WO1994020523A1 - Tumor targeting with l-enantiomeric oligonucleotide conjugates of immunoreagents and of chelated radionuclides - Google Patents
Tumor targeting with l-enantiomeric oligonucleotide conjugates of immunoreagents and of chelated radionuclides Download PDFInfo
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- WO1994020523A1 WO1994020523A1 PCT/US1994/002610 US9402610W WO9420523A1 WO 1994020523 A1 WO1994020523 A1 WO 1994020523A1 US 9402610 W US9402610 W US 9402610W WO 9420523 A1 WO9420523 A1 WO 9420523A1
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- 0 CCO[C@@](C)(C=*CO)NC(N=C(*(C)*)N)=C(*)N=* Chemical compound CCO[C@@](C)(C=*CO)NC(N=C(*(C)*)N)=C(*)N=* 0.000 description 3
- CWXACBSDDAXDBT-UHFFFAOYSA-N NC1NNC1CO Chemical compound NC1NNC1CO CWXACBSDDAXDBT-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2121/00—Preparations for use in therapy
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2123/00—Preparations for testing in vivo
Definitions
- This invention relates to sequential targeting and delivery of immunoreagent compositions which find particular utility in the therapy and diagnostic imaging of cancer by means of a tumor targeted sequential delivery system comprising a primary non-radioactive targeting immunoreagent and a secondary radioactive delivery agent.
- This invention also relates to novel methods for the attachment of L-enantiomeric
- oligonucleotides and chelates, to proteins, and to bifurcated tumor targeting and delivery vectors for the treatment and diagnostic imaging of tumors.
- radioimmunotherapy and diagnostic imaging with the various currently available radiopharmaceuticals which include radionuclide-containing immunoreactive proteins can be less than optimal because these radionuclide-containing immunoreactive proteins
- radiopharmaceuticals may bind to non-target normal tissue. This binding can result in undesirable toxicity to normal tissue during therapeutic applications as well as in high background signals during diagnostic imaging applications. Inefficient covalent bonding of the radioactive component with protein in conjugate
- radionuclide preparation can be another. problem due to release of the radioactive component which may then deposit in healthy tissue. Also, long plasma half-lives of currently available radionuclide-containing immunoreactive proteins and slow clearance of radionuclide from the body can result in prolonged exposure of normal tissue to damaging effects of radiation and can produce unacceptable toxic effects in otherwise normal and disease free tissues in the body, especially in those tissues and cells most sensitive to radiation damage, e.g., the stem cells of the bone marrow and
- immunoreactive protein is restricted by the number of sites of chelation available, an increase in that number which can be achieved by increasing the number of chelating agents conjugated to the protein can produce a decrease in the immunoreactivity of the protein. This can limit the number of such chelating agents that can be attached to the protein.
- the number of chelating agents that can be attached to an immunoreactive protein is also limited by the number of available groups such as, for example, amino groups suitable for use in attachment of the chelating agents and by the potential immunogenicity of the thus modified protein which, being highly derivatized, could be recognized by a host immune system as being haptenated.
- Nucleic acids in the form of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) encode and transfer genetic information for the cellular synthesis of proteins and enzymes.
- Naturally occuring nucleic acids are composed of nucleosides such as 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), and thymidine (T) in DNA and of adenosine (A), guanosine (G), cytidine (C), and uridine (U) in RNA.
- dA 2'-deoxyadenosine
- dG 2'-deoxyguanosine
- dC 2'-deoxycytidine
- T thymidine
- A adenosine
- G guanosine
- C cytidine
- U uridine
- modified nucleosides such as those containing 2'-O-methylribosyl groups and those containing bases such as N 4 ,N 4 -dimethyladenine and N 7 -methylguanidine are found in messenger, transfer, and ribosomal RNA.
- nucleosidyl purine and pyrimidine bases adenine with thymine and guanine with cytosine in DNA, and adenine with uracil and guanine with cytosine in RNA.
- sequences of bases in two separate oligonucleotide strands or in two regions of a single strand are complementary to each other, the complementary sequences can hybridize with each other via hydrogen bonds between complementary base pairs to form a right-handed double helix with a B-type conformation, the two phosphate-ribose ester backbones of which are antiparallel.
- each nucleoside unit is a D-enantiomer whose structure is defined by the chirality of the D-deoxyribose ring which has
- each nucleoside unit is also a D-enantiomer whose structure is defined by the chirality of the D-ribose ring which has substituents at the 1'-(b), 2'-(a), 3'-(a), and 4'- (b) positions as represented schematically in Structure I wherein R 1 is OH and "Base” represents an adenine, guanine, uracil or cytosine moiety.
- the phosphate diesters and the bases do not comprise chiral centers.
- oligonucleotides composed of naturally configured D- enantiomers have been used as primers for nucleic acid polymerizing enzymes, as synthons in the construction of artificial genes for the preparation of proteins by recombinant DNA techniques, and as diagnostic probes to detect and characterize cellular nucleic acid sequences.
- synthetically prepared oligonucleotides have been investigated for use in the control of gene expression in living cells, and as therapeutic agents in the inhibition of viral replication and in the treatment of cancer. These applications rely on the sequence specific complementary binding properties of the
- Synthetically prepared oligonucleotides composed of naturally configured D-enantiomers can be generated by a variety of methods, the currently most useful of which include solid phase synthesis via phosphoramidite intermediates and solid phase synthesis via H- phosphonate intermediates as described by E. Uhlmann and A. Peyman in Chemical Reviews, 1990, 90, 544.
- a 5' hydroxyl group of a growing DNA oligomer chain of D-enantiomers having amide protecting groups on the exocylic amine groups of the bases therein and which is attached to a solid phase support reacts with an activated D-nucleoside 5'-O- dimethoxytrityl-3'-(2-cyanoethyl N,N-diisopropyl)- phosphoramidite in the presence of 1H-tetrazole as a catalyst.
- Commonly used protecting groups include the benzoyl group for the protection of the exocyclic amino groups of adenine and cytosine and the isobutyryl group for the protection of the exocyclic amino group of guanine. Any unreacted 5'-hydroxyl groups are capped with acetate groups by reaction with acetic anhydride in the presence of 4-N,N-dimethylaminopyridine. The resulting phosphite is then oxidized with iodine to form a phosphotriester.
- the 5'-O-dimethoxyxrityl group is removed under acid conditions using dichloroacetic acid, and the reaction sequence is repeated using another activated D-nucleoside 5'-O-dimethoxytrityl-3'-(2- cyanoethyl N,N-diisopropyl) phosphoramidite.
- the oligonucleotide is freed from amide protecting groups and cleaved from the support by treatment with ammonium hydroxide. The oligonucleotide is then purified or isolated by methods such as precipitation, electrophoresis, or
- L-d(AAAAAA) hexameric L-DNA
- L-d(AAAAAA) L-d(AAAAAA)
- D-d(AAAAAA) D-poly(U)
- D-poly(U) a natural D-RNA enantiomer
- L-DNA has been shown to hybridize in vitro with natural enantiomeric D-RNA and to be stable to nuclease resistance, in practice such a duplex formation with targeted D-RNA is not readily achievable in vivo since it is necessary for the L-DNA to cross at least one cell membrane to find the intact D-RNA.
- the present invention is directed to a non- radioactive targeting immunoreagent that comprises a tumor antigen recognizing moiety, one or more L- enantiomeric oligonucleotides comprising non-self- associating L-enantiomeric oligonucleotide sequences, and one or more linking groups.
- the present invention is also directed to a
- radioactive targeting immunoreagent that comprises an L- enantiomeric oligonucleotide comprising an L- enantiomeric oligonucleotide sequence that is
- the present invention is also directed to
- compositions comprising one or more of the above-described immunoreagents and a
- the present invention is further directed to methods for treating and imaging disease sites such as tumor sites in a patient.
- Said methods comprise administration to the patient of an effective amount of the above-described non-radioactive targeting
- the present invention provides many advantages compared to conventional targeting immunoreagents.
- the non-radioactive targeting immunoreagent can accumulate at a tumor site in vivo while it is not accumulated at normal tissue sites.
- the in vivo residence half life of the non- radioactive targeting immunoreagent is long enough to permit its accumulation at a tumor site.
- the in vivo residence half life of the radioactive targeting reagent is shorter than the residence half life of the non-radioactive targeting immunoreagent.
- the portion of the radioactive targeting reagent that does not hybridize to tumor associated non- radioactive targeting reagent is rapidly cleared from the patient.
- radionuclide or a chelate containing a radionuclide an amplification of the number of radionuclides per site of modification per targeting immunoreagent can be
- targeting reagent can hybridize in vitro and in vivo.
- oligonucleotides of the non-radioactive targeting immunoreagent and the radioactive targeting reagent will not hybridize with isomeric, complementary sequenced, naturally occurring D-enantiomeric oligonucleotides.
- oligonucleotides of the non-radioactive targeting immunoreagent and the radioactive targeting reagent are stable to nuclease activity.
- oligonucleotides of the non-radioactive targeting immunoreagent and the radioactive targeting reagent and the hybrid formed between the complementary L- enantiomeric oligonucleotides will not bind to enzymes which are specific for binding to isomeric D- enantiomeric oligonucleotides.
- the hybrid formed between complementary sequenced L-enantiomeric oligonucleotides of the non-radioactive targeting immunoreagent and the radioactive targeting reagent is stable to nuclease activity.
- oligonucleotides of the non-radioactive targeting immunoreagent and the radioactive targeting reagent do not self hybridize.
- the non-radioactive targeting immunoreagent and the radioactive targeting reagent can comprise a wide variety of spacing, linking, and chelating groups, L- enantiomeric oligonucleotide sequences and
- oligonucleotides of the non-radioactive targeting immunoreagent can comprise L-enantiomeric
- oligonucleotide sequences which can be tandemly linked by spacing groups, wherein a segment of each L- enantiomeric oligonucleotide sequence can hybridize with a segment of the radioactive targeting reagent.
- oligonucleotides of the non-radioactive targeting immunoreagent can be linked to an antibody by means of either a 5'- or a 3'-substituent such as a 5'-amine or 3'-amine.
- Reagents are provided that have a specificity for tumors and a wide variety of compositions can be prepared in accordance with the present invention.
- This invention describes various novel
- bioconjugates which possess utility in therapeutic and diagnostic imaging compositions and methods.
- This invention further describes novel methods of preparing bioconjugates by the attachment of various L- enantiomeric oligonucleotide sequences to chelating agents, preferably terpyridine containing chelating agents, and to immunoreagents such as proteins,
- this invention describes novel bioconjugates useful for sequential targeting and amplified delivery of novel radioactive immunoreagent compositions which find particular utility in
- this invention describes the preparation and use of targeting immunoreagents that comprise a tumor antigen recognizing moiety, one or more L-enantiomeric oligonucleotides comprising non-self- associating L-enantiomeric oligonucleotide sequences, and one or more linking groups.
- These targeting immunoreagents react with a radioactive sequential targeting reagent that comprises an L-enantiomeric oligonucleotide sequence that is complementary in sequence to and capable of hybridization with one or more fragments of the said non-self-associating L- enantiomeric oligonucleotide sequences, one or more chelating agents, one or more linking groups, and having one or more radionuclides associated therewith.
- the L-enantiomeric nucleotides are L- enantiomers of natural D-deoxyribonucleotides.
- the above-described targeting immunoreagents form a compound that comprises moieties represented by the structure IV: Structure IV
- Z is the residue of an immunoreactive protein
- L z and L Q are independently a chemical bond or a linking group
- I is an L-enantiomeric oligonucleotide comprising a contiguous sequence of from 12 to about 50 L- enantiomeric nucleotide units wherein said contiguous sequence contains one or more members of a family of homologous contiguous sequences, the individual homologs of said family comprising from 12 to about 30 L- enantiomeric nucleotide units, and provided that
- contiguous sequences of six or more L-enantiomeric nucleotide units of said L-enantiomeric oligonucleotide do not hybridize with any other contiguous sequences of six or more contiguous L-enantiomeric nucleotide units anywhere in structure IV;
- Q l is a spacing group
- a is 0 or an integer from 1 to about 6;
- I i is an L-enantiomeric oligonucleotide comprising a contiguous sequence of from 12 to about 50 L- enantiomeric nucleotide units, a contiguous sequence therein comprising a portion of I;
- E is an end capping group
- p is an integer from 1 to about 10.
- a is an integer from 1 to about 6.
- the above- described targeting reagent comprises moieties
- described targeting reagent comprises moieties
- cl is a contiguous sequence of from 12 to about 50 L-enantiomeric nucleotide units wherein said contiguous sequence contains one or more members of a family of homologous contiguous sequences, the individual homologs of said family comprising from 12 to about 30 L- enantiomeric nucleotide units, where the nucleotide sequences of said homologs are complementary to the nucleotide sequences of members of the set of L- enantiomeric oligonucleotides in a co-administerable targeting immunoreagent, and where contiguous sequences of six or more L-enantiomeric nucleotide units of said complementary L-enantiomeric oligonucleotide do not hybridize with any other contiguous sequences of six or more contiguous L-enantiomeric nucleotide units anywhere in structure V;
- Q cI is a spacing group
- L 1 , L 2 , and L 3 are independently a chemical bond or a linking group
- W 1 , W 2 , and W 3 are each a residue of a chelating group;
- M 1 , M 2 , and M 3 comprise elements with oxidation states equal to or greater than +1, and at least one of M 1 , M 2 and M 3 is a radionuclide;
- x, y, and z are independently zero or one provided that at least one of x, y, or z is one;
- w and b are independently zero or an integer from 1 to about 4.
- an L-enantiomeric nucleotide unit is defined as the mirror image of the naturally occurring nucleotide unit
- an L- enantiomeric nucleoside is defined as the mirror image of the naturally occurring, isomeric D-enantiomeric nucleoside
- an L-enantiomeric oligonucleotide sequence is defined as the mirror image of the naturally occurring, isomeric D-enantiomeric oligonucleotide sequence.
