U.S. patent number 6,685,933 [Application Number 09/744,754] was granted by the patent office on 2004-02-03 for interferon .alpha. hybrids.
This patent grant is currently assigned to The United States of America as represented by the Department of Health and Human Services, The United States of America as represented by the Department of Health and Human Services. Invention is credited to Joseph B. Beiksz, Mark P. Hayes, Renqiu Hu, Kathryn C. Zoon.
United States Patent |
6,685,933 |
Zoon , et al. |
February 3, 2004 |
**Please see images for:
( Certificate of Correction ) ** |
Interferon .alpha. hybrids
Abstract
Hybrid human interferon-.alpha. polypeptides, and the
corresponding nucleic acid molecules, are disclosed. Pharmaceutical
compositions comprising these peptides, and the use of these
polypeptides to treat viral disease and regulate cell growth are
also provided.
Inventors: |
Zoon; Kathryn C. (Kensington,
MD), Hu; Renqiu (Bethesda, MD), Beiksz; Joseph B.
(Hyattsville, MD), Hayes; Mark P. (Hopkinton, MA) |
Assignee: |
The United States of America as
represented by the Department of Health and Human Services
(Washington, DC)
|
Family
ID: |
30447864 |
Appl.
No.: |
09/744,754 |
Filed: |
January 24, 2001 |
PCT
Filed: |
July 06, 1999 |
PCT No.: |
PCT/US99/15284 |
PCT
Pub. No.: |
WO00/06735 |
PCT
Pub. Date: |
February 10, 2000 |
Current U.S.
Class: |
424/85.4;
424/185.1; 424/278.1; 435/252.3; 435/320.1; 435/69.1; 530/350;
530/351; 536/23.1; 536/23.52 |
Current CPC
Class: |
C07K
14/56 (20130101); A61K 38/00 (20130101); C07K
2319/00 (20130101) |
Current International
Class: |
C07K
14/435 (20060101); C07K 14/56 (20060101); A61K
38/00 (20060101); C12N 005/00 (); C07K 014/00 ();
C07H 021/04 (); A61K 038/00 (); A61K 038/21 () |
Field of
Search: |
;435/69.1,69.5,69.51,69.7,70.1,71.2,325,252.3,320.1 ;530/350,351
;536/23.1,23.4,23.5,23.52 ;424/85.4,185.1,278.1 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Other References
Goeddel et al., "The structure of eight distinct cloned human
leukocyte interferon cDNAs," Nature, GB, Macmillan Journal Ltd.
London, 290:20-26, Mar. 5, 1981. .
Streuli et al., "Target cell specificity of two species of human
interferon-alpha produced in Escherichia coli and of hybrid
molecules drived from them," Proc. Natl. Acad. Sci. USA,
78(5):2848-2852, May 1981. .
Weck et al., "Antiviral activities of hybrids of two major human
leukocyte interferons," Nucleic acids. Res., 9(22):6153-6156, Nov.
25, 1981. .
Rehberg et al., "Specific molecular activities of recombinant and
hybrid leukocyte interferons," J. Biol. Chem., 257(19):11497-11502,
Oct. 10, 1982. .
Fish et al., "Human leukocyte interferon subtypes have different
antiproliferative and antiviral activities on human cells,"
Biochem. Biophys. Res. Commun., 112(2):537-546, Apr. 29, 1983.
.
Meister et al., "Biological activites and receptor binding of two
human recombinant interferons and their hybrids," J. Gen. Virol.,
67(8):1633-1643, Aug. 1986. .
Fidler et al., "Direct antiproliferative effects of recombinant
human interferon-alpha B/D hybrids on human tomor cell lines,"
Cancer Res., 47(8):2020-2027, Apr. 15, 1987. .
Horton et al., "Engineering hybrid genes without the use of
restriction enzymes: gene splicing by overlap extension," Gene, NL,
Elservier Biomedical Press, Amsterdam, 77:61-68, 1989. .
Alexanko et al., "Reconstruction of an epitope cabaple of binding
murine monoclonal antibodies NK2 within the sequence of human
leukocyte interferon alpha F by site-directed mutagenesis,"
Biochem. Biophys. Res. Commun., 169(3):1061-1067, Jun. 29, 1990.
.
Alexanko et al., "Mapping of an epitope of human leukocyte alpha
interferon A which is recognized by the murine monoclonal antibody
NK2," Biomed. Sci., 2(4):403-409, 1991. .
Sperber et al., "Anti-rhonoviral activity of recombinant and hybrid
species of interferon alpha," Antiviral Res., 22(2-3):121-129, Oct.
1993. .
Di Marco et al., "Mutational analysis of the structure-function
relationship in interferon-alpha," Biochem. Biophys. Res. Comm.,
202(3):1445-1451, Aug. 15, 1994. .
Hu et al., "HuIFNalpha21 gene expression and properties of
recombinant IFNalpha21," J. Interferon Res., 14(suppl1):S98, Sep.
1994. .
Horisberger and Di Marco., "Interferon-alpha hybrids," Pharmac.
Ther. 66(3):507-534, 1995. .
Allen et al., "Nomenclature of the Human Interferon Proteins," J.
Interferon Cytokine Res., 16:181-184, 1996. .
Hu et al., "Divergence of Binding, Signaling, and Biological
Responses to Recombinant Human Hybrid IFN," J. Immunol.,
16(2):854-860, Jul. 15, 1999..
|
Primary Examiner: Spector; Lorraine
Assistant Examiner: Seharaseyon; Jegatheesan
Attorney, Agent or Firm: Klarquist Sparkman LLP.
Parent Case Text
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a U.S. National Stage application of
International Application No. PCT/US99/15284, filed Jul. 6, 1999,
which was published in English under PCT Article 21(2), and claims
the benefit of U.S. Provisional Application No. 60/094,407, filed
Jul. 28, 1998. Both applications are incorporated herein in their
entirely.
Claims
What is claimed is:
1. A purified hybrid interferon-.alpha. polypeptide, comprising a
first amino acid sequence consisting of residues 1-75 of
interferon-.alpha.2c; a second amino acid sequence consisting of
residues 76-81 of interferon-.alpha.2c or residues 76-81 of
interferon-.alpha.21a; a third amino acid sequence consisting of
the sequence LDKFXTELXQQLND or the sequence LEKFXTELXQQLND, wherein
X is any amino acid residue; and a fourth amino acid sequence
consisting of residues 96-166 of interferon-.alpha.21a; wherein the
C-terminal residue of the first amino acid sequence is fused to the
N-terminal residue of the second amino acid sequence; and the
C-terminal residue of the second amino acid sequence is fused to
the N-terminal residue of the third amino acid sequence; and the
C-terminal residue of the third amino acid sequence is fused to the
N-terminal residue of the fourth amino acid sequence; and wherein
the hybrid interferon-.alpha. polypeptide has interferon-.alpha.
protein biological activity.
2. A nucleic acid molecule encoding a polypeptide according to
claim 1.
3. A recombinant vector comprising the nucleic acid molecule
according to claim 2.
4. A cell transformed with the recombinant vector according to
claim 3.
5. A pharmaceutical composition comprising: a pharmaceutically
acceptable vehicle or carrier; and at least one hybrid
interferon-.alpha. polypeptide according to claim 1.
6. The hybrid interferon-.alpha. polypeptide according to claim 1,
comprising the amino acid sequence as set forth in SEQ ID NO:
13.
7. A nucleic acid molecule encoding the hybrid interferon-.alpha.
polypeptide according to claim 6.
8. A recombinant vector comprising the nucleic acid molecule
according to claim 7.
9. A cell transformed with the recombinant vector according to
claim 9.
10. A pharmaceutical composition comprising: a pharmaceutically
acceptable vehicle or carrier; and the hybrid interferon-.alpha.
polypeptide according to claim 6.
11. The nucleic acid molecule according to claim 6, comprising the
nucleic acid sequence as set forth in SEQ ID NO: 12.
Description
FIELD OF THE INVENTION
This invention relates to human interferon-.alpha. hybrids and
nucleic acid molecules that encode these hybrids.
BACKGROUND OF THE INVENTION
Interferons arc cytokines produced by a variety of eukaryotic cells
upon exposure to certain environmental stimuli, including mitogens,
endotoxins, double stranded RNA, and viral infection. In addition
to having antiviral properties, interferons have been shown to
affect a wide variety of cellular functions. These effects include
inhibition of cell proliferation, immune regulatory functions and
activation of multiple cellular genes. Interferons (IFNs) have been
classified into four groups according to their chemical,
immunological, and biological characteristics: .alpha. (leukocyte),
.beta. (fibroblast), .gamma., and .omega.. IFNs are further
identified by the eukaryote in which they originated, with HuIFN
indicating human interferon, for instance.
HuIFN-.alpha.s are encoded by a multigene family consisting of
about 20 genes; each gene encodes a single subtype of the
HuIFN-.alpha.. Amino acid sequence identity among IFN-.alpha.
subtypes is generally 80-85% (Horisberger and Di Marco 1995).
HuIFN-.alpha. polypeptides are produced by a number of human cell
lines and human leukocyte cells after exposure to viruses or
double-stranded RNA, or in transformed leukocyte cell lines (e.g.,
lymphoblastoid lines).
IFN-.alpha.s act through interaction with cell-surface receptors
and induce the expression, primarily at the transcriptional level,
of a broad but specific set of cellular genes. Several IFN-induced
gene products have been used as markers for the biological activity
of interferons. These include, for instance, ISG15, ISG54, IRF1,
GBP, and IP10.
Individual IFN-.alpha. subtypes have different biological
activities. For instance, it was recognized early in interferon
research that IFN-.alpha.1 and IFN-.alpha.2 have distinct
target-cell specificities. Human IFN-.alpha.2 shows high specific
activity on bovine and human cells (similar to most
HuIFN-.alpha.s), whereas human IFN-.alpha.1 shows high activity
only on bovine cells.
Interferon activities were first characterized in relation to viral
infections, and IFN-.alpha.s have proven to be remarkably effective
antiviral agents. The current definition of IFN activity units is
expressed in virological terms. There are many assays known to
those skilled in the art that measure the degree of resistance of
cells to viruses (McNeill, 1981). These assays generally can be
categorized into three types: inhibition of cytopathic effect;
virus plaque formation; and reduction of virus yield. Viral
cytopathic effect assays measure the degree of protection induced
in cell cultures pretreated with IFN and subsequently infected with
viruses. Vesicular stomatitis virus, for instance, is an
appropriate virus for use in such an assay. This type of assay is
convenient for screening numerous different IFNs, as it can be
performed in 96-well plates (Rubinstein et al., 1981).
Plaque-reduction assays measure the resistance of IFN-treated cell
cultures to a plaque-forming virus (for instance, measles virus).
One benefit to this assay is that it allows precise measurement of
a 50% reduction in plaque formation. Finally, virus yield assays
measure the amount of virus released from cells during, for
instance, a single growth cycle. Such assays are useful for testing
the antiviral activity of IFNs against viruses that do not cause
cytopathic effects, or that do not build plaques in target-cell
cultures. The multiplicity of infection (moi) is an important
factor to consider when using either plaque-reduction or
virus-yield assays.
Other clinically important interferon characteristics are also
easily assayed in the laboratory setting. One such characteristic
is the ability of an interferon polypeptide to bind to specific
cell-surface receptors. For instance, some IFN-.alpha.s exhibit
different cell-surface properties compared to IFN-.alpha.2b, the
IFN most widely used in clinical trials. While IFN-.alpha.2b is an
effective antiviral agent, it causes significant adverse side
effects. Interferons that exhibit distinct binding properties from
IFN-.alpha.2b may not cause the same adverse effects. Therefore,
interferons that compete poorly with IFN-.alpha.2b for binding
sites on cells are of clinical interest. Competitive interferon
binding assays are well known in the art (Hu et al., 1993; Di Marco
et al., 1994). In general, such assays involve incubation of cell
culture cells with a mixture of .sup.125 I-labeled IFN-.alpha.2b
and an unlabeled interferon of interest. Unbound interferon is then
removed, and the amount of bound label (and by extension, bound
.sup.125 I-labeled IFN-.alpha.2b) is measured. By comparing the
amount of label that binds to cells in the presence or absence of
competing interferons, relative binding affinities can be
calculated.
Another prominent effect of IFN-.alpha.s is their ability to
inhibit cell growth, which is of major importance in determining
anti-tumor action. Growth inhibition assays are well established,
and usually depend on cell counts or uptake of tritiated thymidine
([.sup.3 H]thymidine) or another radiolabel. The human
lymphoblastoid Daudi cell line has proven to be extremely sensitive
to IFN-.alpha.s, and it has been used to measure antiproliferative
activity in many IFN-.alpha.s and derived hybrid polypeptides
(Meister et al., 1986). Use of this cell line has been facilitated
by its ability to be grown in suspension cultures (Evinger and
Pestka, 1981).
IFN-.alpha.s also exhibit many immunomodulatory activities (Zoon et
al., 1986).
Although IFNs were first discovered by virologists, their first
clinical use (in 1979) was as therapeutic agents for myeloma
(Joshua et al., 1997). IFN-.alpha.s have since been shown to be
efficacious against a myriad of diseases of viral, malignant,
angiogenic, allergic, inflammatory, and fibrotic origin (Tilg,
1997). For instance, IFN-.alpha. is the only drug that is currently
approved for treatment of hepatitis C in Europe and North America
(Moussalli et al., 1998), and is the treatment of choice for
chronic acute hepatitis B and AIDS-related Karposi's sarcoma. It
has also proven efficacious in the treatment of metastatic renal
carcinoma and chronic myeloid leukemia (Williams and Linch, 1997).
Clinical uses of IFNs are reviewed in Gresser (1997) and Pfeffer
(1997).
Standard recombinant techniques have become useful methods for the
production and modification of IFN-.alpha. proteins (Streuli et
al., 1981; Horisberger and Di Marco 1995; Rehberg et al., 1982;
Meister et al., 1986; Fidler et al., 1987; Sperber et al., 1993;
Mitsui et al., 1993; Muller et al., 1994; and Zav'Yalov and
Zav'Yalov 1997). One such recombinant modification is the formation
of hybrid IFN molecules. Hybrid IFNs contain fragments of two or
more different interferon polypeptides, functionally fused
together. The first IFN-.alpha. hybrids were designed to study
molecular structure-function relationships. Much research has since
been directed toward the production of hybrid IFNs that combine
different biological properties of the parental proteins. Some
hybrid IFNs display biological activity that is significantly
different from that of both parent molecules (Horisberger and Di
Marco 1995). For instance, certain early IFN-.alpha./IFN-.alpha.
hybrids acquired the novel property of very high activity on mouse
cells (Streuli et al., 1980; Rehberg et al., 1982).
