METHODS AND COMPOSITIONS FOR IMPROVED LABELING OF TARGETING PEPTIDES

Inventors: William J. McBride and David M. Goldenberg

ASSIGNEE: IMMUNOMEDICS, INC.

Related Applications

[001] This application claims the benefit under 35 U.S.C.119(e) of provisional U.S. Patent Appl. No.62/078,657, filed 11/12/14. This application is a continuation-in-part of U.S. Patent Appl. No.14/755,712, filed 6/30/15. The text of each priority application is incorporated herein by reference in its entirety.

Sequence Listing

[002] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 9, 2015 is named IMM351WO1_SL and is 14,481 bytes in size.

Field

[003] The present invention concerns novel compounds comprising octreotide, octreotate, or another somatostatin analog, of use for targeted delivery to cells or tissues expressing somatostatin receptors. The compounds may be used to deliver diagnostic agents for detection, diagnosis or imaging, or therapeutic agents for treatment of diseased cells or tissues that express somatostatin receptor, such as neuroendocrine tumors. The compounds further comprise one or more chelator moieties, which may be used to attach diagnostic or therapeutic radionuclides, paramagnetic ions or other diagnostic or therapeutic agents. In more preferred embodiments, the compounds may be labeled with metal- ¹⁸ F or metal- ¹⁹ F complexes that are of use, for example, in PET, MRI or SPECT in vivo imaging. Preferably, the ¹⁸ F or ¹⁹ F is attached as a complex with aluminum or another Group IIIA metal. The chelating moiety may be attached to the targeting peptide either before or after binding to the metal- ¹⁸ F or metal- ¹⁹ F complex. Although labeling may occur at an elevated temperature, such as 70˚C, 80˚C, 90˚C, 95˚C, 100˚C, 105˚C, 110˚C, or any temperature in between, preferably labeling of heat sensitive molecules may occur at a lower temperature, such as room temperature. Although fluorine isotopes are preferred, in alternative embodiments other therapeutic or diagnostic metals and/or isotopes may be bound to the chelating moieties discussed below, including but not limited to isotopes of aluminum, gallium, indium, copper and yttrium. Using the techniques described herein, labeled molecules of high specific activity may be prepared in 30 minutes or less and with minimal or no need for purification of labeled molecules. Labeling may occur in a saline medium suitable for direct use in vivo. Alternatively, an organic solvent may be added to improve the labeling efficiency. The labeled targeting peptides are stable under physiological conditions, although for certain purposes, such as kit formulations, a stabilizing agent such as ascorbic acid, trehalose, sorbitol or mannitol may be added.

Background

[004] Octreotate is an octapeptide that mimics somatostatin and binds with high affinity to somatostatin receptors. Octreotide is a structurally similar peptide wherein the C-terminal threonine moiety has been reduced to the corresponding amino alcohol. Both have been extensively used for detection, imaging or treatment of diseased tissues that express high levels of somatostatin receptor, particularly neuroendocrine tumors or other somatostatin- receptor positive endocrine tumors (e.g., Bodei et al., 2014, Thorac Surg Clin 24:333-49; van Essen et al., 2009, Nat Rev Endocrinol 5:382-93; De Jong et al., 2002, Semn Nucl Med 32:133-40). Therapeutic or diagnostic agents that have been attached to octreotide or octreotate for delivery to targeted tissues have included ⁹⁰ Y, ¹⁷⁷ Lu, ¹¹¹ In, ⁶⁸ Ga, ¹²³ I, ¹¹ C, ²¹³ Bi, and ²¹¹ At, (see, Bodei et al., 2014, Thorac Surg Clin 24:333-49; van Essen et al., 2009, Nat Rev Endocrinol 5:382-93; Kwekkeboom et al., 2010, Endocr Relat Cancer 17:R53-73; Chin et al., 2013, Amino Acids 45:1097-108; Dadachova, 2010, Semin Nucl Med 40:204-8).

[005] Although symptomatic improvement has been reported with such conjugated somatostatin analogs, large variation in antitumor effects has been reported in different studies with, for example, an objective response achieved in 9% to 33% of patients treated with ⁹⁰ Y-octreotide (van Essen et al., 2009, Nat Rev Endocrinol 5:382-93). A need exists for more effective somatostatin analogs that have better labeling characteristics, improved stability and/or increased therapeutic or diagnostic efficiency with better imaging.

Summary

[006] In various embodiments, the present invention concerns compositions and methods relating to labeled octreotide, octreotate or other somatostatin analogs, of use for targeted delivery to cells or tissues expressing somatostatin receptors (sst). The major subtype of sst is sst ₂ , and therapeutic or diagnostic uses of octreotide or octreotate have been primarily directed to sst ⁺

2 cancers (e.g., Lustig et al., 2003, J Clin Endocrinol 88:2586-92; Uhl et al., 1999, Digestion 60(Suppl 2):23-31). Within the scope of the present invention for therapy and/or diagnosis, labeled octreotide or octreotate may be applied to tumors including, but not limited to, sst ⁺

2 neuroendocrine tumors (NET), gastroenteropancreatic NET, meningiomas, well-differentiated brain tumors, malignant lymphomas, renal cell carcinoma, breast carcinoma and lung carcinoma. Any other cancer that is sst ⁺

2 may also be treated or diagnosed.

[007] Exemplary radionuclides or stable isotopes that may be attached to the subject peptides for therapy and/or diagnosis include, but are not limited to, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, 1 ⁸ F, ¹⁹ F, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² Ga, ¹¹¹ In, ¹⁷⁷ Lu, ⁴⁴ Sc, ⁴⁷ Sc, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ⁴⁵ Ti and ⁸⁹ Zr.

[008] In preferred embodiments, the radionuclides or other diagnostic or therapeutic agents may be attached to octreotide, octreotate or other somatostatin analogs using a chelating moiety, such as NOTA, NETA, DOTA, DTPA or derivatives thereof. Exemplary chelating moieties of particular use are shown below and in the Examples section. The person of ordinary skill will understand that the chelating moieties of use are not limited to the specific embodiments disclosed herein, but rather may include other chelating moieties known in the art to bind therapeutic and/or diagnostic agents.

[009] In particularly preferred embodiments, targeting peptides of use may include, but are not limited to the NOTA-octreotate derivatives shown below. The underlined portion of the peptide may be cyclized by disulfide bond formation between the two cysteine residues.

[0010] Although the Examples below show use of radionuclide-labeled chelator-peptide complexes, the invention is not limited and other diagnostic or therapeutic agents known to bind to chelating moieties may be utilized within the scope of the claimed methods and compositions.

[0011] In preferred embodiments, the chelating moieties are used to attach metal- ¹⁸ F or metal- ¹⁹ F complexes to octreotide, octreotate or other somatostatin analogs. In an exemplary approach, the ¹⁸ F is bound to a metal and the ¹⁸ F-metal complex is attached to a chelator on the peptide. As described below, the metals of group IIIA (aluminum, gallium, indium, and thallium) are suitable for ¹⁸ F or ¹⁹ F binding, although aluminum is preferred. Lutetium may also be of use. The chelating moiety be selected from NOTA, NETA, DOTA, DTPA and other chelating groups discussed in more detail below. Alternatively, one can attach the metal to a molecule first and then add the ¹⁸ F to bind to the metal. In still other embodiments, one may attach an ¹⁸ F-metal to a chelating moiety first and then attach the labeled chelating moiety to the peptide. In this way, the ¹⁸ F-metal may be attached to a chelating moiety at a higher temperature, such as between 90° to 110° C, more preferably between 95° to 105° C, and the ¹⁸ F-labeled chelating moiety may be attached to the peptide at a lower temperature, such as at room temperature. In preferred embodiments, the labeling method uses a biofunctional chelator that forms a physiologically stable complex with metal- ¹⁸ F, which contains reactive groups that can bind to peptides at, e.g., room temperature. More preferably, labeling can be accomplished in 10 to 15 minutes in aqueous medium, with a total synthesis time of about 30 minutes.

[0012] Certain alternative embodiments involve the use of“click” chemistry for attachment of ¹⁸ F-labeled moieties to targeting molecules. Preferably, the click chemistry involves the reaction of a targeting peptide comprising a functional group such as an alkyne, nitrone or an azide group, with a ¹⁸ F-labeled moiety comprising the corresponding reactive moiety such as an azide, alkyne or nitrone. Where the targeting molecule comprises an alkyne, the chelating moiety or carrier will comprise an azide, a nitrone or similar reactive moiety. [0013] In other alternative embodiments, a prosthetic group, such as a NOTA-maleimide moiety, may be labeled with ¹⁸ F-metal and then conjugated to a targeting molecule, for example by a maleimide-sulfhydryl reaction. Exemplary NOTA-maleimide moieties include, but are not limited to, NOTA-MPAEM, NOTA-PM, NOTA-PAEM, NOTA-BAEM, NOTA- BM, NOTA-MPM, and NOTA-MBEM.

[0014] The claimed compounds may be used in combination with other standard therapeutic modalities, such as surgery, chemotherapy, radiation therapy, immunotherapy and the like. Labeled somatostatin analogs may also be utilized in adjuvant or neoadjuvant settings. The therapeutic efficacy of the labeled somatostatin analogs may be enhanced by combination therapy with other therapeutic agents, administered either before, concurrently with or after the labeled somatostatin analogs. Agents of use in combination therapy may include, but are not limited to, canertinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, leflunomide, nilotinib, pazopanib, semaxinib, sorafenib, sunitinib, vatalanib, temsirolimus, rapamycin, ridaforolimus everolimus, ibrutinib, 5-fluorouracil, capecitabine, temozolomide,

lambrolizumab, pidilizumab, ipilimumab and tremelimumab. Brief Description of the Drawings [0015] The following Figures are included to illustrate particular embodiments of the invention and are not meant to be limiting as to the scope of the claimed subject matter.

[0016] FIG.1. Biodistribution of ¹⁸ F-labeled agents in tumor-bearing nude mice by microPET imaging. Coronal slices of 3 nude mice bearing a small, subcutaneous LS174T tumor on each left flank after being injected with either (A) ¹⁸ F-FDG, (B) Al ¹⁸ F(IMP449) pretargeted with the anti-CEA x anti-HSG bsMAb, (C) Al ¹⁸ F(IMP449) alone (not pretargeted with the bsMAb). Biodistribution data expressed as percent-injected dose per gram (% ID/g) are given for the tissues removed from the animals at the conclusion of the imaging session. Abbreviations: B, bone marrow; H, heart; K, kidney; T, tumor.

[0017] FIG.2. Dynamic imaging study of pretargeted Al ¹⁸ F(IMP449) given to a nude mouse bearing a 35-mg LS174T human colorectal cancer xenograft in the upper flank. The top 3 panels show coronal, sagittal, and transverse sections, respectively, taken of a region of the body centering on the tumor’s peripheral location at 6 different 5-min intervals over the 120-min imaging session. The first image on the left in each sectional view shows the positioning of the tumor at the intersection of the crosshairs, which is highlighted by arrows. The animal was partially tilted to its right side during the imaging session. The bottom 2 panels show additional coronal and sagittal sections that focus on a more anterior plane in the coronal section to highlight distribution in the liver and intestines, while the sagittal view crosses more centrally in the body. Abbreviations: Cor, coronal; FA, forearms; H, heart; K, kidney; Lv, liver; Sag, sagittal; Tr, transverse; UB, urinary bladder.

[0018] FIG.3. In vivo tissue distribution with Al ¹⁸ F(IMP466) bombesin analogue.

[0019] FIG.4. Comparison of biodistribution of Al ¹⁸ F(IMP466) and ⁶⁸ Ga(IMP466) at 2 hours post-injection in AR42J tumor-bearing mice (n=5). As a control, mice in separate groups (n=5) received an excess of unlabeled octreotide to demonstrate receptor specificity.

[0020] FIG.5. Coronal slices of PET/CT scan of Al ¹⁸ F(IMP466) and ⁶⁸ Ga(IMP466) at 2 hours post-injection in mice with an s.c. AR42J tumor in the neck. Accumulation in tumor and kidneys is clearly visualized.

[0021] FIG.6. Biodistribution of 6.0 nmol ¹²⁵ I-TF2 (0.37 MBq) and 0.25 nmol

6 ⁸ Ga(IMP288) (5 MBq), 1 hour after i.v. injection of ⁶⁸ Ga(IMP288) in BALB/c nude mice with a subcutaneous LS174T and SK-RC52 tumor. Values are given as means ± standard deviation (n=5).

[0022] FIG.7. Biodistribution of 5 MBq FDG and of 5 MBq ⁶⁸ Ga(IMP288) (0.25 nmol) 1 hour after i.v. injection following pretargeting with 6.0 nmol TF2. Values are given as means ± standard deviation (n=5).

[0023] FIG.8. PET/CT images of a BALB/c nude mouse with a subcutaneous LS174T tumor (0.1 g) on the right hind leg (light arrow) and a inflammation in the left thigh muscle (dark arrow), that received 5 MBq ¹⁸ F-FDG, and one day later 6.0 nmol TF2 and 5 MBq ⁶⁸ Ga(IMP288) (0.25 nmol) with a 16 hour interval. The animal was imaged one hour after the ¹⁸ F-FDG and ⁶⁸ Ga(IMP288) injection. The panel shows the 3D volume rendering (A), transverse sections of the tumor region (B) of the FDG-PET scan, and the 3D volume rendering (C), transverse sections of the tumor region (D) of the pretargeted immunoPET scan.

[0024] FIG.9. Biodistribution of 0.25 nmol Al ¹⁸ F(IMP449) (5 MBq) 1 hour after i.v.

injection, following 6.0 nmol TF2 administered 16 hours earlier. Biodistribution of

Al ¹⁸ F(IMP449) without pretargeting, or biodistribution of [Al ¹⁸ F]. Values are given as means ± standard deviation.

[0025] FIG.10. Static PET/CT imaging study of a BALB/c nude mouse with a

subcutaneous LS174T tumor (0.1 g) on the right side (arrow), that received 6.0 nmol TF2 and 0.25 nmol Al ¹⁸ F(IMP449) (5 MBq) intravenously with a 16 hour interval. The animal was imaged one hour after injection of Al ¹⁸ F(IMP449) . The panel shows the 3D volume rendering (A) posterior view, and cross sections at the tumor region, (B) coronal, (C) sagittal.

[0026] FIG.11. Structure of IMP479 (SEQ ID NO:24).

[0027] FIG.12. Structure of IMP485 (SEQ ID NO:25).

[0028] FIG.13A. Structure of IMP487 (SEQ ID NO:26).

[0029] FIG.13B. Structure of IMP490 (SEQ ID NO:22).

[0030] FIG.13C. Structure of IMP493 (SEQ ID NO:23).

[0031] FIG.13D. Structure of IMP495 (SEQ ID NO: 27).

[0032] FIG.13E. Structure of IMP496 (SEQ ID NO: 28).

[0033] FIG.13F. Structure of IMP500.

[0034] FIG.14. Synthesis of bis-t-butyl-NOTA-MPAA.

[0035] FIG.15. Synthesis of maleimide conjugate of NOTA.

[0036] FIG.16. Chemical structure of exemplary NOTA-based bifunctional chelators.

[0037] FIG.17. Chemical structures of NOTA-BM derived bifunctional chelators.

[0038] FIG.18. Further exemplary structures of NOTA-based bifunctional chelators: (A) NOTA-HA, (B) NOTA-MPN, (C) NOTA-EPN, (D) NOTA-MBA, (E) NOTA-EPA, (F) NOTA-MPAA, (G) NOTA-BAEM, (H) NOTA-MPAEM, (I) NOTA-BM, (J) NOTA- MBEM, (K) NOTA moiety with maleimide reactive group, (L) alternative NOTA moiety with maleimide reactive group, (M) NOTA-BA, (N) NOTA-EA, (O) NOTA-MPH, (P)

NOTA-butyne, (Q) NOTA-MPAPEG ₃ N ₃ , (R) NOTA moiety with carboxyl reactive group, (S) NOTA moiety with nitrophenyl reactive group, (T) NOTA moiety with carboxyl and nitrophenyl reactive groups, (U) another NOTA moiety with carboxyl reactive group, (V) another NOTA moiety with carboxyl reactive group, (W) another NOTA moiety with carboxyl reactive group, (X) another NOTA moiety with carboxyl reactive group, (Y) another NOTA moiety with carboxyl reactive group, (Z) another NOTA moiety with carboxyl reactive group, (AA) another NOTA moiety with carboxyl reactive group, (BB) another NOTA moiety with carboxyl reactive group, (CC) another NOTA moiety with carboxyl reactive group.

[0039] FIG.19(A) and 19(B). Radiochromatograms of the ¹⁸ F-labeled functionalized TACN ligands.

[0040] FIG.20(A) and 20(B). Radiochromatograms of ¹⁸ F-hMN14-Fab’, its stability in human serum and immunoreactivity with CEA.

[0041] FIG.21. Schematic diagram of automated synthesis module for ¹⁸ F-labeling via [Al ¹⁸ F]-chelation. [0042] FIG.22. NOTA-propyl amine derived bifunctional chelating moieties.

[0043] FIG.23A. Structure of IMP 508 (SEQ ID NO: 29).

[0044] FIG.23B. Structure of IMP517 (SEQ ID NO: 30).

[0045] FIG.23C. Structure of NOTA-2-nitroimidazole.

[0046] FIG.23D. Structure of NOTA-DUPA-Peptide.

[0047] FIG.24. Labeling efficiency as a function of temperature. DETAILED DESCRIPTION

[0048] The following definitions are provided to facilitate understanding of the disclosure herein. Terms that are not explicitly defined are used according to their plain and ordinary meaning.

[0049] As used herein, the term“somatostatin analog(s)” refers to octreotide, octreatate, or other derivatives or analogs of somatostatin.

[0050] As used herein,“a” or“an” may mean one or more than one of an item.

[0051] As used herein, the terms“and” and“or” may be used to mean either the conjunctive or disjunctive. That is, both terms should be understood as equivalent to“and/or” unless otherwise stated.

[0052] As used herein,“about” means within plus or minus ten percent of a number. For example,“about 100” would refer to any number between 90 and 110.

[0053] As used herein, a“peptide” refers to any sequence of naturally occurring or non- naturally occurring amino acids of between 2 and 100 amino acid residues in length, more preferably between 2 and 10, more preferably between 4 and 8 amino acids in length. An “amino acid” may be an L-amino acid, a D-amino acid, an amino acid analogue, an amino acid derivative or an amino acid mimetic.

[0054] As used herein, a“radiolysis protection agent” refers to any molecule, compound or composition that may be added to a radionuclide-labeled complex or molecule to decrease the rate of breakdown of the radiolabeled complex or molecule by radiolysis. Any known radiolysis protection agent, including but not limited to ascorbic acid, may be used.

Peptides

[0055] The targeting peptides used are conveniently synthesized on an automated peptide synthesizer using a solid-phase support and standard techniques of repetitive orthogonal deprotection and coupling. Free amino groups in the peptide, that are to be used later for conjugation of chelating moieties or other agents, are advantageously blocked with standard protecting groups such as a Boc group, while N-terminal residues may be acetylated to increase serum stability. Such protecting groups are well known to the skilled artisan. See Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, N.Y.). Peptides are advantageously cleaved from the resins to generate the corresponding C-terminal amides, in order to inhibit in vivo carboxypeptidase activity. Exemplary structures of use and methods of peptide synthesis are disclosed in the Examples below. Chelating moieties may be conjugated to peptides using bifunctional chelating moieties as discussed below.

Amino Acid Substitutions

[0056] Certain embodiments may involve production and use of targeting peptides with one or more substituted amino acid residues. The skilled artisan will be aware that amino acid substitutions typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions). The properties of the various amino acids and effect of amino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.

[0057] For example, the hydropathic index of amino acids may be considered (Kyte & Doolittle, 1982, J. Mol. Biol., 157:105-132). The relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (- 0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). In making conservative substitutions, the use of amino acids whose hydropathic indices are within ± 2 is preferred, within ± 1 are more preferred, and within ± 0.5 are even more preferred.

[0058] Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Pat. No.4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0); glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). Replacement of amino acids with others of similar hydrophilicity is preferred.

[0059] Other considerations include the size of the amino acid side chain. For example, it would generally not be preferred to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine. The effect of various amino acid residues on protein secondary structure is also a

consideration. Through empirical study, the effect of different amino acid residues on the tendency of protein domains to adopt an alpha-helical, beta-sheet or reverse turn secondary structure has been determined and is known in the art (see, e.g., Chou & Fasman, 1974, Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys. J., 26:367-384).

[0060] Based on such considerations and extensive empirical study, tables of conservative amino acid substitutions have been constructed and are known in the art. For example:

arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R) gln, asn, lys; Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys (C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser; Val (V) ile, leu, met, phe, ala.

[0061] Some embodiments may involve substitution of one or more D-amino acids for the corresponding L-amino acids. Peptides comprising D-amino acid residues are more resistant to peptidase activity than L-amino acid comprising peptides. Such substitutions may be readily performed using standard amino acid synthesizers, as discussed in the Examples below.

[0062] In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.

[0063] Methods of substituting any amino acid for any other amino acid in an encoded protein sequence are well known and a matter of routine experimentation for the skilled artisan, for example by the technique of site-directed mutagenesis or by synthesis and assembly of oligonucleotides encoding an amino acid substitution and splicing into an expression vector construct.

Chelating Moieties

[0064] In some embodiments, Al ¹⁸ F or another radiolabel may bind to a hydrophilic chelating moiety, which can bind metal ions and also help to ensure rapid in vivo clearance. Chelators may be selected for their particular metal-binding properties, and substitution by known chemical cross-linking techniques or by use of chelators with side-chain reactive groups (such as bifunctional chelating moieties) may be performed with only routine experimentation.

[0065] Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as ¹²⁵ I, ¹³¹ I, ¹²³ I, ¹²⁴ I, ⁶² Cu, ⁶⁴ Cu, ¹⁸ F, ¹¹¹ In, ⁶⁷ Ga, ⁶⁸ Ga, ^99m Tc, ^94m Tc, ¹¹ C, ¹³ N, ¹⁵ O, ⁷⁶ Br , for radioimaging. The same chelates, when complexed with non- radioactive metals, such as manganese, iron and gadolinium are useful for MRI. Macrocyclic chelates such as NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), DOTA, TETA (p- bromoacetamido-benzyl-tetraethylaminetetraacetic acid) and NETA are of use with a variety of diagnostic or therapeutic metals and radiometals, most particularly with radionuclides of gallium, yttrium and copper. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as ²²³ Ra for RAIT are encompassed. The person of ordinary skill will understand that, by varying the groups attached to a macrocyclic ring structure such as NOTA, the binding characteristics and affinity for different metals and/or radionuclides may change and such derivatives or analogs of, e.g. NOTA, may therefore be designed to bind any of the metals, radionuclides and/or paramagnetic species discussed herein.

[0066] DTPA and DOTA-type chelators, where the ligand includes hard base chelating functions such as carboxylate or amine groups, are most effective for chelating hard acid cations, especially Group IIa and Group IIIa metal cations. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelators such as macrocyclic polyethers are of interest for stably binding nuclides. Porphyrin chelators may be used with numerous metal complexes. More than one type of chelator may be conjugated to a peptide to bind multiple metal ions. Chelators such as those disclosed in U.S. Pat. No.5,753,206, especially thiosemicarbazonylglyoxylcysteine (Tscg-Cys) and thiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelators are advantageously used to bind soft acid cations of Tc, Re, Bi and other transition metals, lanthanides and actinides that are tightly bound to soft base ligands. One example of such a peptide is Ac-Lys(DTPA)-Tyr- Lys(DTPA)-Lys(Tscg-Cys)-NH ₂ (core peptide disclosed as SEQ ID NO:5). Other hard acid chelators such as DOTA, TETA and the like can be substituted for the DTPA and/or Tscg- Cys groups. [0067] Another useful chelator may comprise a NOTA-type moiety, for example as disclosed in Chong et al. (J. Med. Chem., 2008, 51:118-25). Chong et al. disclose the production and use of a bifunctional C-NETA ligand, based upon the NOTA structure, that when complexed with ¹⁷⁷ Lu or ^205/206 Bi showed stability in serum for up to 14 days. The chelators are not limiting and these and other examples of chelators that are known in the art and/or described in the following Examples may be used in the practice of the invention.

Click Chemistry

[0068] In various embodiments, targeting peptide conjugates may be prepared using click chemistry technology. The click chemistry approach was originally conceived as a method to rapidly generate complex substances by joining small subunits together in a modular fashion. (See, e.g., Kolb et al., 2004, Angew Chem Int Ed 40:3004-31; Evans, 2007, Aust J Chem 60:384-95.) Various forms of click chemistry reaction are known in the art, such as the Huisgen 1,3-dipolar cycloaddition copper catalyzed reaction (Tornoe et al., 2002, J Organic Chem 67:3057-64), which is often referred to as the "click reaction." Other alternatives include cycloaddition reactions such as the Diels-Alder, nucleophilic substitution reactions (especially to small strained rings like epoxy and aziridine compounds), carbonyl chemistry formation of urea compounds and reactions involving carbon-carbon double bonds, such as alkynes in thiol-yne reactions.

[0069] The azide alkyne Huisgen cycloaddition reaction uses a copper catalyst in the presence of a reducing agent to catalyze the reaction of a terminal alkyne group attached to a first molecule. In the presence of a second molecule comprising an azide moiety, the azide reacts with the activated alkyne to form a 1,4-disubstituted 1,2,3-triazole. The copper catalyzed reaction occurs at room temperature and is sufficiently specific that purification of the reaction product is often not required. (Rostovstev et al., 2002, Angew Chem Int Ed 41:2596; Tornoe et al., 2002, J Org Chem 67:3057.) The azide and alkyne functional groups are largely inert towards biomolecules in aqueous medium, allowing the reaction to occur in complex solutions. The triazole formed is chemically stable and is not subject to enzymatic cleavage, making the click chemistry product highly stable in biological systems. However, the copper catalyst is toxic to living cells, precluding biological applications.

[0070] A copper-free click reaction has been proposed for covalent modification of biomolecules in living systems. (See, e.g., Agard et al., 2004, J Am Chem Soc 126:15046- 47.) The copper-free reaction uses ring strain in place of the copper catalyst to promote a [3 + 2] azide-alkyne cycloaddition reaction (Id.) For example, cyclooctyne is a 8-carbon ring structure comprising an internal alkyne bond. The closed ring structure induces a substantial bond angle deformation of the acetylene, which is highly reactive with azide groups to form a triazole. Thus, cyclooctyne derivatives may be used for copper-free click reactions, without the toxic copper catalyst (Id.)

[0071] Another type of copper-free click reaction was reported by Ning et al. (2010, Angew Chem Int Ed 49:3065-68), involving strain-promoted alkyne-nitrone cycloaddition. To address the slow rate of the original cyclooctyne reaction, electron-withdrawing groups are attached adjacent to the triple bond (Id.) Examples of such substituted cyclooctynes include difluorinated cyclooctynes, 4-dibenzocyclooctynol and azacyclooctyne (Id.) An alternative copper-free reaction involved strain-promoted alkyne-nitrone cycloaddition to give N- alkylated isoxazolines (Id.) The reaction was reported to have exceptionally fast reaction kinetics and was used in a one-pot three-step protocol for site-specific modification of peptides and proteins (Id.) Nitrones were prepared by the condensation of appropriate aldehydes with N-methylhydroxylamine and the cycloaddition reaction took place in a mixture of acetonitrile and water (Id.)

[0072] The Diels-Alder reaction has also been used for in vivo labeling of molecules. Rossin et al. (2010, Angew Chem Int Ed 49:3375-78) reported a 52% yield in vivo between a tumor- localized anti-TAG72 (CC49) antibody carrying a trans-cyclooctene (TCO) reactive moiety and an ¹¹¹ In-labeled tetrazine DOTA derivative. The TCO-labeled CC49 antibody was administered to mice bearing colon cancer xenografts, followed 1 day later by injection of ¹¹¹ In-labeled tetrazine probe (Id.) The reaction of radiolabeled probe with tumor localized antibody resulted in pronounced radioactivity localized in the tumor, as demonstrated by SPECT imaging of live mice three hours after injection of radiolabeled probe, with a tumor- to-muscle ratio of 13:1 (Id.) The results confirmed the in vivo chemical reaction of the TCO and tetrazine-labeled molecules.

[0073] Modifications of click chemistry reactions are suitable for use in vitro or in vivo. Reactive targeting molecule may be formed either by either chemical conjugation or by biological incorporation. The targeting peptide may be activated with an azido moiety, a substituted cyclooctyne or alkyne group, or a nitrone moiety. Where the targeting peptide comprises an azido or nitrone group, the corresponding chelator will comprise a substituted cyclooctyne or alkyne group, and vice versa. Such activated molecules may be made by metabolic incorporation in living cells, as discussed above. Alternatively, methods of chemical conjugation of such moieties to biomolecules are well known in the art, and any such known method may be utilized. The disclosed techniques may be used in combination with the diagnostic radionuclide (e.g., ¹⁸ F) labeling methods described below for PET, SPECT or MRI imaging, or alternatively may be utilized for delivery of any therapeutic and/or diagnostic agent that may be attached to a suitable activated targeting peptide.

