U.S. patent application number 12/395159 was filed with the patent office on 2010-11-25 for enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents.
This patent application is currently assigned to MERCK PATENT GMBH. Invention is credited to STEPHEN D. GILLIES, SYLVIA HOLDEN, YAN LAN.
Application Number | 20100297060 12/395159 |
Document ID | / |
Family ID | 22801380 |
Filed Date | 2010-11-25 |
United States Patent
Application |
20100297060 |
Kind Code |
A1 |
GILLIES; STEPHEN D. ; et
al. |
November 25, 2010 |
ENHANCEMENT OF ANTIBODY-CYTOKINE FUSION PROTEIN MEDIATED IMMUNE
RESPONSES BY COMBINED TREATMENT WITH IMMUNOCYTOKINE UPTAKE
ENHANCING AGENTS
Abstract
Disclosed are methods and compositions for treating tumors.
Disclosed methods and compositions enhance the uptake of
immunocytokines into tumors, and are based on a combination of an
immunocytokine with an immunocytokine uptake enhancing agent.
Disclosed methods and compositions are particularly useful for
reducing tumor size and metastasis in a mammal.
Inventors: |
GILLIES; STEPHEN D.;
(CARLISLE, MA) ; LAN; YAN; (BELMONT, MA) ;
HOLDEN; SYLVIA; (WOBURN, MA) |
Correspondence
Address: |
GOODWIN PROCTER LLP;PATENT ADMINISTRATOR
53 STATE STREET, EXCHANGE PLACE
BOSTON
MA
02109-2881
US
|
Assignee: |
MERCK PATENT GMBH
DARMSTADT
DE
|
Family ID: |
22801380 |
Appl. No.: |
12/395159 |
Filed: |
February 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09896909 |
Jun 29, 2001 |
7517526 |
|
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12395159 |
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60215038 |
Jun 29, 2000 |
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Current U.S.
Class: |
424/85.1 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; A61K 38/193 20130101; A61K 2300/00 20130101; A61K
38/2013 20130101; A61K 31/282 20130101; A61K 45/06 20130101; A61K
38/193 20130101; C07K 2319/00 20130101; A61K 47/6813 20170801; A61K
38/208 20130101; A61K 31/675 20130101; A61K 2039/505 20130101; A61P
43/00 20180101; A61K 38/191 20130101; A61K 31/337 20130101; A61K
38/2013 20130101; A61K 39/39558 20130101; C07K 2317/24 20130101;
A61K 31/337 20130101; C07K 16/30 20130101; A61K 38/208 20130101;
A61P 37/04 20180101; A61K 31/675 20130101; A61P 35/00 20180101;
A61K 39/39558 20130101; A61K 31/282 20130101; A61K 47/6851
20170801; A61K 38/191 20130101 |
Class at
Publication: |
424/85.1 |
International
Class: |
A61K 38/19 20060101
A61K038/19; A61P 37/04 20060101 A61P037/04; A61P 35/00 20060101
A61P035/00 |
Claims
1. A method of inducing a cytocidal immune response against a tumor
in a mammal, the method comprising the steps of administering to a
mammal: (i) an immunocytokine comprising an antibody binding site
and a cytokine; and, (ii) an immunocytokine uptake enhancing agent
that enhances an immune response induced by the immunocytokine.
2. The method of claim 1, wherein the antibody binding site binds
to a cancer cell.
3. The method of claim 1, wherein the antibody binding site binds
to a tumor specific antigen.
4. The method of claim 1, wherein the immunocytokine uptake
enhancing agent is co-administered with the immunocytokine.
5. The method of claim 1, wherein the immunocytokine uptake
enhancing agent is administered prior to administration of the
immunocytokine.
6. The method of claim 1, wherein the antibody binding site
comprises, in an amino-terminal to carboxy-terminal direction, an
immunoglobulin variable region, a CH1 domain, and a CH2 domain.
7. The method of claim 6, wherein the antibody binding site further
comprises a CH3 domain attached to the carboxy terminal end of the
CH2 domain.
8. The method of claim 1, wherein the immunocytokine is a fusion
protein comprising, in an amino-terminal to carboxy-terminal
direction, (i) the antibody binding site comprising an
immunoglobulin variable region capable of binding a cell surface
antigen on a preselected cell type, an immunoglobulin CH1 domain,
an immunoglobulin CH2 domain, and (ii) the cytokine.
9. The method of claim 8, wherein the antibody binding site further
comprises a CH3 domain interposed between the CH2 domain and the
cytokine.
10. The method of claim 1, wherein the cytokine of the
immunocytokine is selected from the group consisting of a tumor
necrosis factor, an interleukin, a colony stimulating factor, and a
lymphokine.
11. The method of claim 1, wherein said immunocytokine uptake
enhancing agent is a taxane.
12. The method of claim 11, wherein said taxane is selected from
the group consisting of Taxol, docetaxel, 10-deacetyl Baccatin III,
and derivatives thereof.
13. The method of claim 1, wherein said immunocytokine uptake
enhancing agent is an alkylating chemotherapeutic agent.
14. The method of claim 13, wherein said alkylating
chemotherapeutic agent is selected from the group consisting of
cyclophosphamide, carboplatin, and derivatives thereof.
15. The method of claim 1, wherein two or more different
immunocytokine uptake enhancing agents are administered to said
mammal.
16. The method of claim 1, wherein two or more different
immunocytokines are administered to said mammal.
17. The method of claim 1, wherein said immunocytokine uptake
enhancing agent is administered about 24 hours before said
immunocytokine.
18. A composition for inducing an immune response against a tumor
in a mammal, the composition comprising: (i) an immunocytokine
comprising an antibody binding site and a cytokine; and, (ii) an
immunocytokine uptake enhancing agent.
19. The composition of claim 18, wherein the antibody binding site
comprises in an amino-terminal to carboxy-terminal direction, an
immunoglobulin variable region, a CH1 domain and a CH2 domain.
20. The composition of claim 19, wherein the antibody binding site
further comprises a CH3 domain attached to the C-terminal end of
the CH2 domain.
21-27. (canceled)
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/896,909, filed Jun. 29, 2001, which claims
priority to, and the benefit of 60/215,038 filed Jun. 29, 2000, the
disclosures of each of which are incorporated by reference
herein.
FIELD OF THE INVENTION
[0002] The present invention relates to antibody-cytokine fusion
proteins useful for targeted immune therapy. In general, the
invention relates to the use of immunocytokine uptake enhancing
agents in combination therapy to enhance an antibody-cytokine
fusion protein mediated immune response against a preselected
target, for example, cells in a tumor. In particular, the invention
relates to the administration of antibody-cytokine fusion proteins
in combination with chemotherapeutics such as taxanes and/or
alkylating agents to treat tumor cells and other cancerous or
diseased cells.
BACKGROUND OF THE INVENTION
[0003] Effective treatment of diseases such as cancer require
robust immune responses by one or more effector cell types such as
natural killer (NK) cells, macrophage and T lymphocytes. In animals
and patients bearing tumors, the immune system has not effectively
dealt with the growing tumor due, in large part, to specific
mechanisms the tumor has elaborated to suppress the immune
response. In many cases, potentially tumor-destructive monocytic
cells, e.g. macrophages, migrate into growing tumor beds, but the
secretion of factors such as prostaglandins, TGF-.beta. and IL-10
by the tumor cells modulate their cytotoxic activity (see, for
example, Sharma et al., 1999, J. IMMUNOL. 163:5020-5028). Likewise,
lymphocytic cells migrating into tumors, such as NK and T cells,
can be suppressed by factors secreted by tumors as well as by
interactions with receptors expressed on the surface of tumor cells
that activate apoptosis of the immune cells (see, for example,
Villunger, et al, 1997, BLOOD 90:12-20). The exposure of these
lymphocytes to immunosuppressive monocytic cells within the tumor
bed can further reduce their ability to mount an effective
anti-tumor response.
[0004] Efforts made to overcome the immune suppressive effects of
the local tumor microenvironment include targeted immune
stimulation, such as treatment with tumor-specific
antibody-cytokine fusion proteins. Effective treatment with this
approach has been demonstrated in several mouse tumor metastasis
models, however, treatment is far less effective as the size of the
tumors increases. This is likely due to the increased level of
suppressive factors secreted by the tumor mass as well as other
factors, such as the increase in tumor interstitial fluid pressure
(Griffon-Etienne et al. 1999, CANCER RES. 59:3776-3782), a barrier
to penetration of solid tumors by therapeutic agents.
[0005] While most cancer patients are still treated with one or
more courses of chemotherapy, it is well known that cytotoxic
therapy of cancer is damaging to the immune system. Immune cells
are among the most rapidly dividing cells in the human body, and
any treatment that kills dividing cells will also kill immune
cells. Thus, treatments including radiation, DNA-damaging
chemicals, inhibitors of DNA synthesis, and inhibitors of
microtubule function all cause damage to the immune system. Bone
marrow transplants are needed as an adjunct to cancer therapy
precisely because the immune system becomes damaged and needs to be
replenished. Methotrexate and other anti-cancer drugs are often
used as immunosuppressants. There is also evidence that anti-cancer
treatments can specifically inhibit T cell function. For example,
patients who have been treated for Hodgkin's disease with
whole-body irradiation suffer from an apparently permanent loss of
naive T cells (Watanabe et al., 1997, Blood 90:3662).
[0006] Based on current knowledge it would appear unlikely that
standard treatments (chemotherapy and radiation) and local immune
stimulation would be a useful combination approach for effective
treatment of cancer. Therefore, there is a need in the art for
methods that enhance antibody-cytokine fusion protein mediated
immune responses against pre-selected cell types, for example,
tumor cells, and compositions employed in such methods.
SUMMARY OF THE INVENTION
[0007] It has been discovered that when an antibody-cytokine fusion
protein (immunocytokine) is administered to a mammal bearing a
tumor or tumor metastases, it is possible to create a more potent
anti-tumor response if it is administered before, simultaneously
with, or after treatment of the mammal with an immunocytokine
uptake enhancing agent that increases or enhances the therapeutic
effect of the antibody-cytokine fusion protein by enhancing or
increasing its uptake by the tumor. It has been found that useful
immunocytokine uptake enhancing agents comprise alkylating
chemotherapeutic agents and taxanes such as paclitaxel. In
particular, it has been found that such combinations are useful in
mediating the immune destruction of the pre-selected cell type,
such as tumor cells or virus-infected cells.
[0008] In one aspect, the invention provides a method of inducing a
cytocidal immune response against a preselected cell-type in a
mammal. The method comprises administering to the mammal (i) an
immunocytokine comprising an antibody binding site capable of
binding the preselected cell-type and a cytokine capable of
inducing such an immune response against the preselected cell-type,
and (ii) an immunocytokine uptake enhancing agent in an amount
sufficient to enhance the immune response relative to the immune
response stimulated by the immunocytokine alone.
[0009] In a preferred embodiment, the preselected cell-type can be
a cancer cell present, for example, in a solid tumor, more
preferably in a larger, solid tumor (i.e., greater than about 100
mm.sup.3). Alternatively, the preselected cell-type can be a cancer
cell present in the form of small metastases.
[0010] In another preferred embodiment, the immunocytokine uptake
enhancing agent can be administered simultaneously with the
immunocytokine. Alternatively, the immunocytokine uptake enhancing
agent can be administered prior to administration of the
immunocytokine. Furthermore, it is contemplated that the
immunocytokine can be administered together with a plurality of
different immunocytokine uptake enhancing agents. Alternatively, it
is contemplated that an immunocytokine uptake enhancing agent can
be administered together with a plurality of different
immunocytokines.