- Non-limiting examples of L-enantiomeric nucleosides are set forth below.
- oligonucleotides from isomeric D-enantiomeric nucleotide intermediates. Examples of such methods include those discussed in "Chemistry of Nucleosides and Nucleotides", edited by Leroy B. Townsend, Plenum Press, N.Y., 1988.
- the synthesis of L-enantiomeric oligonucleotides by such methods requires the use of L-enantiomeric reagents such as L-enantiomeric nucleoside and nucleotide derivatives.
- the preparations of various L-enantiomeric nucleosides are well documented by Robins, M. et al (1970), J. Org. Chem., 35, p636-639; Holy, A. (1972), Coll. Czechoslov.
- a preferred method of synthesis of L-enantiomeric oligonucleotides of this invention comprises solid phase synthesis utilizing L-enantiomeric nucleotide phosphoramidite intermediates which contain blocking groups on the hydroxyl groups therein.
- the blocking of a 5'-hydroxyl group in a ribonucleoside moiety and in a deoxyribonucleoside moiety is
- an acid labile trityl group such as, for example, a monomethoxytrityl group (sometimes hereafter referred to as an MMT group) or a
- dimethoxytrityl group (sometimes hereafter referred to as a DMT group) employing the respective trityl
- trityl- blocked 5'-hydroxyl group is preferably done with an acid such as, for example, acetic acid in water, a chloroacetic acid in a solvent such as dichloromethane, or benzenesulfonic in a solvent such as chloroform or methanol as a reagent.
- an acid such as, for example, acetic acid in water, a chloroacetic acid in a solvent such as dichloromethane, or benzenesulfonic in a solvent such as chloroform or methanol as a reagent.
- the blocking of a 2'-hydroxyl group in a ribonucleic acid moiety is preferably done with a silyl chloride reagent such as, for example, t- butyldimethylchlorosilane which reacts with a ribosyl 2'-hydroxyl group to form a t-butyldimethylsilyl ether.
- a silyl chloride reagent such as, for example, t- butyldimethylchlorosilane which reacts with a ribosyl 2'-hydroxyl group to form a t-butyldimethylsilyl ether.
- the deblocking of such a t-butyldimethylsilyl ether can be achieved by treatment with, for example, sodium hydroxide dissolved in a solvent such as, for example, methanol.
- Preferred L-enantiomeric nucleotides are L-2- deoxyribonucleotides, and preferred L-enantiomeric oligonucleotides are oligo-L-2-deoxyribonucleotides.
- L-enantiomeric deoxyribonucleotides that are useful in the synthesis of the L-enantiomeric oligo- L-deoxyribonucleotides of this invention are L- enantiomeric deoxyribonucleosides such as, for example, derivatives of compounds 1 to 5 which have the 5'-OH in each blocked, for example, with a dimethoxytrityl (DMT) group, each of which is activated at the 3'-OH for phosphate bond formation in the oligonucleotide, for example, by treatment with 2-cyanoethyl-N,N- diisopropylphosphoryl chloride to produce the respective 3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidite intermediates.
- DMT dimethoxytrityl
- 2-cyanoethyl-N,N- diisopropylphosphoryl chloride to produce the respective 3'-(2-cyanoethyl-N
- L-adenosine 3'-O-phosphoramidite derivative, A10 that is suitable for use in the synthesis of the L- enantiomeric oligo-L-deoxyribonucleotide materials of this invention is outlined in Scheme 1.
- Scheme 1 is prepared from L-arabinal (A1) by treatment with p-toluoyl chloride and methanol according to the method described by Smejkal, I. et al (1964), Coll.
- A6 The exocyclic amine of A6 is then blocked by diacylation with benzoyl chloride to form A7 which contains two benzoate esters in addition to the blocked amine.
- the benzoate esters are removed by saponification with sodium hydroxide in water to form A8.
- This diol is then treated with dimethoxytrityl chloride (available from Aldrich Chemical Company) to form A9 which has a DMT ether at the 5'-position.
- A9 is then treated with 2- cyanoethyl N,N-diisopropylchlorophosphoramidite
- A10 the desired diprotected 5'-O-dimethoxytrityl-2'- deoxy-L-adenosine 3'-O-2-cyanoethylphosphoramidite.
- A10 is used as an intermediate in the synthesis of L- oligonucleotide sequences of the compositions of
- Production of such sequences can be done, for example, using an automated oligonucleotide synthesizer using procedures described by the manufacturer for the
- a preferred method of preparing a blocked 2'-deoxy- L-guanosine 3'-O-phosphoramidite derivative, G8, that is suitable for use in the synthesis of the L-enantiomeric oligo-L-deoxyribonucleotide materials of this invention is outlined in Scheme 2.
- 1-O-Methyl-3,5-di-O-p-toluyl- 2-deoxy-L-erythro-pentofuranose (A2 in Scheme 1) is prepared from L-arabinal (A1) by treatment with p- toluoyl chloride and methanol according to the method described by Smejkal, I. et al (1964), Coll. Czechoslov. Chem.
- the methyl acetal (A2) is hydrolyzed in dilute hydrochloric acid to provide the hemiacetal (G2) which is acylated with acetic anhydride to form 1-acetyl 3,5-di-O-p-toluyl-beta-L-erythro- pentofuranose (G3).
- Fusion of G3 and 2-fluoro-6- benzyloxypurine (G4) provides G5 which is then treated with alcoholic ammonia to provide the desired product (G6) which is purified by chromatography.
- the 6-benzyl protecting group of G6 is removed by .hydrogenolysis with palladium on carbon in ammonium hydroxide to yield 2- amino-9-(2-deoxy-L-erythro-pentofuranosyl)purin-6-one (G7; L-dG).
- G7; L-dG 2- amino-9-(2-deoxy-L-erythro-pentofuranosyl)purin-6-one
- the exocyclic 2-amino group of G7 is then protected by acylation with isobutyryl chloride, the 5'- hydroxyl group is protected as a DMT ether using
- Structure V of this invention Production of such sequences can be done, for example, using an automated oligonucleotide synthesizer using procedures described by the manufacturer for the synthesis and purification of isomeric D-oligonucleotides.
- a preferred method of preparing a blocked 2'-deoxy- L-uridine 3'-O-phosphoramidite derivative, U7, that is suitable for use in the synthesis of the L-enantiomeric oligo-L-deoxyribonucleotide materials of this invention is outlined in Scheme 3.
- the reaction of L-arabinose (U1) with cyanamide yields 2'-amino-1,2-oxazoline (U2) which upon treatment with methyl propiolate (U3) affords anhydro-L-uridine (U4).
- the anhydro derivative U4 is opened with HBr to give the 2'-bromo-nucleoside (U5), which upon catalytic hydrogenation affords 2-deoxy-L- uridine (U6).
- U6 The 5'-hydroxyl group of U6 is protected as a DMT ether using dimethoxytrityl chloride, and the 3'-hydroxyl is treated with 2-cyanoethyl N,N- diisopropylchlorophosphoramidite to provide U7, the desired protected 5'-O-dimethoxytrityl-2'-deoxy-L- uridine 3'-O-2-cyanoethylphosphoramidite.
- U7 is used as an intermediate in the synthesis of L-oligonucleotide sequences of the compositions of Structure IV and
- Structure V of this invention Production of such sequences can be done, for example, using an automated oligonucleotide synthesizer using procedures described by the manufacturer for the synthesis and purification of isomeric D-oligonucleotides.
- a preferred method of preparing a blocked L- thymidine 3'-O-phosphoramidite derivative, T7, that is suitable for use in the synthesis of the L-enantiomeric oligo-L-deoxyribonucleotide materials of this invention is outlined in Scheme 4.
- the synthetic method is analogous to the preparation of the 2'-deoxy-L-uridine 3'-O-phosphoramidite derivative, U7, described in Scheme 3 using L-arabinose as a chiral starting material.
- L-arabinose is treated with cyanamide to provide 2'-amino-1,2-oxazoline (U2) which is then reacted with methyl methacrylate with heating to yield the anhydro-nucleoside (T4).
- T4 is opened with
- T5 2-bromo-L-thymidine
- T6 L-thymidine
- T6 The 5'-hydroxyl group of T6 is protected as a DMT ether using dimethoxytrityl chloride, and the 3'-hydroxyl is treated with 2-cyanoethyl N,N- diisopropylchlorophosphoramidite to provide T7, the desired protected 5'-O-dimethoxytrityl-L-thymidine 3'-O- 2-cyanoethylphosphoramidite.
- T7 is used as an
- Structure V of this invention Production of such sequences can be done, for example, using an automated oligonucleotide synthesizer using procedures described by the manufacturer for the synthesis and purification of isomeric D-oligonucleotides.
- a preferred method of preparing a blocked 2'-deoxy- L-cytidine 3'-O-Ohosphoramidite derivative, C7, that is suitable for use in the synthesis of the L-enantiomeric oligo-L-deoxyribonucleotide materials of this invention is outlined in Scheme 5.
- L-arabinal, A1 is treated with HCl and toluyl chloride to provide the 3,5- di-O-p-toluyl-2-deoxy-L-ribofuranosyl chloride (C2).
- dimethoxytrityl chloride and the 3'-hydroxyl is treated with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to provide C7, the desired protected 5'-O- dimethoxytrityl-2'-deoxy-L-cytidine 3'-O-2- cyanoethylphosphoramidite.
- C7 is used as an
- Structure V of this invention Production of such sequences can be done, for example, using an automated oligonucleotide synthesizer using procedures described by the manufacturer for the synthesis and purification of isomeric D-oligonucleotides.
- L-dA the 2'-deoxy-L- guanosine (G7) is sometimes hereinafter referred to as L-dG; the 2'-deoxy-L-uridine (U6) is sometimes
- L-dU L-thymidine
- T6 L-thymidine
- C5 2'- deoxy-L-cytidine
- the term “residue” is used herein in context with a chemical entity.
- Said chemical entity comprises, for example, a chelating group, or a linking group, or a protein reactive group, or an immunoreactive group, or an immunoreactive protein, or an antibody, or an antibody fragment, or a cross-linking agent such as a heterobifunctional cross-linking agent, or an L- enantiomeric oligonucleotide, or a spacing group, or an end capping group.
- the term “residue” is defined as that portion of the chemical entity which exclusively remains when one or more chemical bonds therein when considered as an independent chemical entity, is altered, modified, or replaced to comprise one or more covalent bonds to one or more other chemical entities.
- a "residue of an L- enantiomeric oligonucleotide" in the context of, for example, I and I i in Structure IV or of cl in Structure V comprises an L-enantiomeric oligonucleotide modified at least for divalent attachment to the residue of another chemical entity, i.e., the residue of said L- enantiomeric oligonucleotide comprises at least a divalent L-enantiomeric oligonucleotidyl sequence.
- the residue of a chelating group in the context of W 1 , W 2 or W 3 of Structure V comprises a chelating group which is at least
- Z preferably is an antibody or antibody fragment which recognizes and is specific for a tumor associated antigen.
- the above-described protein can contain an
- immunoreactive group covalently bonded thereto through a chemical bond or a linking group derived from the residue of a protein reactive group and the residue of a reactive group on the protein.
- immunoreactive protein which can be abbreviated by "IRP” also includes an organic compound which is capable of covalently bonding to the protein and which is found in a living organism or is useful in the diagnosis, treatment or genetic engineering of cellular material or living organisms, and which has a capacity for
- the immunoreactive group can be selected from a wide variety of naturally occurring or synthetically prepared materials, including, but not limited to enzymes, amino acids, peptides, polypeptides, proteins, lipoproteins, glycoproteins, lipids, phospholipids. hormones, growth factors, steroids, vitamins,
- polysaccharides polysaccharides, viruses, protozoa, fungi, parasites, rickettsia, molds, and components thereof, blood components, tissue and organ components,
- haptens lectins
- toxins nucleic acids (including oligonucleotides), antibodies (monoclonal and polyclonal), anti-antibodies, antibody fragments, antigenic materials (including proteins and
- an immunoreactive group can be any substance which when presented to an immunocompetent host will result in the production of a specific antibody capable of binding with that substance, or the antibody so produced, which participates in an antigen- antibody reaction.
- Preferred immunoreactive groups are antibodies and various immunoreactive fragments thereof, as long as they contain at least one reactive site for reaction with a protein reactive group as described herein on the residue of the L-enantiomeric oligonucleotide or with linking groups as described herein. That site can be inherent to the immunoreactive species or it can be introduced through appropriate chemical modification of the immunoreactive species.
- other immunoreactive species include antibodies produced by the techniques outlined above, other immunoreactive species produced by the techniques outlined above, other immunoreactive fragments thereof, as long as they contain at least one reactive site for reaction with a protein reactive group as described herein on the residue of the L-enantiomeric oligonucleotide or with linking groups as described herein. That site can be inherent to the immunoreactive species or it can be introduced through appropriate chemical modification of the immunoreactive species.
- antibodies produced by the techniques outlined above other immunoreactive fragments thereof, as long as they contain at least one reactive site for reaction with a protein reactive group as described herein on the residue of the
- the immunoreactive group does not bind to the residue of an L-enantiomeric oligonucleotide in structure IV so as to inhibit the binding of the L- enantiomeric oligonucleotide to a complementary
- antibody fragment refers to an immunoreactive material which comprises a residue of an antibody, which antibody characteristically exhibits an affinity for binding to an antigen.
- affinity for binding to an antigen refers to the thermodynamic expression of the strength of interaction or binding between an antibody combining site and an antigenic determinant and, thus, of the stereochemical compatibility between them. As such, it is the expression of the equilibrium or association constant for the antibody-antigen interaction.
- affinity as used herein also refers to the
- thermodynamic expression of the strength of interaction or binding between a ligand and a receptor and, thus, of the stereochemical compatibility between them. As such, it is the expression of the equilibrium or association constant for the ligand-receptor interaction.
- antibody fragments exhibit a percentage of said affinity for binding to said antigen, that percentage being in the range of 0.001 per cent to 1,000 per cent, preferably 0.01 per cent to 1,000 per cent, more preferably 0.1 per cent to 1,000 per cent, and most preferably 1.0 per cent to 100 per cent, of the relative affinity of said antibody for binding to said antigen.
