WO1998018820A1 - Procedes et compositions relatifs a des immunotoxines de la proteine de fusion du ricin, pour le traitement du cancer et de maladies auto-immunes - Google Patents

Procedes et compositions relatifs a des immunotoxines de la proteine de fusion du ricin, pour le traitement du cancer et de maladies auto-immunes Download PDF

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WO1998018820A1
WO1998018820A1 PCT/US1997/019577 US9719577W WO9818820A1 WO 1998018820 A1 WO1998018820 A1 WO 1998018820A1 US 9719577 W US9719577 W US 9719577W WO 9818820 A1 WO9818820 A1 WO 9818820A1
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fusion protein
ricin
amino acid
chain
ligand
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PCT/US1997/019577
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English (en)
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Arthur Edward Frankel
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Medical University Of South Carolina
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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
    • C07K14/435—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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • 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
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • 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

Definitions

  • the present invention relates to a ricin fusion protein immunotoxin and methods for treating a cancer or autoimmune disease with this immunotoxin.
  • the present invention provides a ricin fusion protein immunotoxin comprising a ricin toxin B chain from which three lectin binding sites have been removed, conjugated to a ligand specific for a particular cell surface receptor and associated with a ricin toxin A chain.
  • methods for administering the ricin fusion protein immunotoxin to treat a cancer or autoimmune disease in a patient diagnosed with a cancer or autoimmune disease are also provided.
  • Ricin toxin a glycoprotein produced in the seeds ⁇ Ricinus communis plants consists of a galactose-binding B chain (RTB) disulfide linked to an rRNA N-glycosidase A chain (RTA).
  • RTB galactose-binding B chain
  • RTA rRNA N-glycosidase A chain
  • the 65 kilodalton heterodimeric glycoprotein binds to cell surface galactose-terminated oligosaccharides via lectin binding sites in RTB (1) and undergoes receptor-mediated endocytosis (2). After trafficking to the Golgi (3), the toxin is transported to a distal compartment (4) from which the intersubunit disulfide bond is reduced (5).
  • RTA then translocates to the cytosol and catalytically inactivates protein synthesis by hydrolysis of a specific adenine base from the 26S ribosomal RNA (6).
  • Galactose binding is important for cell binding and may be needed for internalization and intracellular trafficking of ricin (34).
  • Ricin toxin is one of the most toxic substances known to man. A single molecule is capable of causing cell death (7), and the LD 50 of ricin in 20 gram C57 B16 mice is reported to be 60 nanograms or 6 x 10 11 molecules (8). Histopathological examination of mice given toxic doses of ricin failed to show any definite abnormalities (8). Thus, the critical target organ for ricin is unknown.
  • the x-ray crystallographic structure of ricin revealed two domains each with three subdomains each with similar folding and primary amino acid sequence (13). While all six subdomains had ⁇ -carbon chains forming a loop, twist and hook, only four of the subdomains (l ⁇ , l ⁇ , 2 ⁇ , and 2 ⁇ ) contained tripeptide kinks. Co-crystallization of 5 mM ⁇ -lactose with ricin showed sugar binding in the tripeptide kinks of subdomains l ⁇ and 2 ⁇ .
  • Fusion toxins have been developed in an effort to treat these diseases. Fusion toxins are hybrid proteins composed of peptide ligands reactive with malignant cells (antibody fragments or cytokines) fused to polypeptide toxins [diphtheria toxin (DT), Pseudomonas exotoxin (PE) or ricin].
  • DT diphtheria toxin
  • PE Pseudomonas exotoxin
  • ricin ricin
  • the toxin- ligand-receptor complex internalizes into intracellular compartments from which the catalytic domain of the toxin translocates to the cytosol and inactivates protein synthesis.
  • L2R interleukin-2 receptors
  • IL2R bearing cells (31).
  • Ricin' s extreme potency has led to its use in immunotoxins consisting of monoclonal antibodies chemically coupled to a modified ricin moiety.
  • the large 200 kilodalton Mr immunoco ⁇ jugates showed significant vascular endothelial toxicity (32).
  • Three groups of investigators have chemically or genetically modified lectin sites on ricin and used covalently attached ligands to study cell intoxication (33-35). In each case, reductions in lectin function led to profound decreases in cytotoxic potency.
  • novel therapeutic modalities with minimal toxicities and no cross-resistance with current cytotoxic treatments are still needed.
  • the present invention overcomes previous shortcomings by providing a ricin fusion protein immunotoxin comprising a ricin toxin B chain fusion protein having a modification in a lectin binding site in each of the l , l ⁇ and 2 ⁇ subunits and a ligand specific for a cell surface receptor in association with a ricin toxin A chain that selectively targets and intoxicates very specific cell populations for the treatment of cancer and autoimmune disease.
  • the present invention provides a plant holotoxin comprising 1 ⁇ , 1 ⁇ and 2 ⁇ subdomains and having a modification in a lectin binding site in each of the
  • a ricin toxin B chain having a modification in a lectin binding site in each of the l , l ⁇ and 2 ⁇ subdomains is provided.
  • the present invention provides a plant holotoxin fusion protein comprising a moiety consisting of a plant holotoxin comprising l ⁇ , l ⁇ and 2 ⁇ subdomains and having a modification in a lectin binding site in each of the l ⁇ , l ⁇ and 2 ⁇ subdomains and a moiety consisting of a ligand specific for a cell surface receptor.
  • a ricin toxin B chain fusion protein comprising a moiety consisting of a ricin toxin B chain having a modification in a lectin binding site in each of the l ⁇ , l ⁇ and 2 ⁇ subdomains and a moiety consisting of a ligand specific for a cell surface receptor is provided.
  • a method of constructing a ricin fusion protein immunotoxin comprising expressing the nucleic acid in a vector in a eukaryotic cell expression system to produce a fusion protein; isolating and purifying the fusion protein; and contacting the fusion protein of with a ricin toxin A chain under conditions which permit the association of the fusion protein with the ricin toxin A chain.
  • the present invention provides a method of treating a cancer or an autoimmune disease in a patient diagnosed with a cancer or an autoimmune disease comprising constructing a ricin fusion protein immunotoxin, wherein the ligand is specific for a particular cell surface receptor present only on the surfaces of the cancer cells or on the surfaces of the cells causing the patient's autoimmune disease; and administering the ricin fusion protein immunotoxin in a pharmaceutically acceptable carrier to the patient, whereby the ricin fusion protein immunotoxin treats the patient's cancer or autoimmune disease.
  • a ricin protein immunotoxin comprising a ricin toxin A chain associated with a ricin toxin B chain fusion protein comprising a ricin toxin B chain having a W to S substitution at amino acid position 37 in the l ⁇ subdomain, a Y to H substitution at amino acid position 248 in the 1 ⁇ subdomain and a Y to H substitution at position 78 in the 2 ⁇ subdomain and a ligand specific for a cell surface receptor.
  • LL2R is a heterotrimeric glycoprotein complex on the cell membrane with a 55 kD ⁇ subunit, a 75 kD ⁇ subunit and a 64 kD ⁇ subunit (16)
  • the only normal human tissues expressing IL2R ⁇ and IL2R ⁇ are activated T cells, B cells, LGL cells and monocytes and some liver Kupffer cells, lung macrophages and skin Langerhans' cells
  • an immunotoxin targeted to this receptor is expected to be reasonably selective
  • a variety of hematologic neoplasms may show high affinity LL2R expression including hairy cell leukemia, adult T cell leukemia and a fraction of cutaneous T cell lymphomas and B-cell chronic lymphocytic leukemias (17)
  • DT and PE have been fused to either LL2 or antibody Fv anti-IL2R peptides (18-25) All reagents showed potent selective cytoplasm
  • Ricin-based fusion proteins are attractive candidates for development for several reasons
  • the toxin inactivates cell protein synthesis by a mechanism independent of that used by DT or PE
  • the RNA N-glycosidase activity of ricin cripples 1500 ribosomes/minute and a single molecule of ricin in the cytosol can cause cell death (6,27)
  • ricin fusion toxins may be used in combination with bacterial fusion toxins or when bacterial fusion toxin resistance is encountered
  • there is no immunologic cross- reactivity between ricin and the bacterial toxins Patients who have been immunized with DT or had previous exposure to PE do not show amnestic immune responses to ricin (28)
  • the present invention provides a plant holotoxin comprising 1 ⁇ , 1 ⁇ and 2 ⁇ subdomains and having a modification in a lectin binding site in each of the
  • the plant holotoxin can be, but is not limited to, ricin, mistletoe toxin, abrin, volkensin as well as any other compound now known or identified in the future to be a plant holotoxin
  • the preferred plant holotoxin will be characterized as a 65 kD glycoprotein with a galactose lectin B chain and RNA N-glycosidase A chain
  • the plant holotoxin with the modification described herein can have various functional attributes, such as, for example, the ability to be produced in high yields as an expressed protein in a eukaryotic gene expression system, specific reactivity with antibodies against the plant holotoxin, proper folding to retain the stability and functional characteristics of the wild type holotoxin, proper association with additional moieties which are normally associated with the wild type holotoxin, a 50% lethal dose (LD 50 ) value of greater than ten micrograms in mice, at least a one thousand fold reduction in sugar binding as
  • LD 50 50% lethal dose
  • the modification in a lectin binding site in each of the 1 ⁇ , 1 ⁇ and 2 ⁇ subdomains of the plant holotoxin can be an amino acid substitution, such as, for example, a substitution of an amino acid having an aromatic ring residue with an amino acid lacking an aromatic ring residue
  • Other modifications in the lectin binding sites can include, but are not limited to, for example, substitution of an amino acid with a polar hydrogen binding residue with an amino acid with a nonpolar residue, as well as other deletions, additions or amino acid substitutions or any other modifications now known or later discovered that result in either complete or significant removal of the sugar binding activity of the lectin binding site.