[0011] In another aspect, the invention provides a composition for
inducing a cytocidal immune response against a preselected
cell-type in a mammal. The composition comprises in combination:
(i) an immunocytokine comprising an antibody binding site capable
of binding the preselected cell-type, and a cytokine capable of
inducing such an immune response against the preselected cell-type
in the mammal, and (ii) an immunocytokine uptake enhancing agent in
an amount sufficient to enhance the cytocidal response induced by
the immunocytokine of the combination relative to the cytocidal
response stimulated by the immunocytokine alone.
[0012] In a preferred embodiment, the antibody binding site of the
immunocytokine preferably comprises an immunoglobulin heavy chain
or an antigen binding fragment thereof. The immunoglobulin heavy
chain preferably comprises, in an amino-terminal to
carboxy-terminal direction, an immunoglobulin variable (V.sub.H)
region domain capable of binding a preselected antigen, an
immunoglobulin constant heavy 1 (CH1) domain, an immunoglobulin
constant heavy 2 (CH2) domain, and optionally may further include
an immunoglobulin constant heavy 3 (CH3) domain. In a more
preferred embodiment, the immunocytokine is a fusion protein
comprising an immunoglobulin heavy chain or an antigen binding
fragment thereof fused via a polypeptide bond to the cytokine.
Accordingly, a preferred antibody-cytokine fusion protein
comprises, in an amino-terminal to carboxy-terminal direction, (i)
the antibody binding site comprising an immunoglobulin variable
region capable of binding a cell surface antigen on the preselected
cell-type, an immunoglobulin CH1 domain, an immunoglobulin CH2
domain (optionally a CH3 domain), and (ii) the cytokine. Methods
for making and using such fusion proteins are described in detail
in Gillies et al. (1992) Proc. Natl. Acad. Sci. USA 89: 1428-1432;
Gillies et al. (1998) J. Immunol. 160: 6195-6203; and U.S. Pat. No.
5,650,150.
[0013] The immunoglobulin constant region domains (i.e., the CH1,
CH2 and/or CH3 domains) may be the constant region domains normally
associated with the variable region domain in a naturally occurring
antibody. Alternatively, one or more of the immunoglobulin constant
region domains may be derived from antibodies different from the
antibody used as a source of the variable region domain. In other
words, the immunoglobulin variable and constant region domains may
be derived from different antibodies, for example, antibodies
derived from different species. See, for example, U.S. Pat. No.
4,816,567. Furthermore, the immunoglobulin variable regions may
comprise framework region (FR) sequences derived from one species,
for example, a human, and complementarity determining region (CDR)
sequences interposed between the FRs, derived from a second,
different species, for example, a mouse. Methods for making and
using such chimeric immunoglobulin variable regions are disclosed,
for example, in U.S. Pat. Nos. 5,225,539 and 5,585,089.
[0014] The antibody-based immunocytokines preferably further
comprise an immunoglobulin light chain which preferably is
covalently bonded to the immunoglobulin heavy chain by means of,
for example, a disulfide bond. The variable regions of the linked
immunoglobulin heavy and light chains together define a single and
complete binding site for binding the preselected antigen. In other
embodiments, the immunocytokines comprise two chimeric chains, each
comprising at least a portion of an immunoglobulin heavy chain
fused to a cytokine. The two chimeric chains preferably are
covalently linked together by, for example, one or more interchain
disulfide bonds.
[0015] The invention thus provides fusion proteins in which the
antigen-binding specificity and activity of an antibody is combined
with the potent biological activity of a cytokine. A fusion protein
of the present invention can be used to deliver the cytokine
selectively to a target cell in vivo so that the cytokine can exert
a localized biological effect in the vicinity of the target cell.
In a preferred embodiment, the antibody component of the fusion
protein specifically binds an antigen on or within a cancer cell
and, as a result, the fusion protein exerts localized anti-cancer
activity. In an alternative preferred embodiment, the antibody
component of the fusion protein specifically binds a virus-infected
cell, such as an HIV-infected cell, and, as a result, the fusion
protein exerts localized anti-viral activity.
[0016] Cytokines that can be incorporated into the immunocytokines
of the invention include, for example, tumor necrosis factors,
interleukins, colony stimulating factors, and lymphokines, as well
as others known in the art. Preferred tumor necrosis factors
include, for example, tissue necrosis factor .alpha. (TNF.alpha.).
Preferred interleukins include, for example, interleukin-2 (IL-2),
interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-7 (IL-7),
interleukin-12 (IL-12), interleukin-15 (IL-15) and interleukin-18
(IL-18). Preferred colony stimulating factors include, for example,
granulocyte-macrophage colony stimulating factor (GM-CSF) and
macrophage colony stimulation factor (M-CSF). Preferred lymphokines
include, for example, lymphotoxin (LT). Other useful cytokines
include interferons, including IFN-.alpha., IFN-.beta. and
IFN-.gamma., all of which have immunological effects, as well as
anti-angiogenic effects, that are independent of their anti-viral
activities.
[0017] It has been found that several types of chemotherapeutic
agents are effective immunocytokine uptake enhancing agents. In
particular, useful immunocytokine uptake enhancing agents include
taxanes and alkylating chemotherapeutic agents. Several taxanes are
known in the art (see Bissery and Lavelle, 1997, in Cancer
Therapeutics: Experimental and Clinical Agents, Chapter 8, B.
Teicher, ed.). In a preferred embodiment, the taxane is Taxol, also
known as paclitaxel. Other embodiments include the semisynthetic
taxane, docetaxel, which in some tumor models and clinical
indications is more efficacious than paclitaxel. Further
embodiments include additional taxane derivatives, such as those
derived from the natural starting material, 10-deacetyl Baccatin
III, extracted from the needles of the European Yew tree. One such
example is the orally available compound, IDN5109, which is also a
poor substrate for P-glycoprotein and generally more active against
multidrug resistant tumors. In addition to being orally
bioavailable, it also has a higher tolerated dose and exhibits less
neurotoxic side effects (Polizzi et al., 1999, Cancer Res.
59:1036-1040).
[0018] Also provided are preferred dosages and administration
regimes for administering the immunocytokines in combination with
the immunocytokine uptake enhancing agents.
DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a schematic representation of a cytokine.
[0020] FIG. 2 shows the effect of paclitaxel and an immunocytokine
on LLC/KSA tumor volume over time.
[0021] FIG. 3 shows the effect of multiple doses of paclitaxel and
an immunocytokine on mean tumor volume of time.
[0022] FIG. 4 shows the effect of paclitaxel and an immunocytokine
on tumor weight in a lung metastasis assay.
[0023] FIG. 5 shows the effect of paclitaxel and an immunocytokine
on CT26/KSA tumor volume over time.
[0024] FIG. 6 shows the effect of paclitaxel and an immunocytokine
on tumor weight in a liver metastasis assay.
[0025] FIGS. 7A and 7B show the effect of paclitaxel on
immunocytokine uptake by a tumor.
[0026] FIG. 8 shows the effect of cyclophosphamide on
immunocytokine uptake by a tumor.
[0027] FIG. 9 shows the effect of cyclophosphamide and an
immunocytokine on tumor weight in a lung metastasis assay.
[0028] FIG. 9B shows the effect of cyclophosphamide and an
immunocytokine on tumor volume in tumor growth assay.
[0029] FIG. 9C shows the effect of cyclophosphamide and an
immunocytokine on tumor volume in tumor growth assay.
[0030] FIG. 10 shows the effect of carboplatin and an
immunocytokine on tumor volume in a tumor growth assay.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Studies have shown that large, solid tumors are much more
refractory to antibody-mediated therapeutic intervention, and to
immune therapies in general than are disseminated metastatic foci
(Sulitzeanu et al. (1993) Adv. Cancer Res. 60: 247-267). It is
believed that low responsiveness to antibody-based therapies is
based, in part, upon the production of immunosuppressive factors by
the tumors.
[0032] Although the mechanism for tumor eradication is not
completely understood, it is contemplated that cytotoxic T
lymphocyte (CTL) responses can lead to destruction of cancer cells
and provide immune memory. Furthermore, it is contemplated that
under certain circumstances natural killer (NK) cells are
responsible for tumor eradication in the absence of CTLs. The
different immune responses may result from the fact that certain
tumors produce different types or amounts of substances capable of
down-regulating T cells. This is especially true for solid tumors,
rather than micrometastatic foci, that have reached a critical mass
and are capable of producing and secreting immunosuppressive
factors at levels sufficient to modulate an immune response against
the tumors.
[0033] It has now been discovered that cytocidal immune responses
initiated by an immunocytokine against a preselected cell-type can
be enhanced significantly by administering the immunocytokine
together with an immunocytokine uptake enhancing agent. The
combined therapy is particularly effective in mediating the immune
destruction of a diseased tissue, such as, an established tumor.
Without wishing to be bound by theory, it is contemplated that the
immunocytokine uptake enhancing agent increases the penetration of
the immunocytokine into the tumor microenvironment thus making it
capable of overcoming the immune suppressive effect and more
effective at activating cellular immune responses against the
tumor. Similarly, it is contemplated that such a method may be
useful for the treatment of certain viral diseases where a similar
immune suppressive mechanism prevents effective cellular immunity,
for example, in HIV infection. It is contemplated that the
immunocytokine uptake enhancing agent acts synergistically with the
immunocytokine to mediate the immune destruction of a diseased
tissue such as an established tumor or virally-infected cells. The
present invention also describes methods for making and using
useful immunocytokines, as well as assays useful for testing their
pharmacokinetic activities in pre-clinical in vivo animal models
when combined with suitable immunocytokine uptake enhancing
agents.
[0034] As used herein, the term "immunocytokine uptake enhancing
agent" is understood to mean any agent that enhances a cytocidal
immune response induced by an immunocytokine against a pre-selected
cell type. More specifically, a preferred immunocytokine uptake
enhancing agent is a tumor uptake enhancing agent that increases
the penetration of an immunocytokine into a tumor. Examples of
immunocytokine uptake enhancing agents include, but are not limited
to, chemotherapeutic agents such as taxanes, DNA damaging agents
including alkylating chemotherapeutic agents, radiation therapy
agents, and agents that modulate blood pressure. Preferred taxanes
are taxol, docetaxel, 10-deacetyl Baccatin III, and derivatives
thereof. Preferred alkylating agents are cyclophosphamide,
carboplatin, cisplatin, and derivatives thereof. A preferred form
of radiation is gamma irradiation. A preferred blood pressure
modulating agent is an angiotensin II agonist, such as angiotensin
II itself, preferably administered periodically according to the
general principles described by Netti et al. (Cancer Research
[1995] 55:5451-8) and Netti et al (Proc. Nat. Acad. Sci. [1999]
96:3137-3142). Immune response may be determined by methods known
to one of ordinary skill in the art and/or as described herein.
[0035] As used herein, the term "cytocidal immune response" is
understood to mean any immune response in a mammal, either humoral
or cellular in nature, that is stimulated by an immunocytokine and
which either kills or otherwise reduces the viability of a
preselected cell-type in the mammal. The immune response may
include one or more cell types, including T cells, NK cells and
macrophages.
