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WO1991016069A1 - Reactifs cytotoxiques selectifs comprenant des fractions toxiques derivees de proteines de mammiferes - Google Patents

Reactifs cytotoxiques selectifs comprenant des fractions toxiques derivees de proteines de mammiferes Download PDF

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WO1991016069A1
WO1991016069A1 PCT/US1991/001587 US9101587W WO9116069A1 WO 1991016069 A1 WO1991016069 A1 WO 1991016069A1 US 9101587 W US9101587 W US 9101587W WO 9116069 A1 WO9116069 A1 WO 9116069A1
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rnase
moiety
reagent according
cytotoxic reagent
cytotoxic
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Susanna M. Rybak
Richard J. Youle
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United States Department of Commerce
US Department of Health and Human Services
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US Department of Health and Human Services
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2881—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD71
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to directed cytotoxic reagents, including immunotoxins, that selectively kill cells having a given surface marker, consisting essentially of a toxic moiety that is derived from a mammalian protein, linked to a recognition moiety capable of specific binding with a chosen cell surface marker.
  • the present invention relates to such cytotoxic reagents comprising mammalian proteins with ribonucleolytic activity and antibodies or receptor binding ligands that recognize tumor cells or virus- infected cells.
  • Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and pseudomonas toxin have been coupled to antibodies or receptor binding ligands to generate cell-type-specific-killing reagents (Youle, R. and D. Neville, 1980, "Anti-Thy 1.2 monoclonal antibody linked to ricin is a potent cell-type-specific toxin.” Proc Natl Acad Sci USA 77:5483-5486; Gilliland, D. , Z. Steplewski, R. Collier et al .
  • hybrid proteins kill tumor cells, for example, which express the receptor that the antibody or ligand portion of the molecule recognizes. Under appropriate conditions, conferred by the particular receptor system, the toxin enters the cytosol, inactivates the protein synthesis machinery and causes death of the target cell.
  • Immunotoxins are highly cytotoxic to cancer cells growing in cell culture and animal models demonstrate the potential of these reagents to treat blood borne malignancies as well as solid tumors in restricted compartments such as the intraperitoneal cavity (reviewed in Griffin, T. W. et al . , 1988, Immunotoxins.
  • Transferrin is a serum glycoprotein that binds and delivers iron to cells by receptor mediated endocytosis reviewed in (Youle, R. and D. Neville, 1987, Immunoconiugates: Antibody Conjugates in Radioimaging and Therapy of Cancer. Oxford, Oxford University Press) . After relinquishing its iron apo-Tfn- receptor recycles to the cell surface where apo-Tfn is released to continue the cycle. Monoclonal antibodies originally isolated based upon selectivity for tumor cells have been found to react with the human transferrin receptor. Transferrin (Raso, V. and M.
  • Toxic ribosome inactivating proteins from plants inactivate protein synthesis by enzymatically cleaving a single N-glycosidic bond of the 28S ribosomal RNA (Endo, Y. and K. Tsurugi, 1987, "Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. RNA-N- glycosidase activity of ricin A-chain.” J Biol Chem. 262: 8128-8130).
  • Other cytotoxic proteins that inactive ribosomes include ⁇ -sarcin, which is produced by a fungus (Endo, T. and I. Wool, 1982, J Biol Chem.
  • a human serum ribonuclease (angiogenin) was shown to abolish cell-free protein synthesis by inactivating the small ribosomal subunit of rabbit reticulocyte ribosomes (St. Clair, D. , S. Rybak, J. Riordan and B. Vallee, 1987, "Angiogenin abolishes cell-free protein synthesis by specific ribonucleolytic inactivation of ribosomes.”
  • the present invention stems from research of the inventors aimed at identifying mammalian proteins with potential for use as toxic moieties in cytotoxic reagents that selectively kill cells having a given surface marker. More specifically, the present invention resulted from efforts to determine whether or not mammalian RNAase, delivered to cells via the transferrin receptor-mediate endocytosis pathway, would be toxic to cells.
  • cytotoxic reagents that selectively kill cells having a given surface marker, comprising a toxic moiety that is a mammalian protein which is endogenous to the species in which the reagent is intended for use or is otherwise minimally immunogenic.
