CZ74797A3 - Composition for specific intracellular introduction of a chemical agent and method of such introduction - Google Patents

Abstract

A composition for specific intracellular delivery of a chemical agent into a CR2 receptor-bearing cell comprises a CR2 receptor binding and endocytosis inducing ligand (CBEL) coupled to the chemical agent. The CBEL binds to the CR2 receptor 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. Methods of using these compositions for delivering a chemical agent in vivo or in vitro are also disclosed.

Description

The invention relates to the introduction of chemical agents into cells. In particular, the invention provides compositions and methods for intracellular delivery of chemical agents into a specific cell type that expresses the CR2 receptor.

BACKGROUND OF THE INVENTION

In terms of usefulness in the treatment of cancer, toxins that target cell surface receptors or antigens on tumor cells have attracted considerable attention. Such toxins are described, for example, by I. Pastan & D. FitzGerald in Recombinant Toxins for Cancer Treatment, 254 Science 1173 (1991); Anderson et al. in U.S. Pat. U.S. Patent Nos. 5,169,933 and 5,135,736; Thorpe et al. in U.S. Pat. No. 5,165,923; Jansen et al. in U.S. Pat. U.S. Patent No. 4,906,469; Frankel in U.S. Pat. U.S. Patent No. 4,962,188; Uhr et al. in U.S. Pat. U.S. Patent No. 4,792,447; Masu et al. in U.S. Pat. No. 4,450,154 and 4,350,626. These agents include a moiety such as a cell growth factor or a protein capable of binding to a cell antigen that binds to a plant or bacterial toxin. The mechanism of cell killing differs from conventional chemotherapy, thus potentially reducing or eliminating cross-resistance to conventional chemotherapeutic agents.

CR2 membrane glycoprotein, also known as CD21, occurs on mature lymphocytes (B cells) and on certain epithelial cells such as human pharynx epithelial cells, human follicular dendritic cells, and cervical epithelium. This membrane glycoprotein functions as a receptor for Epstein-Barr virus (EBV) and for C3d / C3dg complement fragments as described by N. Miller & L.M. Hutt-Fletcher, 66 J. Virol. 3409 (1990). Said receptor is a 145 kD large membrane glycoprotein which, in addition to its binding function, is also involved in B cell activation. The receptor is described, for example, in G.R. Nemerow, et al., Identification and Characterization of Epstein-Barr Virus Receptor on Human B lymphocytes and its Relationship to the C3d Complement Receptor (CR2), 55 J. Virol. 347 (1985). Epstein-Barr virus (EBV) B cell infection is initiated by binding of the viral envelope glycoprotein to the CR2 receptor, followed by internalization of the CR2 receptor and endocytosis of the virions. This process is described in Tedder et al. (1986), Epstein-Barr virus binding inducing internalization of the C3d receptor: a novel immunotoxin delivery system, 137 J. Immunol. 1387 (1986). Epithelial cells that carry the CR2 receptor also bind to Epstein-Barr virus (EBV), but the infection appears to be through a different mechanism than that of receptor-mediated endocytosis.

Nemerow et al. in Identification of gp350 as a viral glycoprotein mediating attachment of Epstein-Barr virus (EBV) to the EBV / C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d, 61 J. Virol. 1416 (1987), determined the region of amino acid sequence similarity between the C3dg complement fragment and the gp350 / 220 viral envelope glycoprotein. This region includes a region lying near the N-terminus of the gp350 / 220 viral envelope glycoprotein (Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu; SEQ ID NO: 2) that corresponds to the C3dg complement fragment sequence (Glu- AspPro-Gly-Lys-Gln-Leu-Tyr-Asn-Val-Glu (SEQ ID NO: 2). Nemerow et al. in the publication Epitope-Barr virus epitope in the major envelope protein that mediates viral binding to the B lymphocyte EBV receptor (CR2), 56 Cell 369 (1989), describes the binding of a synthetic tetradecapeptide comprising the amino acid sequence designated SEQ ID NO. 1, to purified CR2 receptor and to B cells that express CR2 receptor. The synthetic peptide also blocks binding of the recombinant viral envelope glycoprotein gp350 / 220 or C3dg complement fragment to the CR2 receptor of B cells. A similar synthetic peptide inhibits EpsteinBarr virus infection in vitro. Analysis of garbled and substituted peptide analogs showed that the Epstein-Barr virus (EBV) epitope that allows binding to the CR2 receptor is included in the sequence GluAsp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID NO: 1). ). The shorter peptide exhibits limited binding ability, although the peptide with the Glu-Asp-Pro-Gly sequence (SEQ ID NO: 3) retains significant binding activity. A peptide having a single amino acid swap in this region, proline replaces glycine, also exhibits reduced CR2 receptor binding activity.

SUMMARY OF THE INVENTION

The invention provides compositions for intracellular delivery of selected chemical agents to a specific cell type, that is, cells expressing the CR2 receptor. Binding to this receptor triggers receptor-mediated endocytosis.

The invention also provides methods for preparing and using compositions for intracellular delivery of selected chemical agents to cells that express the CR2 receptor.

The invention further provides compositions and methods for intracellularly selected selected chemical agents, such as cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like, into cells that express the CR2 receptor.

The invention provides peptide ligands that can be coupled to selected chemical agents and allow binding of chemical agents to CR2 receptors and endocytosis of these chemical agents.

These and other embodiments of the invention are accomplished by providing a composition for specific intracellular delivery of a chemical agent to a cell that carries a CR2 receptor in a population of cells comprising cells without the expressed CR2 receptor. This composition comprises a ligand (CBEL) that is capable of binding to the CR2 receptor and inducing receptor-mediated endocytosis. Further, the composition comprises a ligand-bound chemical agent, wherein the chemical agent is capable of producing the desired effect upon its intracellular introduction into a cell that carries the CR2 receptor. Mature B cells are cells that carry the CR2 receptor and are target cells for these compositions. Chemical agents that can be introduced into such cells include cytotoxins, transforming nucleic acids, gene regulators, labeling agents, antigens, and drugs. The ligand (CBEL) and the chemical agent may bind to each other and / or may bind to other functional moieties through spacer. The spacer may be either biodegradable, such as certain peptides, or non-biodegradable. The composition may further comprise a carrier system selected from the group consisting of water-soluble polymers, liposomes and particles.

The compositions are used in vitro, wherein the cell population is treated with an effective amount of the composition under conditions where the α ligand (CBEL) binds to the CR2 receptor and induces endocytosis of the composition that is bound to the receptor. When used in vivo, it systemically administers an effective amount of the composition such that the ligand (CBEL) contacts, binds to, and binds to the CR2 receptor of mature B cells, and then induces endocytosis of the composition.

