TARGETING MACROMOLECULAR PRODRUGS TO T LYMPHOCYTES

Abstract: A composition for intracellular delivery of a chemical agent into a T cell comprises a receptor-binding and endocytosis-inducing ligand and a chemical agent coupled to a water soluble polymer. The ligand binds to a receptor on T lymphocytes and elicits endocytosis of the composition. The composition also includes a spacer for coupling the chemical agent and the ligand to the polymer. Chemical agents can include cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like. A preferred water soluble polymer is a copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA). The composition can further comprise a carrier such as a water soluble polymer, liposome, or particulate. Methods of using these compositions for delivering a chemical agent in vivo or in vitro are also disclosed.

Description: TARGETING MACROMOLECULAR PRODRUGS TO T LYMPHOCYTES Background of the Invention 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. E.g. , 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. 4,906,469; Frankel, U.S. Patent No. 4,962,188; Uhr et al . , U.S. Patent No. 4,792,447; Masuho et al . , U.S. Patent Nos. 4,450,154 and 4,350,626. These agents include a cell- targeting moiety, such as a growth factor or an antigen- binding protein, linked to a plant or bacterial toxin. They kill cells by mechanisms different from conventional chemotherapy, thus potentially reducing or eliminating cross resistance to conventional chemotherapeutic agents. 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. CD. Tsoukas & J.D. Lambris, Expression of EBV/D3d Receptors on I Cells: Biological Si nificance. 14 Immunology Today 56 (1993) . The reactivities of these molecules with ligands or antibodies vary from those of B cells, however, which suggested that there are structural differences between such receptors on T-cells and B-cells. J.A. Hedrick et al . , Interaction between Epstein-Barr Virus and a T Cell Line (HSB-2) via a Receptor Phenotypicallv Distinct from Complement Receptor Type 2. 22 Eur. J. Immunol. 1123 (1992) . More recently, EBV was discovered to bind and infect HSB-2 T cells via a receptor distance from CR2. J.A. Hedrick et al., Characterization of a 70-kDa. EBC gp350/220-Bindinα Protein on HSB-2 T Cells. 153 J. Immunol. 4418 (1994) . 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. E.g.. G.R. Nemerow, et al . , Identification and Characterization of the Epstein-Barr Virus Receptor on Human B Lymphocytes and its Relationship fco. the. 2ύ Complement Receptor ( R2) . 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 . E.g.. Tedder et al. (1986) , Epstein-Barr Virus Binding Induces Tnternalization of the C3d Receptor: A Novel Immunotoxin Delivery System. 137 J. Immunol. 1387 (1986) . 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 . CD. Tsoukas & J.D. Lambris, Expression of EBV/C3d Receptors QQ I Cells: Biological Significance. 14 Immunology Today 56 (1993) . For example, although 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) . Nemerow et al . , Identification of gp350 as the Viral Glycoprotein Mediating Attachment of Epstein-Barr Virus (EBV) to the EBV/C3d Receptor of B Cells: Sequence Homoloαy of gp350 and C3 Complement Fragment C3d. 61 J. Virol. 1416 (1987) , have identified domains of amino acid sequence similarity between C3dg and gp350/220, including a domain near the N-terminus of gp350/220 (Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu; SEQ ID N0:1) that corresponds to a sequence in C3dg (Glu-Asp-Pro-Gly-Lys- Gln-Leu-Tyr-Asn-Val-Glu; SEQ ID N0:2) . Nemerow et al . , Identification o_£ an SpitPpe in tJi≤ Manor Envelope Protein o_ Epstein-Barr Virus that Mediates Viral Binding to the B Lymphocyte EBV Receptor (CR2) . 56 Cell 369 (1989) , have also described binding of a synthetic tetradecapeptide containing the amino acid sequence identified as SEQ ID NO:1 both to the purified CR2 receptor and to CR2-expressing B cells, but this tetradecapeptide failed to bind to HSB-2 T cells. 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. Analysis of truncation and substitution peptide analogs showed that the EBV epitope involved in CR2 binding is contained within the Glu-Asp-Pro-Gly-Phe-Phe- Asn-Val-Glu sequence (SEQ ID N0:1) . Reduced levels of binding were observed with shorter peptides, although a Glu-Asp-Pro-Gly (SEQ ID NO:3) peptide retained significant CR2 binding activity. A peptide containing a single amino acid substitution of glycine for proline within this region also exhibited significantly reduced CR2 binding activity. Copending U.S. Patent Application Serial No. 08/305,770, filed September 13, 1994, describes compositions and methods for specific intracellular delivery of a chemical agent into a CR2-receptor-bearing cell, e.g. B lymphocytes. The compositions comprise a CR2-receptor-binding and endocytosis-inducing ligand (CBEL) coupled to the chemical agent. The CBEL binds to the CR2 receptor on the surface of B lymphocytes and elicits endocytosis of the composition. Optionally, the composition can include a spacer, which can be either biodegradable or non-biodegradable, for coupling the CBEL to the chemical agent. 