141 Ce,  111 In,  168 Yb,  175 Yb,  140 La,  90 Y,  88 Y,  153 Sm,  166 Ho,  165 Dy,  166 Dy,  62 Cu,  64 Cu,  67 Cu,  97 Ru,  103 Ru,  186 Re,  188 Re,  203 Pb,  211 Bi,  212 Bi,  213 Bi  214 Bi,  105 Rh  109 Pd,  117 mSn,  149 Pm,  161 Tb,  198 Au,  199 Au,  18 F,  89 Zr,  124 I,  86 Y,  94m Tc,  110m In,  11 C, or  76 Br. 
     6. The composition of paragraph 4 or 5, wherein the radioisotope is  177 Lu or  68 Ga. 
     7. The composition of any one of paragraphs 1-6, wherein the first component is toxic to a cell, to an organ, or to both. 
     8. The composition of paragraph 7, wherein the separation of the first component from the second component reduces toxic effect of the first component to the cell, the organ, a subject containing the cell, the organ, or both, or a combination thereof, compared to the toxic effect of the uncleaved composition. 
     9. The composition of paragraph 8, wherein the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to a tumor compared to the delivery percentage of the uncleaved composition. 
     10. The composition of paragraph 8, wherein the reduction in the toxic effect is at least partially due to an increased delivery rate of the separated first component to a tumor compared to the delivery rate of the uncleaved composition. 
     11. The composition of paragraph 8, wherein the reduction in the toxic effect is at least partially due to an increased rate of clearance of the separated first component from the subject compared to the rate of clearance of the uncleaved composition. 
     12. The composition of paragraph 8, wherein the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to a second cell, to a second organ, or to both compared to the delivery percentage of the uncleaved composition. 
     13. The composition of paragraph 8, wherein the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to the cell, to the organ, or to both compared to the delivery percentage of the uncleaved composition. 
     14. The composition of any one of paragraphs 1-13, wherein the second component comprises a ligand. 
     15. The composition of paragraph 14, wherein the ligand can bind to a target. 
     16. The composition of any one of paragraphs 1-15, wherein the second component comprises a biligand. 
     17. The composition of any one of paragraphs 1-16, wherein the second component comprises a heterobiligand. 
     18. The composition of paragraph 16 or 17, wherein the biligand and the heterobiligand each comprise two ligands, wherein both of the two ligands of the biligand and heterobiligand can bind either two separate parts of the same target or two different targets. 
     19. The composition of paragraph 18, wherein each target is, independently, a detection target, a therapeutic target, both a detection target and a therapeutic target, or a combination thereof. 
     20. The composition of any one of paragraphs 1-19, wherein one or more of the second component, the linking component, and the first component further comprise an albumin binding moiety. 
     21. The composition of any one of paragraphs 1-20, wherein the albumin binding moiety is 4-methylphenyl butyric acid (4-MPBA) or 4-iodophenyl butyric acid (IPBA). 
     22. The composition of any one of paragraphs 1-21, wherein one or more of the second component, the linking component, and the first component further comprise a reporter moiety. 
     23. The composition of any one of paragraphs 1-22, wherein the composition comprises the structure (I): 
     
       
         
         
             
             
         
       
     
     or a salt, tautomer, prodrug or stereoisomer thereof, wherein: 
     L 1  and L 2  are each individually a bond or an optionally substituted linker moiety, wherein each linker moiety optionally comprises a linkage to the NEP cleavage site and the first component, a linkage to the first component, a linkage to a ligand, a linkage to a reporter moiety, a linkage to an albumin binding moiety, a linkage to a peptide ligand, or combinations thereof; 
     G is a triazole, a carbon-carbon double bond or an amide; 
     M is methionine; 
     R is H or an optionally substituted linker moiety, wherein each linker moiety optionally comprises a linkage to the NEP cleavage site and the first component, a linkage to the first component, a linkage to a ligand, a linkage to a reporter moiety, a linkage to an albumin binding moiety, a linkage to a peptide ligand, or combinations thereof; 
     R 1  is H or C 1 -C 6  alkyl; 
     Y 1  and Y 2  are each individually 0 or 1; and 
     SEQ is an amino acid sequence comprising from 2 to 20 amino acids selected from natural and non-natural amino acids. 
     24. The composition of paragraph 23, wherein L 1  is —C(HR 2 )— wherein R 2  is H, —R 5 -L 3 -A 1 , —R 5 —C(═O)-L 3 -A 1 , —R 5 -A 2 -L 3 -A 1 , —R 5 —C(═O)-A 2 -L 3 -A 1 , —R 5 -L 3 (-A 2 )-A 1 , or —R 5 —C(═O)-L 3 (-A 2 )-A 1 , where —R 5  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 3  is a linker moiety, and where A 1  and A 2  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     25. The composition of paragraph 23 or 24, wherein L 2  is —C(HR 4 )—, wherein R 4  is H, —R 6 -L 5 -A 3 , —R 6 —C(═O)-L 5 -A 3 , —R 6 -A 4 -L 5 -A 3 , —R 6 —C(═O)-A 4 -L 5 -A 3 , —R 6 -L 5 (-A 4 )-A 3 , or —R 6 —C(═O)-L 5 (-A 4 )-A 3 , where —R 6  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 5  is a linker moiety, and where A 3  and A 4  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     26. The composition of any one of paragraphs 23-25, wherein R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     27. The composition of any one of paragraphs 23-26, wherein L 1  is —C(HR 2 )—, wherein R 2  is H, —R 5 -L 3 -A 1 , —R 5 —C(═O)-L 3 -A 1 , —R 5 -A 2 -L 3 -A 1 , —R 5 —C(═O)-A 2 -L 3 -A 1 , —R 5 -L 3 (-A 2 )-A 1 , or —R 5 —C(═O)-L 3 (-A 2 )-A 1 , where —R 5  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 3  is a linker moiety, and where A 1  and A 2  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     28. The composition of any one of paragraphs 23-27, wherein L 2  is —C(HR 4 )—, wherein R 4  is H, —R 6 -L 5 -A 3 , —R 6 —C(═O)-L 5 -A 3 , —R 6 -A 4 -L 5 -A 3 , —R 6 —C(═O)-A 4 -L 5 -A 3 , —R 6 -L 5 (-A 4 )-A 3 , or —R 6 —C(═O)-L 5 (-A 4 )-A 3 , where —R 6  is absent, —C(═O)—NH—, or —CH 2 —C(═O)—NH—, where L 5  is a linker moiety, and where A 3  and A 4  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     29. The composition of any one of paragraphs 23-28, wherein R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     30. The composition of any one of paragraphs 23-29, wherein one or more of A 1 , A 2 , A 3 , A 4 , A 5 , and A 6  individually and independently comprise a combination of one or more of the following: the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, and a peptide ligand. 
     31. The composition of any one of paragraphs 23-30, wherein the composition has one of the following structures (Ia) or (Ib): 
     
       
         
         
             
             
         
       
     
     wherein: 
     R 3  is H, -L 3 -A 1 , —C(═O)-L 3 -A 1 , -A 2 -L 3 -A 1 , —C(═O)-A 2 -L 3 -A 1 , -L 3 (-A 2 )-A 1 , or —C(═O)-L 3 (-A 2 )-A 1 , where L 3  is a linker moiety and A 1  and A 2  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof; and x and y are each independently an integer from 1 to 8. 
     32. The composition of paragraph 31, wherein R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently comprise the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     33. The composition of paragraph 31 or 32, wherein R 3  is H, -L 3 -A 1 , —C(═O)-L 3 -A 1 , -A 2 -L 3 -A 1 , —C(═O)-A 2 -L 3 -A 1 , -L 3 (-A 2 )-A 1 , or —C(═O)-L 3 (-A 2 )-A 1 , where L 3  is a linker moiety and A 1  and A 2  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     34. The composition of any one of paragraphs 31-33, wherein R is H, -L 7 -A 5 , —C(═O)-L 7 -A 5 , -A 6 -L 7 -A 5 , —C(═O)-A 6 -L 7 -A 5 , -L 7 (-A 6 )-A 5 , or —C(═O)-L 7 (-A 6 )-A 5 , where L 7  is a linker moiety and A 5  and A 6  independently are the NEP cleavage site and the first component, the first component, a linkage to a ligand, a reporter moiety, an albumin binding moiety, a peptide ligand, a linker moiety, or combinations thereof. 
     35. The composition of any one of paragraphs 31-34, wherein the composition has one of the following structures: 
     
       
         
         
             
             
         
       
     
