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WO1996000087A2 - Materiel de preciblage et nouveaux conjugues de preciblage - Google Patents

Materiel de preciblage et nouveaux conjugues de preciblage Download PDF

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Publication number
WO1996000087A2
WO1996000087A2 PCT/EP1995/002425 EP9502425W WO9600087A2 WO 1996000087 A2 WO1996000087 A2 WO 1996000087A2 EP 9502425 W EP9502425 W EP 9502425W WO 9600087 A2 WO9600087 A2 WO 9600087A2
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Prior art keywords
conjugate
antibody
group
targeting
zinc finger
Prior art date
Application number
PCT/EP1995/002425
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English (en)
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WO1996000087A3 (fr
Inventor
Ebo Sybren Bos
Franciscus Michael Kaspersen
Original Assignee
Akzo Nobel N.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Akzo Nobel N.V. filed Critical Akzo Nobel N.V.
Priority to AU28871/95A priority Critical patent/AU2887195A/en
Publication of WO1996000087A2 publication Critical patent/WO1996000087A2/fr
Publication of WO1996000087A3 publication Critical patent/WO1996000087A3/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
    • A61K47/665—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells the pre-targeting system, clearing therapy or rescue therapy involving biotin-(strept) avidin systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891—Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897—Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • 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

Definitions

  • the invention relates to a novel kit for the delivery of pharmaceutical compounds or diagnostics to target cells in the body.
  • This kit comprises a targeting conjugate and an effector conjugate, these conjugates being able to form a specifically interacting binding pair by means of a zinc finger and its cognate nucleotide sequence.
  • the invention relates to targeting cytotoxic drugs or radioisotopes to tumor cells, preferably with a human antibody.
  • a major problem has been the inability to attain a sufficient difference in uptake of the immunoconjugates between the target tissues as compared to the non-target tissues. As a result it has been difficult for instance to effect an increase in therapeutic index of clinically used anti-cancer drugs by administration in the form of a drug-immunoconjugate. Similarly, due to the lack of operational selectivity of therapeutic radio-immunoconjugates when administered in vivo, normal tissues are unduly exposed to destructive radiation.
  • the therapeutic and diagnostic- limitations of immunoconjugates apart from a number of factors adversely affecting antibody delivery, are amongst others due to changes in antibody affinity, specificity and immunogenicity upon chemically linking a diagnostic or therapeutic agent to an antibody.
  • the bifunctional antibody approach suffers from the fact that the antibody molecule is composed of two monovalent antibody fragments with different specificities.
  • the avidity of monovalent antibody fragments such as Fab fragments is orders of magnitude lower than that of bivalent antibody molecules.
  • the efficacy of the two step bifunctional antibody approach is dependent on high avidity binding of the bifunctional antibody to the toxin-conjugated hapten and to extracellular or cell surface antigens at the target site.
  • a period of 4 to 6 days is required. Using monovalent antibody fragments, complete dissociation of bound antibody molecules from the target site is expected in this period of time.
  • Bifunctional antibodies have also been prepared with specificity for both tumor antigens and (radiolabelled) chelates (Goodwin, D.A. et al., J. Nucl. Med. _29, 226-234, 1988; Stickney, D.R. et al., Cancer Res. 5 ⁇ , 6650-6655, 1991/ Le Doussal, J.-M., J. Cancer Suppl. 1_, 58-62, 1992).
  • Major drawback in this approach is the low affinity of the bispecific antibodies for the chelating moiety.
  • target site selective antibodies have been endowed with a second specificity by linkage, through either chemical or biological methods, to a member of a specifically interacting binding pair.
  • the high affinity system avidin-biotin has been adopted in such an approach (European Patent Appl. No. 0 251 494) .
  • the pretargeting system used in said patent application employs an antibody that has been linked to the protein avidin or streptavidin and a biotinylated metal chelate.
  • a major problem with the pretargeting system mentioned above is the immunogenicity of the antibody-conjugate. First of all immunogenic activity is caused by the murine antibodies, which are often used in this kind of conjugates. These, like all other foreign proteins, are highly immunogenic in man.
  • HAMA human anti mouse antibodies
