CELL INTERNALIZABLE CONJUGATES AND COMPLEXES INCLUDING INTRACELLULARLY CLEAVABLE MOIETIES

Abstract: Cell internalizable and intracellularly cleavable conjugates and complexes of therapeutically or diagnostically effective moieties are described. A novel system for delivery of such moieties is provided.

Description: CELL INTERNALIZABLE CONJUGATES AND COMPLEXES INCLUDING INTRACELLULARLY CLEAVABLE MOIETIES CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation-in-part of application Serial No. 07/523,334 filed May 14, 1990. This application is also a continuation-in- part of application Serial No. 07/635,084, filed December 28, 1990, which is a continuation-in-part of application Serial No. 576,084, filed August 30, 1990, which is a continuation of application Serial No. 210,594, filed June 23, 1988, now abandoned, and a continuation-in-part of application Serial No. 367,751 filed June 21, 1989, all of which are incorporated herein by reference. TECHNICAL FIELD This invention relates to cell internalizable, intracellularly cleavable compositions. More particularly, the invention relates to intracellularly cleavable peptide-toxin conjugates, to complexes for delivery of such conjugates to target cells, and to the therapeutic and diagnostic use of such conjugates and complexes. Such uses frequently entail intracellular cleavage of the toxin from peptide-toxin conjugate. BACKGROUND OF THE INVENTION Practical therapeutic and diagnostic uses of many cytotoxic and che otherapeutic compounds including ribozymes and anti-sense oligonucleotides await the development of improved cell delivery systems. Liposomal and biodegradable microsphere bloodstream delivery systems have been proposed for cancer chemotherapeutics and antifungal agents such as Amphotericin B. Limitations on these delivery systems include unduly rapid release of the encapsulated drug and insipid uptake of blood circulating liposomes by circulating and fixed macrophages of the reticuloendothelial system. SUMMARY OF THE INVENTION This invention provides cell internalizable and intracellularly cleavable conjugates of diagnostically or therapeutically useful ligands. In its more broad aspects, the invention provides conjugates which may have the formula E-Z-Q in which E is an effector moiety, preferably a toxin, Z is an intracellularly cleavable linkage and Q is any organic moiety. Figure 4 illustrates a conjugate having an adriamycin effector, E, a disulfide cleavable linkage Z and a peptide moiety Q. The invention includes complexes of the E-Z-Q conjugates with a moiety A which may facilitate delivery of the conjugates to and internalization of the entire complex or of the conjugate by target cells. Such complexes may have the formula (E-Z-Q) A in which A may, for example be a discrete molecule or an MHC glycoprotein molecule which includes a protein forming the Q component of a conjugate. Such complexes are referenced and illustrated, for example, by Figures 8 to 21. Figure 23 illustrates one series of events related to the invention. Toxin is conjugated to cognate peptide via a cleavable disulfide link. The cleavable conjugate is complexed with the relevant MHC II molecule (IA k in the examples described hereinafter) . The MHC II-(peptide toxin) complex is incubated with the appropriate T cell as shown in step 1. As shown in step 2, only the peptide-toxin moiety of the ternary complex, [MHC II-(peptide- toxin) ]-TCR, on the T cell surface is internalized. The internalized conjugate is then cleaved, for example, by reduction with reduced glutathione. The released toxin pills the cell as shown in step 3. Another aspect of the invention is embodied in the cell internalizable, intracellularly cleavable derivatives of diagnostically and therapeutically useful ligands described in copending application Serial No. 07/523,334. That application explains that "The novel cell internalizable derivatives of the invention may have the formula + NH 2 II L-NH-C-(CH 2 ) 3 -S-S-X in which L is or is included in an intracellularly releasable ligand moiety and X is any organic radical. Preferably, X provides a function to be internalized by or attached to cells. X may enable detection, modify cellular function or serve some other diagnostic or therapeutic purpose." (p. 2, 11. 12-20). These "derivatives" are a subset of the conjugates E-Z-Q, in which "L" corresponds to the effector E and "X" corresponds to the Q moiety of the conjugate. Thus in one aspect the invention is directed to compositions of matter which are E-Z-Q conjugates and (E-Z-A) A complexes. Each component of the conjugate may be associated with or coupled to another conjugate component by, for example, noncovalent or covalent association. These linkages may be effective when Q and A are different parts of the same molecule. Another aspect of the invention comprises methods for preparing the E-Z-Q conjugates and the (E-Z-Q) A complexes. The invention also includes the diagnostic and therapeutic use of the conjugates and complexes of the invention and of compositions which include or contain such conjugates or complexes. The conjugates and complexes of the invention may be parenterally administered as such or as associated with liposomes or biodegradable microspheres. DEFINITIONS Effector—A moiety which modifies cell metabolism upon internalization. Cell Internalizable—Capable of entry into a cell. Intracellular Cleavable Linkage—A linkage that is cleaved upon cell internalization. Conjugate—An effector associated to another chemical moiety by a cell internalizable linkage. Complex—An association of a conjugate with a moiety that facilitates conjugate delivery to or conjugate internalization by a cell. MHC—Major histocompatability complex. Glycoproteins encoded by the MHC have been classified as Class I glycoproteins found on the surfaces of all cells and primarily recognized by cytotoxic T cells and Class II glycoproteins which are found on the surfaces of several cells including accessory cells such as macrophages and are involved in the presentation of antigens to helper T cells. Isolated MHC Component—An MHC glycoprotein or an effective portion of an MHC glycoprotein (i.e., a portion comprising an antigen binding pocket and sequences necessary for recognition by the appropriate T cell receptor) which is in other than its native state, for example, not associated with the cell membrane that normally expresses MHC. IA-^—A protein form the IA subregion of the murine MHC. MBP—Myelin basic protein. AJ1.2}—Murine T cell clones prepared by 4B3.4) immunization of mice against rat MBP 4B3.9} peptide (1-11) and characterized for antigen specificity. Obtained from Dr. Patricia Jones of Stanford University, Palo Alto, California. IA k -MBP Peptide—A conjugate of an IA*^ protein complexed with an MBP peptide fragment. IA k -MBP Peptide-Adriamycin—An IA k protein complexed with an MBP peptide-Adriamycin conjugate. IA-^-OVA-Adriamycin—An IA k protein complexed with an hen egg albumin peptide adriamycin conjugate. Ligand—A moiety which binds to a cell surface receptor. DESCRIPTION OF THE FIGURES Figure 1 illustrates one group of antineoplastic anthraquinones useful as ligands in the compound of the invention. Figure 2 illustrates another group of antineoplastic anthraquinones useful as ligands in the compound of the invention. Figures 3A to 3D set forth the sequences of four peptides AcMBP(1-14)ALA 4 , AcMBP(l-14ALA 4 CYS 14 , AcMBP(1014)ALA 3 ALA ALA 6 , and OVA(3240336)CYS 336 . Figure 4 is