WO1996041608A2 - Thionine pyrularia contenant des immunotoxines ainsi que des conjugues similaires aux immunotoxines - Google Patents

Thionine pyrularia contenant des immunotoxines ainsi que des conjugues similaires aux immunotoxines Download PDF

Info

Publication number
WO1996041608A2
WO1996041608A2 PCT/US1996/008811 US9608811W WO9641608A2 WO 1996041608 A2 WO1996041608 A2 WO 1996041608A2 US 9608811 W US9608811 W US 9608811W WO 9641608 A2 WO9641608 A2 WO 9641608A2
Authority
WO
WIPO (PCT)
Prior art keywords
cells
molecule
toxin
immunotoxin
amino acid
Prior art date
Application number
PCT/US1996/008811
Other languages
English (en)
Other versions
WO1996041608A3 (fr
Inventor
Leo P. Vernon
Eppie D. Rael
Sardar E. Gasanov
Original Assignee
Thera Pro
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Thera Pro filed Critical Thera Pro
Priority to AU59817/96A priority Critical patent/AU5981796A/en
Publication of WO1996041608A2 publication Critical patent/WO1996041608A2/fr
Publication of WO1996041608A3 publication Critical patent/WO1996041608A3/fr

Links

Classifications

    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • 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
    • A61K47/6817—Toxins
    • A61K47/6819—Plant toxins
    • 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
    • A61K47/6835—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—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 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant

