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WO1997033618A1 - Promedicaments macromoleculaire de ciblage des lymphocytes t - Google Patents

Promedicaments macromoleculaire de ciblage des lymphocytes t Download PDF

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Publication number
WO1997033618A1
WO1997033618A1 PCT/US1997/003832 US9703832W WO9733618A1 WO 1997033618 A1 WO1997033618 A1 WO 1997033618A1 US 9703832 W US9703832 W US 9703832W WO 9733618 A1 WO9733618 A1 WO 9733618A1
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Prior art keywords
composition
chemical agent
ligand
cells
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PCT/US1997/003832
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English (en)
Inventor
Ramesh K. Prakash
Jindrich Kopecek
Pavla Kopeckova
Vladimir Omelyanenko
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Theratech, Inc.
University Of Utah Research Foundation
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Application filed by Theratech, Inc., University Of Utah Research Foundation filed Critical Theratech, Inc.
Priority to AU22057/97A priority Critical patent/AU708304B2/en
Priority to EP19970915001 priority patent/EP0904109A4/fr
Priority to JP53278097A priority patent/JP2001519762A/ja
Priority to KR1019980709230A priority patent/KR19990087839A/ko
Priority to BR9711081-7A priority patent/BR9711081A/pt
Publication of WO1997033618A1 publication Critical patent/WO1997033618A1/fr

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    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere

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. T lymphocytes .
  • 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) ; Anderson et al. , U.S. Patent Nos . 5,169,933 and 5,135,736; Thorpe et al . , U.S. Patent No. 5,165,923; Jansen et al . , U.S. Patent No.
  • 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) . Thymocytes, peripheral T cells, and T-cell lines have also been found to express CR2 or CR2-like molecules.
  • B cells mature B lymphocytes
  • epithelial cells such as human pharyngeal epithelial cells, human follicular dendritic cells, and cervical epithelium
  • EBV Epstein-Barr Virus
  • C3d/C3dg a receptor for both Epstein-Barr Virus
  • the CR2 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 fco. the. 2 ⁇ Complement Receptor
  • Epithelial cells containing the CR2 receptor also bind EBV, but apparently such cells are infected by a mechanism other than receptor-mediated endocytosis. Some T-cell lines can be infected by EBV, while infection of other T-cell lines is variable or undetectable .
  • HSB-2 T cells lack the CR2 receptor, such cells are infected by EBV. J.A. Hedrick et al. , 22 Eur. J. Immunol. 1123 (1992) .
  • This synthetic peptide also blocked binding of recombinant gp350/220 or C3dg to the CR2 receptor on B cells, and a similar synthetic peptide inhibited EBV infection in vitro.
  • the compositions comprise a
  • the composition can include a spacer, which can be either biodegradable or non-biodegradable, for coupling the
  • Chemical agents can include cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like.
  • the composition can further comprise a carrier such as a water soluble polymer, liposome, or particulate.
  • T lymphocytes are specifically targeted to other cell types.
  • targeting of T lymphocytes would enable therapeutic applications for T-cell-associated diseases and tissue graft rejection.
  • T-cell-associated diseases include arthritis, T-cell lymphoma, skin cancers, and diseases resulting from HIV infection.
  • compositions for intracellular delivery of chemical agents to T 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. T lymphocytes .
  • selected chemical agents such as cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like
  • compositions for intracellular delivery of a chemical agent capable of eliciting a selected effect when delivered intracellularly into a T lymphocyte having the formula:
  • L-S] a -C- [S-A] b wherein L is a ligand capable of binding to a receptor on the T lymphocyte and stimulating receptor-mediated endocytosis of the composition; A is the chemical agent;
  • a preferred water soluble polymer is a copolymer of N- (2-hydroxypropyl)methacrylamide (HPMA) .
  • the ligand is preferably a member of the group consisting of a peptide with the amino acid sequence identified as SEQ ID N0:1 and peptides substantially homologous thereto, with a peptide having the amino acid sequence of SEQ ID NO:1 being especially preferred.
  • the chemical agent is preferably a member selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs.
  • the spacer is biodegradable such that the chemical agent is detachable from the polymer inside a cell. More preferably, the spacer comprises a peptide, and most preferably the spacer is a peptide with the amino acid sequence Gly-Phe-Leu-Gly (SEQ ID NO:4) .
  • the composition can further comprise a carrier 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 ligand binds to a receptor on the T lymphocyte and elicits endocytosis of the receptor-bound composition.
  • an effective amount of the composition is systemically administered such that the ligand contacts and binds to receptors on T lymphocytes and then stimulates endocytosis of the composition.
  • the chemical agent elicits its selected effect, although some agents may be active at the cell membrane.
  • FIG. 1 shows comparative in vi tro cytotoxic effects of different concentrations of ligand-HPMA copolymer- adriamycin composition No. 177 on HSB-2 T cells (D) ,
  • FIG. 2 shows the in vi tro cytotoxic effects of exposure to different concentrations of a ligand-HPMA copolymer-adriamycin composition (No. 177; ⁇ ) and a HPMA copolymer-adriamycin control composition (No. 237A; ⁇ ) on CCRF-CEM human T cells.
