the present invention provides simple and efficient methods for incorporating the f - 18 radionuclide into peptide - containing targeting vectors , such as proteins , antibodies , antibody fragments and receptor - targeted peptides . for convenience , the term “ peptide ” is used below and in the claims to refer to proteins , antibodies , antibody fragments and receptor - targeted peptides . the methods of the present invention makes such targeting vectors available for routine clinical positron emission tomography . of all nucleophiles present on peptides , only the free thiol group can be rapidly alkylated at neutral ph and moderate temperature . the present invention takes advantage of this unique property of free thiol groups , and provides methods for labelling thiol - containing peptides with f - 18 . in accordance with one embodiment , the method of the present invention comprises the following reaction : wherein n is 0 , 1 or 2 , m is 0 , 1 or 2 , and n + m is 0 , 1 , or 2 , and x is a substitutable leaving group such as iodide , bromide , chloride , azide , tosylate , mesylate , nosylate , triflate and the like . alternatively , x is maleimide or a substituted maleimide , substituted , for example with one or two alkyl groups or a sulfonate group . examples of suitable substituted maleimides include 3 - methylmaleimide , 3 , 4 - dimethylmaleimide and 3 - sulfo - maleimide . r 1 and r 2 can be the same or different and , as discussed in more detail below , are chosen for the desirable physical properties they bring to the reagent . in general , r 1 and r 2 can be selected from the same groups as x , and can be the same as or different from x . alternatively , r 1 and r 2 each independently can be hydrogen , a substituted or unsubstituted linear or branched alkyl group , or a carbonyl function such as an ester , amide or ketone , for example , — coor ′, — conr ′ 2 , or cor ′, where r ′ is a c 1 - c 6 alkyl or phenyl . examples of suitable r 1 and r 2 groups or substituents thereon also include groups which impart aqueous solubility , such as — conh 2 , carboxyl , hydroxyl , sulfonic acid and tertiary amine or quaternary ammonium . in accordance with another embodiment of the invention , the peptide is labeled with an f - 18 fluorinated alkene , wherein at least one of the two double - bonded carbon atoms bears at least one leaving group selected from the group consisting of iodide , bromide , chloride , azide , tosylate , mesylate , nosylate and triflate . examples of suitable fluorinated alkenes include 18 f — ch ═ ci 2 , 18 f — ci ═ ch 2 , or 18 f — ci ═ ci 2 . the labeling reaction is analogous to the one described above . the methods of present invention can be used to label any thiol - containing peptide . of particular interest are peptides useful as targeting vectors . examples of such targeting vectors include antibodies , f ( ab ′) 2 , f ( ab ) 2 , fab ′ and fab fragments , single - chain sub - fragments such as sfvs , divalent constructs such as dsfvs , and polypeptides containing one or more free thiol groups . see choi et al ., cancer res ., 55 : 5323 - 29 ( 1995 ). further examples include antibody constructs such as antibodies comprising igg 3 or igg 3 - f ( ab ′) 2 frameworks . igg 3 &# 39 ; s have multiple hinge - region disulfide groups which can be reduced to generate multiple free thiol groups . peptides that originally do not comprise a free thiol group can be labelled in accordance with the present invention by first modifying the peptide to add a free thiol group by methods known to those skilled in the art . for example , the peptide can be thiolated with reagents such as 2 - iminothiolane , or intrinsic disulfide bonds such as cystine residues can be reduced . a combination of both modifications also can be performed , such as the acylation of lysine residues with n - succinimidyl - 3 -( 2 - pyridylthio )- propionate ( spdp ) followed by the controlled reduction of the appended disulfide bond . in one embodiment of the present invention , the peptide is a fab or fab ′ fragment . these peptides have free thiol groups in their hinge - region , a site which is both specific and remote from the antigen - targeting sites . to optimize the reaction with the thiol - containing peptides , the labelling reagent preferably has the following physical and chemical properties : ( 2 ) the reagent has adequate aqueous solubility in the neutral ( 4 - 8 ) ph range . by “ adequate aqueous solubility ” is meant that the reagent readily dissolves at up to a concentration comparable to a stoichiometric amount of the thiol - containing peptide used . if , for example , an antibody is being labeled , a typical