U.S. patent application number 12/417277 was filed with the patent office on 2009-12-24 for chimeric proteins with cell-targeting specificity and apoptosis-inducing activities.
This patent application is currently assigned to Yissum Research Development Company of the Hebrew University of Jerusalem. Invention is credited to Rami Ishaq Aqeilan, Yehudith Azar, Ruth Belostotsky, Ahmi Ben-Yehudah, Haya Lorberboum-Galski, Shai Yarkoni.
Application Number | 20090317358 12/417277 |
Document ID | / |
Family ID | 21870910 |
Filed Date | 2009-12-24 |
United States Patent
Application |
20090317358 |
Kind Code |
A1 |
Yarkoni; Shai ; et
al. |
December 24, 2009 |
CHIMERIC PROTEINS WITH CELL-TARGETING SPECIFICITY AND
APOPTOSIS-INDUCING ACTIVITIES
Abstract
The present invention relates to chimeric proteins with
cell-targeting specificity and apoptosis-inducing activities. In
particular, the invention is illustrated by a recombinant chimeric
protein between human interleukin-2 (IL2) and Bax. The chimeric
protein specifically targets IL2 receptor (IL2R)-expressing cells
and induces cell-specific apoptosis.
Inventors: |
Yarkoni; Shai; (Kfar-Saba,
IL) ; Ben-Yehudah; Ahmi; (D.N. Harpi Yehuda, IL)
; Azar; Yehudith; (Jerusalem, IL) ; Aqeilan; Rami
Ishaq; (Jerusalem, IL) ; Belostotsky; Ruth;
(Maale Adumim, IL) ; Lorberboum-Galski; Haya;
(Jerusalem, IL) |
Correspondence
Address: |
VOLPE AND KOENIG, P.C.
UNITED PLAZA, SUITE 1600, 30 SOUTH 17TH STREET
PHILADELPHIA
PA
19103
US
|
Assignee: |
Yissum Research Development Company
of the Hebrew University of Jerusalem
Jerusalem
IL
|
Family ID: |
21870910 |
Appl. No.: |
12/417277 |
Filed: |
April 2, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10640668 |
Aug 13, 2003 |
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12417277 |
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09033525 |
Mar 2, 1998 |
6645490 |
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10640668 |
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Current U.S.
Class: |
424/85.2 ;
424/134.1; 424/94.63; 435/219; 514/1.1; 530/350; 530/351;
530/387.3; 530/399 |
Current CPC
Class: |
C07K 14/55 20130101;
A61P 35/00 20180101; C07K 14/4747 20130101; C07K 2319/00 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
424/85.2 ;
530/351; 530/387.3; 530/399; 530/350; 435/219; 514/12; 424/134.1;
424/94.63 |
International
Class: |
A61K 38/19 20060101
A61K038/19; C07K 14/52 20060101 C07K014/52; C07K 16/18 20060101
C07K016/18; C07K 14/575 20060101 C07K014/575; C07K 14/00 20060101
C07K014/00; C12N 9/50 20060101 C12N009/50; A61K 38/16 20060101
A61K038/16; A61K 38/22 20060101 A61K038/22; A61K 39/395 20060101
A61K039/395; A61K 38/48 20060101 A61K038/48; A61P 35/00 20060101
A61P035/00 |
Claims
1. A chimeric protein comprising a cell-specific targeting moiety
and an apoptosis-inducing moiety, wherein the chimeric protein is
produced by a recombinant DNA method, the targeting moiety is
selected from the group consisting of a cytokine, a growth factor,
a hormone, an antibody and a binding fragment of an antibody, and
the apoptosis-inducing moiety is a caspase or a caspase subunit
that induces apoptosis, cytochrome C, a DNA fragmentation factor
(DFF), a domain of an apoptotic protein of the Bcl-2 family which
domain has apoptosis-inducing activity, or a domain of DFF which
domain has apoptosis-inducing activity.
2. The chimeric protein of claim 1 in which the apoptosis-inducing
moiety is a protein of human origin.
3. The chimeric protein of claim 1 in which the apoptosis-inducing
moiety is DFF 40.
4. The chimeric protein of claim 1 in which the apoptosis-inducing
moiety is a caspase.
5. The chimeric protein of claim 1 in which the apoptosis-inducing
moiety is caspase 3.
6. The chimeric protein of claim 1 in which the apoptosis-inducing
moiety is cytochrome C.
7. The chimeric protein of claim 1 in which the apoptosis-inducing
moiety is a fragment of an apoptotic protein of the Bcl-2
family.
8. The chimeric protein of claim 1 in which the cell-specific
targeting moiety binds interleukin 2 receptor-expressing cells.
9. The chimeric protein of claim 1 in which the cell-specific
targeting moiety is an interleukin.
10. The chimeric protein of claim 9 in which the cell-specific
targeting moiety is an interleukin 2.
11. The chimeric protein of claim 1 in which the cell-specific
targeting moiety is myelin basic protein.
12. The chimeric protein of claim 1 in which the cell-specific
targeting moiety binds tumor cells.
13. The chimeric protein of claim 1 in which the cell-specific
targeting moiety is an antibody or a fragment thereof.
14. The chimeric protein of claim 13 in which the cell-specific
targeting moiety is a single chain antibody.
15. The chimeric protein of claim 13 in which the cell-specific
targeting moiety is a Fc fragment of an IgE antibody.
16. The chimeric protein of claim 1 in which the cell-specific
targeting moiety is a cytokine.
17. The chimeric protein of claim 1 in which the cell-specific
targeting moiety is epidermal growth factor.
18. The chimeric protein of claim 1 in which the cell-specific
targeting moiety is insulin-like growth factor.
19. The chimeric protein of claim 1 in which the two moieties are
connected by a polylinker.
20. A pharmaceutical composition comprising a chimeric protein
according to claim 1 and a pharmaceutically acceptable carrier.
21. A pharmaceutical composition comprising a chimeric protein
according to claim 2 and a pharmaceutically acceptable carrier.
22. A pharmaceutical composition comprising a chimeric protein
according to claim 5 and a pharmaceutically acceptable carrier.
23. A pharmaceutical composition comprising a chimeric protein
according to claim 7 and a pharmaceutically acceptable carrier.
24. A pharmaceutical composition comprising a chimeric protein
according to claim 8 and a pharmaceutically acceptable carrier.
25. A pharmaceutical composition comprising a chimeric protein
according to claim 11 and a pharmaceutically acceptable
carrier.
26. A pharmaceutical composition comprising a chimeric protein
according to claim 12 and a pharmaceutically acceptable
carrier.
27. A pharmaceutical composition comprising a chimeric protein
according to claim 13-15 and a pharmaceutically acceptable
carrier.
28. A pharmaceutical composition comprising a chimeric protein
according to claim 16 and a pharmaceutically acceptable
carrier.
29. A pharmaceutical composition comprising a chimeric protein
according to claim 19 and a pharmaceutically acceptable
carrier.
30. A method of treating a pathological disorder associated with
undesirable target cells, comprising administering to a patient in
need thereof a chimeric protein comprising a targeting moiety
specific for the undesirable cells and an apoptosis-inducing
moiety, wherein the apoptosis-inducing moiety is a caspase,
cytochrome C, a DNA fragmentation factor (DFF), a fragment of an
apoptotic protein of the Bcl-2 family which fragment has
apoptosis-inducing activity, or a domain of caspase, cytochrome C
or DFF which domain has apoptosis-inducing activity.
31. A method of treating a malignant or pre-malignant condition,
comprising administering to a patient in need thereof a chimeric
protein of claim 1 or a pharmaceutical composition of claim 20.
32. A method of treating an immune disorder associated with cells
expressing and IL-2 receptor comprising administering to a patient
in need thereof a chimeric protein of claim 10.
33. A method of treating a hypersensitivity condition, comprising
administering to a patient in need thereof a chimeric protein of
claim 1 or a pharmaceutical composition of claim 20.
34. A method of treating an infectious disease comprising
administering to a patient in need thereof a chimeric protein of
claim 1 or a pharmaceutical composition of claim 20.
35. A pharmaceutical composition comprising a chimeric protein
according to claim 4 and a pharmaceutically acceptable carrier.
36. A pharmaceutical composition comprising a chimeric protein
according to claim 9 and a pharmaceutically acceptable carrier.
37. A pharmaceutical composition comprising a chimeric protein
according to claim 10 and a pharmaceutically acceptable
carrier.
38. The chimeric protein of claim 1 wherein the apoptosis-inducing
moiety is the domain of an apoptotic protein of the Bcl-2 family
which domain has apoptosis-inducing activity and the domain is a
BH3 domain.
39. A chimeric protein comprising a cell-specific targeting moiety
and an apoptosis-inducing moiety, wherein the chimeric protein is
produced by a recombinant DNA method, the targeting moiety is
selected from the group consisting of a cytokine, a growth factor,
a hormone, an antibody and a binding fragment of an antibody, and
the apoptosis-inducing moiety is a caspase or a caspase subunit
that induces apoptosis.
40. The Chimeric protein of claim 39 wherein the targeting moiety
is an IL-2.
Description
1. INTRODUCTION
[0001] The present invention relates to chimeric proteins with
cell-targeting specificity and apoptosis-inducing activities. In
particular, the invention is illustrated by a recombinant chimeric
protein between human interleukin-2 (IL2) and Bax. The chimeric
protein specifically targets IL2 receptor (IL2R)-expressing cells
and induces cell-specific apoptosis. In accordance with the
invention, chimeric proteins may be generated between any molecule
that binds a specific cell type and an apoptosis-inducing protein.
Such chimeric proteins are useful for selectively eliminating
specific cell types in vitro and in vivo, and may be used in the
treatment of autoimmunity, cancer and infectious diseases such as
viral infections.
2. BACKGROUND OF THE INVENTION
2.1. Immunotoxins
[0002] The advent of the monoclonal antibody technology and
recombinant DNA technology have led to the discovery of numerous
cell surface molecules associated with specific cell populations.
