EP0781139A1 - Intracellular delivery of chemical agents to a specific cell type - Google Patents

Intracellular delivery of chemical agents to a specific cell type

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Publication number
EP0781139A1
EP0781139A1 EP95932472A EP95932472A EP0781139A1 EP 0781139 A1 EP0781139 A1 EP 0781139A1 EP 95932472 A EP95932472 A EP 95932472A EP 95932472 A EP95932472 A EP 95932472A EP 0781139 A1 EP0781139 A1 EP 0781139A1
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EP
European Patent Office
Prior art keywords
chemical agent
seq
receptor
composition
ligand
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP95932472A
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German (de)
English (en)
French (fr)
Inventor
Ramesh K. Prakash
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Actavis Laboratories UT Inc
Original Assignee
Theratech Inc
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Filing date
Publication date
Application filed by Theratech Inc filed Critical Theratech Inc
Publication of EP0781139A1 publication Critical patent/EP0781139A1/en
Withdrawn legal-status Critical Current

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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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
    • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to delivery of chemical agents to cells. More particularly, this invention relates to compositions and methods for intracellular delivery of chemical agents to a specific cell type, i.e. cells expressing the CR2 receptor.
  • Toxins that target cell surface receptors or antigens on tumor cells have attracted considerable attention for treatment of cancer.
  • I. Pastan & D. FitzGerald Recombinant Toxins for Cancer Treatment, 254 Science 1173 (1991); U.S. Patent Nos. 5,169,933 and 5,135,736 to Anderson et al. ; U.S. Patent No. 5,165,923 to Thorpe et al. ; U.S. Patent No. 4,906,469 to Jansen et al.; U.S. Patent No. 4,962,188 to Frankel; U.S. Patent No. 4,792,447 to Uhr et al. ; U.S. Patent Nos.
  • the membrane glycoprotein CR2 also known as CD21, occurs on mature B lymphocytes (B cells) and certain epithelial cells, such as human pharyngeal epithelial cells, human follicular dendritic cells, and cervical epithelium, and is a receptor for both Epstein-Barr Virus (EBV) and complement fragments C3d/C3dg. N. Miller & L.M.
  • Hutt-Fletcher, 66 J. Virol. 3409 (1990) This receptor is a 145 kD membrane glycoprotein that, in addition to its binding function, is also involved in a pathway of B cell activation.
  • G.R. Nemerow, et al . Identification and characterization of the Epstein- Barr virus receptor on human B lymphocytes and its relationship to the C3d complement receptor (CR2) , 55 J. Virol 347 (1985) .
  • Infection of B cells by EBV is initiated by selective binding of the gp350/220 envelope glycoprotein of the virus to the CR2 receptor, followed by internalization of the CR2 receptor and endocytosis of the receptor-bound virions.
  • compositions for intracellular delivery of chemical agents to CR2 receptor-bearing B cells and methods of use thereof would be significant advancements in the art.
  • compositions for intracellular delivery of selected chemical agents to a specific cell type, i.e. cells expressing the CR2 receptor, to which binding triggers receptor-mediated endocytosis. It is also an object of the invention to provide methods of making and methods of using compositions for intracellular delivery of selected chemical agents to cells expressing the CR2 receptor.
  • selected chemical agents such as cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like
  • compositions for specific intracellular delivery of a chemical agent into a CR2 receptor-bearing cell in a population of cells including non-CR2- receptor-bearing cells comprising a ligand (CBEL) capable of binding to the CR2 receptor and inducing receptor-mediated endocytosis and a chemical agent coupled to the ligand, wherein the chemical agent is capable of eliciting a selected effect when delivered intracellularly into the CR2 receptor-bearing cell .
  • Mature B lymphocytes are CR2 receptor-bearing cells targeted by these compositions.
  • Chemical agents that can be delivered to such cells include cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs.
  • the CBEL and chemical agent can be bound to each other and/or to other functional moieties through a spacer, which can either be biodegradable, such as certain peptides, or non-biodegradable.
  • the composition can further comprise a carrier-type system selected from the group consisting of water soluble polymers, liposomes, and particulates.