  • FIG. 3 shows the in vitro cytotoxic effects of exposure to different concentrations of a ligand-HPMA copolymer-adriamycin composition (No. 177; ⁇ ) and a HPMA copolymer-adriamycin control composition (No. 237A; ⁇ ) on human epithelial cells (HeLa cells) .
  • FIG. 4 shows the in vi tro cytotoxic effects of a ligand-HPMA copolymer-adriamycin composition (No. 177; O) and a HPMA copolymer-adriamycin control composition (No. 237A; ⁇ ) on human monocyte cells (U 937 cells) .
  • FIG. 5 shows in vi tro cytotoxic effects of a ligand-HPMA copolymer-adriamycin composition (No. 177) on human CCRF-CEM T cells in the presence of anti-CR2 monoclonal antibody OKB7 : 0KB7 (D) ; no antibody (dotted line) .
  • FIG. 6 shows in vi tro cytotoxic effects of a ligand-HPMA copolymer-adriamycin composition (No. 177) on human Raji B cells in the presence of anti-CR2 monoclonal antibody OKB7: 0KB7 (D) ; no antibody (dotted line) .
  • FIG. 7 shows in vi tro cytotoxic effects of a ligand-HPMA copolymer-adriamycin composition (No. 177) on human CCRF-CEM T cells in the presence of the anti- CR2 monoclonal antibody B-Ly 4 : B-Ly 4 (D) ; no antibody (dotted line) .
  • FIG. 8 shows in vi tro cytotoxic effects of a ligand-HPMA copolymer-adriamycin composition (No. 177) on human Raji B cells in the presence of the anti-CR2 monoclonal antibody B-Ly 4: B-Ly 4 (D) ; no antibody (dotted line) .
  • 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.
  • ligand means a composition capable of binding to a receptor on a T lymphocyte and stimulating internalization by endocytosis of the receptor and receptor-bound ligand.
  • ligands are coupled to various functional molecules so that upon endocytosis of the ligands the various functional molecules coupled thereto are also internalized by the T cells.
  • Preferred ligands for binding to a receptor on a T lymphocyte and inducing internalization by endocytosis of the receptor and receptor-bound ligand are a peptide having the amino acid sequence identified as SEQ ID NO:l and peptides substantially homologous thereto.
  • substantially homologous means peptides that retain functionality in binding T-cell receptors and eliciting receptor-mediated endocytosis although they may be truncations, deletion variants, or substitution variants of SEQ ID NO:1 or include additional amino acid residues attached thereto. 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 preserved, in this case, functionality in binding a T-cell 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 classification of such conservative substitution groups is as follows: (a) Pro; (b) Ala, Gly; (c) Ser, Thr; (d) Asn, Gin; (e) Asp, Glu; (f) His; (g) Lys, Arg; (h) Cys; (I) lie, Leu, Met, Val; and (j) Phe, Trp, Tyr. M. Jimenez-Montano & 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) . Another classification of such groups is described in M. Dayhoff et al . , Atlas of Protein Sequence and Structure (Nat ' 1 Biomed. Res. Found., Washington, D.C, 1978), hereby incorporated by reference. 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.
  • compositions comprising a CR2- receptor-binding and endocytosis-inducing ligand (CBEL) , i.e. SEQ ID NO:1, coupled to a chemical agent, i.e.
  • CBEL CR2- receptor-binding and endocytosis-inducing ligand
  • ricin A were specifically delivered intracellularly into B cells, but not T cells.
  • a spacer peptide e.g. a peptide having the sequence of SEQ ID NO:4
  • the composition binds preferentially to T cells and is delivered intracellularly into such T cells.
  • the presence of multiple ligands may modify the specificity such that T cells are preferentially recognized.
  • Binding and uptake of the composition by T cells may be mediated by the EBV receptor described by J.A. Hedrick et al . , supra .
  • a fraction of B cells exposed to this composition also binds and takes up the composition, but in vivo the composition appears to be taken up by T cells, and not at all or only slightly by B cells.
  • acromolecule means a composition comprising a water soluble polymer with at least 2 ligands and a chemical agent bound thereto.
  • the polymer is an HPMA copolymer and the ligand is an oligopeptide.
  • the chemical agent can be from many different classes of molecules, as explained in more detail herein.
  • prodrug means a chemical agent that is chemically modified to overcome a biological barrier.
  • a chemical agent When a chemical agent is converted into its prodrug form, its biological activity is eliminated or substantially reduced, but the biological barrier that inhibited its effectiveness is no longer problematic.
  • the chemical group that is attached to the chemical agent to form the prodrug, i.e. the "pro-moiety" is removed from the prodrug by enzymatic or nonenzymatic means to release the active form of the chemical agent. See A. Albert, Chemical Aspects of Selective Toxicity. 182 Nature 421 (1958) .
  • compositions are prodrugs because the chemical agent that has the selected effect when internalized in T lymphocytes is modified with ligands, water soluble polymer, and spacers such that the composition is delivered into the T lymphocytes, thus penetrating the cell membrane thereof.
