There is disclosed an oligonucleotide strand or duplex-targeting protein conjugate, a linking group to link an oligonucleotide to a targeting protein, and a method for producing targeting protein conjugates containing oligonucleotide strands or duplexes. A suitable linker is a bifunctional phosphoramidite ester that binds to both the targeting protein and the oligonucleotide strand or duplex. The invention can be used for delivering chemotherapeutic agents to target sites based upon the specificity of the targeting protein and to deliver specific oligonucleotide sequences to a target site to hybridize and inhibit the expression of complementary sequences within the target cell genome.

TECHNICAL FIELD 
The present invention relates to oligonucleotide-targeting protein 
conjugates in general, and more particularly, to such conjugates joined 
through a linking group and optionally having a chemotherapeutic agent 
associated therewith. 
BACKGROUND OF THE INVENTION 
Recent efforts have been devoted to the conjugation of cytotoxic agents or 
neoplastic drugs to specific antibodies, such as monoclonal antibodies, to 
produce targeting protein conjugates that can selectively kill target 
cells (e.g., tumor cells) while sparing normal tissues. A large number of 
different cytotoxic agents (including beta- and alpha-emitting isotopes, 
plant and bacterial toxins) and a variety of drugs (including antibiotics, 
antiviral agents, intercalating agents, antimalarial agents, 
antimetabolites, antineoplastic agents, antifungal agents, and alkylating 
agents) have been contemplated for this purpose. 
Conjugation of chemotherapeutic drugs to antibodies is undertaken for the 
following reasons: 
1. It has recently been shown that up to a 1,000-fold increase in the 
amount of drug delivered to tumor cells can be attained when the drug is 
conjugated to an antigen-specific monoclonal antibody over the amount of 
delivered drug attained by the addition of free drug. 
2. Pleiotropic drug resistance may arise following treatment with one of a 
number of chemotherapeutic drugs, resulting in the inducement of 
resistance to drugs of several classes. The mechanism(s) of pleiotropic 
drug resistance are not entirely understood, but it is known that this 
resistance can be partially overcome by antibody targeting of drugs. 
3. Even though current chemotherapeutic drugs are active against only some 
of the major tumor types, the chemotherapeutic response rate in 
drug-insensitive tumor types may be increased by antibody-mediated 
delivery. 
4. Many dose limiting toxicities, which are now seen with chemotherapeutic 
drugs, can be decreased by conjugation to an antibody. A decrease in 
toxicity with at least equal efficacy would give a superior product, and 
such a product would have a higher therapeutic index. 
Many chemotherapeutic drugs have been conjugated to monoclonal antibodies 
using a variety of covalent bonds (see, for example, Ghose et al., Meth. 
Enzymol. 93:280 (1983); Ram et al., Pharm. Res. 4:181 (1987)). One problem 
with covalent conjugation of drugs to antibodies is a concomitant loss of 
drug activity. It is believed that the strength of the covalent bond 
results in an inability to separate drug from antibody at the target site. 
Thus, there is a need in the art for a method for binding drugs to 
targeting agents that permits efficient release of the conjugated drug at 
the target site. 
SUMMARY OF THE INVENTION 
The present invention provides targeting protein conjugates wherein an 
oligonucleotide, as a single-strand or a duplex, is attached to the 
targeting protein via a linking group as described herein. These 
conjugates optionally facilitate delivery of a therapeutic agent to a 
target site. 
The present invention also provides, in one aspect, linkers which can 
covalently bind an oligonucleotide strand or duplex to a targeting 
protein. 
In other words, the oligonucleotide-targeting protein conjugates of this 
aspect of the present invention are as follows: 
##STR1## 
Therapeutic oligonucleotide-targeting protein conjugates of the present 
invention may be depicted as follows: 
##STR2## 
Each conjugate of the present invention comprises a targeting protein bound 
(preferably covalently) to a linking group, which in turn is bound 
(preferably covalently) to an oligonucleotide strand that can be a single 
strand or a duplex. The oligonucleotide strand can bind a therapeutic 
agent directly, such as by complementary strand binding of the therapeutic 
agent. Alternatively, the oligonucleotide strand can bind to a 
complementary strand to form a duplex that is capable of binding 
therapeutic agents, such as through intercalation or by major or minor 
groove binding. Furthermore, the oligonucleotide strand can be bound to a 
second linking group that, in turn, binds to a therapeutic agent. 
In yet another aspect, the present invention provides a DNA oligonucleotide 
that acts as an oligonucleotide probe, thereby supplementing the targeting 
capability of the oligonucleotide-targeting protein conjugate. In this 
aspect of the invention, the oligonucleotide (probe) can improve 
specificity of therapeutic agent delivery, and/or can serve to 
intracellularly target the conjugate, subsequent to extracellular delivery 
to target cells by the targeting protein component of the conjugate. 
Hybridization of the oligonucleotide (probe) and the target cell DNA will 
inhibit expression of specific nucleotide sequences within the target cell 
(by oligonucleotide binding to target cell DNA sequences). This inhibition 
of DNA translation can be enhanced by the synergistic action of a 
chemotherapeutic agent whose mechanism of action is inhibition of DNA 
expression. Synergism is achieved by attaching such chemotherapeutic agent 
to the oligonucleotide, which can be single- or double-stranded DNA, RNA 
or analogs thereof.

