Note: Descriptions are shown in the official language in which they were submitted.
W095/05398 ~ 3 8 ~l~S911~292
S3N CHELATING COMPOUNDS FOR THE RADIOLABELING
OF LIGANDS, ANTI-LIGANDS CR OTHER P~OTEINS
Backaround
Radiolabeled proteins such as antibodies are used in a
variety of diagnostic and therapeutic medical procedures. The
increased specificity of monoclonal antibodies, compared to
polyclonal antibodies, makes them even more useful for
delivering diagnostic or therapeutic agents such as
radioisotopes to desired target sites in vivo. A monoclonal
antibody specific for a desired type of target cells such as
tumor cells may be used to deliver a therapeutic radionuclide
attached to the antibody to the target cells, thereby causing
the eradication of the undesired target cells. Alternatively,
a monoclonal antibody having a diagnostically effective
radionuclide attached thereto may be administered, whereupon
the radiolabelèd antibody localizes on the target tissue.
Conventional diagnostic procedures then may be used to detect
the presence of the target sites within the patient. In
contrast to such "chelate-labeled antibody" proceures,
pretargeting approaches may be used to achieve therapeutic or
diagnostic goals, which pretargeting approaches involve the
interaction of two members of a hign affinity binding pair
such as a ligand-anti-ligand binding pair.
One method for radiolabeling proteins such as antibodies
as well as proteinaceous and non-proteinaceous binding pair
216553~
WO95/05398 ~1/U~91~'~3292
members involves attachment of radionuclide metal chelates to
the proteins or binding pair members. Chelates having a
variety of chemical structures have been developed for this
purpose. The usefulness of such chelates is dependent upon
a number of factors such as the stability of radionuclide
binding within the chelate and the reactivity of the chelating
compound with the desired protein or bindir.g pair member. The
efficiency of radiolabeling of the chelating compound to
produce the desired radionuclide metal chelate also is
important. Another consideration is the biodistribution of
the radiolabeled protein or binding pair member and
catabolites thereof in vivo. Localization in non-target
tissues limits the total dosage of a therapeutic radiolabeled
protein or binding pair membe- that can be administered,
thereby decreasing the therapeutic effect. In diagnostic
- procedures, localization in non-target tissues may cause
undesirable background and/or result in misdiagnosis. The
need remains for improvement in these and other
characteristics of radionuclide metal chelate compounds used
for radiolabeling of proteins such as antibodies. The use of
pretargeting approaches diminishes non-target tissue
localization of radiolabel; however, the need rem~in~ for
improvement in molecules incorporating chelates and binding
pair members of proteinaceous or non-proteinaceous structure.
Summarv of the Invention
The present invention provides chelating com?ounds useful
as protein or binding pair member labeling reagents, the
corresponding radionuclide metal chelates, and targeting
molecules such as proteins or binding pair members
radiolabeled therewith. The radiolabeled proteins or binding
pair members of the present invention have use in various
assays as well as ln vivo diagnostic and therapeutic
procedures. The protein may be a monoclonal antibody that
binds to cancer cells, for example. The binding pair member
may be a ligand or an anti-ligand, for example.
~ -- 2165~38
WOgS~ 38 PCT~S94/09292
The chelating compounds or chelate-binding pair member
conjugates of the present invention include compounds of the
formulas:
T T
H C/SR S~R- (I)
Q-N~ "SR
~ l\
R R' R" R
and
/SR /R
I (II)
HN~ SR
./1 ~\
R R' Rn R"
WOss/05398 2 1 6 S ~ 3 ~ PCT~S91~32$2
-- 4
wherein:
each R is a protecting group;
Q is hydrogen or a protecting group;
each T is independently chosen from hydrogen, lower alkyl
groups of from l to about 6 carbon atoms, electron withdrawing
groups, and lower alkyl groups of from l to about 6 carbon
atoms substituted with electron withdrawing group(s);
X represents O, S, or NH;
each R' is independently selected from:
-(CH2)n-COOH with n=0 to about 4,
-(CH2)n-Z, wherein Z represents a conjugation group
reactive with a protein, a ligand or an anti-ligand; a ligand;
or an anti-ligand; a ligand-linker moiety or an anti-ligand
linker moiety wherein the linker moiety is a portion of a
ligand or an anti-ligand conjugation group and wherein n=0 to
about 4,
hydrogen, and
a lower alkyl group of from l to about 6 carbon
atoms;
each R" is independently selected from:
-(CH2)n-COOH, with n=0 to about 4,
hydrogen, and
a lower alkyl group of from l to about 6 carbon
atoms; and
the compound comprises at least one -(CH2)n-Z substituent.
The conjugation group Z serves to react with a protein or
binding pair member to bind the chelating compound thereto.
Alternatively, Z may itself constitute or include a binding
pair member.
The compounds of formulas I and II are reacted with
molecules such as proteins or binding pair members to attach
the compounds to the proteins or binding pair members. The
compounds may be radiolabeled before or after attachment to
the protein or binding pair member. The resulting
radiolabeled proteins or binding pair members have diagnostic
`` 216~538
WO9S~38 PCT~S~ 3252
or therapeutic use, depending on the particular radionuclide
employed.
The nitrogen atom and three sulfur atoms shown in
formulas (I) and (II) are believed to function as donor atoms
that are bonded to the radionuclide metal in the corresponding
chelate. The compounds of formulas (I) and (II) thus may be
designated S3N chelating compounds.
Radiolabeling of the chelating compounds of formulas I
and II produces the radionuclide metal chelates of formulas
III and IV, respectively:
T T T T
15 2l ~ M ~ x=c/S~ S R~
R' ~R" R~ R' ~R" R~
III IV
wherein M represents a radionuclide metal or oxide thereof and
the other symbols are as defined above. Preferred
radionuclide metals include 99~Tc, ~8aRe, and ~6Re.
The present invention also provides protein-chelating
compound conjugates resulting from reaction of a Z group of
compounds I or II with a protein. In addition, ligand- or
anti-ligand-chelating compound conjugates resulting from
reaction of an appropriate Z conjugation group of certain
embodiments of the present invention with a ligand or anti-
ligand are contemplated as additional embodiments of the
present invention. Radiolabeled proteins, ligands or anti-
ligands comprising a radionuclide metal chelate of formula III
or IV attached to a targeting protein, ligand or anti-ligand
also are provided by the present invention. An example of a
ligand is biotin, with the complementary anti-ligand thereof
being avidin or streptavidin, wherein biotin and avidin or
streptavidin together form a ligand-anti-ligand binding pair.
-
W09s/0s398 2 1 6 ~ ~ 3 8 PCT~S94/09292
Brief Descri~ton of the Drawinas
Figure 1 depicts the tumor uptake profile of NR-LU-10-
streptavidin conjugate (Lu-10-StrAv) in comparison to a
control profile of nature NR-LU-10 whole antibody.
s
Detailed DescriPtion of the Tnvention
Prior to setting forth the invention, it may be helpful
to set forth definitions of certain terms to be used within
the disclosure.
Taraetinc moiety or Tarcetinq molecule: A molecule that
binds to a defined population of cells. The targeting moiety
may bind a receptor, an oligonucleotide, an enzymatic
substrate, an antigenic determinant, or other binding site
present on or in the target cell population. Targeting
moieties that -re proteins are referred to herein as
"targeting proteins." Antibody is used throughout the
specification as a prototypical example of a targeting moiety
and a targeting protein. Tumor is used as a prototypical
example of a target in describing the present invention.
Licand/anti-liqand ~air: A complementary/anti-
complementary set of molecules that demonstrate specific
binding, generally of relatively high affinity. Exemplary
ligand/anti-ligand pairs include zinc finger protein/dsDNA
fragment, hapten/antibody, lectin/carbohydrate,
ligand/receptor, and biotin/avidin. Biotin/avidin is used
throughout the specification as a prototypical example of a
ligand/anti-ligand pair.
Anti-liaand: As defined herein, an l'anti-ligand"
demonstrates high affinity, and preferably, multivalent
binding of the complementary ligand. Preferably, the anti-
ligand is large enough to avoid rapid renal clearance, and
contains sufficient multivalency to accomplish crosslinking
and aggregation of targeting moiety-ligand conjugates.
Univalent anti-ligands are also contemplated by the p_esent
invention. Anti-ligands of the present invention may exhibit
or be derivitized to exhibit structural features that direct
~ W09s/05398 2 1 6 ~ 5 ~ 8 PCT~S~ 3292
the uptake thereof, e.c., galactose residues that direct liver
uptake. Avidin and streptavidln are used herein as
prototypical anti-ligands.
Avidin and Stre~tavidin: 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.
Liaand: As defined herein, a ~ligand~ is a relatively
small, soluble molecule that exhibits rapid serum, blood
and/or whole body clearance when administered intravenously
in an animal or human. Biotin is used as the prototypical
ligand.
Pretaraetinq: As defined herein, pretargeting involves
target site localization of a targeting moiety th~ is
conjugated with one member of a ligand/anti-ligand pair; after
a time period sufficient for optimal target-to-non-target
accumulation of this targeting moiety conjugate, active agent
conjugated to the opposite member of the ligand/anti-ligand
pair is administered and is bound (directly or indirectly) to
the targeting moiety conjugate at the target site (two-step
pretargeting). Three-step and other related methods described
herein are also encompassed.
Linker MoietY: A moiety that is a portion of a protein,
ligand or anti-ligand conjugation group that rem~; n~ part of
the structure of a protein-chelate, ligand-chelate or anti-
ligand-chelate conjugate following the conjugation step. For
example, the linker moiety of an active ester chelate
derivative includes, for example, a carbonyl (-CO-) moiety.
The present invention provides chelating compounds useful
as protein or binding pair member labeling reagents, methods
for radiolabeling proteins or binding pair members using these
reagents, and the resulting radiolabeled proteins or binding
pair members having use in diagnostic or therapeutic
procedures. The protein or binding pair member labeling
reagents and chelate-binding pair conjugates are of the
following formulas I and II:
09s/05398 2 1 6 5 ~ 3 ~ PCT~S91l03252
a
H2C/SR S~RI
Q-N SR
R' ~ (I)
R R" R'
X=C SR S~R.
lo HN SR (II)
R' ~<R"
wherein:
each R is a protecting group;
Q is hydrogen or a protecting group;
each T is ir.dependently chosen from hydrogen, lower alkyl
groups of from 1 to about 6 carbon atoms, electron withdrawing
groups (e.g., nitro, sulfonate, or carboxylic acid groups),
and lower alkyl groups of from 1 to about 6 carbon atoms0 substituted with electron withdrawing group(s);
X represents 0, S, or NH;
each R' is independently selected from:
-(CH2)n-COOH with n=0 to about 4,
-(CH2)n-Z, wherein Z represents a conjugation group
reactive with a protein, a ligand or an anti-ligand; a ligandi
or an anti-ligandi or a ligand-linker moiety or an anti-
ligand-linker moiety wherein the linker moiety is a portion
of a ligand or an anti-ligand conjugation group and wherein
n=0 to about 4,
hydrogen, and
a lower alkyl group of from 1 to about 6 carbon
atoms (preferably 1 or 2 car~on atoms);
each R" is independently selected from:
-(CH2)n-COOH, with n=0 to about 4,
hydrogen, and
~ W095/05398 2 1 6 5 ~ 3 8 PCT~S94/09292
a lower alkyl group of from 1 to about 6 carbon
atoms (preferably 1 or 2 carbon atoms); and
the compound comprises at least one -(CH2)n-Z substituent.
For the reagents of formulas (I) and (II), the two T
substituents preferably are identical (preferably, both are
methyl groups, most preferably, both are hydrogen).
Further, the compounds preferably comprise only one
-(CH2)n-COOH substituent and only one -(CH) 2) n~Z substituent.
The -(CH2)~-COOH substituent generally increases the water
solubility of the compound.
The conjugation group Z is a functional group that will
react with a functional group on a molecule to be radiolabeled
(e.g., a targeting protein, a ligand or an anti-ligand)
thereby attaching the chelating compound thereto.
Radiolabeling of the chelating compound produces a
radionuclide metal chelate attacheQ to the targeting protein,
ligand or anti-ligand. The chelating compounds of the present
invention each comprise at least one conjugation group, as
described in more detail below.
In the compound of formula I, Q represents hydrogen or
any suitable nitrogen protecting group (a number of which are
known) such as an alkyl group of 1 to 6 carbon atoms.
Q preferably is hydrogen or a methyl group.