  • a second problem is caused by the use of biotin, which is an endogenous factor (vitamin H) in the human body, especially in the liver.
  • biotin which is an endogenous factor (vitamin H) in the human body, especially in the liver.
  • Injection with a conjugate with avidin, which has a very high affinity to biotin will lead to accumulation of the avidin-conjugate in the liver and other organs where biotin can be found.
  • Zinc fingers are parts of transcription factors and were firstly discovered in 1985 (Miller, J. et al., EMBO Journal, _4(6), 1609-1614, 1985). Zinc fingers are amino acid sequences of about 25-30 amino acids in a protein which form a tertiary structure able to chelate a zinc ion in a loop protruding from the rest of the protein. These zinc fingers are able to bind to a specific ds (double strand) DNA nucleotide sequence.
  • steroid receptors One large group of molecules which naturally contain zinc fingers are steroid receptors. In each DNA binding domain of a steroid receptor two zinc fingers can be found which are able to bind to a genomic DNA sequence, generally consisting of about 15 nucleotides, which sequence is also known as hormone responsive element (HRE) .
  • HRE hormone responsive element
  • Zinc fingers of steroid receptors are relatively highly conserved sequences (Fuller, P.J., FASEB Journal, 5_, 3092- 3099, 1991) : for all receptors across species, 18 of 68 amino acid residues are invariant and for human receptors this figure rises to 36 of 68. This conservation of structure extends to function; the DNA-binding domain interacts with hormone responsive elements which are enhancer elements in the regulatory (usually 5' flanking) region of the specific steroid-induced gene.
  • HREs appears from the fact that in the mouse mammary tumor virus long-terminal repeat (MMTV-LTR) the same HRE is recognized by androgen, progesterone, glucocorticoid and mineralocorticoid receptors.
  • GRE glucocorticoid receptor responsive element
  • the tyrosine response element which also binds to the vitamin D receptor and the retinoic acid receptor, is the same as the ERE, only lacking the three intervening variable bases.
  • Variations from the above given consensus sequences can either increase or decrease the affinity, of a response element for the receptor.
  • Next to the response elements which exert in vivo gene- activating effects also negative response elements have been found. They also bind to the DNA binding domain of the receptor albeit with a lower affinity than the positive HREs.
  • Our invention now is a kit, comprising a targeting conjugate comprising a targeting moiety that can bind to a target cell, and an effector conjugate which can bind to said targeting conjugate and which also comprises an effector moiety, characterised in that either the targeting moiety of the targeting conjugate or the effector moiety of the effector conjugate is conjugated to a zinc finger, while the other conjugate comprises a nucleotide sequence which can bind with the zinc finger.
  • the present invention proposes to administer to a patient a targeting conjugate in a dose, or in successive doses, sufficient to maximise target site accumulation of the targeting moiety. At some optimum time later, when most of the non-bound targeting moiety has been cleared, a therapeutic or diagnostic effector molecule carried by the effector conjugate or a cross-reactive analogue thereof, is administered.
  • a zinc finger is defined as a peptide whose amino acid sequence comprises one or more than one amino acid sequence corresponding with a zinc binding domain, or a functional derivative of the zinc binding domain.
  • a functional derivative is considered any modified zinc finger sequence wherein the ability to bind to the hormone responsive element is retained.
  • Zinc fingers and their HRE are thus very useful for this kind of pretargeting methods on the basis of their high affinity, but they have also the disadvantage that both zinc finger and HRE are already endogenously present in the human body. However, this presence is only intracellularly in case of the zinc fingers or intranuclearly for the HRE. Interaction of conjugates with circulating, endogenous zinc fingers or HRE's will be minimal and only limited to cases of circulating cell components from degenerating cells.
  • the zinc finger is derived from a steroid receptor. From most steroid receptors the amino acid sequence and the underlying DNA sequence for the transcription domain, which harbours the zinc fingers, is known (Fuller 1991, supra) .
  • Conjugates of zinc finger with either targeting moiety or with effector moiety can be produced by normal chemical means, but it is also possible to make fusion proteins.
  • Chemical conjugation can, for example, be obtained through conjugation of the zinc finger to an antibody through a normal peptide bond or via disulphide or thiether bonds.
