CA2165052A1

CA2165052A1 - Radionuclide metal chelates for the radiolabeling of ligands, anti-ligands or other proteins

Info

Publication number: CA2165052A1
Application number: CA 2165052
Authority: CA
Inventors: Linda M. Gustavson; Sudhakar Kasina; Alan R. Fritzberg
Original assignee: Individual
Current assignee: Poniard Pharmaceuticals Inc
Priority date: 1993-07-12
Filing date: 1994-07-12
Publication date: 1995-01-26
Also published as: EP0792170A1; EP0792170A4; WO1995002418A1

Abstract

Chelating compounds of specified structure are useful for radiolabeling targeting proteins such as antibodies as well as proteinaceous and non-proteinaceous ligands and anti-ligands. The radiolabeled antibodies, ligands or anti-ligands, or catabolites thereof, demonstrate improved biodistribution properties, including reduced localization within the intestines.

Description

~Wo 95/02418216 ~i O S ~ PCT/US94/07733 \ RADIONUCLIDE METAL CHELATES
FOR THE RADIOLABELING OF LIGANDS, ANTI-LIGANDS OR Ol~K PROTEINS

5 Background l~diol~bel~d antibodies are used in a variety of ~ gnostic and thela~eulic meAi~-~l procedures. The increased srPrifirity of monoclonal antibodies, CO~ )a~'t;d to polyclonal antibodies, makes them even more useful for deliveringgnostic or th~apeuLic agents such as r~rlioi~otopes to desired target sites in 10 vivo. A monoclc n~l antibody specific for a desired type of target cells such as tumor cells may be used to deliver a thel~apeulic r~-liom~çli~le ~tt~-hçd to theantibody to the target cells, thereby causing the er~rlic~tion of the undesired target cells. ~ltp-rn~tively~ a monoclonal antibody having a ~ gnosti~ ~lly ~rrecliv~ iionllcli~le ~tt~hed thereto may be ~1mini~tered~ whel~upoll the 15 r~-liol~kelP~ antibody loc~li7Ps on the target tissue. Conventional diagnostic procedur~s then may be used to detect the presence of the target sites within the patient. In contrast to such "chelate-labeled antibody" pr~celules, pr~ elillg approaches may be used to achieve th~.~p~ul;c or diagnostic goals, which y~ lh~g approaches involve the intpractioll of two members of a high affinity 20 binding pair such as a ligand-anti-ligand binding pair.
One method for r~-liol~heling proteins such as antibodies as well as pn~teinaceous and non-~roleillaceous binding pair members involves ~tt~chm~Pnt of radionuclide metal chPl~tPs to the proteins or binding pair members. ChPl~tPShaving a variety of c-hemir~l structures have been developed for this pul~ose.
25 The usefulness of such çhPl~tPS is dependent upon a number of factors such asthe stability of radionuclide binding within the chelate and the reactivity of the chelate with the desired protein or binding pair member. The çfficiçnçy of ~iiol~heling of the çh~l~tin~ colllpoulld to produce the desired radionuclide metal chelate also is important. Another consideration is the biodistribution of30 the r~-1iol~heled antibody or binding pair member and catabolites thereof in vivo.

SUBSTIME SHEET (~.VEE 2~J

wo 95/02418 PcT/uss4/07733 ~
21B~5~
. i Loc~1i7~tion in non-target tissues limits the total dosage of a the~dlJeuLic ra-lio1~heled antibody or binding pair member that can be ~-lmini~tPred, therebydecreasing the th~ ulic effect. In ~ gnostic procedures, loc~li7~ticn in non-target tissues may cause 1lnrlP~ h1e bacL~ r~ulld and/or result in mi~ gnosi~.
5 The need remains for improvement in these and other characteri~ti~s of r~licn1)c1id~P metal chelate co~ ou.lds used for ra-lio1~hP1ing of proteins such as antibodies. The use of ~ e~ g approaches ~imini~hPs non-target tissue loc~1i7~tion of r~-lin1~bel; however, the need remains for i.l.plove--.ent in mol~cul~s inccl~ oldling c*P1~tes and binding pair members of proteinaceous or 10 non-protein~ceQus structure.

Su-----l~y of the Invention The present invention provides a co-..pound of the ftlrmul~

R I R
\r(c~

R~/ N N ~R
",(R-C) (GR)m S S
T T
wherein:
each R independent1y l~Lesents =O, H2, a lower alkyl group, -(CH2)~-15 COOH, -(CH2)n-CO-s~c ch~ricle or s~cch~ride derivative, or -(CH2)n-NH-~cch~ride or s~ch~ritle derivative, or Rl-Z;
nisOtoabout3;
Rl re~lt;sell~s a lower alkyl or substituted lower alkyl group;
Z re~Lesellts a protein conjugation group, a ligand conjugation group, an 20 anti-ligand conjugation group or a targeting protein, ligand or anti-ligand; or a SUBSTITUTE SHEET (Rl ILE 26) ~WO 95/02418 2 1 6 ~ n ~j 2 PCT/US94/07733 ligand-linker moiety or an anti-ligand-linker moiety wherein the linker moiety is derived from a ligand or anti-ligand conjugation group;
each R2 independçntly rq?resellts H2, a lower alkyl group, -(CH2)n-COOH, -(CH2)n-CO-~ch~nde or ~cch~ri~e derivative, or -(CH2)n-NH-5 ~h~ritle or ~ ch~ride deliv~L~ive, or Rl-Z;
each m is 0 or 1, with at most one m = 1;
each T .epl~sellls a sulfur pn~cLing group; and the co...Lou.ld comp~i~es at least one (CH2)n-COOH substituent or one -(CH2)n-CO-sacch~nde or s~cch~ le derivative or -(CH~)n-NH-sa(ch~ride or 10 ~rch~ricle derivative substituent and one -Rl-Z substituent The present invention also provides radionuclide metal chelate compounds of the formtll~

R I R
\l (C),"~/

R~/ \ / \rR

R2~ / \ ~2 wherein: S S

M ~ resen~s a radionuclide metal or oxide thereof and the other symbols 15 are as described above.
These compounds comprise a targeting protein such as an antibody, or a conjugation group for ~tt~chm~nt of the co~ oulld to a targeting protein.
Allær.~ti~ely, the colllpounds include a ligand or an anti-ligand or a conjugation group for the ~ttachmPnt of the compound to a ligand or to an anti-ligand. The 20 ch~l~ting colllpoulld may be ~tt~ hçd to a ~ge~il g protein, ligand or anti-ligand and subsequently r~liol~heled. ~ltern~tively, the r~io~u-~li(le metal chelate co.l.pound may be pr~pared and then ~tt~h~ to a targeting protein, ligand o anti-ligand. The res~-lting radiolabeled targeting proteins, ligands or anti-ligands SUBSTITUTE SHEET tRULE 26) WO 95/02418 PCT/US94/07733 ~

2~0~ ~ _ ,, , ~ ~

are useful in diagnostic and th~ldp~;u~ic me~lic~l procedures. An example of a targeting protein is a monoclonal antibody that binds to cancer cells. An example of a ligand is biotin, with the complP~mPnt~ry anti-ligand thereof beingavidin or streptavidin, wherein biotin and avidin or streptavidin together form a S ligand-anti-ligand binding pair.
Some ~ ition~l colll~wlds of the present invention incol~oldle an ester cleavable Rl moietv exhibiting, for example, ester and/or amide functi- n~litiPs.
An example of a chelate-biotin conjugate of this aspect of the present invention, involving a succinate mono-ester mono-amide, is shown below:

ON~ \ CH2~(CH2~2 C NH-C~CH~--N~C--(CH~ S
NH~O - - ~
H~NH

Y S S/\Y Each Y - H or CH2COOH
aCM EOE

10 wherein X is H or COOH.
The carboxylic acid substihuPnt(s) on the compounds of the present invention are believed to assist in chtql~tion of a r~ionu~lide and to contribute to improved biodistribution plop~llies of catabolites of the r~diol~heled targetingproteins, ligands or anti-lig~n~ RP~uced loc~li7~tion of radio~tivity within the15 intestinps is achieved using the r~(liol~beled targeting proteins, ligands or anti-ligands of the present invention.

Brief Description of the Drawings Figures 1-7 depict chemic~l synthesis procedures that may be used to t; certain chpl~tin~ co,ll~ounds of the present invention.

SUBSTIME SHET (RULE 26) ~ WO 9~/02418 PCTIUS94/07733 21ssn~2 Figure 8 depicts the tumor uptake profile of NR-LU-10 streptavidin conjugate (LU-10-StrAv) in colllp~ on to a control profile of native NR-LU-10 whole antibody.

Detailed Desc~ ion of the Invention Prior to setting forth the invention, it may be helpful to set forth ~çfinition~ of certain terms to be used within the disclosure.
Targeting moiety: A molecule that binds to a defined population of cells.
The l~u~eLiilg moiety may bind a rece~or, an oligonucleotide, an enzymatic substrate, an antigenic delel."i~ -l, or other binding site present on or in thetarget cell population. Targeting moi~ti~s that are proteins are referred to herein as "~ ,e~ g proteins." Antibody is used throughout the spe~ific~tion as a prot~"ypical example of a ~r~,eLing moiety and a ~ ing protein. Tumor is used as a pf~o~y~3ical ~Y~mple of a target in describing the present invention.
T.i~nd/anti-ligand pair: A complem~nt~ry/anticomplem~nt~ry set of molecules that d~mon~tr~tç specific binding, generally of relatively high affinity.
FYem~ ry ligand/anti-ligand pairs include zinc finger protein/dsDNA fragm~nt hapten/antibody, lectin/carbohydrate, ligand/rece~lor, and biotin/avidin.
Biotin/avidin is used throughout the spe~ific~tio~ as a ~n~toly~ical example of a ligand/anti-ligand pair.
Anti-ligand: As defined herein, an "anti-ligand" demon~tr~tes high affinlty, and preferably, multivalent binding of the complem~-nt~ry ligand.
Preferably, the anti-ligand is large enough to avoid rapid renal çl~r~nçe, and contains s~fficient multivalency to accomplish cros~linking and agg~egaLion of ~r~li"g moiety-ligand conjugates. Univalent anti-ligands are also co~ ",pl~tçd 25 by the present invention. Anti-ligands of the present invention may exhibit or be derivitized to exhibit structural featu,es that direct the uptake thereof, e.g.,galactose residues that direct liver uptake. Avidin and streptavidin are used herein as prototypical anti-lig~nds.

SUBSTITUTE SHEET ~RULE 26) 2~5~2 i . :

Avidin and Streptavidin: As defined herein, both of the terms "avidin"
and "streptavidin" include avidin, streptavidin and derivatives and analogs thereof that are capable of high affinity, multivalent or univalent binding of biotin.
Tig~nd: As defined herein, a "ligand" is a relatively small, soluble S molecllle that eYhihit~ rapid serum, blood and/or whole body clearance when ~lmini~t~red intravenously in an animal or human. Biotin is used as the proL()Ly~ical ligand.
Pl~L~u~etillg: As defined herein, ~l~Lalg~ling involves target site loc~li7~ti~ n of a targeting moiety that is conjugated with one member of a 10 ligand/anti-ligand pair; after a time period s~-ffici~nt for optimal target-to-non-target ~ccuml-l~tic-n of this L~geLillg moiety conjugate, active agent conjugat~d to the o~;Le llenlbe~ of the ligand/anti-ligand pair is ~rlmini~tered and is bound(di~ ;lly or indirectly) to the targeting moiety conjugate at the target site (two-step ~.c~t;ting). Three-step and other related methods described herein are 15 also encomp~e~l.
~ .ink~r Moiety: A moiety that is a portion of a protein, ligand or anti-ligand conjugation group that remains part of the structure of a protein-chelate, ligand-chelate or anti-ligand-chelate conjugate following the conjugation step.
For eY~mple, the lirLker moiety of an active ester chelate derivative inrlll-les, for 20 eY~mple, a carbonyl (-CO-) moiety.
The present invention provides chpl~tinE compounds and radionuclide metal chelate compounds pf~paled the.~f O.ll, as well as radiolabeled proteins, ligands or anti-ligands having the chel~tes attached thereto. The r~rlionllrlicle metal ch~l~tes of the present invention are ~tt~rhed to targeting proteins such as 25 antibodies to form r~(liol~beled targeting proteins having diagnostic or the.~ll.e~ll;c use. The col-li)ounds each comprise a targeting protein or a protein conjugationgroup for ~tt~rhment of the compound to a targeting protein. ~ltPrn~tively, the r~-lionu~lide metal chrl~tes of the present invention are attached to ligands oranti-ligands to form r~-liol~heled ligands or anti-ligands having diagnostic or 30 the-~euLic use. Such compounds include a ligand or anti-ligand conjugation SllBSTITUTE SHEET ~RULE 26) Wo 9510241& 2 1 6 5 n s ~ PCT/US94/07733 group to f~cilit~te ~tt~hm~nt of the co~ ound to a ligand or anti-ligand. The compounds also comprise at least one carboxylic acid substihlçnt The good radiolabeling yields (i.e., chelate formation) achieved with these compounds arebelieved to be attributable, at least in part, to the presence of the carboxylic acid S subs~ tont(s). The improved biodistribution prop~llies of the r~iol~heled pl`Ok',illS of the invention also are believed to be at least in part attributable to the carboxylic acid substituçnt(s) on the ch~l~te.
Provided by the present invention are ch~l~tin~ co,--pounds of the following formula:

