CN103221072A - In vivo copper-free click chemistry for delivery of therapeutic and/or diagnostic agents - Google Patents

Abstract

The present application discloses compositions and methods of synthesis and use involving click chemistry reactions for in vivo or in vitro formation of therapeutic and/or diagnostic complexes. Preferably, the diagnostic complex is of use for 18F imaging, while the therapeutic complex is of use for targeted delivery of chemotherapeutic drugs or toxins. More preferably, a chelating moiety or targetable construct may be conjugated to a targeting molecule, such as an antibody or antibody fragment, using a click chemistry reaction involving cyclooctyne, nitrone or azide reactive moieties. In most preferred embodiments, the click chemistry reaction occurs in vivo. In vivo click chemistry is not limited to 18F labeling but can be used for delivering a variety of therapeutic and/or diagnostic agents.

Description

The interior catalytic click chemistry of no copper of body that is used for delivering therapeutic agents and/or diagnostic agent

Related application

The application requires the U.S. Patent application No.12/958 of December in 2010 submission on the 2nd, the U.S. Patent application No.61/419 of 889 and 2010 on Decembers submission in 2,, 082 priority.Each priority application integral body is by reference incorporated this paper into.

Sequence table

The application contains by EFS-Web and submits to ASCII fromat and the whole by reference in view of the above sequence table of incorporating into.Described ASCII copy called after IMM310WO.txt and the size created on November 17th, 2011 are 24,970 bytes.

The field

The present invention relates to (for example) useful in PET or NMR in-vivo imaging ¹⁸F or ¹⁹The method of F labelling peptide or other molecule.Preferably, by making with the covalently bound chelating moiety of protein, peptide or other molecule ¹⁸F or ¹⁹F and aluminum or another kind of metal connect into conjugate [complex].Use technology described herein, can be at 30min or preparation tool high specific activity in the time still less ¹⁸F or ¹⁹F labelled molecule and being suitable in the imaging technique need not HPLC purification labelled molecule.Carry out labelling in the salt medium that can in being fit to body, directly use.In alternate embodiment, can add organic solvent to improve labeling effciency.Although for for example test kit preparation of some purpose, but stabilizing agent added, for example ascorbic acid, trehalose, Sorbitol or mannitol, but labelled molecule is stable under the condition in vivo.

In preferred embodiments, can for example use antibody, antibody fragment or comprise activated partial in vivo, for example other targeted molecular of cyclooctyne, nitrone or azide generates ¹⁸F or ¹⁹The F labelled molecule.To experimenter's administration of antibodies or other targeted molecular and reserve the enough time, use and comprise corresponding active group, for example azide, nitrone or cyclooctyne with after being positioned target tissue ¹⁸F or ¹⁹But the targeting construct of F labelling.But targeting construct and targeted molecular form covalent bond in position and can be the covalent complex imaging.The technical staff will recognize, described compositions and method be not limited to and ¹⁸F or ¹⁹The F mark part uses together, but can be used for sending and can be connected with the suitable active group, keeps any diagnostic agent and/or the therapeutic agent of functional activity simultaneously.Limiting examples comprises medicine, toxin, radiosiotope, hormone, enzyme, immunomodulator, cytokine, siRNA, anti-angiogenic agent, somatomedin, short apoptosis agent, cytotoxic agent, photosensitive therapeutic agent, chemotherapeutics, dyestuff, contrast agent, fluorescent labeling, chemiluminescent labeling, reinforcing agent and paramagnetic ion.The click chemistry reaction also is not limited to use in vivo, but also can be in external use to generate highly stable conjugate.

Background of invention

Positron emission tomography (PET) has become one of functional imaging mode the most outstanding in the diagnostic medicine, sensitivity very high (fmol), resolution height (4-10mm) and can fully quantize hamartoplasia (Volkow etc., 1988, Am.J.Physiol.Imaging3:142).Though in the oncology [ ¹⁸F] 2-deoxidation-2-fluoro-D-glucose ([ ¹⁸F] FDG) be the most widely used functional image agent (Fletcher etc., 2008, J.Nucl.Med.49:480), but research and development are used for functional imaging great interest (Torigian etc. are arranged with other labelled compound additional and enhancing anatomy imaging method, 2007, CA Cancer J.Clin.57:206), particularly with the hybrid PET/computed tomography imaging system that uses at present.Therefore, need the short-cut method that the radionuclide and various biology and the medical science target molecule that make the emission positron puted together.

Available positron emitter ¹⁸F, ⁶⁴Cu, ¹¹C, ⁶⁶Ga, ⁶⁸Ga, ⁷⁶Br, ^94mTc, ⁸⁶Y and ¹²⁴I labelling peptide or other micromolecule.The isotopic low emission energy of PET is satisfied the demand and is reduced to minimum with the distance of before producing by 2 511keV gamma-rays of PET camera imaging at it positron being left target site.Many isotopes of emission positron also have other emitting substance for example gamma-rays, alpha-particle or beta-particle in its decay chain.Also need to have PET isotope, so that will minimize all dosiology problems for pure electron emitter.The isotopic half-life is also very important because the half-life must long enough so that isotope is connected with targeted molecular, it is injected in patient's body, make the product location, from non-target tissue, remove, then imaging.If the half-life is oversize, specific activity may be not high enough obtaining enough photons of blur-free imaging, and if the half-life too short, production, commercial distribution and chorologic required time may be not enough.Because its positron emission can be low, no side emission and the suitable half-life is arranged, so ¹⁸F (β ⁺635keV97%, t _1/2110min) be one of the most widely used PET emission isotope.

Routinely, by making ¹⁸F combines with carbon atom and makes ¹⁸F is connected (Miller etc., 2008, Angew Chem Int Ed47:8998-9033) with chemical compound, but has also reported and silicon (Shirrmacher etc., 2007, Bioconj Chem18:2085-89; Hohne etc., 2008, Bioconj Chem19:1871-79) and the connection of boron (Ting etc., 2008, Fluorine Chem129:349-58).Usually to involve multistep synthetic with combining of carbon, comprises a plurality of purification steps, existing problems for this isotope that is 110min for the half-life. ¹⁸The current method of F labelling peptide involves the labelling of low specific activity reagent usually, and the HPLC purification of reagent is puted together with target peptide then.After puting together usually the repurity conjugate to obtain the required specific activity of labelling peptide.

Example is the labeling method (J.Nucl.Med.2004 of Poethko etc.; 45:892-902), wherein at first synthetic and purification 4-[ ¹⁸F] fluorobenzaldehyde (Wilson etc., J.Labeled Compounds and Radiopharm.1990; XXVIII:1189-1199), put together with peptide then.Be used to drive with removal by HPLC purified peptide conjugate then and put together the excessive peptide of finishing.Other example comprise with succinyl [ ¹⁸F] and fluobenzoic acid ester (SFB) (for example, Vaidyanathan etc., 1992, Int.J.Rad.Appl.Instrum.B19:275), other acyl compounds (Tada etc., 1989, Labeled Compd.Radiopharm.XXVII:1317; Wester etc., 1996, Nucl.Med.Biol.23:365; Guhlke etc., 1994, Nucl.Med.Biol21:819) or click chemistry addition product (Li etc., 2007, Bioconjugate Chem.18:1987) labelling.The total synthetic and preparation time range of these methods is between 1-3h, and the most of the time is exclusively used in the HPLC purification of labelling peptide to obtain the required specific activity of targeting in the body.Half-life is 2h, makes ¹⁸F is connected required all operations with peptide be very big burden.It is also very dull and need to use and to be designed for the equipment that generates marked product and/or specialized scholar's effort especially to carry out these methods.They also are unfavorable for the kit preparation that can use routinely in the clinical setting.

Need simple fast ¹⁸F labelling targeting moiety is the method for protein or peptide for example, described method produces has suitable specific activity and body internal stability so that detect and/or the targeting construct of imaging, will minimize and reduce the exposure of operator to high levels of radiation to special equipment or height start-up's demand simultaneously.More preferably, the needs preparation is used for pre-targeting technology ¹⁸The F labels targets is to the method for peptide.Further needing to provide the pre-package kit of carrying out the required compositions of this type of new method.

Summary of the invention

In each embodiment, the present invention relates to and be used for PET or NMR imaging ¹⁸F or ¹⁹Compositions and method that the F labelled molecule is relevant.Discuss as this paper, the application mentions ¹⁸During F, the technical staff will recognize and can utilize ¹⁸F, ¹⁹The radionuclide of F or another kind of bond.In illustrative methods, ¹⁸F and melts combine and ¹⁸The F-metal complex is connected with part on peptide or other molecule.As described below, though preferred aluminum, IIIA family metal (aluminum, gallium, indium and thallium) is suitable for ¹⁸The F combination.Lutecium also comes in handy.The melts combine part is preferably chelating agen, for example NOTA, NETA, DOTA, DTPA and following other chelating class discussed in detail.Alternatively, people can at first make metal be connected with molecule, add then ¹⁸F with melts combine.

The technical staff will recognize, for the purpose of imaging, in fact can make any send molecule with ¹⁸F connects, as long as it contains and can modify, but and does not influence the deriveding group of sending the ligand-receptor binding interactions between molecule and the cell or tissue target receptor.Though following examples relate generally to ¹⁸F labelling peptide moiety, but available ¹⁸The F labelling can be with the molecule of sending of other type, for example oligonucleotide, hormone, somatomedin, cytokine, chemotactic factor, angiogenesis factor, anti-angiogenesis, immunomodulator, protein, nucleic acid, antibody, antibody fragment, medicine, interleukin, interferon, oligosaccharide, polysaccharide, lipid etc., and can be used for the imaging purpose.

That describes in following examples is right ¹⁸But the useful exemplary targeting construct peptide of the pre-targeted delivery of F or other reagent includes but not limited to comprise IMP449, IMP460, IMP461, IMP467, IMP469, IMP470, IMP471, IMP479, IMP485 and the MP487 of chelating moiety, and described chelating moiety includes but not limited to alkyl or aryl derivant, NODA-GA, C-NETA, succinyl group-C-NETA and two-tert-butyl group-NODA of DTPA, NOTA, benzyl-NOTA, NOTA.

In certain embodiments, exemplary ¹⁸But F labelling peptide can be used as the targeting construct in utilizing the pre-targeted approach of bispecific or multi-specificity antibody or antibody fragment.In this case, antibody or fragment will comprise the target relevant with disease or condition of illness, for example tumor is relevant the autoimmune disease related antigen or by causal organism for example virus, antibacterial, fungus or other microorganism generate or antigenic one or more binding sites of displaying.But second binding site will combine with targeting construct specificity.Use the pre-targeted approach of bispecific or multi-specificity antibody well-known in this area (see, for example, U.S. Patent No. 6,962,702, the embodiment part is incorporated this paper by reference into).Similarly, but also well-known with the bonded antibody of targeting construct or its fragment in this area, for example (see U.S. Patent No. 7,429 with HSG (histidine succinyl glycine) bonded 679 monoclonal antibodies or with bonded 734 antibody of In-DTPA, 381,7,563,439,7,666,415 and 7,534,431, embodiment part is separately incorporated this paper by reference into).Usually, in pre-targeted approach, at first use bispecific or multi-specificity antibody and it is combined with the cell or tissue target antigen.Behind the appropriate time that binding antibody is not removed from circulation, for example use to the patient ¹⁸But the targeting construct of F labelling and with the antibodies that is positioned at target cell or tissue, (for example) obtains image by PET scanning then.

In alternate embodiment, but labelling and receptor for example Somat, octreotide (octreotide), bombesin (bombesin), folate or folate analog, RGD peptide or the direct bonded molecule of other known receptor part and be used for imaging.Receptor targeting agent can comprise (for example) TA138, beta 2 integrin alpha _vβ ₃The non-peptide antagonists of receptor (Liu etc., 2003, Bioconj.Chem.14:1052-56).Be known in the art other receptor target formation method that uses metallo-chelate and can be used in the practice of claimed method (see, for example, Andre etc., 2002, J.Inorg.Biochem.88:1-6; Pearson etc., 1996, J.Med., Chem.39:1361-71).

Imageable disease or condition of illness type only are used for the availability limitations that is fit to send molecule of the targeting cell or tissue relevant with disease or condition of illness.Known many this type of sent molecule.For example, can use by disclosed method ¹⁸Bonded any protein of F labelling and diseased tissue or target (for example cancer) or peptide also are used for detecting and/or imaging.In certain embodiments, this proteinoid or peptide can include but not limited to and bonded antibody of tumor associated antigen (TAA) or antibody fragment.Use by described method ¹⁸Any known TAA binding antibody of F labelling or fragment and (for example) are used for the imaging and/or the detection of tumor by PET scanning or other known technology.

Some alternate embodiment involves uses catalytic some striking delivering therapeutic agents of no copper and/or diagnostic agent, and for example radionuclide (for example, ¹⁸F), medicine, cytotoxic agent, toxin, hormone, enzyme, immunomodulator, cytokine, siRNA, anti-angiogenic agent, somatomedin, short apoptosis agent, cytotoxic agent, photosensitive therapeutic agent, chemotherapeutics, dyestuff, contrast agent, fluorescent labeling, chemiluminescent labeling, reinforcing agent or paramagnetic ion.Preferably, click chemistry involve comprise activated partial (for example cyclooctyne, nitrone or azido group) targeted molecular for example antibody or conjugated antigen antibody fragment with comprise corresponding active part (for example azide, nitrone or cyclooctyne) but the reaction of targeting construct.When targeted molecular comprises cyclooctyne, but the targeting construct will comprise azide or nitrone or similar active part.When targeted molecular comprises azide or nitrone, but the targeting construct will comprise cyclooctyne, alkynes or similar active part.Available ¹⁸But F labelling targeting construct or can with any substituting diagnostic agent and/or therapeutic agent, for example chemotherapeutics is puted together.But click chemistry reaction makes the highly stable covalent bond of formation between targeted molecular and targeting construct.

The click chemistry reaction can be in external generation to form the high stability labelling targeted molecular that is administered to the experimenter then.In preferred alternate embodiment, the click chemistry reaction can take place in vivo.At first, to experimenter's administration of antibodies or comprise other targeted molecular of activated partial and it is positioned on target cell, tissue, causal organism or other target.But use the targeting construct that comprises the suitable active part to the experimenter then.Reaction between activated partial and the active part has enough specificitys, but consequently the targeting construct does not combine with intravital other katakinetomeres of experimenter.But targeting construct and the targeted molecular irreversible fixation that is arranged in target tissue.

The accompanying drawing summary

Following accompanying drawing is included particular embodiment of the present invention to be described and not to mean that the scope that is limited to claimed theme.

Fig. 1. in the tumor bearing nude mice body by little PET imaging ¹⁸The bio distribution of F labelled reagent.The injection (A) [ ¹⁸F] FDG, (B) be through the Al[of anti-CEA * pre-targeting of anti-HSG bsMAb ¹⁸F] IMP449, (C) independent Al[ ¹⁸F] have the crown sections of 3 nude mices of little subcutaneous LS174T tumor on each left side, IMP449 (without the pre-targeting of bsMAb) back.Provide when the imaging phase finishes the bio distribution data of the tissue of extracing in the animal body, be expressed as the injected dose percentage ratio (%ID/g) of every gram.Abbreviation: B, bone marrow; H, heart; K, kidney; T, tumor.

Fig. 2. to giving to have the pre-targeting Al[of the nude mice of 35mg LS174T people colorectal cancer xenograft at upside ¹⁸F] Study on dynamic imaging of IMP449.Above 3 figure show respectively during the 120min imaging at interval with 6 different 5min, taking from the tumor peripheral position is crown, the sagittal and the cross section at the body position at center.First image in left side shows the location of place, cross-hair cross point tumor in every sectional view, and it indicates with arrow.Animal part tilts to its right side during the imaging.Below 2 illustrate the planar other crown and sagittal plane that concentrates on front more in the coronalplane indicating the distribution in liver and intestinal, and sagittal plane in vivo more the concentrated area intersect.Abbreviation: Cor, crown; FA, forearm; H, heart; K, kidney; Lv, liver; Sag, sagittal; Tr, the cross section; UB, bladder.

Fig. 3. ¹⁸The in-vivo tissue of the IMP468 bombesin analog of F labelling distributes.

Fig. 4. mice (n=5) 2h after injection of band AR42J tumor ¹⁸F-IMP466 and ⁶⁸The bio distribution of Ga-IMP466 relatively.In contrast, the mice of organizing separately in (n=5) has been accepted excessive unlabelled octreotide with the proof receptor-specific.

Fig. 5. cervical region has mice 2h after injection of subcutaneous AR42J tumor ¹⁸F-IMP466 and ⁶⁸The PET/CT of Ga-IMP466 scans crown section.Clearly visiblely in tumor and kidney, gather.

Fig. 6. the BALB/c nude mice with subcutaneous LS174T and SK-RC52 tumor exists ⁶⁸1h after the Ga-IMP288 intravenous injection, 6.0nmol ¹²⁵I-TF2 (0.37MBq) and 0.25nmol ⁶⁸The bio distribution of Ga-IMP288 (5MBq).Value provides (n=5) as meansigma methods ± standard deviation.

Fig. 7. with injection back 1h5MBq FDG and 5MBq in the pre-targeting posterior vein of 6.0nmol TF2 ⁶⁸The bio distribution of Ga-IMP288 (0.25nmol).Value provides (n=5) as meansigma methods ± standard deviation.

Fig. 8. having subcutaneous LS174T tumor (0.1g) on the right hind (white arrow) and in left thigh muscle (black arrow), inflammation being arranged, accepted 5MBq ¹⁸F-FDG and after 1 day at interval 16h accepted 6.0nmol TF2 and 5MBq ⁶⁸The PET/CT image of the BALB/c nude mice of Ga-IMP288 (0.25nmol). ¹⁸F-FDG and ⁶⁸Ga-IMP288 injection back 1h is the animal imaging.There is shown the 3D volume drawing (A) of FDG-PET scanning, the cross section (B) of tumor region and the 3D volume drawing (C) of pre-targeting immunity PET scanning, the cross section (D) of tumor region.

6.0nmol TF2 injection back 1h0.25nmol Al before Fig. 9 .16h ¹⁸The bio distribution of F-IMP449 (5MBq), the not Al of pre-targeting ¹⁸F-IMP449 bio distribution or Al[ ¹⁸F] bio distribution.Value provides as meansigma methods ± standard deviation.

Figure 10. right side (arrow) has subcutaneous LS174T tumor (0.1g), and the 16h intravenous has been accepted 6.0nmol TF2 and 0.25nmol Al at interval ¹⁸The static PET/CT imaging research of the BALB/c nude mice of F-IMP449 (5MBq).Al ¹⁸Injection 1h is the animal imaging behind the F-IMP449.There is shown 3D volume drawing (A) tumor region rearview and cross section, (B) crown, (C) sagittal.

Figure 11. use the chelating moiety and the azide activation targeted molecular of end of tape alkynes, click chemistry is puted together chelating agen part and targeted molecular.

Figure 12. use the chelating moiety of band azide part and the targeted molecular of end of tape alkynes, click chemistry is puted together chelating agen part and targeted molecular.

Figure 13. be used for the cyclooctyne derivant of click chemistry.

Figure 14. be used for the azide derivatives of click chemistry.

Figure 15. be used for the nitrone derivative of click chemistry.

Figure 16. be used for the alternate rings octyne part of click chemistry.

Figure 17. be used for the substituting azide part of click chemistry.

The structure of Figure 18 .IMP479 (SEQ ID NO:52).

The structure of Figure 19 .IMP485 (SEQ ID NO:53).

The structure of Figure 20 .IMP487 (SEQ ID NO:54).

Figure 21. two-tert-butyl group-NODA-MPAA's is synthetic.

Synthesizing of the maleimide conjugate of Figure 22 .NOTA.

Detailed Description Of The Invention

Provide to give a definition so that understand the disclosure of this paper.Clearly the term of definition does not use according to its simple its ordinary meaning.

As used herein, " a kind of (a) " or " a kind of (an) " can refer to more than one or one.

As used herein, term " with " and " or " can be used for referring to conjunction or adversative conjunction.That is, except as otherwise noted, two terms be interpreted as being equal to " and/or ".

As used herein, " pact " refers in the plus or minus 10% of numeral.For example, " about 100 " will refer to any numeral between 90 and 110.

As used herein, " peptide " refers to length between 2-100 amino acid residue, and length is more preferably between 2-10, more preferably between any sequence of the natural or alpha-non-natural amino acid of 2-6 amino acid residue." aminoacid " can be L-aminoacid, D-aminoacid, amino acid analogue and amino acid derivativges or amino acid analog thing.

As used herein, term " pathogen " includes but not limited to fungus, virus, parasite and antibacterial include but not limited to HIV (human immunodeficiency virus) (HIV), herpesvirus, cytomegalovirus, rabies virus, influenza virus, hepatitis B virus, Sendai virus (Sendai virus), the cat leukemia virus, reovirus (Reo virus), poliovirus (polio virus), the tiny sample virus of human serum, simian virus 40, respiratory syncytial virus, mouse mammary adenoma virus, varicella zoster virus (Varicella-Zoster virus), dengue virus (Dengue virus), rubella virus, Measles virus, adenovirus, HTL's poison, Ai Bositan-epstein-Barr virus (Epstein-Barr virus), murine leukemia virus, mumps virus, the blister stomatitis virus, Xin Dehuasi virus (Sindbis virus), lymphocytic choriomeningitis virus, Verrucosis poison (wart virus), blue tongue rims, streptococcus agalactiae (Streptococcus agalactiae), legionella pneumophilia (Legionella pneumophila), streptococcus pyogenes (Streptococcus pyogenes), escherichia coli (Escherichia coli), Neisseria gonorrhoeae (Neisseria gonorrhoeae), Neisseria meningitidis (Neisseria meningitidis), streptococcus pneumoniae (Pneumococcus), Type B hemophilus influenza (Hemophilus influenzae B), Treponoma palladium (Treponema pallidum), lyme disease spirochete (Lyme disease spirochete), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Mycobacterium leprae (Mycobacterium leprae), Bacillus abortus (Brucella abortus), Mycobacterium tuberculosis (Mycobacterium tuberculosis) and Clostridium tetani (Clostridium tetani).

As used herein, " radiolysis protective agent " refers to and can add to ¹⁸In F labelling complex or the molecule to reduce ¹⁸F labelling complex or molecule are subjected to any molecule, chemical compound or the compositions of the decomposition rate of radiolysis.Can use any known radiolysis protective agent, include but not limited to ascorbic acid.

Click chemistry

At first the click chemistry method is contemplated to by small subunit being connected together and generates the method for complex fast with modular form.(see, for example, Kolb etc., 2004, Angew Chem Int Ed40:3004-31; Evans, 2007, Aust J Chem60:384-95.) be known in the art the reaction of various forms of click chemistries, the catalytic Huisgen1 of copper for example, 3-Dipolar Cycloaddition (Tornoe etc., 2002, J Organic Chem67:3057-64) usually also is referred to as " click-reaction ".Other replacement scheme comprises cycloaddition reaction, the carbonylation formation of for example Diels-Alder reaction, nucleophilic substitution (particularly with as epoxide and aziridine cpd small tension receiving coil ring), carbamide compound and involve the reaction of carbon-to-carbon double bond, for example alkynes in sulfydryl-alkyne reaction.

The reaction that azide-alkynes Huisgen cycloaddition reaction uses copper catalyst to be connected with first molecule with the terminal alkynyl of catalysis in the presence of Reducing agent.In the presence of second molecule that comprises the azide part, azide forms 1, the dibasic 1,2,3-triazoles of 4-with the activation alkyne reaction.Catalytic being reflected under the room temperature of copper taken place and the enough specificitys of tool, so that usually do not need the purification reaction product.(Rostovstev etc., 2002, Angew Chem Int Ed41:2596; Tornoe etc., 2002, J Org Chem67:3057.) there is inertia in azide and alkynes functional group to the biomolecule in the water-bearing media to a great extent, make in complex solution, to react.The triazole chemically stable that forms and not cut by enzyme action makes the click chemistry product highly stable in biosystem.Yet copper catalyst is toxic to living cells, has hindered biological applications.

Advised not have the covalent modification that the catalytic click-reaction of copper is used for the biosystem biomolecule.(see, for example, Agard etc., 2004, J Am Chem Soc126:15046-47.) the catalytic reaction of no copper uses ring strain Alloy instead of Copper catalyst to promote [3+2] azide-alkynes cycloaddition reaction (the same).For example, cyclooctyne is the 8-carbocyclic ring structure that comprises inner acetylene bond.Closed-loop structure is impelled has the height reactivity and the bond angle that forms the acetylene of triazole is out of shape substantially to azido group.Therefore, the cyclooctyne derivant can be used for need not the catalytic click-reaction of no copper (the same) of toxicity copper catalyst.

Ning etc. have reported the catalytic click-reaction of no copper (2010, Angew Chem Int Ed49:3065-68) of another type, involve alkynes-nitrone cycloaddition that tension force promotes.For the speed that solves the reaction of former cyclooctyne is slow, electron withdraw group is connected (the same) with adjacent triple bond.The example of the cyclooctyne that this type of is substituted comprises bifluoride cyclooctyne, the pure and mild azacyclo-octyne of 4-cyclohexyl biphenyl octyne (the same).Substituting no copper reaction involves the alkynes-nitrone cycloaddition of tension force promotion to generate N-alkylation isoxazoline (the same).It is reported that reaction has unusually fast kinetics and is used for an one-pot three-step with pointed decoration peptide and protein (the same).By making suitable aldehyde and N-methyl hydroxylamine condensation prepared nitrone and initial ring additive reaction (the same) in the mixture of acetonitrile and water.Yet, attempt to use reaction and the metabolic marker in the Jurkat cell unsuccessful (the same) with the monosaccharide derivatives of nitrone labelling.

In some cases, the activated group that can use the endogenous route of synthesis of cell will be used for the click chemistry reaction is incorporated biomolecule into.For example; Agard etc. (2004, J Am Chem Soc126:15046-47) prove that the recombinant glycoprotein of expressing causes corresponding N-nitrine acetyl sialic acid biology to be incorporated in the carbohydrate of glycoprotein in Chinese hamster ovary celI in the presence of full acetylated N-nitrine acetylmannosamine.Azido derivitised carbohydrate albumen and biotinylation cyclooctyne specific reaction to be forming the biotinylation glycoprotein, and do not have the contrast glycoprotein still unmarked (the same) of azido part.Laughlin etc. (2008, Science320:664-667) use cell surface polysaccharide in the zebrafish embryo that the similar techniques metabolic marker hatches with full acetylated N-nitrine acetylgalactosamine.Azido derivatization polysaccharide and bifluoride cyclooctyne (DIFO) reagent reacting are so that the interior polysaccharide of body is visual.

The Diels-Alder reaction also has been used for the body internal labeling of molecule.Rossin etc. (2010, Angew Chem Int Ed49:3375-78) reported the anti-TAG72 of tumor by local (CC49) antibody that carries trans-cyclo-octene (TCO) active part and ¹¹¹Productive rate in 52% the body between the tetrazine DOTA derivant of In labelling.Use the CC49 antibody of TCO labelling to the mice that has the colon cancer xenograft, then injection after 1 day ¹¹¹The tetrazine probe (the same) of In labelling.By 3h behind injection radio-labeled probe the SPECT imaging of the Mus that lives is proved that the reaction of radio-labeled probe and tumor by local antibody causes being positioned at the obvious radioactivity of tumor, tumor and muscle ratios are 13:1 (the same).The result has confirmed the interior chemical reaction of the body of TCO and tetrazine labelled molecule.

In U.S. Patent No. 6,953, the antibody labeling technology that usage flag biology is partly incorporated into was further disclosed in 675 (the embodiment part is incorporated this paper by reference into).This type of " is beautified (landscaped) ", and Antibody Preparation becomes to have active ketone groups at glycosylation site.Described method is involved in to be expressed in the culture medium of the ketone derivatives that comprises sugar or sugar precursor with being coded in the expression vector cells transfected that has the antibody of one or more N-glycosylation sites in CH1 or the V κ domain.One derivative sugar or precursor comprise N-levulinic acidic group mannosamine and N-levulic acid base fucose.Make subsequently and beautify antibody and comprise the ketone active part, the reagent reacting of hydrazides, hydrazine, hydroxylamino or thiosemicarbazides group for example is to form the labelling targeted molecular.With beautify the peptide that exemplary agents that antibody is connected comprises chelating agen (as DTPA), medicine macromole (for example amycin-glucosan) and contains acyl group-hydrazides.As discussing in more detail in following examples, beautify technology and be not limited to generate the antibody that comprises the ketone part, but can replace being used for the click chemistry active group, for example nitrone, azide or cyclooctyne are introduced on antibody or other biomolecule.

Following examples provide the modification of the click chemistry reaction that is fit to use in external or the body.Can be by chemically conjugated or form the active targeting molecule by incorporating into biology.Available azido part, the octyne that is substituted or alkynyl or nitrone partly activate targeted molecular, for example antibody or antibody fragment.When targeted molecular comprises azido or nitrone base, but corresponding targeting construct will comprise octyne or the alkynyl that is substituted, and vice versa.As discussed above, can incorporate this type of bioactive molecule of generation into by metabolism in living cells.Alternatively, as further discussing in following examples, the chemically conjugated method to biomolecule of this type of part is well known in the art, and can utilizes any this type of known method.Disclosed technology can be used for PET or NMR imaging with described below ¹⁸F or ¹⁹The F labeling method is used in combination, or alternatively, but can be used for sending can be fit to any therapeutic agent and/or the diagnostic agent that activatory targeting construct and/or targeted molecular are puted together.

¹⁸The F labelling technique

Known usefulness ¹⁸The multiple technologies of F labelled molecule.Table 1 has been listed the characteristic of the fluorination process of several more normal reports.Involve by the peptide-labeled of carbon ¹⁸F combines with prothetic group by nucleophilic displacement of fluorine, divides 2 or 3 step marks and purification prothetic group usually, be connected with chemical compound, and then purification.This universal method has been used for being connected prothetic group (Marik etc., 2006, Bioconjug Chem17:1017-21 with " click chemistry " by amido link, aldehyde; Poethko etc., 2004, J Nucl Med45:892-902; Li etc., 2007, Bioconjug Chem18:989-93).It is 4-that the most frequently used amido link forms reagent ¹⁸F-fluobenzoic acid N-succinimide ester ( ¹⁸F-SFB), many other classifications (Marik etc., 2006) have still been tested.In some cases, for example when using ¹⁸When the active ester amide of F labelling forms group, may during coupling reaction, after its cutting, protect some group on the peptide.Synthetic this ¹⁸F-SFB reagent and subsequently with peptide put together the many synthesis steps of needs and the cost about 2-3h.

Poethko etc. (2004) have researched and developed a kind of simpler, more effective ¹⁸The peptide-labeled method of F-, the 4-that wherein in about 75-90min, (comprises drying steps) ¹⁸F-fluorobenzaldehyde reagent connects with peptide by oxime and puts together." click chemistry " method of upgrading will with acetylene or azide in the presence of copper catalyst ¹⁸The F labelled molecule is connected to (Li etc., 2007 on the peptide; Glaser and Arstad, 2007, Bioconjug Chem18:989-93).Reaction between azide and the acetenyl forms triazole and connects, and triazole connects very stable and forms very effectively on peptide, need not protecting group.Click chemistry (comprising drying steps) generates high yield (～50%) in about 75-90min ¹⁸F labelling peptide.

Table 1. is selected ¹⁸The summary of the peptide-labeled method of F-

^aComprise drying steps

^bRevise decay

Make ¹⁸The bonded another kind of method of F and silicon uses isotopic Exchange to use ¹⁸The F displacement ¹⁹F (Shirrmacher etc., 2007).At room temperature carry out 10min, this reaction generation tool high specific activity ¹⁸F aldehyde prothetic group (225-680GBq/ μ mol; 6,100-18,400Ci/mmol).Make subsequently ¹⁸The aldehyde of F labelling and peptide are puted together and by the HPLC purification, and (comprise drying) and obtain the purification labelling peptide of～55% productive rate in 40min.This is changed into single-step process (Hohne etc., 2008) by before labeled reactant, silicon being incorporated in the peptide subsequently.Yet, use ¹⁸F-silicon-bombesin derivant shows to the biodistribution research of mice that the bone uptake ratio increases in time and (when 1.35 ± 0.47% injected dose (ID)/g and 4.0h 5.14 ± 2.71%ID/g), shows during 0.5h ¹⁸F discharges from peptide, because known unconjugated ¹⁸F is arranged in bone (Hohne etc., 2008).Have a large amount of in the HPLC analysis demonstration voidage of urine ¹⁸The F activity, this may be because discharge from peptide ¹⁸The F fluoride anion.Therefore as if ¹⁸F-silicon labelled molecule is unstable in serum.Also reported a large amount of liver and gall drainages, owing to ¹⁸The lipophilic character of F-silicon bound substrates, and derivant has more hydrophilic in the future.Also studied and made ¹⁸The direct-connected method of F and boron; Yet current technology generates the conjugate (Ting etc., 2008) with low specific activity.

Usually in 15min with quantitative yield, routinely with radiometal coupling antibody and peptide (Meares etc., 1984, Acc Chem Res17:202-209; Scheinberg etc., 1982, Science215:1511-13).For the PET imaging, make ⁶⁴Cu and ⁶⁸Ga combines with peptide by chelate, and has demonstrated goodish PET imaging character (Heppler etc., 2000, Current Med Chem7:971-94).Because fluoride and most melts combine, so we attempt to determine ¹⁸Whether the F-metal complex can combine (Tewson, 1989, Nucl Med Biol.16:533-51 with the chelating agen on the targeted molecular; Martin, 1996, Coordination Chem Rev141:23-32).We have concentrated on Al ¹⁸The combination of F complex is because aluminum-fluoride in vivo can relatively stable (Li, 2003, Crit Rev Oral Biol Med14:100-114; Antonny etc., 1992, J Biol Chem267:6710-18).The initial this method that studies confirm for preparing ¹⁸F labelling peptide so that with the feasibility of targeting cancer in the pre-targeted system body of bi-specific antibody (bsMAb), this be in some cases than ¹⁸F-FDG (fluorodeoxyglucose) better highly sensitive and high specific technology (McBride etc., 2008, J Nucl Med (suppl) 49:97P; Wagner, 2008, J Nucl Med49:23N-24N; Karacay etc., 2000, Bioconj Chem11:842-54; Sharkey etc., 2008, Cancer Res68; 5282-90; Gold etc., 2008, Cancer Res68:4819-26; Sharkey etc., 2005, Nature Med11:1250-55; Sharkey etc., 2005, Clin Cancer Res11:7109s-7121s; McBride etc., 2006, J Nucl Med47:1678-88; Sharkey etc., 2008, Radiology246:497-508).These study announcement, Al ¹⁸The F complex can be stably with 1,4,7-7-triazacyclononane-1,4, and 7-triacetic acid (NOTA), but productive rate is very low.

In following examples, checked new flag condition and several new chelating moiety, productive rate rises to about 80% from about 10%, provide ¹⁸F labelling peptide and the feasible method that is used for other molecule of PET imaging.

But targeting construct

In certain embodiments, use ¹⁸But the part of F or other diagnostic agent and/or therapeutic agent labelling can comprise peptide or other targeting construct.But selected marker peptide (or protein) is directly to combine with other target of target cell, tissue, causal organism or confession imaging and/or treatment.In other embodiments, but the selected marker peptide has one or more binding sites and the relevant target antigen of disease or condition of illness is had the bi-specific antibody of one or more binding sites targeting construct peptide but for example use with indirect combination.For example, can at first in the pre-targeting technology of experimenter's administration of antibodies, can use bi-specific antibody therein.Can reserve enough time confession bi-specific antibodys and combine and supply not binding antibody removing from circulation with target antigen.But can use the targeting construct to the experimenter then, for example labelling peptide and make it combine and be positioned sick cell or tissue with bi-specific antibody.Preferably, but that the targeting construct comprises is one or more by the bonded hapten of bi-specific antibody, for example HSG or DTPA.In conjunction with after, can measure by PET scanning or other known technology ¹⁸But the distribution of the targeting construct of F labelling.

