CA2198361A1 - Intracellular delivery of chemical agents to a specific cell type - 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

~ W096/08263' '2 T 9 8 3 6 ~ pcTruss~llsl5 5T~TT~rT~T.T.~T.~TI DELIVERY OF r~MT~T. AGENTS

g_-k.-iround of the Invention This invention relates to delivery of chemical agents to cells. More particularly, this invention relates to compositions and methods for intracellular delivery of chemical agents to a specific cell type, i.e. cells expressing the CR2 receptor.
Toxins that target cell surface receptors or antigens on tumor cells have attracted considerable attention for treatment of cancer E.~., I. Pastan & D.
FitzGerald, Recombinant Toxins for Cancer Treatment, 254 Science 1173 (1991); U.S. Patent Nos. 5,169,933 and 5,135,736 to Anderson et al.; U.S. Patent Mo. 5,165,923 to Thorpe et al.; U.S. Patent No. 4,906,469 to Jansen et al.; U.S. Patent No. 4,962,188 to Frankel; U.S. Patent No. 4,792,447 to Uhr et al.; U.S. Patent Nos. 4,450,154 and 4,350,626 to Masuho et al. These agents include a cell-targeting moiety, such as a growth factor or an antigen binding protein, linked to a plant or bacterial toxin. They kill cells by me-~An;l different from conv-nt;nnAl chemotherapy, thus pot-nt;Ally reducing or _1; m; nAt; ng cross resistance to conv_nt;nnAl chemotherapeutic agents.
The membrane glycoprotein CR2, also known as CD21, occurs on mature B lymphocytes (B cells) and certain epithelial cells, such as human pharyngeal epithelial cells, human follicular dendritic cells, and cervical epithelium, and is a receptor for~both Epstein-Barr Virus (B V) and complement fragments C3d/C3dg. N.
Miller & ~.M. ~utt-Fletcher, 66 J. Virol. 3409 (1990).
This receptor is a 145 kD I ' - glycoprotein that, in addition to its binding function, is also involved in a pathway of B cell activation. E.~., G.R. Nemerow, et al., I~-nt;f;~ ;nn and characterization of the Epstein-Barr virus receptor= on human B lymphocytes and its relationship to the C3d complement receptor (CR2), 55 J.
Virol 347 (1985). Infection of B cell~ by EBV is .

W096~8263 2 1 9 8 3 6 1 PCT~S95/11515 initiated by selective binding of the gp350/220 envelope glycoprotein of the virus to the CR2 receptor, ~ollowed by ;ntPrn~l;7at;nn of the CR2 receptor and endocytosis of the receptor-bound virions. E.c., Tedder et al.
(1986), Epstein-Barr ~ virus binding induce~
;ntPrn~l;7~tion of the C3d receptor: a novel immunotoxin delivery system, 137 J. Immunol. 1387 tl986).
Epithelial cells cnnt~;nlng the CR2 receptor also bind EBV, but apparently such cells are infected by a ~h~n;rm other than receptor-mediated endocytosis.
Nemerow et al., Identification of gp350 as the viral glycoprotein mediating attachment of Epstein-Barr virus (EBV) to the EBV/C3d receptor of B cells: se~uence homology of gp350 and C3 complement fragment C3d, 61~J.
Virol. 1416 (1987), have identified domains of amino acid sequence similarity between C3dg and gp350/220, ;nrltl~;ng a domain near~the: N-terminus of gp350/220 (Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu; SEQ ID NO:l) that corresponds to a sequence in C3dg (Glu-Asp-Pro-Gly-Bys-Gln-~eu-Tyr-Asn-Val-Glu; SEQ ID NO.2). Nemerow et al., Identification of an epitope in the ma~or envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2), 56 Cell 369 (1989), have also described binding of a synthetic tetr~Pr~pPrtide rQnt~;n;ng the amino acid ser1uence ;~Pnt;f;P~ as SEQ ID NO:l both to the purified CR2 receptor and to CR2-expressing B cells. This synthetic peptide also blocked binding of recombinant gp350/220 or C3dg to the CR2 receptor on B cells, and a similar synthetic peptide inhibited EBV infection in vitro.
Analysis of trnnrAt;nn and substitution peptide analogs showed that the EBV epitope involved in CR2 binding is rnnt~;nPd within the Glu-Agp-Pro-Gly-Phe-Phe-Asn-Val-Glu ser~uence (SBQ ID NO:l). Reduced levels of binding were observed with shorter peptides, although the Glu-Asp-~0 Pro-Gly (SEQ ID NO:3) peptide retained Significant CR2 binding activity. A peptide cnnt~n;ng a single amino ~ W096t08263 2 1 9 8 3 6 1 PCT~595/11515 acid substitution of glycine for proline within thi3 region also exhibited significantly reduced CR2 binding activity.
In view of the foregoing, it will be appreciated that ~ a;~;ons for intracellular delivery of chemical agents to CR2=~receptor-bearing B cells and methods of use thereof would be significant adv~nr a in the art.

Obiects and SummarY of the Invention It is an object of the presen~ invention to provide compositions for intracellular delivery of selected chemical agents to a specific cell type, i.e. cells expressing the CR2 receptor, to which binding triggers receptor-mediated endocytosis.
It is also an object of the invention to provide methods of making and methods of using compositions for intracellular delivery of selected chemical agents to cells expressing the CR2 receptor.
It is another object of the invention to provide compositions and methods for intracellularly delivering selected chemical agents, such as cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like, to cells expressing the CR2 receptor.
It i8 still another object of the invention to provide peptide ligands that can be attached to selected chemical agents for binding of the chemical agents to CR2 receptors and endocytosis of the chemical agents.
These and other objects can be sc ~1; ah~ by providing a composition for specific intracellular delivery of a ~h~m; ~1 agent into a CR2 receptor-bearing cell in a population of cells including non-CR2-receptor-bearing cells, comprising a ligand (CBEL) capable of binding to the CR2 receptor and inducing receptor-~~~;~t~d endocytosis and a chemical agent coupled to the ligand, wherein the chemical agent is WO9C/08263 2 1 9 3 3 6 l PCT~S95111515 capable of eliciting a selected effect when delivered ;ntr~n~llularly into the CR2 receptor-bearing cell.
Mature B lymphocytes are CR2 recep~or-bearing cells targeted by these compositions. Chemical agents that can be delivered to such :celIs include cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs. The CBEL and chemical agent can be bound to each other and/or to other functional moieties through a spacer, which can either be biodegradable, such as certain peptides, or non-biodegradable. The composition can further comprise a carrier-type system selected from the group consisting of water soluble polymers, liposomes, and particulates.
The compositions are used in vitro by cnntact;ng populations of cells with an effective amount of composition under conditions wherein the CR2 receptor binding and endocytosis-;n~nn;ng ligana (CBEL) binds the CR2 receptor and elicits endocytosis o~ the receptor-bound composition. For in vivo use, an effective amount of the composition is systemically administered so that the CBEL contacts and binds to CR2 receptors on mature B lymphocytes and then induces endocytosis of the composition. Once inside the cells, the chemical agent elicits its selected effect.

Brief Descri~tion of the Drawinas FIGS. lA and lB illustratively depict chemical conjugation of a CBEL with a chemical agent having a free sulfhydryl group to form a composition according to the present invention.
FIGS. 2A-2D show steps in constructing a plasmid for expressing a fusion protçin~cnnt~in;n~ a CBEL and a chemical agent peptide according to the present invention.
FIG. 3 shows a comparison of the~=effects on CR2' B
cells (darker bars) and CR2- T cells (lighter bars) ceIls of exposing the cells in vitro to various~concentrations .

W096/08263 2 1 9 8 3 6 1 PCT~S95/ll5l5 ,5 of ar,:C3EL-rici~ A fusion protein according to the present invention.