Therapeutic Agents

[0074] In varioius embodiments, the labeled targeting peptides may be administered in combination with one or more additional therapeutic or diagnostic agents. Such additional agents may be administered before, concurrently with, or after the labeled peptide.

Therapeutic agents of use may include cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes, antibodies, antibody fragments, immunoconjugates, immunomodulators, oligonucleotides, siRNA, RNAi or other known agents.

[0075] Drugs of use may possess a pharmaceutical property selected from the group consisting of antimitotic, antikinase, alkylating, antimetabolite, antibiotic, alkaloid, anti-angiogenic, pro- apoptotic agents and combinations thereof. Exemplary drugs of use include, but are not limited to, 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101, AVL-291, bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatinum, Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2- pyrrolinodoxorubicine (2P-DOX), pro-2P-DOX, cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3',5'-O-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl-protein transferase inhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101, gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L-asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6- mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341, raloxifene, semustine, sorafenib, streptozocin, SU11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD1839. [0076] Toxins of use may include ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.

[0077] Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.

[0078] In certain embodiments, anti-angiogenic agents, such as angiostatin, baculostatin, canstatin, maspin, anti-VEGF antibodies, anti-PlGF peptides and antibodies, anti-vascular growth factor antibodies, anti-Flk-1 antibodies, anti-Flt-1 antibodies and peptides, anti-Kras antibodies, anti-cMET antibodies, anti-MIF (macrophage migration-inhibitory factor) antibodies, laminin peptides, fibronectin peptides, plasminogen activator inhibitors, tissue metalloproteinase inhibitors, interferons, interleukin-12, IP-10, Gro-ß, thrombospondin, 2- methoxyoestradiol, proliferin-related protein, carboxiamidotriazole, CM101, Marimastat, pentosan polysulphate, angiopoietin-2, interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment, Linomide (roquinimex), thalidomide, pentoxifylline, genistein, TNP-470, endostatin, paclitaxel, accutin, angiostatin, cidofovir, vincristine, bleomycin, AGM-1470, platelet factor 4 or minocycline may be of use.

[0079] Immunomodulators of use may be selected from a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and a combination thereof. Specifically useful are

lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors, such as interleukin (IL), colony stimulating factor, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF), interferon, such as interferons-α, - β or - γ, and stem cell growth factor, such as that designated "S1 factor".

Included among the cytokines are growth hormones such as human growth hormone, N- methionyl human growth hormone, and bovine growth hormone; parathyroid hormone;

thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor; prolactin; placental lactogen, OB protein; tumor necrosis factor-α and - ß; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor;

integrin; thrombopoietin (TPO); nerve growth factors such as NGF-ß; platelet-growth factor; transforming growth factors (TGFs) such as TGF- α and TGF- ß; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-α, - β, and - γ; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); interleukins (ILs) such as IL-1, IL-1 ^, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT.

[0080] Radionuclides of use include, but are not limited to- ¹¹¹ In, ¹⁷⁷ Lu, ²¹² Bi, ²¹³ Bi, ²¹¹ At, ⁶² Cu, ⁶⁷ Cu, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ³² P, ³³ P, ⁴⁷ Sc, ¹¹¹ Ag, ⁶⁷ Ga, ¹⁴² Pr, ¹⁵³ Sm, ¹⁶¹ Tb, ¹⁶⁶ Dy, ¹⁶⁶ Ho, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹⁸⁹ Re, ²¹² Pb, ²²³ Ra, ²²⁵ Ac, ⁵⁹ Fe, ⁷⁵ Se, ⁷⁷ As, ⁸⁹ Sr, ⁹⁹ Mo, ¹⁰⁵ Rh, ¹⁰⁹ Pd, ¹⁴³ Pr, ¹⁴⁹ Pm, ¹⁶⁹ Er, ¹⁹⁴ Ir, ¹⁹⁸ Au, ¹⁹⁹ Au, ²²⁷ Th, and ²¹¹ Pb. The therapeutic radionuclide preferably has a decay-energy in the range of 20 to 6,000 keV, preferably in the ranges 60 to 200 keV for an Auger emitter, 100-2,500 keV for a beta emitter, and 4,000-6,000 keV for an alpha emitter. Maximum decay energies of useful beta-particle-emitting nuclides are preferably 20- 5,000 keV, more preferably 100-4,000 keV, and most preferably 500-2,500 keV. Also preferred are radionuclides that substantially decay with Auger-emitting particles. For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119, 1-125, Ho-161, Os-189m and Ir-192. Decay energies of useful beta-particle-emitting nuclides are preferably <1,000 keV, more preferably <100 keV, and most preferably <70 keV. Also preferred are radionuclides that substantially decay with generation of alpha-particles. Such radionuclides include, but are not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213, Th-227 and Fm-255. Decay energies of useful alpha- particle-emitting radionuclides are preferably 2,000-10,000 keV, more preferably 3,000- 8,000 keV, and most preferably 4,000-7,000 keV. Additional potential radioisotopes of use include ¹¹ C, ¹³ N, ¹⁵ O, ⁷⁵ Br, ¹⁹⁸ Au, ²²⁴ Ac, ¹²⁶ I, ¹³³ I, ⁷⁷ Br, ^113m In, ⁹⁵ Ru, ⁹⁷ Ru, ¹⁰³ Ru, ¹⁰⁵ Ru, ¹⁰⁷ Hg, ²⁰³ Hg, ^121m Te, ^122m Te, ^125m Te, ¹⁶⁵ Tm, ¹⁶⁷ Tm, ¹⁶⁸ Tm, ¹⁹⁷ Pt, ¹⁰⁹ Pd, ¹⁰⁵ Rh, ¹⁴² Pr, ¹⁴³ Pr, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁹⁹ Au, ⁵⁷ Co, ⁵⁸ Co, ⁵¹ Cr, ⁵⁹ Fe, ⁷⁵ Se, ²⁰¹ Tl, ²²⁵ Ac, ⁷⁶ Br, ¹⁶⁹ Yb, and the like. Some useful diagnostic nuclides may include ¹⁸ F, ⁵² Fe, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁸⁹ Zr, ⁹⁴ Tc, ^94m Tc, ^99m Tc, or ¹¹¹ In.

[0081] Therapeutic agents may include a photoactive agent or dye. Fluorescent

compositions, such as fluorochrome, and other chromogens, or dyes, such as porphyrins sensitive to visible light, have been used to detect and to treat lesions by directing the suitable light to the lesion. In therapy, this has been termed photoradiation, phototherapy, or photodynamic therapy. See Jori et al. (eds.), PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto 1985); van den Bergh, Chem. Britain (1986), 22:430. Moreover, targeting molecules have been coupled with photoactivated dyes for achieving phototherapy. See Mew et al., J. Immunol. (1983),130:1473; idem., Cancer Res. (1985), 45:4380; Oseroff et al., Proc. Natl. Acad. Sci. USA (1986), 83:8744; idem.,

Photochem. Photobiol. (1987), 46:83; Hasan et al., Prog. Clin. Biol. Res. (1989), 288:471; Tatsuta et al., Lasers Surg. Med. (1989), 9:422; Pelegrin et al., Cancer (1991), 67:2529.

[0082] Other useful therapeutic agents may comprise oligonucleotides, especially antisense oligonucleotides that preferably are directed against oncogenes and oncogene products, such as bcl-2 or p53. A preferred form of therapeutic oligonucleotide is siRNA.

Diagnostic Agents

[0083] Diagnostic agents are preferably selected from the group consisting of a radionuclide, a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent label, a

chemiluminescent label, an ultrasound contrast agent and a photoactive agent. Such diagnostic agents are well known and any such known diagnostic agent may be used. Non- limiting examples of diagnostic agents may include a radionuclide such as ¹¹⁰ In, ¹¹¹ In, ¹⁷⁷ Lu, ¹⁸ F, ⁵² Fe, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁷ Ga, ⁶⁸ Ga, ⁸⁶ Y, ⁹⁰ Y, ⁸⁹ Zr, ^94m Tc, ⁹⁴ Tc, ^99m Tc, ¹²⁰ I, ¹²³ I, ¹²⁴ I, ¹²⁵ I, ¹³¹ I, ^154-158 Gd, ³² P, ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸⁶ Re, ¹⁸⁸ Re, ⁵¹ Mn, ^52m Mn, ⁵⁵ Co, ⁷² As, ⁷⁵ Br, ⁷⁶ Br, ^82m Rb, ⁸³ Sr, or other gamma-, beta-, or positron-emitters. Paramagnetic ions of use may include chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III). Metal contrast agents may include lanthanum (III), gold (III), lead (II) or bismuth (III). Radiopaque diagnostic agents may be selected from compounds, barium compounds, gallium compounds, and thallium compounds. A wide variety of fluorescent labels are known in the art, including but not limited to fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o- phthaldehyde and fluorescamine. Chemiluminescent labels of use may include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt or an oxalate ester. Methods of Administration

[0084] The labeled targeting peptides may be formulated to obtain compositions that include one or more pharmaceutically suitable excipients, one or more additional ingredients, or some combination of these. These can be accomplished by known methods to prepare

pharmaceutically useful dosages, whereby the active ingredients (i.e., the labeled peptides) are combined in a mixture with one or more pharmaceutically suitable excipients. Sterile phosphate-buffered saline is one example of a pharmaceutically suitable excipient. Other suitable excipients are well known to those in the art. See, e.g., Ansel et al.,

PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, 5th Edition (Lea & Febiger 1990), and Gennaro (ed.), REMINGTON’S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Company 1990), and revised editions thereof.

[0085] The preferred route for administration of the compositions described herein is parenteral injection. Injection may be intravenous, intraarterial, intralymphatic, intrathecal, subcutaneous or intracavitary (i.e., parenterally). In parenteral administration, the compositions will be formulated in a unit dosage injectable form such as a solution, suspension or emulsion, in association with a pharmaceutically acceptable excipient. Such excipients are inherently nontoxic and nontherapeutic. Examples of such excipients are saline, Ringer's solution, dextrose solution and Hank's solution. Nonaqueous excipients such as fixed oils and ethyl oleate may also be used. A preferred excipient is 5% dextrose in saline. The excipient may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, including buffers and preservatives. Other methods of administration, including oral administration, are also contemplated.

[0086] Formulated compositions comprising labeled targeting peptides can be used for intravenous administration via, for example, bolus injection or continuous infusion.

Compositions for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. Compositions can also take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the compositions can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0087] The compositions may be administered in solution. The pH of the solution should be in the range of pH 5 to 9.5, preferably pH 6.5 to 7.5. The formulation thereof should be in a solution having a suitable pharmaceutically acceptable buffer such as phosphate, TRIS (hydroxymethyl) aminomethane-HCl or citrate and the like. In certain preferred

embodiments, the buffer is potassium biphthalate (KHP), which may act as a transfer ligand to facilitate ¹⁸ F-labeling. Buffer concentrations should be in the range of 1 to 100 mM. The formulated solution may also contain a salt, such as sodium chloride or potassium chloride in a concentration of 50 to 150 mM. An effective amount of a stabilizing agent such as glycerol, albumin, a globulin, a detergent, a gelatin, a protamine or a salt of protamine may also be included. The compositions may be administered to a mammal subcutaneously, intravenously, intramuscularly or by other parenteral routes. Moreover, the administration may be by continuous infusion or by single or multiple boluses. [0088] In general, the dosage of ¹⁸ F or other radiolabel to administer to a human subject will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition and previous medical history. Preferably, a saturating dose of the labeled molecule is administered to a patient. For administration of radiolabeled molecules, the dosage may be measured by millicuries. A typical range for imaging studies would be five to 10 mCi.

Administration of Peptides

[0089] Various embodiments of the claimed methods and/or compositions may concern one or more ¹⁸ F- or other radiolabeled peptides to be administered to a subject. Administration may occur by any route known in the art, including but not limited to oral, nasal, buccal, inhalational, rectal, vaginal, topical, orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intraarterial, intrathecal or intravenous injection.

[0090] In certain embodiments, the standard peptide bond linkage may be replaced by one or more alternative linking groups, such as CH ₂ -NH, CH ₂ -S, CH ₂ -CH ₂ , CH=CH, CO-CH ₂ , CHOH-CH ₂ and the like. Methods for preparing peptide mimetics are well known (for example, Hruby, 1982, Life Sci 31:189-99; Holladay et al., 1983, Tetrahedron Lett.24:4401- 04; Jennings-White et al., 1982, Tetrahedron Lett.23:2533; Almquiest et al., 1980, J. Med. Chem.23:1392-98; Hudson et al., 1979, Int. J. Pept. Res.14:177-185; Spatola et al., 1986, Life Sci 38:1243-49; U.S. Patent Nos.5,169,862; 5,539,085; 5,576,423, 5,051,448,

5,559,103.) Peptide mimetics may exhibit enhanced stability and/or absorption in vivo compared to their peptide analogs.

[0091] Peptide stabilization may also occur by substitution of D-amino acids for naturally occurring L-amino acids, particularly at locations where endopeptidases are known to act. Endopeptidase binding and cleavage sequences are known in the art and methods for making and using peptides incorporating D-amino acids have been described (e.g., U.S. Patent Application Publication No.20050025709, McBride et al., filed June 14, 2004, the Examples section of which is incorporated herein by reference).

Imaging Using Labeled Molecules

[0092] Methods of imaging using labeled molecules are well known in the art, and any such known methods may be used with the labeled targeting peptides disclosed herein. See, e.g., U.S Patent Nos.6,241,964; 6,358,489; 6,953,567 and published U.S. Patent Application Publ. Nos.20050003403; 20040018557; 20060140936, the Examples section of each incorporated herein by reference. See also, Page et al., Nuclear Medicine And Biology, 21:911-919, 1994; Choi et al., Cancer Research 55:5323-5329, 1995; Zalutsky et al., J. Nuclear Med., 33:575- 582, 1992; Woessner et. al. Magn. Reson. Med.2005, 53: 790-99.

[0093] Methods of diagnostic imaging with labeled peptides are well-known. For example, in the technique of immunoscintigraphy, peptide ligands are labeled with a gamma-emitting radioisotope and introduced into a patient. A gamma camera is used to detect the location and distribution of gamma-emitting radioisotopes. See, for example, Srivastava (ed.),

RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY

(Plenum Press 1988), Chase, "Medical Applications of Radioisotopes," in REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, Gennaro et al. (eds.), pp.624-652 (Mack Publishing Co., 1990), and Brown, "Clinical Use of Monoclonal Antibodies," in

BIOTECHNOLOGY AND PHARMACY 227-49, Pezzuto et al. (eds.) (Chapman & Hall 1993). Also preferred is the use of positron-emitting radionuclides (PET isotopes), such as with an energy of 511 keV, such as ¹⁸ F, ⁶⁸ Ga, ⁶⁴ Cu, and ¹²⁴ I. Such radionuclides may be imaged by well-known PET scanning techniques.

Kits

[0094] Various embodiments may concern kits containing components suitable for imaging, diagnosing and/or detecting diseased tissue in a patient using labeled compounds. Exemplary kits may contain a targeting peptide of use as described herein.

[0095] A device capable of delivering the kit components may be included. One type of device, for applications such as parenteral delivery, is a syringe that is used to inject the composition into the body of a subject. Inhalation devices may also be used for certain applications.

[0096] The kit components may be packaged together or separated into two or more containers. In some embodiments, the containers may be vials that contain sterile, lyophilized formulations of a composition that are suitable for reconstitution. A kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents. Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers.

Another component that can be included is instructions to a person using a kit for its use. EXAMPLES

Example 1. ¹⁸ F-Labeling of Peptide IMP272

[0097] The first peptide that was prepared and ¹⁸ F-labeled was IMP272: DTPA-Gln-Ala-Lys(HSG)-D-Tyr-Lys(HSG)-NH ₂ ( SEQ ID NO:6)

[0098] Acetate buffer solution - Acetic acid, 1.509 g was diluted in ~ 160 mL water and the pH was adjusted by the addition of 1 M NaOH then diluted to 250 mL to make a 0.1 M solution at pH 4.03.

[0099] Aluminum acetate buffer solution - A solution of aluminum was prepared by dissolving 0.1028 g of AlCl ₃ hexahydrate in 42.6 mL DI water. A 4 mL aliquot of the aluminum solution was mixed with 16 mL of a 0.1 M NaOAc solution at pH 4 to provide a 2 mM Al stock solution.

[00100] IMP272 acetate buffer solution - Peptide, 0.0011 g, 7.28 x 10 ^-7 mol IMP272 was dissolved in 364 μL of the 0.1 M pH 4 acetate buffer solution to obtain a 2 mM stock solution of the peptide.

[00101] ¹⁸ F-Labeling of IMP272 - A 3 μL aliquot of the aluminum stock solution was placed in a REACTI-VIAL™ and mixed with 50 μL ¹⁸ F (as received) and 3 μL of the IMP272 solution. The solution was heated in a heating block at 110˚C for 15 min and analyzed by reverse phase HPLC. The HPLC trace (not shown) showed 93 % free ¹⁸ F and 7 % bound to the peptide. An additional 10 μL of the IMP272 solution was added to the reaction and it was heated again and analyzed by reverse phase HPLC (not shown). The HPLC trace showed 8 % ¹⁸ F at the void volume and 92 % of the activity attached to the peptide. The remainder of the peptide solution was incubated at room temperature with 150 μL PBS for ~ 1hr and then examined by reverse phase HPLC. The HPLC (not shown) showed 58 % ¹⁸ F unbound and 42 % still attached to the peptide. The data indicate that Al ¹⁸ F(DTPA) complex may be unstable when mixed with phosphate.

Example 2. IMP272 ¹⁸ F-Labeling with Other Metals

[00102] A ~3 μL aliquot of the metal stock solution (6 x 10 ^-9 mol) was placed in a polypropylene cone vial and mixed with 75 μL ¹⁸ F (as received), incubated at room temperature for ~ 2 min and then mixed with 20 μL of a 2 mM (4 x 10 ^-8 mol) IMP272 solution in 0.1 M NaOAc pH 4 buffer. The solution was heated in a heating block at 100˚C for 15 min and analyzed by reverse phase HPLC. IMP272 was labeled with indium (24%), gallium (36%), zirconium (15%), lutetium (37 %) and yttrium (2 %) (not shown). These results demonstrate that the ¹⁸ F metal labeling technique is not limited to an aluminum ligand, but can also utilize other metals as well. With different metal ligands, different chelating moieties may be utilized to optimize binding of an ¹⁸ F-metal conjugate. Example 3. Production and Use of a Serum-Stable ¹⁸ F-Labeled Peptide IMP449

NOTA- benzyl-ITC-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH ₂ (SEQ ID NO:7)

[00103] The peptide, IMP448 D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH ₂ (SEQ ID NO:8) was made on Sieber Amide resin by adding the following amino acids to the resin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved, Fmoc-D- Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved, Fmoc-D-Ala- OH with final Fmoc cleavage to make the desired peptide. The peptide was then cleaved from the resin and purified by HPLC to produce IMP448, which was then coupled to ITC-benzyl NOTA.

[00104] IMP448 (0.0757g, 7.5 x 10 ^-5 mol) was mixed with 0.0509 g (9.09 x 10 ^-5 mol) ITC benzyl NOTA and dissolved in 1 mL water. Potassium carbonate anhydrous (0.2171 g) was then slowly added to the stirred peptide/NOTA solution. The reaction solution was pH 10.6 after the addition of all the carbonate. The reaction was allowed to stir at room temperature overnight. The reaction was carefully quenched with 1 M HCl after 14 hr and purified by HPLC to obtain 48 mg of IMP449.

1 ⁸ F-Labeling of IMP449

[00105] IMP449 (0.002 g, 1.37 x 10 ^-6 mol) was dissolved in 686 μL (2 mM peptide solution) 0.1 M NaOAc pH 4.02. Three microliters of a 2 mM solution of Al in a pH 4 acetate buffer was mixed with 15 μL, 1.3 mCi of ¹⁸ F. The solution was then mixed with 20 μL of the 2 mM IMP449 solution and heated at 105 ºC for 15 min. Reverse Phase HPLC analysis showed 35 % (t _R ~ 10 min) of the activity was attached to the peptide and 65 % of the activity was eluted at the void volume of the column (3.1 min, not shown) indicating that the majority of activity was not associated with the peptide. The crude labeled mixture (5 μL) was mixed with pooled human serum and incubated at 37 ºC. An aliquot was removed after 15 min and analyzed by HPLC. The HPLC showed 9.8 % of the activity was still attached to the peptide (down from 35 %). Another aliquot was removed after 1 hr and analyzed by HPLC. The HPLC showed 7.6 % of the activity was still attached to the peptide (down from 35 %), which was essentially the same as the 15 min trace (data not shown).

High Dose ¹⁸ F-Labeling of IMP449

[00106] Further studies with purified IMP449 demonstrated that the ¹⁸ F-labeled peptide was highly stable (91%, not shown) in human serum at 37 °C for at least one hour and was partially stable (76%, not shown) in human serum at 37 °C for at least four hours. Additional studies were performed in which the IMP449 was prepared in the presence of ascorbic acid as a stabilizing agent. In those studies (not shown), the ¹⁸ F-metal-peptide complex showed no detectable decomposition in serum after 4 hr at 37 °C. The mouse urine 30 min after injection of ¹⁸ F-labeled peptide was found to contain ¹⁸ F bound to the peptide (not shown). These results demonstrate that the ¹⁸ F-labeled peptides disclosed herein exhibit sufficient stability under approximated in vivo conditions to be used for ¹⁸ F imaging studies.

[00107] Since IMP449 peptide contains a thiourea linkage, which is sensitive to radiolysis, several products are observed by RP-HPLC. However, when ascorbic acid is added to the reaction mixture, the side products generated are markedly reduced.

Example 4. Preparation of DNL Constructs for ¹⁸ F Imaging by Pretargeting

[00108] The DNL technique may be used to make dimers, trimers, tetramers, hexamers, etc. comprising virtually any antibodies or fragments thereof or other effector moieties. For certain preferred embodiments, IgG antibodies, Fab fragments or other proteins or peptides may be produced as fusion proteins containing either a DDD (dimerization and docking domain) or AD (anchoring domain) sequence. Bispecific antibodies may be formed by combining a Fab-DDD fusion protein of a first antibody with a Fab-AD fusion protein of a second antibody. Alternatively, constructs may be made that combine IgG-AD fusion proteins with Fab-DDD fusion proteins. For purposes of ¹⁸ F detection, an antibody or fragment containing a binding site for an antigen associated with a target tissue to be imaged, such as a tumor, may be combined with a second antibody or fragment that binds a hapten on a targetable construct, such as IMP 449, to which a metal- ¹⁸ F can be attached. The bispecific antibody (DNL construct) is administered to a subject, circulating antibody is allowed to clear from the blood and localize to target tissue, and the ¹⁸ F-labeled targetable construct is added and binds to the localized antibody for imaging.

[00109] Independent transgenic cell lines may be developed for each Fab or IgG fusion protein. Once produced, the modules can be purified if desired or maintained in the cell culture supernatant fluid. Following production, any DDD ₂ -fusion protein module can be combined with any corresponding AD-fusion protein module to generate a bispecific DNL construct. For different types of constructs, different AD or DDD sequences may be utilized. The following DDD sequences are based on the DDD moiety of PKA RIIα, while the AD sequences are based on the AD moiety of the optimized synthetic AKAP-IS sequence (Alto et al., Proc. Natl. Acad. Sci. USA.2003;100:4445).

DDD1: SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:9) DDD2: CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:10)

AD1: QIEYLAKQIVDNAIQQA (SEQ ID NO:11)

AD2: CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO:12)

[00110] The plasmid vector pdHL2 has been used to produce a number of antibodies and antibody-based constructs. See Gillies et al., J Immunol Methods (1989), 125:191-202;

Losman et al., Cancer (Phila) (1997), 80:2660-6. The di-cistronic mammalian expression vector directs the synthesis of the heavy and light chains of IgG. The vector sequences are mostly identical for many different IgG-pdHL2 constructs, with the only differences existing in the variable domain (VH and VL) sequences. Using molecular biology tools known to those skilled in the art, these IgG expression vectors can be converted into Fab-DDD or Fab- AD expression vectors. To generate Fab-DDD expression vectors, the coding sequences for the hinge, CH2 and CH3 domains of the heavy chain are replaced with a sequence encoding the first 4 residues of the hinge, a 14 residue Gly-Ser linker and the first 44 residues of human RII ^ (referred to as DDD1). To generate Fab-AD expression vectors, the sequences for the hinge, CH2 and CH3 domains of IgG are replaced with a sequence encoding the first 4 residues of the hinge, a 15 residue Gly-Ser linker and a 17 residue synthetic AD called AKAP-IS (referred to as AD1), which was generated using bioinformatics and peptide array technology and shown to bind RII ^ dimers with a very high affinity (0.4 nM). See Alto, et al. Proc. Natl. Acad. Sci., U.S.A (2003), 100:4445-50.

[00111] Two shuttle vectors were designed to facilitate the conversion of IgG-pdHL2 vectors to either Fab-DDD1 or Fab-AD1 expression vectors, as described below.

Preparation of CH1

[00112] The CH1 domain was amplified by PCR using the pdHL2 plasmid vector as a template. The left PCR primer consisted of the upstream (5’) end of the CH1 domain and a SacII restriction endonuclease site, which is 5’ of the CH1 coding sequence. The right primer consisted of the sequence coding for the first 4 residues of the hinge followed by four glycines and a serine (SEQ ID NO: 31), with the final two codons (GS) comprising a Bam HI restriction site. The 410 bp PCR amplimer was cloned into the pGemT PCR cloning vector (Promega, Inc.) and clones were screened for inserts in the T7 (5’) orientation.

[00113] A duplex oligonucleotide was synthesized by to code for the amino acid sequence of DDD1 preceded by 11 residues of a linker peptide, with the first two codons comprising a BamHI restriction site. A stop codon and an EagI restriction site are appended to the 3’end. The encoded polypeptide sequence is shown below, with the DDD1 sequence underlined. GSGGGGSGGGGSHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA

(SEQ ID NO:13)

[00114] Two oligonucleotides, designated RIIA1-44 top and RIIA1-44 bottom, that overlap by 30 base pairs on their 3’ ends, were synthesized (Sigma Genosys) and combined to comprise the central 154 base pairs of the 174 bp DDD1 sequence. The oligonucleotides were annealed and subjected to a primer extension reaction with Taq polymerase. Following primer extension, the duplex was amplified by PCR. The amplimer was cloned into pGemT and screened for inserts in the T7 (5’) orientation.

[00115] A duplex oligonucleotide was synthesized to code for the amino acid sequence of AD1 preceded by 11 residues of the linker peptide with the first two codons comprising a BamHI restriction site. A stop codon and an EagI restriction site are appended to the 3’end. The encoded polypeptide sequence is shown below, with the sequence of AD1 underlined.

GSGGGGSGGGGSQIEYLAKQIVDNAIQQA (SEQ ID NO:14)

[00116] Two complimentary overlapping oligonucleotides encoding the above peptide sequence, designated AKAP-IS Top and AKAP-IS Bottom, were synthesized and annealed. The duplex was amplified by PCR. The amplimer was cloned into the pGemT vector and screened for inserts in the T7 (5’) orientation.

Ligating DDD1 with CH1

[00117] A 190 bp fragment encoding the DDD1 sequence was excised from pGemT with BamHI and NotI restriction enzymes and then ligated into the same sites in CH1-pGemT to generate the shuttle vector CH1-DDD1-pGemT.

Ligating AD1 with CH1

[00118] A 110 bp fragment containing the AD1 sequence was excised from pGemT with BamHI and NotI and then ligated into the same sites in CH1-pGemT to generate the shuttle vector CH1-AD1-pGemT.

Cloning CH1-DDD1 or CH1-AD1 into pdHL2-based vectors

[00119] With this modular design either CH1-DDD1 or CH1-AD1 can be incorporated into any IgG construct in the pdHL2 vector. The entire heavy chain constant domain is replaced with one of the above constructs by removing the SacII/EagI restriction fragment (CH1-CH3) from pdHL2 and replacing it with the SacII/EagI fragment of CH1-DDD1 or CH1-AD1, which is excised from the respective pGemT shuttle vector.

