U.S. patent application number 13/459867 was filed with the patent office on 2012-08-23 for doxorubicin immunoassay.
Invention is credited to Jodi Blake Courtney, Shu He, Salvatore J. Salamone.
Application Number | 20120214184 13/459867 |
Document ID | / |
Family ID | 37053697 |
Filed Date | 2012-08-23 |
United States Patent
Application |
20120214184 |
Kind Code |
A1 |
Salamone; Salvatore J. ; et
al. |
August 23, 2012 |
DOXORUBICIN IMMUNOASSAY
Abstract
Novel conjugates of doxorubicin and novel doxorubicin immunogens
derived from the 13 and 14 positions of doxorubicin and antibodies
generated by these doxorubicin linked immunogens all of which are
useful in immunoassays for the quantification and monitoring of
doxorubicin in biological fluids.
Inventors: |
Salamone; Salvatore J.;
(Stockton, NY) ; Courtney; Jodi Blake;
(Doylestown, PA) ; He; Shu; (Allentown,
PA) |
Family ID: |
37053697 |
Appl. No.: |
13/459867 |
Filed: |
April 30, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13242124 |
Sep 23, 2011 |
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13459867 |
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12433111 |
Apr 30, 2009 |
8053205 |
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13242124 |
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11389934 |
Mar 27, 2006 |
7569358 |
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12433111 |
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60666288 |
Mar 30, 2005 |
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Current U.S.
Class: |
435/7.93 ;
530/395; 536/6.4 |
Current CPC
Class: |
A61K 47/646 20170801;
Y10S 530/807 20130101; G01N 33/9446 20130101; G01N 33/94 20130101;
C07K 16/44 20130101 |
Class at
Publication: |
435/7.93 ;
536/6.4; 530/395 |
International
Class: |
G01N 33/53 20060101
G01N033/53; C07K 14/00 20060101 C07K014/00; C07H 15/252 20060101
C07H015/252 |
Claims
1. A compound of the formula: ##STR00030## wherein A is
##STR00031## Y is an organic spacing group; X is a functional group
capable of binding to a thiol or amino group; and p is an integer
from 0 to 1.
2. The compound of claim 1, wherein X is ##STR00032## wherein
R.sub.3 is hydrogen or taken together with its attached oxygen atom
forms a reactive ester and R.sub.4 is oxygen or sulfur.
3. The compound of claim 2, wherein X is ##STR00033## and R.sub.3
is hydrogen.
4. The compound of claim 2, wherein X is ##STR00034## and R.sub.3
forms a reactive ester.
5. The compound of claim 4, wherein the ester formed is a lower
alkyl ester, imidoester or amidoester.
6. The compound of claim 1, wherein X is a functional group capable
of binding to a thiol group.
7. The compound of claim 6, wherein X is ##STR00035##
8. The compound of claim 7, wherein Y is alkylene containing from 1
to 10 carbon atoms.
9. A compound of the formula: ##STR00036## wherein Y is an organic
spacing group; X is a functional group capable of binding to a
thiol or an amino group; and B is --CH.sub.2-- or ##STR00037## or
mixtures thereof.
10. The compound of claim 9, wherein p is 0.
11. The compound of claim 10, wherein X is ##STR00038## wherein
R.sub.3 is hydrogen or taken together with its attached oxygen atom
forms a reactive ester and R.sub.4 is oxygen or sulfur.
12. The compound of claim 11, wherein X is ##STR00039## and R.sub.3
is hydrogen.
13. The compound of claim 12, wherein X is ##STR00040## and R.sub.3
forms a reactive ester.
14. The compound of claim 13, wherein the ester formed is a lower
alkyl ester, imidoester or amidoester.
15. The compound of claim 9, wherein p is 1.
16. The compound of claim 15, wherein X is ##STR00041## wherein
R.sub.3 is hydrogen or taken together with its attached oxygen atom
forms a reactive ester and R.sub.4 is oxygen or sulfur.
17. The compound of claim 16, wherein Y is alkylene containing from
1 to 10 carbon atoms, ##STR00042## wherein n and o are integers
from 0 to 6, and m is an integer from 1 to 6.
18. A conjugate of a carrier containing a functional thiol or amino
group with a compound of the formula: ##STR00043## wherein A is
##STR00044## Y is an organic spacing group; X is a functional group
capable of binding to said thiol or amino group; and p is an
integer from 0 to 1.
19. The conjugate of claim 18, wherein p is 0.
20. The conjugate of claim 19, wherein X is ##STR00045## wherein
R.sub.3 is hydrogen or taken together with its attached oxygen atom
forms a reactive ester and R.sub.4 is oxygen or sulfur.
21. The conjugate of claim 18, wherein p is 1 and Y is alkylene
containing from 1 to 10 carbon atoms, ##STR00046## wherein n and o
are integers from 0 to 6, and m is an integer from 1 to 6.
22. The conjugate of claim 21, wherein said carrier has a reactive
functional thiol group and X is a functional group capable of
binding to said thiol group.
23. The conjugate of claim 22, wherein X is ##STR00047##
24. The conjugate of claim 23, wherein Y is lower alkylene.
25. The conjugate of claim 24, wherein said carrier contains a
polymeric polyamine containing a functional group of the formula:
##STR00048## wherein v is an integer from 1 to 6.
26. A conjugate of a carrier having a thiol or amine group with a
compound of the formula: ##STR00049## wherein B is --CH.sub.2-- or
##STR00050## Y is an organic spacing group; X is a functional group
capable of binding to said thiol or amino group in said carrier;
and p is an integer from 0 to 1.
27. The conjugate of claim 26, wherein p is 0.
28. The conjugate of claim 26, wherein p is 1.
29. The conjugate of claim 28, wherein Y is alkylene containing
from 1 to 10 carbon atoms, ##STR00051## wherein n and o are
integers from 0 to 6, and m is an integer from 1 to 6.
30. The conjugate of claim 29, wherein the carrier contains an
immunogenic polymeric polymer containing more amino groups linked
by ##STR00052## wherein R.sub.4 is oxygen or sulfur.
31. A kit for determining the presence of doxorubicin in a patient
sample comprising reagents in separate containers, one of the
reagents being a conjugate of a carrier containing a functional
amino or thiol group with a compound selected from the groups
consisting of compounds of the formula: ##STR00053## wherein A is
##STR00054## Y is an organic spacing group; X is a functional group
capable of binding to the amino or thiol group in said carrier; p
is an integer from 0 to 1; and or a 14-substituted doxorubicin of
the formula: ##STR00055## wherein X, Y and p are as above and B is
--CH.sub.2-- or ##STR00056## or mixtures thereof; and the second
container containing an antibody substantially selectively reactive
with doxorubicin and not substantially cross-reactive to
doxorubicin aglycone.
32. The kit of claim 31, wherein said conjugate is present in a
predetermined amount in said first container.
33. The kit of claim 32, wherein said kit is used to determine the
amount of doxorubicin in said sample.
34. The kit of claim 31, wherein, said antibody is generated from a
conjugate of immunogenic carrier having a terminal functional amino
or thiol group linked to said 13-substituted or 14-substituted
doxorubicin.
35. The kit of claim 34, wherein said conjugate is said
13-substituted doxorubicin.
36. The kit of claim 35, wherein said carrier has a reactive
terminal functional thiol group and X is a terminal functional
group capable of binding to said thiol group.
37. The kit of claim 36, wherein X is ##STR00057##
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of Ser. No. 13/242,124,
filed Sep. 23, 2011, entitled "Doxorubicin Immunoassay", which is a
divisional of Ser. No. 12/433,111, filed Apr. 30, 2009, entitled
"Doxorubicin Immunoassay", which is a divisional of Ser. No.
11/389,934, filed Mar. 27, 2006, entitled "Doxorubicin
Immunoassay", issued as U.S. Pat. No. 7,569,358 on Aug. 4, 2009;
which claimed priority from Provisional Application Ser. No.
60/666,288, filed Mar. 30, 2005, entitled "Adriamycin derivatives,
immunogens, and antibodies and their use in determining therapeutic
drug levels in biological samples". Each of these applications is
hereby incorporated by reference herein in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the field of immunological assays
for determining the presence and/or quantifying the amount of
doxorubicin and pharmaceutically active metabolites in human
biological fluids in order to rapidly determine optimal drug
concentrations during chemotherapy.
BACKGROUND OF THE INVENTION
[0003] Cancer is a term used to describe a group of malignancies
that all share the common trait of developing when cells in a part
of the body begin to grow out of control. Most cancers form as
tumors, but can also manifest in the blood and circulate through
other tissues where they grow. Cancer malignancies are most
commonly treated with a combination of surgery, chemotherapy,
and/or radiation therapy. The type of treatment used to treat a
specific cancer depends upon several factors including the type of
cancer malignancy and the stage during which it was diagnosed.
[0004] Doxorubicin, also known as Adriamycin, is one of the more
common cytotoxic agents used for the treatment of breast cancer.
Adriamycin which is the commercial hydrochloride salt of
doxorubicin has the formula:
##STR00001##
This compound has been associated with debilitating side effects
such as cardiotoxicity, myelosuppression, hypersensitivity, nausea
and vomiting. By monitoring the levels of doxorubicin in the body
and adjusting the dose these side effects can be better controlled
and limited in patients.
[0005] At the same time, there is often highly variable
relationship between the dose of doxorubicin and the resulting
serum drug concentration that affects therapeutic effect. The
degree of intra- and inter-individual pharmacokinetic variability
of doxorubicin can be as high as 5-fold and is impacted by many
factors, including: [0006] Organ function [0007] Genetic regulation
[0008] Disease state [0009] Age [0010] Drug-drug interaction [0011]
Time of drug ingestion, [0012] Mode of drug administration, and
[0013] Technique-related administration. As a result of this
variability, equal doses of the same drug in different individuals
can result in dramatically different clinical outcomes (Hon et. al.