the structural formula of an adriamycin-peptide conjugate. Figure 5 is the structural formula of a mycophenolic-peptide conjugate. Figure 6 illustrates the LD50 of AJ1.2 T cells in the presence of free and peptide bound adriamycin. Figure 7 illustrates the killing of AJ 1.2 T cells with IA k -[MBP (1-14)A4-adriamycin] complex. Figure 8 illustrates the killing of 4R3.9 T cells with IA k -[MBP (1-14)A4-adriamycin] complex. Figure 9 illustrates LD50 of AJ1.2 and 4R3.9 cells in the presence of mycophenolic-MBP peptide. Figure 10 illustrates killing of AJ1.2 cells with IA k -[MBP(1-14) )A4-mycophenolic] comple . Figure 11 illustrates internalization by T cells of the peptide moiety "MPB(l-14)Ala4Tyrl4" compared with internalization of the MHC moiety of MHC II-peptide complex radiolabelled in the peptide or MHC II respectively. Figure 12 illustrates the concentration of the toxin alone and IA k [peptide-toxin] conjugate complex required for the killing of T cells iτι vitro. See Figures 5, 7, 9 and 10. Figure 13 illustrates one experimental design for the killing of T cells with IA k (peptide) adriamycin complex. Figure 14 illustrates the stability of IAk-[1-125]MPB(1-14) complex in phosphate buffer or 50% mouse serum at 37 degrees. Figure 15 illustrates the uptake of peptide and MHC II moieties of dual labeled [S-35]IAk-{I-125]MBP(l-14) complex by T cell clone or by T lymphoma cells which have no T cell receptor for MHC II complex. Figure 16 illustrates the azide inhibition of uptake of peptide and MHC II moieties of doubly labeled IAk-MBP(l-14) complex by clone 4R3.9. Figure 17 illustrates the cytochalasin B inhibition of uptake of peptide and MHC II moieties of doubly labeled IAk-MBP(l-14) complex by T cell clone 4R3.9. Figure 18 illustrates the stability of MBP(1-14)-(1-125)Adriamycin conjugate and IAk-[MBP(1-14)-(1-125)Adriamycin] complex in buffer alone. Figure 19 illustrates the stability of MBP(1-14)A4-(1-125)Adriamycin conjugate and IAk-[MBP(l-14)A4-(I-125)Adriamycin] complex in the presence of dithiothreitol (DTT) . Figure 20 illustrates the stability of MBP(1-14)A4-(1-125)Adriamycin conjugate and IAK-[MBP(l-14)A4-(I-l25)Adriamycin] complex in the presence of mouse serum. Figure 21 illustrates the internalized forms of radiolabeled adriamycin. Figure 22A is a bar graph showing elimination of AJ1.2 T cells using a complex containing I-A k and MB (1014)A4 peptide conjugated to adriamycin. Figure 22B shows the results of the same experiment using 4R3.9T cells. In each figure, No. 1 represents proliferation of cells alone, No. 2 represents proliferation of cells incubated with unconjugated complexes, No. 3 represents proliferation of cells incubated with complexes comprising ovalbumin peptide conjugated to adriamycin, and No. 4 represents cells incubated with complexes comprising MBP(1-14)A4 conjugated to adriamycin. Figure 23 illustrates the targeted T cell deletion using cognate MHC II-[peptide toxin] complex. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS This invention provides internalizable conjugates of an effector and another moiety. The conjugates are intracellularly cleavable. Preferably the effectors are toxins. Complexes of these conjugates with moieties that facilitate bloodstream delivery to the target cells and cell internalization are provided. In one preferred embodiment of the invention, only the conjugates are internalized. Thereafter, the toxin is cleaved by the cell from the moiety with which it was associated. A. The Effector Conjugates E-Z-Q The E, Z and Q moieties may be bound covalently, by non-covalent association, or by covalently or non-covalently bound linkers. The selection of the effector component E is determined by the function it is to serve after intracellular cleavage. In general, effectors include, but are not limited to, receptor agonists, receptor antagonists, growth factors, antineoplastic agents such as doxorubicin, peptides that are ligands for natural receptors, poly and monoclonal antibodies, polynucleic acids, low density lipoprotein, α-2-macroglobin, antitoxins, antifungal agents, enzyme inhibitors and effectors useful to provide mucosal tissue retention. The various cancer chemotherapeutic and antifungal ligands are described in the relevant patents and publications. Anthracycline glycoside effectors useful in the invention include anthraquinone structures having one quinone and hydroquinone group on adjacent rings of the anthracene ring structure. Two groups of antineoplastic anthraquinones having these features are illustrated in Figures 1 and 2. Many other compounds of this type, including idarubicin and bromoidarubicin, are described in the prior art. Included in the Figure 1 group are a number of clinically important antineoplastic drugs, such as doxorubicin, daunomycin, carcinomycin, N-acetyladriamycin, N-acetyldaunomycin, rubidasone, idarubicin, bromoidarubicin, and 5-imidodaunomycin. Table I gives the structure variations of several of these drugs, in terms of the R*j_, R2 and R 3 groups in Figure 1. 5-Iminodaunomycin =NH -CO-CH 3 _ NH 2 Drugs in this class are known to have antineoplastic effects against a variety of cancers, including acute leukemia, breast cancer, Hodgkin disease, non-Hodgkin lymphomas and sarcomas. A second group of anthracene glycosides, which are distinguished from the Table I compounds by more complex (multimeric) amino glycoside residues, as seen in Figure 2. These compounds share the same general therapeutic and toxicity properties of their Table I counterparts. Representative anthracene aminoglycosides are listed in Table II, with reference to the R^, R and R 3 groups shown in Figure 2. TABLE II Preferred conjugates include effectors which are toxins, including toxic peptides such as ricin, diptheria, gelonin, pseudomonar toxin and abrin. Oligodeoxynucleotides which destroy cells or mediate cell function such as antisense and ribozyme RNA constitute valuable effector moieties for the conjugates of this invention. Intracellular cleavage of the effectors is preferably achieved by a disulfide linkage, an ester linkage or other cleavable linkage, which joins the effector to the balance of the conjugate. The chemistry for the production of such cleavable linkages is known. Appropriate chemistry can be selected for use with any desired ligand functionality. An appropriate functionality, e.g., a primary amine (-NH2) substituent, an hydroxyl or a carboxyl may be provided in known chemical manner to ligands which lack such functionality. For the purposes of this invention, effectors which have or which have been provided with amine, preferably primary amine, or a carboxyl functionality are preferred. Q (corresponds to X in application Serial No. 07/523,334) is appropriately chosen to facilitate delivery of the conjugate to the target cells. Q (X) moieties may provide a function to be internalized or attached to cells. Q (X) may enable or facilitate detection, i.e., function as a label, modify cellular function or, in addition to E, serve a diagnostic or therapeutic function. Liposomal conjugate delivery is facilitated by an Q (X) moiety which is a lipid thus facilitating anchoring of the conjugate to the liposome bilayer. Intracellular cleavage linkages between E and Q (X) are provided by known chemical procedures. Synthesis of Intracellularly Cleavable Linkages To provide a disulfide linkage, the ligand amine functionality may be reacted with 2,2 / -dithiodipyridine and 2 -iminothiolane in solution in dimethylacetamide