Based on the expression pattern of these molecules, recombinant
immunotoxins have been constructed to specifically target and
destroy the cells that express such molecules. Recombinant
immunotoxins are a class of targeted molecules designed to
recognize and specifically destroy cells expressing specific
receptors, such as cancer cells and cells involved in many
disorders of the immune system. Generally, immunotoxins utilize a
bacterial or plant toxin to destroy the unwanted cells. These
molecules are designed and constructed by gene fusion techniques
and are composed of both the cell targeting and cell killing
moieties, a combination that makes these agents potent molecules
for treatment. Examples of immunotoxins are growth factors or
antigen-binding domains of antibody, including the Fv portion of an
antibody (single-chain immunotoxins) fused to various mutant forms
of toxin molecules. However, over the years it has become clear
that treatment with such "magic bullets" for targeted immunotherapy
possesses still many problems and new approaches are needed to
produce improved recombinant immunotoxins.
[0003] Each recombinant immunotoxin displays some nonspecific
toxicity and at sufficiently high concentrations damages normal
cells that do not express the specific target antigen. This
non-specific toxicity of immunotoxins is the dose-limiting factor
in immunotoxin therapy. Which tissues are affected by nonspecific
toxicity is dependent on the particular toxin used for immunotoxin
preparation, and the ability of immunotoxins to penetrate into
tissues and tumors is largely dependent on the size of the
immunotoxins.
[0004] Large stable conjugated immunotoxins persist for long
periods in blood vessels (T.sub.1/25-15 hour), thus endothelial
cells are exposed to high toxin concentrations which may lead to
endothelial cell damage. Smaller molecules, such as recombinant
immunotoxins which rapidly leave the vascular system, would
presumably have different toxicity. In humans, immunotoxins made
with ricin and other ribotoxins, as well as with Pseudomonas
exotoxin A (PE), Diphtheria toxin (DT) and their truncated
derivatives have produced a variety of toxicities. These include
vascular leak syndrome (mainly ricin immunotoxins) as well as liver
toxicity (PE-derived immunotoxins). Vascular leak syndrome observed
with ricin immunotoxins in animals and man may be explained by
specific binding of ricin A-chain to endothelial cells and
subsequent killing of the cells and damage to the vessels. The
nonspecific liver-toxicity of PE immunotoxins is likely to be due
to easy access and very rapid nonspecific uptake and
internalization of proteins by hepatocytes. However, it is also
possible that PE contains, in addition to the specific cell-binding
site (Domain I) which is removed in most immunotoxins, an
additional site which could be recognized with low affinity by
hepatocytes, thus accounting for liver toxicity.
[0005] Another major impediment with immunotoxins in their clinical
application is the human immune response against them, mainly
toward the toxin moiety. Bacterial toxins like PE and DT are highly
immunogenic and cannot be humanized with standard techniques. Usage
of DT-derived immunotoxins is limited because most people in
developed countries have been vaccinated against DT and many adults
have neutralizing antibodies to DT. Immunogenicity is a problem to
which so far no practical solution has been found. Reduced
immunogenicity of these molecules would greatly improve the
clinical application of immunotoxins.
[0006] An example of the successful use of an immunotoxin is the
elimination of activated T cells which express high affinity IL2
receptors (IL2R), whereas normal resting T cells and their
precursors do not. An immunotoxin made of IL2 could theoretically
eliminate IL2R-expressing leukemia cells or IL2R-expressing immune
cells involved in various disease states while not destroying IL2R
negative normal cells, thereby preserving the full repertoire of
antigen receptors required for T cell immune responses.
[0007] A chimeric protein, IL2-PE40, was produced and shown to
eliminate activated T cells (Lorberboum-Galski et al., 1988, Proc.
Natl. Acad. Sci. U.S.A. 85:1922). IL2-PE40 was extremely cytotoxic
to IL2R-expressing cell lines of human, ape and murine origin. It
was also extremely cytotoxic to Con A-stimulated mouse and rat
spleen cells, and had a suppressive effect against
antigen-activated mouse cells and the generation of cytotoxic T
cells in mixed lymphocyte cultures (Lorberboum-Galski et al., 1988,
J. Biol. Chem. 263:18650-18656; Ogata et al., 1988, J. Immunol.
41:4224-4228; Lorberboum-Galski et al., 1990, J. Bio. Chem.
265:16311-16317).
[0008] A highly purified IL2-PE40 preparation (Bailon et al., 1988,
Biotechnol. 6:1326-1329) was shown to (a) delay and mitigate
adjuvant induced arthritis in rats (Case et al., 1989, Proc. Natl.
Acad. Sci. U.S.A. 86:287-291), (b) significantly prolong the
survival of vascularized heart allograft in mice (Lorberboum-Galski
et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:1008-1012) and
corneal allografts in rats (Herbort et al., 1991, Transplant.
52:470-474), (c) reduce the incidence and severity of experimental
autoimmune uveoretinitis in rats (Roberge et al., 1989, J. Immunol.
143:3498-3502), (d) suppress the growth of an IL2R bearing T cell
lymphoma in mice (Kozak et al., 1990, J. Immunol. 145:2766-2771)
and (e) prevent the development of experimental allergic
encephalomyelitis, a T cell mediated disease of the central nervous
system, in rats and mice (Beraud et al., 1991, Cell. Immunol.
133:379-389; Rose et al., 1991, J. Neuroimmunol. 32:209-217).
However, such immunotoxin still suffers from the same deficiencies
outlined above, particularly non-specific toxicity and
immunogenicity in the human host.
2.2. Apoptosis-Inducing Proteins
[0009] The development of multilineage organisms and the
maintenance of homeostasis within tissues both require tightly
regulated cell death. The ability of an individual cell to execute
a suicidal response following a death stimulus varies markedly
during its differentiation. Both positive and negative regulators
of programmed cell death (apoptosis) have been identified.
[0010] A high percentage of follicular lymphomas have a
characteristic chromosomal translocation, which places the
proto-oncogene, Bcl-2 next to the immunoglobulin heavy chain locus,
resulting in deregulation of Bcl-2 expression. Bcl-2 was found to
function as a repressor of programmed cell death (Vaux et al.,
1988, Nature 334:440-442). Recently, other Bcl-2 homologues were
shown to inhibit apoptosis. However, one such homologue, Bax,
mediates an opposite effect by accelerating apoptosis. An expanding
family of Bcl-2 related proteins has recently been noted to share
homology that is principally, but not exclusively, clustered within
two conserved regions known as Bcl-2 homology domains 1 and 2 (BH1
and BH2) (Oltvai et al., 1993, Cell 74:609-619; Boise et al., 1993,
Cell 74:597-608; Kozopas et al., 1993, Proc. Natl. Acad. Sci.
U.S.A. 90:3516-3520; Lin et al., 1993, J. Immunol. 151:1979-1988).
Members of the Bcl family include Bax, Bcl-X.sub.L, Mcl-1, A1 and
several open reading frames in DNA viruses. Another conserved
domain in Bax, distinct from BH1 and BH2 was identified and termed
BH3. This domain mediates cell death and protein binding functions
(Chittenden et al., 1995, EMBO J. 14:5589-5596). Another member of
the pro-apoptotic proteins contains only the BH3 domain, implying
that this particular domain may be uniquely important in the
promotion of apoptosis (Diaz et al., 1997, J. Biol. Chem.
272:11350-11355).
[0011] Bax homodimerizes and forms heterodimers with BCL-2 in vivo.
Overexpressed Bax overcomes the death repressor activity of Bcl-2
(Oltvai et al., 1993, Cell 74:609-619). It was found that levels of
Bax expression higher than Bcl-2 in bladder tumors was correlated
with a better outcome for patients. Early relapses were much more
frequently observed in patients whose tumors expressed more Bcl-2
than Bax mRNA (Gazzaniga et al., 1996, Int. J. Cancer
69:100-104).
[0012] Recently it was reported that Bax-alpha, a splice variant of
Bax was expressed in high amount in normal breast epithelium,
whereas only weak or no expression could be detected in 39 out of
40 cancer tissue samples examined (Bargou et al., 1996, J. Clin.
Invest. 97:2651-2659). Of interest, downregulation of Bax-alpha was
found in different histological subtypes. Furthermore, when
Bax-alpha was transfected into breast cancer cell lines under the
control of a tetracycline-dependent expression system, Bax restored
sensitivity of the cancer cells toward both serum starvation and
APO-I/Fas-triggered apoptosis, and significantly reduced tumor
growth in SCID mice. Therefore, it was proposed that dysregulation
of apoptosis might contribute to the pathogenesis of breast cancer
at least in part due to an imbalance between members of the Bcl-2
gene family (Bargou et al., 1996, J. Clin. Invest.
97:2651-2659).
[0013] In another study, the expression of Bax was investigated in
52 cases of Hodgkin's disease in parallel with Epstein-Barr virus,
and was compared with the immunodetection of other
apoptosis-regulating proteins, Mcl-1, Bcl-2 and Bcl-x. Bax
expression was frequently detected in Hodgkin's disease, providing
an explanation for the good chemoresponses generally obtained for
patients with this neoplastic disorder (Rigal-Haguet et al., 1996,
Blood 87:2470-2475).
[0014] Additional members of this growing family of apoptosis
inducing proteins have been cloned and identified. Bak is a new
member of the Bcl-2 family which is expressed in a wide variety of
cell types and binds to the Bcl-2 homologue Bcl-x2 in yeast (Farrow
et al., 1995, Nature 374:731-733; Chittenden et al., 1995, Nature
374:733). A domain in Bak was identified as both necessary and
sufficient for cytotoxicity activity and binding to Bcl-x1.
Sequences similar to this domain that are distinct from BH1 and BH2
have been identified in Bax and Bip1. This domain was found to be
of central importance in mediating the function of multiple cell
death-regulatory proteins that interact with Bcl-2 family members
(Chittenden et al., 1995, EMBO J. 14:5589-5596).
[0015] Overexpression of Bak in sympathetic neurons deprived of
nerve growth factor accelerated apoptosis and blocked the
protective effect of co-injected E1B 19K. The adenovirus E1B 19K
protein is known to inhibit apoptosis induced by E1A,
tumor-necrosis factor-alpha, FAS antigen and nerve growth factor
deprivation (Farrow et al., 1995, Nature 374:731-733). Expression
of Bak induced rapid and extensive apoptosis of serum-deprived
fibroblasts, thus raising the possibility that Bak is directly
involved in activating the cell death machinery (Chittenden et al.,
1995, Nature 374:733-736). It was also reported that in the normal
and neoplastic colon, expression of immunoreactive Bak co-localized
with sites of epithelial cell apoptosis. Induction of apoptosis in
the human colon cancer cell line HT29 and the rat normal small
intestinal cell line 1EC 18 in culture was accompanied by increased
Bak expression without consistent changes in expression of other
Bcl-2 homologous proteins (Moss et al., 1996, Biochem. Biophys.