R I R
\r(c)~/
R--I' N N `rR

m(R-C) (GR)m R21\ ~LR2 wherein:
each R indepen~çntly l~.esents =O, H2, a lower alkyl group, -(CH2)n-COOH, -(CH2)n-CO-~t~çh~ride or s~ch~ri~le derivative, or -(CH2)n-NH-s~cch~ride or sacçh~ride derivative, or R,-Z;
n is 0 to 3;
R~ t;sents a lower alkyl or substituted lower alkyl group;
Z f~lc;se-l~ a protein conjugation group, a ligand conjugation group, an anti-ligand conjugation group or a ~gelillg protein, ligand or anti-ligand; or a ligand-linker moiety or an anti-ligand-linker moiety wherein the linker moiety is derived from a ligand or an anti-ligand conjugation group;
each R2 independently r~r~sel-~ H2, a lower alkyl group, -(CH2),l-COOH, -(cH2)n-co-s~çh~ride or s~çch~nde derivative, or -(CH2)n-NH-~cçh~ e or .~cçh~ le derivative, or Rl-Z;

SU~STITUTE SHEET (~ILE 26) WO 95/02418 PCT/US94/07733 ~
216~0~

each m is O or 1, with at most one m = l;
each T l~resellts a sulfur ~rote~;ling group; and the co~ uu,ld compri~Ps at least one -(CH2)n-COOH substituent -(CH2)n-CO~ ch~ride or s~cch~ri~e derivative, or -(cH2)n-NH-s~(ch~ride or ~çh~ri~e S derivative s~sLi~uent and one -Rl-Z substit~-ent The above pl`e;sen~cd çhPl~ting compounds are r~(linl~heled to form the cor~onding radionuclide metal chPl~tes of the following form R R R
~(I)m~
N N
R~/ \M/ `rR

R2~ / \ ~2 wherein: S S

M le~lcsellt~ a r~tlionuçli~ie metal or an oxide thereof and all the other 10 symbols are as described above.
Some ~ition~l con,~unds of the present invention incorporate este cleavable Rl moieties, incorporating, for eY~mple, ester and/or amide-cont~iningRl groups. An example of such co...~ounds of the present invention employing a cleavable succinate mono-ester mono-amide linkage has the formula shown 15 below:

SUBSTITUTE SllEET (R~LE 2~) ~\WO 95/02418 216 5 0 S 2 PCT/US94/07733 _9_ CH2~(CH2)2--C--NH-CH--~CH2)4--NH--C--(CH~)~S~
NH O - -HOOC-- \~ ~
H~ ~NH

Y S SAY Each Y - H or CH2COOH
~CM EOE

wherein X is H or COOH. The advantage of an ester cleavable Rl group is a reduction in non-target cell retentioll- Also, ester function~lities often improve water solubility and overall polarity of small mole~cules. p,~,~H;on of compounds having ester cleavable linkers is discussed in the Ex~mrl~s set forth S below.
A protein conjugation group is a chPmi~lly reactive functional group that will react with a protein under con~litioll~ that do not denature or otherwise adversely affect the protein. The protein conjugation group llle.efo,o is s..fficiently reactive with a fim~ tion~l group on a protein so that the reaction can 10 be con~ucted in subst~nti~lly aqueous solutions and does not have to be forced, e.g. by heating to high ~,~.p.c,i,l..,c:s, which may denature the protein. E~mples of suitable protein conjugation groups include but are not limited to active esters, isothiocy~lates, ~mines~ hy~lr~7in~s, thiols, and m~leimi~es Among the ~,~fe.,ed active esters are thiophenyl ester, 2,3,5,6-tetr~fluorophenyl ester, and 15 2,3,5,6-tetr~fluQrothiophenyl ester. The preferred active esters may comprise a group that enh~nces water solubility, at the para (i.e., 4) position on the phenyl ring. Examples of such groups are CO2H, S03-, po32- and oPO32-, and O(CH[2CH20)~CH3 groups.
A ligand or anti-ligand conjugation group is a chemic~lly reactive 20 functional group that will react with a ligand or anti-ligand under conditions that do not adversely affect the ligand or anti-ligand, including the capacity of theligandl or anti-ligand to bind to its complement~ry binding pair member. Ligand SUBSTIME SHEET ~RllLE 2B) WO 95/02418 PCT/US94/07733 1~
2l~sn~ 2 or anti-ligand conjugation groups therefore are sllfficiently reactive with a filnctio~l group on a ligand or anti-ligand so that the re~action can be co~ducted under relatively mild reaction conditions inclllding those described above for protein-chelate conjug~tion For çlu~ei~ eous ligands or anti-lig~n-ls, such as 5 ~Ll~lavidin, protein conjugaLion groups may correspond to ligand or anti-ligand conjugation groups. FY~mples of suitable ligand or anti-ligand conjugation groups therefore incl~lde, but are not limited to, active esters, isothiocyanates, ~mineS, hy~ .os, thiols, and m~l~imi~1~s Among the p~ d active esters are thiophenyl ester, 2,3,5,6-tetr~fl~-crophenyl ester, and 2,3,5,6-10 tetr~fluorothiophenyl ester. The pfer~fed active esters may comprise a groupthat çnh~nces water solubility, at the para (i.e., 4) position on the phenyl ring.
Fy~mrles of such groups are CO2H, S03-, po32~, oPO32-, and O(CH2CH20),,CH3 groups.
For non-~r~teh-~reous ligand or anti-ligand moieties, such as biotin, 15 suitable conjugations groups are those functional groups that react with a ligand or anti-ligand functional group (e.~., a terminal carboxy group) or a functit n~l group which the ligand or anti-ligand has been derivatized to contain ~, an alcohol or an amine group produced by the reduction of a terminal carboxy moiety). As a result, conjugation groups, such as those recited above, that are 20 capable of reacting with -COOH, -OH or -NH2 groups are useful conjugation groups for producing biotin-chelate molecules of this aspect of the present invention. Fxempl~ry biotin-COOH conjugation groups are ~mines, hy~ 7ines, alcohols and the like. Flremrl~ry biotin-OH conjugation groups are tosylates Cls), active esters, halides and the like, with exemplary groups being reactive 25 with biotin-O-Ts incl~l~ing ~mines, hy(l,~7;i~es~ thiols and the like. F.xempl~ry biotin-NH2 conjugation groups are active esters, acyl chlorides, tosylates, isothiocyanates and the like.
The protein conjugation group, ligand conjugation group, or anti-ligand conjugation group (l~reselltPA as Z in the above-presented formulas) is ~tt~ h~A30 to the chel~ting col,lpoulld core through the linkage representeA as Rl. Rl is a SUBSTITUTE SHEET (RULE 26) 2~6S~2 lower alkyl or substituted lower alkyl group. By "lower alkyl" is meant an alkylgroup of preferably one to four carbon atoms. Most preferably, Rl is a methylene chain comprising from two to three carbon atoms. The lower alkyl group may be substituted with hetero atoms such as oxygen or nitrogen atoms.
5 When the protein conjugation group, ligand conjugation group, or anti-ligand conjugation group is a primary amine, the Rl linkage comprises a methylene group imm~Ai~trly ~Aj~ent to the tPrmin~l primary amine protein conjugation group.
The "t~,eting moiety" of the present invention binds to a defined target 10 cell population, such as tumor cells. Preferred targeting moieties useful in this regard include antibody and antibody fr~m~nt~, p~te;n~reous or non-ot~in~eQus ligands or anti-lig~nAs, peptides, and hormonrs. Proteins col,esl.onding to known cell surface receplo,s (in~lurling low density li~plu~ s~ tr~n~fr~rrin and insulin), fihrinolytic enzymes, anti-HER2, platelet 15 binrtin~ proteins such as ~nnr~Yin~, and biological response modifiiers (inrlurtin~
intPrleukin, intelr~oil, elyt~r~oietin and colony-stim~ ting factor) are also pr~relled tafgeLillg moieties. Also, anti-EGF rece~tc.r antibodies, which intr1~li7e following binding to the receptor and traffic to the n~çlPu$ to an extent, are pl~;re"~d t~eling moieties for use in the present invention to 20 f~ilit~t~ delivery of Auger e"~itle~ s and nucleus binding drugs to target cell nuclei. Oligonucleotides, e.g., ~nti~n~e oligonucleotides that are complement~ryto portions of target cell nucleic acids (DNA or RNA), are also useful as ting moieties in the practice of the present invention. Oligonucleotides binding to cell surfaces are also useful. Analogs of the above-listed targeting 25 moietir s that retain the capacity to bind to a defineA target cell population may also be used within the claimed invention. In addition, synthetic t~gt;ting moietir s may be ~e~ignrA
Functional equivalents of the aforementioned molecules are also useful as l~eling moir~ties of the present invention. One ~eling moiety functional 30 equivalent is a mimPtic compound, an organic chemical construct designed to SUBSTITUTE SHEET (RULE 26) WO 95/02418 PCT/US94/07733 ~

2165~

mimic the proper configuration and/or c rient~tic)n for targeting moiety-target cell binding. Another targeting moiety functional equivalent is a short polypeptide ~ecipn~t~ as a "minim~l" polypeptide, constructed using~'co~pul~r-~cci~t~
molecular modeling and ."~ having altered binding affinity, which minim~l S polypeptides exhibit the binding affinity of the targeting moiety.
The term "t~eling protein" as used herein refers to proteins which are capable of binding to a desired target site in vivo. The targeting protein may bind to a ,~ceplor, sul~sL.~e, antigenic delel",inallt, complement~ry binding pair member or other binding site on a target cell or other target site. The ~g~;li,lg 10 protein serves to deliver the radionuclide att~t~h~d thereto to the desired target site in vivo. F.Y~mples of ~eling proteins include, but are not limited to, antibodies and antibody fr~m~ntc, ~lu~ eQus ligands or anti-lig~n~s, hormon~s, fibrinolytic enzymes, and biologic response modifiers. The term "~geling protein" in~ des proteins, polypeptides, and fr~m~ntc thereof. In 15 ~ldit~ other molecul~s that localize in a desired target site in vivo, although not strictly proteins, are incl~lded within the definitiQn of the term "~f~,elillg proteins" as used herein. For eY~mrl~, certain carbohydrates or glycoproleins may be used in the present invention. The proteins may be modified, e.g., to produce variants and fr~gm~ntc thereof, as long as the desired biological ~r~t;lly 20 (i.~, the ability to bind to the target site) is ret~ined. The proteins may be modified by using various genetic engin~ring or protein engin~ring techniques.
Among the p,~relled targeting proteins are antibodies, most preferably monoclonal antibodies. A number of monoclonal antibodies that bind to a specific type of cell have been developed, inclll-ling monoclonal antibodies 25 spe~ific for tumor-~ccoc;~ted antigens in h~lm~nc. Among the many such monoclonal antibodies that may be used are anti-TAC, or other interleukin-2 ,~ce~lor antibodies; 9.2.27 and NR-ML-05 to the 250 kilodalton human melanoma-~cco- i~ted proteoglycan; and NR-LU-10 to a pancarcinoma glycop~tein. The antibody employed in the present invention may be an intact 30 (whole) molecule, a fragment thereof, or a functional equivalent thereof.
SUBSTI~E SHEET (RVLE 26) ~ WO 95/02418 PCT/US94/07733 2l6~nS2 PY~mr]es of antibody fr~gmPnt~ are F(ab')2, Fab', Fab, and Fv fr~gmPnt$, which may be produced by conventional methods or by genetic or protein enginPPring.
Human monoclonal antibodies or "h~1m~ni7PA" murine antibodies are also useful as t~,eLing moieties in accord~lce with the present invention. Por 5 eY~mple, murine monoclon~l antibody may be "h~ PA" by gPnPtic~lly recomhining the nucleotide sequence encoding the murine Fv region (~, corli1;ning the antigen binding site) or the comrlem~-l;";Iy dele~ Ill;nillg regions thereof with the nucleotide sequence encoding at least a human constant domain region and an Fc region, e.g., in a manner similar to that disclosed in Eu~
Patent Application No. 0,411,893 A2. Some additional murine residues may also be retained within the human variable region framework domains to ensure proper target site binding characteri~tics~ T-TIlm~ni7P~ ~gt;~ing moi~Ptips are recognized to decrease the immlmoreactivity of the antibody or polypeptide in the host re~irient~ lliUillg an increase in the half-life and a reduction in the 15 possibility of adverse imm~lne re~ction~.
Targeting proteins are rarely completely specific for a desired target site.
T oc~ii7~tiolt in non-target tissues may occur through cross-reactivity or non-spe~ ific uptake, for eY~mplç. In the case of r~rliol~beled L~eling proteins, such loc~l17~tion at non-target sites may result in decreased clarity of rli~gnostic 20 images (due to the increased "back~roulld") and mi~ gnosis. E~os.lle of non-target tissues to r~tliation also occurs, which is especially und~Psir~hle in th~ C;ulic procedures. The improved biodistribution L.rope,lies of the ~ ol~heled ~,E;eLiilg proteins of the present invention are believed to be attributable to the effect of the chelate, most likely on the biodistribution of25 catabolites of the r~dic)l~heled proteins.
T.igan-l~ suitable for use within the present invention include biotin, haptens, lectins, ~it~es, dsDNA fr~gm~Pnt~ and analogs and derivatives thereof.
Useful complement~ry anti-ligands include avidin (for biotin), carbohydrates (for lectins), antibody, fr~gmPnt~ or analogs thereof, inc~ ling mimetics (for haptens 30 and epitopes) and zinc finger proteins (for dsDNA fr~gmPnt~). Preferred ligands SUBSTITUTE SHEET (RULE 26~