  • the plant holotoxin with modifications will maintain a functional conformation and be able to associate normally with the intoxication-imparting moiety. Whether a given modification results in the complete or significant removal of the sugar binding activity of a lectin binding site can be determined according to the protocols provided in the Examples herein.
  • a ricin toxin B chain having a modification in a lectin binding site in each of the l , l ⁇ and 2 ⁇ subdomains.
  • the ricin toxin B chain with such modification can have various functional attributes, such as, for example, the ability to be produced in high yields as an expressed protein in a eukaryotic gene expression system; specific reactivity with antibodies against the ricin toxin B chain; proper folding to retain the stability and functional characteristics of the wild type ricin toxin B chain; proper association with ricin toxin A chain; a 50% lethal dose (LD 50 ) value of greater than ten micrograms in mice; at least a one thousand fold reduction in sugar binding as compared with wild type ricin toxin B chain, at least a one hundred fold reduction in toxicity in mice as compared with wild type ricin toxin B chain; and the ability to selectively intoxicate a target cell with a two hundred fold reduction in the ability to intoxicate a non-
  • the modification in a lectin binding site in each of the l , l ⁇ and 2 ⁇ subdomains in the ricin toxin B chain of this invention can be an amino acid substitution, such as, for example, wherein the amino acid substitution consists of substitution of an amino acid having an aromatic ring residue with an amino acid lacking an aromatic ring residue.
  • Other modifications in the lectin binding sites can include, but are not limited to, for example, substitution of an amino acid with a polar hydrogen binding residue with an amino acid with a nonpolar residue, as well as other deletions, additions or amino acid substitutions or any other modifications now known or later discovered that result in either complete or significant removal of the sugar binding activity of the lectin binding site. Whether a given modification results in the complete or significant removal of the sugar binding activity of a lectin binding site can be determined according to the protocols provided in the Examples herein.
  • the amino acid substitutions can consist of a W to S substitution at amino acid position 37 in the l ⁇ subdomain, a Y to H substitution at amino acid position 248 in the 1 ⁇ subdomain and a Y to H substitution at position 78 in the 2 ⁇ subdomain.
  • Other modifications in the lectin binding sites can include, but are not limited to, for example, substitution of an amino acid with a polar hydrogen binding residue with an amino acid with a nonpolar residue, as well as other deletions, additions or amino acid substitutions or any other modifications now known or later discovered that result in either complete or significant removal of the sugar binding activity of the lectin binding site. Whether a given modification results in the complete or significant removal of the sugar binding activity of a lectin binding site can be determined according to the protocols provided in the Examples herein.
  • a plant holotoxin comprising 1 ⁇ , 1 ⁇ and 2 ⁇ subdomains and having a modification in a lectin binding site in one or more or the subdomains, including at least a modification in the 1 ⁇ subdomain is also contemplated in the present invention.
  • the modification in the lectin binding site in one or more of the subdomains can be an amino acid substitution, such as for example, a substitution of an amino acid having an aromatic ring residue with an amino acid lacking an aromatic ring residue (e.g., a Y to H substitution at amino acid position 248 in the 1 ⁇ subdomain).
  • a ricin toxin B chain fusion protein having a modification in a lectin binding site in one or more subdomain, including at least a modification in the 1 ⁇ subdomain.
  • the modification in one or more of the subdomains can be an amino acid substitution, such as, for example, a substitution of an amino acid having an aromatic ring residue with an amino acid lacking an aromatic ring residue.
  • the amino acid substitution can be, but is not limited to, a Y to H substitution at position 248 in the 1 ⁇ subdomain.
  • a plant holotoxin fusion protein comprising a plant holotoxin comprising l ⁇ , l ⁇ and 2 ⁇ subdomains and having a modification in a lectin binding site in each of the l ⁇ , l ⁇ and 2 ⁇ subdomains and a ligand specific for a cell surface receptor.
  • the ligand of the plant holotoxin fusion protein can be, but is not limited to, interleukin-2, granulocyte/macrophage colony stimulating factor, an antibody or antibody fragment to CD3, an antibody or antibody fragment to GD2, epidermal growth factor, IGF2, GRF, substance P, MSH, as well as any other molecular entity now known or identified in the future to be a ligand specific for a cell surface receptor as determined by assaying a potential ligand for selective binding avidity for a particular cell surface receptor by protocols standard in the art for measuring binding avidities.
  • the present invention additionally provides a ricin toxin B chain fusion protein comprising a ricin toxin B chain having a modification in a lectin binding site in each of the l ⁇ , l ⁇ and 2 ⁇ subdomains as described above and a ligand specific for a cell surface receptor.
  • the ligand of the ricin toxin B chain fusion protein can be, but is not limited to, interleukin-2, granulocyte/macrophage colony stimulating factor, an antibody to CD3, an antibody to GD2, epidermal growth factor, an antibody or antibody fragment to CD3, an antibody or antibody fragment to GD2, epidermal growth factor, IGF2, GRF, substance P, MSH, as well as any other molecular entity now known or identified in the future to be a ligand specific for a cell surface receptor as determined by assaying a potential ligand for selective binding avidity for a particular cell surface receptor by protocols standard in the art for measuring binding avidities.
  • the present invention also provides a ricin toxin B chain fusion protein comprising a ricin toxin B chain having a W to S substitution at amino acid position 37 in the l ⁇ subdomain, a Y to H substitution at amino acid position 248 in the 1 ⁇ subdomain and a Y to H substitution at position 78 in the 2 ⁇ subdomain and a ligand specific for a cell surface receptor.
  • a ricin fusion protein immunotoxin comprising this ricin toxin B chain fusion protein associated with a ricin toxin A chain.
  • a plant holotoxin fusion protein immunotoxin is also provided in the present invention, comprising a plant holotoxin fusion protein (which imparts a binding function to the immunotoxin) as described above, associated with a moiety imparting an intoxicating function to the immunotoxin.
  • a ricin fusion protein immunotoxin is provided in the present invention, comprising a ricin toxin B chain fusion protein consisting of a ricin toxin B chain having a modification in a lectin binding site in each of the l ⁇ , l ⁇ and 2 ⁇ subdomains and a ligand specific for a cell surface receptor, associated with a ricin toxin A chain.
  • Nucleic acids encoding the plant holotoxins and fusion proteins of this invention are also contemplated, as well as vectors comprising the nucleic acids and hosts comprising the vectors.
  • the present invention also provides nucleic acids complementary to or capable of hybridizing with the nucleic acids encoding the plant holotoxins and fusion proteins.
  • the nucleic acid of the plant holotoxin or fusion protein can encode the intact plant holotoxin with the modifications described herein or an active fragment thereof.
  • An active fragment of a plant holotoxin is a fragment which is capable of maintaining a functional conformation and associating with the moiety which imparts an intoxicating function to the plant holotoxin.
  • the nucleic acid of the fusion protein can also encode the intact ligand or an active fragment thereof.
  • An active fragment of a ligand of this invention is a fragment which is capable of maintaining a functional conformation and specifically binding to its corresponding cell surface receptor.
  • Protocols for construction of a vector containing a nucleic acid encoding a plant holotoxin fusion protein such as the ricin toxin B chain fusion protein are well known in the art and are described in the Examples provided herein.
