Note: Descriptions are shown in the official language in which they were submitted.
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TAR~ G~CROMOLECU~AR PRODRUGS TO T LYn~PHOCYTES
ackground of the Invention
This invention relates to delivery of chemical
agents to cells. More particularly, this invention
relates to compositions and methods for intracellular
delivery of chemical agents to a specific cell type,
i.e. T lymphocytes.
Toxins that target cell surface receptors or
antigens on tumor cells have attracted considerable
attention for treatment of cancer. E.g., I. Pastan & D.
FitzGerald, Recomb;nant Toxins for Cancer Treatment, 25
Science 1173 (1991); Anderson et al., U.S. Patent Nos.
5,169,933 and 5,135,736; Thorpe et al., U.S. Patent No.
5,165,923; Jansen et al., U.S. Patent No. 4,906,469;
Frankel, U.S. Patent No. 4,962,188; Uhr et al., U.S.
Patent No. 4,792,447; Masuho et al., U.S. Patent Nos.
4,450,154 and 4,350,626. These agents include a cell-
targeting moiety, such as a growth factor or an antigen
binding protein, linked to a plant or bacterial toxin.
They kill cells by mechanisms different from
conventional chemotherapy, thus potentially reducing or
eliminating cross resistance to conventional
chemotherapeutic agents.
The membrane glycoprotein CR2, also known as CD21,
occurs on mature B lymphocytes (B cells) and certain
epithelial cells, such as human pharyngeal epithelial
cells, human follicular dendritic cells, and cervical
epithelium, and is a receptor for both Epstein-Barr
Virus (EBV) and complement fragments C3d/C3dg. N.
~iller & L.M. Hutt-Fletcher, 66 J. Virol. 3409 (1990).
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Thymocytes, peripheral T cell~, and T-cell lines have
also been found to express CR2 or CR2-like molecules.
C.D. Tsoukas & J.D. Lambris, Expression of ~RV/~3
Receptors on T Cel~s: Biological Signi~;cance, 14
Immunology Today 56 ( 1993). The reactivities of these
molecules with ligands or antibodies vary from those of
B cells, however, which suggested that there are
structural differences between such receptors on T-cells
and B-cells. J.A. Hedrick et al., Interact;on betwe~n
Epstein-R~3l~r Virus i3n~l a T Cel1 Tj ~e (~.~ ) v;a a
Receptor Phenoty~ically Distinct from Co~p~ement
Rece~tor Type 2, 22 Eur. J. Immunol. 1123 (1992). More
recently, EBV was discovered to bind and in~ect HSB-2 T
cells via a receptor distance from CR2. J.A. Hedrick et
al., ~h~r~cteriz~tion of a 70-kn~, RRC g~350/~0-R;n~;ng
Prote;n on HSB-2 T Cells, 153 J. Immunol. 4418 (1994).
The CR2 receptor is a 145 kD membrane glycoprotein
that, in addition to its binding function, is also
involved in a pathway of B cell activation. E.a., G.R.
Nemerow, et al., I~ntification ~n~ ~h~cter;~ on of
the ~stein-Barr V;rlls Rece~tor on Hl~m~n B Ly~phocytes
~n~ its RelatiQn~h;p to the C3~ Com~lem~nt Rece~tor
(C~), 55 J. Virol 347 (1985). Infection of B cells by
EBV is initiated by selective binding of the gp350/220
envelope glycoprotein of the virus to the CR2 receptor,
followed by internalization of the CR2 receptor and
endocytosis of the receptor-bound virions. E.a., Tedder
et al. (1986), Epstein-R~r- Virn~ Rin~;ng Induces
Intern~l;zat;on of the C3d Rece~tor: A Novel Immunotoxin
nelivery System, 137 J. Immunol. 1387 (1986).
Epithelial cells containing the CR2 receptor also bind
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EBV, but apparently such cells are infected by a
mechanism other than receptor-mediated endocytosis.
Some T-cell lines can be infected by EBV, while
infection of other T-cell lines is variable or
undetectable. C.D. Tsoukas & J.D. Lambris, ~Xpression
of ~RV/C3d Receptors on T Cells Riological
Signi~icance, 14 Immunology Today 56 (1993)- For
example, although HSB-2 T cells lack the CR2 receptor,
such cells are infected by EBV. J.A. Hedrick et al., 22
Eur. J. Immunol. 1123 (1992).
Nemerow et al., Ident;f~cation of gp350 as the
Viral Glycoprotein Mediating Attachment of Epste;n-Barr
Vlrus (EBV) to the ERV/C3d Receptor of B Cells: Sequence
Homology of g~350 and C3 Com~lement Fragment C3d, 61 J.
Virol. 1416 (1987), have identified domains of amino
acid sequence similarity between C3dg and gp350/220,
including a domain near the N-terminus of gp350/220
(Glu-Asp-Pro-Gly-Phe-Phe-Asn-Val-Glu; SEQ ID NO:1) that
corresponds to a sequence in C3dg ~Glu-Asp-Pro-Gly-Lys-
Gln-Leu-Tyr-Asn-Val-Glui SEQ ID NO:2). Nemerow et al.,
I~entificat;on of an ~,~ito~e ;n the Major ~nvelope
Protein of E~stein-Barr Virus that Mediates Viral
s;n~l;ng to the R T~ymphocyte ~BV Receptor (C~), 56 Cell
369 ~1989), have also described binding of a synthetic
tetradecapeptide containing the amino acid sequence
identified as SEQ ID NO:1 both to the purified CR2
receptor and to CR2-expressing B cells, but this
tetradecapeptide ~ailed to bind to HSB-2 T cells. This
synthetic peptide also blocked binding of recombinant
gp350/220 or C3dg to the CR2 receptor on B cells, and a
similar synthetic peptide inhibited EBV infection in
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vitro. Analysis o~ truncation and substitution peptide
analogs showed that the EBV epitope involved in CR2
binding is contained within the Glu-Asp-Pro-Gly-Phe-Phe-
Asn-Val-Glu sequence (SEQ ID NO:l). Reduced levels of
binding were observed with shorter peptides, although a
Glu-Asp-Pro-C~ly (SEQ Il~ NO: 3) peptide retained
signi~icant CR2 binding activity. A peptide containing
a single amino acid substitution o~ glycine ~or proline
within this region also exhibited signi~icantly reduced
CR2 binding activity.
Copending U.S. Patent Application Serial No.
08/305,770, ~iled September 13, 1994, describes
compositions and methods ~or speci~ic intracellular
delivery o~ a chemical agent into a CR2-receptor-bearing
cell, e.g. B lymphocytes. The compositions comprise a
CR2-receptor-binding and endocytosis-inducing ligand
(CBEL) coupled to the chemical agent. The CBEL binds to
the CR2 receptor on the sur~ace o~ B lymphocytes and
elicits endocytosis o~ 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, trans~orming nucleic acids, gene regulators,
labels, antigens, drugs, and the like. The composition
can further comprise a carrier such as a water soluble
polymer, liposome, or particulate.
It would be advantageous to develop prodrugs that
are speci~ically targeted to other cell types. For
example, targeting of T lymphocytes would enable
therapeutic applications ~or T-cell-associated diseases
and tissue gra~t rejection. Such T-cell-associated
-
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diseases include arthritis, T-cell lymphoma, skin
cancers, and diseases resulting from HIV infection.