WO 9~/02418 PCT/US94/07733 ~
21~05~
.. ..

and anti-ligands bind to each other with an affinity of at least about kD 2 10-9 M.
The ch~ tin~ compounds of the present invention comprise two nitrogen and two sulfur donor atoms, and thus may be termed "N2S2" chçl~ting S compounds. The r~-liol~heled targeting proteins of the present invention exhibit certain i~ uved biodistribution p.opellies co---;?a~ed to targeting proteins r~diol~heled with certain other N2S2 ch~l~tes. Most notably, lc!c~li7~tiQn of r~tliol~beled targeting proteins (or catabolites thereof) within the intestin~s is reduced Targeting proteins r~-liol~beled with certain N2S2 r~t1ionurlide metal çh~l~tPs are described, for elr~mrl~, in European Patent Appliç~tin~ PublicationNumber 188,256. When the r~-liol~heled proteins of EP 188,256 are ~lmini~tpred in vivo, a pe~el ~dge of the injected dosage of the r~-lionuclide becomes loc~li7ed within the intestin~s (i.e., becomes part of the intestin~l 15 contel.~, rather than binding to intestin~l epithelial tissue per se). Although stable att~hm~nt of r~r1ionuçlides to antibodies and effective lcc~1i7~tion thereof on target tumors has been achieved using the EP 188,256 system, reducti~ ~ of the il~te~ l loc~li7~tiQn would be bçnefici~l A portion of the non-target-bound ~-lmini~tt~red r~iol~heled proteins (e.g., antibodies or fr~gm~nt~ thereof) most20 likely is first metabolized to produce radiolabeled catabolites that subsequently enter the intestin~s, probably through hepatobiliary excretion. When the chelateis ~tt~ch~d to lysine residues of the targeting protein, a major catabolite may be the lysine adduct of the chelate. Tntestin~l loc~li7~tion of r~dio~ctivity may be confused with (or obstruct) target sites in the abdominal area. For theldl~eu~ic25 procedures, the dosage that can be safely ~mini~tPred is reduced when intçstin~l loc~li7~tion occurs (due to exposure of normal tissues to the radiation). The thel~euLic effect on the target sites therefore also is reduced.
As illustrated in the examples below, th.e biodistribution p~tt~rn~ in vivo differ when targeting proteins (e.g., antibody fr~gm~nt~) are r~iol~heled with a30 chelate of the present invention, co---~ared to radiolabeling using certain other SlJBsrlTuTE Sl IEET (RVLE 26) ~ WO 95/02418 PCT/US94/07733 21~5(~52 N2S2 çhPl~tPS The advantage of reduced intestin~l loc~li7~tion is demonstrated for the radiolabeled l~ ~Lillg proteins of the present invention. While not wishing to be bound by theory, it is believed that the carboxylic acid substituçnt(s) on the chelate confer the advantageous biodistribution p~ul)elLies on S catabolites of the ra-liol~heled protein (most likely lysine adducts of the chelate).
The carboxylic acid substituçnt(s) on the col"~ounds of the present invention increase the polarity, and therefore the water solubility, of the compounds. Theincreased water solubility is believed to promote excretion of the catabolites by the lddneys, resulting in efficient elimin~tioll of the radioactive catabolites in the 10 urine. Other subsl;lue~ that e-nh~nce polarity (e.g., sulfate groups) may be used on the chPl~ting cG.l.pounds, in addition to (or instead of) the COO
, subs~ el-ts.
Another advantage of the chPl~tes of the present invention is the co---L~tively good r~(liol~heling yields. The free carboxylic acid substih-ent(s) 15 are believed to assist in the chPl~tioll of the r~io~uçli~e. l~-liQl~heled ligands and anti-ligands also exhibit these favorable biodistAbution and çhPl~tio ~ro~ellies.
DuAng radiolabeling, bonds form between the four donor atoms and the radionuclide metal to form the coll~;s~onding r~ionuçlicle metal chPl~t~. Any 20 suitable conventio~l sulfur ylo~ec~ g group(s) may be ~tt~chP~d to the sulfurdonor atoms of the compounds of the present invention. The protPcting groups should be removable, either pAor to or duAng the radiolabeling reaction. The protecting groups ~tt~chPA to the two sulfur donor atoms may be the same or dirrerent. ~ltern~tively, a single protec~ g group, e.g. a thio~çet~l group, may25 protect both sulfur donor atoms. Among the pl~Çell~d sulfur l~rotecling groups are ~cet~midomethyl and hernithio~çet~l protecting groups, which are displacablefrom the ch~pl~ting co---pou-ld duAng the r~-liol~heling reaction. Preferably, at least one sulfur protecting group is a hçmithio~çet~l group, and at most one sulfur prote~;ling group is an ~ et~midomethyl group.

SUBST~ SHEET (~ULE 26) WO 9$/02418 , . ! . PCT/US94/07733 ~
216~05 `2 An ~cet~midomethyl sulfur-protecting group is e~resellled by the following form~ , wherein the sulfur atom shown is a sulfur donor atom of the ch~l~tin~ co~ und: ~

1~ 0 H~C--C~

The ~-et~midomethyl group is ~ pl~ced from the çh~ ting compound 5 during r~-liol~heling conductçd at about 50C in a reaction ~ u.c; having a pH
of about 3 to 6.
When h~mithio~ret~l protective groups are used, each sulfur atom to be pr~L~led has a sep~r~tP pLo~ec~ e group ~tt~h~i to it, which together with the sulfur atom defines a htomithio~-et~l group. The hemithio~et~l groups contain a 10 carbon atom bonded di~ly (i.e., without any intervening atoms) to a sulfur atom and an oxygen atom, i.e., I

Preferred hçmithio~-et~l~ generally are of the following formula, wherein the sulfur atom is a sulfur atom of the ch~l~ting col"pound, and a sep~le protecting group is ~tt~ h~A to each of the sulfur atoms on the chPl~ting 15 co"lpow~d:

SUBSTITUTE SHEET ~RULE 26) ~ WO 95/02418 2 1 6 5 ~ ~ 2 PCT/US94/07733 IP~
R4 I n~

wherein R3 is a lower alkyl group, preferably of from two to five carbon atoms, and ]R4 is a lower alkyl group, preferably of from one to three carbon atoms.
ely, R3 and R4 may be taken together with the carbon atom and the oxygen atom shown in the formula to define a nonalu,.latic ring, preferably 5 comprising from three to seven carbon atoms in ~ tion to the carbon and oYygen atoms shown in the formul~ R5 ~Lesents hydrogell or a lower alkyl group wherein the alkyl group preferably is of from one to three carbon atoms.
FY~mrles of such p~ert;ll~d hlomithio~et~l~ include, but are not limited to:
(SEE TOP OF PAGE 18) These sulfur-protective groups are rli~pl~ee~ during the radiolabeling reaction, conducted at acidic pH, in what is believed to be metal-~si~ted acid cleavage. Covalent bonds form between the sulfur atoms and the metal r~-lio~ucli~e. A sep~dle step for removal of the sulfur-protective groups is notneceS.~-y. The r~iol~beling procedure thus is simplified. In ~d~lition~ the basic 15 pH conditions and harsh con~ition~ associated with certain known r~iol~helingprocedures or procedures for removal of other sulfur protective groups are avoidled. Thus, base-sensitive groups on the chel~ting compound survive the radiolabeling step intact. Such base labile groups include any group which may be destroyed, hydrolyzed, or otherwise adversely affected by exposure to basic 20 pH. In general, such groups include esters, m~leimi~es, and isothiocyanates, among others. Such groups may be present on the chel~ting co,l,l~ound as protein, ligand or anti-ligand conjugation groups.

SUBSTITUTE SHEET (I~ULE 26) WO 95/02418 PCT/US94/07733 ~
2 1 ~ t i'"'`';~ ~.'' `-18-,C--fH2 o f H~C~5 H C~C~S

T~ ~"~1~ùfuranyl 2-methyl tetrahydro~uranyl ~C~ H3C~CIH2 ~C~C~

H2C~C~ H3C~C~H2C~C~
H I ~CH3 Tetrahydropyranyl~ IuA~cthyl 2-methyl tetrahydropyranyl The coll,poullds of the present invention preferably compAse at least substitl-ent, most preferably two =O sub~tituPnt~ In one embodiment of the invention at least one and preferably two R2 substi~lent~ are -(CH2)n-COOH, withn preferably equal to 1.
FY~mples of the ch~ ting coll-poullds of the present invention are the compounds of the following formulas:
(SEE TOP OF PAGE 19) wherein the symbols are as described above. Procedures for synthesi~ing these colll~uw~ds are presented in the examples below. In one embodiment of the ~0 invention, these ch~ ting compounds comprise either two h~mithio~cet~l, or one htomithio~cet~l and one ~(~et~midomethyl sulfur plvteclillg groups.