Res. Commun. 223:199-203). Therefore, Bak was also suggested to be
the endogenous Bcl-2 family member best correlated with intestinal
cell apoptosis (Moss et al., 1996, Biochem. Biophys. Res. Commun.
223:119-203).
[0016] Unlike Bax, however, Bak can inhibit cell death in an
Epstein-Barr-virus-transformed cell line. Tissues with unique
distribution of Bak messenger RNA include those containing
long-lived, terminally differentiated cell types (Krajewski et al.,
1996, Cancer Res. 56:2849-2855), suggesting that
cell-death-inducing activity is broadly distributed, and that
tissue-specific modulation of apoptosis is controlled primarily by
regulation of molecules that inhibit apoptosis (Kiefer et al.,
1995, Nature 374:736-739).
[0017] Another member of the Bcl2 family is Bad that possesses the
key amino acid motifs of BH1 and BH2 domains. Bad lacks the
classical C-terminal signal-anchor sequence responsible for the
integral membrane positions of other family members. Bad
selectively dimerizes with Bcl-x.sub.L as well as Bcl-2, but not
with Bax, Bcl-Xs-Mcl1, A1 or itself. Bad reverses the death
repressor activity of Bcl-X.sub.L, but not that of Bcl-2 (Yang et
al., 1995, Cell 80:285-291; Ottilie et al., 1997, J. Biol. Chem.
272:30866-30872; Zha et al., 1997, J. Biol. Chem.
272:24101-24104).
[0018] Another member is Bik which interacts with the cellular
survival-promoting proteins, Bcl-2 and Bcl-X.sub.L as well as the
viral survival-promoting proteins, Epstein Barr virus-BHRF1 and
adenovirus E1B-19 kDa. In transient transfection assays, Bik
promotes cell death in a manner similar to other death-promoting
members of the Bcl-2 family, Bax and Bak. This death-promoting
activity of Bik can be suppressed by coexpression of Bcl-2,
Bcl-X.sub.L, EBV-BHRF1 and E1B-19 kDa proteins suggesting that Bik
may be a common target for both cellular and viral anti-apoptotic
proteins. While Bik does not contain overt homology to the BH1 and
BH2 conserved domains characteristic of the Bcl-2 family, it shares
a 9 amino acid domain (BH3) with Bax and Bak which may be a
critical determinant for the death-promoting activity of these
proteins (Boyd et al., 1995, Oncogene 11:1921-1928; Han et al.,
1996, Mol. Cell. Biol. 16:5857-5864).
[0019] The Bcl-2 family is composed of various pairs of antagonist
and agonist proteins that regulate apoptosis. Whether their
function is interdependent is uncertain. Using a genetic approach
to address this question, Knudson et al. (1997, Nature Genetics
16:358-363), recently utilized gain- and loss of function models of
Bcl-2 and Bax, and found that apoptosis and thymic hypoplasia,
characteristic of Bcl-2-deficient mice, are largely absent in mice
also deficient in Bax. A single copy of Bax promoted apoptosis in
the absence of Bcl-2. In contrast, overexpression Bcl-2 still
repressed apoptosis in the absence of Bax. While an in vivo
competition exists between Bax and Bcl-2, each is able to regulate
apoptosis independently. Bax has been shown to form channels in
lipid membranes and trigger the release of liposome-encapsulated
carboxyluorescein at both neutral and acidic pH. At physiological
pH, release could be blocked by Bcl-2. In planer lipid bilayers,
Bax formed pH- and voltage-dependent ion-conduction channels. Thus,
the pro-apoptotic effects of Bax may be elicited through an
intrinsic pore-forming activity that can be antagonized by Bcl-2
(Antonsson et al., 1997, Science 277:370-372). Two other members of
this family, Bcl-2 and Bcl-1, were also shown to form pores in
lipid membranes (Schendel et al., 1997, Proc. Natl. Acad. Sci.
U.S.A. 94:5113-5118).
[0020] Prior to the present invention, a fusion protein containing
a Bcl-2 pro-apoptotic member was not reported, nor was it
predictable if such a molecule could retain biological activities
when added to a cell exogenously to induce apoptosis.
3. SUMMARY OF THE INVENTION
[0021] The present invention relates to chimeric proteins with
cell-targeting specificity and apoptosis-inducing activities. The
chimeric proteins of the invention are composed of a cell-specific
targeting moiety and an apoptosis-inducing moiety. The
cell-specific targeting moiety provides cell-specific binding
properties to the chimeric protein, while the apoptosis-inducing
moiety induces programmed cell death upon entry into a target cell.
It is preferred that the chimeric proteins of the invention be
produced by recombinant expression of a fusion polynucleotide
between a coding sequence of a cell-targeting moiety and a coding
sequence of an apoptosis-inducing protein. Such chimeric proteins
are likely to be superior to the immunotoxins currently used in the
art because they are of human origin and thus are expected to have
reduced immunogenicity in a human recipient. In addition, chimeric
proteins kill target cells by inducing apoptosis which does not
cause a release of cellular organelles into the extracellular
environment to result in an inflammatory response. When cells die
by the apoptotic pathway, they shrink and condense, but the
organelles and plasma membranes retain their integrity, and the
dead cells are rapidly phagocytosed by neighboring cells or
macrophages before there is leakage of the cells' contents, thereby
eliciting minimal tissue or systemic response.
[0022] The invention also relates to pharmaceutical compositions of
the chimeric proteins, methods of producing such proteins, and
methods of using the same in vitro and in vivo, especially for
eliminating specific undesirable target cells, and for the
treatment of a variety of disease conditions as well as the use of
the proteins for disease diagnosis.
[0023] The invention is based, in part, on the Applicants'
discovery that a partially purified recombinant chimeric protein,
IL2-Bax, specifically targets IL2R.sup.+ cells, which include but
are not limited to, T cells, B cells, monocytes and natural killer
cells. The protein kills target cells by inducing apoptosis of
these cells. A wide variety of uses are encompassed by the present
invention, including but not limited to, the treatment of
autoimmunity, transplantation rejection, graft-versus-host disease,
cancer, hypersensitivity, and infectious diseases.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1: Construction of the pSY1 plasmid that encodes a
chimeric protein composed of IL2 and Bax-.alpha. (designated
IL2-Bax) under the control of the T7 promoter. The numbers
represent the corresponding amino acids.
[0025] FIG. 2: Nucleotide sequence (SEQ ID NO:1) of a coding
sequence for chimeric protein, IL2-Bax, and its deduced amino acid
sequence (SEQ ID NO:2).
[0026] FIG. 3: SDS-PAGE analysis of cell fractions containing the
IL2-Bax chimeric protein. IL2-Bax was overexpressed in E. coli BL21
(.lamda.DE3) and subfractionated as described in Section 6.1.2.,
infra. Samples of each subfraction were mixed with Laemmli sample
buffer and loaded on a 10% polyacrylamide gel.
[0027] Lanes: 1, insoluble fraction treated with extraction buffer
B containing SDS. 2; insoluble fraction treated with extraction
buffer C containing urea. 3, insoluble fraction treated with
extraction buffer A, containing Gu-HCL. 4, soluble fraction. M,
markers. Arrow indicates the position of the IL2-Bax chimeric
protein.
[0028] FIG. 4: Immunobloting of fractions containing IL2-Bax with
antibodies to the Bax protein.
[0029] Lanes: 1, soluble fraction. 2, insoluble fraction treated
with extraction buffer A containing Gu-HCL. 3, insoluble fraction
treated with extraction buffer C containing urea. 4, insoluble
fraction treated with extraction buffer B containing SDS. 5, A
protein extract of MCF-7 cells known to express the Bax protein
(indicated by the *) Arrow indicates the position of the IL2-Bax
chimeric protein.
[0030] FIGS. 5A & B: Effect of IL2-Bax on protein synthesis in
target (5A) and non-target (5B) cell lines. IL2-Bax (insoluble
fraction treated with Gu-HCL) was added at different concentrations
to the various cell lines, [.sup.3H] leucine incorporation into
cellular protein was measured. Results are expressed as percent of
control cells not exposed to IL2-Bax.
[0031] FIG. 6A-D: FACS analysis of fresh lymphocytes exposed to
IL2-Bax. Fresh lymphocytes were separated, exposed to the IL2-Bax
chimeric protein and apoptotic cells were analyzed by FACS. The
cells were untreated (6A) or treated with dexamethasone (6B),
IL2-Bax at 1 .mu.g/ml (6C) or IL2-Bax at 10 .mu.g/ml (6D).
[0032] FIG. 7A-C: FACS analysis of fresh lymphocytes exposed to
IL2-Bax. Fresh lymphocytes were separated, exposed to the IL2-Bax
chimeric protein and apoptotic cells were analyzed by FACS. The
cells were untreated (7A) or treated with IL2-Bax at 1 .mu.g/ml
(7B) or IL2-Bax at 10 .mu.g/ml (7C).
[0033] FIG. 8A-E: FACS analysis of HUT102 exposed to IL2-Bax.
HUT102 cells were exposed to IL2-Bax chimeric protein and analyzed
by FACS to characterize apoptotic cells. Results are expressed in a
logarithmic mode. The cells were untreated (8A) or treated with
IL2-Bax at 1 .mu.g/ml (8B), IL2-Bax at 2 .mu.g/ml (8C), IL2-Bax at
4 .mu.g/ml (8D) or IL2-Bax at 5 .mu.g/ml (8E).
[0034] FIG. 9A-C: FACS analysis of CEM cells exposed to IL2-Bax.
CEM cells were exposed to IL2-Bax chimeric protein and analyzed by
FACS to characterize apoptotic cells. Results are expressed in a
logarithmic mode. The cells were untreated (9A) or treated with
IL2-Bax at 1 .mu.g/ml (9B) or IL2-Bax at 5 .mu.g/ml (9C).
5. DETAILED DESCRIPTION OF THE INVENTION
[0035] The present invention relates to chimeric proteins,
pharmaceutical compositions of chimeric proteins, methods of
producing a chimeric protein and methods of using the protein. For
clarity of discussion, the specific compositions, procedures and
methods described herein are exemplified using IL2 and Bax; they
are merely illustrative for the practice of the invention.