[0036] As used herein, the term "immunocytokine" is understood to
mean a fusion of (i) an antibody binding site having binding
specificity for, and capable of binding a pre-selected antigen, for
example, a cell-type specific antigen, and (ii) a cytokine that is
capable of inducing or stimulating a cytocidal immune response
typically against a cancer or virally-infected cell. Examples of
pre-selected antigens include cell surface antigens such as on
cancer cells or virally-infected cells, and insoluble intracellular
antigens, for example, of necrotic cells, which can remain attached
to the cell membrane. Preferred antigens are target antigens that
are characteristic of tumor cells, such as tumor specific antigens.
Accordingly, the immunocytokine is capable of selectively
delivering the cytokine to a target (which typically is a cell) in
vivo so that the cytokine can mediate a localized immune response
against a target cell. For example, if the antibody component of
the immunocytokine selectively binds an antigen on a cancer cell,
such as a cancer cell in a solid tumor, and in particular a larger
solid tumor of greater than about 100 mm.sup.3, the immunocytokine
exerts localized anti-cancer activity. Alternatively, if the
antibody component of the immunocytokine selectively binds an
antigen on a virally-infected cell, such as a HIV infected cell,
the immunocytokine exerts localized anti-viral activity.
[0037] As used herein, the term "antibody binding site" is
understood to mean at least a portion of an immunoglobulin heavy
chain, for example, an immunoglobulin variable region capable of
binding a pre-selected antigen such as a cell type. The antibody
binding site also preferably comprises at least a portion of an
immunoglobulin constant region including, for example, a CH1
domain, a CH2 domain, and optionally, a CH3 domain, or at least a
CH2 domain, or one or more portions thereof. Furthermore, the
immunoglobulin heavy chain may be associated, either covalently or
non-covalently, to an immunoglobulin light chain comprising, for
example, an immunoglobulin light chain variable region and
optionally light chain constant region. Accordingly, it is
contemplated that the antibody binding site may comprise an intact
antibody or a fragment thereof, or a single chain antibody, capable
of binding the preselected antigen.
[0038] With regard to the immunocytokine, it is contemplated that
the antibody fragment may be linked to the cytokine by a variety of
ways well known to those of ordinary skill in the art. For example,
the antibody binding site preferably is linked via a polypeptide
bond or linker to the cytokine in a fusion protein construct.
Alternatively, the antibody binding site may be chemically coupled
to the cytokine via reactive groups, for example, sulfhydryl
groups, within amino acid sidechains present within the antibody
binding site and the cytokine.
[0039] As used herein, the term "cytokine" is understood to mean
any protein or peptide, analog or functional fragment thereof,
which is capable of stimulating or inducing a cytocidal immune
response against a preselected cell-type, for example, a cancer
cell or a virally-infected cell, in a mammal. Accordingly, it is
contemplated that a variety of cytokines can be incorporated into
the immunocytokines of the invention. Useful cytokines include, for
example, tumor necrosis factors (TNFs), interleukins (ILs),
lymphokines (Ls), colony stimulating factors (CSFs), interferons
(IFNs) including species variants, truncated analogs thereof which
are capable of stimulating or inducing such cytocidal immune
responses. Useful tumor necrosis factors include, for example, TNF
.alpha.. Useful lymphokines include, for example, LT. Useful colony
stimulating factors include, for example, GM-CSF and M-CSF. Useful
interleukins include, for example, IL-2, IL-4, IL-5, IL-7, IL-12,
IL-15 and IL-18. Useful interferons, include, for example,
IFN-.alpha., IFN-.beta. and IFN-.gamma..
[0040] The gene encoding a particular cytokine of interest can be
cloned de novo, obtained from an available source, or synthesized
by standard DNA synthesis from a known nucleotide sequence. For
example, the DNA sequence of LT is known (see, for example, Nedwin
et al. (1985) NUCLEIC ACIDS RES. 13: 6361), as are the sequences
for IL-2 (see, for example, Taniguchi et al. (1983) NATURE 302:
305-318), GM-CSF (see, for example, Gasson et al. (1984) SCIENCE
266: 1339-1342), and TNF .alpha. (see, for example, Nedwin et al.
(1985) NUCLEIC ACIDS RES. 13: 6361).
[0041] In a preferred embodiment, the immunocytokines are
recombinant fusion proteins produced by conventional recombinant
DNA methodologies, i.e., by forming a nucleic acid construct
encoding the chimeric immunocytokine. The construction of
recombinant antibody-cytokine fusion proteins has been described in
the prior art. See, for example, Gillies et al. (1992) Proc. Natl.
Acad. Sci. USA 89: 1428-1432; Gillies et al. (1998) J. Immunol.
160: 6195-6203; and U.S. Pat. No. 5,650,150. Preferably, a gene
construct encoding the immunocytokine of the invention includes, in
5' to 3' orientation, a DNA segment encoding an immunoglobulin
heavy chain variable region domain, a DNA segment encoding an
immunoglobulin heavy chain constant region, and a DNA encoding the
cytokine. The fused gene is assembled in or inserted into an
expression vector for transfection into an appropriate recipient
cell where the fused gene is expressed. The hybrid polypeptide
chain preferably is combined with an immunoglobulin light chain
such that the immunoglobulin variable region of the heavy chain
(V.sub.H) and the immunoglobulin variable region of the light chain
(V.sub.L) combine to produce a single and complete site for binding
a preselected antigen. In a preferred embodiment, the
immunoglobulin heavy and light chains are covalently coupled, for
example, by means of an interchain disulfide bond. Furthermore, two
immunoglobulin heavy chains, either one or both of which are fused
to a cytokine, can be covalently coupled, for example, by means of
one or more interchain disulfide bonds.
[0042] Accordingly, methods of the invention are useful to enhance
the anti-tumor activity of an immunocytokine used in a therapeutic
method to treat a tumor, including immunocytokine compositions and
methods disclosed in WO99/29732, WO99/43713, WO99/52562,
WO99/53958, and WO01/10912, and antibody-based fusion proteins with
an altered amino acid sequence in the junction region. In one
embodiment, methods of the invention are useful in combination with
Fc fusion proteins such as Fc-interferon-.alpha..
[0043] FIG. 1 shows a schematic representation of an exemplary
immunocytokine 1. In this embodiment, cytokine molecules 2 and 4
are peptide bonded to the carboxy termini 6 and 8 of CH3 regions 10
and 12 of antibody heavy chains 14 and 16. V.sub.L regions 26 and
28 are shown paired with V.sub.H regions 18 and 20 in a typical IgG
configuration, thereby providing two antigen binding sites 30 and
32 at the amino terminal ends of immunocytokine 1 and two cytokine
receptor-binding sites 40 and 42 at the carboxy ends of
immunocytokine 1. Of course, in their broader aspects, the
immunocytokines need not be paired as illustrated or only one of
the two immunoglobulin heavy chains need be fused to a cytokine
molecule.
[0044] Immunocytokines of the invention may be considered chimeric
by virtue of two aspects of their structure. First, the
immunocytokine is chimeric in that it includes an immunoglobulin
heavy chain having antigen binding specificity linked to a given
cytokine. Second, an immunocytokine of the invention may be
chimeric in the sense that it includes an immunoglobulin variable
region (V) and an immunoglobulin constant region (C), both of which
are derived from different antibodies such that the resulting
protein is a V/C chimera. For example, the variable and constant
regions may be derived from naturally occurring antibody molecules
isolatable from different species. See, for example, U.S. Pat. No.
4,816,567. Also embraced are constructs in which either or both of
the immunoglobulin variable regions comprise framework region (FR)
sequences and complementarity determining region (CDR) sequences
derived from different species. Such constructs are disclosed, for
example, in Jones et al. (1986) Nature 321: 522-525, Verhoyen et
al. (1988) SCIENCE 239: 1534-1535, and U.S. Pat. Nos. 5,225,539 and
5,585,089. Furthermore, it is contemplated that the variable region
sequences may be derived by screening libraries, for example, phage
display libraries, for variable region sequences that bind a
preselected antigen with a desired affinity. Methods for making and
screening phage display libraries are disclosed, for example, in
Huse et al. (1989) Science 246: 1275-1281 and Kang et al. (1991)
Proc. Natl. Acad. Sci. USA 88: 11120-11123.
[0045] The immunoglobulin heavy chain constant region domains of
the immunocytokines can be selected from any of the five
immunoglobulin classes referred to as IgA (Ig.alpha.), IgD
(Ig.delta.), IgE (Ig.epsilon.), IgG (Ig.gamma.), and IgM (Ig.mu.).
However, immunoglobulin heavy chain constant regions from the IgG
class are preferred. Furthermore, it is contemplated that the
immunoglobulin heavy chains may be derived from any of the IgG
antibody subclasses referred to in the art as IgG1, IgG2, IgG3 and
IgG4. As is known, each immunoglobulin heavy chain constant region
comprises four or five domains. The domains are named sequentially
as follows: CH1-hinge-CH2--CH3-(--CH4). CH4 is present in IgM,
which has no hinge region. The DNA sequences of the heavy chain
domains have cross homology among the immunoglobulin classes, for
example, the CH2 domain of IgG is homologous to the CH2 domain of
IgA and IgD, and to the CH3 domain of IgM and IgE. The
immunoglobulin light chains can have either a kappa (.kappa.) or
lambda (.lamda.) constant chain. Sequences and sequence alignments
of these immunoglobulin regions are well known in the art (see, for
example, Kabat et al., "Sequences of Proteins of Immunological
Interest," U.S. Department of Health and Human Services, third
edition 1983, fourth edition 1987, and Huck et al. (1986) NUC.
ACIDS RES. 14: 1779-1789).
[0046] In preferred embodiments, the variable region is derived
from an antibody specific for a preselected cell surface antigen
(an antigen associated with a diseased cell such as a cancer cell
or virally-infected cell), and the constant region includes CH1,
and CH2 (and optionally CH3) domains from an antibody that is the
same or different from the antibody that is the source of the
variable region. In the practice of this invention, the antibody
portion of the immunocytokine preferably is non-immunogenic or is
weakly immunogenic in the intended recipient. Accordingly, the
antibody portion, as much as possible, preferably is derived from
the same species as the intended recipient. For example, if the
immunocytokine is to be administered to humans, the constant region
domains preferably are of human origin. See, for example, U.S. Pat.
No. 4,816,567. Furthermore, when the immunoglobulin variable region
is derived from a species other than the intended recipient, for
example, when the variable region sequences are of murine origin
and the intended recipient is a human, then the variable region
preferably comprises human FR sequences with murine CDR sequences
interposed between the FR sequences to produce a chimeric variable
region that has binding specificity for a preselected antigen but
yet while minimizing immunoreactivity in the intended host. The
design and synthesis of such chimeric variable regions are
disclosed in Jones et al. (1986) Nature 321: 522-525, Verhoyen et
al. (1988) SCIENCE 239: 1534-1535, and U.S. Pat. Nos. 5,225,539 and
5,585,089. The cloning and expression of a humanized
antibody-cytokine fusion protein, KS-1/4 anti-EpCAM antibody-IL-12
fusion protein, as well as its ability to eradicate established
colon carcinoma metastases has been described in Gillies et al.
(1998) J. Immunol. 160: 6195-6203.
[0047] The gene encoding the cytokine is joined, either directly or
by means of a linker, for example, by means of DNA encoding a
(Gly.sub.4-Ser).sub.3 linker in frame to the 3' end of the gene
encoding the immunoglobulin constant region (e.g., a CH2 or CH3
exon). In certain embodiments, the linker can comprise a nucleotide
sequence encoding a proteolytic cleavage site. This site, when
interposed between the immunoglobulin constant region and the
cytokine, can be designed to provide for proteolytic release of the
cytokine at the target site. For example, it is well known that
plasmin and trypsin cleave after lysine and arginine residues at
sites that are accessible to the proteases. Many other
site-specific endoproteases and the amino acid sequences they
cleave are well-known in the art. Preferred proteolytic cleavage
sites and proteolytic enzymes that are reactive with such cleavage
sites are disclosed in U.S. Pat. Nos. 5,541,087 and 5,726,044.
