U.S. patent application number 10/718534 was filed with the patent office on 2004-04-22 for antibody/receptor targeting moiety for enhanced delivery of armed ligand.
This patent application is currently assigned to Center of Molecular Medicine and Immunology, Center of Molecular Medicine and Immunology. Invention is credited to Burton, Jack D., Goldenberg, David M..
Application Number | 20040077843 10/718534 |
Document ID | / |
Family ID | 31890709 |
Filed Date | 2004-04-22 |
United States Patent
Application |
20040077843 |
Kind Code |
A1 |
Burton, Jack D. ; et
al. |
April 22, 2004 |
Antibody/receptor targeting moiety for enhanced delivery of armed
ligand
Abstract
A method for intracellular delivery of drugs or other agents for
diagnosis and therapy of malignancies or immune-mediated or
inflammatory conditions. A targeting moiety of a an antibody and
the ligand-binding region of a selected cytokine receptor is used.
The targeting moiety targets surface antigen on a specific cell
population. The targeting moiety is administered to a subject, and
then, after a specified interval, therapeutic or diagnostic agents
linked to the cognate cytokine then are given. The invention
provides rapid, efficient internalization of the cytokine receptor
antibody/antigen complexes. Targeting of a high-level cell surface
antigen with such bispecific fusion molecules substantially
increases the number of cytokine receptors over their low
background level.
Inventors: |
Burton, Jack D.; (Long
Island City, NY) ; Goldenberg, David M.; (Mendham,
NJ) |
Correspondence
Address: |
FOLEY AND LARDNER
SUITE 500
3000 K STREET NW
WASHINGTON
DC
20007
US
|
Assignee: |
Center of Molecular Medicine and
Immunology
|
Family ID: |
31890709 |
Appl. No.: |
10/718534 |
Filed: |
November 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10718534 |
Nov 24, 2003 |
|
|
|
09231642 |
Jan 15, 1999 |
|
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60071520 |
Jan 15, 1998 |
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Current U.S.
Class: |
530/391.1 ;
530/350 |
Current CPC
Class: |
B82Y 5/00 20130101; C07K
2317/24 20130101; A61K 2039/505 20130101; A61K 47/6813 20170801;
A61K 38/00 20130101; C07K 14/7155 20130101; A61K 47/67 20170801;
C07K 2317/77 20130101; C07K 7/08 20130101; C07K 16/3007 20130101;
A61K 47/65 20170801; C07K 2317/622 20130101; C07K 2319/00
20130101 |
Class at
Publication: |
530/391.1 ;
530/350 |
International
Class: |
C07K 016/46; C07K
014/715 |
Goverment Interests
[0002] The U.S. Governrnent has a paid-up license in this invention
and the right in limited circumstances to require the patent owner
to license others on reasonable terms as provided for by the terms
of grant numbers CA 39841-13 and RR 12603, awarded by the National
Institutes of Health.
Claims
What is claimed is:
1. A targeting moiety comprising a conjugate of an antibody linked
to a ligand-binding region of a receptor subunit selected from the
group consisting of interleukin-2 receptor .alpha.(IL-2.alpha.),
interleukin-4 receptor .alpha.(IL-4.alpha.), and interleukin-15
receptor .alpha.(IL-15R.alpha.), which antibody is specific for a
cell marker specific to a targeted cell.
2. A targeting moiety as claimed in claim 1, comprising a covalent
conjugate in which the antibody is covalently linked to the
ligand-binding region of the receptor.
3. A targeting moiety as claimed in claim 1, comprising a fusion
protein of the antibody and the ligand-binding region.
4. A targeting moiety as claimed in claim 1, comprising a
bispecific antibody that has a first specificity for a cell marker
specific to a targeted cell and a second specificity for the
ligand-binding region.
5. A targeting moiety as claimed in claim 1, wherein the antibody
is specific to CEA.
6. A targeting moiety as claimed in claim 1, wherein the antibody
is specific to HLA-DR.
7. A targeting moiety as claimed in claim 1, wherein the receptor
subunit is IL-4.alpha..
8. A targeting moiety as claimed in claim 1, wherein the receptor
subunit is IL-15R.alpha..
9. A targeting moiety as claimed in claim 1, wherein the antibody
is specific to HLA-DR and is linked to the ligand-binding region of
IL-15R.alpha..
10. A targeting moiety as claimed in claim 1, wherein the antibody
is specific to CEA and is linked to the ligand-binding region of
IL-15R.alpha..
11. A targeting moiety as claimed in claim 1, wherein the antibody
is specific to HLA-DR and is linked to the ligand-binding region of
IL-4R.alpha..
12. A targeting moiety as claimed in claim 1, wherein the antibody
is specific to CEA and is linked to the ligand-binding region of
IL-4R.alpha..
13. A targeting moiety as claimed in claim 1, wherein the antibody
is specific for a cell marker specific to a B-cell.
14. A targeting moiety as claimed in claim 1, wherein the antibody
is specific for a cell marker specific to a malignant B-cell.
15. A targeting moiety as claimed in claim 1, wherein the antibody
is specific for a cell marker specific to an activated B-cell.
16. A targeting moiety as claimed in claim 1, wherein the antibody
is specific for a cell marker specific to a normal B-cell.
17. A targeting moiety as claimed in claim 1, wherein the antibody
is specific for a cell marker specific to a T-cell.
18. A targeting moiety as claimed in claim 1, wherein the antibody
is specific for a cell marker specific to a malignant T-cell.
19. A targeting moiety as claimed in claim 1, wherein the antibody
is specific for a cell marker specific to an activated T-cell.
20. A composition comprising a targeting moiety according to claim
1, and a pharmaceutically acceptable carrier.
21. A composition comprising a targeting moiety according to claim
7, and a pharmaceutically acceptable carrier.
22. A composition comprising a targeting moiety according to claim
8, and a pharmaceutically acceptable carrier.
23. A kit comprising a conjugate of IL-15 linked to a drug,
radionuclide or toxin, and a targeting moiety comprising an
antibody specific for a cell marker specific to a targeted cell,
linked to the ligand-binding region of IL-15R.alpha..
24. A kit as claimed in claim 23, wherein the antibody is specific
for a cell marker specific to a B-cell.
25. A kit as claimed in claim 23, wherein the antibody is specific
for a cell marker specific to a malignant B-cell.
26. A kit as claimed in claim 23, wherein the antibody is specific
for a cell marker specific to an activated B-cell.
27. A kit as claimed in claim 23, wherein the antibody is specific
for a cell marker specific to a normal B-cell.
28. A kit as claimed in claim 23, wherein the antibody is specific
for a cell marker specific to a T-cell.
29. A kit as claimed in claim 23, wherein the antibody is specific
for a cell marker specific to a malignant T-cell.
30. A method of treatment for cancer or an immunologically-mediated
or infectious disease, comprising: first administering to a subject
in need of such treatment a targeting moiety comprising a antibody
specific for a cell marker specific to a targeted cell, linked to
the ligand-binding region of IL-15R.alpha., and then administering
to the subject a therapeutically effective amount of a conjugate of
IL-15 linked to a drug, radionuclide or toxin.
31. A method as claimed in claim 30, wherein the antibody is
specific for a cell marker specific to a B-cell.
32. A method as claimed in claim 30, wherein the antibody is
specific for a cell marker specific to a malignant B-cell.