Analogous procedures and techniques are equally applicable to
constructing other chimeric proteins between any cell-specific
targeting moiety and apoptosis-inducing moiety.
5.1. Construction of Chimeric Molecules
[0036] While the chimeric proteins of the present invention may be
produced by chemical synthetic methods or by chemical linkage
between the two moieties, it is preferred that they are produced by
fusion of a coding sequence of a cell-specific targeting moiety and
a coding sequence of an apoptosis-inducing protein under the
control of a regulatory sequence which directs the expression of
the fusion polynucleotide in an appropriate,host cell. The fusion
of two full length coding sequences can be achieved by methods well
known in the art of molecular biology. It is preferred that a
fusion polynucleotide contain only the AUG translation initiation
codon at the 5' end of the first coding sequence without the
initiation codon of the second coding sequence to avoid the
production of two encoded product. In addition, a leader sequence
may be placed at the 5' end of the polynucleotide in order to
target the expressed product to a specific site or compartment
within a host cell to facilitate secretion or subsequent
purification after gene expression. The two coding sequences can be
fused directly without any linker or by using a flexible polylinker
composed of the pentamer Gly-Gly-Gly-Gly-Ser repeated 1 to 3 times.
Such linker has been used in constructing single chain antibodies
(scFv) by being inserted between V.sub.H and V.sub.L (Bird et al.,
1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad.
Sci. U.S.A. 85:5979-5883). The linker is designed to enable the
correct interaction between two beta-sheets forming the variable
region of the single chain antibody. Other linkers which may be
used include
Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (Chaudhary
et al., 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1066-1070) and
Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp
(Bird et al., 1988, Science 242:423-426).
5.1.1. Cell-Specific Targeting Moieties
[0037] The chimeric proteins of the invention are composed of a
cell-specific targeting moiety and an apoptosis-inducing moiety.
The cell-specific targeting moiety confers cell-type specific
binding to the molecule, and it is chosen on the basis of the
particular cell population to be targeted. A wide variety of
proteins are suitable for use as cell-specific targeting moieties,
including but not limited to, ligands for receptors such as growth
factors, hormones and cytokines, and antibodies or antigen-binding
fragments thereof.
[0038] Since a large number of cell surface receptors have been
identified in hematopoietic cells of various lineages, ligands or
antibodies specific for these receptors may be used as
cell-specific targeting moieties. In a specific embodiment
illustrated by working examples in Section 6, infra, IL2 was used
as the cell-specific targeting moiety in a chimeric protein to
target IL2R.sup.+ cells. In addition, other molecules such as B7-1,
B7-2 and CD40 may be used to specifically target activated T cells
(The Leucocyte Antigen Facts Book, 1993, Barclay et al. (eds.),
Academic Press). On the other hand, B cells express CD19, CD40 and
IL4 receptor and may be targeted by moieties that bind these
receptors. Examples of such moieties include CD40 ligand, IL4, IL5,
IL6 and CD28. The elimination of immune cells such as T cells and B
cells is particularly useful in the treatment of autoimmunity,
hypersensitivity, transplantation rejection responses and in the
treatment of lymphoid tumors. Examples of autoimmune diseases are
multiple sclerosis, rheumatoid arthritis, insulin-dependent
diabetes mellitus, systemic lupus erythematosis, scleroderma,
uviatis, and the like. More specifically, since myelin basic
protein is known to be the major target of immune cell attack in
multiple sclerosis, this protein may be used as a cell-specific
targeting moiety for the treatment of multiple sclerosis (WO
97/19179; Becker et al., 1997, Proc. Natl. Acad. Sci. U.S.A.
94:10873).
[0039] Other cytokines which may be used to target specific cell
subsets include the interleukins (IL1-IL15), granulocyte-colony
stimulating factor, macrophage-colony stimulating factor,
granulocyte-macrophage colony stimulating factor, leukemia
inhibitory factor, tumor necrosis factor, transforming growth
factor, epidermal growth factor, insulin-like growth factors,
fibroblast growth factor and the like (Thompson (ed.), 1994, The
Cytokine Handbook, Academic Press, San Diego).
[0040] Additionally, certain cell surface molecules are highly
expressed in tumor cells, including hormone receptors such as human
chorionic gonadotropin receptor and gonadotropin releasing hormone
receptor (Nechushtan et al., 1997, J. Biol. Chem. 272:11597).
Therefore, the corresponding hormones may be used as the
cell-specific targeting moieties in cancer therapy.
[0041] Antibodies are the most versatile cell-specific targeting
moieties because they can be generated against any cell surface
antigen of interest. Monoclonal antibodies have been generated
against cell surface receptors, tumor-associated antigens, and
leukocyte lineage-specific markers such as CD antigens. Antibody
variable region genes can be readily isolated from hybridoma cells
by methods well known in the art. However, since antibodies are
assembled between two heavy chains and two light chains, it is
preferred that a scFv be used as a cell-specific targeting moiety
in the present invention. Such scFv are comprised of V.sub.H and
V.sub.L domains linked into a single polypeptide chain by a
flexible linker peptide. Furthermore, the Fc portion of the heavy
chain of an antibody may be used to target Fc receptor-expressing
cells such as the use of the Fc portion of an IgE antibody to
target mast cells and basophils. The specific targeting of these
cell types is useful for treating IgE-mediated hypersensitivity in
humans and animals (Helm et al., 1988, Nature 331:180-183;
PCT/IL96/00181)
5.1.2. Apoptosis-Inducing Moieties
[0042] The pro-apoptotic proteins in the BCL2 family are
particularly suitable for use as the apoptosis-inducing moieties in
the present invention. Such human proteins are expected to have
reduced immunogenicity over many immunotoxins composed of bacterial
toxins. In a specific embodiment illustrated by working examples in
Section 6, infra, the bax coding sequence is fused with an IL2
coding sequence for the production of a chimeric protein IL2-Bax.
While Bax is the preferred apoptosis-inducing moiety, other members
in this family suitable for use in the present invention include
Bak (Farrow et al., 1995, Nature 374:731; Chittenden et al., 1995,
Nature 374:733; Kiefer et al., 1995, Nature 374:736), Bcl-X.sub.s
(Boise et al., 1993, Cell 74:597; Fang et al., 1994, J. Immunol.
153:4388), Bad (Yang et al., 1995, Cell 80:285), Bid (Wang et al.,
1996, Genes Develop. 10:2859-2869), Bik (Bovd et al., 1995,
Oncogene 11:1921-1928), Hrk (Inohara et al., 1997, EMBO J.
16:1686-1694) and Bok (Hsu et al., 1997, Proc. Natl. Acad. Sci. USA
94: 12401-12406). The nucleotide sequences encoding these proteins
are known in the art, and thus cDNA clones can be readily obtained
for fusion with a coding sequence for a cell-specific targeting
moiety in an expression vector.
[0043] Specific domains of certain of the Bcl-2 family members have
been studied with respect to their apoptosis-inducing activities.
For example, the GD domain of Bak is involved in the apoptosis
function (U.S. Pat. No. 5,656,725). In addition, Bax and Bipla are
shown to share a homologous domain. Therefore, any biologically
active domains of the Bcl-2 family may be used as an
apoptosis-inducing moiety for the practice of the present
invention.
[0044] Caspases also play a central role in apoptosis and may well
constitute part of the consensus core mechanism of apoptosis.
Caspases are implicated as mediators of apoptosis. Since the
recognition that CED-3, a protein required for developmental cell
death, has sequence identity with the mammalian cysteine protease
interleukin-1 beta-converting enzyme (ICE), a family of at least 10
related cysteine proteases has been identified. These proteins are
characterized by almost absolute specificity for aspartic acid in
the P1 position. All the caspases (ICE-like proteases) contain a
conserved QACXG (where X is R, Z or G) pentapeptide active-site
motif. Caspases are synthesized as inactive proenzymes comprising
an N-terminal peptide (Prodomain) together with one large and one
small subunit. The crystal structures of both caspase-1 and
caspase-3 show that the active enzyme is a heterotetramer,
containing two small and two large subunits. Activation of caspases
during apoptosis results in the cleavage of critical cellular
substrates, including poly (ADP-ribose) polymerase and lamins, so
precipitating the dramatic morphological changes of apoptosis
(Cohen, 1997, Biochem. J. 326:1-16). Therefore, it is also within
the scope of the present invention to use a caspase as an
apoptosis-inducing moiety.
[0045] Recently a few new proteins were cloned and identified as
factors required for mediating activity of proteins, mainly
caspases, involved in the apoptosis pathway. One factor was
identified as the previously known electron transfer protein,
cytochrome c (Lin et al., 1996, Cell 86;147-157), designed as
Apaf-2. In addition to cytochrome c the activation of caspase-3
requires two other cytosolic factors-Apaf-1 and Apaf-3. Apaf-1 is a
protein homologous to C. elegans CED-4, and Apaf-3 was identified
as a member of the caspase family, caspase-9. Both factors bind to
each other via their respective NH2-terminal CED-3 homologous
domains, in the presence of cytochrome c, an event that leads to
caspase-9 activation. Activated caspase-9 in turn cleaves and
activates caspase-3 (Liu et al., 1996, Cell 86:147-157; Zou et al.,
1997, Cell 90:405-413; Li et al., 1997, Cell 91:479-489). Another
protein involved in the apoptotic pathway is DNA fragmentation
factor (DFF), a heterodimer of 45 and 40 kd subunits that functions
downstream of caspase-3 to trigger fragmentation of genomic DNA
into nucleosomal segments (Liu et al., 1997, Cell 89:175-184).
5.2. Expression of Chimeric Proteins
[0046] In accordance with the invention, a polynucleotide which
encodes a chimeric protein, mutant polypeptides, biologically
active fragments of chimeric protein, or functional equivalents
thereof, may be used to generate recombinant DNA molecules that
direct the expression of the chimeric protein, chimeric peptide
fragments, or a functional equivalent thereof, in appropriate host
cells.
[0047] Due to the inherent degeneracy of the genetic code, other
DNA sequences which encode substantially the same or a functionally
equivalent amino acid sequence, may be used in the practice of the
invention for the cloning and expression of the chimeric protein.
