U.S. patent number 5,614,191 [Application Number 08/404,685] was granted by the patent office on 1997-03-25 for il-13 receptor specific chimeric proteins and uses thereof.
This patent grant is currently assigned to The United States of America as represented by the Department of Health. Invention is credited to Waldemar Debinski, Nicholas Obiri, Ira Pastan, Raj K. Puri.
United States Patent |
5,614,191 |
Puri , et al. |
March 25, 1997 |
IL-13 receptor specific chimeric proteins and uses thereof
Abstract
The present invention provides a method and compositions for
specifically delivering an effector molecule to a tumor cell. The
method involves providing a chimeric molecule that comprises an
effector molecule attached to a targeting molecule that
specifically binds an IL-13 receptor and contacting a tumor cell
with the chimeric molecule.
Inventors: |
Puri; Raj K. (North Potomac,
MD), Debinski; Waldemar (Hummelstown, PA), Pastan;
Ira (Potomac, MD), Obiri; Nicholas (Gaithersburg,
MD) |
Assignee: |
The United States of America as
represented by the Department of Health (Washington,
DC)
|
Family
ID: |
23600619 |
Appl.
No.: |
08/404,685 |
Filed: |
March 15, 1995 |
Current U.S.
Class: |
424/178.1;
424/134.1; 435/7.23; 424/138.1; 424/183.1; 530/391.7; 530/391.3;
530/387.7; 530/387.3; 435/69.6 |
Current CPC
Class: |
C07K
14/5437 (20130101); C07K 14/21 (20130101); A61P
35/00 (20180101); A61K 47/6849 (20170801); C07K
16/2866 (20130101); A61K 47/642 (20170801); A61K
2039/505 (20130101); C07K 2319/00 (20130101); A61K
38/00 (20130101) |
Current International
Class: |
C07K
14/54 (20060101); C07K 16/28 (20060101); C07K
16/18 (20060101); A61K 47/48 (20060101); C07K
14/21 (20060101); C07K 14/195 (20060101); C07K
14/435 (20060101); A61K 38/00 (20060101); A61K
039/395 (); C07K 016/46 (); G01N 033/53 () |
Field of
Search: |
;424/134.1,143.1,138.1,155.1,172.1,173.1,178.1,174.1,183.1,7.23
;435/69.6,69.7,91.4,188,240.37,252.33
;530/387.3,387.7,388.8,389.7,391.7,391.3 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Thrush et al 1996 Ann Rev Immunol vol. 14 49-71. .
Obiri et al. (1995). J. Biol. Chem vol. 270, No. 15:8797. .
Debinski et al. (1993) J. Biol. Chem vol. 268 No. 19:14065. .
Debinski et al. (1994). Bioconjugate Chem vol. 5:40-46. .
Chester et al (1995) Tibtech vol. 13:294-300. .
Gottstein et al (1994) Annab of Oncology vol. 5 Supplement 1:
S97-103. .
Vita et al., Journ. of Biol. Chem., 270:3512 (1995). .
Chaudhary et al., Nature, 339: 394-397 (1989). .
McKenzie et al. Proc. Natl. Acad. Sci. USA, 90: 3735 (1993). .
Pastan et al., Ann. Rev. Biochem., 61: 331-354 (1992). .
Minty et al., Nature, 362: 248 (1993)..
|
Primary Examiner: Nucker; Christine M.
Assistant Examiner: Reeves; Julie E.
Attorney, Agent or Firm: Townsend and Townsend and Crew
LLP
Claims
What is claimed is:
1. A method for specifically delivering an effector molecule to a
solid tumor cell bearing an IL-13 receptor, said method
comprising:
providing a chimeric molecule comprising said effector molecule
attached to a targeting molecule selected from the group consisting
of an IL-13, and an anti-IL-13 receptor antibody; and
contacting said chimeric molecule with said tumor cell to
specifically bind said chimeric molecule to said tumor cell.
2. The method of claim 1, wherein said targeting molecule is
IL-13.
3. The method of claim 1, wherein said tumor is a carcinoma.
4. The method of claim 1, wherein said effector molecule is
selected from the group consisting of a cytotoxin, a label, a
radionuclide, a drug, a liposome, a ligand, and an antibody.
5. The method of claim 4, wherein said effector molecule is a
Pseudomonas exotoxin.
6. The method of claim 5, wherein chimeric molecule is a fusion
protein.
7. The method of claim 6, wherein said fusion protein is
IL-13-PE38QQR.
8. The method of claim 1, wherein said effector molecule is a
detectable label and said method further comprises detecting said
label and thereby detecting said tumor cell.
9. The method of claim 8, wherein said detectable label is selected
from the group consisting of a radiolabel, an enzyme, a
colorimetric label, a fluorescent label, and a magnetic bead.
10. The method of claim 8, wherein said label is a fluorescent
label.
11. The method of claim 8, wherein said detecting is by
scintillography.
12. The method of claim 8, wherein said tumor cell is a carcinoma
cell.
13. The method of claim 11, wherein said carcinoma is selected from
the group consisting of colon carcinoma, skin carcinoma, and
gastric carcinoma.
14. A method for impairing growth of a solid tumor cell bearing an
IL-13 receptor, said method comprising contacting said tumor cell
with a chimeric molecule comprising:
a targeting molecule selected from the group consisting of an
IL-13, and an anti-IL-13 receptor antibody; and
an effector molecule selected from the group consisting of a
Pseudomonas exotoxin, a Diphtheria toxin, and a radionuclide,
wherein said effector molecule may be linked to the targeting
molecule by a linker consisting of a ligand or an antibody; and
wherein said contacting specifically binds said chimeric molecule
to said tumor cell.
15. The method of claim 14, wherein said targeting molecule is a
human IL-13.
16. The method of claim 15, wherein said effector molecule is a
Pseudomonas exotoxin or a Diphtheria toxin.
17. The method of claim 16, wherein chimeric molecule is a
single-chain fusion protein.
18. The method of claim 17, wherein said effector molecule is a
Pseudomonas exotoxin.
19. The method of claim 18, wherein said Pseudomonas exotoxin is
PE38QQR.
20. The method of claim 15, wherein said tumor cell growth is tumor
cell growth in a human.
21. The method of claim 20, wherein said contacting comprises
administering said chimeric molecule to the human intravenously,
into a body cavity, or into a lumen or an organ.
Description
FIELD OF THE INVENTION
This invention relates to methods of specifically delivering an
effector molecule to a tumor cell. In particular this invention
relates to chimeric molecules that specifically bind to IL-13
receptors and their use to deliver molecules having a particular
activity to tumors overexpressing IL-13 receptors.
BACKGROUND OF THE INVENTION
In a chimeric molecule, two or more molecules that exist separately
in their native state are joined together to form a single molecule
having the desired functionality of all of its constituent
molecules. Frequently, one of the constituent molecules of a
chimeric molecule is a "targeting molecule". The targeting molecule
is a molecule such as a ligand or an antibody that specifically
binds to its corresponding target, for example a receptor on a cell
surface. Thus, for example, where the targeting molecule is an
antibody, the chimeric molecule will specifically bind (target)
cells and tissues beating the epitope to which the antibody is
directed.
Another constituent of the chimeric molecule may be an "effector
molecule". The effector molecule refers to a molecule that is to be
specifically transported to the target to which the chimeric
molecule is specifically directed. The effector molecule typically
has a characteristic activity that is desired to be delivered to
the target cell. Effector molecules include cytotoxins, labels,
radionuclides, ligands, antibodies, drugs, liposomes, and the
like.
In particular, where first effector molecule is a cytotoxin, the
chimeric molecule may act as a potent cell-killing agent
specifically targeting the cytotoxin to cells bearing a particular
target molecule. For example, chimeric fusion proteins which
include interleukin 4 (IL-4) or transforming growth factor
(TGFc.alpha.) fused to Pseudomonas exotoxin (PE) or interleukin 2
(IL-2) fused to Diphtheria toxin (DT) have been shown to
specifically target and kill cancer cells (Pastan et al., Ann. Rev.
Biochem., 61: 331-354 (1992)).
Generally, it is desirable to increase specificity and affinity and
decrease cross-reactivity of the chimeric cytotoxins in order to
increase their efficacy. To the extent the chimeric molecule
preferentially selects and binds to its target (e.g. a tumor cell)
and not to a non-target (e.g. a healthy cell), side effects of the
chimeric molecule will be minimized. Unfortunately, many targets to
which chimeric molecules have been directed (e.g. the IL-2 and IL-4
receptors), while showing elevated expression on tumor cells, are
also expressed at significant levels on healthy cells. Thus,
chimeric molecules directed to these targets (e.g. cytotoxins) show
some adverse side-effects as they bind non-target cells that also
express the targeted receptor.
SUMMARY OF THE INVENTION
The present invention provides methods and compositions for
specifically delivering an effector molecule to a tumor cell. In
particular, the present invention provides chimetic molecules that
specifically target tumor cells with less binding to healthy cells
than other analogous chimetic molecules known in the prior art.
The improved specific targeting of this invention is premised, in
part, on the discovery that tumor cells, especially carcinomas such
as renal cell carcinoma, overexpress IL-13 receptors at extremely
high levels. The extremely high level of IL-13 receptor expression
on target tumor cells permits the use of lower dosages of chimeric
molecule to deliver the same amount of effector molecule to the
target cells and also results in reduced binding of non-tumor
cells.
In a preferred embodiment, this invention provides for a method for
specifically delivering an effector molecule to a tumor cell
bearing an IL-13 receptor. The method involves providing a chimeric
molecule comprising an effector molecule attached to a targeting
molecule that specifically binds to an IL-13 receptor and
contacting the tumor with the chimeric molecule resulting in
binding of the chimeric molecule to the tumor cell.
The targeting molecule is preferably either a ligand, such as
IL-13, that specifically binds an IL-13 receptor or an anti-IL-13
receptor antibody. The targeting molecule may be conjugated to the
effector molecule, or where both targeting and effector molecules
are polypeptides, the targeting molecule may be joined to the
effector molecule through one or more peptide bonds thereby forming
a fusion protein. Suitable effector molecules include a cytotoxin,
a label, a radionuclide, a drug, a liposome, a ligand, and an
antibody. In a particularly preferred embodiment, the effector is a
cytotoxin, more specifically a Pseudomonas exotoxin such as
PE38QQR. Where the Pseudomonas exotoxin is fused to an IL-13
targeting molecule, a preferred fusion protein is
IL-13-PE38QQR.