to produce an N-(4-pyridyldithiobutyrimido) derivative of the ligand pursuant to Equation I in which E represents any effector. E-NH 2 + H 2 N + = C CH 2 ) 3 SH Compound A Preferably E-NH 2 is an antineoplastic anthraquinone such as doxorubicin of the kind shown by Figure 1 and Figure 2. E-NH 2 may also be amphotericin B, another antifungal agent or a peptide toxin or any other drug having an -NH 2 functionality. The Compound A is utilized to produce a disulfide linked peptide or lipid derivative (conjugate) of the ligand (effector) . For example, the peptide derivative may be produced pursuant to Equation II by reacting Compound A with a mercaptopeptide Q-SH in which Q is any peptide. E- Q-SH Compound A Dimethylacetamide Solvent Equation II + NH 2 II Q-S-S-(CH 2 ) 3 -C-NH-L Compound B Q is preferably a peptide having from about 5 to 50 residues. Q may also be an alkyl group R. R groups having from about 12 to 18 carbon atoms are miscible in liposome bilayers and serve as anchors for minimizing leakage when the derivatives of this invention are administered in the form of liposome delivery systems. Because E is preferably toxic, compounds having the formula of Compound B are toxic to cells when internalized. The toxicity is enhanced by cleavage of the disulfide or other appropriate linkage with consequent internal release of one or both of the toxic moiety. Synthesis of Chemotherapeutic Conjugates EXAMPLE I Synthesis of a Disulfide-Linked Doxorubicin (Adriamycin) Peptide Derivative (See Figure 4) Adriamycin hydrochloride (Aldrich Chemical Co., 10 mg. , 17.2 μmoles) was dissolved in 3.0 ml dry dimethylacetamide containing 10 μl diisopropylethylamine, 2,2 -dithiodipyridine (Aldrich, 50 mg. 227 μmoles) and 2-iminothiolane hydrochloride (Pierce, 10 mg, 72.7 moles) were dissolved together in 1.0 ml and mixed 5 min. on a Vortex mixer. The Adriamycin solution was added dropwise with Vortex mixing. After 6 hrs. at room temperature, the reaction product was purified by reverse-phase HPLC on a C18 column using linear gradient elution (solvent A:0.1% trifluoroacetic acid in H 2 O; solvent B:0.1% trifluoroacetic acid in 70% acetonitrile in H 2 O) . Cleavage of the purified product with ^-mercaptoethanol gave the expected two peaks on HPLC. Reactions: S +NH 2 / II Adriamycin - NH 2 + H 2 N + = C V -y Adria- NH-C-CH 2 CH 2 SH 2-Iminothiolane Adrιa-NH Product N-(4-pyridyldithiobutyrimido)Adriamycin HS-CH2CH20H Adria /3-mercaptoethanol N-(4-mercaptobutyrimido) Adriamycin The HPLC-purified, lyophilized mixed disulfide Adriamycin derivative was dissolved in 0.5 ml degassed H 2 O in a 15 ml polypropylene centrifuge tube. HPLC-pure synthetic mercaptopeptide (1.0 mg) with the structure: 0 .-SH CH 3 C 11-NH-Ala-Ser-Gln-Ala-Arg-Pro-Gln-Arg-His-Gly-Ser-Lys-C rys-0H (A4 peptide sequence) was dissolved in 0.5 ml. degassed H 2 O and added to the mixed disulfide of Adriamycin with Vortex mixing. After 6 hrs. at room temperature, the disulfide-linked peptide-Adriamycin derivative was purified by gradient elution, reverse phase HPLC on a C18 column, as above. Reductive cleavage of the disulfide link with dithiothreitol followed by HPLC analysis, gave the expected starting mercaptopeptide and the 4-mercaptobutyrimido Adriamycin derivative. Reactions: 2-pyridinethione dithiothreitol on peptide-SH+Adriamyc 2 CH 2 CH2-SH N-(4-mercaptobutyrimido)Adriamycin The HPLC-purified disulfide linked peptide-Adriamycin derivative was toxic to cells that internalized the derivative, presumably by reductive cleavage of the disulfide link and internal release of the toxic Adriamycin moiety. EXAMPLE II Synthesis of a Disulfide-Linked Doxorubicin (Adriamycin)-Lipid Derivative The reactions are similar to those described for the disulfide-linked peptide-Adriamycin derivative. The Adriamycin intermediate described in Example I is used: +NH 2 Adriamycin -NH-C-CH 2 CH 2 CH 2 -S-S- Q j + HS-(CH 2 )ι 5 CH 3 N-(4-pyridyldithobutyrimido) N hexadecyl mercaptan Adriamycin 6 hrs. dimethylacetamide solvent Adriamycin-NH N-(4-hexadecy Adriamycin pyridinethion The purified Adriamycin-lipid derivative is incorporated into liposomes by standard techniques. See, generally, Pozansky, M.J., et al. (1984) Pharmacol.Rev. 3^5:277-336. After uptake of the liposomes by cells, the internalized derivative is reductively cleaved by reduced glutathione, and the released, toxic Adriamycin is cytocidal. EXAMPLE III Treatment of AJ1.2 T Cell Clone With Adriamycin and Peptide-Adriamycin Conjugate 10 5 myelin basic protein(MBP)-specific T cells (clone AJ1.2, obtained from Dr. Patricia Jones, Stanford University) were incubated with adriamycin or intracellularly cleavable AcMBP(l-14)cys 1 -S-S-adriamycin conjugate as described in Example I for 24 hours at 37°. The cells were washed and plated in microtiter plates with rlL-2 (5 units/ml) . After 72 hours, cell survival as measured by cell proliferation was determined by the MTT colorimetric assay (Mossmann, T., Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, J. Immunol. Methods 65:55 (1983)). The data (absorbance at 650 nm from the MTT assay) are shown with the corresponding concentrations of adriamycin or adriamycin conjugate in the cell incubation medium. The LD 50 data, obtained from these data, are depicted by Figure 6. LD5 0 values: 0.25μMolar(Adriamycin) and 5.0μMolar[MBP(l-14)- Adriamycin] EXAMPLE IV Synthesis of Mycophenolic-Peptide Conjugate (See Figure 5) Mycophenolic acid (Sigma Chemical Co., St. Louis, MO, 32 mg, 100 μmoles) and bromoacetic acid N-hydroxysuccinimide ester (Sigma Chemical Co. , 12 mg, 50 μmoles) were dissolved in 200 μl of dry dimethylsulfoxide (DMSO, Sigma Chemical Co.) containing 9 μl diisopropylethylamine (DIEA, Aldrich chemical Co., 50 μmoles). Reactions: N- N-hydroxysuccinimidyl mycophenoloxyacetate After standing overnight at room temperature, the solution was added with vortex mixing to a solution of 5 mg (2.5 μmoles) of the peptide: NH 2 CH 3 CO-Ala-Ser-Gln-Ala-Arg-Pro-Ser-Gin-Arg-His-Gly-Ser-Lys-Tyr- in 100 μl DMSO containing 50 μl DIEA (278 μmoles) . After 2 hours at room temperature, the mycophenoloxyacetylated peptide was precipitated by addition of 50 volumes of dry ethyl acetate (Aldrich Chemical Co.). The peptide derivative was collected by centrifugation, washed several times with ethyl acetate and dried. The dried residue was dissolved in 0.1% TFA and immediately applied to a preparative C18 reverse-phase column. Gradient elution HPLC was used to obtain pure derivative (solvent A: 0.1% aqueous TFA; solvent B: 0.1% TFA in 70% aqueous acetonitrile) . The structure of the purified mycophenoloxyacetylated peptide derivative (attachment at the N e -amino group of Lys) : CH 3 CONH-Ala-Ser-Gln-Ala-Arg-Pro-Ser-Gln-Arg-His-Gly- Ser-Lys-Tyr-OH I NH 2 was verified by mass spectroscopy. The HPLC-pure peptide-mycophenolic acid derivative was toxic to cells that internalized the derivative, presumably via mtracellular enzymatic hydrolysis of the ester linkage with release of the toxic mycophenolic acid moiety. Reactions: peptide-NH2 + N-0-C-CH 2 -0-C peptide-NH- CH 3 Figure 9 depicts the LD 50 of AJ1.2 and 4R3.9 T cells in the presence of this mycophenolic MBP peptide. The results presented in Figure 10 show that proliferation of cells (as an indication of cell survival) incubated with IA k -MBP(l-14)A4-mycophenolate complexes (No. 3) was significantly less than that of cells alone (No. 1) or cells incubated with unconjugated complexes (No. 2) . The results further demonstrate