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WO1992005200A1 - Conjugues d'anticorps xenobiotiques - Google Patents

Conjugues d'anticorps xenobiotiques Download PDF

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Publication number
WO1992005200A1
WO1992005200A1 PCT/US1990/005297 US9005297W WO9205200A1 WO 1992005200 A1 WO1992005200 A1 WO 1992005200A1 US 9005297 W US9005297 W US 9005297W WO 9205200 A1 WO9205200 A1 WO 9205200A1
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WO
WIPO (PCT)
Prior art keywords
tubercidin
antibody
linker
xenobiotic
composition
Prior art date
Application number
PCT/US1990/005297
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English (en)
Inventor
Charles G. Smith
Bernard Loev
Original Assignee
Chemex Pharmaceuticals, Inc.
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 Chemex Pharmaceuticals, Inc. filed Critical Chemex Pharmaceuticals, Inc.
Priority to PCT/US1990/005297 priority Critical patent/WO1992005200A1/fr
Publication of WO1992005200A1 publication Critical patent/WO1992005200A1/fr

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Classifications

    • 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
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A61K47/6809—Antibiotics, e.g. antitumor antibiotics anthracyclins, adriamycin, doxorubicin or daunomycin

Definitions

  • This invention relates to the general area of antibody conjugates capable of in vivo delivery of compounds to target sites. More specifically it relates to a method of delivery of potent cytotoxins to diseased cells while sparing healthy cells. Finally, it describes delivery systems for unique cytotoxic compounds that are trapped within target cells.
  • cytotoxic agent a cytotoxic agent to be considered a good candidate for target directed immunotoxic therapy are the following: 1) highly potent cytotoxicity to human tumor cells when the agent is absorbed into the tumor cell; 2) sufficient chemical stability to allow chemical coupling to the antibody? 3) sufficient hydrophylicity to allow adequate solubility in the serum while being delivered to the tumor site; and 4) adequate "metabolic stability" to allow penetration of the drug into the cells (e.g., little or no enzymatic degradation at the surface of the tumor cell or in the serum, and adequate residence time within the tumor cell) .
  • Tubercidin is a cytotoxic analog of adenosine that has been shown to simultaneously inhibit RNA, DNA and protein synthesis in essentially every living mammalian cell line studied in which macromolecule synthesis is taking place, once the compound has been absorbed into the cells. This finding is quite contrary to that of most inhibitors of macromolecule synthesis as the others show a preference for either RNA synthesis, DNA synthesis or protein synthesis but do not inhibit all three simultaneously.
  • tubercidin is incorporated into both RNA and DNA in multiplying cells. This is most unusual if not totally unique among substances that can be incorporated into nucleic acids.
  • tubercidin is indiscriminate in its ability to kill living cells, it has been studied topically as well as systemically. For example, Klein et al. (Cancer 34: 250-253) in 1974 reported on the treatment of basal cell carcinoma and actinic keratosis with topical tubercidin. The use of nitrobenzylthioinosinate has been reported to reduce tubercidin toxicity when it is applied to basal cell tumors or warts. DeClercq and his co-workers [Antibiotic Agents and Chemotherapy 30: 719-724 (1986)] reported activity of xylo-tubercidin against herpetic skin lesions in the hairless mouse.
  • Tubercidin has also been used for the treatment, in animals, of parasitic diseases. Jaffe and co-worker (J. Parasitol 62: 910-913 (1976) reported on the treatment of liver flukes in female mice with tubercidin. The same investigators reported treatment of schistosomiasis (both S_ ⁇ mansonii and S ⁇ japonicum) through a single dose of tubercidin by iv drip to iv baboons. El Kouni et al. Biochem. Pharmcrol 36: 3816-3821 (1987) reported that a combination of tubercidin and nitrobenzylthioinosine provided a highly selective combination therapy for treatment of S ⁇ mansonii in mice.
  • tubercidin in human (or animal) medicine is its toxicity to normal tissue, particularly the veins and the kidneys.
  • tubercidin was administered to human cancer patients after it had been taken up in vitro into the red blood cells of the patient being treated (it does not kill red blood cells because they are not synthesizing RNA, DNA or protein) and the red blood cells were reinfused into the patient [Cancer Research 30: 79-81 (1970)].
  • the degree of selectivity was inadequate to achieve the goal of adequate antitumor activity although venous and nephro- toxicities were reduced.