In another embodiment, this invention provides a method for
impairing the growth of tumor cells, more preferably solid tumor
cells, bearing an IL-13 receptor. The method involves contacting
the tumor with a chimeric molecule comprising an effector molecule
selected from the group consisting of a cytotoxin, a radionuclide,
a ligand and an antibody; said effector molecule being attached to
a targeting molecule that specifically binds a human IL-13
receptor. The targeting molecule is preferably a ligand (such as
IL-13) that binds the IL-13 receptor or an anti-IL-13 receptor
antibody. Preferred cytotoxic effector molecules include
Pseudomonas exotoxin, Diphtheria toxin, ricin and abrin.
Psuedomonas exotoxins, such as PE38QQR, are particularly preferred.
The targeting molecule may be conjugated or fused to the effector
molecule with attachment by fusion preferred for cytotoxic effector
molecules. The tumor growth that is impaired may be tumor growth in
a human. Thus the method may further comprise administering the
chimeric molecule to a human intravenously into a body cavity, or
into a human or an organ.
In yet another embodiment, this invention provides for a method of
detecting the presence or absence of a tumor. The method involves
contacting the tumor with a chimetic molecule comprising a
detectable label attached to a targeting molecule that specifically
binds a human IL-13 receptor and detecting the presence or absence
of the label. In a preferred embodiment, the label is selected from
the group consisting of a radioactive label, an enzymatic label, an
electron dense label, and a fluorescent label.
This invention also provides for vectors comprising a nucleic acid
sequence encoding a chimefie polypeptide fusion protein comprising
an IL-13 attached to a second polypeptide. The chimetic polypeptide
fusion protein specifically binds to a tumor cell beating an IL-13
receptor. A preferred vector encodes an IL-13-PE fusion protein and
more preferably encodes an IL-13-PE38QQR fusion protein.
This invention also provides for host cells comprising a nucleic
acid sequence encoding a chimeric polypeptide fusion protein
comprising an IL-13 attached to a second polypeptide. A preferred
host cell comprises a nucleic acid encoding an IL-13-PE fusion
protein, more preferably encoding an IL-13-PE38QQR fusion protein.
The encoded fusion protein specifically binds to a tumor cell
bearing an IL-13 receptor. Particularly preferred host cells are
bacterial host cells, especially E. coli cells.
In still yet another embodiment, this invention provides chimeric
molecules that specifically bind a tumor cell bearing an IL-13
receptor. In one preferred embodiment, the chimeric molecule
comprises a cytotoxic molecule attached to a targeting molecule
that specifically binds an IL-13. The targeting molecule may be
conjugated or fused to the cytotoxic molecule. In a preferred
embodiment, the targeting molecule is fused to the cytotoxin
thereby forming a single-chain fusion protein. Particularly
preferred targeting molecules are IL-13 or an antibody that
specifically binds to the IL-13 receptor. Preferred cytotoxic
molecules include Pseudomonas exotoxin, Diphtheria toxin, ricin,
and abrin, with Pseudomonas exotoxins (especially PE38QQR) being
most preferred.
In another preferred embodiment, the chimeric molecule comprises an
effector molecule attached to an antibody that specifically binds
to an IL-13 receptor. Effector molecules include a cytotoxin, a
label, a radionuclide, a drug, liposome, a ligand and an antibody.
The effector molecule may be fused or conjugated to the
antibody.
The invention additionally provides for pharmacological
compositions comprising a pharmaceutically acceptable carrier and a
chimeric molecule where the chimeric molecule comprises and
effector molecule attached to a targeting molecule that
specifically binds to an IL-13 receptor. The targeting and effector
molecules may be conjugated or fused to each other. Particularly
preferred targeting molecules include IL-13 and anti-IL-13 receptor
antibodies, while preferred effector molecules include a cytotoxin,
a label, a radionuclide, a drug, a liposome, a ligand and an
antibody. A preferred pharmacological composition includes an
IL-13-PE fusion protein, more preferably an IL-13-PE38QQR fusion
protein.
Definitions
The term "specifically deliver" as used herein refers to the
preferential association of a molecule with a cell or tissue
beating a particular target molecule or marker and not to cells or
tissues lacking that target molecule. It is, of course, recognized
that a certain degree of non-specific interaction may occur between
a molecule and a non-target cell or tissue. Nevertheless, specific
delivery, may be distinguished as mediated through specific
recognition of the target molecule. Typically specific delivery
results in a much stronger association between the delivered
molecule and cells bearing the target molecule than between the
delivered molecule and cells lacking the target molecule. Specific
delivery typically results in greater than 2 fold, preferably
greater than 5 fold, more preferably greater than 10 fold and most
preferably greater than 100 fold increase in amount of delivered
molecule (per unit time) to a cell or tissue bearing the target
molecule as compared to a cell or tissue lacking the target
molecule or marker.
The term "residue" as used herein refers to an amino acid that is
incorporated into a polypeptide. The amino acid may be a naturally
occurring amino acid and, unless otherwise limited, may encompass
known analogs of natural amino acids that can function in a similar
manner as naturally occurring amino acids.
A "fusion protein" refers to a polypeptide formed by the joining of
two or more polypeptides through a peptide bond formed between the
amino terminus of one polypeptide and the carboxyl terminus of
another polypeptide. The fusion protein may be formed by the
chemical coupling of the constituent polypeptides or it may be
expressed as a single polypeptide from nucleic acid sequence
encoding the single contiguous fusion protein. A single chain
fusion protein is a fusion protein having a single contiguous
polypeptide backbone.
A "spacer" as used herein refers to a peptide that joins the
proteins comprising a fusion protein. Generally a spacer has no
specific biological activity other than to join the proteins or to
preserve some minimum distance or other spatial relationship
between them. However, the constituent amino acids of a spacer may
be selected to influence some property of the molecule such as the
folding, net charge, or hydrophobicity of the molecule.
A "ligand", as used herein, refers generally to all molecules
capable of reacting with or otherwise recognizing or binding to a
receptor on a target cell. Specifically, examples of ligands
include, but are not limited to, antibodies, lymphokines,
cytokines, receptor proteins such as CD4 and CD8, solubilized
receptor proteins such as soluble CD4, hormones, growth factors,
and the like which specifically bind desired target cells.
DETAILED DESCRIPTION
Chimeric Molecules Targeted to the IL-13 Receptor
The present invention provides a method for specifically delivering
an effector molecule to a tumor cell. This method involves the use
of chimeric molecules comprising a targeting molecule attached to
an effector molecule. The chimeric molecules specifically target
tumor cells while providing reduced binding to non-target cells as
compared to other targeted chimeric molecules known in the art.
The improved specific targeting of this invention is premised, in
part, on the discovery that solid tumors, especially carcinomas,
overexpress IL-13 receptors at extremely high levels. While the
IL-13 receptors are overexpressed on tumor cells, expression on
other cells (e.g. monocytes and T cells) appears negligible. Thus,
by specifically targeting the IL-13 receptor, the present invention
provides chimeric molecules that are specifically directed to solid
tumors while minimizing targeting of other cells or tissues.
In a preferred embodiment, this invention provides for compositions
and methods for impairing the growth of tumors. The methods involve
providing a chimeric molecule comprising a cytotoxic effector
molecule attached to a targeting molecule that specifically binds
an IL-13 receptor. The cytotoxin may be a native or modified
cytotoxin such as Pseudomonas exotoxin (PE), Diphtheria toxin (DT),
ricin, abrin, and the like.
The chimeric cytotoxin is administered to an organism containing
tumor cells which are then contacted by the chimeric molecule. The
targeting molecule component of the chimeric molecule specifically
binds to the overexpressed IL-13 receptors on the tumor cells. Once
bound to the IL-13 receptor on the cell surface, the cytotoxic
effector molecule mediates internalization into the cell where the
cytotoxin inhibits cellular growth or kills the cell.
The use of chimeric molecules comprising a targeting moiety joined
to a cytotoxic effector molecules to target and kill tumor cells is
known in the prior art. For example, chimeric fusion proteins which
include interleukin 4 (IL-4) or transforming growth factor
(TGF.alpha.) fused to Pseudomonas exotoxin (PE) or interleukin 2
(IL-2) fused to Diphtheria toxin (DT) have been tested for their
ability to specifically target and kill cancer cells (Pastan et
al., Ann. Rev. Biochem., 61: 331-354 (1992)).
Although chimeric IL-4-cytotoxin molecules are known in the prior
art, and IL-4 shows some sequence similarity to IL-13, it was an
unexpected discovery of the present invention that cytotoxins
targeted by a moiety specific to the IL-13 receptor show
significantly increased efficacy as compared to IL-4 receptor
directed cytotoxins. Without being bound to a particular theory, it
is believed that the improved efficacy of the IL-13 chimeras of the
present invention is due to at least three factors.
First, IL-13 receptors are expressed at much lower levels, if at
all on non-tumor cells (e.g. monocytes, T cells, B cells). Thus
cytotoxins directed to IL-13 receptors show reduced binding and
subsequent killing of healthy cells and tissues as compared to
cytotoxins directed to IL-4 receptors.
Second, the receptor component that specifically binds IL-13
appears to be expressed at significantly higher levels on solid
tumors than the receptor component that binds IL-4. Thus, tumor
cells bind higher levels of cytotoxic chimeric molecules directed
against IL-13 receptors than cytotoxic chimeric molecules directed
against IL-4 receptors.
Finally, IL-4 receptors are up-regulated when immune system cells
(e.g. T-cells) are activated. This results in healthy cells, for
example T-cells and B-cells, showing greater susceptibility to IL-4
receptor directed cytotoxins. Thus, the induction of an immune
response (as against a cancer), results in greater susceptiblity of
cells of the immune system to the therapeutic agent. In contrast,
IL-13 receptors are not up-regulated in activated cells. Thus IL-13
receptor targeted cytotoxins have no greater effect on activated
cells and thereby minimize adverse effects of the therapeutic
composition on cells of the immune system.
In another embodiment, this invention also provides for
compositions and methods for detecting the presence or absence of
tumor cells. These methods involve providing a chimeric molecule
comprising an effector molecule, that is a detectable label
attached to a targeting molecule that specifically binds an IL-13
receptor. The IL-13 receptor targeting moiety specifically binds
the chimetic molecule to tumor cells which are then marked by their
association with the detectable label. Subsequent detection of the
cell-associated label indicates the presence of a tumor cell.
In yet another embodiment, the effector molecule may be another
specific binding moiety such as an antibody, a growth factor, or a
ligand. The chimetic molecule will then act as a highly specific
bifunctional linker. This linker may act to bind and enhance the
interaction between cells or cellular components to which the
fusion protein binds. Thus, for example, where the "targeting"
component of the chimefie molecule comprises a polypeptide that
specifically binds to an IL-13 receptor and the "effector"
component is an antibody or antibody fragment (e.g. an Fv fragment
of an antibody), the targeting component specifically binds cancer
cells, while the effector component binds receptors (e.g., IL-2 or
IL-4 receptors) on the surface of immune cells. The chimeric
molecule may thus act to enhance and direct an immune response
toward target cancer cells.