EXAMPLE 1 
Synthesis of a Phosphine Linking Group 
Methyl-6-hydroxyhexanoate was made as follows. To a solution of 25 g (220 
mmole) of .epsilon.-caprolactone in 250 ml of methanol was added 12.3 g 
(220 mmole) of sodium methoxide. The resulting solution was stirred at 
25.degree. C. for 12 hours, and then evaporated to dryness. The residue 
was partitioned between saturated aqueous ammonium chloride and 
dichloromethane, and the organic layer was washed with saturated aqueous 
sodium bicarbonate. Drying over magnesium sulfate and removal of the 
solvent in vacuo yielded methyl-6-hydroxyhexanoate as a clear, colorless 
oil (21 g; 65% unoptimized). 
To a solution of 0.786 g (5.37 mmole) of methyl-6-hydroxyhexanoate and 0.48 
g (2.8 mmole) of diisopropylammonium tetrazolide in 25 ml of 
dichloromethane were added dropwise 1.62 g (5.37 mmole) of bis(diisopropyl 
amino)-2-cyanoethyoxyphosphine (Aldrich). The resulting solution was 
stirred at 25.degree. C. for 2 hours and extracted with phosphate buffer 
at pH 7. Drying over magnesium sulfate and fast chromatography on silica 
gel (40% ethylacetate-hexanes, trace triethylamine) yielded the 
diisopropylamino-5-(carbomethoxy)pentyloxy-2-cyanoethoxy phosphine linking 
group as a colorless oil (1.30 g: 70%). 
EXAMPLE 2 
Synthesis of a Monoclonal Antibody-DNA Probe Conjugate 
For conjugation to a monoclonal antibody (MAb), to the 5'-phosphodiester of 
dodecathymine (T.sub.12) and omega hydroxycaproic acid (C.sub.5 --COOH), 
dubbed T.sub.12 -C.sub.5 --COOH (i.e., DNA probe), in approximately 20 
.mu.l water was added 16.8 micromoles of 1-ethyl-3-(dimethylamino 
propylcarbodiimide) (hydrochloride) in 5.5 .mu.l of water. After 10 
minutes at room temperature, 18.6 micromoles of N-hydroxysuccinimide (NHS) 
was added in 8.7 .mu.l over a course of 70 minutes. To this was added 0.96 
mg (6 nmoles) of MAb 9.2.27, which reacts with an antigen present on 
melanoma cells, in 90 .mu.l of 67 mM carbonate buffer, pH 9.6, followed by 
an additional 80 .mu.l of 0.2M carbonate buffer for pH adjustment to 9.5. 
The reaction mix was incubated for 2 hours at 4.degree. C., applied to a 
0.8.times.13 cm Sephacryl S-200 (Pharmacia Fine Chemicals, Piscataway, 
N.J.) column pre-equilibrated with 6.7 mM phosphate buffer, pH 7.3, in 
0.15M NaCl (PBS), and eluted with PBS. Ultraviolet spectra of the eluate 
reveal that the native antibody had an absorbance maximum at 277 nm and 
the T.sub.12 -C.sub.5 --COOH had an absorbance maximum at 265 nm. However, 
the spectrum of the antibody-DNA probe conjugate showed a 5 nm shift to 
272 nm as a result of covalent linkage of the DNA probe (FIG. 1). From the 
absorbance of the conjugate at two wavelengths, 260 and 290 nm, the 
concentrations of MAb and oligonucleotide probe can be calculated, and a 
probe/MAb ratio of 1.6 was obtained. 
This conjugate was also analyzed by isoelectric focusing (IEF) gel 
electrophoresis. In this procedure, a pH gradient was established in an 
agarose gel in an electric field. When a protein was applied to the gel, 
it migrates until it reaches the region of the gel where the pH was equal 
to the isoelectric point of the protein. At this pH, the negative charges 
on the protein exactly balanced the positive charges, thus it was 
electrically neutral, and, as such, will not migrate in an electric field. 
While each monoclonal antibody was a homogeneous protein that had a single 
amino acid sequence, a monoclonal antibody did not focus on an IEF gel 
into a single band, but rather displayed a closely spaced family of bands. 
This microheterogeneity was due to differences in the post-translational 
glycosylation of the antibody, specifically to differences in the number 
of sialic acid residues. Thus, the family of bands represented proteins 
differing from each other by an integral number of charge units. Since the 
T.sub.12 -C.sub.5 --COOH probe contained thirteen negative charges, a 
large shift towards the anode in an IEF gel would be expected with the DNA 
probe-MAb conjugate. The IEF gel of the conjugate (FIG. 2) indicated the 
expected large shift towards the anode. Using other derivatives of the MAb 
as standards, a shift of approximately 25 charge units, or approximately 2 
probes/antibody, was found, which is in agreement with the results from UV 
spectroscopy. 