  • the biological effect of the chemical agent is greatly reduced or eliminated until the composition is delivered intracellularly and the chemical agent is released from the remainder of the composition by biodegradation of the spacer.
  • chemical agent means and includes any substance that has a selected effect when internalized into a T lymphocyte. Certain chemical agents have a physiological effect, such as a cytotoxic effect or an effect on gene regulation, on a T cell when internalized into the cell.
  • a "transforming nucleic acid” (RNA or DNA)
  • RNA or DNA when internalized into a cell, can be replicated and/or expressed within the cell.
  • Other nucleic acids can 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.
  • 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.
  • drug or “pharmacologically active agent” means any chemical material or compound suitable for intracellular administration in a T lymphocyte that stimulates a desired biological or pharmacological effect in such cell.
  • carrier means water soluble polymers, particulates, or liposomes to which a composition according to the instant invention can be coupled.
  • Such carriers increase the molecular size of the compositions and may provide 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. 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 . , Polysaccharides as Drug Carriers, in Controlled-Release Technology 188 (P.I. Lee & W.R. Good, eds . , 1987) ; F.
  • illustrative water soluble polymers include dextran, inulin, poly(L-lysine) with modified epsilon amino groups, poly(L-glutamic acid) , N-substituted methacrylamide-containing synthetic polymers and copolymers, and the like.
  • an effect 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 T cells within a selected time period.
  • An effect 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 intracellular delivery of a chemical agent capable of eliciting a selected effect when delivered intracellularly into a T lymphocyte, the composition having the formula:
  • L is a ligand capable of binding to a receptor on the T lymphocyte and stimulating receptor-mediated endocytosis of the composition
  • A is the chemical agent
  • S is a spacer
  • C is a water soluble polymer having functional groups compatible with forming covalent bonds with the ligand, chemical agent, and spacer
  • a is an integer of at least 2
  • b is an integer of at least 1.
  • a is an integer of 2 to about 1000
  • b is an integer of 1 to about 1000.
  • the spacers are preferably biodegradable such that the chemical agent is detached from the composition by hydrolysis and/or enzymatic cleavage inside T cells.
  • CD4 + T cells are targeted by these compositions, but it remains to be determined whether CD8* T cells are also targeted.
  • the chemical agent is selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like.
  • the coupling of a ligand to a chemical agent can be, without limitation, by covalent bond, electrostatic interaction, hydrophobic interaction, physical encapsulation, and the like.
  • the compositions of the present invention can further comprise a carrier selected from the group consisting of water soluble polymers, liposomes, and particulates .
  • Such water soluble polymers are selected from the group consisting of dextran, inulin, poly(L-lysine) (PLL) with modified epsilon amino groups, poly(L-glutamic acid) (PGA) , N-substituted methacrylamide-containing polymers and copolymers, and the like.
  • a preferred water soluble polymer is a copolymer of N- (2- hydroxypropyl)methacrylamide (HPMA) .
  • the composition provides means for preferential binding to a receptor on T cells, thus triggering internalization of the composition by endocytosis.
  • the chemical agent provides means for achieving a selected effect in the T cells.
  • chemical agents comprise cytotoxins, including radionuclides, for selective killing or disabling of T cells; nucleic acids for genetically transforming or regulating gene expression in T 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 are constructed by chemically conjugating the ligand and chemical agent to the water soluble polymer.
  • "Chemically conjugating" the ligand and the chemical agent to the water soluble polymer means covalently bonding the ligand and chemical agent to the polymer by way of a spacer moiety.
  • a spacer moiety is used to form a linkage between functional groups on the polymer and the chemical agent .
  • Peptide portions of the compositions of the present invention can 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 ligand, spacer, or peptide chemical agent 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. Peptides containing a ligand, spacer, or peptide chemical agent can also be constructed by chemical synthesis.
  • 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 stimulating internalization of the chemical agent by endocytosis.
  • compositions according to the present invention preferably 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 remainder of the composition by proteolysis of the digestion site.
  • a protease susceptible spacer can be added regardless of whether the peptide portions of the composition are synthesized chemically or as expression peptides in a genetically engineered organism.
  • nucleotides encoding the protease susceptible spacer can be inserted into the hybrid gene encoding the ligand and or a peptide chemical agent by techniques well known in the art.
  • Another aspect of the present invention features a method for specifically effecting a desired activity in T lymphocytes contained in a heterogeneous population of cells, by steps of contacting the population of cells with a composition, prepared according to the present invention, that directs such activity intracellularly.
  • the compositions of the invention are selectively bound to T cells in the mixed population, whereupon endocytosis of the composition into the T cells is stimulated, and the chemical agent effects its activity within such T cells.
  • the invention features peptides, employed as ligands, spacers, and/or chemical agents.
  • the peptides according to the invention can 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 (1982) ; Houghten, 82 Proc. Nat'l Acad. Sci. USA 5131 (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 ligand and/or spacer, or a chemical agent and/or spacer.