In still yet another embodiment the effector molecule may be a
pharmacological agent (e.g. a drug) or a vehicle containing a
pharmacological agent. This is particularly suitable where it is
merely desired to invoke a non-lethal biological response. Thus the
moiety that specifically binds to an IL-13 receptor may be
conjugated to a drug such as vinblastine, doxirubicin, genistein (a
tyrosine kinase inhibitor), an antisense molecule, and other
pharmacological agents known to those of skill in the art, thereby
specifically targeting the pharmacological agent to tumor cells
over expressing IL-13 receptors.
Alternatively, the targeting molecule may be bound to a vehicle
containing the therapeutic composition. Such vehicles include, but
are not limited to liposomes, micelles, various synthetic beads,
and the like.
One of skill in the art will appreciate that the chimeric molecules
of the present invention may include multiple targeting moieties
bound to a single effector or conversely, multiple effector
molecules bound to a single targeting moiety. In still other
embodiment, the chimeric molecules may include both multiple
targeting moieties and multiple effector molecules. Thus, for
example, this invention provides for "dual targeted" cytotoxic
chimeric molecules in which targeting molecule that specifically
binds to IL-13 is attached to a cytotoxic molecule and another
molecule (e.g. an antibody, or another ligand) is attached to the
other terminus of the toxin. Such a dual-targeted cytotoxin might
comprise an IL-13 substituted for domain Ia at the amino terminus
of a PE and anti-TAC(fv) inserted in domain III, between amino acid
604 and 609. Other antibodies may also be suitable.
The Targeting Molecule
In a preferred embodiment, the targeting molecule is a molecule
that specifically binds to the IL-13 receptor. The term
"specifically binds", as used herein, when referring to a protein
or polypeptide, refers to a binding reaction which is determinative
of the presence of the protein or polypeptide in a heterogeneous
population of proteins and other biologics. Thus, under designated
conditions (e.g. immunoassay conditions in the case of an
antibody), the specified ligand or antibody binds to its particular
"target" protein (e.g. an IL-13 receptor protein) and does not bind
in a significant amount to other proteins present in the sample or
to other proteins to which the ligand or antibody may come in
contact in an organism.
A variety of immunoassay formats may be used to select antibodies
specifically immunoreactive with an IL-13 receptor protein. For
example, solid-phase ELISA immunoassays are routinely used to
select monoclonal antibodies specifically immunoreactive with a
protein. See Harlow and Lane (1988) Antibodies, A Laboratory
Manual, Cold Spring Harbor Publications, New York, for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity.
Similarly, assay formats for detecting specific binding of ligands
(e.g. IL-13) with their respective receptor are also well known in
the art. Example 1 provides a detailed protocol for assessing
specific binding of labeled IL-13 by and IL-13 receptor.
The IL-13 receptor is a cell surface receptor that specifically
binds IL-13 and mediates a variety of physiological responses in
various cell types as described below in the description of IL-13.
The IL-13 receptor may be identified by contacting a cell or other
sample with labeled IL-13 and detecting the amount of specific
binding of IL-13 according to methods well known to those of skill
in the art. Detection of IL-13 receptors by labeled IL-13 binding
is described in detail in Example 1.
Alternatively, an anti-IL-13 receptor antibody may also be used to
identify IL-13 receptors. The antibody will specifically bind to
the IL-13 receptor and this binding may be detected either through
detection of a conjugated label or through detection of a labeled
second antibody that binds the anti-IL-13 receptor antibody.
In a preferred embodiment, the moiety utilized to specifically
target the IL-13 receptor is either an antibody that specifically
binds the IL-13 receptor (an anti-IL-13R antibody) or a ligand,
such as IL-13, that specifically binds to the receptor.
IL-13
IL-13 is a pleiotropic cytokine that is recognized to share many of
the properties of IL-4. IL-13 has approximately 30% sequence
identity with IL-4 and exhibits IL-4-like activities on
monocytes/macrophages and human B cells (Minty et al., Nature, 362:
248 (1993), McKenzie et al. Proc. Natl. Acad. Sci. USA, 90: 3735
(1987)). In particular, IL-13 appears to be a potent regulator of
inflammatory and immune responses. Like IL-4, IL-13 can up-regulate
the monocyte/macrophage expression of CD23 and MHC class I and
class II antigens, down-regulate the expression of Fc.gamma., and
inhibit antibody-dependent cytotoxicity. IL-13 can also inhibit
nitric oxide production as well as the expression of
pro-inflammatory cytokines (e.g. IL-1, IL-6, IL-8, IL-10 and IL-12)
and chemokines (MIP-1, MCP), but enhance the production of IL-1ra
(Minty supra.; Mckenzie et al., supra.; Zurawski et al. Immunol.
Today, 15: 19 (1994); de Wall Malefyt et al. J. lmmunol., 150: 180A
(1993); de Wall Malefyt et al. J. Immunol., 151: 6370 (1993);
Doherty et al. J. lmmunol., 151: 7151 (1993); and Minty et al. Eur.
cytokine Netw., 4: 99 (1993)).
Recombinant IL-13 is commercially available from a number of
sources (see, e.g. R & D Systems, Minneapolis, Minn., U.S.A.,
and Sanoff Bio-Industries, Inc., Tervose, Pa., U.S.A.).
Alternatively, a gene or a cDNA encoding IL-13 may be cloned into a
plasmid or other expression vector and expressed in any of a number
of expression systems according to methods well known to those of
skill in the art. Methods of cloning and expressing IL-13 and the
nucleic acid sequence for IL-13 are well kown (see, for example,
Minty et al. (1993) supra. and McKenzie (1987), supra). In
addition, the expression of IL-13 as a component of a chimeric
molecule is detailed in Example 4.
One of skill in the art will appreciate that analogues or fragments
of IL-13 bearing will also specifically bind to the IL-13 receptor.
For example, conservative substitutions of residues (e.g., a serine
for an alaninc or an aspartic acid for a glutamic acid) comprising
native IL-13 will provide IL-13 analogues that also specifically
bind to the IL-13 receptor. Thus, the term "IL-13", when used in
reference to a targeting molecule, also includes fragments,
analogues or peptide mimetics of IL-13 that also specifically bind
to the IL-13 receptor.
Anti-IL-13 Receptor Antibodies
One of skill will recognize that other molecules besides IL-13 will
specifically bind to IL-13 receptors. Polyclonal and monoclonal
antibodies directed against IL-13 receptors provide particularly
suitable targeting molecules in the chimeric molecules of this
invention. The term "antibody", as used herein, includes various
forms of modified or altered antibodies, such as an intact
immunoglobulin, various fragments such as an Fv fragment, an Fv
fragment containing only the light and heavy chain variable
regions, an Fv fragment linked by a disulfide bond (Brinkmann, et
al. Proc. Natl. Acad. Sci. USA, 90: 547-551 (1993)), an Fab or
(Fab)'.sub.2 fragment containing the variable regions and parts of
the constant regions, a single-chain antibody and the like (Bird et
al., Science 242: 424-426 (1988); Huston et al., Proc. Nat. Acad.
Sci. USA 85: 5879-5883 (1988)). The antibody may be of animal
(especially mouse or rat) or human origin or may be chimeric
(Morrison et al., Proc Nat. Acad. Sci. USA 81: 6851-6855 (1984)) or
humanized (Jones et al., Nature 321: 522-525 (1986), and published
UK patent application #8707252). Methods of producing antibodies
suitable for use in the present invention are well known to those
skilled in the art and can be found described in such publications
as Harlow & Lane, Antibodies: A Laboratory Manual, Cold Spring
Harbor Laboratory (1988), and Asai, Methods in Cell Biology Vol.
37: Antibodies in Cell Biology, Academic Press, Inc. New York
(1993).
Antibodies that specifically bind the IL-13 receptor may be
produced by a number of means well known to those of skill in the
art. Generally, this involves using an antigenic component of the
IL-13 receptor as an antigen to induce the production of antibodies
in an organism (e.g. a sheep, mouse, rabbit, etc.). One of skill in
the art will recognize that there are numerous methods of isolating
all or components of the IL-13 receptor for use as an antigen. For
example, IL-13 receptors may be isolated by crosslinking the
receptor to a labeled IL-13 by the exposure to 2 mM disuccinimidyl
subcrate (DSS). The labeled receptor may then be isolated according
to routine methods and the isolated receptor may be used as an
antigen to raise anti-IL-13 receptor antibodies as described below.
Cross-linking and isolation of components of the IL-13 receptor is
described in Example 3.
In a preferred embodiment, however, IL-13 receptors may be isolated
by means of affinity chromatography. It was a surprising discovery
of the present invention that solid tumor cells overexpress IL-13
receptors. This discovery of cells overexpressing IL-13 receptor
greatly simplifies the receptor isolation. Generally,
approximately, 100 million renal carcinoma cells, may be
solubilized in detergent with protease inhibitors according to
standard methods. The resulting lysate is then run through an
affinity column bearing IL-13. The receptor binds to the IL-13 in
the column thereby effecting an isolation from the lysate. The
column is then eluted with a low pH buffer to dissociate the IL-13
ligand from the IL-13 receptor resulting in isolated receptor. The
isolated receptor may then be used as an antigen to raise
anti-IL-13 receptor antibodies.
Antibody Production
Methods of producing polyclonal antibodies are known to those of
skill in the art. In brief, an immunogen, preferably an isolated
IL-13 receptor or receptor epitope is mixed with an adjuvant and
animals are immunized with the mixture. The animal's immune
response to the immunogen preparation is monitored by taking test
bleeds and determining the tiler of reactivity to the polypeptide
of interest. When appropriately high titers of antibody to the
immunogen are obtained, blood is collected from the animal and
antisera are prepared. Further fractionation of the antisera to
enrich for antibodies reactive to the polypeptide is performed
where desired. See, e.g., Coligan (1991) Current Protocols in
Immunology Wiley/Greene, New York; and Harlow and Lane (1989)
Antibodies: A Laboratory Manual Cold Spring Harbor Press, New York,
which are incorporated herein by reference.
Monoclonal antibodies may be obtained by various techniques
familiar to those skilled in the art. Description of techniques for
preparing such monoclonal antibodies may be found in, e.g., Stites
et al. (eds.) Basic and Clinical Immunology (4th ed.) Lange Medical
Publications, Los Altos, Calif., and references cited therein;
Harlow and Lane (1988) Antibodies: A Laboratory Manual CSH Press;
Goding (1986) Monoclonal Antibodies: Principles and Practice (2d
ed.) Academic Press, New York, N.Y.; and particularly in Kohler and
Milstein (1975) Nature 256: 495-497, which discusses one method of
generating monoclonal antibodies.