33. A method as claimed in claim 30, wherein the antibody is
specific for a cell marker specific to an activated B-cell.
34. A method as claimed in claim 30, wherein the antibody is
specific for a cell marker specific to a normal B-cell.
35. A method as claimed in claim 30, wherein the antibody is
specific for a cell marker specific to a T-cell.
36. A method as claimed in claim 30, wherein the antibody is
specific for a cell marker specific to a malignant T-cell.
37. A targeting moiety comprising a conjugate of an antibody linked
to a ligand-binding region of interleukin-13 receptor
.alpha.(IL-13R.alpha.), which antibody is specific to HLA-DR.
38. A composition comprising a targeting moiety according to claim
18, and a pharmaceutically acceptable carrier.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/231,642, filed Jan. 15, 1999, which claims benefit from U.S.
Provisional Application No. 60/071,520, filed Jan. 15, 1998, both
of which are incorporated herein by reference in their
entirety.
BACKGROUND OF THE INVENTION
[0003] The present invention relates to a conjugate, preferably a
fusion protein, of a component of an internalizing receptor complex
and a monoclonal antibody (mAb) that binds to a specific surface
antigen on a cell, to a conjugate of a radionuclide or toxin and a
ligand for the internalizing receptor system, and to a method of
diagnosis or therapy using such genetic or chemical conjugates.
[0004] There is now a fairly large and growing body of experience
in the use of mAbs for tumor therapy. Several studies targeting
different antigens have shown promising results. These studies have
used radiolabeled mAbs and, to a lesser extent, mAb-toxin
conjugates.
[0005] mAbs used in tumor diagnosis and therapy differ in their
ability to bind cognate antigen and to become internalized. For
example, CD22 exhibits efficient internalization as well as
reexpression of this antigen after internalization. It suffers,
however, from relatively low expression levels on some B-cell
malignancies, e.g., it is expressed on only 30-50% of cases of
B-cell lymphocytic leukemia (B-CLL).
[0006] Other cell surface antigens such as HLA-DR and the CD20
antigen, in contrast to the CD22 antigen, are quite highly
expressed B-cell antigens that are expressed on a wide range of
B-cell malignancies, ranging from acute lymphocytic leukemia (ALL)
to the more differentiated B-Cell (B-CLL) and non-Hodgkin's
lymphoma (NHL), and even to hairy cell leukemia (HCL). These
antigens are generally expressed on cells in the vast majority of
cases of these malignancies at a high antigen density. A major
disadvantage of these antigens is that they slowly internalizing.
This feature militates significantly against targeting HLA-DR and
CD20 for toxin-based therapy.
[0007] A further problem of HLA-DR and CD20 is the fact that B-cell
malignancies exhibit a more rapid dissociation of bound anti-HLA-DR
and anti-CD20 mAbs from the surface as compared to nonlymphoma
tumor cells. This suggests that a therapy that targets a B-cell
restricted antigen, particularly those characterized by slow
internalization, could be enhanced by addressing these issues.
[0008] A variety of mAb-toxin constructs have been tested in both
in vitro experiments and human trials. These studies have
demonstrated potent and specific effects of these reagents. Most of
the toxin molecules that have been used derive from either plant or
bacterial sources and hence produce neutralizing anti-toxin
antibody responses in patients. This severely limits the duration
of therapy.
SUMMARY OF THE INVENTION
[0009] It is therefore an object of the present invention to
provide more effective methods of diagnosis and/or therapy for
cancer and immunologically-mediated or infectious diseases.
[0010] It is another object of the invention to improve the value
as antigenic targets of slowly internalizing surface antigens.
[0011] It is a further object of the invention to reduce the
tendency of antibodies bound to the surface of tumor cells to
dissociate from the surface of the cells.
[0012] These and other objects of the invention are achieved by
providing a targeting moiety comprising a conjugate of an antibody
linked to a ligand-binding region of a receptor subunit selected
from the group consisting of IL-2R.alpha., IL-4R.alpha.,
IL-13R.alpha. and IL-15R.alpha., which antibody is specific for a
cellular antigen specific to a targeted cell. The targeting moiety
may comprise a covalent conjugate in which the antibody is
covalently linked to the ligand-binding region, a fusion protein of
the antibody and the ligand-binding region, or a bispecific
antibody that has a first specificity for a cellular antigen
specific to a targeted cell and a second specificity for a rapidly
internatlizing receptor complex. In one embodiment, the antibody is
specific to an antigen expressed by solid tumors, for example, CEA,
and is linked to the ligand-binding region of IL-13R.alpha.. In an
alternative embodiment, the antibody is specific to HLA-DR and is
linked to the ligand-binding region of IL-15R.alpha.. A composition
comprising a targeting moiety according to the invention and a
pharmaceutically acceptable carrier also is provided.
[0013] A kit comprising a conjugate of IL-13 linked to a drug,
radionuclide or toxin, and a targeting moiety comprising an
antibody specific for a cell marker specific to a targeted cell,
linked to the ligand-binding region of IL-13R.alpha., is provided.
A second kit comprising a conjugate of IL-15 linked to a drug,
radionuclide or toxin, and a targeting moiety comprising an
antibody specific for a cell marker specific to a targeted cell,
linked to the ligand-binding region of IL-15R.alpha., also is
provided.
[0014] The invention provides a method of treatment for cancer,
comprising first administering to a subject in need of such
treatment a targeting moiety comprising an antibody specific for an
antigen specific to a targeted cell, linked to the ligand-binding
region of IL-13R.alpha., and then, after a predetermined time
interval, administering to the subject a therapeutically effective
amount of a conjugate of IL-13 linked to a drug, radionuclide or
toxin. Another method of treatment for cancer or an
immunologically-mediated or infectious disease comprises first
administering to a subject in need of such treatment a targeting
moiety comprising an antibody specific for an antigen specific to a
targeted cell, linked to the ligand-binding region of
IL-15R.alpha., and then administering to the subject a
therapeutically effective amount of a conjugate of IL-15 linked to
a drug, radionuclide or toxin.
[0015] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0016] It has been discovered, surprisingly, that the value of
surface antigens as antigenic targets can be improved significantly
by functionally linking them to a high affinity, internalizing
receptor system. The present invention is of particular advantage
in the case of useful cell surface antigens that internalize
slowly.
[0017] The present invention is based on a fundamental property of
cytokine and growth factor receptors, viz., their ability to
rapidly and efficiently internalize. Examples of rapid
internalization of receptor and ligand include intracellular
transport of nutrients, as with the transferrin and low-density
lipoprotein (LDL) receptors. Receptors for growth factors like
insulin and epidermal growth factor (EGF) as well as cytokine
receptors such as IL-1R, IL-2R and IL-4R also internalize rapidly.
In all cases studied, except that of transferrin, the ligand
undergoes proteolysis as a consequence of trafficking to the low
pH, protease/acid hydrolase-containing lysosomal compartment.
Molecules associated with the ligands, such as cholesterol bound to
LDL or drugs, toxins, and radionuclides, linked to other ligands
can exit to extra-lysosomal compartments where they can exert their
effects.
[0018] The fate of a receptor following internalization varies
depending on the receptor system. For example, it may recycle to
the cell surface, as with the transferrin and LDL receptors, or it
may itself be degraded. Lysosomal degradation of receptors has been
reported for receptors such as the EGF receptor and contributes to
receptor down-regulation and desensitization to subsequent ligand
stimulation.