When a chemical agent enters the cell, it becomes effective.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and 1B depict chemical conjugation of ligand (CBEL) with a chemical agent having a free mercapto group to form a composition of the invention. TM indicates room temperature.

Figures 2A to 2B depict the individual steps of constructing a plasmid for expressing a fusion protein comprising a ligand (CBEL) and a chemical agent, which is a peptide of the invention.

Figure 3 shows a comparison of the effect of in vitro treatment of the CBEL-ricin A fusion protein at various concentrations on CR2-bearing B cells (dark bars) and T-cells not carrying CR2 receptor (light bars) according to the invention. The x-axis plots the concentration of the fusion protein in µg / ml and the y-axis plots the% of cells capable of self-existence after exposure.

The term "peptide" refers to peptides of any length and includes proteins.

The terms polypeptide and oligopeptide are used herein without size limitations, unless a particular size is specified otherwise.

The terms CR2 receptor-binding ligand and endocytosis inducing or CBEL refer to a composition capable of binding to the CR2 receptor (CD21) and causing internalization by receptor-bound ligand (CBEL) endocytosis. According to the invention, ligands (CBELs) associate with a variety of functional molecules such that, in endocytosis of ligands (CBELs), a number of functional molecules attached are also internalized into cells that carry the CR2 receptor.

Recent evidence suggests that the ligand (CBEL) may be derived from the epstein-Barr virus gp350 / 220 membrane glycoprotein and comprises the sequence shown in SEQ ID NO: 1 and the flanking sequence; C3dg complement fragment peptides comprising the sequence shown in SEQ ID NO: 2 and flanking sequences; or from peptides that exhibit substantial homology to these agents. By substantially homologous peptides is meant peptides that retain the ability to bind to CR2 receptors and induce receptor-mediated endocytosis, although they may include flanking sequences or may form truncations, deletion variants, or substitution variants of the sequences described in SEQ ID NO: 1 or SEQ. ID No: 2. The sequence described as SEQ ID NO: 3 appears to be minimally necessary for binding to the receptor and for inducing receptor-mediated endocytosis. Substitution variants are those peptides wherein one or more amino acid residues are conservatively replaced. A conservative replacement is defined as the replacement of one amino acid residue for another, while the peptide retains its functionality. In this case, it is the ability to bind to the CR2 receptor and induce endocytosis of the receptor-bound composition. Amino acid residues belonging to certain conservative substitution groups may sometimes be replaced by another amino acid residue from the same group. One such substitution moiety is described in M. Jimenez-Montano & L. Zamora-Cortina, Evalutionary model for the generation of amino acid sequences and its application to the study of mammalian alpha-hemoglobin chains, Proc. Vllth Int'l Biophysics Congress, Mexico City (1981), include: Pro, Ala, Gly, Ser, Thr, Asn, Gin, Asp, Glu, His, Lys, Arg, Cys, Ile, Leu, Met, Val, and Phe, Trp, Tyr. For other variants considered to be substantially homologous, the D-amino acids are replaced by the naturally occurring L-amino acid, the amino acid derivatives such as those containing other side chains are replaced, and the non-standard amino acids, which are α - amino acids that are sparse or absent from proteins. Only functionality limits the primary ligand structure (CBEL).

The term chemical agent describes and includes any substance that exhibits the desired effect upon internalization into the B lymphocyte by endocytosis. Upon internalization into B cells, certain chemical agents have the following physiological effects: a cytotoxic effect or affect the regulation of the genes of these cells.

The transforming nucleic acid (RNK or DNA) replicates and / or is expressed in the cell upon internalization into the cell. Other nucleic acids may interact with cells in conjunction with regulatory sequences or regulatory factors, affecting the expression of the gene in the cell in a prescribed manner. The detection of the labeling agent upon its introduction into the cell allows the identification of those cells in which the compositions of the invention are internalized. Antigens that have been introduced into the interior of a cell can elicit a specific immune response. Drugs or pharmacologically active substances are used to improve pathogenic effects or other types of disorders. Particularly preferred chemical agents include polypeptides, some such chemical agents are active fragments of biologically active proteins or are specifically antigenic fragments (e.g. epitopes) of antigenic proteins. Chemical agents include cytotoxins, gene regulators, transforming nucleic acids, labels, antigens, drugs, and the like.

The term carrier refers to water-soluble polymers, particles or liposomes. Such carriers include several sites to which one or more ligands (CBELs) and / or a chemical agent may attach. Said carriers increase the size of the molecule of the compositions and can provide them with added selectivity and / or stability. Selectivity increases because the carrier-containing compositions are too large to enter the cell by passive diffusion and are therefore restricted to enter the cells by receptor-mediated endocytosis. Carriers containing water-soluble polymers can be used to bind peptide ligands, chemical agents, and other functional molecules. The potential for using such carriers for targeted drug delivery has been established, as described, for example, in J. Kopecek, 5 Biomaterials 19 (1984); E. Schacht et al., Polysaccharides as Drug Carriers, in ControlledRelease Technology 188 (P.I. Lee & W.R. Good, eds., 1987); F. Hudecz et al., Carrier design: cytotoxicity and immunogenicity of synthetic branched polypeptides with (L-lysine) backbone poles, 19 J. Controlled Release 231 (1992); Z. Břich et al., Preparation and characterization of water solubla dextran immunoconjugate of doxorubicin and monoclonal antibody (ABL364), 19 J. Controlled Release 245 (1992). Illustrative water-soluble polymers include dextran, inulin, poly (L-lysine), methacrylamide-containing synthetic polymers, and the like.

The term drug or pharmacologically active agent refers to any chemical material or compound that is suitable for intracellular introduction into a B cell bearing the CR2 receptor and which produces the desired biological or pharmacological effect in such a cell.

By "effective amount" is meant an amount that allows the desired effect to be achieved. For example, the desired effect of a composition comprising a cytotoxin as a chemical agent may be to kill a selected proportion of mature B cells over a given period of time. An effective amount of the composition is an amount at which this desired result is achieved, and such amount can be determined routinely by one skilled in the art.