Summarized briefly, this method involves injecting an animal with
an immunogen. The animal is then sacrificed and cells taken from
its spleen, which are then fused with myeloma cells (See, Kohler
and Milstein (1976) Eur. J. Immunol. 6: 511-519, incorporated
herein by reference). The result is a hybrid cell or "hybridoma"
that is capable of reproducing in vitro.
Colonies arising from single immortalized cells are screened for
production of antibodies of the desired specificity and affinity
for the antigen, and yield of the monoclonal antibodies produced by
such cells is enhanced by various techniques, including injection
into the peritoneal cavity of a vertebrate host. Alternatively, one
may isolate DNA sequences which encode a monoclonal antibody or a
binding fragment thereof by screening a DNA library from human B
cells according to the general protocol outlined by Huse et al.
(1989) Science 246: 1275-1281. In this manner, the individual
antibody species obtained are the products of immortalized and
cloned single B cells from the immune animal generated in response
to a specific site recognized on the immunogenic substance.
Other suitable techniques involve selection of libraries of
antibodies in phage or similar vectors. See, Huse et al. Science
246: 1275-1281 (1989); and Ward, et al. Nature 341: 544-546 (1989).
In general suitable monoclonal antibodies will usually bind their
target epitope with at least a K.sub.D of about 1 mM, more usually
at least about 300 .mu.M, and most preferably at least about 0.1
.mu.M or better.
Other Targeting Antibodies
Where the chimeric molecule contains more than one targeting
molecule (e.g. a dual-targeted cytotoxin), the molecule may contain
targeting antibodies directed to tumor markers other than the
overexpressed IL-13 receptor. A number of such antibodies are known
and have even been converted to form suitable for incorporation
into fusion proteins. These include anti-erbB2, B3, BR96, OVB3,
anti-transferrin, Mik-.beta.1 and PR1 (see Batra et at., Mol. Cell.
Biol., 11: 2200-2205 (1991); Batra et at., Proc. Natl. Acad. Sci.
USA, 89: 5867-5871 (1992); Brinkmann, et al. Proc. Natl. Acad. Sci.
USA, 88: 8616-8620 (1991); Brinkmann et al., Proc. Natl. Acad. Sci.
USA, 90: 547-551 (1993); Chaudhary et al., Proc. Natl. Acad. Sci.
USA, 87: 1066-1070 (1990); Friedman et al., Cancer Res. 53: 334-339
(1993); Kreitman et al., J. Immunol., 149: 2810-2815 (1992);
Nicholls et al., J. Biol. Chem., 268: 5302-5308 (1993); and Wells,
et al., Cancer Res., 52: 6310-6317 (1992), respectively).
The Effector Molecule
As described above, the effector molecule component of the chimeric
molecules of this invention may be any molecule whose activity it
is desired to deliver to cells that overexpress IL-13 receptors.
Particularly preferred effector molecules include cytotoxins such
as PE or DT, radionuclides, ligands such as growth factors,
antibodies, detectable labels such as fluorescent or radioactive
labels, and therapeutic compositions such as liposomes and various
drugs.
Cytotoxins
Particularly preferred cytotoxins include Pseudomonas exotoxins,
Diphtheria toxins, ricin, and abrin. Pseudomonas exotoxin and
Dipthteria toxin are most preferred.
Pseudornonas exotoxin (PE)
Pseudomonas exotoxin A (PE) is an extremely active monomeric
protein (molecular weight 66 kD), secreted by Pseudomonas
aeruginosa, which inhibits protein synthesis in eukaryotic cells
through the inactivation of elongation factor 2 (EF-2) by
catalyzing its ADP-ribosylation (catalyzing the transfer of the ADP
ribosyl moiety of oxidized NAD onto EF-2).
The toxin contains three structural domains that act in concert to
cause cytotoxicity. Domain Ia (amino acids 1-252) mediates cell
binding. Domain II (amino acids 253-364) is responsible for
translocation into the cytosol and domain III (amino acids 400-613)
mediates ADP ribosylation of elongation factor 2, which inactivates
the protein and causes cell death. The function of domain Ib (amino
acids 365-399) remains undefined, although a large part of it,
amino acids 365-380, can be deleted without loss of cytotoxicity.
See Siegall et al., J. Biol. Chem. 264: 14256-14261 (1989),
incorporated by reference herein.
Where the targeting molecule (e.g. IL-13) is fused to PE, a
preferred PE molecule is one in which domain Ia (amino acids 1
through 252) is deleted and amino acids 365 to 380 have been
deleted from domain Ib. However all of domain Ib and a portion of
domain II (amino acids 350 to 394) can be deleted, particularly if
the deleted sequences are replaced with a linking peptide such as
four glycine residues followed by a serine.
In addition, the PE molecules can be further modified using
site-directed mutagenesis or other techniques known in the art, to
alter the molecule for a particular desired application. Means to
alter the PE molecule in a manner that does not substantially
affect the functional advantages provided by the PE molecules
described here can also be used and such resulting molecules are
intended to be covered herein.
For maximum cytotoxic properties of a preferred PE molecule,
several modifications to the molecule are recommended. An
appropriate carboxyl terminal sequence to the recombinant molecule
is preferred to translocate the molecule into the cytosol of target
cells. Amino acid sequences which have been found to be effective
include, arginine, followed by EDL and optionally followed by
lysine (as in native PE), or DEL preceded by either an arginine or
a lysine repeats of those, or other sequences that function to
maintain or recycle proteins into the endoplasmic reticulum,
referred to here as "endoplasmic retention sequences". See, for
example, Chaudhary et al, Proc. Natl. Acad. Sci. USA 87: 308-312
and Seetharam et al, J. Biol. Chem. 266: 17376-17381 (1991) and
commonly assigned, U.S. Ser. No. 07/459,635 filed Jan. 2, 1990, now
abandoned, all of which are incorporated by reference herein.
Deletions of amino acids 365-380of domain Ib can be made without
loss of activity. Further, a substitution of methionine at amino
acid position 280 in place of glycine to allow the synthesis of the
protein to begin and of serine at amino acid position 287 in place
of cysteine to prevent formation of improper disulfide bonds is
beneficial. In a preferred embodiment, the targeting molecule is
inserted in replacement for domain Ia. A similar insertion has been
accomplished in what is known as the TGF.alpha.-PE40 molecule (also
referred to as TP40) described in Heimbrook et al., Proc. Natl.
Acad. Sci., USA, 87: 4697-4701 (1990) and in commonly assigned U.S.
Ser. No. 07/865,722filed Apr. 8, 1992 and in U.S. Ser. No.
07/522,563 filed May 14, 1990, now abandonded, all of which are
incorporated by reference.
Preferred forms of PE contain amino acids 253-364 and 381-608, and
are followed by the native sequences arginine, followed by EDL and
further followed by lysine (as in native PE), or the mutant
sequences DEL preceded by either an arginine or a lysine. Lysines
at positions 590 and 606 may or may not be mutated to
glutamine.
In a particularly preferred embodiment, the IL-13 receptor targeted
cytotoxins of this invention comprise the PE molecule designated
PE38QQR. This PE molecule is a truncated form of PE composed of
amino acids 253-364 and 381-608. The lysine residues at positions
509 and 606 are replaced by glutamine and at 613 are replaced by
arginine (Debinski et al. Bioconj. Chem., 5: 40 (1994) which is
incorporated herein by reference).
The targeting molecule (e.g. IL-13 or anti-IL-13R antibody) may
also be inserted at a point within domain III of the PE molecule.
Most preferably the targeting molecule is fused between about amino
acid positions 607 and 609 of the PE molecule. This means that the
targeting molecule is inserted after about amino acid 607 of the
molecule and an appropriate carboxyl end of PE is recreated by
placing amino acids about 604-613 of PE after the targeting
molecule. Thus, the targeting molecule is inserted within the
recombinant PE molecule after about amino acid 607 and is followed
by amino acids 604-613of domain III. The targeting molecule may
also be inserted into domain Ib to replace sequences not necessary
for toxicity. Debinski, et al. Mol. Cell. Biol., 11: 1751-1753
(1991).
In a preferred embodiment, the PE molecules will be fused to the
targeting molecule by recombinant means. The genes encoding protein
chains may be cloned in cDNA or in genomic form by any cloning
procedure known to those skilled in the art. See for example
Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold
Spring Harbor Laboratory, (1989), incorporated by reference herein.
Methods of cloning genes encoding PE fused to various ligands are
well known to those of skill in the art. See, for example, Siegall
et al., FASEB J., 3: 2647-2652 (1989); Chaudhary et al. Proc. Natl.
Acad. Sci. USA, 84: 4538-4542 (1987), which are incorporated herein
by reference.
Those skilled in the art will realize that additional
modifications, deletions, insertions and the like may be made to
the chimeric molecules of the present invention or to the nucleic
acid sequences encoding IL-13 receptor-directed chimeric molecules.
Especially, deletions or changes may be made in PE or in a linker
connecting an antibody gene to PE, in order to increase
cytotoxicity of the fusion protein toward target cells or to
decrease nonspecific cytotoxicity toward cells without antigen for
the antibody. All such constructions may be made by methods of
genetic engineering well known to those skilled in the art (see,
generally, Sambrook et al., supra) and may produce proteins that
have differing properties of affinity, specificity, stability and
toxicity that make them particularly suitable for various clinical
or biological applications.
Diphtheria TOxin (DT)
Like PE, diphtheria toxin (DT) kills cells by ADP-ribosylating
elongation factor 2 thereby inhibiting protein synthesis.
Diphtheria toxin, however, is divided into two chains, A and B,
linked by a disulfide bridge. In contrast to PE, chain B of DT,
which is on the carboxyl end, is responsible for receptor binding
and chain A, which is present on the amino end, contains the
enzymatic activity (Uchida et al., Science, 175: 901-903 (1972);
Uchida et al. J. Biol. Chem., 248: 3838-3844 (1973)).
In a preferred embodiment, the targeting molecule-Diphtheria toxin
fusion proteins of this invention have the native receptor-binding
domain removed by truncation of the Diphtheria toxin B chain.
Particularly preferred is DT388, a DT in which the carboxyl
terminal sequence beginning at residue 389 is removed. Chaudhary,
et al., Bioch. Biophys. Res. Comm., 180: 545-551 (1991).
Like the PE chimeric cytotoxins, the DT molecules may be chemically
conjugated to the IL-13 receptor targeting molecule, but, in a
preferred embodiment, the targeting molecule will be fused to the
Diphtheria toxin by recombinant means. The genes encoding protein
chains may be cloned in cDNA or in genomic form by any cloning
procedure known to those skilled in the art. Methods of cloning
genes encoding DT fused to various ligands are also well known to
those of skill in the art. See, for example, Williams et al. J.
Biol. Chem. 265: 11885-11889 (1990) and copending patent
application (U.S. Ser. No. 07/620,939) which describe the
expression of a number of growth-factor-DT fusion proteins.