  • a toxic moiety that is a mammalian protein which is endogenous to the species in which the reagent is intended for use or is otherwise minimally immunogenic.
  • it is an object of this invention to provide toxic moieties with less systemic toxicity than presently known toxins used in directed cytotoxic reagents.
  • direct cytotoxic reagents comprising mammalian proteins with ribonucleolytic activity and antibodies or receptor binding ligands that recognize specific markers on tumor cells or virus-infected cells.
  • Bovine pancreatic RNase A is used in an initial embodiment of the present invention because of its mammalian origin and its ready availability. Further, it is known that administration of bovine RNase A to human patients (in the treatment f tick-borne encephalitis, for example; Glukhov, B. N. , Jerusalimsky, A.P., Canter, V. M. , and Salganik, R. I., 1976, Arch. Neurol. 38:598-603) does not produce any allergic reactions, and this seem to be connected with its weak antigenic activity. This application discloses a new use for RNAse.
  • the complex When it is chemically linked to a receptor-specific ligand, such as transferrin (Tfn) , the complex specifically kills target cells bearing the Tfn-receptor.
  • a receptor-specific ligand such as transferrin (Tfn)
  • Tfn transferrin
  • This particular directed toxin complex is but one convenient exemplary embodiment of the broader concept of the present invention where a mammalian protein is used as a toxic moiety in a directed cytotoxin.
  • This complex further comprises a recognition moiety capable of specific binding with a chosen cell surface marker, where preferably both moieties are endogenous to the species in which the cytotoxin complex is to be used, thereby minimizing the immunogenicity of the complex and extending the period of useful administration.
  • the present invention relates to a selective cytotoxic reagent consisting essentially of: a toxic moiety that is a mammalian protein or a modified form thereof; a recognition moiety that binds a specific cellular surface marker; and a means of linking said toxic moiety and said recognition moiety. wherein binding of said recognition moiety to said surface marker on a cell thereby causes said toxic moiety to kill said cell.
  • the toxic mammalian protein in this cytotoxic reagent is endogenous to the species in which the reagent is intended for use.
  • the toxic mammalian protein has ribonucleolytic activity, for example, bovine ribonuclease A or, most preferably for human applications, human angiogenin.
  • ribonucleolytic activity for example, bovine ribonuclease A or, most preferably for human applications, human angiogenin.
  • other members of the ribonuclease superfamily in mammals have biological properties related to ribonuclease A and angiogenin and, therefore, are useful as the toxic moiety in the present invention, including bovine seminal ribonuclease, cytotoxic eosinophile granule proteins, and human eosinophil- derived neurotoxin (EDN) which is identical to the nonsecretory ribonuclease from human urine.
  • EDN human eosinophil- derived neurotoxin
  • the recognition moiety of the cytotoxic reagent according to the present invention is preferably a mammalian protein or a modified form thereof.
  • the recognition moiety of the cytotoxic reagent is endogenous to the species in which said reagent is intended for use.
  • the recognition moiety is human transferrin or a modified form thereof, which preferentially binds cells with high levels of the transferrin receptor, for example, certain tumor cells. This embodiment is preferred for human applications in compartments where endogenous levels of transferrin are sufficiently low to avoid competitive interference with binding of the reagent to the transferrin receptor.
  • the recognition moiety may also be an antibody or a modified form thereof which binds a specific cellular surface marker on the type of cells that are to be killed.
  • the recognition moiety is an antibody or portion thereof which recognizes the transferrin receptor in such a way that it is not competitively blocked by transferrin.
  • a modified form of a protein includes chemically modified forms as well as mutant forms created through genetic engineering. Chemical modifications include, for example, derivitization for the purpose of linking the moieties to each other, either directly or through a linking compound, by methods that are well known in the art of protein chemistry.
  • the means of linking the toxic moiety and the recognition moiety comprises a heterobifunctional coupling reagent which ultimately contributes to formation of an intermolecular disulfide bond between the two moieties.
  • Other types of coupling reagents that are useful in this capacity for the present invention are described, for example, in U.S. patent 4,545,985, Pseudomonas exotoxin conjugate immunotoxins.
