EP0584251A1 - Molecules ciblees sur un recepteur de cytokine par le traitement de la croissance de cellules neoplasiques - Google Patents

Molecules ciblees sur un recepteur de cytokine par le traitement de la croissance de cellules neoplasiques

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Publication number
EP0584251A1
EP0584251A1 EP92912726A EP92912726A EP0584251A1 EP 0584251 A1 EP0584251 A1 EP 0584251A1 EP 92912726 A EP92912726 A EP 92912726A EP 92912726 A EP92912726 A EP 92912726A EP 0584251 A1 EP0584251 A1 EP 0584251A1
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Prior art keywords
receptor
molecule
interleukin
cells
dab
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German (de)
English (en)
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EP0584251A4 (fr
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Cory Ann Waters
Louis R. Poisson
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Seragen Inc
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Seragen Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • 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
    • 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
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5406—IL-4
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • 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
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • This invention relates to the treatment of neoplastic cell growth, e.g., cancer.
  • the invention features a method for treating neoplastic cell growth in a patient, wherein the neoplastic cell is of non-lymphoid and non-monocytic origin ' .
  • the method includes administering to the patient a molecule which is capable of specifically binding to a cytokine receptor expressed on the neoplastic cell, which molecule is capable of decreasing the viability of the neoplastic cell.
  • the cytokine receptor is a receptor normally expressed on cells of lymphoid or monocytic origin.
  • the neoplastic cell is not of hematopoietic stem cell origin; the neoplastic cell is a sarcoma; the neoplastic cell is a osteosarcoma; the neoplastic cell is a fibrosarcoma; the neoplastic cell is a leiomyosarcoma; the neoplastic cell is a carcinoma; and the neoplastic cell is a rhabdomyosarcoma.
  • the carcinoma is a lung carcinoma; and the carcinoma is hepatocellular carcinoma.
  • the cytokine receptor is an interleukin receptor.
  • the interleukin receptor is an interleukin- 2 receptor; the interleukin receptor is an interleukin-4 receptor; arid the interleukin receptor is an interleukin- 6 receptor.
  • the interleukin-2 receptor is a high affinity interleukin-2 receptor.
  • the cytokine receptor is not expressed on non-neoplastic cells of non- lyphoid and non-monocytic origin.
  • the molecule kills cells bearing the cytokine receptor;
  • the molecule is a hybrid molecule includes a first and a second portion joined together covalently, the first portion includes a molecule capable of decreasing cell viability and the second portion includes a molecule capable of specifically binding to the cytokine receptor.
  • the second portion includes all or a binding portion of an antibody specific for the cytokine receptor; the second portion includes all or a binding portion of a ligand for the cytokine receptor; and the first portion includes a cytotoxin; and the antibody is a complement activating antibody.
  • the cytokine receptor is an interleukin receptor.
  • the ligand is an interleukin.
  • the cytotoxin is a fragment of a peptide toxin which is enzymatically active but which does not possess generalized eukaryotic receptor binding activity.
  • the fragment of a peptide toxin includes fragment A of diphtheria toxin and enough of fragment B of diphtheria toxin to facilitate translocation into the cytosol.
  • the molecule is DAB 389 IL-2; the molecule is DAB 389 IL-4; the molecule is DAB 389 IL-6; the molecule is DAB 48g IL-2; the molecule is DAB 486 IL-4; and the molecule is DAB 486 IL-6.
  • non-lymphoid and non-monocytic origin is meant all cells that are not descended from lymphoid stem cells or monocytes, e.g., cells other than T lymphocytes, B lymphocytes, and macrophages.
  • lymphoid and monocytic origin is meant cells descended from lymphoid stem cells or monocytes, e.g., T lymphocytes,
  • B lymphocytes, plasma cells, and macrophages B lymphocytes, plasma cells, and macrophages.
  • specifically binding is meant that the molecule does not substantially bind to other cell surface receptors.
  • not of he atopoietic stem cell origin is meant all cell are not descended from pluripotent stem cells.
  • DAB 3Cg IL-4 and DAB 40C IL-2 Reduce the Viability of Several Cancer Cell Types
  • interleukin-4 or interleukin-2 receptor targeted cytotoxins can provide a means by which to decrease the viability of cancer cells.
  • various cancer cell lines are shown to be sensitive to a fusion protein (DAB 389 IL-4) in which the receptor binding domain of diphtheria toxin (a single molecule of which, when present intracellularly, can block protein synthesis) has been replaced by a portion of human interleukin-4.