Detc;1ed Descri~tion of the Invention Before the present compositions and methods for intracellular delivery of chemical agents to a specific cell type are disclosed and described, it is to be understood that this invention is not limited to the particular l ~; c, process steps, and materials disclosed herein as such embodiments, process steps, and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular ~ 8 only and is not ;nt~n~d to be limiting since the scope of the present invention will be limited only by the appended claims ana equivalents thereof.
It must be noted that, as used in this specification and the ~pr~n~P~ claims, the singular forms "a, n "an," and ~the~ include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a compositio~ r~nt~in;ng "a ligandn i n~l p~c two or more ligands, reference to "a chemical agent" includes reference to one or more of such chemical agents which may be the same or different chemical agents, and reference to "a gpacer" in~ c reference to two or more spacers.
In describing and claiming the present invention, the following terminology will be used in accordance with the de~i~itions set out below.
As used herein, "peptide" means peptides of any length and ;n~ln~Pc proteins. The terms "polypeptide"
~ and ~'oligopeptide"= are used herein without any particular int~n~d size limitatior., unless a particular size is otherwise stated.
As used herei~, "CR2 receptor birding and endocytosis-inducing ligand" or "C3EB" means a composition capable of binding to the CR2(CD21) receptor W096/08263 2 t 9 8 3 6 1 PCT~S95/11515 and ;n~nc;ng intPrnAl;zation by endocytosis of the receptor and receptor-bound~C3EL. According to the present invention, CBELs are coupled to various functional molecules so that upon endocytosis of the C.3ELs, the various functional molecules coupled thereto are also intPrnv1ized by the CR2-bearing cells.
According to current understanding, a C~3EL can be derived from the EBV gp350/220 glycoprotein, including SEQ ID NO:l and flvnking sequences; the C3dg peptides, including SEQ ID NO:2 and fl~nk;ng sequences; or peptides substvnti~11y homologous therçto. As used herein, "substantially homologous" means peptides that retain functionality in binding CR2 receptors and tn~nrin~ receptor-mediated endocytosis although they may include fl~nk;ng sequences or be truncations, deletion variants, or substitution variants of SEQ ID NO:l or SEQ
ID NO:2. The minimum requirement for binding and ;n~llr;ng receptor mediated endocytosis appears to be the sequence ;~nt;f;ed as SEQ ID NO:3. Substitution variants are those that contain a conservative substitution of one or more amino acid residues. A
conservative substitution is a substitution of one amino acid residue for another wherein functionality of the peptide is retained, in this case, functionality in binding the CR2 receptor and Pl;c;tins endocytosis of the receptor-bound composition. Amino acid~ residues bPl~ng;ng to certain conservative substitution groups can s -t; -- substitute for another amino acid residue in the~same group. One such grouping is as follows:
Pro; Ala, Gly; Ser, Thr; Asn, Gln; Asp, Glu; ~is; Lys, Arg; Cys; Ile, Leu, Met, Val; and Phe, Trp, Tyr. M.
Jimenez-Montano ~ B. Zamora-Cortina, Evolutionary model for the generation of amino acid sequences and its application to the study of mammal alpha-hemoglobin chains, Proc. VlIth Int'l Biophysics Congress, Mexico City (1981). Other variations that are to be considered substantially homologous include substitution of D-amino ~ W096/08~3 2 t 9 8 3 6 I PCT~S951~l5l5 ~ 7 acids for t'he naturally occurring B-amino acids, substitution of amino acid derivatives such as those ~nnt~;n;ng additional side chains, and substitution of non-standard amino acids, i.e. ~-amino acids that are rare or do not occur in proteins. The primary structure of a CBEL is'limited only by functionality.
As used herein, "chemical agent" means and includes any substance that has a selected effect when intPrn~1; 7e~ into a B lymphocyte by endocytosis.
Certain chemical agents have a physiological ef~ect, such as a cytotoxic effect or zan effect on gene regulation, on a B cell when intrrn~l i 7Gd into the cell.
A ~transforming nucleic acid~ ~R~A or DNA), when int~rn~l;7ed into a cell, may be replicated and/or expressed within the cell. Other nucleic acids may interact with regulatory seruences or regulatory factors within the cell to ;nf11l~nre gene expression within the cell in a selected manner. A detectable label delivered intrac~1lnl~rly can permit identification of cells that have ;n~rn~l;7ed the compositions of the present invention by detection of the label. Antigens that are delivered to the interior of a cell can elicit an immune response specific to the antigen. Drugs or phar--cnlogically active ~ r can be used to ameliorate pathogenic effects or other types of disorders. Particularly useful rh~m;r~1 agents include polypeptides, and some such chemical agents are active fL~ of biologically active proteins, or are specific antigenic fragments (e.g., epitopes) of antigenic proteins. Thus, rh~m;r~l agents include cytotoxins, gene regulators, transforming nucleic acids, ~ labels, antigens, drugs, and the like.
As used herein, "carrier" means water soluble polymers, par~iculates, or liposomes. Such carriers may contain multiple sites to which one or more CBE~ and/or chemical agent can be coupled. ~uch carriers increase the molecular size of the compositions and may provide W096/08263 2 1 ~ 8 3 6 ~ PCT~S95/11515 added selectivity andtor stability. Such selectivity arises because carrier-containing compositions are too large to enter cells by passive diffusion, and thus are limited to ~nt~r; ng cells :through receptor-mediated endocytosis. Carriers comprising water soluble polymers can be used for linking of peptide ligands, chemical agents, and other functional molecules. The potential for use of such carriers for targeted drug delivery has been est~hl;~h~ See, e.g., J. Kopecek, 5 Biomaterials 19 (1984); E. Schacht et al~, Polysaccharides as Drug Carriers, in Controlled-Release Technology 188 (P.I. Lee & W.R. Good, eds., 1987); F. ~udecz et al., Carrier design: cytotoxicity and immunogenicity of synthetic hr~nchPd polypeptides with poly(L-lysine) backbone, 19 J. Controlled Reiease 231 (1992); Z. Brich et al., Preparation and characterization of a water soluble dextran ; ncnnjugate of doxorubicin and the monoclonal antibody (ABL364), 19 J. rnntrnlled Release 245 (1992). Thus, illustrative water soluble polymers include dextran, inulin, poly(L-lysine), methacrylamide-cnnt~;n;ng synthetic polymers, and the like.
As used herein, "drug" or "pharmacologically active agent" means any chemical material or compound suitable for intracellular administration in a CR2-bearing cell B which induces a desired biological or pharmacological effect in such cell.
As used herein, "effective amount" is an amount that produces a selected effect. For example, a selected effect of a composition ~nnt~;n;ng a=cytotoxin as the chemical agent could be to kill a selected proportion of mature B cells within a selected time period An effective amount of the composition would~be the amount that achieves this selected result, and such an amount could be determined as a matter of routine by a person skilled in the art.
The compositions of the present invention provide speciftc intracellular delivery of a chemical agent into ~ WogC/08263 2 ~ 9 8 3 6 ~ PCT~S95/11515 ; 5 a CR2 receptor-bearing cell in a population of cells including non-CR2-receptor-bearing cells, the compositions comprising a CBEL capable of binding to the CR2 receptor and ;n~n~;ng receptor-mediated endocytosis and a chemical agent coupled to the CBE~, wherein the chemical agent is capable of eliciting a selected effect when delivered intracellularly into the CR2 receptor-bearing cell ~Mature B cells are especially targeted by these compositions. The c~emical agents are selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, drugs, and the like. The coupling of a CBEL to a chemical agent can be, without limitation, by covalent bond, electrostatic interaction, hydrophobic interaction, physical encapsulation, and the like. The coupling of a C!3E~ to a chemlcal agent can also be direct or through another functional moiety. Thus, the compositions can further comprise a spacer coupled to and interposed between the CBEL and the chemical agent. Such spacers ca~ be biodegradable or non-biodegradable, and peptide spacers are preferred. The compositions of the present invention can further comprise a carrier selected from the group consisting of water~ soluble polymers, lipoaomes, and part; Clll ~t~c. When the carrier is a water soluble polymer, the composition has the formula:
[I.-S~I]b-C-[Se-A] r wherein L is the ligand (C3E~) capable of binding to the CR2 receptor and ;n~ ;ng endocytosis thereof; A is the chemical agent; S is a spacer; C is the water soluble polymer having functional groups compatible with forming covalent bonds with ligand, chemical agent, and spacer;
a and e are D or 1; and b and f are integers of at least 1. Such water soluble polymers are selected from the yroup consisting of dextran, inulin, poly(~-lysine), methacrylamide-r~nt~;n;ng polymers, and the like.
Thus, according to the invention, the CBEB provides means for the composition to bind the CR2 receptor on _, ~ _ _ _ _ _ _ _ _ _ . _ _ _ _ _ . . _ .