  • a suitable host cell of a nucleic acid containing an oligonucleotide encoding a ligand and/or spacer, or a chemical agent and/or spacer.
  • compositions according to the present invention will now be described, with particular reference to examples in which a peptide ligand coupled to a biodegradable spacer (SEQ ID NO:4) and a cytotoxic chemical agent, adriamycin, are coupled to a copolymer of HPMA.
  • SEQ ID NO:4 a biodegradable spacer
  • a cytotoxic chemical agent adriamycin
  • Example 1 a composition according to the present invention was prepared by coupling an EBV- derived ligand, SEQ ID N0:1, and/or the cytotoxic chemical agent, adriamycin, to an HPMA copolymer via a protease-sensitive spacer (Gly-Phe-Leu-Gly; SEQ ID N0:4) .
  • Adriamycin intercalates with DNA and inhibits DNA replication, thus exerting a toxic effect on cells.
  • Compositions according to this example are lysosomotropic, and the degree of cytotoxicity depends on the biodegradability of the drug-polymer linkage within the lysoso es .
  • the protease-sensitive spacer Gly-Phe-Leu-Gly (SEQ ID NO:4) is biodegradable in lysosomes, but is resistant to proteolysis in the bloodstream.
  • the construction of these compositions is described in detail below.
  • HPMA copolymer-adriamycin compositions were employed in this example, namely a ligand-containing composition and a control composition lacking a ligand.
  • N- methacryloylglycylphenylalanylleucylglycine p- nitrophenyl ester (MA-Gly-Phe-Leu-Gly-ONp) was synthesized by aminolysis of N- methacryloylglycylphenylalanine p-nitrophenyl ester (MA- Gly-Phe-ONp) with leucylglycine (Leu-Gly-OH) , followed by esterification with p-nitrophenol, as described in Kopecek et al . , U.S. Patent No. 5,037,883, which is hereby incorporated by reference.
  • the polymer precursors (copolymers of HPMA and methacryloylated oligopeptide active esters) were prepared by radical precipitation polymerization in acetone at 50°C for 24 h using 2,2'- azobisisobutyronitrile (AIBN) as the initiator.
  • AIBN 2,2'- azobisisobutyronitrile
  • the polymerization mixture was 618
  • the composition of the polymerization mixtures for polymer precursor HPMA copolymer-Gly-Phe-Leu-Gly-ONp was 2.000 g HPMA, 0.706 g MA-Gly-Phe-Leu-Gly-ONp (SEQ ID NO:4) , 0.130 g AIBN, and 18.8 g acetone.
  • the mole ratio of ONp to HPMA was 8/92.
  • the weight-average molecular weight and polydispersity were determined after aminolysis with l-amino-2-propanol by size exclusion chromatography on a Superose 6 column using the Pharmacia FPLC system (0.05 M Tris, 0.5 M NaCl, pH 8.0 as eluent) calibrated with poly(HPMA) fractions.
  • Solution behavior (molecular characterization) of polymers (a inolysed precursors) was also evaluated by light scattering methods (see below) .
  • Aminolysed precursors were prepared by adding a ten-fold excess of l-amino-2-propanol to a solution of polymer precursors in DMSO (20 wt %) and precipitating with an excess of acetone.
  • the characterization of polymer precursors is presented in Table 1. Table 1
  • a ligand can be coupled to a polymeric precursor, made for example as described above, in any of a variety of ways that are well known in the art .
  • the composition and the analysis of two conjugates are shown in Table 2.
  • P represents the HPMA copolymer backbone
  • ADR represents adriamycin
  • L represents the ligand SEQ ID NO:l
  • Gly-Phe-Leu-Gly (SEQ ID NO:4) is a spacer susceptible to proteolytic degradation.
  • composition no. 177 was synthesized as follows.
  • HPMA copolymer precursor 100 mg containing 6.2 mol% of Gly-Phe-Leu-Gly (SEQ ID NO:4) side chains (36 ⁇ mol ONp) was dissolved in 0.5 ml anhydrous dimethylformamide (DMF) and 14.0 mg (24 ⁇ mol) of adriamycin hydrochloride (solid) and a solution of 12.7 mg (12 ⁇ mol) ligand (SEQ ID NO:l) in 100 ⁇ l DMF were added. Then, 27 ⁇ l of a diluted solution of triethylamine (1:1) in DMF (96 ⁇ mol, 9.8 mg) was added in three portions within 30 min.
  • reaction mixture was stirred for 6 h at room temperature, then 10 ⁇ l of aminopropanol diluted with DMF (10:1) was added to aminolyze unreacted ONp groups.
  • the reaction mixture was precipitated into 200 ml acetone, the polymeric product was filtered off, washed with acetone and reprecipitated from methanolic solution (20 wt%) into acetone. Unbound adriamycin was removed on a Sephadex LH-20 column (16/50) in methanol. The polymer fractions were collected and evaporated under reduced pressure.
  • the pure conjugate in powdered form was obtained after precipitating (0.8 ml) into 300 ml acetone and drying in vacuo (yield 75 mg) .