The term "Diphtheria toxin" (DT) as used herein refers to full
length native DT or to a DT that has been modified. Modifications
typically include removal of the targeting domain in the B chain
and, more specifically, involve truncations of the carboxyl region
of the B chain.
Detectable Labels
Detectable labels suitable for use as the effector molecule
component of the chimeric molecules of this invention include any
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels in the present invention include magnetic beads (e.g.
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein isothiocyanate,
texas red, rhodamine, green fluorescent protein, and the like),
radiolabels (e.g., .sup.3 H, .sup.125 I, .sup.35 S, .sup.14 C, or
.sup.32 P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric
labels such as colloidal gold or colored glass or plastic (e.g.
polystyrene, polypropylene, latex, etc.) beads.
Means of detecting such labels are well known to those of skill in
the art. Thus, for example, radiolabels may be detected using
photographic film or scintillation counters, fluorescent markers
may be detected using a photodetector to detect emitted
illumination. Enzymatic labels are typically detected by providing
the enzyme with a substrate and detecting the reaction product
produced by the action of the enzyme on the substrate, and
colorimetric labels are detected by simply visualizing the colored
label.
Ligands
As explained above, the effector molecule may also be a ligand or
an antibody. Particularly preferred ligand and antibodies are those
that bind to surface markers of immune cells. Chimetic molecules
utilizing such antibodies as effector molecules act as bifunctional
linkers establishing an association between the immune cells
bearing binding partner for the ligand or antibody and the tumor
cells overexpressing the IL-13 receptor. Suitable antibodies and
growth factors are known to those of skill in the art and include,
but are not limited to, IL-2, IL-4, IL-6, IL-7, tumor necrosis
factor (TNF), anti-Tac, TGF.alpha., and the like.
Other Therapeutic Moleties
Other suitable effector molecules include pharmacological agents or
encapsulation systems containing various pharmacological agents.
Thus, the targeting molecule of the chimeric molecule may be
attached directly to a drug that is to be delivered directly to the
tumor. Such drugs are well known to those of skill in the art and
include, but are not limited to, doxirubicin, vinblastine,
genistein, an antisense molecule, and the like.
Alternatively, the effector molecule may be an encapsulation
system, such as a liposome or micelie that contains a therapeutic
composition such as a drug, a nucleic acid (e.g. an antisense
nucleic acid), or another therapeutic moiety that is preferably
shielded from direct exposure to the circulatory system. Means of
preparing liposomes attached to antibodies are well known to those
of skill in the art. See, for example, U.S. Pat. No. 4,957,735,
Connor et al., Pharm. Ther., 28: 341-365 (1985)
Attachment of the Targeting Molecule to the Effector Molecule
One of skill will appreciate that the targeting molecule and
effector molecules may be joined together in any order. Thus, where
the targeting molecule is a polypeptide, the effector molecule may
be joined to either the amino or carboxy termini of the targeting
molecule. The targeting molecule may also be joined to an internal
region of the effector molecule, or conversely, the effector
molecule may be joined to an internal location of the targeting
molecule, as long as the attachment does not interfere with the
respective activities of the molecules.
The targeting molecule and the effector molecule may be attached by
any of a number of means well known to those of skill in the art.
Typically the effector molecule is conjugated, either directly or
through a linker (spacer), to the targeting molecule. However,
where both the effector molecule and the targeting molecule are
polypeptides it is preferable to recombinantly express the chimeric
molecule as a single-chain fusion protein.
Conjugation of the Effector Molecule to the Targeting Molecule
In one embodiment, the targeting molecule (e.g. IL-13 or
anti-IL-13R antibody) is chemically conjugated to the effector
molecule (e.g. a cytotoxin, a label, a ligand, or a drug or
liposome). Means of chemically conjugating molecules are well known
to those of skill.
The procedure for attaching an agent to an antibody or other
polypeptide targeting molecule will vary according to the chemical
structure of the agent. Polypeptides typically contain variety of
functional groups; e.g., carboxylic acid (COOH) or free amine
(--NH.sub.2) groups, which are available for reaction with a
suitable functional group on an effector molecule to bind the
effector thereto.
Alternatively, the targeting molecule and/or effector molecule may
be derivatized to expose or attach additional reactive functional
groups. The derivatization may involve attachment of any of a
number of linker molecules such as those available from Pierce
Chemical Company, Rockford Ill.
A "linker", as used herein, is a molecule that is used to join the
targeting molecule to the effector molecule. The linker is capable
of forming covalent bonds to both the targeting molecule and to the
effector molecule. Suitable linkers are well known to those of
skill in the art and include, but are not limited to, straight or
branched-chain carbon linkers, heterocyclic carbon linkers, or
peptide linkers. Where the targeting molecule and the effector
molecule are polypeptides, the linkers may be joined to the
constituent amino acids through their side groups (e.g., through a
disulfide linkage to cysteine). However, in a preferred embodiment,
the linkers will be joined to the alpha carbon amino and carboxyl
groups of the terminal amino acids.
A bifunctional linker having one functional group reactive with a
group on a particular agent, and another group reactive with an
antibody, may be used to form the desired immunoconjugate.
Alternatively, derivatization may involve chemical treatment of the
targeting molecule, e.g., glycol cleavage of the sugar moiety of a
the glycoprotein antibody with periodate to generate free aidehyde
groups. The free aldehyde groups on the antibody may be reacted
with free amine or hydrazine groups on an agent to bind the agent
thereto. (See U.S. Pat. No. 4,671,958). Procedures for generation
of free sulfhydryl groups on polypeptide, such as antibodies or
antibody fragments, are also known (See U.S. Pat. No.
4,659,839).
Many procedure and linker molecules for attachment of various
compounds including radionuclide metal chelates, toxins and drugs
to proteins such as antibodies are known. See, for example,
European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958,
4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and
4,589,071; and Borlinghaus et al. Cancer Res. 47: 4071-4075 (1987)
which are incorporated herein by reference. In particular,
production of various immunotoxins is well-known within the art and
can be found, for example in "Monoclonal Antibody-Toxin Conjugates:
Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in
Clinical Medicine, Academic Press, pp. 168-190 (1982), Waldmann,
Science, 252: 1657 (1991), U.S. Pat. Nos. 4,545,985 and 4,894,443
which are incorporated herein by reference.
In some circumstances, it is desirable to free the effector
molecule from the targeting molecule when the chimeric molecule has
reached its target site. Therefore, chimeric conjugates comprising
linkages which are cleavable in the vicinity of the target site may
be used when the effector is to be released at the target site.
Cleaving of the linkage to release the agent from the antibody may
be prompted by enzymatic activity or conditions to which the
immunoconjugate is subjected either inside the target cell or in
the vicinity of the target site. When the target site is a tumor, a
linker which is cleavable under conditions present at the tumor
site (e.g. when exposed to tumor-associated enzymes or acidic pH)
may be used.
A number of different cleavable linkers are known to those of skill
in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014.
The mechanisms for release of an agent from these linker groups
include, for example, irradiation of a photolabile bond and
acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958, for example,
includes a description of immunoconjugates comprising linkers which
are cleaved at the target site in vivo by the proteolytic enzymes
of the patient's complement system. In view of the large number of
methods that have been reported for attaching a variety of
radiodiagnostic compounds, radiotherapeutic compounds, drugs,
toxins, and other agents to antibodies one skilled in the art will
be able to determine a suitable method for attaching a given agent
to an antibody or other polypeptide.
Production of Fusion Proteins
Where the targeting molecule and/or the effector molecule is
relatively short (i.e., less than about 50 amino acids) they may be
synthesized using standard chemical peptide synthesis techniques.
Where both molecules are relatively short the chimeric molecule may
be synthesized as a single contiguous polypeptide. Alternatively
the targeting molecule and the effector molecule may be synthesized
separately and then fused by condensation of the amino terminus of
one molecule with the carboxyl terminus of the other molecule
thereby forming a peptide bond. Alternatively, the targeting and
effector molecules may each be condensed with one end of a peptide
spacer molecule thereby forming a contiguous fusion protein.
Solid phase synthesis in which the C-terminal amino acid of the
sequence is attached to an insoluble support followed by sequential
addition of the remaining amino acids in the sequence is the
preferred method for the chemical synthesis of the polypeptides of
this invention. Techniques for solid phase synthesis are described
by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284
in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special
Methods in Peptide Synthesis, Part A., Merrifield, et al. J. Am.
Chem. Soc., 85: 2149-2156 (1963), and Stewart et al., Solid Phase
Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984)
which are incorporated herein by reference.
In a preferred embodiment, the chimeric fusion proteins of the
present invention are synthesized using recombinant DNA
methodology. Generally this involves creating a DNA sequence that
encodes the fusion protein, placing the DNA in an expression
cassette under the control of a particular promoter, expressing the
protein in a host, isolating the expressed protein and, if
required, renaturing the protein.
DNA encoding the fusion proteins (e.g. IL-13-PE38QQR) of this
invention may be prepared by any suitable method, including, for
example, cloning and restriction of appropriate sequences or direct
chemical synthesis by methods such as the phosphotriester method of
Narang et al. Meth. Enzymol. 68: 90-99 (1979); the phosphodiester
method of Brown et al., Meth. Enzymol. 68: 109-151 (1979); the
diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:
1859-1862 (1981); and the solid support method of U.S. Pat. No.
4,458,066, all incorporated by reference herein.
Chemical synthesis produces a single stranded oligonucleotide. This
may be converted into double stranded DNA by hybridization with a
complementary sequence, or by polymerization with a DNA polymerase
using the single strand as a template. One of skill would recognize
that while chemical synthesis of DNA is limited to sequences of
about 100 bases, longer sequences may be obtained by the ligation
of shorter sequences.
Alternatively, subsequences may be cloned and the appropriate
subsequences cleaved using appropriate restriction enzymes. The
fragments may then be ligated to produce the desired DNA
sequence.
In a preferred embodiment, DNA encoding fusion proteins of the
present invention may be cloned using DNA amplification methods
such as polymerase chain reaction (PCR). Thus, in a preferred
embodiment, the gene for IL-13 is PCR amplified, using a sense
primer containing the restriction site for NdeI and an antisense
primer containing the restriction site for HindIII. In a
particularly preferred embodiment, the primers are selected to
amplify the nucleic acid starting at position 19, as described by
McKenzie et al. (1987), supra. This produces a nucleic acid
encoding the mature IL-13 sequence and having terminal restriction
sites. A PE38QQR fragment may be cut out of the plasmid
pWDMH4-38QQR or plasmid pSGC242FdN1 described by Debinski et al.
Int. J. Cancer, 58: 744-748 (1994), and by Debinski et al. Clin.