  • IC 50 is defined as the concentration of drug required to cause a 50% decrease in protein synthesis.
  • Cancer cell lines from striated muscle, smooth muscle, liver, bone, and lung are sensitive to DAB 389 IL-4; the IC50 for these cell lines was found to be 10 "10 M to 10 ⁇ 8 M.
  • Cytotoxicity was evaluated by measuring protein synthesis.
  • [ 14 C]leucine incorporation in the presence and absence of DAB 389 IL-4 (or DAB 486 IL-2) as follows. Cells were plated 96 well microtiter plates in growth medium appropiate to the cell type. DAB 389 IL-4 ((or DAB 48g IL-2) was added at various concentrations and the cultures were incubated for 20 hours prior to pulse labelling with [ 14 C]leucine. Cells were then trypsinized, harvested onto glass fiber filter mats and counted. The IC 50 is the concentration of cytotoxin which leads to a 50% decrease in [ 14 C]leucine incorporation.
  • Viability can also be measured using any standard viability assay appropriate to the cell type being studied. For example, viability can be determined using a trypan blue dye exclusion assay (Kruse et al., eds. Tissue Culture: Methods and Applications, Academic Press, 1989) .
  • the molecules useful in the method of the invention are tarageted to a cytokine receptor.
  • the molecules useful in the invention can act: (1) the molecule can kill a cell because the molecule has a cytotoxic domain; (2) the molecule (an antibody) can cause cell lysis by inducing complement fixation; and (3) the molecule can block binding or uptake of receptor's ligand.
  • the molecule must be targeted to receptor bearing cells; this is accomplished by including the receptor's ligand (or a portion or derivative thereof) or an anti-receptor antibody as part of the molecule.
  • Interleukin-2 (IL-2) receptor targeted molecules provide examples of each of these three approaches.
  • a fusion molecule which includes the IL-2 receptor binding portion of IL-2 and a cytotoxin can be used to kill neoplastic cells bearing the interleukin-2 receptor.
  • the second type"of molecule described above a complement fixing antibody, in this instance directed against the IL-2 receptor, can eliminate IL-2 receptor- bearing cells.
  • the third type of molecule could be a molecule that blocks binding of IL-2 to its receptor. This molecule would prevent neoplastic cells that bear the interleukin-2 receptor from receiving a proliferation signal from IL-2.
  • Molecules useful for treating neoplastic cell growth can take a number of forms.
  • the molecule can be a cytotoxic hybrid molecule in which IL-2 is fused to a toxin molecule, preferably a polypeptide toxin.
  • Derivatives of IL-2 which bind to IL-2R, lack IL-2 activity and block binding and/or uptake of bona fide IL-2 are useful in the method of the invention because they will prevent IL-2- induced proliferation of IL-2R bearing cells.
  • an anti-IL-2R antibody is the targeting agent
  • a cytotoxic hybrid molecule can be formed by fusing all or part of the antibody to a cytotoxin.
  • Anti-IL-2R antibodies which block binding and/or uptake of IL-2 are also useful in the method of the invention. Lytic anti-IL-2R antibodies are useful in the invention because they can cause complement-mediated lysis of IL-2R-bearing cells.
  • the molecules can be hybrid molecules formed by the fusion of all or part of two or more molecules.
  • the hybrid molecule can be a hybrid protein encoded by a reco binant DNA molecule, in which case the two domains are joined (directly or through an intermediary domain) by a peptide bond.
  • two domains can be produced separately and joined by a covalent bond in a separate chemical linkage step.
  • the cytotoxic domain of a hybrid molecule may itself be derived from two separate molecules.
  • Interleukin-2 as a Targeting Agent
  • Interleukin-2 or any IL-2 receptor binding derivative thereof can be used as a targeting agent for a cytotoxin.
  • the DNA and amino acid sequences of IL-2 are known (Tadatsugu et al.. Nature 302:305, 1983), and its structure has been predicted by x-ray crystallography (Brandhuber et al. , Science 238:1707, 1987).
  • Analysis of genetically engineered variants of IL-2 has provided some information concerning which residues are important for IL-2R binding (Collins et al., Proc. Natl . Acad. Sci . USA 85:7709, 1988) and bioactivity (Cohen et al. Science 234:349, 1989; Collins et al.