  • compositions are used in vi tro by contacting populations of cells with an effective amount of composition under conditions wherein the CR2 receptor binding and endocytosis-inducing ligand (CBEL) binds the CR2 receptor and elicits endocytosis of the receptor- bound composition.
  • CBEL CR2 receptor binding and endocytosis-inducing ligand
  • an effective amount of the composition is systemically administered so that the CBEL contacts and binds to CR2 receptors on mature B lymphocytes and then induces endocytosis of the composition. Once inside the cells, the chemical agent elicits its selected effect.
  • FIGS. IA and IB illustratively depict chemical conjugation of a CBEL with a chemical agent having a free sulfhydryl group to form a composition according to the present invention.
  • FIGS. 2A-2D show steps in constructing a plasmid for expressing a fusion protein containing a CBEL and a chemical agent peptide according to the present invention.
  • FIG. 3 shows a comparison of the effects on CR2 * B cells (darker bars) and CR2 " T cells (lighter bars) cells of exposing the cells in vitro to various concentrations of an CBEL-ricin A fusion protein according to the present invention.
  • peptide means peptides of any length and includes proteins.
  • polypeptide and oligopeptide are used herein without any particular intended size limitation, unless a particular size is otherwise stated.
  • CR2 receptor binding and endocytosis-inducing ligand means a composition capable of binding to the CR2 (CD21) receptor
  • CBELs are coupled to various functional molecules so that upon endocytosis of the CBELs, the various functional molecules coupled thereto are also internalized by the CR2-bearing cells.
  • a CBEL can be derived from the EBV gp350/220 glycoprotein, including SEQ ID N0:1 and flanking sequences; the C3dg peptides, including SEQ ID NO:2 and flanking sequences; or peptides substantially homologous thereto.
  • substantially homologous means peptides that retain functionality in binding CR2 receptors and inducing receptor-mediated endocytosis although they may include flanking sequences or be truncations, deletion variants, or substitution variants of SEQ ID N0:1 or SEQ ID NO:2.
  • the minimum requirement for binding and inducing receptor mediated endocytosis appears to be the sequence identified as SEQ ID N0:3.
  • Substitution variants are those that contain a conservative substitution of one or more amino acid residues.
  • a conservative substitution is a substitution of one amino acid residue for another wherein functionality of the peptide is retained, in this case, functionality in binding the CR2 receptor and eliciting endocytosis of the receptor-bound composition.
  • Amino acid residues belonging to certain conservative substitution groups can sometimes substitute for another amino acid residue in the same group.
  • One such grouping is as follows: Pro; Ala, Gly; Ser, Thr; Asn, Gin; Asp, Glu; His; Lys, Arg; Cys; lie, Leu, Met, Val; and Phe, Trp, Tyr. M. Jimenez-Montano S. L.
  • Zamora-Cortina Evolutionary model for the generation of amino acid sequences and its application to the study of mammal alpha-hemoglobin chains, Proc. Vllth Int' 1 Biophysics Congress, Mexico City (1981) .
  • Other variations that are to be considered substantially homologous include substitution of D-amino acids for the naturally occurring L-amino acids, substitution of amino acid derivatives such as those containing additional side chains, and substitution of non-standard amino acids, i.e. ⁇ -amino acids that are rare or do not occur in proteins.
  • the primary structure of a CBEL is limited only by functionality.
  • chemical agent means and includes any substance that has a selected effect when internalized into a B lymphocyte by endocytosis. Certain chemical agents have a physiological effect, such as a cytotoxic effect or an effect on gene regulation, on a B cell when internalized into the cell.
  • a "transforming nucleic acid” (RNA or DNA)
  • RNA or DNA when internalized into a cell, may be replicated and/or expressed within the cell.
  • Other nucleic acids may interact with regulatory sequences or regulatory factors within the cell to influence gene expression within the cell in a selected manner.
  • a detectable label delivered intracellularly can permit identification of cells that have internalized the compositions of the present invention by detection of the label.
  • Antigens that are delivered to the interior of a cell can elicit an immune response specific to the antigen.
  • Drugs or pharmacologically active compounds can be used to ameliorate pathogenic effects or other types of disorders.
  • Particularly useful chemical agents include polypeptides, and some such chemical agents are active fragments of biologically active proteins, or are specific antigenic fragments (e.g., epitopes) of antigenic proteins.