Definitions

  • the present invention relates to immunotoxins and immunotoxin like compounds. More particularly, the invention relates to molecules containing a protein which is specific for an antigen or ligand on the cell surface conjugated to the toxin Pyrularia thionin.
  • Cancer develops when a cell in ones body undergoes uncontrolled growth. Unregulated cell proliferation is primarily a result of irreversible damage to particular classes of genes within the cell. As genetic mutations accumulate in the DNA of the cell, each successive generation of daughter cells becomes less responsive to growth inhibitory and regulatory signals. Eventually, a daughter cell will no longer respond to these signals and begins to display the signs of malignancy. The resulting cellular mass, or tumor, damages the surrounding healthy tissue. With time, the cancer can metastasize and breakdown organ tissue barriers, establishing new colonies at distant sites.
  • Immunotoxins and immunotoxin-like compounds are anticancer agents that are highly selective for cancer cells.
  • Immunotoxins are protein conjugates comprised of a monoclonal antibody and a toxic peptide or small protein.
  • the monoclonal antibody portion of the immunotoxin recognizes a specific antigen expressed only, or primarily, by a particular type of cells. When the antibody binds to the antigen, it brings the toxin in close proximity to the cancer cell were it exerts its cytotoxic effect.
  • Immunotoxin-like compounds derive their target specificity from growth factors that bind to a particular receptor, such as erbB2, EGF, IL2, IL4, and IL6 receptors; other antigens which are predominantly found on carcinomas; or certain oligosaccharides, such as derivatives of the Lewis Y type carbohydrate which are abundantly expressed by carcinomas.
  • a particular receptor such as erbB2, EGF, IL2, IL4, and IL6 receptors
  • other antigens which are predominantly found on carcinomas
  • certain oligosaccharides such as derivatives of the Lewis Y type carbohydrate which are abundantly expressed by carcinomas.
  • Immunotoxins and immunotoxin-like compounds which are composed of monoclonal antibodies or growth factors coupled to plant or bacterial toxins have been shown to be highly cytotoxic to, and specific for, the targeted cancer cell. Most of these toxins rely on arresting cellular protein synthesis for their cytotoxicity.
  • the plant toxins ricin and abrin inacti- vate ribosomes by cleaving a specific sequence in 60S rRNA, while the bacterial toxins diphtheria toxin and Pseudomonas exotoxin A inactivate the elongation factor of the protein synthesis mechanism.
  • immunotoxins are themselves immunogenic. Upon entering the host, the immunotoxins illicit an immune response which ultimately leads to the sequestration of many of the immunotoxins before they ever reach their target cell.
  • the immunogenicity of the immunotoxins may be reduced by producing chimeric monoclonal antibodies that contain a mouse variable region and a human constant region. This strategy, however, will not affect the immunogenicity of the conjugated toxin.
  • Another problem is that most of the conjugated toxins used must be internalized by receptor-mediated endocytosis in order to exert their cytotoxic effect.
  • the bulky, sterically hindered antibody which is conjugated to the toxin results in slower internalization rates and the inability of the toxin to penetrate large tumor burdens.
  • many of the toxins contain two subunits, one subunit which binds to a receptor on the cell and mediates endocytosis, and another which is toxic.
  • Another immunotoxin which has been developed contains the toxin purothionin, isolated from barley flour, conjugated to the monoclonal antibody 225.28S. Unlike the other toxins used to date, purothionin does not need to be internalized to be cytotoxic. Instead, purothionin binds to the cell membrane where it disrupts the phospholipid bilayer causing cell death. Thus, purothionin is never exposed to the proteases found in the cytosol and lysosomes of the host cell.
  • conjugated purothionin is approximately 10,000 times less toxic than the ribosome inactivating toxins (1).
  • the purothionin immunotoxin is only able to moderately suppress the growth of melanoma cells in nude mice at the early stages of growth, and thus has limited human therapeutic potential (55).
  • Immunotoxins and immunotoxin-like molecules consist of proteins that have been chemically coupled to a toxin, usually from bacteria or plants.
  • the protein typically a monoclonal antibody or growth factor, is chosen such that it delivers the toxin to a selective group of cells which express the corresponding antigen or ligand.
  • the molecules display a high degree of cellular specificity and related cytotoxicity.
  • the present invention relates to an immunotoxin, which in one embodiment, comprises a mouse antihuman-CD5 monoclonal antibody conjugated to the toxin Pyrularia thionin ("PT" or "P. thionin”).
  • the anti-CD5 monoclonal antibody recognizes the antigen CD5 found on all human T-cells and activated B-cells.
  • PT is a small, highly basic and stable, 47 amino acid residue peptide.
  • PT belongs to a family of peptides which, with the exception of crambin, exhibit some degree of toxicity towards animals and cultured cells including yeast, fungi, bacteria, and mammalian cells. PT, however, does not display any toxicity towards bacteria. At moderate concentrations it is toxic to cancer cells in culture and at higher concentrations it is toxic to mice.
  • PT causes a number of damaging cellular responses including hemolysis of erythrocytes, depolarization of the cellular membrane, activation of a calcium ion channel, and activation of endogenous phospholipase A 2 enzyme.
  • anti-CD5 conjugated PT was found to have the same mechanism of action as native PT, the conjugated PT was significantly more cytotoxic than native PT. In all three measures of toxicity, including trypan blue exclusion, release of radio- activity from T cells loaded with 51 Cr, and inhibition of mito- gen-stimulated cell proliferation, the PT immunotoxin was approximately > 10 4 times more active than native PT, with an average ID 50 of approximately 1.8 X 10 -11 M.
  • the PT immunotoxin also displayed a high degree of specificity.
  • the PT immunotoxin's specificity was shown by its inactivity towards either human or sheep erythrocytes or to mouse lymphocytes which do not express the human CD5 antigen.
  • the anti-CD5 antibody alone was not toxic to human lymphocytes.
  • CD5 + After treating human lymphocytes with the PT immunotoxin and guinea pig complement, only 1.5% of the remaining cells were CD5 + , which is specific for T cells. In contrast, 92.5% were positive for anti-human IgM-FITC, which is specific for human B cells.
  • PT is membrane-active, obviating the need for PT to be internalized in order to exert its cytotoxic effect. PT, therefore, is never exposed to the proteases found in the cytosol of the host cell.
  • PT is a very stable, compact peptide which is resistant to most proteases.
  • PT is not immunogenic. PT goes undetected by the host's immune system, further increasing its efficacy and cytotoxicity. Fourth, the cytotoxicity of PT does not decrease, and in fact unexpectedly increases, when it is conjugated to the monoclonal antibody. Fifth, the small size of PT facilitates the immunotoxin's entry into solid tumors.
  • PT's cytotoxicity is lost after it is incorporated into the lipid bilayer of the host cell. Therefore, the PT immunotoxin will not produce "second round" cytotoxicity towards macrophages and other cells that come in contact with the membrane of cells containing the PT immunotoxin.
  • Figure 1 illustrates the purification profile of Pyrularia thionin on a Sephadex G-75 column , monitored by A 278 and cytotoxicity.
  • the cytotoxicity was rated between 0-4+.
  • a score of 0 indicates that the fraction was not cytotoxic, while a score of 4+ indicates the fraction was highly cytotoxic. Both absor-ettee and cytotoxicity peaked at tube 34.
  • the dashed line shows absorbance readings obtained from a solution containing cytochrome c (12.4 kDa), bovine trypsin inhibitor (6.2 kDa), and insulin (3.0 kDa).