  • the intermolecular disulfide may conveniently be formed between cysteines in each moiety which occur naturally or are inserted by genetic engineering.
  • the means of linking moieties may also use thioether linkages between heterobifunctional crosslinking reagents or specific low pH cleavable crosslinkers or specific protease cleavable linkers or other cleavable or noncleavable chemical linkages.
  • the means of linking moieties of the cytotoxic reagent may also comprise a peptidyl bond formed between moieties which are separately synthesized by standard peptide synthesis chemistry or recombinant means.
  • Possible chemical modifications of the protein moieties of the present invention also include derivatization with polyethylene glycol (PEG) to extend time of residence in the circulatory system and reduce immunogenicity, according to well known methods (see for examples, Lisi, P. J., Van Es, T., Abuchowski. A., et al ., 1982, Enzyme Therapy, Applied Biochem. 4.:19-33; Beauchamp, C. 0., Gonias, S. L. , Menapace, D. P. et al . , 1982, Anal. Biochem. 131:25-33; and Goodson, R. J., and Katre, N.V. , 1990, Bio/Technolo ⁇ y 8:343-346).
  • PEG polyethylene glycol
  • Possible genetic engineering modifications of the proteins of the cytotoxic reagent include combination of the relevant functional domains of each into a single chain multi-functional biosynthetic protein expressed from a single gene derived by recombinant DNA techniques.
  • a single chain multi-functional biosynthetic protein expressed from a single gene derived by recombinant DNA techniques.
  • the cytotoxic agent of the present invention may be directed toward various different types of cells by appropriate selection of a recognition moiety that binds to a specific cellular surface marker found specifically or predominantly on the type of cell that is to be selectively killed.
  • the cytotoxic reagent of this invention includes those with a recognition moiety that binds to a tumor cell-specific surface marker, of which many are known in the art.
  • the recognition moiety is human transferrin or a modified form thereof, which preferentially binds cells with high levels of the transferrin receptor, particularly certain tumor cells, as noted in the Background.
  • the recognition moiety advantageously binds to a marker of that infectious agent on the surface of an infected cell.
  • reagents comprising recognition moieties for cells infected by a virus, especially including latent or chronic virus infections, for examples, Epstein-Barr Virus, herpesviruses (herpes simple types I and II) , hepatitis viruses (B, non-A-non-B, and delta) , herpes zoster, and cytomegalovirus.
  • the recognition moiety of virus-specific cytotoxic reagents of this invention conveniently may be an antibody, advantageously the anti-viral recognition moiety may be a cellular receptor for a virus or a modified form thereof.
  • the recognition moiety of the present cytotoxic agent is advantageously the CD4 receptor protein.
  • This receptor protein is the point of recognition between the HIV-1 virus and its specific target cell and, therefore, the envelope proteins of all immunologically distinct HIV-1 strains must nevertheless be able to bind to the CD4 protein.
  • use of the CD4 protein in the present invention will direct the toxic moiety to kill cells infected by HIV-1 and expression on their surface the envelope protein which normally becomes embedded in the surface of cells producing that viral protein.
  • the recognition moiety is directed to a component of the agent which appears on the surface of infected cells, such as a marker for the meroz ⁇ ite form of a malaria parasite.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a cytotoxic reagent of the present invention and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is suitable for parenteral administration.
  • the cytotoxic reagent of the present invention may be administered by various means appropriate for different purposes, for example, for treating tumors in various parts of the body, according to methods known in the art for other immunotoxins. (See, for example, Rybak, S.M. and Youle, R.J., 1990, "Human Cancer Immunology", in Immunology and Allergy Clinics of America, W. B. Saunders, and references cited therein) .
  • these means of administration include, for example, injection by intravenous, intrathecal, intratumoral, intraocular, and intraarterial routes, to bathe particular tumor beds or the brain in high concentrations of the reagent; administration directly into the wound during surgery (intracranially, for example) ; administration in an enema (for colon cancer, for instance) ; administration in the form a spray into the bronchi or nasopharyngeal cavity; administration topically (for example, for treating melanoma) ; administration by drip into the peritoneal cavity (e.g., for certain forms of ovarian cancer); and administration transvaginally or transurethrally.
