AU2014259570B2 - Improved methods and compositions for F-18 labeling of proteins, peptides and other molecules - Google Patents

Abstract

The present application discloses compositions and methods of synthesis and use of F 18 labeled molecules of use, for example, in PET imaging techniques. In particular embodiments, the labeled molecules may be peptides or proteins, although other types of molecules including but not limited to aptamers, oligonucleotides and nucleic acids may be labeled and utilized for such imaging studies. In preferred embodiments, the F 18 label may be conjugated to a targeting molecule by formation of a metal complex and binding of the F-I 8-metal complex to a chelating moiety, such as DOTA, NOTA, DTPA, TETA or NETA. In other embodiments, the metal may first be conjugated to the chelating group and subsequently the F-18 bound to the metal. In other preferred embodiments, the F- 18 labeled moiety may comprise a targetable conjugate that may be used in combination with a bispecific or multispecific antibody to target the F- 18 to an antigen expressed on a cell or tissue associated with a disease, medical condition, or pathogen. Exemplary results show that F- 18 labeled targetable conjugate peptides are stable in human serum at 370 C for several hours, sufficient time to perform PET imaging analysis.

Description

IMPROVED METHODS AND COMPOSITIONS FOR F-18 LABELING OF PROTEINS, PEPTIDES AND OTHER MOLECULES

Related Applications [001] This application is a divisional of Australian Patent Application No. 2008338917, the entire content of which is incorporated herein by reference.

Field (002j la certain embodiments, the present invention concerns a simple method of labeling peptides with F-18, which are of use for in-vivo imaging. The preferred specific activity of the P- i 8 labeled peptide would be about 1,000 to 2,000 Ci/mmol at the time of administration to the patient. Specific activities that are in the range oflOO to lens of thousands of Ci/mmol would also be of use. Although higher specific activities are preferred for certain imaging applications, mother alternative embodiments a lower specific activity of a metot-F-18 complex with ΝΟΤΑ (1,4 J-triaza-cyclononatie-N,Ni,N,’-triacetic acid) or another chelating moiety could be of use, for example, as a renal Row imaging agent or for heart and brain imaging agents to image blood flow. Preferably, P-1 8 labeling is accomplished without need for a purification step to separate milabeled from labeled peptide. More preferably, F-18 labeled peptides are stable under in vivo conditions, such as in human serum.

Bachground {003] Positron Emission Tomography (PET) imaging provides high resolution and quantitation from the PET images. Peptides or other small molecules can be labeled with the positron emitters ,sF,wCu, wGa, **Ga, ,6Br„ ^“Tc, **Y, and IWI to name a few. The positron emitted from the nucleus of the isotope is ejected with, different energies depending on the isotope used. When the positron reacts with an electron two 51.1 keVgamma rays are emitted in opposite directions. The energy of the ejected positron controls the average distance that a positron travels before it is annihilated by hitting an electron. The higher tire ejection energy the further the positron travels before the collision with an electron. A low ejection energy for a PET isotope is desirable to minimize the distance that the positron travels from the target site before it generates the two 511 keV gamma rays that are imaged by the PET camera.

Many isotopes that emit positrons also have other emissions such as gamma rays, alpha panicles or beta panicles in their decay chain, it is desirable to have a PET isotope that is a pure positron etui Per so that any dosi metry problems will be minimized.

[604j The half-life of the isotope is also important, since the half-life must be karg enough to attach the isotope to a targeting molecule, analyze the product, inject it into the patient, and allow the product to localize, clear from non-target tissues and then image. If the half-life is too long the specific activity may not be high enough to obtain enough photons for a clear image and if it is too short the time needed for manufacturing, commercial distribution and biodistribution may not be sufficient. F-l 8 (ff 635 keV 97%, tia 110 min) is one of the most widely used PET emitting isotopes because of its low positron emission energy, tack of side emissions and suitable half-life. The F-l 8 is produced with a high specific activity. When an isotope is attached to a molecule for targeting it is usually accompanied by some unreacted targeting agent, which is often present in a large molar excess compared to the radiolabeled product. Usually, the labeled product and the unlabeled product can compete for the same target in-vivo so the presence of the cold targeting agent lowers the effective specific activity of the targeting agen t. If the F-18 is attached to a molecule which has a very high uptake such as 2-fiuoro-2-deoxy glucose (FDG) then effective specific activity is not as important. However, if one is targeting a receptor with a labeled peptide or performing an immunoPET pretargeting study with a limited number of binding sites available, the cold targeting agent could potentially block the uptake of the radiolabeled targeting agent if the cold targeting agent is present in excess. 1005] Conventional F-18 labeling of peptides involves the labeling of a reagent at low specific activity, HPLC purification of the reagent and then conjugation to the peptide of interest. The conjugate is often repurified after conjugation to obtain the desired specific activity of labeled peptide. An example is the labeling method of Poethko et al. (./ Nucl. Med. 2004; 45: 892-902) in which 4-[lsF|fIuorobenxaldehyde is first synthesized and purified (Wilson el al,./. Labeled Compounds andRadiopharm. 1990; XXVIH: 1189-1199) and then conjugated io the peptide. The peptide conjugate is then, puri fied by HPLC to remove excess peptide that was used to drive the conjugation to completion. The two reactions and purification would not be a problem if F-l 8 had along half-life. However the half-life oi'F-18 is only 2 hr so all of the manipulations that are needed to attach the F-l8 to the peptide are a significant burden.

[0061 These methods are tedious to perform and require the use of equipment designed specifically to produce the labeled product and/or the efforts of specialized professional chemists. They are not kit formulations that could routinely be used in a clinical setting. A need exists for a rapid, simple method of 18-.F-labeling of targeting moieties, such as proteins or peptides, that results in targeting constructs of suitable specific activity and in vivo stability for detection and/or imaging, while minimizing the requirements for specialized equipment or highly trained personnel and reducing operator exposure io high levels of radiation. A further need exists for prepackaged kits that could provide compositions required for performing such novel methods.

Summary |007| Fluoride binds to practically all other elements and some of those bonds are relatively stable. Peptides, bearing metal binding ligands, arc known to bind radiometals stably and at very high specific activity. The approach utilized in the present method was to first hind the F-,18 to a metal and then chelate the F-18 metal complex with a ligand on the peptide. The question was then, which metal (or other element, e.g. boron) to choose. The elements in group 1IIA (boron, aluminum, gallium, indium, and thallium) were the first choice based on a quick search of the literature. Lutetium may also be of use.

[008 j Alternatively, one might attach the metal or other atom to the peptide first and then add the F-i 8. The second approach might work better, for example, for a boron fluoride connection. |009| Aluminum fluoride complexes are reported to be stable in-vUro (Martinez et al, Inorg. Chm. 1999; 38: 4765-4660: Antonny etal. J. Biol. Ckem. 1992:267:6710-67(8),

Aluminum fluoride becomes incorporated into bone and into the enamel of teeih so the complexes can also be stable in-vivo (Li, Crit. Bev. Oral Biol. Med. 2003; 14: 100-114).

[00.10 j The skilled artisan will realize that virtually any delivery molecule can be used to attach the P-18 for imaging purposes, so long as it contains derivaiizabie groups that may be modified without affecting the ligand-receptor binding interaction between the delivery molecule and the cellular or tissue target receptor. Although the Examples below concern F-18 labeled peptide moieties, many other types of delivery molecules, such as oligonucleotides, hormones, growth factors, cytokines, chemokines, angiogenic factors, anti-angiogenic factors, imnuinomodulators, proteins, nucleic acids, antibodies, antibody fragments, drugs, interleukins, interferons, oligosaccharides, polysaccharides, lipids, etc. may be F-18 labeled and utilized for imaging purposes. Similarly, the type of diseases or conditions that may be imaged is limited only by the availability of a suitable delivery molecule for targeting a cell or tissue associated with the disease or condition. Many such delivery molecules are known, as exemplified in the Examples below. For example, any protein or peptide that binds to a diseased tissue or target, such as cancer, may be labeled with F-18 by the disclosed methods and used for detection and/or imaging, la certain embodiments, such proteins or peptides may include, but are not limited to, antibodies or antibody fragments that bind to utinor-associated antigens (TAAs). Any known TAA-binding antibody or fragment may be labeled with F-18 by the described methods and used for imaging and/or detection of tumors, for example by PET scanning or other known techniques. |Of)H j In certain Examples below', the exemplary F-18 labeled peptides may be of use for imaging purposes as targetable constructs in a pre-targeiing method, utilizing bispecific or multbpecific antibodies or antibody fragments. lathis case, the antibody or fragment will comprise one or more binding sites for a target associated with a disease or condition, such as a tumor-associated or autoimmune disease-associated antigen or an antigen produced or displayed by a pathogenic organism, such as a virus, bacterium, fungus or other microorganism, A second binding site will specifically bind to the targetable construct. Methods for pre-targeting using bispecific or muHispecific antibodies are well known in the art (see, e.g,,U.S. Patent No, 6,062,702, the entire contents of which are incorporated herein by reference.) Similarly, antibodies or fragments thereof that bind to targetable constructs tue also well known in the art. (/</.), such as (he 679 monoclonal antibody that binds to HSQ (histamine succinyl glycine). Generally, m pretargeting methods the («specific or multispecific antibody is administered first and allowed to bind to cell or tissue target antigens. After an appropriate amount of time for unbound antibody to dear from circulation, the e g. F-18 labeled targetable construct is administered to the patient and binds to the antibody localized to target cells or tissues, then an image is taken for example by PET scanning.

[0012] In an exemplary embodiment, a non-peptide receptor targeting agent such as folic acid may be conjugated to ΝΟΤΑ and then labeled with, for example, an F-18 metal complex that binds to NOTA, Such non-peptide receptor targeting agents may include, for example, TAi 38, a non-peptide antagonist, for the imegrin οφ? receptor (Liu et ah, 2003, Bioconi. Chem. 14:1052-56). Similar non-peptide targeting agents known in the art that can be conjugated to DGTA, ΝΟΤΑ or another chelating agent for P-18 metal complexes may be utilized in the claimed methods. Other receptor targeting agents are known in the art, such as the somatostatin receptor targeting agent In-DTPA octreotide (TYCO#). As discussed below, an F-] 8-metal complex could potentially be chelated using DTP A and used for imaging purposes. The NODAGATOC peptide could be labeled with AIF-18 for somatostatin receptor targeting (Eisenwiener ei. al. Bioconj. Chem. 2002,13(3.):530-41). Other methods of receptor targeting imaging using metal chelates are known in the art and may be utilized in the practice of the claimed methods (see, e.g., Andre et »1„ 2002, J. Inorg. Biochem. 88:1-6; Pearson etal„ 1996,3. Med.,Cheni. 39:1361-71).

[0013 j Imaging techniques and apparatus for F-l 8 imaging by PET scanning are also well known in the art (see, e.g., U.S. Patent Nos. 6,358,489; 6,953,567; Page et al., Nuclear Medicine And Biology, 21:91 i-919,1994; Choi et al.. Cancer Research 55:5323-5329, 1995; Zalulsky ei al., J. Nuclear Med., 33:575-582, 1992) and any such known PET imaging technique or apparatus may be utilized.

[0014] Although the Examples below demonstrate the use of F-18 metal complexes for PET imaging, the skilled artisan will realize that stable metal-fluorine complexes, such as (he non-Tadioaciive Ai-27 and F-19 complex, could also be bound to ΝΟΤΑ or other chelators and attached to peptides or other targeting agents for use as an MR1 contrast agent. The A1F ΝΟΤΑ complexes could also be attached to polymers for MR! imaging, The A.IF ΝΟΤΑ derivatives could be used as P.ARACEST MRI imaging agents (Woessner et. ai. Magn. Reson. Med. 2005, 53; 790-99).

Br ief Description of the Drawings [OOISj The following Figures are included to illustrate particular embodiments of the invention and are not meant io be limiting as to the scope of foe claimed subject matter.

[0016] FIG, 1. Exemplary peptide IMP 272.

[0017( FIG. 2 . Exemplary peptide IMP 288.

[0018] FIG. 3. Exemplary peptide IMP 326.

[0019] FIG. 4, .Exemplar)·'peptide IMP 329.

[0020] FIG. 5. Exemplary peptide IMP 331.

[0021] FIG. 6. Exemplary peptide IMP 332.

[0022} FIG. 7. Exemplary pepiide IMP 333.

[0023} FIG. 8. Exemplary peptide IMP 334.

[0024} FIG. 9. Exemplary pepiide IMP 349.

[0025] FIG. 10. Exemplary peptide IMP 368.

[0026] FIG. 1.1. Exemplary peptide IMP 375.

[0027] FIG. 12. Exemplary peptide IMP 384, [0028] FIG. 13. Exemplary peptide IMP 386. 10029] FIG. 14. Exemplary peptide IMP 389.

[0030] FiG. 15. Exemplary peptide IMP 449.

[0031} FIG. 16. Additional exemplar)' peptides IMP 422, IMP426 and IMP 428.

[0032} FIG. 17. Exemplary ΝΟΤΑ derivative. (0033} FIG, 18. Exemplary NODA-peptide structure.

[0034] FIG. 19, Comparative biodistributioti of In-111 and F-18 labeled IMP 449 in mice will) or without TP2 bispecifie antibody.

[0035 j FIG, 20. In vivo imaging of tumors using an 151 In-labeled diHSG peptide (IMP 288) with or without pvetargeting IF 10 bispecific anti-MUC 1 antibody.

[0036] FIG, 21. PEI' imaging of micrometastatic human colon cancer in lungs of nude mice, using '^-labeled peptide and pretargeting with TF2 bispecific anti-CFA antibody.

[0037] FIG, 22A-22.D, Additional exemplary chelating moieties for use with F-18 labeling. DETAILED DESCRIPTION

[0038] In the description thru follows, a number of terms are used-and 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, [0039 j As used herein, “a” or “an” may mean one or more than one of an item.

[0040] 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.

[0041) As used herein, “about” means within plus or minus ten percent of a number. For example, “about 100” would refer to nny number between 90 anti i 10.

[0042] As used herein, a “pept ide” refers to any sequence of naturally occurring or non-naturaliy occurring amino acids of between 2 and 100 amino acid residues in length, more preferably between 2 and 10, more preferably between 2 and <5 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.

[0043) As used herein, a labeled molecule is “purified” when the labeled molecule is partially or wholly separated from unlabeled molecules, so that tire fraction of labeled molecules is enriched compared to the starting mixture. A “purified” labeled molecule may comprise a mixture of labeled and unlabeled molecules in almost any ratio, including but not limited to about 5:95; 10:90; 15:85; 20:80; 25:75; 30:70; 40:60; 50:50; 60:40; 70:30; 75:25; 80:20; 85:15; 90:10; 95:5; 97:3; 98:2; 99:1 or 100:0.

[0044] As used herein, the term “pathogen” includes, but is not limited to fungi, viruses, parasites and bacteria, including but not limited to human immunodeficiency vims (HIV), herpes virus, cytomegalovirus, rabies virus, influenza vims, hepatitis B virus, Sendai virus, feline leukemia virus, Reo virus, polio virus, human serum parvo-like virus, simian vims 40, respiratory syncytial virus, mouse mammary tumor virus, Varicella-Zoster virus, Dengue virus, rubella virus, measles virus, adenovirus, human T-ceil leukemia viruses, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocytic choriomeningitis virus, wart vims, blue tongue virus, Streptococcus agaluctiae. Legionella pmumophilla, Streptococcus pyogenes. Escherichia, coll. Neisseria gonorrhoea«, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Ly me disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis and Chbstridium tetani. j 0045] As used herein, a “radiolysis protection agent” refers to any molecule, compound or composition that may be added to an F-18 labeled complex or molecule to decrease the rate of breakdown of the F-18 labeled complex or molecule by radiolysis. Any known radiolysis protection agent, including but not limited to ascorbic acid, may be used..