In view of the foregoing, it will be appreciated
that compositions for intracellular delivery of chemical
agents to T cells and methods of use thereof would be
significant advancements in the art.
Ob~ects ~nd Sl~mm~ry of the ~nvent~on
It is an object of the present invention to provide
compositions for intracellular delivery of selected
chemical agents to a specific cell type, i.e. T
lymphocytes.
It is also an object of the invention to provide
methods of making and methods of using compositions for
intracellular delivery o~ selected chemical agents to T
lymphocytes.
It is another ob~ect of the invention to provide
compositions and methods for intracellularly delivering
selected chemical agents, such as cytotoxins,
transforming nucleic acids, gene regulators, labels,
antigens, drugs, and the like, to T lymphocytes.
These and other ob~ects can be accomplished by
providing a composition for intracellular delivery of a
chemical agent capable of eliciting a selected effect
when delivered intracellularly into a T lymphocyte, the
composition having the formula:
[L-S]a-C-[s-A] b
wherein L is a ligand capable of binding to a receptor
on the T lymphocyte and stimulating receptor-mediated
endocytosis of the composition; A is the chemical agent;
S is a spacer; C is a water soluble polymer having
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functional groups compatible with forming covalent bonds
with the ligand, chemical agent, and spacer; a is an
integer of at least 2; and b is an integer of at least
1. A preferred water soluble polymer is a copolymer o~
N-(2-hydroxypropyl)methacrylamide (HPMA). The ligand is
preferably a member of the group consisting of a peptide
with the amino acid sequence identified as SEQ ID NO:1
and peptides substantially homologous thereto, with a
peptide having the amino acid sequence of SEQ ID NO:1
being especially preferred. The chemical agent is
preferably a member selected from the group consisting
o~ cytotoxins, transforming nucleic acids, gene
regulators, labels, antigens, and drugs. Preferably,
the spacer is ~iodegradable such that the chemical agent
is detachable from the polymer inside a cell. More
preferably, the spacer comprises a peptide, and most
preferably the spacer is a peptide with the amino acid
sequence Gly-Phe-Leu-Gly (SEQ ID NO:4). The composition
can further comprise a carrier selected from the group
consisting o~ water soluble polymers, liposomes, and
particulates.
The compositions are used in vi tro by contacting
populations of cells with an effective amount of
composition under conditions wherein the ligand binds to
a receptor on the T lymphocyte and elicits endocytosis
of the receptor-bound composition. For in vivo use, an
effective amount of the composition is systemically
administered such that the ligand contacts and binds to
receptors on T lymphocytes and then stimulates
endocytosis of the composition. Once inside the cells,
-
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the chemical agent elicits its selected effect, although
some agents may be active at the cell membrane.
Brief Descript;on of the Drawings
FIG. 1 shows comparative in vi tro cytotoxic effects
of different concentrations of ligand-HPMA copolymer-
adriamycin composition No 177 on HSB-2 T cells (~),
CCRF-CEM T cells (0), MOLT-3 T cells (~), Raji B cells
(Q), and Daudi B cells (--).
FIG. 2 shows the in vi tro cytotoxic effects of
exposure to different concentrations of a ligand-HPMA
copolymer-adriamycin composition (No. 177; ~) and a HPMA
copolymer-adriamycin control composition (No. 237A; ~)
on CCRF-CEM hl1m~n T cells.
FIG. 3 shows the in vitro cytotoxic effects of
exposure to di~ferent concentrations of a ligand-HPMA
copolymer-adriamycin composition (No. 177; ~) and a HPMA
copolymer-adriamycin control composition (No. 237A; ~)
on human epithelial cells (HeLa cells).
FIG. 4 shows the in vitro cytotoxic effects of a
ligand-HPMA copolymer-adriamycin composition (No. 177;
~) and a HPMA copolymer-adriamycin control composition
(No. 237A; A) on human monocyte cells (U 937 cells).
FIG. 5 shows in vitro cytotoxic ef~ects of a
ligand-HPMA copolymer-adriamycin composition (No. 177)
on human CCRF-CEM T cells in the presence of anti-CR2
monoclonal antibody OKB7: OKB7 (~); no antibody (dotted
line).
FIG. 6 shows in vitro cytotoxic effects o~ a
ligand-HPMA copolymer-adriamycin composition (No. 177)
on human Raji B cells in the presence of anti-CR2
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monoclonal antibody OKB7: OKB7 (a); no antibody (dotted
line).
FIG. 7 shows in vi~ro cytotoxic effects of a
ligand-HPMA copolymer-adriamycin composition (No. 177)
on human CCRF-CEM T cells in the presence of the anti-
CR2 monoclonal antibody B-Ly 4: B-Ly 4 (n); no antibody
(dotted line).
FIG. 8 shows in vitro cytotoxic effects of a
ligand-HPMA copolymer-adriamycin composition (No. 177)
on human Raji B cells in the presence of the anti-CR2
monoclonal antibody B-Ly 4: B-Ly 4 (~); no antibody
(dotted line).
Detaile~ Descr;pt;on of the Tnv~ntion
Before the present compositions and methods for
targeting of macromolecular prodrugs to T lymphocytes
are disclosed and described, it is to be understood that
this invention is not limited to the particular
embodiments, process steps, and materials disclosed
herein as such embodiments, process steps, and materials
may vary somewhat. It is also to be understood that the
terminology used herein is used for the purpose of
describing particular embodiments only and is not
intended to be limiting since the scope of the present
invention will be limited only by the appended claims
and e~uivalents thereof.
It must be noted that, as used in this
specification and the appended claims, the singular
forms "a," "an," and "the" include plural referents
unless the context clearly dictates otherwise. Thus,
for example, reference to a composition containing "a
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ligand" includes two or more ligands, reference to "a
chemical agent" includes reference to one or more of
such chemical agents that may be the same or di~ferent
chemical agents, and reference to "a spacer'l includes
reference to two or more spacers.
In describing and claiming the present invention,
the following terminology will be used in accordance
with the definitions set out below.
As used herein, "peptidel' means peptides of any
length and includes proteins. The terms l'polypeptide"
and "oligopeptide" are used herein without any
particular intended size limitation, unless a particular
size is otherwise stated.
As used herein, 1l ligand" means a composition
capable o~ binding to a receptor on a T lymphocyte and
stimulating internalization by endocytosis of the
receptor and receptor-bound ligand. According to the
present invention, ligands are coupled to various
functional molecules so that upon endocytosis of the
ligands the various functional molecules coupled thereto
are also internalized by the T cells.