SUBSTI~UTE SHEET (RULE 26) ~WO 95/02418 PCT/US94/07733 Z

~~~'' HN NU

HOO COOH HOOO ( ~ ~COOH

Oq f z q / z HN NH HJ~ NH
HOOC~/ `F HOOO~/ `F

Sl IS COOH~Sl I
T T T T

Other chPl~tin~ colllpounds of the present invention incorporate one or more ~-ch~ri~e resi~ues. A ~ lled number of ~cch~ride residues ranges from 1 to about 10, although when polymeric s~crh~ri-les are employed the number of c~c~h~ri~le residues therein may be higher.
S .Saçrh~ri~es, such as h~xoses (~g., glucose) and pentoses (~, fructose)and polymers of such sacçh~ri(les are hydrophilic and, consequently, are genelally excreted efficitontly into the urine by glomular filtration. Inulin, a 5 kD
polymer of fructose, is the gold standard for glomular filtr~tio~ studies.
Derivatization of a ch~ ting çoll,pou.ld with one or more hexose resi~ues, such as ghJcose re~i-lues, for example, is expeçted to increase the water solubility and hydluphilicity of the chel~tin~ çolllpound and conjugates conL~ining the same.
Consequently, glucose-bearing çh~l~ting compounds and conjugates will exhibit çnh~nced renal excretion.
S~cçh~ride or sacch~ride derivative-bearing conjugates can be pr~d in accordance with procedures discussed in the examples below for glucose derivative-bearing conjugates. Exemplary glucose derivative-cont~ining SllBSTITUrE SHEET (RULE 26) WO 95/02418 PCT/US94/07733 ~, 2l~sns2 conjugates of the present invention can be pr~a.c;d from a variety of intPrmPAi~tPS inr~ ing glucose, plucos~minP, gluconate, gl~cohPptonate, ~lucoslic acid, glucs~nolactone~ gll~r~ric acid C~, s~crh~ric acid), D-sacçh~ric1,4-lactone monohydl~te, glucuronic acid, and the like. other sugars and sugar 5 derivatives of similar function~li7~tion are also commercially available and useful in the practice of the present invention.
The choice of sugar derivative (unmodified, amino functionalized or carboxy function~li7P~) depends on the conjugating group of the che1~tinf~
compound. For example, amino sugars are ~ t;lled for conjugation to çhPl~ting 10 conl~ound carboxyl groups. Glucosamine, for example, is useful for thi ~ul~ose, as it allows amino group reaction with applopliate deliv~ rcs of chPl~ting conl~ullds such as active esters, active h~ es, aldehydes.
l;v~ly~ ~rçh~ride colll~!.unds bearing carboxyl residues are commercially available and can be reacted with amine derivatives of chPl~ting 15 conl~uunds. Glucuronic acid, for example, is useful for this purpose, as it bears a carboxy residue available for reaction with a chel~ting compound amine.
Also, native s~cçh~r;~e colllpounds, such as glucose for example, can be reacted with chPl~ting c~sl..p~,u,ld amines to form amine-linked sugar-chel~tingco...~uulld conjugates. Subsequent or concurrent imine reduction results in a stable amine linkage.
Sugar l~tonPs may be employed in the preparation of amide-linked sugar-chPl~ting cGlllpoulld conjugates. The lactone serves as an activated carboxylic acid which undergoes nucleophilic, ring opening upon reaction with amine bearing çhPl~ting colllpouilds.
The following table summ~ri7es examples of saccharide (sugar)-chPl~ting compound (non-sugar) chemic~l conjugates ~r~ar~d via nucleophile-electrophile reaction:

SUBSTITUTE SHEET (RULE 26) ,~ WO 95/02418 PCTIUS94/07733 2l~sn~

F~' ~.i.. le ~ SugarI)~ Iiie I,ir~age sugar amine h~ I carbo~yl yl.. ~ ...... ;. F- amide arnino sugar aldehyde native sugar amine amino activated sugar carbo~cyl ~lu.;u.~.l.. c acid amide S amino æugar lactone ~ c .I,)"r amide l~.L~de sugar aldehyde native ~ugar hydrazide The chPl~ting compounds of the present invention are r~liol~beled, using col,velllional procedures, with any of a variety of r~dionllrlide metals to form the corr~sponding r~-liotlu(li~e metal çh~l~t~s These r~ionucli~e metals include7 but are not limited to, copper (e.g., 67Cu and 64Cu); tec-hnetillm (e.g., 99~Tc);
i"", (e.g., l86Re and ~88Re); lead (e.g., 2l2Pb); bismuth (e.g, 2l2Bi); and p~ lm (e.g., 109Pd). Methods for ~r~alillg these isotopes are known.
Molybcl~nllm/techn~otium generators for producing 99mTc are commercially available. Procedures for producin~ l86Re include the procedures described by Deutsch et al., (Nucl. Med. Biol., Vol. 13:4:465-477, 1986) and Vanderheyden et al. (Inorganic Chemistry, Vol. 24:1666-1673, 1985), and methods for production of l88Re have been described by Blachot et al. (Intl. J. of Applied Radiation and Isotopes, Vol. 20:467~70, 1969) and by Klofutar et al. (J. of Radioanalytical Chem., Vol. 5:3-10, 1970). Production of 2lZPd is described in Fawwaz et al., J. Nucl. Med. (1984), 25:796. Production of Zl2Pb and 2l2Bi is described in Gansow et al., Amer. Chem. Soc. Symp. Ser. (1984), 241:215-217, and Kozah et al., Proc. Nat'l. Acad. Sci. USA (January 1986), 83:474-478.
99mTc is pl~re l~d for diagnostic use, and the other radionuclides listed above have thel~peuLic use.
In one embodiment of the present invention, ch~l~ting colllpou,lds of the - invention comprising ~-et~mi~omethyl and/or hPmithio~cet~l sulfur protective groups are r~-liol~heled with a metal radionuclide by reacting the compound withthe radionuclide under con-litions of acidic pH. It is believed that the acidic pH
SUBSTITUTE SHEET ~RULE 2~) W O 95/02418 PCTrJS94/07733 ~

;-22-and the presence of the metal both contribute to the di~pl~-emPnt of the sulfur plvlecLi~e groups from the chPl~ting co~pou~ld. The radionuclide is in chPl~t~hle form when reacted with the chPl~ting compounds of the invention.
In the case of teçhnPtinm and rhtonium, being in "çhPl~t~hle form"
5 generally l~Uil~S a reduçin~ step. A reducing agent will be employed to reducethe r~-lionuçli~es (e.g., in the form of pe.LP~hllPt~tP and perrhPn~tP, le~eclively) to a lower oxitl~tion state at which chel~tiorl will occur. Many suitable reducin agents, and the use thereof, are known. (See, for eY~mple, U.S. Patents 4,440,738; 4,434,151; and 4,652,440.) Such reducing agents include, but are 10 not limited to, stannous ion (e.g., in the form of stannous salts such as stannous chlori-lP or stannous fluc-ri~P), mPt~llic tin, ferrous ion (e.g., in the form of ferrous salts such as ferrous çhlc~ride~ ferrous sulfate, or ferrous ascorbate) and many others. Sodium pel~eç~ pt~t~p (i.e., 99mTc04- which is in the +7 oxi~tion level) or sodium pPrrhPn~tP (i.e., l88ReO4-, l86ReOi) may be combined 15 ~imlllt~np~usly with a redu~ing agent and a chPl~ting co---pound of the invention in accordance with the r~-liol~heling method of the invention, to form a çhplatePreferably, the r~lio~llcli(le is treated with a reduçing agent and a complexing agent to form an interlnPAi~te complex (i.e., an "eYch~nge complex"). ComplPYinp agents are compounds which bind the radionuclide more 20 wealcly than do the chelate co---~ou-lds of the invention, and may be weak chel~tors. Any of the suitable known complP ~ing agents may be used, incluriing but not limited to gluconic acid, glucoheptonic acid, methylene disphosphonate, glyceric acid, glycolic acid, .,.~l~nilQl, oxalic acid, malonic acid, succinic acid, bicine, N,N'-bis(2-hydru~y ethyl) ethylene ~ mine~ citric acid, ascorbic acid and 25 gentisic acid. Good results are obtained using gluconic acid or glucoheptonic acid as the Tc-complexing agent and citric acid for rhçnillm When the radionuclide in the form of such an exch~nge complex is reacted with the ch~l~hng compounds of the invention, the radionuclide will transfer to these co---~ounds which bind the r~ion~ e more strongly to form chel~t~s of the 30 invention. ~ting is often required to promote transfer of the radiom~çlide.
SUBS~ITUTE SHEET (RULE 26) ~WO 95/02418 21 B S O ~ ~ PCT/US94/07733 R~iom~cli~les in the form of such complPYes also are co~idç~ed to be in "chPl~t~hle form" for the purposes of the present invention.
ChPl~tPS of 2l2Pb, 2l2Bi, l09Pd may be pr~c~d by combining the appropliate salt of the radionuclide with the chPl~ting compound and incubating S the reaction ~ LulG at room telllpeldLulG or at higher telll~GldlurGs. It is not l-ece~ y to treat the lead, bi~mllth, p~ lm, and copper r~-lioi~otopes with a re~urinp agent prior to ch~ tion, as such isotopes are already in an oxitl~tion state suitable for rhPl~tion (i.e., in chel~t~hle form). The specific r~-liol~heling reaction conditions may vary somewhat according to the particular r~-lion~lclide10 and chpl~ting compound involved.
The chPl~ting colll~uund may be radiolabeled to form a radionuclide metal chel~tP~ which then is reacted with a ~ eLing protein, ligand or anti-ligand.
ely, the unlabeled c~hPl~ting colllpouild may be ~tt~rhp~1 to the ~ Gling protein, ligand or anti-ligand and subsequently r~tliol~heled. Proteins and 15 prot~in~eous ligands or anti-ligands (~, avidin or streptavidin) as well as non-~fol~ ceous ligands or anti-ligands (~, biotin) contain one or more of a variety of functional groups; e.g., carboxylic acid (COOH) or free amine (-NH2) groups, which are available for reaction with a suitable protein, ligand or anti-ligand conjugation group "Z" on a chelator to bind the chelator to the protein, 20 ligand or anti-ligand. For example, an active ester on the chelator reacts with primary amine groups on lysine residues of proteins to form amide bonds.
~lle.n~t;~ely, the protein, ligand or anti-ligand and/or chelator may be derivatized to expose or attach additional reactive functional groups. The derivatization may involve ~thchmPnt of any of a number of linker molecules 25 such as those available from Pierce Ch~mic~l Company, Rockford, Tllinoi~. (See the Pierce 1986-87 General Catalog, pages 313-54.) ~ltem~tively, the derivatization may involve chemic~l tre~tmPnt of the protein (which may be an antibody), ligand or anti-ligand. Procedures for g~n~tion of free sulfhydryl groups on antibodies or antibody fr~gmPnt~ are also known. (See U.S. Patent SUBSI ITUTE SHEET (RULE 26) WO 95/02418 PCT/US94/07733 ~
~l65os~

No. 4,659,839.) M~leimide conjugation groups on a chelator are reactive with the sulnly~lyl (thiol) groups.
;vely~ when the targeting compound is a carbohydrate or glyc~n~leill, deriv~ti7~tic-n may involve chemic~l tre~tm~nt of the carbohydrate;
e.g., glycol cleavage of the sugar moiety of a glyco~l~Jteill antibody with pçrio~ to genPr~tP free aldehyde groups. The free aldehyde groups on the antibody may be reacted with free amine or hydrazine conjugation groups on the chPl~tor to bind the chPl~tor thereto. (See U.S. Patent No. 4,671,958.) Biotin has a terminal carboxy moiety which may be reacted with a s-lit~hle ligand conjugation group, such as an amine, hydroxyl in the presence of a coupling agent such as DCC or the like. In ~rlr~ition, the tPrmin~l carboxy moiety may be derivatized to form an active ester, which is suitable for reaction with a suitable ligand conjugation group, such as an amine, a hydru~yl, another nucleophile, or the like. ~ltPrn~tively, the terminal carboxy moiety may be reduced to a hydloAy moiety for reaction with a suitable ligand conjugation group, such as a halide (~, iodide, bromide or chloride), toxylate, mesylate, other good leaving groups or the like. The hydroxy moiety may be chemic~lly motlifi~d to form an amine moiety, which may be reacted with a suitable ligand conjugation group, such as an active ester or the like.
The r~-liol~bel~d targeting proteins, ligands and anti-ligands of the present invention have use in diagnostic and thel~eu~ic procedures, both for in vitro assays and for in vo m~Ai~l procedures. One type of thel~peulic or ~i~gnostic procedure in which the conlpounds of the present invention may be employed is l,le~e~ g ~n~tocol. Generally, ~ g encomp~ces two ~lotocols, ter-m--ed the three-step and the two-step. In the three-step protocol, shown schem~tic~lly below, targeting moiety-ligand is ~mini~tered and permitted to localize to target.