TmgetabSe Construct Peptides [0046) In certain embodiments, the F-18 labeled moiety may comprise a.peptide or other targe-table construct. F-18 labeled peptides (or proteins) may be selected to bind directly to a targeted cell, tissue, pathogenic organism or other target lor imaging and/or detection. In other embodiments, F-18 labeled peptides may be selected to bind indirectly, for example using a bispecific antibody with one or more binding sites for a targetable construct peptide and one or more binding sites for a target antigen associated with a disease or condition. Bispecific antibodies may be used, for example, in a pretnrgeling technique wherein die antibody may be administered first io a subject. Sufficient time may be allowed for the bispecific antibody to bind to a target antigen and for unbound antibody to clear from circulation. Then a targetable construct, such as an F-I8 labeled peptide, may be administered to the subject and allowed to bind to the bispecific antibody and localize to the diseased cell or tissue, after which the distribution of the F-i 8 labeled targetable construct may be determined by 'PET scanning or other known techniques. (0047] Such targetable constructs can be of diverse structure and arc selected not only for the availability of an antibody or fragment that binds with high affinity io the targetable construct, but also for rapid in viva clearance when used within the pre-targeting method and bispecific antibodies (bsAb) or multispecific antibodies. Hydrophobic agents are best at eliciting strong immune responses, whereas hydrophilic agents are preferred for rapid in vivo clearance. Thus, &amp; balance between hydrophobic and. hydrophilic character is established. This may be accomplished, in part, by using hydrophilic chelating agents to offset the inherent hydrophobicity of many organic moieties. Also, sub-units of the targetable construct may be chosen which have opposite solution properties, for example, peptides, which contain amino acids, some of which are hydrophobic and some of which are hydrophilic. Aside from peptides, carbohydrates may also be used, [0048( Peptides having as few as two amino acid residues, preferably two to ten residues, may be used and may also be coupled to other moieties, such as chelating agents. The linker should be a low molecular weight conjugate, preferably having a molecular weight of less titan 50,000 daltons, and advantageously less than about 20,000 daltons, 10,000 daltons or 5,000 da!tons, including the metal ions in the chelates. More usually, the targetable construct peptide will have four or more residues, such as the peptide DOTA-Phe-Lys(HSG)-Tyr-Lys(HSG)-NH2 (SEQID NO: 1). wherein DOTA is 1,4,7,10-tetraazacyclododecanetetraacetic acid and HSG is the histamine succinyi givey! group. Alternatively, the DOTA may be replaced by a ΝΟΤΑ U,4,7-tTiaza.-cyckmonan.e-N,N’!N”-triacetic acid) or TF.TA (p-bromoacetamidO'benz,y1-tetraethylaminetetra.acetic acid) moiety.

[00491 The targe fable construct may also comprise unnatural amino acids, e.g., D-amino acids, in the backbone structure to increase the stability of the peptide in vivo. 3n alternative embodiments, other backbone structures such os those constructed from non-natural amino acids and peptoids. 10050] The peptides used as targelabie constructs are synthesized conveniently 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 tor chelate conjugation, are advantageously blocked with standard protecting groups such as a Boo group, while .N-terminal residues may be acetylated to increase serum stability. Such protecting groups will be known to the skilled, artisan. See Greene and. Wots Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, N.Y.). When the peptides are prepared for later use within the bispecific antibody system, they are advantageously cleaved from the resins to generate the corresponding C-terminal amides, in order to inhibit in vivo carboxypeptidase activity.

[0051} The haptens of the immunogen comprise a recognition moiety, for example, a chemical hapten. Using a chemical hapten, preferably the HSG hapten, high specificity of the linker for the antibody is exhibited. Antibodies raised to the HSG hapten, are known and can be easily incorporated into the appropriate bispecific antibody (see, e.g., U.S. Patent »Nos. 6,962,702; 7,138,103 and 7,300,644, the entire text of each of which is incorporated herein by reference). Thus, binding of the linker with the attached hapten would be highly specific for tire antibody or antibody fragment.

Chelate Moieties [0052| In some embodiments, an F-l 8 labeled molecule may comprise one or more hydrophilic chelate moieties, 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 may be readily interchanged. (0053 ] Particular ly useful metal-chelate combinations include 2-benzyl-DTP A and its tnonomeihyl and cyclohexyl analogs. MacrocycHc chelators such as ΝΌΤΑ (!,4,74i'ia/n-cyc 1 otronane~N,N ’ ,N”-iri a eetic acid), DOTA, and TE-TA (p-brotnoacetamido-benzyl-tetraethy laminetetraacetic acid) are also of use with a variety of metals, that may potentially be used as ligands forF-18 conjugation.

[0054[ DT.PA 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 Ha and Group Ilia metal cations. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of i nterest. Other ring-type chelators such as roacrocyclic polyeihers are of interest lor stably binding nuclides. Porphyrin chelators may be used with numerous metal complexes. More than one type of chelator may be conjugated to a carrier to bind multiple metal ions. Chelators such as those disclosed in Ü.S. Pat. No. 5,753,206, especially thiosemicarbazonylglyoxylcysteine (Tscg-Cys) and thiosemicarbazinyl-acetylcysteine (Tsca-Cys) chelators are advantageously ased to bind soft acid cations of Tc, Re, Bi and other transition metals, lanthanides and actinides that are tightly bound to soft base ligands. It can be useful to link more (han one type of chelator to a peptide. Because antibodies to a di-DTPA hapten, are known (Barbel et al.> US. Pal. Nos. 5,256,395) and are readily coupled to a targeting antibody to fonn a bispecific antibody, it is possible to use a peptide hapten with cold diDTPA chelator and another chelator lor binding an F-18 complex, in a pretargeting protocol. One example of such a peptide is Ac-

Lys(DTPA)''ryr*Lys(DTPA)-Lys(Tscg-Cys)-NH?. (SBQ ID NG:2). Other hard acid chelators such as DOT A, TETA and the tike can be substituted for the DTP A and/or Tscg-Cys groups, and M Abs specific to them can be produced using analogous techniques to those used to generate the. anti-di-DTPA MAI). (0055] Another useful chelator may comprise a NOTA-lype moiety, for example as disclosed in Chong el at. (Rational design and generat ion of a bimodal bifunctional ligand for antibody* targeted radiation cancer therapy, J. Med. Chem., e-published on 12-7-07, incorporated herein by reference). Chong et al. disclose the production and use of a. bifunctional C-NETA ligand, based upon the ΝΟΤΑ structure, that when comp lexer! with !77Lu or 305 J0<’Bi showed stability in serum lor up to 14 days. (0056] It will be appreciated that two different hard acid or soft acid chelators can be incorporated into the targetable construct, e.g., wit!) different chelate ring sixes, to bind preferentially to two different hard acid or soft acid cations, due to the differing sixes of the cations, the geometr ies of the chelate rings ami the preferred complex ion structures of the cations. This will permit two different metals, one or both of which may be attached to F* 18, to be incorporated into a targetable construct tor eventual capture by a pretargeted bispeciftc antibody.

Methods of Administration [0()57] In various embodiments, bispecific antibodies and targetable constructs maybe used for imaging normal or diseased tissue and organs (see, e.g. U.S. Pat. Nos, 6,126,916; 6,077,499; 6,010,680; 5,776,095; 5,776,094; 5,776,093; 5,772,951; 5,753,206; 5,746,996; 5,697,902; 5,328,679; 5,128,119; 5,10.1,827; and 4,735,210, each incorporatedherein by reference in its entirety). (0058] The administration of a bispeciftc antibody (bsAb) and an F-1K labeled targetable construct may be conducted by administering tire bsAb antibody at some time prior to administration of the targetable construct The doses and timing of the reagents can be readily devised by a skilled artisan, and are dependent on the specific nature of the reagents employed. If a bsAb-.F(ab’)’ derivative is given first, then a waiting time of 24-72 hr (alternatively 48-96 horn's) before administration of the targetable construct would be appropriate, if an IgO-Fab’ bsAb conjugate is the primary targeting vector, (hen a longer waiting period before administration of the targetable construct would be indicated, in the range of 3-10 days. After sufficient lime has passed for the bsAb to target to tire diseased tissue, the F48 labeled tavgetable construct is administered. Subsequent to administration of the targetable construct, imaging can be performed. )0059j Certain embodiments concern the use of multivalent target binding proteins which have at least three diilerent target binding sites as described in patent, application Ser. No. 60/220,782. Multivalent target binding proteins have been made by cross-linking several Fab-like fragments via chemical linkers. See U.S. Pat. Nos. 5,262,524; 5,091,542 and Landsdorp el al. Euro. J. Immunol. 16: 679-83 (1986). Multivalent target binding proteins also have been made by covalently linking Severn! single chain Fv molecules (scFv) to form a single polypeptide. See U.S. Pat. No. 5,892,020. A multivalent target binding protein which is basically an aggregate of scFv molecules has been disclosed in U.S. Pat. Nos. 6,025, .165 and 5,837,242. A trivalem target binding protein comprising three scFv molecules has been described in Krott et al. Protein Engineering 10(4): 423-433 (1997). )0060] Alternatively, a technique known as “dock-and-lock” (DNL) has been demonstrated for the simple and repr oducible construction of a variety ofmttUivalei.it complexes, including complexes comprising two or more different antibodies or antibody fragments. (See, e.g„ U.S. Patent Application Serial Nos. 11/389,358, filed March 24,2006; 11/391,584, filed March 28, 2006; 11/478,021, filed June 29,2006; 11/633,729, filed December 5,2006; and 11/925,408, filed October 26,2007, the text of each of which is incorporated herein by reference in its entirety.) Such constructs are also of use for the practice of the claimed methods and compositions described herein.

[0061] A clearing agent may be used which is given between doses of tire bispecific antibody (bs.Ab) and the targetable construct. A clearing agent of novel mechanistic action may be used, namely a glycosylated anti-idiotypic Fab’ fragment targeted against the disease targeting arm(s) of the bsAb. In one example, anli-CEA (MN-14 Ab) x anti-peptide bsAb is given and allowed to accrete in disease targets to its maximum extent. To clear residual bsAb, an anti-idiotypic Ab to MN-14, termed W12, is given, preferably as a glycosylated Fab* fragment. The clearing agent binds to the bsAb in a monovalent manner, while its appended glycosyl residues direct the entire complex to the liver, where rapid metabolism takes place. Then the F-18 labeled targetable construct is given to (be subject. Tbc W12 Ab to the MN-14 arm of the. bsAb has a high affinity and the clearance mechanism differs from other disclosed mechanisms (see Goodwin et al, ibid), as it does not involve cjfoss-1 inking, because the W12-Fab’ is a monovalent moiety. However, alternative methods and compositions for clearing agents are known and any such known clearing agents may be used.

Formulation and Administration [0062 j The F-18 labeled molecules may be formulated to obtain compositions that include one or more pharmaceutically suitable excipients, one or more additional ingredients, or some combination of these. T hese cm» be accomplished by known methods to prepare pharmaceutically useful dosages, whereby the active ingredients (i.e., the F-18 labeled molecules) are combined in a mixture with one or more pharmaceutically suitable excipients. Sterile phosphate-buflered 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 &amp; Febiger 1990), and Gennaro (ed.),.REMINGTON'S PHARMACEUTICAL SCIENCES, ]8th Edition (Mack Publishing Compauv 1.990), and revised editions thereof, [0063] The preferred route for administration of the compositions described herein is parental injection. Injection may be intravenous, intraarterial, intralympliatic, intrathecal, 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 nontax ie and nontherapeuiic. Examples of such excipients are saline, Ringer's solution, dextrose solution and Hank's solution. Nonaqneous excipients such as fixed oils and ethyl oleaie may also be used. A preferred excipient is 5% dextrose in saline. The excipient may contain minor amounts of additi ves such as substances that enhance isotonicity and chemical stability, including buffers and preservatives. Other methods of administration, including oral administration, are also contemplated, (0064) Formulated compositions comprising F-18 labeled molecules 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 cat) contain formulafory 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.

[O065| The compositions may be administered in solution. The pi I 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) aminomeihane-HCl or citrate and the like. Buffer concentrations should be in the range of l to ! 00 raM. 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- Tire 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. (0066) Where bispecific antibodies are administered, for example in a preiargeting technique, the dosage of an administered antibody for humans will vary depending upon such factors as tire patient’s age, weight, height, sex, general medical condition and previous medical history. Typically, for imaging purposes it is desirable to provide the recipient with a dosage of bispecific antibody hat is in the range of from about 1 mg to 200 mg as a single intravenous infusion, although a lower or higher dosage also may be administered as circumstances dictate. Typically, it is desirable to provide the recipient will) a dosage that is in the range of from about 10 mg per square meter of body surface area or 17 to 18 rag of the antibody for the typical adult, although a lower or higher dosage also may be administered as circumstances dictate, Examples of dosages ofbispeciftc antibodies that may be administered to a human subject for imaging purposes are 1 to 200 mg, more preferably .1 to 70 mg, most preferably 1 to 20 mg, although higher or lower doses may be used. (00671 In general, the dosage of F-18 label to administer 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 F-I8 labeled molecules is administered to a patient. For administration of F-18 labeled molecules, the dosage may be measured by roiliieuries, A typical range for F-18 imaging studies would be five to 10 vnCi.

Administration of Peptides [0068] Variolas embodiments of the claimed methods and/or compositions may concern one or more F-l 8 labeled 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, orlhotopic, intradermal, subcutaneous, intramuscular, intraperitoneai. intraarterial, intrathecal or intravenous injection. Where, for example, F-18 labeled peptides are administered in a preiargeting protocol, the peptides would preferably be administered i.v.

[0069] Unmodified peptides administered orally to a subject can be degraded in the digestive tract and depending on sequence and structure may exhibit poor absorption across the intestinal lining. However, methods for chemically modifying peptides to render them less susceptible to degradation by endogenous proteases or more absorbable through the alimentary tract are well known (see, for example, Blondelle et al., 1995, Biophys. .1. 69:604-11; Ecker and Crooke, 1995, Biotechnology 13:351-69; Goodman and Ro, 1995, BURGER'S MEDICINAL CHEMIS TRY AND DRUG DISCOVERY, VOL. 1, ed. Wolff, lohn Wiley &amp; Sons; Goodman and Shao, 1996, Pure &amp; Appl. Chem. 68:1303-08). Methods for preparing libraries of peptide analogs, such as peptides containing D-amino acids; peptidomimetics consisting of organic molecules that mimic the structure of a peptide; or peptoids such as vinylogons peptoids, have also been described and may be used to construct peptide based F» 18 labeled molecules suitable for oral administration to a subject, [0070J in certain embodiments, the standard peptide bond linkage may be replaced by one or more alternative linking groups, such as CI-fj-NH, CIL-S, CHrCHj, CH>:€H, CO-C1L, CHOH-CHj and the like. Methods for preparing peptide mimetics are well known (for example, Hruby, 1982, Life Sei 31:189-99; Holladay etal., 1983, Tetrahedron lea. 24:4401-04; Jennings-White et ah, 1982, Tetrahedron hat. 23:2533; Almquiesl et al., 1980, J. Med. Chem. 23:1392-98; Hudson etal., 1979, Ini../. Pep!. Res. 14:177-185; Spatola etal, 1986, Life Sei 38:1243-49; U.S. Patent Nos. 5,169,862; 5,53.9,085; 5,576,423, 5,051,448,5,559,103, each incorporated herein by reference,) Peptide mimetics may exhibit enhanced stability and/or absorption in vivo compared to their peptide analogs, [0071 j Alternatively, peptides may be administered by oral delivery using N-ierrainal and/or C-tenninal capping to prevent exopeptidase activity. For example, tire C-ferminus may be capped using amide pept ides and (he N-termimis may be capped by acetylation of the peptide. Peptides may also be cyclized to block exopeptidases, for example by formation of cyclic amides, disulfides, ethers, sulfides and the like.

[0072] 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 ait and methods for making and using peptides incorporating D-amino acids have been described (e.g., U.S, Patent

Application Publication No. 20050025709, McBride el a!., filed June 14,2004, incorporated herein by reference). In certain embodiments, peptides and/or proteins may be orally administered by co-formulation with proteinase- and/or peptidase-inhibitors.

[0073 i Other methods for oral deli very of therapeutic peptides are disclosed in Mehta (“Oral delivery and recombinant production of peptide hormones,” June 2004, BfoPharm International). The peptides are administered in an enteric-coaied solid dosage form with excipients that modulate intestinal proteolytic activity and enhance peptide transport across the intestinal wall. Relative bioavailability of intact peptides using (his technique ranged from 1 % to 10% of the administered dosage. Insulin has been successfully administered in dogs using enteric-coated microcapsules with sodium choline and a protease inhibitor (Ziv et ah, 1994,./. Bone Miner. Res. 18 (Supp!. 2):792-94. Oral administration of peptides has been performed vising acylcamitine as a permeation enhancer and an enteric coating (Eudragit L30D-55, Rohm Pharma Polymers, see Mehta, 2004). Excipients of use for orally administered peptides may generally include one or more inhibitors of intestinal proteases/peptidases along with detergents or other agents to improve solubility or absorption of the peptide, which may be packaged within mr enteric-coated capsule or tablet (Mehta, 2004). Organic acids may lie included in the capsule to acidify the intestine and inhibit intestinal protease activity once the capsule dissolves in the intestine {Mehta, 2004). Another alternative for oral delivery of peptides would include conjugation to polyethylene glycol (PEG)-based amphiphilic oligomers, increasing absorption and resistance to enzymatic degradation (Soltero and Kkwuribe, 2001, Pharm. Technol. 6:110).