[0019] In some ligand/receptor systems, there is re-expression of
receptors via de novo mRNA and protein synthesis. For example, CD22
is internalized rapidly after binding of the cognate LL2 mAb and is
re-expressed as soon as 2 hours after a complete cycle of antigen
saturation binding of cognate antigen by the specific mAb followed
by internalization at 37.degree. C. Further evidence of
re-expression is found in the ability of a wide array of
cytokine/growth factor-dependent cell lines to be maintained for
months or even years, suggesting ongoing re-synthesis and
re-expression of the requisite ligand binding as well as the
associated signaling proteins. Taken together, these observations
show that cytokine receptors are capable of multiple, rapid cycles
of internalization and re-expression and hence have a high capacity
for intracellular delivery of ligands.
[0020] In accordance with the present invention, it is possible to
induce rapid internalization of a slowly internalizing antigen by
bringing it in juxtaposition with a more rapidly internalizing
complex. For example, the IL-2 receptor system consists of an alpha
(IL-2R.alpha., formerly Tac antigen), beta (IL-2R.beta.) and gamma
(.gamma..sub.c) chain. IL-2R.alpha. internalizes slowly, but once
it becomes physically associated to IL-2R.beta. and .gamma..sub.c
by the presence of the IL-2 ligand the entire trimeric protein
complex becomes internalized at the rapid intrinsic rate of the
IL-2R.beta./.gamma..sub.c dimer. This follows a recurring pattern
in cytokine biochemistry, in which a functional receptor consists
of two or more subunits, one of which is typically a private,
specific alpha chain. The IL-6-IL-6 system is particularly notable
in that the extracellular domain of the alpha subunit has an
intrinsic ability to associate with the gp130 signaling molecule
such that, when IL-6 plus a soluble form of IL-6R.alpha. are added
to cells that express only gp130, a signaling response occurs. The
IL-2 and IL-6 receptor systems are exemplary of two major cytokine
signaling subunits, .gamma..sub.c (utilized by receptor complexes
for ILs-2, 4, 7, 9 and 15) and gp130 (utilized by receptor
complexes for ILs-6 and 11, CNTF, LIF, OSM and cardiotrophin-1),
respectively.
[0021] Receptor systems can be harnessed according to the present
invention to provide enhanced intracellular delivery of armed
ligands. Cytokine receptors can be targeted to the surface of cells
that normally lack such receptors by the use of mAb-receptor
conjugates. For example, the alpha chains of the IL-6 and the
ciliary neurotrophic factor (CNTF) receptors have been targeted to
the surface of previously negative cells by way of mAbs directed
against the CD34, CD45 and CD64 cell surface antigens. Addition of
such mAb-R.alpha. conjugates to factor-dependent cells conferred de
novo, specific responsiveness to IL-6 or CNTF.
[0022] A targeting moiety according to the invention comprises a
receptor linked to a mAb fragment up to F(ab').sub.2 size. Suitable
antibody fragments include F(ab').sub.2, F(ab).sub.2, Fab', Fab, Fv
and the like, including hybrid fragments. Also useful are any
subfragments that retain the hypervariable, antigen-binding region
of an immunoglobulin. This will include genetically engineered
and/or recombinant proteins, whether single-chain or
multiple-chain, which incorporate an antigen binding site and
otherwise function in vivo as targeting vehicles in substantially
the same way as natural immunoglobulin fragments. Single-chain
binding molecules are disclosed in U.S. Pat. No. 4,946,778, which
is hereby incorporated by reference. Fab' antibody fragments may be
conveniently made by reductive cleavage of F(ab').sub.2 fragments,
which themselves may be made by pepsin digestion of intact
immunoglobulin. Fab antibody fragments may be made by papain
digestion of intact immunoglobulin, under reducing conditions, or
by cleavage of F(ab).sub.2 fragments which result from careful
papain digestion of whole Ig. The fragments may also be produced by
genetic engineering. When the term "antibody" is used herein, all
the above types of fragments are included therein.
[0023] mAb fragments such as the single chain antibody scF.sub.v
have the added advantages of rapid blood and organ clearance and
improved penetration into tumor nodules and, in a preferred
embodiment, scF.sub.v of a mAb to a desired target antigen is
linked to the ligand-binding region of a receptor. mAb molecular
engineering techniques can be used to produce scF.sub.v. This
molecule can be produced by cloning the V.sub.H and V.sub.L
segments from the mAb of interest and splicing them together with a
short linker region interposed between them. These molecules, after
proper design and renaturation, retain the antigen binding activity
of the parent mAb and can be expressed at high levels in E.
coli-based insect or mammalian expression systems. These constructs
then can provide a platform for the engineering of bifunctional
single chain molecules that can link a second moiety (receptor or a
single chain antibody) to the first to retarget effector cells or
molecules.
[0024] Mixtures of antibodies, as well as hybrid antibodies, can be
used. The hybrids can have two specificities, e.g., one arm binding
to one antigen on the target cell and another arm binding to
another antigen on the target, or one arm could possess a ligand
binding region of a receptor subunit. Hybrid antibody fragments
with dual specificities can be prepared analogously to the
anti-tumor marker hybrids disclosed in U.S. Pat. No. 4,361,544.
Other techniques for preparing hybrid antibodies are disclosed in,
e.g., U.S. Pat. Nos. 4,479,895, 4,474,893, 4,714,681, and in
Milstein et al., Immunol. Today 5:299 (1984), all incorporated
herein by reference. The foregoing are merely illustrative, and
other combinations of specificities can be envisioned that also
fall within the scope of the invention.
[0025] The antibody is linked to the extracellular domain of a
receptor. Since the entire extracellular domain is large, truncated
versions of the domain that contain the ligand-binding site can be
used. The antibody/receptor conjugate can be formed by covalently
linking the antibody to the receptor, directly or through a short
or long linker moiety, through one or more functional groups on the
anti-body and/or the enzyme, e.g., amine, carboxyl, phenolic, thiol
or hydroxyl groups, to form a covalent conjugate. Various
conventional linkers can be used, e.g., diisocyanates,
diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides,
bismaleimides, dithiols, maleimide-hydroxysuccinimide esters,
glutaraldehyde and the like. The antibody construct may bind one
arm to either the ligand binding region or a site that is remote
from the ligand-binding site depending on whether ligand will be
employed in a given application.
[0026] A simple method is to mix the antibody with the
ligand-binding region in the presence of glutaraldehyde to form the
antibody/receptor conjugate. The initial Schiff base linkages can
be stabilized, e.g., by borohydride reduction to secondary amines.
This method is conventionally used to prepare, e.g.,
peroxidase-antibody conjugates for immunohistochemical uses or for
immunoassays. A diisothiocyanate, a bishydroxysuccinimide ester,
carbodiimide or other biofunctional crosslinkers can be used in
place of glutaraldehyde.
[0027] More selective linkage can be achieved by using a
heterobifunctional linker such as a maleimide-hydroxysuccinimide
ester. Reaction of the latter with the ligand-binding region of the
receptor will derivatize amine groups on the receptor, and the
derivative can then be reacted with, e.g., an antibody Fab fragment
with free sulfhydryl groups (or a larger fragment with sulfhydryl
groups appended thereto by, e.g., Traut's Reagent).