  • Such conjugation techniques (and others) have been described in a review article of J. Goodchild, Bioconjug. Che . 1 , 165-187, 1990), which is encorporated herein by reference.
  • Fusion proteins can be made by standard biotechnological techniques. In short, DNA coding for the light and heavy chains of an antibody is cloned into a plasmid.
  • DNA encoding the zinc finger is cloned in a position that enables it to be expressed as an extension of the Fc part of the heavy chain.
  • the plasmid is brought into a host capable of expressing and joining the light and heavy chains from this plasmid.
  • any pair of zinc finger and HRE is equally suited in the pretargeting scheme, preferably the glucocorticoid receptor and the corresponding GRE are used.
  • the nucleotide sequences constituting the HRE are chemically modified for protection against enzymatic degradation by nucleases.
  • the main nuclease activity in the blood origins from 3'-exonucleases. Therefore, modification of the phosphodiester at the 3'-end of the oligonucleotide is indicated. This also enhances the stability of the nucleotide sequence.
  • oligonucleotide When the oligonucleotide is bound to the targeting moiety it should be stable for quite a long period (2-3 days) .
  • nucleotide analogues which are backbone-modified such as phosphorothioate, methylphosphonate and alkyl phosphotriester (inter alia phosphate-methylated DNA) oligonucleotides.
  • introduction of modified phosphate groups should not lead to poorer solubility of the oligonucleotide in water or to a decrease of the stability of the complex with the zinc finger.
  • Methylphosphonate linkage is most preferred because this can be easily introduced into DNA fragments using standard (phosphoroamidite) DNA chemistry.
  • this modification is stable towards the chemical conditions necessary for conjugations, and the introduction of neutral internucleoside linkages diminishes the non-specific binding of the highly charged oligonucleotides to positively charged blood proteins.
  • Partially phosphate-methylated DNA fragments can as well be applied.
  • At least one of the nucleotide strands is equipped at either the 3' or 5' end, with a functional group that allows for coupling to a targeting moiety or effector moiety.
  • a functional group like a thiol- group, a maleimido-group, a pyridyldithio-group or an amino-group, are well known in the art of conjugation chemistry.
  • an amino group is introduced at either end, or both ends, of a nucleotide strand, during or after the nucleotide synthesis process. This can, for instance, be done by esterification of the terminal phosphate groups with an amino-group containing alcohol, such as, for example 3-amino-2-hydroxy-propanol.
  • the amino group can subsequently be derivatized with one of a number of bifunctional reagents that are known in the art for introducing a functional group that is suitable for conjugation.
  • Preferred bifunctional reagents are SPDP (3- (2- pyridyldithio)propionic acid N-hydroxy-succinimide ester), SMCC (N-succinimidyl-4-maleimidomethyl-cyclohexane-l- carboxylate) , iminothiolane (Traut's reagent), 2-(acetyl- thio) succinic anhydride or SATA (S-acetylthioglycolic acid N- hydroxysuccinimide ester) .
  • the amino-group is derivatized with SPDP to introduce a pyridyldithio-group, which can subsequently be reduced, for instance with the use of dithioerythritol (DTE) , dithiothreitol (DTT) or tributyl- phosphine, to a thiol group.
  • DTE dithioerythritol
  • DTT dithiothreitol
  • tributyl- phosphine tributyl- phosphine
  • Targeting moieties which can be used in the present invention are compounds which after administration are predominantly found to be present at or in the neighbourhood of the target cells.
  • compounds which adhere to a structure present at the site of the target cells are especially useful.
  • Such compounds can be specific antibodies, ligands or enzymes.
  • an antibody is used as targeting moiety.
  • antibodies, and/or analogues of these antibodies or parts thereof should be comprised.
  • antibodies directed to a tumor related epitope are preferred.
  • Monospecific antibodies to a tumor related epitope can be obtained by affinity purification from polyspecific antisera by a modification of the method of Hall et al. (Nature 311, 379-387 1984) .
  • Polyspecific antisera can be obtained by immunizing rabbits according to standard immunization schemes.
  • Monospecific antibody as used herein is defined as a single antibody species or multiple antibody species with homogeneous binding characteristics for the relevant antigen.
  • Homogeneous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope.
  • the antibody is preferably a monoclonal antibody, more preferably a humanised or human monoclonal antibody.