  • tubercidin has not, to date, been found to be useful as a therapeutic composition.
  • tubercidin and other potent cytotoxins can be therapeutically useful in the treatment of malignant disease.
  • This invention describes therapeutically useful antibody conjugates of tubercidin or other xenobiotic agents for delivery of drug compounds to target cells.
  • This invention involves the chemical coupling of cytotoxic agents to appropriate antibodies using linkers that are sufficiently resistant to biological degradation in vivo so as to deliver the agents directly to the surface of the target cells in the human body.
  • a monoclonal antibody produced against a tumor cell antigen is attached to a cytotoxic agent.
  • the cytotoxic agents which have been shown to have a broad spectrum of cytotoxic effects in vitro and shown to be able to kill tumors growing in experimental animals, and, in the case of tubercidin, in humans as well, will kill the tumor tissue in a highly selective way that will lead to far better antitumor activity than is possible upon administration of the non-conjugated compound to an animal.
  • the whole animal may be exposed to products of tumor cell disintegration in such a manner that these products will induce an immunologic response on the part of the host that will aid the host in rejecting tumor tissue and preventing metastatic spread or growth of cells that have already lodged in a site distal to the original tumor.
  • a very clear advantage of the conjugation of the cytotoxic agent with an appropriate antibody is that the antibody will prevent these compounds from entering normal cells as they are capable of doing when administered in free form into the blood stream of animals, thereby reducing whole animal toxicity and markedly increasing selective kill of tumor tissue.
  • tubercidin and other xenobiotic compounds to antibodies is conceived to be of considerable value in the treatment of the cancer patient since these potent compounds have, heretofore, been unavailable for use in the treatment of disease.
  • the selectivity of the antibody will assure the delivery of these highly cytotoxic compounds directly and specifically to the tumor site and will minimize its absorption into other normal tissues, including the local veins and the kidneys.
  • Each of the cytotoxic agents after coupling to antibodies that are selective for attachment to the surfaces of various tumors, will be administered by the intravenous, intracavitary, topical or intratumoral routes to patients suffering malignant disease and the MoAb's will affix the cytotoxic agents to the surface of the tumor and they will be absorbed in a highly concentrated and highly selective manner.
  • each of these unique agents will exert its selective and distinctive biochemical mechanisms that will result in the death of the tumor cells, thereby ⁇ uring the patient or offering significant amelioration of the course of the disease to give relief and to prolong life and well being of the treated individual.
  • conjugates disclosed herein are particularly useful for the treatment of malignant disease, it will be recognized by those skilled in the art that these compounds may be useful in the treatment of other diseases, where the object is the destruction of macromolecule-synthesizing entities within the body.
  • a tubercidin-containing conjugate could be used against certain viruses, parasites, bacteria, fungi, non-malignant hyperproliferative disorders such as fibromatosis, certain brain tumors, psoriasis, etc.
  • FIG. 1 is a plot showing the effect of tubercidin on KB cells.
  • potent cytotoxic agents are linked to antibodies that are directed against particular constituents of target cells.
  • cytotoxic compounds contemplated by the present invention have unique biochemical properties and modes of action that make them particularly suitable for cancer therapy.
  • tubercidin methyl ester of tubercidin-5,-phosphate (MepTu) , xylotubercidin, toyoca ycin and sangivamycin or analogs with substantially the same structure or xenobiotic activity.
  • MepTu tubercidin
  • xylotubercidin xylotubercidin
  • toyoca ycin sangivamycin or analogs with substantially the same structure or xenobiotic activity.
  • Tubercidin is most preferred, as it inhibits RNA, DNA and protein synthesis simultaneously.
  • tubercidin kills mammalian cells that are synthesizing macromolecules and especially kills reproducing cells. Tubercidin is less cytotoxic to resting cells, although it also eventually kills them.
  • tubercidin kills the tumor cell involves its phosphorylation through normal metabolic pathways, using enzyme kinases and ATP in exactly the same way that normal nucleosides are metabolized. If circulating kinases modify tubercidin to give the mono-, di- and triphosphates in the blood stream, the phosphoryl groups will nevertheless be hydrolyzed at cell surfaces (as are all phosphorylated nucleosides) to liberate free tubercidin.
  • this compound is more refractory to degradation in the human body and more likely to be absorbed and to deliver intact tubercidin inside the tumor cell even if it is not conjugated to an antibody.