     36. The composition of any one of paragraphs 23-35, wherein the linker moieties independently comprise ethylene glycol, triazole, lysine, ethylene diamine, or combinations thereof. 
     37. The composition of any one of paragraphs 23-36, wherein SEQ comprises from 2 to 9 amino acids. 
     38. The composition of any one of paragraphs 23-37, wherein SEQ comprises from 5 to 7 amino acids. 
     39. The composition of any one of paragraphs 23-38, wherein SEQ comprise natural amino acids. 
     40. The composition of any one of paragraphs 23-38, wherein SEQ comprises non-natural amino acids. 
     41. The composition of any one of paragraphs 23-38, wherein SEQ comprises natural and non-natural amino acids. 
     42. A method of treating a subject having a tumor, the method comprising administering to the subject a composition according to any one of paragraphs 1-41, wherein the first component is toxic to a cell, to an organ, or to both, wherein the separation of the first component from the second component reduces toxic effect of the first component to the cell, the organ, a subject containing the cell, the organ, or both, or a combination thereof, compared to the toxic effect of the uncleaved composition. 
     43. The composition of paragraph 42, wherein the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to a tumor compared to the delivery percentage of the uncleaved composition. 
     44. The composition of paragraph 42, wherein the reduction in the toxic effect is at least partially due to an increased delivery rate of the separated first component to a tumor compared to the delivery rate of the uncleaved composition. 
     45. The composition of paragraph 42, wherein the reduction in the toxic effect is at least partially due to an increased rate of clearance of the separated first component from the subject compared to the rate of clearance of the uncleaved composition. 
     46. The composition of paragraph 42, wherein the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to a second cell, to a second organ, or to both compared to the delivery percentage of the uncleaved composition. 
     47. The composition of paragraph 42, wherein the reduction in the toxic effect is at least partially due to an increased delivery percentage of the separated first component to the cell, to the organ, or to both compared to the delivery percentage of the uncleaved composition. 
     48. The organ of any one of paragraphs 42-47, wherein the organ is the kidney, lung or heart. 
     EXAMPLES 
     Example 1: Analysis of Heterobiligands Having Different Combinations of Components 
     Materials and Methods 
     Library Synthesis 
     Screens were performed using a triazole-cyclized OBOC library of the form H 2 N-Pra-(Pra-X 1 X 2 X 3 X 4 X 5 -Az4)-Met-TG, where TG=TentaGel® S NH 2  resin (S 30 902, Rapp Polymere), X 1 =one of sixteen D-amino acids (D-Ala, D-Arg, D-Asn, D-Asp, D-Glu, Gly, D-His, D-Leu, D-Lys, D-Phe, D-Pro, D-Ser, D-Thr, D-Trp, D-Tyr, D-Val), Pra=L-propargylglycine, and ( )=triazole cyclization via flanking Pra and Az4 (=L-azidolysine) residues. An encoded cyclic peptide library (ECPL) was created where 20% of the peptide on each bead is a linear tag for MALDI-TOF/TOF sequencing, while 80% remains the cyclic peptide for ligand discovery. The OBOC library was synthesized using Fmoc-based solid-phase synthesis on a Titan 357 automated peptide synthesizer (AAPPTEC). When synthesizing the ECPL, methionine was first coupled to the TentaGel beads as a cyanogen bromide (CNBr)-selective cleavage handle. Then, Az4 and the 5-residue variable region were coupled via the split-and-mix technique, respectively. A mixture of 80:20 Pra/Gly (mol/mol) was subsequently coupled onto the resins. Then, Cu(I) was added to cyclize the Pra-coupled peptide with the Az4, while the Gly-terminated peptides remained linear for MALDI-TOF/TOF sequencing. Finally, Pra was coupled onto the N-terminus of the library as a click handle for screening. Global side chain deprotection was achieved by treating the library for 2 h with 92.5% TFA, 2.5% H 2 O, 2.5% TIS (triisopropylsilane), and 2.5% DODT (3,6-dioxa-1,8-octanedithiol). The library resin was then neutralized with 1-methyl-2-pyrrolidinone (NMP), and washed thoroughly with NMP (5×), water (5×), methanol (MeOH, 5×), and methylene chloride (DCM, 5×), and then dried under vacuum before equilibrating in the screening buffer. 
     Screening a Macrocycle Library Against FOLR1 Epitopes—Library C4 
     Macrocyclic peptide ligands were identified by screening the library against a cocktail of three FOLR1 epitopes using the following steps: 1) pre-clear and anti-screens to eliminate non-specific binders, 2) a product screen to identify hits resulting from FOLR1 epitope-templated in situ click chemistry, and 3) target screens against His-tagged FOLR1 human recombinant protein to identify peptides that bind to the protein as well as the epitope. 
     Epitopes 1, 2, and 3 were selected due to their close proximity to the FOLR1 active site. The short distance between these epitopes and the active site enabled the attachment of folate ligand to macrocycle hits to yield heterobiligands. 
     Pre-clear. Swelled library beads (500 mg) were blocked overnight with Blocking Buffer (25 mM Tris-HCl, 150 mM NaCl, 1% (w/v) BSA, and 0.05% (v/v) Tween-20, pH 7.6) at 4° C., then washed with Blocking Buffer three times. In 5 mL Blocking Buffer, 1:1000 Atto565-labeled Streptavidin-Alkaline Phosphatase and 1:10,000 Atto565-labeled Anti-6× His tag antibody [HIS-1] (Alkaline Phosphatase-conjugated) were added to the beads and incubated with gentle shaking at room temperature for 1 h. The beads were subsequently washed with Blocking Buffer (3×1 min) and TBST (25 mM Tris-HCl, 150 mM NaCl, pH 7.6+0.05% Tween-20) (6×3 min). Automated sorting was performed on a Union Biometrica BioSorter based on the red signal and object size. The brightest, most non-selective beads were eliminated as background binders (approx. 25% of total). The remaining clear beads were collected and stripped with 0.1 M glycine pH 2.8 buffer for 15 min, washed six times with water, and incubated in TBST overnight. 
     Anti-screen. Beads remaining from the pre-clear were incubated in Blocking Buffer for 2 h, then subjected to anti-screening against 50 nM His-tagged CD8α human recombinant protein (10980-H08H, SinoBiological) in Blocking Buffer for 1 h at room temperature. The beads were washed five times with Blocking Buffer and then incubated with 1:10,000 Anti-6× His tag antibody [HIS-1] (Alkaline Phosphatase-conjugated) (ab49746, Abcam) in Blocking Buffer for 1 h at room temperature. The beads were subsequently washed with Blocking Buffer (3×1 min), TBST (3×3 min), TBS (3×3 min), then Alkaline Phosphatase buffer (100 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl 2 , pH 9) buffer (3×1 min). Binding was visualized by incubating the beads in the presence of 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) substrate (S3771, Promega). Purple beads indicated background binders and were removed by pipet and discarded. The remaining clear beads were collected and stripped with 0.1 M glycine pH 2.8 buffer for 15 min, washed ten times with water, and incubated in NMP overnight to decolorize. 
     Beads were washed with water ten times and TBS three times. Anti-screening was then repeated against 50 nM His-tagged PSMA human recombinant protein (15877-H07H, SinoBiological) using the same protocol as above. 
     Target screen with 50 nM His-tagged FOLR1 protein. Beads remaining from the anti-screen were washed with water ten times and TBS three times. Beads were incubated in Blocking Buffer for 2 h, then subjected to screening against 50 nM His-tagged FOLR1 human recombinant protein (11241-H08H, SinoBiological) in Blocking Buffer for 1 h at room temperature. The beads were washed three times with Blocking Buffer and then incubated with 1:10,000 Anti-6× His tag antibody [HIS-1] (Alkaline Phosphatase-conjugated) (ab49746, Abcam) in Blocking Buffer for 1 h at room temperature. The beads were subsequently washed with Blocking Buffer (5×3 min), TBST (3×3 min), TBS (3×3 min), then Alkaline Phosphatase buffer (pH 9) buffer (3×1 min). Binding was visualized by incubating the beads in the presence of BCIP/NBT substrate (S3771, Promega). Hundreds of purple hit beads bound to FOLR1 protein were selected by pipet. Hits were treated with 0.1 M glycine pH 2.8 buffer for 15 min to remove bound proteins, washed ten times with water, and incubated in NMP overnight to decolorize. 
     Product screen with FOLR1 epitopes. Hit beads selected in the target screen were washed with water ten times and PBST (0.05% Tween-20 in PBS) three times. They were re-swelled in PBST overnight. Beads were then incubated with a cocktail of three epitopes—50 μM FOLR1 epitope 1 (Biotin-PEG 3 -HHKEKP[Az4]PEDKLHE; (SEQ ID NO:3), 50 μM FOLR1 epitope 2 (Biotin-PEG 3 -GPWIQQVDQ[Az4]WRKERVLN; (SEQ ID NO:4), and 50 μM FOLR1 epitope 3 (Biotin-PEG 3 -R[S]IQMWFDPA[Az4]GNPNEEVAR; (SEQ ID NO:5)—in PBST at 25° C. overnight to allow for an in situ click reaction to occur. The beads were washed with 3% DMSO in PBST (5×3 min) and Blocking Buffer (3×1 min) and re-blocked with Blocking Buffer for 2 h. A 1:1000 dilution of Streptavidin-Alkaline Phosphatase (V5591, Promega) in 5 mL Blocking Buffer was added for 1 h to detect the presence of FOLR1 epitope clicked to beads. The beads were subsequently washed with Blocking Buffer (5×3 min), TBST (3×3 min), TBS (3×3 min), then Alkaline Phosphatase (pH 9) buffer (3×1 min). After this, the beads were developed with BCIP/NBT as outlined in the target screen. Purple epitope-conjugated hit beads were selected by pipet. These 63 hits were treated with 0.1 M glycine pH 2.8 buffer for 15 min to remove attached streptavidin, washed ten times with water, and incubated in NMP overnight to decolorize. 
     Target screen with 10 nM His-tagged FOLR1 protein. The 63 hits obtained from the product screen were washed with water ten times and TBS three times. They were then transferred to Corning® 8162 Costar® Spin-X® centrifuge tube filters (cellulose acetate membrane) and incubated with Blocking Buffer at 4° C. overnight. Beads were rinsed three times with Blocking Buffer and then incubated with 10 nM His-tagged FOLR1 human recombinant protein (11241-H08H, SinoBiological) in Blocking Buffer for 1 h at room temperature. The beads were washed three times with Blocking Buffer and then incubated with 1:10,000 Anti-6× His tag antibody [HIS-1] (Alkaline Phosphatase-conjugated) (ab49746, Abcam) in Blocking Buffer for 1 h at room temperature. The beads were subsequently washed with PBST (3×3 min) and PBS (3×3 min) and rinsed once with Alkaline Phosphatase (pH 9) buffer (centrifuging at 7000 rpm for each wash). After this, the beads were developed with BCIP/NBT. Purple hit beads bound to FOLR1 protein were selected by pipet and saved. Of the 63 product hits isolated for the FOLR1 epitopes, 2 beads were purple indicating strong binding to both FOLR1 epitope and protein. The 2 target hits were treated with 0.1 M glycine pH 2.8 buffer for 15 min to remove bound proteins, washed ten times with water, and incubated in NMP overnight to decolorize. The hits were finally washed with water ten times to prepare for sequencing analysis. 
     Heterobiligands Via In Situ Click Screening—Library D1 
     Folate-PCC heterobiligands were templated by screening the library and biotinylated azido-folate anchor against FOLR1 protein using the following steps: 1) pre-clear and anti-screen to eliminate non-specific binders, and 2) in situ click screen with biotinylated azido-folate anchor and FOLR1 protein to identify heterobiligand hits resulting from protein-templated in situ click chemistry. 
     Pre-clear. Swelled library beads (500 mg) were blocked overnight with 2% DMSO in PBST. They were then incubated with 1 μM biotinylated azido-folate anchor (Folate-PEG 3 -Az4-PEG 3 -Biotin) in 8 mL of 2% DMSO in PBST at 25° C. overnight. The library/anchor complex was washed with 2% DMSO in PBST (2×1 min), PBST (3×1 min), and 1% BSA in PBST (3×1 min), then blocked overnight with 1% BSA in PBST at 25° C. Binding was detected by incubating the beads in 1% BSA in PBST with 1:1000 Atto565-labeled Streptavidin-Alkaline Phosphatase and 1:10,000 Atto565-labeled Anti-6× His tag antibody [HIS-1] (Alkaline Phosphatase-conjugated) with gentle shaking for 1 h. The beads were subsequently washed with 1% BSA in PBST (3×1 min) and PBST (6×3 min). Automated sorting was then performed on a Union Biometrica BioSorter based on the red signal and object size. The brightest, most non-selective beads were eliminated as background binders. The remaining clear beads were collected and stripped with 0.1 M glycine pH 2.8 buffer (3×5 min), washed five times with water, and incubated in NMP for 1 h to decolorize. 
     Anti-screen. Beads remaining from the pre-clear were washed with water ten times and 1% BSA in PBST (3×3 min). Beads were incubated with 1% BSA in PBST at 4° C. overnight, then subjected to anti-screening against 50 nM His-tagged PSMA human recombinant protein (15877-H07H, SinoBiological) in 1% BSA in PBST for 1 h at room temperature. The beads were washed with 1% BSA in PBST (5×3 min) and then incubated with 1:10,000 Anti-6× His tag antibody [HIS-1] (Alkaline Phosphatase-conjugated) (ab49746, Abcam) and 1:1000 Streptavidin-Alkaline Phosphatase (V5591, Promega) in 1% BSA in PBST for 1 h at room temperature. The beads were subsequently washed with 1% BSA in PBST (3×5 min), PBST (3×3 min), PBS (3×3 min), then Alkaline Phosphatase buffer (pH 9) buffer (3×1 min). Binding was visualized by incubating the beads in the presence of BCIP/NBT substrate (S3771, Promega). Purple beads indicated background binders and were removed by pipet and discarded. The remaining clear beads were collected and stripped with 0.1 M glycine pH 2.8 buffer (3×5 min), washed ten times with water, incubated in NMP for 1 h to decolorize, and washed ten times with water. 
     In situ click screen with biotinylated azido-folate anchor and FOLR1 protein. Library beads were treated with biotinylated azido-folate anchor and FOLR1 protein under conditions promoting protein-templated in situ click chemistry between ligands. Here, the clear beads from the anti-screen were first blocked with 2% DMSO in PBST. Biotinylated azido-folate anchor (100 nM) and 1 nM FOLR1 human recombinant protein (11241-H08H, SinoBiological) were mixed in 10 mL of 2% DMSO in PBST overnight at 4° C. in a conical tube. The PBST was then drained from the beads and the 10 mL of anchor/FOLR1 solution was added to the beads and incubated for 4 h at 25° C. under rotation. The library/anchor/protein complex was washed with PBST (5×1 min) and 1% BSA in PBST (3×1 min), then blocked with 1% BSA in PBST for 30 min at 25° C. A 1:1000 dilution of Streptavidin-Alkaline Phosphatase (V5591, Promega) in 1% BSA in PBST (10 mL) was added for 1 h to detect the presence of biotinylated azido-folate anchor clicked to beads. The beads were subsequently washed with 1% BSA in PBST (3×5 min), PBST (3×3 min), PBS (3×3 min), then Alkaline Phosphatase (pH 9) buffer (3×). After this, the beads were developed with BCIP/NBT as outlined above. Purple heterobiligand-linked hit beads were selected by pipet. These 9 hits were treated with 0.1 M glycine pH 2.8 buffer (3×5 min) to remove attached streptavidin, washed ten times with water, incubated in NMP for 1 h to decolorize, and washed ten times with water to prepare for sequencing analysis. 
     Heterobiligands Via In Situ Click Screening—Library D2 
     Screens for additional heterobiligands were completed following the above protocol, except that:
         Biotinylated azido-folate anchor (500 nM) and 5 nM FOLR1 human recombinant protein (11241-H08H, SinoBiological) were used.   The library/anchor/protein complex was washed with PBST (5×1 min), 0.1 M glycine pH 2.8 buffer (2×5 min), PBST (5×1 min), and 1% BSA in PBST (3×1 min), then blocked with 1% BSA in PBST before detection with Streptavidin-Alkaline Phosphatase.       