The techniques used by researchers to generate hybrid IFN
polypeptides have evolved through time (Horisberger and Di Marco
1995). Early researchers took advantage of the presence of
naturally occurring restriction endonuclease (RE) cleavage sites
within IFN-encoding sequences to piece together homologous coding
fragments. (See, for instance, U.S. Pat. No. 5,071,761 "Hybrid
Interferons"). Though convenient, this was a limited method in that
only so many of such pre-existing RE sites occurred in each IFN
coding sequence. In addition, the location of each restriction site
was fixed, making the possible combinations relatively small. More
recently, researchers have used PCR amplification to create
specific desired nucleic acid fragments, thereby gaining the
ability to piece together new pieces of different IFNs (Horton et
al., 1989).
A number of U.S. patents discuss various hybrid IFNs, how to
produce them, and how to use them to treat patients. Many such
patents relate to inter-group (multi-class) hybrid IFNs, wherein
portions of the final hybrid are taken from at least two different
interferon classification groups (e.g., .alpha. and .beta.). For
instance, U.S. Pat. No. 4,758,428 ("Multiclass hybrid interferons")
describes the multi-class hybrid IFN
HuIFN-.alpha.1(1-73)/HuIFN-.beta.1(74-166), and its use in
pharmaceutical compositions to treat viral infections and tumorous
growths in animal patients. Another such patent (U.S. Pat. No.
4,914,033 "Structure and properties of modified interferons")
discloses the making of constructs that encode hybrid interferons
comprising amino- and carboxy-terminal fragments of HuIFN-.beta.
fused to an internal sequence (amino acid residues 36-46) of a
HuIFN-.alpha.. This patent also discloses the purification of the
encoded hybrid IFN polypeptide and its use in pharmaceutical
formulations.
Intra-group hybrid interferons (e.g., .alpha.1/.alpha.8 hybrids)
have also been described. U.S. Pat. No. 5,071,761 ("Hybrid
interferons") provides a good example of such intra-group hybrids.
This patent discloses the construction, purification, use, and
pharmaceutical preparation of various fusions hybrids between
HuIFN-.alpha.1 and HuIFN-.alpha.8, where as many as four distinct
IFN-.alpha. fragments have been used to construct the fusion. The
construction, purification, and use of similar IFN-.alpha. hybrids
to treat animal patients are disclosed in U.S. Pat. No. 5,137,720
("Antiviral combination, and method of treatment").
It is to such engineered, recombinant intra-group hybrid interferon
molecules that the present invention is directed.
SUMMARY OF THE INVENTION
The present invention provides hybrid interferons constructed by
combining portions of two or more interferon-.alpha.s, and mutant
and mutant hybrid interferons constructed by point mutagenesis.
These interferon molecules have good antiviral and
antiproliferative activities. Thus, they may be used clinically to
treat viral infections (such as influenza, rabies, and hepatitis B)
and tumors, including but not limited to osteogenic sarcoma,
multiple myeloma, nodular, poorly differentiated lymphoma,
leukemia, carcinoma, melanoma, and papilloma, as well to modulate
the immune system.
Six of the hybrids provided by this invention are termed HY-1,
HY-2, HY-3, HY-4, HY-5, and HY-6, and are composed as follows:
HY-1: IFN-.alpha.21a(1-75)/IFN-.alpha.2c(76-166); HY-2:
IFN-.alpha.21a(1-95)/IFN-.alpha.2c(96-166); HY-3:
IFN-.alpha.2c(1-95)/IFN-.alpha.21a(96-166); HY-4:
IFN-.alpha.-21a(1-75)/IFN-.alpha.2c(76-81)/IFN-.alpha.21a(82-95)/
IFN-.alpha.2c(96-166); HY-5:
IFN-.alpha.-21a(1-75)/IFN-.alpha.21a(76-81)/IFN-.alpha.2c(82-95)/
IFN-.alpha.2c(96-166); HY-6:
IFN-.alpha.21a(1-75)/IFN-.alpha.2c(76-95)/IFN-.alpha.21a(96-166).
This nomenclature indicates that HY-1 is comprised of amino acids
1-75 of IFN-.alpha.21a fused to amino acids 76-166 of
IFN-.alpha.2c; HY-2 is comprised of amino acids 1-95 of
IFN-.alpha.21a fused to amino acids 96-166 of IFN-.alpha.2c; HY-3
is comprised of amino acids 1-95 of IFN-.alpha.2c fused to amino
acids 96-166 of IFN-.alpha.21a; and so forth for the remaining
mutants. HY-3 is 165 amino acids long due to facilitated alignment
numbering, as explained below.
Further aspects of the invention include the hybrid IFNs HY-1,
HY-2, HY-3, HY-4, HY-5, and HY-6 and nucleic acid molecules that
encode these hybrid interferons. Also encompassed within the scope
of the invention are recombinant vectors that comprise such a
nucleic acid molecule. Such vectors can be transformed into various
cells to gain expression of these hybrid interferons. Accordingly,
the invention also encompasses a cell transformed with a
recombinant vector comprising such a nucleic acid molecule.
While each of these hybrids has particular advantages for clinical
use, HY-3 in particular shows striking antiproliferative activity.
This activity is associated with the combination of the 76-95
region of IFN-.alpha.2c and the 96-166 region of IFN-.alpha.21a.
Accordingly, another aspect of the invention comprises HY-3-like
molecules comprising such IFN components. Such molecules may be
represented as X-A-B wherein "X" comprises about amino acid
residues 1-75 of any IFN-.alpha., "A" comprises about amino acid
residues 76-95 of IFN-.alpha.2c and "B" comprises about amino acid
residues 96-166 of IFN-.alpha.21a. While precise numerical
limitations for the size of these sub-regions are provided (e.g.,
about amino acid residues 76-95), one of ordinary skill in the art
will appreciate that these biological molecules may be varied in
exact size. In certain embodiments, such variations will be no
greater than plus or minus five amino acids from the specified
termination points. Likewise, residues 81-90 or 81-95 of
IFN-.alpha.2c comprise about the same amino acid residues as
residues 76-95.
Strong antiproliferative activity may also be obtained by combining
about IFN-.alpha.2c(76-95) with amino- and carboxy-regions of other
IFNs. In such hybrids, the amino--(about residues 1-75) and
carboxy--(about residues 96-166) regions may be provided from a
single IFN-.alpha., or from two different IFN-.alpha.s. These
hybrid IFN molecules may be represented as X-A-Y, wherein "X"
comprises about amino acid residues 1-75 of any IFN-.alpha., "A"
comprises about amino acid residues 76-95 of IFN-.alpha.2c, and "Y"
comprises about amino acid residues 96-166 of any IFN-.alpha..
Another aspect of the invention is a recombinant IFN hybrid protein
comprising first, second, and third domains, wherein the first
domain comprises the amino-region of an IFN-.alpha., the second
domain comprises the middle region of IFN-.alpha.2c (about residues
76-95), and the third domain comprises the carboxy-region of an
IFN-.alpha..
In certain embodiments of the invention, a shorter region of
IFN-.alpha.2c contained within the region from residue 76 to
residue 95 will be sufficient to confer substantial
antiproliferative activity on a hybrid interferon containing such a
fragment. The amino- and carboxy-terminal regions are provided from
a single IFN-.alpha. or from two different IFN'.alpha.s. Such a
hybrid interferon-.alpha. molecule with a short IFN-.alpha.2c
middle region may be represented as V-C-Y, wherein "V" comprises
about amino acid residues 1-81 of an interferon-.alpha., "C"
comprises about amino acid residues 82-95 of IFN-.alpha.2c, and "Y"
comprises about amino acid residues 96-166 of an
interferon-.alpha..
In particular embodiments of the invention, the third domain of the
protein comprises about amino acid residues 96-166 of
IFN-.alpha.21a. In these embodiments, the first domain of the
protein comprises the amino-region of any IFN-.alpha.. Such a
hybrid IFN can be represented generally as X-A-B, wherein "X"
comprises about amino acid residues 1-75 of an interferon-.alpha.,
"A" comprises about amino acid residues 76-95 of IFN-.alpha.2c, and
"B" comprises about amino acid residues 96-166 of
IFN-.alpha.21a.
Hybrid interferon molecules according to the present invention can
also contain more than three segments or domains of different
parental interferons. Such multiple domains are taken from at least
two different source or parental interferons, and may be taken from
up to as many different interferon-.alpha.s as there are segments
assembled to construct the hybrid. For instance, a four-domain
hybrid interferon-.alpha. will be constructed from as few as two or
as many as four different interferon-.alpha.s.
One four domain hybrid interferon-.alpha. molecule encompassed
within the current invention can be designated M-N-O-P, wherein "M"
comprises about amino acid residues 1-75 of interferon .alpha.21a,
"N" comprises about amino acid residues 76 to 81 of
interferon-.alpha.2c, "O" comprises about amino acid residues 82 to
95 of interferon-.alpha.21a, and "P" comprises about amino acid
residues 96 to 166 of interferon-.alpha.2c. A representative four
domain hybrid interferon-.alpha. of this type is HY-4.
If a parental interferon that has one or more point or short
deletions (as found with the 44.sup.th position in IFN-.alpha.2c)
is used in construction of any of the hybrid interferons disclosed
herein (e.g., those represented generally as X-A-B, X-A-Y, V-C-Y,
or M-N-O-P), the numbering of the resultant hybrid fusions should
be carried out using the facilitated alignment system.
The invention also provides nucleic acid molecules that encode any
of the multi-domain hybrid IFN proteins disclosed herein, including
those that can be represented generally as X-A-B, X-A-Y, V-C-Y, and
M-N-O-P, as well as recombinant vectors that comprise such a
nucleic acid molecule and cells transformed with such a vector.
One of ordinary skill in the art will also appreciate that minor
modifications to the IFN-.alpha. sequences described herein may
also be employed, such as amino acid substitutions, additions, and
deletions, to create a mutant hybrid interferon-.alpha.. Thus, it
is entirely possible that hybrid IFNs having greater than or fewer
than 166 amino acids may be produced. Substitutions will typically
be conservative in nature (e.g., one aliphatic amino acid for
another), and such modifications will generally be designed not to
have a significant effect on the biological properties of the
hybrid IFN.
Also encompassed are purified or isolated interferon-.alpha.s (such
as IFN-.alpha.2c) that contain point mutations at either residue 86
or residue 90, thereby changing these residues to tyrosine. Such
mutant interferon-.alpha.s may be mutant hybrid molecules, and such
mutant hybrids can contain short or long segments of IFN-.alpha.2c,
IFN-.alpha.21a, or both of these parental interferons. Specific
representatives of these mutant hybrid interferons include SDM-1
and SDM-2. Additional mutations can be made to replace existing
tyrosine residues at 86 or 90 with other amino acids; specific
representatives of this type of mutant hybrid interferon are SDM-3,
and SDM-4.
Further aspects of the invention include nucleic acid molecules
that encode the mutant hybrid interferons as disclosed herein, and
particularly SDM-1, SDM-2, SDM-3, and SDM-4. Recombinant vectors
that comprise such a nucleic acid molecule are also encompassed.
Such vectors can be transformed into various cells to gain
expression of these mutant interferons. Accordingly, the invention
also encompasses a cell transformed with a recombinant vector
comprising such a nucleic acid molecule.
The invention further provides pharmaceutical compositions
comprising a pharmaceutically acceptable vehicle or carrier and at
least one hybrid IFN-.alpha. polypeptide as described above. Such
hybrid IFN-.alpha.s include those generally represented as X-A-Y,
as X-A-B, as V-C-Y, and as M-N-O-P, as well as the specific hybrids
HY-1, HY-2, HY-3, HY-4, HY-5, and HY-6. Mutant hybrid IFN-.alpha.s
(e.g., SDM-1, SDM-2, SDM-3, or SDM-4) may also be included in such
pharmaceutical compositions, either singly, in combinations with
other mutant hybrid interferons, or in combination with hybrids
IFNs as listed above.
These pharmaceutical compositions can be administered to humans or
other animals on whose cells they are effective, in various manners
such as topically, orally, intravenously, intramuscularly,
intraperitoneally, intranasally, intradermally, intrathecally, and
subcutaneously. Accordingly, a further aspect of the invention is
such a pharmaceutical composition that is an injectable
composition.
The invention also encompasses methods for treating a patient for a
viral disease, comprising administering to the patient a
therapeutically effective, viral disease-inhibiting amount of one
or more hybrid or mutant hybrid interferon-.alpha.s as described
above. One specific aspect of this invention is a method of
treatment, wherein the hybrid interferon-.alpha. is administered to
the patient by injection.
Another aspect of the invention encompasses methods for regulating
cell growth in a patient, comprising administering to the patient a
therapeutically effective, cell growth-regulating amount of one or
more hybrid or mutant hybrid interferon-.alpha.s as described
above. The cell growth regulated by this treatment may be, for
instance, tumor cell growth. One specific aspect of this invention
is a method of regulating cell growth, wherein the hybrid or mutant
hybrid interferon-.alpha. is administered to the patient by
injection.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C shows the general PCR strategy used to construct
interferon-.alpha. hybrids. FIG. 1(A) shows the strategy for
construction of HY-1; FIG. 1(B) shows the strategy for construction
of HY-2; and FIG. 1(C), that for HY-3 construction.
FIGS. 2A-2C shows the antiproliferative effects of IFN-.alpha.2c
(.quadrature.) and IFN-.alpha.21a (.gradient.) compared to that of
hybrid IFNs HY-1 (.DELTA.), HY-2 (O) and HY-3 (*). Panel A, Daudi
cells, Panel B, WISH cells, and Panel C, primary human
lymphocytes.
FIG. 3 shows the antiviral activities of IFN-.alpha.2c and
IFN-(.alpha.21a compared to that of hybrid IFNs HY-2 and HY-3 on
primary human lymphocytes. Legend: a, No IFN; b, IFN-.alpha.2c; c,
IFN-.alpha.21a; d, HY-2; e, HY-3.
FIGS. 4A-4B shows the competitive binding curves for .sup.125
I-labeled IFN-.alpha.2b using native interferons IFN-.alpha.2c
(.quadrature.) and IFN-.alpha.21a (.gradient.), and hybrid
interferons HY-1 (.DELTA.), HY-2 (O) and HY-3 (*) as competitors.