  • variants of IL-2 which are useful in the invention include deletion mutants (Genbauffe et al. , USSN 388,557, hereby incorporated by reference) which lack one or more amino acid residues in the region between residue 74 and residue 79 (numbering according to Williams et al., Nucl . Acids Res. 16:1045, 1988). These mutants effectively target toxins to IL-2R-bearing cells (Genbauffe et al., ' supra) . Generally, IL-2 variants useful for targeting a cytotoxin must efficiently bind IL-2R and be endocytosed.
  • the ability of various derivatives to bind to the IL-2 receptor can be tested with an IL-2R binding assay described below.
  • the human interleukin-2 receptor has a high-, an intermediate-, and a low-affinity form.
  • the high affinity receptor has an apparent K d of ⁇ 10 ⁇ 10 M and is composed of two subunits, p55 and p75 (also called p70) . When expressed on the cell surface, both the p75 and p55 subunits are capable of binding IL-2.
  • the p75 subunit corresponds to the intermediate affinity receptor (K d - 8.2 x 10 "10 M)
  • p55 subunit corresponds to the low affinity receptor (K d ⁇ 1-3 x 10 ⁇ 8 M)
  • the p75 subunit is expressed on the surface of resting T cells, natural killer cells, monocytes/macrophages, and lymphokine- 5 activated killer (LAK) cell precursors, while the high affinity receptor is expressed on activated T- and B- cells.
  • selectivity can be accomplished by administering the molecule at a concentration which does not permit significant binding to cells bearing lower affinity receptors.
  • a hybrid molecule may have altered receptor affinities compared to
  • Such hybrid molecules may be more or less selective for cells bearing the high affinity IL-2 receptor.
  • cells bearing the high-affinity receptor are 500-1000 times more sensitive to DAB 48g IL-2, a fusion protein consisting of part of diphtheria toxin
  • a cytotoxin can be attached to an IL-2 derivative in a number of ways.
  • an IL-2/toxin hybrid is •> 30 a hybrid protein produced by the expression of a fused gene.
  • the cytotoxin and the IL-2 derivative can be produced separately and later coupled by means of a non-peptide covalent bond. Linkage methods are described below.
  • Useful cytotoxins are preferably significantly cytotoxic only when present intracellularly and are substantially excluded from any given cell in the absence of a targeting domain. Peptide toxins fulfill both of these criteria and are readily incorporated into hybrid molecules.
  • a mixed cytotoxin, a cytotoxin composed of all or part of two or more toxins, can also be used.
  • Interleukin-4 is a cytokine which acts on a variety of cell types. Its receptor is expressed on a number of cell types, including CD4+ T cells and monocytes. IL-4 can act as a T cell growth factor and it is thought to have an influence on IL-2 induced lymphocyte proliferation.
  • a cytotoxin directed against IL-4 receptor-bearing cells or IL-6 receptor-bearing cells may enhance the effectiveness of molecules directed against IL-2R-bearing cells.
  • the protein and DNA sequence of IL-4 and IL-6 are known (Lee et al., ⁇ Biol . Chem. 263:10817, 1988; Hirano et al., Nature 324:73, 1986). These lymphokines can be used to create hybrid lymphokine/toxin molecules similar to IL-2/toxin hybrid molecules.
  • Monoclonal Antibodies as Targeting Agents Monoclonal antibodies directed against the lymphokine receptor of choice can be used to direct toxins to cells bearing that receptor.
  • antibodies or antibody fragments can be fused to a cytotoxin either by virtue of the toxin and the antibody being encoded by a fused gene which encodes a hybrid protein molecule, or by means of a non-peptide covalent bond which is used to join separately produced ligand and toxin molecules.
  • cytotoxin Several useful toxins are described below.
  • Antibody/toxin hybrids can be tested for their ability to kill receptor bearing cells using a toxicity assay similar to that which is described below for IL-2R bearing cells.
  • Monoclonal antibodies useful in the method of the invention can be made by immunizing mice with human IL- 2R or cultured T-lymphocytes, fusing the murine splenocytes with appropriate myeloma cells, and screening the antibodies produced by the resultant hybridoma lines for the requisite IL-2R binding properties by means of an ELISA assay.
  • Antibody production and screening can be performed according to Uchiyama et al. (J. Immunol .
  • useful antibodies may be isolated from a combinatorial library produced by the method of Huse et al. (Science 246:1275, 1989).