Methods for Raising Antibodies [0074 j Abs to peptide backbones may be generated by well-known methods for Ab production. For example, injection of an immunogen, such as (pepiide)n-K.LH, wherein KLH is keyhole limpet hemocyanin, and n-1-30, in complete Freund’s adjuvant, followed by two subsequent injections of the same immunogen suspended in incomplete Freund’s adjuvant into immunocompetent animals, is .followed three days after an i.v. boost of antigen, by spleen ceil harvesting. Harvested spleen cells are then fused, with Sp2/0-Ag.l4 myeloma cells and culture supernatants ofthe resulting clones analyzed for anti-peptide reactivity using a direct-binding ELISA. Specificity of generated Abs can be analyzed for by using peptide fragments of the original immunogen. These fragments can be prepared readily using an automated peptide synthesizer. For Ab production, enzyme-deficient hybridomas are isolated [o enable selection of fused cell lines. This technique also can be used to raise antibodies to one or more of the chelates comprising the targetüble construct, e.g., in(ni}~DTPA chelates. Monoclonal mouse antibodies to an in(OI)*di-DTPA are known (Barbel ‘395 supra).

[0075] Targeting antibodies of use, for example as components of bispecific antibodies, may be specific to a variety of cell surface or intracellular tumor-associated antigens as marker substances. These markers may be substances produced by the tumor or may be substances which accumulate at a tumor site, on tumor cell surfaces or within tumor cells, whether in Lhe cytoplasm, the nucleus or in various organelles or sub-cellular structures. Among such tumor-associated markers are those disclosed by Merberman, “Immunodiagnosis o f Cancer”, in Fleisher ed., “The Clinical Biochemistry of Cancer”, page 347 (American Association of Clinical Chemists, 1979) aud in U.S. Pat. Nos. 4,150,149; 4,361,544; and 4,444,744, each incorporated herein by reference. Recent reports on tumor associated antigens include Mizukami et aL (2005, Nature Med 11:992-97); Hatfield et a!., (2005, Omr. Cancer Drug Targets 5:229-48); Vallbohmer el al. (2005,./. Clin. Oncol 23:3536-44); and Ren et al (2005, Am. Surg. 242:55-63), each incorporated herein by reference. (0076( Tumor-associated markers have been categorized by Herbennan, supra, in a number of categories includ ing oncofetal antigens, placental antigens, oncogenic or tumor virus associated antigens, tissue associated antigens, organ, associated antigens, ectopic hormones and normal antigens or variants thereof. Occasionally, a sub-unit of a tumor-associated marker is advantageously used to raise antibodies ha ving higher tumor-specificity, e.g., the beta-subunit of human chorionic gonadotropin (BCG) or the gamma region of carcinoembryonic antigen (CEA), which stimulate the production of antibodies having a greatly reduced cross-reactivity to non-tumor substances as disclosed in US. Pat, Nos. 4,361,644 and 4,444,744.

[0077 ( Another marker of interest is Iransmembrane activator and CAML-interaclor (TACI). See Yu et al. Nat, Immunol. 1:252-256 (2000). Briefly, TACI is a marker for B-ceil malignancies (e.g., lymphoma). Further it is known that TACI and B cell maturation antigen (BCMA) are bound bv the tumor necrosis factor homolog - a proliferation-inducing ligand (APRIL.). APRIL stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL also competes with TALL-I (also called BLyS or BAF.F) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APR.II, and block APRIL-stimulated proliferation of primary B cells. BCM A-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and

Pneumovax in mice, indicating that APRIL and/αν TALL-I signaling via BCMA and/or ΤΛΟΪ are required for generation of humoral immunity. Thus, APRU.-TAU.-i and BCMA-TACI form a two ligand-two receptor pathway involved in stimulation of B and T cd I function.

[0078] Exemplary target antigens of use for imaging various diseases or conditions, such as a malignant disease, a cardiovascular disease, an infectious disease, an inflammatory disease, an autoimmune- disease, or a neurological disease may include colon-specific antigeu-p (CSAp), carcinoembrvonic antigen (CEA), CD4, CD3, CDS, CD 14, CD 15, CD 19, CD20, CD2I, CD22, CD23, CD25, CD30, CD45, CD74, CD79a, CD80, HLA-DR, fa, 1i, MUC 1, MUC 2, MUC 3, MUC 4, MCA (CEACAM6 or CD66a*d and CD67, as well as CD138), EGFR, HER 2/neu, TAG-72, EGP-1, EGP-2, A3, KS-i, Le(y), Si Oft, PSMA, PSA, tenascin, folate receptor, VEGFR, PlGF, ILGF-I, necrosis antigens, IL-2,1L-6, Tl 01, MAGE, or a combination of these antigens. In particular, antigens may include camnoembryunic antigen (CEA), tenascin, epidermal growth factor receptor, platelet derived growth factor receptor, fibroblast growth factor receptors, vascular endothelial growth factor receptors, gangliosides, HER/2neu receptors and combinations of t hese antigens.

[00791 Where imaging or detection involves a lymphoma, leukemia or autoimmune disorder, targeted antigens may be selected from the group consisting of CD4, CD5, CD8, CD 14, CD! 3, CD 19, CD20, CD21, CD22, CD23, CD25, C033, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD67, CD74, CD79a, CD80, CD12G, CD138, CD 154, B7, MUC 1, la, li, HM 1.24, HLA-DR, tenascin, V.EGF, PlGF, ED-B fibronectin, an oncogene, an oncogene product, CD66a-d, necrosis antigens, IL-2, T1.Ü1, TAG, IL-6, .Ml.F, T.RAiL-Rl (DR4) and TRA1L-R2 (DR5).

[0080) After the initial raising of antibodies lo the immunogen, the antibodies can be sequenced and subsequently prepared bv recombinant techniques. Humanization and chiraerizalion of murine antibodies and antibody fragments are well known to those skilled in the art. For example, humanized monoclonal antibodies «reproduced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, and then, substituting human residues in the framework regions of the murine counterparts. The use of antibody components derived from humanized'monoclonal antibodies obviates potential problems associated with the inununogeniciiy of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by the publication of Oriandi et aL Proc, Natl Acad, Set. USA 86: 3833 (1989), which is incorporated by reference in its entirety. Techniques lor producing humanized MAbs are described, lor example, by Jones et aL Nature 321: 522 (1986), Riechmann et.ah, Nature 332: 323 (1988), Verhoeyen et a!., Science 239: 1534 {1988), Carter etal., Proc. Nat’) Acad. Sei. USA 89:4285 (1992), Sandbu, Crit. Rev. Biotech. 12: 437 {1992), and Singer et aL, J. immun. 150:2844(1993), each of which is incorporated herein by reference in its entirety. (0081] Alternatively, Fully human antibodies can be obtained from transgenic non-human animals. See, e.g., Mendez et al., Nature Genetics, 15: 140-156 (1997); U.S. Pat. No. 5,633,425. For example, human antibodies can be recovered from transgenic mice possessing human immunoglobulin loci. The mouse humoral immune system is humanized by inactivating the endogenous immunoglobulin genes and introducing human immunoglobulin loci. The human immunoglobulin loci are exceedingly complex and comprise a large number of discrete segments which together occupy almost 0.2% of the human genome. To ensure that transgenic mice are capable of producing adequate repertoires of antibodies, large portions of human heavy- and light-chain loci must be introduced into the mouse genome. This is accomplished in a stepwise process beginning with the formation of yeast artificial chromosomes (YACs) containing either human heavy- or light-chain immunoglobulin loci in germline configuration. Since each insert is approximately 1 Mb in size, YAC construction requires homologous recombination of overlapping fragments of the immunoglobulin loci. The- two YACs, one containing the heavy-chain loci and one containing the light-chain loci, are introduced separately into mice via fusion of YAC-containing yeast spheroblasls with mouse embryonic stem cells. Embryonic stem cell clones are then microinjeaed into mouse blastocysts. Resulting chimeric males are screened for iheir ability to transmit the YAC.' through their germline and tue bred with mice deficient in murine antibody production. Breeding ihe two transgenic strains, one containing the human heavy-chain loci and the other containing the human light-chain loci, creates progeny which produce human antibodies in response to immunization.

[0082] Unrearranged immun immunoglobulin genes also can be introduced into mouse embryonic stem cells via tnicrocell-mediated chromosome transfer (MMCT). See. e.g., Tomizuka et al., Nature Genetics, 1.6: 133 (1997). In this methodology microccils containing human chromosomes are fused with mouse embryonic stem cells. Transferred chromosomes are stably retained, ami adult chimeras exhibit proper tissue-specific expression.

[0083 j As an alternative, an antibody or antibody fragment may be derived .from human antibody fragments isolated from a combinatorial immunoglobulin library. See, e.g., Barbas et al., METHODS: A Companion to Methods in Enzymology 2: i 19 (1991), and Winter et al., Ann. Rev. Immunol. 12: 433 (1994), which are incorporated herein by reference. Many of the difficulties associated with generating monoclonal antibodies by B-cell immortalization can be overcome by engineering and expressing antibody fragments in E, coli, using phage display. (0084) A similar strategy can be employed io obtain high-affinity scFv. See, e.g., Vaughn et at., Nat. BiotechnoL, 14: 309-314 (1996). An scFv library with a large repertoire can be constructed by isolating V-genes from non-immunized human donors using PCR primers corresponding io all known Vif, ν^ρί, and Vg» gene families. Following amplification, the Vfcflppa and Vtsmbdii pools am combined to form one pool. These fragments are ligated into a phagemid vector. The scFv linker, (Gly*. Ser):,: is then ligated into the phagemid upstream of the Vi, fragment. The Vu and 1 inker-Vi, f ragments are amplified and assembled on the Ju region. The resulting Vu -linker-V), fragments are ligated into a phagemid. vector. The phagemid library can be panned using filters, as described above, or using immunotubes (NUNC®; M AXISORP®), Similar results can be achieved by constructing a combinatorial immunoglobulin library from lymphocytes or spleen cells of immunized rabbits and by expressing the scFv constructs in 1\ pastaris. See, e.g., Ridder et al.. Biotechnology, 13: 255-260 (1995). Additionally, following isolation of an. appropriate scFv, antibody fragments with higher binding affinities and slower dissociation rales can be obtained through affinity maturation processes such as CD.R3 mutagenesis and chain shuffling. See, e.g,, Jackson et al,, Br. 3. Cancer, 78: 181-188 (1998); Osbourn et al., Immtmotechnology, 2: 181-196 (1996). (0085) Another form of an antibody fragment is a peptide coding for a single C.DR. CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells, See, for example, Larrick et al.. Methods: A Companion to Methods in Enzymology 2:106 (1991); Courtenay-Luck, “Genet ic Manipulation of Monoclonal Antibodies,” in MONOCLONAL ANTIBODIES; PRODUCTION, ENGINEERING AND CLINICAL APPLICATION, Ritter et al. (eds.), pages 166-179 (Cambridge University Press 1995); and Ward et al., “Genetic Manipulation and Expression of Antibodies," in MONOCLONAL ANTIBODIES: PRINCIPLES AND APPLICATIONS, Birch et al., (eds,), pages 137-185 (Wiley-Liss, Inc. 1995).

[00861 Bispecific antibodies can be prepared by techniques known in the an, for example, an anti-CEA tumor Ab and an anti-peptide Ah are both separately digested with pepsin to their respective Ffab’lj fragments. The auti-CEA-Ab-FiabOs is reduced with cysteine to generate Fab' monomeric units which are further reacted with the cross-linker bisfmaleimido) hexane to produce Fab’-maleimide moieties. The anti-peptide Ab-Fiab’fe is reduced with cysteine and the purified, recovered anti-peptide Fab’-SH is reacted with the anti-CFA-Fab’-maleimide to generate the Fab' x Fab' bi-speciftc Ab. Alternatively, the anti-peptide Fab’-SH fragment may be coupled with Ute anti-CEA F{ab’)j to generate a F(ab’)j x Fab’ construct, or with anti-CEA IgG to generate an IgG x Fab1 bi-specific construct. In one embodiment, the IgG x Fab’ construct can be prepared in a site-specific manner by attaching the antipeptide Fab’ thiol group to anti-CEA IgG heavy-chain carbohydrate which has been periodate-oxidized, and subsequently activated by reaction with a commercially available hydrazide-jmaleimide cross-linker. The component Abs used, can be chimerized or humanized by known techniques. A chimeric antibody1 is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody. Humanized antibodies are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and tight variable chains of the murine immunoglobulin into a human variable domain. (0087.] A chimeric Ab is constructed by ligating the cDNA fragment encoding die mouse light variable and heavy variable domains to fragment encoding the C domains from a human antibody. Because the C domains do not contribute to antigen binding, the chimeric antibody will retain the same antigen specificity as the original mouse Ab but will be closer to human antibodies in sequence. Chimeric Abs still contain some mouse sequences, however, and may still be immunogenic. A humanized Ab contains only those mouse amino acids necessary to recognize the antigen, This product is constructed by building into a human antibody framework the amino acids from mouse complementarity determining regions.

[0088] Other recent methods .for producing bispecific antibodies include engineered recombinant Abs which have additional cysteine residues so that they crosslink more strongly than the more common immunoglobulin isotypes. See, e.g., FitzGerald e-f al., Protein Eng. 10:1221-1225, 1997. Another approach is to engineer recombinant fusion proteins linking two or more different single-chain antibody or antibody fragment segments with the needed dual specificities. See, e.g„Coioma et ai., Nature Biotech. 15:159-163,1997. A variety of bispecific fusion proteins can lie produced using molecular engineering. In one form, the bi-specific fusion protein is .monovalent, consisting of, for example, a scFv with a single binding site for one antigen and a Fab fragment with a single binding site for a second antigen, in another form, the bi-specific fusion protein is divalent, consisting of, for example, an IgG with two binding sites for one antigen and two scFv with two binding sites for a second antigen.

[0089] Functional bi-specific single-chain antibodies (bscAb), also called diabodies, can be produced in mammalian cells using recombinant methods. See, e,g., Mack et ol., Proc. Natl. Acad. Sei., 92: 7021-7025, 1995.

[0090] Preferred bispeeific antibodies are those which incorporate the Fv of MAb Mu-9 and the Fv of MAb 679 or the Fv of MAb MN-14 and the Fv of MAb 679, and their human, chimerized or humanized counterparts. The MN-14, as well as its chi merited and humanized counterparts, are disclosed in U.S. Pat. No. 5,874,540. Also preferred are bispecific antibodies which incorporate one or more of the CDRs of Mu-9 or 679. The antibody can also be a fusion protein or a bispecific antibody that incorporates a Class III anti-CEA antibody and the Fv of 679. Class ΠΙ antibodies, including Class HI anti-CEA are discussed in detail in U.S. Pat. No, 4,818,709.

[00911 Tlie skilled artisan will realize that bispeeific antibodies may incorporate any antibody or fragment known in the art that has binding specificity for a target antigen that is known to be associated with a disease state or condition. Such known antibodies include, but are not limited to, 1X1 (ami-CD74), .1X2 and RFB4 (anti«CD22), hA20(ami-CD20), RS7 (anti-epithelial glycoprotein-1 (EGP-1)}, PAM-4 and KC4 (both anti-mucin), MN-14 (ami-carcinoembryonic antigen (CEA, also known as CD66e)), MN-3 or MN-15 (NCA or CEACAM6), Mu-9 (anti-colon-specific antigen-p), Immu 3i (an anti-alpba-fetopvotein), TAG-72 (e.g., CC49), Tn, J591 (ami-PSMA prostate-specific membrane antigen)), G250 (an anti-carbonic anhvclrase IX M Ab) and L243 (anti-HLA-DR). Such antibodies are known in the art (e.g., U.S. Patent Nos. 5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730.300; 6,89.9,864; 6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004; 7,282,567; 7,300,655; 7,312,318; and U.S. Patent Application Publ.