Panel A, Daudi cells; Panel B, WISH cells.
FIG. 5 shows the amino acid sequences of IFN-.alpha.2c (accession
number P01563), IFN-.alpha.21 (accession number X00145) and
IFN-.alpha. hybrids, HY-1 (SEQ ID NO: 9; accession number
AF085803), HY-2 (SEQ ID NO: 11; accession number AF085804) and HY-3
(SEQ ID NO: 13; accession number AF085805). Shaded residues differ
between IFN-.alpha.2c and IFN-.alpha.21a. The asterisk (*) at
apparent position 44 in the sequences of IFN-.alpha.2c and HY-3 has
been inserted to facilitate alignment of the hybrid sequences and
make subsequent position numbering consistent.
Table 1 summarizes the results of antiproliferative, antiviral, and
competitive binding activity assays using parental IFNs .alpha.2
and .alpha.21a, hybrids HY-1, HY-2, HY-3, HY-4, and HY-5, and
interferon mutant hybrids SDM-1, SDM-2, SDM-3, and SDM-4.
Antiproliferative activities are reported as the amount (ng/ml) of
each IFN species needed to inhibit cell growth by 50%. N/D: No
Data.
SEQUENCE LISTING
The nucleic and amino acid sequences listed in the accompanying
sequence listing are shown using standard letter abbreviations for
nucleotide bases, and three letter code for amino acids. Only one
strand of each nucleic acid sequence is shown, but it is understood
that the complementary strand is included by any reference to the
displayed strand.
SEQ ID NO: 1 shows the outside PCR primer used for synthesis of
hybrids HY-1, HY-2, HY-3, HY-4, HY-5, and HY-6, and mutant hybrids
SDM-1, SDM-2, SDM-3, and SDM-4. This primer contains an engineered
BamHI restriction site, and was used as the upstream primer during
IFN-.alpha.21a (HY-1 and HY-2) and IFN-.alpha.2c (HY-3)
amplification. This primer was also used as the upstream primer for
amplification of IFN-.alpha.21a from cDNA.
SEQ ID NO: 2 shows the inside PCR primer used for synthesis of
HY-1. This primer was used as the downstream primer during
IFN-.alpha.21a amplification.
SEQ ID NO: 3 shows the inside PCR primer used for synthesis of
HY-1. This primer was used as the upstream primer during
IFN-.alpha.2c amplification.
SEQ ID NO: 4 shows the outside PCR primer used for synthesis of
HY-1 and HY-2. This primer contains an engineered SpHI restriction
site, and was used as the downstream primer during IFN-.alpha.2c
amplification.
SEQ ID NO: 5 shows the inside PCR primer used for synthesis of HY-2
and HY-3. This primer was used as the downstream primer during
IFN-.alpha.21a amplification.
SEQ ID NO: 6 shows the inside PCR primer used for synthesis of HY-2
and HY-3. This primer was used as the downstream primer during
IFN-.alpha.21a amplification.
SEQ ID NO: 7 shows the outside PCR primer used for synthesis of
HY-3. This primer contains the engineered SpHI restriction site,
and was used as the downstream primer during IFN-.alpha.2c (HY-2)
and IFN-.alpha.21a (HY-3) amplification. This primer was also used
as the downstream primer for amplification of IFN-.alpha.21a from
cDNA.
SEQ ID NO: 8 shows the DNA coding sequence and corresponding amino
acid sequence of HY-1.
SEQ ID NO: 9 shows the amino acid sequence of HY-1.
SEQ ID NO: 10 shows the DNA coding sequence and corresponding amino
acid sequence of HY-2.
SEQ ID NO: 11 shows the amino acid sequence of HY-2.
SEQ ID NO: 12 shows the DNA coding sequence and corresponding amino
acid sequence of HY-3.
SEQ ID NO: 13 shows the amino acid sequence of HY-3.
SEQ ID NOs: 14 and 15 show the inside primers used for synthesis of
HY-4.
SEQ ID NOs: 16 and 17 show the inside primers used for synthesis of
HY-5.
SEQ ID NOs: 18 and 19 show the inside primers used for synthesis of
HY-6.
SEQ ID NOs: 20 and 21 show the inside primers used for synthesis of
SDM-1.
SEQ ID NOs: 22 and 23 show the inside primers used for synthesis of
SDM-2.
SEQ ID NOs: 24 and 25 show the inside primers used for synthesis of
SDM-3.
SEQ ID NOs: 26 and 27 show the inside primers used for synthesis of
SDM-4.
SEQ ID NO: 28 Shows the outside primer used with SEQ ID NO: 1 for
synthesis of HY-4, HY-5, HY-6, SDM-1, SDM-2, SDM-3, and SDM-4.
SEQ ID NO: 29 shows the DNA coding sequence and corresponding amino
acid sequence of HY-4.
SEQ ID NO: 30 shows the amino acid sequence of HY-4.
SEQ ID NO: 31 shows the DNA coding sequence and corresponding amino
acid sequence of HY-5.
SEQ ID NO: 32 shows the amino acid sequence of HY-5.
SEQ ID NO: 33 shows the DNA coding sequence and corresponding amino
acid sequence of HY-6.
SEQ ID NO: 34 shows the amino acid sequence of HY-6.
SEQ ID NO: 35 shows the DNA coding sequence and corresponding amino
acid sequence of SDM-1.
SEQ ID NO: 36 shows the amino acid sequence of SDM1.
SEQ ID NO: 37 shows the DNA coding sequence and corresponding amino
acid sequence of SDM-2.
SEQ ID NO: 38 shows the amino acid sequence of SDM-2.
SEQ ID NO: 39 shows the DNA coding sequence and corresponding amino
acid sequence of SDM-3.
SEQ ID NO: 40 shows the amino acid sequence of SDM-3.
SEQ ID NO: 41 shows the DNA coding sequence and corresponding amino
acid sequence of SDM-4.
SEQ ID NO: 43 shows a consensus amino acid sequence for a hybrid
interferon-.alpha. polypeptide.
The amino acid sequences of the hybrid IFNs are depicted without
leader sequences. Such leader sequences are typically present on
IFNs produced in eukaryotic cells, but are generally cleaved off to
produce the mature form of the protein. The nomenclature for IFNs
used herein is based on the amino acid sequences of mature
IFNs.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions and Abbreviations
A. Abbreviations IFN: interferon IFN-.alpha.: interferon-.alpha.
HuIFN-.alpha.: human interferon-.alpha. IU: international units
MDBK: Madin-Darby bovine kidney cells ATCC: American Type Culture
Collection E. coli: Escherichia coli PHA: phytohemagglutinin
SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis
poly-DI-DC: polydeoxyinosine-deoxycytosine RNase: ribonuclease RE:
restriction endonuclease moi: multiplicity of infection
B. Definitions
Unless otherwise noted, technical terms are used according to
conventional usage. Definitions of common terms in molecular
biology may be found in Lewin, Genes V published by Oxford
University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.),
The Encyclopedia of Molecular Biology, published by Blackwell
Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers
(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8). The nomenclature for DNA bases and the three-letter
code for amino acid residues, as set forth at 37 CFR .sctn.1.822,
are used herein. All interferon units are expressed with reference
to the NIH human lymphoblastoid IFN standard Ga 23-901-532.
In order to facilitate review of the various embodiments of the
invention, the following definitions of terms are provided. These
definitions are not intended to limit such terms to a scope
narrower than would be known to a person of ordinary skill in the
field.
Interferons: A family of secreted polypeptides produced by a
variety of eukaryotic cells upon exposure to various environmental
stimuli, including virus infection or exposure to a mitogen. In
addition to having antiviral properties, interferons have been
shown to affect a wide variety of cellular functions. Interferons
(IFNs) have been classified into four major groups according to
their chemical, immunological, and biological characteristics:
.alpha., .beta., .gamma., and .omega.. Each IFN is further
identified by the eukaryote in which it originated, with HuIFN
indicating human interferon. For the purposes of this disclosure,
any interferon that lacks a specific eukaryote source designation
is presumed to be that set of equivalent interferons from any
source. In other words, IFN-.alpha.2c would refer to the
interferon-.alpha.2c from any eukaryotic source, while
HuIFN-.alpha.2c refers specifically to human interferon-.alpha.2c.
Throughout this specification, the IFN nomenclature provided by
Allen and Diaz (1996) is employed unless otherwise noted.
Interferon-.alpha. (IFN-.alpha.) polypeptides are produced in, for
instance, human leukocyte cells after exposure to viruses or
double-stranded RNA, or in transformed leukocyte cell lines (e.g.,
lymphoblastoid lines). Most IFN-.alpha.s are non-glycosylated
polypeptides of 165 or 166 amino acids, encoded for by a multigene
family of at least 20 genes. The difference in length is due to an
amino acid deletion at the 44.sup.th position in certain IFNs, for
instance IFN-.alpha.2c. Each gene (termed IFNA1, IFNA2, etc.)
encodes a single IFN-.alpha. polypeptide subtype (termed
IFN-.alpha.1, IFN-.alpha.2, etc., respectively). Amino acid
sequence identity among IFN-.alpha. subtypes is generally 80-85%
(Horisberger and Di Marco 1995). Within each subtype, individual
sequence variants (IFN species) are further denoted with an
additional letter designation, e.g., IFN-.alpha.2a, IFN-.alpha.2b,
and IFN-.alpha.2c. The sequence differences between these species
are often very small (1-3 amino acids).
Hybrid interferons: Recombinant interferon molecules that combine
various segments from parental interferon molecules. Hybrids may be
constructed using portions of two (or more) IFNs from different IFN
groups (e.g., one segment from an IFN-.alpha. polypeptide and
another segment from an IFN-.beta. polypeptide) (see, for instance,
U.S. Pat. No. 4,758,428 "Multiclass hybrid interferons"; U.S. Pat.
No. 4,914,033 "Structure and properties of modified interferons";
and U.S. Pat. No. 4,917,887 "Hybrid interferons, their use as
pharmaceutical compositions and as intermediate products for the
preparation of antibodies and the use thereof and processes for
preparing them"). These are referred to as inter-group or
multi-class hybrids. Alternatively, hybrids can be formed using
portions of two different IFN species from one IFN group (e.g., one
segment from each of two IFN-.alpha. polypeptides) (see, for
instance, U.S. Pat. No. 4,806,347 "Interferon combinations"; U.S.
Pat. No. 4,892,743 "Novel hybrid interferon species"; U.S. Pat. No.
5,071,761 "Hybrid interferons"; U.S. Pat. No. 5,137,720 "Antiviral
combination, and method of treatment"; and U.S. Pat. No. 5,609,868
"Pharmaceutical compositions comprising hybrid
.alpha.-interferon"). These are referred to as intra-group hybrids.
The construction and properties of certain IFN-.alpha./IFN-.alpha.
hybrids has been reviewed (Horisberger and Di Marco 1995).
Herein, hybrid interferon protein nomenclature is used largely as
proposed in Allen and Diaz (1996). For instance, the hybrid
interferon fusion HY-1 is fully designated as
IFN-.alpha.21a(1-75)/IFN-.alpha.2c(76-166), wherein the
amino-terminal end of the polypeptide consists of amino acids 1-75
of IFN-.alpha.21a and the carboxy-terminal end consists of amino
acids 76-166 of IFN-.alpha.2c. Hybrids HY-2
[IFN-.alpha.21a(1-95)/IFN-.alpha.2c(96-166)], HY-4
[IFN-.alpha.-21a(1-75)/IFN-.alpha.2c(76-81)/IFN-.alpha.21a(82-95)/
IFN-.alpha.2c(96-166)], and HY-6
[IFN-.alpha.21a(1-75)/IFN-.alpha.2c(76-95)/IFN-.alpha.21a(96-166)]
are designated similarly. The same terminology can be used where
otherwise contiguous regions of the same parental interferon
molecule are re-joined in a final construct, as is true in HY-5
[IFN-.alpha.-21a(1-75)/IFN-.alpha.21a(76-81)/IFN-.alpha.2c(82-95)/
IFN-.alpha.2c(96-166)].
One modification has been made to the nomenclature method of Allen
and Diaz (1996), to facilitate consistent and simple numbering of
hybrids constructed from interferons of different lengths where the
length difference is due to relatively short internal insertions or
deletions. For instance, IFN-.alpha.2c is one amino acid shorter
than IFN-.alpha.21a due to the absence of an aspartate at the
44.sup.th position in the sequence. The hybrid fusion interferon
HY-3 [IFN-.alpha.2c(1-95)/IFN-.alpha.21a(96-166)], illustrates the
"facilitated alignment" modification to the standard numbering
system. HY-3 is in fact only 165 amino acids long, due to the
"empty" place-saving designation at position 44.
This "facilitated alignment" numbering system is illustrated in
FIG. 5. The asterisk (*) at apparent position 44 in the sequences
of IFN-.alpha.2c and HY-3 has been inserted to facilitate alignment
of the hybrid sequences and make subsequent residue position
numbering consistent. In other words, this asterisk serves as a
"place-saver" in the numbering of these sequences. This numbering
system also could be used for sequences that differ by more than
one residue in length, simply by inserting the appropriate number
of "spacers" to force alignment of the remaining sequence.
If a parental interferon that has one or more point or short
deletions (as found with the 44.sup.th position in IFN-.alpha.2c)
is used in construction of a hybrid, the numbering of the resultant
hybrid fusions should be carried out using the facilitated
alignment system.
It is possible to refer to general classes of hybrid IFN molecules
by designating domains within the protein that are of interest. For
instance, data indicate that it is likely the carboxy-terminal
portion (residues 76-166) of HY-3 that is important for the
observed high antiproliferative activity. A class of HY-3-like
molecules that contains this carboxy-terminal portion can be
represented generally as X-A-B, wherein "X" comprises about amino
acid residues 1-75 of an interferon-.alpha., "A" comprises about
amino acid residues 76-95 of IFN-.alpha.2c, and "B" comprises about
amino acid residues 96-166 of IFN-.alpha.21a.
One of ordinary skill in the art will appreciate that these
elements may be combined to produce HY-3-like molecules without
necessarily splicing the components in the same place. It is
possible to use shorter or longer fragments of IFN-.alpha.2c, fused
to correspondingly longer or shorter fragments of IFN-.alpha.21a.
In such instances, the middle element of IFN-.alpha.2c used to
construct the hybrid molecule comprises residues 76-96, 76-97 or
76-98, while the carboxy-terminal element of IFN-.alpha.21a would
correspondingly comprise residues 97-166, 98-166, or 99-166,
respectively. Any component that is spliced within 5 amino acid
residues of the residue specified comprises about the same region.