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. It would be advantageous to develop prodrugε that are specifically targeted to other cell types. For example, targeting of T lymphocytes would enable therapeutic applications for T-cell-associated diseases and tissue graft rejection. Such T-cell-associated diseases include arthritis, T-cell lymphoma, skin cancers, and diseases resulting from HIV infection. In view of the foregoing, it will be appreciated that compositions for intracellular delivery of chemical agents to T cells and methods of use thereof would be significant advancements in the art. Objects and Summary of the Invention It is an object of the present invention to provide compositions for intracellular delivery of selected chemical agents to a specific cell type, i.e. T lymphocytes . 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 T lymphocytes. It is another object of the invention to provide compositions and methods for intracellularly delivering selected chemical agents, such as cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like, to T lymphocytes. These and other objects can be accomplished by providing a composition for 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-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; S is a spacer; C is a water soluble polymer having 618 6 functional groups compatible with forming covalent bonds with the ligand, chemical agent, and spacer; a is an integer of at least 2; and b is an integer of at least 1. 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. Preferably, 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 . The 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. For in vivo use, 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. Once inside the cells, the chemical agent elicits its selected effect, although some agents may be active at the cell membrane. Brief Description of the Drawings 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) , CCRF-CEM T cells (0) , MOLT-3 T cells (O) , Raji B cells (Δ) , and Daudi B cells (■) . 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; Δ) 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; Δ) 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; Δ) 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) . Detailed Description of the Invention Before the present compositions and methods for targeting of macromolecular prodrugs to T lymphocytes are disclosed and described, it is to be understood that this invention is not limited to the particular embodiments, process steps, and materials disclosed herein as such embodiments, process steps, and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof. It must be noted that, as used in this specification and the appended claims, the singular forms "a, " "an, " and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a composition containing "a ligand" includes two or more ligands, reference to "a chemical agent" includes reference to one or more of such chemical agents that may be the same or different chemical agents, and reference to "a spacer" includes reference to two or more spacers . In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. As used herein, "peptide" means peptides of any length and includes proteins. The terms "polypeptide" and "oligopeptide" are used herein without any particular intended size limitation, unless a particular size is otherwise stated. As used herein, "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. According to the present invention, 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. As used herein, "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. α-amino acids that are rare or do not occur in proteins. Thus, the primary structure of a ligand is limited only by functionality. It was unexpected and surprising to discover that a composition having at least 2 ligands comprising a peptide with an amino acid sequence of SEQ ID N0:1 and peptides substantially homologous thereto are preferentially targeted to T cells rather than to B cells. This result is surprising because in copending U.S. Patent Application Serial No. 08/305,770, filed September 13, 1994, compositions comprising a CR2- receptor-binding and endocytosis-inducing ligand (CBEL) , i.e. SEQ ID NO:1, coupled to a chemical agent, i.e. ricin A, were specifically delivered intracellularly into B cells, but not T cells. Without being limited to any particular theory of operation, it appears that coupling the ligand to a spacer peptide, e.g. a peptide having the sequence of SEQ ID NO:4, modifies the specificity of the ligand such that the composition binds preferentially to T cells and is delivered intracellularly into such T cells. Alternatively, 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 . In in vi tro experiments, 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. As used herein, " acromolecule" means a composition comprising a water soluble polymer with at least 2 ligands and a chemical agent bound thereto. Preferably 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. As used herein, "prodrug" means a chemical agent that is chemically modified to overcome a biological barrier. 