Construction of h679-Fd-AD1-pdHL2

[00120] h679-Fd-AD1-pdHL2 is an expression vector for production of h679 Fab with AD1 coupled to the carboxyl terminal end of the CH1 domain of the Fd via a flexible Gly/Ser peptide spacer composed of 14 amino acid residues. A pdHL2-based vector containing the variable domains of h679 was converted to h679-Fd-AD1-pdHL2 by replacement of the SacII/EagI fragment with the CH1-AD1 fragment, which was excised from the CH1-AD1- SV3 shuttle vector with SacII and EagI.

Construction of C-DDD1-Fd-hMN-14-pdHL2

[00121] C-DDD1-Fd-hMN-14-pdHL2 is an expression vector for production of a stable dimer that comprises two copies of a fusion protein C-DDD1-Fab-hMN-14, in which DDD1 is linked to hMN-14 Fab at the carboxyl terminus of CH1 via a flexible peptide spacer. The plasmid vector hMN14(I)-pdHL2, which has been used to produce hMN-14 IgG, was converted to C-DDD1-Fd-hMN-14-pdHL2 by digestion with SacII and EagI restriction endonucleases to remove the CH1-CH3 domains and insertion of the CH1-DDD1 fragment, which was excised from the CH1-DDD1-SV3 shuttle vector with SacII and EagI.

[00122] The same technique has been utilized to produce plasmids for Fab expression of a wide variety of known antibodies, such as hLL1, hLL2, hPAM4, hR1, hRS7, hMN-14, hMN- 15, hA19, hA20 and many others. Generally, the antibody variable region coding sequences were present in a pdHL2 expression vector and the expression vector was converted for production of an AD- or DDD-fusion protein as described above. The AD- and DDD-fusion proteins comprising a Fab fragment of any of such antibodies may be combined, in an approximate ratio of two DDD-fusion proteins per one AD-fusion protein, to generate a trimeric DNL construct comprising two Fab fragments of a first antibody and one Fab fragment of a second antibody.

C-DDD2-Fd-hMN-14-pdHL2

[00123] C-DDD2-Fd-hMN-14-pdHL2 is an expression vector for production of C-DDD2- Fab-hMN-14, which possesses a dimerization and docking domain sequence of DDD2 appended to the carboxyl terminus of the Fd of hMN-14 via a 14 amino acid residue Gly/Ser peptide linker. The fusion protein secreted is composed of two identical copies of hMN-14 Fab held together by non-covalent interaction of the DDD2 domains.

[00124] Two overlapping, complimentary oligonucleotides, which comprise the coding sequence for part of the linker peptide and residues 1-13 of DDD2, were made synthetically. The oligonucleotides were annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3' ends that are compatible for ligation with DNA digested with the restriction endonucleases BamHI and PstI, respectively.

[00125] The duplex DNA was ligated with the shuttle vector CH1-DDD1-pGemT, which was prepared by digestion with BamHI and PstI, to generate the shuttle vector CH1-DDD2- pGemT. A 507 bp fragment was excised from CH1-DDD2-pGemT with SacII and EagI and ligated with the IgG expression vector hMN14(I)-pdHL2, which was prepared by digestion with SacII and EagI. The final expression construct was designated C-DDD2-Fd-hMN-14- pdHL2. Similar techniques have been utilized to generated DDD2-fusion proteins of the Fab fragments of a number of different humanized antibodies.

H679-Fd-AD2-pdHL2

[00126] h679-Fab-AD2, was designed to pair as B to C-DDD2-Fab-hMN-14 as A. h679-Fd- AD2-pdHL2 is an expression vector for the production of h679-Fab-AD2, which possesses an anchor domain sequence of AD2 appended to the carboxyl terminal end of the CH1 domain via a 14 amino acid residue Gly/Ser peptide linker. AD2 has one cysteine residue preceding and another one following the anchor domain sequence of AD1.

[00127] The expression vector was engineered as follows. Two overlapping, complimentary oligonucleotides (AD2 Top and AD2 Bottom), which comprise the coding sequence for AD2 and part of the linker sequence, were made synthetically. The oligonucleotides were annealed and phosphorylated with T4 PNK, resulting in overhangs on the 5' and 3' ends that are compatible for ligation with DNA digested with the restriction endonucleases BamHI and SpeI, respectively.

[00128] The duplex DNA was ligated into the shuttle vector CH1-AD1-pGemT, which was prepared by digestion with BamHI and SpeI, to generate the shuttle vector CH1-AD2- pGemT. A 429 base pair fragment containing CH1 and AD2 coding sequences was excised from the shuttle vector with SacII and EagI restriction enzymes and ligated into h679-pdHL2 vector that prepared by digestion with those same enzymes. The final expression vector is h679-Fd-AD2-pdHL2.

Example 5. Generation of TF2 DNL Construct

[00129] A trimeric DNL construct designated TF2 was obtained by reacting C-DDD2-Fab- hMN-14 with h679-Fab-AD2. A pilot batch of TF2 was generated with >90% yield as follows. Protein L-purified C-DDD2-Fab-hMN-14 (200 mg) was mixed with h679-Fab-AD2 (60 mg) at a 1.4:1 molar ratio. The total protein concentration was 1.5 mg/ml in PBS containing 1 mM EDTA. Subsequent steps involved TCEP reduction, HIC chromatography, DMSO oxidation, and IMP 291 affinity chromatography. Before the addition of TCEP, SE- HPLC did not show any evidence of a ₂ b formation. Addition of 5 mM TCEP rapidly resulted in the formation of a ₂ b complex consistent with a 157 kDa protein expected for the binary structure. TF2 was purified to near homogeneity by IMP 291 affinity chromatography (not shown). IMP 291 is a synthetic peptide containing the HSG hapten to which the 679 Fab binds (Rossi et al., 2005, Clin Cancer Res 11:7122s-29s). SE-HPLC analysis of the IMP 291 unbound fraction demonstrated the removal of a ₄ , a ₂ and free kappa chains from the product (not shown).

[00130] Non-reducing SDS-PAGE analysis demonstrated that the majority of TF2 exists as a large, covalent structure with a relative mobility near that of IgG (not shown). The additional bands suggest that disulfide formation is incomplete under the experimental conditions (not shown). Reducing SDS-PAGE shows that any additional bands apparent in the non-reducing gel are product-related (not shown), as only bands representing the constituent polypeptides of TF2 are evident. MALDI-TOF mass spectrometry (not shown) revealed a single peak of 156,434 Da, which is within 99.5% of the calculated mass (157,319 Da) of TF2.

[00131] The functionality of TF2 was determined by BIACORE assay. TF2, C-DDD1- hMN-14+h679-AD1 (used as a control sample of noncovalent a ₂ b complex), or C-DDD2- hMN-14+h679-AD2 (used as a control sample of unreduced a ₂ and b components) were diluted to 1 μg/ml (total protein) and passed over a sensorchip immobilized with HSG. The response for TF2 was approximately two-fold that of the two control samples, indicating that only the h679-Fab-AD component in the control samples would bind to and remain on the sensorchip. Subsequent injections of WI2 IgG, an anti-idiotype antibody for hMN-14, demonstrated that only TF2 had a DDD-Fab-hMN-14 component that was tightly associated with h679-Fab-AD as indicated by an additional signal response. The additional increase of response units resulting from the binding of WI2 to TF2 immobilized on the sensorchip corresponded to two fully functional binding sites, each contributed by one subunit of C- DDD2-Fab-hMN-14. This was confirmed by the ability of TF2 to bind two Fab fragments of WI2 (not shown).

Example 6. Production of TF10 DNL Construct

[00132] A similar protocol was used to generate a trimeric TF10 DNL construct, comprising two copies of a C-DDD2-Fab-hPAM4 and one copy of C-AD2-Fab-679. The TF10 bispecific ([hPAM4] ₂ x h679) antibody was produced using the method disclosed for production of the (anti CEA) ₂ x anti HSG bsAb TF2, as described above. The TF10 construct bears two humanized PAM4 Fabs and one humanized 679 Fab.

[00133] The two fusion proteins (hPAM4-DDD2 and h679-AD2) were expressed independently in stably transfected myeloma cells. The tissue culture supernatant fluids were combined, resulting in a two-fold molar excess of hPAM4-DDD2. The reaction mixture was incubated at room temperature for 24 hours under mild reducing conditions using 1 mM reduced glutathione. Following reduction, the DNL reaction was completed by mild oxidation using 2 mM oxidized glutathione. TF10 was isolated by affinity chromatography using IMP 291-affigel resin, which binds with high specificity to the h679 Fab.

Example 7. In Vivo Imaging Using ¹⁸ F-Labeled Peptides and Comparison with ¹⁸ F-FDG

[00134] In vivo imaging techniques using pretargeting with bispecific antibodies and labeled targeting peptides were used to successfully detect tumors of relatively small size. The ¹⁸ F was purified on a WATERS® ACCELL ^TM Plus QMA Light cartridge. The ¹⁸ F- eluted with 0.4 M KHCO ₃ was mixed with 3 μL 2 mM Al ³⁺ in a pH 4 acetate buffer. The Al ¹⁸ F solution was then injected into the ascorbic acid IMP449 labeling vial and heated to 105 °C for 15 min. The reaction solution was cooled and mixed with 0.8 mL DI water. The reaction contents were loaded on a WATERS® OASIS® 1cc HLB Column and eluted with 2 x 200 μL 1:1 EtOH/H ₂ O. TF2 was prepared as described above. TF2 binds divalently to carcinoembryonic antigen (CEA) and monovalently to the synthetic hapten, HSG (histamine- succinyl-glycine).

Biodistribution and microPET imaging.

[00135] Six-week-old NCr nu-m female nude mice were implanted s.c. with the human colonic cancer cell line, LS174T (ATCC, Manassas, VA). When tumors were visibly established, pretargeted animals were injected intravenously with 162 µg (~1 nmole/0.1 mL) TF2 or TF10 (control non-targeting tri-Fab bsMAb), and then 16-18 h later, ~0.1 nmol of Al ¹⁸ F(IMP449) (84 µCi, 3.11 MBq/0.1 mL) was injected intravenously. Other non- pretargeted control animals received ¹⁸ F alone (150 µCi, 5.5 MBq), Al ¹⁸ F complex alone (150 µCi, 5.55 MBq), the Al ¹⁸ F(IMP449) peptide alone (84 µCi, 3.11 MBq), or ¹⁸ F-FDG (150 µCi, 5.55 MBq). ¹⁸ F and ¹⁸ F-FDG were obtained on the day of use from IBA Molecular (Somerset, NJ). Animals receiving ¹⁸ F-FDG were fasted overnight, but water was given ad libitum.

[00136] At 1.5 h after the radiotracer injection, animals were anesthetized, bled

intracardially, and necropsied. Tissues were weighed and counted together with a standard dilution prepared from each of the respective products. Due to the short physical half-life of ¹⁸ F, standards were interjected between each group of tissues from each animal. Uptake in the tissues is expressed as the counts per gram divided by the total injected activity to derive the percent-injected dose per gram (% ID/g).

[00137] Two types of imaging studies were performed. In one set, 3 nude mice bearing small LS174T subcutaneous tumors received either the pretargeted Al ¹⁸ F(IMP449),

Al ¹⁸ F(IMP449) alone (not pretargeted), both at 135 µCi (5 MBq; 0.1 nmol), or ¹⁸ F-FDG (135 µCi, 5 MBq). At 2 h after the intravenous radiotracer injection, the animals were anesthetized with a mixture of O ₂ /N ₂ O and isoflurane (2%) and kept warm during the scan, performed on an INVEON® animal PET scanner (Siemens Preclinical Solutions, Knoxville, TN).

[00138] Representative coronal cross-sections (0.8 mm thick) in a plane located

approximately in the center of the tumor were displayed, with intensities adjusted until pixel saturation occurred in any region of the body (excluding the bladder) and without background adjustment.

[00139] In a separate dynamic imaging study, a single LS174T bearing nude mouse that was given the TF2 bsMAb 16 h earlier was anesthetized with a mixture of O ₂ /N ₂ O and isoflurane (2%), placed supine on the camera bed, and then injected intravenously with 219 µCi (8.1 MBq) Al ¹⁸ F(IMP449) (0.16 nmol). Data acquisition was immediately initiated over a period of 120 minutes. The scans were reconstructed using OSEM3D/MAP. For presentation, time- frames ending at 5, 15, 30, 60, 90, and 120 min were displayed for each cross-section (coronal, sagittal, and transverse). For sections containing tumor, at each interval the image intensity was adjusted until pixel saturation first occurred in the tumor. Image intensity was increased as required over time to maintain pixel saturation within the tumor. Coronal and sagittal cross-sections without tumor taken at the same interval were adjusted to the same intensity as the transverse section containing the tumor. Background activity was not adjusted.

Results

[00140] While ¹⁸ F alone and [Al ¹⁸ F] complexes had similar uptake in all tissues, considerable differences were found when the complex was chelated to IMP449 (Table 1). The most striking differences were found in the uptake in the bone, where the non-chelated ¹⁸ F was 60- to nearly 100-fold higher in the scapula and ~200-fold higher in the spine. This distribution is expected since ¹⁸ F, or even a metal-fluoride complex, is known to accrete in bone (Franke et al.1972, Radiobiol. Radiother. (Berlin) 13:533). Higher uptake was also observed in the tumor and intestines as well as in muscle and blood. The chelated Al ¹⁸ F(IMP449) had significantly lower uptake in all the tissues except the kidneys, illustrating the ability of the chelate-complex to be removed efficiently from the body by urinary excretion.

[00141] Pretargeting the Al ¹⁸ F(IMP449) using the TF2 anti-CEA bsMAb shifted uptake to the tumor, increasing it from 0.20 ± 0.05 to 6.01 ± 1.72% injected dose per gram at 1.5 h, while uptake in the normal tissues was similar to the Al ¹⁸ F(IMP449) alone. Tumor/nontumor ratios were 146 ± 63, 59 ± 24, 38 ± 15, and 2.0 ± 1.0 for the blood, liver, lung, and kidneys, respectively, with other tumor/tissue ratios >100:1 at this time. Although both ¹⁸ F alone and [Al ¹⁸ F] alone had higher uptake in the tumor than the chelated Al ¹⁸ F(IMP449), yielding tumor/blood ratios of 6.7 ± 2.7 and 11.0 ± 4.6 vs.5.1 ± 1.5, respectively, tumor uptake and tumor/blood ratios were significantly increased with pretargeting (all P values <0.001).

[00142] Biodistribution was also compared to the most commonly used tumor imaging agent, [ ¹⁸ F]FDG, which targets tissues with high glucose consumption and metabolic activity (Table 1). Its uptake was appreciably higher than the Al ¹⁸ F(IMP449) in all normal tissues, except the kidney. Tumor uptake was similar for both the pretargeted Al ¹⁸ F(IMP449) and ¹⁸ F- FDG, but because of the higher accretion of [ ¹⁸ F]FDG in most normal tissues,

tumor/nontumor ratios with ¹⁸ F-FDG were significantly lower than those in the pretargeted animals (all P values <0.001).

Table 1. Biodistribution of TF2-pretargeted Al ¹⁸ F(IMP449) and other control ¹⁸ F-labeled agents in nude mice bearing LS174T human colonic xenografts. For pretargeting, animals were given TF216 h before the injection of the Al ¹⁸ F(IMP449). All injections were administered intravenously.

[00143] Several animals were imaged to further analyze the biodistribution of

Al ¹⁸ F(IMP449) alone or Al ¹⁸ F(IMP449) pretargeted with TF2, as well [ ¹⁸ F]FDG. Static images initiated at 2.0 h after the radioactivity was injected corroborated the previous tissue distribution data showing uptake almost exclusively in the kidneys (FIG.1). A 21-mg tumor was easily visualized in the pretargeted animal, while the animal given the Al ¹⁸ F(IMP449) alone failed to localize the tumor, having only renal uptake. No evidence of bone accretion was observed, suggesting that the Al ¹⁸ F was bound firmly to IMP 449. This was confirmed in another pretargeted animal that underwent a dynamic imaging study that monitored the distribution of the Al ¹⁸ F(IMP449) in 5-min intervals over 120 minutes (FIG.2). Coronal and sagittal slices showed primarily cardiac, renal, and some hepatic uptake over the first 5 min, but heart and liver activity decreased substantially over the next 10 min, while the kidneys remained prominent throughout the study. There was no evidence of activity in the intestines or bone over the full 120-min scan. Uptake in a 35-mg LS174T tumor was first observed at 15 min, and by 30 min, the signal was very clearly delineated from background, with intense tumor activity being prominent during the entire 120-min scanning.

[00144] In comparison, static images from an animal given ¹⁸ F-FDG showed the expected pattern of radioactivity in the bone, heart muscle, and brain observed previously (McBride et al., 2006, J. Nucl. Med.47:1678; Sharkey et al., 2008, Radiology 246:497), with considerably more background activity in the body (FIG. 1). Tissue uptake measured in the 3 animals necropsied at the conclusion of the static imaging study confirmed much higher tissue ¹⁸ F radioactivity in all tissues (not shown). While tumor uptake with ¹⁸ F-FDG was higher in this animal than in the pretargeted one, tumor/blood ratios were more favorable for pretargeting; and with much less residual activity in the body, tumor visualization was enhanced by pretargeting.

[00145] These studies demonstrate that a hapten-peptide used in pretargeted imaging can be rapidly labeled (60 min total preparation time) with ¹⁸ F by simply forming an aluminum- fluoride complex that can then be bound by a suitable chelate and incorporated into the hapten-peptide. This can be made more general by simply coupling the [Al ¹⁸ F]-chelate to any molecule that can be attached to the chelating moiety and be subsequently purified.

[00146] This report describes a direct, facile, and rapid method of binding ¹⁸ F to various compounds via an aluminum conjugate. The [Al ¹⁸ F] peptide was stable in vitro and in vivo when bound by a NOTA-based chelate. Yields were within the range found with

conventional ¹⁸ F labeling procedures. These results further demonstrate the feasibility of PET imaging using metal ¹⁸ F chelated to a wide variety of targeting molecules.

Example 8. Preparation and Labeling of IMP460 with Al- ¹⁸ F

[00147] IMP460 NOTA-Ga-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH ₂ (SEQ ID NO:15) was chemically synthesized. The NOTA-Ga ligand was purchased from

CHEMATECH® and attached on the peptide synthesizer like the other amino acids. The peptide was synthesized on Sieber amide resin with the amino acids and other agents added in the following order Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc removal, Fmoc-D- Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc removal, Fmoc-D-Ala-OH, and NOTA-GA(tBu) ₃ . The peptide was then cleaved and purified by HPLC to afford the product..

Radiolabeling of IMP460

[00148] IMP 460 (0.0020 g) was dissolved in 732 μL, pH 4, 0.1 M NaOAc. The ¹⁸ F was purified as described above, neutralized with glacial acetic acid and mixed with the Al solution. The peptide solution, 20 μL was then added and the solution was heated at 99 °C for 25 min. The crude product was then purified on a WATERS® HLB column. The [Al ¹⁸ F] labeled peptide was in the 1:1 EtOH/H ₂ O column eluent. The reverse phase HPLC trace in 0.1 % TFA buffers showed a clean single HPLC peak at the expected location for the labeled peptide (not shown).

Example 9. Synthesis and Labeling of IMP461 and IMP462 NOTA-Conjugated

Peptides

[00149] The simplest possible NOTA ligand (protected for peptide synthesis) was prepared and incorporated into two peptides for pretargeting– IMP461 and IMP462.

Synthesis of Bis-t-butyl-NOTA

[00150] NO2AtBu (0.501 g 1.4 x 10 ^-3 mol) was dissolved in 5 mL anhydrous acetonitrile. Benzyl-2-bromoacetate (0.222 mL, 1.4 x 10 ^-3 mol) was added to the solution followed by 0.387 g of anhydrous K ₂ CO ₃ . The reaction was allowed to stir at room temperature overnight. The reaction mixture was filtered and concentrated to obtain 0.605 g (86 % yield) of the benzyl ester conjugate. The crude product was then dissolved in 50 mL of isopropanol, mixed with 0.2 g of 10 % Pd/C (under Ar) and placed under 50 psi H ₂ for 3 days. The product was then filtered and concentrated under vacuum to obtain 0.462 g of the desired product ESMS [M-H]- 415.

Synthesis of IMP461

[00151] The peptide was synthesized on Sieber amide resin with the amino acids and other agents added in the following order Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc removal, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc removal, Fmoc-D-Ala- OH, and Bis-t-butylNOTA. The peptide was then cleaved and purified by HPLC to afford the product IMP461 ESMS MH ⁺ 1294 NOTA-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH ₂ ; SEQ ID NO:32).

Synthesis of IMP 462

[00152] The peptide was synthesized on Sieber amide resin with the amino acids and other agents added in the following order Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc removal, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, Aloc removal, Fmoc-D- Asp(But)-OH, and Bis-t-butyl NOTA. The peptide was then cleaved and purified by HPLC to afford the product IMP462 ESMS MH ⁺ 1338 (NOTA-D-Asp-D-Lys(HSG)-D-Tyr-D- Lys(HSG)-NH ₂ ; SEQ ID NO:16).

1 ⁸ F Labeling of IMP461 & IMP462

[00153] The peptides were dissolved in pH 4.13, 0.5 M NaOAc to make a 0.05 M peptide solution, which was stored in the freezer until needed. The F-18 was received in 2 mL of water and trapped on a SEP-PAK® Light, WATERS® ACCELL ^TM Plus QMA Cartridge. The ¹⁸ F was eluted from the column with 200 μL aliquots of 0.4 M KHCO ₃ . The bicarbonate was neutralized to ~ pH 4 by the addition of 10 μL of glacial acetic acid to the vials before the addition of the activity. A 100 μL aliquot of the purified ¹⁸ F solution was removed and mixed with 3 μL, 2 mM Al in pH 4, 0.1 M NaOAc. The peptide, 10 μL (0.05 M) was added and the solution was heated at ~ 100 ºC for 15 min. The crude reaction mixture was diluted with 700 μL DI water and placed on an HLB column and after washing the ¹⁸ F was eluted with 2 x 100 μL of 1:1 EtOH/H ₂ O to obtain the purified ¹⁸ F-labeled peptide.

Example 10. Preparation and ¹⁸ F-Labeling of IMP467

IMP467 C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH ₂ (SEQ ID NO:17)

[00154] Tetra tert-butyl C-NETA-succinyl was produced. The tert-Butyl {4-[2-(Bis-( tert- butyoxycarbonyl)methyl-3-(4-nitrophenyl)propyl]-7-tert-butyo xycarbonyl[1,4,7]triazanonan- 1-yl} was prepared as described in Chong et al. (J. Med. Chem.2008, 51:118-125).

[00155] The peptide, IMP467 C-NETA-succinyl-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH ₂ (SEQ ID NO:17) was made on Sieber Amide resin by adding the following amino acids to the resin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved, tert- Butyl{4-[Bis-(tert-butoxycarbonylmethyl)amino)-3-(4-succinyl amidophenyl)propyl]-7-tert- butoxycarbonylmethyl[1,4,7]triazanonan-1-yl}acetate. The peptide was then cleaved from the resin and purified by RP-HPLC to yield 6.3 mg of IMP467. The crude peptide was purified by high performance liquid chromatography (HPLC) using a C18 column.

Radiolabeling

[00156] A 2 mM solution of IMP467 was prepared in pH 4, 0.1 M NaOAc. The ¹⁸ F-, 139 mCi , was eluted through a WATERS® ACCELL ^TM Plus SEP-PAK® Light QMA cartridge and the ¹⁸ F- was eluted with 1 mL of 0.4 M KHCO ₃ .The labeled IMP467 was purified by HLB RP-HPLC. The RP-HPLC showed two peaks eluting (not shown), which are believed to be diastereomers of Al ¹⁸ F(IMP467). Supporting this hypothesis, there appeared to be some interconversion between the two HLB peaks when IMP467 was incubated at 37°C (not shown). In pretargeting techniques as discussed below, since the [Al ¹⁸ F]-chelator complex is not part of the hapten site for antibody binding, the presence of diastereomers does not appear to affect targeting of the ¹⁸ F-labeled peptide to diseased tissues.

Comparison of Yield of Radiolabeled Peptides

[00157] In an attempt to improve labeling yields while maintaining in vivo stability, 3 NOTA derivatives of pretargeting peptide were synthesized (IMP460, IMP461 and IMP467). Of these, IMP467 nearly doubled the labeling yields of the other peptides (Table 2). All of the labeling studies in Table 2 were performed with the same number of moles of peptide and aluminum. The results shown in Table 2 represent an exemplary labeling experiment with each peptide.

[00158] The ¹⁸ F-labeling yield of IMP467 was ~70% when only 40 nmol (~13-fold less than IMP449) was used with 1.3 GBq (35 mCi) of ¹⁸ F, indicating this ligand has improved binding properties for the Al ¹⁸ F complex. By enhancing the kinetics of ligand binding, yields were substantially improved (average 65-75% yield), while using fewer moles of IMP467 (40 nmol), relative to IMP449 (520 nmol, 44% yield).

Table 2. Comparison of yields of different NOTA containing peptides

Example 11. Factors Affecting Yield and Stability of IMP467 Labeling

Peptide concentration

[00159] To examine the effect of varying peptide concentration on yield, the amount of binding of Al ¹⁸ F to peptide was determined in a constant volume (63 μL) with a constant amount of Al ³⁺ (6 nmol) and ¹⁸ F, but varying the amount of peptide added. The yield of labeled peptide IMP467 decreased with a decreasing concentration of peptide as follows: 40 nmol peptide (82% yield); 30 nmol (79% yield); 20 nmol (75% yield); 10 nmol (49% yield). Thus, varying the amount of peptide between 20 and 40 nmol had little effect on yield with IMP467. However, a decreased yield was observed starting at 10 nmol of peptide in the labeling mix.

Aluminum concentration

[00160] When IMP467 was labeled in the presence of increasing amounts of Al ³⁺ (0, 5, 10, 15, 20 μL of 2 mM Al in pH 4 acetate buffer and keeping the total volume constant), yields of 3.5%, 80%, 77%, 78% and 74%, respectively, were achieved. These results indicated that (a) non-specific binding of ¹⁸ F to this peptide in the absence of Al ³⁺ is minimal, (b) 10 nmol of Al ³⁺ was sufficient to allow for maximum ¹⁸ F-binding, and (c) higher amounts of Al ³⁺ did not reduce binding substantially, indicating that there was sufficient chelation capacity at this peptide concentration.

Kinetics of Al ¹⁸ F(IMP467) radiolabeling

[00161] Kinetic studies showed that binding was complete within 5 min at 107 ºC (5 min, 68%; 10 min, 61%; 15 min, 71%; and 30 min, 75%) with only moderate increases in isolated yield with reaction times as long as 30 min. A radiolabeling reaction of IMP467 performed at 50 ^o C showed that no binding was achieved at the lower temperature. Additional experiments, disclosed in the Examples below, show that under some conditions a limited amount of labeling can occur at reduced temperatures.

Effect of pH

[00162] The optimal pH for labeling was between 4.3 and 5.5. Yield ranged from 54% at pH 2.88; 70-77% at pH 3.99; 70% at pH 5; 41% at pH 6 to 3% at pH 7.3. The process could be expedited by eluting the ¹⁸ F- from the anion exchange column with nitrate or chloride ion instead of carbonate ion, which eliminates the need for adjusting the eluent to pH 4 with glacial acetic acid before mixing with the AlCl ₃ .

High-dose radiolabeling of IMP467

[00163] Five microliters of 2 mM Al ³⁺ stock solution were mixed with 50 μL of ¹⁸ F 1.3 GBq (35 mCi) followed by the addition of 20 μL of 2 mM IMP467 in 0.1 mM, pH 4.1 acetate buffer. The reaction solution was heated to 104 °C for 15 min and then purified on an HLB column (~10 min) as described above, isolating 0.68 GBq (18.4 mCi) of the purified peptide in 69% radiochemical yield with a specific activity of 17 GBq/ ^mol (460 Ci/mmol). The reaction time was 15 min and the purification time was 12 min. The reaction was started 10 min after the 1.3 GBq (35 mCi) ¹⁸ F- was purified, so the total time from the isolation of the ¹⁸ F- to the purified final product was 37 min with a 52% yield without correcting for decay. Human Serum Stability Test

[00164] An aliquot of the HLB purified peptide (~ 30 μL) was diluted with 200 μL human serum (previously frozen) and placed in the 37ºC HPLC sample chamber. Aliquots were removed at various time points and analyzed by HPLC. The HPLC analysis showed very high stability of the ¹⁸ F-labeled peptides in serum at 37°C for at least five hours (not shown). There was no detectable breakdown of the ¹⁸ F-labeled peptide after a five hour incubation in serum (not shown).

[00165] The IMP461 and IMP462 ligands have two carboxyl groups available to bind the aluminum whereas the NOTA ligand in IMP467 had four carboxyl groups. The serum stability study showed that the complexes with IMP467 were stable in serum under conditions replicating in vivo use. In vivo biodistribution studies with labeled IMP467 show that the Al ¹⁸ F-labeled peptide is stable under actual in vivo conditions (not shown).