The compositions of the invention allow specific intracellular delivery of a chemical agent to a CR2 receptor-bearing cell in a population of cells including CR2 receptor-free cells. The composition comprises a ligand (CBEL) that is capable of binding to the CR2 receptor and inducing receptor-mediated endocytosis, and a ligand-associated chemical (CBEL), wherein the chemical agent is capable of producing the desired effect upon intracellular introduction into a CR2 receptor-bearing cell. Particularly mature B cells are the target cells of these compositions. The chemical agents are selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like. The ligand (CBEL) can be attached to a chemical agent without limitation by covalent bonding, electrostatic interaction, hydrophobic interaction, physical capsulation, and the like. The ligand (CBEL) may be coupled to the chemical agent directly or through other functional moieties. The compositions may then further comprise a spacer that links the ligand (CBEL) and the chemical reagent and is interposed therebetween. Such a spacer may be biodegradable or non-biodegradable. A peptide spacer is preferred. The compositions of the invention may further comprise a carrier selected from the group consisting of water-soluble polymers, liposomes and particles. Where the carrier is a water-soluble polymer, the composition is characterized by the formula:

[LS _a ] _b - C - [S _e -A] _f in which

L is a ligand (CBEL) capable of binding to the CR2 receptor and inducing its endocytosis,

A is a chemical agent,

S indicates a space,

C is a water-soluble polymer whose functional groups are compatible with the ligand, the chemical reagent and the spacer by forming covalent bonds, and has a value of 0 or 1, e has a value of 0 or 1, b is an integer having a value of at least 1 , f is an integer of at least 1.

Such a water-soluble polymer is selected from the group consisting of dextran, inulin, poles (L-lysine), polymers containing methacrylamide, and the like.

The ligand (CBEL) of the invention mediates binding of the composition to the CR2 receptor on mature B cells, thereby triggering the internalization of the compositions by endocytosis. The chemical agent provides agents for achieving the desired effect in B cells. For example, chemical agents include cytotoxins, including radionucleotides, to selectively kill or attenuate cells that carry the CR2 receptor; further comprising antigens to elicit the desired immune response; nucleic acids that genetically transform or regulate gene expression in B cells; drugs or pharmacologically active agents to achieve the selected therapeutic effect; labeling substances which function on the principles of fluorescence, radioactivity and magnetism and allow to locate cells that have taken up compositions and the like.

The compositions of the invention further comprise water-soluble polymeric carriers that allow most ligands (CBELs) and / or chemical agents with selected functions to bind together to form complex molecules. At least one chemical agent and at least one ligand (CBEL) are bound to such carriers, a preferred number of CBELs associated with the chemical agents is in the range of 1 to about 1000. Such carriers increase the size of the molecule molecules and can provide functional selectivity and or stability, which is not achieved without the carrier. Biodegradable or non-biodegradable spacers preferably mediate the coupling of ligands (CBELs) and / or chemical agents. Their relative susceptibility or resistance to hydrolysis and / or enzymatic cleavage within B cells plays an important role in their selection. This selectivity provides one of skill in the art to select a spacer based on whether it is most advantageous to have a chemical agent still attached to the carrier or to be released from the carrier by intracellular enzymatic activity. The use of liposomes and particles as carriers does not depend on the use of water-soluble polymeric carriers.

In some embodiments of the invention, the compositions are formulated by chemical coupling of a ligand (CBEL) and a chemical reagent. By chemical coupling of a ligand (CBEL) and a chemical reagent is meant covalent coupling of a ligand (CBEL) and a chemical reagent either directly or via linking moieties. In particular embodiments of the invention where the chemical agent is a peptide, linking moieties is used to form a bond between functional groups on ligands (CBEL) and the chemical agent. For example, a composition comprising a ligand (CBEL) and a chemical agent that is a peptide is formed by coupling a mercapto group carried by a chemical agent and an amino group on a ligand (CBEL) via a heterobifunctional cross linker. For example, Figures 1A and IB show the chemical scheme of the chemical coupling of ligand (CBEL) and ricin A via a maleimide cross linker. In addition to the maleimide cross-linkers used in the chemical coupling of ligands (CBEL) and the mercapto containing chemical reagent, haloacetyl, alkyl halide, alkyl sulfonate, and, β-unsaturated carbonyl or α, β-unsaturated sulfone moieties can also be used as cross linkers. In the case of chemical coupling of ligands and chemical reagents containing amino groups, active esters can be used as cross-linkers. Other linkers also apply, depending on which functional group (s) carried by the ligands (CBEL) and the chemical agent are available.

The compositions of the invention also form in a gene manipulated organism, such as E. coli, in the form of a fusion protein. That is, by means of genetic engineering, a hybrid gene can be generated comprising a nucleotide sequence encoding a ligand (CBEL) and a nucleotide sequence encoding a chemical agent peptide. This hybrid gene can be introduced into an organism by expressing a fusion protein that is encoded by the hybrid gene. The fusion protein is then purified by standard methods, including affinity chromatography.

Fusion proteins containing the ligand (CBEL) and the chemical agent peptide of the invention can also be produced by chemical synthesis.

When the compositions of the invention are produced in the form of fusion proteins, the ligand (CBEL) and the chemical agent peptide may be immediately adjacent, meaning that the ligand end (CBEL) binds directly to the N-terminus of the chemical agent and vice versa. Alternatively, the fusion protein may also contain additional amino acid residues that are located between the ligand (CBEL) and the chemical agent. These added amino acid residues serve as a spacer between the ligand (CBEL) and the chemical reagent peptide. Generally, short peptide ligands are preferred because short peptides are handled because the presence of the added and particularly a substantial number of these is easier to handle and amino acid residues, amino acid residues, can interfere with the function of the peptide ligand to induce internalization of the chemical agent by endocytosis.

The compositions of the invention may further comprise protease cleavage sites that are positioned such that upon introduction of the composition into the cell, the chemical agent may be separated from the ligand (CBEL) by proteolysis of the cleavage site. Such cleavage sites naturally occur in the viral membrane glycoprotein gp350 / 220 adjacent to a segment of the sequence shown in SEQ ID NO: 1, so that the ligand portion of the composition can be suitably expanded to include these cleavage sites. A protease-sensitive spacer may be added to the composition regardless of whether the composition is synthesized chemically or is an expressed peptide of a genetically manipulated organism. If the composition is an expressed peptide, nucleotides encoding a protease-sensitive spacer may be inserted into a hybrid gene between segments encoding a ligand (CBEL) and a chemical reagent peptide by a method well known in the art.

Further, the invention provides a method by which the desired activity is specifically affected in CR2 receptor expressing cells included in a population of cells without CR2 receptor expression. The cell population is gradually contacted with a composition comprising a ligand (CBEL) associated with a chemical agent that intracellularly controls such activity. The composition of the invention selectively binds to cells expressing the CR2 receptor in a mixed population of cells, inducing endocytosis of the composition and the chemical agent actively acting in cells expressing the CR2 receptor.