     Purple heterobiligand-linked hit beads were selected by pipet. These 4 hits were treated with 0.1 M glycine pH 2.8 buffer (3×5 min) to remove attached streptavidin, washed ten times with water, incubated in NMP for 1 h to decolorize, and washed ten times with water to prepare for sequencing analysis. 
     Sequencing Cyclic Peptide Hits by MALDI-TOF/TOF 
     Peptides on hit beads were selectively cleaved from the resin using CNBr and sequenced by MALDI-MS/MS. 
     Cleavage of hit peptides from single beads with CNBr. Each hit bead was transferred in pure water (10 μL) to a single well of a 96-well conical-bottom polypropylene microplate. After addition of CNBr (10 μL, 0.50 M in 0.2 N HCl solution) to each well, the plate was purged with argon and then placed under microwave for 1 min. Acidic aq. CNBr results in methionine-specific cleavage at the C-terminus, resulting in cleavage of the cyclic and linear peptides from the beads. The resulting solution was concentrated under centrifugal vacuum for 2 hours at 45° C. 
     Sequencing of peptides cleaved from single beads by MALDI-MS and MS/MS. To each sample was added α-cyano-4-hydroxycinnamic acid (CHCA) (0.5 μL, 5 mg/mL matrix solution in acetonitrile/water (70:30) containing 0.1% TFA (v/v)). The mixture was taken up to be spotted onto a 384-well MALDI plate, which was allowed to stand for 15 min to dry naturally. Samples were then analyzed by matrix-assisted laser-desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) using a Bruker ultrafleXtreme™ TOF/TOF instrument (Bruker Daltonics; Bremen, Germany) operated in reflectron mode. MS/MS spectra were acquired for each linear peptide in LIFT™ mode. BioTools™ was used to assign the sequence based on analysis of the MS/MS spectra. 
     Peptide Synthesis 
     The peptides were synthesized using standard SPPS Fmoc chemistry. CTC resin was loaded with Fmoc-Lys(Dde)-OH. Each subsequent amino acid coupling was achieved using the amino acid (3.0 equiv), HBTU coupling reagent (2.85 equiv.), DIPEA (6.0 equiv.) in DMF. DOTA was installed by deprotecting the Dde protecting group (2% hydrazine in DMF) followed by coupling DOTA(3×tBu). The peptide was cleaved from the resin and precipitated using cold isopropyl ether. The crude peptides were purified by preparative-HPLC. 
     Fluorine-18 Radiochemistry 
     Heterobiligands were synthesized as aminooxy conjugates using Fmoc solid-phase peptide synthesis. The aminooxy linker was appended on the side chain of the C-terminal lysine. After acidic resin cleavage and deprotection of the side chains, heterobiligands were purified by C18 HPLC. Purity and mass were confirmed before entering into the labeling reaction. 
     Fluorine-18 labeling of heterobiligands. 4-[ 18 F]fluorobenzaldehyde ([ 18 F]FBA) in methanol (MeOH) was obtained from the UCLA Crump Institute for Molecular Imaging. The  18 F-fluorobenzaldehyde oxime was prepared by reacting the aminooxy-conjugated heterobiligand (5 mM in 50% MeOH and 50% potassium dihydrogen phosphate solution with phosphoric acid pH 3) with [ 18 F]FBA for 7 min at room temperature (reaction volume=70 μL). The [ 18 F]FBA-labeled heterobiligand was purified from the reaction mixture by C 18  HPLC and then co-injected with reference standard to confirm its identity. The solvent was evaporated from the purified fraction, and the dried product was dissolved in phosphate-buffered saline (PBS) prior to mouse injection. 
     Gallium-68 and Lutetium-177 Radiochemistry 
     Heterobiligands were synthesized as DOTA conjugates using Fmoc solid-phase peptide synthesis. The DOTA chelator was appended on the side chain of the C-terminal lysine. After acidic resin cleavage and deprotection of the side chains, heterobiligands were purified by C 18  HPLC. Purity and mass were confirmed before entering into the labeling reaction. 
     Gallium-68 labeling of heterobiligands.  68 GaCl 3  was obtained from the UCLA Biomedical Cyclotron Facility. The DOTA-conjugated heterobiligand and  68 GaCl 3  (1:1) were reacted for 5 min at 90° C. in buffer comprised of 1.5 mL NaCl and 0.5 mL of 0.1 M NaOAc pH 4. The efficiency was &gt;95% according to HPLC, and the product was diluted with PBS prior to mouse injection. 
     Lutetium-177 labeling of heterobiligands.  177 LuCl 3  in 0.05 M HCl was obtained from the Missouri University Research Reactor (MURR). A specific activity of 0.2 mCi of  177 Lu was added per nmol of DOTA conjugated heterobiligand. The solution was warmed to 95° C. for 15 minutes in 0.4 M sodium acetate buffer (pH 4.5). Conversion was 100% according to both radioTLC and HPLC, and purity was confirmed to be 100% on HPLC. The undiluted product in 0.4 M sodium acetate buffer (pH 4.5) was used directly or formulated in buffered saline. 
     Mice—Tumor Imaging and Therapeutic Studies 
     Female, 7-week-old NSG mice were housed under pathogen-free conditions. Water and food were provided ad libitum. To create mice with FOLR1+ ovarian xenograft tumors, mice were injected subcutaneously with OVCAR3 cells (1×10 7  cells per mouse) resuspended in 50% Matrigel in PBS into the shoulder region. Tumor growth was monitored by caliper measurements. OVCAR3 is a human ovarian adenocarcinoma known to over-express FOLR1. 
     In Vivo  8 F PET/CT Imaging 
       18 F positron emission tomography (PET) imaging was used to study in vivo biodistribution of heterobiligands in OVCAR3 tumor bearing mice. 40-60 μCi of [ 18 F]FBA-labeled heterobiligand was administered intravenously (i.v.) via the tail vein of each mouse. Dynamic PET imaging was performed from 0-1 h, followed by acquisition of static PET images at 2 h and 4 h post-injection. CT scans were acquired for anatomical reference. Imaging was performed on a GENISYS 8  microPET/CT small animal scanner. The PET signal was quantitated by three-dimensional region of interest (ROI) analysis and represented as percent injected dose (% ID) vs. time (min) for major organs including tumor, liver, heart, lung, bladder, and kidney. 
     In Vivo  68 Ga PET/CT Imaging 
       68 Ga PET imaging was used to study in vivo biodistribution of heterobiligands in non-tumor bearing (healthy) mice and OVCAR3 tumor bearing mice. 100-200 μCi of [ 68 Ga]DOTA-labeled heterobiligand was administered intravenously (i.v.) via the tail vein of each mouse. Dynamic PET imaging was performed from 0-1 h, followed by acquisition of static PET images at 2 h and 4 h post-injection. CT scans were acquired for anatomical reference. Imaging was performed on an Inveon microPET/CT small animal scanner. The PET signal was quantitated by three-dimensional ROI analysis and represented as percent injected dose (% ID) vs. time (min) for major organs including tumor, liver, heart, lung, bladder, and kidney. 
       77 Lu FOLR1 Therapy 
     OVCAR3 tumor bearing mice were used to study FOLR1-directed radioligand therapy (RLT) for treatment of ovarian cancer. Animals were administered a single dose of heterobiligand (vehicle control) or 111, 37, 18.5, 9.25, or 3.7 MBq [ 177 Lu]DOTA-labeled heterobiligand. Mice received the dose intravenously (i.v.) via the tail vein. To monitor the effect of the RLT, tumor size measurements were taken twice weekly and body weights were measured. 
     At endpoint, mice were sacrificed and plasma and tissues (tumor, kidney, liver, lung, muscle) were collected. Plasma samples were assayed for urea and creatinine biomarkers indicative of nephrotoxicity. Tissue samples were fixed in 10% buffered formalin, transferred to 70% ethanol, and embedded in paraffin for immunohistochemistry. 
     Plasma Stability 
     Plasma was thawed in a 37° C. water bath. Residual clots were removed via centrifugation at 4000 rpm for 5 minutes. The pH of the plasma was adjusted to 7.4 if required. Test articles were diluted to 100 μM by diluting a 1 mM working solution (in DMSO) with 45% methanol in water. To 98 μL of plasma was added 2 μL of the 100 μM solution, resulting in 2 μM final concentration of the test article. Samples were incubated at 37° C. in a water bath. At the indicated time points (0, 10, 30, 60, 120 minutes) 100 μL of 4% H 3 PO 4  was added followed by 800 μL of stop solution (200 ng/mL tolbutamide and 200 ng/mL labetalol in 100% acetonitrile). The samples were centrifuged and the plasma protein-free supernatant (100 μL) was subjected to LC-MS/MS analysis. 
     Plasma Protein Binding 
     HTD 96 a/b regenerated cellulose membrane strips with mass cutoff of 12-14 kDa were soaked in ultra-pure water at room temperature for 1 hour. Each swelled membrane was then soaked in 20:80 ethanol:water for 20 minutes. 
     Just prior to the experiment, the membranes were re-soaked in ultra-pure water. Plasma was prepared by thawing under cold water. The plasma was centrifuged to remove any clots and pH was adjusted to 7.0-8.0. Test articles and control compounds were diluted to 2 μM. The dialysis device was loaded by transferring 150 μL of the test article (in triplicate) to the donor side. The dialysis device was placed in a humidified incubator at 37° C. with 5% CO 2  in a shaking platform that rotated slowly for 4 hours. After incubation, 50 μL of the supernatant was taken from both sides and analyzed by LC-MS/MS. 
     Results 
     Folate receptor 1 (FOLR1) is a cell membrane protein with significant overexpression in carcinomas (most notably ovarian and breast cancers). FOLR1 is responsible for trafficking folic acid into cells via receptor-mediated endocytosis. The protein is anchored to the cell via a glycosylphosphatidylinositol (GPI) attached to the C terminus of FOLR1. FOLR1 is overexpressed in approximately 80% of ovarian cancers. Other organs have very low FOLR1 expression with the exception of the kidney to facilitate folate resorption. Prior FOLR1 therapeutic strategies have relied on a modified folate conjugated to a standard-of-care chemotherapeutic. Efficacy was no better that standard-of-care chemotherapeutic arm. The example heterobiligands—folate linked to a receptor-specific ligand—can use a theranostic platform to image and treat cancer using radioisotopes. Some example heterobiligands display cooperative binding that yields very high EC50s compared to native folate. The heterobiligands can be modified based on medicinal chemistry principles to diminish off target binding and alter pharmacokinetics for specific applications, such as imaging or therapy.  177 Lu provides predictable tumor killing while limiting bystander injury. 
     The extracellular domain of FOLR1 is 234 amino acids (M1-S234) and is glycosylated at N69, N161, and N201. S234 is coupled to glycosylphosphatidylinositol (GPI). The FOLR1 monomer coordinates (PDB: 4LRH) were minimized and equilibrated using periodic boundary conditions employing Particle Mesh Ewald electrostatics. The monomer was solvated in a water box with at least 10 water molecules separating the protein and unit cell wall. The system was warmed to 310° K, minimized for 1000 steps, and equilibrated for 1000 ps. The minimized monomer has a solvent accessible surface of 11,279.917 Å 2 . 
     Pocket analysis was undertaken to identify cavities present on the FOLR1 surface. The modeling software fPocket was used to calculate the volumes of putative pockets. Pockets large enough to accommodate protein catalyzed capture agents (PCCs) are located adjacent to the active site (itself a large rigid cavity). This information informed the epitope design and click handle placement. 
     Epitope 1 is H20-H33 of FOLR1 (HHKEKPGPEDKLHE; SEQ ID NO:3). Epitope 1 is composed of a largely unstructured loop near the N terminus of the protein. This epitope has high solvent exposure (1141.512 Å 2 ) and a low net charge (−1). The RMSD of the side chains and backbone (average equilibrated backbone RMSD 0.794 (0.175); average equilibrated total RMSD 1.450 (0.272)) suggests that this portion of the protein is flexible. Glycine-26 was selected as the Az4 substitution. The epitope 1 molecule used for catalyzed selection with G26 substituted with Az4 is biotin-PEG3-HHKEKP[G→Az4]PEDKLHE (SEQ ID NO:3). The structure is shown below. 
     Epitope 2 is G92-N109 of FOLR1 (GPWIQQVDQSWRKERVLN; SEQ ID NO:4). Epitope 2 is an epitope that is located adjacent to the active site. It was realized that the ability to attach folic acid itself to any ligand identified from this screen would further increase potency and selectivity of the final heterobiligand. While many constituent amino acids are charged, the overall epitope has only a +1 net charge. The RMSD of this epitope (average equilibrated backbone RMSD 0.576 (0.103); average equilibrated total RMSD 0.882 (0.150)) suggests that this region is flexible. Epitope 2 has a solvent accessible surface of 976.372 Å 2 . Serine-101 was selected as the Az4 substitution. The epitope 2 molecule used for catalyzed selection with S101 substituted with Az4 is biotin-PEG3-GPWIQQVDQ[S→Az 4 ]WRKERVLN (SEQ ID NO:4). The structure is shown below. 
     Epitope 3 is R186-R205 of FOLR1 (RCIQMWFDPAQGNPNEEVAR; SEQ ID NO:5). Epitope 3 is a contiguous sequence near the C terminus of the protein. The epitope is made up of an alpha helix connected to an unstructured loop. Epitope 3 has a solvent accessible surface of 1029.471 Å 2  and a net charge of −1. The average equilibrated backbone RMSD is 1.085 (0.205) and the average equilibrated total RMSD is 1.548 (0.207). Glutamine-196 was selected for Az4 substitution. The epitope 3 molecule used for catalyzed selection with Q196 substituted with Az4 is biotin-PEG3-R[C→S]IQMWFDPA[Q→Az 4 ]GNPNEEVAR (SEQ ID NO:5). The structure is shown below. 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     FOLR1 is small enough to allow heterobiligand preparation regardless of which epitope is targeted. Screens were performed using a triazole-cyclized OBOC library of the form H 2 N-Pra-(Pra-X 1 X 2 X 3 X 4 X 5 -Az4)-Met-TG, where TG=TentaGel® S NH 2  resin (S 30 902, Rapp Polymere), X 1 =one of sixteen D-amino acids, Pra=L-propargylglycine, and ( )=triazole cyclization via flanking Pra and Az4 (=L-azidolysine) residues. An encoded cyclic peptide library (ECPL) was created where 20% of the peptide on each bead is a linear tag for MALDI-TOF/TOF sequencing, while 80% remains the cyclic peptide for ligand discovery. 
     
       
         
         
             
             
         
       
     
     Macrocyclic peptide ligands were identified by screening the library against a cocktail of three FOLR1 epitopes ( FIG. 1 ) using the following steps: (1) pre-clear and anti-screens to eliminate non-specific binders, (2) a product screen to identify hits resulting from FOLR1 epitope-templated in situ click chemistry, and (3) target screens against His-tagged FOLR1 human recombinant protein to identify peptides that bind to the protein as well as the epitope ( FIG. 2 ). Two hits were isolated with strong binding to both FOLR1 epitope and protein: hshta (SEQ ID NO:6) and slyyk (SEQ ID NO:7). 
     Heterobiligands were also developed via in situ click screening ( FIG. 3 ). FOLR1 is a GPI-anchored cell surface protein that binds to folate ligand with high affinity (K D ≈1 nM). The FOLR1 protein provides a template for selectively promoting the azide-alkyne cycloaddition between folate ligand and library peptide. Folate-PCC heterobiligands were templated by screening the library and biotinylated azido-folate anchor against FOLR1 protein using the following steps: (1) pre-clear and anti-screen to eliminate non-specific binders, and (2) in situ click screen with biotinylated azido-folate anchor and FOLR1 protein to identify heterobiligand hits resulting from protein-templated in situ click chemistry ( FIG. 4 ). Two forms of folate anchor were used and were based on the fact that folate conjugates typically incorporate a PEG3 linker at the Glu side chain. 
     
       
         
         
             
             
         
       
     
     The short distance between FOLR1 epitope and the active site enabled the attachment of folate ligand to macrocycle hits to yield heterobiligands. The general structure of the heterobiligands is shown below, with the folate on the left, the peptide ligand on the right, and the linker in the middle. 
     
       
         
         
             
             
         
       
     
     Nine heterobiligand hits were found when the library was screened against 100 nM biotinylated azido-folate anchor and 1 nM FOLR1 protein (Library D1). Ghwef (SEQ ID NO:8), kyeet (SEQ ID NO:9), ltdwh (SEQ ID NO:10), hepff (SEQ ID NO:11), wGlhk (SEQ ID NO:12), wwprG (SEQ ID NO:13), nnyl (SEQ ID NO:14), twsw (SEQ ID NO:15), and yfytw (SEQ ID NO:16). Another four heterobiligand hits were found when the library was screened against 500 nM biotinylated azido-folate anchor and 5 nM FOLR1 protein (Library D2). wkhef (SEQ ID NO:17), tyGeh (SEQ ID NO:18), anGel (SEQ ID NO:19), and deryt (SEQ ID NO:20). 
     Two forms of heterobiligand were produced using the strong ligand hits. Tz heterobiligands were produced using ligands hshta (SEQ ID NO:6), kyeet (SEQ ID NO:9), and deryt (SEQ ID NO:20) and are characterized by a PEG linker, a Tz attachment of the ligand to the linker, and biotin attached to the N-terminus of the ligand. Tz heterobiligands had the general structure: 
     
       
         
         
             
             
         
       
     
     NTerm heterobiligands were produced using the ligand hshta (SEQ ID NO:6) and are characterized by direct attachment of the N-terminus of the ligand to the folate and biotin attached to the C-terminus of the ligand. NTerm heterobiligands have the general structure: 
     
       
         
         
             
             
         
       
     
     The specific structures of the tested PCCs and heterobiligands are shown below. 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Following the identification of promising hits, the compounds were evaluated by ELISA ( FIG. 5 ). Tested alone, kyeet (SEQ ID NO:9) demonstrated only minimal binding affinity for FOLR1 (EC 50 &gt;10,000 nM). However, once conjugated to folic acid, the heterobiligand is a highly potent FOLR1 binder (EC 50 =0.149 nM). This is over an order of magnitude greater than folic acid alone (EC 50 =2.8 nM). 
     A second PCC with sequence hshta (SEQ ID NO:6) was also evaluated in an analogous workflow. Alone, hshta (SEQ ID NO:6) bound to FOLR1 with an EC 50  of 3917 nM. Attaching the natural ligand increased the affinity to sub-nanomolar levels. Two heterobiligand constructs were tested, one with folic acid attached to the PCC via click reaction (Tz heterobiligand) and the other attached directly to the N-terminus of the peptide (NTerm heterobiligand). Both constructs were superior to folic acid alone (EC 50 =2.3 nM), with the NTerm heterobiligand (EC 50 =0.065 nM) outperforming the Tz construct (EC 50 =0.107 nM). 
     Both the kyeet (SEQ ID NO:9) and hshta (SEQ ID NO:6)-derived biligands were tested by flow cytometry for binding to FOLR1 (+) and FOLR1 (−) cell lines ( FIGS. 6 and 7 ). The human ovarian adenocarcinoma-derived cell line OVCAR3 has robust FOLR1 expression and was selected as the FOLR1 (+) cell line. The human prostate adenocarcinoma-derived cell line PC-3 was selected as a FOLR1 (−) cell line. Alone, kyeet (SEQ ID NO:9) showed slight binding selectivity for FOLR1 (+) cells over FOLR1 (−) cells ( FIG. 6 ). The heterobiligand construct containing folic acid conjugated to kyeet demonstrated high selectivity for FOLR1 (+) cells in a dose-dependent manner. Virtually complete selectivity was observed at 40 nM of the test article. 
     The alternative PCC hshta (SEQ ID NO:6) alone demonstrated higher selectivity for FOLR1 than kyeet (SEQ ID NO:9). While both the Tz and NTerm heterobiligands containing folic acid conjugated to hshta (SEQ ID NO:6) demonstrated high selectivity for FOLR1 (+) cells, the NTerm construct was superior ( FIG. 7 ). The selectivity of the best performing folate-hshta (SEQ ID NO:6) NTerm heterobiligand was superior to a commercially available FOLR1 antibody. Non-specific PC3 signal can be titrated away while maintaining OVCAR3 binding at low test concentrations. 
     To optimize the distance and physiochemical properties of the linker connecting folic acid to the PCC hshta (SEQ ID NO:6), a small linker screen was undertaken. Rather than direct linkage of the PCC to folate, linkers of PEG7, Pro, and Gly were used. Increasing the spacer distance or adding peptidic character were detrimental to the heterobiligand when profiled by ELISA. This trend was also reflected in a cellular context: the parent compound with direct attachment of folic acid to the N-terminus of the peptide was optimal. The folate-deryt (SEQ ID NO:20) heterobiligand also shows good binding affinity against FOLR1 (EC 50 =0.28 nM).
         Folate-hshta Nterm Ec50=0.18 nM   Folate-deryt Ec50=0.28 nM   Folate-PEG7-hshta Ec50=1.0 nM   Folate-Pro-hshta Ec50=0.9 nM   Folate-Gly-hshta Ec50=0.9 nM       

     These varied linker heterobiligands were also tested by flow cytometry against OVCAR3 FOLR1+ and PC3 FOLR1− cells  FIG. 8 ). Similar to ELISA, our lead heterobiligand demonstrates the best binding against OVCAR3 FOLR1+ cells. All compounds show little to no binding of PC3 FOLR1− cells. The specific structures of the tested heterobiligands are shown below. 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     A competition experiment was conducted to further understand the binding kinetics of the lead heterobiligand (folate-hshta (SEQ ID NO:6) Nterm heterobiligand). The natural ligand folic acid was doped into the Nterm heterobiligand dilution series at increasing concentrations to identify a concentration of folic acid that would worsen the apparent affinity of the heterobiligand for FOLR1. At concentrations of folic acid from 0 to 500 nM the binding affinity of the heterobiligand is virtually unchanged. Higher concentration of folic acid (greater than 500 nM) can partially compete away the heterobiligand. 
     Competition at the cellular level was also probed. Biotinylated heterobiligand or biotinylated folic acid was incubated with OVCAR3 or PC-3 cells ( FIG. 9 ). At increasing levels of exogenous folic acid, the heterobiligand was more resilient to folic acid displacement than biotinylated folic acid. The heterobiligand and biotinylated folic acid maintained high selectivity for FOLR1 (+) cells. An inverse trend of increased binding of the ligands with the reduction of free folic acid was observed. The percentage of OVCAR3 cells binding to biotinylated heterobiligand was reduced from 76.6% (no folate) to 57% (0.1 μg/mL folate). 
     The folate-hshta (SEQ ID NO:6) Nterm heterobiligand was also evaluated in cell penetration imaging assays ( FIG. 10 ). OVCAR3 and PC-3 cells were incubated with the test article at 37° C. for 1 hour. Flow cytometry analysis of the resulting treated cells indicated strong binding to FOLR1 (+) cells. To understand the proportion bound to the surface but not internalized, the cells were briefly washed with acid to liberate the non-internalized peptide. Following acid treatment, the percentage of positively stained cells decreases modestly from 73.4% to 54.2%. This suggests that a larger portion of the positive staining is the result of internalization and not cell surface binding. 
     To further understand intracellular trafficking of the Nterm heterobiligand, these dye-labeled compounds were imaged by fluorescence microscopy. The cells were counterstained with BioTracker 490, which stains membranes, and Rab5a+, an endosomal stain. The heterobiligand labeled with DyLight 650 demonstrated internalization with clear preference for early endosomes. This colocalization with the endosomal dye suggests receptor-mediated endocytosis. Low levels of DyLight650 was detected within PC-3 (FOLR1−) cells. Taken together, internalization of the heterobiligand appears to be receptor driven and selective for FOLR1 (+) cells. 
     