  • chemical agents include cytotoxins, gene regulators, transforming nucleic acids, labels, antigens, drugs, and the like.
  • carrier means water soluble polymers, particulates, or liposomes. Such carriers may contain multiple sites to which one or more CBEL and/or chemical agent can be coupled. Such carriers increase the molecular size of the compositions and may provide 8 added selectivity and/or stability. Such selectivity arises because carrier-containing compositions are too large to enter cells by passive diffusion, and thus are limited to entering cells through receptor-mediated endocytosis. Carriers comprising water soluble polymers can be used for linking of peptide ligands, chemical agents, and other functional molecules. The potential for use of such carriers for targeted drug delivery has been established. See, e.g., J. Kopecek, 5 Biomaterials 19 (1984) ; E. Schacht et al.
  • illustrative water soluble polymers include dextran, inulin, poly(L-lysine) , methacrylamide- containing synthetic polymers, and the like.
  • drug or “pharmacologically active agent” means any chemical material or compound suitable for intracellular administration in a CR2-bearing cell B which induces a desired biological or pharmacological effect in such cell.
  • an effective amount is an amount that produces a selected effect.
  • a selected effect of a composition containing a cytotoxin as the chemical agent could be to kill a selected proportion of mature B cells within a selected time period.
  • An effective amount of the composition would be the amount that achieves this selected result, and such an amount could be determined as a matter of routine by a person skilled in the art.
  • compositions of the present invention provide specific intracellular delivery of a chemical agent into a CR2 receptor-bearing cell in a population of cells including non-CR2-receptor-bearing cells, the compositions comprising a CBEL capable of binding to the CR2 receptor and inducing receptor-mediated endocytosis and a chemical agent coupled to the CBEL, wherein the chemical agent is capable of eliciting a selected effect when delivered intracellularly into the CR2 receptor- bearing cell .
  • Mature B cells are especially targeted by these compositions.
  • the chemical agents are selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like.
  • the coupling of a CBEL to a chemical agent can be, without limitation, by covalent bond, electrostatic interaction, hydrophobic interaction, physical encapsulation, and the like.
  • the coupling of a CBEL to a chemical agent can also be direct or through another functional moiety.
  • the compositions can further comprise a spacer coupled to and interposed between the CBEL and the chemical agent .
  • spacers can be biodegradable or non-biodegradable, and peptide spacers are preferred.
  • the compositions of the present invention can further comprise a carrier selected from the group consisting of water soluble polymers, liposomes, and particulates.
  • the composition has the formula: [L-S b -C-[S.-A] f wherein L is the ligand (CBEL) capable of binding to the CR2 receptor and inducing endocytosis thereof; A is the chemical agent; S is a spacer; C is the water soluble polymer having functional groups compatible with forming covalent bonds with ligand, chemical agent, and spacer; a and e are 0 or 1; and b and f are integers of at least 1.
  • Such water soluble polymers are selected from the group consisting of dextran, inulin, poly(L-lysine) , methacrylamide-containing polymers, and the like.
  • the CBEL provides means for the composition to bind the CR2 receptor on mature B cells, thus triggering internalization of the compositions by endocytosis.
  • the chemical agent provides means for achieving a selected effect in the B cells.
  • chemical agents comprise cytotoxins, including radionuclides, for selective killing or disabling of cells displaying the CR2 receptor; antigens for eliciting a selected immune response; nucleic acids for genetically transforming or regulating gene expression in B cells; drugs or other pharmacologically active agents for achieving a selected therapeutic effect; labels, including fluorescent, radioactive, and magnetic labels, for permitting detection of cells that have taken up the compositions; and the like.
  • compositions of the present invention further comprise a water soluble polymeric carrier so that a plurality of CBELs and/or chemical agents with selected functionalities can be bound together in a complex molecule.
  • At least 1 chemical agent and at least 1 CBEL are bound to such carriers, with the preferred number of CBELs coupled to chemical agents in the range of 1 to about 1000.
  • Such carriers increase the molecular size of the compositions and may provide added selectivity and/or stability to the functional moieties beyond what would be achieved without the carrier.
  • CBELs and/or chemical agents is accomplished by means of biodegradable or non-biodegradable spacers .