  • the vector can be expressed in any in vitro eukaryotic cell expression system, such as, for example, the Spodoptera frugiperda insect cell line which expresses proteins in a baculovirus vector, as described in the Examples herein.
  • Isolation and purification of the expressed fusion protein can be carried out by protocols well known to those of skill in the art, e.g., as described in the Examples herein.
  • a method of constructing a ricin fusion protein immunotoxin comprising expressing a nucleic acid encoding the ricin toxin B fusion protein, in a vector in a eukaryotic cell expression system, to produce a ricin toxin B chain fusion protein; isolating and purifying the ricin toxin B chain fusion protein; and contacting the ricin toxin B chain fusion protein with a ricin toxin A chain under conditions which permit the association of the ricin toxin B chain fusion protein with the ricin toxin A chain.
  • the present invention further contemplates a method of constructing a plant holotoxin fusion protein immunotoxin comprising expressing a nucleic acid encoding the plant holotoxin fusion protein described above, in a vector in a eukaryotic cell expression system to produce a plant holotoxin fusion protein; isolating and purifying the plant holotoxin fusion protein; and contacting the plant holotoxin fusion protein with a moiety which imparts an intoxicating function, under conditions which permit the association of the plant holotoxin fusion protein with the intoxicating moiety to yield a plant holotoxin fusion protein immunotoxin.
  • vectors and expression systems described in the Examples herein a variety of vectors and eukaryotic expression systems such as yeast, filamentous fungi, insect cell lines, bird, fish, transgenic plants and mammalian cells, among others, as are known to those of ordinary skill in the art, can also be used in the present invention.
  • the vectors of the invention can be in a host (e.g., cell line or transgenic animal) that can express the nucleic acid contemplated by the present invention.
  • a host e.g., cell line or transgenic animal
  • the vector e.g., a plasmid, which is used to transform the host cell, preferably contains DNA sequences to initiate transcription and sequences to control the translation of the protein. These sequences are referred to as expression control sequences.
  • Suitable vectors for expression systems usually have expression control sequences, such as promoters, including 3-phosphoglycerate kinase or other glycolytic enzymes, an origin of replication, termination sequences and the like, as desired.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40 virus, adenovirus, bovine papilloma virus, etc, as are well known in the art.
  • gene sequences to control replication in the host cell may be incorporated into the vector such as those found in bovine papilloma virus-type vectors (64).
  • suitable vectors are described in the literature (see, for example, 65,66).
  • Appropriate vectors for expressing proteins in insect cells are usually derived from baculovirus.
  • suitable insect cell lines include, but are not limited to, mosquito larvae, silkworm, armyworm, moth and Drosophila cell lines such as a Schneider cell line (67), as well as any other insect cell line now known or identified in the future to be a suitable host cell line for baculovirus or other insect cell expression vectors.
  • polyadenylation or transcription terminator sequences from known mammalian genes can be incorporated into the vector.
  • An example of a terminator sequence is the polyadenylation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript may also be included.
  • An example of a splicing sequence is the VP1 intron from SV40 (68).
  • the nucleic acid sequences can be expressed in hosts after the sequences have been operably linked to, i.e., positioned, to ensure the functioning of an expression control sequence.
  • These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors can contain selection markers, e.g., tetracycline resistance, hygromycin resistance, gentamicin resistance or methotrexate resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences (see, e.g., U.S. Patent 4,704,362).
  • the presence of the vector RNA in transformed cells can be confirmed by Northern blot analysis and production of a cDNA or opposite strand RNA corresponding to the antigen coding sequence can be confirmed by Southern and Northern blot analysis, respectively.
  • Polynucleotides encoding a variant polypeptide may include sequences that facilitate transcription (expression sequences) and translation of the coding sequences such that the encoded polypeptide product is produced. Construction of such polynucleotides is well known in the art. For example, such polynucleotides can include a promoter, a transcription termination site (polyadenylation site in eukaryotic expression hosts), a ribosome binding site and optionally, an enhancer for use in eukaryotic expression hosts as well as any sequences necessary for replication of a vector.
  • the host cells are rendered competent for transformation by various means known in the art. There are several well-known methods of introducing
  • DNA into eukaryotic cells include, but are not limited to, calcium phosphate precipitation, fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the recipient cells with liposomes containing the DNA DEAE dextran, electroporation, micro-injection of the DNA directly into the cells, as well as any other technique now known or developed in the future for introducing nucleic acid into cells.
  • the transformed cells are cultured by means well known to one of ordinary skill in the art (69).
  • the expressed polypeptides are isolated from cells grown as suspensions or monolayers. The latter are recovered by well- known mechanical, chemical, or enzymatic means and purified according to standard methods well known in the art.
  • yeast cells are first converted into protoplasts using zymolase, lytiacase, or glusulase, followed by addition of DNA and polyethylene glycol (PEG).
  • PEG polyethylene glycol
  • the PEG- treated protoplasts are then regenerated in a 3% agar medium under selective conditions. Details of this procedure are described by Beggs, J.D. (71) and Hinnen et al. (72).
  • the second procedure does not involve removal of the cell wall. Instead, the cells are treated with lithium chloride or acetate and PEG and put on selective plates (73).
  • Plant holotoxins and fusion proteins once expressed, can be isolated from yeast by lysing the cells and applying standard protein isolation and purification techniques to the lysates.
  • the monitoring of the purification process can be accomplished by using Western blot techniques or radioimmunoassay or other standard immunoassay techniques.
  • sequences encoding plant holotoxins and fusion proteins of the present invention can also be ligated to various expression vectors for use in transforming cell cultures of, for example, mammalian, insect, plant, bird or fish origin.
  • Illustrative of cell cultures useful for the production of polypeptides are mammalian cells. Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures and secretion of active protein.
  • Mammalian cell systems can be in the form of monolayers of cells, although mammalian cell suspensions may also be used.
  • a number of suitable host cell lines capable of expressing intact proteins have been developed in the art, and include the HEK293, BHK21 and CHO cell lines, as well as various human cells such as COS cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc., as are known in the art.
  • Other animal cells useful for the production of proteins are available, for example, from the American Type Culture Collection
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter (e.g., the CMV promoter, a HSC tk promoter or pgk [phosphoglycerate kinase] promoter), an enhancer (74) and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T antigen poly A addition site) and transcriptional terminator sequences.
  • a promoter e.g., the CMV promoter, a HSC tk promoter or pgk [phosphoglycerate kinase] promoter
  • an enhancer e.g., the CMV promoter, a HSC tk promoter or pgk [phosphoglycerate kinase] promoter
  • necessary processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an
  • Efficient post translational glycosylation and expression of recombinant proteins can also be achieved in insect cell expression systems employing baculovirus vectors, as described in the Examples herein.
  • the plant holotoxin of this invention can also be expressed in transgenic plant expression systems known in the art, such as, for example, soy bean cells or Nicotiana tabacum cells (83).
  • the nucleic acids of the present invention can be used to generate transgenic nonhuman animals in which the nucleic acid encoding a plant holotoxin or fusion protein of the present invention is added to the germ line of the animal.
  • a cell of the invention containing an nucleic acid of this invention is contemplated to include a cell in a transgenic animal.
  • the plant holotoxin or fusion protein can be isolated and purified from materials secreted by the animal, such as for example, milk secreted from nonhuman mammals.
  • Transgenic animals are generated by standard means known to those skilled in the art (see, for example, 84)
  • Ricin toxin A chain can be obtained commercially or by expression of a nucleic acid encoding ricin toxin A chain in prokaryotic or eukaryotic in vitro expression systems according to standard protocols known in the art and associated with the ricin toxin B chain fusion protein to produce the immunotoxin (82).
  • the nucleic acid encoding the ricin toxin A chain can also be included in a vector which also comprises the nucleic acid encoding the ricin toxin B chain fusion protein of this invention. This vector can be introduced into a eukaryotic expression system, such as a plant cell expression system
  • the terms “selective” or “selectively” and “specific” or “specifically” all have the same meaning and are thus used interchangeably to mean either that the ligand binds to only one type of cell surface receptor which has been identified as binding that particular ligand and does not randomly bind to other cell surface molecules, that the antibody binds its corresponding antigen which has been identified and does not randomly bind with other antigens, or that the immunotoxin binds its intended target cell which has been identified and does not randomly bind to other nontarget cells.
