Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Reexamination Certificate
1998-04-03
2001-10-02
Eyler, Yvonne (Department: 1646)
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
C424S185100, C424S192100, C530S351000, C530S350000, C514S002600
Reexamination Certificate
active
06296843
ABSTRACT:
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 entity (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, other ligands, antibodies, drugs, prodrugs, liposomes, and the like.
In particular, where the effector component 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 (IL4) or transforming growth factor (TGF&agr;) fused to Pseudomonas exotoxin (PE), interleukin 2 (IL2) 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)).
The targeting moiety of these chimeric cytotoxins is often selected to pacifically target and bind to growth factor receptors, particularly those receptors that are overexpressed on cancer cells as compared to normal cells (e.g., Debinski et al. (1993)
J. Biol. Chem
., 268: 14065-14070, Phillips et al. (1994)
Cancer Res
., 54: 1008-1015, Debinski et al. (1994)
Int. J. Cancer
, 58: 744-748). However, even where the target receptor is overexpressed on cancer cells there is typically a significant level of receptor expression on normal cells as well. Therefore, even though one can obtain a therapeutic window for the cytotoxins, toxicities related to the presence of growth factor receptors on normal cells are dose-limiting for their administration (Phillips et al. (1994)
Cancer Res
., 54: 1008-1015, Debinski et al. (1994)
Int. J. Cancer
, 58: 744-748). It is thus desirable to identify targets or targeting ligands that show provide increased specificity for cancer cells as compared to normal cells and thereby improve the dosage levels that can be administered with diminished or no toxic side-effects.
SUMMARY OF THE INVENTION
This invention provides novel targeting ligands (specific binding moieties) that have increased specificity for cancer cells as compared to normal cells and therefore extremely effective for specifically delivering effector molecules to various neoplasias. The targeting ligands are mutagenized IL13 molecules having one or more mutations in the domain that interacts with the hIL4 receptor subunit designated the 140 kDa hIL4R
&bgr;
subunit.
Particularly preferred mutagenized IL13 molecules (specific binding moieties) of this invention are mutagenized human IL13 molecules. These molecules can be mutated at one or more of a variety of residues including at residues 12, 13, 14, 65, 66, 67, 68, 69, 70, 109, or 112. In one particular embodiment, residue 13 is a basic amino acid. Other preferred mutagenized IL13 molecules include lysine or arginine at residue 13 and/or aspartic acid at residue 66 and/or aspartic acid at residue 69 and or aspartic acid at residue 109 or 112. When a native human IL13 is mutated these mutations can include hIL13.E13R, hIL13.R66D, hIL13.S69D, hIL13.E13K, hIL13.R109D, and hIL13.R112D. Preferred double mutations include hIL13.E13K/R66D or hIL13.E13K/S69D. The specific binding moieties (mutagenized IL13) can be attached to and therefore comprise an effector molecule as described herein.
In one embodiment, any of the mutagenized IL13 molecules described herein is a component of a chimeric molecule having the formula:
R
1
—(L)
j
—(R
2
)
n
in which R
1
is the mutagenized human interleukin 13, j and n are independently 0 or 1; R
2
is an effector molecule; and L is an optional linker. The effector molecule can be virtually any molecule that can be attached to the mutagenized IL 13. Effector molecules include, but are not limited to cytotoxins, labels, antibodies, liposomes, lipids, DNA or RNA nucleic acids, DNA or RNA vector, recombinant viruses, chemotherapeutics, anti-cancer antibiotics, photosensitizers, and the like. Particularly preferred cytotoxins include a Pseudomonas exotoxin or a Diphtheria toxin. The Pseudomonas exotoxin can be modified such that it substantially lacks domain Ia, and most preferred Pseudomonas exotoxins include PE38QQR and PE4E. It will be appreciated that the effector molecule can be attached to either the amino terminus, the carboxyl terminus or to an internal residue of the mutagenized IL13 molecule although terminal attachment is preferred.
Preferred cytotoxic chimeric molecules are fuision proteins and include any of the mutagenized IL13 molecules described herein fused to the cytotoxin. Particularly preferred cytotoxins include any of the above mutagenized IL13 molecules fused to a Pseudomonas exotoxin (e.g., hIL13.E13K-PE38QQR, hIL13.E13K-PE4E, etc.). Particularly preferred cytotoxic molecules include, but are not limited to hIL13.E13K-PE38QQR, hIL13.E13K-PE4E, hIL13.R66D-PE38QQR, hIL13.R66D-PE4E, hIL13.S69D-PE38QQR, hIL13.S69D-PE4E, hIL13.R109D-PE38QQR, hIL13.R112D-PE38QQR, hIL13.R109D-PE4E, hIL13.R112D-PE4E hIL13.E13K/R109D-PE38QQR, hIL13.E13K/R112D-PE38QQR, hIL13.E13K/R66D-PE4E, hIL13.E13K/RS69D-PE4E, DT390-hIL13.E13K, DT390-hIL13.R66D, DT390-hIL13.S69D, DT390-hIL13.R109D, DT390-hIL13.R112D, DT390-hIL13.E13K/R109D, and DT390-hIL13.E13K/R112D.
In another embodiment, this invention provides methods of delivering an effector molecule to a cell bearing an interleukin 13 receptor (IL13R). The methods involve contacting the cell with a chimeric molecule comprising the effector molecule attached to any of the mutagenized interleukin 13 (IL13) molecules described herein. The methods can involve any of the chimeric molecules described herein.
Where the effector molecule is a cytotoxin, this invention provides methods of killing a cell or inhibiting the growth (and/or proliferation) of a cell expressing an IL13 receptor (IL13R). Again, these methods involve contacting the cell any of the mutagenized IL13-cytotoxin chimeric molecules described herein. In a preferred embodiment, the cell is a neoplastic cell (e.g. a glioma).
The cytotoxic chimeric molecules described herein can be used as components of a pharmacological composition. In this embodiment, the composition comprises any one or more of the cytotoxic chimeric molecules of this invention and a pharmacologically acceptable excipient,
The mutagenized IL13 can also be attached to a detectable label. The chimeric label can be used to detect and/or localize and/or quantify a cell or cells expressing an IL13 receptor. The label when administered to a subject will localize at the site(s) of cells expressing or overexpressing IL13 receptors and detection of the label provides an indication of the presence, absence, quantity or location of such cells. Similarly ex vivo detection can be accomplished e.g. using a biological sample taken from the subject.
This invention also provides kits for the detection of cells expressing IL13 receptors or for inhibiting the growth and/or proliferation of such cells. The kits preferably include one or more containers containing a mutagenized IL13 of this invention. The mutagenized IL13 can be attached to either label (e.g. for detection of an IL13R bearing cell) or a cytotoxin (e.g. for inhibiting the growth of an IL13R bearing cell). Any of the cytotoxic or label (or o
Andres Janet L.
Eyler Yvonne
Senterfitt Akerman
The Penn State Research Foundation
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