For the compounds of formula II, X preferably is 0.
R represents any suitable sulfur protecting group. A
number of protecting groups, including but not limited to
acyl, ar~l, and alkyl groups, are known for use in protecting
sulfur atoms. The protecting groups should be removable,
either prior to or during the radiolabeling reaction. The
protecting groups on the three sulfur atoms may be the same
or different. In some cases, a single protecting group (e.g.,
a thioacetal) may serve to protect two sulfur atoms, as shown
below.
Among the suitable sulfur protecting groups are
hemithioacetal, thioacetal, benzyl, and acetamidomethyl
-- 216553~
WOss/05398 PCT~S~ 3292
- 10 -
protecting groups. Also useful are acyl type groups such as
those of the formula O
-S-C-R, wherein the S is a sulfur atom of
the chelating compound and R is an alkyl or aryl group.
Examples are isobutyryl, benzoyl, and acetyl protecting
groups.
When hemithioacetal protective groups are used, each
sulfur atom to be protected has a separate protective group
attached to it, which together with the sulfur atom defines
a hemithioacetal group. The hemithioacetal groups contain
acarbon atom bonded directly (i.e., without any intervening
atoms) to a sulfur atom and an oxygen atom, i.e.,
S - C - O -
I
Preferred hemithioacetals generally are of the following
formula, wherein the sulfur atom is a sulfur atom of the
chelating compound:
oR3
R4-C--Rs
S
wherein R3 is a lower alkyl group, preferably of from two to
five carbon atoms, and R~ is a lower alkyl group, preferably
of from one to three carbon atoms. Alternatively, R3 and R4
may be taken together with the carbon atom and the oxygen atom
shown in the formula to define a nonaromatic ring, preferably
comprising from three to seven carbon atoms in addition to the
carbon and oxygen atoms shown in the formula. R5 represents
hydrogen or a lower alkyl group wherein the alkyl group
preferably is of from one to three carbon atoms. Examples of
~` WO9S~3J3~ 2 1 6 ~ ~ 3 8 PCT~S~/03292
such preferred hemithioacetals include, but are not limited
to:
~H2 CH2
--CH2 / --CH2
0 0
\C~CH2 \,C`--CH2
H S H3C S
Tetrahydrofuranyl 2-methyl tetrahydrofuranyl
o CH2 H3C CH2
H2f lcH2 ~f H2 H2f f H2
2 \C~ 3 \CH~ 2 \C~
~ H ~ S CH3
I
Tetrahydropyranyl ethoxyethyl 2-methyl tetrahydropyranyl
Other hemithioacetal sulfur protecting groups include
those derived from monosaccarides, such as the following,
wherein the sulfur atom is a sulfur donor atom of the
chelating compound:
CH20H CH3
2 5 HO~ j/ HO~Y
HO~ yS-- HO~S--
JOH OH
Examples of thioacetal protecting groups are as follows,
wherein two sulfur atoms (the two sulfur atoms attached to
adjacent carbon atoms in the chelating compound) are attached
35 to a single protecting group:
~ WOgs/0s398 2 1 6 5 ~ 3 ~ PCT~S91/03~52
Y\ /S--
y~C~s--
wherein each Y is independently selected from hydrogen, alkyl
groups of 1 to 6 carbons (preferably methyl or ethyl), alkoxy
groups of 1 to 6 carbons (preferably 1 or 2 carbon atoms),
phenyl groups, and phenyl rings having an electron donating
group (e.g., hydroxy, methoxy, or ethoxy group) bonded
directly thereto. The two sulfur atoms shown are sulfur donor
atoms of the chelating compound which, together with the
protecting group, form the thioacetal group. Suitable
thioacetals include, but are not limited to, the following:
~C~ ~ C S
Representative examples of the compounds of formula (I)
include, but are not limited to:
H2C SR SRZH2CI SR SRZ H2lC SR SRZ
HN SR HN SR HN~ ~SR
HOOC~H <HHOOC~<CH3 H/r7H \COOH
H2C SR SRZH2C SR SRZ H2CI/~COOH
HN SR HN SR HN ~SR
3 5 COOH H ?OOH HH Z~ H
W09S~5 21 6 5 5 3 8 PCT~S911'~3292
- 13 -
Representative examples of the compounds of formula (II)
include but are not limited to:
O=C SR SRZ O--C SR SRZ
HN~ SR HN SR
HOOC H H3C COOH
O=C SR SR COOH O=CI SR SR Z
HN SR HN~ SR
H/r~COOH
Z H H
O=C/r~R\Z
HN SR
H>~COOH
H H3C
In preferred chelating compounds of the present
invention, Z represents an active ester (described below).
The two sulfur atoms that are attached to immediately adjacent
carbon atoms (i.e., the vicinal dithiol portion of the
compound) preferably are attached to a single protecting group
(e.g., a thioacetal). The rem~in;ng sulfur atom preferably
is protected by a different group such as a benzyl group.
The compounds of formulas I and II are useful as reagents
for radiolabeling other molecules. The chelating compounds
may be attached to the molecule to be rad~olabeled either
before or after the radiolabeling reaction. The molecule
should contain (or be modified to contain) a functional group
such as a primary amine or sulfhydryl that will react with the
WO95/OS398 2 1 6 ~ 5 3 ~ PCT~S94/09292
conjugation group on the chelating compound. The molecule may
be any such molecule to be radiolabeled for use in in vitro
assays, diagnostic or therapeutic procedures ln vivo, or other
such purpose.
In one embodiment of the invention, the molecule to be
radiolabeled is a targeting molecule. The targeting molecule
is any molecule that will serve to deliver the radionuclide
metal chelate to a desired target site (e.g., target cells)
ln vitro or in vlvo. Examples of targeting molecules include,
but are not limited to, steroids, lymphokines, and those drugs
and proteins that bind to a desired target site.
The "targeting moiety" of the present invention binds to
a defined target cell population, such as tumor cells.
Preferred targeting moieties useful in this regard include
antibody and antibody fragments, proteinaceous and non-
proteinaceous ligands or anti-ligands, peptides, and hormones.
Proteins corresponding to or binding to known cell surface
receptors (including low density lipoproteins, transferrin and
insulin), fibrinolytic enzymes, anti-HER2, platelet binding
proteins such as znnexins, and biological response modifiers
(including interleukin, interferon, erythropoietin andcolony-
stimulating factor) are also preferred targeting moieties.
Also, anti-EGF receptor antibodies, which internalize
following binding to the EGF receptor and which traffic to the
nucleus, are preferred targeting moieties for use in the
present invention to facilitate delivery of Auger emitters and
nucleus binding drugs to target cell nuclei.
Oligonucleotides, e.q., antisense oligonucleotides that are
complementary to portions of target cell nucleic acids (DNA
or RNA), are also useful as targeting moieties in the practice
of the present invention. Oligonucleotides binding to cell
surfaces are also useful. Analogs of the above-listed
targeting moieties that retain the capacity to bind to a
de~ined target cell population may also be used within the
claimed invention. In addition, synthetic targeting moieties
may be designed.
~ WO95/05398 2 1 6 5 ~ 3 8 PCT~S91,~3292
Functional equivalents of the aforementioned molecules
are also useful as targeting moieties of the present
invention. One targeting moiety functional equivalent is a
~'mimetic" compound, an organic chemical construct designed to
mimic a proper configuration and/or orientation for targeting
moiety-target cell binding. Another targeting moiety
functional equivalent is a short polypeptide designated as a
"minimal" polypeptide, constructed using computer-assisted
molecular modeling and mutants having altered binding
affinity, which minimal polypeptides exhibit the binding
affinity of the targeting moiety.
The targeting molecule may be a targeting protein, which
is capable of binding to a desired target site. The term
"protein" as used herein includes proteins, polypeptides, and
fragments thereof, including proteinaceous ligands and anti-
ligands. The targeting protein may bind to a receptcr,
substrate, antigenic determinant, complementary binding pair
member or other binding site on a target cell or other target
site. The targeting protein serves to deliver the
radionuclide attached thereto to a desired target site in
vi~o. Examples of targeting proteins include, but are not
limited to, antibodies and antibody fragments, proteinaceous
ligands or anti-ligands, hormones, fibrinolytic enzymes, and
biologic response modifiers. In addition, other polymeric
molecules that localize in a desired target site ln vivo,
although not strictly proteins, are included within the
definition of the term "targeting proteins" as used herein.
For example, certain carbohydrates or glycoproteins may be
used in the present invention. The proteins may be modified,
e.g., to produce variants and fragments thereof, as long as
the desired biological property (i.e., the ability to localize
at the target site) is retained. The proteins may be modified
by using various genetic engineering or protein engineering
techniques, for example.
Among the preferred targeting proteins are antibodies,
most preferably monoclonal antibodies. A number of monoclonal
-
W095/OS398 ~ 1 6 ~ 5 3 8 PCT~S94/09292
- 16 -
antibodies that bind to a specific type of cell have been
developed, including monoclonal antibodies specific for tumor-
associated antigens in humans. Among the many such monoclonal
antibodies that may be used are anti-TAC, or other
interleukin-2 receptor antibodies; 9.2.27 and NR-ML-05,
reactive with the 250 kilodalton human melanoma-associated
proteoglycan; and NR-LU-10, reactive with a pancarcinoma
glycoprotein. The antibody employed in the present invention
may be an intact ~whole) molecule, a fragment thereof, or a
functional equivalent thereof. Examples of antibody fragments
are F(ab') 2/ Fab', Fab, and Fv fragments, which may be
produced by conventional methods or by genetic or protein
engineering.
Human monoclonal antibodies or ~humanized~ murine
antibodies are also useful as targeting moieties in accordance
with the present invention. For example, murine monoclonal
antibody may be "humanized" by genetically recombining a
nucleotide sequence encoding the murine Fv region (i.e.,
containing the antigen binding site which antibodies are also
known as chimeric antibodies) or the complementarity
determining regions thereof with a nucleotide secuence
encoding at least a human constant domain region and an
Fc region, e.g., in a manner similar to that disclosed in
European 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
characteristics. Humanized targeting moieties are recognized
to decrease the i~ml~noreactivity of the antibody or
polypeptide in the host recipient, permitting an increase in
the half-life and a reduction in the possibility of adverse
immune reactions.
Ligands suitable for use within the present invention
include biotin, haptens, lectins, epitopes, dsDNA fragments
and analogs and derivatives thereof. Useful complementary
anti-ligands include avidin (for biotin), carbohydrates (for
lectins), antibody, fragments or analogs thereof, including
. WOss/05398 PCT~S~ g292
216 ~ 5 3 8
mimetics (for haptens and epitopes) and zinc finger proteins
(for dsDNA fragments). Preferred ligands and anti-ligands
bind to each other with an affinity of at least about kD 2
109 M.
The chelating compounds of the present invention comprise
at least one (and preferably only one) conjugation group Z.
A conjugation group is a chemically reactive functional group
that will react with a molecule to be radiolabeled to bind the
chelating co~.pound thereto. When the targeting molecule is
a protein, the conjugation group is reactive under conditions
that do not denature or otherwise adversely affect the
protein. Examples of suitable conjugation groups include but
are not limited to active esters, isothiocyanates, amines,
hydrazines, maleimides or other Michael-type acceptors,
thiols, and activated halides. Among the preferred active
esters are N-hydroxysuccinimidyl ester, sulfosuccinimidyl
ester, thiophenyl ester, 2,3,5,6-tetrafluorophenyl ester, and
2,3,5,6-tetrafluorothiophenyl ester. The latter three
preferred active esters may comprise a group that enhances
water solubility, at the para (i.e., 4) or the ortho position
on the phenyl ring. Examples of such groups are CO2H, S03-,
po32-, Gpo32-, CS03-, NtR3 wherein each R represents H or an alkyl
group, and O(CH2CH2O)nCH3 groups.
A ligand or anti-ligand conjugation group (l.e., a group
located on a chelate compound that is reactive with a ligand
or an anti-ligand) is a chemically reactive 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 the ligand or anti-ligand to bind
to its complementary binding pair member. Ligand or anti-
ligand conjugation groups therefore are sufficiently reactive
with a functional group on a ligand or anti-ligand so that the
reaction can be conducted under relatively mild reaction
conditions including those described above for protein-chelate
conjugation. For proteinaceous ligands or anti-ligands, such
as streptavidin, protein conjugation groups may correspond to
~` W095/05398 2 1 6 5 i~ 3 8 PCT~S~ 3~92
- 18 -
ligand or anti-ligand conjugation groups. Examples of
suitable ligand or anti-ligand conjugation groups therefore
include, but are not limited to, active esters,
isothiocyanates, amines, hydrazines, thiols, and maleimides.