SUBSTITUTE SHEET ~I~ULE 26 ~ WO 95/02418 216 5 0 ~ 2 PCTrUS94/07733 Blood Tumor 10 1~ r~ ~ 0 o ~ *
~ I

3) ' *

Ta~getlng moiety * Anti-ligand o Ligand ~5 Ligand-active agent Binding site (i.~., receptor, antigenic dete_minant) 30 ~ Liver ~ Kidney SUBSTITUTE SHEET t~ULE 26) WO 95/02418 PCT/US94/07733 ~
21~5~5 ~2 Targeting moiety-ligand conjugates may be pr~?ared in accordance with known techniques therefor. Anti-ligand is then ~lmini~t~red to act as a clP~ring agentand to f~ ilit~te and direct the excretion of circulating ~ elillg moiety-ligand.
The anti-ligand also binds to target-associated targeting moiety-ligand. Next, a5 conjugate employing a co~ uulld of the present invention is ~mini~tPred, having the following structure:

Ligand - - - - Chelate - - - - l~ionuçlitle The r~-liol~heled ligand conjugate either binds to target-associated targeting moiety-ligand-anti-14-gand or is rapidly excreted, with the excretion prl!cee ling 10 prim~rily through the renal pathway. Consequently, the target-non-target ratio of active agent is improved, and lmd~psir~ble hepatobiliary excretion and intestin~l uptake of the active agent are sulJs~ lly decreased.
Two-step PÇ~ ing involves ~dmini~tration of targeting moiety-anti-ligand, which may be ~r~d in accordance with known techniques therefor.
15 After pe~ l;ng the ~lminist~pred agent to localize to target, a r~diQl~heled ligand of the present invention is ~mini~tPred. Preferably, as a "step l.S," a clç~ringagent is a~lmini~tered to remove circul~ting targeting moiety-anti-ligand without binding of clP~rin~ agent to target-associated targeting moiety-anti-ligand. In this manner, the target-non-target ratio of the ra~ heled ligand is increased, and 20 undesirable hepatobiliary excretion and i~tk~ l uptake of the M~liol~heled ligand are subst~nti~lly decreased.
The radiol~heled proteins, ligands or anti-ligands may be a(lmini~tPred in~l~venously, i~ .pe. ;lolleally, intralymphatically, locally, or by other suitable means, depending on such factors as the type of target site. The amount to be 25 ~mini~tPred will vary according to such factors as the type of r~icml~lide (e.g., whether it is a diagnostic or the~ ulic radionuclide), the route of ~dmini~tr~tion, the type of target site(s), the affinity of the targeting protein for the target site of interest, the affinity of the ligand and anti-ligand for each other SUBSII~UJE SHEET ~RVLE 2~) ~WO 95/02418 216 ~ Q S 2 PCT/US94/07733 and any cross-reactivity of the L~eLing protein, ligand or anti-ligand with normal tissues. Appropriate dosages may be established by convention~l procedures and a physician skilled in the field to which this invention pertainswill be able to determine a suitable dosage for a patient. A di~gnostic~lly 5 effective dose for a chelate labeled antibody embodiment of the present invention is generally from about 5 to about 35 and typically from about 10 to about 30 mCi per 70 kg body weight. A th~l~elllic~lly effective dose is generally from about 20 mCi to about 300 mCi. Elevated doses, e.g., r~nging from about 2 to aboul: 10 times higher, can be used when pre~,eLi-lg procedures are employed, 10 because of the decoupling of L~eLillg moiety loc~li7~tion and r~-lionuçlide loc~li7~tion. For diagnosis, conv~-l;on~l non-invasive procedures (e.g., gamma c~m~ ) are used to detect the biotli~tribution of the diagnostic radionllclide, thereby dele~lllil~ing the presence or absence of the target sites of interest (e.g., tumors).
15The co"~ ;vely low intestin~l loc~li7~tion of the thel~euLic heled antibodies of the present invention or catabolites thereof permits increased dosages, since intestin~l tissues are exposed to less radiation. The clarity and accuracy of diagnostic images also is ill,pr~ved by the reduced loc~li7~tion of r~iol~heled antibodies or catabolites thereof in normal tissues.20 These advantages are also expçriençed in the practice of the pr~L~,e~ing aspects of the present invention.
The following examples are p-c~sellLed to illllstr~te certain embodimçrlt~ of the present invention.

l~xample I
Synthesis of S-~cet~mitl-methyl-N-t-BOC Isocyste-ne Trichloroethyl Ester The synthesis procedure is outlined in Figure 1. Pl~ ;on of S-~çet~midQmethyl-N-t-BOC isocysteine 6 from 1:

SUBSTITUTE-SHEET ~RULE 26) wo 95/02418 PCT/US94/07733 ~
2l~sas2 Me~al)Losucçinic acid 1 (commercially available) was reacted with cyclopç~ none in TosOH to form 2-oxathiolone_.
To a sol~l~ion of 2-oxathiolone 2 in benzene (40 mL) and triethylamine (3.28 mL, 23.55 mmol) at 0C, was added a solution of diphenyl phosphorylazide (5.08 mL, 23.55 mmol) in benzene (5.0 mL). The ice bath was removed and the reaction solution was stirred at room ~ell.peld~u~e for 1 hour.
The sollltion was washed with water. The water was extracted with benzene.
The combined benzene eYtract~ were dried, concçntr~t~d to half the origin~l volume, and heated under reflux in an oil bath ~ lly raised in le~ eldlule from 50C to 80C over 1 hour. The reaction solution was cooled to room ~elllpeldLule, diluted with ethyl acetate (50 mL) and washed twice with a s~tllr~t~A solution of NaHCO3 (30 mL). The organic eYt-~t~ were dried (MgSO4) and tv~oldted to give the crude iso~ya.lale ~ as a brown oil (4.92 g).
A suspension of 3 in 6N HCl (45 mL) was heated under reflux for 40 ~ es. The reaction sol~ltion was cooled, washed twice with ethyl acetate (50 mL). Evaporation of the aqueous extract gave crude isocy~Leine 4 as an amber oil (4.92 g, theoretical 3.64 g). NMR shows iso~;y~Lehle plus an aliphatic cont~min~nt To half of the crude isocysteine_ (2.42 g, theoretical 11.61 mmol) in water (3.0 mL) at 0C was added N-hydroxy~ret~mide (1.14 g). To this solution was added dropwise concentr~ted HCl (0.45 mL). The solution was stored at 0C for 3 days. The solution was e:v~?ol~lled to give S-acm isocy~L~ine as a cok)rlPss liquid NMR (D20) 1.95 (S, 3H), 3.35 (dd, 2H), 3.8 (t, lH), 4.4 (dd, 2H). TLC (c-18, 15% meOH/H2O 1% HOAc, one spot 0.4 Rf.
To a solution of 5 (theoretical 11.61 mmol) in DMF/H20 3:2, 25 mL) and triethylamine (3.60 mL, 25-54 mmol) was added di-t-butyl dicarbonate (3.04 g, 13.9 mmol). The reaction was stirred at room le~ el~tu~ for 3 hours and then e~/~pol~ted. The residue was partitioned between water and ethyl acetate.
The water layer was ~ejrlifie~ to pH 3.0 with 1.0 M HCl and further extracted with ethyl acetate (3 x 30 mL) and methylene chloride (2 x 50 mL). The SUBSTITUTE SHEET (I~ULE 2~) combined organic extracts were dried (MgSO4) and evaporated to give an oil.
pllrifit~tion by c~ atography (15% iso~lulrdnoVmethylene ehloride 2% acetic acid) afforded 6 as an oil which cryst~lli7Pd from acelonil,ile. Yield from 2-o~thiolonP 2 was 1.90 g (6.21 mmol) = 53%.

5 Conversion of S-acm N-T-BOC isocysteine ~ to S-acm N-T-BOC isocysteine trichloroethyl ester_:
To an ice cold solution of 6 (1.90 g, 6.21 mmol) and trichloroethanol (0.71 mL, 7.45 mmol) in aceloni~ile (12 mL) and methylene chloride (2 mL) was added dicyclohexylcarbo~liimide (DCC) (1.47 g, 7.14 mmol) and dime&ylamino pyridine (76 mg, 0.62 mmol). The ice bath was allowed to melt and tlhe reaction solution was stirred for 16 hours at room le~ tul~. The reaction was cooled to 0C, filtered, and e~/~oldted to give an oil which was purified by clw~ dlog~hy (1:1 EtOAc/~Y~nes 1% HOAc) to give_ as an oil (1.25 g, 2.95 mmol) in 47% yield.

Examplé I~
Synthesis of N-T-BOC Am;no~-l;r;~ Acid ~t-butyl Ester lx-snc~;n;mi(lyl Ester 12 The synthesis procedure is outlined in Figure 2. Conversion of N-t-BOC
oY~7oli~line ~mino~-liric acid (9) to N-t-BOC o~701ic~ine ~mino~ipic acid t-butyl 20 ester ClQ):
To an ice cold sol~tion of 2 (3.23 g, 12.4 mmol) in ~ Qn jll ;le (12 mL) and t-butanol (1.75 mL, 18.6 mmol) were added dimethylaminopyridine (151 mg, 1.24 mmol) and DCC (3.07 g, 14.9 mmol). The reaction was stirred at 0C
for 69 .~in~l~es and then stored at 0C for 60 hours. The ~ Lure was filtered.
25 The filtrate was evaporated to give a solid which was chromatogr~phed (25%
EtOAc/~eY~n~s). The t-butyl ester 10 was obtained as a white solid (2.85 g, 8.66 mmol) in 70% yield.

SUBSTITUTE SHEET ~RULE 26J

wo 95/02418 PCTIUS94/07733 2165~
.-30-Conversion of 10 to N-t-BOC ~mino~(liric acid ~-t-butyl ester ~
To a solution of 10 (100 mg, 0.30 mmol) in methanol (2.0 mL) was added lN NaOH (0.33 mL) dropwise. The solution was stirred for 1 hour and then treated with eth~n~l~min~ (0.02 mL, 0.33 mmol). To this solution was added lN NaOH (0.32 mL, 0.32 mmol). The reaction solution was stirred for 48 hours, conc~ntr~ted, and then neutr~li7ed by the addition of lN HCl (0.33 mL). The aqueous phase was ext~ted with EtOAc (25 mL). The aqueous phase was ~cidifi~d with 1.0 N HCl to pH 1 and further extracted with EtOAc (2 x 50 mL). The combined EtOAc ~rtracte were dried (MgSO4), and evaporated to give an oil. Chromatography (40% EtOAc/~Y~nes 1% HOAc) gave 11 as a colorl~ss oil (60 mg, 0.19 mmol) in 63% yield.

Conversion of 11 to N-t-BOC ~mino~ )ic acid ~-t-butyl ester cr-succinimidyl ester 12:
To an ice cold solution of 11 (0.97 g, 3.06 mmol) in ace~onillile (6.0 mL) was added N-hy-lluAy~lcçinimide (422 mg, 3.67 mmol) and DCC (747 mg, 3.67 mmol). The ice bath was allowed to melt and the reaction solution was stirred atroom ~lllpel~urc for 5 hours. The nliAlure was cooled to 0C, treated with a few drops acetic acid, and filtered. Evaporation of the filtrate provided 12 as a white solid (1.19 g, 3.06 mmol) in 100% yield.

Example m Synthesis of Sllcc;n~te Reagent 16 Two procedures for synthe~i7ing compound 16 are outlined in Figure 3.
Procedure #1: Synthesis of succinate reagent 16 via base opening of o~thiolone:

25 Conversion of 2 to 2-mercaptosuccinic acid oxathiolone B-t-butyl ester L~:
Col,lpound _ was pl~aled from 1 as described in Example I.
SUBSTITUTE SHEET (RULE 26) _ WO 95/0241~ PCT/US94/07733 -- 216~ 0~ 2 To an ice cold solution of 2 (1.45 g, 6.30 mmol) in ace~oniLiile (6.5 mL) and t-butanol (0.89 mL) were added dimethyl aminopyridine (77 mg, 0.63 mmol) and ]DCC 1.55 g, 7.56 mmol). The reaction was stirred for 1 hour at 0C and then stored at 0C for 4 days. The product was filtered. The filtrate was c:va~of~led. Chromatography (10% EtOAc/~Y~nes) provided 13 as a yellow oil (1.76 g, 6.15 mmol) in 98% yield.

Conversion of 13 to 2-mercaptosuccinic acid B-t-butyl ester ~L):
To a solution of 13 (0.58 g, 1.82 mmol) in acetone (2.0 mL) was added lN NAOH (1.82 mL, 1.82 mmol). After the reaction solution was stirred for 4 hours, ~ ition~l lN NaOH (1.82 mL, 1.82 mmol) was added. The reaction solution was stirred for 20 hours, and then neutr~li7ed by the addition of 1.0 MHCl (3.6 mL). The aqueous phase was eYtr~cted with EtOAc (3 x 25 mL). The combined EtOAc extracts were washed with brine, dried and evaporated to give an oil. The product was chromatogr~I~hç~ (first 10% EtOAc/~e~r~nPs 10%
HOA.c, 300 mL, then 33% EtOAc/~Y~nPs 1% HOAc, 300 mL) to give 14 as colorless oil (0.24 g, 1.16 mmol) in 64% yield.

Conversion of 14 to S-tetrahydr~yl~lylmercaptosuccinic acid B-t-butyl ester C~) and NHS ester 16:
To a solution of 14 (240 mg, 1.16 mmol) and tosic acid monohydrate (7 mg, 0.03 mmol) in methylene chloride at -40C was added dihydro-2H-pyran (0.11 mL, 1.16 mmol). After the ~d~ition, the reaction was warmed to -5C and stirred for 30 minutes. The solvent was evaporated. The residue was dissolved in EtOAc (30 mL) and washed with pH 4.0 buffer. The aqueous phase was extracted with EtOAc (2 x 20 mL). The combined EtOAc extracts were washed with brine, dried and evaporated to give an oil which was used without pllrifi~tion. The oil was dissolved in acetonitrile (2.0 mL), cooled to 0C, andtreated with N-hydro~y~ucc-inimide (160 mg, 1.39 mmol) and DCC (287 mg, 1.39 mmol). The ice bath was allowed to melt and the reaction mixture was SUBSTITUTE SHEET (RULE 26) wo 95/02418 PCT/US94t07733 ~
2i65~2 stirred at room te~ e~ for 20 hours. The mixture was filtered. The filtrate was ev~pol~t~d. Chrul-lalog,d~hy provided 16 as a white solid (145 mg, 0.37 mmol) in 32% yield.

Procedure #2: Synthesis of succinate reagent 16 using LDA

5 Conversion of S-tetrahydl~yl~nylme~ (,acetic acid (~7) to S-tetrahydru~y~ ylmel~aL,lu~uccinic acid B-t-butyl ester 15 and NHS ester ~:
A solution of lithium diisopropylamide (LDA) was pl~c~d by adding a 1.30 M solution of n-butyl lithium in hP~n~s (13.2 mL, 17.2 mmol) to a solution of diisopropyl amine (2.52 mL, 18.0 mmol) in 'l~ (10.0 mL) at -10 78C. The sol~ltion was stirred for 20 minllt~s~ To this was added dropwise asolution of S-tetrahydl~yl~lylme~a~>toacetic acid (1.32 g, 7.50 mmol) in l~F
(5.0 mL). The reaction became cloudy. It was stirred at -78C for 25 ,-i-,utes,warmed to 0C, and stirred for 25 ..~ PS. The reaction was then cooled to -78C and treated with a solution of t-butyl bromo~et~te (3.2 mL) in THF (2.0 mL). The reaction solution was stirred for 1 hour at -78C, and for 30 minutes at 0C. The reaction was quench~d by the addition of acetic acid (1.0 mL) in methylene çhlori~e. The .lli~u~e was collcen~ ted, diluted with water and ethyl acetate. The aqueous layer was se~ idified with 1.0 M HCl to pH 3.0, and further extracted with EtOAc (2 x 75 mL). The combined EtOAc extracts 20 were washed with brine, dried, and e~/;.pol~ted to give 15 as a canary yellow oil.
The oil was dissolved in acetonitrile (10.0 mL) and methylene chloride (1.5 mL), cooled to 0C, and treated with N-hydroxysucçinimi~le (1.03 g, 9.0 mmol) and DCC (1.86 g, 9.0 mmol). The ice bath was allowed to melt and the reaction Illi~lule was stirred for 4 hours. The ~ tule was cooled to 0C and 25 filtered. The filtrate was e~/~ul~ed to give an oil which was chromatographed(305 EtOAc/~e~nes) to give 16 as a white foam (1.36 g, 3.51 mmol) in 47%
yield.

SUBSTITUTE SHEET (RVLE 26) ~ WO 95/0241& 216 5 0 5 2 PCT/US94/07733 Example IV
Synthesis of Isocys-~mino~iri~-merc~rtosl-crin~te ~h~l~tin~ C~ ulld 21 The synthesis procedure is outlined in Figure 4. Co~ n~tion of cysteine 5 8 with ~mino~liric acid derivative 12 to give 17:
To an ice cold solution of S-acm N-T-BOC isocysteine trichloroethyl ester 7, yl~yared in FY~mp]e I, (1008 mg, 2.38 mmol) in methylene chlori-le (7. 0 mL) was added trifluoroacetic acid (6.0 mL) dropwise. The solution was stirred at room t~lllyc;ldlulc for 1 hour. The sol~ltion was evaporated from carbon tlot~hloricie (3 x 50 mL). The residue was dried in vacuo for 18 hours. To an ice cold sol~ltion of the residue 8 in DMF (2.5 mL) was added a solution of 12, ey~cd in FY~mrle II, (867 mg, 2.22 mmol) in DMF (3.5 mL). To this was added triethylamine (0.73 mL, 5.24 mmol). The reaction was stirred at room lelllyeldlule for 6 hours and then ev~yoldled. The residue was partitioned between water and EtOAc. The aqueous phase was extracted with EtOAc (2 x 50 mL). The combined EtOAc extracts were washed with brine, dried, and e~yol~ed. The product was chn,lllalogl~yhed (50% EtOAc/~e~nes 1%
HOAc) to give 17 as a white foam (1005 mg, 1.61 mmol) in 68% yield.

Conden~tion of 17 with succin~t~ reagent 16 to give tripeptide L8:
To an ice cold solution 17 (500 mg, 0.81 mmol) in methylene chloride (4.3 mL) was added trifluoroacetic acid (4.3 mL). The ice bath was removed and ~he reaction was stirred for 1 hour. The sol~ltion was evaporated from carbon tetr~chlc~ride (3 x 30 mL). The residue was dissolved in DMF (1.0 mL) and cooled to 0C. To this was added a solution of 16, yrt;y~ed in Example m, (376 mg, 0.97 mmol) in DMF (2 mL). Lastly triethylamine was added (0.22 mL, 1.62 mmol). The ice was allowed to melt. The reaction was stirred at room ~elllye~ for 21 hours. The solvent was evaporated. The residue was dissolved in EtOAc and washed with pH 4.0 buffer. The aqueous phase was SU8SrlTUTE SHEET (RULE 26) WO 95/02418 PCT/US94/07733 ~

~1650~

extracted with EtOAc, then ~itlifiecl with 1.0 M HCl to pH 3Ø further extracted with EtOAc (2 x 30 mL). The combined EtOAc extracts were washed with brine, dried, and cv~LJGldted. The residue was chr~ atographed (99: 1 EtOAc:HOAc). The product 18 was obtained as a white solid in 80% yield (480 S mg, 0.65 mmol).

Conversion of 1~ to TFP ester 19:
To an ice cold sol~ltion of 18 (480 mg, 0.65 mmol) in ace~o,-it~ ;le (l.S
mL) and methylene chlnride (0.5 mL) were added tPtr~fluorophenol (140 mg, 0.84 mmol) and DCC (161 mg, 0.78 mmol). The ice bath was allowed to melt 10 and the reaction was stirred at room ~ )eld~ufe for 20 hours. The reaction was cooled to 0C, ~eated with 2 drops acetic acid, and filtered. The filtrate was t;vd~oldlcd. The residue was cLlu-lldtop.~lh~d to give 19 as an oil (240 mg, 0.27 mmol) in 42% yield.

Cleavage of TCE ester 19 to give 20:
To a solution of 19 (190 mg, 0.21 mmol) in THF (1.4 mL) and 1.0 M
KH2PO4 (0.28 mL) was added Zn dust (137 mg, 2.10 mmol). The mixture was stirred for 30 ,--in~-les. ~d-lition~l phosphate buffer (0.28 mL) and Zn dust (137 mg, 2.10 mmol) were added. The reaction was stirred for 80 I.~ s~
Ad~liti~n~l phosphate buffer (0.25 mL), l~F (1.0 mL), and Zn dust (137 mg, 2.10 mmol) were added. The reaction was filtered. The filtrate was e~o,~ted.
The residue was chr~---atûgl~æhed to give in the first fractions recovered 19 (60 mg, 0.07 mmol), then in the later fr~-tio~s 20 as a white foam (40 mg, 0.05 mmol) in 25 % yield.

Cleavage of t-butyl ester 20 to give 21:
A solution of 20 (40 mg, 0.05 mmol) in formic acid (1.5 mL) was stirred for S hours. The sollltinn was evaporated. The product was purified by pa~ e LC on reverse phase semi-prep C-18 column with 45% CH3CN/H20 SUBSTITUTE SHEET ~IJLE 26) 216~2 1% HOAc as mobilephase. Theproduct 21 was obtained as a film (6 mg, 0.01 mmol) in 16% yield. The compound 21 is a chel~ting compound of the present mvention.

~ C V
S Synthesis of Cysteine Monocarboxylate Ch~l~/t;n~ Compound 28 The synthesis procedure is outlined in Figure 5. t-BOC cleavage and condçn~tic)n of cysteine 22 with ~mino~dipic acid derivative 12:
To an ice cold solution of 22 (0.97 g, 2.30 mmol) in methylene chloride (6.0 mL) was added trifluoroacetic acid (6.0 mL). The reaction was stirred at room ~ )el~lure, then coev~ol~ted with carbon tetr~çhloride (3 x 15 mL) and dried in vacuo. The residue (23) was dissolved in dimethyl form~mitle (1.0 mL) and triethylamine (0.35 mL, 2.53 mmol). To this was added a suspension of N-t-BOC ~minoa~liric acid-a-NHS-~-t-butyl ester 12, ~lepared in Fx~mple II, (897 mg, 2.30 mmol) in DMF (2.5 mL). Triethylamine (0.35 mL, 2.53 mmol) was added and the reaction was stirred for 18 hours. The solution was concentr~tPA.
The residue was dissolved in EtOAc and washed with pH 4.0 buffer. The aqueous phase was further extracted with EtOAc (2 x 30 mL). The combined EtOAc extracts were washed with brine, dried, and evaporated to give an oil.
Chrollla~o~l~hy (75æ EtOAc/~nPs 1% HOAc) gave 24 as a white solid (1.40 g, 2.30 mmol) in 100% yield. FAB MS parent ions 622 and 624.

D~ ol~;lion of ~ and con~çn~tion with S-ethoxyethyl mer~a~lo~çetic acid NHS ester to give 26:
To an ice cold solution of 24 (690 mg, 1.12 mmol) in methylene chloride (6.0 mL) was added trifluoroacetic acid (6.0 mL). The ice bath was removed 25 and the reaction was stirred at room temperature for 2 hours. The solution was coe~apoldled with carbon tetrachloride (3 x 10 mL). The residue was dissolved in DMF and triethylamine (0.15 mL, 1.12 mmol). To this solution at 0C was SUBSTITUTE SHEET (RULE 26) 21~5~

added a solution of S-ethoxyethyl me~aploacetic acid NHS ester (322 mg, 1.23 mmol) in DMF 2.0 mL). Lastly triethylamine (0.31 mL, 2.24 mmol) was added. The ice bath was allowed to melt and the reaction was stirred at room le~ eldlule for 18 hours. The solvent was e~ ol~ted. The residue was dissolved in EtOAc (30 mL) and washed with pH 4.0 buffer. The aqueous phase was eYtract~P~l with EtOAc (2 x 25 mL). The combined EtOAc extracts were dried and e~oldted. The residue was chromatographed (50% EtOAc/~PY~nPs 1 % HOAc). The product 26 was obtained as an oil (380 mg, 0.55 mmol) in 50% yield.

Conversion of ~_ to TFP ester 27:
To a soluti~ n of 26 (190 mg, 0.31 mmol) in THF (1.8 mL) was added tet~flnorophenol (65 mg, 0.35 mmol) and DCC (73 mg, 0.35 mmol). The reaction was stirred for 20 hours, cooled to 0C, and filtered. The filtrate wase~/~poldted. The residue was chromatographed (99: 1 EtOAc:HOAc). The product ~Z was obtained as colorlP~s oil (150 mg, 0.20 mmol) in 64% yield.

TCE ester cleavage of 27 to give cysteine ligand 28:
To a solution of 27 (90 mg, 0.12 mmol) in THF (0-8 mL) and 1.0 M
KH2PO4 buffer (0.16 mL) was added Zn dust (78 mg, 1.20 mmol). The suspension was stirred for 40 minutps~ Acl-lition~l phosphate buffer (0.16 mL) and Zn dust (78 mg, 1.20 mmol) were added. The reaction was stirred for 40 minutes, filtered, and rinsed with 50% aqueous acetonitrile (30 mL). The filtrate was e~ol~led. The residue was chrol"atogl~hed (15% isopr~anol/methylene chloride 2% HOAc). The product 28 was obtained as an oil (60 mg, 0.10 mmol) in 80% yield. Compound 28 is a ch~l~tin~ col"pound of the present invention.

SUBSTITUTE SHEET (~UEE 26) WO 95/02418 216 5 0 ~i ~ PCT/US94/07733 Example VI
Synthesis of Cysteine S~lcrin~te ~h~l~tin~ Compound 32 The ~y-lthesis procedure is outlined in Figure 6. t-BOC and t-butyl clea~age of 24 and con-le-n~tion with sucein~t~ reagent 16 to give protec~ed 5 tripeptide 29:
To an ice cold sol~ltion of ~, pre~cd as in Example V, (708 mg, 1.16 mmol) in methylene chloride (6.2 mL) was added trifluoroacetic acid (6.2 mL).
The sollltioll was stirred at room tempcldlu~c for 1.5 hours and then evaporatedfrom carbon ~etraehlori~e (3 x 15 mL). To the residue dissolved in DMF (2.0 mL) at 0C was added a solution of L6, ~lcpaled in F.~r~mple m, (450 mg, 1.16 mmol) in DMF (2.0 mL). The reaction was stirred for 18 hours, and concP~ ted. The residue was partitioned between EtOAc and pH 4.0 buffer.
The aqueous phase was extracted with EtOAc (2 x 25 mL). The combined EtoAc ext~ct~ were washed with brine, dried, and cv~L)ol~ted to give an oil.
Chfu~ ogl~?hy (99:1 EtOAclHOAc) provided 29 as a white foam (0.39 g, 0.53 mmol) in 46% yield.

Conversion of 29 to TFP ester 30:
To an ice cold solution of 29 (390 mg, 0.53 mmol) in ~celoni~ ;le (1.0 mL) were added tetrafluorophenol (115 mg, 0.69 mmol) and DCC (131 mg, 0.63 20 mmol) The reaction was stirred for 18 hours, cooled to 0C, filtered, and the filtrate was e~/~pold~ed. Chromatography (75% EtOAc/Hexanes 1 %; HOAc) gave 30 as an oil (400 mg, 0.45 mmol) in 85% yield.

Cleavage of t-butyl and trichloroethylester protecting groups to give 32:
A solution of 30 (200 mg, 0.22 mmol) in formic acid (7.5 mL) was 25 stirred for 3 hours and then evaporated. The residue was chromatographed (99:1, EtOAclHOAc) to give 31 as a white foam. To a solution of 31 (180 mg, 0.22 mmol) in TH}~ (1.44 mL) were added Zn (144 mg, 2.20 mmol) and 1.0 M

SUBSTITUTE SHEET (RULE 26) WO 95/02418 PCT/US94/07733 ~

2~ 650~