Unless otherwise specified, the invention utilizes standard methods of manipulating peptides and nucleic acids to express peptides. Methods for conjugating 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); Rigaudy et al., 49 Cancer Res. 1836 (1989).

As mentioned above, the invention provides peptides that act as ligands (CBELs) in a composition comprising also chemical agents. The chemical agents of the composition may also be peptides or may contain peptides. The peptides of the invention can be produced by any of the methods including organic synthesis and genetic engineering. For example, methods of chemical peptide synthesis are described in B. Merrifield et al., 21 Biochemistry 5020-31 (1982); Houghten, 82 Why. Nat'l Acad. Sci. USA 5131-35 (1985). Methods of chemical conjugation of peptides to other molecules are well known in the art.

The fusion protein of the invention can be produced by expressing the nucleic acid in a suitable host cell. The nucleic acid comprises an oligonucleotide encoding a ligand (CBEL) as described above and an oligonucleotide encoding a chemical reagent peptide. These methods for producing recombinant fusion proteins are well known in the art and are generally described, for example, in J. Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989). Reagents suitable for the application of these methods, such as restriction enzymes and the like, are well known in the art and are commercially available from several suppliers.

Further disclosed is the formation of compositions comprising a ligand (CBEL) and a chemical agent peptide of the invention with reference to examples wherein the ligand (CBEL) is associated with a cytotoxic chemical agent that is a ricin A peptide. Ricin is a toxic glycoprotein produced by Ricinus communis. . It consists of two subunits, chain A and chain B, each having a molecular weight of about 30 kD, linked together by a disulfide bond. Ricin is synthesized as a large precursor to produce mature ricin subunits, chains A and B. The A chain is an enzyme that cleaves a glycosidic linkage in the 28S ribosomal RNK, thereby abolishing the ability of ribosomes to synthesize protein. In S. Olsen et al., Ribosome inactivation by the toxic lectins abrin and ricin, 60 Eur. J. Biochem. 281 (1975) discloses that the A chain can inactivate about 1500 ribosomes per minute, meaning that the only internalized ricin A molecule is lethal to the cell. The B chain binds to a portion of the cell surface galactose, a phenomenon necessary for the internalization of ricin. Removing the B chain prevents the A chain from entering the cell, thereby rendering it inactive. Linking the ricin A chain to the ligand (CBEL) allows the A chain of the ricin molecule to enter the cell by receptor-mediated endocytosis, resulting in an active cytotoxin.

DETAILED DESCRIPTION OF THE INVENTION

In Example 1, the composition is formed by chemical conjugation; and in Example 2, the composition is formed as a recombinant fusion protein.

Example 1

Chemical conjugation of ligand (CBEL) and ricin A.

Chemical conjugation of the ligand (CBEL) having the amino acid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID NO: 1) with the cytotoxic chemical agent ricin A peptide was illustrated in two steps for illustration as shown in Figures IA and IB.

In the first step, the ligand (CBEL) (prepared by Peptide International, Kentucky, USA) is activated by reaction with m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo-MBS; Pierce, Rockford, Illinois) essentially following the manufacturer's instructions. Briefly, the ligand (CBEL) is mixed with sulfo-MBS at a molar ratio of 1:10 in PBS buffer (20 mM sodium phosphate, 0.15 M sodium chloride, pH7). The mixture was allowed to stand at room temperature for two hours and the resulting activated peptide was purified by standard liquid chromatography (FPLC) methods using Superose 12 (Pharmacia).

In the second step, the maleimide-activated peptide was reacted with deglycosylated ricin A (Sigma Chemical Co., St. Louis, Missouri, USA). These reagents are mixed at a molar ratio of 1: 5 in buffered PBS and allowed to stand for 2 hours at room temperature. Unreacted peptide molecules are removed from the ligand (CBEL) -ricin A conjugate by dialysis overnight at 4 ° C across a 6 to 8 kilodalton cut-off membrane.

The molecular weight of the resulting composition is about 32 kD and was estimated by gel electrophoresis on a 10% SDS-PAGE gel (10% agarose dissolved in a buffered sodium dodecyl sulfate solution) as described in U. Laemmli, 227 Nature 680 ( 1970). The composition prepared according to this example contains one ligand (CBEL) per ricin A molecule. Three-dimensional computer modeling shows that the ligand (CBEL) is most likely bound to the Cys residue at the C-terminus of the ricin A molecule. AND.

Example 2

Recombinant protein containing ligand (CBEL) and ricin A

A fusion protein comprising a ligand (CBEL) having the amino acid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID NO: 1) and a cytotoxic chemical agent, the peptide ricin A, is engineered by genetic engineering. . Briefly, the fusion protein of this example is produced by introducing a synthetic oligonucleotide encoding a ligand (CBEL) into an expression vector suitable for an E. coli microorganism downstream of a polynucleotide (downstream) that encodes ricin A, followed by expression with a high yield of fusion protein. in an E. coli host organism. The fusion protein is purified from the cell lysate by affinity chromatography.

Referring to Figures 2A-2D, the ligand-containing fusion protein (CBEL) and ricin A are produced by genetic engineering as described below. The pTrcHis B expression vector suitable for the E. coli microorganism (Invitrogen, San Diego, California) (Figure 2A), which contains a downstream translational cloning site (MCS), is cleaved with Ncol restriction endonucleases and BamHI and the resulting cohesive ends changed to blunt ends by treatment with T4 DNA polymerase. This linear fragment was re-ligated to give the vector pTrc B (Fig. 2B). From the plasmid pAKG (plasmid pAKG provided by Robert Weaver, University of Kansas, Lawrence; and described in RC Halling et al., 13 Nucleic Acids Res. 8019 (1985)), a BamHI-Kpn1 fragment that is 863 bp large is isolated and This fragment was cloned into plasmid pTrc B, which had been digested with BamHI and KpnI. The resulting construct was digested with BamHI and T4 DNA polymerase blunt-ended, and the linear fragment re-ligated to provide the correct reading frame for translation of the cloned gene (Fig. 2C).

The resulting engineered pTrc B vector containing the ricin A coding sequence was further digested with KpnI and EcoRI to ligate the following synthetic oligonucleotide containing the nucleotide residues encoding the ligand (CBEL) and cohesive ends compatible with KpnI and EcoRI cloning sites.