But but but this type of targeting construct can have different structure and select this type of targeting construct not still because with high-affinity and bonded antibody of targeting construct or segmental availability, and be because when being used for pre-targeted approach and bi-specific antibody (bsAb) or multi-specificity antibody, remove in vivo fast.Water-repelling agent is good at causing strong immunoreation aspect most, yet preferred hydrophilizing agent is used for removing in the quick body.Therefore, set up balance between hydrophobicity and the hydrophilic.This can be partly by using hydrophilic chelating agen to realize to offset the intrinsic hydrophobicity of many organic moiety.Equally, but the targeting construct subunit that can select to have opposite SOLUTION PROPERTIES for example contains some of them and is hydrophobicity and some of them are hydrophilic amino acid whose peptide.Except that peptide, also can use carbohydrate.

Can use only has 2 amino acid residues, the peptide of preferred 2-10 residue and also can with other parts coupling, for example chelating agen.Connexon should be the low-molecular-weight conjugate, and preferred molecular weight is less than 50,000Da, and advantageously less than about 20,000Da, 10,000Da or 5,000Da.More generally, but targeting construct peptide will have 4 or more a plurality of residue, for example peptide DOTA-Phe-Lys (HSG)-Tyr-Lys (HSG)-NH ₂(SEQ ID NO:1), wherein DOTA is 1,4,7,10- tetraazacyclododecanand 1,4,7,10-tetraacethyl and HSG are histidine succinyl glycyl.Alternatively, available NOTA (1,4,7-7-triazacyclononane-1,4,7-triacetic acid), TETA (to acetyl bromide amido-benzyl-triethylammonium tetrakis tetraacethyl), NETA ([2-(and 4,7-two carboxymethyl [l, 4,7] three azacyclo-s ninth of the ten Heavenly Stems-1-base-ethyl]-2-carbonyl methyl-amino] acetic acid) or other known chelating moiety replace DOTA.

But the targeting construct also can comprise alpha-non-natural amino acid in framing structure, and D-aminoacid for example is to strengthen peptide stability in vivo.In alternate embodiment, can use other framing structure, for example the framing structure that makes up by alpha-non-natural amino acid or class peptide.But known preparation contains the method (seeing that for example, U.S. Patent No. 7,172,751,7,521,416 and 776,311, separately embodiment part is incorporated this paper by reference into) of the amino acid whose targeting construct of D-in this area.

On the automated peptide synthesizer, but use solid phase support and repetition quadrature to go to protect and synthetic easily the peptide of link coupled standard technique as the targeting construct.Advantageously use the standard protecting group, for example be used to put together the free amine group of chelating moiety or other reagent in the Boc group retardance peptide after a while, but and acetyl group N-holds residue to strengthen serum stability.This type of protecting group is that the technical staff is well-known.See Greene and Wuts Protective Groups in Organic Synthesis, 1999 (John Wiley and Sons, N.Y.).When the preparation peptide when being used for the bispecific system after a while, peptide advantageously from resin cleavage to generate corresponding C-end amide, so that suppress the interior carboxypeptidase activity of body.The synthetic illustrative methods of peptide is disclosed in following examples.

When using the pre-targeting of bi-specific antibody, but described antibody will contain being generated by target tissue or associated antigenic first binding site and to haptenic second binding site on the targeting construct.Exemplary hapten includes but not limited to HSG and In-DTPA.The known antibody (for example 679 antibody) that the HSG hapten is produced and can be easy to incorporate in the suitable bi-specific antibody (seeing that for example, U.S. Patent No. 6,962,702,7,138,103 and 7,300,644, the embodiment part is incorporated this paper by reference into).Yet other hapten and bonded with it antibody are known in the art and can use, for example In-DTPA and 734 antibody (for example, U.S. Patent No. 7,534,431, embodiment part are incorporated this paper by reference into).

In alternate embodiment, the specificity of click chemistry reaction can be used as substituting of antibody-hapten binding interactions of being used for the pre-targeting of bispecific antibody.As discussed above, for example the cyclooctyne part can be used in the body intramolecular cycloaddition reaction nitrone specific reaction partly azide part or alkynyl moiety.Partly activate antibody or other targeted molecular by incorporating the cyclooctyne, azide or the nitrone that are substituted into.With ¹⁸But F or other diagnostic agent or therapeutic agent and complementary activity part labelling targeting construct.That is, when targeted molecular comprises cyclooctyne, but the targeting construct will comprise azide; When targeted molecular comprises nitrone, but the targeting construct will comprise alkynes etc.As disclosed, to experimenter's administration of activated targeted molecular and make it be positioned target cell, tissue or cause of disease to pre-targeted approach.But use the activity targeting construct of labelling then.But because the cyclooctyne on the targeting construct, nitrone or azide not with the endogenous biomolecular reaction with targeted molecular on complementary portion height reaction, so but the specificity of binding interactions causes the targeting construct to combine with the high degree of specificity of the targeted molecular that is positioned to organize.

The technical staff will recognize, though but in following examples disclosed major part targeting construct be peptide, but the molecule that can use other type is as the targeting construct.For example, available chelating moiety is the derivatization polymer molecule easily, and Polyethylene Glycol (PEG) for example is with combination ¹⁸F-Al or other diagnostic agent or therapeutic agent.Connect the suitable active group, after the cyclooctyne that for example is substituted, nitrone or the azide, can utilize tagged polymers to send ¹⁸F-Al or other diagnostic agent or therapeutic agent.Be known in the art many examples of examples of such carriers molecule and can utilize, include but not limited to polymer, nano-particle, microsphere, liposome and micelle.

Chelating moiety

In some embodiments, ¹⁸But the hydrophilic chelate part that the F labelled molecule can comprise one or more bind metal ions and help to guarantee to remove in the quick body.Can select chelating agen in conjunction with character according to its particulate metal, and can easily exchange.

Useful especially metal-chelate combination comprises 2-benzyl-DTPA and monomethyl and cyclohexyl analog.Macrocyclic chelants, for example NOTA (1,4,7-three azepines-cyclononane-1,4,7-triacetic acid), DOTA, TETA (to acetyl bromide amido-benzyl-triethylammonium tetrakis tetraacethyl) and NETA also may be used as with multiple ¹⁸The metal of the part that F puts together uses together.

Wherein part comprises the hard base chelating function, and for example the DTPA of carboxylate or amido and DOTA class chelating agen are the most effective to chelating hard acid cation, particularly IIa family and IIIa family metal cation.Can make that by making ring size be fit to metal target this metalloid-chelate complex is highly stable.Other lopps chelating agen, Macrocyclic polyester for example is stably in conjunction with the target of nucleic.The porphyrin chelating agen can use with many metal complexs.The chelating agen of more than one types is puted together to combine multiple metal ion with carrier.Chelating agen; for example U.S. Patent No. 5; 753; disclosed chelating agen in 206; particularly the glyoxyl-based cysteine of thiosemicarbazones (Tscg-Cys) and thiosemicarbazones-acetylcysteine (Tsca-Cys) chelating agen are beneficial to and are used in conjunction with Tc, Re, Bi and other transition metal, the group of the lanthanides of combining closely with the soft base part and the soft acid cation of actinium series.Can be used for making the chelating agen of more than one types to be connected with peptide.Because the haptenic antibody of known two-DTPA (Barbet etc., U.S. Patent No. 5,256,395) and be easy to the targeting antibodies coupling forming bi-specific antibody, so may be in pre-targeted approach with cold two DTPA chelating agen be used to combine ¹⁸The another kind of chelating agen of F complex uses the peptide hapten together.An example of this type of peptide is Ac-Lys (DTPA)-Tyr-Lys (DTPA)-Lys (Tscg-Cys)-NH ₂(SEQ ID NO:2).Available other hard acid chelating agen, for example DOTA, TETA etc. replace DTPA and/or Tscg-Cys group, and can use the similar techniques of the technology that is used to generate anti-two-DTPA MAb to generate it is had specific MAb.

Another kind of useful chelating agents can comprise NOTA class part, for example (J.Med.Chem., 2008,51:118-25) disclosed NOTA class part such as Chong.Chong etc. disclose the structure based on NOTA, generate and use when with ¹⁷⁷Lu or ^205/206During the Bi complexation in serum exhibit stabilization reach 14 days difunctionality C-NETA part.These and other chelating agen example of describing in known and/or the following example in the unrestricted and this area of chelating agen can be used in the practice of the present invention.

To understand, because cationic size difference, the geometry character and the cationic preferred complicated ions structure of chela ring, can incorporate two kinds of different hard acid or soft acid chelating agen into (for example) but have in the targeting construct of different chela ring sizes, with preferentially with two kinds of different hard acid or the combination of soft acid cation.This will allow wherein one or both can with ¹⁸But two kinds of different metals that F connects are incorporated in the targeting construct and are finally caught for pre-targeting bi-specific antibody.

Antibody

Target antigen

Useful targeting antibodies may have specificity or selectivity to various kinds of cell surface or disease association antigen.To imaging or treatment various diseases or condition of illness, for example malignant disease, the cardiovascular diseases, infectious disease, diseases associated with inflammation, autoimmune disease, the useful exemplary target antigen of metabolic disease or neuropathy (for example neurodegenerative diseases) comprises carbonic anhydrase IX, CCCL19, CCCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CXCR4, CXCR7, CXCL12, HIF-1 α, AFP, CEACAM5, CEACAM6, c-met, B7, the ED-B of fibronectin, factor H, FHL-1, Flt-3, folate receptor, GRO-β, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-i (IGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5a-c, MUC16, NCA-95, NCA-90, Ia, PAM4 antigen, the cancer of pancreas mucin, placental growth factor, p53, PLAGL2, prostate acid phosphatase, PSA, PRAME, PSMA, PlGF, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le (y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigen, neoplasm necrosis antigen, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, PlGF, complement factor C3, C3a, C3b, C5a, C5 and oncogene product.

In certain embodiments, for example imaging or the treatment tumor, but useful antibody target tumor related antigen.These antigenic marks can be the material that tumor generates or can be material in tumor locus, tumor cell surface or tumor cell inner accumulated.Wherein this type of tumor mark of correlation is Herberman, " Immunodiagnosis of Cancer ", in " The Clinical Biochemistry of Cancer " that Fleisher edits, the 347th page of (American Association of Clinical Chemists, 1979) and U.S. Patent No. 4,150,149,4,361,544 and 4, disclosed tumor mark of correlation in 444,744, described document embodiment part is separately incorporated this paper by reference into.The report of relevant tumor associated antigen (TAA) comprises Mizukami etc., (2005, Nature Med.11:992-97); Hatfield etc., (2005, Curr.Cancer Drug Targets5:229-48); Vallbohmer etc. (2005, J.Clin.Oncol.23:3536-44); With Ren etc. (2005, Ann.Surg.242:55-63), separately by incorporating this paper into about the quoting of TAA of identifying.

Herberman (seeing above) is divided into many classifications with the tumor mark of correlation, comprises carcinoembryonic antigen, pregniotin, carcinogenic or oncovirus related antigen, organizes related antigen, organ related antigen, ectopic hormone and normal antigen or its variant.Sometimes, the subunit of tumor mark of correlation is advantageously used in and produces the antibody with higher tumour-specific, the γ zone of the β subunit of human chorionic gonadotropin (HCG) or carcinoembryonic antigen (CEA) for example, it stimulates as U.S. Patent No. 4,361,644 and 4,444, the antibody that the cross reactivity of 744 disclosed non-tumor materials reduces greatly generates.

Another kind of target label is for striding film activation factor and CAML interaction factor (TACI).See Nat.Immunol.1:252-256 such as Yu (2000).Briefly, TACI is the labelling of B cell malignancies (for example, lymphoma).Tumor necrosis factor homologue-proliferation-inducing ligand (APRIL) is in conjunction with TACI and B cell maturation antigen (BCMA).APRIL stimulates former generation B and T cells in vitro to breed and increases spleen weight owing to the B cell gathers in vivo.APRIL also competes receptors bind with TALL-I (being also referred to as BLyS or BAFF).Solubility BCMA and TACI specificity prevent APRIL in conjunction with and former generation B cell proliferation of stimulating of retardance APRIL.The antibody that BCMA-Fc also suppresses interior anti-keyhole limpet hemocyanin (keyhole limpet hemocyanin) of mice body and Pneumovax 23 (Pneumovax) generates, and shows that the generation humoral immunization need be via APRIL and/or the TALL-I signal of BCMA and/or TACI.Therefore, APRIL-TALL-I and BCMA-TACI form two parts-two receptor pathway that involves in B and the stimulation of T cell function.

Disease involves lymphoma, when leukemia or autoimmune disorder, target antigen can be selected from CD4, CD5, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38, CD40, CD40L, CD46, CD52, CD54, CD67, CD74, CD79a, CD80, CD126, CD138, CD154, B7, MUC1, Ia, Ii, HM1.24, HLA-DR, tenascin (tenascin), VEGF, PlGF, the ED-B fibronectin, oncogene (for example, c-met or PLAGL2), the oncogene product, CD66a-d, downright bad antigen, IL-2, T101, TAG, IL-6, MIF, TRAIL-R1 (DR4) and TRAIL-R2 (DR5).

Produce the method for antibody

Can from the hybridoma culture, separate and purification MAb by multiple definite technology.This type of isolation technics comprises affinity chromatography, size exclusion chromatography (SEC) and the ion exchange chromatography with a-protein or protein G agarose.See, for example, Coligan 2.7.1-2.7.12 page or leaf and 2.9.1-2.9.3 page or leaf.Equally, see Baines etc., " Purification of ImmunoglobulinG (IgG), " in METHODS IN MOLECULAR BIOLOGY, the 10th volume, the 79-104 page or leaf (The Humana Press, Inc.1992).Initial produce immunogenic antibody after, can be the antibody order-checking and prepare by recombinant technique subsequently.As described below, the humanization of well-known murine antibody of those skilled in the art and antibody fragment and chimeric.

Chimeric antibody

Chimeric antibody is the variable region of the variable region of wherein people's antibody through (for example) mouse antibodies, comprises the metathetical recombiant protein of complementary determining region (CDR) of mouse antibodies.When chimeric antibody is administered to the experimenter, shows immunogenicity and reduce and the stability rising.For example, at Orlandi etc., the general technology of clone's rat immune globulin variable domains is disclosed among the Proc.Nat'l Acad.Sci.USA6:3833 (1989).Those skilled in the art makes up the technology of chimeric antibody as everyone knows.For example, Leung etc., Hybridoma13:469 (1994) is by merging the V of coding Mus LL2 (a kind of anti-CD22 monoclonal antibody) _κAnd V _HThe DNA sequence of domain and each one κ and IgG ₁The constant region domain generates the LL2 chimera.

Humanized antibody

The technology that generates humanization MAb well-known in this area (see, for example, Jones etc., Nature321:522 (1986), Riechmann etc., Nature332:323 (1988), Verhoeyen etc., Science239:1534 (1988), Carter etc., Proc.Nat'l Acad.Sci.USA89:4285 (1992), Sandhu, Crit.Rev.Biotech.12:437 (1992) and Singer etc., J.Immun.150:2844 (1993)).Can be by mice CDR be shifted the chimeric or mouse monoclonal antibody of humanization the corresponding variable domains of the pure man antibody from the variable heavy chain of mouse immuning ball protein and light chain.Also the choose FR sequence displacement of mice skeleton district (FR) in the chimeric mAb.Because only mice CDR is transferred to usually cause among the people FR reducing or even lose affinity of antibody, for the original affinity that recovers murine antibody may need other modification.This can be by realizing with the one or more people's residues in its Mus counter pair displacement FR district, to obtain its epitope is had the antibody of good combination affinity.See, for example, Tempest etc., Biotechnology9:266 (1991) and Verhoeyen etc., Science239:1534 (1988).Preferred for the residue that replaces comprise be positioned at CDR residue side chain 1,2 or

Interior FR residue is positioned near the FR residue of CDR sequence, or estimates meeting and the interactional FR residue of CDR residue.

People's antibody

Use combined method known in the art or generate method (for example, Mancini etc., 2004, the New Microbiol.27:315-28 of human antibody through the transgenic animal that human immunoglobulin gene's seat transforms; Conrad and Scheller, 2005, Comb.Chem.High Throughput Screen.8:117-26; Brekke and Loset, 2003, Curr.Opin.Pharmacol.3:544-50).Also can be by whole known genes or chromosome infection protocol and display technique of bacteriophage make up human antibody in the art.For example see McCafferty etc., Nature348:552-553 (1990).Expect that this type of human antibody shows side effect still less and plays endogenous in essence people's antibody in vivo than chimeric or humanized antibody.

In an alternative, can use display technique of bacteriophage generate people's antibody (for example, Dantas-Barbosa etc., 2005, Genet.Mol.Res.4:126-40).Can or show the special disease state by normal human subject, for example the mankind of cancer generate people's antibody (Dantas-Barbosa etc., 2005).The advantage that is made up people's antibody by diseased individuals is that the circulating antibody spectrum can be partial to the antigenic antibody of anti-disease association.

In a limiting examples of this method, Dantas-Barbosa etc. (2005) have been made up the phage display storehouse of human Fab's antibody fragment by the osteosarcoma patient.Usually, obtain total RNA (the same) from the blood circulation lymphocyte.Clone reorganization Fab and insert (the same) in the phage display storehouse by μ, γ and κ chain antibody spectrum.RNA is converted into cDNA and be used to use heavy chain and the Auele Specific Primer of light chain immunoglobulin sequences obtain Fab cDNA storehouse (Marks etc., 1991, J.Mol.Biol.222:581-97).According to Andris-Widhopf etc. (2000, in Phage Display Laboratory Manual, Barbas etc. (editor), the 1st edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 9.1-9.22 page or leaf) carrying out the storehouse makes up.Digest final Fab fragment and insert in the phage genome with restriction endonuclease to obtain the phage display storehouse.As known in the art, can screen this class libraries by standard phage display method.Can carry out phage display in a variety of forms,, for example see, Johnson and Chiswell, Current Opinion in Structural Biology3:5564-571 (1993) for its comment.

Also can generate people's antibody by external activating B cell.See the whole by reference U.S. Patent No. 5,567,610 and 5,229,275 of incorporating this paper into.The technical staff will recognize that these technology are exemplary and can utilize preparation and any known method of screening people's antibody or antibody fragment.

In another alternative, can use the transgenic animal of the genetically engineered adult's antibody of making a living, use the standard immunoassay method to generate the antibody of anti-any immunogen target basically.Green etc., Nature Genet.7:13 (1994), Lonberg etc., Nature368:856 (1994) and Taylor etc., Int.Immun.6:579 (1994) disclose the method that is obtained people's antibody by transgenic mice.The limiting examples of this type systematic be from Abgenix (Fremont, CA) (for example, Green etc., 1999, J.Immunol.Methods231:11-23 incorporates this paper by reference into).

In similar animal body, deactivation mouse antibodies gene and with the displacement of functional human immunoglobulin gene, and all the other genes of mouse immune system are kept perfectly.

With containing groups of people IgH and Ig kappa gene seat, comprise most of variable region sequences, the YAC (yeast artificial chromosome) that makes up together with the kind of subsidiary gene and regulating and controlling sequence system transforms

People variable region spectrum can be used for generating the B cell that can be processed into the generation antibody of hybridoma by known technology.Through the target antigen immunity

To generate standard technique results that can be from what has been discussed above and/or the people's antibody that generates by normal immunoreaction.Multiple Bacterial strain can be used, and it all can generate different classes of antibody separately.Confirmed that people's antibody that transgenic generates has the treatment potentiality, keeps the pharmacokinetic property (Green etc., 1999) of normal person's antibody simultaneously.The technical staff will recognize that compositions required for protection and method are not limited to use

System, but can utilize genetically engineered make a living adult antibody any transgenic animal.

Known antibodies

The technical staff will recognize, to imaging and/or treat useful targeted molecular and can have any antibody or the fragment of binding specificity to incorporate into to morbid state or the relevant target antigen of condition of illness with as known in the art.This type of known antibodies includes but not limited to hR1 (anti-IGF-1R, the U.S. Patent Application Serial Number No.12/772 that on March 12nd, 2010 submitted to, 645), hPAM4 (anti-cancer of pancreas mucin, U.S. Patent No. 7,282,567), hA20 (anti-CD20, U.S. Patent No. 7,251,164), hA19 (anti-CD19, U.S. Patent No. 7,109,304), hIMMU31 (anti-AFP, U.S. Patent No. 7,300,655), hLL1 (anti-CD74, U.S. Patent No. 7,312,318), hLL2 (anti-CD22, U.S. Patent No. 7,074,403), hMu-9 (anti-CSAp, U.S. Patent No. 7,387,773), hL243 (anti-HLA-DR, U.S. Patent No. 7,612,180), hMN-14 (anti-CEACAM5, U.S. Patent No. 6,676,924), hMN-15 (anti-CEACAM6, U.S. Patent No. 7,662, the U.S. Patent Application Serial Number No.12/846 that on July 29th, 378,2010 submitted to, 062), hRS7 (anti-EGP-1, U.S. Patent No. 7,238,785), hMN-3 (anti-CEACAM6, U.S. Patent No. 7,541,440), Ab124 and Ab125 (anti-CXCR4, U.S. Patent No. 7,138,496) each patent, quoted or the embodiment of application part are incorporated this paper by reference into.Other known useful antibody includes but not limited to anti-TAG-72 (for example, CC49), Tn, J591 or HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX), alemtuzumab (anti-CD52), bevacizumab (anti-VEGF), Cetuximab (anti-EGFR), gemtuzumab Ozogamicin Mylotarg CDP 771 (anti-CD 33), ibritumomab tiuxetan (anti-CD20); Handkerchief Buddhist nun monoclonal antibody (anti-EGFR); Rituximab (anti-CD20); Tositumomab (anti-CD20); GA101 (anti-CD20); And Herceptin (anti-ErbB).

Known useful antibody can with cause of disease, for example HIV generates or associated antigen combination.This antibody-like can be used for detecting, diagnoses and/or the treatment infectious disease.The anti-HIV antibody of candidate comprises (AIDS.2006 October 3 such as Johansson; 20 (15): 1911-5) anti-peplos antibody of Miao Shuing and Polymun (Vienna, Austria) the anti-HIV antibody of describing and selling, at United States Patent (USP) 5,831,034, United States Patent (USP) 5,911,989 and Vcelar etc., AIDS2007; 21 (16): 2161-2170 and Joos etc., Antimicrob.Agents Chemother.2006; 50 (5): description is also arranged among the 1773-9, and it all incorporates this paper by reference into.

The parasitic antibody of malaria can resist sporinite, merozoite, schizont and gametocyte stage.Generated the monoclonal antibody of anti-sporinite (circumsporozoite protein antigen), and verified and the external and intravital sporinite of rodent combines (N, Yoshida etc., Science207:71-73,1980).Several groups have have researched and developed antibody (Kasper etc., J.Immunol.129:1694-1699,1982 of the primary parasite toxoplasma (T.gondii) that is involved in the anti-toxoplasmosis; The same, 30:2407-2412,1983).Researched and developed the antibody of schistosomicide surface antigen and have been found that with body in or external schistosomulum (schistosomulae) combine (Simpson etc., Parasitology, 83:163-177,1981; Smith etc., Parasitology, 84:83-91,1982:Gryzch etc., J.Immunol., 129:2739-2743,1982; Zodda etc., J.Immunol.129:2326-2328,1982; Dissous etc., J.immunol., 129:2232-2234,1982).

Schizotrypanum cruzi (Trypanosoma cruzi) is the pathogen of american trypanosomiasis (Chagas'disease), and by the entomochory of triatoma sanguisuga section.Generated parasite that specificity suppresses a kind of form vitro differentiation be another kind of form (epimastigote is to the trypomastigote phase) and with the antibody of cell surface glycoprotein reaction; Yet this antigen is not present in the parasite of mammal (blood flow) form (Sher etc., Nature, 300:639-640,1982).

Be known in the art anti fungal antibody, for example anti-Sclerotinia (Sclerotinia) antibody (United States Patent (USP) 7,910,702), anti-glucuronic acid xylose mannan antibody (Zhong and Priofski, 1998, Clin Diag Lab Immunol5:58-64), anti-candida belong to (Candida) antibody (Matthews and Burnie, 2001,2:472-76) with anti-glycosyl sphingolipid antibody (Toledo etc., 2010, BMC Microbiol10:47).

Known various other useful antibody (for example, U.S. Patent No. 5,686,072,5,874 in this area, 540,6,107,090,6,183,744,6,306,393,6,653,104,6,730,300,6,899,864,6,926,893,6,962,702,7,074,403,7,230,084,7,238,785,7,238,786,7,256,004,7,282,567,7,300,655,7,312,318,7,585,491,7,612,180,7,642,239 and U.S. Patent Application Publication No.20060193865; Incorporate this paper separately by reference into).This type of known antibodies is useful to the detection and/or the imaging of various disease states or condition of illness (for example, hMN-14 or TF2 (CEA expresses cancer), hA20 or TF-4 (lymphoma), hPAM4 or TF-10 (cancer of pancreas), RS7 (pulmonary carcinoma, breast carcinoma, ovarian cancer, carcinoma of prostate), hMN-15 or hMN3 (inflammation), anti-gp120 and/or anti-gp41 (HIV), antiplatelet and antithrombase (clot imaging), anti-myosin (heart necrosis), anti-CXCR4 (cancer and diseases associated with inflammation)).

When using bi-specific antibody, can from antihapten antibody known in the art, select the 2nd MAb, include but not limited to h679 (U.S. Patent No. 7,429,381) and 734 (U.S. Patent No.s 7,429,381,7,563,439,7,666,415 and 7,534,431), its embodiment part is separately incorporated this paper by reference into.

Useful antibody can be bought from various known sources and obtain.For example, can be from American type culture collection (ATCC, Manassas, VA) the hybridoma system of the multiple secretory antibody of acquisition.Deposited a lot of anti-various diseases targets at ATCC, included but not limited to the antibody of tumor associated antigen, and/or announced variable region sequences and can be used in the method and composition required for protection.See, for example, U.S. Patent No. 7,312,318,7,282,567,7,151,164,7,074,403,7,060,802,7,056,509,7,049,060,7,045,132,7,041,803,7,041,802,7,041,293,7,038,018,7,037,498,7,012,133,7,001,598,6,998,468,6,994,976,6,994,852,6,989,241,6,974,863,6,965,018,6,964,854,6,962,981,6,962,813,6,956,107,6,951,924,6,949,244,6,946,129,6,943,020,6,939,547,6,921,645,6,921,645,6,921,533,6,919,433,6,919,078,6,916,475,6,905,681,6,899,879,6,893,625,6,887,468,6,887,466,6,884,594,6,881,405,6,878,812,6,875,580,6,872,568,6,867,006,6,864,062,6,861,511,6,861,227,6,861,226,6,838,282,6,835,549,6,835,370,6,824,780,6,824,778,6,812,206,6,793,924,6,783,758,6,770,450,6,767,711,6,764,688,6,764,681,6,764,679,6,743,898,6,733,981,6,730,307,6,720,155,6,716,966,6,709,653,6,693,176,6,692,908,6,689,607,6,689,362,6,689,355,6,682,737,6,682,736,6,682,734,6,673,344,6,653,104,6,652,852,6,635,482,6,630,144,6,610,833,6,610,294,6,605,441,6,605,279,6,596,852,6,592,868,6,576,745,6,572; 856,6,566,076,6,562,618,6,545,130,6,544,749,6,534,058,6,528,625,6,528,269,6,521,227,6,518,404,6,511,665,6,491,915,6,488,930,6,482,598,6,482,408,6,479,247,6,468,531,6,468,529,6,465,173,6,461,823,6,458,356,6,455,044,6,455,040,6,451,310,6,444,206 ', 6,441,143,6,432,404,6,432,402,6,419,928,6,413,726,6,406,694,6,403,770,6,403,091,6,395,276,6,395,274,6,387,350,6,383,759,6,383,484,6,376,654,6,372,215,6,359,126,6,355,481,6,355,444,6,355,245,6,355,244,6,346,246,6,344,198,6,340,571,6,340,459,6,331,175,6,306,393,6,254,868,6,187,287,6,183,744,6,129,914,6,120,767,6,096,289,6,077,499,5,922,302,5,874,540,5,814,440,5,798,229,5,789,554,5,776,456,5,736,119,5,716,595,5,677,136,5,587,459,5,443,953,5,525,338.These only are exemplary and are known in the art diversified other antibody and hybridoma thereof.The technical staff will recognize, can resist the data base of the antibody of the relevant target of selected target disease to obtain the hybridoma of anti-antigenic antibody sequence of almost any disease association or secretory antibody by simple search ATCC, NCBI and/or USPTO.Can use the antigen binding structural domain of standard technique amplification well-known in the art, excision clonal antibody, be connected in the expression vector, transfection extremely is fit in the host cell and is used for protein generate.

Antibody fragment

Can generate the antibody fragment at identification specific antigen decision position by known technology.Described antibody fragment is the antigen-binding portion thereof of antibody, for example F (ab') ₂, Fab', F (ab) ₂, Fab, Fv, sFv etc.Can generate F (ab') by the pepsin digested antibody molecule ₂Fragment and can by the reduction F (ab') ₂Segmental disulphide bridges generates the Fab' fragment.Alternatively, can make up the Fab' expression library (Huse etc., 1989, Science is 246:1274-1281) to allow easily to identify the required specific monoclonal Fab' fragment of tool fast.Can express the described segmental DNA of coding by the Proteolytic enzyme full length antibody or in escherichia coli (E.coli) or other host and prepare antibody fragment.For example, Goldenberg, U.S. Patent No. 4,036,945 and 4,331,647 reach wherein contained reference has described these methods, and described patent integral body is by reference incorporated this paper into.Equally, see Nisonoff etc., Arch Biochem.Biophys.89:230 (1960); Porter, Biochem.J.73:119 (1959), Edelman etc., the 1st volume in METHODS IN ENZYMOLOGY, the 422nd page (Academic Press1967) and Coligan 2.8.1-2.8.10 and 2.10.-2.10.4 page or leaf.

Strand Fv molecule (scFv) comprises V _LDomain and V _HDomain.V _LAnd V _HDomain associates and forms the target binding site.These two domains are further covalently bound by peptide connexon (L).The U.S. Patent No. 4 of incorporating this paper by reference into, 704,692, U.S. Patent No. 4,946,778, R.Raag and M.Whitlow, " Single Chain Fvs. " FASEB the 9th volume: 73-80 (1995) and R.E.Bird and B.W.Walker, " Single Chain Antibody Variable Regions, " TIBTECH, the 9th volume: described the method for preparing scFv molecule and the suitable peptide connexon of design among the 132-137 (1991).

Can use and all known V by from the human donor of non-immunity, separating the V gene _H, V _κAnd V ₈₀The corresponding PCR primer of gene family makes up the scFv storehouse with big spectrum.See, for example, Vaughn etc., Nat.Biotechnol., 14:309-314 (1996).After the amplification, merge V _κAnd V _λThe storehouse is to form a storehouse.These fragments are connected in the phasmid carrier.The scFv connexon can be connected to V then _LIn the phasmid of fragment upstream.Amplification V _HAnd connexon-V _LFragment also is assembled in J _HIn the district.With gained V _H-connexon-V _LFragment is connected in the phasmid carrier.Elutriation phasmid storehouse is to combine with selected antigen.

Be known in the art other antibody fragment, for example single domain antibody fragment and can be used in the claimed construct.For example, can obtain single domain antibody (VHH) by camel, alpaca or yamma by the standard immunoassay technology.(see, for example, Muyldermans etc., TIBS26:230-235,2001; Yau etc., J Immunol Methods281:161-75,2003; Maass etc., J Immunol Methods324:13-25,2007).VHH may have the strong antigen binding ability and can interact at right novel antigens decision position with being difficult to approaching conventional VH-VL.(Muyldermans etc., 2001) alpaca serum IgG contains the unique IgG antibody of 50% camel heavy chain (Cabs) (Maass etc., 2007) of having an appointment.Available known antigens makes in alpaca immunity and the separable combination also and the VHH (Maass etc., 2007) of target antigen.Identified the PCR primer of all VHH coded sequences that in fact increase and can be used for structure and can be used for the isolating camel VHH of antibody fragment phage display storehouse (Maass etc., 2007) by standard biological panning technique well-known in the art.In claimed method and composition, can utilize these and other known antigen binding antibody fragment.

The general technology of antibody cloning and generation

Various technology, the generation of for example chimeric or humanized antibody can involve antibody cloning and construction procedures.Can be by a plurality of molecular cloning programs, for example the screening of RT-PCR, 5'-RACE and cDNA storehouse obtains the V of the conjugated antigen of target antibody _κ(variable light chain) and V _H(variable heavy chain) sequence.Can be by V gene and the order-checking of pcr amplification clone from the MAb of the cell of expressing Mus MAb.For confirming its verity, can make clone's V _LAnd V _HGene is expressed as Orlandi in cell culture etc., the chimeric Ab that (Proc.Natl.Acad.Sci, USA, 86:3833 (1989)) describes.Based on the V gene order, then can be of (Mol.Immunol, 32:1413 (1995)) such as Leung, design also makes up humanization MAb.

Can prepare cDNA (Sambrook etc., Molecular Cloning, A laboratory manual, the 2nd edition (1989)) by any known hybridoma system or the transfectional cell series that generate Mus MAb by general molecule clone technology.Can use the V of the extension primer collection amplification MAb of (BioTechniques, 15:286 (1993)) descriptions such as primer VK1BACK and VK1FOR (Orlandi etc., 1989) or Leung _κSequence.Can use primer to VH1BACK/VH1FOR (Orlandi etc., 1989) or be annealed into the primer amplification V of the Mus IgG constant region that Leung etc. (Hybridoma, 13:469 (1994)) describes _HSequence.Can combination synthetic by the long oligonucleotide template that (Mol.Immunol, 32:1413 (1995)) such as Leung describes and pcr amplification make up humanization V gene.

Can be with V _κPCR product sub-clone to staging vector, for example based on the staging vector of pBR327, contain the VKpBR of Ig promoter, signal peptide sequence and accessible restriction site.Can be with V _HPCR product sub-clone to the similar staging vector, for example based on the VHpBS of pBluescript.Can from VKpBR and VHpBS, excise and contain V _κAnd V _HSequence is together with the expression cassette of promoter and signal peptide sequence and be connected respectively in the suitable expression vector, for example pKh and pG1g (Leung etc., Hybridoma, 13:469 (1994)).Can be to suitable cell and monitor the generation of chimeric in the supernatant, humanization or people MAb with the expression vector cotransfection.Alternatively, can excise V _κAnd V _HExpression cassette and sub-clone are to the single expression vector, and pdHL2 for example is as also illustrating among the Gillies etc. (as described in the J.Immunol.Methods125:191 (1989) and at Losman etc., Cancer, 80:2660 (1997)).

In an alternate embodiment, can be to the host cell of pre-adaptation transfection in serum-free medium, growth and expression with the expression vector transfection.Available exemplary cells system comprise Sp/EEE, Sp/ESF and Sp/ESF-X cell line (see, for example, U.S. Patent No. 7,531,327,7,537,930 and 7,608,425; Its embodiment part is separately incorporated this paper by reference into).These exemplary cells system is based on the Sp2/0 myeloma cell line, through sudden change Bcl-EEE gene transfection, be exposed to methotrexate (methotrexate) with amplification rotaring redyeing gene sequence and pre-adaptation serum-free cell line so that protein expression.