- An antibody fragment can be produced from an antibody by a chemical reaction comprising one or more chemical bond cleaving reactions; by a chemical reaction comprising of one or more chemical bond forming
- reactions employing as reactants one or more chemical components selected from a group comprising amino acids, peptides, carbohydrates, linking groups as defined herein, spacing groups as defined herein, protein reactive groups as defined herein, and antibody
- An antibody fragment such as are produced as described herein and by a molecular biological process, a bacterial process, or by a process comprising or resulting from the genetic engineering of antibody genes.
- An antibody fragment can be derived from an antibody by a chemical reaction comprising one or more of the following reactions:
- bonds being selected from, for example, carbon-nitrogen bonds, sulfur-sulfur bonds, carbon-carbon bonds, carbon-sulfur bonds, and carbon-oxygen bonds, and wherein the method of said cleavage is selected from:
- a catalysed chemical reaction comprising the action of a biochemical catalyst such as an enzyme such as papain or pepsin which enzymes to those skilled in the art are known to produce antibody fragments commonly referred to as Fab and Fab'2, respectively;
- a biochemical catalyst such as an enzyme such as papain or pepsin which enzymes to those skilled in the art are known to produce antibody fragments commonly referred to as Fab and Fab'2, respectively;
- a catalysed chemical reaction comprising the action of an electrophilic chemical catalyst such as a hydronium ion which, for example, favorably occurs at a pH equal to or less than 7;
- a catalysed chemical reaction comprising the action of a nucleophilic catalyst such as a hydroxide ion which, for example, favorably occurs at a pH equal to or greater than 7;
- a nucleophilic catalyst such as a hydroxide ion which, for example, favorably occurs at a pH equal to or greater than 7;
- an anionic sulfide group comprising an -S- group in the form of a salt such as a -S- Na + group
- a chemical reaction comprising an oxidation reaction such as the oxidation of a carbon-oxygen bond of a hydroxyl group or the oxidation of a carbon-carbon bond of a vicinal diol group such as occurs in a carbohydrate moiety; or
- one or more reagents comprising amino acids, peptides, carbohydrates, linking groups as defined herein, spacing groups as defined herein, protein reactive groups as defined herein, and antibody fragments such as are produced as described in (a), above; or
- an antibody fragment can be derived by
- non-covalent bonds comprise hydrophobic interactions such as occur in an aqueous medium between chemical species that independently comprise mutually accessible regions of low polarity such as regions comprising aliphatic and carbocyclic groups, and of hydrogen bond interactions such as occur in the binding of an oligonucleotide with a
- an antibody fragment can be produced as a result of the methods of molecular biology or by genetic engineering of antibody genes, for example, in the genetic engineering of a single chain immunoreactive group or a Fv fragment.
- An antibody fragment can be produced as a result a combination of one or more of the above methods.
- the immunoreactive group can be an enzyme which has a reactive group for
- Representative enzymes include, but are not limited to, aspartate aminotransaminase, alanine aminotransaminase, lactate dehydrogenase, creatine phosphokinase, gamma glutamyl transferase, alkaline acid phosphatase, prostatic acid phosphatase, horseradish peroxidase and various esterases.
- the immunoreactive group can be modified or chemically altered to provide a reactive group for use in the attachment to the residue of the L- enantiomeric oligonucleotide, I, through a linking group as described below by techniques known to those skilled in the art.
- Such techniques include the use of linking moieties and chemical modification such as described in WO-A-89/02931 and WO-A-89/2932, which are directed to modification of oligonucleotides, and U.S. Patent No. 4,719,182.
- compositions of this invention are for the diagnostic imaging of tumors and the radiological treatment of tumors.
- Preferred immunological groups therefore include antibodies to tumor-associated antigens.
- An antibody is sometimes hereinafter referred to as Ab.
- Specific non-limiting examples of antibodies include B72.3 and related antibodies
- colorectal tumors Pan-carcinoma
- 7E11C5 and related antibodies which recognize prostate tumors
- CC49 and related antibodies which recognize colorectal tumors
- TNT and related antibodies which recognize necrotic tissue
- PR1A3 and related antibodies which recognize colon carcinoma
- ING-1 and related antibodies which are described in International Patent Publication WO-A- 90/02569
- B174, C174 and related antibodies which recognize squamous cell carcinomas
- B43 and related antibodies which are reactive with certain lymphomas and leukemias
- An especially preferred antibody is ING-1.
- L z and L Q are independently a chemical bond or the residue of a linking group.
- the phrase "residue of a linking group” as used herein refers to a moiety that remains, results, or is derived from the reaction of a protein reactive group with a reactive site on the protein.
- the phrase "protein reactive group” as used herein refers to any group which can react with a functional group typically found on a protein. However, it is specifically
- a protein reactive groups can also react with a functional group typically found on a non- protein biomolecule.
- a linking group useful in the practice of this invention derives from a group which can react with any biological molecule containing an immunoreactive group, whether or not the biological molecule is a protein, to form a linking group between the immunoreactive group and the L-enantiomeric
- oligonucleotide containing species as described below.
- linking groups are derived from protein reactive groups selected from but not limited to:
- vinylsulfonyl vinylcarbonyl; epoxy; isocyanato; isothiocyanato; aldehyde; aziridine;
- succinimidoxycarbonyl activated acyl groups such as carboxylic acid halides; mixed anhydrides and the like; and other groups known to be useful in conventional photographic gelatin hardening agents;
- immunoreactive group i.e., proteins or biological molecules containing the immunoreactive group modified to contain reactive groups such as those mentioned in
- the "linking group” can be derived from protein reactive groups selected from amino, alkylamino, arylamino, hydrazino, alkylhydrazino, arylhydrazino, carbazido, semicarbazido, thiocarbazido, thiosemicarbazido, sulfhydryl, sulfhydrylalkyl,
- the alkyl portions of said linking groups can contain from 1 to about 20 carbon atoms.
- the aryl portions of said linking groups can contain from about 6 to about 20 carbon atoms;
- hardeners, bisepoxides, and bisisocyanates can become a part of, i.e., a linking group in, the protein-(L- enantiomeric oligonucleotide-containing species) conjugate during the crosslinking reaction.
- Other useful crosslinking agents can facilitate the crosslinking, for example, as consumable catalysts, and are not present in the final conjugate. Examples of such crosslinking agents are carbodiimide and carbamoylonium crosslinking agents as disclosed in U.S. Patent No. 4,421,847 and the ethers of U.S. Patent No. 4,877,724.
- one of the reactants such as the immunoreactive group
- the other such as the L-enantiomeric oligonucleotide-containing species, must have a reactive amine, alcohol, or sulfhydryl group.
- the crosslinking agent first reacts
- Preferred useful linking groups are derived from various heterobifunctional cross-linking reagents such as those listed in the Pierce Chemical Company
- reagents include:
- Sulfo-SMCC Sulfosuccinimidyl 4-(N- maleimidomethyl)cyclohexane-1- carboxylate;
- Sulfo-SIAB Sulfosuccinimidyl (4- iodoacetyl)aminobenzoate
- Sulfo-SMPB Sulfosuccinimidyl 4-(p- maleimidophenyl)butyrate
- linking groups in whole or in part, can also comprise and be derived from nucleotides and residues of
- nucleotides both naturally occurring and modified.
- linking groups of this invention are derived from the reaction of a reactive functional group such as an amine or sulfhydryl group as are available in the above Clonetech reagents, one or more of which has been incorporated into an L-enantiomeric oligonucleotide sequence of this invention, with, for example, one or more of the previously described protein reactive groups such as heterobifunctional protein reactive groups, one or more of which has been incorporated into an
- I and I i each independently comprise an L-enantiomeric oligonucleotide of a contiguous sequence of from 12 to about 50 L- enantiomeric nucleotide units wherein said contiguous sequence contains one or more members of a family of homologous contiguous sequences; wherein the individual homologs of said family comprise from 12 to about 30 L- enantiomeric nucleotide units; wherein the homologs of said family of sequences, both individually or as a set of homologous sequences being hereinafter sometimes referred to as "the Sequence"; and wherein any
- contiguous sequence of six or more L-enantiomeric nucleotide units does not hybridize with any other contiguous sequence of six or more contiguous L- enantiomeric nucleotide units anywhere in structure IV.
- Members of the set of homologous contiguous sequences which comprise "the Sequence" can be found in both the sequence I and the sequence I i , and at least one such sequence is common to both I and I i .
- the L-enantiomeric oligonucleotide sequence of I and I i in Structure IV can comprise L-DNA, L-RNA, purine and pyrimidine base-modified L-DNA or L-RNA, backbone- modified L-DNA or L-RNA such as methyl phosphonate or thiophosphonate or carbohydrate modified L-DNA or L-RNA analogs, whole or partially modified, or combinations thereof as long as a complementary L-enantiomeric oligonucleotide sequence incorporated into the
- radioactive targeting moiety of Structure V described below can hybridize to said L-enantiomeric
- L-enantiomeric oligonucleotide sequence to form a hybrid which exhibits a Tm (melting temperature) greater than about 37 °C.
- Preferred L-enantiomeric oligonucleotides are non-base- modified and non-backbone-modified L-DNA and L-RNA, more preferred are L-DNA comprising L-dA, L-T, L-dG, L-dU and L-dC L-enantiomeric nucleotide units.
- L-enantiomeric oligonucleotides are L-DNA comprising L-dA, L-T, L-dG, and L-dC L- enantiomeric nucleotide units.
- the L-enantiomeric oligonucleotide sequence I and I i can comprise double stranded L-DNA or L-RNA. That is, the L-enantiomeric oligonucleotide sequence may comprises complementary L- DNA or L-RNA which forms a double helix molecule.
- complementary L-enantiomeric oligonucleotide sequence incorporated into the radioactive targeting moiety interacts with the duplex L-DNA or L-RNA of I and I i in such a way as to form triplex (triple helix) L-DNA, triplex L-RNA, or a triplex L-DNA:L-RNA hybrid.
- L-enantiomeric oligonucleotide sequences comprising the "Sequence" are shown below.
- the following sequences comprise a set of homologous L-enantiomeric oligonucleotide sequences which when considered individually or in any combination comprise a set herein defined as the "Sequence":
- sequence (viii) contains sequence (vii) which contains sequence (vi) which contains sequence (v), and so on.
- I contains, for example sequence (iii)
- I i contains, for example, sequence (v)
- Another I i in structure IV can contain (viii), in which case it would also contain (i) through (vii) as well as (viii).
- all three sequences would contain at least (iii) [as well as (i) and (ii)], and the two I i 's would contain at least (v) [as well as (i) through (iv)].
- an L-enantiomeric oligonucleotide that comprises a
- contiguous sequence of L-enantiomeric nucleotides which sequence being complementary to at least sequence (i), would hybridize to all sequences (i) through (viii) as would any member of a set of contiguous complementary sequences, the individual members of which comprise the sequence complementary to any of (i) through (viii).
- a set of contiguous complementary sequences can comprise cl as will be described below.
- An especially preferred sequence is: L- d(TTATGGACGGAGAAGCTAA) (SEQ ID NO:8).
- L-enantiomeric oligonucleotide sequences of this invention can be tandemly linked by means of chemical bonds, by linking groups such as described above, or by spacing groups as described below.
- the sequential order of L-enantiomeric oligonucleotide sequences of this invention can be tandemly linked by means of chemical bonds, by linking groups such as described above, or by spacing groups as described below.
- nucleotides in the L-enantiomeric oligonucleotide sequences of this invention can be from the 5' to the 3' end or from the 3' to the 5' end. Attachment of the L- enantiomeric oligonucleotide sequences of this invention via linking groups as described above to the immune reactive group as described above can be accomplished via 3' or via 5' sites or via derivatives attached to 3' or 5' sites of the L-enantiomeric oligonucleotide.
- the "Sequence” may also be composed of a double stranded L-DNA or L-RNA. That is, the "Sequence” may consist of complementary L- enantiomeric oligonucleotides which non-covalently interact to form double stranded L-DNA or L-RNA.
- Q I is a spacing group which separates and links two or more L- enantiomeric oligonucleotide sequences of this
- Q I can comprise a linking group as defined above, alone, or in combination with an L-enantiomeric nucleotide or an L-enantiomeric oligonucleotide
- each spacing group can be linked to from two to about six L-enantiomeric oligonucleotide sequences, at least two of which contain the Sequence of this invention.
- the spacing group is linked to two L-enantiomeric oligonucleotide sequences each of which contains the Sequence of this invention.
- the spacing group is an L-enantiomeric oligonucleotide sequence.
- Non limiting examples of preferred spacing groups are L-enantiomeric oligonucleotides comprising the following sequences: L-d(ACT);
- An especially preferred spacing group is an L- enantiomeric oligonucleotide: L-d(ACTCTC).
- the condition described above i.e., that the L-enantiomeric oligonucleotide sequence of this invention comprises a non-self associating sequence, still applies when considering the selection of L- enantiomeric oligonucleotide spacing groups linked in combination with the L-enantiomeric oligonucleotide Sequence groups.
- a is from 0 to about 6, preferably an integer from 1 to about 6, more preferably one to about 4, and most preferably one or two.
- p is an integer from 1 to 10, preferably 1 to about 6, and more preferably 1 to 3. It is also contemplated that mixtures of immunoreactive proteins comprising mixtures of Z modified as defined in structure IV together with Z not so modified will also be useful in this invention. In this case, the bulk mixture properties of "p" of such mixtures would be useful in this invention.
- the bulk p values would be from about 0.1 to about 10.0, more preferably from about 0.2 to about 5.0, and most preferably from about 0.4 to about 3.
- E is an end capping group.
- E is preferably an L-enantiomeric nucleotide group or a group of one or more D-oligonucleotides that is modified so as to reduce or prevent the action of exonuclease enzymatic activity on the D-enantiomeric oligonucleotide sequence therein.