Res., 42: 251A (abstract (1994) respectively. Ligation of the IL-13
and PE38QQR sequences and insertion into a vector produces a vector
encoding IL-13 joined to the amino terminus of PE38QQR (position
253 of PE). The two molecues are joined by a three amino acid
junction consaisting of glutamic acid, alanine, and phenylalanine
introduced by the restriction site.
While the two molecules are preferrably essentially directly joined
together, one of skill will appreciate that the molecules may be
separated by a peptide spacer consisting of one or more amino
acids. Generally the spacer will have no specific biological
activity other than to join the proteins or to preserve some
minimum distance or other spatial relationship between them.
However, the constituent amino acids of the spacer may be selected
to influence some property of the molecule such as the folding, net
charge, or hydrophobicity.
The nucleic acid sequences encoding the fusion proteins may be
expressed in a variety of host cells, including E. coli, other
bacterial hosts, yeast, and various higher eukaryotic cells such as
the COS, CHO and HeLa cells lines and myeloma cell lines. The
recombinant protein gene will be operably linked to appropriate
expression control sequences for each host. For E. coli this
includes a promoter such as the T7, trp, or lambda promoters, a
ribosome binding site and preferably a transcription termination
signal. For eukaryotic cells, the control sequences will include a
promoter and preferably an enhancer derived from immunoglobulin
genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence,
and may include splice donor and acceptor sequences.
The plasmids of the invention can be transferred into the chosen
host cell by well-known methods such as calcium chloride
transformation for E. coli and calcium phosphate treatment or
electroporation for mammalian cells. Cells transformed by the
plasmids can be selected by resistance to antibiotics conferred by
genes contained on the plasmids, such as the amp, gpt, neo and hyg
genes.
Once expressed, the recombinant fusion proteins can be purified
according to standard procedures of the art, including ammonium
sulfate precipitation, affinity columns, column chromatography, gel
electrophoresis and the like (see, generally, R. Scopes, Protein
Purification, Springer-Verlag, New York (1982), Deutscher, Methods
in Enzymology Vol. 182: Guide to Protein Purification., Academic
Press, Inc. New York (1990)). Substantially pure compositions of at
least about 90 to 95% homogeneity are preferred, and 98 to 99% or
more homogeneity are most preferred for pharmaceutical uses. Once
purified, partially or to homogeneity as desired, the polypeptides
may then be used therapeutically.
One of skill in the art would recognize that after chemical
synthesis, biological expression, or purification, the IL-13
receptor targeted fusion protein may possess a conformation
substantially different than the native conformations of the
constituent polypeptides. In this case, it may be necessary to
denature and reduce the polypeptide and then to cause the
polypeptide to re-fold into the preferred conformation. Methods of
reducing and denaturing proteins and inducing re-folding are well
known to those of skill in the art. (See Debinski et al. J. Biol.
Chem., 268: 14065-14070 (1993); Kreitman and Pastan, Bioconjug.
Chem., 4: 581-585 (1993); and Buchner, et al., Anal. Biochem., 205:
263-270 (1992) which are incorporated herein by reference.)
Debinski et al., for example, describe the denaturation and
reduction of inclusion body proteins in guanidine-DTE. The protein
is then refolded in a redox buffer containing oxidized glutathione
and L-arginine.
One of skill would recognize that modifications can be made to the
IL-13 receptor targeted fusion proteins without diminishing their
biological activity. Some modifications may be made to facilitate
the cloning, expression, or incorporation of the targeting molecule
into a fusion protein. Such modifications are well known to those
of skill in the art and include, for example, a methionine added at
the amino terminus to provide an initiation site, or additional
amino acids placed on either terminus to create conveniently
located restriction sites or termination codons.
Identification of Target Cells
It was a surprising discovery of the present invention that tumor
cells overexpress IL-13 receptors. In particular, carcinoma tumor
cells (e.g. renal carcinoma cells) overexpress IL-13 receptors at
levels ranging from about 2100 sites/cell to greater than 150,000
sites per cell.
One of skill in the art will appreciate that identification of
other cells that overexpress IL-13 receptors requires only routine
screening using well-known methods. Typically this involves
providing a labeled molecule that specifically binds to the IL-13
receptor. The cells in question are then contacted with this
molecule and washed. Quantification of the amount of label
remaining associated with the test cell provides a measure of the
amount of IL-13 receptor (IL-13R) present on the surface of that
cell.
In a preferred embodiment, IL-13 receptor may be quantified by
measuring the binding of .sup.125 I-labeled IL-13 (.sup.125
I-IL-13) to the cell in question. Details of such a binding assay
are provided in Example 1.
Pharmaceutical Compositions
The chimeric molecules of this invention are useful for parenteral,
topical, oral, or local administration, such as by aerosol or
transdermally, for prophylactic and/or therapeutic treatment. The
pharmaceutical compositions can be administered in a variety of
unit dosage forms depending upon the method of administration. For
example, unit dosage forms suitable for oral administration include
powder, tablets, pills, capsules and lozenges. It is recognized
that the fusion proteins and pharmaceutical compositions of this
invention, when administered orally, must be protected from
digestion. This is typically accomplished either by complexing the
protein with a composition to render it resistant to acidic and
enzymatic hydrolysis or by packaging the protein in an
appropriately resistant carder such as a liposome. Means of
protecting proteins from digestion are well known in the art.
The pharmaceutical compositions of this invention are particularly
useful for parenteral administration, such as intravenous
administration or administration into a body cavity or lumen of an
organ. The compositions for administration will commonly comprise a
solution of the chimeric molecule dissolved in a pharmaceutically
acceptable carrier, preferably an aqueous carrier. A variety of
aqueous carriers can be used, e.g., buffered saline and the like.
These solutions are sterile and generally free of undesirable
matter. These compositions may be sterilized by conventional, well
known sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, for
example, sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration of
chimeric molecule in these formulations can vary widely, and will
be selected primarily based on fluid volumes, viscosities, body
weight and the like in accordance with the particular mode of
administration selected and the patient's needs.
Thus, a typical pharmaceutical composition for intravenous
administration would be about 0.1 to 10 mg per patient per day.
Dosages from 0.1 up to about 100 mg per patient per day may be
used, particularly when the drug is administered to a secluded site
and not into the blood stream, such as into a body cavity or into a
lumen of an organ. Actual methods for preparing parenterally
administrable compositions will be known or apparent to those
skilled in the art and are described in more detail in such
publications as Remington's Pharmaceutical Science, 15th ed., Mack
Publishing Company, Easton, Pa. (1980).
The compositions containing the present fusion proteins or a
cocktail thereof (i.e., with other proteins) can be administered
for therapeutic treatments. In therapeutic applications,
compositions are administered to a patient suffering from a
disease, in an amount sufficient to cure or at least partially
arrest the disease and its complications. An amount adequate to
accomplish this is defined as a "therapeutically effective dose."
Amounts effective for this use will depend upon the severity of the
disease and the general state of the patient's health.
Single or multiple administrations of the compositions may be
administered depending on the dosage and frequency as required and
tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the proteins of this invention to
effectively treat the patient.
Among various uses of the cytotoxic fusion proteins of the present
invention are included a variety of disease conditions caused by
specific human cells that may be eliminated by the toxic action of
the protein. One preferred application is the treatment of cancer,
such as by the use of an IL-13 receptor targeting molecule (e.g.
IL-13 or anti-IL-13R antibody) attached to a cytotoxin.
Where the chimeric molecule comprises an IL-13 receptor targeting
molecule attached to a ligand, ligand portion of the molecule is
chosen according to the intended use. Proteins on the membranes of
T cells that may serve as targets for the ligand includes CD2
(T11), CD3, CD4 and CD8. Proteins found predominantly on B cells
that might serve as targets include CD10 (CALLA antigen), CD19 and
CD20. CD45 is a possible target that occurs broadly on lymphoid
cells. These and other possible target lymphocyte target molecules
for the chimeric molecules bearing a ligand effector are described
in Leukocyte Typing III, A. J. McMichael, ed., Oxford University
Press (1987). Those skilled in the art will realize ligand
effectors may be chosen that bind to receptors expressed on still
other types of cells as described above, for example, membrane
glycoproteins or ligand or hormone receptors such as epidermal
growth factor receptor and the like.
Diagnostic Kits
In another embodiment, this invention provides for kits for the
treatment of tumors or for the detection of cells overexpressing
IL-13 receptors. Kits will typically comprise a chimeric molecule
of the present invention (e.g. IL-13-label, IL-13-cytotoxin,
IL-13-ligand, etc.). In addition the kits will typically include
instructional materials disclosing means of use of chimeric
molecule (e.g. as a cytotoxin, for detection of tumor cells, to
augment an immune response, etc.). The kits may also include
additional components to facilitate the particular application for
which the kit is designed. Thus, for example, where a kit contains
a chimeric molecule in which the effector molecule is a detectable
label, the kit may additionally contain means of detecting the
label (e.g. enzyme substrates for enzymatic labels, filter sets to
detect fluorescent labels, appropriate secondary labels such as a
sheep anti- mouse-HRP, or the like). The kits may additionally
include buffers and other reagents routinely used for the practice
of a particular method. Such kits and appropriate contents are well
known to those of skill in the art.
EXAMPLES
The following examples are offered to illustrate, but not to limit
the claimed invention. All references cited in the foregoing
discussion and the following examples are incorporated herein by
reference.
Example 1
Identification of Cell that Overexpress IL-13
Recombinant human IL-4 and IL-13 were labeled with .sup.125 I
(Amersham Research Products, Arlington Heights, Ill., U.S.A.) by
using the IODO-GEN reagent (Pierce, Rockford, Ill., U.S.A.)
according to the manufacturer's instructions. The specific activity
of the radiolabeled cytokines was estimated to range from 20-100
.mu.Ci/.mu.g protein. For binding experiments, typically,
1.times.10.sup.6 renal cell carcinoma (RCC) tumor cells were
incubated at 4.degree. C. for 2 hours with .sup.125 I-IL-13 (100
pM) with or without increasing concentrations (up to 500 nM) of
unlabeled IL-13. In some experiments, IL-13R expression was
examined as previously described (Obiri et al. J. Clin. Invest.,
91: 88-93 (1993))). The data were analyzed with the LIGAND program
(Munson et al. Anal. Biochem., 107: 220-239 (1980)) to determine
receptor number and binding affinity.
Four human renal cell carcinoma (RCC) cell lines (WS-RCC, HL-RCC,
PM-RCC, and MA-RCC) bound .sup.125 I-IL-13 specifically and the
density of IL-13 R varied from 2100 sites per cell in WS-RCC cells
to 150,000 sites per cell in HL-RCC cells (Table 1). The represents
an increase in IL-13 receptor expression ranging from 15 to about
500 fold as compared to normal immune cells. In contrast, IL-4
receptors overexpressed on cancers have been reported at
concentrations as high as 4000 sites per cell. Scatchard analyses
(Scatchard, Ann. N.Y. Acad. Sci., 51: 660-663 (1949)) revealed that
only one affinity class of receptors was expressed on each cell
line. The binding affinities (Kd) ranged between 100 pM to 400 pM
in three RCC cell lines while HL-RCC cells expressed lower affinity
receptors (Kd.about.3 nM).