Clinical Chemistry 44, pp 388-400, 1998). The effectiveness of the
same doxorubicin dosage varies significantly based upon individual
drug clearance and the ultimate serum drug concentration in the
patient. Therapeutic drug management would provide the clinician
with insight on patient variation in intravenous drug
administration. With therapeutic drug management, drug dosages
could be individualized to the patient, and the chances of
effectively treating the cancer, without the unwanted side effects,
would be much higher.
[0014] In addition, therapeutic drug management of doxorubicin
would serve as an excellent tool to ensure compliance in
administering chemotherapy with the actual prescribed dosage and
achievement of the effective serum concentration levels. It has
been found that variability in serum concentration is not only due
to physiological factors, but can also result from variation in
administration technique.
[0015] Routine therapeutic drug management of doxorubicin would
require the availability of simple automated tests adaptable to
general laboratory equipment. Tests that best fit these criteria
are immunoassays such as a radioimmunoassay and an enzyme-linked
immunosorbent assay. However the corresponding antibodies used in
these immunoassays must demonstrate a broad cross-reactivity to
doxorubicin, without any substantial activity to
non-pharmaceutically active doxorubicin metabolites. In order to be
effective in monitoring drug levels of doxorubicin, the antibody
should be most specific to the active compound, doxorubicin and
display very low cross-reactivity to no cross-reactivity to the
non-pharmaceutically active metabolites of doxorubicin particularly
doxorubicin aglycone which has the formula:
##STR00002##
SUMMARY OF INVENTION
[0016] In accordance with this invention, a new class of antibodies
have been produced which are substantially selectively reactive to
doxorubicin so as to bind to doxorubicin without any substantial
cross reactivity to non-pharmaceutically active doxorubicin
metabolites, particularly doxorubicin aglycone. By selectively
reactive, it is meant that this antibody only reacts with the
pharmaceutically active doxorubicin molecule and does not
substantially react with the non-pharmaceutically active
doxorubicin metabolites, the most important blocking metabolites
being doxorubicin aglycone.
[0017] It has been found that by using immunogens which are
conjugates of an immunogenic carrier having a reactive thiol or
amino functional group with 13 substituted doxorubicin compounds of
the formula:
##STR00003## [0018] or [0019] wherein A is
[0019] ##STR00004## [0020] Y is an organic spacing group; [0021] X
is a functional group capable of binding to said carrier through
said amino or thiol group; and [0022] p is an integer from 0 to 1
or compounds of the formula:
[0022] ##STR00005## [0023] wherein p, Y and X are as above and B
is
##STR00006##
[0023] or mixtures thereof; produce antibodies which are specific
for doxorubicin and do not substantially react with or bind with
non-pharmaceutical active metabolites particularly doxorubicin
aglycone. The provision of these antibodies which substantially
selectively react with doxorubicin and do not cross react with
pharmaceutically inactive metabolites particularly doxorubicin
aglycone allows one to produce an immunoassay which can
specifically detect and monitor doxorubicin in the fluid samples of
patients being treated with doxorubicin. Also included within this
invention are reagents and kits for said immunoassay.
DETAILED DESCRIPTION
[0024] In accordance with this invention, a new class of antibodies
is provided which substantially selectively react with doxorubicin
and do not substantially react or cross react with pharmaceutically
inactive doxorubicin metabolites mentioned hereinabove. It has been
discovered that through the use of these derivatives of 13-oxo
substituted doxorubicin of formula II-A and/or of the 14-hydroxy
substituted doxorubicin of formula II-B or mixtures thereof, as
immunogens, this new class of antibodies of this invention are
provided. It is through the use of these antibodies that an
immunoassay, including reagents and kits for such immunoassay for
detecting and/or quantifying doxorubicin in blood, plasma or other
body fluid samples has been developed. By use of this immunoassay,
the presence and amount of doxorubicin in body fluid samples,
preferably a blood or plasma sample, can be detected and/or
quantified. In this manner, a patient being treated with
doxorubicin can be monitored during therapy and his treatment
adjusted in accordance with said monitoring. By means of this
invention one achieves the therapeutic drug management of
doxorubicin in cancer patients being treated with doxorubicin as a
chemotherapeutic agent.
[0025] The reagents utilized in the assay of this invention are
conjugates of a carrier containing a reactive thiol or amino group
with the compounds of formula II-A and II-B or mixtures thereof.
Preferably the carriers contain a polyamine polymer, which contains
a reactive thiol or amino group. In the immunogens the carriers
preferably contain a polyamine polymer, which contains a reactive
thiol or amino group. These conjugates are competitive binding
partners with the doxorubicin present in the sample for the binding
with the antibodies of this invention. Therefore, the amount of
conjugate reagent which binds to the antibody will be inversely
proportional to the amount of doxorubicin in the sample. In
accordance with this invention, the assay utilizes any conventional
measuring means for detecting and measuring the amount of said
conjugate which is bound or unbound to the antibody. Through the
use of said means, the amount of the bound or unbound conjugate can
be determined. Generally, the amount of doxorubicin in a sample is
determined by correlating the measured amount of the bound or
unbound conjugate produced by the doxorubicin in the sample with
values of the bound or unbound conjugate determined from a standard
or calibration curve obtained from samples containing known amounts
of doxorubicin, which known amounts are in the range expected for
the sample to be tested. These studies for producing calibration
curves are determined using the same immunoassay procedure as used
for the sample.
[0026] The conjugates which include the immunogens, are prepared
from compounds of the formula II-A or II-B or mixtures thereof. The
carriers including the immunogens having a reactive terminal amino
or thiol group are linked to the ligand portions which have the
formula:
##STR00007## [0027] wherein Y, A and p are as above; and [0028] x'
is --CH.sub.2-- or a functional linking group; compounds of the
formula:
[0028] ##STR00008## [0029] wherein x', Y, B and p are as above.
These ligand portions may be linked to one or more active thiol or
amino sites on the carrier containing the polyamine polymer.
Preferably these carriers contain a polymer, most preferred a
polyamine polymer, containing a reactive thiol or amino group.
DEFINITIONS
[0030] Throughout this description the following definitions are to
be understood:
The term doxorubicin includes doxorubicin as well as the
pharmaceutically acceptable salts of doxorubicin.
[0031] The terms "immunogen" and "immunogenic" refer to substances
capable of eliciting, producing, or generating an immune response
in an organism.
[0032] The term "conjugate" refers to any substance formed from the
joining together of two parts. Representative conjugates in
accordance with the present invention include those formed by the
joining together of a small molecule, such as the compound of
formula II-A and II-B, and a large molecule, such as a carrier or a
polyamine polymer, particularly protein. In the conjugate the small
molecule maybe joined at one or more active sites on the large
molecule. The term conjugate includes the term immunogen.
[0033] "Haptens" are partial or incomplete antigens. They are
carrier-free substances, mostly low molecular weight substances,
which are not capable of stimulating antibody formation, but which
do react with antibodies. The latter are formed by coupling a
hapten to a high molecular weight immunogenic carrier and then
injecting this coupled product, i.e., immunogen, into a human or
animal subject. The hapten of this invention is doxorubicin.
[0034] As used herein, a "spacing group" or "spacer" refers to a
portion of a chemical structure which connects two or more
substructures such as haptens, carriers, immunogens, labels, or
tracers through a CH.sub.2 or functional linking group. These
spacer groups will be enumerated hereinafter in this application.
The atoms of a spacing group and the atoms of a chain within the
spacing group are themselves connected by chemical bonds. Among the
preferred spacers are straight or branched, saturated or
unsaturated, carbon chains. Theses carbon chains may also include
one or more heteroatoms within the chain or at termini of the
chains. By "heteroatoms" is meant atoms other than carbon which are
chosen from the group consisting of oxygen, nitrogen and sulfur.
Spacing groups may also include cyclic or aromatic groups as part
of the chain or as a substitution on one of the atoms in the
chain.
[0035] The number of atoms in the spacing group is determined by
counting the atoms other than hydrogen. The number of atoms in a
chain within a spacing group is determined by counting the number
of atoms other than hydrogen along the shortest route between the
substructures being connected. A functional linking group may be
used to activate, e.g., provide an available functional site on, a
hapten or spacing group for synthesizing a conjugate of a hapten
with a label or carrier or polyamine polymer.
[0036] An "immunogenic carrier," as the terms are used herein, is
an immunogenic substance, commonly a protein or a protein modified
to carry a reactive thiol or amino group, that can join with a
hapten, in this case doxorubicin, thereby enabling these hapten
derivatives to induce an immune response and elicit the production
of antibodies that can bind specifically with these haptens. The
immunogenic carriers and the linking groups will be enumerated
hereinafter in this application. Among the immunogenic carrier
substances are included proteins, glycoproteins, complex
polyamino-polysaccharides, particles, and nucleic acids that are
recognized as foreign and thereby elicit an immunologic response
from the host. The polyamino-polysaccharides may be prepared from
polysaccharides using any of the conventional means known for this
preparation.
[0037] Also various protein types may be employed as a poly(amino
acid) immunogenic carrier. These types include albumins, serum
proteins, lipoproteins, etc. Illustrative proteins include bovine
serum albumin (BSA), keyhole limpet hemocyanin (KLH), egg
ovalbumin, bovine thyroglobulin (BTG) etc. Alternatively, synthetic
poly(amino acids) may be utilized. Alternatively these proteins can
be modified so as to contain a reactive thiol group.