  • the conjugate was dissolved in 20 ml of deionized water and dialyzed against water at 4°C for 24 hours in the dark.
  • the purified conjugate was isolated by freeze-drying.
  • the quantity of free adriamycin in the resulting product was determined by extraction assay. Two mg of polymer conjugate in a test tube was dissolved in 0.5 ml water, then 0.5 ml of 0.2 M sodium carbonate/bicarbonate buffer, pH 10.5, was added and the solution was extracted with 2 ml ethylacetate. The organic layer was separated, dried with magnesium sulfate, and absorption spectrum (350-550 nm) was measured at a range of 0 to 0.02 A. There was no detectable ADR in the conjugate.
  • compositions No. 177 and 237A prepared according to the procedure of Example 1 were tested on several human T and B cell lines as follows. Triplicate samples of 1 x 10 5 cells each were mixed with different concentrations of the purified compositions in 0.1 ml of culture medium (RPMI 1640, 10% fetal calf serum) in the wells of a 96-well microtiter plate (Falcon Microtest 111) , and incubated for 18-48 hours at 37°C in a humidified, 5% C0 2 atmosphere.
  • culture medium RPMI 1640, 10% fetal calf serum
  • MTS tetrazolium compound
  • 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) . According to this method, 20 ⁇ l of MTS/PMS solution (Promega No. G-5421) was added to each well of the assay plate. The plate was then further incubated at 37°C in a humidified, 5% C0 2 atmosphere for 4 hours. The absorbance of each well was then measured at 490 nm with an EL311 Microplate Autoreader (Bio-Tek Instruments) . The mean absorbance for each treatment was then calculated, and the percent cytotoxicity was determined using the formula:
  • a s represents the mean absorbance for each treatment and A c represents mean absorbance of the control treatment, i.e. cells not exposed to a conjugate.
  • the following cell lines were tested according to this procedure: HSB-2 (ATCC No. CCL 120.1; CD4* human T cells) ; CCRF-CEM (ATCC No. CCL 119; CD4 + human T cells) ; MOLT-3 (ATCC No. CRL 1552; CD4 + human T cells) ; Raji (ATCC No. CCL 86; CR2 + human B lymphoblastoid cells) ; and Daudi (ATCC No. CCL 213; CR2 + human B cells) .
  • FIG. 1 summarizes the results of this experiment wherein it is shown that the adriamycin-containing composition No. 177 exhibited greater cytotoxicity in the three T cell lines than in the B cell lines. This suggests that the T cells were able to internalize more of composition No. 177 than were the B cells. All of the T cells tested internalized the composition approximately equally as judged by the similar levels of cytotoxicity, particularly at 50 ⁇ g/ml and 100 ⁇ g/ml of composition No. 177. The Raji and Daudi B cell lines also exhibited moderate ability to take up composition No. 177. The levels of cytotoxicity to these B cell lines was markedly reduced, however, as compared to the three T cell lines at all concentrations tested. All of the cell lines internalized some of control composition No.
  • composition No. 237A which lacks a ligand but contains adriamycin, shown for example in FIG. 2 with respect to CCRF-CEM T cells.
  • the level of cytotoxicity in response to composition No. 237A was less than the level of cytotoxicity in response to composition No. 177 at all concentrations tested.
  • Example 3 In this example, the procedure of Example 2 was followed with the exception that the cells used were human epithelial cells (HeLa cells) .
  • the results of this test are shown in FIG. 3, wherein at every concentration of conjugate tested, the ligand-containing and control compositions yielded substantially identical results. At 100 ⁇ g/ml of conjugate there was virtually no cytotoxicity. Only when the concentration was increased to 500 ⁇ g/ml was cytotoxicity observed, and then only about 25% of the cells were killed.
  • Example 2 the procedure of Example 2 was followed with the exception that the cells used were human monocyte cells (U 937 cells, ATCC No. CRL 15393) .
  • FIG. 4 shows that composition No. 177 and control composition No. 237A were substantially similar with respect to cytotoxic effects on human monocyte cells, i.e. very little or no cytotoxicity at conjugate concentrations at or below 10 ⁇ g/ml and increasing levels of cytotoxicity at higher conjugate concentrations.
  • compositions according to the present invention can be employed for targeted delivery of a chemical agent to T cells, generally by contacting the T cells with the composition under conditions in which binding of the ligand to a receptor stimulates endocytosis of the composition into the T cells.
  • the chemical agent then acts on or within the targeted cell into which the composition is internalized, and the desired effect of the active agent can be confined to those cells having the receptor.
  • a composition according to the invention can be employed as an effective antitumor agent in vivo for killing T 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 can be contacted with the cells in vi tro or in vivo .
  • the T cells constitute a subpopulation of a mixed population of cell types; the ligand according to the invention can provide for endocytosis of the conjugate into T cells and possibly into a small proportion of other cells having a closely related receptor.
  • the chemical agent can 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 5 In Vivo Targeted Delivery £o ⁇ _ cells
  • compositions according to the present invention can be administered to a warm-blooded animal for targeted delivery to T cells.