Preferred ligands for binding to a receptor on a T
lymphocyte and inducing internalization by endocytosis
of the receptor and receptor-bound ligand are a peptide
having the amino acid sequence identified as SEQ ID NO:1
and peptides substantially homologous thereto. As used
herein, llsubstantially homologousll means peptides that
retain functionality in binding T-cell receptors and
eliciting receptor-mediated endocytosis although they
may be truncations, deletion variants, or substitution
variants of SEQ ID NO:1 or include additional amino acid
CA 02247432 1998-08-26
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residues attached thereto. Substitution variants are
those that contain a conservative substitution of one or
more amino acid residues. A conservative substitution
is a substitution of one amino acid residue ~or another
wherein ~unctionality o~ the peptide is preserved, in
this case, ~unctionality in binding a T-cell receptor
and eliciting endocytosis o~ the receptor-bound
composition. Amino acid residues belonging to certain
conservative substitution groups can sometimes
substitute ~or another amino acid residue in the same
group. One classification o~ such conservative
substitution groups is as follows: ~a) Pro; (b) Ala,
Gly; (c) Ser, Thr; (d) Asn, Gln; (e) Asp, Glu; (~) His;
~g) Lys, Arg; (h) Cysi (I) Ile, Leu, Met, Val; and (j)
Phe, Trp, Tyr. M. Jimenez-Montano & L. Zamora-Cortina,
Evolutionary model ~or the generation o~ amino acid
sequences and its application to the study of mammal
alpha-hemoglobin ch~; n-~, Proc. VIIth Int'1 Biophysics
Congress, Mexico City (1981). Another classi~ication o~
such groups is described in M. Dayho~ et al., Atlas of
Protein Se~uence and Structure (Natll Biomed. Res.
Found., Washington, D.C., 1978), hereby incorporated by
re~erence. Other variations that are to be considered
substantially homologous include substitution of D-amino
acids ~or the naturally occurring L-amino acids,
substitution o~ amino acid derivatives such as those
containing additional side chains, and substitution of
non-standard amino acids, i.e. ~-amino acids that are
rare or do not occur in proteins. Thus, the primary
structure o~ a ligand is limited only by ~unctionality.
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11
It was unexpected and surprising to discover that
a composition having at least 2 ligands comprising a
peptide with an amino acid sequence of SEQ ID NO:1 and
peptides substantially homologous thereto are
preferentially targeted to T cells rather than to B
cells. This result is surprising because in copending
U.S. Patent Application Serial No. 08/305,770, filed
~eptember 13, 1994, compositions comprising a CR2-
receptor-binding and endocytosis-inducing ligand (CBEL),
i. e . SEQ ID NO:1, coupled to a chemical agent, i. e.
ricin A, were specifically delivered intracellularly
into B cells, but not T cells. Without being limited to
any particular theory of operation, it appears that
coupling the ligand to a spacer peptide, e.g. a peptide
having the sequence o~ SEQ ID NO:4, modifies the
specificity of the ligand such that the composition
binds preferentially to T cells and is delivered
intracellularly into such T cells. Alternatively, the
presence of multiple ligands may modify the speci~icity
such that T cells are pre~erentially recognized.
Binding and uptake of the composition by T cells may be
mediated by the EBV receptor described by J.A. Hedrick
et al., supra . In in vi tro experiments, a ~raction o~
B cells exposed to this composition also binds and takes
up the composition, but in vivo the composition appears
to be taken up by T cells, and not at all or only
slightly by B cells.
As used herein, "macromolecule" means a composition
comprising a water soluble polymer with at least 2
ligands and a chemical agent bound thereto. Pre~erably
the polymer is an HPMA copolymer and the ligand is an
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12
oligopeptide. The chemical agent can be from many
different classes of molecules, as explained in more
detail herein.
As used herein, "prodrug" means a chemical agent
that is chemically modified to overcome a biological
barrier. When a chemical agent is converted into its
prodrug form, its biological activity is eliminated or
substantially reduced, but the biological barrier that
inhibited its effectiveness is no longer problematic.
The chemical group that is attached to the chemical
agent to form the prodrug, i.e. the "pro-moiety", is
removed from the prodrug by enzymatic or nonenzymatic
means to release the active form of the chemical agent.
~ A. Albert, Chemical ~s~ects of Selective Tox;c;ty,
182 Nature 421 (1958). The instant compositions are
prodrugs because the chemical agent that has the
selected effect when internalized in T lymphocytes is
modified with ligands, water soluble polymer, and
spacers such that the composition is delivered into the
T lymphocytes, thus penetrating the cell membrane
thereof. The biological effect of the chemical agent is
greatly reduced or eliminated until the composition is
delivered intracellularly and the chemical agent is
released from the remainder of the composition by
biodegradation of the spacer.
As used hereln, "chemical agent" means and includes
any substance that has a selected effect when
internalized into a T lymphocyte. Certain chemical
agents have a physiological effect, such as a cytotoxic
effect or an effect on gene regulation, on a T cell when
internalized into the cell. A "transforming nucleic
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acid" (RNA or DNA), when internalized into a cell, can
be replicated and/or expressed within the cell. Other
nucleic acids can interact with regulatory se~uences or
regulatory factors within the cell to influence gene
expression within the cell in a selected manner. A
detectable label delivered intracellularly can permit
identification of cells that have internalized the
compositions of the present invention by detection of
the label. Drugs or pharmacologically active compounds
can be used to ameliorate pathogenic effects or other
types of disorders. Particularly useful chemical agents
include polypeptides, and some such chemical agents are
active fragments of biologically active proteins, or are
specific antigenic fragments (e.g., epitopes) of
antigenic proteins. Thus, chemical agents include
cytotoxins, gene regulators, transforming nucleic acids,
labels, antigens, drugs, and the like.
As used herein, I'drug'' or "pharmacologically active
agent" means any chemical material or compound suitable
for intracellular administration in a T lymphocyte that
stimulates a desired biological or pharmacological
effect in such cell.
As used herein, "carrier" means water soluble
polymers, particulates, or liposomes to which a
composition according to the instant invention can be
coupled. Such carriers increase the molecular size of
the compositions and may provide added selectivity
and/or stability. Such selectivity arises because
carrier-containing compositions are too large to enter
cells by passive diffusion, and thus are limited to
entering cells through receptor-mediated endocytosis.
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~he potential ~or use of such carriers for targeted drug
delivery has been established. See, e.g., J. Kopecek,
5 Biomaterials 19 (1984); E. Schacht et al.,
Po~ysacch~rides ~s Drug Carr;ers, in Controlled-Release
Technology 188 (P.I. Lee ~ W.R. Good, eds., 1987); F.
Hudecz et al., ~rr;er des;qn: Cytoto~-c~ty ~nd
Immllnogenic;ty of Synthetic Br~nche~ Poly~ept;~es with
Poly(T,-lysine~ B~ckbo~e, 19 J. Controlled Release 231
(1992~; Z. Brich et al., Prep~r~tion and
Character;~t;on of a Water Soll7h~e nextr~n
~mllnoconjugate of Doxorllh;c; n ~nd ~he Monoclo~l
~nt;hody (~RL~64), 19 J. Controlled Release 245 (1992).
Thus, illustrative water soluble polymers include
dextran, inulin, poly(L-lysine) with modi~ied epsilon
amino groups, poly(L-glutamic acid), N-substituted
methacrylamide-containing synthetic polymers and
copolymers, and the like.
As used herein, "effective amount" is an amount
that produces a selected effect. For example, a
selected effect of a composition containing a cytotoxin
as the chemical agent could be to kill a selected
proportion of T cells within a selected time period. An
effect amount of the composition would be the amount
that achieves this selected result, and such an amount
could be determined as a matter o~ routine by a person
skilled in the art.