[0038] Immunogenic carriers can also include poly
amino-polysaccharides, which are a high molecular weight polymer
built up by repeated condensations of monosaccharides. Examples of
polysaccharides are starches, glycogen, cellulose, carbohydrate
gums such as gum arabic, agar, and so forth. The polysaccharide may
also contain polyamino acid residues and/or lipid residues.
[0039] The immunogenic carrier can also be a poly(nucleic acid)
either alone or conjugated to one of the above mentioned poly(amino
acids) or polysaccharides.
[0040] The immunogenic carrier can also include solid particles.
The particles are generally at least about 0.02 microns (.mu.m) and
not more than about 100 .mu.m, and usually about 0.05 .mu.m to 10
.mu.m in diameter. The particle can be organic or inorganic,
swellable or non-swellable, porous or non-porous, optimally of a
density approximating water, generally from about 0.7 to 1.5 g/mL,
and composed of material that can be transparent, partially
transparent, or opaque. The particles can be biological materials
such as cells and microorganisms, including non-limiting examples
such as erythrocytes, leukocytes, lymphocytes, hybridomas,
Streptococcus, Staphylococcus aureus, E. coli, and viruses. The
particles can also be comprised of organic and inorganic polymers,
liposomes, latex, phospholipid vesicles, or lipoproteins.
[0041] "Poly(amino acid)" or "polypeptide" is a polyamide formed
from amino acids. Poly(amino acids) will generally range from about
2,000 molecular weight, having no upper molecular weight limit,
normally being less than 10,000,000 and usually not more than about
600,000 daltons. There will usually be different ranges, depending
on whether an immunogenic carrier or an enzyme is involved.
[0042] A "peptide" is any compound formed by the linkage of two or
more amino acids by amide (peptide) bonds, usually a polymer of
.alpha.-amino acids in which the .alpha.-amino group of each amino
acid residue (except the NH.sub.2 terminus) is linked to the
.alpha.-carboxyl group of the next residue in a linear chain. The
terms peptide, polypeptide and poly(amino acid) are used
synonymously herein to refer to this class of compounds without
restriction as to size. The largest members of this class are
referred to as proteins. These polymer peptides can be modified by
conventional means to convert the reactive NH.sub.2 terminal group
into a terminal SH group.
[0043] A "label," "detector molecule," or "tracer" is any molecule
which produces, or can be induced to produce, a detectable signal.
The label can be conjugated to an analyte, immunogen, antibody, or
to another molecule such as a receptor or a molecule that can bind
to a receptor such as a ligand, particularly a hapten. Non-limiting
examples of labels include radioactive isotopes, enzymes, enzyme
fragments, enzyme substrates, enzyme inhibitors, coenzymes,
catalysts, fluorophores, dyes, chemiluminescers, luminescers, or
sensitizers; a non-magnetic or magnetic particle, a solid support,
a liposome, a ligand, or a receptor.
[0044] The term "antibody" refers to a specific protein binding
partner for an antigen and is any substance, or group of
substances, which has a specific binding affinity for an antigen to
the exclusion of other substances. The generic term antibody
subsumes polyclonal antibodies, monoclonal antibodies and antibody
fragments.
[0045] The term "derivative" refers to a chemical compound or
molecule made from a parent compound by one or more chemical
reactions.
[0046] The term "carrier" refers to solid particles and/or
polymeric polymers such as immunogenic polymers such as those
mentioned above. Where the carrier is a solid particle, the solid
particle may be bound, coated with or otherwise attached to a
polyamine polymer to provide one or more reactive sites for bonding
to the functional group X in the compounds of the formula II-A and
II-B.
[0047] The term "reagent kit," or "test kit," refers to an assembly
of materials that are used in performing an assay. The reagents can
be provided in packaged combination in the same or in separate
containers, depending on their cross-reactivities and stabilities,
and in liquid or in lyophilized form. The amounts and proportions
of reagents provided in the kit can be selected so as to provide
optimum results for a particular application. A reagent kit
embodying features of the present invention comprises antibodies
specific for doxorubicin. The kit may further comprise ligands of
the analyte and calibration and control materials. The reagents may
remain in liquid form or may be lyophilized.
[0048] The phrase "calibration and control materials" refers to any
standard or reference material containing a known amount of a drug
to be measured. The concentration of drug is calculated by
comparing the results obtained for the unknown specimen with the
results obtained for the standard. This is commonly done by
constructing a calibration curve.
[0049] The term "biological sample" includes, but is not limited
to, any quantity of a substance from a living thing or formerly
living thing. Such living things include, but are not limited to,
humans, mice, monkeys, rats, rabbits, horses, and other animals.
Such substances include, but are not limited to, blood, serum,
plasma, urine, cells, organs, tissues, bone, bone marrow, lymph,
lymph nodes, synovial tissue, chondrocytes, synovial macrophages,
endothelial cells, and skin.
Reagents and Immunogens
[0050] In constructing an immunoassay, a conjugate of doxorubicin
is constructed to compete with the doxorubicin in the sample for
binding sites on the antibodies. In the immunoassay of this
invention, the reagents are the 13-substituted doxorubicin
derivatives of the compounds of formula III-A and the
14-substituted doxorubicin derivatives of formula III-B. In the
compounds of formula III-A and III-B, the linker spacer constitutes
the --CH.sub.2--(Y).sub.p--X'-- or --B--(Y).sub.p--X' portion of
this molecule. In these linkers X' and the spacer
--CH.sub.2--(Y).sub.p-- or --B--(Y).sub.p--X' are conventional in
preparing conjugates and immunogens. Any of the conventional
spacer-linking groups utilized to prepare conjugates and immunogens
for immunoassays can be utilized in the compounds of formula III-A
and III-B. Such conventional linkers and spacers are disclosed in
U.S. Pat. No. 5,501,987 and U.S. Pat. No. 5,101,015.
[0051] Among the preferred spacer groups are included the spacer
groups hereinbefore mentioned. Particularly preferred spacing
groups are groups such as alkylene containing from 1 to 10 carbon
atoms,
##STR00009##
wherein n and o are integers from 0 to 6, and m is an integer from
1 to 6 with alkylene being the especially preferred spacing group.
With respect to the above structures of the spacing group
designated by Y, the functional group X is connected at the
terminal position at the right side of the structure i.e. where
(CH.sub.2).sub.m and (CH.sub.2)o are located.
[0052] In the compounds of formula III-A and III-B, X' is
--CH.sub.2-- or a functional group linking the spacer, to an amine
or thiol group on the polymeric carrier. The group X' is the result
of the terminal functional group X in the compounds of Formula II-A
and II-B which is capable of binding to the amino or thiol group in
the polyamine polymer used as either the carrier or the immunogen.
Any terminal functional group capable of reacting with an amine or
thiol group can be utilized as the functional group X in the
compounds of formula II-A and II-B. These terminal functional
groups preferably included within X are:
##STR00010##
wherein R.sub.3 is hydrogen or taken together with its attached
oxygen atom forms a reactive ester and R.sub.4 is oxygen or sulfur.
The radical --N.dbd.C.dbd.R.sub.4, can be an isocyanate or as
isothiocyanate. The active esters formed by OR.sub.3 include
imidoester, such as N-hydroxysuccinamide, 1-hydroxy benzotriazole
and p-nitrophenyl ester. However any active ester which can react
with an amine or thiol group can be used.
[0053] The carboxylic group and the active esters are coupled to
the carrier or immunogenic polymer by conventional means. The amine
group on the polyamine polymer, such as a protein, produces an
amide group which connects the spacer to the polymeric immunogens
or carrier to form the conjugates of this invention.
When X in the compound of formula II-A or II-B is
##STR00011##
these compounds preferably react with the free amino group of the
polymeric or immunogenic carrier. On the other hand, when X in the
compound of formula II-A or II-B is the maleimide radical of the
formula
##STR00012##
this compound preferably reacts with the thiol (or SH) group which
may be present on the polymeric or protein carrier, including the
immunogens, to produce X' in the compounds of the formula III-A and
III-B having the structure:
##STR00013##
In accordance with a preferred embodiment, these compounds of
formula III-A1 and III-B1 are attached to a polymeric protein which
has been modified to convert an amino group to a thiol group. This
can be done by the reacting a free amino group of a polymeric
protein carrier with a compound of the formula
##STR00014## [0054] wherein R.sub.15 is a thiol protecting group;
[0055] R.sub.3 is as above; and [0056] v is an integer of from 1 to
4. In this manner, the thiol group, SH-- becomes the functional
group of the carrier bonded to the remainder of the carrier.
[0057] This reaction is carried out in an aqueous medium by mixing
the protein containing carrier with the compound of formula V in an
aqueous medium. In this reaction temperature and pressure are not
critical and the reaction can be carried out at room temperature
and atmospheric pressure. Temperatures of from 10.degree. C. to
25.degree. C. are generally preferred. In the next step before the
thiol modified carrier is reacted with the compound of formula II-A
and II-B after the thiol protecting group of carrier is removed by
conventional means from the resulting reaction product of the
compound of formula V with the carrier.
[0058] Any conventional means for removing a thiol protecting group
can be utilized in carrying out this reaction. However, in
utilizing a means to remove the thiol protecting group, care must
be taken that the reactants be soluble in the aqueous medium and do
not in any way destroy or harm the polyamine polymer contained in
the carrier. A preferred means for removing this protecting group
is by the use of dithiothreitol as an agent to reduce the resultant
condensation product. This reduction can be carried out by simply
adding the reducing agent to the reaction medium without utilizing
higher pressures or temperatures. This reduction can be carried out
at room temperature and atmospheric pressure. Any conventional
thiol protecting agent can be utilized in carrying out this in the
compound of formula V. The thiol protecting groups are well known
in the art with 2-pyridyldithio being the preferred protecting
group.