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) . The instant 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. As used herein, "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) , 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. Thus, chemical agents include cytotoxins, gene regulators, transforming nucleic acids, labels, antigens, drugs, and the like. As used herein, "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. As used herein, "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. Hudecz et al., Carrier design: Cytotoxicity and Immunogenicitv of Synthetic Branched Polypeptides with Poly(L-lysine) Backbone. 19 J. Controlled Release 231 (1992) ; Z. Brich et al . , Preparation and Characterization o_ a Water Soluble Dextran Immunoconjugate g_f. DoxQrub cjn and. the Monoclonal Antibody (ABL364) . 19 J. Controlled Release 245 (1992) . Thus, 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. As used herein, "effective amount" is an amount that produces a selected effect. For example, 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. The 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-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; 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; and b is an integer of at least 1. Preferably, a is an integer of 2 to about 1000, and 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) . Thus, according to the invention, 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. Accordingly, for example, 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. In some embodiments, the 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, as that term is used herein, means covalently bonding the ligand and chemical agent to the polymer by way of a spacer moiety. In particular embodiments, 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. Such 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. In the latter case, 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. This application employs, except where otherwise indicated, standard techniques for manipulation of peptides and for manipulation of nucleic acids for expression of peptides. Techniques for conjugation of oligopeptides and oligonucleotides are known in the art, and are described for example in T. Zhu et al . , 3 Antisense Res. Dev. 265 (1993) ; T. Zhu et al . , 89 Proc . Nat'l Acad. Sci . USA 7934 (1992) ; P. Rigaudy et al . , 49 Cancer Res. 1836 (1989) . As is noted above, 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. 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 endonucleases and the like, are widely known in the art and commercially available from any of several vendors. Construction of 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. Example 1 In this example, 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. Two HPMA copolymer-adriamycin compositions were employed in this example, namely a ligand-containing composition and a control composition lacking a ligand. Synthesis of HPMA is described in 70 Angew. Makromol. Chem. 109 (1978) ; N. Krinick et al . , Synthesis of N- (2- Hydroxypropyl)Methacrylamide Copolymer-Anti-Thy 1.2 Antibody-Chlorin e. Conjugates and a Preliminary Study of Their Pho oriynamic Effect on Mouse Splenocγtes in vi tro . 191 Makrol. Chem. 839 (1990) ; U.S. Patent No. 5,037,883; U.S. Patent No. 5,258,453; N.L. Krinick, Combination Polymeric Drugs as Anticancer Agents (Doctoral Dissertation, University of Utah, 1992) , hereby incorporated by reference . 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. Copolymerization 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. The ratio of monomers to initiator and solvent was 12.5:0.6:86.9 by weight. The polymerization mixture was 618 21 poured into an ampoule, bubbled with nitrogen, and sealed. After the polymerization, the precipitated polymer was collected by filtration, washed with acetone, and reprecipitated from methanolic solution (20 wt %) into an excess of acetone. The composition of the polymerization mixtures for polymer precursor HPMA copolymer-Gly-Phe-Leu-Gly-ONp (SEQ ID NO:4) 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. Characterization of Polymeric Precursors The content of reactive p-nitrophenoxy groups (ONp) was determined spectrophotometrically using e 75nm = 9500 [1 mol^cm 1 ] in DMSO. 