  • Monoclonal antibodies can be prepared by immunizing inbred mice, preferably Balb/c with the appropriate protein by techniques known in the art (Kohler and Milstein, Nature 256; 495-497, 1975) .
  • Hybridoma cells are subsequently selected by growth in hypoxanthine, thymidine and aminopterin in an appropriate cell culture medium such as Dulbecco's modified Eagle's medium (DMEM) .
  • Antibody producing hybridomas are cloned, preferably using the soft agar technique of MacPherson, (Soft Agar Techniques, Tissue Culture Methods and Applications, Kruse and Paterson, eds.
  • Discrete colonies are transferred into individual wells of culture plates for cultivation in an appropriate culture medium.
  • Antibody producing cells are identified by screening with the appropriate immunogen.
  • Immunogen positive hybridoma cells are maintained by techniques known in the art. Specific anti-monoclonal antibodies are produced by cultivating the hybridomas in vitro or preparing ascites fluid in mice following hybridoma injection by procedures known in the art.
  • human or humanized antibodies It is preferred to use human or humanized antibodies.
  • Methods for humanizing antibodies, such as CDR-grafting, are known (Jones et al., Nature 321, 522-525, 1986) .
  • Another possibility to avoid antigenic response to antibodies reactive with polypeptides according to the invention is the use of human antibodies or fragments or derivatives thereof.
  • Human antibodies can be produced by in vitro stimulation of isolated B-lymphocytes, or they can be isolated from (immortalized) B-lymphocytes which have been harvested from a human being immunized with an autologous tumor cell vaccine (Haspel, M.V. et. al., Cancer Res. 4_5, 3951-3961, 1985).
  • a preferred human monoclonal antibody is MoAb 16-88 produced by a hybridoma cell line which is deposited under number HB 8495 with the American Type Culture Collection on January 30, 1984. Reactivity of this monoclonal antibody has been specified in US Patent No. 5,106,738.
  • the effector moiety can be either a therapeutic drug or a diagnostic compound.
  • a diagnostic compound preferably a gamma-emitter is used, such as ⁇ Iii or 131 I.
  • radioisotopes When using a therapeutic drug as effector moiety preferably a compound selected from the group consisting of ⁇ - and ⁇ - emitting radioisotopes, boron addends and radiosensitizers is used.
  • radioisotopes preferably 90 Y, 131 I, l 53 Sm, 186 Re, 188 Re, 211 At, 212 Bi, 213 Bi or 225 Ac are used.
  • a diagnostic compound is linked to the antibody of the targeting conjugate.
  • a diagnostic moiety linked to the targeting moiety enables the detection of the amount of targeting conjugate localized at the target site and detection of the amount of circulating or eliminated material.
  • a diagnostic compound preferably a ⁇ -emitter is used, such as 99m Tc, 11] -In or 123 I.
  • effector conjugates for therapeutical use comprising a zinc finger.
  • These zinc finger-conjugates can be used for bringing a therapeutic or diagnostic effector moiety to the cognate hormone responsive element of the zinc finger.
  • the effector moiety can be either a therapeutic drug or a diagnostic compound.
  • a diagnostic compound preferably a ⁇ - emitter is used, such as 99m Tc, Ulln or 1 23 I.
  • a therapeutic drug preferably a compound selected from the group consisting of ⁇ - and ⁇ - emitting radioisotopes, boron addends and radiosensitizers is used.
  • radioisotopes preferably 90 Y, 131 I, 153 Sm, 186 Re, 188 Re, 211 At, 1 Bi, 13 Bi or 25 Ac are used.
  • the targeting conjugate and the effector conjugate should be in a pharmaceutically acceptable form, which may be administered parentally.
  • the useful dosage to be administered will vary depending on the age and the weight of the patient and on the mode of administration.
  • the conjugates are mixed with pharmaceutically suitable auxiliaries, pharmaceutically suitable liquids, and one or more pharmaceutically acceptable carriers, e.g. as described in the standard reference Gennaro et.al., Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., 1990, see especially Part 8: pharmaceutical Preparations and Their Manufacture) .
  • the conjugates may be processed to an injection preparation in the form of a solution, or the conjugates may be processed to a preparation in lyophilized form.
  • pharmaceutically acceptable carriers or diluents useful in the present invention include stabilizers such as carbohydrates (e.g.
  • sorbitol mannitol-, starch, sucrose, glucose, trehalose, dextran
  • proteins such as albumin, preferably human serum albumin, scavengers for radiolysis, and buffers (e.g. phosphate buffer) .
  • buffers e.g. phosphate buffer
  • any pharmaceutically acceptable additive which does not interfere with the function of the active compounds can be used.