Such DNA sequences include those which are capable of hybridizing
to the chimeric sequences or their complementary sequences under
stringent conditions. The phrase "stringent conditions" as used
herein refers to those hybridizing conditions that (1) employ low
ionic strength and high temperature for washing, for example, 0.015
M NaCl/0.0015 M sodium citrate/0.1% SDS at 50.degree. C.; (2)
employ during hybridization a denaturing agent such as formamide,
for example, 50% (vol/vol) formamide with 0.1% bovine serum
albumin/0.1%. Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate
at 42.degree. C.; or (3) employ 50% formamide, 5.times.SSC (0.75 M
NaCl, 0.075 M Sodium pyrophosphate, 5.times.Denhardt's solution,
sonicated salmon sperm DNA (50 .mu.g/ml), 0.1% SDS, and 10% dextran
sulfate at 42.degree. C., with washes at 42.degree. C. in
0.2.times.SSC and 0.1% SDS.
[0048] Altered DNA sequences which may be used in accordance with
the invention include deletions, additions or substitutions of
different nucleotide residues resulting in a sequence that encodes
the same or a functionally equivalent fusion gene product. The gene
product itself may contain deletions, additions or substitutions of
amino acid residues within a chimeric sequence, which result in a
silent change thus producing a functionally equivalent chimeric
protein. Such amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues
involved. For example, negatively charged amino acids include
aspartic acid and glutamic acid; positively charged amino acids
include lysine, histidine and arginine; amino acids with uncharged
polar head groups having similar hydrophilicity values include the
following: glycine, asparagine, glutamine, serine, threonine,
tyrosine; and amino acids with nonpolar head groups include
alanine, valine, isoleucine, leucine, phenylalanine, proline,
methionine, tryptophan.
[0049] The DNA sequences of the invention may be engineered in
order to alter a chimeric coding sequence for a variety of ends,
including but not limited to, alterations which modify processing
and expression of the gene product. For example, mutations may be
introduced using techniques which are well known in the art, e.g.,
site-directed mutagenesis, to insert new restriction sites, to
alter glycosylation patterns, phosphorylation, etc.
[0050] In an alternate embodiment of the invention, the coding
sequence of the chimeric protein could be synthesized in whole or
in part, using chemical methods well known in the art. See, for
example, Caruthers et al., 1980, Nuc. Acids Res. Symp. Ser.
7:215-233; Crea and Horn, 180, Nuc. Acids Res. 9(10):2331;
Matteucci and Caruthers, 1980, Tetrahedron Letter 21:719; and Chow
and Kempe, 1981, Nuc. Acids Res. 9(12):2807-2817. For example,
active domains of the moieties can be synthesized by solid phase
techniques, cleaved from the resin, and purified by preparative
high performance liquid chromatography followed by chemical linkage
to form a chimeric protein. (e.g., see Creighton, 1983, Proteins
Structures And Molecular Principles, W.H. Freeman and Co., N.Y. pp.
50-60). The composition of the synthetic peptides may be confirmed
by amino acid analysis or sequencing (e.g., the Edman degradation
procedure; see Creighton, 1983, Proteins, Structures and Molecular
Principles, W.H. Freeman and Co., N.Y., pp. 34-49). Alternatively,
the two moieties of the chimeric protein produced by synthetic or
recombinant methods may be conjugated by chemical linkers according
to methods well known in the art (Brinkmann and Pastan, 1994,
Biochemica et Diophysica Acta 1198:27-45).
[0051] In order to express a biologically active chimeric protein,
the nucleotide sequence coding for a chimeric protein, or a
functional equivalent, is inserted into an appropriate expression
vector, i.e., a vector which contains the necessary elements for
the transcription and translation of the inserted coding sequence.
The chimeric gene products as well as host cells or cell lines
transfected or transformed with recombinant chimeric expression
vectors can be used for a variety of purposes. These include but
are not limited to generating antibodies (i.e., monoclonal or
polyclonal) that bind to epitopes of the proteins to facilitate
their purification.
[0052] Methods which are well known to those skilled in the art can
be used to construct expression vectors containing the chimeric
protein coding sequence and appropriate
transcriptional/translational control signals. These methods
include in vitro recombinant DNA techniques, synthetic techniques
and in vivo recombination/genetic recombination. See, for example,
the techniques described in Sambrook et al., 1989, Molecular
Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.
and Ausubel et al., 1989, Current Protocols in Molecular Biology,
Greene Publishing Associates and Wiley Interscience, N.Y.
[0053] A variety of host-expression vector systems may be utilized
to express the chimeric protein coding sequence. These include but
are not limited to microorganisms such as bacteria transformed with
recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression
vectors containing the chimeric protein coding sequence; yeast
transformed with recombinant yeast expression vectors containing
the chimeric protein coding sequence; insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus)
containing the chimeric protein coding sequence; plant cell systems
infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or
transformed with recombinant plasmid expression vectors (e.g., Ti
plasmid) containing the chimeric protein coding sequence; or animal
cell systems. It should be noted that since most apoptosis-inducing
proteins cause programmed cell death in mammalian cells, it is
preferred that the chimeric protein of the invention be expressed
in prokaryotic or lower eukaryotic cells. Section 6 illustrates
that IL2-Bax may be efficiently expressed in E. coli.
[0054] The expression elements of each system vary in their
strength and specificities. Depending on the host/vector system
utilized, any of a number of suitable transcription and translation
elements, including constitutive and inducible promoters, may be
used in the expression vector. For example, when cloning in
bacterial systems, inducible promoters such as pL of bacteriophage
.lamda.; plac, ptrp, ptac (ptrp-lac hybrid promoter;
cytomegalovirus promoter) and the like may be used; when cloning in
insect cell systems, promoters such as the baculovirus polyhedrin
promoter may be used; when cloning in plant cell systems, promoters
derived from the genome of plant cells (e.g., heat shock promoters;
the promoter for the small subunit of RUBISCO; the promoter for the
chlorophyll .alpha./.beta. binding protein) or from plant viruses
(e.g., the 35S RNA promoter of CaMV; the coat protein promoter of
TMV) may be used; when cloning in mammalian cell systems, promoters
derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the adenovirus late
promoter; the vaccinia virus 7.5K promoter) may be used; when
generating cell lines that contain multiple copies of the chimeric
DNA, SV40-, BPV- and EBV-based vectors may be used with an
appropriate selectable marker.
[0055] In bacterial systems a number of expression vectors may be
advantageously selected depending upon the use intended for the
chimeric protein expressed. For example, when large quantities of
chimeric protein are to be produced, vectors which direct the
expression of high levels of protein products that are readily
purified may be desirable. Such vectors include but are not limited
to the pHL906 vector (Fishman et al., 1994, Biochem. 33:6235-6243),
the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
2:1791), in which the chimeric protein coding sequence may be
ligated into the vector in frame with the lacZ coding region so
that a hybrid AS-lacZ protein is produced; pIN vectors (Inouye
& Inouye, 1985, Nucleic acids Res. 13:3101-3109; Van Heeke
& Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the
like.
[0056] An alternative expression system which could be used to
express chimeric protein is an insect system. In one such system,
Autographa californica nuclear polyhidrosis virus (AcNPV) is used
as a vector to express foreign genes. The virus grows in Spodoptera
frugiperda cells. The chimeric protein coding sequence may be
cloned into non-essential regions (for example the polyhedrin gene)
of the virus and placed under control of an AcNPV promoter (for
example the polyhedrin promoter). Successful insertion of the
chimeric protein coding sequence will result in inactivation of the
polyhedrin gene and production of non-occluded recombinant virus
(i.e., virus lacking the proteinaceous coat coded for by the
polyhedrin gene). These recombinant viruses are then used to infect
Spodoptera frugiperda cells in which the inserted gene is
expressed. (e.g., see Smith et al., 1983, J. Viol. 46:584; Smith,
U.S. Pat. No. 4,215,051).
[0057] Specific initiation signals may also be required for
efficient translation of the inserted chimeric protein coding
sequence. These signals include the ATG initiation codon and
adjacent sequences. In cases where the entire chimeric gene,
including its own initiation codon and adjacent sequences, is
inserted into the appropriate expression vector, no additional
translational control signals may be needed. However, in cases
where the chimeric protein coding sequence does not include its own
initiation codon, exogenous translational control signals,
including the ATG initiation codon, must be provided. Furthermore,
the initiation codon must be in phase with the reading frame of the
chimeric protein coding sequence to ensure translation of the
entire insert. These exogenous translational control signals and
initiation codons can be of a variety of origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of appropriate transcription enhancer elements,
transcription terminators, etc. (see Bittner et al., 1987, Methods
in Enzymol. 153:516-544).
[0058] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function of the
protein. The presence of consensus N-glycosylation sites in a
chimeric protein may require proper modification for optimal
chimeric protein function. Different host cells have characteristic
and specific mechanisms for the post-translational processing and
modification of proteins. Appropriate cell lines or host systems
can be chosen to ensure the correct modification and processing of
the chimeric protein. To this end, eukaryotic host cells which
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the chimeric
protein may be used. Such mammalian host cells include but are not
limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, WI38, and the
like.
[0059] For long-term, high-yield production of recombinant chimeric
proteins, stable expression is preferred. For example, cell lines
which stably express the chimeric protein may be engineered. Rather
than using expression vectors which contain viral origins of
replication, host cells can be transformed with a chimeric coding
sequence controlled by appropriate expression control elements
(e.g., promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of foreign DNA, engineered cells may be allowed to
grow for 1-2 days in an enriched media, and then are switched to a
selective media. The selectable marker in the recombinant plasmid
confers resistance to the selection and allows cells to stably
integrate the plasmid into their chromosomes and grow to form foci
which in turn can be cloned and expanded into cell lines.
[0060] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567;
O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg,
1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,
1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to
hygromycin (Santerre et al., 1984, Gene 30:147) genes. Additional
selectable genes have been described, namely trpB, which allows
cells to utilize indole in place of tryptophan; hisD, which allows
cells to utilize histinol in place of histidine (Hartman &
Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85:8047); and ODC
(ornithine decarboxylase) which confers resistance to the ornithine
decarboxylase inhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO
(McConlogue L., 1987, In: Current Communications in Molecular
Biology, Cold Spring Harbor Laboratory ed.).