wosc/o8263 2 1 ~ 8 3 6 1 PCT~S95/11515 mature B cells, thus triggering ;n~rn~l i7~ticll of the compositions by endocytosis. The chemical agent provides means for achieving a selected effect in the B
cells. Accordingly, for example, chemical agents comprise cytotoxins, including rA~;nnnrl;des, for selective killing or ~ hl ;nr~ of cells displaying the CR2 receptor; antigens for eliciting a selected immune response; nucleic acids for genetically transforming~or regulating gene expression ~in B cells; drugs or other pharmacologically active agents for achieving a selected therapeutic effect; labels, including fluorescent, radioactive, and magnetic labels, for permitting detection of cells that have taken up the compositions;
and the like.
Optionally, the compositions of the present invention further comprise a water soluble polymeric carrier 80 that a plurality of CB~s and/or rh~m; r~l agents with selected functionalities can be bound together in a complex molecule. At least 1 chemical agent and at least l CBEL are bound to such carriers, with the preferred number of CBE~s coupled to chemical agents in the range of 1 to about lOOO. Such carriers increase the molecular size of the compositions and may provide added selectivity ana/or stability to the fllnr~;nnAl moietieg beyond what would be achieved without the carrier. Advantageously, coupling of the CBELs and/or rh~m;r~1 agents is accomplished by means of biodegradable or non-biodegradable spacers. Such spacers can be selected for their relative susceptibility or resistance to hydrolysis and/or enzymatic cleavage inside B cells. This selectivity provides a practitioner of this art the ability to choose a spacer based on whether it would be most advantageous to have the chemical agent remain coupled to the carrier within the cells or to be released from the carrier by intracellular enzymatic activity. Use of ~ W096/08263 2 1 ~ 8 3 ~ 1 PCT~S95/11515 .

11 .
liposomes and particulates as carriers is independent of use of a water soluble polymer carrier.
In some embodi~ents, the compositions are constructed by chemically conjugating a CBEL to a chemical agent. "Chemically conjugating~' the CBEL to the chemical agent, as that term is used herein, means covalently bonding the CBEL to the chemical agent, either directly or by way of a coupling moiety. In particular embodiments, where the chemical agent is a peptide, a coupling moiety can be used to form a linkage between functional groups on the CBEL and the chemical agent. For example, compositions cnnt~;n;n, a CBEL and a chemical agent peptide can be formed by coupling a sulfhydryl group on the chemical agent and an amino group on the CBEL through a heterobifnnrt;nnAl crosslinker. A reaction scheme for chemically conjugating a CBEL and ricin A through a maleimide crosslinker is shown in FIGS. lA and lB, for example.
Besides maleimide crnrRl;nkPrs for chemically conjugating CBELs to chemical agentg rnnt~;n;ng sulfhydryl groups, haloace~yl, ~alkyl halide, alkyl sulfonate, ~,B-unsaturated carbonyl, or ~,B-unsaturated sulfone moieties can be used as crosslinkers. For chemically conjugating CBELs and rhPm;r~l agents rnnt~;n;ng amine groupg, active esters can be used as crnRRl;nk~rs. ~ther coupling moieties may be employed depending upon which functional group(s) on the CBELs and on the chemical agent are available.
The compositions of the present invention can also be produced in a genetically engineered organism, such as E. coli, as a "fusion protein." That is, a hybrid gene rnnt~;n;nr~ a se~uence o~ nucleotides encoding a CBEL and a seguence of nucleotides ~nro~;ng a chemical agent peptide can be constructed by recombinant DNA
technology. This hybrid gene can be inserted into an organism such that the "fusion protein" encoded by the hybrid gene is exp~essed. The fusion protein can then . . _ .

W096/08263 PCT~S95/11~15 be purified by standard methods, including affinity ChL ~ torraphy.
Eusion proteins rrntAininr a CBE~ and a chemical agent peptide according to the present invention can also be constructed by chemical synthesis.
W4ere the compositions of the present invention are produced as fusion proteins, the CBEL and the chemical agent peptide can be immediately adjacent~ to one another, that is, the carboxyl end of the CBE~ can be bonded directly to the amino end of the chemical agent, or vice ver~a. Alternatively, the iusion protein can also include A~;tion=1 amino acid resiaues between the CBE~ and the rh~m; rAl agent, such that these additional amino acid residues serve as a spacer between the CBEL
and the chemical agent peptide. Short peptide ligands are generally preferred, both because short peptides can be manipulated more readily and because the presence of additional amino acids rrqi~7l~r, and particularly of substantial numbers of additional amino acids residues, may interfere with the function of the peptide ligand in in~nr;nr; ;nt~rnA1;7~t;~n of the chemical agent by endocytosis.
Compositions according to the present invention can also iurther include a protease digestion site situated 80 that once the composition is within the cell, the chemical agent can be separated from the CBEL by proteolysis of the digestion site. Suc4 digestion sites occur naturally in the gp350/220 glycoprotein adjacent to the SEQ ID ~O:l segment, thus the CBE~ portion of the composition can conveniently extend to include such sites. Such a protease susceptible spacer can be added regardless of whether the composition is synthesized chemically or as an expression peptide in a genetically engineered organism. In the latter case, nucleotides encoding the protease susceptible spacer can be inserted into the hybrid gene between the CBEB-encoding segment ~ W096/08263 2~98361 pcT~ssslll5ls and the chemical agent peptide-encoding segment by technir1ues well known in the art.
Another aspect of the present invention features a method for specifically effecting a desired activity in CR2-expressing cells rnnt~;nr~ in a population of non-CR2-expressing cells, by steps of contacting the population of cells with a composition rnnt~;n;ng a CBEL
coupled to a rhrm;r~l agent that directs such activity intracellularly. The compositions of the invention are selectively bound to CR2-expressing cells in the mixed population, whereupon endocytosis of the composition into the cells is induced, and the chemical agent effects its activity within the CR2-expressing cells.
This application employs, except where otherwise indicated, standard technir~ues for ~-n;p~ tion of peptides and for manipulation of nucleic acids for expression of peptides. Technir~ues for conjugation of oligopeptides and oligonuclçotides are known in the art, and are described for example in T. Zhu et al., 3 Antisense Res. Dev. 265 (1993); T. Zhu et al., 89 Proc_ Nat'l Acad. Sci. USA 7934 (1992); P. Rigaudy et al., 49 Cancer Res. 1836 ~1989).
As is noted above, the invention features peptides, employed as CBELs in compositions also cnntA;n;nS
rh~m;r~l agentg, which chemical agents may also be peptideE or contain peptides. The peptides according to the invention may be made by any of a variety of techniriues, including organic synthegig and re~ ~;n~n~
DNA methods. Technir~ues for chemical synthesis of peptides are described, for example, in B. Merrifield et al., 21 Biochemistry 5020-31 (1982); Houghten, 82 Proc.
Nat'l Acad. Sci. USA 513~-B5 (1985), incorporated herein by reference. Terhn;rll-rr for chemical conjugation of peptides with other molecules are known in the art.
A fusion protein according to the invention can be made by expression in a suitable host cell of a nucleic acid rnnt~;n;n9 an oligonucleotide rnro~;n~ a CBEB, as _ _ _ _ _ . . . . , ~ :, . . ~ .