  • Also contemplated for the present invention is a method for treating or preventing a cancer or an autoimmune disease in a patient comprising constructing the plant holotoxin fusion protein immunotoxin of the present invention as described above, wherein the ligand is specific for a particular cell surface receptor present on the surfaces of the cells to be targeted for intoxication and killing for the purpose of treating or preventing a cancer or autoimmune disease; and administering the plant holotoxin fusion protein immunotoxin in a pharmaceutically acceptable carrier to the patient, whereby the plant holotoxin fusion protein immunotoxin treats or prevents a cancer or autoimmune disease in the patient.
  • the present invention provides a method of treating or preventing a cancer or an autoimmune disease in a patient comprising constructing the ricin fusion protein immunotoxin of the present invention as described above, wherein the ligand is specific for a particular cell surface receptor present on the surfaces of the cells to be targeted for intoxication and killing; and administering the ricin fusion protein immunotoxin in a pharmaceutically acceptable carrier to the patient, whereby the ricin fusion protein immunotoxin treats or prevents a cancer or autoimmune disease in the patient.
  • the treatment or prevention of the cancer or autoimmune disease is by the specific intoxication and killing of the cells associated, or potentially associated, with a cancer or autoimmune disease, resulting in prevention of or remission of the cancer, or prevention, elimination or reduction in severity of the symptoms of autoimmune disease.
  • That a given plant holotoxin fusion protein immunotoxin is effective in treating or preventing a cancer or autoimmune disease in a patient can be determined by evaluating the particular aspects of the medical history, the signs, symptoms and objective laboratory tests that have a documented utility in evaluating disease activity. These signs, symptoms and objective laboratory tests will vary depending on the particular cancer or autoimmune disease being treated or prevented as will be well known to any clinician in this field.
  • Such methods can include, but are not limited to, x-rays, biopsies of biological samples, palpation of masses and measurements of blood and body fluid components.
  • autoimmune disease e.g., multiple sclerosis
  • clinical parameters that can be monitored can include the severity and number of attacks, or for continuously progressive disease, the worsening of symptoms and signs, the cumulative development of disability, the number or extent of brain lesions as determined by magnetic resonance imaging and the need for continued use of immunosuppressive medications (78,79).
  • the cancer to be treated or prevented by administration of the plant holotoxin fusion protein immunotoxin can be, but is not limited to, a human leukemia or lymphoma having cancer cells expressing interleukin-2 receptors on the surfaces, wherein the ligand is interleukin-2, acute myelogenous leukemia, wherein the ligand is granulocyte/macrophage-colony stimulating factor and melanoma/neuroblastoma, wherein the ligand is an antibody to GD2, as well as brain neoplasms, epithelial malignancies, sarcomas or any other cancer now known or identified in the future which can be treated or prevented by administration of the plant holotoxin fusion protein immunotoxin of the present invention.
  • a human leukemia or lymphoma having cancer cells expressing interleukin-2 receptors on the surfaces, wherein the ligand is interleukin-2, acute myelogenous leukemia, wherein the ligand is
  • Such a cancer would express on the surface of the tumor cells or potential tumor cells an antigen to which the ligand of the present immunotoxin can selectively bind, for selective intoxication of the tumor cells or potential tumor cells by the ricin protein immunotoxin
  • an antigen to which the ligand of the present immunotoxin can selectively bind for selective intoxication of the tumor cells or potential tumor cells by the ricin protein immunotoxin
  • tumor antigens or potential tumor antigens such as, but not limited to, growth factor receptors, adhesion molecules, oncogene products, differentiation antigens and oncofetal antigens on the surface of a subject's tumor cells or potential tumor cells
  • autoimmune disease describes a disease state or syndrome whereby a subject's body produces a dysfunctional immune response against the subject's own body components, with adverse effect
  • the autoimmune disease to be treated or prevented by administration of the ricin protein immunotoxin of the present invention can be, but is not limited to, graft-versus-host disease, wherein the ligand can be an antibody to CD3
  • Other examples of autoimmune diseases that can be treated or prevented include ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, diabetes mellitus, pernicious anemia, autoimmune gastritis, psoriasis, Bechet's disease, idiopathic thrombocytopenic purpura, Wegener's granulomatosis, autoimmune thyroiditis, autoimmune oophoritis, bullous pemphigoid, pemphigus, polyendocrinopathies, Still's disease, Lambert-Eaton myasthenia syndrome, myas
  • allergic disease describes a disease state or syndrome whereby the body produces a dysfunctional immune response composed of IgE antibodies to environmental antigens and which evoke allergic symptoms.
  • allergic diseases include, but are not limited to, asthma, ragweed pollen hayfever, allergy to food substances and allergic reactions.
  • the treatment or prevention of the allergic disease is by the specific intoxication and killing of the cells associated with ,or potentially associated with, the allergic disease, resulting in the prevention of symptoms or the elimination or reduction in severity of the symptoms of the allergic disease. That a given plant holotoxin fusion protein immunotoxin is effective in treating or preventing an allergic disease in a patient can be determined by evaluating the particular aspects of the medical history, the signs, symptoms and objective laboratory test that have a documented utility in evaluating disease activity.
  • allergic diseases can include, but are not limited to, x-rays, biopsies of biological samples, palpation of masses and measurements of blood and other body fluid components.
  • clinical parameters that can be monitored for an allergic disease e.g., asthma
  • the measurement of airway resistance by the use of respiratory spirometry, the extent of disability and the dependence on immunosuppressive medications or bronchodilators can also be determined (80,81).
  • Also contemplated for the present invention is a method of inducing immune tolerance in the patient, comprising constructing the plant holotoxin fusion protein immunotoxin of the present invention as described above, wherein the ligand is specific for a particular cell surface receptor present on the surfaces of certain immune cells to be targeted for intoxication and killing; and administering the plant holotoxin fusion protein immunotoxin in a pharmaceutically acceptable carrier to the patient, whereby the plant holotoxin fusion protein immunotoxin induces immune tolerance.
  • the ligand can be IL2 or an antibody or fragment thereof (e.g., Fv region) to the CD3 antigen.
  • the induction of immune tolerance is by the specific intoxication and killing of certain immune cells, resulting in the elimination or reduction in severity of a particular immune response. That a given plant holotoxin fusion protein immunotoxin is effective in inducing an immune tolerance in a patient can be determined by evaluating the particular aspects of the medical history, the signs, symptoms and objective laboratory tests that have a documented utility in evaluating immune activity. These signs, symptoms and objective laboratory tests will vary depending on the particular immune response being reduced or eliminated, as will be well known to any clinician in this field. Examples of such methods include, but are not limited to, x-rays, biopsies of biological samples, palpation of masses and measurements of blood and other body fluid components. In particular, cytokine assays, routine clinical chemistries, immune function assays, complete blood counts and the like, as would be known to the clinician, can be measured at various intervals during treatment.
  • immune tolerance e.g., transplantation rejection
  • This time interval may be large, with respect to the development of cancer, autoimmune or allergic diseases (years/decades) or short (weeks/months) with respect to the development of a need for induction of immune tolerance.
  • the determination of who would be at risk for the development of a cancer, autoimmune disease, allergic disease or in need of induction of immune tolerance would be made based on current knowledge of the known risk factors for a particular disease or immune response familiar to a clinician in this field, such as a particularly strong family history of disease or need for a transplant.
  • the plant holotoxins, fusion proteins and immunotoxins of the present invention are preferably provided in a pharmaceutically acceptable carrier and can be parenterally administered to the subject.
  • Suitable carriers for parenteral administration of the immunotoxin in a sterile solution or suspension can include sterile saline that may contain additives, such as ethyl oleate or isopropyl myristate, and can be injected, for example, intravenously, as well as into subcutaneous or intramuscular tissues.
  • the plant holotoxin fusion protein immunotoxin can be administered to the subject in amounts sufficient to treat or prevent a cancer, autoimmune disease or allergic disease, or to induce immune tolerance. Optimal dosages used will vary according to the individual, as well as the particular cancer or autoimmune or allergic disease being treated or the type of immune response being induced. Typically, for treatment of humans, plant holotoxin fusion protein immunotoxin, (e.g., ricin toxin fusion protein immunotoxin) would be administered intravenously in a dosage range between 1 ⁇ g and 10 mg/kg of body weight and most preferably in a dose of 0.5 mg/kg, either as a single bolus or as a continuous infusion ranging in time from a day to a month. Treatment can be continued for an indefinite period of time, as indicated by monitoring of the signs, symptoms and clinical parameters associated with a particular cancer, autoimmune disease, allergic disease or immune response induction.
  • plant holotoxin fusion protein immunotoxin e.g., ricin
  • the amount of plant holotoxin fusion protein immunotoxin administered will also vary among individuals on the basis of age, size, weight, condition, etc.