  • the composition provides for receptor-mediated internalization of the composition into the T cells.
  • mice About 1 x 10 7 HSB-2 human T-cell leukemia cells in 500 ⁇ l of PBS were injected intraperitoneally into mice, and the cells were allowed to colonize the mice for 48 hours.
  • the human T-cell leukemia cells were found to preferentially colonize the spleen and liver.
  • the mice were injected intraperitoneally with 500 ⁇ g of either composition No. 177 or control composition No. 237A in 500 ⁇ l of PBS.
  • the spleen and liver were harvested, and PCR assay of genomic DNA and cDNA prepared from these organs was used to determine the relative numbers of mouse and human cells therein.
  • Genomic DNA and cDNA were prepared from mouse spleen and liver according to methods that are generally well known in the art. See, e.g.. J. Sambrook et al . , Molecular Cloning: A Laboratory Manual (2d ed. , 1989) ; T. Maniatis et al . , Molecular Cloning: A Laboratory Manual (1982) ; F. Ausubel et al . , Current Protocols in Molecular Biology (1987) . Illustrative methods for preparation of cDNA and genomic DNA are briefly described below. Preparation o_f cDNA
  • the excised spleen and liver were disrupted and the resulting cells were washed in PBS.
  • the cells were then resuspended in a buffer containing 100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 25 mM EDTA, 0.5% SDS, and 0.1 mg/ml proteinase K and incubated overnight at 37°C
  • the disrupted cells were then centrifuged and washed once in cold PBS.
  • the cell pellet was then resuspended in RNAzol B reagent (Tel-Test, Friendwood, Texas) .
  • the resulting lysate was then extracted with chloroform, and RNA was precipitated with isopropanol .
  • RNA was washed with 75% ethanol, dried briefly, and dissolved in 0.5% SDS. An aliquot of RNA was then mixed with reverse transcriptase, random primers, buffer, and deoxynucleotide triphosphates, and the mixture was incubated at 37°C for 1 hour. The reaction was stopped by RNase digestion, and then the cDNA was extracted in succession with phenol/chloroform and chloroform, precipitated with ethanol, and resuspended in water. Preparation o_£ Genomic DNA
  • PCR of cDNA and. Generi ⁇ h PCR is well known in the art for determining the presence of selected sequences in genomic DNA and cDNA samples.
  • the following references illustrate PCR methodology: PCR Technology: Principles and Applications for DNA Amplification (H. Erlich ed., Stockton Press, New York, 1989) ; PCR Protocols: A Guide to Methods and Applications (Innis et al . eds, Academic Press, San Diego, Calif., 1990) ; U.S. Patent Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188.
  • PCR reactions were carried out in glass capillary tubes in 10 ⁇ l volumes containing 0.8 mM of each of the four deoxynucleotide triphosphates, 2.5 ⁇ Ci of 32 P-dCTP (3000 Ci/mmol) , 0.72 units of Thermus aquaticus (Taq) DNA polymerase, 35-70 pmol of each primer (20-23 nucleotides in length) , 200 ng cDNA, and a reaction buffer containing 50 mM Tris-HCl, pH 8.3, 3 mM MgCl 2 , 20 mM KC1, and 0.5 mg/ml of bovine serum albumin.
  • the amount of cDNA and number of cycles of amplification can be determined empirically by a person of ordinary skill in the art without undue experimentation. Titration of cDNA from about 1 ng to about 500 ⁇ g, and titration of cycles from about 12 to about 36 gives a good indication of amounts of cDNA and numbers of cycles needed. For example, using ⁇ -actin primers, 12-15 cycles and about 200 ng of cDNA generally give good results.
  • the reaction mixtures were sealed in capillary tubes and then the capillaries were placed in a Model 1605 Air Thermocycler (Idaho Technology, Idaho Falls, Idaho) . Parameters of annealing temperature, elongation time, and number of cycles were selected. Increasing the annealing temperature increases the specificity of PCR amplification reactions and decreases the amounts of nonspecific products. Annealing temperature can be estimated from thermal melting temperature according to the formula:
  • T m 4°C(no. of G and C residues in primer) + 2°C(no. of A and T residues in primer) .
  • the elongation time depends on the size of product to be amplified. As a rule of thumb, about 4 seconds is sufficient for products of about 100-150 bp, about 8 seconds is sufficient for products of about 200-300 bp, and about 20 seconds is needed for products larger than about 500 bp. Increasing elongation times may result in amplification of nonspecific products.
  • reaction mixture is removed from the capillary, mixed with an equal volume of stop solution (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol FF) , and either stored frozen or immediately heated at 95°C for 5 minutes and subjected to polyacrylamide gel electrophoresis.
  • stop solution 95% formamide, 20 mM EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol FF
  • the fractionated products are then quantitated according to the amount of radioactivity in each fraction, such as by autoradiography.
  • An illustrative method of determining the relative amounts of human and mouse cells in spleen and liver tissues involves comparison of amplified products from reactions with mouse ⁇ -actin and human ⁇ -actin specific primers.