antibody concentration is about 50 mg / ml , which corresponds to a molar concentration of about 3 × 10 − 4 m . in this example , the reagent should be soluble at a concentration of about 3 × 10 − 4 m . with lower molecular weight peptide species , more peptide will dissolve without precipitation , and more reagent can be used . because f - 18 is carrier - free , lower concentrations of fluorination agents also might be effective . ( 3 ) the active halides of the reagent are not immediately hydrolyzed by water at neutral ph ( ph 4 - 8 ). thus , the halides should react more readily with sh or s − than with h 2 o . as long as the reagent is not immediately hydrolyzed by water ( or by neutral buffer solutions ), the selectivity and reactivity of the thiol group ensures an efficient peptide labeling reaction . ( 4 ) the leaving group x can be displaced rapidly , specifically , and near - quantitatively by free thiol moieties . a carbo - cationic center can be developed at the carbon atom which is attacked by the nucleophile , for example , r 1 and r 2 can be electron - withdrawing groups . the presence of electron - withdrawing groups alpha to the — c — x functional group also facilitates fast displacement of the x moiety . examples of useful electron - withdrawing groups include — cor ′, — conr ′, — co 2 r ′, — cooh , — conh 2 , and — so 3 h , where r ′ is a c 1 - c 6 alkyl or phenyl . in addition , the presence of more than one leaving group in the labelling reagent can be advantageous . multiple leaving groups , such as iodo groups , attached to the same carbon atom produce steric strain . when a reaction comprises the departure of a single leaving group , this steric strain is relieved , imparting faster reaction kinetics to the thiol displacement of the x group . thus , in accordance with one embodiment of the invention , the labeling reagent comprises at least two leaving groups , such as two iodo groups . in accordance with one embodiment of the present invention , the peptide is labeled with a labelling reagent of the general formula 18 f —( ch 2 ) m — cr 1 r 2 —( ch 2 ) n — x , wherein n is 0 , 1 or 2 , m is 0 , 1 or 2 , and n + m is 0 , 1 , or 2 , and x is a substitutable leaving group such as iodide , bromide , chloride , azide , tosylate , mesylate , nosylate , triflate , and the like . alternatively , x is maleimide or a substituted maleimide , substituted , for example with one or two alkyl groups . examples of suitable substituted maleimides include 3 - methylmaleimide , 3 , 4 - dimethylmaleimide and 3 - sulfo - maleimide . r 1 and r 2 can be the same or different and , as discussed above , are chosen for the desirable physical properties they bring to the reagent . in general , r 1 and r 2 can be selected from the same groups as x , and can be the same as or different from x . alternatively , r 1 and r 2 each independently can be hydrogen , a substituted or unsubstituted linear or branched alkyl group , or a carbonyl function such as an ester , amide or ketone , for example , — coor ′, — conr ′ 2 , or cor ′, where r ′ is a c 1 - c 6 alkyl or phenyl . examples of suitable r 1 and r 2 groups or substituents thereon also include those which impart aqueous solubility , such as — conh 2 , carboxyl , hydroxyl , sulfonic acid and tertiary amine or quaternary ammonium . examples of suitable labelling reagents include 18 f - ci 3 ; 18 f - chi 2 ; 18 f - ci 2 cooh ; 18 f - ci 2 cooch 3 ; 18 f - ci 2 ch 2 oh ; 18 f - chich 2 oh ; 18 f - chicooch 3 ; 18 f - ci 2 ch 2 cooh ; 18 f - ci 2 ch 2 n + ( ch 3 ) 3 ; 18 f - ci 2 ch 2 - maleimide ; 18 f - ci 2 - conh 2 ; 18 f - ci 2 - co 2 ch 3 ; 18 f - chbr 2 ; 18 f - cbr 2 ch 2 ch 2 - so 3 h ; 18 f - ch 2 ci 2 cooh ; 18 f - ch 2 ci 2 conh 2 ; 18 f - chico 2 ch 3 ; 18 f - ci 2 conh 2 ; 18 f - chiconh 2 ; 18 f - cbr 2 ch 2 oh ; ch 3 cocbr 2 - 18 f ; cbrf 2 - 18 f ; 18 f - cbr ( conh 2 ) 2 , and c 6 h 5 - cocbr 2 - 18 f . other suitable labeling reagents will be apparent to those skilled in the art . the labeling reagent can be made by the f - 18 fluorination of a corresponding compound . the following are examples of compounds which can be fluorinated to make the labeling reagents set forth above : ci 4 ; chi 3 ; chi 2 cooch 3 ; ci 3 cooh ; ci 3 cooch 3 ; ci 3 ch 2 oh ; chi 2 ch 2 oh ; ci 3 ch 2 cooh ; ci 3 ch 2 n + ( ch 3 ) 3 ; ci 3 ch 2 - maleimide ; ci 3 - conh 2 ; ci 3 - co 2 ch 3 ; chibr 2 ; clbr 2 ch 2 ch 2 - so 3 h ; ch 2 ci 3 cooh ; ch 2 ci 3 conh 2 ; chi 2 co 2 ch 3 ; ci 3 conh 2 ; chi 2 conh 2 ; cbr 3 ch 2 oh ; cf 3 coci 3 ; ch 3 cocbr 3 ; br 2 chcn ; ci 3 chcn ; cbr 2 f 2 ; cbr 2 ( conh 2 ) 2 and c 6 h 5 - cocbr 3 . other suitable compounds will be apparent to those skilled