  • tubercidin An extremely interesting property of tubercidin is the resistance of its 4- amino group to deamination by nucleoside or nucleotide deaminases and the resistance of its nucleoside linkage to cleavage by nucleoside phosphorylase, the enzymes that normally convert nucleosides to the corresponding hydroxy analogues or to simple purines or pyrimidines, respectively. Since the preponderence of evidence indicates that tubercidin cannot be degraded by nucleoside phosphorylase and it is known not to be deaminated by adenosine deaminase, it provides a kind of residually active form of cytotoxic compound to the tumor cell.
  • tubercidin is rapidly and completely converted to its phosphorylated forms (nucleotides) as soon as it is adsorbed into mammalian cells, it cannot exit from the cells because nucleotides do not cross cell membranes. As a result, tubercidin remains trapped inside the cell in its undegraded nuclectide form such that its killing impact is maximized.
  • the cytotoxicity of tubercidin is not reversed in mammalian cells by added purine or pyrimidine compounds and it is not cross-resistant with 6-MP.
  • CH_Tu 5'-phosphonate isostere
  • the compounds can show enhanced effects due to their unique properties, which include 1) high degree of cytotoxicity to all cells into which they penetrate that are synthesizing macromolecules, 2) good chemical stability, 3) refractoriness to reversal of cytotoxicity by normal purine and pyrimidine compounds 4) lack of cross- resistance with 6-MP, 5) resistance to the key catabolic enzymes in the body (e.g., nucleoside phosphorylase and adenosine deaminase) , 6) "metabolic entrapment" within cells once it gains entry by virtue of being phosphorylated by cellular kinases, and 7) unique, and still unidentified mechanism of action.
  • the key catabolic enzymes in the body e.g., nucleoside phosphorylase and adenosine deaminase
  • Nogalamycin is an anthracyclinone, as are adriamycin and daunomycin.
  • nogalamycin represents a very unique xenobiotic that differs from other available agents in its ability to kill tumor cells.
  • Porfiromycin is a xenobiotic chemically related to mitomycin C.
  • the latter antibiotic has been coupled with monoclonal antibodies and reported to show some selective antitumor activity in certain animal systems.
  • Porfiromycin is conceived to be a good candidate for coupling with antibodies because of its unique chemical structure.
  • Sparsomycin is a very unique antibiotic, having as its mechanism of action a very unusual and specific inhibition of one of the sites of elongation of the peptide chain during protein biosynthesis in animal cells. In early studies, it showed a very broad spectrum of cytotoxicity against a variety of animal cells grown in vitro and a broad spectrum of antitumor activity in experimental animal models. When studied in human beings, sparsomycin caused a most unusual toxic reaction that limited its further investigation in man; namely, the production of so-called "ring scatoma" in the human retina [Cancer Chemo. Repts. 43:29-31
  • Pactamycin like sparsomycin, is a very unique molecule unlike any other of its kind or any other under investigation in cancer research today. It inhibits, as does sparmocycin, protein synthesis in animal cells but by a mechanism different from that of sparsomycin. Most of the clinically useful antitumor agents (e.g., alkylating agents, adria ycin, daunomycin, pyrimidine antagonists, purine antagonists, etc.) inhibit nucleic acid, rather than protein, synthesis (either RNA or DNA synthesis) . No anitumor agent proven to be useful in the treatment of human cancer is available today for direct administration to human beings that has, as its primary mechanism of action, the inhibition of protein synthesis.
  • antitumor agents e.g., alkylating agents, adria ycin, daunomycin, pyrimidine antagonists, purine antagonists, etc.
  • Streptozotocin is a very unique antibiotic/xenobiotic that alkylates various sensitive centers in macro olecules. It was discovered some years ago as an antibacterial agent but its development as an antibiotic was curtailed because I) it was not absorbed orally and 2) in experimental rodent systems the substance, when injected intravenously, produced permanent diabetes by killing the beta cells of the pancreas after a single dose. It was next screened broadly in animal systems against a variety of tumors and shown to have antileukemic activity in a mouse model. In clinical studies involving the treatment of human leukemia, it was found to be inactive as an antitumor agent but, surprisingly, it was also shown not to affect the pancreas of patients suffering leukemic disease.