[0028] It is advantageous to link the ligand-binding region of the
receptor subunit to a site on the antibody remote from the antigen
binding site. This can be accomplished by, e.g., linkage to cleaved
interchain sulfhydryl groups, as noted above. Another method
involves reacting an antibody whose carbohydrate portion has been
oxidized, with a ligand-binding region which has at least one free
primary amino group. This results in an initial Schiff base (imine)
linkage, which is preferably stabilized by reduction to a secondary
amine, e.g., by borohydride reduction, to form the final
conjugate.
[0029] Alternatively, the antibody/receptor conjugate comprises a
bispecific antibody conjugate which is linked immunologically to
the ligand-binding region of a receptor. The bispecific antibody or
antibody fragment has a first specificity for a cellular antigen
specific to a targeted cell and a second specificity for an
extracellular antigen specific to a targeted cell and a second
specificity for an extracellular retion of the ligand-binding
subunit or another subunit of the receptor complex.
[0030] Most preferably, the antibody/receptor conjugate comprises a
fusion protein, in which a fusion sequence comprising antibody
linked to a ligand-binding region of the receptor is expressed in a
recombinant virion-based, mammalian expression system or other
mammalian, insect, yeast or E. coli-based expression system.
Suitable linkers for linking the antibody to the ligand-binding
region are, for example, (GGSBS).sub.3 (SEQ ID NO: 1) or the
23-amino acid linker disclosed in Kurucz et al., J Immunol. 154:
4576-4582 (1995).
[0031] Any of the antibody/receptor conjugates can be labeled with,
or conjugated or adapted for conjugation to, a radioisotope or
magnetic resonance image enhancing agent. In a preferred
embodiment, the antibody/receptor bifunctional construct is
unlabeled. It is administered and then, after a predetermined
interval sufficient for localization to the target site and also
clearance from the circulatory system of the mammal, the armed
cognate ligand will be given. This is particularly important when
boron complexes are used therapeutically. Alternatively, the
conjugate is tagged with a label, e.g., a radiolabel, a fluorescent
label or the like, that permits its detection and quantitation in
body fluids, e.g., blood and urine, so that targeting and/or
clearance can be measured and/or inferred.
[0032] Any conventional method of radiolabeling which is suitable
for labeling proteins for in vivo use, generally is suitable for
labeling these antibody/receptor conjugates. This can be achieved
by direct labeling with, e.g., I-131, I-124 or I-123. Labeling with
either I-131, I-124 or I-123, is readily effected using an
oxidative procedure wherein a mixture of radioactive potassium or
sodium iodide and the antibody is treated with chloramine-T, e.g.,
as reported by Greenwood et al., Biochem. J, 89: 114 (1963) and
modified by McConahey et al., Int. Arch. Allergy Appl. Immunol.,
29: 185 (1969). This results in direct substitution of iodine atoms
for hydrogen atoms on the antibody molecule, presumably mostly on
tyrosine residues, possibly also on tryptophan and even on
phenylalanine residues, depending on the proportions of reagents
and the reaction conditions. Alternatively, either lodogen-based
methods as described by Fraker et al., Biochem Biophys Res. Commun
80:849-857, 1978, or lactoperoxidase iodination may be used, as
described by Feteanu, supra, page 303, and references cited
therein.
[0033] Instead of I-131, I-124 or I-123, the conjugate can be
labeled by metallation with, e.g., Tc-99m or Cu ions or the like,
by conventional techniques, or by attaching a chelator for a
radiometal or paramagnetic ion. Such chelators and their modes of
attachment to antibodies are well known to the ordinary skilled
artisan.
[0034] The antibody/receptor conjugate acts as a targeting moiety
according to the invention when administered to a patient. This is
followed by a targeting and clearance interval that allows for
binding of the targeting moiety to intended target cells and its
clearance from normal tissues, after which armed ligand is
administered. The armed ligand comprises the cognate ligand for the
receptor of the targeting moiety, conjugated to a radionuclide,
drug or toxin. The radionuclide can be either a diagnostic or
therapeutic radionuclide.
[0035] Examples of diagnostic radionuclides include iodine-123,
iodine-124, iodine-131, indium-111, gallium-67, gallium-68,
ruthenium-97, technetium-94, technetium-99m, copper-64, copper-67,
cobalt-57, cobalt-58, chromium-51, iron-59, yttrium-86,
selenium-75, thallium-201, and ytterbium-169. Examples of
therapeutic radionuclides include alpha-emitters, e.g., bismuth-212
and astatine-211; beta-emitters, e.g., yttrium-90, rhenium-186,
rhenium-188, copper-67 and iodine-131; and alternatively, electron
capture or Auger conversion electronemitting radionuclides such as
iodine-125, indium-111 and gallium-67. Preferably the radionuclide
will emit in the 10-5000 keV range, more preferably in the 50-1500
keV range, and most preferably in the 50-500 keV range. The
radionuclides may be incorporated into the specific antibody by the
labeling techniques discussed above, as well as other conventional
techniques well known to the art.
[0036] Many drugs and toxins are known which have a cytotoxic
effect on tumor cells or microorganisms that may afflict humans and
mammals in general. They are to be found in compendia of drugs and
toxins, such as the Merck Index and the like. A preferred toxin is
a ribonuclease, such as onconase. Onconase is a non-mammalian RNAse
purified from Rana pipiens oocytes. It has been shown in clinical
trials to have anti-tumor activity against human pancreatic cancer,
but has been found to have minimal anti-tumor activity against
B-cell malignancies such as B-cell lymphoma or leukemia.
[0037] Addition of the armed ligand (specifically IL-2 and IL-15)
results in the formation of a trimeric complex comprising the
.beta./.gamma..sub.c chain subunits present on the surface of the
targeted cell and the .alpha.-chain subunit of receptor, which is
attached to the surface of the targeted cell by means of the
antibody/receptor conjugate. This leads to rapid internalization of
toxin and/or radionuclide into the targeted cells. An analogous
dimeric or trimeric complex assembles after the addition of IL-4 or
IL-13. While internalization is not necessary for a therapeutic
radionuclide to be effective, these multimeric complexes provide a
tighter, more stable binding of the ligand to the targeted cells,
and also facilitates internalizations.
[0038] The same ligand can be armed with both radionuclide, drug
and toxin, or separate ligands can be armed with radionuclide, drug
and toxin. Where separate ligands armed with radionuclide, drug and
toxin are used, these may be administered together or
sequentially.
[0039] The antibody/receptor conjugate and armed ligand conjugate
are administered in a composition with a pharmaceutically
acceptable carrier. In this regard, a pharmaceutically acceptable
carrier is a material that can be used as a vehicle for
administering the fusion protein or armed ligand because the
material is inert or otherwise medically acceptable, as well as
compatible with the fusion protein or armed ligand.
[0040] Preferred high affinity, internalizing receptor systems to
which the antibody can be linked include the IL-2, IL-4, IL-13 and
IL-15 receptor systems. Internalization rates for IL-2/IL-2R
(related to the IL-15/IL-15R system) and for IL-4/IL-4R (related to
the IL-13/IL-13R system) have t.sub.1/2 values of approximately
15-30 minutes. Particularly preferred receptor systems for use in
the present invention are the IL-13 and IL-15 receptor systems.