[0059] While the above method represents one means for converting a
reactive terminal amino group on the polyamine polymeric containing
carrier to a thiol group, any conventional means for carrying out
this conversion can be utilized. Methods for converting terminal
amino groups on polyamine polymeric containing carriers are well
known in the art and can be employed in accordance with this
invention. It has been found that in accordance with the preferred
embodiment of this invention, when the compounds of formula III-A
and III-B having X' bound to a thiol group carried by the
immunogenic polymeric polyamine containing carrier produce
antibodies of greater specificity to doxorubicin. Therefore, the
use of the compound of formula II-A and II-B where X is bonded to a
terminal thiol group of the immunogenic polymeric polyamine
polymeric containing carrier constitutes the preferred embodiment
of the immunogens of this invention.
[0060] The reaction of the polymeric polyamine containing carrier
having a terminal reactive thiol group with the compound of formula
II-A or II-B where X is a functional group capable of binding to
the terminal thiol group carried by the carrier can be carried out
by conventional means. In the preferred embodiment the maleimide of
formula III-A1 and III-B1 is reacted with the thiol group carried
by the polyamine polymeric carrier. Any well known means for
addition of a thiol across a maleimide double bond can be utilized
in producing the conjugates of formula II-A and II-B which are
conjugated through a thiol bridge.
[0061] In the conjugates, bonded through amide bonds which
conjugates include the immunogens of the present invention, the
chemical bond between the carboxyl group containing doxorubicin
haptens and the amino groups on the carrier or immunogen can be
obtained using a variety of methods known to one skilled in the
art. It is frequently preferable to form amide bonds by first
activating the carboxylic acid moiety of the doxorubicin hapten in
the compounds of formula II-A and II-B by reacting the carboxy
group with a leaving group reagent (e.g., N-hydroxysuccinimide,
1-hydroxybenzotriazole, p-nitrophenol and the like). An activating
reagent such as dicyclohexylcarbodiimide, diisopropylcarbodiimide
and the like can be used. The activated form of the carboxyl group
in the doxorubicin hapten of formula II-A or II-B is then reacted
in a buffered solution containing the protein carrier.
[0062] In preparing the amino bonded conjugates where the
doxorubicin derivative of formula II-A or II-B contains a primary
or secondary amino group as well as the carboxyl group, it is
necessary to use an amine protecting group during the activation
and coupling reactions to prevent the conjugates from reacting with
themselves. Typically, the amines on the doxorubicin derivative of
formula II-A or II-B are protected by forming the corresponding
N-trifluoroacetamide, N-tertbutyloxycarbonyl urethane (N-t-BOC
urethane), N-carbobenzyloxy urethane or similar structure. Once the
coupling reaction to the immunogenic polymer or carrier has been
accomplished, as described above, the amine protecting group can be
removed using reagents that do not otherwise alter the structure of
the immunogen or conjugate. Such reagents and methods are known to
one skilled in the art and include weak or strong aqueous or
anhydrous acids, weak or strong aqueous or anhydrous bases,
hydride-containing reagents such as sodium borohydride or sodium
cyanoborohydride and catalytic hydrogenation. Various methods of
conjugating haptens and carriers are also disclosed in U.S. Pat.
No. 3,996,344 and U.S. Pat. No. 4,016,146, which are herein
incorporated by reference.
[0063] On the other hand in preparing amino conjugates where X is a
terminal isocyanate or thioisocyanate radical in the compound of
formula II-A or II-B, these radicals when reacted with the free
amine of a polyamine polymer produce the conjugate or immunogen of
formula III-A or III-B
##STR00015##
where X' is, where R.sub.4 is as above, which functionally connects
with the amino group on the polyamine carrier or the immunogenic
polypeptide.
[0064] In preparing the amino conjugates of the compounds of
formula II-A and II-B, where X is an aldehyde group these compounds
may be connected to the amine group of the polyamine polypeptide or
carrier through an amine linkage by reductive amination. Any
conventional method of condensing an aldehyde with an amine such as
through reductive amination can be used to form this linkage. In
this case, X' in the ligand portions of formula III-A and III-B is
--CH.sub.2--.
Doxorubicin of the compound of formula I, and its 13-keto group can
be represented by the formula:
##STR00016##
where
##STR00017##
represents doxorubicin with its 13-keto group shown. The 13-keto
doxorubicin can be converted to the compound of formula II-A where
A is .dbd.N--O-- by reacting doxorubicin with a methoxyamine of the
formula:
NH.sub.2--O--CH.sub.2--(Y).sub.p--X VI-A
to produce the compound of the formula:
##STR00018## [0065] wherein p, Y and X are as above.
[0066] The compound of formula I is reacted at its 13-oxo group
with a methoxyamine of formula VI-A to form the compounds of
formula VI-B by conventional means of condensing methoxyamine with
a carbonyl group to form an oxylamine of formula VI-B such as
disclosed in U.S. Pat. No. 4,039,385. If the compound of formula
VI-A contains any functional substituents, these substituents can
be reacted with conventional protecting groups prior to the
reaction of doxorubicin with a compound of VI-A. After the
conjugate is produced from the compound of formula VI-B, these
protecting groups can be removed by procedures well known in the
art for removing such protecting groups while retaining the
oxylamine linkage in the compound of formula VI-B. The compound of
formula II-A where A is
##STR00019##
can be prepared by first converting the 13-oxo group on doxorubicin
to 13-amino group and then condensing this 13-amino doxorubicin
with an acid halide of the formula:
##STR00020## [0067] wherein Y, p and X are as above. The 13-oxo
group on doxorubicin can be converted to the 13-amino group by
reductive amination utilizing ammonium chloride and a reducing
agent such as sodium cyanoborohydride.
[0068] Any of the conditions conventional in reductive amination
can be utilized to convert the 13-oxo group on doxorubicin to an
amino group. The 13-amino doxorubicin is reacted with the acid
halide by condensation to form the amide of formula II-A where A
is
##STR00021##
[0069] Any method of condensing an acid halide with an amine to
form an amide can be utilized to carry out his condensation.
The compound of formula II-A where A is a hydrazone of the
formula
##STR00022##
can be prepared by reacting the 13-oxo in the doxorubicin of
formula I with a hydrazide of the formula
##STR00023## [0070] where p, Y and X are as above.
[0071] Any method of reacting a ketone with a hydrazide to produce
a hydrazone can be used to carry out this conversion. Generally
this reaction is carried out by reacting the ammonium salt of the
compound of IX-B with the 13-oxo group on the compound of formula
I, in an inert organic solvent medium such as a lower alkanol at a
pH of from 3 to 6, with acid pHs being generally preferred. In
carrying out this reaction temperature and pressure are not
critical and this reaction can be carried out at room temperature
and atmospheric pressure.
The 14-substituted compounds of formula II-B where B is
--CH.sub.2-- are formed by reacting the 14-hydroxy group of
doxorubicin with a halide of the formula:
halo-CH.sub.2--(Y).sub.p--X VIII-B [0072] wherein p, Y and X are as
above.
[0073] In forming the compound of formula II-B from doxorubicin,
any conventional means of reacting an alcohol to form an ether can
be utilized to condense the compound of formula VIII-B with the
14-hydroxy position on the doxorubicin. The use of a halide in the
compound of formula VIII-B provides an efficient means for forming
such an ether by condensing with the alcohol. On the other hand,
where Y in the compound of formula VIII-B contains functional
groups, which may interfere with this reaction to form the compound
of formula II-B, these functional groups can be protected by means
of suitable protecting groups which can be removed after this
reaction as described hereinabove.
The 14-substituted compounds of formula II-B where B is
##STR00024##
is produced by reacting 14-hydroxy group on doxorubicin with an
amino compound of the formula:
NH.sub.2--CH.sub.2--(Y).sub.p--X IX [0074] wherein X, Y and p are
as above. After first converting the 14-hydroxy group on
doxorubicin to the chloroformatic group
##STR00025##
[0075] Any conventional means of converting a hydroxy group to a
chloroformatic group can be used. After the formulation of a
chloroformate, the halo group of the chloroformate is condensed
with the amine group in the compound of formula IX. Prior to this
reaction, the reactive group on doxorubicin and/or on the compound
of formula IX are protected as described hereinabove with a
conventional protecting group. These protecting groups can be
removed after this halide condensation by conventional means such
as described hereinbefore.
[0076] The compound of formula II-A and II-B can be converted into
the immunogens and/or the conjugate reagents of this invention by
reacting these compounds with a polyamine or a polypeptide carrier
which contains a terminal amino group. The same polypeptide can be
utilized as the carrier and as the immunogenic polymer carrier in
the immunogen of this invention provided that the polyamine or
polypeptide carrier used to generate the antigen is immunologically
active. However, to form the conjugates, these polymers need not
produce an immunological response as needed for the immunogens. In
accordance with this invention, the various functional groups
represented by X in the compounds of formula II-A and II-B can be
conjugated to the polymeric material by conventional means of
attaching a functional group to an amine or thiol group contained
within the polymeric carrier.
Antibodies
[0077] The present invention also relates to novel antibodies
including monoclonal antibodies to doxorubicin produced by
utilizing the aforementioned immunogens. In accordance with this
invention it has been found that these antibodies produced in
accordance with this invention are selectively reactive with
doxorubicin and unlike the prior art antibodies, do not react with
non-pharmaceutically active metabolites which would interfere with
immunoassays for doxorubicin. The most problematic of these
doxorubicin metabolites is doxorubicin aglycone. The ability of the
antibodies of this invention not to react with these inactive
metabolites makes these antibodies particularly valuable in
providing an immunoassay for doxorubicin.