[0048] The nucleic acid construct optionally can include the
endogenous promoter and enhancer for the variable region-encoding
gene to regulate expression of the chimeric immunoglobulin chain.
For example, the variable region encoding genes can be obtained as
DNA fragments comprising the leader peptide, the VJ gene
(functionally rearranged variable (V) regions with joining (J)
segment) for the light chain, or VDJ gene for the heavy chain, and
the endogenous promoter and enhancer for these genes.
Alternatively, the gene encoding the variable region can be
obtained apart from endogenous regulatory elements and used in an
expression vector which provides these elements.
[0049] Variable region genes can be obtained by standard DNA
cloning procedures from cells that produce the desired antibody.
Screening of the genomic library for a specific functionally
rearranged variable region can be accomplished with the use of
appropriate DNA probes such as DNA segments containing the J region
DNA sequence and sequences downstream. Identification and
confirmation of correct clones is achieved by sequencing the cloned
genes and comparison of the sequence to the corresponding sequence
of the full length, properly spliced mRNA.
[0050] The target antigen can be a cell surface antigen of a tumor
or cancer cell, a virus-infected cell or another diseased cell. The
target antigen may also be an insoluble intracellular antigen of a
necrotic cell. (see, for example, U.S. Pat. No. 5,019,368) Genes
encoding appropriate variable regions can be obtained generally
from immunoglobulin-producing lymphoid cell lines, For example,
hybridoma cell lines producing immunoglobulin specific for tumor
associated antigens or viral antigens can be produced by standard
somatic cell hybridization techniques well known in the art (see,
for example. U.S. Pat. No. 4,196,265). These immunoglobulin
producing cell lines provide the source of variable region genes in
functionally rearranged form. The variable region genes typically
will be of murine origin because this murine system lends itself to
the production of a wide variety of immunoglobulins of desired
specificity. Furthermore, variable region sequences may be derived
by screening libraries, for example, phage display libraries, for
variable region sequences that bind a preselected antigen with a
desired affinity. Methods for making and screening phage display
libraries are disclosed, for example, in Huse et al. (1989) Science
246: 1275-1281 and Kang et al. (1991) Proc. Natl. Acad. Sci. USA
88: 11120-11123.
[0051] The DNA fragment encoding the functionally active variable
region gene is linked to a DNA fragment containing the gene
encoding the desired constant region (or a portion thereof).
Immunoglobulin constant regions (heavy and light chain) can be
obtained from antibody-producing cells by standard gene cloning
techniques. Genes for the two classes of human light chains
(.kappa. and .lamda.) and the five classes of human heavy chains
(.alpha., .delta., .epsilon., .gamma. and .mu.) have been cloned,
and thus, constant regions of human origin are readily available
from these clones.
[0052] The fused gene encoding the hybrid immunoglobulin heavy
chain is assembled or inserted into an expression vector for
incorporation into a recipient cell. The introduction of the gene
construct into plasmid vectors can be accomplished by standard gene
splicing procedures. The chimeric immunoglobulin heavy chain can be
co-expressed in the same cell with a corresponding immunoglobulin
light chain so that a complete immunoglobulin can be expressed and
assembled simultaneously. For this purpose, the heavy and light
chain constructs can be placed in the same or separate vectors.
[0053] Recipient cell lines are generally lymphoid cells. The
preferred recipient cell is a myeloma (or hybridoma). Myelomas can
synthesize, assemble, and secrete immunoglobulins encoded by
transfected genes and they can glycosylate proteins. Particularly
preferred recipient or host cells include Sp2/0 myeloma which
normally does not produce endogenous immunoglobulin, and mouse
myeloma NS/0 cells. When transfected, the cell produces only
immunoglobulin encoded by the transfected gene constructs.
Transfected myelomas can be grown in culture or in the peritoneum
of mice where secreted immunocytokine can be recovered from ascites
fluid. Other lymphoid cells such as B lymphocytes can be used as
recipient cells.
[0054] There are several methods for transfecting lymphoid cells
with vectors containing the nucleic acid constructs encoding the
chimeric immunoglobulin chain. For example, vectors may be
introduced into lymphoid cells by spheroblast fusion (see, for
example, Gillies et al. (1989) BIOTECHNOL. 7: 798-804). Other
useful methods include electroporation or calcium phosphate
precipitation (see, for example, Sambrook et al. eds (1989)
"Molecular Cloning: A Laboratory Manual," Cold Spring Harbor
Press).
[0055] Other useful methods of producing the immunocytokines
include the preparation of an RNA sequence encoding the construct
and its translation in an appropriate in vivo or in vitro
expression system. It is contemplated that the recombinant DNA
methodologies for synthesizing genes encoding antibody-cytokine
fusion proteins, for introducing the genes into host cells, for
expressing the genes in the host, and for harvesting the resulting
fusion protein are well known and thoroughly documented in the art.
Specific protocols are described, for example, in Sambrook et al.
eds (1989) "Molecular Cloning: A Laboratory Manual," Cold Spring
Harbor Press.
[0056] It is understood that the chemically coupled immunocytokines
may be produced using a variety of methods well known to those
skilled in the art. For example, the antibody or an antibody
fragment may be chemically coupled to the cytokine using chemically
reactive amino acid side chains in the antibody or antibody
fragment and the cytokine. The amino acid side chains may be
covalently linked, for example, via disulfide bonds, or by means of
homo- or hetero-bifunctional crosslinking reagents including, for
example, N-succinimidyl 3(-2-pyridyylditio)propionate,
m-maleimidobenzoyl-N-hydroxysuccinate ester,
m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, and
1,4-di-[3'(2'-pyridylthio)propionamido]butane, all of which are
available commercially from Pierce, Rockford, Ill.
[0057] According to methods of the invention, the combination of
immunocytokines with immunocytokine uptake enhancing agents is
useful for enhanced stimulation of the immune system, thereby
resulting in a cytotoxic response at the site of the targeted cell
type, for example, tumor or other disease cells. A combination of
an immunocytokine and an immunocytokine uptake enhancing agent
would be expected to have no combined or synergistic anti-tumor
effect in vitro since the immunocytokine alone is
non-cytotoxic.
[0058] Without wishing to be bound by any particular theory, it is
believed that the effects of combined therapy in vivo may include
enhanced uptake of one of the agents by the action of the other
resulting in either or both (1) increased chemotherapeutic
cytotoxicity (if the immunocytokine increased the uptake of the
chemotherapeutic immunocytokine uptake enhancing agent into tumor
cells); and/or (2) increased immune stimulation (if the
immunocytokine uptake enhancing agent in some way increased uptake
of the immunocytokine into the tumor). With respect to mechanism
(1), earlier studies have shown that it is possible to increase the
uptake of radiolabeled antibodies (and presumably, small molecule
drugs) into tumors by prior treatment with high doses of an
antibody-IL2 immunoconjugate that induces a local vascular leak
(see for example, Hornick et al., 1999, CLIN CANCER RES 5:51-60).
If this particular mechanism is operative in the combination
therapy of immunocytokines and immunocytokine uptake enhancing
agents, it would be necessary to first treat the tumor-bearing
animal with the immunocytokine. However, if a single dose of an
immunocytokine uptake enhancing agent given prior to treatment with
an immunocytokine resulted in a synergistic effect on anti-tumor
activity, then such a mechanism could not be operative. Rather, a
more likely explanation would be that treatment with an
immunocytokine uptake enhancing agent increased the uptake of the
immunocytokine by mechanism (2). This hypothesis could be further
supported by demonstrating that co-administration with an
immunocytokine uptake enhancing agent increases the uptake of a
radiolabeled immunocytokine into a solid tumor.
[0059] According to methods of the invention, an advantage of the
combination therapy is that the administration of an immunocytokine
enhances the cytotoxic effect of a chemotherapeutic agent that acts
as immunocytokine uptake enhancing agent. Therefore, a lower dosage
of the chemotherapeutic agent may be administered to a patient.
Accordingly, the suppression of some aspects of a patient's immune
system, often associated with treatment using a chemotherapeutic
agent, is reduced. In one embodiment of the invention, a single
dose of chemotherapeutic immunocytokine uptake enhancing agent is
administered to a patient before an immunocytokine is administered.
The chemotherapeutic immunocytokine uptake enhancing agent is
administered preferably between about 4 days and about 4 hours, and
most preferably about 24-48 hours, before the immunocytokine. In
another embodiment of the invention several doses of the
chemotherapeutic immunocytokine uptake enhancing agent are
administered to a patient before the immunocytokine is
administered. In further embodiments of the invention, the
chemotherapeutic immunocytokine uptake enhancing agent may be
administered before, at the same time, and/or after the
immunocytokine.
[0060] Paclitaxel is an example of a chemotherapeutic
immunocytokine uptake enhancing agent that can suppress or
compromise aspects of a patient's immune system. While most immune
potentiating effects of paclitaxel are mediated through
macrophage/monocyte cells, many studies on lymphocyte function
indicate a detrimental effect of paclitaxel on this subset. For
example, paclitaxel treatment was found to severely compromise the
proliferative capacity of lymphocytes in both normal and
tumor-bearing mice (Mullins et al., 1998, IMMUNOPHARMACOL
IMMUNOTOXICOL 20:473-492), and to impair both the cytotoxicity of
NK cells and the generation of lymphokine-activated cytotoxicity in
cell cultures containing IL-2 (Chuang et al., 1993, GYNECOL ONCOL
49:291-298). In fact, the available evidence points to the
lymphocyte subset of cells as the essential effector population in
the anti-tumor activity of immunocytokines (Lode et al, 1998,
PHARMACOL THER 80:277-292. Experimental evidence contained within
the present invention has revealed several novel findings that
would not have been predicted by the prior art, especially with
respect to the order of drug administration.
[0061] Taxanes may be co-administered simultaneously with the
immunocytokine, or administered separately by different routes of
administration. Compositions of the present invention may be
administered by any route that is compatible with the particular
molecules. Thus, as appropriate, administration may be oral or
parenteral, including intravenous and intraperitoneal routes of
administration.
[0062] The compositions of the present invention may be provided to
an animal by any suitable means, directly (e.g., locally, as by
injection, implantation or topical administration to a tissue
locus) or systemically (e.g., parenterally or orally). Where the
composition is to be provided parenterally, such as by intravenous,
subcutaneous, ophthalmic, intraperitoneal, intramuscular, buccal,
rectal, vaginal, intraorbital, intracerebral, intracranial,
intraspinal, intraventricular, intrathecal, intracisternal,
intracapsular, intranasal or by aerosol administration, the
composition preferably comprises part of an aqueous or
physiologically compatible fluid suspension or solution. Thus, the
carrier or vehicle is physiologically acceptable so that in
addition to delivery of the desired composition to the patient, it
does not otherwise adversely affect the patient's electrolyte
and/or volume balance. The fluid medium for the agent thus can
comprise normal physiologic saline (e.g., 9.85% aqueous NaCl, 0.15
M, pH 7-7.4). For many taxanes, the formulations are generally more
complex, due to their generally unfavorable solubility properties.