EXAMPLE 3 
Synthesis of an Oligonucleotide-Linking Group-Monoclonal Antibody Conjugate 
In this example, the tetrafluorophenyl (TFP) ester of T.sub.12 -C.sub.5 
--COOH was synthesized and purified before reaction with MAb. Briefly, to 
a solution of 0.25 micromoles of T.sub.12 -C.sub.5 --COOH in 0.310 .mu.l 
of PBS (phosphate-buffered saline) were added 10 micrograms of TFP in 20 
.mu.l of acetonitrile: water (9:1) and 12.5 micrograms of EDAC in 25 .mu.l 
of water, and the solution was incubated at 75.degree. C. for 20 minutes. 
After purification on a reverse phase C-18 silica column, 1 ml of 30% 
acetonitrile/water eluate was evaporated to about 20 .mu.l. To this was 
added 0.67 mg MAb in 40 .mu.l PBS, 0.46 ml phosphate buffer, pH 9.5, and 
40 .mu.l acetonitrile. After 2 hours incubation at 37.degree. C., the 
conjugate was purified on a Sephacryl S-200 column as in Example 1. 
Spectral analysis indicated an average of 2.2 oligonucleotides per MAb. 
EXAMPLE 4 
Formation of an Intercalating Drug/Double-Stranded DNA/Monoclonal Antibody 
Conjugate 
A duplex oligonucleotide-MAb complex may be formed in several ways. One 
method involves hybridization of an oligonucleotide-linker intermediate 
with its complementary oligonucleotide, and then conjugation of the 
double-stranded intermediate to targeting protein. A more specialized 
example involves synthesis of a self-complementary, oligonucleotide-linker 
derivative, which has the effect of providing two linking groups per 
duplex DNA. This oligonucleotide-linker derivative is used to improve 
yields of the conjugation reaction, or to provide another functional group 
for linking a therapeutic agent to the conjugate. 
This example describes the hybridization of oligonucleotide-linker-MAb 
conjugates (described above) with a complementary oligonucleotide. A 
special example of this methodology involves derivatization of the 
complementary oligonucleotide with a drug or a linker that reacts with a 
therapeutic agent to form a therapeutic agent-(linker)-duplex 
DNA-linker-MAb conjugate. 
To the conjugate synthesized in Example 3 was added 50 nmoles of 
dodecaadenosine phosphate (A.sub.12) in 0.5 ml PBS containing 5 mM EDTA, 
and the hybridization mixture was incubated at 37.degree. C. for 44 hours. 
To this reaction mix at room temperature was added 60 .mu.g (30 .mu.l) 
adriamycin (Adr). After 2 hours at room temperature, the mixture was 
dialyzed against 50 mM sodium phosphate, 1M NaCl, pH 5.5, for 72 hours, 
concentrated on a centrifugal ultrafilter ("Centricon") with a 30 Kdalton 
molecular weight cutoff, and washed three times with 3 ml of the same 
buffer on the ultrafilter. 
Spectral analysis revealed that this conjugate contains 7 molecules of Adr 
per MAb. 
EXAMPLE 5 
Synthesis of an Oligonucleotide-Monoclonal Antibody Conjugate Using a 
Thioether Bond 
A T.sub.12 -C.sub.2 --NH.sub.2 oligonucleotide derivative was synthesized 
on an Applied Biosystems DNA synthesizer using standard methods 
(B-cyano-ethyl phosphoramidite ester of thymidine and "Aminolink-1" 
[Applied Biosystems, Foster City, Calif.]). 
To about 50 nmoles (34 .mu.l) T.sub.12 -C.sub.2 --NH.sub.2 were added 2.4 
.mu.moles (80 .mu.l) 
N-succinimidyloxy-4(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) in 
dimethylsulfoxide (DMSO) and 25 .mu.l 1M carbonate buffer, pH 9.5. After 
incubation for 1 hour at room temperature, the reaction mix was applied to 
a 9.5 ml Sephadex G-25 column (PD-10, Pharmacia, Piscataway, N.J.) and 
eluted with PBS. A control reaction mix contained underivatized T.sub.12 
(Pharmacia) and SMCC. Simultaneously, a monoclonal antibody, NR-ML-05 
(reactive with an antigen on melanoma cells), was incubated with 50 mM 
dithiothreitol to produce thiol groups from native antibody disulfides. 
The reduced antibody was purified on a PD-10 column at 0.degree.-4.degree. 
C., the two fractions containing antibody (by absorbance at 280 nm) were 
pooled, and 1.5 mg (0.33 ml) aliquots were added to the 1.0 ml fractions 
containing the T.sub.12 -C.sub.2 NHCO-SMCC, T.sub.12, or the same fraction 
from the SMCC control. 
These reaction mixes were incubated for 25 hours at room temperature and 
then separated on a Superose 12 (Pharmacia, Piscataway, N.J.) gel 
permeation column. The antibody fraction contained an average of 0.32 
T.sub.12 probes per MAb as compared to the control, which contained none. 
From the foregoing, it will be appreciated that, although specific 
embodiments of the invention have been described herein for purposes of 
illustration, various modifications may be made without deviating from the 
spirit and scope of the invention.