5.3. Protein Purification
[0061] The chimeric proteins of the invention can be purified by
art-known techniques such as high performance liquid
chromatography, ion exchange chromatography, gel electrophoresis,
affinity chromatography and the like. The actual conditions used to
purify a particular protein will depend, in part, on factors such
as net charge, hydrophobicity, hydrophilicity, etc., and will be
apparent to those having skill in the art.
[0062] For affinity chromatography purification, any antibody which
specifically binds the protein may be used. For the production of
antibodies, various host animals, including but not limited to
rabbits, mice, rats, etc., may be immunized by injection with a
chimeric protein or a fragment thereof. The protein may be attached
to a suitable carrier, such as bovine serum albumin (BSA), by means
of a side chain functional group or linkers attached to a side
chain functional group. Various adjuvants may be used to increase
the immunological response, depending on the host species,
including but not limited to, Freund's (complete and incomplete),
mineral gels such as aluminum hydroxide, surface active substances
such as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, dinitrophenol, and
potentially useful human adjuvants such as BCG (bacilli
Calmette-Guerin) and Corynebacterium parvum.
[0063] Monoclonal antibodies to a chimeric protein may be prepared
using any technique which provides for the production of antibody
molecules by continuous cell lines in culture. These include but
are not limited to the hybridoma technique originally described by
Koehler and Milstein (1975, Nature 256:495-497), the human B-cell
hybridoma technique, (Kosbor et al., 1983, Immunology Today 4:72;
Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) and
the EBV-hybridoma technique (Cole et al., 1985, Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In
addition, techniques developed for the production of "chimeric
antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. U.S.A.
81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et
al., 1985, Nature 314:452-454) by splicing the genes from a mouse
antibody molecule of appropriate antigen specificity together with
genes from a human antibody molecule of appropriate biological
activity can be used. Alternatively, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778) can
be adapted to produce chimeric protein-specific single chain
antibodies for chimeric protein purification and detection.
5.4. Uses of Chimeric Proteins
[0064] Once a chimeric protein is expressed and purified, its
identity and functional activities can be readily determined by
methods well known in the art. For example, antibodies to the two
moieties of the protein may be used to identify the protein in
Western blot analysis. In addition, the chimeric protein can be
tested for specific binding to target cells in binding assays using
a fluorescent-labeled or radiolabelled secondary antibody.
5.4.1. In Vitro and Ex Vivo Uses
[0065] The chimeric proteins of the invention are useful for
targeting specific cell types in a cell mixture, and eliminating
the target cells by inducing apoptosis. For example, IL2-Bax may be
used to purge IL2R.sup.+ leukemic cells in a bone marrow
preparation or mobilized peripheral blood prior to infusion of the
cells into a recipient following ablative therapy. In addition,
this chimeric protein may be used to deplete IL2R.sup.+ cells in a
donor cell preparation prior to allogeneic or xenogeneic bone
marrow transplantation in order to reduce the development of
graft-versus-host disease. It can also be used for ex vivo purging
of specific cell subsets in any body fluids such as cerebral spinal
fluid, pleural fluid and sinovial fluid.
[0066] The chimeric protein of the invention is also useful as a
diagnostic reagent. For example, IL2-Bax may be used to detect the
presence of autoimmune IL2R-expressing cells in a body fluid or to
detect the tissue origin of an IL2R.sup.+ lymphoma. The binding of
a chimeric protein to a target cell can be readily detected by
using a secondary antibody specific for the apoptosis-inducing
moiety. In that connection, the secondary antibody or the chimeric
protein can be linked to a detectable label such as fluorescein, an
enzyme or a radioisotope to facilitate the detection of binding of
the chimeric protein to a cell.
5.4.2. In Vivo Uses
[0067] The chimeric proteins of the invention may be administered
to a subject per se or in the form of a pharmaceutical composition
for the treatment of cancer, autoimmunity, transplantation
rejection, post-traumatic immune responses and infectious diseases
by targeting viral antigens such as gp120 of HIV. More
specifically, IL2-Bax is useful for eliminating activated
IL2R.sup.+ cells involved in immune cell-mediated disorder,
including lymphoma; autoimmunity, transplantation rejection,
graft-versus-host disease, ischemia and stroke. Pharmaceutical
compositions comprising the proteins of the invention may be
manufactured by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping or lyophilizing processes. Pharmaceutical compositions
may be formulated in conventional manner using one or more
physiologically acceptable carriers, diluents, excipients or
auxiliaries which facilitate processing of the proteins into
preparations which can be used pharmaceutically. Proper formulation
is dependent upon the route of administration chosen.
[0068] For topical administration the proteins of the invention may
be formulated as solutions, gels, ointments, creams, suspensions,
etc. as are well-known in the art.
[0069] Systemic formulations include those designed for
administration by injection, e.g. subcutaneous, intravenous,
intramuscular, intrathecal or intraperitoneal injection, as well as
those designed for transdermal, transmucosal, inhalation, oral or
pulmonary administration.
[0070] For injection, the proteins of the invention may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hanks's solution, Ringer's solution, or
physiological saline buffer. The solution may contain formulatory
agents such as suspending, stabilizing and/or dispersing
agents.
[0071] Alternatively, the proteins may be in powder form for
constitution with a suitable vehicle, e.g., sterile pyrogen-free
water, before use.
[0072] For transmucosal administration, penetrants appropriate to
the barrier to be permeated are used in the formulation. Such
penetrants are generally known in the art.
[0073] For oral administration, the proteins can be readily
formulated by combining the proteins with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
proteins of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions and
the like, for oral ingestion by a patient to be treated. For oral
solid formulations such as, for example, powders, capsules and
tablets, suitable excipients include fillers such as sugars, e.g.
lactose, sucrose, mannitol and sorbitol; cellulose preparations
such as maize starch, wheat starch, rice starch, potato starch,
gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,
and/or polyvinylpyrrolidone (PVP); granulating agents; and binding
agents. If desired, disintegrating agents may be added, such as the
cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0074] If desired, solid dosage forms may be sugar-coated or
enteric-coated using standard techniques.
[0075] For oral liquid preparations such as, for example,
suspensions, elixirs and solutions, suitable carriers, excipients
or diluents include water, glycols, oils, alcohols, etc.
Additionally, flavoring agents, preservatives, coloring agents and
the like may be added.
[0076] For buccal administration, the proteins may take the form of
tablets, lozenges, etc. formulated in conventional manner.
[0077] For administration by inhalation, the proteins for use
according to the present invention are conveniently delivered in
the form of an aerosol spray from pressurized packs or a nebulizer,
with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of gelatin for use in an inhaler or insufflator may be
formulated containing a powder mix of the protein and a suitable
powder base such as lactose or starch.
[0078] The proteins may also be formulated in rectal or vaginal
compositions such as suppositories or retention enemas, e.g,
containing conventional suppository bases such as cocoa butter or
other glycerides.
[0079] In addition to the formulations described previously, the
proteins may also be formulated as a depot preparation. Such long
acting formulations may be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the proteins may be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0080] Alternatively, other pharmaceutical delivery systems may be
employed. Liposomes and emulsions are well known examples of
delivery vehicles that may be used to deliver proteins of the
invention. Certain organic solvents such as dimethylsulfoxide also
may be employed, although usually at the cost of greater toxicity.
Additionally, the proteins may be delivered using a
sustained-release system, such as semipermeable matrices of solid
polymers containing the therapeutic agent. Various of
sustained-release materials have been established and are well
known by those skilled in the art. Sustained-release capsules may,
depending on their chemical nature, release the proteins for a few
weeks up to over 100 days. Depending on the chemical nature and the
biological stability of the chimeric protein, additional strategies
for protein stabilization may be employed.
[0081] As the proteins of the invention may contain charged side
chains or termini, they may be included in any of the
above-described formulations as the free acids or bases or as
pharmaceutically acceptable salts. Pharmaceutically acceptable
salts are those salts which substantially retain the biologic
activity of the free bases and which are prepared by reaction with
inorganic acids. Pharmaceutical salts tend to be more soluble in
aqueous and other protic solvents than are the corresponding free
baser forms.
5.4.3. Effective Dosages
[0082] The proteins of the invention will generally be used in an
amount effective to achieve the intended purpose. For use to treat
or prevent a disease condition, the proteins of the invention, or
pharmaceutical compositions thereof, are administered or applied in
a therapeutically effective amount. A therapeutically effective
amount is an amount effective to ameliorate or prevent the
symptoms, or prolong the survival of, the patient being treated.
Determination of a therapeutically effective amount is well within
the capabilities of those skilled in the art, especially in light
of the detailed disclosure provided herein.
[0083] For systemic administration, a therapeutically effective
dose can be estimated initially from in vitro assays. For example,
a dose can be formulated in animal models to achieve a circulating
concentration range that includes the IC.sub.50 as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans.
[0084] Initial dosages can also be estimated from in vivo data,
e.g., animal models, using techniques that are well known in the
art. One having ordinary skill in the art could readily optimize
administration to humans based on animal data.
[0085] Dosage amount and interval may be adjusted individually to
provide plasma levels of the proteins which are sufficient to
maintain therapeutic effect. Usual patient dosages for
administration by injection range from about 0.1 to 5 mg/kg/day,
preferably from about 0.5 to 1 mg/kg/day. Therapeutically effective
serum levels may be achieved by administering multiple doses each
day.
[0086] In cases of local administration or selective uptake, the
effective local concentration of the proteins may not be related to
plasma concentration. One having skill in the art will be able to
optimize therapeutically effective local dosages without undue
experimentation.
[0087] The amount of protein administered will, of course, be
dependent on the subject being treated, on the subject's weight,
the severity of the affliction, the manner of administration and
the judgment of the prescribing physician.
[0088] The therapy may be repeated intermittently while symptoms
detectable or even when they are not detectable. The therapy may be
provided alone or in combination with other drugs. In the case of
autoimmune disorders, the drugs that may be used in combination
with IL2-Bax of the invention include, but are not limited to,
steroid and non-steroid anti-inflammatory agents.
5.4.4. Toxicity
[0089] Preferably, a therapeutically effective dose of the chimeric
proteins described herein will provide therapeutic benefit without
causing substantial toxicity.