  • dosages are best optimized by the practicing physician and methods for determining dosage are described, for example, in Remington 's Pharmaceutical Sciences (11). That a given dosage amount or regimen is effective in treating a cancer, autoimmune disease, or allergic disease or inducing immune tolerance, can be readily determined by using the parameters described above.
  • the present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.
  • EXAMPLE I Construction of a ricin toxin containing a modification in a lectin binding site in each of the l ⁇ , l ⁇ and 2 ⁇ subdomains.
  • DNAs (4 ⁇ g) were co-transfected with 0.5 ⁇ g of BaculoGold AcNPV DNA (PharMingen) into 2 x 10 6 Sf9 Spodoptera frugiperda insect cells as recommended by the supplier.
  • media were centrifuged and supernatants tested in limiting dilution assays with Sf9 cells as previously described (40). Positive wells were identified and supernatants reassayed by limiting dilution until all wells up to 10 "8 dilution were positive. Two rounds of selection were required for each mutant.
  • Recombinant viruses in the supernatants were then amplified by infecting Sf9 cells at a multiplicity of infection (moi) of 0.1 , followed by collection of day 7 supernatants.
  • moi multiplicity of infection
  • mutant RTBs Recombinant baculoviruses were used to infect Sf9 cells at an moi of 5 in EX-CELL400 media (JRH Scientific, Lexena, KS) with 25 mM ⁇ -lactose in spinner flasks. Media supernatants and cell pellets containing mutant RTBs were collected day
  • Cell pellets were dissolved in 20 mM Tris HCL( pH 8), 50 mM NaCl, 1% NP40, 1 mM PMSF, 2 ug/ml aprotinin, 1.5 ug/ml pepstatin and 1.5 ug/ml leupeptin, frozen at -70°C, thawed, centrifuged at 22,000g for 15 minutes at 4°C , dialyzed into NTEAL at 4°C and treated identically to dialyzed concentrated cell supernatants.
  • mutant RTBs Reassociation of mutant RTBs with plant RTA to form heterodimers.
  • 0.25 ml of mutant RTBs (5 - 15 ⁇ g) was mixed with a 4 - fold molar excess of plant RTA (Inland Laboratories) in 0.1 M triethylamine-sodium phosphate pH 7 overnight at room temperature.
  • the reaction mixture was then analyzed by a ricin ELISA utilizing P2 monoclonal anti-RTB coated wells, biotin conjugated ⁇ BR12 monoclonal anti-RTA and alkaline phosphatase-conjugated streptavidin detection reagents, as previously described (15).
  • W37S/Y248H/W160S RTB The yields were not dissimilar from the yields for the double-site mutant W37S/Y248H (220 ug/liter from supernatant and 250 ug/liter from cell pellet) or wild-type RTB (400 ug/liter for supernatants) in this expression system and may reflect proper folding for the triple-site mutants.
  • the conservative modification of surface residues may have contributed to protein stability.
  • the double-site RTB mutant, W37S/Y248H, bound asialofetuin 4.8 ⁇ 2% (n ⁇ ) relative to recombinant or plant RTB.
  • the triple-site RTB mutant, W37S/Y248H/W160S, bound asialofetuin similarly at 1.1 ⁇ 0.27% (n 7) relative to plant RTB.
  • mutant RTB binding to glycoproteins was made by detecting mutant RTB bound to cell surfaces. Only W37S/Y248H and W37S/Y248H/W160S showed significant binding to KB cells at 4°C. Sugar binding of triple-site mutants. W37S/Y248H/W160S RTB retained binding to immobilized asialofetuin and KB cell surface glycoproteins. In both cases, the binding was competed with soluble saccharides. In contrast, W37S/Y248H/Y78H had minimal to negligible sugar binding.
  • the IC 50 of ricin on HUT 102 human leukemia cells was 4 x 10 '12 M.
  • the IC 50 for W37S/Y248H was 2 x 10 "10 M; the IC 50 for W37S/Y248H/W160S was 1 x 10 "10 M; and the IC 50 for W37S/Y248H/Y78H was 5 x 10 "9 M.
  • Plant RTA alone had a 20-fold higher
  • ricin lectin sites Widely separated ricin lectin sites.
  • the 1 ⁇ and 2 ⁇ sites are separated by 36 Angstroms, l ⁇ and 2 ⁇ sites are 44 Angstroms apart.
  • the l ⁇ and l ⁇ distance is 19 Angstroms.
  • These inter-binding site distances are much larger than the inter-site spacing for the hepatic Gal/GalNac receptor and its triantennary N-glycoside ligand (10 -20 Angstroms) (41).
  • the ricin geometry resembles the spacing of sites on surface binding lectins including mammalian mannose-binding protein, influenza virus hemagglutinin, pertussis toxin and cholera toxin (42).
  • proteins are phylogenetically unrelated based on lack of primary or tertiary structure homology. Nevertheless, in all these proteins, the sugar combining sites are multiple widely spaced and project toward a single plane. Thus, they are ideally suited for binding to eukaryotic cell surfaces.
  • the three binding sites on ricin may provide the optimal geometry for binding to the uneven galactosyl oligosaccharide-rich surface of mammalian cells similar to camera tripods or stools.
  • the RTB l ⁇ and 2 ⁇ subdomains are unlikely to contribute additional sugar binding as they lack the tripeptide ⁇ -carbon kink, aromatic residues or charged residues for hydrogen bond formation. Further, no RGD-like domains exist in RTB—unlike discoidin I from the slime mold, Dictylostelium discoideum (46).
  • EXAMPLE II In vivo toxicity of a ricin toxin containing a modification in a lectin binding site in each of the l ⁇ , l ⁇ and 2 ⁇ subdomains.
  • Ricin and recombinant heterodimers Purified castor bean ricin (3.9 mg/ml in 0.15 M NaCl, 0.015 M potassium phosphate pH 7, 0.1% sodium azide ) was purchased from Sigma (St. Louis, MO). Purified deglycosylated RTA (5 mg/ml in PBS) was a gift of Dr. Jerry Fulton, Inland Laboratories, Dallas, Texas. Partially purified recombinant mutant RTBs were prepared and reassociated with plant RTA as previously described (15,40). The proteins tested and their characteristics are shown in Table 1. Ricin was stored at 4°C and all other tested proteins were stored at -20°C until used.
  • mice Pathogen-free C57B/6 female mice (16-18 grams) were purchased from Jackson Laboratories (Bar Harbor, ME) or Harlan Sprague- Dawley (Indianapolis, IN), culled on each experiment to obtain 24 animals each
  • mice 18 + 1 grams, and housed in groups of four in specific pathogen-free environment in Micro-Isolator cages (Lab Products, Maywood, NY).
  • Experimental Design Mice were injected intraperitoneally with dilutions of toxins in phosphate buffered saline (PBS) plus 0 5% bovine serum albumin (BSA) so that each animal received 0 25 - 0 5 milliliters solution Animals were then observed twice daily for mortality Six different concentrations were tested on groups of four mice each for each protein Graphs of animal survival versus time were prepared for each protein Once the LD 50 was determined for each protein, single animals received the LD 50 and were collected at death or when severely morbid and autopsies were performed
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • mice Postmortem examinations were carried out on mice given LD 50 doses on moribund animals
  • Samples from lungs, thymus, heart, esophagus, trachea, lymph node, liver, spleen, pancreas, kidneys, adrenals, gallbladder, stomach, duodenum, jejunum, colon, ovaries, uterus, brain, spinal cord, and skeletal muscle were taken for microscopic examination
  • the tissues were fixed in 4% buffered formaldehyde and embedded in paraffin Sections were stained with hematoxylin and eosin
  • the 1 ⁇ g dose killed two mice— one each on day 4 and 5
  • the 500 ng dose led to the death of two mice—one each on days 7 and 10 None of the mice receiving 100 ng died
  • mice receiving 0.1 ⁇ g, 0.5 ⁇ g, 1 ⁇ g, 2 ⁇ g, or 5 ⁇ g died and only one of four mice receiving 10 ⁇ g died on day 3 post-injection.
  • the LD 50 was estimated at >10 micrograms.
  • Plant RTA was significantly less toxic than either the wild-type ricin or mutant heterodimers.
  • mice treated with RTA and the triple-site mutant ricin were calculated to be significantly different (p ⁇ 0.001) than the survival rates of wild-type, single-site mutant or double-site mutant ricins over a range of doses.
  • mice treated with intraperitoneal ricin or RTA showed apoptosis in the thymus and spleen. Peritoneal inflammation was observed in some animals presumably due to intraperitoneal injection of toxin. There was no evidence of apoptosis based on nuclear morphology in the other organs examined. The triple-site mutant heterodimer treated mouse showed no histopathologic abnormalities.