For instance, amino acid residues 1-80 or 1-70 of IFN-.alpha.2c
comprise about the same amino acid residues as the component with
residues 1-75. Likewise, residues 81-90 or 81-95 of IFN-.alpha.2c
comprise about the same amino acid residues as this component with
residues 76-95.
Further, hybrid interferon molecules can be constructed in which
the middle region is defined as being from a specific source, for
instance residues 76-95 of IFN-.alpha.2c, but the amino- and
carboxy-regions can be chosen from any IFN-.alpha.. In such
hybrids, the amino--(about residues 1-75) and carboxy--(about
residues 96-166) regions may be provided from any single
IFN-.alpha., or from two different IFN-.alpha.s. These hybrid IFN
molecules may be represented as X-A-Y, wherein "X" comprises about
amino acid residues 1-75 of any IFN-.alpha., "A" comprises about
amino acid residues 76-95 of IFN-.alpha.2c, and "Y" comprises about
amino acid residues 96-166 of any IFN-.alpha.. As above, any
component that is spliced within 5 amino acid residues of the
residue specified comprises about the same region. For instance,
residues 81-90 or 81-95 of IFN-.alpha.2c, serving as the "A"
component of this construct, comprise about the same amino acid
residues as "A" with residues 76-95 of IFN-.alpha.2c.
Parenteral: Administered outside of the intestine, e.g., not via
the alimentary tract. Generally, parenteral formulations are those
that will be administered through any possible mode except
ingestion. This term especially refers to injections, whether
administered intravenously, intrathecally, intramuscularly,
intraperitoneally, or subcutaneously, and various surface
applications including intranasal, intradermal, and topical
application, for instance.
Pharmaceutically acceptable carriers: The pharmaceutically
acceptable carriers useful in this invention are conventional.
Remington's Pharmaceutical Sciences, by E. W. Martin, Mack
Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of the hybrid interferons herein disclosed.
In general, the nature of the carrier will depend on the particular
mode of administration being employed. For instance, parenteral
formulations usually comprise injectable fluids that include
pharmaceutically and physiologically acceptable fluids such as
water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(e.g., powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
Injectable composition: A pharmaceutically acceptable fluid
composition comprising at least an active ingredient. The active
ingredient is usually dissolved or suspended in a physiologically
acceptable carrier, and the composition can additionally comprise
minor amounts of one or more non-toxic auxiliary substances, such
as emulsifying agents, preservatives, and pH buffering agents and
the like. Such injectable compositions that are useful for use with
the hybrid interferons of this invention are conventional;
appropriate formulations are well known in the art, and examples
may be found in U.S. Pat. No. 5,609,868 ("Pharmaceutical
compositions comprising hybrid .alpha.-interferon").
Therapeutically effective amount of IFN-.alpha.: A quantity of
interferon-.alpha. sufficient to achieve a desired effect in a
subject being treated. For instance, this can be the amount
necessary to inhibit viral proliferation or to regulate cell, and
more specifically tumor cell, proliferation. See, U.S. Pat. No.
4,089,400 ("Polypeptides and process for the production thereof")
and U.S. Pat. No. 5,503,828 ("Alpha interferon composition and
method for its production from human peripheral blood leukocytes")
for general disclosure as to the amounts of IFN-.alpha. that have
proven efficacious in clinical settings. The same dose levels as
are used in conventional (non-hybrid) interferon therapy may be
used with hybrid interferons. In general, a dose of about 10.sup.5
to 10.sup.8 IU will be appropriate and may be administered more
than once, for example daily, during a course of treatment.
However, the effective amount of hybrid IFN-.alpha. will be
dependent on the subject being treated, the severity of the
affliction, and the manner of administration of the interferon.
The hybrid interferons disclosed in the present invention have
equal application in medical and veterinary settings. Therefore,
the general term "subject being treated" is understood to include
all animals that produce interferon polypeptides, including humans
or other simians, dogs, cats, horses, and cows.
Probes and primers: A nucleic acid probe comprises an isolated
nucleic acid attached to a detectable label or reporter molecule.
Typical labels include radioactive isotopes, ligands,
chemiluminescent agents, and enzymes. Methods for labeling and
guidance in the choice of labels appropriate for various purposes
are discussed, e.g., in Sambrook et al., (1989) and Ausubel et al.,
(1987).
Primers are short nucleic acids, preferably DNA oligonucleotides 15
nucleotides or more in length. Primers may be annealed to a
complementary target DNA strand by nucleic acid hybridization to
form a hybrid between the primer and the target DNA strand. The
primer may be then extended along the target DNA strand through the
use of a DNA polymerase enzyme. Primer pairs (one on either side of
the target nucleic acid sequence) can be used for amplification of
a nucleic acid sequence, eg., by the polymerase chain reaction
(PCR) or other nucleic-acid amplification methods known in the
art.
Methods for preparing and using probes and primers are described,
for example, in Sambrook et a., (1989), Ausubel et al., (1987), and
Innis et al., (1990). PCR primer pairs can be derived from a known
sequence, for example, by using computer programs intended for that
purpose such as Primer (Version 0.5, .COPYRGT. 1991, Whitehead
Institute for Biomedical Research, Cambridge, Mass.). One of skill
in the art will appreciate that the specificity of a particular
probe or primer increases with its length. Thus, for example, a
primer comprising 20 consecutive nucleotides of one human
IFN-.alpha. subtype cDNA or gene will anneal to a target sequence
(e.g., a different human IFN-.alpha. subtype or an IFN-.alpha. from
another species) with a higher specificity than a corresponding
primer of only 15 nucleotides. Thus, in order to obtain greater
specificity, probes and primers may be selected that comprise 20,
25, 30, 35, 40, 50 or more consecutive nucleotides of one
IFN-.alpha. subtype cDNA or gene sequence.
Vector: A nucleic acid molecule as introduced into a host cell,
thereby producing a transformed host cell. A vector may include
nucleic acid sequences that permit it to replicate in a host cell,
such as an origin of replication. A vector may also include one or
more selectable marker genes and other genetic elements known in
the art.
Transformed: A transformed cell is a cell into which has been
introduced a nucleic acid molecule by molecular biology techniques.
As used herein, the term transformation encompasses all techniques
by which a nucleic acid molecule might be introduced into such a
cell, including transfection with viral vectors, transformation
with plasmid vectors, and introduction of naked DNA by
electroporation, lipofection, particle gun acceleration, and the
like.
Isolated: An "isolated" biological component (such as a nucleic
acid or protein) has been substantially separated or purified away
from other biological components in the cell of the organism in
which the component naturally occurs, ie., other chromosomal and
extra-chromosomal DNA and RNA, and proteins. Thus, nucleic acids
and proteins that have been "isolated" include nucleic acids and
proteins purified by standard purification methods. The term also
embraces nucleic acids and proteins prepared by recombinant
expression in a host cell as well as chemically synthesized nucleic
acids.
Purified: The term purified does not require absolute purity;
rather, it is intended as a relative term. Thus, for example, a
purified IFN-.alpha. preparation is one in which the
interferon-alpha is more enriched than the protein is in its
natural environment within a cell. Preferably, a preparation of
IFN-.alpha. is purified such that the IFN-.alpha. represents at
least 50% of the total protein content of the preparation.
Operably linked: A first nucleic acid sequence is operably linked
with a second nucleic acid sequence when the first nucleic acid
sequence is placed in a functional relationship with the second
nucleic acid sequence. For instance, a promoter is operably linked
to a coding sequence if the promoter affects the transcription or
expression of the coding sequence. Generally, operably linked DNA
sequences are contiguous and, where necessary to join two
protein-coding regions, in the same reading frame.
Recombinant: A recombinant nucleic acid is one that has a sequence
that is not naturally occurring, or has a sequence that is made by
an artificial combination of two otherwise separated segments of
sequence. This artificial combination is often accomplished by
chemical synthesis or, more commonly, by the artificial
manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques, or a combination of these
techniques. Similarly, a recombinant protein is one that is encoded
by a recombinant nucleic acid.
II. Construction of Hybrid and Mutant Hybrid
Interferon-.alpha.s
A. General Approaches to Hybrid Interferon-.alpha. Construction
The production of a number of hybrid IFNs has been reviewed by
Horisberger and Di Marco (1995); this article provides a good
overview of the process of construction of such molecules. Specific
examples of methods for construction of hybrid interferons can be
found, for example, in U.S. Pat. No. 4,892,743 ("Novel hybrid
interferon species"); U.S. Pat. No. 5,071,761 "Hybrid
Interferons"); U.S. Pat. No. 4,758,428 ("Multiclass hybrid
interferons"); and U.S. Pat. No. 4,716,217 ("Hybrid
lymphoblastoid-leukocyte human interferons").
Generally, two procedures are used to create hybrid IFN-.alpha.s.
First, some researchers have taken advantage of the presence of
naturally occurring RE cleavage sites within IFN-encoding sequences
to piece together homologous coding fragments. (See, for instance,
U.S. Pat. No. 5,071,761 "Hybrid Interferons"). The second general
procedure for construction of hybrid IFN-.alpha.s uses PCR
amplification to create specific desired nucleic acid fragments,
thereby gaining the potential to piece together new pieces of
different IFNs (Horton et al., 1989). It is this second technique
that has been employed herein to generate novel and useful
IFN-.alpha. hybrids.
B. Construction of Parental Interferon-bearing Plasmids
A plasmid bearing the IFN-.alpha.2c coding sequence
(pBluescript/A2) was constructed as previously described (Hayes and
Zoon, 1993).
To construct a plasmid bearing the coding sequence of
IFN-.alpha.21a, a pair of oligonucleotides, SEQ ID NO: 1 and SEQ ID
NO: 7, with BamHI and PstI restriction sites, were synthesized
based on the cDNA coding region for mature human IFN-.alpha.21a
protein (Genentech, South San Francisco, Calif.). These were used
as primers in a standard polymerase chain reaction (PCR) (Innis et
al., 1990), and the entire coding region for mature human
IFN-.alpha.21a protein amplified. The resulting products were
cleaved with restriction endonucleases (REs) BamHI and PstI and
cloned into the E. coli expression vector pQE30 (QIAGEN,
Chatsworth, Calif.) cleaved with REs BamHI and PstI, to form
pQE30/A21. The final construct was verified by DNA sequence
analysis (Sanger et al., 1977).
C. Construction of HY-1
The hybrid IFN cDNAs presented in this invention were constructed
by PCR technology (Horton et al., 1989). The procedure used to
construct HY-1 is illustrated in FIG. 1(A). In reaction one,
primers 1 (SEQ ID NO: 1) and 2 (SEQ ID NO: 2) were used to amplify
the amino-terminal portion of IFN-.alpha.21a (encoding amino acids
1-75), using linearized pQE30/A21 as template. In reaction two,
primers 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 4) were used to amplify
the carboxy-terminal portion of IFN-.alpha.2c (encoding amino acids
76-166), using linearized pBluescript/A2 as template. Purified DNA
fragments from the first pair of PCR reactions were then mixed as
templates for the second round of PCR, and the fused sequence
amplified using primers 1 (SEQ ID NO: 1) and 4 (SEQ ID NO: 4). This
reaction generated the full-length HY-1 fusion coding sequence (SEQ
ID NO: 8), which encodes IFN-.alpha.21a(1-75)/IFN-.alpha.2c(76-166)
(SEQ ID NO: 9).
Purified DNA fragments from the second round of PCR amplification
were digested with REs BamHI and SpHI and ligated into pQE30. The
final HY-1 construct (pHY-1) was verified by DNA sequence analysis
(Sanger et a., 1977). HY-1 has been submitted to GenBank for
publication on or after Jul. 7, 1999, accession number
AF085803.
D. Construction of HY-2
The procedure used to construct HY-2 is illustrated in FIG. 1(B).
In reaction one, primers 1 (SEQ ID NO: 1) and 5 (SEQ ID NO: 5) were
used to amplify the amino-terminal portion of IFN-.alpha.21a
(encoding amino acids 1-95), using linearized pQE30/A21 as
template. In reaction two, primers 6 (SEQ ID NO: 6) and 4 (SEQ ID
NO: 4) were used to amplify the carboxy-terminal portion of
IFN-.alpha.2c (encoding amino acids 96-166), using linearized
pBluescript/A2 as template. Purified DNA fragments from the first
pair of PCR reactions were then mixed as templates for the second
round of PCR, and the fused sequence amplified using primers 1 (SEQ
ID NO: 1) and 4 (SEQ ID NO: 4). This reaction generated the
full-length HY-2 fusion coding sequence (SEQ ID NO: 10), which
encodes IFN-.alpha.21a(1-95)/IFN-.alpha.2c(96-166) (SEQ ID NO:
11).
Purified DNA fragments from the second round of PCR amplification
were digested with BamHI and SpHI and ligated into pQE30. The final
HY-2 construct (pHY-2) was verified by DNA sequence analysis
(Sanger et al., 1977). HY-2 has been submitted to GenBank for
publication on or after Jul. 7, 1999, accession number
AF085804.
E. Construction of HY-3
The procedure used to construct HY-3 is illustrated in FIG. 1(C).
In reaction one, primers 1 (SEQ ID NO: 1) and 5 (SEQ ID NO: 5) were
used to amplify the amino-terminal portion of IFN-.alpha.2c
(encoding amino acids 1-95; because of the facilitated alignment
method that assigns the "absent" position 44 a number, this
fragment is only 94 amino acids long), using linearized pQE30/A21
as template. In reaction two, primers 6 (SEQ ID NO: 6) and 7 (SEQ
ID NO: 7) were used to amplify the carboxy-terminal portion of
IFN-.alpha.21a (encoding amino acids 96-166), using linearized
pBluescript/A2 as template. Purified DNA fragments from the first
pair of PCR reactions were then mixed as templates for the second
round of PCR, and the fused sequence amplified using primers 1 (SEQ
ID NO: 1) and 7 (SEQ ID NO: 7). This generated the full-length HY-3
fusion coding sequence (SEQ ID NO: 12), which encodes
IFN-.alpha.2c(1-95)/IFN-.alpha.21a(96-166) (SEQ ID NO: 13) (which
is in fact only 165 amino acids long).
Purified DNA fragments from the second round of PCR amplification
was digested with BamHI and SpHI and ligated into pQE30. The final
HY-3 construct (pHY-3) was verified by DNA sequence analysis
(Sanger et al., 1977). HY-3 has been submitted to GenBank for
publication on or after Jul. 7, 1999, accession number
AF085805.