The compositions of the present invention provide
intracellular delivery of a chemical agent capable of
eliciting a selected e~fect when delivered
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intracellularly into a T lymphocyte, the composition
having the formula:
[L-S]a-C-[S-A] b
wherein L is a ligand capable of binding to a receptor
on the T lymphocyte and stimulating receptor-mediated
endocytosis of the composition; A is the chemical agent;
S is a spacer; C is a water soluble polymer having
functional groups compatible with forming covalent bonds
with the ligand, chemical agent, and spacer; a is an
integer of at least 2; and b is an integer of at least
1. Preferably, a is an integer of 2 to about 1000, and
b is an integer of 1 to about lO00. The spacers are
preferably biodegradable such that the chemical agent is
detached from the composition by hydrolysis and/or
enzymatic cleavage inside T cells. CD4~ T cells are
targeted by these compositions, but it remains to be
determined whether CD8~ T cells are also targeted. The
chemical agent is selected from the group consisting of
cytotoxins, trans~orming nucleic acids, gene regulators,
labels, antigens, drugs, and the like. The coupling of
a ligand to a chemical agent can be, without limitation,
by covalent bond, electrostatic interaction, hydrophobic
interaction, physical encapsulation, and the like. The
compositions of the present invention can further
comprise a carrier selected from the group consisting of
water soluble polymers, liposomes, and particulates.
Such water soluble polymers are selected from the group
consisting of dextran, inulin, poly(L-lysine) (PLL) with
modified epsilon amino groups, poly(L-glutamic acid)
~PGA), N-substituted methacrylamide-containing polymers
and copolymers, and the like. A preferred water soluble
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16
polymer is a copolymer of N-(2-
hydroxypropyl)methacrylamide (HPMA).
Thus, according to the invention, the composition
provides means for preferential binding to a receptor on
T cells, thus triggering internalization of the
composition by endocytosis. The chemical agent provides
means for achieving a selected e~fect in the T cells.
Accordingly, for example, chemical agents comprise
cytotoxins, including radionuclides, for selective
killing or disabling of T cells; nucleic acids for
genetically trans~orming or regulating gene expression
in T cellsi drugs or other pharmacologically active
agents ~or achieving a selected therapeutic e~fect;
labels, including fluorescent, radioactive, and magnetic
labels, for permitting detection of cells that have
taken up the compositions; and the like.
In some embodiments, the compositions are
constructed by chemically conjugating the ligand and
chemical agent to the water soluble polymer.
"Chemically conjugating" the ligand and the chemical
agent to the water soluble polymer, as that term is used
herein, means covalently bonding the ligand and chemical
agent to the polymer by way of a spacer moiety. In
particular embodiments, a spacer moiety is used to form
a linkage between functional groups on the polymer and
the chemical agent.
Peptide portions of the compositions of the present
invention can be produced in a genetically engineered
organism, such as E. coli, as a "~usion protein." That
is, a hybrid gene containing a sequence o~ nucleotides
encoding a ligand, spacer, or peptide chemical agent can
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17
be constructed by recombinant DNA technology. This
hybrid gene can be inserted into an organism such that
the "fusion protein" encoded by the hybrid gene is
expressed. The fusion protein can then be purified by
standard methods, including affinity chromatography.
Peptides containing a ligand, spacer, or peptide
chemical agent can also be constructed by chemical
synthesis. Short peptide ligands are generally
preferred, both because short peptides can be
manipulated more readily and because the presence of
additional amino acids residues, and particularly of
substantial numbers of additional amino acids residues,
may interfere with the function of the peptide ligand in
stimulating internalization of the chemical agent by
endocytosis.
Compositions according to the present invention
pre~erably also further include a protease digestion
site situated so that once the composition is within the
cell, the chemical agent can be separated from the
remainder of the composition by proteolysis o~ the
digestion site. Such a protease susceptible spacer can
be added regardless of whether the peptide portions of
the composition are synthesized chemically or as
expression peptides in a genetically engineered
organism. In the latter case, nucleotides encoding the
protease susceptible spacer can be inserted into the
hybrid gene encoding the ligand and or a peptide
chemical agent by techniques well known in the art.
Another aspect of the present invention features a
method ~or specifically ef~ecting a desired activity in
T lymphocytes contained in a heterogeneous population of
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18
cells, by steps of contacting the population of cells
with a composition, prepared according to the present
invention, that directs such activity intracellularly.
The compositions of the invention are selectively bound
to T cel~s in the mixed population, whereupon
endocytosis of the composition into the T cells is
stimulated, and the chemical agent effects its activity
within such T cells.
This application employs, except where otherwise
indicated, standard techniques for manipulation of
peptides and for manipulation of nucleic acids for
expression of peptides. Techniques for conjugation of
oligopeptides and oligonucleotides are known in the art,
and are described for example in T. Zhu et al., 3
Antisense Res. Dev. 265 (1993), T. Zhu et al., 89 Proc.
Nat'l Acad. Sci. USA 7934 (1992); P. Rigaudy et al., 49
Cancer Res. 1836 (1989).
As is noted above, the invention features peptides,
employed as ligands, spacers, and/or chemical agents.
The peptides according to the invention can be made by
any of a variety of techniques, including organic
synthesis and recombinant DNA methods. Techniques for
chemical synthesis o~ peptides are described, for
example, in B. Merrifield et al., 21 Biochemistry 5020
(1982); Houghten, 82 Proc. Nat'l Acad. Sci. USA 5131
~1985), incorporated herein by reference. Techniques
for chemical conjugation of peptides with other
molecules are known in the art.
A fusion protein according to the invention can be
made by expression in a suitable host cell of a nucleic
acid containing an oligonucleotide encoding a ligand
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19
and/or spacer, or a chemical agent and/or spacer. Such
techniques for producing recombinant fusion proteins are
well-known in the art, and are described generally in,
e.g., J. Sambrook et al., Molecular Cloning: A
Laboratory Manual (2d ed., 19~9), the pertinent part~ of
which are hereby incorporated herein by reference.
Reagents useful in applying such techniques, such as
restriction endonucleases and the like, are widely known
in the art and commercially available from any of
several vendors
Construction of compositions according to the
present invention will now be described, with particular
reference to examples in which a peptide ligand coupled
to a biodegradable spacer (SEQ ID N0:4) and a cytotoxic
che...ical age~t, ad~iar.,ycin, are coupled to a copoly~ner
o~ HPMA
Example 1
In this example, a composition according to the
present invention was prepared by coupling an EBV-
derived ligand, SEQ ID NO:l, and/or the cytotoxic
chemical agent, adriamycin, to an HPMA copolymer via a
protease-sensitive spacer (Gly-Phe-Leu-Gly; SEQ ID
NO:4). Adriamycin intercalates with DNA and inhibits
DNA replication, thus exerting a toxic effect on cells.
Compositions according to this example are
lysosomotropic, and the degree of cytotoxicity depends
on the biodegradability of the drug-polymer linkage
within the lysosomes. The protease-sensitive spacer,
Gly-Phe-Leu-Gly (SEQ ID NO:4) is biodegradable in
lysosomes, but is resistant to proteolysis in the
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bloodstream. The construction of these compositions is
described in detail below.