For example, the standard formulation for paclitaxel is 10%
Cremophor, 10% ethanol, and 80% saline (0.9% NaCl), while the
formulation for docetaxel is a 1:1 ethanol:polysorbate 80 solution
that is diluted 1:10 into 5% glucose solution prior to
administration (Bissery and Lavelle, 1999). However, other
formulations including taxanes and newly synthesized analogs will
be recognized and/or routinely developed by those skilled in the
art.
[0063] Preferred dosages of the immunocytokine per administration
are within the range of 0.1 mg/m.sup.2-100 mg/m.sup.2, more
preferably, 1 mg/m.sup.2-20 mg/m.sup.2, and most preferably 2
mg/m.sup.2-6 mg/m.sup.2. Preferred dosages of the immunocytokine
uptake enhancing agent will depend generally upon the type of
immunocytokine uptake enhancing agent used, however, optimal
dosages may be determined using routine experimentation.
Administration of the immunocytokine and/or the immunocytokine
uptake enhancing agent may be by periodic bolus injections, or by
continuous intravenous or intraperitoneal administration from an
external reservoir (for example, from an intravenous bag) or
internal (for example, from a bioerodable implant). Furthermore, it
is contemplated that the immunocytokine of the invention may also
be administered to the intended recipient together with a plurality
of different immunocytokine uptake enhancing agents. It is
contemplated, however, that the optimal combination of
immunocytokines and immunocytokine uptake enhancing agents, modes
of administration, dosages may be determined by routine
experimentation well within the level of skill in the art.
[0064] A variety of methods can be employed to assess the efficacy
of combined therapy using antibody-cytokine fusion proteins and
immunocytokine uptake enhancing agents on immune responses. For
example, the animal model described in the examples below, or other
suitable animal models, can be used by a skilled artisan to test
which immunocytokine uptake enhancing agents, or combinations of
immunocytokine uptake enhancing agents, are most effective in
acting synergistically with an immunocytokine (for example, an
antibody-IL2 fusion protein) to enhance the immune destruction of
established tumors. The immunocytokine uptake enhancing agent, or
combination of immunocytokine uptake enhancing agents, can be
administered prior to, or simultaneously with, the course of
immunocytokine therapy and the effect on the tumor can be
conveniently monitored by volumetric measurement. Further, as novel
immunocytokine uptake enhancing agents are identified, a skilled
artisan will be able to use the methods described herein to assess
the potential of these novel compounds to enhance or otherwise
modify the anti-cancer activity of antibody-cytokine fusion
proteins.
[0065] Alternatively, following therapy, tumors can be excised,
sectioned and stained via standard histological methods, or via
specific immuno-histological reagents in order to assess the effect
of the combined therapy on the immune response. For example, simple
staining with hematoxolin and eosin can reveal differences in
lymphocytic infiltration into the solid tumors which is indicative
of a cellular immune response. Furthermore, immunostaining of
sections with antibodies to specific classes of immune cells can
reveal the nature of an induced response. For example, antibodies
that bind to CD45 (a general leukocyte marker), CD4 and CD8 (for T
cell subclass identification), and NK1.1 (a marker on NK cells) can
be used to assess the type of immune response that has been
mediated by the immunocytokines of the invention.
[0066] Alternatively, the type of immune response mediated by the
immunocytokines can be assessed by conventional cell subset
depletion studies described, for example, in Lode et al. (1998)
Blood 91: 1706-1715. Examples of depleting antibodies include those
that react with T cell markers CD4 and CD8, as well as those that
bind the NK markers NK1.1 and asialo GM. Briefly, these antibodies
are injected to the mammal prior to initiating antibody-cytokine
treatment at fairly high doses (for example, at a dose of about 0.5
mg/mouse), and are given at weekly intervals thereafter until the
completion of the experiment. This technique can identify the
cell-types necessary to elicit the observed immune response in the
mammal.
[0067] In another approach, the cytotoxic activity of splenocytes
isolated from animals having been treated with the combination
therapy can be compared with those from the other treatment groups.
Splenocyte cultures are prepared by mechanical mincing of
recovered, sterile spleens by standard techniques found in most
immunology laboratory manuals. See, for example, Coligan et al.
(eds) (1988) "Current Protocols in Immunology," John Wiley &
Sons, Inc. The resulting cells then are cultured in a suitable cell
culture medium (for example, DMEM from GIBCO) containing serum,
antibiotics and a low concentration of IL-2 (.about.10 U/mL). For
example, in order to compare NK activity, 3 days of culture
normally is optimal, whereas, in order to compare T cell cytotoxic
activity, 5 days of culture normally is optimal. Cytotoxic activity
can be measured by radioactively labeling tumor target cells (for
example, LLC cells) with .sup.51Cr for 30 min. Following removal of
excess radiolabel, the labeled cells are mixed with varying
concentrations of cultured spleen cells for 4 hr. At the end of the
incubation, the .sup.51Cr released from the cells is measured by a
gamma counter which is then used to quantitate the extent of cell
lysis induced by the immune cells. Traditional cytotoxic T
lymphocyte (or CTL) activity is measured in this way.
[0068] The invention is illustrated further by the following
non-limiting examples.
Example 1
Animal Models
[0069] Murine cancer models were developed to study the effect of
combining immunocytokines and taxanes in mediating effective
cytotoxic responses against a tumor. The immunocytokines used in
the following examples bind EpCAM, a human tumor antigen found on
most epithelial derived tumors. (see, Perez and Walker (1989) J.
Immunol. 142: 3662-3667). In order to test the efficacy in an
immuno-competent murine model, it was necessary to express the
human antigen on the surface of a mouse tumor cell that is
syngeneic with the mouse host. Lewis lung carcinoma (LLC) cells, a
well known mouse lung cancer cell line, was the first cell line
selected for this purpose. This cell line is known to produce high
levels of inhibitors of the immune system and to induce IL-10
production from immune cells in the tumor microenvironment leading
to localized immune suppression (Sharma et al., 1999, J IMMUNOL
163:5020-5028). The human tumor antigen, EpCAM (also referred to as
KSA), was expressed on the surface of LLC cells so that it could be
targeted in vivo with immunocytokines derived from the mouse
anti-EpCAM antibody, KS-1/4. This was accomplished by transducing
the EpCAM cDNA sequence with a recombinant retroviral vector as
described (Gillies, U.S. patent application Ser. No. 09/293,042)
resulting in a cell line designated LLC/KSA. These cells were
maintained in DMEM, supplemented with 10% heat inactivated fetal
bovine serum, L-glutamine, penicillin/streptomycin and Geneticin
(GIBCO) at 37.degree. C. and 7.0% CO.sub.2.
[0070] Additional cell lines representing carcinoma of different
tissue origins were engineered in a similar manner. 4T1, a
non-immunogenic murine mammary carcinoma cell line, was provided by
Dr. Paul Sondel (Univ. of Wisconsin). This line grows slowly and
progressively after subcutaneous implantation and spontaneously
metastasizes to many organs even prior to surgical removal of the
primary tumor. It is also possible to induce experimental
metastases in the lung by intravenous injection. CT26, a murine
colon carcinoma cell line, derived by intrarectal injection of
N-nitroso-N-methylurethane in BALB/C mice, was provided by Dr. I.
J. Fidler (MD Anderson Cancer Center, Houston, Tex.). 4T1 and CT26
cells were transfected with Ep-CAM as described (Gillies et al.,
1998, J IMMUNOL 160:6195-6203). 4T1/KSA cells were maintained in
RPMI, supplemented with 10% heat inactivated fetal bovine serum,
L-glutamine, penicillin/streptomycin and Geneticin (GIBCO) at
37.degree. C. and 7.0% CO.sub.2. CT26/KSA cells were maintained in
DMEM, supplemented with 10% heat inactivated fetal bovine serum,
L-glutamine, vitamins, sodium pyruvate, non-essential amino acids,
penicillin/streptomycin and Geneticin (GIBCO, Gaithersberg, Md.) at
37.degree. C. and 7.0% CO.sub.2. Geneticin was added to the
transfected cells to maintain KSA expression. All of the
transfected cell lines grow progressively as skin tumors (after
subcutaneous injection) or as metastases (after intravenous
injection) and kill the mice, despite their expression of the human
EpCAM molecule (a potential foreign antigen) on their cell
surface.
[0071] For tumor growth studies either LLC/KSA or CT26/KSA tumors
were implanted subcutaneously on the backs of mice. For LLC/KSA
studies, tumors were transplanted from several stock tumors that
had been injected with a single cell suspension of 1.times.10.sup.6
cells in 100 ul of PBS. After about two weeks, tumors were
aseptically collected, passed through a sieve fitted with a 150
.mu.m screen. Cells were then passed through a syringe and 23 gauge
needle tow or three times, washed twice, and resuspended in PBS. A
single cell suspension of 1.times.10.sup.6 LLC/KSA cells in 100 ul
of PBS was injected subcutaneously using a 301/2 gauge needle on
the backs of mice. For CT26/KSA studies, cells growing
exponentially in culture were injected as a single cell suspension
of 1.times.10.sup.6 cells in 100 .mu.l of PBS. After tumors had
become established, about 2 weeks after implantation, dosing was
initiated on Day 0. Tumors were measured with calipers in three
dimensions twice weekly. Tumor volumes were calculated using the
equation:
Volume=1/2.times.4/3.pi.(L/2.times.W/2.times.H)
where L=length, W=width and H=height of the tumor. Animals were
weighed and general health was monitored during the course of the
study. When tumors became necrotic or if animals became moribund,
the animals were euthanized by CO.sub.2 asphyxiation.
[0072] Data are presented in graphic form. Graphs depict individual
or average tumor volumes (+/-SEM) during and after dosing. Data are
also expressed as the percent of control of average tumor volumes
from treated mice relative to vehicle treated mice. Student's t
test was performed on the individual tumor volumes to determine
significant differences.
[0073] For experimental hepatic metastases studies, mice were
anesthetized using 80 mg/kg ketamine HCL (Fort Dodge Animal Health,
Fort Dodge, Iowa) and 5 mg/kg xylazine (Bayer, Shawnee Mission,
Kans.). A single cell suspension of 1.times.10.sup.5 CT26/KSA cells
in 100 .mu.l of DMEM containing 25 mM HEPES (GIBCO) was injected
using a 271/2 gauge needle beneath the splenic capsule over a
period of 60 seconds on Day 0. After another 2 minutes the splenic
vessels were cauterized with a cautery unit (Roboz, Rockville, Md.)
and the spleen removed. Animals were sutured using autoclips. Three
weeks after inoculation the animals were sacrificed; their livers
were removed and weighed. The livers were then fixed and stained in
Bouin's solution (Sigma, St. Louis Mo.).
[0074] Data are presented in graphic form. Graphs depict average
tumor burdens (+/-SEM) at the time of sacrifice. Tumor burdens were
determined by subtracting the weight of a normal liver from the
weight of the experimental livers. Data are also expressed as the
percent of control of the average tumor burden from treated mice
relative to vehicle treated mice. Student's t test was performed on
the individual tumor burdens to determine significant
differences.
[0075] For experimental lung metastases studies, a single cell
suspension of 2.5.times.10.sup.5 4T1/KSA cells in 100 .mu.l of PBS
was slowly injected using a 271/2 gauge needle into the lateral
tail vein on Day 0. About 3 weeks-after inoculation animals were
sacrificed; their lungs were removed and weighed. The lungs were
then fixed and stained in Bouin's solution (Sigma). Data are
presented in graphic form. Graphs depict average tumor burdens
(+/-SEM) at time of sacrifice. Tumor burden was determined by
subtracting the weight of a normal lung from the weight of the
experimental lungs. Data are also expressed as the percent of
control of average tumor burden from treated mice relative to
vehicle treated mice. Student's t test was performed on the
individual tumor burdens to determine significant differences.