Affine body

Affine body be the effect of antibody analog and in binding target molecule useful small protein.By the integration engineeringization of α coilin support having been researched and developed affine body (Nord etc., 1995, Protein Eng8:601-8; Nord etc., 1997, Nat Biotechnol15:772-77).Affine body design is based on triple helical bundle structure (Nord etc., 1995 of the IgG binding structural domain that comprises a-protein; 1997).Can involve in the Fc of bacterioprotein A by randomization and generate affine body (Nord etc., 1995 with various binding affinities in conjunction with active 13 aminoacid; 1997).Behind the randomization, the pcr amplification storehouse is cloned in the phasmid carrier so that the phage display by mutain screens.

Verified HER2/neu is had specific ¹⁷⁷The xenograft (Tolmachev etc., 2007, Cancer Res67:2773-82) of the Lu labelling is affine body targeting expression in vivo HER2.Though because the gathering of low-molecular-weight radio-labelled compound, initial nephrotoxicity is a problem, combine with albuminous reversibility and reduced kidney and gather, make the therapy based on radionuclide feasible (the same) of the affine body of usefulness labelling.

Proved the feasibility (Tolmachev etc., 2011, Bioconjugate Chem22:894-902) of using radiolabeled affine body to carry out the in-vivo tumour imaging recently.Maleimide derivative NOTA is connected with the affine body of anti-HER2 and uses ¹¹¹In radioactive label (the same).Use to the mice that has the DU-145 xenograft of expressing HER2, then the γ camera imaging makes xenograft visual (the same).

The technical staff will recognize that affine body can be used as targeted molecular in the practice of claimed method and composition.Can described in following examples, carry out puting together with metal ¹⁸The F labelling.Can be from Affibody AB (Solna, Sweden) the affine body of purchase acquisition.

Bispecific and multi-specificity antibody

But some embodiment relates to bi-specific antibody and the pre-targeted approach that is loaded with haptenic targeting construct.The method of known many generation bispecifics or multi-specificity antibody, open in 320 for example in U.S. Patent No. 7,405, the embodiment part is incorporated this paper by reference into.Can generate bi-specific antibody by four tumor methods, described method involves two kinds of different hybridomas of fusion, and every kind of hybridoma generates monoclonal antibody (Milstein and Cuello, Nature, 1983 of the different antigen sites of identification; 305:537-540).

The another kind of method that generates bi-specific antibody uses allos bi-functional cross-linking agent chemistry to connect two kinds of different monoclonal antibodies (Staerz etc., Nature.1985; 314:628-631; Perez etc., Nature.1985; 316:354-356).Also can be by each of two kinds of parent's monoclonal antibodies being reduced to half point separately, mix then and it is reoxidized obtaining hybrid structure, thereby generate bi-specific antibody (Staerz and Bevan.Proc Natl Acad Sci USA.1986; 83:1453-1457).Other method comprise by through retrovirus source shuttle vector with different selectable marker gene transfer to parent's hybridoma separately, merge parent's hybridoma (DeMonte etc. subsequently, Proc Natl Acad Sci USA.1990,87:2941-2945), improve the efficient that generates the hybrid hybridoma or with the expression plasmid transfection hybridoma cell line of heavy chain that contains different antibodies and light chain gene.

Available suitable composition is connected homology V with the peptide connexon of length (being made up of the amino acid residue more than 12 usually) _HAnd V _LDomain is to form strand Fv (scFv) as discussed above.Peptide connexon length is reduced to less than 12 amino acid residues prevents same the V on the chain _HAnd V _LThe domain pairing also forces V _HAnd V _LComplementary structure territory pairing on domain and other chain causes forming functional polymer.The V that uses the connexon between 3 to 12 amino acid residues to connect _HAnd V _LThe polypeptide chain of domain mainly forms dimer (being called double-stranded antibody).Use connexon, help the trimer (being called three chain antibodies) and the tetramer (being called four chain antibodies), but except that connexon length, as if the oligomerization pattern also depends on the composition and the direction (V of V domain accurately between 0 to 2 amino acid residue _H-connexon-V _LOr V _L-connexon-V _H).

These technology that generate polyspecifics or bi-specific antibody are low at productive rate, the purification necessity, stability is low or the labor intensity of technology aspect show all difficulties.Recently, utilized the combination of technology production almost any required antibody, antibody fragment and other effector molecule (DNL) of following discussed in detail being called " butt joint locking " (to see, for example, U.S. Patent Application Publication No.20060228357,20060228300,20070086942,20070140966 and 20070264265, embodiment part is separately incorporated this paper by reference into).The DNL technology allow with monospecific, bispecific or multi-specificity antibody be assembled into exposed antibody moiety or with the combination of multiple other effector molecule, for example immunomodulator, enzyme, chemotherapeutics, chemotactic factor, cytokine, diagnostic agent, therapeutic agent, radionuclide, developer, anti-angiogenic agent, somatomedin, oligonucleotide, hormone, peptide, toxin, short apoptosis agent or its combination.In the practice of at present claimed method, can utilize any technology of preparation bispecific known in the art or multi-specificity antibody.

Butt joint locking (DNL)

In preferred embodiments, can use the butt joint lock-in techniques generate bispecific or multi-specificity antibody or other construct (see, for example, U.S. Patent No. 7,550,143,7,521,056,7,534,866,7,527,787,7,666,400,7,906,118 and 7,901,680, embodiment part is separately incorporated this paper by reference into).The DNL method has been utilized specific protein/protein interaction (Baillie etc., the FEBS Letters.2005 that takes place between the anchoring structure territory (AD) of adjusting (R) subunit of cAMP dependent protein kinase (PKA) and A kinases anchorin (AKAP); 579:3264.Wong and Scott, Nat.Rev.Mol.Cell Biol.2004; 5:959).Nineteen sixty-eight, isolate PKA from rabbit skeletal muscle first, it is by central role (Walsh etc., the J.Biol.Chem.1968 in conjunction with the signal transduction pathway of studying the most thoroughly that cause of second message,second messenger cAMP with the R subunit; 243:3763).The structure of holoenzyme is formed (Taylor, J.Biol.Chem.1989 by two catalytic subunits that the R subunit keeps being inactive form; 264:8443).Find the R subunit (RI and RII) that the isozyme of PKA has 2 types, and every type have α and β isotype (Scott, Pharmacol.Ther.1991; 50:123).Therefore, there are 4 isotype PKA-RI α, RI β, RII α and RII β.Only isolate to be and stablize dimeric R subunit and confirmed that the dimerization domain forms (Newlon etc., Nat.Struct.Biol.1999 by preceding 44 aminoterminal residues; 6:222).The active catalytic subunit that cAMP and combining of R subunit cause discharging large-scale activity of serine/threonine kinases docks with AKAP by it, and compartmentalization PKA makes the active catalytic subunit towards selected substrate orientation (Scott etc., J.Biol.Chem.1990; 265; 21561).

Characterized the first AKAP from 1984, (Lohmann etc., Proc.Natl.Acad.Sci USA.1984 since the microtubule-associated protein-2; 81:6723), in the species of scope from the yeast to the mankind, identified have different structure more than 50 kinds be confined to the subcellular fraction position, the AKAP (Wong and Scott, the Nat.Rev.Mol.Cell Biol.2004 that comprise plasma membrane, actin cytoskeleton, nuclear, mitochondrion and endoplasmic reticulum; 5:959).AKAP is amphiphatic molecule spiral (Carr etc., the J.Biol.Chem.1991 of 14-18 residue to the AD of PKA; 266:14188).The aminoacid sequence of AD is different fully in indivedual AKAP, it is reported that the binding affinity scope to RII is 2-90nM (Alto etc., Proc.Natl.Acad.Sci.USA.2003; 100:4445).AKAP will only combine with dimerization R subunit.For people RII α, AD is in conjunction with the hydrophobic surface (Colledge and Scott, the Trends CellBiol.1999 that are formed by 23 aminoterminal residues; 6:216).Therefore, the dimerization domain of people RII α all is arranged in identical N-with the AKAP binding structural domain and holds 44 aminoacid sequences (Newlon etc., Nat.Struct.Biol.1999; 6:222; Newlon etc., EMBO are J.2001; 20:1651), this paper also is referred to as DDD.

The AD that we have have researched and developed the DDD that utilizes people PKA to regulate subunit and AKAP is right as good connexon module, any two entities of A and B are hereinafter referred to as butted up against platform technology in the non-covalent complex, can non-covalent complex further be locked as stable syndeton by the key position of cysteine residues being introduced in DDD and the AD, form to promote disulfide bond.The conventional method opinion of " butt joint locking " method is as follows.Make up entity A by the precursor that the DDD sequence is connected to A, produce first component of a hereinafter referred to as.Because the DDD sequence can influence dimeric spontaneous formation, A will be by a ₂Form.Make up entity B by the precursor that the AD sequence is connected to B, produce second component of b hereinafter referred to as.a ₂In contained DDD dimerization motif will create with b in the bonded site of docking of contained AD sequence, thereby be beneficial to a ₂Associate rapidly to form with b by a ₂The binary trimerization complex that b constitutes.This binding events carries out to pass through two entities of disulphide bridges Covalent Immobilization with the subsequent reaction irreversibility, because initial binding interactions answers bridge joint to place sulfhydryl-group activity on DDD and the AD near (Chmura etc., Proc.Natl.Acad.Sci.USA.2001; 98:8480) connect, so according to the principle of effective local concentration, binding events carries out very effectively with locus specificity.Use the various combinations of connexon, adaptive submodule and precursor, can generate and use the DNL construct of multiple different chemical metering, include but not limited to that dimer, trimer, the tetramer, pentamer and six aggressiveness DNL constructs (see, for example, U.S. No.7,550,143,7,521,056,7,534,866,7,527,787 and 7,666,400).

By connecting DDD and AD, estimate that also this locus specificity connects original activity of having protected two kinds of precursors away from the functional group of two kinds of precursors.This method is modularity and may be applied to locus specificity and covalently bound multiple material in essence, comprises peptide, protein, antibody, antibody fragment and has other effect part of various active.In fact the fusion rotein method of utilizing the construct AD that describes in following examples and DDD to put together effector can be incorporated any protein or peptide in the DNL construct into.Yet technology is not limit and can be utilized other conjugation methods.

The several different methods of known preparation fusion rotein comprises that nucleic acid synthesizes, hybridizes and/or increases to generate the proteic synthetic double-strandednucleic acid of coding Target Fusion.Can this type of double-strandednucleic acid be inserted by standard molecular biological technique and be used for generating Expression of Fusion Protein carrier (seeing Sambrook etc. for example, Molecular Cloning, A laboratory manual, the 2nd edition, 1989).In this type of preferred embodiment, AD and/or DDD part is connected with the N-terminal or the C-terminal of effect protein or peptide.Yet the technical staff will recognize that the part that site that AD or DDD part and effect partly are connected can partly involve its physiologically active according to the chemical constitution and the effect of effect part changes.Can use technology known in the art, the locus specificity that for example uses bivalence cross-linking agent and/or other chemically conjugated technology to carry out multiple effect part connects.

In other alternate embodiment, can use the electric shock chemical reaction to generate AD or the DDD peptide of partly puting together with effect, or even make AD and DDD part covalently bound mutually so that irreversible covalent bond to be provided so that the DNL complex is stable.

Pre-targeting

In pre-targeting technology, can utilize bispecific or multi-specificity antibody.Pre-targeting is that the blood that initial research and development are used to solve direct targeting antibodies is removed multistep technology slowly, and blood is removed and slowly facilitated normal structure, for example harmful toxicity of bone marrow.Use pre-targeting, radionuclide or other therapeutic agent are connected with the micromolecule of removing from blood in a few minutes (but targeting construct) of sending.But at first use the pre-targeting bispecific or the multi-specificity antibody of binding site, free antibodies is removed from circulation, but used the targeting construct then with targeting construct and target antigen.

For example, in Goodwin etc., U.S. Patent No. 4,863,713; Goodwin etc., J.Nucl.Med.29:226,1988; Hnatowich etc., J.Nucl.Med.28:1294,1987; Oehr etc., J.Nucl.Med.29:728,1988; Klibanov etc., J.Nucl.Med.29:1951,1988; Sinitsyn etc., J.Nucl.Med.30:66,1989; Kalofonos etc., J.Nucl.Med.31:1791,1990; Schechter etc., Int.J.Cancer48:167,1991; Paganelli etc., Cancer Res.51:5960,1991; Paganelli etc., Nucl.Med.Commun.12:211,1991; U.S. Patent No. 5,256,395; Stickney etc., Cancer Res.51:6650,1991; Yuan etc., CancerRes.51:3119,1991; U.S. Patent No. 6,077,499,7,011,812,7,300,644,7,074,405,6,962,702,7,387,772,7,052,872,7,138,103,6,090,381,6,472,511,6,962, disclose pre-targeted approach in 702 and 6,962,702, described document is incorporated this paper separately by reference into.

Can pass through: (1) uses bi-specific antibody or antibody fragment to the experimenter; (2) randomly use the removing compositions, and described compositions is removed antibody from circulation to the experimenter; And (3) but use the pre-targeted approach that the targeting construct that contains one or more chelatings or chemically combined therapeutic agent or diagnostic agent provides treatment or diagnosis experimenter's disease or disease to the experimenter.

Therapeutic agent and diagnostic agent

In certain embodiments, but targeted molecular disclosed herein or targeting construct can be connected with one or more therapeutic agents and/or diagnostic agent, for example ¹⁸F.Therapeutic agent is preferably selected from radionuclide, immunomodulator, anti-angiogenic agent, cytokine, chemotactic factor, somatomedin, hormone, medicine, prodrug, enzyme, oligonucleotide, short apoptosis agent, RNA interfering, photosensitive therapeutic agent, cytotoxic agent (can be chemotherapeutics or toxin) and combination thereof.Useful medicine can have the pharmaceutical properties that is selected from antimitotic agent, antikinase agent, alkylating agent, antimetabolite, antibiotic, alkaloid, anti-angiogenic agent, short apoptosis agent and combination thereof.

Useful illustrative drug includes but not limited to 5-fluorouracil, aplidin, triacetyl azauridine (azaribine), Anastrozole (anastrozole), anthracycline antibiotics, bendamustine (bendamustine), bleomycin, bortezomib (bortezomib), bryostatin-1, busulfan (busulfan), calicheamycin (calicheamycin), camptothecine, carboplatin (carboplatin), 10-hydroxycamptothecine, carmustine, celecoxib (Celebrex), Chlorambucil (chlorambucil), cisplatin (cisplatin) (CDDP), the Cox-2 inhibitor, irinotecan (irinotecan) (CPT-11), SN-38, carboplatin, cladribine (cladribine), camptothecin derivative (camptothecan), cyclophosphamide, cytosine arabinoside (cytarabine), dacarbazine (dacarbazine), docetaxel (docetaxel), radiating streptozotocin D (dactinomycin), daunorubicin (daunorubicin), amycin, 2-pyrrolin amycin (2P-DOX), cyano group-morpholine amycin, the amycin glucuronic acid, epirubicin (epirubicin) glucuronic acid, estramustine (estramustine), epipodophyllotoxin, the estrogen receptor bonding agent, etoposide (VP16), the etoposide glucuronic acid, the phosphoric acid etoposide, floxuridine (floxuridine) (FUdR), 3', 5'-O-two oleoyls-FudR (FUdR-dO), fludarabine (fludarabine), flutamide (flutamide), inhibition of farnesyl protein transferase, gemcitabine (gemcitabine), hydroxyurea, idarubicin (idarubicin), ifosfamide (ifosfamide), the altheine enzyme, lenalidomide (lenolidamide), folinic acid, lomustine (lomustine), chlormethine (mechlorethamine), melphalan (melphalan), neck base purine (mercaptopurine), 6-neck base purine, methotrexate, mitoxantrone (mitoxantrone), mithramycin (mithramycin), mitomycin (mitomycin), rice holder load (mitotane), nvelbine (navelbine), nitroso ureas, plicamycin (plicomycin), procarbazine, paclitaxel (paclitaxel), pentostatin (pentostatin), PSI-341, raloxifene (raloxifene), semustine (semustine), streptozotocin (streptozocin), zitazonium (tamoxifen), taxol, temozolomide (temazolomide) (the moisture form of DITC), trans platinum (transplatinum), Thalidomide (thalidomide), thioguanine (thioguanine), thiophene is for sending (thiotepa), teniposide (teniposide), topotecan (topotecan), uracil mustard (uracil mustard), vinorelbine (vinorelbine), vinblastine (vinblastine), vincristine and vinca alkaloids.

Useful toxin can comprise ricin, abrin, alpha toxin, saporin (saporin), ribonuclease (RNA enzyme) (for example ranpirnase), DNA enzyme I, Staphylococcus aureus enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, diphtherotoxin, Pseudomonas exotoxin and pseudomonas endotoxin.

Useful immunomodulator can be selected from cytokine, stem cell factor, lymphotoxin, Hemopoietic factor, colony stimulating factor (CSF), interferon (IFN), erythropoietin, thrombopoietin and combination thereof.Useful especially be lymphotoxin (for example tumor necrosis factor (TNF)), Hemopoietic factor (for example interleukin (IL)), colony stimulating factor (for example granulocyte-colony stimulating factor (G-CSF) or CM-CSF (GM-CSF)), interferon (for example interferon-' alpha ' ,-β or-γ) and stem cell factor (stem cell factor that for example is called " the S1 factor ").Comprise growth hormone in the cytokine, for example human growth hormone, N-methionyl human growth hormone and bovine growth hormone; Parathyroid hormone; Thyroxine; Insulin; Proinsulin; Relaxin; Relaxation precipitinogen; Glycoprotein hormones, for example follicle stimulating hormone (FSH), thyrotropin (TSH) and lutropin (LH); The liver somatomedin; Prostaglandin, fibroblast growth factor; Prolactin antagonist; Galactagogin; OB albumen; Tumor necrosis factor-alpha and-β; Miao Le Shi manages inhibiting substances (mullerian-inhibiting substance); Mice promoting sexual gland hormone related peptides; Inhibin; Activin; VEGF; Integrin; Thrombopoietin (TPO); Nerve growth factor, for example NGF-β; PDGF; Transforming growth factor (TGF), for example TGF-α and TGF-β; Insulin like growth factor-1 and II; Erythropoietin (EPO); Osteogenesis induction factor; Interferon, for example interferon-' alpha ', β and γ; Colony stimulating factor (CSF), for example macrophage-CSF (M-CSF); Interleukin (IL), for example IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, test kit part or FLT-3, angiostatin (angiostatin), thrombospondin (thrombospondin), Endostatin (endostatin), tumor necrosis factor and LT.

Useful chemotactic factor comprises RANTES, MCAF, MIP1-α, MIP1-β and IP-10.

The useful radiosiotope of treatment pathological tissues is included but not limited to ¹¹¹In, ¹⁷⁷Lu, ²¹²Bi, ²¹³Bi, ²¹¹At, ⁶²Cu, ⁶⁷Cu, ⁹⁰Y, ¹²⁵I, ¹³¹I, ³²P, ³³P, ⁴⁷Sc, ¹¹¹Ag, ⁶⁷Ga, ¹⁴²Pr, ¹⁵³Sm, ¹⁶¹Tb, ¹⁶⁶Dy, ¹⁶⁶Ho, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁸⁹Re, ²¹²Pb, ²²³Ra, ²²⁵Ac, ⁵⁹Fe, ⁷⁵Se, ⁷⁷As, ⁸⁹Sr, ⁹⁹Mo, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹⁴³Pr, ¹⁴⁹Pm, ¹⁶⁹Er, ¹⁹⁴Ir, ¹⁹⁸Au, ¹⁹⁹Au and ²¹¹Pb.For Auger emitter (Auger emitter), the decay energy of therapeutic radiation nucleic is preferably at 20-6, in the scope of 000keV, preferably in the scope of 60-200keV, for beta emitter at 100-2, in the 500keV scope, and for alpha emitter 4,000-6 is in the 000keV scope.The highest decay energy of the nucleic of useful emission beta-particle is preferably 20-5,000keV, and 100-4 more preferably, 000keV, and most preferably be 500-2,500keV.The radionuclide of Yin Exie emitted particle decay on the preferred general also.For example, Co-58, Ga-67, Br-80m, Tc-99m, Rh-103m, Pt-109, In-111, Sb-119,1-125, Ho-161, Os-189m and Ir-192.The decay energy of the nucleic of useful emission beta-particle is preferred＜and 1,000keV, more preferably＜100keV, and most preferably＜70keV.Also on the preferred general because of generating the radionuclide of alpha-particle decay.This type of radionuclide includes but not limited to: Dy-152, At-211, Bi-212, Ra-223, Rn-219, Po-215, Bi-211, Ac-225, Fr-221, At-217, Bi-213 and Fm-255.The decay energy of the radionuclide of useful emission alpha-particle is preferably 2,000-10, and 000keV, more preferably 3,000-8,000keV, and most preferably be 4,000-7,000keV.Useful potential radiosiotope in addition comprises ¹¹C, ¹³N, ¹⁵O, ⁷⁵Br, ¹⁹⁸Au, ²²⁴Ac, ¹²⁶I, ¹³³I, ⁷⁷Br, ^113mIn, ⁹⁵Ru, ⁹⁷Ru, ¹⁰³Ru, ¹⁰⁵Ru, ¹⁰⁷Hg, ²⁰³Hg, ^121mTe, ^122mTe, ^125mTe, ¹⁶⁵Tm, ¹⁶⁷Tm, ^I68Tm, ¹⁹⁷Pt, ¹⁰⁹Pd, ¹⁰⁵Rh, ¹⁴²Pr, ¹⁴³Pr, ¹⁶¹Tb, ¹⁶⁶Ho, ¹⁹⁹Au, ⁵⁷Co, ⁵⁸Co, ⁵¹Cr, ⁵⁹Fe, ⁷⁵Se, ²⁰¹Tl, ²²⁵Ac, ⁷⁶Br, ¹⁶⁹Yb etc.

Therapeutic agent can comprise photosensitizer or dyestuff.Fluorescent composition (for example fluorescent dye and other chromogen) or the dyestuff porphyrin of visible light sensitivity (for example to) have been used for by suitable light being pointed to damage check and treatment damages.In treatment, this is called light radiation, phototherapy or photodynamic therapy.See (editors) such as Jori, PHOTODYNAMIC THERAPY OF TUMORS AND OTHER DISEASES (Libreria Progetto1985); Van den Bergh, Chem.Britain (1986), 22:430.And, made monoclonal antibody and photosensitive dye coupling to realize phototherapy.See Mew etc., J.Immunol. (1983), 130:1473; The same, Cancer Res. (1985), 45:4380; Oseroff etc., Proc.Natl.Acad.Sci.USA (1986), 83:8744; The same, Photochem.Photobiol. (1987), 46:83; Hasan etc., Prog.Clin.Biol.Res. (1989), 288:471; Tatsuta etc., Lasers Surg.Med. (1989), 9:422; Pelegrin etc., Cancer (1991), 67:2529.

Corticosteroid hormone can increase the effectiveness of other chemotherapeutics, and therefore, usually uses corticosteroid hormone in therapeutic alliance.Prednisone and dexamethasone are the example of corticosteroid hormone.

In certain embodiments, anti-angiogenic agent comes in handy, for example angiostatin, baculostatin, blood vessel energy chalone (canstatin), the mammary gland silk presses down albumen (maspin), anti-plgf (PlGF) peptide and antibody, anti-angiogene factor antibody (for example anti-VEGF and anti-PlGF), anti-Flk-1 antibody, anti-Flt-1 antibody and peptide, anti-Kras antibody, anti-cMET antibody, anti-MIF (macrophage migration inhibition factor) antibody, the laminin peptide, the fibronectin peptide, plasminogen activator inhibitor, tissue inhibitor of metalloproteinase, interferon, interleukin 12, IP-10, Gro-β, thrombospondin, the 2-methoxyestradiol, proliferin (proliferin) associated protein, the Methanamide triazole, CM101, marimastat (Marimastat), many sulfur pentosan (pentosan polysulphate), angiopoietin-2 (angiopoietin-2), interferon-' alpha ', Antibiotic TAN 420F (herbimycin A), PNU145156E, 16K prolactin antagonist fragment, linomide (Linomide), Thalidomide, pentoxifylline (pentoxifylline), genistein (genistein), TNP-470, Endostatin, paclitaxel, Kistin (accutin), angiostatin, cidofovir (cidofovir), vincristine, bleomycin, AGM-1470, platelet factor or minocycline (minocycline).

Other useful therapeutic agent comprises oligonucleotide, and especially preferred pin is to oncogene and oncogene product, for example antisense oligonucleotide of bcl-2.

Diagnostic agent is preferably selected from radionuclide, radiopaque contrast medium, paramagnetic ion, metal, fluorescent labeling, chemiluminescent labeling, acoustic contrast agent and photosensitizer.This type of diagnostic agent is well-known and can use known any this type of diagnostic agent.The limiting examples of diagnostic agent can comprise radionuclide, for example ¹⁸F, ⁵²Fe, ¹¹⁰In, ¹¹¹In, ¹⁷⁷Lu, ¹⁸F, ⁵²Fe, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁷Ga, ⁶⁸Ga, ⁸⁶Y, ⁹⁰Y, ⁸⁹Zr, ^94mTc, ⁹⁴Tc, ^99mTc, ¹²⁰I, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^154-158Gd, ³²P, ¹¹C, ¹³N, ¹⁵O, ¹⁸⁶Re, ¹⁸⁸Re, ⁵¹Mn, ^52mMn, ⁵⁵Co, ⁷²As, ⁷⁵Br, ⁷⁶Br, ^82mRb, ⁸³Sr or other γ-, β-or positron-emitter.

Useful paramagnetic ion comprises chromium (III), manganese (II), ferrum (III), ferrum (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) or erbium (III).The metal contrast agent can comprise lanthanum (III), gold (III), plumbous (II) or bismuth (III).

Acoustic contrast agent can comprise liposome, for example inflates liposome.Radiopaque diagnostic agent can be selected from chemical compound, barium compound, gallium compound and thallium compound.Known diversified fluorescent labeling in this area includes but not limited to Fluorescein isothiocyanate, rhodamine, phycoerythrin (phycoerytherin), phycocyanin (phycocyanin), allophycocyanin (allophycocyanin), o-phthalaldehyde(OPA) and fluorescamine (fluorescamine).Useful chemiluminescent labeling can comprise luminol (luminol), different luminol (isoluminol), fragrant acridinium ester, imidazoles, acridinium salt or oxalate.

Application process

Can prepare ¹⁸The molecule that tried of F or other diagnostic agent or therapeutic agent labelling comprises one or more pharmaceutically compositionss of suitable excipient, one or more other compositions or these some combinations with acquisition.These can realize dosage with the preparation pharmaceutically useful by known method, thereby with one or more excipient that pharmaceutically are fit to active component (that is labelled molecule) are merged into mixture.The sterile phosphate buffer is an example of the excipient that pharmaceutically is fit to.Other suitable excipient is that those skilled in the art are well-known.See, for example, Ansel etc., PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY SYSTEMS, the 5th edition (Lea and Febiger1990) and Gennaro (editor), REMINGTON'S PHARMACEUTICAL SCIENCES, the 18th edition (Mack Publishing Company1990) and revised edition thereof.

The preferred route of administration of compositions described herein is the parenteral injection.Injection can be in subcutaneous, intramuscular, intravenous, intra-arterial, intralymphatic, sheath or intracavity.Unite pharmaceutically acceptable excipient, compositions is mixed with the unit dose injection form, for example solution, suspension or emulsion.Nontoxic and the non-therapeutic of this type of excipient itself.The example of this type of excipient is saline, Ringer's mixture (Ringer's solution), glucose solution and Hank solution (Hank's solution).Also can use non-water excipient, for example fixed oil and ethyl oleate.Preferred excipient is 5% glucose in saline.Excipient can contain a small amount of additive, for example strengthens the material of isotonicity and chemical stability, comprises buffer and antiseptic.Also contained other application process, comprised Orally administered.

The compositions that comprises labelled molecule of preparation can be used for intravenous through (for example) fast injection or lasting infusion and use.The compositions that is used to inject can for example present with unit dosage forms in ampoule or multi-dose container with the antiseptic that adds.Compositions also can be for example forms such as suspension, solution or emulsion in oiliness or aqueous vehicles, and can contain preparaton, for example suspending agent, stabilizing agent and/or dispersant.Alternatively, compositions can be powder type so that before use with being fit to vehicle, for example aseptic apirogen water is restored.

Compositions can be used in solution.The pH of solution should be at pH5-9.5, in the scope of preferred pH6.5-7.5.Its preparation should have the pharmaceutically acceptable buffer of being fit to, for example in phosphate, TRIS (methylol) aminomethane HCl or the citrate etc.Buffer concentration should be in the scope of 1-100mM.Obtain solution also can contain the salt that concentration is 50-150mM, for example sodium chloride or potassium chloride.Also can comprise effective amount of stabilizer, for example the salt of mannitol, trehalose, Sorbitol, glycerol, albumin, globulin, detergent, gel, protamine or protamine.Can or pass through other parenteral route through subcutaneous, intravenous, intramuscular to the administration compositions.And, can inject by lasting infusion or by single or multiple and use.

For example in pre-targeting technology, when using bi-specific antibody, for the dosage of human administration of antibodies will be different according to for example factors such as patient's age, body weight, height, sex, general health and medical history.Usually, and though depend on the circumstances, use higher or than low dosage, but for the purpose of imaging, need be provided at bi-specific antibody dosage in about 1mg to 200mg scope as the single venoclysis for receptor.Usually, and though depend on the circumstances, use higher or than low dosage, but for the adult, need provide dosage in every square metre of body surface area of about 10mg or the 17-18mg antibody scope for receptor.Though can use higher or than low dosage, the example of the dosage of the bi-specific antibody that can use to the human experimenter for the imaging purpose is 1-200mg, more preferably 1-70mg, most preferably 1-20mg.The dosage of therapeutic bi-specific antibody can be higher, for example 1-200,1-100,100-1000,100-500,200-750mg or between any scope.

Usually, labelled molecule dosage to be administered will be according to factors such as for example patient's age, body weight, height, sex, general health and medical histories and is different.Preferably, use the labelled molecule of Sa to the patient.For ¹⁸Using of F labelled molecule, dosage can be measured by millicurie.Be used for ¹⁸The typical range of F imaging research will be 5-10mCi.

Using of peptide

Each embodiment of claimed method and/or compositions can relate to one or more labelling peptides of the experimenter of giving to be administered.Can use by any approach known in the art, include but not limited in oral, nose, cheek, suction, rectum, vagina, part, original position, Intradermal, subcutaneous, intramuscular, intraperitoneal, intra-arterial, the sheath or intravenous injection.For example, when in pre-targeted approach, using the labelling peptide, preferably use described peptide through intravenous.

In certain embodiments, available one or more substituting linking groups, for example CH ₂-NH, CH ₂-S, CH ₂-CH ₂, CH=CH, CO-CH ₂, CHOH-CH ₂Connect Deng displacement standard peptide bond.The method well-known (for example, Hruby, 1982, the Life Sci31:189-99 that prepare peptide mimics; Holladay etc., 1983, Tetrahedron Lett.24:4401-04; Jennings-White etc., 1982, Tetrahedron Lett.23:2533; Almquiest etc., 1980, J.Med.Chem.23:1392-98; Hudson etc., 1979, Int.J.Pept.Res.14:177-185; Spatola etc., 1986, Life Sci38:1243-49; U.S. Patent No. 5,169,862,5,539,085,5,576,423,5,051,448,5,559,103).Compare with its peptide analogues, peptide mimics can show stronger stability in vivo and/or absorb.

Alternatively, can use N-end and/or C-end capization to make stabilized peptide to hinder the exopeptidase activity.For example, can use amidated peptide capping C-end and can be by the acetylation capping N-end of peptide.For example, also can make the peptide cyclisation with the retardance exopeptidase by forming cyclic amides, disulphide, ether, sulfide etc.

Also can particularly, carry out stabilized peptideization by with the natural L-aminoacid of D-aminoacid replacement in the position of known endopeptidase effect.Be known in the art endopeptidase combination and cutting sequence and described preparation and used peptide to incorporate the amino acid whose method of D-into that (for example, U.S. Patent No. 7,172,751,7,521,416 and 7,776,311, its embodiment part is separately incorporated this paper by reference into).In certain embodiments, can be by prepare Orally administered peptide and/or protein jointly with protease and/or peptidase inhibitors.

Morbid state

In preferred embodiments, the peptide of labelling, protein and/or antibody are to the imaging of cancer or treat useful.The example of cancer includes but not limited to cancer, lymphoma, blastoma, sarcoma and leukemia or lymph malignant tumor.Below pointed out more specifically example and comprising of this type of cancer: squamous cell carcinoma (for example epithelium squamous cell carcinoma), pulmonary carcinoma (comprises small cell lung cancer, nonsmall-cell lung cancer, adenocarcinoma of lung and lung squamous cancer), peritoneal cancer, hepatocarcinoma, gastric cancer or stomach cancer (comprising human primary gastrointestinal cancers), cancer of pancreas, glioblastoma, cervical cancer, ovarian cancer, hepatocarcinoma, bladder cancer, hepatoma, breast carcinoma, colon cancer, rectal cancer, colorectal cancer, carcinoma of endometrium or uterus carcinoma, salivary-gland carcinoma, kidney or renal carcinoma, carcinoma of prostate, carcinoma vulvae, thyroid carcinoma, hepatocarcinoma, anus cancer, carcinoma of penis and head and neck cancer.Term " cancer " (for example comprises primary malignant tumor cell or tumor, the subject inner cell is not moved to the primary malignant tumor cell or the tumor at the position except that original malignant tumor or original tumor locus) and second malignant neoplasm cell or tumor are (for example, by transfer, second malignant neoplasm cell or tumor that malignant cell or tumor cell migration produce to the Secondary cases position that is different from original tumor locus).