  • Figure 2 illustrates the purification profile of Pyrularia thionin on a carboxymethyl cellulose CM52 column with a 0.1 - 1.0 M NaCl gradient, monitored by A 278 and cytotoxicity which peaked at tube 24.
  • Figure 3 is an elution profile of approximately 25 ⁇ g of purified Pyrularia thionin from an Ultrasphere C3 column, monitored at 214 nm. Superimposed on the elution profile is a line designating the percentage of buffer B, containing 60% acetonitrile, in the developing buffer.
  • Figure 4 illustrates the isolation of anti-CD5-PT conjugate from the reaction mixture.
  • Figure 4A illustrates the fractionation of the mixture of anti-CD5 and PT in phosphate buffer (pH 6.9, 1 mM EDTA) by cation exchange HPLC.
  • SCX 83- C13-ETI Hydropore column was equilibrated with 0.02 M Na 2 HPO 4 , pH 6.9, 1 mM EDTA.
  • a salt gradient was established with 1 M
  • Fraction a is anti-CD5
  • fraction b is PT
  • Figure 4B illustrates the fractionation of the reaction mixture of derivatized anti-CD5 and anti-CD5 conjugated to PT upon the conditions as noted with respect to Figure 4A.
  • the material eluting first at 3 minutes, designated fraction a is unmodified anti-CD5.
  • the material eluting at 4 minutes, designated fraction b is anti-CD5 SPDP.
  • the active immunotoxin eluted with a peak at 20 minutes, and is designated fraction c. This is the fraction used in the other experiments.
  • Figure 5 illustrates the results of SDS-polyacrylamide gel electrophoresis of fractions a (lane 3), b (lane 4), and c (lane 5) as described in Figure 4B.
  • Lane 1 is standard high molecular weight proteins. Molecular weights are indicated in kilodaltons. Lane 2 is native anti-CD5.
  • Figure 6 illustrates the results of isoelectric focusing of fractions a (lane 3), b (lane 4), and c (lane 5) as
  • Lane 1 is standard pI reference proteins. Lane 2 is native anti-CD5.
  • Figure 7 illustrates the EPR spectra of 5-doxylstearic acid in oriented phospholipid-multibilayers having a spin probe/lipid mole ratio 1/100. Magnetic field parallel (solid line) and perpendicular (broken line) to the bilayer normal.
  • Figure 8 illustrates endotherms of DMPC phospholipid bilayers (upper curves) and DMPC + 5 mol% PS (lower curves) without added reagents (1), and with the addition of PT (2) and immunotoxin (3).
  • the reagent/lipid mole ratio was 0.075.
  • Figure 9 illustrates the cytotoxicity of fractions a (open square), b (black squares), and c (black circles) of Figure 4B, as measured by trypan blue uptake, on human peripheral blood lymphocytes. Cytotoxicity is expressed as the percent of viable cells of control samples.
  • Figure 10 is a graph illustrating the effect of PT immunotoxin on lymphocyte proliferation as measured by 3 H thymidine uptake.
  • Human lymphocytes were stimulated by pokeweed mitogen (open squares), concanavalin A (black circles), and allogenic antigens in the mixed lymphocyte reaction (open circles).
  • Figure 11 is a graph illustrating the cytotoxic effect of the PT immunotoxin on human lymphocytes as measured by 51 Cr release.
  • Curve 1 black circles
  • Curve 2 open circles
  • Curve 3 black triangles
  • Curve 3 was run under the same conditions as curve 2 except that the PT immunotoxin was replaced with native anti-CD5 antibody.
  • the present invention relates to immunotoxins and immunotoxin-like molecules.
  • the invention comprises an anti-CD5 monoclonal antibody conjugated to the toxin Pyrularia thionin.
  • Immunotoxin - is defined as a toxin coupled to a monoclonal antibody or any fragment of an antibody which confers specificity.
  • Immunotoxin-like molecules - is defined as a toxin coupled to a target specific molecule which confers specificity.
  • Target specific toxin - is defined as a toxin coupled to a target specific molecule which confers specificity.
  • Target specific molecule - is defined as any molecule which is capable of conferring specificity, such as growth factors, cell adhesion molecules, or carbohydrates.
  • Specificity - is the ability of a molecule to recognize and bind to one particular ligand on the surface of a cell in the presence of other ligands.
  • Conservative amino acids - are defined as natural or synthetic amino acids which have a similar size, charge, polarity or conformation, and therefore can readily substitute for one another.
  • TSM target specific molecules
  • the present invention employs the mouse antihuman-CD5 monoclonal antibody described in United States Patent No. 4,680,383 issued to Kung et al., which is hereby incorporated by reference.
  • CD5 is a 65-kDa membrane glycoprotein expressed on T cells and activated B cells. The glycoprotein is also expressed on most T-cell derived tumors, and on B cells in chronic B-lymphocytic leukemia.
  • the anti-CD5 was isolated by standard techniques well known in the art. Briefly, the anti-CD5 was obtained from a known hybridoma cell line (OKT1, ATCC CRL 8000) purchased from American Type Culture Collection, Rockville, MD. The cells were propagated by injection into pristane-primed Balb c mice. After 6-10 days the ascites fluid was collected and the antibody purified on a protein A-Sepharose column, followed by absorption chromatography on hydroxyapatite. Similarly, other IgGs specific for an antigen expressed by a particular carcinoma could be isolated and conjugated to PT. Monoclonal antibodies such as HB21, C242, LL2, or B3 have all been successfully conjugated to plant and bacterial toxins to confer specificity.
  • the immunogenicity of the monoclonal antibodies could be reduced by producing chimeric monoclonal antibodies that contain a mouse variable region and a human constant region (41-46).
  • a PT immunotoxin composed of a "humanized" monoclonal antibody should go virtually undetected by the host's immune system.
  • IgG conjugated toxins are their size.
  • the large molecules have difficulty penetrating large solid tumors.
  • Preferential proteolytic cleavage of the IgG generates two Fab fragments (Fab') 2 .
  • Each Fab' is comprised of two heter- odimers which, in turn, are comprised of a heavy chain Fd and a light chain covalently linked by disulfide bonds at their C- terminus.
  • (Fab') 2 fragments can be conjugated to toxins in the same manner as IgGs.
  • (Fab') 2 fragments can be preferentially reduced to generate Fab' molecules. Like (Fab') 2 fragments, Fab' fragments can be conjugated to toxins in the same manner as IgGs. It has also been shown that the free mercapto group generated during reduction of the disulfide bond can be used for conjugation without affecting the Fab' fragment's full binding activity. Moreover, Fab' fragments, unlike (Fab') 2 fragments and IgGs, produce a homogeneous population of conjugates because the conjugation reaction occurs at a defined position. Several Fab' ricin and Pseudomonas exotoxin conjugates have been successfully constructed (2, 3-7).
  • Fv fragments Yet another subunit of IgGs that can be used to convey specificity are Fv fragments.
  • An Fv is the smallest subunit of an IgG that can mediate specific binding.
  • An Fv is comprised of a heavy and light variable region. Proteolytically derived Fv fragments cannot be obtained because they are unstable.
  • Fv fragments which are stabilized by either a peptide bond (scFv) or a disulfide bond (dsFv) display good specificity and stability.
  • the scFv frag- ments are produced as a single chain.
  • the peptide bond connects the C- and N-terminal regions of the heavy and light variable regions much like a anti-parallel ⁇ -sheet structure (11-12).
  • scFv conjugated to bacterial toxins such as anti- Tac (Fv)-PE40 directed at IL2 receptor, have been shown to display excellent antitumor activity in animal models (13-27) .
  • the dsFv fragments are generated by recombinant expression of the variable heavy and light chains separately in E. coli and combined during refolding. While the dsFv display similar properties as the svFv fragments, they typically give higher yields and are more resistant to thermal and chemical denatur- ation. Examples of dsFv immunotoxins are B3 (dsFv) -PE38,