Example 2
Preparation of Antibody-Fusion Proteins (Immunocytokines)
[0076] Several antibody-cytokine fusion proteins are discussed in
the following examples.
[0077] huKS-hu.gamma.l-huIL2 (Abbreviated, KS-IL2)
[0078] A gene encoding huKS-hu.gamma.1-huIL2 fusion protein was
prepared and expressed essentially as described in Gillies et al.
(1998) J. Immunol. 160: 6195-6203 and U.S. Pat. No. 5,650,150.
Briefly, humanized variable regions of the mouse KS1/4 antibody
(Varki et al., (1984) Cancer Res. 44: 681-687) were modeled using
the methods disclosed in Jones et al. (1986) Nature 321: 522-525,
which involved the insertion of the CDRs of each KS1/4 variable
region into the consensus framework sequences of the human variable
regions with the highest degree of homology. Molecular modeling
with a Silicon Graphics Indigo work station implementing BioSym
software confirmed that the shapes of the CDRs were maintained. The
protein sequences then were reverse translated, and genes
constructed by the ligation of overlapping oligonucleotides.
[0079] The resulting variable regions were inserted into an
expression vector containing the constant regions of the human
.kappa. light chain and the human C.gamma.1 heavy chain essentially
as described in Gillies et al. (1992) Proc. Natl. Acad. Sci. USA
89: 1428-1432, except that the metallothionein promoters and
immunoglobulin heavy chain enhancers were replaced by the CMV
promoter/enhancer for the expression of both chains. Fusions of the
mature sequences of IL-2 to the carboxy terminus of the human heavy
chains were prepared as described in Gillies et al. (1992) Proc.
Natl. Acad. Sci. USA 89:1428-1432, except that the 3' untranslated
regions of the IL-2 gene was derived from the SV40 poly(A)
region.
[0080] The IL-2 fusion protein was expressed by transfection of the
resulting plasmid into NS/0 myeloma cell line with selection medium
containing 0.1 .mu.M methotrexate (MTX). Briefly, in order to
obtain stably transfected clones, plasmid DNA was introduced into
the mouse myeloma NS/0 cells by electroporation. NS/0 cells were
grown in Dulbecco's modified Eagle's medium supplemented with 10%
fetal bovine serum. About 5.times.10.sup.6 cells were washed once
with PBS and resuspended in 0.5 mL PBS. Ten .mu.g of linearized
plasmid DNA then was incubated with the cells in a Gene Pulser
Cuvette (0.4 cm electrode gap, BioRad) on ice for 10 min.
Electroporation was performed using a Gene Pulser (BioRad,
Hercules, Calif.) with settings at 0.25 V and 500 .mu.F. Cells were
allowed to recover for 10 min. on ice, after which they were
resuspended in growth medium and then plated onto two 96 well
plates. Stably transfected clones were selected by growth in the
presence of 100 nM methotrexate, which was introduced two days
post-transfection. The cells were fed every 3 days for three more
times, and MTX-resistant clones appeared in 2 to 3 weeks.
[0081] Expressing clones were identified by Fc or cytokine ELISA
using the appropriate antibodies (see, for example, Gillies et al.
(1989) Biotechnol. 7: 798-804). The resulting fusion protein was
purified by binding, and elution from protein A Sepharose
(Pharmacia), in accordance with the manufacturer's
instructions.
huKS-hu.gamma.4-huIL2
[0082] A gene encoding the huKS-hu.gamma.4-huIL2 fusion protein was
constructed and expressed essentially as described in U.S. Ser. No.
09/256,156, filed Feb. 24, 1999, which claims priority to U.S. Ser.
No. 60/075,887, filed Feb. 25, 1998.
[0083] Briefly, an Ig.gamma.4 version of the huKS-hu.gamma.1-huIL2
fusion protein, described above, was prepared by removing the
immunoglobulin constant region C.gamma.1 gene fragment from the
huKS-hu.gamma.1-huIL2 expression vector and replacing it with the
corresponding sequence from the human C.gamma.4 gene. Sequences and
sequence alignments of the human heavy chain constant regions
C.gamma.1, C.gamma.2, C.gamma.3, and C.gamma.4 are disclosed in
Huck et al. (1986) NUC. ACIDS RES. 14: 1779-1789.
[0084] The swapping of the C.gamma.1 and C.gamma.4 fragments was
accomplished by digesting the original C.gamma.1-containing plasmid
DNA with Hind III and Xho I and purifying a large 7.8 kb fragment
by agarose gel electrophoresis. A second plasmid DNA containing the
Cy4 gene was digested with Hind III and Nsi I and a 1.75 kb
fragment was purified. A third plasmid containing the human IL-2
cDNA and SV40 polyA site, fused to the carboxyl terminus of the
human C.gamma.1 gene, was digested with Xho I and Nsi I and the
small 470 by fragment was purified. All three fragments were
ligated together in roughly equal molar amounts. The ligation
product was used to transform competent E. coli and colonies were
selected by growth on plates containing ampicillin. Correctly
assembled recombinant plasmids were identified by restriction
analyses of plasmid DNA preparations from isolated transformants
and digestion with Fsp I was used to discriminate between the
C.gamma.1 (no Fsp I) and Cy4 (one site) gene inserts.
[0085] The final vector, containing the C.gamma.4-IL2 heavy chain
replacement, was introduced into NS/0 mouse myeloma cells by
electroporation (0.25 V and 500 .mu.F) and transfectants were
selected by growth in medium containing methotrexate (0.1 .mu.M).
Cell clones expressing high levels of the huKS-hu.gamma.4-huIL2
fusion protein were identified, expanded, and the fusion protein
purified from culture supernatants using protein A Sepharose
chromatography. The purity and integrity of the Cy4 fusion protein
was determined by SDS-polyacrylamide gel electrophoresis. IL-2
activity was measured in a T-cell proliferation assay (Gillis et
al. (1978) J. Immunol. 120: 2027-2032) and was found to be
identical to that of the .gamma.1-construct.
huKS-mu.gamma.2a-muIL2
[0086] A gene encoding the huKS-mu.gamma.2a-muIL2 fusion protein
was constructed by replacing the human antibody constant regions
and human IL-2 of the huKS-hu.gamma.1-huIL2 fusion protein, as
described above, with the corresponding murine sequences.
Specifically, the human C.gamma.1-IL2 DNA was replaced with a
murine C.gamma.2a cDNA fragment fused to a DNA encoding murine
IL-2. Briefly, the V.sub.H region of the huKS was joined in frame
to the murine .gamma.2a cDNA by performing overlapping PCR using
overlapping oligonucleotide primers:
TABLE-US-00001 (SEQ ID NO: 1) (sense) 5' CC GTC TCC TCA GCC AAA ACA
ACA GCC CCA TCG GTC; (SEQ ID NO: 2) (antisense) 5' GG GGC TGT TGT
TTT GGC TGA GGA GAC GGT GAC TGA CG; (SEQ ID NO: 3) (sense) 5' C TTA
AGC CAG ATC CAG TTG GTG CAG; and (SEQ ID NO: 4) (antisense) 5' CC
CGG GGT CCG GGA GAA GCT CTT AGT C.
[0087] The oligonucleotides of SEQ ID NOS: 1 and 2 were designed to
hybridize to the junction of the V.sub.H domain of huKS and the
constant region of murine .gamma.2a cDNA (in italics). In the first
round of PCR, there were two separate reactions. In one reaction,
the V.sub.H of huKS DNA was used as the template with the
oligonucleotides of SEQ ID NOS: 2 and 3. The primer of SEQ ID NO: 3
introduced an AflII (CTTAAG) restriction site upstream of the
sequence encoding the mature amino terminus of huKS V.sub.H (in
bold). In another reaction, murine .gamma.2a cDNA was used as the
template with the oligonucleotides SEQ ID NOS: 1 and 4. The primer
of SEQ ID NO: 4 hybridized to the cDNA encoding the region around
the C-terminus of .gamma.2a and introduced a XmaI (CCCGGG)
restriction site for subsequent ligation to the muIL2 cDNA. PCR
products from the two reactions were mixed and subjected to a
second round of PCR, using the oligonucleotides of SEQ ID NOS: 3
and 4. The resulting PCR product was cloned, and upon sequence
verification, the AflII-XmaI fragment encoding the V.sub.H of huKS
and the murine .gamma.2a constant region was used for ligation to
the DNA encoding the signal peptide at the AflII site and the muIL2
cDNA at the XmaI site.
[0088] The murine IL2 cDNA was cloned from mRNA of murine
peripheral blood mononuclear cells using the oligonucleotides set
forth in SEQ ID NOS: 5 and 6, namely:
TABLE-US-00002 (SEQ ID NO: 5) (sense) 5' GGC CCG GGT AAA GCA CCC
ACT TCA AGC TCC; and (SEQ ID NO: 6) (antisense) 5'
CCCTCGAGTTATTGAGGGCTTGTTG.
[0089] The primer of SEQ ID NO: 5 adapted the muIL2 (sequence in
bold) to be joined to mu .gamma.2a at the XmaI restriction site
(CCCGGG). The primer of SEQ ID NO: 6 introduced an XhoI restriction
site (CTCGAG) immediately after the translation termination codon
(antisense in bold).
[0090] Similarly, the variable light (V.sub.L) domain of huKS was
joined to the mu .kappa. cDNA sequence by overlapping PCR. The
overlapping oligonucleotides used included
TABLE-US-00003 (SEQ ID NO: 7) (sense) 5' G GAA ATA AAA CGG GCT GAT
GCT GCA CCA ACT G; (SEQ ID NO: 8) (antisense) 5' GC AGC ATC AGC
CCGTT TTA TTT CCA GCT TGG TCC; (SEQ ID NO: 9) (sense) 5' C TTA AGC
GAG ATC GTG CTG ACC CAG; and (SEQ ID NO: 10) (antisense) 5' CTC GAG
CTA ACA CTC ATT CCT GTT GAA GC.
[0091] The oligonucleotides were designed to hybridize to the
junction of the V.sub.L of huKS and the constant region of murine
.kappa. cDNA (in italics). In the first round of PCR, there were
two separate reactions. In one reaction, the V.sub.L of huKS DNA
was used as template, with the oligonucleotides set forth in SEQ ID
NOS: 8 and 9, which introduced an AflII (CTTAAG) restriction site
upstream of the sequence encoding the mature amino terminus of huKS
V.sub.L (in bold). In the other reaction, murine .kappa. cDNA was
used as template, with the oligonucleotides set forth in SEQ ID
NOS: 7 and 10, which introduced an XhoI restriction site after the
translation termination codon (antisense in bold).
[0092] PCR products from the two reactions were mixed and subjected
to a second round of PCR using the oligonucleotide primers set
forth in SEQ ID NOS: 9 and 10. The resultant PCR product was
cloned, and upon sequence verification, the AflII-XhoI fragment
encoding the V.sub.L of huKS and the murine .kappa. constant region
was ligated to the DNA encoding the signal peptide at the AflII
site.