KH2PO4 (0.29 mL). The reachon was shrred 40 minutes. Addihional Zn (150 mg, 2.29 mmol) and 1.0 M KH2PO4 (0.29 mL~ were added. The reaction was shrred for 30 .~ les. Addihonal Zn (150 mg, 2.29 mmol) and 1.0 M KH2PO4 (0.29 mL) were added. The reaction was stirred 20 minutes, filtered, rinsed S with ~-etonitrile (25 mL), 50% aqueous acetoniL ile (10 mL), and evaporated to give a solid (140 mg). One third of the crude product was purified by ~r~aLive LC on a semi-analytical C-18 reverse LC column with 45%
~-~elon;l~;le/water 1% acehc acid as the mobile phase. The final ch~l~ting colllpound 32 was obtained as a white film (17 mg, 0.025 mmol). Thus 10 projected yield if all of the crude product had been LC prepped is 34% for the two deL~rolecLion steps. Compound 32 is a chPl~ting colll~ound of the present invenhon.

Example VI~
Synthesis of DAP~ c- in~fe 36 The synthesis procedure is outlined in Figure 7. Contl~n~tinn of 4, 5 -minope~ nQic acid (DAP) with s~lc~in~te reagent 16:
To an ice cold suspension of DAP (338 mg, 1.65 mmol) and 16, ~r~paled in Example m, (1160 mg, 3.0 mmol) in DMF (3.5 mL) was added triethylamine (1.03 mL, 5.77 mmol). The ice bath was allowed to melt and the reaction was stirred at room Len~ldLure for 18 hours. The solution was conrentr~ted. The residue was partitioned between EtOAc and pH 4.0 buffer. The aqueous phase was washed with EtOAc (2 x 50 mL). The combined EtOAc e~rt~tc were washed with brine, dried, and e~d~oldled. The residue was chromatographed (50% EtOAc/~elr~nes 1% HOAc, 400 mL, then 65% EtOAc/~ nes 1%
HOAc) to give 34 as a white solid (770 mg, 1.13 mmol) in 69% yield.

SUBSTITUTE SHEET (RULE 26) 216~0~2 Conversion of 34 to TFP ester 35:
To an ice cold solution of 34 (363 mg, 0.50 mmol) in acelonil, ;le (1.0 mL) and methylene ehlori~e (0.1 mL) were added tetr~fl~lorophenol (113 mg, 0.68 mmol) and DCC (129 mg, 0. 62 mmol). The ice bath was allowed to melt 5 and the reaction was stirred at room le~ e~ule for 18 hours. The reaction was cooled to 0C, treated with 2 drops acetic acid, and filtered. The filtr~t~A wast:va~u~led. The residue was chroll-ato~rarh~i (30% EtOAc/~PY~nes) to give 35 as a white foam (350 mg, 0.41 mmol) in 80% yield.

Conversion of 35 to discuccinate ligand 36:
A soll-tion of 35 (230 mg, 0.27 mmol) was stirred for 2 hours. The soll-til-n was coe-v~û~led with toluene and dried in vacuo. Crude 36 was obtained as a white solid (200 mg). Half of the product was purified by live LC on a C-18 semi-prep reverse phase column. The first eluting major peak, reîe~f~d to as "A", was obtained in 22% yield as a white solid (19 15 mg, û.03 mmol). The second eluting major peak, referred to as "B" was obtairled in 3906 yield (30 mg, 0.05 mmol). High resol~ltion FAB-MS showed parent ions and similar ~gmPnt~tion p~ttprn~ for both isomers "A" and "B".
Compound 36 (both isomers thereof) is a cllel~ting compound of the present invention.

F~ IC VIII
Preparation of }?~lliom-- lide Metal Chelates and A1t~-~hmPnt of the C~hel~tPs to Targeting I`loteills 1. 99mTc ChPl~t~s- Each of the four chel~ting compounds synthesi7ed in Examples I-VII (Compounds 21, 28, 32, and 36) was radiolabeled with 99mTc 25 according to the following procedure:
One mL of sterile water for injection was added to a sterile vial cont~ining a stannous gluconate complex (50 mg sodium ghlcQ~te and 1.2 mg SUBSTITUTE SHEET lRULE 26) 2~L650~ ~

~o-stannous chloride dehydrate, available from Merck Frosst, C~n~ , in dry solid form) and the vial was gently ~git~t~l until the contents were dissolved. A
sterile insulin syringe was used to inject 0.1 mL of the resl-lting stannous glucon~tç solntion into an empty sterile vial. Sodium ~ eclmetate (0.75 mL, 5 75-100 mCi, eluted from a "Mo/"Tc gen~r~tor purchased from DuPont, Mediphysics, M~llinckrodt or E.R. Squibb) was added, and the vial was ~git~tçd gently to mix the colltellls, then incub~t~A at room lelll~ldLulc for 10 ..~h~ s to form a 99mTc-gluconate complex.
In an ~lt~rn~tive procedure for providing the 99mTc-gluconate toy~ h~nge 10 complex, the kit in~ des a vial cont~ining a lyophilized ylc~aldtion comprising 5 mg sodium gluconate, 0.12 mg stannous chloritle dehydrate, about 0.1 mg gentisic acid as a stabilizer colll~oulld, and about 20 mg lactose as a filler colll~ound. The amount of gentisic acid may vary, with the stabilizing effect generally increasing up to about 0.1 mg. Inlelrelel ce with the desired re~ctionC
15 may occur when about 0.2 mg or more gentisic acid is added. The amount of lactose also may vary, with amounts belweell 20 and 100 mg, for example, being ~LræLive in aiding lyophili7~tion. Addition of stabilizer and a filler colll~ound is especi~lly illlpol~ult when the vial contained these relatively small amounts ofsodium gluconate and stannous çhlc)ri(le (col"pared to the ~ltern~tive embodiment 20 above). One mL of sodium perte~hn~t~t~- (about 100 mCi) was added directly tothe lyophilized ~lc~ ;on The vial was ~git~tçd gently to mix the col.~e.
then incubated as described above to form the 99~c-gluconate complex.
A separate vial co~ .ining 0.3 mg of a chPl~ting agent in dry solid form was p-~ared by ~i~pen~ing a solution of 0.3 mg chel~ting agent in acc~onillile 25 into the vial, then removing the solvent under N2 gas. To this vial was then added 0.87 mL of 100% isopropyl alcohol, and the vial was gently shaken for about 2 minutes to completely dissolve the chPl~ting colllpound. Next, 0.58 mL
of this solution of the ch~l~tin~ agent was transferred to a vial cont~ining 0.16 mL of glacial acetic acid/0.2 N HCl (2:14), and the vial was gently ~it~ted. Of 30 this aciciified solution, 0.5 mL was transferred to the vial cont~ining the 99mTc-SUBSTITUTE SHEET (Rl~LE 26) ~ WO 95/02418 21~ ~ O ~ 2 PCT/US94tO7733 gluconate complex, ~lt;pa~ed above. After gentle ~git~tion to mix, the vial was incubatP~ in a 75C+2C water bath for 15 ."in"~es, then imme(1i~tPly t~nsfPrred to a 0C ice bath for 2 i~ es~
To a separate vial col.l;.ininP 10 mg of the Fab fr~gmfnt of a monoclonal antibody in 0.5 mL of phosphate-buffered saline, was added 0.37 mL of 1.0 M
sodiu~m bicarbonate buffer, pH 10Ø The Fab fragment was genPr~tf~d by treating the monoclonal antibody with papain according to convention~l techniques. The monoclonal antibody, ctesign~tPd NR-LU-10, recognizes a panca.~eino.l.a antigen. The vial was gently ~git~tP,d. Other ~eLillg proteins may be substituted for the NR-LU-10 Fab fr~gmPnt The vial cont~inin,~ the ~ei~lifiPcl solution of the 99mTc-labeled chelate (see above) was removed from the ice bath, 0.1 mL of the sodium bicarbonate buffer was added, and the vial was ~git~tPA to mix. TmmeAi~t~ly, the buffered antibody solution (above) was added, gently ~git~ted to mix and incubated at room te -~ ,e for 20 ~ nu~es to allow conjugation of the r~rtiol~beled chelate to theantibody.
A column cof.ti.ini.-g an anion e~ch~nger, either DEAE-Sephadex or QAE-Seph~f~, was used to purify the conjugate. The column was plcpa.ed under aseptic conditions as follows. Five 1 mL QAE-Sephadex columns were connfxted end-to-end to form a single column. ~ltçrn~tively, a single 5 mL
QAE-Sephadex column may he used. The column was washed with 5 mL of 37 mM sodium phosphate buffer, pH 6.8. A 1.2~ filter (available from Millipore) was ~t~f hed to the column, and a 0.2 ~ filter was ~tt~hçd to the 1.2 ~ filter. A
22-gauge sterile, nonpyrogenic needle was ~tt~hçd to the 0.2 ~ filter.
The reaction llli~lUlt; was drawn up into a 3 mL or S mL syringe, and any air bubbles were removed from the solution. After removal of the needle, the syringe was connected to the QAE-Sephadex column on the end opposite the filters. The needle cap was removed from the 22-gauge needle ~tt~çhed to the filter end of the column and the needle tip was inserted into a sterile, nonpylogellic test tube. Slowly, over 2 ~ n~lçs~ the reaction Illi~lUlt~ was SUBSTITUTE Sl I~ET (RULE 26) WO 95/02418 PCT/US94/07733 ~

2165~5~2 injected into the column. The eluant collected in the test tube was discarded.
The now empty syringe on top of the column was replaced with a 5 mL syringe g 5 mL of 75 mM (0.45%) sodium chloritle solution (from which air bubbles had been removed). The needle at the other end of the column was 5 inserted aseptically into a sterile, nonpyrogenic 10 mL serum vial. Slowly, over 2 ...;i.~le5, the NaCl solution was injected into the column, and the eluent wascollectçd in the serum vial.
The re~nlting radiolabeled antibody fr~gm~nt~ may be r~resented as follows:

CO--Fab ~Fab C--( Tc ~F =~/ Tc/ ~F
HOO S S COOH HOOC~J~S S COOH

o I--CO--Fab ~ ~CO--Fab 10 2. I88Re Cht l~tes The same ch~l~tinP: compounds may be radiolabeled with l88Re by a procedure similar to the 99mTc labeling procedure. Sodium pçrrhen~te produced from a W-188/Re-188 lesea~ch scale generator is combined with citric acid (a ~refellcd comrle~ring agent for l88Re), a reduçing agent, and preferably gentisic 15 acid and lactose. The res--ltin~ l88Re-citrate ç~ch~nge complex is heated with the desired chPl~ting coll.poùlld, as above. A Cl8 reversed phase low ~res~ule m~tPri~l (13aker Cl8 cartridges) may be used to purify the l88Re-chelate. A

SUBSTITUTE SHEET (RULE 26) ~ WO 95/02418 21~ 5 ~ 5 2 PCT/US94/07733 monoclonal antibody or fr~gmçnt thereof is reacted with the chelate in a buffered solution to bind the chelate thereto, as described for the 99mTc ~roce lule. A
Sephadex G-25 column may be used to purify the radiolabeled antibody.

Ex~u,~le IX

S Biodistribution of the four 99mTc-labeled antibody fr~mçnt~ p~a~ed in FY~mple Vm was analyzed in a rat model. 100 ,ug of protein (about 0.5 mCi) was ~-lmini~tPred intravenously into Sprague-Dawley rats. Each of the four typesof radiolabeled antibody fr~mPnt~ (i.e., NR-LU-10 Fab fr~mçnt~ radiolabeled with one of the four different chpl~ting co~ uunds) was injected into three rats.
Rio~ tribution was analyzed at 6 hours post-injection by i~ol~ting intestines and kidneys and de~ ;ni~ the mCi of injected radioactivity per gram of these tissues, using a dose calibrator. The ~e~ell~ge of injected dose per gram of inte:jl;n~l and kidney tissue was calculated and averaged to give the mean valuefor each group of three ~nim~l~
The results were compared with data on intestin~l loc~li7~tion of r~rlio~ctivity for radiolabeled antibody fr~gment~ of the following formula I
(wherein the fr~gmPnt~ are labeled with an N2S2 chelate that lacks carboxylic acid substit~lent~):

~~C~Fab 0' 1 =(S S

A reduction in inte~l;n~l loc~li7~tion of radioactivity was demonstrated for each of the four radiolabeled antibody fr~gm~nt~ of the present invention, col,lpared to the radiolabeled fr~mçnt of formula (I).

SUBSTITUTE SHEET tRUEE 26) WO 95/02418 PCT/US94/07733 ~
2 1 ~ 2 . 1~ ..

Example X
Preparation of l~liol~heled Antibody F~ s 1. 99mTc Ch~l~t~c: Ch~ ting compounds 21 and 36 (synthe~i7ed in FY~mrlçs IV and VII, respectively) were r~-liol~heled with 99mTc according to the following procedure (a ~refelfed procedure for these two çhPl~ting compounds):
One mL of sterile water for injection was added to a sterile vial co~ nil-g a stannous gluconate complex (50 mg sodium glncoll~te and 1.2 mg stannous chlori~e dehydl~Le, available from Merck Frosst, C~n~d~, in dry solid form) and the vial was gently ~p~it~tK~ until the contents were dissolved. A
sterile insulin syringe was used to inject 0.1 mL of the resl-ltinp ~I;.n~ouc gluconate sollltir~l~ into an empty ste~ile vial. Sodium perte~hn~t~t~- (0.75 mL, 75-100 mCi, eluted from a 99Mo/9~c generator purchased from DuPont, M~Airhysics, M~llinckrodt or E.R. Squibb) was added, and the vial was ~git~t~d gently to mix the contçnt~, then incubated at room temperature for 10 minlltes to form a 99mTc-gl-lcon~te complex.