  • Growth factors make good TSMs because many of the receptors for growth factors are overexpressed in carcinoma cells.
  • Growth factor conjugates are constructed under the same principle as scFv fragments, as a single chain fusion protein.
  • growth factor PT conjugates can be constructed using basic recombinant DNA technology. Indeed, many growth factor fusions display a high degree of specificity and cytotoxicity (30-40).
  • TGF ⁇ -PE40 binds specifically to EGF receptors overexpressed in many carcinomas.
  • CD4-Pe40 in which the HIVl-gp41 binding portion of CD4 is selectively cytotoxic to HIV1 infected cells.
  • the immunotoxin of this invention employs a cleavable disulfide linker well known in the art.
  • the PT was conjugated to the anti-CD5 antibody by treating PT with 2-Iminothiolane- HCl to provide sulfhydryl groups, and anti-CD5 with N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to provide pyridyl disulfide residues.
  • SPDP N-Succinimidyl 3-(2-pyridyldithio) propionate
  • Step 1 anti-CD5-NH 2 + SPDP ⁇ anti-CD5-NH-CO-CH 2 -CH 2 -S-S-PY
  • Step 2 anti-CD5-NH-CO-CH 2 -CH 2 -S-S-PY + PT-SH ⁇
  • linkages which are commonly utilized and expected to be useful for conjugating PT to target specific molecules are imminothiolane/succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1-carboxylate and carbodiimide linkages.
  • linkages for conjugating proteins of varying lengths, stability, flexibility and chemical reactivity are well known in the art and in many cases are commercially available.
  • PT PT Purification and Recombinant Expression of PT PT was purified from nuts of Pyrularia pubera that were ground in cold 0.1 M phosphate buffered saline (PBS) as described previously (53). The insoluble matter was centrifuged and the supernatant was fractionated with ammonium sulfate. The majority of the biologically active material precipitated between 45% ammonium sulfate and saturation as determined by the cytotoxicity of the fractions. Residual ammonium sulfate was removed by dialysis against 0.01 M PBS.
  • PBS phosphate buffered saline
  • the PT containing fractions were then further purified by gel filtration on a Sephadex G-75 column ( Figure 1).
  • the PT was eluted with 0.02 M NaCl/0.02 M phosphate buffer.
  • the biologically active fractions were then subjected to ion exchange chromatography with a carboxymethyl cellulose CM52 column ( Figure 2).
  • PT was eluted with a linear gradient of 0.1 to 1.0 M NaCl.
  • the resulting biologically active fraction was greater than 95% pure as judged by SDS-PAGE (data not shown) and reverse-phase HPLC (Figure 3).
  • Recombinant PT could also be used to make PT immunotoxin.
  • a nucleic acid sequence which codes for the amino acid sequence of PT containing an initiator methionine at the amino terminus and a stop codon at the carboxyl terminus could be synthesized.
  • the double stranded nucleotide sequence could then be subcloned into a eucaryotic or procaryotic expression vector which is commercially available.
  • Systems which allow proteins to be overexpressed in, and purified from, bacteria, baculovirus or mammalian cells are well known in the art.
  • a synthetically engineered PT gene was subcloned into plasmids capable of expressing the gene in E. coli using techniques well known in the art. Briefly, two synthetic primers, each comprising slightly over half of the PT gene, were synthesized and used to create a full length PT gene by polymerase chain reaction.
  • the nucleotide sequence of the synthetic PT gene (SEQ ID NO.1) contains an initiator methionine immediately preceeding the first amino acid of PT, and a stop codon immediately following the last amino acid of PT, both which are Lys.
  • the gene also contains a Nde I restriction endonuclease site (CATATG) flanking the initiator methionine, and a Bam HI restriction site (GGATCC) just 3' of the stop codon.
  • pET21/ptox consists of the PT gene under the control of the T7 promoter. It can be expressed in any bacterial cell that contains the T7 RNA polymerase gene under the control of an inducible promoter, such as BL21DE3 and BL21DE3pLysS.
  • pRE1/ptox consists of the PT gene under the control of the lambda PL promoter.
  • pREl/ptox is expressed in the E. coli strain CJ347 which expresses the CI857 gene, a temperature sensitive lambda repressor.
  • pREl/ptox is repressed at 30° C and derepressed (or induced) at 42° C.
  • PT is expressed by growing the bacteria at 30° C until they reach mid-log phase, and then at 42° C for thirty minutes. Finally, the bacteria are grown at 37° C for approximately two to five hours. SDS or tricine polyacrylamide gel electrophoresis can be used to visualize the recombinant PT peptide.
  • PT is a 47 amino acid, heat stable, highly basic peptide. It has a calculated molecular weight of 5280.
  • the amino acid sequence of PT (SEQ ID NO. 2) as determined by chemical sequencing is shown below:
  • PT and the cereal thionins contain 8 conserved cysteine residues which form four disulfide bonds.
  • a comparison of PT with several other members of the thionin family is shown below.
  • the one-letter code for amino acids used is: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lle; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.
  • Dashes indicate gaps inserted to improve alignment.
  • the shaded regions indicate the amino acids that are conserved between thionins.
  • Protein modifications of PT can be subdivided into three general classes: substitutions, additions and deletions. These general groups apply to both the nucleic acid and amino acid sequence of the PT immunotoxin. While protein modifications may occur naturally, most often protein modifications are deliberately engineered into the nucleic acid sequence that codes for the protein. Protein modification techniques such as site-directed mutagenesis are well known in the art and in many cases are commercially available as kits complete with instructions. Kits of this type are available from, for example, Amersham and Bethesda Research Laboratories.
  • substitutions may be made in a protein without significantly altering the protein's function.
  • Substitutions are modifications made to the nucleic acid or amino acid sequence of the protein which produce a protein with a different amino acid sequence than the native protein without significantly altering the major properties or function of the toxin. The most favorable substitutions occur when amino acids are substituted by "conserved" amino acids. conserveed amino acids are natural or synthetic amino acids which because of size, charge, polarity and conformation can be substituted into the protein without significantly affecting the structure and function of the protein.
  • the non-polar amino acids Gly, Ala, Val, lle and Leu; the non-polar aromatic amino acids Phe, Trp and Tyr; the neutral polar amino acids Ser, Thr, Cys, Gln, Asn and Met; the negatively charged amino acids Lys, Arg and His; the positively charged amino acids Asp and Glu represent conservative groups of amino acids.
  • This list is not exhaustive. For example, it is well known that Ala, Gly, Ser and sometimes Cys can substitute for each other even though they belong to different groups.
  • Conservative amino acid substitutions are not limited to naturally occurring amino acids, but also include synthetic amino acids.
  • Commonly used synthetic amino acids are ⁇ amino acids of various chain lengths and cyclohexyl alanine which are neutral non-polar analogs; citulline and methionine sulfoxide which are neutral non-polar analogs, phenylglycine which is an aromatic neutral analog; cysteic acid which is a positively charged analog and ornithine which is a negatively charged amino acid analog.
  • this list is not exhaustive, but merely exemplary of the substitutions that are well known in the art.
  • an amino acid can be substituted at all, or whether it can only be substituted by a conserved amino acid is best determined by comparing PT with other members of the th-ionin family.