[0078] The present invention relates to novel antibodies and
monoclonal antibodies to doxorubicin. The antisera of the invention
can be conveniently produced by immunizing host animals with the
immunogens of this invention. Suitable host animals include
rodents, such as, for example, mice, rats, rabbits, guinea pigs and
the like, or higher mammals such as goats, sheep, horses and the
like. Initial doses, bleedings and booster shots can be given
according to accepted protocols for eliciting immune responses in
animals, e.g., in a preferred embodiment mice received an initial
dose of 100 ug immunogen/mouse, i.p. and one or more subsequent
booster shots of between 50 and 100 ug immunogen/mouse over a six
month period. Through periodic bleeding, the blood samples of the
immunized mice were observed to develop an antibodies against
doxorubicin utilizing conventional immunoassays. These methods
provide a convenient way to screen for hosts which are producing
antisera having the desired activity. The antibodies were also
screened against the major metabolites of doxorubicin and showed no
substantial binding to these compounds.
[0079] Monoclonal antibodies are produced conveniently by
immunizing Balb/c mice according to the above schedule followed by
injecting the mice with 100 ug immunogen i.p. or i.v. on three
successive days starting four days prior to the cell fusion. Other
protocols well known in the antibody art may of course be utilized
as well. The complete immunization protocol detailed herein
provided an optimum protocol for serum antibody response for the
antibody to doxorubicin.
[0080] B lymphocytes obtained from the spleen, peripheral blood,
lymph nodes or other tissue of the host may be used as the
monoclonal antibody producing cell. Most preferred are B
lymphocytes obtained from the spleen. Hybridomas capable of
generating the desired monoclonal antibodies of the invention are
obtained by fusing such B lymphocytes with an immortal cell line,
which is a cell line that which imparts long term tissue culture
stability on the hybrid cell. In the preferred embodiment of the
invention the immortal cell may be a lymphoblastoid cell or a
plasmacytoma cell such as a myeloma cell. Murine hybridomas which
produce doxorubicin monoclonal antibodies are formed by the fusion
of mouse myeloma cells and spleen cells from mice immunized against
doxorubicin-protein conjugates. Chimeric and humanized monoclonal
antibodies can be produced by cloning the antibody expressing genes
from the hybridoma cells and employing recombinant DNA methods now
well known in the art to either join the subsequence of the mouse
variable region to human constant regions or to combine human
framework regions with complementary determining regions (CDR's)
from a donor mouse or rat immunoglobulin. An improved method for
carrying out humanization of murine monoclonal antibodies which
provides antibodies of enhanced affinities is set forth in
International Patent Application WO92/11018.
[0081] Polypeptide fragments comprising only a portion of the
primary antibody structure may be produced, which fragments possess
one or more immunoglobulin activities. These polypeptide fragments
may be produced by proteolytic cleavage of intact antibodies by
methods well known in the art, or by inserting stop codons at the
desired locations in expression vectors containing the antibody
genes using site-directed mutageneses to produce Fab fragments or
(Fab').sub.2 fragments. Single chain antibodies may be produced by
joining VL and VH regions with a DNA linker (see Huston et al.,
Proc. Natl. Acad. Sci. U.S.A., 85:5879-5883 (1988) and Bird et al.,
Science, 242:423-426 (1988)).
[0082] The antibodies of this invention are selective for
doxorubicin without having any substantial cross-reactivity with
pharmaceutically active metabolites of doxorubicin such as the
metabolites mentioned hereinabove. By having no substantial
cross-reactivity it is meant that the antibodies of this invention
have a cross reactivity relative to doxorubicin with these
metabolites of less than 20%. Those antibodies having a cross
reactivity of less than 15% are preferred. The antibodies of this
invention may be reactive with other pharmaceutically active
doxorubicin like compounds such as doxorubicinol.
Immunoassays
[0083] In accordance with this invention, the conjugates and the
antibodies generated from the immunogens of these compounds of
formula II-A and II-B or mixtures thereof can be utilized as
reagents for the determination of doxorubicin in patient samples.
This determination is performed by means of an immunoassay. Any
immunoassay in which the reagent conjugates formed from the
compounds of formula II-A and II-B compete with the doxorubicin in
the sample for binding sites on the antibodies generated in
accordance with this invention can be utilized to determine the
presence of doxorubicin in a patient sample. The manner for
conducting such an assay for doxorubicin in a sample suspected of
containing doxorubicin, comprises combining an (a) aqueous medium
sample, (b) an antibody to doxorubicin generated in accordance with
this invention and (c) the conjugates formed from the compounds of
formula II-A or II-B or mixtures thereof. The amount of doxorubicin
in the sample can be determined by measuring the inhibition of the
binding to the specific antibody of a known amount of the conjugate
added to the mixture of the sample and antibody. The result of the
inhibition of such binding of the known amount of conjugates by the
unknown sample is compared to the results obtained in the same
assay by utilizing known standard solutions of doxorubicin.
Various means can be utilized to measure the amount of conjugate
formed from the compounds of formula II-A and II-B bound to the
antibody. One method is where binding of the conjugates to the
antibody causes a decrease in the rate of rotation of a fluorophore
conjugate. The amount of decrease in the rate of rotation of a
fluorophore conjugate in the liquid mixture can be detected by the
fluorescent polarization technique such as disclosed in U.S. Pat.
No. 4,269,511 and U.S. Pat. No. 4,420,568.
[0084] On the other hand, the antibody can be coated or absorbed on
nanoparticles so that when these particles react with the
doxorubicin conjugates formed from the compounds of formula II-A
and II-B, these nanoparticles form an aggregate. However, when the
antibody coated or absorbed nanoparticles react with the
doxorubicin in the sample, the doxorubicin from the sample bound to
these nanoparticles does not cause aggregation of the antibody
nanoparticles. The amount of aggregation or agglutination can be
measured in the assay mixture by absorbance.
[0085] On the other hand, these assays can be carried out by having
either the antibody or the doxorubicin conjugates attached to a
solid support such as a microtiter plate or any other conventional
solid support including solid particles. Attaching antibodies and
proteins to such solid particles is well known in the art. Any
conventional method can be utilized for carrying out such
attachments. In many cases, in order to aid measurement, labels may
be placed upon the antibodies, conjugates or solid particles, such
as radioactive labels or enzyme labels, as aids in detecting the
amount of the conjugates formed from the compounds of formula II-A
and II-B which is bound or unbound with the antibody. Other
suitable labels include chromophores, fluorophores, etc.
[0086] As a matter of convenience, assay components of the present
invention can be provided in a kit, a packaged combination with
predetermined amounts of new reagents employed in assaying for
doxorubicin. These reagents include the antibody of this invention,
as well as, the conjugates formed from the compounds of formula
II-A and II-B or mixtures thereof. It is generally preferred that
in a given immunoassay, if a conjugate formed from a compound of
formula II-A is utilized, that the antibody be generated by an
immunogen formed from a compound of formula II-A. In a like manner,
if a conjugate formed from a compound of formula II-B is utilized,
the antibody be generated by the immunogen formed from a compound
of formula II-B. However, this need not be the case and antibodies
and conjugates in a given assay can be derived from either or both
of these conjugates and immunogens.
[0087] In addition to these necessary reagents, additives such as
ancillary reagents may be included, for example, stabilizers,
buffers and the like. The relative amounts of the various reagents
may vary widely to provide for concentrations in solution of the
reagents which substantially optimize the sensitivity of the assay.
Reagents can be provided in solution or as a dry powder, usually
lyophilized, including excipients which on dissolution will provide
for a reagent solution having the appropriate concentrations for
performing the assay.
EXAMPLES
[0088] In the Examples, the following abbreviations are used for
designating the following: [0089] CHCl.sub.3 Chloroform [0090] BMPH
N-[.beta.-maleimidopropionic acid] hydrazide, trifluoroacetic acid
salt [0091] MeOH methanol [0092] DMF Dimethylformamide [0093] TFA
Trifluoroacetic acid [0094] DMSO Dimethylsulfoxide [0095] CAPS
3-(Cyclohexylamino)-1-propanesulfonic acid [0096] NHS N-hydroxy
succinimide [0097] EDC
1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride [0098]
KPi potassium phosphate buffer pH 7.5 [0099] SPDP
3-(2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester [0100]
MES 2-(N-Morpholino)ethanesulfonic acid buffer pH 6 [0101] ANS
8-Anilino-1-naphthalenesulfonic acid [0102] i.p. Intraperitoneal
[0103] HRP horse radish-peroxidase [0104] TFA Trifluoroacetic
[0105] TMB 3,3',5,5'-Tetramethylbenzidine [0106] TRIS
Tris(hydroxymethyl)aminomethane hydrochloride [0107] BSA Bovine
serum albumin [0108] KLH Keyhole Limpet Hemocyanin [0109] BTG
Bovine thyroglobulin [0110] PBS Phosphate buffered saline [0111] di
deionized water In the Examples, Scheme 1 and Scheme 2 below set
forth the specific compounds prepared and referred to by numbers in
the Examples. The schemes are as follows:
##STR00026##
##STR00027## ##STR00028## ##STR00029##
[0111] Example 1
Preparation of Doxorubicin trifluoroacetamide activated ester 4
(scheme 1)
[0112] A stirred suspension of doxorubicin (i) (0.8 g, 1.38 mmol)
in CHCl.sub.3/MeOH (1:1) (15 mL) at 0.degree. C. was treated with
2.76 mL of 0.5 M methanolic sodium methoxide added dropwise
followed by addition of S-ethyltrifluorothioacetate (0.89 mL, 7.02
mmol) under a nitrogen atmosphere. After being stirred in the dark
for 16 h at room temperature, the reaction was concentrated in
vacuo. The residue was dissolved in 10 mL of CHCl.sub.3/MeOH (1:1),
and 4 mL of toluene, and concentrated. Again the residue was
dissolved in 50 mL of CHCl.sub.3/MeOH (9:1) and washed with 10 mL
of 0.1 M citric acid, and brine (2.times.10 mL). It was dried over
magnesium sulfate and evaporation of solvents followed by
trituration in methylene chloride/Ether/Hexanes gave 2 (0.814, 92%)
as a red solid.