No. 20040185053; 20040202666; 20050271671; 20060193865; 200602.10475; 20070087001; each incorporated herein by reference in its entirety.) Such known antibodies are of use for detection and/or imaging of a variety of disease states or conditions (e.g., hMN-14 or TF2 bsMAb (CEA-expressing carcinomas), hA20 bsMab (TF-4-lymphoina), bPAM4 (TP* 10 pancreas cancers), RS7 bs'MAb (lung, breast, ovarian, prostatic cancers), bMN-15 or hMN3 bsMAb (inflammation), human gp! 20 and/or g|>4i bsMAbs (HIV), ami-platelet bsMab and ami-thrombin bsMAb (clot imaging), anii-myosiu bsMAb (cardiac necrosis)).

[0092j Candidate anti-HIV antibodies include the ami-envelope antibody described by Johansson et al (AIDS. 2006 Oct 3;20(15):1911-5), as well as the anti-HIV antibodies described and sold by Polyimin (Vienna, Austria), also described in U.S. Patent 5,831,034, U.S. patent 5,9.1 i,989, and Vcelar etal, AIDS 2007; 21(16):21()1-2170 and Joos et al., Anrimicrob. Agens Chemother, 2006; 50(5): 1773-9, alt incorporated herein in their entirety by reference.

[00931 In certain embodiments, the bsAb P-18 labeled targetabie constnrcts discussed above may be ttsed in intraoperative, intravascular, and/or endoscopic tumor and lesion detection, biopsy and therapy as described in U.S. Pat. No. 6,096.289. imaging Using Labeled Molecules [0094] Methods of imaging using labeled molecules are well known in the art, and any such known methods may be used with the fluoride-labeled molecules 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, each incorporated herein by reference in its entirety. See also, Page et al,. Nuclear Medicine And Biology, 21.:9.11-919, 1994; Choi et a!., Cancer Research 55:5323-5329,1995; Zalutsky et al, J. Nuclear Med., 33:575-582,1992; Woessner et. al Mann. Reson. Med. 2005, 53: 790-99.

[0095] In certain embodiments, F-18 labeled molecules may be of use in imaging normal or diseased tissue and organs, for example using the methods described in U.S. Pat, Nos. 6.126,916; 6,077,499; 6,010,680; 5,776,095; 5,776,094; 5,776,093; 5,772,981; 5,753,206; 5,746,996; 5,697,902; 5,328,679; 5,128,119; 5,101,827; and 4,735,210, each incorporated herein by reference. Additional methods are described in U.S. application Ser. No. 09/337,756 Filed Jun. 22,1999 and in U.S. application Ser. No. 09/823,746, filed Apr. 3, 2001. Such imaging can be conducted by direct F-l 8 label ing of the appropriate targeting molecules, or by a pretargeted imaging method, as described in Goldenberg et al (2007, Update Cancer Thar. 2:19-31); Sharkey et al. (2008, Radiology 246:497-507); Goldenberg et al. (2008, J. Nucl. Med. 49:158-63); Sharkey et al (2007, Clin. Cancer Res. 13:5777s-5585s); McBride et al, (2006, J. Nucl Med. 47:1678-88); Goldenberg et al (2006, j. Clin.

Oncol,24:823-85), see also U.S. Patent Publication Nos, 20050002945,20040018557, 20030148409 and 20050014207, each incorporated herein by reference.

[0096] Methods of diagnostic imaging with labeled peptides or M Abs are well-known. For example, in the technique oi'immimoscimigraphy, ligands or antibodies are labeled with a gamma-emitting radioisotope and introduced into a patient. A gamma camera is used to detect the location arid distribution of gamma-emitting radioisotopes. See, for example, srivamva fed.), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY (Plenum Press 1988), Chase, "Medical Applications of Radioisotopes," in REMINGTON'S PHARMACEUTICAL SCIENCES, I8th Edition, GeinarO et al. (eds.), pp. 624-652 (Made Publishing Co., 1990), and Brown, "Clinical Use of Monoclonal Antibodies," in BIOTECHNOLOGY AND PHARMACY 227-49, Pezmto etai. (eds.) (Chapman &amp; Hall 1993). Also preferred is the use of positron-emitting radionuclides (PET isotopes), such as with an energy of 511 keV, such as <iSGa, wCu, and ,24I. Such rad ionuclides may be imaged by well-known PET scanning techniques.

EXAMPLES

Example 1. F-18 Labeling of Peptide IMP 272 [0l)97j The first peptide that was used was IMP 272: DTPA-Gltt-Ala-Lys(MSG)-D-Tyr-Lys(HS0)-NH? Mlf 1512 [0098j 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 lo afford a 0.1 M solution at pH 4.03.

[0099] Aluminum acetate buffer sohuion - A solution of aluminum was prepared by dissolving 0.1028 g of AlCb 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 jnM Al stock solui,ton.

[0.100] IMP 272 acetate buffer solution - Peptide, 0.0011 g, 7,28 x 10"' mol IMP 272 was dissolved in 364 pL of the 0.1 M pH 4 acetate buffer solution to obtain a 2 mM stock solution of the peptide.

JOlOl] F-18 Labeling of IMP 272 - A 3 uL aliquot of the aluminum stock sohuion was placed in a REACTI-VIAL™ and mixed with 50 μΙ_ F-18 (as received) and 3 pL of the IMP 272 solution. The solution was heated in a heating block at 110“C for 15 min and analyzed by reverse phase HPLC. The HP.LG trace (not shown) showed 93 % free F-18 and 7 % bound to the peptide. An additional 10 μί, of the IMP 272 solution was added to the reaction and it was healed again and analyzed by reverse phase HPLC (not shown). The HPLC mice showed 8 % F-18 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 j.sL PBS for ·“ Ihr and then examined by reverse phase HPLC. The HPLC (not shown) showed 5$ % F~18 unbound and 42 % still attached to the peptide. The data indicate that F-18-AI-DTPA. complex may be unstable when mixed with phosphate. )0102] Reverse Phase HPLC - Reverse phase HPLC analysis was done under the following conditions:

Column: WATERS« XTBRRA™ MS C« 5 pm, 4.6 x 250 mm Flow Rale: 1 mL/rnin

Gradient Buffers: Buffer C, 0.1 % MHfOAc in D1 water. Baffer D, 90 % acetonitrile 10 % water and 0.1 % NH4OAC

Gradient: 100 % Buffer C to 10Ü % Buffer D using a linear gradient over 30 min.

Run Time: 30 min )0103 j Size Exclusion HPLC - The size exclusion HPLC was dune under the following conditions:

Column: BIORAD® BfO-SIL™ SEC 250, 300 x 7.8 mm Gradient: Lsocvatic

Eluent Buffer. 0.2 M Phosphate pH 6.8 Flow Rate: 1 mL/rnin Run Time; 30 min [0104( All radiometric traces were obtained using a PERKIN ELMER« 6.1 OTr to monitor the emission of F-18. Tables 1-3 are tabular representations of the data.

Table 1 F-l 8 + IMP 272 + AlCij heated at 110°C for 15 min, followed by analysis by reverse phase HPLC.

Regions: F-18 Detector: FSA

[Ö105J Table 2 F-18 + excess IMP 272 + Aids heated at! 10°C for 15 min, followed by analysis by reverse phase HPLC. . Redons: F-18 Detector: FSA .....................................

Table 3 Phosphate Challenge in PBS for 90 min at room temp. Aliquot of F-18 +· excess IMP 272 + Aids heated at 1 RFC for 15 min and analyzed, by reverse phase HPLC.

Regions: F-18 Detector: FSA

|f)106] The labeled peptide was purified by applying the labeled pept ide solution onto a 1 cc (30 mg) WATERS® HEB column (Part # 186001879) and washing with 300 p.L water to remove unbound F-18. The peptide was eluted by washing the column with 2 x 100 pL 1:1 MeOH/HjG. The purified peptide was incubated in water at 25CC and analyzed by reverse phase .HPLC (not shown). The HPLC analysis showed that the F-1S labeled IMP 272 was not stable in water. After 40 min incubation in water about 17% of the F-18 was released from the peptide, while 83% was retained (not shown).

Example 2. Immunoreactivi ly of F-18 IMP 272 101071 The peptide (16 pL 2 mM IMP 272,48 pg) was labeled with F-18 and analyzed for antibody binding by size exclusion HPLC. The size exclusion HPLC showed that foe peptide bound hMN-14 x 679 but did not bind to the irrelevant bispecific antibody bMN-14 x 734 (not shown).

Example 3. IMP 272 F-18 Labeling with Other Metals (0108( A -'3 pL aliquot of the metal slock solution (6 x 10'9 mol) was placed in a polypropylene cone vial and mixed with 75 pL F-18 (as received), incubated at room temperature for - 2 min and then mixed with 20 pL of a 2 mM (4 x 1(TS mol) IMP 272 solution in 0.3 M NaOAc pH 4 buffer. The solution was heated in a heating block at 10ÖT for 15 min and analyzed by reverse phase HPLC. IMP 272 was labeled with indium (24%), gallium (36%), zirconium (15%), lutetiura (37 %) and yttrium (2 %) (not shown).

Example 4. Standard F-18 Peptide Labeling Conditions Used to Screen Other Peptides For Al-,SF Binding (0.109( A 3 pL aliquot of foe 2 mM aluminum stock solution was placed in a polypropylene cone vial and mixed with 50 pL F-i 8 (as received), incubated at room temperature for -- 2 min and then mixed with 16 to 20 pL of a 2 mM peptide solution in 0.3 JVf NaOAc pH 4 buffer. The solution was heaied in a heating block at 10ΟΌ for 15 min and analyzed by-reverse phase HPLC (PHENOMENEX™, GEMINI®, 5p, C-18, 110A, 250 x 4.6 mm HPLC Column).

[0:1:10] IMP 272: DTP A- Gin-Aia-Ly s( H S G)-D-Tyr-Ly s(H SG )-N H> Mlf 1512 (FIG, 1) [0111 [ IMP 288 DOTA-D-Tyr-D-Lys(.HSG)-D-GiU'D-LyS(HSG)-NH> MPT 1453 (FIG. 2) 101321 IMP 326 DTPA4TC-NH-NH.Phe-C0«D-Tyr-D-Lys(HSG>D*Gln-D.Lys(HSG>· NHh MH' 1477 (FIG. 3) [0113] IMP 329 DeleLOxiiniii\e-NH-C’S-NB-NH-Pli-CO-D-T>T-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NHj ΜΗ"' 1804 (FIG. 4) J61I4] IMP 331 NTA-iAsp-D-Ala-D-l.ys(HSG)-D-Tyr-l>Lys(9SG}-HH> ΜΗ 1240 (FIG. S3 (01.15] IMP 332 EDTA'Dpr-D-Ala-D-'LysiHSG)-D-Ala-D-Lsy(HSG)-NH2 MB’ 1327 (FIG. 6) 10116) IMP 333 DTPA-Dpr(DTPA)-D-Ala'D-l,ys(HSG)-D-Ala-D-Lys(HSG)-NH» MB' 1845 (FIG. 7) [0117] IMP 334 (H205l>)rC(0H)*(CH2)s-NM-Gly*D-Lys(HSG'>.D“Glu-D»Lys(HSG)-NHa ΜΙΓ 1192 (FIG. 8} |0U8] IMP 337 Ac-D-Sei(PO^i>)-D-Ser(POsH?>D-A!a-D-Lys(HSG)-D-Ala-D-Lys(HSG> NH> MH“ 1291 [0119] IMP 338 Ac-D-Ser(POiH,)-D-Ala4.)-I.ys(HSÜ)4')-A!a'D-Lys(HSG}-NH; ΜΗ 1126 (0120) IMP 345 DTPA-D-Ser(PO-,il-Ij>D-Ala-D-Lys(H8G)-D-Alö-D-Lys(HSG)-NHj MB' 1459 (0121 j IMP 349 'DTPA-D“Cys((H20jP)rCH-CH2-S)-D-Ala-D-Lys(HSG}"D-Ala-D-Lys(HSG)-NHi Mlf 1583 (FIG. 9) (0122] IMP 361 DTPA-Dpv(BrCH?.CO-)-D-Ala*D-Lys{HSG)-D-Ala>D-Lys(HSG)-NH3 MH* 1498 (0123] IMP 366 DTPA-Dpi(Pb-S-CB>CO-)-D-Ala-.D-Lys(HSG)-D-Ala-D-Lys(HSG)-N Ü: MB'" 1528 (0124] IMP 368 SynvDTPA-D-Ala-D-LysiHSü}-D-Aia-D-Lys(HSCS)-NHv MB' 1292 (FIG. 10) [0I25| IMP 369 Sym-DTPA-NH-CH(2-Br*Phe-)-CH2-CO- D-Ala-D-Lys{HSG)-D»AlvD-Lys(ttSG)-Ktfc MH' 1517 [0126] IMP 370 Sym'DTPA-NH-CH(2-02N-Phe-)-CHrC0- D-Ala-D4,ys(HSG)-D-Al,vD~ Lys(HS0)*NH2 MH* 1484 [0127] IMP 371 DTPA-NH-CH{2-0;;N-Phe-)-CHrC0- D-Ala-D-Lys(HSG)-D-Ala-D-LysiIiSG)-NHi MH* 1484 [0128] IMP 372 DTPA>Dpr(Ser)-.l>Al8-D-Lys(HSG)-D-Ala-D»Lys(HSG>NIi2 MH“ 1465 [0129] lMP373D'rPA-Dpr(Sym-DiPA)4>Ala-D-Lvs{HSG)-D-Aia-D-Lys(HSG)-NH, MH" 1753 [0130] IMP 374 DTPA-Dpr(Cl-CH2CO-Cys(Et)-)-D-Ala-D-Lys(HSG)'D-Ala-D-Lys(HSG)-NHi ΜΙΓ 1585 [01.31] lMP375 DTPA'Dpr(2-Br-Phe-Cl:fNHi-CHrCO>D*Ala-I>J..>'s(HSG)-D-Ak-D>

Lys(HSG)-NHi MH* 1603 (RIG, 11) [0132] IMP 376DTPA-Cys(HOjS-S)-D-Tyr'D-Ala-D-Lys{HSG)-D-Ala-D'Lys(HSG)-NH;i MH* 1558 [0133] IMP 379 DTPA-Dpi(2'B?N-Phe'CO-)-D-Alii-D-Lys(.HSG>0'Ala-D4,ysCHSG)-N H> MH* 1497 [0134] IMP 382 DTPA-Dpr(li)-D-ALvD-Lys(HSG)-D-AU>D-Lys(HSG)-NB„> MH* 1378 [0135] IMP 383 DTPA-Dpr(Gla-)-r>-Ak-D-Lys(HSG)-D-Ala-D-Lys(HSG)-NH:; MH* 1507 [0136] IMP 384 DTPA-Dpr(2-HO-Phe-C.HNHrCHrCO~)~D-Aia^D'Lys(HSG)-D-Ala-D-Lys(HSG)~NH> Mil* 1541 (FIG. .1.2) [0137] IMP 385 DTPA47pr(l>prH>AtaM7-Lys(BSG)-DMGa-l>-Lys{HSG}-NH’ ΜΗ' 14A4 [0138] IMP 386 DTPA-Dpr{2-pyridyl-CHrCHNHrCO-)-D-Ala-0-I,ys(HSG)-D-AIft-D-Lys(HSG)-NH;> MH':' 1526 (FIG. 13) [0139] IMP 387 DTPA-Dpr(D-9-aiiihry1alaniiie)-D-Ala-.D-Lys(HSG)-D'Alii-D-Lys(HSG)' MH? MH* 16.25 [0140] IMP 389 DTPA-Dpr(2-carbOxy piperizinyi)-D-A!a-D-Lys{HSG>D-Ala-D-Lys(HSG)-NH.? MH " 1490 (FIG. 14) [0.141] IMP 460 NODA-GA-D-Ala>D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2 MH+ 1366 [0142( Further example« of peptides of possible use are shown in FIG. 15 and 16. FIG. 16 shows the structures of IMP 422, IMP 426 and IMP 428. As discussed below. IMP 449 (FIG. 15) shows particular stability of the F- i 8 conjugated peptide under in vivo conditions, of use for labeling and imaging techniques. (0143] FIG. 17 shows an alternative configuration for a ΝΟΤΑ type ligand. The ΝΟΤΑ moiety could be made from D or L para-nitrophenylalamne and tire iminodiacetic acid portion would come from dianriuopropionic acid, which could be I) or L. Furthermore, the position of the ethylene bridge could be switched with the diaminopropionic acid to give a different configuration of groups on the ligand. All of these modifications could affect bind ing kinet ics and stability of the complex, which is subsequently formed. FIG. 18 illustra tes lire structure of a NODArOa peptide that could be labeled with, For example, Ga-68 orF-18. (0144] In certain embodiments, alternative chelating moieties may be used to bind to lSF-tneial or tsF-boron complexes. FIG. 22A-D illustrates some exemplary potential chelating moieties based on the structure of ΝΕΤΑ. As discussed above, Chong et al. (2007) report that ΝΕΤΑ ligands may show improved serum stability when complexed with various metals. Chelator design may also be optimized to increase the binding affinity of the peptide for ,SF-metal.