  • Amino acids that are identical in all the members of a protein family usually cannot be substituted. This is the case with the 8 cysteine residues which are absolutely conserved between thionins. Except for cysteines which form essential disulfide bonds, amino acids which are conserved between members of the same family of proteins can usually be substituted by other conserved amino acids without significantly affecting the protein's function.
  • the basic amino acids which are required for binding to the cell membrane, including the terminal lysine residue of PT could likely be substituted only by other basic amino acids.
  • Trp amino acids which are not conserved within a family can usually be freely substituted.
  • Trp is not conserved between other thionins, but has been shown to be essential for activity. Trp 8, could probably only be substituted with Phe, Tyr or a large non-polar, non-aromatic amino acid.
  • Additions are modifications made to the nucleic acid or amino acid sequence of the protein which produce a protein containing at least one amino acid more than the native amino acid sequence of the protein without significantly altering the function of the toxin.
  • the mature protein lacks an initiator methionine which may be included if the toxin is recombinantly expressed.
  • an initiator methionine which may be included if the toxin is recombinantly expressed.
  • the addition of a methionine to the amino terminus of the PT, as well as the additions of other amino acids which facilitate the expression of the protein such as stop codons, are not expected to significantly affect the function of the toxin.
  • Protein modifications may also occur through deletions.
  • Deletions as defined herein, are modifications made to the nucleic acid or amino acid sequence of the protein which produce a protein containing at least one amino acid less than the native amino acid sequence of the protein without significantly altering the function of the toxin.
  • PT belongs to a family of peptides which, with the exception of crambin, exhibit some degree of toxicity towards animals and cultured cells including yeast, fungi, bacteria, and mammalian cells (54). PT, however, does not display any toxicity towards bacteria.
  • Purified PT is cytotoxic for all the cell lines tested in tissue culture to date.
  • the ID 50 of several of these cell lines is shown in Table 2 below:
  • thionins act on the cell membrane.
  • PT's action on the cell membrane can be divided into four distinct phases: (1) binding of PT to the cell membrane by electrostatic forces; (2) perturbation of the phospholipid structure of the bilayer; (3) depolarization of the membrane and opening of a calcium channel; and (4) activation of endogenous phospholipase A 2 (PLA 2 ).
  • the binding phase which has been studied extensively in erythrocytes, most likely involves PT binding to phosphatidyl- serine (PS). This is probably accomplished by electrostatic binding of the basic amino acid residues of PT with the acidic headgroup of PS (47).
  • Trp number 8 After binding, PT perturbs the membrane by inserting Trp number 8 into the lipid bilayer. This changes the membrane fluidity of the cell and leads to depolarization of the cellular membrane and rapid changes in membrane permeability. All these responses are rapid, proceeding without any appreciable delay.
  • the third phase involves membrane depolarization and the opening of a calcium channel with an influx of calcium. This has been shown with mouse P388 cells and with rat anterior pituitary cells in which the calcium channel blockers methoxy- verapamil and dopamine inhibited PT release of growth hormone from the cell.
  • the final phase which is the activation of endogenous PLA 2 , displays a distinct 20 minute time lag.
  • PT-induced PLA 2 activity has been demonstrated in N1H 3T3 fibroblast cells by the release of radiolabled arachidonic acid at concentration above 10 ⁇ g/ml (about 2 ⁇ M).
  • the enzyme hydrolyzes the fatty acid at the SN-2 position of the phospholipid, liberating arachidonic acid which serves as a precursor of prostaglandins.
  • PT is able to influence cellular second messenger systems without entering the cell.
  • the PT immunotoxin was prepared by linking PT to mouse antihuman-CD5 through an artificial disulfide bond.
  • 2-Iminothiolane-HCl was used to provide PT with sulfhydryl groups and N-Succinimidyl 3-(2-pyridyldithio) propionate (SPDP) served to provide the anti-CD5 with pyridyl disulfide residues to react with thiols of the derivatized PT.
  • SPDP N-Succinimidyl 3-(2-pyridyldithio) propionate
  • PT was incubated with 2-Iminothiolane-HCl in phosphate buffer.
  • Anti-CD5 was incubated with SPDP in phosphate buffer.
  • Derivatized PT and anti-CD5 were conjugated by incubating the derivatized PT and anti-CD5 together overnight at room temperature in phosphate buffer.
  • CD5 and PT immunotoxin by a slot-blot procedure utilizing anti- mouse IgG-horseradish peroxidase as a secondary antibody.
  • Figure 5 illustrates the integrity of the fractions as determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions
  • Figure 6 illustrates the integrity of the fractions by isoelectric focusing (IEF).
  • the higher pi for fraction c, the immunotoxin, is consistent with the conjugation of the very basic PT to anti-CD5. 5.5.2. Biological Activity
  • the immunotoxin composed of PT and anti-CD5 antibody interacts with phospholipid bilayers in a manner similar to native PT.
  • Previous research has shown that the thionin inter acts with phospholipid bilayers consisting of phosphatidyl- choline and small amounts of phosphatidylserine by binding to the phosphatidylserine, thus perturbing the order of the phospholipid bilayer. This was shown by EPR (electron paramagnetic resonance) measurements on phospholipid bilayer which have the spin probe 5-doxylstearic acid incorporated into the bilayer.
  • Figure 7 shows such data obtained with phosphatidylcholme (PC) bilayers containing 0 and 30 mole % phosphatidylserine (PS) and a spin probe/lipid mole ratio of 1/100.
  • PC phosphatidylcholme
  • PS phosphatidylserine
  • Curve 1 shows the spectra obtained with phospholipid bilayer alone, while for curve 2 PT was added at a PT/lipid mole ratio of 0.075, and for curve 3 immunotoxin was added at a immuno- toxin/lipid mole ratio of 0.075.
  • Complete spectral isotropy was found for the PC + PS membranes treated with PT. Similar membranes treated with immunotoxin retained some spectral anisotropy, but the same general response was shown for both the immunotoxin and PT.
  • PT immunotoxin cytotoxicity was determined by the trypan blue exclusion assay, lymphocyte proliferation, and 51 Cr release. Trypan blue is excluded by normal healthy cells, but is readily taken up by cells with perturbed or disrupted membranes. As expected, fraction c of Figure 4B containing the PT immunotoxin was highly toxic to cells, fraction b which likely contains the anti-CD5 SPDP derivative was only slightly toxic, and fraction a containing unconjugated antibody was not toxic. ( Figure 9). The ID 50 value for conjugated PT was 4 X 10 -11 M, while native PT had an ID 50 value of approximately 1 X 10 -6 M.
  • the influence of cell proliferation was determined by 3 [H] thymidine uptake after stimulation with mitogen or in a mixed lymphocyte reaction using standard procedures.
  • Human lympho- cytes were stimulated with either concanavalin A or mixed lymphocyte reaction, which only stimulate T cells, or pokeweed mitogen, which stimulates both B and T cells. Stimulated cells were then pulsed with 3 [H] thymidine and incorporated 3 [H] thymidine determined by liquid scintillation counting.
  • Figure 10 demonstrates that 1 X 10 -9 M of PT immunotoxin completely suppressed stimulation by concanavalin A and mixed lymphocytes. Since pokeweed mitogen stimulates both B and T cells in the lymphocyte preparation, and because B cells do not contain CD5, the PT immunotoxin was unable to completely inhib- it cell proliferation in the presence of pokeweed mitogen.
  • the ID 50 value for conjugated PT was approximately 5 X 10 -12 M, while native PT had an ID 50 value of approximately 1 X 10 -7 M.
  • Cytotoxicity was also measured by the release of 51 Cr from human lymphocytes loaded with chromate ion containing 51 Cr.