- E can be a 3'- or 5'-phosphate-linked ribose group containing one or more substituents such as an alkyl group of 1 to about 10 carbon atoms.
- E can be a 5'- or 3'-ether group such as an alkyl ether, an aryl ether, an aralkyl ether, a substituted aryl ether or an aralkyl ether wherein the alkyl groups contain from 1 to about 10 carbon atoms and the aryl groups contain from 6 to 10 carbon atoms, and wherein the alkyl or aryl groups may contain oxygen, nitrogen or sulfur atoms or be substituted by alkyl or aryl groups containing oxygen, nitrogen or sulfur atoms.
- E can be a 5'-O- or 3'-O- phosphate ester group such as an alkyl ester, an aryl ester, an aralkyl ester, a substituted.
- aryl ester or an aralkyl ester wherein the alkyl groups contain from 1 to about 10 carbon atoms and the aryl groups contain from 6 to 10 carbon atoms, and wherein the alkyl or aryl groups may contain oxygen, nitrogen or sulfur atoms or be substituted by alkyl or aryl groups containing oxygen, nitrogen or sulfur atoms.
- E can be a poly (alkylene oxidyl) group on the ribosyl moiety, preferably at the 5'- or 3'- position, either as an ether group or linked by a phosphate ester to the 5'- or 3'-oxygen of the L- ribosyl group.
- the poly(alkylene oxidyl) group can be, for example, a poly(ethylene oxidyl) or poly(propylene oxidyl) or a poly(propylene oxidyl-co-ethylene oxidyl) group, each polymer containing from 2 to about 100 repeating units.
- a phosphate ester comprising such entities is also useful, as well as a phosphate ester or modified ribose comprising elements of a suitable linking group as defined above.
- E can also comprise a Z or it can be attached to Q I by elements of L z as defined above to form a cyclic structure.
- E can also comprise compounds with a two carbon-one nitrogen atom internucleoside linkage.
- E comprises a poly (alkylene glycol) phosphate diester.
- the poly(alkylene glycol) moiety can have from 2 to about 100 repeating units.
- the poly(alkylene glycol) moiety can have from 2 to about 100 repeating units.
- poly(alkylene glycol) is a poly(ethyleene glycol).
- a currently preferred poly(alkylene glycol) phosphate diester is a tetra(ethylene glycol) phosphate diester, hereinafter sometimes referred to as a "Teg” or "Teg unit".
- Teg tetra(ethylene glycol) phosphate diester
- Such poly(alkylene glycol) phosphate diesters can be linked in tandem to each other to form a dimer phosphate ester sequence, a trimer sequence, a tetramer sequence, and so forth.
- One or two such units is preferred.
- Such units can also be attached to residues of Q I , L z , L Q , I, I i or Z described above.
- a preferred end group E comprises a Teg unit linked by a phosphate ester bond to an L-enantiomeric nucleotide such as T.
- Other preferred end capping units for an L-DNA sequence comprise a residue of L-dA, L-dG, L-T, L-dC, and L-dU or an oligonucleotide sequence comprised therefrom.
- modified nucleotide moiety is intended to mean a chemical entity which comprises one or more chemical groups that are analogous to one or more portions of a naturally occurring D-enantiomeric nucleotide or of a residue of a naturally occurring D-enantiomeric
- a "modified D-enantiomeric nucleotide moiety" comprises that chemical entity which exclusively remains when one or more chemical bonds, of which said naturally occurring D-enantiomeric nucleotide is
- modified D-enantiomeric nucleotide moieties comprise reactive functional groups, such as amine and sulfhydryl groups. They can be commercially available such as, for example, those modified D-enantiomeric nucleotide moieties and
- linking groups of this invention are derived from the reaction of a reactive functional group such as an amine or sulfhydryl group as are available in the above
- Clonetech reagents one or more of which has been incorporated into an L-enantiomeric oligonucleotide sequence of this invention, with, for example, one or more of the previously described protein reactive groups such as heterobifunctional protein reactive groups, one or more of which has been incorporated into an
- a "modified D-enantiomeric nucleotide moiety” can comprise a D-enantiomeric nucleotide moiety that is modified so as to reduce or prevent the action of exonuclease enzymatic activity on the D-enantiomeric oligonucleotide sequence.
- It can be a 3'- or 5'- phosphate linked ribose or a 3'- or 5'-phosphate linked 2'-deoxyribose group containing one or more substituents such as an alkyl group of 1 to about 10 carbon atoms, or an ether group such as alkyl or aryl or aralkyl or substituted aryl or aralkyl ether wherein the alkyl groups contain from 1 to about 10 carbon atoms and such alkyl or aryl groups may contain or be substituted by substituents containing oxygen, nitrogen or sulfur atoms, or a poly(alkylene oxidyl) group, preferably at the 5'- or 3'-ribose position, respectively, or
- a "modified D-enantiomeric nucleotide moiety" comprising a phosphate ester comprising said
- substituents is also useful, as well as a phosphate ester or modified ribose comprising elements of a suitable linking group as defined above.
- a "modified D- enantiomeric nucleotide moiety" can also comprise a residue of Z or it can be attached to Q I by elements of L z as defined above to form a cyclic structure.
- modified D-enantiomeric nucleotide moiety can also comprise compounds with a two carbon-one nitrogen atom internucleoside linkage.
- a "modified D-enantiomeric nucleotide moiety” comprises a poly(alkylene glycol) phosphate diester.
- the poly(alkylene glycol) moiety can have from 2 to about 100 repeating units.
- the poly(alkylene glycol) is a poly(ethylene glycol).
- a currently preferred poly(alkylene glycol) phosphate diester is a tetra(ethylene glycol) phosphate diester, hereinafter sometimes referred to as a "Teg" or "Teg unit”.
- Such poly(alkylene glycol) phosphate diesters can be linked in tandem to each other to form a dimer phosphate ester sequence, a trimer sequence, a tetramer sequence, and so forth.
- One or two such units is preferred.
- Such units can also be attached to residues of Q I , L z , L Q , I, I i or Z described herein.
- a preferred "modified D-enantiomeric nucleotide moiety" comprises a Teg unit linked by a phosphate ester bond to a D- enantiomeric nucleotide such as L-T.
- oligonucleotides, cl which are complementary to the members of the set of L-enantiomeric oligonucleotides comprising the "Sequence" of I in structure IV include the L-DNA L-enantiomeric oligonucleotides:
- An especially preferred complementary sequence comprises:
- the L-enantiomeric oligonucleotide sequence cl can comprise double stranded L-DNA or L-RNA. That is, the L-enantiomeric
- oligonucleotide sequence may comprise complementary L- DNA or L-RNA which forms a double helix molecule.
- immunoreagent interacts with the duplex L-DNA or L-RNA of cl in such a way as to form triplex (triple helix) L- DNA, triplex L-RNA, or a triplex L-DNA:L-RNA hybrid.
- Q cI in structure V is a spacing group; Q cl can be selected from Q I as described for structure IV.
- Q cl comprises an L-enantiomeric
- Q cI can also comprise an L- enantiomeric oligonucleotide, preferably a sequence such as (xxvii) to (xxxii) above which is complementary to the "Sequence" in structure IV.
- Q cI can also comprise an L- enantiomeric oligonucleotide, preferably a sequence such as (xxvii) to (xxxii) above which is complementary to the "Sequence" in structure IV.
- L 1 , L 2 , and L 3 in structure V are independently a chemical bond, preferably a phosphate ester bond, or a linking group which are defined as L z and L Q in the above structure V.
- L 1' L 2 , and L 3 can also be a chemical bond, preferably a phosphate ester bond, or a linking group which are defined as L z and L Q in the above structure V.
- L 1' L 2 , and L 3 can also
- W 1 , W 2 , and W 3 in structure V are residues of chelating groups.
- a chelating agent is a compound containing donor atoms that can combine by coordinate bonding with a metal atom to form a cyclic structure called a chelation complex or chelate. This class of compounds is described in the Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 5, 339-368.
- residues of suitable chelating agents can be independently selected from polyphosphates, such as sodium tripolyphosphate and hexametaphosphoric acid; aminocarboxylic acids, such as
- nitrilotriacetic acid N,N-di(2-hydroxyethyl)glycine, ethylenebis(hydroxyphhnylglycine) and diethylenetriamine pentacetic acid
- 1,3-diketones such as acetylacetone, trifluoroacetylacetone, and thenoyltrifluoroacetone
- hydroxycarboxylic acids such as tartaric acid, citric acid, gluconic acid, and 5-sulfosalicylic acid
- polyamines such as ethylenediamine, diethylenetriamine, triethylenetetramine, and triaminotriethylamine;
- aminoalcohols such as triethanolamine and N-(2- hydroxyethyl)ethylenediamine
- aromatic heterocyclic bases such as 2,2'-dipyridyl, 2,2'-diimidazole, dipicoline amine and 1,10-phenanthroline
- phenols such as oalicylaldehyde, disulfopyrocatechol, and
- chromotropic acid aminophenols, such as 8- hydroxyquinoline and oximesulfonic acid; oximes, such as dimethylglyoxime and salicylaldoxime; peptides
- proximal chelating functionality such as polycysteine, polyhistidine, polyaspartic acid
- polyglutamic acid or combinations of such amino acids; Schiff bases, such as disalicylaldehyde 1,2- propylenediimine; tetrapyrroles, such as
- macrocylic compounds such as dibenzo[18]crown-6,
- Preferred residues of chelating agents contain polycarboxylic acid groups and include: ethylenediamine- N, NN, N',N'-tetraacetic acid (EDTA); N,N,N',N",N"- diethylene-triaminepentaacetic acid (DTPA); 1,4,7,10- tetraazacyclododecane-N,N',N",N"'-tetraacetic acid
- DOTA 1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid
- DO3A 1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid
- OTTA 1-oxa-4,7,10-triazacyclododecane-N,N',N"- triacetic acid
- CDTPA trans(1,2)- cyclohexanodiethylenetriamine pentaacetic acid
- Preferred residues of chelating agents contain polycarboxylic acid groups and include the following:
- Suitable residues of chelating agents comprise proteins modified for the chelation of metals such as technetium and rhenium as described in U.S. Patent No. 5,078,985, the disclosure of which is hereby incorporated by reference.
- suitable residues of chelating agents are derived from N 3 S and N 2 S 2 containing
- a residue of each of the chelating agents W 1 , W 2 , and W 3 in structure V is independently linked to the complementary L-enantiomeric oligonucleotide moiety cl or spacing group Q cI through a chemical bond or a linking group, i.e., L 1 , L 2 and L 3 in structure V, above.
- Preferred linking groups include nitrogen atoms in groups such as amino, imido, nitrilo and imino groups; alkylene, preferably containing from 1 to 18 carbon atoms such as methylene, ethylene, propylene, butylene and hexylene, such alkylene optionally being interrupted by 1 or more heteroatoms such as oxygen, nitrogen and sulfur or heteroatom-containing groups;
- ester i.e., carbonyloxy and oxycarbonyl
- thioester i.e., carbonylthio, thiocarbonyl
- thioamide i.e., iminothiocarbonyl
- urethane i.e., iminocarbonyloxy, and oxycarbonylimino
- thiourethane i.e., iminothiocarbonyloxy
- an amino acid linkage i.e., a
- k l and X 1 , X 2 , X 3 independently are H, alkyl, containing from 1 to 18, preferably 1 to 6 carbon atoms, such as methyl, ethyl and propyl, such alkyl optionally being interrupted by 1 or more heteroatoms such as oxygen, nitrogen and sulfur, substituted or unsubstituted aryl, containing from 6 to 18, preferably 6 to 10 carbon atoms such as phenyl, hydroxyiodophenyl, hydroxyphenyl, fluorophenyl and naphthyl, aralkyl, preferably containing from 7 to 12 carbon atoms, such as benzyl, heterocyclyl, preferably containing from 5 to 7 nuclear carbon and one or more heteroatoms such as S, N, P or O, examples of preferred heterocyclyl groups being pyridyl, quinolyl, imidazolyl and thienyl;
- heterocyclylalkyl the heterocyclyl and alkyl portions of which preferably are described above;
- linking groups can be used, such as, for example, alkyleneimino and
- linking groups may be suitable for use herein, such as linking groups commonly used in protein heterobifunctional and homobifunctional conjugation and crosslinking chemistry as described for L z or L Q in structure IV.
- Especially preferred linking groups include unsubstituted or substituted phosphate ester groups containing amino groups which when linked to the residue of a chelating agent via an isothiocyanate group on the chelating agent form a thiourea group.
- the linking groups can contain various substituents which do not interfere with the coupling reaction between chelate W 1 , W 2 , or W 3 and L-enantiomeric oligonucleotide cl or the spacing group.
- the linking groups can also contain substituents which can otherwise interfere with such reaction, but which during the coupling reaction, are prevented from so doing with suitable protecting groups commonly known in the art and which substituents are regenerated after the coupling reaction by suitable deprotection.
- the linking groups can also contain substituents that are introduced after the coupling reaction.
- the linking group can be substituted with a group such as a halogen, such as F, Cl, Br or I; an ester group; an amide group;
- alkyl preferably containing from 1 to about 18, more preferably, 1 to 4 carbon atoms such as methyl, ethyl, propyl, i-propyl, butyl, and the like; substituted or unsubstituted aryl, preferably containing from 6 to about 20, more preferably 6 to 10 carbon atoms such as phenyl, naphthyl, hydroxyphenyl, iodophenyl,
- alkoxy the alkyl portion of which preferably contains from 1 to about 18 carbon atoms as described for alkyl above; alkoxyaralkyl, such as ethoxybenzyl; substituted or unsubstituted
- heterocyclyl preferably containing from 5 to 7 nuclear carbon and heteroatoms such as S, N, P or O, examples of preferred heterocyclyl groups being pyridyl, quinolyl, imidazolyl and thienyl; a carboxyl group; a carboxyalkyl group, the alkyl portion of which preferably contains from 1 to 8 carbon atoms; or the residue of a chelating group.