Among the preferred active esters are thiophenyl ester,
2,3,5,6-tetrafluorophenyl ester, and 2,3,5,6-tetrafluoro-
thiophenyl ester. The preferred active esters may comprise
a group that enhances water solubility, at the para (i.e., 4)
position on the phenyl ring. Examples of such groups are
0 C02H, S03-, po32-, opo32-, and O(CH2CH2O)nCH3 groups.
For non-proteinaceous ligand or anti-ligand moieties,
such as biotin, suitable conjugation groups are those
functional groups that react with a ligand or anti-ligand
functional group (e.a., a terminal carboxy group) or a
functional group which the ligand or anti-ligand has been
derivatized to contain (e.a., 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 capable of reacting with -COOH, -OH or -NH2 groups
are useful conjugation groups for producing biotin conjugates
of this aspect of the present invention. Exemplary biotin-
COOH conjugation groups are amines, hydrazines, alcohols and
the like. Exemplary biotin-OH conjugation groups are
tosylates ~Ts), active esters, halides and the like, with
exemplary groups being reactive with biotin-O-Ts including
amines, hydrazines, thiols and the like. Exemplary biotin-NH2
conjugation groups are active esters, acyl chlorides,
tosylates, isothiocyanates and the like.
Proteins contain a variety of functional groups; e.g.,
carboxylic ~cid (COOH) or free amine (-NH2) groups, which are
available for reaction with a suitable conjugation group on
a chelating compound to bind the chelating compound to the
protein. For example, an active ester on the chelating
compound reacts with epsilon amine groups on lysine residues
of proteins to form amide boncs. Alternatively, a targeting
molecule and/or a chelator may be derivatized to expose or
~ WO9s/0s398 2 1 ~ 5 5 3 ~
PCT~S94/09292
- 19 -
attach additional reactive functional groups. The
derivatization may involve attachment of any of a number of
linker molecules such as those available from Pierce Chemical
Company, Rockford, Illinois. Alternatively, the
derivatization may involve chemical treatment of the protein
(which may be an antibody). Procedures for generation of free
sulfhydryl groups on antibodies or antibody fragments by
reducing disulfide bonds are also known. Maleimide
conjugation groups on a chelating compound are reactive with
the sulfhydryl (thiol) groups.
Alternatively, w:en the targeting molecule, ligand or
anti-ligand is a carbohydrate or glycoprotein, derivatization
may involve chemical treatment of the carbohydrate; e.g.,
glycol cleavage of the sugar moiety of a glycoprotein antibody
with periodate to generate free aldehyde groups. The free
aldehyde groups on the antibody may be reacted with free amine
or hydrazine conjugation groups on the chelator to bind the
chelator thereto. ~See U.S. Patent No. 4,671,958.)
Biotin has a terminal carboxy moiety which may be reacted
with a suitable ligand conjugation group, such as an amine,
hydroxyl in the presence of a coupling agent such as ~CC or
the like. In addition, the terminal 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 hydroxyl, another nucleophile, or the like.
Alternatively, the terminal carboxy moiety may be reduced to
a hydroxy moiety for reaction with a suitable ligand
conjugation group, such as a halide (e c., iodide, bromide or
chloride), tosylate, mesylate, other good leaving groups or
the like. The hydroxy moiety may be chemically modified to
form an amine moiety, which may be reacted with a suitable
ligand conjugation group, such as an active ester or the like.
The chelating compounds o~ the present invention are
radiolabeled, using conventional procedures, with any of a
variety of radionuclide metals to form the corresponding
radionuclide metal chelates. The radiolabeling may be
~ W09s/OS398 2 1 6 ~ 5 3 8 PCT~Sg~J~292
- 20 -
conducted before or after the chelating compound is attached
to the molecule to be radiolabeled. These radionuclide metals
include, but are not limited to, copper (e.g., 6'Cu and 64Cu);
technetium (e.g., 99mTc); rhenium (e.g., l96Re and ~B9Re); lead
(e.g., 2~2Pb)i bismuth (e.g., 2'2Bi); palladium (e.g., 109Pd);
and rhodium (e.g., 10sRh). Methods for preparing these
isotopes are known. Molybdenum/technetium generators for
producing 99~rc are commercially available. Procedures for
processing 186Re include the procedures described by Deutsch
et al., (Nucl. Med. ~iol. Vol. 13:4:46~-477~ 1986) and
Vanderheyden et al. (Inoraanic Chemistrv, Vol. 24: 1666 -1673,
1985) ~ and methods for production of 19~Re have been described
by Blachot et al. (Int. J. A~lied Radiation ard Isoto~es Vol.
20 :467-470, 1969) and by Klofutar et al. (J. of
Radioanalytical Chem., Vol. 5:3-10~ 1970) . Production of 109Pd
is described in Fawwaz et al., J. Nucl. Med. (1984), 25:796.
Production of 212Pb and 212Bi is described in Gansow et al.,
Amer. Chem. Soc. SYm~. Ser. (1984) ~ 241:215-217, and Kozah et
al., Proc. Nat'l. Acad. Sci. USA (January 1986) 83 :474-478.
20 99mTC is preferred for diagnostic use, and the other
radionuclides listed above have therapeutic use.
The radionuclide advantageously is in chelatable form
when reacted with the chelating compounds of the invention.
In the case of technetium and rhenium, being in "chelatable
25 form~' generally requires a reducing step. A reducing agent
will be employed to reduce the radionuclides (e.g., in the
form of pertechnetate and perrhenate, respectively) to a lower
oxidation state at which chelation will occur. Many suitable
reducing agents, and the use thereof, are known. (See, for
example, U.S. Patents 4~440t738; 4~434~151; and 4~652~440.)
Such reducing agents include, but are not limited to, stannous
ion (e.g., in the form of stannous salts such as stannous
chloride or stannous fluoride), metallic tin, ferrous ion
(e.g., in the form of ferrous salts such as ferrous chloride,
35 ferrous sulfate, or ferrous ascorbate) and many others.
Sodium pertechnetate (i.e., 99~cG4- which is in the +7
Wog5/05398 ~ 1 6 5 5 3 8 PCT~S9~/~5292
- 21 -
oxidation level) or sodium perrhenate (i.e., l8aReO4-, 1a6ReO4-)
may be combined simultaneously with a reducing agent and a
chelating compound of the invention in accordance with the
radiolabeling method of the invention, to form a chelate.
Preferably, the radionuclide is treated with a reducing
agent and a complexing agent to form an intermediate complex
(i.e., an "exchange complex"). Complexing agents are
compounds which bind the radionuclide more weakly than do the
chelate compounds of the invention, and may be weak chelators.
Any of the suitable known complexing agents may be used,
including but not limited to gluconic acid, glucoheptonic
acid, methylene bi- or di-phosphonate, glyceric acid, glycolic
acid, tartaric acid, mannitol, oxalic acid, malonic acid,
succinic acid, bicine, malic acid, N,N'-bis(2-hydroxy ethyl)
ethylene diamine, citric acid, ascorbic acid and gentisic
acid. Good results are obtained using gluconic acid or
glucoheptonic acid as the technetium-complexing agent and
citric acid for rhenium. When the radionuclide in the form
of such an exchange complex is reacted with the chelating
compounds of the invention (which may be attached to a
targeting protein, ligand, anti-ligand or the like), the
radionuclide will transfer to these chelating compounds which
bind the radionuclide more strongly to form chelates of the
invention. Radionuclides in the form of such exchange
complexes also are considered to be in ~chelatable form" for
the purposes of the present invention.
Chelates of 2l2pb 2~23i, 103Rh, and 109Pd may be prepared by
combining the appropriate salt of the radionuclide with the
chelating compound and incubating the reaction mixture at room
temperature or at higher temperatures. It is not necessary
to treat the lead, bismuth, rhodium, palladium, and copper
radioisotopes with a reducing agent prior to chelation, as
such isotopes are already in an oxidation state suitable for
chelation (i.e., in chelatable form).
The specific radiolabeling reaction conditions may vary
somewhat according to the particular radionuclide and
~ W095/05398 2 1 6 5 5 3 8 PCT~S9~103292
chelating compound involved. When the chelating compound is
attached to a targeting protein or a proteinaceous ligand or
anti-ligand prior to radiolabeling, the radiolabeling reaction
is conducted under physiologically acceptable conditions to
avoid denaturing or otherwise damaging the protein.
The present invention also provides a method for
radiolabeling targeting proteins by attaching a chelating
compound of formula I or II to the protein, then reacting the
resulting protein-chelating compound conjugate with a
radionuclide metal in chelatable form. Alternatively, the
chelating compound is first reacted with a radionuclide metal
in chelatable form, and the resulting radionuclide metal
chelate is reacted with the protein. In either case, a
protein having a radionuclide metal chelate attached thereto
is produced. Analogous methods of production of radiolabeled
chelate-ligand and radiolabeled chelate-anti-ligand are also
contemplated. Details of these reactions are presented in the
examples below.
The invention thus provides ligand-, anti-ligand- and
protein-chelating compound conjugates of formulas V and VI,
produced by reacting a ligand, an anti-ligand or a protein
with a chelating compound of formula I or II, respectively:
T T T T
25H2C SR S~p X--C SR S~p
Q-N ~ SR HN SR
P p R~ Rn P ~Rn R"
V VI
wherein:
one P represents a substituent -(CH2)n-P' with n = 0 to
about 4 and P' representing a ligand, an anti-ligand or a
protein; and
W095/05398 2 1 ~ 5 ~ 3 8 PCT~S~ 3292
- 23 -
the rem~ining substituents P are independently selected
from:
-(CH2)n-COOH with n = 0 to about 4,
hydrogen, and
a lower alkyl group of from l to about 6 carbon
atoms;
with the other symbols having the definition presented
for formulas I and II above.
Also provided by the present invention are radiolabeled
proteins of the following formulas:
2 ~ ~M o--C/S~MS p
P>rR1~<R" P>~R~<R"
wherein:
M represents a radionuclide metal or oxide thereof;
one P represents a substituent -(CH2)n~P' with n = 0 to
about 4 and P' representing a ligand, an anti-ligand or a
protein; and
the rem~in-ng substituents P are independently selected
from:
-(CH2)n-COOH with n = 0 to about 4,
hydrogen, and
a lower alkyl group of from l to about 6 carbon
atoms;
with the other symbols having the definition presented
for formulas I and II above.
Woss/05398 2 1 6 5 5 3 8 PCT~S9~ 252
The protein P~ may be a targeting protein as described
above or a ligand or anti-ligand as described above. It is
to be understood that the protein P' may include a portion of
the conjugation group Z that reacted with the protein.
Similarly, ligand or anti-ligand P' moieties may include a
poration of the conjugation group z that reacted with the
ligand or anti-ligand.
The radiolabeled targeting proteins, ligands or anti-
ligands of the present invention have use in diagnostic and
therapeutic procedures, both for ln vitro assays and for in
vivo medical procedures. One type of therapeutic or
diagnostic procedure in which the compounds of the present
invention may be employed is a pretargeting protocol.
Generally, pretargeting encompasses two protocols, termed the
three-step and the two-step. In the three-step protocol,
shown schematically below, targeting moiety-ligand is
administered and permitted to localize to target.
Blood Tumo.
2) ~ ' ~ ~ *
Wogs/05398 2 1 6 5 5 3 ~ PCT~S94/09292
Blood Tumor
~ ~ ~pid , ~ ~
3) ~ @~_* ~ *-~5
Ta-geting ~oiety
* Anti-ligand
~ Ligand
Ligand-active agent
3inding site (i.e., rece?.or, antigenic deter~inant)
Liver
~ Kidney
Targeting moiety-ligand conjugates may be prepared in
accordance with known techniques therefor. Anti-ligand is
then administered to act as a clearing agent and to facilitate
and direct the excretion of circulating targeting moiety-
ligand. The anti-ligand also binds to target-associated
targeting moiety-ligand. Next, a conjugate employing a
compound of the present invention is administered, having the
following structure:
Ligand - - - - Chelate - - - - Radionuclide
The radiolabeled ligand conjugate either binds to target-
associated targeting moiety-ligand-anti-ligand or is rapidly
excreted, with the excretion proceeding primarily through the
renal pathway. Consequently, the target-non-target ratio of
active agent is improved, and undesirable hepatobiliary
excretion and intestinal uptake of the active agent are
substantially decreased.