W09~08~3 2 t ~ 8 3 6 1 PCT~S95/ll515 described above, and an: oligonucleotide encoding a chemical agent peptide. Such techniques for producing re~ ';n~nt fusion proteins are well-known in the art, and are described generally in, e.g., J. Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., 1989), the pertinent parts :of which are- hereby incorporated herein by reference. Reagents useful in applying such techniques, such as restriction enzymes and the like, are widely known in the art and commercially available from any of several vendors.
Construction of compositions cnnt=;ning a CBEL and chemical agent peptide A~nn~;ng to the invention will now be described, with particular reference to examples in which a CBEL is conjugated with the cytotoxic chemical agent peptide, ricin A. Ricin is a toxic~0 glycoprotein produced by the castor plant (Ricinus i~). It is composed of two subunits, the A chain and the B chain, both about 30 kD in molecular weight, linked together by a disulfide bond. Ricin is ~y~th~R; 7e~ as a large precursor that is processed to yield mature ricin A chain and B chain subunits. The A
chain is an enzyme that cleaves a glycosidic bonq in 285 ribosomal RNA, thereby destroying the ability of ribosomes to synthesize protein. The A chain can inactivate about 1500 ribosomes per minute, which means that a single ;nt~rn~1l7~ molecule of ricin A is lethal to a cell. S. Olsnes et al., Ribosome inactivation by the toxic lectins abrin and ricin, 60 Eur. J. Biochem.
281 (1975). The B chain binds to galactose moieties on the surface of a cell, an event nPc~q~ry for internalization of ricin. Removal of the B chain prevents the A chain from entering a cell, thus rendering the A chain inactive. Coupling the ricin A
chain to a CBEL permits the~ricin A chain to enter a cell by receptor-mediated endocytosis, resulting in an active cytotoxin ~ W096/08263 2 1 9 8 3 6 ~ PcT~sgS/Il5l5 In a first example, the composition is formed by chemical conjugation; and in a second example, the composition is formed as a recombinant fusion protein.

Example.l Ch~m; cal coniuqation of a ~RT'T~ and Ricin A
By way of illustration, chemical CUllj UUdtiOn of a CBEL having the amino acid~se~uence~Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID N~:l) with the cytotoxic chemical agent peptide, ricin A, was performed i~ a two-step process, as shown in FIGS. lA and lB.
In the first step, the CBEL (made by Peptide TntPrnAt;onal, Rentucky, USA) was activated by reaction with m-maleimidobenzoyl-N-hyd,u~yuulfos~ ;n;m;~P ester ("sulfo-MBS"; Pierce, Rockford, Illinois, U5A) essentially as described in the supplier's instructions.
Briefly, the CBEL was mixed with sulfo-MBS in a molar ratio of 1:10 in PBS buffer (20 mM sodium phosphate, 0.15 M NaCl, pH 7.0), for 2 hours at room temperature.
The resulting activated peptide was purified by FPLC
using Superose 12 (Pharmacia) according to standard methods.
In the second step, the maleimide-activated peptide was reacted with deglycosylated ricin A (Sigma Chemical Co., St. Louis, Missouri, USA) by mixing in a molar ratio of 1:5 in PBS buffer for 1 hour at room temperature. Unreacted peptide molecules were removed from the CBEL-Ricin A conjugate by dialysis overnight at 4~C through a membrane having a 6-8 kilodalton cutoff value.
The resulting composition was estimated to have a molecular weight of about 32 kD by gel electrophoresis on a 10~ SDS-PAGE gel, U. Laemmli, 227 Nature 680 (1970). Thus, the composition prepared according to this example c~nt~;nPd one CBEL per molecule of ricin A.
Three-dimensional computer ~-1 ;ng ghows that the CBEL
is most likely coupled to the Cys residue at the C-: . . . , . . . _ W096/08263 PCT~S95111515 2~ 9836 ~

trrm;n~l end of the ricin A molecule, ;nA! rh as the N-terminal Cys residue is buried internally in the folded ricin A molecule.

Example 2 Recomb;nAnt ~rotein contA;n;nn a CBE~ and Ricin A
A fusion protein cnntAln1n~ a CBBL having the amino acid sequence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu ~SE~
ID NO:l) and the cytotoxic chemical agent peptide, ricin A, was formed by means of rer~ ~;nAnt DNA technology.
Briefly, the fusion protein in this example was made by inserting a synthetic oligonucleotidr: that encodes t~he CBEL downstream from a polynucleotide ~nrn~;ng ricin A
in an E. coli expression vector, expressing the fusion protein at a high yield in an E. coli host, and then purifying the fusion protein from a cell lysate by affinity chromatography.
Referring now to FIGS. 2A-2D, a fusion protein rnntA;n;n~ the CBEL and ricin A was produced by r~rnmhirAnt DNA ter~nnlnrJy as follows. The E. coli expression vector pTrcHis B (Invitrogen, San Diego, California) (FIG. 2A), containing a multiple cloning site ~MCS) downstream of a trAnqlAtinn initiation site (ATG), was digested with restriction Pn~rmlrlrA~PA NcoI
and BamHI, and the resulting cohesive ends were converted to blunt ends with T4 DNA polymerase and religated to generate pTrc B (FIG. 2B). A~863 bp Bam~l-KpnI fragment encoding ricin A was isolated from plasmid pAKG (obtained from Robert Weaver, University of Kansa~, Lawrence; described in R. C. Halling et al., 13 Nucleic Acids Res. 8019 (1985)), and cloned into BamHI and KpnI-digested pTrc B. The resulting construct was digested with BamHI, the cohesive ends converted to blunt ends with T4 DNA polymerase, and religated to yield the correct reading frame for translation of the cloned gene (FIG. 2C).

~ W096~8263 2 t 9 8 3 6 1 PCT~S9SIIIS15 The resulting modified pTrc B vector, rnnt~in1ng a ricin A-~nrn~ng sequence, was then digested with KpnI
and EcoRI, and ~he following synthetic oligonucleotide, rnnt~;n;ng nucleotide regidues encoding the C~3EL and cohesive ends ~ ~ t;hle with cloning at KpnI and EcoRI
sites, was ligated thereto.

(SEQ ID NO:4) 5'- CA AAT TTT AAT GAA GAT CCT
(SEQ ID NO:5) 3'- CAT GGT TTA A~A TTA CTT CTA GGA
(SEQ ID NO:6) Asn Phe Asn Glu Asp Pro (SEQ ID NO:4) GGT TTT TTC AAT GTT GAG CAT CAT
(SEQ ID ~0:5) CCA A~A AAG TTA ~AA CTC GTA GTA
(SEQ ID NO:6) Gly Phe Phe Asn Val Glu Xis Xis (SEQ ID NO:4) CAT CAT CAT CAT TAA G -3' (SEQ ID NO:5) GTA GTA GTA GTA ATT CTT AA -5' (SEQ ID NO:6) His Xis Xis Xis The resulting vector (FIG. 2D), cnnt~in~ a hybrid gene ~nrn~ln~ a ricin A-protease site-ligand-Xis 6 fusion protein, wherein "protease site" signifies a protease dige8tion site or protease susceptible spacer as described above, ~ligand" signifies the CBE~, and rXis 6" signifies a region of 6 consecutive His residues, the function of which will be described below.
The resultant piasmid was then used to transform E. coli cells, and transformants were selected and grown in hB
medium, J. Miller, Experiments iL Molecular Genetics, Cold Spring Xarbor Laboratory, Cold Spring Harbor, N.Y.
(1972). The cells were then lysed and the rec~ ' in~nt fusion protein was purified by affinity chromatography on a column rnnt~;n;n~ a nickel-charged re~in ("PROBOND", Invitrogen, San Diego, California). The six His residues at the C-terminus of the fusion protein bound electrostatically to the nickel atoms on the "PROBOND" resin. The resin rnnt~inlng the bound fusion protein was then washed to remove rnnt~minAntp Then, the electrostatic bonds were broken and the fusion protein eiuted in an imidizole-rnnt~1n;ng elution buffer that displaced the His residues from the nickel-charged ~, .