  • spacers can be selected for their relative susceptibility or resistance to hydrolysis and/or enzymatic cleavage inside B cells. This selectivity provides a practitioner of this art the ability to choose a spacer based on whether it would be most advantageous to have the chemical agent remain coupled to the carrier within the cells or to be released from the carrier by intracellular enzymatic activity.
  • Use of liposomes and particulates as carriers is independent of use of a water soluble polymer carrier.
  • the compositions are constructed by chemically conjugating a CBEL to a chemical agent.
  • "Chemically conjugating" the CBEL to the chemical agent means covalently bonding the CBEL to the chemical agent, either directly or by way of a coupling moiety.
  • a coupling moiety can be used to form a linkage between functional groups on the CBEL and the chemical agent.
  • compositions containing a CBEL and a chemical agent peptide can be formed by coupling a sulfhydryl group on the chemical agent and an amino group on the CBEL through a heterobifunctional crosslinker.
  • FIGS. IA and IB A reaction scheme for chemically conjugating a CBEL and ricin A through a maleimide crosslinker is shown in FIGS. IA and IB, for example.
  • maleimide crosslinkers for chemically conjugating CBELs to chemical agents containing sulfhydryl groups haloacetyl, alkyl halide, alkyl sulfonate, ,6-unsaturated carbonyl, or a,6-unsaturated sulfone moieties can be used as crosslinkers.
  • active esters can be used as crosslinkers.
  • Other coupling moieties may be employed depending upon which functional group(s) on the CBELs and on the chemical agent are available.
  • compositions of the present invention can also be produced in a genetically engineered organism, such as E. coli , as a "fusion protein.” That is, a hybrid gene containing a sequence of nucleotides encoding a CBEL and a sequence of nucleotides encoding a chemical agent peptide can be constructed by recombinant DNA technology. This hybrid gene can be inserted into an organism such that the "fusion protein" encoded by the hybrid gene is expressed. The fusion protein can then be purified by standard methods, including affinity chromatography.
  • Fusion proteins containing a CBEL and a chemical agent peptide according to the present invention can also be constructed by chemical synthesis. Where the compositions of the present invention are produced as fusion proteins, the CBEL and the chemical agent peptide can be immediately adjacent to one another, that is, the carboxyl end of the CBEL can be bonded directly to the amino end of the chemical agent, or vice versa . Alternatively, the fusion protein can also include additional amino acid residues between the CBEL and the chemical agent, such that these additional amino acid residues serve as a spacer between the CBEL and the chemical agent peptide.
  • Short peptide ligands are generally preferred, both because short peptides can be manipulated more readily and because the presence of additional amino acids residues, and particularly of substantial numbers of additional amino acids residues, may interfere with the function of the peptide ligand in inducing internalization of the chemical agent by endocytosis.
  • compositions according to the present invention can also further include a protease digestion site situated so that once the composition is within the cell, the chemical agent can be separated from the CBEL by proteolysis of the digestion site.
  • a protease digestion site situated so that once the composition is within the cell, the chemical agent can be separated from the CBEL by proteolysis of the digestion site.
  • Such digestion sites occur naturally in the gp350/220 glycoprotein adjacent to the SEQ ID NO:1 segment, thus the CBEL portion of the composition can conveniently extend to include such sites.
  • a protease susceptible spacer can be added regardless of whether the composition is synthesized chemically or as an expression peptide in a genetically engineered organism. In the latter case, nucleotides encoding the protease susceptible spacer can be inserted into the hybrid gene between the CBEL-encoding segment and the chemical agent peptide-encoding segment by techniques well known in the art.
  • Another aspect of the present invention features a method for specifically effecting a desired activity in CR2-expressing cells contained in a population of non- CR2-expressing cells, by steps of contacting the population of cells with a composition containing a CBEL coupled to a chemical agent that directs such activity intracellularly.
  • the compositions of the invention are selectively bound to CR2-expressing cells in the mixed population, whereupon endocytosis of the composition into the cells is induced, and the chemical agent effects its activity within the CR2-expressing cells.
  • the invention features peptides, employed as CBELs in compositions also containing chemical agents, which chemical agents may also be peptides or contain peptides.