Other example of cancer or malignant tumor includes but not limited to: the children acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, adult's (constitutional) hepatocarcinoma, adult's (constitutional) hepatocarcinoma, adult's acute lymphoblastic leukemia, become human acute myeloid leukemia, adult's lymphogranulomatosis (Hodgkin's Disease), adult's Hodgkin lymphoma (Hodgkin's Lymphoma), adult lymphoid cellularity leukemia, adult's non-Hodgkin lymphoma, become the human primary liver cancer, adult's soft tissue sarcoma, the AIDS lymphoma of being correlated with, the AIDS associated malignancies, anus cancer, astrocytoma, cancer of biliary duct, bladder cancer, osteocarcinoma, the brain stem glioma, cerebroma, breast carcinoma, renal pelvis and carcinoma of ureter, central nervous system's (constitutional) lymphoma, central nervous system lymphoma, cerebellar astrocytoma, big cerebral astrocytoma, cervical cancer, child's (constitutional) hepatocarcinoma, child's (constitutional) hepatocarcinoma, the children acute lymphoblastic leukemia, the children acute marrow series leukemia, child's brain stem glioma, child's cerebellar astrocytoma, the big cerebral astrocytoma of child, child's extracranial germ cell tumor, child's lymphogranulomatosis, child's Hodgkin lymphoma, child's hypothalamus and visual pathway glioma, child's lymphoblastic leukemia, Children Medulloblastoma, Non-Hodgkin Lymphoma in Children, child's pinus and curtain are gone up original neuroectodermal tumors, child's primary hepatocarcinoma, child's rhabdomyosarcoma, child's soft tissue sarcoma, children's vision approach and hypothalamus glioma, chronic lymphocytic leukemia, chronic granulocytic leukemia, colon cancer, cutaneous T cell lymphoma, the endocrine islet-cell carcinoma, carcinoma of endometrium, ependymoma, epithelial cancer, esophageal carcinoma, ewing's sarcoma (Ewing's Sarcoma) and related neoplasms, the exocrine pancreas cancer, the extracranial germ cell tumor, the outer sexual cell tumor of gonad, cholangiocarcinoma, cancer eye, women's breast carcinoma, familial splenic anemia (Gaucher's Disease), carcinoma of gallbladder, gastric cancer, the gastrointestinal carcinoid tumor, gastrointestinal tumor, germinoma, gestational trophoblastic tumor, hairy cell leukemia, head and neck cancer, hepatocarcinoma, lymphogranulomatosis, Hodgkin lymphoma, hypergammaglobulinemia, hypopharynx osteocarcinoma, intestinal cancer, the ophthalmic melanoma, islet-cell carcinoma, the islet cells cancer of pancreas, Kaposi sarcoma (Kaposi's Sarcoma), renal carcinoma, laryngeal carcinoma, lip and oral cancer, hepatocarcinoma, pulmonary carcinoma, lymphocytosis sexually transmitted disease (STD) disease, macroglobulinemia, cancer of male breast, malignant mesothe, malignant thymoma, medulloblastoma, melanoma, mesothelioma, the cervical metastasis scale cancer that primary tumor is not clear, former cervical metastasis scale cancer, the cervical metastasis scale cancer, multiple myeloma, multiple myeloma/plasma cell tumor, myelodysplastic syndrome, myelocytic leukemia, marrow series leukemia, myeloproliferative disease, nasal cavity and nasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, trimester of pregnancy non-Hodgkin lymphoma, non-melanoma skin cancer, nonsmall-cell lung cancer, former the cervical metastasis scale cancer that kitchen range is not clear, the oropharynx cancer, bone/malignant fibrous sarcoma, osteosarcoma // malignant fibrohistiocytoma, osteosarcoma/malignant fibrous histiocytoma of bone, epithelial ovarian cancer, ovarian germ cell tumors, the low potential malignant tumor of ovary, cancer of pancreas, paraproteinemia, purpura, parathyroid carcinoma, carcinoma of penis, pheochromocytoma, pituitary tumor, plasma cell tumor/multiple myeloma, primary central nervous system lymphoma, primary hepatocarcinoma, carcinoma of prostate, rectal cancer, renal cell carcinoma, renal pelvis and carcinoma of ureter, retinoblastoma, rhabdomyosarcoma, salivary-gland carcinoma, the sarcoidosis sarcoma, Sezary syndrome (Sezary Syndrome), skin carcinoma, small cell lung cancer, carcinoma of small intestine, soft tissue sarcoma, neck squamous cell carcinoma, gastric cancer, original neuroderm and pinus tumor on the curtain, t cell lymphoma, carcinoma of testis, thymoma, thyroid carcinoma, renal pelvis and transitional cell carcinoma of ureter, divide a word with a hyphen at the end of a line renal pelvis and carcinoma of ureter, trophoblastic tumor, ureter and renal pelvis cell carcinoma, carcinoma of urethra, uterus carcinoma, sarcoma of uterus, cancer of vagina, visual pathway and hypothalamus glioma, carcinoma vulvae, Fahrenheit macroglobulinemia (Waldenstrom's Macroglobulinemia), wilms' tumor (Wilms'Tumor) and be arranged in any other hyperplasia disease except that neoplasia of above listed tract.

This paper describes and claimed method and composition can be used for treating pernicious or worsen preceding condition of illness.Under known or the situation of the previous progress of suspection for neoplasia or cancer, especially, occurred by hypertrophy, conversion or more particularly, the non-tumor cell that abnormal development is formed has shown this type of purposes when growing, and (Robbins and Angell are seen in the comment of this type of misgrowth situation, Basic Pathology, the 2nd edition, W.B.Saunders Co., Philadelphia, 68-79 page or leaf (1976)).

Abnormal development usually is the omen of cancer, and mainly finds in epithelial cell.This is the most chaotic form of non-growth of tumour cell, involves the forfeiture of individual cells uniformity and cell framework orientation.Abnormal development appears in characteristic when having chronic stimulation or inflammation.Can detected abnormal development disease include but not limited to anhidrotic ectodermal dysplasia, the antarafacial dysplasia, asphyxiating thoracic dysplasia, atriodigital dysplasia, broncho-pulmonary dysplasia, brain development is bad, the cervix uteri dysplasia, chondroectodermal dysplasia, cleidocranial dysplasia, congenital ectodermal dysplasia, the skull DD, cranium wrist metatarsal hypoplasia, craniometaphyseal dysplasia, the dentin dysplasia, DD, ectodermal dysplasia, enamel development is bad, the dysplasia of brain eye, half limb epiphyseal dysplasia, multiple epiphyseal dysplasia, chondrodysplasia punctata, epithelial dysplasia, face refers to that genital development is bad, familial fibrous dysplasia of jaw, familial white folded dysplasia of mucous membrance, fibromuscular dysplasia, fibrous dysplasia, florid osseous dysplasia, heritability kidney retinal dysplasia, the perspiration ectoderm is grown, hypohidrotic ectodermal dysplasia, the lymphopenia thymic hypoplasia, mammary gland dysplasia, faciomandibular dysostosis, metaphyseal dysplasia, cover end Buddhist nun dysplasia (Mondini dysplasia), single fibrous dysplasia of bone, the mucous epithelium dysplasia, multiple epiphyseal dysplasia, OAVD, oculodentodigital dysplasia, eye-atelorachidia, the odontogenic dysplasia, ophthalmomandibulomelic dysplasia, the periapical cemental dysplasia, the boniness fibrodysplasia, the pseudoachondroplasia spondylo epiphyseal dysplasia, retinal dysplasia, look every dysplasia, vertebra epiphyseal dysplasia and ventricular radial dysplasia.

The other pre-tumprigenicity disease that can detect and/or treat includes but not limited to optimum proliferative disorder sexually transmitted disease (STD) disease (for example, benign tumor, Fibrocystic disease shape, tissue hypertrophy, polyp intestinal, polyp of colon and esophagus abnormal development), leukoplakia, seborrheic keratosis, bowen (Bowen's disease), farmersskin (Farmer's Skin), solar cheilitis and solar keratosis.

Other hyperplasia disease, disease and/or condition of illness include but not limited to malignant tumor progress and/or transfer and associated conditions, for example leukemia (comprises that acute leukemia (for example, acute lymphoblastic leukemia, acute myeloid leukemia (comprises myeloblast, promyelocyte, Myelomonocyte, mononuclear cell and erythroleukemia)) and chronic leukemia (for example chronic myeloid (granulocyte) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphoma (for example, lymphogranulomatosis and Fei Huoqijinshi disease), multiple myeloma, the Fahrenheit macroglobulinemia, heavy chain disease and solid tumor, include but not limited to sarcoma and cancer, for example fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, You Wenshi tumor (Ewing's tumor), leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basaloma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cancer of biliary duct, choriocarcinoma, spermocytoma, the embryo cancer, wilms' tumor, cervical cancer, tumor of testis, pulmonary carcinoma, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

But more than the exemplary condition of illness of listed imaging, diagnosis and/or treatment unrestricted.The technical staff will recognize, become known for antibody, antibody fragment or the targeting peptide of multiple condition of illness, for example autoimmune disease, graft versus host disease, organ-graft refection, cardiovascular disease, neurodegenerative diseases, metabolic disease, cancer, infectious disease and hyperplasia disease.

Exemplary autoimmune disease comprises acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigoid, juvenile diabetes, purpura,Henoch-Schonlein, post-streptococcal infection nephritis, erythema nodosum, high iS-One arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, goodpasture syndrome, thromboangiitis obliterans, sjogren syndrome, primary biliary cirrhosis, struma lymphomatosa, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, wegener granulomatosis, membranous nephropathy becomes, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis and fibrosing alveolitis.

In one embodiment, pharmaceutical composition of the present invention can be used for treatment and suffers from metabolic disease (for example amyloidosis) or neurodegenerative diseases, for example Alzheimer, amyotrophic lateral sclerosis (ALS), Parkinson's disease, hungtington's chorea, olivopontocerebellar atrophy, multiple system atrophy, progressive supranuclear plasy, the diffusivity lewy body disease, corticobasal degeneration, progressive familial myoclonic epilepsy, striatonigral degeneration, dystonia musculorum deformans, familial tremor, twitch obscene words comprehensive (Gilles de la Tourette syndrome) or Hallervorden Spatz (Hallervorden-Spatz disease).

Ba Piniu resistance monoclonal antibody is in the clinical trial of treatment of alzheimer.Other antibody that proposition is used for Alzheimer comprises Alz50 (Ksiezak-Reding etc., 1987, J BiolChem263:7943-47), Luo Shi monoclonal antibody and Su Lan pearl monoclonal antibody (solanezumab).Available directed against amyloid-beta protein antibodies on the market, for example MM26-2.1.3 (MILLIPORE), ab2539 (ABCAM), 10H3 (PIERCE) and NBP1-44048 (NOVUSBIOLOGICALS) and useful to imaging/detection Alzheimer.Anti-CD74 antibody, for example Mi Lazhu monoclonal antibody (milatuzumab) also detects useful to Alzheimer.Reported infliximab, anti-TNF-Alpha antibodies makes amyloid plaque reduce and strengthen identification.Proposed the international antibody of preserving the mutation body SOD1 that hybridoma cell line (registration number No.ADI-290806-01, ADI-290806-02, ADI-290806-03) that unit preserves generates of Canada is used for the treatment of ALS, Parkinson's disease and Alzheimer (seeing U.S. Patent Application Publication No.20090068194).Proposed anti-cd 3 antibodies is used for the treatment of type 1 diabetes (Cernea etc., 2010, Diabetes Metab Rev26:602-05).In addition, pharmaceutical composition of the present invention can be used for treatment and suffers from immune disorder disease, for example graft versus host disease or organ-graft refection's experimenter.

In preferred embodiments, but the disease of the compositions of instructions for use protection and method treatment comprises the cardiovascular diseases, for example fibrin clot, atherosclerosis, myocardial ischemia and infraction.The proteic antibody of known fiber (for example, scFv (59D8); T2G1s; MH1) and in clinical trial as the developer that described grumeleuse and pulmonary infarction are appeared, and Anti granulocyte antibody, for example MN-3, MN-15, anti-NCA95 and anti-CD15 antibody, but targeting myocardial infarction and myocardial ischemia.(see, for example, U.S. Patent No. 5,487,892,5,632,968,6,294,173,7,541,440, embodiment part is separately incorporated this paper by reference into).(for example, hLL1) antibody can be used for the targeting atheromatous plaque for anti-macrophage, anti-low density lipoprotein, LDL (LDL) and anti-CD74.Ratified the auxiliary treatment prevention of restenosis that is used for percutaneous coronary intervention (pci) and unstable angina pectoris of abciximab (anti-glycoprotein iib/iiia) (Waldmann etc., 2000, Hematol1:394-408).Reported anti-cd 3 antibodies reduce development of atherosclerosis and progress (Steffens etc., 2006, Circulation114:1977-84).Reported with sealing MIF Antybody therapy and impelled the atherosclerotic lesion of foundation disappear (Sanchez-Madrid and Sessa, 2010, Cardiovasc Res86:171-73).The antibody of antioxidation LDL also impels the Fadeaway of Atherosclerosis (Ginsberg, 2007, J Am Coll Cardiol52:2319-21) of foundation in mouse model.Confirm anti-ICAM-1 antibody reduced the ischemic cell injury behind the cerebral artery occlusion in the rat body (Zhang etc., 1994, Neurology44:1747-51).The monoclonal antibody of commercially available human leucocyte antigen by: with the anti-T cell monoclonal of the bonded OKT of normal T lymphocyte antibody (can from Ortho Pharmaceutical Company obtain); The ATCC registration number is the monoclonal antibody that the hybridoma of HB44, HB55, HB12, HB78 and HB2 generates; G7E11, W8E7, NKP15 and GO22 (Becton Dickinson); NEN9.4 (New England Nuclear); And FMC11 (Sera Labs) representative.At Knight, Semin.Nucl.Med. contains the description of the antibody of antifibrin and platelet antigen among the 20:52-67 (1990).

The usage flag molecular imaging

The method of usage flag molecular imaging is well-known in this area, and can any this type of known method all can be with disclosed herein ¹⁸The F labelled molecule is used together.See, for example, U.S. Patent No. 6,241,964,6,358,489,6,953,567 and laid-open U.S. Patents Shen Qing Publication No.20050003403,20040018557,20060140936, embodiment part is separately incorporated this paper by reference into.See Page etc., Nuclear Medicine And Biology, 21:911-919,1994 equally; Choi etc., Cancer Research55:5323-5329,1995; Zalutsky etc., J.Nuclear Med., 33:575-582,1992; Woessner etc., Magn.Reson.Med.2005,53:790-99.

In certain embodiments, for example can use the U.S. Patent No. 6,126,916,6,077,499,6 of incorporating this paper separately by reference into, 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, the method of describing in 101,827 and 4,735,210 is used ¹⁸The F labelled molecule is normal or pathological tissues and organ imaging.This type of imaging can be passed through ¹⁸The suitable targeted molecular of the direct labelling of F, or undertaken by pre-targeted imaging method, as (2007, Update Cancer Ther.2:19-31) such as Goldenberg; Sharkey etc. (2008, Radiology246:497-507); Goldenberg etc. (2008, J.Nucl.Med.49:158-63); Sharkey etc. (2007, Clin.Cancer Res.13:5777s-5585s); McBride etc. (2006, J.Nucl.Med.47:1678-88); Goldenberg etc. (2006, J.Clin.Oncol.24:823-85), see U.S. Patent Publication No.20050002945,20040018557,20030148409 and 20050014207 equally, incorporate this paper separately by reference into.

Method with labelling peptide or MAb diagnosing image is well-known.For example, in the immunoscintigraphy technology, in gamma-radiation labelled with radioisotope part or antibody and introducing patient body.Use γ phase radioisotopic position of machine testing gamma-radiation and distribution.See, for example, Srivastava (editor), RADIOLABELED MONOCLONAL ANTIBODIES FOR IMAGING AND THERAPY (Plenum Press1988), Chase, " Medical Applications of Radioisotopes; " in REMINGTON'S PHARMACEUTICAL SCIENCES, the 18th edition, Gennaro etc. (editor), 624-652 page or leaf (Mack Publishing Co., 1990), and Brown, " Clinical Use of Monoclonal Antibodies; " in BIOTECHNOLOGY AND PHARMACY227-49, Pezzuto etc. (editor) (Chapman﹠Hall1993).The also preferred radionuclide (PET isotope) that uses the emission positron, for example energy is 511keV, for example ¹⁸F, ⁶⁸Ga, ⁶⁴Cu and ¹²⁴I.Can be this type of radionuclide imaging by well-known PET scanning technique.

Test kit

Each embodiment can relate to the test kit of the component that contains the pathological tissues that is fit to the imaging of usage flag chemical compound, diagnosis and/or treatment patient.Exemplary kit can contain antibody, antibody fragment or fusion rotein, for example described herein in pre-targeted approach useful bi-specific antibody.But other component can comprise the targeting construct that uses with this type of bi-specific antibody.In preferred embodiments, but the targeting construct in advance with can be used for being connected Al ¹⁸F complex or have different metal ¹⁸The chelation group of F complex is puted together.Alternatively, but can be the targeting construct and preload ¹⁸F can bonded with it aluminum or another kind of metal.Also have in other alternate embodiment, but consider that the targeting construct can different therapeutic agents with one or more and/or diagnostic agent connection.

Be not used for sending if prepare the compositions that contains for using component, for example, can will be able to be included by the device of some other approach delivery of agents box components by oral delivery through digestive tract.One class be fit to be used, and for example the device sent of parenteral is injected to the intravital syringe of experimenter for being used for compositions.Also can use suction apparatus for some application.

Can be with reagent constituents packaging together or be divided into two or more containers.In some embodiments, described container can be the bottle that the sterile freeze-drying preparation that is fit to restorative compositions is housed.Test kit can be equipped with one or more buffer that are fit to restore and/or dilute other reagent.Spendable other container includes but not limited to pouch, pallet, box, pipe etc.Can be with reagent constituents packing and aseptic being kept in the described container.The another kind of component that can comprise is the description of human test kit for its use.

Embodiment

Embodiment 1. peptide IMP272's ¹⁸The F labelling

Preparation and usefulness ¹⁸Article one peptide of F labelling is IMP272:DTPA-Gln-Ala-Lys (HSG)-D-Tyr-Lys (HSG)-NH ₂(SEQ ID NO:3).

Regulate pH in acetate buffer solution-1.509g acetic acid is diluted in～160mL water and by adding 1M NaOH, be diluted to the 0.1M solution of 250mL then with preparation pH4.03.

Aluminum acetate buffer solution-by AlC1 with 0.1028g ₃Hexahydrate is diluted in the 42.6mL DI water and prepares aluminum solutions.4mL aluminum solutions aliquot is mixed so that 2mM Al to be provided stock solution with the 0.1M NaOAc solution of 16mL, pH4.

The IMP272 acetate buffer solution-with peptide, 0.0011g, 7.28 * 10 ^-7Mol IMP272 is dissolved in the 0.1M pH4 acetate buffer solution of 364 μ L to obtain 2mM peptide stock solution.

The F-18 labelling of IMP272-3 μ L aluminum stock solution aliquots are placed REACTI-VIAL ^TMIn and with 50 μ L ¹⁸The IMP272 solution of F (pressing sample) and 3 μ L mixes.Heated solution 15min and in the heat block of 110 ° of C by the reversed-phase HPLC analysis.HPLC follows the tracks of (not shown) and shows 93% ¹⁸F is free and 7% combine with peptide.In reaction, add the IMP272 solution of 10 μ L and heating and analyze (not shown) once more again by reversed-phase HPLC.The HPLC tracing display has 8% on voidage ¹⁸F and 92% active substance is connected with peptide.At room temperature hatch residue peptide solution～1h, then by the reversed-phase HPLC inspection with 150 μ L PBS.The HPLC (not shown) shows 58% ¹⁸F not in conjunction with and 42% still be connected with peptide.Data show, when mixing with phosphate ¹⁸F-Al-DTPA complex potentially unstable.

The peptide IMP272 of embodiment 2. other metals of usefulness ¹⁸The F labelling

With 3 μ L metal stock solution aliquots (6 * 10 ^-9Mol) place the polypropylene conical flask and with 75 μ L ¹⁸F (pressing sample) mixes, and at room temperature hatches～2min, then with the 2mM (4 * 10 of 20 μ L in 0.1MNaOAc pH4 buffer ^-8Mol) IMP272 solution mixes.Heated solution 15min and in 100 ℃ heat block by the reversed-phase HPLC analysis.With indium (24%), gallium (36%), zirconium (15%), lutecium (37%) and yttrium (2%) labelling IMP272 (not shown).These results prove, ¹⁸F metal marker technology is not limited to the aluminum part, but also can utilize other metal.The metal ligand difference can be utilized the combination of different chelating moieties with optimization F-18-metal conjugate.

Embodiment 3. serum are stable ¹⁸Generation and the use of F labelling peptide IMP449

IMP449

NOTA-ITC benzyl-D-Ala-D-Lys (HSG)-D-Tyr-D-Lys (HSG)-NH ₂(SEQID NO:4)

On the Sieber amide resin, by in the indicated order following aminoacid addition being prepared peptide IMP448D-Ala-D-Lys (HSG)-D-Tyr-D-Lys (HSG)-NH to resin ₂(SEQID NO:5): Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, cutting Aloc, Fmoc-D-Tyr (But)-OH, Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, cutting Aloc, Fmoc-D-Ala-OH cuts Fmoc at last to prepare required peptide.Make then described peptide from resin cutting and by the HPLC purification to generate IMP448, make IMP448 and ITC-benzyl NOTA coupling then.

Make IMP448 (0.0757g, 7.5 * 10 ^-5Mol) with 0.0509g (9.09 * 10 ^-5Mol) ITC benzyl NOTA mixes and is dissolved in the 1mL water.In peptide/NOTA agitating solution, slowly add Anhydrous potassium carbonate (0.2171g) then.The pH that adds all carbonate afterreaction solution is 10.6.Making reactant at room temperature stir spends the night.Use 1M HC1 cessation reaction behind the 14h carefully and pass through the HPLC purification to obtain the IMP449 of 48mg.

IMP449's ¹⁸The F labelling

With IMP449 (0.002g, 1.37 * 10 ^-6Mol) be dissolved among 686 μ L (2mM peptide solution) 0.1MNaOAc (pH4.02).2mM Al solution and 15 μ Ls, the 1.3mCi of 3 μ L in the pH4 acetate buffer ¹⁸F mixes.Make described solution mix and be heated to 105 ℃ then and reach 15min with the 2mM IMP449 solution of 20 μ L.The reversed-phase HPLC analysis shows 35% active substance (t _R～10min) be connected with peptide and in column void volume eluting 65% active substance (3.1min, not shown), show that most of active substance does not associate with peptide.Thick labelling mixture (5 μ L) mixes with the PHS and hatches under 37 ℃.Get an aliquot behind the 15min and analyze by HPLC.HPLC shows that 9.8% active substance still is connected (descending from 35%) with peptide.Get another aliquot behind the 1h and analyze by HPLC.HPLC shows that 7.6% active substance still is connected (descending from 35%) with peptide, substantially the same with 15min tracking (data not shown goes out).

High dose ¹⁸The F labelling

IMP449 further studies have shown that with purification, ¹⁸F labelling peptide is at 37 ℃ of human serum camber stable (91%, not shown) 1h and partially stabilized in 37 ℃ of human serums (76%, not shown) 4h at least at least.Carried out other research, wherein in the presence of ascorbic acid, prepared IMP449 as stabilizing agent.In those research (not shown), behind 37 ℃ of following 4h, metal- ¹⁸F-peptide complex does not demonstrate detectable decomposition in serum.Injection ¹⁸Find behind the F labelling peptide 30min that the mice urine contains with peptide bonded ¹⁸The F (not shown).These results prove, and are disclosed herein ¹⁸F labelling peptide is being used for ¹⁸Show enough stability under the condition in the approximation of F imaging research.

Because the thiourea that the IMP449 peptide contains the radiolysis sensitivity connects, so observe several products by RP-HPLC.Yet, when in reactant mixture, adding ascorbic acid, significantly reduced the by-product that generates.

Embodiment 4. is used for by pre-targeting preparation ¹⁸The DNL construct of F imaging

DNL can be used for preparing the dimer that comprises almost any antibody or its fragment or other effect part, trimer, the tetramer, six aggressiveness etc.For some preferred embodiment, can generate the IgG antibody, Fab fragment or other protein or the peptide that are the fusion rotein that contains DDD (dimerization and docking structure territory) or AD (anchoring structure territory) sequence.The Fab-DDD fusion rotein that can be by making first antibody and the Fab-AD fusion rotein of second antibody are combined to form bi-specific antibody.Alternatively, can prepare the construct that makes IgG-AD fusion rotein and the combination of Fab-DDD fusion rotein.For ¹⁸The purpose that F detects, can make contain with the antibody for the treatment of the antigenic binding site that imaging target tissue (for example tumor) is relevant or fragment and bond- ¹⁸But the hapten on the targeting construct that F can be attached thereto, for example second antibody of IMP449 or fragment combination.Use bi-specific antibody (DNL construct) to the experimenter, make circulating antibody from blood, remove and be positioned target tissue, and add ¹⁸But the targeting construct of F labelling also combines so that imaging with local antibody.

Can be every kind of Fab or the IgG fusion rotein is researched and developed independent transgenic cell line.In case generate, but if need or be kept in the cell culture supernatant liquid with regard to the purification module.After the generation, can make any DDD ₂-fusion rotein module and corresponding A-fusion rotein module combinations is to generate bispecific DNL construct.For dissimilar constructs, can utilize different AD or DDD sequence.Following DDD sequence is based on the DDD part of PKA RII α, and the AD sequence is based on the AD part (Alto etc., the Proc.Natl.Acad.Sci.USA.2003 that optimize synthetic AKAP-IS sequence; 100:4445).

DDD1:SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?IDNO：6)

DDD2:CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?IDNO：7)

AU1:QIEYLAKQIVDNAIQQA(SEQ?ID?NO:8)

AD2:CGQIEYLAKQIVDNAIQQAGC(SEQ?ID?NO:9)

Plasmid vector pdHL2 has been used to generate many antibody and based on the construct of antibody.See Gillies etc., J Immunol Methods (1989), 125:191-202; Losman etc., Cancer (Phila) (1997), 80:2660-6.The bicistronic mRNA mammalian expression vector instructs the synthetic of IgG heavy chain and light chain.For many different IgG-pdHL2 constructs, carrier sequence major part is identical, only there are differences in variable domains (VH and VL) sequence.Use biology tool well known by persons skilled in the art, these IgG expression vectors can be converted into Fab-DDD or Fab-AD expression vector.Be to generate the Fab-DDD expression vector, replace the coded sequence of hinge, CH2 and the CH3 domain of heavy chain with the sequence of preceding 44 residues (being called DDD1) of preceding 4 residues, 14 residue Gly-Ser connexons and the people RII α of coding hinge.For generating the Fab-AD expression vector, with preceding 4 residues, the 15 residue Gly-Ser connexons of coding hinge be called the sequence of hinge, CH2 and CH3 domain of synthetic AD (being called AD1) the sequence displacement of the 17 residues IgG of AKAP-IS, use bioinformatics and peptide array technique to generate described sequence and confirm with very high affinity (0.4nM) in conjunction with RII α dimer.See Alto etc., Proc.Natl.Acad.Sci., U.S.A (2003), 100:4445-50.

Two kinds of shuttle vectors have been designed to promote the conversion of IgG-pdHL2 carrier as described below to Fab-DDD1 or Fab-AD1 expression vector.

The preparation of CH1

By PCR, use the pdHL2 plasmid as template amplification CH1 domain.Left side PCR primer is by upstream (5') end of CH1 domain with for forming in the SacII restriction endonuclease site of the 5' of CH1 coded sequence.The right side primer is made up of the sequence of preceding 4 residues of coding hinge, is four glycine and serine subsequently, and latter two codon (GS) comprises the BamHI restriction site.With 410bp pcr amplification primer be cloned into pGemT PCR cloning vehicle (Promega, Inc.) in and the insert of T7 (5') direction of screening and cloning.

Synthetic duplex oligonucleotide is the DDD1 aminoacid sequence of 11 residues of connexon peptide with the coding front, and preceding two codons comprise Bam HI restriction site.Termination codon and EagI restriction site invest the 3' end.Below show the encoded polypeptides sequence, the DDD1 sequence is added with underscore.

GSGGGGSGGGG SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA(SEQ?ID?NO：10)

Synthesize (Sigma Genosys) and be combined in its 3' end 30 eclipsed two kinds of oligonucleotide of base pair (being called RIIA1-44 top and RIIA1-44 bottom), 154 base pairs in center to comprise 174bp DDD1 sequence are arranged.Make oligonucleotide annealing and carry out primer extension reaction with the Taq polymerase.After the primer extension, by the pcr amplification duplex.Be cloned into amplimer among the pGemT and screen the insert of T7 (5') direction.

Synthetic duplex oligonucleotide is the AD1 aminoacid sequence of 11 residues of connexon peptide with the coding front, and preceding two codons comprise Bam HI restriction site.Termination codon and EagI restriction site invest the 3' end.Below show the encoded polypeptides sequence, the AD1 sequence is added with underscore.

GSGGGGSGGGGS QIEYLAKQTVDNAIQQA(SEQ?ID?NO:11)

Two overlapping oligonucleotide of complementation of the synthetic and above peptide sequence of annealing coding are called AKAP-IS top and AKAP-IS bottom.By the pcr amplification duplex.Be cloned into amplimer in the pGemT carrier and screen the insert of T7 (5') direction.

Connect DDD1 and CH1

With BamHI and NotI restriction endonuclease 190bp fragment, be connected to same loci among the CH1-pGemT then to generate shuttle vector CH1-DDD1-pGemT from pGemT excision encoding D DD1 sequence.

Connect AD1 and CH1

Contain the 110bp fragment of AD1 sequence with BamHI and NotI from the pGemT excision, be connected to same loci among the CH1-pGemT then to generate shuttle vector CH1-AD1-pGemT.

CH1-DDD1 or CH1-AD1 are cloned in the carrier based on pdHL2

Use this modularized design, CH1-DDD1 or CH1-AD1 can be incorporated in any IgG construct in the pdHL2 carrier.By from pdHL2, removing SacII/EagI restricted fragment (CH1-CH3) and using the CH1-DDD1 that from each pGemT shuttle vector, excises or the SacII/EagI fragment displacement of CH1-AD1, replace whole heavy chain constant domain in order to last arbitrary construct.

The structure of h679-Fd-AD1-pdHL2

H679-Fd-AD1-pdHL2 is the expression vector that is used to generate h679Fab, and wherein AD1 is by the carboxyl terminal coupling of the CH1 domain of the flexible Gly/Ser peptide spacer that is made of 14 amino acid residues and Fd.The carrier based on pdHL2 that will contain the h679 variable domains by the CH1-AD1 fragment displacement SacII/EagI fragment of excising from the CH1-AD1-SV3 shuttle vector with SacII and EagI is converted into h679-Fd-AD1-pdHL2.

The construct of C-DDD1-Fd-hMN-14-pdHL2

C-DDD1-Fd-hMN-14-pdHL2 is the dimeric expression vector of stablizing that is used to generate the fusion rotein C-DDD1-Fab-hMN-14 that comprises two copies, and wherein DDD1 is connected with hMN-14Fab at the carboxyl terminal of CH1 by flexible peptide spacer.By with the digestion of SacII and EagI restriction endonuclease removing the CH1-CH3 domain and to insert the CH1-DDD1 fragment from the excision of CH1-DDD1-SV3 shuttle vector with SacII and EagI, plasmid vector hMN14 (the I)-pdHL2 that is used to generate hMN-14IgG is converted into C-DDD1-Fd-hMN-14-pdHL2.

Utilized constructed generation to be used for multiple known antibodies, for example the plasmid of the Fab of hLL1, hLL2, hPAM4, hR1, hRS7, hMN-14, hMN-15, hA19, hA20 and other antibody expression.Usually, the antibody variable region coded sequence is present in the pdHL2 expression vector and transforms expression vector as mentioned above to generate AD or DDD fusion rotein.Can be by each AD fusion rotein, comprise the segmental AD of Fab or the DDD fusion rotein of any this antibody-like with the combination of the approximate ratio of two DDD fusion rotein, comprise the segmental trimerization DNL of a Fab construct of two the Fab fragments and the second antibody of first antibody with generation.

C-DDD2-Fd-hMN-14-pdHL2

C-DDD2-Fd-hMN-14-pdHL2 is the expression vector that is used to generate C-DDD2-Fab-hMN-14, and C-DDD2-Fab-hMN-14 has the DDD2 dimerization and the docking structure territory sequence of carboxyl terminal that invests the Fd of hMN-14 by 14 amino acid residue Gly/Ser peptide connexons.Excretory fusion rotein is made of the hMN-14Fab of 2 identical copies that the noncovalent interaction by the DDD2 domain keeps together.

Synthetic preparation comprises two overlapping complementary oligonucleotides of the residue 1-13 of the coded sequence of part connexon peptide and DDD2.Make oligonucleotide annealing and use the T4PNK phosphorylation, produce being connected respectively compatible outstanding of the DNA that digests through restriction endonuclease BamHI and PstI in that 5' and 3' are terminal.

Make duplex DNA and be connected, to generate shuttle vector CH1-DDD2-pGemT by digesting the shuttle vector CH1-DDD1-pGemT for preparing with BamHI and PstI.With SacII and EagI from CH1-DDD2-pGemT excision 507bp fragment and be connected by digesting IgG expression vector hMN14 (the I)-pdHL2 for preparing with SacII and EagI.Final expression construct is called C-DDD2-Fd-hMN-14-pdHL2.Utilized similar technique to generate the segmental DDD2 fusion rotein of Fab of many different humanized antibodies.

H679-Fd-AD2-pdHL2

Design h679-Fab-AD2 with as B with as the C-DDD2-Fab-hMN-14 of A pairing.H679-Fd-AD2-pdHL2 is the expression vector that is used to generate h679-Fab-AD2, and h679-Fab-AD2 has the AD2 anchoring structure territory sequence that invests the carboxyl terminal of CH1 domain by 14 amino acid residue Gly/Ser peptide connexons.AD2 had a cysteine residues and another cysteine residues is arranged thereafter before the sequence of the anchoring structure territory of AD1.

Following through engineering approaches expression vector.Synthetic preparation comprises two overlapping complementary oligonucleotides (AD2 top and AD2 bottom) of the coded sequence of AD2 and part connexon peptide.Make oligonucleotide annealing and use the T4PNK phosphorylation, produce being connected respectively compatible outstanding of the DNA that digests through restriction endonuclease BamHI and SpeI in that 5' and 3' are terminal.

Make duplex DNA and be connected, to generate shuttle vector CH1-AD2-pGemT by digesting the shuttle vector CH1-AD1-pGemT for preparing with BamHI and SpeI.From shuttle vector, excise the 429 base pair fragments that contain CH1 and AD2 coded sequence and be connected to SacII and EagI restriction endonuclease by in the h679-pdHL2 carrier with same enzyme digestion preparation.Final expression vector is h679-Fd-AD2-pdHL2.

The generation of embodiment 5.TF2DNL construct

By making C-DDD2-Fab-hMN-14 and h679-Fab-AD2 reaction obtain to be called the trimerization DNL construct of TF2.Following generation productive rate〉TF2 of 90% pilot scale batch.By the 1.4:1 mol ratio protein L-purification C-DDD2-Fab-hMN-14 (200mg) is mixed with h679-Fab-AD2 (60mg).In the PBS that contains 1mM EDTA, total protein concentration is 1.5mg/ml.Subsequent step involves TCEP reduction, HIC chromatography, DMSO oxidation and IMP291 affinity chromatography.Add before the TCEP, SE-HPLC does not show a ₂Any evidence that b forms.Adding 5mM TCEP causes rapidly forming and the consistent a of 157kDa protein that has estimated diadactic structure ₂The b complex.By IMP291 affinity chromatography purification TF2 to homogeneity (not shown) almost.IMP291 contains the haptenic synthetic peptide of the bonded HSG of 679Fab (Rossi etc., 2005, Clin Cancer Res11:7122s-29s).To IMP291 not the SE-HPLC analytical proof of bound fraction from product, removed a ₄, a ₂With free κ chain (not shown).

Non-reduced SDS-PAGE analytical proof, there is (not shown) in most of TF2 as the approaching big covalent structure of relative mobility and IgG.The additional strip carrying means, disulphide forms incomplete (not shown) under experiment condition.Reduction SDS-PAGE confirmation, when the band of the composition polypeptide of only representing TF2 is obvious, the relevant (not shown) of conspicuous any additional band in the non-reduced gel with product.It is 156 that MALDI-TOF mass spectral analysis (not shown) shows unimodal, 434Da, the calculated mass of TF2 (157,319Da) 99.5% in.

Measure the functional of definite TF2 by BIACORE.TF2, C-DDD1-hMN-14+h679-AD1 (are used as non-covalent a ₂The control sample of b complex) or C-DDD2-hMN-14+h679-AD2 (as unreduced a ₂Control sample with the b component) dilution is 1 μ g/ml (total protein) and passes through the fixed sensing chip with HSG.To the response of TF2 is about 2 times of two kinds of control samples, shows in the control sample that only the h679-Fab-AD component can combine and stay on the sensing chip with sensing chip.Inject WI2IgG subsequently, the anti-id AB of hMN-14 proves that only TF2 has the tight associating DDD-Fab-hMN-14 component with h679-Fab-AD shown in the additional signal response.By WI2 be fixed on sensing chip on TF2 in conjunction with the response unit that causes that two complete function binding sites that provide with each subunit by C-DDD2-Fab-hMN-14 additionally are provided is corresponding.This confirms (not shown) by TF2 in conjunction with the segmental ability of two Fab of WI2.