the ability of complexes of the present invention to specifically eliminate particular T cell clones. Synthesis of Peptide-Oligonucleotide Conjugates Synthetic oligodeoxyribonucleotides and oligoribonucleotides can be synthesized in known manner to contain an amino group at the 5 ' end, using standard automated synthesis with addition of commercially available amine or thiol linkers (see linkers on reagent list from MilliGen/Biosearch, Burlington, MA) as the last step of the synthesis. Upon completion of synthesis, the thiol linker is deprotected to yield the thiol directly. The thiol group is then converted to the pyridyldithio derivative by reaction with an excess of 2,2'-dithiodipyridient (Aldrithiol, Aldrich Chemical Co. , Inc. , Milwaukee, WI) , as shown below. Preparation and Use of Thiolinker-Containing Oligonucleotides ' Solid Trityl-Propylthiol Linker (Milligen couple 1, oxidize 2, deprotect OCH 2 CH 2 CH 2 -SH 0 = 1 O 0 Base \ / \/ / O-OLIGO — OH (Aldrithiol) M, O -Peptide O Alternatively, a deprotected amine linker on the 5' end of the synthetic oligo may be converted directly to a pyridyldithio derivative using N-succinimidyl-3-(2-pyiridyldithio)propionate (SPDP, Sigma Chemical Co. , St. Louis, MO) as described by Gaur et al., A simple method for the introduction of thiol group at 5'-termini of oligodeoxynucleotides, Nucleic Acids Research r7(ll):4404 (1989) and shown below: Mercaptans, including cysteine-containing peptides, can then be covalently attached to the linker-containing oligonucleotides by unsymmetrical disulfide exchange as described in Carlsson et al., Protein Thiolation and Reversible Protein-Protein Conjugation, Biochem. J. 173:723-737 (1978) and also as shown below. Preparation and Use of 5 ' Amine Linker-Containing Oligonucleotides r 1. oxidize 2 . deprotect NU O-OLIGO—OH 3' -SH CH2S-S-Peptide o = O-OLIGO—OH 3' In this way, oligonucleotides including antisense oligodeoxyribonucleotides and ribozymes may be conjugated to peptides, e.g., T cell epitopes. The Complexes The complexes are represented by the formula (E-Z-Q) A in which E-Z-Q is a conjugate as previously described and A is a moiety which facilitates delivery of the conjugate to and internalization of the conjugate by target cells. Q and A may either be discrete or component parts of the same molecule. Preferred A moieties include ligands which are internalized after binding to their cell surface receptors, e.g., MHC II molecules, growth factors such as epidermal growth factor, transferrin and transcobalamin, cytokines such as IL-1, IL-2, IL-4, IL-6, and TGFα. The E-Z-Q conjugates may be associated or coupled to an A moiety by, for example, non-covalent covalent association or covalent binding. The Q moiety of the E-Z-Q conjugates may be a discrete moiety which is inserted into or added to a binding site on an A component of a complex. Alternatively, the Q moiety may be derived from and reinserted, after modification if desired, into the A moiety of the complexes of the invention. Thus, a toxic peptide may be synthesized in known manner to provide a Q component of a conjugate which is then associated noncovalently or covalently with a cognitive A component. Alternatively, a peptide moiety may be separated from an MHC molecule and utilized as a conjugate Q moiety or a different peptide may be synthesized which can be used to replace the original peptide moiety in the MHC molecule. Thus another aspect of the invention includes conjugates which may have the formula E-Z-B in which B is a portion or a component of an A moiety of an (E-Z-B) A conjugate complex. The formation of the complexes of such E-Z-B conjugates entails, in effect, a recombination or replacement of the B conjugate moiety with the residual component of the A moiety from which it was derived or with which it was to associate. As noted, conjugates and complexes based on MHC molecules provide an example. MHC molecules include peptide components which may be separated or isolated in known manner, modified if desired, and then used as Q moieties of the conjugates of the invention. Alternatively synthetic peptides which combine with the residual MHC molecule or which bind to empty peptide pockets in MHC molecules may be synthesized and used as Q moieties. Complexes are formed by combining or recombining such peptides, attached to the E-Z component of a conjugate with the residue of the MHC molecule from which it was derived or with an empty pocket of an MHC molecule. The formation of such complexes may be illustrated by the following schematic: 1. Providing an MHC molecule M having a free peptide binding site. 2. Forming an E-Z-B conjugate in which B is a peptide which binds to a free binding site on said MHC molecule M. 3. Forming a complex E-Z-B(M) by combining the E-Z-B conjugate so formed with the MHC molecule M. In general, conjugate-complexes may be prepared by simple incubation if a moiety of the conjugate has affinity for the Q component of the complex. For example, the peptide moiety of a peptide-toxin conjugate can bind noncovalently to an appropriate MHC II molecule by affinity interaction. Alternatively, the conjugate may be covalently attached via amide bonds formed by the action of carbodiimide-mediated dehydration. This is a standard way to couple components containing carboxyl and amine groups. As Figure 23 illustrates, this invention includes the important discovery that administration of E-Z-B(M) complexes results in cell internalization of only the E-Z-B moiety of the complex. Complexes of this type thus provide outstanding and unexpectedly effective delivery systems for effectors of all kinds. See Figure 23. For example, the conjugates including a chemotherapeutic effector may be associated in known manner, e.g., by incubation, with a selected A moiety to provide complexes of the invention. The previously described peptide-oligonucleotide conjugate may be complexed with an appropriate MHC II molecule and incubated with T cells bearing T cell receptors that recognize the peptide only when bound to the appropriate or "restricting" MHC II allele. As shown in ensuing data and examples, peptides and peptide-toxin derivatives are internalized by those T cells that specifically bind the "cognate" MHC II-(peptide-X) complex where X is any chemical moiety conjugated to the cognate peptide. Thus, antisense deoxyribonucleotide analogs and ribozymes may be specifically targeted to, e.g., autoreactive T cells which are thereby inactivated or deleted. Similar reactions may be executed with thiol groups introduced at the 3' end of the oligonucleotide, using technology described in Zuckerman et al., Efficient methods for attachment of thiol specific probes to the 3'-ends of synthetic oligodeoxyribonucleotides, Nucleic Acids Research 15(13) :5305-5321 (1987). Antisense nucleotides may also be attached by the same reaction to sulfhydryl-containing protein ligands of cell surface receptors to give intracellularly cleavable conjugates which are internalized when ligand moiety is bound to its receptor. An example of this is IL-2 which when bound to IL-2 receptor on activated T cells is internalized. Thus, activated T cells may be inactivated by IL-2-targeting an antisense nucleotide to the gene for the IL-2 receptor, using an intracellularly cleavable IL-2-anti-IL-2R nucleotide conjugate. Preparation of IA-^-fPeptide(1-125)Adriamycin] Complex MBP(1-14)A 4 -(125)Adriamycin conjugate was prepared as follows: To 250 ul solution of IA k (0.8 mg/ml) in