[0090] Toxicity of the proteins described herein can be determined
by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., by determining the LD.sub.50 (the dose
lethal to 50% of the population) or the LD.sub.100 (the dose lethal
to 100% of the population). The dose ratio between toxic and
therapeutic effect is the therapeutic index. Proteins which exhibit
high therapeutic indices are preferred. The data obtained from
these cell culture assays and animal studies can be used in
formulating a dosage range that is not toxic for use in human. The
dosage of the proteins described herein lies preferably within a
range of circulating concentrations that include the effective dose
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (See, e.g., Fingl et al., 1975,
In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1).
[0091] The invention having been described, the following examples
are offered by way of illustration and not limitation.
6. EXAMPLE
Production of a Chimeric Protein that Induced IL2R.sup.+
Cell-Specific Apoptosis
6.1. Materials and Methods
6.1.1. Construction of IL2-Bax Coding Sequence
[0092] A plasmid for the expression of IL2-Bax chimeric protein
under the control of the T7 promoter was constructed as shown in
FIG. 1. pHL906 which carried the fusion gene IL2-PE40 was cut with
HindIII and PpuMI to remove the PE sequence, and the vector
fragment was eluted (Fishman et al., 1994, Biochem. 33:6235-6243).
A cDNA encoding-human Bax-.alpha. was obtained by reverse
transcription-polymerase chain reaction (RT-PCR), using RNA
isolated from fresh human lymphocytes. Total RNA was isolated and
was reverse transcribed into first strand cDNA, using the reverse
transcription system (Promega, USA) under conditions recommended by
the manufacturer. The cDNA was diluted to a total volume of 1 ml
with 10 mM Tris-HCl pH 7.6, 1 mM EDTA and stored at 4.degree. C.
The Bax-encoding fragment was generated by PCR using this cDNA and
a pair of synthetic oligonucleotide primers: 5'
CGCAATTCAAGCTTTGGACGGGTCCGGGGGA 3' (SEQ ID NO:3) (sense) and 5'
CGGAATTCAGGTCGTTCAGCCCATCTTCTTC 3' (SEQ ID NO:4) (antisense)
covering the entire coding region. The reaction mixture was
incubated in a DNA thermal cycler (MJ Research Inc., Watertown,
Mass.) for 33 cycles. Each cycle consisted of 1 min. at 95.degree.
C., 1 min. at 65.degree. C. and 2 min. at 72.degree. C. The
Bax-encoding fragment was digested with EcoRI and HindIII enzymes
and ligated with the pHL906 vector. The resulting plasmid,
designated pSY1, contained the human IL2 coding sequence fused to
the 5' end of the human Bax coding sequence. The plasmid was
confirmed by restriction endonuclease digestion and DNA sequence
analysis. The nucleotide and deduced amino acid sequences (SEQ ID
NOS:1 and 2) of the chimeric molecule referred to as IL2-Bax are
disclosed in FIG. 2.
6.1.2. Protein Expression and Partial Purification
[0093] The pSY1 plasmid containing the fused coding sequences was
transformed into E. coli strain BL21 (.lamda.DE3) and the IL2-Bax
chimeric protein was expressed. A pellet of expressing cells was
suspended in 50 mM Tris-HCl pH 8.0, 1 mM EDTA containing 0.2 mg/ml
lysozyme, sonicated (three 30-s bursts) and centrifuged at
30,000.times.g for 30 min. The supernatant (soluble fraction) was
removed and kept for analysis. The pellet was denatured in one of
three extraction buffers:
[0094] 1) Extraction buffer A: 6 M Guanidine-HCl, 0.1 M Tris-HCl pH
8.6, 1 mM EDTA, 0.05 M NaCl, and 10 mM DTT, and stirred for 30 min.
at 4.degree. C. The suspension was cleared by centrifugation at
30,000.times.g for 15 min. and the pellet discarded. The protein
solution was diluted 1:100 in refolding buffer (50 mM Tris-HCl, pH
8.0, 1 mM EDTA, 0.25 M NaCl, 0.25 M L-arginine, and 5 mM
dithiothreitol) and kept at 4.degree. C. for 48 h. The refolded
protein solution was dialyzed against phosphate-buffered saline
(PBS).
[0095] 2) Extraction buffer B: 20 mM Tris-HCl pH 7.4, 150 mM NaCl,
1 mM EDTA, 1% NP-40, 1% deoxycholic acid, 0.1% SDS. Before testing
its activity, the fraction was dialyzed against PBS.
[0096] 3) Extraction buffer C: 8 M Urea, 50 mM Tris HCl pH 8.0, 1
mM EDTA, 10 mM DTT (v/w 1:1) for 1 hr, centrifuged at
35,000.times.g for 15 min. The supernatant was diluted 1:100 with
refolding buffer (see above) without dithiothreitol.
[0097] The protein profile of various fractions (soluble fraction,
insoluble fraction-treated in three different protocols) were
characterized by gel electrophoresis (FIG. 3).
6.1.3. Western Blot Analysis
[0098] The electrophoresis samples were transferred onto
nitrocellulose and immunoblotted as described (Fishman and
Lorberboum-Galski, 1997, Eur. J. Immunol. 27:486-494). The ECL
detection kit (Amersham, Bukinghamshire, UK) was used according to
the manufacturer's instructions. A protein extract from MCF-7 cells
(breast carcinoma cell line), known to express the Bax protein, was
used as a positive control. Anti-human Bax was obtained from
Pharmingen (San Diego, Calif.) and used at a dilution of 1:2,500.
Anti-human IL2 was obtained from Endogen and used at a dilution of
1:5,000.
6.1.4. Assay for Identifying Apoptotic Cells
[0099] Human peripheral blood lymphocytes from healthy donors were
separated using Ficoll-Isopaque gradient (1.077) (Pharmacia) and
used immediately. Lymphocytes were cultured in 5% CO.sub.2 in air
in RPMI 1640 medium supplemented with 10% fetal calf serum, 200
.mu.g/ml L-glutamate, 50 .mu.g/ml penicillin, 50 .mu.g/ml
streptomycin, 50 .mu.g/ml glutamine and 5.times.10.sup.-5 M
2-.beta. mercaptoethanol. Increasing concentrations of IL2-Bax
(insoluble fraction, 6M Gu-HCl treated) were added to the
lymphocytes for 22 hr. Cells were then stained with:
[0100] A. Propidium Iodide (PI, 3.5 .mu.g/ml)
[0101] B. Propidium Iodide with a detergent for measuring cell
cycle (0.7 ml of the PI buffer: 50 .mu.g/ml PI, 0.1% Na-citrate,
0.1% Triton x100, were added to a cell pellet of .about.10.sup.6
cells). Cells were then analyzed by FACS.
[0102] HUT102 cells (target T cells) and CEM cells (non-target T
cells) were incubated overnight with increasing concentrations of
IL2-Bax. The 200.times.g centrifuged cell pellet was fixed in 2 ml
cold 70% ethanol at 4.degree. C. for 60 min. The cells were then
centrifuged, washed in 1 ml PBS and resuspended in 0.5 ml PBS. 0.5
ml RNAse (Type I-A, Sigma, St. Louis, Mo., 1 mg/ml in PBS) was
added to the sample, followed by gentle mixing with 1 ml PI (Sigma,
100 .mu.g/ml PBS) solution. The mixed cells were incubated in the
dark at room temperature for 15 min. and kept at 4.degree. C. in
the dark until measured. The PI fluorescence of the individual
nuclei was measured using FACS flow cytometer. The forward scatter
and side scatter of particles were simultaneously measured. Cell
debris were excluded from analysis by appropriately raising the
forward scatter threshold (Nicoletti et al., 1991, J. Immunol.
Meth. 139:271-279).
6.1.5. Cytotoxicity Assay
[0103] Cells (10.sup.4 in 0.2 ml culture medium) were seeded in
96-well microplates, followed by the addition of various
concentrations of the chimeric protein (diluted with 0.25% BSA in
PBS). After a 24 hour incubation, [.sup.3H]leucine (2-5 uci/well)
were added for 6-13 hr. The plates were then stored at -70.degree.
C. for several hours, followed by quick thawing at 37.degree. C.
This step was omitted with targets cells growing in suspension. The
cells were harvested on filters and the incorporated radioactivity
was measured with a .beta. counter. Results were expressed as the
percent incorporation of the control experiments in which the cells
were not exposed to any protein. All assays were carried out in
triplicates.
6.2. Results
[0104] An expression plasmid encoding an IL2-Bax chimeric protein
was constructed under the control of the T7 promoter. The plasmid
was expressed in E. coli and the chimeric protein was extracted.
The protein was further characterized by Western blot analysis
using antibodies against Bax and IL2 (FIG. 4). The chimeric protein
reacted with the antibodies to Bax and to IL2, confirming the
cloning and production of in-frame full-length IL2-Bax chimeric
protein.
[0105] The cytotoxic activity of the IL2-Bax chimeric protein was
tested on HUT102 and MT-1 cells (human T-cell lines), and 2B4 cells
(mouse T-cells); all known to express the high affinity receptor
for IL2, by a quantitative assay, in which inhibition of protein
synthesis was measured. The soluble and insoluble fractions
generated from treatment under three different conditions were
tested in cytotoxicity assays as described by Lorberboum-Galski et
al. (1988, J. Biol. Chem. 263:18650-18656). As shown in FIG. 5A,
all three IL2R-expressing cell lines were responsive to the
chimeric protein in a dose dependent manner, although with
different sensitivities. This may be attributed to the different
number of IL2R expressed on each cell line. The insoluble fraction
treated with ether extraction buffer containing Gu-HCl or SDS
exhibited the highest activity toward the various cells. Therefore,
further experiments were performed with mainly the partially
purified fractions (insoluble fraction extracted with Gu-HCl or
SDS). In all experiments IL2-PE chimeric proteins, previously shown
to be cytotoxic to IL2R.sup.+ cells, were used as positive
control.
[0106] The effects of IL2-Bax were also tested on various IL2R
negative cells: CEM cells (a human T-cell line lacking the IL2R)
and Km3 (a human non-T, non-B stem cell line). FIG. 5B shows that
these cell lines were unaffected by the IL2-Bax chimeric protein.
Since the fraction treated with Gu-HCl was active without any
non-specific cytotoxicity, this fraction was further used in
subsequent experiments.