Two HPMA copolymer-adriamycin compositions were
employed in this example, namely a ligand-containing
composition and a control composition lacking a ligand.
Synthesis o~ HPMA is described in 70 Angew. Makromol.
Chem. 109 (19783; N. Krinick et al ., Synthes' s o:E N~
Hy~l~oxypropyl)Methacrylami~e ~o~?olymer-~ntl-Thy 1 2
~nt; ho~y-Chlorl n e6 Conjugates ~nd ;3 Prelim; n;~ry Stud~ of
The; r Photo~n;~ml c E:E~ect on Mouse Spl enocytes 7 n vi tro,
191 Makrol. Chem. 839 (1990); U.S. Patent No. 5,037,883;
U.S. Patent No. 5,258,453; N.L. Krinick, Combination
Polymeric Drugs as Anticancer Agents (Doctoral
Dissertation, University oi~ Utah, 1992), hereby
incorporated by re:Eerence . N-
methacryloylglycylphenylalanylleucylglycine p-
nitrophenyl ester (~A-Gly-Phe-Leu-Gly-ONp)was
s y n t h e s i z e d b y a m i n o 1 y s i s o f~ N -
methacryloylglycylphenylalanine p-nitrophenyl ester (MA-
Gly-Phe-ONp) with leucylglycine (Leu-Gly-OH), f~ollowed
by esteri:Eication with p-nitrophenol, as described in
Kopecek et al., U.S. Patent No. 5,037,883, which is
hereby incorporated by re~erence.
Copolymeri zat ion
The polymer precursors (copolymers o~ HPMA and
methacryloylated oligopeptide active esters) were
prepared by radical precipitation polymerization in
acetone at 50~C ~or 24 h using 2, 2 ' -
azobisisobutyronitrile (AIBN) as the initiator. The
3 0 ratio oi~ monomers to initiator and solvent was
12.5:0.6:86.9 by weight . The polymerization mixture was
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21
poured into an ampoule, bubbled with nitrogen, and
sealed. After the polymerization, the precipitated
polymer was collected by filtration, washed with
acetone, and reprecipitated from methanolic solution (20
wt ~) into an excess of acetone. The composition of the
polymerization mixtures ~or polymer precursor HPMA
copolymer-Gly-Phe-Leu-Gly-ONp (SEQ ID NO:4) was 2.000 g
HPMA, 0.706 g MA-Gly-Phe-Leu-Gly-ONp (SEQ ID NO:4),
0.130 g AIBN, and 18.8 g acetone. The mole ratio of ONp
to HPMA was 8/92.
~acterization of Polymeric Precll~sors
The content o~ reactive p-nitrophenoxy groups (ONp)
was determined spectrophotometrically using C275nm = 9500
[1 mol~lcm~1] in DMSO. The weight-average molecular
weight and polydispersity were determined after
aminolysis with 1-amino-2-propanol by size exclusion
chromatography on a Superose 6 column using the
Pharmacia FPLC system (0.05 M Tris, 0.5 M NaCl, pH 8.0
as eluent) calibrated with poly(HPMA) fractions.
Solution behavior (molecular characterization) of
polymers (aminolysed precursors) was also evaluated by
light scattering methods (see below).
Aminolysed precursors were prepared by adding a
ten-~old excess of 1-amino-2-propanol to a solution of
polymer precursors in DMSO (20 wt ~) and precipitating
with an excess of acetone. The characterization of
polymer precursors is presented in Table 1.
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Table 1
Characterization of Polymeric Precursors
HPMA-Gly-Phe-Leu-Gly-ONp
~ yield of 62
polymerization
mol ~ of ONp groups 6.2~
g polymer/mol ONp 2740 g
mol ONp/g polymer 3.6 X 10-4
Mw~ 17000
Mw/Mn 1.3
D~ nm (diameter) b 6 . 9
a Determined after aminolysis of ONp groups by
1-amino- 2 -propanol.
15b Conjugate diameter as determined by dynamic
light scattering.
R; n~l; ng of T; gan~l~ to Polymer;c P~ecl~sors
A ligand can be coupled to a polymeric precursor,
20made for example as described above, in any of a variety
of ways that are well known in the art. The composition
and the analysis of two conjugates are shown in Table 2.
Table 2
25Composition
No. Ligand Adriamycin Aa
wt ~ mmol/g wt ~ mmol/g
177 6.0 0.06 8.0 0.14 0.764
.
237A - - 8.7 0.16 0.832
a Absorbance at 488 nm; 1 cm; 0.5 mg/m_.
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23
The structures of the compositions represented in
Table 2 are as follows:
(Gly-Phe-Leu-Gly-L) 2
\
(Gly-Phe-Leu-Gly-ADR) 3 (No. 177; SEQ ID NO:4)
P-Gly-Phe-Leu-Gly-ADR (No. 237A; SEQ ID NO:4)
In these structures, "P" represents the HPMA
copolymer backbone, "ADR" represents adriamycin, "L"
represents the ligand SEQ ID NO:l, and 'IGly-Phe-Leu-Gly''
(SEQ ID NO:4) is a spacer susceptible to proteolytic
degradation.
By way of example, composition no. 177 was
synthesized as follows. HPMA copolymer precursor (100
mg) containing 6.2 mol~ oE Gly-Phe-Leu-Gly (SEQ ID NO:4)
side chains (36 ~mol ONp) was dissolved in 0.5 ml
anhydrous dimethylEormamide (DMF) and 14.0 mg (24 ,~Lmol)
o~ adriamycin hydrochloride (solid) and a solution of
12.7 mg (12 ~mol) ligand (SEQ ID NO:1) in 100 ~1 DMF
were added. Then, 27 }Ll of a diluted solution of
triethylamine (1:1) in DMF (96 ,umol, 9.8 mg) was added
in three portions within 30 min. The reaction mixture
was stirred for 6 h at room temperature, then 10 ~Ll of
. 30 aminopropanol diluted with DMF (10:1) was added to
aminolyze unreacted ONp groups. The reaction mixture
was precipitated into 200 ml acetone, the polymeric
product was ~iltered off, washed with acetone and
reprecipitated from methanolic solution (20 wt~) into
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acetone. Unbound adriamycin was removed on a Sephadex
LH-20 column tl6/50) in methanol. The polymer fractions
were collected and evaporated under reduced pressure.
The pure conjugate in powdered ~orm was obtained after
precipitating (0.8 ml) into 300 ml acetone and drying in
vacuo (yield 75 mg). The conjugate was dissolved in 20
ml of deionized water and dialyzed against water at 4~C
for 24 hours in the dark. The purified conjugate was
isolated by freeze-drying. The content of adriamycin
was determined spectrophotometrically using ~ = 10,000
M~1cm~l (water, 488 nm). The content of bound ligand was
determined by amino acid analysis after acid hydrolysis.
