IL-13 receptor specific chimeric proteins

This invention provides chimeric molecules useful for killing tumor cells bearing IL13 receptor(s) (IL-13R). The molecules comprise a cytotoxic molecule attached to a targeting molecule that specifically binds an IL-13 receptor. Preferred targeting molecules include IL-13 and anti-IL-13R antibodies.

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 bearing 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 (TGF.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 chimeric molecules that specifically target 
tumor cells with less binding to healthy cells than other analogous 
chimeric 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 chimeric 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 chimeric polypeptide fusion protein comprising an IL-13 
attached to a second polypeptide. The chimeric polypeptide fusion protein 
specifically binds to a tumor cell bearing 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 bearing 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 reaponse (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 chimeric 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 
chimeric 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 chimeric 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. 
Immunol., 150: 180A (1993); de Wall Malefyt et al. J. Immunol., 151: 6370 
(1993); Doherty et al. J. Immunol., 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., USA, and Sanofi Bio-Industries, 
Inc., Tervose, Pa., USA). 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 
alanine 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. N.Y. (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 
cross-linking the receptor to a labeled IL-13 by the exposure to 2 mM 
disuccinimidyl suberate (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 titer 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 al., 
Mol. Cell. Biol., 11: 2200-2205 (1991); Batra et al., 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. 
Pseudomonas 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 GGGGS (SEQ ID NO:1). 
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, REDLK (SEQ ID NO:2) (as in native 
PE), REDL (SEQ ID NO:3), RDEL (SEQ ID NO:4), or KDEL (SEQ ID NO:5), 
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, 
all of which are incorporated by reference herein. 
Deletions of amino acids 365-380 of 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. 07/865,722 filed Apr. 8, 1992 and in U.S. Ser. No. 
07/522,563 filed May 14, 1990, 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 REDLK (SEQ ID NO:2) or the mutant 
sequences KDEL (SEQ ID NO:5) or RDEL (SEQ ID NO:4). 
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 604-613 of 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 32P), 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 on immune cells. Chimeric 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 Moieties 
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 micelle 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 aldehyde 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 carrier 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 Cells that Overexpress IL-13 
Recombinant human IL-4 and IL-13 were labeled with .sup.125 I (Amersham 
Research Products, Arlington Heights, Ill., USA) by using the IODO-GEN 
reagent (Pierce, Rockford, Ill., USA) 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-13R 
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) 
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 (BBV-transformed B cell 
303 .+-. 90 (4) 
--.sup.d 
line) 
2. RAJI (Burkitt's lymphoma) 
UD.sup.c -- 
Monocytes/Premyeloid cells.sup.e 
1. Peripheral blood monocytes 
124 -- 
2. U937 (premonocytic 
UD -- 
3. TF1.J61 (premyeloid) 
130 .+-. 1 (2) 
-- 
T Lymphocytes/LAK cells.sup.f 
1. PHA-activated PBL 
&lt;30 -- 
2. MOLT-4 (T-cell leukemia) 
UD -- 
3. LAK cells UD -- 
______________________________________ 
.sup.a IL13 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 (&gt;90% purity) were isolated 
by ficollhypaque density gradient followed by ellutriation from a 
leukopac; 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: 2822-2830 (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 I-IL-4 
or .sup.125 I-IL-13 in the presence or absence of excess of either 
cytokine. 
Unlabeled IL-4 more efficiently inhibited .sup.125 I-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 I-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 I.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 I-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 I-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 suberate (DSS) (Pierce, Rockford, Ill., USA) 
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-13R 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. USA, 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 NdeII/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., USA). 
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., USA) 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. USA) 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 
chimeric 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 5 
The Activity of an IL13-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 to 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-PE38QOR 
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: 744-748 (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 cells 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. 
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# SEQUENCE LISTING 
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