  • the peptides according to the invention may be made by any of a variety of techniques, including organic synthesis and recombinant DNA methods. Techniques for chemical synthesis of peptides are described, for example, in B. Merrifield et al., 21 Biochemistry 5020-31 (1982) ; Houghten, 82 Proc. Nat'l Acad. Sci. USA 5131-35 (1985), incorporated herein by reference. Techniques for chemical conjugation of peptides with other molecules are known in the art .
  • a fusion protein according to the invention can be made by expression in a suitable host cell of a nucleic acid containing an oligonucleotide encoding a CBEL, as described above, and an oligonucleotide encoding a chemical agent peptide.
  • Such techniques for producing recombinant fusion proteins are well-known in the art, and are described generally in, e.g., J. Sambrook et al . , Molecular Cloning: A Laboratory Manual (2d ed. , 1989) , the pertinent parts of which are hereby incorporated herein by reference. Reagents useful in applying such techniques, such as restriction enzymes and the like, are widely known in the art and commercially available from any of several vendors.
  • compositions containing a CBEL and chemical agent peptide according to the invention will now be described, with particular reference to examples in which a CBEL is conjugated with the cytotoxic chemical agent peptide, ricin A.
  • Ricin is a toxic glycoprotein produced by the castor plant (Ricinus comnunis) . It is composed of two subunits, the A chain and the B chain, both about 30 kD in molecular weight, linked together by a disulfide bond. Ricin is synthesized as a large precursor that is processed to yield mature ricin A chain and B chain subunits.
  • the A chain is an enzyme that cleaves a glycosidic bond in 28S ribosomal RNA, thereby destroying the ability of ribosomes to synthesize protein.
  • the A chain can inactivate about 1500 ribosomes per minute, which means that a single internalized molecule of ricin A is lethal to a cell.
  • S. Olsnes et al. Ribosome inactivation by the toxic lectins abrin and ricin, 60 Eur. J. Biochem. 281 (1975) .
  • the B chain binds to galactose moieties on the surface of a cell, an event necessary for internalization of ricin.
  • the composition is formed by chemical conjugation; and in a second example, the composition is formed as a recombinant fusion protein.
  • sulfo-MBS Pierce, Rockford, Illinois, USA
  • CBEL CBEL
  • sulfo-MBS Pierce, Rockford, Illinois, USA
  • PBS buffer 20 mM sodium phosphate, 0.15 M NaCl, pH 7.0
  • the resulting activated peptide was purified by FPLC using Superose 12 (Pharmacia) according to standard methods.
  • the maleimide-activated peptide was reacted with deglycosylated ricin A (Sigma Chemical Co., St. Louis, Missouri, USA) by mixing in a molar ratio of 1:5 in PBS buffer for 1 hour at room temperature. Unreacted peptide molecules were removed from the CBEL-Ricin A conjugate by dialysis overnight at 4°C through a membrane having a 6-8 kilodalton cutoff value. The resulting composition was estimated to have a molecular weight of about 32 kD by gel electrophoresis on a 10% SDS-PAGE gel, U. Laemmli, 227 Nature 680 (1970) .
  • composition prepared according to this example contained one CBEL per molecule of ricin A.
  • Three-dimensional computer modeling shows that the CBEL is most likely coupled to the Cys residue at the C- terminal end of the ricin A molecule, inasmuch as the N- terminal Cys residue is buried internally in the folded ricin A molecule.
  • Example 2 Recombinant protein containing a CBEL and Ricin A
  • a fusion protein containing a CBEL having the amino acid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID N0:1) and the cytotoxic chemical agent peptide, ricin A was formed by means of recombinant DNA technology.
  • the fusion protein in this example was made by inserting a synthetic oligonucleotide that encodes the CBEL downstream from a polynucleotide encoding ricin A in an E. coli expression vector, expressing the fusion protein at a high yield in an E. coli host, and then purifying the fusion protein from a cell lysate by affinity chromatography.
  • a fusion protein containing the CBEL and ricin A was produced by recombinant DNA technology as follows.