in the art . in accordance with another embodiment of the invention , the labeling reagent is an f - 18 fluorinated alkene , wherein at least one of the two double - bonded carbon atoms bears at least one leaving group selected from the group consisting of iodide , bromide , chloride , azide , tosylate , mesylate , nosylate and triflate . examples of suitable fluorinated alkenes include 18 f - ch ═ ci 2 , 18 f - ci ═ ch 2 , and 18 f - ci ═ ci 2 . these labeling reagents can be made by the f - 18 fluorination of corresponding compounds , such as ich ═ ci 2 ; ci 2 ═ ch 2 ; ci 2 ═ ci 2 . other fluorinated alkenes useful in accordance with the present invention will be apparent to those skilled in the art . f - 18 can be obtained from cyclotrons after bombardment of o - 18 - enriched water with protons . the enriched water containing h - 18 f can be neutralized with a base having a counter - ion that is any alkali metal ( m ), such as potassium or another monovalent ion , and the water can be evaporated off to give a residue of m - 18 f , which can be taken up in an organic solvent for further use . in general , the counter - ion is selected to enable the fluoride ion to react rapidly in an organic phase with a halogen . potassium is generally used as a counter - ion because it is cheaper than cesium . however , with carrier - free f - 18 , trivial amounts of counter - ion are required , and counter - ion cost largely can be ignored . although potassium is useful as a counter - ion in accordance with the present invention , cesium is preferred to potassium because cesium is a larger ion with a more diffuse charge . accordingly , cesium has looser ionic interactions with the small fluoride atom , and therefore does not interfere with the nucleophilic properties of the fluoride ion . for similar reasons , potassium is preferred to sodium , and , in general , the suitability of a la metal as a counter - ion in accordance with the present invention increases as you go down the periodic table . group ib reagents , such as silver , also are useful as counter - ions in accordance with the present invention . further , organic phase transfer - type ions , such as tetraalkylammonium salts , also can be used as counter - ions . because fluoride is the most electronegative element , it has a tendency to become hydrated and lose its nucleophilic character . to minimize this , the labeling reaction is preferably performed under anhydrous conditions . for example , fluoride ( as potassium fluoride or as a complex with any of the other counter - ions discussed above ) can be placed in organic solvents , such as acetonitrile or thf . with the help of agents which bind to the counter - ion , such as kryptofix 2 . 2 . 2 ( 4 , 7 , 13 , 16 , 21 , 24 - hexaoxa - 1 , 10 - diazabicyclo [ 8 . 8 . 8 ]- hexacosane ), the fluoride ion is very nucleophilic in these solvents . as discussed above , the labeling reagent is used to label targeting vectors comprising a thiol - containing peptide with f - 18 according to the following reaction : alternatively , the labeling reagent is a f - 18 fluorinated alkene , wherein at least one of the two double - bonded carbon atoms bears at least one leaving group selected from the group consisting of iodide , bromide , chloride , azide , tosylate , mesylate , nosylate and triflate . this f - 18 fluorinated alkene labels targeting vectors in an analogous manner to the reaction set forth above . directing the reaction of the fluorinated labeling reagent towards free thiol groups on the targeting vector allows near - quantitative incorporation of f - 18 into the targeting vector within a short time period . generally , the reaction will be completed within a few minutes at room temperature , and complicated purification steps will not be necessary . given the very short half - life of f - 18 , the speed of the reaction is very important . moreover , because free f - 18 exchanges readily with hydroxyl ions in hydroxyapatite crystals in bone , and , therefore , is a bone - seeking agent , the reduced amount of free fluoride remaining in the final product also is an important advantage of the present invention . the embodiments of the invention are further illustrated through examples which show aspects of the invention in detail . these examples illustrate specific elements of the invention and are not to be construed as limiting the scope thereof . 100 mci of f - 18 fluoride ( obtained from bombardment of o - 18 - enriched water ) in dry tetrahydrofuran containing kryptofix 2 . 2 . 2 ( 4 , 7 , 13 , 16 , 21 , 24 - hexaoxa - 1 , 10 - diazabicyclo [ 8 . 8 . 