W096/08263 2 1 9 8 3 6 1 PCT~S95lll5l5 resin. This purification :was done according to the supplier~s manual.

Example 3 A fusion protein r~nt~lning the a CBEL having the amino acid 3e~uence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu (SEQ ID N0:1) and ~he cytotoxic chemical agent peptide, ricin A, waE formed by means of recombinant DNA
te~n~l ~gy as in Example 2 with the exception that the region c~nt~ln;ng six consecutive Xis residues was omitted. Thus, after digestion of the modified pTrc B
vector~ with EcoRI and KpnI, the following synthetic oligonucleotide, containing nucleotide regidueg Pnr~ing the CBEL and cohesive ends ~m~t; hle with cloning at KpnI and EcoRI sites, waE ligated thereto.

(SEQ ID N0:7) 5'- CA AAT TTT AAT ATC CAT CTC
(SEQ ID N0:8) 3'- CAT GGT TTA A~A TTA TAG GTA GAG
(SEQ ID N0:9) Asn Phe Asn Ile Xis Leu (SEQ ID N0:7) ACG GGT GAA GAT CCT GGT TTT TTC
(SEQ ID N0:8) TGC CCA CTT CTA GGA CCA A~A AAG
(SEQ ID N0:9) Thr Glu Glu AEP Pro Gly Phe Phe (SEQ ID N0:7) AAT GTT GAG TAA G -3' (SEQ ID N0:8) TTA CAA CTC ATT CTT AA -5' (SEQ ID N0:9) AEn Val Glu The reEulting vector, cnnt~lnP~ a hybrid gene encoding a ricin A-protease~ site-C8EL fusion protein, wherein "protease site" signifies a protease digastion site or protease susceptible~spacer as described above.
The resultant plasmid waE then used to tran3form E. coli cells, and tr~nqf~rr~ntq were selected and grown in ~B
medium. The expressed protein was isolated by lysing the cells with 1 mg/ml of lysozyme in 20 mM sodium phosphate, pX 7.8, and sonicating three times for 1 minute each. The fusion protein was insoluble under these conditions, but most E. coli proteins were soluble. The lysate was centrifuged at 9000 rpm for 30 minutes, and the resulting fusion ~protein-containing __ _ .... _ . __...... . _ .. ... _ .. :_: ::: _ .

~ w096l08263 : 2 ~ 9 8 3 6 1 PCT~S95/11515 pellet was resuspended and sonicated for 1 minute before being centrifuged again. The steps of resuspension, sonication, and centrifugation were repeated three times. The final pellet, rr,nt~;n;ng a relatively pure preparation of fusion protein, was dissolved in a solution rrnt~in1nrJ 6 M urea and 5 M dithiothreitol.
This dissolved fusion protein was renatured by seri~nt;~lly dialyzing against 4 M urea; 2 M urea; and 20 mM sodium phosphate, 500 mM NaCl, pH 7.8.

Example 4 A fuslon protein rrnt~;n;n,r a CBE~ having the amino acid ser~uence Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu ~SEQ
ID N0:1) and the cytotoxic chemical agent peptide, ricin ~, was formed by means of recombinant DNA technology as in Example 2 with the exception that an additional cysteine residue was introduced into the fusion protein.
This construction gave higher yields of recoverable fusion protein than in Example 2 because the ~ree cysteine residue at the C-terminal end of the ricin A
chain could form an intramolecular disulfide bond with the new cysteine residue instead of with E. coli proteins. The E. coll expression vector pTrcHis A
(Invitrogen, San Diego, California), similar to the pTrcHis B vector of FIG. 2A except for having a different reading frame, was digested with restriction rn~nllr1ea5eg NcoI and BamHI. A synthetic DNA encoding 6 consecutive histidine residues and having NcoI and BamHI cohesive ends was then ligated to the vector.
Plasmid pAKG was digested with BamHI, and the released 893 bp fragment was recovered by electroelutio~ after electrophoresis in an agarose gel. This fragment was ligated into the BamHI and calf integtinal Alk~1;n~
phosphatase-digested modified pTrcHis A vector. The ligation mixture was used to transform E. coli strain XL-1 (Stratagene, Da Jolla, Califo-rnia). Transformants were selected and the orientation of the BamHI f . ~ _ . _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ .

W096/08263 2 1 9~3~ 1 PCT~S9~11515 in the vector was determined by digestion with BglII.
DNA from a transformant with the ricin A gene in the correct f,r;f-nt~tion for translation was digested with SacI and EcoRI, and the following synthetic oligonucleotide, ~fnt~;n;ng nucleotide residues f-n~f~;ng the CBE~ and cohesive ends ~ _~t;hle with cloning at SacI and EcoRI sites, was ligated thereto.

(SEQ ID N0:10) 5'- C GAA GAT CCT GGT TTT
(SEQ ID N0:11) 3'- TC GAG CTT CTA GGA CCA A~A
(SEQ ID N0:1) Glu Asp Pro Gly Phe (SEQ ID N0:10) -TTC A~T GTT GAG TAA G -3' (SEQ ID N0:11) ~AG TTA CAA CTC ATT CTT AA -5' (SEG ID N0:1) Phe Asn Val Glu The ligated DNA was transformed into expression host E.
coli strain BLR (Novagen, Madison, Wisconsin~, a eoA
strain also lacking on and om~T proteases.
~f '; n~nt protein was isolated by growing transformed cells in the presence of ampicillin at 37~C.
When the culture reached an optical density of 0.6-0.8 (600 nm), isopropylthiogalactoside (IPTG) was added to a final rnn~ntraticn of 1 mM to induce expression.
After 3 additional hours of growth, the cells were harvested and sn~r~n~ in buffer ~fnt~;n;ng 10 mM
Tris HCl, pH 7.6, 100 mM RCl, 20 mM EDTA, 10 mM 2-mercaptoeth~n~l, 0.05~ Nonidet P-40, and 0.5 mg/ml lysozyme, and was incubated for 15 minutes in an ice bath. The resulting lysate was sonicated in the presence of 0.5 mM phenylmethylsulfonylfluoride and centrifuged at 9,000 rpm for 30 minutes at 4~C.
Ammonium sulfate was added to 40~ saturation to precipitate soluble proteins, and the precipitated pellet was dissolved and dialyzed against 10 mM
Tris XCl, pH 7.4, 100 mM RCl. The dialysate was loaded onto a strong anion exchange column (Q Sepharo~e Fast Flow, Pharmacia) that had been eguilibrated with the same buffer. Under these conaitions, almost all of the ~ W096/08263 PCT~S95/11515 h~rtPri~l proteing were bound to the column and the unbound ~rar~inn rnnt~;n~ essentially purified recnmh;n~nt protein. mlhe recombinant protein was further purified by dialyzing against 4 M urea, 20 mM
sodium phosphate, 500 mM sodium chloride, pH 7.8. The dialysate was passed through a column cnnt~;n;ng nickel-charged "PROBOND" resin and was washed 5 times with 4 M
urea, 20 mM sodium phosphate, 500 mM sodium chloride, 5 mM ;m;~;7nle~ pH 6Ø The 6 histidine residues in the r~ro-~; n~nt protein caused the recombinant protein to bind to the resin by affinity interaction The column was washed twice more with the same buffer except for the imidizole cnnr~ntratiQn being raised to 30 mM. The recombinant protein was eluted in the same buffer except for having an imidizole nnnrPntration of 300 mM. The eluted protein was then renatured and refolded by first dialyzing against 2 M urea and then against 20 mM sodium rhngrh~te, 500 mM NaCl, pH 7.8.