[0113] A mixture of compound 2 (0.49 g, 0.766 mmol),
carboxymethoxylamine hemihydrochloride (0.30 g, 1.38 mmol), and
sodium acetate (0.38 g, 4.60 mmol) in MeOH (15 mL) was stirred in
the dark overnight at room temperature. The solvent was removed
under reduced pressure and the residue was dissolved in water (25
mL) and CHCl.sub.3/MeOH (9:1) (3.times.25 mL). All the combined
organic layers were dried over MgSO.sub.4, evaporated, and
triturated with methylene chloride/Hexanes to afford 3 (0.45 g,
82%).
[0114] To a solution of compound 3 (0.45 g, 0.63 mmol) in methylene
chloride/DMF (1:5) (12 mL) at 0.degree. C., were added EDC (0.11 g,
0.95 mmol) and NHS (0.18 g, 0.95 mmol) under a nitrogen atmosphere.
After being stirred for 18 h at room temperature, the reaction
mixture was diluted with methylene chloride (50 mL) and washed with
water (2.times.15 mL). It was dried over magnesium sulfate, and
evaporation of solvent gave compound 4 (0.445 g, 86%) as a red
solid; this material was directly used in the next step (Examples
3a and 3b).
Example 2
Preparation of (3-Maleimidopropyl) hydrazone of Doxorubicin (scheme
2)
[0115] Doxorubicin [1] was derivatized with
N-[.beta.-maleimidopropionic acid] hydrazide (BMPH) to introduce a
maleimido group for eventual conjugation to protein through a
thio-ether linkage. To a solution of doxorubicin hydrochloride (29
mg, 0.05.times.10-3 mmol), BMPH (50 mg, 3.4 eq.) in 10 ml of
anhydrous MeOH was added 3 .mu.L of TFA. The reaction mixture was
stirred at room temperature for 24 hours while being protected from
light. The methanolic solution was concentrated to a volume of 2 mL
and added to acetonitrile (30 ml) dropwise with stirring. The
resulting suspension was allowed to stand at 4.degree. C. overnight
for crystallization of the doxorubicin C13 hydrazone maleimido
derivative [5]. This product was isolated by centrifugation, washed
with fresh methanol-acetonitrile (1:10), and dried under vacuum to
yield the (6-Maleimidocaproyl) hydrazone of doxorubicin (5). The
structure was confirmed by NMR.
Example 3a
Preparation of BTG Immunogen with Activated Hapten 4
[0116] To 18.8 mL of BTG (7.1 mg/mL) in 1:1 phosphate buffer (50
mM, pH 7.5):DMSO was added 1.3 mL of compound 4 from Example 1 (20
mg/mL in DMSO) while stirring the protein solution on ice. After
addition the pH was again checked to be 8. The mixture was allowed
to stir for 18 hours at room temperature. The trifluoroacetamide
protecting group on the amino sugar was removed by dialysis with
CAPS buffer, pH 11. The first dialysis was performed with 50% 50 mM
CAPS and 50% DMSO at room temperature. Thereafter the DMSO
proportion was reduced stepwise: 40%, 30%, 20%, 10% and 0%. For the
last CAPS dialysis the buffer concentration was reduced to 25 mM
and the dialysis done at 4.degree. C. The immunogenic conjugate was
then purified by dialysis against phosphate buffer (50 mM, pH 7.5).
The conjugate was characterized by UV/VIS spectroscopy.
Example 3b
Preparation of KLH Immunogen with Activated Hapten 4
[0117] To 18.0 mL of KLH (7.35 mg/mL) in 1:1 phosphate buffer (50
mM, pH 7.5):DMSO was added 1.3 mL of compound 4 from Example 1 (20
mg/mL in DMSO) while stirring the protein solution on ice. After
addition the pH was again checked to be 8. The mixture was allowed
to stir for 18 hours at room temperature. The trifluoroacetamide
protecting group on the amino sugar was removed by dialysis with
CAPS buffer, pH 11. The first dialysis was performed with 50% 50 mM
CAPS and 50% DMSO at room temperature. Thereafter the DMSO
proportion was reduced stepwise: 40%, 30%, 20%, 10% and 0%. For the
last CAPS dialysis the buffer concentration was reduced to 25 mM
and the dialysis done at 4.degree. C. The immunogenic conjugate was
then purified by dialysis against phosphate buffer (50 mM, pH 7.5).
The conjugate was characterized by UV/VIS spectroscopy.
Example 4a
Preparation of BTG Immunogen with Activated Hapten 5
[0118] To conjugate the doxorubicin C13 hydrazone maleimido
derivative to protein the lysine residues of the protein were
modified to introduce a sulfhydryl group. To a solution of bovine
thyroglobulin (BTG) in potassium phosphate buffer, pH 7.5 (14.9
mg/mL, 3 mL) was added 4 mg of 3-(2-Pyridyldithio)propionic acid
N-hydroxysuccinimide ester (SPDP) (20 eq.) in 50 .mu.L of DMSO to
derivatize the lysines with propionic pyridyldithio groups. After
1.5 hours stirring at room temperature, 40 mg of dithiothreitol
dissolved in 100 .mu.L of KPi was added to the mixture to generate
the sulfhydryls by reduction of the dithiopyridyl moiety. The
reduction of the pyridyldithio derivative on the protein to release
the sulfhydryl group was performed under nitrogen, with stirring at
room temperature for 30 minutes. The thiolated BTG was then
purified by gel-filtration chromatography.
[0119] The gel-filtration column was prepared with 15 g of Sephadex
G-25 swelled in 50 mM KPi Buffer at room temperature for 1 h,
degassed under vacuum, and loaded in a column (1.5 cm.times.50 cm).
The loaded column was equilibrated with the buffer for 1 hour. The
reaction mixture was loaded onto the column, and eluted with KPi
buffer. Ellman's reagent was used to monitor the elution of the
protein. The fractions containing protein were collected and
pooled. The molar concentration of thiol groups was determined by
the Ellman's procedure (Riddles, P. W. et al., Analytical
Biochemistry, Ellman's reagent: 5,5'-Dithiobis(2-nitrobenzoic
acid)-A reexamination., 94, 75-81 (1979).
[0120] To the purified thiolated-BTG protein (5 mg/mL in KPi, 44.7
mg) in an ice-water bath was added dropwise 3 mL of the doxorubicin
hydrazone derivate 5 prepared in Example 2 (2.33 mg/mL) the
reaction mixture was stirred at 4.degree. C. for 16 hours and
protected from light. The immunogenic conjugate was purified by
gel-filtration as described above. The immunogenic conjugate was
characterized by UV/VIS spectroscopy.
Example 4b
Preparation of KLH Immunogen with Activated Hapten 5
[0121] To a solution of KLH in potassium phosphate buffer, pH 7.5
(5.58 mg/mL, 4 mL) was added 3 mg of SPDP in 50 .mu.L of DMSO.
After 1.5 hours stirring at room temperature, 25 mg of
dithiothreitol dissolved in 50 .mu.L of KPi was added to the
mixture. The reduction was performed under nitrogen, with stirring
at room temperature for 30 minutes. The thiolated KLH was then
purified by gel-filtration chromatography as described in Example
4a.
[0122] To the purified thiolated-KLH protein (4 mg/mL in KPi, 5 mL)
in an ice-water bath was added dropwise 2.124 mL of the doxorubicin
hydrazone derivate 5 prepared in Example 2 (1.41 mg/mL) the
reaction mixture was stirred at 4.degree. C. for 16 hours and
protected from light. The immunogenic conjugate was purified by
gel-filtration as described in Example 4a. The immunogenic
conjugate was characterized by UV/VIS spectroscopy.
Example 5
Preparation of BSA Conjugate (1:1 ratio) with Activated Hapten
4
[0123] To 40 mL of BSA (25 mg/mL) in 1:1 phosphate buffer (50 mM,
pH 7.5):DMSO was added 0.62 mL of compound 4 from Example 1 (20
mg/mL in DMSO) while stirring the protein solution on ice. After
addition the pH was again checked to be 8. The mixture was allowed
to stir for 18 hours at room temperature. The trifluoroacetamide
protecting group on the amino sugar was removed by dialysis with
CAPS buffer, pH 11. The first dialysis was performed with 50% 50 mM
CAPS and 50% DMSO at room temperature. Thereafter the DMSO
proportion was reduced stepwise: 40%, 30%, 20%, 10% and 0%. For the
last CAPS dialysis the buffer concentration was reduced to 25 mM
and the dialysis done at 4.degree. C. The immunogenic conjugate was
then purified by dialysis against phosphate buffer (50 mM, pH 7.5).
The conjugate was characterized by UV/VIS spectroscopy.
Example 6a
Preparation of Thiolated BSA for Reaction with Activated Hapten
5
[0124] To a solution of BSA in potassium phosphate buffer, pH 7.5
(50 mg/mL, 6 mL) was added 4.2 mg of SPDP (3 eq.) in 84 .mu.L of
DMSO. After 1.5 hours stirring at room temperature, 27 mg of
dithiothreitol dissolved in 0.135 mL of KPi was added to the
mixture. The reduction was performed under nitrogen, with stirring
at room temperature for 30 minutes. The thiolated BSA was then
purified by gel-filtration chromatography.