[0093] Both the murine heavy and light chain sequences were used to
replace the human sequences in pdHL7. The resulting antibody
expression vector, containing a dhfr selectable marker gene, was
electroporated (6.25 V, 500 .mu.F) into murine NS/0 myeloma cells
and clones selected by culturing in medium containing 0.1 .mu.M
methotrexate. Transfected clones, resistant to methotrexate, were
tested for secretion of antibody determinants by standard ELISA
methods. The fusion proteins were purified via protein A Sepharose
chromatography according to the manufacturers instructions.
huKS-mu.gamma.2a-muIL12
[0094] A gene encoding the huKS-mu.gamma.2a-muIL12 fusion protein
was constructed and expressed essentially as described in U.S. Ser.
No. 08/986,997, filed Dec. 8, 1997, and Gillies et al. (1998) J.
Immunol. 160: 6195-6203. Briefly, this was accomplished by fusing
the murine p35 IL-12 subunit cDNA to the huKS-mu.gamma.2a heavy
chain coding region prepared previously. The resulting vector then
was transfected into an NS/0 myeloma cell line pre-transfected
with, and capable of expressing p40 IL-12 subunit. In other words,
a cell line was transfected with p40 alone and a stable, high
expressing cell was selected, which was then used as a recipient
for transfection by the p35 containing fusion protein (i.e.,
sequential transfection).
[0095] The murine p35 and p40 IL-12 subunits were isolated by PCR
from mRNA prepared from spleen cells activated with Concanavalin A
(5 .mu.g/mL in culture medium for 3 days). The PCR primers used to
isolate the p35 encoding nucleic acid sequence which also adapted
the p35 cDNA as an XmaI-XhoI restriction fragment included:
TABLE-US-00004 5' CCCCGGGTAGGGTCATTCCAGTCTCTGG; (SEQ ID NO: 11) and
5' CTCGAGTCAGGCGGAGCTCAGATAGC. (SEQ ID NO: 12)
[0096] The PCR primer used to isolate the p40 encoding nucleic acid
sequence included:
TABLE-US-00005 5' TCTAGACCATGTGTCCTCAGAAGCTAAC; (SEQ ID NO: 13) and
5' CTCGAGCTAGGATCGGACCCTGCAG. (SEQ ID NO: 14)
[0097] A plasmid vector (pdHL7-huKS-mu.gamma.2a-p35) was
constructed as described (Gillies et al. J. Immunol. Methods 125:
191) that contained a dhfr selectable marker gene, a transcription
unit encoding a humanized KS antibody light chain, and a
transcription unit encoding a murine heavy chain fused to the p35
subunit of mouse IL-12. The fusion was achieved by ligation of the
XmaI to XhoI fragment of the adapted p35 subunit cDNA, to a unique
XmaI site at the end of the CH3 exon of the murine .gamma.2a gene
prepared previously. Both the H and L chain transcription units
included a cytomegalovirus (CMV) promoter (in place of the
metallothionein promoter in the original reference) at the 5' end
and, a polyadenylation site at the 3' end.
[0098] A similar vector (pNC-p40) was constructed for expression of
the free p40 subunit which included a selectable marker gene
(neomycin resistant gene) but still used the CMV promoter for
transcription. The coding region in this case included the natural
leader sequence of the p40 subunit for proper trafficking to the
endoplasmic reticulum and assembly with the fusion protein. Plasmid
pNC-p40 was electroporated into cells, and cells were plated and
selected in G418-containing medium. In this case, culture
supernatants from drug-resistant clones were tested by ELISA for
production of p40 subunit.
[0099] The pdHL7-huKS-mu.gamma.2a-p35 expression vector was
electroporated into the NS/0 cell line already expressing murine
p40, as described in Gillies et al. (1998) J. Immunol. 160:
6195-6203. Transfected clones resistant to methotrexate were tested
for secretion of antibody determinants and mouse IL-12 by standard
ELISA methods. The resulting protein was purified by binding to,
and elution from a protein A Sepharose column in accordance with
the manufacturers instructions.
Example 3
In Vitro Cytotoxic Activity of Combination Therapy
[0100] The cell lines engineered for use in animal models (example
1) were tested for their sensitivity to taxane-induced cytotoxicity
in cell culture in the presence or absence of the an IL-2 based
immunocytokine consisting of the humanized form of the KS-1/4
antibody fused at the carboxyl terminus of the H chain to human
IL-2 (huKS-hu.gamma.1-huIL2, hereafter abbreviated, KS-IL2). Cells
were seeded at 1000 cell/well in 96 well flat-bottom plates and
incubated for 24 hours at 37.degree. C., 7% CO.sub.2. Paclitaxel,
at 2-fold dilutions from 200 ng/ml to 3.125 ng/ml, KS-IL2, at 200
ng/ml and IL-2, at 33.3 ng/ml (the equivalent amount of IL-2 in
KS-IL2) were added in duplicate to the cell culture plates and
incubated for 6 days at 37.degree. C., 7% CO.sub.2. The MTS
colorimetric assay (Promega), a measure of cell viability based on
the cellular conversion of a tetrazolium salt, was performed
directly in the 96 well plates. After plates were read and
recorded, viable adherent cells were stained with Crystal violet
(Sigma, St. Louis, Mo.). Crystal violet stained plates were used to
verify MTS assay results. Results are expressed in tabular form.
The IC.sub.50 is the concentration of drug that produced
cytotoxicity at a level of 50% of control.
[0101] A cytotoxicity assay was performed with paclitaxel (3 to 200
ng/ml) alone or combined with KS-IL2 (200 ng/ml) or IL-2 (33.3
ng/ml, the equivalent amount of IL-2 in KS-IL2) against CT26/KSA,
LLC/KSA and 4T1/KSA cells. There was little to no cytotoxicity of
KS-IL2 or IL-2 alone on the three cell lines tested (81% to 101% of
control, Table 1). The addition of either KS-IL2 or IL-2 did not
affect the cytotoxicity of Paclitaxel. Therefore, since neither
KS-IL2 nor IL-2 affects the cytotoxicity of paclitaxel, any
enhancement in anti-tumor activity in mice by the combined
treatments must be due to other mechanisms, which occur only in the
tumor-bearing animal.
TABLE-US-00006 TABLE 1 Cytotoxicity of Paclitaxel in combination
with IL-2 or KS-IL2 CT26/KSA.sup.a LLC/KSA.sup.b 4T1/KSA.sup.b
Paclitaxel IC.sub.50 (ng/ml) Taxol 27 6 16 Taxol + IL-2 (33 ng/ml)
30 8 20 Taxol + KS-IL2 (200 ng/ml) 26 5 19 % of Control IL-2 (33
ng/ml) 97 100 95 KS-IL2 (200 ng/ml) 90 101 81 .sup.aAverage of
three experiments .sup.bAverage of two experiments
Example 4
Combination Therapy of LLC Skin Tumors with KS-IL2 and a Taxane
[0102] A tumor growth regression assay was performed using the
aggressively growing tumor, LLC/KSA, in which a single dose of
paclitaxel (80 mg/kg) was followed one week later by KS-IL2 (20
.mu.g) administered by intravenous tail vein injection for 5 days
(FIG. 2). No effect of either the paclitaxel or KS-IL2 given alone
(on Days 0-4) was observed. However, when KS-IL2 was administered
one week following paclitaxel, a large reduction in average tumor
volume (41% of control) and a tumor growth delay (TGD) of about 8
days was observed which was significantly different than paclitaxel
alone (p=0.023). No drug-related gross toxicity was observed except
for a<5% weight loss in the paclitaxel treated groups.
[0103] Next, the effect of multiple doses of paclitaxel, generally
considered a more effective chemotherapy schedule, was compared to
a single dose of paclitaxel in combination with KS-IL2 to determine
how the schedule affects the enhancement. KS-IL2 (20 .mu.g, Days
0-4) alone again had no effect on LLC/KSA tumor growth but
paclitaxel alone, when given in multiple doses (50 mg/kg, every
other day), reduced the average tumor volume to 63% of control and
caused a 4 day tumor growth delay (TGD) (FIG. 3). When the KS-IL2
immunocytokine was administered one week following paclitaxel
treatment, a reduction in tumor volume to 27% of control and a TGD
of 10 days was observed which was significantly different than
paclitaxel alone (p=0.016). No drug-related gross toxicity was
observed except for a<5% weight loss in the paclitaxel treated
groups. The combined therapy group had even less weight loss. These
positive combination therapy results are surprising considering the
relatively short interval between chemotherapeutic (and potentially
immune damaging) treatment and the initiation of a treatment that
is based on the ability to stimulate lymphocyte proliferation and
cytotoxicity.
[0104] One explanation for the combined effect is that
taxane-induced apoptosis of a portion of the growing tumor mass
reduced the interstitial pressure that, in turn, increased the
effective uptake of KS-IL2 into the tumor. Recent studies
(Griffon-Etienne et al. 1999, CANCER RES. 59:3776-3782) indicate
that the effect of a single dose of paclitaxel effectively lowered
interstitial fluid pressures with a maximum effect seen from 24 to
48 hours (Griffon-Etienne et al. 1999, CANCER RES. 59:3776-3782).
Although this may be the best time for uptake of the immunocytokine
into the tumor, it is also a very short time interval after
chemotherapy. Nonetheless, we treated mice bearing LLC/KSA tumors
with KS-IL2 for 5 consecutive days beginning just 24 hr after
receiving a single dose of paclitaxel. Results indicate that there
is an even better combined response when immunocytokine treatment
was initiated earlier than a week following a single dose of
paclitaxel with this tumor line as well as colon carcinoma CT26
(see below).
Example 5
Combination Therapy of 4T1 Metastases with KS-IL2 and a Taxane
[0105] Since we found that treatment intervals between
administration of a taxane and an immunocytokine could be shorter
than expected, we tested combination regimens in which the taxane
and the immunocytokine are given on the same day and compared a
single dose (75 mg/kg) of paclitaxel with a fractionated dose (25
mg/kg.times.3 days) given concurrently with KS-IL2 treatment (15
.mu.g/dose.times.3 days given 4 hr after paclitaxel). For this
experiment we used an experimental lung metastasis model induced
with 4T1/KSA breast carcinoma cells. The doses of the drugs were
selected to be sub-optimal by themselves so that any potential
additive or synergistic activity could be observed.
[0106] Each agent given alone significantly (p<0.02) reduced
average lung weights to a similar extent: 43% reduction for the
single dose of paclitaxel, a 49% reduction for multiple doses of
paclitaxel alone and a 39% reduction with KS-IL2 alone (FIG. 4).
The combination of paclitaxel and KS-IL2 further reduced lung
metastases slightly but was less than additive: 58% reduction for
single dose paclitaxel in combination with KS-IL2 and a 68%
reduction for multiple dose paclitaxel in combination with KS-IL2.
Even though no synergism was observed, the single dose of
paclitaxel in combination with KS-IL2 resulted in a significant
difference compared to paclitaxel given alone (p=0.047).
[0107] Less than 10% weight loss was observed in all groups,
however, the greatest weight loss was obtained with 25 mg/kg of
paclitaxel given 3 times every other day. Based on these data, the
best regimen in this 4T1 lung metastasis assay with respect to the
greatest effect of combination therapy was a single dose of
paclitaxel followed by KS-IL2, as was the case for the LLC/KSA
tumor growth regression model. Since the dosing interval in this
case was only 4 hr, the results might not have been optimal for
efficient tumor uptake.
Example 6
Combination Therapy of CT26 Skin Tumors with KS-IL2 and a
Taxane
[0108] The results described in example 5 suggested that the time
interval of 4 hr between dosing the two agents might be too short.