The generation of embodiment 6.TF10DNL construct

The trimerization TF10DNL construct of the C-DDD2-Fab-hPAM4 that uses similar approach to generate to comprise 2 copies and the C-AD2-Fab-679 of 1 copy.Use and openly be used to generate aforesaid (anti-CEA) ₂The method of * anti-HSG bsAb TF2 generates TF10 bispecific ([hPAM4] ₂* h679) antibody.The TF10 construct has 2 humanization PAM4Fab and 1 humanization 679Fab.

The independent 2 kinds of fusion rotein (hPAM4-DDD2 and h679-AD2) of expressing in the myeloma cell of stable transfection.Merge tissue culture's supernatant, produce the hPAM4-DDD2 of 2 times of molar excess.Under slight reducing condition, at room temperature use 1mM reduced glutathione incubation reaction mixture 24h.After the reduction, finish the DNL reaction by the mild oxidation of using the 2mM oxidized glutathione.Carry out affinity chromatography by use with the bonded IMP291-affigel resin of h679Fab high specific and separate TF10.

The sequence variants of embodiment 7.DNL

In some preferred embodiment, incorporate the aminoacid sequence that AD in cytokine-MAb DNL complex and DDD sequence comprise AD1 as discussed above or AD2 and DDD1 or DDD2 into.Yet, in alternate embodiment, in the structure of DNL complex, can utilize the sequence variants of AD and/or DDD part.For example, 4 people PKA DDD sequence variants are only arranged, corresponding with the DDD part of PKA RI α, RII α, RI β and RII β.RII α DDD sequence is the basis of above disclosed DDD1 and DDD2.Below show described 4 people PKA DDD sequences.The DDD sequence is represented the residue 12-61 of residue 1-44, RI α of residue 1-44, RII β of RII α and the residue 13-66 of RI β.(sequence of attention DDD1 is only modified a little by people PKA RII α DDD part and is formed.)

PKA?RIα

SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK(SEQ?IDNO:12)

PKA?RIβ

SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILA(SEQID?NO:13)

pKA?RIIα

SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ(SEQ?ID?NO:14)

PKA?RIIβ

SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER(SEQ?ID?NO:15)

Structure-the functional relationship of AD and DDD domain has become research theme.(see, for example, Burns-Hamuro etc., 2005, Protein Sci14:2982-92; Carr etc., 2001, J Biol Chem276:17332-38; Alto etc., 2003, Proc Natl Acad Sci USA100:4445-50; Hundsrucker etc., 2006, Biochem J396:297-306; Stokka etc., 2006, Biochem J400:493-99; Gold etc., 2006, Mol Cell24:383-95; Kinderman etc., 2006, Mol Cell24:397-408, its full text is separately incorporated this paper by reference into.)

For example, Kinderman etc. (2006, Mol Cell 24:397-408) checked that the crystal structure of AD-DDD binding interactions and deduction people DDD sequence contain important many conservative amino acid residues in dimer formation or AKAP combination, underline in following SEQ ID NO:6.(see Fig. 1 of Kinderman etc., 2006, incorporate this paper by reference into.) technical staff will recognize, when the sequence variants of design DDD sequence, people will painstakingly avoid changing underlined any residue, and can replace carrying out conservative amino acid for dimerization and AKAP in conjunction with so crucial residue.

SH IQ IPPG LTE LLQG YT VE VLRQQPPD LVEFA VE YFTR LREARA(SEQ?ID?NO:6)

As known in the art, to 20 kinds of common L-amino acid whose each characterized the conservative amino acid replacement.Therefore, the data based on Kinderman (2006) and conservative amino acid replacement have illustrated the potential alternative DDD sequence based on SEQ ID NO:6 in table 2.When design table 2, only considered that the high conservative acidic amino acid replaces.For example, only replace residue,, only use other hydroxyl substituted hydroxy side chain etc. with the residue of the residue replacement with similar size with little side chain with identical charges with charged residue.Because proline is not used other residue substituted prolines to the uniqueness influence of aminoacid secondary structure.Even have this type of conservative to replace, the possible alternative sequence (2 * 3 * 2 * 2 * 2 * 2 * 2 * 2 * 2 * 2 * 2 * 2 * 2 * 2 * 2 * 4 * 2 * 2 * 2 * 2 * 2 * 4 * 2 * 4) more than 20,000,000 also arranged for 44 residue peptide.The technical staff will recognize, by standard technique, for example use commercial peptide synthesizer or well-known side-directed mutagenesis to make up to belong to the alternative kind of almost unlimited amount of the type of DDD part.Can for example described in (2003, Proc Natl Acad Sci USA100:4445-50) such as Alto, easily measure aminoacid replacement by standard in conjunction with mensuration to the bonded influence of AD part.

Conservative amino acid among the table 2.DDD1 (SEQ ID NO:6) replaces.As the disclosed consensus sequence of SEQ IDNO:16

Alto etc. (2003, Proc Natl Acad Sci USA100:4445-50) the proteic AD sequence of various AKAP having been carried out bioinformatic analysis is 0.4nM with the binding constant that designs DDD, is called the RII selectivity AD sequence of AKAP-IS (SEQ ID NO:8).With the AKAP-IS sequential design is AKAP and the bonded peptide antagonists of PKA.In following SEQ IDNO:8, underline for replacing the bonded residue that tends to reduce in the AKAP-IS sequence with DDD.The technical staff will recognize that when the sequence variants of design AD sequence, people will painstakingly avoid changing underlined any residue, and can replace carrying out conservative amino acid for DDD in conjunction with so not crucial residue.The potential conservative amino acid that table 3 shows in the AKAP-IS sequence (AD1, SEQ ID NO:8) replaces, to similar to the replacement of the conservative amino acid shown in the DDD1 (SEQ ID NO:6) in the above table 2.

Even have this type of conservative to replace, the possible alternative sequence (2 * 3 * 2 * 4 * 3 * 2 * 2 * 2 * 2 * 2 * 2 * 4) more than 35,000 also arranged for 17 residue A D1 (SEQ ID NO:8) peptide sequence.Equally, the technical staff can produce, test and use a large amount of kinds of the type that belongs to possibility AD part based on the data of (2003) such as Alto.The Fig. 2 that is to be noted that Alto (2003) illustrates based on actual in conjunction with experiment, is keeping the more substantial potential aminoacid replacement that can carry out in conjunction with the active while of DDD part.

AKAP-IS

QIEYL AKQ IVDN AIQQA(SEQ?ID?NO:8)

Conservative amino acid among the table 3.AD1 (SEQ ID NO:8) replaces.As the disclosed consensus sequence of SEQ IDNO:17

Gold etc. (2006, Mol Cell24:383-95) utilize the SuperAKAP-IS sequence (SEQ ID NO:18) of crystallography and peptide screening research and development to high 5 orders of magnitude of selectivity ratios RI isotype of the RII isotype of PKA.Underlined residue indication strengthens the bonded aminoacid replacement position to the DDD part of RII α with respect to the AKAP-IS sequence.In this sequence, hold the Q residue to be numbered No. 4 residues N-and C-end A residue is No. 20 residues.Can replace with influence is residue 8,11,15,16,18,19 and 20 (Gold etc., 2006) to the residue of the affinity of RII α.Consider that in certain embodiments available SuperAKAP-IS sequence replaces AKAP-IS AD partial sequence with preparation DNL construct.Other alternative sequence that can replace AKAP-IS AD sequence has been shown among the SEQ ID NO:19-21.Replacement with respect to the AKAP-IS sequence is added with underscore.Estimate that the same with the AD2 sequence shown in the SEQ ID NO:9, the AD part also can comprise other N-end residue cysteine and glycine and C-end residue glycine and cysteine.

SuperAKAP-IS

QIEY VAKQIVD YAI HQA(SEQ?ID?NO:18)

Substitute the AKAP sequence

QIEY KAKQIVD HAI HQA(SEQ?ID?NO:19)

QIEY HAKQIVD HAI HQA(SEO?ID?NO:20)

QIEY VAKQIVD HAI HQA(SEQ?ID?NO:2U

Fig. 2 of Gold etc. discloses the proteic additional DDD binding sequence from multiple AKAP, and wherein any sequence all can be used for designing the DNL construct.

Stokka etc. (2006, Biochem J400:493-99) have have also researched and developed AKAP shown in the SEQ IDNO:22-24 and the bonded peptide competitor of PKA.Peptide antagonists called after Ht31 (SEQ ID NO:22), RIAD (SEQ ID NO:23) and PV-38 (SEQ ID NO:24).The Ht-31 peptide shows more high-affinity to the RII isotype of PKA, and RIAD and PV-38 demonstrate more high-affinity to RI.

Ht31

DLIEEAASRIVDAVIEQVKAAGAY(SFQ?ID?NO:22)

RIAD

LEQYANQLADQIIKEATE(SEQ?ID?MO:23)

PV-38

FEELAWKIAKMIWSDVFQQC(SEQ?ID?NO:24)

Hundsrucker etc. (2006, Biochem J 396:297-306) have have also researched and developed other AKAP and the bonded peptide competitor of PKA, and the binding constant of the DDD of the RII form of PKA is low to moderate 0.4nM.The sequence of various AKAP antagonism peptides is provided in the table 1 of Hundsrucker etc., has been replicated in the following table 4.AKAPIS represents synthetic RII subunit binding peptide.The RII binding structural domain of AKAP shown in all other peptides are derived from.

Table 4. AKAP peptide sequence

About AKAP IS sequence (SEQ ID NO:8), below by underlining the residue of pointing out that the proteic AD domain of different AKAP camber is conservative.(2003) such as residue and Alto are observed identical, added C-end alanine residue.(see the Fig. 4 of (2006) such as Hundsrucker, incorporate this paper by reference into.) be the sequence of AKAP-IS, AKAP78-wt-pep, AKAP7 δ-L304T-pep and AKAP7 δ-L308D-pep to the sequence of the extra high peptide antagonists of affinity of RII DDD sequence.

AKAP-IS

QIEYL AKQ IVDN AIQQ A(SEQ?ID?NO:8)

Carr etc. (2001, J Biol Chem 276:17332-38) have checked from people and the proteinic different AKAP of non-human in conjunction with the sequence homology degree between the DDD sequence and identified the conservative residue of topnotch in the seemingly different DDD parts in the DDD sequence.About the people PKA RII α DDD sequence of SEQ ID NO:6, below these residues have been pointed out by underlining.Further pointed out conservative especially residue with italics.(2006) such as described residue and Kinderman propose proteic to combine very important residue overlapping with AKAP, but inequality.The technical staff will recognize, when the sequence variants of design DDD, most preferably avoid changing the most conservative residue (printing in italics), and preferably avoid changing conserved residues (being added with underscore), and be both not underlined the residue that does not also print in italics to have considered the conservative amino acid replacement.

S HIQ IPP GLT ELLQGYTV EVLRQ QPP DLVEFAVE YFTR LR EA RData based on (2001) such as Carr have been shown, to a series of modifications back conservative amino acid replacement of DDD1 (SEQ ID NO:6) sequence in A (the SEQ ID NO:6) table 5.Promptly use a series of still less be substituted sequence, need not undo experimentation, the technical staff just can generate, tests and use and may substitute the DDD partial sequence more than 65,000.The technical staff can easily obtain as above table 2 and disclosed this type of the alternative DDD aminoacid sequence of table 3.

Conservative amino acid among the table 5.DDD1 (SEQ ID NO:6) replaces.As the disclosed consensus sequence of SEQ IDNO:43.

The technical staff will recognize, can utilize these and other aminoacid replacement in DDD or the AD aminoacid sequence, use standard technique and unique normal experiment in this area, generate the alternative kind of the type that belongs to AD or DDD part.

Embodiment 8. uses ¹⁸F labelling peptide in vivo imaging and with ¹⁸F[FDG] relatively

Use in-vivo imaging technology to be used for successfully detecting the relative little tumor of size with bi-specific antibody and the pre-targeting of labelling targeting antibodies.

ACCELL ^TMPurification on the Plus QMALight pillar ¹⁸F.Use 0.4M KHCO ₃Eluting ¹⁸F and the 2mM Al of 3 μ L in the pH4 acetate buffer ³⁺Mix.Then with Al ¹⁸F solution injects ascorbic acid IMP449 labelling bottle and is heated to 105 ℃ and reaches 15min.Cooled reaction solution and mix with 0.8mL DI water.Reaction content is stated from

On the 1cc HLB post and with 2 * 200 μ L1:1EtOH/H ₂The O eluting.Prepare TF2 as mentioned above.TF2 combines with carcinoembryonic antigen (CEA) bivalence and combines with synthetic hapten HSG (histidine-succinyl-glycine) unit price.

Bio distribution and little PET imaging

Be the subcutaneous implantation CCL188 of the 6 week female nude mices of NCr nu-m in age, and LS174T (ATCC, Manassas, VA).When tumor is obviously set up, for pre-targeting animal intravenous injection 162 μ g (～1nmol/0.1mL) TF2 or TF10 (contrast non-targeting three-FabbsMAb), then behind 16-18h, [the Al of intravenous injection～0.1nmol ¹⁸F] and IMP449 (84 μ Ci, 3.11MBq/0.1mL).The control animals received of targeting is not independent in advance for other ¹⁸F (150 μ Ci, 5.5MBq), independent Al ¹⁸The F complex (150 μ Ci, 5.55MBq), independent [Al ¹⁸F] the IMP449 peptide (84 μ Ci, 3.11MBq) or [ ¹⁸F] and FDG (150 μ Ci, 5.55MBq).Use the same day, (Somerset NJ) obtains from IBA Molecular ¹⁸F and [ ¹⁸F] FDG.Accept [ ¹⁸F] FDG the animal fasting whole night, but arbitrarily feedwater.

At radioactive indicator injection back 1.5h, anesthetized animal makes its intracardiac hemorrhage and autopsy.Tissue is with being weighed by the standard diluent of each preparation of product separately and counting.Because ¹⁸The physical half time of F is short, so insert standard substance between every group of tissue from every animal.The counting that uptake ratio in the tissue is expressed as every gram divided by total active substance injected to obtain the injected dose percentage ratio (%ID/g) of every gram.

Carried out two types imaging research.In one group, 3 nude mices that have little LS174T Subcutaneous tumor have accepted to be 135 μ Ci (5MBq; 0.1nmol) pre-targeting [Al ¹⁸F] IMP449, independent [Al ¹⁸F] IMP449 (pre-targeting) or [ ¹⁸F] and FDG (135 μ Ci, 5MBq).2h behind the intravenous injection radioactive indicator uses O ₂/ N ₂The mixture anesthetized animal of O and isoflurane (2%) and

(Siemens PreclinicalSolutions, Knoxville TN) keep warm to animal PET scanner during the enterprising line scanning.

Showed the representative crown cross section (0.8mm is thick) near the plane that is positioned at the tumor center, it is saturated and need not the background adjusting until pixel occurs in any zone of health (except that bladder) to regulate intensity.

In independent Study on dynamic imaging, use O ₂/ N ₂One of the mixture anesthesia of O and isoflurane (2%) has LS174T, has given the nude mice of TF2bsMAb before the 16h, and lying on the back is positioned on the camera pedestal, then intravenous injection 219 μ Ci (8.1MBq) [Al ¹⁸F] IMP449 (0.16nmol).Begin data acquisition behind the 120min immediately.Use OSEM3D/MAP to rebuild scanning.In order to state, for each cross section (crown, sagittal and cross section) showed 5,15,30,60,90 and the time frame that finishes of 120min.For the cross section of containing tumor, it is saturated until occur pixel first in tumor to regulate image intensity at interval at each.It is saturated with the pixel of keeping in the tumor to pass the increase image intensity as required in time.To be adjusted to the intensity identical in the tumor free crown and sagittal cross section that same intervals obtains with the cross section that contains tumor.Activity at the bottom of the uncomfortable abridged edition.

The result

Though it is independent ¹⁸F and [Al ¹⁸F] complex the uptake ratio in a organized way similar, but when complex and IMP449 chelating, find sizable difference (table 6).Uptake ratio in bone is found the most significant difference, wherein non-chelating ¹⁸F in scapula high 60 to nearly 100 times and high in spinal column～200 times.Because it is known ¹⁸F, or even metal-fluoride complex in bone, increase (Franke etc., 1972, Radiobiol.Radiother. (Berlin) 13:533), so expected this distribution.At tumor and intestinal and in muscle and blood, also observe higher uptake ratio.Except that kidney the institute in a organized way in, chelating [Al ¹⁸F] uptake ratio of IMP449 is obviously lower, and the ability that chelate-complex is effectively got rid of in the body by homaluria has been described.

Use the pre-targeting [Al of the anti-CEA bsMAb of TF2 ¹⁸F] IMP449 is transferred in the tumor picked-up thing, makes uptake ratio rise to 6.01 ± 1.72% injected dose/grams from 0.20 ± 0.05% injected dose/gram when 1.5h, and uptake ratio in the normal structure and independent [Al ¹⁸F] IMP449 is similar.For blood, liver, pulmonary and kidney, tumor/non-tumor ratio is respectively 146 ± 63,59 ± 24,38 ± 15 and 2.0 ± 1.0, this moment other tumor/organize ratio 100:1.Though it is independent ¹⁸F and independent [Al ^I8F] uptake ratio in tumor is than chelating [Al ¹⁸F] the IMP449 height, produced the tumor ratio that is respectively 6.7 ± 2.7 and 11.0 ± 4.6 pairs 5.1 ± 1.5, but significantly improved tumor uptake rate and tumor ratio (all P values through pre-targeting＜0.001).

Also with the most frequently used tumor developer of the high tissue of targeting glucose consumption and metabolic activity [ ¹⁸F] FDG compared bio distribution (table 6).In all normal structures except that kidney, [ ¹⁸F] uptake ratio of FDG is slightly higher than [Al ¹⁸F] IMP449.For pre-targeting [Al ¹⁸F] IMP449 and [ ¹⁸F] FDG, the tumor uptake rate is similar, but because in most normal structures [ ¹⁸F] growth of FDG is higher, so have [ ¹⁸F] tumor/non-tumor ratio of FDG is starkly lower than the intravital tumor of pre-targeting animal/non-tumor ratio (all P values＜0.001).

[the Al of table 6. pre-targeting of TF2 in the nude mouse that has LS174T people's colon xenograft ¹⁸F] IMP449 and other contrast ¹⁸The bio distribution of F labelled reagent.For pre-targeting, at injection [Al ¹⁸F] 16h gives animal TF2 before the IMP449.All injections are all used through intravenous.

The injected dose percentage ratio of the every gram of injection back 1.5h (meansigma methods ± SD)

For several animal imagings with the independent [Al of further analysis ¹⁸F] IMP449 the or with [Al of the pre-targeting of TF2 ¹⁸F] IMP449 and [ ¹⁸F] bio distribution of FDG.The still image that 2h begins behind the injection radioactive substance has been proved conclusively and shown the prior structure distributed data (Fig. 1) that almost completely absorbs in kidney.In pre-targeting animal body, be easy to see the 21mg tumor, and given independent [Al ¹⁸F] animal of IMP449 can not positioning tumor, and the kidney picked-up is only arranged.Do not observe the evidence of bone lengthening, show Al ¹⁸F firmly combines with IMP449.This is carrying out Study on dynamic imaging, [Al in the 5min interval in the monitoring 120min ¹⁸F] obtain confirming (Fig. 2) in the pre-targeting animal of another of distribution of IMP449.Mainly be heart, kidney and the picked-up of some livers in the 5min before crown and sagittal slices is presented at, but heart regulating liver-QI dirty work reduces greatly in ensuing 10min, and kidney keep outstanding in whole research process.In whole 120min scanning, in intestinal or bone, there is not active evidence.At first observe the picked-up in the 35mgLS174T tumor when 15min, and during to 30min, most clearly depict signal on background, whole 120min scan period, strong tumor promotion is very outstanding always.

By contrast, from given [ ¹⁸F] still image of animal of FDG shown expection radioactivity pattern (McBride etc., 2006, the J.Nucl.Med.47:1678 in bone, cardiac muscle and the brain of formerly observing; Sharkey etc., 2008, Radiology246:497), have considerable background activity (Fig. 1) in the body.The uptake ratio of organizing of 3 animal in-vivo measurements of autopsy is confirmed to organize in a organized way in institute when quiescent imaging research finishes ¹⁸The much higher (not shown) of F radioactivity.Though in this animal body to [ ¹⁸F] the tumor uptake rate of FDG is than pre-targeting animal height, but the tumor ratio is more conducive to pre-targeting; And intravital residual activity still less, and it is visual to have improved tumor by pre-targeting.

These studies have shown that, only by form then can be fit to chelate in conjunction with and incorporate aluminum-fluoride complex in hapten-peptide into, available ¹⁸The quick labelling of F (total preparation time 60min) is used for the hapten-peptide of pre-targeted imaging.This can be more at large only by making [Al ¹⁸F]-any molecule coupling of subsequent purificn prepares chelate with being connected also with chelating moiety.

This report has been described and has been made ¹⁸F is by bonded direct, the easy and method rapidly of aluminum conjugate and all cpds.When by based on the chelate of NOTA in conjunction with the time, [Al ¹⁸F] peptide is stable in vitro and in vivo.Productive rate is being used routine ¹⁸In the scope that the F labeling method is found.These results have further proved and have used and multiple targeted molecular chelated metal ¹⁸F carries out the feasibility of PET imaging.

Embodiment 9. preparation and use Al- ¹⁸F labelling IMP460

Chemosynthesis IMP460NODA-Ga-D-Ala-D-Lys (HSG)-D-Tyr-D-Lys (HSG)-NH ₂(SEQ IDNO:44).From

Buy the NODA-Ga part and be connected on the peptide synthesizer, as other aminoacid.On the Sieber amide resin, synthesize described peptide: Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, removal Aloc, Fmoc-D-Tyr (But)-OH, Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, removal Aloc, Fmoc-D-Ala-OH and NODA-GA (tBu) with the aminoacid that adds in the following order and other reagent ₃Cut described peptide then and by the HPLC purification to obtain described product.HRMS?C61H92N18O18。

The radioactive label of IMP460

IMP460 (0.0020g) is dissolved among 732 μ L, pH4, the 0.1M NaOAc.Purification as mentioned above ¹⁸F mixes with the glacial acetic acid neutralization and with Al solution.Add 20 μ L peptide solutions and heated solution to 99 then and ℃ reach 25min.Exist then

Purification crude product on the HLB post.[Al ¹⁸F] the labelling peptide is at 1:1EtOH/H ₂In the O post eluent.Reversed-phase HPLC tracing display in the 0.1%TFA buffer, HPLC is clear unimodal at the desired location (not shown) for the labelling peptide.

Embodiment 10.IMP461 and IMP462NOTA-put together the synthetic and labelling of peptide

Prepare the simplest possibility NOTA part (it is synthetic that protection is used for peptide) and incorporate two peptides into and be used for pre-targeting IMP461 and IMP462.

Two-tert-butyl group-NOTA's is synthetic

With NO2AtBu (0.501g, 1.4 * 10 ^-3Mol) be dissolved in the 5mL anhydrous acetonitrile.In solution, add 2-benzyl acetate bromide (0.222mL, 1.4 * 10 ^-3Mol), then add the 0.387g anhydrous K ₂CO ₃Making reactant at room temperature stir spends the night.Filtration and concentrated reaction mixture are to obtain the benzyl ester conjugate of 0.605g (86% productive rate).Then crude product is dissolved in the 50mL isopropyl alcohol, mixes with the 10%Pd/C (under Ar) of 0.2g and place 50psi H ₂Following 3 days.Under vacuum, filter then and enriched product to obtain the required product ESMS MH-415 of 0.462g.

IMP461's is synthetic

On the Sieber amide resin, synthesize described peptide: Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, removal Aloc, Fmoc-D-Tyr (But)-OH, Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, removal Aloc, Fmoc-D-Ala-OH and two-tert-butyl group NOTA-OH with the aminoacid that adds in the following order and other reagent.Cut described peptide then and by the HPLC purification to obtain described product IMP461ESMS MH ⁺1294 (NOTA-D-Ala-D-Lys (HSG)-D-Tyr-D-Lys (HSG)-NH ₂SEQ ID NO:45).

IMP462's is synthetic

On the Sieber amide resin, synthesize described peptide: Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, removal Aloc, Fmoc-D-Tyr (But)-OH, Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, removal Aloc, Fmoc-D-Asp (But)-OH and two-tert-butyl group NOTA-OH with the aminoacid that adds in the following order and other reagent.Cut described peptide then and by the HPLC purification to obtain described product IMP462ESMS MH ⁺1338 (NOTA-D-Asp-D-Lys (HSG)-D-Tyr-D-Lys (HSG)-NH ₂SEQ ID NO:46).

IMP461 and IMP462's ¹⁸The F labelling

Described peptide is dissolved among pH4.13, the 0.5M NaOAc peptide solution with preparation 0.05M, peptide solution is stored in the refrigerator till needs.Be contained in F-18 in the 2mL water and be trapped in

Light,

ACCELL ^TMOn the Plus QMA pillar.0.4M KHCO with 200 μ L ₃Aliquot eluting from the post ¹⁸F.Before adding active substance, heavy carbonate is neutralized to～pH4 by in bottle, adding 10 μ L glacial acetic acid.Remove the purification of 100 μ L ¹⁸F solution aliquot and mix with the 2mM Al of 3 μ L in pH4,0.1M NaOAc.Add 10 μ L (0.05M) peptides and heated solution and reach 15min to～100 ℃.With 700 μ L DI water dilutions crude product mixture and place on the HLB post, and after washing with the 1:1EtOH/H of 2 * 100 μ L ₂The O eluting ¹⁸F is to obtain purification ¹⁸F labelling peptide.

The preparation of embodiment 11.IMP467 and ¹⁸The F labelling

IMP467C-NETA-succinyl-D-Lys (HSG)-D-Tyr-D-Lys (HSG)-NH ₂(SEQ ID NO:47)

Generate tetra-tert C-NETA-succinyl.As Chong etc. (J.Med.Chem.2008,51:118-125) the preparation tert-butyl group 4-[2-(two-(tert-butoxycarbonyl) methyl-3-(4-nitrobenzophenone) propyl group]-7-tert-butoxycarbonyl [1,4,7] three azepines nonane-1-yl.

On the Sieber amide resin, prepare peptide IMP467C-NETA-succinyl-D-Lys (HSG)-D-Tyr-D-Lys (HSG)-NH by in resin, adding following aminoacid in the indicated order ₂(SEQ ID NO:47): Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, cutting Aloc, Fmoc-D-Tyr (But)-OH, Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, cutting Aloc, 4-[two-(tert-butoxycarbonyl methyl) amino)-3-(4-succinoamino phenyl) propyl group]-7-tert-butoxycarbonyl methyl [1,4,7] three azepines nonane-1-yl } tert-butyl acetate.The described peptide of cutting also passes through the RP-HPLC purification to produce the IMP467 of 6.3mg from resin then.By using the thick peptide of high performance liquid chromatography (HPLC) purification of C18 post.

Radioactive label

The 2mM solution of preparation IMP467 in pH4,0.1M NaOAc.By

ACCELL ^TMPlus SEP-

Light QMA pillar eluting 139mCi's ¹⁸F and with the 0.4M KHCO of 1mL ₃Eluting ¹⁸F.IMP467 by HLB RP-HPLC purification labelling.RP-HPLC shows two bimodal eluting (not shown), is considered to Al ¹⁸The diastereomer of F IMP467.In pre-targeting technology discussed below, because Al ¹⁸As if F-chelating agen complex is not the part for the hapten site of antibodies, so the existence of diastereomer does not influence ¹⁸F labelling peptide is to the targeting of pathological tissues.

The comparison of radiolabeled peptides productive rate

When attempting to keep the body internal stability, improving the labelling productive rate, 3 kinds of NOTA derivants (IMP460, IMP461 and IMP467) of synthetic pre-targeting peptide.Certainly, IMP467 almost is 2 times (table 7) of the labelling productive rate of other peptide.All marker research in the table 7 all carry out with the peptide and the aluminum of identical molal quantity.Result shown in the table 7 represents the exemplary indicia experiment to every kind of peptide.

When 40nmol only (than IMP449 low～13 times) with 1.3GBq's (35mCi) ¹⁸When F uses together, IMP467's ¹⁸F labelling productive rate is～70%, shows that this part is to Al ¹⁸The F complex has stronger in conjunction with character.By improving the bonded kinetics of part, productive rate improves (average yield 65-75%) greatly, and with respect to IMP449 (520nmol, 44% productive rate), uses still less the IMP467 of molal quantity (40nmol).

Table 7. contain NOTA different peptides productivity ratio

Peptide	Productive rate
		IMP449	44%
IMP460	5.8%
		IMP461	31%
IMP467	87%

Embodiment 12. influences the productive rate of IMP467 labelling and the factor of stability

Peptide concentration

For checking of the influence of different peptide concentrations, in constant volume (63L), use the Al of constant basis to productive rate ³⁺(6nmol) and ¹⁸F, but not commensurability peptide added, measure Al ¹⁸The binding capacity of F and peptide.Following along with peptide concentration reduces, the productive rate of labelling peptide IMP467 reduces: 40nmol peptide (82% productive rate); 30nmol (79% productive rate); 20nmol (75% productive rate); 10nmol (49% productive rate).Therefore, the amount of peptide 20 and 40nmol between change the productive rate influence of IMP467 very little.Yet, in the labelling mixture, begin to observe productive rate from the 10nmol peptide and descend.

Aluminum concentration

As Al at incremental change ³⁺There is (2mM Al and the maintenance cumulative volume of 0,5,10,15,20 μ L in the pH4 acetate buffer is constant) down, during labelling IMP467, reach 3.5%, 80%, 77%, 78% and 74% productive rate respectively.These results show that (a) is at Al ³⁺When not existing, ¹⁸The non-specific binding of F and this peptide is a bottom line, (b) Al of 10nmol ³⁺Be enough to make maximum ¹⁸F combination, and (c) Al of a large amount more ³⁺Can not reduce combination substantially, show under this peptide concentration, to have enough sequestering powers.

Al ¹⁸The radiolabeled kinetics of F IMP467

Dynamics research shows, is combined in the 5min under 107 ℃ and finishes (5min, 68%; 10min, 61%; 15min, 71%; And 30min, 75%), in the response time that reaches 30min, isolated yield only appropriateness increases.

The IMP467 radioactive label reaction and display of under 50 ℃, carrying out, the combination of being unrealized at a lower temperature.

The influence of pH

The optimum pH of labelling is between 4.3 and 5.5.The productive rate scope is: under pH2.88, and 54%; Under pH3.99,70-77%; Under pH5,70%; Under pH6 41%, under pH7.3,3%.Can be by replacing carbanion from the ion exchange column eluting with eliminating nitrate anion or chloride ion ¹⁸F, eliminate with AlCl ₃With glacial acetic acid eluent is adjusted to the needs of pH4 and quickens described process before mixing.

The high dose radioactive label of IMP467

Make the 2mM Al of 5 μ L ³⁺Stock solution and 50 μ L's ¹⁸F1.3GBq (35mCi) mixes, and then adds the 2mM IMP467 of 20 μ L in 0.1mM, pH4.1 acetate buffer.Reacting by heating solution to 104 ℃ reaches 15min, then as mentioned above on the HLB post purification (～10min), separate 0.68GBq (18.4mCi) purified peptide, the radiochemistry productive rate is 69%, specific activity is 17GBq/ μ mol (460Ci/mmol).Response time is 15min and the purification time is 12min.At 1.3GBq (35mCi) ¹⁸F purification 10min afterreaction begins, so from separating ¹⁸F is 37min to the total time of purification end product, and the productive rate that decay is revised not is 52%.

The human serum stability test

With the HLB purified peptide (～30 μ L) of an aliquot of 200 μ L human serums (before freezing) dilutions and place in 37 ℃ of HPLC sample rooms.Get aliquot and analyze in different time points by HPLC.HPLC analyzes demonstration, 5h at least in 37 ℃ of serum, ¹⁸The very high (not shown) of the stability of F labelling peptide.After in serum, hatching 5h, do not detect ¹⁸F labelling peptide decomposes (not shown).

IMP461 and IMP462 part have two carboxyls that can be used in conjunction with aluminum, and the NOTA part among the IMP467 has 4 carboxyls.Serum stability studies show that, duplicates in vivo under the condition of use, and the complex with IMP467 is stable in serum.Biodistribution research in the body of labelling IMP467 is shown, ¹⁸F-Al labelling peptide is stablized (not shown) under the condition in actual body.

Can by form can with the bonded Al of NOTA part on the peptide ¹⁸The F complex is used ¹⁸F does not need the HPLC purification with high yield and rapid (30min) labelling peptide of specific activity of 17GBq/mol at least.Al ¹⁸F NOTA-peptide is stable in serum and body.The modification of NOTA part can cause productive rate and specific activity to improve, and still keeps Al simultaneously ¹⁸The desired body internal stability of F-NOTA complex, and be connected with the kidney that helps peptide with the hydrophilic connexon and remove.Further, this method has been avoided being usually used in using ¹⁸The drying steps of F labelling peptide.Shown in following embodiment, this new ¹⁸The F labeling method is applicable to the many targeting peptides of labelling.

Al ¹⁸The optimization labelling of F IMP467

Identified ¹⁸The optimal conditions of F labelling IMP467.These optimal conditions are made up of following: with commercial Sterile Saline (pH5-7) eluting ¹⁸The F-fluoride is with the AlCl of 20nmol in the pH4 acetate buffer ₃Mix with 40nmol IMP467, cumulative volume is 100 μ L, heats 102 ℃ and reaches 15min, and carry out SPE and separate.30min process after simple Solid-Phase Extraction (SPE) is separated need not the HPLC purification, obtains high yield (85%) and high specific activity (115GBq/ μ mol) with IMP467 in an independent step. ¹⁸F-IMP467 is stable in PBS or human serum, after in any culture medium, hatching 6h under 37 ℃, ¹⁸F loses 2%.

¹⁸F concentrates and purification

Before the use, the radiochemistry level ¹⁸F needs purification and concentrates.Before the use, we have checked that 4 different SPE purge processes are with processing ¹⁸F.Use prepares by conventional method ¹⁸F carries out most of radioactive label processes.With in the 2mL water ¹⁸F load is to the 0.4M KHCO with 10mL ₃, then wash in advance with 10mL water

Light, Waters Accell ^TMOn the QMA Plus pillar.Will ¹⁸F load to the described pillar after, with the 5mL water washing to remove dissolved all metals and radiometal impurity.Use then～0.4MKHCO of 1mL ₃, divide several parts of eluting isotopes to have the composition of the active substance of maximum concentration with separation.Per 100 μ L solution with in the 5 μ L glacial acetic acid and the eluting composition so that eluent is adjusted to pH4-5.

In the second approach, with 10mL pH8.4,0.5M NaOAc, then use 10mL DIH ₂O washing QMA pillar.Will ¹⁸F load is to the aforesaid post and with 1mL, pH6,0.05M KNO ₃, divide 200 μ L every part of eluting, each part has the 60-70% active substance.Do not need to regulate the pH of this solution.

In the third method,, then use 10mL DIH with 10mL pH8.4,0.5M NaOAc ₂O washing QMA pillar.Will ¹⁸F load is divided 200 μ L every part of eluting to aforesaid post and with 1mL, pH5-7, the commercial normal saline of 0.154M, each part has 80% active substance.Do not need to regulate the pH of this solution.