  • lymphoid apoptosis after ricin administration might suggest that those cells have a lower apoptotic threshold. Deletion of lymphocytes is an important ongoing process for tolerance induction and immune regulation in the lymphoid system (50).
  • the toxicity of RTA alone may be due to the presence of small amounts
  • EXAMPLE ⁇ i Construction of a ricin fusion protein immunotoxin containing a modification in a lectin binding site in each of the l ⁇ , l ⁇ and
  • Transfer vector with mutant RTB was then purified by cesium chloride density centrifugation, restricted with BarnHI, bound and eluted from silica matrix (Promega, Madison, WI), digested with calf intestinal phosphatase (Boehringer- Mannheim, Indianapolis, IN), heat inactivated and repurified on silica matrix.
  • the BamHI fragment encoding IL2 prepared by polymerase chain reaction of pDW27 plasmid DNA as previously described (54) was isolated from pUCl 19-
  • LL2 by digestion of cesium chloride density gradient purified plasmid with BamHI, agarose electrophoresis and binding and elution from silica matrix.
  • the 406 bp fragment was subcloned into pAcGP67A-ADP- RTB[W37S/Y248H/Y78H].
  • the expression vector was maintained in INVaF E. coli using 100 ug/ml ampicillin. Plasmid isolated by alkaline lysis followed by cesium chloride density gradient centrifugation was double-stranded dideoxy sequenced by the Sanger method (33) using the Sequenase kit (USB, Cleveland, OH).
  • fusion toxin Sf9 Spodoptera frugiperda ovarian cells (2 x 10 6 ), maintained in TMNFH medium supplemented with 10% fetal calf serum and 50 ug/ml gentamicin sulfate, were co-transfected with pAcGP67A-ADP- LL2-ADP-RTB[W37S/Y248H/Y78H] DNA (4 ⁇ g) and 0 5 ⁇ g of BaculoGold AcNPV DNA (PharMingen) following the recommendations of the supplier
  • Recombinant virus in the supernatant was then amplified by infection Sf9 cells at an moi of 0 1, followed by collection of day 7 supernatants Recombinant baculovirus was then used to infect 2 x 10 8 Sf9 cells at an moi of 5-10 in 150 ml EXCELL400 medium (JRH Scientific, Lexena, KS) with 25 mM lactose in spinner flasks Media supernatants containing ADP-IL2-ADP-
  • Protein purification Media supernatants were adjusted to 0 01% sodium azide and maintained through all purification steps at 4 n C The supernatants were concentrated 15-fold by vacuum dialysis, centrifuged at 3,000x g for 10 minutes to remove precipitate, dialyzed against 50 mM NaCl, 25 mM Tris (pH 8), 1 mM EDTA 0 01% sodium azide and 25 mM lactose (NTEAL), ultracentrif ⁇ iged at 100,000g for one hour and bound and eluted from a P2 monoclonal antibody-acrylamide matrix as previously described (53)
  • P2 is an anti-RTB monoclonal antibody
  • the affinity matrix was prepared using Ultralink azlactone functionality bis-acrylamide following the recommendations of the manufacturer (Pierce, Rockford, LL) Recombinant protein was absorbed to the column in NTEAL, washed with 0 5 M NaCl, 25 mM Tris pH 9, 1 mM EDTA 0.1% Tween 20, 0.02% sodium azide, 25 mM lactose and eluted with 0.1 M triethylamine hydrochloride (pH 11). The eluant was neutralized with 1/10 volume 1 M sodium phosphate pH 4.25 and stored at -20°C until assayed. Three preparations were made.
  • Costar EIA microtiter wells were coated with 100 ⁇ l of 5 ug/ml of monoclonal antibody P2, P8, or P10 reactive with RTB or monoclonal antibody to LL2, washed with PBS plus 0.1% Tween 20, blocked with 3% BSA rewashed and incubated with samples of ADP-LL2-ADP-RTB[W37S/Y248H/Y78H], human IL2 or plant RTB, rewashed, reacted with 1 :400 rabbit antibody to ricin or 1 :500 rabbit antibody to LL2, washed again, incubated with 1 :5000 alkaline phosphatase conjugated goat anti-(rabbit IgG), rewashed, developed with 1 mg/ml p- nitrophenylphosphate in diethanolamine buffer (pH 9.6) and read on a BioRad 450 Microplate reader at 405 nm. Aliquots of ADP-LL2-ADP-
  • RTB[W37S/Y248H Y78H], bacterial IL2, recombinant RTB, plant RTB, and prestained low molecular weight protein standards were run on a reducing 15% SDS-PAGE, transferred to nitrocellulose, blocked with 10% Carnation's nonfat dry milk/0.1% bovine serum albumin (BSA)/0.1% Tween 20, washed with PBS plus 0 05% Tween 20, reacted with either 1 400 rabbit antibody to ricin or 1 100 mouse monoclonal antibody to IL2 (5 ug/ml), rewashed, incubated with alkaline phosphatase conjugated goat anti-(rabbit IgG) or anti-(mouse IgG), washed again and developed with the Vectastain alkaline phosphatase kit
  • HUT 102 human T leukemia cells bearing the high affinity LL2 receptor, YT2C2 human leukemia cells bearing the intermediate affinity LL2 receptor, MT-1 human leukemia cells bearing the low affinity IL2 receptor and CEM human leukemia cells and KB human epidermoid carcinoma cells lacking the IL2 receptor were washed with
  • the cells were then washed with PBS and reacted with goat anti-(mouse Ig) conjugated to rhodamine (Jackson ImmunoResearch, West Grove, PA) at 25 ug/ml for 30 minutes at 4°C.
  • the cells were washed again in PBS and fixed in
  • Cytotoxicity assay Measurement of protein synthesis inhibition by ricin and ADP-IL2-ADP-RTB[W37S/Y248H/Y78H]-RTA in cultured cells was done as previously described using HUT 102, CEM, YT2C2, MT-1, and KB cells (15). ADP-LL2-ADP-wild-type RTB-RTA and ADP-LL2-ADP- RTB[W37S/Y248H] prepared as described previously were also tested. 18 ' 22 All assays were performed in triplicate. Twelve different concentrations of toxins were used. The LD 50 was the concentration of protein which inhibited protein synthesis by 50% compared with control wells without toxin. There was no purification step after heterodimer reassociation.
  • the free RTA concentration at the highest concentration of heterodimer in the assay was 10 "7 M.
  • the LD 50 for free RTA was 2 - 3 x 10 "6 M. 28
  • the free RTA concentration (2 x 10 "9 M - 2 x 10 "13 M) should not produce cytotoxicity.
  • HUT 102 cells ( 1.5 x 10 4 ) were placed in sterile Eppendorf tubes at 4°C in 100 ⁇ l leucine-poor RPMI
  • 3 H-leucine was added as above and cells were harvested four hours later with a Skatron cell harvester and 3 H-leucine incorporation was measured in a liquid scintillation counter. Blocking of selective cytotoxicity was estimated by comparing the ID 50 of toxins in the presence or absence of
  • P2 antibody ELISA showed the concentration of anti-RTB immunoreactive protein was 102 ⁇ g, 49 ⁇ g, and 75 ⁇ g, respectively. Thus, purity was between 21 and 85 % based on absorbance, BioRad protein assay and densitometry of Coomassie-stained gels. Immunologic cross-reactivity of ADP-IL2-ADP- RTB/W37S/Y248H/Y78H].
  • the ricin Kd was 4 x 10 "9 M and the LL2 fusion toxin Kd was 1 2 x 10 "7 M Specificity for high affinity LL2 receptor was demonstrated on a live cell immunofluorescence assay The LL2 fusion toxin bound to HUT 102, YT2C2, MT-1 but did not bind CEM or KB cells. Binding to HUT 102 cells was inhibited by LL2 but not asialofetuin.
  • the LL2- triple-site RTB mutant-RTA had improved specificity with IC 50 values of 5 x 10 "12 M on HUT 102 cells, 1 x 10 "9 M on CEM cells and 6 x 10 "10 M on KB cells.
  • the in vitro therapeutic window (the ratio of the IC 50 of receptor negative cells to the IC 50 of receptor positive cells) was 1 for IL2-wild-type RTB-RTA 50 for LL2-RTB[W37S/Y248H]-RTA and 120 - 200 for IL2-
  • LL2 receptor-mediated cell toxicity was tested by blocking experiments with excess LL2 or lactose. Excess IL2 reduced LL2-triple-site RTB mutant- RTA toxicity towards HUT 102 cells by 1 , 000-fold (IC 50 was 1 x 10 "8 M with
  • IL2R targeted ricin fusion protein for preclinical and clinical development requires adequate yields, simple purification, adequate stability at room temperature and 37°C, and selective toxicity to IL2R bearing lymphocytes.