Example 5 T~rr~eted deliverv of a cvtotoxin to B cells By way of illustration of targeted delivery of an chemical agent to CR2-expressing B cells, use of compositions of CBEL and chemical agent according to the invention will now be described, with particular reference to an example in which a composition of ricin A and a CBEL is delivered to Raji B ly~rhnhl~ctoid cells in vlt~o for specific cytotoxic effect on the CR2-expressing B cells.
In a preliminary demonstrationr l x l06 CR2' Raji B
lymmhnhl~toid cells were incubated for 24 hours at 37~C
in l ml of RPMI 1640 culture medium (Hyclone, Logan, ~tah) with no additional treatment; in l ml of culture medium containing 20 ~g of the ricin A-CBEL composition of Example l above; or in l ml of culture medium rnnt~;n;nnJ ricin A alone. Cell death was determined following the incubations by trypan blue staining and , ... . . , .. .. . . _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ W096108263 ~l 9836 1 PCT~S9~11515 cell rsnnt; ng with a hemacytometer using an inverted microscope. Trypan blue is taken up by and imparts a blue color intracellularly to dead cells. An aliquot of cells was twice diluted in~o.4~ trypan blue stain (Sigma Chemical Co., St. Louis, Missouri) and ;n~nh~t~ for ~
minutes before c~nnt;ng~ The percentage of viable cells was calculated as the number of unstained cells per unit volume divided by the total number of stained and unstained cells x lO0.
Cells treated with ricin A alone a~d control cells appearea healthy. About 99~ of cells treated with the ricin A-CBEL composition died (l~ survival). Because ricin A is cytotoxic only when ;nt~rn~l ;7~ into the cell, these results show that the ricin A-CBEL
composition according to the invention resulted in int~rnAl;7at;~n of the cytotoxic chemical agent.

Example 6 The effect of the r~ ~;nAnt CB~L-ricin A fusion protein according to Example 2 was tested on CR2t human B lymrh~hlA~toid (Raji) cells and CR2- T (HSB2) cells as follows. A suspension of l x lO6 cells was thoroughly mixed with varying concentratisns of the purified re~- ';nAnt fusion protein in l ml of culture medium, and ;n~lhAt~ for 2~ hours at 37~C. Thereafter cell viability was assessed by staining the cells with trypan blue as in Example 5. As FIG. 3 shows, CR2~ cells (darker bars) responded in a dose-~p~n~nt fashion to treatment with the conjugate, while CR2- cells (lighter bars) were nn~f~t~ hy the treatment. At a fusion protein concentration o~~50 ~g/ml, survival of CR2~ B
cells was less than lO~, and survival sf C~2~ cells was lO0~. Treatment of both types of cells with either the CBEL alone or recombinant ricin A alone had no effect, supporting a conclusion that the toxic effect of the recombinant CBEL-ricin A fusion protein on the CR2' cells ~ W096/08~3 2 1 9 8 3 6 1 PCT~S9~11515 ~sults from int~rn~l;7ation of the re ~in~nt fusion protein via the CR2 receptor on the B cells.

Example 7 The ~,ucedu.~ of Example 6 was followed with the exception that the percentage of viable cells was determined by a colorimetric method using the tetrazoliumcompound(3-(4,5-dimethylthiazol-2-yl)-5-(3-ca-bu~y thn~yphenyl)-2-(4-sulfophenyl)-2H-tetrazolium~
inner salt; MTS) and an electron coupling reagent (phenazine methosulfate; PMS). MTS is bioreduced by cells into a formazan that is soluble in tissue culture medium. The ~hsnrh~nre of the fnrr~7Rn at 490 nm can be measured directly from 96 well assay plates without additional processing. The quantity of formazan product as measured by the Ahsnrh~nnp at 490 nm is directly proportional to the number of living cells in culture.
Reagents for the MTS assay were obtained from Promega Corp. (Madison, Wisconsin). Results obtained by this method were substAnt;~lly identical to Example 6.
~
Example 8 The ef~ect of the recombinant CBEL-ricin A fusion protein according to Example 3 was tested on CR2t human B lyl~hnhl~tcid ~Raji) cells and CR2- human T (HSB2) cells according to the procedure of Example 6. The results were substantially similar to those of Example 6.

Example g The effect o~ the reco~;n~nt CBEL ricin A fusion - protein according to Example 4 was tested on CR2 human B l~ _h~hl~toid (Raji) cells and CR2- human T (HSB2) cells according to the procedure of Example 6. The results were subst~nti~lly similar to those of Example 6. ~ ~

,. , ~

W096/08263 ~ 2 1 9 8 3 6 1 PCT~S9~/1151~ -The CBET-chemical agent compositions according to the present invention may be employed for target-specific delivery of a chemical agent to CR2-expressing cells, generally by contacting the CR2-expressing cells with the composition under conditions in which the CBEL
induces endocytosis o~ the composition into the CR2-expressing cells. The chemical agent then acts on~r within the targeted cell irto which the compobition is ;nt~rn~1;7Pd, and the degired effect of the active agent can be confined to t40se cells having a CR2' p4enotype.
For example, a CBE~-cytotoxic agent composition according to the invention can be employed as an effective antitumor agent in vivo, selectively killing CR2' B cells. Preierably, the compo3ition is administered to the subject by systemic administration, typically by snhr~lt~n~ollr~ intramuscular, or intravenous injection, or intraperitoneal administration.
Injectables for such use can be ~ L~d in convrnt;nn~
~orms, either as a liquid solution or buspension or in a solid form suitable for preparation as a solution or suspension in a liquid prior to injection, or as an emulsion. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and the like; and i~ desired, minor amountg of ~l~r;li~ry substances such as wetting or emulsifying agents, buffers, and the like may be added.
The composition may be contacted with the cells in vitro or In vivo. The CR2-expressing cells may constitute (and in most instances are expected to constitute) a subpopulation of a mixed population~of cell types; the peptide ligand according to the invention can provide for CR2-speci~ic endocytosis of the conjugate into CR2-expressing cells.
The chemical agent may have any of a variety of desired ef~ects in the targeted ceIls. As mentioned above, in some particularly useful embodiments the chemical agent is effective~ on a cell only when, or ~ W096108263 2 t 9 8 3 6 t PCT~S95/11515 pr;n~;pRlly when, the agent is ;ntorn~1;7ed into the cell Example lO
~arcTeted deliverv gf a rTiTTl-antiqpn;c aqent to B cells Compositions cgmprising a CBEL and an antigen according to the invention can be administered to a warm-blooded animal for targeted initiation of an immune response in CR2' cells. Particularly, the CBEL provides for CR2-mediated ;nt~rn~l;7~t;on of the antigen into the cells, and can result in initiation of an antibody-independent pathway for complement actiyation in the targeted cells. That is, according to the invention, the targeted cells can be induced to elicit an immune response against an antigen to which the cells are naive.
~ h~m; c~l conjugation of a CBEL having the amino acid seguence i~pnt;f;~d as SEQ ID N0:2 is activated and then coupled to chicken lysozyme (Sigma Chemical Co., St. ~ouis, Missouri) as in Example l. The CBEL-lysozyme conjugate is then systemically administered to a mouse.
The C3dg CBEL ~SEQ ID N0:2~ provides binding specificity to mouse B cells and induces CR2 receptor-mediated endocytosis of the conjugate. The conjugate then elicits an immune response by the targeted B cells against epitopes borne on the conjugate, including epitopes that are unique to the lysozyme portion of the conjugate. The results of this example can be subst~nt;~lly duplicated by construction of a CBEB-lysozyme fusisn protein.
Example ll A method of treating s cell 1 ~nk~m; A in a human comprises (a) providing a composition according to the present invention including a CBE~,~such as the EBV CBEL
(SEQ ID NO:l) or a peptide subst~nt;~lly homologous thereto, and a cytotoxin, such as ricin A, and (b) ... , . , _, .,, .. .. , . ,, ,, ,, . - , .,,,, . ., ~ ,. .. .. .... .