At last, we have designed a kind of denseer high activity of series connection ion exchange preparation that uses ¹⁸The method of F solution.Briefly, (1.27cm is long, 0.64cmOD) inserts TRICORN with polyethylene tube (Tygon tubing) ^TMAlso fill the AG1-X8 resin of～200 μ L in 5/20 post, the 100-200 order.Use 6mL0.4M K ₂CO ₃, then use 6mL H ₂The O washing resin.Use DIH ₂The O washing

Light Waters ACCELL ^TMPlus CM pillar.Use syringe pump, be contained in the 2mL DI H in the 5mL syringe ₂Thick among the O ¹⁸F slowly flows through CM pillar and TRICORN ^TMPost～5min is then with 6mL DI H ₂O is by two ions binding post washings.At last, divide 50 μ L compositions, force 0.4M K ₂CO ₃Only pass through TRICORN ^TMPost.Usually, in a 50 μ L compositions, 40-60% eluting active substance is arranged.Described composition is collected in the miniature centrifuge tube of 2.0mL self-supporting nut that 5 μ L glacial acetic acid are housed with in and carbonate solution.The eluting bottle that will have maximum active substances then is as reaction bulb.

Embodiment 13. is by adding in the peptide with the aluminum preincubate ¹⁸F carries out labelling

With F-18 success labelling contain and the peptide that is connected with the macro ring NOTA of aluminum complexation (IMP465, NOTA-D-Ala-D-Lys (HSG)-D-Tyr-D-Lys (HSG)-NH ₂) HSG of (SEQ IDNO:48).The IMP465 of use 40nmol incorporates into ¹⁸F is 13.20%.Prepare intermediate peptide IMP461 as mentioned above.Then the IMP461 of 25.7mg is dissolved in 2mL and has added 10.2mg AlCl ₃.3H ₂In the DI water of O and heat 1h to 100 ℃ of gained solution.By the IMP465 of RP-HPLC purification crude product mixture with generation 19.6mg.

For ¹⁸The F labelling heats 50 μ L ¹⁸F solution be [0.702mCi's ¹⁸F] and 20 μ L (40nmol) 2mM IMP465 solution (0.1M NaOAc pH4.18) reaches 17min to 101 ℃.The reversed-phase HPLC analysis shows, 15.38% (the about 8.60min of RT) active substance is connected with peptide and at voidage eluting 84.62% active substance (2.60min) of post.

In testing separately, can improve by changing the amount of adding peptide ¹⁸The yield percentage of F labelling peptide.The yield percentage of observed IMP465 is 0.27% when the 10nmol peptide, is 1.8% and be 49% during at the 40nmol peptide when the 20nmol peptide.

When being exposed to ¹⁸When using aluminum preincubate peptide before the F, IMP467 demonstrates the productive rate higher than IMP461.At room temperature hatch IMP467, freezing then and lyophilizing with aluminum.Add the amount difference of the aluminum that is used for preincubate.

Table 8. adds ¹⁸Use the labelling of the IMP467 of aluminum preincubate before the F

Productive rate is comparable to by adding Al ¹⁸The productive rate that obtains during F complex labelling IMP467.Therefore, by adding to the peptide that has with the bonded aluminum of chelating moiety ¹⁸F carries out ¹⁸The F labelling is to use before adding to chelating moiety ¹⁸The feasible alternative method of F preincubate metal.

Synthetic and the labelling of embodiment 14.IMP468 bombesin

¹⁸The targeting moiety of F labelling is not limited to antibody or antibody fragment, can comprise specificity or selective binding on the contrary and can pass through ¹⁸The morbid state of F PET imaging or other condition of illness are relevant or be used for any molecule of the cell target of its diagnosis.Bombesin is and neuromedin B and homologous 14 amino acid peptides of gastrin releasing peptide, and the tumor marker of cancer (for example pulmonary carcinoma and gastric cancer) and neuroblastoma.Synthetic IMP468 (NOTA-NH-(CH as the bombesin analog ₂) ₇CO-Gln-Trp-Val-Trp-Ala-Val-Gly-His-Leu-Met-NH ₂SEQ ID NO:49) and use ¹⁸The F labelling is with the targeting gastrin releasing peptide receptor.

Use the modification of the synthetic schemes of reporting in the document, synthetic by solid-phase peptide based on Fmoc, synthetic described peptide on the Sieber amide resin (Prasanphanich etc., 2007, PNASUSA104:12463-467).Synthetic difference is, added two-tert-butyl group NOTA part between synthesis stage carrying out peptide on the resin in peptide.

With IMP468 (0.0139g, 1.02 * 10 ^-5Mol) be dissolved in the 0.5M pH4.13NaOAc buffer of 203 μ L.Peptide dissolving when leaving standstill but form gel, so with the 0.5MpH4.13NaOAc buffer of 609 μ L and 406 μ L ethanol dilution peptides to generate 8.35 * 10 ^-3The M peptide solution.Purification on the QMA pillar ¹⁸F also uses 0.4M KHCO ₃, be divided into every part of eluting of 200 μ L, neutralize with 10 μ L glacial acetic acid.Make purification ¹⁸(40 μ L are 1.13mCi) with the 2mM AlC1 of 3 μ L in pH4,0.1M NaOAc buffer for F ₃Mix.To Al ¹⁸Add IMP468 (59.2 μ L, 4.94 * 10 in the F solution ^-7Mol) and place 15min on 108 ℃ of heat blocks.Purification crude product on the HLB post is with the 1:1EtOH/H of 2 * 200 μ L ₂The O eluting is to obtain the purification of 34% productive rate ¹⁸F labelling peptide.

Embodiment 15. uses ¹⁸The bombesin of F labelling is a tumor imaging

Prepare the bombesin derivant (IMP468) that NOTA puts together as mentioned above.Such as (PNAS104:12462-12467,2007) such as Prasanphanich, we begin to test it and block radiolabeled bombesin and the bonded ability of PC-3 cell.Our experiment first is to determine whether but specificity retardance bombesin combines with the PC-3 cell IMP468.We use IMP333 as non-specific contrast.In this experiment, with 3 * 10 ⁶Individual PC-3 cellular exposure in the constant basis of IMP468 that has wherein added incremental change or IMP333 (～50,000cpm) ¹²⁵I-bombesin (Perkin-Elmer).With the scope of 56-0.44nM as inhibition concentration.

The result shows that we can block ¹²⁵I-BBN is with IMP468 but not the combining of control peptide (IMP333) (not shown), thereby proved the specificity of IMP468.Prasanphanich points out the IC to its peptide ₅₀Be 3.2nM, than we IC with the IMP468 discovery ₅₀(21.5nM) low about 7 times.

BBN peptide on sale repeats this experiment on the use market.We are increased to 2nM with retardance with the amount of peptide for inhibiting from 250nM ¹²⁵I-BBN combines with the PC-3 cell.For IMP468 and BBN positive control, we observe closely similar IC ₅₀Value, IC ₅₀Value (35.9nM) is higher than the IC of previous report ₅₀Value (3.2nM), the still IC that reaches near the BBN contrast ₅₀Value (24.4nM).

For checking targeting in the body, checked the intravital Al of male nude mouse that has scPC3 carcinoma of prostate xenograft at us ¹⁸F IMP468 distributes; Independent Al ¹⁸F IMP468 and the Al that blocks with bombesin ¹⁸F IMP468.For radioactive label, 100 ℃ of heating aluminum chloride (10 μ L, 2mM), 51.9mCi ¹⁸The IMP468 (8.45mM is in ethanol/NaOAc) of F (from the QMA pillar), acetic acid and 60 μ L reaches 15min.By reversed-phase HPLC purification reaction mixture.Blending constituent 40 and 41 (3.56,1.91mCi) and be coated onto on the HLB post and be used for solvent exchange.Among the eluted product 800 μ L (3.98mCi) and 910 μ Ci stay on the post.The ITLC that develops in saturated NaCl shows 0.1% not in conjunction with active.

Be 6 tumor-bearing mice injection [Al of one group ¹⁸F] IMP468 (167 μ Ci ,～9 * 10 ^-10Mol) and autopsy behind 1.5h.Using [Al ¹⁸F] 18min injects 100 μ g (6.2 * 10 in 6 mouse veins of another group before the IMP468 ^-8Mol) bombesin.Also 1.5h is second group of autopsy after injection.Data show, [Al ¹⁸F] the selectively targeted tumor of IMP468 (Fig. 3).When at [Al ¹⁸F] when the preceding 18min of IMP468 gives bombesin, the uptake ratio decline (Fig. 3) of peptide.The bio distribution data show, [Al ¹⁸F] the body internal stability of IMP468 continues 1.5h (not shown) at least.

Tumor is big more, [the Al of demonstration ¹⁸F] the IMP468 uptake ratio is high more, may be because in big more tumor the high more (not shown) of expression of receptor.The bio distribution data show, [Al ¹⁸F] the IMP468 cancer target at Prasanphanich etc. to being marked with ⁶⁸(not shown) in the same range as of the identical peptide report of Ga.The result proves, can use in vivo ¹⁸The peptide-labeled method of F is to use the receptor that raises in the targeted molecular target tumor except that antibody.In this case, the IMP468 targeting has utilized native ligand-acceptor interaction.Cancer target is remarkable, and the P value is P=0.0013.Known many this type of ligand-receptors to and any this type of targeting interact all can form and use methods described herein to carry out ¹⁸The basis of F imaging.

Synthetic and the labelling of embodiment 16. Somat analog IMP466

Somat is the another kind of non-antibody targeting peptide useful to the imaging of Somat receptor protein. ¹²³The octreotide of I labelling, the Somat analog has been used to express imaging (for example, Kvols etc., 1993, the Radiology187:129-33 of the tumor of Somat receptor; Leitha etc., 1993, J Nucl Med34:1397-1402).Yet, because ¹²³The I costliness, physical half time is short and be difficult to prepare the radio-labeled chemical compound, so ¹²³I is not widely used in imaging as yet.Preferably as herein described ¹⁸The F labeling method is used to express the imaging of the tumor of Somat receptor.

IMP466NOTA-D-Phe- Cys-Phe-D-Trp-Lys-Thr-Cys-Throl(SEQ?IDNO:50)

By based on the NOTA conjugated derivatives of the standard solid phase peptide of Fmoc synthetic preparation Somat analog octreotide (IMP466) to generate linear peptides.C end Throl residue is Soviet Union's ammonia alcohol.Make the peptide cyclisation whole night by handling with DMSO.With peptide (0.0073g, 5.59 * 10 ^-6Mol) be dissolved in the 0.5M pH4NaOAc buffer of 111.9 μ L with preparation 0.05M IMP466 solution.Solution is passed the formation gel in time, so by adding more 0.5M NaOAc buffer it is diluted to 0.0125M.

With the little column purification of QMA and concentrated ¹⁸F is to provide 200 μ L in 0.4M KHCO ₃In ¹⁸F.With in the 10 μ L glacial acetic acid and heavy carbonate.Make the neutralization of 40 μ L ¹⁸The 2mM AlCl of F eluent aliquot and 3 μ L ₃Mix, then add the 0.0125M IMP466 solution of 40 μ L.Reach 17min at 105 ℃ of heating blends.Then on Waters1cc (30mg) HLB post, by with reaction solution load to post and water (3mL) wash off unconjugated ¹⁸F uses 2 * 200 μ L1:1EtOH water elution radiolabeled peptides and the purification reaction thing then.The productive rate of radiolabeled peptides is 34.6% behind the HLB purification.

The influence of ionic strength

For reducing the ionic strength of reactant mixture, in the labelling mixture, add the acetonitrile (ultimate density: 0-80%) of incremental change.The productive rate of radiolabeled IMP466 increases with the concentration of acetonitrile in the medium.Although volume increases (in 80% acetonitrile in the 500L:0% acetonitrile 200L), acquisition best radioactive label productive rate (98%) in the acetonitrile of ultimate density 80%.In 3 experiments, the radioactive label productive rate scope from 36% to 55% in 0% acetonitrile.

Embodiment 17. usefulness ¹⁸F and ⁶⁸The IMP466 of Ga labelling is the neuroendocrine tumor imaging

Study relatively to use ¹⁸F with ⁶⁸The PET image that the tumor of the Somat analog peptide of Ga labelling and direct targeted expression Somat receptor obtains.

Method

¹⁸ The F labelling-synthetic IMP466 also uses by the modification of the method described among the above embodiment ¹⁸The F labelling.Has 2-6GBq with the metal-free water washing of 3mL ¹⁸The QMA of F (BV Cyclotron VU, Amsterdam, The Netherlands)

The light pillar (Waters, Milford, MA).Use 0.4M KHCO ₃Eluting from pillar ¹⁸F also collects 200 μ L compositions.Is pH4 with the metal-free glacial acetic acid of 10 μ L with the pH regulator of described composition.Add the 2mM AlCl of 3 μ L in 0.1M sodium-acetate buffer (pH4) ₃Then, in 0.5M sodium acetate (pH4.1), add 10-50 μ L IMP466 (10mg/mL).Unless stated otherwise, at 100 ℃ of following incubation reaction mixture 15min.By RP-HPLC purification radiolabeled peptides.Collection contains ¹⁸The composition of F-IMP466 is also used H ₂2 times of O dilutions and at 1-cc Oasis HLB pillar (Waters, Milford MA) go up purification to remove acetonitrile and TFA.Briefly, be coated in described composition on the pillar and use 3mL H ₂O washs described pillar.Use 2 * 200 μ L50% ethanol elution radiolabeled peptides then.In order in the mice body, to inject, dilute described peptide with 0.9%NaCl.Obtain 45, the high specific activity of 000GBq/mmol.

⁶⁸ The Ga labelling-with using the ultrapure HCl of 0.1M (J.T.Baker, Deventer, The Netherlands) from based on TiO ₂1,110MBq ⁶⁸Ge/ ⁶⁸Ga generator (Cyclotron Co.Ltd., Obninsk, Russia) eluting ⁶⁸GaCl ₃Labelling IMP466.IMP466 is dissolved in the 1.0M HEPES buffer (pH7.0).4 volumes of interpolation ⁶⁸Ga eluent (120-240MBq) and reach 20min at 95 ℃ of heating blends.Add then 50mMEDTA to ultimate density be 5mM, do not incorporate into complexation ⁶⁸Ga ³⁺Purification on Oasis HLB pillar ⁶⁸The IMP466 of Ga labelling also uses 50% ethanol elution.

Octanol-water partition coefficient (log P _{Octanol/water} )-for determining the lipotropy of radiolabeled peptides, with about 50, the 000dpm radiolabeled peptides is diluted in the 0.5mL phosphate buffered saline (PBS) (PBS).Add isopyknic capryl alcohol to obtain diphasic system.Behind the stirring system 2min, by centrifugal (100 * g, 5min) be divided into two-layer.From every layer get 3 100L samples and well type gamma counter (Wallac Wizard3 ", Perkin-Elmer, Waltham measures radioactivity in MA).

Stability-in the new human serum of collecting of 500 μ L, hatch 10 μ L ¹⁸The IMP466 of F labelling and under 37 ℃, hatch 4h.The interpolation acetonitrile also stirs the mixture, and follows centrifugal 5min under 1000 * g so that the serum albumin precipitation.Pass through analytically clear liquid of RP-HPLC as mentioned above.

Cell culture-at Du Erbeikeshi MEM (DMEM) (the Gibco Life Technologies that has replenished 4500mg/L D-glucose, 10% (v/v) hyclone, 2mmol/L glutamine, 100U/mL penicillin and 100g/mL streptomycin, Gaithersburg, MD, USA) the middle AR42J pancreas in rat tumor cell line of cultivating.Under 37 ℃ in having 5%CO ₂Wet air in cultured cell.

IC ₅₀ Measure-in the competition combination is measured, use ¹⁹F-IMP466, ⁶⁹Ga-IMP466 or ¹¹⁵In-DTPA-octreotide competition combination ¹¹¹The In-DTPA-octreotide is measured 50% apparent inhibition concentration (IC in conjunction with the Somat receptor on the AR42J cell ₅₀).

By making aluminium fluoride (AlF) solution (the 0.02M AlCl in 0.5M NaAc ₃, pH4 and the 0.1M NaF in 0.5M NaAc pH4) mix with IMP466 and are incorporated in 100 ℃ of heating and reach 15min and form ¹⁹F-IMP466.As mentioned above, on the C-18 post, pass through RP-HPLC purification reaction mixture.

By with Ganite (Fujisawa). (2.3 * 10 ^-8Mol) be dissolved in 10mM NaAc (pH5.5) solution that 30 μ L have mixed 20 μ LIMP466 (1mg/mL) and prepare ⁶⁹Ga-IMP466, and reach 15min 90 ℃ of heating.Use blend sample, need not to be further purified.

By making indium chloride (1 * 10 ^-5Mol) be mixed with the DTPA-octreotide (1mg/mL) of 10 μ L in 50mM NaAc (pH5.5) ¹¹⁵The In-DTPA-octreotide, and under room temperature (RT), hatch 15min.Use this sample, need not to be further purified.Method radioactive label according to the manufacturer ¹¹¹The In-DTPA-octreotide

The AR42J cell is grown to merge on 12 orifice plates and wash 2 times with binding buffer liquid (DMEM) with 0.5% bovine serum albumin.Under RT, in binding buffer liquid, hatch 10min after, add ¹⁹F-IMP466, ⁶⁹Ga-IMP466 or ¹¹⁵The In-DTPA-octreotide, together with trace (10,000cpm) ¹¹¹The In-DTPA-octreotide (radiochemical purity〉95%), the ultimate density scope is 0.1-1000nM.After under RT, hatching 3h, with ice-cold PBS washed cell 2 times.Scrape cell and measure the relevant radioactivity of cell.The internalization that limited extent may occur under these conditions.Therefore we will compete in conjunction with the result who measures and be described as " apparent IC ₅₀" value rather than IC ₅₀With apparent IC ₅₀Reach when being defined as uncontested dose 50% in conjunction with the time peptide concentration.

Biodistribution research-be male BALB/c nude mice (6-8 week) subcutaneous injection 0.2mL (10 on the right side ⁷The AR42J cell suspending liquid of individual cell/mL).Tumor cell inoculation is after about 2 weeks, and when diameter of tumor was 5-8mm, intravenous was used 370kBq ¹⁸F or ⁶⁸The IMP466 of Ga labelling (n=5).For organizing the unmarked IMP466 of 1,000 times of molar excess of (n=5) injection separately.3 injected in mice of a group are the [Al of chelating not ¹⁸F].Injection back (p.i.) 2h passes through CO ₂/ O ₂Suffocate and put to death all mices.Collect Target organ, weigh and in gamma counter, count.Calculate the injected dose percentage ratio (%ID/g) of every gram tissue for each tissue.Zoopery is carried out through local animal welfare committee's approval and according to national legislation.

The PET/CT imaging-for injecting 10MBqAl in the mouse vein with subcutaneous AR42J tumor ¹⁸F-IMP466 or ⁶⁸Ga-IMP466.1h and 2h behind the injection peptide, (Siemens Preclinical Solutions, Knoxville TN) go up the scanning mice, and inherent spatial resolution is 1.5mm (Visser etc., JNM, 2009) at Inveon animal PET/CT scanner.In scanner, animal is placed dorsal position.Obtain the PET emission scan behind the 15min, then carry out CT scan for anatomical reference (spatial resolution 113 μ m, 80kV, 500 μ A).Use 1.2 version Inveon Acquisition Workplace software (Siemens Preclinical Solutions, Knoxville, TN), use has ordered set expectation maximization-3D/ maximum a posteriori probability (OSEM3D/MAP) algorithm of following parameter and rebuilds scanning: matrix 256 * 256 * 159, pixel size 0.43 * 0.43 * 0.8mm ³With previous MAP be 0.5mm.

The result

The influence of buffer-studied the influence of buffer to the labeling effciency of IMP466.By 10mg/mL (7.7mM) IMP466 is dissolved in sodium citrate buffer solution, sodium-acetate buffer, 2-(N-morpholine) ethyl sulfonic acid (MES) or 4-(2-ethoxy)-1-piperazine ethyl sulfonic acid (HEPES) buffer.The molar concentration of all buffer is 1M and pH is 4.1.In the IMP466 of 200 μ g (153nmol), add 100 μ L Al-F-18 (pH4) and under 100 ℃, hatch 15min.Measure radioactive label productive rate and specific activity with RP-HPLC.When using sodium acetate, MES or HEPES, the radioactive label productive rate is respectively 49%, 44% and 46%.In the presence of sodium citrate, do not observe labelling (＜1%).When carrying out labeled reactant in sodium-acetate buffer, the specific activity of preparation is 10,000GBq/mmol, and in MES and HEPES buffer, obtain 20,500 and 16 respectively, the specific activity of 500GBq/mmol.

AlCl ₃ The influence of concentration3 kinds of AlCl in sodium acetate (pH4.1) of-preparation ₃Stock solution: 0.2,2.0 and 20mM.From these solution, add 3 μ L to 200 μ L ¹⁸Among the F to form [Al ¹⁸F].In these samples, add the 153nmol peptide and under 100 ℃, hatch 15min.At 6nmol AlCl ₃Ultimate density under hatch after, the radioactive label productive rate is 49%.Use 0.6nmol AlCl ₃With 60nmol AlCl ₃Hatch and cause the radioactive label productive rate to reduce greatly: be respectively 10% and 6%.

The influence of peptide amount-studied of the influence of peptide amount to labeling effciency.IMP466 is dissolved in the sodium-acetate buffer (pH4.1), concentration be 7.7mM (10mg/mL) and with 38,153 or the IMP466 of 363nmol add 200 μ L[Al to ¹⁸F] (581-603MBq) in.The radioactive label productive rate increases with the peptide amount.Under 38nmol, radioactive label productive rate scope is 4-8%, and under 153nmol, gain in yield radioactive label productive rate to 42-49% and under maximum concentration is 48-52%.

Octanol-water partition coefficient-for determining ¹⁸F and ⁶⁸The lipotropy of the IMP466 of Ga labelling has been measured octanol-water partition coefficient.Al ¹⁸The log P of F-IMP466 _{Octanol/water}Value is-2.44 ± 0.12 ⁶⁸The log P of Ga-IMP466 _{Octanol/water}Value shows for-3.79 ± 0.07 ¹⁸The hydrophilic of the IMP466 of F labelling is low slightly.

Stability-after in human serum, hatching 4h under 37 ℃, ¹⁸The IMP466 of F labelling does not demonstrate ¹⁸F discharges, and shows Al ¹⁸The F-NOTA complex has good stability.

IC ₅₀ Measure-Al ¹⁹The apparent IC of the IMP466 of F labelling ₅₀Be 3.6 ± 0.6nM, and ⁶⁹The apparent IC of the IMP466 of Ga labelling ₅₀Be 13 ± 3nM.With reference to peptide ¹¹⁵The In-DTPA-octreotide

Apparent IC ₅₀Be 6.3 ± 0.9nM.

Biodistribution research-studied injection back 2h, have in the BALB/c nude mouse of subcutaneous AR42J tumor Al ¹⁸F-IMP466 and ⁶⁸The bio distribution of Ga-IMP466 (Fig. 4).With Al ¹⁸F is included in contrast. ¹⁸The cancer target height of F-IMP466,2h accumulation 28.3 ± 5.7%ID/g after injection.Uptake ratio in the presence of excessive unmarked IMP466 is 8.6 ± 0.7%ID/g, shows that tumor uptake is by receptor-mediated.Blood levels very low (injection back 2h, 0.10 ± 0.07%ID/g), causing tumor-blood ratio is 299 ± 88.Uptake ratio in organ is low, at the organ of expressed receptor, and for example specificity picked-up in adrenal gland, pancreas and the stomach.With non-chelating Al ¹⁸The uptake ratio of F is compared, Al ¹⁸The bone uptake ratio of F-IMP466 can ignore that (injection back 2h is respectively 0.33 ± 0.07 and 36.9 ± 5.0%ID/g), shows ¹⁸The body internal stability of the NOTA peptide of F labelling is good.

With Al ¹⁸The bio distribution of F-IMP466 with ⁶⁸The bio distribution of Ga-IMP466 compares (Fig. 4). ⁶⁸The tumor uptake rate of Ga-IMP466 (injection back 2h, 29.2 ± 0.5%ID/g) and Al ¹⁸Similar (the p of F-IMP466＜0.001). ⁶⁸Pulmonary's uptake ratio ratio of Ga-IMP466 ¹⁸F-MP466 (is respectively 4.0 ± 0.9%ID/g and 1.9 ± 0.4%ID/g) for high 2 times.In addition, ⁶⁸The kidney of Ga-IMP466 is detained a little more than Al ¹⁸F-IMP466 (is respectively 16.2 ± 2.86%ID/g and 9.96 ± 1.27%ID/g).

PET and CT have been shown among Fig. 5 have merged scanning.The bio distribution data have been proved conclusively in PET scanning.Al ¹⁸F-MP466 and ⁶⁸Ga-IMP466 demonstrates the uptake ratio height in tumor and is detained in kidney.Active substance in the kidney mainly is arranged in kidney cortex.Equally, because do not observe the bone picked-up, confirm Al ⁸F stablizes chelating by the NOTA chelating agen.

Fig. 5 is clear to be shown, ¹⁸The distribution of the Somat analog (octreotide) of F labelling with ⁶⁸The distribution of the Somat analog of Ga labelling the spitting image of.These results are significant, because verified, compare with diagnosis CT with conventional Somat receptor scintigraphy, in having human experimenter's body of neuroendocrine tumor ⁶⁸The octreotide PET imaging of Ga labelling has significantly higher verification and measurement ratio, and sensitivity is 97%, and specificity is 92% and accuracy is 96% (for example, Gabriel etc., 2007, J Nucl Med48:508-18).It is reported usefulness ⁶⁸The PET imaging that the octreotide of Ga labelling carries out is than using ¹¹¹Even the SPECT that the octreotide of In labelling carries out analyzes and to the detection of very little meningioma highly sensitive (Henze etc., 2001, J Nucl Med42:1053-56).Because and ¹⁸F compares ⁶⁸The energy of Ga is higher, expect based on ¹⁸The PET imaging meeting of F demonstrate than based on ⁶⁸The better spatial resolution of PET imaging of Ga.In Fig. 5 by relatively using ¹⁸The IMP466 of F labelling (Fig. 5, left side) with ⁶⁸The renal image that the IMP466 of Ga labelling (Fig. 5, right side) obtains has illustrated this point.With ⁶⁸The PET pictorial display that Ga obtains goes out than usefulness ¹⁸More dispersive edge of image and lower resolution that F obtains.These results prove, with using methods described herein and preparation of compositions ¹⁸The image that F labelling targeting moiety obtains is better and confirmed described ¹⁸The F labelling technique is to the practicality of non-antibody targeting peptide.

Embodiment 18. relatively uses pre-targeting ⁶⁸Ga and ¹⁸F PET imaging

We have compared the bispecific TF2 antibody pre-targeting of use through preparing as mentioned above ⁶⁸Ga or ¹⁸The PET image that F labelling peptide obtains. ⁶⁸Ga (t _1/2=68min) and ¹⁸F (t _1/2=half-life 110min) and the pharmacokinetics of radiolabeled peptides coupling are because its growth to greatest extent in tumor reached in a few hours.And, be easy to from ⁶⁸Ge/ ⁶⁸The Ga generator obtains ⁶⁸Ga, and ¹⁸F is the most frequently used and radionuclide that can extensively utilize among the PET.

Method

Mice with LS174T tumor of subcutaneous expression CEA is accepted TF2 (6.0nmol through intravenous; 0.94mg) and 5MBq ⁶⁸The IMP288 of Ga labelling (0.25nmol) or ¹⁸The IMP449 of F labelling (0.25nmol), interval 16h between injection bi-specific antibody and the radiolabeled peptides.1h or 2h behind the injection radiolabeled peptides obtain the PET/CT imaging and measure the bio distribution of radiolabeled peptides.Compare uptake ratio and the interior uptake ratio of the negative SK-RC52 tumor of subcutaneous CEA in the LS174T tumor.Relatively have in the mice body of subcutaneous LS174T tumor and the inflammation of offside leg muscle pre-targeting immunity PET imaging with ¹⁸The F-FDG-PET imaging.

IMP288DOTA-D-Tyr-D-Lys(HSG)-D-Glu-D-Lys(HSG)-NH ₂(SEQID?NO:51)

Pre-targeting-prepare bispecific TF2 antibody by aforesaid DNL method.TF2 comprises from one of h679 antibody in conjunction with the Fab fragment of HSG with from the segmental trivalent bi-specific antibody of two Fab in conjunction with CEA of hMN-14 antibody.Synthetic by aforesaid peptide, the peptide IMP288 that contains HSG that synthetic DOTA puts together.Synthetic as mentioned above IMP449 peptide contains 1,4,7-7-triazacyclononane-1,4, and 7-triacetic acid (NOTA) chelating moiety is to promote usefulness ¹⁸The F labelling.As the tracer of antibody component, use by iodogen method (Fraker and Speck, 1978, Biochem Biophys Res Comm80:849-57) ¹²⁵I (Perkin Elmer, Waltham, MA) labelling TF2, to specific activity be 58MBq/nmol.

The labelling of IMP288-under the no metal condition of strictness, use ¹¹¹In (Covidien, Petten, The Netherlands) labelling IMP288 is used for biodistribution research, and specific activity is 32MBq/nmol.With using the ultrapure HCl of 0.1M from based on 1 of TiO, 110MBq ⁶⁸Ge/ ⁶⁸Ga generator (Cyclotron Co.Ltd., Obninsk Russia) eluting ⁶⁸Ga labelling IMP288.Collect 5 1ml compositions and be used for the described peptide of labelling second part.Add the 1.0M HEPES buffer (pH7.0) of 1 volume to 3.4nmol IMP288.4 volumes of interpolation ⁶⁸Ga eluent (380MBq) and heating blends to 95 ℃ reach 20min.Add then 50mMEDTA to ultimate density be 5mM with complexation chelating not ⁶⁸Ga ³⁺(Waters, Milford MA) go up purification at 1mL Oasis HLB pillar ⁶⁸The IMP288 peptide of Ga labelling.After washing described pillar with water, with the described peptide of 25% ethanol elution.In 45min, use ⁶⁸The operation of Ga labelling IMP288 is got ready in the preparation donor and is used.

The labelling of IMP449-use as mentioned above ¹⁸F labelling IMP449.Use 0.4M KHCO ₃From QMA pillar eluting 555-740MBq ¹⁸F (B.V.Cyclotron VU, Amsterdam, The Netherlands).In the bottle that peptide (230 μ g) and ascorbic acid (10mg) are housed, add Al ¹⁸The F active substance.At 100 ℃ of following mixtures incubated 15min.By RP-HPLC purification reaction mixture.After adding the water of 1 volume, the described peptide of purification on 1mL Oasis HLB pillar.After washing with water, with 50% ethanol elution radiolabeled peptides.In 60min, prepare ¹⁸F-IMP449 gets ready in the preparation donor and uses.

Be used to study ¹²⁵I-TF2, ¹¹¹In and ⁶⁸Ga-IMP288 and Al ¹⁸The radiochemical purity of F-IMP449 preparation always surpasses 95%.

Zoopery-be that the male BALB/c nude mice (6-8 week age) of 20-25g experimentizes to weight.Mice 1 * 10 of the 0.2mL that gets an injection under the skin ⁶An individual LS174T cell (expressing the CCL188 of CEA) (American type culture collection, Rockville, MD, suspension USA).(after tumor inoculation 10-14 days) begin one's study when tumor size reaches about 0.1-0.3g.

TF2 and IMP288 injection interval 16h are because be enough to make this period unconjugated TF2 to remove from circulation.In some researchs, ¹²⁵I-TF2 (0.4MBq) injects jointly with unmarked TF2.With ¹¹¹In or ⁶⁸Ga labelling IMP288.With ¹⁸F labelling IMP449.Mice is accepted TF2 and IMP288 (0.2mL) through vein. ⁶⁸Ga labelling peptide injection 1h and ¹⁸F-IMP449 injection back 2h uses CO ₂/ O ₂Make mice euthanasia, and obtain blood and anatomical tissue by cardiac puncture.

(Siemens Preclinical Solutions, Knoxville TN) obtain the PET image with Inveon animal PET/CT scanner.15min acquisition PET emission scan confession anatomy reference after the CT scan (spatial resolution is 113 μ m, 80kV, and 500 μ A, open-assembly time is 300ms).

After the imaging, anatomical object tumor and organ are weighed and are counted in having the gamma counter of suitable energy window ¹²⁵I, ¹¹¹In, ⁶⁸Ga or ¹⁸F.Calculate the injected dose percentage ratio (%ID/g) of each gram tissue.

The result

In the 1h, pre-targeting immunity PET causes in the tumor ⁶⁸The high specific targeting of Ga-IMP288 (10.7 ± 3.6%ID/g), the negative tumor of normal structure (for example, tumor 69.9 ± 32.3), CEA (0.35 ± 0.35%ID/g) and inflammation muscle (uptake ratio in 0.72 ± 0.20%ID/g) is very low.Not with the tumor of the pre-targeting of TF2 ⁶⁸Ga-IMP288 uptake ratio also very low (0.20 ± 0.03%ID/g).[ ¹⁸F] FDG in tumor, effectively increase (7.42 ± 0.20%ID/g), but also inflammation muscle (4.07 ± 1.13%ID/g) and many normal structures in effectively increase and therefore pre-targeting ⁶⁸Ga-IMP288 provides better specificity and susceptiveness.With having accepted behind the pre-targeting of TF2 ⁶⁸Ga-IMP288 or ¹⁸The clear efficient targeting that is presented at radiolabeled peptides in the subcutaneous LS174T tumor of the corresponding PET/CT image of the mice of the IMP449 of F labelling, and inflammation muscle is invisible.Use on the contrary, [ ¹⁸F] clear tumor and the inflammation described of FDG.

Injectivity optimizing-mensuration TF2bsMAb dosage is to the influence of the cancer target of the IMP288 of fixed dosage 0.01nmol (15ng).Group intravenous injection 0.10,0.25,0.50 of 5 mices or 1.0nmol are with micro- ¹²⁵The TF2 of I (0.4MBq) labelling (being respectively 16,40,80 or 160 μ g).Injection ¹¹¹(0.01nmol, 0.4MBq) back 1h measures radiolabeled bio distribution to In-IMP288.

TF2 removes rapidly from blood and normal structure.Injection back 18h, under all TF2 dosage of test, haemoconcentration is lower than 0.45%ID/g.TF2 targeting in the 2h tumor of injection back be 3.5%ID/g and with TF2 dosage indifference (data not shown goes out).Under all TF2 dosage, ¹¹¹In-IMP288 effectively gathers (not shown) in tumor.Under higher TF2 dosage, observe in the tumor ¹¹¹The uptake ratio of In-IMP288 improves: under 1.0nmol TF2 dosage, reach the targeting to greatest extent (26.2 ± 3.8%ID/g) of IMP288.Therefore under 0.01nmol peptide dosage, be issued to the highest cancer target and tumor-blood ratio (TF2:IMP288 mol ratio=100:1) at the highest TF2 dosage of 1.0nmol.In normal structure, kidney ¹¹¹In IMP288 uptake ratio the highest (1.75 ± 0.27%ID/g) and kidney in uptake ratio be not subjected to TF2 dosage to influence (not shown).The uptake ratio of all other normal structures is very low, causes high tumor-non-tumor ratio, surpasses 50:1 under all TF2 dosage of test.

In order to use ⁶⁸The IMP288 of Ga labelling carries out the PET imaging, needs higher peptide dosage, because if 1h carries out the PET imaging after injection, every mice need be injected minimum 5-10MBq ⁶⁸The Ga active substance.When injection ⁶⁸The specific activity of Ga-IMP288 preparation is 50-125MBq/nmol.Therefore, for the PET imaging, must use the IMP288 of 0.1-0.25nmol at least.The identical TF2:IMP288 mol ratio of test under 0.1nmol IMP288 dosage.By injecting 1.0,2.5,5.0 or the pre-targeting LS174T tumor of 10.0nmol TF2 (160,400,800 or 1600 μ g).Opposite with the result under the low peptide dosage, in the tumor ¹¹¹The In-IMP288 uptake ratio is not subjected to TF2 dosage to influence (be 15%ID/g under all test doses, data not shown goes out).Under higher dosage, the TF2 targeting reduction by %ID/g in the tumor (under 1.0nmol and 10.0nmol injected dose, is respectively 3.21 ± 0.61%ID/g and 1.16 ± 0.27%ID/g) (data not shown goes out).The kidney uptake ratio is also irrelevant with bsMAb dosage (2%ID/g).Based on these data, we select the bsMAb dosage of 6.0nmol to be used to make the IMP288 target tumor of 0.1-0.25nmol.