Two-step pretargeting involves administration of
targeting moiety-anti-ligand, which may be prepared in
accordance with known technicues therefor. After permitting
the administered agent to localize to target, a radiolabeled
~ W09S~ 2 1 6 ~ ~ 3 ~ PCT~S~SJ~292
- 26 -
ligand of the present invention is administered. Preferably,
as a "step 1.5," a clearing agent is administered to remove
circulating targeting moiety-anti-ligand without binding of
clearing agent to target-associated targeting moiety-anti-
ligand. In this manner, the target-non-target ratio of the
radiolabeled ligand is increased, and undesirable
hepatobiliary excretion and intestinal uptake of the
radiolabeled ligand are substantially decreased.
The radiolabeled molecules may be administered
intravenously,intraperitoneally,intralymphatically,locally,
or by other suitable means, depending on such factors as the
type of target site. The amount to be administered will vary
according to such factors as the type of radionuclide (e.g.,
whether it is a diagnostic or therapeutic radionuclide), the
lS route of administration, 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 and any cross-reactivity of the targeting protein ligand
or anti-ligand with normal tissues. Appropriate dosages may
be established ~y conventional procedures and a physician
skilled in the field to which this invention pertains will be
able to determine a suitable dosage for a patient. A
diagnostically effective dose is generally from about 5 to
about 35 and typically from about 10 to about 30 mCi per 70
kg body weight. A therapeutically effective dose is generally
from about 20 mCi to about 300 mCi, depending on the
radionuclide. Autologous bone marrow rescue may be required
at the higher dose levels. Higher doses, e.a., ranging to an
order of magnitude or more, may be administered using
pretargeting procedures employed, because of the decoupling
of targeting molety localization and radionuclide
localization, which decoupling results in a reduction in whole
body absorbed dose. For diagnosis, conventional non-invasive
procedures (e.g., gamma cameras) are used to detect the
biodistribution of the diagnostic radionuclide, thereby
WOsS/os398 2 1 6 5 ~ 3 8
PCT~S91~'~3292
- 27 -
determining the presence or absence of the target sites of
interest (e.g., tumors).
The following examples are presented to illustrate
certain embodiments of the invention, and are not to be
construed as limiting the scope of the present invention.
EXAMPLE I: Svnthesis of a Chelatinq Com~ound
The synthesis procedure is generally depicted in the
following reaction scheme:
HsxcooH ~=O/H' \ ~ DCClEt
2 HOURS, 50C /~ 25C, 2 hours
HS' `COOH S~ ~COOH
0 2
NaOCH3/CH30H ~5XCOOCH~ B
;L lo
~5yCOOCH3 H3C{~502CUpyri~ine ySyCOOCH3
S~CH20H 0- - 25-C SJ~CHzOTs
O
~SO2CI 0Yc
S-CH2--O
CH3CH20C ~NH2602~CH3 CH3 ~3CH3
~ NaH, CH31 ~
~S-CH2 0 ~ S-CH2 '0
1~ 15
HBrlHoAc EIOC~NHSr H~, EIOH (Elo)3c~NHcH3
~CH20 1 7
WO 95/0S398 2 1 6 5 ~ ~ 8 PCIIUS~ 3~92
S~COOCH3
SJ~cH2oTs /~COOCH3 /~\COOH
H3C--N~_~S-CH2~ H3C--N~ S-CH
C(OEt)3COOCHzCCI3
1 9
/~COSTFP Zn /~COSTFP
\/ Phosphatt3 but~er \/
DCC/HS-TFP /\ pH 2-4 / \
CH2Cl2
H3C--N~--S-CHz~ H3C--N~ S-CH
COOCH2CCI3 COOCH2CCI3
2Q 21
~5 S,S'-IsoPropYlidene 2,3-dimercaPtosuccinic acid (2~
To 1.0 g of meso 2,3-dimercaptosuccinic acid (DMSA), 50
mL of anhydrous acetone followed by 0.3 mL (6 drops)
perchloric acid were added. The heterogeneous suspension was
heated at 50C for 2 hours. Solvent from the clear light
golden yellow solution was removed under reduced pressure.
To the dried residue 50 mL water was added and extracted with
ethyl acetate three times, each time with 100 mL. The
combined ethyl acetate extracts were dried over anhydrous
sodium sulfate and the solvent was removed in vacuo. The dry
solid was dissolved in ether and the compound was precipitated
by the addition of petroleum ether. The solid was filtered
to give 0.9 g (74%) of 2 as a white compound which was
recrystallized from CHCl3/hexane to give crystals, MP 158-
160C. lH NMR (d6 acetone) ~ 4.85 (S,2H), ~ 1.8, 1.9 (2S,6H).
S,S'-Iso~roPYlidene 2,3-dimercaptosuccinic anhYdride (3)
To 0.8 g (3.6 mmol) of S,S~-isopropylidene 2,3-
dimercaptosuccinic acid (2), 10 mL o~ ethyl acetate was added.
To this solution with stirring was added a solution of 1,3-
dicyclohexylcar~odiimide 0.82 g (0.396 mmol) in 10 mL ethyl
acetate. Afte_ stirring the reaction mixture at room
~ W09s/05398 216 5 5 3 8 PCT~S9q/a~292
- 29 -
temperature for two hours, the reaction mixture was filtered
to remove the dicyclohexyl urea, a byproduct of the reaction.
Removal of the solvent from the filtrate left a white solid.
The crude product was purified by sublimation to give 0.52 g
(71~) of S,S'-isopropylidene 2,3-dimercaptosuccinic anhydride
(3), mp 141-142C. lH NMR (d6 acetone) ~ 5.5 (S,2H), ~ 1.8
(S,6H).
3-Carbomethoxv S,S'-isoProovlidene 2,3-dimercaPtooroDionic
acid (10)
Prepare a fresh 0.011 mol sodium methoxide in methanol
solution by dissolving 0.26 g of sodium in 50 mL of anhydrous
methanol. Then 1.0 g of S,S'-isopropylidene 2,3-dimercapto-
succinic anhydride is added to the reaction mixture and
stirred at room temperature. Completion of the reaction is
followed by thin layer chromatography. Add 25 mL of prewashed
(water followed by methanol) BIORAD AG 50W-X~ (H~) cation
exchange resin and stir for 15 minutes. Then filter off the
resin and wash it thoroughly with 25 mL methanol. Concentrate
the filtrate to a residue on a rotatory evaporator. Add 15
mL heptane and evaporate the solvent to a dry residue to yield
grey solid.
3-CarbomethoxY S,S'-iso~ropvlidene 2,3-dimerca~to~ro~anol (11)
To a solution of S,S'-isopropylidene 3-carbomethoxy 2,3-
dimercaptopropionic acid in tetrahydrofuran at 35-40C is
rapidly added BH3-THF (0.18 mmol). After three hours, an
aliquot is analyzed by thin layer chromatography (ethyl
acetate:hexane = 4:1). Disappearance of the starting material
is an indication of complete conversion to the alcohol. 10
mL of ethanol is added to the reaction mixture and the mixture
is evaporated to dryness. After repeating the procedure twice
with 20 mL of ethanol, the residue is suspended in water,
extracted with ethyl acetate and the organic layer is washed
successively with 2x15 mL of 2~ aqueous bicarbonate and water
followed by drying over anhydrous sodium sulfate. The organic
~ W0951~3~3~ 2 1 6 5 ~ 3 ~ PCT~S911~3292
- 30 -
solvent is then evaporated, the residue dissolved in hexane
and upon cooling gives S,S'-isopropylidene 3-carbomethoxy 2,3-
dimercaptopropanol (11) in high yield. The compound 10 to
compound 11 tranformation may be preferably conducted
employing a two-step synthetic procedure as shown below.
/\ X MeOH /\ X BH3/THF
S COOCH3 5 COOCH3
101 0 1 0b
SXCOOCH3
S CH20H
11
3-Carbomethoxy S,S'-isopropylidene 2,3-dimercaptopropionic
acid 10 is treated with 1.0 equivalent of sodium hydroxide to
hydrolyse the diester to the monoester (lOb). The monoester
is then reduced with borane at O C to form S,S'-isopropylidene
3-carbomethoxy 2,3-dimercaptopropanol 11.
3-CarbomethoxY S.S'-iso~ro~Ylidene 2,3-dimerca~to~ro~anol
tosvlate (12~
The alcohol S,S'-isopropylidene 3-carbomethoxy 2,3-
dimercaptopropanol (11, 1.0 g, 4.5 mmol) prepared above is
dissolved in 5 mL of pyridine (0C-5C) and 0.9 g (4.7 mmol)
of p-toluenesulfonyl chloride added at once. Precipitation
of pyridinium hydrochloride is observed after one hour and the
mixture is stirred for additional two hours, followed by
storage at ~C overnight. The solution is poured with
stirring into 50 mL of ice water and the resulting solid is
isolated by filtration, washed with water and dried under
vacuum in a desicator overnight to yield 60-80~ of the tosyl
ester, S,S'-isopropylidene 3-carbomethoxy 2,3-
dimercaptopropanol tosyl ester, 12.
W095lOS398 2 16 ~ ~ ~ 8 PCT~S~/Og292
N-TosYl-S-benzYlcysteine ethvl ester (14)
S-benzylcysteine ethyl ester hydrochloride, 13 (5.0 g,
18.1 mmol) is dissolved in 25 ml of pyridine (0C-5C) and 3.5
g (18.3 mmol) of tosyl chloride is added at once.
Precipitation of pyridinium hydrochloride is observed after
one hour and the reaction mixture is stirred for an additional
two hours, followed by storage at 4C overnight. The solution
is poured with stirring into 150 mL of ice water and the
resulting solid isolated by filtration, washed with ice cold
water and dried under vacuum in a desicator overnight to yield
75-90~ yield of S-benzyl cysteine ethyl ester tosylamide, 14.
The crude product is recrystallized from ethyl acetate.
N-TosYl-N-methvl S-benzYlcvsteine ethvl ester ~15)
S-benzylcysteine ethyl ester tosylamide, 14 (5.0 g,
12.7 mmol) is dissolved in dimethylformamide. Solid sodium
hydride (0.31 g, 12.9 mmol) is added. Then methyl iodide
(1.9 g, 13.4 mmol) is added and the reaction mixture stirred
at room temperature. Completion of the reaction is monitored
by thin layer chromatography. Solvent from the reaction
mixture is removed under vacuum and dried. The crude product
is purified by flash chromatography to yield 50-75% of S-
benzylcysteine ethyl ester N-methyltosylamide, 15.
S-BenzYl N-methYlcYsteine ethYl ester hYdrobromide (16~
Glacial acetic acid is saturated with hydrogen bromide
gas. To the stirred HBr solution one equivalent of solid S-
benzylcysteine ethyl ester N-methyltosylamide is added. The
reaction mixture is stirred at room temperature for 2 to 4
hours. Solvent from the reaction mixture is removed under
reduced pressure and dried under vacuum to yield 75-80% of S-
benzyl N-Methylcysteine ethyl ester hydrobromide salt 16.
S-BenzYl N-methYlcYsteine triethyl orthoester (17)
S-benzyl N-methylcysteine ethyl ester hydrobromide is
converted to its triethyl orthoesther using acid catalyst by
~ W095~ 2 1 6 5 ~ 3 8 PCT~S9~ 9292
conventional method. Completion of the reaction is monitored
by thin layer chromatography. The crude product is purified
by flash chromatography.
N-MethYl-N-(B-benzYlthio-a-triethoxymethyl)ethYl-S,S~-
iso~ropylidene-2,3-dimerca~to-4-~minohutyric acid meth~1 ester
(18
S-benzyl N-methylcysteine triethylorthoester, 17 (3.0 g,
9.2 mmol) is dissolved in DMF. To the stirred solution at
room temperature triethylamine ~1.3 mL, 9.2 mmol) is added.
Then, 3-carbomethoxy S,S~-isopropylidene 2,3-dimercapto-
propanol tosylate, 12 (3.2 g, 9.2 mmol) is added and the
reaction mixture is stirred at room temperature. The progress
and completion of the reaction is followed by thin layer
chromatography. Sol~ent from the reaction mixture is removed
under vacuum and the resulting solid dried. The crude
compound is purified by flash chromatography, to yield
compound 18 in 60-80~ yield.