W096/08263 2 1 9 8 3 6 1 PCT~S9~11515 systemically administering an effective amount of the composition to an individual. Such composition can be made, for example, as shown above in Example 3. The EBV
CBEL targets mature human B cells and the ricin A~is cytotoxic to any cell into which it is delivered :~An effective amount of the composition is systemically administered to the individual so that the compositIon enters the bloodstream and contacts s cells. The CBEL
causes the composition to bind to the CR2 receptor on the B cells and induces ;nt~rn~l; 7~tion of the composition by endocytosis. The ricin A cytotoxin then kills the cell by destroying ribosomes. This procedure reduces the number of malignant B cells in the body of the individual, thereby having a positive effect in treatment of the disease.
Example 12 A method for treating an autoimmune disease, e.g.
lupus erythematosus or rheumatoid arthritis, follows the procedure of Example ll. Once delivered into s cells, the cytotoxin kills the cells, thus reducing the number of B cells producing ~lt~nt;hcdies~ thereby having a positive effect in treatment of the disease.

2 1 9836 ~
~ W096/08263 PCT~S95/1151 - - Seauence Llstinq (1) GENERAL INFORMATION:
(i) APPLICANT: Ramesh K. Prakash (ii) TITLE OF lh~ . : INTR~rT~TlT~TTn~R DE~rV~RY OF
C~EMICAL AGENTS TO
SPECIFIC CELLS
15 (iii) NUMBER OP S _ : ll (iv) CO~K~ _ ADDRESS:
(A) ~nn~C~ Thorpe, North & Western ~:B) STREET: 9035 South 700 East, Suite 200 20 ' C) CITY: Sandy D) STATE: Utah .E) COUNTRY: USA
:F) ZIP: 84070 25 (v) I ~L~ ~n~T-T~ FORM:
(A) MEDIT~M TYPE: Diskette, 3.5 inch, 720 R~ storage (B) ~ ~: Toshiba Satellite Tl800 (C) OPERATING SYSTEM: DOS 6.0 (D) SOFTWARE: Word Perfect 6.0 (vi) C~RRENT APPLICATION DATA:
(A) APPLI QTION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPT.Tr~ Nlk~ER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INF~ :
(A) NAME: Alan J. ~o~arth (B) REGISTRATION NUMBER: 36,553 (C) Y~r /DOCRET NUNBER: T2361 (iX) TT~'T. - ~T~'' INFORMATION:
(A) TT.!T, (801)566-6633 (B) TELEFAX: (801)566-0750 (2) lN~ 'TION FOR SEQ ID NO:l:
(i) I _ C~ARA~ ~KI~ L l~9:
(A) LENGT~: 9 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) .T~r~T.T~ TYPE: peptide W096108263 ~ 2 1 ~836 1 PCT~S95/11515 5(Xi) ! __ DES~KI~lvN: SEQ ID NO~
Glu Asp Pro Gly Phe Phe Asn Val Glu 10(2) Ih~ORMATION FOR SEQ ID NO:2:
(i) 1 _ r~aR~rT~D TR TICS:
(A) LENGTH: ll amino acids (B) TYPE: amino acid 15(D) TOPOLOGY: linear . .
(ii) .~cm.~ TYPE: peptide (Xi) ~LyU N~: DES~KI~lON: SEQ ID NO:2:
Glu Asp Pro Gly Lys Asn Leu Tyr Asn Val Glu ..

2 5 (2) INFORMATION FOR SEQ ID NO:3:
(i) 1 _ r~ARa~ T.~ll~s (A) LENGTH: 4 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) ~cm.~ TYPE: peptide (Xi) ~yl DES~Kl~ : SEQ In NO:3:
Glu Asp Pro Gly (2) INFORMATION FOR SEQ ID NO:4:
r~aRA-~r~TA ~
(A) LENGT~: 60 base pairs (B) TYPE: nucleic acid (C) STR~ ~A~ single (D) TOPOLOGY: linear (xi) ~yl ~A~KI~ : SEQ ID NO:4:
CaaATTTTAA TGA~GATCCT GGTTTTTTCa ATGTTGAGCA TCATCATC~T CATCATTAAG 60 (2) INF~R~_ FOR SEQ ID NO:5:
(i) Si~:yUL ._~!i rTTARA- ~ ~-~T-~Ll~;b (A) LENGTH: 68 base pairs (B) TYPE: nucleic acid (C) S~RA ~ .AS: cingle (D) TOPOLOGY: linear (xi) ~yl_ ~2~Kl~lu~: SEQ ID NO:5:

W096/08263 2 1 9 8 3 6 PcTn~95/11515 APTTCTTAAT GATGATGATG ATGATGCTCA l~r~T~r.~ AACCAGGATC TTCATTAAAA 60 (2) INFORMATION FOR SEQ ID NO:6:
(i) r~ r~ T.~ S:
(A) LENGT~: 18 amino acids (B) TYPE: amino acid (Dl TOPOLOGY: linear ~ nT~ TYPE: peptide (xi) : __ D~K1~L1U~ SEQ ID NO:6:
20 Asn Phe Asn Glu Asp Pro Gly Phe Phe Asn Val Glu His His Hls ~is His His (2) LNr6 ~- POR SEQ ID NO:7:
(i) ~u~ r~Dr ~ T.~i ~lCS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STR~ n~n~ : single (D) TOPOLOGY: linear (xi) ! _ Dk~Kl~L : SEQ ID NO:7:
ChaATTTTAA TATCCATCTC DrGrrT~ A;~,~ TTTCA~TGTT GAGTAAG 57 (2) INFO~- FOR SEQ ID NO:8:
(i) ~ r~ T~Ll~:
(A) LENGTH: 65 base palrs (B) TYPE: nucleic acid (C) 8r~ S: single (D) TOPOLOGY: linear ~ . ~. ~=.: = =
(xi) I __ D~K1~L- : SEQ ID NO:8:
AaTTcTTAcT CA~CaTTGAA ~ rr~r~.~ TCTTCACCC~ TGaGATGGAT ATTAaAATTT 60 (2) INFORMATION FOR SEQ ID NO:9:
r~ . . -T.~. L lC~g:
(A) LENGTH: 17 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) ,~r~T.~ TYPE: peptide W096~8263 2 1 9 8 3 6 1 PCT~S95/11515 (xi) ~b~UbN~: Db~KI~LU~: SEQ ID NO:9:
Asn Phe Asn Ile His Leu Thr Gly Glu Asp Pro Gly Phe Phe Asn Val Glu (2) INF~~~- FOR SEQ ID NO:lû:
(i) _ r~~~TR~TRTICS:
(A) LENGT~: 32 (B) TYPE: nucleic acid . . _.
(C) STR~r-~ S: single . (D) TOPOLOGY: linear (xi) ~byl ~KI~__ : SEQ ID NO:l0:
CGAAGATCCT G~L ' ' ~ '~ ATGTTGAGTA AG 32 (2) lNFO~M~TT~'- FOR SEQ ID NO:ll:
(i) I:ib~ RI'~ . . ~T~ CS:
(A) LENGTH: 40 (B) TYPE: nucleic acid (C) STR~ F: single (D) TOPOLOGY: linear (xi) ! _ DES~l~L_ : SEQ ID NO:ll:
~TTrTT~rT CAACATTGAA ~ rr~r.r~ TCTTCGAGCT 40

Claims (51)

Claims I claim :

1. A composition for specific intracellular delivery of a chemical agent into a CR2 receptor-bearing cell in a population of cells including non-CR2-receptor-bearing cells, consisting essentially of a ligand capable of binding to said CR2 receptor and inducing receptor-mediated endocytosis and a chemical agent coupled to said ligand, wherein said chemical agent is capable of eliciting a selected effect when delivered intracellularly into said CR2 receptor-bearing cell.