       
         
         
             
             
         
       
     
     Further evaluation of the folate-hshta (SEQ ID NO:6) Nterm heterobiligand depended on cross reactivity with mouse recombinant FOLR1 (rMuFOLR1). The heterobiligand binds to both species&#39; variants of FOLR1 with high affinity. This observation was expected given the high homology between the two variants (approximately 92%). 
     
       
         
           
               
               
               
             
               
                   
               
             
            
               
                 Folate-hshta Nterm 
                 Mouse FOLR1 
                 Ec50 = 0.33 nM 
               
               
                 Folate-hshta Nterm 
                 Human FOLR1 
                 Ec50 = 0.35 nM 
               
               
                 Biotinylated Folate 
                 Mouse FOLR1 
                 Ec50 = 0.46 nM 
               
               
                 Biotinylated Folate 
                 Human FOLR1 
                 Ec50 = 0.82 nM 
               
               
                 Biotinylated hshta 
                 Mouse FOLR1 
                 Ec50 = 2100 nM 
               
               
                 Biotinylated hshta 
                 Human FOLR1 
                 Ec50 = 3400 nM 
               
               
                 Biotinylated Folate 
                 Mouse FOLR1 
                 Ec50 = 0.60 nM 
               
               
                 Biotinylated Folate 
                 Human FOLR1 
                 Ec50 = 0.89 nM 
               
               
                 Biotin 
                 Mouse FOLR1 
                   
               
               
                 Biotin 
                 Human FOLR1 
               
               
                   
               
            
           
         
       
     
     NSG mice were engrafted with OVCAR3 cells subcutaneously. Once the tumor had reached around 0.8 cm diameter, they were treated with parenteral  18 F-labeled folate-hshta (SEQ ID NO:6) Nterm heterobiligand and evaluated by PET/CT imaging.  18 F PET/CT imaging scans were performed at 0-1 h (dynamic), 2 h (static), and 4 h (static). All time points measured out to 4 hours exhibited substantial tumor uptake of the radiotracer ( FIG. 11 ). Absorption of the radiotracer by highly perfused organs including the lungs indicate systemic circulation of the compound. Compound clearance is driven at the very early timepoints by a combination of hepatic and renal excretion. Over time, clearance is exclusively renal with retention of the compound in the tumor, and kidney. Accumulation in the mouse kidney is not surprising given the high expression of FOLR1 in this organ and the demonstrated cross reactivity of the heterobiligand with muFOLR1. The time-course biodistribution of  18 F-labeled folate-hshta (SEQ ID NO:6) Nterm heterobiligand was calculated to quantify the uptake in major organs including the tumor ( FIG. 12 ). Accumulation of  18 F-labeled folate-hshta (SEQ ID NO:6) Nterm heterobiligand is observed in the tumor with ˜4% of the injected dose/cc at 4 h post injection. A cross trial comparison of three mice shows radiotracer accumulation in the tumor ranging from 3.5% and 5.5% of the injected dose/cc at 4 h post injection ( FIG. 13 ). 
     
       
         
         
             
             
         
       
     
     A biotin-labeled albumin binding heterobiligand was synthesized to verify the heterobiligand binds to FOLR1 with acceptable affinity and to confirm albumin binding. A biotin-labeled heterobiligand was also synthesized to verify the DOTA does not interfere with binding. 
     
       
         
         
             
             
         
       
     
     where Tag=Lys(4-MPBA)-PEG10-PEG10-Lys(Biotin) for albumin binder and Lys(DOTA)-PEG10-PEG10-Lys(Biotin) for chelate. 
     
       
         
         
             
             
         
       
     
     Increasing the serum-bound fraction of the peptide may decrease first pass removal of the heterobiligand, and drive compound retention of tumor over kidney. Constructs bearing the known albumin binder 4-methylphenyl butyric acid (4-MPBA) were synthesized. These compounds were evaluated in vitro to verify binding fidelity to FOLR1. The modified heterobiligands were incubated at 400 nM with OVCAR3 and PC-3 cells for 20 minutes at 37° C. in complete media containing 20% FBS. They both bind to OVCAR3 FOLR1+ cells with similar binding affinities as the original heterobiligand studied ( FIG. 14 ). Gratifyingly, the addition of the albumin binding moiety does not impact binding to the target. Similar activity of these heterobiligands is anticipated in vivo. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                   
                 EC 50   
               
               
                   
                   
               
             
            
               
                   
                 Folate-hshta 
                 0.065 nM 
               
               
                   
                 Folate-hshta-MPBA 
                 0.110 nM 
               
               
                   
                 Folate-hshta-DOTA 
                 0.098 nM 
               
               
                   
                 Folate 
                  2.3 nM 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Evaluation of a folate-hshta (SEQ ID NO:6) Nterm heterobiligand bearing both an albumin binder and DOTA chelator in PET was a priority, given that DOTA can accommodate both  68 Ga for imaging and  177 Lu for radiotherapy. A 200 μCi dose of  68 Ga-labeled folate-hshta-PEG3-Lys(MPBA)-Lys(DOTA) (SEQ ID NO:6) (compound #2809) was injected i.v. via tail vein in an NSG mouse bearing s.c. human OVCAR3 (FOLR1+) tumor.  68 Ga PET/CT imaging scans were performed at 0-1 h (dynamic), 2 h (static), and 4 h (static). Images for mouse 4 are shown in  FIG. 15  (tumor size 150 mm 3 ), and images for mouse 6 are shown in  FIG. 16  (tumor size 490 mm 3 ). Kidneys are the major organ of elimination. The time-course biodistribution of  68 Ga-labeled folate-hshta-PEG3-Lys(MPBA)-Lys(DOTA) (SEQ ID NO:6) (compound #2809) was calculated to quantify the uptake in major organs including the tumor ( FIG. 17 ). At early tumor sizes (150 mm 3 ), low decay-corrected dose accumulation is observed in the tumor (˜1.4% of the injected dose/cc at 4 h post injection) ( FIGS. 17A and 17B ). Like the  18 F-labeled peptide, however, renal clearance is the preferred excretion pathway ( FIGS. 17C and 17D ). Larger tumors (490 mm 3 ) were also evaluated with the  68 Ga-chelated PET probe. In these larger models, higher accumulation within the tumor was observed (˜2.2% of the injected dose/cc at 4 h post injection) ( FIGS. 17A and 17B ). Tumor to muscle ratio data indicate that the radiotracer provides sufficient measurable PET signal to identify tumor delineated from background tissue ( FIGS. 17E and 17F ). Clearance of activity from the liver ( FIGS. 17G and 17H ) and heart ( FIGS. 171 and 17J ) is observed over time with elimination through the bladder ( FIGS. 17K and 17L ). Probe accumulation in tumor is significantly associated with tumor size ( FIG. 18 ; data collected at the 4 h time point are used because they are most relevant to the therapeutic effects). 
     For use in imaging and targeted therapy additional forms of the lead heterobiligand were produced. The parent compound, including DOTA for chelating a radionuclide (X) is: 
     
       
         
         
             
             
         
       
     
     To this an albumin binding moiety (MPBA) was added: 
     
       
         
         
             
             
         
       
     
     As an alternative, a GFK linker, susceptible to brush border cleavage, was added between the DOTA and the heterobiligand: 
     
       
         
         
             
             
         
       
     
     As another alternative, both modifications (albumin binding moiety and brush border cleavage linker) can be included in the same heterobiligand construct (e.g., Folate-hshta-Lys(MPBA)-Gly-Phe-Lys(DOTA) (SEQ ID NO:6), not shown). 
     With data suggesting targeted tumor retention, a therapeutic study was initiated by engrafting NSG mice with OVCAR3 cells. The engraftment was allowed to expand until palpable tumor sizes ranging in size from 218-384 mm 3  were detected. Animals were split into groups in a dose rangefinding experiment receiving 111 MBq, 37 MBq, 18.5 MBq, 9.25 MBq, 3.7 MBq, or 0 MBq radiotracer dose in the form of Folate-hshta-PEG3-Lys(MPBA)-Lys(DOTA) (SEQ ID NO:6) (compound #2809) with  177 Lu chelated (Table 1). Animals receiving the peptide alone with no  177 Lu chelated (0 MBq dose) experienced increasing tumor volume until the study endpoints were met at day 25. Gratifyingly, no observable compound-related adverse events were detected in this 0 MBq dose cohort. Animals treated with the three highest doses (111 MBq, 37 MBq, and 18.5 MBq) displayed adverse events that were dose proportional. These observed effects were consistant with radiation overdosing. The two lowest radio-doses (9.25 MBq and 3.7 MBq) were well tolerated. Mice receiving these dosing regimes demonstrated significant tumor reduction proportional to the dose received. From this study, it was determined that doses of 9.25 MBq and 3.7 MBq are well tolerated and efficacious. The best response was observed using 9.25 MBq Lu-177 Heterobiligand (compound #2809). Imaging and treatment feasibility was achieved with the same basic PCC construct, thus confirming that the PCC platform can perform theranostically. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Therapeutic Dose Finding Study in OVCAR3 Ovarian Cancer Xenograft 
               
               
                 Model 
               
            
           
           
               
               
               
            
               
                   
                 DOTA-Heterobiligand 
                   
               
               
                   177 Lu dose 
                 Albumin binder conjugate 
                 Comments 
               
               
                   
               
               
                   3 mCi (111 MBq) 
                 3 mice 
                 2 mice died on day 4 
               
               
                   
                   
                 1 mouse died on day 5 
               
               
                   1 mCi (37 MBq) 
                 2 mice 
                 2 mice died on day 10 
               
               
                  0.5 mCi (18.5 MBq) 
                 2 mice 
                 1 mouse died on day 10 
               
               
                   
                   
                 1 mouse died on day 13 
               
               
                 0.25 mCi (9.25 MBq) 
                 2 mice 
                 Sacrificed on day 24 
               
               
                  0.1 mCi (3.7 MBq) 
                 2 mice 
                 Sacrificed on day 24 
               
               
                   0 
                 3 mice 
                 Sacrificed on day 24 
               
               
                   
               
            
           
         
       
     
     The identical folate-hshta (SEQ ID NO:6) Nterm heterobiligand bearing both an albumin binder and DOTA chelator is amenable to both  68 Ga chelation for imaging or  177 Lu chelation for radiotherapy ( FIG. 19 ). Doses for this repeat experiment were derived from the tolerability study, with treatment cohorts receiving either 9.25 MBq (high dose) or 3.7 MBq (low dose). The two radiotracer doses and the control dose (peptide only with no  177 Lu) were well tolerated, recapitulating the results of the rangefinding study out to 17 days. Over the course of the experiment, a clear differential in tumor growth was observed for both doses compared to the unabated control cohort ( FIG. 20 ). The response was dose-dependent, with the highest dose exhibiting the largest tumor growth suppression ( FIG. 20 ). 
     Subjecting the DOTA-containing heterobiligands to  177 LuCl 3  at 95° C. for 15 minutes in 0.4 M sodium acetate buffer (pH 4.5) provided the chelated adduct in high yields. No further optimization of the labeling conditions was required. Both folate-hshta-PEG3-Lys(DOTA) and folate-hshta-PEG3-Lys(MPBA)-Lys(DOTA) (SEQ ID NO:6) (compound #2809) were successfully labeled with Lu-177 (100% conversion verified by both radioTLC and HPLC). Purity was confirmed to be 100% on HPLC. For mouse injection, the undiluted product in 0.4 M sodium acetate buffer (pH 4.5) was used directly or formulated in buffered saline. 
     The gallium labeling conditions were optimized using Ga(NO 3 ) 3 . The chelation was found to be rapid and efficient, with near quantitative conversion observed after 5 minutes at 90° C. and pH 4. Heating the reaction allowed for nearly 100% conversion of starting material (MW: 1927 m/z) into the gallium chelated product (MW: 1994 m/z) in under 5 min. Analytical HPLC data show the retention time shift from ˜24.5 min (starting material) to ˜25.1 min (product). HPLC conditions: 150×4.6 mm C18 column; 2-20% B, 60 min gradient; 25 μL injection volume. 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Utilizing an azido-folate anchor to template heterobiligands, an additional hit was profiled, This heterobiligand, based on the PCC deryt (SEQ ID NO:20), demonstrated high binding affinity by ELISA and excellent selectivity for FOLR1 (+) cells ( FIG. 21 ). Cells were incubated for 20 min at 37° C. in TC incubator with 400 nM of compound.
         Lys(PEG3-Folate)-deryt EC 50 =0.16 nM   Pra(Az4-PEG3-folate)-deryt EC 50 =0.27 nM   Folate EC 50 =2.3 nM   deryt EC 50 =10 μM       