  • the present invention also relates to pharmaceutical compositions comprising a cytotoxic reagent of this invention and a pharmaceutically acceptable carrier, particularly such compositions which are suitable for the above means of administration.
  • the present invention relates to a method of selectively killing cells using a selective cytotoxic reagent consisting essentially of: a toxic moiety that is a mammalian protein or a modified form thereof; a recognition moiety that binds a specific cellular surface marker; and a means of linking said toxic moiety and said recognition moiety, wherein binding of said recognition moiety to said surface marker on a cell thereby causes said toxic moiety to kill said cell.
  • This method of the present invention may be used for cell separation in vitro by selectively killing unwanted types of cells, for example, in bone marrow prior to transplantation into a patient undergoing marrow ablation by radiation, for killing leukemia cells or T-cells that would cause graft-versus-host disease.
  • the mammalian protein of the reagent used in this method is endogenous to the species in which said reagent is intended for use.
  • Specific in vivo methods of this invention include a method for the chemotherapeutic alleviation of cancer in mammals comprising administering a chemotherapeutically alleviating amount of a selective cytotoxic reagent according to the present invention.
  • This invention further comprises a method for the chemotherapeutic alleviation of an infectious disease in mammals comprising administering a chemotherapeutically alleviating amount of a selective cytotoxic reagent according to the present invention.
  • Fig. 2 Inhibition of protein synthesis in K562 cells by Tfn-RNase conjugate compared with component proteins.
  • K562 cells (1 x 10 5 cells/ml) were plated into 96 well microtiter plates and treated with varying concentrations of Tfn-RNase (filled circles) , RNase (filled squares) , or SPDP derivatized RNase (filled triangles) . Additional sets of wells contained RNase or
  • Fig. 3 Time course of protein synthesis inhibition caused by Tfn-RNase in K562 cells.
  • K562 cells (2 x 10 5 cells/ml) were treated as described in the legend to Fig. 2.
  • Different concentrations of Tfn-RNase were added and the cells were processed for [ 14 C]leucine incorporation as described. The times indicated include a 1 hour pulse with [ 14 C]leucine.
  • the data points were determined as described in the legend to Fig. 2.
  • Fig. 4 Clonogenic growth assay of K562 cells treated with Tfn-RNase.
  • K562 cells (1.4 x 10 5 cells/ml) were treated with buffer A, 0.1M NaCl-O.lM NaP0 4 , pH7.2 or buffer A which contained SPDP-RNase (7xlO "5 M) .
  • Another set in the same experiment contained K562 cells treated with buffer B, 0.1M NaPO*, pH7.2 or buffer B containing Tfn-RNase at 10 "6 or 10 "7 M. After 24 hour treatment the cells were washed, diluted into complete medium and plated into 96 well microtiter plates (10 wells/dilution) .
  • Fig. 5 Inhibition of protein synthesis by a monoclonal antibody against the Tfn receptor coupled to RNAse.
  • Cells were incubated as described in Figure 2 with Fr27, a monoclonal antibody B3-25 against the human transferrin receptor coupled by a disulfide linkage to RNase.
  • Fr28 is a repeat experiment. SPDP-RNase alone was also incubated with K562 cells.
  • a presently preferred embodiment of the present invention is exemplified by a cytotoxic reagent in which the toxic moiety is bovine pancreatic ribonuclease A and the recognition moiety is human transferrin, the target being tumor cells having high levels of the transferrin receptor.
  • RNase A is the first enzyme for which the amino acid sequence was determined and is so well defined chemically and physically that it has been a major test protein in the study of a wide variety of methods in protein chemistry since the 1950s. (Blackburn, P., and Moore, S., The Enzymes XV Part B: 317-433).
  • Bovine RNase A is a monomeric, non-glycosylated basic protein (PI 9.5) with a M r of 13.8 KDa, that cleaves RNA endonucleolytically to yield 3'-phospho-mono- and oligonucleotides ending in Cp or Up. Thus, it is a member of the pyrimidine-specific mammalian neutral and 17 alkaline RNases (reviewed in Beintema, J.J., et al . , 1988, Biochemistry 27:4530-38).