  • the invention can employ not only intact monoclonal or polyclonal antibodies, but also an immunologically-active antibody fragment, for example, a Fab or (Fab) 2 fragment; an antibody heavy chain, an antibody light chain; a genetically engineered single-chain Fv molecule (Ladner et al., U.S. Patent No.
  • the toxin molecules useful in the method of the invention are preferably toxins, such as peptide toxins, which are significantly cytotoxic only when present intracellularly.
  • toxins such as peptide toxins
  • the molecule must be able to enter a cell bearing the targeted receptor. This ability depends on the nature of the molecule and the nature of the cell receptor.
  • cell receptors which naturally allow uptake of a ligand are likely to provide a means for a molecule which includes a toxin to enter a cell bearing that receptor.
  • a peptide toxin is fused to an IL-2R binding domain by producing a recombinant DNA molecule which encodes a hybrid protein molecule. Such an approach ensures consistency of composition.
  • peptide toxins have a generalized eukaryotic receptor binding domain; in these instances the toxin must be modified to prevent intoxication of non-receptor bearing cells. Any such modifications must be made in a manner which preserves the cytotoxic functions of the molecule (see U.S. Department of Health and Human
  • Potentially useful toxins include, but are not limited to: cholera toxin, ricin, O-Shiga-like toxin (SLT-I, SLT-II, SLT II V ) , LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, Pseudomonas exot ⁇ xin, alorin, saporin, modeccin, and gelanin.
  • SLT-I, SLT-II, SLT II V O-Shiga-like toxin
  • Diphtheria toxin can be used to produce molecules useful in the method of the invention. Diphtheria toxin, whose sequence is known, is described in detail in Murphy U.S. Patent 4,675,382, hereby incorporated by reference.
  • the natural diphtheria toxin molecule secreted by Corynebacterium diphtheriae consists of several functional domains which can be characterized, starting at the amino terminal end of the molecule, as enzymatically-active Fragment A (amino acids Gly. - Arg 193 ) and Fragment B (amino acids Ser 194 - Ser 535 ) , which includes a translocation domain and a generalized cell binding domain (amino acid residues 475 through 535) .
  • diphtheria toxin intoxicates sensitive eukaryotic cells involves at least the following steps: (i) the binding domain of diphtheria toxin binds to specific receptors on the surface of a sensitive cell; (ii) while bound to its receptor, the toxin molecule is internalized into an endocytic vesicle; (iii) either prior to internalization, or within the endocytic vesicle, the toxin molecule undergoes a proteolytic cleavage between fragments A and B; (iv) as the pH of the endocytic vesicle decreases to below 6, the toxin crosses the endosomal membrane, facilitating the delivery of Fragment A into the cytosol; (v) the catalytic activity of Fragment A (i.e., the nicotinamide adenine dinucleotide - dependent adenosine diphosphate (ADP) ribosylation of the eukary
  • DAB 48g IL-2 a fusion protein in which the receptor binding domain of diphtheria toxin has been replaced by a portion of human IL-2 (Williams et al., J. Biol . Chem . 35:20673, 1990; see also Williams et al., Protein Eng. 1:493, 1987), is an example of a molecule useful in the method of the invention. This molecule selectively kills IL-2R-expressing tumor cells and lymphocytes (Waters et al., Eur. J. Immunol . 20:785, 1990; Kiyokawa et al., Cancer Res . 49:4042, 1989).
  • DAB 48g IL-2 Because of its ability to kill activated lymphocytes, DAB 48g IL-2 has been used to control graft rejection (Pankewycz et al., Transplantation 47:318, 1989; Kickman et al., Transplantation 47:327, 1989) and to treat certain autoimmune disorders (Forte et al., 2nd International Symposium on Immunotoxins , 1990) .
  • DAB 48g IL-2 is a chimeric molecule consisting of Met followed by amino acid residues 1 through 485 of the mature diphtheria toxin fused to amino acid residues 2 through 133 of IL-2.
  • DAB 48g IL-2 includes all of diphtheria toxin fragment A, which encodes the enzymatically active portion of the molecule, and a portion of fragment B.
  • the portion of fragment B present in DAB 48g IL-2 does not include the generalized receptor binding domain but does include the translocation domain which facilitates delivery of the enzymatically active portion into the cytosol.
  • Preparation of DAB 40C IL-2 and DAB 3CQ IL-2 DAB 48g IL-2 was produced in E.