Results ofPeptide Labeling Screening Study (0145] Most of the DTPA derivatives showed labeling comparable to the labeling of IMP 272. There wore exceptions, IMP 349. bearing die bisphospliotiate group on a cysteine side chain, labeled very poorly. The DOT A ligand did not bind the A1-ISF. The ITC DTPA ligand of IMP 326 did not bind the Al·18? as well as DTPA. The NT A ligand of IMP 331 did not bind tire AI-!iiP. The EDTA ligand of IMP 332 bound the Al-'*F but not as well as the DTPA. Symmetrical DTPA ligand did not bind the Al-!SF. The phosphonaies and phosphate groups tested did not bind Al*iSF well under the conditions tested. The screen did show that a group that was attached near the DTPA could influence the stability of the A1-UF-DTPA complex. The screen showed that IMP 375 labeled better and formed a complex that was significantly more stable than IMP 272. IMP 37.5 labeled well and was stable in water, showing 95.4% remaining bound after 5 hours at. 25°C (not shown). For in vivo use a peptide with high serum stability would be preferred.

[0146 ! The peptide labeling screening study only looked at the binding of AI»1*?. Some of the peptides that did not. label well with A!-**F might label better with another metal binding to the F-18.

Peptide Synthesis J0147] The peptides were synthesized by solid phase peptide synthesis using the Fmoc strategy. Groups were added to the side chains of diamino amino acids by using Fmoe/Aloc protecting groups to allow differentia! deprotection. The Aloe groups were removed by the method of Dangles et. al. (./. Org, Cham. 1987,52:4984-4993) except that piperidine was added in a 1:1 ratio to the acetic acid used. The unsymmeirical teira-l-butyl DTPA was mode as described in McBride et at. (US Patent Application Pub. No,US 2005/0002945 A.1, Appl. No. 10/776,470, Pub, Date, Jan. 6,2005). The tri-t-butyl DOT A, symmetrica! letra-t-butyl DTPA and ITC-benzyl DTPA were obtained from MACROCYCLICS®, The Aloc/Fmoc Lysine and Dap (diaminopropionic acid derivatives (also Dpt)) were obtained from CRBOSALUS® or BACHEMS. The Sieber Amide resin was obtained from NOYABIOCHEM®. The remaining Fmoc amino acids were obtained from CREOSALUS#, BACHEMOb, PEPTECHdb or NOVAB1QCHEM®.

[0148! IMP 272 was synthesized as described (McBride et al.US Patent Application Publ. No. 2004024.1158 Al, Appl. No. 10/768,707, Dec. 2,2004). IMP 288 was made as described (McBride et el.,/. NucL Med. 2006,47:1678-1688). |0149] IMP 326 The hydrazine peptide (IMP 319) was made- on Sieber amide resin using Fmoc-D-Lys(Aloc)~OH, Fmoc-D-Glu(OBut)-Oe, Fmoc-D-Lys(Aloc)-OH, Fmoc-D-Tvr(But)-OH and 4-(Boc-NH-NH-XyFlt-OOiH in that order. The 4-iBoe-NH-NH-)C«H4-COvH was made by adding Boc dicarbonate to 4-hydrazinobenzoic acid in a dioxane sodium hydroxide solution.

[6150) After the addition of the Boc-bydrazide the side chain Aloe groups were removed and the Trityl-HSG-OH groups were added to the side chains of the lysines. The peptide was then cleaved from the resin with TFA and purified by HPLC to obtain the desired hydrazine bis-HSG peptide IMP 319 (MU’ 120.1), The hydrazide peplide (0.0914 g) was (hen mixed with 0.0650 g of lTC-Benzyl DTPA in 3 mL of 0.1 M sodium phosphate pH 8.2. The pH of the solution was adjusted with 1 M NaOH to keep the pH at pH 8.2, After the reaction between the peptide and die ITC-Benzyl DTPA was complete the peptide con jugate was purified by HPLC. |015iI IMP 329 The deferoxamine isothiocyanaie was prepared by mixing i .0422 g of deferoxamine mesylate (1.59 x 10“' mol} with 0.2835 g (1.59 x 10'? mol) of thioearbonyldiinrida/ole in 10 mL of 1: l meihanol/water, Triethylttmine, 0.23 ml. was added and the reaction was purified by reverse phase HPLC after 2.5 hr to obtain the deferoxamine isothiocyanaie MNa* 625. (0152) The hydrazine peptide, IMP 319. (0.0533 g,4.4 x 10"s mol, Mtf1 1201) was mixed with 0.0291 g of deferoxamine isothiocyanate in a sodium phosphate buffer at pH 8.1 for two hours then purified by HPLC to a fiord the desired product MH-r f 804. (0153) IMP 331 The following amino acids were attached to Sieber amide Tesin (0.58 mmol/g) in the order shown; Fmoc-D4;ys(AU>c)-OH, Fmoc-D-Tyr(Biii)OH and Fmoc-D-Lys( Aloc)-OH. The Aloe groups were removed and Trt-HSG-OH was added to the side chains of the lysines. The Fmoc was removed, then Fmoc-D-Ala-OH and Fmoc-Asp-0 But were added in that order (0.5 g of resin). The Fnioc was removed and the nitrogen of the Asp was alkylated overnight with 3 mL i-bulyl bromoaeetate and 3.6 mL diisopropylethySamine in 3,4 mL of NMP, The peptide was cleaved from the resin with IF A and purified by reverse phase HPLC to obtain the desired peptide ΜΙΓ 1240.

[0 J 54 j IMP 332 The peptide was made on 3 g of Sieber amide resin (0,58 nnnot/g). The following amino adds were added to the resin in the order shown: Fmoc-D-Lys(Aloc)-OH, Fmoc-D-Tyr(But)-OH, Fmoc-D-Lys(Aloc)-OH, Fmoc-D-Ala-OH, and Fmoc-Dpr(Fmoc)-OH. The resin was split into portions for subsequent syntheses. One gram of the resin was removed and the Fmoc groups were removed from the diaminopropionic acid. The peptide was alkylated overnight with 3 ml, t- butyl bromoaeetate, .3.6 mL diisopropylethyl amine and 3,4 ml NMP. The side chain Aloe groups were then removed and die Tri-HSG-OH groups were added. The peptide was then cleaved from die resin and purified by HPLC to obtain die product ΜΗ* 1327. 10155 j IMP 333 The peptide was made with 1 g of the same resin that was used to make IMP 332. The DTPA tetra-t-butyl ester (U.S. Publ. No, 20050002945) was added to both of the amines of the Dpr group. The Aloe groups were then removed and theTrt-HSG-OH was added. The peptide was then cleaved and purified by HPLC to obtain the desired product MH* 1845. (0156} IMP 334 The peptide was made on 1 g Rink amide resin (0.7 mmol/g) with the following amino acids added in the order shown: Fm.oc-D~Lys{Aloc)"OH, Fmoc-D~Glu(Bm}- OH, Fmoc-D-Lys(Aloc)-OH, Boc-Ser(But)*OH, The Aloe groups were removed and the Triiyl-HSG-OH was added. The peptide was cleaved from the resin with TFA, The crude peptide was collected by precipitation from ether and dried. Sodium periodate, 0.33 g, was dissolved in 15 mL water. The crude peptide was dissolved in l mL 0,5 M sodium phosphate pH 7,6, 3 mL water and I mL of the periodate solution. 3 mL more periodate in one milliliter increments was added over ~ 2 hr. The mixture was dien purified by reverse phase HPLC and lyophilized to obtain the aldehyde IMP 289 HCQ-CO-D-Lys(HSG)-D'Ght-D-Lys(HSG)-NHj Mil ’ 959. Alendronate (0.0295 g, CALBIOCHEM®} was dissolved in 150 pL 0.1 M NaOAc pH 4. The peptide, IMP 289, (0.0500 g) was dissolved in 100 pL of 13 % isopropauol in water. Sodium cyaiioborohydride was added and the mixture was purified by HPLC to afford the desired product MH' 1192.

[0157) IMP 337 &amp; IMP 338 The peptide was made on Sieber amide resin using the following amino acids added in the order shown: Fmoc-D-Lys(Aloc)-OH, Fmoc-D-Ala-OH, Fmoc-D- LystAl oc)-OH. Fmoc-D-Ala-OH, Fmoc-D-Ser(PO(OBzl)OH)-OH, Fmoc-D-Ser(PO(OBH)OH>OH, and Ac>0. 'Hie Aloe groups were removed and the Trt-HSG-OH groups were added to the side chains of the lysines. The peptide was cleaved from the resin and purified by HPLC to afford the desired products: IMP 337 MH 1291 and IMP 338 MH* 1126.

[0158] IMP 345 The peptide was made on Sieber amide Resin using the following amino acids added in the order shown: Fmoc*D- Ly s( A!oc)-OH, Fmoc-D-Ala-OH. Fmoe-D-Lys(Aloe)-OH. Fmoc-D-Ala-OH, Fmoc-D-SetCPOfOB/lJOHj-OH, and telra-t-butyi DTPA. The Aloe groups were removed and the Τπ-HSG-QH groups were added to the side chains of the lysines. The peptide was cleaved from the resin and purified by HPLC to afford the desired product: IMP 345 ΜΗΊ459. (0159.1 IMP 349 The peptide IMP 347 DTPA-D-Cys-D-Ala-D-Lys(iLSG}-D-Tyr-D-Lys(HSG)-NPh was made on Sieber amide Resin using the following amino acids added in the order shown: Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloe was cleaved, Fmoc-D-Ala-OH, Aloc-D-Lys(Fmoc)-OM, Trt-HSG-OH were added, the Aloe was cleaved Fmoc-D-Ala-OH, Fmoc-D-Cys(Trt)-OH and teua-t-buiyS DTPA were added. The peptide was cleaved from the resin and purified by HPLC to afford the desired product: IMP 347 MH 1395. I he peptide. IMP 347,0.0446 g (3.2 x 10'5 mol) was mixed with 0.4605 g (2,4 x 1 O'3 mol) of efhenylidenebis(phosphonic acid) (Degenhard! et al„ J. Gig. Chew. 1986, 51:3488-3490) in 3 mL of water and the solution was adjusted to pH 6.5 with I M Mu OH added dropwise. The reaction was stirred overnight and the reaction solution was adjusted to pH 1.49 by the addition of excess ethenyüdenebis(phosphonicacid). The mixture was stirred overnight at room temperature and then purified by RPLC to obtain the desired peptide IMP 349 M.H' 1583.

[0160] IMP 36 ! The peptide was made on Siebet: amide resin using the following amino acids added in the order shown: Aloc-D-Lys{Fmoc)~QH, Tri-HSG-OH, the Aloe was cleaved, Fmoc-D-Aia-OH, Atoc-D-Lys(Fmoc>OH, Trt-HSG*OH were added, the Aloe was cleaved, Fmoc-D-Ala-OH, Fmoc-Dap(AIoc)-OH and lelra-l-butyl ΠΊΤΑ were added. The Aloe on the side chain of Cite Dap was removed and bromo acetyl was added with bromo acetic anhydride. The crude product was purified by HPLC to obtain the desired peptide IMP 361 (MfT 1498). )0161] IMP 366 The peptide was made by the same method as IMP 361 with phenvlthioacetic acid added Iasi. Tire crude product was purified by HPLC to afford the product IMP 366 MH’ 1528. 10162} IMP 368 The peptide was as described for IMP 349 except the cysteine residue was not added and symmetrical fetra-t-buty!DTPA (MACROCYCLICS®) was used in place of the unsymmctrical DTPA to obtain the desired product after purification. IMP 368 MH' 1292.

[0163] IMP 369 The peptide was made as described tor IMP 349 with Fmoc-R-3-amino-3-(2-biOmophenyl)propionie acid added in place of the D-Cvs and symmetrical tetra-l-bvstylDTP A added in place of the nnsymmetrical version to the DTPA tetm-t-butyl ester. The crude peptide was purified to obtain the desired product, MH* 1517.

[0164) IMP 370 The peptide was made as described for IMP 369 except Fmoc-R-3-ami no-3-(2-nitrophenyl) propionic acid was used instead of the bromo. The desired prod uct was obtained after purification by H.PLC MB' 1484, 101651 IMP 371 The peptide was made as described for IMP 370 except the «asymmetrical teira-t-butyl DTPA was used in place of the of the symmetrical version. The desired product was obtained aller purification by HPLC MH‘ 1484, [0166] IMP 372 The peptide was made as described for IMP 361 with Fmoc-Serf But)-Oil used to attach the Ser to the Dap side chain. The Fmoc was removed and the peptide was cleaved from the resin and purified to obtain the desired product MH” 1465. {0167} IMP 373 The peptide was made as described lot IMP 361 with syinmetricaMetra-t-buty! ester DTP A used to attach the Sym-DTPA to the Dap side chain. The peptide was cleaved from the resin and purified to obtain the desired product MH1 1753.

[0168] IMP 374 The peptide was made as described for IMP 36! with Fmoc-S-ethyl cysteine added to the Dap side chain followed by chloro acetyl (on the cysteine nitrogen) added via chloroacedc anhydride. The peptide was cleaved from the resin and purified to obtain the desired product M.H* 1585. (0169} IMP 375 'Hie peptide was made as described for IMP 36! with Pmoc-R-3-ammo-3-(3-bromopheny Dpropiontc acid added to the Dap side chain followed by cleavage of the Fmoc group. The peptide was cleaved from, the resin and purified to obtain the desired product MH* 1603.

[0170] IMP 376 The peptide was made as described for IMP 361 with Fmoc-D-Tyr(Btit)~OH added after the second alanine followed by Fmoc-Cys(SOsH) and tetra-t-butyiDTPA. The pep tide was clea ved from the resin and purified to obtain the desired product MH* ! 558.

[01711 IMP 379 The peptide was made as described for IMP 361 with Boc-2-Abz-OH added to the side chain of the Dap. The peptide was cleaved from the resin and purified to obtain the desired product MIT 1497.

[0172[ IMP 382 Hie peptide was made as described for IMP 361 with the Aloe removed from the side chain of the Dap. The peptide was cleaved from the resin and purified to obtain the desired product MH" 1378.

[0173] IMP 383 The peptide was made as described for IMP 361 with Fmoc-Gla(OBut)rOH added to the side chain of the Dap. The peptide was cleaved from the resin and purified to obtain the desired product MH'-COj 1507 (0174} IMP 384 'Hie peptide was made as described for IMP 361 with Fmoc-Boc-S-3-amino-3-(2-hydioxypheny1)propiontc acid added to the side chain of the Dap. The peptide was cleaved from the resin and purified to obtain the desired product MH" 1541. 10175] IMP 385 The peptide was made as described for IMP 361 with. Fnroc~Dpr(.Pmoc)*OH added to the side chain of the Dap, The peptide was cleaved from the resin and purified to obtain tire desired product ΜΗ* 1464.

[0176} IMP 386 The peptide was made as described for IMP 361 with Boc-D-2-pyridyliilanine-OH added to the side chain of the Dap. The peptide was cleaved from the resin and puri fied to obtain the desired product ΜΗ' 1526. |0I77} IMP 387 The peptide was made as described for IMP 36! with Fmoc-D-9-anthryialanine-OH added to the side chain of the Dap. The peptide was cleaved from the resin and purified to obtain the desired product ΜΗΓ 1625.

[0178] IMP 389 The peptide was made as described for IMP 361 with bis-Boc-piperaxme~2-carboxyiate added to the side chain of the Dap. The peptide was cleaved, from the resin and purified to obtain the desired product MIL 1664.

Example 5, Alternative Methods for Preparing and Separating F-18 Labeled Peptides [0379j In certain embodiments, heating is used to get the Al-F-18 complex into the ΝΟΤΑ chelating group. Alternatively, ITC benzyl ΝΟΤΑ. (Macrocyclics) could be labeled with Al-F-.18 and then conjugated to other heat sensitive molecules, such as proteins, after labeling. If high specific activity is needed the ITC Benzyl ΝΟΤΑ complex can be purified away from the cold ligand.