[0041] The IL-13/IL-13 receptor system is a good candidate for
addressing solid tumors due to its widespread expression and
signaling capability on cancers of epithelial origin
(preponderantly CEA+). The IL-13/IL-13 receptor system shares
several features in common with the IL-4/IL-4 receptor system.
Structurally, the IL-4 and IL-13 ligands show approximately 30%
protein sequence homology, which is the highest amongst the
interleukins. Both receptors possess the canonical four conserved
cysteine residues in the N-terminal half of their extracellular
domains as well as a WSXWS motif in the juxtamembrane region of
their extracellular domains, and are members of the hematopoietin
superfamily. IL-13R.alpha. is of smaller overall size than
IL-4R.alpha., but has a somewhat larger extracellular domain and a
correspondingly shorter intracellular domain.
[0042] IL-13 and IL-4 have considerable functional similarity as
well. Both suppress production of pro-inflammatory cytokines by
macrophages, are co-stimulatory for B cell proliferation and induce
immunoglobulin isotype switching. Both induce upregulation of both
CD23 and MHC class II on both monocytes and B cells. IL-13 and IL-4
bind nonhematopoietic cells, such as carcinoma cell lines, with
high affinity and exert biologic effects on them. IL-4 inhibits the
growth of these epithelial cancer cell lines in unmodified form
both in vitro and in vivo. Unmodified IL-13 also has in vitro
growth-inhibitory effects on breast carcinoma cell lines, and thus
shares this property.
[0043] In several cell types, IL-13 competes for IL-4 binding and
vice versa, indicating that IL-4 and IL-13 share receptor
components. In addition, a mutant form of IL-4, Y124D, is capable
of inhibiting both IL-4 and IL-13 biologic responses in lymphoid
and nonlymphoid cell types. The nonlymphoid cell types are
predominantly negative for .gamma..sub.c, while all of these cell
types express varying amounts of IL-13R.alpha. and IL-4R.alpha..
Both IL-4R.alpha. and IL-13R.alpha., when expressed alone, bind
their cognate ligands with a similar high affinity, with K.sub.as
of approximately 10.sup.10 M.sup.-1. The soluble form of
IL-4R.alpha. has been shown to retain this ligand-binding ability.
Soluble forms of IL-13.alpha. have recently been shown to retain
ligand-binding ability both in vitro and in vivo. When IL-4R.alpha.
and IL-13R.alpha. are coexpressed, they are capable of forming a
complex that can be impacted by both ligands as well as
antagonistic ligands like IL-4-Y124D.
[0044] Human IL-13R.alpha. is expressed at either low or moderate
levels at both the mRNA and protein level by a variety of
hematopoietic and epithelial cell lines. In colon carcinoma cells,
specific signaling events, namely, Jak 2 tyrosine kinase
activation, have been shown to occur in conjunction with IL-4's
biologic effects.
[0045] The cytotoxic activity exhibited by IL-4-Pseudomonas
exotoxin (IL-4-PE) and IL-13-PE fusion molecules provides further
evidence of the efficient internalization of these receptor
systems, since a wide range of carcinoma cell lines are quite
sensitive of these cytotoxins. Several different IL-4-PE constructs
have been made that are active in vitro and in a mouse xenograft
model, and analogous IL-13-PE constructs have been shown to possess
equally potent in vitro activity. Likewise, diphtheria-IL-4 fusion
proteins (e.g. DAB.sub.389IL-4) exhibit in vitro cytotoxic
activity.
[0046] IL-13's effects are comparable to IL-4, both in unmodified
form and linked to PE. However, unmodified IL-13 can be given at
higher doses than IL-4 and IL-13-PE shows less toxicity towards
hematopoietic cells than IL-4-PE. Epithelial cancer cell lines can
be targeted and killed by IL-3-toxins as readily as by
corresponding IL-4-toxins, indicating that the IL-13 receptor
complex internalizes as efficiently as the IL-4 receptor complex.
Unlike IL-4, IL-13 has no biologic effect on T cells. For these
reasons, the IL-13 receptor system is preferred to the IL-4
receptor system for diagnostic and therapeutic use in vivo.
[0047] In a preferred embodiment, the IL-13 receptor system is used
in conjunction with a functional antibody, preferably a single
chain mAb (scF.sub.v), to carcinoembryonic antigen (CEA). CEA
represents an attractive antigenic target for several reasons. It
is a tumor-associated antigen that it is absent or poorly expressed
by normal tissues and highly expressed by the vast majority of
carcinomas of colon, lung, breast, pancreatic, gastric, ovarian,
and medullary thyroid origin. High incidence and mortality rates
for these cancers coupled with suboptimal diagnostic and
therapeutic options result in a serious and persistent overall
public health problem.
[0048] CEA is a glycosylated cell surface protein of approximately
180 kDa, and is a solid tumor antigen that has been extensively
studied clinically, both as a circulating tumor marker and as an
antigenic target for radiolabeled mAbs for imaging and therapy. A
number of anti-CEA antibodies have been under study in phase I-III
clinical diagnostic and therapeutic trials. Exemplary of an
anti-CEA mAb is the MN-14mAb. A humanized version of this mAb,
hMN-14, in which human constant and framework regions replace the
corresponding mouse sequences, has been constructed and expressed
and is the mAb used in these clinical trials. A .sup.99mTc-labeled
Fab' fragment of another, related anti-CEA mAb, Immu-4, has
received FDA approval for the detection and staging of colon
cancer.
[0049] Though promising as an imaging agent, radiolabeled anti-CEA
mAbs in the therapeutic mode previously have yielded few responses.
A low response rate resulted even when an anti-CEA mAb was
co-administered with an anti-TAG-72 mAb, recognizing a second,
distinct highly expressed tumor-associated antigen, along with
IFN-.alpha., which upregulates both antigens, on a group of
patients with metastatic colon cancer. Advanced colon cancer
likewise has been quite resistant to all chemotherapeutic
combinations tested to date.
[0050] The present invention seeks to overcome these therapeutic
barriers, which are common to most solid tumors, by enhancing the
internalization of CEA. An anti-CEA targeting moiety, such as
shIL-13R.alpha.-anti-CEA scF.sub.v fusion protein, is administered
to a subject and, after the targeting moiety localizes at the tumor
sites, IL-13 ligand armed with a therapeutic or diagnostic moiety
is delivered. This system provides diagnostic and therapeutic
options for the large number of CEA+ malignancies, including
cancers of the lung, colon, breast, stomach, ovary and pancreas,
most of which express both CEA and IL-13 receptor components. The
IL-13 receptor components enable rapid internalization of the armed
ligand, to selectively deliver high levels of cytotoxic agents to a
large group of tumors. Targeting a highly expressed antigen, such
as CEA, in a sIL-13R.alpha.-anti-CEA scF.sub.v fusion molecule,
increases the typically low number of cytokine receptors by up to
two orders of magnitude.
[0051] The IL-15/IL-15 receptor system is a good candidate for
targeting B-cell and T cell malignancies, normal or activated B
cells, and activated T cells. The IL-15/IL-15 receptor system
shares several features in common with the IL-2/IL-2 receptor
system. In vitro and in vivo, both IL-2R.alpha. and IL-4R.alpha.
have soluble forms, which bind its cognate ligand. The IL-2/IL-2
receptor system has a trimeric receptor. IL-15, like IL-2, uses
IL-2R.beta. and .gamma..sub.c, but not IL-2R.alpha. (formerly Tac).