  • Transferrin-RNase Conjugate A heterobifunctional reagent, N-succinimidyl 3- (2-Pyridyldithio) propionate (SPDP) , was used to introduce 2-pyridyl disulfide groups into bovine pancreatic RNase.
  • the derivatized RNase was reacted with 2-iminothiolane (2-IT) treated human transferrin (Tfn) to form a conjugate via intermolecular disulfide bond formation.
  • RNase-Tfn conjugates were separated from unreacted RNase and Tfn multimers by gel filtration on a TSK-3000 HPLC column.
  • Intact RNase-transferrin conjugate displayed potent RNase activity that was linear in the range from 1 to 10 nM in an assay that measured release of acid soluble nucleic acids from tRNA. This activity was 3-10 times less than that expected from the calculated amount of RNase in different conjugate preparations compared to a known standard.
  • the increased activity of the conjugate compared to RNase depends on the chemical linkage to Tfn since mixtures of Tfn and RNase do not significantly affect protein synthesis compared to RNase alone (Fig. 2) .
  • Protein synthesis in guinea pig L2C cells, monkey Vero cells and human TE671 rhabdomyosarcoma cells was also inhibited by Tfn-RNase (5xl0 "8 M-lxl0 "7 M) .
  • Tfn-RNase inhibits protein synthesis at concentrations 10,000 fold lower than free RNase.
  • transferrin mediates the toxicity of the conjugate via binding the transferrin receptor
  • excess transferrin was incubated with the conjugate to compete for binding.
  • Table 1 shows that 70 ⁇ g/ml of Tfn blocks the action of Tfn-RNase 10-fold.
  • two inhibitors of pancreatic bovine RNase were added to the protein synthesis inhibition assay. The addition of PRI or a new more potent inhibitor of RNase to culture medium along with Tfn-RNase blocked the activity of the conjugate.
  • both protein components of the conjugate are required for the inhibition of protein synthesis by the Tfn-RNase conjugate.
  • Tfn-RNase The time course for Tfn-RNase at four different concentrations is shown in Fig. 3. Like ricin and diphtheria toxin Tfn-RNase exhibits a dose dependent lag time and then protein synthesis decreases according to a first order process. The highest concentration of Tfn- RNase (5xl0 "7 M) presented in Fig. 3 was saturating since higher concentrations did not significantly increase the steepness of the slope (not shown) .
  • the rate of protein synthesis inactivation by both ricin and diphtheria toxin increases with increasing concentration of the toxin.
  • the relationship between the rate of killing by the toxins and concentration of toxin is not linear but increases proportional to the square root of the toxin concentration. This diminishes the achievable extent of killing of target cells.
  • the relationship between the killing rate and the concentration of the Tfn-RNase conjugate does not follow the square root function as does ricin and diphtheria toxin but correlates linearly with concentration. This should enable increasing doses of Tfn-RNase to yield proportionate gains in log target cell kill.
  • Tfn-RNase ricin A-chain conjugated with transferrin
  • Tfn-RNase Clonagenic Assay of Tfn-RNase. The extent of the cell killing in clonagenic assays was performed. After 24 hours in the presence of conjugate K562 cells were washed, resuspended in complete medium, diluted serially and plated into 96 well microtiter plates. The wells that contained surviving cells were scored after 2 wks and the results of a representative experiment is presented in Fig. 4. Tfn-RNase (10 ⁇ 7 ) killed between 2-3 logs of cells and a concentration of 10 "6 M killed at least 6 logs of cells. In contrast to the results in the protein synthesis assay (Fig. 1) the clonogenic assay indicates no toxicity of 7 x 10-5 M SPDP-RNase. The elimination of 6 logs of cells can be compared to 3-4 log cell kill for RTA conjugates.
  • Transferrin in plasma is about 4 mg/ml in man and may block the toxicity of Tfn-RNase in vivo.
  • the central nervous system in an important site of metastases of peripheral tumors such as breast and lung cancer as well as a site of primary tumors.
  • the CSF fluid contains 14 ⁇ g/ml free transferrin, an amount that would block the toxicity of Tfn-RNase only 2 fold.