  • the experiments were performed using the anti-CD5 immunotoxin alone and also with PT immunotoxin and anti-CD5 antibody, both in the presence of guinea pig complement which lyses cells in the presence of antibody independently of the action of the PT immunotoxin.
  • the data obtained are shown in Figure 11.
  • the PT immunotoxin alone was able to lyse about 50% of the cells.
  • Half-maximal activity of the PT immunotoxin alone was observed at about 1 X 10 -17 M.
  • the immunotoxin conjugate in the presence of complement was more effective in terms of lysis, with a ID 50 of 1 X 10 -12 M.
  • concentrations below 10 -11 M of antibody or immunotoxin conjugate added complement was synergistic to the toxic effect of PT immunotoxin alone. This indicates that perturbation of the membrane bilayer by the PT contained on the immunotoxin facilitated the action of complement on the membrane.
  • PT immunotoxin for CD5 + cells was assessed by immunofluorescence. For lymphocytes treated with the PT immunotoxin only, 1.5 ⁇ 0.11 % of the cells tested positive for the CD5 antigen, while 92.5 ⁇ 4.8 % of the cells were positive for anti-human IgM-FITC which is specific for human B cells. In control samples, 75.8 ⁇ 3.9 % of the cells were positive for CD5, while 19.2 ⁇ 1.3 % of the cells tested positive for anti-human IgM-FITC. These data demonstrate that the PT immunotoxin is specific for cells that express the CD5 antigen, killing only the CD5 + T cells.
  • the PT immunotoxin of the present invention may.be used for the treatment of T cell leukemias in vivo .
  • Recent studies have demonstrated that anti-CD5 monoclonal antibodies conjugat- ed to both single and double chain ribosomal inactivating proteins are useful for treating T-cell leukemias.
  • One such study employed an anti-CD5 momordin conjugate (48).
  • the potency of the immunotoxin was high against the T-cell leukemia cell line Jurkat, with an ID 50 of 1-10 pM.
  • the immunotoxin also significantly reduced tumor development in nude mice bearing the Jurkat leukemia.
  • this invention and the latter study use the same monoclonal antibody, and that they display similar ID 50 values, the PT immunotoxin is also expected to be effective against T cell leukemias in vivo .
  • the use of a cocktail of immunotoxins and immunotoxin-like molecules should be more cytotoxic and more effective for killing tumor cells.
  • the other immunotoxins would be those containing ribosomal inactivating proteins (RIP) toxins conjugated to the same anti-CD5 antibody, or could be ribosomal inactivating proteins toxins conjugated to other target specific molecules directed toward other antigens on the same cell.
  • the advantage of using a cocktail of immunotoxins is that the two types of conjugated toxins would react with, and kill, the cell by different mechanisms.
  • the RIP immunotoxins would enter the cytosol and inhibiting protein synthesis, while the PT immunotoxins would disrupt the cell membrane and initiate second messenger systems.
  • the second immunotoxin in the cocktail could be an antibody-drug conjugate containing a drug such as doxorubicin, or doxorubicin alone, whose effectiveness would be increased by the membrane perturbation caused by PT.
  • PT immunotoxin Another approach to increasing the effectiveness of the PT immunotoxin would be to prepare double conjugates which contain both PT and a ribosomal inactivating protein conjugated to the same TSM.
  • the two toxins could be joined together using basic recombinant technology to form a double toxin, and then conjugate this double toxin to the TSM by a single linker.
  • both toxins could be joined directly to the TSM with linkers of sufficient length to allow both toxins to interact with the cell membrane and exert their toxic effects.
  • the PT immunotoxin of the present invention may be used for the in vitro manipulation of donor cells used in tissue and organ grafts, blood transfusions, and bone marrow transplants.
  • One area of particular concern is when the donor and the recipient have different histocompatibility.
  • the allogeneic recipient is unable to identify and reject the T cells.
  • the donor's T cells react against the tissues of the host in a graft-versus-host
  • the recipient receives the tissue or blood from the donor free of T-cells.
  • Immunotoxins directed at T-cells have been previously used to treat graft-versus-host disease (49).
  • the immunotoxin used in the above cited study employed the ricin A chain toxin conjugated to a antihuman-CD5 antibody. The treatment resulted in a rapid reduction of peripheral blood T lymphocytes which persisted for more than a month.
  • Other studies have used T101 Fab fragment (50), anti-CD3 (51) and a combination of all three (52). It is expected that strategies similar to those used to treat T cell leukemias would be effective for graft-versus-host disease.
  • Extraction and ammonium sulfate precipitation Five hundred grams of fresh or frozen Pyrularia leaves, or 300 g of fresh or frozen Pyrularia nuts were ground for approximately 1 minute in 500 ml of 0.1 M phosphate buffer, pH 7.0, in a chilled blender, and the homogenate was frozen overnight. Upon thawing, the homogenate was ground again for 15 seconds in the blender and the resulting homogenate was centrifuged at 1000g for 10 min to remove debris, which was extracted again with 100 ml of buffer solution. To each 100 ml of supernatant fluid was added successively 20, 10, 10, 10, and 10 g of (NH 4 ) 2 SO 4 . After each addition, the suspension stood for 10 min at 25°C and was then centrifuged at 20,000g to remove the precipitated protein. The last 10 g of ammonium sulfate did not completely dissolve at room temperature, since the solution was >95% saturated.
  • the sedimented protein from each stage was dissolved in 50 ml of 0.05 M phosphate buffer, pH 7.2, to give fractions which we designate as I, II, III, IV, and V, respectively.
  • the fractions were dialyzed overnight against 5 mM phosphate buffer, pH 7.2, and tested for cytotoxicity on mouse B16 melanoma cells. The higher-toxicity fractions were used for the further purification.
  • Toxicity assay For L1210 and P388 cells, test fractions were dissolved in growth medium (RPMI) supplemented with 10% fetal calf serum and 20 mM Hepes buffer) at twice the desired final concentration. Ordinarily, compounds were tested over a 3-4 log dilution range. Cells were adjusted to 1x10 5 cells/ml in growth medium and dispensed into 13x100 mm test tubes (1 ml/tube). One ml of test compound was added to each tube with duplicate tubes run at each dose level. Cells were incubated at 37°C for 48 h. Growth was determined by cell count using a Coulter counter. At each dose level, growth was expressed as a percentage of growth in the untreated control.
  • RPMI growth medium
  • fetal calf serum 20 mM Hepes buffer
  • cells were adjusted to 2.5X10 4 cells/ml in growth medium (Eagle's MEM supplemented with 10% FCS and 20 mm Hepes buffer) and plated in 24 well tissue culture plates (1 ml/well). Plates were incubated for 24 h at 37°C in a 5% carbon dioxide atmosphere. Medium was removed and replaced with 1 ml/well of test compound dissolved in fresh growth medium (duplicate wells at each dose level). Cells were grown 72 h at 37°C in 5% carbon dioxide. The medium was then removed and the plate was washed twice with normal saline (1 ml/well). Growth was assessed by a Lowry protein determination, and growth at each dose level was expressed as a percentage of growth in the untreated controls.
  • growth medium Eagle's MEM supplemented with 10% FCS and 20 mm Hepes buffer
  • Hybridoma cells (OKTl, ATCC CRL 8000) that produced a monoclonal antibody specific for the CD5 antigen on human lymphocytes (anti-CD5) were purchased from the American Type Culture Collection (Rochville, MD). The cells were injected into pristane-primed mice to propagate monoclonal antibody production. Six to ten days later, ascites fluid was collected from the peritoneal cavity and the antibodies were purified on a protein A-Sepharose column (Pharmacia, Uppsala, Sweden) followed by adsorption chromatography on hydroxyapatite (Bio-Rad, Hercules, CA). The purity of the monoclonal antibody was determined by the standard techniques of SDS-polyacrylamide gel electrophoresis and isoelectric focusing. EXAMPLE 7