A separate vial co~t~inin~ 0.3 mg of the çhel~ting agent (21 or 36) in dry solid form was prepa.ed by dispensing a solution of 0.3 mg chel~ting agent in ac~;~oniL- ;le into the vial, then removing the solvent under N2 gas. To this vial was then added 0.87 mL of 100% isopropyl alcohol, and the vial was gently shaken for about 2 ~ nules to completely dissolve the ch~l~ting compound.
Next, 0.58 mL of this solution of the chel~ting agent was transferred to a vial cor.l~;ning 0.16 mL of glacial acetic acid/0.2 N HCl (2:14), and the vial ws gently ~git~t~. Of this ~ci~ified solution, 0.5 mL was t~ansferred to the vial con~ the 99~c-gluconate complex, ~l~pal~d above. After gentle agitation to mix, the vial was incubated in a 75C~2C water bath for 15 mimltes, then immyli~tloly tr~nsfçrred to a 0C ice bath for 2 minutes.
For compound 36, and whenever the radiolabeling yield for compound 21 was below 40%, the radiolabeled chelate was purified prior to conjugation to an antibody as follows. An SPE-Cl8 extraction column (a reversed phase column SUBSTITUTE SHEET (RULE 26) ~ Wo 95/0~418 PcTluss4lo7733 216~0~

~s-available from Baker) was co~itic)ned by washing with 2~mL of ethanol followed by 2 mL of sterile water. The reaction llli~Ur~ was then loaded onto the top of the column. The column was washed with 2 mL aliquots of 1% ethanol/0.01 M
phQsph~te (pH=7.0) 6-8 times and dried for 10 minlltçs under vacuum. The 99mTc chtol~tes were then eluted using 0.5 mL of CH3CN for co~ ound 21 and 1 mL of CH3CN for colllpound 36. The CH3CN was e~oldted under a stream of N2 p~rior to the conjugation with antibody.
The 99mTc ch~l~tes thus purified were ~tt~`hed to the Fab fr~.~ment of a monoclonal antibody (~iesign~t~d NR-LU-10) as described in Example VIII.
Other ~eLiilg proteins may be substituted for the NR-LU-10 antibody fr~gm~nt Example XI
Preparation of 99mTc ~hPl~te Using ~hPl~t;n~ Compound 32 Co~ ound 32 (~r~aled in Example VI) was r~(liol~heled by the following procedure, which is pl`t;r~ d for this particular ch~l~ting co~ ouild:One mL of NaTcO4 (~ 100 mCi) was added to a lyophilized prep~r~tion cont~ining 5.0 mg of sodium gluconate, 0.12 mg of stannous chloride dehydrate, 0.1 mg of gentisic acid, and 20 mg of lactose (lyophili7~tic-n pH=3.5). After incubating the vial at room tt;ll~eldlu~ for 2 ~ u~s~ 0.1 mL of co-ll~ouild 32 (1 mg/mL in 90% isoplupyl alcohol) was added. Then 0.300 mL of isopropyl alcohol and 0.060 mL of 0.1 N HCl were added. 2 cc of air was added into the vial and incubated at 75C for 15 ~ es. The vial was then imm~i~t~ly transferred to a 0C ice bath for 2 ,~ es.
The resnlting g9mTc chelate was ~tt~ched to an antibody fr~gm~nt as described in Example VIII. Other targeting proteins may be substituted for the antibody fr~pm~nt SllBSrlT~lTE SHEET (RULE 26) Wo 95/02418 PCT/US94/07733 ~
2~65~2 ~6-F,Y~n~rle XII
R~inl~l-eled ~ n(l I`~ lion A. A synthesis scheme for a N2S2-biotin conjugate is shown below:

o 11 o Il ~ 11 HO--C--CH--(Cl~2)4 Nl 1 GOC + N--O--C--CH2--STHP

o o C HCOOH ~ HO--C--fH--(CH2)4--N~ DMF
NH NH Et3N

S S
THP THP
c d O O
HO--C--CH (CH2)4--NH--C--(CH2)4~ ~
NH ) ( ENDHCSI , \F HN~ NH
S O
THP

SUBSTITUTE SHEET (RV~E 26 I WO 95/02418 216 S O ~ ~ PCT/US94/07733 aCM

~' " CYS \ HO~CH--NH2/~
o DMF, EtSN
Il O O
~N--~C--Cl~ '~ ~111 11 (CH2)~?

~ \N(HC~)4 ~1 C--(C~2) HOOC-- ~
HN~o~NH

\S S/
aCM THP

Epsilon-BOC-lysine a (available from R~h~m Inc.) is acylated with N-hyd~ y succinimidyl-S-tetrahyd,u~y,~nyl merca~o~et~te _ (~re~alable in accol.lance with known procedures for plute~;ling thiols as S-tetrahyd,u~y,~lyl hemithio~et~l~, such as Greene et al., Protective Gruups in Organic Synthesis, 2nd ed., page 291, John Wiley & Sons, Inc., New York, 1990, to give N-alpha-(S-tet~ahydr~y,~lylmercapto acetyl)-N-epsilon-BOC-lysine_. The BOC group is cleaved with formic acid, and the rçsult~nt amine d is acylated with NHS-biotin to give _. The free carboxyl group of ç is activated with NHS and EDCI
to give f, which is then coupled to S-~çet~mi(lomethyl-cysteine to give the result~nt N2S2-biotin conjugate g.

SUBSTIT~ SHEET t~ULE 26) Wo 95/02418 PCT/US94/07733 lg~

2165~3rJ 2 B. Diamino~e.~ oic acid (DAP) co~e N2S2-biotin conjugates of the following general form~ are contel--plated;as embc~lim~nt~ of the present invention:

o o (C~2k--C ~JI I CH--(C~)4--NH--C--(CH~) NH~6~0 X )~( HN~NH

Y~ SAy O
~HP THP
or or EOE EOE

wherein X is H (synthP~i7ed using a 5-biotinam4-do-pentylamine re~ct~nt, 5 wherein the reactant is available from Pierce Chemical Company) or COOH
(synthto~i7.t~A using biocytin as a r~-t~nt, wherein the reactant is available from Sigma Ch~mic~l Company) and wherein Y is H (synth~i7ed using bis-EOE-me~ca~Loacetyl-DAp as a re~ct~nt, wherein the reactant is syntht~si7~hle by known procedures) or CH2COOH (synthP~i7~1 using bis-THP-mercal~osuccinyl-10 DAP as a reactant).

SUBSrlT~lTE SHEET ~RULE 26) ~ WO 95102418 21 ~ ~ ~ S 2 PCT/US94/07733 ~9 A one step synthesis for such DAP core N2S2-biotin conjugates is shown below:

F ~ O
(CH~--C ~ ~ HzN-ICH--(CH~4--N~--C-(CH~~

HN~"NH

\SEO ~COOH
E EOE
O O
o \N(HCH~ C NH-ICH-(CH~4-NH-C-(CH~4 ~

HN~"NH

\SEO ~ COOH O
E EOE

A s~spen~ion of N2S2-tetr~fluolu~hellylester or thioester a and biocytin b is heated at 100C for lO minut~s. The product is purified by C-18 flash 5 chromalogl~hy to afford the N2S2-biotin amide product. The 5-biotin~mido-pentylamine and N2s2-tetr~flllorophenyl ester reaction occurs analogously.
C. In the ~ ;on of a conjugate as shown below, the following procedure may be employed.

ON~ \ (C ~ -O~ (C ~ -C-NH-CH-(CH~-N~-C-(CH~4 S
NH~ O X ~

HN ~ NH

\~sc~ EOE

SlJBSTITUTE SHEET (RULE 26) ., ",~, ,.

Claims

What is Claimed is:

1. A compound of the formula:

wherein:
each R independently represents =O, H2, lower alkyl, -(CH2)n-COOH, -(CH2)n-CO-glucose or glucose derivative, -(CH2)n-NH-glucose or glucose derivative, or R1-Z;
n is 0 to 3;
R1 represents a lower alkyl or substituted lower alkyl group;
Z represents biotin and optionally a linker moiety;
each R2 independently represents H2, a lower alkyl group, -(CH2)n-COOH, -(CH2)n-CO-glucose or glucose derivative, -(CH2)n-NH-glucose or glucose derivative, or R1-Z;
each m is 0 or 1, with at most one m = 1;
each T represents a sulfur protecting group; and the compound comprises at least one (CH2)n-COOH substituent or one -(CH2)n-CO-glucose or glucose derivative or one -(CH2)n-NH-glucose or glucose derivative and one -R1-Z substituent

2. The compound of claim 1 wherein R1 is a methylene chain comprising from two to three carbon atoms.

3. The compound of claim 1 wherein two R substituents are =O.

4. The compound of claim 1 wherein at least one R2 substituent is -(CH2)n-COOH.

5. The compound of claim 1 wherein at least one T represents a hemithioacetal sulfur protecting group.

6. (Deleted)

7. (Deleted)

8. A compound of claim 1 having the following formula:

wherein each T represents a hemithioacetal sulfur protecting group and Z represents biotin and, optionally, a linker moiety that is part of a ligand conjugation group.

9. (Deleted)

10. A compound of claim 1 of the following formula:

wherein A represents an acetamidomethyl sulfur protecting group, T represents a hemithioacetal sulfur protecting group, and Z comprises biotin.

11. A compound of claim 1 of the following formula:

wherein A represents an acetamidomethyl sulfur protecting group, T represents a hemithicacetal sulfur protecting group, and Z comprises biotin.

12. A compound of claim 1 of the following formula:

wherein A represents an acetamidomethyl sulfur protecting group, T represents a hemithioacetal sulfur protecting group, and Z comprises biotin.

13. A compound of the formula:

wherein:
M represents a radionuclide metal or an oxide thereof;
each R independently represents =O, H2, lower alkyl, -(CH2)n-COOH, -(CH2)n-CO-glucose or glucose derivative, -(CH2)n-NH-glucose or glucose derivative, or R1-Z;
n is 0 to 3;
R1 represents a lower alkyl or substituted lower alkyl group;
Z represents a ligand or an anti-ligand and, optionally, a linker moiety that is part of a ligand or an anti-ligand conjugation group;
each R2 independently represents H2 lower alkyl, -(CH2)n, -COOH, -(CH2)n-CO-glucose or glucose derivative, -(CH2)n-NH-glucose or glucose derivative, or R1-Z;

each m is 0 or 1, with at most one m = 1; and the compound comprises at least one -(CH2)n-COOH substituent or one -(CH2)n-CO-glucose or glucose derivative or one -(CH2)n, -NH-glucose or glucose derivative substituent and one -R1-Z substituent.

14. The compound of claim 13 wherein R1 is a methylene chain comprising from two to three carbon atoms.

15. The compound of claim 13 wherein two R substituents are =O.

16. The compound of claim 13 wherein at least one R2 substituent is -(CH2)n-COOH.

17. (Deleted)

18. (Deleted)

19. The compound of claim 13 wherein M represents a radionuclide metal selected from the group consisting of 99mTc, 186Re, 188Re, and oxides thereof.

20. A compound of claim 13 of the formula:

wherein M represents a radionuclide metal selected from the group consisting of 99mTc, 186Re and 188Re; and Z comprises biotin.

21. A compound of claim 13 of the formula:

wherein M represents a radionuclide metal selected from the group consisting of 99mTc, 186Re and 188Re; and Z comprises biotin.

22. A compound of claim 13 of the formula:

wherein M represents a radionuclide metal selected from the group consisting of 99mTc, 116Re and 188Re; and Z comprises biotin.

23. A compound of claim 13 of the formula:

wherein M represents a radionuclide metal selected from the group consisting of 99mTc, 186Re and 188Re; and Z comprises biotin.

74-1 -24. A N2S2-biotin conjugate selected from the group consisting of:

or where:
ACM is acetamidomethyl, EOE is ethoxyethyl, THP is tetrahydropyranayl, Y is H or CH2COOH, and X is H or COOH.

25. A compound of the formula:

wherein:
Each R independently represents =O, H2, lower alkyl, -(CH2)n-COOH, -(CH2)n-CO-glucose or glucose derivative, -(CH2)n-NH-glucose or glucose derivative or R1-Z;
n is 0 to 3;
R1 represents a lower alkyl or substituted lower alkyl group;
Z represents biotin and optionally a linker moiety;

each R2 independently represents H2, a lower alkyl group, -(CH2)n-COOH, (CH2)n-CO-glucose or glucose derivative, -(CH2)n-NH-glucose or glucose derivative, or R1-Z;
each m is 0 or 1, with at most one m= 1;
each T represents a sulfur protecting group; and the compound comprises at least one -(CH2)n-CO-glucose or glucose derivative or one -(CH2)n-NH-glucose or glucose derivative and one -R1-Z substituent

26. A compound of the formula:

wherein:
M represents a radionuclide metal or an oxide thereof;
each R independently represents =O, H2, lower alkyl, -(CH2)n-COOH, -(CH2)n-CO-glucose or glucose derivative, -(CH2)n-NH-glucose or glucose derivative, or R1-Z;
n is 0 to 3;
R1 represents a lower alkyl or substituted lower alkyl group;
Z represents a ligand or an anti-ligand and, optionally, a linker moiety that is part of a ligand or an and-ligand conjugation group;
each R2 independently represents H2 lower alkyl, -(CH2)n, -COOH, -(CH2)n-CO-glucose or glucose derivative, -(CH2)n-NH-glucose or glucose derivative, or R1-Z;
each m is 0 or 1, with at most one m = 1; and the compound comprises at least one -(CH2)n-CO-glucose or glucose derivative or one -(CH2)n, -NH-glucose or glucose derivative substituents and one -R1-Z
substituent.-