PBS containing 0.5% octylglucopyranoside (OG) and 0.02% sodium azide at pH 7.5, 50 ul of freshly prepared PBS solution of [MBP(1-14)A -(1-125)Adriamycin] conjugate (6 mg/ml, 50-fold molar excess over IA k ) was added, and the resulting solution was incubated at 37°C with gentle mixing for 48 hours. The solution was then dialyzed against 1 liter PBS, pH 7.4 at 4°C using 8000-12000 molecular weight cut off dialysis membrane. The dialysis was continued for 48 hours with buffer change at 6 hours and 24 hours. The dialysed sample was removed from dialysis just before it was used to mix with the T cells. In some experiments, the complex was dialyzed further against RPMI culture medium. The same method was used for the preparation of lA k -[MBP(1-14)A 3 A 4 A 6 -(1-125)Adriamycin] complex. All IA -peptide complexes referenced in the Examples were prepared exactly the same way. EXAMPLE V The lA k -[1-125]MBP(1-14)A 4 complex (27.5% peptide loading of the lA k ) was incubated at 9.6 μg/ml in phosphate buffer, pH7.2, or 50% normal AJ mouse serum in phosphate buffer at 37° for the indicated times. Equal aliquots of the incubated samples were subjected to silica gel TLC. After development, the dried spots at the origin corresponding to MHC-peptide complex were excised and counted in a gamma counter. The radioactivity value obtained at zero time was chosen to correspond to 100% intact complex. The stability of this complex under these conditions is depicted by Figure 14. EXAMPLE VI MBP(1-14)A 4 analog was labeled with 125 I by the standard chloramine T method. Affinity-purified IA k was labeled with (S-35)BocMethionine by the supplier's recommended procedure (Amersham Corporation) . Doubly labeled [S-35]IA k -[I-125]MBP(l-14)A 4 complex 8.8 x 10 5 cpm/μg for MBP(1-14)A 4 and 1.98 x 10 5 cpm/μg for lA ) was prepared from the labeled components. Doubly labeled complex (1.9 μg, 18% peptide loading) in RPMI was incubated in 1.0 ml serum-free RPMI medium with 5 x 10 5 MBP-specific clone 4R3.9 murine T cells (10 days post-stimulation) or 5 x 10 5 T cell receptor-negative BW5147 murine T lymphoma cells (both obtained from Dr. Patricia Jones, Stanford University) . After incubation for 5 hours at 37°, the cells were washed extensively with RPMI medium, pelleted and counted in a gamma counter for 1-125 radioactivity. The cells were then transferred into vials containing scintillation liquid and counted in a beta counter for S-35 radioactivity. The number of molecules of MBP(1-14)A 4 peptide or lA k taken up by the cells was calculated from the channel-corrected cpm and the specific activity of each moiety of the doubly labeled complex. Aliquots of cells were also incubated with doubly labeled complex in medium containing 0.2% sodium azide or 4μM cytochalasin B. The results show that only the peptide moiety of the T cell-bound MHC II-peptide complex is internalized. This internalization is inhibited by azide but not by cytochalasin B, suggesting an energy-dependent but not microfilament mediated internalization process. Figure 15 illustrates the uptake of peptide and MHC II moieties of dual labeled [S-35]IAk-[I-125]MBP(l-14) complex by T cell clone or T lymphoma. Figure 16 illustrates the azide inhibition of uptake of peptide and MHC II moieties of doubly labeled IAk-MBP(l-14) complex by clone 4R3.9. Figure 17 illustrates the lack of cytochalasin B inhibition of uptake of peptide and MHC II moieties of doubly labeled IAk-MBP(l-14) complex by T cell clone 4R3.9. EXAMPLE VII Serum Stability of MBP(1-14)A 4 -(1-125)Adriamycin Conjugate and IA k -rMBP(l-14)A 4 -(I-125)Adriamycim Complex Preparation of radiolabelling agent Bolton-Hunter reagent (500 mCi, Amersham Corporation, 2636 South Clearbrook Drive, Arlington Heights, IL 60005) was converted to its hydrazide by incubation with excess hydrazine overnight. The excess hydrazine was removed by drying the reaction mixture with argon gas at room temperature. To the dry residue, 1 ml of 0.1 M hydrochloric acid containing 30% sodium chloride, was added and the product was extracted with 14 ml of ethyl acetate. The extract was then treated with 1 ml of 10% sodium bicarbonate containing 20% sodium chloride. The organic layer was separated, and TFA was added to a final concentration of 0.01%. This solution was stored at 4°C until used. Radiolabelling of Peptide-Adriamycin Conjugate Ethyl acetate was removed form the [1-125] hydrazide reagent by evaporation with argon at room temperature. Peptide-adriamycin conjugate was dissolved in acetic acid, and this solution was added to the dry radiolabelling agent and incubated for 6 hours. The excess reagent was then removed by ethyl acetate precipitation of conjugate. The precipitated and dried labeled peptide-(1-125)adriamycin conjugate was redissolved in acetic acid and reprecipitated with ethyl acetate. The precipitate was washed with heptane and dried. The labeled peptide-adriamycin was stored dry at 4°C. Preparation of IAk-rMBP(l-14)A4-(I-125)Adriamycinl Complex The complex was prepared by mixing IA with excess MBP(1-14)A 4 -(1-125)adriamycin conjugate or IAk with MBP(1-14)A 3 A 4 A 6 (1-125)adriamycin at 37°C for 48 hours in PBS. The excess peptide-(1-125)adriamycin conjugate was removed form the complex by extensive dialysis against PBS. Serum Stability Studies Blood was obtained from AJ mouse and allowed to clot at room temperature. The serum was separated by centrifugation. The MBP(1-14)A 4 -(1-125)adriamycin conjugate or IA k [MBP(l-14)A 4 -(1-125)adriamycin] complex were mixed with serum and incubated at 37°C in a humidified incubator with 5% Carbon dioxide. Aliquot samples were subjected to silica Gel TLC to obtain the % cleavage of peptide-(1-125)adriamycin conjugate. Cleavage was also tested in buffer and buffer containing dithiothreitol (DTT) . Figure 18 shows that MBP(1-14)A 4 -(1-125)adriamycin peptide-toxin conjugate is cleaved at the disulfide bond to a small extent in buffer. The disulfide bond is not cleaved in buffer when this peptide-toxin conjugate is complexed with MHC II to form IA k -[MBP(1-14)A 4 -(1-125)adriamycin] complex. Figure 19 shows that when the buffer contains the reducing agent, dithiothreitol, the MBP(1-14)A 4 -(1-125)adriamycin peptide-toxin conjugate is rapidly cleaved extensively. However, the disulfide bond between toxin and peptide when the peptide-toxin is incorporated in complex, i.e., IA k -[MBP(l-14)A 4 -(1-125)adriamycin] complex is much less susceptible to reduction. Figure 20 shows that the disulfide bond between peptide and toxin moieties in the MHC II-[peptide-toxin] complex is completely stable to reduction in serum, whereas the peptide-toxin conjugate is cleaved at the disulfide bond in the presence of serum. Serum contains low concentrations of cysteine and glutathione which can act as reductants. EXAMPLE VIII Specific Internalization and Cleavage of Peptide-Toxin Moiety of MHC Class II-rPeptide-Toxin] Complex This Example shows that the disulfide linker between peptide and adriamycin is cleaved intracellularly following the binding of relevant MHC class II-peptide-adriamycin complex to T cells and internalization of the peptide-toxin moiety. T Cells: IA k -MBP(l-14)A 4 specific T cell clone 4R3.9. Complexes: 1. MBP(l-l4)A 4 -(I-125)Adriamycin, relevant (MBP) peptide-toxin conjugate. 2. MBP(1-14)A 3 A 4 -A 6 -(1-125)Adriamycin, irrelevant (hen albumin) peptide-toxin conjugate. 3. IA k -[MBP(l-14)A 4 -(I-125)Adriamycin] complex, relevant MHC class II-[peptide-toxin] complex recognized by T cell clone. 