  • the IL2 triple-site mutant RTB fusion protein was obtained at 50% purity in good yields of 0.75 mg/liter culture. This compares with 1 mg/liter for LL2 wild-type RTB and 0.34 mg/liter LL2 double-site mutant RTB fusion protein.
  • the IL2 triple-site mutant RTB molecules reacted with antibodies to LL2 and RTB both by ELISA and Western blots. These results are evidence of proper folding of both the IL2 and RTB domains.
  • the protein was secreted into the insect cell medium, and purification was accomplished by a one-step immunoaffinity absorption. This contrasts with the requirement for denaturation and refolding for bacterial toxin fusion proteins and chemical derivatization and conjugation for immunotoxins.
  • the toxophore domain of ricin (RTA) was added by simply mixing with the LL2 triple-site mutant RTB at 10 "6 M. Extensive ionic and hydrophobic bonds in the RTA-RTB interface promote reassociation and disulfide bond formation (13). The observation of 80% reassociation compares favorably with the 60% reassociation for IL2 wild-type RTB-RTA (54), 55% reassociation for IL2 double-site mutant RTB-RTA and 50% reassociation for plant RTB-RTA under identical conditions (53). The heterodimers were stable at high dilution (10-12 M) suggesting formation of the disulfide bond between RTA Cys-259 and RTB Cys-4 (55).
  • Binding specificity of the lectin-deficient heterodimer was demonstrated in both ELISA and cell immunofluorescence formats.
  • the fusion toxin displayed 0.3% binding to immobilized asialofetuin.
  • the Kd was 1.2 x 10 "7 M versus 4 x 10 "9 M for plant ricin. This weak binding compares to 1% binding
  • Subdomain l ⁇ mutation W37S reduced sugar binding avidity 4-fold, while other 1 ⁇ subdomain mutations (K40M and K40M/N46G) yielded proteins with 7-8-fold reductions in asialofetuin avidity (40).
  • the W37S mutation was used in the LL2-triple-site mutant because of its much better yields.
  • the lack of detectable binding to other human cell lines which still have cell surface galactosides may be due to the insensitivity of the immunofluorescence assay, a two and one-half log reduction in sugar binding may be beneath the immunofluorescence detection limit. Binding to cell lines was blocked by IL2 but not asialofetuin.
  • LL2-lectin-deficient ricin fusion protein The most important property of an LL2-lectin-deficient ricin fusion protein is its selective cytotoxicity to IL2R expressing cells.
  • the triply cross-linked molecules lacked sugar-binding and were unable to intoxicate antigen-bearing cells.
  • the lectin-deficient heterodimer was nontoxic to mouse macrophages in the presence of lactose, even though binding and internalization was mediated by binding to mannose receptors.
  • the lectin-deficient ricin conjugate again had reduced cell cytotoxicity, although cell binding and entry was mediated by non- galactoside mechanisms.
  • the IL2 -lectin-deficient fusion toxin was selectively cytotoxic to hematopoietic neoplastic cell lines with the heterotrimeric high affinity IL2R.
  • the molecules were less toxic to cells with intermediate or low affinity LL2R, and nontoxic to cells without 1L2R. These results are similar to those seen with LL2-PE40 35 but distinct from those seen with DAB 389 LL2. The latter molecule fails to intoxicate low affinity LL2R ⁇ , ⁇ cells perhaps due to steric effects of the N-terminal toxin moiety. As a note of caution, fresh leukemic blasts often display lower levels of IL2R ⁇ and IL2R ⁇ than cell lines and may show lowered sensitivity to the ricin fusion molecule (23).
  • the fusion toxin needs IL2R binding for cell intoxication.
  • EXAMPLE TV Studies of mannose receptor mediated cell cytotoxicity of ricin fusion protein immunotoxins containing a modification in a lectin binding site in each of the l ⁇ , l ⁇ and 2 ⁇ subdomains.
  • toxins Preparation of toxins.
  • Recombinant baculovirus encoding the gp67A leader and the RTB mutant (W37S/Y78H/Y248H) was prepared as previously described and used to infect Sf9 insect cells at an moi of 5-10 in EXCELL400 medium (JRH Scientific, Lexena, KS) supplemented with 25 mM lactose.
  • W37SiY78H/Y248H RTB protein was purified from day 6 post-infection cell supernatants by, sequentially, vacuum dialysis, dialysis into 50 mM NACL, 25 mM Tris HCL pH 8, 1 mM EDTA, 0. 1% sodium azide, and 25 mM lactose, binding and elution from a P2 monoclonal antibody anti-RTB azlactone functionality bis-acrylamide affinity matrix using 0. 1 M triethylamine HCL (pH 11) elution buffer and neutralization with 11 M NAPO, pH 4.
  • Protein was quantitated by P2 antibody ELISA and reassociated overnight at room temperature with a three-fold molar excess of plant RTA (Inland Laboratories, Austin, TX). Heterodimer concentration was determined by a sandwich ELISA employing P2 monoclonal anti-RTB antibody capture and biotinylated ⁇ BR12 anti-RTA monoclonal antibody detection. Plant ricin was obtained from Inland Laboratories.
  • Pansorbin fixed Staphyloccocus aureus Cowan strain was prewashed with 3% BSA in PBS and then incubated with cell extracts to remove non-specifically-binding labeled materials.
  • the metabolically-labeled solutions were then reacted with Pansorbin pretreated with rabbit anti-ricin antibody.
  • the Pansorbin was then pelleted and washed with RJPAL twice and RIPAL plus 500 mM NaCl once.
  • the pellets were then boiled in reducing sample buffer and the samples run along with prestained low molecular standards on a 15% SDS polyacrylamide gel
  • the gel was fixed briefly with 10% acetic acid, soaked for one hour in Enlightening, dried, exposed to x-ray film with enhancing screens for 1-3 days at -70 °C and developed
  • Macrophage cells Mouse peritoneal macrophages were prepared using 12 week old female Balb/c mice house in an IACUC approved facility Briefly, mice were injected intraperitoneally with 1 5 ml Brewer's thioglycoUate medium (Becton Dickinson Microbiology Systems) After five days, mice were sacrificed The mice were then injected into their thigh fatty deposits with 10 ml of cold HEPES buffered Hank's balanced salt solution with 10 U/ml heparin with 11% BSA After injection, the mice were gently shaken and the injected medium withdrawn The cell suspension was centrifuged at l,000x g for 10 minutes and the cell pellet resuspended in 10 ml of RPMI 1640 with 10% fetal calf serum and 0 01 M HEPES Cells were then diluted to 2 x 10 6 / ml and seeded to 96 well plates at 100 ⁇ l/well After incubation for two hours at 37°C/5% CO
  • the J774E mouse macrophage cell line (56) was cultured in ⁇ -MEM with 10% fetal calf serum, 60 ⁇ thioguanine and transferred from flasks to wells and dishes by exposure to trypsin EDTA (Gibco) Cells were plated at 2 x 10 cells/well in 96 well plates and 2 x 10 cells/35 mm dish, and incubated a further 24 hours at 37°C/5% C0 2 prior to assay MMR61 rat fibroblasts transfected with mouse mannose receptor cDNA were grown in Dulbecco's MEM containing 10% fetal calf serum and 400 ug/ml G418 (57). Cells were split and transferred to wells and dishes again by trypsinization and incubated a further 24 hours prior to assay.
  • KB human epidermoid carcinoma cells obtained from the American Type Culture Collection (Rockville, MD) were grown in Dulbecco's MEM with 10% fetal calf serum (58). Cells were removed from flasks by trypsin treatment and seeded for experiments identically to the other cell types.
  • the cells were again incubated four hours at 37°C/5% C0 2 and then harvested with a Skatron cell harvester onto glass fiber filter mats. Filters were dried and counted in Econofluor liquid scintillation fluid in a LKB liquid scintillation counter. The IC 50 was determined for each toxin/cell type/medium condition as the toxin concentration that reduced protein synthesis to 50% of control. Each assay was performed in quadruplicate. The mannose receptor directed toxicity was quantitated by the ratio of the toxin IC 50 in the presence of lactose plus mannan to the IC 50 in the presence of lactose alone. KB cells were assayed identically.
  • J774E and MMR61 were assayed identically except leucine-free DMEM medium was used instead of RPMI 1640.