The specific alpha chain of IL-15 (IL-15R.alpha.) has homology and
a similar structural organization to IL-2R.alpha.. While
IL-15R.alpha. is homologous structurally to IL-2R.alpha., it has a
significantly higher affinity for its cognate ligand, with a
K.sub.a of .about.10.sup.10 m.sup.-1.
[0052] IL-2R.alpha. expressed in the absence of the other two
chains internalizes slowly, but when juxtaposed to the other
subunits by the presence of ligand, the entire
ligand/.alpha..beta..gamma. complex internalizes at the rapid rate
intrinsic to the IL-2R.beta./.gamma..sub.c dimer (t.sub.1/2 of
approximately 15 minutes). By comparison IL-15R.alpha. has an
affinity for its cognate ligand (K.sub.a.gtoreq.10.sup.10 M.sup.-1)
that is at least two orders of magnitude greater than that of
IL-2R.alpha. for IL-2.
[0053] T, B, NK and monocyte populations respond to IL-15 and
correspondingly are positive for
IL-2R.beta./.gamma..sub.c+/-IL-15R.alpha- .. In addition,
B-lymphoma/leukemia expresses IL-2R.beta., and .gamma..sub.c is
nearly uniformly expressed by all normal and malignant
hematopoietic cells. The minimal functional receptor structure for
IL-2 and IL-15 requires co-expression of IL-2R.beta. and
.gamma..sub.c. Therefore, both normal leukocyte populations, which
may be pathogenic and which have been targeted in
immune/inflammatory conditions, as well as the malignant
counterparts of B-cells and T-cells can be targeted according to
the invention. While similar in many respects, the IL-15 receptor
system provides at least one advantage over the IL-2 receptor
system, having somewhat less capacity to induce clinically apparent
vascular permeability.
[0054] In a preferred embodiment, the IL-2 or IL-15 receptor system
is used in conjunction with a functional single chain mAb
(scF.sub.v) to HLA-DR. HLA-DR is a favorable antigenic target. It
is expressed by a range of hematopoietic malignancies
(particularly, B-lymphomas and B-leukemias) as well as certain
normal immune cells, such as B cells, monocytes, dendritic and
activated T cells. Like CEA, it is expressed at levels that can
reach 10.sup.6 sites/cell, and is internalized at a slow rate.
[0055] The absence of HLA-DR antigen from many critical normal cell
populations, such as pluripotent bone marrow stem cells, adds to
its clinical utility. This undifferentiated stem cell population
can replenish any depletions in differentiated normal cell
populations, as has been observed in myelotoxic situations such as
cancer chemotherapy and bone marrow transplantation. Moreover, in
certain severe cases of immune-mediated disease, these cell
populations may need to be eliminated or significantly
attenuated.
[0056] There is a large body of clinical experience in the
treatment of B-cell malignancies with mAbs directed against HLA-DR
(using the Lym-1 mAb), as well as other B-cell-specific mAbs,
including CDs19-22 and CD37. In these studies, radiolabeled mAbs or
mAbs linked to toxin have been used therapeutically against
malignant B cells. Immunotoxins also have been employed to
eliminate pathogenic T cells in unrelated-donor bone marrow
transplantation.
[0057] Effective and selective cellular cytotoxicity via the IL-15
receptor and single chain mAb (scF.sub.v) to HLA-DR according to
the invention yields significant clinical benefits in a wide range
ofcancers, including B-cell lymphomas and leukemias, as well as
many immune-mediated diseases including autoimmune and inflammatory
diseases such as autoimmune diabetic states, inflammatory bowel
disease, systemic lupus erythematosus, rheumatoid arthritis, and
severe psoriasis, and allograft reactions such as organ transplant
rejection, graft-versus-host disease in bone marrow
transplantation. Specific targeting of highly expressed HLA-DR
antigen on an activated T cell population with an
sIL-15R.alpha.-anti-HLA-DR scF.sub.v targeting moiety achieves more
specificity and effectiveness in eliminating reactive T cell
populations.
[0058] In accordance with the invention, an anti-HLA-DR targeting
moiety, for example, a shIL-13R.alpha.-anti-HLA-DR scF.sub.v fusion
protein such as sIL-15R.alpha.-Lym-2 scF.sub.v, is administered to
a subject. After the targeting moiety localizes at the therapy
site, IL-15 ligand armed with a therapeutic or diagnostic moiety is
delivered. The IL-15 receptor is predicted to enhance the
internalization of HLA-DR, to selectively deliver high levels of
cytotoxic agents. Here again, mAb fragments such as scF.sub.v can
be used to provide the added advantages of rapid blood and organ
clearance. By careful selection of components, delivery systems
with minimal immunogenicity can be achieved. The armed ligand, the
R-.alpha. moiety and the mAb scF.sub.v can be tailored to fit the
characteristics of the particular disease.
[0059] The present invention is designed to provide higher
tumor/non-tumor ratios, as can be achieved with traditional
pretargeting systems that utilize avidin or streptavidin and
biotin, while eliminating certain problems associated with these
systems. While the avidin-biotin system has a very high affinity,
clinical experience has shown that approximately 20-30% of patients
mount an antibody response against avidin and up to 70% make
antibodies to streptavidin. The present invention avoids the
immunogenicity of avidin and biotin. A three-step approach can be
implemented by using an anti-idiotypic mAb, W12, that is reactive
to the antigen combining site of MN-14, and thereby any humanized
version of MN-14. The W12 mAbs have been galactosylated, which
allows for rapid blood clearance of unlabeled MN-14-based reagent
through the hepatic asialoglycoprotein receptor. These pretargeting
approaches strive for maximal blood and organ clearance of the
first step unlabeled pretargeting agent prior to administering the
armed second or third step reagent, in order to minimize normal
tissues' exposure to armed diagnostic or therapeutic agents and to
maximize tumor/normal tissue ratios of the armed agent.
[0060] In sum, the present invention approach offers potentially
beneficial alternatives to current approaches for several reasons.
First, it uses agents with decreased immunogenicity. mAb fragments
have inherently lower immunogenicity, particularly hMN-14mAb which
has been humanized. The receptor and ligand components are native
and non-immunogenic. Second, the invention uses a pretargeting
strategy that permits higher specific delivery of armed ligand to
tumor sites or pathogenic cell populations. Third, the invention
maximizes internalization of ligand to allow higher intracellular
concentration of armed ligand and better diagnostic or therapeutic
effects. Fourth, the invention allows a therapeutic approach with
combinations or sequences with various agents including drugs,
toxins, radionuclides, antisense and antigene reagents. To
accomplish this, different armed forms of the same ligand are used.
Finally, the present invention uses cytokines which appear
favorable in their toxicity profiles and can be applied to a wide
range of diseases.
[0061] The following examples are illustrative of the present
invention, but are not to be construed as limiting.
EXAMPLE 1
Construction of a Bifunctional Soluble IL-13R.alpha.-MN-14scF.sub.v
Fusion Protein
[0062] An MN-14scF.sub.v was produced by PCR amplification of cDNA
from the humanized MN-14transfectoma. The linker used for
MN-14scF.sub.v was a 15-amino acid linker (GGSGS).sub.3 (SEQ ID NO:
1) and the orientation was V.sub.L-linker-V.sub.H. After
confirmation of the DNA sequences, the single chain construct was
subcloned into an appropriately restricted containing expression
plasmid used for other scF.sub.vs. This construct then was
subcloned into BL21(.lambda.DE3) E. coli for expression.