The quantity of ~ree adriamycin in the resulting
product was determined by extraction assay. Two mg of
polymer conjugate in a test tube was dissolved in 0.5 ml
water, then 0.5 ml of 0.2 M sodium carbonate/bicarbonate
buffer, pH 10.5, was added and the solution was
extracted with 2 ml ethylacetate. The organic layer was
separated, dried with magnesium sulfate, and absorption
spectrum (350-550 nm) was measured at a range of 0 to
0.02 A. There was no detectable ADR in the conjugate.
Example 2
The in vitro effects of compositions No. 177 and
237A prepared according to the procedure of Example 1
were tested on several human T and B cell lines as
follows. Triplicate samples of 1 x 105 cells each were
mixed with different concentrations of the purified
compositions in 0.1 ml of culture medium (RPMI 1640, 10~
fetal calf serum) in the wells o~ a 96-well microtiter
plate tFalcon Microtest 111), and incubated ~or 18-48
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hours at 37~C in a humidified, 5~ CO2 atmosphere.
Thereafter, cell viability was assessed by a
colorimetric method using the tetrazolium compound MTS
(3 - (4 , 5 - d i m e t h y l t h 1 a z o l - 2 - y l) - 5 - (3 -
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,
inner salt) and an electron coupling reagent, PMS
(phenazine methosulfate). MTS is bioreduced by living
cells into a soluble formazan product. The absorbance
of the formazan at 490 nm can be measured directly from
96 well assay plates without additional processing. The
quantity of formazan product as measured by the
absorbance at 490 nm is directly proportional to the
number of living cells in culture. Reagents for the MTS
assay were obtained from Promega Corp. (Madison,
Wisconsin). According to this method, 20 ~l of MTS/PMS
solution (Promega No. G-5421) was added to each well of
the assay plate. The plate was then further incubated
at 37~C in a humidified, 5~ CO2 atmosphere for 4 hours.
The absorbance of each well was then measured at 490 nm
with an EL311 Microplate Autoreader (Bio-Tek
Instruments). The mean absorbance for each treatment
was then calculated, and the percent cytotoxicity was
determined using the formula:
A
% cytotoxity = (1--As ) x 100
wherein As represents the mean absorbance for each
treatment and A~ represents mean absorbance of the
control treatment, i.e. cells not exposed to a
conjugate.
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26
The following cell lines were tested according to
this procedure: HSB-2 (ATCC No CCL 120.1; CD4+ human T
cells); CCRF-CEM (ATCC No. CCL 119; CD4+ human T cells);
MOLT-3 (ATCC No. CRL 1552; CD4+ human T cells); Ra~i
(ATCC No. CCL 86; CR2' human B lymphoblastoid cells); and
Daudi (ATCC No. CCL 213; C~2+ human B cells).
FIG. 1 summarizes the results of this experiment
wherein it is shown that the adriamycin-containing
composition No. 177 exhibited greater cytotoxicity in
the three T cell lines than in the B cell lines. This
suggests that the T cells were able to internalize more
of composition No. 177 than were the B cells. All of
the T cells tested internalized the composition
approximately equally as judged by the similar levels of
cytotoxicity, particularly at 50 ~g/ml and 100 ~g/ml of
composition No. 177. The Raji and Daudi B cell lines
also exhibited moderate ability to take up composition
No. 177. The levels of cytotoxicity to these B cell
lines was markedly reduced, however, as compared to the
three T cell lines at all concentrations tested All of
the cell lines internalized some of control composition
No. 237A, which lacks a ligand but contains adriamycin,
shown for example in FIG. 2 with respect to CCRF-CBM T
cells. The level of cytotoxicity in response to
composition No. 237A was less than the level of
cytotoxicity in response to composition No. 177 at all
concentrations tested.
Example 3
In this example, the procedure of Example 2 was
followed with the exception that the cells used were
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27
human epithelial cells (HeLa cells~. The results of
this test are shown in FIG. 3, wherein at every
concentration o~ conjugate tested, the ligand-containing
and control compositions yielded substantially identical
results. At 100 ~g/ml of conjugate there was virtually
no cytotoxicity. Only when the concentration was
increased to 500 ~g/ml was cytotoxicity observed, and
then only about 25~ of the cells were killed.
Example 4
In this example, the procedure of Example 2 was
~ollowed with the exception that the cells used were
human monocyte cells (U 937 cells, ATCC No. CRL 15393).
FIG. 4 shows that composition No. 177 and control
composition No. 237A were substantially similar with
respect to cytotoxic effects on human monocyte cells,
i.e. very little or no cytotoxicity at conjugate
concentrations at or below 10 ~g/ml and increasing
levels of cytotoxicity at higher conjugate
concentrations.
The compositions according to the present invention
can be employed for targeted delivery of a chemical
agent to T cells, generally by contacting the T cells
with the composition under conditions in which binding
of the ligand to a receptor stimulates endocytosis of
the composition into the T cells. The chemical agent
then acts on or within the targeted cell into which the
composition is internalized, and the desired e~fect of
the active agent can be confined to those cells having
the receptor.
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28
For example, a composition according to the
inventlon can be employed a~ an e~ective antitumor
agent in vlvo ~or killing T cells. Preferably, the
composition is administered to the subject by systemic
administration, typically by subcutaneous,
intramuscular, or intravenous injection, or
intraperitoneal administration. Injectables ~or such
use can be prepared in conventional ~orms, either as a
liquid solution or suspension or in a solid ~orm
suitable for preparation as a solution or suspension in
a liquid prior to injection, or as an emulsion.
Suitable excipients include, ~or example, water, saline,
dextrose, glycerol, ethanol, and the like; and i~
desired, minor amounts o~ auxiliary substances such as
wetting or emulsifying agents, bu~ers, and the like may
be added.
The composition can be contacted with the cells in
vitro or in vivo. The T cells constitute a
subpopulation o~ a mixed population of cell types; the
ligand according to the invention can provide ~or
endocytosis of the conjugate into T cells and possibly
into a small proportion o~ other cells having a closely
related receptor.
The chemical agent can have any o~ a variety o~
desired effects in the targeted cells. ~s mentioned
above, in some particularly use~ul embodiments the
chemical agent is e~ective on a cell only when, or
principally when, the agent is internalized into the
cell.
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29
Example 5
In vivo Targeted Delivery to T cells
Compositions according to the present invention can
be administered to a warm-blooded animal for targeted
delivery to T cells. Particularly, the composition
provides for receptor-mediated internalization of the
composition into the T cells.
About 1 x 107 HSB-2 human T-cell leukemia cells in
500 ~1 of PBS were injected intraperitoneally into mice,
and the cells were allowed to colonize the mice for 48
hours. The human T-cell leukemia cells were found to
preferentially colonize the spleen and liver. After 48
hours, the mice were injected intraperitoneally with 500
~g of either composition No. 177 or control composition
No. 237A in 500 ~1 of PBS. A~ter an additional 48
hours, the spleen and liver were harvested, and PCR
assay of genomic DNA and cDNA prepared from these organs
was used to determine the relative numbers of mouse and
human cells therein.
Genomic DNA and cDNA were prepared ~rom mouse
spleen and liver according to methods that are generally
well known in the art. See, e.g., J. Sambrook et al.,
Molecular Cloning: A Laboratory Manual (2d ed., 1989);
T. Maniatis et al., Molecular Cloning: A Laboratory
Manual (1982); F. Ausubel et al., Current Protocols in
Molecular Biology (1987). Illustrative methods for
preparation of cDNA and genomic DNA are briefly
described below.