Construction of HY-4,5, and -6
Using methods essentially similar to those discussed above for
HY-1, -2, and -3, three further hybrid interferon molecules were
constructed which incorporate shorter internal segments of the
parent interferons. HY-4 was constructed using HY-2 as a template,
and incorporates the following .alpha.-interferon sequences:
IFN-.alpha.21a(1-75)/IFN-.alpha.2c(76-81)/IFN-.alpha.21a(82-95)/
IFN-.alpha.2c(96-166). The nucleotide sequence of HY-4 is depicted
in SEQ ID NO: 29. Primers 14s and 14as (SEQ ID NO: 14 and 15)
served as the inside primers for construction of this hybrid.
HY-5 was constructed using HY-2 as a template, and incorporates the
following interferon sequences:
IFN-.alpha.21a(1-75)/IFN-.alpha.21a(76-81)/IFN-.alpha.2c(82-95)/
IFN-.alpha.2c(96-166). The nucleotide sequence of HY-5 is depicted
in SEQ ID NO: 31. Primers 15s and 15as (SEQ ID NO: 16 and 17)
served as the inside primers for construction of this hybrid.
HY-6 was constructed using HY-1 and parental IFN-.alpha.2a as
templates, and incorporates the following interferon sequences:
IFN-.alpha.21a(1-75)/IFN-.alpha.2c(76-95)/IFN-.alpha.21a(96-166).
The nucleotide sequence of HY-6 is depicted in SEQ ID NO: 33.
Primers M291 s and M219as (SEQ ID NO: 18 and 19) served as the
inside primers for construction of this hybrid. Primers 28 and 1
(SEQ ID NO: 28 and 1) served as the outside primers for production
of all three of these hybrids.
G. Construction of Mutant Hybrid Interferon-.alpha.s SDM-1, SDM-2,
SDM-3, and SDM4
In addition to the production of hybrid interferons from native
sequences, it is also possible to construct hybrids that have
specific sequence mutations at specific nucleotide and/or amino
acid residues. As an example of this, four mutant interferon
hybrids were constructed using methods essentially similar to those
used above to construct the base hybrids. Mutations in specific
amino acids were introduced into these mutant hybrids by
incorporating desired nucleotide changes into the primers used for
amplification of the relevant hybrid sequences.
SDM1 was constructed using HY-4 as the template, and integrates a
single amino acid mutation at residue 86, which changes the serine
found in the IFN-.alpha.21a sequence to a tyrosine. The nucleotide
sequence of SDM-1 is depicted in SEQ ID NO: 35. Primers SDM1s and
SDM1as (SEQ ID NO: 20 and 21) served as the inside primers for
construction of this mutant.
SDM-2 was constructed using HY-4 as the template, and integrates a
single amino acid mutation at residue 90, which changes the
asparagine found in the IFN-.alpha.21a sequence to a tyrosine. The
nucleotide sequence of SDM-2 is depicted in SEQ ID NO: 37. Primers
SDM2s and SDM2as (SEQ ID NO: 22 and 23) served as the inside
primers for this mutant.
SDM-3 was constructed using HY-5 as the template, and integrates a
single amino acid mutation at residue 86, which changes the
tyrosine found in the IFN-.alpha.2c sequence to a serine. The
nucleotide sequence of SDM-3 is depicted in SEQ ID NO: 39. Primers
SDM3s and SDM3as (SEQ ID NO: 24 and 25) served as the inside
primers for this mutant.
SDM-4 was constructed using HY-5 as the template, and integrates a
single amino acid mutation at residue 90, which changes the
tyrosine found in the IFN-.alpha.2c sequence to an asparagine. The
nucleotide sequence of SDM-4 is depicted in SEQ ID NO: 41. Primers
SDM4s and SDM4as (SEQ ID NO: 26 and 27) served as the inside
primers for this mutant. Primers 2* and 1 (SEQ ID NO: 28 and 1)
served as the outside primers for production of all four
mutants.
III. Expression and Purification of Hybrid and Mutant Hybrid
Interferons
A. Expression of IFN-.alpha. Hybrids and Mutants
The following method of expression of hybrid and mutant interferons
is provided merely by way of example. One skilled in the art will
understand that there are myriad ways to express a recombinant
protein such that it can be subsequently purified. See, for
instance, U.S. Pat. No. 5,089,400 ("Polypeptides and process for
the production thereof"). In general, an expression vector carrying
the nucleic acid sequence that encodes the desired protein will be
transformed into a microorganism for expression. Such
microorganisms can be prokaryotic (bacteria) or eukaryotic (e.g.,
yeast). One appropriate species of bacteria is Escherichia coli (E.
coli), which has been used extensively as a laboratory experimental
expression system. A eukaryotic expression system will be preferred
where the protein of interest requires eukaryote-specific
post-translational modifications such as glycosylation.
The expression vector can include a sequence encoding a targeting
peptide, positioned in such a way as to be fused to the coding
sequence of the IFN. This allows the hybrid IFN to be targeted to
specific locations. In a prokaryotic expression system, a signal
sequence can be used to secrete the newly synthesized hybrid
protein. In a eukaryotic expression system, the targeting peptide
would specify targeting of the hybrid protein to one or more
specific sub-cellular compartments, or to be secreted from the
cell, depending on which peptide is chosen. One such appropriate
targeting peptide is the native IFN signal peptide, which would
direct the hybrid IFN to be secreted from eukaryotic cells.
Vectors suitable for stable transformation of bacterial cells are
well known. Typically, such vectors include a multiple-cloning site
suitable for inserting a cloned nucleic acid molecule, such that it
will be under the transcriptional control of 5' and 3' regulatory
sequences. In addition, transformation vectors include one or more
selectable markers; for bacterial transformation this is often an
antibiotic resistance gene. Such transformation vectors typically
also contain a promoter regulatory region (e.g., a regulatory
region controlling inducible or constitutive expression), a
transcription initiation start site, a ribosome binding site, an
RNA processing signal, and a transcription termination site, each
functionally arranged in relation to the multiple-cloning site. For
production of large amounts of recombinant proteins, an inducible
promoter is preferred. This permits selective production of the
recombinant protein, and allows both higher levels of production
than constitutive promoters, and enables the production of
recombinant proteins that may be toxic to the expressing cell if
expressed constitutively.
In addition to these general guidelines, protein
expression/purification kits have been produced commercially. See,
for instance, the QIAexpress.TM. expression system from QIAGEN
(Chatsworth, Calif.) and various expression systems provided by
INVITROGEN (Carlsbad, Calif.). Depending on the details provided by
the manufactures, such kits can be used for production and
purification of the disclosed hybrid interferons.
The following procedure can be used to express hybrid interferons
as disclosed herein. Plasmid DNA molecules carrying parental
interferons IFNA2 (pBluescript/A2) and IFNA21 (pQE30/A21), and
hybrid interferons HY-2 (pHY-2) and HY-3 (pHY-3) are transformed
into E. coli strain SG13009 [pREP4] (QIAGEN, Chatsworth, Calif.).
pHY-1 plasmid DNA is transformed into E. coli strain DH5.alpha.F'IQ
(Gibco BRL, Gaithersburg, Md.). Bacteria are grown overnight in LB
broth containing 100 .mu.g/ml ampicillin (pHY1) or 100 .mu.g/ml
ampicillin and 25 .mu.g/ml kanamycin (pHY-2, pHY-3, pBluescript/A2
and pQE30/A21) in a 37.degree. C. shaker incubator. The cultures
are diluted 1:50 in LB Broth containing the appropriate
antibiotic(s) and incubated at 37.degree. C. with shaking, to a
cell density of 0.8-0.9 A.sub.600. Protein expression is induced by
the addition of 2 mM isopropyl-1-thio-.beta.-D-galactopyranoside
(IPTG). The bacteria are then incubated at 30.degree. C. for 4-5
hours, after which cells were harvested and lysed by sonication.
Each cell lysate is clarified by centrifugation at 10,000.times.g
for 30 minutes at 4.degree. C. The resultant supernatants are used
for subsequent purification of IFN polypeptides.
B. Purification of Interferon Hybrids and Mutants
One skilled in the art will understand that there are myriad ways
to purify recombinant interferon polypeptides. Typical methods of
protein purification may be used to purify the disclosed
interferons. Such methods include, for instance, monoclonal
antibody affinity chromatography and isolation of insoluble protein
inclusion bodies after over production. In addition, purification
affinity-tags, for instance a hexa-histidine sequence, may be
recombinantly fused to the protein and used to facilitate
polypeptide purification. For further examples of purification of
interferons, see U.S. Pat. No. 5,089,400 ("Polypeptides and process
for the production thereof") and Zoon et al., (1992).
In additional to the general protein purification procedures,
certain modifications specific to interferon purification of may be
beneficial. See for instance U.S. Pat. No. 5,593,667 ("Recombinant
immune interferon having an intact carboxyl terminus"), disclosing
IFN extraction techniques that overcome certain difficulties
associated with degradation of the carboxy-terminal region of
interferons during extraction and purification.
Commercially produced protein expression/purification kits provide
tailored protocols for the purification of proteins made using each
system. See, for instance, the QIAexpress.TM. expression system
from QIAGEN (Chatsworth, Calif.) and various expression systems
provided by INVITROGEN (Carlsbad, Calif.). Where a commercial kit
is employed to produce the hybrid interferons, the manufacturer's
purification protocol is a preferred protocol for purification of
that hybrid. For instance, proteins expressed with an
amino-terminal hexa-histidine tag can be purified by binding to
nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography
matrix (The QIAexpressionist, QIAGEN, 1997)
By way of example only, the following procedure can be used to
purify hybrid interferons. Expression of parental interferons
IFN-.alpha.2c and IFN-.alpha.21a, and three IFN hybrids (HY-1, -2,
and -3) is obtained in E. coli using the QIAexpress.TM. expression
system plasmid pQE30. IFN polypeptide purification is first
performed by Ni-NTA-Agarose resin metal-affinity chromatography
(The QIAexpressionist, QIAGEN, 1997; Janknecht et al., 1991). The
specific antiviral activity of this partially purified material
ranges from 3.times.10.sup.6 IU/mg protein to 4.5.times.10.sup.6
IU/mg protein on Madin-Darby bovine kidney (MDBK) cells (ATCC #:
CCL-22). To attain higher specific activity, the IFN-.alpha.s may
optionally be further purified by monoclonal antibody affinity
chromatography (e.g., 4F2, NK2) (Zoon et al., 1992). Final specific
activities of each IFN species are shown in Table 1. Generally,
activities range from 2.times.10.sup.8 IU/mg protein to
3.7.times.10.sup.8 IU/mg protein on MDBK cells and from
0.1.times.10.sup.8 IU/mg protein to 1.9.times.10.sup.8 IU/mg
protein on WISH cells (ATCC #: CCL-25). Purified recombinant
protein concentrations are determined using the Coomassie Plus
protein assay (PIERCE, Rockford, Ill). Purity of the recombinant
IFN-.alpha.s can be assessed by SDS-PAGE and HPLC analysis.
Similar procedures can be used to produce and purify the interferon
hybrids HY-4, -5, and -6, as well as mutant interferon hybrids
SDM-1, -2, -3, and 4.
IV. Activity of Hybrid and Mutant Hybrid Interferons
A. Antiproliferative Activity
The antiproliferative activities of several purified IFN-.alpha.
hybrids and mutant hybrids were compared to parental interferons
IFN-.alpha.21a and IFN-.alpha.2c. The ability of the IFN-.alpha.
hybrids, mutants and both parents to inhibit the growth of Daudi,
WISH and primary human lymphocyte cells was compared.
Human Daudi cells that are sensitive to IFN were obtained from Dr.
P. Grimley, Dept. of Pathology, USUHS, Bethesda Md. Cells were
grown in suspension using RPM1 1640 with 10% fetal calf serum
(FCS), 2 mM glutamine and 0.2% gentamicin. WISH cells as above were
grown as monolayer cultures using Eagle's minimal essential medium
supplemented with 10% FCS and gentamicin (50 .mu.g/ml). The
cultures were incubated at 37.degree. C. in a humidified atmosphere
containing 5% CO.sub.2. All cultures were free of mycoplasma.
Antiproliferative assays on Daudi cells were performed as
previously described (Hu et al., 1993). The assays on WISH cells
were performed by incubating the cells with various IFN-.alpha.s at
concentrations ranging from 0.0003 ng/ml to 300 ng/ml for 72 hours
at 37.degree. C. A 50 .mu.l aliquot of 2 mg/ml
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT)
was added into each well and incubated for 4 hours at 37.degree. C.
Then, 10% SDS in 0.01 N HCl (250 .mu.l) was added to each well and
incubated overnight at 37.degree. C. The OD.sub.570 of each well
was determined, and the percentage of growth inhibition was
calculated by comparing control (untreated) cultures with the
IFN-treated cultures.
Primary human lymphocytes were treated with phytohemagglutinin
(PHA) (Promega) at 1 .mu.g/ml overnight, and the resultant PHA
blasts treated for 72 hours with the various IFNs at the
concentrations ranging from 0 to 1000 ng/ml. Percent inhibition of
proliferation was calculated from direct cell counts, performed by
Coulter counter analysis. Results are shown in FIG. 2.
The concentrations of IFN-.alpha.s that inhibited Daudi and WISH
cell growth by 50% are shown in Table 1. HY-3 exhibited a higher
antiproliferative activity than parental interferons IFN-.alpha.2c
and IFN-.alpha.21a and the other hybrids on Daudi, WISH and primary
human lymphocyte cells. In comparison, hybrids HY-2 and HY-4 have
lower antiproliferative activities than the other hybrids or either
of the parental IFN-.alpha.s on all these cells. HY-2 displays a
10,000 fold decrease in antiproliferative activity compared to HY-3
on Daudi cells and greater than a 1000 fold decrease on WISH and
primary human cells. The hybrid HY-1 has a two- to eight-fold
greater antiproliferative activity than HY-2 on Daudi and WISH
cells. An intermediate level of antiproliferative activity on Daudi
cells was found in the hybrid interferon HY-5 and the two mutant
interferon hybrids SDM-1 and SDM-2.
B. Antiviral Activity
The antiviral activities of purified IFN-.alpha. hybrids HY-1
through HY-5 and mutant hybrid interferons SDM1 through SDM-4 were
compared to that of parental interferons IFN-.alpha.21a and
IFN-.alpha.2c. MDBK cells (ATCC, Manassas, Va.; ATCC #: CCL-22)
were prepared and maintained as described (Zoon et al., 1992). WISH
cells were prepared and maintained as above.