  • Kits and conjugates according to the present invention can be used for the treatment and detection of tissue specific cell disorders such as cancer, auto-immune diseases, antiviral diseases and inflammatory diseases.
  • tissue specific cell disorders such as cancer, auto-immune diseases, antiviral diseases and inflammatory diseases.
  • Fig. 1 shows chemical linkage possibilities to 3'- and 5'-end modified synthetic ologonucleotide sequences for introduction of (radio) labels or -SH-groups for conjugation purposes.
  • Fig. 2 shows the pretargeting of a zinc finger.
  • Microtiter plate wells were coated with recombinant DNA zinc finger DBD-93. Specific reaction of the zinc finger with increasing concentrations of a conjugate of double-stranded DNA and monoclonal IgM antibody 16-88 is shown.
  • Fig. 2a detection of antibody 16-88 is obtained with the cognate antigen of MoAb 16-88, designated CTA1, coupled to horse radish peroxidase (HRP) which is detected.
  • CTA1 cognate antigen of MoAb 16-88
  • HRP horse radish peroxidase
  • Fig. 3 shows a dot-blot of the same reactions.
  • HRE Hormone responsive element
  • the nucleotide sequence and chemical structure of the ds DNA strands representing the hormone-responsive element (HRE) of the human glucocorticoid receptor (which is also presented in SEQ ID N0:1) are as follows:
  • Both complementary strands A and B contain natural phospho- diester linkages, and were protected at the 3'-end against nuclease degradation by the introduction of a phospho- propylamino (-O-PO2-O-CH2-CHOH-CH2-NH2) group.
  • a phospho- propylamino -O-PO2-O-CH2-CHOH-CH2-NH2
  • this group offers sufficient protection against exonucleases.
  • the terminal amino-group serves as a selective site for labeling (attachment of a diagnostic or therapeutic effector molecule) , and a site through which the oligonucleotide can be conjugated to a targeting moiety, such as an antibody.
  • the oligonucleotides were prepared by automated synthesis on a CPG (controlled glass support) support (P.N. Nelson et.al. Nucleic Acid Research 1_7, 7189, 1989) using a trifluoroacetyl amino protecting group instead of the described fluorenyl- methyloxycarbonyl (Fmoc) group, and using otherwise standard DNA-synthesizing conditions.
  • CPG controlled glass support
  • Fmoc fluorenyl- methyloxycarbonyl
  • a second set of complementary oligonucleotides which do not correspond with a hormone responsive element and which is referred to as irrelevant ds-DNA in the binding assays described under D, were prepared and derivatized at the 3' and 5' ends as described above for the HRE.
  • the sequences of the complementary strands of irrelevant ds-DNA are:
  • Strand A was biotinylated at the 3'-end with NHS-biotin (N- hydroxysuccinimido-biotin) at a molar DNA/biotin ratio of 1:30 in sodium phosphate buffer pH 8.5. Excess NHS-biotin was removed by ion-exchange chromatography on Q-Sepharose. The fraction eluted at 0.6 mol/1 NaCl in phosphate buffer represented the biotinylated oligonucleotide A. To this solution, an equal amount of strand B was added and the mixture was heated for 5 min at 100° au bain marie and allowed to cool down to ambient temperature. The dsDNA thus formed was incubated with SPDP at a 30 molar excess for 30 min.
  • NHS-biotin N- hydroxysuccinimido-biotin
  • a dithiopyridyl-group is introduced at the 3'-end of the B strand of the doublestrand oligonucleotide.
  • dithiothreitol was added to a final concentration of 25 mmol/1 and incubation was continued for 10 min.
  • the reaction mixture was diluted tenfold with phosphate buffer pH 8.5 and applied on a Q-Sepharose column equilibrated in phosphate buffer. The absorbance of the eluate was monitored at 254 nm.
  • a recombinant (expressed in Escherichia Coli) protein fragment, a peptide of 93 residues the sequence of which is represented in SEQ. ID. NO. 2, containing the DNA-binding domain (DBD) of the human glucocorticoid receptor (GR) was purified and checked for DNA-binding activity as described by Freed an et al. (Nature, 334, 543-546, 1988).
  • the peptide is referred to as DBD-93 by Hard et.al. (Biochemistry, 2_9, 9015- 9023, 1990; see Fig.l) and contains an 80 amino acid segment - the sequence 6-85 of SEQ. ID. NO.
  • the peptide further contains an N-terminal linker sequence (1-5 of SEQ. ID. NO. 2) and a C-terminal linker sequence (92-93 of SEQ. ID. NO. 2) 18
  • This zinc-finger peptide corresponds to the the unique rat sequence.
  • This zinc-finger peptide contains two zinc-finger domains, corresponding with the segments 6-26 and 42-61 of SEQ. ID. NO. 2, respectively.
  • the zinc finger (ZnF) peptide DBD-93, described under Cj_ was adsorbed overnight to the wells of a microtitre plate at an initial concentration of 1 ⁇ g/ml in PBS.
  • Lys lie Lys Gly lie Gin Gin Ala Thr Ala Gly Arg Leu 85 90 (2) INFORMATION FOR SEQ ID NO: 3:
  • MOLECULE TYPE other nucleic acid
  • DESCRIPTION: /desc "synthetic DNA"