  • Illustrative mouse ⁇ -actin primers are as follows :
  • GTAACAATGC CATGTTCAAT (SEQ ID NO:5) CTCCATCGTG GGCCGCTCTA G (SEQ ID NO: 6)
  • Illustrative human ⁇ -actin primers are as follows: CTTAGTTGCG TTACACCCTT TC (SEQ ID NO:7) GGGCCATTCT CCTTAGAGAG AAG (SEQ ID NO: 8)
  • mice treated with the control composition no. 237A exhibited the presence of both human and mouse DNA by PCR analysis with specific ⁇ -actin primers. Twelve of twelve mice treated with composition no. 177 exhibited the presence of mouse DNA, however, only three of twelve mice treated with composition no. 177 exhibited the presence of human DNA. These results demonstrate that a ligand and adriamycin-containing composition according to the present invention selectively kills T cells in animals to which it is administered.
  • Example 5 The procedure of Example 5 was followed except that Raji B cells were used instead of HSB-2 T cells. All mice treated with either composition no. 177 or control composition no. 237A exhibited the presence of both human and mouse DNA by PCR analysis with specific ⁇ - actin primers. These results demonstrate that a ligand and adriamycin-containing composition according to the present invention has no detectable effect on B cells in animals to which it is administered.
  • Example 7 Four groups of four mice per group were treated according to the following treatments: (1) no tumor cells, (2) tumor cells, (3) tumor cells plus composition no. 237A, or (4) tumor cells plus composition no. 177.
  • Groups 2-4 were injected intraperitoneally with about 1 x 10 7 HSB-2 human T-cell leukemia cells in 500 ⁇ l of PBS, and the cells were allowed to colonize the mice for 48 hours. After 48 hours, the mice of groups 3 and 4 were injected intraperitoneally with 500 ⁇ g of either composition no. 237A (group 3) or composition no. 177 (group 4) in 500 ⁇ l of PBS, and these injections were repeated on each of the next two days .
  • composition according to the present invention is capable of selectively killing human T cells in vivo whereas a control composition lacking a T-cell specific ligand was not.
  • a method of treating T cell lymphoma in a human comprises (a) providing a composition according to the present invention including a ligand, such as the EBV ligand (SEQ ID N0:1) or a peptide substantially homologous thereto, and a cytotoxin, such as adriamycin, both of which are coupled to an HPMA copolymer by means of a spacer (Gly-Phe-Leu-Gly; SEQ ID NO:4) and (b) systemically administering an effective amount of the composition to an individual.
  • a ligand such as the EBV ligand (SEQ ID N0:1) or a peptide substantially homologous thereto
  • a cytotoxin such as adriamycin
  • the composition binds to a receptor on the T cells and stimulates internalization of the composition by endocytosis.
  • the adriamycin then kills the cell by intercalating with DNA in the cell. This procedure reduces the number of malignant T cells in the body of the individual, thereby having a positive effect in treatment of the disease.
  • Example 9 The ability of anti-CR2 antibodies to competitively inhibit binding and receptor-mediated endocytosis of a composition according to the present invention was investigated in this example.
  • OKB7 antibodies Ortho diagnostic Systems, Raritan, N.J.
  • the 0KB7-reactive epitope seems to be closely related to the epitope or epitopes that interacts with the viral pg350/220 and with C3d. Carel et al . , supra ; CA. Lowell et al.
  • Example 2 The procedure of Example 2 was followed with the exception that the cells were preincubated at 37°C with OKB7 antibodies before administration of composition no. 177.
  • FIG. 5 shows that the 0KB7 anti-CR2 antibodies at the lowest dilution inhibit cytotoxicity of CCRF-CEM T cells by about 30%, but are otherwise ineffective in inhibiting binding and uptake of composition no. 177 and the cytotoxicity associated therewith.
  • FIG. 6 shows that in the absence of OKB7 antibodies, composition no. 177 is ineffectively taken up by Raji B cells, resulting in cytotoxicity to only about 40% of cells.
  • compositions according to the present invention such as composition no. 177, being capable of efficiently binding to T cells via a receptor other than the CR2 receptor, being internalized in such cells, and exhibiting their effects inside the cells.
  • Example 9 The procedure of Example 9 was followed in this example except that the antibodies used were the anti- CR2 monoclonal antibodies B-Ly 4 (Pharmagen) .
  • FIG. 7 shows that this anti-CR2 antibody has little or no effect on inhibiting cytotoxicity from composition no. 177 in CCRF-CEM T cells.
  • FIG. 8 shows, however, that B- Ly 4 antibodies are able to completely inhibit the cytotoxicity of composition no. 177 in Raji B cells.