- M 1 , M 2 and M 3 each comprise elements with oxidation states equal to or greater than +1, and at least one of which is a radionuclide.
- each of M 1 , M 2 and M 3 comprise a metal isotope, preferably a radioactive metal isotope, sometimes herein referred to as a metal radioisotope, which radioisotope is useful in a therapeutic or in a diagnostic imaging application.
- Preferred metal radioisotopes are selected from, for example, Sc, Fe, Pb, Ga, Y, Bi, Mn, Cu, Cr, Zn, Ge, Mo, Tc, Ru, In, Sn, Re, Sr, Sm, Lu, Eu, Ru, Dy, Sb, W, Re, Po, Ta and TI.
- Useful emissions from such radioisotopes include spontaneous alpha emissions, beta emissions, gamma emissions.
- X-ray emissions, positron emissions, and such emissions as are induced by the processes of electron capture and internal conversion Said emissions can be purely of one kind such as pure alpha, pure beta, pure gamma and the like, or of combinations of nuclear emissions such as beta and gamma emissions and the like.
- Radioisotopes with emissions comprising, for example, alpha radiation or beta radiation are useful in therapeutic applications, especially in the therapy of cancer.
- Useful isotopes in therapeutic applications include, for example, alpha radiation emitting isotopes such as, for example, 207 Pb, 211 Pb, 208 Pb, 212 Pb, 212 Bi, 207 Ti, and 223 Ra; beta radiation emitting isotopes such as, for example 47 Sc, 66 Ga, 67 Cu, 77 As, 90 Y, 105 Rh,
- Radioisotopes especially preferred in therapeutic applications include 212 Pb, 212 Bi, 90 ⁇ , 1 77 Lu, 186 Re, and 188 Re. Currently the most preferred radioisotope is 90 Y.
- Radioisotopes with emissions comprising, for example, gamma radiation or positron radiation are useful in diagnostic imaging applications, especially in diagnostic imaging of cancer.
- Useful isotopes in diagnostic imaging applications include, for example, gamma radiation emitting isotopes such as 47 Sc, 51 Cr, 6 7 Cu, 67 Ga, 97 Ru, 99m Tc, 111 In, 117m Sn, 141 Ce, 167 Tm, 199 Au, 87 Y and 203 Pb; and positron radiation emitting isotopes such as 44 Sc, 48 V, 64 Cu, 66 Ga, 69 Ge, 72 As, 86 Y and 89 Zr.
- Radioisotopes especially preferred in diagnostic imaging applications include 64 Cu, 99m Tc, 111 In and 87 Y. Currently, the most preferred are 99m Tc and 111 In.
- radionuclides can be incorporated, for example, by covalent bonding into
- QcI and include radioactive isotopes of halogens such as radioactive isotopes of iodine, for example, 123 I, 124 l, 125 I and 131 I as well as radioactive isotopes of astatine such as 211 At.
- radioactive isotopes of halogens such as radioactive isotopes of iodine, for example, 123 I, 124 l, 125 I and 131 I as well as radioactive isotopes of astatine such as 211 At.
- Methods of generating an image useful in the diagnostic imaging of, for example, cancer in a mammal comprise detecting emissions imagewise from
- radioisotopes as employed in the compositions and methods of this invention.
- Said image generating methods comprise the use of, for example, a collimated camera detector such as a gamma camera commonly employed in radioimmunoscintigraphy (RIS), and the use of linked X-ray detectors commonly employed in positron emission tomography (PET) and in single photon emission
- a collimated camera detector such as a gamma camera commonly employed in radioimmunoscintigraphy (RIS)
- RIS radioimmunoscintigraphy
- PET positron emission tomography
- SPET tomography
- x, y, and z are independently zero or 1 provided that at least one of x, y, or z is one; and w and b are zero or an integer from 1 to about 4.
- compositions can be prepared as outlined in the schemes that follow.
- the protein (antibody such as TNG-1, antibody fragment, enzyme, receptor) is chemically modified for later covalent coupling to a thiolated L- enantiomeric oligonucleotide (to Ab-M) or to a
- oligonucleotide to Ab-SH.
- Chemical modification is effected using a bifunctional cross linking agent, preferably a heterobifunctional cross linking agent having both a group capable of reacting with protein functional groups (e.g. amine in Ab-amine) and also having a further group capable of reacting with thiol groups.
- the latter is selected from haloacetyl, halo- acetamidyl, maleimido, and activated disulfide
- Maleimido and thioalkyl groups are introduced to an antibody by utilizing the heterobifunctional linkers, sulfosuccinimido-4-(M-maleimidomethyl)-cyclohexane-1- carboxylate (Sulfo-SMCC), 2-iminothiolane (2-IT), or succinimidyl-S-acetylthioacetate (SATA).
- the reaction of a sample containing antibody with a linking agent is for a time sufficient to introduce an average of about 0.5-3 linking agent molecules per antibody molecule in the sample.
- the derivatized antibody is purified using a gel filtration column, and more preferably a Sephadex G- 25 column.
- ING-1 is a preferred protein containing amine groups such as lysine amines as represented by Ab-amine.
- TrS-5'-L-d(l-Q I -l)-3'-Biotin TrS-5'-L-d(Oligo)-3'-Biotin
- L-enantiomeric oligonucleotides and modified L- enantiomeric oligonucleotides are synthesized according to standard methods such as solid phase synthesis that are well known in the art for the synthesis of D- enantiomeric oligonucleotides.
- Derivatizations of L- enantiomeric oligonucleotides L-d(I-Q I -I) are achieved using the reaction of 5'-TEG-L-enantiomeric oligomer- NH 2 -3' with SATA or SMCC to afford L-enantiomeric oligomer-3'-SH or L-enantiomeric oligomer-3'-M (see Scheme 7).
- L-enantiomeric oligonucleotides are preferred:
- SEQ ID No: 31 denotes the preferred spacer, -L-d[ACTCTC]-, which separates two sequences each containing the preferred sequence -L-d(TTATGGACGGAGAAGCTAA)- (SEQ ID No: 8).
- L-enantiomeric oligomers are also derivatized to afford a bifunctionalized L-enantiomeric oligomer: 5'-HS-L-d(I-Q I -I)-NH 2 -3' via introduction of
- the modified maleimido antibody (Ab-M, Scheme 6) and the thiolated L-enantiomeric oligonucleotide (5'-HS- L-d(I-Q I -I)-NH 2 -3') can be assembled to yield the modified antibody-L-enantiomeric oligonucleotide conjugate (Ab-M-5 -S-succinimido-L-d (I-Q I -I) -NH 2 -3 ' ) as shown in Scheme 11 .
- Ab-S-M-3 ' -L-d ( I-Q I -I) - TEG-5 ' is prepared from Ab-SH and 5 ' -TEG-L-d ( I-Q I -I) - maleimide-3 ' as shown in Scheme 13.
- L-d(oligo) represents an L- enantiomeric oligodeoxyribonucieotide such as an L- deoxyribonucleotide, L-d(I); d(cl) represents the complementary L-enantiomeric oligodeoxyribonucieotide sequence; L-d(I-Q I -I) is an L-enantiomeric
- TMT represents a member of a class of terpyridine chelates, preferably as described above.
- a preferred TMT is 4'-(3-isothiocyanato-4- methoxyphenyl)-6,6"-bis[N,N-di(carboxymethyl)- aminomethxl]-2,2':6',2"-terpyridine, TMT-NCS.
- an effective dose of a radioactive targeting reagent as described above in a pharmaceutically acceptable medium is prepared by exposing a composition comprising a complementary L- enantiomeric oligonucleotide sequence containing one or more chelating groups such as the
- oligodeoxyribonucleotide sequence as described above to a composition containing a radioactive metal isotope such that the molar amount of the radionuclide metal isotope is less than the molar amount of the chelating groups.
- the exposure lasts an effective time during which uptake of of the radionuclide metal isotope into the chelating agents occurs.
- an effective dose of a non-radioactive targeting immunoreagent as described above in a pharmaceutically acceptable medium is
- the non-radioactive targeting immunoreagent is allowed to accumulate at the target site such as at a tumor site in the patient.
- the radioactive targeting reagent is allowed to accumulate at the target site, said target site being the non-radioactive targeting immunoreagent which has accumulated at the tumor site in the patient.
- the present invention also comprises one or more of the immunoreagents of this invention formulated into compositions together with one or more non-toxic
- physiologically acceptable carriers, adjuvants or vehicles which are collectively referred to herein as carriers, for parenteral injection for oral administration in solid or liquid form, for rectal or topical administration, or the like.
- compositions can be administered to humans and animals either orally, rectally, parenterally
- compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into sterile injectable solutions or dispersions.
- suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propylene glycol, polyethylene glycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
- Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
- compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
- adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
- Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
- isotonic agents for example sugars, sodium chloride and the like.
- Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
- Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
- the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or
- fillers or extenders as for example, starches, lactose, sucrose, glucose, mannitol and silicic acid
- binders as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose and acacia
- humectants as for example, glycerol
- disintegrating agents as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates and sodium carbonate
- solution retarders as for example paraffin
- absorption accelerators as for example, quaternary ammonium compounds
- wetting agents as for example
- lubricants as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate or mixtures thereof.
- the dosage forms may also comprise buffering agents.
- compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols, and the like.
- Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and others well known in the art. They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner.
- embedding compositions which can be used are polymeric substances and waxes.
- the active compounds can also be in micro- encapsulated form, if appropriate, with one or more of the above-mentioned excipients.
- Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
- the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan or mixtures of these substances, and the like.
- inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and e
- composition can also include adjuvants, such as wetting agents,
- Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols,
- microcrystalline cellulose aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
- compositions for rectal administrations are provided.
- suppositories which can be prepared by mixing the compounds of the present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
- suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and, therefore, melt in the rectum or vaginal cavity and release the active component.
- Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays and inhalants.
- the active component is admixed under sterile conditions with a physiologically
- compositions of the present invention may be varied so as to obtain an amount of active ingredient that is effective to obtain a desired therapeutic response for a particular composition and method of administration.
- the selected dosage level therefore depends upon the desired therapeutic effect, on the route of
- the total daily therapeutic dose of the compounds of this invention administered to a host in a single or divided dose may be in amounts, for example, of from about 100 picomol to about 5 micromols per kilogram of body weight. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the daily dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
- the present invention is directed to a method of diagnosis comprising the
- a method for diagnostic imaging for use in medical procedures in accordance with this invention comprises administering to the body of a test subject in need of a diagnostic image an effective diagnostic image producing amount of the above-described compositions.
- an effective diagnostic image producing amount of a non-radioactive targeting immunoreagent as described above in a pharmaceutically acceptable medium is administered to a patient and said non-radioactive targeting immunoreagent is allowed to accumulate at the target site such as at a tumor site in said patient.
- a diagnostic imaging effective dose of a radioactive targeting reagent as described above in a pharmaceutically acceptable medium is administered to said patient, and said radioactive targeting reagent is allowed to accumulate at the target site, said target site being the said non-radioactive targeting immunoreagent accumulated at said tumor site in said patient.
- the image pattern can then be
- the total diagnostic imaging effective dose of the compounds of this invention administered to a host in a single or divided dose may be in amounts, for example, of from about 1 picomol to about 0.5 micromols per kilogram of body weight. Dosage unit compositions may contain such amounts of such submultiples thereof as may be used to make up the effective diagnosting imaging dose. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the body weight, general health, sex, diet, time and route of administration, rates of absorption and excretion, combination with other drugs and the severity of the particular disease being treated.
- test subjects can include mammalian species such as rabbits, dogs. cats, monkeys, sheep, pigs, horses, bovine animals and the like.
- compositions of the present invention After administration of the compositions of the present invention, the subject mammal is maintained for an effective time which is a time period sufficient for the administered compositions to be distributed
- a sufficient time period for the non- radioactive targeting immunoreagent is generally from about 1 hour to about 2 weeks or more and, preferably from about 2 hours to about 1 week.
- a sufficient time period for the radioactive targeting reagent such as the preferred 90 Y is generally measured in terms of half- life of the radionuclide and as such is in the range of from about 1 to about 10 half-lives or more and, preferably from about 2 hours to about 6 half-lives.
- a targeting immunoreagent of this invention as described in Structure IV comprising an antibody Z, linking groups L z and L Q , an L-enantiomeric
- oligonucleotide sequence I is designed as follows utilizing anticipated complementary binding properties between complementary pairs of mirror image D-enantiomeric oligonucleotides as a model for analogous physical properties imputed to the desired L- enantiomeric oligonucleotide.
- a sample mirror image, naturally occurring D- enantiomeric oligodeoxyribonucleotide with the following sequence (herein referred to as SEQ ID NO: 32) is analyzed for conformity to the criteria described previously for Structure IV wherein groups L z , I, Q I , I i , and L Q and E are as represented below as D- enantiomers:
- oligonucleotide are then made in accordance with the criteria outlined for structure IV to replace selected bases in regions of self-complementarity such that excessive base pairing is removed in iterative analysis. Additional D-enantiomeric and additional L-enantiomeric nucleotides are also inserted into the respective spacer sequences to ensure that, in helical conformations, the respective terminal groups of two respective
- D-cI and L-cI when hybridized to the two D-enantiomer sequences I and II and to the two L-enantiomer sequences, respectively are orthogonal to each other.
- oligonucleotide (SEQ ID NO: 33) with the following sequence is designed.
- the mirror image L-enantiomer (SEQ ID NO: 34) is synthesized and the L-enantiomer is conjugated to an immunoreactive molecule (i.e., to an antibody such as ING-1):
- the groups L z and L QE at the 5' or the 3' end comprise groups X and Y.
- X and Y can each independently be an amine-containing group (such as, for example, those available from Clontech Industries, each of which amine- containing groups being sometimes hereinafter
- NH 2 cryptically referred to as "NH 2 " or as "amine”
- one of X and Y is selected from an amine-containing group and the other is selected from one or more TEG groups as described above.