8 ] hexacosane ) and a slurry of potassium carbonate is treated with triiodoacetic acid . after a 30 minute reaction at room temperature , the desired labelling reagent , 18 f - ci 2 cooh , is obtained and purified by reverse - phase column chromatography . this labelling reagent is then used to label a variety of thiol - containing targeting vectors , or is shipped to clinical sites for the same usage . a 1 mg vial of lyophilized fab ′- sh - np4 ( an anti - cacinoembryonic antigen antibody fragment ) is reconstituted with 1 ml of a solution of 18 f - ci 2 cooh in 0 . 1 m sodium acetate buffer at ph 6 . the reaction is allowed to proceed for 30 minutes at room temperature . an aliquot of the mixture is removed for analysis by hplc using a size - exclusion sizing column and by itlc ( instant thin - layer chromatography ) using silica gel - impregnated glass - fiber strips ( gelman sciences ). this analysis reveals that the antibody fragment &# 39 ; s hinge - region thiol groups effect nucleophilic displacement of both iodine atoms of 18 f - ci 2 cooh , and that this reaction proceeds in near - quantitative yield . the f - 18 - labeled fab ′ fragment is therefore ready for injection . a sample of 100 mci of f - 18 fluoride ( obtained from bombardment of o - 8 - enriched water ) in dry acetonitrile containing kryptofix 222 and a slurry of potassium carbonate is treated with triiodomethane . after a 30 minute reaction at room temperature the labelling reagent 18 f - chi 2 is obtained and purified by reverse - phase column chromatography . the labeling reagent is then used to label a variety of thiol - containing targeting vectors , or is shipped to clinical sites for the same usage . a 1 mg vial of lyophilized , reduced octreotide ( d - phe - cys - phe - d - trp - lys - thr - cys - thr - ol ) is reconstituted with 1 ml of a solution of 18 f - chi 2 ( made up first in dmso ) in 0 . 1 m sodium acetate buffer at ph 6 , containing 20 % dmso . the reaction is allowed to proceed for 30 minutes at room temperature . alternatively , can be effected at elevated temperatures , and in non - aqueous solvents , e . g ., dmso , and later cooled and / or diluted for injection . an aliquot of the labeling mixture is removed for analysis by hplc using a size - exclusion sizing column and itlc ( instant thin - layer chromatography ) using silica gel - impregnated glass - fiber strips ( gelman sciences ). this analysis reveals that the two cysteinyl thiol groups of octreotide effect the nucleophilic displacement of both iodo atoms of 18 f - chi 2 and that this reaction proceeds in near - quantitative yield . the f - 18 - labeled , recyclized ( linkage : — s — ch — 18 f — s —) octreotide peptide is therefore ready for injection . fluorodiiodoacetamide ( 18 f - ci 2 conh 2 ) 100 mci of f - 18 fluoride ( obtained from bombardment of o - 18 - enriched water ) in dry tetrahydrofuran containing kryptofix 2 . 2 . 2 ( 4 , 7 , 13 , 16 , 21 , 24 - hexaoxa - 1 , 10 - diazabicyclo [ 8 . 8 . 8 ] hexacosane ) and a slurry of potassium carbonate is treated with triiodoacetamide . after a 30 minute reaction at room temperature , the desired labelling reagent , 18 f - ci 2 conh 2 , is obtained and purified by reverse - phase column chromatography . this labelling reagent is then used to label a variety of thiol - containing targeting vectors , or is shipped to clinical sites for the same usage . a 1 mg vial of lyophilized cys - lhrh ( lhrh whose amine terminus bears an appended cysteine , in reduced , thiol , form ) reconstituted with 1 ml of a solution of 18 f - chiconh 2 in 0 . 1 m sodium acetate buffer at ph 6 . the reaction is allowed to proceed for 2 hours at 50 ° c . the antibody modified peptide &# 39 ; s thiol group effects nucleophilic displacement of the iodo atom of 18 f - cihconh 2 , and the reaction proceeds in near - quantitative yield . the f - 18 - labeled peptide is ready for injection . it will be apparent to those skilled in the art that various modifications and variations can be made to this invention . thus , it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the claims and their equivalents .
thiol - containing peptides can be radiolabeled with fluorine - 18 by reacting a peptide comprising a free thiol group with an 18 f - bound labelling reagent which also has a group that is reactive with thiols . the resulting 18 f - labeled peptides may be targeted to a tissue of interest using bispecific antibodies or bispecific antibody fragments having one arm specific for the 18 f - labeled peptide or a low molecular weight hapten conjugated to the 18 f - labeled peptide , and another arm specific to the targeted tissue . the targeted tissue is subsequently visualized by clinical positron emission tomography .