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Prep~at;on of ~E~a
The excised spleen and liver were disrupted and the
resulting cells were washed in PBS. The cells were then
resuspended in a bu~er containing 100 mM NaCl, 10 mM
TrisHCl, pH 8.0, 25 mM EDTA, 0.5~ SDS, and 0.1 mg/ml
proteinase K and incubated overnight at 37~C. The
disrupted cells were then centrifuged and washed once in
cold PBS. The cell pellet was then resuspended in
RNAzol B reagent (Tel-Test, Friendwood, Texas). The
resulting lysate was then extracted with chloro~orm, and
RNA was precipitated with isopropanol. The RNA was
washed with 75~ ethanol, dried briefly, and dissolved in
0.5~ SDS. An aliquot o~ RNA was then mixed with reverse
transcriptase, random primers, bu~er, and
deoxynucleotide triphosphates, and the mixture was
incubated at 37~C for 1 hour. The reaction was stopped
by RNase digestion, and then the cDNA was extracted in
succession with phenol/chloroform and chloroform,
precipitated with ethanol, and resuspended in water.
Preparation o~ Genomic DNA
Cells that were disrupted, washed, and incubated
overnight in digestion bu~er as described above were
twice extracted with phenol/chloroform/isoamylalcohol.
The DNA in the aqueous phase was then precipitated with
ethanol, washed, dried, and resuspended in a buffer
containing 10 mM TrisHCl, pH 8.0, 1 mM EDTA.
E~ of cDNA and Genomic ~_
PCR is well known in the art ~or determining the
presence of selected sequences in genomic DNA and cDNA
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31
samples. The ~ollowing re~erences illustrate PCR
methodology: PCR Technology: Principles and
Applications ~or DNA Amplification (H. Erlich ed.,
Stockton Press, New York, 1989); PCR Protocols: A Guide
to Methods and Applications (Innis et al. eds, Academic
Press, San Diego, Cali~., 1990); U.S. Patent Nos.
4,683,195; 4,683,202; 4,800,159; 4,965,188. Brief~ly,
PCR reactions were carried out in glass capillary tubes
in 10 ~1 volumes containing 0 .8 mM of each o~ the Eour
deoxynucleotide triphosphates, 2.5 ~lCi of~ 32P-dCTP (3000
Ci/mmol), 0.72 units o~ Thermus aquat;cus (Taq) DNA
polymerase, 35-70 pmol o~ each primer (20-23 nucleotides
in length), 200 ng cDNA, and a reaction bufEer
containing 50 mM TrisHCl, pH 8.3, 3 mM MgCl2, 20 mM KCl,
and 0. 5 mg/ml o~ bovine serum albumin. The amount of
cDNA and number o~ cycles o~ ampli~ication can be
determined empirically by a person o~ ordinary skill in
the art without undue experimentation. Titration o~
cDNA i~rom about 1 ng to about 500 ~Lg, and titration o~
cycles ~rom about 12 to about 36 gives a good indication
o~ amounts o~ cDNA and numbers o~ cycles needed. For
example, using i~-actin primers, 12-15 cycles and about
200 ng of cDNA generally give good results.
The reaction mixtures were sealed in capillary
tubes and then the capillaries were placed in a Model
1605 Air Thermocycler (Idaho Technology, Idaho Falls,
Idaho). Parameters o~ annealing temperature, elongation
time, and number o~ cycles were selected. Increasing
the annealing temperature increases the speci~icity o~
PCR ampli:Eication reactions and decreases the amounts o~
nonspeci~ic products. Anneallng temperature can be
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32
estimated ~rom thermal melting temperature according to
the ~ormula:
Tm = 4~C(no. o~ G and C residues in primer) + 2 DC (no. of
A and T residues in primer). A person o~ ordinary skill
in the art can optimize the annealing temperature
according to known principles. The elongation time
depends on the size of product to be ampli~ied. As a
rule of~ thumb, about 4 seconds i8 suf~icient for
products o~ about 100-150 bp, about 8 seconds is
su~ficient :Eor products oi~ about 200-300 bp, and about
20 seconds is needed for products larger than about 500
bp Increasing elongation times may result in
ampli~ication o~ nonspeci~ic products.
A~ter ampli~ication, the reaction mixture is
removed ~rom the capillary, mixed with an equal volume
o~ stop solution (95~ :Eormamide, 20 mM EDTA, 0.05~
bromphenol blue, 0.05~ xylene cyanol FF), and either
stored ~rozen or immediately heated at 95~C ~or 5
minutes and sub~ected to polyacrylamide gel
electrophoresis. The ~ractionated products are then
quantitated according to the amount o~ radioactivity in
each ~raction, such as by autoradiography.
An illustrative method o~ determining the relative
amounts o~ human and mouse cells in spleen and liver
tissues involves comparison o~ ampli~ied products from
reactions with mouse ~-actin and human ~-actin speci~ic
primers. Illustrative mouse ~-actin primers are as
~ollows:
GTAACAATGC CATGTTCAAT (SEQ ID NO:5)
CTCCATCGTG GGCCGCTCTA G (SEQ ID N0:6)
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33
Illustrative human ~-actin primers are as ~ollows:
CTTAGTTGCG TTACACCCTT TC (SEQ ID NO:7)
GGGCCATTCT CCTTAGAGAG AAG (SEQ ID NO:8)
The results o~ this experiment are presented in
Table 3.
Table 3
Treatment Dose Human Mouse
primers primers
237A 0.5 mg/ml 12/12 12/12
177 0.5 mg/ml 3/12 12/12
These results show that all o~ the mice treated
with the control composition no. 237A exhibited the
presence o~ both human and mouse DNA by PCR analysis
with speci~ic ~-actin primers. Twelve o~ twelve mice
treated with composition no. 177 exhibited the presence
o~ mouse DNA, however, only three o~ twelve mice treated
with composition no. 177 exhibited the presence o~ human
DNA. These results demonstrate that a ligand and
adriamycin-containing composition according to the
present invention selectively kills T cells in animals
to which it is administered.
Example 6
The procedure of Example 5 was ~ollowed except that
Raji B cells were used instead o~ HSB-2 T cells. A11
mice treated with either composition no. 177 or control
composition no. 237A exhibited the presence o~ both
human and mouse DNA by PCR analysis with speci~ic ~-
actin primers. These results demonstrate that a ligand
and adriamycin-containing composition according to the
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34
present invention has no detectable effect on B cells in
animals to which it is administered.
Example 7
Four groups o~ ~our mice per group were treated
according to the ~ollowing treatments: (1) no tumor
cells, ~2) tumor cells, (3) tumor cells plus composition
no. 237A, or (4) tumor cells plus composition no. 177.
Groups 2-4 were injected intraperitoneally with about 1
x 107 HSB-2 human T-cell leukemia cells in 500 ,Ll of PBS,
and the cells were allowed to colonize the mice i~or 48
hours. After 48 hours, the mice of groups 3 and 4 were
injected intraperitoneally with 500 ~g o~ either
composition no. 237A (group 3) or composition no. 177
(group 4) in 500 ~Ll of~ PBS, and these injections were
repeated on each of the next two days.
Within 65 days after injection of the tumor cells,
all of the animals in groups 2 and 3 had died, whereas
all the animals in groups 1 and 4 were still alive at 90
days after injection. These results show that a
composition according to the present invention is
capable of selectively killing human T cells in vivo
whereas a control composition lacking a T-cell specific
ligand was not.