- one of X and Y can also be a terminal hydroxyl group (in which case the 3'-YT in the above sequence becomes a deoxyribosyl-OH group on removal from the solid phase support, and more
- X is a 5'OH group available by removal of a DMT group with acid at the end of the synthetic sequence before deprotecting the amine groups with ammonium hydroxide), or one of X and Y can also be a terminal phosphate ester group (in which case the 3'-YT or the
- 5'-X becomes a -OPO 3 H 2 group or an ionized salt such as a sodium salt thereof), as desired.
- phosphate terminal groups can be made by treatment of the 5'-OH terminal group with 2-cyanoethyl N,N-diisopropyl chlorophosphoramidite at the end of the synthesis.
- a 3'-phosphate group can be introduced after synthesis of the oligomer and its removal from the solid support by treatment of the 3-hydroxyl group with phosphoric acid anhydide followed by hydrolysis in water.
- this L-enantiomeric oligonucleotide cryptically referred to as 5'-Teg-L- d(I-Q I -I)-3'-amine, is:
- oligonucleotide cryptically referred to as 5'-amine-L- d( I-Q I -I )-3'Teg, is:
- oligonucleotide cryptically referred to as 5'-amine-L- d( I-Q I -I )-3'OH, is:
- oligonucleotide cryptically referred to as 5'-amine-L- d( I-Q I -I )-3'OPO 3 H 2 , is:
- This L-enantiomeric oligonucleotide is prepared on an Applied Biosystems oligonucleotide synthesizer originally designed by the manufacturer to be used for the synthesis of naturally occurring D-enantiomeric oligonucleotides.
- the method applicable to the tritylon protocol for the synthesis of naturally occurring D- enantiomeric oligonucleotides is used as directed by the equipment manufacturer but is modified to instead use L- 2-deoxynucleotide phosphoramidite reagent precursors 5'- dimethoxytrityl L-cytidine-3'-O-phosp oramidite, 5'- dimethoxytrityl L-adenosine-3'-O-phosphoramidite, 5'- dimethoxytrityl L-guanosine-3'-O-phosp'oramidite, and 5'-dimethoxytrityl L-thymidine-3'-O-phosphoramidite prepared as described above rather than the D- enantiomers.
- L- 2-deoxynucleotide phosphoramidite reagent precursors 5'- dimethoxytrityl L-cytidine-3'-O-phosp oramidite, 5'- dimethoxy
- TEG group a tetra(ethylene glycol) phosphate diester linked in this invention by a phosphate ester bond to 5'- dimethoxytrityl L-thymidine-3'-O-phosphoramidite [which is the L-enantiomer (mirror image) of the reagent disclosed in WO/92/02534 which refers to a
- oligonucleotide is desalted and further purified by elution down an OPEC Cartridge (Clonetech) with
- polyacrylamide gel is used to further purify the L- enantiomeric oligonucleotide.
- the L-DNA band is
- oligonucleotide is estimated using absorbance at 260 nm.
- This L-enantiomeric oligonucleotide is prepared on an Applied Biosystems oligonucleotide synthesizer by the trityl-off protocol otherwise used for D-enantiomers as directed by the equipment manufacturer but modified to use L-2-deoxynucleotide phosphoramidite reagent
- the L-enantiomeric oligonucleotide sequence of Example 2a minus the Teg group is prepared on an Applied Biosystems oligonucleotide synthesizer by the trityl-on protocol as directed by the equipment manufacturer using L-2-deoxynucleotide phosphoramidite reagent precursors (5'-dimethoxytrityl L-cytidine-3'-O-phosphoramidite, 5'- dimethoxytrityl L-adenosine-3'-O-phosphoramidite, 5'- dimethoxytrityl L-guanosine-3'-O-phosphoramidite, and 5'-dimethoxytrityl L-thymidine-3'-O-phosphoramidite as described above). Clonetech's Uni-link Amino Modifier is used as the precursor to the 3'-amine group.
- Clonetech's C6-ThioModifier is used as the precursor to the 5'-thiol group (which is herein referred to
- Example 2a TEG in Example 2a. Following synthesis of the whole L- enantiomeric oligonucleotide, the base protecting groups and solid support are removed with ammonium hydroxide. The L-enantiomeric oligonucleotide is desalted and further purified by elution down an OPEC Cartridge
- L-enantiomeric oligonucleotide (Clonetech) with deionized water.
- concentration of L-enantiomeric oligonucleotide is estimated using absorbance at 260 nm.
- L-d(cI) containing the L-enantiomeric nucleotide sequence 5'X-L- d(TTAGCTTCTCCGTCCATAAYT)-3' (SEQ ID NO: 38) complementary to the L-enantiomeric oligonucleotide I is prepared on an Applied Biosystems oligonucleotide synthesizer as outlined in Example 2a.
- the 3'- (Y) and 5'- (X) amine- containing groups are incorporated as directed by the equipment manufacturer using Uni-link Amino Modifier (Clonetech) for the precursor to the 3'-amine group, and Clonetech's 6 carbon monomethoxytrityl AminoModifier (N- MMT-C6-AminoModifier: Catalog # 5202) as precursor to the 5'-amine group.
- the protecting groups are removed with ammonium hydroxide, and the amine- functionalized L-enantiomeric oligonucleotide is
- the L-enantiomeric oligonucleotide is further purified by polyacrylamide gel electrophoresis or reverse-phase HPLC.
- concentration of L-enantiomeric oligonucleotide is estimated using absorbance at 260 nm.
- L-enantiomeric oligonucleotide, I containing the L-enantiomeric nucleotide sequence 5'X-L- d(TTATGGACGGAGAAGCTAAYT)-3' (SEQ ID NO: 39) is prepared on an Applied Biosystems oligonucleotide synthesizer as outlined in Example 3a.
- the 3'- (Y) and 5'- (X) amine- containing groups are incorporated as directed by the equipment manufacturer using Uni-link Amino Modifier (Clonetech) for the precursor to the 3'-amine group, and Clonetech's 6 carbon monomethoxytrityl AminoModifier (N- MMT-C6-AminoModifier: Catalog # 5202) as the precursor to the 5'-amine group.
- the protecting groups are removed with ammonium hydroxide, and the amine- functionalized L-enantiomeric oligonucleotide is purified by elution down an OPEC Cartridge (Clonetech) with deionized water.
- oligonucleotide is further purified by polyacrylamide gel electrophoresis or reverse-phase HPLC.
- concentration of L-enantiomeric oligonucleotide is estimated using absorbance at 260 nm.
- oligonucleotide synthesizer as outlined in Example 2a. After final deblocking and cleavage from the solid support, the protecting groups are removed with ammonium hydroxide. The L-enantiomeric oligonucleotide is purified by elution down an OPEC Cartridge (Clonetech) with deionized water. The L-enantiomeric
- oligonucleotide is further purified by polyacrylamide gel electrophoresis.
- concentration of L- enantiomeric oligonucleotide is estimated using
- L-enantiomeric oligonucleotide, I containing the L-enantiomeric nucleotide sequence 5'-L- d(TTATGGACGGAGAAGCTAA) -3 ' (SEQ ID NO: 8) is prepared on an Applied Biosystems oligonucleotide synthesizer as outlined in Example 3c. After final deblocking and cleavage from the solid support, the protecting groups are removed with ammonium hydroxide, and the L- enantiomeric oligonucleotide is purified by elution down an OPEC Cartridge (Clonetech) with deionized water. The L-enantiomeric oligonucleotide is further purified by polyacrylamide gel electrophoresis. The concentration of L-enantiomeric oligonucleotide is estimated using absorbance at 260 nm.
- L-d(cI) concentrations below equimolar with respect to 5*-Teg-L-d( I-Q I -I )-3 , -NH 2 .
- concentrations of L-d(cI) below equimolar with respect to 5*-Teg-L-d( I-Q I -I )-3 , -NH 2 .
- a ternary complex L-d(cI):5'-Teg-L-d(I-Q I - I)-3'-NH 2 :L-d(cI)
- a suspension of about 40 mmoles of TMT amine (PCT US91/08253) in 650 mL methanol is stirred at room temperature and deionized water is added dropwise (about 70 mL) until a clear pale yellow solution develops.
- the solution is cooled in an ice bath to 10°C and about 60 mmoles 35 S-thiophosgene is added dropwise over about 3 minutes.
- a precipate of TMT isothiocyanate forms and the solution is stirred continuously for a further 2.5 hours.
- the solution and precipitate are concentrated to near dryness on a rotovap under reduced pressure ( ⁇ 15mm Hg) at room temperature.
- TMT-NCS TMT isothiocyanate
- oligonucleotides either 5'-H 2 N-L-d(cI)-3'-NH 2 solution of Example 3a or 5'-H 2 N-L-d(I)-3'-NH 2 solution of
- Example 3b in 500 microL of 1.0 M carbonate/bicarbonate buffer at pH 9.0 is added 12 mg of TMT isothiocyanate (PCT US91/08253). The reaction mixture is vortex mixed and kept at 37°C for 2 hours and at room temperature for overnight. The resulting reaction mixture is quenched with ethanolamine (15 microM) and the product is purified by Sephadex G-25 column chromatography using deionized water as the eluting solvent.
- PCT US91/08253 TMT isothiocyanate
- TMT's per molecule of L-enantiomeric oligonucleotide diamine is quantified by an asssay using the time resolved fluorescence of Europium metal chelated to the TMT.
- 6b General procedure for labelling of Ing-1-TMT, 5'- TMT-L-d(cI)-3'-TMT, and 5'-TMT-L-d(I)-3'-TMT conjugates with fluorescent metals.
- Binding of lanthanides such as europium (3 + ) to chelating agents that contain an aromatic moiety held close to the co-ordination sphere can lead to
- the metal then produces emissions characterized by a very large Stokes shift and fluorescence lifetimes of up to several seconds.
- the fluorescence at 615 nm is measured at a time-delay of 400 microseconds after an excitation pulse at 340 nm. This time delay is useful for high sensitivity
- a known amount of Ing-1-TMT, 5'- TMT-L-d(cI)-3'-TMT, or 5'-TMT-L-d(I)-3'-TMT conjugate is titrated with increasing amounts of added EUCl 3 in an aqueous buffer.
- one microliter of a solution containing 1-30 picomoles of the conjugate is added, in duplicate, to wells in a Costar EIA/RIA 96-well plate containing a precalculated amount of Tris.HCl buffer (pH 7.4).
- the buffer volume is derived by subtracting from 99 the volume in microliters of aqueous EUCl 3 (typically 10 -4 M to 10 -6 M in Tris.HCl buffer). The total volume in each well is thereby fixed at 100 microliters.
- Aqueous EUCl 3 is then added to the buffered solution of the conjugate.
- the plate is then covered and shaken at low speed for one hour.
- the time resolved fluorescence is then measured using a Delfia 1232 time-resolved fluorometer (Wallac Inc.) and the data are analyzed. It is found that each conjugated TMT molecule chelates one Europium ion and that both 5'-TMT-L-d(cl)-3'-TMT and 5'- TMT-L-d(I)-3'-TMT conjugates bind two Europium ions per molecule of conjugate.
- TMT-NCS 35 S-labeled TMT-NCS (prepared as in Example 5) is substituted for the 12 mg of TMT isothiocyanate (PCT US91/08253) in the method of Example 6a and the reaction carried out as described above.
- the number of TMT's per molecule of 5'-H2N-L-d(cI)-3'-NH 2 is quantified by counting the TMT-L-d(cl)-TMT product in a liquid
- a solution of the L-enantiomeric oligonucleotide TMT conjugates, either (TMT-L-d(cl)-TMT) from Example 6a or TMT-L-d(I)-TMT from Example 6a, in deionized water at room temperature is treated with a solution of 90 YCl 3 (>500 Ci/mg; from Amersham Corp.) in 0.5 M sodium acetate buffer at pH 6.0 to a specific activity of 0.1 Ci/pmole for one hour at room temperature.
- the labeling efficiency is determined by removing 1.0 microliter of the sample and spotting it on to a Gelman ITLC-SG strip. The strip is developed in a glass beaker
- Example 7(a) is mixed with increasing amounts (0.375 to 12 pmoles) of 5'-Teg-L-d(I-Q I -I)-3'-NH 2 from Example 2a in PBS at 37°C for one hour. A 5 mL aliquot from each of these hybridizations is removed, mixed with SDS- containing buffer, and run on a 12% PAGE gel.
- TMT-L-d(cl)-TMT labeled with 90 Y to a specific activity of 28 mCi/28 mg is injected into a 25 g nude mouse bearing a subcutaneous tumor in its right flank.
- blood samples are taken from the tail and counted for 90 Y radioactivity in a liquid scintillation counter.
- the results reveal that more than 95% of the injected dose of 90 Y is removed from the blood stream in the first 30 minutes following injection. After 2 hours following injection the radioactivity in the blood levels off and a minute fraction ( ⁇ 0.01% of injected dose ) continues to circulate during the next 22 hours.
- a Sulfo-SMCC solution (108 nmoles) in phosphate buffered saline (PBS) is added to a sample of a chimeric antibody (ING-1; 18 nmoles) solution in phosphate buffer (pH 7).
- PBS phosphate buffered saline
- ING-1 18 nmoles
- the resulting mixture is allowed to stand for 30 minutes with occasional mixing at room temperature.
- the reaction is stopped with 60 nmoles basic tris buffer.
- the reaction mixture is diluted with phosphate buffered saline, added to a prewashed PD-10 column, and eluted with PBS to afford ING-1-maleimide. This material is stored on ice until use.
- a sample of a chimeric antibody (ING-1; 5 nmoles) solution in 0.1 M carbonate buffer (pH 8.8) is mixed with 200 nmoles of an aqueous solution of 2- iminothiolane. The resulting mixture is allowed to stand for 30 min with occasional mixing at room
- reaction mixture is diluted with phosphate buffed saline, added to a prewashed PD-10 column (Pharmacia), and eluted with PBS to afford mercaptoalkyl-ING-1. This material is stored on ice until use.