  • Immunotoxin preparation The PT immunotoxin was prepared by linking PT to anti-CD5 through an artificial disulfide bond.
  • 2-Iminothiolane-HCl (Pierce, Rockford, IL) was used to provide PT with sulfhydryl groups and N-Succinimidyl 3-(2-pyridyl- dithio) propionate (SPDP) (Pierce, Rockford, IL) served to provide the anti-CD5 monoclonal antibody with pyridyl disulfide residues.
  • PT at 10 -3 M was incubated at 37°C for 45 min with 6 x 10 -3 M of 2-Iminothiolane-HCl in 0.02 M Na 2 HPO 4 (pH 7.0), 1 mM EDTA.
  • Anti-CD5 at 10 -4 M was incubated at 37°C for 1 hr with 5 X 10 -4 M of SPDP in 0.02 M Na 2 HPO 4 (pH 8.0), 1 mM EDTA.
  • the derivatized proteins were separated from the reactants by dialysis against 0.02 M Na 2 HPO 4 (pH 8.0), 1 mM EDTA.
  • Derivatized PT and anti-CD5 were incubated together overnight at room temperature in 0.02 M Na 2 HPO 4 (pH 8.0), 1 mM EDTA. Unreacted PT was removed by dialysis (14,000 MW cutoff) against water. The reaction mixture was then concentrated with a Centricon 30 microconcen- trator (30,000 MW cutoff; Amicon, Danvers, MA). Aggregated material was removed by centrifugation. Unreacted anti-CD5 was eliminated by cation exchange HPLC with a SCX 83-C13-ETI Hydro- pore column (Rainin Instrument Company, Inc., Woburn, MA) equilibrated with 0.02 M Na 2 HPO 4 (pH 6.9), 1 mM EDTA.
  • Samples for SDS-PAGE were dissolved in buffer (20 mM Tris-HCl, 2 mM EDTA, 5% SDS, 0.01% Bromophenol Blue) and applied to an 8-25% acrylamide gradient gel. IEF was done in isogel agarose gels with a pH gradient from 3.0 to 10.0.
  • Cytotoxicity - Trypan blue exclusion Cytotoxicity was determined by trypan blue uptake of PT immunotoxin treated cells (Figure 9). Lymphocytes from human blood and from spleens of adult BALB/c mice were separated by density gradient centrifugation on LSM solution (Bionetics, Kensington, MD). Cells (1 X 10 6 ) in hepes buffered saline solution (HBSS) containing 2% (by volume) heat-inactivated human serum (56°C for 30 min) were incubated for different time periods at 37°C with a specified concentration of fraction a, b, or c of Figure 4B. Anti-CD5 was used as a control. Each data point reported is the mean of three experiments, each performed in triplicate. The standard deviation was always within ⁇ 6% of means. The lytic activity of the fraction was also tested on human and sheep red blood cells. Hemolysis was determined by OD at 541 nm.
  • Cytotoxicity - 3 [H] Thymidine uptake The influence of the PT immunotoxin on cell proliferation was determined by mitogen stimulation or by the mixed lymphocyte reaction ( Figure 10) following methods described previously (48). Briefly, lymphocytes suspended in RPMI 1640 supplemented with 2% (by volume) heat-inactivated human serum, 2 mM L-glutamine and 10 ⁇ g/ml gentamicine were seeded in 96-well plates (10 5 cells/well).
  • Pokeweed Mitogen PWM; Sigma, St. Louis, MO. The cells were incubated for 48 hr in a 5% CO 2 humidified atmosphere at 37°C, then pulsed with 3 [H] thymidine (1 ⁇ Ci/well). The cells were harvested 5 hr later.
  • 10 5 responder cells were mixed in each well with 10 5 stimulator cells.
  • Stimulator cells had been treated with mitomycin C (25 ⁇ g/10 7 cells) for 20 min at 37°C followed by washing three times in RPMI 1640. The cells were incubated for 120 hr and then pulsed with 3 [H] thymidine (1 ⁇ Ci/well) 5 hr prior to harvesting.
  • test wells were incubated with specified concentrations of PT immunotoxin or anti-CD5, and control wells without PT immunotoxin or anti-CD5.
  • the incorporated radioactivity was measured by liquid scintillation counting. Data are expressed as means of two separate experiments, each performed in triplicate. The standard deviation was always within ⁇ 5% of means.
  • Cytotoxicity - 51 Cr release The cytotoxic activity of the PT immunotoxin with and without added guinea pig complement was examined by a 51 Cr release assay (Figure 11). Cells suspended in Dulbecco's Minimal Essential Media (MEM) were labled with 5 1 Cr (100 ⁇ Ci 51 Cr per 10 7 cells). Radioactivity released from the lysed cells was detected by liquid scintillation spectroscopy. Cytolytic activity was calculated using the formula:
  • Immunotoxin specificity Specificity of the PT immunotoxin was determined by immunofluorescent staining of human blood lymphocytes. Cells (10 7 ) suspended in HBSS containing 2% (by volume) heat inactivated human serum were incubated with 10 -7 M of PT immunotoxin for 2 hr at 37°C followed by incubation with 10 -7 M of anti-CD5 for an additional 2.5 hr. In control samples, 10 7 cells were incubated for 2 hr without PT immunotoxin, then incubated for an additional 2.5 hr with 10 -7 M of anti-CD5.
  • Viable cells were separated on LSM solution and then incubated with either anti-mouse.
  • IgG (Fc specific)-FITC to label the T cells that bound anti-CD5, or anti-human IgM ( ⁇ -chain specific)-FITC to label the B cells.
  • the results obtained were expressed as the percent of labeled cells to the total number of cells in the samples. Data were obtained from four separate experiments, each performed in triplicate. The standard deviation was always within ⁇ 7% of means.
  • Immunotoxin membrane interaction - electron paramagnetic resonance The ability of the PT immunotoxin to interact with lipid membranes was analyzed by EPR of spin probes (Figure 7).
  • Egg yolk L- ⁇ -phosphatidylcholine (PC), bovine brain L- ⁇ -phos- phatidylserine (PS), and dimyristoylphosphatidylserine (DMPS) were used to prepare lipid membranes.
  • Membranes prepared for EPR studies were composed of PC or PC + 30 mol% PS. Lipids were hydrated in 10 mM Tris-HCl buffer (pH 7.5) containing 0.1 M NaCl and 1 mM EDTA. 5-doxylstearic acid was used as a spin probe.
  • Oriented multibilayers were prepared by squeezing 50 ⁇ l of hydrated lipid (5 mg/ml) between two glass plates as previously described. Aripov et al. (1986) Akad Nauk SSSR 288:728. The lipid to probe molar ratio was 100/1. The EPR spectra of spin probes were recorded with a Varian E-4 spectrometer at modulation amplitudes not exceeding 2 x 10 -4 T and a resonator input power not exceeding 20 mW.
  • Immunotoxin membrane interaction - calorimetry The ability of the IT to interact with lipid membranes was analyzed by differential scanning microcalorimetry (Figure 8). Multi- lamellar lipid dispersions for calorimetric studies were prepared from dimyristoylphosphatidylcholine (DMPC) or DMPC + 5 mol% phosphatidylserine (PS). The low proportion of PS used was due to the fact that PS (as any lipid with unsaturated hydrocarbon chains) appreciable disturbs the ordered packing of synthetic saturated lipids, thus notably reducing the degree of transition cooperativity. Lipids were dissolved in chloroform/ethanol/methanol (6/1/3 by volume) solvent.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Toxicology (AREA)
  • Botany (AREA)
  • Genetics & Genomics (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Medical Informatics (AREA)
  • Biotechnology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention se rapporte à une immunotoxine comportant un anticorps monoclonal CD5-antihumain de souris conjugué à la toxine thionine Pyrularia (thionine P.). La thionine P. est un petit peptide très stable dont la membrane est active et qui présente une toxicité pour certaines cellules eucaryotes et pour toutes les cellules procaryotes testées jusqu'à présent. L'immunotoxine de thionine P. présente un taux élevé de cytotoxicité et de spécificité. L'immunotoxine de thionine P. s'est révélée cytotoxique pour des cellules T qui expriment l'antigène CD5, mais elle n'a présenté aucune réactivité vis-à-vis des cellules B humaines, des erythrocytes humains ou du mouton ou encore des lymphocytes de la souris qui n'expriment pas l'antigène CD5 humain.
PCT/US1996/008811 1995-06-07 1996-06-05 Thionine pyrularia contenant des immunotoxines ainsi que des conjugues similaires aux immunotoxines WO1996041608A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU59817/96A AU5981796A (en) 1995-06-07 1996-06-05 Pyrularia thionin containing immunotoxins and immunotoxin-li ke conjugates