N-Methyl-N-~-benz~lthio-a-trichloroethoxycarbonYl)ethYl-S.S'-
iso~ro~Ylidene-2,3-dimerca~to-4-aminobutyric acid (19)
Compound 18 is hydrolyzed conventionally with 1 equivalent
of sodium hydroxide to N-Methyl-N-(~-benzylthio-a-
trichloroethoxycarbonyl)ethyl-S,S'-isopropylidene-2,3-
dimercapto-4-~min~utyric acid, 19. The crude product is
purified by flash chromatography.
N-Methvl-N-(~-benzylthio-a-trichloroethoxycarbonyl)ethyl-S,S'-
is~ul~vlidene-2 3-dimerca~to-4 -~mj no~utyric acid-2~,3~ 5',6'-
tetrafluorothioohenYl ester (20)
To a solution of compound 19 (1.0 g, 18.3 mmol) and2,3,5,6-tetrafluorothiophenol (0.35 g, 21.08 mmol) in 25 m~
dichloromethaneisaddedl,3-dicyclohexylcarbodiimide l0.45 g,
21.9 mmol) with rapid stirring. The mixture is stirred at
room temperature for 18 to 24 hours, or until TLC analysis
indicates complete con~ersion to the tetrafluorothiophenYl
~ ~ WO95/05398 2 1 6 5 5 3 8 PCT~S9~/03252
ester. Then the mixture is cooled to 0C, a few drops of
acetic acid is added, and the mixture is stirred for a few
minutes and then filtered. The filtrate is concentrated under
vacuum to give a solid. The solid is dissolved in mjn;~llm
amount of methylene chloride and allowed to stand at 5C for
two to three hours. The solution is then filtered to remove
any precipitated dicyclohexyl urea, and the filtrate is
concentrated to afford solid compound 20. The solid is then
washed with ether to remove any re~i n ing 2,3,5,6-tetrafluoro-
thiophenol. The crude compound is purified by flash chroma-
tography on silica gel column.
N-MethYl-N-(~-benzvlthio-~-carboxv)ethyl-S S'-iso~ro~vlidene-
2 3-dimerca~to-4-aminobutYric acid-2' 3' 5' 6'-tetrafluoro-
thio~henvl ester (21)
To a solution of N-Methyl-N-($-benzylthio-~-
trichloroethoxycarbonyl)ethyl-S,S'-isopropylidene-2,3-
dimercapto-4-aminobutyric acid-2,3',5',6'-tetrafluoro-
thiophenyl ester 20 (0.5 g, 6.9 mmol) in 75 mL
tetrahydrofuran, 1.4 mL of 1.0 M KH2PO4 is added. To this
solution, zinc dust (0.6 g, 91.9 mmol) is added. After
stirring the reaction mixture at room temperature for 45
minutes, additional buffer (1.4 mL) and zinc dust (0.6 g) are
added. The reaction mixture is sonicated for another hour.
The TLC of the reaction mixture is an indication of completion
of product formation. The mixture is sonicated for an
additional hour. The reaction mixture is filtered, rinsed
with acetonitrile, 50~ CH3CN/H2O with 1~ acetic acid
successively. The solvent is removed under reduced pressure.
The residue is chromatographed on silica gel with 10~
~-OH/CH2Cl2 - 2~ HOAC and then 25% ~-OH/CH2C12-2~ HOAC as
elution solvents.
Compound 21 is a chelating compound of the present
invention, also referred to as a ligand elsewhere herein.
"COSTFP~ represents a 2,3,~,6-tetrafluorothiophenyl ester,
which is a conjugation group.
~ WO ~/OS398 PCT~S9~/Og~52
2165~38
- 34 -
An alternative route to compound 21 is shown below.
HO--C NH2 HCI (CFIC) O HO--C~NH-C-CF3
--~S-CH2-O 3 0C 2 14, S-CH2{~
O CH3
DCC,DMAP o O Cl3CCH20--C N--C-CF3
Cl3CCH20H,CH3CN " -C-CF 1) NaH ~~
a3CCH20--C~NH 3 2~ Mel ~s-CH2{~
S-CH2~0 16'
COOH
S-CH2-O / s COOH H3C--N~S-CH2~0
17'
CO2CH2cc13
~}\COSTFP
DCCIHS-TFP \/
CH2CI2 /\ 1.0 M KH2PO" ~1
H3C--NyS~CH20
CO2CH2CCI3
2Q
Compound a, utilized in the above synthetic scheme, is
formed as shown below.
S COOCH3 S COOH
NaOH \/ ~ TsCI
S CH20H /\S~CH20H
11
\ S ~ COOH
S~CH20Ts
2165~38
WO95/OS39~ PCT~S911'~3292
This alternative route begins with commercially available
S-benzylcysteine (available from Aldrlch Chemical Co.,
Milwaukee, Wisconsin), which is converted to trichloroethyl-N-
methyl-S-benzylcysteine by established peptide
protection/alkylation procedures. The protected N-methyl
cysteine derivative is alkylated with the tosylate a in a step
analogous to the original coupling of 17 and 12 to give 18.
By using a instead of 12, the need to selectively hydrolyze
the methyl ester in the presence of the protected carboxy
group of 18 is avoided. Thus, the new route has the
advantages of (1) incorporating the carboxy protecting group
earlier in the synthesis which facilitates the formation of
the tetrafluorothiophenyl ester and (2) simplifying thioester
formation by protecting the 3-carboxyl propanol prior to
incorporation of the dimercaptosuccinic acid portion of the
molecule.
Trifluoroacetyl-S-benzYl cvsteine (14')
Trifluoroacetic anhydride is added to a solution of S-
benzylcysteine (13') in methylene chloride and triethylamine.
- The reaction mixture is stirred at O C for one hour, acidified
with 1.0 M HCl and extracted with methylene chloride. The
methylene chloride extracts are dried ~MgSO4) and evaporated
to give 14'.
TrichloroethYl N-trifluoroacetvl-S-benzyl cvsteine (15~)
DCC is added to a solution of N-trifluoroacetyl-S-benzyl
cysteine 14, trichloroethanol, and a catalytic amount of
dimethylaminopyridine (DMAP) in acetonitrile. The reaction
mixture is stirred at 23 C for 12 hours, then is filtered to
remove the DCU. The filtrate is evaporated to give 15'.
Trichloroethvl N-methyl-N-trifluoroacetyl-s-benzyl CYSteine
(16')
To a suspension of 15' and sodium hydride in DMF is added
1.O eouivalents methyl iodide. The reaction mixture is
2165538
WO ss~3a PCT~S94/09292
stirred at 23 C for 12 hours and monitored by thin layer
chromatography. The mixture is acidified carefully by the
addition of acetic acid in DMF. The mixture is extracted with
water and CH2Cl2. The CH2Cl2 extracts are dried (MgSO4) and
evaporated to give 16'. The product may be further purified
by flash chromatography.
TrichloroethYl-S-benzYl-N-methvl cvsteine (17')
The trifluoroacetyl group of 16~ is cleaved by
acidolysis. HCl gas is bubbled into a solution of 16' in
methanol. The reaction is stirred at 23 C for 12 hours or
until thin layer chromatography shows that the reaction is
complete. The methanol is evaporated to give 17'.
N-methYl-N-(beta-benzvlthio-al~ha-carboxv)ethyl-~S S'-
iso~roPylidene)-2 3-dimerca~to-4-aminobutyric acid (19)
Trichloroethyl S-benzyl N-methyl cysteine 17' is
dissolved in DMF. To the stirred solution at room temperature
is added triethylamine. The 3-carboxy S,S'-isopropylidene
2,3-dimercapto propanol tosylate a, formed as described below,
is added and the reaction mixture is stirred at room
temperature. The progress and completion of the reaction is
followed by thin layer chromat6graphy. Solvent from the
reaction mixture is removed under vacuum. The crude compound
19 is purified by flash chromatography.
3-Carboxv-(S S'-isopro~lidene)-2 3-dimercaPto~ropanol
tosYlate (a)
To a solution of 3-carbomethoxy-S,S~-isopropylidene-2,3-
dimercaptopropanol 11 in methanol is added 1-2 equivalents
1 N NaOH. The solution is stirred at 23 C for 4 hours or
until the reaction is complete as indicated by thin layer
chromatography. The solution is acidified by the addition of
1.0 M HCl to pH 3, and concentrated. The residue is
partitioned between ethyl acetate and water. The ethyl
acetate is dried (MgSO4) and evaporated to give 3-car~oxy-
~ WOgS/OS~98 ~ 1 6 5 5 3 8 PCT~S~ l~og~92
(S,S'-isopropylidene)-2,3-dimercaptopropanol (a'). The
alcohol a' is dissolved in pyridine at O C and p-toluene
sulfonyl chloride is added at once. The reaction mixture is
stirred at O'C for 4 hours and then is stored over night at
4 C. The reaction solution is poured with stirring into ice
water, and the resulting solid is isolated by filtration,
washed with water and dried under vacuum in a dessicator over
night to give the tosyl ester a.
N-Methvl-N-(~-benzvlthio-~-trichloroethoxycarbonyl)ethvl-S,S'-
iso~ropylidene-2,3-dimerca~to-4 -~mi nc~utyric acid-2~,3',5',6'-
tetrafluorothio~henvl ester (20)
To a solution of compound 19 (1.0 g, 18.3 mmol) and
2,3,5,6-tetrafluorothiophenol (0.35 g, 21.08 mmol) in 25 mL
dichloromethaneisaddedl,3-dicyclohexylcarbodiimide (0.45 g,
21.9 mmol) with rapid stirring. The mixture is stirred at
room temperature for 18 to 24 hours, or until TLC analysis
indicates complete conversion to the tetrafluorothiophenyl
ester. Then the mixture is cooled to 0C, a few drops of
acetic acid is added, and the mixture is stirred for a few
minutes and then filtered. The filtrate is concentrated under
vacuum to give a solid. The solid is dissolved in minimum
amount of methylene chloride and allowed to stand at 5C for
two to three hours. The solution is then filtered to remove
any precipitated dicyclohexyl urea, and the filtrate is
concentrated to afford solid compound 20. The solid is then
washed with ether to remove any re~;ning 2,3,5,6-tetrafluoro-
thiophenol. The crude compound is purified by flash chroma-
tography on silica gel column.
N-MethYl-N-(~-benzvlthio-~-carboxv)ethyl-S,S~-isopro~vlidene-
2,3-dimerca~to-4-aminobutyric acid-2~,3',5',6'-tetrafluoro-
thiophenvl ester (21)
To a solution of N-Methyl-N-(~-benzylthio-~-
trichloroethoxycarbonyl)ethyl-S~S~-isopropylidene-2~3-
dimercapto-4-aminobutyric acid-2,3~,5~,6~-tetrafluoro-
`- 2~ s~æ
Wog5/~3_3~ PCT~S91J'~3~92
- 38 -
thiophenyl ester 20 (0.5 g, 6.9 mmol) in 75 mL
tetrahydrofuran, 1.4 mL of 1.0 M KH~P04 is added, preferably
to pH 4-5. To this solution, zinc dust (0.6 g, 91.9 mmol) is
added. After stirring the reaction mixture at room
S temperature for 45 minutes, additional buffer (1.4 mL) and
zinc dust (0.6 g) are added. T~e reaction mixture is
sonicated for another hour. The TLC of the reaction mixture
is an indication of completion of product formation. The
mixture is sonicated for an additional hour. The reaction
mixture i~ filtered, rinsed with acetonitrile, 50~ CH3CN/~2O
with 1~ acetic acid successively. The solvent is removed
under reduced pressure. The residue is chromatographed on
silica gel with 10~ ~-OH/CH2Cl2 - 2~ HOAC and then
25~ ~-OH/CH2Cl2-2% HOAC as elution solvents.
EXAMPLE II: Svnthesis of a Chelatin~ Compound
A chelating compound of the present invention was
synthesized as generally depicted in the following reaction
scheme: O
HOOC ~NHBOC Cl3CCH20H/DCC/DMAP Cl3CCH2-O-C~NHBOC
CH3CN
~S-CH2~ ~S-CH20
Cl3CCH2--O-C NH3~- OCOCF3\~ ~o
TFA/CH2C12 --~S-CH20
DMAP/DMF
3 0 O--I--\COOH O--/ 5 COOTFP
\/ TFP/DCC \/
/\ EtOAc /\
HNyS-CH2~ HN S-CHz~
COOCH2CCI3 COOCH2CCI3
35 7 8
` - 216553~
; W09s/05398 PCT~S94/09292
O ~ COOTFP
8 Zn pH4s ~
HN~ S-CH20
COOH
N-t-ButYloxvcarbonYl S-benzyl L-cYsteine trichloroethyl ester
(5)
To an ice cold suspension of N-t-Butyloxycarbonyl S-
benzyl cysteine 4 (5.0 g, 16.06 mmol` in 100 mL acetonitrile
was added 4-dimethylaminopyridine ~2.4 g, 19.6 mmol). To the
solution was then added trichloroethanol (2.0 mL, 20.8 mmol).