Antiviral activity was determined as previously described using
MDBK cells and WISH cells (Zoon et al., 1992). All IFN units are
expressed with reference to the NIH human lymphoblastoid IFN
standard Ga 23-901-532.
The specific antiviral activities on MDBK and WISH cells are shown
in Table 1. The antiviral specific activities of the five hybrids
and four mutant hybrids are similar to each other and to
IFN-.alpha.2c and IFN-.alpha.21a on MDBK cells (2.0.times.10.sup.8
IU/mg to 5.0.times.10.sup.8 IU/mg). The specific activities of HY-1
and HY-2 are seven-fold lower than that of HY-3 on WISH cells.
The Edmonston strain of measles virus (low passage, human embryonic
kidney 7, VERO 5) (Albrecht et al., 1981) was plaque-purified and
used to infect 1.times.10.sup.6 primary human lymphocytes that had
been primed for expansion with phytohemagglutinin (PHA). Primary
human lymphocytes were obtained from normal donors by centrifugal
elutriation after Ficoll-Hypaque sedimentation (Lymphocyte
Separation Medium (LSM) Package Insert, ORGANON TEKNIKA, Durham,
N.C.). 1.times.108 cells were resuspended in RPMI-1640 media
supplemented with 10% FCS and fungizone (containing penicillin,
streptomycin, and amphotericin B) with or without with 100 ng/ml of
parental or hybrid interferon for 24 hours prior to infection.
These cells were then either infected with measles virus at moi of
0.1-1.6 TCID.sub.50 /ml, or mock infected with virus-free medium,
and harvested 72 hours post infection. Supernatants were titrated
on VERO cell monolayers. Measles virus cytopathic effect was
evaluated microscopically after 6 days and confirmed by staining
with crystal violet. FIG. 3 shows the results from lymphocytes from
two donors. All experiments were preformed in triplicate and the
results are expressed as percent of control; 100 percent for donors
1 and 2 were 4.95 and 5.7 TCID.sub.50 log.sub.10, respectively.
C. Interferon Binding
Interferon binding assays were performed as previously described on
human Daudi (Hu et al., 1993) and WISH (Zoon et al., 1982) cells.
Human IFN-.alpha.2b was obtained from Schering Corp. (Kenilworth,
N.J.), and has an antiviral specific activity of 2.times.10.sup.8
IU/mg protein. IFN-.alpha.2b was labeled with .sup.125
I-Bolton-Hunter reagent (Amersham, Arlington Heights, Ill.) as
previously described (Hu et al., 1993).
FIG. 4 shows the competitive binding curves for .sup.125 I-labeled
IFN-.alpha.2b using IFN-.alpha.2c, IFN-21a and the three hybrids
(HY-1, HY-2, HY-3) as competitors on Daudi (Panel A) and WISH
(Panel B) cells, respectively. The IFN-.alpha.2c parent and the
hybrid HY-3 compete very well for the .sup.125 I-IFN-.alpha.2b
binding site on Daudi and WISH cells, while hybrids HY-1 and HY-2,
like IFN-.alpha.21a, compete poorly for the .sup.125
I-IFN-.alpha.2b of binding site on Daudi cells. These results are
summarized in Table 1.
V. Production of Hybrid Interferon Variants
Of the hybrid IFN-.alpha.s herein described, HY-3 has by far the
highest antiproliferative activity, exhibiting 1000-10,000-fold
higher activity than HY-2 and HY-4. A possible explanation for this
high activity is the existence of a domain affecting the
antiproliferative activity within the carboxy-region (about
residues 76-166) of HY-3. This region comprises a middle element of
IFN-.alpha.2c (about residues 76-95) fused to the carboxy-terminal
element of IFN-.alpha.21a (about residues 96-166). One of skill in
the art will appreciate that these elements may be combined to
produce HY-3-like molecules without necessarily splicing the
components in the same place. It might be possible to use shorter
or longer fragments of IFN-.alpha.2c, fused to correspondingly
longer or shorter fragments of IFN-.alpha.21a. For instance, the
middle element of IFN-.alpha.2c might comprise residues 76-96,
76-97 or 76-98, while the carboxy-terminal element of
IFN-.alpha.21a would correspondingly comprise residues 97-166,
98-166, or 99-166, respectively. Any component that is spliced
within 5 amino acid residues of the residue specified comprises
about the same region. For instance, amino acid residues 1-80 or
1-70 of IFN-.alpha.2c comprise about the same amino acid residues
as the component with residues 1-75.
Although in the HY-3 fusion the amino-terminal element of the
hybrid comprises amino acid residues 1-75 of IFN-.alpha.2c, one
skilled in the art will appreciate that other IFN-.alpha.s could be
used to provide this element. For instance, a HY-3-like polypeptide
could be constructed that comprised residues 1-75 of IFN-.alpha.21a
fused to amino acid residues 76-166 of HY-3. Beyond this, it would
be possible to use the amino-terminal element (residues 1-75) of
any IFN-.alpha. (e.g., IFN-.alpha.1, -2, -3, -4, etc.). The
designation for a HY-3-like fusion of this type, wherein the
amino-terminal 1-75 region comprises amino acids chosen from any
IFN-.alpha. species, is
IFN-.alpha.X(1-75)/IFN-.alpha.2c(76-95)/IFN-.alpha.21a(96-166),
wherein "IFN-.alpha.X" designates any IFN-.alpha., including but
not limited to IFN-.alpha.2c and IFN-.alpha.21a. Alternately, such
a HY-3-like molecule can be referred to generally as X-A-B, wherein
"X" comprises about amino acid residues 1-75 of an
interferon-.alpha., "A" comprises about amino acid residues 76-95
of IFN-.alpha.2c, and "B" comprises about amino acid residues
96-166 of IFN-.alpha.21a. As above, it can be appreciated also that
these elements may be spliced in different places. For instance,
the amino-terminal element may comprise residues 1-74 or 1-73,
fused to amino acid residues 75-166 or 74-166 of HY-3,
respectively. Amino-terminal fragments shorter than these could
also be employed, with correspondingly longer middle regions. If a
parental interferon that has one or more point or short deletions
(as found with the 44.sup.th position in IFN-.alpha.2c) is used in
construction of a hybrid, the numbering of the resultant hybrid
fusions should be carried out using the facilitated alignment
system.
It is further possible that the element that confers increased
antiproliferative activity is found wholly within the middle
element of IFN-.alpha.2c (about residues 76-95). This is supported
by the finding that HY-1 has higher antiproliferative activity than
HY-2; the sole difference between these two hybrid interferons is
the middle element. One skilled in the art will therefore
appreciate that another HY-3-like polypeptide could be constructed
which comprises an amino-terminal element (about residues 1-75) of
any IFN-.alpha., fused to the middle element (about residues 76-95)
of IFN-.alpha.2c, further fused to the carboxy-terminal element
(about residues 96-166) of any IFN-.alpha.. In such hybrids, the
amino--(about residues 1-75) and carboxy--(about residues 96-166)
regions may be provided from any single IFN-.alpha., or from two
different IFN-.alpha.s. These hybrid IFN molecules are represented
as X-A-Y, wherein "X" comprises about amino acid residues 1-75 of
any IFN-.alpha., "A" comprises about amino acid residues 76-95 of
IFN-.alpha.2c, and "Y" comprises about amino acid residues 96-166
of any IFN-.alpha.. In addition, as discussed above, the amino- and
carboxy-regions may be shorter than those specified herein, for
instance amino-regions of 1-74 or 1-73 residues, or carboxy-regions
of 97-166 or 96-166. The corresponding middle region of
IFN-.alpha.2c will vary correspondingly in these latter hybrid
molecules. If a parental interferon that has one or more point or
short deletions (as found with the 44.sup.th position in
IFN-.alpha.2c) is used in construction of a hybrid, the numbering
of the resultant hybrid fusions should be carried out using the
facilitated alignment system.
Shorter segments of the IFN-.alpha.2c middle region, for instance
residues 82-95, are sufficient to confer a substantial portion of
the antiproliferative activity found in the HY-3 hybrid. This is
evidenced by the high antiproliferative activity of HY-4 (SEQ ID
NO: 30). In hybrid constructs using this shorter region of
IFN-.alpha.2c, the amino--(about residues 1-81) and carboxy--(about
residues 96-166) regions may be provided from any single
IFN-.alpha., or from two different IFN-.alpha.s. Such a hybrid
interferon-.alpha. molecule with a short IFN-.alpha.2c middle
region may be represented as V-C-Y, wherein "V" comprises about
amino acid residues 1-81 of an interferon-.alpha., "C" comprises
about amino acid residues 82-95 of IFN-.alpha.2c, and "Y" comprises
about amino acid residues 96-166 of an interferon-.alpha.. If a
parental interferon that has one or more point or short deletions
(as found with the 44.sup.th position in IFN-.alpha.2c) is used in
construction of a hybrid, the numbering of the resultant hybrid
fusions should be carried out using the facilitated alignment
system.
More than three segments or domains of different parental
interferons can be used to construct the hybrid IFN-.alpha.s of
this invention. Such multiple domains are taken from at least two
different source or parental interferons, and can be taken from up
to as many different interferons as there are fragments assembled
to construct the hybrid. A four-domain hybrid interferon-.alpha.
therefore will be constructed from as few as two or as many as four
different interferon-.alpha.s. The total length of these constructs
will depend on the length(s) of the constituent parental
interferons used.
One four domain hybrid interferon-.alpha. molecule may be
represented as M-N-O-P, wherein "M" comprises about amino acid
residues 1-75 of interferon .alpha.21a, "N" comprises about amino
acid residues 76 to 81 of interferon-.alpha.2c, "O" comprises about
amino acid residues 82 to 95 of interferon-.alpha.21a, and "P"
comprises about amino acid residues 96 to 166 of
interferon-.alpha.2c. A representative four domain hybrid
interferon of this type is HY-4. If a parental interferon that has
one or more point or short deletions (as found with the 44.sup.th
position in IFN-.alpha.2c) is used in construction of a multidomain
hybrid, the numbering of the resultant hybrid fusions should be
carried out using the facilitated alignment system.
At least a substantial portion of the antiproliferative activity
found in HY-3 and the related hybrid and mutant interferons is
linked to the presence of either or both of the tyrosine residues
found at positions 86 and 90 in the fusion proteins. With the
provision herein of the information that these residues are
important in interferon biological activity, and specifically
antiproliferative activity, this invention also encompasses mutant
interferon-.alpha.s and mutant hybrid interferon-.alpha.s that
contain point mutations at either residue 86 or residue 90, thereby
changing these residues either to or from a tyrosine. Such point
mutations can be introduced into interferon-.alpha.s and hybrid
interferons through any available mutagenesis techniques, including
but not limited to site-directed and PCR mediated mutagenesis.
Mutant hybrid interferon polypeptides can for instance contain
short or long segments of IFN-.alpha.2c, IFN-.alpha.21a, or both of
these parental interferons. Specific representatives of these
mutant hybrid interferons include SDM-1, SDM-2, SDM-3, and
SDM4.
VI. Incorporation of Hybrid and Mutant IFN-.alpha.s into
Pharmaceutical Compositions
Pharmaceutical compositions that comprise at least one hybrid or
mutant hybrid interferon as described herein as an active
ingredient will normally be formulated with an appropriate solid or
liquid carrier, depending upon the particular mode of
administration chosen. The pharmaceutically acceptable carriers and
excipients useful in this invention are conventional. For instance,
parenteral formulations usually comprise injectable fluids that are
pharmaceutically and physiologically acceptable fluid vehicles such
as water, physiological saline, other balanced salt solutions,
aqueous dextrose, glycerol or the like. Excipients that can be
included are, for instance, other proteins, such as human serum
albumin or plasma preparations. If desired, the pharmaceutical
composition to be administered may also contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
Other medicinal and pharmaceutical agents, including non-hybrid
interferons, also may be included.
The dosage form of the pharmaceutical composition will be
determined by the mode of administration chosen. For instance, in
addition to injectable fluids, topical and oral formulations can be
employed. Topical preparations can include eye drops, ointments,
sprays and the like. Oral formulations may be liquid (e.g., syrups,
solutions or suspensions), or solid (e.g., powders, pills, tablets,
or capsules). For solid compositions, conventional non-toxic solid
carriers can include pharmaceutical grades of mannitol, lactose,
starch, or magnesium stearate. Actual methods of preparing such
dosage forms are known, or will be apparent, to those skilled in
the art; for example, see Remington's Pharmaceutical Sciences, E.
W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition
(1975).
The pharmaceutical compositions that comprise hybrid interferon
polypeptide will preferably be formulated in unit dosage form,
suitable for individual administration of precise dosages. One
possible unit dosage contains approximately 100 .mu.g of protein.
The amount of active compound administered will be dependent on the
subject being treated, the severity of the affliction, and the
manner of administration, and is best left to the judgment of the
prescribing clinician. Within these bounds, the formulation to be
administered will contain a quantity of the active component(s) in
an amount effective to achieve the desired effect in the subject
being treated.
The herein disclosed hybrid IFNs can also be administered to a
patient using other acceptable delivery systems, including
liposome-mediated delivery systems as disclosed in WO 96/17596
("Liposomal interferon hybrid compositions").
The serum half-life of the administered hybrid IFN polypeptide can
be extended in various ways, for instance, through formation of a
complex with a monoclonal antibody. Such an antibody is usually
directed to the hybrid IFN polypeptide at a site that does not
materially impair its therapeutic activity (U.S. Pat. No. 5,055,289
"Interferon antibody compositions having an extended serum
half-life"). Alternately, interferons can be conjugated to
non-antigenic polymers, such as polyethylene glycol or related
polyakylene glycol moieties, to increase their serum persistence.
See, for instance, Nieforth et al., (1996) and U.S. Pat. No.
5,681,811 ("Conjugation-stabilized therapeutic agent compositions,
delivery and diagnostic formulations comprising same, and method of
making and using same"); U.S. Pat. No. 5,711,944 ("Interferon
polymer conjugates"); and U.S. Pat. No. 5,738,846 ("Interferon
polymer conjugates and process for preparing the same").