  • streptozotocin might possess an organotropic toxicity that was expressed in normal rodent pancreas and that might be expressed in human carcinoma of the pancreas cells, an extremely serious illness that, at the time streptozotocin was being studied, was not treatable in a meaningful way by the chemotherapeutic agents then available.
  • streptozotocin was studied in patients suffering from inoperable islet cell carcinoma (a uniformly fatal disease) , useful antitumor activity was demonstrated.
  • streptozotocin was approved by the FDA for the treatment of human islet cell carcinoma when administered by the intravenous route.
  • conjugation of streptozotocin with antibodies that will target this compound to tumor cells will markedly increase the therapeutic index and permit a much qreater exposure of tumor tissue to this drug.
  • Monoclonal antibodies as produced by hybridoma cells using recognized methodology, are most preferred due to their specificity to a particular target antigen.
  • polyclonal antibodies produced by exposure of an immunocompetent organism to target cells can be purified to homogeneity as regards specific antigenic moieties.
  • the antibody and xenobiotic agent are conjugated or "linked” together in such a manner that they are not directly linked to each other, so as to interfere with each other,s activity; and linked in a manner which is pharmaceutically acceptable.
  • the attachment is accomplished via chemical bonds (e.g. ester or amide linkages) and preferably via linkers which are susceptible to cleavage by one or more activated complement enzymes.
  • the conjugate may be designed so that the compound is delivered to the target but not released.
  • the compound may be attached to antibody molecules via linkers which are not susceptible to cleavage by complement enzymes.
  • linkers may include amino acids, peptides, or other organic compounds which may be modified to include functional groups that can subsequently be utilized in attachment to antibody molecules or antibody fragments by the methods described herein.
  • the compound may be attached to the linker before or after the linker is attached to the antibody molecule. If the compound contains functional groups that would interfere with attachment to the linker, these interfering functional groups can be blocked before attachment of the compound and deblocked once the conjugate is made (as in Example 1) .
  • the linker molecule before conjugation with the antibody or xenobiotic compound, must contain a plurality of functional groups such as carboxylic acid, aldehyde, halide, ester, mercaptan, disulfide, hydroxyl, sulfonyl, amino or hydrazine groups.
  • the linker molecule will not directly react with the functional groups of the cytotoxic agent or of the antibody, e.g., when the linker group contains carboxyl groups.
  • the functional groups of the agent, the antibody, or the union group must first be activated such that they will then undergo reaction.
  • carboxyl groups can be activiated by first reacting with a carbodiimide, or converted to a hydroxylamine ester.
  • EDC l-ethyl- 3(3-dimethylaminopropyl) carbodiimide
  • the reaction between carbodiimide and a linker molecule containing carboxylic acid groups is carried out at room temperature and is a simple addition reaction wherein the carbodiimide adds to the carboxyl group.
  • the carbodiimide "activates" the carboxyl group of the carboxylic acid such that it is now a reactive site for the cytotoxic agent.
  • the carbodiimide is an intermediate activating composition which is a "leaving group".
  • the reaction with cytotoxic a ine- containing agents is a direct addition reaction which can be carried out at room temperature and results in a carboxyamide bond involving the free carboxyl groups of the linker acid and the free amino groups of the cytotoxic agent.
  • the reaction occurs in a short period of time, and has been noted to be substantially complete in as short as five minutes, for small reaction quantities. The reaction does not appear to be process critical.
  • the composition which in its present state may be referred to as the "intermediate conjugate"
  • the intermediate conjugate now has the cytotoxic amine-containing agent attached to it. It now needs to have the tumor specific antibody attached to it.
  • the linker has multiple functional groups, such as carboxylic acids, only some of which have reacted with the cytotoxic agent. Thus, there are additional reactive sites which remain on the carrier molecule for reaction with the antibody.
  • carbodiimide activator may again be added.
  • Carbodiimide functions in the exact manner as described above. After the carbodiimide is allowed to react for about five minutes, the reaction mixture may be diluted with, for example, phosphate buffered saline solution (PBS) and purified antibody is added. Again, the reaction does not appear to be time or temperature dependent. The only important process criteria in the antibody addition reaction is that the reaction mixture be allowed reactive contact for a sufficient period of time to allow carbodiimide groups to leave and be replaced by the linkage between free amine or mercaptan moieties of the antibody and. carboxyl groups of the acid. In laboratory experiments, three hours at room temperature will usually be sufficient.