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Abstract

L'invention concerne un procédé de préciblage de composés thérapeutiques ou diagnostiques permettant de cibler des cellules dans le corps, ainsi qu'un matériel destiné audit procédé. De préférence, le ciblage s'effectue par un anticorps spécifique. La liaison entre la fraction de ciblage préinjectée et la fraction effectrice s'établit à l'aide d'un 'doigt' de zinc et de la séquence de nucléotides parente de celui-ci, conjuguée à la fraction de ciblage ou effectrice. L'invention concerne également des conjugués de 'doigts' de zinc ainsi que des composés thérapeutiques ou diagnostiques.
PCT/EP1995/002425 1994-06-24 1995-06-22 Materiel de preciblage et nouveaux conjugues de preciblage WO1996000087A2 (fr)

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AU28871/95A AU2887195A (en) 1994-06-24 1995-06-22 Kit for pretargeting and novel pretargeting conjugates

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EP94201826 1994-06-24
EP94201826.8 1994-06-24

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WO1996000087A2 true WO1996000087A2 (fr) 1996-01-04
WO1996000087A3 WO1996000087A3 (fr) 1996-03-07

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020082979A (ko) * 2001-04-24 2002-11-01 이명철 질 수축 압력측정기
JP2015513541A (ja) * 2012-02-24 2015-05-14 アルテオジェン インコーポレイテッドＡｌｔｅｏｇｅｎ Ｉｎｃ． システイン残基を含むモチーフが結合した修飾抗体、前記修飾抗体を含む修飾抗体−薬物複合体及びその製造方法

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* Cited by examiner, † Cited by third party
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DK608589D0 (da) * 1989-12-01 1989-12-01 Holm Arne Kemisk fremgangsmaade
EP0564592B1 (fr) * 1990-12-21 1999-10-13 The Rockefeller University Facteur de transcription enrichi par extraits hepatiques
US5578289A (en) * 1992-03-04 1996-11-26 Akzo N.V. In vivo binding pair pretargeting
EP0650735A3 (fr) * 1993-07-09 1999-04-14 Akzo Nobel N.V. Kit et méthode de préciblage

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020082979A (ko) * 2001-04-24 2002-11-01 이명철 질 수축 압력측정기
JP2015513541A (ja) * 2012-02-24 2015-05-14 アルテオジェン インコーポレイテッドＡｌｔｅｏｇｅｎ Ｉｎｃ． システイン残基を含むモチーフが結合した修飾抗体、前記修飾抗体を含む修飾抗体−薬物複合体及びその製造方法
US9814782B2 (en) 2012-02-24 2017-11-14 Alteogen Inc. Modified antibody in which motif comprising cysteine residue is bound, modified antibody-drug conjugate comprising the modified antibody, and production method for same

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