[0180] Λ1 was added to the peptide and its HPLC profile compared to the empty ΝΟΤΑ peptide and the AI-F-18 peptide. The Λ1 peptide and the AI-F-fiS peptides have virtually the same retention time by HPLC, with - i min longer RT for the unlabeled peptide. The peptide was purified on a PHENOMENEX™ ONYX® monolithic CM8 .100 x 4.5 mm column using a 3 mL/min flow rate, Buffer A was 0.1 % TEA in water and Buffer B was 90 % Cf-LCN 10 % water and 0.1 % TFA. The linear gradient went from 100 % buffer A to 75:25 A/B over 15 rai n, Since the AI complex co-elutes with the Al-F-18 complex, the amount of A! and P-18 added will determine the specific activity.

[0181} IMP 449 was prepared according to Example 7 below and labeled as follows. The ΤΙ 8 was received in a 2.0ra.L Fisher Microcentrifuge vial (02-681.-374) containing 15 mCi of F-.1H in -325 μΐ, in water. 3 μΕ of 2 mM AlCh in 0.1 M pH 4 NaOAc was added to the F-18 solution and then vortex mixed. After about 4 min, 10 pL of 0.05 M IMP 449 in pI14 0.5 M NaOAc was added. The sample was vortex mixed again and heated in a 102VC healing block for 17 min. The reaction was then cooled briefly and then the vial contents were removed and purified by HPLC as described above.

[Ό182] Separately, elution conditions were determined on the WATERS® ALLIANCE™ analytical system and the labeled peptide was eluted between 7.5 and 8.5 min. The analytical HPLC showed that the 'labeled peptide contained the Al-F IMP 449 CUV 220 nm) and did not contain the nncomplexed peptide, resulting in an increased specific activity. (0183] The peptide was diluted in water and then pushed through a WATERS® OASIS PLUS HLB™ extraction column. The labeled peptide was eluted with 3 mL of 1; I EtOH/HjO. HPLC analysis of the eluents confirmed that the column efficiently trapped the labeled peptide, which allowed the acetonitrile and TFA to be washed away from the peptide. The HPLC also showed that 1:1 EtOR/IhO eluent contained the desired product free ofioose F-1.8 in a solvent suitable lor injection alter dilution. The apparent yield after purification, was 11 %.

Example 6. In~Vivo Studies 10184] Nude mice bearing GW-39 human colonic xenograft tumors (100-500 mg) are injected with the bispecific antibody hMN-14 x in679 (1.5 x l(fu1 mol). The antibody is allowed to clear for 24 hr before the F-18 labeled HSG-bearing peptide (8.8 pCi, i .5 x 1 O'" mol) is injected. The animals are imaged at 3,24 and 48 hr post injection. The xenograft tumors are dearly imaged by PET scanning detection of the F-18 labeled peptide bound to the bispeci.fic hMN-14 x ra679 that is localized to the tumors by binding of hM.N-14 to tumor antigen.

Example 7. Production and Use of a Seram-Stable F-18 Labeled Peptide (0185] IMP 449 NÖTÄ-1TC bemyl-D-Ala-D-Lys(.] tSG)-D-Tyr-D-Lys(!ISG)-Nl i2 MU’ 1459 (FIG, 1.5) (0186) The peptide, IMP 448 D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH:/ MH' 1009 was made on Sieber Amide resin by adding the following amino acids to the resin in the order shown: Aloe-D-Lys(Fmoc)-OH, Tri-HSG-OH, the Aloe was cleaved, Fmoc-D-Tyf(But)-OK, Aloc-D-Lys(Fmoc)-OH, Trt-HSG-OH, the Aloe was cleaved, Pmoe-D-Ala-OM with final Fmoc cleavage to make the desired peptide. The peptide was then cleaved from the resin and purified by HPLC to produce IMP 448, which was then coupled to ITC-benzyl ΝΟΤΑ. The peptide, IMP 448,0.0757g (7.5 x I0J mol) was mixed with 0,0509 g (9.09 x 1 Ö'J mol) ITC' benzyl ΝΟΤΑ and dissolved in I 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 (he 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 i 4 hr and purified by HPLC to obtain 4ft mg of IMP 449, the desired product (FIG. 15), F-l 8 Labeling of IMP 449 [0187] The peptide IMP 449 ¢0.002 g, 1.37 x I O'6 moi) was dissolved in 686 pL (2 mM peptide solution) 0. .1 M NaOAc pH 4.02. Three microliters of a 2 mM solution of A1 in a pH 4 acetate buffer was mixed with 15 pL, 1,3 raCi of F-l 8. The solution was then mixed with 20 pL of the 2 mM IMP 449 solution and heated at !05"C for 15 min. Reverse Phase HPLC analysis showed 35 % (RT - 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, trot shown) indicating that the majority of activity was not associated with the peptide. The crude labeled mixture (5 pL) was mixed with pooled human serum and incubated at 37"C, An aliquot was removed aller 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 Use peptide (down from 35 %.), which was essentially the same as the .15 min trace (data not shown).

Hieb Dose P-4 8 Labeling [0188] Further studies with purified IMP 449 demonstrated that the F-18 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. These results demonstrate that the F-18 labeled peptides disclosed herein exhibit sufficient stability under approximated in vivo conditions to be used for F-.18 imaging studies.

[0189] F-l 8-21 mCi in - 400 pi. of water was mixed with 9 pL of 2 mM AlCfi in 0.1 M pH 4 NaOAc. The peptide, IMP 449,60 pL ( 0,01 M 6 x IQ"7 mol in 0.5 NaOH pH 4.13) was added and foe solution was heated to l !0”C for 15 min. Hie crude labeled peptide was then purified by placing the reaction solution in the barrel of a 1 cc WATERS'®? HLB column and eluting with water to remove unbound F- l 8 followed by .1:1 F.IOH/H20 to elute foe F-18 labeled peptide. The crude reaction solution was pulled through the column into a waste vial and foe column was washed with three one milliliter fractions of water (18.97 mCi). The HLB column was then placed on a new vial and eluted with iwo x 200 μΐ, 1:1 EtOH/H’O to collect foe labeled peptide (1,83 mCi). The column retained 0.1 mCi of activity after all of foe elutions were complete. An aliquot of the purified F-18 labeled peptide (20 pL) was mixed with 200 |.iL of pooled human serum mid heated at 37UC. Aliquots were analyzed by reverse phase HPLC (as described above). The results showed the relative stability of F- i 8 labeled purified IMP 449 at 37eC at time zero, one hour (91% labeled peptide), two hours {77% labeled peptide) and four hours (76% labeled peptide) of incubation inhuman serum (not shown). It was also observed that F-18 labeled IMP 449 was stable in TFA solution, which is occasionally used during reverse phase HPLC chromatography. There appears to be a general correlation between stability in TFA and stability in human serum observed for the exemplary F-18 labeled molecules described herein. These results demonstrate dial F-18 labeled peptide, produced according to the methods disclosed herein, shows sufficient stability in human serum to be successfully used for in vivo labeling and imaging studies, for example using PET scanning to detect labeled celts or tissues.

Example 8, In Vivo Biodistributiou of F-18 Labeled IMP 449 in SCI® Mice 10190] F-18 labeled IMP 449 was prepared as described above (Example 7). The material was purified on an OASIS® HLB column (WATERS®, Milford, MA). The unbound material was washed out. with water and the labeled peptide (hat was bound to the column was eluted with 1:1 ethanoliwater mixture, Both fractions were analyzed by reverse phase Cl 8 HPLC. The purified peptide eluted as several peaks on the reverse HPLC column (not shown). The unbound fraction collected from the OASIS® column showed poor recovery, 7%, from the C18 column (not shown), |01911 The “unbound” fraction and the purified. ’V-IMP 449 were injected into SOD mice that were previously injected with sc SU-DHL6 lymphoma cells. Only a few of the mice had visible tumors. Biodistributiou data showed a significant difference between the “unbound” F-.I8 fraction and the purified i!>P-iMP 449. Data are shown in fables 4-6 below. Note that in this study, no pretargeting bispecific antibodies were administered to the animals before the labeled peptide. These results demonstrate the distribution of labeled peptide vs. free F-18 In vivo. |(il 92) Unconjugated F-18 shows a high level of distribution to bone tissue in vivo. Uptake 20 minutes after injection was, as expected, seen primarily in the bone (spine), with about 12-15% injected dose per gram (ID/g), followed by the kidneys with about 4% ID/g.

Localization of the F-18 label to bone tissue was .substantially decreased by conjugation to a targeting peptide. When bound to IMP 449, uptake in the bone is reduced to -! % ID/g at 20 min and 0.3 % at. 1 h after injection, with ratal uptake of 11% at 20 min and 3,3% ID/g at I hr. Renal uptake of the peptide alone was similar to (hat of the pretargeted ,i(F-lMP 449 peptide (see following Example), suggesting its uptake was a function of the peptide rather than a consequence of the animals having been give the bsMAb LS h earlier. Relatively low non-specific uptake was observed in the spine and femur with the F-18 labeled peptide compared with unbound F-18.

Table 4. P-18 “unbound” fraction at 20 min post injection: %ID/g mean and the individual animals.

Animal Animal Animal

Tissue n Mean SD 1 2 3

Tumor 1 · - 0.902 - -

Liver 3 2.056 0.244 1.895 2.338 1.937

Spleen 3 1.869 0.434 1.677 2.366 1.564

Kidney 3 4.326 0.5^6 3.931 4.936 4.111 lung 3 2.021 0.149 1.903 2.168 1.972

Blood 3 2.421 0.248 2.355 2.696 2.212 stomach 3 0,777 0.409 0.421 1.224 0.687

Small int. 3 2.185 0.142 2.042 2.325 2.187

Large Int. 3 1.40 3 0.069 1.482 1.356 1.372

Femur 3 11.668 1.619 11.502 13.292 10.270

Spine 3 14.343 2.757 17.506 13.072 12.452

Muscie 3 1.375 0.160 1.191 1.457 1.478

Table 5, κΊ"-ίΜΡ 449 purified, 80 pCi, lx 104 mol at 20 min post injection: %ID/g mean and the individual animals

Animal Animal Animat Animal Anima!

Tissue n Mean SD 1 2 3 4 5

Tumor 1 - - 0.891 ----Liver 5 2.050 0.312 1.672 1.801 2.211 2.129 2.440

Spleen 5 1.297 0.259 0.948 1.348 1.144 1.621 1.425

Kidney 5 12.120 4.128 8.354 7.518 12.492 15.535 16.702

Lung 5 2.580 0.518 2.034 2.103 2.804 2.678 3.278

Blood 5 3.230 0.638 2.608 2.624 3.516 3.512 3,992

Stomach 5 1.017 0,907 0.805 0.775 0.344 0.557 2,605

Small Inf. 5 1.212 0.636 0.896 0.921 0.927 0.967 2.349

Large Int. 5 0.709 0.220 0.526 0.568 0.599 0.793 1.057

Femur 5 0.804 0.389 0.314 0.560 1.280 0.776 1.087

Spine 5 3.915 6.384 0.819 0.923 1.325 1.177 15.330111

Muscle 5 0.668 0.226 0.457 0 439 0.960 0.673 0.814 *High spine uptake in Animal #5 was confirmed by recounting.

Table 6. ISF-IMP 449 purified, 80 μΟΪ, lx 10’s mol at 1 h post injection: %10/g mean and the individual animals

Animal Animal Animal Animal Tissue n Mean SO 1 2 3 4

Tumor 1 0.032 0.064 0.000 0 127 0.000 0.000

Liver 4 0.683 0.308 1.103 0.632 0.604 1.191

Spleen 4 1.061 0.702 1.598 0.631 0.301 1.713

Kidney 4 3.256 0.591 3.606 2.392 3.362 3.666

Lung 4 0.324 0.094 0,411 0.232 0.256 0.399

Blood 4 0.265 0.104 0.378 0.153 0.250 0.358

Stomach 4 0.152 0.082 0.225 0.041 0.199 0.142

Small Int. 4 1.290 0.228 1.124 1.247 1.166 1.624

Large int. 4 0.115 0.035 0.167 0.091 0.094 0.109

Femur 4 1.006 0.876 2.266 0.448 0.939 0.374

Spine 4 0.314 0.076 0.423 0.257 0.268 0-306

Muscle 4 0.591 0.946 0.205 0.077 2.008 0.075 (0193( We conclude that the F-18 labeled peptide showed sufficient in vivo stability to successfully perform labeling and imaging studies.

Example 9. Jin Vivo Studies With Pretargeting Antibody (0194] F' hS labeled IMP 449 was prepared as follows. The F-18S 54.7 mCi in *· 0.5 mL was mixed with 3 μΐ, 2 mM AJ in 0.1 M NaOAc pH 4 buffer. After 3 min,10 pL of 0.05 M IMP 449 in 0,5 M pH 4 NaO Ac buffer was added and the reaction was heated in a 96°€ heating block for 15 min. The contents of the reaction were removed with a syringe. The crude labeled peptide was then purified by HPLC on a Phenomenex. Onyx monolithic CIS, 100x4.6 mm column part. No. CHO-7643. The flow rale was 3 mL/min, Buffer A was 0,1 % TFA in water and Buffer B was 90 % acetonitrile in water with 0.1 % TFA. The gradient went from 100 % A to 75/25 A:B over 15 min, There was about 1. min difference in R.T between the labeled peptide, which eluted first and the unlabeled peptide. The HPLC eluent was collected in 0.5 min fractions. The labeled peptide came out between 6 to 9 min depending on the HPLC used. The HPLC purified peptide sample was further processed by diluting the fractions of interest two fold in water and placing the solution in the barrel of a 1 cc Waters HLB column. The cartridge was eluted with 3 x 1 roL water to remove acetonitrile and TFA followed by 400 μΐ 1:1 EtOIiH2(i to elute the F-18 labeled peptide.

[01951 The puriited^F-lMP 440 eluted as a single peak on an analytical H'PLC Cl 8 column. (01 %| Taconic nude mice bearing the four slow-growing sc CaPanl xenografts were used. Three of the mice were injected with TF10 (162 gg.) followed with ivF-lMP 449 18 h later. TP 10 is a humanized bispecific anybody of me for tumor imaging studies, with divalent binding to the PAM-4 defined MUC 1 tumor antigen and monovalent binding to MSG (see, e.g., Gold et al, 2007, J. Gin. Oncol. 25(.183):4564). One mouse was injected with peptide alone. A ll of the mice were necropsied t h post peptide injection. Tissues were counted immediately. Animal #2 showed high counts in the femur. The femur was transferred into a new vial and was recounted along with the old empty vial Recounting indicated that the counts were on the tissue. This femur was broken and had a large piece of muscle attached to it Comparison of mean distributions showed substantially higher levels of F-l 8-labeled peptide localized in the tumor than in any norma] tissues in the presence of tumor-targeting bispecific antibody.

[01971 Tissue uptake was similar in animals given the li>F-1.MP 449 alone or in a pretargeting setting. Uptake in the human pancreatic cancer xenograft, CaPanl, at 1 h was increased 5-fold in the pretargeted animals as compared to the peptide alone (4,6 ± (),91¾ ID/g vs. 0.89% ID/g), Exceptional tumor/nontumor ratios were achieved at this time (e.g., tumov/blood and liver ratios were 23.4 * 2,0 and 23.5 ±2.8, respectively).

Table 7. Tissue uptake at 1 h post peptide injection, mean and the individual animals:

*H:gh counts in Animal # 2 femur were confirmed by recounting after transferring femur into a new vial. Animal #2 showed higher uptake in normal tissues than Animals it 1 and #/3.