  • the toxicity of Tfn-RNase to animals was examined after direct injection into the CSF fluid. Tfn-RNase was injected into the CSF fluid of rats and guinea pigs to yield an initial concentration of 2 x 10-6 M, twice the concentration that killed 6 logs of cells in culture. No toxicity was observed in these animals.
  • a monoclonal antibody specific for the human transferrin receptor, B3/25 was coupled to RNase by the same method as transferrin resulting in a disulfide coupled conjugate. Incubating K562 cells for 24 hours with the conjugate, B/25-RNase at 1 x 10 "7 M to 1 x 10 "8 M resulted in 50 to 80% inhibition of protein synthesis (Fig. 5) .
  • the antibody is specific for the human transferrin receptor and does not bind the green monkey transferrin receptor.
  • a green monkey cell line, Vero was not affected by the same concentration of B3/25-RNase conjugate.
  • Bovine pancreatic RNase A was purchased from CALBIOCHEM (San Diego, CA) .
  • Human placental ribonuclease inhibitor (PRI) from Promega Biotech (Madison, Wl) and Inhibit-ACE RNase was from 5--3 1 (Paoli, PA).
  • Human transferrin and tRNA type X was from Sigma (St. Louis, MO).
  • Dithiothreitol (DTT) N-Succinimidyl 3-(2-
  • SPDP Pyridyldithio)propionate
  • 2-IT 2-Iminothiolane
  • K562 human erythroleukemia-derived cell line
  • RPMI 1640 medium containing 10% fetal calf serum (FCS) , 2mM glutamine, ImM sodium pyruvate, and 10 ⁇ g/ml gentamycin.
  • Vero monkey kidney cell derived line
  • TE671 human myosarcoma derived cell line
  • the cell lines were grown at 37—C in 5% C0 2 in a humidified atmosphere.
  • I ⁇ C leukemia is a spontaneous transplantable B cell leukemia of Strain 2 guinea pigs.
  • I ⁇ C leukemia cells were harvested from the blood of animals in the terminal stage of the leukemia as previously described (Zovickian, J. and R. Youle, 1988, "Efficacy of intrathecal immunotoxin therapy in an animal model of leptomeningeal neoplasia.” J Neurosurg. 68:767-774) . The prepared cells were used within 24 h.
  • the conjugate was purified by gel filtration on a TSK-3000 high pressure liquid chromatography (HPLC) column. Individual peaks were characterized biologically using inhibition of protein synthesis in K562 cells as an assay. The greatest activity was associated with the peak that contained transferrin and RNase in a 1:1 molar ration which was determined by reducing the conjugate to its individual proteins followed by HPLC analysis. The amount of total protein in the conjugate was quantified by Lowry assay using BSA as a standard. Protein synthesis Assay. Protein synthesis in cells growing in suspension or in adherent cells was measured as previously described (Johnson, V. , D. Wilson, L. Greenfield and R.
  • Clonagenic Cell Assay The number of clonagenic cells surviving treatment with conjugate or other additions was determined by using a limiting dilution assay. Cells were treated with additions in a 1 ml volume in 24 well plates for 18-24 hr under the same culture conditions described in the section on protein synthesis assays. The cells were harvested by centrifugation and washed with complete culture medium. The washed cells were resuspended in complete growth medium and 6 serial 10 fold dilutions were made. Ten aliquots (100 ⁇ l) of each dilution were plated in 96-vell microtiter plates. Plates were incubated for 14 days at 37—C in a humidified atmosphere. Medium was replenished every 3-4 days.
  • RNA was dissolved at 1 mg/ml in water and added to a reaction mixture containing RNase and buffer (Tris, 0.5M, pH 7.5, EDTA 5 mM, human serum albumin, 0.5 mg/ml) in a total volume of 300 ml in polypropylene microfuge tubes. The mixture was incubated for 30 min at 37—C and then placed on ice. Perchloric acid (6%, 700 ml) was added and the mixture was left on ice for 10 min. and then microfuged for 10 min. at 4—C. An aliquot of the supernatant was read at 260nm. The unknowns were compared to a standard curve of bovine pancreatic RNase A.