2. The composition of claim 1 wherein said CR2 receptor-bearing cell is a B lymphocyte.

3. 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, and SEQ ID NO;9.

4, The composition of claim 3 wherein said chemical agent is selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs.

5. The composition of claim 4 further comprising a spacer covalently bonded to and interposed between said ligand and said chemical agent.

6. The composition of claim 5 wherein said spacer is biodegradable.

7. The composition of claim 6 wherein said spacer comprises a peptide.

8. The composition of claim 7 wherein said ligand is the peptide having the sequence given herein as SEQ
ID NO;1 and said chemical agent is ricin A.

9. The composition of claim 5 wherein said spacer is non-biodegradable.

10. The composition of claim 4 further comprising a carrier selected from the group consisting of water soluble polymers, liposomes, and particulates.

11. The composition of claim 10 wherein said carrier is a water soluble polymer and said composition has the formula:
[L-Sa]b-C-[Se-A]f wherein L is said ligand capable of binding to said CR2 receptor and inducing endocytocis thereof; A is said chemical agent; S is a spacer; C is said water soluble polymer having functional groups compatible with forming covalent bonds with said ligand, chemical agent, and spacer; a and e are 0 or 1; and b and f are integers of at least 1.

12. The composition of claim 11 wherein C is selected from the group consisting of dextran, inulin, poly(L-lysine), and methacrylamide-containing polymers.

13. A method of specifically delivering a chemical agent in vitro into a CR2 receptor-bearing cell in a population of cells including non-CR2-receptor-bearing cells, comprising the steps of :
(a) providing a composition for specific intracellular delivery of a chemical agent into a CR2 receptor-bearing cell in a population of cells including non-CR2-receptor-bearing cells, consisting essentially of a ligand capable of binding to said CR2 receptor and inducing receptor-mediated endocytosis and a chemical agent coupled to said ligand, wherein said chemical agent is capable of eliciting a selected effect when delivered intracellularly into said CR2 receptor-bearing cell; and (b) contacting said population of cells with an effective amount of said composition under conditions wherein said ligand binds to said CR2 receptor on CR2 receptor-bearing cells and elicits endocytosis of said composition.

14. The method of claim 13 wherein said CR2 receptor-bearing cell is a B lymphocyte.

15. The method of claim 14 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, and SEQ ID NO:9.

16. The method of claim 15 wherein said chemical agent is selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs.

17. The method of claim 16 wherein said composition further comprises a spacer covalently bonded to and interposed between said ligand and said chemical agent.

18. The method of claim 17 wherein said spacer is biodegradable.

19. The method of claim 18 wherein said spacer comprises a peptide.

20. The method of claim 19 wherein said ligand is the peptide having the sequence given herein as SEQ ID
NO:1 and said chemical agent is ricin A.

21. The method of claim 17 wherein said spacer is non-biodegradable.

22. The method of claim 16 wherein said composition further comprises a carrier selected from the group consisting of water soluble polymers, liposomes, and particulates.

23. The method of claim 22 wherein said carrier is a water soluble polymer and said composition has the formula:
[L-Sa]b-C-[Se-A]f wherein L is said ligand capable of binding to said CR2 receptor and inducing endocytosis thereof; A is said chemical agent; S is a spacer; C is said water soluble polymer having functional groups compatible with forming covalent bonds with said ligand, chemical agent, and spacer; a and e are 0 or 1; and b and f are integers of at least 1.

24. The method of claim 23 wherein C is selected from the group consisting of dextran, inulin, poly(L-lysine), and methacrylamide-containing polymers.

25. A method of specifically delivering a chemical agent intracellularly into a CR2 receptor-bearing cell in a warm-blooded animal, comprising the steps of:
(a) providing a composition for specific intracellular delivery of a chemical agent into a CR2 receptor-bearing cell in a population of cells including non-CR2-receptor-bearing cells, consisting essentially of a ligand capable of binding to said CR2 receptor and inducing receptor-mediated endocytosis and a chemical agent coupled to said ligand, wherein said chemical agent is capable of eliciting a selected effect when delivered intracellularly into said CR2 receptor-bearing cell; and (b) systemically administering to said warm-blooded animal an effective amount of said composition under conditions wherein said ligand contacts and binds to said CR2 receptor on CR2 receptor-bearing cells and elicits endocytosis of said composition.

26. The method of claim 25 wherein said CR2 receptor-bearing cell is a B lymphocyte.

27. The method of claim 26 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, and SEQ ID NO:9.

28. The method of claim 27 wherein said chemical agent is selected from the group consisting of cytotoxins, transforming nucleic acids, gene regulators, labels, antigens, and drugs.

29. The method of claim 28 wherein said composition further comprises a spacer covalently bonded to and interposed between said ligand and said chemical agent.

30. The method of claim 29 wherein said spacer is biodegradable.

31. The method of claim 30 wherein said spacer comprises a peptide.

32. The method of claim 31 wherein said ligand is the peptide having the sequence given herein as SEQ ID
NO:1 and said chemical agent is ricin A.

33. The method of claim 29 wherein said spacer is non-biodegradable.

34. The method of claim 28 wherein said composition further comprises a carrier selected from the group consisting of water soluble polymers, liposomes, and particulates.

35. The method of claim 34 wherein said carrier is a water soluble polymer and said composition has the formula:
[L-Sa]b-C-[Se-A]f wherein L is said ligand capable of binding to said CR2 receptor and inducing endocytosis thereof; A is said chemical agent; S is a spacer; C is said water soluble polymer having functional groups compatible with forming covalent bonds with said ligand, chemical agent, and spacer; a and e are 0 or 1; and b and f are integers of at least 1.

36. The method of claim 35 wherein C is selected from the group consisting of dextran, inulin, poly(L-lysine), and methacrylamide-containing polymers.

37. A recombinant vector adapted for transformation of a host, said vector including a DNA
segment encoding a fusion protein comprising a CR2 receptor binding and endocytosis-inducing ligand and a chemical agent.

38. The recombinant vector of claim 37 wherein said vector comprises a plasmid.

39. The recombinant vector of claim 38 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, and sequences substantially homologous thereto.

40. The recombinant vector of claim 39 wherein said ligand has an amino acid sequence identified herein as SEQ ID NO:1.

41. The recombinant vector of claim 40 wherein said chemical agent comprises a cytotoxin.

42. The recombinant vector of claim 41 wherein said cytotoxin comprises ricin A.

43. The recombinant vector of claim 40 wherein said chemical agent comprises an antigen.

44. The recombinant vector of claim 40 wherein said chemical agent is selected from the group consisting of gene regulators, labels, and drugs.

45. A DNA segment encoding a fusion protein comprising a CR2 receptor binding and endocytosis-inducing ligand and a chemical agent.

46. The DNA segment of claim 45 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, and sequences substantially homologous thereto.

47. The DNA segment of claim 46 wherein said ligand has an amino acid sequence identified herein as SEQ ID NO:l.

48. The DNA segment of claim 47 wherein said chemical agent comprises a cytotoxin.

49. The DNA segment of claim 48 wherein said cytotoxin comprises ricin A.

50. The DNA segment of claim 47 wherein said chemical agent comprises an antigen.

51. The DNA segment of claim 47 wherein said chemical agent is selected from the group consisting of gene regulators, labels, and drugs.