- a sample of a solution of 5'-Teg-L-d(I-Q I -I)-3'-NH 2 (30 nmoles) in water is mixed with 1 M carbonate buffer (pH 9) to give a final buffer concentration of 890 mM.
- 1 M carbonate buffer pH 9
- Into the buffered L-DNA is added 12 mmoles of an aqueous solution of 2-iminothiolane hydrochloride. These reactants are vortex mixed and kept at 37°C for 30 minutes.
- hydrochloride Cl- For use in conjugation to a
- 5'-H 2 N-L-d( I-Q I -I )-3'-Teg is reacted with sulfo- SMCC in the same manner as 5'-Teg-L-d(I-Q I -I)-3'-NH 2 in 9c to afford 5'-Maleimide-L-d( I-Q I -I )-3'Teg.
- the sulfhydryl group of the acetylthioacetylated L-enantiomeric oligonucleotide is deacylated by the addition of 30 mL of a pH 7.5 solution containing 100 mM sodium phosphate, 25 mM EDTA, and 500 mM NH 2 OH. The reaction is allowed to proceed for two hours at room temperature after which time the material is passed down a NAP-5 column using PBS for the elution. The product, 5'-Teg-L-d(I-Q I -I)-3'NH-CO-CH 2 - SH, is used immediately to obviate oxidative
- the sample containing the product is resuspended in fresh PBS, and ultrafiltration, concentration by centrifugation and resuspension are repeated a further 3 times until the ratio of optical densities at 260 nm and 280 nm is constant.
- L-d(I-Q l - I) the number of L-enantiomeric oligonucleotide molecules, L-d(I-Q l - I), is estimated to be between 1 and 2 L-d( I-Q I -I ) per antibody in the sample.
- concentration of ING-1 in a conjugate solution is determined by the BioRad protein assay using bovine immunoglobulin as the protein standard. These data agreed well with the antibody concentrations determined by examination of the optical density of the conjugate at 280 nm once it has been corrected for absorbance due to the conjugated L-d( I-Q I -I ) . Both these sets of data are further confirmed by subjecting the antibody-L-d(I- Q l -I) conjugates to acid digestion and amino acid analysis.
- Antibody-L-d( I-Q I -I ) conjugates are examined for their ability to bind to antigens on the surface of a human tumor cell line to which the antibody is raised.
- the immunoreactivity of the conjugates is compared by flow cytometry with a standard preparation of the antibody before being subjected to modification and conjugation to L-d( I-Q I -I ).
- Target HT29 cells a human adenocarcinoma cell line: ATTC
- the cells After extensive washing to remove unbound antibody, the cells are resuspended in 100 mL flow buffer and incubated at 4°C for 1 hour with goat-anti-human antibody labeled with fluorescene isothiocyanate (FITC). After further washing in flow buffer the samples are analyzed by flow cytometry on a Coulter EPICS 753 flow cytometer.
- FITC fluorescene isothiocyanate
- Fluorescene from Fluorescene isothiocyanite (FITC) and propidium iodide (PI) is excited using the 488 nm emission line of an argon laser. The output is set at 500 mW in light regulation mode. Single cells are identified by 90 degree and forward angle light scatter. Analysis windows are applied to these parameters to separate single cells from aggregates and cell debris. Fluorescence from FITC and propidium are separated with a 550 nm long pass dichroic filter and collected through a 530 nm band pass filter (for FITC), and a 635 nm band pass filter (for PI). Light scatter parameters are collected as integrated pulses and fluorescence is collected as log integrated pulses.
- FITC Fluorescene isothiocyanite
- PI propidium iodide
- Dead cells are excluded from the assay by placing an analysis window on cells negative for PI uptake.
- the mean fluorescence per sample (weighted average from 2500 cells) is calculated for each histogram.
- FITC calibration beads are analysed in each experiment to establish a fluorescence standard curve.
- the average fluorescence intensity for each sample is then expressed as the average FITC equivalents per cell.
- Immunoreactivity is calculated by comparing the average fluorescence intensity of the unknown sample with values from the standard curve. From the
- the SDS PAGE gels of ING-1-L-d( I-Q I -I ) and ING-1 antibody demonstrate that the molecular weight of the ING-1-L-d( I-Q I -I ) conjugates are higher than that of the antibody ING-1 alone.
- the sample is resuspended in fresh PBS and this sequence of concentration by centrifugation is repeated a further 3 times until the ratio of optical densities at 260 nm and 280 nm is constant.
- a 6 nmole sample of ING-1-Maleimide (Ab-M from Example 8a) in PBS is reacted with 40 nmoles of Teg-5'- L-d( I-Q I -I )-3'-NH-CO-CH 2 -SH (from Example 9e) at 4°C for 16 hours.
- the product is concentrated in a Centricon-300 ® device by
- oligonucleotide molecules per antibody is estimated.
- a sample (10 nmoles) of 5'-Trityl(S)-L-d( I-Q I -I )- 3'-NH 2 from Example 2c is diluted into PBS and a
- a 6 nmole sample of ING-1-Maleimide (from Example 8a) in PBS is reacted with 40 nmoles of HS-5'-L-d(I-Q ⁇ - I)-3'-Teg at 4°C for 16 hours.
- the reactants are diluted with PBS and eluted in 4 mL from a pre-washed Econopac 106-DG column (BioRad) to afford ING-1- Maleimide-S-5'-L-d( I-Q I -I )-3'-Teg.
- the product is concentrated in a Centricon-300 ® concentration device by centrifugation at 1000 g for 25 minutes.
- the sample is resuspended in fresh PBS and concentration by
- centrifugation is repeated a further 3 times until the ratio of optical densities at 260 nm and 280 nm is constant.
- optical density of these samples are examined in a spectrophotometer at 260 nm and 280 nm.
- the ratio of optical densities at these two wavelengths is
- the cuvettes are cooled to 20°C, loaded into a Cary 13 instrument, and the absorbance is analysed by UV light (260 nm) while the cuvette temeerature is ramped up from 20°C to 80°C and then back down to 20°C at a rate of 0.5°C/min. Analysis of the data reveals that cl is able to
- Example 10a) and 50 microL 90 Y-TMT-L-d(cI)-TMT- 90 Y solution from Example 9a are mixed with freshly prepared human serum (200 mL) or PBS (200 mL; pH7.2) and
- the gels are autoradiographed on a phosphoimager system to show that 90 Y-TMT-L-d(cI)-TMT- 90 Y is able to hybridize with ING-1-L-d( I-Q I -I ).
- the reaction mixture of each is kept on ice for 10 minutes. Aliquots of each reaction mixture are mixed with SDS buffer and loaded onto two duplicate 8 to 16% polyacrylamide gels. The gels are subjected to electrophoresis at a constant voltage for 2 hours.
- One gel is electroblotted onto nitrocellulose paper using CAPS buffer for 20 minutes according to the manufacturer's protocol (Hoefer semi-dry transfer method). After washing thrice with a solution of 0.05% Tween 20 in PBS, the gel is blocked with a solution of 3% BSA in PBS at room temperature for 1 hour. Following further washing with Tween/PBS, the gel is overlaid with a solution of a murine anti-TMT antibody (10mg/ml in PBS/Tween) and left overnight at room temperature. The western blot is developed using a goat anti-mouse IgG antibody conjugated to horseradish peroxidase (BioRad Western blot kit) and peroxidase substrate. The blot demonstrates that the TMT-L-d(cl)-TMT can be detected via the TMT's as being hybridized to bands that
- TMT-NCS Preparation of Ab-TMT by direct conjugation (ING-1/TMT) TMT-NCS (or a suitable derivative thereof) can be conjugated to an antibody molecule to yield an antibody- TMT conjugate molecule that displays the ability to bind to a target antigen recognized by the antibody variable region.
- a conjugate molecule can be used to deliver metal ions that are chelated by the TMT moiety in order to localize and/or treat the tumor that is targeted by such an immunoconjugate.
- the antibody is selected such that it has a broad reactivity with an antigen molecule expressed on tumor cells, thereby providing an antibody-TMT conjugate that can deliver radionuclides to the tumors for therapeutic or diagnostic purposes.
- the chimeric antibody, ING-1 (International patent publication WO 90/02569) consists of a murine variable region and a human immunoglobulin constant region. The antibody is produced by culturing a mouse myeloma cell line
- ING-1 is used at a concentration of 5.0 mg/mL in 50 mM sodium acetate and 150 mM sodium chloride buffered at pH 5.6.
- the conjugation of ING-1 to TMT-NCS is achieved by first adding 1.0 M carbonate plus 150 mM sodium chloride buffer, pH 9.3, to ING-1 until the antibody solution reaches a pH of 9.0. A sample of that ING-1 solution containing 5 mg of protein is then pipetted into an acid washed, conical, glass reaction vial. A solution of TMT-NCS is prepared by dissolving 100 mg in 10 mL of 1.0 M carbonate plus 150 mM sodium chloride buffer, pH 9.0. The conjugation reaction is started by the
- conjugate solutions are determined by the BioRad protein assay using bovine immunoglobulin as the protein
- ING-1/TMT is reacted with a solution of Europium chloride until saturation of the metal-binding capacity of the TMT occurs.
- a 0.375 mg aliquot of the ING-1/TMT in 2.5 ml in 0.05 M Tris HCl buffer pH 7.5 is pipetted into a 5 ml quartz cuvette.
- a 20 mM Europium chloride (Europium chloride hexahydrate; Aldrich) solution in 0.05 M Tris HCl buffer pH 7.5 is prepared.
- the ratio of TMT molecules per molecule of antibody is in the range from 0.3:1 to 2:1.
- the antigen to which the antibody, ING-1, binds is prepared from LS174T or HT 29 cells (available from American Type Tissue Collection, ATTC) by scraping confluent monolayers of cells from the walls of culture flasks with a cell scraper. The cells from many flasks are combined and a sample is taken and counted to estimate the total number of cells harvested. At all times the cells are kept on ice. Following
- the cells are washed once in 25 mL ice-cold 50 mM sodium phosphate buffer supplemented with 150 mM sodium chloride, pH 7.4 (PBS), pelleted under the same conditions and transfered in 10 mL PBS to an ice-cold glass mortar.
- the cells are homogenized at 4°C using a motor-driven pestle and then centrifuged at 3000 x g for 5 minutes. The antigen-rich supernatant is removed from the other cell debris and subjected to further
- the pellet (antigen fraction) from this final step is suspended in 100 mL of PBS for every million cells harvested. Following an estimate of the protein concentration (BioRad BCA protein assay using bovine immunoglobulin as the protein standard) the antigen is stored at -20°C until use.
- Each well of a 96-well Costar microtiter plates is coated with antigen by adding 100 mL/well of cell lysate (10 mg/ml) prepared as above. The microtiter plates are allowed to dry overnight in a 37°C incubator. After washing the plates five times with 0.05% Tween-20 (Sigma) they are blotted dry. The wells of each plate are blocked by adding 125 mL/well of a 1% BSA (bovine serum albumin, Sigma A-7906) solution in PBS and incubated for 1 hour at room
- Biotinylated ING-1 1.0 mg/mL in 0.1% BSA is added to each well (50mL/well) and the plates are then incubated for 2 hours at room temperature.
- the plates are blotted dry and incubated at room temperature for one hour with dilute (1:2000 in 0.1% BSA) streptavidinalkaline phosphatase (Tago; #6567). After a further five washes, color is developed in each well upon the addition of 100 mL per well of phosphatase substrate reagent (Sigma). After one hour at room temperature, the color is read using a 405 nm filter in a Titertek Multiscan microplate reader.
- the immunoconjugates of ING-1 with TMT are found to have immunoreactivity comparable to native ING-1.
- HT29 cells are grown to confluency in tissue culture flasks using McCoy's media supplemented with 10% fetal calf serum. The cells are harvested by scraping the flask walls with a cell scraper. Cells from many separate flasks are pooled, 'centrifuged to a pellet. resuspended at 5 x 10 5 /mL in a solution of ice-cold 50 mM sodium phosphate with 150 mM sodium chloride buffer pH 7.4 (PBS) supplemented with 0.1% bovine serum albumin (Sigma) and 0.02% sodium azide (Flow buffer).
- PBS sodium chloride buffer pH 7.4
- the cells are washed in this same buffer and then counted.
- An antibody standard curve is constructed by diluting ING-1 with an irrelevant (non binding), isotype-matched control antibody (human IgG1) to give a number of samples ranging in ING-1 content from 10% to 100%.
- the standard curve is made in flow buffer so that each sample contains 1.0 mg protein per mL. Samples from the standard curve and unknowns are then incubated with 5 x 10 5 HT29 cells at 4°C for 1 hour. After extensive washing to remove unbound antibody, the cells are resuspended in 100 mL flow buffer and incubated at 4°C for 1 hour with goat-anti-human antibody labeled with fluorescene isothiocyanate (FITC). After further washing in flow buffer the samples are analyzed by flow cytometry on a Coulter EPICS 753 flow cytometer.
- FITC fluorescene isothiocyanate
- Fluorescene FITC and propidium iodide (PI) are excited using the 488 nm emission line of an argon laser.
- the output is set at 500 mw in light regulation mode.
- Single cells are identified by 90 degree and forward angle light scatter. Analysis windows are applied to these parameters to separate single cells from
- Fluorescence from FITC and propidium are separated with a 550 nm long pass dichroic filter and collected through a 530 nm band pass filter (for FITC), and a 635 nm band pass filter (for PI).
- Light scatter parameters are collected as integrated pulses and fluorescence is collected as log integrated pulses. Dead cells are excluded from the assay by placing an analysis window on cells negative for PI uptake. The mean fluorescence per sample (weighted average from 2500 cells) is calculated for each
- FITC calibration beads are analysed in each experiment to establish a standard curve. The average fluorescence intensity for each sample is then expressed as the average FITC equivalents per cell.
- Immunoreactivity is calculated by comparing the average fluorescence intensity of the unknown sample with values from the standard curve. Samples of ING-1/TMT have immunoreactivity values comparable to the native ING-1 antibody by this method. (13d) Determination of aggregate formation by size- exclusion HPLC.