The PET imaging-for the proof with TF2 and ⁶⁸The pre-targeting immunity PET of Ga-IMP288 is imaged as the effectiveness of CEA expressing tumor imaging, induces Subcutaneous tumor in 5 mice bodies.Induce subcutaneous LS174T tumor on the right side, and simultaneously in the same mouse body, in left side inoculation 1 * 10 ⁶Individual SK-RC52 cell is to induce the negative tumor of CEA.After 14 days, when the tumor size is 0.1-0.2g, use 6.0nmol ¹²⁵I-TF2 is through the pre-targeting mice of vein.Behind the 16h, mice is accepted 5MBq ⁶⁸Ga-IMP288 (0.25nmol, specific activity is 20MBq/nmol).3 mices of group are accepted the independent of same amount separately ⁶⁸Ga-IMP288 is not with the pre-targeting of TF2. ⁶⁸Ga-IMP288 injection back 1h obtains the PET/CT scanning of mice.

Illustrated among Fig. 6 in the mice body ¹²⁵I-TF2 and ⁶⁸The bio distribution of Ga-IMP288.Observe bsMAb in the tumor (2.17 ± 0.50%ID/g) and peptide (10.7 ± 3.6%ID/g) high uptake ratio, the very low (tumor-blood ratio: 64 ± 22) of the uptake ratio in the normal structure equally.In the negative tumor SK-RC52 of CEA ⁶⁸The targeting of Ga-IMP288 very low (0.35 ± 0.35%ID/g).Equally, not with the tumor of the pre-targeting of TF2 ⁶⁸The Ga-IMP288 uptake ratio very low (0.20 ± 0.03%ID/g), show express CEA the LS174T tumor in the IMP288 specific accumulation.

Injection ⁶⁸In the PET image that 1h obtains behind the Ga labelling peptide in the clearly visible CEA expressing tumor with the pre-targeting of TF2 ⁶⁸Ga-IMP288 specificity picked-up (not shown).By using 50% maximum intensity threshold value, the uptake ratio in the qualitative assessment tumor of the target area of drawing (ROI).With abdomen area as background region.Accepted TF2 and ⁶⁸Tumor in the image of the mice of Ga-IMP288-background ratio is 38.2.

Then we checked with [ ¹⁸F] the pre-targeting immunity PET of FDG.In the group of two 5 mices, induce subcutaneous LS174T tumor and induce the inflammation focus at left thigh muscle by intramuscular injection 50 μ L Oleum Terebinthinaes (18) at right hind.The injection Oleum Terebinthinae after 3 days, one group of mice is accepted 6.0nmol TF2, then accepts 5MBq behind 16h ⁶⁸Ga-IMP288 (0.25nmol).Another winding be subjected to [ ¹⁸F] FDG (5MBq).Make the mice fasting in the 10h before the injection and 1h anesthetized mice and warm until euthanasia after injection 37 ℃ of following maintenances.

The inflammation intramuscular ⁶⁸The uptake ratio of Ga-IMP288 is very low, and in the tumor of same animals very high (0.72 ± 0.20%ID/g and 8.73 ± 1.60%ID/g, p of uptake ratio＜0.05, Fig. 7).The intramuscular uptake ratio of inflammation (is respectively 0.50 ± 0.14%ID/g, 0.72 ± 0.07%ID/g, 0.44 ± 0.10%ID/g) in the scope identical with uptake ratio in pulmonary, liver and the spleen.In these mice bodies ⁶⁸The tumor of Ga-IMP288-blood ratio is 69.9 ± 32.3; Inflammation muscle-blood ratio is 5.9 ± 2.9; Tumor-inflammation muscle ratios is 12.5 ± 2.1.In another group mice body, ¹⁸F-FDG increases effectively in tumor that (7.42 ± 0.20%ID/g, tumor-blood ratio is 6.24 ± 1.5, Fig. 4). ¹⁸F-FDG gathers in inflammation muscle mainly also that (4.07 ± 1.13%ID/g), inflammation muscle-blood ratio is 3.4 ± 0.5, and tumor-inflammation muscle ratios is 1.97 ± 0.71.

Behind the pre-targeting of TF2, accepted ⁶⁸The corresponding PET/CT image of the mice of Ga-IMP288 is clear to have shown that radiolabeled peptides effectively increases in the tumor, and inflammation muscle invisible (Fig. 8).On the contrary, accepting ¹⁸In the image of the mice of F-FDG, tumor and inflammation visible (Fig. 8).Accepting ⁶⁸In the mice body of Ga-IMP288, tumor-Inflamed tissue's ratio is 5.4; Tumor-background ratio is 48; Inflammation muscle-background ratio is 8.9.[ ¹⁸F] tumor-inflammation muscle ratios of FDG uptake ratio is 0.83; Tumor-background ratio is 2.4; Inflammation muscle-background ratio is 2.9.

Use Al ¹⁸The IMP449 of F labelling tests pre-targeting immunity PET formation method.5 mices have been accepted 6.0nmol TF2, have then accepted 5MBq Al behind 16h ¹⁸F-IMP449 (0.25nmol).Other 3 mices have been accepted 5MBq Al ¹⁸F-IMP449 before do not used TF2, and 2 control mice has been injected [Al ¹⁸F] (3MBq).Summed up this result of experiment among Fig. 9.Al in the tumor of the pre-targeting of TF2 ¹⁸The uptake ratio of F-IMP449 is very high by (10.6 ± 1.7%ID/g), and in pre-targeting mice body very low (0.45 ± 0.38%ID/g).[Al ¹⁸F] the bone inner accumulated (50.9 ± 11.4%ID/g), and in the bone uptake ratio of radioactive label IMP449 very low (0.54 ± 0.2%ID/g), show Al ¹⁸F-IMP449 is stable in vivo.Al in the pre-targeting mice of the TF2 with subcutaneous LS174T tumor body ¹⁸The bio distribution of F-IMP449 with ⁶⁸Ga-IMP288 is closely similar.

Use Al ¹⁸Intensity in the PET pictorial display tumor of the pre-targeting immunity PET that F-IMP449 carries out with ⁶⁸Ga-IMP288 is identical, still ¹⁸The resolution ratio of F image ⁶⁸Ga image higher (Figure 10).Al ¹⁸The tumor of F-IMP449 signal-background ratio is 66.

Conclusion

Originally studies show that, with anti-CEA * anti-HSG bi-specific antibody TF2, in conjunction with ⁶⁸Ga or ¹⁸The pre-targeting immunity PET that two-HSG-DOTA-peptide of F labelling carries out is the particular technology that is used for the PET imaging of CEA expressing tumor.

Use TF2, in conjunction with ⁶⁸Ga-IMP288 or Al ¹⁸The pre-targeting immunity PET that F-IMP449 carries out involves 2 intravenouss and uses.Between infusion bsMAb and radiolabeled peptides, use 16h at interval.Behind the 16h, most of TF2 removes (haemoconcentration＜1%ID/g), prevented the IMP288 complexation in TF2 and the circulation from blood.

For these research, optimized usefulness ⁶⁸The operation of Ga labelling IMP288 produces a step mark technology.We find, form when 95 ℃ of following specific activitys surpass 150GBq/nmol labelling peptide for removing ⁶⁸The Ga colloid, need be on the C18/HLB pillar purification.If preparation contains the sub-fraction colloid and uses through intravenous, ⁶⁸The Ga colloid gathers in the tissue (liver, spleen and bone marrow) of mononuclear phagocyte system, has reduced picture quality.Can be on the C18 pillar purification rapidly ⁶⁸Ga labelling peptide.Can in 45min, finish radioactive label and purification so that use.

⁶⁸The half-life of Ga mates with the kinetics of the interior IMP288 peptide of pre-targeted system: the growth to greatest extent in tumor reaches in 1h.Every day can from ⁶⁸Ge/ ⁶⁸Ga generator eluting ⁶⁸Ga twice, avoids the needs to on-the-spot formula cyclotron.Yet, ⁶⁸The positron energy height (1.9MeV) of Ga emission makes the spatial resolution that obtains image be limited to 3mm, and the intrinsic resolution of little PET system is low to moderate 1.5mm.

¹⁸F, the most widely used radionuclide among the PET has the half-life (t more favourable to pre-targeting PET imaging _1/2=110min).As mentioned above, use ¹⁸F labelling NOTA puts together peptide IMP449.And usefulness ⁶⁸The Ga labelling is the same, is single stepping.Acquisition is up to 50% labelling productive rate.Al ¹⁸The bio distribution of F-IMP449 with ⁶⁸The IMP288 of Ga labelling is closely similar, shows to use this labeling method, ¹⁸F is the residualize radionuclide.

Opposite with FDG-PET, it is a kind of tumour-specific imaging pattern that pre-targeting radioimmunity detects.Detect the high sensitivity and the specificity (Huebner etc. of recurrent colorectal cancer pathological changes in patient's body though reported FDG-PET, 2000, J Nucl Med41:11277-89), but the FDG-PET image can cause the difficult diagnosis in difference pernicious and benign lesion, as in the inflammation shown in the observed protrude mark level.On the contrary, use TF2 ⁶⁸Ga or ¹⁸Tumor-background ratio height and the CEA positive tumor of the pre-targeting immunity PET that F labelling peptide carries out are high-visible, support described method is used for the clinical imaging of cancer and other condition of illness.Except that detecting transfer and distinguishing CEA positive tumor and other pathological changes, before also being used in pre-targeting radioimmunotherapy, pre-targeting immunity PET assesses the radiation dose that is delivered to tumor and normal structure.Because TF2 is a humanized antibody, its immunogenicity is low, so opened the approach of repeatedly imaging or treatment cycle.

Synthesizing of embodiment 19. folic acid NOTA conjugates

Activate as described folic acid (Wang etc., Bioconjugate Chem.1996,7,56-62.) and and Boc-NH-CH ₂-CH ₂-NH ₂Put together.By the chromatography purification conjugate.Remove the Boc group by handling then with TFA.The AMT salt derivative is mixed with p-SCN-Bn-NOTA (macro ring) in the carbonate buffer solution.Then by the HPLC purified product.With the Al that describes in the previous embodiment ¹⁸F labeled leaf hydrochlorate-NOTA derivant, HPLC purification then.For example in cancer or diseases associated with inflammation, incite somebody to action ¹⁸The folate intravenous injection of F labelling to the subject and successfully be used to folate receptor be scattered in picture (see, for example, Ke etc., Advanced Drug Delivery Reviews, 56:1143-60,2004).

The intravital pre-targeting PET of embodiment 20. people imaging

For having the patient (1.7m of doubtful recurrent tumor ²Body surface area) injection 17mg bispecific monoclonal antibody (bsMab).Make bsMab be positioned target and from blood, remove.When the bsMab when 99% has removed from blood, injection ¹⁸F labelling peptide is (5.7 * 10 ^-9The last 5-10mCi of mol).There is the micrometastasis tumor in PET imaging demonstration.

Embodiment 21. passes through ¹⁸F is labeled as angiogenesis and is subjected to volume imaging

Arg-Gly-Asp (RGD) peptide of labelling is used for (for example) and involves α _vβ ₃The imaging (Jeong etc., J.Nucl.Med.2008 electronic publishing on April 15) that ischemic tissue's medium vessels of integrin generates.According to (2008) such as Jeong RGD and SCN-Bn-NOTA are puted together.As mentioned above, by make the aluminum stock solution with ¹⁸F and derivatization RGD peptide mix and reach 15min 110 ℃ of heating makes [Al ¹⁸F] be connected with NOTA derivatization RGD peptide, use excessive peptide to impel labeled reactant to finish.Disclosed as (2008) such as Jeong, will ¹⁸The RGD peptide of F labelling is used for bio distribution and PET imaging in the body.[Al with RGD-NOTA ¹⁸F] conjugate absorbs to ischemic tissue and the PET imaging of angiogenesis is provided.

Embodiment 22. carbohydrate labellings

By making p-SCN-Bn-NOTA and hydrazine reaction, prepare NOTA thiosemicarbazides derivant by HPLC purification part then.Prepare [Al as described above described in the embodiment ¹⁸F] and with [Al ¹⁸F] add in the NOTA thiosemicarbazides and heat 15min.Randomly, by HPLC purification [Al ¹⁸F] NOTA thiosemicarbazides complex.Make [Al by known method ¹⁸F] NOTA thiosemicarbazides and carbohydrate oxidation using put together.Using PET to scan will ¹⁸The carbohydrate of F labelling successfully is used for imaging research.

Embodiment 23. organic solvents are to the influence of F-18 labelling

(for example NETA and NOTA) is more right than aluminum to the affinity of aluminum for chelating moiety ¹⁸The affinity of F is much higher.Al is right ¹⁸Therefore the affinity of F is subjected to for example factor affecting such as ionic strength of solution, because the existence of other counter ion counterionsl gegenions often makes positively charged aluminum and electronegative fluorion avoid mutually and reduces ions binding intensity.Therefore the low ionic strength medium answer REINFORCED Al and ¹⁸Effective combination of F.

Ethanol is added in discovery to ¹⁸Preliminary study in the F reaction has improved the productive rate of radiolabeled peptides.Prepare IMP461 as mentioned above.

IMP461 in table 9. ethanol ¹⁸The F labelling

#	2mM?AlCl 3	F-18	2mM?IMP461	Solvent	Productive rate *
						1	10μL	741μCi	20μL	EtOH60μL	64.9%
2	10μL	739μCi	20μL	H 2O60μL	21.4%
						3	10μL	747μCi	20μL	EtOH60μL	46.7%
4	5μL	947μCi	10μL	EtOH60μL	43.2%

* the productive rate behind the HLB column purification, Rxn# 1,2 and 4 is heated to 101 ℃ and reaches 5min, and Rxn#3 heats 1min in microwave oven.

PRELIMINARY RESULTS shows, ethanol is added in the reactant mixture to making ¹⁸The productive rate of F labelling peptide rises more than one times.Table 9 also shows, can use microwave irradiation to replace heating to promote [Al ¹⁸F] incorporate in the chelating moiety of IMP461.As if microwave irradiation 60s (#3) reach 5min (#1) efficient low slightly (18%) than being heated to 101 ℃.

Checked of the influence of other solvent to the 19F labelling of peptide.Under different situations, the identical and solvent difference only of concentration of reactants.Reaction condition comprises mixing 25 μ L Na ¹⁹F+20 μ LAlCl ₃+ 20 μ L IMP-461+60 μ L solvents then reach 5min 101 ℃ of heating.Table 10 shows that the existence of solvent has increased substantially [Al really ¹⁹F] productive rate of IMP-461 (IMP473).

IMP461 in table 10. all kinds of solvents ¹⁹The F labelling

Solvent	H 2O	MeOH	EtOH	CH 3CN
					Al-IMP-461	2.97	3.03	2.13	1.54
IMP-465	52.46	34.19	31.58	24.58
					IMP-473	14.99	30.96	33.00	37.48
IMP-473	15.96	31.81	33.29	36.40
					IMP-461	13.63	-	-	-

Solvent	IPA	Acetone	THF	Diox
					Al-IMP-461	2.02	2.05	2.20	16.67
IMP-465	32.11	28.47	34.76	10.35
					IMP-473	27.31	34.35	29.38	27.09
IMP-473	27.97	35.13	29.28	11.62
					IMP-461	10.58	-	4.37	34.27

Solvent	DMF	DMSO	t R(min)
				Al-IMP-461	-	-	9.739

IMP-465	19.97	37.03	10.138
				IMP-473	27.77	31.67	11.729
IMP-473	27.34	31.29	11.952
				IMP-461	-	-	12.535

[Al ¹⁹F]IMP461=IMP473

Embodiment 24. heavy carbonate eluting ¹⁸F

Will ¹⁸F (10.43mCi) is contained in the 2mL syringe.Solution is passed through

Light, ACCELL ^TMPlus QMA pillar.Use the described post of 5mL DI water washing then.As shown in table 11 below, use 0.4M KHCO ₃Divide several parts of eluting ¹⁸F.

Table 11. KHCO ₃Eluting QMA pillar

Bottle	Acetic acid volume μ L	0.4M?KHCO 3Volume μ L	Active substance mCi
				1	7.5	150	0.0208
2	10	200	7.06
				3	5	100	1.653
4	25	500	0.548

Checked other solvent (CH ₃CN) amount is to IMP-461's ¹⁸The influence of F labelling.Under different situations, the identical and amount difference only solvent of concentration of reactants.Reaction condition comprises mixing 10 μ L AlCl ₃+ 20 μ L ¹⁸F+20 μ L IMP-461+CH ₃CN then reaches 5min 101 ℃ of heating.Table 12 shows that after preliminary the improvement, labeling effciency reduces in the presence of excessive solvent.

Table 12. uses not commensurability CH ₃CN carries out IMP461 ¹⁸The F labelling

CH3CN(μL)	F-18mCi	t R2.70min(%)	t R8.70min(%)	RCY%(HLB)
					0	0.642	13.48	86.52	50.7
100	0.645	1.55	98.45	81.8*
					200	0.642	2.85	97.15	80.8
400	0.645	14.51	85.49	57.8

* contain rinsing agent and contain the labelling peptide.Radiochemistry productive rate behind the RCY=HLB purification

The high dose radioactive label of embodiment 25.IMP461

Will ¹⁸F (163mCi) is contained in the 2mL syringe.Solution is passed through

ACCELL ^TMPlus QMA pillar.Use the described post of 5mL DI water washing then.As shown in table 13, use 0.4M K ₂CO ₃Divide several parts of eluting ¹⁸F.

Table 13. high dose labelling

Bottle	Acetic acid volume μ L	0.4M?K 2CO 3Volume μ L	Active substance mCi
				1	18.5	185	5.59
2	5	50	35.8
				3	5	50	59.9
4	5	50	20.5
				5	5	50	5.58
6	50	500	4.21

In No. 3 bottles of table 13, add liquor alumini chloridi (10 μ L, 2mM is in pH4,2mMNaOAc).(20 μ L, 2mM is in pH4,2mM NaOAc) add in the bottle with peptide, then add the CH of 170 μ L ₃CN.Reach 10min and use the dilution of 6mL water at 103 ℃ of heated solutions.Suck solution in the 10mL syringe and inject 2 arranged in series

In the HLB Plus pillar.With the described pillar of 8mL water washing.Use 10mL1:1EtOH/H then ₂O (30.3mCi) eluting radiolabeled peptides Al ¹⁸F IMP461, productive rate are 63.5%, and specific activity is 750Ci/mmol.It is unconjugated by HPLC the labelling peptide not to be contained ¹⁸F.Reaction and purification total time are 20min.

Embodiment 26. ¹⁹The preparation of F labelling peptide

Contain ²⁷Al and/or ¹⁹The product of F is used some, and is useful as the NMR imaging.Researched and developed preparation [Al ¹⁹F] the improving one's methods of labelled compound.Prepare IMP461 and usefulness as mentioned above ¹⁹The F labelling.Make IMP461 and AlCl ₃+ NaF reaction causes forming 3 kinds of product (not shown).Yet, by making IMP461 and AlF ₃.3H ₂The O reaction, we obtain the more [Al of high yield ¹⁹F] IMP461.

IMP473's is synthetic: ([Al ¹⁹F] IMP461) to (2mM, pH4.18) (14.1mg, the 10.90 μ mol) IMP461 in the solution adds (4.51mg, 32.68 μ mol) AlF in 2mL NaOAc ₃.3H ₂O and 500 μ L ethanol.Use 3 μ L1N NaOH with the pH regulator to 4.46 of solution and in boiling water bath, heat 30min.By the IMP473 of preparation RP-HPLC purification crude product mixture with generation 4.8mg (32.9%).HRMS (ESI-TOF) MH ⁺Expected value is 1337.6341; Actual value is 1337.6332.

These results prove, can by form metal- ¹⁹The F complex and make metal- ¹⁹F and as discussed above being used for ¹⁸The chelating moiety of F labelling is in conjunction with preparation ¹⁹The F labelled molecule.Present embodiment shows, can use this method preparation to be used for the targeting peptide of pre-targeting detection, diagnosis and/or imaging.

Synthetic and the labelling of embodiment 27.IMP479, IMP485 and IMP487

Illustrated in addition among Figure 18-Figure 20 and be designed for ¹⁸The structure of the peptide of F labelling (IMP479, IMP485, IMP487).IMP485 has been shown among Figure 19.On the Sieber amide resin, by in the indicated order following aminoacid addition being prepared IMP485:Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, cutting Aloc, Fmoc-D-Tyr (But)-OH, Aloc-D-Lys (Fmoc)-OH, Trt-HSG-OH, cutting Aloc, (tert-butyl group) to resin ₂NODA-MPAA (aminomethyl phenyl acetic acid).Make peptide from the resin cutting and by the IMP485 of RP-HPLC purification then with generation 44.8mg.

Two-tert-butyl group-NODA-MPAA's is synthetic: be used for the synthetic NO2AtBu-MPAA of IMP485

(0.5925g is 2.59mmol) in CH to 4-(bromomethyl) phenylacetic acid (Aldrich310417) under 0 ℃ ₃The solution of CN (anhydrous) in (50mL) drips NO2AtBu, and (1.0087g is 2.82mmol) in CH ₃Solution 1h among the CN (50mL).Behind the 4h, in reactant mixture, add anhydrous K ₂CO ₃(0.1008g, 0.729mmol) and make its stirred overnight at room temperature.Evaporating solvent also passes through preparation RP-HPLC purification crude product to produce white solid (0.7132g, 54.5%).

Though this is an one-step synthesis, because product is by the esterification of 4-(bromomethyl) phenylacetic acid, productive rate is very low.Adopt the NO2AtBu alkylation of using (4-bromomethyl) phenylacetic acid methyl ester to prevent esterification (Figure 21).

¹⁸The F labelling

For in the water ¹⁸The F labelling (uses trehalose+ascorbic acid+AlCl to the IMP-479/485/487 of 40nmol ₃Preparation) adds 250 μ L F-18 solution [F-18 of～919-1112 μ Ci] and be heated to 101 ℃ and reach 15min.In ethanol, (use trehalose+ascorbic acid+AlCl to the IMP-479/485/487 of 40nmol ₃Preparation) adds 250 μ L F-18 solution [F-18 of 1.248-1.693mCi], 100 μ L EtOH and be heated to 101 ℃ and reach 15min.The exemplary experiment of the labelling that shows different peptides has been shown in the table 14.Through minimum optimization, the radioactive label productive rate of having observed IMP485 is up to 88%, and specific activity is 2,500Ci/mmol.Under this specific activity, for PET imaging in the body that uses radiolabeled peptides, do not need HPLC purification radiolabeled peptides.

The labelling of table 14.IMP479, IMP485 and IMP487

Serum stability

With the purification in the 200 μ L saline ¹⁸F makes IMP485 or IMP487, the 20nmol AlCl that contains 40nmol ₃, 0.1mg ascorbic acid and 0.1g trehalose, the test kit that is adjusted to pH3.9 restores and is heated to 106 ℃ and reaches 15min.Then with 800 μ L water diluted reaction mixtures and place on the HLB post.After the washing, with 2 * 200 μ L1:1EtOH/H ₂The described post of O eluting is to obtain the purification of 64.6% isolated yield ¹⁸F-IMP485.The radiolabeled peptides of 50 μ L mixes with 250 μ L Freshman serum in the bottle and hatches under 37 ℃.

The test 4h in, under 37 ℃ in people's fresh serum two kinds of radiolabeled peptides all stablize (not shown).

Filler is to the influence of lyophilizing peptide productive rate

Use has the IMP485 test kit (40nmol) of the different filleies of F-18 (from the F-18 of the same batch) labelling through 2mCi, experimentizes in 200 μ L saline to compare productive rate.Introducing concentration in water is the filler of 5 weight %, and dosage is 200 μ L/ bottles.We have tested Sorbitol, trehalose, sucrose, mannitol and glycine as filler.The results are shown in the table 15.

Table 15. filler is to the influence of radioactive label productive rate

Filler	Active substance mCi	Productive rate %
			Sorbitol	2.17	82.9
Glycine	2.17	41.5
			Mannitol	2.11	81.8
Sucrose	2.11	66.1
			Trehalose	2.10	81.3

The radioactive label product of the identical productive rate of the equal output of Sorbitol, mannitol and trehalose.Mannitol test kit and trehalose test kit all produce good result.The productive rate of sucrose test kit and glycine test kit is obviously lower.We have also tested recently as the 2-hydroxy propyl-Beta-cyclodextrin of filler and for the 40nmol test kit and have obtained 58% productive rate.We have found that radioactive label is very sensitive and need to regulate to be suitable for part and even may to need to regulate to be suitable for peptide+part to pH.Under the situation of IMP485, optimum pH is pH4.0 ± 0.2, and is pH4.5 ± 0.5 for the optimum pH of IMP467.In both cases, outside the ideal pH interval, productive rate descends rapidly.

The time-histories of labelling

Checked the time-histories of labelling IMP485.IMP485 to 40nmol (uses trehalose+AlCl ₃(20nmol)+and ascorbic acid preparation) interpolation～200-250 μ L F-18 (0.9% saline) and be heated to 104 ℃ and reach 5-15min.The labelling yield results is: 5min (28.9%), 10min (57.9%), 15min (83.7%) and 30min (88.9%).Therefore, the time-histories of labelling is about 15min.

The bio distribution of independent IMP485

Checked to have in female Taconic nude mice little or that do not have BXPC3 cancer of pancreas xenograft (the 10 week age) body bio distribution of IMP485 when no any pre-targeting antibodies.For injecting in the mouse vein ¹⁸F-IMP485 (340 μ Ci, 2.2 * 10 ^-9Mol, 100 μ L are in saline).30min and 90min after injection, each time point are 6 mice autopsies.When pre-targeting antibodies lacks, in tumor and most of normal structure, see low-level gathering.The radioactive label of the overwhelming majority is present in the bladder and on less degree and is present in the kidney.Removed most of active substance before the 90min time point.

With the pre-targeting IMP485 of TF2DNL targeted molecular

¹⁸ The F-MP485 radioactive label-purification ¹⁸F (218mCi) is to separate 145.9mCi.With purification ¹⁸F (135mCi) adds in the lyophilizing bottle that the pre-complexation Al-IMP485 of 40nmol is housed.Reach 17min at 110 ℃ of reacting by heating bottles.In reactant mixture, add water (0.8mL) before the HLB purification.Use 0.6mL water: ethanol (1:1) mixture is eluted to product (22mCi) in the bottle that the lyophilizing ascorbic acid is housed.Use the saline cut back.Be used to inject ¹⁸F-Al IMP485 specific activity is 550Ci/mmol.

Separately ¹⁸ The bio distribution of F-Al IMP485-for having the injected in mice of subcutaneous LS174T xenograft ¹⁸F-Al IMP485 (28 μ Ci, 5.2 * 10 ^-11Mol, 100 μ L).After injection 1 and 3h be the mice autopsy, 6 mices of each time point.

BsMAb at 20:1: the TF2+ of pre-targeting under the peptide ratio ¹⁸ The biology of F-Al IMP485 Distribute-for having injected in mice TF2 (163.2 μ g, 1.03 * 10 of subcutaneous LS174T xenograft ^-9Mol, vein) and in injection ¹⁸F-Al IMP485 (28 μ Ci, 5.2 * 10 ^-11Mol, 100 μ L, vein) make it remove 16.3h before.After injection 1 and 3h be the mice autopsy, 7 mices of each time point.

Urine stability-for having the injected in mice of subcutaneous Capan-1 xenograft ¹⁸F-Al-IMP485 (400 μ Ci, in saline, 100 μ L).55min collects the urine of 3 mices after injection.Analyze urine sample by anti-phase and SE HPLC.Observe the stability of radiolabeled IMP485 in urine.

Back 1h is independent in table 16. injection ¹⁸F-IMP485

Back 3h is independent in table 17. injection ¹⁸F-IMP485:

The TF2+ of table 18. peptide injection back 1h ¹⁸F-IMP485

The TF2+ of table 19. peptide injection back 3h ¹⁸F-IMP485:

Conclusion

The IMP485 labelling is the same with IMP467 good or better than IMP467, and the stability in serum quite.Yet IMP485 is easier to synthesize than IMP467.The preliminary study demonstration, lyophilizing IMP485's ¹⁸F labelling the same with the effect of non-lyophilizing peptide good (data not shown goes out).Checked the existence that chelating moiety is connected to alkyl or aryl in the connexon of peptide remainder.With respect to the existence of alkyl in the connexon, as if the existence of aryl has improved the radioactive label productive rate in the connexon.

The bio distribution of IMP485 is similar to the observed bio distribution with IMP467 when pre-targeting antibodies exists or lacks.When pre-targeting antibodies lacked, the bio distribution of radiolabeled peptides was low and by renal excretion peptide is removed from circulation in tumor and the most of normal structure.In the presence of TF2 antibody, radiolabeled IMP485 mainly is present in the tumor, seldom is distributed to normal structure.In the presence of pre-targeting antibodies, the kidney radioactive label reduces significantly.We infer that other peptide that has aryl in IMP485 and the connexon is fit to warp very much ¹⁸The F labelling carries out the PET imaging.

Embodiment 28. is used for the test kit preparation of the IMP485 of imaging

The reagent tabulation

Obtain reagent from following source: acetic acid (JT Baker6903-05 or 9522-02), sodium hydroxide (Aldrich semiconductor grade 99.99%30,657-6), α, α-trehalose (JT Baker4226-04), Aluminum Chloride Hexahydrate (Aldrich99%237078), ascorbic acid (Aldrich25,556-4).

Acetate buffer 2mM-dilute 22.9 μ L (4.0 * 10 with 200mL water ^-4Mol) acetic acid and with 6N NaOH (～15 μ L) neutralization so that solution is adjusted to pH4.22.

Aluminum solutions 2mM-with 0.0225g (9.32 * 10 ^-5Mol) aluminum hexahydrate is dissolved in the 47mL DI water.

α, α-aqueous trehalose-with the α of 4.004g, α-aqueous trehalose is dissolved in the 40mL DI water to prepare 10% solution.

Peptide solution IMP4852mM-peptide IMP485 (0.0020g, 1.52 μ mol) is dissolved in the 2mM acetate buffer of 762 μ L.PH was 2.48 (the peptide lyophilizing is tfa salt).1M NaOH by adding 4.1 μ L with the pH regulator of peptide solution to pH4.56.

Ascorbic acid solution 5mg/mL-with 0.0262g (1.49 * 10 ^-4Mol) ascorbic acid is dissolved in the 5.24mL DI water.

The preparation of peptide reagent box

20 μ L (40nmol) peptides are mixed with the DI water of trehalose, 20 μ L (0.1mg) ascorbic acid and the 900 μ L of Al, the 100 μ L of 12 μ L (24nmol) in 3mL lyophilizing bottle.The final pH of solution should be～pH4.0.Freezing, lyophilizing and in vacuum lower seal bottle.

Also prepared 10 and the 20nmol test kit.With these test kits be prepared into with peptide: Al ³⁺Ratio remains 1 peptide: 0.6 Al ³⁺The 40nmol test kit identical, but be formulated in the 2mL bottle, total loading is 0.5mL.

¹⁸The generation of F

Will be thick ¹⁸F is contained in the 2mL DI water in the syringe.Place syringe on the syringe pump and promote liquid and pass through Waters CM pillar, then by the QMA pillar.With two pillars of 10mL DI water washing.Change the aseptic disposable three-way valve between two pillars and divide 200 μ L every part and promote the commercial Sterile Saline of 1mL by the QMA pillar.No matter use ¹⁸What (test 10-300mCi carrying capacity) F amount be, second part contain usually～80% ¹⁸F.

We are used in the commercialization of purification on the QMA pillar in the saline alternatively ¹⁸F.This is the conc forms of commercial skeletal imaging agent, so be easy to obtain and use in the mankind.200 μ L active substances are provided in the 0.5mL tuberculin syringe.

Radioactive label

Make an addition in the 200 μ L saline by the lyophilizing peptide in the wiredrawn edge air-tight bottle ¹⁸F, heated solution reaches 15min to the peptide radioactive label to 90-105 ℃ then.By in reaction bulb, making an addition to the 800mL DI water in the 1mL syringe, take out liquid and liquid is coated in Waters HLB post (1cc, 30, mg) the last described peptide of purification with the 1mL syringe.The HLB post is placed 5mL wiredrawn edge air-tight bottle and under the vacuum that provides with (using aseptic disposable pipe) 10mL remote controlled injection syringe liquid sucked bottle.With the 1mL DI water washing reaction bulb of 2 aliquots, draw DI water equally by described post washing.And then with the described post of 1mL DI water washing.Then described post is moved to and buffering lyophilizing ascorbic acid is housed (～pH5.5 is in bottle 15mg).1:1EtOH/DI water elution radioactive label product with 3 part of 200 μ L.By in reaction bulb, water lotion and product bottle, measuring determination of activity productive rate on the HLB pillar to obtain yield percentage.

Ethanol added in the radioactive label reaction can improve the labelling productive rate.F-18 and the 200 μ Ls alcoholic acid mixture of available 200 μ L in saline restores the 20nmol test kit.Heated solution reaches 15min to 100-110 ℃ in the wiredrawn edge air-tight bottle then.After the heating, before purification on 3cc (60mg) the HLB extraction pillar, with 3mL water diluting reaction thing.Can make in this way with good yield with up to 4 the described peptide of specific activity labelling of 100Ci/mmol.

When using 1.0mCi's ¹⁸During the F labelling, the productive rate of this test kit and described labelling is 80-90%.When using high dose more ¹⁸F (～100mCi) time, productive rate descends.If yet in the labelling mixture, adding ethanol, productive rate rises.If excessive dilution peptide in saline, productive rate will descend.Labelling is also very sensitive to pH.For peptide, we have found that the optimum pH of final preparation is pH4.0 ± 0.2 with this part.

Embodiment 29. uses Al ¹⁸F or Al ¹⁹Other prothetic group labeling method of F

In certain embodiments, for for heat sensitive molecule, can use the prothetic group labeling method to carry out the aluminium fluoride labeling method.For the thermal sensitivity molecule, can carry out prothetic group at a lower temperature and put together.

Use as mentioned above ¹⁸F or ¹⁹F labelling prothetic group NOTA makes it be connected with targeted molecular then.In a non-limiting example, this carries out with aldehyde NOTA, and aldehyde NOTA is connected with amino-oxy chemical compound on the targeted molecular then.Alternatively, make amino-oxy maleimide and aldehyde reaction, maleimide is connected (Toyokuni etc., 2003, Bioconj Chem14:1253) with cysteine on the targeted molecular then.

In another replacement scheme, AlF-chelating agen complex is connected with targeted molecular by click chemistry.As discussed above, at first use Al ¹⁸F or Al ¹⁹The F tagged ligand.By the click chemistry reaction AlF-chelating agen and targeted molecular are puted together then.For example, according to Marik and Stucliffe (2006, Tetrahedron Lett47:6681) labelling alkynes NOTA and make it be connected (Figure 11) with the targeting agent that contains azide.

In another alternate embodiment (Figure 12), azide is on chelating agen part and alkynes (Glaser and Arstad, 2007, Bioconj Chem18:989) in the targeting agent.

Embodiment 30. is used for ¹⁸The maleimide conjugate of the NOTA of F labelling

As discussed above, in certain embodiments, the maleimide derivatives of NOTA may be marked with usefulness to the low temperature of molecule.The illustrative methods of preparation maleimide derivative NOTA below has been discussed.Details have been shown among Figure 22.