  • DAB 48g IL-2 encoding plasmid, pDW24 (Williams et al., J. Biol. Chem. 265:20673, 1990, except amp r is replaced by kan r ) .
  • the protein was purified by immunoaffinity chromatography and high pressure liquid chromatography (Williams et al., supra) .
  • DAB 389 IL-2 can be prepared as described below for DAB 389 IL-4 by substituting IL-2 for IL-4. Preparation of DAB 30!? IL-4 and DAB 48( -IL-2
  • a synthetic gene encoding human interleukin-4 was synthesized (Milligen/Biosearch 7500 DNA synthesizer) .
  • the IL-4 sequence (Yodota et al. , Proc Nat ' l Acad Sci . USA, 83:58994, 1986) was modified to incorporate E. coli- preferred codon usage (deBoer et al., in Maximizing Gene Expression, Reznikioff et al., eds., 1986, Butterworths, Boston) , and restriction endonuclease cleavage sites were added to facilitate subsequent cloning steps.
  • IL-4 coding sequence (His to Ser ) was inserted into pDW27 plasmid.
  • pDW27 is derived from pDW24 (Williams et al., J. Biol. Chem. 265:11885, 1990) by deleting DNA corresponding to amino acids 388 to 485 of native diphtheria toxin.
  • DAB 48g IL-4 can be prepared as described above for DAB 48g IL-2 by substituting IL-4 for IL-2.
  • Cytotoxicity of DAB 389 IL-4 The ability of DAB 38g IL-4 to reduce viability of various cell types was measured using an inhibition of protein synthesis assay; the results of this assay are presented in Table 3.
  • l c 50 (M) is the concentration of DAB 389 IL-4 required for a 50% decrease in protein synthesis.
  • the rat, Con A-activated, normal splenic lymphocytes were far less sensitive to DAB 389 IL-4 than any of the other cells or cell lines. Since the rat interleukin-4 receptor does not bind human interleukin- 4, this result demonstrates the specificity of DAB 38g IL- 4. These rat cells are sensitive to a diphtheria toxin/rat interleukin-2 hybrid molecule. Preparation of DAB 38 -IL-6 and DAB 486 _5
  • a synthetic gene encoding human interleukin-6 was synthesized (Milligen/Biosearch 7500 DNA synthesizer) .
  • the IL-6 sequence (Revel et al., EPA 8611404.9) was modified to incorporate E. Coli preferred codon usage (deBoer et al., supra) , and restriction endonuclease cleavage sites were added to facilitate subsequent cloning steps.
  • the entire IL-6 coding sequence was inserted into pDW27 plasmid as described above for DAB 38g IL-4.
  • DAB 48g IL-6 can be produced as described above for DAB 48g IL-2 by substituting IL-6 for IL-2.
  • a mixed toxin molecule is a molecule derived from two different polypeptide toxins.
  • polypeptide toxins have, in addition to the domain responsible for generalized eukaryotic cell binding, an enzymatically active domain and a translocation domain.
  • the binding and translocation domains are required for cell recognition and toxin entry respectively.
  • the enzymatically active domain is the domain responsible for cytotoxic activity once the molecule is inside a cell.
  • Naturally-occurring proteins which are known to have a translocation domain include diphtheria toxin, Pseudomonas exotoxin A, and possibly other peptide toxins.
  • the translocation domains of diphtheria toxin and Pseudomonas exotoxin A are well characterized (see, e.g., Hoch et al.- Proc. Natl . Acad. Sci . USA 82:1692, 1985; Colombatti et al., J". i3iol. Chem. 261:3030, 1986; and Deleers et al., FEBS Lett.
  • IL-2/mixed toxin hybrid molecule is formed by fusing the enzymatically active A subunit of E. coli Shiga-like toxin (Calderwood et al., Proc. Natl . Acad. Sci . USA 84:4364, 1987) to the translocation domain (amino acid residues 202 through 460) of diphtheria toxin, and to IL-2.
  • This three-part hybrid molecule is formed by fusing the enzymatically active A subunit of E. coli Shiga-like toxin (Calderwood et al., Proc. Natl . Acad. Sci . USA 84:4364, 1987) to the translocation domain (amino acid residues 202 through 460) of diphtheria toxin, and to IL-2.
  • SLT-A/DTB'/IL-2 is useful in the method of the invention in the same way as DAB 48g IL-2 described above.