  • PBS phosphate buffered saline solution
  • the excess carbodiimide may be quenched with sodium acetate, and the mixture dialyzed to separate low molecular weight reactants from the linked product with has bound to it both the antibody and the xenobiotic compound without either of them being directly bonded to the other.
  • Free sulfhydryl groups may be present in the antibodies or can be generated from the disulfide bonds of the immunoglobulin molecule. This is accomplished by mild reduction of the antibody molecule.
  • the disulfide bonds of IgG which are generally most susceptible to reduction are those that link the two heavy chains.
  • the disulfide bonds located near the antigen binding region of the antibody molecule remain relatively unaffected. Such reduction results in the loss of ability to fix complement but does not interfere with antibody-antigen binding ability (Karush et al., 1979, Biochem. 18: 2226-2232) .
  • the free sulfhydryl groups generated in the intra-heavy chain region can then react with iodoalkyl derivatives of any linker compound containing carboxy or amino groups (e.g., iodoalkyl derivatives of linker groups which are attached to a compound) to form a covalent linkage.
  • iodoalkyl derivatives of any linker compound containing carboxy or amino groups e.g., iodoalkyl derivatives of linker groups which are attached to a compound
  • a linker carboxylic acid may be activated by conversion to a hydroxylamine ester, for example to an O-acyl-N- hydroxysuccinimide.
  • the preparation of such esters are well known, and may be prepared, for example by adding a carbodiimide to a solution containing equivalent amounts of the carboxylic acid and the N-hydroxy compound in an inert solvent such as methylene chloride. After storing at room temperature until the by-product urea has all precipitated (usually 2-14 hours) , the urea is filtered and the activated ester is isolated usually by evaporation of the solvent. The activated ester is then reacted with the amino, hydroxy or mercaptan group in the antibody or the cytotoxic compound in dimethylformamide or other suitable solvent.
  • the xenobiotic compound is attached to the linker molecule through covalent bonds such as those present in the carbonyl, alkyl, thiocarbonyl, sulfonyl, carbamyl, sulfide, disulfide, alkoxy, or alkenyl groups, before or after the linker is covalently bonded to an antibody or fragment thereof by chemical reactions well known to the art.
  • the linker group is attached to the antibodies through covalent bonds such as those present in sulfonyl, carbamyl, sulfide, disulfide, amino, carbonyl, alkyl, hydrazino, hydrazano, and alkene groups by chemical reactions well known to the art and in such a manner that the pharmacological activity of the xenobiotic composition is approximately the same as in the non-substituted form.
  • the linked product is concentrated and prepared for use.
  • the compounds can be administered by intravenous, intra- cavitary r intratumoral routes or they can be applied topically. In either case the product must be sufficiently soluble so as to allow injection, or in the case of topical application, to allow the product to permeate the skin.
  • the dosage in all cases, will vary with the size of the subject and the size and nature of the tumor, or the nature of the viral or parasitic infection.
  • the product is chromatographed on silica gel using 95% ethyl acetate, 95% ethanol, water (94:4:2) to give hemisuccinoyltubercidin -2', 3 ' - acetonide.
  • Monoclonal anti-carcinoembryonic antigen (CEA) antibody 11.285.14, has been shown to have strong anti-CEA binding activity, low reactivity with non ⁇ specific cross reactive antigen and a high degree of gastrointestinal tumour selectivity in comparison with a wide range of normal tissues. It was isolated from ascites fluids using a Protein A affinity column. Ascites fluids were diluted with one volume of 0.1 M phosphate buffer pH 8.0, filtered and adsorbed onto Protein A- Agarose (Sigma Chemical Comp., Poole, England), equilibrated with the pH 8.0 buffer and maintained at 4 ⁇ C.
  • Unadsorbed material was removed by washing with the pH 8.0 buffer, and bound immunoglobulin was then eluted using 0.1 M citrate/phosphate buffer pH 3.5.
  • the eluates were neutralised, concentrated to ca. 25 mg/ml by ultrafiltration, and dialysed against 0.34 M borate buffer, pH 8.6.
  • Hemisuccinoyltubercidin (20 mg) is dissolved in 0.8 ml of dry dimethylformamide (DMF) and a mixture of 3.4 mg of N-hydroxysuccinimide (Sigma) and 9.9 mg of 1- cyclohexyl-3-(2- morpholinoethyl) - carbodiimide metho- p-toluene-sulphonate (Sigma) in 0.2 ml of dry DMF is added. After 48 h at room temperature, a 0.25 ml aliquot is added to a stirred 1 ml aliquot of a 20 mg/ml solution of CEA antibody in 0.34 borate buffer, pH 8.6.