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Abstract

Le couplage de ribonucléase pancréatique A à de la transferrine humaine par l'intermédiaire d'une liaison disulfure avait pour résultat une protéine hybride qui était cytotoxique pour des cellules de mammifères in vitro. Lors de la mesure de la capacité de synthèse de protéines de cellules de myélose érythrémique aiguë humaines K562, le IC50 de différentes préparations de transférine-RNase s'est situé entre 8 x 10-9 et 8 x 10-8 M tandis que le IC¿50? de RNase ou de SPDP RNase seule était 1000 à 10 000 fois supérieur. Dans une analyse clonogénique 1 x 10?-6¿ M du conjugué de transférine-Rnase a tué 6 logs de cellules tandis que 7 x 10-5 M de la RNase modifiée par respect des P n'a pas eu d'effet significatif. L'inhibition de la synthèse de protéines par transferrine-RNase a été bloquée par la transferrine excédentaire ou des inhibiteurs de ribonucléase dans le milieu démontrant que la cytotoxicité dépend à la fois des deux constituants du conjugué. La RNase de sérum humain peut également présenter des toxicités induites par récepteur constituant une nouvelle approche de la possibilité d'élimination sélective de cellules avec une toxicité systémique inférieure et, cela est important, moins d'immunogénicité que les conjugués d'anticorps-toxines actuellement employés.
PCT/US1991/001587 1990-04-17 1991-03-13 Reactifs cytotoxiques selectifs comprenant des fractions toxiques derivees de proteines de mammiferes Ceased WO1991016069A1 (fr)

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WO1994015644A1 (fr) * 1993-01-15 1994-07-21 Imperial Cancer Research Technology Limited Composes de ciblage
GB2289679A (en) * 1993-01-15 1995-11-29 Imp Cancer Res Tech Compounds for targeting
US6074836A (en) * 1993-09-01 2000-06-13 Boehringer Mannheim Gmbh Method of marking eukaryotic cells
US6268214B1 (en) 1996-04-04 2001-07-31 Roche Diagnostics Gmbh Vectors encoding a modified low affinity nerve growth factor receptor
EP1393750A2 (fr) 1992-04-07 2004-03-03 Immunomedics, Inc. Conjugués pour le traitement d'une maladie immunitaire

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1393750A2 (fr) 1992-04-07 2004-03-03 Immunomedics, Inc. Conjugués pour le traitement d'une maladie immunitaire
EP1393750A3 (fr) * 1992-04-07 2004-03-31 Immunomedics, Inc. Conjugués pour le traitement d'une maladie immunitaire
EP1375519A3 (fr) * 1992-04-07 2004-06-30 Immunomedics, Inc. Methode pour influencer les fonctions cellulaires en utilisant des anticorps
US7811570B2 (en) 1992-04-07 2010-10-12 Immunomedics, Inc. Method of affecting a function of or ablating a non-malignant cell
WO1994015644A1 (fr) * 1993-01-15 1994-07-21 Imperial Cancer Research Technology Limited Composes de ciblage
GB2289679A (en) * 1993-01-15 1995-11-29 Imp Cancer Res Tech Compounds for targeting
GB2300859A (en) * 1993-01-15 1996-11-20 Imp Cancer Res Tech Compounds comprising a target cell-specific portion (TCP) and a cytotoxic portion (CP)
GB2289679B (en) * 1993-01-15 1997-02-05 Imp Cancer Res Tech Compounds comprising a target cell-specific portion and a portion which has DNA endonucleolytic activity, and uses thereof
GB2300859B (en) * 1993-01-15 1997-06-18 Imp Cancer Res Tech Compounds comprising a target cell-specific portion and a further portion
EP0815872A3 (fr) * 1993-01-15 2003-12-10 Cancer Therapeutics Limited Composés de ciblage
US6074836A (en) * 1993-09-01 2000-06-13 Boehringer Mannheim Gmbh Method of marking eukaryotic cells
US6268214B1 (en) 1996-04-04 2001-07-31 Roche Diagnostics Gmbh Vectors encoding a modified low affinity nerve growth factor receptor

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