  • the E. coli expression vector pTrcHis B (Invitrogen, San Diego, California) (FIG. 2A) , containing a multiple cloning site (MCS) downstream of a translation initiation site (ATG) , was digested with restriction endonucleases Ncol and BamHI, and the resulting cohesive ends were converted to blunt ends with T4 DNA polymerase and religated to generate pTrc B (FIG. 2B) .
  • a 863 bp BamHI- Kpnl fragment encoding ricin A was isolated from plasmid pAKG (obtained from Robert Weaver, University of Kansas, Lawrence; described in R. C. Hailing et al . , 13 Nucleic Acids Res. 8019 (1985)) , and cloned into BamHI and Kpnl- digested pTrc B. The resulting construct was digested with BamHI, the cohesive ends converted to blunt ends with T4 DNA polymerase, and religated to yield the correct reading frame for translation of the cloned gene (FIG. 2C) .
  • the resulting modified pTrc B vector containing a ricin A-encoding sequence, was then digested with Kpnl and EcoRI, and the following synthetic oligonucleotide, containing nucleotide residues encoding the CBEL and cohesive ends compatible with cloning at Kpnl and EcoRI sites, was ligated thereto.
  • the resulting vector contained a hybrid gene encoding a ricin A-protease site-ligand-His 6 fusion protein, wherein "protease site” signifies a protease digestion site or protease susceptible spacer as described above, "ligand” signifies the CBEL, and "His 6” signifies a region of 6 consecutive His residues, the function of which will be described below.
  • the resultant plasmid was then used to transform E. coli cells, and transformants were selected and grown in LB medium, J. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1972) .
  • the cells were then lysed and the recombinant fusion protein was purified by affinity chromatography on a column containing a nickel-charged resin ("PROBOND", Invitrogen, San Diego, California) .
  • PROBOND nickel-charged resin
  • the six His residues at the C-terminus of the fusion protein bound electrostatically to the nickel atoms on the "PROBOND" resin.
  • the resin containing the bound fusion protein was then washed to remove contaminants. Then, the electrostatic bonds were broken and the fusion protein eluted in an imidizole-containing elution buffer that displaced the His residues from the nickel-charged resin. This purification was done according to the supplier' s manual.
  • Example 3 A fusion protein containing the a CBEL having the amino acid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID N0:1) and the cytotoxic chemical agent peptide, ricin A, was formed by means of recombinant DNA technology as in Example 2 with the exception that the region containing six consecutive His residues was omitted.
  • the following synthetic oligonucleotide containing nucleotide residues encoding the CBEL and cohesive ends compatible with cloning at Kpnl and EcoRI sites, was ligated thereto.
  • the resulting vector contained a hybrid gene encoding a ricin A-protease site-CBEL fusion protein, wherein "protease site” signifies a protease digestion site or protease susceptible spacer as described above.
  • the resultant plasmid was then used to transform E. coli cells, and transformants were selected and grown in LB medium.
  • the expressed protein was isolated by lysing the cells with 1 mg/ml of lysozyme in 20 mM sodium phosphate, pH 7.8, and sonicating three times for 1 minute each. The fusion protein was insoluble under these conditions, but most E. coli proteins were soluble.
  • the lysate was centrifuged at 9000 rpm for 30 minutes, and the resulting fusion protein-containing pellet was resuspended and sonicated for 1 minute before being centrifuged again. The steps of resuspension, sonication, and centrifugation were repeated three times.
  • the final pellet, containing a relatively pure preparation of fusion protein was dissolved in a solution containing 6 M urea and 5 M dithiothreitol . This dissolved fusion protein was renatured by sequentially dialyzing against 4 M urea 2 M urea; and 20 mM sodium phosphate, 500 mM NaCl, pH 7.8.
  • a fusion protein containing a CBEL having the amino acid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID NO:l) and the cytotoxic chemical agent peptide, ricin A was formed by means of recombinant DNA technology as in Example 2 with the exception that an additional cysteine residue was introduced into the fusion protein.
  • This construction gave higher yields of recoverable fusion protein than in Example 2 because the free cysteine residue at the C-terminal end of the ricin A chain could form an intramolecular disulfide bond with the new cysteine residue instead of with E. coli proteins.