1,3-dicyclohexylcarbodiimide (4.0 g, 19.4 mmol) was added as
a solid. The ice ba~h was allowed to melt and the reaction
mixture was stirred at room temperature for 15-18 hours.
Analysis by thin layer chromatography indicated the reaction
was complete. A few drops (8-10) of glacial acetic acid were
added. The reaction mixture was cooled to 0C and the
precipitate was filtered. The solvent from filtrate was
Lel,loved in vacuo. The residue was dissolved in methylene
chloride and washed with saturated sodium bicarbonate, brine,
and water, respectively. Methylene chloride layer was dried
over anhydrous sodium sulfate. After filtration and removal
of solvent, a semi-solid was obtained which was purified by
flash chromatography on a silica gel column using methylene
chloride as an elution solvent to give 3.o g of S: ~H NMR
(d6-DMSO) 1.39 (S,9H), 2.6-2.9 (m,2H), 3.75 (S,2H), 4.15-4.35
(M,lH), 4.75-5.0 (Q,2H), 7.25 (S,5H), 7.40-7.55 (d, lH).
` W09~J~33 2 1 6 5 ~ 3 8 PCT~S~ 3292
- 40 -
S-Benzvlcysteine 1 1 l-trichloroethylester trifluoroacetate
(6)
1,1,1-trichloroethyl N-t-Butyloxycarbonyl S-benzyl
cysteinate (1.5 g, 3.0 mmol) was dissolved in 10 mL methylene
chloride. To the clear solution, 10 mL of trifluoroacetic
acid was added and the reaction mixture was stirred at room
temperature for one hour. Completion of the reaction was also
monitored by thin layer chromatography on silica gel plates
using methylene chloride as developing solvent. The solvent
was removed under reduced pressure. The solid residue was
washed twice with heptane and the solvent was removed under
vacuum to give 1.5 g. The residue was dried under vacuum and
used for further reactions without purification.
N-(3-Carboxv-S S'-iso~ro~vlidene-2 3-dimerca~to)~ro~ionYl-S-
benzYlcYsteine trichloroethYl este (7)
To a solution of trichloroethyl S-benzylcysteine
trifluoroacetate (1.54 g, 3.4 mmol) in 10 mL anhydrous
dimethylformamide was added a solution of isopropylidene 2,3-
dimercaptosuccinic anhydride (0.75 g, 3.7 mmol) in 10 mL of
dimethylformamide. To the reaction mixture was then added
dimethylaminopyridine (1.0 g, 8.2 mmol) as a dry solid. The
reaction mixture was stirred at room temperature overnight.
The DMF from the reaction mixture was removed at low heat
under vacuum. The semi-solid residue was dried and purified
by flash chromatography on a silica gel column using ethyl
acetate and ethyl acetate:acetic acid (98:2) as elution
solvents successively.
N-(3-(2' 3' 5' 6'-tetrafluoro~henoxvcarbonyl)-S,S'-
isopro~ylidene-2 3-dimercasto)~ro~ionyl-S-benzYlcYsteine
trichloroethYl ester (8)
To a solution of compound 7 (0.5 g, 0.0009 mol) and
2,3,5,6-tetrafluorophenol (0.3 g, 0.0018 mol) in 15 mL
dichloromethane is added 1,3-dicyclohexyl carbodiimide (0.3
g, 0.0015 mol) with rapid stirring. The mixture is stirred
~ W095/0S398 2 1 6 5 5 3 $ PCT~S91/0~92
- 41 -
at room temperature for 2 to 4 hours or until TLC analysis
indicated complete conversion to the tetrafluorophenyl ester.
Then the mixture is cooled to OC, a few drops of acetic acid
are added, the mixture stirred for a few minutes, and then is
filtered. The filtrate is concentrated under vacuum to give
solid. The solid is dissolved in minimum amount of methylene
chloride and allowed to stand at 5C for two to three hours.
The solution is then filtered to remove any precipitated
dicyclohexyl urea, and the filtrate is concentrated to afford
solid N-(3-(2',3',5',6'-tetrafluorophenoxycarbonyl-S,S'-
isopropylidene-2~3-dimercapto)propionyl-S-benzylcysteine
trichloroethyl ester. The solid is then either washed with
ether to remove any rem~in;ng 2,3,5,6-tetrafluorophenol or
purified by flash chromatography on silica gel using
CH2Cl2:IPA:HOAC = 90:5:5 as elution solvent. The solvent from
eluent is removed under vacuum and dried.
N-(3-(2',3',5',6'-tetrafluoro~henoxycarbonvl)-S,S'-
iso~ro~Ylidene-2,3-dimercapto~ro~ionvl-s-benzYlcvsteine (9)
To a solution of compound 8 (0.21 g, 0.0003 mol) in 75
mL tetrahydrofuran, 0.56 mL of 1.0 M KH2PO~ is added. To this
solution, zinc dust (0.275 g, 0.004 mol) is added. After
stirring the reaction mixture at room temperature for 45
minutes, additional buffer (0.56 mL) and zinc dust (0.275 g)
are added. The reaction mixture is sonicated for another
hour. The TLC of the reaction mixture is an indication of
completion of product formation. The mixture is sonicated for
an additional hour. The reaction mixture is filtered, then
rinsed with acetonitrile, 50~ CH3CN/H2O with 1~ acetic acid
successively. The solvent is removed under reduced pressure.
The residue is chromatographed on silica gel with 10%
~-OH/CH2Cl2-2% HOAC and then 25~ ~-OH/CH2C12-2~ HOAC as elution
- solvents.
Compound 9 is a chelating compound of the present
invention, also referred to as a ligand elsewhere herein.
"COOTFP" represents a 2,3,5,6-tetrafluorophenyl esterr which
~ W09~ 3~ 2 1 6 S 5 3 8 PCT~S9~3~52
- 42 -
is a conjugation group. It is to be noted that the thioacetal
portion of the chelating compound may also be shown as:
H3C CH3
(the two different representations being equivalent).
EXAMPLE III:
Conju~ation of 2,3,5,6-Tetrafluoro~hen~l S-benzYl
Cvsteino 2 3-Dimerca~tosuccinamidate to an Antibodv
The antibody used for derivatization is a monoclonal
antibody fragment designated NR-LU-10 Fab, a murine antibody
specific for human carcinoma surface antigen. The antibody
is functionalized by dissolving the ligand, 2,3,5,6-tetra-
fluorophenyl S-benzyl cysteino 2,3-dimercaptosuccinamidate
(compound 9 prepared in example II), in dimethylformamide
solvent during derivatization with 70:1 molar offering of
ligand to antibody. 100 ~L of 20 mg/mL NR-LU-10 Fab in
phosphate buffered saline is mixed with 300 ~L of 0.2 M
phosphate buffer, pH 9.5. To the buffered antibody solution,
30 ~L of 2.0 mg/mL ligand solution in DMF is added. The
reaction mixture is incubated at room temperature for one
hour. The resulting antibody-ligand conjugate is purified by
size exclusion chromatography using a sephadex G-25 (PD-10)
reversed phase column equilibrated with 0.2 M sodium acetate
buffer, pH 5Ø The 2.4-4.8 mL fractions off the PD-10 column
are collected and used for radiolabeling with 99mTc.
The anti~ody-ligand (i.e., chelating compound) conjugate
is believed to be of the following chemical structure:
` - 2 1 ~ 8
WO 9S~ 3~ PCr/US9~ ,3292
O=C--S~C--NH-Ab
HN~ S-CH
COOH
o H
wherein Ab represents the antibody fragment and -C-N is the
amide bond formed by reaction of a primary amine on a lysine
residue of the antibody with the active ester conjugation
group on the chelating compound.
Chelating compound 21, produced in Example I, may be
substituted for compound 9 in the procedures above to produce
an antibody-ligand conjugate of the formula:
S~S C--NH-Ab
H3C-NyS-CH
COOH
EXAMPLE IV: Preparation of Radiolabeled Proteins
Tc-99m Radiolabelinq of Antibodv-liaand Coniu~ate
To 100 ~L of a solution containing 5 mg of sodium
gluconate and 0.1 mg of stannous chloride in water, 1.0 mL of
99mTcO~- (pertechnetate) is added. After incubation at room
temperature for one minute to form a 99mTc-gluconate inter-
mediate ~xch~nge complex, 200 ~L of the 99mTc-gluconate
solution is mixed with 525 ~L of freshly prepared PD-10
purified antibody-ligand conjugate (0.44 mg antibody-ligand
conjugate prepared in example III). The reaction mixture is
incubated at 37C for 15 minutes. The percentage of the
Tc-99m from Tc-gluconate bound to the antibody-ligand
conjugate is determined by standard instant thin layer
~` WO9s/OS398 2 1 6 ~ 5 3 3 PCT~S94/09292
chromatography (ITLC) in 12~ trichloroacetic acid as a
developing solvent. The native antibody fragment
underivatized with ligand is used as a control. Minimal Tc-
99m uptake in the control experiment is an indication that the
Tc-99m uptake by the antibody-ligand conjugate is specific for
the ligand and that non-specific Tc-99m uptake is negligible.
Re Chelates
The same chelating compound may be radiolabeled with ~8aRe
by a procedure similar to the 99mTc labeling procedure. Sodium
perrhenate produced from a W-188/Re-188 research scale
generator is combined with citric acid (a preferred complexing
agent for l88Re), a reducing agent, and preferably gentisic
acid and lactose. The resultinglttRe-citrate exchange complex
is reacted with the desired chelating compound-antibody
conjugate, as above. A Sephadex G-25 column may be used to
purify the radiolabeled antibody. A l86Re-citrate exchange
complex may be substituted in the same procedure.
The resulting radiolabeled antibodies (produced using
antibody conjugates of ligands 9 and 21, respectively) are
believed to have the following structures:
O=C~NH-Ab /~\C~--NH-Ab
N~_~S H3C-N~ S
COOH COOH
- 216553~
WO9S~ PCT~S9S~ 292
- 45 -
wherein M represents a radionuclide metal selected from 99~Tc,
~Re, and 186Re. Ab represents the antibody fragment, and
O ~
-C-N- is the amide bond formed by the reaction of the primary
amine on a lysine residue of the antibody with the active
ester conjugation group on the chelating compound.
Other chelating compounds of formulas I and II may be
attached to an antibody fragment and radiolabeled using the
same procedures described above for chelating compounds 9 and
21. Other targeting proteins may be substituted for the
antibody fragment in these procedures.
EXAMPLE V: Radiolabeled Liqand Preparation
A. One synthetic route for a S3N-biotin conjugate is
shown below:
/~S COSTFP H2N~ NH C--(CH2)4~2 DMFlEt3N
~ COOH HN NH
H3C N~ S-CH
COOCH2CCI3
2Q
S C-NH-CH-(CH2)4-NH-C-(CH2)4 ~ ~ Zn
S~ COOH )~ 1.0 M KH2 PO4
/\ HN~NH
H3C-N~ S-CH2~ 0
CO2CH2CCI3
21 '
21~5-3~3
W09~ 33 PCT~S94/09292
- 46 -
O COOH o
/~C--NH-CH-(CH2)4--NH-c--(CH2)4~
H3C-N yS-CH2~ HN~NH
COOH
2?
N-r4-biocytinamido-(S S'-iso~ro~lidine)-2 3-dimerca~tol
butYrYl-S-S-benzYl trichloroethyl cysteine (21')
The tetrafluorothiophenyl ester 20, as previously
described, and biocytin (commercially available from Sigma
Chemical Co., St. Louis, Missouri) are stirred in DMF and
triethylamine. The progress of the reaction is monitored by
thin layer chromatography. If the reaction does not go to
completion, the solution is heated at 80-C for 30 minutes.
After thin layer chromatography indicates reaction completion,
the DMF is evaporated. The residue is purified by
chromatography to afford 21'.