Although IL-13 responsiveness has previously been reported in human
monocytes, B cells and pre-myeloid (TF-1) cells (see, e.g. de Waal
Malefyt, et al. J. Immunol., 151: 6370-6381 (1993), de Waal
Malefyt, et al. J. Immunol., 144: 629-633 (1993)), little was known
about IL-13R structure or its binding characteristics in these, or
any other cells. The present data show that freshly isolated human
monocytes, EBV-transformed B cell line and TF-1 cell line express
very few IL-13 binding sites (100-300/cell) compared to human RCC
cells (Table 1). On the other hand, no binding of .sup.125 I-IL-13
was observed on H9 T cells, LAK cells and resting or PHA activated
PBL. This is compatible with the fact that IL-13 responsiveness has
not been observed in T lymphocytes (Punnonen et al., Proc. Natl.
Acad. Sci. USA, 90: 3730-3734 (1993).
TABLE 1 ______________________________________ Expression of IL-13
receptor by human cells. IL-13 Binding Sites/cell.sup.a Kd(nM) Cell
Types Mean .+-. SD Mean .+-. SD
______________________________________ Renal Cell Carcinoma (RCC)
1. WS-RCC 2,090 .+-. 367 (5) .sup. 0.247 .+-. 0.12 (3).sup.b 2.
MA-RCC 5,013 .+-. 1.347 (5) 0.128 .+-. 0.05 (2) 3. PM-RCC 26,500
.+-. 5.000 (2) 0.394 .+-. 0.26 (2) 4. HL-RCC 150,000 .+-. 15.00 (3)
3.1 .+-. 0.7 (2) B Lymphocytes 1. DH (EBV-trans- 303 .+-. 90 (4)
.sup. --.sup.d formed B cell line) 2. RAJI (Burkitt's UD.sup.c --
lymphoma) Monocytes/ Premyeloid cells.sup.e 1. Peripheral blood 124
-- monocytes 2. U937 (premonocytic UD -- 3. TF1.J61 (premyeloid)
130 .+-. 1 (2) -- T Lymphocytes/LAK cells.sup.f 1. PHA-activated
PBL <30 -- 2. MOLT-4 (T-cell UD -- leukemia) 3. LAK cells UD --
______________________________________ .sup.a IL-13 binding
sites/cell were determined as described in Example 1 .sup.b (n) =
number of experiments used to calculate mean .+-. standard
deviation. .sup.c UC = undetectable .sup.d The Kd could not be
reliably calculated because of low binding of .sup.125 IIL-13
.sup.e The peripheral blood derived monocytes (>90% purity) were
isolated by ficollhypaque density gradient followed by ellutriation
from a leukopa obtained from normal donor. .sup.f LAK cells and
activiated Tlymphocytes were generated by the cultur of donor PBLs
(106/ml) with IL2 (500 Units/ml) for 3 days or PHA (10 .mu.g/ml)
for 3-4 days respectively.
Example 2
IL-13 and IL-4 Bind to Different Receptors
Recently, it was proposed that the IL-2R.gamma..sub.c receptor
subunit is associated with IL-13R (see, e.g., Russell et al.
Science 262: 1880-1883 (1993); Kondo et al. Science, 262: 1874-1877
(1993); Noguchi et al. Science, 262: 1877-1880 (1993); Kondo et al.
Science 263: 1453-1454 (1994); Giri et al. EMBO J. 13: 282-2830
(1994))) and IL-13R may share a common component with IL-4R
(Zurawski et al. EMBO J. 12: 2663-2670 (1993); Aversa et al. J.
Exp. Med. 178: 2213-2218 (1993)). To directly address these
possibilities, radio-ligand binding experiments were performed, as
described in Example 1, on HL-RCC and WS-RCC cells using .sup.125
IL-IL-4 or .sup.125 IL-IL-13 in the presence or absence of excess
of either cytokine.
Unlabeled IL-4 more efficiently inhibited .sup.125 IL-4 from
binding to RCC cells (84%, and 72% displacement of total binding in
WS-RCC and HL-RCC, respectively) than IL-13 which also displaced
.sup.125 I-IL-4 binding to these cells (61% of total binding in
WS-RCC and 51% in HL-RCC) under similar conditions. On the other
hand, while .sup.125 IL-13 binding was effectively displaced by
IL-13 (about 85% of total in both cell types), it was only
minimally displaced by IL-4 (12% of total displacement in WS-RCC,
and 7% in HL-RCC). These results indicate that IL-4 and IL-13 both
interact with each other's receptors, however, the interaction is
not identical since IL-4 inhibition of .sup.125 I-IL-13 binding was
weak and IL-13 inhibition of .sup.125 I-IL-4 binding was not
complete. These results agree with previous observations in which
IL-13 was found to compete with IL-4 binding on TF-1 cells
(Zurawski et al., EMBO J. 12: 2663-2670 (1993)). However, in that
report the converse experiment was not done. Here, the data show
that even though IL-13 competed for IL-4 binding, IL-4 did not
compete for IL-13 binding.
The competition by IL-13 for IL-4 binding sites on lymphoid MLA 144
cells and RAJI cell lines was also investigated. These cells were
incubated with radiolabled IL-4 with or without excess unlabeled
IL-4 or IL-13. Excess unlabeled IL-4 effectively displaced labeled
.sup.125 IL-IL-4 bound to MLA 144 and RAJI cells, while excess
IL-13 could not compete this binding. This observation is at
variance to that seen with RCC cells in which IL-13 competed for
IL-4 binding. The inability of IL-13 to compete for .sup.125 I-IL-4
binding to MLA 144 is consistent with the observation that IL-13
did not bind to peripheral blood T (or MLA 144) cells.
Example 3
Subunit Structure of IL-13 and IL-4 Receptors
The subunit structure of IL-13R on RCC cells was investigated by
crosslinking studies. Cells (5.times.10.sup.6) were labeled with
.sup.125 I-IL-13 or .sup.125 IL-4 in the presence or absence of
excess IL-13 or IL-4 for 2 h at 4.degree. C. The bound ligand was
cross-linked to its receptor with disuccinimidyl subcrate (DSS)
(Pierce, Rockford, Ill., U.S.A.) at a final concentration of 2 mM
for 30 min. Cells were lysed in a buffer containing 1% Triton
X-100, 1 mM phenylmethylsulfonyl fluoride, 0.02 mM leupeptin, 5.0
.mu.M trypsin inhibitor, 10 mM benzamidine HCl, 1 mM phenanthroline
iodoacetamide, 50 mM amino caproic acid, 10 .mu.g/ml pepstatin, and
10 .mu.g/ml aprotinin. The cell lysates were cleared by boiling in
buffer containing 2-mercaptoethanol and analyzed by electrophoresis
through 8% SDS/polyacrylamide gel. The gel was subsequently dried
and autoradiographed. In some experiments, the receptor/ligand
complex was immunoprecipitated from the lysate overnight at
4.degree. C. by incubating with protein A sepharose beads that had
been pre-incubated with P7 anti hIL-4R or anti-.gamma..sub.c
antibody and analyzed as above.
The labeled .sup.125 I-IL-13 cross-linked to one major protein on
all four RCC cell lines and the complex migrated as a single broad
band ranging between 68 and 80 kDa. A single band was also observed
on human pre-myeloid TF-1.J61 cells only after much longer exposure
of the gel. After subtracting the molecular mass of IL-13 (12 kDa),
the size of IL-13 binding protein was estimated at 56 to 68 kDa.
The .sup.125 I-IL-13 cross-linked band was not observed when the
crosslinking was performed in the presence of 200-fold molar excess
of IL-13. In addition to the major band, a faint band of
approximately 45 kDa was also observed in HL-RCC and PM-RCC but not
on MA-RCC cells. This band appeared to be specifically associated
with IL-13R because unlabeled IL-13 competed for the binding of
.sup.125 I-IL-13. This band could represent an IL-13R associated
protein or a proteolytic fragment of the larger band. In contrast
to the displacement of .sup.125 I-IL-13 binding by unlabeled IL-13,
an excess of unlabeled IL-4 did not prevent the appearance of IL-13
R band in RCC cell lines. IL-13 on the other hand competed for
.sup.125 I-IL-4 binding to both major proteins on WS-RCC cells. It
is of interest that .sup.125 I-IL-13-cross-linked protein was
slightly larger in size in TF-1.J61, WS-RCC, PM-RCC, and HL-RCC
cell lines compared to that seen in MA-RCC. Post-translational
modifications, such as glycosylation or phosphorylation, may
account for this difference.
Example 4
Construction of an IL-13-PE Fusion Protein
Construction of a Plasmid Encoding IL-13-PE38QQR
To construct the chimeric toxin a coding region of the human
interleukin 13 (hIL-13) gene (plasmid JFE14-SR.alpha.) (Minty et
al., Nature, 362: 248 (1993), McKenzie et al. Proc. Natl. Acad.
Sci. U.S.A., 90: 3735 (1987) which are incorporated herein by
reference) was fused to a gene encoding PE38QQR, a mutated form of
PE, thereby producing a construct (phuIL-13-Tx) encoding the
chimeric molecule. Specifically, a DNA encoding human IL-13 was
PCR-amplified from plasmid JFE14-SR.alpha.. New sites were
introduced for the restriction endonucleases NdeI and Hind III at
the 5' and 3' ends of the hIL-13 gene, respectively by PCR using a
sense primer that incorporated the NdeI site and an antisense
primer that incorporated the HindIII site.
The NdeI/HindIII fragment containing encoding hIL-13 was subcloned
into a vector obtained by digestion of plasmid pWDMH4-38QQR
(Debinski et al. Int. J. Cancer 58: 744-748 (1994)) or plasmid
pSGC242FdN1 (Debinski et al. Clin. Res. 42: 251A, (abstr.) (1994)
with NdeI and HindIII, to produce plasmid phuIL-13-Tx. The 5' end
of the gene fusion was sequenced and showed the correct DNA of
hIL-13.
Human interleukin 4 (hIL-4) was cloned into an expression vector in
a similar way to hIL-13 using plasmid pWDMH4 (Debinski et al. J.
Biol. Chem. 268: 14065-14070 (1993)) as a template for PCR
amplification. Recombinant proteins were expressed in E. coli BL21
(.lambda.DE3) under control of the T7 late promoter (Id.). In
addition to the T7 bacteriophage late promoter, the plasmids also
carried a T7 transcription terminator at the end of the open
reading frame of the protein, an f1 origin of replication and gene
for ampicillin resistance (Debinski et al. J. Clin. Invest. 90:
405-411 (1992)). The plasmids were amplified in E. coli (HB101 or
DH5.alpha. high efficiency transformation) (BRL) and DNA was
extracted using Qiagen kits (Chatsworth, Calif., U.S.A.).