[00166] Peptides can be labeled with ¹⁸ F rapidly (30 min) and in high yield by forming Al ¹⁸ F complexes that can be bound to a NOTA ligand on a peptide and at a specific activity of at least 17 GBq/μmol, without requiring HPLC purification. The Al ¹⁸ F(NOTA)-peptides are stable in serum and in vivo. Modifications of the NOTA ligand can lead to improvements in yield and specific activity, while still maintaining the desired in vivo stability of the Al ¹⁸ F(NOTA) complex, and being attached to a hydrophilic linker aids in the renal clearance of the peptide. Further, this method avoids the dry-down step commonly used to label peptides with ¹⁸ F. As shown in the following Examples, this new ¹⁸ F-labeling method is applicable to labeling of a broad spectrum of targeting peptides.

Optimized Labeling of Al ¹⁸ F(IMP467)

[00167] Optimized conditions for ¹⁸ F-labeling of IMP467 were identified. These consisted of eluting ¹⁸ F- with commercial sterile saline (pH 5-7), mixing with 20 nmol of AlCl ₃ and 40 nmol IMP467 in pH 4 acetate buffer in a total volume of 100 μL, heating to 102 ºC for 15 min, and performing SPE separation. High-yield (85%) and high specific activity (115 GBq/ μmol) were obtained with IMP467 in a single step, 30-min procedure after a simple solid-phase extraction (SPE) separation without the need for HPLC purification.

Al ¹⁸ F(IMP467) was stable in PBS or human serum, with 2% loss of ¹⁸ F- after incubation in either medium for 6 h at 37º C.

Concentration and purification of ¹⁸ F- [00168] Radiochemical-grade ¹⁸ F- needs to be purified and concentrated before use. We examined 4 different SPE purification procedures to process the ¹⁸ F- prior to its use. Most of the radiolabeling procedures were performed using ¹⁸ F- prepared by a conventional process. The ¹⁸ F- in 2 mL of water was loaded onto a SEP-PAK ^® Light, Waters Accell ^TM QMA Plus Cartridge that was pre-washed with 10 mL of 0.4M KHCO _3, followed by 10 mL water. After loading the ¹⁸ F- onto the cartridge, it was washed with 5 mL water to remove any dissolved metal and radiometal impurities. The isotope was then eluted with ~ 1 mL of 0.4M KHCO ₃ in several fractions to isolate the fraction with the highest concentration of activity. The eluted fractions were neutralized with 5 μL of glacial acetic acid per 100 μL of solution to adjust the eluent to pH 4-5.

[00169] In the second process, the QMA cartridge was washed with 10 mL pH 8.4, 0.5 M NaOAc followed by 10 mL DI H ₂ O. ¹⁸ F- was loaded onto the column as described above and eluted with 1 mL, pH 6, 0.05 M KNO ₃ in 200- μL fractions with 60-70% of the activity in one of the fractions. No pH adjustment of this solution was needed.

[00170] In the third process, the QMA cartridge was washed with 10 mL pH 8.4, 0.5 M NaOAc followed by 10 mL DI H ₂ O. The ¹⁸ F- was loaded onto the column as described above and eluted with 1 mL, pH 5-7, 0.154 M commercial normal saline in 200- μL fractions with 80% of the activity in one of the fractions. No pH adjustment of this solution was needed.

[00171] Finally, we devised a method to prepare a more concentrated and high-activity ¹⁸ F- solution, using tandem ion exchange. Briefly, Tygon tubing (1.27 cm long, 0.64 cm OD) was inserted into a TRICORN ^TM 5/20 column and filled with ~200 μL of AG 1-X8 resin, 100-200 mesh. The resin was washed with 6 mL 0.4 M K ₂ CO ₃ followed by 6 mL H ₂ O. A SEP-PAK ^® light Waters ACCELL ^TM Plus CM cartridge was washed with DI H ₂ O. Using a syringe pump, the crude ¹⁸ F- that was received in 5-mL syringe in 2 mL DI H ₂ O flowed slowly through the CM cartridge and the TRICORN ^TM column over ~5 min followed by a 6 mL wash with DI H ₂ O through both ion-binding columns. Finally, 0.4 M K ₂ CO ₃ was pushed through only the TRICORN ^TM column in 50- μL fractions. Typically, 40 to 60% of the eluted activity was in one 50- μL fraction. The fractions were collected in 2.0 mL free-standing screw-cap microcentrifuge tubes containing 5 μL glacial acetic acid to neutralize the carbonate solution. The elution vial with the most activity was then used as the reaction vial.

Example 12. Labeling by Addition of ¹⁸ F- to a Peptide Complexed With Aluminum

[00172] An HSG containing peptide (IMP 465, Al(NOTA)-D-Ala-D-Lys(HSG)-D-Tyr-D- Lys(HSG)-NH ₂ ) (SEQ ID NO:18) linked to macrocyclic NOTA complexed with aluminum, was successfully labeled with F-18. ¹⁸ F incorporation using 40 nmol of IMP 465 was 13.20%. An intermediate peptide, IMP 461, was made as described above. Then 25.7 mg of IMP461 was dissolved in 2 mL DI water to which was added 10.2 mg AlCl ^.

33H ₂ O and the resultant solution heated to 100 ^o C for 1 h. The crude reaction mixture was purified by RP-HPLC to yield 19.6 mg of IMP465.

[00173] For ¹⁸ F-labeling, 50 μL ¹⁸ F solution [0.702 mCi of ¹⁸ F-] and 20 μL (40 nmol) 2 mM IMP465 solution (0.1 M NaOAc, pH 4.18) was heated to 101 ^o C for 17 minutes. Reverse Phase HPLC analysis showed 15.38% (RT about 8.60 min) of the activity was attached to the peptide and 84.62% of the activity eluted at the void volume of the column (2.60 min).

[00174] In a separate experiment, the percent yield of ¹⁸ F-labeled peptide could be improved by varying the amount of peptide added. The percent yield observed for IMP465 was 0.27% at 10 nmol peptide, 1.8% at 20 nmol of peptide and 49% at 40 nmol of peptide.

[00175] IMP467 showed higher yield than IMP461 when peptide was pre-incubated with aluminum before exposure to ¹⁸ F. IMP467 was incubated with aluminum at room temperature and then frozen and lyophilized. The amount of aluminum added for the pre- incubation was varied.

Table 3. Labeling of IMP467 Pre-Incubated with Aluminum Before ¹⁸ F- is Added

IMP467 + Al Premixed, Frozen and Lyophilized Isolated Labeling Yield 40 nmol IMP467 + 10 nmol Al Premix 82%

40 nmol IMP467 + 20 nmol Al Premix 64%

40 nmol IMP467 + 30 nmol Al Premix 74%

40 nmol IMP467 + 6 nmol Al Normal Labeling (Mix Al+ ¹⁸ F first) 77%

[00176] The yields were comparable to those obtained when IMP467 is labeled by addition of an Al ¹⁸ F complex. Thus, ¹⁸ F labeling by addition of ¹⁸ F to a peptide with aluminum already bound to the chelating moiety is a feasible alternative approach to pre-incubating the metal with ¹⁸ F- prior to addition to the chelating moiety.

Example 13. Synthesis and Labeling of IMP468 Bombesin Peptide

[00177] The ¹⁸ F labeled targeting moieties are not limited to antibodies or antibody fragments, but rather can include any molecule that binds specifically or selectively to a cellular target that is associated with or diagnostic of a disease state or other condition that may be imaged by ¹⁸ F PET. Bombesin is a 14 amino acid peptide that is homologous to neuromedin B and gastrin releasing peptide, as well as a tumor marker for cancers such as lung and gastric cancer and neuroblastoma. IMP468 (NOTA-NH-(CH ₂ ) ₇ CO-Gln-Trp-Val- Trp-Ala-Val-Gly-His-Leu-Met-NH ₂ ; SEQ ID NO:19) was synthesized as a bombesin analogue and labeled with ¹⁸ F to target the gastrin-releasing peptide receptor.

[00178] The peptide was synthesized by Fmoc based solid phase peptide synthesis on Sieber amide resin, using a variation of a synthetic scheme reported in the literature (Prasanphanich et al., 2007, PNAS USA 104:12463-467). The synthesis was different in that a bis-t-butyl NOTA ligand was add to the peptide during peptide synthesis on the resin.

[00179] IMP468 (0.0139 g, 1.02 x 10 ^-5 mol) was dissolved in 203 μL of 0.5 M pH 4.13 NaOAc buffer. The peptide dissolved but formed a gel on standing so the peptide gel was diluted with 609 μL of 0.5 M pH 4.13 NaOAc buffer and 406 μL of ethanol to produce an 8.35 x 10 ^-3 M solution of the peptide. The ¹⁸ F was purified on a QMA cartridge and eluted with 0.4 M KHCO ₃ in 200 μL fractions, neutralized with 10 μL of glacial acetic acid. The purified ¹⁸ F, 40 μL, 1.13 mCi was mixed with 3 μL of 2 mM AlCl ₃ in pH 4, 0.1 M NaOAc buffer. IMP468 (59.2 μL, 4.94 x 10 ^-7 mol) was added to the Al ¹⁸ F solution and placed in a 108 ºC heating block for 15 min. The crude product was purified on an HLB column, eluted with 2 x 200 μL of 1:1 EtOH/H ₂ O to obtain the purified ¹⁸ F-labeled peptide in 34% yield. Example 14. Imaging of Tumors Using ¹⁸ F Labeled Bombesin

[00180] A NOTA-conjugated bombesin derivative (IMP468) was prepared as described above. We began testing its ability to block radiolabeled bombesin from binding to PC-3 cells as was done by Prasanphanich et al. (PNAS 104:12462-12467, 2007). Our initial experiment was to determine if IMP468 could specifically block bombesin from binding to PC-3 cells. We used IMP333 as a non-specific control. In this experiment, 3x10 ⁶ PC-3 cells were exposed to a constant amount (~50,000 cpms) of ¹²⁵ I-Bombesin (Perkin-Elmer) to which increasing amounts of either IMP468 or IMP333 was added. A range of 56 to 0.44 nM was used as our inhibitory concentrations.

[00181] The results showed that we could block the binding of ¹²⁵ I-BBN with IMP468 but not with the control peptide (IMP333) (not shown), thus demonstrating the specificity of IMP468. Prasanphanich indicated an IC ₅₀ for their peptide at 3.2 nM, which is approximately 7-fold lower than what we found with IMP468 (21.5 nM).

[00182] This experiment was repeated using a commercially available BBN peptide. We increased the amount of inhibitory peptide from 250 to 2 nM to block the ¹²⁵ I-BBN from binding to PC-3 cells. We observed very similar IC ₅₀ -values for IMP468 and the BBN positive control with an IC ₅₀ -value higher (35.9 nM) than what was reported previously (3.2 nM) but close to what the BBN control achieved (24.4 nM). [00183] To examine in vivo targeting, the distribution of Al ¹⁸ F(IMP468) was examined in scPC3 prostate cancer xenograft bearing nude male mice; alone vs. blocked with bombesin. For radiolabeling, aluminum chloride (10 µL, 2mM), 51.9 mCi of ¹⁸ F (from QMA cartridge), acetic acid, and 60 µL of IMP468 (8.45 mM in ethanol/NaOAc) were heated at 100 ºC for 15 min. The reaction mixture was purified on reverse phase HPLC. Fractions 40 and 41 (3.56, 1.91 mCi) were pooled and applied to HLB column for solvent exchange. The product was eluted in 800 µL (3.98 mCi) and 910 µCi remained on the column. iTLC developed in saturated NaCl showed 0.1 % unbound activity.

[00184] A group of six tumor-bearing mice were injected with Al ¹⁸ F(IMP468) (167 µCi, ~9 x10 ^-10 mol) and necropsied 1.5 h later. Another group of six mice were injected iv with 100 µg (6.2x10 ^-8 mol) of bombesin 18 min before administering Al ¹⁸ F(IMP468). The second group was also necropsied 1.5 h post injection. The data shows specific targeting of the tumor with [Al ¹⁸ F] IMP 468 (FIG.3). Tumor uptake of the peptide is reduced when bombesin was given 18 min before the Al ¹⁸ F(IMP468) (FIG.3). Biodistribution data indicates in vivo stability of Al ¹⁸ F(IMP468) for at least 1.5 h (not shown).

[00185] Larger tumors showed higher uptake of Al ¹⁸ F(IMP468), possibly due to higher receptor expression in larger tumors (not shown). The biodistribution data showed

Al ¹⁸ F(IMP468) tumor targeting that was in the same range as reported for the same peptide labeled with ⁶⁸ Ga by Prasanphanich et al. (not shown). The results demonstrate that the ¹⁸ F peptide labeling method can be used in vivo to target receptors that are upregulated in tumors, using targeting molecules besides antibodies. In this case, the IMP468 targeting took advantage of a naturally occurring ligand-receptor interaction. The tumor targeting was significant with a P value of P=0.0013. Many such ligand-receptor pairs are known and any such targeting interaction may form the basis for ¹⁸ F imaging, using the methods described herein.

Example 15. Synthesis and Labeling of Somatostatin Analog IMP466

[00186] Somatostatin is another non-antibody targeting peptide that is of use for imaging the distribution of somatostatin receptor protein. ¹²³ I-labeled octreotide, a somatostatin analog, has been used for imaging of somatostatin receptor expressing tumors (e.g., Kvols et al., 1993, Radiology 187:129-33; Leitha et al., 1993, J Nucl Med 34:1397-1402). However, ¹²³ I has not been of extensive use for imaging because of its expense, short physical half-life and the difficulty of preparing the radiolabeled compounds. The ¹⁸ F-labeling methods described herein are preferred for imaging of somatostatin receptor expressing tumors. IMP466 NOTA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl (SEQ ID NO:20)

[00187] A NOTA-conjugated derivative of the somatostatin analog octreotide (IMP466) was made by standard Fmoc based solid phase peptide synthesis to produce a linear peptide. The C-terminal Throl residue is threoninol. The peptide was cyclized by treatment with DMSO overnight. The peptide, 0.0073 g, 5.59 x 10 ^-6 mol was dissolved in 111.9 μL of 0.5 M pH 4 NaOAc buffer to make a 0.05 M solution of IMP466. The solution formed a gel over time so it was diluted to 0.0125 M by the addition of more 0.5 M NaOAc buffer.

[00188] ¹⁸ F was purified and concentrated with a QMA cartridge to provide 200 μL of ¹⁸ F in 0.4 M KHCO ₃ . The bicarbonate solution was neutralized with 10 μL of glacial acetic acid. A 40 μL aliquot of the neutralized ¹⁸ F eluent was mixed with 3 μL of 2 mM AlCl ₃ , followed by the addition of 40 μL of 0.0125 M IMP466 solution. The mixture was heated at 105ºC for 17 min. The reaction was then purified on a Waters 1 cc (30 mg) HLB column by loading the reaction solution onto the column and washing the unbound ¹⁸ F away with water (3 mL) and then eluting the radiolabeled peptide with 2 x 200 μL 1:1 EtOH water. The yield of the radiolabeled peptide after HLB purification was 34.6 %.

Effect of Ionic Strength

[00189] To lower the ionic strength of the reaction mixture escalating amounts of acetonitrile were added to the labeling mixture (final concentration: 0-80%). The yield of radiolabeled IMP466 increased with increasing concentration of acetonitrile in the medium. The optimal radiolabeling yield (98%) was obtained in a final concentration of 80% acetonitrile, despite the increased volume (500 μL in 80% vs.200 μL in 0% acetonitrile). In 0% acetonitrile the radiolabeling yield ranged from 36% to 55% in three experiments.

Example 16. Imaging of Neuroendocrine Tumors with an ¹⁸ F- and ⁶⁸ Ga-Labeled IMP466

[00190] Studies were performed to compare the PET images obtained using an ¹⁸ F versus ⁶⁸ Ga-labeled somatostatin analogue peptide and direct targeting to somatostatin receptor expressing tumors.

Methods

[00191] ¹⁸ F labeling - IMP466 was synthesized and ¹⁸ F-labeled by a variation of the method described in the Example above. A QMA SEPPAK® light cartridge (Waters, Milford, MA) with 2-6 GBq ¹⁸ F- (BV Cyclotron VU, Amsterdam, The Netherlands) was washed with 3 mL metal-free water. ¹⁸ F- was eluted from the cartridge with 0.4 M KHCO ₃ and fractions of 200 µL were collected. The pH of the fractions was adjusted to pH 4, with 10 µL metal-free glacial acid. Three µL of 2 mM AlCl ₃ in 0.1 M sodium acetate buffer, pH 4 were added. Then, 10-50 µL IMP 466 (10 mg/mL) were added in 0.5 M sodium acetate, pH 4.1. The reaction mixture was incubated at 100° C for 15 min unless stated otherwise. The radiolabeled peptide was purified on RP-HPLC. The Al ¹⁸ F(IMP466) containing fractions were collected and diluted two-fold with H ₂ O and purified on a 1-cc Oasis HLB cartridge (Waters, Milford, MA) to remove acetonitrile and TFA. In brief, the fraction was applied on the cartridge and the cartridge was washed with 3 mL H ₂ O. The radiolabeled peptide was then eluted with 2 x 200 µL 50% ethanol. For injection in mice, the peptide was diluted with 0.9% NaCl. A maximum specific activity of 45,000 GBq/mmol was obtained.

[00192] ⁶⁸ Ga labeling - IMP466 was labeled with ⁶⁸ GaCl ₃ eluted from a TiO ₂ -based 1,110 MBq ⁶⁸ Ge/ ⁶⁸ Ga generator (Cyclotron Co. Ltd., Obninsk, Russia) using 0.1 M ultrapure HCl (J.T. Baker, Deventer, The Netherlands). IMP466 was dissolved in 1.0 M HEPES buffer, pH 7.0. Four volumes of ⁶⁸ Ga eluate (120-240 MBq) were added and the mixture was heated at 95°C for 20 min. Then 50 mM EDTA was added to a final concentration of 5 mM to complex the non-incorporated ⁶⁸ Ga ³⁺ . The ⁶⁸ Ga-labeled IMP466 was purified on an Oasis HLB cartridge and eluted with 50% ethanol.

[00193] Octanol-water partition coefficient (log P _oct/water ) - To determine the lipophilicity of the radiolabeled peptides, approximately 50,000 dpm of the radiolabeled peptide was diluted in 0.5 mL phosphate-buffered saline (PBS). An equal volume of octanol was added to obtain a binary phase system. After vortexing the system for 2 min, the two layers were separated by centrifugation (100xg, 5 min). Three 100 μL samples were taken from each layer and radioactivity was measured in a well-type gamma counter (Wallac Wizard 3”, Perkin-Elmer, Waltham, MA).

[00194] Stability - Ten µL of the ¹⁸ F-labeled IMP466 was incubated in 500 µL of freshly collected human serum and incubated for 4 h at 37 °C. Acetonitrile was added and the mixture was vortexed followed by centrifugation at 1000xg for 5 min to precipitate serum proteins. The supernatant was analyzed on RP-HPLC as described above.

[00195] Cell culture - The AR42J rat pancreatic tumor cell line was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium (Gibco Life Technologies, Gaithersburg, MD, USA) supplemented with 4500 mg/L D-glucose, 10% (v/v) fetal calf serum, 2 mmol/L glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin. Cells were cultured at 37 °C in a humidified atmosphere with 5% CO ₂ . [00196] IC ₅₀ determination - The apparent 50% inhibitory concentration (IC ₅₀ ) for binding the somatostatin receptors on AR42J cells was determined in a competitive binding assay using Al ¹⁹ F(IMP466), ⁶⁹ Ga(IMP466) or ¹¹⁵ In(DTPA-octreotide) to compete for the binding of ¹¹¹ In(DTPA-octreotide).

[00197] Al ¹⁹ F(IMP466) was formed by mixing an aluminium fluoride (Al ¹⁹ F) solution (0.02 M AlCl ₃ in 0.5 M NaAc, pH 4, with 0.1 M NaF in 0.5 M NaAc, pH 4) with IMP466 and heating at 100º C for 15 min. The reaction mixture was purified by RP-HPLC on a C-18 column as described above.

[00198] ⁶⁹ Ga(IMP466) was prepared by dissolving gallium nitrate (2.3x10 ^-8 mol) in 30 µL mixed with 20 µL IMP466 (1 mg/mL) in 10 mM NaAc, pH 5.5, and heated at 90º C for 15 min. Samples of the mixture were used without further purification.

[00199] ¹¹⁵ In(DTPA-octreotide) was made by mixing indium chloride (1x10 ^-5 mol) with 10 µL DTPA-octreotide (1 mg/mL) in 50 mM NaAc, pH 5.5, and incubated at room temperature (RT) for 15 min. This sample was used without further purification. ¹¹¹ In(DTPA-octreotide) (OCTREOSCAN ^® ) was radiolabeled according to the manufacturer’s protocol.

[00200] AR42J cells were grown to confluency in 12-well plates and washed twice with binding buffer (DMEM with 0.5% bovine serum albumin). After 10 min incubation at RT in binding buffer, Al ¹⁹ F(IMP466), ⁶⁹ Ga(IMP466) or ¹¹⁵ In(DTPA-octreotide) was added at a final concentration ranging from 0.1-1000 nM, together with a trace amount (10,000 cpm) of ¹¹¹ In(DTPA-octreotide) (radiochemical purity >95%). After incubation at RT for 3 h, the cells were washed twice with ice-cold PBS. Cells were scraped and cell-associated radioactivity was determined. Under these conditions, a limited extent of internalization may occur. We therefore describe the results of this competitive binding assay as“apparent IC ₅₀ ” values rather than IC ₅₀ . The apparent IC ₅₀ was defined as the peptide concentration at which 50% of binding without competitor was reached.

[00201] Biodistribution studies - Male nude BALB/c mice (6-8 weeks) were injected subcutaneously in the right flank with 0.2 mL AR42J cell suspension of 10 ⁷ cells/mL.

Approximately two weeks after tumor cell inoculation when tumors were 5-8 mm in diameter, 370 kBq ¹⁸ F or ⁶⁸ Ga-labeled IMP466 was administered intravenously (n=5).

Separate groups (n=5) were injected with a 1,000-fold molar excess of unlabeled IMP466. One group of three mice was injected with unchelated [Al ¹⁸ F]. All mice were killed by CO ₂ /O ₂ asphyxiation 2 h post-injection (p.i.). Organs of interest were collected, weighed and counted in a gamma counter. The percentage of the injected dose per gram tissue (%ID/g) was calculated for each tissue. The animal experiments were approved by the local animal welfare committee and performed according to national regulations.

[00202] PET/CT imaging - Mice with s.c. AR42J tumors were injected intravenously with 10 MBq Al ¹⁸ F(IMP466) or ⁶⁸ Ga(IMP466). One and two hours after the injection of peptide, mice were scanned on an Inveon animal PET/CT scanner (Siemens Preclinical Solutions, Knoxville, TN) with an intrinsic spatial resolution of 1.5 mm (Visser et al, JNM, 2009). The animals were placed in a supine position in the scanner. PET emission scans were acquired over 15 minutes, followed by a CT scan for anatomical reference (spatial resolution 113 µm, 80 kV, 500 µA). Scans were reconstructed using Inveon Acquisition Workplace software version 1.2 (Siemens Preclinical Solutions, Knoxville, TN) using an ordered set expectation maximization-3D/maximum a posteriori (OSEM3D/MAP) algorithm with the following parameters: matrix 256 x 256 x 159, pixel size 0.43 x 0.43 x 0.8 mm ³ and MAP prior of 0.5 mm.

Results

[00203] Effect of buffer - The effect of the buffer on the labeling efficiency of IMP466 was investigated. IMP466 was dissolved in sodium citrate buffer, sodium acetate buffer, 2-(N- morpholino)ethanesulfonic acid (MES) or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer at 10 mg/mL (7.7 mM). The molarity of all buffers was 1 M and the pH was 4.1. To 200 µg (153 nmol) of IMP466 was added 100 µL [Al ¹⁸ F] (pH 4) and incubated at 100°C for 15 min. Radiolabeling yield and specific activity was determined with RP-HPLC. When using sodium acetate, MES or HEPES, radiolabeling yield was 49%, 44% and 46%, respectively. In the presence of sodium citrate, no labeling was observed (<1%). When the labeling reaction was carried out in sodium acetate buffer, the specific activity of the preparations was 10,000 GBq/mmol, whereas in MES and HEPES buffer a specific activity of 20,500 and 16,500 GBq/mmol was obtained, respectively.

[00204] Effect of AlCl ₃ concentration - Three stock solutions of AlCl ₃ in sodium acetate, pH 4.1 were prepared: 0.2, 2.0 and 20 mM. From these solutions, 3 µL was added to 200 µL of ¹⁸ F- to form [Al ¹⁸ F]. To these samples, 153 nmol of peptide was added and incubated for 15 min at 100°C. Radiolabeling yield was 49% after incubation at a final concentration of 6 nmol AlCl ₃ . Incubation with 0.6 nmol AlCl ₃ and 60 nmol AlCl ₃ resulted in a strong reduction of the radiolabeling yield: 10% and 6%, respectively.

[00205] Effect of amount of peptide - The effect of the amount of peptide on the labeling efficiency was investigated. IMP466 was dissolved in sodium acetate buffer, pH 4.1 at a concentration of 7.7 mM (10 mg/mL) and 38, 153 or 363 nmol of IMP466 was added to 200 µL [Al ¹⁸ F] (581-603 MBq). The radiolabeling yield increased with increasing amounts of peptide. At 38 nmol, radiolabeling yield ranged from 4-8%, at 153 nmol, the yield had increased to 42-49% and at the highest concentration the radiolabeling yield was 48-52%.

[00206] Octanol-water partition coefficient - To determine the lipophilicity of the ¹⁸ F and ⁶⁸ Ga-labeled IMP466, the octanol-water partition coefficients were determined. The log P _{octanol/water} value for the Al ¹⁸ F(IMP466) was -2.44 ± 0.12 and that of ⁶⁸ Ga(IMP466) was - 3.79 ± 0.07, indicating that the ¹⁸ F-labeled IMP 466 was slightly less hydrophilic.

[00207] Stability - The ¹⁸ F-labeled IMP466 did not show release of ¹⁸ F after incubation in human serum at 37 °C for 4 h, indicating excellent stability of the Al[ ¹⁸ F]NOTA complex.

[00208] IC ₅₀ determination - The apparent IC ₅₀ of Al ¹⁹ F(IMP466) was 3.6 ± 0.6 nM, whereas that for ⁶⁹ Ga(IMP466) was 13 ± 3 nM. The apparent IC ₅₀ of the reference peptide, ¹¹⁵ In(DTPA-octeotride) (OCTREOSCAN ^® ), was 6.3 ± 0.9 nM.

[00209] Biodistribution studies - The biodistribution of both Al ¹⁸ F(IMP466) and

6 ⁸ Ga(IMP466) was studied in nude BALB/c mice with s.c. AR42J tumors at 2 h p.i. (FIG.4). Al ¹⁸ F was included as a control. Tumor targeting of the Al ¹⁸ F(IMP466) was high with 28.3 ± 5.7 %ID/g accumulated at 2 h p.i. Uptake in the presence of an excess of unlabeled IMP466 was 8.6 ± 0.7 %ID/g, indicating that tumor uptake was receptor-mediated. Blood levels were very low (0.10 ± 0.07 %ID/g, 2 h pi), resulting in a tumor-to-blood ratio of 299 ± 88. Uptake in the organs was low, with specific uptake in receptor expressing organs such as adrenal glands, pancreas and stomach. Bone uptake of Al ¹⁸ F(IMP466) was negligible as compared to uptake of non-chelated Al ¹⁸ F (0.33 ± 0.07 vs.36.9 ± 5.0 %ID/g at 2 h p.i., respectively), indicating good in vivo stability of the ¹⁸ F-labeled NOTA-peptide.

[00210] The biodistribution of Al ¹⁸ F(IMP466) was compared to that of ⁶⁸ Ga(IMP466) (FIG. 4). Tumor uptake of ⁶⁸ Ga(IMP466) (29.2 ± 0.5 %ID/g, 2 h pi) was similar to that of Al ¹⁸ F- IMP 466 (p<0.001). Lung uptake of ⁶⁸ Ga(IMP466) was two-fold higher than that of

Al ¹⁸ F(IMP466) (4.0 ± 0.9 %ID/g vs.1.9 ± 0.4%ID/g, respectively). In addition, kidney retention of ⁶⁸ Ga(IMP466) was slightly higher than that of Al ¹⁸ F(IMP466) (16.2 ± 2.86 %ID/g vs.9.96 ± 1.27 %ID/g, respectively.