  • the IL-2 portion of the three-part hybrid causes the molecule to attach specifically to IL-2R-bearing cells, and the diphtheria toxin translocation portion acts to insert the enzymatically active A subunit of the Shiga-like toxin into the targeted cell.
  • the enzymatically active portion of Shiga-like toxin acts on the protein synthesis machinery of the cell to prevent protein synthesis, thus killing the cell.
  • the binding ligand and the cytotoxin of useful hybrid molecules can be linked in several ways. If the hybrid molecule is produced by expression of a fused gene, a peptide bond serves as the link between the cytotoxin and the binding ligand. Alternatively, the toxin and the binding ligand can be produced separately and later coupled by means of a non-peptide covalent bond.
  • the covalent linkage may take the form of a disulfide bond.
  • the DNA encoding IL-2 can be engineered to contain an extra cysteine codon as described in Murphy et al. U.S. Serial No. 313,599, hereby incorporated by reference.
  • the cysteine must be positioned so as to not interfere with the IL-2R binding activity of the molecule.
  • the cysteine codon can be inserted just upstream of the DNA encoding Pro 2 of the mature form of IL-2.
  • the toxin molecule must be derivatized with a sulfhydryl group reactive with the cysteine the modified IL-2.
  • a sulfhydryl group reactive with the cysteine the modified IL-2.
  • this can be accomplished by inserting a cysteine codon into the DNA sequence encoding the toxin.
  • a sulfhydryl group either by itself or as part of a cysteine residue, can be introduced using solid phase polypeptide techniques. For example, the introduction of sulfhydryl groups into peptides is described in Hiskey (Peptides 3:137, 1981). Derivatization can also be carried out according to the method described for the derivatization of a peptide hormone in Bacha et al. U.S. Patent No.
  • proteins can be derivatized at the DNA or protein chemistry level.
  • the introduction of sulfhydryl groups into proteins is described in Maasen et al. (Eur. J. Biochem. 134:32, 1983) .
  • the cytotoxin and the IL-2R binding ligand are then produced and purified, and the disulfide bond between the purified molecules formed by reducing both sulfur groups, mixing toxin and ligand, in a ratio of about 1:5 to 1:20, and allowing disulfide bond formation to proceed to completion (generally 20 to 30 minutes) at room temperature.
  • the mixture is then dialyzed against phosphate buffered saline to remove unreacted ligand and toxin molecules.
  • Sephadex chromatography or the like is then carried out to separate on the basis of size the desired toxin-ligand conjugates from toxin-toxin and ligand-ligand conjugates.
  • the IL-2R binding ability of various molecules can be measured using an IL-2R assay for high affinity (Ju et al., J. Biol . Chem . 262:5723, 1987) or intermediate affinity receptors (Rob et al. , Proc. Natl . Acad . Sci . USA 84:2002, 1987).
  • the IL-4R binding activity of various molecules can be measured using the assay described by Park et al. (J " . Exp. Med. 166:176, 1984) or the assay described by Foxwell et al. (Eur. J. Immunol . 19:1637, 1989).
  • Molecules of the invention can be screened for the ability to decrease viability of cells bearing the targeted receptor by means of assays such as those described below. Toxicity towards IL-2R bearing cells can be tested as follows. Cultured HUT 102/6TG (Tsudo et al., Proc . Natl . Acad . Sci . USA 83:9694, 1986) or YT2C2 (Teshigiwari et al., J. Exp. Med.
  • 165:223, 1987 cells are maintained in RPMI 1640 medium (Gibco, Grand Island, NY) supplemented with 25 mM HEPES (pH 7.4), 2mM 1-glutamine, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 10% fetal calf serum (Hazelton, Lenexa, KS) .
  • Cells are seeded in 96-well V-bottomed plates (Linbro-Flow Laboratories, McLean, VA) at a concentration of 1 x 10 5 per well in complete medium. Putative toxins are added to varying concentrations (10 "12 M to 10 "6 M) and the cultures are incubated for 18 hrs.
  • the plates are centrifuged for 5 min. at 170 x g, and the medium removed and replaced with 100 ⁇ l leucine-free medium (MEM, Gibco) contai .ning 8 ⁇ Ci./ml (3H-leucm. e; New England Nuclear, Boston, MA) .
  • MEM leucine-free medium
  • the plates are centrifuged for 5 min. at 170 x g, the medium is removed, and the cells are collected on glass fiber filters using a cell harvester (Skatron, Sterling, VA) . Filters are washed, dried, and counted according to standard methods. Cells cultured with medium alone serve as the control.