  • coli strain XL-1 (Stratagene, La Jolla, California) . Transformants were selected and the orientation of the BamHI fragment in the vector was determined by digestion with Bglll. DNA from a transformant with the ricin A gene in the correct orientation for translation was digested with Sad and EcoRI, and the following synthetic oligonucleotide, containing nucleotide residues encoding the CBEL and cohesive ends compatible with cloning at Sad and EcoRI sites, was ligated thereto.
  • the ligated DNA was transformed into expression host E. coli strain BLR (Novagen, Madison, Wisconsin) , a recA strain also lacking ion and ompT proteases. Recombinant protein was isolated by growing transformed cells in the presence of ampicillin at 37°C.
  • IPTG isopropylthiogalactoside
  • Ammonium sulfate was added to 40% saturation to precipitate soluble proteins, and the precipitated pellet was dissolved and dialyzed against: 10 mM Tris-HCl, pH 7.4, 100 mM KC1.
  • the dialysate was loaded onto a strong anion exchange column (Q Sepharose Fast Flow, Pharmacia) that had been equilibrated with the same buffer. Under these conditions, almost all of the bacterial proteins were bound to the column and the unbound fraction contained essentially purified recombinant protein.
  • the recombinant protein was further purified by dialyzing against 4 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, pH 7.8.
  • the dialysate was passed through a column containing nickel- charged "PROBOND" resin and was washed 5 times with 4 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, 5 mM imidizole, pH 6.0.
  • the 6 histidine residues in the recombinant protein caused the recombinant protein to bind to the resin by affinity interaction.
  • the column was washed twice more with the same buffer except for the imidizole concentration being raised to 30 mM.
  • the recombinant protein was eluted in the same buffer except for having an imidizole concentration of 300 mM. The eluted protein was then renatured and refolded by first dialyzing against 2 M urea and then against 20 mM sodium phosphate, 500 mM NaCl, pH 7.8.
  • Example 5 Targeted delivery of a cytotoxin to B cells
  • compositions of CBEL and chemical agent according to the invention will now be described, with particular reference to an example in which a composition of ricin A and a CBEL is delivered to Raji B lymphoblastoid cells in vi tro for specific cytotoxic effect on the CR2- expressing B cells.
  • Example 6 The effect of the recombinant CBEL-ricin A fusion protein according to Example 2 was tested on CR2 * human B lymphoblastoid (Raji) cells and CR2 " T (HSB2) cells as follows. A suspension of 1 x 10 6 cells was thoroughly mixed with varying concentrations of the purified recombinant fusion protein in 1 ml of culture medium, and incubated for 24 hours at 37°C. Thereafter cell viability was assessed by staining the cells with trypan blue as in Example 5. As FIG. 3 shows, CR2 * cells
  • Example 7 The procedure of Example 6 was followed with the exception that the percentage of viable cells was determined by a colorimetric method using the tetrazoliumcompound (3- (4, 5-dimethylthiazol-2-yl) -5- (3- carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt; MTS) and an electron coupling reagent (phenazine methosulfate; PMS) .
  • MTS is bioreduced by cells into a formazan that is soluble in tissue culture medium. The absorbance of the formazan at 490 nm can be measured directly from 96 well assay plates without additional processing.
  • the quantity of formazan product as measured by the absorbance at 490 nm is directly proportional to the number of living cells in culture.
  • Reagents for the MTS assay were obtained from Promega Corp. (Madison, Wisconsin) . Results obtained by this method were substantially identical to Example 6.
  • Example 8 The effect of the recombinant CBEL-ricin A fusion protein according to Example 3 was tested on CR2* human B lymphoblastoid (Raji) cells and CR2 " human T (HSB2) cells according to the procedure of Example 6. The results were substantially similar to those of Example 6.
  • Example 9 The effect of the recombinant CBEL-ricin A fusion protein according to Example 4 was tested on CR2 + human B lymphoblastoid (Raji) cells and CR2 " human T (HSB2) cells according to the procedure of Example 6. The results were substantially similar to those of Example 6.
  • the CBEL-chemical agent compositions according to the present invention may be employed for target- specific delivery of a chemical agent to CR2-expressing cells, generally by contacting the CR2-expressing cells with the composition under conditions in which the CBEL induces endocytosis of the composition into the CR2- expressing cells.