[00211] Fused PET and CT scans are shown in FIG.5. PET scans corroborated the biodistribution data. Both Al ¹⁸ F(IMP466) and ⁶⁸ Ga(IMP466) showed high uptake in the tumor and retention in the kidneys. The activity in the kidneys was mainly localized in the renal cortex. Again, the [Al ¹⁸ F] proved to be stably chelated by the NOTA chelator, since no bone uptake was observed. [00212] FIG.5 clearly shows that the distribution of an ¹⁸ F-labeled analog of somatostatin (octreotide) mimics that of a ⁶⁸ Ga-labeled somatostatin analog. These results are significant, since ⁶⁸ Ga-labeled octreotide PET imaging in human subjects with neuroendocrine tumors has been shown to have a significantly higher detection rate compared with conventional somatostatin receptor scintigraphy and diagnostic CT, with a sensitivity of 97%, a specificity of 92% and an accuracy of 96% (e.g., Gabriel et al., 2007, J Nucl Med 48:508-18). PET imaging with ⁶⁸ Ga-labeled octreotide is reported to be superior to SPECT analysis with ¹¹¹ In- labeled octreotide and to be highly sensitive for detection of even small meningiomas (Henze et al., 2001, J Nucl Med 42:1053-56). Because of the higher energy of ⁶⁸ Ga compared with ¹⁸ F, it is expected that ¹⁸ F based PET imaging would show even better spatial resolution than ⁶⁸ Ga based PET imaging. This is illustrated in FIG.5 by comparing the kidney images obtained with ¹⁸ F-labeled IMP466 (FIG.5, left) vs. ⁶⁸ Ga-labeled IMP466 (FIG.5, right). The PET images obtained with ⁶⁸ Ga show more diffuse margins and lower resolution than the images obtained with ¹⁸ F. These results demonstrate the superior images obtained with ¹⁸ F- labeled targeting moieties prepared using the methods and compositions described herein and confirm the utility of the described ¹⁸ F-labeling techniques for non-antibody targeting peptides.

Example 17. Comparison of ⁶⁸ Ga and ¹⁸ F PET Imaging Using Pretargeting

[00213] We compared PET images obtained using ⁶⁸ Ga- or ¹⁸ F-labeled peptides that were pretargeted with the bispecific TF2 antibody, prepared as described above. The half-lives of ⁶⁸ Ga (t _½ = 68 minutes) and ¹⁸ F (t _½ = 110 minutes) match with the pharmacokinetics of the radiolabeled peptide, since its maximum accretion in the tumor is reached within hours. Moreover, ⁶⁸ Ga is readily available from ⁶⁸ Ge/ ⁶⁸ Ga generators, whereas ¹⁸ F is the most commonly used and widely available radionuclide in PET.

Methods

[00214] Mice with s.c. CEA-expressing LS174T tumors received TF2 (6.0 nmol; 0.94 mg) and 5 MBq ⁶⁸ Ga(IMP288) (0.25 nmol) or Al ¹⁸ F(IMP449) (0.25 nmol) intravenously, with an interval of 16 hours between the injection of the bispecific antibody and the radiolabeled peptide. One or two hours after the injection of the radiolabeled peptide, PET/CT images were acquired and the biodistribution of the radiolabeled peptide was determined. Uptake in the LS174T tumor was compared with that in an s.c. CEA-negative SK-RC 52 tumor.

Pretargeted immunoPET imaging was compared with ¹⁸ F-FDG PET imaging in mice with an s.c. LS174T tumor and contralaterally an inflamed thigh muscle. IMP288 DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH ₂ (SEQ ID NO:21)

[00215] Pretargeting– The bispecific TF2 antibody was made by the DNL method, as described above. TF2 is a trivalent bispecific antibody comprising an HSG-binding Fab fragment from the h679 antibody and two CEA-binding Fab fragments from the hMN-14 antibody. The DOTA-conjugated, HSG-containing peptide IMP288 was synthesized by peptide synthesis as described above. The IMP449 peptide, synthesized as described above, contains a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelating moiety to facilitate labeling with ¹⁸ F. As a tracer for the antibody component, TF2 was labeled with ¹²⁵ I (Perkin Elmer, Waltham, MA) by the iodogen method (Fraker and Speck, 1978, Biochem Biophys Res Comm 80:849-57), to a specific activity of 58 MBq/nmol.

[00216] Labeling of IMP288 - IMP288 was labeled with ¹¹¹ In (Covidien, Petten, The Netherlands) for biodistribution studies at a specific activity of 32 MBq/nmol under strict metal-free conditions. IMP288 was labeled with ⁶⁸ Ga eluted from a TiO-based 1,110 MBq ⁶⁸ Ge/ ⁶⁸ Ga generator (Cyclotron Co. Ltd., Obninsk Russia) using 0.1 M ultrapure HCl. Five 1 ml fractions were collected and the second fraction was used for labeling the peptide. One volume of 1.0 M HEPES buffer, pH 7.0 was added to 3.4 nmol IMP 288. Four volumes of ⁶⁸ Ga eluate (380 MBq) were added and the mixture was heated to 95 ^o C for 20 min. Then 50 mM EDTA was added to a final concentration of 5 mM to complex the non-chelated ⁶⁸ Ga ³⁺ . The ⁶⁸ Ga(IMP288) peptide was purified on a 1-mL Oasis HLB-cartridge (Waters, Milford, MA). After washing the cartridge with water, the peptide was eluted with 25% ethanol. The procedure to label IMP288 with ⁶⁸ Ga was performed within 45 minutes, with the preparations being ready for in vivo use.

[00217] Labeling of IMP449 - IMP449 was labeled with ¹⁸ F as described above. 555-740 MBq ¹⁸ F (B.V. Cyclotron VU, Amsterdam, The Netherlands) was eluted from a QMA cartridge with 0.4 M KHCO ₃ . The Al ¹⁸ F activity was added to a vial containing the peptide (230 µg) and ascorbic acid (10 mg). The mixture was incubated at 100 °C for 15 min. The reaction mixture was purified by RP-HPLC. After adding one volume of water, the peptide was purified on a 1-mL Oasis HLB cartridge. After washing with water, the radiolabeled peptide was eluted with 50% ethanol. Al ¹⁸ F(IMP449) was prepared within 60 minutes, with the preparations being ready for in vivo use.

[00218] Radiochemical purity of ¹²⁵ I-TF2, ¹¹¹ In(IMP288) and ⁶⁸ Ga(IMP288) and

Al ¹⁸ F(IMP449) preparations used in the studies always exceeded 95%. [00219] Animal experiments - Experiments were performed in male nude BALB/c mice (6- 8 weeks old), weighing 20-25 grams. Mice received a subcutaneous injection with 0.2 mL of a suspension of 1 x 10 ⁶ LS174T-cells, a CEA-expressing human colon carcinoma cell line (American Type Culture Collection, Rockville, MD, USA). Studies were initiated when the tumors reached a size of about 0.1-0.3 g (10-14 days after tumor inoculation).

[00220] The interval between TF2 and IMP288 injection was 16 hours, as this period was sufficient to clear unbound TF2 from the circulation. In some studies ¹²⁵ I-TF2, (0.4 MBq) was co-injected with unlabeled TF2. IMP288 was labeled with either ¹¹¹ In or ⁶⁸ Ga. IMP449 was labeled with ¹⁸ F. Mice received TF2 and IMP288 intravenously (0.2 mL). One hour after the injection of ⁶⁸ Ga-labeled peptide, and two hours after injection of Al ¹⁸ F(IMP449), mice were euthanized by CO ₂ /O ₂ , and blood was obtained by cardiac puncture and tissues were dissected.

[00221] PET images were acquired with an Inveon animal PET/CT scanner (Siemens Preclinical Solutions, Knoxville, TN). PET emission scans were acquired for 15 minutes, preceded by CT scans for anatomical reference (spatial resolution 113 µm, 80 kV, 500 µA, exposure time 300 msec).

[00222] After imaging, tumor and organs of interest were dissected, weighed and counted in a gamma counter with appropriate energy windows for ¹²⁵ I, ¹¹¹ In, ⁶⁸ Ga or ¹⁸ F. The percentage-injected dose per gram tissue (% ID/g) was calculated.

Results

[00223] Within 1 hour, pretargeted immunoPET resulted in high and specific targeting of ⁶⁸ Ga-IMP288 in the tumor (10.7 ± 3.6 % ID/g), with very low uptake in the normal tissues (e.g., tumor/blood 69.9 ± 32.3), in a CEA-negative tumor (0.35 ± 0.35 % ID/g), and inflamed muscle (0.72 ± 0.20 % ID/g). Tumors that were not pretargeted with TF2 also had low ⁶⁸ Ga(IMP288) uptake (0.20 ± 0.03 % ID/g). [ ¹⁸ F]FDG accreted efficiently in the tumor (7.42 ± 0.20% ID/g), but also in the inflamed muscle (4.07 ± 1.13 % ID/g) and a number of normal tissues, and thus pretargeted ⁶⁸ Ga-IMP 288 provided better specificity and sensitivity. The corresponding PET/CT images of mice that received ⁶⁸ Ga(IMP288) or Al ¹⁸ F(IMP449) following pretargeting with TF2 clearly showed the efficient targeting of the radiolabeled peptide in the subcutaneous LS174T tumor, while the inflamed muscle was not visualized. In contrast, with ¹⁸ F-FDG the tumor as well as the inflammation was clearly delineated.

[00224] Dose optimization - The effect of the TF2 bsMAb dose on tumor targeting of a fixed 0.01 nmol (15 ng) dose of IMP288 was determined. Groups of five mice were injected intravenously with 0.10, 0.25, 0.50 or 1.0 nmol TF2 (16, 40, 80 or 160 µg respectively), labeled with a trace amount of ¹²⁵ I (0.4 MBq). One hour after injection of ¹¹¹ In(IMP288) (0.01 nmol, 0.4 MBq), the biodistribution of the radiolabels was determined.

[00225] TF2 cleared rapidly from the blood and the normal tissues. Eighteen hours after injection the blood concentration was less than 0.45 % ID/g at all TF2 doses tested. Targeting of TF2 in the tumor was 3.5% ID/g at 2 h p.i. and independent of TF2 dose (data not shown). At all TF2 doses ¹¹¹ In(IMP288) accumulated effectively in the tumor (not shown). At higher TF2 doses enhanced uptake of ¹¹¹ In(IMP288) in the tumor was observed: at 1.0 nmol TF2 dose maximum targeting of IMP288 was reached (26.2 ± 3.8% ID/g). Thus at the 0.01 nmol peptide dose highest tumor targeting and tumor-to-blood ratios were reached at the highest TF2 dose of 1.0 nmol (TF2:IMP288 molar ratio = 100:1). Among the normal tissues, the kidneys had the highest uptake of ¹¹¹ In(IMP288) (1.75 ± 0.27% ID/g) and uptake in the kidneys was not affected by the TF2 dose (not shown). All other normal tissues had very low uptake, resulting in extremely high tumor-to-nontumor ratios, exceeding 50:1 at all TF2 doses tested (not shown).

[00226] For PET imaging using ⁶⁸ Ga-labeled IMP288, a higher peptide dose is required, because a minimum activity of 5-10 MBq ⁶⁸ Ga needs to be injected per mouse if PET imaging is performed 1 h after injection. The specific activity of the ⁶⁸ Ga(IMP288) preparations was 50-125 MBq/nmol at the time of injection. Therefore, for PET imaging at least 0.1-0.25 nmol of IMP288 had to be administered. The same TF2:IMP288 molar ratios were tested at 0.1 nmol IMP288 dose. LS174T tumors were pretargeted by injecting 1.0, 2.5, 5.0 or 10.0 nmol TF2 (160, 400, 800 or 1600 µg). In contrast to the results at the lower peptide dose, ¹¹¹ In(IMP288) uptake in the tumor was not affected by the TF2 doses (15% ID/g at all doses tested, data not shown). TF2 targeting in the tumor in terms of % ID/g decreased at higher doses (3.21 ± 0.61% ID/g versus 1.16 ± 0.27% ID/g at an injected dose of 1.0 nmol and 10.0 nmol, respectively) (data not shown). Kidney uptake was also independent of the bsMAb dose (2% ID/g). Based on these data we selected a bsMAb dose of 6.0 nmol for targeting 0.1-0.25 nmol of IMP288 to the tumor.

[00227] PET imaging - To demonstrate the effectiveness of pretargeted immunoPET imaging with TF2 and ⁶⁸ Ga(IMP288) to image CEA-expressing tumors, subcutaneous tumors were induced in five mice. In the right flank an s.c. LS174T tumor was induced, while at the same time in the same mice 1 x 10 ⁶ SK-RC 52 cells were inoculated in the left flank to induce a CEA-negative tumor. Fourteen days later, when tumors had a size of 0.1-0.2 g, the mice were pretargeted with 6.0 nmol ¹²⁵ I-TF2 intravenously. After 16 hours the mice received 5 MBq ⁶⁸ Ga(IMP288) (0.25 nmol, specific activity of 20 MBq/nmol). A separate group of three mice received the same amount of ⁶⁸ Ga-IMP 288 alone, without pretargeting with TF2.

PET/CT scans of the mice were acquired 1 h after injection of the ⁶⁸ Ga(IMP288).

[00228] The biodistribution of ¹²⁵ I-TF2 and [ ⁶⁸ Ga]IMP288 in the mice are shown in FIG.6. Again high uptake of the bsMAb (2.17 ± 0.50 % ID/g) and peptide (10.7 ± 3.6 % ID/g) in the tumor was observed, with very low uptake in the normal tissues (tumor-to-blood ratio: 64 ± 22). Targeting of ⁶⁸ Ga(IMP288) in the CEA-negative tumor SK-RC 52 was very low (0.35 ± 0.35 % ID/g). Likewise, tumors that were not pretargeted with TF2 had low uptake of ⁶⁸ Ga(IMP288) (0.20 ± 0.03 % ID/g), indicating the specific accumulation of IMP288 in the CEA-expressing LS174T tumor.

[00229] The specific uptake of ⁶⁸ Ga(IMP288) in the CEA-expressing tumor pretargeted with TF2 was clearly visualized in a PET image acquired 1 h after injection of the ⁶⁸ Ga-labeled peptide (not shown). Uptake in the tumor was evaluated quantitatively by drawing regions of interest (ROI), using a 50% threshold of maximum intensity. A region in the abdomen was used as background region. The tumor-to-background ratio in the image of the mouse that received TF2 and ⁶⁸ Ga(IMP288) was 38.2.

[00230] We then examined pretargeted immunoPET with ¹⁸ F-FDG. In two groups of five mice a s.c. LS174T tumor was induced on the right hind leg and an inflammatory focus in the left thigh muscle was induced by intramuscular injection of 50 µL turpentine (18). Three days after injection of the turpentine, one group of mice received 6.0 nmol TF2, followed 16 h later by 5 MBq ⁶⁸ Ga(IMP288) (0.25 nmol). The other group received ¹⁸ F-FDG (5 MBq). Mice were fasted during 10 hours prior to the injection and anaesthetized and kept warm at 37 ^o C until euthanasia, 1 h postinjection.

[00231] Uptake of ⁶⁸ Ga(IMP288) in the inflamed muscle was very low, while uptake in the tumor in the same animal was high (0.72 ± 0.20 % ID/g versus 8.73 ± 1.60 % ID/g, p<0.05, FIG.7). Uptake in the inflamed muscle was in the same range as uptake in the lungs, liver and spleen (0.50 ± 0.14 % ID/g, 0.72 ± 0.07 % ID/g, 0.44 ± 0.10 % ID/g, respectively).

Tumor-to-blood ratio of ⁶⁸ Ga(IMP288) in these mice was 69.9 ± 32.3; inflamed muscle-to- blood ratio was 5.9 ± 2.9; tumor-to-inflamed muscle ratio was 12.5 ± 2.1. In the other group of mice ¹⁸ F-FDG accreted efficiently in the tumor (7.42 ± 0.20% ID/g, tumor-to-blood ratio 6.24 ± 1.5, Figure 4). ¹⁸ F-FDG also substantially accumulated in the inflamed muscle (4.07 ± 1.13 % ID/g), with inflamed muscle-to-blood ratio of 3.4 ± 0.5, and tumor-to-inflamed muscle ratio of 1.97 ± 0.71.

[00232] The corresponding PET/CT image of a mouse that received ⁶⁸ Ga(IMP288), following pretargeting with TF2, clearly showed the efficient accretion of the radiolabeled peptide in the tumor, while the inflamed muscle was not visualized (FIG.8). In contrast, on the images of the mice that received ¹⁸ F-FDG, the tumor as well as the inflammation was visible (FIG.8). In the mice that received ⁶⁸ Ga(IMP288), the tumor-to-inflamed tissue ratio was 5.4; tumor-to-background ratio was 48; inflamed muscle-to-background ratio was 8.9. ¹⁸ F-FDG uptake had a tumor-to-inflamed muscle ratio of 0.83; tumor-to-background ratio was 2.4; inflamed muscle-to-background ratio was 2.9.

[00233] The pretargeted immunoPET imaging method was tested using the Al ¹⁸ F(IMP449). Five mice received 6.0 nmol TF2, followed 16 h later by 5 MBq Al[ ¹⁸ F]IMP449 (0.25 nmol). Three additional mice received 5 MBq Al ¹⁸ F(IMP449) without prior administration of TF2, while two control mice were injected with [Al ¹⁸ F] (3 MBq). The results of this experiment are summarized in FIG.9. Uptake of Al ¹⁸ F(IMP449) in tumors pretargeted with TF2 was high (10.6 ± 1.7 % ID/g), whereas it was very low in the non-pretargeted mice (0.45 ± 0.38 %ID/g). [Al ¹⁸ F] accumulated in the bone (50.9 ± 11.4 %ID/g), while uptake of the radiolabeled IMP449 peptide in the bone was very low (0.54 ± 0.2 % ID/g), indicating that the Al ¹⁸ F(IMP449) was stable in vivo. The biodistribution of Al ¹⁸ F(IMP449) in the TF2 pretargeted mice with s.c. LS174T tumors were highly similar to that of ⁶⁸ Ga(IMP288).

[00234] The PET-images of pretargeted immunoPET with Al ¹⁸ F(IMP449) show the same intensity in the tumor as those with ⁶⁸ Ga(IMP288), but the resolution of the ¹⁸ F PET images were superior to those of the ⁶⁸ Ga. (FIG.10). The tumor-to-background ratio of the

Al ¹⁸ F(IMP449) signal was 66.

Conclusions

[00235] The present study showed that pretargeted immunoPET with the anti-CEA x anti- HSG bispecific antibody TF2 in combination with a ⁶⁸ Ga- or ¹⁸ F-labeled di-HSG-DOTA- peptide is a rapid and specific technique for PET imaging of CEA-expressing tumors.

[00236] Pretargeted immunoPET with TF2 in combination with ⁶⁸ Ga(IMP288) or

Al ¹⁸ F(IMP449) involves two intravenous administrations. An interval between the infusion of the bsMAb and the radiolabeled peptide of 16 h was used. After 16 h most of the TF2 had cleared from the blood (blood concentration <1% ID/g), preventing complexation of TF2 and IMP288 in the circulation.

[00237] For these studies the procedure to label IMP288 with ⁶⁸ Ga was optimized, resulting in a one-step labeling technique. We found that purification on a C18/HLB cartridge was needed to remove the ⁶⁸ Ga colloid that is formed when the peptide was labeled at specific activities exceeding 150 GBq/nmol at 95 ^o C. If a preparation contains a small percentage of colloid and is administered intravenously, the ⁶⁸ Ga colloid accumulates in tissues of the mononuclear phagocyte system (liver, spleen, and bone marrow), deteriorating image quality. The ⁶⁸ Ga-labeled peptide could be rapidly purified on a C18-cartridge. Radiolabeling and purification for administration could be accomplished within 45 minutes.

[00238] The half-life of ⁶⁸ Ga matches with the kinetics of the IMP288 peptide in the pretargeting system: maximum accretion in the tumor is reached within 1 h. ⁶⁸ Ga can be eluted twice a day form a ⁶⁸ Ge/ ⁶⁸ Ga generator, avoiding the need for an on-site cyclotron. However, the high energy of the positrons emitted by ⁶⁸ Ga (1.9 MeV) limits the spatial resolution of the acquired images to 3 mm, while the intrinsic resolution of the microPET system is as low as 1.5 mm.

[00239] ¹⁸ F, the most widely used radionuclide in PET, has an even more favorable half-life for pretargeted PET imaging (t _½ = 110 min). The NOTA-conjugated peptide IMP449 was labeled with ¹⁸ F, as described above. Like labeling with ⁶⁸ Ga, it is a one-step procedure. Labeling yields as high as 50% were obtained. The biodistribution of Al ¹⁸ F(IMP449) was highly similar to that of ⁶⁸ Ga-labeled IMP288, suggesting that with this labeling method ¹⁸ F is a residualizing radionuclide.

[00240] In contrast with FDG-PET, pretargeted radioimmunodetection is a tumor specific imaging modality. Although a high sensitivity and specificity for FDG-PET in detecting recurrent colorectal cancer lesions has been reported in patients (Huebner et al., 2000, J Nucl Med 41:11277-89), FDG-PET images could lead to diagnostic dilemmas in discriminating malignant from benign lesions, as indicated by the high level of labeling observed with inflammation. In contrast, the high tumor-to-background ratio and clear visualization of CEA-positive tumors using pretargeted immunoPET with TF2 ⁶⁸ Ga- or ¹⁸ F-labeled peptides supports the use of the described methods for clinical imaging of cancer and other conditions. Apart from detecting metastases and discriminating CEA-positive tumors from other lesions, pretargeted immunoPET could also be used to estimate radiation dose delivery to tumor and normal tissues prior to pretargeted radioimmunotherapy. As TF2 is a humanized antibody, it has a low immunogenicity, opening the way for multiple imaging or treatment cycles.

Example 18. Synthesis of Folic Acid NOTA conjugate

[00241] Folic acid is activated as described (Wang et. al. Bioconjugate Chem.1996, 7, 56- 62.) and conjugated to Boc-NH-CH ₂ -CH ₂ -NH ₂ . The conjugate is purified by

chromatography. The Boc group is then removed by treatment with TFA. The amino folate derivative is then mixed with p-SCN-Bn-NOTA (Macrocyclics) in a carbonate buffer. The product is then purified by HPLC. The folate-NOTA derivative is labeled with Al ¹⁸ F as described in the preceding Examples and then HPLC purified. The ¹⁸ F-labeled folate is injected i.v. into a subject and successfully used to image the distribution of folate receptors, for example in cancer or inflammatory diseases (see, e.g., Ke et al., Advanced Drug Delivery Reviews, 56:1143-60, 2004).

Example 19. Imaging of Angiogenesis Receptors by ¹⁸ F-Labeling

[00242] Labeled Arg-Gly-Asp (RGD) peptides have been used for imaging of angiogenesis, for example in ischemic tissues, where α _v β ₃ integrin is involved. (Jeong et al., J. Nucl. Med. 2008, Apr.15 epub). RGD is conjugated to SCN-Bn-NOTA according to Jeong et al. (2008). [Al ¹⁸ F] is attached to the NOTA-derivatized RGD peptide as described above, by mixing aluminum stock solution with ¹⁸ F and the derivatized RGD peptide and heating at 110 ˚C for 15 min, using an excess of peptide to drive the labeling reaction towards completion. The ¹⁸ F labeled RGD peptide is used for in vivo biodistribution and PET imaging as disclosed in Jeong et al. (2008). The [Al ¹⁸ F] conjugate of RGD-NOTA is taken up into ischemic tissues and provides PET imaging of angiogenesis.

Example 20. Effect of Organic Solvents on F-18 Labeling

[00243] The affinity of chelating moieties such as NETA and NOTA for aluminum is much higher than the affinity of aluminum for ¹⁸ F. The affinity of Al for ¹⁸ F is affected by factors such as the ionic strength of the solution, since the presence of other counter-ions tends to shield the positively charged aluminum and negatively charged fluoride ions from each other and therefore to decrease the strength of ionic binding. Therefore low ionic strength medium should increase the effective binding of Al and ¹⁸ F.

[00244] An initial study adding ethanol to the ¹⁸ F reaction was found to increase the yield of radiolabeled peptide. IMP461 was prepared as described above. Table 4. ¹⁸ F-labeling of IMP461 in ethanol

*Yield after HLB column purification, Rxn # 1,2 and 4 were heated to 101 ^o C for 5 minutes, Rxn # 3 was heated for 1 minute in a microwave oven. [00245] Preliminary results showed that addition of ethanol to the reaction mixture more than doubled the yield of ¹⁸ F-labeled peptide. Table 4 also shows that microwave irradiation can be used in place of heating to promote incorporation of [Al ¹⁸ F] into the chelating moiety of IMP461. Sixty seconds of microwave radiation (#3) appeared to be slightly less (18%) effective than heating to 101°C for 5 minutes (#1).

[00246] The effect of additional solvents on Al ¹⁹ F complexation of peptides was examined. In each case, the concentration of reactants was the same and only the solvent varied.

Reaction conditions included mixing 25 μL Na ¹⁹ F + 20 μL AlCl ₃ + 20 μL IMP461 + 60 μL solvent, followed by heating at 101°C for 5 min. Table 5 shows that the presence of a solvent does improve the yields of Al ¹⁹ F(IMP461) (i.e., IMP473) considerably. Table 5. Complexation of IMP 461 with Al ¹⁹ F in various solvents

=

Example 21. Elution of ¹⁸ F- with Bicarbonate

[00247] ¹⁸ F, 10.43 mCi, was received in 2 mL in a syringe. The solution was passed through a SEP-PAK® Light, WATERS® ACCELL ^TM Plus QMA Cartridge. The column was then washed with 5 mL of DI water. The ¹⁸ F was eluted with 0.4 M KHCO ₃ in fractions as shown in Table 6 below. T l El i n f MA r ri i h KH

[00248] The effects of the amount of additional solvent (CH ₃ CN) on ¹⁸ F-labeling of IMP461 was examined. In each case, the concentration of reactants was the same and only the amount of solvent varied. Reaction conditions included mixing 10 μL AlCl ¹⁸

3 + 20 μL F + 20 μL IMP461 + CH ₃ CN followed by heating at 101°C for 5 min. Table 7 shows that following an initial improvement the labeling efficiency decreases in the presence of excess solvent.

Table 7. ¹⁸ F-labeling of IMP461 using varying amounts of CH ₃ CN

Example 22. High Dose Radiolabeling of IMP461

[00249] ¹⁸ F-, 163 mCi, was received in 2 mL in a syringe. The solution was passed through a SEP-PAK® Light, WATERS® ACCELL ^TM Plus QMA Cartridge. The column was then washed with 5 mL of DI water. The ¹⁸ F- was eluted with 0.4 M K ₂ CO ₃ in fractions as shown in Table 8. Table 8. High Dose Labeling

[00250] An aluminum chloride solution (10 μL, 2 mM in pH 4, 2 mM NaOAc) was added to vial number 3 from Table 8. The peptide (20 μL, 2 mM in pH 4, 2 mM NaOAc) was added to the vial followed by the addition of 170 μL of CH ₃ CN. The solution was heated for 10 min at 103ºC the diluted with 6 mL of water. The solution was pulled into a 10 mL syringe and injected onto two WATERS® HLB Plus Cartridges arranged in tandem. The cartridges were washed with 8 mL water. The radiolabeled peptide Al ¹⁸ F(IMP461) was then eluted with 10 mL 1:1 EtOH/H ₂ O, 30.3 mCi, 63.5% yield, specific activity 750 Ci/mmol. The labeled peptide was free of unbound ¹⁸ F by HPLC. The total reaction and purification time was 20 min.

Example 23. Preparation of Al ¹⁹ F Peptides

[00251] Products containing ²⁷ Al and/or ¹⁹ F are useful for certain applications like MR imaging. An improved method for preparing [Al ¹⁹ F] compounds was developed. IMP461 was prepared as described above and labeled with ¹⁹ F. Reacting IMP461 with AlCl ₃ + NaF resulted in the formation of three products (not shown). However, by reacting IMP461 with AlF ^.

33H ₂ O we obtained a higher yield of Al ¹⁹ F(IMP461).

[00252] Synthesis of IMP 473: [Al ¹⁹ F(IMP461)] To (14.1 mg, 10.90 ^mol) IMP461 in 2 mL NaOAc (2 mM, pH 4.18) solution added (4.51 mg, 32.68 ^mol) AlF ^.