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 Characterization of Polymeric Precursors HPMA-Gly-Phe-Leu-Gly-ONp % yield of 62% polymerization mol % of ONp groups 6.2% g polymer/mol ONp 2740 g mol ONp/g polymer 3.6 X 10 "4 M w a 17000 „/M n 1.3 J D H nm (diameter) 6.9 Determined after aminolysis of ONp groups by l-amino-2-propanol . b Conjugate diameter as determined by dynamic light scattering. Binding of Ligands to Polymeric Precursors 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. Table 2 Composition No. Ligand Adriamycin A a t % mmol/g wt % mmol/g 177 6.0 0.06 8.0 0.14 0.764 237A - - 8.7 0.16 0.832 Absorbance at 488 nm; 1 cm; 0.5 mg/ml The structures of the compositions represented in Table 2 are as follows: (Gly-Phe-Leu-Gly-L) 2 \ (Gly-Phe-Leu-Gly-ADR) (No. 177; SEQ ID NO:4) P-Gly-Phe-Leu-Gly-ADR (No. 237A; SEQ ID N0:4) In these structures, "P" represents the HPMA copolymer backbone, "ADR" represents adriamycin, "L" represents the ligand SEQ ID NO:l, and "Gly-Phe-Leu-Gly" (SEQ ID NO:4) is a spacer susceptible to proteolytic degradation. By way of example, 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 μmol ONp) was dissolved in 0.5 ml anhydrous dimethylformamide (DMF) and 14.0 mg (24 μmol) of adriamycin hydrochloride (solid) and a solution of 12.7 mg (12 μmol) ligand (SEQ ID NO:l) in 100 μl DMF were added. Then, 27 μl of a diluted solution of triethylamine (1:1) in DMF (96 μmol, 9.8 mg) was added in three portions within 30 min. The reaction mixture was stirred for 6 h at room temperature, then 10 μl 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 content of adriamycin was determined spectrophotometrically using e = 10,000 M^cm "1 (water, 488 nm) . The content of bound ligand was determined by amino acid analysis after acid hydrolysis. 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. Example 2 The in vi tro effects of 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. Thereafter, cell viability was assessed by a colorimetric method using the tetrazolium compound MTS ( 3 - ( 4 , 5 - dime t hyl t hi a z o l - 2 - yl ) - 5 - ( 3 - carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium, inner salt) and an electron coupling reagent, PMS (phenazine ethosulfate) . MTS is bioreduced by living cells into a soluble formazan product. 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) . According to this method, 20 μl 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 % cytotoxi ty = (l-_£) x 100 A c wherein 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 μg/ml and 100 μg/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. 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 μg/ml of conjugate there was virtually no cytotoxicity. Only when the concentration was increased to 500 μg/ml was cytotoxicity observed, and then only about 25% of the cells were killed. Example 4 In this example, 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 μg/ml and increasing levels of cytotoxicity at higher conjugate concentrations. The 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. For example, a composition according to the invention can be employed as an effective antitumor agent in vivo for killing T cells. Preferably, 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 τ_ cells Compositions according to the present invention can be administered to a warm-blooded animal for targeted delivery to T cells. Particularly, the composition provides for receptor-mediated internalization of the composition into the T cells. About 1 x 10 7 HSB-2 human T-cell leukemia cells in 500 μl 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. After 48 hours, the mice were injected intraperitoneally with 500 μg of either composition No. 177 or control composition No. 237A in 500 μl of PBS. After an additional 48 hours, 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 . The 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 Cells that were disrupted, washed, and incubated overnight in digestion buffer as described above were twice extracted with phenol/chloroform/isoamylalcohol . The DNA in the aqueous phase was then precipitated with ethanol, washed, dried, and resuspended in a buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM EDTA. PCR of cDNA and. Generi Ω 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. Briefly, PCR reactions were carried out in glass capillary tubes in 10 μl volumes containing 0.8 mM of each of the four deoxynucleotide triphosphates, 2.5 μCi 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 μg, 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 β-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) . A person of ordinary skill in the art can optimize the annealing temperature according to known principles. 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. After amplification, the 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. 