N-Meth~l-N-r4-bioc~tinamido-(S S'-iso~ro~Ylidene)-2,3-
dimerca~tol butYrvl-S-benzYl cYsteine (22)
To a solution of 21' in THF and 1.0 M KH2PO4 is added
zinc dust. After 30 minutes, additional 1.0 M KH2PO~ and zinc
dust are added. The mixture is sonicated for 1 hour, then
filtered, rinsed with CH3CN, 50~ CH3CN/H20. The filtrate is
evaporated under vacuum. The residue is chromatographed on
silica gel to afford the product 22.
B. Another synthetic route for a S3N-biotin conjugate is
shown below. o
COOCH3 S COOH S C--O--N~
\/ ~ NaOH \/ ~NHS \ / ~ V
/\ M~30H /\ .DCC /\ ll
~S~ ~COOCH3 ~ ~S~ ~COOCH3 S~ ~COOCI 13
lOb 23
WO9S/05398 ~ 1 6 5 5 3 8 PCT~S~ 3292
- 47 -
" S ~ S C--Nll--(cl12h~ c--(c)~2l4~ N~O~
2~ 1I,N--(C~I,),--NH-C--(C)1,),~ ? /\5Xcooctl, ~IN~N~1
Il 11 S S C--NN--(C112)~--N1~3 /CH~b--~ ~ s.CI
~5XC--N11--IC~),--N~i-C--ICH,)~ \/ X HNb~NII
~ 29 0
7C O
Il N~--(CH~,-NII-C--(CH,).~ 2~ I HO--C~NHCH3 DMF, E~,N
/\SXCI~20T~ HNyN11 S-
O 1 6'
/~S C--NH-(cH2)s--NH C--~cH2)~
H3C-NyS~CH2~~0 HN~NH
COOH 2~
This route is preferred over that described in Section
A of this Example for the following reasons: tl) the route
has fewer synthetic steps; (2) biotin is introduced earlier
in the synthesis and, therefore, orthogonal protection of the
cysteine carboxylic acid is not required; and (3) the amide
bond linking biocytin to succinic acid is formed from a
monocarboxylate 10b, rather than by selective acylation of the
diester 20 of the previously discussed route.
Monomethvl-(S S'-iso~ro~lidene)-2 3-dimerca~tosuccinate (10b)
To a solution of dimethyl-S,S'-isopropylidene-2,3-
dimercaptosuccinate 10 in methanol is added 1-2 equivalents
lN NaOH. The solution is stirred at 23-C for 4 hours or until
WOsS/oS398 2 1 6 ~ 5 3 8 ~ 5~ 292
- 48 -
the reaction is complete as lndicated by thin layer
chromatography. The solution is acidified by the addition of
1.0 M HCl to pH 3 and then concentrated. The residue is
partitioned between ethyl acetate and water. The ethyl
acetate is dried (MgSO4) and evaporated to give 10b.
N-hYdroxYsuccinimidYl-methyl-(s,S~-iso~ro~ylidene)-2~3
dimerca~to succinate (23~
To a solution of 10b in acetonitrile is added NHS and
DCC. The reaction is stirred at 23 C for 4 hours and filtered
to remove DCU. The filtrate is evaporated to give the NHS
ester product 23.
(5-Biotinamido)-~entylamido-methyl-(S,S'-iso~ro~vlidene)-2.3-
dimerca~tosuccinate (24)
A solution of 5-biotinamido-pentylamine (available from
Pierce Chemical Company) and 23 in DMF and triethylamine is
stirred at 2' C for 4 hours. The progress of the reaction is
monitored by thin layer chromatography. If the reaction is
not progressing, the reaction mixture is heated at 80 C for
minutes. The solvent is removed under vacuum upon
completion of the reaction. The residue is purified by flash
chromatography.
5-biotinamido)-pentylamido-(S,S~-iso~ropYlidene)-2,3-
dimerca~tosuccinate 25
To a solution of 24 in methanol is added 1-2 equivalents
of 1 N NaOH. The solution is stirred at 23-C for 4 hours or
until the reaction is complete as analyzed by thin layer
chromatography. The solution is acidified by the addition of
1.0 M HCl to pH 3 and then concentrated. The residue is dried
and used without further purification.
3- r (5-Biotinamido)-~entvlamidol-(s~s~-isopropylidene)-2~3
dimerca~to-pro~anol 26
To an ice cold solution of 25 in THF is added 1.0 M
borane in THF. The reaction is stirred at 0 C for 4 hours and
~ W09s/0s398 2 1 6 ~ 5 3 ~ PCT~S94/09292
- 49 -
then quenched by the addition of methanol. The solution is
evaporated. The residue is dissolved in methanol and
evaporated. The residue is redissolved in methanol and
evaporated. The residue is purified by flash chromatography
to afford the product 26.
3-~(5-Biotinamido)-~entYlamidol-(S,S'-isopro~Ylidene)-2,3-
dimerca~to-~ro~anol toluene sulfonate 27
Para-toluene sulfonyl chloride is added to an ice cold
solution of 26 in pyridine. The reaction is stirred at O C
for 4 hours and then stored over night at 4 C. The reaction
mixture is poured with stirring into ice water, and the
resulting solid is isolated by filtration, washed with water
and dried under vacuum in a dessicator over night to give the
tosyl ester 27.
N-~[(5-Biotinamido)-PentYlamidol-S,S'-(isopro~Ylidene)-2,3-
dimerca~tolbutyrvl-S benzyl cvsteine 28
In a manner analogous to the procedure described above
for the preparation of trichloroethyl-N-methyl-S-benzyl
cysteine 17', N-methyl-S-benzyl cysteine 16'' is prepared and
is employed to produce 28 as shown and then described below.
~ 2 (CF3C ) O HO--8~NH-8--CF3 11 N H
Et3N, DMF S-CH
~ 14'
o CH3 o
3 HO--C ~N--C--CF3 H HO--C~NHcH3
S-CI 12~ S-CH
15~ 16
~ W09Sl~3~ 2 1 ~ 5 5 3 8 PCT~S94/09292
- 50 -
To a suspension of N-trifluoroacetyl-S-benzyl cysteine
14' and sodium hydride in DMF is added 1.0 equivalents methyl
iodide. The reaction mixture is stirred at 23 C for 12 hours
and monitored by thin layer chromatography. The mixture is
quenched by the addition of water and acidified by the
addition of 1.0 M HCl. The solution is evaporated. The
residue is partitioned between ethyl acetate and water. The
ethyl acetate extracts are dried (MgSO4), filtered and
evaporated. If necessary, the product, N-methyl-N-
trifluoroacetyl-S-benzyl cysteine 15'', is further purified
by column chromatography.
The trifluoroacetyl group of 15'' is cleaved by
acidolysis. ~Cl gas is bubbled into a solution of 15'' in
methanol. The reaction is stirred at 23 C for 12 hours or
until thin layer chromatography shows that the reaction is
complete. The methanol is evaporated to give S-benzyl-N-
methyl cysteine 16''.
16'' is dissolved in DMF and triethylamine. To the
stirred solution at 23-C is added the tosylate 27. The
progress and completion of the reaction is followed by thin
layer chromatography. The reaction solution is concentrated
under vacuum. The crude product is purified by flash
chromatography.
25C. A third S3N-biotin conjugate synthetic scheme is
shown below.
\/ ~ O + H2N-(cH2)s-NHc tC 2)4 ~ DMF,DMAP
O
O
HO--C--fH--CH C-NH -(CH2)s-NH-C-(CH2),, ~ DMF
S~ ~S HN~NH
/ \ o
2~
W09~ 3~ ~ 1 6 S ~ 3 8PCT~S94/09292
Il O O O
[~N--O--C--CH CH-C-NH--(CH2)s - NH-c--(CH2)4~ HOOC~NH2
S~S HN NH S-CH
~Q
o=~~~C--NH-(CH2)s--NH-C--(CH2)4 S
DMF ~ ~ 1
Et3N S-CH2~ Hl` ~ ~IH
Y O
COOH31
r (5-Biotinamido)-~entvlamidol-(S,S/-iso~ro~vlidene)-2.3-
dimerca~tosuccinate 29
To a solution of 3 in DMF is added a solution of 5-
biotinamido-pentylamine (available from Pierce Chemical
Co~r~ny) in DMF. To the reaction mixture is then added
dimethylaminopyridine as a dry solid. The mixture is stirred
at 23 C for 12 hours. The DMF is removed under vacuum. The
residue is purified by flash chromatosraphy on silica gel.
N-hYdroxYsuccinimidYl- r ( s -biotinamido)-~entYlamidol-(s~s~-
isopro~Ylidene)-2,3-dimerca~tosuccinate 30
To a solution of the carboxylic acid 29 in DMF is added
NXS and DCC. The reaction is stirred at 23 C for 4 hours and
is then filtered to remove DCU. The DMF is e~aporated. The
crude NHS ester is used without purification.
(S-benzYl)-cysteinyl-[(5-biotinamido)-~entylamidol-(
iso~ro~ylidene)-2,3-dimerca~tosuccinate 31
To an equimolar solution of S-benzylcysteine (available
from Aldrich Chemical Company, Milwaukee, Wisconsin) the N-
hydroxy-succinimidyl ester 30 in DMF is added triethylamine.
W095/0539~ 21 ~ ~ S 3 ~ PCT~S91~'03292
- 52 -
The solution is stirred at 23 C for 12 hours and is then
concentrated under vacuum. The residue is purified by flash
chromatography to give the final product.
D. A fourth reachtion scheme useful in the production
of S3N-biotin conjugates is shown below.
COOH O
O=C/I--s\COOTFP + H2N--CH-(CH2),-NH-C--(CH2),~ DMF, E!3N
10 HN~ S-CH2~ HN~NH
COOH
O=CC--NH-CH--(CH2)~--NH-C--(CH2)-~
HN~NH
5 HN~_~S-CH
COOH ;12
BiocYtinamido-(S-benzvl)cysteinYl-(S,S'-iso~ro~ylidene)-2,3-
dimerca~tosuccinate 32
A solution of biocytin (available from Sigma Chemical
Company) and 9 in DMF and triethylamine is stirred at 23 C for
4 hours. The progress of the reaction is monitored by thin
layer chromatography. If the reaction is not progressing, the
reaction solution is heated at 80 C for 30 minutes. The DMF
is Le."oved under vacuum. The residue is purified by
2 5 chromatography.
Exam~le VI: Pre~aration of Tarqetinq Moietv-Liqand and
Tarqetina Moiet~-Anti-Liqand Coniuqates
A Pre~aration and Characterization of Biotinylated
Antibody
Biotinylated NR-LU-lO was prepared according to either
of the following procedures. The first procedure involved
derivitization of antibody via lysine ~-amino groups. NR-LU-
35 10 was radioiodinated at tyrosines using chloramine T and
either l2sI or l3lI sodium iodide. The radioiodinated antibody
~ W095/05398 2 16 ~ 5 3 8 PCT~S91~0~252
(5-10 mg/ml) was then biotinylated using biotinamido caproate
NHS ester in carbonate buffer, pH 8.5, containing 5~ DMS0,
according to the scheme below.
~N--O--C--(CH2 )s--N--C--(CH2 ) NH p(~ n NH2
o
H
Ab-N-C-(CH2)s-N~C-~cH2)4 N ~l1
The impact of lysine biotinylation on antibody
;mmllnoreactivity was P~m;ned. As the molar offering of
biotin:antibody increased from 5:1 to 40:1, biotin
incorporation increased as expected (measured using the HABA
assay and pronase-digested product) (Table 1, below). Percent
of biotinylated antibody i~mnnoreactivity as compared to
native antibody was assessed in a limiting antigen ELISA
assay. The immunoreactivity percentage dropped below 70~ at
a measured derivitization of 11.1:1; however, at this level
of derivitization, no decrease was observed in antigen-
positive cell binding (performed with LS-180 tumor cells at
antigen excess). Subsequent experiments used antibody
derivitized at a biotin:antibody ratio of 10:1.