  • Toxicity towards cells bearing IL-4R may be tested by an assay similar to that described above for IL-2R bearing cells, but utilizing MLA144 cells (Rabin et al. J. Immunol . 127:1852/1981) or HUT 102/6TG cells, seeded at 1 x 10 5 cells per well and incubated for 40 hours. Therapy
  • the molecules of the invention will be administered by intravenous infusion. They may also be administered subcutaneously. Dosages of molecules useful in the methods of the invention will vary, depending on factors such as whether the substance is a cytotoxin, a lytic antibody, or an IL-2R blocking molecule. In the case of toxic molecules that act intracellularly, the extent of cell uptake is an important factor; less permeable molecules must be administered at a higher dose.
  • MTD maximum tolerated dose
  • Anti-tumor effects have been seen in approximately 40% of patients; responses were noted in B- cell leukemias and lymphomas, cutaneous T-cell lymphoma and Hodgkin's disease (LeMaistre et al., Blood 360a:abstract 1429, 1990; Woodworth et al.. Fourth International Conference on Human Retrovirology, 1991) .
  • Serum concentrations of 10 "8 M DAB 48g IL-2 have been achieved in patients with IL-2 receptor expressing malignancies.
  • drugs targeted to IL-2 receptor will be administered immediately after (e.g., within several minutes or less) vascular injury.
  • treatment begins before the accumulation of platelets and leukocytes. Animal models of denuding balloon catheter injury have been used to show that platelet aggregation and thrombus formation occurs immediately after injury and that leukocyte adhesion begins within several hours.
  • endothelial regeneration is complete within one to two weeks. Since re- endothelialization of the vessel wall appears to inhibit smooth muscle cell proliferation (Ip et al., J. Amer . Coll . Cardiol . 15:1667, 1990), treatment may need to last for only a few weeks. Accordingly, it is desirable to administer the compounds of the invention periodically over a period adequate to allow regeneration of the endothelium.
  • the hybrid molecule can be administered as an unmodified molecule or in the form of a pharmaceutically acceptable salt, admixed with a therapeutically acceptable carrier, e.g., saline.
  • a therapeutically acceptable carrier e.g., saline.
  • preferred salts are therapeutically acceptable organic acids, e.g., acetic, lactic, aleic, citric, or salicylic.
  • the hybrid molecule may be purified and sterile filtered using 2 micron filters and suspended in sterile phosphated buffer saline (0.15M NaCl; 0.02M phosphate buffer, pH 7.2) .
  • Embodiments Derivatives of IL-2 which block utilization of endogenous IL-2 are useful for preventing proliferation of IL-2R bearing cells. Activated cells deprived of IL- 2 fail to proliferate and, in the absence of the essential anabolic stimulus provided by IL-2, will eventually die. The ability of a given IL-2 derivative to interfere with IL-2 function can be tested in an IL-2 bioactivity assay such as the one described by Ju et al. (J. Biol . Chem. 262:5723, 1987). IL-2R/toxin hybrids in which the toxin has been rendered inactive can be also used to block IL-2 receptors.
  • a non-toxic mutant diphtheria toxin molecule has been described (Uchida et al. J. Biol . Chem. 248:3838, 1973), and this molecule can be used to produce a non-toxic IL-2/diphtheria toxin hybrid. See Svrluga et al. U.S. Serial No. 590,113, hereby incorporated by reference, for an example of such a hybrid molecule.
  • Monoclonal antibodies which interfere with the binding and/or uptake of IL-2 are useful, in the method of the invention because IL-2R bearing cells deprived of IL- 2 fail to proliferate. Blocking monoclonal antibodies can be tested for their ability to interfere with IL-2 bioactivity using the method of Ju et al. , (supra) .
  • Monoclonal antibodies which induce complement can be used to destroy IL-2R-bearing cells.
  • Complement inducing antibodies are generally those of the IgGl, IgG2, IgG3, and IgM isotypes.
  • Monoclonal anti-IL-2R antibodies can be screened for those able to induce complement using a complement-dependent cytotoxicity test, as follows.