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Abstract

La présente invention concerne une composition destinée à l'apport intracellulaire d'un agent chimique dans un lymphocyte T. Cette composition est constituée d'un ligand se liant à un récepteur et induisant l'endocytose et d'un agent chimique couplé à un polymère hydrosoluble. Le ligand se lie à un récepteur présent sur le lymphocyte T et provoque l'endocytose de la composition. La composition comporte également une séquence intercalaire assurant le couplage de l'agent chimique et du ligand avec le polymère. Les agents chimiques sont notamment des cytotoxines, acides nucléiques de transformation, régulateurs de gènes, marqueurs, antigènes, médicaments, etc. L'un des polymères hydrosolubles préférés est un copolymère du N-(2-hydroxypropyl)méthacrylamide (HPMA). La composition peut également inclure un vecteur tel qu'un polymère hydrosoluble, un liposome ou une matière particulaire. L'invention concerne également des procédés d'utilisation de ces compositions pour l'apport in vivo ou in vitro d'un agent chimique.
PCT/US1997/003832 1996-03-15 1997-03-12 Promedicaments macromoleculaire de ciblage des lymphocytes t WO1997033618A1 (fr)

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AU22057/97A AU708304B2 (en) 1996-03-15 1997-03-12 Targeting macromolecular prodrugs to T lymphocytes
EP19970915001 EP0904109A4 (fr) 1996-03-15 1997-03-12 Promedicaments macromoleculaire de ciblage des lymphocytes t
JP53278097A JP2001519762A (ja) 1996-03-15 1997-03-12 Ｔリンパ球への巨大分子プロドラッグのターゲティング
KR1019980709230A KR19990087839A (ko) 1996-03-15 1997-03-12 T임파세포로 거대분자 프로드럭을 표적 수송
BR9711081-7A BR9711081A (pt) 1996-03-15 1997-03-12 Direcionamento de pró-drogas macromoleculares para linfócitos t.

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WO2000007543A3 (fr) * 1998-08-04 2000-05-11 Watson Lab Inc Utah Conjugues cibles apportes aux recepteurs cibles et/ou aux recepteurs de l'interleukine 2
EP1011705A4 (fr) * 1997-08-05 2000-09-20 Watson Pharmaceuticals Inc Conjugues cibles apportes au recepteur de l'interleukine-2
WO2009036368A3 (fr) * 2007-09-14 2009-07-09 Nitto Denko Corp Vecteurs de médicament
WO2010014117A1 (fr) * 2008-07-30 2010-02-04 Nitto Denko Corporation Vecteurs de médicament
EP2210616A1 (fr) * 2009-01-21 2010-07-28 Centre National de la Recherche Scientifique Nanoparticules furtives polyvalentes pour une utilisation biomédicale
US8715685B2 (en) 2009-07-14 2014-05-06 Lucia Irene Gonzalez Stereoisomer peptides and their polymer conjugates for HIV disease
US8715986B2 (en) 2009-10-29 2014-05-06 Lucia Irene Gonzalez Stereoisomer peptides, ligand-targeted multi- stereoisomer peptide polymer conjugates, and uses thereof
US9572886B2 (en) 2005-12-22 2017-02-21 Nitto Denko Corporation Agent for treating myelofibrosis

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BR. J. CANCER, Volume 57, issued 1988, R. DUNCAN et al., "Anticancer Agents Coupled to N-(2-hydroxypropyl)methacrylamide Copolymers. II. Evaluation of Daunomycin Conjugates in Vivo Against L1210 Leukaemia", pages 147-156. *
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1011705A4 (fr) * 1997-08-05 2000-09-20 Watson Pharmaceuticals Inc Conjugues cibles apportes au recepteur de l'interleukine-2
US6251866B1 (en) 1997-08-05 2001-06-26 Watson Laboratories, Inc. Conjugates targeted to the interleukin-2 receptor
US6693083B2 (en) 1997-08-05 2004-02-17 Watson Pharmaceuticals, Inc. Conjugates targeted to the interleukin-2 receptor
WO2000007543A3 (fr) * 1998-08-04 2000-05-11 Watson Lab Inc Utah Conjugues cibles apportes aux recepteurs cibles et/ou aux recepteurs de l'interleukine 2
US9572886B2 (en) 2005-12-22 2017-02-21 Nitto Denko Corporation Agent for treating myelofibrosis
WO2009036368A3 (fr) * 2007-09-14 2009-07-09 Nitto Denko Corp Vecteurs de médicament
WO2010014117A1 (fr) * 2008-07-30 2010-02-04 Nitto Denko Corporation Vecteurs de médicament
EP2210616A1 (fr) * 2009-01-21 2010-07-28 Centre National de la Recherche Scientifique Nanoparticules furtives polyvalentes pour une utilisation biomédicale
WO2010084157A3 (fr) * 2009-01-21 2010-11-04 Centre National De La Recherche Scientifique (Cnrs) Nanoparticules furtives multifonctionnelles pour usage biomédical
US8715685B2 (en) 2009-07-14 2014-05-06 Lucia Irene Gonzalez Stereoisomer peptides and their polymer conjugates for HIV disease
US8715986B2 (en) 2009-10-29 2014-05-06 Lucia Irene Gonzalez Stereoisomer peptides, ligand-targeted multi- stereoisomer peptide polymer conjugates, and uses thereof

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BR9711081A (pt) 2000-01-11
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EP0904109A1 (fr) 1999-03-31
JP2001519762A (ja) 2001-10-23

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