[0125] The gel-filtration column was prepared with 12 g of Sephadex
G-25 swelled in 10 mM MES Buffer (pH 6) at room temperature for 1
h, degassed under vacuum, and loaded in a column (1.5 cm.times.50
cm). The loaded column was equilibrated with the buffer for 1 hour.
The reaction mixture was loaded onto the column, and eluted with
MES buffer. Ellman's reagent was used to monitor the elution of the
protein. The fractions containing protein were collected and
pooled. The molar concentration of thiol groups was determined by
the Ellman's procedure.
Example 6b
Preparation of BSA Conjugate (3:1 Ratio) with Activated Hapten
5
[0126] To the purified thiolated-BSA protein prepared in Example 6a
(5 mg/mL in MES, 35 mg) in an ice water bath was added dropwise
0.135 mL of the doxorubicin hydrazone derivate 5 prepared in
Example 2 (8 mg/mL) the reaction mixture was stirred at 4.degree.
C. for 16 hours and protected from light. The immunogenic conjugate
was purified by gel-filtration as described in Example 6a. The
immunogenic conjugate was characterized by UV/VIS spectroscopy.
Example 6c
Preparation of BSA Conjugate (1:1 Ratio) with Activated Hapten
5
[0127] To the purified thiolated-BSA protein prepared in Example 6a
(5 mg/mL in MES, 80 mg) in an ice water bath was added dropwise
0.107 mL of the doxorubicin hydrazone derivate 5 prepared in
Example 2 (8 mg/mL) the reaction mixture was stirred at 4.degree.
C. for 16 hours and protected from light. The immunogenic conjugate
(6 mL) was purified by gel-filtration as described in Example 6a.
The purified immunogenic conjugate was characterized by UV/VIS
spectroscopy. The rest of immunogenic conjugate reaction mixture
was used for the capping reaction without further purification.
Example 6d
Capping of Doxorubicin Hydrazone 5
BSA Conjugate (1:1 ratio)
[0128] To 5 mL of 1:1 doxorubicin [5]--BSA conjugate prepared in
Example 6c (5 mg/mL in MES buffer) was added 0.047 mL of
N-Ethylmaleimide (2 equivalents, 2 mg/mL in MES buffer) to cap
thiol groups not modified by doxorubicin. The reaction was stirred
for 3 hours at room temperature and then purified as in Example
6a.
Example 7
Preparation of Doxorubicin [4] Antibodies
[0129] Two groups of ten Female BALB/c mice were immunized i.p. one
with 100 .mu.g/mouse of doxorubicin [4]--BTG immunogen prepared in
Example 3a and the other with 100 .mu.g/mouse of doxorubicin
[4]--KLH immunogen prepared in Example 3b emulsified in Complete
Freund's adjuvant. The mice were boosted once four weeks after the
initial injection with 100 .mu.g/mouse of the same immunogens
emulsified in Incomplete Freund's Adjuvant. Ten days after the
boost test bleeds from each mouse were obtained by orbital bleed.
For monoclonal antibodies depending on the immunogen, age and rest
period of the mouse starting four days before the fusion, the mice
were injected i.p. with either 400 .mu.g (3 days before fusion),
200 .mu.g (2 days before fusion), and 200 .mu.g (1 day before
fusion) of doxorubicin [4]--BTG immunogen prepared in Example 3a in
PBS or 100 ug on each day of doxorubicin [4]--KLH immunogen
prepared in Example 3b in PBS on three successive days. According
to the protocol of Coligan et al. spleen cells were isolated from
the selected mice and fused with 2.times.10.sup.7 cells of the
myeloma fusion partner cell line (SP2/0) using 50% polyethylene
glycol 1500 [Coligan, J. E. et al., eds., Current Protocols in
Immunology, 2.5.1-2.5.8, (1992), Wiley & Sons, NY.] To grow the
fused cells into antibody producing colonies according to the
method of Coligan et al. the fused cells were plated on 10 96-well
plates in a conventional HAT (hypoxanthine, aminopterin and
thymidine) selective growth medium such as DMEM/F12 (Dulbecco's
Modified Eagle's Medium 1:1 with L-glutamine and HEPES)
supplemented with 20% fetal bovine serum alternative, and
containing 2% L-glutamine (100 mM) and 2% 50X HAT. Two weeks later,
the hybridoma supernatant was assayed for the presence of
anti-doxorubicin antibodies by ELISA as described in Example 10b.
Positive wells were expanded and again screened by the same method.
The positive clones were confirmed for doxorubicin binding by a
competitive ELISA as described in Example 11 or subcloned directly.
Clones positive by ELISA were subcloned once or twice by limiting
dilution according to the method disclosed in Coligan, J. E. et
al., eds., Current Protocols in Immunology, 2.5.8-2.5.17, (1992),
Wiley & Sons, NY. Only the monoclonal antibodies which were
selective for doxorubicin and had a cross reactivity relative to
doxorubicin with the aglycone of doxorubicin of 15% or less as
determined by these screening procedures were selected.
Example 8
Preparation of Doxorubicin [5] Antibodies
[0130] Two groups of ten Female BALB/c mice were immunized i.p. one
group with 100 .mu.g/mouse of doxorubicin [5]--BTG immunogen
prepared in Example 4a and the other with 100 .mu.g/mouse
doxorubicin [5]--KLH immunogen prepared in Example 4b emulsified in
Complete Freund's Adjuvant. Mice were boosted once four weeks after
the initial injection with 100 .mu.g/mouse of the same immunogens
emulsified in Incomplete Freund's Adjuvant. Ten and 28 days after
the boost test bleeds from each mouse were obtained by orbital
bleed. The anti-serum from the day 28 test bleeds contained
doxorubicin antibodies evaluated in Examples 10a and 11. Only the
antiserum having antibodies which were selective for doxorubicin
and had a cross reactivity relative to doxorubicin with the
aglycone of doxorubicin of 15% or less as determined by these
screening procedures were selected.
Example 9a
Microtiter Plate Sensitization Procedure with Doxorubicin [4]--BSA
1:1 Conjugate
[0131] For the purpose of screening antibodies and measuring
doxorubicin concentration by the Enzyme-Linked Immunosorbent Assay
(ELISA) method polystyrene microtiter plates (Nunc MaxiSorp F8
Immunomodules) optimized for protein binding and containing 96
wells per plate were used. Each well was coated with doxorubicin
[4]--BSA 1:1 conjugate (prepared as in Example 5) by adding 300
.mu.L of doxorubicin [4]--BSA conjugate at 10 .mu.g/mL in 0.01M
MES, pH=6, and incubating for three hours at room temperature. The
wells were washed with 0.005M MES, pH 6 and then were blocked with
375 .mu.L of 5% sucrose, 0.2% sodium caseinate solution for 30
minutes at room temperature. After removal of the post-coat
solution the plates were dried at 37.degree. C. overnight.
Example 9b
Microtiter Plate Sensitization Procedure with Doxorubicin [5]--BSA
3:1 Conjugate
[0132] For the purpose of screening antibodies and measuring
doxorubicin concentration by the Enzyme-Linked Immunosorbent Assay
(ELISA) method polystyrene microtiter plates (Nunc MaxiSorp F8
Immunomodules) optimized for protein binding and containing 96
wells per plate were used. Each well was coated with doxorubicin
[5]--BSA 3:1 conjugate (prepared as in Example 6b) by adding 300
.mu.L of doxorubicin [5]--BSA conjugate at 10 .mu.g/mL in 0.01M
MES, pH=6, and incubating for three hours at room temperature. The
wells were washed with 0.005M MES, pH 6 and then were blocked with
375 .mu.L of 5% sucrose, 0.2% sodium caseinate solution for 30
minutes at room temperature. After removal of the post-coat
solution the plates were dried at 37.degree. C. overnight.
Example 9c
[0133] Microtiter Plate Sensitization Procedure with Doxorubicin
[5]--BSA 1:1 Conjugate
[0134] For the purpose of screening antibodies and measuring
doxorubicin concentration by the Enzyme-Linked Immunosorbent Assay
(ELISA) method polystyrene microtiter plates (Nunc MaxiSorp F8
Immunomodules) optimized for protein binding and containing 96
wells per plate were used. Each well was coated with doxorubicin
[5]--BSA 1:1 conjugate (prepared as in Example 6c) by adding 300
.mu.L of doxorubicin [5]--BSA conjugate at 10 .mu.g/mL in 0.01M
MES, pH=6, and incubating for three hours at room temperature. The
wells were washed with 0.005M MES, pH 6 and then were blocked with
375 .mu.L of 5% sucrose, 0.2% sodium caseinate solution for 30
minutes at room temperature. After removal of the post-coat
solution the plates were dried at 37.degree. C. overnight.
Example 9d
Microtiter Plate Sensitization Procedure with Doxorubicin [5]--BSA
1:1 Conjugate (thiol capped)
[0135] For the purpose of screening antibodies and measuring
doxorubicin concentration by the Enzyme-Linked Immunosorbent Assay
(ELISA) method polystyrene microtiter plates (Nunc MaxiSorp F8
Immunomodules) optimized for protein binding and containing 96
wells per plate were used. Each well was coated with doxorubicin
[5]--BSA 1:1 capped conjugate (prepared as in Example 6d) by adding
300 .mu.L of doxorubicin [5]--BSA conjugate at 10 .mu.g/mL in 0.01M
MES, pH=6, and incubating for three hours at room temperature. The
wells were washed with 0.005M MES, pH 6 and then were blocked with
375 .mu.L of 5% sucrose, 0.2% sodium caseinate solution for minutes
at room temperature. After removal of the post-coat solution the
plates were dried at 37.degree. C. overnight.