  • DMF dry dimethylformamide
  • the conjugation reaction is allowed to proceed at room temperature for 4 h, after which time it is adjusted to pH 7.2, clarified by centrifugation if necessary, and chromatoqraphed on a 1.5 x 110 cm column of Sephadex G- 200 (Pharmacia Milton Keynes, England) (IgG conjugates) or Sephadex G-100 (Pharmacia) (Fab conjugates) equilibrated with 0.01 M phosphate, 0.15 M sodium chloride, pH 7.2 (PBS). Fractions corresponding to conjugate are combined. Drug and protein content of the conjugate may be determined by spectrophotometry at 270 and 280 nm, relating the absorbances obtained to predetermined extinction coefficients for free drug and unconjugated antibody at the two wavelengths.
  • the immunoconjugates are purified with Sephadex G-25 Fine (Pharmacia) and dialyzed against isotonic saline at 4 ⁇ C.
  • the immunoconjugate is lyophilized in the presence of 10% lactose and stored at -20°C until use.
  • This conjugate remains stable at the intravascular pH of 7, but once endocytosed by the target cells the tubercidin is cleaved in the intralysosomal acid environment and enters into the cytoplasm.
  • Dextran T-40 is oxidized to polyaldehyde dextran (PAD) using sodium periodate, as described by Manabe, et al, Bioche . and Biophys. Res. Co m. 1009 (1983).
  • PAD polyaldehyde dextran
  • 60 mg of the polyaldehyde is incubated with 20 mg of monoclonal antibody H-l, an anti-HLA IgG produced by a hybridoma cell line (Manabe, v.s.), in 10 ml of 0.15 M phosphate buffered saline.
  • HLA are antigens of the major histocompatibility antigen system A, B and C. The condensation is allowed to proceed for 24 h at pH 7.2 at 4°C.
  • the Schiff base solution is reduced by treating for 2 h with 0.3 ml of a sodium borohydride solution containing 10 mg borohydride in 10 ml of the phosphate buffer.
  • the resulting mixture may be purified over the Sephadex column as described above.
  • Tubercidin Conjugated to a Monoclonal Antibody Via Human Serum Albumin (HSA) as the Link HSA (Sigma Chemical Co., St. Louis, MO) (111 mg of protein/ml, 9 ml) was first freed of its dimer by gel filtration on Sephadex G-150 in PBS, and then reduced with lOmM dithiothreitol and dialyzed against PBS to obtain a preparation with 1.65 mole of thiol group per mole of HSA.
  • the HSA was kept at 4"C to allow the thiol group:HSA molar ratio to decrease to 0.72 ( 6 days) .
  • the thiol group was determined with 5, 5'-dithiobis(2- nitrobenzoic acid) .
  • Murine monoclonal antibody aMM46 prepared as in Endo, et al, Cancer Research, 47_, 1076 (1987) , is an antibody to an antigen on syngeneic ascitic C3H/He mouse mammary tumor MM46 cells. 74.3 mM of the maleimidosuccinyl derivative of this antibody in phosphate buffered saline is added to the solution above in the same buffer. The mixture is kept at 4°C for 18 hours. Any unreacted maleimide groups are blocked by treating the solution with 71 mM of L-cysteine in 30 ml of the buffer for 1 hour and then subjecting to gel filtration on a Sephadex G-150 column to give the conjugated product.
  • tumor cells are injected into B6CF1 mice and 6 hours later a series of ip treatments are begun. The percentage survival of mice in each group is plotted as a function of time.
  • tumor cells are injected sc into the abdominal wall of the mouse and allowed to develop into palpable tumors measuring 0.5 cm in diameter before treatment is begun. Mice are then subjected to a series of ip treatments and the size of the tumors are measured daily with a caliper square; the data is recorded as mean tumor size (product of two diameters + standard error of the mean) .

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Abstract

Cette invention concerne des conjugués d'anticorps xénobiotiques et leur procédé d'utilisation in vivo. On obtient les conjugués par couplage covalent d'un composé xénobiotique tel que la tubercidine, la toyocamycine, la sangivamycine et des analogues avec un anticorps ou des fragments d'anticorps, par l'intermédiaire d'un liant qui peut être clivable ou non par des composants du sérum.