[0063] The protein was solubilized and renatured from inclusion
bodies and was purified by sequential anion exchange and gel
filtration chromatography. After functional evaluation, the
scF.sub.v fragment was ligated to a DNA fragment encoding PE40.
This immunotoxin was shown to have specific cytotoxicity for CEA+
cell lines.
[0064] Another single chain construct also was made. This was made
with the opposite 5'-3' orientation of the heavy and light chains,
was assembled in pCANTABE5E (Pharmacia Biotech, Piscataway, N.J.)
and expressed in phage. Specific binding of recombinant phage
expressing this scF.sub.v was demonstrated by ELISA.
[0065] The V.sub.L-linker-V.sub.H sequence is used for construction
of the IL-13R.alpha.-MN-14 fusion protein, as diagrammed below. A
23-amino acid linker is used between shIL-13R.alpha. and the
scF.sub.v. Kurucz et al. (1995). Alternatively, the (GGSGS).sub.3
(SEQ ID NO: 1) linker which was used in construction of the MN-14
scF.sub.v described above is used. The configuration of this fusion
protein is:
[0066] shIL-s13R.alpha.-linker--V.sub.L-(GGSGS).sub.3--V.sub.H
[0067] The DNA fragment encoding the soluble, extracellular domain
of IL-13R.alpha. is obtained by PCR amplification from positive
cell lines, currently Caki-1, HuT 102B2 and A549. PCR primer pairs
for RT-PCR are synthesized, including a primer pair for cloning.
The primer pairs span almost the entire extracellular domain of the
receptor.
[0068] The primers include unique restriction enzyme sites to allow
for directional cloning into the expression vector, pSinrep 5. This
vector is part of a high-level Sindbis virus, mammalian expression
system (Invitrogen, San Diego, Calif.). The recombinant plasmid
will include the wild-type signal sequence and hence can be
secreted into the cell culture medium. The ligand-binding region is
predicted to lie in the N-terminal half of the molecule, a
considerable distance away from the scF.sub.v domains, and thus the
above configuration was selected. To allow unhindered folding of
individual domains a 23-amino acid linker is included, as has been
described for a bispecific single chain protein, i.e., a fusion of
two scF.sub.vs.
[0069] In order to retain sequences potentially important for
interactions of IL-13R with partner proteins such as IL-4R, it is
preferable to retain the WSXWS domain and all of the conserved
cysteine residues, along with nearly all of the extracellular
domain. This maximizes the possibility of interaction with
associated proteins in the membrane, thereby facilitating the
binding and internalization of the IL-13/IL-13 receptor
complex.
EXAMPLE 2
Expression and Purification of the Bifunctional Soluble
IL-13R.alpha.-MN-14 scF.sub.v Fusion Protein
[0070] Bacterial clones containing recombinant pSinRep5 plasmids
are screened and those with correct sequences are used for
expression. Recombinant virions are produced to provide higher
expression and a stable reusable stock for multiple transductions.
This is accomplished by co-transfection of in vitro transcribed RNA
from the recombinant plasmid plus a replication-deficient helper
virus DNA template (as per manufacturer's instructions).
Alternatively, an E. coli-based expression system, similar to the
one used for production of MN-14scF.sub.v is used.
[0071] When recombinant Sindbis virions are used, RNA transcription
is performed from the recombinant plasmid (prepared from the
initial pSinRep5 plasmids) and a helper virus plasmid that is
included in a standard kit. (Invitrogen, Inc., San Diego, Calif.)
RNA yields are assessed by agarose gel electrophoresis and the RNAs
then are used to co-transfect the BHK cell line. This is done using
cationic liposomes and/or electroporation. After 3 days in culture
the supernatant from the transfection is collected and used to
transduce fresh BHK cells to assess viral titer and to assess the
level of recombinant protein expression. For all transductions,
cells are plated initially in FCS-containing medium. After
approximately 20 hours the medium is changed to serum-free medium.
After 3 days, supernatants from recombinant and non-recombinant
controls are collected and aliquots are concentrated by centrifugal
ultrafiltration for total protein determination (Coomassie
Plus.TM., Pierce, Rockford, Ill.).
[0072] Aliquots of the concentrated supernatant are fractionated by
SDS-PAGE. The gel is divided for Coomassie staining and
electroblotting onto PVDF for Western blotting.
sIL-13R.alpha.-MN-14 scF.sub.v expression is detected with goat
anti-human IgG-peroxidase to detect Hmn-14 sequences and
development by chemiluminescence. After exposure to photographic
film, the blot is examined for a specifically stained band of
approximately 70 kDa.
[0073] Once expression is confirmed the transductions are scaled
up. For large-scale expression runs, the purification scheme
includes anion exchange, gel filtration and/or other HPLC modes
with determination of final recovery and purity. In initial genetic
constructs a C-terminal hexahistidine or related inert affinity tag
sequence will be added to assist purification.
EXAMPLE 3
Assay of Antigen and Ligand Binding Activity of Soluble
IL-13R.alpha.-MN-14 scF.sub.v Fusion Protein
[0074] Purified sIL-13R.alpha.-MN-14 scF.sub.v is .sup.125I-labeled
by the lodogen (Pierce) method to approximately 5 .mu.Ci/.mu.g. The
LS174T cell line is used for binding studies since it expresses
high levels of CEA and has low to moderate IL-13 binding.
[0075] An amount of 1.times.10.sup.6 washed LS 174T cells/tube is
suspended in 100 .mu.l of binding buffer (RPMI 1640/10% FCS).
Either labeled sIL-13R.alpha.-MN-14 sc.sub.v at 10 nM alone or
labeled sIL-13R.alpha.-MN-14 scF.sub.v at 10 nM together with a 200
fold molar excess of the unlabeled protein is added to replicate
tubes. The overall ability to bind antigen is assessed by using
tracer concentrations (50-200 pM) of labeled fusion protein and
larger numbers of LS174T cells. The bindable fraction should be
greater than 50% in antigen excess.
[0076] IL-13 binding ability is assessed using .sup.125I-IL-13
(Iodogen labeled as before). To replicate tubes containing
1.times.10.sup.6 LS174T cells is added either unlabeled fusion
protein or buffer. After 30-40 min at 4.degree. C. cells are washed
and .sup.125I-IL-13 is added, either in the presence or absence of
cold IL-13. A significant positive increment in specific IL-13
binding indicates a functional IL-13R.alpha. moiety.
EXAMPLE 4
Assay of Ability of Soluble IL-13R.alpha.-MN-14 scF.sub.v Fusion
Protein to Internalize CEA
[0077] sIL-13R.alpha.-MN-14 scF.sub.v is radioiodinated to a
specific activity of approximately 5-10 .mu.Ci/.mu.g. Ten nM ligand
is added to replicate tubes of 2.5.times.10.sup.5 LS174T
cells/tube, and incubated at 4.degree. C. for one hour. Cells are
washed twice with binding buffer and plated in 24 well plates. To
some tubes a 200-fold molar excess of cold ligand is added to
assess specific binding. To other tubes, 1 nM unlabeled rhIL-13 is
added to assess effects on internalization and processing.