     Two versions of the folate-deryt (SEQ ID NO:20) heterobiligand were synthesized. The first construct, utilizing a Tz linker, mimics the attachment structure from the in situ click screen. A second construct, utilizing a lysine in place of the Tz linker, maintains the correct distance between folic acid and the PCC but replaces the triazole with an amide bond. 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Further experiments were used to determine that the addition of an improved albumin binder and brush border linker would attenuate the high kidney signal we observed in  68 Ga imaging experiment described above. As described above, compound #2809 (folate-hshta-PEG3-Lys(MPBA)-Lys(DOTA) (SEQ ID NO:6)) was tested in tumor bearing NSG mice. This compound contains the folate-PCC heterobiligand with the albumin binder 4-methylphenyl butyric acid (MPBA) and DOTA chelator connected via PEG3. 
     A new compound (#1307; folate-hshta-Lys(IPBA)-Gly-Phe-Lys(DOTA) (SEQ ID NO:6)) contains the identical FOLR1 binding motif with the addition of a superior albumin binder 4-iodophenyl butyric acid (IPBA). IPBA has a high affinity for albumin, a large component of mammalian serum. By binding to albumin, the heterobiligand-albumin complex is shielded from the kidney&#39;s filtering system. The bound and unbound equilibrium allows for the serum-free fraction to participate in FOLR1(+) tumor binding ( FIG. 22 ). 
     In addition to the albumin binder, a brush border cleavable linker Gly-L-Phe-L-Lys was employed to connect the DOTA chelator. This allows enzymes in the kidney to cleave off the radionuclide-chelator complex which is eliminated through the bladder. 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     To optimize the albumin binding potential of the heterobiligand, a plasma protein binding (PPB) experiment was performed. This experiment measured the amount of test article bound and free after equilibrating with plasma. Compound #6305 (folate-hshta-Lys(MPBA)-Gly-Phe-Lys(DOTA) (SEQ ID NO:6)), which contains the MPBA albumin binder, was 96.26% bound to mouse plasma and 98.34% bound to human plasma. Compound #1307 (folate-hshta-Lys(IPBA)-Gly-Phe-Lys(DOTA) (SEQ ID NO:6)), which is identical except for the use of IPBA instead of MPBA, demonstrated a significantly higher level of plasma binding. In mouse and human plasmas 99.6% and 99.5% was bound, respectively. The alternative brush border compound L-Met-L-Val-L-Lys (compound #1306; folate-hshta-Lys(IPBA)-M-V-Lys(DOTA) (SEQ ID NO:6)) was roughly 97% bound to plasma, demonstrating that the linker also plays a role in plasma affinity. The absence of an albumin binder (compound #7327; folate-hshta-Gly-Phe-Lys(DOTA) (SEQ ID NO:6)) demonstrated comparatively low binding in both mouse (40.87%) and human (40.29%) plasma. Plasma Protein Binding indicates that IPBA is a superior albumin binder than MPBA. Compound #1307 possesses the optimized Alb binder and neprilysin (NEP) cleavage linker and has been chosen for further in vivo study. The results are summarized in Table 2. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Summary of FOLR1 Constructs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                 Plasma % Bound 
                   
               
               
                 # 
                 Compound 
                 Alb 
                 BB 
                 in vivo 
                 % MeCN 
                 (% Recovery) 
                 in vitro Assay Data 
               
               
                   
               
               
                 2809 
                 Fol-hshta-PEG3- 
                 MPBA 
                 N/A 
                 RVDx 
                  29.6% 
                 Hu: 83.28% (85.8% 
                 Confirmed to be 
               
               
                   
                 Lys(4-MPBA)- 
                   
                   
                   
                   
                 recovered) 
                 resistant to NEP 
               
               
                   
                 Lys(DOTA) 
                   
                   
                   
                   
                 Mu: 92.09% 
                 cleavage. 
               
               
                   
                   
                   
                   
                   
                   
                 (107.7% recovered) 
                   
               
               
                   2 
                 Fol-hshta-PEG3- 
                 N/A 
                 N/A 
                 Dx 
                 17.45% 
                 Hu: 43.01% (85.0% 
                   
               
               
                   
                 Lys(FB) 
                   
                   
                   
                   
                 recovered) 
                   
               
               
                   
                   
                   
                   
                   
                   
                 Mu: 20.22% 
                   
               
               
                   
                   
                   
                   
                   
                   
                 (115.0% recovered) 
                   
               
               
                 1189 
                 Fol-hshta-PEG3- 
                 N/A 
                 N/A 
                   
                  25.2% 
                 Hu: 46.02% (102.5% 
                 Confirmed to be 
               
               
                   
                 Lys(DOTA) 
                   
                   
                   
                   
                 recovered) 
                 resistant to NEP 
               
               
                   
                   
                   
                   
                   
                   
                 Mu: 29.51% (97.4% 
                 cleavage. 
               
               
                   
                   
                   
                   
                   
                   
                 recovered) 
                   
               
               
                 7327 
                 Fol-hshta-Gly- 
                 N/A 
                 GFK 
                   
                  23.8% 
                 Hu: 40.29% (112.7% 
                 Cleavage of -G-F-K- 
               
               
                   
                 Phe-Lys(DOTA) 
                   
                   
                   
                   
                 recovered) 
                 linker was confirmed 
               
               
                   
                   
                   
                   
                   
                   
                 Mu: 40.87% 
                 in NEP assay. 
               
               
                   
                   
                   
                   
                   
                   
                 (102.4% recovered) 
                   
               
               
                 6305 
                 Fol-hshta-Lys(4- 
                 MPBA 
                 GFK 
                 Rx/Dx 
                  32.3% 
                 Hu: 98.34% (86.2% 
                 Cleavage of -G-F-K- 
               
               
                   
                 MPBA)-Gly-Phe- 
                   
                   
                   
                   
                 recovered) 
                 linker was confirmed 
               
               
                   
                 Lys(DOTA) 
                   
                   
                   
                   
                 Mu: 96.26% 
                 in NEP assay. 
               
               
                   
                   
                   
                   
                   
                   
                 (107.1% recovered) 
                   
               
               
                 1307 
                 Fol-hshta-Lys(4- 
                 IPBA 
                 GFK 
                 Rx/Dx 
                  34.1% 
                 Hu: 99.50% (84.4% 
                 Cleavage of -G-F-K- 
               
               
                   
                 IPBA)-Gly-Phe- 
                   
                   
                   
                   
                 recovered) 
                 linker was confirmed 
               
               
                   
                 Lys(DOTA) 
                   
                   
                   
                   
                 Mu: 99.60% (87.7% 
                 in NEP assay. 
               
               
                   
                   
                   
                   
                   
                   
                 recovered) 
                   
               
               
                 1306 
                 Fol-hshta-Lys(4- 
                 IPBA 
                 MVK 
                   
                  33.4% 
                 Hu: 97.35% (84.8% 
                 Cleavage of -M-V-K- 
               
               
                   
                 IPBA)-Met-Val- 
                   
                   
                   
                   
                 recovered) 
                 linker was confirmed 
               
               
                   
                 Lys(DOTA) 
                   
                   
                   
                   
                 Mu: 97.12% (82.7% 
                 in NEP assay. 
               
               
                   
                   
                   
                   
                   
                   
                 recovered) 
                   
               
               
                 3302 
                 Fol-hshta-Lys(4- 
                 MPBA 
                 GFK 
                 Dx 
                  33.2% 
                 Hu: 94.9% (88.1% 
                 Cleavage of -G-F-K- 
               
               
                   
                 MPBA)-Gly-Phe- 
                   
                   
                   
                   
                 recovered) 
                 linker was confirmed 
               
               
                   
                 Lys(PEG3-DOTA) 
                   
                   
                   
                   
                 Mu: 96.5% (102.8% 
                 in NEP assay. 
               
               
                   
                   
                   
                   
                   
                   
                 recovered) 
                   
               
               
                 6307 
                 Fol-hshta-PEG3- 
                 N/A 
                 GFK 
                   
                  26.1% 
                 Hu: 41.3% (81.6% 
                 Cleavage of -G-F-K- 
               
               
                   
                 Gly-Phe- 
                   
                   
                   
                   
                 recovered) 
                 linker was confirmed 
               
               
                   
                 Lys(DOTA) 
                   
                   
                   
                   
                 Mu: 40.6% (68.9% 
                 in NEP assay. 
               
               
                   
                   
                   
                   
                   
                   
                 recovered) 
                   
               
               
                   
                 Octreotide Acetate 
                   
                   
                   
                   
                 Hu: 59.41% (84.3% 
                 Control (Cyclic 
               
               
                   
                   
                   
                   
                   
                   
                 recovered) 
                 peptide) 
               
               
                   
                   
                   
                   
                   
                   
                 Mu: 62.36% (84.5% 
                   
               
               
                   
                   
                   
                   
                   
                   
                 recovered) 
               
               
                   
               
               
                 Alb: Albumin Binder; BB: Brush Border Cleavable linker; in vivo: compound has been dosed; PPB: plasma protein binding assay (Human/CD-1 Mouse); % MeCN: percent Acetonitrile that elutes the compound (HPLC). 
               
            
           
         
       
     
     
       
         
         
             
             
         
       
     
     Following the injection of four female NSG mice with the improved construct (compound #1307 chelated with  68 Ga) the animals were imaged by PET/CT to profile the time-course biodistribution. Images were captured for compound #1307 at the time points 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, and 4 h. PET/CT images were taken following the injection of 4 female NSG mice with 68Ga-chelated compound #2809. In contrast to compound #2809, compound #1307 demonstrated very low kidney absorption. Instead, this compound initially accumulates in the lung, a highly perfused organ that also expresses FOLR1. Images for mouse 4 are shown in  FIG. 23  (Compounds #1307 and #2809) and  FIG. 24  (Compound #1307). 
     The biodistribution of  68 Ga-chelated compound #1307 was also calculated and compared to the biodistribution of  68 Ga-chelated compound #2809 ( FIG. 25 ). The kidney profile of compound #1307 demonstrates a seven-fold reduction in dose when compared to compound #2809 at the last time point (4 hours) ( FIG. 25B ). Both compounds demonstrate robust elimination through the bladder ( FIG. 25D ). Gratifyingly, the low kidney signal for compound #1307 did not result in an offsetting increase in liver clearance ( FIG. 25E ). This is in contrast with other FOLR1-targeted PET imaging agents. 
     Higher accumulation of compound #1307 in the heart was also observed ( FIG. 25C ). The increased dose to this highly perfused organ is most likely due to the increased plasma binding contributed by the IPBA moiety. The lung also demonstrates increased accumulation of compound #1307 ( FIG. 25A ). The greater than three-fold increase in lung accumulation when compared to the less than two-fold increase in heart exposure suggests that this accumulation is the result of FOLR1-mediated accumulation. 
     Kidney retention of radiotracers is a source of nephrotoxicity that can hinder development of theranostic agents via radiation-mediated DNA damage. To reduce the radioactive dose to the kidney, compounds were constructed to incorporate a linker that is susceptible to cleavage by enzymes found predominately in the kidney&#39;s brush border. These enzymes cleave off the radionuclide-chelator complex, which is quickly eliminated by the kidney. 
     The zinc metalloprotease neprilysin (NEP) is expressed in a variety of tissue but has high abundance in the kidney. Two tripeptide sequences are recognized and cleaved by NEP: L-Met-L-Val-L-Lys and Gly-L-Phe-L-Lys. These sequences were used as cleavable linkers in the disclosed compounds. 
     In order to test the susceptibility of GFK and MVK linkers to NEP-mediated cleavage, the peptide (Folate-hshta-Lys(MPBA)-Gly-Phe-Lys(DOTA) (SEQ ID NO:6)) was incubated with human recombinant NEP and analyzed the solution for the presence of cleaved adducts. MALDI was utilized to monitor the cleavage of a PCC heterobiligand that contains a NEP protease recognizable sequence (G-F-K). The PCC concentration was constant and the NEP concentration was titrated down from 10 nM to 1 nM, 0.1 nM, 0.01 nM, and no NEP. The NEP-mediated cleaved adduct was identified by mass. 10 μM PCC containing the GFK linker was combined with varying amounts of NEP. At 10 nM NEP, complete consumption of the starting peptide was observed. At 1 nM and 0.1 nM NEP, partial cleavage was observed. Below 0.1 nM no cleavage was observed.
         Folate-hshta-Lys(MPBA)-Gly-Phe-Lys(DOTA) (SEQ ID NO:6)
           Molecular weight 2231.42   
               

     
       
         
         
             
             
         
       
     
     The NEP-mediated cleavage was also monitored by HPLC. In agreement with the MALDI spectra, the HPLC chromatograms show depletion of the parent brush-border heterobiligand (retention time 17.1 minutes), dose-dependent on the amount of NEP spiked into the sample (10 nM, 1 nM, 0.1 nM, 0.01 nM, and no NEP). An appearance of a new peak (retention time 16.5 minutes) in concert with the depletion of the starting material is presumed to be the cleaved folate-containing peptide. 
     In order to test the specificity of NEP, a non-cleavable linker (PEG3) was also assayed. The NEP enzymatic cleavage assay was run using hshta-folate heterobiligands with and without the cleavable linker.
         Folate-hshta-Lys(MPBA)-Gly-Phe-Lys(DOTA) (SEQ ID NO:6)
           Molecular weight 2231.42   
           Folate-hshta-PEG3-Lys(MPBA)-Lys(DOTA) (SEQ ID NO:6)
           Molecular weight 2215.42   
               

     In both MALDI and HPLC, the PEG3 compound was unchanged when incubated with NEP, while the GFK linker cleaved at the predicted site. In MALDI, the heterobiligand containing the -G-F-K- linker cleaved at the designed amide bond, producing an observable fragment with the predicted m/z (Folate-hshta-Lys(MPBA)-G-OH (SEQ ID NO:6); Molecular weight 1569.67). The heterobiligand containing a PEG3 in place of the -G-F-K-, however, did not cleave in the presence of NEP. In addition to confirming the selectivity of the NEP enzyme for the -G-F-K- peptide linker, it is also important to note that the remaining portions of the peptide were impervious to NEP. 
     The NEP-mediated cleavage was also monitored by HPLC. In agreement with the MALDI spectra, the HPLC chromatograms show depletion of the parent brush-border heterobiligand (retention time 17.1 minutes). The related non-cleavable analog remained unchanged (retention time 15.6 minutes). An appearance of a new peak (retention time 16.5 minutes) in concert with the depletion of the starting material is presumed to be the cleaved folate-containing peptide. 
     Two new heterobiligands were tested for NEP-mediated cleavage. One possesses an L-Met-L-Val-L-Lys linker, the other a Gly-L-Phe-L-Lys linker. Both are also appended with an improved albumin binder, 4-iodophenyl butyric acid (IPBA).
         Folate-hshta-Lys(IPBA)-M-V-Lys(DOTA) (SEQ ID NO:6)
           Molecular weight 2369.39   
           Folate-hshta-Lys(IPBA)-Gly-Phe-Lys(DOTA) (SEQ ID NO:6)
           Molecular weight 2343.29   
               

     At 1 nM NEP, substantial (and in the case of GFK complete) cleavage of the test article was observed by MALDI-TOF MS. For the MVK linker, two species were identified, differing by mass of one methionine. This experiment confirms that both cleavable linkers are susceptible to NEP cleavage.
         Folate-hshta-Lys(IPBA)-M-OH (SEQ ID NO:6)
           Molecular weight 1755.68   
           Folate-hshta-Lys(IPBA)-OH (SEQ ID NO:6)
           Molecular weight 1624.48   
           Folate-hshta-Lys(IPBA)-G-OH (SEQ ID NO:6) Molecular weight 1681.54       

     Key compounds were subjected to a plasma stability assay to determine the liability of the heterobiligand (and the NEP linker in particular) towards secreted peptidases found in mammalian plasma. Plasma stability of compounds #2809, 1307, 2 and 1306 and a propantheline bromide control were measured. Gratifyingly, we observed robust stability toward both rodent (CD-1 mouse) and human plasma for all compounds tested. This suggests that the PCCs with or without the L-amino acid NEP linker are stable in plasma. The results are shown in Table 3. All of the compounds showed stability in both human and mouse up to the limit of the assay (&gt;289.1 min). 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Plasma Stability of Select Compounds 
               
            
           
           
               
               
               
            
               
                 # 
                 Compound 
                 Plasma Stability (t 1/2) 
               
               
                   
               
               
                 2809 
                 Fol-hshta-PEG3-Lys(4-MPBA)-Lys(DOTA) 
                 Hu: &gt;289.1 min 
               
               
                   
                   
                 Mu: &gt;289.1 min 
               
               
                   2 
                 Fol-hshta-PEG3-Lys(FB) 
                 Hu: &gt;289.1 min 
               
               
                   
                   
                 Mu: &gt;289.1 min 
               
               
                 1307 
                 Fol-hshta-Lys(4-IPBA)-Gly-Phe-Lys(DOTA) 
                 Hu: &gt;289.1 min 
               
               
                   
                   
                 Mu: &gt;289.1 min 
               
               
                 1306 
                 Fol-hshta-Lys(4-IPBA)-Met-Val-Lys(DOTA) 
                 Hu: &gt;289.1 min 
               
               
                   
                   
                 Mu: &gt;289.1 min 
               
               
                 7327 
                 Fol-hshta-Gly-Phe-Lys(DOTA) 
                 Hu: &gt;6937.7 min 
               
               
                   
                   
                 Mu: &gt;6937.7 min 
               
               
                 6305 
                 Fol-hshta-Lys(4-MPBA)-Gly-Phe-Lys(DOTA) 
                 Hu: &gt;6937.7 min 
               
               
                   
                   
                 Mu: &gt;6937.7 min 
               
               
                 3303 
                 Fol-hshta-Gly-Phe-Lys(AO) 
                 Hu: &gt;6937.7 min 
               
               
                   
                   
                 Mu: &gt;6937.7 min 
               
               
                   
                   
                 (FB) 
               
               
                   
                 Propantheline bromide (Control) 
                 Hu: 11.6 min 
               
               
                   
                   
                 Mu: 44.8 min 
               
               
                   
               
            
           
         
       
     
     The heterobiligand dosed in these studies is Folate-hshta-PEG3-Lys(MPBA)-Lys(DOTA) (Compound #2809; SEQ ID NO:6)) labeled with  177 Lu. Plasma samples taken from tumor-bearing mice (at each dose) were assayed for biomarkers indicative of nephrotoxicity. These samples were compared to plasma taken from normal mice. Elevated urea levels in plasma could indicate kidney damage. Endogenous urea is converted to ammonia and carbon dioxide by the addition of urease. Berthelot&#39;s reagent reacts with the dissolved ammonia. The resulting green coloration can be used to back-calculate the amount of urea present in the original plasma sample. 
     