PCT/US1990/005297 1990-09-18 1990-09-18 Conjugues d'anticorps xenobiotiques WO1992005200A1 (fr)

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* Cited by examiner, † Cited by third party
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US6689803B2 (en) 1996-12-02 2004-02-10 Angiotech Pharmaceuticals, Inc. Compositions and methods for treating surgical adhesions
AU771994B2 (en) * 1996-12-02 2004-04-08 Ipxmedical, Llc Compositions and methods for treating or preventing inflammatory diseases
AU2004200715B2 (en) * 1996-12-02 2006-08-31 Angiotech International Ag Compositions and methods for treating or preventing inflammatory diseases
WO2017107817A1 (fr) * 2015-12-21 2017-06-29 江苏恒瑞医药股份有限公司 Procédé de préparation d'un conjugué anticorps-médicament
US9701706B2 (en) 2015-08-06 2017-07-11 Chimerix, Inc. Pyrrolopyrimidine nucleosides and analogs thereof
WO2020219598A1 (fr) * 2019-04-22 2020-10-29 The Board Of Trustees Of The Leland Stanford Junior University Méthodes de modulation de l'activité d'acétyltransférases et applications associées comprenant des traitements
US11111264B2 (en) 2017-09-21 2021-09-07 Chimerix, Inc. Morphic forms of 4-amino-7-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0088695A2 (fr) * 1982-03-09 1983-09-14 Cytogen Corporation Liaisons d'anticorps
US4814438A (en) * 1986-12-24 1989-03-21 Eli Lilly And Company Immunoglobulin conjugates of 2',2'-difluronucleosides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0088695A2 (fr) * 1982-03-09 1983-09-14 Cytogen Corporation Liaisons d'anticorps
US4814438A (en) * 1986-12-24 1989-03-21 Eli Lilly And Company Immunoglobulin conjugates of 2',2'-difluronucleosides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AUTIMICROBIAL AGENTS AND CHEMOTHERAPY, Volume 30, No. 5, issued November 1986, E. DECLERCQ et al., "Xylotubercidin against Herpes simplex virus Type 2 in mice", pages 719-724. *
JOURNAL OF IMMUNOLOGICAL METHODS, Volume 59, issued 1983, A. H. BLAIR et al., "Linkage of Cytotoxic Agents to Immunoglobulins", pages 129-143. *

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US6689803B2 (en) 1996-12-02 2004-02-10 Angiotech Pharmaceuticals, Inc. Compositions and methods for treating surgical adhesions
AU771994B2 (en) * 1996-12-02 2004-04-08 Ipxmedical, Llc Compositions and methods for treating or preventing inflammatory diseases
AU2004200715B2 (en) * 1996-12-02 2006-08-31 Angiotech International Ag Compositions and methods for treating or preventing inflammatory diseases
US9701706B2 (en) 2015-08-06 2017-07-11 Chimerix, Inc. Pyrrolopyrimidine nucleosides and analogs thereof
US9708359B2 (en) 2015-08-06 2017-07-18 Chimerix, Inc. Pyrrolopyrimidine nucleosides and analogs thereof
US10407457B2 (en) 2015-08-06 2019-09-10 Chimerix, Inc. Pyrrolopyrimidine nucleosides and analogs thereof
US10941175B2 (en) 2015-08-06 2021-03-09 Chimerix, Inc. Pyrrolopyrimidine nucleosides and analogs thereof
US11981700B2 (en) 2015-08-06 2024-05-14 Chimerix, Inc. Pyrrolopyrimidine nucleosides and analogs thereof
WO2017107817A1 (fr) * 2015-12-21 2017-06-29 江苏恒瑞医药股份有限公司 Procédé de préparation d'un conjugué anticorps-médicament
US11111264B2 (en) 2017-09-21 2021-09-07 Chimerix, Inc. Morphic forms of 4-amino-7-(3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-methyl-7H-pyrrolo[2,3-d]pyrimidine-5-carboxamide and uses thereof
WO2020219598A1 (fr) * 2019-04-22 2020-10-29 The Board Of Trustees Of The Leland Stanford Junior University Méthodes de modulation de l'activité d'acétyltransférases et applications associées comprenant des traitements

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