VII. Clinical Usage of Hybrid and Mutant Hybrid Interferons
The cell growth-regulating activity exhibited by the disclosed
hybrid interferons makes these hybrids useful for treating tumors
and cancers such as osteogenic sarcoma; multiple myeloma; Hodgkin's
disease; nodular, poorly differentiated lymphoma; acute lymphocytic
leukemia; acute myeloid leukemia; breast carcinoma; melanoma;
papilloma; and nasopharyngeal carcinoma. In addition, the antiviral
activity exhibited makes the disclosed hybrid interferons useful
for treating viral infections in human and other animal patients.
Possibly susceptible virus infections include, but are not limited
to, encephalomyocarditis virus infection, influenza and other
respiratory tract virus infections, rabies and other viral
zoonoses, and arbovirus infections, as well as herpes simplex
keratitis, acute hemorrhagic conjunctivitis, varicella zoster, and
hepatitis B and C.
The hybrid and mutant hybrid interferons of this invention may be
administered to humans, or other animals on whose cells they are
effective, in various manners such as topically, orally,
intravenously, intramuscularly, intraperitoneally, intranasally,
intradermally, intrathecally, and subcutaneously. Administration of
hybrid interferon composition is indicated for patients with
malignancies or neoplasms, whether or not immunosuppressed, or
those patients requiring immunomodulation, or for antiviral
treatment. The particular mode of administration and the dosage
regimen will be selected by the attending clinician, taking into
account the particulars of the case (e.g., the patient, the
disease, and the disease state involved). For instance, tumor or
cancer treatment typically involves daily or multi-daily doses of
hybrid interferon over a period of months or even years. In
contrast, viral infections are usually treated by daily doses of
hybrid IFN over a few days to weeks. The same dose levels as are
used in conventional (non-hybrid) interferon therapy may be used.
See U.S. Pat. No. 4,089,400 ("Polypeptides and process for the
production thereof") and U.S. Pat. No. 5,503,828 ("Alpha interferon
composition and method for its production from human peripheral
blood leukocytes") for general disclosure as to the amounts of
IFN-.alpha. that have proven efficacious in clinical settings. In
general, approximately 10.sup.5 to 10.sup.8 IU will be
appropriate.
In addition to their individual use, a hybrid and mutant hybrid
interferon as disclosed in the current invention may be combined
with or used in association with other chemotherapeutic or
chemopreventive agents for providing therapy against neoplasms or
other conditions against which it is effective. See, for instance,
U.S. Pat. No. 4,805,347 ("Interferon combinations"), which
discloses various compositions and methods for treating tumors and
viruses in humans by administering a combination of IFN-.alpha. and
an IFN-.alpha.2/IFN-.alpha.1 hybrid.
The foregoing examples are provided by way of illustration only.
One of skill in the art will appreciate that numerous variations on
the biological molecules and methods described above may be
employed to make and use IFN-.alpha.21a/IFN-.alpha.2c hybrid
interferons. We claim all such subject matter that falls within the
scope and spirit of the following claims.
TABLE 1 Binding Activity Specific Antiviral Activity (competes with
.sup.125 I- Antiproliferative Activity (ng/ml).sup.a (IU/mg)
IFN-.alpha.2b) Ratio Ratio (ng/ml).sup.b Daudi WISH WISH/ MDBK WISH
MDBK/ Daudi Interferon Cells Cells Daudi Cells Cells WISH cells
WISH Cells IFN-.alpha.2c 0.005 80 1.6 .times. 10.sup.4 3.3 .times.
10.sup.8 1.9 .times. 10.sup.8 1.7 35 25 IFN-.alpha.21a 0.0008 95
1.2 .times. 10.sup.5 3.7 .times. 10.sup.8 0.7 .times. 10.sup.8 5.3
>400 >200 HY-1 0.5 110 2.2 .times. 10.sup.2 2.0 .times.
10.sup.8 0.1 .times. 10.sup.8 20.0 >400 >200 HY-2 1.0
>1000 >1 .times. 10.sup.3 3.0 .times. 10.sup.8 0.1 .times.
10.sup.8 30.0 >400 >200 HY-3 0.0001 8 8 .times. 10.sup.4 2.0
.times. 10.sup.8 0.7 .times. 10.sup.8 2.9 30 30 HY-4 1.0 N/D 4.8
.times. 10.sup.8 N/D N/D N/D HY-5 0.03 N/D 3.0 .times. 10.sup.8 N/D
N/D N/D SDM-1 0.01 N/D 2.7 .times. 10.sup.8 N/D N/D N/D SDM-2 0.013
N/D 4.6 .times. 10.sup.8 N/D N/D N/D SDM-3 N/D N/D 4.6 .times.
10.sup.8 N/D N/D N/D SDM-4 N/D N/D 5.0 .times. 10.sup.8 N/D N/D N/D
.sup.a Concentration of IFN species that inhibits cell growth by
50% .sup.b Concentration of IFN species that inhibits binding of
.sup.125 I-IFN-.alpha.2b by 50%
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SEQUENCE LISTING <100> GENERAL INFORMATION: <160>
NUMBER OF SEQ ID NOS: 42 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 1 <211> LENGTH: 27 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 1 tccggatcct gtgatctgcc tcagacc 27
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 2
<211> LENGTH: 27 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 2
agcagatgag tcctttgtgc tgaagag 27 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 3 <211> LENGTH: 27
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 3 ctcttcagca caaaggactc
atctgct 27 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID
NO 4 <211> LENGTH: 36 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
4 gagctcgcat gctcatcatt ccttacttct taaact 36 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 5 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 5 cacgcaggcc tcgaggtcat tcag
24 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 6
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 6
ctgaatgacc tcgaggcctg cgtg 24 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 7 <211> LENGTH: 36 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 7 gagctcgcat gctcatcatt ccttcctcct taatct 36
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 8
<211> LENGTH: 500 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Gene Fusion <400> SEQUENCE: 8 tgt gat ctg cct
cag acc cac agc ctg ggt aat agg agg gcc ttg ata 48 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 ctc ctg
gca caa atg gga aga atc tct cct ttc tcc tgc ctg aag gac 96 Leu Leu
Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30
aga cat gac ttt gga ttc ccc caa gag gag ttt gat ggc aac cag ttc 144
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35
40 45 cag aag gct caa gcc atc tct gtc ctc cat gag atg atc cag cag
acc 192 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln
Thr 50 55 60 ttc aat ctc ttc agc aca aag gac tca tct gct gct tgg
gat gag acc 240 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 ctc cta gac aaa ttc tac act gaa ctc tac cag
cag ctg aat gac ctg 288 Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln
Gln Leu Asn Asp Leu 85 90 95 gaa gcc tgt gtg ata cag ggg gtg ggg
gtg aca gag act ccc ctg atg 336 Glu Ala Cys Val Ile Gln Gly Val Gly
Val Thr Glu Thr Pro Leu Met 100 105 110 aag gag gac tcc att ctg gct
gtg agg aaa tac ttc caa aga atc act 384 Lys Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 ctc tat ctg aaa gag
aag aaa tac agc cct tgt gcc tgg gag gtt gtc 432 Leu Tyr Leu Lys Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 aga gca gaa
atc atg aga tct ttt tct ttg tca aca aac ttg caa gaa 480 Arg Ala Glu
Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160
agt tta aga agt aag gaa tg 500 Ser Leu Arg Ser Lys Glu 165
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 9
<211> LENGTH: 166 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Gene Fusion <400> SEQUENCE: 9 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu
Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35
40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln
Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Gly Val Gly
Val Thr Glu Thr Pro Leu Met 100 105 110 Lys Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Lys Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160
Ser Leu Arg Ser Lys Glu 165 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 10 <211> LENGTH: 500 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Gene Fusion <400> SEQUENCE: 10
tgt gat ctg cct cag acc cac agc ctg ggt aat agg agg gcc ttg ata 48
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5
10 15 ctc ctg gca caa atg gga aga atc tct cct ttc tcc tgc ctg aag
gac 96 Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys
Asp 20 25 30 aga cat gac ttt gga ttc ccc cag gag gag ttt gat ggc
aac cag ttc 144 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly
Asn Gln Phe 35 40 45 cag aag gct caa gcc atc tct gtc ctc cat gag
atg atc cag cag acc 192 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu
Met Ile Gln Gln Thr 50 55 60 ttc aat ctc ttc agc aca aag gac tca
tct gct act tgg gaa cag agc 240 Phe Asn Leu Phe Ser Thr Lys Asp Ser
Ser Ala Thr Trp Glu Gln Ser 65 70 75 80 ctc cta gaa aaa ttt tcc act
gaa ctt aac cag cag ctg aat gac ctc 288 Leu Leu Glu Lys Phe Ser Thr
Glu Leu Asn Gln Gln Leu Asn Asp Leu 85 90 95 gag gcc tgt gtg ata
cag ggg gtg ggg gtg aca gag act ccc ctg atg 336 Glu Ala Cys Val Ile
Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met 100 105 110 aag gag gac
tcc att ctg gct gtg agg aaa tac ttc caa aga atc act 384 Lys Glu Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 ctc
tat ctg aaa gag aag aaa tac agc cct tgt gcc tgg gaa gtt gtc 432 Leu
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135
140 aga gca gaa atc atg aga tct ttt tct ttg tca aca aac ttg caa gaa
480 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu
145 150 155 160 agt tta aga agt aag gaa tg 500 Ser Leu Arg Ser Lys
Glu 165 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
11 <211> LENGTH: 166 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Gene Fusion <400> SEQUENCE: 11 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu
Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Thr
Trp Glu Gln Ser 65 70 75 80 Leu Leu Glu Lys Phe Ser Thr Glu Leu Asn
Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Gly Val
Gly Val Thr Glu Thr Pro Leu Met 100 105 110 Lys Glu Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Lys
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 12 <211> LENGTH: 497
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Gene Fusion
<400> SEQUENCE: 12 tgt gat ctg cct cag acc cac agc ctg ggt
agc agg agg acc ttg atg 48 Cys Asp Leu Pro Gln Thr His Ser Leu Gly
Ser Arg Arg Thr Leu Met 1 5 10 15 ctc ctg gca cag atg agg aga atc
tct ctt ttc tcc tgc ttg aag gac 96 Leu Leu Ala Gln Met Arg Arg Ile
Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 aga cgt gac ttt gga ttt
ccc cag gag gag ttt ggc aac cag ttc caa 144 Arg Arg Asp Phe Gly Phe
Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 aag gct gaa acc
atc cct gtc ctc cat gag atg atc cag cag atc ttc 192 Lys Ala Glu Thr
Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 aat ctc
ttc agc aca aag gac tca tct gct gct tgg gat gag acc ctc 240 Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80
cta gac aaa ttc tac act gaa ctc tac cag cag ctg aat gac ctc gag 288
Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85
90 95 gcc tgc gtg ata cag gag gtt ggg gtg gaa gag act ccc ctg atg
aat 336 Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met
Asn 100 105 110 gtg gac tcc atc ctg gct gtg aag aaa tac ttc caa aga
atc act ctt 384 Val Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125 tat ctg aca gag aag aaa tac agc cct tgt gcc
tgg gag gtt gtc aga 432 Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140 gca gaa atc atg aga tcc ttc tct tta
tca aaa att ttt caa gaa aga 480 Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Lys Ile Phe Gln Glu Arg 145 150 155 160 tta agg agg aag gaa tg
497 Leu Arg Arg Lys Glu 165 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 13
<211> LENGTH: 165 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Gene Fusion <400> SEQUENCE: 13 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu
Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30
Arg Arg Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35
40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile
Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln
Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Glu Val Gly Val
Glu Glu Thr Pro Leu Met Asn 100 105 110 Val Asp Ser Ile Leu Ala Val
Lys Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Thr Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Lys Ile Phe Gln Glu Arg 145 150 155 160
Leu Arg Arg Lys Glu 165 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 14 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 14 gctgcttggg atgagaccct ccta 24 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 15 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 15 taggagggtc
tcatcccaag cagc 24 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 16 <211> LENGTH: 30 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 16 ctagacaaat tctacactga actctaccag 30
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 17
<211> LENGTH: 30 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 17
ctggtagagt tcagtgtaga atttgtctag 30 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 18 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 18 ctgaatgacc tcgaggcctg cgtg
24 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 19
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 19
cacgcaggcc tcgaggtcat tcag 24 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 20 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 20 gaaaaatttt acactgaact t 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 21 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 21 aagttcagtg
taaaattttt c 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 22 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 22 actgaacttt accagcagct g 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 23 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synethetic Oligonucleotide <400> SEQUENCE: 23 cagctgctgg
taaagttcag t 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 24 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 24 gacaaattct ccactgaact c 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 25 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 25 gagttcagtg
gagaatttgt c 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 26 <211> LENGTH: 21 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 26 actgaactca accagcagct g 21 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 27 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 27 cagctgctgg
ttgagttcag t 21 <200> SEQUENCE CHARACTERISTICS: <210>
SEQ ID NO 28 <211> LENGTH: 36 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 28 gagctcgcat gctcatcatt ccttacttct taaact 36
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 29
<211> LENGTH: 500 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Gene Fusion <400> SEQUENCE: 29 tgt gat ctg cct
cag acc cac agc ctg ggt aat agg agg gcc ttg ata 48 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 ctc ctg
gca caa atg gga aga atc tct cct ttc tcc tgc ctg aag gac 96 Leu Leu
Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30
aga cat gac ttt gga ttc ccc cag gag gag ttt gat ggc aac cag ttc 144
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35
40 45 cag aag gct caa gcc atc tct gtc ctc cat gag atg atc cag cag
acc 192 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln
Thr 50 55 60 ttc aat ctc ttc agc aca aag gac tca tct gct gct tgg
gat gag acc 240 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 ctc cta gaa aaa ttt tcc act gaa ctt aac cag
cag ctg aat gac ctc 288 Leu Leu Glu Lys Phe Ser Thr Glu Leu Asn Gln
Gln Leu Asn Asp Leu 85 90 95 gag gcc tgt gtg ata cag ggg gtg ggg
gtg aca gag act ccc ctg atg 336 Glu Ala Cys Val Ile Gln Gly Val Gly
Val Thr Glu Thr Pro Leu Met 100 105 110 aag gag gac tcc att ctg gct