33H ₂ O and 500 μL ethanol. The pH of the solution to adjusted to 4.46 using 3 μL 1 N NaOH and heated in a boiling water bath for 30 minutes. The crude reaction mixture was purified by preparative RP-HPLC to yield 4.8 mg (32.9%) of IMP 473. HRMS (ESI-TOF) MH ⁺ expected

1337.6341; found 1337.6332

[00253] These results demonstrate that ¹⁹ F labeled molecules may be prepared by forming metal- ¹⁹ F complexes and binding the metal- ¹⁹ F to a chelating moiety, as discussed above for 1 ⁸ F labeling. The instant Example shows that a targeting peptide of use for pretargeting detection, diagnosis and/or imaging may be prepared using the instant methods.

Example 24. Synthesis and Labeling of IMP479, IMP485 and IMP487 [00254] The structures of additional peptides (IMP479, IMP485, and IMP487) designed for ¹⁸ F-labeling are shown in FIG.11 to FIG.13. IMP485 is shown in FIG.12. IMP485 was made on Sieber Amide resin by adding the following amino acids to the resin in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved, Fmoc-D-Tyr(But)-OH, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloc was cleaved, (tert-Butyl) ₂ NOTA-MPAA (methyl phenyl acetic acid). The peptide was then cleaved from the resin and purified by RP- HPLC to yield 44.8 mg of IMP485.

Synthesis of Bis-t-butyl-NOTA-MPAA: (tBu) ₂ NOTA-MPAA for IMP485 Synthesis

[00255] To a solution of 4-(bromomethyl) phenyl acetic acid (Aldrich 310417) (0.5925 g, 2.59 mmol) in CH ₃ CN (anhydrous) (50 mL) at 0 ^o C was added dropwise over 1 h a solution of NO2AtBu (1.0087 g, 2.82 mmol) in CH ₃ CN (50 mL). After 4 h anhydrous K ₂ CO ₃ (0.1008 g, 0.729 mmol) was added to the reaction mixture and allowed to stir at room temperature overnight. Solvent was evaporated and the crude mixture was purified by preparative RP- HPLC to yield a white solid (0.7132 g, 54.5%).

[00256] Although this is a one step synthesis, yields were low due to esterification of the product by 4-(bromomethyl)phenylacetic acid. Alkylation of NO2AtBu using methyl(4- bromomethyl) phenylacetate was employed to prevent esterification (FIG.14).

1 ⁸ F-Labeling

[00257] For ¹⁸ F labeling studies in water, to 40 nmol of IMP479/485/487 (formulated using trehalose + ascorbic acid + AlCl ₃ ) was added 250 μL ¹⁸ F- solution [~ 919 - 1112 ^Ci of ¹⁸ F-] and heated to 101 ^o C for 15 minutes. In ethanol, to 40 nmol of IMP479/485/487 (formulated using trehalose + ascorbic acid + AlCl ₃ ) was added 250 μL ¹⁸ F- solution [1.248– 1.693 mCi of ¹⁸ F-], 100 μL EtOH and heated to 101 ^o C for 15 minutes. An exemplary experiment showing labeling of different peptides is shown in Table 9. With minimal optimization, radiolabeling of IMP485 has been observed with up to an 88% yield and a specific activity of 2,500 Ci/mmol. At this specific activity, HPLC purification of the radiolabeled peptide is not required for in vivo PET imaging using the radiolabeled peptide. Table 9. Labeling of IMP479, IMP485 and IMP487

Stability in Serum

[00258] A kit containing 40 nmol of IMP485 or IMP487, 20 nmol AlCl ₃ , 0.1 mg ascorbic acid and 0.1 g trehalose adjusted to pH 3.9 was reconstituted with purified ¹⁸ F- in 200 μL saline and heated 106 ºC for 15 min. The reaction mixture was then diluted with 800 μL water and placed on an HLB column. After washing, the column was eluted with 2 x 200 μL 1:1 EtOH/H ₂ O to obtain the purified Al ¹⁸ F(IMP485) in 64.6 % isolated yield. The radiolabeled peptide in 50 μL was mixed with 250 μL of fresh human serum in a vial and incubated at 37ºC.

[00259] Both radiolabeled peptides were stable at 37ºC in fresh human serum over the four hours tested (not shown).

Effect of Bulking Agents on Yield of Lyophilized Peptide

[00260] An experiment was performed to compare yield using IMP485 kits (40 nmol) with different bulking agents labeled with 2 mCi of F-18 (from the same batch of F-18) in 200 microliters of saline. The bulking agents were introduced at a concentration of 5% by weight in water with a dose of 200 microliters/vial. We tested sorbitol, trehalose, sucrose, mannitol and glycine as bulking agents. Results are shown in Table 10 Table 10. Effects of Bulking Agents on Radiolabeling Yield

[00261] Sorbitol, mannitol and trehalose all gave radiolabeled product in the same yield. The mannitol kit and the trehalose kit both formed nice cakes. The sucrose kit and the glycine kit both had significantly lower yields. We also recently tested 2-hydroxypropyl-beta- cyclodextrin as a bulking agent and obtained a 58 % yield for the 40 nmol kit. We have found that radiolabeling is very pH sensitive and needs to be tuned to the ligand and possibly even to the peptide + the ligand. In the case of IMP485 the optimal pH is pH 4.0 ± 0.2 whereas the optimal pH for IMP467 was pH 4.5 ± 0.5. In both cases the yields drop off rapidly outside the ideal pH zone.

Time Course of Labeling

[00262] The time course for labeling of IMP485 was examined. To 40 nmol of IMP485 (formulated using trehalose + AlCl ₃ (20 nmol ) + ascorbic acid) was added ~ 200– 250 μL ¹⁸ F- solution (0.9% saline) and heated to 104 ^o C for 5 to 15 minutes. The results for labeling yield were: 5 min (28.9%), 10 min (57.9%), 15 min (83.7%) and 30 min (88.9%). Thus, the time course for labeling was approximately 15 minutes.

Biodistribution of IMP485 Alone

[00263] The biodistribution of IMP485 in the absence of any pretargeting antibody was examined in female Taconic nude mice (10 week old) bearing small or no BXPC3 pancreatic cancer xenografts. The mice were injected i.v. with Al ¹⁸ F(IMP485), (340 µCi, 2.29x 10 ^-9 mol, 100 µL in saline). The mice, 6 per time point, were necropsied at 30 min and 90 min post injection. In the absence of pretargeting antibody a low level of accumulation was seen in tumor and most normal tissues. The substantial majority of radiolabel was found in the bladder and to a lesser extent in kidney. Most of the activity was cleared before the 90 min time point.

Pretargeting of IMP485 with TF2 DNL Targeting Molecule

[00264] IMP485 Radiolabeling - ¹⁸ F- (218 mCi) was purified to isolate 145.9 mCi. The purified ¹⁸ F- (135 mCi) was added to a lyophilized vial containing 40 nmol of pre-complexed Al(IMP485). The reaction vial was heated at 110º C for 17 min. Water (0.8 mL) was added to the reaction mixture before HLB purification. The product (22 mCi) was eluted with 0.6 mL of water:ethanol (1:1) mixture into a vial containing lyophilized ascorbic acid. The product was diluted with saline. The Al ¹⁸ F(IMP485)specific activity used for injection was 550 Ci/mmol.

[00265] Biodistribution of Al ¹⁸ F(IMP485) alone - Mice bearing sc LS174T xenografts were injected with Al ¹⁸ F(IMP485) (28 µCi, 5.2x10 ^-11 mol, 100 µL. Mice were necropsied at 1 and 3 h post injection, 6 mice per time point.

[00266] Biodistribution of TF2 + Al ¹⁸ F(IMP485) With Pretargeting at 20:1 bsMAb to peptide ratio - Mice bearing sc LS174T xenografts were injected with TF2 (163.2 µg, 1.03x10 ^-9 mol, iv) and allowed 16.3 h for clearance before injecting Al ¹⁸ F(IMP485) (28 µCi, 5.2x10 ^-11 mol, 100 µL iv). Mice were necropsied at 1 and 3 h post injection, 7 mice per time point.

[00267] Urine stability - Ten mice bearing s.c. Capan-1 xenografts were injected with Al ¹⁸ F(IMP485) (400 µCi, in saline, 100 µL). Urine was collected from 3 mice at 55 min post injection. The urine samples were analyzed by reverse phase and SE-HPLC. Stability of the radiolabeled IMP485 in urine was observed. 1 ⁸

¹⁸

1 ⁸

Conclusions

[00268] The IMP485 labels as well as or better than IMP467, with equivalent stability in serum. However, IMP485 is much easier to synthesize than IMP467. Preliminary studies have shown that ¹⁸ F-labeling of lyophilized IMP485 works as well as non-lyophilized peptide (data not shown). The presence of alkyl or aryl groups in the linker joining the chelating moiety to the rest of the peptide was examined. The presence of aryl groups in the linker appears to increase the radiolabeling yield relative to the presence of alkyl groups in the linker.

[00269] Biodistribution of IMP485 in the presence or absence of pretargeting antibody resembles that observed with IMP467. In the absence of pretargeting antibody, distribution of radiolabeled peptide in tumor and most normal tissues is low and the peptide is removed from circulation by kidney excretion. In the presence of the TF2 antibody, radiolabeled IMP485 is found primarily in the tumor, with little distribution to normal tissues. Kidney radiolabeling is substantially decreased in the presence of the pretargeting antibody. We conclude that IMP485 and other peptides with aryl groups in the linker are highly suitable for PET imaging with ¹⁸ F-labeling.

Example 25. Kit Formulation of IMP485 for Imaging

[00270] We report a simple, general kit formulation for labeling peptides with ¹⁸ F. A ligand that contains 1,4,7-triazacyclononane-N, N’, N”-triacetic acid (NOTA) attached to a methyl phenylacetic acid (MPAA) group was used to form a single stable complex with (AlF) ²⁺ . The lyophilized kit contained IMP485, a di-HSG hapten-peptide used for pretargeting. The kit was reconstituted with an aqueous solution of ¹⁸ F-, heated at 100-110ºC for 15 min, followed by a rapid purification by solid-phase extraction (SPE). In vitro and in vivo stability and tumor targeting of the Al ¹⁸ F(IMP485) were examined in nude mice bearing human colon cancer xenografts pretargeted with an anti-CEACAM5 bispecific antibody. ¹⁸ F-labeling of MPAA-bombesin and somatostatin peptides also was evaluated.

[00271] The HSG peptide was labeled with ¹⁸ F- as a single isomer complex, in high yield (50-90%) and high specific activity (up to 153 GBq/μmol), within 30 min. It was stable in human serum at 37ºC for 4 h, and in vivo showed low uptake (0.06% ± 0.02 ID/g) in bone. At 3 h, pretargeted animals had high Al ¹⁸ F(IMP485) tumor uptake (26.5% ± 6.0 ID/g), with ratios of 12 ± 3, 189 ± 43, 1240 ± 490 and 502 ± 193 for kidney, liver, blood and bone, respectively. Bombesin and octreotide analogs were labeled with comparable yields. In conclusion, ¹⁸ F-labeled peptides can be produced as a stable, single [Al ¹⁸ F] complex with good radiochemical yields and high specific activity in a simple one-step kit.

Reagents List

[00272] Reagents were obtained from the following sources: Acetic acid (JT Baker 6903-05 or 9522-02), Sodium hydroxide (Aldrich semiconductor grade 99.99% 30,657-6), ^, ^- Trehalose (JT Baker 4226-04), Aluminum chloride hexahydrate (Aldrich 99% 237078), Ascorbic acid (Aldrich 25,556-4).

[00273] Acetate Buffer 2 mM - Acetic acid, 22.9 μL (4.0 x 10 ^-4 mol) was diluted with 200 mL water and neutralized with 6 N NaOH (~ 15 to adjust the solution to pH 4.22.

[00274] Aluminum Solution 2 mM - Aluminum hexahydrate, 0.0225 g (9.32 x 10 ^-5 mol) was dissolved in 47 mL DI water.

[00275] α, α-Trehalose Solution - α, α-Trehalose, 4.004 g was dissolved in 40 mL DI water to make a 10 % solution. [00276] Peptide Solution, IMP4852 mM - The peptide IMP485 (0.0020 g, 1.52 μmol) was dissolved in 762 μL of 2 mM acetate buffer. The pH was 2.48 (the peptide was lyophilized as the TFA salt). The pH of the peptide solution was adjusted to pH 4.56 by the addition of 4.1 μL of 1 M NaOH.

[00277] Ascorbic Acid Solution 5 mg/mL - Ascorbic acid, 0.0262 g (1.49 x 10 ^-4 mol) was dissolved in 5.24 mL DI water.

Formulation of Peptide Kit

[00278] The peptide, 20 μL (40 nmol) was mixed with 12 μL (24 nmol) of Al, 100 μL of trehalose, 20 μL (0.1 mg) ascorbic acid and 900 μL of DI water in a 3 mL lyophilization vial. The final pH of the solution was about pH 4.0. The vial was frozen, lyophilized and sealed under vacuum. Ten and 20 nmol kits have also been made. These kits are made the same as the 40 nmol kits keeping the peptide to Al ³⁺ ratio of 1 peptide to 0.6 Al ³⁺ but formulated in 2 mL vials with a total fill of 0.5 mL.

Purification of ¹⁸ F- [00279] The crude ¹⁸ F- was received in 2 mL of DI water in a syringe. The syringe was placed on a syringe pump and the liquid pushed through a Waters CM cartridge followed by a QMA cartridge. Both cartridges were washed with 10 mL DI water. A sterile disposable three way valve between the two cartridges was switched and 1 mL commercial sterile saline was pushed through the QMA cartridge in 200 μL fractions. The second fraction usually contains ~ 80 % of the ¹⁸ F- regardless of the amount of ¹⁸ F- applied (10-300 mCi loads were tested).

[00280] We alternatively use commercial ¹⁸ F- in saline, which has been purified on a QMA cartridge. This is a concentrated version of the commercial bone imaging agent so it is readily available and used in humans. The activity is supplied in 200 μL in a 0.5 mL tuberculin syringe.

Radiolabeling

[00281] The peptide was radiolabeled by adding ¹⁸ F- in 200 μL saline to the lyophilized peptide in a crimp sealed vial and then heating the solution to 90-110°C for 15 min. The peptide was purified by adding 800 mL of DI water in a 1 mL syringe to the reaction vial, removing the liquid with the 1 mL syringe and applying the liquid to a Waters HLB column (1cc, 30 mg). The HLB column was placed on a crimp sealed 5 mL vial and the liquid was drawn into the vial under vacuum supplied by a remote (using a sterile disposable line) 10 mL syringe. The reaction vial was washed with two one mL aliquots of DI water, which were also drawn through the column. The column was then washed with 1 mL more of DI water. The column was then moved to a vial containing buffered lyophilized ascorbic acid (~ pH 5.5, 15 mg). The radiolabeled product was eluted with three 200 μL portions of 1:1 EtOH/DI water. The yield was determined by measuring the activity on the HLB cartridge, in the reaction vial, in the water wash and in the product vial to get the percent yield.

[00282] Adding ethanol to the radiolabeling reaction can increase the labeling yield. A 20 nmol kit can be reconstituted with a mixture of 200 μL ¹⁸ F- in saline and 200 μL ethanol. The solution is then heated to 110°C in the crimp sealed vial for 16 min. After heating, 0.8 mL of water was added to the reaction vial and the activity was removed with a syringe and placed in a dilution vial containing 2 mL of DI water. The reaction vial was washed with 2 x 1 mL DI water and each wash was added to the dilution vial. The solution in the dilution vial was applied to the HLB column in 1-mL aliquots. The column and the dilution vial were then washed with 2 x 1-mL water. The radiolabeled peptide was then eluted from the column with 3 x 200 μL of 1:1 ethanol/water in fractions. The peptide can be labeled in good yield and up to 4,100 Ci/mmol specific activity using this method.

[00283] The yield for this kit and label as described was 80-90 % when labeled with 1.0 mCi of ¹⁸ F-. When higher doses of ¹⁸ F- (~ 100 mCi) were used the yield dropped. However if ethanol is added to the labeling mixture the yield goes up. If the peptides are diluted too much in saline the yields will drop. The labeling is also very sensitive to pH. For our peptide with this ligand we have found that the optimal pH for the final formulation was pH 4.0 ± 0.2.

[00284] The purified radiolabeled peptide in 50 μL 1:1 EtOH/H ₂ O was mixed with 150 μL of human serum and placed in the HPLC autosampler heated to 37ºC and analyzed by RP- HPLC. No detectable ¹⁸ F above background at the void volume was observed even after 4 h. Table 15. IMP 485 Labeling

Synthesis and Radiolabeling of IMP486: Al-OH(IMP485) [00285] IMP485 (21.5 mg, 0.016 mmol) was dissolved in 1 mL of 2 mM NaOAc, pH 4.4 and treated with AlCl ₃ .6H ₂ O (13.2 mg, 0.055 mmol). The pH was adjusted to 4.5-5.0 and the reaction mixture was refluxed for 15 minutes. The crude mixture was purified by preparative RP-HPLC to yield a white solid (11.8 mg).

[00286] The pre-filled Al(NOTA) complex (IMP486) was also radiolabeled in excellent yield after formulating into lyophilized kits. The labeling yields with IMP486 (Table 16) were as good as or better than IMP485 kits (Table 15) when labeled in saline. This high efficiency of radiolabeling with chelator preloaded with aluminum was not observed with any of the other Al(NOTA) complexes tested (data not shown). The equivalency of labeling in saline and in 1:1 ethanol/water the labeling yields was also not observed with other chelating moieties (not shown).

Table 16. IMP486 Labeling

Effect of Bulking Agents

[00287] An experiment was performed to compare yield using IMP485 kits (40 nmol) with different bulking agents. The peptide was labeled with 2 mCi of ¹⁸ F- from the same batch of 1 ⁸ F- in 200 microliters saline. The bulking agents were introduced in water at a concentration of 5% by weight, with a dose of 200 microliters/vial. We tested sorbitol, trehalose, sucrose, mannitol and glycine as bulking agents. Results are shown in Table 17.

Table 17. Effects of Bulking Agents on Radiolabeling Yield

[00288] Sorbitol, mannitol and trehalose all gave radiolabeled product in the same yield. The sucrose kit and the glycine kit both had significantly lower yields. Trehalose was formulated into IMP485 kits at concentrations ranging from 2.5% to 50% by weight when reconstituted in 200 μL. The same radiolabeling yield, ~83 %, was obtained for all concentrations, indicating that the ¹⁸ F-radiolabeling of IMP485 was not sensitive to the concentration of the trehalose bulking agent. IMP 485 kits were formulated and stored at 2- 8°C under nitrogen for up to three days before lyophilization to assess the impact of lyophilization delays on the radiolabeling. The radiolabeling experiments indicated that yields were all ~ 80 % at time zero, and with 1, 2, and 3 days of delay before lyophilization.

[00289] Ascorbic or gentisic acid often are added to radiopharmaceuticals during preparation to minimize radiolysis. When IMP485 (20 nmol) was formulated with 0.1, 0.5 and 1.0 mg of ascorbic acid at pH 4.1-4.2 and labeled with ¹⁸ F- in 200 μL saline, final yields were 51, 31 and 13% isolated yields, respectively, suggesting 0.1 mg of ascorbic acid was the maximum amount that could be included in the formulation without reducing yields.

Formulations containing gentisic acid did not label well. Ascorbic acid was also included in vials used to isolate the HLB purified product as an additional means of ensuring stability post-labeling. The IMP485 to Al ³⁺ ratio appeared to be optimal at 1:0.6, but good yields were obtained from 1:0.5 of up to a ratio of 1:1. The radiolabeling reaction was also sensitive to peptide concentration, with good yields obtained at concentrations of 1 x 10 ^-4 M and higher.

Effect of pH on radiolabeling

[00290] The effect of pH on radiolabeling of IMP485 is shown in Table 18. The efficiency of labeling was pH sensitive and decreased at either higher or lower pH relative to the optimal pH of about 4.0.

Table 18. Effect of pH on IMP485 Radiolabeling Efficiency.

[00291] Collectively, these studies led to a final formulated kit that contained 0.5 mL of a sterile solution with 20 nmol IMP485, 12 nmoles Al ³⁺ , 0.1 mg ascorbic acid, and 10 mg trehalose adjusted to 4.0 ± 0.2, which was then lyophilized Biodistribution

[00292] Biodistribution studies were performed in Taconic nude mice bearing subcutaneous LS174T tumor xenografts.

[00293] Al ¹⁸ F(IMP485) alone: Mice bearing sc LS174T xenografts were injected with Al ¹⁸ F(IMP485) (28 μCi, 5.2 x 10 ^-11 mol, 100 μL, iv). Mice were necropsied at 1 and 3 h post injection, 6 mice per time point.

[00294] TF2 + Al ¹⁸ F(IMP485) Pretargeting at 20:1 bsMab to peptide ratio: Mice bearing sc LS174T xenografts were injected with TF2 (163.2 μg, 1.03 x 10 ^-9 mol, iv) and allowed 16.3 h for clearance before injecting Al ¹⁸ F(IMP485) (28 μCi, 5.2 x 10 ^-11 mol, 100 μL, iv). Mice were necropsied at 1 and 3 h post injection, 7 mice per time point. Table 19. Biodistribution of TF2 pretargeted Al ¹⁸ F(IMP485) or Al ¹⁸ F(IMP485) alone at 1 and 3 h after peptide injection in nude mice bearing LS174T human colonic cancer xeno rafts.

Synthesis of IMP492 or Al ¹⁹ F(IMP485)

[00295] IMP485 (16.5 mg, 0.013 mmol) was dissolved in 1 mL of 2 mM NaOAc, pH 4.43, 0.5 mL ethanol and treated with AlF ₃ .3H ₂ O (2.5 mg, 0.018 mmol). The pH was adjusted to 4.5-5.0 and the reaction mixture was refluxed for 15 minutes. On cooling the pH was once again raised to 4.5-5.0 and the reaction mixture refluxed for 15 minutes. The crude was purified by preparative RP-HPLC to yield a white solid (10.3 mg).

Synthesis of IMP490

NODA-MPAA-D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Throl (SEQ ID NO:22)

[00296] The peptide was synthesized on threoninol resin with the amino acids added in the following order: Fmoc-Cys(Trt)-OH, Fmoc-Thr(But)-OH, Fmoc-Lys(Boc)-OH, Fmoc-D- Trp(Boc)-OH, Fmoc-Phe-OH, Fmoc-Cys(Trt)-OH, Fmoc-D-Phe-OH and (tBu) ₂ NODA- MPAA. The peptide was then cleaved and purified by preparative RP-HPLC. The peptide was cyclized by treatment of the bis-thiol peptide with DMSO.

Synthesis of IMP491 or Al ¹⁹ F(IMP490)

[00297] The Al ¹⁹ F(IMP490) was prepared as described above (IMP492) to produce the desired peptide after HPLC purification.

Synthesis of IMP493

NODA-MPAA-(PEG) ₄ -Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH ₂ (SEQ ID NO:23)

[00298] The peptide was synthesized on Sieber amide resin with the amino acids added in the following order: Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-NH-(PEG) ₃ - COOH and (tBu) ₂ NODA-MPAA. The peptide was then cleaved and purified by preparative RP-HPLC.

[00299] The affinity of the Al ¹⁹ F complex of IMP493 was EC ₅₀ = 183 nm versus EC ₅₀ = 59 nm for ¹²⁵ I-bombesin. The IMP493 kit radiolabeled with ~100 MBq of ¹⁸ F- had a 70% yield. Radiolabeling IMP490 with 100 MBq of ¹⁸ F- resulted in 80% yield, which was reduced to 65% when 2.11 GBq ¹⁸ F- was used. The peptide is eluted as a single radiolabeled peak at 15.4 min using HPLC (not shown).

Synthesis of IMP494 or Al ¹⁹ F(IMP493)

[00300] The Al ¹⁹ F(IMP493) was prepared as described above (IMP492) to produce the desired peptide after HPLC purification. Example 26. Other Prosthetic Group Labeling Methods Using Al ¹⁸ F

[00301] In certain embodiments, the aluminum fluoride labeling method may be performed using prosthetic group labeling methods for molecules that are sensitive to heat. Prosthetic group conjugation may be carried out at lower temperatures for heat-sensitive molecules. [00302] The prosthetic group NOTA is labeled with ¹⁸ F as described above and then it is attached to the targeting molecule. In one non-limiting example, this is performed with an aldehyde NOTA that is then attached to an amino-oxy compound on a targeting molecule. Alternatively an amino-oxy maleimide is reacted with the aldehyde and then the maleimide is attached to a cysteine on a targeting molecule (Toyokuni et al., 2003, Bioconj Chem

14:1253).

[00303] In another alternative, the AlF-chelator complexes are attached to targeting molecules through click chemistry. The ligands are first labeled with Al ¹⁸ F as discussed above. The Al ¹⁸ F-chelate is then conjugated to a targeting molecule through a click chemistry reaction. For example, an alkyne NOTA is labeled according to Marik and Stucliffe (2006, Tetrahedron Lett 47:6681) and conjugated to an azide containing targeting agent.

Radiolabeling of Kits With ¹⁸ F- in Saline

[00304] The ¹⁸ F- (0.01 mCi or higher) is received in 200 μL of saline in a 0.5 mL syringe and the solution is mixed with 200 μL of ethanol and injected into a lyophilized kit as described above. The solution is heated in the crimp sealed container at 100-110°C for 15 min. The solution is diluted with 3 mL water and eluted through an HLB cartridge. The reaction vial and the cartridge are washed with 2 x 1 mL portions of water and then the product is eluted into a vial containing buffered ascorbic acid using 1:1 ethanol water in 0.5 mL fractions. Some of the ethanol may be evaporated off under a stream of inert gas. The solution is then diluted in saline and passed through a 0.2 ^m sterile filter prior to injection. Example 27. Maleimide Conjugates of NOTA for ¹⁸ F-Labeling

[00305] The Examples above describe a novel method of ¹⁸ F-labeling, which captures a ([ ¹⁸ F]AlF) ²⁺ complex, using a NOTA-derived ligand bound on a peptide. These labeled peptides were stable in vivo and retained their binding abilities (McBride et al., 2009, J. Nucl. Med.50, 991-998; McBride et al., 2010, Bioconjug. Chem.21, 1331-1340; Laverman et al., 2010, J. Nucl. Med.51, 454-461; McBride et al.2011, J. Nucl. Med.52 (Suppl.1), 313-314P (abstract 1489)). Although this procedure allows peptides to be radiofluorinated in one simple step within 30 min, it requires agents to be heated to ~100 ^o C, which is unsuitable for most proteins and some peptides. We and others have found that an aromatic group attached to one of the nitrogen atoms of the triazacylcononane ring of NOTA can enhance the yield for the ([ ¹⁸ F]AlF) ²⁺ complexation compared to some alkyl and carboxyl substituents (D'Souza et al., 2011, Bioconjug. Chem.1793-1803 ; McBride et al., 2010, Bioconjug. Chem.21, 1331- 1340; Shetty et al.2011, Chem. Comm. DOI: 10.1039/c1cc13151f). In the present Example, we explored the potential for labeling heat-labile compounds with ([ ¹⁸ F]AlF) ²⁺ , using a new ([ ¹⁸ F]AlF) ²⁺ -binding ligand that contains NOTA attached to a methyl phenylacetic acid group (MPAA). This was conjugated to N-(2-aminoethyl)maleimide (N-AEM) to form NOTA- MPAEM. (Details of the synthesis are shown in FIG.15.) The NOTA-MPAEM was labeled with ([ ¹⁸ F]AlF) ²⁺ at 105 °C in 49-82% yield and conjugated at room temperature to an antibody Fab’ fragment in 69-80% yield (total time ~ 50 min) and with retention of immunoreactivity. These data indicate that the rapid and simple [Al ¹⁸ F]-labeling method can be easily adapted for preparing heat-sensitive compounds with ¹⁸ F quickly and in high yields.

Synthesis of Bis-t-butyl-NOTA-MPAA NHS ester: (tBu) ₂ NOTA-MPAA NHS ester

[00306] To a solution of (tBu) ₂ NOTA-MPAA (175.7 mg, 0.347 mmol) in CH ₂ Cl ₂ (5 mL) was added 347 μL (0.347 mmol) DCC (1 M in CH ₂ Cl ₂ ), 42.5 mg (0.392 mmol) N- hydroxysuccinimide (NHS), and 20 μL N,N-diisopropylethylamine (DIEA). After 3 h DCU was filtered off and solvent evaporated. The crude mixture was purified by flash

chromatography on (230-400 mesh) silica gel (CH ₂ Cl ₂ :MeOH, 100:0 to 80:20) to yield (128.3 mg, 61.3%) of the NHS ester. The HRMS (ESI) calculated for C ₃₁ H ₄₆ N ₄ O ₈ (M+H) ⁺ was 603.3388, observed was 603.3395.