4. IA -[MBP(l-14)A 3 A 4 -A 6 -(1-125)Adriamycin] , irrelevant MHC class II-[peptide-toxin] complex not recognized by T cell clone. 5. IA k -[MBP(1-14)A 4 -(1-125)Adriamycin] complex + 10 million cells. 6. IA k -[MBP(1-14)A 3 A 4 -A 6 -(1-125)Adriamycin] complex + 10 million cells. The complexes (19,000 CPM) were incubated with or without cells in 0.5 ml RPMI 1640 for 5 hours at 37°C in a humidified incubator with 5% Carbon dioxide. Cells incubated with complex were then washed with 10 ml of PBS three times. To the solutions or cells (in 300 μl) was added, 800 ul of acetonitrile containing 10 μl TCA. The samples were then mixed well and centrifuged in a microcentrifuge at high speed. The supernatant was saved and the pellet was extracted again with 1 ml of 80% aqueous acetonitrile containing 0.1% TCA. The supernatant was then subjected to vacuum centrifugation. The residue was dissolved in 5 μl aqueous acetonitrile and counted. The samples were then applied to Silica gel TLC sheets developed with butanol:acetic acid:water = 100:10:30 upper phase. Figure 21 reflects data concerning forms of radiolabeled adriamycin and adriamycin. Referring to Figure 21, bars 1 and 2 were calculated from the TLC of cell extracts obtained after incubation of the relevant IA k -[MBP(1-14)A 4 -(1-125)Adriamycin] complex with 4R3.9 T cell clones: 1. Free (1-125)adriamycin derivative (Post cleavage structure of the derivative appears at page 14 hereof) 2. MBP(1-14)A 4 -(1-125)Adriamycin conjugate (uncleaved) . Control bars 3 and 4 were calculated from TLC of cell extracts obtained after incubation of the irrelevant complex, IA k -[MBP(1-14)A 3 A 4 A 6 -(1-125)Adriamycin] with 4R3.9 T cell clones: 3. (1-125)Adriamycin derivative (Post cleavage structure of this derivative appears at page 14 hereof) . 4. MBP(1-14)A 3 A -A 6 -(1-125)Adriamycin conjugate (uncleaved) . Figure 21 shows that the T cell clone only internalizes the relevant peptide-toxin conjugate moiety of the cognate MHC II-[peptide-toxin] complex. Furthermore, the internalized peptide-toxin conjugate, MBP(1-14)A 4 -(1-125)adriamycin, is completely cleaved at the disulfide bond to generate free adriamycin derivative (see page 14) intracellularly. EXAMPLE IX T Cell Internalization of MHC II-Peptide Complex To study internalization of the IA k -peptide complex, the MHC II and peptide moieties were separately labeled with 125 I, and two complexes were prepared, each with one or the other of the labeled moieties, and dialyzed extensively against RPMI to remove free peptide. T cells, ten days after antigen stimulation, were then incubated with labeled complex for five hours after which the cells were washed extensively with culture medium. The bound radioactivity was measured by scintigraphy. The cells were then washed with pH 3 buffer at 4° (with no loss of cell viability) to remove complex bound to the cell surface, and the cells were reexamined by scintigraphy. If complex is bound but not internalized, the number of bound complexes per T cell was expected to be less than or equal to the number of T cell receptors on the cell surface (estimated by others to be between 20,000 and 40,000 per cell) . The results indicate that the cognate peptide moiety of the complex is internalized preferentially over the IA k moiety. The internalization is specific for the cognate peptide, since control, noncognate peptide complexed with IA k is much less preferentially internalized. The experiment was repeated several times with the same result. In addition, an experiment with doubly labeled MHC Il-peptide complex was performed in which the IA k moiety was radiolabeled with 35 S and the peptide moiety was radiolabeled with 125 I. The results of this experiment shown in Figure 15 clearly confirm the previous experiments, i.e., that the peptide moiety of the cognate MHC Il-peptide complex is internalized but the MHC II moiety is not. Figure 22 is a schematic representation of this phenomenon. The uptake process appears to be energy dependent, as shown by azide inhibition at azide concentrations that result in no loss of cell viability. The results of the internalization experiments suggested that small antimetabolite toxins such as Adriamycin and mycophenolic acid have been be attached to peptides which are then complexed with MHC II molecules. The resultant complexes have been be used to specifically target toxins into the cytoplasm of autoreactive T cells involved in autoimmunity. EXAMPLE X Killing of AJ1.2 T Cells With lA k -MBP(1-14)A4Adriamycin Complex 100 ug of affinity-purified IA k (or IA*-) was incubated with 167 ug of respective adriamycin- peptide (fifty-fold molar excess) in 1 ml at 37°C for 48 hours. The excess unbound peptide was removed by extensive dialysis against RPMI media. The dialysed complex was then mixed with 1 x 10 6 AJ 1.2 T cells (1 ml total volume) and incubated at 37°C/C02 for 24 hours. The unbound complex was removed by washing of cells. Treated cells were plated in a 96 well microtiter plate at a cell density of 10 5 cells/well in 200 ul volume and in presence of 5 units/ml final concentration of rIL2. Proliferation was measured after 72 hours by the MTT colorimetric assay. Figure 7 shows that effective killing of AJ1.2T cells was achieved only with the lA k -MBP-Ad complex. The OVA (hen) albumin (peptide) used in the bar 3 experiment is not recognized by AJ1.2T cells. EXAMPLE XI Killing of 4R3.9 T Cells With lA -MBP(1-14)A4Adriamycin Complex 100 ug of affinity-purified IA k (or Ih-) was incubated with 167 ug of respective adriamycin- peptide (fifty-fold molar excess) in 1 ml at 37°C for 48 hours. The excess unbound peptide was removed by extensive dialysis against RPMI media. The dialysed complex was then mixed with 1 x 10 6 4R3.9 T cells (1 ml total volume) and incubated at 37°C/C02 for 24 hours. The unbound complex was removed by washing of cells. Treated cells were plated in a 96 well microtiter plate at a cell density of 10 5 cells/well in 200 ul volume and in presence of 5 units/ml final concentration of rIL2. Proliferation was measured after 72 hours by the MTT colorimetric assay. Figure 8 illustrates that effective killing of 4R3.9 T cells was achieved only with the lAk-MBP(1-14)A4Adriamycin complex. EXAMPLE XII Elimination of Autoreactive T Cell Clones In Vitro Using a Complex 1-A k And MBP Peptide Conjugated to Adriamycin This example demonstrates in vitro deletion of MBP-specific AJ 1.2 and 4R 3.9 T cells using adriamycin conjugated with MBP(1-14)A4 via an intracellularly cleavable linkage. IA k was incubated with a 50-fold molar excess of MBP(1-14)A4-Adriamycin conjugate for 24 hours at 37°C. As an irrelevant control, OVA(324-335)-Adriamycin was similarly complexed with IA d . The nonbound peptide-adriamycin conjugates were removed by extensive dialysis against RPMI medium. AJ 1.2 or 4R 3.9 cells were incubated with IA d or IA k -(peptide-Adriamycin) complex for 16 hours at 37"C. The cells were washed to remove nonbound complex and plated in culture medium containing IL-2 as described above. After 72 hours, cell proliferation was measured by the MTT spectrophotometric method as described above. The peptide-adriamycin conjugates used in the complexes were prepared with an intracellularly cleavable disulfide bond between the peptide and the adriamycin moiety. The preparation of intracellularly cleavable compounds was performed generally as taught in commonly owned copending application Serial No. 07/523,334, fled May 14, 1990, which is incorporated herein by reference. The preparation of the conjugates of the present invention was as follows: Doxorubicin hydrochloride (Aldrich Chemical Co., Milwaukee, WI, 20 mg, 34.4 μmoles) was dissolved in 1 ml dry dimethylsulfoxide (DMSO, Sigma Chemical Co., St. Louis, MO) containing 100 μl collidine (Aldrich Chemical Co., 757 μmoles). 