       
         
         
             
             
         
       
     
     Mouse plasma samples (five mice each from the 0 MBq control group, 3.7 MBq group, and 9.25 MBq group) taken at endpoint and normal mouse plasma were evaluated in urea assays to gain information on renal function. Data show that the 9.25 MBq treated mice have 1.5 times elevated levels of blood urea nitrogen (BUN) level than the normal mice (Tables 4 and 5). The BUN value for normal mice has a range of 17-20 mg/dL, while mice with kidney injuries have 2×-4× higher BUN levels. Animals receiving either 0 or 3.7 MBq doses fell within nominal plasma urea levels. The highest dose, 9.25 MBq, yielded increased plasma urea levels. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                   177 Lu dose 
                 [BUN] (mg/dL) 
               
               
                   
                   
               
             
            
               
                   
                 Normal Mice 
                 20.7 ± 4.3 
               
               
                   
                   0 MBq 
                 22.6 ± 2.7 
               
               
                   
                  3.7 MBq 
                 25.4 ± 4.3 
               
               
                   
                 9.25 MBq 
                 30.4 ± 7.8 
               
               
                   
                   
               
            
           
         
       
     
     
       
         
           
               
               
               
             
               
                   
                 TABLE 5 
               
               
                   
                   
               
               
                   
                 Mice 
                 [BUN] (mg/dL) 
               
               
                   
                   
               
             
            
               
                   
                 Normal C57BL/6 a   
                 17 
               
               
                   
                 Normal C57BL/6 b   
                 20 
               
               
                   
                 2 day post Ischemia b   
                 51 
               
               
                   
                 Normal DBA/2J c   
                 19 
               
               
                   
                 3 day post 5/6-nephrectomy c   
                 42 
               
               
                   
                 Normal C57BL/6 d   
                 19 
               
               
                   
                 1 day post Renal 
                 82 
               
               
                   
                 Ischemia/Reperfusion d   
               
               
                   
                   
               
               
                   
                 References:  a Rodrigues et. al., Biomed Res Int. 2014; 872827. 
               
               
                   
                   b Han et. al., Stem Cell Res Ther. 2013; 4(3) 74. 
               
               
                   
                   c Grindle, et. al., Comp Med. 2006; 56(6) 482. 
               
               
                   
                   d Jouret et. al., PLoS One 2016; 11(9) e0163021. 
               
            
           
         
       
     
     Creatinine is another biomarker indicative of kidney stress. This compound is a byproduct of metabolism that is removed from circulation by the kidney. An elevated plasma creatinine level is indicative of poorly functioning kidneys. Mouse plasma samples (five mice each from the 0 MBq control group, 3.7 MBq group, and 9.25 MBq group) taken at endpoint and normal mouse plasma were evaluated in creatinine assays to gain information on renal function (Table 6). In the tumor therapeutic experiment, the animals dosed with 0 MBq and 3.7 MBq demonstrated elevated dissolved creatinine. Animals dosed with 9.25 MBq of the compound demonstrated substantially higher creatinine concentration. 
     
       
         
           
               
               
               
             
               
                   
                 TABLE 6 
               
               
                   
                   
               
               
                   
                   177 Lu dose 
                 [Creatinine] (μmol/L) 
               
               
                   
                   
               
             
            
               
                   
                 Normal Mice 
                  7.7 ± 3.6 
               
               
                   
                   0 MBq 
                 16.2 ± 12.0 
               
               
                   
                  3.7 MBq 
                 15.5 ± 6.1 
               
               
                   
                 9.25 MBq 
                 43.9 ± 27.0 
               
               
                   
                   
               
            
           
         
       
     
     Tumor growth, body weight, and gamma counting data were compiled at the endpoint of the  177 Lu-#6305 therapy study of OVCAR3 xenografts. Tumor sizes at time of treatment were compared for the  177 Lu-#6305 and previous therapy studies. The  68 Ga and  177 Lu labeling of heterobiligand #3302 is being tested. This heterobiligand is similar to compound #6305 but has a PEG3 spacer between the GFK linker and DOTA to improve the solubility and reactivity. Chelation of  175 Lu proceeds more efficiently for #3302 than #6305, which indicates that this enhancement in labeling will translate to the  177 Lu reaction. 
     Lu177-#6305 treatments were tested to determine the tolerated and most effective doses. Single dose treatments at 18.5 MBq/nmol specific activity. OVCAR3 mouse model was used. 10 7  cell tumors were implanted in the mice one month before administration of  177 Lu-#6305. Five mice each were administered Cold #6305, 9.25 MBq Lu177-#6305, and 14.8 MBq Lu177-#6305, and two mice were administered 29.6 MBq Lu177-#6305. Tumor growth and body weight were then monitored. 
     No significant body weight change was detected in groups treated with 9.25 MBq and 14.8 MBq Lu177-#6305 ( FIG. 26 ). The 29.6 MBq Lu177-#6305 treatment group showed substantial body weight decrease after injection ( FIG. 26 ). 14.8 MBq Lu177-#6305 treatment significantly slowed down OVCAR3 tumor growth ( FIG. 27 ). 
     7 days after Lu177-#6305 injection, the 29.6 MBq group lost 20% body weight. These mice were sacrificed. Tissues and plasma were collected for evaluation of radioactivity by gamma counting. The toxicity may have resulted from its accumulation in the kidneys and liver ( FIG. 28 ). At study endpoint (Day 28 post-injection), plasma, tumors, kidneys, lungs, hearts, livers, and muscles from 9.25 MBq and 14.8 MBq treatment groups were collected for evaluation of radioactivity by gamma counting ( FIG. 29 ). 
     Lu177-#6305 at 14.8 MBq significantly slowed down OVCAR3 xenograft tumor growth. Both 9.25 MBq and 14.8 MBq doses of Lu177-#6305 were tolerated without significant body weight change detected. The 29.6 MBq Lu177-#6305 dose caused substantial body weight decrease after injection. Based on the gamma counting data from the 29.6 MBq group on Day 7 post-injection, high radioactivity was detected in the kidney and the liver (394,834 CPM/g and 107,374 CPM/g, respectively). 42,674 CPM/g was detected in the tumor. Based on the gamma counting data from the groups of 9.25 MBq and 14.8 MBq on Day 28 post-injection, the liver radioactivity (685 CPM/g in the 9.25 MBq group and 1,264 CPM/g in the 14.8 MBq group) was higher than that in the kidney (414 CPM/g in the 9.25 MBq group and 653 CPM/g in the 14.8 MBq group). The tumor radioactivity was 33 CPM/g and 60 CPM/g, respectively, for the 9.25 MBq group and the 14.8 MBq group. 
     In the gamma counting data, the tumor/kidney ratio was 0.108 and 0.093 in the 29.6 MBq group and the 14.8 MBq group, which are quite comparable. There was a ˜6-fold increase in tumor mass between Day 7 and Day 28, suggesting that the decrease in radioactivity in the tumor was slower than that in the kidney (the values were normalized to mass, CPM/g). There was a substantial increase in the liver/kidney radio from 0.27 to 1.94 (29.6 MBq group on Day 7 vs. 14.8 MBq group on Day 28). 
     Tumor size on day −2 for the  177 Lu-#2809 treatment is shown in  FIG. 30 . Heterobiligand #2809 has a PEG3 linker and MPBA albumin binder. A single dose at 81.4 MBq/nmol specific activity was used. Tumor size (avg.) at treatment was 203 mm 3 . Significant tumor growth control at 9.25 MBq and partial response at 3.7 MBq was observed. 
     Tumor size on day 0 for the  177 Lu-#1307 treatment is shown in  FIG. 31 . Heterobiligand #1307 has IPBA albumin binder and GFK linker (for kidney cleavage). A single dose at 1.73 MBq/nmol specific activity was used. Tumor size (avg.) at treatment was 117 mm 3 . A partial response was observed at 6.29-7.03 MBq (small n). 
     Tumor size on day 0 for the  177 Lu-#6305 treatment is shown in  FIG. 32 . Heterobiligand #6305 has MPBA albumin binder and GFK linker (for kidney cleavage). A single dose at 18.5 MBq/nmol specific activity was used. Tumor size (avg.) at treatment was 148 mm 3 . A partial response was observed at 14.8 MBq and nonresponse at 9.25 MBq. 
     The greatest tumor growth control was observed when treating at the highest  177 Lu specific activity and largest tumor sizes. Also, the linkers attached to a given heterobiligand would have influenced its exposure to the tumor. 
     The heterobiligands can also be tested in in vivo therapy studies designed to investigate the specific activity of  177 Lu labeling, tumor size at time of treatment, and protein binding contributions to therapy response.  18 F-FBA PET imaging studies can also be used to profile the biodistribution and clearance with iteration of BB cleavable linker designs for reduction of renal uptake. SKOV3 can be employed as a second FOLR+ ovarian cancer xenograft model for PET imaging. 
     A summary of the results of in vivo response to heterobiligand treatment in OVCAR3 xenografts is shown in  FIG. 33 .  FIG. 33A  shows the results for OVCAR3 xenografts treated with Lu177-#2809 (SA=81.4 MBq/nmol).  FIG. 33B  shows results for OVCAR3 xenografts treated with Lu177-#1307 (SA=1.73 MBq/nmol).  FIG. 33C  shows results for OVCAR3 xenografts treated with Lu177-#6305 (SA=18.5 MBq/nmol). 
     Select FOLR constructs using AO instead of DOTA were tested. The results are summarized in Table 7. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 Summary of AO-containing FOLR1 Constructs 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                 # 
                 Compound 
                 Alb 
                 BB 
                 in vivo 
                 % MeCN 
                 Plasma % Bound (% Recovery) 
                 in vitro Assay Data 
               
               
                   
               
               
                 2808 
                 Fol-hshta-PEG3-Lys(4- 
                 MPBA 
                 N/A 
                   
                 — 
                   
                   
               
               
                   
                 MPBA)-Lys(AO) 
                   
                   
                   
                   
                   
                   
               
               
                 0328 
                 Fol-hshta-PEG3-Lys(AO) 
                 N/A 
                 N/A 
                 Dx 
                 17.45% 
                 Hu: 43.01% (85.0% recovered)  
                   
               
               
                   
                   
                   
                   
                   
                 (FB) 
                 Mu: 20.22% (115.0% recovered) (FB)  
                   
               
               
                 9416 
                 Fol-hshta-Lys(4-IPBA)- 
                 IPBA 
                 GFK 
                   
                 — 
                   
                   
               
               
                   
                 Gly-Phe-Lys(AO) 
                   
                   
                   
                   
                   
                   
               
               
                 3304 
                 Fol-hshta-Lys(4-MPBA)- 
                 MPBA 
                 GFK 
                   
                 40.1% 
                   
                 Cleavage of —G—F—K- 
               
               
                   
                 Gly-Phe-Lys(AO) 
                   
                   
                   
                 (FB) 
                   
                 linker was confirmed in NEP assay. 
               
               
                 3303 
                 Fol-hshta-Gly-Phe- 
                 N/A 
                 GFK 
                 Dx 
                 33.5% 
                 Hu: 81.0% (80.6% recovered) 
                 Cleavage of —G—F—K- 
               
               
                   
                 Lys(AO) 
                   
                   
                   
                 (FB) 
                 Mu: 75.5% (77.7% recovered) (FB) 
                 linker was confirmed in NEP assay. 
               
               
                   
               
               
                 Alb: Albumin Binder; 
               
               
                 BB: Brush Border Cleavable linker; 
               
               
                 in vivo: compound has been dosed; 
               
               
                 % MeCN: percent Acetonitrile that elutes the compound (HPLC). 
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                   
                     
                     
                         
                         
                     
                   
                   
                     
                     
                         
                         
                     
                   
                   
                     
                     
                         
                         
                     
                   
                   
                     
                     
                         
                         
                     
                   
                 
               
            
           
         
       
     
     Example 2: In Vivo Studies of Heterobiligand Compounds 
     In order to establish a maximum tolerated dose (MTD), a tolerability study was undertaken testing increasing amounts of the 177-Lu heterobiligand 7327 (folate-hshta-Gly-Phe-Lys(DOTA)). Animals were monitored for signs of toxicity and weighed twice weekly. Repeated doses of 14.8 MBq, 22.2 MBq, 29.6 MBq, and 37 MBq were well tolerated ( FIGS. 34 and 35 ). The second and third doses were administered 21 days following the previous dose. Doses of 111 MBq and 185 MBq were not tolerated ( FIG. 35 ). At these high doses, animals lost substantial body weight following the first dose, which worsened following the second and third doses. This experiment established an MTD for 177-Lu 7327 of between 37 and 111 MBq. The tolerance of the 0 MBq arm suggests that the radionuclide drives the toxicity profile and not the peptide alone. 
     Female NSG mice were implanted subcutaneously with OVCAR3 cells. Once tumors reached volumes of 200 mm 3 , the animals were treated with 177-Lu 7327. Three treatment arms of 9.25 MBq, 14.8 MBq, and 29.6 MBq were studied. The first dose was well tolerated and the animals displayed no visible signs of radiotoxicity. Climbing body weights of all animals also suggest each dosing arm was well tolerated ( FIG. 36A ). 
     A second dose was administered 21 days following the first dose. Body weight loss of animals within the 9.25 MBq and 14.8 MBq arms was minimal ( FIG. 36A ). Animals receiving the highest dose exhibited modest loss of body weight that stabilized and then rebounded 10 days following the second dose. Caliper-measured tumor volume stabilized and then fell following the second dose of the highest arm ( FIGS. 36B and 37 ). Statistically significant differential between treated and untreated was reached 12 days post injection for the 29.6 MBq arm (* for p≤0.05). P values were determined by ordinary one-way ANOVA with Tukey&#39;s multiple comparisons test. 
     At 45 days post injection, the animals bearing the smallest tumors were randomized and given elevated doses of 177-Lu 7327. Animals received 46.3 MBq, 74 MBq, or 148 MBq doses. The first dose was well tolerated for all groups ( FIG. 38A ). The two highest doses, 74 MBq and 148 MBq, caused modest body weight loss ( FIG. 38A ). Tumor volumes for these doses decreased on average 50% following this single therapeutic dose ( FIG. 38B ). Animals in the 46.3 MBq group experienced weight gain and stable tumor volumes ( FIG. 38B ). On day 24, each arm received an additional treatment. Animals in all three groups exhibited shrinking tumor sizes ( FIG. 38B ). This data suggests that 177-Lu 7327 can stabilize and in some cases reverse highly aggressive FOLR1 expressing tumors. 
     In order to understand the toxicity-limiting tissues and organs, plasma was collected from the therapy studies and analyzed for blood urea nitrogen (BUN) and creatinine. BUN is often utilized in a series of tests to assess kidney function. Elevated BUN levels are an indication of reduced kidney function. Creatinine is another important kidney biomarker. Elevated creatinine is a sign of poor renal function. 
     Normal BUN levels were observed in mice receiving doses at or below 29.6 MBq (Table 9). Those receiving doses of 46.3 MBq and 74 MBq exhibited slightly elevated BUN levels (Table 9). Mice that received the highest dose of 148 MBq had elevated BUN levels (74.1 mg/dL) (Table 9). 
     