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US47979995A 1995-06-07 1995-06-07
US08/479,799 1995-06-07

Publications (2)

Publication Number Publication Date
WO1996041608A2 true WO1996041608A2 (fr) 1996-12-27
WO1996041608A3 WO1996041608A3 (fr) 1997-02-06

Family

ID=23905495

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/008811 WO1996041608A2 (fr) 1995-06-07 1996-06-05 Thionine pyrularia contenant des immunotoxines ainsi que des conjugues similaires aux immunotoxines

Country Status (2)

Country Link
AU (1) AU5981796A (fr)
WO (1) WO1996041608A2 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1322323A1 (fr) * 2000-09-11 2003-07-02 Therapro Technologies, Inc. Thionine en tant qu'agent antineoplastique et immunostimulant
AU781677B2 (en) * 1999-11-12 2005-06-02 Syntaxin Limited Use of lytic toxins and toxin conjugates
WO2020023561A1 (fr) * 2018-07-23 2020-01-30 Magenta Therapeutics, Inc. Utilisation d'un conjugué anticorps anti-cd5 -médicament (adc) dans une thérapie cellulaire allogénique
WO2020216947A1 (fr) 2019-04-24 2020-10-29 Heidelberg Pharma Research Gmbh Conjugués anticorps-médicaments d'amatoxine et leurs utilisations

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ARCH. BIOCHEM. BIOPHYS., April 1985, Vol. 238, No. 1, VERNON et al., "A Toxic Thionin from Pyrularia Pubera: Purification, Properties and Amino Acid Sequence", pages 18-29. *
BIOCHIM. BIOPHYS. ACTA, 1994, Vol. 1198, BRINKMANN et al., "Immunotoxins Against Cancers", pages 27-45. *
BLOOD, 01 April 1990, Vol. 75, No. 7, BYERS et al., "Use of an Anti-Pan T-Lymphocyte Ricin A Chain Immunotoxin in Steroid-Resistant Acute Graft-Versus-Host Disease", pages 1426-1432. *
CANCER IMMUNOL. IMMUNOTHER., 1983, Vol. 15, IMAI et al., "Selective in Vitro Toxicity of Purothionin Conjugate to the Monoclonal Antibody 225.28S to a Human High-Molecular-Weight Melanoma-Associated Antigen", pages 206-209. *
CANCER IMMUNOL. IMMUNOTHER., 1993, Vol. 36, PORRO et al., "In Vitro and in Vivo Properties of an Anti-CD5-Momordin Immunotoxin on Normal and Neoplastic T Lymphocytes", pages 346-350. *
J. TOXICOL., 1992, Vol. 11, No. 3, VERNON L.P., "Pyrularia Thionin: Physical Properties, Biological Response and Comparison to Other Thionins and Cardiotoxin", pages 169-191. *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU781677B2 (en) * 1999-11-12 2005-06-02 Syntaxin Limited Use of lytic toxins and toxin conjugates
AU781677C (en) * 1999-11-12 2006-09-07 Syntaxin Limited Use of lytic toxins and toxin conjugates
US7422740B1 (en) 1999-11-12 2008-09-09 Health Protection Agency Use of lytic toxins and toxin conjugates
EP1322323A1 (fr) * 2000-09-11 2003-07-02 Therapro Technologies, Inc. Thionine en tant qu'agent antineoplastique et immunostimulant
EP1322323A4 (fr) * 2000-09-11 2004-07-28 Therapro Technologies Inc Thionine en tant qu'agent antineoplastique et immunostimulant
US7572436B1 (en) 2000-09-11 2009-08-11 Therapro Technologies, Inc. Thionin as an antineoplastic and immunostimulant
WO2020023561A1 (fr) * 2018-07-23 2020-01-30 Magenta Therapeutics, Inc. Utilisation d'un conjugué anticorps anti-cd5 -médicament (adc) dans une thérapie cellulaire allogénique
WO2020216947A1 (fr) 2019-04-24 2020-10-29 Heidelberg Pharma Research Gmbh Conjugués anticorps-médicaments d'amatoxine et leurs utilisations

Also Published As

Publication number Publication date
WO1996041608A3 (fr) 1997-02-06
AU5981796A (en) 1997-01-09

Similar Documents

Publication Publication Date Title
EP0710723B1 (fr) Clonage et expression d'un gène codant pour bryodine-1 de bryonia dioica
US5744580A (en) Immunotoxins comprising ribosome-inactivating proteins
US5376546A (en) Analogs of ribosome-inactivating proteins
Theuer et al. Immunotoxins made with a recombinant form of Pseudomonas exotoxin A that do not require proteolysis for activity
Ozawa et al. Selective killing of squamous carcinoma cells by an immunotoxin that recognizes the EGF receptor
AU684571B2 (en) A new ribosome-inactivating protein isolated from the plant (bryonia dioica)
Ghetie et al. Chemical construction of immunotoxins
US4985541A (en) Novel cytotoxic protein
JP3340127B2 (ja) 異常増殖性疾患措置のための抗体接合体
JP3949158B2 (ja) Ｃｄ３３関連表面抗原に対する免疫毒素
Choe et al. B3 (Fab)-PE38M: a recombinant immunotoxin in which a mutant form of Pseudomonas exotoxin is fused to the Fab fragment of monoclonal antibody B3
Marsh et al. A flexible peptide spacer increases the efficacy of holoricin anti-T cell immunotoxins.
Bjorn et al. Characterization of translational inhibitors from Phytolacca americana Amino-terminal sequence determination and antibody-inhibitor conjugates
WO1991007437A3 (fr) Anticorps cd-30 ameliores et fragments de ces derniers
Porro et al. In vitro and in vivo properties of an anti-CD5—momordin immunotoxin on normal and neoplastic T lymphocytes
Siegall et al. Characterization of ribosome-inactivating proteins isolated from Bryonia dioica and their utility as carcinoma-reactive immunoconjugates
WO1996041608A2 (fr) Thionine pyrularia contenant des immunotoxines ainsi que des conjugues similaires aux immunotoxines
MATSUI et al. Suppression of human melanoma growth in nude mice injected with anti high-molecular-weight melanoma-associated antigen monoclonal antibody 225.28 S conjugated to purothionin
Batra et al. Insertion of constant region domains of human IgG, into CD4-PE40 increases its plasma half-life
Gasanov et al. In vitro evaluation of Pyrularia thionin—anti-CD5 immunotoxin
Bolognesi et al. Anti-CD30 immunotoxins with native and recombinant dianthin 30
Tsai et al. Growth suppression of human colorectal carcinoma in nude mice by monoclonal antibody C27-abrin A chain conjugate
Debinski et al. Recombinant F (ab') C242-Pseudomonas exotoxin, but not the whole antibody-based immunotoxin, causes regression of a human colorectal tumor xenograft.
US5149528A (en) Cytotoxic protein from Trichosanthes kirilowii
Tazzari et al. An immunotoxin containing a rat IgM monoclonal antibody (Campath 1) and saporin 6: effect on T lymphocytes and hemopoietic cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN

AK Designated states

Kind code of ref document: A3

Designated state(s): AL AM AT AU AZ BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU IL IS JP KE KG KP KR KZ LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK TJ TM TR TT UA UG UZ VN AM AZ BY KG KZ MD RU TJ TM

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): KE LS MW SD SZ UG AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: CA