Example 10. Comparison of'Biodistribution o f Iuln-IMP 44? w, iSF»TMP 449 With Pretargeting Antibody 10198] The goal of the study was to compare biodistribution of mIn-IMP 449 tmd tsF4MP 449 in nude mice bearing sc LS I 74 T xenografts after pretargeting with bispecific antibody TF2. TF2 antibody was made by the dock-and-lock method and contains binding sites for the CEA tumor antigen and the HSG hapten (see, e.g., Sharkey etal., Radiology 2008,246:497-507; Rossi et a!., PNAS USA 2006,103:6841-46). Since there were insufficient numbers of mice with tumors at one time, the study was performed on 2 different weeks. (0199] ntIn-iM.P 449: m!n labeling was performed using a procedure similar to the one used for labeling IMP 288, except at lower specific activity. 1.TLC and C-1S RP HPLC showed --30 % unbound (not shown). The labeled peptide was purified on an HUB column (1 inL, 30 mg). The analyses of the purified product again showed 33 % unbound (top 20% of strip) by ITLC developed in saturated sodium chloride·. RP HPLC showed multiple peaks before and after purification (not shown). SE HPLC after purification showed 47% of the activity shift to HMW when mixed with 20x molar excess of TF2 (not shown). |0200] ISK- IMP 449: Labeling was performed as described above except the F-18 was purified on a QMA cartridge before labeling as described by others (Kim et. ol. Applied Radiation and Isotopes 6.1,2004,1241-46). Briefly, the Sep-Pak® Light Waters Accel!)M Plus QMA Cartridge was prepared flushed with it) ml, 0.4 M KHCOj and then washed with 10 mL D1 water. The tsF (42 mCi) in 2 mL water was loaded onto the QMA cartridge. The cartridge was eluted with 10 mL DI water to remove impurities. Tire column was then eluted with i mL 0,4 M K.HCO-, in 200 pL fractions. Fraction number two contained the bulk of the activity, 33 mCi. The pH of the F-18 solution was then adjusted with 10 pL of glacial acetic acid. The ,tfF from fraction if-2 was then mixed with 3 μΐ of 2 mM ΛΙ in 0.1 M pH 4 NaOAc buffer. The sample was then mixed with 10 μϊ.. of 0.05 M IMP 449 in 0.5 M NaOAc buffer at pi 14 and the reaction solution was heated at 94°C for 15 min. The 1SF-1MP 449 was purified by RP HPLC. The fraction containing the product was put through an HLB column to exchange the buffer. The column was washed with water after loading the sample. The product was eluted with 1:1 wateretharsol in a 400 μΐ, volume. RP HPLC of the product showed one major peak with a shoulder (not. shown). Since the yield was low, the specific activity was low and. more peptide was injected into mice, resulting in a bsMAhtpeptide ratio of 6.9'. i instead of l 0:1. tefllm [0201] The labeling of IMP 449 with ln-111 resulted in multiple products. Possibly some might be binuclear complexes. The 1 ’1 In-1MP 449 showed high kidney uptake and high blood concentration. However, even as multiple species,1 "itt-IMT 449 showed localization to the tumor when pretargeted with TF2 (FIG. 19).

[02(12 j FIG. 1.9 shows the comparative biodisiribution ofin-111 and F-18 labeled IMP 449 in mice. Both labeled peptides showed similarly high levels of localization to tumor tissues in the presence of the bispecific TF2 antibody. The In-111 labeled species showed higher concentration in kidney titan the F-18 labeled species in the presence or absence of TF2 antibody. The data are summarized in Tables 8-11. below.

Table 8. Mice were injected with TF2 (163.2ug, 1.035x1 iv^mof) iv followed with 11 ’in IMP 449 0 ,035x10’’° mol) 16 h later. Peptide tissue uptake (%JD/g)at 1 hpost peptide injection is shown below.

Table 9. Λ group oi'2 mice were injected with nsIu IMP 449 (1.035x10',B mol) without pretargeting antibody. Peptide tissue uptake (%ID/g) at 1 h post peptide injection is shown below.

Table 10. Mice were injected with TF2 (163.2tig, 1.03 5x1mol) iv followed with tsF- IMP 449 (1.5x10''” mol) 16 h later. Peptide tissue uptake t'%ID/g) at 1 h post peptide injection is shown below.

Table 11. Mice were injected with '“F** IMF 449 (1.5xl(T,n mo!) without pretargeting antibody. Peptide tissue uptake (%ID/g) at 1 b post peptide injection is shown below.

(0203) In summary, a simple, reproducible method and compositions are described herein for producing IMS labeled targeting peptides that are suitable for use in in v/vo imaging of a variety of disease states. The skilled, artisan will realize that thebispeciiic antibodies disclosed above are not limiting, but may comprise any known antibodies against a wide variety of disease or pathogen target antigens. Not is the method limited to pretargeting with bispeciitc antibodies. hi oilier embodiments, molecules or complexes that directly bind to target cells, tissues or organisms to be imaged may be labeled with F-.i 8 using the methods disclosed herein and administered to a subject for PE T imaging (see Examples below), 10204| The Ai-F-1.8 labeled peptides, exemplified by IMP 449, are sufficiently stable under in viva conditions to be utilized in known imaging protocols, such as PET scanning. The present yield of radiolabeled peptide prepared as described above varies between 5 and 20%, and even with a briei'HPLC purification step to separate labeled from unlaheled peptide the final yield is about 5%. Further, the claimed methods result in preparation ofF-!8 labeled targeting peptides that are ready for injection within .1 hour of preparation time, well within the decay time of F48 to allow» suitable imaging procedures to be performed. Finally, the described and claimed methods result in minimal exposure of the operator to radioisotope exposure, compared with known methods of preparing F«18 labeled compounds for imaging studies.

Example 1:1. F-18 Labeling Kit.

[9205) An F* 18 labeling kit was made by mixing 8.0 mg of IMP 449 with 0.1549 g of ascorbic acid. The two reagents vvere dissolved in 10.5 mL water and the solution was dispensed in TO mL aliquots into 10 vials. The pH was not adjusted. The solutions were frozen, lyoph il.iz.ed and sea led under vacuum.

Example 12. i maging of Tumors In Vivo Using Labeled Peptides and Pretargeting with Bispecific Antibodies [9206] The present Examples show that in vivo imaging techniques using pretargeting with bispecific antibodies and labeled targeting peptides may be used to successfully detect tumors of relatively small size. The p retargeting antibodies utilized were either TF2, described above, or the TF10 antibody.

Et?mMl.ation..bMife.r,: [0207) The formulation buffer was made by mixing 0.3023 g ascorbic acid, 18.4 mL D1 water and 1.6 mL 1 M NnOH to adjust the pH to pH 6.61, The buffer was dispensed in l m'L aliquots into 20 vials and lyophilized. |0208] The F-18 was purified on a WATERS® ACCELL™ Plus QMA Light cartridge according to the literature procedure, wherein the cartridge was washed with 10 mL 0.4 M KHCO.i followed by a 10 ml. wash with D1 water. T he F-18 in 2 mL of water was pushed through the cartridge and then washed with 10 mL of water. The F-18 was then eluted from the cartridge in 5 x 200 pL aliquots with 0.4 M KHCOj. Most of the activity was eluted in the second fraction, 'Hie activity in the second fraction was mixed with 3 pL 2 mM A1 in a pH 4 acetate buffer. The Al-F-1.8 solution was then injected into the ascorbic acid IMP 449 labeling vial and heated to 105eC for 15 min. The reaction solution was cooled and mixed with 0.8 mL D1 water. The reaction contents were placed on a WATERS® OASIS® lee HX..B Column and eluted into a waste vial. The column was washed with 3 x 1 ml. DI water. The column was transferred to a formulation vial containing ascorbic acid. The column was eluted with 2 x 200 μΕ 1: l EtOH/HjO to elute the labeled peptide.

Production of T.F10 Bisnecific Antibody Using DNL Technology (0209( The cancer-targeting antibody component in TFiO is derived from hPAM4, a humanized anti-MUC 1 MAb that has been studied as a radiolabeled MAb in detail (e.g..

Gold etah. Clin. Cancer Res. 13:7380-7387,2007). The hapten-binding component is derived from h67(), a humanized anii-histammyl-succmyl-glycine (HSG) MAb discussed above. The TF iO bispecific (fhPAM4|2 x li679) antibody was produced using the method disclosed for production of the (anti CEAjj x anti HSG bsAb TF2 (Rossi etal., 2006). The TFT 0 construct bears two humanized PAM4 Fabs and one humanized 679 Fab. (021.0 j For TFIO, a Fab of the humanized hPAM4 antibody was linked using a peptide spacer to an α-sequertce. The a-sequence is unique because it spontaneously associates with another α,-sequenee to form a dimer, ln TF10, the structure contains 2 KPAM4 anti-MUC1 Fabs finked together by the 2 cx-sequences (called hPAM4-DDD), The other component of TFIO is produced by linking a ß-sequence to the Fab’ of the humanized ami-HSG antibody. Unlike the α-sequence, the (3-sequence does not self-associate, but instead binds to the dimeric structure formed by the 2 «-sequences (h679~AD), Thus, when these 2 separately produced proteins are mixed together, they immediately form an ‘ihb’ structure, with each Fab’ oriented in a manner to allow unimpeded binding to its antigen, The stability of this binding interaction has been further improved by strategically positioning cysteine in each of the a-a«d (3-sequences (2 in the (3-sequence and 1 in the α-sequence). Because ‘b’ binds ro ‘a/ in a highly specific orientation, once ajb is assembled, disulfide bridges can form between ihe u-and ß-moieties, thereby covalently attaching these 2 proteins. Both the a- and (3-sequences are found in human proteins, and therefore are not expected to add to the iramunogenicky of the complex.

[02HJ Theanti-MüCi fusion protein hPAM4-a was generated by fusion of the a sequence to the C-terminal end of the Pel chain. The anti-HSG fusion protein h679-ß was formed by linking the p sequence to the C-teaminal end of the Fd chain. The stably tethered, multivalent bsMAb TF10 was formed by pairing the hPAM4-a with the h679-ß. 10212] The two fusion proteins (hPAM4-DDD«nd h679-AD2) were expressed independently in stably transfected myeloma ceils. The tissue culture supernatant fluids were combined, resulting in a two-fold molar excess of hPAM4-ot. The reaction mixture was incubated at room temperature for 24 hours under mild reducing conditions using I mM reduced glutathione. Following reduction, Are DNL reaction was completed by mild oxidation using 2 mM oxidized glutathione. TF.l 0 was isolated by affinity chromatography using IMP 291-afiigel resin, which binds with high specificity to the h679 Fab. (0213] A full tissue histology and blood cell binding panel has already been examined for hPAM4 IgO and for an anti-CEA x anti-HSG bsMAb that is entering clinical trials. hPAM4 binding was restricted to very weak binding to the urinary bladder and stomach in. 1/3 specimens (no binding was seen in vivo), and no binding to normal tissues was attributed to the anti-CEA x anti-HSG bsMAb. Furthermore, in vitro studies against cell fines bearing the HI and H2 histamine receptors showed no antagonistic or agonistic activity with the ΓΜΡ-288 di-HSG peptide, and animal studies in 2 different species showed no pharmacologic activity of the peptide related to the histamine component at doses 20,000 times higher than that used for imaging. Thus, the HSG-histamine derivative does not have pharmacologic activity.

Biodistrihution. Taraetina and Dosage Studies of TF10 Bispecific Antibody (0214] The biodistribution and tumor targeting of TF10 with increasing TF10 doses is determined. These studies provide basic Pk data for l'F 10 over a range of doses. The primary dose range simulates human equivalent doses (HED) between 1.0 to 50 mg given to a 70 kg patient. Based on FDA guidelines for converting a dose given to an animal to a HED j i.e., (mg/kg in a mo«se/l 2.3} = mg/kg HED], a 1 mg (6.37 nmol) TFI0 dose given to a 70 kg human would be equivalent to a 3.5 gg (0.022 nmol) dose in a 20 g mouse. (0215] Briefly, animals are given iv injections of 3.5, 17,5, 35, and 70 TF10 (trace 1 ‘Ί-TF10 added). Animals given .17.5, 35, and 70 pg doses (HED -1,5,10 and 20 rag) are necropsied at .1.6,16,48, and 72 h (n - 5 per observation; total N = 75 animals/cell line). Studies with the current lot oi'TFlO have indicated a very rapid clearance in mice, similar to that of the TP2 anti-CEA construct described above. 102.16] Pk studies are also performed with ,,nT-TFH) in rabbits. Prior studies with TF2 anti-CEA bsMAb have indicated that rabbits might better predict the Pk behavior that is observed in patients, since they clear humanized anti-CEA IgG in an identical manner as that found in patients, white mice dear humanized IgG at a faster rate. These studies would involve 4 rabbits, 2 given a 5-mg HED and 2 given a20-mg HED oi'TFlO spiked with i VtTTFI0 ( -700 uCi). Rabbits are bled at 5 min, 1, 3, 6,24,48,72, %, 120, and 168 h. Whole-body images are also taken using an ADAC Solus gamma camera equipped with a high-energy collimator. An ^-standard (-20 μΟί in a 10 ml. syringe) is placed in. the field of view with each rabbit during each imaging session taken at 3,24,48,120, and 168 h. The standard is then used to provide semi-quantitative data on the distribution, of olJ«TF10.

Imaging Studies Using Pretaraetim» WiihTF2 and TF10 Bisneciltc Antibodies and Labeled

Peptides }0217{ The following studies demonstrate the feasibility of A? viva imaging using the pretargeting technique with bispecific antibodies and labeled, peptides. While the images were not obtained using an lsF-metai labeled peptide as described above, the pretargeting technique with bispecific antibodies may be generally adapted to use with any type of label Thus, tiie studies are representative of results that would be obtained using the claimed F-l 8 labeled peptides.

[0218] FIG. 20 and FIG. 21 show examples of how clearly delineated tumors can be detected in animal models using a bsMAb preturgeiiog method, with an iHIn-labei.ed di-HSG peptide, IMP-288. In FIG. 20, nude mice bearing 0.2 to 0.3 g human pancreatic cancer xenografts were imaged, usingpreUugetitig with TF10 and 11 Ίη-ΙΜΡ-288 peptide. The six animals in the top of the Figure received 2 different doses of TP 10(10:1 and 20:1 mole ratio to the moles of peptide given), and (he next day they were given an mfn-labeied diJHSG peptide (IMP 288). The 3 other animals received only the11 *ΙηΊΜΡ-288 (no pretargeting). The images were taken 3 h after the injection of the labeled peptide and show dear localization of 0.2 - 0.3 g mmol's in the pretargeted animals, with no localization in the animals given the n>In-peptide alone.

[0219j In this study, tumor uptake averaged 20-23% ID/g with nrmor/blood ratios exceeding 2000: l, Uimor/liver ratios of 170:1, and tumor/kidtiey ratios of 18/1. Since tumor uptake shown in (he'Examples above for the Λ I-^P* labeled IMP 449 averaged only about 4-5% ID/g in the same CaPanl xenograft model, it is believed the lower uptake with ’^-labeled peptide merely reflects the lower specific activity of (he A]-lsF*l.abeled IMP 449, Nevertheless, the AJ-tsF-IMP 449 data show an extraordinary potential for tire pretargeted, fhiorinated peptide that when combined with the- specificity ofthe TF1Ö bsMAb would be an excellent tool for imaging pancreatic or oiher cancers. The biodistribution data for lsF-labeled peptides far exceed the targeting ability of directly radiolabeled antibodies and small engineered antibody constructs directly labeled with iSF (Cai et ai.. J. Nticl Med. 48:304-310,2007).

[0220{ The data shown in FIG. 21 further highlights the sensitivity of the pretargeting method for detecting cancer. Here, a panel of microPRT images was obtained from mule mice injected intravenously with a human colon cancer cell line and bearing 0.2-0.3 mm micrometastaiic tumors in the lungs. Animals were administered the anti-CEA bsMAb TF2, followed with a preiargeted ‘^I-Uibeled peptide. The images show intense uptake in both the transverse and coronal sections at 1.5 h that persisted even at 21 h. 'lire coronal section is a more posterior view to illustrate that the i:Mi-pepUde was also seen in (he stomach and kidneys 1.5 h after its injection. The images show what appear to be individual lesions in the lungs (arrows) that when necropsied were no larger than 0.3 nun in diameter (top panel, transverse sections through the chest) (Sharkey et ah, Radiology, 246(2): 497-507,2008). A control animal pretargeted with an anti-CD22 TF6 bsMAb and given the same -labeled peptide (left side, middle panel) is shown to illustrate the specificity of the localization by, in this case, an anti-CEA bsMAb. The coronal section of the anti-CEA-pretargeted animals shows the uptake in the chest, as well as in (he kidneys and some activity in the stomach. Significantly, the same sized lesions in the lungs were not seen in animals given ,SF-FDG, Thus, use of pretargeting antibodies provides greater specificity and sensitivity of detection comparing to the standard F-l 8-labled flnorodeoxyglucose probe currently used for FET imaging of cancer. |022i] These data further demonstrate the feasibility of imaging using pretargeting with bispectfic antibodies and 1ÄF-labe!ed peptides.

Example 13. Synthesis of Folic Acid ΝΟΤΑ conjugate [0222j Folic acid is activated as described (Wang ei, al. Bioconjugate Citem. 1996,7,56-62.) and conjugated to Boo-NH-CHrCHj-Nlfe. The conjugate is purified by chromatography. The Boc group is then removed bv treatment with TFA. The amino folate derivative is then mixed with gj-SCN-Bn-NÖTA (Macrocyclics) in a carbonate buffer. The product is theu purified by BPLC. The foiate-NOTA derivative is labeled with Al-isF as described in Example! 0 and then HPLC purified. The iSP*!abe1ed 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 el a)., Advanced Drug Delivery Reviews, 5(5:1143*60,2004).