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 β-actin and human β-actin specific primers. Illustrative mouse β-actin primers are as follows : GTAACAATGC CATGTTCAAT (SEQ ID NO:5) CTCCATCGTG GGCCGCTCTA G (SEQ ID NO: 6) Illustrative human β-actin primers are as follows: CTTAGTTGCG TTACACCCTT TC (SEQ ID NO:7) GGGCCATTCT CCTTAGAGAG AAG (SEQ ID NO: 8) The results of this experiment are presented in Table 3. Table 3 Treatment Dose Human Mouse primers primers 237A 0.5 mg/ml 12/12 12/12 177 0.5 mg/ml 3/12 12/12 These results show that all of the mice treated with the control composition no. 237A exhibited the presence of both human and mouse DNA by PCR analysis with specific β-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 6 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 β- 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 μl 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 μg of either composition no. 237A (group 3) or composition no. 177 (group 4) in 500 μl of PBS, and these injections were repeated on each of the next two days . Within 65 days after injection of the tumor cells, all of the animals in groups 2 and 3 had died, whereas all the animals in groups 1 and 4 were still alive at 90 days after injection. These results show that a 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. Example 8 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. Such composition can be made, for example, as shown above in Example 1. An effective amount of the composition is systemically administered to the individual such that the composition enters the bloodstream and contacts T cells. 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.) are thought to recognize distinct epitopes of the B-cell CR2 receptor that are localized at the amino-terminal portion of the molecule. J.C Carel et al . , 265 J. Biol . Chem. 12293 (1990) . 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. , 170 J. Exp. Med. 1931 (1989) . 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. Moreover, the presence of 0KB7 antibodies has little, if any, effect on uptake and cytotoxicity in Raji B cells. This results is consistent with the results of J.A. Hedrick et al., supra . Therefore, these results are consistent with 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 10 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. These results are consistent with compositions according to the present invention, such as composition no. 177, being capable of efficiently binding to T cells via a 618 37 receptor other than the CR2 receptor, being internalized in such cells, and exhibiting their effects inside the cells. Sequence Listing (1) GENERAL INFORMATION: (I) APPLICANT: Ramesh K. Prakash, Jindrich Kopecek, Pavla Kopeckova, Vladimir G. O elyanenko, (ii) TITLE OF INVENTION: TARGETING MACROMOLECULAR PRODRUGS TO T LYMPHOCYTES (iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Thorpe, North & Western, L.L.P. (B) STREET: 9035 South 700 East, Suite 200 (C) CITY: Sandy (D) STATE: Utah (E) COUNTRY: USA (F) ZIP: 84070 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 3.5 inch, 1.44 Mb storage (B) COMPUTER: AST Ascentia 900 N (C) OPERATING SYSTEM: DOS 6.22 (D) SOFTWARE: Word Perfect 6.0 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Alan J. Howarth (B) REGISTRATION NUMBER: 36,553 (C) REFERENCE/DOCKET NUMBER: T3216 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (801)566-6633 (B) TELEFAX: (801)566-0750 (2) INFORMATION FOR SEQ ID NO:l: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 Glu Asp Pro Gly Phe Phe Asn Val Glu 1 5 (2) INFORMATION FOR SEQ ID NO:2: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 : Glu Asp Pro Gly Lys Asn Leu Tyr Asn Val Glu 1 5 10 (2) INFORMATION FOR SEQ ID NO:3: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 Glu Asp Pro Gly (2) INFORMATION FOR SEQ ID NO:4: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : Gly Phe Leu Gly (2) INFORMATION FOR SEQ ID NO:5: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5 : GTAACAATGC CATGTTCAAT 20 (2) INFORMATION FOR SEQ ID NO:6: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6 CTCCATCGTG GGCCGCTCTA G 21 (2) INFORMATION FOR SEQ ID NO:7: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7 : CTTAGTTGCG TTACACCCTT TC 22 (2) INFORMATION FOR SEQ ID NO: 8: (I) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 : GGGCCATTCT CCTTAGAGAG AAG 23

Claims: Claims We claim: 1. A composition for intracellular delivery of a chemical agent capable of eliciting a selected effect when delivered intracellularly into a T lymphocyte, said composition having the formula: [L-S] a -C- [S-A] b wherein L is a ligand capable of binding to a receptor on said T lymphocyte and stimulating receptor-mediated endocytosis of said composition; A is said chemical agent; S is a spacer; C is a water soluble polymer having functional groups compatible with forming covalent bonds with said ligand, chemical agent, and spacer; a is an integer of at least 2; and b is an integer of at least 1. 2. The composition of claim 1 wherein C is an HPMA copolymer. 3. The composition of claim 2 wherein said ligand is a member selected from the group consisting of a peptide with an amino acid sequence identified as SEQ ID NO:l and peptides substantially homologous thereto. 4. The composition of claim 3 wherein said chemical agent is selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs. 5. The composition of claim 4 wherein said spacer is biodegradable. 6. The composition of claim 5 wherein said spacer comprises a peptide. 7. The composition of claim 6 wherein said spacer comprises Gly-Phe-Leu-Gly (SEQ ID NO:4) . 8. The composition of claim 7 wherein said ligand is the peptide having the sequence identified herein as SEQ ID N0:1 and said chemical agent is adriamycin. 