Note: Descriptions are shown in the official language in which they were submitted.
~ W096/08263' '2 T 9 8 3 6 ~ pcTruss~llsl5
5T~TT~rT~T.T.~T.~TI DELIVERY OF r~MT~T. AGENTS
TO A SPECIFIC CELL TYP3
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
. . _ .
2 1 9836 1
W096/08263 PCT~S95/11~15
12
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 ~
16
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
17
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
18
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
- 2198361
21
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
22
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
23
~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~ -
24
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
26
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
27
- - 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
28
5(Xi) ! __ DES~KI~lvN: SEQ ID NO~
Glu Asp Pro Gly Phe Phe Asn Val Glu
1 5
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 ..
1 5 10
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
TTTGGTAC 68
(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
1 5 10
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
GGTAC 65
(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
1 0
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