  • Immunofluorescence assay Mouse peritoneal macrophages, J774E, MMR61 and KB cells were attached for 24 hours at 37° C in 5%C0 2 to petri dishes, fixed in 3.7% formaldehyde in PBS (15 minutes), washed with 2 mg/ml BSA in PBS and 0.1% saponin and incubated in PBS plus BSA with rabbit anti-mouse mannose receptor antibody (56) for 30 minutes at 4°C.
  • the cells were rewashed with PBS and reacted with goat anti-(rabbit Ig) conjugated to rhodamine (Jackson ImmunoResearch, West Grove, PA) at 25 ug/ml with 0.1% saponin for 30 minutes at 23 °C. After a final wash with PBS, the cells were fixed in 3.7% formaldehyde in PBS, mounted under a #1 coverslip in glycerol-PBS (90: 10) and examined using a Zeiss Axioplan epifluorescence microscope (63 x, NA. 1.4 planapochromat objective). Fluorescence images were recorded using Tri-X film and negatives were digitized and edited using Adobe Photoshop software and a PowerMac 8500/120 computer. Relative intensities for the brightness of mannose receptor reactions in the different cell types were, respectively, for K-B, for J774E cells, and (+) for MMR61 and mouse peritoneal macrophages [scale (-) - (++++)].
  • ricin with lactose on mouse peritoneal macrophages, J774E cells and MMR61 rat fibroblasts transfected with mouse mannose receptor were 8 x 10 "11 M, 1 x 10 " 10 M, and 8 x 10 "10 M, respectively.
  • the triple-site mutant RTB-RTA with lactose showed IC 50 's on mouse peritoneal macrophages, J774E cells and MM61 cells of 1 x 10 "9 M, 3 x 10 "10 M, and 7 x 10 "9 M, respectively.
  • the receptor-positive cells were 4 - 36 fold more sensitive to ricin and 30-100 fold more sensitive to triple-site mutant RTB-RTA heterodimer than receptor- negative cells.
  • Ricin binds cell surface galactosides in the absence of lactose and intoxicates cells with an IC 50 of 1 X 10 "12 , 11 x 10 '11 M and 2 x 10 "11 M on mouse peritoneal macrophages, J774E cells and MMR61 cells, respectively.
  • ricin was 10-80 fold more potent in the absence of lactose.
  • the lectin-deficient mutant ricin had minimal residual galactoside mediated cytotoxicity with IC 50 'S of 5 x l0 " M, 7 x 10 " M, and 3 x 10 " M on mouse peritoneal macrophages,
  • KB cells showed no effects of mannan on lectin deficient ricin toxicity in the absence of lactose (IC 50 of 1 x 10 " M for both) or presence of lactose (IC 50 of 3 x l0 "8 M for both).
  • Lectin-deficient ricin behaved like ricin with significant mannan inhibition of toxicity on these cell lines. Mannan increased the IC 50 to 5 x 10 "9 M, 2 x 10 "8 M, and 3 x 10 "8 M for mouse peritoneal macrophages, J774E cells and MMR61 cells, respectively. This yielded a 4 - 50 fold reduction in toxicity.
  • the lower potency of mannose receptor-mediated toxicity for both lactose blocked ricin and lectin-deficient ricin may be due to reduced surface receptor content or lower avidity of cell surface binding for the D-mannose receptors and may not reflect altered intracellular processing.
  • the number of galactosyl-terminated cell surface glycoprotein receptors for ricin has been reported to be about 10 7 /mammalian cell (59) versus 105 /cell for D-mannose receptors (56). Avidities for each were similar with Ka's of 10 9 M "1 (56,59).
  • J774E cells The greater sensitivity of J774E cells relative to MMR61 or mouse peritoneal macrophages may be due to higher cell surface mannose receptor density on J774E cells or different intracellular metabolism.
  • the immunofluorescence assay suggests higher receptor content is the cause for the
  • RTB lectin function for mannose receptor has also been documented for the IL2 receptor using an insect-derived lectin-deficient LL2 ricin fusion molecule.
  • RTB enhancement for other receptors may be due to inefficient internalization by the ligand-receptor complex or misrouting intracellularly away from a translocation competent compartment (61,62).
  • IC 50 is the concentration of toxin reducing protein synthesis by 50% after 24 hour incubation.
  • LL2- W.T. RICIN LL2-wild-type RTB-RTA;
  • IL2-D.S. RIC ⁇ N LL2-RTB[W37S/Y248H]- RTA;
  • IL2-T.S. RICIN IL2-RTB[W37S/Y248H/Y78H]-RTA.

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Abstract

L'invention concerne une holotoxine de végétal comprenant les sous-domaines 1α, 1β et 2η et présentant une modification, dans un site de fixation de la lectine, dans chacun des sous-domaines 1α, 1β et 2η. De plus, elle concerne une protéine de fusion de l'holotoxine de végétal, comprenant un fraction consistant en une holotoxine de végétal comprenant les sous-domaines 1α, 1β et 2η et ayant une modification, dans le site de fixation de la lectine, dans chacun desdits sous-domaines 1α, 1β et 2η et une fraction consistant en un ligand spécifique d'un récepteur de surface. Elle porte aussi sur un procédé de production d'une immunotoxine de protéine de fusion de ricin, qui consiste à exprimer l'acide nucléique contenu dans un vecteur d'un système d'expression de cellules eucaryotes de sorte qu'une protéine de fusion soit produite; à isoler et à purifier la protéine de fusion; et à mettre ladite protéine de fusion en contact avec une chaîne de toxine A de ricin dans des conditions permettant l'association de la protéine de fusion avec la chaîne de toxine A de ricin. Elle porte sur un procédé de traitement d'un cancer ou d'une maladie auto-immune chez un patient souffrant de cancer ou d'une maladie auto-immune, qui consiste à produire une immunotoxine de protéine de fusion de ricin, le ligand étant spécifique d'un récepteur de surface présent seulement à la surface des cellules cancéreuses ou des cellules induisant la maladie auto-immune; et à administrer au patient l'immunotoxine de protéine de fusion dans un véhicule pharmaceutiquement acceptable, l'immunotoxine de protéine de ricin traitant le cancer ou la maladie auto-immune dont souffre le patient.
PCT/US1997/019577 1996-10-28 1997-10-28 Procedes et compositions relatifs a des immunotoxines de la proteine de fusion du ricin, pour le traitement du cancer et de maladies auto-immunes WO1998018820A1 (fr)

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WO1998029540A2 (fr) * 1997-01-02 1998-07-09 B.R.A.I.N. Biotechnology Research And Information Network Gmbh Proteines recombinantes de fusion a base de proteines du gui viscum album qui desactivent les ribosomes
WO2000058456A2 (fr) * 1999-03-30 2000-10-05 Board Of Regents, The University Of Texas System Compositions et methodes permettant de modifier les effets toxiques de composes proteiques
WO2000066755A2 (fr) * 1999-04-28 2000-11-09 Plant Bioscience Limited Fusions pesticides

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998029540A2 (fr) * 1997-01-02 1998-07-09 B.R.A.I.N. Biotechnology Research And Information Network Gmbh Proteines recombinantes de fusion a base de proteines du gui viscum album qui desactivent les ribosomes
WO1998029540A3 (fr) * 1997-01-02 1998-11-12 B R A I N Biotechnology Resear Proteines recombinantes de fusion a base de proteines du gui viscum album qui desactivent les ribosomes
WO2000058456A2 (fr) * 1999-03-30 2000-10-05 Board Of Regents, The University Of Texas System Compositions et methodes permettant de modifier les effets toxiques de composes proteiques
WO2000058456A3 (fr) * 1999-03-30 2001-02-15 Univ Texas Compositions et methodes permettant de modifier les effets toxiques de composes proteiques
US6566500B1 (en) 1999-03-30 2003-05-20 Board Of Regents, The University Of Texas System Compositions and methods for modifying toxic effects of proteinaceous compounds
US7829668B2 (en) 1999-03-30 2010-11-09 Board Of Regents, The University Of Texas System Compositions and methods for modifying toxic effects of proteinaceous compounds
WO2000066755A2 (fr) * 1999-04-28 2000-11-09 Plant Bioscience Limited Fusions pesticides
WO2000066755A3 (fr) * 1999-04-28 2001-01-18 Plant Bioscience Ltd Fusions pesticides
AU777669B2 (en) * 1999-04-28 2004-10-28 Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Pesticidal fusions
US7019197B1 (en) 1999-04-28 2006-03-28 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E. V Pesticidal fusions

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