Synthesis of NOTA-MPAEM: (MPAEM = methyl phenyl acetamido ethyl maleimide)

[00307] To a solution of (tBu) ₂ NOTA-MPAA NHS ester (128.3 mg, 0.213 mmol) in CH ₂ Cl ₂ (5 mL) was added a solution of 52.6 mg (0.207 mmol) N-(2-aminoethyl) maleimide trifluoroacetate salt in 250 μL DMF and 20 μL DIEA. After 3 h the solvent was evaporated and the concentrate was treated with 2 mL TFA. The crude product was diluted with water and purified by preparative RP-HPLC to yield (49.4 mg, 45%) of the desired product. HRMS (ESI) calculated for C ₂₅ H ₃₃ N ₅ O ₇ (M+H) ⁺ was 516.2453, observed was 516.2452.

1 ⁸ F-Labeling of NOTA-MPAEM

[00308] The NOTA-MPAEM ligand (20 nmol; 10 μL), dissolved in 2 mM sodium acetate (pH 4), was mixed with AlCl ₃ (5 μL of 2 mM solution in 2 mM acetate buffer, 200 μL of ¹⁸ F- (0.73 and 1.56 GBq) in saline, and 200 μL of acetonitrile. After heating at 105-109°C for 15- 20 min, 800 μL of deionized (DI) water was added to the reaction solution, and the entire contents removed to a vial (dilution vial) containing 1 mL of deionized (DI) water. The reaction vial was washed with 2 x 1 mL DI water and added to the dilution vial. The crude product was then passed through a 1-mL HLB column, which was washed with 2 x 1 mL fractions of DI water. The labeled product was eluted from the column using 3 x 200 μL of 1:1 EtOH/water.

Conjugation of Al ¹⁸ F(NOTA-MPAEM) to hMN-14 Fab’

[00309] Fab’ fragments of humanized MN-14 anti-CEACAM5 IgG (labetuzumab) were prepared by pepsin digestion, followed by TCEP (Tris(2-carboxyethyl)phosphine) reduction, and then formulated into a lyophilized kit containing 1 mg (20 nmol) of the Fab’ (2.4 thiols/Fab’) in 5% trehalose and 0.025 M sodium acetate, pH 6.72. The kit was reconstituted with 0.1 mL PBS, pH 7.01, and mixed with the Al ¹⁸ F(NOTA-MPAEM) (600 μL 1:1

EtOH/H ₂ O). After incubating for 10 min at room temperature, the product was purified on a 3-mL SEPHADEX G50-80 spin column in a 0.1 M, pH 6.5 sodium acetate buffer (5 min). The isolated yield was calculated by dividing the amount of activity in the eluent by the total activity in the eluent and the activity on the column.

[00310] Immunoreactivity of the purified product was analyzed by adding an excess of CEA and separating on SE-HPLC, comparing to a profile of the product alone. The product was also analyzed by RP-HPLC to assess percent-unbound Al ¹⁸ F(NOTA-MPAEM).

9 ^9m Tc-CEA-Scan®

[00311] A CEA-SCAN® kit containing 1.2 mg of IMMU-4, a murine anti-CEACAM5 Fab’ (anti-CEA, 2.4 x 10 ^-2 μmol), was labeled with 453 MBq ^99m TcO - 4 Na ⁺ in 1 mL saline according to manufacturer’s instructions and used without further purification.

Animal Study

[00312] Nude mice were inoculated subcutaneously with CaPan-1 human pancreatic adenocarcinoma (ATCC Accession No. HTB-79, Manassas, VA). When tumors were visible, the animals were injected intravenously with 100 μL of the radiolabeled Fab’. The

Al ¹⁸ F(NOTA-MPAES)-hMN-14 Fab’ was diluted in saline to 3.7 MBq/100 μL containing ~2.8 μg of Fab’. A ^99m Tc-IMMU-4 Fab’ aliquot (16.9 MBq) was removed and diluted with saline (0.85 MBq/100 μL containing ~2.8 μg of Fab’). The animals were necropsied at 3 h post injection, tissues and tumors removed, weighed, and counted by gamma scintillation, together with standards prepared from the injected products. The data are expressed as percent injected dose per gram.

Results

Synthesis and reagent preparation [00313] The NOTA-MPAEM was produced as shown in FIG.15, where the (tBu) ₂ NOTA- MPAA was coupled to 2-aminoethyl-maleimide and then deprotected to form the desired product. The crude product was diluted with water and purified by preparative RP-HPLC to yield (49.4 mg, 45%) of the desired product [HRMS (ESI) calculated for C ₂₅ H ₃₃ N ₅ O ₇ (M+H) ⁺ 516.2453, found 516.2452].

Radiolabeling

[00314] The NOTA-MPAEM (20 nmol) was mixed with 10 nmol of Al ³⁺ and labeled with 0.73 GBq and 1.56 GBq of ¹⁸ F- in saline. After SPE purification, the isolated yields of Al ¹⁸ F(NOTA-MPAEM) were 82% and 49%, respectively, with a synthesis time of about 30 min. The Al ¹⁸ F(NOTA-MPAES)-hMN-14 Fab’ conjugate was isolated in 74 % and 80% yields after spin-column purification for the low and high dose protein labeling, respectively. The total process was completed within 50 min. The specific activity for the purified

Al ¹⁸ F(NOTA-MPAES)-hMN-14 Fab’ was 19.5 GBq/μmol for the high-dose label and 10.9 GBq/μmol for the low dose label.

[00315] SE-HPLC analysis of the labeled protein for the 0.74-GBq run showed the ¹⁸ F- labeled Fab’ as a single peak and all of the activity shifted when excess CEA was added (not shown). RP-HPLC analysis on a C4 column showed the labeled maleimide standard eluting at 7.5 min, while the purified ¹⁸ F-protein eluted at 16.6 min (not shown). There was no unbound Al ¹⁸ F(NOTA-MPAEM) in the spin-column purified product.

Serum stability

[00316] The Al ¹⁸ F(NOTA-MPAES)-hMN-14 Fab’ was mixed with fresh human serum and incubated at 37 °C. SE-HPLC analysis over a 3-h period, with and without CEA showed that the product was stable and retained binding to CEA (not shown).

Biodistribution

[00317] The biodistribution of the Al ¹⁸ F(NOTA-MPAES)-hMN-14 Fab’ and the ^99m Tc- IMMU-4 murine Fab’ was assessed in nude mice bearing Capan-1 pancreatic cancer xenografts. At 3 h post-injection, both agents showed an expected elevated uptake in the kidneys, since Fab’ is renally filtered from the blood (Table 20). The [Al ¹⁸ F]-Fab’ concentration in the blood was significantly (P < 0.0001) lower than the ^99m Tc-Fab’, with a correspondingly elevated uptake in the liver and spleen. The faster blood clearance of the [Al ¹⁸ F]-Fab’ likely contributed to the lower tumor uptake as compared to the ^99m Tc-Fab’ (2.8 ± 0.3 vs.6.8 ± 0.7, respectively), but it also resulted in a more favorable tumor/blood ratio for the fluorinated Fab’ (5.9 ± 1.3 vs.0.9 ± 0.1, respectively). Bone uptake for both products was similar, suggesting the Al ¹⁸ F(NOTA) was tightly held by the Fab’. Table 20. Biodistribution of Al ¹⁸ F(NOTA-MPAES)-hMN-14 Fab’ and ^99m Tc-IMMU-4 Fab’ at 3 h after injection with 0.37 MBq (~3 μg) of each conjugate in nude mice bearing Capan-1 human pancreatic cancer xenografts (N = 6).

Discussion

[00318] We prepared a simple NOTA-MPAEM ligand for attachment to thiols on temperature-sensitive proteins or other molecules bearing a sulfhydryl group. To avoid exposing the heat-labile compound to high temperatures, the NOTA-MPAEM was first mixed with Al ³⁺ and ¹⁸ F- in saline and heated at 100-115°C for 15 min to form the

Al ¹⁸ F(NOTA-MPAEM) intermediate. This intermediate was rapidly purified by SPE in 49- 82% isolated yield (67.7 ± 13.0%, n=5), depending on the amount of activity added to a fixed amount (20 nmol) of the NOTA-MPAEM. The Al ¹⁸ F(NOTA-MPAEM) was then efficiently (69-80% isolated yield, 74.3 ± 5.5, n=3) coupled to a reduced Fab’ in 10-15 min, using a spin column gel filtration procedure to isolate the radiolabeled protein, in this case an antibody Fab’ fragment. The entire two-step process was completed in ~50 min, and the labeled product retained its molecular integrity and immunoreactivity. Thus, the feasibility of extending the simplicity of the [Al ¹⁸ F]-labeling procedure to heat-sensitive compounds was established.

[00319] The [Al ¹⁸ F]-ligand complex has been shown to be very stable in serum in vitro, and in animal testing, minimal bone uptake is seen (McBride et al., 2009, J. Nucl. Med.50, 991- 998; D’Souza et al., 2011a, J. Nucl. Med.52 (Suppl.1), 171P (abstract 577)). In this series of studies, ¹⁸ F associated with the NOTA-MPAEM compound conjugated to a Fab’ was stable in serum in vitro, and the conjugate retained binding to CEA. When injected into nude mice, there was selective localization in the tumor, providing a ~6:1 tumor/blood ratio. Bone uptake was similar for the Al ¹⁸ F(NOTA-MPAES)-hMN-14 Fab’ and the ^99m Tc-IMMU-4 murine Fab’, again reflecting in vivo stability of the ¹⁸ F or Al ¹⁸ F complex. However, [Al ¹⁸ F]- Fab’ hepatic and splenic uptake was higher as compared to the ^99m Tc-IMMU-4. The specific NOTA derivative can be modified in different ways to accommodate conjugation to other reactive sites on peptides or proteins. However, use of this particular derivative showed that the Al ¹⁸ F-labeling procedure can be adapted for use with heat-labile compounds.

Conclusions

[00320] NOTA-MPAEM was labeled rapidly with ¹⁸ F- in saline and then conjugated to the immunoglobulin Fab’ protein in high yield. The labeling method uses only inexpensive disposable purification columns, and while not requiring an automated device to perform the labeling and purification, it can be easily adapted to such systems. Thus, the NOTA-MPAEM derivative established that this or other NOTA-containing derivatives can extend the capability of facile ([ ¹⁸ F]AlF) ²⁺ fluorination to heat-labile compounds.

Example 28. Improved ¹⁸ F-Labeling of NOTA-Octreotide

[00321] The aim of this study was to further improve the rapid one-step method for ¹⁸ F- labeling of NOTA-conjugated octreotide. Octreotide was conjugated with a NOTA ligand and was labeled with ¹⁸ F in a single- step, one-pot method. Aluminum (Al ³⁺ ) was added to ¹⁸ F- and the AlF ²⁺ was incorporated into NOTA-octreotide, as described in the Examples above. The labeling procedure was optimized with regard to aluminum:NOTA ratio, ionic strength and temperature. Radiochemical yield and specific activity were determined.

[00322] Under optimized conditions, NOTA-octreotide was labeled with Al ¹⁸ F in a single step with 98% yield. The radiolabeling, including purification, was performed in 45 min. Optimal labeling yield was observed with Al:NOTA ratios around 1:20. Lower ratios led to decreased labeling efficiency. Labeling efficiencies in the presence of 0%, 25%, 50%, 67% and 80% acetonitrile in Na-acetate pH 4.1 were 36%, 43%, 49%, 70% and 98%, respectively, indicating that increasing concentrations of the organic solvent considerably improved labeling efficiency. Similar results were obtained in the presence of ethanol, DMF and THF. Labeling in the presence of DMSO failed. Labeling efficiencies in 80% MeCN at 40°C, 50°C and 60°C were 34%, 65%, 83%, respectively. Labeling efficiency was >98% at 80°C and 100°C. Specific activity of the ¹⁸ F-labeled peptide was higher than 45,000 GBq/mmol.

[00323] Optimal ¹⁸ F-labeling of NOTA-octreotide with Al ¹⁸ F was performed at 80-100 °C in Na-acetate buffer with 80% (v/v) acetonitrile and a Al:NOTA ratio between 1:20 and 1:50. Labeling efficiency was typically >98%. Since labeling efficiency at 60°C was 83%, this method may also allow ¹⁸ F-labeling of temperature-sensitive biomolecules such as proteins and antibody fragments. These conditions allow routine ¹⁸ F-labeling of peptides without the need for purification prior to administration and PET imaging.

Example 29. Functionalized Triazacyclononane Ligands for Molecular Imaging

[00324] The present Example relates to synthesis and use of a new class of triazacyclonane derived ligands and their complexes useful for molecular imaging. Exemplary structures are shown in FIG.16 to FIG.18. The ligands may be functionalized with a ¹⁹ F moiety selected from the group consisting of fluorinated alkyls, fluorinated acetates, fluoroalkyl

phosphonates, fluoroanilines, trifluoromethyl anilines, and trifluoromethoxy anilines in an amount effective to provide a detectable ¹⁹ F NMR signal. The complexation of these ligands with radioisotopic or paramagnetic cations renders them useful as diagnostic agents in nuclear medicine and magnetic resonance imaging (MRI). Preferably, the Al ¹⁸ F and ⁶⁸ Ga complexes of these ligands are useful for PET imaging, while the ¹¹¹ In complexes can be used in SPECT imaging. Methods for conjugating these radiolabeled ligands to a targeting molecule like antibody, protein or peptide are also disclosed.

[00325] The disclosed bifunctional chelators (BFCs) can be radiolabeled with ¹¹¹ In, ⁶⁸ Ga, ⁶⁴ Cu, ¹⁷⁷ Lu, Al ¹⁸ F, ^99m Tc or ⁸⁶ Y or complexed with a paramagnetic metal like manganese, iron, chromium or gadolinium, and subsequently attached to a targeting molecule

(biomolecule). The labeled biomolecules can be used to image the hematological system, lymphatic reticuloendothelial system, nervous system, endocrine and exocrine system, skeletomuscular system, skin, pulmonary system, gastrointestinal system, reproductive system, immune system, cardiovascular system, urinary system, auditory or olfactory system or to image affected cells or tissues in various medical conditions.

Synthesis of Bifunctional Chelators

2-{4-(carboxymethyl)-7-[2-(4-nitrophenyl)ethyl]-1,4,7-triaza cyclononan-1-yl)acetic acid. NOTA-EPN

[00326] To a solution of 4-nitrophenethyl bromide (104.5 mg, 0.45 mmol) in anhydrous CH ₃ CN at 0 ^o C was added dropwise over 20 min a solution of (tBu) ₂ NOTA (167.9 mg, 0.47 mmol) in CH ₃ CN (10 mL). After 1 h, anhydrous K ₂ CO ₃ (238.9 mg, 1.73 mmol) was added to the reaction mixture and allowed to stir at room temperature overnight. Solvent was evaporated and the concentrate was acidified with 4 mL TFA. After 5 h, the reaction mixture was diluted with water and purified by preparative RP-HPLC to yield a pale yellow solid (60.8 mg, 32.8%). HRMS (ESI) calculated for C ₁₈ H ₂₆ N ₄ O ₆ (M+H) ⁺ 395.1925; found 395.1925.

2-{4-(carboxymethyl)-7-[2-(4-nitrophenyl)methyl]-1,4,7-triaz acyclononan-1-yl)acetic acid. NOTA-MPN

[00327] To a solution of 4-nitrobenzyl bromide (61.2 mg, 0.28 mmol) in anhydrous CH ₃ CN at 0 ^o C was added dropwise over 20 min a solution of (tBu) ₂ NOTA (103.6 mg, 0.29 mmol) in CH ₃ CN (10 mL). After 1 h, anhydrous K ₂ CO ₃ (57.4 mg, 0.413 mmol) was added to the reaction mixture and allowed to stir at room temperature overnight. Solvent was evaporated and the concentrate was acidified with 3 mL TFA. After 5 h, the reaction mixture was diluted with water and purified by preparative RP-HPLC to yield a pale yellow solid (19.2 mg, 17.4%). HRMS (ESI) calculated for C ₁₇ H ₂₄ N ₄ O ₆ (M+H) ⁺ 381.1769; found 381.1774.

6-(4,7-bis{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacycl ononan-1-yl)hexanoic acid. (tBu) ₂ NOTA-HA

[00328] To a solution of (tBu) ₂ NOTA (208.3 mg, 0.58 mmol) in 10 mL CH ₃ CN was added 6-bromohexanoic acid (147.3 mg, 0.755 mmol) and K ₂ CO ₃ (144.5 mg, 1.05 mmol). The reaction flask was placed in a warm water-bath for 48 h. Solvent was evaporated and the concentrate was diluted with water and purified by preparative RP-HPLC to yield a white solid (138.5 mg, 50.1%). ESMS calculated for C ₂₄ H ₄₅ N ₃ O ₆ (M+H) ⁺ 472.3381; found 472.27.

4-[(4,7-bis{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacyc lononan-1-yl) methyl]benzoic acid. (tBu) ₂ NOTA-MBA [00329] To a solution of ^-bromo-p-toluic acid (126.2 mg, 0.59 mmol) in anhydrous CH ₃ CN was added dropwise over 20 min a solution of (tBu) ₂ NOTA (208 mg, 0.58 mmol) in CH ₃ CN (10 mL) and allowed to stir at room temperature for 48 h. Solvent was evaporated and the concentrate was diluted with water/DMF and purified by preparative RP-HPLC to yield a white solid (74.6 mg). HRMS (ESI) calculated for C ₂₆ H ₄₁ N ₃ O ₆ (M+H) ⁺ 492.3068; found 492.3071.

4-[(4,7-bis{[(tert-butyl)oxycarbonyl]methyl}-1,4,7-triazacyc lononan-1- yl)ethyl]benzoic acid. (tBu) ₂ NOTA-EBA

[00330] To a solution of 4-(2-bromoethyl)benzoic acid (310.9 mg, 1.36 mmol) in anhydrous CH ₃ CN was added dropwise over 20 min a solution of (tBu) ₂ NOTA (432.3 mg, 1.21 mmol) in CH ₃ CN (10 mL) and K ₂ CO ₃ (122.4 mg, 0.89 mmol). The reaction was stirred at room temperature for 72 h. Solvent was evaporated and the concentrate was diluted with water/DMF and purified by preparative RP-HPLC to yield a white solid (35.1 mg). HRMS (ESI) calculated for C ₂₇ H ₄₃ N ₃ O ₆ (M+H) ⁺ 506.3225; found 506.3234.

2-[7-but-3-ynyl-4-(carboxymethyl]-1,4,7-triazacyclononan-1-y l)acetic acid. NOTA- Butyne

[00331] To a solution of (tBu) ₂ NOTA (165.8 mg, 0.46 mmol) in 5 mL CH ₃ CN was added 4- bromo-1-butyne (44 μL, 62.3 mg, 0.47 mmol) and reaction mixture was stirred at room temperature for 72 h. Solvent was evaporated and the concentrate was purified by preparative RP-HPLC to yield an oil. HRMS (ESI) calculated for C ₂₂ H ₃₉ N ₃ O ₄ (M+H) ⁺ 410.3013; found 410.3013. The purified product was acidified with 2 mL TFA and after 5 h diluted with water, frozen and lyophilized. HRMS (ESI) calculated for C ₁₄ H ₂₃ N ₃ O ₆ (M+H) ⁺ 298.1761; found 298.1757.

tert-butyl-2-(7-(4-aminobutyl)-4-{[(tert-butyl)oxycarbonyl]m ethyl}-1,4,7- triazacyclononan-1-yl)acetic acid. (tBu) ₂ NOTA-BA

[00332] To a solution of (tBu) ₂ NOTA (165.2 mg, 0.46 mmol) in 5 mL CH ₃ CN was added 4- (Boc-amino)butyl bromide (124.7 mg, 0.49 mmol), a pinch of K ₂ CO ₃ and reaction mixture was stirred at room temperature for 72 h. Solvent was evaporated and the concentrate was treated with 1 mL CH ₂ Cl ₂ and 0.5 mL TFA. After 5 min the solvents were evaporated and the crude oil was diluted with water/DMF and purified by preparative RP-HPLC to yield a white solid (137.2 mg, 69.3%). HRMS (ESI) calculated for C ₂₂ H ₄₄ N ₄ O ₄ (M+H) ⁺ 429.3435; found 429.3443. NOTA-BAEM: (BAEM = butyl amido ethyl maleimide)

[00333] To a solution of (tBu) ₂ NOTA-BM (29.3 mg, 0.068 mmol) in CH ₂ Cl ₂ (3 mL) was added a ^-maleimido propionic acid NHS ester (16.7 mg, 0.063 mmol), 20 μL DIEA and stirred at room temperature overnight. Solvent was evaporated and the concentrate was acidified with 1 mL TFA. After 3 h, the reaction mixture was diluted with water and purified by preparative RP-HPLC to yield a white solid. HRMS (ESI) calculated for C ₂₁ H ₃₃ N ₅ O ₇ (M+H) ⁺ 468.2453; found 468.2441.

2-{4-[(4,7-bis-tert-butoxycarbonylmethyl)-[1,4,7]-triazacycl ononan-1- yl)methyl]phenyl}acetic acid. (tBu) ₂ NOTA-MPAA

[00334] To a solution of 4-(bromomethyl)phenylacetic acid (593 mg, 2.59 mmol) in anhydrous CH ₃ CN (50 mL) at 0 ^o C were added dropwise over 1 h a solution of (tBu) ₂ NOTA (1008 mg, 2.82 mmol) in CH ₃ CN (50 mL). After 4 h, anhydrous K ₂ CO ₃ (100.8 mg, 0.729 mmol) was added to the reaction mixture and allowed to stir at room temperature overnight. Solvent was evaporated and the crude was purified by preparative RP-HPLC (Method 5) to yield a white solid (713 mg, 54.5%). ¹ H NMR (500 MHz, CDCl ₃ , 25 ^o C, TMS) ^ 1.45 (s, 18 H), 2.64-3.13 (m, 16 H), 3.67 (s, 2 H), 4.38 (s, 2 H), 7.31 (d, 2H), 7.46 (d, 2H); ¹³ C (125.7 MHz, CDCl ₃ ) ^ 28.1, 41.0, 48.4, 50.9, 51.5, 57.0, 59.6, 82.3, 129.0, 130.4, 130.9, 136.8, 170.1, 173.3. HRMS (ESI) calculated for (M+H) ⁺ 506.3225; found 506.3210.

2-(4-(carboxymethyl)-7-{[4-(carboxymethyl)phenyl]methyl}-1,4 ,7-triazacyclononan- 1-yl)acetic acid. NOTA-MPAA

[00335] To a solution of 4-(bromomethyl)phenylacetic acid (15.7 mg, 0.068 mmol) in anhydrous CH ₃ CN at 0 ^o C was added dropwise over 20 min a solution of (tBu) ₂ NOTA (26 mg, 0.073 mmol) in CH ₃ CN (5 mL). After 2 h, anhydrous K ₂ CO ₃ (5 mg) was added to the reaction mixture and allowed to stir at room temperature overnight. Solvent was evaporated and the concentrate was acidified with 2 mL TFA. After 3 h, the reaction mixture was diluted with water and purified by preparative RP-HPLC to yield a white solid (11.8 mg, 43.7%). ¹ H NMR (500 MHz, DMSO-d ₆ , 25 ^o C) ^ 2.65-3.13 (m, 12 H), 3.32 (d, 2H), 3.47 (d, 2H), 3.61 (s, 2 H), 4.32 (s, 2 H), 7.33 (d, 2H), 7.46 (d, 2H); ¹³ C (125.7 MHz, DMSO-d ₆ ) 40.8, 47.2, 49.6, 50.7, 55.2, 58.1, 130.4, 130.5, 130.9, 136.6, 158.4, 158.7, 172.8, 172.9. HRMS (ESI) calculated for C ₁₉ H ₂₇ N ₃ O ₆ (M+H) ⁺ 394.1973; found 394.1979.

(tBu) ₂ NOTA-MPAA NHS ester. [00336] To a solution of (tBu) ₂ NOTA-MPAA (175.7 mg, 0.347 mmol) in CH ₂ Cl ₂ (5 mL) was added (1 M in CH ₂ Cl ₂ ) DCC (347 μL, 0.347 mmol), N-hydroxysuccinimide (NHS) (42.5 mg, 0.392 mmol), and 20 μL N,N-diisopropylethylamine (DIEA). After 3 h, dicyclohexylurea (DCU) was filtered off and solvent evaporated. The crude product was purified by flash chromatography on (230-400 mesh) silica gel (CH ₂ Cl ₂ :MeOH (100:0 to 80:20) to yield the NHS ester (128.3 mg, 61.3%). HRMS (ESI) calculated for C ₃₁ H ₄₆ N ₄ O ₈ (M+H) ⁺ 603.3388; found 603.3395.

NOTA-MPAEM: (MPAEM = methyl phenyl acetamido ethyl maleimide)

[00337] To a solution of (tBu) ₂ NOTA-MPAA NHS ester (128.3 mg, 0.213 mmol) in CH ₂ Cl ₂ (5 mL) was added a solution of N-(2-aminoethyl) maleimide trifluoroacetate salt (52.6 mg, 0.207 mmol) in 250 μL DMF and 20 μL DIEA. After 3 h, the solvent was evaporated and the concentrate treated with 2 mL TFA. The crude product was diluted with water and purified by preparative RP-HPLC to yield a white solid (49.4 mg, 45%). HRMS (ESI) calculated for (M+H) ⁺ 516.2453; found 516.2452.

tert-butyl-2-(7-(4-aminopropyl)-4-{[(tert-butyl)oxycarbonyl] methyl}-1,4,7- triazacyclononan-1-yl)acetic acid. (tBu) ₂ NOTA-PA

[00338] To a solution of (tBu) ₂ NOTA (391.3 mg, 1.09 mmol) in 5 mL CH ₃ CN was added Benzyl-3-bromo propyl carbamate (160 µL) and reaction mixture was stirred at room temperature for 28 h. Solvent was evaporated and the concentrate was dissolved in 40 mL 2- propanol, mixed with 128.7 mg of 10% Pd-C and placed under 43 psi H ₂ overnight. The product was then filtered and the filtrate concentrated.The crude product was diluted with water/DMF and purified by preparative RP-HPLC to yield a white solid (353 mg). HRMS (ESI) calculated for C ₂₁ H ₄₂ N ₄ O ₄ (M+H) ⁺ 415.3291; found 415.3279.

NOTA-PAEM: (PAEM = propyl amido ethyl maleimide)

[00339] To a solution of (tBu) ₂ NOTA-PM (109.2 mg, 0.263 mmol) in CH ₂ Cl ₂ (3 mL) was added a ^-maleimido propionic acid NHS ester (63.6 mg, 0.239 mmol), 20 μL DIEA and stirred at room temperature overnight. Solvent was evaporated and the concentrate was acidified with 1 mL TFA. After 3 h, the reaction mixture was diluted with water and purified by preparative RP-HPLC to yield a white solid (79 mg). HRMS (ESI) calculated for C ₂₀ H ₃₁ N ₅ O ₇ (M+H) ⁺ 454.2319; found 454.2296.

1 ⁸ F-Labeling of Functionalized Triazacyclononane Ligands [00340] The functionalized triazacyclononane ligand (20 nmol; 10 μL), dissolved in 2 mM sodium acetate (pH 4), was mixed with AlCl ₃ (5 μL of 2 mM solution in 2 mM acetate buffer, 25 - 200 μL of ¹⁸ F- in saline, and 25 - 200 μL of ethanol. After heating at 90-105°C for 15-20 min, 800 μL of deionized (DI) water was added to the reaction solution, and the entire contents removed to a vial (dilution vial) containing 1 mL of deionized (DI) water. The reaction vial was washed with 2 x 1 mL DI water and added to the dilution vial. The crude product was then passed through a 1-mL HLB column, which was washed with 2 x 1 mL fractions of DI water. The labeled product was eluted from the column using 3 x 200 μL of 1:1 EtOH/water. Radiochromatograms of the ¹⁸ F-labeling of functionalized TACN ligands are shown in FIG.19.

1 ⁸ F-labeling of NOTA-MPAEM

[00341] To 10 μL (20 nmol) 2 mM NOTA-MPAEM solution was added 5 μL 2 mM AlCl ₃ , 200 μL ¹⁸ F- solution [15.94 mCi, Na ¹⁸ F, PETNET] followed by 200 μL CH ₃ CN and heated to 110 ^o C for 15 minutes. The crude reaction mixture was purified by transferring the resultant solution into a Oasis ^{^} HLB 1 cc (30 mg) cartridge (P# 186001879, L# 099A30222A) and eluting with DI H ₂ O to remove unbound ¹⁸ F- followed by 1:1 EtOH/H ₂ 0 to elute the ¹⁸ F- labeled peptide. The crude reaction solution was pulled through the HLB cartridge into a 10 mL vial and the cartridge washed with 6 x 1 mL fractions of DI H ₂ O (4.34 mCi). The HLB cartridge was then placed on a new 3 mL vial and eluted with 4×150 μL 1:1 EtOH/H ₂ O to collect the labeled peptide (7.53 mCi). The reaction vessel retained 165.1 ^Ci, while the cartridge retained 270 ^Ci of activity.7.53 mCi ^ 61.2% of Al[ ¹⁸ F]NOTA-MPAEM.