[0107] The ability of IL2-Bax to induce apoptosis in IL2R.sup.+
cells was determined in an apoptosis assay. FIG. 6A-6D shows an
increase of apoptotic cells in a freshly isolated lymphocyte
population treated with IL2-Bax, and the effects of the chimeric
protein were dose dependent. The apoptotic cells ranged from 2% to
14% (Table 1A, B) of the total cell population (FIG. 7A-7C). In
that regard, it should be noted that freshly isolated lymphocytes
from healthy donors usually contain only low levels of cells
expressing the IL2R, thus the percentage of cells expected to be
targeted by IL2-Bax in fresh lymphocytes is low.
[0108] Dexamethasone (10.sup.-7 M), a known inducer for apoptosis
in various cells, was used in all experiments to follow apoptosis.
However, it is well known that various cells respond differently
to, if at all, to this reagent. Dexamethasone was also shown to be
a weak inducer of apoptosis in fresh lymphocytes (FIG. 6B). 2B4
cells, known to react very strongly to this agent were used as
control cells to detect apoptosis. In conclusion, recombinant
chimeric protein IL2-Bax was specifically cytotoxic to IL2R.sup.+
cells, but did not affect IL2R.sup.- cells. Furthermore, the
cytotoxic effects of the chimeric protein were mediated by an
induction of apoptosis, as evidenced by its ability to induce
programmed cell death in freshly isolated human lymphocytes.
TABLE-US-00001 TABLE 1 Effect of IL2-Bax on Fresh Lymphocytes
Analyzed by FACS A. Experiment No. 1 Treatment M1 M2 M1 - 2 control
7.1 1.9 5.2 dexamethasone 9.5 2.8 6.7 IL2-Bax, 1 .mu.g 17.5 4 13.5
IL2-Bax, 5 .mu.g 24.3 4.3 20 IL2-Bax, 10 .mu.g 42.1 9.1 35 B.
Experiment No. 2 Treatment UL UR UL + UR control 2.36 2.12 4.48
IL2-Bax, 1 .mu.g 4.98 5.56 10.54 IL2-Bax, 10 .mu.g 14.71 16.84
31.55 Each M1 or M2 values are the mean of duplicates. Each UL or
UR value is the mean of duplicates.
[0109] FIG. 8A-8E demonstrates the increase of an apoptotic-cell
population in HUT102 cells exposed to IL2-Bax in a dose dependent
manner (M1 values represent the sub-G1 apoptotic-cell population).
At the highest concentration tested, IL2-Bax induced a 3.6-fold
increase in the percentage of the apoptotic-cell population. In
contrast, CEM cells which lacked IL2R expression did not show an
increase in the apoptotic cell population (FIG. 9A-9C), confirming
the specificity of the effects of IL2-Bax.
[0110] The present invention is not to be limited in scope by the
exemplified embodiments which are intended as illustrations of
single aspects of the invention and any sequences which are
functionally equivalent are within the scope of the invention.
Indeed, various modifications of the invention in addition to those
shown and described herein will become apparent to those skilled in
the art from the foregoing description and accompanying drawings.
Such modifications are intended to fall within the scope of the
appended claims.
[0111] All publications cited herein are incorporated by reference
in their entirety.
Sequence CWU 1
1
101996DNAHomo sapiensCDS(1)...(993) 1atg gca gat cct act tca agt
tct aca aag aaa aca cag cta caa ctg 48Met Ala Asp Pro Thr Ser Ser
Ser Thr Lys Lys Thr Gln Leu Gln Leu 1 5 10 15gag cat tta ctg ctg
gat tta cag atg att ttg aat gga att aat aat 96Glu His Leu Leu Leu
Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 20 25 30tac aag aat ccc
aaa ctc acc agg atg ctc aca ttt aag ttt tac atg 144Tyr Lys Asn Pro
Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 35 40 45ccc aag aag
gcc aca gaa ctg aaa cat ctt cag tgt cta gaa gaa gaa 192Pro Lys Lys
Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 50 55 60ctc aaa
cct ctg gag gaa gtg cta aat tta gct caa agc aaa aac ttt 240Leu Lys
Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 65 70 75
80cac tta aga ccc agg gac tta atc agc aat atc aac gta ata gtt ctg
288His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu
85 90 95gaa cta aag gga tct gaa aca aca ttc atg tgt gaa tat gct gat
gag 336Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp
Glu 100 105 110aca gca acc att gta gaa ttt ctg aac aga tgg att acc
ttt tgt caa 384Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr
Phe Cys Gln 115 120 125agc atc atc tca aca atc ccc gag ggc gaa gct
ttg gac ggg tcc ggg 432Ser Ile Ile Ser Thr Ile Pro Glu Gly Glu Ala
Leu Asp Gly Ser Gly 130 135 140gag cag ccc aga ggc ggg ggg ccc acc
agc tct gag cag atc atg aag 480Glu Gln Pro Arg Gly Gly Gly Pro Thr
Ser Ser Glu Gln Ile Met Lys145 150 155 160aca ggg gcc ctt ttg ctt
cag ggt ttc atc cag gat cga gca ggg cga 528Thr Gly Ala Leu Leu Leu
Gln Gly Phe Ile Gln Asp Arg Ala Gly Arg 165 170 175atg ggg ggg gag
gca ccc gag ctg gcc ctg gac ccg gtg cct cag gat 576Met Gly Gly Glu
Ala Pro Glu Leu Ala Leu Asp Pro Val Pro Gln Asp 180 185 190gcg tcc
acc aag aag ctg agc gag tgt ctc aag cgc atc ggg gac gaa 624Ala Ser
Thr Lys Lys Leu Ser Glu Cys Leu Lys Arg Ile Gly Asp Glu 195 200
205ctg gac agt aac atg gag ctg cag agg atg att gcc gcc gtg gac aca
672Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile Ala Ala Val Asp Thr
210 215 220gac tcc ccc cga gag gtc ttt ttc cga gtg gca gct gac atg
ttt tct 720Asp Ser Pro Arg Glu Val Phe Phe Arg Val Ala Ala Asp Met
Phe Ser225 230 235 240gac ggc aac ttc aac tgg ggc cgg gtt gtc gcc
ctt ttc tac ttt gcc 768Asp Gly Asn Phe Asn Trp Gly Arg Val Val Ala
Leu Phe Tyr Phe Ala 245 250 255agc aaa ctg gtg ctc aag gcc ctg tgc
acc aag gtg ccg gaa ctg atc 816Ser Lys Leu Val Leu Lys Ala Leu Cys
Thr Lys Val Pro Glu Leu Ile 260 265 270aga acc atc atg ggc tgg aca
ttg gac ttc ctc cgg gag cgg ctg ttg 864Arg Thr Ile Met Gly Trp Thr
Leu Asp Phe Leu Arg Glu Arg Leu Leu 275 280 285ggc tgg atc caa gac
cag ggt ggt tgg gac ggc ctc ctc tcc tac ttt 912Gly Trp Ile Gln Asp
Gln Gly Gly Trp Asp Gly Leu Leu Ser Tyr Phe 290 295 300ggg acg ccc
acg tgg cag acc gtg acc atc ttt gtg gcg gga gtg ctc 960Gly Thr Pro
Thr Trp Gln Thr Val Thr Ile Phe Val Ala Gly Val Leu305 310 315
320acc gcc tcg ctc acc atc tgg aag aag atg ggc tga 996Thr Ala Ser
Leu Thr Ile Trp Lys Lys Met Gly 325 3302331PRTHomo sapiens 2Met Ala
Asp Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 1 5 10
15Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn
20 25 30Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr
Met 35 40 45Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu
Glu Glu 50 55 60Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser
Lys Asn Phe65 70 75 80His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile
Asn Val Ile Val Leu 85 90 95Glu Leu Lys Gly Ser Glu Thr Thr Phe Met
Cys Glu Tyr Ala Asp Glu 100 105 110Thr Ala Thr Ile Val Glu Phe Leu
Asn Arg Trp Ile Thr Phe Cys Gln 115 120 125Ser Ile Ile Ser Thr Ile
Pro Glu Gly Glu Ala Leu Asp Gly Ser Gly 130 135 140Glu Gln Pro Arg
Gly Gly Gly Pro Thr Ser Ser Glu Gln Ile Met Lys145 150 155 160Thr
Gly Ala Leu Leu Leu Gln Gly Phe Ile Gln Asp Arg Ala Gly Arg 165 170
175Met Gly Gly Glu Ala Pro Glu Leu Ala Leu Asp Pro Val Pro Gln Asp
180 185 190Ala Ser Thr Lys Lys Leu Ser Glu Cys Leu Lys Arg Ile Gly
Asp Glu 195 200 205Leu Asp Ser Asn Met Glu Leu Gln Arg Met Ile Ala
Ala Val Asp Thr 210 215 220Asp Ser Pro Arg Glu Val Phe Phe Arg Val
Ala Ala Asp Met Phe Ser225 230 235 240Asp Gly Asn Phe Asn Trp Gly
Arg Val Val Ala Leu Phe Tyr Phe Ala 245 250 255Ser Lys Leu Val Leu
Lys Ala Leu Cys Thr Lys Val Pro Glu Leu Ile 260 265 270Arg Thr Ile
Met Gly Trp Thr Leu Asp Phe Leu Arg Glu Arg Leu Leu 275 280 285Gly
Trp Ile Gln Asp Gln Gly Gly Trp Asp Gly Leu Leu Ser Tyr Phe 290 295
300Gly Thr Pro Thr Trp Gln Thr Val Thr Ile Phe Val Ala Gly Val
Leu305 310 315 320Thr Ala Ser Leu Thr Ile Trp Lys Lys Met Gly 325
330331DNAHomo sapiens 3cgcaattcaa gctttggacg ggtccggggg a
31431DNAHomo sapiens 4cggaattcag gtcgttcagc ccatcttctt c
3155PRTArtificial SequenceFlexible polylinker 5Gly Gly Gly Gly Ser
1 5614PRTArtificial SequenceLinker 6Glu Gly Lys Ser Ser Gly Ser Gly
Ser Glu Ser Lys Val Asp 1 5 10718PRTArtificial SequenceLinker 7Lys
Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10
15Leu Asp85PRTArtificial SequenceConserved active-site motif 8Gln
Ala Cys Xaa Gly 1 5914DNAArtificial SequencePortion of pSY1 plasmid
9aagctttgga cggg 141013DNAArtificial SequencePortion of pSY1
plasmid 10ggctgaagga cct 13
* * * * *