- a 30 cm x 7.5 mm TSK-G3000SW size-exclusion HPLC column (Supelco) fitted with a guard column of the same material is equilibrated with 12 column volumes of 10 mM sodium phosphate buffer pH 6.0 supplemented with 150 mM sodium chloride using a Waters 600E HPLC system with a flow rate of 1.0 mL per minute at 400-600 PSI.
- a sample (25 mL) of BioRad gel filtration protein standards is injected on to the column. The retention time of each standard is monitored by a Waters 490 UV detector set at 280 nm.
- ING-1/TMT has a major peak also at 9.1 minutes but a minor peak, attributable to aggregates, can sometimes be seen at 7.3 minutes. By comparison of the peak areas, the aggregate peak is less than 5% of the total.
- a volume of radioactive Yttrium chloride ( 90 ⁇ in 0.04 M hydrochloric acid at a specific activity of >500 Ci/mg; Amersham-Mediphysics) is neutralized using two volumes of 0.5 M sodium acetate pH 6.0.
- the neutralized 9 0 Y (1.0 mCi) is added to 1.0 mL of ING-1/TMT (1 mg/mL) in 50 mM sodium acetate buffer containing 150 mM sodium chloride at pH 5.6.
- the labelling is allowed to proceed for one hour and then the reaction mixture is loaded onto a PD-10 chromatography column which has been prewashed and equilibrated in a buffer containing 50 mM sodium phosphate with 150 mM sodium chloride pH 7.4 (PBS). The sample is eluted from the column with 1.5 mL of PBS. Fractions of radiolabeled ING-1/TMT (0.5 mL) are collected, assayed for radioactivity, and pooled.
- the labeling efficiency is determined by removing 1.0 mL of the sample and spotting it on to a Gelman ITLC-SG strip.
- the strip is developed in a glass beaker containing 0.1 M sodium citrate, pH 6.0, for a few minutes until the solvent front has reached three- quaters of the way to the top of the paper.
- the strip is inserted into a System 200 Imaging Scanner (Bioscan) which has been optimized for 90 ⁇ and is controlled by a Compaq 386/20e computer. In this system free 90 Y migrates at the solvent front while the ING-1/TMT/ 90 Y remains at the origin.
- Binding of lanthanides such as europium (3+) to chelating agents that contain an aromatic moiety held close to the co-ordination sphere can lead to
- a 0.5 mg aliquot of the ING-1/TMT in 2.5 mL in 0.05 M Tris HCl buffer pH 7.5 is pipetted into a 4 mL conical reaction vial containing a small stirring bar.
- a 250 mM europium chloride (europium chloride hexahydrate: Aldrich) solution in 0.05 M Tris HCl buffer pH 7.5 is prepared.
- An aliquot (50 mL) of this europium chloride solution is added to the reaction vial containing ING-1/TMT, and the resulting solution is stirred very slowly on a magnetic stirrer at room temperature.
- the labelling is allowed to proceed for one hour and then the reaction mixture is loaded on to a PD-10 chromatography column which had been pre-washed and equilibrated in a buffer containing 10 mM sodium phosphate and 150 mM sodium chloride at pH 6.0 (PBS).
- PBS pH 6.0
- the sample is eluted from the column with 3.5 mL of PBS.
- the fluorescence of a 50 mL sample of the metal-ING-1/TMT complex is determined in a Perkin Elmer LS 50 spectrofluorometer using an excitation wavelength of 340 nm (10 nm slit width).
- fluorescent emission is recorded at 618 nm using a 10 nm slit width and a 430 nm cutoff filter.
- Each functional chelating site on the ING-1/TMT conjugate binds one europium ion.
- an average of between 0.1 and 3 fluorescent europium ions are bound per molecule of antibody in solution.
- Fluorescently labeled CY5.18-L-d(I- Q l -I) is prepared as in Example 2d.
- Fluorescently labeled CY5.18-L-d(cI) and CY5.18-L-d(I) are prepared as in Example 3e.
- Flow cytometry is carried out
- HT-29 cells (0.5 x 10 6 ) are incubated on ice for 30 min with 1 mg each of the ING-1-L-d( I-Q I -I ) samples. The cells are washed twice with flow buffer and pelleted at 1400 rpm for 5 minutes between washes. Next the cells in each sample are incubated with 5 mg CY5.18-L-d(cI) or CY5.18- L-d(I) for 3 hours on ice. Some cells are incubated with CY5.18-L-d(cI) and CY5.18-L-d(I) alone. After extensive washing with flow buffer, the cells are subjected to analysis on a fluorescence activated cell sorter. CY5-18 calibration beads are analysed to establish a standard curve of relative fluorescence intensity versus CY5-18 concentration. The mean
- fluorescence per sample (weighted average from 2500 cells) is calculated for each histogram. The average fluorescence intensity for each sample is then expressed as the average CY5-18 equivalents per cell. Identical experiments are carried out in which the medium used for incubation of the cells with the components is 100% fetal calf serum in place of flow buffer.
- TMT-L-d(cl)-TMT is radiolabeled to a specific activity of 0.1 mCi/pmole as described in Example 7.
- ING-1-TMT- 90 Y is prepared as in Example 13e.
- Three tubes each containing 1 x 10 5 HT-29 cells in DMEM medium supplemented with 10% fetal calf serum are prepared and kept at 4°C
- McCoys media containing 10% FCS and 50 microgram/ml of gentamyacin The cells are washed with phosphate buffered saline, and 5 ml of Trypsin Versene is added. The HT-29 cells are then incubated at 37°C in 5% CO 2 for 15 minutes, complete media (5 ml) is added, and the cells are removed and washed in PBS. The HT-29 cells are then blocked with 10 micrograms of sheared salmon sperm (natural D-enantiomer) DNA per 10 6 cells at 4°C for 30 minutes, washed in PBS and used in the hybridization assay as follows.
- Delfia 1232 time-resolved fluorimeter by aliquoting four 100 microL portions into separate wells in a Costar
- EIA/RIA 96-well plate from each tube after vortexing.
- the results are processed as described in Example 15f.
- the counts per seconds (cps) of the Eu-TMT-L-d(cI)-TMT- Eu treated cells are considered as background and are subtracted from the cps data from the hybridization experiments (Eu-TMT-L-d(cI)-TMT-Eu/Ing-1-L-d( I-Q I -I )).
- This result and the cps data from the Ing-1/TMT-Eu binding experiment are translated as picomoles of bound TMT molecules per cell from the individual standard curves created independently.
- ING-1/TMT-Eu and ING-conjugates 0.25 microgram or 1.65 picomoles; Eu-TMT-L-d(cI)-TMT-Eu: 100 ng or 15 picomoles per 5 X 10 5 cells.
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- FEATURE FEATURE
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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AU64025/94A AU6402594A (en) | 1993-03-10 | 1994-03-10 | Tumor targeting with l-enantiomeric oligonucleotide conjugates of immunoreagents and of chelated radionuclides |
CA002157902A CA2157902A1 (en) | 1993-03-10 | 1994-03-10 | Tumour targeting with l-enantiomeric oligonucleotide conjugates of immunoreagents and of chelated radionuclides |
JP6520313A JPH08512019A (en) | 1993-03-10 | 1994-03-10 | Tumor targeting using L-enantiomeric oligonucleotide conjugates consisting of immunoreactive agent and chelated radionuclide |
EP94911542A EP0691981A1 (en) | 1993-03-10 | 1994-03-10 | Tumor targeting with l-enantiomeric oligonucleotide conjugates of immunoreagents and of chelated radionuclides |
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US3308393A | 1993-03-10 | 1993-03-10 | |
US08/033,083 | 1993-03-10 |
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WO1994020523A1 true WO1994020523A1 (en) | 1994-09-15 |
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ID=21868470
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PCT/US1994/002610 WO1994020523A1 (en) | 1993-03-10 | 1994-03-10 | Tumor targeting with l-enantiomeric oligonucleotide conjugates of immunoreagents and of chelated radionuclides |
Country Status (5)
Country | Link |
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EP (1) | EP0691981A1 (en) |
JP (1) | JPH08512019A (en) |
AU (1) | AU6402594A (en) |
CA (1) | CA2157902A1 (en) |
WO (1) | WO1994020523A1 (en) |
Cited By (12)
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EP0639083A1 (en) * | 1992-05-07 | 1995-02-22 | Nycomed Imaging As | Complexing agents and targeting immunoreagents |
US6395716B1 (en) * | 1998-08-10 | 2002-05-28 | Novirio Pharmaceuticals Limited | β-L-2′-deoxy-nucleosides for the treatment of hepatitis B |
US6444652B1 (en) * | 1998-08-10 | 2002-09-03 | Novirio Pharmaceuticals Limited | β-L-2'-deoxy-nucleosides for the treatment of hepatitis B |
EP1264603A1 (en) * | 2001-06-10 | 2002-12-11 | Noxxon Pharma AG | Use of L-polynucleotides for in vivo imaging |
EP1288309A1 (en) * | 2001-08-30 | 2003-03-05 | Noxxon Pharma AG | Methods for labeling L-nucleic acids |
WO2003035665A1 (en) * | 2001-10-26 | 2003-05-01 | Noxxon Pharma Ag | Modified l-nucleic acid |
EP1306382A1 (en) * | 2001-10-26 | 2003-05-02 | Noxxon Pharma AG | Modified L-nucleic acids |
US6787526B1 (en) | 2000-05-26 | 2004-09-07 | Idenix Pharmaceuticals, Inc. | Methods of treating hepatitis delta virus infection with β-L-2′-deoxy-nucleosides |
EP1485500A2 (en) * | 2001-10-24 | 2004-12-15 | Beckman-Coulter, Inc. | Efficient synthesis of protein-oligonucleotide conjugates |
US6875751B2 (en) | 2000-06-15 | 2005-04-05 | Idenix Pharmaceuticals, Inc. | 3′-prodrugs of 2′-deoxy-β-L-nucleosides |
US7186700B2 (en) | 2002-09-13 | 2007-03-06 | Idenix Pharmaceuticals, Inc. | β-L-2′-deoxynucleosides for the treatment of resistant HBV strains and combination therapies |
US7323451B2 (en) | 2002-08-06 | 2008-01-29 | Idenix Pharmaceuticals, Inc. | Crystalline and amorphous forms of beta-L-2′-deoxythymidine |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4863713A (en) * | 1986-06-23 | 1989-09-05 | The Board Of Trustees Of Leland Stanford Jr. Univ. | Method and system for administering therapeutic and diagnostic agents |
WO1992008494A2 (en) * | 1990-11-08 | 1992-05-29 | Sterling Winthrop Inc. | Targeting radioactive immunoreagents |
US5158880A (en) * | 1988-09-23 | 1992-10-27 | E. I. Du Pont De Nemours And Company | Process for preparing solid perfluorocarbon polymer supports having attached perfluorocarbon-substituted ligand or binder |
-
1994
- 1994-03-10 AU AU64025/94A patent/AU6402594A/en not_active Abandoned
- 1994-03-10 CA CA002157902A patent/CA2157902A1/en not_active Abandoned
- 1994-03-10 WO PCT/US1994/002610 patent/WO1994020523A1/en not_active Application Discontinuation
- 1994-03-10 EP EP94911542A patent/EP0691981A1/en not_active Withdrawn
- 1994-03-10 JP JP6520313A patent/JPH08512019A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4863713A (en) * | 1986-06-23 | 1989-09-05 | The Board Of Trustees Of Leland Stanford Jr. Univ. | Method and system for administering therapeutic and diagnostic agents |
US5158880A (en) * | 1988-09-23 | 1992-10-27 | E. I. Du Pont De Nemours And Company | Process for preparing solid perfluorocarbon polymer supports having attached perfluorocarbon-substituted ligand or binder |
WO1992008494A2 (en) * | 1990-11-08 | 1992-05-29 | Sterling Winthrop Inc. | Targeting radioactive immunoreagents |
Non-Patent Citations (1)
Title |
---|
Journal of the American Chemical Society, Volume 113, No. 21, issued 09 October 1991, H. URATA et al., "Mirror-Image DNA", pages 8174-8175, see entire document. * |
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US9290533B2 (en) * | 1998-08-10 | 2016-03-22 | Novartis Ag | β-L-2′-deoxy-nucleosides for the treatment of hepatitis B |
US20110257120A1 (en) * | 1998-08-10 | 2011-10-20 | Idenix Pharmaceuticals, Inc. | Beta-l-2'-deoxy-nucleosides for the treatment of hepatitis b |
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WO2002100442A2 (en) * | 2001-06-10 | 2002-12-19 | Noxxon Pharma Ag | Use of l-polynucleotides and derivatives thereof for in vivo imaging |
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EP1485500A4 (en) * | 2001-10-24 | 2005-12-07 | Beckman Coulter Inc | Efficient synthesis of protein-oligonucleotide conjugates |
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US7629456B2 (en) | 2001-10-26 | 2009-12-08 | Noxxon Pharma Ag | Modified L-nucleic acid |
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US7589079B2 (en) | 2002-08-06 | 2009-09-15 | Novartis Ag | Crystalline and amorphous forms of beta-L-2′-deoxythymidine |
US7323451B2 (en) | 2002-08-06 | 2008-01-29 | Idenix Pharmaceuticals, Inc. | Crystalline and amorphous forms of beta-L-2′-deoxythymidine |
US7858594B2 (en) | 2002-08-06 | 2010-12-28 | Novartis Pharma Ag | Crystalline and amorphous forms of beta-L-2′-deoxythymidine |
US7186700B2 (en) | 2002-09-13 | 2007-03-06 | Idenix Pharmaceuticals, Inc. | β-L-2′-deoxynucleosides for the treatment of resistant HBV strains and combination therapies |
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Also Published As
Publication number | Publication date |
---|---|
EP0691981A1 (en) | 1996-01-17 |
JPH08512019A (en) | 1996-12-17 |
CA2157902A1 (en) | 1994-09-15 |
AU6402594A (en) | 1994-09-26 |
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