(SEQ ID C: 4) 5 '- CA AAT TTT AAT GAA GAT CCT (SEQ ID C: 5) 3 '- CAT GGT TTA AAA TTA CTT CTA GGA (SEQ ID C: 6) Asn Phe Asn Glu Asp For (SEQ ID C: 4) GGT TTT TTC AAT GTT GAG CAT CAT (SEQ ID C: 5) CCA AAA AAG TTA CAA CTC GTA GTA (SEQ ID C: 6) Gly Phe Phe Asn Wall Glu His His (SEQ ID C: 4) CAT CAT CAT CAT TAA G -3 ' (SEQ ID C: 5) GTA GTA GTA GTA ATT CTT AA -5 ' (SEQ ID C: 6) His His His His Resultant vector (Giant . 2D) contain e hybrid gene coding

ricin A-protease site-ligand-His 6 fusion protein, where the protease site indicates a protease cleavage site or protease-sensitive spacer as described above, the ligand indicates a ligand (CBEL) and His 6 indicates a region of six consecutive His residues whose functions will be described below. The resulting plasmid is used to transform E. coli cells and selected transformants are cultured in LB medium as described by J. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1972). The cells are then lysed and the recombinant fusion protein is purified by affinity chromatography on a column containing nickel-enriched resin (PROBOND, Invitrogen, San Diego, California). The six His residues at the C-terminus of the fusion protein bind electrostatically to the nickel atoms found on the PROBOND resin. The resin containing the bound fusion protein is washed to remove impurities. The electrostatic bonds are then disrupted and the fusion protein eluted with an elution buffer containing imidizole. His residues were displaced from the nickel-enriched resin with this buffer. The purification of the fusion protein is carried out according to the manufacturer's instructions.

Example 3

A fusion protein comprising a ligand (CBEL) having the amino acid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID NO: 1) and a cytotoxic chemical agent, the peptide ricin A, is engineered by genetic engineering. as in Example 2 except that the region containing six consecutive His residues is omitted. The following synthetic oligonucleotide, containing the nucleotide residues encoding the ligand (CBEL) and cohesive ends compatible with KpnI and EcoRI cloning sites, was ligated into the EcoRI and KpnI restriction digested vector pTrc B.

(SEQ ID No: 7) 5 '- CA AAT TTT AAT ATC CAT CTC (SEQ ID No: 8) 3'- CAT GGT TTA AAA TTA TAG GTA GAG (SEQ ID No: 9) Asn Phe Asn Ile His Leu (SEQ ID No: 7) ACG GGT GAA GAT CCT GGT TTT TTC (SEQ ID No: 8) TGC CCA CTT CTA GGA CCA AAA AAG (SEQ ID No: 9) Thr Glu Glu Asp For Gly Phe Phe (SEQ ID No: 7) AAT GTT GAG TAA G -3 ' (SEQ ID No: 8) TTA CAA CTC ATT CTT AA -5 ' (SEQ ID No: 9) Asn Wall Glu

The resulting vector comprises a hybrid gene encoding a ricin A protease site-ligand (CBEL) fusion protein, wherein the protease site designates a protease cleavage site or protease sensitive spacer as described above. The resulting plasmid is used to transform E. coli cells and selected transformants are cultured in LB medium. To isolate the expressed protein, cells are lysed by culturing with lysozyme (1 mg / ml) in 20 mM sodium phosphate, pH 7.8, and sonication at three minute intervals. The fusion protein is insoluble under these conditions unlike most proteins found in E. coli cells. The lysate is centrifuged at 9,000 rpm for 30 minutes. The resulting fusion protein-containing sediment is resuspended and sonicated for 1 minute before centrifugation again. The re-suspension, sonication and centrifugation steps are repeated three times. The final sediment containing the relatively pure fusion protein is dissolved in a solution containing 6 M urea and 5 M dithiothreitol. The dissolved fusion protein is renatured by sequential dialysis against 4M urea; 2 M urea; and a buffer containing 20 mM sodium phosphate, 500 mM sodium chloride and having a pH of 7.8.

Example 4

A fusion protein comprising a ligand (CBEL) having the amino acid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID NO: 1) and a cytotoxic chemical agent, the peptide ricin A, is engineered by genetic engineering. as in Example 2 except that another cysteine residue is introduced into the fusion protein. This construction provides higher yields of obtainable fusion protein compared to Example 2, since the free cysteine residue at the C-terminus of the ricin A chain may form an intramolecular disulfide bond with the new cysteine residue instead of the E. coli protein. The expression vector pTrcHis A (Invitrogen, San Diego, California), which is suitable for E. coli, and which is similar to the vector pTrcHis B in Figure 2A except for a different reading frame, is digested with the restriction endonucleases NcoI and BamHI. Synthetic

The DNA that encodes six consecutive histidine residues and has NcoI and BamHI cohesive ends is then ligated into this vector. Plasmid pAKG was digested with BamHI and the released 893 bp fragment was obtained by electroelution after agarose gel electrophoresis. This fragment was ligated into the engineered vector pTrcHis A, which was digested with BamHI and calf intestinal alkaline phosphatase. The ligation mix was used for E. coli XL-1 (Stratagene, La Jolla, transformants were isolated, and the orientation of the BamHI fragment in the vector was determined by digestion) and the DNA from the transformant carrying the ricin A gene in the correct translation orientation was digested with SacI and EcoRI, the following synthetic oligonucleotide is ligated into this DNA, which contains nucleotide residues encoding a ligand (CBEL) and cohesive ends compatible with SacI and EcoRI cloning sites.

California strain). Further, BglII

(SEQ ID C: 10) 5 '- C GAA GAT CCT GGT TTT (SEQ ID C: 11) 3 '- TC GAG CTT CTA GGA CCA AAA (SEQ ID C: 1) Glu Asp For Gly Phe (SEQ ID C: 10) TTC AAT GTT GAG TAA G -3 ' (SEQ ID C: 11) AAG TTA CAA CTC ATT CTT AA (SEQ ID C: 1) Phe Asn Wall Glu

The ligated DNA is transformed into an expression host E. coli strain BLR (Novagen, Madison, Wisconsin) that is recA positive but lacks the protease genes ion and ompt.