       
         
           
               
             
               
                 TABLE 9 
               
             
            
               
                   
               
               
                 Assessment of Blood Urea Nitrogen (BUN) After Therapy 
               
            
           
           
               
               
               
            
               
                   
                   177 Lu-#7327 
                 [BUN] 
               
               
                   
                 dose 
                 (mg/dL) 
               
               
                   
                   
               
               
                   
                   0 MBq 
                 27.3 ± 2.4 
               
               
                   
                 9.25 MBq 
                 27.5 ± 2.6 
               
               
                   
                 14.8 MBq 
                 30.6 ± 3.6 
               
               
                   
                 29.6 MBq 
                 30.3 ± 2.1 
               
               
                   
                 46.3 MBq 
                 32.4 ± 1.3 
               
               
                   
                   74 MBq 
                 34.6 ± 1.0 
               
               
                   
                  148 MBq 
                 74.1 ± 21.3 
               
               
                   
                   
               
            
           
         
       
     
     The creatinine trends largely recapitulate the healthy renal functions uncovered in the BUN assay. Animals receiving up to 29.6 MBq had normal creatinine levels (Table 10). One mouse in the 46.3 MBq and 74 MBq groups had elevated creatinine plasma concentrations. As a group, animals receiving 148 MBq had elevated creatinine levels (Table 10). 
     
       
         
           
               
             
               
                 TABLE 10 
               
             
            
               
                   
               
               
                 Assessment of Creatinine After Therapy 
               
            
           
           
               
               
               
            
               
                   
                   177 Lu-#7327 
                   
               
               
                   
                 dose 
                 [Creatinine] (μmol/L) 
               
               
                   
                   
               
               
                   
                   0 MBq 
                 12.2 ± 2.0 
               
               
                   
                 9.25 MBq 
                 15.3 ± 2.7 
               
               
                   
                 14.8 MBq 
                 16.4 ± 1.9 (Mouse #1) 
               
               
                   
                   
                 41.6 ± 2.8 (Mouse #2) 
               
               
                   
                 29.6 MBq 
                 12.4 ± 0.3 
               
               
                   
                 46.3 MBq 
                 13.9 ± 1.7 
               
               
                   
                   74 MBq 
                 11.8 ± 3.3 (Mice #1 &amp; 2) 
               
               
                   
                   
                 37.6 ± 2.6 (Mouse #3) 
               
               
                   
                  148 MBq 
                 19.0 ± 1.6 
               
               
                   
                   
               
            
           
         
       
     
     Efforts were undertaken to evaluate 177-Lu 6305 (folate-hshta-Lys(MPBA)-Gly-Phe-Lys(DOTA)), a compound with a brush border cleavable linker in addition to an albumin binder. The albumin binding moiety increases the circulation time of the compound by shielding it from renal proteases and elimination. Two separate MTD experiments were undertaken. Animals receiving doses of 29.6 MBq, 37 MBq, 111 MBq, and 185 MBq began to experience weight loss after the first dose ( FIG. 39 ). A second dose 7 days after the first was not tolerated ( FIG. 39 ). A second experiment interrogated lower doses. Animals receiving a single dose of 14.8 MBq, 18.5 MBq, and 22.2 MBq exhibited only minimal loss in body weight that quickly stabilized ( FIG. 40 ). A second dose administered 21 days was not tolerated ( FIG. 40 ). The tolerance of the 0 MBq arm indicates that the radionuclide drives the toxicity profile and not the peptide alone ( FIG. 40 ). 
     Example 3: Analysis of Heterobiligands for Therapeutic Use Introduction 
     Folate receptor is highly overexpressed in ovarian cancer and presents an attractive target to specifically deliver therapeutic radiation. Indi  177 Lu-7327 is a low molecular weight (˜2100 Da), water soluble, ultra-high affinity synthetic peptide-folate conjugate targeting the folate receptor alpha (FOLR). This construct is internalized into ovarian cancer cells by the cell&#39;s folate receptor, outcompeting native folate for receptor binding by approximately 500-fold. FOLR-targeted drugs can present significant renal toxicity unless rapidly cleared. Since FOLR is highly expressed in the kidney, the kidney exposure of Indi  177 Lu-7327 is mitigated by inclusion of a 3-amino acid linker that is enzymatically cleaved at the renal brush border membrane. Upon cleavage, the intact  177 Lu-DOTA fragment is released from the primary drug conjugate and passed to the bladder for excretion. The drug is highly stable in human plasma and due to the use of d-amino acids and its low molecular weight, it is unlikely to be immunogenic. 
     Indi  177 Lu-7327 is a radiolabeled peptide-folate conjugate is especially useful in the treatment of patients with epithelial ovarian, primary peritoneal or fallopian tube cancers that are platinum resistant and FOLR positive. FOLR is a folate-binding protein located on cellular membranes that contributes to folate uptake by cells. It is an attractive anticancer drug target owing to its overexpression in a range of solid tumors, including ovarian, lung, and breast cancers (Scaranti et al.,  Nature Reviews Clinical Oncology.  2020; 17(6):349-359). PCC technology is a powerful screening strategy to rapidly discover high-avidity, synthetic peptide ligands to judiciously selected epitopes of proteins (Agnew et al.,  Chemical Reviews.  2019; 119(17):9950-9970). Heterobiligands designed for multivalent interactions with human FOLR were developed by conjugating folate ligand to macrocyclic peptide ligands selected by our PCC platform. The close proximity of targeted FOLR epitopes to the active site and chemical linker optimization were leveraged in generating ultra-high affinity binding that outcompetes the native folate ligand. 
     The FOLR targeting ligand in Indi  177 Lu-7327 is chemically attached via a DOTA chelator to lutetium-177 ( 177 Lu), a therapeutic radioactive atom which releases an energetic beta particle to precisely deliver cell killing radiation to the tumor. To reduce treatment related kidney exposure, the chemically attached G-F-K linker is susceptible to enzymatic cleavage at the renal brush border membrane. When Indi  177 Lu-7327 enters the renal brush border cells, enzymatic cleavage results in liberation of the intact  177 Lu-DOTA fragment and excretion in the urine. 
     Ovarian cancer can only be definitively diagnosed with a tissue biopsy. Ovarian cancer is suspected when there are certain findings on a clinical pelvic exam, symptoms that may be concerning for a malignancy, or abnormalities that may be seen incidentally on imaging studies for other purposes. Once suspected, bloodwork, such as cancer antigen CA-125, is conducted in combination with imaging which is often ultrasound (US) followed by computed tomography (CT) or magnetic resonance imaging (MRI). When possible, positron emission tomography combined with CT (PET-CT) can be used to further quantify the likelihood of an ovarian tumor. The role of imaging in ovarian cancer involves detection, characterization, and staging. Imaging plays an important role in characterization of ovarian masses, as the number of benign ovarian masses greatly exceeds the number of malignant masses (Balachandran and Iyer, Applied Radiology. 2005; 34(9):19-29). If someone is considered at elevated risk for having an ovarian tumor, risk-based protocols that combine surgery, sometimes with neoadjuvant chemotherapy, are used to both stage and treat the patient. 
     Women who have completed initial treatment for advanced stage ovarian carcinoma are monitored closely for evidence of recurrence. Follow up consists of physical exams, CA-125 blood level monitoring, and imaging (CT or PET-CT being the most common modalities). Typically, if CA-125 levels are obtained and an elevation from post-treatment baseline is seen, this would determine the need for radiologic imaging to try and identify location of tumor recurrence. Some women have interval debulking procedures scheduled either during or immediately following completion of first-line treatment. Women can also have interval debulking procedures as part of the initial treatment for recurrent or suspected recurrent ovarian cancer. 
     PET-CT is used to diagnose recurrence of ovarian cancer. A meta-analysis comparing techniques for detection of recurrence determined that PET-CT performed better than CT or MRI with sensitivities of 95% vs 79% and 75%, respectively, and specificities of 88% vs 84% and 78%, respectively (Gu et al.,  European Journal of Radiology.  2009; 71(1):164-174). 
     If epithelial ovarian cancer is diagnosed early (Stage I or Localized), the 5-year survival rate is 92%. Stage II or Regional 5-year survival rate is approximately 76%. Stage III/IV or Distant 5-year survival rate is around 30%. Approximately 66-80% of women with epithelial ovarian cancer are diagnosed at Stage III or higher (American Cancer Society). 
     All ovarian cancer patients, except those going immediately to palliative care, require surgery, with most women having surgery soon after the diagnosis is suspected or confirmed. For premenopausal women with early stage (Stage I) disease, an individualized approach that includes fertility preserving options is sometimes possible. When fertility is not a concern in younger women, or in women who are no longer of reproductive age, surgery for early-stage disease (Stage I-II) most commonly removes the uterus, cervix, both fallopian tubes, and both ovaries. Pelvic washings are always done prior to any surgical manipulation to determine the presence of cancer cells in the peritoneal fluid. Additional biopsies and/or lymph node dissections may also be performed if the intraoperative findings differ from the preoperative imaging studies. 
     For later stage cancers, Stage III-IV, women may be given the option of clinical trials in addition to traditional therapy. Most Stage III cancer is managed initially with a debulking cytoreductive surgery with the goal being to reduce the tumor burden as much as possible allowing the patient the most optimal outcome from chemotherapy. The debulking surgery includes removing as many diseased organs as possibly including the uterus, cervix, tubes, and ovaries along with any diseased organs or tissue including pelvic lymph nodes, the peritoneal lining, part of the diaphragm, bowel, spleen, and portions of the liver. Optimal surgical debulking is defined as removing the tumor and leaving residual implants that are &lt;1 cm in greatest diameter. Optimal surgical debulking has been shown to improve survival outcomes. 
     The most common chemotherapeutic agents used to treat epithelial ovarian cancer after initial surgery are cisplatin or carboplatin plus paclitaxel or docetaxel. These two drugs are most commonly given IV three to four weeks apart for a total of six treatments (Matulonis et al.,  Nature Reviews Disease Primers.  2016; 2(1):16061). Approximately 70-80% of women with epithelial ovarian cancer will relapse after initial therapy is completed (Lorusso et al.,  International Journal of Surgical Oncology.  2012; 2012:613980). Once a woman has relapsed with ovarian cancer, she also has a high likelihood of her cancer becoming platinum resistant. A woman who has relapsed with epithelial ovarian cancer is not considered curable. This fact drives second-line therapy research toward non-platinum-based drugs that could extend median survival times beyond the ˜12-months post recurrence generally observed (Davis et al., Gynecologic Oncology. 2014; 133(3):624-631). 
     Often, a woman with recurrent epithelial ovarian cancer will have subsequent surgeries done for further cytoreduction, commonly called interval debulking, or for complications commonly seen with metastatic ovarian cancer including most commonly bowel obstructions. 
     Materials and Methods 
     Folate-hshta-Lys(MPBA)-Gly-Phe-Lys(DOTA) 
     Folate-hshta-G-F-K( 177 Lu-DOTA)-OH has four components: Folate-hshta is the targeting vector (where h=d-histidine, s=d-serine, t=d-threonine, and a=d-alanine), G-F-K (Glycine-Phenylalanine-Lysine) is a L-amino acid linker that is susceptible to enzymatic cleavage at the renal brush border membrane for kidney protection, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) is a radiometal chelator, and  177 Lu is the beta emitter that upon internalization delivers radiation to the nucleus of tumor cells to cause DNA damage. The targeting vector utilizes folate conjugated to hshta, a d-amino-acid-containing cyclic peptide, resulting in high affinity for the folate receptor alpha (FOLR). 
     
       
         
         
             
             
         
       
     
     Indi 7327 Precursor Synthesis 
     Standard solid-phase peptide synthesis (SPPS) coupling conditions using fluorenylmethoxycarbonyl (Fmoc)-protected amino acids and chlorotrityl chloride (CTC) resin were employed to synthesize the linear compound (Table 11, Scheme 1). The click macrocyclization reaction was catalyzed by copper iodide on resin. The C-terminus lysine was selectively deprotected to reveal a free amine, which was used to conjugate the DOTA chelating moiety. Finally, the peptide was cleaved from the resin and deprotected in one step. All components (amino acids, pteroic acid, and DOTA) were obtained from commercial sources. 
     
       
         
           
               
             
               
                 TABLE 11 
               
             
            
               
                   
               
               
                 Solid Phase Peptide Synthesis (SPPS) Steps 
               
            
           
           
               
               
               
            
               
                 Step 
                   
                   
               
               
                 Number 
                 Materials 
                 Coupling Reagents 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 Fmoc-Lys(Dde)-OH (1.00 eq) 
                 DIEA (4.00 eq) 
               
               
                 2 
                 Fmoc-Phe-OH (3.00 eq) 
                 HBTU (2.85 eq) and DIEA (6.00 eq) 
               
               
                 3 
                 Fmoc-Gly-OH (3.00 eq) 
                 HBTU (2.85 eq) and DIEA (6.00 eq) 
               
               
                 4 
                 Fmoc-Lys(N 3 )-OH (2.00 eq) 
                 HATU (1.90 eq) and DIEA (4.00 eq) 
               
               
                 5 
                 Fmoc-D-Ala-OH (3.00 eq) 
                 HBTU (2.85 eq) and DIEA (6.00 eq) 
               
               
                 6 
                 Fmoc-D-Thr(tBu)-OH (3.00) 
                 HBTU (2.85 eq) and DIEA (6.00 eq) 
               
               
                 7 
                 Fmoc-D-His(Trt)-OH (3.00 eq) 
                 HBTU (2.85 eq) and DIEA (6.00 eq) 
               
               
                 8 
                 Fmoc-D-Ser(tBu)-OH (3.00 eq) 
                 HATU (2.85 eq) and DIEA (6.00 eq) 
               
               
                 9 
                 Fmoc-D-His(Trt)-OH (3.00 eq) 
                 HATU (2.85 eq) and DIEA (6.00 eq) 
               
               
                 10 
                 Fmoc-Pra-OH (2.00 eq) 
                 HATU (1.90 eq) and DIEA (4.00 eq) 
               
               
                 11 
                 Fmoc-Glu-OtBu (3.00 eq) 
                 HBTU (2.85 eq) and DIEA (6.00 eq) 
               
               
                 12 
                 PTEROIC ACID (CAS: 119-24- 
                 EDCI (1.5 eq), HOBT (1.5 eq), and DIEA 
               
               
                   
                 4) (1.50 eq) 
                 (4.00 eq), 36 hours 
               
               
                   
                 (dissolve in DMSO 6 mg/mL) 
                   
               
               
                 13 
                 Click Reaction 
                 CuI (0.75 eq), L-Ascorbic Acid (5.0 eq), 
               
               
                   
                   
                 piperidine (6 mL) in NMP (30 mL), 10 hours 
               
               
                 14 
                 De-Dde 
                 2% NH 2 NH 2  in DMF 
               
               
                 15 
                 DOTA(3tBu)(CAS: 137076-54- 
                 HBTU (1.90 eq) and DIEA (4.00 eq) 
               
               
                   
                 1)(2.00 eq) 
               
               
                   
               
               
                 CuI: copper (I) iodide; 
               
               
                 DIEA: N,N-diisopropylethylamine; 
               
               
                 DMF: dimethylformamide; 
               
               
                 EDCI: 1-ethy1-3-(3-dimethylaminopropyl)carbodiimide; 
               
               
                 HATU: 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium; 
               
               
                 HBTU: 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, Hexafluorophosphate Benzotriazole Tetramethyl Uronium; 
               
               
                 HOBT: 1-Hydroxybenzotriazole hydrate; 
               
               
                 NMP: N-methylpyrrolidone; 
               
               
                 SPPS: solid phase peptide synthesis. 
               