9. The composition of claim 4 further comprising a carrier selected from the group consisting of water soluble polymers, liposomes, and particulates . 10. The composition of claim 9 wherein said carrier is a water soluble polymer selected from the group consisting of dextran, inulin, poly(L-lysine) with modified epsilon amino groups, poly(L-glutamic acid) , and N-substituted methacrylamide-containing polymers. 11. A method of delivering a chemical agent in vi tro into a T lyphocyte in a heterogeneous population of cells, comprising the steps of: (a) providing a composition for intracellular delivery of a chemical agent capable of eliciting a selected effect when delivered intracellularly into a T lymphocyte, said composition having the formula: [L-S] a -C-[S-A] b wherein L is a ligand capable of binding to a receptor on said T lymphocyte and stimulating receptor-mediated endocytosis of said composition; A is said chemical agent; S is a spacer; C is a water soluble polymer having functional groups compatible with forming covalent bonds with said ligand, chemical agent, and spacer; a is an integer of at least 2; and b is an integer of at least 1; and (b) contacting said population of cells with an effective amount of said composition under conditions wherein said ligand binds to said receptor on T lymphocytes and elicits endocytosis of said composition. 12. The method of claim 11 wherein C is an HPMA copolymer. 13. The method of claim 12 wherein said ligand is a member selected from the group consisting of a peptide with an amino acid sequence identified as SEQ ID NO:l and peptides substantially homologous thereto. 14. The method of claim 13 wherein said chemical agent is selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs. 15. The method of claim 14 wherein said spacer is biodegradable. 16. The method of claim 15 wherein said spacer comprises a peptide. 17. The method of claim 16 wherein said spacer comprises Gly-Phe-Leu-Gly (SEQ ID N0:4) . 18. The method of claim 17 wherein said ligand is the peptide having the sequence given herein as SEQ ID N0:1 and said chemical agent is adriamycin. 19. The method of claim 14 further comprising a carrier selected from the group consisting of water soluble polymers, liposomes, and particulates. 20. The method of claim 19 wherein said carrier is a water soluble polymer selected from the group consisting of dextran, inulin, poly(L-lysine) with modified epsilon amino groups, poly(L-glutamic acid) , and N-substituted methacrylamide-containing polymers. 21. A method of delivering a chemical agent intracellularly into a T lymphocyte in a warm-blooded animal, comprising the steps of: (a) providing a composition for intracellular delivery of a chemical agent capable of eliciting a selected effect when delivered intracellularly into a T lymphocyte, said composition having the formula: [L-S] a -C- [S-A] b wherein L is a ligand capable of binding to a receptor on said T lymphocyte and stimulating receptor-mediated endocytosis of said composition; A is said chemical agent; S is a spacer; C is a water soluble polymer having functional groups compatible with forming covalent bonds with said ligand, chemical agent, and spacer; a is an integer of at least 2; and b is an integer of at least 1; and (b) systemically administering to said warm¬ blooded animal an effective amount of said composition under conditions wherein said ligand contacts and binds to said receptor on T lymphocyte and elicits endocytosis of said composition. 22. The method of claim 21 wherein C is an HPMA copolymer. 23. The method of claim 22 wherein said ligand is a member of the group consisting of a peptide with an amino acid sequence identified as SEQ ID NO: 1 and peptides substantially homologous thereto. 24. The method of claim 23 wherein said chemical agent is selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs. 25. The method of claim 24 wherein said spacer is biodegradable. 26. The method of claim 25 wherein said spacer comprises a peptide. 27. The method of claim 26 wherein said spacer comprises Gly-Phe-Leu-Gly (SEQ ID N0:4) . 28. The method of claim 27 wherein said ligand is the peptide having the sequence identified herein as SEQ ID N0:1 and said chemical agent is adriamycin. 29. The method of claim 24 wherein further comprising a carrier selected from the group consisting of water soluble polymers, liposomes, and particulates. 30. The method of claim 29 wherein said carrier is a water soluble polymer selected from the group consisting of dextran, inulin, poly(L-lysine) with modified epsilon amino groups, poly(L-glutamic acid) , and N-substituted methacrylamide-containing polymers.

Inventors: KOPECEK JINDRICH, KOPECKOVA PAVLA, OMELYANENKO VLADIMIR, PRAKASH RAMESH K, KOPECEK, JINDRICH, KOPECKOVA, PAVLA, OMELYANENKO, VLADIMIR, PRAKASH, RAMESH, K.
Applicants: THERATECH INC, UNIV UTAH RES FOUND, THERATECH, INC., UNIVERSITY OF UTAH RESEARCH FOUNDATION
Application year: 1997
Main classification: A61K39
IPC codes: A61K9, A61K31, A61K47, C07K14, C07K16