To isolate the recombinant protein, transformed cells are cultured in the presence of ampicillin at 37 ° C. When the optical density (measured at 600 nm) of the culture reaches a range of 0.6 to 0.8, an amount of isopropylthiogalactoside (IPTG) is added such that the final concentration is 1 mM. Isopropylthiogalactoside (IPTG) induces gene expression. After an additional three hours of growth, cells are recovered from the medium and suspended in a buffer solution containing 10 mM Tris HCl pH7.6, 100 mM potassium chloride, mM EDTA, 10 mM 2-mercaptoethanol, 0.05% Nonidet P-40. and 0.5 mg / ml lysozyme. After a 15 minute incubation in an ice bath, the resulting lysate is sonicated in the presence of 0.5 mM phenylmethylsulfonyl fluoride and centrifuged at 9,000 rpm for 30 minutes at 4 ° C. The soluble proteins are precipitated with ammonium sulfate (added to 40% saturation), the precipitate is dissolved and dialyzed against a buffer containing 10 mM Tris HCl pH 7.4 and 100 mM potassium chloride. The dialysate is applied to a strong anion exchange column (Q Sepharose Fast Flow, Pharmacia), which is equilibrated with the same buffer. Under these conditions, most bacterial proteins bind to the column and the fraction that does not bind contains substantially pure recombinant protein. The recombinant protein is further purified by dialysis against a solution containing 4 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, whose pH is 7.8. The dialysate is passed through a column containing a nickel-enriched resin (PROBOND resin) and is washed five times with a solution containing 4 M urea, 20 mM sodium phosphate, 500 mM sodium chloride, 5 mM imidizole, the pH of the solution is 6.0. The six histidine residues in the rembinant protein cause the recombinant protein to bind by affinity interaction to the resin. The column is washed two more times with buffer, which differs only in the concentration of imidizole, which increases to 30 mM. The recombinant protein is eluted into a buffer that differs only in that the concentration of imidizole is 300 mM. The eluted protein is then renatured and spun by first dialysis against 2 M urea and then against a solution containing 20 mM sodium phosphate, 500 mM sodium chloride having a pH of 7.8.

Example 5

Targeted introduction of cytotoxin into B cells.

To illustrate the targeted introduction of a chemical agent into CR2 receptor expressing B cells, the use of the ligand (CBEL) and chemical agent compositions of the invention is described herein with reference to examples wherein ricin A and ligand (CBEL) compositions are introduced into Paradise B lymphoblastoid cells in vitro . The result is a specific cytotoxic effect on B cells expressing the CR2 receptor.

In a preliminary demonstration, 1 x 10 ^fe CR2 * Paradise B lymphoblastoid cells (CR2 ⁺ indicates that the cells express the CR2 receptor) are cultured for 24 hours at 37 ° C in 1 ml RPMI 1640 medium (Hyclone, Logan, Utah) without further treatment or in 1 ml medium containing 20 gg of ricin A-ligand composition (CBEL) as described in Example 1 or in 1 ml medium containing ricin A alone. After incubations, dead cells are determined by trypan blue staining and counted by hemacytometer using invert microscope. Trypan blue penetrates into dead cells and stains blue. A portion of the cells is diluted twice in 0.4% trypan blue dye (Sigma Chemical Co., St. Louis, Missouri) and incubated for 5 minutes before counting. The percentage of cells capable of self-existence is calculated according to the algorithm: the number of unstained cells per unit volume divided by the total number of stained and unstained cells multiplied by 100.

Cells treated with ricin A alone and control cells appear undamaged. About 99% of the cells treated with the ricin A-ligand (CBEL) composition die (1% of the cells survive). Since ricin A only acts toxic when it is internalized into a cell, these results indicate that the ricin A-ligand (CBEL) composition of the invention results in the internalization of a cytotoxic chemical agent.

Example 6

The effect of recombinant fusion protein ligand (CBEL) of ricin A, according to Example 2, was tested on CR2 ⁺ human B lymphoblastoid cells (Paradise) (CR2 ⁺ indicates cells expressing the CR2 receptor) and CR2 T (HSB2) cells (CR2 'symbol) means that the cells do not express the CR2 receptor) as described below. The cell suspension (cell number in suspension is 1 x 10 ^b ) is mixed intimately with varying concentrations of purified recombinant fusion protein in 1 ml of medium and incubated for 24 hours at 37 ° C. Thereafter, the ability of cells to exist alone by cell trypan blue staining was assessed as in Example 5. As shown in Figure 3, the response of cells expressing the Cr2 receptor (CR2 ⁺ ) (dark bars) to the conjugate depends on its dose, whereas cells without CR2 receptor expression (CR2 ^- ) (light bars) are not affected by this effect. When the fusion protein concentration is 50 pg / ml, less than 10% of the CR2 receptor expressing B cells and 100% of the CR2 receptor expression-free cells survive. Treatment of both cell types with either ligand alone (CBEL) or recombinant ricin A has no effect, supporting the conclusion that the toxic effect of recombinant ligand fusion protein (CBEL) -ricin A on cells expressing the CR2 receptor results from internalization of the recombinant fusion protein via the CR2 receptor. on B cells.

Example 7

The procedure described in Example 6 is followed except that the percentage of cells capable of self-existence is determined by a colorimetric method using the tetrazolium compound (3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4- sulfophenyl) -2H-tetrazolium inner salt (MTS)) and electron binding agents (phenazine methosulfate; PMS). The MTS is biologically reduced by the cells to formazan, which is soluble in tissue culture medium. Absorbance of formazan is measured at 490 nm directly on 96-well assay plates without further processing. The amount of formazan measured by absorbance at 490 nm is directly proportional to the number of viable cells in culture. Reagents used in the MTS assay were supplied by Promega Corp. (Madison, Wisconsin). The results obtained in this way are essentially the same as those described in Example 6.

Example 8

The effect of recombinant ligand fusion protein (CBEL) ricin A, according to Example 3, is tested on CR2 ⁺ human B lymphoblastoid cells (Paradise) (CR2 ⁺ indicates that the cells express the CR2 receptor) and CR2-human T (HSB2) cells (symbol CR2 means that the cells do not express the CR2 receptor according to the procedure described in Example 6. The results are substantially similar to those shown in Example 6.

Example 9

The effect of recombinant ligand fusion protein (CBEL) ricin A, according to Example 4, is tested on CR2 ⁺ human B lymphoblastoid cells (Paradise) (CR2 ⁺ indicates that the cells express the CR2 receptor) and CR2-human T (HSB2) cells (symbol CR2 - means that the cells do not express the CR2 receptor according to the procedure described in Example 6. The results are substantially similar to those shown in Example 6.

The ligand (CBEL) -chemical agent composition of the invention is used to target specific delivery of a chemical agent to cells expressing the CR2 receptor. Typically, this occurs when the CR2 receptor expressing cells are in contact with the composition under conditions where the CBEL ligand induces endocytosis of the composition into the CR2 receptor expressing cells. The chemical agent then acts on the target cells or in the target cells into which the composition is internalized. The desired effect of the active agent may be limited to those cells having a CR2 ⁺ phenotype (expressing the CR2 receptor).