Expression and purification of recombinant proteins
E. coli BL21 (.lambda.DE3) cells were transformed with plasmids of
interest and cultured in 1.0 liter of Super broth. Expressed
recombinant human IL-13 and human IL-13-PE38QQR were localized in
inclusion bodies. The recombinant proteins were isolated from the
inclusion bodies as described by Debinski et al., J. Biol. Chem.
268: 14065-14070 (1993), which is incorporated herein by reference.
After dialysis, the renatured protein of human IL-13-PE38QQR was
purified on Q-Sepharose Fast Flow and by size exclusion
chromatography on Sephacryl S-200HR (Pharmacia, Piscataway, N.J.,
U.S.A.) The initial step of hIL-13 or hIL-4 purification was
conducted on SP-Sepharose Fast Flow (Pharmacia).
Protein concentration was determined by the Bradford assay (Pierce
"Plus", Rockford, Ill., U.S.A.) using BSA as a standard.
Human IL-13 and IL-13-PE38QQR were expressed at high levels in
bacteria as seen in SDS-PAGE analysis of the total cell extract.
After initial purification on SP-Sepharose (hIL-13) or Q-Sepharose
(hIL-13-PE38QQR) the renatured recombinant proteins were applied
onto a Sephacryl S-200 HR Pharmacia column. Human IL-13 and
hIL-13-PE38QQR appeared as single entities demonstrating the very
high purity of the final products. The chimefie toxin migrated
within somewhat lower than expected for 50 kDa protein M.sub.r
range which may be related to the hydrophobicity of the molecule.
The biologic activity of the rhIL-13 was exactly the same as
commercially obtained hIL-13.
Example 4
The Activity of an IL-13-PE Fusion Protein on Human Carcinoma
Cells
Cytotoxic Activity of hIL-13-PE38QQR
The cytotoxic activity of chimeric toxins, such as hIL-13-PE38QQR,
were tested by measuring inhibition of protein synthesis. Protein
synthesis was assayed by plating about 1.times.10.sup.4 cells per
in a 24-well tissue culture plate in 1 ml of medium. Various
concentrations of the chimeric toxins were added 20-28 h following
cell plating. After 20 h incubation with chimeric toxins, [.sup.3
H]-leucine was added to cells for 4 h, and the cell-associated
radioactivity was measured. For blocking studies, rhIL-2, 4 or 13
was added to cells for 30 min before the chimeric toxin addition.
Data were obtained from the average of duplicates and the assays
were repeated several times.
Several established cancer cell lines were tested to determine if
hIL-13 PE38QQR is cytotoxic to them. In particular, cancers derived
from colon, skin and stomach were examined. The cancer cells were
sensitive to hIL-13-PE38QQR with ID.sub.50 s ranging from less than
1 ng/ml to 300 ng/ml (20 pM to 6.0 nM) (ID.sub.50 indicates the
concentration of the chimeric toxin at which the protein synthesis
fell by 50% when compared to the sham-treated cells). A colon
adenocarcinoma cell line, Colo201, was very responsive with an
IC.sub.50 of 1 ng/ml. A431 epidermoid carcinoma cells were also
very sensitive to the action of hIL-13-toxin; the ID.sub.50 for
hIL-13-PE38QQR ranged from 6 10 ng/ml. A gastric carcinoma CRL1739
cell line responded moderately to the hIL-13-toxin with an
ID.sub.50 of 50 ng/ml. Another colon carcinoma cell line, Colo205,
had a poorer response with an ID.sub.50 of 300 ng/ml.
The cytotoxic action of hIL-13-PE38QQR was specific as it was
blocked by a 10-fold excess of hIL-13 on all cells. These data
suggest that a spectrum of human cancer cells possess hIL-13
binding sites and such cells are sensitive to hIL-13-PE38QQR
chimeric toxin.
Because the hIL-13R has been suggested to share the .lambda..sub.c
subunit of the IL-2R (Russell et al. Science 262: 1880-1883
(1993)), the specificity of hIL-13-PE38QQR action on A431 and
CRL1739 cells, the two cell lines with different sensitivities to
the chimeric toxin was further explored. The cells were treated
with hIL-13-PE38QQR with or without rhIL-2 at a concentration of
1.0 .mu.g/ml or 10 .mu.g/ml. The rhIL-2 did not have any blocking
action on hIL-13-PE38QQR on the two cell lines, even at 10,000 fold
molar excess over the chimeric toxin. These results indicate that
the cell killing by the hIL-13-toxin is independent of the presence
of hIL-2.
IL-4, unlike IL-2, blocks the action of IL-13-PE38QQR
Native hIL-4 was added to cells which were then treated with hIL-13
PE38QQR. Unexpectedly, it was found that hIL-4 inhibited the
cytotoxic activity of the hIL-13-toxin. This phenomenon was seen on
all the tested cell lines, including Colo201, A431 and CRL1739. To
investigate the possibility that hIL-13 and hIL-4 may compete for
the same binding site, the cells were also treated with the
hIL-4-based recombinant toxin, hIL-4-PE38QQR (Debinski et al. Int.
J. Cancer 8: 74-748 (1994)). The cytotoxic action of hIL-4-PE38QQR
had already been shown to be blocked by an excess of hIL-4 but not
of hIL-2 (Id.). In the present experiment hIL-13 potently blocked
the cytotoxic activity of hIL-4-PE38QQR. Also, the action of
another hIL-4-based chimeric toxin, hIL-4-PE4E (Debinski et al. J.
Biol. Chem. 268: 14065-14070 (1993)), was blocked by an excess of
hIL-13 on Colo201 and A431 cells. Thus, the cytotoxicity of
hIL-13-PE38QQR is blocked by an excess of hIL-13 or hIL-4, and the
cytotoxic action of hIL-4-PE38QQR is also blocked by the same two
growth factors. However, IL-2 does not block the action of either
chimeric toxin. These results strongly suggest that hIL-4 and
hIL-13 have affinities for a common binding site.
This conclusion was supported by the observation of one cytokine
blocking the effect of a mixture of the two chimeric toxins. When
A431 cells were incubated with both hIL-3- and hIL-4-PE38QQR
chimeric toxins concomitantly the cytotoxic action was preserved
and additive effect was observed as expected. An excess of hIL-13
efficiently blocked the action of a mixture of the two chimeric
toxins. Moreover, neither hIL-13 nor hIL-4 blocked cell killing by
another mixture composed of hIL-13-PE38QQR and TGF.alpha.-PE40, a
chimeric toxin which targets the EGFR (TGF.alpha.-based chimeric
toxin, TGF.alpha.-PE40) (Siegall et al. FASEB J. 3, 2647-2652
(1992)). The same was observed on Colo201 cells.
Reciprocal Blocking of Chimeric Toxins by IL-13 and IL-4 is due to
competition for binding sites
The binding ability of human IL-13 was compared to human
IL-4-PE38QQR in competitive binding assays. Recombinant
hIL-4-PE38QQR was labeled with .sup.125 I using the lactoperoxidase
method as described by Debinski et al., J. Clin. Invest. 90,
405-411 (1992). Binding assays were performed by a standard
saturation and displacement curves analysis. A431 epidermoid
carcinoma cells were seeded at 10.sup.5 cells per well in a 24-well
tissue culture plates at 24 h before the experiment. The plates
were placed on ice and cells were washed with ice-cold PBS without
Ca++, Mg++ in 0.2% BSA, as described (Id.). Increasing
concentrations of hIL-13 or hIL-4-PE38QQR were added to cells and
incubated 30 min prior to the addition of fixed amount of .sup.125
I-hIL-4-PE38QQR (specific activity 6.2 .mu.Ci/.mu.g protein) for 2
to 3 h. After incubation, the cells were washed twice and lysed
with 0.1N NaOH, and the radioactivity was counted in a
.gamma.-counter.
Human IL-4-PE38QQR competed for the binding of .sup.125
I-hIL-4-PE38QQR to A431 cells with an apparent ID.sub.50 of
4.times.10.sup.-8 M. In addition, hIL-13 also competed for the
.sup.125 I-hIL-4-PE38QQR binding site with a comparable potency to
that exhibited by the chimeric protein. More extensive binding
studies have shown that hIL-13 also competes for hIL-4 binding
sites on human renal carcinoma cell lines.
The possibility of an influence of hIL-13 or hIL-4 on the process
of receptor-mediated endocytosis and post-binding PE cellular
toxicity steps was excluded by adding to cells: (i) native PE (PE
binds to the .alpha..sub.2 -macroglobulin receptor), (ii)
TGF.alpha.-PE40, and (iii) a recombinant immunotoxin
C242rF(ab)-PE38QQR (Debinski et al. Clin. Res. 42, 251A, (Abstr.)
(1994)). C242rF(ab)-PE38QQR binds a tumor-associated antigen that
is a sialylated glycoprotein (Debinski et al. J. Clin. Invest. 90:
405-411 (1992)). The expected cytotoxic actions of these
recombinant toxins were observed and neither hIL-13 nor hIL-4
blocked these actions on A431 and Colo205 cells.
hIL-4 and hIL-13 compete for a common binding site on carcinoma
cell but evoke different biological effects
Even though hIL-13 and hIL-4 compete for a common binding site,
they induce different cellular effects. Protein synthesis was
inhibited in A431 epidermoid carcinoma cells in a dose-dependent
manner by hIL-4 alone, or by a ADP-ribosylation deficient chimeric
toxin containing hIL-4 (Debinski et al. Int. J. Cancer 58: 744-748
(1994)). This effect of hIL-4 or enzymatically deficient chimeric
toxin can be best seen with a prolonged time of incubation
(.gtoreq.24 h) and requires concentrations of hIL-4 many fold
higher than that of the active chimeric toxin in order to cause a
substantial decrease in tritium incorporation. However, when A431
cells were treated with various concentrations of hIL-13, no
inhibition (or stimulation) of protein synthesis was observed, even
at concentrations as high as 10 .mu.g/ml of hIL-13 for a 72 h
incubation. The same lack of response to hIL-13 was found on renal
cell carcinoma cells PM-RCC. Thus, while hIL-13 and hIL-4 may
possess a common binding site, they appear to transduce differently
in carcinoma cells expressing this common site, such as A431 and
PM-RCC cells.
The above examples are provided to illustrate the invention but not
to limit its scope. Other variants of the invention will be readily
apparent to one of ordinary skill in the art and are encompassed by
the appended claims. All publications, patents, and patent
applications cited herein are hereby incorporated by reference.
* * * * *