Perhaps the levels of paclitaxel still remaining in the animal at
the time of KS-IL2 dosing could interfere directly with lymphocyte
activation, thus reducing its potential anti-tumor activity in the
combination setting. Also, at the 4 hr time point, the maximum
effect on the tumor interstitial pressure would not have been
reached. Therefore, we designed another experiment, this time using
established skin tumors of the CT26/KSA colon carcinoma, in which
we combined a single dose of paclitaxel (75 mg/kg) with a 5-day
course of KS-IL2 beginning 24 hr after administration of the
taxane. Paclitaxel alone had no effect on tumor growth (FIG. 5).
Treatment with sub-optimal doses of KS-IL2 (10 .mu.g, Days 1-5)
resulted in tumor volumes that were 71% of control. A dramatic and
synergistic reduction of tumor volume to 8% of control was observed
with the combination of paclitaxel and KS-IL2, which was
significantly different from paclitaxel treatment alone
(p<0.001). A minimal weight loss of .about.5% was observed for
both paclitaxel treated groups.
[0109] A second experiment was performed using the CT26/KSA model,
this time testing the effect of combined therapy on established
liver metastases and again using the 24 hr delay between paclitaxel
administration and KS-IL2 treatment. We also compared the dose
response of paclitaxel in the combination therapy. Mice were
injected with 25, 50, or 75 mg/kg of paclitaxel on Day 5 after
metastasis induction, alone or followed one day later with KS-IL2
(7 ug) for 5 days. A dose response effect was observed for
paclitaxel alone, in which 25, 50, 75 mg/kg resulted in tumor
burdens of 49%, 23%, 10% of control, respectively (FIG. 6).
Combining paclitaxel with KS-IL2 further reduced lung metastases to
12%, 9%, and 6% of control for the same respective doses of
paclitaxel. The lowest dose of paclitaxel (25 mg/kg) in combination
with KS-IL2 resulted in the greatest and most significant
(p<0.001) reduction in tumor burden compared to the higher doses
of paclitaxel with KS-IL2. Therefore, the combination of KS-IL2
preceded by paclitaxel resulted in a greater anti-tumor effect than
either agent alone. Further, the lowest dose of paclitaxel in
combination with KS-IL2 resulted in similar anti-tumor efficacy as
the highest dose of paclitaxel alone. Hence, using a lower dose of
paclitaxel in combination with KS-IL2 would reduce toxicity while
maintaining good efficacy.
Example 7
Measuring Uptake of KS-IL2 into Tumors
[0110] If the effect of single doses of cytotoxic drug treatment,
prior to immunocytokine therapy, is to decrease tumor interstitial
pressure and increase penetration of tumors, this should be
measurable using radioactively labeled immunocytokine, e.g. KS-IL2.
Purified KS-IL2 was labeled with .sup.125I by standard procedures
(reference) through contract to a commercial vendor (New England
Nuclear, Billerica, Mass.). Skin tumors of CT26/KSA were implanted
subcutaneously as described in Example 1 and allowed to grow until
they reached from 100-200 mm.sup.3. Two groups of 4 mice were
injected with either paclitaxel (50 mg/kg) in vehicle or vehicle
alone followed in 1 hr (Experiment 1) or 24 hr (Experiment 2) by 10
.mu.g of .sup.125I-KS-IL2 (95 .mu.Ci). Six hours after injecting
the radiolabeled immunocytokine, the mice were sacrificed and their
tumors were surgically removed. As a control, livers of the animals
were also collected and all tissues were weighed and then counted
in a gamma counter. Results were expressed as the counts per minute
(CPM) per gram of tissue by dividing the total CPM in the tissue by
the weight.
[0111] When labeled KS-IL2 was injected 1 hr after paclitaxel
treatment (FIG. 7A), only a small increase in the amount of
radioactivity was seen in the excised tumors from animals receiving
the drug. In contrast, when labeled KS-IL2 was injected 24 hr after
paclitaxel treatment, a dramatic increase in uptake was seen
(>200 percent) relative to the vehicle control (FIG. 7B). This
great difference in tumor uptake between the 1 hr and 24 hr time
points is in agreement with the data on taxane-induced changes in
interstitial pressure (Griffon-Etienne et al. 1999, CANCER RES.
59:3776-3782), and is consistent with the data in our tumor models
showing that treatment beginning 24 hr after paclitaxel is more
efficient than treatment at earlier times (4 hr).
[0112] We also tested whether other classes of drugs could increase
the uptake of labeled immunocytokine into solid tumors. In this
case, mice were injected with a single dose of cyclophosphamide (40
mg/kg) either 24 hr or 3 days prior to the experiment.
.sup.125I-labeled KS-IL2 was injected into all mice, including
control mice pre-treated with PBS, and the amount of radioactivity
in excised tumors was determined 16 hr later. Results (FIG. 8) show
that pre-treatment with cyclophosphamide increased the uptake of
KS-IL2 by 48% in mice pre-treated 24 hr earlier and by 70% in mice
pre-treated for 3 days.
Example 8
Combination Therapy with huKS-hu.gamma.4-IL2 and a Taxane
[0113] New forms of immunocytokines have been described recently
that have increased circulating half-lives and improved efficacy
due to a reduced affinity for Fc receptors (see Gillies et. 1999,
CANCER RES. 59:2159-2166). One representative of these improved
IL-2 immunocytokines, huKS-hu.gamma.4-IL2, was tested in
combination therapy with a single dose of paclitaxel. Again, there
was improved efficacy when the two drugs were given sequentially in
mice bearing CT26/KSA skin tumors.
Example 9
Combination Therapy with huKS-mu.gamma.2a-muIL12 and a Taxane
[0114] In order to test whether the synergistic therapeutic effect
is specific only for IL-2 based immunocytokines, we treated
established CT26/KSA bulky tumors first with paclitaxel (single
dose of 75 mg/kg) followed 24 hr later with a 5-day course of
huKS-mu.gamma.2a-muIL12 (5 .mu.g per day). This immunocytokine
represents a fusion between the murine form of the HuKS antibody
(i.e. the constant regions were reverted to murine C kappa and C
gamma 2a) and murine IL-12. It was necessary to use murine IL-12
sequences because, unlike IL-2, this cytokine is highly species
specific and the human form is not very active in the mouse.
Results show that treatment with paclitaxel alone had very little
effect on tumor growth. Treatment with sub-optimal doses of
huKS-mu.gamma.2a-muIL12 had an anti-tumor effect and this was
increased in mice that were treated first with a single dose of
paclitaxel.
Example 10
Combination Therapy with huKS-IL2 and an Alkylating Agent
[0115] i. The improved therapeutic effect of the combination of
huKS-IL2 with cyclophosphamide, a chemotherapy drug in the
alkylating agent class, was also demonstrated. 4T1 breast carcinoma
cells were injected intravenously into immuno-competent mice to
establish pulmonary metastases 3 days before treatment. Mice were
treated with a single dose of cyclophosphamide (15, 40, or 80
mg/kg) followed three days later with a 5-day course of huKS-IL2
(15 ug/day). Even though the two lowest doses alone caused only a
modest reduction in lung metastasis tumor burden, the combination
with huKS-IL2 resulted in a significantly large decrease in tumor
burden compared to cyclophosphamide alone (p<0.05, FIG. 9).
However, at the highest dose (80 mg/kg) no synergy occurs.
[0116] ii. The improved therapeutic effect of the combination of
huKS-IL2 with cyclophosphamide was also demonstrated in a tumor
growth assay, in immuno-competent mice bearing established breast
carcinoma subcutaneous tumors. Mice were treated with a single dose
of 80 mg/kg cyclophosphamide, either alone or in combination with 5
daily doses of huKS-IL2 (30 .mu.g) 3 days following the
cyclophosphamide treatment. Average tumor volumes for huKS-IL2 and
80 mg/kg of cyclophosphamide alone were reduced by 31% and 69%,
respectively (FIG. 9B). The combination treatment reduced average
tumor volumes by 100% on Day 25 which was significantly different
than either huKS-IL2 alone or cyclophosphamide alone (p<0.05)
and completely eliminated tumors in six out of eight mice up to at
twelve weeks after the initial treatment. Animals tolerated these
treatments well with less than 10% weight loss observed in all
groups.
[0117] iii. The improved therapeutic effect of the combination of
huKS-IL2 with cyclophosphamide was also demonstrated in a tumor
growth assay, in immuno-competent mice bearing established lung
carcinoma subcutaneous tumors. Mice were treated with a single dose
of 80 mg/kg cyclophosphamide, either alone or in combination with 5
daily doses of huKS-IL2 (20 .mu.g) 3 days following the
cyclophosphamide treatment. Average tumor volumes for huKS-IL2 and
80 mg/kg of cyclophosphamide alone were reduced by 2% and 27%,
respectively (FIG. 9C). The combination treatment reduced average
tumor volumes by 48% on Day 20 which was significantly different
than either huKS-IL2 alone or cyclophosphamide alone (p<0.05).
Animals tolerated these treatments well with less than 10% weight
loss observed in all groups.
Example 11
Combination Therapy with huKS-IL2 and an Alkylating Agent
[0118] The improved therapeutic effect of the combination of
huKS-IL2 with Carboplatin, another chemotherapy agent in the
alkylating agent class, was demonstrated. Mice bearing established
non-small cell lung carcinoma subcutaneous tumors (LLC/KSA) were
treated with Carboplatin (75 mg/kg) on Day 0 followed by three days
later with a 5-day course of KS-IL2 (20 ug per day). Carboplatin
and KS-IL2 treatment alone each resulted in a modest decrease in
tumor growth, however, only the combination treatment significantly
reduced the average tumor volume on Day 20 (p<0.05, FIG. 10).
Further, the growth of tumors in which mice were treated with the
combination compared to Carboplatin treatment alone was
significantly different (p<0.05).
[0119] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather then limiting on the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes which come within the meaning and range of equivalency of
the claims are intended to be embraced therein.
[0120] Each of the patent documents and scientific publications
disclosed hereinabove is incorporated by reference herein.
Sequence CWU 1
1
15135DNAArtificial Sequencesense primer for junction of the
huKS-mouse gamma 2a cDNA 1ccgtctcctc agccaaaaca acagccccat cggtc
35237DNAArtificial Sequenceantisense primer for huKS-gamma 2a cDNA
2ggggctgttg ttttggctga ggagacggtg actgacg 37325DNAArtificial
Sequencesense primer including an AflII site 3cttaagccag atccagttgg
tgcag 25427DNAArtificial Sequenceantisense primer including an XmaI
site 4cccggggtcc gggagaagct cttagtc 27530DNAArtificial
Sequencesense primer 5ggcccgggta aagcacccac ttcaagctcc
30625DNAArtificial Sequenceantisense primer 6ccctcgagtt attgagggct
tgttg 25732DNAArtificial Sequencesense primer 7ggaaataaaa
cgggctgatg ctgcaccaac tg 32834DNAArtificial Sequenceantisense
primer 8gcagcatcag cccgttttat ttccagcttg gtcc 34925DNAArtificial
Sequencesense primer 9cttaagcgag atcgtgctga cccag
251029DNAArtificial Sequenceantisense primer 10ctcgagctaa
cactcattcc tgttgaagc 291128DNAArtificial Sequencep35 PCR primer
11ccccgggtag ggtcattcca gtctctgg 281226DNAArtificial Sequencep35
PCR primer 12ctcgagtcag gcggagctca gatagc 261328DNAArtificial
Sequencep40 PCR primer 13tctagaccat gtgtcctcag aagctaac
281425DNAArtificial Sequencep40 PCR primer 14ctcgagctag gatcggaccc
tgcag 251515PRTArtificial SequencePeptide linker 15Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15
* * * * *