For example, the ligand (CBEL) -cytotoxic agent composition of the invention is used in vivo as potent antitumor agents that selectively kill CR2 ⁺ B cells (B cells expressing the CR2 receptor). Systemic administration of the composition to a subject is preferred. Typical routes of administration of the composition are subcutaneous, intramuscular or intravenous injection or intraperitoneal administration. Injectable preparations for this purpose are prepared in conventional forms, either as liquid solutions or suspensions or as solid forms suitable for the preparation of liquid solutions or suspensions before use or as an emulsion. For example, water, saline, dextrose, glycerol, ethanol and the like; in addition, if desired, a small amount of excipients such as wetting or emulsifying agents, buffering agents and the like can be added.

Contact of the cells with the composition may occur in vivo or in vitro. Cells expressing the CR2 receptor may form (and in most cases are expected to form) a subpopulation of a mixed population of cell blunts; the ligand of the peptides of the invention may mediate the CR2 receptor-specific endocytosis of the conjugate to cells expressing the CR2 receptor.

The chemical agent may exhibit any of a variety of desired effects in the target cells. As mentioned previously, in some particularly preferred embodiments of the invention, the chemical agent acts on the cell only when, or mainly, when it is internalized into the cell.

Example 10

Targeted introduction of ligand (CBEL) -antigenic agent into B cells.

The ligand-containing composition (CBEL) and antigen of the invention are administered to warm-blooded animals to target an immune response in CR2 ⁺ cells (CR2 ⁺ indicates cells expressing the CR2 receptor). The ligand (CBEL) secures the CR2 receptor-mediated internalization of atigene into cells and can induce complement activation in target cells, the course of which is not dependent on antibodies. Which means that

according to the invention, the target cells They could be induced for a purpose induction of immune nesetkaly. responses to antigen, with whom never Chemical conjugation ligand (CBEL) who has amino acid the sequence described as SEQ ID No: 2, se

activates, and then the ligand is coupled to chicken lysozyme (Sigma Chemical Co., St. Louis, Missouri) as described in Example 1. The ligand (CBEL) -lysozyme conjugate is administered to the mouse systemically. C3dg CBEL (SEQ ID NO: 2) (C3dg CBEL is a ligand derived from C3dg complement fragment peptides) provides binding specificity to mouse B cells and induces CR2 receptor-mediated endocytosis of the conjugate. The conjugate then elicits an immune response to target conjugate epitopes, including epitopes found only on the lysozyme portion of the conjugate. The results of this example can be substantially duplicated by assembling a ligand (CBEL) -lysyme fusion protein.

Example 11

A method of treating human B cell leukemia comprises (a) obtaining a composition of the invention comprising a ligand (CBEL) such as a ligand derived from an Epstein-Barr virus membrane glycoprotein (EBV CBEL; SEQ ID NO: 1) or a peptide exhibiting substantial homology therewith , and a cytotoxin such as ricin A, and (b) systemically administering an effective amount of the composition. For example, the composition is prepared as described in Example 3. EBV CBEL is directed to mature human B cells and ricin A exhibits a cytotoxic effect on any cell into which it is introduced. An effective amount of the composition is administered to the subject in a systemic manner. Thus, the composition enters the bloodstream and associates with B cells. The ligand (CBEL) causes the composition to bind to the CR2 receptor on B cells and induce internalization of the composition by endocytosis. The cytotoxin ricin A then kills the cells by disrupting the ribosomes. This process reduces the number of malignant B cells in the body of the individual, thereby showing a positive effect in the treatment of the disease.

Example 12

The treatment of autoimmune diseases, such as erythematous lupus or rheumatoid arthritis, is carried out in the same manner as in Example 11. Cytotoxin acts upon its death in B cells, thereby reducing the number of autoantibody-producing B cells and showing a positive effect in the treatment of said diseases.

Sequence list

Sequence information # 1 (i) sequence characteristics:

(A) length: 9 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) representation of SEQ ID NO: 1

Glu Asp Gly Phe Phe Asn Val Glu 1 5

Sequence Information No. 2 (i) sequence characteristics:

(A) length: 11 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) sequence illustration SEQ ID NO: 2

Glu Asp Gly Lys Asn Leu Tyr Asn Val Glu 15 10

Sequence information No. 3 (i) sequence characteristics:

(A) length: 4 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) representation of SEQ ID NO: 3

Glu Asp For Gly

Sequence Information No. 4 (i) sequence characteristics:

(A) length: 60 base pairs (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear (xi) representation of SEQ ID NO: 4

CAAATTTTAA TGAAGATCCT GGTTTTTTCA ATGTTGAGCA TCATCATCAT CATCATTAAG 60

Sequence Information No. 5 (i) sequence characteristics:

(A) length: 68 base pairs (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear (xi) representation of SEQ ID NO: 5

AATTCTTAAT GATGATGATG ATGATGCTCA ACATTGAAA AACCAGGATC TTCATTAAAA 60 TTTGGTAC 68

Sequence Information No. 6 (i) sequence characteristics:

(A) length: 18 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: peptide (iii) sequence illustration SEQ ID NO: 6

Asn Phe Asn Glu His His 15 10

His His His His

Sequence Information No. 7 (i) sequence characteristics:

(A) length: 57 bp (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear (xi) representation of SEQ ID NO: 7

CAAATTTTAA TATCCATCTC ACGGGTGAAG ATCCTGGTTT TTTCAATGTT GAGTAAG 57

Sequence Information No. 8 (i) sequence characteristics:

(A) (B) (C) (D) length: 65 base pairs type: nucleic acid number of strands: single chain topology: linear (xi) representation of SEQ ID NO: 8

AATTCTTACT CAACATTGAA AAAACCAGGA TCTTCACCCG TGAGATGGAT ATTAAAATTT 60

GGTAC 65

Sequence Information No. 9 (i) sequence characteristics:

(A) (B) (D) length: 17 amino acids type: amino acid topology: linear (ii) molecule type: peptide (iii) representation of SEQ ID NO: 9

Asn Phe Asn Phle Phle Phle Phle

Asn Val Glu

Sequence # 10 (i) sequence characteristics:

(A) length: 32 base pairs (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear (xi) representation of SEQ ID NO: 10

CGAAGATCCT GGTTTTTTCA ATGTTGAGTA AG 31

Sequence Information No. 11 (i) sequence characteristics:

(A) length: 40 base pairs (B) type: nucleic acid (C) number of strands: single-stranded (D) topology: linear (xi) representation of SEQ ID NO: 11