Example 1.4 Pretargeted PET Imaging in humans (02231 A patient (1.7 nr' body surface area) with a suspected recurrent tumor is injected with 17 mg of bispecific monoclonal antibody (bsMab). The bsMab is allowed to localize to the target and clear from the blood. The F-18 labeled peptide (5-10 mCi on 5.7 x 1 (f9 mol) is injected when 99 % of the bsMab has cleared from the blood. PET imaging shows the presence of micrometastaiic tumors.

Example IS, Imaging of Angiogenesis Receptors by F-18 Labeling (0224) Labeled Arg-GIy-Asp (RGD) peptides have been used for imaging of angiogenesis, for example in ischemic tissues, where ctvßs integral is involved. (Jeong et al., J. Nucl. Med. 2008, Apr, 15 epub). RGD is conjugated to SCM-Bz-NOTA according to Jeong etai. (2008). AI-ISF is attached to the NOTA-derivaiized RGD peptide as described in Example. 10 above, by mixing aluminum stock solution with F-t 8 and the derivatized RGD peptide and healing nt 11(PC for 15 min, using an excess of peptide to drive the labeling reaction towards completion as disclosed in Example 10. The F-l 8 labeled RGD peptide is used for in vivo biodistribution and P.ET imaging as disclosed in Jeong et al. (2008). The Al-iSF conjugate of RGD-NOTA. is taken up into ischemic tissues and provides PET imaging of angiogenesis.

Example Hi. I maging of Tumors Using F-18 Labeled Bombesin (0225( A NOTA-conjugated bombesin derivative (NOTA-8-Aoc-BBNT(7*14)NH2) is prepared according to Prasanphanich et at. (Proc. Natl. Acad. Sei, USA 2007, 104:12462-467), The NOTA-bombesin derivative is labeled with AI-! 'F according to Example 10 above. The P-18 labeled bombesin derivative is separated from unlabeled bombesin on an OASJSdt' column (Waters, Milford, MA), as described in Example 10. The Al-iKF labeled NOTA-bombesin conjugate is successfully used lor PET imaging of gastrin-releasing peptide receptor expressing tumors, according to Prasanphanich et al. (2()07).

Example 17. Imaging of Tumors Using F-l 8 Labeled Targctnble Conjugates (0226( ΝΟΤΑ derivatives of peptides, polypeptides, proteins, carbohydrates, cytokines, hormones or cell receptor-binding agents are prepared according io U.S. Patent No. 7,0Π ,816 (incorporated herein by reference in its entirety). The NOTA-derivatized targetable conjugates are labeled with At-l1liF as disclosed in Example 10. The conjugates are administered in vivo and successfully used for F-l Si PET imaging of tumors.

Example 18. imaging of Tumors Using Blspecilic Antibodies j0227) Bispeciftc antibodies having at least one arm that specifically binds a targeted tissue and at least one other arm that specifically binds a targefabie conjugate are prepared according to U.S. Pa tent No. 7,052,872, incorporated herein by reference in its entirety. The targetable conjugate comprises one or more ΝΟΤΑ chelating moieties. The targetable conjugate is labeled with Al·'*? as described in Example 10. A subject with a disease condition is injected with bispecific antibody. After allowing a sufficient time for free 'bispecific antibody to clear from the circulation, the subject is injected with F-18 labeled targetable conjugate. Imaging of the distribution of the F* 18 label is performed by PET scanning, )0228) In another exemplary embodiment, humanized or chimeric internalizing ami-CD74 antibody is prepared as described in U.S. Patent No. 7,312,318. The />-SCN-bn~NO'T'A precursor is labeled, with Al-'*F as described in Example 7, TbeA1-l8F ΝΟΤΑ is then conjugated to the antibody using standard techniques. Upon i.v. injection into a subject with a CD74-expressing tumor, the ami-CD74 antibody localizes to die tumor, allowing imaging of the tumor by PET scanning. In alternative embodiments, F-l8 labeled antibodies are prepared using the alpha-fetoprotein binding antibody lmmu31, hPAM4, ePAM4, RS7, anii-CD20, anti«CD'19, anti-CEA and anti-CD22, as described in U.S. Patent Nos. 7,300,655; 7,282,567; 7,238,786; 7,238,785; 7,151,164; 7,109,304; 6,676,924; 6,306393 and 6,183,744. The antibodies are conjugated to ΝΟΤΑ using standard techniques and labeled with A!-l8F as described for anti-CD74 antibody. The F-l 8 labeled antibodies at e injected into subjects and provide successful imaging of tumors by PET scanning.

Example 19. Use of lsF*Labeled ΝΟΤΑ for Renal Flow Imaging. (0229) Aluminum stock solution (20 μΐ 0,05 M in pH 4 NaOAc buffer) is mixed with 200 pL of QMA purified F»18 (as in Example 10). The A1F-18 solution is then mixed with 500 pt pH 4,0.2 Μ ΝΟΤΑ and heated Ibr 15 min. The sample is then diluted in 5 mL PBS for injection. The F-l 8 labeled ΝΟΤΑ is used directly for successful renal flow imaging.

Example 20. Further Peptide Labeling Studies with AI-ieF 10230! IMP 460 NOüA-ÖA*D-Ala“D*Lys(HSö)-D-Tyr“D-Lys{.HSG)-NH; was synthesized in a similar manner as described above for IMP 361. Ute NODA-Ga ligand was purchased from Chematecii and attached on (he peptide synthesizer like the other amino acids. The crude peptide was purified to afford the desired peptide MM+ 1366.

[0231 j IMP 460 {0.0020 g) was dissolved in 732 μ[„ pH 4,0.1 M NaOAe, The F-18 was purified as described in Example 10, neutralized with glacial acetic acid and mixed with the AI solution. The peptide solution, 20 pL was then added and the solution was heated at W''C for 25 min. The crude product was then purified on a Waters HIB column as described above. The Al-F-18 labeled peptide was in the 1:1 EtOH/l-LO column eluent. The reverse phase HPLC trace in 0.1 % TFA buffers showed a dean single MPLC peak at the expected location for the labeled peptide.

Example 21, Carbohydrate labeling [0232) A ΝΟΤΑ thiosemicarbazide derivative is prepared by reacting the p-SCN-bn-NQTA with hydrazine and then purifying the ligand by MPLC. AI-F-IS is prepared as described in Example 10 and the Al-F-18 is added to the ΝΟΤΑ thiosemicarbazide and heated for 15 niin. Optionally the Al-F-18-ihiosemicarbazide complex is purified by HPLC. The Al-F-18* thiosemicarbazide is conjugated to oxidized carbohydrates by known methods. The F-18 labeled carbohydrate is successfully used for imaging studies using PET scanning.

Example 22. Lipid labeling [0233] A lipid comprising an aldehyde is conjugated to the Al-F-18 ΝΟΤΑ thiosemicarbazide of Example 2! and the F-18 labeled lipid is used for successful imaging studies using PET scanning.

[0234( in an alternative embodiment, a lipid comprising an amino group is reacted with p-SCN-bn-NOTA. The NOTA-iabe1ed lipid is reacted with Al-F-18 as described in the Examples above. The F-18 labeled lipid is used for successful imaging studies using PET scanning.

Example 23. Aptamer labeling [0235[ An aptamer comprising an aldehyde is conjugated to the Al-F-18 ΝΟΤΑ thiosemicarbazide· of Example 21, The F-18 labeled aptamer is administered to a subject and used for successful imaging studies using PET scanning.

[0236] Throughout this specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or 5 steps.

[0237] The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or 10 information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (39)

1. What is Claimed is:

   1. A method of labeling a molecule with F-19 comprising: a) reacting the F-19 with a metal to form an F-19 metal complex; and b) attaching the F-19 metal complex to a molecule to form one or more F-19 labeled molecules to be administered to a subject; wherein the metal is selected from the group consisting of aluminium, gallium, indium, lutetium and thallium.
2. 2. The method of claim 1, wherein the complex attaches to a chelating moiety on the molecule.
3. 3. The method of claim 1 or claim 2, wherein the molecule is a protein or peptide.
4. 4. The method of any one of claims 1-3, wherein the F-19 labeled molecule is stable in aqueous solution.
5. 5. The method of claims 1-4, wherein the F-19 labeled molecule is stable in serum.
6. 6. The method of any one of claims 2-5, wherein the chelating moiety is selected from the group consisting of DOT A, TETA, ΝΟΤΑ, ΝΕΤΑ or a derivative of ΝΟΤΑ.
7. 7. The method of any one of claims 3-6, wherein the peptide is IMP 449 (NOTA-ITC benzyl-D-Ala-D-Lys(HSG)-D-Tyr-D-Lys(HSG)-NH2).
8. 8. The method of any one of claims 1-7, further comprising administering the F-19 labeled molecules to a subject without separating the F-19 labeled molecule from unlabeled molecules.
9. 9. The method of any one of claims 1-7, further comprising: c) separating the F-19 labeled molecules from unlabeled molecules to produce purified F-19 labeled molecules; and d) administering the purified F-19 labeled molecules to a subject.
10. 10. The method of claim 9, wherein the purified F-19 labeled molecules are produced in less than one hour from the start of the method.
11. 11. The method of claim 9 or claim 10, further comprising using MRI scanning to image the distribution of the purified F-19 labeled molecules in the subject.
12. 12. A method of labeling a molecule with F-19 comprising adding F-19 to a metal-labeled molecule under conditions allowing the F-19 to bind to the metal, wherein the metal is attached to a ehe lating moiety on the molecule and wherein the metal is selected from the group consisting of aluminium, gallium, indium, lutetium and thallium..
13. 13. An F-19 labeled protein or peptide comprising an F-19 metal complex attached to the protein or peptide, wherein the metal is selected from the group consisting of aluminium, gallium, indium, lutetium. and thallium.
14. 14. A method of F-19 imaging by magnetic resonance imaging (MRI) comprising: a) reacting the F-19 with aluminium, gallium, indium, lutetium, or thallium to form an F-19 complex; b) attaching the F-19 complex to a molecule to form one or more F-19 labeled molecules; c) administering the F-19 labeled molecules to a subject under conditions where the labeled molecule is localized to one or more cells, tissues or organs; and d) imaging the distribution of the F-19 labeled molecule by MRI scanning.
15. 15. The method of claim 14, wherein the F-19 labeled molecule is administered to the subject without separating the F-19 labeled molecules from unlabeled molecules.
16. 16. The method of claim 14, further comprising: e) separating the F-19 labeled molecules from unlabeled molecules to produce purified F-19 labeled molecules before the F-19 labeled molecules are administered to the subject.
17. 17. The method of any one of claims 14-16, wherein the imaged distribution of F-19 labeled molecules is indicative of the presence or absence of a disease.
18. 18. The method of claim 17, wherein the disease is selected from the group consisting of solid cancers (carcinomas, sarcomas, melanomas, gliomas, breast cancer, lung cancer, pancreatic cancer, ovarian cancer, colorectal cancer, prostatic cancer), hematopoietic cancers (leukemias, lymphomas, myelomas), autoimmune disease, neurodegenerative disease, Alzheimer's disease, heart disease, myocardial infarction, congestive heart failure, cardiac necrosis, thrombosis, stroke, inflammation, atherosclerosis, rheumatoid arthritis, lupus erythematosus, AIDS and infection with a pathogen.
19. 19. The method of any one of claims 14-18, wherein the F-19 labeled molecule is used to image receptors.
20. 20. The method of claim 19, wherein the receptors are angiogenesis receptors and the F-19 labeled molecule is A1-19F conjugated RGD-NOTA.
21. 21. The method of any one of claims 14-20, wherein the F-19 labeled molecule is used to image tumors.
22. 22. The method of any one of claims 14-21, wherein the F-19 labeled molecule is F-19 labeled bombesin.
23. 23. The method of any one of claims 14-21, wherein the F-19 labeled molecule is an antibody or fragment thereof, selected from the group consisting of hLLl, hLL2, hlmmu31, hPAM4, hRS7, hA19, hA20, hMN-14, hMu-9, hMN-3, hMN-15 and hL243.
24. 24. The method of any one of claims 14-21 or 23, wherein the F-19 labeled molecule is F-19 labeled ΝΟΤΑ and the F-19 labeled ΝΟΤΑ is used to image renal flow.
25. 25. The method of claim 14, wherein the F-19 labeled molecule is targeted to sites of interest using antibodies, antibody fragments, or antibody constructs.
26. 26. The method of claim 14, where the F-19 labeled molecule is targeted to sites of interest using' bispecific antibodies.
27. 27. The method of claim 26, further comprising: i) administering a bispecific antibody to a subject, said bispecific antibody having at least one binding site for a targetable construct and at least one binding site for a targeted antigen, wherein the presence of the targeted antigen is indicative of a disease or condition and the targetable construct is the F-19 labeled molecule; and ii) allowing a sufficient amount of time for bispecific antibody that is not bound to the targeted antigen to clear from circulation prior to the administration of the targetable construct.
28. 28. The method of claim 27, wherein the targeted antigen is a tumor-associated antigen.
29. 29. The method of claim 27, wherein the targeted antigen is present on a pathogenic organism.
30. 30. The method of claim 29, wherein the pathogen is a virus, bacterium, fungus, yeast or microorganism.
31. 31. The method of claim 30, wherein the virus is selected from the group consisting of human immunodeficiency virus (HIV), herpes virus, cytomegalovirus, rabies virus, influenza virus, hepatitis B virus, Sendai virus, feline leukemia vims, Reo virus, polio virus, human serum parvo-like vims, simian vims 40, respiratory syncytial virus, mouse mammary tumor virus, Varicella-Zoster vims, Dengue vims, rubella virus, measles virus, adenovims, human T-cell leukemia viruses, Epstein-Barr vims, murine leukemia vims, mumps vims, vesicular stomatitis vims, Sindbis vims, lymphocytic choriomeningitis virus, wart vims and blue tongue vims; or the bacterium is selected from the group consisting of Streptococcus agalactiae, Legionella pneumophilia, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis and Chlostridium tetani.
32. 32. The method of claim 28, wherein the targeted antigen is selected from the group consisting of colon-specific antigen-p (CSAp), carcinoembryonic antigen (CEA), CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45, CD66a-d, CD67, CD74, CD79a, CD80, CD138, HLA-DR, la, li, MUC 1, MUC 2, MUC 3, MUC 4, NCA, EGFR, HER 2/neu receptor, TAG-72, EGP-1, EGP-2, A3, KS-1, Le(y), S100, PSMA, PSA, tenascin, folate receptor, VEGFR, PDGFR, FGFR, P1GF, ILGF-1, necrosis antigens, IL-2, IL-6, T101, MAGE, or a combination of these antigens.
33. 33. The method of claim 30, wherein the bispecific antibody comprises an antibody or fragment thereof selected from the group consisting of hLLl, hLL2, hlmmu31, hPAM4, hRS7, hA19, hA20, hMN-14, hMu-9, hMu-3, hMN-15 and hL243.
34. 34. Method of preparing a kit for F-19 peptide labeling, wherein the method comprises combining: (a) a metal selected from aluminium, gallium, indium, lutetium, and thallium; and (b) a targeting peptide comprising one or more chelating moieties to bind to the F-19 complex.
35. 35. The method of claim 34, further comprising combining a radiolysis protection agent.
36. 36. The method of claim 35, wherein the radiolysis protection agent is ascorbic acid.
37. 37. The method of any one of claims 34-36, further comprising combining a bispecific antibody, said antibody with one binding specificity for the targeting peptide and another binding specificity for a target antigen.
38. 38. The method of claim 37, wherein the target antigen is a tumor-associated antigen.
39. 39. The method of claim 38, wherein the target antigen is selected from the group consisting of colon-specific antigen-p (CSAp), carcinoembryonic antigen (CEA), CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD45, CD66a-d, CD67, CD74, CD79a, CD80, CD138, HLA-DR, la, li, MUC 1, MUC 2, MUC 3, MUC 4, NCA, EGFR, HER 2/neu receptor, TAG-72, EGP-1, EGP-2, A3, KS-1, Le(y), S100, PSMA, PSA, tenascin, folate receptor, VEGFR, PDGFR, FGFR, P1GF, ILGF-1, necrosis antigens, IL-2, IL-6, T101, MAGE, or a combination of these antigens.