gtg agg aaa tac ttc caa aga atc act 384 Lys Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 ctc tat ctg aaa gag
aag aaa tac agc cct tgt gcc tgg gag gtt gtc 432 Leu Tyr Leu Lys Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 aga gca gaa
atc atg aga tct ttt tct ttg tca aca aac ttg caa gaa 480 Arg Ala Glu
Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160
agt tta aga agt aag gaa tg 500 Ser Leu Arg Ser Lys Glu 165
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 30
<211> LENGTH: 166 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Gene Fusion <400> SEQUENCE: 30 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu
Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35
40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Ser Thr Glu Leu Asn Gln
Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Gly Val Gly
Val Thr Glu Thr Pro Leu Met 100 105 110 Lys Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Lys Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160
Ser Leu Arg Ser Lys Glu 165 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 31 <211> LENGTH: 500 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Gene Fusion <400> SEQUENCE: 31
tgt gat ctg cct cag acc cac agc ctg ggt aat agg agg gcc ttg ata 48
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5
10 15 ctc ctg gca caa atg gga aga atc tct cct ttc tcc tgc ctg aag
gac 96 Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys
Asp 20 25 30 aga cat gac ttt gga ttc ccc cag gag gag ttt gat ggc
aac cag ttc 144 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly
Asn Gln Phe 35 40 45
cag aag gct caa gcc atc tct gtc ctc cat gag atg atc cag cag acc 192
Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50
55 60 ttc aat ctc ttc agc aca aag gac tca tct gct act tgg gaa cag
agc 240 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Thr Trp Glu Gln
Ser 65 70 75 80 ctc cta gac aaa ttc tac act gaa ctc tac cag cag ctg
aat gac ctc 288 Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu
Asn Asp Leu 85 90 95 gag gcc tgt gtg ata cag ggg gtg ggg gtg aca
gag act ccc ctg atg 336 Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr
Glu Thr Pro Leu Met 100 105 110 aag gag gac tcc att ctg gct gtg agg
aaa tac ttc caa aga atc act 384 Lys Glu Asp Ser Ile Leu Ala Val Arg
Lys Tyr Phe Gln Arg Ile Thr 115 120 125 ctc tat ctg aaa gag aag aaa
tac agc cct tgt gcc tgg gag gtt gtc 432 Leu Tyr Leu Lys Glu Lys Lys
Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 aga gca gaa atc atg
aga tct ttt tct ttg tca aca aac ttg caa gaa 480 Arg Ala Glu Ile Met
Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 agt tta
aga agt aag gaa tg 500 Ser Leu Arg Ser Lys Glu 165 <200>
SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 32 <211>
LENGTH: 166 <212> TYPE: PRT <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION: Gene
Fusion <400> SEQUENCE: 32 Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu Ala Gln Met Gly Arg
Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala
Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 Phe
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Thr Trp Glu Gln Ser 65 70
75 80 Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp
Leu 85 90 95 Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr
Pro Leu Met 100 105 110 Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr
Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Lys Glu Lys Lys Tyr Ser
Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser
Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser
Lys Glu 165 <200> SEQUENCE CHARACTERISTICS: <210> SEQ
ID NO 33 <211> LENGTH: 500 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Gene Fusion <400> SEQUENCE: 33 tgt gat ctg
cct cag acc cac agc ctg ggt aat agg agg gcc ttg ata 48 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 ctc
ctg gca caa atg gga aga atc tct cct ttc tcc tgc ctg aag gac 96 Leu
Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 aga cat gac ttt gga ttc ccc caa gag gag ttt gat ggc aac cag ttc
144 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45 cag aag gct caa gcc atc tct gtc ctc cat gag atg atc cag
cag acc 192 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln
Gln Thr 50 55 60 ttc aat ctc ttc agc aca aag gac tca tct gct gct
tgg gat gag acc 240 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 ctc cta gac aaa ttc tac act gaa ctc tac
cag cag ctg aat gac ctg 288 Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
Gln Gln Leu Asn Asp Leu 85 90 95 gaa gcc tgc gtg ata cag gag gtt
ggg gtg gaa gag act ccc ctg atg 336 Glu Ala Cys Val Ile Gln Glu Val
Gly Val Glu Glu Thr Pro Leu Met 100 105 110 aat gtg gac tcc atc ttg
gct gtg aag aaa tac ttc caa aga atc act 384 Asn Val Asp Ser Ile Leu
Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr 115 120 125 ctt tat ctg aca
gag aag aaa tac agc cct tgt gct tgg gag gtt gtc 432 Leu Tyr Leu Thr
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 aga gca
gaa atc atg aga tcc ttc tct tta tca aaa att ttt caa gaa 480 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Lys Ile Phe Gln Glu 145 150 155
160 aga tta agg agg aag gaa tg 500 Arg Leu Arg Arg Lys Glu 165
<200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO 34
<211> LENGTH: 166 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Gene Fusion <400> SEQUENCE: 34 Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu
Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35
40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln
Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Glu Thr 65 70 75 80 Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln
Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Glu Val Gly
Val Glu Glu Thr Pro Leu Met 100 105 110 Asn Val Asp Ser Ile Leu Ala
Val Lys Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Thr Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu
Ile Met Arg Ser Phe Ser Leu Ser Lys Ile Phe Gln Glu 145 150 155 160
Arg Leu Arg Arg Lys Glu 165 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 35 <211> LENGTH: 500 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Gene Fusion <400> SEQUENCE: 35
tgt gat ctg cct cag acc cac agc ctg ggt aat agg agg gcc ttg ata 48
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5
10 15 ctc ctg gca caa atg gga aga atc tct cct ttc tcc tgc ctg aag
gac 96 Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys
Asp 20 25 30 aga cat gac ttt gga ttc ccc cag gag gag ttt gat ggc
aac cag ttc 144 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly
Asn Gln Phe 35 40 45 cag aag gct caa gcc atc tct gtc ctc cat gag
atg atc cag cag acc 192 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu
Met Ile Gln Gln Thr 50 55 60 ttc aat ctc ttc agc aca aag gac tca
tct gct gct tgg gat gag acc 240 Phe Asn Leu Phe Ser Thr Lys Asp Ser
Ser Ala Ala Trp Asp Glu Thr 65 70 75 80 ctc cta gaa aaa ttt tac act
gaa ctt aac cag cag ctg aat gac ctc 288 Leu Leu Glu Lys Phe Tyr Thr
Glu Leu Asn Gln Gln Leu Asn Asp Leu 85 90 95 gag gcc tgt gtg ata
cag ggg gtg ggg gtg aca gag act ccc ctg atg 336 Glu Ala Cys Val Ile
Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met 100 105 110 aag gag gac
tcc att ctg gct gtg agg aaa tac ttc caa aga atc act 384 Lys Glu Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 ctc
tat ctg aaa gag aag aaa tac agc cct tgt gcc tgg gag gtt gtc 432 Leu
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135
140 aga gca gaa atc atg aga tct ttt tct ttg tca aca aac ttg caa gaa
480 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu
145 150 155 160 agt tta aga agt aag gaa tg 500 Ser Leu Arg Ser Lys
Glu 165 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
36 <211> LENGTH: 166 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Gene Fusion <400> SEQUENCE: 36 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu
Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr 65 70 75 80 Leu Leu Glu Lys Phe Tyr Thr Glu Leu Asn
Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Gly Val
Gly Val Thr Glu Thr Pro Leu Met 100 105 110 Lys Glu Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Lys
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 37 <211> LENGTH: 500
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Gene Fusion
<400> SEQUENCE: 37 tgt gat ctg cct cag acc cac agc ctg ggt
aat agg agg gcc ttg ata 48 Cys Asp Leu Pro Gln Thr His Ser Leu Gly
Asn Arg Arg Ala Leu Ile 1 5 10 15 ctc ctg gca caa atg gga aga atc
tct cct ttc tcc tgc ctg aag gac 96 Leu Leu Ala Gln Met Gly Arg Ile
Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 aga cat gac ttt gga ttc
ccc cag gag gag ttt gat ggc aac cag ttc 144 Arg His Asp Phe Gly Phe
Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 cag aag gct caa
gcc atc tct gtc ctc cat gag atg atc cag cag acc 192 Gln Lys Ala Gln
Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 ttc aat
ctc ttc agc aca aag gac tca tct gct gct tgg gat gag acc 240 Phe Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 65 70 75 80
ctc cta gaa aaa ttt tcc act gaa ctt tac cag cag ctg aat gac ctc 288
Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85
90 95 gag gcc tgt gtg ata cag ggg gtg ggg gtg aca gag act ccc ctg
atg 336 Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
Met 100 105 110 aag gag gac tcc att ctg gct gtg agg aaa tac ttc caa
aga atc act 384 Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr 115 120 125 ctc tat ctg aaa gag aag aaa tac agc cct tgt
gcc tgg gag gtt gtc 432 Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val 130 135 140 aga gca gaa atc atg aga tct ttt tct
ttg tca aca aac ttg caa gaa 480 Arg Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 agt tta aga agt aag gaa
tg 500 Ser Leu Arg Ser Lys Glu 165 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 38 <211> LENGTH: 166
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Gene Fusion
<400> SEQUENCE: 38 Cys Asp Leu Pro Gln Thr His Ser Leu Gly
Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu Ala Gln Met Gly Arg Ile
Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe
Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45
Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50
55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu
Thr 65 70 75 80 Leu Leu Glu Lys Phe Ser Thr Glu Leu Tyr Gln Gln Leu
Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr
Glu Thr Pro Leu Met 100 105 110 Lys Glu Asp Ser Ile Leu Ala Val Arg
Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Lys Glu Lys Lys
Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met
Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu
Arg Ser Lys Glu 165 <200> SEQUENCE CHARACTERISTICS:
<210> SEQ ID NO 39 <211> LENGTH: 500 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Gene Fusion <400> SEQUENCE: 39
tgt gat ctg cct cag acc cac agc ctg ggt aat agg agg gcc ttg ata 48
Cys Asp Leu Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5
10 15 ctc ctg gca caa atg gga aga atc tct cct ttc tcc tgc ctg aag
gac 96 Leu Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys
Asp 20 25 30 aga cat gac ttt gga ttc ccc cag gag gag ttt gat ggc
aac cag ttc 144 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly
Asn Gln Phe 35 40 45 cag aag gct caa gcc atc tct gtc ctc cat gag
atg atc cag cag acc 192 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu
Met Ile Gln Gln Thr 50 55 60 ttc aat ctc ttc agc aca aag gac tca
tct gct act tgg gaa cag agc 240 Phe Asn Leu Phe Ser Thr Lys Asp Ser
Ser Ala Thr Trp Glu Gln Ser 65 70 75 80 ctc cta gac aaa ttc tcc act
gaa ctc tac cag cag ctg aat gac ctc 288 Leu Leu Asp Lys Phe Ser Thr
Glu Leu Tyr Gln Gln Leu Asn Asp Leu 85 90 95 gag gcc tgt gtg ata
cag ggg gtg ggg gtg aca gag act ccc ctg atg 336 Glu Ala Cys Val Ile
Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met 100 105 110 aag gag gac
tcc att ctg gct gtg agg aaa tac ttc caa aga atc act 384 Lys Glu Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 ctc
tat ctg aaa gag aag aaa tac agc cct tgt gcc tgg gag gtt gtc 432 Leu
Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135
140 aga gca gaa atc atg aga tct ttt tct ttg tca aca aac ttg caa gaa
480 Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu
145 150 155 160 agt tta aga agt aag gaa tg 500 Ser Leu Arg Ser Lys
Glu 165 <200> SEQUENCE CHARACTERISTICS: <210> SEQ ID NO
40 <211> LENGTH: 166 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Gene Fusion <400> SEQUENCE: 40 Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu
Leu Ala Gln Met Gly Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp 20 25
30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45 Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Ile Gln
Gln Thr 50 55 60 Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Thr
Trp Glu Gln Ser 65 70 75 80 Leu Leu Asp Lys Phe Ser Thr Glu Leu Tyr
Gln Gln Leu Asn Asp Leu 85 90 95 Glu Ala Cys Val Ile Gln Gly Val
Gly Val Thr Glu Thr Pro Leu Met 100 105 110 Lys Glu Asp Ser Ile Leu
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr 115 120 125 Leu Tyr Leu Lys
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 130 135 140 Arg Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu 145 150 155
160 Ser Leu Arg Ser Lys Glu 165 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 41 <211> LENGTH: 500
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Gene Fusion
<400> SEQUENCE: 41 tgt gat ctg cct cag acc cac agc ctg ggt
aat agg agg gcc ttg ata 48 Cys Asp Leu Pro Gln Thr His Ser Leu Gly
Asn Arg Arg Ala Leu Ile 1 5 10 15 ctc ctg gca caa atg gga aga atc
tct cct ttc tcc tgc ctg aag gac 96 Leu Leu Ala Gln Met Gly Arg Ile
Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 aga cat gac ttt gga ttc
ccc cag gag gag ttt gat ggc aac cag ttc 144 Arg His Asp Phe Gly Phe
Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 cag aag gct caa
gcc atc tct gtc ctc cat gag atg atc cag cag acc 192 Gln Lys Ala Gln
Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 ttc aat
ctc ttc agc aca aag gac tca tct gct act tgg gaa cag agc 240 Phe Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Thr Trp Glu Gln Ser 65 70 75 80
ctc cta gac aaa ttc tac act gaa ctc aac cag cag ctg aat gac ctc 288
Leu Leu Asp Lys Phe Tyr Thr Glu Leu Asn Gln Gln Leu Asn Asp Leu 85
90 95 gag gcc tgt gtg ata cag ggg gtg ggg gtg aca gag act ccc ctg
atg 336 Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
Met 100 105 110 aag gag gac tcc att ctg gct gtg agg aaa tac ttc caa
aga atc act 384 Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr 115 120 125 ctc tat ctg aaa gag aag aaa tac agc cct tgt
gcc tgg gag gtt gtc 432 Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val 130 135 140 aga gca gaa atc atg aga tct ttt tct
ttg tca aca aac ttg caa gaa 480 Arg Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 agt tta aga agt aag gaa
tg 500 Ser Leu Arg Ser Lys Glu 165 <200> SEQUENCE
CHARACTERISTICS: <210> SEQ ID NO 42 <211> LENGTH: 166
<212> TYPE: PRT <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Gene Fusion
<400> SEQUENCE: 42 Cys Asp Leu Pro Gln Thr His Ser Leu Gly
Asn Arg Arg Ala Leu Ile 1 5 10 15 Leu Leu Ala Gln Met Gly Arg Ile
Ser Pro Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe
Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe 35 40 45 Gln Lys Ala Gln
Ala Ile Ser Val Leu His Glu Met Ile Gln Gln Thr 50 55 60 Phe Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Thr Trp Glu Gln Ser 65 70 75 80
Leu Leu Asp Lys Phe Tyr Thr Glu Leu Asn Gln Gln Leu Asn Asp Leu 85
90 95 Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
Met 100 105 110 Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr 115 120 125 Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val 130 135 140 Arg Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu 145 150 155 160 Ser Leu Arg Ser Lys Glu
165
* * * * *