Example 8
A method of treating T cell lymphoma in a human
comprises (a) providing a composition according to the
present invention including a ligand, such as the EBV
ligand (SEQ ID NO:1) or a peptide substantially
homologous thereto, and a cytotoxin, such as adriamycin,
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both of which are coupled to an HPMA copolymer by means
of a spacer (Gly-Phe-Leu-Gly; SEQ ID NO:4) and (b)
systemically administering an effective amount of the
composition to an individual. Such composition can be
made, for example, as shown above in Example 1. An
effective amount of the composition is systemically
- administered to the individual such that the composition
enters the bloodstream and contacts T cells. The
composition binds to a receptor on the T cells and
stimulates internalization of the composition by
endocytosis. The adriamycin then kills the cell by
intercalating with DNA in the cell. This procedure
reduces the number of malignant T cells in the body of
the individual, thereby having a positive effect in
treatment of the disease.
Example 9
The ability of anti-CR2 antibodies to competitively
inhibit binding and receptor-mediated endocytosis of a
composition according to the present invention was
investigated in this example. OK37 antibodies (Ortho
diagnostic Systems, Raritan, N.J.) are thought to
recognize distinct epitopes of the B-cell CR2 receptor
that are localized at the amino-terminal portion of the
molecule. J.C. Carel et al., 265 J. Biol. Chem. 12293
(1990). The OKB7-reactive epitope seems to be closely
related to the epitope or epitopes that interacts with
the viral pg350/220 and with C3d. Carel et al., supra;
C.A. Lowell et al., 170 J. Exp. Med. 1931 (1989).
The procedure o~ Example 2 was followed with the
exception that the cells were preincubated at 37OC with
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36
OKB7 antibodies before administration of composition no.
177. FIG. 5 shows that the OKB7 anti-CR2 antibodies at
the lowest dilution inhibit cytotoxicity of CCRF-CEM T
cells by about 30~, but are otherwise ine~fective in
inhibiting binding and uptake of composition no. 177 and
the cytotoxicity associated therewith. FIG. 6 shows
that in the absence of OKB7 antibodies, composition no.
177 is inef~ectively taken up by Raji B cells, resulting
in cytotoxicity to only about 40~ of cells. Moreover,
the presence of OKB7 antibodies has little, if any,
effect on uptake and cytotoxicity in Raji B cells. This
results is consistent with the results o~ J.A. Hedrick
et al., supra . There~ore, these results are consistent
with compositions according to the present invention,
such as composition no. 177, being capable of
efficiently binding to T cells via a receptor other than
the CR2 receptor, being internalized in such cells, and
exhibiting their ef~ects inside the cells.
Example 10
The procedure o~ Example 9 was followed in this
example except that the antibodies used were the anti-
CR2 monoclonal antibodies B-Ly 4 (Pharmagen). FIG. 7
shows that this anti-CR2 antibody has little or no
ef~ect on inhibiting cytotoxicity from composition no.
177 in CCRF-CEM T cells. FIG. 8 shows, however, that B-
Ly 4 antibodies are able to completely inhibit the
cytotoxicity of composition no. 177 in Raji B cells.
These results are consistent with compositions according
to the present invention, such as composition no. 177,
being capable of e~ficiently binding to T cells via a
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37
receptor other than the CR2 receptor, being internalized
in such cells, and exhibiting their e~ects inside the
~ cells.
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38
Se~uence T.;s t; n~
'~N~R 2~T- lNr l~K IATION:
(I) APPLICANT: Ramesh K. Prakash, Jind~ich Kopecek,
Pavla Kopeckova, Vladimir G.
Omelyanenko,
(ii) TITLE OF lNv~ lON: TARGETING MACROMOLECULAR
PRODRUGS TO T LYMPHOCYTES
(iii) NL.~ iK OF SEQIJ~ 8
(iv) CORRESPGh~N~-~ Ann~F~cs
(A) z~nn~2F~:gEE: Thorpe, North & Western, L.L.P.
(B) ~ : 9035 South 70D East, Suite 200
(C) CITY: Sandy
(D) STATE: Utah
(E) ~)U~l~Y: USA
(F) ZIP: 84070
(v) ~I~u~ ~K ~T'~n~T~T-~ FO~M:
(A) D~lu.I TYPE: Diskette, 3.5 inch, 1.44
Mb storage
(B) ~;O11~U~;K: AST Ascentia 900 N
(C) OPERATING ~Y~l~l: DOS 6.22
(D) S~r- -~RF: Word Per~ect 6.0
(vi) ~u~nl_~ APPLICATION DATA:
( A) APPLICATION NU.-~r;~:
(B) FILING DATE:
(C) ChASSIFICATION:
(vii) PRIOR APPLICATION DATA:
3 5 (A) APPI-ICATION NL.-
(B) FILING DATE:
(viii) A OKNr;r/AGENT l~rOKMATION:
(A) NAME: Alan J. Howarth
(B) l~T~'GT~TRATION NUl~ ~: 3 6,553
(C) ~r~K~/~K~. N- Ir~ ~ T3216
( iX) TFT-I<:~ 'Q~ JNlcATIoN lN"'~K.~ATION:
. (A) TEL~:~P: (801)566-6633
(B) TELEFAX: (801)566-0750
CA 02247432 1998-08-26
W O97/~3618 PCTrUS97/03832
39
(2) INFORMATION FOR SEQ ID NO:l:
(I) SEQ~:~ CF~R~CTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Glu Asp Pro Gly Phe Phe Asn Val Glu
1 5
(2) INFORMATION FOR SEQ ID NO:2:
(I) SEQu~N~ CHARACTERISTICS:
(A) L~ Ltl: 11 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO:2:
Glu Asp Pro Gly Lys Asn Leu Tyr Asn Val Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO:3:
(I) SEQu~N~ ~CTERISTICS:
(A) L~l~: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Glu Asp Pro Gly
CA 02247432 1998-08-26
W O 97/~3618 PCTrUS97/03832
(2) INFORMATION FOR SEQ ID NO:4:
(I) SEQUENCE C~A~CTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Gly Phe Leu Gly
(2) INFO~MATION FOR SEQ ID NO:5:
(I) SEQu~N~ C~CTERISTICS:
(A) L~ln: 20 base pairs
~B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQ~N~ DESCRIPTION: SEQ ID NO:5:
GTAACAATGC CATGTTCAAT 20
(2) INFORMATION FOR SEQ ID NO:6:
~I) SEQ~N~ ~ CTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQu~N~ DESCRIPTION: SEQ ID NO:6:
CTCCATCGTG GGCCGCTCTA G 21
(2) INFORMATION FOR SEQ ID NO:7:
CA 02247432 1998-08-26
W O 97/~3618 PCTrUS97/03832
41
(I) SEQu~N~ C~ARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
CTTAGTTGCG TTACACCCTT TC 22
(2) INFORMATION FOR SEQ ID NO:8:
(I) SEQUENCE ~R~CTERISTICS:
(A) LENGTH: 23 ~ase pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQ~N~h: DESCRIPTION: SEQ ID NO:8:
GGGCCATTCT CCTTAGAGAG AAG 23