2-Iminothiolane (Pierce Chemical Co. , Rockford, IL, 10 mg, 72 μmoles) and 2,2-dithiodipyridine (Aldrich Chemical Co., 75 mg, 340 μmoles) were dissolved in 1 ml dry dimethylsulfoxide. The latter solution was added dropwise with stirring on a Vortex mixer to the doxorubicin solution. After 6 hours at room temperature and in the dark, the reaction mixture was directly purified by reverse-phase HPLC on a preparative C18 column using linear gradient elution (solvent A: 0.1% aqueous trifluoroacetic (TFA); solvent B: 0.1% TFA in 70% aqueous acetonitrile). Cleavage of the purified product with excess 9-merceptoethanol gave the expected products by HPLC analysis. Mass spectroscopy of the purified product confirmed the expected structure. The HPLC-purified, lyophilized adriamycin-mixed-disulfide derivative (2.3 mg) was dissolved in 0.5 ml degassed water in a 15 ml polypropylene centrifuge tube. HPLC-pure synthetic mercaptopeptide (1 mg) with the structure: SH CH 3 C0NH-Ala-Ser-Gln-Ala-Arg-Pro-Ser-Gln-Arg-His-Gly-Ser-Lys I- Cys-OH was dissolved in 0.5 ml degassed water and added with vortex mixing to the solution of adriamycin-mixed- disulfide. After 6 hours at room temperature in the dark, the disulfide linked peptide-adriamycin derivative was purified by gradient elution reverse-phase PHLC on a preparative C18 column, as described above. Reductive cleavage of the disulfide link with excess dithiothreitol gave the expected mercaptopeptide and the 4-mercaptobutyrimidodoxo- rubicin derivative upon analysis by reverse-phase HPLC. Mass spectroscopy confirmed the structure expected for the peptide-adriamycin derivative. The HPLC-purified disulfide linked peptide-adriamycin derivative was toxic to cells that internalized the derivative, presumably by mtracellular reductive cleavage of the disulfide link with release of the toxic adriamycin moiety. The results of in vitro T cell deletion with IA k -(peptide-Adriamycin) complex are shown in Figures 22A and 22B. About 85% cell deletion of clone AJ 1.2 (No. 4, Figure 22A) and about 50% deletion of the 4R 3.9 clone (No. 4, Figure 22B) was achieved at a dose in humans equivalent to about 120 mg of actual IA k -(peptide-Adriamycin) complex (assuming about 20% loading of the IA k with peptide-Adriamycin) which contains about 1 mg Adriamycin. Adriamycin is administered orally at doses up to 15 mg daily in cancer patients. Formulation and Administration The conjugates and complexes of this invention are conveniently administered as such or in the form of liposomes or miscelles. Methods for preparing such compositions generally follow conventional liposome preparation methods. Unilamellar and multilamellar liposomes formed in conventional manner are useful in the invention. Vesicle forming lipids which generally include neutral and negatively charged phospholipids and a sterol such as cholesterol are appropriate. Vesicles comprising dipalmitoylphosphatidylcholine (DPPC) or distearoylphosphatidylcholine (DSPC) are preferred and may be prepared in known manner. See, e.g., Tomita, T., et al. (1989), Biochim.Biophys. Acta 978:185-190. In one preferred method, vesicle forming lipids are taken up in a suitable organic solvent or solvent system and dried in vacuum or in an inert gas to a lipid film. The derivative is included in the lipids forming the film. The concentration of the derivative in the lipid solution is preferably in molar excess of the final maximum concentration of the drug in the liposome. The dried lipid/drug film is hydrated with a physiologically compatible medium, preferably physiological saline. The lipids hydrate to form a suspension of multilamellar vesicles (MLVs) whose size typically range from about 0.5 microns to at least about 10 microns. In general, the size distribution of MLVs in the above procedures can be shifted toward smaller sizes by hydrating the lipid film more rapidly, with shaking. The liposome suspension may be sized to achieve a selective size distribution of vesicles in a size range less than about 1 micron and preferably between about 0.05 to 0.5 microns, and most preferably between about 0.005 and 0.2 microns. The sizing serves to eliminate larger liposomes and to produce a defined size range having optimal pharmacokinetic properties. Several known techniques are available for reducing the sizes and size heterogeneity of liposomes. Sonicating a liposome suspension either by bath or probe sonication produces a progressive size reduction down to small unilamellar vesicles (SUVs) less than about 0.05 microns in size. A known sonicating procedure is preferably used in reducing liposome sizes to about 0.2 microns or less. Homogenization is another method which relies on shearing energy to fragment large liposomes into smaller ones. In a typical homogenization procedures, MVLs are recirculated through a standard emulsion homogenizer until selected liposome sizes, typically between about 0.1 and 0.5 microns, are observed. In both methods, the particle size distribution can be monitored by conventional laser-beam particle size discrimination. Extrusion of liposomes through a small-pore polycarbonate membrane is an effective method for reducing liposome sizes down to a relatively well-defined size distribution whose average in the range between about 0.1 and 1 micron, depending on the pore size of the membrane. Typically, the suspension is cycled through the membrane several times until the desired liposome size distribution is achieved. The liposomes may be extruded through successively smaller pore membranes, to achieve a gradual reduction in liposome size. Centrifugation and molecular size chromatography are other methods which are available for producing a liposome suspension with particle sizes below a selected threshold less than 1 micron: These two methods both involve preferential removal of larger liposomes, rather than conversion of large particles to smaller ones. Liposome yields are correspondingly reduced. Administration is systemic and is effected by injection, preferably intravenous,thus formulations compatible with the injection route of administration may be used. Suitable formulations are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985) , which is incorporated herein by reference. A variety of pharmaceutical compositions comprising conjugates or complexes of the present invention and pharmaceutically effective carriers can be prepared. The pharmaceutical compositions are suitable in a variety of drug delivery systems. For a brief review of present methods of drug delivery, see, Langer, Science 249:1527-1533 (1990) which is incorporated herein by reference. A dosage level of 10-500 ug for murine subjects is effective; thus about 0.5 mg/kg to 25 mg/kg is suggested. For pharmaceutical compositions which comprise the conjugates or complexes of the present invention, the dose will vary according to, e.g., the particular complex, the manner of administration, the particular disease being treated and its severity, the overall health and condition of the patient, and the judgment of the prescribing physician. The pharmaceutical compositions are intended for parenteral, topical, oral or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered in the variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, and capsules. Preferably, the pharmaceutical compositions are administered intravenously. Thus, this invention provides compositions for intravenous administration which comprise a solution of the complex dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, postassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. The concentration of the conjugate or complex can vary widely, i.e., from less than 0.05%, usually at or at least about 1% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected. As described above, the complexes may be delivered via liposome preparations. For solid compositions, conventional nontoxic solid carriers may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceuti¬ cally acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient. For aerosol administration, the conjugates or complexes are preferably supplied in finely divided form along with a surfactant and propellant. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, lineolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters. Mixed esters, such as mixed or natural glycerides may be employed. The surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%. The balance of the composition is ordinarily propellant. Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure. Among suitable liquefied propellants are the lower alkanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes. Mixtures of the above may also be employed. In producing the aerosol, a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided compounds and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve. The compositions containing the conjugates or complexes can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as "therapeutically effective dose". Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient. In prophylactic applications, compositions containing the conjugate complexes of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an a ount is defined to be a "prophylaσtically effective dose". In this use, the precise amounts again depend on the patient's state of health and weight. For the oral mode of administration, conjugates and complexes of this invention can be used in the form of tablets, capsules, lozenges, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like. In the case of tablets, carriers which can be used include lactose, sodium citrate, and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, certain sweetening and/or flavoring agents can be added. The conjugates of this invention may also be used in diagnostic assays; in this case the amount of the composition used will depend on the sensitivity of the liposome-coupled derivative to the target components in the sample.

Claims: CLAIMS : 1. A cell internalizable, intracellularly cleavable conjugate having the formula E-Z-Q in which E is an effector moiety, Z is an intracellularly cleavable linkage and Q is an organic moiety. 2. A conjugate as defined by claim 1 in which the effector E is a receptor agonist, a receptor antagonist, a growth factor, an antineoplastic agent, an oligonucleotide molecule, an antibody, an antifungal agent, or an inhibitor of enzyme or protein function. 3. A conjugate as defined by claim 1 in which said effector E is a chemotherapeutic agent. 4. A conjugate as defined by claim 1 in which Q is an oligonucleotide molecule. 5. A conjugate as defined by claim 1 in which Q is an antisense molecule or a ribozyme. 6. A conjugate as defined by claim 2 in which Q is a component recognized by a binding site of another organic moiety, said Q component as it is present in said conjugate being combinable with said binding site of said other organic moiety. 7. A conjugate as defined by claim 1 in which said Q component is a peptide and said other organic moiety is an MHC molecule. 8. A conjugate as defined by claim 1 in which Q is a moiety recognized by a noncovalent or covalent binding site of an MHC molecule, an IL-2 molecule, an IL-4 molecule and an IL-6 molecule or a TGFα molecule. 9. A conjugate as defined by claim 1 in which Q is a peptide recognized by a binding site of an MHC molecule. 10. A conjugate as defined by claim 9 in which Q is a synthetic peptide. 11. A conjugate as defined by claim 1 in which Q is a component derived from an organic moiety, said Q component, as present in said conjugate, being recombinable either per se or after modification with the residue of the moiety from which it was derived. 12. A conjugate as defined by claim 11 in which said other organic moiety is an MHC molecule. 13. A conjugate as defined by claim 11 in which Q is a peptide component of an MHC molecule. 14. A conjugate as defined by claim 11 in which Q is a chemically modified peptide component of an MHC molecule. 15. A complex having the formula (E-Z-Q) A in which E-Z-Q is a conjugate as defined by claim 1 and A is any organic moiety. 16. A complex as defined by claim 15 in which A is an organic moiety that facilitates delivery of said E-Z-Q conjugate to and internalization of said conjugate by a target cell. 17. A complex as defined by claim 16 in which A is any ligand which is internalized after binding to its cell receptor surface. 18. A complex as defined by claim 15 in which A is a growth factor, a cytokine, a low density lypoprotein or alpha-2-macroglobulin. 19. A complex as defined by claim 18 in which said growth factor is an epidermal growth factor, transferrin or transcobalamin, and in which said cytokine is IL-1, IL-2, IL-4, IL-6 or TGFα. 20. A complex as defined by claim 15 in which the Q component of said E-Z-Q conjugate is or may also be a component of the moiety of said complex, said Q component, as present in said conjugate, being combinable with the portion of said A moiety of which it is or may be a component. 21. A complex as defined by claim 20 in which said Q component is a peptide and said A component is an MHC molecule which includes a binding site for said Q component. 22. A complex as defined by claim 15 in which the effector component E is a chemotherapeutic and the A component is an MHC moiety which includes a ligand for the surface receptors of target cells. 23. A method for the in vivo killing of target cells which comprises selecting an effector moiety E which is cytotoxic with respect to said target cells; forming a conjugate having the formula (E-Z-Q) in which E is said effector, Z is an intracellularly cleavable linkage, Q is a peptide recognized by a binding site of an MHC molecule and which, as a component of said peptide, is combinable with said MHC molecule; forming a complex of said conjugate, said complex having the formula (E-Z-Q) A in which E-Z-Q is said conjugate and A is an MHC molecule having a binding site for Q, and administering said complex in need of treatment by said effector; said complex being effective to deliver said conjugate to said target cells; said conjugate being internalized by said target cells after delivery; said conjugate being intracellularly cleaved by said target cells after internalization to release said target cell cytotoxic effector E.

Inventors: CLARK BRIAN R, DESHPANDE SHRIKANT, NAG BISHWAJIT, CLARK, BRIAN, R., DESHPANDE, SHRIKANT, NAG, BISHWAJIT
Applicants: BIOSPAN CORP, BIOSPAN CORPORATION
Application year: 1991
Main classification: C07K7
IPC codes: A61K9, A61K38, A61K47, C07K9, C07K14, C07K16, C07K17, C07K19