1 ⁸ F-labeling of NOTA-MPAEM:

[00342] To 10 μL (20 nmol) 2 mM NOTA-MPAEM solution was added 5 μL 2 mM AlCl ₃ , 200 μL ¹⁸ F- solution [15.94 mCi, Na ¹⁸ F, PETNET] followed by 200 μL CH ₃ CN and heated to 110 ^o C for 15 minutes. The crude reaction mixture was purified by transferring the resultant solution into a Oasis ^{^} HLB 1 cc (30 mg) cartridge (P# 186001879, L# 099A30222A) and eluting with DI H ₂ O to remove unbound ¹⁸ F- followed by 1:1 EtOH/H ₂ 0 to elute the ¹⁸ F- labeled peptide. The crude reaction solution was pulled through the HLB cartridge into a 10 mL vial and the cartridge washed with 6 x 1 mL fractions of DI H ₂ O (4.34 mCi). The HLB cartridge was then placed on a new 3 mL vial and eluted with 4×150 μL 1:1 EtOH/H ₂ O to collect the labeled peptide (7.53 mCi). The reaction vessel retained 165.1 ^Ci, while the cartridge retained 270 ^Ci of activity.7.53 mCi ^ 61.2% of Al[ ¹⁸ F]NOTA-MPAEM. Table 21. ¹⁸ F-labeling of 20 nmol NOTA-MPAEM + 10 nmol Al ³⁺

Conjugation of hMN14-Fab’-SH with Al ¹⁸ F(NOTA-MPAEM):

[00343] To the vial containing hMN14-Fab’-SH (1 mg, ~ 20 nmoles) L # 112310 was added 200 μL PBS, pH 7.38 and 600 μL of the HLB purified Al ¹⁸ F(NOTA-MPAEM) (EtOH:H ₂ O::1:1). The crude reaction mixture was passed through a sephadex (G-50/80, 0.1 M NaOAc, pH 6.5) 3 mL spin column. The activity in the eluate was 4.27 mCi, while 1.676 mCi was retained on the spin column and 0.178 mCi in the empty reaction vial. 4.27 mCi ^ 68.6% of [Al ¹⁸ F]-hMN14-Fab.

To 800 μL of PBS was added 1 μL of eluate ^ injected 40 μL (SEC-HPLC) at 2.08 p.m. Major product at 10.312 min.

Serum Stability:

[00344] In an autosampler vial 200 μL of fresh human serum + 50 μL of eluate ^ 149.6 ^Ci at 2.45 p.m. Incubated at 37 ^o C ^ [Al ¹⁸ F]-hMN14-Fab in serum.

To 4 μL of [Al ¹⁸ F]-hMN14-Fab in serum added 280 μL of buffer B (PBS) ^ injected 40 μL (SEC-HPLC) at 3.51 p.m. Major product at 10.331 min.

Immunoreactivity of ¹⁸ F-hMN14-Fab:

[00345] To 100 ^g carcinoembryonic antigen (CEA) [L # 2371505, Scripp’s Labs] was added 200 μL 1% HSA in PBS, pH 7.38 + 100 μL PBS, pH 7.38 ^ CEA in PBS. Added 4 μL of [Al ¹⁸ F]-hMN14-Fab in serum at 37 ^o C to 150 μL CEA in PBS ^ injected 40 μL (SEC- HPLC) at 4.33 p.m. Major product at 7.208 min.

Radiochromatograms of spin column purified [Al ¹⁸ F]-hMN14-Fab, stability of [Al ¹⁸ F]- hMN14-Fab in human serum and its immunoreactivity with CEA are shown in FIG.20. [00346] Exemplary synthetic schemes for the bifunctional chelators are shown below.

Scheme 1: Synthesis of NOTA-MBA

Scheme 2: Synthesis of NOTA-EBA

Scheme 3: Synthesis of NOTA-MPN

S _{cheme 4: Synthesis of NOTA-EPN}

Scheme 5: Synthesis of NOTA-Butyne

Scheme 6: Synthesis of NOTA-MPH

Scheme 7: Synthesis of NOTA-Ethanol

Scheme 8: Synthesis of NOTA-PBr

Scheme 9: Synthesis of NOTA-MBEM

2. (AcO) ₂ O/CH ₃ COONa 3. TFA

_{Scheme 10: Synthesis of NOTA-BM}

2. TFA

Scheme 11: Synthesis of NOTA-BAEM

Scheme 12: Synthe sis of NOT A-MPAPE G ₃ N ₃

Scheme 13: Synthe sis of Al ¹⁸ F (NOTA-M PAA NHS ester)

Scheme 14: Synthe sis of Al ¹⁸ F (NOTA-B AEM)

Scheme 15: Synthe sis of NOT A-BAMA

Scheme 16: Synthesis of NOTA-BAO

Scheme 17: Synthesis of NOTA-MPAFO

Scheme 18: Synthe sis of NOT A-MPAFS A

[00347] Exempla ry structure s of ¹⁸ F-lab eled probes are shown below.

Conclusion s

[00348] We have found that a novel clas s of triazac yclononane (TACN) d erived BFC s, possess ing a functi onality that provides fo r an easy l inkage onto biomolecu les via soli d phase or in so lution, form stable com plexes wit h a variety of metals. T hese BFCs also form remarka bly stable A l ¹⁸ F chela tes. Most ¹⁸ F-labeling methods ar e tedious to perform, re quire the effo rts of a spe cialized che mist, invol ve multiple purificatio ns of the in termediates , anhydro us conditio ns, and gen erally end up with low RCYs. An advantage of this new class of BFC s is that the y can be rad iofluorinat ed rapidly in one step with high s pecific activ ity in an aque ous medium .

Examp le 30. Furt her Optim ization of Kit Formu lation

[00349] The effec t of varying buffer com position o n labeling e fficiency w as determi ned. Kits we re formulat ed with 20 nmol IMP485 and 10 n mol AlCl ₃ ·6H ₂ O in 5 % ^, ^-treh alose. The buf fers and as corbic acid were varied in the diff erent formu lations. Th e peptide an d trehalos e were diss olved in DI water and the AlCl ₃ ·6 H ₂ O was d issolved in the buffer t ested. [00350] MES Buffer - 4-morpholineethanesulfonic acid (MES, Sigma M8250), 0.3901 g (0.002 mol) was dissolved in 250 mL of DI H ₂ O and adjusted to pH 4.06 with acetic acid (8 mM buffer).

[00351] KHP Buffer - Potassium biphthalate (KHP, Baker 2958-1), 0.4087 g (0.002 mol) was dissolved in 250 mL DI H ₂ O pH 4.11 (8 mM buffer).

[00352] HEPES Buffer - N-2 hydroxyethylpiperazine-N’-2-ethane-sulfonic acid (HEPES, Calbiochem 391338) 0.4785 g (0.002 mol) dissolved in 250 mL DI H ₂ O and adjusted to pH 4.13 with AcOH (8 mM buffer).

[00353] HOAc Buffer - Acetic acid (HOAc, Baker 9522-02), 0.0305 g (0.0005 mol) was dissolved in 250 mL DI H ₂ O and adjusted to pH 4.03 with NaOH (2 mM buffer).

[00354] The AlCl ₃ ·6H ₂ O (Aldrich 23078) was dissolved in the buffers to obtain a 2 mM solution of Al ³⁺ in 2 mM buffer. IMP 4850.0011 g (MW 1311.67, 8.39 x 10 ^-7 mol) was dissolved in 419 μL DI H ₂ O. Ascorbic acid, 0.1007 g (Aldrich 25,556-4, 5.72 x 10 ^-4 mol) was dissolved in 20 mL DI H ₂ O.

[00355] A variety of kits (summarized in Table 22) were prepared and adjusted to the proper pH by the addition of NaOH or HOAc as needed. The solution was then dispensed in 1 mL aliquots into 4, 3 mL lyophilization vials, frozen on dry ice and lyophilized. The initial shelf temperature for the lyophilization was -10°C. The samples were placed under vacuum and the shelf temperature was increased to 0°C. The samples were lyophilized for 15 hr and the shelf temperature was increased to 20°C for 1 h before the vials were sealed under vacuum and removed from the lyophilizer. The kits were prepared with different buffers, at different pH values, with or without ascorbic acid and with or without acetate. After lyophilization, the kits were dissolved in 400 μL of saline and the pH was measured with a calibrated pH meter with a micro pH probe.

[00356] Radiolabeling - The kits were all labeled with ¹⁸ F- in saline (200 μL, PETNET) with ethanol (200 μL) and heated to ~105°C for 15 min. The labeled peptides were diluted with 0.6 mL DI H ₂ O and then added to a dilution vial containing 2 mL DI H ₂ O. The reaction vial was washed with 2 x 1 mL portions of DI H ₂ O, which were added to the dilution vial. The diluted solution was filtered through a 1 mL (30 mg) HLB cartridge (1 mL at a time) and washed with 2 mL DI H ₂ O. The cartridge was moved to an empty vial and eluted with 3 x 200 μL 1:1 EtOH/DI H ₂ O. The Al[ ¹⁸ F]IMP485 was in the 1:1 EtOH/DI H ₂ O fractions. The isolated yield was determined by counting the activity in the reaction vial, the dilution vial, the HLB cartridge, the DI H ₂ O column wash and the 1:1 EtOH/DI H ₂ O wash adding up the total and then dividing the amount in the 1:1 EtOH/DI H ₂ O fraction by the total and multiplying by 100.

Results

[00357] The results of the studies are shown in Table 22. All the labeling in the presence of 0.1 mg of ascorbic acid went well. The ascorbic acid appears to serve as a significant non- volatile buffer that keeps the pH the same before and after lyophilization (kits 1-4). When ascorbic acid is not used (kits 5-8) the pH can change significantly along with the radiolabeling yield. The KHP buffer, kit 8, was the best kit in the second batch. Higher levels of ascorbate might also stabilize the Al ¹⁸ F complex in solution and act as a transfer ligand for Al ¹⁸ F. The KHP buffer might also act as a transfer ligand for Al ¹⁸ F so the amount of KHP was increased from 5 x 10 ^-7 mol/kit for kit 8 to 6 x 10 ^-6 mol/kit for kit 11. The increase in KHP stabilized the pH better than kit 8 and gave a much better labeling yield. The kits with KHP + ascorbate (kit 12) and KHP + MES (kit 13) had slightly higher labeling yields. It may be that the higher levels of KHP and ascorbate act both as buffers and as transfer ligands to increase the labeling yields with those excipients. Citric acid is not a good buffer for [Al ¹⁸ F]- labeling (kit 14), it gives low labeling yields even when only 50 μL of 2 mM citrate was used in the presence 0.1 mg of ascorbate. Increasing amounts of KHP, 0.1 M and above (kits 16- 18) lead to lower labeling yields with more activity found in the aqueous wash from the HLB column. Table 22. Results of labeling and pH studies

[00358] It appears from these results that potassium biphthalate is an optimal buffer for labeling. The peptide labeling kits were therefore reformulated to utilize KHP in the labeling buffer. The reformulated kits gave very high isolated labeling yields of about 97 % when 100 nmol of peptide was labeled in 1:1 ethanol/saline. The labeling and purification time was also simplified and reduced to 20 min. In addition to using the new buffering agent, potassium biphthalate (KHP), we also added more moles of buffer, which may help stabilize the pH during labeling. The peptide is purified through an Alumina N cartridge by adding more saline to the reaction after heating and pushing crude product through the cartridge. The unbound ¹⁸ F- and Al ¹⁸ F stick to the alumina and the labeled peptide is eluted very efficiently from the cartridge with saline. The formulation shown below is for a 20 nmol peptide kit but the same formulation is used for a 100 nmol peptide kit by adding more peptide and more Al ³⁺ (60 nmol Al ³⁺ for the 100 nmol peptide kit).

[00359] 2 mM Al ³⁺ in 2 mM KHP - Aluminum chloride hexahydrate, 0.0196 g (8.12 x 10 ^-5 mol, Aldrich 23078, MW 241.43) was dissolved in 40.6 mL of 2 mM potassium biphthalate (KHP, JT Baker 2958-1, MW 204.23). This can be stored at room temperature for months.

[00360] Ascorbic Acid - Ascorbic acid, 0.100 g was dissolved in 20 mL DI H ₂ O. This is made fresh on the day of use.

[00361] 5% Trehalose ^ ^ ^, ^-Trehalose dihydrate, 2.001 g (JT Baker, 4226-04, MW 378.33) was dissolved in 20 mL DI H ₂ O. This can be stored at room temperature for weeks.

[00362] KHP Kit Buffer - KHP, 0.2253 g was dissolved in 18 mL DI H ₂ O (0.06 M). This solution can be kept for months at room temperature.

[00363] IMP485 solution - IMP485, 0.0049 g (3.74 x 10 ^-6 mol, MW 1311.67) was dissolved in 1.494 mL DI H ₂ O (2.5 x 10 ^-3 M). This solution can be stored for months at -20 ºC.

[00364] 1M KOH - Potassium hydroxide (99.99 % semiconductor grade, MW 56.11, Aldrich 306568) was dissolved in DI H2O to make a 1 M solution.

[00365] Kit Formulation (20 nmol kit, 40 kits) - The peptide, IMP485 (320 μL, 8 x 10 ^-7 mol) was placed in a 50 mL sterile polypropylene centrifuge tube (metal free) and mixed with 240 μL of the 2 mM Al ³⁺ solution (4.8 x 10 ^-7 mol) 800 μL of the ascorbic acid solution, 1600 μL of the 0.06 M KHP solution, 8 mL of the 5 % trehalose solution and the mixture was diluted to 40 mL with DI H ₂ O. The solution was adjusted to pH 3.99-4.03 with a few microliters of 1 M KOH. The peptide solution was dispensed 1 mL/vial with a 1 mL pipette into 3 mL glass lyophilization vials (unwashed).

[00366] Lyophilization - The vials were frozen on dry ice, fitted with lyophilization stoppers and placed on a -20 ºC shelf in the lyophilizer. The vacuum pump was turned on and the shelf temperature was raised to 0 ºC after the vacuum was below 100 mtorr. The next morning the shelf temperature was raised to 20 ºC for 4 hr before the samples were closed under vacuum and crimp sealed.

[00367] Radiolabeling - The ¹⁸ F- in saline was received from PETNET in 200 μL saline in a 0.5 mL tuberculin syringe. Ethanol, 200 μL, was pulled into the ¹⁸ F- solution and then the mixture was injected into a lyophilized kit containing the peptide. The solution was then heated in a 105 ºC heating block for 15 min. Sterile saline, 0.6 mL was then added to the reaction vial and the solution was removed from the vial and pushed through an alumina N cartridge (SEP-PAK light, WAT023561, previously washed with 5 mL sterile saline) into a collection vial. The reaction vial was washed with 2 x 1 mL saline and the washes were pushed through the alumina column. The total labeling and purification time was about 20 min.

Example 31. Labeling at Reduced Temperature

[00368] The effect of varying the chelator structure on efficiency of labeling at reduced temperature was examined. A comparison of low temperature labeling of IMP466 (NOTA- Octreotide) with IMP485 showed that the simple NOTA ligand labels much better at low temperature than the NOTA-MPAA ligand.

[00369] In one embodiment, a temperature sensitive molecule, such as a protein, may be conjugated to multiple copies of a simple NOTA ligand. The protein can then be purified and formulated for Al ¹⁸ F-labeling (e.g., lyophilized). The protein kit was reconstituted with ¹⁸ F- in saline, heated for the appropriate length of time and purified by gel filtration or an alumina column. Tables 27 and 28 show the temperature effects of labeling IMP466 vs. IMP485. Table 23. Temperature-dependent labeling for Al ¹⁸ F(IMP466)

Table 24. Temperature-dependent labeling for Al ¹⁸ F(IMP485)

[00370] The data show that by switching to a different chelating moiety, the efficiency of low temperature labeling with Al ¹⁸ F may be tripled at 50 ºC. Further modification of the chelating moiety may provide additional improvement of low temperature labeling.

However, the 37% efficiency observed with IMP466 is sufficient to enable ¹⁸ F PET imaging with temperature sensitive molecules if a sufficient number of chelating moieties are attached to the molecule.

[00371] We have also examined the effect of peptide concentration on low temperature labeling of IMP485. Kits were made with 10, 20, 40, 100 and 200 nmol of peptide and 0.6 equivalents of Al ³⁺ respectively. The rest of the formulation was the same for all of the kits. The kits were labeled with 400 μL saline/EtOH and heated at 50-110 ºC for 15 min and then purified through the Alumina N cartridge. The labeling results are reported as isolated yields in Table 25. At any temperature, increasing the concentration of peptide increased the efficiency of labeling. The results indicate that if the reaction volume can be decreased with the use of a microfluidics device then we can greatly reduce the amount of peptide and ¹⁸ F- needed to prepare a single dose of labeled peptide for PET imaging. Table 25. Effect of peptide concentration on efficiency of labeling as a function of temperature.

Example 32. Automated synthesis of ¹⁸ F-labeled molecules [00372] This Exa mple comp ared the aut omated syn thesis of ¹⁸ F-FBEM p ublished by Kiesew etter et al., (2011, App l Radiat Iso t 69:410-4) to that of A l ¹⁸ F(NOTA -MPAEM ). The automat ed synthes is of ¹⁸ F-FB EM was ac complished using a so phisticated synthesis m odule (see bel ow), with a RCY of 17 % in 95 m in. Our synt hesis modu le (FIG.21 ) would in clude a heating device and a HLB cart ridge or HP LC colum n. With NO TA-MPAE M we were able to get 67-79% RCY ( decay corre cted) in 40 min in one single step .

Scheme 19: Synth esis of Al ¹⁸ F(NOTA-M PAEM) [00373] In both ¹⁸ F-FBEM a nd ¹⁸ F-FDG -MHO, the ¹⁸ F is intro duced first followed b y a maleim ide (Schem e 20 and 21 ). While N OTA-MPA EM - a mal eimide con taining BFC - is ¹⁸ F-labe led in one final step (S cheme 19) .

Scheme 20: S nthesis of ¹⁸ F-FBEM

Scheme 21: Synthesis of ¹⁸ F-FDG-MHO

* not decay corrected. Example 33. Room Temperature Labeling of Targeting Molecules Using Bifunctional Chelator (BFC) Moieties [00374] The objective of this Example was to perform ¹⁸ F-labeling of temperature sensitive molecules at reduced temperatures, such as room temperature, with high radiochemical yield and high specific activity of the labeled molecule. Preferably, the labeling reaction is accomplished in 10 to 15 minutes in aqueous medium, with a total synthesis time of 30 minutes or less. More preferably, the labeling technique involves the initial reaction of a metal- ¹⁸ F or metal- ¹⁹ F with a bifunctional chelating (BFC) moiety at elevated temperature (e.g., 90 to 105˚C), followed by site-specific attachment of the BFC to the targeting molecule at a reduced temperature (e.g., room temperature). In certain embodiments, the BFC may be derived from the structure of NOTA-propyl amine (FIG.22).

[00375] IMP508 (FIG.23A) and IMP517 (FIG.23B) were synthesized as disclosed below. The NOTA chelating moiety formed according to schemes 22 and 23 was attached to a bis- HSG peptide (IMP508), formulated into 20 nmol peptide kits and labeled with ¹⁸ F. Scheme 22. Synthesis of the pyridyl methyl ester NOTA derivative

Scheme 23. Hydrolysis of the methyl ester

[00376] The methyl ester was synthesized as follows. The NO ₂ AtBu, 1.0033g (2.807 x 10 ^-3 mol) was mixed with 0.4638g (2.810 x 10 ^-3 mol) of the methyl 6-formylnicotinate and dissolved in 10 mL THF. Triacetoxyborohydride, 0.6248 g (2.948 x 10 ^-3 mol) was added and the reaction was stirred at room temperature for two days and an additional 0.3044 g of the borohydride was added. The reaction was quenched with H ₂ O after stirring 6.5 hr more at room temp. The product was extracted with dichloromethane, dried over Na ₂ SO ₄ , filtered and concentrated under reduced pressure to obtain the crude brown product. The product was purified by flash chromatography eluting with hexanes, 25% EtOAc/hexanes, 50%

EtOAc/hexanes, 75% EtOAc/hexanes, 100% EtOAc, dichloromethane, 5% MeOH/94% dichloromethane/1% triethylamine and 10% MeOH/89% dichloromethane/1% triethylamine. The product, was isolated as a brown tar 0.455 g and was in the MeOH/ dichloromethane/ triethylamine fractions.

[00377] To synthesize the acid, the methyl ester (0.411 g, 8.12 x 10 ^-4 mol) was dissolved in 5 mL dioxane and stirred with 0.8 mL of 1 M NaOH. The reaction was stirred for 18 hr at room temperature and another 1.3 mL of NaOH was added in portions as the reaction stirred at room temperature for another 8 hr. The reaction was quenched with 1 M citric acid and adjusted to pH 4.91 with 1 M NaOH. The product was extracted with dichloromethane. Some saturated NaCl solution was added to the aqueous layer and the solution was again extracted with dichloromethane. The organic layers were combined, dried over Na ₂ SO ₄ , and concentrated to obtain 0.3421 g of the product (85% yield).

[00378] IMP517 was produced as disclosed in Scheme 24. The methyl ester triazole precursor was hydrolyzed and conjugated to the bis-HSG peptide to obtain IMP517 (FIG. 23B).

Scheme 24

[00379] IMP517 was test labeled with different concentrations of peptide in 400 μL of saline. IMP485 was also labeled in 400 μL of saline for comparison.

[00380] IMP517 was labeled with F-18 in 400 μL of 1:1 EtOH/saline at different temperatures for 15 min.

[00381] FIG.24 compares the labeling of IMP51720 nmol kits in 400 μL of 1:1 EtOH/saline heated for 15 min. IMP517 gave the highest labeling yields of the ligands tested so far and also gave high yields in saline alone. New NOTA derivatives with different functional groups in the vicinity of the 1,4,7-triazacyclononane ring were prepared and attached to a standard test peptide. The peptides were radiolabeled over a range of temperatures from 50 to110°C with and without a co-solvent. Two of these derivatives containing a pyridyl or a triazole group showed improved labeling yields at lower temperatures as well as labeling equal or better than the benzyl-NOTA standard at higher temperatures. Adding ethanol to the triazole derivative did not increase yields as much as the other derivatives, indicating that it may be possible to improve the radiolabeling yield at lower temperatures and reduce or eliminate the need for a co-solvent.

[00382] Alterations to the NOTA/NOTA ligand on a peptide can have a positive effect on the radiolabeling yield of the peptide, and may lead to ligands that can be used for direct one- step ¹⁸ F labeling of some temperature-sensitive molecules.

Example 34. Non-Peptide, Small Molecule-Imaging Agents

[00383] A NOTA-2-nitroimidazole derivative (50 nmol, I mL) (FIG.23C) used for hypoxia imaging was labeled in 0.1 M, pH 4, NaOAc buffer by mixing with 22.5 µL of 2 mM AlCl ₃ ·6H ₂ O (45 nmol) in 0.1 M pH 4 NaOAc, and 50 µL of ¹⁸ F- in saline, then heating at 110°C for 10 min to obtain the labeled complex in 85% yield. In vivo studies with the Al ¹⁸ F- NOTA-2-nitroimidazole showed the expected biodistribution and tumor targeting, with no evidence of product instability. The NOTA-DUPA-Pep molecule (FIG.23D) was made for targeting the prostate-specific membrane antigen (PSMA). The ¹⁸ F-labeled molecule was synthesized in 79% yield after HPLC purification to remove the unlabeled targeting agent. Example 35. Large Peptide and Protein Labeling

[00384] NOTA-N-ethylmaleimide was attached to a cysteine side chain of the 40 amino acid exendin-4 peptide, which targets the glucagon-like peptide type-1 receptor (GLP-1 receptor) (Kiesewetter et al., 2012, Tharanostics 2:999-1009). The peptide was labeled with ¹⁸ F-, using unpurified cyclotron target water to obtain the labeled peptide in 23.6 ± 2.4% uncorrected yield in 35 min. The Al ¹⁸ F-labeled peptide had 15.7 ± 1.4% ID/g in the tumor and 79.25 ± 6.20% ID/g in the kidneys at 30 min, with low uptake in all other tissues.

[00385] The NOTA-affibody Z _HER2:2395 (58-amino acid, 7 kDa) was labeled at 90°C for 15 min with Al ¹⁸ F, the affibody, and acetonitrile (Heskamp et al., 2012, J Nucl Med 53:146-53). The labeling and purification process took about 30 min and the yield was 21 ± 5.7%. Again, biodistribution studies supported the stability of the product with negligible bone uptake.

[00386] We also examined a two-step labeling method for temperature-sensitive molecules. The NOTA-MPAA ligand was attached to N-ethylmaleimide to make NOTA-MPAEM. The NOTA-MPAEM (20 nmol in 10 µL 2 mM, pH 4, NaOAc) was mixed with 5 µL 2 mM AlCl ₃ in 2 mM, pH 4, NaOAc followed by 200 µL ¹⁸ F- in saline and 200 µL of acetonitrile. The solution was heated at 105-109°C for 15 min and purified by SPE to produce the Al ¹⁸ F- NOTA-MPAEM in 80% yield. This product was then coupled to a pre-reduced antibody Fab’ fragment (20 nmol) by mixing the purified Al ¹⁸ F-NOTA-MPAEM at room temperature for 10 min, followed by isolation of the labeled Fab’ by gel filtration. The labeled protein was obtained in an 80% yield. The total synthesis time for both steps combined was about 50 min, with an overall decay-corrected yield of about 50-60%.

Example 36. Residualization and In Vivo Clearance of Al ¹⁸ F Complexes

[00387] Lang et al. compared the biodistribution of ¹⁸ F on carbon, Al ¹⁸ F and ⁶⁸ Ga attached to the same NOTA-PRGD2 peptide in the U-87 MG human glioblastoma model (Lang et al., 2011, Bioconjugate Chem 22:2415-22). They found that tumor uptake of the ¹⁸ F-PPRGD2 peptide was 3.65 ± 0.51% ID/g at 30 min PI compared to 1.85 ± 0.30% ID/g at 2 h, indicating that the ¹⁸ F activity was slowly clearing from the tumor between 30 min and 2 h (51% retention). The metal-complexed RGD peptides had higher tumor retention [4.20 ± 0.23% ID/g (30 min), 3.53 ± 0.45% ID/g (2 h) or 84% retention for Al ¹⁸ F-NOTA-PRGD2, and 3.25 ± 0.62% ID/g (30 min), 2.66 ± 0.32% ID/g (2 h), or 82% retention ⁶⁸ Ga-NOTA-PRGD2] over the same period. These data show that the chelated AlF complex may be retained better in the tumor than the radiofluorinated compound with ¹⁸ F bound to a carbon atom. The retention of activity also was seen with the exendin peptide and the affibody, where the activity cleared from the kidneys when the ¹⁸ F was attached to a carbon atom (Kiesewetter et al., 2012, Eur J Nucl Med Mol Imaging 39:463-73; Kramer et al., 2008, Eur J Nucl Med Mol Imaging 35:1008-18), but was retained with the Al ¹⁸ F complex (Kiesewetter et al., 2012, Theranostics 2:999-1009; Heskamp et al., 2012, J Nucl Med 53:146-53). Retention of the radionuclide in a tissue could provide a targeting advantage, particularly in rapidly metabolizing tissues, such as damaged heart tissue.

Example 37. Labeling of NOTA-derivatized Octreotate

[00388] Exemplary targeting peptides of use in the claimed methods and compositions are disclosed below. The peptides are produced by standard synthesis techniques and conjugated to chelating moieties as disclosed in the Examples above. The NOTA-octreotate derivatives are labeled with Al- ¹⁸ F as described above and administered to patients with suspected neuroendocrine tumors. PET imaging is used to detect sst ⁺

2 tumors by standard PET techniques, as disclosed above. The labeled targeting peptides provide high resolution images of both primary and metastatic tumors.

[00389] Labeling with other isotopic species, such as ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ¹⁸ F, ¹⁹ F, ⁶⁶ Ga, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² Ga, ¹¹¹ In, ¹⁷⁷ Lu, ⁴⁴ Sc, ⁴⁷ Sc, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ⁴⁵ Ti and ⁸⁹ Zr, show that the methods and compositions are not limited to Al- ¹⁸ F labeling, but rather are applicable to any radionuclide or other diagnostic agent that can bind to NOTA or a derivatized NOTA.

Numerous exemplary species of derivatized NOTA are disclosed in the Examples above and any such chelating moiety may be utilized.

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