            
           
         
       
     
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     Indi 7327 Precursor Purification and Analytical Testing 
     The crude material was purified using reverse phase high-performance liquid chromatography (HPLC) purification. HPLC analysis of the purified material reveals high isolated purity of Indi 7327. This method estimates purity to be 97%. 
     Purity and identity were determined by analytical HPLC and liquid chromatography coupled with mass spectrometry (LC/MS) analysis. LC/MS analysis of the purified fractions exhibits polycationic species consistent with Indi 7327. The most abundant ion is 648.6, corresponding to a [M+3H] 3+  molecular species. Other identifiable charged species include 972.4, [M+2H] 2+  and 486.7, [M+4H] 4+ . 
       77 Lu Labeling Procedures 
       177 Lu chloride (in 0.05 M HCl solution) was ordered from the National Isotope Development Center (NIDC). The material is produced weekly from the University of Missouri Research Reactor Center (MURR). Buffered gentisic acid (260-330 μL) is added to the desired quantity of  177 Lu chloride (typically 814-1665 MBq of activity which corresponds to 10-20 μL of the solution as received) in a metal-free Eppendorf. The gentisic acid solution is prepared by diluting gentisic acid to a concentration of 10 mg/mL in 0.4 M NaOAc (pH=4.5). Gentisic acid is utilized to minimize radiolysis of the peptide. A volume of peptide solution is added to the reaction corresponding to the desired specific activity (typically 60-120 μL). After all additions are complete, a final pH measurement is taken. The measured reaction pH has consistently fallen within the acceptable pH of 4.5-4.8 and has never required adjustment. The reaction is aged for 15 minutes at 95° C. Labeling efficiency and yield of the  177 Lu chelation reaction are determined by reverse phase HPLC coupled to an in-line radio detector. The  177 Lu labeling of Indi 7327 occurs in near quantitative yield. No un-chelated  177 Lu is present. 
     Results Nonclinical studies were first conducted with Folate-hshta-PEG 10 -PEG 10 -K(Biotin), where Folate-hshta is the targeting heterobiligand, PEG 10  is a deca(ethylene glycol) linker, and K(Biotin) is the detection label. This peptide-folate heterobiligand showed low picomolar affinity (EC 50 =65 μM) in enzyme-linked immunosorbent assay (ELISA), enhanced binding to human FOLR protein compared to folate alone, and selective binding in human OVCAR3 (FOLR+) epithelial ovarian cancer cells ( FIG. 6 ). Tested alone, the macrocyclic peptide ligand hshta demonstrated low micromolar affinity for recombinant human FOLR protein in ELISA. Conjugation of hshta to folate ligand yielded a highly potent heterobiligand with ˜30-fold greater affinity than folate itself. This heterobiligand was resilient to displacement by exogenous folate in a competitive ELISA. Cross reactivity with the murine folate receptor was also observed. Dose-dependent binding to human OVCAR3 (FOLR+) ovarian cancer cells and low non-specific binding to PC3 (FOLR−) cells were demonstrated by flow cytometry. Selective internalization into OVCAR3 (FOLR+) cells and limited uptake into PC3 (FOLR−) cells were demonstrated by live cell imaging of DyLight650-labeled peptide-folate heterobiligand on a confocal microscope. In the merged image, purple puncta were observed indicating co-localization of internalized heterobiligand with early endosomes (Rab5a+ vesicles) in OVCAR3 cells. An acid wash (pH 3.0) assay (Kamen et al.,  Journal of Biological Chemistry.  1988; 263(27):13602-13609) analyzed by flow cytometry provided further evidence that a high percentage of DyLight650-labeled peptide-folate heterobiligand was internalized in the OVCAR3 cell line. 
     To reduce treatment-related kidney exposure, the heterobiligand was chemically modified with a 3-amino acid (Gly-Phe-Lys) linker that is susceptible to cleavage by neprilysin, an abundant endopeptidase on the renal brush border membrane. In vitro cleavage studies of Indi 7327 (Folate-hshta-G-F-K(DOTA)-OH) confirmed that the G-F-K sequence is selectively recognized by neprilysin and cleaved at the amide bond between Gly and Phe, thus releasing the DOTA chelator fragment containing Lys. In vitro bioanalysis studies showed that Indi 7327 is stable in both human and mouse plasma (T 1/2 &gt;6900 min), 40.87% bound to mouse plasma proteins, and 40.29% bound to human plasma proteins. In vivo positron emission tomography (PET) imaging of Indi  68 Ga-7327 showed a significant reduction in kidney uptake with rapid drainage to the bladder in human OVCAR3 (FOLR+) tumor implanted female NOD scid gamma (NSG) mice. This is consistent with the mechanism that cleavage in the renal brush border membrane liberates the intact radionuclide-chelator complex, which is then excreted in the urine. Similar tumor uptake of Indi  68 Ga-7327 was observed when compared to constructs that lack the renal brush border cleavage site. 
     A series of in vivo studies examining the tolerated doses and anti-tumor activity of Indi  177 Lu-7327 were conducted using IV administration. Healthy, non-tumor bearing female NSG mice tolerated three treatments of Indi  177 Lu-7327 at radioactive doses up to 111 MBq. In OVCAR3 tumor implanted female NSG mice, two treatments of Indi  177 Lu-7327 at a radioactive dose of 29.6 MBq resulted in tumor stasis. Tumor stasis was also observed in OVCAR3 tumor implanted female NSG mice that received two treatments of Indi  177 Lu-7327 at a radioactive dose of 74 MBq. Tumor regression was then observed in OVCAR3 tumor implanted female NSG mice that received two treatments of Indi  177 Lu-7327 at 29.6 MBq followed by a third treatment at 74 MBq. Kidney injury biomarker levels in mouse plasma collected at study endpoints were determined to be largely in the normal range. Indi  177 Lu-7327 treated mice demonstrated normal body weights (+/−10%) and behavior (no observable change). 
     Biodistribution was initially evaluated by PET imaging, as the time frame for a significant portion of clearance is roughly similar to the time frame of PET imaging experiments. Indi  68 Ga-7327 was injected via the tail vein into OVCAR3 (FOLR+) tumor implanted female NSG mice once the tumor size reached 200 mm 3 , and microPET/CT scans were acquired at 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, and 4 h post-injection. PET images were corrected for CT-based photon attenuation, detector normalization and radioisotope decay and converted to units of percent injected dose per cc (% ID/cc). The imaging study revealed that the biodistribution of Indi  68 Ga-7327 is based in the tumor and clearance organs (kidney, bladder). Rapid accumulation of Indi  68 Ga-7327 was observed in the tumor, with 1.55% ID/cc at 4 h post-injection. Radiation that has found the tumor folate receptor is internalized and no longer subject to clearance mechanisms. Indi  68 Ga-7327 clears via the kidneys to the bladder for excretion in the urine. 
     Therapy response based on Indi  177 Lu-7327 has been demonstrated in a number of tumor stasis or regression studies using IV administration. Female NSG mice were implanted subcutaneously with human OVCAR3 (FOLR+) ovarian cancer cells. Once tumors reached volumes of 200 mm 3 , the animals were treated with Indi  177 Lu-7327. One treatment arm of 74 MBq and a control group of 0 MBq were studied ( FIGS. 41 and 38A ). Following two doses of Indi  177 Lu-7327 at 74 MBq, there was a decrease in tumor volume. The second dose was administered 21 days following the first dose. Statistically significant differential between treated and control was reached 24 days post-injection (three days following the second dose) for the 74 MBq arm. P values (* for p≤0.05, ** for p≤0.01) were determined by ordinary one-way ANOVA with Tukey&#39;s multiple comparisons test. Normal body weights and behavior (no observable change) indicated that the treatments were well tolerated. 
     Another therapy response study in OVCAR3 tumor-bearing NSG mice evaluated three treatment arms of 9.25, 14.8, and 29.6 MBq and a control group of 0 MBq ( FIGS. 36 and 37 ). Following two doses of Indi  177 Lu-7327, there was a dose dependent decrease in tumor volume. The second dose was administered 21 days following the first dose. Animals receiving the 29.6 MBq dose of Indi  177 Lu-7327 exhibited modest (5-10%) loss of body weight that stabilized and then recovered 10 days following the second dose. Normal body weights and behavior (no observable change) indicated that the treatments were well tolerated. 
     Statistically significant differential between treated and control was reached 12 days post-injection for the 29.6 MBq arm. P values (* for p≤0.05) were determined by ordinary one-way ANOVA with Tukey&#39;s multiple comparisons test. At 45 days post-injection, the animals bearing the smallest tumors were re-randomized and given elevated doses of Indi  177 Lu-7327. Three treatment arms of 46.3, 74, and 148 MBq were studied ( FIG. 38A ). Tumor volumes for the 74 and 148 MBq arms decreased by 50%, on average, following this single elevated therapeutic dose. On day 24, the mice received an additional treatment that maintained the therapy response. These data show that Indi  177 Lu-7327 can stabilize and, at augmented doses, reverse the growth of FOLR-expressing tumors. 
     We have been using mouse body weight, mouse behavior, and kidney injury biomarkers (at end of study) to assess toxicology. Weight loss provides information on longer time horizons for doses at or below the maximum tolerated dose (MTD). In order to establish a MTD, dose range finding studies were undertaken testing increasing amounts of Indi  177 Lu-7327 in healthy, non-tumor bearing female NSG mice using IV administration ( FIGS. 34 and 35 ). Mouse body weight and behavior were monitored. Multiple dose administration of 14.8, 22.2, 29.6, and 37 MBq were well tolerated. The second and third doses were administered 21 days following the previous dose. At the high doses of 111 and 185 MBq, animals lost substantial body weight following the first dose, which worsened following the second and third doses. These studies established an MTD for Indi  177 Lu-7327 that lies between 37 and 111 MBq. The high tolerability observed in the 0 MBq group (treated with Indi 7327) indicates that the radionuclide drives the toxicity profile and not the peptide alone. 
     To reduce the radioactive dose to the kidney, Indi  177 Lu-7327 was constructed to incorporate a GFK linker that is susceptible to cleavage by enzymes found predominately in the kidney&#39;s brush border. These enzymes cleave off the radionuclide-chelator complex, which is quickly eliminated by the kidney for excretion in the urine. The zinc metalloprotease neprilysin (NEP) is expressed with high abundance in the kidney. Preclinical studies by Suzuki et al. showed that the tripeptide GFK sequence is recognized as a substrate and cleaved by NEP on the renal brush border membrane (Suzuk et al.,  Journal of Medicinal Chemistry.  2018; 61(12):5257-5268). To confirm NEP-mediated cleavage of the GFK linker in Indi 7327, the peptide was incubated with recombinant human NEP at 37° C. for 1 h and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the evolution of cleaved adducts ( FIG. 42 ). Titration of NEP (10.0 to 0 nM) resulted in partial to complete cleavage of Indi 7327. The expected cleaved adduct (mass: 1283.16 m/z) appears in the samples containing 10, 1.0, and 0.1 nM NEP. At 10 nM NEP, no intact Indi 7327 remains indicating complete consumption of the peptide. At 1.0 and 0.1 nM NEP, partial cleavage was observed. Below 0.1 nM NEP, no cleavage was observed. In addition to confirming the selectivity of NEP for the GFK linker, it is also important to note that the remaining portions of the Indi 7327 were impervious to the enzyme. 
     In vitro bioanalysis studies showed that Indi 7327 is stable in plasma (T 1/2 2&gt;6900 min), 40.87% bound to mouse plasma proteins, and 40.29% bound to human plasma proteins (Table 2). 
     Kidney injury biomarkers were evaluated from mouse plasma collected at the end of the therapy response studies of Indi  177 Lu-7327. The blood urea nitrogen (BUN) and creatinine levels were evaluated to gain information on renal function. After Indi  177 Lu-7327 therapy, mouse plasma from the treatment arms of 9.25, 14.8, and 29.6 MBq and the control group of 0 MBq showed normal BUN (Table 9) and creatinine levels (Table 10). After augmenting the dose of Indi  177 Lu-7327, BUN and creatinine levels were normal in the treatment arms of 46.3 and 74 MBq and elevated at the highest dosing (148 MBq) (Tables 9 and 10). 
     It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. 
     Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, is this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials. These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed. 
     It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a ligand” includes a plurality of such ligands, reference to “the ligand” is a reference to one or more ligands and equivalents thereof known to those skilled in the art, and so forth. 
     Throughout the description and claims of this specification, the word “comprise” and variations of the word, such as “comprising” and “comprises,” means “including but not limited to,” and is not intended to exclude, for example, other additives, components, integers or steps. 
     “Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present. 
     Unless the context clearly indicates otherwise, use of the word “can” indicates an option or capability of the object or condition referred to. Generally, use of “can” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of the word “may” indicates an option or capability of the object or condition referred to. Generally, use of “may” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of “may” herein does not refer to an unknown or doubtful feature of an object or condition. 
     Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. It should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. Finally, it should be understood that all ranges refer both to the recited range as a range and as a collection of individual numbers from and including the first endpoint to and including the second endpoint. In the latter case, it should be understood that any of the individual numbers can be selected as one form of the quantity, value, or feature to which the range refers. In this way, a range describes a set of numbers or values from and including the first endpoint to and including the second endpoint from which a single member of the set (i.e. a single number) can be selected as the quantity, value, or feature to which the range refers. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. 
     Although the description of materials, compositions, components, steps, techniques, etc. can include numerous options and alternatives, this should not be construed as, and is not an admission that, such options and alternatives are equivalent to each other or, in particular, are obvious alternatives. Thus, for example, a list of different ligands does not indicate that the listed ligands are obvious one to the other, nor is it an admission of equivalence or obviousness. 
     Every compound disclosed herein is intended to be and should be considered to be specifically disclosed herein. Further, every subgroup that can be identified within this disclosure is intended to be and should be considered to be specifically disclosed herein. As a result, it is specifically contemplated that any compound, or subgroup of compounds can be either specifically included for or excluded from use or included in or excluded from a list of compounds. 
     Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.