  • Human T-lymphocytes and EBV transformed B- lymphocytes are labeled with Cr sodium chromate and used as target cells; these cells are incubated with hybridoma culture supernatants and with complement, and then the supernatants are collected and counted with a gamma counter. Those supernatants exhibiting toxicity against activated T-lymphocytes, but not resting T- or B- lymphocytes, are selected, and then subjected to a further screening step to select those supernatants containing antibody which precipitates (i.e., is specifically reactive with) the 50 kd glycoprotein IL-2 receptor (described in detail in Leonard et al. (Proc . Natl . Acad . Sci . USA 80:6957, 1983). The desired anti- IL-2 receptor antibody is purified from the supernatants using conventional methods.

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Abstract

Procédé de traitement de la croissance de cellules néoplasiques chez un patient, lesdites cellules néoplasiques étant d'origine non lymphoïde et non monocytaire. Ledit procédé consiste à administrer au patient une molécule capable de se lier spécifiquement à un récepteur de cytokine exprimé sur la cellule néoplasique, ladite molécule étant capable de diminuer la viabilité de la cellule néoplasique. Le récepteur de cytokine est un récepteur normalement exprimé sur les cellules d'origine lymphoïde ou monocytaire.
EP92912726A 1991-05-17 1992-05-15 Molecules ciblees sur un recepteur de cytokine par le traitement de la croissance de cellules neoplasiques. Withdrawn EP0584251A4 (fr)

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WO1991001004A1 (fr) * 1989-07-06 1991-01-24 Seragen, Inc. Molecules hybrides
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WO1991019745A1 (fr) * 1990-06-13 1991-12-26 The University Hospital Toxines chimeriques a geometrie inter-domaine amelioree

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US5082927A (en) * 1986-09-24 1992-01-21 The United States Of America As Represented By The Department Of Health And Human Services Selectively cytotoxic IL-4-PE40 fusion protein
US5084556A (en) * 1987-10-23 1992-01-28 Genetics Institute, Inc. Composition of M-CSF conjugated to cytotoxic agents and a method for treating cancers characterized by over-expression of the c-fms proto-oncogene

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WO1991001004A1 (fr) * 1989-07-06 1991-01-24 Seragen, Inc. Molecules hybrides
WO1991002000A1 (fr) * 1989-08-02 1991-02-21 Seragen, Inc. Mutants de il-2 a action deletive
WO1991019745A1 (fr) * 1990-06-13 1991-12-26 The University Hospital Toxines chimeriques a geometrie inter-domaine amelioree

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DATABASE BIOSIS BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US AN=91:424373, & ISITITUTO SUP. DI SANITA. VII INTERNATIONAL CONFERENCE ON AIDS: SCIENCE CHALLENGING AIDS; FLORENCE, ITALY, 16 June 1991 - 21 June 1991 page 79 C. F. LEMAISTRE ET AL. 'PHASE I-II EVALUATION OF AN IL-2 RECEPTOR TARGETED FUSION TOXIN DAB-486 IL-2 FOR TREATMENT OF AIDS-ASSOCIATED KAPOSI'S SARCOMA.' *
INT. J. CANCER (1991), 49(1), 96-101 CODEN: IJCNAW;ISSN: 0020-7136, 1991 BACHA, PATRICIA A. ET AL 'Impact of interleukin -2- receptor -targeted cytotoxins on a unique model of murine interleukin -2- receptor -expressing malignancy' *
LANCET THE, vol. 337, 11 May 1991 LONDON GB, pages 1124-1125, CHARLES F. LEMAISTRE ET AL. 'THERAPEUTIC EFFECTS OF GENETICALLY ENGINEERED TOXIN (DAB486IL-2) IN PATIENT WITH CHRONIC LYMPHOCYTIC LEUKAEMIA.' *
PROTEIN ENGINEERING, vol. 4,no. 4, 1991 ENGLAND GB, pages 463-468, TESUYUKI KIYOKAWA ET AL. 'PROTEIN ENGEERING OF DIPHTERIA-TOXIN-RELATED INTERLEUKIN-2 FUSION TOXINS TO INCREASE CYTOTOXIC POTENCY FOR HIGH-AFFINITY IL-2-RECEPTOR-BEARING TARGET CELLS.' *
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ZENTRALBL. BAKTERIOL., SUPPL. (1990), 19(BACT. PROTEIN TOXINS), 479-87 CODEN: ZBASE2, 1990 NICHOLS, J. C. ET AL 'Development of IL-2-toxin ( DAB486 -IL-2) for human clinical trials in interleukin -2 receptor positive malignancies' *

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