  • a CBEL-cytotoxic agent composition according to the invention can be employed as an effective antitumor agent in vivo, selectively killing CR2" B cells.
  • the composition is administered to the subject by systemic administration, typically by subcutaneous, intramuscular, or intravenous injection, or intraperitoneal administration.
  • injectables for such use can be prepared in conventional forms, either as a liquid solution or suspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion.
  • Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and if desired, minor amounts of auxiliary substances such as wetting or emulsifying agents, buffers, and the like may be added.
  • the composition may be contacted with the cells in vi tro or in vivo .
  • the CR2-expressing cells may constitute (and in most instances are expected to constitute) a subpopulation of a mixed population of cell types; the peptide ligand according to the invention can provide for CR2-specific endocytosis of the conjugate into CR2-expressing cells.
  • the chemical agent may have any of a variety of desired effects in the targeted cells. As mentioned above, in some particularly useful embodiments the chemical agent is effective on a cell only when, or principally when, the agent is internalized into the cell.
  • Example 10 Targeted delivery of a CBEL-antigenic agent to B cells
  • Compositions comprising a CBEL and an antigen according to the invention can be administered to a warm-blooded animal for targeted initiation of an immune response in CR2 + cells.
  • the CBEL provides for CR2-mediated internalization of the antigen into the cells, and can result in initiation of an antibody- independent pathway for complement activation in the targeted cells. That is, according to the invention, the targeted cells can be induced to elicit an immune response against an antigen to which the cells are naive.
  • Chemical conjugation of a CBEL having the amino acid sequence identified as SEQ ID NO:2 is activated and then coupled to chicken lysozyme (Sigma Chemical Co . , St. Louis, Missouri) as in Example 1.
  • the CBEL-lysozyme conjugate is then systemically administered to a mouse.
  • the C3dg CBEL (SEQ ID NO:2) provides binding specificity to mouse B cells and induces CR2 receptor-mediated endocytosis of the conjugate.
  • the conjugate then elicits an immune response by the targeted B cells against epitopes borne on the conjugate, including epitopes that are unique to the lysozyme portion of the conjugate.
  • the results of this example can be substantially duplicated by construction of a CBEL- lysozyme fusion protein.
  • a CBEL such as the EBV CBEL (SEQ ID NO:l) or a peptide substantially homologous thereto
  • a cytotoxin such as ricin A
  • Such composition can be made, for example, as shown above in Example 3.
  • the EBV CBEL targets mature human B cells and the ricin A is cytotoxic to any cell into which it is delivered.
  • An effective amount of the composition is systemically administered to the individual so that the composition enters the bloodstream and contacts B cells.
  • the CBEL causes the composition to bind to the CR2 receptor on the B cells and induces internalization of the composition by endocytosis.
  • the ricin A cytotoxin then kills the cell by destroying ribosomes. This procedure reduces the number of malignant B cells in the body of the individual, thereby having a positive effect in treatment of the disease.
  • Example 12 A method for treating an autoimmune disease, e.g. lupus erythematosus or rheumatoid arthritis, follows the procedure of Example 11. Once delivered into B cells, the cytotoxin kills the cells, thus reducing the number of B cells producing autoantibodies, thereby having a positive effect in treatment of the disease.
  • an autoimmune disease e.g. lupus erythematosus or rheumatoid arthritis
  • MOLECULE TYPE peptide
  • SEQUENCE DESCRIPTION SEQ ID NO: 1 :

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US6251866B1 (en) 1997-08-05 2001-06-26 Watson Laboratories, Inc. Conjugates targeted to the interleukin-2 receptor
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AU759164C (en) * 1997-11-19 2007-03-29 Georgetown University Targeted liposome gene delivery
DE19926154A1 (de) * 1999-06-09 2000-12-14 Ktb Tumorforschungs Gmbh Verfahren zur Herstellung einer injizierbaren Arzneimittelzubereitung
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US10806803B2 (en) 2014-07-17 2020-10-20 Ohio State Innovation Foundation Compositions for targeting macrophages and other CD206 high expressing cells and methods of treating and diagnosis
US11007272B1 (en) 2016-10-07 2021-05-18 Navidea Biopharmaceuticals, Inc. Compounds and methods for diagnosis and treatment of viral infections
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