~ ~ WOsS/05398 2 1 6 5 5 3 8 PCT~S94/09292
TABLE 1
Effect of Lysine Biotinylation
on Immunoreactivity
5 Molar Measured Immunoassessment (%)
Offering Derivitization
(Biotins/Ab) (Biotins~Ab) ELISA Cell Bindinc
5:1 3.4 a6
10:1 8.5 73 100
1013:1 11.1 69 102
20:1 13.4 36 106
40:1 23.1 27
Alternatively, NR-LU-10 was biotinylated using thiol
groups generated by reduction of cystines. Derivitization of
thiol groups was hypothesized to be less compromising to
anti~ody im~llnoreactivity. N~-LU-10 was radioiodinated using
p-aryltin phenylate NHS ester (PIP-NHS) and either l2sI or l3lI
sodium iodide. Radioiodinated NR-LU-10 was incubated with 25
mM dithiothreitol and purified using size exclusion
chromatography. The reduced antibody (containing free thiol
groups) was then reacted with a 10- to 100-fold molar excess
of N-iodoacetyl-n'-biotinyl hexylene diamine in phosphate-
buffered saline (PBS), pH 7.5, containing 5% DMSO (v/v).
TABLE 2
Effect of Thiol Biotinylation
on Immunoreactivity
30 Molar Measured Immunoassessment (%)
Offering Derivitization
(Biotins/Ab) tBiotlns/Ab) ELISA Cell Bindinq
10:1 4.7 114
50:1 6.5 102 100
100-1 6.1 95 100
As shown in Table 2, at a 50:1 or greater biotin:antibody
molar offering, only 6 biotins per antibody were incorporated.
No significant impact on immunoreactivity was observed.
Woss/0s398 2 1 6 ~ 5 3 8 PCT~S94/09292
The lysine- and thiol-derivitizedbiotinylated antibodies
("antibody ~lysine)" and "antibody (thiol)", respectively)
were compared. Molecular sizing on size exclusion FPLC
demonstrated that both biotinylation protocols yielded
monomolecular (monomeric) IgGs. Biotinylated antibody
(lysine) had an apparent molecular weight of 160 kD, while
biotinylated antibody (thiol) had an apparent molecular weight
of 180 kD. Reduction of endogenous sulfhydryls to thiol
groups, followed by conjugation with biotin, may produce a
somewhat unfolded macromolecule. If so, the antibody (thiol)
may display a larger hydrodynamic radius and exhibit an
apparent increase in molecular weight by chromatographic
analysis. Both biotinylated antibody species exhibited 9B~
specific binding to immobilized avidin-agarose.
Further comparison of the biotinylated antibody species
was performed using non-reducing SDS-PAGE, using a 4% stacking
gel and a 5% resolving gel. Biotinylated samples were either
radiolabeled or un]abeled and were combined with either
radiolabeled or unlabeled avidin or streptavidin. Samples
were not boiled prior to SDS-PAGE analysis. The native
antibody and biotinylated antibody (lysine) showed similar
migrations; the biotinylated antibody (thiol) produced two
species in the 50-75 kD range. These species may represent
two thiol-capped species. Under these SDS-PAGE conditions,
radiolabeled streptavidin migrates as a 60 kD tetramer. When
400 ~g/ml radiolabeled streptavidin was combined with 50 ~g/ml
biotinylated antibody (analogous to ~sandwiching" conditions
in vivo), both antibody species formed large molecular weight
complexes. However, only the biotinylated antibody (thiol)-
streptavidin complex moved from the stacking gel into theresolving gel, indicating a decreased molecular weight as
compared to the biotinylated antibody (lysine)-streptavidin
complex.
W09s/0s398 216 5 5 3 ~ PCT~S~ g~92
- 56 -
B. Blood Clearance of Biotinylated Antibodv SPeCies
Radioiodinated biotinylated NR-LU-10 (lysine or thiol)
was intravenously administered to non-tumored nude mice at a
dose of 100 ~g. At 24 h post-administration of radioiodinated
biotinylated NR-LU-10, mice were intravenously injected with
either saline or 400 ~g of avidin. With saline
administration, blood clearances for both biotinylated
antibody species were biphasic and similar to the clearance
of native NR-LU-10 antibody.
In the animals that received avidin intravenously at 24
h, the biotinylated antibody (lysine) was cleared (to a level
of 5~ of injected dose) within 15 min of avidin administration
(avidin:biotin = 10:1). With the biotinylated antibody
(thiol), avidin administration ~10:1 or 25:1) reduced the
circulating antibody level to about 35~ of injected dose after
two hours. Residual radiolabeled antibody activity in the
circulation after avidin administration was examined in vitro
using immobilized biotin. This analysis revealed that 85~ of
the biotinylated antibody was complexed with avidin. These
data suggest that the biotinylated antibody (thiol)-avidin
complexes that were formed were insufficiently crosslinked to
be cleared by the RES.
Blood clearance and biodistribution studies of
biotinylated antibody (lysine) 2 h post-avidin or post-saline
administration were performed. Avidin ~mi ni stration
significantly reduced the level of biotinylated antibody in
the blood, and increased the level of biotinylated antibody
in the liver and spleen. Kidney levels of biotinylated
antibody were similar.
C. Pre~aration of Biotinvlated Antibodv (Thiol)
Throuch Endoqenous AntibodY SulfhYdrYl Grou~s
Or Sulfhydrvl-Generatinq Com~ounds
Certain antibodies have available for reaction endogenous
sulfhydryl groups. If the antibody to be biotinylated
contains endogenous sulfhydryl groups, such antibody is
- Wog5/05398 ~ 1 6 5 5 3 8 PCT~S~ 3~52
reacted with N-iodoacetyl-n'-biotinyl hexylene diamine. The
availability of one or more endogenous sulfhydryl groups
obviates the need to expose the antibody to a reducing agent,
such as DTT, which can have other detrimental effects on the
biotinylated antibody.
Alternatively, one or more sulfhydryl groups are attached
to a targeting moiety through the use of chemical compounds
or linkers that contain a terminal sulfhydryl group. An
exemplary compound for this purpose is iminothiolane. As with
endogenous sulfhydryl groups (discussed above), the
detrimental effects of reducing agents on antibody are thereby
avoided.
D. Tar~etinq Moiety-Anti-Liqand Coniuqate for Two-SteD
Pretaraetinq
l. Preparation of SMCC-derivitized streptavidin.
31 mg (0.48 ~mol) streptavidin was dissolved in 9.0 ml
PBS to prepare a final solution at 3.5 mg/ml. The pH of the
solution was adjusted to 8.5 by addition of 0.9 ml of 0.5 M
borate buffer, pH 8.5. A DMSO solution of SMCC (3.5 mg/ml)
was prepared, and 477 ~l (4.8 ~mol) of this solution was added
dropwise to the vortexing protein solution. After 30 minutes
of stirring, the solution was purified by G-25 (PD-10,
Pharmacia, Piscataway, New Jersey) column chromatography to
remove unreacted or hydrolyzed SMCC. The purified SMCC-
derivitized streptavidin was isolated (28 mg, 1.67 mg/ml).
2. Preparation of DTT-reduced NR-LU-10. To 77 mg NR-LU-
10 (0.42 ~mol) in 15.0 ml PBS was added 1.5 ml of 0.5 M borate
buffer, p~ 8.5. A DTT solution, at 400 mg/ml (165 ~l) was
added to the protein solution. After stirring at room
temperature for 30 minutes, the reduced antibody was purified
by G-25 size exclusion chromatography. Purified DTT-reduced
NR-LU-10 was obtained (74 mg, 2.17 mg/ml).
3. Conjugation of SMCC-streptavidin to DTT- reduced NR-
35 LU-10. DTT-reduced NR-LU-10 (63 mg, 29 ml, 0.42 ~mol) was
diluted with 44.S ml PBS. The solution of sMcc-streptavidin
W09S/05398 2 1 6 a ~ 3 ~ PCT~S91~292
- sa -
(28 mg, 17 ml, 0.42 ~mol) was added rapidly to the stirring
solution of NR-LU-10. Total protein concentration in the
reaction mixture was 1.0 mg/ml. The progress of the reaction
was monitored by HPLC (Zorbax~ GF-250, available from MacMod).
After approximately 45 minutes, the reaction was quenched by
adding solid sodium tetrathionate to a final concentration of
5 mM.
4. Purification of conjugate. For small scale
reactions, monosubstituted conjugate was obtained using HPLC
Zorbax (preparative) size exclusion chromatography. The
desired monosubstituted conjugate product eluted at 14.0-14.5
min (3.0 ml/min flow rate), while unreacted NR-LU-10 eluted
at 14.5-15 min and unreacted derivitized streptavidin eluted
at 19-20 min.
For larger scale conjugation reactions, monosubstituted
adduct is isolatable using DEAE ion exchange chromatography.
After concentration of the crude conjugate mixture, free
streptavidin was removed therefrom by eluting the column with
2.5~ xylitol in sodium borate buffer, pH 8.6. The bound
unreacted antibody and desired conjugate were then
sequentially eluted from the column using an increasing salt
gradient in 20 mM diethanolamine adjusted to pH 8.6 with
sodium hydroxide.
5. Characterization of Conjugate.
a. HPLC size exclusion was conducted as described
above with respect to small scale purification.
b. SDS-PAGE analysis was performed using 5
polyacrylamide gels under non-denaturing conditions.
Conjugates to be evaluated were not boiled in sample buffer
containing SDS to avoid dissociation of streptavidin into its
15 kD subunits. Two product bands were observed on the gel,
which correspond to the mono- and di- substituted conjugates.
c. Immunoreactivity was assessed, for example, by
competitive binding ELISA as compared to free antibody.
Values obtained were within 10~ of those for the free
antibody.
WOss/0s398 2 1 ~ ~ ~ 3 8 PCT~S~IU~292
- 59 -
d. Biotin binding capacity was assessed, for
example, by titrating a known quantity of conjugate with p-[I-
125]iodobenzoylbiocytin. Saturation of the biotin binding
sites was observed upon addition of 4 equivalences of the
labeled biocytin.
e. In vivo studies are useful to characterize the
reaction product, which studies include, for example, serum
clearance profiles, ability of the conjugate to target
antigen-positive tumors, tumor retention of the conjugate over
time and the ability of a biotinylated molecule to bind
streptavidin conjugate at the tumor. These data facilitate
determination that the synthesis resulted in the formation of
a 1:1 streptavidin-NR-LU-10 whole antibody conjugate that
exhibits blood clearance properties similar to native NR-LU-10
whole antibody, and tumor uptake and retention properties at
least equal to native NR-LU-10.
For example, Figure 1 depicts the tumor uptake profile
of the NR-LU-10-streptavidin conjugate (LU-10-StrAv) in
comparison to a control profile of native NR-LU-10 whole
antibody. LU-10-StrAv was radiolabeled on the streptavidin
component only, giving a clear indication that LU-10-StrAv
localizes to target cells as efficiently as NR-LU-10 whole
antibody itself.
Exam~le VII: Three-Ste~ Pretarqetinq
A patient presents with ovarian cancer. A monoclonal
antibody (MAb) directed to an ovarian cancer cell antigen is
conjugated to biotin to form a MAb-biotin conjugate. The MAb-
biotin conjugate is administered to the patient in an amount
in excess of the maximum tolerated dose of conjugate
administerable in a targeted, chelate labeled molecule
protocol (e.a., administrationof monoclonal antibody-chelate-
radionuclide conjugate) and is permitted to localize to targetcancer cells for 24-48 hours. Next, an amount of avidin
~ W095/05398 2 1 6 ~ ~ ~ 8 PCT~S~103~92
- 60 -
sufficient to clear non-targeted MAb-biotin conjugate and to
bind to the targeted biotin is administered. A biotin-
radionuclide chelate conjugate of the type discussed in
Example V(A) above is dispersed in a pharmaceutically
acceptable diluent and administered to the patient in a
therapeutically effective dose. The biotin-radionuclide
chelate conjugate localizes to the targeted MAb-biotin-avidin
moiety or is removed from the patient via the renal pathway.
~xam~le VIII: Two-Ste~ Pretarqetinq
A patient presents with colon cancer. A monoclonal
antibody (MAb) directed to a colon cancer cell antigen is
conjugated to streptavidin to form a MAb-streptavidin
conjugate. The MAb-streptavidin conjugate is administered to
the patient in an amount in excess of the maximum tolerated
dose of conjugate administerable in a targeted, chelate
labeled molecule protocol (e.c., administration of monoclonal
antibody-chelate-radionuclide conjugate) and is permitted to
localize to target cancer cells for 24-48 hours. A biotin-
radionuclide chelate conjugate of the type discussed in
Example V(B) above is dispersed in a pharmaceutically
acceptable diluent and administered to the patient in a
therapeutically effective dose. The biotin-radionuclide
chelate conjugate localizes to the targeted MAb-streptavidin
moiety or is removed from the patient via the renal pathway.