Two-tert-butyl group NODA-MPAA NHS ester: (tBu) ₂Synthesizing of NODA-MPAA NHS ester

To (tBu) ₂(175.7mg is 0.347mmol) in CH for NODA-MPAA ₂C1 ₂Solution (5mL) adds 347 μ L (0.347mmol) DCC, and (1M is in CH ₂C1 ₂In), 42.5mg (0.392mmol) N-hydroxy-succinamide (NHS) and 20 μ L N, N-diisopropylethylamine (DIEA).Behind the 3h, filter out DCU and evaporating solvent.By at (230-400 order) silica gel (CH ₂Cl ₂: carry out flash column chromatography purification crude mixture MeOH:100:0-to 80:20) to produce the NHS ester of (128.3mg, 61.3%).C ₃₁H ₄₆N ₄O ₈(M+H) ⁺HRMS (ESI) value of calculation be 603.3388, observation is 603.3395.

NODA-MPAEM's is synthetic: (MPAEM=aminomethyl phenyl acetamido ethyl maleimide)

To (tBu) ₂(128.3mg is 0.213mmol) in CH for NODA-MPAA NHS ester ₂C1 ₂Solution (5mL) adds the solution of 52.6mg (0.207mmol) N-(2-aminoethyl) maleimide trifluoroacetate in 250 μ L DMF and 20 μ L DIEA.Evaporating solvent and handle concentrated solution behind the 3h with 2mL TFA.The dilute with water crude product also passes through preparation RP-HPLC purification to produce the required product of (49.4mg, 45%).C ₂₅H ₃₃N ₅O ₇(M+H) ⁺HRMS (ESI) value of calculation be 516.2453, observation is 516.2452.

Synthesizing of embodiment 31.NOTA cyclooctyne reagent

Substitute technology can be used for the click chemistry reaction so that but chelating moiety or targeting construct are connected with targeted molecular, for example antibody or antibody fragment.In other replacement scheme, click chemistry can be used for irreversibility and puts together AD or DDD part to generate permanent DNL construct.Can use the click chemical technology is connected to any molecule or construct on any other molecule or the construct.

In an exemplary, activation cyclooctyne part is puted together with the NOTA chelation group, to connect with the targeted molecular that comprises azide or nitrone active group (for example, antibody).Figure 13 illustrated limiting examples.

Mix a normal protection NOTA part (Figure 13,1) and a normal cyclooctyne (Ning etc., 2010, Angew Chemie49:3065-68) (Figure 13,2) in the Yu diox.At room temperature N-hydroxy-succinamide (HONSu, 1.2 equivalents) and 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide (EDC, 1 equivalent) are added in the reaction then.Behind the 4h, solvent is removed in decompression on Rotary Evaporators.Be dissolved in reactant mixture in the chloroform and wash with water.Use the dried over sodium sulfate chloroform layer, filter, on Rotary Evaporators, concentrate then so that two-tert-butyl ester crude product (Figure 13,3) to be provided.At room temperature handle crude product 2h with TFA, decompression is removed TFA so that crude product (Figure 13,4) to be provided then, is further purified crude product by carry out reversed-phase HPLC with the 0.01%HCl buffer on the C-18 preparative column.The technical staff will recognize, reaction can be used for making cyclooctyne with targeted molecular (for example antibody) but, chelating moiety or targeting construct or AD part, DDD partly or any other protein, peptide or the molecule that comprise carboxyl functional group be connected.

The radioactive label of NOTA cyclooctyne reagent

A1F-18 part (Figure 13,4) is dissolved in preparation 2mM ligand solution in pH4, the 2mM acetate buffer, ligand solution is adjusted to pH4 by adding several 1M NaOH (99.99% electrochemical classes and grades in school).By ascorbic acid being dissolved in preparation 5mg/mL ascorbic acid solution in an amount of DI water.By Aluminum Chloride Hexahydrate being dissolved in the Al of preparation 2mM in the suitable quantity of water ³⁺Solution.By being dissolved in, trehalose prepares 5% (by weight) α, α-aqueous trehalose in an amount of DI water.

The preparation of freeze-dried reagent box

Prepare test kit in bulk and be dispensed in the 2mL lyophilizing bottle by pipet at mix reagent with the pH regulator of solution in bulk to pH4.0 ± 0.2 back.Following mix reagent box (by every bottle): 10 μ L part (Figure 13,4) solution, 6 μ L Al ³⁺Solution, 20 μ L ascorbic acid solutions, 200 μ L α, α-aqueous trehalose and 264 μ L DI water.By adding several 1M NaOH solution in bulk is adjusted to pH4.0 ± 0.2.Be divided into the 0.5mL aliquot solution is dispensed in the 2mL bottle, lyophilizing is then at the vacuum lower seal.

In saline, use ¹⁸F radioactive label test kit

Will ¹⁸F (0.01mCi or higher) is contained in the 200 μ L saline in the 0.5mL syringe and solution is mixed with 200 μ L ethanol and injects aforesaid freeze-dried reagent box.100-110 ℃ in the wiredrawn edge sealed container heated solution reach 15min.With 3mL water dilute solution and by HLB pillar eluting.With 2 * 1mL water washing reaction bulb and pillar, use 1:1 ethanol then: water, divide every part of 0.5mL that product is eluted in the bottle that the buffering ascorbic acid is housed.Can flow down at noble gas and evaporate some ethanol.In saline and before injection, make it pass through 0.2 μ m sterilizing filter solution dilution then.

The preparation of embodiment 32.NOTA azide reagent

In other alternate embodiment, but the activation azido group can be connected with chelating moiety or targeting construct, and with targeted molecular on corresponding alkynes, for example cyclooctyne reacts.Figure 14 shows the exemplary arrangement that NOTA chelating moiety and azide are puted together.The part that is designated as " R " is the spacer group that can be alkyl, aromatic group, peg moiety or similar compound.As mentioned above, shielded NOTA part mixes with amino azide and coupling.Remove protecting group by handling with TFA.The chemical compound that the HPLC purification is new (Figure 14,6) is mixed with the freeze-dried reagent box and carries out radioactive label in the same manner as described above.

The technical staff will recognize that described reaction can be used for making azido group and targeted molecular (for example antibody), but and chelating moiety or the connection of targeting construct.Alternatively, same reaction other molecule of can be used for making azido group and AD part, DDD part or any other protein, peptide or comprising suitable carboxyl is connected.

The preparation of embodiment 33.NOTA nitrone

In another alternate embodiment, but activation nitrone part is connected with chelation group or targeting construct, but the cyclooctyne on chelation group or targeting construct and the targeted molecular reacts then.Figure 15 shows shielded as mentioned above NOTA part and mixes also link coupled reaction with amino serine.Remove protecting group by mixing with TFA.

React to form nitrone (Figure 15,9) with the new chemical compound of periodate oxidation (Figure 15,8) and with the N-methyl hydroxylamine then.The HPLC purified product is mixed with the freeze-dried reagent box and carries out radioactive label in the same manner as described above.The technical staff will recognize that described reaction can be used for making nitrone and targeted molecular (for example antibody), but and chelating moiety or the connection of targeting construct.Alternatively, same reaction can be used for making nitrone to be connected with AD part, DDD part or other protein or peptide moiety.

Embodiment 34. is through the pre-targeting of click chemistry

The synthetic antibody that has nitrone, azide or cyclooctyne part is as mentioned above injected in patient's body.Before but injection has the targeting construct of isotope or medicine, make antibody be positioned target and from blood, remove.If use the antibody that contains azide or nitrone part, but then the targeting construct will contain alkynes, for example cyclooctyne.If antibody contains alkynes, but then can use the targeting construct that contains azide or nitrone.But the targeting construct can comprise and Al- ¹⁸The chelating moiety that the F complex connects, or can put together with one or more other diagnostic agents and/or therapeutic agent.But the click chemistry reaction has enough specificitys so that localized antibody in the targeting construct targeting body in the body, but need not to use with the targeting construct on the bonded bi-specific antibody of hapten.

Embodiment 35. antibody are puted together with the activation cyclooctyne

In another exemplary, antibody is puted together (Figure 16) with the active ester 1 or 2 (R=NHS) that is substituted cyclooctyne.With the anti-CEACAM5 monoclonal antibody of humanization (MAb), hMN-14 has illustrated described process.At room temperature, be in the 0.1M phosphate buffer of 7.5-8.5 in the pH scope, use the active ester 1 or 2 (Figure 16) of 10 times of molar excess and put together 2h as the 5-10%v/v DMF of cosolvent.By equilibrated in pH7,0.1M phosphate buffer

Carry out size exclusion chromatography (SEC) purification conjugate on 50/80.The conjugate by MALDI-TOF analytical reagent composition purification and the hMN-14 of unmodified are to measure octyne/IgG substitute proportion.

Cyclooctyne put together antibody can be in vivo or external and as above embodiment described in the NOTA-azide or the reaction of NOTA-nitrone that prepare.

The preparation of embodiment 36. azidos-PEG-P (glu) block copolymer

Use benzyl L-glutamate, Glu N-carboxylic acid anhydrides and the isodigeranyl function PEG (Figure 17) that contains azide and amine, the process (Nishiyama etc., 2003, the Cancer Res63:8977-83 that use this area to describe; Koizumi etc., 2006, Cancer Res66:1004-56) block copolymer of preparation azido PEG and benzyl-L-glutamate, Glu.Bifunctional PEG is on sale on market, and the PEG molecular weight is up to 3400Da, and by the more high-molecular weight PEG of bifunctional PEG preparation on sale on other market.In block copolymer, remove benzyl protecting group (Figure 17) by mild hydrolysis.

The preparation of azido-PEG-P (glu) block copolymer that embodiment 37. medicines are puted together

Use SN-38, the active medicine form of cancer prodrug, the irinotecan illustration medicine put together.Described process is not limited to SN-38, however applicable to and other medicines, for example amycin, paclitaxel etc. are puted together.SN-38 is converted into SN-38-20-O-glycinate derivant and uses EDC to make side chain COOH group coupling on itself and the polymer as solvent as coupling agent and DMF.According to the repetitive in the polymer, optimize medicine and replace to produce replacement to 5-20 SN-38 molecule.The drug-polymer substitute proportion is measured in integration by PEG selectivity and SN-38 selectivity signal in the proton N MR spectrum.

The glucosan of embodiment 38. attached COOH (40kD MW) is derived successively and is azido amine and chemotherapeutics (SN-38)

As disclosed among the U.S. Patent Application Publication No.20080171067, with bromocaproic acid and sodium hydroxide derivatization glucosan (40kD), so that each glucosan has～60 COOH, the embodiment part is incorporated this paper by reference into.At first with N on sale on the market ₃-CH ₂-CH ₂-(OCH ₂CH ₂) ₁₀-O-CH ₂CH ₂NH ₂The azido amine of form uses EDC as some hydroxy-acid groups of coupling agent derivatization.The mol ratio of regulating azido amine is to introduce 1-10mol azide/mol polymer.Short PEG guarantee azido be easy to pre-targeting antibodies on cyclooctyne put together.Yet other modification of azido amine is also applicable.For example, also can use 11-azido-3,6,9-trioxa hendecane-1-amine or other similar reagents.By using the 10KDa filter to carry out ultrafiltration-diafiltration purified product.Next, use aqueous conditions, use EDC as coupling agent and DMSO as cosolvent (～5%v/v), with SN-38-20-O-glycinate derivatization residue hydroxy-acid group.By the diafiltration purified product.Measure glucosan concentration and by the spectrophotometry 366nm under and with the dependency acquisition SN-38 content of SN-38 standard curve.

Embodiment 39. has the antibody of nitrine carboxylic acid NHS ester and has the puting together of polymer of the attached medicine of cyclooctyne

As mentioned above, antibody is hMN-14 for example, with azido carboxylic acid active ester for example 6-azido carboxylic acid N-hydroxy-succinamide ester put together.In a similar manner, use cyclooctyne derivatization PEG to replace azido-PEG to carry out the block copolymer preparation and to the azide derivatization of glucosan with using the cyclooctyne derivatization replacement that contains amine.The latter usually by Figure 16 (1 or 2) by with single protection diamidogen or the coupling of BOC-hydrazides, then go protection to prepare expediently.Azido activation antibody can with the cyclooctyne part coupling on the polymer of attached medicine.

Embodiment 40. uses the pre-targeting of electric shock chemistry

Use the suspension of GW-39 human colon cancer cell for the nude mice intravenous.After 14 days, begin by use the pre-targeted therapy of hMN-14 cyclooctyne conjugate through intravenous.Location and removing after date, intravenous gives the micellar nanoparticles compositions of SN-38/PEG-PG or the glucosan conjugate of SN-38.Weightlessness and the survival rate of monitoring animal.The contrast therapy is involved in independent saline or the micelle composition of use in second step.Determine that pre-targeted therapies is obviously compared according to therapy better in this human colon carcinoma pulmonary metastasis model of nude mice.Repeat identical experiment to obtain pre-targeting comparison with antibody and medicine substrate according to obviously better tumor growth control of therapy.

Embodiment 41. azido high lactamine metabolism are incorporated among the IgG

Use the stable transgenic mouse myeloma cell line that generates reorganization hMN-14IgG (the anti-CEACAM5 monoclonal antibody of humanization) to generate azido high lactamine derivatization hMN-14IgG.Before to rat bone marrow tumour host cell Sp2/0, researched and developed described cell line by the pdHL2 plasmid expression vector stable transfection that will contain hMN-14 heavy chain and light chain expression box.With the methionine residues among the azido high lactamine replacement hMN-14IgG.Making hMN-14IgG cellulation system grow to viable cell density in the roller bottle culture in 1L H-SFM culture medium (Invitrogen) is 1.5 * 10 ⁶Individual cell/mL.By the centrifugal cell precipitation that makes, and be suspended in once more in the H-SFM culture medium of having replenished 30mg/L azido high lactamine, no methionine.Make roller bottle culture at 37 ℃ and 5%CO ₂Following growth is reduced to below 30% until cell viability.Filter culture supernatants and also be coated on the 10mL a-protein affinity chromatographic column, with the described post of phosphate buffered saline (PBS) (PBS) washing to baseline and with the azido-hMN-14IgG of pH3.5,0.1M sodium citrate elution of bound.Because 8 methionine residues/hMN-14IgG molecule is arranged, so≤8 azido groups can be incorporated among the IgG.

The technical staff will recognize that described technology is not limited to hMN-14 or IgG, but can use with any antibody, antibody fragment or other protein that can clone in host cell and express.

Embodiment 42. alkynyl metabolism are incorporated among the IgG

Can the alkynyl metabolism be incorporated among the hMN-14IgG by in culture medium, replacing methionine with 2-amino-5-hexynic acid.Making hMN-14IgG cellulation system grow to viable cell density in the roller bottle culture in 1L H-SFM culture medium is 1.5 * 10 ⁶Individual cell/mL.By the centrifugal cell precipitation that makes, and be suspended in once more in the H-SFM culture medium of having replenished 30mg/L2-amino-5-hexynic acid, no methionine.Make roller bottle culture at 37 ℃ and 5%CO ₂Following growth is reduced to below 30% until cell viability.Filter culture supernatants and also be coated on the 10mL a-protein affinity chromatographic column, wash described post to baseline and with the alkynes-hMN-14IgG of pH3.5,0.1M sodium citrate elution of bound with PBS.Because 8 methionine residues/hMN-14IgG molecule is arranged, so≤8 alkynyls can be incorporated among the IgG.

The technical staff will recognize that described technology is not limited to hMN-14 or IgG, but can use with any antibody, antibody fragment or other protein that can clone in host cell and express.

Embodiment 43. azido high lactamine metabolism are incorporated in the Fab-DDD2DNL module

Dimerization and docking structure territory (DDD) used in the DNL method mediate the proteinic stable dimerization that merges with it.Use and express the hPAM4-Fab-DDD2 that the DDD2 that is derived from the anti-stick protein monoclonal antibody hPAM4 of humanization merges the segmental stable transgenic cell line generation azide-modified of Fab.Before the pdHL2 plasmid expression vector stable transfection of the expression cassette by will containing hPAM4Fd-DDD2 and hPAM4 κ light chain had been researched and developed described cell line to rat bone marrow tumour host cell SpESFX.With the methionine residues among the azido high lactamine replacement hPAM4-Fab-DDD2.Make the growth of cellulation system reach 1.5 * 10 in the roller bottle culture in 1L H-SFM culture medium until viable cell density ⁶Individual cell/mL.By the centrifugal cell precipitation that makes, and be suspended in once more in the H-SFM culture medium of having replenished 30mg/L azido high lactamine, no methionine.Make roller bottle culture at 37 ℃ and 5%CO ₂Following growth is reduced to below 30% until cell viability.Filter culture supernatants and also be coated on the 10mLKappaSelect affinity chromatographic column, wash described post to baseline and with the azido-hPAM4-Fab-DDD2 of pH3.5,0.1M sodium citrate elution of bound with PBS.Each hPAM4Fab can have 2 azide and replace (2 methionine residues/hPAM4Fab), therefore stablize the hPAM4Fab-DDD2 dimer and can incorporate into≤4 azido groups.

Embodiment 44. alkynyl metabolism are incorporated in the Fab-DDD2DNL module

Can the alkynyl metabolism be incorporated among the hPAM4-Fab-DDD2 by in culture medium, replacing methionine with 2-amino-5-hexynic acid.Making hPAM4-Fab-DDD2 cellulation system grow to viable cell density in the roller bottle culture in 1L H-SFM culture medium is 1.5 * 10 ⁶Individual cell/mL.By the centrifugal cell precipitation that makes, and be suspended in once more in the H-SFM culture medium of having replenished 30mg/L2-amino-5-hexynic acid, no methionine.Make roller bottle culture at 37 ℃ and 5%CO ₂Following growth is reduced to below 30% until cell viability.Filter culture supernatants and also be coated on the 10mL KappaSelect affinity chromatographic column, wash described post to baseline and with the alkynes-hPAM4-Fab-DDD2 of pH3.5,0.1M sodium citrate elution of bound with PBS.Each hPAM4Fab can have 2 alkynes and replace (2 methionine residues/hPAM4Fab), therefore stablize the hPAM4Fab-DDD2 dimer and can incorporate into≤4 azido groups.

Claims (40)

1. One kind with ¹⁸The method of F labelled molecule comprises:

   A) make metal- ¹⁸The F complex is connected with chelating moiety; And

   B) described chelating moiety and molecule are connected to form ¹⁸The F labelled molecule,

   Wherein described chelating moiety is connected with described molecule by the click chemistry reaction.
2. 2. method according to claim 1, wherein said click chemistry reaction is selected from: (i) nitrone and cycloalkyne; (ii) azide and cycloalkyne.
3. 3. method according to claim 2, wherein said cycloalkyne are cyclooctyne.
4. 4. method according to claim 1, wherein said molecule are protein or peptide.
5. 5. method according to claim 1, wherein said molecule are the targeted molecular that is selected from antibody, monoclonal antibody, bi-specific antibody, multi-specificity antibody, antibody fusion protein, antigen binding antibody fragment and affine body.
6. 6. method according to claim 1, but wherein said molecule is the targeting construct.
7. 7. method according to claim 6 further comprises:

   C) use targeted molecular to the experimenter;

   D) reserving the described targeted molecular of enough time confessions combines with target antigen; And

   E) but use described targeting construct to described experimenter subsequently, but wherein said targeting construct combines with described targeted molecular.
8. 8. method according to claim 1, wherein said metal is selected from aluminum, gallium, indium, lutecium and thallium.
9. 9. method according to claim 8, wherein said metal are aluminum.
10. 10. method according to claim 1, wherein said chelating moiety are selected from DOTA, TETA, NOTA, NODA, NODA derivant, (tert-butyl group) ₂NODA, NETA, C-NETA, L-NETA, S-NETA, NODA-MPAA, NODA-MPAEM and NOTA derivant.
11. 11. method according to claim 1 further comprises:

    C) use to the experimenter described ¹⁸The F labelled molecule; And

    D) use PET scanning intravital described for described experimenter ¹⁸The F labelled molecule be scattered in picture.
12. 12. method according to claim 5 further comprises:

    C) use described targeted molecular to described experimenter;

    D) described targeted molecular is positioned on target cell, tissue, organ or the pathogen; And

    E) to described experimenter use with described metal- ¹⁸The bonded described chelating moiety of F complex;

    Wherein said chelating moiety combines with described targeted molecular by click chemistry reaction in the body.
13. 13. method according to claim 1, wherein in culture medium, add organic solvent so that described metal- ¹⁸The F complex is connected with chelating moiety.
14. 14. method according to claim 1, wherein by the heating or microwave irradiation make described metal- ¹⁸The F complex is connected with described chelating moiety.
15. 15. a method of sending diagnostic agent and/or therapeutic agent to target cell, tissue or pathogen comprises:

    But a) at least a diagnostic agent and/or therapeutic agent are connected with the targeting construct;

    B) use targeted molecular to the experimenter; And

    C) but use described targeting construct to described experimenter, but wherein said targeting construct combines with described targeted molecular by click chemistry reaction.
16. 16. method according to claim 15, wherein said click chemistry reaction is selected from: (i) nitrone and cycloalkyne; (ii) azide and cycloalkyne.
17. 17. method according to claim 17, wherein said cycloalkyne are cyclooctyne.
18. 18. method according to claim 15, wherein said targeted molecular are selected from antibody, monoclonal antibody, bi-specific antibody, multi-specificity antibody, antibody fusion protein, antigen binding antibody fragment and affine body.
19. 19. method according to claim 15, wherein said diagnostic agent or therapeutic agent are selected from radionuclide, cytotoxic agent, medicine, toxin, oligonucleotide, siRNA, short apoptosis agent, anti-angiogenic agent, enzyme, hormone, immunomodulator, boron compound, photosensitizer, fluorescent chemicals, chemiluminescence compound, bioluminescent compound, dyestuff, paramagnetic ion and contrast agent.
20. 20. method according to claim 15, wherein said diagnostic agent is ¹⁸The F-metal complex.
21. 21. a method for the treatment of disease comprises:

    But a) at least a therapeutic agent is connected with the targeting construct;

    B) use targeted molecular to the experimenter, wherein said targeted molecular with combine with the antigen of described disease association; And

    C) but use described targeting construct to described experimenter, but wherein said targeting construct combines with described targeted molecular by click chemistry reaction.
22. 22. method according to claim 21, but wherein said targeting construct comprises chelating moiety and described therapeutic agent combines with described chelating moiety.
23. 23. method according to claim 21, wherein said click chemistry reaction is selected from: (i) nitrone and cycloalkyne; (ii) azide and cycloalkyne.
24. 24. method according to claim 23, wherein said cycloalkyne are cyclooctyne.
25. 25. method according to claim 21, wherein said targeted molecular are selected from antibody, monoclonal antibody, bi-specific antibody, multi-specificity antibody, antibody fusion protein, antigen binding antibody fragment and affine body.
26. 26. method according to claim 21, wherein said disease are selected from cancer, cardiovascular diseases, metabolic disease, infectious disease, diseases associated with inflammation, autoimmune disease, immunologic function disorder disease, graft versus host disease, organ-graft refection, neuropathy, amyloidosis, 1 type or type 2 diabetes mellitus, amyotrophic lateral sclerosis (ALS), Parkinson's disease, hungtington's chorea, fibrin clot, atherosclerosis, myocardial ischemia, myocardial infarction, pulmonary infarction and Alzheimer.
27. 27. method according to claim 21, wherein said targeted molecular be selected from following antigen and combine: AFP, APRIL, B7, BCMA, BLyS, carbonic anhydrase IX, CCCL19, CCCL21, CCR4, CSAp, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM-6, complement factor C3, C3a, C3b, C5a, C5, CXCR2, CXCR4, CXCR7, CXCL12, the ED-B of fibronectin, EGFR, EGP-1 (TROP2), EGP-2, factor H, FHL-1, Flt-3, folate receptor, GRO-β, HLA-DR, HM1.24, HMGB-1, hypoxia inducible factor (HIF), HIF-1 α, HM1.24, Ia, insulin-like growth factor-i (IGF-1), IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, Le (y), MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5a-c, MUC16, NCA-95, NCA-90, PlGF, PSMA, RANTES, T101, TAC, TACI, TAG, tenascin, Tn antigen, Thomson-Friedenreich antigen, neoplasm necrosis antigen, TNF-α, TRAIL receptor (R1 and R2), VEGF, the oncogene product, gp41, gp120, amyloid-beta, SOD1, fibrin, LDL, glycoprotein iib/iiia, ICAM-1, beta 2 integrin alpha _νβ ₃, folate receptor 1 and myocardium myo globulin.
28. 28. method according to claim 21, wherein said therapeutic agent is selected from immunomodulator, hormone, cytotoxic agent, medicine, toxin, enzyme, antisense oligonucleotide, boron compound, photosensitizer and radionuclide.
29. 29. method according to claim 28, wherein said immunomodulator are selected from cytokine, stem cell factor, lymphotoxin, tumor necrosis factor, Hemopoietic factor, colony stimulating factor, interferon, erythropoietin, thrombopoietin or its combination.
30. 30. method according to claim 28, wherein said immunomodulator are selected from IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, IL-21, granulocyte-colony stimulating factor (G-CSF) and CM-CSF (GM-CSF).
31. 31. method according to claim 29, wherein said radionuclide is selected from ²²⁵Ac, ⁶⁸Ga, ⁶⁷Ga, ⁹⁰Y, ⁸⁶Y, ¹¹¹In, ¹³¹I, ¹²⁵I, ¹²³I, ^99mTc, ^94mTc, ¹⁸⁶Re, ¹⁸⁸Re, ^I77Lu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ²¹²Bi, ²¹³Bi, ³²P, ¹¹C, ¹³N, ¹⁵O, ⁷⁶Br and ²¹¹At.
32. 32. method according to claim 28, wherein said medicine is selected from vinca alkaloids, anthracycline antibiotics, epipodophyllotoxin (epipodophyllotoxin), taxane (taxane), antimetabolite, alkylating agent, antibiotic, cox 2 inhibitor, antimitotic agent, anti-angiogenic agent, apoptosis agent, amycin (doxorubicin), methotrexate (methotrexate), taxol (taxol), CPT-11, camptothecine (camptothecin), chlormethine, alkylsulfonate, nitroso ureas, triazenes (triazene), folacin, cox 2 inhibitor, pyrimidine analogue, purine analogue, platinum coordination complex, cyclophosphamide, etoposide (etoposide), carmustine (carmustine), vincristine (vincristine), procarbazine (procarbazine), prednisone (prednisone), methotrexate, bleomycin (bleomycin), dexamethasone (dexamethasone), folinic acid (leucovorin), phenyl butyrate, bryostatin-1 (bryostatin-1) and hormone.
33. 33. method according to claim 28, wherein said toxin are selected from ricin (ricin), abrin (abrin), DNA enzyme I, Staphylococcus aureus enterotoxin-A (Staphylococcal enterotoxin-A), pokeweed antiviral protein, gelonin (gelonin), diphtheria toxin, diphtherotoxin (diphtheria toxin), Pseudomonas exotoxin (Pseudomonas exotoxin) and pseudomonas endotoxin (Pseudomonas endotoxin).
34. 34. method according to claim 26, wherein said autoimmune disease is selected from immune-mediated thrombocytopenia, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, sjogren syndrome (Sjogren's syndrome), multiple sclerosis, Sydenham's chorea (Sydenham's chorea), myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, polyglandular syndrome, bullous pemphigoid, diabetes, purpura,Henoch-Schonlein (Henoch-Schonlein purpura), post-streptococcal infection nephritis, erythema nodosum, high iS-One arteritis (Takayasu's arteritis), Addison's disease (Addison's disease), rheumatoid arthritis, sarcoidosis, ulcerative colitis, erythema multiforme, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, pneumorrhagia nephritis syndrome (Goodpasture's syndrome), thromboangiitis obliterans, primary biliary cirrhosis, struma lymphomatosa (Hashimoto's thyroiditis), thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis/dermatomyositis, polychondritis, pemphigus vulgaris, wegener granulomatosis (Wegener's granulomatosis), membranous nephropathy becomes, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis/polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis and fibrosing alveolitis.
35. 35. method according to claim 25, wherein said antibody are selected from hR1 (anti-IGF-1R), hPAM4 (anti-cancer of pancreas mucin), hA20 (anti-CD20), hA19 (anti-CD19), hIMMU31 (anti-AFP), hLL1 (anti-CD74), hLL2 (anti-CD22), hMu-9 (anti-CSAp), hL243 (anti-HLA-DR), hMN-14 (anti-CEACAM5), hMN-15 (anti-CEACAM6), hRS7 (anti-EGP-1), hMN-3 (anti-CEACAM6), Ab124 (anti-CXCR4), Ab125 (anti-CXCR4), CC49 (anti-TAG-72), Tn (anti-PSMA), J591 (anti-PSMA), HuJ591 (anti-PSMA), AB-PG1-XG1-026 (anti-PSMA dimer), D2/B (anti-PSMA), G250 (anti-carbonic anhydrase IX), alemtuzumab (alemtuzumab) (anti-CD52), bevacizumab (bevacizumab) (anti-VEGF), Cetuximab (cetuximab) (anti-EGFR), gemtuzumab Ozogamicin Mylotarg CDP 771 (gemtuzumab) (anti-CD 33), ibritumomab tiuxetan (ibritumomab tiuxetan) (anti-CD20), handkerchief Buddhist nun monoclonal antibody (panitumumab) (anti-EGFR), Rituximab (rituximab) (anti-CD20), tositumomab (tositumomab) (anti-CD20), GA101 (anti-CD20), Herceptin (trastuzumab) (anti-ErbB), h679 (anti-HSG), h734 (anti-DTPA), A Bafu monoclonal antibody (abagovomab) (anti-CA 125), abciximab (abciximab) (anti-CD41), adalimumab (adalimumab) (anti-TNF-α), Afelimomab (afelimomab) (anti-TNF-α), A De wood monoclonal antibody (adecatumumab) (anti-EpCAM), trainingization A Zhu monoclonal antibody (alacizumab pegol) (anti-VEGFR2), ALD518 (anti-IL6), anatumomab mafenatox (anatumomab mafenatox) (anti-TAG72), A Sai pearl monoclonal antibody (aselizumab) (anti-CD 6 2L), A Li pearl monoclonal antibody (atlizumab) (anti-IL-6R), Ba Piniu hinders monoclonal antibody (bapineuzumab) (anti-directed against amyloid-beta albumen), basiliximab (basiliximab) (anti-CD25), Bei Nali pearl monoclonal antibody (benralizumab) (anti-CD125), bevacizumab (bevacizumab) (anti-VEGF-A), biciromab (biciromab) (antifibrin II), appropriate monoclonal antibody-the Wei Duoting in Bel (brentuximab vedotin) (anti-CD30), brodalumab (anti-IL-17), block appropriate rope monoclonal antibody (catumaxomab) (anti-EpCAM), ground, west pearl monoclonal antibody (cedelizumab) (anti-CD4), match trastuzumab (certolizumab pegol) (anti-TNF-α), Cetuximab (cetuximab) (anti-EGFR), clenoliximab (clenoliximab) (anti-CD4), but that wooden monoclonal antibody (conatumumab) (anti-TRAIL-R 2), decetuzumab (anti-CD40), Dary pearl monoclonal antibody (daclizumab) (anti-CD25), reach thunder wood monoclonal antibody (daratumumab) (anti-CD38), Yi Kuli monoclonal antibody (eculizumab) (anti-C5), Ai Ximo monoclonal antibody (elsilimomab) (anti-IL-6), E Masuo monoclonal antibody (ertumaxomab) (anti-HER2/neu), dust daclizumab (etaracizumab) (anti-alpha 2 integrin α _νβ ₃), method rope monoclonal antibody (fanolesomab) (anti-CD15), defend material monoclonal antibody (farletuzumab) (anti-folate receptor 1), fexakinumab (anti-IL-22), virtue trastuzumab (fontolizumab) (anti-IFN-γ), husband Lignum Sappan monoclonal antibody (fresolimumab) (anti-TGF-beta), Luo Shi monoclonal antibody (gantenerumab) (directed against amyloid-beta albumen), Jia Weimo monoclonal antibody (gavilimomab) (anti-CD147), gemtuzumab Ozogamicin Mylotarg CDP 771 azoles rice star (gemtuzumab ozogamicin) (anti-CD 33) difficult to understand, the sharp wooden monoclonal antibody (golimumab) (anti-TNF-α) of dagger-axe, Shandong former times monoclonal antibody (gomiliximab) (anti-CD23), Eibar pearl monoclonal antibody (ibalizumab) (anti-CD4), imciromab (imciromab) (anti-myosin), infliximab (infliximab) (anti-TNF-α), the appropriate wooden monoclonal antibody of English (intetumumab) (anti-CD51), Inolimomab (inolimomab) (anti-CD25), her monoclonal antibody (ipilimumab) (anti-CD152), her appropriate wooden monoclonal antibody (iratumumab) (anti-CD30), itolizumab (anti-CD 6), lebrikizumab (anti-il-13), lintuzumab (lintuzumab) (anti-CD 33), Shandong card wood monoclonal antibody (lucatumumab) (anti-CD40), horse handkerchief wood monoclonal antibody (mapatumumab) (anti-TRAIL-R 1), horse trastuzumab (matuzumab) (anti-EGFR), mepolizumab (mepolizumab) (anti-IL-5), mogamulizumab (anti-CCR4), muromonab-CD3 (muromonab-CD3) (anti-CD3), how former times wood monoclonal antibody (necitumumab) (anti-EGFR), ponezumab (directed against amyloid-beta albumen), PRO140 (anti-CCR5), thunder is Lu Dankang (remucirumab) (anti-VEGFR2) not, Raleigh pearl monoclonal antibody (rontalizumab) (anti-IFN-α), ruplizumab (ruplizumab) (anti-CD 40 L), the former times monoclonal antibody (teneliximab) (anti-CD40) for how, for sharp pearl monoclonal antibody (teplizumab) (anti-CD3), TGN1412 (anti-CD28), trastuzumab (tocilizumab) (anti-IL-6R), tositumomab (tositumomab) (anti-CD20), Herceptin (trastuzumab) (anti-HER2/neu), Qu Jiazhu monoclonal antibody (tregalizumab) (anti-CD4), tie up western pearl monoclonal antibody (visilizumab) (anti-CD3) and prick wooden monoclonal antibody (zanolimumab) (anti-CD4).
36. 36. method according to claim 21 further comprises:

    D) but described targeted molecular or targeting construct are expressed in cell;

    E) in the culture medium that comprises nitrine acetylglucosamine, nitrine high lactamine or 2-amino-5-hexynic acid, cultivate described cell; And

    F) but described nitrine acetylglucosamine, nitrine high lactamine or 2-amino-5-hexynic acid are incorporated in described targeted molecular or the targeting construct.
37. 37. a DNL construct comprises:

    A) with first albumen or the peptide that are connected from the proteic anchoring structure of AKAP territory (AD) part; With

    B) with second albumen or the peptide that are connected with the domain that berths (DDD) part from the dimerization of protein kinase A (PKA);

    Wherein the described DDD of 2 copies partly forms dimer and combines to form targeted delivery complex and wherein said DDD part covalently bound with described AD part by the click chemistry reaction with described AD part.
38. 38. according to the described DNL construct of claim 37, wherein said click chemistry reaction is selected from: (i) nitrone and cycloalkyne; (ii) azide and cycloalkyne.
39. 39. according to the described DNL construct of claim 38, wherein said cycloalkyne is a cyclooctyne.
40. 40. according to the described DNL construct of claim 37, wherein said albumen or peptide are selected from monoclonal antibody, bi-specific antibody, multi-specificity antibody, antibody fusion protein, antigen binding antibody fragment, cytokine and affine body.

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