AATTCTTACT caacattgaa aaaaccagga tcttcgagct, 40

Claims (32)

PATENT CLAIMS A composition for specific intracellular introduction of a chemical agent into a CR2 receptor-bearing cell in a population of cells comprising cells without CR2 receptor, the composition comprising substantially a ligand capable of binding to said CR2 receptor and inducing receptor-mediated endocytosis and a chemical agent associated with said ligand, by causing said chemical to produce the desired effect when introduced intracellularly into said cell bearing the CR2 receptor. the indicating agent is capable 2. The composition of claim 2, wherein the CR2 receptor is a B cell. of claim 1, said cell carrying The composition of claim 2, wherein said ligand comprises a peptide with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO. : 9. The composition of the agent is selected according to claim 3, wherein said chemical is selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labeling agents, antigens and drugs. The composition of claim 4 further comprising a spacer covalently bound to said ligand and said chemical agent, being interposed therebetween. SUBSTITUTE SHEET 6. The composition of claim 1, wherein the composition is biodegradable. of claim 5, wherein said spacebar is 7. Composition according to claim 6, characterized by it contains the peptide. and is that e mentioned mezerník 8. Composition according to claim 7, characterized by and is that e ligand is peptide, which has a sequence here listed as SEQ ID NO: 1 a mentioned the chemical agent is ricin A. 9. The composition of claim 5, wherein said spacer is not biodegradable. The composition of claim 4 further comprising a carrier selected from the group consisting of water-soluble polymers, liposomes and particles. The composition of claim 10, wherein said carrier is a water-soluble polymer and said composition is characterized by the formula: [L-Sa] b - c - [Se-A] _f in which L is said ligand capable of binding to the CR2 receptor and inducing its endocytosis, And it represents the chemical agent, S indicates a space, SUBSTITUTE SHEET -----..... C is said water-soluble polymer whose functional groups are compatible with said ligand, chemical agent and spacer by forming covalent bonds, and has a value of 0 or 1, e has a value of 0 or 1, b is an integer having a value of at least 1, f is an integer having a value of at least 1. Composition according to claim 11, characterized in that C is selected from the group consisting of dextran, inulin, poles (L-lysine), methacrylamide-containing synthetic polymers. A method specifically introducing a chemical agent in vitro into a cell bearing comprising cells characterized by the CR2 receptor in a cell population The CR2 receptor without (a) provides compositions for specifically intracellular delivery of a chemical agent to a CR2 receptor-bearing cell in a population of cells comprising cells without CR2 receptor, the composition substantially comprising a ligand capable of binding to said CR2 receptor and inducing receptor-mediated endocytosis; the chemical agent associated with said ligand is capable of producing the desired effect upon its internalization into said cell bearing the CR2 receptor; and (b) allowing said population of cells to come into contact with an effective amount of the composition under conditions in which the ligand binds to said CR2 receptor found on cells bearing the CR2 receptor and induces endocytosis of said composition. SUBSTITUTE SHEET 14. The method of claim 2, wherein the CR2 receptor is a B cell. of claim 13, said cell carrying 15. The method of claim 14, wherein said ligand comprises a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO. : 9. 16. The method of claim 15, wherein said chemical is selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labeling agents, antigens, and drugs. The method of claim 16, said composition further comprising a spacer covalently bound to said ligand and said chemical agent, wherein the spacer is interposed. 18. The method of claim 1, wherein the biodegradable material is biodegradable. of claim 17, said spacebar being 19. The method of claim 1, comprising the peptide. claim mentioned 18, spacebar 20. The method of claim 19, wherein said ligand is A SUBSTITUTE SHEET peptide having the sequence set forth herein as SEQ ID NO: 1 and said chemical agent is ricin A. 21. A method characterized by biodegradability. according to claim 17, wherein said spacer is not 22. The method of claim 16, wherein said composition further comprises a carrier selected from the group consisting of water-soluble polymers, liposomes, and particles. 23. The method of claim 22 wherein said carrier is a water-soluble polymer and said composition is characterized by the formula: [LS _a ] b - C - [S _e -A] _f in which L is said ligand capable of binding to the CR2 receptor and inducing its endocytosis, And it represents the chemical agent, S indicates a space, C is said water-soluble polymer whose functional groups are compatible with said ligand, chemical agent and spacer by forming covalent bonds, and has a value of 0 or 1, e has a value of 0 or 1, b is an integer having a value of at least 1, f is an integer having a value of at least 1. The method of claim 23, wherein C is selected from SUBSTITUTE SHEET groups containing polymers containing dextran, inulin methacrylamide. · <Ό ~ 73 es K> cr- with —1 - <OO G i m i > J WHAT" O O * 'O O CL C m σ | c / x r— O o < ro 25. A recombinant vector adapted for host transformation, said vector carrying a DNA segment encoding a fusion protein comprising a CR2 receptor-binding ligand and inducing endocytosis and a chemical agent. 26. Recombinant vector according to claim 37, characterized in that it comprises a plasmid. ž e mentioned vector 27. The recombinant vector of claim 38, wherein said ligand comprises a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO. SEQ ID NO: 9 and sequences exhibiting substantial homology therewith. 28. A recombinant amino acid sequence vector according to claim 1, wherein said ligand is shown herein as SEQ ID NO: 1. 39, has 29. A recombinant vector characterized in that the agent comprises a cytotoxin. according to claim 40, wherein said chemical 30. A recombinant protein comprising castor A. The vector of claim 41, wherein said cytotoxin 31. The agent comprises a vector antigen according to claim 40, said chemical - 39 32. The recombinant vector of claim 40, wherein said chemical agent is selected from the group consisting of gene regulators, labels, and drugs. 33. A DNA segment encoding a fusion protein comprising a CR2 receptor binding ligand and inducing endocytosis and a chemical agent. 34. The DNA segment of claim 45, wherein said ligand comprises a peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO. SEQ ID NO: 9 and sequences exhibiting substantial homology therewith. 35. The DNA segment of claim 46, wherein said ligand has the amino acid sequence set forth herein as SEQ ID NO: 1. 36. Segment DNK according to claim 47, characterized s e t and m that mentioned chemical the agent comprises a cytotoxin. 37. Segment DNK according to claim 48, characterized s e t and m that mentioned cytotoxin contains ricin A. 38. Segment DNK according to claim 47, characterized s e t and m that mentioned chemical the agent comprises an antigen. 39. Segment DNK according to claim 47, characterized s e t and m that mentioned chemical the agent is selected from the group consisting of gene regulators, labels and drugs.