Example Boa
Antibody Screening Procedure
Titer
[0136] Antibodies were screened by Enzyme-Linked Immunosorbent
Assay (ELISA) method. This method for screening the doxorubicin
antibodies (produced in Examples 7 and 8) was performed with the
microtiter plates that were sensitized with doxorubicin [5]--BSA
prepared in Examples 9b, c, d. The antibody screening assay was
performed by diluting the antisera containing doxorubicin
antibodies to 1:1,000, 1:10,000, 1:100,000 and 1:1,000,000 in
phosphate buffered saline containing 0.1% BSA and 0.01% thimerosal.
For evaluation of monoclonal antibodies, hybridoma supernatants, of
Example 7 found to be positive for presence of antibodies by the
procedure of Example 10b, were diluted 1:2, 1:4, 1:8, 1:16, etc. To
each well of doxorubicin [5]--BSA sensitized wells (prepared in
Examples 9b, c, d) 100 .mu.L of diluted antibody was added and
incubated for 10 minutes at room temperature with shaking. During
this incubation antibody binds to the doxorubicin [5]--conjugate in
the well. The wells of the plates were washed three times with 0.02
M TRIS, 0.9% NaCl, 0.5% Tween-80 and 0.001% Thimerosal, pH 7.8 to
remove any unbound antibody. To detect the amount of doxorubicin
antibody bound to the doxorubicin [5]--BSA conjugate in the wells,
100 .mu.L of a goat anti-mouse antibody--HRP enzyme conjugate
(Jackson Immunoresearch) diluted to a specific activity
(approximately 1/2800) in PBS with 0.1% BSA, 0.05% ANS, 0.01%
thimerosal, capable of binding specifically with murine
immunoglobulins and producing a colored product when incubated with
a substrate, were added to each well. After an incubation of 10
minutes at room temperature with shaking, during which the goat
anti-mouse antibody--HRP enzyme conjugate binds to doxorubicin
antibodies in the wells, the plates were again washed three times
to remove unbound goat anti-mouse antibody--HRP enzyme conjugate.
To develop a measurable color in the wells washing was followed by
the addition of 100 .mu.L of TMB (TMB Liquid Substrate), a
substrate for HRP, to develop color during a 10 minute incubation
with shaking at room temperature. Following the incubation for
color development, 50 .mu.L of stop solution (1.5% sodium fluoride
in di H.sub.2O) was added to each well to stop the color
development and after 10 seconds of shaking the absorbance was
determined at 650 nm with a 96-well plate reader. The amount of
antibody in a well was proportional to the absorbance measured and
was expressed as the dilution (titer) resulting in an absorbance of
1.5. Titers were determined by graphing log antibody dilution of
the antibody measured (x-axis) vs. absorbance 650 nm (y-axis) and
extrapolating the titer at an absorbance of 1.5. The titer
determined the concentration (dilution) of antibody used in the
indirect competitive Microtiter plate assay described in Example
11.
Example 10b
Antibody Screening Procedure
Monoclonal Screening
[0137] Antibodies were screened by Enzyme-Linked Immunosorbent
Assay (ELISA) method. This method for screening doxorubicin
monoclonal antibodies (produced in Example 7) was performed with
the microtiter plates that were sensitized with doxorubicin
[5]--BSA as described in Example 9b. To each well of doxorubicin
[5]--BSA sensitized wells (prepared in Example 9b) 50 .mu.L
phosphate buffered saline containing 0.1% BSA and 0.01% thimerosal
and then 50 .mu.L of monoclonal culture supernatant were added and
incubated for 10 minutes at room temperature with shaking. During
this incubation antibody binds to the doxorubicin [5]--conjugate in
the well. The wells of the plates were washed three times with 0.02
M TRIS, 0.9% NaCl, 0.5% Tween-80 and 0.001% Thimerosal, pH 7.8 to
remove any unbound antibody. To detect the amount of doxorubicin
antibody bound to the doxorubicin [5]--BSA conjugate in the wells,
100 .mu.L of a goat anti-mouse antibody--HRP enzyme conjugate
(Jackson Immunoresearch) diluted to a predetermined specific
activity (approximately 1/2800) in PBS with 0.1% BSA, 0.05% ANS,
0.01% thimerosal, capable of binding specifically with murine
immunoglobulins and producing a colored product when incubated with
a substrate, were added to each well. After an incubation of 10
minutes at room temperature with shaking, during which the goat
anti-mouse antibody--HRP enzyme conjugate binds to doxorubicin
antibodies in the wells, the plates were again washed three times
to remove unbound goat anti-mouse antibody--HRP enzyme conjugate.
To develop a measurable color in the wells washing was followed by
the addition of 100 .mu.L of TMB (TMB Liquid Substrate), a
substrate for HRP, to develop color during a 10 minute incubation
with shaking at room temperature. Following the incubation for
color development, 50 .mu.L of stop solution (1.5% sodium fluoride
in di H.sub.2O) was added to each well to stop the color
development and after 10 seconds of shaking the absorbance was
determined at 650 nm with a 96-well plate reader. The amount of
antibody in a well was proportional to the absorbance measured.
Samples with an absorbance of greater than three or more times
background were designated as positive.
Example 11
Indirect Competitive Microtiter Plate Immunoassay Procedure
Determining IC.sub.50 and Cross-Reactivity for Antibodies to
Doxorubicin
[0138] Doxorubicin concentrations were measured by an indirect
competitive Enzyme-Linked Immunosorbent Assay (ELISA) method This
method for measuring doxorubicin concentrations was performed with
the microtiter plates that were sensitized with doxorubicin
[5]--BSA described in Examples 9b, c, d. Doxorubicin, and
doxorubicin aglycone diluted 10 fold in PBS containing 0.1% BSA and
0.01% Thimerosal over a concentration range of 0.01 to 10,000
ng/mL. The assay was performed by incubating 50 .mu.L of the
analytes to be measured with 50 .mu.L of antibody (produced in
Examples 7 and 8 with immunogens of Examples 3a, 3b, 4a and 4b)
diluted to a titer determined in Example boa. During the 10 minute
incubation (R.T., with shaking) there is a competition of antibody
binding for the doxorubicin conjugate in the well and the analyte
in solution. Following this incubation the wells of the plate were
washed three times with 0.02 M TRIS, 0.9% NaCl, 0.5% Tween-80 and
0.001% Thimerosal, pH 7.8 to remove any material that was not
bound. To detect the amount of doxorubicin antibody bound to the
doxorubicin [5]--BSA conjugate in the wells, 100 .mu.L of a goat
anti-mouse antibody--HRP enzyme conjugate (Jackson Immunoresearch)
diluted to a predetermined specific activity (approximately 1/2800)
in PBS with 0.1% BSA, 0.05% ANS, 0.01% thimerosal, capable of
binding specifically with murine immunoglobulins and producing a
colored product when incubated with a substrate, were added to each
well. After an incubation of 10 minutes at room temperature with
shaking, during which the goat anti-mouse antibody--HRP enzyme
conjugate binds to doxorubicin antibodies in the wells, the plates
were again washed three times to remove unbound secondary
conjugate. To develop a measurable color in the wells washing was
followed by the addition of 100 .mu.L of TMB (TMB Liquid
Substrate), a substrate for HRP, to develop color in a 10 minute
incubation with shaking at room temperature. Following the
incubation for color development, 50 .mu.L of stop solution (1.5%
sodium fluoride in di H.sub.2O) was added to each well to stop the
color development and after 10 seconds of shaking the absorbance
was determined at 650 nm with a 96-well plate reader. The amount of
antibody in a well was proportional to the absorbance measured and
inversely proportional to the amount of doxorubicin in the sample.
The absorbance of the color in the wells containing analyte was
compared to that with no analyte and a standard curve was
generated. The IC.sub.50 value for a given analyte was defined as
the concentration of analyte that is required to inhibit 50% of the
absorbance for the wells containing no analyte. The
cross-reactivity of a given analyte was calculated as the ratio of
the IC.sub.50 for doxorubicin to the IC.sub.50 for doxorubicin
aglycone and expressed as a percent. When measured with an antibody
as produced in Examples 7 and 8 with immunogen of Examples 3a, 3b,
4a & b the percent cross-reactivates relative to doxorubicin
for doxorubicin aglycone was less than or equal to 10%. Results are
in tables 1 & 2 below.
TABLE-US-00001 TABLE 1 Cross-Reactivity of Competitive Immunoassay
using antibodies to doxorubicin [5]-BTG and KLH (Example 8) with
plate coatings doxorubicin [5]-BSA conjugate (Examples 9b, 9c, 9d).
Immunogen Example 4a Immunogen Example 4b Plate % cross-reactivity
% cross-reactivity sensitized as doxorubicin- doxorubicin- in
Example doxorubicin aglycone doxorubicin aglycone 9b 100% 5.7% 100%
8.6% 9c 100% 10.0% 100% not measured 9d 100% 8.8% 100% not
measured
TABLE-US-00002 TABLE 2 Cross-Reactivity of Competitive Immunoassay
using a monoclonal antibody to doxorubicin [4]-BTG and -KLH
(Example 7) with plate coating doxorubicin [5]-BSA conjugate
(Examples 9b, 9c, 9d). Immunogen Example 3a Immunogen Example 3b
Plate % cross-reactivity % cross-reactivity sensitized as
doxorubicin- doxorubicin- in Example doxorubicin aglycone
doxorubicin aglycone 9b 100% 10.6% 100% 1.5% 9c 100% 12.9% 100%
3.9% 9d not tested 100% 3.0%
As seen from these tables, the antibodies of this invention are
substantially selectively reactive with the active, parent form of
doxorubicin and are not substantially cross-reactive with the
inactive aglycone metabolite of doxorubicin.
* * * * *