[0078] Supernatants are removed at multiple time points and brought
to 10% TCA to precipitate intact the label, so as to distinguish
label which dissociates versus label that is internalized,
catabolized and released. Plates are washed three times with
binding buffer. Cells are solubilized with 0.4 ml 2 N NaOH for
counting.
EXAMPLE 5
Biodistribution of Radiolabeled IL-13
[0079] Biodistribution of IL-13 is studied in CEA+ tumor-bearing
nude mice, using the LS174T model system. Five week old female nude
mice are injected with 5.times.10.sup.6 cells from the LS1747T or
HT-29 cell lines, both of which are colon cancer cell line,
resulting in development of subcutaneous tumors. Biodistribution of
.sup.125I-IL-13 in mice simultaneously injected with
.sup.131I-labeled MN-14Fab' or MN-14scF.sub.v having similar
K.sub.ds and MWs is compared.
[0080] Dual label biodistribution experiments using radioiodinated
IL-13 with IL-4 and MN-14 Fab' as controls have been performed in
both the LS174T and HT-29 tumor xenograft model systems to assess
tumor and normal organ uptake of these agents. Uptake values
observed were consistent with both the molecular mass of the agents
and the level of expression of the corresponding binding proteins
in the tumor (cognate receptor proteins for the cytokines and CEA
for MN-14Fab'). In summary, IL-13 uptake in these colon tumors was
typically in the range of 0.2-1.0 percent of injected dose/gram
tissue (%ID/g), while the corresponding uptake values for MN-14Fab'
were 4.0-7.5 for HT-29 and 7.0 for >20 for the LS174T tumors.
The IL-4 control behaved similarly IL-13. These results are
consistent with the low levels of cognate cytokine receptor in both
of these tumors (as assessed by radiotracer binding to harvested
tumor cells), the intermediate levels of CEA on HT-29 and the high
levels of CEA on LS 174T. Tumor uptake values were maximal for both
cytokines and Fab' at 5 hours post-injection, which is consistent
with their rapid clearance that is a consequence of their low
molecular masses. Renal uptake of all agents was high and also
peaked at 1-5 hours, which is consistent with the known renal
clearance mechanism of such agents. Liver showed the highest uptake
of IL-14 and IL-14 amongst other normal organs, which is consistent
with the known preclinical and clinical hepatotoxicity seen with
higher doses of these agents.
[0081] A parallel set of experiments using the Ramos B cell tumor
xenograft model were performed using radioiodinated IL-15, with
IL-2 as cytokine control and LL2 FAB' (recognizing the CD22 B cell
antigen) as their control. Overall results were similar in this
model system, except that liver intake vales were lower with these
cytokines compared to IL-13 and IL-14. These experiments confirmed
the expected pharmacokinetic and pharmacodynamic behavior of the
IL-4, IL-13, IL-2 and IL-15 cytokine agents. The results indicate
that tumor uptake reflects the low basal level of expression of the
cognate cytokine receptors in both CEA+ carcinomas as well as
lymphomas, which can be increased by first targeting tumors with
antibody-R.alpha. fusion proteins. Once targeted with this
bifunctional agent, administration of armed, cognate cytokine is
predicted to result in higher uptake than would occur with either
the antibody or cytokine without pretargeting with the
antibody-R.alpha. agent.
EXAMPLE 6
Construction of a IL-13/Onconase Immunotoxin
[0082] A fusion protein consisting of IL-13 and onconase is
genetically engineered following procedures outlined by Rybak for
the production of mAb-onconase fusion proteins. Tumor Targeting
1:141-147 (1995). Briefly, a sequence-confirmed fragment
corresponding to the mature IL-13 protein is ligated to the
sequence of onconase with the IL-13 sequence lying 5', though the
other orientation also can be evaluated. Onconase genes are cloned
from two or more frog species. Authentic fragments representing the
fusion sequence are subcloned into the pET21d vector again using a
C-terminal hexahistidine tag. The complete sequence encoding the
entire IL-13-onconase fusion protein is confirmed in the pET vector
in the XL1 Blue strain as above. Appropriate clones are expanded to
produce plasmid for transformation of the AD494 (DE3) E. coli
expression strain.
[0083] Transformed clones are picked and grown in small scale
culture, induced with IPTG, lysed in SDS sample buffer and
separated on a SDS-PAGE gel for Coomassie staining and
transblotting for detection both with anti-IL-13 antibodies and
anti-onconase antibodies. Isolation and washing of inclusion
bodies, their solubilization, renaturation and subsequent
purification is performed using the steps outlined above. The final
product is tested for its ability to bind the IL-13 receptor by
labeling with .sup.125I and comparing it with equimolar
concentrations of similarly labeled IL-13 in the cell binding assay
described above. Conjugates with the ability to bind IL-13 are
tested for cytotoxicity on cell lines known to express receptors
for IL-13.
[0084] The conjugate is tested in the presence and absence of the
sIL-13R.alpha.-hMN-14scF.sub.v fusion protein to determine toxicity
and the ability to bind and internalize greater amounts of the
immunotoxin. A dose response curve for each experimental and
control condition is generated.
EXAMPLE 7
Therapy of Colon, Lung, Breast, Pancreatic, Gastric, Ovarian and
Medullary Thyroid Carcinoma
[0085] A patient having carcinoma of the colon, lung, pancreas,
stomach, ovary, breast or medullary thyroid is infused
intravenously with a sterile, pyrogen-free solution containing
sIL-13R.alpha.-hMN-14scF.sub.v fusion protein in phosphate-buffered
saline (PBS), prepared according to Examples 1 and 2. Time is
allowed for the fusion protein to bind to malignant tumor cells and
to clear substantially from the circulation of the patient.
[0086] The patient then is infused intravenously on pre-determined
schedule with a sterile, pyrogen-free solution that contains
radioisotopically-labled or drug-conjugated IL-13 or IL-13/onconase
immunotoxin conjugate, prepared according to Example 6. Subsequent
radiologic or radioimmunodetection methods are then used to
evaluate antitumor responses.
[0087] While the invention has been described in detail with
respect to particular preferred embodiments, it should be
understood that such description is presented by way of
illustration and not limitation. Many changes and modifications
within the scope of the present invention may be made without
departing from the spirit thereof, and the invention includes all
such modifications. For example, sIL-15R.alpha.-Lym-2 scF.sub.v
fusion protein can be constructed, expressed, purified and
evaluated using similar methodology to that described for soluble
IL-13R.alpha.-MN-14scF.sub.v fusion protein in Examples 1 through
3. The cognate ligand for receptor moiety, IL-15, can be similarly
armed or labeled with drug, toxin or radionuclide. In this case,
the B-cell lines Ramos and RL can be used as the in vitro and
preclinical mouse tumor xenograft model. The ultimate clinical
malignancies that would be addressed are B and T cell
lymphoma/leukemia, Hodgkin's disease and other HLA-DR+ cancers.
This approach can also be used to suppress HLA-DR+ cell
populations, which mediate immunologic or inflammatory diseases.
Sequence CWU 1
1
1 1 15 PRT Artificial Sequence Description of Artificial Sequence
Linker 1 Gly Gly Ser Gly Ser Gly Gly Ser Gly Ser Gly Gly Ser Gly
Ser 1 5 10 15
* * * * *